Defining the cytoplasmic sequestration of Caenorhabditis elegans orphan nuclear receptor 49 on trafficking vesicles

定义秀丽隐杆线虫孤儿核受体 49 在运输囊泡上的细胞质隔离

基本信息

批准号：
10313887
负责人：
Lexus Marc Tatge
金额：
$ 3.27万
依托单位：
UT SOUTHWESTERN MEDICAL CENTER
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-08-01 至 2024-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10313887
关键词：
Acetyl Coenzyme A Affinity Alanine Binding Biochemical Biological Assay Biological Process C-terminal Caenorhabditis elegans Carbon Cell Nucleus Cells Complex Confocal Microscopy Cysteine Cytoplasm Cytoplasmic Tail Cytosol DNA Data Development Disease Drug Targeting Exhibits Fatty Acids Fractionation Genes Genetic Genetic Transcription Glucocorticoid Receptor Heat-Shock Proteins 90 Hepatic Hookworms Human Hydrophobicity Hyperinsulinism Immunoprecipitation Impairment Insulin-Dependent Diabetes Mellitus Label Ligand Binding Ligand Binding Domain Ligands Link Lipids Measures Membrane Metabolic Control Metabolic Diseases Molecular Chaperones Monitor Mutate Mutation Non-Insulin-Dependent Diabetes Mellitus Nuclear Nuclear Hormone Receptors Nuclear Orphan Receptor Nuclear Receptors Nuclear Translocation Organism Orphan Orthologous Gene PF4 Gene Pathway interactions Pharmaceutical Preparations Pharmacologic Substance Pharmacotherapy Post-Translational Protein Processing Production Proteins Recycling Regulation Reporting Research Scintillation Counting Sex Differentiation Specificity Structure Syndrome Tertiary Protein Structure Testing Therapeutic Intervention Time Transgenic Organisms Up-Regulation Vesicle Work cancer type experimental study gain of function gain of function mutation geranylgeraniol geranylgeranyl pyrophosphate hormone regulation isoprenoid knock-down lipid metabolism mevalonate mutant prenyl protein geranylgeranyltransferase rab GTP-Binding Proteins receptor receptor binding response targeted treatment trafficking transcription factor vesicle transport

Acetyl Coenzyme A Affinity Alanine Binding Biochemical Biological Assay Biological Process C-terminal Caenorhabditis elegans Carbon Cell Nucleus Cells Complex Confocal Microscopy Cysteine Cytoplasm Cytoplasmic Tail Cytosol DNA Data Development Disease Drug Targeting Exhibits Fatty Acids Fractionation Genes Genetic Genetic Transcription Glucocorticoid Receptor Heat-Shock Proteins 90 Hepatic Hookworms Human Hydrophobicity Hyperinsulinism Immunoprecipitation Impairment Insulin-Dependent Diabetes Mellitus Label Ligand Binding Ligand Binding Domain Ligands Link Lipids Measures Membrane Metabolic Control Metabolic Diseases Molecular Chaperones Monitor Mutate Mutation Non-Insulin-Dependent Diabetes Mellitus Nuclear Nuclear Hormone Receptors Nuclear Orphan Receptor Nuclear Receptors Nuclear Translocation Organism Orphan Orthologous Gene PF4 Gene Pathway interactions Pharmaceutical Preparations Pharmacologic Substance Pharmacotherapy Post-Translational Protein Processing Production Proteins Recycling Regulation Reporting Research Scintillation Counting Sex Differentiation Specificity Structure Syndrome Tertiary Protein Structure Testing Therapeutic Intervention Time Transgenic Organisms Up-Regulation Vesicle Work cancer type experimental study gain of function gain of function mutation geranylgeraniol geranylgeranyl pyrophosphate hormone regulation isoprenoid knock-down lipid metabolism mevalonate mutant prenyl protein geranylgeranyltransferase rab GTP-Binding Proteins receptor receptor binding response targeted treatment trafficking transcription factor vesicle transport

展开

项目摘要

PROJECT SUMMARY Nuclear hormone receptors are ligand-gated transcription factors that primarily reside in the nucleus. Upon ligand binding, nuclear hormone receptors bind to DNA and initiate elaborate transcriptional responses involved in sexual differentiation, hormonal regulation, metabolic control and other biological processes specific to metazoans. Nuclear receptors are considered one of the most ancestral receptors that are hypothesized to have aided in the development of multicellular organisms, yet half of all known nuclear hormone receptors do not have defined endogenous ligands. These receptors are referred to as orphan nuclear receptors. Hepatic Nuclear Factor 4 (HNF4) is an orphan receptor, and it is involved in diseases such as Type 1 Diabetes, Fanconi Renotubular Syndrome 4, Type-2 Diabetes (non-insulin dependent), hyperinsulinemia, and multiple types of cancer. My preliminary studies have elucidated a single Caenorhabditis elegans nuclear hormone receptor, NHR-49, a functional ortholog to HNF4, that is sequestered in the cytosol. A small percentage of receptors have been reported in the cytosol, including the glucocorticoid receptor, which is sequestered in the cytosol through direct interactions with molecular chaperone HSP90. Upon ligand binding, the glucocorticoid receptor dissociates from HSP90 and translocates to the nucleus to initiate transcription. In an opposing manner, I hypothesize that an endogenous fatty acid, geranylgeranyl, sequesters NHR-49 in the cytosol through binding it to trafficking vesicles. When intracellular carbon levels are low, the geranylgeranyl moiety is no longer synthesized. This in turn, abolishes the inhibitory sequestration of NHR-49 to cytosolic trafficking vesicle, promotes nuclear localization, and induces transcriptional upregulation of genes to re-establish homeostatic vesicular trafficking. Therefore, the central hypothesis of this proposal is that one of the two covalently linked geranylgeranyl moieties on RAB-11.1(a recycling endosomal protein) is bound to NHR-49, while the other geranylgeranyl tethers the RAB-11.1/NHR-49 complex to the trafficking vesicle. To test this hypothesis, I will use multiple transgenic C. elegans strains that are fluorescently labeled for different ablated forms of NHR-49 and RAB-11.1. Experiments proposed in Aim 1 will define the structural boundaries of NHR-49’s subcellular localization and vesicular association. Aim 2 will identify the endogenous ligand sequestering NHR-49 in the cytoplasm. Additionally, 13% of commercially available drugs target the 50% of human nuclear receptors that have been deorphanized. NHR-49’s mammalian ortholog, HNF4, has been studied time and time again without identification of a sole endogenous ligand responsible for its transcriptional activity. The identification of a post- translational modification as an endogenous ligand for NHR-49 provides potential translatability to the HNF4, which is responsible for a myriad of lipid metabolism disorders.

项目摘要核马酮受体是原发核中的配体门控转录因子。配体结合后，核马酮受体与DNA结合并启动详尽的转录性别差异，荷尔蒙调节，代谢控制和其他生物学的反应特定于后生动物的过程。核受体被认为是最祖先的受体之一假设有助于多细胞生物的发展，但已知的一半核马酮受体没有定义内源配体。这些受体被提及作为孤儿核受体。肝核因子4（HNF4）是孤儿受体，它涉及在诸如1型糖尿病等疾病中依赖性），高胰岛素血症和多种类型的癌症。我的初步研究已经阐明了秀丽隐杆线虫核马受体NHR-49，HNF4的功能性直系同源物，在细胞质中被隔离。在细胞质中报告了一小部分接收器，包括糖皮质激素受体，该受体通过直接相互作用在细胞质中隔离与分子伴侣HSP90。配体结合，糖皮质激素受体解离从 HSP90并易位到核以启动转录。我以相反的方式假设内源性脂肪酸，黄烷基凝酰基，通过将其结合到细胞质中的NHR-49 贩运蔬菜。当细胞内碳水平较低时，黄烷基果仁部分不再是合成。反过来，取消了NHR-49的抑制作用到胞质贩运囊泡，促进核定位并诱导基因转录上调以重新建立稳态囊泡贩运。因此，该提议的核心假设是在RAB-11.1（回收内体蛋白）上，两个共价连接的黄烷基果仁基部分连接到 NHR-49，而另一个geranylgeranyl将RAB-11.1/NHR-49复合物归为贩运囊泡。为了检验这一假设，我将使用多种荧光型的转基因秀丽隐杆线虫菌株标记为NHR-49和RAB-11.1的不同消融形式。 AIM 1中提出的实验将定义 NHR-49亚细胞定位和囊泡关联的结构边界。 AIM 2意志识别细胞质中的内源性配体隔离NHR-49。另外，有13％市售药物的目标是已被脱字的50％的人类核接收器。 NHR-49的哺乳动物直系同源物HNF4已经进行了研究，并且没有识别唯一负责其转录活性的内源配体。识别后转化修饰作为NHR-49的内源配体，为对 HNF4，负责无数脂质代谢疾病。