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1
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Wang J, Kjellgren A, DeMartino GN. PI31 is a positive regulator of 20S immunoproteasome assembly. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2025:2025.01.15.633194. [PMID: 39868238 PMCID: PMC11761684 DOI: 10.1101/2025.01.15.633194] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Download PDF] [Subscribe] [Scholar Register] [Indexed: 01/28/2025]
Abstract
PI31 (Proteasome Inhibitor of 31,000 Da) is a 20S proteasome-binding protein originally identified as an inhibitor of in vitro 20S proteasome activity. Although recent studies have provided a detailed structural basis for this activity, the physiologic significance of PI31-mediated proteasome inhibition remains uncertain and alternative cellular roles for PI31 have been described. Here we report a role for PI31 as a positive regulator for the assembly of the 20S immuno-proteasome (20Si), a compositionally and functionally distinct isoform of the proteasome that is poorly inhibited by PI31. Genetic ablation of PI31 in mammalian cells had no effect on the cellular content or activity of constitutively expressed proteasomes but reduced the cellular content and activity of interferon-γ-induced immuno-proteasomes. This selective effect is a consequence of defective 20Si assembly, as indicated by the accumulation of 20Si assembly intermediates. Our results highlight a distinction in the assembly pathways of constitutive and immuno-proteasomes and indicate that PI31 plays a chaperone-like role for the selective assembly of 20S immunoproteasomes.
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Affiliation(s)
| | - Abbey Kjellgren
- Department of Physiology, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX 75390-9040
| | - George N. DeMartino
- Department of Physiology, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX 75390-9040
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2
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Cascio P. PA28γ, the ring that makes tumors invisible to the immune system? Biochimie 2024; 226:136-147. [PMID: 38631454 DOI: 10.1016/j.biochi.2024.04.003] [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: 02/07/2024] [Revised: 03/29/2024] [Accepted: 04/12/2024] [Indexed: 04/19/2024]
Abstract
PA28γ is a proteasomal interactor whose main and most known function is to stimulate the hydrolytic activity of the 20 S proteasome independently of ubiquitin and ATP. Unlike its two paralogues, PA28α and PA28β, PA28γ is largely present in the nuclear compartment and plays pivotal functions in important pathways such as cellular division, apoptosis, neoplastic transformation, chromatin structure and organization, fertility, lipid metabolism, and DNA repair mechanisms. Although it is known that a substantial fraction of PA28γ is found in the cell in a free form (i.e. not associated with 20 S), almost all of the studies so far have focused on its ability to modulate proteasomal enzymatic activities. In this respect, the ability of PA28γ to strongly stimulate degradation of proteins, especially if intrinsically disordered and therefore devoid of three-dimensional tightly folded structure, appears to be the main molecular mechanism underlying its multiple biological effects. Initial studies, conducted more than 20 years ago, came to the conclusion that among the many biological functions of PA28γ, the immunological ones were rather limited and circumscribed. In this review, we focus on recent evidence showing that PA28γ fulfills significant functions in cell-mediated acquired immunity, with a particular role in attenuating MHC class I antigen presentation, especially in relation to neoplastic transformation and autoimmune diseases.
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Affiliation(s)
- Paolo Cascio
- Department of Veterinary Sciences, University of Turin, Largo P. Braccini 2, 10095, Grugliasco, Turin, Italy.
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3
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Basler M, Schliehe C. In memory of Prof. Dr. Marcus Groettrup (1964-2022). Eur J Immunol 2024; 54:e2451341. [PMID: 39540575 DOI: 10.1002/eji.202451341] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/27/2024] [Accepted: 07/01/2024] [Indexed: 11/16/2024]
Affiliation(s)
- Michael Basler
- Institute of Cell Biology and Immunology Thurgau (BITG) at the University of Konstanz, Kreuzlingen, Switzerland
- Division of Immunology, Department of Biology, University of Konstanz, Konstanz, Germany
| | - Christopher Schliehe
- Department of Immunology, Erasmus MC, University Medical Center Rotterdam, Rotterdam, The Netherlands
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4
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Santamorena MM, Tischer-Zimmermann S, Bonifacius A, Mireisz CNM, Costa B, Khan F, Kulkarni U, Lauruschkat CD, Sampaio KL, Stripecke R, Blasczyk R, Maecker-Kolhoff B, Kraus S, Schlosser A, Cicin-Sain L, Kalinke U, Eiz-Vesper B. Engineered HCMV-infected APCs enable the identification of new immunodominant HLA-restricted epitopes of anti-HCMV T-cell immunity. HLA 2024; 103:e15541. [PMID: 38923358 DOI: 10.1111/tan.15541] [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: 10/27/2023] [Revised: 04/24/2024] [Accepted: 05/07/2024] [Indexed: 06/28/2024]
Abstract
Complications due to HCMV infection or reactivation remain a challenging clinical problem in immunocompromised patients, mainly due to insufficient or absent T-cell functionality. Knowledge of viral targets is crucial to improve monitoring of high-risk patients and optimise antiviral T-cell therapy. To expand the epitope spectrum, genetically-engineered dendritic cells (DCs) and fibroblasts were designed to secrete soluble (s)HLA-A*11:01 and infected with an HCMV mutant lacking immune evasion molecules (US2-6 + 11). More than 700 HLA-A*11:01-restricted epitopes, including more than 50 epitopes derived from a broad range of HCMV open-reading-frames (ORFs) were identified by mass spectrometry and screened for HLA-A*11:01-binding using established prediction tools. The immunogenicity of the 24 highest scoring new candidates was evaluated in vitro in healthy HLA-A*11:01+/HCMV+ donors. Thus, four subdominant epitopes and one immunodominant epitope, derived from the anti-apoptotic protein UL36 and ORFL101C (A11SAL), were identified. Their HLA-A*11:01 complex stability was verified in vitro. In depth analyses revealed highly proliferative and cytotoxic memory T-cell responses against A11SAL, with T-cell responses comparable to the immunodominant HLA-A*02:01-restricted HCMVpp65NLV epitope. A11SAL-specific T cells were also detectable in vivo in immunosuppressed transplant patients and shown to be effective in an in vitro HCMV-infection model, suggesting their crucial role in inhibiting viral replication and improvement of patient's outcome. The developed in vitro pipeline is the first to utilise genetically-engineered DCs to identify naturally presented immunodominant HCMV-derived epitopes. It therefore offers advantages over in silico predictions, is transferable to other HLA alleles, and will significantly expand the repertoire of viral targets to improve therapeutic options.
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Affiliation(s)
- Maria Michela Santamorena
- Institute of Transfusion Medicine and Transplant Engineering, Hannover Medical School (MHH), Hannover, Germany
| | - Sabine Tischer-Zimmermann
- Institute of Transfusion Medicine and Transplant Engineering, Hannover Medical School (MHH), Hannover, Germany
| | - Agnes Bonifacius
- Institute of Transfusion Medicine and Transplant Engineering, Hannover Medical School (MHH), Hannover, Germany
- German Center for Infection Research (DZIF), Site Hannover-Braunschweig, Hannover, Germany
| | - Chiara Noemi-Marie Mireisz
- Rudolf Virchow Center for Integrative and Translational Bioimaging, University of Wuerzburg, Wuerzburg, Germany
| | - Bibiana Costa
- Institute for Experimental Infection Research, TWINCORE, Centre for Experimental and Clinical Infection Research, Hannover, Germany
| | - Fawad Khan
- Immune Ageing and Chronic Infection, Helmholtz Centre for Infection Research, Braunschweig, Germany
| | - Upasana Kulkarni
- Immune Ageing and Chronic Infection, Helmholtz Centre for Infection Research, Braunschweig, Germany
| | | | | | - Renata Stripecke
- University of Cologne, Faculty of Medicine and University Hospital Cologne, Department I of Internal Medicine, Center for Integrated Oncology Aachen Bonn Cologne Düsseldorf, Center for Molecular Medicine Cologne (CMMC), Institute of Translational Immuno-oncology, Cologne, Germany
- German Center for Infections Research (DZIF) Bonn-Cologne, Cologne, Germany
| | - Rainer Blasczyk
- Institute of Transfusion Medicine and Transplant Engineering, Hannover Medical School (MHH), Hannover, Germany
| | - Britta Maecker-Kolhoff
- German Center for Infection Research (DZIF), Site Hannover-Braunschweig, Hannover, Germany
- German Center for Infections Research (DZIF) Bonn-Cologne, Cologne, Germany
- Department of Pediatric Hematology and Oncology, Hannover Medical School, Hannover, Germany
| | - Sabrina Kraus
- Department of Internal Medicine II, University Hospital Wuerzburg, Wuerzburg, Germany
| | - Andreas Schlosser
- Rudolf Virchow Center for Integrative and Translational Bioimaging, University of Wuerzburg, Wuerzburg, Germany
| | - Luka Cicin-Sain
- German Center for Infection Research (DZIF), Site Hannover-Braunschweig, Hannover, Germany
- Immune Ageing and Chronic Infection, Helmholtz Centre for Infection Research, Braunschweig, Germany
- Cluster of Excellence - Resolving Infection Susceptibility (RESIST, EXC 2155), Hannover Medical School, Hannover, Germany
| | - Ulrich Kalinke
- Institute for Experimental Infection Research, TWINCORE, Centre for Experimental and Clinical Infection Research, Hannover, Germany
- Cluster of Excellence - Resolving Infection Susceptibility (RESIST, EXC 2155), Hannover Medical School, Hannover, Germany
| | - Britta Eiz-Vesper
- Institute of Transfusion Medicine and Transplant Engineering, Hannover Medical School (MHH), Hannover, Germany
- German Center for Infection Research (DZIF), Site Hannover-Braunschweig, Hannover, Germany
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5
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Leister H, Krause FF, Gil B, Prus R, Prus I, Hellhund-Zingel A, Mitra M, Da Rosa Gerbatin R, Delanty N, Beausang A, Brett FM, Farrell MA, Cryan J, O’Brien DF, Henshall DC, Helmprobst F, Pagenstecher A, Steinhoff U, Visekruna A, Engel T. Immunoproteasome deficiency results in age-dependent development of epilepsy. Brain Commun 2024; 6:fcae017. [PMID: 38317856 PMCID: PMC10839634 DOI: 10.1093/braincomms/fcae017] [Citation(s) in RCA: 2] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/20/2023] [Revised: 11/17/2023] [Accepted: 01/25/2024] [Indexed: 02/07/2024] Open
Abstract
The immunoproteasome is a central protease complex required for optimal antigen presentation. Immunoproteasome activity is also associated with facilitating the degradation of misfolded and oxidized proteins, which prevents cellular stress. While extensively studied during diseases with increasing evidence suggesting a role for the immunoproteasome during pathological conditions including neurodegenerative diseases, this enzyme complex is believed to be mainly not expressed in the healthy brain. In this study, we show an age-dependent increase in polyubiquitination in the brains of wild-type mice, accompanied by an induction of immunoproteasomes, which was most prominent in neurons and microglia. In contrast, mice completely lacking immunoproteasomes (triple-knockout mice), displayed a strong increase in polyubiquitinated proteins already in the young brain and developed spontaneous epileptic seizures, beginning at the age of 6 months. Injections of kainic acid led to high epilepsy-related mortality of aged triple-knockout mice, confirming increased pathological hyperexcitability states. Notably, the expression of the immunoproteasome was reduced in the brains of patients suffering from epilepsy. In addition, the aged triple-knockout mice showed increased anxiety, tau hyperphosphorylation and degeneration of Purkinje cell population with the resulting ataxic symptoms and locomotion alterations. Collectively, our study suggests a critical role for the immunoproteasome in the maintenance of a healthy brain during ageing.
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Affiliation(s)
- Hanna Leister
- Institute for Medical Microbiology and Hygiene, Philipps-University, 35043 Marburg, Germany
| | - Felix F Krause
- Institute for Medical Microbiology and Hygiene, Philipps-University, 35043 Marburg, Germany
| | - Beatriz Gil
- Department of Physiology and Medical Physics, Royal College of Surgeons in Ireland, University of Medicine and Health Sciences, D02 YN77 Dublin, Ireland
| | - Ruslan Prus
- Department of Physiology and Medical Physics, Royal College of Surgeons in Ireland, University of Medicine and Health Sciences, D02 YN77 Dublin, Ireland
| | - Inna Prus
- Department of Physiology and Medical Physics, Royal College of Surgeons in Ireland, University of Medicine and Health Sciences, D02 YN77 Dublin, Ireland
| | - Anne Hellhund-Zingel
- Institute for Medical Microbiology and Hygiene, Philipps-University, 35043 Marburg, Germany
| | - Meghma Mitra
- Department of Physiology and Medical Physics, Royal College of Surgeons in Ireland, University of Medicine and Health Sciences, D02 YN77 Dublin, Ireland
| | - Rogerio Da Rosa Gerbatin
- FutureNeuro, SFI Research Centre for Chronic and Rare Neurological Diseases, Royal College of Surgeons in Ireland, University of Medicine and Health Sciences, D02 YN77 Dublin, Ireland
| | - Norman Delanty
- FutureNeuro, SFI Research Centre for Chronic and Rare Neurological Diseases, Royal College of Surgeons in Ireland, University of Medicine and Health Sciences, D02 YN77 Dublin, Ireland
- Department of Neurology, Beaumont Hospital, D09V2N0 Dublin, Ireland
| | - Alan Beausang
- Department of Neuropathology, Beaumont Hospital, D09V2N0 Dublin, Ireland
| | - Francesca M Brett
- Department of Neuropathology, Beaumont Hospital, D09V2N0 Dublin, Ireland
| | - Michael A Farrell
- Department of Neuropathology, Beaumont Hospital, D09V2N0 Dublin, Ireland
| | - Jane Cryan
- Department of Neuropathology, Beaumont Hospital, D09V2N0 Dublin, Ireland
| | - Donncha F O’Brien
- Department of Neurosurgery, Beaumont Hospital, D09V2N0 Dublin, Ireland
| | - David C Henshall
- Department of Physiology and Medical Physics, Royal College of Surgeons in Ireland, University of Medicine and Health Sciences, D02 YN77 Dublin, Ireland
- FutureNeuro, SFI Research Centre for Chronic and Rare Neurological Diseases, Royal College of Surgeons in Ireland, University of Medicine and Health Sciences, D02 YN77 Dublin, Ireland
| | - Frederik Helmprobst
- Institute of Neuropathology, Philipps-University, 35043 Marburg, Germany
- Core Facility for Mouse Pathology and Electron Microscopy, Philipps-University, 35043 Marburg, Germany
| | - Axel Pagenstecher
- Institute of Neuropathology, Philipps-University, 35043 Marburg, Germany
- Core Facility for Mouse Pathology and Electron Microscopy, Philipps-University, 35043 Marburg, Germany
| | - Ulrich Steinhoff
- Institute for Medical Microbiology and Hygiene, Philipps-University, 35043 Marburg, Germany
| | - Alexander Visekruna
- Institute for Medical Microbiology and Hygiene, Philipps-University, 35043 Marburg, Germany
| | - Tobias Engel
- Department of Physiology and Medical Physics, Royal College of Surgeons in Ireland, University of Medicine and Health Sciences, D02 YN77 Dublin, Ireland
- FutureNeuro, SFI Research Centre for Chronic and Rare Neurological Diseases, Royal College of Surgeons in Ireland, University of Medicine and Health Sciences, D02 YN77 Dublin, Ireland
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6
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Dafun AS, Živković D, Leon-Icaza SA, Möller S, Froment C, Bonnet D, de Jesus AA, Alric L, Quaranta-Nicaise M, Ferrand A, Cougoule C, Meunier E, Burlet-Schiltz O, Ebstein F, Goldbach-Mansky R, Krüger E, Bousquet MP, Marcoux J. Establishing 20S Proteasome Genetic, Translational and Post-Translational Status from Precious Biological and Patient Samples with Top-Down MS. Cells 2023; 12:cells12060844. [PMID: 36980185 PMCID: PMC10047880 DOI: 10.3390/cells12060844] [Citation(s) in RCA: 2] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/05/2022] [Revised: 02/23/2023] [Accepted: 02/27/2023] [Indexed: 03/11/2023] Open
Abstract
The mammalian 20S catalytic core of the proteasome is made of 14 different subunits (α1-7 and β1-7) but exists as different subtypes depending on the cell type. In immune cells, for instance, constitutive catalytic proteasome subunits can be replaced by the so-called immuno-catalytic subunits, giving rise to the immunoproteasome. Proteasome activity is also altered by post-translational modifications (PTMs) and by genetic variants. Immunochemical methods are commonly used to investigate these PTMs whereby protein-tagging is necessary to monitor their effect on 20S assembly. Here, we present a new miniaturized workflow combining top-down and bottom-up mass spectrometry of immunopurified 20S proteasomes that analyze the proteasome assembly status as well as the full proteoform footprint, revealing PTMs, mutations, single nucleotide polymorphisms (SNPs) and induction of immune-subunits in different biological samples, including organoids, biopsies and B-lymphoblastoid cell lines derived from patients with proteasome-associated autoinflammatory syndromes (PRAAS). We emphasize the benefits of using top-down mass spectrometry in preserving the endogenous conformation of protein modifications, while enabling a rapid turnaround (1 h run) and ensuring high sensitivity (1–2 pmol) and demonstrate its capacity to semi-quantify constitutive and immune proteasome subunits.
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Affiliation(s)
- Angelique Sanchez Dafun
- Institut de Pharmacologie et de Biologie Structurale (IPBS), Université de Toulouse, CNRS, Université Toulouse III—Paul Sabatier (UPS), 31077 Toulouse, France
| | - Dušan Živković
- Institut de Pharmacologie et de Biologie Structurale (IPBS), Université de Toulouse, CNRS, Université Toulouse III—Paul Sabatier (UPS), 31077 Toulouse, France
| | - Stephen Adonai Leon-Icaza
- Institut de Pharmacologie et de Biologie Structurale (IPBS), Université de Toulouse, CNRS, Université Toulouse III—Paul Sabatier (UPS), 31077 Toulouse, France
| | - Sophie Möller
- Institute of Medical Biochemistry and Molecular Biology, University Medicine Greifswald, 17475 Greifswald, Germany
| | - Carine Froment
- Institut de Pharmacologie et de Biologie Structurale (IPBS), Université de Toulouse, CNRS, Université Toulouse III—Paul Sabatier (UPS), 31077 Toulouse, France
| | - Delphine Bonnet
- IRSD, Université de Toulouse, INSERM, INRAE, ENVT, Université de Toulouse III—Paul Sabatier (UPS), 31300 Toulouse, France
- Internal Medicine Department of Digestive Disease, Rangueil Hospital, Université de Toulouse III—Paul Sabatier (UPS), 31400 Toulouse, France
| | - Adriana Almeida de Jesus
- Translational Autoinflammatory Diseases Section, LCIM, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA
| | - Laurent Alric
- Internal Medicine Department of Digestive Disease, Rangueil Hospital, Université de Toulouse III—Paul Sabatier (UPS), 31400 Toulouse, France
| | - Muriel Quaranta-Nicaise
- IRSD, Université de Toulouse, INSERM, INRAE, ENVT, Université de Toulouse III—Paul Sabatier (UPS), 31300 Toulouse, France
| | - Audrey Ferrand
- IRSD, Université de Toulouse, INSERM, INRAE, ENVT, Université de Toulouse III—Paul Sabatier (UPS), 31300 Toulouse, France
| | - Céline Cougoule
- Institut de Pharmacologie et de Biologie Structurale (IPBS), Université de Toulouse, CNRS, Université Toulouse III—Paul Sabatier (UPS), 31077 Toulouse, France
| | - Etienne Meunier
- Institut de Pharmacologie et de Biologie Structurale (IPBS), Université de Toulouse, CNRS, Université Toulouse III—Paul Sabatier (UPS), 31077 Toulouse, France
| | - Odile Burlet-Schiltz
- Institut de Pharmacologie et de Biologie Structurale (IPBS), Université de Toulouse, CNRS, Université Toulouse III—Paul Sabatier (UPS), 31077 Toulouse, France
| | - Frédéric Ebstein
- Institute of Medical Biochemistry and Molecular Biology, University Medicine Greifswald, 17475 Greifswald, Germany
| | - Raphaela Goldbach-Mansky
- Translational Autoinflammatory Diseases Section, LCIM, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA
| | - Elke Krüger
- Institute of Medical Biochemistry and Molecular Biology, University Medicine Greifswald, 17475 Greifswald, Germany
| | - Marie-Pierre Bousquet
- Institut de Pharmacologie et de Biologie Structurale (IPBS), Université de Toulouse, CNRS, Université Toulouse III—Paul Sabatier (UPS), 31077 Toulouse, France
- Correspondence: (M.-P.B.); (J.M.)
| | - Julien Marcoux
- Institut de Pharmacologie et de Biologie Structurale (IPBS), Université de Toulouse, CNRS, Université Toulouse III—Paul Sabatier (UPS), 31077 Toulouse, France
- Correspondence: (M.-P.B.); (J.M.)
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7
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Targeting immunoproteasome in neurodegeneration: A glance to the future. Pharmacol Ther 2023; 241:108329. [PMID: 36526014 DOI: 10.1016/j.pharmthera.2022.108329] [Citation(s) in RCA: 8] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/03/2022] [Revised: 12/02/2022] [Accepted: 12/05/2022] [Indexed: 12/15/2022]
Abstract
The immunoproteasome is a specialized form of proteasome equipped with modified catalytic subunits that was initially discovered to play a pivotal role in MHC class I antigen processing and immune system modulation. However, over the last years, this proteolytic complex has been uncovered to serve additional functions unrelated to antigen presentation. Accordingly, it has been proposed that immunoproteasome synergizes with canonical proteasome in different cell types of the nervous system, regulating neurotransmission, metabolic pathways and adaptation of the cells to redox or inflammatory insults. Hence, studying the alterations of immunoproteasome expression and activity is gaining research interest to define the dynamics of neuroinflammation as well as the early and late molecular events that are likely involved in the pathogenesis of a variety of neurological disorders. Furthermore, these novel functions foster the perspective of immunoproteasome as a potential therapeutic target for neurodegeneration. In this review, we provide a brain and retina-wide overview, trying to correlate present knowledge on structure-function relationships of immunoproteasome with the variety of observed neuro-modulatory functions.
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8
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Scalavino V, Piccinno E, Valentini AM, Mastronardi M, Armentano R, Giannelli G, Serino G. A Novel Mechanism of Immunoproteasome Regulation via miR-369-3p in Intestinal Inflammatory Response. Int J Mol Sci 2022; 23:ijms232213771. [PMID: 36430249 PMCID: PMC9691197 DOI: 10.3390/ijms232213771] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/29/2022] [Revised: 11/04/2022] [Accepted: 11/05/2022] [Indexed: 11/11/2022] Open
Abstract
The immunoproteasome is a multi-catalytic protein complex expressed in hematopoietic cells. Increased expression of immuno-subunits followed by increased proteasome activities is associated with the pathogenesis of IBD. Therefore, the identification of molecules that could inhibit the activities of this complex has been widely studied. microRNAs are small molecules of non-coding RNA that regulate the expression of target genes. Our purpose was to demonstrate that miR-369-3p is able to reduce the expression of the PSMB9 subunit and consequently modulate the catalytic activities of immunoproteasome. After bioinformatics prediction of the gene target of miR-369-3p, we validated its modulation on PSMB9 expression in the RAW264.7 cell line in vitro. We also found that miR-369-3p indirectly reduced the expression of other immunoproteasome subunits and that this regulation reduced the catalytic functions of the immunoproteasome. Increased levels of PSMB9 were observed in colon samples of acute IBD patients compared to the remission IBD group and control group. Our data suggest that miR-369-3p may be a future alternative therapeutic approach to several compounds currently used for the treatment of inflammatory disorders including IBD.
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9
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Lahman MC, Schmitt TM, Paulson KG, Vigneron N, Buenrostro D, Wagener FD, Voillet V, Martin L, Gottardo R, Bielas J, McElrath JM, Stirewalt DL, Pogosova-Agadjanyan EL, Yeung CC, Pierce RH, Egan DN, Bar M, Hendrie PC, Kinsella S, Vakil A, Butler J, Chaffee M, Linton J, McAfee MS, Hunter DS, Bleakley M, Rongvaux A, Van den Eynde BJ, Chapuis AG, Greenberg PD. Targeting an alternate Wilms' tumor antigen 1 peptide bypasses immunoproteasome dependency. Sci Transl Med 2022; 14:eabg8070. [PMID: 35138909 DOI: 10.1126/scitranslmed.abg8070] [Citation(s) in RCA: 11] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022]
Abstract
Designing effective antileukemic immunotherapy will require understanding mechanisms underlying tumor control or resistance. Here, we report a mechanism of escape from immunologic targeting in an acute myeloid leukemia (AML) patient, who relapsed 1 year after immunotherapy with engineered T cells expressing a human leukocyte antigen A*02 (HLA-A2)-restricted T cell receptor (TCR) specific for a Wilms' tumor antigen 1 epitope, WT1126-134 (TTCR-C4). Resistance occurred despite persistence of functional therapeutic T cells and continuous expression of WT1 and HLA-A2 by the patient's AML cells. Analysis of the recurrent AML revealed expression of the standard proteasome, but limited expression of the immunoproteasome, specifically the beta subunit 1i (β1i), which is required for presentation of WT1126-134. An analysis of a second patient treated with TTCR-C4 demonstrated specific loss of AML cells coexpressing β1i and WT1. To determine whether the WT1 protein continued to be processed and presented in the absence of immunoproteasome processing, we identified and tested a TCR targeting an alternative, HLA-A2-restricted WT137-45 epitope that was generated by immunoproteasome-deficient cells, including WT1-expressing solid tumor lines. T cells expressing this TCR (TTCR37-45) killed the first patients' relapsed AML resistant to WT1126-134 targeting, as well as other primary AML, in vitro. TTCR37-45 controlled solid tumor lines lacking immunoproteasome subunits both in vitro and in an NSG mouse model. As proteasome composition can vary in AML, defining and preferentially targeting these proteasome-independent epitopes may maximize therapeutic efficacy and potentially circumvent AML immune evasion by proteasome-related immunoediting.
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Affiliation(s)
- Miranda C Lahman
- Program in Immunology, Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA.,Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA.,Department of Laboratory Medicine and Pathology, University of Washington School of Medicine, Seattle, WA 98115, USA
| | - Thomas M Schmitt
- Program in Immunology, Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA.,Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA
| | - Kelly G Paulson
- Program in Immunology, Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA.,Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA.,University of Washington School of Medicine, Seattle, WA 98115, USA
| | - Nathalie Vigneron
- Ludwig Institute for Cancer Research, 1200 Brussels, Belgium.,de Duve Institute, Université Catholique de Louvain, Brussels, Belgium
| | - Denise Buenrostro
- Program in Immunology, Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA.,Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA
| | - Felecia D Wagener
- Program in Immunology, Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA.,Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA
| | - Valentin Voillet
- Vaccine and Infectious Disease Division, Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA.,Hutchinson Centre Research Institute of South Africa, Cape Town 8001, South Africa
| | - Lauren Martin
- Program in Immunology, Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA.,Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA
| | - Raphael Gottardo
- Vaccine and Infectious Disease Division, Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA.,Public Health Sciences Division, Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA
| | - Jason Bielas
- Department of Laboratory Medicine and Pathology, University of Washington School of Medicine, Seattle, WA 98115, USA.,Public Health Sciences Division, Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA.,Human Biology Division, Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA
| | - Julie M McElrath
- Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA.,University of Washington School of Medicine, Seattle, WA 98115, USA.,Vaccine and Infectious Disease Division, Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA
| | - Derek L Stirewalt
- Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA.,University of Washington School of Medicine, Seattle, WA 98115, USA
| | | | - Cecilia C Yeung
- Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA.,Department of Laboratory Medicine and Pathology, University of Washington School of Medicine, Seattle, WA 98115, USA.,University of Washington School of Medicine, Seattle, WA 98115, USA
| | - Robert H Pierce
- Program in Immunology, Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA.,Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA.,Department of Laboratory Medicine and Pathology, University of Washington School of Medicine, Seattle, WA 98115, USA
| | - Daniel N Egan
- Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA.,University of Washington School of Medicine, Seattle, WA 98115, USA
| | - Merav Bar
- Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA.,University of Washington School of Medicine, Seattle, WA 98115, USA
| | - Paul C Hendrie
- University of Washington School of Medicine, Seattle, WA 98115, USA
| | - Sinéad Kinsella
- Program in Immunology, Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA.,Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA
| | - Aesha Vakil
- Program in Immunology, Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA.,Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA
| | - Jonah Butler
- Program in Immunology, Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA.,Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA
| | - Mary Chaffee
- Program in Immunology, Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA.,Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA
| | - Jonathan Linton
- Program in Immunology, Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA.,Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA
| | - Megan S McAfee
- Program in Immunology, Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA.,Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA
| | - Daniel S Hunter
- Program in Immunology, Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA.,Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA
| | - Marie Bleakley
- Program in Immunology, Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA.,Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA.,Department of Pediatrics, University of Washington School of Medicine, Seattle, WA 98115, USA
| | - Anthony Rongvaux
- Program in Immunology, Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA.,Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA.,Department of Immunology, University of Washington, Seattle, WA 98115, USA
| | - Benoit J Van den Eynde
- de Duve Institute, Université Catholique de Louvain, Brussels, Belgium.,Ludwig Institute for Cancer Research, Nuffield Department of Medicine, University of Oxford, Oxford OX3 7DQ, UK.,Walloon Excellence in Life Sciences and Biotechnology (WELBIO), 1300 Wavre, Belgium
| | - Aude G Chapuis
- Program in Immunology, Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA.,Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA.,Department of Laboratory Medicine and Pathology, University of Washington School of Medicine, Seattle, WA 98115, USA.,University of Washington School of Medicine, Seattle, WA 98115, USA
| | - Philip D Greenberg
- Program in Immunology, Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA.,Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA.,University of Washington School of Medicine, Seattle, WA 98115, USA.,Department of Immunology, University of Washington, Seattle, WA 98115, USA
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10
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On the Role of the Immunoproteasome in Protein Homeostasis. Cells 2021; 10:cells10113216. [PMID: 34831438 PMCID: PMC8621243 DOI: 10.3390/cells10113216] [Citation(s) in RCA: 32] [Impact Index Per Article: 8.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/29/2021] [Revised: 11/12/2021] [Accepted: 11/15/2021] [Indexed: 12/28/2022] Open
Abstract
Numerous cellular processes are controlled by the proteasome, a multicatalytic protease in the cytosol and nucleus of all eukaryotic cells, through regulated protein degradation. The immunoproteasome is a special type of proteasome which is inducible under inflammatory conditions and constitutively expressed in hematopoietic cells. MECL-1 (β2i), LMP2 (β1i), and LMP7 (β5i) are the proteolytically active subunits of the immunoproteasome (IP), which is known to shape the antigenic repertoire presented on major histocompatibility complex (MHC) class I molecules. Furthermore, the immunoproteasome is involved in T cell expansion and inflammatory diseases. In recent years, targeting the immunoproteasome in cancer, autoimmune diseases, and transplantation proved to be therapeutically effective in preclinical animal models. However, the prime function of standard proteasomes and immunoproteasomes is the control of protein homeostasis in cells. To maintain protein homeostasis in cells, proteasomes remove proteins which are not properly folded, which are damaged by stress conditions such as reactive oxygen species formation, or which have to be degraded on the basis of regular protein turnover. In this review we summarize the latest insights on how the immunoproteasome influences protein homeostasis.
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11
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Tundo GR, Sbardella D, Oddone F, Kudriaeva AA, Lacal PM, Belogurov AA, Graziani G, Marini S. At the Cutting Edge against Cancer: A Perspective on Immunoproteasome and Immune Checkpoints Modulation as a Potential Therapeutic Intervention. Cancers (Basel) 2021; 13:4852. [PMID: 34638337 PMCID: PMC8507813 DOI: 10.3390/cancers13194852] [Citation(s) in RCA: 15] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/31/2021] [Revised: 09/20/2021] [Accepted: 09/21/2021] [Indexed: 01/22/2023] Open
Abstract
Immunoproteasome is a noncanonical form of proteasome with enzymological properties optimized for the generation of antigenic peptides presented in complex with class I MHC molecules. This enzymatic property makes the modulation of its activity a promising area of research. Nevertheless, immunotherapy has emerged as a front-line treatment of advanced/metastatic tumors providing outstanding improvement of life expectancy, even though not all patients achieve a long-lasting clinical benefit. To enhance the efficacy of the currently available immunotherapies and enable the development of new strategies, a broader knowledge of the dynamics of antigen repertoire processing by cancer cells is needed. Therefore, a better understanding of the role of immunoproteasome in antigen processing and of the therapeutic implication of its modulation is mandatory. Studies on the potential crosstalk between proteasome modulators and immune checkpoint inhibitors could provide novel perspectives and an unexplored treatment option for a variety of cancers.
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Affiliation(s)
| | | | | | - Anna A. Kudriaeva
- Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry of the Russian Academy of Sciences, Miklukho-Maklaya 16/10, 117997 Moscow, Russia; (A.A.K.)
| | - Pedro M. Lacal
- Laboratory of Molecular Oncology, IDI-IRCCS, 00167 Rome, Italy;
| | - Alexey A. Belogurov
- Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry of the Russian Academy of Sciences, Miklukho-Maklaya 16/10, 117997 Moscow, Russia; (A.A.K.)
- Lomonosov Moscow State University, Leninskie Gory, 119991 Moscow, Russia
| | - Grazia Graziani
- Laboratory of Molecular Oncology, IDI-IRCCS, 00167 Rome, Italy;
- Department of Systems Medicine, University of Rome Tor Vergata, 00133 Rome, Italy
| | - Stefano Marini
- Department of Clinical Sciences and Translational Medicine, University of Rome Tor Vergata, 00133 Rome, Italy;
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12
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Cecarini V, Cuccioloni M, Zheng Y, Bonfili L, Gong C, Angeletti M, Mena P, Del Rio D, Eleuteri AM. Flavan-3-ol Microbial Metabolites Modulate Proteolysis in Neuronal Cells Reducing Amyloid-beta (1-42) Levels. Mol Nutr Food Res 2021; 65:e2100380. [PMID: 34318994 PMCID: PMC9285603 DOI: 10.1002/mnfr.202100380] [Citation(s) in RCA: 25] [Impact Index Per Article: 6.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/20/2021] [Revised: 07/01/2021] [Indexed: 11/20/2022]
Abstract
INTRODUCTION Alzheimer's disease (AD) is a progressive neurodegeneration characterized by extensive protein aggregation and deposition in the brain, associated with defective proteasomal and autophagic-lysosomal proteolytic pathways. Since current drugs can only reduce specific symptoms, the identification of novel treatments is a major concern in AD research. Among natural compounds, (poly)phenols and their derivatives/metabolites are emerging as candidates in AD prevention due to their multiple beneficial effects. This study aims to investigate the ability of a selection of phenyl-γ-valerolactones, gut microbiota-derived metabolites of flavan-3-ols, to modulate the functionality of cellular proteolytic pathways. METHODS AND RESULTS Neuronal SH-SY5Y cells transfected with either the wild-type or the 717 valine-to-glycine amyloid precursor protein mutated gene are used as an AD model and treated with 5-(4'-hydroxyphenyl)-γ-valerolactone, 5-(3',4'-dihydroxyphenyl)-γ-valerolactone and 5-(3'-hydroxyphenyl)-γ-valerolactone-4'-sulfate. Combining in vitro and in silico studies, it is observed that the phenyl-γ-valerolactones of interest modulated cellular proteolysis via proteasome inhibition and consequent autophagy upregulation and inhibited cathepsin B activity, eventually reducing the amount of intra- and extracellular amyloid-beta (1-42) peptides. CONCLUSION The findings of this study establish, for the first time, that these metabolites exert a neuroprotective activity by regulating intracellular proteolysis and confirm the role of autophagy and cathepsin B as possible targets of AD preventive/therapeutic strategies.
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Affiliation(s)
- Valentina Cecarini
- School of Biosciences and Veterinary MedicineUniversity of CamerinoCamerinoItaly
| | | | - Yadong Zheng
- School of Biosciences and Veterinary MedicineUniversity of CamerinoCamerinoItaly
| | - Laura Bonfili
- School of Biosciences and Veterinary MedicineUniversity of CamerinoCamerinoItaly
| | - Chunmei Gong
- School of Biosciences and Veterinary MedicineUniversity of CamerinoCamerinoItaly
| | - Mauro Angeletti
- School of Biosciences and Veterinary MedicineUniversity of CamerinoCamerinoItaly
| | - Pedro Mena
- Human Nutrition UnitDepartment of Food and DrugsUniversity of ParmaParmaItaly
- Microbiome Research HubUniversity of ParmaParmaItaly
| | - Daniele Del Rio
- Human Nutrition UnitDepartment of Food and DrugsUniversity of ParmaParmaItaly
- Microbiome Research HubUniversity of ParmaParmaItaly
| | - Anna Maria Eleuteri
- School of Biosciences and Veterinary MedicineUniversity of CamerinoCamerinoItaly
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13
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NetCleave: an open-source algorithm for predicting C-terminal antigen processing for MHC-I and MHC-II. Sci Rep 2021; 11:13126. [PMID: 34162981 PMCID: PMC8222286 DOI: 10.1038/s41598-021-92632-y] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/04/2021] [Accepted: 06/10/2021] [Indexed: 11/13/2022] Open
Abstract
Antigens presented on the cell surface have been subjected to multiple biological processes. Among them, C-terminal antigen processing constitutes one of the main bottlenecks of the peptide presentation pathways, as it delimits the peptidome that will be subjected downstream. Here, we present NetCleave, an open-source and retrainable algorithm for the prediction of the C-terminal antigen processing for both MHC-I and MHC-II pathways. NetCleave architecture consists of a neural network trained on 46 different physicochemical descriptors of the cleavage site amino acids. Our results demonstrate that prediction of C-terminal antigen processing achieves high accuracy on MHC-I (AUC of 0.91), while it remains challenging for MHC-II (AUC of 0.66). Moreover, we evaluated the performance of NetCleave and other prediction tools for the evaluation of four independent immunogenicity datasets (H2-Db, H2-Kb, HLA-A*02:01 and HLA-B:07:02). Overall, we demonstrate that NetCleave stands out as one of the best algorithms for the prediction of C-terminal processing, and we provide one of the first evidence that C-terminal processing predictions may help in the discovery of immunogenic peptides.
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14
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Basler M, Christ M, Goebel H, Groettrup M. Immunoproteasome Upregulation Is Not Required to Control Protein Homeostasis during Viral Infection. THE JOURNAL OF IMMUNOLOGY 2021; 206:1697-1708. [PMID: 33731337 DOI: 10.4049/jimmunol.2000822] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Received: 07/14/2020] [Accepted: 02/01/2021] [Indexed: 12/28/2022]
Abstract
The prime function of proteasomes is the control of protein homeostasis in cells (i.e., the removal of proteins that are not properly folded, damaged by stress conditions like reactive oxygen species formation, or degraded on the basis of regular protein turnover). During viral infection, the standard proteasome is replaced by the so-called immunoproteasome (IP) in an IFN-γ-dependent manner. It has been proposed that the IP is required to protect cell viability under conditions of IFN-induced oxidative stress. In this study, we investigated the requirement for IP to cope with the enhanced need for protein degradation during lymphocytic choriomeningitis virus (LCMV) infection in mice lacking the IP subunit LMP7. We found that IP are upregulated in the liver but not in the spleen during LCMV infection, although the total proteasome content was not altered. The expression of standard proteasome subunits is not induced in LMP7-deficient mice, indicating that enhanced proteasomal activity is not required during viral infection. Furthermore, ubiquitin accumulation, apoptosis induction, and viral titers were similar in LCMV-infected mice lacking LMP7 compared with wild-type mice. Taken together, these data indicate that the IP is not required to regulate protein homeostasis during LCMV infection.
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Affiliation(s)
- Michael Basler
- Division of Immunology, Department of Biology, University of Konstanz, 78457 Konstanz, Germany; and .,Biotechnology Institute Thurgau at the University of Konstanz, 8280 Kreuzlingen, Switzerland
| | - Marleen Christ
- Division of Immunology, Department of Biology, University of Konstanz, 78457 Konstanz, Germany; and
| | - Heike Goebel
- Division of Immunology, Department of Biology, University of Konstanz, 78457 Konstanz, Germany; and
| | - Marcus Groettrup
- Division of Immunology, Department of Biology, University of Konstanz, 78457 Konstanz, Germany; and.,Biotechnology Institute Thurgau at the University of Konstanz, 8280 Kreuzlingen, Switzerland
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15
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Ware CA, Buhimschi CS, Zhao G, El Helou Y, Buhimschi IA. Amniotic Fluid Proteasome and Immunoproteasome in the Setting of Intra-Amniotic Infection, Inflammation, and Preterm Birth. Reprod Sci 2021; 28:2562-2573. [PMID: 33665784 DOI: 10.1007/s43032-021-00512-7] [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: 11/10/2020] [Accepted: 02/21/2021] [Indexed: 11/30/2022]
Abstract
Preterm birth is an important determinant of neonatal morbidity and mortality and intra-amniotic infection (IAI) and inflammation play a causative role. The constitutive proteasome and immunoproteasome are key players in maintenance of proteostasis and their alteration outside pregnancy has been linked to pathogenesis of numerous inflammatory diseases. Our goal was to evaluate the levels, activities, and potential origin of amniotic fluid (AF) proteasome in women with preterm birth induced by infection and/or inflammation. Total proteasome and immunoproteasome concentrations were measured in AF retrieved by trans-abdominal amniocentesis from 155 pregnant women. Proteasome activities were measured with fluorogenic substrates targeting caspase-like (CAS-L), trypsin-like (TRY-L), or chymotrypsin-like (CHE-L) lytic activities. We found that IAI significantly upregulated AF concentrations of total proteasome and of the immunoproteasome (P<0.001 for both) with no differences based on gestational age. Based on substrate preference and profile of pharmacologic inhibition, we identified the CHE-L activity of the immunoproteasome as the primary lytic activity upregulated in AF of pregnancies complicated by IAI. When compared with matched maternal blood and cord blood, proteasome activity was by far the highest in AF and this was further elevated in IAI. Western blot confirmed β5 (PSMB5) and β5i (PSMB8) subunits of the constitutive proteasome and immunoproteasome are present in AF and IHC staining of fetal membranes pointed to chorio-decidua as a potential source. In conclusion, IAI is associated with increased AF immunoproteasome activity that by analogy with other inflammatory diseases may generate antigenic oligopeptides and may play a role in triggering preterm birth.
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Affiliation(s)
- Courtney A Ware
- Department of Obstetrics and Gynecology, The Ohio State University College of Medicine, Columbus, OH, 43210, USA
| | - Catalin S Buhimschi
- Department of Obstetrics and Gynecology, University of Illinois College of Medicine, Chicago, IL, 60612, USA
| | - Guomao Zhao
- Department of Obstetrics and Gynecology, University of Illinois College of Medicine, Chicago, IL, 60612, USA.,Center for Perinatal Research, The Research Institute at Nationwide Children's Hospital, Columbus, OH, 43215, USA
| | - Yara El Helou
- Department of Obstetrics and Gynecology, University of Illinois College of Medicine, Chicago, IL, 60612, USA
| | - Irina A Buhimschi
- Department of Obstetrics and Gynecology, University of Illinois College of Medicine, Chicago, IL, 60612, USA. .,Center for Perinatal Research, The Research Institute at Nationwide Children's Hospital, Columbus, OH, 43215, USA.
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16
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Breczko W, Lemancewicz D, Dzięcioł J, Kłoczko J, Bołkun Ł. High immunoproteasome concentration in the plasma of patients with newly diagnosed multiple myeloma treated with bortezomib is predictive of longer OS. Adv Med Sci 2021; 66:21-27. [PMID: 33246214 DOI: 10.1016/j.advms.2020.11.003] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/12/2020] [Revised: 10/23/2020] [Accepted: 11/17/2020] [Indexed: 11/28/2022]
Abstract
PURPOSE Proteasome inhibitors (PI) bortezomib or carfilzomib among them, play a crucial role in the modern standard therapy for multiple myeloma (MM). In this study, we intended to evaluate whether immunoproteasome (IMP) concentration could act as an effective biomarker which determines the probability of response to treatment with bortezomib, in order to detect groups of patients who are more likely to respond to treatment with PI. MATERIALS AND METHODS In our study, we evaluated IMP concentration in the plasma of 40 patients with monoclonal gammopathy of undetermined significance (MGUS) and 116 patients with newly diagnosed MM during treatment with or without PI. RESULTS The values of all the studied parameters after the applied chemotherapy in the responders' group of patients declined considerably during the consecutive cycles of chemotherapy compared to their initial levels. On the contrary, in the group of non-responders, we observed no change in the measured IMP parameters during the consecutive cycles of therapy. We also showed that higher baseline IMP concentration might indicate longer overall survival (OS) in all patients. CONCLUSIONS Our results indicate that assessing plasma IMP concentration can be applied as a strong biomarker for predicting clinical response to treatment and OS in patients with newly diagnosed MM.
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Affiliation(s)
- Wioletta Breczko
- Department of Hematology, Medical University of Bialystok, Bialystok, Poland
| | - Dorota Lemancewicz
- Department of Hematology, Medical University of Bialystok, Bialystok, Poland; Department of Human Anatomy, Medical University of Bialystok, Bialystok, Poland
| | - Janusz Dzięcioł
- Department of Human Anatomy, Medical University of Bialystok, Bialystok, Poland
| | - Janusz Kłoczko
- Department of Hematology, Medical University of Bialystok, Bialystok, Poland
| | - Łukasz Bołkun
- Department of Hematology, Medical University of Bialystok, Bialystok, Poland.
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17
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Cascio P. PA28γ: New Insights on an Ancient Proteasome Activator. Biomolecules 2021; 11:228. [PMID: 33562807 PMCID: PMC7915322 DOI: 10.3390/biom11020228] [Citation(s) in RCA: 40] [Impact Index Per Article: 10.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/02/2021] [Revised: 02/02/2021] [Accepted: 02/03/2021] [Indexed: 02/06/2023] Open
Abstract
PA28 (also known as 11S, REG or PSME) is a family of proteasome regulators whose members are widely present in many of the eukaryotic supergroups. In jawed vertebrates they are represented by three paralogs, PA28α, PA28β, and PA28γ, which assemble as heptameric hetero (PA28αβ) or homo (PA28γ) rings on one or both extremities of the 20S proteasome cylindrical structure. While they share high sequence and structural similarities, the three isoforms significantly differ in terms of their biochemical and biological properties. In fact, PA28α and PA28β seem to have appeared more recently and to have evolved very rapidly to perform new functions that are specifically aimed at optimizing the process of MHC class I antigen presentation. In line with this, PA28αβ favors release of peptide products by proteasomes and is particularly suited to support adaptive immune responses without, however, affecting hydrolysis rates of protein substrates. On the contrary, PA28γ seems to be a slow-evolving gene that is most similar to the common ancestor of the PA28 activators family, and very likely retains its original functions. Notably, PA28γ has a prevalent nuclear localization and is involved in the regulation of several essential cellular processes including cell growth and proliferation, apoptosis, chromatin structure and organization, and response to DNA damage. In striking contrast with the activity of PA28αβ, most of these diverse biological functions of PA28γ seem to depend on its ability to markedly enhance degradation rates of regulatory protein by 20S proteasome. The present review will focus on the molecular mechanisms and biochemical properties of PA28γ, which are likely to account for its various and complex biological functions and highlight the common features with the PA28αβ paralog.
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Affiliation(s)
- Paolo Cascio
- Department of Veterinary Sciences, University of Turin, Largo P. Braccini 2, 10095 Grugliasco, Italy
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18
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Cryo-EM of mammalian PA28αβ-iCP immunoproteasome reveals a distinct mechanism of proteasome activation by PA28αβ. Nat Commun 2021; 12:739. [PMID: 33531497 PMCID: PMC7854634 DOI: 10.1038/s41467-021-21028-3] [Citation(s) in RCA: 17] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/12/2020] [Accepted: 01/08/2021] [Indexed: 02/07/2023] Open
Abstract
The proteasome activator PA28αβ affects MHC class I antigen presentation by associating with immunoproteasome core particles (iCPs). However, due to the lack of a mammalian PA28αβ-iCP structure, how PA28αβ regulates proteasome remains elusive. Here we present the complete architectures of the mammalian PA28αβ-iCP immunoproteasome and free iCP at near atomic-resolution by cryo-EM, and determine the spatial arrangement between PA28αβ and iCP through XL-MS. Our structures reveal a slight leaning of PA28αβ towards the α3-α4 side of iCP, disturbing the allosteric network of the gatekeeper α2/3/4 subunits, resulting in a partial open iCP gate. We find that the binding and activation mechanism of iCP by PA28αβ is distinct from those of constitutive CP by the homoheptameric TbPA26 or PfPA28. Our study sheds lights on the mechanism of enzymatic activity stimulation of immunoproteasome and suggests that PA28αβ-iCP has experienced profound remodeling during evolution to achieve its current level of function in immune response. The proteasome activator PA28αβ affects MHC class I antigen presentation by associating with immunoproteasome core particles (iCPs). Cryo-EM structures of the mammalian PA28αβ -iCP immunoproteasome and free iCP, combined with cross-linking data, reveal the complex architecture and suggest a distinct immunoproteasome activation mechanism.
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19
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Zhao Z, Sun C, Chen L, Qin J, Yuan X, Li W. Inorganic nitrite increases the susceptibility of tilapia (Oreochromis niloticus) leucocytes to Streptococcus agalactiae. FISH & SHELLFISH IMMUNOLOGY 2020; 97:1-11. [PMID: 31846770 DOI: 10.1016/j.fsi.2019.12.039] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 08/10/2019] [Revised: 12/08/2019] [Accepted: 12/13/2019] [Indexed: 06/10/2023]
Abstract
Deteriorating water quality, especially from high concentrations of nitrite, is currently largely blamed for disease outbreaks in farmed tilapia (Oreochromis niloticus). In this study, the underlying mechanism of nitrite on the susceptibility of tilapia leucocytes to Streptococcus agalactiae (S. agalactiae) was studied. We found that a high dose of heat-killed S. agalactiae decreased tilapia leucocytes cell viability, whereas nitrite decreased the cell viability of leucocytes exposed to a low dose of bacteria. Bacterial challenge increased the production of nitric oxide (NO), whereas nitrite and bacteria coexposure caused higher NO production than nitrite or bacterial exposure alone. Cell viability increased after elimination of NO, and negative correlations existed between cell viability and the NO content, suggesting that nitrite increased the susceptibility of the leucocytes against S. agalactiae was NO-dependent. For a more comprehensive understanding of the mechanism of nitrite affecting disease resistance in tilapia leucocytes, an RNA-Seq-based transcriptome was generated. The results showed that 6173 transcripts were differently expressed, and the differentially expressed transcripts (DETs) of the bacterial group, nitrite group and bacteria-nitrite co-treatment group compared to the control group were selected for GO and KEGG analyses. The DETs in the bacterial group and bacteria-nitrite cotreatment group were highly involved with the membrane component, signal transduction, and immune responses. KEGG analysis showed that the protein processing in the endoplasmic reticulum and the AMPK signaling pathway, which are related to autophagy, were significantly enriched in the cotreatment group but not in bacterial group. In addition, the mRNA expression of ten DETs and several autophagy and apoptosis related genes validated by q-PCR showed the high reliability of the RNA-seq. Taken together, the results of this study suggest that nitrite may increase the susceptibility of tilapia leucocytes to S. agalactiae by generating excess NO to affect the autophagy and apoptosis process.
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Affiliation(s)
- Zaoya Zhao
- State Key Laboratory of Biocontrol, Institute of Aquatic Economic Animals and Guangdong Province Key Laboratory for Aquatic Economic Animals, Guangdong Provincial Engineering Technology Research Center of Healthy Breeding in Important Economic Fish, School of Life Sciences, Sun Yat-Sen University, Guangzhou, 510006, PR China
| | - Caiyun Sun
- State Key Laboratory of Biocontrol, Institute of Aquatic Economic Animals and Guangdong Province Key Laboratory for Aquatic Economic Animals, Guangdong Provincial Engineering Technology Research Center of Healthy Breeding in Important Economic Fish, School of Life Sciences, Sun Yat-Sen University, Guangzhou, 510006, PR China
| | - Limin Chen
- State Key Laboratory of Biocontrol, Institute of Aquatic Economic Animals and Guangdong Province Key Laboratory for Aquatic Economic Animals, Guangdong Provincial Engineering Technology Research Center of Healthy Breeding in Important Economic Fish, School of Life Sciences, Sun Yat-Sen University, Guangzhou, 510006, PR China
| | - Jingkai Qin
- State Key Laboratory of Biocontrol, Institute of Aquatic Economic Animals and Guangdong Province Key Laboratory for Aquatic Economic Animals, Guangdong Provincial Engineering Technology Research Center of Healthy Breeding in Important Economic Fish, School of Life Sciences, Sun Yat-Sen University, Guangzhou, 510006, PR China
| | - Xi Yuan
- State Key Laboratory of Biocontrol, Institute of Aquatic Economic Animals and Guangdong Province Key Laboratory for Aquatic Economic Animals, Guangdong Provincial Engineering Technology Research Center of Healthy Breeding in Important Economic Fish, School of Life Sciences, Sun Yat-Sen University, Guangzhou, 510006, PR China
| | - Wensheng Li
- State Key Laboratory of Biocontrol, Institute of Aquatic Economic Animals and Guangdong Province Key Laboratory for Aquatic Economic Animals, Guangdong Provincial Engineering Technology Research Center of Healthy Breeding in Important Economic Fish, School of Life Sciences, Sun Yat-Sen University, Guangzhou, 510006, PR China.
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20
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Solanki KS, Gandham RK, Thomas P, Chaudhuri P. Transcriptome analysis of Brucella abortus S19∆ per immunized mouse spleen revealed activation of MHC-I and MHC-II pathways. Access Microbiol 2019; 2:acmi000082. [PMID: 33062939 PMCID: PMC7525051 DOI: 10.1099/acmi.0.000082] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/26/2019] [Accepted: 10/23/2019] [Indexed: 12/24/2022] Open
Abstract
The mouse (Mus musculus) has been extensively used for studying brucellosis, regarding pathogenesis, immunity and the evaluation of vaccines and therapeutics. In this work, RNA-seq was applied to explore the immunological potential of a live Brucella abortus S19∆per, a perosamine synthetase gene mutant of B. abortus S19. Comparison of transcriptome data was carried out for identifying differentially expressed genes among PBS (control) and B. abortus S19∆per immunized mice at 15 days post-immunization. Functional analysis revealed 545 significant differentially expressed genes related to mouse immunity. Specific activation of MHC-I and MHC-II antigen-processing pathways were identified as the highly enriched pathways based on Kyoto Encyclopedia of Genes and Genomes annotation. Other major immune response pathways regulated within the host were NF-kappa B signalling, chemokine signalling, T-cell receptor pathway, apoptosis, TNF signalling and nucleotide-binding oligomerization domain-like receptor signalling. These data provided new insights into the molecular mechanisms of B. abortus S19∆per-induced immune response in mice spleen that might facilitate the development of a highly immunogenic vaccine against brucellosis.
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Affiliation(s)
- Khushal Singh Solanki
- Division of Veterinary Biotechnology, ICAR-Indian Veterinary Research Institute, Izatnagar, Bareilly, Uttar Pradesh, 243122, India
| | - Ravi Kumar Gandham
- Division of Veterinary Biotechnology, ICAR-Indian Veterinary Research Institute, Izatnagar, Bareilly, Uttar Pradesh, 243122, India.,National Institute of Animal Biotechnology, Opp. Journalist Colony, Near Gowlidoddy Extended, Q City Road, Gachibowli, Hyderabad, Telangana 500032, India
| | - Prasad Thomas
- Division of Bacteriology and Mycology, ICAR-Indian Veterinary Research Institute, Izatnagar, Bareilly, Uttar Pradesh, 243122, India
| | - Pallab Chaudhuri
- Division of Bacteriology and Mycology, ICAR-Indian Veterinary Research Institute, Izatnagar, Bareilly, Uttar Pradesh, 243122, India
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21
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Morozov AV, Karpov VL. Proteasomes and Several Aspects of Their Heterogeneity Relevant to Cancer. Front Oncol 2019; 9:761. [PMID: 31456945 PMCID: PMC6700291 DOI: 10.3389/fonc.2019.00761] [Citation(s) in RCA: 77] [Impact Index Per Article: 12.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/23/2019] [Accepted: 07/29/2019] [Indexed: 01/19/2023] Open
Abstract
The life of every organism is dependent on the fine-tuned mechanisms of protein synthesis and breakdown. The degradation of most intracellular proteins is performed by the ubiquitin proteasome system (UPS). Proteasomes are central elements of the UPS and represent large multisubunit protein complexes directly responsible for the protein degradation. Accumulating data indicate that there is an intriguing diversity of cellular proteasomes. Different proteasome forms, containing different subunits and attached regulators have been described. In addition, proteasomes specific for a particular tissue were identified. Cancer cells are highly dependent on the proper functioning of the UPS in general, and proteasomes in particular. At the same time, the information regarding the role of different proteasome forms in cancer is limited. This review describes the functional and structural heterogeneity of proteasomes, their association with cancer as well as several established and novel proteasome-directed therapeutic strategies.
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Affiliation(s)
- Alexey V. Morozov
- Laboratory of Regulation of Intracellular Proteolysis, W.A. Engelhardt Institute of Molecular Biology RAS, Moscow, Russia
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22
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Hemming ML, Lawlor MA, Andersen JL, Hagan T, Chipashvili O, Scott TG, Raut CP, Sicinska E, Armstrong SA, Demetri GD, Bradner JE, Ganz PA, Tomlinson G, Olopade OI, Couch FJ, Wang X, Lindor NM, Pankratz VS, Radice P, Manoukian S, Peissel B, Zaffaroni D, Barile M, Viel A, Allavena A, Dall'Olio V, Peterlongo P, Szabo CI, Zikan M, Claes K, Poppe B, Foretova L, Mai PL, Greene MH, Rennert G, Lejbkowicz F, Glendon G, Ozcelik H, Andrulis IL, Thomassen M, Gerdes AM, Sunde L, Cruger D, Birk Jensen U, Caligo M, Friedman E, Kaufman B, Laitman Y, Milgrom R, Dubrovsky M, Cohen S, Borg A, Jernström H, Lindblom A, Rantala J, Stenmark-Askmalm M, Melin B, Nathanson K, Domchek S, Jakubowska A, Lubinski J, Huzarski T, Osorio A, Lasa A, Durán M, Tejada MI, Godino J, Benitez J, Hamann U, Kriege M, Hoogerbrugge N, van der Luijt RB, van Asperen CJ, Devilee P, Meijers-Heijboer EJ, Blok MJ, Aalfs CM, Hogervorst F, Rookus M, Cook M, Oliver C, Frost D, Conroy D, Evans DG, Lalloo F, Pichert G, Davidson R, Cole T, Cook J, Paterson J, Hodgson S, Morrison PJ, Porteous ME, Walker L, Kennedy MJ, Dorkins H, Peock S, et alHemming ML, Lawlor MA, Andersen JL, Hagan T, Chipashvili O, Scott TG, Raut CP, Sicinska E, Armstrong SA, Demetri GD, Bradner JE, Ganz PA, Tomlinson G, Olopade OI, Couch FJ, Wang X, Lindor NM, Pankratz VS, Radice P, Manoukian S, Peissel B, Zaffaroni D, Barile M, Viel A, Allavena A, Dall'Olio V, Peterlongo P, Szabo CI, Zikan M, Claes K, Poppe B, Foretova L, Mai PL, Greene MH, Rennert G, Lejbkowicz F, Glendon G, Ozcelik H, Andrulis IL, Thomassen M, Gerdes AM, Sunde L, Cruger D, Birk Jensen U, Caligo M, Friedman E, Kaufman B, Laitman Y, Milgrom R, Dubrovsky M, Cohen S, Borg A, Jernström H, Lindblom A, Rantala J, Stenmark-Askmalm M, Melin B, Nathanson K, Domchek S, Jakubowska A, Lubinski J, Huzarski T, Osorio A, Lasa A, Durán M, Tejada MI, Godino J, Benitez J, Hamann U, Kriege M, Hoogerbrugge N, van der Luijt RB, van Asperen CJ, Devilee P, Meijers-Heijboer EJ, Blok MJ, Aalfs CM, Hogervorst F, Rookus M, Cook M, Oliver C, Frost D, Conroy D, Evans DG, Lalloo F, Pichert G, Davidson R, Cole T, Cook J, Paterson J, Hodgson S, Morrison PJ, Porteous ME, Walker L, Kennedy MJ, Dorkins H, Peock S, Godwin AK, Stoppa-Lyonnet D, de Pauw A, Mazoyer S, Bonadona V, Lasset C, Dreyfus H, Leroux D, Hardouin A, Berthet P, Faivre L, Loustalot C, Noguchi T, Sobol H, Rouleau E, Nogues C, Frénay M, Vénat-Bouvet L, Hopper JL, Daly MB, Terry MB, John EM, Buys SS, Yassin Y, Miron A, Goldgar D, Singer CF, Dressler AC, Gschwantler-Kaulich D, Pfeiler G, Hansen TVO, Jønson L, Agnarsson BA, Kirchhoff T, Offit K, Devlin V, Dutra-Clarke A, Piedmonte M, Rodriguez GC, Wakeley K, Boggess JF, Basil J, Schwartz PE, Blank SV, Toland AE, Montagna M, Casella C, Imyanitov E, Tihomirova L, Blanco I, Lazaro C, Ramus SJ, Sucheston L, Karlan BY, Gross J, Schmutzler R, Wappenschmidt B, Engel C, Meindl A, Lochmann M, Arnold N, Heidemann S, Varon-Mateeva R, Niederacher D, Sutter C, Deissler H, Gadzicki D, Preisler-Adams S, Kast K, Schönbuchner I, Caldes T, de la Hoya M, Aittomäki K, Nevanlinna H, Simard J, Spurdle AB, Holland H, Chen X, Platte R, Chenevix-Trench G, Easton DF. Enhancer Domains in Gastrointestinal Stromal Tumor Regulate KIT Expression and Are Targetable by BET Bromodomain Inhibition. Cancer Res 2019. [PMID: 18483246 DOI: 10.1158/0008-5472] [Show More Authors] [Citation(s) in RCA: 750] [Impact Index Per Article: 125.0] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/07/2023]
Abstract
Gastrointestinal stromal tumor (GIST) is a mesenchymal neoplasm characterized by activating mutations in the related receptor tyrosine kinases KIT and PDGFRA. GIST relies on expression of these unamplified receptor tyrosine kinase (RTK) genes through a large enhancer domain, resulting in high expression levels of the oncogene required for tumor growth. Although kinase inhibition is an effective therapy for many patients with GIST, disease progression from kinase-resistant mutations is common and no other effective classes of systemic therapy exist. In this study, we identify regulatory regions of the KIT enhancer essential for KIT gene expression and GIST cell viability. Given the dependence of GIST upon enhancer-driven expression of RTKs, we hypothesized that the enhancer domains could be therapeutically targeted by a BET bromodomain inhibitor (BBI). Treatment of GIST cells with BBIs led to cell-cycle arrest, apoptosis, and cell death, with unique sensitivity in GIST cells arising from attenuation of the KIT enhancer domain and reduced KIT gene expression. BBI treatment in KIT-dependent GIST cells produced genome-wide changes in the H3K27ac enhancer landscape and gene expression program, which was also seen with direct KIT inhibition using a tyrosine kinase inhibitor (TKI). Combination treatment with BBI and TKI led to superior cytotoxic effects in vitro and in vivo, with BBI preventing tumor growth in TKI-resistant xenografts. Resistance to select BBI in GIST was attributable to drug efflux pumps. These results define a therapeutic vulnerability and clinical strategy for targeting oncogenic kinase dependency in GIST. SIGNIFICANCE: Expression and activity of mutant KIT is essential for driving the majority of GIST neoplasms, which can be therapeutically targeted using BET bromodomain inhibitors.
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Affiliation(s)
- Matthew L Hemming
- Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, Massachusetts. .,Center for Sarcoma and Bone Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts
| | - Matthew A Lawlor
- Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, Massachusetts
| | - Jessica L Andersen
- Center for Sarcoma and Bone Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts
| | - Timothy Hagan
- Department of Oncologic Pathology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts
| | - Otari Chipashvili
- Department of Oncologic Pathology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts
| | - Thomas G Scott
- Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, Massachusetts
| | - Chandrajit P Raut
- Department of Surgery, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts
| | - Ewa Sicinska
- Department of Oncologic Pathology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts
| | - Scott A Armstrong
- Department of Pediatric Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts
| | - George D Demetri
- Center for Sarcoma and Bone Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts.,Ludwig Center at Dana-Farber Cancer Institute and Harvard Medical School, Boston, Massachusetts
| | - James E Bradner
- Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, Massachusetts
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23
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Basler M, Lindstrom MM, LaStant JJ, Bradshaw JM, Owens TD, Schmidt C, Maurits E, Tsu C, Overkleeft HS, Kirk CJ, Langrish CL, Groettrup M. Co-inhibition of immunoproteasome subunits LMP2 and LMP7 is required to block autoimmunity. EMBO Rep 2018; 19:e46512. [PMID: 30279279 PMCID: PMC6280796 DOI: 10.15252/embr.201846512] [Citation(s) in RCA: 61] [Impact Index Per Article: 8.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/31/2018] [Revised: 09/04/2018] [Accepted: 09/10/2018] [Indexed: 12/11/2022] Open
Abstract
Cells of hematopoietic origin express high levels of the immunoproteasome, a cytokine-inducible proteasome variant comprising the proteolytic subunits LMP2 (β1i), MECL-1 (β2i), and LMP7 (β5i). Targeting the immunoproteasome in pre-clinical models of autoimmune diseases with the epoxyketone inhibitor ONX 0914 has proven to be effective. ONX 0914 was previously described as a selective LMP7 inhibitor. Here, we show that PRN1126, developed as an exclusively LMP7-specific inhibitor, has limited effects on IL-6 secretion, experimental colitis, and experimental autoimmune encephalomyelitis (EAE). We demonstrate that prolonged exposure of cells with ONX 0914 leads to inhibition of both LMP7 and LMP2. Co-inhibition of LMP7 and LMP2 with PRN1126 and LMP2 inhibitors LU-001i or ML604440 impairs MHC class I cell surface expression, IL-6 secretion, and differentiation of naïve T helper cells to T helper 17 cells, and strongly ameliorates disease in experimental colitis and EAE. Hence, co-inhibition of LMP2 and LMP7 appears to be synergistic and advantageous for the treatment of autoimmune diseases.
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Affiliation(s)
- Michael Basler
- Biotechnology Institute Thurgau (BITg) at the University of Konstanz, Kreuzlingen, Switzerland
- Division of Immunology, Department of Biology, University of Konstanz, Konstanz, Germany
| | | | | | | | | | - Christian Schmidt
- Division of Immunology, Department of Biology, University of Konstanz, Konstanz, Germany
- Konstanz Research School Chemical Biology (KoRS-CB), University of Konstanz, Konstanz, Germany
| | - Elmer Maurits
- Leiden Institute of Chemistry, Leiden University, Leiden, The Netherlands
| | - Christopher Tsu
- Takeda Pharmaceuticals International Co., Cambridge, MA, USA
| | | | | | | | - Marcus Groettrup
- Biotechnology Institute Thurgau (BITg) at the University of Konstanz, Kreuzlingen, Switzerland
- Division of Immunology, Department of Biology, University of Konstanz, Konstanz, Germany
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24
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Schmidt C, Berger T, Groettrup M, Basler M. Immunoproteasome Inhibition Impairs T and B Cell Activation by Restraining ERK Signaling and Proteostasis. Front Immunol 2018; 9:2386. [PMID: 30416500 PMCID: PMC6212513 DOI: 10.3389/fimmu.2018.02386] [Citation(s) in RCA: 42] [Impact Index Per Article: 6.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/30/2018] [Accepted: 09/26/2018] [Indexed: 12/19/2022] Open
Abstract
Immunoproteasome (IP) inhibition holds potential as a novel treatment option for various immune-mediated pathologies. The IP inhibitor ONX 0914 reduced T cell cytokine secretion and Th17 polarization and showed pre-clinical efficacy in a range of autoimmune disorders, transplant-allograft rejection, virus-mediated tissue damage, and colon cancer progression. However, the molecular basis of these effects has remained largely elusive. Here, we have analyzed the effects of ONX 0914 in primary human and mouse lymphocytes. ONX 0914-treatment impaired primary T cell activation in vitro and in vivo. IP inhibition reduced ERK-phosphorylation sustainment, while leaving NF-κB and other signaling pathways unaffected. Naïve T and B cells expressed nearly exclusively immuno- or mixed proteasomes but no standard proteasomes and IP inhibition but not IP-deficiency induced mild proteostasis stress, reduced DUSP5 expression and enhanced DUSP6 protein levels due to impaired degradation. However, accumulation of DUSP6 did not cause the reduced ERK-phosphorylation in a non-redundant manner. We show that broad-spectrum proteasome inhibition and immunoproteasome inhibition have distinct effects on T cell activation at the molecular level. Notably, ONX 0914-treated T cells recovered from proteostasis stress without apoptosis induction, apparently via Nrf1-mediated up-regulation of standard proteasomes. In contrast, B cells were more susceptible to apoptosis after ONX 0914-treatment. Our data thus provide mechanistic insights how IP inhibition functionally impedes T and B cells likely accounting for its therapeutic benefits.
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Affiliation(s)
- Christian Schmidt
- Chair of Immunology, Department of Biology, University of Konstanz, Konstanz, Germany.,Konstanz Research School Chemical Biology, University of Konstanz, Konstanz, Germany
| | - Thilo Berger
- Chair of Immunology, Department of Biology, University of Konstanz, Konstanz, Germany
| | - Marcus Groettrup
- Chair of Immunology, Department of Biology, University of Konstanz, Konstanz, Germany.,Konstanz Research School Chemical Biology, University of Konstanz, Konstanz, Germany.,Biotechnology Institute Thurgau at the University of Konstanz, Kreuzlingen, Switzerland
| | - Michael Basler
- Chair of Immunology, Department of Biology, University of Konstanz, Konstanz, Germany.,Konstanz Research School Chemical Biology, University of Konstanz, Konstanz, Germany.,Biotechnology Institute Thurgau at the University of Konstanz, Kreuzlingen, Switzerland
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25
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Immunoproteasome Subunits Are Required for CD8 + T Cell Function and Host Resistance to Brucella abortus Infection in Mice. Infect Immun 2018; 86:IAI.00615-17. [PMID: 29263103 DOI: 10.1128/iai.00615-17] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/27/2017] [Accepted: 12/12/2017] [Indexed: 01/18/2023] Open
Abstract
The immunoproteasome is a specific proteasome isoform composed of three subunits, termed β1i, β2i, and β5i. Its proteolytic activity enhances the quantity and quality of peptides to be presented by major histocompatibility complex class I (MHC-I) molecules to CD8+ T cells. However, the role of the combined deficiency of the three immunoproteasome subunits in protective immunity against bacterial pathogens has not been investigated. In this study, we addressed the role of the immunoproteasome during infection by Brucella abortus, an intracellular bacterium that requires CD8+ T cell responses for the control of infection. Here, we demonstrate that immunoproteasome triple-knockout (TKO) mice were more susceptible to Brucella infection. This observed susceptibility was accompanied by reduced interferon gamma (IFN-γ) production by mouse CD4+ and CD8+ T lymphocytes. Moreover, the absence of the immunoproteasome had an impact on MHC-I surface expression and antigen presentation by dendritic cells. CD8+ T cell function, which plays a pivotal role in B. abortus immunity, also presented a partial impairment of granzyme B expression and, consequently, reduced cytotoxic activity. In conclusion, these results strongly suggest that immunoproteasome subunits are important components in host resistance to B. abortus infection by impacting both the magnitude and quality of CD8+ T cell responses.
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26
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The 20S immunoproteasome and constitutive proteasome bind with the same affinity to PA28αβ and equally degrade FAT10. Mol Immunol 2017; 113:22-30. [PMID: 29208314 DOI: 10.1016/j.molimm.2017.11.030] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/31/2017] [Revised: 11/27/2017] [Accepted: 11/28/2017] [Indexed: 11/22/2022]
Abstract
The 20S immunoproteasome (IP) is an interferon(IFN)-γ - and tumor necrosis factor (TNF) -inducible variant of the 20S constitutive proteasome (CP) in which all its peptidolytically active subunits β1, β2, and β5 are replaced by their cytokine inducible homologues β1i (LMP2), β2i (MECL-1), and β5i (LMP7). These subunit replacements alter the cleavage specificity of the proteasome and the spectrum of proteasome-generated peptide ligands of MHC class I molecules. In addition to antigen processing, the IP has recently been shown to serve unique functions in the generation of pro-inflammatory T helper cell subtypes and cytokines as well as in the pathogenesis of autoimmune diseases, but the mechanistic involvement of the IP in these processes has remained elusive. In this study we investigated whether the IP differs from the CP in the interaction with two IFN-γ/TNF inducible factors: the 11S proteasome regulator PA28αβ and the ubiquitin-like modifier FAT10 (ubiquitin D). Using thermophoresis, we determined the affinity of PA28αβ for the CP and IP to be 12.2nM +/- 2.8nM and 15.3nM +/- 2.7nM, respectively, which is virtually identical. Also the activation of the peptidolytic activities of the IP and CP by PA28αβ did not differ. For FAT10 we determined the degradation kinetics in cycloheximide chase experiments in cells expressing almost exclusively IP or CP as well as in IFN-γ stimulated and unstimulated cells and found no differences between the degradation rates. Taken together, we conclude that neither differences in the binding strength to, nor activation by PA28αβ, nor a difference in the rate of FAT10-mediated degradation can account for distinct functional capabilities of the IP as compared to the CP.
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27
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Basler M, Maurits E, de Bruin G, Koerner J, Overkleeft HS, Groettrup M. Amelioration of autoimmunity with an inhibitor selectively targeting all active centres of the immunoproteasome. Br J Pharmacol 2017; 175:38-52. [PMID: 29034459 DOI: 10.1111/bph.14069] [Citation(s) in RCA: 29] [Impact Index Per Article: 3.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/25/2017] [Revised: 09/27/2017] [Accepted: 09/27/2017] [Indexed: 01/04/2023] Open
Abstract
BACKGROUND AND PURPOSE Multicatalytic endopeptidase complex-like-1 (β2i), low molecular mass polypeptide (LMP) 2 (β1i) and LMP7 (β5i) are the proteolytically active subunits of the immunoproteasome, a special type of proteasome mainly expressed in haematopoietic cells. Targeting LMP7 has been shown to be therapeutically effective in preclinical models of autoimmune diseases. In this study, we investigated the selectivity and biological activity of LU-005i, a recently described inhibitor of the immunoproteasome. EXPERIMENTAL APPROACH The specificity of LU-005i and other immunoproteasome-selective inhibitors was characterized using fluorogenic peptide substrates. The effect of proteasome inhibition on cytokine release was investigated in endotoxin-stimulated mouse splenocytes or human peripheral blood mononuclear cells (PBMCs). The effect of proteasome inhibition on inflammatory bowel disease in the dextran sulfate sodium (DSS)-induced colitis model was assessed by measuring weight loss and colon length. KEY RESULTS LU-005i is the first human and mouse immunoproteasome-selective inhibitor that targets all three proteolytically active immunoproteasome subunits. LU-005i inhibited cytokine secretion from endotoxin-stimulated mouse splenocytes or human PBMCs. Furthermore, differentiation of naïve T helper cells to T helper 17 cells was impaired in the presence of LU-005i. Additionally, LU-005i ameliorated DSS-induced colitis. CONCLUSION AND IMPLICATIONS This study with a novel pan-immunoproteasome inhibitor substantiates that the immunoproteasome is a promising drug target for the treatment of inflammatory diseases and that exclusive inhibition of LMP7 is not necessary for therapeutic effectiveness. Our results will promote the design of new generations of immunoproteasome inhibitors with optimal therapeutic efficacy for clinical use in the treatment of autoimmunity and cancer.
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Affiliation(s)
- Michael Basler
- Biotechnology Institute Thurgau (BITg) at the University of Konstanz, Kreuzlingen, 8280, Switzerland.,Department of Biology, Division of Immunology, University of Konstanz, Konstanz, 78457, Germany
| | - Elmer Maurits
- Leiden Institute of Chemistry, Leiden University, Leiden, 2333 CC, The Netherlands
| | - Gerjan de Bruin
- Leiden Institute of Chemistry, Leiden University, Leiden, 2333 CC, The Netherlands
| | - Julia Koerner
- Department of Biology, Division of Immunology, University of Konstanz, Konstanz, 78457, Germany
| | - Herman S Overkleeft
- Leiden Institute of Chemistry, Leiden University, Leiden, 2333 CC, The Netherlands
| | - Marcus Groettrup
- Biotechnology Institute Thurgau (BITg) at the University of Konstanz, Kreuzlingen, 8280, Switzerland.,Department of Biology, Division of Immunology, University of Konstanz, Konstanz, 78457, Germany
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28
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Matuszczak E, Sankiewicz A, Debek W, Gorodkiewicz E, Milewski R, Hermanowicz A. Immunoproteasome in the blood plasma of children with acute appendicitis, and its correlation with proteasome and UCHL1 measured by SPR imaging biosensors. Clin Exp Immunol 2017; 191:125-132. [PMID: 28940383 DOI: 10.1111/cei.13056] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 09/19/2017] [Indexed: 12/19/2022] Open
Abstract
The aim of this study was to determinate the immunoproteasome concentration in the blood plasma of children with appendicitis, and its correlation with circulating proteasome and ubiquitin carboxyl-terminal hydrolase L1 (UCHL1). Twenty-seven children with acute appendicitis, managed at the Paediatric Surgery Department, were included randomly into the study (age 2 years 9 months up to 14 years, mean age 9·5 ± 1 years). There were 10 girls and 17 boys; 18 healthy, age-matched subjects, admitted for planned surgeries served as controls. Mean concentrations of immunoproteasome, 20S proteasome and UCHL1 in the blood plasma of children with appendicitis before surgery 24 h and 72 h after the appendectomy were higher than in the control group. The immunoproteasome, 20S proteasome and UCHL1 concentrations in the blood plasma of patients with acute appendicitis were highest before surgery. The immunoproteasome, 20S proteasome and UCHL1 concentration measured 24 and 72 h after the operation decreased slowly over time and still did not reach the normal range (P < 0·05). There was no statistical difference between immunoproteasome, 20S proteasome and UCHL1 concentrations in children operated on laparoscopically and children after classic appendectomy. The immunoproteasome concentration may reflect the metabolic response to acute state inflammation, and the process of gradual ebbing of the inflammation may thus be helpful in the assessment of the efficacy of treatment. The method of operation - classic open appendectomy or laparoscopic appendectomy - does not influence the general trend in immunoproteasome concentration in children with appendicitis.
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Affiliation(s)
- E Matuszczak
- Paediatric Surgery Department, Medical University of Bialystok, Bialystok, Poland
| | - A Sankiewicz
- Electrochemistry Department, University of Bialystok, Bialystok, Poland
| | - W Debek
- Paediatric Surgery Department, Medical University of Bialystok, Bialystok, Poland
| | - E Gorodkiewicz
- Electrochemistry Department, University of Bialystok, Bialystok, Poland
| | - R Milewski
- Statistics Department, Medical University of Bialystok, Bialystok, Poland
| | - A Hermanowicz
- Paediatric Surgery Department, Medical University of Bialystok, Bialystok, Poland
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29
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Kovacsics CE, Gill AJ, Ambegaokar SS, Gelman BB, Kolson DL. Degradation of heme oxygenase-1 by the immunoproteasome in astrocytes: A potential interferon-γ-dependent mechanism contributing to HIV neuropathogenesis. Glia 2017; 65:1264-1277. [PMID: 28543773 DOI: 10.1002/glia.23160] [Citation(s) in RCA: 21] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/23/2017] [Revised: 03/28/2017] [Accepted: 04/07/2017] [Indexed: 12/16/2022]
Abstract
Induction of the detoxifying enzyme heme oxygenase-1 (HO-1) is a critical protective host response to cellular injury associated with inflammation and oxidative stress. We previously found that HO-1 protein expression is reduced in brains of HIV-infected individuals with HIV-associated neurocognitive disorders (HAND) and in HIV-infected macrophages, where this reduction associates with enhanced glutamate release and neurotoxicity. Because HIV-infected macrophages are a small component of the cellular content of the brain, the reduction of macrophage HO-1 expression likely accounts for a small portion of brain HO-1 loss in HIV infection. We therefore investigated the contribution of astrocytes, the major pool of brain HO-1. We identified immunoproteasome-mediated HO-1 degradation in astrocytes as a second possible mechanism of brain HO-1 loss in HIV infection. We demonstrate that prolonged exposure of human fetal astrocytes to interferon-gamma (IFNγ), an HIV-associated CNS immune activator, selectively reduces expression of HO-1 protein without a concomitant reduction in HO-1 RNA, increases expression of immunoproteasome subunits, and decreases expression of constitutive proteasome subunits, consistent with a shift towards increased immunoproteasome activity. In HIV-infected brain HO-1 protein reduction also associates with increased HO-1 RNA expression and increased immunoproteasome expression. Finally, we show that IFNγ treatment of astrocytic cells reduces HO-1 protein half-life in a proteasome-dependent manner. Our data thus suggest unique causal links among HIV infection, IFNγ-mediated immunoproteasome induction, and enhanced HO-1 degradation, which likely contribute to neurocognitive impairment in HAND. Such IFNγ-mediated HO-1 degradation should be further investigated for a role in neurodegeneration in inflammatory brain conditions. BRIEF SUMMARY Kovacsics et al. identify immunoproteasome degradation of heme oxygenase-1 (HO-1) in interferon gamma-stimulated astrocytes as a plausible mechanism for the observed loss of HO-1 protein expression in the brains of HIV-infected individuals, which likely contributes to the neurocognitive impairment in HIV-associated neurocognitive disorders.
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Affiliation(s)
- Colleen E Kovacsics
- Department of Neurology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, 19104
| | - Alexander J Gill
- Department of Neurology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, 19104
| | - Surendra S Ambegaokar
- Department of Botany & Microbiology, Robbins Program in Neuroscience, Ohio Wesleyan University, Delaware, Ohio, 43015
| | - Benjamin B Gelman
- Department of Pathology, University of Texas Medical Branch, Galveston, Texas, 77555
| | - Dennis L Kolson
- Department of Neurology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, 19104
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Immunoproteasome subunit deficiency has no influence on the canonical pathway of NF-κB activation. Mol Immunol 2017; 83:147-153. [PMID: 28157553 DOI: 10.1016/j.molimm.2017.01.019] [Citation(s) in RCA: 33] [Impact Index Per Article: 4.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/19/2016] [Revised: 01/13/2017] [Accepted: 01/20/2017] [Indexed: 01/02/2023]
Abstract
Activation of the pro-inflammatory transcription factor NF-κB requires signal-induced proteasomal degradation of the inhibitor of NF-κB (IκB) in order to allow nuclear translocation. Most cell types are capable of expressing two types of 20S proteasome core particles, the constitutive proteasome and immunoproteasome. Inducible under inflammatory conditions, the immunoproteasome is mainly characterized through an altered cleavage specificity compared to the constitutive proteasome. However, the question whether immunoproteasome subunits affect NF-κB signal transduction differently from constitutive subunits is still up for debate. To study the effect of immunoproteasomes on LPS- or TNF-α-induced NF-κB activation, we used IFN-γ stimulated peritoneal macrophages and mouse embryonic fibroblasts derived from mice deficient for the immunoproteasome subunits low molecular mass polypeptide (LMP) 2, or LMP7 and multicatalytic endopeptidase complex-like 1 (MECL-1). Along the canonical signaling pathway of NF-κB activation no differences in the extent and kinetic of IκB degradation were observed. Neither the nuclear translocation and DNA binding of NF-κB nor the production of the NF-κB dependent cytokines TNF-α, IL-6, and IL-10 differed between immunoproteasome deficient and proficient cells. Hence, we conclude that immunoproteasome subunits have no specialized function for canonical NF-κB activation.
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Expression of immunoproteasome genes is regulated by cell-intrinsic and -extrinsic factors in human cancers. Sci Rep 2016; 6:34019. [PMID: 27659694 PMCID: PMC5034284 DOI: 10.1038/srep34019] [Citation(s) in RCA: 67] [Impact Index Per Article: 7.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/06/2016] [Accepted: 09/06/2016] [Indexed: 01/07/2023] Open
Abstract
Based on transcriptomic analyses of thousands of samples from The Cancer Genome Atlas, we report that expression of constitutive proteasome (CP) genes (PSMB5, PSMB6, PSMB7) and immunoproteasome (IP) genes (PSMB8, PSMB9, PSMB10) is increased in most cancer types. In breast cancer, expression of IP genes was determined by the abundance of tumor infiltrating lymphocytes and high expression of IP genes was associated with longer survival. In contrast, IP upregulation in acute myeloid leukemia (AML) was a cell-intrinsic feature that was not associated with longer survival. Expression of IP genes in AML was IFN-independent, correlated with the methylation status of IP genes, and was particularly high in AML with an M5 phenotype and/or MLL rearrangement. Notably, PSMB8 inhibition led to accumulation of polyubiquitinated proteins and cell death in IPhigh but not IPlow AML cells. Co-clustering analysis revealed that genes correlated with IP subunits in non-M5 AMLs were primarily implicated in immune processes. However, in M5 AML, IP genes were primarily co-regulated with genes involved in cell metabolism and proliferation, mitochondrial activity and stress responses. We conclude that M5 AML cells can upregulate IP genes in a cell-intrinsic manner in order to resist cell stress.
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McCarthy MK, Malitz DH, Molloy CT, Procario MC, Greiner KE, Zhang L, Wang P, Day SM, Powell SR, Weinberg JB. Interferon-dependent immunoproteasome activity during mouse adenovirus type 1 infection. Virology 2016; 498:57-68. [PMID: 27560373 DOI: 10.1016/j.virol.2016.08.009] [Citation(s) in RCA: 19] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/24/2016] [Revised: 08/04/2016] [Accepted: 08/11/2016] [Indexed: 12/13/2022]
Abstract
The immunoproteasome is an inducible host mechanism that aids in the clearance of damaged proteins. The immunoproteasome also influences immune function by enhancing peptide presentation by MHC class I and promotes inflammation via IκB degradation and activation of NF-κB. We used mouse adenovirus type 1 (MAV-1) to characterize the role of the immunoproteasome in adenovirus pathogenesis. Following intranasal infection of mice, immunoproteasome activity in the heart and lung was significantly increased in an IFN-γ-dependent manner. Absence of the β5i immunoproteasome subunit and pharmacological inhibition of β5i activity had minimal effects on viral replication, virus-induced cellular inflammation, or induction of cytokine expression. Likewise, the establishment of protective immunity following primary infection was not significantly altered by β5i deficiency. Thus, although immunoproteasome activity is robustly induced during acute infection with MAV-1, our data suggest that other mechanisms are capable of compensating for immunoproteasome activity to maintain antiviral immunity and appropriate inflammatory responses.
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Affiliation(s)
- Mary K McCarthy
- Department of Microbiology and Immunology, University of Michigan, Ann Arbor, MI, USA
| | - Danielle H Malitz
- Department of Pediatrics and Communicable Diseases, University of Michigan, Ann Arbor, MI, USA
| | - Caitlyn T Molloy
- Department of Pediatrics and Communicable Diseases, University of Michigan, Ann Arbor, MI, USA
| | - Megan C Procario
- Department of Pediatrics and Communicable Diseases, University of Michigan, Ann Arbor, MI, USA
| | - Kaitlyn E Greiner
- Department of Pediatrics and Communicable Diseases, University of Michigan, Ann Arbor, MI, USA
| | - Luna Zhang
- Department of Pediatrics and Communicable Diseases, University of Michigan, Ann Arbor, MI, USA
| | - Ping Wang
- Feinstein Institute for Medical Research, Manhasset, New York, USA
| | - Sharlene M Day
- Department of Internal Medicine, University of Michigan, Ann Arbor, MI, USA
| | - Saul R Powell
- Feinstein Institute for Medical Research, Manhasset, New York, USA
| | - Jason B Weinberg
- Department of Microbiology and Immunology, University of Michigan, Ann Arbor, MI, USA; Department of Pediatrics and Communicable Diseases, University of Michigan, Ann Arbor, MI, USA.
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Kammerl IE, Meiners S. Proteasome function shapes innate and adaptive immune responses. Am J Physiol Lung Cell Mol Physiol 2016; 311:L328-36. [PMID: 27343191 DOI: 10.1152/ajplung.00156.2016] [Citation(s) in RCA: 52] [Impact Index Per Article: 5.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/20/2016] [Accepted: 06/17/2016] [Indexed: 11/22/2022] Open
Abstract
The proteasome system degrades more than 80% of intracellular proteins into small peptides. Accordingly, the proteasome is involved in many essential cellular functions, such as protein quality control, transcription, immune responses, cell signaling, and apoptosis. Moreover, degradation products are loaded onto major histocompatibility class I molecules to communicate the intracellular protein composition to the immune system. The standard 20S proteasome core complex contains three distinct catalytic active sites that are exchanged upon stimulation with inflammatory cytokines to form the so-called immunoproteasome. Immunoproteasomes are constitutively expressed in immune cells and have different proteolytic activities compared with standard proteasomes. They are rapidly induced in parenchymal cells upon intracellular pathogen infection and are crucial for priming effective CD8(+) T-cell-mediated immune responses against infected cells. Beyond shaping these adaptive immune reactions, immunoproteasomes also regulate the function of immune cells by degradation of inflammatory and immune mediators. Accordingly, they emerge as novel regulators of innate immune responses. The recently unraveled impairment of immunoproteasome function by environmental challenges and by genetic variations of immunoproteasome genes might represent a currently underestimated risk factor for the development and progression of lung diseases. In particular, immunoproteasome dysfunction will dampen resolution of infections, thereby promoting exacerbations, may foster autoimmunity in chronic lung diseases, and possibly contributes to immune evasion of tumor cells. Novel pharmacological tools, such as site-specific inhibitors of the immunoproteasome, as well as activity-based probes, however, hold promises as innovative therapeutic drugs for respiratory diseases and biomarker profiling, respectively.
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Affiliation(s)
- Ilona E Kammerl
- Comprehensive Pneumology Center, University Hospital, Ludwig-Maximilians University and Helmholtz Zentrum München, Member of the German Center for Lung Research (DZL), Munich, Germany
| | - Silke Meiners
- Comprehensive Pneumology Center, University Hospital, Ludwig-Maximilians University and Helmholtz Zentrum München, Member of the German Center for Lung Research (DZL), Munich, Germany
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Emerging role of immunoproteasomes in pathophysiology. Immunol Cell Biol 2016; 94:812-820. [PMID: 27192937 DOI: 10.1038/icb.2016.50] [Citation(s) in RCA: 38] [Impact Index Per Article: 4.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/23/2015] [Revised: 05/15/2016] [Accepted: 05/16/2016] [Indexed: 11/08/2022]
Abstract
The immunoproteasome is a proteasome variant that is found only in jawed vertebrates. It is responsible for degrading intracellular proteins to generate a major source of peptides with substantial major histocompatibility complex I binding affinity. The immunoproteasome also has roles in T-cell survival, differentiation and proliferation in various pathological conditions. In humans, any alteration in the expression, assembly or function of the immunoproteasome can lead to cancer, autoimmune disorders or inflammatory diseases. Although the roles of the immunoproteasome in cancer and neurodegenerative disorders have been extensively studied, its significance in other disease conditions has only recently become known. Therefore, there is renewed interest in the development of drugs, vaccines and biomarkers that target the immunoproteasome. The current review highlights the involvement of this complex in disease pathology in addition to the advances made in immunoproteasome research.
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Herrington FD, Carmody RJ, Goodyear CS. Modulation of NF-κB Signaling as a Therapeutic Target in Autoimmunity. ACTA ACUST UNITED AC 2015; 21:223-42. [PMID: 26597958 DOI: 10.1177/1087057115617456] [Citation(s) in RCA: 111] [Impact Index Per Article: 11.1] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/04/2015] [Accepted: 10/26/2015] [Indexed: 01/04/2023]
Abstract
Autoimmune diseases arise from the loss of tolerance to endogenous self-antigens, resulting in a heterogeneous range of chronic conditions that cause considerable morbidity and mortality worldwide. In Western countries, over 5% of the population is affected by some form of autoimmune disease, with enhanced or inappropriate activation of nuclear factor (NF)-κB implicated in a number of these conditions. Although treatment strategies for autoimmunity have improved significantly in recent years, current therapeutics are still not capable of achieving satisfactory disease management in all patients, and as such, the therapeutic modulation of NF-κB is an attractive target in autoimmunity. To date, no NF-κB inhibitors have progressed to the clinic for the treatment of autoimmunity, but a variety of promising approaches targeting multiple stages of the NF-κB pathway are currently being explored. This review focuses on the current strategies being investigated for the inhibition of the NF-κB pathway in autoimmune diseases and considers potential future strategies for the therapeutic targeting of this crucial transcription factor.
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Affiliation(s)
- Felicity D Herrington
- University of Glasgow, Institute of Infection, Immunity and Inflammation, Glasgow, UK
| | - Ruaidhrí J Carmody
- University of Glasgow, Institute of Infection, Immunity and Inflammation, Glasgow, UK
| | - Carl S Goodyear
- University of Glasgow, Institute of Infection, Immunity and Inflammation, Glasgow, UK GLAZgo Discovery Centre, University of Glasgow, Institute of Infection, Immunity and Inflammation, Glasgow, UK
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Characterization of the Interaction between the Matrix Protein of Vesicular Stomatitis Virus and the Immunoproteasome Subunit LMP2. J Virol 2015; 89:11019-29. [PMID: 26311888 DOI: 10.1128/jvi.01753-15] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/09/2015] [Accepted: 08/17/2015] [Indexed: 11/20/2022] Open
Abstract
UNLABELLED The matrix protein (M) of vesicular stomatitis virus (VSV) is involved in virus assembly, budding, gene regulation, and cellular pathogenesis. Using a yeast two-hybrid system, the M globular domain was shown to interact with LMP2, a catalytic subunit of the immunoproteasome (which replaces the standard proteasome catalytic subunit PSMB6). The interaction was validated by coimmunoprecipitation of M and LMP2 in VSV-infected cells. The sites of interaction were characterized. A single mutation of M (I96A) which significantly impairs the interaction between M and LMP2 was identified. We also show that M preferentially binds to the inactive precursor of LMP2 (bearing an N-terminal propeptide which is cleaved upon LMP2 maturation). Furthermore, taking advantage of a sequence alignment between LMP2 and its proteasome homolog, PSMB6 (which does not bind to M), we identified a mutation (L45R) in the S1 pocket where the protein substrate binds prior to cleavage and a second one (D17A) of a conserved residue essential for the catalytic activity, resulting in a reduction of the level of binding to M. The combination of both mutations abolishes the interaction. Taken together, our data indicate that M binds to LMP2 before its incorporation into the immunoproteasome. As the immunoproteasome promotes the generation of major histocompatibility complex (MHC) class I-compatible peptides, a feature which favors the recognition and the elimination of infected cells by CD8 T cells, we suggest that M, by interfering with the immunoproteasome assembly, has evolved a mechanism that allows infected cells to escape detection and elimination by the immune system. IMPORTANCE The immunoproteasome promotes the generation of MHC class I-compatible peptides, a feature which favors the recognition and the elimination of infected cells by CD8 T cells. Here, we report on the association of vesicular stomatitis virus (VSV) matrix protein (M) with LMP2, one of the immunoproteasome-specific catalytic subunits. M preferentially binds to the LMP2 inactive precursor. The M-binding site on LMP2 is facing inwards in the immunoproteasome and is therefore not accessible to M after its assembly. Hence, M binds to LMP2 before its incorporation into the immunoproteasome. We suggest that VSV M, by interfering with the immunoproteasome assembly, has evolved a mechanism that allows infected cells to escape detection and elimination by the immune system. Modulating this M-induced immunoproteasome impairment might be relevant in order to optimize VSV for oncolytic virotherapy.
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Sei JJ, Haskett S, Kaminsky LW, Lin E, Truckenmiller ME, Bellone CJ, Buller RM, Norbury CC. Peptide-MHC-I from Endogenous Antigen Outnumber Those from Exogenous Antigen, Irrespective of APC Phenotype or Activation. PLoS Pathog 2015; 11:e1004941. [PMID: 26107264 PMCID: PMC4479883 DOI: 10.1371/journal.ppat.1004941] [Citation(s) in RCA: 26] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/20/2015] [Accepted: 05/08/2015] [Indexed: 12/19/2022] Open
Abstract
Naïve anti-viral CD8+ T cells (TCD8+) are activated by the presence of peptide-MHC Class I complexes (pMHC-I) on the surface of professional antigen presenting cells (pAPC). Increasing the number of pMHC-I in vivo can increase the number of responding TCD8+. Antigen can be presented directly or indirectly (cross presentation) from virus-infected and uninfected cells, respectively. Here we determined the relative importance of these two antigen presenting pathways in mousepox, a natural disease of the mouse caused by the poxvirus, ectromelia (ECTV). We demonstrated that ECTV infected several pAPC types (macrophages, B cells, and dendritic cells (DC), including DC subsets), which directly presented pMHC-I to naïve TCD8+ with similar efficiencies in vitro. We also provided evidence that these same cell-types presented antigen in vivo, as they form contacts with antigen-specific TCD8+. Importantly, the number of pMHC-I on infected pAPC (direct presentation) vastly outnumbered those on uninfected cells (cross presentation), where presentation only occurred in a specialized subset of DC. In addition, prior maturation of DC failed to enhance antigen presentation, but markedly inhibited ECTV infection of DC. These results suggest that direct antigen presentation is the dominant pathway in mice during mousepox. In a broader context, these findings indicate that if a virus infects a pAPC then the presentation by that cell is likely to dominate over cross presentation as the most effective mode of generating large quantities of pMHC-I is on the surface of pAPC that endogenously express antigens. Recent trends in vaccine design have focused upon the introduction of exogenous antigens into the MHC Class I processing pathway (cross presentation) in specific pAPC populations. However, use of a pantropic viral vector that targets pAPC to express antigen endogenously likely represents a more effective vaccine strategy than the targeting of exogenous antigen to a limiting pAPC subpopulation. To induce a protective cell type (CD8+ T cells) following virus infection, it is necessary to present degraded fragments of viral protein in complex with self molecules on the surface of so-called antigen presenting cells (APC). This process can occur in infected or uninfected APC and has been studied and quantified extensively in experimental setups in the lab. However, the extent to which presentation by infected or uninfected cells contribute to the induction of a protective CD8+ T cell response has not been studied extensively during a natural infection in a mouse model. Here we use a natural mouse virus to examine importantly, quantify, the contribution of presentation of the fragments of viral protein by infected or uninfected cells. We find that the presentation by infected cells dwarfs that seen by uninfected cells. The importance of this work lies in the fact that, if infected cells present way more antigen than uninfected cells, successful vaccine design should utilize this observation to make a vaccine where infected cells expressing virus proteins are the prevalent mode of induction of CD8+ T cells.
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Affiliation(s)
- Janet J. Sei
- Department of Microbiology and Immunology, College of Medicine, Pennsylvania State University, Hershey, Pennsylvania, United States of America
| | - Scott Haskett
- Department of Molecular Microbiology and Immunology, Saint Louis University Health Sciences Center, St. Louis, Missouri, United States of America
| | - Lauren W. Kaminsky
- Department of Microbiology and Immunology, College of Medicine, Pennsylvania State University, Hershey, Pennsylvania, United States of America
| | - Eugene Lin
- Department of Microbiology and Immunology, College of Medicine, Pennsylvania State University, Hershey, Pennsylvania, United States of America
| | - Mary E. Truckenmiller
- Department of Microbiology and Immunology, College of Medicine, Pennsylvania State University, Hershey, Pennsylvania, United States of America
| | - Clifford J. Bellone
- Department of Molecular Microbiology and Immunology, Saint Louis University Health Sciences Center, St. Louis, Missouri, United States of America
| | - R. Mark Buller
- Department of Molecular Microbiology and Immunology, Saint Louis University Health Sciences Center, St. Louis, Missouri, United States of America
| | - Christopher C. Norbury
- Department of Microbiology and Immunology, College of Medicine, Pennsylvania State University, Hershey, Pennsylvania, United States of America
- * E-mail:
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McCarthy MK, Weinberg JB. The immunoproteasome and viral infection: a complex regulator of inflammation. Front Microbiol 2015; 6:21. [PMID: 25688236 PMCID: PMC4310299 DOI: 10.3389/fmicb.2015.00021] [Citation(s) in RCA: 135] [Impact Index Per Article: 13.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/03/2014] [Accepted: 01/08/2015] [Indexed: 11/13/2022] Open
Abstract
During viral infection, proper regulation of immune responses is necessary to ensure successful viral clearance with minimal host tissue damage. Proteasomes play a crucial role in the generation of antigenic peptides for presentation on MHC class I molecules, and thus activation of CD8 T cells, as well as activation of the NF-κB pathway. A specialized type of proteasome called the immunoproteasome is constitutively expressed in hematopoietic cells and induced in non-immune cells during viral infection by interferon signaling. The immunoproteasome regulates CD8 T cell responses to many viral epitopes during infection. Accumulating evidence suggests that the immunoproteasome may also contribute to regulation of proinflammatory cytokine production, activation of the NF-κB pathway, and management of oxidative stress. Many viruses have mechanisms of interfering with immunoproteasome function, including prevention of transcriptional upregulation of immunoproteasome components as well as direct interaction of viral proteins with immunoproteasome subunits. A better understanding of the role of the immunoproteasome in different cell types, tissues, and hosts has the potential to improve vaccine design and facilitate the development of effective treatment strategies for viral infections.
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Affiliation(s)
- Mary K McCarthy
- Department of Microbiology and Immunology, University of Michigan Ann Arbor, MI, USA
| | - Jason B Weinberg
- Department of Microbiology and Immunology, University of Michigan Ann Arbor, MI, USA ; Department of Pediatrics and Communicable Diseases, University of Michigan Ann Arbor, MI, USA
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Erath S, Groettrup M. No evidence for immunoproteasomes in chicken lymphoid organs and activated lymphocytes. Immunogenetics 2014; 67:51-60. [PMID: 25403261 DOI: 10.1007/s00251-014-0814-1] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/21/2014] [Accepted: 11/05/2014] [Indexed: 11/29/2022]
Abstract
The proteasome is the main protein-degrading machine within the cell, producing ligands for MHC class I molecules. It is a cylindrical multicatalytic protease complex, and the catalytic activity is mediated by the three subunits β1, β2, and β5 which possess caspase-, trypsin-, and chymotrypsin-like activities, respectively. By stimulation with interferon (IFN)-γ the replacement of these subunits by β1i, β2i, and β5i is induced leading to formation of immunoproteasomes with altered proteolytic and antigen processing properties. The genes coding for these immunosubunits are restricted to jawed vertebrates but have so far not been found in the genomes of birds, e.g., chicken, turkey, quail, black grouse and zebra finch. However, the chicken genome sequences are not completely assigned; therefore, we investigated the presence of immunoproteasome on protein level. 20S proteasome was purified from the chicken brain, blood, spleen, and bursa of Fabricius, followed by separation via two-dimensional (2D) gel electrophoresis. We analyzed the protein spots derived from the spleen and brain by mass spectrometry and could identify all 14 proteasomal subunits, but there were no differences detectable in the spot patterns. Moreover, we stimulated the chicken spleen cells with phorbol 12-myristate 13-acetate (PMA) and ionomycin aiming at the induction of immunoproteasome, but in spite of the induction of proliferation and IFN-γ, no evidence for immunoproteasome formation in chicken could be obtained. This result was substantiated by the finding that 20S proteasomes isolated from immune and non-immune tissues showed very similar peptidolytic activities. Taken together, our results indicate that chicken lack immunoproteasomes also on protein level.
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Affiliation(s)
- Sonja Erath
- Department of Immunology, University of Konstanz, Universitaetsstrasse 10, 78464, Konstanz, Germany
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40
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Donohue TM, Thomes PG. Ethanol-induced oxidant stress modulates hepatic autophagy and proteasome activity. Redox Biol 2014; 3:29-39. [PMID: 25462063 PMCID: PMC4297932 DOI: 10.1016/j.redox.2014.10.006] [Citation(s) in RCA: 31] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/12/2014] [Revised: 10/27/2014] [Accepted: 10/28/2014] [Indexed: 02/07/2023] Open
Abstract
In this review, we describe research findings on the effects of alcohol exposure on two major catabolic systems in liver cells: the ubiquitin-proteasome system (UPS) and autophagy. These hydrolytic systems are not unique to liver cells; they exist in all eukaryotic tissues and cells. However, because the liver is the principal site of ethanol metabolism, it sustains the greatest damage from heavy drinking. Thus, the focus of this review is to specifically describe how ethanol oxidation modulates the activities of the UPS and autophagy and the mechanisms by which these changes contribute to the pathogenesis of alcohol-induced liver injury. Here, we describe the history and the importance of cellular hydrolytic systems, followed by a description of each catabolic pathway and the differential modulation of each by ethanol exposure. Overall, the evidence for an involvement of these catabolic systems in the pathogenesis of alcoholic liver disease is quite strong. It underscores their importance, not only as effective means of cellular recycling and eventual energy generation, but also as essential components of cellular defense.
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Affiliation(s)
- Terrence M Donohue
- Research Service (151), VA-Nebraska-Western Iowa Health Care System, Omaha, NE 68105, USA; Department of Internal Medicine, College of Medicine, USA; Department of Biochemistry and Molecular Biology, College of Medicine, USA; Department of Pathology and Microbiology, College of Medicine, USA; The Center for Environmental Health and Toxicology, College of Public Health, University of Nebraska Medical Center, Omaha, NE, 68198, USA.
| | - Paul G Thomes
- Research Service (151), VA-Nebraska-Western Iowa Health Care System, Omaha, NE 68105, USA; Department of Internal Medicine, College of Medicine, USA
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Krzych U, Zarling S, Pichugin A. Memory T cells maintain protracted protection against malaria. Immunol Lett 2014; 161:189-95. [PMID: 24709142 PMCID: PMC6499475 DOI: 10.1016/j.imlet.2014.03.011] [Citation(s) in RCA: 24] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/20/2014] [Revised: 03/25/2014] [Accepted: 03/27/2014] [Indexed: 10/25/2022]
Abstract
Immunologic memory is one of the cardinal features of antigen-specific immune responses, and the persistence of memory cells contributes to prophylactic immunizations against infectious agents. Adequately maintained memory T and B cell pools assure a fast, effective and specific response against re-infections. However, many aspects of immunologic memory are still poorly understood, particularly immunologic memory inducible by parasites, for example, Plasmodium spp., the causative agents of malaria. For example, memory responses to Plasmodium antigens amongst residents of malaria endemic areas appear to be either inadequately developed or maintained, because persons who survive episodes of childhood malaria remain vulnerable to intermittent malaria infections. By contrast, multiple exposures of humans and laboratory rodents to radiation-attenuated Plasmodium sporozoites (γ-spz) induce sterile and long-lasting protection against experimental sporozoite challenge. Multifactorial immune mechanisms maintain this protracted and sterile protection. While the presence of memory CD4 T cell subsets has been associated with lasting protection in humans exposed to multiple bites from Anopheles mosquitoes infected with attenuated Plasmodium falciparum, memory CD8 T cells maintain protection induced with Plasmodium yoelii and Plasmodium berghei γ-spz in murine models. In this review, we discuss our observations that show memory CD8 T cells specific for antigens expressed by P. berghei liver stage parasites as an indispensable component for the maintenance of protracted protective immunity against experimental malaria infection; moreover, the provision of an Ag-depot assures a quick recall of memory T cells as IFN-γ-producing effector CD8 T cells and IL-4- producing CD4 T cells that collaborate with B cells for an effective antibody response.
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Affiliation(s)
- Urszula Krzych
- Department of Cellular Immunology, Branch of Malaria Vaccine Development, Walter Reed Army Institute of Research, Silver Spring, MD 20910, United States.
| | - Stasya Zarling
- Department of Cellular Immunology, Branch of Malaria Vaccine Development, Walter Reed Army Institute of Research, Silver Spring, MD 20910, United States
| | - Alexander Pichugin
- Department of Cellular Immunology, Branch of Malaria Vaccine Development, Walter Reed Army Institute of Research, Silver Spring, MD 20910, United States
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Kisselev AF, Groettrup M. Subunit specific inhibitors of proteasomes and their potential for immunomodulation. Curr Opin Chem Biol 2014; 23:16-22. [PMID: 25217863 DOI: 10.1016/j.cbpa.2014.08.012] [Citation(s) in RCA: 52] [Impact Index Per Article: 4.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/30/2014] [Revised: 08/15/2014] [Accepted: 08/22/2014] [Indexed: 11/20/2022]
Abstract
Specialized variants of the constitutive 20S proteasome in the immune system like the immunoproteasomes and the thymoproteasome contain active site-bearing subunits which differ in their cleavage priorities and substrate binding pockets. The immunoproteasome plays a crucial role in antigen processing and for the differentiation of pro-inflammatory T helper cells which are involved in the pathogenesis of autoimmunity. Selective inhibitors of the immunoproteasome and constitutive proteasome have recently been generated which interfere with the development and progression of autoimmune diseases. Here we describe these inhibitors and their therapeutic potential as predicted from preclinical models.
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Affiliation(s)
- Alexei F Kisselev
- Department of Pharmacology & Toxicology, Norris Cotton Cancer Center, Geisel School of Medicine at Dartmouth, One Medical Center Drive, Lebanon, NH 03756, USA.
| | - Marcus Groettrup
- Division of Immunology, Department of Biology, University of Konstanz, D-78457 Konstanz, Germany; Biotechnology Institute Thurgau (BITg) at the University of Konstanz, CH-8280 Kreuzlingen, Switzerland.
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Siciliano NA, Hersperger AR, Lacuanan AM, Xu RH, Sidney J, Sette A, Sigal LJ, Eisenlohr LC. Impact of distinct poxvirus infections on the specificities and functionalities of CD4+ T cell responses. J Virol 2014; 88:10078-91. [PMID: 24965457 PMCID: PMC4136331 DOI: 10.1128/jvi.01150-14] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/23/2014] [Accepted: 06/13/2014] [Indexed: 12/14/2022] Open
Abstract
UNLABELLED The factors that determine CD4+ T cell (TCD4+) specificities, functional capacity, and memory persistence in response to complex pathogens remain unclear. We explored these parameters in the C57BL/6 mouse through comparison of two highly related (>92% homology) poxviruses: ectromelia virus (ECTV), a natural mouse pathogen, and vaccinia virus (VACV), a heterologous virus that nevertheless elicits potent immune responses. In addition to elucidating several previously unidentified major histocompatibility complex class II (MHC-II)-restricted epitopes, we observed many qualitative and quantitative differences between the TCD4+ repertoires, including responses not elicited by VACV despite complete sequence conservation. In addition, we observed functional heterogeneity between ECTV- and VACV-specific TCD4+ at both a global and individual epitope level, particularly greater expression of the cytolytic marker CD107a from TCD4+ following ECTV infection. Most striking were differences during the late memory phase where, in contrast to ECTV, VACV infection failed to elicit measurable epitope-specific TCD4+ as determined by intracellular cytokine staining. These findings illustrate the strong influence of epitope-extrinsic factors on TCD4+ responses and memory. IMPORTANCE Much of our understanding concerning host-pathogen relationships in the context of poxvirus infections stems from studies of VACV in mice. However, VACV is not a natural mouse pathogen, and therefore, the relevance of results obtained using this model may be limited. Here, we explored the MHC class II-restricted TCD4+ repertoire induced by mousepox (ECTV) infection and the functional profile of the responding epitope-specific TCD4+, comparing these results to those induced by VACV infection under matched conditions. Despite a high degree of homology between the two viruses, we observed distinct specificity and functional profiles of TCD4+ responses at both acute and memory time points, with VACV-specific TCD4+ memory being notably compromised. These data offer insight into the impact of epitope-extrinsic factors on the resulting TCD4+ responses.
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Affiliation(s)
- Nicholas A Siciliano
- Department of Microbiology and Immunology, Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, Pennsylvania, USA
| | - Adam R Hersperger
- Department of Microbiology and Immunology, Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, Pennsylvania, USA Department of Biology, Albright College, Reading, Pennsylvania, USA
| | - Aimee M Lacuanan
- Department of Microbiology and Immunology, Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, Pennsylvania, USA
| | - Ren-Huan Xu
- Fox Chase Cancer Center, Immune Cell Development and Host Defense Program, Philadelphia, Pennsylvania, USA
| | - John Sidney
- Division of Vaccine Discovery, La Jolla Institute for Allergy and Immunology, La Jolla, California, USA
| | - Alessandro Sette
- Division of Vaccine Discovery, La Jolla Institute for Allergy and Immunology, La Jolla, California, USA
| | - Luis J Sigal
- Fox Chase Cancer Center, Immune Cell Development and Host Defense Program, Philadelphia, Pennsylvania, USA
| | - Laurence C Eisenlohr
- Department of Microbiology and Immunology, Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, Pennsylvania, USA
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Claud EC, McDonald JAK, He SM, Yu Y, Duong L, Sun J, Petrof EO. Differential expression of 26S proteasome subunits and functional activity during neonatal development. Biomolecules 2014; 4:812-26. [PMID: 25177858 PMCID: PMC4192673 DOI: 10.3390/biom4030812] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/06/2014] [Revised: 05/02/2014] [Accepted: 08/19/2014] [Indexed: 01/21/2023] Open
Abstract
Proteasomes regulate many essential cellular processes by degrading intracellular proteins. While aging is known to be associated with dysfunction of the proteasome, there are few reports detailing activity and function of proteasomes in the early stages of life. To elucidate the function and development of mammalian proteasomes, 26S proteasomes were affinity-purified from rat intestine, spleen and liver. The developmental expression of core, regulatory and immunoproteasome subunits was analyzed by immunoblotting and reverse-transcriptase PCR of mRNA subunits, and proteasome catalytic function was determined by fluorogenic enzymatic assays. The expression of core (β2, β5, α7 and β1) and regulatory (Rpt5) subunits was found to be present at low levels at birth and increased over time particularly at weaning. In contrast, while gradual developmental progression of proteasome structure was also seen with the immunoproteasome subunits (β1i, β5i, and β2i), these were not present at birth. Our studies demonstrate a developmental pattern to 26S proteasome activity and subunit expression, with low levels of core proteasome components and absence of immunoproteasomes at birth followed by increases at later developmental stages. This correlates with findings from other studies of a developmental hyporesponsiveness of the adaptive immune system to allow establishment of microbial colonization immediately after birth.
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Affiliation(s)
- Erika C Claud
- Departments of Pediatrics and Medicine, University of Chicago, Chicago, IL 60611, USA.
| | - Julie A K McDonald
- Gastrointestinal Diseases Research Unit, Department of Medicine, Queen's University, Kingston, ON K7L 2V7, Canada.
| | - Shu-Mei He
- Gastrointestinal Diseases Research Unit, Department of Medicine, Queen's University, Kingston, ON K7L 2V7, Canada.
| | - Yueyue Yu
- Departments of Pediatrics and Medicine, University of Chicago, Chicago, IL 60611, USA.
| | - Lily Duong
- Gastrointestinal Diseases Research Unit, Department of Medicine, Queen's University, Kingston, ON K7L 2V7, Canada.
| | - Jun Sun
- Department of Biochemistry, Rush University, Chicago, IL 60612, USA.
| | - Elaine O Petrof
- Gastrointestinal Diseases Research Unit, Department of Medicine, Queen's University, Kingston, ON K7L 2V7, Canada.
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Mechanisms of HIV protein degradation into epitopes: implications for vaccine design. Viruses 2014; 6:3271-92. [PMID: 25196483 PMCID: PMC4147695 DOI: 10.3390/v6083271] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/11/2014] [Revised: 08/06/2014] [Accepted: 08/11/2014] [Indexed: 12/02/2022] Open
Abstract
The degradation of HIV-derived proteins into epitopes displayed by MHC-I or MHC-II are the first events leading to the priming of HIV-specific immune responses and to the recognition of infected cells. Despite a wealth of information about peptidases involved in protein degradation, our knowledge of epitope presentation during HIV infection remains limited. Here we review current data on HIV protein degradation linking epitope production and immunodominance, viral evolution and impaired epitope presentation. We propose that an in-depth understanding of HIV antigen processing and presentation in relevant primary cells could be exploited to identify signatures leading to efficient or inefficient epitope presentation in HIV proteomes, and to improve the design of immunogens eliciting immune responses efficiently recognizing all infected cells.
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Kasthuri SR, Umasuthan N, Whang I, Lim BS, Jung HB, Oh MJ, Jung SJ, Yeo SY, Kim SY, Lee J. Molecular characterization and expressional affirmation of the beta proteasome subunit cluster in rock bream immune defense. Mol Biol Rep 2014; 41:5413-27. [PMID: 24867079 DOI: 10.1007/s11033-014-3413-1] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/17/2013] [Accepted: 05/13/2014] [Indexed: 11/27/2022]
Abstract
Immunoproteasomes are primarily induced upon infection and formed by replacing constitutive beta subunits with inducible beta subunits which possess specific cleavage properties that aid in the release of peptides necessary for MHC class I antigen presentation. In this study, we report the molecular characterization and expression analysis of the inducible immunosubunits PSMB8, PSMB9, PSMB9-L, and PSMB10 from rock bream, Oplegnathus fasciatus. The three subunits shared common active site residues and were placed in close proximity to fish homologues in the reconstructed phylogenetic tree, in which the mammalian homologues formed separate clades, indicating a common ancestral origin. The rock bream immunosubunits possessed higher identity and similarity with the fish homologues. RbPSMB8, RbPSMB9, RbPSMB9-L, and RbPSMB10 were multi-exonic genes with 6, 6, 7 and 8 exons, respectively. These four genes were constitutively expressed in all the examined tissues. Immunostimulants such as lipopolysaccharide and poly I:C induced RbPSMB8, RbPSMB9, RbPSMB9-L, and RbPSMB10 in liver and head kidney, suggesting their possible involvement in immune defense in rock bream.
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Affiliation(s)
- Saranya Revathy Kasthuri
- Department of Marine Life Sciences, School of Marine Biomedical Sciences, Jeju National University, Jeju, 690-756, Jeju Self-Governing Province, Republic of Korea
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Cascio P. PA28αβ: the enigmatic magic ring of the proteasome? Biomolecules 2014; 4:566-84. [PMID: 24970231 PMCID: PMC4101498 DOI: 10.3390/biom4020566] [Citation(s) in RCA: 70] [Impact Index Per Article: 6.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/04/2014] [Revised: 05/15/2014] [Accepted: 06/08/2014] [Indexed: 11/16/2022] Open
Abstract
PA28αβ is a γ-interferon-induced 11S complex that associates with the ends of the 20S proteasome and stimulates in vitro breakdown of small peptide substrates, but not proteins or ubiquitin-conjugated proteins. In cells, PA28 also exists in larger complexes along with the 19S particle, which allows ATP-dependent degradation of proteins; although in vivo a large fraction of PA28 is present as PA28αβ-20S particles whose exact biological functions are largely unknown. Although several lines of evidence strongly indicate that PA28αβ plays a role in MHC class I antigen presentation, the exact molecular mechanisms of this activity are still poorly understood. Herein, we review current knowledge about the biochemical and biological properties of PA28αβ and discuss recent findings concerning its role in modifying the spectrum of proteasome's peptide products, which are important to better understand the molecular mechanisms and biological consequences of PA28αβ activity.
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Affiliation(s)
- Paolo Cascio
- Department of Veterinary Sciences, University of Turin, Grugliasco 10095, Italy.
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Karpova ID, Lyupina IV, Astakhova TM, Stepanova AA, Erokhov PA, Abramova EB, Sharova NP. [Immune proteasomes in the development of rat immune system]. RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY 2014; 39:400-10. [PMID: 24707720 DOI: 10.1134/s1068162013040092] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/23/2022]
Abstract
The dynamics of the expression of LMP7 and LMP2 proteasome subunits in embryonic and early postnatal development of rat spleen and liver is investigated in comparison with the dynamics of chymotrypsin-like and caspase-like proteasome activities and expression of MHC (major histocompatibility complex) class I molecules. The immune subunits LMP7 and LMP2 distribution in spleen and liver cells in the development process is also studied. A mutual for both organs tendency to the increase of the expression of both LMP7 subunit and LMP2 one on P21 (the 21st postnatal day) as compared to the embryonic period is discovered. However, the total proteasome level is shown to be constant. At definite development stages, the dynamics of immune subunits expression in the spleen and liver was different. In the spleen gradual enhancement of both immune subunits level being detected on P1, P18 and P21, in the liver gradual enhancement periods on E16 (the 16th embryonic day) and E18 changed to the stage of the shrink of immune subunits level on P5. This level did not reliably change till P18 and was augmented on P21. The alterations revealed were accompanied by chymotrypsin-like activity raise and caspase-like activity drop in spleen by P21 as compared with the embryonic period, which proves the enlargement of proteasome ability to form antigenic epitopes for MHC class I molecules. In the liver, both activities increased by P21 in comparison with the embryonic period. Such dynamics of caspase-like activity can be explained not only by the change of proteolytic constitutive and immune subunits, but also by additional regulatory mechanisms. Besides, it is discovered that the increment of immune subunits expression in the early spleen development is connected with the process of successive forming the white pulp by B- and T-lymphocytes enriched by immune subunits. In the liver, the growth of immune subunits level by P21 was accompanied by their expression expansion in hepatocytes, while their plunge by P5 may be related to the loss of liver function of a primary lymphoid organ of the immune system by this stage and disappearance of B-lymphocytes enriched by immune proteasomes in it. In the spleen and liver, MHC class I molecules were revealed at the periods of the raise of proteasome immune subunits level. On E21 , the liver was enriched by neuronal NO-synthase, its level decreased after birth and enhanced to P18. This fact indicates the possibility of the induction of the immune subunits LMP7 [character: see text] LMP2 expression in hepatocytes in signal way with neuronal NO-synthase participation. The results obtained prove that T-cell immune response with spleen participation as regards rat liver cells is possible starting with P19-P21 stage. First, at this period, white pulp T-area is formed in the spleen. Second, enhanced immune proteasomes and MHC class I molecules levels in hepatocytes can procure antigenic epitopes formation from foreign proteins and their delivery to cell surface for their subsequent presentation for cytotoxic T-lymphocytes.
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Gohlke S, Mishto M, Textoris-Taube K, Keller C, Giannini C, Vasuri F, Capizzi E, D’Errico-Grigioni A, Kloetzel PM, Dahlmann B. Molecular alterations in proteasomes of rat liver during aging result in altered proteolytic activities. AGE (DORDRECHT, NETHERLANDS) 2014; 36:57-72. [PMID: 23690132 PMCID: PMC3889881 DOI: 10.1007/s11357-013-9543-x] [Citation(s) in RCA: 18] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 10/12/2012] [Accepted: 05/08/2013] [Indexed: 06/02/2023]
Abstract
Aging induces alterations of tissue protein homoeostasis. To investigate one of the major systems catalysing intracellular protein degradation we have purified 20S proteasomes from rat liver of young (2 months) and aged (23 months) animals and separated them into three subpopulations containing different types of intermediate proteasomes with standard- and immuno-subunits. The smallest subpopulation ΙΙΙ and the major subpopulation Ι comprised proteasomes containing immuno-subunits β1i and β5i beside small amounts of standard-subunits, whereas proteasomes of subpopulation ΙΙ contained only β5i beside standard-subunits. In favour of a relative increase of the major subpopulation Ι, subpopulation ΙΙ and ΙΙΙ were reduced for about 55 % and 80 %, respectively, in aged rats. Furthermore, in all three 20S proteasome subpopulations from aged animals standard-active site subunits were replaced by immuno-subunits. Overall, this transformation resulted in a relative increase of immuno-subunit-containing proteasomes, paralleled by reduced activity towards short fluorogenic peptide substrates. However, depending on the substrate their hydrolysing activity of long polypeptide substrates was significantly higher or unchanged. Furthermore, our data revealed an altered MHC class I antigen-processing efficiency of 20S proteasomes from liver of aged rats. We therefore suggest that the age-related intramolecular alteration of hepatic proteasomes modifies its cleavage preferences without a general decrease of its activity. Such modifications could have implications on protein homeostasis as well as on MHC class I antigen presentation as part of the immunosenescence process.
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Affiliation(s)
- Sabrina Gohlke
- />Institute of Biochemistry, Charité-Universitätsmedizin Berlin, CCM, CharitéCrossOver, Charitéplatz 1, 10117 Berlin, Germany
| | - Michele Mishto
- />Institute of Biochemistry, Charité-Universitätsmedizin Berlin, CCM, CharitéCrossOver, Charitéplatz 1, 10117 Berlin, Germany
- />Centro Interdipartimentale di Ricerca sul Cancro “Giorgio Prodi”, University of Bologna, Bologna, Italy
| | - Kathrin Textoris-Taube
- />Institute of Biochemistry, Charité-Universitätsmedizin Berlin, CCM, CharitéCrossOver, Charitéplatz 1, 10117 Berlin, Germany
| | - Christin Keller
- />Institute of Biochemistry, Charité-Universitätsmedizin Berlin, CCM, CharitéCrossOver, Charitéplatz 1, 10117 Berlin, Germany
| | - Carolin Giannini
- />Institute of Biochemistry, Charité-Universitätsmedizin Berlin, CCM, CharitéCrossOver, Charitéplatz 1, 10117 Berlin, Germany
| | - Francesco Vasuri
- />“F. Addarii” Institute of Oncology and Transplant Pathology, S.Orsola-Malpighi Hospital, University of Bologna, Bologna, Italy
| | - Elisa Capizzi
- />“F. Addarii” Institute of Oncology and Transplant Pathology, S.Orsola-Malpighi Hospital, University of Bologna, Bologna, Italy
| | - Antonia D’Errico-Grigioni
- />“F. Addarii” Institute of Oncology and Transplant Pathology, S.Orsola-Malpighi Hospital, University of Bologna, Bologna, Italy
| | - Peter-Michael Kloetzel
- />Institute of Biochemistry, Charité-Universitätsmedizin Berlin, CCM, CharitéCrossOver, Charitéplatz 1, 10117 Berlin, Germany
| | - Burkhardt Dahlmann
- />Institute of Biochemistry, Charité-Universitätsmedizin Berlin, CCM, CharitéCrossOver, Charitéplatz 1, 10117 Berlin, Germany
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Tacchi L, Misra M, Salinas I. Anti-viral immune responses in a primitive lung: characterization and expression analysis of interferon-inducible immunoproteasome subunits LMP2, LMP7 and MECL-1 in a sarcopterygian fish, the Nigerian spotted lungfish (Protopterus dolloi). DEVELOPMENTAL AND COMPARATIVE IMMUNOLOGY 2013; 41:657-665. [PMID: 23932981 PMCID: PMC3963498 DOI: 10.1016/j.dci.2013.07.023] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 06/15/2013] [Revised: 07/30/2013] [Accepted: 07/31/2013] [Indexed: 06/02/2023]
Abstract
Lungfishes (Dipnoi) represent the closest ancestor of tetrapods. Dipnoi have dual breathing modes extracting oxygen from water and air. The primitive lungs of lungfishes are exposed to external antigens including viruses. To date, the immune response of lungfishes against viruses has not been investigated. During viral immune responses, cell exposure to type I interferon induces the replacement of the constitutive proteasome with LMP2, LMP7 and MECL-1 beta subunits forming the immunoproteasome and enhancing antigen presentation to MHC class I molecules. In order to study the immune defense system of the lungfish lung, we have characterized for the first time the three immunoproteasome subunits in the sarcopterygian fish, the Nigerian spotted lungfish (Protopterus dolloi). LMP2, LMP7 and MECL-1 were identified in P. dolloi and their sequences encoded predicted proteins of 216, 275 and 278 amino acids, respectively. The mRNA of these three genes was expressed in multiple tissues, including the lung, with the highest abundance observed in kidney and post-pyloric spleen. In vitro stimulation of lungfish lung and kidney primary cell cultures with PolyI:C for 4 and 12 h resulted in increased LMP2, LMP7 and MECL-1 expression in both tissues. These results suggest a central role of these genes in the activation of an antiviral immune response in lungfish. Importantly, they indicate that the primitive lung of the common ancestor of all tetrapods is capable of inducing the expression of these genes in response to viral stimulation.
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Affiliation(s)
- Luca Tacchi
- Center for Evolutionary and Theoretical Immunology (CETI), Department of Biology, MSC03 2020, 1 University of New Mexico, Albuquerque, NM 87131, USA
| | - Milind Misra
- Center for Evolutionary and Theoretical Immunology (CETI), Department of Biology, MSC03 2020, 1 University of New Mexico, Albuquerque, NM 87131, USA
| | - Irene Salinas
- Center for Evolutionary and Theoretical Immunology (CETI), Department of Biology, MSC03 2020, 1 University of New Mexico, Albuquerque, NM 87131, USA
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