Molecular Cloning And Enzymatic Activity Analysis Of Ubiquitin Specific Protease26(USP26)

Objective： Ubiquitin-proteasome pathway (UPP) participates inregulating many important processes by Ubiquitination modification, namelythe combination of the ubiquitin with amino acid residues of target proteins,such as cell cycle progression, membrane proteins translocation,transcriptional regulation, function of histones, immune response and DNAreplication-repair, etc. It is very important in maintaining normal life forcells.However, the modification for proteins is a reversible and dynamicprocess, involving ubiquitination and deubiquitination. Dubiquitinatingenzymes play a pivotal role in ubiquintin-mediated cell process by negativelyregulating ubiquitination of proteins. The dysfunction of dubiquitinatingenzymes (DUBs) can lead to many diseases, including hereditary cancer andneurodegenerative disease.Deubiquitinating enzymes (DUBs) belongs to the homocysteinehydrolase, and they are divided into four subtypes: ubiquitin specific proteasefamily (USPs), Ubiquitin C-terminal Hydrolases (UCHs), the Ovarian tumourProteases (OTUs) and the Machado-Joseph Disease Protein Domain Proteases(MJDs).There are numerous members in the family of USP, which containsseveral highly conserved regions in their amino acid sequence, including theCys, His, and Asp/Asn residues. This paper research to dubiquitinatingenzyme usp26is one of the families of USP.This paper research Ubiquitin specific protease (USP26) belings to amember of USPs superfamily.The relationship about USP26genepolymorphism with male infertility is studied to be confined to populationgenotyping, but it’s under the influence of factors such as ethnic, regional, andsample size. So whether these SNPs are associated with male infertility is notclear. Through this study we obtain usp26gene, and build expression plasmids of usp26-wild type and six SNPs. Meanwhile, we set up two systems fordetecting the activity of dubiquitinating enzymes, analyzing whether theseSNPS influence enzyme activity, thereby, which strongly verifies if usp26gene polymorphism can cause male infertility and provides theoretical supportfor further study.Methods：(1) The molecular cloning method was used to clone the USP26gene ofhuman. Genomic DNA was extracted from normal human blood using DNAextraction kit. Method of segmented amplification was used to amplificateusp26-5′and usp26-3′with Polymerase Chain Reaction (PCR), respectively.Then the fragment of usp26-5’and usp26-3’ were cloned into pGEM-T easycarrier.Blue white screening experiment was adopted to define the positiveclone and the plasmids were extracted by alkaline lysis method. After theidentification of the plasmids in double enzyme was right, then sequencingthem.We would get pGEX-6p-1-usp26according to the right recombinantplasmids, pGEM-T-usp26-5’and pGEM-T-usp26-3’ and the plasmid pGEX-6p-1was the media.(2) Expression of GST-USP26fusion protein and identification ofsolubility. The recombinant plasmid pGEX-6p-1-usp26was transfected intoDH5α E. coli. GST-USP26fusion protein (about130kDA) was expressedunder the IPTG induction.Meanwhile, we detected whether it was soluble ornot.(3) Using recombinant plasmids pGEX-6p-1-usp26-WT as templates,wehave built successfully the recombinant plasmids of six SNPs （G170R、E192E、F348L、T659M、K734N、E749D）and C304S by site-directedmutagenesis method.We changed the vecter by restriction enzymes, then weabtained the recombinant plasmids, which made pAC-T7as the vecter inpreparation for the activity analysis behind.(4) Building two systems for deubiquitinating enzyme activity assay todetect the deubiquitinating enzyme activity of USP26wild-type, six SNPs andC304S.①T7-USP26/GST-Ub52system: The recombinant plasmids which made pAC-T7as the vecter, USP26-WT, six SNPs, C304S, USP46(positivecontrol), pAC-T7(negative control) were co-expressed with pGEX-Ub52respectively. GST-Ub52fusion protein became the model substrate.UsingGSH-Sepharose-TM Resin purified protein to purify GST fusion protein, andthey were detected by10%SDS-PAGE electrophoresis. Deubiquitinatingenzyme activity positive means there were36kDa proteins product generatedby45kDa substrate protein being cut off.②GST-USP/Ub-Met-β-gal system:The recombinant plasmids which made pGEX-6p-1as the vecter, usp26-WT,six SNPs, C304S, usp46(positive control), pGEX-6p-1(negative control)were co-expressed with pAC-ub-β-gal respectively.Plasmid pAC-M-β-galexpresses the Ub-Met-β-gal fusion protein substrate and total protein extractswere analyzed by Western blotting with mouse monoclonal anti-β-galantibody, and detected by Odyssey infrared fluorescence scanning imagingsystem. Ub-Met-β-gal fusion protein substrate was cut into little Met-β-galprotein, incating deubiquitinating enzyme activity was positive. Active sizewas determined by the proportion of them.Results:(1) Ubiquitin specific protease USP26encoding gene was cloned fromhuman blood, total length of2742bp.(2) Construct recombinant plasmids about six SNPs of usp26: pGEX-usp26-G170R、 pGEX-usp26-E192E、 pGEX-usp26-F348L、 pGEX-usp26-T659M、 pGEX-usp26-K734N、 pGEX-usp26-E749D and inactive mutantplamid pGEX-usp26-C304S,which were identified by enzyme digestion andsequencing.(3) The GST-USP26fusion protein, expressed in the E. coli DH5α with amolecular weight of approximately130kDa, corresponding with thetheoretical molecular weight of usp26(molecular weight is about104kDa)and GST (molecular weight is about26kDa). The fusion protein wasindissoluable when induced with IPTG at37℃and15℃for4hours.(4)Two deubiquitinating enzyme activity assays were consistent witheach other. Empty plasmid pGEX-6p-1(negative control) and C304S mutation was inactive, and USP46(positive control) had a certain activity; six SNPs ofUSP26were similar with wild type, but stronger than the USP46. Imagesdisplaying that the empty plasmid pGEX-6p-1and C304S couldn’t cut GST-Ub52(45Kda) into36Kda product, but USP26wild type and its six SNPScould cut GST-Ub52into36Kda product completely, and USP46could cutGST-Ub52into36Kda product partly; Another system, the empty plasmidpAC-T7and C304S had two bands, the substrate Ub-Met-β-gal (the top),endogenousβ-gal (the bottom). USP46had three bands, not only two bandsabove, but Met-β-gal (the middle), the product of Ub-Met-β-galdeubiquitinated. Six SNPS had two bands the middle and the bottom.Conclusions:(1) We have successfully cloned a novel human ubiquitin specificprotease, USP26.(2) USP26gene coding sequence is2742bp and encodes913aminoacids, thus we speculate that the molecular weight is104kDa, and themolecular weight of GST is26kDa, so130kDa stripe is GST-USP26fusionprotein and is unsolvable.(3) We have successfully constructed expression plasmids about sixSNPs of usp26(G170R, E192E, F348L, T659M, K734N, E749D) and C304S.(4) Two deubiquitinating enzyme activity assay systems were establishedwell. The deubiquitinating enzyme activity of USP26were not affected by sixSNPs.The activity was still strong.