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Nadendla EK, Tweedell RE, Kasof G, Kanneganti TD. Caspases: structural and molecular mechanisms and functions in cell death, innate immunity, and disease. Cell Discov 2025; 11:42. [PMID: 40325022 PMCID: PMC12052993 DOI: 10.1038/s41421-025-00791-3] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/28/2024] [Accepted: 03/05/2025] [Indexed: 05/07/2025] Open
Abstract
Caspases are critical regulators of cell death, development, innate immunity, host defense, and disease. Upon detection of pathogens, damage-associated molecular patterns, cytokines, or other homeostatic disruptions, innate immune sensors, such as NLRs, activate caspases to initiate distinct regulated cell death pathways, including non-lytic (apoptosis) and innate immune lytic (pyroptosis and PANoptosis) pathways. These cell death pathways are driven by specific caspases and distinguished by their unique molecular mechanisms, supramolecular complexes, and enzymatic properties. Traditionally, caspases are classified as either apoptotic (caspase-2, -3, -6, -7, -8, -9, and -10) or inflammatory (caspase-1, -4, -5, and -11). However, extensive data from the past decades have shown that apoptotic caspases can also drive lytic inflammatory cell death downstream of innate immune sensing and inflammatory responses, such as in the case of caspase-3, -6, -7, and -8. Therefore, more inclusive classification systems based on function, substrate specificity, or the presence of pro-domains have been proposed to better reflect the multifaceted roles of caspases. In this review, we categorize caspases into CARD-, DED-, and short/no pro-domain-containing groups and examine their critical functions in innate immunity and cell death, along with their structural and molecular mechanisms, including active site/exosite properties and substrates. Additionally, we highlight the emerging roles of caspases in cellular homeostasis and therapeutic targeting. Given the clinical relevance of caspases across multiple diseases, improved understanding of these proteins and their structure-function relationships is critical for developing effective treatment strategies.
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Affiliation(s)
- Eswar Kumar Nadendla
- Department of Immunology, St. Jude Children's Research Hospital, Memphis, TN, USA
| | - Rebecca E Tweedell
- Department of Immunology, St. Jude Children's Research Hospital, Memphis, TN, USA
| | - Gary Kasof
- Cell Signaling Technology, Danvers, MA, USA
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2
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Fuller JL, Shi K, Pockes S, Finzel BC, Ashe KH, Walters MA. Reengineering of Circularly Permuted Caspase-2 to Enhance Enzyme Stability and Enable Crystallographic Studies. ACS Chem Biol 2025; 20:845-857. [PMID: 40117490 DOI: 10.1021/acschembio.4c00795] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 03/23/2025]
Abstract
Caspase activation has been linked to several diseases, including cancer, neurodegeneration, and inflammatory conditions, generating interest in targeting this family of proteases for drug development. Caspase-2 (Casp2) in particular has been implicated in Alzheimer's Disease (AD) by cleaving tau protein into fragment Δtau314, which reversibly impairs cognitive and synaptic function. Thus, Casp2 inhibition could be a useful strategy for therapeutic treatment of AD. To that end, we have previously synthesized and characterized various series of peptide and peptidomimetic inhibitors that demonstrate potency and selectivity for Casp2 over caspase-3 (Casp3). Despite promising developments in the design of selective Casp2 inhibitors, low expression yields of Casp2 hinder crystallographic experiments and make structure-based design challenging. The design of circularly permuted (cp) Casp2 increased protein yields considerably; however, this protein could not be crystallized. This article describes the characterization of ten novel cpCasp2 mutants, designed with the goal of increasing stability and facilitating crystallization. Gratifyingly, engineered mutant JF1cpCasp2 displayed high relative stability and was readily crystallizable with the canonical Casp2 inhibitor AcVDVAD-CHO, leading to what we believe to be the first crystal structures of any reverse caspase in the PDB. Moreover, we have reported the structure of JF1cpCasp2 with our recently described Casp2-selective inhibitor MUR-65, which revealed a unique interaction with Arg417 in the binding pocket. Overall, JF1cpCasp2 has proven valuable for structure-based design and expanding understanding of Casp2 inhibition, with potential implications for drug discovery and the development of more selective compounds.
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Affiliation(s)
- Jessica L Fuller
- Department of Medicinal Chemistry, University of Minnesota, Minneapolis, MN 55455, United States
- Department of Neurology, University of Minnesota, Minneapolis, MN 55455, United States
| | - Ke Shi
- College of Biological Sciences at University of Minnesota, Nanoliter Crystallization Facility, Minneapolis, MN 55455, United States
| | - Steffen Pockes
- Institute of Pharmacy, University of Regensburg, Regensburg 93053, Germany
| | - Barry C Finzel
- Department of Medicinal Chemistry, University of Minnesota, Minneapolis, MN 55455, United States
| | - Karen H Ashe
- Department of Neurology, University of Minnesota, Minneapolis, MN 55455, United States
| | - Michael A Walters
- Department of Medicinal Chemistry, University of Minnesota, Minneapolis, MN 55455, United States
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3
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Farag M, Guedeney N, Schwalen F, Zadoroznyj A, Barczyk A, Giret M, Antraygues K, Wang A, Cornu M, Suzanne P, Since M, Sophie Voisin-Chiret A, Dubrez L, Leleu-Chavain N, Kieffer C, Sopkova-de Oliveira Santos J. Towards New Anti-Inflammatory Agents: Design, Synthesis and Evaluation of Molecules Targeting XIAP-BIR2. ChemMedChem 2025; 20:e202400567. [PMID: 39364702 DOI: 10.1002/cmdc.202400567] [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: 07/23/2024] [Revised: 09/19/2024] [Accepted: 10/02/2024] [Indexed: 10/05/2024]
Abstract
The X-chromosome-linked inhibitor of apoptosis protein (XIAP) plays a crucial role in controlling cell survival across multiple regulated cell death pathways and coordinating a range of inflammatory signalling events. The discovery of selective inhibitors for XIAP-BIR2, able to disrupt the direct physical interaction between XIAP and RIPK2, offer promising therapeutic options for NOD2-mediated diseases like Crohn's disease, sarcoidosis, and Blau syndrome. The objective of this study was to design, synthesize, and evaluate small synthetic molecules with binding selectivity to XIAP-BIR2 domain. To achieve this, we applied an interdisciplinary drug design approach and firstly we have synthesized an initial fragment library to achieve a first XIAP inhibition activity. Then using a growing strategy, larger compounds were synthesized and one of them presents a good selectivity for XIAP-BIR2 versus XIAP-BIR3 domain, compound 20 c. The ability of compound 20 c to block the NOD1/2 pathway was confirmed in cell models. These data show that we have synthesized molecules capable of blocking NOD1/2 signalling pathways in cellulo, and ultimately leading to new anti-inflammatory compounds.
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Affiliation(s)
- Marc Farag
- Department, Normandie Univ, UNICAEN, CERMN, bd Becquerel, F-14000, Caen, Cedex, France
| | - Nicolas Guedeney
- Department, Normandie Univ, UNICAEN, CERMN, bd Becquerel, F-14000, Caen, Cedex, France
| | - Florian Schwalen
- Department, Normandie Univ, UNICAEN, CERMN, bd Becquerel, F-14000, Caen, Cedex, France
| | - Aymeric Zadoroznyj
- Institut National de la Santé et de la Recherche Médicale (Inserm), LNC UMR1231, Dijon, France
- Université de Bourgogne Franche-Comté, LNC UMR1231, Dijon, France
| | - Amélie Barczyk
- Univ. Lille, Inserm, CHU Lille, U1286 - INFINITE - Institute for Translational Research in Inflammation, F-59000, Lille, France
| | - Martin Giret
- Department, Normandie Univ, UNICAEN, CERMN, bd Becquerel, F-14000, Caen, Cedex, France
| | - Kevin Antraygues
- Department, Normandie Univ, UNICAEN, CERMN, bd Becquerel, F-14000, Caen, Cedex, France
| | - Alice Wang
- Department, Normandie Univ, UNICAEN, CERMN, bd Becquerel, F-14000, Caen, Cedex, France
| | - Marie Cornu
- Department, Normandie Univ, UNICAEN, CERMN, bd Becquerel, F-14000, Caen, Cedex, France
| | - Peggy Suzanne
- Department, Normandie Univ, UNICAEN, CERMN, bd Becquerel, F-14000, Caen, Cedex, France
| | - Marc Since
- Department, Normandie Univ, UNICAEN, CERMN, bd Becquerel, F-14000, Caen, Cedex, France
| | | | - Laurence Dubrez
- Institut National de la Santé et de la Recherche Médicale (Inserm), LNC UMR1231, Dijon, France
- Université de Bourgogne Franche-Comté, LNC UMR1231, Dijon, France
| | - Natascha Leleu-Chavain
- Univ. Lille, Inserm, CHU Lille, U1286 - INFINITE - Institute for Translational Research in Inflammation, F-59000, Lille, France
| | - Charline Kieffer
- Department, Normandie Univ, UNICAEN, CERMN, bd Becquerel, F-14000, Caen, Cedex, France
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4
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Castellón JO, Ofori S, Burton NR, Julio AR, Turmon AC, Armenta E, Sandoval C, Boatner LM, Takayoshi EE, Faragalla M, Taylor C, Zhou AL, Tran K, Shek J, Yan T, Desai HS, Fregoso OI, Damoiseaux R, Backus KM. Chemoproteomics Identifies State-Dependent and Proteoform-Selective Caspase-2 Inhibitors. J Am Chem Soc 2024; 146:14972-14988. [PMID: 38787738 PMCID: PMC11832190 DOI: 10.1021/jacs.3c12240] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 05/26/2024]
Abstract
Caspases are a highly conserved family of cysteine-aspartyl proteases known for their essential roles in regulating apoptosis, inflammation, cell differentiation, and proliferation. Complementary to genetic approaches, small-molecule probes have emerged as useful tools for modulating caspase activity. However, due to the high sequence and structure homology of all 12 human caspases, achieving selectivity remains a central challenge for caspase-directed small-molecule inhibitor development efforts. Here, using mass spectrometry-based chemoproteomics, we first identify a highly reactive noncatalytic cysteine that is unique to caspase-2. By combining both gel-based activity-based protein profiling (ABPP) and a tobacco etch virus (TEV) protease activation assay, we then identify covalent lead compounds that react preferentially with this cysteine and afford a complete blockade of caspase-2 activity. Inhibitory activity is restricted to the zymogen or precursor form of monomeric caspase-2. Focused analogue synthesis combined with chemoproteomic target engagement analysis in cellular lysates and in cells yielded both pan-caspase-reactive molecules and caspase-2 selective lead compounds together with a structurally matched inactive control. Application of this focused set of tool compounds to stratify the functions of the zymogen and partially processed (p32) forms of caspase-2 provide evidence to support that caspase-2-mediated response to DNA damage is largely driven by the partially processed p32 form of the enzyme. More broadly, our study highlights future opportunities for the development of proteoform-selective caspase inhibitors that target nonconserved and noncatalytic cysteine residues.
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Affiliation(s)
- José O Castellón
- Biological Chemistry Department, David Geffen School of Medicine, UCLA, Los Angeles, California 90095, United States
| | - Samuel Ofori
- Biological Chemistry Department, David Geffen School of Medicine, UCLA, Los Angeles, California 90095, United States
| | - Nikolas R Burton
- Biological Chemistry Department, David Geffen School of Medicine, UCLA, Los Angeles, California 90095, United States
- Department of Chemistry and Biochemistry, UCLA, Los Angeles, California 90095, United States
| | - Ashley R Julio
- Biological Chemistry Department, David Geffen School of Medicine, UCLA, Los Angeles, California 90095, United States
- Department of Chemistry and Biochemistry, UCLA, Los Angeles, California 90095, United States
| | - Alexandra C Turmon
- Biological Chemistry Department, David Geffen School of Medicine, UCLA, Los Angeles, California 90095, United States
- Department of Chemistry and Biochemistry, UCLA, Los Angeles, California 90095, United States
| | - Ernest Armenta
- Biological Chemistry Department, David Geffen School of Medicine, UCLA, Los Angeles, California 90095, United States
- Department of Chemistry and Biochemistry, UCLA, Los Angeles, California 90095, United States
| | - Carina Sandoval
- Department of Microbiology, Immunology, and Molecular Genetics, University of California, Los Angeles, California 90095, United States
| | - Lisa M Boatner
- Biological Chemistry Department, David Geffen School of Medicine, UCLA, Los Angeles, California 90095, United States
- Department of Chemistry and Biochemistry, UCLA, Los Angeles, California 90095, United States
| | - Evan E Takayoshi
- Biological Chemistry Department, David Geffen School of Medicine, UCLA, Los Angeles, California 90095, United States
- Department of Chemistry and Biochemistry, UCLA, Los Angeles, California 90095, United States
| | - Marina Faragalla
- Biological Chemistry Department, David Geffen School of Medicine, UCLA, Los Angeles, California 90095, United States
- Department of Chemistry and Biochemistry, UCLA, Los Angeles, California 90095, United States
| | - Cameron Taylor
- California NanoSystems Institute (CNSI), UCLA, Los Angeles, California 90095, United States
| | - Ann L Zhou
- Biological Chemistry Department, David Geffen School of Medicine, UCLA, Los Angeles, California 90095, United States
- Department of Chemistry and Biochemistry, UCLA, Los Angeles, California 90095, United States
| | - Ky Tran
- Biological Chemistry Department, David Geffen School of Medicine, UCLA, Los Angeles, California 90095, United States
- Department of Chemistry and Biochemistry, UCLA, Los Angeles, California 90095, United States
| | - Jeremy Shek
- Biological Chemistry Department, David Geffen School of Medicine, UCLA, Los Angeles, California 90095, United States
- Department of Chemistry and Biochemistry, UCLA, Los Angeles, California 90095, United States
| | - Tianyang Yan
- Biological Chemistry Department, David Geffen School of Medicine, UCLA, Los Angeles, California 90095, United States
- Department of Chemistry and Biochemistry, UCLA, Los Angeles, California 90095, United States
| | - Heta S Desai
- Biological Chemistry Department, David Geffen School of Medicine, UCLA, Los Angeles, California 90095, United States
| | - Oliver I Fregoso
- Department of Microbiology, Immunology, and Molecular Genetics, University of California, Los Angeles, California 90095, United States
| | - Robert Damoiseaux
- Jonsson Comprehensive Cancer Center, UCLA, Los Angeles, California 90095, United States
- Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research, UCLA, Los Angeles, California 90095, United States
- California NanoSystems Institute (CNSI), UCLA, Los Angeles, California 90095, United States
- Department of Molecular and Medical Pharmacology, UCLA, Los Angeles, California 90095, United States
- Department of Bioengineering, Samueli School of Engineering, UCLA, Los Angeles, California 90095, United States
| | - Keriann M Backus
- Biological Chemistry Department, David Geffen School of Medicine, UCLA, Los Angeles, California 90095, United States
- Department of Chemistry and Biochemistry, UCLA, Los Angeles, California 90095, United States
- DOE Institute for Genomics and Proteomics, UCLA, Los Angeles, California 90095, United States
- Jonsson Comprehensive Cancer Center, UCLA, Los Angeles, California 90095, United States
- Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research, UCLA, Los Angeles, California 90095, United States
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5
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Bibo-Verdugo B, Salvesen G. Evolution of Caspases and the Invention of Pyroptosis. Int J Mol Sci 2024; 25:5270. [PMID: 38791309 PMCID: PMC11121540 DOI: 10.3390/ijms25105270] [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: 04/16/2024] [Revised: 05/08/2024] [Accepted: 05/10/2024] [Indexed: 05/26/2024] Open
Abstract
The protein scaffold that includes the caspases is ancient and found in all domains of life. However, the stringent specificity that defines the caspase biologic function is relatively recent and found only in multicellular animals. During the radiation of the Chordata, members of the caspase family adopted roles in immunity, events coinciding with the development of substrates that define the modern innate immune response. This review focuses on the switch from the non-inflammatory cellular demise of apoptosis to the highly inflammatory innate response driven by distinct members of the caspase family, and the interplay between these two regulated cell death pathways.
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Affiliation(s)
- Betsaida Bibo-Verdugo
- Instituto Tecnológico de La Paz, Boulevard Forjadores de Baja California Sur 4720, La Paz 23080, Mexico;
| | - Guy Salvesen
- Sanford Burnham Prebys Medical Discovery Institute, 10901 North Torrey Pines Road, La Jolla, CA 92037, USA
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6
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Obaha A, Novinec M. Regulation of Peptidase Activity beyond the Active Site in Human Health and Disease. Int J Mol Sci 2023; 24:17120. [PMID: 38069440 PMCID: PMC10707025 DOI: 10.3390/ijms242317120] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/17/2023] [Revised: 12/01/2023] [Accepted: 12/02/2023] [Indexed: 12/18/2023] Open
Abstract
This comprehensive review addresses the intricate and multifaceted regulation of peptidase activity in human health and disease, providing a comprehensive investigation that extends well beyond the boundaries of the active site. Our review focuses on multiple mechanisms and highlights the important role of exosites, allosteric sites, and processes involved in zymogen activation. These mechanisms play a central role in shaping the complex world of peptidase function and are promising potential targets for the development of innovative drugs and therapeutic interventions. The review also briefly discusses the influence of glycosaminoglycans and non-inhibitory binding proteins on enzyme activities. Understanding their role may be a crucial factor in the development of therapeutic strategies. By elucidating the intricate web of regulatory mechanisms that control peptidase activity, this review deepens our understanding in this field and provides a roadmap for various strategies to influence and modulate peptidase activity.
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Affiliation(s)
| | - Marko Novinec
- Faculty of Chemistry and Chemical Technology, Department of Chemistry and Biochemistry, University of Ljubljana, Večna pot 113, SI-1000 Ljubljana, Slovenia;
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7
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Castellón JO, Ofori S, Armenta E, Burton N, Boatner LM, Takayoshi EE, Faragalla M, Zhou A, Tran K, Shek J, Yan T, Desai HS, Backus KM. Chemoproteomics identifies proteoform-selective caspase-2 inhibitors. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2023:2023.10.25.563785. [PMID: 37961563 PMCID: PMC10634807 DOI: 10.1101/2023.10.25.563785] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/15/2023]
Abstract
Caspases are a highly conserved family of cysteine-aspartyl proteases known for their essential roles in regulating apoptosis, inflammation, cell differentiation, and proliferation. Complementary to genetic approaches, small-molecule probes have emerged as useful tools for modulating caspase activity. However, due to the high sequence and structure homology of all twelve human caspases, achieving selectivity remains a central challenge for caspase-directed small-molecule inhibitor development efforts. Here, using mass spectrometry-based chemoproteomics, we first identify a highly reactive non-catalytic cysteine that is unique to caspase-2. By combining both gel-based activity-based protein profiling (ABPP) and a tobacco etch virus (TEV) protease activation assay, we then identify covalent lead compounds that react preferentially with this cysteine and afford a complete blockade of caspase-2 activity. Inhibitory activity is restricted to the zymogen or precursor form of monomeric caspase-2. Focused analogue synthesis combined with chemoproteomic target engagement analysis in cellular lysates and in cells yielded both pan-caspase reactive molecules and caspase-2 selective lead compounds together with a structurally matched inactive control. Application of this focused set of tool compounds to stratify caspase contributions to initiation of intrinsic apoptosis, supports compensatory caspase-9 activity in the context of caspase-2 inactivation. More broadly, our study highlights future opportunities for the development of proteoform-selective caspase inhibitors that target non-conserved and non-catalytic cysteine residues.
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8
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Sahoo G, Samal D, Khandayataray P, Murthy MK. A Review on Caspases: Key Regulators of Biological Activities and Apoptosis. Mol Neurobiol 2023; 60:5805-5837. [PMID: 37349620 DOI: 10.1007/s12035-023-03433-5] [Citation(s) in RCA: 87] [Impact Index Per Article: 43.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/17/2021] [Accepted: 06/06/2023] [Indexed: 06/24/2023]
Abstract
Caspases are proteolytic enzymes that belong to the cysteine protease family and play a crucial role in homeostasis and programmed cell death. Caspases have been broadly classified by their known roles in apoptosis (caspase-3, caspase-6, caspase-7, caspase-8, and caspase-9 in mammals) and in inflammation (caspase-1, caspase-4, caspase-5, and caspase-12 in humans, and caspase-1, caspase-11, and caspase-12 in mice). Caspases involved in apoptosis have been subclassified by their mechanism of action as either initiator caspases (caspase-8 and caspase-9) or executioner caspases (caspase-3, caspase-6, and caspase-7). Caspases that participate in apoptosis are inhibited by proteins known as inhibitors of apoptosis (IAPs). In addition to apoptosis, caspases play a role in necroptosis, pyroptosis, and autophagy, which are non-apoptotic cell death processes. Dysregulation of caspases features prominently in many human diseases, including cancer, autoimmunity, and neurodegenerative disorders, and increasing evidence shows that altering caspase activity can confer therapeutic benefits. This review covers the different types of caspases, their functions, and their physiological and biological activities and roles in different organisms.
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Affiliation(s)
- Gayatri Sahoo
- Department of Zoology, PSSJ College, Banarpal, 759128, Odisha, India
| | - Dibyaranjan Samal
- Department of Biotechnology, Academy of Management and Information Technology (AMIT, affiliated to Utkal University), Khurda, 752057, Odisha, India
| | | | - Meesala Krishna Murthy
- Department of Allied Health Sciences, Chitkara School of Health Sciences, Chitkara University, Rajpura, Punjab, 140401, India.
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9
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Farag M, Kieffer C, Guedeney N, Voisin-Chiret AS, Sopkova-de Oliveira Santos J. Computational Tool to Design Small Synthetic Inhibitors Selective for XIAP-BIR3 Domain. Molecules 2023; 28:5155. [PMID: 37446817 DOI: 10.3390/molecules28135155] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/01/2023] [Revised: 06/26/2023] [Accepted: 06/27/2023] [Indexed: 07/15/2023] Open
Abstract
X-linked inhibitor of apoptosis protein (XIAP) exercises its biological function by locking up and inhibiting essential caspase-3, -7 and -9 toward apoptosis execution. It is overexpressed in multiple human cancers, and it plays an important role in cancer cells' death skipping. Inhibition of XIAP-BIR3 domain and caspase-9 interaction was raised as a promising strategy to restore apoptosis in malignancy treatment. However, XIAP-BIR3 antagonists also inhibit cIAP1-2 BIR3 domains, leading to serious side effects. In this study, we worked on a theoretical model that allowed us to design and optimize selective synthetic XIAP-BIR3 antagonists. Firstly, we assessed various MM-PBSA strategies to predict the XIAP-BIR3 binding affinities of synthetic ligands. Molecular dynamics simulations using hydrogen mass repartition as an additional parametrization with and without entropic term computed by the interaction entropy approach produced the best correlations. These simulations were then exploited to generate 3D pharmacophores. Following an optimization with a training dataset, five features were enough to model XIAP-BIR3 synthetic ligands binding to two hydrogen bond donors, one hydrogen bond acceptor and two hydrophobic groups. The correlation between pharmacophoric features and computed MM-PBSA free energy revealed nine residues as crucial for synthetic ligand binding: Thr308, Glu314, Trp323, Leu307, Asp309, Trp310, Gly306, Gln319 and Lys297. Ultimately, and three of them seemed interesting to use to improve XIAP-BR3 versus cIAP-BIR3 selectivity: Lys297, Thr308 and Asp309.
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Affiliation(s)
- Marc Farag
- Normandie Univ., UNICAEN, CERMN, 14000 Caen, France
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10
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Structural insights into the regulation of Cas7-11 by TPR-CHAT. Nat Struct Mol Biol 2023; 30:135-139. [PMID: 36471056 PMCID: PMC9935389 DOI: 10.1038/s41594-022-00894-5] [Citation(s) in RCA: 9] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/27/2022] [Accepted: 11/04/2022] [Indexed: 12/12/2022]
Abstract
The CRISPR-guided caspase (Craspase) complex is an assembly of the target-specific RNA nuclease known as Cas7-11 bound to CRISPR RNA (crRNA) and an ancillary protein known as TPR-CHAT (tetratricopeptide repeats (TPR) fused with a CHAT domain). The Craspase complex holds promise as a tool for gene therapy and biomedical research, but its regulation is poorly understood. TPR-CHAT regulates Cas7-11 nuclease activity via an unknown mechanism. In the present study, we use cryoelectron microscopy to determine structures of the Desulfonema magnum (Dm) Craspase complex to gain mechanistic insights into its regulation. We show that DmTPR-CHAT stabilizes crRNA-bound DmCas7-11 in a closed conformation via a network of interactions mediated by the DmTPR-CHAT N-terminal domain, the DmCas7-11 insertion finger and Cas11-like domain, resulting in reduced target RNA accessibility and cleavage.
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11
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Khezri MR, Ghasemnejad-Berenji M. The Role of Caspases in Alzheimer's Disease: Pathophysiology Implications and Pharmacologic Modulation. J Alzheimers Dis 2023; 91:71-90. [PMID: 36442198 DOI: 10.3233/jad-220873] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/24/2022]
Abstract
Alzheimer's disease (AD) is the most common neurodegenerative disorder worldwide. Although the main cause of the onset and development of AD is not known yet, neuronal death due to pathologic changes such as amyloid-β (Aβ) deposition, tau aggregation, neuroinflammation, oxidative stress, and calcium dyshomeostasis are considered to be the main cause. At the present, there is no cure for this insidious disorder. However, accurate identification of molecular changes in AD can help provide new therapeutic goals. Caspases are a group of proteases which are known because of their role in cellular apoptosis. In addition, different caspases are involved in other cellular responses to the environment, such as induction of inflammation. Emerging evidence suggest that these proteases play a central role in AD pathophysiology due to their role in the processing of amyloid-β protein precursor, tau cleavage, and neuroinflammation. Therefore, it seems that targeting caspases may be a suitable therapeutic option to slow the progression of AD. This review focuses on the role of caspases in AD pathophysiology and introduce results from studies targeted caspases in different models of AD.
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Affiliation(s)
| | - Morteza Ghasemnejad-Berenji
- Department of Pharmacology and Toxicology, Faculty of Pharmacy, Urmia University of Medical Sciences, Urmia, Iran.,Research Center for Experimental and Applied Pharmaceutical Sciences, Urmia University of Medical Sciences, Urmia, Iran
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12
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Bibo-Verdugo B, Salvesen GS. Caspase mechanisms in the regulation of inflammation. Mol Aspects Med 2022; 88:101085. [PMID: 35248371 DOI: 10.1016/j.mam.2022.101085] [Citation(s) in RCA: 6] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/30/2021] [Revised: 02/22/2022] [Accepted: 02/26/2022] [Indexed: 12/31/2022]
Abstract
Regulated cell death is defined as genetically encoded pathways that lead towards the demise of cells. In mammals, cell demise can be either inflammatory or non-inflammatory, depending on whether the mechanism of death results in cell rupture or not. Inflammatory cell death can lead towards acute and chronic disease. Therefore, it becomes important to distinguish the mechanisms that result in these different inflammatory cell death outcomes. Apoptosis is a non-inflammatory form of cell death where cells resist rupture. In contrast, pyroptosis and necroptosis are inflammatory forms of cell death principally because of release of pro-inflammatory mediators from cells undergoing lysis. This review focusses on the mechanisms of these different cell death outcomes with specific emphasis on the caspase family of proteolytic enzymes.
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Affiliation(s)
- Betsaida Bibo-Verdugo
- Graduate School of Biomedical Sciences, Sanford Burnham Prebys Medical Discovery Institute, 10901 North Torrey Pines Road, La Jolla, CA, 92037, USA.
| | - Guy S Salvesen
- Graduate School of Biomedical Sciences, Sanford Burnham Prebys Medical Discovery Institute, 10901 North Torrey Pines Road, La Jolla, CA, 92037, USA.
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13
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Conchou L, Doumèche B, Galisson F, Violot S, Dugelay C, Diesis E, Page A, Bienvenu AL, Picot S, Aghajari N, Ballut L. Structural and molecular determinants of Candida glabrata metacaspase maturation and activation by calcium. Commun Biol 2022; 5:1158. [PMID: 36316540 PMCID: PMC9622860 DOI: 10.1038/s42003-022-04091-4] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/09/2021] [Accepted: 10/11/2022] [Indexed: 11/25/2022] Open
Abstract
Metacaspases are caspase-like homologs which undergo a complex maturation process involving multiple intra-chain cleavages resulting in a composite enzyme made of a p10 and a p20 domain. Their proteolytic activity involving a cysteine-histidine catalytic dyad, show peptide bond cleavage specificity in the C-terminal to lysine and arginine, with both maturation- and catalytic processes being calcium-dependent. Here, we present the structure of a metacaspase from the yeast Candida glabrata, CgMCA-I, in complex with a unique calcium along with a structure in which three magnesium ions are bound. We show that the Ca2+ ion interacts with a loop in the vicinity of the catalytic site. The reorganization of this cation binding loop, by bringing together the two catalytic residues, could be one of the main structural determinants triggering metacaspase activation. Enzymatic exploration of CgMCA-I confirmed that the maturation process implies a trans mechanism with sequential cleavages. Structural and functional analyses of yeast metacaspase reveal unique Ca2+ and Mg2+ binding sites and provide insights into Ca2+-dependent maturation of metacaspases along with the inhibitory effects of Mg2+ and Zn2+.
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Affiliation(s)
- Léa Conchou
- grid.25697.3f0000 0001 2172 4233Molecular Microbiology and Structural Biochemistry, UMR 5086, CNRS-Université de Lyon, F-69367 Lyon, France
| | - Bastien Doumèche
- grid.25697.3f0000 0001 2172 4233Université de Lyon, Université Lyon 1, Institut de Chimie et Biochimie Moléculaires et Supramoléculaire, ICBMS UMR 5246, CNRS, F-69622 Lyon, France
| | - Frédéric Galisson
- grid.25697.3f0000 0001 2172 4233Molecular Microbiology and Structural Biochemistry, UMR 5086, CNRS-Université de Lyon, F-69367 Lyon, France
| | - Sébastien Violot
- grid.25697.3f0000 0001 2172 4233Molecular Microbiology and Structural Biochemistry, UMR 5086, CNRS-Université de Lyon, F-69367 Lyon, France
| | - Chloé Dugelay
- grid.25697.3f0000 0001 2172 4233Molecular Microbiology and Structural Biochemistry, UMR 5086, CNRS-Université de Lyon, F-69367 Lyon, France
| | - Eric Diesis
- grid.15140.310000 0001 2175 9188University of Lyon, INSERM, ENS Lyon, CNRS, Protein Science Facility, SFR BioSciences, UAR3444/US8, F-69366 Lyon, France
| | - Adeline Page
- grid.15140.310000 0001 2175 9188University of Lyon, INSERM, ENS Lyon, CNRS, Protein Science Facility, SFR BioSciences, UAR3444/US8, F-69366 Lyon, France
| | - Anne-Lise Bienvenu
- grid.25697.3f0000 0001 2172 4233Université de Lyon, Université Lyon 1, Institut de Chimie et Biochimie Moléculaires et Supramoléculaire, ICBMS UMR 5246, CNRS, F-69622 Lyon, France ,grid.413852.90000 0001 2163 3825Service Pharmacie, Groupement Hospitalier Nord, Hospices Civils de Lyon, F-69004 Lyon, France
| | - Stéphane Picot
- grid.25697.3f0000 0001 2172 4233Université de Lyon, Université Lyon 1, Institut de Chimie et Biochimie Moléculaires et Supramoléculaire, ICBMS UMR 5246, CNRS, F-69622 Lyon, France ,grid.413306.30000 0004 4685 6736Institute of Parasitology and Medical Mycology, Hôpital de la Croix-Rousse, Hospices Civils de Lyon, F-69004 Lyon, France
| | - Nushin Aghajari
- grid.25697.3f0000 0001 2172 4233Molecular Microbiology and Structural Biochemistry, UMR 5086, CNRS-Université de Lyon, F-69367 Lyon, France
| | - Lionel Ballut
- grid.25697.3f0000 0001 2172 4233Molecular Microbiology and Structural Biochemistry, UMR 5086, CNRS-Université de Lyon, F-69367 Lyon, France
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14
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Zhang B, Xu D, Shao L, Liang H, Li J, Huang C. Toxicity mechanism of patulin on 293 T cells and correlation analysis of Caspase family. Toxicol Res (Camb) 2022; 11:758-764. [PMID: 36337240 PMCID: PMC9618098 DOI: 10.1093/toxres/tfac053] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/17/2022] [Revised: 05/25/2022] [Accepted: 06/21/2022] [Indexed: 08/27/2023] Open
Abstract
Patulin (PAT), a kind of mycotoxin, is a widely disseminated mycotoxin found in agricultural products. Although the existing research results show that PAT can cause nerve, immune, and skin toxicities, resulting in heart, liver, and kidney damages. However, evidence on the underlying mechanisms of PAT is still lacking. Present study aims to investigate the renal toxicity and related mechanisms of PAT on 293 T cells. Cell Counting Kit-8 method was used to reveal the dose-effect relationship and the time-effect relationship of PAT toxicity. Trypan blue staining and Hoechst 33342 staining were used to analyze PAT, which induced apoptosis on 293 T cells. Superoxide-dismutase (SOD), GSH, and malondialdehyde (MDA) were used to measure the changes of oxidative stress status of 293 T cells induced by PAT. The changes of reactive oxygen species (ROS) and ATP in mitochondria indicate the role of mitochondria when PAT induced cell damage and apoptosis. Through Cyt-C release assay analysis, caspase activity change, and correlation analysis, the potential mechanism of mitochondrial apoptosis pathway was proved. Results demonstrated that PAT significantly induced cell injury, and with the increase of time and concentration, the cell survival rate decreased significantly. Hoechst 33342 staining and Trypan blue staining showed that apoptosis rate was elevated by PAT. As PAT concentration increased, intracellular SOD, glutathion peroxidase activities were decreased and the MDA content was increased. The decrease of intracellular ATP level and accumulation of ROS content indicated an increased permeability of the mitochondrial membrane. Overexpression of Cyt-C activated the cascade reaction of caspase enzyme, leading to apoptosis. The results of enzyme activity assay and correlation analysis indicated that caspase 3 was the most critical caspase in the cascade system and that it was most correlated with caspase 8 and caspase 9.
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Affiliation(s)
- Baigang Zhang
- School of Life Science and Engineering, Lanzhou University of Technology, Lanzhou, Gansu 730050, China
| | - Dongmei Xu
- School of Life Science and Engineering, Lanzhou University of Technology, Lanzhou, Gansu 730050, China
| | - Lin Shao
- School of Life Science and Engineering, Lanzhou University of Technology, Lanzhou, Gansu 730050, China
| | - Hairong Liang
- School of Life Science and Engineering, Lanzhou University of Technology, Lanzhou, Gansu 730050, China
| | - Jinliang Li
- School of Life Science and Engineering, Lanzhou University of Technology, Lanzhou, Gansu 730050, China
| | - Chenghui Huang
- School of Life Science and Engineering, Lanzhou University of Technology, Lanzhou, Gansu 730050, China
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15
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Affiliation(s)
- Douglas R Green
- St. Jude Children's Research Hospital, Memphis, Tennessee 38105, USA
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16
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Russell LG, Davis LAK, Hunter JE, Perkins ND, Kenneth NS. Increased migration and motility in XIAP-null cells mediated by the C-RAF protein kinase. Sci Rep 2022; 12:7943. [PMID: 35562367 PMCID: PMC9106734 DOI: 10.1038/s41598-022-11438-8] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/04/2019] [Accepted: 04/11/2022] [Indexed: 11/18/2022] Open
Abstract
The product encoded by the X-linked inhibitor of apoptosis (XIAP) gene is a multi-functional protein which not only controls caspase-dependent cell death, but also participates in inflammatory signalling, copper homeostasis, response to hypoxia and control of cell migration. Deregulation of XIAP, either by elevated expression or inherited genetic deletion, is associated with several human disease states. Reconciling XIAP-dependent signalling pathways with its role in disease progression is essential to understand how XIAP promotes the progression of human pathologies. In this study we have created a panel of genetically modified XIAP-null cell lines using TALENs and CRISPR/Cas9 to investigate the functional outcome of XIAP deletion. Surprisingly, in our genetically modified cells XIAP deletion had no effect on programmed cell death, but instead the primary phenotype we observed was a profound increase in cell migration rates. Furthermore, we found that XIAP-dependent suppression of cell migration was dependent on XIAPdependent control of C-RAF levels, a protein kinase which controls cell signalling pathways that regulate the cytoskeleton. These results suggest that XIAP is not necessary for control of the apoptotic signalling cascade, however it does have a critical role in controlling cell migration and motility that cannot be compensated for in XIAP-knockout cells.
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Affiliation(s)
- Lauren G Russell
- Newcastle University Biosciences Institute, Faculty of Medical Sciences, Newcastle University, Newcastle upon Tyne, NE2 4HH, UK
| | - Lydia A K Davis
- Newcastle University Biosciences Institute, Faculty of Medical Sciences, Newcastle University, Newcastle upon Tyne, NE2 4HH, UK
| | - Jill E Hunter
- Newcastle University Biosciences Institute, Faculty of Medical Sciences, Newcastle University, Newcastle upon Tyne, NE2 4HH, UK
| | - Neil D Perkins
- Newcastle University Biosciences Institute, Faculty of Medical Sciences, Newcastle University, Newcastle upon Tyne, NE2 4HH, UK
| | - Niall S Kenneth
- Newcastle University Biosciences Institute, Faculty of Medical Sciences, Newcastle University, Newcastle upon Tyne, NE2 4HH, UK.
- Department of Molecular Physiology and Cell Signalling, Institute of Systems, Molecular and Integrative Biology, University of Liverpool, Liverpool, L69 7ZB, UK.
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Abstract
Blocking host cell death is an important virulence strategy employed by many bacterial pathogens. We recently reported that Shigella flexneri inhibits host pyroptosis by delivering a type III secretion system (T3SS) effector OspC3 that catalyzes a novel arginine ADP-riboxanation modification on caspase-4/11. Here, we investigated the OspC3 homologue CopC from Chromobacterium violaceum, an opportunistic but sometimes deadly bacterial pathogen. CopC bears the same arginine ADP-riboxanase activity as OspC3, but with a different substrate specificity. Through proteomic analysis, we first identified host calmodulin (CaM) as a binding partner of CopC. The analyses additionally revealed that CopC preferably modifies apoptotic caspases including caspase-7, -8 and -9. This results in suppression of both extrinsic and intrinsic apoptosis programs in C. violaceum-infected cells. Biochemical reconstitution showed that CopC requires binding to CaM, specifically in the calcium-free state, to achieve efficient ADP-riboxanation of the caspases. We determined crystal structure of the CaM-CopC-CASP7 ternary complex, which illustrates the caspase recognition mechanism and a unique CaM-binding mode in CopC. Structure-directed mutagenesis validated the functional significance of CaM binding for stimulating CopC modification of its caspase substrates. CopC adopts an ADP-ribosyltransferase-like fold with a unique His-Phe-Glu catalytic triad, featuring two acidic residues critical for site-specific arginine ADP-riboxanation. Our study expands and deepens our understanding of the OspC family of ADP-riboxanase effectors.
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18
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Characterization of caspase-7 interaction with RNA. Biochem J 2021; 478:2681-2696. [PMID: 34156061 DOI: 10.1042/bcj20210366] [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: 05/18/2021] [Revised: 06/18/2021] [Accepted: 06/22/2021] [Indexed: 11/17/2022]
Abstract
Apoptosis is a regulated form of cell death essential to the removal of unwanted cells. At its core, a family of cysteine peptidases named caspases cleave key proteins allowing cell death to occur. To do so, each caspase catalytic pocket recognizes preferred amino acid sequences resulting in proteolysis, but some also use exosites to select and cleave important proteins efficaciously. Such exosites have been found in a few caspases, notably caspase-7 that has a lysine patch (K38KKK) that binds RNA, which acts as a bridge to RNA-binding proteins favoring proximity between the peptidase and its substrates resulting in swifter cleavage. Although caspase-7 interaction with RNA has been identified, in-depth characterization of this interaction is lacking. In this study, using in vitro cleavage assays, we determine that RNA concentration and length affect the cleavage of RNA-binding proteins. Additionally, using binding assays and RNA sequencing, we found that caspase-7 binds RNA molecules regardless of their type, sequence, or structure. Moreover, we demonstrate that the N-terminal peptide of caspase-7 reduces the affinity of the peptidase for RNA, which translates into slower cleavages of RNA-binding proteins. Finally, employing engineered heterodimers, we show that a caspase-7 dimer can use both exosites simultaneously to increase its affinity to RNA because a heterodimer with only one exosite has reduced affinity for RNA and cleavage efficacy. These findings shed light on a mechanism that furthers substrate recognition by caspases and provides potential insight into its regulation during apoptosis.
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19
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Sharma A, Gogoi P, Chandravanshi M, Kanaujia SP. Water-mediated structural rearrangement establishes active conformation of caspases for apoptosis and inflammation. J Biomol Struct Dyn 2021; 40:6013-6026. [PMID: 33491574 DOI: 10.1080/07391102.2021.1875884] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/22/2022]
Abstract
Caspases are cysteine-dependent aspartate-specific proteases that play a crucial role in apoptosis (or programmed cell death) and inflammation. Based on their function, caspases are majorly categorized into apoptotic (initiator/apical and effector/executioner) and inflammatory caspases. Caspases undergo transition from an inactive zymogen to an active caspase to accomplish their function. This transition demands structural rearrangements which are most prominent at the active site loops and are imperative for the catalytic activity of caspases. In effector caspase-3, the structural rearrangement in the active site loop is shown to be facilitated by a set of invariant water (IW) molecules. However, the atomic details involving their role in stabilizing the active conformation have not been reported yet. Moreover, it is not known whether water molecules are essential for the active conformation in all caspases. Thus, in this study, we located IW molecules in initiator, effector, and inflammatory caspases to understand their precise role in rendering the structural arrangement of active caspases. Furthermore, IW molecules involved in anchoring the fragments of the protomer and rendering regulated flaccidity to caspases were identified. Location and identification of IW molecules interacting with amino acid residues involved in establishing the active conformation in the caspases might facilitate the design of potent inhibitors during up-regulated caspase activity in neurodegenerative and immune disorders. Communicated by Ramaswamy H. Sarma.
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Affiliation(s)
- Anjaney Sharma
- Department of Biosciences and Bioengineering, Indian Institute of Technology Guwahati, Guwahati, Assam, India
| | - Prerana Gogoi
- Department of Biosciences and Bioengineering, Indian Institute of Technology Guwahati, Guwahati, Assam, India
| | - Monika Chandravanshi
- Department of Biosciences and Bioengineering, Indian Institute of Technology Guwahati, Guwahati, Assam, India
| | - Shankar Prasad Kanaujia
- Department of Biosciences and Bioengineering, Indian Institute of Technology Guwahati, Guwahati, Assam, India
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20
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Makoni NJ, Nichols MR. The intricate biophysical puzzle of caspase-1 activation. Arch Biochem Biophys 2021; 699:108753. [PMID: 33453207 DOI: 10.1016/j.abb.2021.108753] [Citation(s) in RCA: 19] [Impact Index Per Article: 4.8] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/11/2020] [Revised: 12/22/2020] [Accepted: 01/05/2021] [Indexed: 12/13/2022]
Abstract
This review takes a closer look at the structural components of the molecules involved in the processes leading to caspase-1 activation. Interleukins 1β and 18 (IL-1β, IL-18) are well-known proinflammatory cytokines that are produced following cleavage of their respective precursor proteins by the cysteine protease caspase-1. Active caspase-1 is the final step of the NLRP3 inflammasome, a three-protein intracellular complex involved in inflammation and induction of pyroptosis (a proinflammatory cell-death process). NLRP3 activators facilitate assembly of the inflammasome complex and subsequent activation of caspase-1 by autoproteolysis. However, the definitive structural components of active caspase-1 are still unclear and new data add to the complexity of this process. This review outlines the historical and recent findings that provide supporting evidence for the structural aspects of caspase-1 autoproteolysis and activation.
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Affiliation(s)
- Nyasha J Makoni
- Department of Chemistry & Biochemistry, University of Missouri-St. Louis, St. Louis, MO, USA
| | - Michael R Nichols
- Department of Chemistry & Biochemistry, University of Missouri-St. Louis, St. Louis, MO, USA.
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21
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Wang J, Wang F, Mai D, Qu S. Molecular Mechanisms of Glutamate Toxicity in Parkinson's Disease. Front Neurosci 2020; 14:585584. [PMID: 33324150 PMCID: PMC7725716 DOI: 10.3389/fnins.2020.585584] [Citation(s) in RCA: 123] [Impact Index Per Article: 24.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/31/2020] [Accepted: 10/28/2020] [Indexed: 01/07/2023] Open
Abstract
Parkinson’s disease (PD) is a common neurodegenerative disease, the pathological features of which include the presence of Lewy bodies and the neurodegeneration of dopaminergic neurons in the substantia nigra pars compacta. However, until recently, research on the pathogenesis and treatment of PD have progressed slowly. Glutamate and dopamine are both important central neurotransmitters in mammals. A lack of enzymatic decomposition of extracellular glutamate results in glutamate accumulating at synapses, which is mainly absorbed by excitatory amino acid transporters (EAATs). Glutamate exerts its physiological effects by binding to and activating ligand-gated ion channels [ionotropic glutamate receptors (iGluRs)] and a class of G-protein-coupled receptors [metabotropic glutamate receptors (mGluRs)]. Timely clearance of glutamate from the synaptic cleft is necessary because high levels of extracellular glutamate overactivate glutamate receptors, resulting in excitotoxic effects in the central nervous system. Additionally, increased concentrations of extracellular glutamate inhibit cystine uptake, leading to glutathione depletion and oxidative glutamate toxicity. Studies have shown that oxidative glutamate toxicity in neurons lacking functional N-methyl-D-aspartate (NMDA) receptors may represent a component of the cellular death pathway induced by excitotoxicity. The association between inflammation and excitotoxicity (i.e., immunoexcitotoxicity) has received increased attention in recent years. Glial activation induces neuroinflammation and can stimulate excessive release of glutamate, which can induce excitotoxicity and, additionally, further exacerbate neuroinflammation. Glutamate, as an important central neurotransmitter, is closely related to the occurrence and development of PD. In this review, we discuss recent progress on elucidating glutamate as a relevant neurotransmitter in PD. Additionally, we summarize the relationship and commonality among glutamate excitotoxicity, oxidative toxicity, and immunoexcitotoxicity in order to posit a holistic view and molecular mechanism of glutamate toxicity in PD.
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Affiliation(s)
- Ji Wang
- Department of Neurology, Nanfang Hospital, Southern Medical University, Guangzhou, China.,Guangdong-Hong Kong-Macao Greater Bay Area Center for Brain Science and Brain-Inspired Intelligence, Guangzhou, China.,Key Laboratory of Mental Health of the Ministry of Education, Southern Medical University, Guangzhou, China
| | - Fushun Wang
- Institute of Brain and Psychological Science, Sichuan Normal University, Chengdu, China.,Department of Neurosurgery, Baylor Scott & White Health, Temple, TX, United States
| | - Dongmei Mai
- Department of Neurology, Nanfang Hospital, Southern Medical University, Guangzhou, China.,Guangdong-Hong Kong-Macao Greater Bay Area Center for Brain Science and Brain-Inspired Intelligence, Guangzhou, China.,Key Laboratory of Mental Health of the Ministry of Education, Southern Medical University, Guangzhou, China
| | - Shaogang Qu
- Department of Neurology, Nanfang Hospital, Southern Medical University, Guangzhou, China.,Guangdong-Hong Kong-Macao Greater Bay Area Center for Brain Science and Brain-Inspired Intelligence, Guangzhou, China.,Key Laboratory of Mental Health of the Ministry of Education, Southern Medical University, Guangzhou, China
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22
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Cserjan-Puschmann M, Lingg N, Engele P, Kröß C, Loibl J, Fischer A, Bacher F, Frank AC, Öhlknecht C, Brocard C, Oostenbrink C, Berkemeyer M, Schneider R, Striedner G, Jungbauer A. Production of Circularly Permuted Caspase-2 for Affinity Fusion-Tag Removal: Cloning, Expression in Escherichia coli, Purification, and Characterization. Biomolecules 2020; 10:E1592. [PMID: 33255244 PMCID: PMC7760212 DOI: 10.3390/biom10121592] [Citation(s) in RCA: 21] [Impact Index Per Article: 4.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/22/2020] [Revised: 11/13/2020] [Accepted: 11/20/2020] [Indexed: 02/08/2023] Open
Abstract
Caspase-2 is the most specific protease of all caspases and therefore highly suitable as tag removal enzyme creating an authentic N-terminus of overexpressed tagged proteins of interest. The wild type human caspase-2 is a dimer of heterodimers generated by autocatalytic processing which is required for its enzymatic activity. We designed a circularly permuted caspase-2 (cpCasp2) to overcome the drawback of complex recombinant expression, purification and activation, cpCasp2 was constitutively active and expressed as a single chain protein. A 22 amino acid solubility tag and an optimized fermentation strategy realized with a model-based control algorithm further improved expression in Escherichia coli and 5.3 g/L of cpCasp2 in soluble form were obtained. The generated protease cleaved peptide and protein substrates, regardless of N-terminal amino acid with high activity and specificity. Edman degradation confirmed the correct N-terminal amino acid after tag removal, using Ubiquitin-conjugating enzyme E2 L3 as model substrate. Moreover, the generated enzyme is highly stable at -20 °C for one year and can undergo 25 freeze/thaw cycles without loss of enzyme activity. The generated cpCasp2 possesses all biophysical and biochemical properties required for efficient and economic tag removal and is ready for a platform fusion protein process.
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Affiliation(s)
- Monika Cserjan-Puschmann
- ACIB-Austrian Centre of Industrial Biotechnology, Muthgasse 18, 1190 Vienna, Austria; (M.C.-P.); (P.E.); (C.K.); (J.L.); (A.F.); (F.B.); (A.-C.F.); (C.Ö.); (C.O.); (R.S.); (G.S.)
- Department of Biotechnology, University of Natural Resources and Life Sciences, Vienna, Muthgasse 18, 1190 Vienna, Austria
| | - Nico Lingg
- ACIB-Austrian Centre of Industrial Biotechnology, Muthgasse 18, 1190 Vienna, Austria; (M.C.-P.); (P.E.); (C.K.); (J.L.); (A.F.); (F.B.); (A.-C.F.); (C.Ö.); (C.O.); (R.S.); (G.S.)
- Department of Biotechnology, University of Natural Resources and Life Sciences, Vienna, Muthgasse 18, 1190 Vienna, Austria
| | - Petra Engele
- ACIB-Austrian Centre of Industrial Biotechnology, Muthgasse 18, 1190 Vienna, Austria; (M.C.-P.); (P.E.); (C.K.); (J.L.); (A.F.); (F.B.); (A.-C.F.); (C.Ö.); (C.O.); (R.S.); (G.S.)
- Institute of Biochemistry, Center for Molecular Biosciences Innsbruck (CMBI), University of Innsbruck, 6020 Innsbruck, Austria
| | - Christina Kröß
- ACIB-Austrian Centre of Industrial Biotechnology, Muthgasse 18, 1190 Vienna, Austria; (M.C.-P.); (P.E.); (C.K.); (J.L.); (A.F.); (F.B.); (A.-C.F.); (C.Ö.); (C.O.); (R.S.); (G.S.)
- Institute of Biochemistry, Center for Molecular Biosciences Innsbruck (CMBI), University of Innsbruck, 6020 Innsbruck, Austria
| | - Julian Loibl
- ACIB-Austrian Centre of Industrial Biotechnology, Muthgasse 18, 1190 Vienna, Austria; (M.C.-P.); (P.E.); (C.K.); (J.L.); (A.F.); (F.B.); (A.-C.F.); (C.Ö.); (C.O.); (R.S.); (G.S.)
| | - Andreas Fischer
- ACIB-Austrian Centre of Industrial Biotechnology, Muthgasse 18, 1190 Vienna, Austria; (M.C.-P.); (P.E.); (C.K.); (J.L.); (A.F.); (F.B.); (A.-C.F.); (C.Ö.); (C.O.); (R.S.); (G.S.)
| | - Florian Bacher
- ACIB-Austrian Centre of Industrial Biotechnology, Muthgasse 18, 1190 Vienna, Austria; (M.C.-P.); (P.E.); (C.K.); (J.L.); (A.F.); (F.B.); (A.-C.F.); (C.Ö.); (C.O.); (R.S.); (G.S.)
| | - Anna-Carina Frank
- ACIB-Austrian Centre of Industrial Biotechnology, Muthgasse 18, 1190 Vienna, Austria; (M.C.-P.); (P.E.); (C.K.); (J.L.); (A.F.); (F.B.); (A.-C.F.); (C.Ö.); (C.O.); (R.S.); (G.S.)
- Department of Biotechnology, University of Natural Resources and Life Sciences, Vienna, Muthgasse 18, 1190 Vienna, Austria
| | - Christoph Öhlknecht
- ACIB-Austrian Centre of Industrial Biotechnology, Muthgasse 18, 1190 Vienna, Austria; (M.C.-P.); (P.E.); (C.K.); (J.L.); (A.F.); (F.B.); (A.-C.F.); (C.Ö.); (C.O.); (R.S.); (G.S.)
- Institute of Molecular Modeling and Simulation, University of Natural Resources and Life Sciences, Vienna, Muthgasse 18, 1190 Vienna, Austria
| | - Cécile Brocard
- Biopharma Process Science Austria, Boehringer Ingelheim RCV GmbH & Co KG, Dr. Boehringer-Gasse 5-11, 1121 Vienna, Austria; (C.B.); (M.B.)
| | - Chris Oostenbrink
- ACIB-Austrian Centre of Industrial Biotechnology, Muthgasse 18, 1190 Vienna, Austria; (M.C.-P.); (P.E.); (C.K.); (J.L.); (A.F.); (F.B.); (A.-C.F.); (C.Ö.); (C.O.); (R.S.); (G.S.)
- Institute of Molecular Modeling and Simulation, University of Natural Resources and Life Sciences, Vienna, Muthgasse 18, 1190 Vienna, Austria
| | - Matthias Berkemeyer
- Biopharma Process Science Austria, Boehringer Ingelheim RCV GmbH & Co KG, Dr. Boehringer-Gasse 5-11, 1121 Vienna, Austria; (C.B.); (M.B.)
| | - Rainer Schneider
- ACIB-Austrian Centre of Industrial Biotechnology, Muthgasse 18, 1190 Vienna, Austria; (M.C.-P.); (P.E.); (C.K.); (J.L.); (A.F.); (F.B.); (A.-C.F.); (C.Ö.); (C.O.); (R.S.); (G.S.)
- Institute of Biochemistry, Center for Molecular Biosciences Innsbruck (CMBI), University of Innsbruck, 6020 Innsbruck, Austria
| | - Gerald Striedner
- ACIB-Austrian Centre of Industrial Biotechnology, Muthgasse 18, 1190 Vienna, Austria; (M.C.-P.); (P.E.); (C.K.); (J.L.); (A.F.); (F.B.); (A.-C.F.); (C.Ö.); (C.O.); (R.S.); (G.S.)
- Department of Biotechnology, University of Natural Resources and Life Sciences, Vienna, Muthgasse 18, 1190 Vienna, Austria
| | - Alois Jungbauer
- ACIB-Austrian Centre of Industrial Biotechnology, Muthgasse 18, 1190 Vienna, Austria; (M.C.-P.); (P.E.); (C.K.); (J.L.); (A.F.); (F.B.); (A.-C.F.); (C.Ö.); (C.O.); (R.S.); (G.S.)
- Department of Biotechnology, University of Natural Resources and Life Sciences, Vienna, Muthgasse 18, 1190 Vienna, Austria
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23
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Structural basis for Ca 2+-dependent activation of a plant metacaspase. Nat Commun 2020; 11:2249. [PMID: 32382010 PMCID: PMC7206013 DOI: 10.1038/s41467-020-15830-8] [Citation(s) in RCA: 34] [Impact Index Per Article: 6.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/21/2020] [Accepted: 03/26/2020] [Indexed: 12/20/2022] Open
Abstract
Plant metacaspases mediate programmed cell death in development, biotic and abiotic stresses, damage-induced immune response, and resistance to pathogen attack. Most metacaspases require Ca2+ for their activation and substrate processing. However, the Ca2+-dependent activation mechanism remains elusive. Here we report the crystal structures of Metacaspase 4 from Arabidopsis thaliana (AtMC4) that modulates Ca2+-dependent, damage-induced plant immune defense. The AtMC4 structure exhibits an inhibitory conformation in which a large linker domain blocks activation and substrate access. In addition, the side chain of Lys225 in the linker domain blocks the active site by sitting directly between two catalytic residues. We show that the activation of AtMC4 and cleavage of its physiological substrate involve multiple cleavages in the linker domain upon activation by Ca2+. Our analysis provides insight into the Ca2+-dependent activation of AtMC4 and lays the basis for tuning its activity in response to stresses for engineering of more sustainable crops for food and biofuels. Plant metacaspases mediate immune response following activation by Ca2+. Here, via crystallography and functional analyses, the authors show that a linker domain in Arabidopsis Metacaspase 4 blocks substrate access to the active site but is cleaved multiple times in the presence of Ca2+ to allow enzyme activation.
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Boon L, Ugarte-Berzal E, Vandooren J, Opdenakker G. Protease propeptide structures, mechanisms of activation, and functions. Crit Rev Biochem Mol Biol 2020; 55:111-165. [PMID: 32290726 DOI: 10.1080/10409238.2020.1742090] [Citation(s) in RCA: 49] [Impact Index Per Article: 9.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022]
Abstract
Proteases are a diverse group of hydrolytic enzymes, ranging from single-domain catalytic molecules to sophisticated multi-functional macromolecules. Human proteases are divided into five mechanistic classes: aspartate, cysteine, metallo, serine and threonine proteases, based on the catalytic mechanism of hydrolysis. As a protective mechanism against uncontrolled proteolysis, proteases are often produced and secreted as inactive precursors, called zymogens, containing inhibitory N-terminal propeptides. Protease propeptide structures vary considerably in length, ranging from dipeptides and propeptides of about 10 amino acids to complex multifunctional prodomains with hundreds of residues. Interestingly, sequence analysis of the different protease domains has demonstrated that propeptide sequences present higher heterogeneity compared with their catalytic domains. Therefore, we suggest that protease inhibition targeting propeptides might be more specific and have less off-target effects than classical inhibitors. The roles of propeptides, besides keeping protease latency, include correct folding of proteases, compartmentalization, liganding, and functional modulation. Changes in the propeptide sequence, thus, have a tremendous impact on the cognate enzymes. Small modifications of the propeptide sequences modulate the activity of the enzymes, which may be useful as a therapeutic strategy. This review provides an overview of known human proteases, with a focus on the role of their propeptides. We review propeptide functions, activation mechanisms, and possible therapeutic applications.
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Affiliation(s)
- Lise Boon
- Rega Institute for Medical Research, Department of Microbiology, Immunology and Transplantation, Laboratory of Immunobiology, KU Leuven, Leuven, Belgium
| | - Estefania Ugarte-Berzal
- Rega Institute for Medical Research, Department of Microbiology, Immunology and Transplantation, Laboratory of Immunobiology, KU Leuven, Leuven, Belgium
| | - Jennifer Vandooren
- Rega Institute for Medical Research, Department of Microbiology, Immunology and Transplantation, Laboratory of Immunobiology, KU Leuven, Leuven, Belgium
| | - Ghislain Opdenakker
- Rega Institute for Medical Research, Department of Microbiology, Immunology and Transplantation, Laboratory of Immunobiology, KU Leuven, Leuven, Belgium
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25
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Xu JH, Eberhardt J, Hill-Payne B, González-Páez GE, Castellón JO, Cravatt BF, Forli S, Wolan DW, Backus KM. Integrative X-ray Structure and Molecular Modeling for the Rationalization of Procaspase-8 Inhibitor Potency and Selectivity. ACS Chem Biol 2020; 15:575-586. [PMID: 31927936 PMCID: PMC7370820 DOI: 10.1021/acschembio.0c00019] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/24/2022]
Abstract
Caspases are a critical class of proteases involved in regulating programmed cell death and other biological processes. Selective inhibitors of individual caspases, however, are lacking, due in large part to the high structural similarity found in the active sites of these enzymes. We recently discovered a small-molecule inhibitor, 63-R, that covalently binds the zymogen, or inactive precursor (pro-form), of caspase-8, but not other caspases, pointing to an untapped potential of procaspases as targets for chemical probes. Realizing this goal would benefit from a structural understanding of how small molecules bind to and inhibit caspase zymogens. There have, however, been very few reported procaspase structures. Here, we employ X-ray crystallography to elucidate a procaspase-8 crystal structure in complex with 63-R, which reveals large conformational changes in active-site loops that accommodate the intramolecular cleavage events required for protease activation. Combining these structural insights with molecular modeling and mutagenesis-based biochemical assays, we elucidate key interactions required for 63-R inhibition of procaspase-8. Our findings inform the mechanism of caspase activation and its disruption by small molecules and, more generally, have implications for the development of small molecule inhibitors and/or activators that target alternative (e.g., inactive precursor) protein states to ultimately expand the druggable proteome.
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Affiliation(s)
- Janice H. Xu
- Department of Molecular Medicine, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037
- Department of Structural and Computational Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037
| | - Jerome Eberhardt
- Department of Structural and Computational Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037
| | - Brianna Hill-Payne
- Departments of Biological Chemistry and Chemistry and Biochemistry, David Geffen School of Medicine, University of California, Los Angeles, 405 Hilgard Avenue, Los Angeles, CA 90095
| | - Gonzalo E. González-Páez
- Department of Molecular Medicine, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037
- Department of Structural and Computational Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037
| | - José Omar Castellón
- Departments of Biological Chemistry and Chemistry and Biochemistry, David Geffen School of Medicine, University of California, Los Angeles, 405 Hilgard Avenue, Los Angeles, CA 90095
| | - Benjamin F. Cravatt
- Department of Chemistry, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037
| | - Stefano Forli
- Department of Structural and Computational Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037
| | - Dennis W. Wolan
- Department of Molecular Medicine, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037
- Department of Structural and Computational Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037
| | - Keriann M. Backus
- Departments of Biological Chemistry and Chemistry and Biochemistry, David Geffen School of Medicine, University of California, Los Angeles, 405 Hilgard Avenue, Los Angeles, CA 90095
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26
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Boice A, Bouchier-Hayes L. Targeting apoptotic caspases in cancer. BIOCHIMICA ET BIOPHYSICA ACTA-MOLECULAR CELL RESEARCH 2020; 1867:118688. [PMID: 32087180 DOI: 10.1016/j.bbamcr.2020.118688] [Citation(s) in RCA: 226] [Impact Index Per Article: 45.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Received: 09/16/2019] [Revised: 01/20/2020] [Accepted: 02/15/2020] [Indexed: 12/30/2022]
Abstract
Members of the caspase family of proteases play essential roles in the initiation and execution of apoptosis. These caspases are divided into two groups: the initiator caspases (caspase-2, -8, -9 and -10), which are the first to be activated in response to a signal, and the executioner caspases (caspase-3, -6, and -7) that carry out the demolition phase of apoptosis. Many conventional cancer therapies induce apoptosis to remove the cancer cell by engaging these caspases indirectly. Newer therapeutic applications have been designed, including those that specifically activate individual caspases using gene therapy approaches and small molecules that repress natural inhibitors of caspases already present in the cell. For such approaches to have maximal clinical efficacy, emerging insights into non-apoptotic roles of these caspases need to be considered. This review will discuss the roles of caspases as safeguards against cancer in the context of the advantages and potential limitations of targeting apoptotic caspases for the treatment of cancer.
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Affiliation(s)
- Ashley Boice
- Department of Pediatrics, Division of Hematology-Oncology and Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, TX 77030, USA; William T. Shearer Center for Human Immunobiology, Texas Children's Hospital, Houston, TX 77030, USA
| | - Lisa Bouchier-Hayes
- Department of Pediatrics, Division of Hematology-Oncology and Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, TX 77030, USA; William T. Shearer Center for Human Immunobiology, Texas Children's Hospital, Houston, TX 77030, USA.
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27
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Tuna M, Amos CI, Mills GB. Molecular mechanisms and pathobiology of oncogenic fusion transcripts in epithelial tumors. Oncotarget 2019; 10:2095-2111. [PMID: 31007851 PMCID: PMC6459343 DOI: 10.18632/oncotarget.26777] [Citation(s) in RCA: 16] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/14/2018] [Accepted: 02/22/2019] [Indexed: 02/07/2023] Open
Abstract
Recurrent fusion transcripts, which are one of the characteristic hallmarks of cancer, arise either from chromosomal rearrangements or from transcriptional errors in splicing. DNA rearrangements include intrachromosomal or interchromosomal translocation, tandem duplication, deletion, inversion, or result from chromothripsis, which causes complex rearrangements. In addition, fusion proteins can be created through transcriptional read-through. Fusion genes can be transcribed to fusion transcripts and translated to chimeric proteins, with many having demonstrated transforming activities through multiple mechanisms in cells. Fusion proteins represent novel therapeutic targets and diagnostic biomarkers of diagnosis, disease status, or progression. This review focuses on the mechanisms underlying the formation of oncogenic fusion genes and transcripts and their impact on the pathobiology of epithelial tumors.
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Affiliation(s)
- Musaffe Tuna
- Department of Epidemiology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA
- Department of Medicine, Baylor College of Medicine, Houston, TX, USA
| | - Christopher I. Amos
- Department of Medicine, Baylor College of Medicine, Houston, TX, USA
- Institute for Clinical and Translational Research, Baylor College of Medicine, Houston, TX, USA
| | - Gordon B. Mills
- Department of Systems Biology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA
- Department of Cell, Developmental and Cancer Biology, School of Medicine, Oregon Health Science University, Portland, OR, USA
- Precision Oncology, Knight Cancer Institute, Portland, OR, USA
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28
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Khan C, Muliyil S, Rao BJ. Genome Damage Sensing Leads to Tissue Homeostasis in Drosophila. INTERNATIONAL REVIEW OF CELL AND MOLECULAR BIOLOGY 2019; 345:173-224. [PMID: 30904193 DOI: 10.1016/bs.ircmb.2018.12.001] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/12/2022]
Abstract
DNA repair is a critical cellular process required for the maintenance of genomic integrity. It is now well appreciated that cells employ several DNA repair pathways to take care of distinct types of DNA damage. It is also well known that a cascade of signals namely DNA damage response or DDR is activated in response to DNA damage which comprise cellular responses, such as cell cycle arrest, DNA repair and cell death, if the damage is irreparable. There is also emerging literature suggesting a cross-talk between DNA damage signaling and several signaling networks within a cell. Moreover, cell death players themselves are also well known to engage in processes outside their canonical function of apoptosis. This chapter attempts to build a link between DNA damage, DDR and signaling from the studies mainly conducted in mammals and Drosophila model systems, with a special emphasis on their relevance in overall tissue homeostasis and development.
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Affiliation(s)
- Chaitali Khan
- Department of Biological Sciences, Tata Institute of Fundamental Research, Mumbai, India
| | - Sonia Muliyil
- Department of Biological Sciences, Tata Institute of Fundamental Research, Mumbai, India
| | - B J Rao
- Department of Biological Sciences, Tata Institute of Fundamental Research, Mumbai, India.
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29
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Bi X, Liu X, Bi D, Sun Y. Identification of Caspase-6 and Caspase-7 from miiuy croaker and evolution analysis in fish. FISH & SHELLFISH IMMUNOLOGY 2018; 83:406-409. [PMID: 30240802 DOI: 10.1016/j.fsi.2018.09.050] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 05/23/2018] [Revised: 09/14/2018] [Accepted: 09/17/2018] [Indexed: 06/08/2023]
Abstract
Apoptosis is a basic biological phenomenon of cells, which is an important component in the evolution of organisms, the stabilization of the internal environment and the development of multiple systems. In addition, the caspase protein family plays an important role in these pathways of apoptosis. Among them, apoptotic executors can directly act on specific substrates to complete the apoptotic response. In this study, we identified the Caspase-6 and Caspase-7 genes of miiuy croaker, and then analyzed the evolution of the whole Caspase family, furthermore described the evolutionary selection sites of the caspase-6 and caspase-7 genes in fish. The results showed that Caspase-6 gene appeared earlier than Caspase-7 in species evolution and gene duplication in teleost fish. Moreover, we also found that caspase-6 gene had no potential positive selection sites in the evolution of fish. Unlike the caspase-6 gene, the caspase-7 gene did not appear to be missed or replicated during the evolution of the species, while, it to be found two potential positive selection sites.
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Affiliation(s)
- Xueyi Bi
- Laboratory of Fish Biogenetics & Immune Evolution, College of Marine Science, Zhejiang Ocean University, Zhoushan, 316022, China
| | - Xuezhu Liu
- Laboratory of Fish Biogenetics & Immune Evolution, College of Marine Science, Zhejiang Ocean University, Zhoushan, 316022, China.
| | - Dekun Bi
- Laboratory of Fish Biogenetics & Immune Evolution, College of Marine Science, Zhejiang Ocean University, Zhoushan, 316022, China
| | - Yuena Sun
- Laboratory of Fish Biogenetics & Immune Evolution, College of Marine Science, Zhejiang Ocean University, Zhoushan, 316022, China.
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30
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Bingöl EN, Serçinoğlu O, Ozbek P. How do mutations and allosteric inhibitors modulate caspase-7 activity? A molecular dynamics study. J Biomol Struct Dyn 2018; 37:3456-3466. [DOI: 10.1080/07391102.2018.1517611] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/09/2023]
Affiliation(s)
- Elif Naz Bingöl
- Department of Bioengineering, Institute of Pure and Applied Sciences, Marmara University, Istanbul, Turkey
| | - Onur Serçinoğlu
- Department of Bioengineering, Institute of Pure and Applied Sciences, Marmara University, Istanbul, Turkey
- Faculty of Engineering, Department of Bioengineering, Marmara University, Istanbul, Turkey
| | - Pemra Ozbek
- Faculty of Engineering, Department of Bioengineering, Marmara University, Istanbul, Turkey
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31
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Regulating Apoptosis by Degradation: The N-End Rule-Mediated Regulation of Apoptotic Proteolytic Fragments in Mammalian Cells. Int J Mol Sci 2018; 19:ijms19113414. [PMID: 30384441 PMCID: PMC6274719 DOI: 10.3390/ijms19113414] [Citation(s) in RCA: 25] [Impact Index Per Article: 3.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/13/2018] [Revised: 10/24/2018] [Accepted: 10/27/2018] [Indexed: 12/13/2022] Open
Abstract
A pivotal hallmark of some cancer cells is the evasion of apoptotic cell death. Importantly, the initiation of apoptosis often results in the activation of caspases, which, in turn, culminates in the generation of proteolytically-activated protein fragments with potentially new or altered roles. Recent investigations have revealed that the activity of a significant number of the protease-generated, activated, pro-apoptotic protein fragments can be curbed via their selective degradation by the N-end rule degradation pathways. Of note, previous work revealed that several proteolytically-generated, pro-apoptotic fragments are unstable in cells, as their destabilizing N-termini target them for proteasomal degradation via the N-end rule degradation pathways. Remarkably, previous studies also showed that the proteolytically-generated anti-apoptotic Lyn kinase protein fragment is targeted for degradation by the UBR1/UBR2 E3 ubiquitin ligases of the N-end rule pathway in chronic myeloid leukemia cells. Crucially, the degradation of cleaved fragment of Lyn by the N-end rule counters imatinib resistance in these cells, implicating a possible linkage between the N-end rule degradation pathway and imatinib resistance. Herein, we highlight recent studies on the role of the N-end rule proteolytic pathways in regulating apoptosis in mammalian cells, and also discuss some possible future directions with respect to apoptotic proteolysis signaling.
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32
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Lu G, Yu Z, Lu M, Liu D, Wang F, Wu Y, Liu Y, Liu C, Wang L, Song L. The self-activation and LPS binding activity of executioner caspase-1 in oyster Crassostrea gigas. DEVELOPMENTAL AND COMPARATIVE IMMUNOLOGY 2017; 77:330-339. [PMID: 28888538 DOI: 10.1016/j.dci.2017.09.002] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 05/06/2017] [Revised: 09/05/2017] [Accepted: 09/05/2017] [Indexed: 06/07/2023]
Abstract
Executioner caspases play important roles in apoptotic pathway and immune defense, which is considered to coordinate the execution phase of apoptosis by cleaving multiple structural and repair proteins. However, the knowledge about the activation mechanism and function of executioner caspases in mollusks, especially marine bivalves is limited. In the present study, the full-length cDNA sequence of caspase-1 was cloned from oyster Crassostrea gigas, which encoded a predicted protein containing a small subunit (p10) and large subunit (p20) with a conserved caspase active site QACRG similar to that of human executioner caspase-3/7. SDS-polyacrylamide gel electrophoresis and western blot results demonstrated that the CgCaspase-1 zymogen could be cleaved into p20p10, p20 and p10 in prokaryotic expression systems, and the C-terminus of CgCaspase-1 was also cleaved into p20 and p10. Both of the recombinant CgCaspase-1 (rCgCaspase-1) and the C-terminus of CgCaspase-1 (rCgCaspase-1-C) exhibited similar caspase activity towards proteolytic substrate Ac-DMQD-pNA and Ac-DEVD-pNA. However, the recombinant N-terminus of CgCaspase-1 (rCgCaspase-1-N) did not display any caspase activity. Moreover, the inhibitor of both caspase-3/7 and pan-caspase could significantly inhibit the proteolytic activity of rCgCaspase-1. The strong binding activities towards lipopolysaccharide (LPS) of both rCgCaspase-1 and rCgCaspase-1-C were revealed by ELISA techniques and western blotting. A high level of CgCaspase-1 mRNA transcripts was detected in the gills and hemocytes by quantitative real-time PCR, and the CgCaspase-1 protein was mainly located in the cytoplasm of oyster hemocytes by immunofluorescence assay. These results collectively suggested that CgCaspase-1 was a homolog of executioner caspase-3/7, which could be self-activated through proteolytic cleavage in prokaryotic expression systems, and performed caspase and LPS binding activities in the innate immune response of oyster.
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Affiliation(s)
- Guangxia Lu
- Liaoning Key Laboratory of Marine Animal Immunology and Disease Control, Dalian Ocean University, Dalian 116023, China; Laboratory of Marine Fisheries Science and Food Production Process, Qingdao National Laboratory for Marine Science and Technology, Qingdao 266071, China
| | - Zichao Yu
- Liaoning Key Laboratory of Marine Animal Immunology and Disease Control, Dalian Ocean University, Dalian 116023, China
| | - Mengmeng Lu
- Liaoning Key Laboratory of Marine Animal Immunology and Disease Control, Dalian Ocean University, Dalian 116023, China
| | - Dongyang Liu
- Liaoning Key Laboratory of Marine Animal Immunology and Disease Control, Dalian Ocean University, Dalian 116023, China
| | - Feifei Wang
- Liaoning Key Laboratory of Marine Animal Immunology and Disease Control, Dalian Ocean University, Dalian 116023, China
| | - Yichen Wu
- Liaoning Key Laboratory of Marine Animal Immunology and Disease Control, Dalian Ocean University, Dalian 116023, China
| | - Yu Liu
- Liaoning Key Laboratory of Marine Animal Immunology and Disease Control, Dalian Ocean University, Dalian 116023, China
| | - Chao Liu
- Liaoning Key Laboratory of Marine Animal Immunology and Disease Control, Dalian Ocean University, Dalian 116023, China
| | - Lingling Wang
- Liaoning Key Laboratory of Marine Animal Immunology and Disease Control, Dalian Ocean University, Dalian 116023, China; Laboratory of Marine Fisheries Science and Food Production Process, Qingdao National Laboratory for Marine Science and Technology, Qingdao 266071, China
| | - Linsheng Song
- Liaoning Key Laboratory of Marine Animal Immunology and Disease Control, Dalian Ocean University, Dalian 116023, China; Laboratory of Marine Fisheries Science and Food Production Process, Qingdao National Laboratory for Marine Science and Technology, Qingdao 266071, China.
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Engineering a light-activated caspase-3 for precise ablation of neurons in vivo. Proc Natl Acad Sci U S A 2017; 114:E8174-E8183. [PMID: 28893998 DOI: 10.1073/pnas.1705064114] [Citation(s) in RCA: 44] [Impact Index Per Article: 5.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/18/2022] Open
Abstract
The circuitry of the brain is characterized by cell heterogeneity, sprawling cellular anatomy, and astonishingly complex patterns of connectivity. Determining how complex neural circuits control behavior is a major challenge that is often approached using surgical, chemical, or transgenic approaches to ablate neurons. However, all these approaches suffer from a lack of precise spatial and temporal control. This drawback would be overcome if cellular ablation could be controlled with light. Cells are naturally and cleanly ablated through apoptosis due to the terminal activation of caspases. Here, we describe the engineering of a light-activated human caspase-3 (Caspase-LOV) by exploiting its natural spring-loaded activation mechanism through rational insertion of the light-sensitive LOV2 domain that expands upon illumination. We apply the light-activated caspase (Caspase-LOV) to study neurodegeneration in larval and adult Drosophila Using the tissue-specific expression system (UAS)-GAL4, we express Caspase-LOV specifically in three neuronal cell types: retinal, sensory, and motor neurons. Illumination of whole flies or specific tissues containing Caspase-LOV-induced cell death and allowed us to follow the time course and sequence of neurodegenerative events. For example, we find that global synchronous activation of caspase-3 drives degeneration with a different time-course and extent in sensory versus motor neurons. We believe the Caspase-LOV tool we engineered will have many other uses for neurobiologists and others for specific temporal and spatial ablation of cells in complex organisms.
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34
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Alves J, Garay-Malpartida M, Occhiucci JM, Belizário JE. Modulation of procaspase-7 self-activation by PEST amino acid residues of the N-terminal prodomain and intersubunit linker. Biochem Cell Biol 2017; 95:634-643. [PMID: 28658581 DOI: 10.1139/bcb-2016-0220] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022] Open
Abstract
Procaspase-7 zymogen polypeptide is composed of a short prodomain, a large subunit (p20), and a small subunit (p10) connected to an intersubunit linker. Caspase-7 is activated by an initiator caspase-8 and -9, or by autocatalysis after specific cleavage at IQAD198↓S located at the intersubunit linker. Previously, we identified that PEST regions made of amino acid residues Pro (P), Glu (E), Asp (D), Ser (S), Thr (T), Asn (N), and Gln (Q) are conserved flanking amino acid residues in the cleavage sites within a prodomain and intersubunit linker of all caspase family members. Here we tested the impact of alanine substitution of PEST amino acid residues on procaspase-7 proteolytic self-activation directly in Escherichia coli. The p20 and p10 subunit cleavage were significantly delayed in double caspase-7 mutants in the prodomain (N18A/P26A) and intersubunit linker (S199A/P201A), compared with the wild-type caspase-7. The S199A/P201A mutants effectively inhibited the p10 small subunit cleavage. However, the mutations did not change the kinetic parameters (kcat/KM) and optimal tetrapeptide specificity (DEVD) of the purified mutant enzymes. The results suggest a role of PEST-amino acid residues in the molecular mechanism for prodomain and intersubunit cleavage and caspase-7 self-activation.
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Affiliation(s)
- Juliano Alves
- a Department of Pharmacology, Institute of Biomedical Sciences, Avenida Lineu Prestes, 1524, São Paulo, SP, 05508-900, Brazil
| | - Miguel Garay-Malpartida
- b School of Arts, Communication and Humanity, University of São Paulo, Rua Arlindo Béttio, 1000, São Paulo, SP, 03828-000, Brazil
| | - João M Occhiucci
- a Department of Pharmacology, Institute of Biomedical Sciences, Avenida Lineu Prestes, 1524, São Paulo, SP, 05508-900, Brazil
| | - José E Belizário
- a Department of Pharmacology, Institute of Biomedical Sciences, Avenida Lineu Prestes, 1524, São Paulo, SP, 05508-900, Brazil
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Dagbay KB, Bolik-Coulon N, Savinov SN, Hardy JA. Caspase-6 Undergoes a Distinct Helix-Strand Interconversion upon Substrate Binding. J Biol Chem 2017; 292:4885-4897. [PMID: 28154009 DOI: 10.1074/jbc.m116.773499] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/20/2016] [Revised: 02/01/2017] [Indexed: 12/22/2022] Open
Abstract
Caspases are cysteine aspartate proteases that are major players in key cellular processes, including apoptosis and inflammation. Specifically, caspase-6 has also been implicated in playing a unique and critical role in neurodegeneration; however, structural similarities between caspase-6 and other caspase active sites have hampered precise targeting of caspase-6. All caspases can exist in a canonical conformation, in which the substrate binds atop a β-strand platform in the 130's region. This caspase-6 region can also adopt a helical conformation that has not been seen in any other caspases. Understanding the dynamics and interconversion between the helical and strand conformations in caspase-6 is critical to fully assess its unique function and regulation. Here, hydrogen/deuterium exchange mass spectrometry indicated that caspase-6 is inherently and dramatically more conformationally dynamic than closely related caspase-7. In contrast to caspase-7, which rests constitutively in the strand conformation before and after substrate binding, the hydrogen/deuterium exchange data in the L2' and 130's regions suggested that before substrate binding, caspase-6 exists in a dynamic equilibrium between the helix and strand conformations. Caspase-6 transitions exclusively to the canonical strand conformation only upon substrate binding. Glu-135, which showed noticeably different calculated pK a values in the helix and strand conformations, appears to play a key role in the interconversion between the helix and strand conformations. Because caspase-6 has roles in several neurodegenerative diseases, exploiting the unique structural features and conformational changes identified here may provide new avenues for regulating specific caspase-6 functions for therapeutic purposes.
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Affiliation(s)
| | | | - Sergey N Savinov
- Biochemistry and Molecular Biology, University of Massachusetts, Amherst, Massachusetts 01003
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36
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Tunable allosteric library of caspase-3 identifies coupling between conserved water molecules and conformational selection. Proc Natl Acad Sci U S A 2016; 113:E6080-E6088. [PMID: 27681633 DOI: 10.1073/pnas.1603549113] [Citation(s) in RCA: 30] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/19/2022] Open
Abstract
The native ensemble of caspases is described globally by a complex energy landscape where the binding of substrate selects for the active conformation, whereas targeting an allosteric site in the dimer interface selects an inactive conformation that contains disordered active-site loops. Mutations and posttranslational modifications stabilize high-energy inactive conformations, with mostly formed, but distorted, active sites. To examine the interconversion of active and inactive states in the ensemble, we used detection of related solvent positions to analyze 4,995 waters in 15 high-resolution (<2.0 Å) structures of wild-type caspase-3, resulting in 450 clusters with the most highly conserved set containing 145 water molecules. The data show that regions of the protein that contact the conserved waters also correspond to sites of posttranslational modifications, suggesting that the conserved waters are an integral part of allosteric mechanisms. To test this hypothesis, we created a library of 19 caspase-3 variants through saturation mutagenesis in a single position of the allosteric site of the dimer interface, and we show that the enzyme activity varies by more than four orders of magnitude. Altogether, our database consists of 37 high-resolution structures of caspase-3 variants, and we demonstrate that the decrease in activity correlates with a loss of conserved water molecules. The data show that the activity of caspase-3 can be fine-tuned through globally desolvating the active conformation within the native ensemble, providing a mechanism for cells to repartition the ensemble and thus fine-tune activity through conformational selection.
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Salvesen GS, Hempel A, Coll NS. Protease signaling in animal and plant-regulated cell death. FEBS J 2016; 283:2577-98. [PMID: 26648190 PMCID: PMC5606204 DOI: 10.1111/febs.13616] [Citation(s) in RCA: 74] [Impact Index Per Article: 8.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/06/2015] [Revised: 11/23/2015] [Accepted: 11/30/2015] [Indexed: 12/26/2022]
Abstract
This review aims to highlight the proteases required for regulated cell death mechanisms in animals and plants. The aim is to be incisive, and not inclusive of all the animal proteases that have been implicated in various publications. The review also aims to focus on instances when several publications from disparate groups have demonstrated the involvement of an animal protease, and also when there is substantial biochemical, mechanistic and genetic evidence. In doing so, the literature can be culled to a handful of proteases, covering most of the known regulated cell death mechanisms: apoptosis, regulated necrosis, necroptosis, pyroptosis and NETosis in animals. In plants, the literature is younger and not as extensive as for mammals, although the molecular drivers of vacuolar death, necrosis and the hypersensitive response in plants are becoming clearer. Each of these death mechanisms has at least one proteolytic component that plays a major role in controlling the pathway, and sometimes they combine in networks to regulate cell death/survival decision nodes. Some similarities are found among animal and plant cell death proteases but, overall, the pathways that they govern are kingdom-specific with very little overlap.
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Affiliation(s)
- Guy S. Salvesen
- Sanford Burnham Prebys Medical Discovery Institute, 10901 North Torrey Pines Road, La Jolla, CA 92037, USA
| | - Anne Hempel
- Sanford Burnham Prebys Medical Discovery Institute, 10901 North Torrey Pines Road, La Jolla, CA 92037, USA
| | - Nuria Sanchez Coll
- Centre for Research in Agricultural Genomics, Campus UAB, Edifici CRAG, Bellaterra 08193, Barcelona, Spain
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Quantitative MS-based enzymology of caspases reveals distinct protein substrate specificities, hierarchies, and cellular roles. Proc Natl Acad Sci U S A 2016; 113:E2001-10. [PMID: 27006500 DOI: 10.1073/pnas.1524900113] [Citation(s) in RCA: 99] [Impact Index Per Article: 11.0] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/18/2022] Open
Abstract
Proteases constitute the largest enzyme family, yet their biological roles are obscured by our rudimentary understanding of their cellular substrates. There are 12 human caspases that play crucial roles in inflammation and cell differentiation and drive the terminal stages of cell death. Recent N-terminomics technologies have begun to enumerate the diverse substrates individual caspases can cleave in complex cell lysates. It is clear that many caspases have shared substrates; however, few data exist about the catalytic efficiencies (kcat/KM) of these substrates, which is critical to understanding their true substrate preferences. In this study, we use quantitative MS to determine the catalytic efficiencies for hundreds of natural protease substrates in cellular lysate for two understudied members: caspase-2 and caspase-6. Most substrates are new, and the cleavage rates vary up to 500-fold. We compare the cleavage rates for common substrates with those found for caspase-3, caspase-7, and caspase-8, involved in apoptosis. There is little correlation in catalytic efficiencies among the five caspases, suggesting each has a unique set of preferred substrates, and thus more specialized roles than previously understood. We synthesized peptide substrates on the basis of protein cleavage sites and found similar catalytic efficiencies between the protein and peptide substrates. These data suggest the rates of proteolysis are dominated more by local primary sequence, and less by the tertiary protein fold. Our studies highlight that global quantitative rate analysis for posttranslational modification enzymes in complex milieus for native substrates is critical to better define their functions and relative sequence of events.
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Abstract
The role of caspase proteases in regulated processes such as apoptosis and inflammation has been studied for more than two decades, and the activation cascades are known in detail. Apoptotic caspases also are utilized in critical developmental processes, although it is not known how cells maintain the exquisite control over caspase activity in order to retain subthreshold levels required for a particular adaptive response while preventing entry into apoptosis. In addition to active site-directed inhibitors, caspase activity is modulated by post-translational modifications or metal binding to allosteric sites on the enzyme, which stabilize inactive states in the conformational ensemble. This review provides a comprehensive global view of the complex conformational landscape of caspases and mechanisms used to select states in the ensemble. The caspase structural database provides considerable detail on the active and inactive conformations in the ensemble, which provide the cell multiple opportunities to fine tune caspase activity. In contrast, the current database on caspase modifications is largely incomplete and thus provides only a low-resolution picture of global allosteric communications and their effects on the conformational landscape. In recent years, allosteric control has been utilized in the design of small drug compounds or other allosteric effectors to modulate caspase activity.
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Affiliation(s)
- A Clay Clark
- Department of Biology, University of Texas at Arlington , Arlington, Texas 76019, United States
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Abstract
Apoptosis is an energy-dependent enzymatic cell suicide process. It almost always involves the activation of caspases. In this chapter, we systemically introduce methodologies to assay caspases dependent biochemical and morphological changes in vitro breast cancer cell lines and in vivo breast cancer tissues. In addition, mitochondrial involvement is crucial to distinguish two different apoptotic pathways. Methodology to assay dissipation of mitochondrial transmembrane potential, an early event of mitochondrial involvement, is also included. Of note, since apoptotic features may not appear to the same extent depending on the context of cell types and the death-inducing insults, a common practice is to use more than one method to assess apoptosis, qualitatively and quantitatively.
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Affiliation(s)
- Yu Sun
- Department of Chemical Biology, Rutgers University, 164 Frelinghuysen Road, Piscataway, NJ, 08854, USA
| | - Wei-Xing Zong
- Department of Chemical Biology, Rutgers University, 164 Frelinghuysen Road, Piscataway, NJ, 08854, USA.
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Poreba M, Szalek A, Kasperkiewicz P, Rut W, Salvesen GS, Drag M. Small Molecule Active Site Directed Tools for Studying Human Caspases. Chem Rev 2015; 115:12546-629. [PMID: 26551511 DOI: 10.1021/acs.chemrev.5b00434] [Citation(s) in RCA: 57] [Impact Index Per Article: 5.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/21/2022]
Abstract
Caspases are proteases of clan CD and were described for the first time more than two decades ago. They play critical roles in the control of regulated cell death pathways including apoptosis and inflammation. Due to their involvement in the development of various diseases like cancer, neurodegenerative diseases, or autoimmune disorders, caspases have been intensively investigated as potential drug targets, both in academic and industrial laboratories. This review presents a thorough, deep, and systematic assessment of all technologies developed over the years for the investigation of caspase activity and specificity using substrates and inhibitors, as well as activity based probes, which in recent years have attracted considerable interest due to their usefulness in the investigation of biological functions of this family of enzymes.
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Affiliation(s)
- Marcin Poreba
- Department of Bioorganic Chemistry, Faculty of Chemistry, Wroclaw University of Technology , Wyb. Wyspianskiego 27, 50-370 Wroclaw, Poland
| | - Aleksandra Szalek
- Department of Bioorganic Chemistry, Faculty of Chemistry, Wroclaw University of Technology , Wyb. Wyspianskiego 27, 50-370 Wroclaw, Poland
| | - Paulina Kasperkiewicz
- Department of Bioorganic Chemistry, Faculty of Chemistry, Wroclaw University of Technology , Wyb. Wyspianskiego 27, 50-370 Wroclaw, Poland
| | - Wioletta Rut
- Department of Bioorganic Chemistry, Faculty of Chemistry, Wroclaw University of Technology , Wyb. Wyspianskiego 27, 50-370 Wroclaw, Poland
| | - Guy S Salvesen
- Program in Cell Death and Survival Networks, Sanford Burnham Prebys Medical Discovery Institute , La Jolla, California 92037, United States
| | - Marcin Drag
- Department of Bioorganic Chemistry, Faculty of Chemistry, Wroclaw University of Technology , Wyb. Wyspianskiego 27, 50-370 Wroclaw, Poland
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Gianazza E, Parravicini C, Primi R, Miller I, Eberini I. In silico prediction and characterization of protein post-translational modifications. J Proteomics 2015; 134:65-75. [PMID: 26436211 DOI: 10.1016/j.jprot.2015.09.026] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/02/2015] [Revised: 07/17/2015] [Accepted: 09/23/2015] [Indexed: 01/06/2023]
Abstract
This review outlines the computational approaches and procedures for predicting post translational modification (PTM)-induced changes in protein conformation and their influence on protein function(s), the latter being assessed as differential affinity in interaction with either low (ligands for receptors or transporters, substrates for enzymes) or high molecular mass molecules (proteins or nucleic acids in supramolecular assemblies). The scope for an in silico approach is discussed against a summary of the in vitro evidence on the structural and functional outcome of protein PTM.
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Affiliation(s)
- Elisabetta Gianazza
- Dipartimento di Scienze Farmacologiche e Biomolecolari, Università degli Studi di Milano, Gruppo di Studio per la Proteomica e la Struttura delle Proteine, Sezione di Scienze Farmacologiche, Via Balzaretti 9, I-20133 Milan, Italy.
| | - Chiara Parravicini
- Dipartimento di Scienze Farmacologiche e Biomolecolari, Università degli Studi di Milano, Laboratorio di Biochimica e Biofisica Computazionale, Sezione di Biochimica, Biofisica, Fisiologia ed Immunopatologia, Via Trentacoste, 2, I-20134 Milan, Italy
| | - Roberto Primi
- Dipartimento di Scienze Farmacologiche e Biomolecolari, Università degli Studi di Milano, Laboratorio di Biochimica e Biofisica Computazionale, Sezione di Biochimica, Biofisica, Fisiologia ed Immunopatologia, Via Trentacoste, 2, I-20134 Milan, Italy
| | - Ingrid Miller
- Institut für Medizinische Biochemie, Veterinärmedizinische Universität Wien, Veterinärplatz 1, A-1210 Vienna, Austria
| | - Ivano Eberini
- Dipartimento di Scienze Farmacologiche e Biomolecolari, Università degli Studi di Milano, Laboratorio di Biochimica e Biofisica Computazionale, Sezione di Biochimica, Biofisica, Fisiologia ed Immunopatologia, Via Trentacoste, 2, I-20134 Milan, Italy
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McLuskey K, Mottram J. Comparative structural analysis of the caspase family with other clan CD cysteine peptidases. Biochem J 2015; 466:219-32. [PMID: 25697094 PMCID: PMC4357240 DOI: 10.1042/bj20141324] [Citation(s) in RCA: 66] [Impact Index Per Article: 6.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/22/2014] [Revised: 11/19/2014] [Accepted: 12/08/2014] [Indexed: 11/29/2022]
Abstract
Clan CD forms a structural group of cysteine peptidases, containing seven individual families and two subfamilies of structurally related enzymes. Historically, it is most notable for containing the mammalian caspases, on which the structures of the clan were founded. Interestingly, the caspase family is split into two subfamilies: the caspases, and a second subfamily containing both the paracaspases and the metacaspases. Structural data are now available for both the paracaspases and the metacaspases, allowing a comprehensive structural analysis of the entire caspase family. In addition, a relative plethora of structural data has recently become available for many of the other families in the clan, allowing both the structures and the structure-function relationships of clan CD to be fully explored. The present review compares the enzymes in the caspase subfamilies with each other, together with a comprehensive comparison of all the structural families in clan CD. This reveals a diverse group of structures with highly conserved structural elements that provide the peptidases with a variety of substrate specificities and activation mechanisms. It also reveals conserved structural elements involved in substrate binding, and potential autoinhibitory functions, throughout the clan, and confirms that the metacaspases are structurally diverse from the caspases (and paracaspases), suggesting that they should form a distinct family of clan CD peptidases.
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Key Words
- caspase
- clan cd
- crystallography
- metacaspase
- peptidase
- protein structure
- ap, activation peptide
- card, caspase recruitment domain
- chf, caspase/haemoglobinase fold
- cpd, cysteine peptidase domain
- csd, c-terminal subdomain
- dd, death domain
- ded, death effector domain
- insp6, myo-inositol hexakisphosphate
- lsam, legumain stabilization and activity modulation
- lsd1, lesion-simulating disease 1
- malt1, mucosa-associated lymphoid tissue translocation protein 1
- martx, multi-functional, autoprocessing repeat in toxin
- rmsd, root-mean-square deviation
- sse, secondary structural element
- xiap, x-linked inhibitor of apoptosis
- z-vrpr-fmk, benzoxycarbonyl-val-arg-pro-arg-fluoromethylketone
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Affiliation(s)
- Karen McLuskey
- *Wellcome Trust Centre for Molecular Parasitology, Institute of Infection, Immunity and Inflammation, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow G12 8TA, UK
| | - Jeremy C. Mottram
- *Wellcome Trust Centre for Molecular Parasitology, Institute of Infection, Immunity and Inflammation, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow G12 8TA, UK
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Wang XJ, Cao Q, Zhang Y, Su XD. Activation and regulation of caspase-6 and its role in neurodegenerative diseases. Annu Rev Pharmacol Toxicol 2014; 55:553-72. [PMID: 25340928 DOI: 10.1146/annurev-pharmtox-010814-124414] [Citation(s) in RCA: 74] [Impact Index Per Article: 6.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/09/2022]
Abstract
Caspases, a family of cysteine proteases, are major mediators of apoptosis and inflammation. Caspase-6 is classified as an apoptotic effector, and it mediates nuclear shrinkage during apoptosis, but it possesses unique activation and regulation mechanisms that differ from those of other effector caspases. Furthermore, increasing evidence has shown that caspase-6 is highly involved in axon degeneration and neurodegenerative diseases, such as Huntington's disease and Alzheimer's disease. Cleavage at the caspase-6 site in mutated huntingtin protein is a prerequisite for the development of the characteristic behavioral and neuropathological features of Huntington's disease. Active caspase-6 is present in early stages of Alzheimer's disease, and caspase-6 activity is associated with the disease's pathological lesions. In this review, we discuss the evidence relevant to the role of caspase-6 in neurodegenerative diseases and summarize its activation and regulation mechanisms. In doing so, we provide new insight about potential therapeutic approaches that incorporate the modulation of caspase-6 function for the treatment of neurodegenerative diseases.
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Affiliation(s)
- Xiao-Jun Wang
- State Key Laboratory of Protein and Plant Gene Research and
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45
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Paul I, Jones JM. Apoptosis block as a barrier to effective therapy in non small cell lung cancer. World J Clin Oncol 2014; 5:588-594. [PMID: 25302163 PMCID: PMC4129524 DOI: 10.5306/wjco.v5.i4.588] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 01/10/2014] [Revised: 02/28/2014] [Accepted: 06/11/2014] [Indexed: 02/06/2023] Open
Abstract
Lung cancer, is the most common cause of cancer death in men and second only to breast cancer in women. Currently, the first line therapy of choice is platinum-based combination chemotherapy. A therapeutic plateau has been reached with the prognosis for patients with advanced non-small cell lung cancer (NSCLC) remaining poor. New biomarkers of prognosis as well as new therapies focusing on molecular targets are emerging helping to identify patients who are likely to benefit from therapy. Despite this, drug resistance remains the major cause for treatment failure. In this article we review the role of apoptosis in mediating drug resistance in NSCLC. Better understanding of this fundamental biological process may provide a rationale for overcoming the current therapeutic plateau.
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46
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Combined inhibition of caspase 3 and caspase 7 by two highly selective DARPins slows down cellular demise. Biochem J 2014; 461:279-90. [PMID: 24779913 DOI: 10.1042/bj20131456] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/17/2023]
Abstract
Caspases play important roles during apoptosis, inflammation and proliferation. The high homology among family members makes selective targeting of individual caspases difficult, which is necessary to precisely define the role of these enzymes. We have selected caspase-7-specific binders from a library of DARPins (designed ankyrin repeat proteins). The DARPins D7.18 and D7.43 bind specifically to procaspase 7 and active caspase 7, but not to other members of the family. Binding of the DARPins does not affect the active enzyme, but interferes with its activation by other caspases. The crystal structure of the caspase 7-D7.18 complex elucidates the high selectivity and the mode of inhibition. Combining these caspase-7-specific DARPins with the previously reported caspase-3-inhibitory DARPin D3.4S76R reduces the activity of caspase 3 and 7 in double-transfected HeLa cells during apoptosis. In addition, these cells showed less susceptibility to TRAIL (tumour-necrosis-factor-related apoptosis-inducing ligand)-induced apoptosis in living cell experiments. D7.18 and D7.43 are therefore novel tools for in vitro studies on procaspase 7 activation as well as for clarifying the role of its activation in different cellular processes. If applied in combination with D3.4S76R, they represent an excellent instrument to increase our understanding of these enzymes during various cellular processes.
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47
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McStay GP, Green DR. Measuring apoptosis: caspase inhibitors and activity assays. Cold Spring Harb Protoc 2014; 2014:799-806. [PMID: 25086023 DOI: 10.1101/pdb.top070359] [Citation(s) in RCA: 18] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/24/2022]
Abstract
Caspases are proteases that initiate and execute apoptotic cell death. These caspase-dependent events are caused by cleavage of specific substrates that propagate the proapoptotic signal. A number of techniques have been developed to follow caspase activity in vitro and from apoptotic cellular extracts. Many of these techniques use molecules that are based on optimal peptide motifs for each caspase and on our understanding of caspase cleavage events that occur during apoptosis. Although these approaches are useful, there are several drawbacks associated with them. The optimal peptide motifs are not unique recognition sites for each caspase, so techniques that use them may yield information about more than one caspase. Furthermore, caspase cleavage does not take into account the different caspase activation mechanisms. Recently, probes having greater specificity for individual caspases have been developed and are being used successfully. This introduction provides background on the various caspases and introduces a set of complementary techniques to examine the activity, substrate specificity, and activation status of caspases from in vitro or cell culture experiments.
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Affiliation(s)
- Gavin P McStay
- Department of Life Sciences, New York Institute of Technology, Old Westbury, New York 11568
| | - Douglas R Green
- Department of Immunology, St. Jude Children's Research Hospital, Memphis, Tennessee 38105
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48
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Ma C, MacKenzie SH, Clark AC. Redesigning the procaspase-8 dimer interface for improved dimerization. Protein Sci 2014; 23:442-53. [PMID: 24442640 DOI: 10.1002/pro.2426] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/15/2013] [Revised: 01/10/2014] [Accepted: 01/10/2014] [Indexed: 01/04/2023]
Abstract
Caspase-8 is a cysteine directed aspartate-specific protease that is activated at the cytosolic face of the cell membrane upon receptor ligation. A key step in the activation of caspase-8 depends on adaptor-induced dimerization of procaspase-8 monomers. Dimerization is followed by limited autoproteolysis within the intersubunit linker (IL), which separates the large and small subunits of the catalytic domain. Although cleavage of the IL stabilizes the dimer, the uncleaved procaspase-8 dimer is sufficiently active to initiate apoptosis, so dimerization of the zymogen is an important mechanism to control apoptosis. In contrast, the effector caspase-3 is a stable dimer under physiological conditions but exhibits little enzymatic activity. The catalytic domains of caspases are structurally similar, but it is not known why procaspase-8 is a monomer while procaspase-3 is a dimer. To define the role of the dimer interface in assembly and activation of procaspase-8, we generated mutants that mimic the dimer interface of effector caspases. We show that procaspase-8 with a mutated dimer interface more readily forms dimers. Time course studies of refolding also show that the mutations accelerate dimerization. Transfection of HEK293A cells with the procaspase-8 variants, however, did not result in a significant increase in apoptosis, indicating that other factors are required in vivo. Overall, we show that redesigning the interface of procaspase-8 to remove negative design elements results in increased dimerization and activity in vitro, but increased dimerization, by itself, is not sufficient for robust activation of apoptosis.
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Affiliation(s)
- Chunxiao Ma
- Department of Molecular and Structural Biochemistry, North Carolina State University, Raleigh, North Carolina, 27695
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49
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Chai J, Shi Y. Apoptosome and inflammasome: conserved machineries for caspase activation. Natl Sci Rev 2014. [DOI: 10.1093/nsr/nwt025] [Citation(s) in RCA: 39] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/13/2022] Open
Abstract
Abstract
Apoptosome and inflammasome are multimeric protein complexes that mediate the activation of specific caspases at the onset of apoptosis and inflammation. The central component of apoptosome or inflammasome is a tripartite scaffold protein, exemplified by Apaf-1 and NLRC4, which contains an amino-terminal homotypic interaction motif, a central nucleotide-binding oligomerization domain and a carboxyl-terminal ligand-sensing domain. In the absence of death cue or an inflammatory signal, Apaf-1 or NLRC4 exists in an auto-inhibited, monomeric state, which is stabilized by adenosine diphosphate (ADP). Binding to an apoptosis- or inflammation-inducing ligand, together with replacement of ADP by adenosine triphosphate (ATP), results in the formation of a multimeric apoptosome or inflammasome. The assembled apoptosome and inflammasome serve as dedicated machineries to facilitate the activation of specific caspases. In this review, we describe the structure and functional mechanisms of mammalian inflammasome and apoptosomes from three representative organisms. Emphasis is placed on the molecular mechanism of caspase activation and the shared features of apoptosomes and inflammasomes.
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Affiliation(s)
- Jijie Chai
- Center for Life Sciences, School of Life Sciences and School of Medicine, Tsinghua University, Beijing 100084, China
| | - Yigong Shi
- Center for Life Sciences, School of Life Sciences and School of Medicine, Tsinghua University, Beijing 100084, China
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50
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Morgan CW, Julien O, Unger EK, Shah NM, Wells JA. Turning on caspases with genetics and small molecules. Methods Enzymol 2014; 544:179-213. [PMID: 24974291 DOI: 10.1016/b978-0-12-417158-9.00008-x] [Citation(s) in RCA: 23] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/11/2022]
Abstract
Caspases, aspartate-specific cysteine proteases, have fate-determining roles in many cellular processes including apoptosis, differentiation, neuronal remodeling, and inflammation (for review, see Yuan & Kroemer, 2010). There are a dozen caspases in humans alone, yet their individual contributions toward these phenotypes are not well understood. Thus, there has been considerable interest in activating individual caspases or using their activity to drive these processes in cells and animals. We envision that such experimental control of caspase activity can not only afford novel insights into fundamental biological problems but may also enable new models for disease and suggest possible routes to therapeutic intervention. In particular, localized, genetic, and small-molecule-controlled caspase activation has the potential to target the desired cell type in a tissue. Suppression of caspase activation is one of the hallmarks of cancer and thus there has been significant enthusiasm for generating selective small-molecule activators that could bypass upstream mutational events that prevent apoptosis. Here, we provide a practical guide that investigators have devised, using genetics or small molecules, to activate specific caspases in cells or animals. Additionally, we show genetically controlled activation of an executioner caspase to target the function of a defined group of neurons in the adult mammalian brain.
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Affiliation(s)
- Charles W Morgan
- Department of Pharmaceutical Chemistry, University of California, San Francisco, California, USA; Graduate Group in Chemistry and Chemical Biology, University of California, San Francisco, California, USA
| | - Olivier Julien
- Department of Pharmaceutical Chemistry, University of California, San Francisco, California, USA
| | - Elizabeth K Unger
- Department of Anatomy, University of California, San Francisco, California, USA; Program in Biomedical Sciences, University of California, San Francisco, California, USA
| | - Nirao M Shah
- Department of Anatomy, University of California, San Francisco, California, USA.
| | - James A Wells
- Department of Pharmaceutical Chemistry, University of California, San Francisco, California, USA; Department of Cellular and Molecular Pharmacology, University of California, San Francisco, California, USA.
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