Browsing by Subject "Polyubiquitin"
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Item Biochemical Mechanism of Protein Kinase Activation by the Ubiquitination System(2008-05-13) Xia, Zong-Ping; Chen, ZhijianThe NF-κB signaling pathway is important for immune, inflammatory and stress responses of cells and can be activated by a variety of extracellular stimuli. In the IL1R/TLR signaling pathway, NF-κB is activated through activation of TAK1-IKK cascade by TRAF6 and Ubc13/Uev1A in a polyubiquitinationdependent manner. Mechanistically how TAK1 is activated by the TRAF6- Ubc13/Uev1A dependent polyubiquitination system is unknown. Whether TAK1 and IKK kinases can be activated by other than TRAF6-Ubc13/Uev1A is an open question. By inactivating ubiquitin activating enzyme E1 and ubiquitin conjugating enzyme E2 using NEM to further dissect the system, studies on how TAK1 is activated by the TRAF6-Ubc13/Uev1A system have revealed that 1) polyubiquitination step and kinase activation step can be un-coupled; 2) Polyubiquitination step generates unanchored K63-linked polyubiquitin chains as the kinase activators; 3) Coiled-coil domain of TRAF6 is required for synthesis of active polyubiquitin chains. Through biochemical fractionation, I purified UbcH5 as another E2 that works with TRAF6 to directly activate IKK. Mechanistic analysis on how TRAF6/UbcH5 activates IKK reveals that TRAF6-UbcH5 synthesize mixedlinkage- linked unanchored polyubiquitin chains and this polyubiquitin chains function as direct activators for IKK activation.Item Mechanism of Ubiquitin Induced Activation of RIG-I Like Receptors in Antiviral Innate Immunity(2012-07-20) Jiang, Xiaomo; Chen, Zhijian J.The innate immune response is the first line of defense against viral infections. Innate immune recognition is mediated by a set of host pattern recognition receptors that can recognize pathogen associated molecular patterns, and trigger the expression of immune response genes. Two proteins of the RIG-I like receptor family, RIG-I and MDA5, are essential for the detection of viral RNA in the cytoplasm. Upon viral recognition, RIG-I and MDA5 activate the mitochondrial signaling protein AVS, and initiate downstream signaling pathways that activate the transcription factors NF-kappaB and IRF3. These transcription factors regulate the production of type I interferons and proinflammatory cytokines, which serve to keep virus infections under control. RIG-I and MDA5 contain N-terminal tandem CARD domains. CARD domains in other proteins are known to mediate protein-protein interactions. MAVS also contain one CARD domain. It has been proposed that the activation of MAVS by RIG-I is mediated by homotypic interaction between their CARD domains. However, it is hard to detect stable interaction between the CARD domains of RIG-I and that of MAVS. Therefore, the underlying biochemical mechanisms of how activation signals are transduced from a cytosolic receptor, RIG-I, to a mitochondria signaling protein, MAVS, is not clear. Protein ubiquitination have pivotal roles in diverse cell signaling pathways, including those in the immune system. A ubiquitin E3 ligase, TRIM25, promotes Lysine 63-linked polyubiquitination of RIG-I, and is essential for the RIG-I antiviral pathway. However, the biochemical mechanisms by which RIG-I is activated by ubiquitin remain poorly understood. In addition, whether ubiquitination is involved in MDA5 activation is unknown. Here, I describe the development of an in vitro biochemical assay to detect MAVS activation by a cytosolic activator. Purification of this activator revealed an important role of ubiquitin in RIG-I activation. Biochemical characterization of the ubiquitin induced RIG-I activation process revealed a new mechanism by which ubiquitin regulates cell signaling that involves the formation of a polyubiquitin-induced tetrameric complex. In addition, polyubiquitin-induced oligomerization appears to be a conserved mechanism for both MDA5 and RIG-I activation. [Keywords: antiviral innate immunity, RIG-I, MDA5, ubiquitin, oligomerization]