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

项目摘要 核激素受体是主要存在于细胞核中的配体门控转录因子。 在配体结合后，核激素受体与DNA结合并启动精细的转录， 参与性分化、激素调节、代谢控制和其他生物学反应。 后生动物特有的过程。核受体被认为是最古老的受体之一 据推测，这些蛋白质有助于多细胞生物的发育，但已知的蛋白质中有一半是 核激素受体没有确定的内源性配体。这些受体被称为 作为孤儿核受体。肝核因子4（HNF 4）是一种孤儿受体，它参与 在诸如1型糖尿病、Fanconi肾管综合征4、2型糖尿病（非胰岛素依赖性）、糖尿病性肾病、糖尿病性肾病等疾病中， 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，HNF 4的功能性直系同源物， 被隔离在细胞质中据报道，小部分受体存在于胞质溶胶中， 包括糖皮质激素受体，其通过直接相互作用被隔离在胞质溶胶中 分子伴侣HSP 90在配体结合时，糖皮质激素受体从 HSP 90和易位到细胞核启动转录。我以相反的方式假设 内源性脂肪酸，香叶基香叶基，通过结合NHR-49在胞质溶胶中螯合NHR-49， 运输囊泡当细胞内碳水平低时，香叶基香叶基部分不再是 合成了这反过来又消除了NHR-49对胞质运输的抑制性螯合作用 囊泡，促进核定位，并诱导基因的转录上调，以重新建立 稳态囊泡运输。因此，这一建议的核心假设是， RAB-11.1（一种再循环内体蛋白）上的两个共价连接的香叶基香叶基部分结合到 NHR-49，而另一种香叶基香叶基将RAB-11.1/NHR-49复合物与贩运联系在一起。 囊泡为了验证这一假设，我将使用多个转基因C。elegans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 鉴定在细胞质中螯合NHR-49的内源性配体。此外，13%的 市售药物靶向50%的已去铁蛋白化的人核受体。 NHR-49的哺乳动物直系同源物HNF 4已经被一次又一次地研究，但没有鉴定出一种 负责其转录活性唯一内源性配体。一个岗位的认定-- 作为NHR-49的内源性配体的翻译修饰为NHR-49提供了潜在的可翻译性。 HNF 4是导致多种脂质代谢紊乱的原因。

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.