Regulation of RNA Polymerase II by the Ess1 Prolyl Isomerase

Ess1 脯氨酰异构酶对 RNA 聚合酶 II 的调节

• 批准号: 10158493
• 负责人: Steven D. HANES
• 金额: $ 32.4万
• 依托单位: UPSTATE MEDICAL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-07-01 至 2023-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10158493
• 关键词: Address; Affect; Affinity; Antifungal Agents; Antineoplastic Agents; Area; Attenuated; Back; Binding; Binding Sites; Biochemical; Biological Assay; Biological Models; Biophysics; Candida albicans; Catalytic Domain; Cell Cycle Regulation; Cell Death; Cell Extracts; Cell physiology; Cells; ChIP-seq; Chemicals; Chromatin; Chromatin Remodeling Factor; Code; Complex; Consensus; Cryptococcus neoformans; Crystallization; Cyclic Amino Acids; DNA; Data; Development; Disease; Elongation Factor; Enzymes; Eukaryotic Cell; Gene Expression Regulation; Genes; Genetic; Genetic Transcription; Genomic approach; Goals; Growth; HIV; Human; Immunocompromised Host; Immunosuppressive Agents; In Vitro; Instruction; Interferometry; Knowledge; Laboratories; Laboratory Study; Ligands; Link; Malignant Neoplasms; Maps; Mitotic Cell Cycle; Modeling; Modification; Molecular; Molecular Conformation; Mutation; Mycoses; Neurodegenerative Disorders; Organism; Orthologous Gene; Pathogenicity; Patients; Pattern; Peptides; Peptidylprolyl Isomerase; Phosphorylation; Physiological; Play; Positioning Attribute; Proline; Property; Proteins; RNA; RNA Polymerase II; RNA Processing; RNA Splicing; RNA polymerase II largest subunit; Reading; Regulation; Repetitive Sequence; Reporter; Research; Resolution; Roentgen Rays; Role; Saccharomyces cerevisiae; Series; Specific qualifier value; Structure; Substrate Domain; Substrate Specificity; Tertiary Protein Structure; Testing; Transcription Elongation; Transcriptional Regulation; Transplantation; Virulence; Work; Writing; Yeasts; analytical ultracentrifugation; cis trans isomerization; cis-trans-Isomerases; cofactor; experimental study; genome-wide; human disease; in vivo; inhibitor/antagonist; pathogenic fungus; protein protein interaction; recruit; sedimentation velocity; seryl-proline; stoichiometry

Project Summary The long-term goal is to understand the mechanism by which peptidyl-prolyl cis/trans isomerases (PPIases) function as molecular switches to regulate gene activity. PPIases catalyze the isomerization of the peptide bond that precedes the cyclic amino acid proline, causing conformational changes that facilitate the folding of newly-synthesized proteins and that regulate the activity of mature proteins by altering their activity or protein- protein interactions. PPIases are found in all organisms, and are best known as the targets of immunosuppressive drugs. However, their normal function in cells is poorly understood. We study a PPIase called Ess1, which is essential for growth in Saccharomyces cerevisiae. Ess1 and its human ortholog, Pin1 (which can substitute for Ess1 in yeast), are implicated in transcription regulation and mitotic cell cycle control. In the pathogenic fungi, Candida albicans and Cryptococcus neoformans, Ess1 is essential for virulence. In humans, misexpression of Pin1 may contribute to cancer and neurodegenerative disease. These findings make clear the importance of prolyl isomerization for cellular function. The goal of the proposed research is to understand the mechanism by which Ess1 recognizes and regulates RNA polymerase II (RNAPII) in eukaryotic cells. Work from this laboratory has shown that Ess1 binds and isomerizes peptidyl-prolyl bonds within the carboxy-terminal domain (CTD) of the large subunit of RNAPII. Ess1 acts by throwing a conformational “proline switch” thus plays an integral role in specifying the so-called “CTD code” that helps coordinate the recruitment of protein co-factors to the transcribing RNAPII complex. Ess1-induced conformational changes in the CTD are likely to be important for multiple steps in the transcription cycle. The goal of Aim1 is to understand how Ess1 “reads the CTD code,” a critical first step in its regulation of the RNAPII transcription cycle. To determine how Ess1 recognizes and binds the CTD a combination of structural, biochemical, biophysical and in vivo studies will be used. The results will be important for understanding how the mammalian ortholog (Pin1) recognizes RNAPII and a variety of other substrates. This information will also be useful for efforts to develop antifungal inhibitors (Ess1) or anti-cancer drugs (Pin1). The goal of Aim2 is to understand how Ess1 “writes the CTD code” by focusing on its role in transcription elongation. The working hypothesis is that Ess1 controls the recruitment and/or activity of elongation factors, and/or chromatin modifiers, thus regulating RNAPII elongation. This will be tested using biochemical and genomic approaches. These results will be important for two main reasons. First, they will help us understand how non-covalent proline switches (versus covalent modification such as phosphorylation – an area already heavily studied) regulate recruitment of RNAPII co-factors to the CTD. Second, they will help us understand how Ess1 regulates elongation, a mechanism that is increasingly recognized as critical in gene regulation. To our knowledge, this is the only proposal, and we are the only laboratory, studying the role of this key peptidyl-prolyl isomerase in transcription.

项目摘要 长期目标是了解肽基脯氨酰顺/反式 异构酶（PPIases）作为分子开关调节基因活性。 PPIases催化环氨基之前的肽键的异构化， 酸性脯氨酸，引起构象变化，促进新合成的 蛋白质，并通过改变其活性或蛋白质来调节成熟蛋白质的活性， 蛋白质相互作用PPIases存在于所有生物体中，并且最为人所知的是作为靶标 免疫抑制药物。然而，它们在细胞中的正常功能却知之甚少。 我们研究了一种名为PPIase 1的PPIase，它是酿酒酵母生长所必需的。 PIN 1及其人类直系同源物Pin 1（可在酵母中替代PIN 1）与 转录调控和有丝分裂细胞周期控制。在致病真菌中，念珠菌属 白念珠菌和新型隐球菌中，EST 1是毒力所必需的。在人类中， Pin 1的错误表达可能导致癌症和神经退行性疾病。这些 这些发现清楚地表明脯氨酰异构化对细胞功能的重要性。 这项研究的目的是了解BMP 1 识别并调节真核细胞中的RNA聚合酶II（RNAPII）。从这个开始工作 实验室已经表明，BMP 1结合并异构化了肽基-脯氨酰键， RNAPII大亚基的羧基末端结构域（CTD）。101通过抛出一个 因此，构象“脯氨酸开关”在指定所谓的“CTD”中起着不可或缺的作用 帮助协调蛋白质辅因子向转录RNAPII的募集的”编码 复杂. CTD 1诱导的CTD构象变化可能对 转录循环中的多个步骤。 Aim 1的目标是了解Aim 1如何“读取CTD代码”，这是关键的第一步 在RNAPII转录周期的调节中。为了确定EST 1如何识别和 结合CTD，结构、生物化学、生物物理和体内研究的组合将 被利用这些结果对于理解哺乳动物直系同源基因（Pin 1） 识别RNAPII和各种其他底物。这些信息也将有助于 努力开发抗真菌抑制剂（PIN 1）或抗癌药物（Pin 1）。 Aim 2的目标是通过关注其 在转录延伸中的作用。目前的假设是，RP 1控制着招募， 和/或延伸因子的活性，和/或染色质修饰剂，从而调节RNAPII 伸长率这将使用生物化学和基因组方法进行测试。这些结果 有两个主要原因。首先，它们将帮助我们理解非共价键 脯氨酸开关（相对于共价修饰，如磷酸化-一个领域已经 大量研究）调节RNAPII辅因子向CTD的募集。第二，他们会帮助 我们了解了BMP 1是如何调节伸长的，这一机制越来越多地被认识到， 对基因调控至关重要。 据我们所知，这是唯一的建议，我们是唯一的实验室，研究的作用 这种关键的肽基脯氨酰异构酶的转录。

项目成果

Sec61 channel subunit Sbh1/Sec61β promotes ER translocation of proteins with suboptimal targeting sequences and is fine-tuned by phosphorylation.

• DOI: 10.1016/j.jbc.2023.102895
• 发表时间: 2023-03
• 期刊: JOURNAL OF BIOLOGICAL CHEMISTRY
• 影响因子: 4.8
• 作者: Barbieri, Guido;Simon, Julien;Lupusella, Cristina R.;Pereira, Fabio;Elia, Francesco;Meyer, Hadar;Schuldiner, Maya;Hanes, Steven D.;Nguyen, Duy;Helms, Volkhard;Roemisch, Karin
• 通讯作者: Roemisch, Karin