Transporters for Glucocorticoids: Exploring a New Paradigm for Steroid Hormone Regulation, and a Potential Strategy for Identification of Toxin/Disruptor Transporter Machineries

糖皮质激素转运蛋白：探索类固醇激素调节的新范式以及识别毒素/干扰物转运蛋白机制的潜在策略

• 批准号: 9120396
• 负责人: KEITH Robert YAMAMOTO
• 金额: $ 23.78万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-08-05 至 2018-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9120396
• 关键词: ATP-Binding Cassette Transporters; Active Biological Transport; Adverse effects; Affect; Affinity; Affinity Labels; Alkynes; Alleles; Appearance; Aromatic Hydrocarbons; Azides; Binding; Biochemical Genetics; Biological; Biophysics; Biotin; Blood Circulation; Breathing; CCL21 gene; CRISPR/Cas technology; Caenorhabditis elegans; Carrier Proteins; Cell Extracts; Cell Line; Cell membrane; Cells; Cellular Membrane; Cessation of life; Chemistry; Cholesterol; Cloning; Code; Copper; Coupling; Cytoplasm; Defect; Detection; Development; Dexamethasone; Diagnosis; Diffuse; Diffusion; Disease; Elements; Endocrine; Endocrine Disruptors; Endocrine System Diseases; Endocytosis; Environmental Estrogen; Estradiol; Etiology; Evaluation; Gene Family; Genes; Genetic Transcription; Glucocorticoid Receptor; Glucocorticoids; Health; Hormones; Human; Human Development; Hydrocortisone; Intestines; Investigation; Lead; Libraries; Ligands; Liver; Lung; Lymphoma; Mammalian Cell; Mass Spectrum Analysis; Mediating; Membrane; Membrane Proteins; Mus; Neurons; Nuclear Receptors; Oral; Outcome; Oxidative Phosphorylation; P-Glycoprotein; Partition Coefficient; Pathologic; Pharmaceutical Preparations; Physiological; Pinocytosis; Process; Proteins; Proteome; Proteomics; RNA Interference; Reaction; Receptor Gene; Regulation; Regulator Genes; Reporter; Reporting; Role; Route; Signal Transduction; Specific qualifier value; Specificity; Staging; Steroids; Technology; Testing; Therapeutic; Tissues; Toxic Environmental Substances; Toxin; Translations; Work; Yeasts; absorption; adduct; affinity labeling; aqueous; base; crosslink; cycloaddition; forward genetics; genome editing; hormone regulation; in vivo; interest; mutant; novel; prophylactic; receptor; reverse genetics; small molecule; steroid hormone; success; uptake

 DESCRIPTION (provided by applicant): Transporters for Glucocorticoids: Exploring a New Paradigm for Steroid Hormone Regulation and a Potential Strategy for Identification of Toxin/Disruptor Transporter Machineries A well-entrenched paradigm holds that steroid hormones, like glucocorticoids (GCs), diffuse freely across plasma membranes in order to access their intracellular receptors and influence gene transcription. This view persists despite biochemical, genetic, and cell biological evidence, albeit sporadic, consistent with mediated transport (herein referred to as any process that moves molecules across plasma membranes, including active transport, endocytosis/pinocytosis, passage through pores or channels, and/or coupling to carrier proteins) of steroids. Nearly two decades ago, for example, we identified a conserved ATP-binding-cassette transporter that selectively exports dex in yeast, and showed that a drug that inhibits the yeast activity also leads to increased intracellular dex in mammalian cells. Nevertheless, the widely held assumption that steroids diffuse passively through membranes precluded a focused inquiry into possible transporters. Here, we propose a tripartite project to identify steroid transporters. A distinctive element of our strategy is its sensitivity, achieved either by specific binding of intracellular GCs to the glucocorticoid receptor (GR), which in turn stimulates increased transcription and translation of fluorescent reporter proteins, or by covalent affinity labeling of steroid-interacting proteins and identification by ultrasensitie mass spectrometry. Our specific approaches are (i) to identify candidate machineries for GC transport via reverse and forward genetics, (ii) to identify GC-interacting proteins via chemistry and proteomics, and (iii) to test and validate roles for candidate proteins in GC transport via targeted genome editing in mice. Success of this exploratory project would provoke future research extending in at least two major directions. First, machineries that specify the transit of steroids across plasma membranes will likely participate in novel gene regulatory mechanisms, uncover new avenues to cell specificity of hormone action, and contribute to both the cause and the understanding of endocrine diseases. Second, identification of transport machinery for steroids will motivate re-evaluation of cellular access for other lipophilic small molecules - physiologic, pathologic and pharmacologic, as well as environmental toxins. Indeed, transporters for other such molecules are likely to reside in the same or related gene families as those discovered for glucocorticoids. A simple re- execution of our GC-based strategy using a different hormone or receptor-dependent toxin/endocrine disruptor (for example, estradiol, an environmental estrogen, or an aromatic hydrocarbon) would reveal quickly whether this molecule utilizes similar and/or distinct machinery components. At this exploratory stage, either conclusion would be interesting and informative.

 描述（由申请人提供）：糖皮质激素转运蛋白：探索类固醇激素调节的新范式和识别毒素/干扰物转运机制的潜在策略 一个根深蒂固的范式认为类固醇激素，如糖皮质激素（GC），可以自由扩散尽管存在生化、遗传和细胞生物学，但这种观点仍然存在。证据虽然零星，但与类固醇的介导运输（本文指的是使分子穿过质膜的任何过程，包括主动运输、内吞作用/胞饮作用、通过孔或通道和/或与载体蛋白偶联）一致。例如，几十年前，我们发现了一种保守的 ATP 结合盒转运蛋白，可以选择性地输出酵母中的 dex，并表明抑制酵母活性的药物也会导致哺乳动物细胞内 dex 的增加 然而，普遍认为类固醇通过细胞膜被动扩散的假设排除了对可能的转运蛋白的集中研究，我们的策略的一个独特要素是其敏感性。 通过细胞内 GC 与糖皮质激素受体 (GR) 的特异性结合（进而刺激荧光报告蛋白的转录和翻译增加）或通过类固醇相互作用蛋白的共价亲和标记并通过超灵敏质谱法进行鉴定来实现。 (i) 通过反向和正向遗传学鉴定 GC 运输的候选机制，(ii) 通过化学和蛋白质组学鉴定 GC 相互作用蛋白，以及 (iii) 测试和验证通过对小鼠进行靶向基因组编辑来确定 GC 转运中的候选蛋白，这一探索性项目的成功将引发未来至少在两个主要方向上的研究。 跨质膜的类固醇可能会参与新的基因调控机制，揭示激素作用的细胞特异性的新途径，并有助于内分泌疾病的病因和理解。 其次，类固醇转运机制的识别将促进对内分泌疾病的重新评估。其他亲脂性小分子的细胞通路——生理学、病理学和药理学以及环境毒素事实上，其他此类分子的转运蛋白可能位于与发现的那些相同或相关的基因家族中。使用不同的激素或受体依赖性毒素/内分泌干扰物（例如雌二醇、环境雌激素或芳香烃）简单地重新执行我们基于GC的策略将快速揭示该分子是否利用相似的和/或不同的机械部件。在这个探索阶段，任何一个结论都是有趣且信息丰富的。