抗肥胖因子WDTC1抑制脂肪细胞分化的分子机制研究

Despite growing recognition of the problem, the obesity epidemic continues in the world, and the prevalence of obesity is increasing around the world. It has become a serious problem of how to tackle obesity and related metabolic diseases in the future. In essence, the content of adipose tissue in the body of obesity increases, and the adipogenesis plays an important role in it. The CUL4-DDB1-WDTC1 E3 ligase complex has been reported to play an important role in suppressing adipogenesis, but the physiological substrate of this complex has not been identified yet. To address the issue, we performed preliminary experiments and identified Med18, a subunit of the Mediator complex, as a potential substrate of the CUL4-DDB1-WDTC1 complex. We went on to show that Med18 was indeed required for the adipogenesis of 3T3-L1 preadipocytes. Furthermore, we showed that CUL4-DDB1-WDTC1 complex regulate the stability of Med18-Med8-Med20, the head module of the Mediator complex. We aim to further identify how the CUL4-DDB1-WDTC1 complex affects the stability of Med18, uncover the molecular mechanism of how Med18 is required for adipogenesis, and elucidate the physiological function of Med18 through knockout animal models. Our work will provide a novel mechanism of the regulation of adipogenesis.

近年来，我国肥胖患病率逐年增长，呈流行态势，如何有效防控肥胖等代谢疾病是重要的社会问题。肥胖的实质系体内脂肪组织含量增加，脂肪细胞分化起重要作用。CUL4-DDB1-WDTC1泛素连接酶E3复合物能够抑制脂肪细胞分化，但其生理底物尚未鉴定。针对这一问题，我们通过候选基因筛选的方法，发现在转录激活中起重要作用的Mediator复合物的Med18亚基是CUL4-DDB1-WDTC1复合物的潜在底物。并进一步证实了Med18对于脂肪细胞分化是必需的，且发现CUL4-DDB1-WDTC1复合物对于Med18-Med8-Med20组成的Mediator头部复合物的稳定性具有重要调控作用。我们将进一步从生化、分子和生理水平上明确CUL4-DDB1-WDTC1对Med18蛋白稳定性调控的分子基础，阐明Med18影响脂肪细胞分化的机制，揭示Med18的生理功能，有望从新的角度揭示脂肪细胞分化的调控机制。

WDTC1作为一个抗肥胖因子，通过形成CUL4-DDB1-WDTC1 E3 泛素连接酶复合体的形式来抑制脂肪细胞分化，但其作用底物及其分子机制尚不清楚。因此本课题以CUL4-DDB1-WDTC1及其作用底物蛋白为研究对象，探讨其影响脂肪细胞分化的分子机制，并在动物水平上揭示该底物蛋白的分子生理功能。在细胞水平，我们利用3T3-L1脂肪前体细胞以及Rosa-CreERT2;Cul4af/f 和 Rosa-CreERT2;Cul4bf/y 小鼠白色脂肪组织的原代细胞，发现WDTC1不影响C/EBPβ，KLF5等在脂肪细胞分化前期发挥重要作用的转录因子，但是能够明显抑制脂肪细胞分化晚期转录因子C/EBPα，PPARγ，KLF15的表达；我们通过候选基因筛选的方法，发现Meditor复合体的亚基MED20是CUL4-DDB1-WDTC1复合物的底物蛋白，并在分子机制上验证了CUL4-DDB1-WDTC1复合物通过降解MED20蛋白水平而不是调控其RNA水平抑制脂肪细胞分化起作用。在动物实验方面，我们构建了Pdgfra-Cre;Med20f/f基因型小鼠，发现Med20对于小鼠棕色脂肪组织的发育必不可少。我们的研究揭示了WDTC1的抑制脂肪细胞分化的分子机制，鉴定了其作用底物MED20在脂肪分化中的作用及其机制，为开发防治肥胖的靶向药物提供了理论基础。