Apoptotic cell death is essential for normal development and maintenance of normal tissue homeostasis. There is growing evidence that dysregulation of apoptosis may lead to several human diseases including cancer, autoimmune disorders and degenerative neuronal diseases such as Alzheimer's and Parkinson's diseases. Emerging evidence suggests that protease activation might be the central mechanism leading to the execution of apoptosis. Unlike in C. elegans where CED-3 cysteine protease is the major executioner of apoptosis, recent observations suggest that execution of apoptosis in higher eukaryotes may involve multiple cysteine proteases related to CED-3 and ICE. These proteases, might be components of an amplifiable apoptotic protease cascade similar to the cascade of complement activation. The number of cysteine proteases is still growing, however their mechanism of activation, role in apoptosis and relevant apoptotic substrates are still largely unknown. To isolate and characterize novel cysteine proteases we developed a PCR technique to enrich for DNA sequences which encode the highly conserved pentapeptides QACRG and GSWFI/GSWYI present in CED-3/ICE-like apoptotic cysteine proteases. We were able to clone several CED-3/ICE-like proteases that could be components of the molecular mechanism of vertebrate apoptosis. Consequently, it is proposed to overexpress full length, and truncated and mutated derivatives of these cysteine proteases in Spodoptera frugiperda (Sf9) cells and in E. coli. This will avail large quantities of these proteins for functional and structural studies. Mammalian apoptotic cysteine proteases cloned in our laboratory, will be characterized. Their genes will be analyzed for tissue specific and temporal expression and the presence of alternatively spliced isoforms. Potential substrates and inhibitors of these proteases will be identified using biochemical and molecular biology techniques. It is anticipated that these studies will contribute to elucidation of the mechanism of activation of this important class of proteases. This will enhance the efforts to identify their relevant endogenous substrates and to design specific drugs that will regulate their activity. In addition to the value which will be gained from understanding the molecular mechanism of apoptosis in development and homeostasis, the proposed studies will also generate significant knowledge that could be applied in the treatment of many human diseases such as cancer and many other degenerative diseases.

National Institute of Health (NIH)
National Institute on Aging (NIA)
Research Project (R01)
Project #
Application #
Study Section
Human Embryology and Development Subcommittee 1 (HED)
Program Officer
Sierra, Felipe
Project Start
Project End
Budget Start
Budget End
Support Year
Fiscal Year
Total Cost
Indirect Cost
Thomas Jefferson University
Schools of Medicine
United States
Zip Code
Yu, J-W; Wu, J; Zhang, Z et al. (2006) Cryopyrin and pyrin activate caspase-1, but not NF-kappaB, via ASC oligomerization. Cell Death Differ 13:236-49
Gupta, Sanjeev; Singh, Rajesh; Datta, Pinaki et al. (2004) The C-terminal tail of presenilin regulates Omi/HtrA2 protease activity. J Biol Chem 279:45844-54
Hegde, Ramesh; Srinivasula, Srinivasa M; Datta, Pinaki et al. (2003) The polypeptide chain-releasing factor GSPT1/eRF3 is proteolytically processed into an IAP-binding protein. J Biol Chem 278:38699-706
Cilenti, Lucia; Lee, Younghee; Hess, Sibylle et al. (2003) Characterization of a novel and specific inhibitor for the pro-apoptotic protease Omi/HtrA2. J Biol Chem 278:11489-94
Srinivasula, Srinivasa M; Datta, Pinaki; Kobayashi, Masatomo et al. (2002) sickle, a novel Drosophila death gene in the reaper/hid/grim region, encodes an IAP-inhibitory protein. Curr Biol 12:125-30
Hegde, Ramesh; Srinivasula, Srinivasa M; Zhang, ZhiJia et al. (2002) Identification of Omi/HtrA2 as a mitochondrial apoptotic serine protease that disrupts inhibitor of apoptosis protein-caspase interaction. J Biol Chem 277:432-8
Madesh, Muniswamy; Antonsson, Bruno; Srinivasula, Srinivasa M et al. (2002) Rapid kinetics of tBid-induced cytochrome c and Smac/DIABLO release and mitochondrial depolarization. J Biol Chem 277:5651-9
Komata, T; Kondo, Y; Kanzawa, T et al. (2001) Treatment of malignant glioma cells with the transfer of constitutively active caspase-6 using the human telomerase catalytic subunit (human telomerase reverse transcriptase) gene promoter. Cancer Res 61:5796-802
Srinivasula, S M; Datta, P; Fan, X J et al. (2000) Molecular determinants of the caspase-promoting activity of Smac/DIABLO and its role in the death receptor pathway. J Biol Chem 275:36152-7
Saleh, A; Srinivasula, S M; Balkir, L et al. (2000) Negative regulation of the Apaf-1 apoptosome by Hsp70. Nat Cell Biol 2:476-83

Showing the most recent 10 out of 26 publications