The Role of Citrullination in Gonadotrope Function

瓜氨酸化在促性腺激素功能中的作用

• 批准号: 9387993
• 负责人: Brian D. Cherrington
• 金额: $ 18.06万
• 依托单位: UNIVERSITY OF WYOMING
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-07-14 至 2019-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9387993
• 关键词: Address; Amino Acids; Anterior Pituitary Gland; Architecture; Arginine; Biology; Cell Line; Cells; Citrulline; Clinical; Contraceptive methods; Cytoskeletal Filaments; Cytoskeletal Modeling; Cytoskeletal Proteins; Cytoskeleton; DNA; Data; Disease; Endoplasmic Reticulum; Enzymes; Epigenetic Process; Estrus; Event; Fertility; Fluorescence-Activated Cell Sorting; Gene Expression; Gene Targeting; Genes; Goals; Golgi Apparatus; Gonadotrope Cell; Gonadotropin Hormone Releasing Hormone; Gonadotropins; Health; Histones; Hormones; Human; In Vitro; Infertility; Intracellular Transport; Knowledge; Label; Luteinizing Hormone; Mammals; Modification; Molecular; Molecular Target; Multiple Sclerosis; Mus; Ovulation; Physiological; Physiology; Positioning Attribute; Post-Translational Protein Processing; Protein-arginine deiminase; Proteomics; Publishing; Reaction; Reproduction; Research; Rheumatoid Arthritis; Role; Tail; Testing; Untranslated RNA; Vesicle; Work; base; beta Actin; beta Tubulin; chromatin immunoprecipitation; citrullinated protein; histone modification; in vivo; inhibitor/antagonist; innovation; insight; malignant breast neoplasm; novel; programs; protein structure function; reproductive function; response; therapeutic target; trafficking; transcriptome sequencing

Gonadotropin Releasing Hormone (GnRH) stimulates peptidylarginine deiminase (PAD) catalyzed citrullination of histones and cytoskeletal filaments in gonadotropes, yet the physiological consequence of this on reproductive function is lacking. This gap in knowledge is an important problem because proper gonadotrope function is critical for all vertebrate reproduction and a major therapeutic target for infertility and contraception. Our long-term goal is to develop a detailed understanding of the molecular targets of citrullination and its physiological implications in gonadotropes. The objective of this proposal is to identify citrullination as a novel regulator of gonadotrope specific gene programs and cytoskeletal function in vivo. Our published data demonstrates that GnRH induces PAD catalyzed citrullination of arginine residues on histones to regulate expression of the luteinizing hormone (LH) β subunit gene. Preliminary RNA-seq data suggests that citrullination also regulates the of expression endoplasmic reticulum processing and golgi vesicle trafficking gene networks not previously characterized in gonadotropes. In addition to histones, our data shows that PADs citrullinate β-tubulin and β-actin to regulate cytoskeletal reorganization following GnRH stimulation. Our central hypothesis is that GnRH utilizes PAD catalyzed citrullination of histones and cytoskeletal proteins to implement critical molecular changes necessary for LH synthesis, spatial repositioning of cells, and effective hormone secretion. The central hypothesis will be tested with the following specific aims: (1) To test whether genes governing gonadotropin synthesis and processing are regulated by GnRH induced PAD catalyzed histone citrullination in vitro and in vivo. (2) To identify citrullinated cytoskeletal filaments and determine how these modifications regulate gonadotrope function. In Aim 1, the expression of target genes identified by RNA-seq in LβT2 gonadotropes will be examined following GnRH and PAD inhibitor treatment using chromatin immunoprecipitation (ChIP). Target genes will next be examined in gonadotropes purified by fluorescence activated cell sorting (FACS) from GRIC-GFP mice. Primary gonadotropes will be treated with GnRH and PAD inhibitor and target genes examined by qPCR and ChIP. Proposed studies in Aim 2 will identify citrullinated proteins from LβT2 and primary gonadotropes using a proteomic approach. The physiologic importance of citrullination on cellular architecture and LH secretion will be examined in primary gonadotropes following treatment with GnRH and a PAD inhibitor. The proposed research is innovative because we plan to determine how GnRH acting through the unexplored mechanism of citrullination can temporally initiate an epigenetic event and cytoskeletal reorganization to control gonadotrope physiology, which represents a new and substantial departure from current studies in the gonadotrope field. The work is significant because it is an important step to characterize a completely novel, unexplored mechanism stimulated by GnRH that is critical for gonadotrope function.

促性腺激素释放激素（GnRH）刺激肽基精氨酸脱亚胺酶（PAD）催化的 促性腺激素细胞中组蛋白和细胞骨架丝的瓜氨酸化，但这一生理后果 缺乏生殖功能。这种知识差距是一个重要的问题，因为适当的 促性腺激素功能对于所有脊椎动物的生殖是至关重要的，并且是不育症的主要治疗靶点， 避孕。我们的长期目标是详细了解 瓜氨酸及其在促性腺激素中的生理意义。本建议的目的是确定 瓜氨酸作为促性腺激素特异性基因程序和体内细胞骨架功能的新型调节剂。我们 已发表的数据表明，GnRH诱导PAD催化的组蛋白上精氨酸残基的瓜氨酸化， 调节促黄体生成素（LH）β亚基基因的表达。初步的RNA-seq数据表明， 瓜氨酸还调节内质网加工和高尔基体小泡运输的表达 以前在促性腺激素细胞中没有发现的基因网络。除了组蛋白，我们的数据显示， 瓜氨酸化β-微管蛋白和β-肌动蛋白调节GnRH刺激后的细胞骨架重组。我们 中心假设是GnRH利用PAD催化的组蛋白和细胞骨架蛋白的瓜氨酸酶， 实施LH合成所需的关键分子变化，细胞的空间重新定位， 激素分泌中心假设将被测试与以下具体目标：（1）测试是否 控制促性腺激素合成和加工的基因由GnRH诱导的PAD催化的组蛋白调节 瓜氨酸在体外和体内。(2)为了鉴定瓜氨酸化的细胞骨架丝，并确定这些 修饰调节促性腺激素功能。在目的1中，通过RNA-seq鉴定的靶基因在大肠杆菌中的表达。 在GnRH和PAD抑制剂治疗后，将使用染色质检查LβT2促性腺激素 免疫沉淀（ChIP）。下一步将在荧光纯化的促性腺激素细胞中检测靶基因 活化细胞分选（FACS）。原发性促性腺激素将接受GnRH和PAD治疗 通过qPCR和ChIP检测抑制剂和靶基因。目标2中的拟议研究将确定瓜氨酸 使用蛋白质组学方法从LβT2和初级促性腺激素细胞中提取蛋白质。的生理重要性 瓜氨酸对初级促性腺激素细胞结构和LH分泌的影响， 用GnRH和PAD抑制剂治疗。这项研究是创新的，因为我们计划确定 GnRH如何通过瓜氨酸的未探索机制发挥作用， 事件和细胞骨架重组来控制促性腺激素生理学，这代表了一种新的， 这与目前促性腺激素领域的研究有很大不同。这项工作意义重大，因为它是一项 重要的一步，以表征一个全新的，未探索的机制刺激GnRH是至关重要的 促性腺激素功能。