蛋白质棕榈酰化对小鼠棕色脂肪组织产热功能的影响及其调控机制OA

AIM:To investigate the regulatory role and molecular mechanisms of protein palmitoylation in the thermogenic function of mouse brown adipose tissue(BAT).METHODS:Eight-week-old male C57BL/6J mice were ran-domly assigned to control group(housed at room temperature;n=6)and acute cold exposure group(4℃for 8 h;n=6).Interscapular BAT was collected for subsequent analyses.Primary preadipocytes isolated from C57BL/6J mice were in-duced to differentiate into mature brown adipocytes and were then divided into vehicle control(DMSO)group,β3-adrener-gic receptor agonist CL316243(10 μmol/L)group,and CL316243 combined with palmitoylation inhibitor 2-bromopalmitic acid(2-BP;50 or 100 μmol/L)groups,followed by treatment for 24 h(n=3).qPCR was performed to determine the mRNA expression levels of zinc finger DHHC domain-containing(ZDHHC)family members and thermogenesis-related factors,uncoupling protein 1(UCP1),peroxisome proliferator-activated receptor gamma coactivator 1 alpha(PPARGC1A),PR/SET domain 16(PRDM16)and cell death-inducing DFFA-like effector A(CIDEA).Western blot was used to assess UCP1 protein expression.Acyl-biotin exchange assay coupled with mass spectrometry was used to analyze protein palmi-toylation levels and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway enrichment.Co-immunoprecipitation was performed to verify protein-protein interactions.RESULTS:Compared with control group,the mRNA expression levels of ZDHHC5,ZDHHC7,ZDHHC13 and ZDHHC23 in mouse BAT after cold exposure were significantly increased(P<0.01),while ZDHHC2 and ZDHHC12 expression levels were significantly decreased(P<0.05).Treatment with 2-BP sig-nificantly suppressed CL316243-induced up-regulation of thermogenic factors,including UCP1,PPARGC1A,PRDM16 and CIDEA(P<0.05 or P<0.01).Following cold exposure,2 055 palmitoylation sites in BAT exhibited significant altera-tions(fold change>2.0,P<0.05),among which the palmitoylation level of the thermogenic protein UCP1 was markedly increased.Treatment with 2-BP reduced UCP1 palmitoylation in a time-dependent manner,with near-complete inhibition observed after 48 and 72 h.Mutation of Cys305 resulted in an almost complete loss of the UCP1 palmitoylation signal.Overexpression of ZDHHC7 significantly enhanced UCP1 palmitoylation and promoted a specific interaction with UCP1.The UCP1 protein levels were significantly reduced after 48 and 72 h of 2-BP treatment(P<0.05 or P<0.01).CONCLU-SION:In mouse brown adipocytes,protein palmitoylation promotes thermogenesis,and palmitoylation at Cys305 of UCP1 is essential for maintaining its protein stability.