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Scavone F, Lian S, Eskelinen EL, Cohen RE, Yao T. Trafficking of K63-polyubiquitin-modified membrane proteins in a macroautophagy-independent pathway is linked to ATG9A. Mol Biol Cell 2025; 36:ar42. [PMID: 39969968 DOI: 10.1091/mbc.e24-12-0535] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/20/2025] Open
Abstract
Cytoplasmic K63-linked polyubiquitin signals have well-established roles in endocytosis and selective autophagy. However, how these signals help to direct different cargos to different intracellular trafficking routes is unclear. Here we report that, when the K63-polyubiquitin signal is blocked by intracellular expression of a high-affinity sensor (named Vx3), many proteins originating from the plasma membrane are found trapped in clusters of small vesicles that colocalize with ATG9A, a transmembrane protein that plays an essential role in autophagy. Importantly, whereas ATG9A is required for cluster formation, other core autophagy machinery as well as selective autophagy cargo receptors are not required. Although the cargos are sequestered in the vesicular clusters in an ATG9-dependent manner, additional signals are needed to induce LC3 conjugation. Upon removal of the Vx3 block, K63-polyubiquitylated cargos are rapidly delivered to lysosomes. These observations suggest that ATG9A plays an unexpected role in the trafficking of K63-polyubiquitin-modified membrane proteins.
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Affiliation(s)
- Francesco Scavone
- Department of Biochemistry and Molecular Biology, Colorado State University, Fort Collins, CO 80523, USA
| | - Sharon Lian
- Department of Biochemistry and Molecular Biology, Colorado State University, Fort Collins, CO 80523, USA
| | - Eeva-Liisa Eskelinen
- Molecular and Integrative Biosciences Research Programme, University of Helsinki, Helsinki, 00014, Finland
- Institute of Biomedicine, University of Turku, Turku, FI-20520, Finland
| | - Robert E Cohen
- Department of Biochemistry and Molecular Biology, Colorado State University, Fort Collins, CO 80523, USA
| | - Tingting Yao
- Department of Biochemistry and Molecular Biology, Colorado State University, Fort Collins, CO 80523, USA
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Liu C, Sui H, Li Z, Sun Z, Li C, Chen G, Ma Z, Cao H, Xi H. THBS1 in macrophage-derived exosomes exacerbates cerebral ischemia-reperfusion injury by inducing ferroptosis in endothelial cells. J Neuroinflammation 2025; 22:48. [PMID: 39994679 PMCID: PMC11854006 DOI: 10.1186/s12974-025-03382-x] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/31/2024] [Accepted: 02/17/2025] [Indexed: 02/26/2025] Open
Abstract
Macrophages play a critical role in the development of acute ischemic stroke (AIS). Cerebral ischemia-reperfusion injury (CIRI) is a pivotal pathological process that exacerbates AIS, with exosomes act as crucial mediators. However, the effects and mechanisms of action of macrophage-derived exosomes on CIRI remain unclear. This study demonstrated that macrophage-derived exosomes induce endothelial ferroptosis and barrier disruption during CIRI. Through proteomic sequencing and the reanalysis of transcriptomic and single-cell sequencing data, thrombospondin-1 (THBS1) was identified as a key exosomal molecule. Elevated THBS1 was observed in exosomes and monocytes from the peripheral blood of patients with AIS in oxygen-glucose deprivation/reoxygenation (OGD/R)-stimulated THP-1 and RAW264.7, in their secreted exosomes, and in macrophages within the brains of transient middle cerebral artery occlusion (tMCAO) mice. Additionally, THBS1 expression in exosomes was positively correlated with vascular barrier injury biomarkers, including MMP-9 and S100B. Modulation of THBS1 in macrophage-derived exosomes affected exosome-induced ferroptosis in endothelial cells. The mechanism by which THBS1 binds directly to OTUD5 and promotes GPX4 ubiquitination was elucidated using RNA interference, adeno-associated virus transfection, and endothelial-specific Gpx4 knockout mice. High-throughput screening of small-molecule compounds targeting THBS1 was performed. Molecular docking, molecular dynamics simulations, and cellular thermal shift assays further confirmed that salvianolic acid B (SAB) has a potent binding affinity for THBS1. SAB treatment inhibited the interaction between THBS1 and OTUD5, leading to reduced GPX4 ubiquitination. Further research revealed that SAB treatment enhanced the cerebral protective effects of THBS1 inhibition. In conclusion, this study explored the role of exosome-mediated signaling between macrophages and cerebral vascular endothelial cells in CIRI, highlighting the THBS1-OTUD5-GPX4 axis as a driver of endothelial ferroptosis and brain injury. Targeting this signaling axis represents a potential therapeutic strategy for treating CIRI.
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Affiliation(s)
- Chang Liu
- Department of Anesthesiology, Harbin Medical University Cancer Hospital, Harbin, 150081, People's Republic of China
- The Key Laboratory of Anesthesiology and Intensive Care Research of Heilongjiang Province, Harbin, 150001, People's Republic of China
- The Key Laboratory of Myocardial Ischemia Organization, Chinese Ministry of Education, Harbin, 150001, People's Republic of China
- State Key Laboratory of Frigid Zone Cardiovascular Diseases, Harbin, 150001, People's Republic of China
| | - Haijing Sui
- The Key Laboratory of Anesthesiology and Intensive Care Research of Heilongjiang Province, Harbin, 150001, People's Republic of China
- Department of Anesthesiology, Second Affiliated Hospital of Harbin Medical University, Harbin, 150001, People's Republic of China
| | - Zhixi Li
- The Key Laboratory of Anesthesiology and Intensive Care Research of Heilongjiang Province, Harbin, 150001, People's Republic of China
- The Key Laboratory of Myocardial Ischemia Organization, Chinese Ministry of Education, Harbin, 150001, People's Republic of China
- State Key Laboratory of Frigid Zone Cardiovascular Diseases, Harbin, 150001, People's Republic of China
- Department of Anesthesiology, Second Affiliated Hospital of Harbin Medical University, Harbin, 150001, People's Republic of China
| | - Zhenyu Sun
- The Key Laboratory of Anesthesiology and Intensive Care Research of Heilongjiang Province, Harbin, 150001, People's Republic of China
- Department of Anesthesiology, Second Affiliated Hospital of Harbin Medical University, Harbin, 150001, People's Republic of China
| | - Chenglong Li
- Department of Anesthesiology, Fourth Affiliated Hospital of Harbin Medical University, 37 Yiyuan Road, Harbin, 150001, People's Republic of China
| | - Guangmin Chen
- Department of Anesthesiology, First Affiliated Hospital of Harbin Medical University, 199 Dazhi Road, Harbin, 150001, People's Republic of China
| | - Zhaoxue Ma
- The Key Laboratory of Anesthesiology and Intensive Care Research of Heilongjiang Province, Harbin, 150001, People's Republic of China
- Department of Anesthesiology, Second Affiliated Hospital of Harbin Medical University, Harbin, 150001, People's Republic of China
| | - Hang Cao
- The Key Laboratory of Anesthesiology and Intensive Care Research of Heilongjiang Province, Harbin, 150001, People's Republic of China
- Department of Anesthesiology, Second Affiliated Hospital of Harbin Medical University, Harbin, 150001, People's Republic of China
| | - Hongjie Xi
- The Key Laboratory of Anesthesiology and Intensive Care Research of Heilongjiang Province, Harbin, 150001, People's Republic of China.
- Department of Anesthesiology, Second Affiliated Hospital of Harbin Medical University, Harbin, 150001, People's Republic of China.
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Schlötzer J, Schmalix A, Hügelschäffer S, Rieger D, Sauer F, Tully MD, Rudel T, Wiesner S, Kisker C. Linkage-specific ubiquitin binding interfaces modulate the activity of the chlamydial deubiquitinase Cdu1 towards poly-ubiquitin substrates. PLoS Pathog 2024; 20:e1012630. [PMID: 39432525 PMCID: PMC11527256 DOI: 10.1371/journal.ppat.1012630] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/09/2024] [Revised: 10/31/2024] [Accepted: 10/01/2024] [Indexed: 10/23/2024] Open
Abstract
The chlamydial deubiquitinase Cdu1 of the obligate intracellular human pathogenic bacterium Chlamydia trachomatis plays important roles in the maintenance of chlamydial infection. Despite the structural similarities shared with its homologue Cdu2, both DUBs display remarkable differences in their enzymatic activity towards poly-UB chain substrates. Whereas Cdu1 is highly active towards K48- and K63- poly-UB chains, Cdu2 activity is restricted mostly to mono-UB substrates. Here, we shed light on the molecular mechanisms of the differential activity and the substrate specificity of Cdu1 to better understand the cellular processes it is involved in, including infection-related events. We found that the strikingly elevated activity of Cdu1 relative to its paralogue Cdu2 can be attributed to an N-terminally extended α-helix, which has not been observed in Cdu2. Moreover, by employing isothermal titration calorimetry and nuclear magnetic resonance spectroscopy, we demonstrate the differential recognition of K48- and K63-linked poly-UB substrates by Cdu1. Whereas K63-linked poly-UB substrates appear to be recognized by Cdu1 with only two independent ubiquitin interaction sites, up to four different binding interfaces are present for K48-linked ubiquitin chains. Combined, our data suggest that Cdu1 possesses a poly-UB chain directed activity that may enable its function as a multipurpose DUB with a broad substrate specificity.
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Affiliation(s)
- Jan Schlötzer
- Institute for Structural Biology, Rudolf-Virchow-Zentrum—Center for Integrative and Translational Bioimaging, University of Würzburg, Würzburg, Germany
| | - Alexander Schmalix
- Institute of Biophysics and Physical Biochemistry, University of Regensburg, Regensburg, Germany
| | - Sophie Hügelschäffer
- Department of Microbiology, Biocenter, University of Würzburg, Würzburg, Germany
| | - Dominic Rieger
- Institute for Structural Biology, Rudolf-Virchow-Zentrum—Center for Integrative and Translational Bioimaging, University of Würzburg, Würzburg, Germany
| | - Florian Sauer
- Institute for Structural Biology, Rudolf-Virchow-Zentrum—Center for Integrative and Translational Bioimaging, University of Würzburg, Würzburg, Germany
| | - Mark D. Tully
- Structural Biology Group, European Synchrotron Radiation Facility, Grenoble, France
| | - Thomas Rudel
- Department of Microbiology, Biocenter, University of Würzburg, Würzburg, Germany
| | - Silke Wiesner
- Institute of Biophysics and Physical Biochemistry, University of Regensburg, Regensburg, Germany
| | - Caroline Kisker
- Institute for Structural Biology, Rudolf-Virchow-Zentrum—Center for Integrative and Translational Bioimaging, University of Würzburg, Würzburg, Germany
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Wu Y, Mohd Sani SB, Peng K, Lin T, Tan C, Huang X, Li Z. Research progress of the Otubains subfamily in hepatocellular carcinoma. Biomed Pharmacother 2024; 179:117348. [PMID: 39208669 DOI: 10.1016/j.biopha.2024.117348] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/14/2024] [Revised: 08/14/2024] [Accepted: 08/22/2024] [Indexed: 09/04/2024] Open
Abstract
In cancer research, oncogenesis can be affected by modulating the deubiquitination pathway. Ubiquitination regulates proteins post-translationally in variety of physiological processes. The Otubain Subfamily includes OTUB1 (ovarian tumor-associated proteinase B1) and OTUB2(ovarian tumor-associated proteinase B2). They are deubiquitinating enzymes, which are research hotspots in tumor immunotherapy, with their implications extending across the spectrum of tumor development. Understanding their important role in tumorigenesis, includ-ing hepatocellular carcinoma (HCC) is crucial. HCC has alarming global incidence rates and mortality statistics, ranking among the top five prevalent cancers in Malaysia1. Numerous studies have consistently indicated significant expression of OTUB1 and OTUB2 in HCC cells. In addition, OTUB1 has important biological functions in cancer, suggesting its important role in tumorigenesis. However, the mechanism underlying the action of OTUB1 and OTUB2 in liver cancer remains inadequately explored. Therefore, Otubain Subfamily, as potential molecular target, holds promise for advancing HCC treatments. However, further clinical studies are required to verify its efficacy and application prospects.
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Affiliation(s)
- Yanming Wu
- Department of Biomedical Sciences, Advanced Medical and Dental Institute, Universiti Sains Malaysia, Bertam, Kepala Batas, Penang 13200, Malaysia.
| | - Sa'udah Badriah Mohd Sani
- Department of Biomedical Sciences, Advanced Medical and Dental Institute, Universiti Sains Malaysia, Bertam, Kepala Batas, Penang 13200, Malaysia.
| | - Ke Peng
- Department of Neurology, School of Clinical Medicine, The First Affiliated Hospital of Yangtze University, Jingzhou, Hubei 434000, China.
| | - Tao Lin
- Department of General Surgery, Anyang People's Hospital, Anyang, Henan 450000, China.
| | - Chenghao Tan
- Department of Social Science, Universiti Sain Malaysia, Gelugor, Penang 11700, Malaysia.
| | | | - Zhengrui Li
- Shanghai Jiao Tong University School of Medicine, Shanghai 200240, China.
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5
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Su Y, Xu T, Sun Y. Evolutionarily conserved Otub1 suppresses antiviral immune response by promoting Irf3 proteasomal degradation in miiuy croaker, Miichthys miiuy. DEVELOPMENTAL AND COMPARATIVE IMMUNOLOGY 2024; 159:105218. [PMID: 38914152 DOI: 10.1016/j.dci.2024.105218] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 03/18/2024] [Revised: 06/02/2024] [Accepted: 06/21/2024] [Indexed: 06/26/2024]
Abstract
Increasing evidence has been shown that OTUB1, a member of OTU deubiquitinases, is of importance in regulating the immune system. However, its molecular identification and functional characterization in teleosts are still rarely known. In this work, we cloned the otub1 of miiuy croaker (Miichthys miiuy), analyzed its sequence, structure, and evolution at genetic and protein levels, and determined its function in the antiviral immune response. The complete open reading frame (ORF) of miiuy croaker otub1 is 843 bp in length, encoding 280 amino acids. Miiuy croaker Otub1 has an OTU domain at the carboxyl terminus, which is a common functional domain that exists in OTU deubiquitinases. Molecular characteristics and evolution analysis results indicated that miiuy croaker Otub1, especially its functional domain, is highly conserved during evolution. The luciferase reporter assays showed that miiuy croaker Otub1 could significantly inhibit the poly(I:C) and Irf3-induced IFN1 and IFN-stimulated response element (ISRE) activation. Further experiments showed that miiuy croaker Otub1 decreases Irf3 protein abundance by promoting its proteasomal degradation. These data suggest that the evolutionarily conserved Otub1 acts as a suppressor in controlling antiviral immune response by promoting Irf3 proteasomal degradation in miiuy croaker.
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Affiliation(s)
- Yanli Su
- Laboratory of Fish Molecular Immunology, College of Fisheries and Life Science, Shanghai Ocean University, Shanghai, China
| | - Tianjun Xu
- Laboratory of Fish Molecular Immunology, College of Fisheries and Life Science, Shanghai Ocean University, Shanghai, China; Laboratory for Marine Biology and Biotechnology, Qingdao Marine Science and Technology Center, Qingdao, China; Marine Biomedical Science and Technology Innovation Platform of Lin-gang Special Area, Shanghai, China.
| | - Yuena Sun
- Laboratory of Fish Molecular Immunology, College of Fisheries and Life Science, Shanghai Ocean University, Shanghai, China; National Pathogen Collection Center for Aquatic Animals, Shanghai Ocean University, China; Key Laboratory of Freshwater Aquatic Genetic Resources, Ministry of Agriculture and Rural Affairs, Shanghai Ocean University, China.
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6
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Zimmermann T, Feng J, de Campos LJ, Knight LA, Schlötzer J, Ramirez YA, Schwickert K, Zehe M, Adler TB, Schirmeister T, Kisker C, Sotriffer C, Conda-Sheridan M, Decker M. Structure-Based Design and Synthesis of Covalent Inhibitors for Deubiquitinase and Acetyltransferase ChlaDUB1 of Chlamydia trachomatis. J Med Chem 2024; 67:10710-10742. [PMID: 38897928 DOI: 10.1021/acs.jmedchem.4c00230] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 06/21/2024]
Abstract
Upon infection by an intracellular pathogen, host cells activate apoptotic pathways to limit pathogen replication. Consequently, efficient proliferation of the obligate intracellular pathogen Chlamydia trachomatis, a major cause of trachoma and sexually transmitted diseases, depends on the suppression of host cell apoptosis. C. trachomatis secretes deubiquitinase ChlaDUB1 into the host cell, leading among other interactions to the stabilization of antiapoptotic proteins and, thus, suppression of host cell apoptosis. Targeting the bacterial effector protein may, therefore, lead to new therapeutic possibilities. To explore the active site of ChlaDUB1, an iterative cycle of computational docking, synthesis, and enzymatic screening was applied with the aim of lead structure development. Hereby, covalent inhibitors were developed, which show enhanced inhibition with a 22-fold increase in IC50 values compared to previous work. Comprehensive insights into the binding prerequisites to ChlaDUB1 are provided, establishing the foundation for an additional specific antichlamydial therapy by small molecules.
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Affiliation(s)
- Thomas Zimmermann
- Pharmazeutische und Medizinische Chemie, Institut für Pharmazie und Lebensmittelchemie, Julius-Maximilians-Universität Würzburg (JMU), Am Hubland, 97074 Würzburg, Germany
| | - Jiachen Feng
- Department of Pharmaceutical Sciences, College of Pharmacy, University of Nebraska Medical Center, Omaha, Nebraska 68198, United States
| | - Luana Janaína de Campos
- Department of Pharmaceutical Sciences, College of Pharmacy, University of Nebraska Medical Center, Omaha, Nebraska 68198, United States
| | - Lindsey A Knight
- Department of Pathology, Microbiology and Immunology, University of Nebraska Medical Center, Omaha, Nebraska 68198, United States
| | - Jan Schlötzer
- Rudolf Virchow Center for Integrative and Translational Bioimaging, Institute for Structural Biology, Julius-Maximilians-Universität Würzburg (JMU), 97080 Wurzburg, Germany
| | - Yesid A Ramirez
- Pharmazeutische und Medizinische Chemie, Institut für Pharmazie und Lebensmittelchemie, Julius-Maximilians-Universität Würzburg (JMU), Am Hubland, 97074 Würzburg, Germany
| | - Kevin Schwickert
- Institute of Pharmaceutical and Biomedical Sciences (IPBS), Johannes Gutenberg University Mainz, Staudingerweg 5, 55128 Mainz, Germany
| | - Markus Zehe
- Pharmazeutische und Medizinische Chemie, Institut für Pharmazie und Lebensmittelchemie, Julius-Maximilians-Universität Würzburg (JMU), Am Hubland, 97074 Würzburg, Germany
| | - Thomas B Adler
- Pharmazeutische und Medizinische Chemie, Institut für Pharmazie und Lebensmittelchemie, Julius-Maximilians-Universität Würzburg (JMU), Am Hubland, 97074 Würzburg, Germany
| | - Tanja Schirmeister
- Institute of Pharmaceutical and Biomedical Sciences (IPBS), Johannes Gutenberg University Mainz, Staudingerweg 5, 55128 Mainz, Germany
| | - Caroline Kisker
- Rudolf Virchow Center for Integrative and Translational Bioimaging, Institute for Structural Biology, Julius-Maximilians-Universität Würzburg (JMU), 97080 Wurzburg, Germany
| | - Christoph Sotriffer
- Pharmazeutische und Medizinische Chemie, Institut für Pharmazie und Lebensmittelchemie, Julius-Maximilians-Universität Würzburg (JMU), Am Hubland, 97074 Würzburg, Germany
| | - Martin Conda-Sheridan
- Department of Pharmaceutical Sciences, College of Pharmacy, University of Nebraska Medical Center, Omaha, Nebraska 68198, United States
| | - Michael Decker
- Pharmazeutische und Medizinische Chemie, Institut für Pharmazie und Lebensmittelchemie, Julius-Maximilians-Universität Würzburg (JMU), Am Hubland, 97074 Würzburg, Germany
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Majumder S, Srivastava M, Alam P, Saha S, Kumari R, Chand AK, Asthana S, Sen S, Maiti TK. Hotspot site microenvironment in the deubiquitinase OTUB1 drives its stability and aggregation. J Biol Chem 2024; 300:107315. [PMID: 38663827 PMCID: PMC11154711 DOI: 10.1016/j.jbc.2024.107315] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/27/2024] [Revised: 03/30/2024] [Accepted: 04/15/2024] [Indexed: 05/30/2024] Open
Abstract
Lewy bodies (LB) are aberrant protein accumulations observed in the brain cells of individuals affected by Parkinson's disease (PD). A comprehensive analysis of LB proteome identified over a hundred proteins, many co-enriched with α-synuclein, a major constituent of LB. Within this context, OTUB1, a deubiquitinase detected in LB, exhibits amyloidogenic properties, yet the mechanisms underlying its aggregation remain elusive. In this study, we identify two critical sites in OTUB1-namely, positions 133 and 173-that significantly impact its amyloid aggregation. Substituting alanine at position 133 and lysine at position 173 enhances both thermodynamic and kinetic stability, effectively preventing amyloid aggregation. Remarkably, lysine at position 173 demonstrates the highest stability without compromising enzymatic activity. The increased stability and inhibition of amyloid aggregation are attributed mainly to the changes in the specific microenvironment at the hotspot. In our exploration of the in-vivo co-occurrence of α-synuclein and OTUB1 in LB, we observed a synergistic modulation of each other's aggregation. Collectively, our study unveils the molecular determinants influencing OTUB1 aggregation, shedding light on the role of specific residues in modulating aggregation kinetics and structural transition. These findings contribute valuable insights into the complex interplay of amino acid properties and protein aggregation, with potential implications for understanding broader aspects of protein folding and aggregation phenomena.
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Affiliation(s)
- Sushanta Majumder
- Functional Proteomics Laboratory, Regional Centre for Biotechnology, NCR Biotech Science Cluster, Faridabad, India
| | - Mitul Srivastava
- Translational Health Science and Technology Institute, NCR Biotech Science Cluster, Faridabad, India
| | - Parvez Alam
- Spectroscopy Laboratory, School of Physical Sciences, Jawaharlal Nehru University, New Delhi, India
| | - Sandhini Saha
- Functional Proteomics Laboratory, Regional Centre for Biotechnology, NCR Biotech Science Cluster, Faridabad, India
| | - Raniki Kumari
- Functional Proteomics Laboratory, Regional Centre for Biotechnology, NCR Biotech Science Cluster, Faridabad, India
| | - Ajay Kumar Chand
- Spectroscopy Laboratory, School of Physical Sciences, Jawaharlal Nehru University, New Delhi, India
| | - Shailendra Asthana
- Translational Health Science and Technology Institute, NCR Biotech Science Cluster, Faridabad, India
| | - Sobhan Sen
- Spectroscopy Laboratory, School of Physical Sciences, Jawaharlal Nehru University, New Delhi, India
| | - Tushar Kanti Maiti
- Functional Proteomics Laboratory, Regional Centre for Biotechnology, NCR Biotech Science Cluster, Faridabad, India.
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Zhao X, Lv S, Li N, Zou Q, Sun L, Song T. YTHDF2 protein stabilization by the deubiquitinase OTUB1 promotes prostate cancer cell proliferation via PRSS8 mRNA degradation. J Biol Chem 2024; 300:107152. [PMID: 38462165 PMCID: PMC11002313 DOI: 10.1016/j.jbc.2024.107152] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/22/2023] [Revised: 02/23/2024] [Accepted: 03/04/2024] [Indexed: 03/12/2024] Open
Abstract
Prostate cancer is a leading cause of cancer-related mortality in males. Dysregulation of RNA adenine N-6 methylation (m6A) contributes to cancer malignancy. m6A on mRNA may affect mRNA splicing, turnover, transportation, and translation. m6A exerts these effects, at least partly, through dedicated m6A reader proteins, including YTH domain-containing family protein 2 (YTHDF2). YTHDF2 is necessary for development while its dysregulation is seen in various cancers, including prostate cancer. However, the mechanism underlying the dysregulation and function of YTHDF2 in cancer remains elusive. Here, we find that the deubiquitinase OUT domain-containing ubiquitin aldehyde-binding protein 1 (OTUB1) increases YTHDF2 protein stability by inhibiting its ubiquitination. With in vivo and in vitro ubiquitination assays, OTUB1 is shown to block ubiquitin transfer to YTHDF2 independent of its deubiquitinase activity. Furthermore, analysis of functional transcriptomic data and m6A-sequencing data identifies PRSS8 as a potential tumor suppressor gene. OTUB1 and YTHDF2 decrease mRNA and protein levels of PRSS8, which is a trypsin-like serine protease. Mechanistically, YTHDF2 binds PRSS8 mRNA and promotes its degradation in an m6A-dependent manner. Further functional study on cellular and mouse models reveals PRSS8 is a critical downstream effector of the OTUB1-YTHDF2 axis in prostate cancer. We find in prostate cancer cells, PRSS8 decreases nuclear β-catenin level through E-cadherin, which is independent of its protease activity. Collectively, our study uncovers a key regulator of YTHDF2 protein stability and establishes a functional OTUB1-YTHDF2-PRSS8 axis in prostate cancer.
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Affiliation(s)
- Xuefeng Zhao
- Department of Biochemistry and Molecular Biology, School of Basic Medicine, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China
| | - Suli Lv
- Department of Biochemistry and Molecular Biology, School of Basic Medicine, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China
| | - Neng Li
- Department of Biochemistry and Molecular Biology, School of Basic Medicine, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China
| | - Qingli Zou
- Department of Biochemistry and Molecular Biology, School of Basic Medicine, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China
| | - Lidong Sun
- Department of Biochemistry and Molecular Biology, School of Basic Medicine, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China; Cell Architecture Research Institute, Huazhong University of Science and Technology, Wuhan, Hubei, China.
| | - Tanjing Song
- Department of Biochemistry and Molecular Biology, School of Basic Medicine, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China; Cell Architecture Research Institute, Huazhong University of Science and Technology, Wuhan, Hubei, China.
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Zheng LL, Wang LT, Pang YW, Sun LP, Shi L. Recent advances in the development of deubiquitinases inhibitors as antitumor agents. Eur J Med Chem 2024; 266:116161. [PMID: 38262120 DOI: 10.1016/j.ejmech.2024.116161] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/01/2023] [Revised: 01/12/2024] [Accepted: 01/16/2024] [Indexed: 01/25/2024]
Abstract
Ubiquitination is a type of post-translational modification that covalently links ubiquitin to a target protein, which plays a critical role in modulating protein activity, stability, and localization. In contrast, this process is reversed by deubiquitinases (DUBs), which remove ubiquitin from ubiquitinated substrates. Dysregulation of DUBs is associated with several human diseases, such as cancer, inflammation, neurodegenerative disorders, and autoimmune diseases. Thus, DUBs have become promising targets for drug development. Although the physiological and pathological effects of DUBs are increasingly well understood, the clinical drug discovery of selective DUB inhibitors has been challenging. Herein, we summarize the structures and functions of main classes of DUBs and discuss the recent progress in developing selective small-molecule DUB inhibitors as antitumor agents.
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Affiliation(s)
- Li-Li Zheng
- Department of Medicinal Chemistry, School of Pharmacy, China Pharmaceutical University, Nanjing, 211198, China
| | - Li-Ting Wang
- Department of Medicinal Chemistry, School of Pharmacy, China Pharmaceutical University, Nanjing, 211198, China
| | - Ye-Wei Pang
- Department of Medicinal Chemistry, School of Pharmacy, China Pharmaceutical University, Nanjing, 211198, China
| | - Li-Ping Sun
- Department of Medicinal Chemistry, School of Pharmacy, China Pharmaceutical University, Nanjing, 211198, China.
| | - Lei Shi
- Department of Medicinal Chemistry, School of Pharmacy, China Pharmaceutical University, Nanjing, 211198, China.
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Wu M, Sun L, Song T. OTUB1-mediated inhibition of ubiquitination: a growing list of effectors, multiplex mechanisms, and versatile functions. Front Mol Biosci 2024; 10:1261273. [PMID: 38264570 PMCID: PMC10803509 DOI: 10.3389/fmolb.2023.1261273] [Citation(s) in RCA: 5] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/19/2023] [Accepted: 12/19/2023] [Indexed: 01/25/2024] Open
Abstract
Protein ubiquitination plays a pivotal role in protein homeostasis. Ubiquitination may regulate the stability, activity, protein-protein interaction, and localization of a protein. Ubiquitination is subject to regulation by two groups of counteracting enzymes, the E3 ubiquitin ligases and deubiquitinases. Consistently, deubiquitinases are involved in essentially all biological processes. OTUB1, an OTU-family deubiquitinase, is a critical regulator of development, cancer, DNA damage response, and immune response. OTUB1 antagonizes the ubiquitination of a wide-spectrum of proteins through at least two different mechanisms. Besides direct deubiquitination, OTUB1 can also inhibit ubiquitination by non-canonically blocking ubiquitin transfer from certain ubiquitin-conjugases (E2). In this review, we start with a general background of protein ubiquitination and deubiquitination. Next, we introduce the basic characteristics of OTUB1 and then elaborate on the updated biological functions of OTUB1. Afterwards, we discuss potential mechanisms underlying the versatility and specificity of OTUB1 functions. In the end, we discuss the perspective that OTUB1 can be a potential therapeutic target for cancer.
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Affiliation(s)
- Miaomiao Wu
- Deparment of Obstetrics and Gynecology, Shuyang Hospital of Traditional Chinese Medicine, Suqian, China
| | - Lidong Sun
- Department of Biochemistry and Molecular Biology, School of Basic Medicine, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, China
- Cell Architecture Research Institute, Huazhong University of Science and Technology, Wuhan, Hubei, China
| | - Tanjing Song
- Department of Biochemistry and Molecular Biology, School of Basic Medicine, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, China
- Cell Architecture Research Institute, Huazhong University of Science and Technology, Wuhan, Hubei, China
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11
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Luo VM, Shen C, Worme S, Bhagrath A, Simo-Cheyou E, Findlay S, Hébert S, Wai Lam Poon W, Aryanpour Z, Zhang T, Zahedi RP, Boulais J, Buchwald ZS, Borchers CH, Côté JF, Kleinman CL, Mandl JN, Orthwein A. The Deubiquitylase Otub1 Regulates the Chemotactic Response of Splenic B Cells by Modulating the Stability of the γ-Subunit Gng2. Mol Cell Biol 2024; 44:1-16. [PMID: 38270191 PMCID: PMC10829841 DOI: 10.1080/10985549.2023.2290434] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/15/2023] [Accepted: 11/28/2023] [Indexed: 01/26/2024] Open
Abstract
The ubiquitin proteasome system performs the covalent attachment of lysine 48-linked polyubiquitin chains to substrate proteins, thereby targeting them for degradation, while deubiquitylating enzymes (DUBs) reverse this process. This posttranslational modification regulates key features both of innate and adaptative immunity, including antigen presentation, protein homeostasis and signal transduction. Here we show that loss of one of the most highly expressed DUBs, Otub1, results in changes in murine splenic B cell subsets, leading to a significant increase in marginal zone and transitional B cells and a concomitant decrease in follicular B cells. We demonstrate that Otub1 interacts with the γ-subunit of the heterotrimeric G protein, Gng2, and modulates its ubiquitylation status, thereby controlling Gng2 stability. Proximal mapping of Gng2 revealed an enrichment in partners associated with chemokine signaling, actin cytoskeleton and cell migration. In line with these findings, we show that Otub1-deficient B cells exhibit greater Ca2+ mobilization, F-actin polymerization and chemotactic responsiveness to Cxcl12, Cxcl13 and S1P in vitro, which manifests in vivo as altered localization of B cells within the spleen. Together, our data establishes Otub1 as a novel regulator of G-protein coupled receptor signaling in B cells, regulating their differentiation and positioning in the spleen.
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Affiliation(s)
- Vincent M. Luo
- Department of Microbiology and Immunology, McGill University, Montréal, Québec, Canada
- Lady Davis Institute for Medical Research, Jewish General Hospital, Montréal, Québec, Canada
| | - Connie Shen
- Department of Microbiology and Immunology, McGill University, Montréal, Québec, Canada
- McGill Research Centre for Complex Traits, McGill University, Montréal, Québec, Canada
| | - Samantha Worme
- Lady Davis Institute for Medical Research, Jewish General Hospital, Montréal, Québec, Canada
- Division of Experimental Medicine, McGill University, Montréal, Québec, Canada
| | - Aanya Bhagrath
- McGill Research Centre for Complex Traits, McGill University, Montréal, Québec, Canada
- Department of Physiology, McGill University, Montréal, Québec, Canada
| | - Estelle Simo-Cheyou
- Department of Microbiology and Immunology, McGill University, Montréal, Québec, Canada
| | - Steven Findlay
- Lady Davis Institute for Medical Research, Jewish General Hospital, Montréal, Québec, Canada
- Division of Experimental Medicine, McGill University, Montréal, Québec, Canada
| | - Steven Hébert
- Lady Davis Institute for Medical Research, Jewish General Hospital, Montréal, Québec, Canada
- Department of Human Genetics, McGill University, Montréal, Québec, Canada
| | - William Wai Lam Poon
- Lady Davis Institute for Medical Research, Jewish General Hospital, Montréal, Québec, Canada
- Division of Experimental Medicine, McGill University, Montréal, Québec, Canada
| | - Zahra Aryanpour
- Lady Davis Institute for Medical Research, Jewish General Hospital, Montréal, Québec, Canada
| | - Thomas Zhang
- Lady Davis Institute for Medical Research, Jewish General Hospital, Montréal, Québec, Canada
| | - René P. Zahedi
- Manitoba Centre for Proteomics & Systems Biology, University of Manitoba, Winnipeg, Manitoba, Canada
- Department of Internal Medicine, University of Manitoba, Winnipeg, Manitoba, Canada
- Department of Biochemistry and Medical Genetics, University of Manitoba, Winnipeg, Manitoba, Canada
- CancerCare Manitoba Research Institute, Winnipeg, Manitoba, Canada
| | - Jonathan Boulais
- Institut de Recherches Cliniques de Montréal (IRCM), Montreal, Québec, Canada
| | - Zachary S. Buchwald
- Department of Radiation Oncology, Winship Cancer Institute, Emory University, Atlanta, Georgia, USA
| | - Christoph H. Borchers
- Segal Cancer Proteomics Centre, Lady Davis Institute for Medical Research, McGill University, Montréal, Québec, Canada
- Gerald Bronfman Department of Oncology, Lady Davis Institute for Medical Research, Jewish General Hospital, Montréal, Québec, Canada
- Department of Pathology, McGill University, Montreal, Québec, Canada
| | - Jean-Francois Côté
- Institut de Recherches Cliniques de Montréal (IRCM), Montreal, Québec, Canada
- Department of Anatomy and Cell Biology, McGill University, Montreal, Québec, Canada
- Département de Biochimie et Médecine Moléculaire, Université de Montréal, Montreal, Québec, Canada
- Département de Médecine (Programmes de Biologie Moléculaire), Université de Montréal, Montreal, Québec, Canada
| | - Claudia L. Kleinman
- Lady Davis Institute for Medical Research, Jewish General Hospital, Montréal, Québec, Canada
- Department of Human Genetics, McGill University, Montréal, Québec, Canada
| | - Judith N. Mandl
- Department of Microbiology and Immunology, McGill University, Montréal, Québec, Canada
- McGill Research Centre for Complex Traits, McGill University, Montréal, Québec, Canada
- Department of Physiology, McGill University, Montréal, Québec, Canada
| | - Alexandre Orthwein
- Department of Microbiology and Immunology, McGill University, Montréal, Québec, Canada
- Lady Davis Institute for Medical Research, Jewish General Hospital, Montréal, Québec, Canada
- Division of Experimental Medicine, McGill University, Montréal, Québec, Canada
- Department of Radiation Oncology, Winship Cancer Institute, Emory University, Atlanta, Georgia, USA
- Gerald Bronfman Department of Oncology, Lady Davis Institute for Medical Research, Jewish General Hospital, Montréal, Québec, Canada
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12
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Bailey-Elkin BA, Knaap RCM, De Silva A, Boekhoud IM, Mous S, van Vught N, Khajehpour M, van den Born E, Kikkert M, Mark BL. Demonstrating the importance of porcine reproductive and respiratory syndrome virus papain-like protease 2 deubiquitinating activity in viral replication by structure-guided mutagenesis. PLoS Pathog 2023; 19:e1011872. [PMID: 38096325 PMCID: PMC10754444 DOI: 10.1371/journal.ppat.1011872] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/07/2023] [Revised: 12/28/2023] [Accepted: 11/28/2023] [Indexed: 12/29/2023] Open
Abstract
Deubiquitination of cellular substrates by viral proteases is a mechanism used to interfere with host cellular signaling processes, shared between members of the coronavirus- and arterivirus families. In the case of Arteriviruses, deubiquitinating and polyprotein processing activities are accomplished by the virus-encoded papain-like protease 2 (PLP2). Several studies have implicated the deubiquitinating activity of the porcine reproductive and respiratory syndrome virus (PRRSV) PLP2 in the downregulation of cellular interferon production, however to date, the only arterivirus PLP2 structure described is that of equine arteritis virus (EAV), a distantly related virus. Here we describe the first crystal structure of the PRRSV PLP2 domain both in the presence and absence of its ubiquitin substrate, which reveals unique structural differences in this viral domain compared to PLP2 from EAV. To probe the role of PRRSV PLP2 deubiquitinating activity in host immune evasion, we selectively removed this activity from the domain by mutagenesis and found that the viral domain could no longer downregulate cellular interferon production. Interestingly, unlike EAV, and also unlike the situation for MERS-CoV, we found that recombinant PRRSV carrying PLP2 DUB-specific mutations faces significant selective pressure to revert to wild-type virus in MARC-145 cells, suggesting that the PLP2 DUB activity, which in PRRSV is present as three different versions of viral protein nsp2 expressed during infection, is critically important for PRRSV replication.
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Affiliation(s)
- Ben A. Bailey-Elkin
- Department of Microbiology, University of Manitoba, Winnipeg, Manitoba, Canada
| | - Robert C. M. Knaap
- Molecular Virology Laboratory, Department of Medical Microbiology, Leiden University Center of Infectious Diseases (LU-CID), Leiden University Medical Center, Leiden, The Netherlands
| | - Anuradha De Silva
- Department of Microbiology, University of Manitoba, Winnipeg, Manitoba, Canada
| | - Ilse M. Boekhoud
- Molecular Virology Laboratory, Department of Medical Microbiology, Leiden University Center of Infectious Diseases (LU-CID), Leiden University Medical Center, Leiden, The Netherlands
| | - Sandra Mous
- Molecular Virology Laboratory, Department of Medical Microbiology, Leiden University Center of Infectious Diseases (LU-CID), Leiden University Medical Center, Leiden, The Netherlands
| | - Niek van Vught
- Molecular Virology Laboratory, Department of Medical Microbiology, Leiden University Center of Infectious Diseases (LU-CID), Leiden University Medical Center, Leiden, The Netherlands
| | - Mazdak Khajehpour
- Department of Chemistry, University of Manitoba, Winnipeg, Manitoba, Canada
| | | | - Marjolein Kikkert
- Molecular Virology Laboratory, Department of Medical Microbiology, Leiden University Center of Infectious Diseases (LU-CID), Leiden University Medical Center, Leiden, The Netherlands
| | - Brian L. Mark
- Department of Microbiology, University of Manitoba, Winnipeg, Manitoba, Canada
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13
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Lee J, Roh JL. Epigenetic modulation of ferroptosis in cancer: Identifying epigenetic targets for novel anticancer therapy. Cell Oncol (Dordr) 2023; 46:1605-1623. [PMID: 37438601 DOI: 10.1007/s13402-023-00840-7] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 06/27/2023] [Indexed: 07/14/2023] Open
Abstract
Ferroptosis is a newly recognized form of oxidative-regulated cell death resulting from iron-mediated lipid peroxidation accumulation. Radical-trapping antioxidant systems can eliminate these oxidized lipids and prevent disrupting the integrity of cell membranes. Epigenetic modifications can regulate ferroptosis by altering gene expression or cell phenotype without permanent sequence changes. These mechanisms include DNA methylation, histone modifications, RNA modifications, and noncoding RNAs. Epigenetic alterations in cancer can control the expression of ferroptosis regulators or related pathways, leading to changes in cell sensitivity to ferroptosis inducers or cancer progression. Epigenetic alterations in cancer are influenced by a wide range of cancer hallmarks, contributing to therapeutic resistance. Targeting epigenetic alterations is a promising approach to overcoming cancer resilience. However, the exact mechanisms involved in different types of cancer remain unresolved. Discovering more ferroptosis-associated epigenetic targets and interventions can help overcome current barriers in anticancer therapy. Many papers on epigenetic modifications of ferroptosis have been continuously published, making it essential to summarize the current state-of-the-art in the epigenetic regulation of ferroptosis in human cancer.
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Affiliation(s)
- Jaewang Lee
- Department of Otorhinolaryngology-Head and Neck Surgery, CHA Bundang Medical Center, CHA University, Seongnam, Gyeonggi-do, 13496, Republic of Korea
- Department of Biomedical Science, General Graduate School, CHA University, Seongnam, Republic of Korea
| | - Jong-Lyel Roh
- Department of Otorhinolaryngology-Head and Neck Surgery, CHA Bundang Medical Center, CHA University, Seongnam, Gyeonggi-do, 13496, Republic of Korea.
- Department of Biomedical Science, General Graduate School, CHA University, Seongnam, Republic of Korea.
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14
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Shestoperova EI, Strieter ER. Uncovering DUB Selectivity through an Ion Mobility-Based Assessment of Ubiquitin Chain Isomers. Anal Chem 2023; 95:17416-17423. [PMID: 37962301 PMCID: PMC11103383 DOI: 10.1021/acs.analchem.3c04622] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/15/2023]
Abstract
Ubiquitination is a reversible post-translational modification that maintains cellular homeostasis and regulates protein turnover. Deubiquitinases (DUBs) are a large family of proteases that catalyze the removal of ubiquitin (Ub) along with the dismantling and editing of Ub chains. Assessing the activity and selectivity of DUBs is critical for defining physiological functions. Despite numerous methods for evaluating DUB activity, none are capable of assessing activity and selectivity in the context of multicomponent mixtures of native unlabeled Ub conjugates. Here, we report an ion mobility (IM)-based approach for measuring DUB selectivity in the context of unlabeled mixtures of Ub chains. We show that IM-mass spectrometry (IM-MS) can be used to assess the selectivity of DUBs in a time-dependent manner. Moreover, using the branched Ub chain selective DUB UCH37/UCHL5 along with a mixture of Ub trimers, a strong preference for branched Ub trimers bearing K6 and K48 linkages is revealed. Our results demonstrate that IM-MS is a powerful method for evaluating DUB selectivity under conditions more physiologically relevant than single-component mixtures.
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Affiliation(s)
- Elizaveta I Shestoperova
- Department of Chemistry, University of Massachusetts Amherst, Amherst, Massachusetts 01003, United States
| | - Eric R Strieter
- Department of Chemistry, University of Massachusetts Amherst, Amherst, Massachusetts 01003, United States
- Molecular & Cellular Biology Graduate Program, University of Massachusetts Amherst, Amherst, Massachusetts 01003, United States
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15
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Shestoperova EI, Strieter ER. Uncovering DUB Selectivity Through Ion-Mobility-Based Assessment of Ubiquitin Chain Isomers. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2023:2023.10.11.561976. [PMID: 37873305 PMCID: PMC10592704 DOI: 10.1101/2023.10.11.561976] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 10/25/2023]
Abstract
Ubiquitination is a reversible posttranslational modification that maintains cellular homeostasis and regulates protein turnover. Deubiquitinases (DUBs) are a large family of proteases that catalyze the removal of ubiquitin (Ub) along with the dismantling and editing of Ub chains. Assessing the activity and selectivity of DUBs is critical for defining physiological function. Despite numerous methods for evaluating DUB activity, none are capable of assessing activity and selectivity in the context of multicomponent mixtures of native, unlabeled ubiquitin conjugates. Here we report on an ion mobility (IM)-based approach for measuring DUB selectivity in the context of unlabeled mixtures of Ub chains. We show that IM-MS can be used to assess the selectivity of DUBs in a time-dependent manner. Moreover, using the branched Ub chain selective DUB UCH37/UCHL5 along with a mixture of Ub trimers, a strong preference for branched Ub trimers bearing K6 and K48 linkages is revealed. Our results demonstrate that IM coupled with mass spectrometry (IM-MS) is a powerful method for evaluating DUB selectivity under conditions more physiologically relevant than single component mixtures.
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16
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Li Y, Li R, Qin H, He H, Li S. OTUB1's role in promoting OSCC development by stabilizing RACK1 involves cell proliferation, migration, invasion, and tumor-associated macrophage M1 polarization. Cell Signal 2023; 110:110835. [PMID: 37532135 DOI: 10.1016/j.cellsig.2023.110835] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/24/2023] [Revised: 07/19/2023] [Accepted: 07/30/2023] [Indexed: 08/04/2023]
Abstract
Ovarian tumor domain, ubiquitin aldehyde binding 1 (OTUB1), a deubiquitinating enzyme known to regulate the stability of downstream proteins, has been reported to regulate various cancers tumorigenesis, yet its direct effects on oral squamous cell carcinoma (OSCC) progression are unclear. Bioinformatics analysis was performed to screen for genes of interest, and in vitro and in vivo studies were carried out to investigate the function and mechanism of OTUB1 in OSCC. We found that OTUB1 was abnormally elevated in OSCC tissues and positively associated with the pathological stage and tumor stage. Knockdown of OTUB1 impaired the malignance of OSCC cells - suppressed cell proliferation, invasion, migration, and xenografted tumor growth. OTUB1 silencing also drove tumor-associated macrophage M1 polarization but suppressed M2 polarization, and the induction of M1 polarization inhibited the survival of OSCC cells. However, OTUB1 overexpression exerted the opposite effects. Furthermore, the protein network that interacted with the OTUB1 protein was constructed based on the GeneMANIA website. Receptor for activated C kinase 1 (RACK1), a facilitator of OSCC progression, was identified as a potential target of the OTUB1 protein. We revealed that OTUB1 positively regulated RACK1 expression and inhibited RACK1 ubiquitination. Additionally, RACK1 upregulation reversed the effects of OTUB1 knockdown on OSCC progression. Overall, we demonstrated that OTUB1 might regulate OSCC progression by maintaining the stability of the RACK1 protein. These findings highlight the potential roles of the OTUB1/RACK1 axis as a potential therapeutic target in OSCC.
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Affiliation(s)
- Yunyun Li
- Department of Pathology, School of Basic Medical Sciences, Zhengzhou University, Zhengzhou, China; Department of Stomatology, the First Affiliated Hospital of Zhengzhou University, Zhengzhou, China
| | - Ruizhe Li
- Department of Pathology, School of Basic Medical Sciences, Zhengzhou University, Zhengzhou, China; Department of Pathology, the First Affiliated Hospital of Zhengzhou University, Zhengzhou, China
| | - Hui Qin
- Department of Pathology, School of Basic Medical Sciences, Zhengzhou University, Zhengzhou, China; Department of Pathology, the First Affiliated Hospital of Zhengzhou University, Zhengzhou, China
| | - Hongliu He
- Department of Pathology, School of Basic Medical Sciences, Zhengzhou University, Zhengzhou, China; Department of Pathology, the First Affiliated Hospital of Zhengzhou University, Zhengzhou, China
| | - Shanshan Li
- Department of Pathology, School of Basic Medical Sciences, Zhengzhou University, Zhengzhou, China; Department of Pathology, the First Affiliated Hospital of Zhengzhou University, Zhengzhou, China.
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17
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Aboushousha R, van der Velden J, Hamilton N, Peng Z, MacPherson M, Erickson C, White S, Wouters EFM, Reynaert NL, Seward DJ, Li J, Janssen-Heininger YMW. Glutaredoxin attenuates glutathione levels via deglutathionylation of Otub1 and subsequent destabilization of system x C. SCIENCE ADVANCES 2023;9:eadi5192. [PMID: 37703360 PMCID: PMC10499329 DOI: 10.1126/sciadv.adi5192] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 05/02/2023] [Accepted: 08/10/2023] [Indexed: 09/15/2023]
Abstract
Glutathione (GSH) is a critical component of the cellular redox system that combats oxidative stress. The glutamate-cystine antiporter, system xC-, is a key player in GSH synthesis that allows for the uptake of cystine, the rate-limiting building block of GSH. It is unclear whether GSH or GSH-dependent protein oxidation [protein S-glutathionylation (PSSG)] regulates the activity of system xC-. We demonstrate that an environment of enhanced PSSG promotes GSH increases via a system xC--dependent mechanism. Absence of the deglutathionylase, glutaredoxin (GLRX), augmented SLC7A11 protein and led to significant increases of GSH content. S-glutathionylation of C23 or C204 of the deubiquitinase OTUB1 promoted interaction with the E2-conjugating enzyme UBCH5A, leading to diminished ubiquitination and proteasomal degradation of SLC7A11 and augmentation of GSH, effects that were reversed by GLRX. These findings demonstrate an intricate link between GLRX and GSH via S-glutathionylation of OTUB1 and system xC- and illuminate a previously unknown feed-forward regulatory mechanism whereby enhanced GSH protein oxidation augments cellular GSH.
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Affiliation(s)
- Reem Aboushousha
- Department of Pathology and Laboratory Medicine, College of Medicine, University of Vermont, Burlington, VT 05405, USA
| | - Jos van der Velden
- Department of Pathology and Laboratory Medicine, College of Medicine, University of Vermont, Burlington, VT 05405, USA
| | - Nicholas Hamilton
- Department of Chemistry, University of Vermont, Burlington, VT 05405, USA
| | - Zhihua Peng
- Department of Pathology and Laboratory Medicine, College of Medicine, University of Vermont, Burlington, VT 05405, USA
| | - Maximilian MacPherson
- Department of Pathology and Laboratory Medicine, College of Medicine, University of Vermont, Burlington, VT 05405, USA
| | - Cuixia Erickson
- Department of Pathology and Laboratory Medicine, College of Medicine, University of Vermont, Burlington, VT 05405, USA
| | - Sheryl White
- Department of Neurological Sciences, University of Vermont, Burlington, VT 05405, USA
| | - Emiel F. M. Wouters
- Department of Respiratory Medicine, NUTRIM School of nutrition and translational research in metabolism, Maastricht University Medical Center, Maastricht, Netherlands
- Ludwig Boltzmann Institute for Lung Research, Vienna, Austria
| | - Niki L. Reynaert
- Department of Respiratory Medicine, NUTRIM School of nutrition and translational research in metabolism, Maastricht University Medical Center, Maastricht, Netherlands
| | - David J. Seward
- Department of Pathology and Laboratory Medicine, College of Medicine, University of Vermont, Burlington, VT 05405, USA
| | - Jianing Li
- Department of Pathology and Laboratory Medicine, College of Medicine, University of Vermont, Burlington, VT 05405, USA
- Department of Medicinal Chemistry and Molecular Pharmacology, Purdue University, West Lafayette, IN 47907, USA
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18
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Zhao Y, Ruan J, Li Z, Su X, Chen K, Lin Y, Cai Y, Wang P, Liu B, Schlüter D, Liang G, Wang X. OTUB1 inhibits breast cancer by non-canonically stabilizing CCN6. Clin Transl Med 2023; 13:e1385. [PMID: 37608493 PMCID: PMC10444971 DOI: 10.1002/ctm2.1385] [Citation(s) in RCA: 2] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/22/2023] [Revised: 08/10/2023] [Accepted: 08/14/2023] [Indexed: 08/24/2023] Open
Abstract
BACKGROUND CCN6 is a matricellular protein that critically regulates the tumourigenesis and progression of breast cancer. Although the tumour-suppressive function of CCN6 has been extensively studied, molecular mechanisms regulating protein levels of CCN6 remain largely unclear. This study aims to investigate the regulation of CCN6 by ubiquitination and deubiquitinating enzymes (DUBs) in breast cancer. METHODS A screening assay was performed to identify OTUB1 as the DUB for CCN6. Various biochemical methods were applied to elucidate the molecular mechanism of OTUB1 in the regulation of CCN6. The role of OTUB1-CCN6 interaction in breast cancer was studied with cell experiments and the allograft model. The correlation of OTUB1 and CCN6 in human breast cancer was determined by immunohistochemistry and Western blot. RESULTS We found that CCN6 protein levels were controlled by the ubiquitin-proteasome system. The K48 ubiquitination and degradation of CCN6 was inhibited by OTUB1, which directly interacted with CCN6 through its linker domain. Furthermore, OTUB1 inhibited the ubiquitination of CCN6 in a non-canonical manner. Deletion of OTUB1, concomitant with reduced CCN6 abundance, increased the migration, proliferation and viability of breast cancer cells. Supplementation of CCN6 abolished the effect of OTUB1 deletion on breast cancer. Importantly, OTUB1 expression was downregulated in human breast cancer and positively correlated with CCN6 levels. CONCLUSION This study identified OTUB1 as a novel regulator of CCN6 in breast cancer.
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Affiliation(s)
- Ying Zhao
- Chemical Biology Research CenterSchool of Pharmaceutical SciencesWenzhou Medical UniversityWenzhouChina
- Oujiang Laboratory (Zhejiang Lab for Regenerative Medicine, Vision and Brain Health)Department of Neurological RehabilitationThe Second Affiliated Hospital and Yuying Children's Hospital of Wenzhou Medical UniversityWenzhouChina
| | - Jing Ruan
- Department of PathologyThe First Affiliated HospitalWenzhou Medical UniversityWenzhouChina
| | - Zhongding Li
- Chemical Biology Research CenterSchool of Pharmaceutical SciencesWenzhou Medical UniversityWenzhouChina
| | - Xian Su
- Chemical Biology Research CenterSchool of Pharmaceutical SciencesWenzhou Medical UniversityWenzhouChina
| | - Kangmin Chen
- Chemical Biology Research CenterSchool of Pharmaceutical SciencesWenzhou Medical UniversityWenzhouChina
| | - Yimin Lin
- Chemical Biology Research CenterSchool of Pharmaceutical SciencesWenzhou Medical UniversityWenzhouChina
| | - Yuepiao Cai
- School of Pharmaceutical SciencesWenzhou Medical UniversityWenzhouChina
| | - Peng Wang
- School of Pharmaceutical SciencesWenzhou Medical UniversityWenzhouChina
| | - Baohua Liu
- Oujiang Laboratory (Zhejiang Lab for Regenerative Medicine, Vision and Brain Health)Department of Neurological RehabilitationThe Second Affiliated Hospital and Yuying Children's Hospital of Wenzhou Medical UniversityWenzhouChina
| | - Dirk Schlüter
- Institute of Medical Microbiology and Hospital EpidemiologyHannover Medical SchoolHannoverGermany
- Cluster of Excellence RESIST (EXC 2155)Hannover Medical SchoolHannoverGermany
| | - Guang Liang
- Chemical Biology Research CenterSchool of Pharmaceutical SciencesWenzhou Medical UniversityWenzhouChina
- School of Pharmaceutical SciencesHangzhou Medical CollegeHangzhouChina
| | - Xu Wang
- Chemical Biology Research CenterSchool of Pharmaceutical SciencesWenzhou Medical UniversityWenzhouChina
- Oujiang Laboratory (Zhejiang Lab for Regenerative Medicine, Vision and Brain Health)Department of Neurological RehabilitationThe Second Affiliated Hospital and Yuying Children's Hospital of Wenzhou Medical UniversityWenzhouChina
- Institute of Medical Microbiology and Hospital EpidemiologyHannover Medical SchoolHannoverGermany
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19
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Kang S, Kim G, Choi M, Jeong M, van der Heden van Noort GJ, Roh SH, Shin D. Structural insights into ubiquitin chain cleavage by Legionella ovarian tumor deubiquitinases. Life Sci Alliance 2023; 6:e202201876. [PMID: 37100438 PMCID: PMC10133868 DOI: 10.26508/lsa.202201876] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/15/2022] [Revised: 04/17/2023] [Accepted: 04/17/2023] [Indexed: 04/28/2023] Open
Abstract
Although ubiquitin is found only in eukaryotes, several pathogenic bacteria and viruses possess proteins that hinder the host ubiquitin system. Legionella, a gram-negative intracellular bacterium, possesses an ovarian tumor (OTU) family of deubiquitinases (Lot DUBs). Herein, we describe the molecular characteristics of Lot DUBs. We elucidated the structure of the LotA OTU1 domain and revealed that entire Lot DUBs possess a characteristic extended helical lobe that is not found in other OTU-DUBs. The structural topology of an extended helical lobe is the same throughout the Lot family, and it provides an S1' ubiquitin-binding site. Moreover, the catalytic triads of Lot DUBs resemble those of the A20-type OTU-DUBs. Furthermore, we revealed a unique mechanism by which LotA OTU domains cooperate together to distinguish the length of the chain and preferentially cleave longer K48-linked polyubiquitin chains. The LotA OTU1 domain itself cleaves K6-linked ubiquitin chains, whereas it is also essential for assisting the cleavage of longer K48-linked polyubiquitin chains by the OTU2 domain. Thus, this study provides novel insights into the structure and mechanism of action of Lot DUBs.
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Affiliation(s)
- Sangwoo Kang
- Department of Systems Biology, College of Life Science and Biotechnology, Yonsei University, Seoul, Republic of Korea
| | - Gyuhee Kim
- School of Biological Science, Institute of Molecular Biology and Genetics, Seoul National University, Seoul, Republic of Korea
| | - Minhyeong Choi
- Department of Systems Biology, College of Life Science and Biotechnology, Yonsei University, Seoul, Republic of Korea
| | - Minwoo Jeong
- Department of Systems Biology, College of Life Science and Biotechnology, Yonsei University, Seoul, Republic of Korea
| | | | - Soung-Hun Roh
- School of Biological Science, Institute of Molecular Biology and Genetics, Seoul National University, Seoul, Republic of Korea
| | - Donghyuk Shin
- Department of Systems Biology, College of Life Science and Biotechnology, Yonsei University, Seoul, Republic of Korea
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20
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Sheryazdanova A, Amoedo ND, Dufour S, Impens F, Rossignol R, Sablina A. The deubiquitinase OTUB1 governs lung cancer cell fitness by modulating proteostasis of OXPHOS proteins. Biochim Biophys Acta Mol Basis Dis 2023:166767. [PMID: 37245529 DOI: 10.1016/j.bbadis.2023.166767] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/30/2023] [Revised: 05/04/2023] [Accepted: 05/23/2023] [Indexed: 05/30/2023]
Abstract
Aerobic glycolysis is a hallmark of cancer development, but this dogma has been challenged by reports showing a key role of oxidative phosphorylation (OXPHOS) in cancer cell survival. It has been proposed that increased levels of intramitochondrial proteins in cancer cells are associated with high OXPHOS activity and increased sensitivity to OXPHOS inhibitors. However, the molecular mechanisms leading to the high expression of OXPHOS proteins in cancer cells remain unknown. Multiple proteomics studies have detected the ubiquitination of intramitochondrial proteins, suggesting the contribution of the ubiquitin system to the proteostatic regulation of OXPHOS proteins. Here, we identified the ubiquitin hydrolase OTUB1 as a regulator of the mitochondrial metabolic machinery essential for lung cancer cell survival. Mitochondria-localized OTUB1 modulates respiration by inhibiting K48-linked ubiquitination and turnover of OXPHOS proteins. An increase in OTUB1 expression is commonly observed in one-third of non-small-cell lung carcinomas and is associated with high OXPHOS signatures. Moreover, OTUB1 expression highly correlates with the sensitivity of lung cancer cells to mitochondrial inhibitors.
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Affiliation(s)
- Aidana Sheryazdanova
- VIB-KU Leuven Center for Cancer Biology, VIB, Leuven, Belgium; Department of Oncology, KULeuven, Leuven, Belgium
| | - Nivea Dias Amoedo
- INSERM U1211 Rare Diseases, Genetics and Metabolism, University of Bordeaux, Bordeaux, France
| | - Sara Dufour
- Department of Biomolecular Medicine, Ghent University, Ghent, Belgium; VIB Center for Medical Biotechnology, Ghent, Belgium; VIB Proteomics Core, Ghent, Belgium
| | - Francis Impens
- Department of Biomolecular Medicine, Ghent University, Ghent, Belgium; VIB Center for Medical Biotechnology, Ghent, Belgium; VIB Proteomics Core, Ghent, Belgium
| | - Rodrigue Rossignol
- INSERM U1211 Rare Diseases, Genetics and Metabolism, University of Bordeaux, Bordeaux, France
| | - Anna Sablina
- VIB-KU Leuven Center for Cancer Biology, VIB, Leuven, Belgium; Department of Oncology, KULeuven, Leuven, Belgium.
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21
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Furuhata T, Devadasan Racheal PA, Murayama I, Toyoda U, Okamoto A. One-Pot, Photocontrolled Enzymatic Assembly of the Structure-Defined Heterotypic Polyubiquitin Chain. J Am Chem Soc 2023; 145:11690-11700. [PMID: 37200097 DOI: 10.1021/jacs.3c01912] [Citation(s) in RCA: 2] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 05/20/2023]
Abstract
Heterotypic polyubiquitins are an emerging class of polyubiquitins that have attracted interest because of their potential diversity of structures and physiological functions. There is an increasing demand for structure-defined synthesis of heterotypic chains to investigate the topological factors underlying the intracellular signals that are characteristically mediated by the heterotypic chain. However, the applicability of chemical and enzymatic polyubiquitin synthesis developed to date has been limited by laborious rounds of ligation and purification or by a lack of modularity of the chain structure with respect to the length and the branch position. Here, we established a one-pot, photocontrolled synthesis of structurally defined heterotypic polyubiquitin chains. We designed ubiquitin derivatives with a photolabile protecting group at a lysine residue used for polymerization. Repetitive cycles of linkage-specific enzymatic elongation and photoinduced deprotection of the protected ubiquitin units enabled stepwise addition of ubiquitins with appropriate functionalities to control the length and branching positions. The positional control of branching was achieved without isolation of intermediates, allowing one-pot synthesis of K63 triubiqutin chains and a K63/K48 heterotypic tetraubiquitin chain with defined branching positions. The present study provides a chemical platform for the efficient construction of long polyubiquitin chains with defined branch structures that will facilitate the understanding of the essential relationships between functions and structures of the heterotypic chain that have hitherto been overlooked.
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Affiliation(s)
- Takafumi Furuhata
- Department of Chemistry and Biotechnology, Graduate School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan
| | - Phebee Angeline Devadasan Racheal
- Department of Advanced Interdisciplinary Studies, Graduate School of Engineering, The University of Tokyo, 4-6-1 Komaba, Meguro-ku, Tokyo 153-8904, Japan
| | - Iori Murayama
- Department of Chemistry and Biotechnology, Graduate School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan
| | - Usano Toyoda
- Department of Chemistry and Biotechnology, Graduate School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan
| | - Akimitsu Okamoto
- Department of Chemistry and Biotechnology, Graduate School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan
- Department of Advanced Interdisciplinary Studies, Graduate School of Engineering, The University of Tokyo, 4-6-1 Komaba, Meguro-ku, Tokyo 153-8904, Japan
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22
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van Tol BDM, van Doodewaerd BR, Lageveen-Kammeijer GSM, Jansen BC, Talavera Ormeño CMP, Hekking PJM, Sapmaz A, Kim RQ, Moutsiopoulou A, Komander D, Wuhrer M, van der Heden van Noort GJ, Ovaa H, Geurink PP. Neutron-encoded diubiquitins to profile linkage selectivity of deubiquitinating enzymes. Nat Commun 2023; 14:1661. [PMID: 36966155 PMCID: PMC10039891 DOI: 10.1038/s41467-023-37363-6] [Citation(s) in RCA: 5] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/08/2022] [Accepted: 03/13/2023] [Indexed: 03/27/2023] Open
Abstract
Deubiquitinating enzymes are key regulators in the ubiquitin system and an emerging class of drug targets. These proteases disassemble polyubiquitin chains and many deubiquitinases show selectivity for specific polyubiquitin linkages. However, most biochemical insights originate from studies of single diubiquitin linkages in isolation, whereas in cells all linkages coexist. To better mimick this diubiquitin substrate competition, we develop a multiplexed mass spectrometry-based deubiquitinase assay that can probe all ubiquitin linkage types simultaneously to quantify deubiquitinase activity in the presence of all potential diubiquitin substrates. For this, all eight native diubiquitins are generated and each linkage type is designed with a distinct molecular weight by incorporating neutron-encoded amino acids. Overall, 22 deubiquitinases are profiled, providing a three-dimensional overview of deubiquitinase linkage selectivity over time and enzyme concentration.
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Affiliation(s)
- Bianca D M van Tol
- Department of Cell and Chemical Biology, Chemical Biology and Drug Discovery, Leiden University Medical Center, 2333 ZC, Leiden, The Netherlands
| | - Bjorn R van Doodewaerd
- Department of Cell and Chemical Biology, Chemical Biology and Drug Discovery, Leiden University Medical Center, 2333 ZC, Leiden, The Netherlands
| | | | - Bas C Jansen
- Center for Proteomics and Metabolomics, Leiden University Medical Center, 2333 ZA, Leiden, The Netherlands
| | - Cami M P Talavera Ormeño
- Department of Cell and Chemical Biology, Chemical Biology and Drug Discovery, Leiden University Medical Center, 2333 ZC, Leiden, The Netherlands
| | - Paul J M Hekking
- Department of Cell and Chemical Biology, Chemical Biology and Drug Discovery, Leiden University Medical Center, 2333 ZC, Leiden, The Netherlands
| | - Aysegul Sapmaz
- Department of Cell and Chemical Biology, Chemical Biology and Drug Discovery, Leiden University Medical Center, 2333 ZC, Leiden, The Netherlands
| | - Robbert Q Kim
- Department of Cell and Chemical Biology, Chemical Biology and Drug Discovery, Leiden University Medical Center, 2333 ZC, Leiden, The Netherlands
| | - Angeliki Moutsiopoulou
- Department of Cell and Chemical Biology, Chemical Biology and Drug Discovery, Leiden University Medical Center, 2333 ZC, Leiden, The Netherlands
| | - David Komander
- Ubiquitin Signalling Division, Walter and Eliza Hall Institute of Medical Research, 1G Royal Parade, Parkville, 3052, Melbourne, Victoria, Australia
| | - Manfred Wuhrer
- Center for Proteomics and Metabolomics, Leiden University Medical Center, 2333 ZA, Leiden, The Netherlands
| | - Gerbrand J van der Heden van Noort
- Department of Cell and Chemical Biology, Chemical Biology and Drug Discovery, Leiden University Medical Center, 2333 ZC, Leiden, The Netherlands
| | - Huib Ovaa
- Department of Cell and Chemical Biology, Chemical Biology and Drug Discovery, Leiden University Medical Center, 2333 ZC, Leiden, The Netherlands
| | - Paul P Geurink
- Department of Cell and Chemical Biology, Chemical Biology and Drug Discovery, Leiden University Medical Center, 2333 ZC, Leiden, The Netherlands.
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23
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Santelices J, Ou M, Maegawa GHB, Hercik K, Edelmann MJ. USP8 inhibition regulates autophagy flux and controls Salmonella infection. Front Cell Infect Microbiol 2023; 13:1070271. [PMID: 37026055 PMCID: PMC10072284 DOI: 10.3389/fcimb.2023.1070271] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/14/2022] [Accepted: 02/13/2023] [Indexed: 04/08/2023] Open
Abstract
Introduction Ubiquitination is an important protein modification that regulates various essential cellular processes, including the functions of innate immune cells. Deubiquitinases are enzymes responsible for removing ubiquitin modification from substrates, and the regulation of deubiquitinases in macrophages during infection with Salmonella Typhimurium and Yersinia enterocolitica remains unknown. Methods To identify deubiquitinases regulated in human macrophages during bacterial infection, an activity-based proteomics screen was conducted. The effects of pharmacological inhibition of the identified deubiquitinase, USP8, were examined, including its impact on bacterial survival within macrophages and its role in autophagy regulation during Salmonella infection. Results Several deubiquiitnases were differentially regulated in infected macrophages. One of the deubiquitinases identified was USP8, which was downregulated upon Salmonella infection. Inhibition of USP8 was associated with a decrease in bacterial survival within macrophages, and it was found to play a distinct role in regulating autophagy during Salmonella infection. The inhibition of USP8 led to the downregulation of the p62 autophagy adaptor. Discussion The findings of this study suggest a novel role of USP8 in regulating autophagy flux, which restricts intracellular bacteria, particularly during Salmonella infection.
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Affiliation(s)
- John Santelices
- Department of Microbiology and Cell Science, Institute of Food and Agricultural Sciences, University of Florida, Gainesville, FL, United States
| | - Mark Ou
- Department of Microbiology and Cell Science, Institute of Food and Agricultural Sciences, University of Florida, Gainesville, FL, United States
| | - Gustavo H. B. Maegawa
- Department of Pediatrics and Genetics, Columbia University Irving Medical Center, Vagelos Physicians and Surgeons College of Medicine, New York, NY, United States
| | - Kamil Hercik
- Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, Prague, Czechia
- Department of Molecular Pathology and Biology, Faculty of Military Health Sciences, University of Defense, Hradec Kralove, Czechia
| | - Mariola J. Edelmann
- Department of Microbiology and Cell Science, Institute of Food and Agricultural Sciences, University of Florida, Gainesville, FL, United States
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24
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Xing X, Xu F, Wang Y, Liu H. Role of the OTUB1/IRF7/NOX4 axis in oxidative stress injury and inflammatory responses in mice with Parkinson's disease. Psychogeriatrics 2023; 23:32-44. [PMID: 36332656 DOI: 10.1111/psyg.12900] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 06/27/2022] [Revised: 09/21/2022] [Accepted: 10/10/2022] [Indexed: 11/06/2022]
Abstract
BACKGROUND Parkinson's disease (PD) is one of the most devastating neurodegenerative disorders and is associated with oxidative stress injury (OSI) and inflammatory responses. This study sought to investigate the mechanism of ovarian tumour domain-containing ubiquitin aldehyde binding 1 (OTUB1) in OSI and inflammatory responses in PD, providing a theoretical foundation for PD treatment. METHODS The PD mouse model was established by an intraperitoneal injection of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, followed by behavioural tests, observation of brain pathological changes, and quantification of inflammatory (TNF-α, IL-1β, and IL-10) and OS (ROS, SOD, and MDA) factors. Next, the expression levels of OTUB1, interferon regulatory factor 7 (IRF7), and NADPH oxidase 4 (NOX4) levels were determined by real-time quantitative polymerase chain reaction and western blot assay, the binding of OTUB1 to IRF7 was analysed by co-immunoprecipitation, and the ubiquitination level of IRF7 and the enrichment and binding of IRF7 and the NOX4 promoter were measured by chromatin immunoprecipitation and dual-luciferase assays. Afterwards, rescue experiments were performed with IRF7 or NOX4 overexpression in OTUB1 knockout PD mice. RESULTS OTUB1 was upregulated in brain tissues of PD mice. Inhibition of OTUB1 alleviated PD progression, OSI, and inflammatory responses. OTUB1 stabilized IRF7 through deubiquitination, and IRF7 bound to the NOX4 promoter to promote NOX4 expression. IRF7 or NOX4 overexpression reversed the effects of silencing OTUB1 on OSI and inflammatory responses in PD mice. CONCLUSION OTUB1-mediated deubiquitination stabilized IRF7 and upregulated NOX4 expression, thereby promoting OSI and inflammatory responses in PD mice.
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Affiliation(s)
- Xiaolian Xing
- Department of Neurology, Taiyuan Central Hospital, Shanxi Medical University, Taiyuan, China
| | - Fei Xu
- Department of Neurology, Taiyuan Central Hospital, Shanxi Medical University, Taiyuan, China
| | - Yu Wang
- Departments of Emergency Internal Medicine, Shanxi Bethune Hospital, Shanxi Academy of Medical Sciences, Tong ji Shanxi Hospital, Third Hospital of Shanxi Medical University, Taiyuan, China
| | - Hongwei Liu
- Department of Neurology, Taiyuan Central Hospital, Shanxi Medical University, Taiyuan, China
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25
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Jin S, Kudo Y, Horiguchi T. The Role of Deubiquitinating Enzyme in Head and Neck Squamous Cell Carcinoma. Int J Mol Sci 2022; 24:ijms24010552. [PMID: 36613989 PMCID: PMC9820089 DOI: 10.3390/ijms24010552] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/07/2022] [Revised: 12/20/2022] [Accepted: 12/24/2022] [Indexed: 12/30/2022] Open
Abstract
Ubiquitination and deubiquitination are two popular ways for the post-translational modification of proteins. These two modifications affect intracellular localization, stability, and function of target proteins. The process of deubiquitination is involved in histone modification, cell cycle regulation, cell differentiation, apoptosis, endocytosis, autophagy, and DNA repair after damage. Moreover, it is involved in the processes of carcinogenesis and cancer development. In this review, we discuss these issues in understanding deubiquitinating enzyme (DUB) function in head and neck squamous cell carcinoma (HNSCC), and their potential therapeutic strategies for HNSCC patients are also discussed.
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26
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Li Z, Wang W, Xiao Y, Du S, Chen Z, Li B, Zhou ZW, Liu K, Gao F, Sun L. Discovery of a small-molecule inhibitor targeting the ovarian tumor domain of a novel Tamdy orthonairoviruse associated with human febrile illness. J Med Virol 2022; 94:5954-5964. [PMID: 36002383 DOI: 10.1002/jmv.28089] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/16/2022] [Revised: 07/31/2022] [Accepted: 08/19/2022] [Indexed: 01/06/2023]
Abstract
Tick-borne orthonairoviruses have been characterized as a global health threat to humans and animals. Tacheng Tick virus 1 (TcTV-1) from this family was provided as evidence that is associated with the febrile illness syndrome. Here, we first identify and demonstrate that the ovarian tumor (OTU) domain of TcTV-1 has remarkable deubiquitinating activity both in vitro and in vivo. By solving the crystal structure of TcTV-1 OTU (tcOTU) domain and comparing it to that of human deubiquitinating enzymes, we found that overall structures of tcOTU and human OTU family are similar, but the residues involved in the catalytic pocket vary widely. Based on the tcOTU domain we screened 5090 bioactive compounds and found mecobalamin had a good effect on suppressing the deubiquitinating activity. The structural model of tcOTU and mecobalamin suggests that mecobalamin occupies the site of the substrate Ub, by blocking the substrate binding to the enzyme. Thus, our results showed OTU domain of TcTV-1 has a robust deubiquitinating activity and mecobalamin or its derivatives might be promising candidates for the treatment or prevention of disease caused by the TcTV-1 virus.
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Affiliation(s)
- Zan Li
- School of Public Health (Shenzhen), Shenzhen Campus of Sun Yat-sen University, Shenzhen, China
| | - Weijia Wang
- School of Public Health (Shenzhen), Shenzhen Campus of Sun Yat-sen University, Shenzhen, China
| | - Yanshuang Xiao
- School of Public Health (Shenzhen), Shenzhen Campus of Sun Yat-sen University, Shenzhen, China
| | - Shan Du
- School of Public Health (Shenzhen), Shenzhen Campus of Sun Yat-sen University, Shenzhen, China
| | - Zhuohang Chen
- School of Public Health (Shenzhen), Shenzhen Campus of Sun Yat-sen University, Shenzhen, China
| | - Bing Li
- School of Public Health (Shenzhen), Shenzhen Campus of Sun Yat-sen University, Shenzhen, China
| | - Zhong-Wei Zhou
- School of Medicine, Shenzhen Campus of Sun Yat-sen University, Shenzhen, China
| | - Kuancheng Liu
- School of Public Health (Shenzhen), Shenzhen Campus of Sun Yat-sen University, Shenzhen, China
| | - Feng Gao
- Laboratory of Protein Engineering and Vaccines, Tianjin Institute of Industrial Biotechnology Chinese Academy of Sciences (CAS), Tianjin, China
| | - Litao Sun
- School of Public Health (Shenzhen), Shenzhen Campus of Sun Yat-sen University, Shenzhen, China
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27
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Tan L, Shan H, Han C, Zhang Z, Shen J, Zhang X, Xiang H, Lu K, Qi C, Li Y, Zhuang G, Chen G, Tan L. Discovery of Potent OTUB1/USP8 Dual Inhibitors Targeting Proteostasis in Non-Small-Cell Lung Cancer. J Med Chem 2022; 65:13645-13659. [PMID: 36221183 DOI: 10.1021/acs.jmedchem.2c00408] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/28/2022]
Abstract
Deubiquitinating enzymes (DUBs) are key regulatory components of the ubiquitination system. Many DUBs have been revealed to play key roles in normal physiology and diseases. However, only very limited DUB members have well-characterized inhibitors. OTUB1 and USP8 are two DUBs reported to promote both immune evasion and tumorigenesis in tumor models, yet their targeted inhibitors are in the early stages of development. Here, we describe the lead identification and optimization of an OTUB1/USP8 dual inhibitor, 61, which exhibits highly potent and selective inhibition of both targets with subnanomolar IC50s in vitro. By inhibiting both DUBs, 61 phenocopies the double knockdown of OTUB1/USP8 and exerts pronounced antiproliferative effects in H1975 and other non-small-cell lung cancer (NSCLC) cell lines. Moreover, 61 efficaciously mitigates tumor growth in vivo. Collectively, our results provide a useful tool for pharmacological perturbation of OTUB1/USP8 and introduce a promising therapeutic strategy of dual DUB inhibition for treating NSCLC.
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Affiliation(s)
- Lingli Tan
- School of Pharmacy, Fudan University, Shanghai 201203, China.,Interdisciplinary Research Center on Biology and Chemistry, Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, Shanghai 201210, China
| | - Hengyue Shan
- Interdisciplinary Research Center on Biology and Chemistry, Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, Shanghai 201210, China.,University of Chinese Academy of Sciences, Beijing 100049, China
| | - Chao Han
- Interdisciplinary Research Center on Biology and Chemistry, Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, Shanghai 201210, China.,University of Chinese Academy of Sciences, Beijing 100049, China
| | - Zhenfeng Zhang
- State Key Laboratory of Oncogenes and Related Genes, Shanghai Cancer Institute, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai 200240, China
| | - Jiali Shen
- Interdisciplinary Research Center on Biology and Chemistry, Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, Shanghai 201210, China.,University of Chinese Academy of Sciences, Beijing 100049, China
| | - Xiao Zhang
- Interdisciplinary Research Center on Biology and Chemistry, Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, Shanghai 201210, China
| | - Huaijiang Xiang
- Interdisciplinary Research Center on Biology and Chemistry, Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, Shanghai 201210, China.,University of Chinese Academy of Sciences, Beijing 100049, China
| | - Kuankuan Lu
- Interdisciplinary Research Center on Biology and Chemistry, Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, Shanghai 201210, China.,University of Chinese Academy of Sciences, Beijing 100049, China
| | - Chunting Qi
- Interdisciplinary Research Center on Biology and Chemistry, Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, Shanghai 201210, China
| | - Ying Li
- Interdisciplinary Research Center on Biology and Chemistry, Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, Shanghai 201210, China
| | - Guanglei Zhuang
- State Key Laboratory of Oncogenes and Related Genes, Shanghai Cancer Institute, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai 200240, China
| | - Gang Chen
- School of Pharmacy, Fudan University, Shanghai 201203, China
| | - Li Tan
- Interdisciplinary Research Center on Biology and Chemistry, Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, Shanghai 201210, China
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28
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Kumar P, Kumar P, Mandal D, Velayutham R. The emerging role of Deubiquitinases (DUBs) in parasites: A foresight review. Front Cell Infect Microbiol 2022; 12:985178. [PMID: 36237424 PMCID: PMC9552668 DOI: 10.3389/fcimb.2022.985178] [Citation(s) in RCA: 7] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/03/2022] [Accepted: 08/30/2022] [Indexed: 11/30/2022] Open
Abstract
Before the discovery of the proteasome complex, the lysosomes with acidic proteases and caspases in apoptotic pathways were thought to be the only pathways for the degradation of damaged, unfolded, and aged proteins. However, the discovery of 26S and 20S proteasome complexes in eukaryotes and microbes, respectively, established that the degradation of most proteins is a highly regulated ATP-dependent pathway that is significantly conserved across each domain of life. The proteasome is part of the ubiquitin-proteasome system (UPS), where the covalent tagging of a small molecule called ubiquitin (Ub) on the proteins marks its proteasomal degradation. The type and chain length of ubiquitination further determine whether a protein is designated for further roles in multi-cellular processes like DNA repair, trafficking, signal transduction, etc., or whether it will be degraded by the proteasome to recycle the peptides and amino acids. Deubiquitination, on the contrary, is the removal of ubiquitin from its substrate molecule or the conversion of polyubiquitin chains into monoubiquitin as a precursor to ubiquitin. Therefore, deubiquitylating enzymes (DUBs) can maintain the dynamic state of cellular ubiquitination by releasing conjugated ubiquitin from proteins and controlling many cellular pathways that are essential for their survival. Many DUBs are well characterized in the human system with potential drug targets in different cancers. Although, proteasome complex and UPS of parasites, like plasmodium and leishmania, were recently coined as multi-stage drug targets the role of DUBs is completely unexplored even though structural domains and functions of many of these parasite DUBs are conserved having high similarity even with its eukaryotic counterpart. This review summarizes the identification & characterization of different parasite DUBs based on in silico and a few functional studies among different phylogenetic classes of parasites including Metazoan (Schistosoma, Trichinella), Apicomplexan protozoans (Plasmodium, Toxoplasma, Eimeria, Cryptosporidium), Kinetoplastidie (Leishmania, Trypanosoma) and Microsporidia (Nosema). The identification of different homologs of parasite DUBs with structurally similar domains with eukaryotes, and the role of these DUBs alone or in combination with the 20S proteosome complex in regulating the parasite survival/death is further elaborated. We propose that small molecules/inhibitors of human DUBs can be potential antiparasitic agents due to their significant structural conservation.
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Affiliation(s)
- Prakash Kumar
- Department of Biotechnology, National Institute of Pharmaceutical Education and Research, Hajipur, India
| | - Pawan Kumar
- Department of Biotechnology, National Institute of Pharmaceutical Education and Research, Hajipur, India
| | - Debabrata Mandal
- Department of Biotechnology, National Institute of Pharmaceutical Education and Research, Hajipur, India
- *Correspondence: Ravichandiran Velayutham, ; Debabrata Mandal,
| | - Ravichandiran Velayutham
- Department of Biotechnology, National Institute of Pharmaceutical Education and Research, Hajipur, India
- National Institute of Pharmaceutical Education and Research, Kolkata, India
- *Correspondence: Ravichandiran Velayutham, ; Debabrata Mandal,
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29
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Breyer E, Zhao Z, Herndl GJ, Baltar F. Global contribution of pelagic fungi to protein degradation in the ocean. MICROBIOME 2022; 10:143. [PMID: 36050758 PMCID: PMC9434897 DOI: 10.1186/s40168-022-01329-5] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 01/26/2022] [Accepted: 07/19/2022] [Indexed: 05/31/2023]
Abstract
BACKGROUND Fungi are important degraders of organic matter responsible for reintegration of nutrients into global food chains in freshwater and soil environments. Recent evidence suggests that they are ubiquitously present in the oceanic water column where they play an active role in the degradation of carbohydrates. However, their role in processing other abundant biomolecules in the ocean in comparison with that of prokaryotes remains enigmatic. Here, we performed a global-ocean multi-omics analysis of all fungal-affiliated peptidases (main enzymes responsible for cleaving proteins), which constitute the major fraction (> 50%) of marine living and detrital biomass. We determined the abundance, expression, diversity, taxonomic affiliation, and functional classification of the genes encoding all pelagic fungal peptidases from the epi- and mesopelagic layers. RESULTS We found that pelagic fungi are active contributors to protein degradation and nitrogen cycling in the global ocean. Dothideomycetes are the main fungi responsible for protease activity in the surface layers, whereas Leotiomycetes dominate in the mesopelagic realm. Gene abundance, diversity, and expression increased with increasing depth, similar to fungal CAZymes. This contrasts with the total occurrence of prokaryotic peptidases and CAZymes which are more uniformly distributed in the oceanic water column, suggesting potentially different ecological niches of fungi and prokaryotes. In-depth analysis of the most widely expressed fungal protease revealed the potentially dominating role of saprotrophic nutrition in the oceans. CONCLUSIONS Our findings expand the current knowledge on the role of oceanic fungi in the carbon cycle (carbohydrates) to the so far unknown global participation in nitrogen (proteins) degradation, highlighting potentially different ecological niches occupied by fungi and prokaryotes in the global ocean. Video Abstract.
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Affiliation(s)
- Eva Breyer
- Department of Functional and Evolutionary Ecology, University of Vienna, Djerassiplatz 1, 1030 Vienna, Austria
| | - Zihao Zhao
- Department of Functional and Evolutionary Ecology, University of Vienna, Djerassiplatz 1, 1030 Vienna, Austria
| | - Gerhard J. Herndl
- Department of Functional and Evolutionary Ecology, University of Vienna, Djerassiplatz 1, 1030 Vienna, Austria
- NIOZ, Department of Marine Microbiology and Biogeochemistry, Royal Netherlands Institute for Sea Research, Utrecht University, AB Den Burg, The Netherlands
- Vienna Metabolomics Center, University of Vienna, Djerassiplatz 1, 1030 Vienna, Austria
| | - Federico Baltar
- Department of Functional and Evolutionary Ecology, University of Vienna, Djerassiplatz 1, 1030 Vienna, Austria
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Lu L, Zhai X, Li X, Wang S, Zhang L, Wang L, Jin X, Liang L, Deng Z, Li Z, Wang Y, Fu X, Hu H, Wang J, Mei Z, He Z, Wang F. Met1-specific motifs conserved in OTUB subfamily of green plants enable rice OTUB1 to hydrolyse Met1 ubiquitin chains. Nat Commun 2022; 13:4672. [PMID: 35945250 PMCID: PMC9363410 DOI: 10.1038/s41467-022-32364-3] [Citation(s) in RCA: 28] [Impact Index Per Article: 9.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/05/2021] [Accepted: 07/26/2022] [Indexed: 01/28/2023] Open
Abstract
Linear (Met1-linked) ubiquitination is involved inflammatory and innate immune signaling. Previous studies have characterized enzymes regulating the addition and removal of this modification in mammalian systems. However, only a few plant-derived deubiquitinases targeting Met1-linked ubiquitin chains have been reported and their mechanism of action remains elusive. Here, using a dehydroalanine-bearing Met1-diubiquitin suicide probe, we discover OTUB1 from Oryza sativa (OsOTUB1) as a Met1-linked ubiquitin chain-targeting deubiquitinase. By solving crystal structures of apo OsOTUB1 and an OsOTUB1/Met1-diubiquitin complex, we find that Met1 activity is conferred by Met1-specific motifs in the S1’ pocket of OsOTUB1. Large-scale sequence alignments and hydrolysis experiments provide evidence that these motifs are a general determinant of Met1 activity in the OTUB subfamily across species. Analysis of the species distribution of OTUBs capable of hydrolysing Met1-linked ubiquitin chains shows that this activity is conserved in green plants (Viridiplantae) and does not exist in metazoans, providing insights into the evolutionary differentiation between primitive plants and animals. Deubiquitinases (DUBs) targeting Met1-linked ubiquitin chains have important functions in mammals but are barely studied in plants. Here, the authors identify rice OTUB1 as a Met1-targeting DUB, characterize the structural determinants of this activity, and show that these features are conserved in green plants.
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Affiliation(s)
- Lining Lu
- State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources, College of Life Science and Technology, Guangxi University, Nanning, 530004, PR China. .,Tsinghua-Peking Center for Life Sciences, Key Laboratory of Bioorganic Phosphorus Chemistry & Chemical Biology (Ministry of Education), Center for Synthetic and Systems Biology, State Key Laboratory of Chemical Oncogenomics (Shenzhen), Department of Chemistry, Tsinghua University, Beijing, 100084, PR China.
| | - Xiaoguo Zhai
- State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources, College of Life Science and Technology, Guangxi University, Nanning, 530004, PR China
| | - Xiaolong Li
- Key Laboratory of Molecular Medicine and Biotherapy in the Ministry of Industry and Information Technology, Department of Biology, School of Life Science, Beijing Institute of Technology, Beijing, 100081, PR China
| | - Shuansuo Wang
- Shanxi Key Laboratory of Minor Crops Germplasm Innovation and Molecular Breeding, Shanxi Agricultural University, Taiyuan, 030031, PR China.,The State Key Laboratory of Plant Cell and Chromosome Engineering, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, 100101, PR China
| | - Lijun Zhang
- State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources, College of Life Science and Technology, Guangxi University, Nanning, 530004, PR China
| | - Luyang Wang
- State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources, College of Life Science and Technology, Guangxi University, Nanning, 530004, PR China
| | - Xi Jin
- State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources, College of Life Science and Technology, Guangxi University, Nanning, 530004, PR China
| | - Lujun Liang
- Tsinghua-Peking Center for Life Sciences, Key Laboratory of Bioorganic Phosphorus Chemistry & Chemical Biology (Ministry of Education), Center for Synthetic and Systems Biology, State Key Laboratory of Chemical Oncogenomics (Shenzhen), Department of Chemistry, Tsinghua University, Beijing, 100084, PR China
| | - Zhiheng Deng
- Tsinghua-Peking Center for Life Sciences, Key Laboratory of Bioorganic Phosphorus Chemistry & Chemical Biology (Ministry of Education), Center for Synthetic and Systems Biology, State Key Laboratory of Chemical Oncogenomics (Shenzhen), Department of Chemistry, Tsinghua University, Beijing, 100084, PR China
| | - Zichen Li
- Tsinghua-Peking Center for Life Sciences, Key Laboratory of Bioorganic Phosphorus Chemistry & Chemical Biology (Ministry of Education), Center for Synthetic and Systems Biology, State Key Laboratory of Chemical Oncogenomics (Shenzhen), Department of Chemistry, Tsinghua University, Beijing, 100084, PR China
| | - Yanfeng Wang
- Key Laboratory of Molecular Medicine and Biotherapy in the Ministry of Industry and Information Technology, Department of Biology, School of Life Science, Beijing Institute of Technology, Beijing, 100081, PR China
| | - Xiangdong Fu
- The State Key Laboratory of Plant Cell and Chromosome Engineering, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, 100101, PR China
| | - Honggang Hu
- Tsinghua-Peking Center for Life Sciences, Key Laboratory of Bioorganic Phosphorus Chemistry & Chemical Biology (Ministry of Education), Center for Synthetic and Systems Biology, State Key Laboratory of Chemical Oncogenomics (Shenzhen), Department of Chemistry, Tsinghua University, Beijing, 100084, PR China.,Institute of Translational Medicine, Shanghai University, Shanghai, 200444, PR China
| | - Jiawei Wang
- State Key Laboratory of Membrane Biology, Tsinghua University, Beijing, 100084, PR China
| | - Ziqing Mei
- School of Chemistry and Biological Engineering, University of Science and Technology Beijing, Beijing, 100083, PR China.
| | - Zhengguo He
- State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources, College of Life Science and Technology, Guangxi University, Nanning, 530004, PR China.
| | - Feng Wang
- Key Laboratory of Molecular Medicine and Biotherapy in the Ministry of Industry and Information Technology, Department of Biology, School of Life Science, Beijing Institute of Technology, Beijing, 100081, PR China.
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Jeong M, Jeon H, Shin D. Ubiquitin-regulating effector proteins from Legionella. BMB Rep 2022. [PMID: 35651329 PMCID: PMC9340081 DOI: 10.5483/bmbrep.2022.55.7.054] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/03/2022] Open
Abstract
Ubiquitin is relatively modest in size but involves almost entire cellular signaling pathways. The primary role of ubiquitin is maintaining cellular protein homeostasis. Ubiquitination regulates the fate of target proteins using the proteasome- or autophagy-mediated degradation of ubiquitinated substrates, which can be either intracellular or foreign proteins from invading pathogens. Legionella, a gram-negative intracellular pathogen, hinders the host-ubiquitin system by translocating hundreds of effector proteins into the host cell’s cytoplasm. In this review, we describe the current understanding of ubiquitin machinery from Legionella. We summarize structural and biochemical differences between the host-ubiquitin system and ubiquitin-related effectors of Legionella. Some of these effectors act much like canonical host-ubiquitin machinery, whereas others have distinctive structures and accomplish non-canonical ubiquitination via novel biochemical mechanisms.
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Affiliation(s)
- Minwoo Jeong
- Department of System Biology, College of Life Sciences and Biotechnology, Yonsei University, Seoul 03722, Korea
| | - Hayoung Jeon
- Department of System Biology, College of Life Sciences and Biotechnology, Yonsei University, Seoul 03722, Korea
| | - Donghyuk Shin
- Department of System Biology, College of Life Sciences and Biotechnology, Yonsei University, Seoul 03722, Korea
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Jeong M, Jeon H, Shin D. Ubiquitin-regulating effector proteins from Legionella. BMB Rep 2022; 55:316-322. [PMID: 35651329 PMCID: PMC9340081] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/20/2022] [Revised: 05/05/2022] [Accepted: 05/30/2022] [Indexed: 02/21/2025] Open
Abstract
Ubiquitin is relatively modest in size but involves almost entire cellular signaling pathways. The primary role of ubiquitin is maintaining cellular protein homeostasis. Ubiquitination regulates the fate of target proteins using the proteasome- or autophagymediated degradation of ubiquitinated substrates, which can be either intracellular or foreign proteins from invading pathogens. Legionella, a gram-negative intracellular pathogen, hinders the host-ubiquitin system by translocating hundreds of effector proteins into the host cell's cytoplasm. In this review, we describe the current understanding of ubiquitin machinery from Legionella. We summarize structural and biochemical differences between the host-ubiquitin system and ubiquitin-related effectors of Legionella. Some of these effectors act much like canonical host-ubiquitin machinery, whereas others have distinctive structures and accomplish non-canonical ubiquitination via novel biochemical mechanisms. [BMB Reports 2022; 55(7): 316-322].
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Affiliation(s)
- Minwoo Jeong
- Department of System Biology, College of Life Sciences and Biotechnology, Yonsei University, Seoul 03722, Korea
| | - Hayoung Jeon
- Department of System Biology, College of Life Sciences and Biotechnology, Yonsei University, Seoul 03722, Korea
| | - Donghyuk Shin
- Department of System Biology, College of Life Sciences and Biotechnology, Yonsei University, Seoul 03722, Korea
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Zhao J, Guo J, Wang Y, Ma Q, Shi Y, Cheng F, Lu Q, Fu W, Ouyang G, Zhang J, Xu Q, Hu X. Research Progress of DUB Enzyme in Hepatocellular Carcinoma. Front Oncol 2022; 12:920287. [PMID: 35875077 PMCID: PMC9303014 DOI: 10.3389/fonc.2022.920287] [Citation(s) in RCA: 7] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/14/2022] [Accepted: 05/24/2022] [Indexed: 11/13/2022] Open
Abstract
According to GLOBOCAN 2021 cancer incidence and mortality statistics compiled by the International Agency for Research on Cancer, hepatocellular carcinoma (HCC) is the most common malignancy in the human liver and one of the leading causes of cancer death worldwide. Although there have been great advances in the treatment of HCC, such as regofenib, sorafenib, and lomvatinib, which have been developed and approved for the clinical treatment of advanced or metastatic HCC. However, they only prolong survival by a few months, and patients with advanced liver cancer are susceptible to tumor invasion metastasis and drug resistance. Ubiquitination modification is a type of post-translational modification of proteins. It can affect the physiological activity of cells by regulating the localization, stability and activity of proteins, such as: gene transcription, DNA damage signaling and other pathways. The reversible process of ubiquitination is called de-ubiquitination: it is the process of re-releasing ubiquitinated substrates with the participation of de-ubiquitinases (DUBs) and other active substances. There is growing evidence that many dysregulations of DUBs are associated with tumorigenesis. Although dysregulation of deuquitinase function is often found in HCC and other cancers, The mechanisms of action of many DUBs in HCC have not been elucidated. In this review, we focused on several deubiquitinases (DUBs) associated with hepatocellular carcinoma, including their structure, function, and relationship to hepatocellular carcinoma. hepatocellular carcinoma was highlighted, as well as the latest research reports. Among them, we focus on the USP family and OTU family which are more studied in the HCC. In addition, we discussed the prospects and significance of targeting DUBs as a new strategy for the treatment of hepatocellular carcinoma. It also briefly summarizes the research progress of some DUB-related small molecule inhibitors and their clinical application significance as a treatment for HCC in the future.
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Affiliation(s)
- Jie Zhao
- College of Biotechnology and Bioengineering, Zhejiang University of Technology, Hangzhou, China
- Laboratory of Tumor Molecular Diagnosis and Individualized Medicine of Zhejiang Province, Zhejiang Provincial People’s Hospital, Affiliated People’s Hospital, Hangzhou Medical College, Hangzhou, China
| | - Jinhui Guo
- Qingdao Medical College, Qingdao University, Qingdao, China
| | - Yanan Wang
- College of Biotechnology and Bioengineering, Zhejiang University of Technology, Hangzhou, China
| | - Qiancheng Ma
- College of Biotechnology and Bioengineering, Zhejiang University of Technology, Hangzhou, China
| | - Yu Shi
- College of Biotechnology and Bioengineering, Zhejiang University of Technology, Hangzhou, China
| | - Feng Cheng
- College of Biotechnology and Bioengineering, Zhejiang University of Technology, Hangzhou, China
| | - Qiliang Lu
- Qingdao Medical College, Qingdao University, Qingdao, China
| | - Wen Fu
- Qingdao Medical College, Qingdao University, Qingdao, China
| | | | - Ji Zhang
- College of Biotechnology and Bioengineering, Zhejiang University of Technology, Hangzhou, China
| | - Qiuran Xu
- Laboratory of Tumor Molecular Diagnosis and Individualized Medicine of Zhejiang Province, Zhejiang Provincial People’s Hospital, Affiliated People’s Hospital, Hangzhou Medical College, Hangzhou, China
- *Correspondence: Xiaoge Hu, ; Qiuran Xu,
| | - Xiaoge Hu
- Department of Hepatobiliary and Pancreatic Surgery, Zhejiang Provincial People’s Hospital, Affiliated People’s Hospital, Hangzhou Medical CollegeHangzhou, China
- The Key Laboratory of Tumor Molecular Diagnosis and Individualized Medicine of Zhejiang Province, Zhejiang Provincial People’s Hospital, Affiliated People’s Hospital, Hangzhou Medical College, Hangzhou, China
- *Correspondence: Xiaoge Hu, ; Qiuran Xu,
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Hou L, Du J, Ren Q, Zhu L, Zhao X, Kong X, Gu W, Wang L, Meng Q. Ubiquitin-modified proteome analysis of Eriocheir sinensis hemocytes during Spiroplasma eriocheiris infection. FISH & SHELLFISH IMMUNOLOGY 2022; 125:109-119. [PMID: 35500876 DOI: 10.1016/j.fsi.2022.04.046] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 02/14/2022] [Revised: 04/20/2022] [Accepted: 04/26/2022] [Indexed: 06/14/2023]
Abstract
Spiroplasma eriocheiris, the pathogen of Eriocheir sinensis tremor disease (TD), has bring a huge economic loss to China aquaculture. The hemocytes of crab as the first target cells of S. eriocheiris, but the interactive relationship between the E. sinensis and this pathogen not particularly clear. The present study is the first time to analysis the role of protein ubiquitination in the process of E. sinensis hemocytes response S. eriocheiris infection. By applying label-free quantitative liquid chromatography with tandem mass spectrometry proteomics, 950 lysine ubiquitination sites and 803 ubiquitination peptides on 458 proteins were identified, of which 48 ubiquitination sites on 40 proteins were quantified as significantly changed after the S. eriocheiris infection. Bioinformatics analysis of ubiquitination different proteins suggested many biological process and pathways were participated in the interaction between S. eriocheiris and host cell, such as ubiquitin system, endocytosis, prophenoloxidase system (proPO system), cell apoptosis, glycolysis. Our study can enhance our understanding of interaction between the crab and S. eriocheiris, and also provides basis to study the role of protein ubiquitination in other crustacean innate immune system.
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Affiliation(s)
- Libo Hou
- Engineering Lab of Henan Province for Aquatic Animal Disease Control, College of Fisheries, Henan Normal University, Xinxiang, 453007, China
| | - Jie Du
- Animal Husbandry and Veterinary College, Jiangsu Vocational College of Agriculture and Forestry, Jurong, Jiangsu, 212400, China
| | - Qiulin Ren
- Engineering Lab of Henan Province for Aquatic Animal Disease Control, College of Fisheries, Henan Normal University, Xinxiang, 453007, China
| | - Lei Zhu
- Engineering Lab of Henan Province for Aquatic Animal Disease Control, College of Fisheries, Henan Normal University, Xinxiang, 453007, China
| | - Xianliang Zhao
- Engineering Lab of Henan Province for Aquatic Animal Disease Control, College of Fisheries, Henan Normal University, Xinxiang, 453007, China
| | - Xianghui Kong
- Engineering Lab of Henan Province for Aquatic Animal Disease Control, College of Fisheries, Henan Normal University, Xinxiang, 453007, China
| | - Wei Gu
- Jiangsu Key Laboratory for Aquatic Crustacean Diseases, College of Marine Science and Engineering, Nanjing Normal University, 2 Xuelin Road, Nanjing, 210046, China
| | - Li Wang
- College of Animal & Veterinary Sciences, Southwest Minzu University, Chengdu, 610041, China.
| | - Qingguo Meng
- Jiangsu Key Laboratory for Aquatic Crustacean Diseases, College of Marine Science and Engineering, Nanjing Normal University, 2 Xuelin Road, Nanjing, 210046, China.
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Kumari R, Kumar R, Dey AK, Saha S, Maiti TK. S-Nitrosylation of OTUB1 Alters Its Stability and Ubc13 Binding. ACS Chem Neurosci 2022; 13:1517-1525. [PMID: 35500217 DOI: 10.1021/acschemneuro.1c00855] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/28/2022] Open
Abstract
S-Nitrosylation is a reversible post-translational modification that regulates protein function involving the covalent attachment of the nitric oxide (NO) moiety to sulfhydryl residues of the protein. It is an important regulator in the cell signaling process under physiological conditions. However, the release of an excess amount of NO due to dysregulated NOS machinery causes aberrant S-nitrosylation of proteins, which affects protein folding, localization, and activity. Here, we have shown that OTUB1, a deubiquitinating enzyme, undergoes S-nitrosylation under redox stress conditions in vivo and in vitro. Previously, we have shown that OTUB1 forms an amyloid-like structure that promotes phosphorylation of α-synuclein and neuronal toxicity. However, the mechanistic insight into OTUB1 aggregation remains elusive. Here, we identified that OTUB1 undergoes S-nitrosylation in SH-SY5Y neuroblastoma cells under rotenone-induced stress, as well as excitotoxic conditions, and in rotenone-treated mouse brains. The in vitro S-nitrosylation of OTUB1 followed by mass-spectrometry analysis has identified cysteine-23 and cysteine-91 as S-nitrosylation sites. S-Nitrosylated OTUB1 (SNO-OTUB1) diminished its catalytic activity, impaired its native structure, promoted amyloid-like aggregation, and compromised its binding with Ubc13. Thus, our results demonstrated that nitrosylation of OTUB1 might play a crucial role in regulating the ubiquitin signaling and Parkinson's disease pathology.
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Affiliation(s)
- Raniki Kumari
- Functional Proteomics Laboratory, Regional Centre for Biotechnology (RCB), NCR Biotech Science Cluster, 3rd Milestone Gurgaon-Faridabad Expressway, Faridabad 121001, India
- Kalinga Institute of Industrial Technology (KIIT), Bhubaneswar, Odisha 751024, India
| | - Roshan Kumar
- Functional Proteomics Laboratory, Regional Centre for Biotechnology (RCB), NCR Biotech Science Cluster, 3rd Milestone Gurgaon-Faridabad Expressway, Faridabad 121001, India
| | - Amit Kumar Dey
- Functional Proteomics Laboratory, Regional Centre for Biotechnology (RCB), NCR Biotech Science Cluster, 3rd Milestone Gurgaon-Faridabad Expressway, Faridabad 121001, India
| | - Sandhini Saha
- Functional Proteomics Laboratory, Regional Centre for Biotechnology (RCB), NCR Biotech Science Cluster, 3rd Milestone Gurgaon-Faridabad Expressway, Faridabad 121001, India
| | - Tushar Kanti Maiti
- Functional Proteomics Laboratory, Regional Centre for Biotechnology (RCB), NCR Biotech Science Cluster, 3rd Milestone Gurgaon-Faridabad Expressway, Faridabad 121001, India
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Kang JB, Shah MA, Park DJ, Koh PO. Retinoic acid regulates the ubiquitin-proteasome system in a middle cerebral artery occlusion animal model. Lab Anim Res 2022; 38:13. [PMID: 35562751 PMCID: PMC9102573 DOI: 10.1186/s42826-022-00123-6] [Citation(s) in RCA: 7] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/06/2022] [Accepted: 05/03/2022] [Indexed: 11/10/2022] Open
Abstract
Background Retinoic acid is a major metabolite of vitamin A and exerts beneficial effects including anti-oxidant and anti-inflammatory activities in neurons. The ubiquitin–proteasome system is an important biological system that regulates cell survival. Ubiquitination regulates protein degradation and plays an important role in oxidative stress. Deubiquitinating enzymes cleave ubiquitin from proteins and control ubiquitination-induced degradation. We detected decreases in ubiquitin carboxy-terminal hydrolase L1, ubiquitin thioesterase OTUB1, and proteasome subunit alpha types 1 and 3 in cerebral ischemic damage. In this study, we investigated whether retinoic acid regulates the expression of deubiquitinating enzymes ubiquitin carboxy-terminal hydrolase L1, ubiquitin thioesterase OTUB1, and proteasome subunit alpha types 1 and 3 in cerebral ischemic injury. Right middle cerebral artery occlusion (MCAO) was performed to induce cerebral ischemic damage in male rats. Retinoic acid (5 mg/kg) or vehicle was intraperitoneally injected every day from 4 days before surgery. Neurological behavioral tests were performed 24 h after MCAO, and right cerebral cortical tissues were collected. Results MCAO damage caused neurological behavioral dysfunction, and retinoic acid alleviated these deficits. The identified proteins decreased in MCAO animals with vehicle, while retinoic acid treatment attenuated these decreases. The results of proteomic study were confirmed by a reverse transcription-PCR technique. Expressions of ubiquitin carboxy-terminal hydrolase L1, ubiquitin thioesterase OTUB1, and proteasome subunit alpha types 1 and 3 were decreased in MCAO animals treated with vehicle. Retinoic acid treatment alleviated these MCAO-induced reductions. The ubiquitin–proteasome system plays an essential role in maintaining cell function and preserving cell shape against ischemic damage. Conclusions These findings suggest that retinoic acid regulates ubiquitin- and proteasome-related proteins including ubiquitin carboxy-terminal hydrolase L1, ubiquitin thioesterase OTUB1, and proteasome subunit alpha types 1 and 3 in a brain ischemia model. Changes in these proteins are involved in the neuroprotective effects of retinoic acid.
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Affiliation(s)
- Ju-Bin Kang
- Department of Anatomy, College of Veterinary Medicine, Research Institute of Life Science, Gyeongsang National University, 501 Jinju-daero, Jinju, 52828, South Korea
| | - Murad-Ali Shah
- Department of Anatomy, College of Veterinary Medicine, Research Institute of Life Science, Gyeongsang National University, 501 Jinju-daero, Jinju, 52828, South Korea
| | - Dong-Ju Park
- Department of Anatomy, College of Veterinary Medicine, Research Institute of Life Science, Gyeongsang National University, 501 Jinju-daero, Jinju, 52828, South Korea
| | - Phil-Ok Koh
- Department of Anatomy, College of Veterinary Medicine, Research Institute of Life Science, Gyeongsang National University, 501 Jinju-daero, Jinju, 52828, South Korea.
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Zhang JL, Du BB, Zhang DH, Li H, Kong LY, Fan GJ, Li YP, Li PC, Liang C, Wang Z, Yang LL, Hao ZY, Wu LM, Huang Z, Dong JZ, Zhang JY, Yao R, Wang SJ, Zhang YZ. OTUB1 alleviates NASH through inhibition of the TRAF6-ASK1 signaling pathways. Hepatology 2022; 75:1218-1234. [PMID: 34591986 DOI: 10.1002/hep.32179] [Citation(s) in RCA: 18] [Impact Index Per Article: 6.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 06/23/2021] [Revised: 09/09/2021] [Accepted: 09/22/2021] [Indexed: 12/26/2022]
Abstract
BACKGROUND AND AIMS NAFLD is considered as the hepatic manifestation of the metabolic syndrome, which includes insulin resistance, obesity and hyperlipidemia. NASH is a progressive stage of NAFLD with severe hepatic steatosis, hepatocyte death, inflammation, and fibrosis. Currently, no pharmacological interventions specifically tailored for NASH are approved. Ovarian tumor domain, ubiquitin aldehyde binding 1 (OTUB1), the founding member of deubiquitinases, regulates many metabolism-associated signaling pathways. However, the role of OTUB1 in NASH is unclarified. METHODS AND RESULTS We demonstrated that mice with Otub1 deficiency exhibited aggravated high-fat diet-induced and high-fat high-cholesterol (HFHC) diet-induced hyperinsulinemia and liver steatosis. Notably, hepatocyte-specific overexpression of Otub1 markedly alleviated HFHC diet-induced hepatic steatosis, inflammatory responses, and liver fibrosis. Mechanistically, we identified apoptosis signal-regulating kinase 1 (ASK1) as a key candidate target of OTUB1 through RNA-sequencing analysis and immunoblot analysis. Through immunoprecipitation-mass spectrometry analysis, we further found that OTUB1 directly bound to tumor necrosis factor receptor-associated factor 6 (TRAF6) and suppressed its lysine 63-linked polyubiquitination, thus inhibiting the activation of ASK1 and its downstream pathway. CONCLUSIONS OTUB1 is a key suppressor of NASH that inhibits polyubiquitinations of TRAF6 and attenuated TRAF6-mediated ASK1 activation. Targeting the OTUB1-TRAF6-ASK1 axis may be a promising therapeutic strategy for NASH.
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Affiliation(s)
- Jie-Lei Zhang
- Department of Endocrinologythe First Affiliated Hospital of Zhengzhou UniversityZhengzhou UniversityZhengzhouChina
| | - Bin-Bin Du
- Cardiovascular Hospitalthe First Affiliated Hospital of Zhengzhou UniversityZhengzhou UniversityZhengzhouChina
| | - Dian-Hong Zhang
- Cardiovascular Hospitalthe First Affiliated Hospital of Zhengzhou UniversityZhengzhou UniversityZhengzhouChina
| | - Huan Li
- Cardiovascular Hospitalthe First Affiliated Hospital of Zhengzhou UniversityZhengzhou UniversityZhengzhouChina
| | - Ling-Yao Kong
- Cardiovascular Hospitalthe First Affiliated Hospital of Zhengzhou UniversityZhengzhou UniversityZhengzhouChina
| | - Guang-Jian Fan
- Precision Research Center for Refractory Diseases, Institute for Clinical ResearchShanghai General HospitalShanghai Jiao Tong University School of MedicineShanghaiChina
| | - Ya-Peng Li
- Cardiovascular Hospitalthe First Affiliated Hospital of Zhengzhou UniversityZhengzhou UniversityZhengzhouChina
| | - Peng-Cheng Li
- Cardiovascular Hospitalthe First Affiliated Hospital of Zhengzhou UniversityZhengzhou UniversityZhengzhouChina
| | - Cui Liang
- Cardiovascular Hospitalthe First Affiliated Hospital of Zhengzhou UniversityZhengzhou UniversityZhengzhouChina
| | - Zheng Wang
- Cardiovascular Hospitalthe First Affiliated Hospital of Zhengzhou UniversityZhengzhou UniversityZhengzhouChina
| | - Lu-Lu Yang
- Cardiovascular Hospitalthe First Affiliated Hospital of Zhengzhou UniversityZhengzhou UniversityZhengzhouChina
| | - Zheng-Yang Hao
- Cardiovascular Hospitalthe First Affiliated Hospital of Zhengzhou UniversityZhengzhou UniversityZhengzhouChina
| | - Lei-Ming Wu
- Cardiovascular Hospitalthe First Affiliated Hospital of Zhengzhou UniversityZhengzhou UniversityZhengzhouChina
| | - Zhen Huang
- Cardiovascular Hospitalthe First Affiliated Hospital of Zhengzhou UniversityZhengzhou UniversityZhengzhouChina
| | - Jian-Zeng Dong
- Cardiovascular Hospitalthe First Affiliated Hospital of Zhengzhou UniversityZhengzhou UniversityZhengzhouChina
| | - Jin-Ying Zhang
- Cardiovascular Hospitalthe First Affiliated Hospital of Zhengzhou UniversityZhengzhou UniversityZhengzhouChina
| | - Rui Yao
- Cardiovascular Hospitalthe First Affiliated Hospital of Zhengzhou UniversityZhengzhou UniversityZhengzhouChina
| | - Shou-Jun Wang
- Department of Endocrinologythe First Affiliated Hospital of Zhengzhou UniversityZhengzhou UniversityZhengzhouChina
| | - Yan-Zhou Zhang
- Cardiovascular Hospitalthe First Affiliated Hospital of Zhengzhou UniversityZhengzhou UniversityZhengzhouChina
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Teh WP, Zhu H, Marto JA, Buhrlage SJ. DUB to the rescue. Mol Cell 2022; 82:1411-1413. [PMID: 35452613 DOI: 10.1016/j.molcel.2022.03.039] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/18/2022]
Abstract
Henning et al. (2022) report development of a novel class of agents, bivalent deubiquitinase (DUB)-targeting chimeras (DUBTACs), that can selectively stabilize protein targets. These findings encourage further pursuit of targeted protein stabilization as a new paradigm in chemical biology and drug discovery.
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Affiliation(s)
- Wei Pin Teh
- Department of Cancer Biology, Dana-Farber Cancer Institute, Boston, MA 02215, USA; Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115, USA
| | - He Zhu
- Department of Cancer Biology, Dana-Farber Cancer Institute, Boston, MA 02215, USA; Department of Pathology, Brigham and Women's Hospital and Harvard Medical School, Boston, MA 02215, USA
| | - Jarrod A Marto
- Department of Cancer Biology, Dana-Farber Cancer Institute, Boston, MA 02215, USA; Department of Pathology, Brigham and Women's Hospital and Harvard Medical School, Boston, MA 02215, USA
| | - Sara J Buhrlage
- Department of Cancer Biology, Dana-Farber Cancer Institute, Boston, MA 02215, USA; Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115, USA.
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Zhou H, Carpenter T, Fu X, Jin B, Ody B, Hassan MS, Jacobs SE, Cheung J, Nicholson EM, Turlington M, Zhao B, Lorenz S, Cropp TA, Yin J. Linkage-Specific Synthesis of Di-ubiquitin Probes Enabled by the Incorporation of Unnatural Amino Acid ThzK. Chembiochem 2022; 23:e202200133. [PMID: 35263494 PMCID: PMC9129888 DOI: 10.1002/cbic.202200133] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/07/2022] [Indexed: 11/08/2022]
Abstract
Di-ubiquitin (diUB) conjugates of defined linkages are useful tools for probing the functions of UB ligases, UB-binding proteins and deubiquitinating enzymes (DUBs) in coding, decoding and editing the signals carried by the UB chains. Here we developed an efficient method for linkage-specific synthesis of diUB probes based on the incorporation of the unnatural amino acid (UAA) Nϵ -L-thiaprolyl-L-Lys (L-ThzK) into UB for ligation with another UB at a defined Lys position. The diUB formed by the UAA-mediated ligation reaction has a G76C mutation on the side of donor UB for conjugation with E2 and E3 enzymes or undergoing dethiolation to generate a covalent trap for DUBs. The development of UAA mutagenesis for diUB synthesis provides an easy route for preparing linkage-specific UB-based probes to decipher the biological signals mediated by protein ubiquitination.
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Affiliation(s)
- Han Zhou
- Department of Chemistry, Center for Diagnostics and Therapeutics, Georgia State University, Atlanta, GA 30303, USA
| | - Tomaya Carpenter
- Department of Chemistry, Center for Diagnostics and Therapeutics, Georgia State University, Atlanta, GA 30303, USA
| | - Xuan Fu
- Department of Chemistry, Center for Diagnostics and Therapeutics, Georgia State University, Atlanta, GA 30303, USA
| | - Bo Jin
- Department of Chemistry, Center for Diagnostics and Therapeutics, Georgia State University, Atlanta, GA 30303, USA
- Engineering Research Center of Cell and Therapeutic Antibody, Ministry of Education, and School of Pharmacy, Shanghai Jiao Tong University, Shanghai, China
| | - Britton Ody
- Department of Chemistry, Center for Diagnostics and Therapeutics, Georgia State University, Atlanta, GA 30303, USA
| | - Mohammad Sazid Hassan
- Department of Chemistry, Center for Diagnostics and Therapeutics, Georgia State University, Atlanta, GA 30303, USA
| | - Savannah E Jacobs
- Department of Chemistry, Center for Diagnostics and Therapeutics, Georgia State University, Atlanta, GA 30303, USA
| | - Jenny Cheung
- Department of Chemistry, Virginia Commonwealth University, Richmond, VA 23284, USA
| | - Eve M Nicholson
- Department of Chemistry, Virginia Commonwealth University, Richmond, VA 23284, USA
| | - Mark Turlington
- Department of Chemistry and Biochemistry, Berry College, Mount Berry, GA 30149, USA
| | - Bo Zhao
- Engineering Research Center of Cell and Therapeutic Antibody, Ministry of Education, and School of Pharmacy, Shanghai Jiao Tong University, Shanghai, China
| | - Sonja Lorenz
- Max Planck Institute for Multidisciplinary Sciences, 37077, Göttingen, Germany
| | - T Ashton Cropp
- Department of Chemistry, Virginia Commonwealth University, Richmond, VA 23284, USA
- Institute for Structural Biology, Drug Discovery and Development, Virginia Commonwealth University, Richmond, VA 23219, USA
| | - Jun Yin
- Department of Chemistry, Center for Diagnostics and Therapeutics, Georgia State University, Atlanta, GA 30303, USA
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Zhong X, Wang T, Zhang W, Wang M, Xie Y, Dai L, He X, Madhusudhan T, Zeng H, Wang H. ERK/RSK-mediated phosphorylation of Y-box binding protein-1 aggravates diabetic cardiomyopathy by suppressing its interaction with deubiquitinase OTUB1. J Biol Chem 2022; 298:101989. [PMID: 35490780 PMCID: PMC9163515 DOI: 10.1016/j.jbc.2022.101989] [Citation(s) in RCA: 15] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/14/2021] [Revised: 04/22/2022] [Accepted: 04/25/2022] [Indexed: 12/22/2022] Open
Abstract
Diabetic cardiomyopathy (DCM) is a major complication of diabetes, but its underlying mechanisms still remain unclear. The multifunctional protein Y-box binding protein-1 (YB-1) plays an important role in cardiac pathogenesis by regulating cardiac apoptosis, cardiac fibrosis, and pathological remodeling, whereas its role in chronic DCM requires further investigation. Here, we report that the phosphorylation of YB-1 at serine102 (S102) was markedly elevated in streptozotocin-induced diabetic mouse hearts and in high glucose-treated cardiomyocytes, whereas total YB-1 protein levels were significantly reduced. Coimmunoprecipitation experiments showed that YB-1 interacts with the deubiquitinase otubain-1, but hyperglycemia-induced phosphorylation of YB-1 at S102 diminished this homeostatic interaction, resulting in ubiquitination and degradation of YB-1. Mechanistically, the high glucose-induced phosphorylation of YB-1 at S102 is dependent on the upstream extracellular signal-regulated kinase (ERK)/Ras/mitogen-activated protein kinase (p90 ribosomal S6 kinase [RSK]) signaling pathway. Accordingly, pharmacological inhibition of the ERK pathway using the upstream kinase inhibitor U0126 ameliorated features of DCM compared with vehicle-treated diabetic mice. We demonstrate that ERK inhibition with U0126 also suppressed the phosphorylation of the downstream RSK and YB-1 (S102), which stabilized the interaction between YB-1 and otubain-1 and thereby preserved YB-1 protein expression in diabetic hearts. Taken together, we propose that targeting the ERK/RSK/YB-1 pathway could be a potential therapeutic approach for treating DCM.
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Affiliation(s)
- Xiaodan Zhong
- Division of Cardiology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, PR China; Hubei Key Laboratory of Genetics and Molecular Mechanisms of Cardiological Disorders, Wuhan, PR China
| | - Tao Wang
- Division of Cardiology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, PR China; Department of Cardiology, Affiliated Hospital of Weifang Medical University, Weifang, Shandong, PR China
| | - Wenjun Zhang
- Division of Cardiology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, PR China; Hubei Key Laboratory of Genetics and Molecular Mechanisms of Cardiological Disorders, Wuhan, PR China
| | - Mengwen Wang
- Division of Cardiology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, PR China; Hubei Key Laboratory of Genetics and Molecular Mechanisms of Cardiological Disorders, Wuhan, PR China
| | - Yang Xie
- Division of Cardiology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, PR China; Hubei Key Laboratory of Genetics and Molecular Mechanisms of Cardiological Disorders, Wuhan, PR China
| | - Lei Dai
- Division of Cardiology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, PR China; Hubei Key Laboratory of Genetics and Molecular Mechanisms of Cardiological Disorders, Wuhan, PR China
| | - Xingwei He
- Division of Cardiology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, PR China; Hubei Key Laboratory of Genetics and Molecular Mechanisms of Cardiological Disorders, Wuhan, PR China
| | - Thati Madhusudhan
- Center for Thrombosis and Hemostasis, University Medical Center Mainz, Mainz, Germany
| | - Hesong Zeng
- Division of Cardiology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, PR China; Hubei Key Laboratory of Genetics and Molecular Mechanisms of Cardiological Disorders, Wuhan, PR China.
| | - Hongjie Wang
- Division of Cardiology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, PR China; Hubei Key Laboratory of Genetics and Molecular Mechanisms of Cardiological Disorders, Wuhan, PR China.
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41
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Li J, Zhang N, Li M, Hong T, Meng W, Ouyang T. The Emerging Role of OTUB2 in Diseases: From Cell Signaling Pathway to Physiological Function. Front Cell Dev Biol 2022; 10:820781. [PMID: 35309903 PMCID: PMC8926145 DOI: 10.3389/fcell.2022.820781] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/23/2021] [Accepted: 02/03/2022] [Indexed: 11/13/2022] Open
Abstract
Ovarian tumor (OTU) domain-containing ubiquitin aldehyde-binding protein Otubain2 (OTUB2) was a functional cysteine protease in the OTU family with deubiquitinase activity. In recent years, with the wide application of molecular biology techniques, molecular mechanism regulation at multiple levels of cell signaling pathways has been gradually known, such as ubiquitin-mediated protein degradation and phosphorylation-mediated protein activation. OTUB2 is involved in the deubiquitination of many key proteins in different cell signaling pathways, and the effect of OTUB2 on human health or disease is not clear. OTUB2 is likely to cause cancer and other malignant diseases while maintaining normal human development and physiological function. Therefore, it is of great value to comprehensively understand the regulatory mechanism of OTUB2 and regard it as a target for the treatment of diseases. This review makes a general description and appropriate analysis of OTUB2's regulation in different cell signaling pathways, and connects OTUB2 with cancer from the research hotspot perspective of DNA damage repair and immunity, laying the theoretical foundation for future research.
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Affiliation(s)
- Jun Li
- Department of Neurosurgery, The First Affiliated Hospital of Nanchang University, Jiangxi, China.,Department of the Second Clinical Medical College of Nanchang University, Jiangxi, China
| | - Na Zhang
- Department of Neurology, The First Affiliated Hospital of Nanchang University, Jiangxi, China
| | - Meihua Li
- Department of Neurosurgery, The First Affiliated Hospital of Nanchang University, Jiangxi, China
| | - Tao Hong
- Department of Neurosurgery, The First Affiliated Hospital of Nanchang University, Jiangxi, China
| | - Wei Meng
- Department of Neurosurgery, The First Affiliated Hospital of Nanchang University, Jiangxi, China
| | - Taohui Ouyang
- Department of Neurosurgery, The First Affiliated Hospital of Nanchang University, Jiangxi, China
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42
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Liu Q, Yan T, Tan X, Wei Z, Li Y, Sun Z, Zhang H, Chen J. Genome-Wide Identification and Gene Expression Analysis of the OTU DUB Family in Oryza sativa. Viruses 2022; 14:v14020392. [PMID: 35215984 PMCID: PMC8878984 DOI: 10.3390/v14020392] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/24/2022] [Revised: 02/03/2022] [Accepted: 02/11/2022] [Indexed: 12/10/2022] Open
Abstract
Ovarian tumor domain (OTU)-containing deubiquitinating enzymes (DUBs) are an essential DUB to maintain protein stability in plants and play important roles in plant growth development and stress response. However, there is little genome-wide identification and analysis of the OTU gene family in rice. In this study, we identified 20 genes of the OTU family in rice genome, which were classified into four groups based on the phylogenetic analysis. Their gene structures, conserved motifs and domains, chromosomal distribution, and cis elements in promoters were further studied. In addition, OTU gene expression patterns in response to plant hormone treatments, including SA, MeJA, NAA, BL, and ABA, were investigated by RT-qPCR analysis. The results showed that the expression profile of OsOTU genes exhibited plant hormone-specific expression. Expression levels of most of the rice OTU genes were significantly changed in response to rice stripe virus (RSV), rice black-streaked dwarf virus (RBSDV), Southern rice black-streaked dwarf virus (SRBSDV), and Rice stripe mosaic virus (RSMV). These results suggest that the rice OTU genes are involved in diverse hormone signaling pathways and in varied responses to virus infection, providing new insights for further functional study of OsOTU genes.
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Affiliation(s)
- Qiannan Liu
- College of Plant Protection, Northwest Agriculture and Forestry University, Yangling, Xianyang 712100, China; (Q.L.); (T.Y.); (X.T.)
- State Key Laboratory for Managing Biotic and Chemical Threats to the Quality and Safety of Agro-Products, Key Laboratory of Biotechnology in Plant Protection of Ministry of Agriculture and Zhejiang Province, Institute of Plant Virology, Ningbo University, Ningbo 315211, China; (Z.W.); (Y.L.); (Z.S.)
| | - Tingyun Yan
- College of Plant Protection, Northwest Agriculture and Forestry University, Yangling, Xianyang 712100, China; (Q.L.); (T.Y.); (X.T.)
- State Key Laboratory for Managing Biotic and Chemical Threats to the Quality and Safety of Agro-Products, Key Laboratory of Biotechnology in Plant Protection of Ministry of Agriculture and Zhejiang Province, Institute of Plant Virology, Ningbo University, Ningbo 315211, China; (Z.W.); (Y.L.); (Z.S.)
| | - Xiaoxiang Tan
- College of Plant Protection, Northwest Agriculture and Forestry University, Yangling, Xianyang 712100, China; (Q.L.); (T.Y.); (X.T.)
- State Key Laboratory for Managing Biotic and Chemical Threats to the Quality and Safety of Agro-Products, Key Laboratory of Biotechnology in Plant Protection of Ministry of Agriculture and Zhejiang Province, Institute of Plant Virology, Ningbo University, Ningbo 315211, China; (Z.W.); (Y.L.); (Z.S.)
| | - Zhongyan Wei
- State Key Laboratory for Managing Biotic and Chemical Threats to the Quality and Safety of Agro-Products, Key Laboratory of Biotechnology in Plant Protection of Ministry of Agriculture and Zhejiang Province, Institute of Plant Virology, Ningbo University, Ningbo 315211, China; (Z.W.); (Y.L.); (Z.S.)
| | - Yanjun Li
- State Key Laboratory for Managing Biotic and Chemical Threats to the Quality and Safety of Agro-Products, Key Laboratory of Biotechnology in Plant Protection of Ministry of Agriculture and Zhejiang Province, Institute of Plant Virology, Ningbo University, Ningbo 315211, China; (Z.W.); (Y.L.); (Z.S.)
| | - Zongtao Sun
- State Key Laboratory for Managing Biotic and Chemical Threats to the Quality and Safety of Agro-Products, Key Laboratory of Biotechnology in Plant Protection of Ministry of Agriculture and Zhejiang Province, Institute of Plant Virology, Ningbo University, Ningbo 315211, China; (Z.W.); (Y.L.); (Z.S.)
| | - Hehong Zhang
- State Key Laboratory for Managing Biotic and Chemical Threats to the Quality and Safety of Agro-Products, Key Laboratory of Biotechnology in Plant Protection of Ministry of Agriculture and Zhejiang Province, Institute of Plant Virology, Ningbo University, Ningbo 315211, China; (Z.W.); (Y.L.); (Z.S.)
- Correspondence: (H.Z.); (J.C.)
| | - Jianping Chen
- College of Plant Protection, Northwest Agriculture and Forestry University, Yangling, Xianyang 712100, China; (Q.L.); (T.Y.); (X.T.)
- State Key Laboratory for Managing Biotic and Chemical Threats to the Quality and Safety of Agro-Products, Key Laboratory of Biotechnology in Plant Protection of Ministry of Agriculture and Zhejiang Province, Institute of Plant Virology, Ningbo University, Ningbo 315211, China; (Z.W.); (Y.L.); (Z.S.)
- Correspondence: (H.Z.); (J.C.)
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43
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The Deubiquitinase OTUB1 Is a Key Regulator of Energy Metabolism. Int J Mol Sci 2022; 23:ijms23031536. [PMID: 35163456 PMCID: PMC8836018 DOI: 10.3390/ijms23031536] [Citation(s) in RCA: 6] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/05/2022] [Revised: 01/26/2022] [Accepted: 01/27/2022] [Indexed: 11/17/2022] Open
Abstract
Dysregulated energy metabolism is a major contributor to a multitude of pathologies, including obesity and diabetes. Understanding the regulation of metabolic homeostasis is of utmost importance for the identification of therapeutic targets for the treatment of metabolically driven diseases. We previously identified the deubiquitinase OTUB1 as substrate for the cellular oxygen sensor factor-inhibiting HIF (FIH) with regulatory effects on cellular energy metabolism, but the physiological relevance of OTUB1 is unclear. Here, we report that the induced global deletion of OTUB1 in adult mice (Otub1 iKO) elevated energy expenditure, reduced age-dependent body weight gain, facilitated blood glucose clearance and lowered basal plasma insulin levels. The respiratory exchange ratio was maintained, indicating an unaltered nutrient oxidation. In addition, Otub1 deletion in cells enhanced AKT activity, leading to a larger cell size, higher ATP levels and reduced AMPK phosphorylation. AKT is an integral part of insulin-mediated signaling and Otub1 iKO mice presented with increased AKT phosphorylation following acute insulin administration combined with insulin hypersensitivity. We conclude that OTUB1 is an important regulator of metabolic homeostasis.
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44
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Schulze-Niemand E, Naumann M, Stein M. Substrate-assisted activation and selectivity of the bacterial RavD effector deubiquitinylase. Proteins 2021; 90:947-958. [PMID: 34825414 DOI: 10.1002/prot.26286] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/26/2021] [Revised: 11/02/2021] [Accepted: 11/22/2021] [Indexed: 11/12/2022]
Abstract
Deubiquitinylases (DUBs) catalyze the peptide bond cleavage of specific ubiquitin linkages at distinct protein substrates. Pathogens from viruses and bacteria independently developed effector proteins with DUB activity to mimic host DUB functions and circumvent immune responses. The effector protein RavD from Legionella pneumophila cleaves linear ubiquitin chains with an exclusive methionine-1 selectivity. It thus performs as a functional analogue of the human DUB OTULIN, which achieves its selectivity only via a specialized proximal ubiquitin S1' binding site as well as a substrate-assisted activation of the catalytic triad. An analysis of the crystal structures of bacterial RavD in its free and di-ubiquitin-bound forms, in order to rationalize the structural basis for its selectivity and activation mechanism, is not fully conclusive. As these ambiguities might arise from the introduced double mutation of the di-ubiquitin substrate in the RavD-di-ubiquitin complex crystal structure, biomolecular modeling, and molecular dynamics sampling (1-2 μs for each system of RavD and OTULIN) were employed to reconstitute the physiological RavD-di-ubiquitin complex. The simulations show that the distal S1 ubiquitin binding sites of RavD and OTULIN are similar in terms of interface area, composition, and ubiquitin binding affinity. The proximal S1' site of RavD, in contrast, is significantly smaller and ubiquitin binding is weaker and more flexible than in OTULIN. Upon substrate access, the residues of the catalytic triad of RavD show a reduction of flexibility and a conformational transition toward a catalytically active state. Thus, the enzymatic activation of RavD is presumably also substrate-assisted and a clear rationale for the common M1-substrate selectivity.
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Affiliation(s)
- Eric Schulze-Niemand
- Institute for Experimental Internal Medicine, Medical Faculty, Otto-von-Guericke University, Magdeburg, Germany.,Molecular Simulations and Design Group, Max Planck Institute for Dynamics of Complex Technical Systems, Magdeburg, Germany
| | - Michael Naumann
- Institute for Experimental Internal Medicine, Medical Faculty, Otto-von-Guericke University, Magdeburg, Germany
| | - Matthias Stein
- Molecular Simulations and Design Group, Max Planck Institute for Dynamics of Complex Technical Systems, Magdeburg, Germany
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45
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Kim S, Park K, Oh JM, Kim H. RNF126 is a positive regulator of TRAF3 ubiquitination. Biosci Biotechnol Biochem 2021; 85:2420-2428. [PMID: 34643674 DOI: 10.1093/bbb/zbab177] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/09/2021] [Accepted: 10/06/2021] [Indexed: 11/14/2022]
Abstract
Ubiquitination and deubiquitination of signaling molecules are critical regulatory mechanisms in various biological contexts such as inflammatory signaling and the DNA damage response. Thus, finely tuned regulation of protein ubiquitination is essential for maintaining cellular homeostasis. Here, we showed that the RING finger protein RNF126 interacts with TRAF3 and promotes its K63-linked polyubiquitination, which is a crucial step in the TRAF3-dependent antiviral response. We found that RNF126 also interacts with OTUB1, a deubiquitinating enzyme that negatively regulates K63-linked ubiquitination of TRAF3. RNF126 promotes ubiquitination of OTUB1, leading to reduced deubiquitinating activity toward TRAF3. Moreover, RNF126 promotes ubiquitination of OTUB1 on cysteine 91, which is reportedly required for its catalytic activity. Taken together, our results suggest that RNF126 positively regulates the antiviral response by directly promoting K63-linked polyubiquitination of TRAF3 and by reducing OTUB1 activity.
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Affiliation(s)
- Soomi Kim
- Department of Life Sciences, Ulsan National Institute of Science and Technology (UNIST), Ulsan, Republic of Korea
| | - Kibeom Park
- Department of Life Sciences, Ulsan National Institute of Science and Technology (UNIST), Ulsan, Republic of Korea
| | - Jung-Min Oh
- Department of Oral Biochemistry, Dental and Life Science Institute, School of Dentistry, Pusan National University, Yangsan, Republic of Korea
| | - Hongtae Kim
- Department of Life Sciences, Ulsan National Institute of Science and Technology (UNIST), Ulsan, Republic of Korea
- Center for Genomic Integrity Institute for Basic Science (IBS), UNIST, Ulsan, Republic of Korea
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46
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Ruiz-Serrano A, Monné Rodríguez JM, Günter J, Sherman SPM, Jucht AE, Fluechter P, Volkova YL, Pfundstein S, Pellegrini G, Wagner CA, Schneider C, Wenger RH, Scholz CC. OTUB1 regulates lung development, adult lung tissue homeostasis, and respiratory control. FASEB J 2021; 35:e22039. [PMID: 34793600 DOI: 10.1096/fj.202100346r] [Citation(s) in RCA: 17] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/23/2021] [Revised: 09/17/2021] [Accepted: 10/28/2021] [Indexed: 12/30/2022]
Abstract
OTUB1 is one of the most highly expressed deubiquitinases, counter-regulating the two most abundant ubiquitin chain types. OTUB1 expression is linked to the development and progression of lung cancer and idiopathic pulmonary fibrosis in humans. However, the physiological function of OTUB1 is unknown. Here, we show that constitutive whole-body Otub1 deletion in mice leads to perinatal lethality by asphyxiation. Analysis of (single-cell) RNA sequencing and proteome data demonstrated that OTUB1 is expressed in all lung cell types with a particularly high expression during late-stage lung development (E16.5, E18.5). At E18.5, the lungs of animals with Otub1 deletion presented with increased cell proliferation that decreased saccular air space and prevented inhalation. Flow cytometry-based analysis of E18.5 lung tissue revealed that Otub1 deletion increased proliferation of major lung parenchymal and mesenchymal/other non-hematopoietic cell types. Adult mice with conditional whole-body Otub1 deletion (wbOtub1del/del ) also displayed increased lung cell proliferation in addition to hyperventilation and failure to adapt the respiratory pattern to hypoxia. On the molecular level, Otub1 deletion enhanced mTOR signaling in embryonic and adult lung tissues. Based on these results, we propose that OTUB1 is a negative regulator of mTOR signaling with essential functions for lung cell proliferation, lung development, adult lung tissue homeostasis, and respiratory regulation.
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Affiliation(s)
| | - Josep M Monné Rodríguez
- Laboratory for Animal Model Pathology (LAMP), Institute of Veterinary Pathology, University of Zurich, Zurich, Switzerland
| | - Julia Günter
- Institute of Physiology, University of Zurich, Zurich, Switzerland.,National Centre of Competence in Research 'Kidney.CH', Zurich, Switzerland
| | | | | | - Pascal Fluechter
- Institute of Physiology, University of Zurich, Zurich, Switzerland
| | - Yulia L Volkova
- Institute of Physiology, University of Zurich, Zurich, Switzerland
| | | | - Giovanni Pellegrini
- Laboratory for Animal Model Pathology (LAMP), Institute of Veterinary Pathology, University of Zurich, Zurich, Switzerland
| | - Carsten A Wagner
- Institute of Physiology, University of Zurich, Zurich, Switzerland.,National Centre of Competence in Research 'Kidney.CH', Zurich, Switzerland
| | | | - Roland H Wenger
- Institute of Physiology, University of Zurich, Zurich, Switzerland.,National Centre of Competence in Research 'Kidney.CH', Zurich, Switzerland
| | - Carsten C Scholz
- Institute of Physiology, University of Zurich, Zurich, Switzerland.,National Centre of Competence in Research 'Kidney.CH', Zurich, Switzerland
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47
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Snyder NA, Silva GM. Deubiquitinating enzymes (DUBs): Regulation, homeostasis, and oxidative stress response. J Biol Chem 2021; 297:101077. [PMID: 34391779 PMCID: PMC8424594 DOI: 10.1016/j.jbc.2021.101077] [Citation(s) in RCA: 147] [Impact Index Per Article: 36.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/17/2021] [Revised: 08/10/2021] [Accepted: 08/11/2021] [Indexed: 12/17/2022] Open
Abstract
Ubiquitin signaling is a conserved, widespread, and dynamic process in which protein substrates are rapidly modified by ubiquitin to impact protein activity, localization, or stability. To regulate this process, deubiquitinating enzymes (DUBs) counter the signal induced by ubiquitin conjugases and ligases by removing ubiquitin from these substrates. Many DUBs selectively regulate physiological pathways employing conserved mechanisms of ubiquitin bond cleavage. DUB activity is highly regulated in dynamic environments through protein-protein interaction, posttranslational modification, and relocalization. The largest family of DUBs, cysteine proteases, are also sensitive to regulation by oxidative stress, as reactive oxygen species (ROS) directly modify the catalytic cysteine required for their enzymatic activity. Current research has implicated DUB activity in human diseases, including various cancers and neurodegenerative disorders. Due to their selectivity and functional roles, DUBs have become important targets for therapeutic development to treat these conditions. This review will discuss the main classes of DUBs and their regulatory mechanisms with a particular focus on DUB redox regulation and its physiological impact during oxidative stress.
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Affiliation(s)
- Nathan A Snyder
- Department of Biology, Duke University, Durham, North Carolina, USA
| | - Gustavo M Silva
- Department of Biology, Duke University, Durham, North Carolina, USA.
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48
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Iskandar A, Zulkifli NW, Ahmad MK, Theva Das K, Zulkifle N. OTUB1 expression and interaction network analyses in MCF-7 breast cancer cells. GENE REPORTS 2021. [DOI: 10.1016/j.genrep.2021.101273] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/24/2022]
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49
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Koschel J, Nishanth G, Just S, Harit K, Kröger A, Deckert M, Naumann M, Schlüter D. OTUB1 prevents lethal hepatocyte necroptosis through stabilization of c-IAP1 during murine liver inflammation. Cell Death Differ 2021; 28:2257-2275. [PMID: 33712742 PMCID: PMC8257688 DOI: 10.1038/s41418-021-00752-9] [Citation(s) in RCA: 29] [Impact Index Per Article: 7.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/20/2020] [Revised: 01/25/2021] [Accepted: 02/04/2021] [Indexed: 01/31/2023] Open
Abstract
In bacterial and sterile inflammation of the liver, hepatocyte apoptosis is, in contrast to necroptosis, a common feature. The molecular mechanisms preventing hepatocyte necroptosis and the potential consequences of hepatocyte necroptosis are largely unknown. Apoptosis and necroptosis are critically regulated by the ubiquitination of signaling molecules but especially the regulatory function of deubiquitinating enzymes (DUBs) is imperfectly defined. Here, we addressed the role of the DUB OTU domain aldehyde binding-1 (OTUB1) in hepatocyte cell death upon both infection with the hepatocyte-infecting bacterium Listeria monocytogenes (Lm) and D-Galactosamine (DGal)/Tumor necrosis factor (TNF)-induced sterile inflammation. Combined in vivo and in vitro experiments comprising mice lacking OTUB1 specifically in liver parenchymal cells (OTUB1LPC-KO) and human OTUB1-deficient HepG2 cells revealed that OTUB1 prevented hepatocyte necroptosis but not apoptosis upon infection with Lm and DGal/TNF challenge. Lm-induced necroptosis in OTUB1LPC-KO mice resulted in increased alanine aminotransferase (ALT) and lactate dehydrogenase (LDH) release and rapid lethality. Treatment with the receptor-interacting serine/threonine-protein kinase (RIPK) 1 inhibitor necrostatin-1s and deletion of the pseudokinase mixed lineage kinase domain-like protein (MLKL) prevented liver damage and death of infected OTUB1LPC-KO mice. Mechanistically, OTUB1 reduced K48-linked polyubiquitination of the cellular inhibitor of apoptosis 1 (c-IAP1), thereby diminishing its degradation. In the absence of OTUB1, c-IAP1 degradation resulted in reduced K63-linked polyubiquitination and increased phosphorylation of RIPK1, RIPK1/RIPK3 necrosome formation, MLKL-phosphorylation and hepatocyte death. Additionally, OTUB1-deficiency induced RIPK1-dependent extracellular-signal-regulated kinase (ERK) activation and TNF production in Lm-infected hepatocytes. Collectively, these findings identify OTUB1 as a novel regulator of hepatocyte-intrinsic necroptosis and a critical factor for survival of bacterial hepatitis and TNF challenge.
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Affiliation(s)
- Josephin Koschel
- Institute of Medical Microbiology and Hospital Epidemiology, Hannover Medical School, Hannover, Germany
- Institute of Experimental Internal Medicine, Otto von Guericke University Magdeburg, Magdeburg, Germany
| | - Gopala Nishanth
- Institute of Medical Microbiology and Hospital Epidemiology, Hannover Medical School, Hannover, Germany.
- Institute of Medical Microbiology and Hospital Hygiene, Otto von Guericke University Magdeburg, Magdeburg, Germany.
| | - Sissy Just
- Institute of Medical Microbiology and Hospital Epidemiology, Hannover Medical School, Hannover, Germany
| | - Kunjan Harit
- Institute of Medical Microbiology and Hospital Epidemiology, Hannover Medical School, Hannover, Germany
| | - Andrea Kröger
- Institute of Medical Microbiology and Hospital Hygiene, Otto von Guericke University Magdeburg, Magdeburg, Germany
- Innate Immunity and Infection Group, Helmholtz Centre for Infection Research, Braunschweig, Germany
| | - Martina Deckert
- Department of Neuropathology, Faculty of Medicine and University Hospital Cologne, University of Cologne, Cologne, Germany
| | - Michael Naumann
- Institute of Experimental Internal Medicine, Otto von Guericke University Magdeburg, Magdeburg, Germany
| | - Dirk Schlüter
- Institute of Medical Microbiology and Hospital Epidemiology, Hannover Medical School, Hannover, Germany.
- Institute of Medical Microbiology and Hospital Hygiene, Otto von Guericke University Magdeburg, Magdeburg, Germany.
- Cluster of Excellence RESIST (EXC 2155), Hannover Medical School, Hannover, Germany.
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Mulas F, Wang X, Song S, Nishanth G, Yi W, Brunn A, Larsen PK, Isermann B, Kalinke U, Barragan A, Naumann M, Deckert M, Schlüter D. The deubiquitinase OTUB1 augments NF-κB-dependent immune responses in dendritic cells in infection and inflammation by stabilizing UBC13. Cell Mol Immunol 2021; 18:1512-1527. [PMID: 32024978 PMCID: PMC8167118 DOI: 10.1038/s41423-020-0362-6] [Citation(s) in RCA: 44] [Impact Index Per Article: 11.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/29/2019] [Accepted: 01/01/2020] [Indexed: 01/09/2023] Open
Abstract
Dendritic cells (DCs) are indispensable for defense against pathogens but may also contribute to immunopathology. Activation of DCs upon the sensing of pathogens by Toll-like receptors (TLRs) is largely mediated by pattern recognition receptor/nuclear factor-κB (NF-κB) signaling and depends on the appropriate ubiquitination of the respective signaling molecules. However, the ubiquitinating and deubiquitinating enzymes involved and their interactions are only incompletely understood. Here, we reveal that the deubiquitinase OTU domain, ubiquitin aldehyde binding 1 (OTUB1) is upregulated in DCs upon murine Toxoplasma gondii infection and lipopolysaccharide challenge. Stimulation of DCs with the TLR11/12 ligand T. gondii profilin and the TLR4 ligand lipopolysaccharide induced an increase in NF-κB activation in OTUB1-competent cells, resulting in elevated interleukin-6 (IL-6), IL-12, and tumor necrosis factor (TNF) production, which was also observed upon the specific stimulation of TLR2, TLR3, TLR7, and TLR9. Mechanistically, OTUB1 promoted NF-κB activity in DCs by K48-linked deubiquitination and stabilization of the E2-conjugating enzyme UBC13, resulting in increased K63-linked ubiquitination of IRAK1 (IL-1 receptor-associated kinase 1) and TRAF6 (TNF receptor-associated factor 6). Consequently, DC-specific deletion of OTUB1 impaired the production of cytokines, in particular IL-12, by DCs over the first 2 days of T. gondii infection, resulting in the diminished production of protective interferon-γ (IFN-γ) by natural killer cells, impaired control of parasite replication, and, finally, death from chronic T. encephalitis, all of which could be prevented by low-dose IL-12 treatment in the first 3 days of infection. In contrast, impaired OTUB1-deficient DC activation and cytokine production by OTUB1-deficient DCs protected mice from lipopolysaccharide-induced immunopathology. Collectively, these findings identify OTUB1 as a potent novel regulator of DCs during infectious and inflammatory diseases.
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Affiliation(s)
- Floriana Mulas
- Institute of Medical Microbiology and Hospital Hygiene, Otto-von-Guericke University Magdeburg, 39120, Magdeburg, Germany
- Institute of Medical Microbiology and Hospital Epidemiology, Hannover Medical School, 30625, Hannover, Germany
| | - Xu Wang
- Institute of Medical Microbiology and Hospital Hygiene, Otto-von-Guericke University Magdeburg, 39120, Magdeburg, Germany.
- Institute of Medical Microbiology and Hospital Epidemiology, Hannover Medical School, 30625, Hannover, Germany.
- Chemical Biology Research Center, School of Pharmaceutical Sciences, Wenzhou Medical University, 325035, Wenzhou, China.
| | - Shanshan Song
- Institute of Medical Microbiology and Hospital Hygiene, Otto-von-Guericke University Magdeburg, 39120, Magdeburg, Germany
| | - Gopala Nishanth
- Institute of Medical Microbiology and Hospital Hygiene, Otto-von-Guericke University Magdeburg, 39120, Magdeburg, Germany
- Institute of Medical Microbiology and Hospital Epidemiology, Hannover Medical School, 30625, Hannover, Germany
| | - Wenjing Yi
- Institute of Medical Microbiology and Hospital Hygiene, Otto-von-Guericke University Magdeburg, 39120, Magdeburg, Germany
- Institute of Medical Microbiology and Hospital Epidemiology, Hannover Medical School, 30625, Hannover, Germany
| | - Anna Brunn
- Department of Neuropathology, Faculty of Medicine and University Hospital Cologne, University of Cologne, 50931, Cologne, Germany
| | - Pia-Katharina Larsen
- Institute for Experimental Infection Research, TWINCORE Centre for Experimental and Clinical Infection Research, a joint venture between the Hannover Medical School and the Helmholtz Centre for Infection Research, 30625, Hannover, Germany
| | - Berend Isermann
- Institute for Clinical Chemistry and Pathobiochemistry, Otto-von-Guericke University Magdeburg, 39120, Magdeburg, Germany
| | - Ulrich Kalinke
- Institute for Experimental Infection Research, TWINCORE Centre for Experimental and Clinical Infection Research, a joint venture between the Hannover Medical School and the Helmholtz Centre for Infection Research, 30625, Hannover, Germany
- Cluster of Excellence-Resolving Infection Susceptibility (RESIST), Hannover Medical School, 30625, Hannover, Germany
| | - Antonio Barragan
- Department of Molecular Biosciences, Stockholm University, 10691, Stockholm, Sweden
| | - Michael Naumann
- Institute for Experimental Internal Medicine, Otto-von-Guericke University Magdeburg, 39120, Magdeburg, Germany
| | - Martina Deckert
- Department of Neuropathology, Faculty of Medicine and University Hospital Cologne, University of Cologne, 50931, Cologne, Germany
| | - Dirk Schlüter
- Institute of Medical Microbiology and Hospital Hygiene, Otto-von-Guericke University Magdeburg, 39120, Magdeburg, Germany.
- Institute of Medical Microbiology and Hospital Epidemiology, Hannover Medical School, 30625, Hannover, Germany.
- Cluster of Excellence-Resolving Infection Susceptibility (RESIST), Hannover Medical School, 30625, Hannover, Germany.
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