本文關鍵詞：mTORC1調控乙酰轉移酶p300活性的機制及功能研究　出處：《浙江大學》2017年博士論文　論文類型：學位論文

  更多相關文章： p300 mTORC1 自噬 基因轉錄

【摘要】：細胞代謝通過調控細胞內的能量和生物大分子的合成與分解影響細胞的生長、分化和衰老。細胞內外的營養(yǎng)狀態(tài)和應激壓力通過影響位于各個代謝通路的重要蛋白激酶的活性,調節(jié)細胞的代謝水平。然而,近年來的研究發(fā)現,細胞代謝通路中大量蛋白的活性受到乙�；揎椀恼{控,表明蛋白質乙酰轉移酶/去乙�；冈诩毎x中發(fā)揮重要作用。乙酰轉移酶p300及其同源蛋白CBP(CREB-binding protein),是研究較為廣泛的乙酰轉移酶。最初,它們被發(fā)現發(fā)揮轉錄共激活子的作用。p300通過乙�；M蛋白和轉錄因子等,加強轉錄起始復合物的組裝,促進基因轉錄。除了在細胞核內發(fā)揮作用,p300也能定位細胞質,通過乙�；毎|蛋白,參與調控眾多的細胞生理病理過程。最近的研究發(fā)現,p300在細胞內重要的分解代謝過程一自噬中發(fā)揮作用。p300通過乙�；允上嚓P蛋白(autophagy related gene,Atg)LC3、Atg5和Atg7等抑制自噬的發(fā)生。LC3的去乙�；瞧湓诩毎|中完成脂質化修飾,參與自噬起始的前提。葡萄糖饑餓條件下,細胞質中的糖酵解酶GAPDH被激活的蛋白激酶AMPK磷酸化后進入細胞核,通過與核內的去乙�；窼irt1相互作用而激活Sirt1,進而導致LC3的去乙�；妥允傻钠鹗�。但是,無論是在細胞水平還是在動物水平,抑制Sirt1的活性或者敲除Sirt1,均不影響氨基酸饑餓或者雷帕霉素(rapamycin)處理誘導的自噬。提示在上述條件下,可能存在其他的信號通路參與調控LC3的乙�；妥允傻钠鹗�。本研究中我們發(fā)現,氨基酸饑餓或者雷帕霉素處理的細胞中,乙酰轉移酶p300的活性受到顯著抑制,提示氨基酸和雷帕霉素主要的作用靶點mTORC1(mammalian target of rapamycin complex 1)與 p300 的關系。我們進一步發(fā)現,在細胞內敲低mTORC1的重要組分Raptor,能降低p300的活性;高表達mTORC1的激活蛋白Rheb或者敲除mTORC1的抑制蛋白TSC1/2能顯著激活p300,證實了mTORC1對p300活性的調控作用。我們還發(fā)現,p300能定位溶酶體膜上,并與mTORC1發(fā)生直接的相互作用。利用體外激酶反應實驗,我們證實mTORC1能直接磷酸化p300,利用質譜分析我們鑒定了位于p300羧基端的被mTORC1磷酸化的4個絲氨酸位點。利用模擬磷酸化和模擬去磷酸化的突變體,我們驗證了mTORC1介導的磷酸化對p300活性的調控作用。機制研究表明,p300的磷酸化會解除其分子內RING結構域對酶活HAT結構域的抑制作用,從而導致p300的激活。功能研究中我們發(fā)現,依賴mTORC1的p300磷酸化能顯著抑制氨基酸饑餓誘導的LC3去乙�；妥允傻陌l(fā)生;而且,p300的磷酸化能提高其轉錄共激活子的活性,通過乙酰化和激活轉錄因子SREBP-1c促進脂肪酸的合成。通過本研究中,我們揭示了一個調控乙酰轉移酶p300活性的新機制。mTORC1-p300通路通過調控細胞自噬和脂質代謝等,在協(xié)調細胞分解代謝和合成代謝中發(fā)揮關鍵作用。
[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.
【學位授予單位】：浙江大學
【學位級別】：博士
【學位授予年份】：2017
【分類號】：Q25

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本文編號：1360056