Mechanistic Study On The Modification And Stability Of Insig-2 Protein

Cholesterol,the most abundant steroid of animal tissues,performs several essential functions in the body.In mammals,the de novo synthesis of cholesterol,from acetyl-CoA through a cascade of enzymatic reactions,is mainly regulated by the known feedback control systems:sterol-regulated degradation of 3-hydroxy-3-methylglutaryl(HMG)CoA reductase and maturation of sterol-regulatory element-binding protein(SREBP)(Goldstein et al.,2006).Insigs(Insulin-induced genes)are key proteins in regulating the degradation of HMGCR and maturing of SREBP.Insigs,include Insig-1 and Insig-2,are closely related polytopic membrane proteins of the endoplasmic reticulum(ER).They regulate lipid homeostasis by binding to two ER proteins,HMG-CoA reductase(HMGCR)and SCAP(SREBP cleavage-activating protein),in a sterol-dependent manner(Gong et al.,2006).In cholesterol biosynthesis,Insigs mediate the sterol-accelerated proteasomal degradation of HMGCR,the rate-limiting enzyme,by the recruitment of some E3s,such as,gp78,RNF145 and TRC8.Besides,Insigs bind the SCAP/SREBP complex,inhibiting the maturing of SREBP.The stabilities of both Insig-1 and Insig-2 are under the control of the ER-membrane-bound E3,gp78,while Insig-1 is much short-lived than Insig-2 in cultured cells.Deficiency of gp78 in liver dramatically increases the stability of Insig-2(Liu et al.,2012).The degradation of Insig-1 has been well studied.However,it remains to be explored about the protein modification and stability regulatory mechanism of Insig-2.Our previous work showed that constitutive knockout gp78 mice exhibit markedly increased Insig-2 protein level in several tissues,including livern WAT,kidney.Interestingly,the Insig-2 protein amounts remained unaltered in the skeletal muscle of global gp78-deficient mice compared with that of the wild-type(WT)littermates.It reminds us that there may be a distinct regulatory mechanism of Insig-2 in muscle.We found that Cys215(C215)is the ubiquitination site of Insig-2,and the YEC215K motif is unique in Insig-2 compared to Insig-1.Consistently,Insig-2,but not Insig-1,was oxidized as measured by the DCP-Biol probe on the C215 residue,which competes with the ubiquitination of Insig-2,suggesting that the cellular redox state was responsible for the regulatory mechanism on the stability of Insig-2.In this study,we unveil the mechanism of muscle-specific resistance of the proteasomal degradation of Insig-2 mediated by gp78.The oxidation of cysteine215 competes with the conjugation of ubiquitin chains on the same amino acid residue,and thus stabilized Insig-2.Furthermore,we discover elevated Insig-2 during the myogenic differentiation of myoblast,accompanied with increased ROS(Reactive oxygen species)level.Moreover,genetic disruption of Insig-2 alters the expression of relative genes on fatty acid synthesis,and induces severe lipid accumulation in differentiated myotubes.Taken together,our study reports a novel tissue-specific competitive oxidative modification on the stability of Insig-2,modulating lipid metabolism.Our findings provide new ideas for further exploration about therapeutic approaches against lipid metabolic diseases in muscle by targeting Insig-2.