Analysis of Proline Isomerization in Epigenetics

表观遗传学中脯氨酸异构化分析

• 批准号: 8305832
• 负责人: Rob Carl Oslund
• 金额: $ 4.71万
• 依托单位: PRINCETON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-05-01 至 2015-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8305832
• 关键词: Acetylation; Address; Amides; Amino Acids; Avidin; Binding; Binding Proteins; Biochemical; Biological; Biological Assay; Biological Testing; Biotin; C-terminal; Cell division; Cell physiology; Cells; Chemicals; Chromatin; Chromatin Structure; DNA; Dipeptides; Enzymes; Epigenetic Process; Eukaryotic Cell; Event; Fluorescence Anisotropy; Foundations; Genome; Goals; Hand; Histone H3; Histones; Homeostasis; Human; Incubated; Investigation; Isomerism; Label; Lysine; Mass Spectrum Analysis; Methylation; Methyltransferase; Modification; Molecular Conformation; Nature; Nucleosomes; Peptide Synthesis; Peptides; Peptidylprolyl Isomerase; Phase; Phosphorylation; Physiological; Post-Translational Protein Processing; Proline; Protein Engineering; Proteins; Publishing; Reader; Recombinants; Recruitment Activity; Regulation; Research; Research Proposals; Role; Route; Solid; Structure; Tail; Techniques; Testing; Training; Work; Yeasts; base; cancer cell; cis trans isomerization; demethylation; design; histone modification; human DNA; novel; peptide chemical synthesis; physical state; research study; sedimentation velocity; tool

DESCRIPTION (provided by applicant): The proposed research in this training plan will focus on studying the effects of cis-trans proline isomerization on chromatin structure and function. The physiological state of DNA in eukaryotic cells occurs in the form of chromatin. As a result, access to the genome for fundamental cellular processes such as cell division, differentiation, and homeostasis must occur at the chromatin level. This has led to a widespread investigation of chromatin regulatory mechanisms. An important type of chromatin regulatory mechanism takes place on the tails of histone proteins in nucleosomes. These histone tail post-translational modifications (PTMs) are essential for normal cellular function and their misregulation has been shown to contribute to cancer cell formation. Histone tail PTMs are covalent in nature and, among others, include: phosphorylation, methylation, ubiquitylation, and acetylation. However, the one exception to the list of covalent histone tail modifications is cis-trans proline isomerization. In yeast, it has been shown that cis-trans proline isomerization on histone H3 proline 38 (H3-Pro38) can influence the methylation of a neighboring lysine residue. In addition, a yeast proline isomerase enzyme has been identified that can catalyze the interconversion of this proline isomerization event. This has led to the hypothesis that H3-Pro38 can act as a switch to control other histone modifications, recruit effector proteins, and alter the structure o chromatin. However, the ability to study proline isomerization has been severely limited by the lack of chemical tools that effectively mimic the cis and trans conformational states of proline. This proposed research will address this issue by developing H3-Pro38 peptide and protein mimics that are conformationally locked in either the cis or trans states. Such mimics do not currently exist and would be valuable in studying the effects of cis and trans proline on histone structure and function. For these studies, we will use chemical and peptide synthesis along with protein engineering techniques to develop proline peptide and protein mimics. With these chemical tools in hand, we will then use biochemical and biophysical approaches to explore the proposed biological roles of H3-Pro38 isomerization described above. The specific aims of this research are: (1) To synthesize conformationally locked peptide mimics of histone H3-Pro38. (2) To characterize the role of H3-Pro38 isomerization on H3-Lys36 methylation/demethylation. (3) To identify human reader and writer proteins of cis and trans H3-Pro38. (4) To elucidate the structural role of cis and trans Pro38 isomerization on chromatin compaction. This proposed research is designed to provide much needed tools for studying H3-Pro38 cis-trans isomerization and, if successful, we expect this work to lay a foundation for understanding how proline switches regulate chromatin structure and function in humans. PUBLIC HEALTH RELEVANCE: Human DNA is packaged into cells in the form of chromatin which means that access to DNA for cellular processes must occur at the chromatin level, and that the regulation of chromatin has important consequences on physiological and pathophysiological cellular events including cancer cell formation. The goal of this proposed research is to investigate a poorly understood chromatin regulatory mechanism where proline amino acids act as a switch on histone protein tails to regulate chromatin structure and function. Efforts will focus on developing novel chemical tools that mimic different proline conformations and then using such tools to understand the effects of proline isomerization on chromatin regulation.

描述（由申请人提供）：本培训计划中的拟议研究将重点研究顺式跨性别脯氨酸异构化对染色质结构和功能的影响。真核细胞中DNA的生理状态以染色质形式出现。结果，必须在染色质水平上访问基因组，例如细胞分裂，分化和稳态。这导致了对染色质调节机制的广泛研究。染色质调节机制的一种重要类型发生在核小体中组蛋白的尾巴上。这些组蛋白尾后翻译后修饰（PTM）对于正常细胞功能至关重要，已证明它们的正常调节有助于癌细胞的形成。组蛋白的尾巴在本质上是共价的，其中包括：磷酸化，甲基化，泛素化和乙酰化。但是，共价组蛋白尾部修饰列表的一个例外是顺式透射脯氨酸异构化。在酵母中，已经表明，在组蛋白H3脯氨酸38（H3-PRO38）上顺式经透射脯氨酸异构化可以影响邻近的赖氨酸残基的甲基化。另外，已经确定了酵母脯氨酸异构酶酶可以催化该脯氨酸异构化事件的互连。这导致了以下假设：H3-Pro38可以充当控制其他组蛋白修饰，募集效应蛋白并改变结构O染色质的转换。但是，研究脯氨酸异构化的能力受到缺乏有效模仿顺式和脯氨酸的反式构象状态的化学工具的严重限制。这项拟议的研究将通过开发H3-Pro38肽和蛋白质模拟物来解决这个问题，这些肽和蛋白质模拟物在顺式或跨状态下构型锁定。此类模拟物当前不存在，并且对于研究CI和反式脯氨酸对组蛋白结构和功能的影响将很有价值。在这些研究中，我们将使用化学和肽合成以及蛋白质工程技术来开发脯氨酸肽和蛋白质模仿。借助这些化学工具，我们将使用生化和生物物理方法来探索上述H3-Pro38异构化所提出的生物学作用。这项研究的具体目的是：（1）合成组蛋白H3-PRO38的构象锁定肽模仿。 （2）表征H3-PRO38异构化在H3- lys36甲基化/脱甲基化中的作用。 （3）确定顺式和反式H3-Pro38的人类读者和作者蛋白。 （4）阐明CIS和Trans Pro38异构化在染色质压实中的结构作用。这项拟议的研究旨在提供急需的工具来研究H3-PRO38顺式传播异构化，如果成功的话，我们希望这项工作为理解脯氨酸开关如何调节人类的染色质结构和功能奠定了基础。 公共卫生相关性：人类DNA以染色质的形式包装到细胞中，这意味着必须在染色质水平上访问DNA进行DNA，并且染色质的调节对生理和病理生理细胞的重要后果对包括癌细胞形成在内的生理和病理生理细胞事件产生了重要影响。这项拟议的研究的目的是研究一种鲜为人知的染色质调节机制，其中脯氨酸氨基酸起到组蛋白蛋白尾巴的开关以调节染色质结构和功能。努力将着重于开发新型化学工具，这些化学工具模仿不同的脯氨酸构象，然后使用此类工具来了解脯氨酸异构化对染色质调节的影响。