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1
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Alotaibi G, Alkhammash A. Pharmacological landscape of endoplasmic reticulum stress: Uncovering therapeutic avenues for metabolic diseases. Eur J Pharmacol 2025; 998:177509. [PMID: 40089262 DOI: 10.1016/j.ejphar.2025.177509] [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: 11/02/2024] [Revised: 03/11/2025] [Accepted: 03/12/2025] [Indexed: 03/17/2025]
Abstract
The endoplasmic reticulum (ER) plays a fundamental role in maintaining cellular homeostasis by ensuring proper protein folding, lipid metabolism, and calcium regulation. However, disruptions to ER function, known as ER stress, activate the unfolded protein response (UPR) to restore balance. Chronic or unresolved ER stress contributes to metabolic dysfunctions, including insulin resistance, non-alcoholic fatty liver disease (NAFLD), and neurodegenerative disorders such as Parkinson's disease and Alzheimer's disease. Recent studies have also highlighted the importance of mitochondria-ER contact sites (MERCs) and ER-associated inflammation in disease progression. This review explores the current pharmacological landscape targeting ER stress, focusing on therapeutic strategies for rare metabolic and neurodegenerative diseases. It examines small molecules such as tauroursodeoxycholic acid (TUDCA) and 4-phenylbutyric acid (4-PBA), repurposed drugs like 17-AAG (17-N-allylamino-17demethoxygeldanamycin (tanespimycin)) and berberine, and phytochemicals such as resveratrol and hesperidin. Additionally, it discusses emerging therapeutic areas, including soluble epoxide hydrolase (sEH) inhibitors for metabolic disorders and MERCs modulation for neurological diseases. The review emphasizes challenges in translating these therapies to clinical applications, such as toxicity, off-target effects, limited bioavailability, and the lack of large-scale randomized controlled trials (RCTs). It also highlights the potential of personalized medicine approaches and pharmacogenomics in optimizing ER stress-targeting therapies.
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Affiliation(s)
- Ghallab Alotaibi
- Department of Pharmacology, College of Pharmacy, Shaqra University, Shaqra, 11961, Saudi Arabia.
| | - Abdullah Alkhammash
- Department of Pharmacology, College of Pharmacy, Shaqra University, Shaqra, 11961, Saudi Arabia.
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2
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Kuo CW, Gök C, Fulton H, Dickson-Murray E, Adu S, Gallen EK, Mary S, Robertson AD, Jordan F, Dunning E, Mullen W, Smith GL, Fuller W. Nanobody-thioesterase chimeras to specifically target protein palmitoylation. Nat Commun 2025; 16:1445. [PMID: 39920166 PMCID: PMC11805987 DOI: 10.1038/s41467-025-56716-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: 02/18/2024] [Accepted: 01/27/2025] [Indexed: 02/09/2025] Open
Abstract
The complexity of the cellular proteome is massively expanded by a repertoire of chemically distinct reversible post-translational modifications (PTMs) that control protein localisation, interactions, and function. The temporal and spatial control of these PTMs is central to organism physiology, and mis-regulation of PTMs is a hallmark of many diseases. Here we present an approach to manipulate PTMs on target proteins using nanobodies fused to enzymes that control these PTMs. Anti-GFP nanobodies fused to thioesterases (which depalmitoylate protein cysteines) depalmitoylate GFP tagged substrates. A chemogenetic approach to enhance nanobody affinity for its target enables temporal control of target depalmitoylation. Using a thioesterase fused to a nanobody directed against the Ca(v)1.2 beta subunit we reduce palmitoylation of the Ca(v)1.2 alpha subunit, modifying the channel's voltage dependence and arrhythmia susceptibility in stem cell derived cardiac myocytes. We conclude that nanobody enzyme chimeras represent an approach to specifically manipulate PTMs, with applications in both the laboratory and the clinic.
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Affiliation(s)
- Chien-Wen Kuo
- School of Cardiovascular & Metabolic Health, College of Medical Veterinary and Life Sciences, University of Glasgow, Glasgow, UK
| | - Caglar Gök
- School of Cardiovascular & Metabolic Health, College of Medical Veterinary and Life Sciences, University of Glasgow, Glasgow, UK
- School of Natural Sciences, College of Health and Science, University of Lincoln, Lincoln, UK
| | - Hannah Fulton
- School of Cardiovascular & Metabolic Health, College of Medical Veterinary and Life Sciences, University of Glasgow, Glasgow, UK
| | - Eleanor Dickson-Murray
- School of Cardiovascular & Metabolic Health, College of Medical Veterinary and Life Sciences, University of Glasgow, Glasgow, UK
| | - Samuel Adu
- School of Cardiovascular & Metabolic Health, College of Medical Veterinary and Life Sciences, University of Glasgow, Glasgow, UK
| | - Emily K Gallen
- School of Cardiovascular & Metabolic Health, College of Medical Veterinary and Life Sciences, University of Glasgow, Glasgow, UK
- Harvard Medical School, Boston, MA, USA
| | - Sheon Mary
- School of Cardiovascular & Metabolic Health, College of Medical Veterinary and Life Sciences, University of Glasgow, Glasgow, UK
| | - Alan D Robertson
- School of Cardiovascular & Metabolic Health, College of Medical Veterinary and Life Sciences, University of Glasgow, Glasgow, UK
| | - Fiona Jordan
- School of Cardiovascular & Metabolic Health, College of Medical Veterinary and Life Sciences, University of Glasgow, Glasgow, UK
| | - Emma Dunning
- School of Cardiovascular & Metabolic Health, College of Medical Veterinary and Life Sciences, University of Glasgow, Glasgow, UK
| | - William Mullen
- School of Cardiovascular & Metabolic Health, College of Medical Veterinary and Life Sciences, University of Glasgow, Glasgow, UK
| | - Godfrey L Smith
- School of Cardiovascular & Metabolic Health, College of Medical Veterinary and Life Sciences, University of Glasgow, Glasgow, UK
| | - William Fuller
- School of Cardiovascular & Metabolic Health, College of Medical Veterinary and Life Sciences, University of Glasgow, Glasgow, UK.
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3
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Chen J, Ye H. Expanding horizons: genetic code expansion technology in the study of PTM functions. Bioorg Med Chem 2025; 118:118049. [PMID: 39729921 DOI: 10.1016/j.bmc.2024.118049] [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: 10/01/2024] [Revised: 12/14/2024] [Accepted: 12/18/2024] [Indexed: 12/29/2024]
Abstract
Recent advancements in Genetic Code Expansion (GCE) have significantly enhanced our understanding of post-translational modifications (PTMs), which are critical for protein regulation. GCE facilitates the precise incorporation of unnatural amino acids (UAAs) at specific sites within proteins of interest (POIs), making it a powerful tool for modulating PTMs in vivo. This review summarizes the various UAAs utilized to directly incorporate PTMs into proteins through GCE, with a focus on their applications in both histone and non-histone PTMs research. We also discuss the challenges associated with incorporating certain PTMs into target proteins via GCE and provide an overview of the latest strategies developed to overcome these hurdles.
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Affiliation(s)
- Jingzhuo Chen
- Jiangsu Provincial Key Laboratory of Drug Metabolism and Pharmacokinetics, State Key Laboratory of Natural Medicines, China Pharmaceutical University, Tongjiaxiang No. 24, Nanjing 210009, Jiangsu, China
| | - Hui Ye
- Jiangsu Provincial Key Laboratory of Drug Metabolism and Pharmacokinetics, State Key Laboratory of Natural Medicines, China Pharmaceutical University, Tongjiaxiang No. 24, Nanjing 210009, Jiangsu, China.
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4
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Ding W, Gu J, Xu W, Wu J, Huang Y, Zhang S, Lin S. The Biosynthesis and Applications of Protein Lipidation. Chem Rev 2024; 124:12176-12212. [PMID: 39441663 DOI: 10.1021/acs.chemrev.4c00419] [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: 10/25/2024]
Abstract
Protein lipidation dramatically affects protein structure, localization, and trafficking via remodeling protein-membrane and protein-protein interactions through hydrophobic lipid moieties. Understanding the biosynthesis of lipidated proteins, whether natural ones or mimetics, is crucial for reconstructing, validating, and studying the molecular mechanisms and biological functions of protein lipidation. In this Perspective, we first provide an overview of the natural enzymatic biosynthetic pathways of protein lipidation in mammalian cells, focusing on the enzymatic machineries and their chemical linkages. We then discuss strategies to biosynthesize protein lipidation in mammalian cells by engineering modification machineries and substrates. Additionally, we explore site-specific protein lipidation biosynthesis in vitro via enzyme-mediated ligations and in vivo primarily through genetic code expansion strategies. We also discuss the use of small molecule tools to modulate the process of protein lipidation biosynthesis. Finally, we provide concluding remarks and discuss future directions for the biosynthesis and applications of protein lipidation.
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Affiliation(s)
- Wenlong Ding
- Life Sciences Institute, Institute of Fundamental and Transdisciplinary Research, Zhejiang University, Hangzhou 310058, China
- Center for Oncology Medicine, the Fourth Affiliated Hospital of School of Medicine, and International School of Medicine, International Institutes of Medicine, Zhejiang University, Yiwu 322000, China
| | - Jiayu Gu
- Department of Medical Oncology, State Key Laboratory of Transvascular Implantation Devices, The Second Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou 310058, China
| | - Wenyuan Xu
- Life Sciences Institute, Institute of Fundamental and Transdisciplinary Research, Zhejiang University, Hangzhou 310058, China
| | - Jing Wu
- Hubei Hongshan Laboratory, College of Biomedicine and Health, Huazhong Agricultural University, Wuhan 430070, China
| | - Yiwen Huang
- Hubei Hongshan Laboratory, College of Biomedicine and Health, Huazhong Agricultural University, Wuhan 430070, China
| | - Shuai Zhang
- Hubei Hongshan Laboratory, College of Biomedicine and Health, Huazhong Agricultural University, Wuhan 430070, China
| | - Shixian Lin
- Life Sciences Institute, Institute of Fundamental and Transdisciplinary Research, Zhejiang University, Hangzhou 310058, China
- Zhejiang Provincial Key Laboratory for Cancer Molecular Cell Biology, Shaoxing Institute, Zhejiang University, Shaoxing 321000, China
- Department of Medical Oncology, State Key Laboratory of Transvascular Implantation Devices, The Second Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou 310058, China
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5
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Dunkelmann DL, Chin JW. Engineering Pyrrolysine Systems for Genetic Code Expansion and Reprogramming. Chem Rev 2024; 124:11008-11062. [PMID: 39235427 PMCID: PMC11467909 DOI: 10.1021/acs.chemrev.4c00243] [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: 04/01/2024] [Revised: 07/29/2024] [Accepted: 07/31/2024] [Indexed: 09/06/2024]
Abstract
Over the past 16 years, genetic code expansion and reprogramming in living organisms has been transformed by advances that leverage the unique properties of pyrrolysyl-tRNA synthetase (PylRS)/tRNAPyl pairs. Here we summarize the discovery of the pyrrolysine system and describe the unique properties of PylRS/tRNAPyl pairs that provide a foundation for their transformational role in genetic code expansion and reprogramming. We describe the development of genetic code expansion, from E. coli to all domains of life, using PylRS/tRNAPyl pairs, and the development of systems that biosynthesize and incorporate ncAAs using pyl systems. We review applications that have been uniquely enabled by the development of PylRS/tRNAPyl pairs for incorporating new noncanonical amino acids (ncAAs), and strategies for engineering PylRS/tRNAPyl pairs to add noncanonical monomers, beyond α-L-amino acids, to the genetic code of living organisms. We review rapid progress in the discovery and scalable generation of mutually orthogonal PylRS/tRNAPyl pairs that can be directed to incorporate diverse ncAAs in response to diverse codons, and we review strategies for incorporating multiple distinct ncAAs into proteins using mutually orthogonal PylRS/tRNAPyl pairs. Finally, we review recent advances in the encoded cellular synthesis of noncanonical polymers and macrocycles and discuss future developments for PylRS/tRNAPyl pairs.
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Affiliation(s)
- Daniel L. Dunkelmann
- Medical
Research Council Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge CB2 0QH, England, United Kingdom
- Max
Planck Institute of Molecular Plant Physiology, Am Mühlenberg 1, 14476 Potsdam-Golm, Germany
| | - Jason W. Chin
- Medical
Research Council Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge CB2 0QH, England, United Kingdom
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6
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Pei S, Piao HL. Exploring Protein S-Palmitoylation: Mechanisms, Detection, and Strategies for Inhibitor Discovery. ACS Chem Biol 2024; 19:1868-1882. [PMID: 39160165 DOI: 10.1021/acschembio.4c00110] [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: 08/21/2024]
Abstract
S-palmitoylation is a reversible and dynamic process that involves the addition of long-chain fatty acids to proteins. This protein modification regulates various aspects of protein function, including subcellular localization, stability, conformation, and biomolecular interactions. The zinc finger DHHC (ZDHHC) domain-containing protein family is the main group of enzymes responsible for catalyzing protein S-palmitoylation, and 23 members have been identified in mammalian cells. Many proteins that undergo S-palmitoylation have been linked to disease pathogenesis and progression, suggesting that the development of effective inhibitors is a promising therapeutic strategy. Reducing the protein S-palmitoylation level can target either the PATs directly or their substrates. However, there are rare clinically effective S-palmitoylation inhibitors. This review aims to provide an overview of the S-palmitoylation field, including the catalytic mechanism of ZDHHC, S-palmitoylation detection methods, and the functional impact of protein S-palmitoylation. Additionally, this review focuses on current strategies for expanding the chemical toolbox to develop novel and effective inhibitors that can reduce the level of S-palmitoylation of the target protein.
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Affiliation(s)
- Shaojun Pei
- Dalian Institute of Chemical Physics, Chinese Academy of Sciences, 116023, Dalian, China
- University of Chinese Academy of Sciences, 100049 Beijing, China
| | - Hai-Long Piao
- Dalian Institute of Chemical Physics, Chinese Academy of Sciences, 116023, Dalian, China
- University of Chinese Academy of Sciences, 100049 Beijing, China
- Department of Biochemistry & Molecular Biology, School of Life Sciences, China Medical University, 110122 Shenyang, China
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7
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Yi HB, Lee S, Seo K, Kim H, Kim M, Lee HS. Cellular and Biophysical Applications of Genetic Code Expansion. Chem Rev 2024; 124:7465-7530. [PMID: 38753805 DOI: 10.1021/acs.chemrev.4c00112] [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: 05/18/2024]
Abstract
Despite their diverse functions, proteins are inherently constructed from a limited set of building blocks. These compositional constraints pose significant challenges to protein research and its practical applications. Strategically manipulating the cellular protein synthesis system to incorporate novel building blocks has emerged as a critical approach for overcoming these constraints in protein research and application. In the past two decades, the field of genetic code expansion (GCE) has achieved significant advancements, enabling the integration of numerous novel functionalities into proteins across a variety of organisms. This technological evolution has paved the way for the extensive application of genetic code expansion across multiple domains, including protein imaging, the introduction of probes for protein research, analysis of protein-protein interactions, spatiotemporal control of protein function, exploration of proteome changes induced by external stimuli, and the synthesis of proteins endowed with novel functions. In this comprehensive Review, we aim to provide an overview of cellular and biophysical applications that have employed GCE technology over the past two decades.
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Affiliation(s)
- Han Bin Yi
- Department of Chemistry, Sogang University, 35 Baekbeom-ro, Mapo-gu, Seoul 04107, Republic of Korea
| | - Seungeun Lee
- Department of Chemistry, Sogang University, 35 Baekbeom-ro, Mapo-gu, Seoul 04107, Republic of Korea
| | - Kyungdeok Seo
- Department of Chemistry, Sogang University, 35 Baekbeom-ro, Mapo-gu, Seoul 04107, Republic of Korea
| | - Hyeongjo Kim
- Department of Chemistry, Sogang University, 35 Baekbeom-ro, Mapo-gu, Seoul 04107, Republic of Korea
| | - Minah Kim
- Department of Chemistry, Sogang University, 35 Baekbeom-ro, Mapo-gu, Seoul 04107, Republic of Korea
| | - Hyun Soo Lee
- Department of Chemistry, Sogang University, 35 Baekbeom-ro, Mapo-gu, Seoul 04107, Republic of Korea
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8
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Gan Q, Fan C. Orthogonal Translation for Site-Specific Installation of Post-translational Modifications. Chem Rev 2024; 124:2805-2838. [PMID: 38373737 PMCID: PMC11230630 DOI: 10.1021/acs.chemrev.3c00850] [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: 02/21/2024]
Abstract
Post-translational modifications (PTMs) endow proteins with new properties to respond to environmental changes or growth needs. With the development of advanced proteomics techniques, hundreds of distinct types of PTMs have been observed in a wide range of proteins from bacteria, archaea, and eukarya. To identify the roles of these PTMs, scientists have applied various approaches. However, high dynamics, low stoichiometry, and crosstalk between PTMs make it almost impossible to obtain homogeneously modified proteins for characterization of the site-specific effect of individual PTM on target proteins. To solve this problem, the genetic code expansion (GCE) strategy has been introduced into the field of PTM studies. Instead of modifying proteins after translation, GCE incorporates modified amino acids into proteins during translation, thus generating site-specifically modified proteins at target positions. In this review, we summarize the development of GCE systems for orthogonal translation for site-specific installation of PTMs.
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Affiliation(s)
- Qinglei Gan
- Department of Chemistry and Biochemistry, University of Arkansas, Fayetteville, Arkansas 72701, United States
| | - Chenguang Fan
- Department of Chemistry and Biochemistry, University of Arkansas, Fayetteville, Arkansas 72701, United States
- Cell and Molecular Biology Program, University of Arkansas, Fayetteville, Arkansas 72701, United States
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9
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Xu Y, Ding K, Peng T. Chemical Proteomics Reveals N ε-Fatty-Acylation of Septins by Rho Inactivation Domain (RID) of the Vibrio MARTX Toxin to Alter Septin Localization and Organization. Mol Cell Proteomics 2024; 23:100730. [PMID: 38311109 PMCID: PMC10924143 DOI: 10.1016/j.mcpro.2024.100730] [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: 10/31/2023] [Revised: 01/16/2024] [Accepted: 01/31/2024] [Indexed: 02/06/2024] Open
Abstract
Vibrio species, the Gram-negative bacterial pathogens causing cholera and sepsis, produce multiple secreted virulence factors for infection and pathogenesis. Among these is the multifunctional-autoprocessing repeats-in-toxin (MARTX) toxin that releases several critical effector domains with distinct functions inside eukaryotic host cells. One such effector domain, the Rho inactivation domain (RID), has been discovered to catalyze long-chain Nε-fatty-acylation on lysine residues of Rho GTPases, causing inactivation of Rho GTPases and disruption of the host actin cytoskeleton. However, whether RID modifies other host proteins to exert additional functions remains to be determined. Herein, we describe the integration of bioorthogonal chemical labeling and quantitative proteomics to globally profile the target proteins modified by RID in living cells. More than 246 proteins are identified as new RID substrates, including many involved in GTPase regulation, cytoskeletal organization, and cell division. We demonstrate that RID extensively Nε-fatty-acylates septin proteins, the fourth cytoskeletal component of mammalian cells with important roles in diverse cellular processes. While affinity purification and mass spectrometry analysis show that RID-mediated Nε-fatty-acylation does not affect septin-septin interactions, this modification increases the membrane association of septins and confers localization to detergent-resistant membrane rafts. As a result, the filamentous assembly and organization of septins are disrupted by RID-mediated Nε-fatty-acylation, further contributing to cytoskeletal and mitotic defects that phenocopy the effects of septin depletion. Overall, our work greatly expands the substrate scope and function of RID and demonstrates the role of RID-mediated Nε-fatty-acylation in manipulating septin localization and organization.
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Affiliation(s)
- Yaxin Xu
- State Key Laboratory of Chemical Oncogenomics, School of Chemical Biology and Biotechnology, Peking University Shenzhen Graduate School, Shenzhen, China
| | - Ke Ding
- State Key Laboratory of Chemical Oncogenomics, School of Chemical Biology and Biotechnology, Peking University Shenzhen Graduate School, Shenzhen, China
| | - Tao Peng
- State Key Laboratory of Chemical Oncogenomics, School of Chemical Biology and Biotechnology, Peking University Shenzhen Graduate School, Shenzhen, China; Shenzhen Bay Laboratory, Institute of Chemical Biology, Shenzhen, China.
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10
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Giltrap A, Yuan Y, Davis BG. Late-Stage Functionalization of Living Organisms: Rethinking Selectivity in Biology. Chem Rev 2024; 124:889-928. [PMID: 38231473 PMCID: PMC10870719 DOI: 10.1021/acs.chemrev.3c00579] [Citation(s) in RCA: 1] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/15/2023] [Revised: 11/14/2023] [Accepted: 11/15/2023] [Indexed: 01/18/2024]
Abstract
With unlimited selectivity, full post-translational chemical control of biology would circumvent the dogma of genetic control. The resulting direct manipulation of organisms would enable atomic-level precision in "editing" of function. We argue that a key aspect that is still missing in our ability to do this (at least with a high degree of control) is the selectivity of a given chemical reaction in a living organism. In this Review, we systematize existing illustrative examples of chemical selectivity, as well as identify needed chemical selectivities set in a hierarchy of anatomical complexity: organismo- (selectivity for a given organism over another), tissuo- (selectivity for a given tissue type in a living organism), cellulo- (selectivity for a given cell type in an organism or tissue), and organelloselectivity (selectivity for a given organelle or discrete body within a cell). Finally, we analyze more traditional concepts such as regio-, chemo-, and stereoselective reactions where additionally appropriate. This survey of late-stage biomolecule methods emphasizes, where possible, functional consequences (i.e., biological function). In this way, we explore a concept of late-stage functionalization of living organisms (where "late" is taken to mean at a given state of an organism in time) in which programmed and selective chemical reactions take place in life. By building on precisely analyzed notions (e.g., mechanism and selectivity) we believe that the logic of chemical methodology might ultimately be applied to increasingly complex molecular constructs in biology. This could allow principles developed at the simple, small-molecule level to progress hierarchically even to manipulation of physiology.
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Affiliation(s)
- Andrew
M. Giltrap
- The
Rosalind Franklin Institute, Oxfordshire OX11 0FA, U.K.
- Department
of Pharmacology, University of Oxford, Oxford OX1 3QT, U.K.
| | - Yizhi Yuan
- The
Rosalind Franklin Institute, Oxfordshire OX11 0FA, U.K.
- Department
of Pharmacology, University of Oxford, Oxford OX1 3QT, U.K.
| | - Benjamin G. Davis
- The
Rosalind Franklin Institute, Oxfordshire OX11 0FA, U.K.
- Department
of Pharmacology, University of Oxford, Oxford OX1 3QT, U.K.
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11
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Ding W, Zhao H, Chen Y, Lin S. New Strategies for Probing the Biological Functions of Protein Post-translational Modifications in Mammalian Cells with Genetic Code Expansion. Acc Chem Res 2023; 56:2827-2837. [PMID: 37793174 DOI: 10.1021/acs.accounts.3c00460] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/06/2023]
Abstract
Protein post-translational modification (PTM) is a major mechanism for functional diversification of the human genome and plays a crucial role in almost every aspect of cellular processes, and the dysregulation of the protein PTM network has been associated with a variety of human diseases. Using high-resolution mass spectrometry, protein PTMs can be efficiently discovered and profiled under various biological and physiological conditions. However, it is often challenging to address the biological function of PTMs with biochemical and mutagenesis-based approaches. Specifically, this field lacks methods that allow gain-of-function studies of protein PTMs to understand their functional consequences in living cells. In this context, the genetic code expansion (GCE) strategy has made tremendous progress in the direct installation of PTMs and their analogs in the form of noncanonical amino acids (ncAAs) for gain-of-function investigations.In addition to studying the biological functions of known protein PTMs, the discovery of new protein PTMs is even more challenging due to the lack of chemical information for designing specific enrichment methods. Genetically encoded ncAAs in the proteome can be used as specific baits to enrich and subsequently identify new PTMs by mass spectrometry.In this Account, we discuss recent developments in the investigation of the biological functions of protein PTMs and the discovery of protein PTMs using new GCE strategies. First, we leveraged a chimeric design to construct several broadly orthogonal translation systems (OTSs). These broad OTSs can be engineered to efficiently incorporate different ncAAs in both E. coli and mammalian cells. With these broad OTSs, we accomplish the following: (1) We develop a computer-aided strategy for the design and genetic incorporation of length-tunable lipidation mimics. These lipidation mimics can fully recapitulate the biochemical properties of natural lipidation in membrane association for probing its biological functions on signaling proteins and in albumin binding for designing long-acting protein drugs. (2) We demonstrate that the binding affinity between histone methylations and their corresponding readers can be substantially increased with genetically encoded electron-rich Trp derivatives. These engineered affinity-enhanced readers can be applied to enrich, image, and profile the interactome of chromatin methylations. (3) We report the identification and verification of a novel type of protein PTM, aminoacylated lysine ubiquitination, using genetically encoded PTM ncAAs as chemical probes. This approach provides a general strategy for the identification of unknown PTMs by increasing the abundance of PTM bait probes.
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Affiliation(s)
- Wenlong Ding
- Department of Medical Oncology, The Second Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou 310058, China
- Zhejiang Provincial Key Laboratory for Cancer Molecular Cell Biology, Life Sciences Institute, Zhejiang University, Hangzhou 310058, China
| | - Hongxia Zhao
- Zhejiang Provincial Key Laboratory for Cancer Molecular Cell Biology, Life Sciences Institute, Zhejiang University, Hangzhou 310058, China
| | - Yulin Chen
- Zhejiang Provincial Key Laboratory for Cancer Molecular Cell Biology, Life Sciences Institute, Zhejiang University, Hangzhou 310058, China
- Shaoxing Institute, Zhejiang University, Shaoxing 321000, China
| | - Shixian Lin
- Department of Medical Oncology, The Second Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou 310058, China
- Zhejiang Provincial Key Laboratory for Cancer Molecular Cell Biology, Life Sciences Institute, Zhejiang University, Hangzhou 310058, China
- Shaoxing Institute, Zhejiang University, Shaoxing 321000, China
- Cancer Center, Zhejiang University, Hangzhou 310058, China
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12
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Kitamura N, Galligan JJ. A global view of the human post-translational modification landscape. Biochem J 2023; 480:1241-1265. [PMID: 37610048 PMCID: PMC10586784 DOI: 10.1042/bcj20220251] [Citation(s) in RCA: 26] [Impact Index Per Article: 13.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/05/2023] [Revised: 07/26/2023] [Accepted: 08/07/2023] [Indexed: 08/24/2023]
Abstract
Post-translational modifications (PTMs) provide a rapid response to stimuli, finely tuning metabolism and gene expression and maintain homeostasis. Advances in mass spectrometry over the past two decades have significantly expanded the list of known PTMs in biology and as instrumentation continues to improve, this list will surely grow. While many PTMs have been studied in detail (e.g. phosphorylation, acetylation), the vast majority lack defined mechanisms for their regulation and impact on cell fate. In this review, we will highlight the field of PTM research as it currently stands, discussing the mechanisms that dictate site specificity, analytical methods for their detection and study, and the chemical tools that can be leveraged to define PTM regulation. In addition, we will highlight the approaches needed to discover and validate novel PTMs. Lastly, this review will provide a starting point for those interested in PTM biology, providing a comprehensive list of PTMs and what is known regarding their regulation and metabolic origins.
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Affiliation(s)
- Naoya Kitamura
- Department of Pharmacology and College of Pharmacy, University of Arizona, Tucson, Arizona 85721, U.S.A
| | - James J. Galligan
- Department of Pharmacology and College of Pharmacy, University of Arizona, Tucson, Arizona 85721, U.S.A
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13
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Congreve SD, Main A, Butler AS, Gao X, Brown E, Du C, Choisy SC, Cheng H, Hancox JC, Fuller W. Palmitoylation regulates the magnitude of HCN4-mediated currents in mammalian cells. Front Physiol 2023; 14:1163339. [PMID: 37123274 PMCID: PMC10133559 DOI: 10.3389/fphys.2023.1163339] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/10/2023] [Accepted: 03/31/2023] [Indexed: 05/02/2023] Open
Abstract
The sinoatrial node (SAN) and subsidiary pacemakers in the cardiac conduction system generate spontaneous electrical activity which is indispensable for electrical and therefore contractile function of the heart. The hyperpolarisation-activated cyclic nucleotide-gated channel HCN4 is responsible for genesis of the pacemaker "funny" current during diastolic depolarisation. S-palmitoylation, the reversible conjugation of the fatty acid palmitate to protein cysteine sulfhydryls, regulates the activity of key cardiac Na+ and Ca2+ handling proteins, influencing their membrane microdomain localisation and function. We investigated HCN4 palmitoylation and its functional consequences in engineered human embryonic kidney 293T cells as well as endogenous HCN4 in neonatal rat ventricular myocytes. HCN4 was palmitoylated in all experimental systems investigated. We mapped the HCN4 palmitoylation sites to a pair of cysteines in the HCN4 intracellular amino terminus. A double cysteine-to-alanine mutation CC93A/179AA of full length HCN4 caused a ∼67% reduction in palmitoylation in comparison to wild type HCN4. We used whole-cell patch clamp to evaluate HCN4 current (IHCN4) in stably transfected 293T cells. Removal of the two N-terminal palmitoylation sites did not significantly alter half maximal activation voltage of IHCN4 or the activation slope factor. IHCN4 was significantly larger in cells expressing wild type compared to non-palmitoylated HCN4 across a range of voltages. Phylogenetic analysis revealed that although cysteine 93 is widely conserved across all classes of HCN4 vertebrate orthologs, conservation of cysteine 179 is restricted to placental mammals. Collectively, we provide evidence for functional regulation of HCN4 via palmitoylation of its amino terminus in vertebrates. We suggest that by recruiting the amino terminus to the bilayer, palmitoylation enhances the magnitude of HCN4-mediated currents, but does not significantly affect the kinetics.
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Affiliation(s)
- Samitha Dilini Congreve
- School of Cardiovascular & Metabolic Health, University of Glasgow, Glasgow, United Kingdom
- School of Physiology, Pharmacology and Neuroscience, University of Bristol, Bristol, United Kingdom
| | - Alice Main
- School of Cardiovascular & Metabolic Health, University of Glasgow, Glasgow, United Kingdom
| | - Andrew S. Butler
- School of Physiology, Pharmacology and Neuroscience, University of Bristol, Bristol, United Kingdom
| | - Xing Gao
- School of Cardiovascular & Metabolic Health, University of Glasgow, Glasgow, United Kingdom
| | - Elaine Brown
- School of Cardiovascular & Metabolic Health, University of Glasgow, Glasgow, United Kingdom
| | - Chunyun Du
- School of Physiology, Pharmacology and Neuroscience, University of Bristol, Bristol, United Kingdom
| | - Stephanié C. Choisy
- School of Physiology, Pharmacology and Neuroscience, University of Bristol, Bristol, United Kingdom
| | - Hongwei Cheng
- School of Physiology, Pharmacology and Neuroscience, University of Bristol, Bristol, United Kingdom
| | - Jules C. Hancox
- School of Physiology, Pharmacology and Neuroscience, University of Bristol, Bristol, United Kingdom
| | - William Fuller
- School of Cardiovascular & Metabolic Health, University of Glasgow, Glasgow, United Kingdom
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14
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Peng T, Das T, Ding K, Hang HC. Functional analysis of protein post-translational modifications using genetic codon expansion. Protein Sci 2023; 32:e4618. [PMID: 36883310 PMCID: PMC10031814 DOI: 10.1002/pro.4618] [Citation(s) in RCA: 19] [Impact Index Per Article: 9.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/05/2023] [Revised: 02/23/2023] [Accepted: 03/02/2023] [Indexed: 03/09/2023]
Abstract
Post-translational modifications (PTMs) of proteins not only exponentially increase the diversity of proteoforms, but also contribute to dynamically modulating the localization, stability, activity, and interaction of proteins. Understanding the biological consequences and functions of specific PTMs has been challenging for many reasons, including the dynamic nature of many PTMs and the technical limitations to access homogenously modified proteins. The genetic code expansion technology has emerged to provide unique approaches for studying PTMs. Through site-specific incorporation of unnatural amino acids (UAAs) bearing PTMs or their mimics into proteins, genetic code expansion allows the generation of homogenous proteins with site-specific modifications and atomic resolution both in vitro and in vivo. With this technology, various PTMs and mimics have been precisely introduced into proteins. In this review, we summarize the UAAs and approaches that have been recently developed to site-specifically install PTMs and their mimics into proteins for functional studies of PTMs.
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Affiliation(s)
- Tao Peng
- State Key Laboratory of Chemical OncogenomicsSchool of Chemical Biology and Biotechnology, Peking University Shenzhen Graduate SchoolShenzhenChina
- Institute of Chemical Biology, Shenzhen Bay LaboratoryShenzhenChina
| | - Tandrila Das
- Departments of Immunology and Microbiology and ChemistryScripps ResearchLa JollaCaliforniaUSA
| | - Ke Ding
- State Key Laboratory of Chemical OncogenomicsSchool of Chemical Biology and Biotechnology, Peking University Shenzhen Graduate SchoolShenzhenChina
| | - Howard C. Hang
- Departments of Immunology and Microbiology and ChemistryScripps ResearchLa JollaCaliforniaUSA
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15
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Applications of genetic code expansion in studying protein post-translational modification. J Mol Biol 2021; 434:167424. [PMID: 34971673 DOI: 10.1016/j.jmb.2021.167424] [Citation(s) in RCA: 25] [Impact Index Per Article: 6.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/15/2021] [Revised: 12/19/2021] [Accepted: 12/21/2021] [Indexed: 01/18/2023]
Abstract
Various post-translational modifications can naturally occur on proteins, regulating the activity, subcellular localization, interaction, or stability of the proteins. However, it can be challenging to decipher the biological implication or physiological roles of site-specific modifications due to their dynamic and sub-stoichiometric nature. Genetic code expansion method, relying on an orthogonal aminoacyl-tRNA synthetase/tRNA pair, enables site-specific incorporation of non-canonical amino acids. Here we focus on the application of genetic code expansion to study site-specific protein post-translational modification in vitro and in vivo. After a brief introduction, we discuss possibilities of incorporating non-canonical amino acids containing post-translational modifications or their mimics into target proteins. This approach is applicable for Ser/Thr/Tyr phosphorylation, Tyr sulfation and nitration, Lys acetylation and acylation, Lys/His mono-methylation, as well as Arg citrullination. The next section describes the use of a precursor non-canonical amino acid followed by chemical and/or enzymatic reactions to afford the desired modification, such as Cys/Lys acylation, ubiquitin and ubiquitin-like modifications, as well as Lys/Gln methylation. We also discuss means for functional regulation of enzymes involving in post-translational modifications through genetically incorporated non-canonical amino acids. Lastly, the limitations and perspectives of genetic code expansion in studying protein post-translational modification are described.
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16
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Cheng WX, Ren Y, Lu MM, Xu LL, Gao JG, Chen D, Kalyani FS, Lv ZY, Chen CX, Ji F, Lin HN, Jin X. Palmitoylation in Crohn’s disease: Current status and future directions. World J Gastroenterol 2021; 27:8201-8215. [PMID: 35068865 PMCID: PMC8717020 DOI: 10.3748/wjg.v27.i48.8201] [Citation(s) in RCA: 12] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 03/19/2021] [Revised: 05/08/2021] [Accepted: 12/10/2021] [Indexed: 02/06/2023] Open
Abstract
S-palmitoylation is one of the most common post-translational modifications in nature; however, its importance has been overlooked for decades. Crohn’s disease (CD), a subtype of inflammatory bowel disease (IBD), is an autoimmune disease characterized by chronic inflammation involving the entire gastrointestinal tract. Bowel damage and subsequent disabilities caused by CD are a growing global health issue. Well-acknowledged risk factors for CD include genetic susceptibility, environmental factors, such as a westernized lifestyle, and altered gut microbiota. However, the pathophysiological mechanisms of this disorder are not yet comprehensively understood. With the rapidly increasing global prevalence of CD and the evident role of S-palmitoylation in CD, as recently reported, there is a need to investigate the relationship between CD and S-palmitoylation. In this review, we summarize the concept, detection, and function of S-palmitoylation as well as its potential effects on CD, and provide novel insights into the pathogenesis and treatment of CD.
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Affiliation(s)
- Wei-Xin Cheng
- Department of Gastroenterology, The First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou 310003, Zhejiang Province, China
| | - Yue Ren
- Department of Gastroenterology, The First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou 310003, Zhejiang Province, China
| | - Miao-Miao Lu
- Department of Gastroenterology, The First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou 310003, Zhejiang Province, China
| | - Ling-Ling Xu
- Department of Gastroenterology, The First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou 310003, Zhejiang Province, China
| | - Jian-Guo Gao
- Department of Gastroenterology, The First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou 310003, Zhejiang Province, China
| | - Dong Chen
- Department of Colorectal Surgery, The First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou 310003, Zhejiang Province, China
| | - Farhin Shaheed Kalyani
- Department of Gastroenterology, The First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou 310003, Zhejiang Province, China
| | - Zi-Yan Lv
- Wenzhou Medical University Renji College, Wenzhou 325035, Zhejiang Province, China
| | - Chun-Xiao Chen
- Department of Gastroenterology, The First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou 310003, Zhejiang Province, China
| | - Feng Ji
- Department of Gastroenterology, The First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou 310003, Zhejiang Province, China
| | - He-Ning Lin
- Department of Chemistry and Chemical Biology, Howard Hughes Medical Institute, Cornell University, Ithaca, NY 14853, United States
| | - Xi Jin
- Department of Gastroenterology, The First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou 310003, Zhejiang Province, China
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17
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Meineke B, Heimgärtner J, Craig AJ, Landreh M, Moodie LWK, Elsässer SJ. A Genetically Encoded Picolyl Azide for Improved Live Cell Copper Click Labeling. Front Chem 2021; 9:768535. [PMID: 34858945 PMCID: PMC8632528 DOI: 10.3389/fchem.2021.768535] [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: 08/31/2021] [Accepted: 10/13/2021] [Indexed: 11/13/2022] Open
Abstract
Bioorthogonal chemistry allows rapid and highly selective reactivity in biological environments. The copper-catalyzed azide–alkyne cycloaddition (CuAAC) is a classic bioorthogonal reaction routinely used to modify azides or alkynes that have been introduced into biomolecules. Amber suppression is an efficient method for incorporating such chemical handles into proteins on the ribosome, in which noncanonical amino acids (ncAAs) are site specifically introduced into the polypeptide in response to an amber (UAG) stop codon. A variety of ncAA structures containing azides or alkynes have been proven useful for performing CuAAC chemistry on proteins. To improve CuAAC efficiency, biologically incorporated alkyne groups can be reacted with azide substrates that contain copper-chelating groups. However, the direct incorporation of copper-chelating azides into proteins has not been explored. To remedy this, we prepared the ncAA paz-lysine (PazK), which contains a picolyl azide motif. We show that PazK is efficiently incorporated into proteins by amber suppression in mammalian cells. Furthermore, PazK-labeled proteins show improved reactivity with alkyne reagents in CuAAC.
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Affiliation(s)
- Birthe Meineke
- Science for Life Laboratory, Department of Medical Biochemistry and Biophysics, Division of Genome Biology, Karolinska Institutet, Stockholm, Sweden.,Ming Wai Lau Centre for Reparative Medicine, Stockholm Node, Karolinska Institutet, Stockholm, Sweden
| | - Johannes Heimgärtner
- Science for Life Laboratory, Department of Medical Biochemistry and Biophysics, Division of Genome Biology, Karolinska Institutet, Stockholm, Sweden.,Ming Wai Lau Centre for Reparative Medicine, Stockholm Node, Karolinska Institutet, Stockholm, Sweden
| | - Alexander J Craig
- Drug Design and Discovery, Department of Medicinal Chemistry, Biomedical Centre, Uppsala University, Uppsala, Sweden
| | - Michael Landreh
- Department of Microbiology, Tumor and Cell Biology, Science for Life Laboratory, Karolinska Institutet, Stockholm, Sweden
| | - Lindon W K Moodie
- Drug Design and Discovery, Department of Medicinal Chemistry, Biomedical Centre, Uppsala University, Uppsala, Sweden.,Uppsala Antibiotic Centre, Uppsala University, Uppsala, Sweden
| | - Simon J Elsässer
- Science for Life Laboratory, Department of Medical Biochemistry and Biophysics, Division of Genome Biology, Karolinska Institutet, Stockholm, Sweden.,Ming Wai Lau Centre for Reparative Medicine, Stockholm Node, Karolinska Institutet, Stockholm, Sweden
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18
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Garst EH, Das T, Hang HC. Chemical approaches for investigating site-specific protein S-fatty acylation. Curr Opin Chem Biol 2021; 65:109-117. [PMID: 34333222 PMCID: PMC8671186 DOI: 10.1016/j.cbpa.2021.06.004] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/15/2021] [Revised: 06/09/2021] [Accepted: 06/18/2021] [Indexed: 12/27/2022]
Abstract
Protein S-fatty acylation or S-palmitoylation is a reversible and regulated lipid post-translational modification (PTM) in eukaryotes. Loss-of-function mutagenesis studies have suggested important roles for protein S-fatty acylation in many fundamental biological pathways in development, neurobiology, and immunity that are also associated with human diseases. However, the hydrophobicity and reversibility of this PTM have made site-specific gain-of-function studies more challenging to investigate. In this review, we summarize recent chemical biology approaches and methods that have enabled site-specific gain-of-function studies of protein S-fatty acylation and the investigation of the mechanisms and significance of this PTM in eukaryotic biology.
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Affiliation(s)
- Emma H Garst
- Laboratory of Chemical Biology and Microbial Pathogenesis, The Rockefeller University, New York, NY 10065, United States; Tri-Institutional Ph.D. Program in Chemical Biology, New York, NY 10065, United States
| | - Tandrila Das
- Laboratory of Chemical Biology and Microbial Pathogenesis, The Rockefeller University, New York, NY 10065, United States; Tri-Institutional Ph.D. Program in Chemical Biology, New York, NY 10065, United States
| | - Howard C Hang
- Laboratory of Chemical Biology and Microbial Pathogenesis, The Rockefeller University, New York, NY 10065, United States; Departments of Immunology and Microbiology and Chemistry, Scripps Research, La Jolla, CA 92037, United States.
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19
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Lin H. Protein cysteine palmitoylation in immunity and inflammation. FEBS J 2021; 288:7043-7059. [PMID: 33506611 PMCID: PMC8872633 DOI: 10.1111/febs.15728] [Citation(s) in RCA: 46] [Impact Index Per Article: 11.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/01/2020] [Revised: 12/24/2020] [Accepted: 01/25/2021] [Indexed: 07/24/2023]
Abstract
Protein cysteine palmitoylation, or S-palmitoylation, has been known for about 40 years, and thousands of proteins in humans are known to be modified. Because of the large number of proteins modified, the importance and physiological functions of S-palmitoylation are enormous. However, most of the known physiological functions of S-palmitoylation can be broadly classified into two categories, neurological or immunological. This review provides a summary on the function of S-palmitoylation from the immunological perspective. Several important immune signaling pathways are discussed, including STING, NOD1/2, JAK-STAT in cytokine signaling, T-cell receptor signaling, chemotactic GPCR signaling, apoptosis, phagocytosis, and endothelial and epithelial integrity. This review is not meant to be comprehensive, but rather focuses on specific examples to highlight the versatility of palmitoylation in regulating immune signaling, as well as the potential and challenges of targeting palmitoylation to treat immune diseases.
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Affiliation(s)
- Hening Lin
- Howard Hughes Medical Institute, Department of Chemistry and Chemical Biology, Cornell University, Ithaca, NY, USA
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20
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Mills EM, Barlow VL, Jones AT, Tsai YH. Development of mammalian cell logic gates controlled by unnatural amino acids. CELL REPORTS METHODS 2021; 1:100073. [PMID: 35474893 PMCID: PMC9017196 DOI: 10.1016/j.crmeth.2021.100073] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 01/04/2021] [Revised: 07/20/2021] [Accepted: 08/13/2021] [Indexed: 11/11/2022]
Abstract
Mammalian cell logic gates hold great potential for wide-ranging applications. However, most of those currently available are controlled by drug(-like) molecules with inherent biological activities. To construct truly orthogonal circuits and artificial regulatory pathways, biologically inert molecules are ideal molecular switches. Here, we applied genetic code expansion and engineered logic gates controlled by two biologically inert unnatural amino acids. Genetic code expansion relies on orthogonal aminoacyl-tRNA synthetase/tRNA pairs for co-translational and site-specific unnatural amino acid incorporation conventionally in response to an amber (UAG) codon. By screening 11 quadruplet-decoding pyrrolysyl tRNA variants from the literature, we found that all variants decoding CUAG or AGGA tested here are functional in mammalian cells. Using a quadruplet-decoding orthogonal pair together with an amber-decoding pair, we constructed logic gates that can be successfully controlled by two different unnatural amino acids, expanding the scope of genetic code expansion and mammalian cell logic circuits.
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Affiliation(s)
- Emily M. Mills
- School of Chemistry, Cardiff University, Main Building, Park Place, Cardiff, Wales CF10 3AT, UK
| | - Victoria L. Barlow
- School of Chemistry, Cardiff University, Main Building, Park Place, Cardiff, Wales CF10 3AT, UK
| | - Arwyn T. Jones
- School of Pharmacy and Pharmaceutical Sciences, Cardiff University, Redwood Building, Cardiff, Wales CF10 3NB, UK
| | - Yu-Hsuan Tsai
- School of Chemistry, Cardiff University, Main Building, Park Place, Cardiff, Wales CF10 3AT, UK
- Institute of Molecular Physiology, Shenzhen Bay Laboratory, Shenzhen, Guangdong 518132, China
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21
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Li Z, Chen Q, Wang J, Pan X, Lu W. Research Progress and Application of Bioorthogonal Reactions in Biomolecular Analysis and Disease Diagnosis. Top Curr Chem (Cham) 2021; 379:39. [PMID: 34590223 DOI: 10.1007/s41061-021-00352-8] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/27/2021] [Accepted: 09/14/2021] [Indexed: 12/14/2022]
Abstract
Bioorthogonal reactions are rapid, specific and high yield reactions that can be performed in in vivo microenvironments or simulated microenvironments. At present, the main biorthogonal reactions include Staudinger ligation, copper-catalyzed azide alkyne cycloaddition, strain-promoted [3 + 2] reaction, tetrazine ligation, metal-catalyzed coupling reaction and photo-induced biorthogonal reactions. To date, many reviews have reported that bioorthogonal reactions have been used widely as a powerful tool in the field of life sciences, such as in target recognition, drug discovery, drug activation, omics research, visualization of life processes or exogenous bacterial infection processes, signal transduction pathway research, chemical reaction dynamics analysis, disease diagnosis and treatment. In contrast, to date, few studies have investigated the application of bioorthogonal reactions in the analysis of biomacromolecules in vivo. Therefore, the application of bioorthogonal reactions in the analysis of proteins, nucleic acids, metabolites, enzyme activities and other endogenous molecules, and the determination of disease-related targets is reviewed. In addition, this review discusses the future development opportunities and challenges of biorthogonal reactions. This review presents an overview of recent advances for application in biomolecular analysis and disease diagnosis, with a focus on proteins, metabolites and RNA detection.
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Affiliation(s)
- Zilong Li
- School of Pharmacy, Health Science Center, Xi'an Jiaotong University, Xi'an, 710061, China
| | - Qinhua Chen
- Department of Pharmacy, Shenzhen Baoan Authentic TCM Therapy Hospital, Shenzhen, 518101, China
| | - Jin Wang
- School of Pharmacy, Health Science Center, Xi'an Jiaotong University, Xi'an, 710061, China
| | - Xiaoyan Pan
- School of Pharmacy, Health Science Center, Xi'an Jiaotong University, Xi'an, 710061, China
| | - Wen Lu
- School of Pharmacy, Health Science Center, Xi'an Jiaotong University, Xi'an, 710061, China.
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22
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Garst EH, Lee H, Das T, Bhattacharya S, Percher A, Wiewiora R, Witte IP, Li Y, Peng T, Im W, Hang HC. Site-Specific Lipidation Enhances IFITM3 Membrane Interactions and Antiviral Activity. ACS Chem Biol 2021; 16:844-856. [PMID: 33887136 DOI: 10.1021/acschembio.1c00013] [Citation(s) in RCA: 22] [Impact Index Per Article: 5.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/15/2022]
Abstract
Interferon-induced transmembrane proteins (IFITMs) are S-palmitoylated proteins in vertebrates that restrict a diverse range of viruses. S-palmitoylated IFITM3 in particular engages incoming virus particles, prevents their cytoplasmic entry, and accelerates their lysosomal clearance by host cells. However, how S-palmitoylation modulates the structure and biophysical characteristics of IFITM3 to promote its antiviral activity remains unclear. To investigate how site-specific S-palmitoylation controls IFITM3 antiviral activity, we employed computational, chemical, and biophysical approaches to demonstrate that site-specific lipidation of cysteine 72 enhances the antiviral activity of IFITM3 by modulating its conformation and interaction with lipid membranes. Collectively, our results demonstrate that site-specific S-palmitoylation of IFITM3 directly alters its biophysical properties and activity in cells to prevent virus infection.
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Affiliation(s)
- Emma H. Garst
- Laboratory of Chemical Biology and Microbial Pathogenesis, The Rockefeller University, New York, New York 10065, United States
- Tri-Institutional Ph.D. Program in Chemical Biology, New York, New York 10065, United States
| | - Hwayoung Lee
- Department of Biological Sciences, Chemistry, and Bioengineering, Lehigh University, Bethlehem, Pennsylvania 18015, United States
| | - Tandrila Das
- Laboratory of Chemical Biology and Microbial Pathogenesis, The Rockefeller University, New York, New York 10065, United States
- Tri-Institutional Ph.D. Program in Chemical Biology, New York, New York 10065, United States
| | | | - Avital Percher
- Laboratory of Chemical Biology and Microbial Pathogenesis, The Rockefeller University, New York, New York 10065, United States
| | - Rafal Wiewiora
- Tri-Institutional Ph.D. Program in Chemical Biology, New York, New York 10065, United States
- Memorial Sloan Kettering Cancer Center, New York, New York 10065, United States
| | - Isaac P. Witte
- Laboratory of Chemical Biology and Microbial Pathogenesis, The Rockefeller University, New York, New York 10065, United States
| | - Yumeng Li
- State Key Laboratory of Chemical Oncogenomics, School of Chemical Biology and Biotechnology, Peking University Shenzhen Graduate School, Shenzhen 518055, China
| | - Tao Peng
- State Key Laboratory of Chemical Oncogenomics, School of Chemical Biology and Biotechnology, Peking University Shenzhen Graduate School, Shenzhen 518055, China
| | - Wonpil Im
- Department of Biological Sciences, Chemistry, and Bioengineering, Lehigh University, Bethlehem, Pennsylvania 18015, United States
| | - Howard C. Hang
- Laboratory of Chemical Biology and Microbial Pathogenesis, The Rockefeller University, New York, New York 10065, United States
- Departments of Immunology and Microbiology and Chemistry, Scripps Research, La Jolla, California 92037, United States
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23
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Chen JJ, Fan Y, Boehning D. Regulation of Dynamic Protein S-Acylation. Front Mol Biosci 2021; 8:656440. [PMID: 33981723 PMCID: PMC8107437 DOI: 10.3389/fmolb.2021.656440] [Citation(s) in RCA: 21] [Impact Index Per Article: 5.3] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/20/2021] [Accepted: 02/16/2021] [Indexed: 12/20/2022] Open
Abstract
Protein S-acylation is the reversible addition of fatty acids to the cysteine residues of target proteins. It regulates multiple aspects of protein function, including the localization to membranes, intracellular trafficking, protein interactions, protein stability, and protein conformation. This process is regulated by palmitoyl acyltransferases that have the conserved amino acid sequence DHHC at their active site. Although they have conserved catalytic cores, DHHC enzymes vary in their protein substrate selection, lipid substrate preference, and regulatory mechanisms. Alterations in DHHC enzyme function are associated with many human diseases, including cancers and neurological conditions. The removal of fatty acids from acylated cysteine residues is catalyzed by acyl protein thioesterases. Notably, S-acylation is now known to be a highly dynamic process, and plays crucial roles in signaling transduction in various cell types. In this review, we will explore the recent findings on protein S-acylation, the enzymatic regulation of this process, and discuss examples of dynamic S-acylation.
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24
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Abstract
S-palmitoylation is a reversible posttranslational lipid modification of proteins. It controls protein activity, stability, trafficking and protein–protein interactions. Recent global profiling of immune cells and targeted analysis have identified many S-palmitoylated immunity-associated proteins. Here, we review S-palmitoylated immune receptors and effectors, and their dynamic regulation at cellular membranes to generate specific and balanced immune responses. We also highlight how this understanding can drive therapeutic advances to pharmacologically modulate immune responses.
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Affiliation(s)
- Tandrila Das
- Laboratory of Chemical Biology and Microbial Pathogenesis, The Rockefeller University, New York, NY 10065, USA
| | - Jacob S Yount
- Department of Microbial Infection and Immunity, The Ohio State University, Columbus, OH 43210, USA
| | - Howard C Hang
- Laboratory of Chemical Biology and Microbial Pathogenesis, The Rockefeller University, New York, NY 10065, USA.,Departments of Immunology and Microbiology, Chemistry, Scripps Research, La Jolla, CA 92037, USA
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25
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Main A, Fuller W. Protein S-Palmitoylation: advances and challenges in studying a therapeutically important lipid modification. FEBS J 2021; 289:861-882. [PMID: 33624421 DOI: 10.1111/febs.15781] [Citation(s) in RCA: 52] [Impact Index Per Article: 13.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/06/2020] [Revised: 02/01/2021] [Accepted: 02/22/2021] [Indexed: 12/11/2022]
Abstract
The lipid post-translational modification S-palmitoylation is a vast developing field, with the modification itself and the enzymes that catalyse the reversible reaction implicated in a number of diseases. In this review, we discuss the past and recent advances in the experimental tools used in this field, including pharmacological tools, animal models and techniques to understand how palmitoylation controls protein localisation and function. Additionally, we discuss the obstacles to overcome in order to advance the field, particularly to the point at which modulating palmitoylation may be achieved as a therapeutic strategy.
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Affiliation(s)
- Alice Main
- Institute of Cardiovascular and Medical Sciences, University of Glasgow, UK
| | - William Fuller
- Institute of Cardiovascular and Medical Sciences, University of Glasgow, UK
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