Functional and structural characterization of spliceosomal cyclophilins

剪接体亲环蛋白的功能和结构表征

基本信息

  • 批准号:
    7961107
  • 负责人:
  • 金额:
    $ 7.85万
  • 依托单位:
  • 依托单位国家:
    美国
  • 项目类别:
  • 财政年份:
    2010
  • 资助国家:
    美国
  • 起止时间:
    2010-08-20 至 2012-07-31
  • 项目状态:
    已结题

项目摘要

DESCRIPTION (provided by applicant): The spliceosome is a complex and dynamic collection of RNA and proteins that removes introns from precursor mRNA transcripts. Alterations in the splicing machinery are associated with a diverse set of human diseases, ranging from cancer to retinitis pigmentosa. Insight into the mechanisms by which splicing leads to these pathological states requires an understanding of the functions of individual components within the spliceosome. In this proposal are plans to elucidate the role of the cyclophilin class of peptdyl-prolyl isomerases in splicing. Cyclophilins are highly conserved proteins and the target of the drug cyclosporin but their physiological functions remain enigmatic. I have solved the structures of several cyclophilins to atomic resolution as part of a structural genomics initiative and generated large numbers of soluble protein expression constructs. I also characterized these proteins in terms of their catalytic activities in solution and hypothesized potential substrate specificity based on in silico modeling. However, in vivo substrates for this enzyme class are not defined, making validation of my previous results difficult. Based on the finding that several nuclear cyclophilins are enriched in purified human splicing complexes, it is likely that the targets of spliceosomal cyclophilins will provide a great deal of information concerning cyclophilin:substrate specificity. Additionally, the sheer number of the cyclophilin family members found within the spliceosomal machinery and their unique distribution throughout splicing complexes indicate that these proteins are likely to be crucial for proper splicing activity. In order to test this hypothesis I will first be trained in the use of an in vitro splicing assay optimized in the Jurica lab to reconstitute spliceosomes with recombinant versions of potential splicing factors. I have already begun to use this assay to test for splicing activity in the presence of the cyclophilin PPIE, and can show that indeed this protein is necessary for proper splicing function. Next I will be trained in the Jurica lab in follow-up studies designed to find the stage of splicing at which PPIE exerts its effect, and learn how to purify spliceosomal complexes for use in mass spectrometric analysis. These studies will provide the first insight into the functional importance of the individual spliceosome-associated cyclophilins in pre-mRNA splicing and reveal the stage of spliceosome assembly that they target. After mastering these techniques I will carry on my work in spliceosomal cyclophilins in my own lab, where I will isolate individual components of the spliceosome found to associate with cyclophilins. I will then perform biophysical assays on the cyclophilin and protein of interest, and direct my efforts to solving complex structures of these protein complexes utilizing x-ray crystallography. The results of these structure/function studies of spliceosomal cyclophilins will expand our understanding of splicing mechanism to include the roles of cyclophilin specific protein:protein interactions and proline isomerization within the spliceosome. PUBLIC HEALTH RELEVANCE: Alterations in the splicing machinery are associated with a diverse set of human diseases ranging from cancer to retinitis pigmentosa. Insight into the mechanisms by which splicing leads to these pathological states requires an understanding of the functions of individual components within the spliceosome. This work will serve as a platform for developing small molecule reagents or protein mutants that specifically target key spliceosome components. These reagents will be used to elucidate the spliceosome's involvement in disease pathologies.
描述(申请人提供):剪接体是一个复杂的和动态的RNA和蛋白质的集合,它从前体mRNA转录本中去除内含子。剪接机制的改变与多种人类疾病有关,从癌症到视网膜色素变性。要了解剪接导致这些病理状态的机制,需要了解剪接体中各个组件的功能。在这项提案中,计划阐明亲环素类的肽基-脯氨基异构酶在剪接中的作用。亲环素是高度保守的蛋白质,也是药物环孢素的靶点,但其生理功能仍是个谜。作为结构基因组学计划的一部分,我已经将几种亲环素的结构解析为原子分辨率,并生成了大量可溶的蛋白质表达结构。我还根据它们在溶液中的催化活性和基于电子计算机模拟的假设的潜在底物专一性来表征这些蛋白质。然而,这类酶的体内底物还没有定义,这使得我之前的结果很难得到验证。根据在纯化的人类剪接复合体中富含几种亲环素的发现,剪接体亲环素的靶标可能会提供大量关于亲环素的信息:底物特异性。此外,在剪接体机制中发现的亲环素家族成员的数量及其在剪接复合体中的独特分布表明,这些蛋白质可能对正确的剪接活性至关重要。为了验证这一假设,我将首先接受培训,使用在Jurica实验室优化的体外剪接试验,用潜在剪接因子的重组版本重建剪接体。我已经开始使用这种方法来测试亲环素PPIE存在时的剪接活性,并可以证明这种蛋白质确实是正确剪接功能所必需的。接下来,我将在Jurica实验室接受后续研究的培训,旨在找到PPIE发挥作用的剪接阶段,并学习如何提纯剪接体复合体用于质谱分析。这些研究将首次深入了解单个剪接体相关的亲环素在前mRNA剪接中的功能重要性,并揭示它们所针对的剪接体组装阶段。在掌握了这些技术之后,我将在我自己的实验室里继续我的剪接体亲环素的工作,在那里我将分离被发现与亲环素相关的剪接体的个别成分。然后,我将对亲环素和感兴趣的蛋白质进行生物物理分析,并利用X射线结晶学将我的努力引导到解决这些蛋白质复合体的复杂结构上。这些剪接体亲环素的结构/功能研究结果将扩大我们对剪接机制的理解,包括亲环素特异性蛋白的作用:蛋白质相互作用和剪接体内的脯氨酸异构化。 公共卫生相关性:剪接机制的改变与多种人类疾病有关,从癌症到视网膜色素变性。要了解剪接导致这些病理状态的机制,需要了解剪接体中各个组件的功能。这项工作将成为开发专门针对关键剪接体组件的小分子试剂或蛋白质突变体的平台。这些试剂将用于阐明剪接体在疾病病理中的作用。

项目成果

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Tara L Davis其他文献

Tara L Davis的其他文献

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{{ truncateString('Tara L Davis', 18)}}的其他基金

Functional and structural characterization of spliceosomal cyclophilins
剪接体亲环蛋白的功能和结构表征
  • 批准号:
    8641391
  • 财政年份:
    2010
  • 资助金额:
    $ 7.85万
  • 项目类别:
Functional and structural characterization of spliceosomal cyclophilins
剪接体亲环蛋白的功能和结构表征
  • 批准号:
    8132566
  • 财政年份:
    2010
  • 资助金额:
    $ 7.85万
  • 项目类别:
Functional and structural characterization of spliceosomal cyclophilins
剪接体亲环蛋白的功能和结构表征
  • 批准号:
    8441043
  • 财政年份:
    2010
  • 资助金额:
    $ 7.85万
  • 项目类别:
Functional and structural characterization of spliceosomal cyclophilins
剪接体亲环蛋白的功能和结构表征
  • 批准号:
    8460129
  • 财政年份:
    2010
  • 资助金额:
    $ 7.85万
  • 项目类别:

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