The Vanderbilt University
Grant: $193,438 - National Institutes of Health - May. 21, 2009
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Award Description: Immunoreceptor-mediated activation of transcription factor NF-kB and MAP kinases (MAPKs) is essential for proper regulation of multiple genes involved in the innate and adaptive immune response to microbial pathogens. Prior experiments with transformed cell lines suggest that Lys-63 (K63)-linked polyubiquitination of receptor-interacting protein 1 (RIP1), a serine/threonine protein kinase, is required for activation of NF-kB/MAPK by members of the tumor necrosis factor receptor (TNFR) and Toll-like receptor (TLR) families. According to recent in vitro data, signal-dependent attachment of K63-linked ubiquitin (Ub) chains to RIP1 creates a temporary docking site for the Ub-binding subunits of cytosolic protein kinases that relay immunoreceptor signals to NF-kB and MAPKs. Deubiquitinating enzymes then remove Ub from RIP1 and terminate the response. If Ub-conjugated RIP1 is left unchecked, chronic activation of these signaling pathways may lead to excessive cytokine production and inflammation-driven diseases such as arthritis, lupus, and cancer. Despite these advances with in vitro models, the physiologic function and clinical relevance of RIP1 ubiquitination remains untested. In this regard, prior experiments with transformed T cells suggested that K63-linked ubiquitination of NEMO, a noncatalytic subunit of IkB kinase, regulates antigen receptor (AgR) signaling to NF-kB. However, in vivo experiments reported here reveal impaired TLR rather than AgR signaling in ?knock-in? mice harboring a point mutation that removes the Ub acceptor site of NEMO. This discovery underscores the need to test downstream consequences of Ub conjugation in a physiologic setting and establishes the feasibility of a complementary project to determine the in vivo significance of RIP1 ubiquitination. Using a similar knock-in approach, mice will be engineered to harbor a mutation that selectively blocks K63-linked ubiquitination of RIP1 but not its kinase activity (Aim 1). Subsequent phenotypic studies will focus on the role of RIP1 ubiquitination in hematopoiesis and immune cell development (Aim 1), innate immunity versus pathologic inflammation (Aim 2), and adaptive immunity (Aim 3). Biochemical studies of immunoreceptor signaling to NF-kB and MAPKs will be conducted with primary cells affected by the germline mutation. Importantly, this analysis will define the function of K63-linked ubiquitination at the level of RIP1 under conditions that ensure its correct spatial, temporal, and quantitative expression, which cannot be achieved via conventional transfection approaches. In vivo data accrued from the proposed project will advance knowledge about the biological workscope of RIP1-Ub conjugates well beyond the current limitations of in vitro systems, a prerequisite for assessing the potential value of K63-linked polyubiquitination as a therapeutic target in inflammation-based disease.
Project Description: Immunoreceptor-mediated activation of transcription factor NF-kB and MAP kinases (MAPKs) is essential for proper regulation of multiple genes involved in the innate and adaptive immune response to microbial pathogens. Prior experiments with transformed cell lines suggest that Lys-63 (K63)-linked polyubiquitination of receptor-interacting protein 1 (RIP1), a serine/threonine protein kinase, is required for activation of NF-kB/MAPK by members of the tumor necrosis factor receptor (TNFR) and Toll-like receptor (TLR) families. In this regard, prior experiments with transformed T cells suggested that K63-linked ubiquitination of NEMO, a noncatalytic subunit of IkB kinase, regulates antigen receptor (AgR) signaling to NF-kB. However, in vivo experiments reported here reveal impaired TLR rather than AgR signaling in ?knock-in? mice harboring a point mutation that removes the Ub acceptor site of NEMO. This discovery underscores the need to test downstream consequences of Ub conjugation in a physiologic setting and establishes the feasibility of a complementary project to determine the in vivo significance of RIP1 ubiquitination. Biochemical studies of immunoreceptor signaling to NF-kB and MAPKs will be conducted with primary cells affected by the germline mutation. Importantly, this analysis will define the function of K63-linked ubiquitination at the level of RIP1 under conditions that ensure its correct spatial, temporal, and quantitative expression, which cannot be achieved via conventional transfection approaches. In vivo data accrued from the proposed project will advance knowledge about the biological workscope of RIP1-Ub conjugates well beyond the current limitations of in vitro systems, a prerequisite for assessing the potential value of K63-linked polyubiquitination as a therapeutic target in inflammation-based disease.
Jobs Summary: The recovery act funds for this award helped to create or retain the following types of positions such as professor, research fellow, assistant professor and research assistant. (Total jobs reported: 1)
Project Status: Less Than 50% Completed
This award's data was last updated on May. 21, 2009. Help expand these official descriptions using the wiki below.
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