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、肾脏和肺中 在离子稳态和细胞体积调节中发挥核心作用。 Slc26a4 基因突变 导致彭德雷综合征和前庭导水管扩大综合征 (EVAS)，这两种综合征都是遗传性的 以儿童早期听力损失为特征的疾病，占遗传性听力的 5-10% 损失且目前无法治愈。先前的研究阐明了 pendrin 在生理学中的作用 耳蜗、甲状腺、肾脏，提出它可以运输碘离子（I-）、碳酸氢根离子（HCO3-）、 根据电中性，氯离子 (Cl-) 和氢氧根离子 (OH-) 穿过上皮细胞膜 交换（反向）反应。然而，由于缺乏对潘德林的了解，我们对潘德林的理解仍然很初级。 纯化的蛋白质可实现精确的功能研究，而不会受到天然蛋白质的潜在干扰 在细胞/天然系统和结构研究中。为了克服我们理解上的差距，我们有 成功表达和纯化了人 pendrin 的哺乳动物同源物，并开发了结合和 转运测定以确定底物选择性和转运。初步研究证实，纯化的 在脂质膜中重构的 pendrin 转运 I- 或 HCO3- 与 Cl- 或 OH- 交换，并揭示了 传输过程是生电的，即离子交换的化学计量不是之前假设的 1:1 对于电中性反端口。我们通过冷冻电子显微镜确定了 I- 和 HCO3- 结合的 pendrin 结构， 我们的初步分析表明pendrin有两个阴离子结合位点，这可能提供了一个解释 用于生电传输过程。该结构揭示了跨膜之间的新颖相互作用 结构域（TMD）和胞质结构域，即硫酸盐转运蛋白和抗 σ 因子拮抗剂结构域 （STAS）似乎与运输机制相关，因为 STAS 和 众所周知，TMD 会导致彭德雷综合症。 Pendrin 也是一个有前途的减弱气道的药物靶点 哮喘高反应性和降低高血压，以及许多 pendrin 抑制剂，例如非 据报道，类固醇抗炎药尼氟酸可以靶向 pendrin，但其作用机制 抑制作用仍未知。虽然这些抑制剂可以重新用于靶向 pendrin，但它们的作用 pendrin 也可能引起不良副作用，因此强调需要阐明其机制 pendrin 通过小分子抑制。我们确定了与抗-pendrin复合物的结构 炎症药物YS-01和尼氟酸，我们初步分析表明这两种抑制剂占据 不同的结合位点，为进一步确定抑制机制提供了动力。对此 最后，该项目的总体目标是在原子水平上了解pendrin的机制和药理学 水平，以帮助开发专门针对 pendrin 的有效药物，以改进治疗方法 彭德雷综合症和 EVAS。