The members of nuclear factor (NF-kB) transcription factor family play central roles in various biological processes by regulating expression of genes involved in such diverse processes as inflammation, immune responses, differentiation, proliferation, and apoptosis. The primary objective of our research is to characterize the function and regulation of NF-kB, specifically the characterization of the mechanisms of regulation and termination of IKK/NF-kB activation in response to upstream signaling by Tumor Necrosis Factor-a (TNF-a) and Interleukin-1 (IL-1). Towards that end we have followed different experimental approaches. We have identified two proteins that not been reported to modulate NF-kB activity. Both these proteins contain phosphoinositol-3-phosphate (PI3P) binding FYVE domain and ubiquitin ligase RING (E3) domain. These proteins CARP-1 and -2 (caspase-8 and -10-associated RING proteins) bind to and negatively regulate DED caspases. These proteins are localized to different compartments in endocytic pathway and negatively regulate the TNF-a induced NF-kB. Our studies also indicated that CARPs downregulate TNF-R1 signaling by directly binding to and significantly downregulating RIP protein expression, a critical component of the TNF-R1 receptosome. siRNA-mediated downregulation of CARPs in two different cell lines enhanced TNF-a induced NF-kB activation. Studies thus far suggest that CARPs might regulate TNF induced NF-?B activation by binding to and downregulating RIP. Yeast two-hybrid screens using CARP-1 as bait 20 different CARP-1 interacting proteins. One of them, TRIM32, is a member of the tripartite motif family of proteins, a mutation in which (D487N) has been linked to Limb-Girdle Muscular Dystrophy type 2H (LGMD2H). We showed that TRIM32 activates NF-?B in myocytes and notably the D487N mutation abrogates its ability to activate NF-?B. The other gene TMBIM1, codes for a novel seven transmembrane protein with the consensus sorting signal motif """"""""NPLY"""""""". This motif conforms to the tyrosine based sorting signal NPXY, that is involved in sorting of transmembrane proteins to endosomes and lysosomes. siRNA-mediated downregulation of TMBIM1 enhanced TNF induced NF-?B activation. We have found that TMBIM1 binds to TNF receptor-1 (TNF-R1) and downregulates TNF-R1 and TNF induced NF-?B activation, possibly by affecting receptor internalization and trafficking.

National Institute of Health (NIH)
Division of Basic Sciences - NCI (NCI)
Intramural Research (Z01)
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Basic Sciences
United States
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Fujita, Ken-Ichi; Srinivasula, Srinivasa (2009) Ubiquitination and TNFR1 Signaling. Results Probl Cell Differ :
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