Modulation of the activity of a key metabolic regulator Small Heterodimer Partner by post-translational modifications

Small Heterodimer Partner (SHP);Protein Arinine Methyltransferase 5 (PRMT5);arginine methylation;phosphorylation;protein kinase C zeta (PKC zeta);obesity and diabetes

Kanamaluru, Deepthi

关键词: Small Heterodimer Partner (SHP);    Protein Arinine Methyltransferase 5 (PRMT5);    arginine methylation;    phosphorylation;    protein kinase C zeta (PKC zeta);    obesity and diabetes;   

Others  :  https://www.ideals.illinois.edu/bitstream/handle/2142/24491/Kanamaluru_Deepthi.pdf?sequence=1&isAllowed=y

美国|英语

来源: The Illinois Digital Environment for Access to Learning and Scholarship

【 摘 要 】

Small Heterodimer Partner (SHP, NR0B2), a member of the nuclear receptor superfamily, is anorphan receptor that lacks a DNA binding domain but contains a putative ligand binding domain.SHP forms non-functional heterodimers with DNA binding transcriptional factors and, thereby, functions as a transcriptional corepressor in diverse biological processes, including cellular metabolism, cell proliferation, apoptosis, and sexual maturation. Of these reported functions of SHP, maintaining cholesterol and bile acid levels by negative feedback regulation of hepatic conversion of cholesterol to bile acids is well established. Cholesterol is essential in many biological activities in mammalian cells. Conversion of hepatic cholesterol into bile acids is a major pathway to eliminate cholesterol from the body. However, excess amounts of cholesterol and bile acids are pathogenic. Therefore, the levels of cholesterol and bile acids need to be tightly regulated. Cholesterol 7α-hydroxylase (CYP7A1), a liver specific P450 enzyme, is the first and rate-limiting enzyme in this process. Increased levels of bile acids repress transcription of CYP7A1 in a feedback manner. In response to elevated bile acid levels, the nuclear bile acid receptor Farnesoid X Receptor (FXR) increases the transcriptionof SHP. SHP interacts with the hepatic DNA-binding activators, hepatic nuclear factor-4α (HNF-4α) or liver receptor homologue-1 (LRH-1) on the CYP7A1 promoter, and represses transcription of the CYP7A1 gene. In addition to regulating cholesterol and bile acid levels, SHP is known to mediate inhibition of fatty acid synthesis, hepatic lipogenesis, and glucose production in response to elevated bile acid levels.Posttranslational modifications profoundly regulate protein stability and activity.Recently, bile acids have been reported to function as signaling molecules that activate kinasepathways. We recently found that SHP stability is increased by bile acid-activated ERK-mediated phosphorylation through inhibition of ubiquitination. We now show that the activity of SHP is increased by post-translational methylation of SHP at Arg-57 by protein arginine methyltransferase 5 in response to bile acids. The overall aim of this study is to delineate the molecular mechanism by which the post-translational modification of SHP regulates SHPfunctional activity.In recent years, several naturally-occurring mutations in the SHP gene have been reportedin human subjects that are associated with mild obesity and diabetes. About 30% of thesereported mutations were Arg mutations, including the R57W mutation. Though it is known thatthe mutations lead to metabolic disorders, the molecular basis underlying the mechanism bywhich the mutations lead to metabolic disease is unknown. By mass spectrometry, we identifiedArg 57 as a site of methylation in SHP catalyzed by Protein Arginine Methyltransferase 5(PRMT5). Functional activity assays showed that methylation of SHP at Arg-57 by PRMT5 isimportant for SHP inhibition of LRH1 and HNF-4α transactivation. Our lab previously showed the molecular mechanism of SHP-mediated repression involving the coordinate recruitment of chromatin modifying repressive cofactors, mSin3A/HDAC1, NCoR1/HDAC3, methyltransferase G9a, and the Swi/Snf-Brm remodelingcomplex, to the CYP7A1 promoter. Mutation of the Arg-57 site to Trp (R57W is the naturallyoccurringmutant) decreased SHP interaction with corepressors that we had previously identified, and severely impaired inhibition of gene expression by SHP. Overexpression of wild type SHPin mouse liver resulted in decreased lipogenic, bile acid synthetic and gluconeogenic geneexpression, and mutation of Arg-57 blocked SHP function, but remarkably in a gene-selectivemanner. Overexpression of the R57W mutant resulted in elevated levels of triglycerides and bile acids in liver compared to that of wild type SHP. Differential interaction and recruitment of corepressors by SHP in a promoter-specific manner may contribute to gene-selective repression by the R57W mutant.Our studies have shown that SHP is methylated by PRMT5 after bile acid treatment. Tandem mass spectrometry revealed that in addition to methylation at Arg-57, SHP is also phosphorylated at Thr-55 after bile acid treatment. Studies with kinase inhibitors showed that a signaling pathway involving PI3K and PKC ζ is involved in SHP Thr phosphorylation, and also regulates arginine methylation of SHP. The close proximity of the phosphorylation (Thr-55) andmethylation (Arg-57) sites suggested a possible interplay between them. Studies withphosphorylation- and methylation-defective mutants demonstrated crosstalk between SHP Thrphosphorylation and Arg methylation. This study demonstrates a critical role for Arg-57 methylation by PRMT5 in SHP function, and suggests a possible mechanism for association of the reported R57W mutation with obesity. This study also reveals Thr-55 phosphorylation of SHP by upstream kinase signaling pathways to be important for SHP functional activity. Targeting post-translational modificationsof SHP may be an effective strategy to develop new therapeutic agents to treat SHP-related human diseases, such as metabolic syndrome, cancer, and infertility.