小鼠肝脏中丝氨酸、苏氨酸和赖氨酸乙酰化修饰的蛋白质组学研究
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摘要
蛋白质中氨基酸的乙酰化修饰是一种可逆的在真核细胞和原核细胞中都能发生的翻译后修饰形式,参与调控多种生物学过程,如DNA-蛋白质相互作用、蛋白质-蛋白质相互作用、基因转录、蛋白质的稳定性、细胞的迁移和分化等。自1968年Vidali发现了第一个乙酰化蛋白质以来,科学家对氨基酸的乙酰化修饰进行了大量的研究,研究重点主要集中于赖氨酸残基的乙酰化修饰,已经发现了300多个赖氨酸乙酰化修饰蛋白质,然而,相比生物体内的蛋白质而言,发现的乙酰化蛋白质仍然只是冰山一角,赖氨酸乙酰化底物的缺乏已经成了研究赖氨酸乙酰化修饰的瓶颈,限制了与赖氨酸乙酰化修饰相关的生物学功能的研究。近年来,蛋白质组学的方法已被作为一种高通量的有效的方法用于研究蛋白质及其翻译后修饰形式。用HPLC分离免疫亲和纯化得到乙酰化蛋白质或肽段,然后用MS/MS进行鉴定,是一个理想的研究赖氨酸乙酰化修饰的方法。
除了赖氨酸残基的乙酰化修饰,2006年Orth等人在研究耶尔森菌属中的细菌毒力因子YopJ(Yersinia species effector protein)时,发现YopJ是一种乙酰基转移酶,能够使mitogen-activated protein kinase kinases(MAPKK)中的丝氨酸和苏氨酸残基发生乙酰化修饰。发生乙酰化修饰的丝氨酸和苏氨酸残基位于MAPKK的活性链上,这两个位点发生乙酰化修饰后,同样的位点上便不能发生磷酸化修饰,从而无法激活mitogen-activated protein kinase(MAPK)信号通路,抑制免疫应答。因此,丝氨酸和苏氨酸残基的乙酰化修饰具有重要的生物学功能。目前研究丝氨酸和苏氨酸乙酰化修饰的方法有限,由于难以得到丝氨酸、苏氨酸乙酰化修饰的抗体,运用免疫亲和纯化技术预先分离和富集丝氨酸和苏氨酸乙酰化蛋白或肽段的方法暂时难以实现。
针对目前乙酰化修饰研究的现状及存在的问题,本论文从蛋白质组学的角度对小鼠肝脏中的丝氨酸、苏氨酸和赖氨酸乙酰化修饰蛋白质进行了研究,共包括以下四部分内容:
第一章前言部分概述了目前蛋白质组学和翻译后修饰蛋白质组学的研究现状,尤其对蛋白质乙酰化修饰的研究进展做了很好的综述,说明了本论文的选题目的及意义。
第二章运用shotgun技术对小鼠肝脏中的丝氨酸和苏氨酸残基的乙酰化修饰蛋白质进行分析,这是首次运用蛋白质组学的方法对丝氨酸和苏氨酸残基的乙酰化修饰进行分析。共在60个乙酰化蛋白质中鉴定到63个乙酰化位点,其中包括丝氨酸乙酰化修饰蛋白质30个,共33个位点;苏氨酸乙酰化蛋白质22个,共22个位点。这些丝氨酸和苏氨酸乙酰化蛋白质均为首次发现可以发生丝氨酸和苏氨酸残基的乙酰化修饰。借助生物信息学工具对这些乙酰化修饰蛋白质进行功能分析、氨基酸序列进行分析等。挑选具有重要生物学功能的乙酰化蛋白质进行克隆表达,证明丝氨酸、苏氨酸的乙酰化修饰在生物体内的存在。我们的发现表明与赖氨酸残基的乙酰化修饰一样,丝氨酸和苏氨酸残基的乙酰化修饰可能是小鼠肝脏中广泛存在的翻译后修饰形式,具有潜在的重要的生物学功能。
第三章运用免疫亲和纯化技术结合nano-HPLC/MS/MS技术,对小鼠肝脏中的赖氨酸残基乙酰化修饰肽段进行富集、鉴定,并对鉴定到的赖氨酸乙酰化修饰蛋白质进行了功能分析、氨基酸序列分析。共在20个赖氨酸乙酰化修饰蛋白质中鉴定到21个赖氨酸乙酰化修饰位点,其中12个蛋白质和16个位点为首次被鉴定到可以发生乙酰化修饰。另外,发现了三个能够发生乙酰化修饰的乙酰基转移酶,对于进一步研究赖氨酸的乙酰化修饰在多种细胞过程中的生物学功能有重要意义。
第四章是全文总结和展望。总结了主要研究结论,提出了进一步研究设想。
Acetylation of proteins on lysine is a reversible post-translational modification with important significance in cells.This modification plays a vital role in the regulation of many cellular processes including,but not limited to,DNA-protein interactions,protein-protein interactions,transcription,protein stability,migration and differentiation.The functional importance of acetylation has been described in many reports.However,the unwitnessed and unspecified substrate of lysine acetylation has become a major bottleneck and thus blocked the development of the functional studies in biology processes associated with lysine acetylation.Proteomics approach could offer a high-throughput and efficient tool to explore the global profile of proteins and their posttranslational modifications,including lysine acetylation.Isolation the proteins and/or peptides with lysine acetylation by HPLC followed by MS/MS is a novel strategy for identification of acetylated sites.
More recently,it was demonstrated that YopJ,a bacterial effector from Yersinia, could acetylate serine and threonine residues.YopJ acts as an acetyltransferase using acetyl-coenzyme(CoA) to modify the critical serine and threonine residues in the activation loop of MAPKK6,thereby blocking phosphorylation.The acetylation of MAPKK6 directly competes with the process of phosphorylation,preventing activation of the modified protein.Therefore,acetylation can compete with phosphorylation at the same residues,leading to alternate regulation of signaling pathways.
This dissertation consists of 4 parts and the contents are summarized as follows:
In the first chapter,a review of proteomic study and post-translational proteomic study was summarized,specially,in the separation and identification of acetylated proteins and peptides.Furthermore,the aims and significance of this dissertation were presented.
In the second chapter,the excellent strategy combining shotgun technology and nano-HPLC/MS/MS analysis was used to discover acetylation of serine and threonine in mouse livers for the first time.Consequently,63 acetylation sites from 60 proteins from mouse livers were identified by the proteomics survey.Of these,33 serine-acetylated sites from 30 proteins and 22 threonine-acetylated sites from 22 proteins have not been previously described.These observations imply that the acetylation of serine and threonine residues may be widespread in mouse livers and play a potential role in altering the course of signaling pathways.
In the third chapter,by combining the method of immunoaffinity purification of lysine-acetylated peptides with nano-HPLC/MS/MS analysis,we detected lysine-acetylated proteins in mouse livers.Total of 20 lysine-acetylated proteins including 21 lysine-acetylated sites have been identified.Among these,12 lysine-acetylated proteins and 16 lysine-acetylated sites have never been reported elsewhere.
In the last chapter,a summary was made and a prospect was presented.
除了赖氨酸残基的乙酰化修饰,2006年Orth等人在研究耶尔森菌属中的细菌毒力因子YopJ(Yersinia species effector protein)时,发现YopJ是一种乙酰基转移酶,能够使mitogen-activated protein kinase kinases(MAPKK)中的丝氨酸和苏氨酸残基发生乙酰化修饰。发生乙酰化修饰的丝氨酸和苏氨酸残基位于MAPKK的活性链上,这两个位点发生乙酰化修饰后,同样的位点上便不能发生磷酸化修饰,从而无法激活mitogen-activated protein kinase(MAPK)信号通路,抑制免疫应答。因此,丝氨酸和苏氨酸残基的乙酰化修饰具有重要的生物学功能。目前研究丝氨酸和苏氨酸乙酰化修饰的方法有限,由于难以得到丝氨酸、苏氨酸乙酰化修饰的抗体,运用免疫亲和纯化技术预先分离和富集丝氨酸和苏氨酸乙酰化蛋白或肽段的方法暂时难以实现。
针对目前乙酰化修饰研究的现状及存在的问题,本论文从蛋白质组学的角度对小鼠肝脏中的丝氨酸、苏氨酸和赖氨酸乙酰化修饰蛋白质进行了研究,共包括以下四部分内容:
第一章前言部分概述了目前蛋白质组学和翻译后修饰蛋白质组学的研究现状,尤其对蛋白质乙酰化修饰的研究进展做了很好的综述,说明了本论文的选题目的及意义。
第二章运用shotgun技术对小鼠肝脏中的丝氨酸和苏氨酸残基的乙酰化修饰蛋白质进行分析,这是首次运用蛋白质组学的方法对丝氨酸和苏氨酸残基的乙酰化修饰进行分析。共在60个乙酰化蛋白质中鉴定到63个乙酰化位点,其中包括丝氨酸乙酰化修饰蛋白质30个,共33个位点;苏氨酸乙酰化蛋白质22个,共22个位点。这些丝氨酸和苏氨酸乙酰化蛋白质均为首次发现可以发生丝氨酸和苏氨酸残基的乙酰化修饰。借助生物信息学工具对这些乙酰化修饰蛋白质进行功能分析、氨基酸序列进行分析等。挑选具有重要生物学功能的乙酰化蛋白质进行克隆表达,证明丝氨酸、苏氨酸的乙酰化修饰在生物体内的存在。我们的发现表明与赖氨酸残基的乙酰化修饰一样,丝氨酸和苏氨酸残基的乙酰化修饰可能是小鼠肝脏中广泛存在的翻译后修饰形式,具有潜在的重要的生物学功能。
第三章运用免疫亲和纯化技术结合nano-HPLC/MS/MS技术,对小鼠肝脏中的赖氨酸残基乙酰化修饰肽段进行富集、鉴定,并对鉴定到的赖氨酸乙酰化修饰蛋白质进行了功能分析、氨基酸序列分析。共在20个赖氨酸乙酰化修饰蛋白质中鉴定到21个赖氨酸乙酰化修饰位点,其中12个蛋白质和16个位点为首次被鉴定到可以发生乙酰化修饰。另外,发现了三个能够发生乙酰化修饰的乙酰基转移酶,对于进一步研究赖氨酸的乙酰化修饰在多种细胞过程中的生物学功能有重要意义。
第四章是全文总结和展望。总结了主要研究结论,提出了进一步研究设想。
Acetylation of proteins on lysine is a reversible post-translational modification with important significance in cells.This modification plays a vital role in the regulation of many cellular processes including,but not limited to,DNA-protein interactions,protein-protein interactions,transcription,protein stability,migration and differentiation.The functional importance of acetylation has been described in many reports.However,the unwitnessed and unspecified substrate of lysine acetylation has become a major bottleneck and thus blocked the development of the functional studies in biology processes associated with lysine acetylation.Proteomics approach could offer a high-throughput and efficient tool to explore the global profile of proteins and their posttranslational modifications,including lysine acetylation.Isolation the proteins and/or peptides with lysine acetylation by HPLC followed by MS/MS is a novel strategy for identification of acetylated sites.
More recently,it was demonstrated that YopJ,a bacterial effector from Yersinia, could acetylate serine and threonine residues.YopJ acts as an acetyltransferase using acetyl-coenzyme(CoA) to modify the critical serine and threonine residues in the activation loop of MAPKK6,thereby blocking phosphorylation.The acetylation of MAPKK6 directly competes with the process of phosphorylation,preventing activation of the modified protein.Therefore,acetylation can compete with phosphorylation at the same residues,leading to alternate regulation of signaling pathways.
This dissertation consists of 4 parts and the contents are summarized as follows:
In the first chapter,a review of proteomic study and post-translational proteomic study was summarized,specially,in the separation and identification of acetylated proteins and peptides.Furthermore,the aims and significance of this dissertation were presented.
In the second chapter,the excellent strategy combining shotgun technology and nano-HPLC/MS/MS analysis was used to discover acetylation of serine and threonine in mouse livers for the first time.Consequently,63 acetylation sites from 60 proteins from mouse livers were identified by the proteomics survey.Of these,33 serine-acetylated sites from 30 proteins and 22 threonine-acetylated sites from 22 proteins have not been previously described.These observations imply that the acetylation of serine and threonine residues may be widespread in mouse livers and play a potential role in altering the course of signaling pathways.
In the third chapter,by combining the method of immunoaffinity purification of lysine-acetylated peptides with nano-HPLC/MS/MS analysis,we detected lysine-acetylated proteins in mouse livers.Total of 20 lysine-acetylated proteins including 21 lysine-acetylated sites have been identified.Among these,12 lysine-acetylated proteins and 16 lysine-acetylated sites have never been reported elsewhere.
In the last chapter,a summary was made and a prospect was presented.
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