The Role Of The Active Cysteine In The Regulation Of The Ubiquitination Of Ubiquitin-conjugating Enzyme CDC34 And The Identification Of CDC34-interacting Proteinsand Its Functional Study

Background and purpose:The ubiquitin proteasome system(UPS)performs very important roles in regulating many cellular processes.There are three kinds of important enzymes in the ubiquitination system,ubiquitin-activating enzymes(Els),ubiquitin-conjugating enzymes(E2s),ubiquitin ligases(E3s).The deregulation of UPS has been implicated in a wide range of human pathologies including cancer,neurodegenerative and immunological disorders,and viral infections.Recent studies were mainly focused on the E3 ligase function and the identification of their substrates.Ubiquitin-conjugating enzymes are more than the middle men in the trio of enzymes responsible for the attachment of ubiquitin to cellular proteins.Humans have about 40 E2s that are involved in transferring ubiquitin(Ub)or Ub-like(Ubl)proteins to substrates.Although many E2s are only twice of Ub in their size,this remarkable family of enzymes performs an irreplaceable role.Little is known about their other roles except their ubiquitin-conjugating function.The object of my study,CDC34(cell division cycle 34),is a member of ubiquitin-conjugating enzymes.It can facilitate the formation of K48 linked polyubiquitin chains and cooperate with an E3 ligase complex(SCF,Skpl-Cullin 1-F-box protein)to lead the ubiquitination of target proteins.At last these ubiquitinated proteins will be degraded through the ubiquitin proteasome pathway.In recent years,studies have found that CDC34 can modulate the proliferation of human diploid fibroblasts by regulating the level of protein p27Kip1.Low levels of p27Kip1 protein correlate with poor prognosis and increase tumor growth.Recent studies have shown that CDC34 is highly expressed in the cytoplasm and the nucleus of HCC(hepatocellular carcinomas)cell lines and patient samples and is also closely-linked to cell proliferation and differentiation.Studies on CDC34 and its interacting proteins are scarce and how CDC34 regulates the function of its substrates remains elusive.As we know,CDC34 is responsible for the proliferation of HCC cells.How CDC34 affects the function of its interacting proteins in tumor cells is still elusive.In this work,we mutated the active cysteine of CDC34 and explored the effect of this mutation on its ubiquitination and stability.We purified the CDC34-interacting proteins and identified them using LC-MS/MS,then validated the interaction biochemically,and studied their regulation.Methods:We constructed Strep-FLAG CDC34 and CDC34 C93S plasmids and expressed them in HEK293T cells.The effect of the active site mutation was examined by immunoprecipitation and immunoblotting.The CDC34-interacting proteins were isolated by anti-FLAG M2 affinity gel and then digested for proteomic identification.To screen proteins that have a difference between control groups,wild-type groups,and mutant groups by searching the UniProt protein database.We obtained potential proteins interacting with CDC34 and validated their interaction by Western blotting.Then we analyzed the potential function of these proteins by bioinformatics and studied their regulation.Results:(1)CDC34 C93S can markedly inhibit its own ubiquitination and increase its stability.(2)CDC34 C93S can increase the stability of some of its interacting proteins.(3)Many potential CDC34 interacting proteins,such as MCM7,PRKDC,and GNL3,were identified by mass spectrometry.CDC34 can upregulate MCM7 and downregulate GNL3 protein level.Conclusions:The C93S mutation can enhance the stability of CDC34.The active site mutation has blocked CDC34 ubiquitination.Therefore,it can reduce the degradation and enhance the level of the CDC34 mutant.From the bioinformatic analysis,we found that the CDC34-interacting proteins have functions in DNA replication and mRNA transport.It indicates these interacting proteins may involve in the cell cycle regulation and cell proliferation.