mTORC1调控乙酰转移酶p300活性的机制及功能研究

发布时间：2017-12-31 14:02

  本文关键词：mTORC1调控乙酰转移酶p300活性的机制及功能研究　出处：《浙江大学》2017年博士论文　论文类型：学位论文

  更多相关文章： p300 mTORC1 自噬 基因转录

【摘要】：细胞代谢通过调控细胞内的能量和生物大分子的合成与分解影响细胞的生长、分化和衰老。细胞内外的营养状态和应激压力通过影响位于各个代谢通路的重要蛋白激酶的活性,调节细胞的代谢水平。然而,近年来的研究发现,细胞代谢通路中大量蛋白的活性受到乙酰化修饰的调控,表明蛋白质乙酰转移酶/去乙酰化酶在细胞代谢中发挥重要作用。乙酰转移酶p300及其同源蛋白CBP(CREB-binding protein),是研究较为广泛的乙酰转移酶。最初,它们被发现发挥转录共激活子的作用。p300通过乙酰化组蛋白和转录因子等,加强转录起始复合物的组装,促进基因转录。除了在细胞核内发挥作用,p300也能定位细胞质,通过乙酰化细胞质蛋白,参与调控众多的细胞生理病理过程。最近的研究发现,p300在细胞内重要的分解代谢过程一自噬中发挥作用。p300通过乙酰化自噬相关蛋白(autophagy related gene,Atg)LC3、Atg5和Atg7等抑制自噬的发生。LC3的去乙酰化是其在细胞质中完成脂质化修饰,参与自噬起始的前提。葡萄糖饥饿条件下,细胞质中的糖酵解酶GAPDH被激活的蛋白激酶AMPK磷酸化后进入细胞核,通过与核内的去乙酰化酶Sirt1相互作用而激活Sirt1,进而导致LC3的去乙酰化和自噬的起始。但是,无论是在细胞水平还是在动物水平,抑制Sirt1的活性或者敲除Sirt1,均不影响氨基酸饥饿或者雷帕霉素(rapamycin)处理诱导的自噬。提示在上述条件下,可能存在其他的信号通路参与调控LC3的乙酰化和自噬的起始。本研究中我们发现,氨基酸饥饿或者雷帕霉素处理的细胞中,乙酰转移酶p300的活性受到显著抑制,提示氨基酸和雷帕霉素主要的作用靶点mTORC1(mammalian target of rapamycin complex 1)与 p300 的关系。我们进一步发现,在细胞内敲低mTORC1的重要组分Raptor,能降低p300的活性;高表达mTORC1的激活蛋白Rheb或者敲除mTORC1的抑制蛋白TSC1/2能显著激活p300,证实了mTORC1对p300活性的调控作用。我们还发现,p300能定位溶酶体膜上,并与mTORC1发生直接的相互作用。利用体外激酶反应实验,我们证实mTORC1能直接磷酸化p300,利用质谱分析我们鉴定了位于p300羧基端的被mTORC1磷酸化的4个丝氨酸位点。利用模拟磷酸化和模拟去磷酸化的突变体,我们验证了mTORC1介导的磷酸化对p300活性的调控作用。机制研究表明,p300的磷酸化会解除其分子内RING结构域对酶活HAT结构域的抑制作用,从而导致p300的激活。功能研究中我们发现,依赖mTORC1的p300磷酸化能显著抑制氨基酸饥饿诱导的LC3去乙酰化和自噬的发生;而且,p300的磷酸化能提高其转录共激活子的活性,通过乙酰化和激活转录因子SREBP-1c促进脂肪酸的合成。通过本研究中,我们揭示了一个调控乙酰转移酶p300活性的新机制。mTORC1-p300通路通过调控细胞自噬和脂质代谢等,在协调细胞分解代谢和合成代谢中发挥关键作用。
[Abstract]:Cell metabolism affects cell growth by regulating intracellular energy and biosynthesis and decomposition of biological macromolecules. Differentiation and senescence. Nutritional status and stress stress in and out of cells regulate the metabolic level of cells by affecting the activity of important protein kinases located in various metabolic pathways. The activity of a large number of proteins in the cellular metabolic pathway is regulated by acetylation modification. It is suggested that protein acetyltransferase / deacetylase plays an important role in cell metabolism. Acetyltransferase p300 and its homologous protein CBP(CREB-binding protein. Initially, they have been found to act as transcriptional co-activators. P300 enhances the assembly of transcription initiation complexes through acetylated histone and transcription factors. Promote gene transcription. In addition to play a role in the nucleus of the p300 can also be located in the cytoplasm, through acetylated cytoplasmic protein, involved in the regulation of numerous cellular physiological and pathological processes. Recent research has found. P300 plays a role in autophagy related gene, an important catabolism process in cells. P300 plays a role in autophagy related gene via acetylated autophagy. Inhibition of autophagy. Deacetylation of LC3 is the precondition of lipid modification in the cytoplasm and participation in the initiation of autophagy. The glycolytic enzyme GAPDH in the cytoplasm is phosphorylated by activated protein kinase AMPK and then enters the nucleus and activates Sirt1 by interacting with deacetylase Sirt1 in the nucleus. This in turn leads to the deacetylation of LC3 and the initiation of autophagy. However, both at the cellular level and at the animal level, the activity of Sirt1 is inhibited or Sirt1 is knocked out. Neither of them affected the autophagy induced by amino acid starvation or rapamycin treatment. There may be other signaling pathways involved in regulating the initiation of acetylation and autophagy in LC3. In this study we found that amino acid starvation or rapamycin treated cells. The activity of acetyltransferase p300 was significantly inhibited. It is suggested that the main target of amino acid and rapamycin is mTORC1(mammalian target of rapamycin complex 1). Relationship with p300. We find out further. Raptor, an important component of mTORC1, could reduce the activity of p300. Overexpression of mTORC1 activator protein Rheb or knockout of mTORC1 inhibitor TSC1/2 could significantly activate p300. The effect of mTORC1 on the activity of p300 was confirmed. We also found that P300 can locate lysosomal membrane and interact directly with mTORC1. We confirm that mTORC1 can directly phosphorylate p300. We identified four sites of serine phosphorylated by mTORC1 at the end of p300 carboxyl group by mass spectrometry, using simulated phosphorylation and simulated dephosphorylation of mutants. We have verified the regulation of phosphorylation mediated by mTORC1 on the activity of p300. The phosphorylation of p300 can depress the inhibitory effect of its intramolecular RING domain on the active HAT domain, which leads to the activation of p300. MTORC1 dependent p300 phosphorylation significantly inhibited LC3 deacetylation and autophagy induced by amino acid starvation. Moreover, phosphorylation of p300 can increase the activity of transcriptional co-activator and promote the synthesis of fatty acids through acetylation and activation of transcription factor SREBP-1c. We have revealed a new mechanism for regulating the activity of acetyltransferase p300. The mTORC1-p300 pathway regulates autophagy and lipid metabolism. Play a key role in coordinating cellular catabolism and biosynthesis.
【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：Q25

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