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.

 DESCRIPTION (provided by application): Transporters for Glucocorticoids: Exploring a New Paradigm for Steroid Hormone Regulation and a Potential Strategy for Identification of Toxin/Disruptor Transporter Machinies 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转录。尽管生化，遗传和细胞生物学证据，尽管偶发，但仍与介导的运输一致（此处被称为跨质膜分子的任何过程，包括活跃的转运，包括主动转运，内吞/生物细胞吞噬，通过毛孔或通道或carterier proteeins），但这种观点仍然存在。例如，大约二十年前，我们确定了一个构成的ATP结合式转运蛋白，在酵母中有选择地导出DEX，并表明一种抑制酵母活性的药物也会导致哺乳动物的细胞内DEX增加 细胞。然而，普遍认为类固醇通过膜被动扩散的假设排除了对可能转运蛋白的重点调查。在这里，我们提出了一个三方项目，以识别类固醇转运蛋白。我们策略的一个独特因素是它的敏感性， 通过特异性结合细胞内GC与糖皮质激素受体（GR），这又可以刺激荧光报告基因蛋白的转录和翻译，或者是通过超级质体质谱法的鉴定蛋白和鉴定的立体相互作用蛋白的共价标记。我们的具体方法是（i）通过反向和前向遗传学鉴定GC转运的候选机器，（ii）通过化学和蛋白质组学鉴定GC相互作用的蛋白质，以及（iii），以测试和确认候选蛋白在小鼠中通过靶向基因组编辑的GC转运中候选蛋白的作用。这个探索项目的成功将引起未来的研究至少两个主要方向。首先，指定运输的机械 跨等离子体机制的类固醇可能会参与新的基因调节机制，发现新的骑马作用细胞特异性途径，并有助于内分泌疾病的原因和理解。第二，用于类固醇的转运机械的鉴定将激发其他亲脂性小分子的细胞访问 - 生理，病理和药理学以及环境毒素。实际上，其他此类分子的转运蛋白可能与糖皮质激素发现的转运蛋白可能存在于相同或相关的基因家族中。简单地使用不同的horsene或接收器依赖性的毒素/内分泌干扰器（例如，雌二醇，环境雌激素或芳族烃）对基于GC的策略进行简单执行，该分子是否会迅速揭示该分子是否利用相似和/或不同的机械组件。在这个探索性阶段，结论将是有趣且内容丰富的。