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：
10674775
负责人：
Lexus Marc Tatge
金额：
$ 3.43万
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/10674775
关键词：
Ablation 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 Dissociation Drug Targeting Endosomes Exhibits Fatty Acids Fractionation Gene Expression Regulation 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 Marketing 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 Syndrome Tertiary Protein Structure Testing Therapeutic Intervention Time Transcription Initiation 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

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.

项目概要核激素受体是主要存在于细胞核中的配体门控转录因子。配体结合后，核激素受体与 DNA 结合并启动复杂的转录涉及性别分化、激素调节、代谢控制和其他生物反应后生动物特有的过程被认为是最古老的受体之一。被认为有助于多细胞生物的发展，但已知的有一半核激素受体没有明确的内源配体，这些受体被称为。肝核因子 4 (HNF4) 是一种孤儿受体，它参与其中。 1 型糖尿病、范可尼肾管综合征 4、2 型糖尿病（非胰岛素依赖）、高胰岛素血症和多种类型的癌症。单一秀丽隐杆线虫核激素受体 NHR-49，HNF4 的功能直系同源物，据报道，一小部分受体被隔离在细胞质中，包括糖皮质激素受体，它通过直接相互作用被隔离在细胞质中与分子伴侣 HSP90 结合后，糖皮质激素受体从其上解离。 HSP90 并易位至细胞核以启动转录。内源性脂肪酸香叶基香叶基通过与 NHR-49 结合而将 NHR-49 隔离在细胞质中当细胞内碳水平较低时，香叶基香叶基部分不再存在。这反过来又消除了 NHR-49 对胞质运输的抑制性隔离。囊泡，促进核定位，并诱导基因转录上调以重建因此，该提案的中心假设是其中之一。 RAB-11.1（一种内体蛋白）上的两个共价连接的回收香叶基香叶基部分与 NHR-49，而其他香叶基香叶基将 RAB-11.1/NHR-49 复合物束缚在贩运上为了检验这个假设，我将使用多种带有荧光的转基因线虫菌株。目标 1 中提出的实验将定义不同消融形式的 NHR-49 和 RAB-11.1。 NHR-49 的亚细胞定位和囊泡关联的结构边界将被确定。另外，还鉴定了细胞质中 13% 的内源配体隔离 NHR-49。市售药物靶向 50% 已脱孤儿的人类核受体。 NHR-49 的哺乳动物直系同源物 HNF4 已被一次又一次地研究，但尚未鉴定出负责其转录活性的唯一内源配体。作为 NHR-49 内源配体的翻译修饰为 NHR-49 提供了潜在的可翻译性 HNF4，它是导致多种脂质代谢紊乱的原因。