The Functional Role And Mechanism Of Deubiquitylase OTUB2 Regulating PCNA Protein Level

Ubiquitin(Ub)-proteasome system(UPS)is responsible for the degradation of most cellular soluble proteins.In the UPS,substrates for degradation are firstly ubiquitylated via an enzymatic cascade involving E1 Ub-activating enzyme,E2 Ub-conjugating enzyme,and E3 Ub ligase.Deubiquitylases(DUBs)remove ubiquitin modifications to regulate virtually all ubiquitin-dependent processes.More than 100 DUBs are predicted to be active in human cells and many of them have been implicated in human diseases including cancer and neurodegeneration disease.Among them,the subfamily of ovarian tumor domain(OTU)DUBs have emerged as regulators of important signaling cascades in part due to their specificity for recognition of poly-ubiquitin chain linkage.For example,OTUD7 B regulates NF-?B signaling through deubiquitylating TRAF3.OTUB1 inhibits non-canonical DNA damage-dependent ubiquitylation,while OTUB2 regulates p97-mediated processes.Linear ubiquitin chain-specific DUB OTULIN is essential for preventing TNF-associated systemic inflammation in human and mice.There are 16 members found with deubiquitinating enzyme catalytic activity in human OTU family DUBs,which aredivided into three subfamilies: Otubains,A20-like OTUs and OTUDs.The members of this family mainly exercise their highly conserved OTU domains.The OTU DUBs play important roles in DNA damage repair,inflammatory immune response,metabolic homeostasis,and tumorigenesis.The ubiquitination enzyme OTUB2(OTU Deubiquitinase,also named Ubiquitin Aldehyde Binding 2)was first discovered in Hela cells in 2003,and it is very abundant in the Otubain subfamily.Similar to other DUBs,OTUB2 has a typical caspase living center and the catalytic core domains are Cys51,His224,Asn226.It is known that OTUB2 is involved in the regulation of inflammatory responses,and knocking out OTUB2 promotes homologous recombination repair in the early stages of DNA damage repair.Moreover,OTUB2 is known to be highly expressed in cancers such as testicular cancer,but it is not clear how to function specifically.Proliferation Cell Nuclear Antigen(PCNA)was first discovered and named by Miyachi et al.in the serum of patients with SLE(systemic lupus erythematosus)in 1978,and was found to be highly expressed in proliferating cells and tumor cells.PCNA is a molecular marker of cell proliferation,and its protein level changes with the cell cycle,peaking at the G1 and S phase protein levels.As a tumor marker,PCNA is able to inhibit post-translational signaling and is subject to a variety of post-translational modifications.In the past decade,more and more studies have found that PCNA has important functions such as promoting cell growth and inhibiting death.DNA damage is a phenomenon in which DNA nucleotide sequences that occur during replication are permanently altered and cause changes in genetic characteristics.In a 2002 issue of Nature,the RAD6 pathway was the core of DNA repair during replication in eukaryotic cells,but its mechanisms and regulation remain poorly understood.Two key factors critical to this pathway are the ubiquitin-binding enzyme RAD6,MMS2-UBC13 heterodimer,and are recruited into the chromatin by the RING-finger proteins RAD18 and RAD5.PCNA is a sliding clamp for DNA polymerase and is an important substrate involved in DNA synthesis and repair.PCNA is monoubiquitinated by RAD6 and RAD18,and polyubiquitination is modified by modification of lysine-63 ubiquitin chain,and ubiquitination of PCNA is crucial in DNA damage repair.And through literature research,it is found that the K164 ubiquitination modification of PCNA is essential for DNA damage repair,and the role of polyubiquitination of PCNA in DNA damage repair is much more efficient than the single ubiquitination modification of PCNA.Scientists have found that KNA-type ubiquitination of PCNA is critical in RAD6-dependent DNA damage repair.However,it is unclear whether E3 affects the ubiquitin chain and thus affects DNA damage repair or whether DUB affects the ubiquitin chain and thus affects DNA damage repair.This prompted us to study the interest of E3 and DUB in repairing DNA damage.Our study found that OTUB2 regulate PCNA protein stability by de-ubiquitinating enzymes.In order to determine the relationship between PCNA and OTUB2 and the effect of OTUB2 on cell proliferation,we conducted a series of experiments.We found that the deubiquitinating enzyme OTUB2 interacts with PCNA in cells by immunoprecipitation and immunoblotting.OTUB2 knockdown increased the polyubiquitination of PCNA in Hela cell.However,Overexpression of OTUB2,but not OTUB2-C51 S in 293 T cells removed the ubiquitination of PCNA.,OTUB2 expression has no influence in PCNA protein level in cells,indicating that OTUB2 did not affect the stability of PCNA.Additionally,we detected the effect of OTUB2 on cell proliferation using CCK8 Assay.The results showed that OTUB2 significantly inhibit the proliferation of Hela tumor cells in vitro and this inhibitory effect is dependent on PCNA.In summary,the interaction between OTUB2 and PCNA affects the proliferation of cervical cancer cells,which provides a new direction for us to study new drugs for cervical cancer and the important role of OTU DUBs in the process of DNA damage repair.Deubiquitinating enzymes and ubiquitinase participate in numerous biological processes in life.A typical feature of the OTU subfamily members of the deubiquitinating enzyme is that its functions primarily as a deubiquitinating enzyme through the highly conserved OTU domain.It is known that it has a catalytically active site composed of Cys,His,and Asp,and the substrate ubiquitination process is completed by catalytically cleaving the isopeptide bond between two ubiquitin units in the polyubiquitin chain of the substrate protein.The modified ubiquitin chain types that we now know are K63-type ubiquitin chains and K48-type ubiquitin chains.Among them,OTUD4 is one of them,and current research is few.Studies have found that scientists use zebrafish as a model organism to knock down Rnf216 or Otud4 in zebrafish embryos,and found that zebrafish eyes,visual caps and cerebellar development defects suggest that OTUD4 may play a crucial role in embryonic development.To study the function of OTUD4 in the overall level of animals,I worked with sisiter Liu Wen to construct a systemic knockout mouse of OTUD4 based on the Loxp-Cre system.The Loxp-Cre system was used to construct the deubiquitinating enzyme Otud4 knockout mouse.Using the Loxp-Cre system principle,the exon 4 of the Otud4 gene was selected as the targeting sequence,and the two Lox P sites were set in E4.At the end,the target vector is transferred into ES cells,positive clone screening,blastocyst injection and the like to obtain chimeric mice,and then the chimeric mice are mated with C57BL/6 mice for breeding,and finally the heterozygous mice are obtained and subjected to Self-crossing gave newborn F1 mice.Genotype identification was then performed using PCR,and the expression of OTUD4 in various tissues and organs was detected from the protein level using immunoblotting(WB)technology to determine whether the knockout mice were successfully constructed.A statistical analysis of the number of mice in each genotype confirmed whether it met the Mendelian law of inheritance and whether it had an embryonic lethal phenotype.The dissected mice were analyzed for major dysplasia in the main tissues and organs,and HE staining was used to analyze whether there was spontaneous pathological changes.Whether the knockout mouse has metabolic abnormality or the like is analyzed by a glucose tolerance test.Genotyping and WB assays showed that the Otud4 knockout mouse model was successfully established.The proportion of genotypes in heterozygous mice after self-crossing accords with Mendel's law,suggesting that Otud4 knockout mice are not embryonic lethal.There was no significant difference in organ size between the tissues,and there was no abnormality in HE staining,and the Otud4 gene knockout did not affect the blood glucose homeostasis of the mice.Otud4 knockout mice were successfully constructed using Loxp-Cre system,and the mice were born normally without any embryonic development defects.There were no significant pathological changes in all organs,and there was no abnormal blood glucose metabolism.In summary,the OTUD4 mouse was successfully constructed using the Loxp-Cre system,which laid a foundation for further study of the function of OTUD4 in vivo.