Difference: HaoWu (1 vs. 5)

Grant Number: 5R01AI045937-07 Project Title: Structural Studies of TNF Receptor Associated Factors (TRAFs) PI Information: Name Email Title WU, HAO haowu@med.cornell.edu PROFESSOR

Abstract: DESCRIPTION (provided by applicant): A long-term goal of my laboratory is to elucidate the molecular basis of signal transduction by Tumor necrosis factor (TNF) receptor associated factors (TRAFs), which are the major signal transducers of TNF receptors, IL-1 receptors, Toll-like receptors, and T-cell receptors. During the previous funding period, we have focused our effort on the molecular interactions of the TRAF C-terminal domain with receptors and other upstream signaling proteins. In this application, we propose the new initiative of elucidating the molecular mechanisms of TRAF downstream signaling, which centers on the activation of the TAK1 kinase complex composed of TAK1 and the adapter proteins TAB1 and TAB2. Two modes of TAK1 activation mechanism have been implicated. While TRAFs activate TAK1 via TAB2 in a Lys63-linked polyubiquitination pathway, XIAP activates TAK1 via TAB1 in an unknown ubiquitination-independent pathway. Our preliminary data support an emerging unifying principle of oligomerization in TAK1 activation by both TRAFs and XIAP. In this application, we propose a series of crystallographic, biochemical, biophysical and cell biological studies to address the molecular mechanisms of TAK1 activation and inhibition. The following specific aims are proposed. Aim 1. Role of TRAF trimerization and K63-linked ubiquitination in TAK1 activation Aim 2. Role of TRAF6 deubiquitination in terminating TAK1 activation: specificity of the deubiquitinase A20 for TRAF6 Aim 3. Role of dimerization in XIAP and TAB1 mediated TAK1 activation

Public Health Relevance: This Public Health Relevance is not available.

Thesaurus Terms: biological signal transduction, cytokine receptor, protein structure function, structural biology, tumor necrosis factor alpha binding site, protein protein interaction, receptor binding X ray crystallography, crystallization, site directed mutagenesis

Institution: WEILL MEDICAL COLLEGE OF CORNELL UNIV 1300 YORK AVENUE NEW YORK, NY 10065 Fiscal Year: 2008 Department: BIOCHEMISTRY Project Start: 01-JUL-1999 Project End: 29-FEB-2012 ICD: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES IRG: ZRG1

Abstract: DESCRIPTION (provided by applicant): 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: This Public Health Relevance is not available.

Thesaurus Terms: cysteine endopeptidase, enzyme complex, enzyme inhibitor, enzyme structure, structural biology, virus protein apoptosis, chemical kinetics, computer simulation, crystallization, molecular dynamics, mutant Baculoviridae, X ray crystallography, mass spectrometry, site directed mutagenesis

Institution: WEILL MEDICAL COLLEGE OF CORNELL UNIV 1300 YORK AVENUE NEW YORK, NY 10065 Fiscal Year: 2009 Department: BIOCHEMISTRY Project Start: 01-JAN-2002 Project End: 29-FEB-2012 ICD: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES IRG: MSFB

Abstract: DESCRIPTION (provided by applicant): Apoptosis is initiated via assembly of multimeric caspase-activating signaling complexes. One such important complex is the PIDDosome for caspase-2 activation, which contains the p53-inducible protein PIDD, RAIDD and caspase-2. Caspase-2 is an initiator caspase and the most evolutionarily conserved caspase. In response to DNA damage and other cellular stress, caspase-2 acts upstream of the mitochondria to mediate cell death by inducing Bid cleavage, Bax translocation and cytochrome c release. Like other caspase activating complexes and many kinase activating complexes, assembly of the PIDDosome is dependent on proteins of the death domain (DD) superfamily. The DD superfamily comprises four subfamilies, the DD subfamily, the caspase recruitment domain (CARD) subfamily, the death effector domain (DED) subfamily and the pyrin domain (PYD) subfamily and shares the similar six helical bundle fold. Full length PIDD contains 910 residues with seven leucine rich repeats (LRRs), two ZU-5 domains and a C-terminal DD. RAIDD contains an N-terminal CARD and a C-terminal DD. Caspase-2 has a CARD prodomain at its N-terminal end and a caspase domain at its C-terminal end. The PIDDosome is assembled via a DD: DD interaction between RAIDD and PIDD and a CARD: CARD interaction between RAIDD and caspase-2. PIDD DD is not only essential for the activation of caspase-2 it can also interact with the DD of RIP1, a kinase implicated in the activation of NF-B. PIDD acts as a molecular switch, controlling the balance between life and death. No structural information is available on the PIDDosome. In this application, we propose a series of structural and biological studies to elucidate the molecular basis of PIDDosome formation. The studies will not only contribute to our understanding of PIDDosome formation but also DD superfamily interactions in other assemblies in general. PUBLIC HEALTH RELEVANCE: Apoptosis plays an important role in human physiology. Deregulation of apoptosis is related to many human diseases. The current application seeks to address a critical question in apoptosis regulation and has a strong implication in understanding and treatment of apoptosis-related human diseases.

Public Health Relevance: This Public Health Relevance is not available.

Thesaurus Terms:

There are no thesaurus terms on file for this project.

Institution: WEILL MEDICAL COLLEGE OF CORNELL UNIV 1300 YORK AVENUE NEW YORK, NY 10065 Fiscal Year: 2008 Department: BIOCHEMISTRY Project Start: 01-JUL-2008 Project End: 30-JUN-2013 ICD: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES IRG: MSFC

Abstract: DESCRIPTION (provided by applicant): In this proposal, funds are requested to acquire automated crystallization and imaging systems to support biomedical research at Weill Medical College of Cornell University (WMCCU) and the neighboring Rockefeller University (RU). The instrument will directly support 14 NIH funded researchers and 41 NIH funded research grants and serve as a core resource in the well established X-ray crystallography core facility of the WMCCU. Currently, the X-ray crystallography core facility is equipped with excellent capability for in-house crystal screening and data collection, but lacks any resources for crystallization, a bottleneck for crystallographic pursuit. To allow medium throughput and small volume crystallization of multiple constructs and multiple complexes of challenging macromolecular targets and to facilitate initiation of crystallographic studies by members of a broader scientific community, there is a critical need for a crystallization robotic device at WMCCU and RU. The instruments requested include a semi-automatic solution-handling robot capable of setting crystallization trials using as little as 50 nl protein solution per drop and a semi-automatic imaging system for the analysis and documentation of crystallization trials. The instruments will provide capabilities that do not currently exist in the WMCCU as well as the neighboring RU and the Sloan-Kettering Institute. Both the crystallization and the imaging system will be located in the 200C temperature controlled room in the X-ray crystallography core facility and will be made accessible to both participating PIs in this application and other researchers in the tri-institutional (WMCCU, RU and Sloan-Kettering Institute) area. PUBLIC HEALTH RELEVANCE: X-ray crystallography is one of the most powerful methods for determination of macromolecular structures at atomic level. The acquisition of semi-automated crystallization and imaging systems will directly support NIH sponsored research aimed at understanding basic biological processes such as signal transduction, transcription, ligand transport across membranes and bacterial pathogenesis and at understanding human diseases such as cancer, Alzheimer's disease, Parkinson's disease and microbial infection.

Public Health Relevance: This Public Health Relevance is not available.

Thesaurus Terms:

There are no thesaurus terms on file for this project.

Institution: WEILL MEDICAL COLLEGE OF CORNELL UNIV 1300 YORK AVENUE NEW YORK, NY 10065 Fiscal Year: 2009 Department: BIOCHEMISTRY Project Start: 01-MAR-2009 Project End: 28-FEB-2010 ICD: NATIONAL CENTER FOR RESEARCH RESOURCES IRG: ZRG1

• Location: WMCCU, phone (212) 746-6451

• Location: WMCCU

• Comment:

• Comment: phone (212) 746-6451

• Location: WMCCU

• Location: WMCCU, phone (212) 746-6451

• Name: Hao Wu
• Email: haowu@med.cornell.edu
• Company Name: WMCCU
• Company URL: http://www.med.cornell.edu
• Location: (Please specify office location)
• Country: USA
• Comment:

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• Name: Hao Wu
• Email: haowu@med.cornell.edu
• Company Name: WMCCU
• Company URL: http://www.med.cornell.edu
• Location: WMCCU
• Country: USA
• Comment:

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