组蛋白读体Sp100C及微管乙酰化酶αTAT1的结构功能研究

发布时间：2018-04-16 10:12

本文选题：组蛋白修饰 + PML核体　；参考：《清华大学》2016年博士论文

【摘要】：结合染色质的读体蛋白对组蛋白翻译后修饰的识别是表观遗传调控的一个主要机制。细胞核抗原Sp100(斑点状、分子量为100 kDa的蛋白)是早幼粒细胞性白血病核体(PML-NB)的组成型成分,在固有免疫及转录抑制中发挥重要作用。其剪接亚型Sp100C的羧基端具有独特的植物同型结构域锌指(PHD)和溴区结构域(Bromo)串联模块(Sp100CPB),且可以被干扰素刺激特异性的上调。运用结构生物学、定量结合实验及细胞共定位分析的方法,我们揭示Sp100C的PHD结构域不仅可以识别组蛋白H3第4位赖氨酸非甲基化状态(H3K4me0),还可以容忍组蛋白H3第3位苏氨酸的磷酸化(H3T3ph)、第9位赖氨酸的三甲基化(H3K9me3)及第10位丝氨酸的磷酸化(H3S10ph)。这些修饰类型都与细胞分裂期染色体相关,我们的实验也表明Sp100C可通过对组蛋白的识别调控细胞周期。与之对比,旁系同源蛋白Sp140的PHD结构域也可以识别H3K4me0,但丧失了对H3T3ph、H3K9me3、H3S10ph的容忍能力。Sp100CPB-H3K4me0复合物晶体结构表明,Sp100C延伸的氨基端可形成高度协调的“配位层”类似结构来识别H3K4的ε-氨基,且二硫键固化后的口袋对H3K4me0的识别增强。Sp100CPB-H3T3ph复合物晶体结构表明,H3T3ph的识别是通过Sp100C的PHD结构域特有的精氨酸实现的。本研究揭示出Sp100C有直接靶向染色质的能力,并可能通过这种方式响应干扰素刺激,在PML-NB介导的固有免疫和基因沉默中发挥限制性作用。而细胞质内的微管骨架上也存在着类似的微管蛋白密码。目前唯一位于微管腹腔一侧的翻译后修饰是α微管蛋白第40位赖氨酸的乙酰化(K40ac),由物种间高度保守的酶αTAT1催化,但其催化反应的分子机制尚不清楚。在本文中,我们解析了鼠αTAT1催化结构域和辅因子乙酰辅酶A(AcCoA)复合物的晶体结构。除了采用GNAT乙酰基转移酶超家族核心结构域相似的折叠外,αTAT1具有额外的底物识别相关的β4/β5插入片段、独特的正电荷区及必需的氨基端疏水区。AcCoA腺苷部分被αTAT1残基侧链以特殊的“三明治”构型紧紧结合在核心结构域中央沟槽内。αTAT1的催化活性依赖于K40所在区域的环形构象及微管原丝间的侧向相互作用。电镜实验表明αTAT1除结合K40位点外,还可能有位于微管外侧的结合位点。本研究阐释了αTAT1的结构特征,为进一步探讨αTAT1与微管识别模式及其对微管相关生命活动的调控作用提供了重要依据。
[Abstract]:The recognition of posttranslational modification of histone by chromatin reading proteins is a major mechanism of epigenetic regulation.Nuclear antigen Sp100 (spotted protein with molecular weight of 100 kDa) is a constituent of PML-NBs in promyelocytic leukemia and plays an important role in innate immunity and transcription inhibition.The carboxyl terminal of splicing subtype Sp100C has a unique phyto-homotypic domain (PHD) and bromo-bromo-bromodomain (SP100CPBN), which can be specifically up-regulated by interferon stimulation.Using the methods of structural biology, quantitative combination experiment and cell co-localization analysis,We reveal that the PHD domain of Sp100C can recognize not only the non-methylated state of histone H3, but also the phosphorylation of threonine at H3, trimethylation of lysine, H3K9me3, and phosphorylation of H3S10, respectively, of histone H3, H3K9me3 and H3K4me0, respectively.These modification types are related to the chromosomes of cell division phase. Our experiments also show that Sp100C can regulate cell cycle through recognition of histone.By contrast, the PHD domain of the homologous protein Sp140 can also recognize H3K4me0, but the crystal structure of the complex. Sp100CPB-H3K4me0 complex shows that the extended amino terminal of Sp100C can form a highly coordinated "coordination layer" similar structure to recognize the 蔚 -amino of H3K4.Moreover, the enhanced recognition of H3K4me0 in the cured pocket of disulfide bond. The crystal structure of Sp100CPB-H3T3ph complex indicates that the recognition of H3T3ph is realized by the arginine specific to the PHD domain of Sp100C.This study revealed that Sp100C has the ability to target chromatin directly and may play a restrictive role in innate immunity and gene silencing mediated by PML-NB in response to interferon stimulation in this way.A similar tubulin code was found in the microtubule skeleton of the cytoplasm.At present, the only post-translational modification located on the celiac side of microtubule is acetylated K40acan of lysine at position 40 of 伪 tubulin, which is catalyzed by highly conserved enzyme 伪 TAT1 among species, but the molecular mechanism of the catalytic reaction is not clear.In this paper, we have elucidated the crystal structure of mouse 伪 TAT1 catalytic domain and cofactor acetyl coenzyme acCoA complex.In addition to the similar folding of the core domain of the GNAT acetyltransferase superfamily, 伪 TAT1 has additional substrates to recognize the associated 尾 _ 4 / 尾 _ 5 inserts.The unique positive charge region and the necessary amino terminal hydrophobic region. AcCoA adenosine is partially bound by the side chain of 伪 TAT1 residues in a special "sandwich" configuration in the central groove of the core domain. The catalytic activity of 伪 TAT1 depends on the ring of the K40 region.Shape conformation and lateral interaction between microtubule filaments.The results of electron microscopy showed that the binding site of 伪 TAT1 to K40 may be located on the outside of microtubule.In this study, the structural characteristics of 伪 TAT1 were elucidated, which provided an important basis for the further study of 伪 TAT1 and microtubule recognition patterns and their role in the regulation of microtubule-related life activities.
【学位授予单位】：清华大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：Q75

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