Man Ubiquitin-activating Enzyme Ube1dc1 Ube1 And Ubiquitin Binding Enzyme Ube2f The Function

Ubiquitin(UB) and Ubiquitin-like proteins(UBLs) as a kind of protein modifications are activated in the first step of ubiquitination pathway.Ubiquitination pathway plays major roles in a variety of basic physiological functions involved in cell cycle,apoptosis,antigen presentation,transcription,DNA damage and repair.The system of ubiquitination conjugation involves 3 steps.The ubiquitin-activating enzyme(E1) plays a key role in the first step of ubiquitination pathway to activate ubiquitin or ubiquitin-like proteins.The ATP-dependent ubiquitin-activating enzyme (E1) forms a covalent bond with ubiquitin(UB) or ubiquitin-like proteins(UBLs) in the first step.Activated UB or UBLs were transferred to ubiquitin-conjugating enzyme E2,and then transferred to ubiquitin-protein ligases E3 or substrates by ubiquitin-eonjugating enzyme E2.The function of ubiquitin-activating enzyme E1-domain containing 1(UBE1DC1),ubiquitin-activating enzyme E1(UBE1) and ubiquitin-conjugating enzyme E2F(UBE2F) were discussed in the paper.In this report,full length of UBE1DC1A and a novel splice variant of full length gene of UBE1DC1A,UBE1DC1B,were cloned and purified.The cDNAs of UBE1DC1A and UBE1DC1B contain an open reading frame encoding 404 amino acids and 348 amino acids respectively with analogical conserved ThiF domain and ATP-binding domain(GXGXXG) by blast analysis.UBE1DC1A,UBE1DC1B,UB and UBLs were expressed in E.coli and purified by chromatography of Ni-NTA His-Bind Superflow.UBE1DC1 is proved to activate another ubiquitin-like protein, SUMO2,besides Ufm1,both in vitro and in vivo by immunological analysis.It indicated that UBE1DC1 could activate two different ubiquitin-like proteins,SUMO2 and Ufm1,which have no significant similarity with each other.It was porved that Cys250 of UBE1DC1 was active site by comparing with homologous proteins and mutation analyzing.Subceilular localization in AD293 cells revealed that UBE1DC1 was especially distributed in the cytoplasm;whereas UBE1DC1 was mainly distributed in the nucleus when it was cotransfected with SUMO2.It presumed that UBE1DC1 transferred activated-SUMO2 to nucleus after it conjugated SUMO2 in the cytoplasm.UBE1 is an ubiquitin-activating enzyme as the same as UBE1DC1.UBE1 as the first member of ubiquitin-activating enzyme plays a key role in the first step of ubiquitin-proteasome pathway to activate ubiquitin.Reaserching the structure and biochemical property of protein,and analyzing the activity of enzymes which were related with ubiquitin-proteasome pathway,are based on obtaining high purity of UBE1 protein.After synthesized full length of UBE1gene,6His-UBE1 was highly expressed in E.coli,and purified by chromatography of Ni-NTA His-Bind Superflow and Strep-Tactin Sepharose,according to 6His-tag of pET28b vector and the characteristics that UBE1 could activate UB to forming UB-UBE1 intermediate complex.We identified that high purity of UBE1 was isolated by the method,and it could activated UB to ubiquitin-conjugating enzyme E2s.On the other hand,the mechanisms how UBE1 activated UB to form UB-UBE1 intermediate complex in tertiary structure is not clearly.Analyzed by circular dichroism spectra(CD),it found that the conformation of UB-UBE1 intermediated complex was changed visiblily compared with UBE1,and the contents ofα-helix increased especially.The ubiquitin-conjugating enzyme(E2) plays a key role in the second step of ubiquitination pathway to conjugate ubiquitin or ubiquitin-like proteins,and transfers them to ubiquitin-protein ligases E3.The ubiquitin-conjugating enzyme E2F(named UBE2F) was selected through large-scale cDNA cloning and sequencing.The cDNAs of UBE2F contains an open reading frame encoding 185 amino acids with analogical conserved UBCc domain and active site Cys by blast analysis.UBE2F is proved to conjugat UB in vitro by immunological analysis,and the active site was Cys117 because UBE2F^C117A could not conjugat activated-UB.Subcellular localization in AD293 cells revealed that UBE2F was especially distributed in the cytoplasm and nucleus like UBE2F^C117A.It presumed that Cys117 of UBE2F was unrelated with localization when it was mutated to Ala.