Ca2+-mediated higher-order assembly of b0,+AT-rBAT is a key step for system b0,+ biogenesis and cystinuria
Ca2+-mediated higher-order assembly of b0,+AT-rBAT is a key step for system b0,+ biogenesis and cystinuria
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Ca2 介导的 b0、AT-rBAT 高阶组装是系统 b0、生物发生和胱氨酸尿症的关键步骤
DOI：
10.1101/2021.05.06.443019
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发表时间：
2021
期刊：
影响因子：
--
通讯作者：
Nagamori Shushi
中科院分区：
文献类型：
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作者：
Lee Yongchan;Wiriyasermkul Pattama;Moriyama Satomi;Mills Deryck J.;Kuehlbrandt Werner;Nagamori Shushi
胱氨酸尿症是一种遗传性疾病，其特征是双碱性氨基酸和胱氨酸的过度排泄，可引起复发性肾结石和偶尔严重的肾衰竭。组成系统b0，+的两种负责蛋白rBAT和b0，+AT的突变分别与I型和非I型胱氨酸尿有关，由于蛋白质运输缺陷或催化失活，它们表现出不同的表型。尽管最近对人类b0 + AT-rBAT的结构研究提出了一种运输失活突变的模型，但I型突变触发运输缺陷的机制尚不清楚。在这里，使用电子冷冻显微镜和生物化学，我们发现Ca2+介导的系统b0，+的高阶组装是其在细胞表面运输的关键。我们发现Ca2+稳定了两个rBAT分子之间的界面以介导超二聚化，这反过来促进了系统b0，+的n -聚糖成熟。常见的胱氨酸尿突变体T216M和破坏rBAT中Ca2+位点的突变导致高阶组装的丢失，导致蛋白质运输缺陷。超二聚体界面的突变再现了错误运输表型，表明超二聚体对细胞功能至关重要。基于细胞的转运实验证实了Ca2+位点和超二聚化的重要性，并进一步表明哪些残基参与了阳离子氨基酸识别。总之，我们的研究结果提供了I型胱氨酸尿的分子基础，并为开发新的治疗策略提供了指导。更广泛地说，我们的研究结果揭示了转运蛋白寡聚物组装与贩运疾病之间前所未有的联系。
Cystinuria is a genetic disorder characterized by overexcretion of dibasic amino acids and cystine, which causes recurrent kidney stones and occasionally severe kidney failure. Mutations of the two responsible proteins, rBAT and b0,+AT, which comprise system b0,+, are linked to type I and non-type I cystinuria respectively and they exhibit distinct phenotypes due to protein trafficking defects or catalytic inactivation. Although recent structural insights into human b0,+AT–rBAT suggested a model for transport-inactivating mutations, the mechanisms by which type I mutations trigger trafficking deficiencies are not well understood. Here, using electron cryo-microscopy and biochemistry, we discover that Ca2+-mediated higher-order assembly of system b0,+is the key to its trafficking on the cell surface. We show that Ca2+stabilizes the interface between two rBAT molecules to mediate super-dimerization, and this in turn facilitates the N-glycan maturation of system b0,+. A common cystinuria mutant T216M and mutations that disrupt the Ca2+site in rBAT cause the loss of higher-order assemblies, resulting in protein trafficking deficiency. Mutations at the super-dimer interface reproduce the mis-trafficking phenotype, demonstrating that super-dimerization is essential for cellular function. Cell-based transport assays confirmed the importance of the Ca2+site and super-dimerization, and additionally suggested which residues are involved in cationic amino acid recognition. Taken together, our results provide the molecular basis of type I cystinuria and serve as a guide to develop new therapeutic strategies against it. More broadly, our findings reveal an unprecedented link between transporter oligomeric assembly and trafficking diseases in general.