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

• 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
• 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

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。核受体被认为是最古老的受体之一 假设它们有助于多细胞生物的发育，但已知的有一半 核激素受体没有确定的内源配体。这些受体被称为 作为孤儿核受体。肝核因子 4 (HNF4) 是一种孤儿受体，它参与 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。我的初步研究阐明了 单一秀丽隐杆线虫核激素受体 NHR-49，HNF4 的功能直系同源物， 被隔离在细胞质中。据报道，一小部分受体存在于细胞质中， 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。配体结合后，糖皮质激素受体从 HSP90 并易位至细胞核以启动转录。以相反的方式，我假设 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 贩运囊泡。当细胞内碳水平较低时，香叶基香叶基部分不再 合成的。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 稳态囊泡运输。因此，该提案的中心假设之一是 RAB-11.1（一种回收内体蛋白）上的两个共价连接的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 囊泡。为了验证这个假设，我将使用多种带有荧光的转基因线虫菌株 标记不同消融形式的 NHR-49 和 RAB-11.1。目标 1 中提出的实验将定义 NHR-49 的亚细胞定位和囊泡关联的结构边界。目标2将 鉴定细胞质中隔离 NHR-49 的内源配体。此外，13% 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。岗位身份识别 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。