【作者】 张晓辉；

【导师】 窦好；

【作者基本信息】 山东大学 ， 遗传学， 2020， 硕士

【摘要】 [研究背景]在我国,肥胖及Ⅱ型糖尿病已成为严重影响人们生命健康及生活质量的重大疾病,由此引发的以胰岛素抵抗为核心的高血糖、高血压、高血脂等并发症也已经给人类带来了沉重的负担。脂肪细胞分化是正常体内脂肪贮存的生理基础,脂肪细胞的增殖和分化异常会引起脂肪组织过多堆积,导致能量代谢失衡,引起肥胖以及与肥胖有关的一系列疾病。过氧化物酶体增殖物激活受体(PPARs)属核受体超家族成员,PPARs家族有PPARα、PPARβ、PPARy三个成员。它们在脂肪细胞中高表达,对脂肪分化、能量平衡和脂类生物合成意义重大。其中,PPARγ作为脂肪细胞分化的主要调节者,分为两个亚型:PPARγ1和PPARγ2。PPARγ2在鼠中比PPARy1多28个氨基酸,在人类中多30个氨基酸。PPARγ的表达和活性受到翻译后修饰的调节,包括磷酸化、类泛素化、泛素化、乙酰化和糖基化修饰等。CUL4B属于Cullin基因家族,在肿瘤的发生发展中发挥着重要的作用。CUL4B的作用机制包括:参与细胞周期调控、维持基因组稳定性、调控相关通路、参与免疫调节、调控表观遗传学修饰等。此外,CUL4B是泛素连接酶E3的骨架蛋白,其氨基端与DDB1结合,羧基端与ROC1结合共同构成CUL4B-RING泛素连接酶(CRL4B)的核心结构。而DDB1则通过结合不同的底物受体DCAFs(DDB1 and CUL4-associated factors),特异性招募底物,并对底物进行泛素化修饰。近年来,本实验室研究发现,PPARγ可被CRL4B泛素化修饰,从而影响脂肪细胞分化和机体对胰岛素的敏感性。但其泛素化修饰机制尚不清楚,比如:PPARγ发生泛素化的赖氨酸位点以及PPARγ的泛素化与乙酰化之间的关系[研究目的]确认泛素化位点将有助于深入理解PPARγ的泛素化降解机制,为治疗肥胖相关的疾病提供理论基础。本课题发现PPARγ被CRL4B泛素化修饰的赖氨酸位点,明确PPARγ泛素化与乙酰化之间的关系,研究AhR的入核机制对PPARy泛素化水平的影响。[研究方法]利用邻位连接技术检测小鼠脂肪组织中PPARγ与AhR间的相互作用;利用定点突变和细胞内泛素化实验证明AhR所参与的PPARγ的泛素化修饰机制;利用蛋白质质谱分析、细胞内泛素化实验以及半衰期实验确定PPARγ的泛素化修饰位点。[研究结果]1、AhR负向调节脂肪分化。为了研究AhR对脂肪分化的影响,发现小鼠在cul4b缺失时,小鼠脂肪组织内PPARγ蛋白水平升高;利用邻位连接技术发现cul4b缺失时,PPARγ与AhR间的相互作用也明显增强;并且在脂肪分化过程中,AhR蛋白水平总体呈下降的趋势。2、AhR参与PPARγ的泛素化修饰。为研究AhR对PPARγ泛素化修饰的影响,发现CRL4BAhR介导的PPARγ泛素化修饰发生在细胞核内,并且AhR的激动剂3MC明显增强PPARγ泛素化水平。发现点突变(AhR F287A)和入核抑制剂(MDL-28170)降低PPARγ的泛素化水平。3、PPARγ的两个泛素化修饰位点为K268、K293,并且这两个位点的乙酰化和泛素化之间存在位点竞争。为了研究PPARγ的两个泛素化修饰位点,利用蛋白质质谱法筛选潜在的泛素化位点。根据质谱结果构建不同类型的突变型质粒,发现PPARγ上的两个赖氨酸被泛素化修饰,分别是K268、K293。这两个位点同时也是PPARγ乙酰化位点,通过加入去乙酰化酶的抑制剂NAM或敲低AhR,发现乙酰化和泛素化之间存在位点竞争。[研究结论]在CRL4B连接酶复合物中,AhR作为底物受体蛋白招募PPARγ,参与介导PPARy泛素化修饰。该泛素化修饰和蛋白质降解过程均发生在细胞核内,并且会受到其乙酰化修饰的影响。更多还原

【Abstract】 BackgroundIn China,obesity and type 2 diabetes are becoming the most common diseases that seriously affect human,health and quality of life.High blood glucose with insulin resistance is the main cause of blood pressure and hyperlipidemia.Adipocyte differentiation is the physiological basis of normal fat storage.Abnormal proliferation and differentiation of adipocyte can cause excessive accumulation in adipose tissue,imbalance in energy metabolism,and a series of diseases related to obesity.Peroxisome proliferator-activated receptors(PPARs)belong to nuclear receptor superfamily,which includes PPARα,PPARβ,and PPARy.They are highly expressed in adipocytes and play important roles in adipose differentiation,energy balance,and lipid biosynthesis.Among them,PPARγ is a key regulator of adipocyte differentiation.There are two isoforms of PPARy:PPARy 1 and PPARγ2.Compared to PPARγ1,PPARγ2 has additional 28 amino acids in mice and additional 30 amino acids in human.The expression and activity of PPARγ are regulated by post-translational modifications.These post-translational modifications include phosphorylation,ubiquitination,acetylation,and glycosylation.CUL4B plays an important role in tumorigenesis and development.CUL4B has many cellular roles,such as:participating in cell cycle regulation,maintaining genome stability,participating in immune regulation,and regulating epigenetic modification.In addition,CUL4B functions as a scaffold protein in ubiquitin ligase E3.Its N-terminus binds DDB1,and C-terminus binds ROC1 to form the core structure of CUL4B-RING ubiquitin ligase(CRL4B).DDB1 specifically recruits substrates receptors,named DCAFs(DDB1 and CUL4-associated factors)which directly interact with substrates.In recent years,we found that PPARy is ubiquitinated by CRL4B,which affects adipocyte differentiation and body sensitivity to insulin.However,the detailed ubiquitination mechanism of PPARy is unclear,such as the ubiquitinated lysine site of PPARy and the relationship between PPARγ ubiquitination and acetylation.ObjectiveUbiquitination sites will help us to understand the ubiquitination degradation mechanism of PPARγ and provide a theoretical basis for the treatment of obesity-related diseases.We investigate the lysine sites of PPARγ modified by CRL4B ubiquitination,clarify the relationship between PPARy ubiquitination and acetylation,and illustrate the effect of AhR localization on the ubiquitination level of PPARy.Research methodsUsing the proximity ligation assay to detect the interactions between PPARγ and AhR in mouse adipose tissue;using site-directed mutagenesis and ubiquitination assays to illustrate the ubiquitination modification mechanism of PPARγ facilitated by AhR;using protein mass spectrometry analysis and protein stability assay to identify the ubiquitination modification sites on PPARγ.Results1.AhR negatively regulates fat differentiation.In order to study the effect of AhR on fat differentiation,it was found that the level of PPARγ protein in mouse adipose tissue increased when cu14b was deleted;the interaction between PPARy and AhR was also significantly enhanced when cul4b was deleted using the ortho-linking technique;During the process of fat differentiation,the level of AhR protein generally showed a downward trend.2.AhR is involved in the ubiquitination modification of PPARγ.To study the effect of AhR on PPARy ubiquitination modification,it was found that CRL4BAhR-mediated PPARγ ubiquitination modification occurred in the nucleus,and AhR agonist 3MC significantly enhanced PPARγ ubiquitination level.Discovery of point mutation(AhR F287A)and nuclear inhibitor(MDL-28170)reduces PPARy ubiquitination3.The two ubiquitination modification sites of PPARγ are K268 and K293,and there is site competition between the acetylation and ubiquitination of these two sites.In order to study the two ubiquitination modification sites of PPARγ,protein mass spectrometry was used to screen potential ubiquitination sites.According to the results of mass spectrometry,different types of mutant plasmids were constructed,and it was found that the two lysines on PPARγ were modified by ubiquitination,namely K268 and K293.These two sites are also PPARγ acetylation sites.By adding the deacetylase inhibitor NAM or knocking down AhR,it is found that there is site competition between acetylation and ubiquitination.ConclusionIn the CRL4B ligase complex,AhR as a substrate receptor protein recruits PPARγand participates in mediating PPARγ ubiquitination.Both ubiquitination modification and protein degradation occur in the nucleus.Besides,the ubiquitination of PPARγ is affected by its acetylation.更多还原