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

Pendrin（SLC 26 A4）在上皮组织中表达，在内耳，甲状腺，肾脏和肺部， 在离子体内平衡和细胞体积调节中起核心作用。Slc 26 a4基因突变 导致Pendred综合征和前庭水管扩大综合征（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% 目前还无法治愈。以前的研究阐明了pendrin在脑组织生理学中的作用， 耳蜗，甲状腺，肾脏，并提出它可以运输碘离子（I-），碳酸氢根离子（HCO 3-）， 氯离子（Cl-）和氢氧根离子（OH-）穿过上皮细胞膜， 交换（逆向）反应。然而，我们对pendrin的了解仍然是初步的， 纯化的蛋白质，使精确的功能研究，而没有潜在的干扰天然蛋白质充满 在细胞/天然系统和结构研究中。为了克服我们理解上的这一差距，我们必须 成功地表达和纯化了人pendrin的哺乳动物同系物， 转运测定以确定底物选择性和转运。初步研究证实， 在脂质膜中重构的pendrin转运I-或HCO 3-，与Cl-或OH-交换，并揭示， 运输过程是产电的，即，离子交换化学计量不是如先前假定的1：1 用于电中性反向转运。我们通过冷冻电子显微镜确定了I-和HCO 3-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，这可能提供了一种解释， 用于产电运输过程。该结构揭示了跨膜之间的新的相互作用， 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），这似乎是相关的运输机制，因为突变的界面STAS和 TMD被认为是导致Pendred综合征的原因。Pendrin也是一种很有前途的药物靶点， 哮喘中的高反应性和降低高血压，以及许多pendrin抑制剂，例如，非 据报道，甾体抗炎药尼氟灭酸靶向pendrin，但其机制 抑制作用仍然未知。虽然这些抑制剂可以重新用于靶向彭德林，但它们对彭德林的作用 pendrin也可能引起不希望的副作用，因此强调需要阐明的机制， pendrin抑制小分子。我们确定了pendrin与反式- 炎症药物YS-01和尼氟灭酸，我们的初步分析表明，这两种抑制剂占据了 不同的结合位点，为进一步确定抑制机制提供了动力。本 最终，本项目的总体目标是了解pendrin在原子水平上的机制和药理学。 水平，以帮助开发有效的药物，专门针对pendrin在改善治疗， 悬垂综合征和EVAS。

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.