It has been well known that polyubiquitination is essential modification for most of protein degradation by proteasome. Therefore, protein ubiquitination is closely associated with protein stability. The ubiquitin protein itself consists of 76 amino acids and has 7 lysine (abbreviated as K) residues in its sequence including K6, K11, K27, K29, K33, K48, and K63. The first ubiquitin is linked to the single lysine residue on the target protein, and following ubiquitin are made by linking the C-terminal glycine residue of a ubiquitin molecule to a lysine residue of previous ubiquitin. If following ubiquitin links to lysine 48 of previous ubiquitin, it’s called K48-linkage. The K48-linkage are the best-characterised type of ubiquitin chain and associated with protein degradation. Recent studies have demonstrated that protein polyubiquitiation play an important role in cell signal transduction. For example, K63-linkage ubiquitination of Akt is important for Akt phosphorylation and activation induced by growth factor. During the NFB activation by TNF, multiple type linkages of ubiquitination, such as K11-linkage, K63-linkage, and new finding M1-linkage ubiquitination are found in RIP1 (receptor-interacting protein 1) for recruiting IKK complexes and then phosphorylating IB by IKK. The M1-linkage also called linear-linkage which linkage occurs between the N-terminal methonine (M1) of one ubiquitin and the C-terminal glycine of the next ubiquitin. MCPIPs (MCPIP1-4) belongs to C3H-type zinc finger protein superfamily and were identified as a transcription factor. All 4 MCPIPs have same CCCH zinc finger domain and NYN domain which is associated with RNA binding and RNase activity, respectively. MCPIP1 was firstly identified in the monocyte chemotactic protein-1 (MCP-1)-activated monocytes and plays a negative role in inflammation. On the other hand, MCPIP4 is originally reported as a putative tumor suppressor due to it is deregulated or mutated in several types of tumor. Our previous studies found that MCPIP1 and MCPIP4 were downregulation in human stage T3 colorectal cancer tissues. In addition, overexpression of MCPIP1 or MCPIP4 induced cell cycle arrest and even cell death. Recent studies indicated that MCPIP1 and MCPIP4 may have deubiquitinase activity. Our preliminary results indicated that MCPIP4 might inhibit Akt activation through removing the polyubiquitin chain from Akt, and MCPIP1 might inhibit TNF-induced NFB activation by deubiquitinating RIP1. However, further studies are necessary to explore the detailed mechanisms of how MCPIP1 and MCPIP4 regulate the ubiquitination in signal transduction. Based on our recent findings and hypothesis, we hope to achieve the following Specific Goals. Year 1. To investigate the abilities of MCPIP1 and MCPIP4 on the removing different linkage types of ubiquitin chains. We will use many wild-type and mutant plasmids to evaluate the deubiquitinating abilities of MCPIP1 and MCPIP4 in tube and in cells. In addition, we will find out the possible target proteins and partner proteins of MCPIP1 and MCPIP4 by immunoprecipitation and LC-MS/MS techniques. Year 2. To fully understand the molecular mechanisms of MCPIP1 and MCPIP4 on the inhibition of Akt ubiquitination and tumor cell growth. We will investigate whether Akt has other linkage types of the ubiquitin chain besides K63, and whether MCPIP1 and MCPIP4 can deubiquitinate these types of ubiquitin chains. Moreover, we will evaluate the anti-tumor activity of MCPIP1 and MCPIP4 in nude mice and understand their underlying molecular mechanisms. Year 3. To fully understand the molecular mechanisms of MCPIP1 and MCPIP4 on the inhibition of ubiquitination related to TNF/NFB signal pathways. We will examine whether MCPIP1 and MCPIP4 can enhance TNFα-induced apoptosis through inhibition of NFB, and whether MCPIP1 and MCPIP4 inhibit NFB through deubiquitinating RIP1 and/or TRAF2/6 induced by TNFα.
|Effective start/end date||8/1/15 → 7/31/16|