Triad3A regulates ubiquitination and proteasomal degradation of RIP1 following disruption of Hsp90 binding.
Toll-like receptors (TLRs) play a crucial role in innate immunity by recognizing microbial pathogens. Triad3A is an E3 ubiquitin-protein ligase that interacts with the Toll/interleukin-1 receptor domain of TLRs and promotes their proteolytic degradation. In the present study, we further investigated its activity on signaling molecules downstream of TLRs and tumor necrosis factor (TNF) receptor 1. Triad3A promoted down-regulation of two TIR domain-containing adapter proteins, TIRAP and TRIF, as well as a RIP1 but had no effect on other adapter molecules in either the TLRs or TNF-alpha signaling pathways. Multiple sequence alignment analysis suggested that RIP1 contains a TIR homologous domain, and mutation of amino acid residues in this domain identified three residues critical for its interaction with Triad3A. Moreover, Triad3A acted as a negative regulator in TNF-alpha signaling. Reduction of Triad3A expression by small interference RNAs rendered cells hyperresponsive to TNF-alpha stimulation. Conversely, overexpression of Triad3A in cells blocked TNF-alpha-induced cell activation. This negative regulation was effected independently of changes in the cellular protein level of RIP1. Further studies indicated that RIP1 formed a complex with Triad3A and heat shock protein 90 (Hsp90), which is a chaperone protein capable of maintaining the stability of its client proteins. Treatment of cells with geldanamycin to disrupt the Hsp90 complex led to proteasomal degradation of RIP1. Depletion of Triad3A by small interference RNA treatment inhibited geldanamycin-activated ubiquitination and proteolytic degradation of RIP1. These results suggest that Triad3A is an E3 ubiquitin-protein ligase to RIP1 and that Hsp90 and Triad3A cooperatively maintain the homeostasis of RIP1.
Fearns
Colleen
C
Department of Immunology, The Scripps Research Institute, La Jolla, California 92037, USA.
Pan
Qilin
Q
Mathison
John C
JC
Chuang
Tsung-Hsien
TH
D033942:Adaptor Proteins, Vesicular Transport / Q000378:metabolism; D000595:Amino Acid Sequence; D016227:Benzoquinones / Q000494:pharmacology; D002478:Cells, Cultured; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D018841:HSP90 Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D015151:Immunoblotting; D047468:Immunoprecipitation; D047908:Intracellular Signaling Peptides and Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D007668:Kidney / Q000166:cytology / Q000378:metabolism; D047029:Lactams, Macrocyclic / Q000494:pharmacology; D008562:Membrane Glycoproteins / Q000378:metabolism; D008969:Molecular Sequence Data; D016328:NF-kappa B / Q000235:genetics / Q000378:metabolism; D028861:Nuclear Pore Complex Proteins / Q000235:genetics / Q000378:metabolism*; D010957:Plasmids; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism*; D011485:Protein Binding; D011499:Protein Processing, Post-Translational; D034741:RNA, Small Interfering / Q000494:pharmacology; D016601:RNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D017472:Receptors, Interleukin-1 / Q000378:metabolism; D018124:Receptors, Tumor Necrosis Factor / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction; D051193:Toll-Like Receptors / Q000235:genetics / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology; D025801:Ubiquitin / Q000378:metabolism*; D044767:Ubiquitin-Protein Ligases
C119724:AGFG1 protein, human; D033942:Adaptor Proteins, Vesicular Transport; D016227:Benzoquinones; D015853:Cysteine Proteinase Inhibitors; D018841:HSP90 Heat-Shock Proteins; D047908:Intracellular Signaling Peptides and Proteins; D047029:Lactams, Macrocyclic; D008562:Membrane Glycoproteins; D016328:NF-kappa B; D028861:Nuclear Pore Complex Proteins; D034741:RNA, Small Interfering; D016601:RNA-Binding Proteins; D017472:Receptors, Interleukin-1; D018124:Receptors, Tumor Necrosis Factor; C470409:TICAM1 protein, human; C434984:TIRAP protein, human; D051193:Toll-Like Receptors; D014409:Tumor Necrosis Factor-alpha; D025801:Ubiquitin; C458527:RNF216 protein, human; D044767:Ubiquitin-Protein Ligases; D046988:Proteasome Endopeptidase Complex; C001277:geldanamycin