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

垂蛋白(SLC26A4)在上皮组织中表达，例如，在内耳、甲状腺、肾脏和肺中表达，其中 在离子动态平衡和细胞体积的调节中起着核心作用。SLC26A4基因的突变 引起Pendred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，目前无法治愈。先前的研究阐明了吊环蛋白在生理中的作用。 耳蜗腺、甲状腺、肾脏，认为它能转运碘离子(I-)、碳酸氢根离子(HCO3-)、 氯离子(Cl-)和氢氧离子(OH-)通过上皮细胞膜的电子中和 交换(反端口)反应。然而，由于缺乏对垂丝蛋白的了解，我们对它的了解仍处于初级阶段。 纯化的蛋白质能够在不受天然蛋白质潜在干扰的情况下进行精确的功能研究 在细胞/自然系统和结构研究方面。为了克服我们在理解上的差距，我们有 成功表达和纯化了人垂垂蛋白的哺乳动物同源物，并开发了结合和 转运试验，以确定底物的选择性和转运。初步研究证实，提纯的 在脂膜中重组的侧耳环转运I-或HCO3-与Cl-或OH-交换，揭示了 传输过程是电生的，即离子交换的化学计量比不是先前假设的1：1 用于电子中和反端口。我们用冷冻电子显微镜测定了I-和HCO3-结合的吊环结构， 我们的初步分析表明，侧翼蛋白有两个阴离子结合部位，这可能为解释 用于产生电的传输过程。该结构揭示了跨膜之间的新的相互作用 结构域(TMD)和胞浆结构域，即硫酸盐转运体和抗Sigma因子拮抗剂结构域 (STAS)似乎与运输机制相关，因为STAS和STAS界面上的突变 众所周知，TMD会导致Pendred综合征。吊环蛋白也是一种很有希望的减弱呼吸道的药物靶点。 哮喘的高反应性和用于降低高血压，以及许多吊环蛋白抑制剂，例如非 类固醇抗炎药物尼氟米酸，已有报道针对侧耳蛋白，但其作用机制。 抑制作用仍不清楚。虽然这些抑制物可以被重新用于靶向支链蛋白，但它们对 吊环蛋白也可能引起不良副作用，因此需要阐明其作用机制。 小分子对吊环的抑制作用。确定了该化合物的结构，确定了其结构。 炎性药物YS-01和尼氟米酸，我们的初步分析表明这两种抑制剂占据了 不同的结合部位，为进一步确定抑制机制提供了动力。对这件事 最后，本项目的总体目标是从原子的角度了解吊环蛋白的作用机制和药理作用。 水平，以帮助开发有效的药物，特别是在改进的治疗中靶向支链蛋白 Pendred综合征和动静脉曲张

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.