The overall objective of this project is to elucidate the mechanisms that control the activation of caspase-9. Caspase-9 is a major initiator caspase in mammalian cells. It exists in healthy cells as a latent precursor and becomes activated in response to a wide range of cell-intrinsic apoptosis stimuli such as developmental lineage information, oncogene activation, DNA damage, and nutritional deprivation. The intrinsic apoptosis pathway that activates caspase-9 is the major mechanism by which the formation of tumor cells is prevented in the first place, and by which tumor cells are killed by chemo- and radiation therapy. The activation of caspase- 9 occurs in a large cytosolic complex known as the apoptosome, the formation of which is triggered by the release of mitochondrial cytochrome c to the cytosol and the subsequent binding of cytochrome c to Apaf-1. This binding enables Apaf-1 to assemble into the heptameric apoptosome, which recruits procaspase-9 and permits its auto-activation. Previous studies by us and others demonstrated that the activation of initiator caspases is induced by their oligomerization. We will determine the mechanism for oligomerization-induced procaspase-9 activation (Aim 1). In addition, we found that tRNAs inhibit caspase-9 activation through direct interaction with cytochrome c and preventing the formation of the apoptosome. We will determine this important function of tRNAs which is beyond their well-established role in the transmission of genetic information. We will also examine this function of tRNAs in apoptosis resistance of tumor cells (Aim 2). Finally, the activation of caspase-9 in the apoptosome is inhibited by the inhibitor of apoptosis proteins (IAPs). This inhibitory effect of IAPs is countered during apoptosis by mitochondrial IAP-binding proteins. Compounds mimicking the effect of IAP-binding proteins show promise in tumor therapy. We recently identified a novel IAP- interacting protein residing in mitochondria. We will elucidate the role of this protein in caspase-9 activation (Aim 3). These three aims represent a concerted effort to elucidate the intricate control of caspase-9 activation at multiple levels. We believe that they will help the development of more effective treatments for apoptosis- related diseases, particularly cancer.

Public Health Relevance

Apoptosis is critical for development and for protection against cancer. The intrinsic/mitochondrial pathway is a major apoptosis pathway in mammalian cells, which leads to the activation of caspase-9 and subsequent cell death. In this proposal, we plan to study the regulation of caspase-9 activation. We believe that this study will further the understanding of the intrinsic apoptosis pathway and have practical implications for treating diseases related to this pathway, including cancer, autoimmune disorders, and immunodeficiency.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM060911-09
Application #
8366894
Study Section
Cellular Signaling and Regulatory Systems Study Section (CSRS)
Program Officer
Maas, Stefan
Project Start
2000-09-01
Project End
2013-11-30
Budget Start
2012-12-01
Budget End
2013-11-30
Support Year
9
Fiscal Year
2013
Total Cost
$290,427
Indirect Cost
$108,910
Name
University of Pennsylvania
Department
Biology
Type
Schools of Medicine
DUNS #
042250712
City
Philadelphia
State
PA
Country
United States
Zip Code
19104
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