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
• 负责人: Tara L Davis
• 金额: $ 24.03万
• 依托单位: DREXEL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-08-20 至 2015-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8460129
• 关键词: Affinity; Architecture; Binding; Biological Assay; Collection; Complex; Computer Simulation; Crystallization; Crystallography; Cyclophilins; Cyclosporins; Data; Development; Disease; Drug Targeting; Family; Family member; Fingerprint; Future; Human; In Vitro; Individual; Introns; Isomerase; Learning; Malignant Neoplasms; Mass Spectrum Analysis; Mentors; Methods; Modeling; Mutate; Nuclear; Nuclear Extract; Pathology; Pattern; Peptidylprolyl Isomerase; Phase; Physiological; Positioning Attribute; Process; Proline; Proteins; Protocols documentation; RNA; RNA Splicing; Reagent; Recombinants; Research; Research Design; Research Personnel; Resolution; Retinitis Pigmentosa; Role; Solutions; Spliceosome Assembly Pathway; Spliceosomes; Staging; Structure; Substrate Specificity; System; Techniques; Tertiary Protein Structure; Testing; Training; Transcript; Validation; Work; base; enzyme substrate; experience; follow-up; human disease; in vitro activity; in vivo; insight; interest; mRNA Precursor; mutant; post-doctoral training; protein complex; protein expression; protein protein interaction; reconstitution; research study; small molecule; structural genomics

PROJECT SUMMARY 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.

项目摘要 剪接体是RNA和蛋白质的复杂且动态的集合，其从RNA中移除内含子。 前体mRNA转录物。剪接机制的改变与一组不同的人类 从癌症到色素性视网膜炎。深入了解剪接导致 这些病理状态需要了解细胞内各个成分的功能， 剪接体在这一建议中，计划阐明亲环素类peptdyl-prolyl的作用， 剪接中的异构酶。 亲环素是高度保守的蛋白质，并且是药物环孢菌素的靶标，但它们的生理活性是通过它们的结合来实现的。 功能仍然是谜。我已经解决了几个亲环素的结构，原子分辨率的一部分， 结构基因组学的倡议，并产生了大量的可溶性蛋白质表达构建体。我也 根据它们在溶液中的催化活性和假设的潜在底物来表征这些蛋白质 基于计算机建模的特异性。然而，这类酶的体内底物没有定义， 使得我之前的结果难以验证基于发现几种核亲环素是 由于富含纯化的人类剪接复合物，剪接体亲环蛋白的靶点很可能会 提供了大量关于亲环蛋白：底物特异性的信息。此外， 在剪接体机制中发现的亲环蛋白家族成员及其独特的分布 在整个剪接复合物中，这些蛋白质表明这些蛋白质可能对适当的剪接活性至关重要。 为了检验这一假设，我将首先接受使用体外剪接测定法的培训，该测定法是在 Jurica实验室用潜在剪接因子的重组版本重建剪接体。我已经 开始使用该测定来测试在亲环素PPIE存在下的剪接活性，并且可以表明， 实际上，这种蛋白质是正确剪接功能所必需的。接下来我将在Jurica实验室接受后续培训 研究旨在找到PPIE发挥作用的剪接阶段，并学习如何纯化 用于质谱分析的剪接体复合物。这些研究将提供第一个洞察力， 单个剪接体相关亲环蛋白在前mRNA剪接中的功能重要性， 揭示了它们所针对的剪接体组装阶段。 在掌握这些技术后，我将在自己的实验室里继续进行剪接体亲环蛋白的研究， 将分离发现与亲环素相关的剪接体的各个组分。然后我会表演 对亲环蛋白和感兴趣的蛋白质进行生物物理测定，并将我的努力引向解决复杂结构 这些蛋白质复合物的结构。这些结构/功能研究的结果 剪接体亲环素将扩大我们对剪接机制的理解，包括 亲环蛋白特异性蛋白：剪接体内的蛋白质相互作用和脯氨酸异构化。