164 results on '"Guy ’ s '
Search Results
2. Granzyme Release and Caspase Activation in Activated Human T-Lymphocytes*
- Author
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Zapata, Juan M., Takahashi, Ryosuke, Salvesen, Guy S., and Reed, John C.
- Abstract
Recently it has been reported that caspase-3 activation occurs in stimulated T-lymphocytes without associated apoptosis (Miossec, C., Dutilleul, V., Fassy, F., and Diu-Hercend, A. (1997) J. Biol. Chem. 272, 13459-13462). To explore this phenomenon, human peripheral blood lymphocytes (PBLs) were stimulated with mitogenic lectins or anti-CD3 antibody, and the proteolytic processing of different caspases and caspase substrates was analyzed by immunoblotting. Proteolytic processing of caspases-3 and -7 and the caspase substrates poly(ADP-ribose) polymerase, GDP dissociation inhibitor, and PKCdelta was observed when PBLs were activated in vitro, and lysates were prepared using RIPA buffer which contains 1% Nonidet P-40, 0.5% deoxycholate, and 0.1% SDS. In contrast, when a lysis buffer containing 2% SDS was used, the caspases remained in their zymogen pro-forms, and no proteolytic processing of caspase substrates was detected. Moreover, in experiments using intact cells and a cell-permeable fluorigenic caspase substrate, no caspase activity was observed in activated T-cells, whereas it was clearly detected when PBLs were treated with the apoptosis-inducing anticancer drug etoposide. Since the granzyme B is a direct activator of caspase-3 and its expression is induced following T-cell activation, we tested the effects of anti-GraB, an engineered serpin that specifically inhibits GraB. When the activated T-lymphocytes were lysed in RIPA buffer containing anti-GraB, no proteolytic processing or activation of caspase-3 was observed, strongly suggesting that release of GraB or similar proteases from their storage sites in cytotoxic granules during the lysis procedure is responsible for caspase activation. These findings demonstrate that T-cells do not process caspases upon activation and caution about the method of cell lysis used when studying granzyme-expressing cells.
- Published
- 1998
3. Resurrection of an ancient inflammatory locus reveals switch to caspase-1 specificity on a caspase-4 scaffold
- Author
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Bibo-Verdugo, Betsaida, primary, Joglekar, Isha, additional, Karadi Giridhar, Mithun N., additional, Ramirez, Monica L., additional, Snipas, Scott J., additional, Clark, A. Clay, additional, Poreba, Marcin, additional, and Salvesen, Guy S., additional
- Published
- 2022
- Full Text
- View/download PDF
4. Extended subsite profiling of the pyroptosis effector protein gasdermin D reveals a region recognized by inflammatory caspase-11
- Author
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Betsaida Bibo-Verdugo, Marcin Poreba, Sonia Kołt, Guy S. Salvesen, and Scott J. Snipas
- Subjects
0301 basic medicine ,Caspase 1 ,Caspase-11 ,Cleavage (embryo) ,Biochemistry ,Catalysis ,Substrate Specificity ,Mice ,03 medical and health sciences ,Pyroptosis ,Animals ,Humans ,Molecular Biology ,Caspase ,Inflammation ,Innate immune system ,030102 biochemistry & molecular biology ,biology ,Effector ,Chemistry ,Intracellular Signaling Peptides and Proteins ,Cell Biology ,Phosphate-Binding Proteins ,Cysteine protease ,Immunity, Innate ,Cell biology ,030104 developmental biology ,Caspases ,Proteolysis ,Enzymology ,biology.protein - Abstract
Pyroptosis is the caspase-dependent inflammatory cell death mechanism that underpins the innate immune response against pathogens and is dysregulated in inflammatory disorders. Pyroptosis occurs via two pathways: the canonical pathway, signaled by caspase-1, and the noncanonical pathway, regulated by mouse caspase-11 and human caspase-4/5. All inflammatory caspases activate the pyroptosis effector protein gasdermin D, but caspase-1 mostly activates the inflammatory cytokine precursors prointerleukin-18 and prointerleukin-1β (pro-IL18/pro-IL1β). Here, in vitro cleavage assays with recombinant proteins confirmed that caspase-11 prefers cleaving gasdermin D over the pro-ILs. However, we found that caspase-11 recognizes protein substrates through a mechanism that is different from that of most caspases. Results of kinetics analysis with synthetic fluorogenic peptides indicated that P1′–P4′, the C-terminal gasdermin D region adjacent to the cleavage site, influences gasdermin D recognition by caspase-11. Furthermore, introducing the gasdermin D P1′–P4′ region into pro-IL18 enhanced catalysis by caspase-11 to levels comparable with that of gasdermin D cleavage. Pro-IL1β cleavage was only moderately enhanced by similar substitutions. We conclude that caspase-11 specificity is mediated by the P1′–P4′ region in its substrate gasdermin D, and similar experiments confirmed that the substrate specificities of the human orthologs of caspase-11, i.e. caspase-4 and caspase-5, are ruled by the same mechanism. We propose that P1′–P4′-based inhibitors could be exploited to specifically target inflammatory caspases.
- Published
- 2020
5. Structural basis of trehalose recognition by the mycobacterial LpqY-SugABC transporter
- Author
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Furze, Christopher M., primary, Delso, Ignacio, additional, Casal, Enriqueta, additional, Guy, Collette S., additional, Seddon, Chloe, additional, Brown, Chelsea M., additional, Parker, Hadyn L., additional, Radhakrishnan, Anjana, additional, Pacheco-Gomez, Raul, additional, Stansfeld, Phillip J., additional, Angulo, Jesus, additional, Cameron, Alexander D., additional, and Fullam, Elizabeth, additional
- Published
- 2021
- Full Text
- View/download PDF
6. NETosis occurs independently of neutrophil serine proteases
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Kasperkiewicz, Paulina, primary, Hempel, Anne, additional, Janiszewski, Tomasz, additional, Kołt, Sonia, additional, Snipas, Scott J., additional, Drag, Marcin, additional, and Salvesen, Guy S., additional
- Published
- 2020
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7. Extended subsite profiling of the pyroptosis effector protein gasdermin D reveals a region recognized by inflammatory caspase-11
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Bibo-Verdugo, Betsaida, primary, Snipas, Scott J., additional, Kolt, Sonia, additional, Poreba, Marcin, additional, and Salvesen, Guy S., additional
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- 2020
- Full Text
- View/download PDF
8. Selective inhibition of matrix metalloproteinase 10 (MMP10) with a single-domain antibody
- Author
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Razai, Amir S., primary, Eckelman, Brendan P., additional, and Salvesen, Guy S., additional
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- 2020
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9. Regulation of Histone Acetylation by Autophagy in Parkinson Disease
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Goonho Park, Guy S. Salvesen, Yunyi Kang, Zhuohua Zhang, Jieqiong Tan, and Guillermina Garcia
- Subjects
Male ,0301 basic medicine ,Programmed cell death ,Nerve Tissue Proteins ,Tissue Banks ,Biochemistry ,Gene Expression Regulation, Enzymologic ,Histone Deacetylases ,Cell Line ,Histones ,Mice ,03 medical and health sciences ,Mesencephalon ,Autophagy ,Animals ,Humans ,Epigenetics ,Enzyme Inhibitors ,Molecular Biology ,Aged ,Histone Acetyltransferases ,biology ,Histone deacetylase 2 ,Dopaminergic Neurons ,Molecular Bases of Disease ,Acetylation ,Parkinson Disease ,Cell Biology ,Histone acetyltransferase ,Molecular biology ,HDAC1 ,Cell biology ,Histone Deacetylase Inhibitors ,030104 developmental biology ,Histone ,biology.protein ,Female ,RNA Interference ,Protein Processing, Post-Translational - Abstract
Parkinson disease (PD) is the most common age-dependent neurodegenerative movement disorder. Accumulated evidence indicates both environmental and genetic factors play important roles in PD pathogenesis, but the potential interaction between environment and genetics in PD etiology remains largely elusive. Here, we report that PD-related neurotoxins induce both expression and acetylation of multiple sites of histones in cultured human cells and mouse midbrain dopaminergic (DA) neurons. Consistently, levels of histone acetylation are markedly higher in midbrain DA neurons of PD patients compared to those of their matched control individuals. Further analysis reveals that multiple histone deacetylases (HDACs) are concurrently decreased in 1-methyl-4-phenylpyridinium (MPP(+))-treated cells and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-treated mouse brains, as well as midbrain tissues of human PD patients. Finally, inhibition of histone acetyltransferase (HAT) protects, whereas inhibition of HDAC1 and HDAC2 potentiates, MPP(+)-induced cell death. Pharmacological and genetic inhibition of autophagy suppresses MPP(+)-induced HDACs degradation. The study reveals that PD environmental factors induce HDACs degradation and histone acetylation increase in DA neurons via autophagy and identifies an epigenetic mechanism in PD pathogenesis.
- Published
- 2016
10. Biochemical Characterization and Substrate Specificity of Autophagin-2 from the Parasite Trypanosoma cruzi
- Author
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Aleksandra Wilk, Robert Vidmar, Vito Turk, Marcin Poreba, Boris Turk, Agata Borowik, Jelena Rajković, Marcin Drag, Dejan Caglič, and Guy S. Salvesen
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Proteases ,Trypanosoma cruzi ,Proteolysis ,Molecular Sequence Data ,Biology ,Biochemistry ,Substrate Specificity ,chemistry.chemical_compound ,parasitic diseases ,Autophagy ,medicine ,Aromatic amino acids ,Animals ,Humans ,Amino Acid Sequence ,Molecular Biology ,Peptide sequence ,chemistry.chemical_classification ,Sequence Homology, Amino Acid ,medicine.diagnostic_test ,Cell Biology ,Cysteine protease ,Amino acid ,Cysteine Endopeptidases ,Kinetics ,chemistry ,Protein Synthesis and Degradation ,Autophagin ,Cysteine - Abstract
The genome of the parasite Trypanosoma cruzi encodes two copies of autophagy-related cysteine proteases, Atg4.1 and Atg4.2. T. cruzi autophagin-2 (TcAtg4.2) carries the majority of proteolytic activity and is responsible for processing Atg8 proteins near the carboxyl terminus, exposing a conserved glycine. This enables progression of autophagy and differentiation of the parasite, which is required for successful colonization of humans. The mechanism of substrate hydrolysis by Atg4 was found to be highly conserved among the species as critical mutations in the TcAtg4.2, including mutation of the conserved Gly-244 residue in the hinge region enabling flexibility of the regulatory loop, and deletion of the regulatory loop, completely abolished processing capacity of the mutants. Using the positional scanning-substrate combinatorial library (PS-SCL) we determined that TcAtg4.2 tolerates a broad spectrum of amino acids in the P4 and P3 positions, similar to the human orthologue autophagin-1 (HsAtg4B). In contrast, both human and trypanosome Atg4 orthologues exhibited exclusive preference for aromatic amino acid residues in the P2 position, and for Gly in the P1 position, which is absolutely conserved in the natural Atg8 substrates. Using an extended P2 substrate library, which also included the unnatural amino acid cyclohexylalanine (Cha) derivative of Phe, we generated highly selective tetrapeptide substrates acetyl-Lys-Lys-Cha-Gly-AFC (Ac-KKChaG-AFC) and acetyl-Lys-Thr-Cha-Gly-AFC (Ac-KTChaG-AFC). Althoughthese substrates were cleaved by cathepsins, making them unsuitable for analysis of complex cellular systems, they were recognized exclusively by TcAtg4.2, but not by HsAtg4B nor by the structurally related human proteases SENP1, SENP2, and UCH-L3.
- Published
- 2015
11. Staphylococcal SplB Serine Protease Utilizes a Novel Molecular Mechanism of Activation
- Author
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Natalia Stach, Jan Potempa, Benedykt Wladyka, Marcin Drag, Adam Dubin, Paweł Mak, Anna Czarna, Grzegorz M. Popowicz, Michal Zdzalik, Justyna Stec-Niemczyk, Przemyslaw Cichon, Grzegorz Dubin, Guy S. Salvesen, and Katarzyna Pustelny
- Subjects
Models, Molecular ,crystal structure ,Staphylococcus aureus ,Stereochemistry ,serine protease ,medicine.medical_treatment ,Chymotrypsinogen ,Protein Sorting Signals ,Crystallography, X-Ray ,Biochemistry ,Structure-Activity Relationship ,Bacterial Proteins ,Catalytic Domain ,Zymogen ,medicine ,Chymotrypsin ,signal peptide ,zymogen activation ,Molecular Biology ,Serine protease ,Enzyme Precursors ,Protease ,biology ,Chemistry ,Proteolytic enzymes ,Active site ,protease ,Hydrogen Bonding ,Cell Biology ,Protein Structure, Tertiary ,Crystal Structure ,Enzyme Inactivation ,Serine Protease ,Staphylococcus Aureus ,Signal Peptide ,Zymogen Activation ,enzyme inactivation ,Zymogen activation ,Protein Structure and Folding ,biology.protein ,Serine Proteases - Abstract
Staphylococcal SplB protease belongs to the chymotrypsin family. Chymotrypsin zymogen is activated by proteolytic processing at the N terminus, resulting in significant structural rearrangement at the active site. Here, we demonstrate that the molecular mechanism of SplB protease activation differs significantly and we characterize the novel mechanism in detail. Using peptide and protein substrates we show that the native signal peptide, or any N-terminal extension, has an inhibitory effect on SplB. Only precise N-terminal processing releases the full proteolytic activity of the wild type analogously to chymotrypsin. However, comparison of the crystal structures of mature SplB and a zymogen mimic show no rearrangement at the active site whatsoever. Instead, only the formation of a unique hydrogen bond network, distant form the active site, by the new N-terminal glutamic acid of mature SplB is observed. The importance of this network and influence of particular hydrogen bond interactions at the N terminus on the catalytic process is demonstrated by evaluating the kinetics of a series of mutants. The results allow us to propose a consistent model where changes in the overall protein dynamics rather than structural rearrangement of the active site are involved in the activation process.
- Published
- 2014
12. Structural and functional determination of homologs of the Mycobacterium tuberculosis N-acetylglucosamine-6-phosphate deacetylase (NagA)
- Author
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Ahangar, Mohd Syed, primary, Furze, Christopher M., additional, Guy, Collette S., additional, Cooper, Charlotte, additional, Maskew, Kathryn S., additional, Graham, Ben, additional, Cameron, Alexander D., additional, and Fullam, Elizabeth, additional
- Published
- 2018
- Full Text
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13. Extensive peptide and natural protein substrate screens reveal that mouse caspase-11 has much narrower substrate specificity than caspase-1
- Author
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Ramirez, Monica L. Gonzalez, primary, Poreba, Marcin, additional, Snipas, Scott J., additional, Groborz, Katarzyna, additional, Drag, Marcin, additional, and Salvesen, Guy S., additional
- Published
- 2018
- Full Text
- View/download PDF
14. Activity, Specificity, and Probe Design for the Smallpox Virus Protease K7L
- Author
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Robert C. Liddington, Naran Gombosuren, Marcin Drag, Arnold C. Satterthwait, Alexander E. Aleshin, Guy S. Salvesen, Ge Wei, Alex Y. Strongin, and Jowita Mikolajczyk
- Subjects
Proteases ,viruses ,medicine.medical_treatment ,Amino Acid Motifs ,Biology ,complex mixtures ,Antiviral Agents ,Biochemistry ,Cell Line ,Gene product ,Viral Proteins ,Viral life cycle ,Peptide Library ,Cricetinae ,medicine ,Animals ,Protease Inhibitors ,Smallpox virus ,Peptide library ,Molecular Biology ,Protease ,virus diseases ,RNA ,Variola virus ,Cell Biology ,NS2-3 protease ,Drug Design ,Molecular Probes ,Enzymology ,Peptide Hydrolases ,Smallpox - Abstract
The K7L gene product of the smallpox virus is a protease implicated in the maturation of viral proteins. K7L belongs to protease Clan CE, which includes distantly related cysteine proteases from eukaryotes, pathogenic bacteria, and viruses. Here, we describe its recombinant high level expression, biochemical mechanism, substrate preference, and regulation. Earlier studies inferred that the orthologous I7L vaccinia protease cleaves at an AG-X motif in six viral proteins. Our data for K7L suggest that the AG-X motif is necessary but not sufficient for optimal cleavage activity. Thus, K7L requires peptides extended into the P7 and P8 positions for efficient substrate cleavage. Catalytic activity of K7L is substantially enhanced by homodimerization, by the substrate protein P25K as well as by glycerol. RNA and DNA also enhance cleavage of the P25K protein but not of synthetic peptides, suggesting that nucleic acids augment the interaction of K7L with its protein substrate. Library-based peptide preference analyses enabled us to design an activity-based probe that covalently and selectively labels K7L in lysates of transfected and infected cells. Our study thus provides proof-of-concept for the design of inhibitors and probes that may contribute both to a better understanding of the role of K7L in the virus life cycle and the design of novel anti-virals.
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- 2012
15. Structural and functional determination of homologs of the Mycobacterium tuberculosis N-acetylglucosamine-6-phosphate deacetylase (NagA)
- Author
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Charlotte Cooper, Mohammad Syed Ahangar, Kathryn S. Maskew, Ben Graham, Collette S. Guy, Christopher M. Furze, Elizabeth Fullam, and Alexander D. Cameron
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0301 basic medicine ,chemistry.chemical_classification ,Amidohydrolase ,biology ,Mycobacterium smegmatis ,Active site ,Cell Biology ,biology.organism_classification ,Biochemistry ,QR ,03 medical and health sciences ,chemistry.chemical_compound ,030104 developmental biology ,Enzyme ,chemistry ,Acetylation ,Hydrolase ,biology.protein ,Peptidoglycan ,Molecular Biology ,Cysteine - Abstract
The Mycobacterium tuberculosis (Mtb) pathogen encodes a GlcNAc-6-phosphate deacetylase enzyme, NagA (Rv3332), that belongs to the amidohydrolase superfamily. NagA enzymes catalyze the deacetylation of GlcNAc-6-phosphate (GlcNAc6P) to glucosamine-6-phosphate (GlcN6P). NagA is a potential antitubercular drug target because it represents the key enzymatic step in the generation of essential amino-sugar precursors required for Mtb cell wall biosynthesis and also influences recycling of cell wall peptidoglycan fragments. Here, we report the structural and functional characterization of NagA from Mycobacterium smegmatis (MSNagA) and Mycobacterium marinum (MMNagA), close relatives of Mtb. Using a combination of X-ray crystallography, site-directed mutagenesis, and biochemical and biophysical assays, we show that these mycobacterial NagA enzymes are selective for GlcNAc6P. Site-directed mutagenesis studies revealed crucial roles of conserved residues in the active site that underpin stereoselective recognition, binding, and catalysis of substrates. Moreover, we report the crystal structure of MSNagA in both ligand-free form and in complex with the GlcNAc6P substrate at 2.6 and 2.0 A resolutions, respectively. The GlcNAc6P complex structure disclosed the precise mode of GlcNAc6P binding and the structural framework of the active site, including two divalent metals located in the α/β binuclear site. Furthermore, we observed a cysteine residue located on a flexible loop region that occludes the active site. This cysteine is unique to mycobacteria and may represent a unique subsite for targeting mycobacterial NagA enzymes. Our results provide critical insights into the structural and mechanistic properties of mycobacterial NagA enzymes having an essential role in amino-sugar and nucleotide metabolism in mycobacteria.
- Published
- 2018
16. Vaccinia Virus Protein F1L Is a Caspase-9 Inhibitor
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John C. Reed, Chaofang Jin, Eric Chi-Wang Yu, Guy S. Salvesen, Dayong Y. Zhai, Robert C. Liddington, Chung-wei Shiau, Kate Welsh, and Lili Chen
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Proteases ,Programmed cell death ,Apoptosis ,Vaccinia virus ,Mitochondrion ,Caspase 8 ,Biochemistry ,Viral Proteins ,Animals ,Humans ,Molecular Biology ,Caspase ,Caspase-9 ,biology ,Intrinsic apoptosis ,Cell Biology ,Caspase Inhibitors ,Molecular biology ,Caspase 9 ,Recombinant Proteins ,Mitochondria ,Protein Structure, Tertiary ,Proto-Oncogene Proteins c-bcl-2 ,biology.protein ,Cattle ,HeLa Cells ,Protein Binding - Abstract
Apoptosis plays important roles in host defense, including the elimination of virus-infected cells. The executioners of apoptosis are caspase family proteases. We report that vaccinia virus-encoded F1L protein, previously recognized as anti-apoptotic viral Bcl-2 family protein, is a caspase-9 inhibitor. F1L binds to and specifically inhibits caspase-9, the apical protease in the mitochondrial cell death pathway while failing to inhibit other caspases. In cells, F1L inhibits apoptosis and proteolytic processing of caspases induced by overexpression of caspase-9 but not caspase-8. An N-terminal region of F1L preceding the Bcl-2-like fold accounts for caspase-9 inhibition and significantly contributes to the anti-apoptotic activity of F1L. Viral F1L thus provides the first example of caspase inhibition by a Bcl-2 family member; it functions both as a suppressor of proapoptotic Bcl-2 family proteins and as an inhibitor of caspase-9, thereby neutralizing two sequential steps in the mitochondrial cell death pathway.
- Published
- 2010
17. Streptolysin O Promotes Group A Streptococcus Immune Evasion by Accelerated Macrophage Apoptosis
- Author
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John C. Timmer, Anjuli M. Timmer, Morgan A. Pence, Mariam Ghochani, Victor Nizet, Terrence G. Frey, Michael Karin, Li-Chung Hsu, and Guy S. Salvesen
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Programmed cell death ,Time Factors ,Streptococcus pyogenes ,Apoptosis ,Biology ,Biochemistry ,Cell Line ,Microbiology ,Mice ,Bacterial Proteins ,Animals ,Humans ,Macrophage ,Molecular Biology ,Membrane Potential, Mitochondrial ,Macrophages ,Cytochrome c ,Mechanisms of Signal Transduction ,Intrinsic apoptosis ,Cytochromes c ,Cell Biology ,Mitochondria ,Cell biology ,Enzyme Activation ,Mice, Inbred C57BL ,Caspases ,Streptolysins ,biology.protein ,Female ,Cytokine secretion ,Streptolysin ,Cytolysin - Abstract
Group A Streptococcus (GAS) is a leading human bacterial pathogen capable of producing invasive infections even in previously healthy individuals. As frontline components of host innate defense, macrophages play a key role in control and clearance of GAS infections. We find GAS induces rapid, dose-dependent apoptosis of primary and cultured macrophages and neutrophils. The cell death pathway involves apoptotic caspases, is partly dependent on caspase-1, and requires GAS internalization by the phagocyte. Analysis of GAS virulence factor mutants, heterologous expression, and purified toxin studies identified the pore-forming cytolysin streptolysin O (SLO) as necessary and sufficient for the apoptosis-inducing phenotype. SLO-deficient GAS mutants induced less macrophage apoptosis in vitro and in vivo, allowed macrophage cytokine secretion, and were less virulent in a murine systemic infection model. Ultrastructural evidence of mitochondrial membrane remodeling, coupled with loss of mitochondrial depolarization and cytochrome c release, suggests a direct attack of the toxin initiates the intrinsic apoptosis pathway. A general caspase inhibitor blocked SLO-induced apoptosis and enhanced macrophage killing of GAS. We conclude that accelerated, caspase-dependent macrophage apoptosis induced by the pore-forming cytolysin SLO contributes to GAS immune evasion and virulence.
- Published
- 2009
18. Cysteine cathepsin strigger caspase-dependent cell death through cleavage of bid and antiapoptotic Bcl-2 homologues
- Author
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Lea Bojič, Saška Ivanova, Ana Petelin, Vito Turk, Boris Turk, Gabriela Droga-Mazovec, Veronika Stoka, Urska Repnik, Rok Romih, and Guy S. Salvesen
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Programmed cell death ,bcl-X Protein ,Apoptosis ,X-Linked Inhibitor of Apoptosis Protein ,Mitochondrion ,Inhibitor of apoptosis ,Biochemistry ,03 medical and health sciences ,0302 clinical medicine ,Proto-Oncogene Proteins ,Humans ,Protease Inhibitors ,Molecular Biology ,Caspase ,030304 developmental biology ,Cathepsin ,0303 health sciences ,biology ,Bcl-2-Like Protein 11 ,Membrane Proteins ,Cell Biology ,Cysteine protease ,Molecular biology ,Cathepsins ,3. Good health ,Cell biology ,XIAP ,Mitochondria ,bcl-2 Homologous Antagonist-Killer Protein ,Proto-Oncogene Proteins c-bcl-2 ,030220 oncology & carcinogenesis ,biology.protein ,Myeloid Cell Leukemia Sequence 1 Protein ,Caco-2 Cells ,Apoptosis Regulatory Proteins ,Lysosomes ,BH3 Interacting Domain Death Agonist Protein - Abstract
As a model for defining the role of lysosomal cathepsins in apoptosis, we characterized the action of the lysosomotropic agent LeuLeuOMe using distinct cellular models. LeuLeuOMe induces lysosomal membrane permeabilization, resulting in release of lysosomal cathepsins that cleave the proapoptotic Bcl-2 family member Bid and degrade the antiapoptotic member Bcl-2, Bcl-xL, or Mcl-1. The papain-like cysteine protease inhibitor E-64d largely prevented apoptosis, Bid cleavage, and Bcl-2/Bcl-xL/Mcl-1 degradation. The pancaspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp(OMe)fluoromethyl ketone failed to prevent Bid cleavage and degradation of anti-apoptotic Bcl-2 homologues but substantially decreased cell death, suggesting that cathepsin-mediated apoptosis in these cellular models mostly follows a caspase-dependent pathway. Moreover, in vitro experiments showed that one or more of the cysteine cathepsins B, L, S, K, and H could cleave Bcl-2, Bcl-xL, Mcl-1, Bak, and BimEL, whereas no Bax cleavage was observed. On the basis of inhibitor studies, we demonstrate that lysosomal disruption triggered by LeuLeuOMe occurs before mitochondrial damage. We propose that degradation of anti-apoptotic Bcl-2 family members by lysosomal cathepsins synergizes with cathepsin-mediated activation of Bid to trigger a mitochondrial pathway to apoptosis. Moreover, XIAP (X-chromosome-linked inhibitor of apoptosis) was also found to be a target of cysteine cathepsins, suggesting that cathepsins can mediate caspase-dependent apoptosis also downstream of mitochondria.
- Published
- 2008
- Full Text
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19. Small Ubiquitin-related Modifier (SUMO)-specific Proteases
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Marcin Drag, John T. Cao, Guy S. Salvesen, Miklós Békés, Ze'ev Ronai, and Jowita Mikolajczyk
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Proteases ,SENP1 ,biology ,Chemistry ,SUMO protein ,SUMO enzymes ,Cell Biology ,environment and public health ,Biochemistry ,Endopeptidase ,Isopeptidase activity ,Endopeptidase activity ,Ubiquitin ,biology.protein ,Molecular Biology - Abstract
SENPs are proteases that participate in the regulation of SUMOylation by generating mature small ubiquitin-related modifiers (SUMO) for protein conjugation (endopeptidase activity) and removing conjugated SUMO from targets (isopeptidase activity). Using purified recombinant catalytic domains of 6 of the 7 human SENPs, we demonstrate the specificity of their respective activities on SUMO-1, -2, and -3. The primary mode of recognition of substrates is via the SUMO domain, and the C-terminal tails direct endopeptidase specificity. Broadly speaking, SENP1 is the most efficient endopeptidase, whereas SENP2 and -5-7 have substantially higher isopeptidase than endopeptidase activities. We developed fluorogenic tetrapeptide substrates that are cleaved by SENPs, enabling us to characterize the environmental profiles of each enzyme. Using these synthetic substrates we reveal that the SUMO domain enhances catalysis of SENP1, -2, -5, -6, and -7, demonstrating substrate-induced activation of SENPs by SUMOs.
- Published
- 2007
20. The Human Anti-apoptotic Proteins cIAP1 and cIAP2 Bind but Do Not Inhibit Caspases
- Author
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Brendan P. Eckelman and Guy S. Salvesen
- Subjects
Immunoblotting ,Molecular Sequence Data ,Caspase 2 ,Apoptosis ,X-Linked Inhibitor of Apoptosis Protein ,Inhibitor of apoptosis ,Biochemistry ,Cell Line ,Inhibitor of Apoptosis Proteins ,Escherichia coli ,Humans ,Amino Acid Sequence ,Enzyme Inhibitors ,Molecular Biology ,Caspase ,Caspase 7 ,Sequence Homology, Amino Acid ,biology ,Caspase 3 ,NLRP1 ,Cell Biology ,Caspase Inhibitors ,Caspase 9 ,Recombinant Proteins ,Protein Structure, Tertiary ,XIAP ,Cell biology ,Kinetics ,Caspases ,biology.protein ,Caspase 10 ,Baculoviral IAP repeat-containing protein 3 ,Protein Binding - Abstract
cIAPs (cellular inhibitor of apoptosis proteins) 1 and 2 are able to regulate apoptosis when ectopically expressed in recipient cells and probably also in vivo. Previous work suggested that this is at least partially due to direct caspase inhibition, mediated by two of the three baculovirus IAP repeat (BIR) domains that are contained in these proteins. In support of this we show that the BIR domains 2 and 3 of the two cIAPs are able to bind caspases-7 and -9. However, we demonstrate that neither of these BIR domains is able to inhibit caspases because of critical substitutions in the regions that target caspase inhibition in the X-linked IAP, a tight binding caspase inhibitor. The cIAP BIR domains can be converted to tight binding caspase inhibitors by substituting these critical residues with XIAP residues. Thus, cIAPs maintain protein scaffolds suitable for direct caspase inhibition but have lost or never acquired specific caspase inhibitory interaction sites. Consequently, although the binding function of the cIAP BIRs may be important for their physiologic function, caspase inhibition is not.
- Published
- 2006
21. Yersinia Phosphatase Induces Mitochondrially Dependent Apoptosis of T Cells
- Author
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Jean-Bernard Denault, Shane Bruckner, Souad Rhamouni, Andres Alonso, Tomas Mustelin, Lutz Tautz, Guy S. Salvesen, and Barbara Becattini
- Subjects
Programmed cell death ,Yersinia pestis ,T-Lymphocytes ,bcl-X Protein ,Apoptosis ,Cell Separation ,DNA Fragmentation ,Protein tyrosine phosphatase ,Transfection ,Biochemistry ,Jurkat cells ,Jurkat Cells ,Immune system ,Humans ,Annexin A5 ,Phosphorylation ,Molecular Biology ,Caspase ,Cell Death ,biology ,Cell Biology ,Flow Cytometry ,Molecular biology ,Mitochondria ,Nucleosomes ,Cell biology ,Enzyme Activation ,Proto-Oncogene Proteins c-bcl-2 ,Caspases ,Mutagenesis, Site-Directed ,biology.protein ,Protein Tyrosine Phosphatases ,Signal transduction ,Tyrosine kinase ,Bacterial Outer Membrane Proteins ,HeLa Cells ,Signal Transduction - Abstract
To evade the immune system, the etiologic agent of plague, Yersinia pestis, injects an exceptionally active tyrosine phosphatase called YopH into host cells using a type III secretion system. We recently reported that YopH acutely inhibits T cell antigen receptor signaling by dephosphorylating the Lck tyrosine kinase. Here, we show that prolonged presence of YopH in primary T cells or Jurkat T leukemia cells causes apoptosis, detected by annexin V binding, mitochondrial breakdown, caspase activation, and internucleosomal fragmentation. YopH also causes cell death when expressed in HeLa cells, and this cell death was inhibited by YopH-specific small molecule inhibitors. Cell death induced by YopH was also prevented by caspase inhibition or co-expression of Bcl-xL. We conclude that YopH not only paralyzes T cells acutely, but also ensures that the cells will not recover to induce a protective immune response but instead undergo mitochondrially regulated programmed cell death.
- Published
- 2005
22. Neutralization of Smac/Diablo by Inhibitors of Apoptosis (IAPs)
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Fiona L. Scott, Guy S. Salvesen, Rebecca A. Csomos, John C. Wilkinson, Colin S. Duckett, and Amanda S. Wilkinson
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musculoskeletal diseases ,biology ,Effector ,viruses ,Caspase independent ,Cell Biology ,Inhibitory postsynaptic potential ,Biochemistry ,Neutralization ,XIAP ,Cell biology ,body regions ,Ubiquitin ,Apoptosis ,biology.protein ,biological phenomena, cell phenomena, and immunity ,Molecular Biology ,Caspase - Abstract
Numerous members of the IAP family can suppress apoptotic cell death in physiological settings. Whereas certain IAPs directly inhibit caspases, the chief proteolytic effectors of apoptosis, the protective effects of other IAPs do not correlate well with their caspase inhibitory activities, suggesting the involvement of alternative cytoprotective abilities. To examine this issue, we have characterized the protective effects of an ancestral, baculoviral IAP (Op-IAP) in mammalian cells. We show that although Op-IAP potently inhibited Bax-mediated apoptosis in human cells, Op-IAP failed to directly inhibit mammalian caspases. However, Op-IAP efficiently bound the IAP antagonist Smac/Diablo, thereby preventing Smac/Diablo-mediated inhibition of cellular IAPs. Whereas reduction of Smac/Diablo protein levels in the absence of Op-IAP prevented Bax-mediated apoptosis, overexpression of Smac/Diablo neutralized Op-IAP-mediated protection, and an Op-IAP variant unable to bind Smac/Diablo failed to prevent apoptosis. Finally, Op-IAP catalyzed the ubiquitination of Smac/Diablo, an activity that contributed to Op-IAP-mediated inhibition of apoptosis. These data show that cytoprotective IAPs can inhibit apoptosis through the neutralization of IAP antagonists, rather than by directly inhibiting caspases.
- Published
- 2004
23. An IAP-IAP Complex Inhibits Apoptosis
- Author
-
John C. Reed, Hua Zou, Shu-ichi Matsuzawa, Kazuya Okada, Kate Welsh, Hiroyouki Marusawa, Dario C. Altieri, Temesgen Samuel, Robert C. Armstrong, Fang Xia, Takehiko Dohi, Guy S. Salvesen, and Casey E. Wilford
- Subjects
Time Factors ,Survivin ,viruses ,Apoptosis ,Biochemistry ,Inhibitor of Apoptosis Proteins ,Mice ,Transgenes ,Glutathione Transferase ,Inhibitor of apoptosis domain ,Caspase-9 ,Cell Death ,Caspase 9 ,Recombinant Proteins ,Neoplasm Proteins ,Cell biology ,XIAP ,Cysteine Endopeptidases ,Caspases ,biological phenomena, cell phenomena, and immunity ,Baculoviridae ,Microtubule-Associated Proteins ,Protein Binding ,musculoskeletal diseases ,Proteasome Endopeptidase Complex ,Programmed cell death ,Immunoblotting ,Mice, Transgenic ,X-Linked Inhibitor of Apoptosis Protein ,Biology ,Transfection ,Inhibitor of apoptosis ,Cell Line ,Multienzyme Complexes ,Cell Line, Tumor ,Animals ,Humans ,Molecular Biology ,Dose-Response Relationship, Drug ,Ubiquitin ,Proteins ,Cell Biology ,Fibroblasts ,Precipitin Tests ,body regions ,Gene Expression Regulation ,biology.protein ,Baculoviral IAP repeat-containing protein 3 - Abstract
Regulators of apoptosis are thought to work in concert, but the molecular interactions of this process are not understood. Here, we show that in response to cell death stimulation, survivin, a member of the inhibitor of apoptosis (IAP) gene family, associates with another IAP protein, XIAP, via conserved baculovirus IAP repeats. Formation of a survivin-XIAP complex promotes increased XIAP stability against ubiquitination/proteasomal destruction and synergistic inhibition of apoptosis, which is abolished in XIAP(-/-) cells. Therefore, orchestration of an IAP-IAP complex regulates apoptosis.
- Published
- 2004
24. Selective Disruption of Lysosomes in HeLa Cells Triggers Apoptosis Mediated by Cleavage of Bid by Multiple Papain-like Lysosomal Cathepsins
- Author
-
John C. Reed, Vito Turk, Kristina Oresic, Boris Turk, Tina Cirman, Richard M. Myers, Guy S. Salvesen, and Gabriela Droga Mazovec
- Subjects
Models, Molecular ,Proteases ,Apoptosis ,Biology ,Transfection ,Caspase 8 ,Cleavage (embryo) ,Models, Biological ,Biochemistry ,Protein Structure, Secondary ,Cell Line ,Mice ,Cytosol ,Cell Line, Tumor ,Lysosome ,Papain ,medicine ,Animals ,Humans ,Molecular Biology ,Cathepsin ,Myocardium ,Cytochrome c ,Temperature ,Cytochromes c ,Cell Biology ,Hydrogen-Ion Concentration ,Flow Cytometry ,Cathepsins ,Recombinant Proteins ,Mitochondria ,Cell biology ,Protein Transport ,medicine.anatomical_structure ,Liver ,Caspases ,biology.protein ,Carrier Proteins ,Lysosomes ,BH3 Interacting Domain Death Agonist Protein ,HeLa Cells - Abstract
Increasing evidence suggests that lysosomal proteases are actively involved in apoptosis. Using HeLa cells as the model system, we show that selective lysosome disruption with L-leucyl-L-leucine methyl ester results in apoptosis, characterized by translocation of lysosomal proteases into the cytosol and by the cleavage of a proapoptotic Bcl-2-family member Bid. Apoptosis and Bid cleavage, but not translocation of lysosomal proteases to the cytosol, could be prevented by 15 microM L-trans-epoxysuccinyl(OEt)-Leu-3-methylbutylamide, an inhibitor of papain-like cysteine proteases. Incubation of cells with 15 microM N-benzoyloxycarbonyl-VAD-fluoromethyl ketone prevented apoptosis but not Bid cleavage, suggesting that cathepsin-mediated apoptosis in this system is caspase-dependent. In vitro experiments performed at neutral pH showed that papain-like cathepsins B, H, L, S, and K cleave Bid predominantly at Arg(65) or Arg(71). No Bid cleavage was observed with cathepsins C and X or the aspartic protease cathepsin D. Incubation of full-length Bid treated with cathepsins B, H, L, and S resulted in rapid cytochrome c release from isolated mitochondria. Thus, Bid may be an important mediator of apoptosis induced by lysosomal disruption.
- Published
- 2004
25. Human Caspase-7 Activity and Regulation by Its N-terminal Peptide
- Author
-
Guy S. Salvesen and Jean-Bernard Denault
- Subjects
DNA, Complementary ,Time Factors ,Recombinant Fusion Proteins ,Immunoblotting ,Molecular Sequence Data ,Apoptosis ,DNA Fragmentation ,Plasma protein binding ,Transfection ,Biochemistry ,Caspase 7 ,Cell Line ,Substrate Specificity ,Cytosol ,In vivo ,Catalytic Domain ,Zymogen ,Animals ,Humans ,Amino Acid Sequence ,Molecular Biology ,Peptide sequence ,Caspase ,Cell Nucleus ,Sequence Homology, Amino Acid ,biology ,Caspase 3 ,Cell Biology ,Recombinant Proteins ,Protein Structure, Tertiary ,Cell biology ,Kinetics ,Microscopy, Fluorescence ,Caspases ,Zymogen activation ,COS Cells ,Mutation ,biology.protein ,Electrophoresis, Polyacrylamide Gel ,Ectopic expression ,Peptides ,Dimerization ,Protein Binding - Abstract
Central to the execution phase of apoptosis are the two closely related caspase-3 and -7. They share common substrate specificity and structure, but differ completely in the sequence of their respective N-terminal regions including their N-peptides, a 23-28 residue segment that are removed during zymogen activation. We show that the N-peptide of caspase-7 plays no role in the fundamental activation or properties of the active protease in vitro. However, the N-peptide modifies the properties of caspase-7 in vivo. In ectopic expression experiments, caspase-7 constructs with no N-peptide are far more lethal than constructs that have an uncleavable peptide. Moreover, the N-peptide of caspase-7 must be removed before efficient activation of the zymogen can occur in vivo. These disparate requirements for the N-peptide argue that it serves to physically sequester the caspase-7 zymogen in a cytosolic location that prevents access by upstream activators (caspase-8, -9, and -10). The N-peptide must first be removed, probably by caspase-3, before efficient conversion and activation of the zymogen can occur in vivo.
- Published
- 2003
26. Ionomycin-activated Calpain Triggers Apoptosis
- Author
-
Andreas Holloschi, Hans Fritz, Shirley Gil-Parrado, Irmgard Assfalg-Machleidt, Eberhard Spiess, Ennes A. Auerswald, John C. Reed, Werner Machleidt, Oliver Popp, Amaury Ernesto Fernandez-Montalvan, Pablo Fuentes-Prior, Tobias Knoch, Guy S. Salvesen, Felix Bestvater, and Katherine Welsh
- Subjects
Programmed cell death ,biology ,Cytochrome c ,Bcl-2 family ,Calpain ,Cell Biology ,Biochemistry ,law.invention ,Cell biology ,chemistry.chemical_compound ,chemistry ,law ,Apoptosis ,Ionomycin ,Recombinant DNA ,biology.protein ,Molecular Biology ,Caspase - Abstract
Ubiquitous calpains (μ- and m-calpain) have been repeatedly implicated in apoptosis, but the underlying mechanism(s) remain(s) to be elucidated. We examined ionomycin-induced cell death in LCLC 103H cells, derived from a human large cell lung carcinoma. We detected hallmarks of apoptosis such as membrane blebbing, nuclear condensation, DNA ladder formation, caspase activation, and poly-(ADP-ribose)polymerase cleavage. Apoptosis was prevented by preincubation of the cells with the calpain inhibitor acetyl-calpastatin 27-peptide and the caspase inhibitor Z-DEVD-fmk, implicating both the calpains and caspases in the apoptotic process. The apoptotic events correlated in a calpastatin-inhibitable manner with Bid and Bcl-2 decrease and with activation of caspases-9, -3, and -7. In vitroboth ubiquitous calpains cleaved recombinant Bcl-2, Bid, and Bcl-xL at single sites truncating their N-terminal regions. Binding studies revealed diminished interactions of calpain-truncated Bcl-2 and Bid with immobilized intact Bcl-2 family proteins. Moreover, calpain-cleaved Bcl-2 and Bid induced cytochrome c release from isolated mitochondria. We conclude that ionomycin-induced calpain activation promotes decrease of Bcl-2 proteins thereby triggering the intrinsic apoptotic pathway.
- Published
- 2002
27. TRAF1 Is a Substrate of Caspases Activated during Tumor Necrosis Factor Receptor-α-induced Apoptosis
- Author
-
Henning R. Stennicke, Eugen Leo, John C. Reed, Kate Welsh, Quinn Deveraux, Christian Buchholtz, Guy S. Salvesen, and Shu-ichi Matsuzawa
- Subjects
Programmed cell death ,TRAF1 ,Apoptosis ,Lymphocyte Activation ,Biochemistry ,Tumor Cells, Cultured ,medicine ,Humans ,Staurosporine ,Lymphocytes ,fas Receptor ,Molecular Biology ,Caspase ,biology ,Tumor Necrosis Factor-alpha ,Intrinsic apoptosis ,NF-kappa B ,Proteins ,Cell Biology ,Fas receptor ,TNF Receptor-Associated Factor 1 ,Molecular biology ,Cell biology ,Caspases ,biology.protein ,Female ,Tumor necrosis factor alpha ,medicine.drug - Abstract
TRAF family proteins are signal-transducing adapter proteins that interact with the cytosolic domains of tumor necrosis factor (TNF) family receptors. Here we show that TRAF1 (but not TRAF2-6) is cleaved by certain caspases in vitro and during TNF-alpha- and Fas-induced apoptosis in vivo. (160)LEVD(163) was identified as the caspase cleavage site within TRAF1, generating two distinct fragments. Significant enhancement of TNF receptor-1 (CD120a)- and, to a lesser extent, Fas (CD95)-mediated apoptosis was observed when overexpressing the C-terminal TRAF1 fragment in HEK293T and HT1080 cells. The same fragment was capable of potently suppressing TNF receptor-1- and TRAF2-mediated nuclear factor-kappaB activation in reporter gene assays, providing a potential mechanism for the enhancement of TNF-mediated apoptosis. Cell death induced by other death receptor-independent stimuli such as cisplatin, staurosporine, and UV irradiation did not result in cleavage of TRAF1, and overexpression of the C-terminal TRAF1 fragment did not enhance cell death in these cases. TRAF1 cleavage was markedly reduced in cells that contain little procaspase-8 protein, suggesting that this apical protease in the TNF/Fas death receptor pathway is largely responsible. These data identify TRAF1 as a specific target of caspases activated during TNF- and Fas-induced apoptosis and illustrate differences in the repertoire of protease substrates cleaved during activation of different apoptotic pathways.
- Published
- 2001
28. Lysosomal Protease Pathways to Apoptosis
- Author
-
Magnus Abrahamson, Stanislaw Krajewski, Sharon L. Schendel, Hudson H. Freeze, Guy S. Salvesen, Lisa M. Ellerby, Dale E. Bredesen, Tae-Hyoung Kim, Scott J. Snipas, John C. Reed, Tina Cirman, Veronika Stoka, Vito Turk, Boris Turk, Dieter Brömme, and Xiao Ming Yin
- Subjects
Cathepsin ,Proteases ,biology ,NLRP1 ,Cytochrome c ,Caspase 2 ,Cell Biology ,Biochemistry ,Cell biology ,medicine.anatomical_structure ,Apoptosis ,Lysosome ,biology.protein ,medicine ,Molecular Biology ,Caspase - Abstract
We investigated the mechanism of lysosome-mediated cell death using purified recombinant pro-apoptotic proteins, and cell-free extracts from the human neuronal progenitor cell line NT2. Potential effectors were either isolated lysosomes or purified lysosomal proteases. Purified lysosomal cathepsins B, H, K, L, S, and X or an extract of mouse lysosomes did not directly activate either recombinant caspase zymogens or caspase zymogens present in an NT2 cytosolic extract to any significant extent. In contrast, a cathepsin L-related protease from the protozoan parasiteTrypanosoma cruzi, cruzipain, showed a measurable caspase activation rate. This demonstrated that members of the papain family can directly activate caspases but that mammalian lysosomal members of this family may have been negatively selected for caspase activation to prevent inappropriate induction of apoptosis. Given the lack of evidence for a direct role in caspase activation by lysosomal proteases, we hypothesized that an indirect mode of caspase activation may involve the Bcl-2 family member Bid. In support of this, Bid was cleaved in the presence of lysosomal extracts, at a site six residues downstream from that seen for pathways involving capase 8. Incubation of mitochondria with Bid that had been cleaved by lysosomal extracts resulted in cytochrome c release. Thus, cleavage of Bid may represent a mechanism by which proteases that have leaked from the lysosomes can precipitate cytochrome c release and subsequent caspase activation. This is supported by the finding that cytosolic extracts from mice ablated in the bid gene are impaired in the ability to release cytochrome c in response to lysosome extracts. Together these data suggest that Bid represents a sensor that allows cells to initiate apoptosis in response to widespread adventitious proteolysis.
- Published
- 2001
29. Cleavage of Automodified Poly(ADP-ribose) Polymerase during Apoptosis
- Author
-
Guy G. Poirier, El Bachir Affar, Marc Germain, Vishva M. Dixit, Guy S. Salvesen, and Damien D'Amours
- Subjects
biology ,medicine.diagnostic_test ,Poly ADP ribose polymerase ,Proteolysis ,Cell Biology ,Cleavage (embryo) ,Biochemistry ,Caspase 7 ,Molecular biology ,Apoptosis ,biology.protein ,medicine ,Staurosporine ,Molecular Biology ,Polymerase ,Caspase ,medicine.drug - Abstract
The abundant nuclear enzyme poly(ADP-ribose) polymerase (PARP) synthesizes poly(ADP-ribose) in response to DNA strand breaks. During almost all forms of apoptosis, PARP is cleaved by caspases, suggesting the crucial role of its inactivation. A few studies have also reported a stimulation of PARP during apoptosis. However, the role of PARP stimulation and cleavage during this cell death process remains poorly understood. Here, we measured the stimulation of endogenous poly(ADP-ribose) synthesis during VP-16-induced apoptosis in HL60 cells and found that PARP was cleaved by caspases at the time of its poly(ADP-ribosyl)ation. In vitro experiments showed that PARP cleavage by caspase-7, but not by caspase-3, was stimulated by its automodification by long and branched poly(ADP-ribose). Consistently, caspase-7 exhibited an affinity for poly(ADP-ribose), whereas caspase-3 did not. In addition, caspase-7 was activated and accumulated in the nucleus of HL60 cells in response to the VP-16 treatment. Furthermore, caspase-7 activation was concommitant with PARP cleavage in the caspase-3-deficient cell line MCF-7 in response to staurosporine treatment. These results strongly suggest that, in vivo, it is caspase-7 that is responsible for PARP cleavage and that poly(ADP-ribosyl)ation of PARP accelerates its proteolysis. Cleavage of the active form of caspase substrates could be a general feature of the apoptotic process, ensuring the rapid inactivation of stress signaling proteins.
- Published
- 1999
30. Caspase-9 Can Be Activated without Proteolytic Processing
- Author
-
John C. Reed, Henning R. Stennicke, Quinn Deveraux, Eric W. Humke, Guy S. Salvesen, and Vishva M. Dixit
- Subjects
Mutant ,Apoptosis ,Cytochrome c Group ,Biochemistry ,Cell Line ,law.invention ,Enzyme activator ,Cytosol ,Deoxyadenine Nucleotides ,Coumarins ,law ,Zymogen ,Humans ,Molecular Biology ,Caspase ,Caspase-9 ,Enzyme Precursors ,biology ,Cytochrome c ,Cell Biology ,Caspase 9 ,Recombinant Proteins ,Enzyme Activation ,Caspases ,Mutation ,Recombinant DNA ,biology.protein ,Oligopeptides - Abstract
The recombinant form of the proapoptotic caspase-9 purified following expression in Escherichia coli is processed at Asp315, but largely inactive; however, when added to cytosolic extracts of human 293 cells it is activated 2000-fold in the presence of cytochrome c and dATP. Thus, the characteristic activities of caspase-9 are context-dependent, and its activation may not recapitulate conventional caspase activation mechanisms. To explore this hypothesis we produced recombinant forms of procaspase-9 containing mutations that disabled one or both of the interdomain processing sites of the zymogen. These mutants were able to activate downstream caspases, but only in the presence of cytosolic factors. The mutant with both processing sites abolished had 10% of the activity of wild-type, and was able to support apoptosis, with equal vigor to wild-type, when transiently expressed in 293 cells. Thus caspase-9 has an unusually active zymogen that does not require proteolytic processing, but instead is dependent on cytosolic factors for expression of its activity.
- Published
- 1999
31. Granzyme B Mimics Apical Caspases
- Author
-
Xiaohe Yang, Reiner U. Jänicke, Henning R. Stennicke, Anu Srinivasan, Christopher J. Froelich, Baikun Wang, Prem Seth, Douglas R. Green, and Guy S. Salvesen
- Subjects
Proteases ,biology ,Caspase 3 ,Cell Biology ,Transfection ,Biochemistry ,Cell biology ,Granzyme B ,Granzyme ,Apoptosis ,Zymogen ,biology.protein ,Molecular Biology ,hormones, hormone substitutes, and hormone antagonists ,Caspase - Abstract
Granzyme B (GrB) is predicted to trigger apoptosis by activating preferred caspases, but the zymogens that are directly processed by the granzyme and the requirements for these interactions remain unclarified. We examined this dilemma by comparing the kinetics and pattern of GrB-mediated activation of the executioner caspase-7 in vitro and in vivo. GrB rapidly activates procaspase-7 in vitro by cleaving between the large and small subunits leaving the propeptide intact. During GrB-mediated apoptosis, the caspase-7 propeptide is removed and cleavage occurs between the subunits. Strikingly, caspase-7 is unprocessed in caspase-3-deficient MCF-7 cells exposed to GrB but is rapidly activated when the cells are solubilized. Transfection with caspase-3 restores the removal of the caspase-7 propeptide and the capacity of GrB to subsequently activate the caspase. The data suggest that GrB activates caspase-3, which then removes the propeptide of caspase-7 allowing activation by GrB. Thus GrB initiates the death pathway by processing the accessible caspase-3, and the caspase-7 propeptide regulates trans-activation of the zymogen by granzyme. As a consequence, two proteases, caspase-3 and GrB, are required to activate procaspase-7.
- Published
- 1998
32. Caspase-14 Is a Novel Developmentally Regulated Protease
- Author
-
Guy S. Salvesen, Vishva M. Dixit, Claudius Vincenz, Shimin Hu, and Scott J. Snipas
- Subjects
Programmed cell death ,Proteases ,DNA, Complementary ,Molecular Sequence Data ,Biochemistry ,Gene Expression Regulation, Enzymologic ,Cell Line ,Substrate Specificity ,Mice ,Zymogen ,Animals ,Caspase 14 ,Humans ,Amino Acid Sequence ,Cloning, Molecular ,Molecular Biology ,Caspase ,Sequence Homology, Amino Acid ,biology ,Intrinsic apoptosis ,Gene Expression Regulation, Developmental ,Cell Biology ,Molecular biology ,Cell biology ,Apoptosis ,Caspases ,biology.protein ,Signal transduction ,Signal Transduction - Abstract
Caspases are a family of cysteine proteases related to interleukin-1 converting enzyme (ICE) and represent the effector arm of the cell death pathway. The zymogen form of all caspases is composed of a prodomain plus large and small catalytic subunits. Herein we report the characterization of a novel caspase, MICE (for mini-ICE), also designated caspase-14, that possesses an unusually short prodomain and is highly expressed in embryonic tissues but absent from all adult tissues examined. In contrast to the other short prodomain caspases (caspase-3, caspase-6, and caspase-7), MICE preferentially associates with large prodomain caspases, including caspase-1, caspase-2, caspase-4, caspase-8, and caspase-10. Also unlike the other short prodomain caspases, MICE was not processed by multiple death stimuli including activation of members of the tumor necrosis factor receptor family and expression of proapoptotic members of the bcl-2 family. Surprisingly, however, overexpression of MICE itself induced apoptosis in MCF7 human breast cancer cells, which was attenuated by traditional caspase inhibitors.
- Published
- 1998
33. Pro-caspase-3 Is a Major Physiologic Target of Caspase-8
- Author
-
Quinn Deveraux, Henning R. Stennicke, Xiaohe Yang, Douglas R. Green, Beni B. Wolf, Qiao Zhou, Lisa M. Ellerby, Hwain Shin, Guy S. Salvesen, Dale E. Bredesen, H. Michael Ellerby, John C. Reed, Juliane M. Jürgensmeier, and Christopher J. Froelich
- Subjects
Apoptosis ,Caspase 3 ,Caspase 8 ,Models, Biological ,Biochemistry ,Granzymes ,Cytosol ,In vivo ,Caspase 10 ,Receptor ,Molecular Biology ,Caspase ,Caspase-9 ,Enzyme Precursors ,biology ,Serine Endopeptidases ,Cell Biology ,Molecular biology ,Caspase 9 ,Enzyme Activation ,Kinetics ,Caspases ,biology.protein ,Signal transduction ,Protein Processing, Post-Translational ,Signal Transduction - Abstract
The apoptotic signal triggered by ligation of members of the death receptor family is promoted by sequential activation of caspase zymogens. We show here that in a purified system, the initiator caspases-8 and -10 directly process the executioner pro-caspase-3 with activation rates (kcat/Km) of 8.7 x 10(5) and 2.8 x 10(5) M-1 s-1, respectively. These rates are of sufficient magnitude to indicate direct processing in vivo. Differentially processed forms of caspase-3 that accumulate during its activation have similar rates of activation, activities, and specificities. The pattern and rate of caspase-8 induced activation of pro-caspase-3 in cytosolic extracts was the same as in a purified system. Moreover, immunodepletion of a putative intermediary in the pathway to activation, pro-caspase-9, was without consequence. Taken together these data demonstrate that the initiator caspase-8 can directly activate pro-caspase-3 without the requirement for an accelerator. The in vitro data thus help to deconvolute previous in vivo transfection studies which have debated the role of a direct versus indirect transmission of the apoptotic signal generated by ligation of death receptors.
- Published
- 1998
34. A Single BIR Domain of XIAP Sufficient for Inhibiting Caspases
- Author
-
John C. Reed, Kate Welsh, Quinn Deveraux, Ryosuke Takahashi, Guy S. Salvesen, Ingo Tamm, and Nuria Assa-Munt
- Subjects
Apoptosis ,X-Linked Inhibitor of Apoptosis Protein ,Transfection ,Inhibitor of apoptosis ,Biochemistry ,Cell Line ,Escherichia coli ,Animals ,Humans ,Molecular Biology ,Caspase ,Caspase 7 ,Inhibitor of apoptosis domain ,biology ,Caspase 3 ,Proteins ,Cell Biology ,Molecular biology ,Peptide Fragments ,Recombinant Proteins ,XIAP ,Cysteine Endopeptidases ,Gene Expression Regulation ,HtrA serine peptidase 2 ,Caspases ,biology.protein ,IAP Family Protein ,Baculoviral IAP repeat-containing protein 3 - Abstract
The inhibitor of apoptosis proteins (IAPs) constitute an evolutionarily conserved family of homologous proteins that suppress apoptosis induced by multiple stimuli. Some IAP family proteins, including XIAP, cIAP-1, and cIAP-2, can bind and directly inhibit selected caspases, a group of intracellular cell death proteases. These caspase-inhibiting IAP family proteins all contain three tandem BIR domains followed by a RING zinc finger domain. To determine the structural basis for caspase inhibition by XIAP, we analyzed the effects of various fragments of this IAP family protein on caspase activity in vitro and on apoptosis suppression in intact cells. The RING domain of XIAP failed to inhibit the activity of recombinant caspases-3 or -7, whereas a fragment of XIAP encompassing the three tandem BIR domains potently inhibited these caspases in vitro and blocked Fas (CD95)-induced apoptosis when expressed in cells. Further dissection of the XIAP protein demonstrated that only the second of the three BIR domains (BIR2) was capable of binding and inhibiting these caspases. The apparent inhibition constants (K i) for BIR2-mediated inhibition of caspases-3 and -7 were 2–5 nm, indicating that this single BIR domain possesses potent anti-caspase activity. Expression of the BIR2 domain in cells also partially suppressed Fas-induced apoptosis and blocked cytochrome c-induced processing of caspase-9 in cytosolic extracts, whereas BIR1 and BIR3 did not. These findings identify BIR2 as the minimal caspase-inhibitory domain of XIAP and indicate that a single BIR domain can be sufficient for binding and inhibiting caspases.
- Published
- 1998
35. Zinc Is a Potent Inhibitor of the Apoptotic Protease, Caspase-3
- Author
-
Patrick J. Duriez, Henning R. Stennicke, Yusuf A. Hannun, Guy G. Poirier, Mirrian J. Smyth, David K. Perry, and Guy S. Salvesen
- Subjects
inorganic chemicals ,Zinc finger ,Protease ,medicine.diagnostic_test ,medicine.medical_treatment ,Proteolysis ,Poly ADP ribose polymerase ,Caspase 3 ,Cell Biology ,Biology ,Biochemistry ,Molecular biology ,law.invention ,Apoptosis ,law ,biological sciences ,medicine ,Recombinant DNA ,bacteria ,Molecular Biology ,IC50 - Abstract
The prevention of apoptosis by Zn2+ has generally been attributed to its inhibition of an endonuclease acting in the late phase of apoptosis. In this study we investigated the effect of Zn2+ on an earlier event in the apoptotic process, the proteolysis of the "death substrate" poly(ADP-ribose) polymerase (PARP). Pretreatment of intact Molt4 leukemia cells with micromolar concentrations of Zn2+ caused an inhibition of PARP proteolysis induced by the chemotherapeutic agent etoposide. Using a cell-free system consisting of purified bovine PARP as a substrate and an apoptotic extract or recombinant caspase-3 as the PARP protease, Zn2+ inhibited PARP proteolysis in the low micromolar range. To rule out an effect of Zn2+ on PARP, a protein with two zinc finger domains, we used recombinant caspase-3 and a chromogenic tetrapeptide substrate containing the caspase-3 cleavage site. In this system, Zn2+ inhibited caspase-3 with an IC50 of 0.1 microM. These results identify caspase-3 as a novel target of Zn2+ inhibition in apoptosis and suggest a regulatory role for Zn2+ in modulating the upstream apoptotic machinery.
- Published
- 1997
36. Target Protease Specificity of the Viral Serpin CrmA
- Author
-
Kim Orth, Vishva M. Dixit, Marta Muzio, Scott J. Snipas, Qiao Zhou, and Guy S. Salvesen
- Subjects
Caspase-9 ,Proteases ,Programmed cell death ,Protease ,biology ,medicine.medical_treatment ,Cell Biology ,Serpin ,Caspase 8 ,Biochemistry ,Cell biology ,Apoptosis ,biology.protein ,medicine ,Molecular Biology ,Caspase - Abstract
When ectopically expressed in animal cells, cytokine response modifier A (CrmA), a product of the cowpox virus, prevents programmed cell death initiated by a variety of stimuli. Since CrmA is a proteinase inhibitor, its target is probably a protease that promotes cell death. The identification of this target is crucial in delineating essential regulation points that modulate the apoptotic program. We have compared the kinetics of interaction of CrmA with five proteases that may play a role in apoptosis. Four of the proteases, all members of the caspase family, are inhibited with widely different rates and affinities ranging over 5 orders of magnitude. One is not inhibited at all under the experimental conditions. CrmA is quite selective in its ability to inhibit caspases, showing the highest affinity for interleukin-1beta-converting enzyme and the second highest for the caspase FLICE (Ki = 0.95 nM), identified as a component of the intracellular signaling complex recruited by ligation of the death receptor Fas. On the basis of comparative inhibitor kinetics, we propose that CrmA is unlikely to inhibit the caspases Yama, Mch2, or LAP3 in vivo but that its inhibition of FLICE is of a magnitude for this protease to be a key target of CrmA during Fas-mediated apoptosis. Therefore, our results support the hypothesis that FLICE catalyzes a crucial step in the promotion of cell death.
- Published
- 1997
37. FLICE Induced Apoptosis in a Cell-free System
- Author
-
Marta Muzio, Guy S. Salvesen, and Vishva M. Dixit
- Subjects
Programmed cell death ,Proteases ,biology ,Cell Biology ,Serpin ,Caspase 8 ,Fas receptor ,Biochemistry ,Molecular biology ,Apoptosis ,biology.protein ,Signal transduction ,Molecular Biology ,Caspase - Abstract
Engagement of CD95 or tumor necrosis factor 1 receptor (TNFR-1) by ligand or agonist antibodies is capable of activating the cell death program, the effector arm of which is composed of mammalian interleukin-1beta converting enzyme (ICE)-like cysteine proteases (designated caspases) that are related to the Caenorhabditis elegans death gene, CED-3. Caspases, unlike other mammalian cysteine proteases, cleave their substrates following aspartate residues. Furthermore, proteases belonging to this family exist as zymogens that in turn require cleavage at internal aspartate residues to generate the two-subunit active enzyme. As such, family members are capable of activating each other. Remarkably, both CD95 and TNFR-1 death receptors initiate apoptosis by recruiting a novel ICE/CED-3 family member, designated FLICE/MACH, to the receptor signaling complex. Therefore, FLICE/MACH represents the apical triggering protease in the cascade. Consistent with this, recombinant FLICE was found capable of proteolytically activating downstream caspases. Furthermore, CrmA, a pox virus-encoded serpin that inhibits Fas and tumor necrosis factor-induced cell death attenuates the ability of FLICE to activate downstream caspases.
- Published
- 1997
38. Inhibition of interleukin-1 beta converting enzyme by the cowpox virus serpin CrmA. An example of cross-class inhibition
- Author
-
Caroline A. Ray, A.D. Howard, Tomoko Komiyama, N.A. Thornberry, David J. Pickup, Guy S. Salvesen, and E.P. Peterson
- Subjects
chemistry.chemical_classification ,Cowpox virus ,Cell Biology ,Biology ,Serpin ,Biochemistry ,Serine ,Enzyme ,chemistry ,Enzyme inhibitor ,biology.protein ,Denaturation (biochemistry) ,Binding site ,Molecular Biology ,Cysteine - Abstract
We reported previously that human interleukin-1 beta converting enzyme (ICE) is regulated by the CrmA serpin encoded by cowpox virus. We now report the mechanism and kinetics of this unusual inhibition of a cysteine proteinase by a member of the serpin superfamily previously thought to inhibit serine proteinase only. CrmA possesses several characteristics typical of a number of inhibitory serpins. It is conformationally unstable, unfolding around 3 M urea, and stable to denaturation in 8 M urea upon complex formation with ICE. CrmA rapidly inhibits ICE with an association rate constant (kon) of 1.7 x 10(7) M-1 s-1, forming a tight complex with an equilibrium constant for inhibition (Ki) of less than 4 x 10(-12) M. These data indicate that CrmA is a potent inhibitor of ICE, consistent with the dramatic effects of CrmA on modifying host responses to virus infection. The inhibition of ICE by CrmA is an example of a "cross-class" interaction, in which a serpin inhibits a non-serine proteinase. Since CrmA possesses characteristics shared by inhibitors of serine proteinases, we presume that ICE, though it is a cysteine proteinase, has a substrate binding geometry strikingly close to that of serine proteinases. We reason that it is the substrate binding geometry, not the catalytic mechanism of a proteinase, that dictates its reactivity with protein inhibitors.
- Published
- 1994
39. Kinetics and physiologic relevance of the inactivation of alpha 1-proteinase inhibitor, alpha 1-antichymotrypsin, and antithrombin III by matrix metalloproteinases-1 (tissue collagenase), -2 (72-kDa gelatinase/type IV collagenase), and -3 (stromelysin)
- Author
-
Jan J. Enghild, K Suzuki, Alan E. Mast, H Nagase, Salvatore V. Pizzo, and Guy S. Salvesen
- Subjects
animal structures ,biology ,Chemistry ,Antithrombin ,Alpha (ethology) ,Cell Biology ,Serpin ,Matrix metalloproteinase ,Biochemistry ,Molecular biology ,Alpha 1-antichymotrypsin ,carbohydrates (lipids) ,Enzyme inhibitor ,embryonic structures ,Collagenase ,medicine ,biology.protein ,Gelatinase ,Molecular Biology ,medicine.drug - Abstract
Serpins encompass a superfamily of proteinase inhibitors that regulate many of the serine proteinases involved in inflammation and hemostasis. In vitro, many serpins are catalytically inactivated by proteinases that they do not inhibit, leading to the concept of proteolytic down-regulation of serpin inhibitory capacity. The extent to which down-regulation of serpin activity occurs in vivo is debated, since little is known of the rates at which the process occurs. To address this debate, we have measured the rates of inactivation of three serpins, alpha 1-proteinase inhibitor (alpha 1PI), alpha 1-antichymotrypsin (alpha 1ACT), and antithrombin III (ATIII), by three human matrix metalloproteinases (MMPs-1, -2, and -3) thought to be involved in tissue destruction and repair. Our object was to establish a working kinetic model which can be used to predict whether serpin inactivation by these proteinases is likely to occur in vivo. We determined the rates of inactivation of these three serpins by each of the MMPs and compared these to rates of inhibition of the MMPs by an endogenous inhibitor, alpha 2-macroglobulin. An equation designed to predict the extent of substrate hydrolyzed by an enzyme in the presence of an enzyme inhibitor gave the following predictions of the inactivation in vivo: (i) ATIII is unlikely to be inactivated by the MMPs. (ii) MMP-2 (72-kDa gelatinase/type IV collagenase) is unlikely to inactivate any of the three serpins. (iii) MMP-1 (tissue collagenase) will inactivate alpha 1PI and alpha 1ACT only when its concentration saturates that of its controlling inhibitors. (iv) MMP-3 (stromelysin) may inactivate small amounts of alpha 1PI and more significant amounts of alpha 1ACT, even in the presence of its controlling inhibitors. Any physiologic or pathologic inactivation of these serpins by these MMPs that occurs in vivo will probably be due to MMP-3, and will likely only take place in tissues and inflammatory loci where the concentration of MMP inhibitors is depressed.
- Published
- 1991
40. Chondroitin 4-sulfate covalently cross-links the chains of the human blood protein pre-alpha-inhibitor
- Author
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S A Hefta, Salvatore V. Pizzo, Ida B. Thøgersen, Shane M. Rutherfurd, Guy S. Salvesen, and Jan J. Enghild
- Subjects
integumentary system ,Binding protein ,Cell Biology ,Carbohydrate ,Immunoglobulin light chain ,Biochemistry ,carbohydrates (lipids) ,Glycosaminoglycan ,chemistry.chemical_compound ,chemistry ,Covalent bond ,Chondroitin ,Chondroitin sulfate ,Molecular Biology ,Inter-alpha-trypsin inhibitor - Abstract
The human blood protein pre-alpha-inhibitor is composed of one heavy and one light protein chain. The chains are covalently linked to each other by a structure that has not previously been described, which we designate a protein-glycosaminoglycan-protein (PGP) cross-link. A combination of protein and carbohydrate analytical techniques indicates that the interchain linkage is mediated by a chondroitin 4-sulfate glycosaminoglycan that originates from a typical O-glycosidic link to Ser-10 of the light chain. The heavy chain is esterified, via the alpha-carbon of its C-terminal Asp, to C-6 of an internal N-acetylgalactosamine of the glycosaminoglycan chain. This PGP cross-link may be present in other proteins, but could have been overlooked due to the heterogeneous behavior of proteins containing glycosaminoglycan.
- Published
- 1991
41. Staphylococcal SplB Serine Protease Utilizes a Novel Molecular Mechanism of Activation
- Author
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Pustelny, Katarzyna, primary, Zdzalik, Michal, additional, Stach, Natalia, additional, Stec-Niemczyk, Justyna, additional, Cichon, Przemyslaw, additional, Czarna, Anna, additional, Popowicz, Grzegorz, additional, Mak, Pawel, additional, Drag, Marcin, additional, Salvesen, Guy S., additional, Wladyka, Benedykt, additional, Potempa, Jan, additional, Dubin, Adam, additional, and Dubin, Grzegorz, additional
- Published
- 2014
- Full Text
- View/download PDF
42. Inducible dimerization and inducible cleavage reveal a requirement for both processes in caspase-8 activation.
- Author
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Oberst, Andrew, primary, Pop, Cristina, additional, Tremblay, Alexandre G., additional, Blais, Véronique, additional, Denault, Jean-Bernard, additional, Salvesen, Guy S., additional, and Green, Douglas R., additional
- Published
- 2014
- Full Text
- View/download PDF
43. Activity, Specificity, and Probe Design for the Smallpox Virus Protease K7L
- Author
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Aleshin, Alexander E., primary, Drag, Marcin, additional, Gombosuren, Naran, additional, Wei, Ge, additional, Mikolajczyk, Jowita, additional, Satterthwait, Arnold C., additional, Strongin, Alex Y., additional, Liddington, Robert C., additional, and Salvesen, Guy S., additional
- Published
- 2012
- Full Text
- View/download PDF
44. Cathepsin D Primes Caspase-8 Activation by Multiple Intra-chain Proteolysis
- Author
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Conus, Sébastien, primary, Pop, Cristina, additional, Snipas, Scott J., additional, Salvesen, Guy S., additional, and Simon, Hans-Uwe, additional
- Published
- 2012
- Full Text
- View/download PDF
45. The Dynamics and Mechanism of SUMO Chain Deconjugation by SUMO-specific Proteases
- Author
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Békés, Miklós, primary, Prudden, John, additional, Srikumar, Tharan, additional, Raught, Brian, additional, Boddy, Michael N., additional, and Salvesen, Guy S., additional
- Published
- 2011
- Full Text
- View/download PDF
46. Inducible Dimerization and Inducible Cleavage Reveal a Requirement for Both Processes in Caspase-8 Activation
- Author
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Oberst, Andrew, primary, Pop, Cristina, additional, Tremblay, Alexandre G., additional, Blais, Véronique, additional, Denault, Jean-Bernard, additional, Salvesen, Guy S., additional, and Green, Douglas R., additional
- Published
- 2010
- Full Text
- View/download PDF
47. Vaccinia Virus Protein F1L Is a Caspase-9 Inhibitor
- Author
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Zhai, Dayong, primary, Yu, Eric, additional, Jin, Chaofang, additional, Welsh, Kate, additional, Shiau, Chung-wei, additional, Chen, Lili, additional, Salvesen, Guy S., additional, Liddington, Robert, additional, and Reed, John C., additional
- Published
- 2010
- Full Text
- View/download PDF
48. Aminopeptidase Fingerprints, an Integrated Approach for Identification of Good Substrates and Optimal Inhibitors
- Author
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Drag, Marcin, primary, Bogyo, Matthew, additional, Ellman, Jonathan A., additional, and Salvesen, Guy S., additional
- Published
- 2010
- Full Text
- View/download PDF
49. Human Caspases: Activation, Specificity, and Regulation
- Author
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Pop, Cristina, primary and Salvesen, Guy S., additional
- Published
- 2009
- Full Text
- View/download PDF
50. Streptolysin O Promotes Group A Streptococcus Immune Evasion by Accelerated Macrophage Apoptosis
- Author
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Timmer, Anjuli M., primary, Timmer, John C., additional, Pence, Morgan A., additional, Hsu, Li-Chung, additional, Ghochani, Mariam, additional, Frey, Terrence G., additional, Karin, Michael, additional, Salvesen, Guy S., additional, and Nizet, Victor, additional
- Published
- 2009
- Full Text
- View/download PDF
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