Association Between Ubiquitin-specific Protease USP46Missense Mutation A81V T187P And ADHD

Objective：Ubiquitin-proteasome pathway (UPP) which plays a pivotalrole in protein homeostasis, is critical in regulating numerous cellularprocesses including membrane trafficking, histone function, transcriptionregulation, DNA repair, and DNA replication through the combination ofubiquitin and the lysine residues of target protein. Ubiquitination is areversible reaction, and the removal of the ubiquitin is catalyzed bydeubiquitinating enzymes (DUBs). The largest group of these subfamilies isubiquitin-specific proteases (USPs). Ubiquitin specific proteases (USP) canremove ubiquitin from protein substrates and allow protein salvage fromproteasome degradation. The current study found that many of the humanubiquitin-specific protease is closely related to the occurrence of the nervousand mental disease. USP46that we researched is a member of USPs.Recent study has found that usp46is a quantitative trait gene regulatingmouse immobile behavior in the tail suspension and forced swimming tests.They found that one3-bp deletion (coding a lysine residue) in usp46is thereason that mice show shorter negligible immobility in inescapable situations.And they made a hypothesis that USP46may implicate in depression of mice.In this study, we aim to analysis the impaction of USP46A81V andT187P mutants on deubiquitinating enzyme activity by constructing theexpression plasmids of USP46missense mutation A81V, T187P andestablishing the deubiquitinating enzyme activity detection system. We alsotake a Preliminary screening on Chinese Han population and children withattention deficit hyperactivity disorder using USP46missense mutationgenotyping method and lay the foundation for further molecular epidemicstudies.Methods： (1) Using pACT7-usp46-WT and pGEX-usp46-WT plasmids those ourresearch group has cloned successfully as templates to build usp46mutantplasmid pACT7-usp46-T187P, pGEX-usp46-T187P, pGEX-usp46-A81V, andpGEX-usp46-C44S by site-directed mutagenesis method. And they wereverified by sequencing analysis to prepare for USP46mutant activity (usp46mutant C44S is44th cysteine site-directed mutagenesis to serine; A81V,81stalanine to valine; T187P,187th threonine to proline).(2) Using GST-Ub52as model substrate to detect the deubiquitinatingenzyme activity of C44S, A81V, T187P USP46mutants. USP46expressingplasmid was co-expressed with pGEX-Ub52. Deubiquitinating enzyme UBP2was used as a positive control. Using GSH-Sepharose-TM Resin purifiedprotein to purify GST fusion protein, and they were detected by10%SDS-PAGE electrophoresis. Using the activity of the USP46wild-type as astandard (the ratio between the brightness of36kDa cut off product proteinand the45kDa substrate protein), to analysis the differet activities betweenwild type and mutants by Odyssey two-color infrared laser imaging system.Rabbit anti-T7polyclonal antibody was used to detect the fusion protein T7-USP expression.(3) Using Ub-Met-β-gal as model substrate to detect the deubiquitinatingenzyme activity of C44S, A81V, T187P USP46mutants. Plasmids expressingGST fusion protein and Ub-Met-β-gal model substrate were co-transformedinto BL21competent cells. The deubiquitinating enzyme activities of wild-type and mutant proteins were compared. The deubiquitinating enzymeactivity was detected by USP cleavage assay using Ub-Met-β-gal fusionprotein as a model substrate. UBP2was used as a positive control. Mouse anti-β-gal monoclonal antibody was used to test substrate Ub-Met-beta-galexpression and rabbit anti-GST polyclonal antibody to detect GST-USP fusionprotein. Using the activity of USP46wild-type as a standard (the ratio betweenthe brightness of Met-β-gal cut off product protein and Ub-Met-β-gal substrateprotein), to analysis the deubiquitinating enzyme activity differences betweenwild-type and mutants by Odyssey two-color infrared laser imaging system. (4) Collecting blood samples of healthy population and ADHD children.(5) The usp46missense mutation detection. Genomic DNA was isolatedfrom blood sample. A81V, T187P mutations specific primers weresynthesized by Huada Gene Inc.(6) Sending specific amplification USP46A81V, T187P purposefragments PCR products to sequencing directly.(7) Using DNAStar and Chromas software to analyze the results returnedfrom the sequencing company.Results:(1) We had constructed usp46mutant plasmids pACT7-usp46T187P,pGEX-usp46T187P, pGEX-usp46A81V, pGEX-usp46C44S. The sequenceswere confirmed by restriction enzyme analysis and DNA sequencing.(2) Using GST-Ub52as model substrate to detect deubiquitinatingenzyme activities of USP46C44S, A81V and T187P mutants. USP46expression plasmid was co-expressed with pGEX-Ub52. The size of modelsubstrate GST-Ub52was about42kDa and it was reduced to the expected sizeof36kDa. USP46wild-type and A81V mutant were successful to cut off GST-Ub52to a product about36kDa, while the ability of mutant A81V to cut offthe substrate reduced, showing that the deubiquitinating enzyme activitydecreased. Mutant C44S, T187P did not cut off the model substrate,suggesting that they did not have deubiquitinating enzyme activity.Compared with the enzyme activity of wild-type, the USP46A81Vmutant decreased to42.91%±1.42%(mean±SD; n=3, P=0.000).(3) Using Ub-Met-β-gal as model substrate to detect deubiquitinatingenzyme activity USP46of C44S, A81V, T187P mutants. Negative control canbe seen as two bands: the upper side was the model substrate Ub-Met-β-gal,the lower side was the endogenous β-gal protein. Positive control showingthree bands, and Met-β-gal was between Ub-Met-β-gal and endogenous β-gal,the product of deubiquitinating enzyme removing the ubiquitin from Ub-Met-β-gal; USP46wild-type and mutant A81V were the same as the positivecontrol, while the ability of mutant A81V to cut off the substrate reduced suggesting that the deubiquitinating enzyme activity decreased. Mutants C44S,T187P can not cut off the model substrate showing that they did not havedeubiquitinating enzyme activity.Compared with the enzymatic activity of wild-type, the USP46A81Vmutant decreased to28.5%±5.96%(mean±SD; n=3, P=0.002).(4) Characteristics of the subjects: A total of122healthy adults and87ADHD children (aged5-14years) were enrolled in this study. The average ageof the ADHD children was8±1.85years (mean±SD). Among them, there were42males and45females accounting for48.28%and51.72%, respectively.(5) Genomic DNA Extraction: TIANGEN blood/cell/tissue genomicDNA kit was used for genomic DNA extracted from the anticoagulated wholeblood samples. Using1μl DNA product for1%agarose gel electrophoresis,the results showed a single band suggesting the integrity of genomic DNA wasgood.(6) Establishment of deubiquitinating enzyme usp46missense mutationdetection method.221bp A81V target fragment and347bp T187P targetfragment were amplified.(7) PCR products sequencing: Using DNAStar software to compare thesequence of PCR products with reference sequence. The healthy control group(122cases) and patient group (87cases) were sequenced, and sequencingresults showed that no mutation was found at the expected mutation basepoints.Conclusions:(1) We have successful constructed the expression plasmids of USP46A81V and USP46T187P mutants.(2) Using both GST-Ub52and Ub-Met-β-gal as model substrates todetect the deubiquitinating enzyme activities of USP46mutants, we find thatthe T187P mutant lost the deubiquitinating enzyme activity, while the A81Vmutant decreased to28.5%-42.91%compared with the wild-type.(3) We have successful established gene typing method for ubiquitin-specific protease USP46missense mutation (A81V, T187P). (4) We have not detected the USP46A81V and T187P mutations inhealthy blood donors and patients with ADHD in initial screen.