|Our lab is investigating the role of mitochondria in cell death, and the regulation of caspase activation during apoptosis and inflammation by cellular proteins such as Apaf-1 (Apoptotic protease activating factor-1), Ipaf (ICE protease activating factor), IAPs (Inhibitor of apoptosis proteins) and several pyrin domain-containing proteins. In addition, we are involved in characterizing NF-kB regulatory complexes in the Bcl-10 pathway that regulates antigen receptor signaling in B and T cell proliferation.|
Role Of Mitochondria In Cell Death
We recently characterized the dual role of the mitochondrial serine protease Omi/HtrA2 in apoptosis and in cell survival during stress. Signaling mechanisms that regulate apoptosis and cellular responses to stress are crucial to normal development and the elimination of damaged cells. We discovered that Omi/HtrA2 is released together with Smac and cytochrome c from the mitochondria into the cytoplasm upon disruption of the outer mitochondrial membrane during apoptosis. We also found that Omi/HtrA2 induces apoptosis in human cells in a caspase-independent manner through its protease activity, and in a caspase-dependent manner via its ability to disrupt caspase-IAP interactions and to degrade IAPs. Our recent finding that a single amino acid substitution in Omi/HtrA2 is the cause of the motoneuron disease mnd2 in mice provides an excellent model system to study the role of Omi/HtrA2 in cell survival.
In addition to extensive biochemical analysis, we are currently studying the proteome of the diseased mouse mitochondria, using 2-D PAGE analysis of normal versus homozygous mutant mouse mitochondria. The objective is to identify critical substrates of Omi/HtrA2 that are differentially processed in the mutant mouse in response to stress stimuli. We are also performing proteomic analysis to identify mitochondrial proteins that interact with the PDZ domain of Omi/HtrA2. The PDZ-interacting proteins might be important regulators of the protease activity of Omi/HtrA2, since preliminary results suggest that short PDZ-binding peptides are excellent activators of Omi/HtrA2.
Protein Regulation of Caspase Activation
A second project is directed toward understanding the mechanism and regulation of caspase-1 activation in inflammation by the caspase-associated recruitment domain (CARD)-containing protein IPAF and the pyrin-containing proteins ASC/CARD-5, NALP1/CARD-7, and cryopyrin/Pypaf1. Our results so far indicate that IPAF and ASC interact with procaspase-1 via CARD-CARD interactions. We are carrying out experiments to test the hypothesis that upstream Pyrin-containing proteins recruit ASC into an inflammasome complex that acts as a platform for activation of caspase-1 in response to stimulation of monocytes with LPS and other proinflammatory stimuli. Experiments are also carried out to investigate the role of IPAF, NALP1 and cryopyrin in activation of caspase-1 and to identify and characterize additional novel proteins, which trigger the formation of the large inflammatosome complex in response to pro-inflammatory cytokine stimulation.
A related project is directed toward identifying and characterizing potential cellular IAP inhibitors. Using proteomic approaches, we have identified two XIAP-binding proteins: the mitochondrial Omi/HtrA2 protease, and a processed isoform of the polypeptide chain-releasing factor GSPT1/eRF3. The two proteins harbor conserved N-terminal IAP-binding motifs and they function to regulate apoptosis by liberating caspases from IAPs. We are currently using different human IAP molecules as baits to isolate other cellular IAP-binding proteins.
Regulatory Complexes In B And T Cell Proliferation
We are also investigating several NF-kB regulatory complexes in the BCL10 pathway. BCL10 is a mediator of NF-kB signaling by the antigen receptors in B and T cells. Several caspase-associated recruitment domain (CARD) proteins were characterized in our lab. Some of these CARD proteins are specific regulators of BCL10 function and bind to BCL10 through a CARD/CARD interaction. They constitute a subclass of CARD proteins that likely function to transduce distinct upstream stimuli to the activation of BCL10 and NF-kappaB. A unique feature of these CARD proteins is the presence of central coiled-coil domains and C-terminal PDZ/SH3/GUK domains, which are believed to mediate interactions with upstream cellular proteins. We are carrying out experiments to characterize the upstream regulators of BCL10, especially proteins, which engage the BCL-10-binding proteins CARD-9, 10, 11 and 14. The identification and characterization of components of the antigen receptor/BCL10 pathway has significant implications for understanding the mechanism of NF-kappaB activation by BCL10 and for future therapeutic development in a range of pathological conditions involving T- or B-cell proliferation.
Keywords: caspases, apoptosis, cell death, death receptors, cytochrome c, APAF-1, mitochondria
Saleh M, Vaillancourt JP, Graham RK, Huyck M, Srinivasula SM, Alnemri ES, Steinberg MH, Nolan V, Baldwin CT, Hotchkiss RS, Buchman TG, Zehnbauer BA, Hayden MR, Farrer LA, Roy S, Nicholson DW. Differential modulation of endotoxin responsiveness by human caspase-12 polymorphisms. Nature 2004 May 6;429(6987):75-9.. ( Abstract )
Blink E, Maianski NA, Alnemri ES, Zervos AS, Roos D, Kuijpers TW. Intramitochondrial serine protease activity of Omi/HtrA2 is required for caspase-independent cell death of human neutrophils. Cell Death Differ 2004 Mar 26;.. ( Abstract )
Guo Y, Cheong N, Zhang Z, De Rose R, Deng Y, Farber SA, Fernandes-Alnemri T, Alnemri ES. Tim50, a component of the mitochondrial translocator, regulate mitochondrial integrity and cell death. J Biol Chem 2004 Mar 25;. ( Abstract )
Maianski NA, Geissler J, Srinivasula SM, Alnemri ES, Roos D, Kuijpers TW. Functional characterization of mitochondria in neutrophils: a role restricted to apoptosis. Cell Death Differ 2004 Feb;11(2):143-53.. ( Abstract )
Jones JM, Datta P, Srinivasula SM, Ji W, Gupta S, Zhang Z, Davies E, Hajnoczky G, Saunders TL, Van Keuren ML, Fernandes-Alnemri T, Meisler MH, Alnemri ES. Loss of Omi mitochondrial protease activity causes the neuromuscular disorder of mnd2 mutant mice. Nature 2003 Oct 16;425(6959):721-7. Epub 2003 Oct 8.. ( Abstract )
Zeuner A, Eramo A, Testa U, Felli N, Pelosi E, Mariani G, Srinivasula SM, Alnemri ES, Condorelli G, Peschle C, De Maria R. Control of erythroid cell production via caspase-mediated cleavage o transcription factor SCL/Tal-1. Cell Death Differ 2003 Aug;10(8):905-13. ( Abstract )
Hegde R, Srinivasula SM, Datta P, Madesh M, Wassell R, Zhang Z, Cheong N, Nejmeh J, Fernandes-Alnemri T, Hoshino S, Alnemri ES. The polypeptide chain-releasing factor GSPT1/eRF3 is proteolytically processed into an IAP-binding protein. J Biol Chem 2003 Oct 3;278(40):38699-706. Epub 2003 Jul 15.. ( Abstract )
Srinivasula SM, Gupta S, Datta P, Zhang Z, Hegde R, Cheong N, Fernandes-Alnemri T, Alnemri ES. Inhibitor of apoptosis proteins are substrates for the mitochondria serine protease Omi/HtrA2. J Biol Chem 2003 Aug 22;278(34):31469-72. Epub 2003 Jun 30. ( Abstract )
Kandasamy K, Srinivasula SM, Alnemri ES, Thompson CB, Korsmeyer SJ, Bryant JL, Srivastava RK. Involvement of proapoptotic molecules Bax and Bak in tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced mitochondrial disruption and apoptosis: differential regulation of cytochrome c and Smac/DIABLO release. Cancer Res 2003 Apr 1;63(7):1712-21.. ( Abstract )
Jia L, Patwari Y, Kelsey SM, Srinivasula SM, Agrawal SG, Alnemri ES, Newland AC. Role of Smac in human leukaemic cell apoptosis and proliferation. Oncogene 2003 Mar 20;22(11):1589-99. ( Abstract )
Chang DW, Ditsworth D, Liu H, Srinivasula SM, Alnemri ES, Yang X. Oligomerization is a general mechanism for the activation of apoptosi initiator and inflammatory procaspases. J Biol Chem 2003 May 9;278(19):16466-9. Epub 2003 Mar 13. ( Abstract )
Shiozaki EN, Chai J, Rigotti DJ, Riedl SJ, Li P, Srinivasula SM, Alnemri ES, Fairman R, Shi Y. Mechanism of XIAP-mediated inhibition of caspase-9. Mol Cell 2003 Feb;11(2):519-27.. ( Abstract )
Cilenti L, Lee Y, Hess S, Srinivasula S, Park KM, Junqueira D, Davis H, Bonventre JV, Alnemri ES, Zervos AS. Characterization of a novel and specific inhibitor for the pro-apoptotic protease Omi/HtrA2. J Biol Chem 2003 Mar 28;278(13):11489-94. Epub 2003 Jan 15.. ( Abstract )
Pistritto G, Jost M, Srinivasula SM, Baffa R, Poyet JL, Kari C, Lazebnik Y, Rodeck U, Alnemri ES. Expression and transcriptional regulation of caspase-14 in simple an complex epithelia. Cell Death Differ 2002 Sep;9(9):995-1006. ( Abstract )
Li W, Srinivasula SM, Chai J, Li P, Wu JW, Zhang Z, Alnemri ES, Shi Y. Structural insights into the pro-apoptotic function of mitochondrial serine protease HtrA2/Omi. Nat Struct Biol 2002 Jun;9(6):436-41.. ( Abstract )
Srinivasula SM, Poyet JL, Razmara M, Datta P, Zhang Z, Alnemri ES. The PYRIN-CARD protein ASC is an activating adaptor for caspase-1. J Biol Chem 2002 Jun 14;277(24):21119-22. Epub 2002 Apr 19.. ( Abstract )
Zhan Y, Hegde R, Srinivasula SM, Fernandes-Alnemri T, Alnemri ES. Death effector domain-containing proteins DEDD and FLAME-3 form nuclear complexes with the TFIIIC102 subunit of human transcription factor IIIC. Cell Death Differ 2002 Apr;9(4):439-47.. ( Abstract )
Guo Y, Srinivasula SM, Druilhe A, Fernandes-Alnemri T, Alnemri ES. Caspase-2 induces apoptosis by releasing proapoptotic proteins from mitochondria. J Biol Chem 2002 Apr 19;277(16):13430-7. Epub 2002 Feb 6.. ( Abstract )
Razmara M, Srinivasula SM, Wang L, Poyet JL, Geddes BJ, DiStefano PS, Bertin J, Alnemri ES. CARD-8 protein, a new CARD family member that regulates caspase- activation and apoptosis. J Biol Chem 2002 Apr 19;277(16):13952-8. Epub 2002 Jan 30. ( Abstract )
Srinivasula SM, Datta P, Kobayashi M, Wu JW, Fujioka M, Hegde R, Zhang Z, Mukattash R, Fernandes-Alnemri T, Shi Y, Jaynes JB, Alnemri ES. sickle, a novel Drosophila death gene in the reaper/hid/grim region encodes an IAP-inhibitory protein. Curr Biol 2002 Jan 22;12(2):125-30. ( Abstract )
Madesh M, Antonsson B, Srinivasula SM, Alnemri ES, Hajnoczky G. Rapid kinetics of tBid-induced cytochrome c and Smac/DIABLO release and mitochondrial depolarization. J Biol Chem 2002 Feb 15;277(7):5651-9. Epub 2001 Dec 6.. ( Abstract )
Chai J, Wu Q, Shiozaki E, Srinivasula SM, Alnemri ES, Shi Y. Crystal structure of a procaspase-7 zymogen: mechanisms of activation and substrate binding. Cell 2001 Nov 2;107(3):399-407.. ( Abstract )
Hegde R, Srinivasula SM, Zhang Z, Wassell R, Mukattash R, Cilenti L, DuBois G, Lazebnik Y, Zervos AS, Fernandes-Alnemri T, Alnemri ES. Identification of Omi/HtrA2 as a mitochondrial apoptotic serine proteas that disrupts inhibitor of apoptosis protein-caspase interaction. J Biol Chem 2002 Jan 4;277(1):432-8. Epub 2001 Oct 17. ( Abstract )
Druilhe A, Srinivasula SM, Razmara M, Ahmad M, Alnemri ES. Regulation of IL-1beta generation by Pseudo-ICE and ICEBERG, two dominant negative caspase recruitment domain proteins. Cell Death Differ 2001 Jun;8(6):649-57.. ( Abstract )
Poyet JL, Srinivasula SM, Tnani M, Razmara M, Fernandes-Alnemri T, Alnemri ES. Identification of Ipaf, a human caspase-1-activating protein related t Apaf-1. J Biol Chem 2001 Jul 27;276(30):28309-13. Epub 2001 Jun 4. ( Abstract )
Bertin J, Wang L, Guo Y, Jacobson MD, Poyet JL, Srinivasula SM, Merriam S, DiStefano PS, Alnemri ES. CARD11 and CARD14 are novel caspase recruitment domain (CARD)/membrane-associated guanylate kinase (MAGUK) family members that interact with BCL10 and activate NF-kappa B. J Biol Chem 2001 Apr 13;276(15):11877-82. Epub 2001 Jan 12.. ( Abstract )
Chai J, Shiozaki E, Srinivasula SM, Wu Q, Datta P, Alnemri ES, Shi Y, Dataa P. Structural basis of caspase-7 inhibition by XIAP. Cell 2001 Mar 9;104(5):769-80. ( Abstract )
Srinivasula SM, Hegde R, Saleh A, Datta P, Shiozaki E, Chai J, Lee RA, Robbins PD, Fernandes-Alnemri T, Shi Y, Alnemri ES. A conserved XIAP-interaction motif in caspase-9 and Smac/DIABLO regulate caspase activity and apoptosis. Nature 2001 Mar 1;410(6824):112-6. ( Abstract )
Poyet JL, Srinivasula SM, Lin JH, Fernandes-Alnemri T, Yamaoka S, Tsichlis PN, Alnemri ES. Activation of the Ikappa B kinases by RIP via IKKgamma /NEMO-mediate oligomerization. J Biol Chem 2000 Dec 1;275(48):37966-77. ( Abstract )
Srinivasula SM, Datta P, Fan XJ, Fernandes-Alnemri T, Huang Z, Alnemri ES. Molecular determinants of the caspase-promoting activity of Smac/DIABL and its role in the death receptor pathway. J Biol Chem 2000 Nov 17;275(46):36152-7. ( Abstract )
Saleh A, Srinivasula SM, Balkir L, Robbins PD, Alnemri ES. Negative regulation of the Apaf-1 apoptosome by Hsp70. Nat Cell Biol 2000 Aug;2(8):476-83. ( Abstract )
Alnemri ES. Hidden powers of the mitochondria. Nat Cell Biol 1999 Jun;1(2):E40-2.. ( Abstract )
De Maria R, Zeuner A, Eramo A, Domenichelli C, Bonci D, Grignani F, Srinivasula SM, Alnemri ES, Testa U, Peschle C. Negative regulation of erythropoiesis by caspase-mediated cleavage o GATA-1. Nature 1999 Sep 30;401(6752):489-93. ( Abstract )
Qin H, Srinivasula SM, Wu G, Fernandes-Alnemri T, Alnemri ES, Shi Y. Structural basis of procaspase-9 recruitment by the apoptoti protease-activating factor 1. Nature 1999 Jun 10;399(6736):549-57. ( Abstract )
Srinivasula SM, Ahmad M, Fernandes-Alnemri T, Alnemri ES. Autoactivation of procaspase-9 by Apaf-1-mediated oligomerization. Mol Cell 1998 Jun;1(7):949-57.. ( Abstract )
Li P, Nijhawan D, Budihardjo I, Srinivasula SM, Ahmad M, Alnemri ES, Wang X. Cytochrome c and dATP-dependent formation of Apaf-1/caspase-9 comple initiates an apoptotic protease cascade. Cell 1997 Nov 14;91(4):479-89. ( Abstract )
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