Activity Detection And Associated Proteins Screening By 2D-DIGE Of Ubiquitin-specific Protease USP13

Ubiquitin-specific protease 13(USP13) is a well-characterized member of the ubiquitin-specific proteases(USPs). USP13 has been identi?ed on human chromosome 3q26.2–q26.3. The gene coding region of USP13 consists of 2592 bases, encoding 863 amino acids. USP13 is an ortholog of USP5 in human genome, which shares about 80% sequence similarity with USP5. Both proteins contain four ubiquitin-associated(UBA) domains and an ubiquitinspecific processing protease(UBP) domain. The UBP domain of USP13 harbors a catalytic site, a zinc finger(Zn F) domain, and two Ub associated(UBA) domains. But USP13 is different from USP5 in both catalytic activity and activation. In order to understand the enzymatic activity of USP13, we detected the deubiquitinating enzyme activity of USP13 by the USP cleavage assay using GST-Ub52, Ub-Met-β-gal and Ub-AMC as model substrates in vitro. In the other hand, in order to elucidate the substrate specificities or associated proteins of USP13, we also took a proteomic approach by using two-dimensional difference gel electrophoresis(2D-DIGE) to detect cellular proteins with significantly altered expression levels in HEK293 T cells after the expression of USP13 or the C345 S mutant(the catalytically inactive form)of it, providing a new method to screen the candidate substrates and associated proteins for USP13.Part one: Activity detection of ubiquitin specific protease USP13Objective: USP13 contains two short and highly conserved fragments,lysine domain(Cys box) and histidine domain(his box). Its sequence contains catalytic triad residues Cys, His, and Asp/Asn conserved domains, which includes the residues critical for catalysis, but its enzymatic properties and catalytic regulation remain to be explored. So in this study, we performed a USP cleavage assay using GST-Ub52, Ub-Met-β-gal and Ub-AMC as modelsubstrates to detecte the deubiquitinating enzyme activity of USP13. At the same time, we constructed four SNPs missense mutation of USP13 to detecte their effects on the activity of USP13.Methods:1 Using p GEX-USP13 as a template, site-directed mutagenese was carried out to generate USP13(C345S) mutation. The plasmids of p GEXUSP13(C345S) and p AC-T7-USP13(C345S) were constructed.2 Escherichia coli DH.5α harboring p GEX-USP13 was incubated in LB medium at 37℃ or 15℃ overnight to express GST-USP13 fusion protein.GST fusion proteins were purified by GSH-Sepharose TM Resin(Sangon Biotech, Shanghai, China) and detected by 10% SDS-PAGE.3 Using p GEX-USP13 as a template, site-directed mutagenese was carried out to generate USP13(A107V), USP13(R217Q), USP13(G465S)and USP13(E669G) mutations. The plasmids of p GEX-USP13(A107V),p GEX-USP13(R217Q), p GEX-USP13(G465S) and p GEX-USP13(E669G)were constructed.4 USP cleavage assay: We performed a USP cleavage assay using three different types of model substrates(GST-Ub52, Ub-Met-β-gal and Ub-AMC)to detect the deubiquitinating enzyme activity of USP13.Results:1 p GEX-USP13(C345S) and p AC-T7-USP13(C345S) were constructed as expected.2 GST-USP13 fusion protein induced with IPTG at 15°C was mostly existed in supernatant, thereby enabling us to purify the fusion proteins by GSH-Sepharose TM Resin.3 USP cleavage assay: We confirmed that USP13 proteins have no detectable deubiquitinating enzyme activity detected by USP cleavage assay using three different types of model substrates(GST-Ub52, Ub-Met-β-gal and Ub-AMC).4 p GEX-USP13(A107V), p GEX-USP13(R217Q), p GEX-USP13(G465S) and p GEX-USP13(E669G) were constructed as expected. Theresults of USP cleavage assay suggested that USP13(A107V), USP13(R217Q), USP13(G465S) and USP13(E669G) mutations have no detectable deubiquitinating enzyme activity. These results were detected by USP cleavage assay using Ub-Met-β-gal as substrate.Conclusions:1 USP13 proteins have no detectable deubiquitinating enzyme activity detected by USP cleavage assay using three different types of model substrates(GST-Ub52, Ub-Met-β-gal and Ub-AMC).2 USP13(A107V), USP13(R217Q), USP13(G465S) and USP13(E669G) mutations have no detectable deubiquitinating enzyme activity, as same as USP13 wild type, detected by USP cleavage assay using Ub-Met-β-gal as substrate.Part two: Using 2D-DIGE to screen associated proteins of ubiquitin-specific protease USP13Objective: In part one, the results suggest that USP13 proteins have no detectable deubiquitinating enzyme activity detected by USP cleavage assay using three different types of model substrates(GST-Ub52, Ub-Met-β-gal and Ub-AMC). In addition to understanding the enzymatic activity of USP13,elucidating the substrate specificities of USP13 is vital to understanding its biological function. The aims of this part are to discover additional substrates or associated proteins of USP13. We took a proteomic approach by using twodimensional difference gel electrophoresis(2D-DIGE) to detect cellular proteins that change significantly in HEK293 T cell lines after expressing USP13 or its C345 S mutant(the catalytically inactive form), which can provide a new method to screen the candidate substrates and associated proteins for USP13.Methods:1 To generate the plasmids p EGFP-USP13 and p EGFP-USP13(C345S),the vectors of p GEX-USP13 and p GEX-USP13(C345S) were digested by Sal I and Not I, respectively. And the products were inserted into the p EGFP-C1 vector which was linearized by Eco R I.2 Purified recombinant plasmids p EGFP-C1, p EGFP-USP13 and p EGFP-USP13(C345S) were transfected into the HEK293 T cells with Lipofectamine 2000 transfection reagent according to the manufacturer’s instructions. After incubation for 24 hours, cells were collected by centrifugation.3 The proteins were extracted from the cells transfected with p EGFPUSP13, p EGFP-USP13(C345S) or p EGFP-C1 plasmids by SDS extraction buffer.4 Total protein extracts were labeled with Cy5- and Cy3-, respectively,and then pooled together with the treated samples prior to 2D-DIGE. For each treatment, images from at least three biological repeats were used for statistical analysis of protein abundance.Results:1 p EGFP-USP13 and p EGFP-USP13(C345S) were constructed as expected. Purified recombinant plasmids p EGFP-C1, p EGFP-USP13 and p EGFP-USP13(C345S) were transfected into the cells, and the green fluorescent protein GFP was expressed in three kinds of transfected cells.2 A total of 11 spots displayed a ≥1.1 average-fold change in abundance with P<0.05 after USP13 over-expression compared with GFP. 3 of the 11 spots still displayed a ≥1.1 average-fold increased in abundance after USP13over-expression compared with USP13(C345S).Conclusions:1 The 2D-DIGE approach provides a useful avenue by which to identify new USP substrates.2 The expression of several proteins changed significantly in HEK293 T cells after the overexpression of USP13 and its mutant.Part three: Identification and verification of differentially expressedproteins by LC-MS/MS combination with Western blotting andq RT-PCRObjective: LC-MS/MS was used to identify the differential protein spots isolating by 2D-DIGE. The m RNA levels of the identified proteins weremeasured by q RT-PCR. And the altered expression level of the identified proteins was proved by western blot.Methods:1 In order to get the samples, the protein spots isolated from part one was digest in-gel. Then, samples were analyzed by LC-MS/MS. The operating software was Xcalibur 2.0 Software and the SEQUEST method in Bio Works3.3.1 software was used for database retrieval.2 HEK293 T cell lines were transfected with p EGFP-USP13, p EGFPUSP13(C345S) or p EGFP-C1. 24 h after transfection, total m RNA extracts were analyzed by q RT-PCR to detect the m RNA expression of the identified proteins.3 HEK293 T cell lines were transfected with p EGFP-USP13, p EGFPUSP13(C345S) or empty vector(p EGFP-C1). 24 h after transfection, total protein extracts were analyzed by western blot to detect the expression of the identified proteins.Results:1 7 of the 11 species were identified by LC-MS/MS. These proteins included four increased proteins, and three decreased proteins. In addition,compared with the samples of HEK293 T cell lines after expression of USP13 and USP13(C345S), vinculin exhibited increased expression, suggesting that it may be a candidate substrate of USP13.2 Total protein extracts were analyzed by western blot to detect the expression of endogenous vinculin. Results from three independent experiments showed that over-expression of USP13, but not the enzyme-dead mutant USP13(C345S) and the empty vector, lead to a prominent increase protein levels of vinculin. On the other hand, the vinculin transcript levels were not significantly different in cells with altered USP13 expression.Conclusions:1 Mass spectroscopy analysis of gel spots identified seven proteins,including four increased proteins, namely vinculin, thimet oligopeptidase(THOP1), cleavage and polyadenylation specific factor 3(CPSF3) andmethylosome protein 50(WDR77), and three decreased proteins, namely PSMD11, TOR1 A and PGAM. In addition, compared with the samples of HEK293 T cell lines after expression of USP13 and USP13(C345S), vinculin exhibited increased expression, suggesting that it may be a candidate substrate of USP13.2 USP13 up-regulates vinculin protein levels but not m RNA level through an enzymatically mediated mechanism.