Structure and mechanism of pendrin and the mutations that cause Pendred's Syndrome

pendrin的结构和机制以及引起Pendred综合征的突变

• 批准号: 10719603
• 负责人: Matthias Quick
• 金额: $ 38.76万
• 依托单位: COLUMBIA UNIVERSITY HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-09-01 至 2027-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10719603
• 关键词: Affect; Anions; Anti-Inflammatory Agents; Asthma; Bicarbonates; Binding; Binding Sites; Biological Assay; Cell Volumes; Cell membrane; Chemical Structure; Child; Chloride Ion; Cholesterol; Clinical; Cochlea; Complex; Cryoelectron Microscopy; Dedications; Development; Disease; Drug Targeting; Electrons; Environment; Epithelial Cells; Epithelium; Foundations; Functional disorder; Genes; Genetic Diseases; Goals; Health; Homeostasis; Homologous Gene; Human; Hydroxide Ion; Hypertension; Iodides; Ion Exchange; Ion Transport; Ions; Kidney; Knowledge; Labyrinth; Lipids; Lung; Membrane Lipids; Molecular Target; Motivation; Mutation; Niflumic Acid; Non-Steroidal Anti-Inflammatory Agents; Pendred Syndrome; Pharmaceutical Preparations; Pharmacology; Physiological; Physiology; Play; Proteins; Reaction; Regulation; Reporting; Role; Sigma Factor; Solid; Structure; Syndrome; System; Thyroid Gland; Time; Tissues; Transmembrane Domain; Transport Process; Variant; Vestibular Aqueduct; Work; airway hyperresponsiveness; antagonist; antiport; causal variant; childhood hearing loss; early childhood; efficacious treatment; experimental study; hereditary hearing loss; improved; inhibitor; insight; new therapeutic target; novel; novel therapeutics; pH Homeostasis; protein expression; protein purification; reconstitution; side effect; small molecule; small molecule inhibitor; stoichiometry; sulfate transporter

Pendrin (SLC26A4) is expressed in epithelial tissues, e.g., in the inner ear, thyroid, kidney, and lung where it plays a central role in ion homeostasis and the regulation of the cell volume. Mutations in the Slc26a4 gene cause Pendred Syndrome and enlarged vestibular aqueduct syndrome (EVAS), both of which are genetic disorders characterized by childhood early hearing loss in children and account for 5-10% of hereditary hearing loss and are currently not curable. Previous studies illuminated the role of pendrin in the physiology of the cochlea, thyroid gland, kidney and proposed that it can transport iodide ions (I-), bicarbonate ions (HCO3-), chloride ions (Cl-), and hydroxide ions (OH-) across epithelial cell membranes according to an electroneutral exchange (antiport) reaction. However, our understanding of pendrin remains rudimentary due to a lack of purified protein that enables precise functional studies without the potential interference of native proteins replete in cellular/native systems and structural studies. To overcome this gap in our understanding, we have successfully expressed and purified a mammalian homolog of human pendrin and developed binding and transport assays to determine substrate selectivity and transport. Preliminary studies confirmed that purified pendrin reconstituted in lipid membranes transports I- or HCO3- in exchange with Cl- or OH- and revealed that the transport process is electrogenic, i.e., the stoichiometry of ion exchange is not 1:1 as previously postulated for electroneutral antiport. We determined I-- and HCO3--bound pendrin structures by cryo-electron microscopy, and our preliminary analysis suggests that pendrin has two anion binding sites, which may provide an explanation for the electrogenic transport process. The structure reveals novel interactions between the transmembrane domain (TMD) and the cytosolic domain, i.e., the sulphate transporter and anti-sigma factor antagonist domain (STAS) that appears to be relevant for the transport mechanism because mutations at the interface of STAS and TMD are known to cause Pendred Syndrome. Pendrin is also a promising drug target for attenuating airway hyperresponsiveness in asthma and for reducing hypertension, and many pendrin inhibitors, e.g., the non- steroidal anti-inflammatory drug niflumic acid, has been reported to target pendrin, but the mechanisms of inhibition remain unknown. Whereas these inhibitors could be repurposed to target pendrin, their action on pendrin may also cause undesired side-effects, thus highlighting the need to elucidate the mechanisms of pendrin inhibition by small molecules. We determined the structures of pendrin in complex with the anti- inflammatory drugs YS-01 and niflumic acid, and our preliminary analysis shows that the two inhibitors occupy different binding sites, providing motivation for the further determination of the mechanisms of inhibition. To this end, the overall goal of this project is to understand the mechanism and pharmacology of pendrin at the atomic level to aid in the development of efficacious drugs that specifically target pendrin in improved therapies against Pendred Syndrome and EVAS.

Pendrin（SLC26A4）在上皮组织中表达，例如在内耳，甲状腺，肾脏和肺部 在离子稳态和细胞体积的调节中起着核心作用。 SLC26A4基因中的突变 导致Pendred综合征和扩大前庭渡槽综合征（EVA），这两种都是遗传 儿童童年早期听力损失的特征疾病，占遗传听证会的5-10％ 损失，目前无法治愈。先前的研究阐明了pendrin在生理学中的作用 耳蜗，甲状腺，肾脏，并提出它可以运输碘化离子（I-），碳酸氢盐离子（HCO3-）， 氯离子（Cl-）和氢氧化物离子（OH-）跨上皮细胞膜，根据电荷 交换反应。但是，由于缺乏 纯化的蛋白质可以实现精确的功能研究，而无需天然蛋白质的潜在干扰 在细胞/天然系统和结构研究中。为了克服这一差距，我们有 成功地表达并纯化了人类彭德蛋白的哺乳动物同源物，并形成结合和 运输分析以确定底物选择性和运输。初步研究证实了纯化 Pendrin在脂质膜中重构I-或HCO3-与Cl-或OH-交换，并揭示 运输过程是电源的，即离子交换的化学计量指定不是1：1，如前所述 用于电自动物。我们通过低温电子显微镜确定了I-和HCO3结合的Pendrin结构， 我们的初步分析表明，Pendrin具有两个阴离子结合位点，这可能会提供一个解释 用于电源运输过程。该结构揭示了跨膜之间的新型相互作用 结构域（TMD）和胞质结构域，即硫酸盐转运蛋白和抗sigma因子拮抗剂结构域 （Stas）似乎与运输机制有关，因为Stas和 已知TMD会引起Pendred综合征。 Pendrin也是衰减气道的有前途的药物目标 哮喘和降低高血压的过度反应性，以及许多Pendrin抑制剂，例如非 - 据报道，类固醇抗炎药硝酸靶向pendrin，但是 抑制仍然未知。尽管这些抑制剂可以重新使用以靶向pendrin，但它们的作用 pendrin也可能引起不希望的副作用，从而突出了阐明机制的需求 小分子抑制pendrin。我们确定了与抗 - 炎症药YS-01和硝酸酸，我们的初步分析表明，这两个抑制剂占据 不同的结合位点，为进一步确定抑制机制提供了动机。对此 最后，该项目的总体目标是了解原子蛋白的机制和药理学 有助于开发有效药物的水平，这些药物专门针对pendrin，以改善疗法 Pendred综合征和EVA。