Evaluation of different E3 ligases on NEMO ubiquitination

• Main Authors: Xiu Han, 韓滫
• Other Authors: Lih-Hwa Hwang
• Format: Others
• Language: zh-TW
• Published: 2011
• Online Access: http://ndltd.ncl.edu.tw/handle/38599682386264868231

碩士 === 國立陽明大學 === 微生物及免疫學研究所 === 99 === Many pathogens in surroundings threaten our health and lives. In order to defense the invasion of pathogens, host cells employed many different receptors, called pattern-recognition receptors (PRRs), to recognize pattern-association molecular patterns (PAMPs) presented on the pathogens and activate the immune system. Retinoic acid-inducible gene I-like receptors (RLRs), including retinoic acid inducible–gene I (RIG-I) and melanoma differentiation-associated protein 5 (MDA5), are major sensors in the cytosol detecting viral nucleic acids. These receptors trigger signaling pathways to activate three transcriptional factors, interferon regulatory factor 3 (IRF3), NF-κB and MAPK, which function together to induce the production of type Ι interferons (IFNs). The rapid secretion of type Ι IFNs is a critical event in innate immunity and is delicately regulated by many cellular proteins. Recently, ubiquitination and deubiquitination have been shown to play important roles in regulating the activities of many proteins involved in the RLR signaling pathway. In the first part of this study, we characterized the roles of a de-ubiquitinating (DUB) enzyme, OTU domain-containing ubiquitin aldehyde-binding protein 2 (OTUB2), in the RLR signaling pathway. It was found that OTUB2 overexpression significantly inhibited the N-RIG-induced activation of IRF3 and the expression of IRF3 downstream genes such as RANTES and IFN-β. In contrast, OTUB2 knockdown enhanced the IRF3 activation. Further studies indicated that OTUB2 could de-ubiquitinate the K63-linked polyubiquitin chains from many molecules involved in the RLR signaling pathway, e.g. RIG-I, TRAF3, TRAF6, NAP1, IKKε, NEMO, and RIP1. OTUB2 overexpression also inhibited the TNF-α- and the IL-1β-induced NF-κB signaling. In the second part of this study, we demonstrated that NEMO could be ubiquitinated by three E3 ligases, TRAF6, TRIM25, and TRIM23. The ubiquitin chains conjugated to NEMO were mostly through K27 linkage instead of K63 linkage, which was also observed for endogenous NEMO under SeV infection. Co-immunoprecipitation results demonstrated that NEMO associated with TRAF6, TRIM25, and TRIM23, supporting the notion that NEMO can be the substrate of these E3 ligases.