| The ubiquitin-proteasome system, which is highly conserved from yeastto man as the principal means of targeting cytosolic proteins for degradation,has been implicated in wide variety biological processes, including cell cycle,immune response, tumorigenesis and inflammation. Covalent linkage ofubiquitin to target proteins is directed by the orchestrated activity of aubiquitin activating enzyme (E1), a ubiquitin conjugating enzyme (E2) and asubstrate specific ubiquitin ligase (E3) that mediates the transfer of ubiquitinto the target. Similar to phosphorylation, ubiquitylation is a reversible processthat is counter-regulated by deubiquitylating enzymes (DUBs).Despite the identification of a large number of DUBs, our knowledge ofthe function and activities of this family of enzymes is just starting toaccumulate.Ubiquitin-specific protease46(Usp46) and ubiquitin-specific protease26(Usp26) were belonging to Ubiquitin-specific protease (USP), a subfamily ofdeubiquitinating enzymes (DUBs). However, their function is not wellunderstood.Recently, Usp46was detected as an quantitative trait gene responsible forthe immobility in the tail suspension and forced swimming tests in mice. Inthe study, mice with a lysine codon (Lys92) deletion of Usp46showed loss of‘behavioral despair’ under inescapable stresses in addition to abnormalities incircadian behavioral rhythms and the GABAergic system. However, themolecular mechanism of the regulation of GABAergic system is unknown.This study aims to detect the influence of Lys92deletion and the missenseSNPs in coding region of Usp46on deubiquitinating enzyme activity by USPcleavage assay, and develop genotyping methods of cSNPs in population, laying the foundation for further exploration of the associated diseases bydeveloped genotyping methods of these SNPs.Usp26, is an X-linked gene located at Xq26.2. Recently, it has beenreported that mutations in the Usp26gene may be associated with maleinfertility. This study aims to assess the strength of the association betweenfive frequent mutations (370–371ins ACA,494T>C,1423C>T,1090C>Tand1737G>A) and male infertility and to evaluate the impact of thosemutations on enzymatic activity of the USP26.Part One: The impact of mutations of Usp46gene on its deubiquitinatingenzyme activity and could not be detected in Chinese people and ADHDpatients.Objective: Deubiquitinating enzymes (DUBs) regulate diverse cellularfunctions by their activity of cleaving ubiquitin from specific proteinsubstrates, which have been implicated in wide variety biological processes.The abnormal of DUBs were involved in the pathogenesis of numerousdiseases. Recently, ubiquitin-specific peptidase46(Usp46), a newquantitative trait gene responsible for immobility in the tail suspension testand forced swimming test in mice, has been described. This study aims todetect whether the Lys92deletion mutant and the V96I missense SNP incoding region of Usp46influence its deubiquitinating enzyme activity. Thisstudy also aims to develop the genotyping methods of V96I cSNP inpopulation，and explore the association between V96I mutant and ADHD.Methods: The USP cleavage assay, using GST-ub52and ub-Met-β-gal asmodel substrates, was conducted to assess enzymatic activity of the mutants.Genotyping methods were developed by amplified SNP loci spreadthroughout a genome using designed specific primers followed by accuratelysequencing.122healthy individuals and100ADHD patients were genotypedby this method.Results: We found that, compared to wild type, the Lys92deletionmutant resulted in a decreased deubiquitinating enzyme activity of27.04%±7.50%by GST-Ub52assay and42.78%±6.47%by Ub-Met-β-gal assay. Interestingly, the V96I cSNP also resulted in a decreased enzymatic activity of38.51%±1.40%by GST-Ub52assay and50.79%±2.60%by Ub-Met-β-galassay. The genotyping methods of V96I cSNP worked well and achieved100%accuracy in clinical samples. We did not detected this cSNP in100ADHD patients and122healthy individuals.Conclusions: These results suggest that the Lys92deletion of Usp46could influence enzyme activity and thereby provide a molecular clue how theenzyme regulating the pathogenesis of psychiatric disorder.Our results suggest that V96I cSNP of human Usp46like Lys92deletionalso influences deubiquitinating enzyme activity. However, we did notdetected this cSNP in ADHD patients and healthy individuals, suggesting thatV96I cSNP of human Usp46may be not involved in the pathology of ADHD.This study lays the foundation for further exploration of associated psychiatricdisorder.Part Two: The impact of370–371ins ACA,494T>C,1423C>T,1090C>T and1737G>A mutations on enzymatic activity of USP26.Objective: The association between mutations in Usp26gene and maleinfertility has been studied intensively. However, the results from differentgroups are controversial. In particular, biological function of the mutantproteins remains to be elucidated. While meta-analysis in the first part of thisstudy found no association between the the Usp26gene mutations and maleinfertility, this part of study aims to evaluate the impact of those mutations onenzymatic activity of the USP26in order to provide new clues on thephysiologic function of USP26and its role in male infertility.Materials:Plasmid construction of pGEX-Usp26. Using pGEX-Usp26as a template,site-directed mutageneses were carried out to generate pGEX-Usp26(370–371ins ACA,494T>C,1423C>T,1090C>T and1737G>A) plasmids.Mutations370–371insACA (T123–Q124ins),1423C> T (H475Y) and494T>C (L165S) of Usp26were always identified together in the same allele, andwere termed ‘cluster’ mutation. We also generate the cluster haplotype mutation. Mutations were confirmed by DNA sequencing.Plasmid construction of pAC-T7-Usp26. pAC-T7-Usp26plasmid wasproduced by inserting the complete coding sequence of Usp26from pGEX-Usp26into pAC-T7plasmid at the BamHI site, and confirmed by byrestriction enzyme digestion.USP cleavage assay. Using GST-ub52and ub-Met-β-gal as modelsubstrates, the USP cleavage assay were conducted to assess enzymaticactivity of the mutants. Then the results were detected by Odyssey InfraredImaging System.Results:The370–371ins ACA,494T>C,1423C>T,1090C>T and1737G>Amutations of Usp26, which result in amino acid changes T123–Q124ins,L165S, H475Y, L364F, M579I, have the same activity compared to the wildtype, detected by USP cleavage assay using both GST-Ub52and Ub-Met-β-gal as substrates.Furthermore we constructed the plasmid of cluster mutation, which weremutated in370–371ins ACA,494T>C and1423C>T. As same as the singlemutation, the cluster mutants of USP26did not affect the deubiquitinatingenzyme activity either.Conclusion: Taken all together, these results suggest that mutations ofUsp26,370–371ins ACA,494T>C,1423C>T,1090C>T,1737G>A andcluster, do not influence the deubiquitinating enzyme activity of USP26. Thisassay provides biological evidence for the lack of association in meta-analysis.Part Three: Evidence from meta-analysis does not support a directassociation between Usp26gene variants and male infertilityObjective: Male factor infertility is a complex disorder that affects alarge sector of the population and accounts for approximately50%of causesfor infertile couples, who make up15%of all couples. Many factors in thecausation of male infertility are being recognized, such as occupation,environmental and particularly genetic factors. Especially, geneticabnormalities are thought to account for15%–30%of male factor infertility, by influencing a variety of physiological processes including hormonalhomeostasis, spermatogenesis, and sperm quality. Several studies havedemonstrated the possible association between the infertile male phenotypeand selected gene variants, including chromosomal aberrations, Ychromosome microdeletions, gene mutation, and gene polymorphisms.Recently, it has been reported that mutations in the testis-specific ubiquitinprotease26(Usp26) gene may be associated with male infertility. However,the results from different groups are controversial. This study aims to assessthe strength of the association between five frequent mutations (370–371insACA,494T>C,1423C>T,1090C>T and1737G>A) and male infertility.Materials:Selection criteria and Identifying studies. Studies were selected througha computerized search of PubMed, HuGNet, and Chinese NationalKnowledge Infrastructure databases by using the keywords ‘‘male infertility,’’‘‘ubiquitin specific protease26’’ and ‘‘Usp26’’ The scope of the computerizedliterature search was expanded on the basis of the reference lists of retrievedarticles. Case-control studies testing the association of Usp26polymorphismswith male infertility were included. When there were multiple publicationsfrom the same study group, the most complete and recent results were used.Statistical analysis. All data were analyzed by Stata11.0software. Tomeasure the strength of genetic association, pooled odds ratios (ORs) withcorresponding95%confidence intervals (CIs) were calculated. Thesignificance of the pooled OR was determined by the Z-test and P<0.05wasconsidered as statistically significant. Heterogeneity was tested by theCochran Q statistic and I2. We conducted subgroup analysis of the clustermutations (370–371ins ACA,494T>C and1423C>T). considered to bestatistically significant. Meanwhile，we did the sensitivity test to examingthe stabilization of the meta-analysis. Funnel plot and Harbord test weredrafted to estimate the publication bias.Result:The initial search identified22potentially relevant studies, of which9 case-control studies met all our inclusion criteria. Thus9full papers, allpublished in English, were included in the current review.Cluster. Seven studies with1297cases and2293controls investigatingthe association between the cluster mutations of Usp26and male infertilitywere included in the analysis. Formal testing for heterogeneity among studiesyielded a Cochran’ s Q test P-value of0.29with an I2value of17.2%,indicating slightly heterogeneity. The pooled random-effects ORs(corresponding95%CI) was1.55(95%CI:0.82-2.93), indicating that clustermutations are not significantly associated with infertility.Categorical meta-analyses also indicated that there is little evidence foran association with the cluster mutations in ethnic groups. The odds ratio formeta-analysis under fixed-effects was not significant neither for populationsof Caucasian descent (OR=1.625,95%CI:0.664,3.977) nor Asian descent(OR=2.252,95%CI:0.865,5.865).A cumulative meta-analysis by year of publication showed a trend of noobvious association between cluster mutations and fertility status asinformation accumulated.Funnel plots were sysmetric, and Harbord test verified that there were nopublication bias(P>0.05).Our analyses using the logit method confirmed that prevalence values ofcluster mutations among controls were highly heterogeneous. The I2valuewas53.4%, with a P-value of0.018for the overall Q statistic, suggesting thatthe individual studies were too different to combine to generate a pooledestimate for prevalence.In the stratified analyses by control sources, still, no significantassociations were observed. The pooled random-effects ORs and95%CIwere1.876(95%CI：0.812，4.333)，1.481，（95%CI：0.613-3.579）and1.597（95%CI：0.230-11.097）of men with normozoospermia, provenfertility and unknown fertility status，respectively).1090C>T. A total of four studies with866cases and558controls wereincluded in the analysis of1090C>T. The pooled random-effects ORs (and corresponding95%CI) was1.598(0.933-2.735), indicating that the1090C>Tmutation was not significantly associated with infertility. The heterogeneityamong these included studies did not show significance (P=0.49, I2=0). Acumulative meta-analysis by year of publication showed a trend of no obviousassociation between1090C>T mutation and fertility status. Funnel plotswere sysmetric, and Harbord test verified that there were no publicationbias(P>0.05).1737G>A. Three eligible studies with688cases and504controls wereincluded in the analysis of1090C>T. There was moderate heterogeneityamong the studies (I2=32.2%, P=0.229). The association with male infertilitywas not significant under random effects model (OR=2.641,95%CI:0.969,7.194). The cumulative meta-analysis by year of publication also showed atrend of no obvious association between1737G>A mutation and fertilitystatus. Funnel plots were sysmetric, and Harbord test verified that there wereno publication bias(P>0.05).Conclusion： Evidence from meta-analysis does not support a directassociation between Usp26variants and male infertility. |
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