Primary Research On TNF Induced Necrosis Mechanism Regulated By USP13

Objective:Using a deubiquitinating enzyme genes exprssion library we established, we tend to find out novle genes that regulates TNF induced necroptosis signaling pathway.Our research will provide new insights in necroptosis signaling pathway.Content :In deubiquitinating enzyme genes exprssion library we established to screen out the novle genes that regulates TNF induced necroptosis signaling pathway.whether the enzyme activity of USP13 and two domains are important for its function in necroptosis preliminary research.Method:1. We found out the list of the human deubiquitinating enzymes, designed the specific cloning primer of these genes in this process, amplified the gene fragments using high-fidelity DNA polymerase, and finally link to pbob-c-flag lentivirus backbone expression vector.2. Following the lentivirus packing method, we generated the lentivirus contaning the deubiquitinating enzymes. Then we infected L929 cells with lentivirus.48 hours later we checked the cell survival rate of L929 infected with indicated lentivirus upon the stimulation of TNF, and found out genes involved in TNF induced necroptosis signaling pathway.3. Constructed USP13 enzyme inactive mutant as well as the USP13 domain deletion lentivirus expression cloning, and packed indicated lentivirus. Infected L929 cells with the indicated lentivrus, 48 hours later we checked the cell survival rate of L929 infected with indicated lentivirus upon the stimulation of TNF.Results:1. We susecessed to construct 71 deubiquitinating enzyme gene expression vectors in lentivirus backbone.2. We found out several genes including USP13 that regulated TNF induced necroptosis.3. We found out the ability of USP13 to faciliate TNF induced necroptosis rely on its zf-UBP and UBA domains but not its enzyme activity.Conclusion:1. Overexpression of deubiquitinating enzymes USP13 accelerate TNF induced necroptosis.2. USP13 zf-UBP and UBA domains are important for its function in necroptosis.3. The enzyme activity of USP13 is dispensable for its function in necroptosis.