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.

项目总结 核激素受体是配体门控的转录因子，主要存在于细胞核中。 在配体结合后，核激素受体与DNA结合，启动精细的转录 反应涉及性分化、激素调节、代谢控制和其他生物 后生动物特有的过程。核受体被认为是最古老的受体之一 它们被认为帮助了多细胞生物体的发展，但已知的 核激素受体没有明确的内源性配体。这些受体指的是 作为孤儿核受体。肝细胞核因子4(HNF4)是一种孤儿受体，它参与了 在1型糖尿病、Fanconi肾小管综合征4、2型糖尿病(非胰岛素)等疾病中 依赖)、高胰岛素血症和多种癌症。我的初步研究阐明了一个 单个秀丽线虫核激素受体，NHR-49，HNF4的功能同源物， 被隔离在细胞质中。据报道，胞浆中有一小部分受体， 包括糖皮质激素受体，它通过直接相互作用隔离在细胞质中 与分子伴侣HSP90结合。一旦与配体结合，糖皮质激素受体就会从 HSP90并转位到细胞核启动转录。以一种相反的方式，我假设 一种内源性脂肪酸香叶醇通过与NHR-49结合而将其隔离在细胞质中 贩卖水泡。当细胞内碳水平较低时，香叶基香叶基部分不再 合成的。这反过来又取消了NHR-49对胞浆运输的抑制隔离 囊泡，促进核定位，并诱导基因转录上调重建 动态平衡的囊泡运输。因此，这一提议的中心假设是， RAB-11.1(一种循环内体蛋白)上的两个共价连接的香叶基香叶基部分将结合到 NHR-49，而另一个香叶基将RAB-11.1/NHR-49复合体与贩运联系在一起 水泡。为了验证这一假设，我将使用多种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‘S的结构边界：亚细胞定位和囊泡缔合。目标2将 鉴定NHR-49在细胞质中的内源性配体。此外，13%的 商业上可以买到的药物针对的是50%已经去孤儿的人类核受体。 NHR-49‘S哺乳动物直系基因HNF4已被反复研究，但尚未鉴定出 唯一的内源性配体，负责其转录活性。职位的识别- 翻译修饰作为NHR-49的内源性配体提供了潜在的可译性 HNF4，它导致了无数的脂代谢紊乱。