Apoptbsis plays critical roles in mammalian biology including embryonic development, cellular homeostasis and immune regulation. Genetic mutations or abnormal expression of apoptosis pathway proteins are associated with many human diseases. Apoptosis is initiated via assembly of multimeric caspase-activating signaling complexes. In the extrinsic cell death pathway, death receptors (members of the tumor necrosis factor (TNF) receptor superfamily) such as Fas (also known as CD95 or APO-1) assemble into an oligomeric death inducing signaling complex (DISC) upon ligand stimulation. Similarly, in the caspase-2 activation pathway, the p53-inducible protein PIDD assembles into an oligomeric complex known as the PIDDosome. Death domain (DD) and death effector domains (DED) play important roles in both DISC and PIDDosome formation. For DISC assembly, the intracellular DD of Fas interacts with the C-terminal DD of the adapter protein FADD and the N-terminal DED of FADD interacts with the tandem DED in caspase-8 (or caspase-10) to form the ternary complex of Fas, FADD and caspase-8. In addition to caspase-8 and -10, cellular and viral FLIPs (cFLIPs and vFLIPs) are also tandem DED-containing proteins that interact with FADD DED and inhibit the function of the DISC. For PIDDosome assembly, one important interaction is between the DD of PIDD and the DD of the adapter protein RAIDD. Caspase recruitment into these complexes initiates proteolytic auto-processing and caspase activation. In this application, we propose to use a combination of biochemical and cell biological approaches study the molecular mechanisms that govern the assembly of these complexes.
The specific aims are: 1) to reconstitute these apoptotic signaling complexes in vitro and to characterize them in cellular systems;2) to elucidate the molecular basis of the Fas DD:FADD DD interaction;3) to elucidate the molecular basis of the PIDD DD:RAIDD DD interaction;4) to elucidate the interaction of FADD DED with tandem DEDs.

Public Health Relevance

Engagement of the TLR/IL-1R pathway in appropriate physiological contexts initiates the development of protective immune responses. However, the complexity of this pathway also renders itself susceptible to interruption and dysregulation, leading to its association with many human diseases. For example, inherited mutations or polymorphisms in TIR-adapters and IRAKs may cause either extreme sensitivity to or protection against infections. Other types of dysregulation in the pathway contribute to both diseases in the immune system such as inflammatory disorders, autoimmune diseases and allergy, and diseases beyond the immune system such as cancer, insulin resistance, atherosclerosis, and painful neuropathy.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Method to Extend Research in Time (MERIT) Award (R37)
Project #
5R37AI050872-14
Application #
8628024
Study Section
Cellular and Molecular Immunology - A Study Section (CMIA)
Program Officer
Quill, Helen R
Project Start
2002-01-01
Project End
2017-03-31
Budget Start
2014-04-01
Budget End
2015-03-31
Support Year
14
Fiscal Year
2014
Total Cost
Indirect Cost
Name
Children's Hospital Boston
Department
Type
DUNS #
City
Boston
State
MA
Country
United States
Zip Code
02115
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Fu, Tian-Min; Li, Yang; Lu, Alvin et al. (2016) Cryo-EM Structure of Caspase-8 Tandem DED Filament Reveals Assembly and Regulation Mechanisms of the Death-Inducing Signaling Complex. Mol Cell 64:236-250
Onizawa, Michio; Oshima, Shigeru; Schulze-Topphoff, Ulf et al. (2015) The ubiquitin-modifying enzyme A20 restricts ubiquitination of the kinase RIPK3 and protects cells from necroptosis. Nat Immunol 16:618-27
Wei, Shuo; Kozono, Shingo; Kats, Lev et al. (2015) Active Pin1 is a key target of all-trans retinoic acid in acute promyelocytic leukemia and breast cancer. Nat Med 21:457-66
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Ru, Heng; Chambers, Melissa G; Fu, Tian-Min et al. (2015) Molecular Mechanism of V(D)J Recombination from Synaptic RAG1-RAG2 Complex Structures. Cell 163:1138-52
Li, Lingyin; Yin, Qian; Kuss, Pia et al. (2014) Hydrolysis of 2'3'-cGAMP by ENPP1 and design of nonhydrolyzable analogs. Nat Chem Biol 10:1043-8
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Lu, Alvin; Kabaleeswaran, Venkataraman; Fu, Tianmin et al. (2014) Crystal structure of the F27G AIM2 PYD mutant and similarities of its self-association to DED/DED interactions. J Mol Biol 426:1420-7

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