Structural and functional investigations of two lysosomal transporters implicated in developmental disorders

项目摘要 溶酶体贮积病（LSD）包括一组超过70种遗传性常染色体隐性疾病， 是由溶酶体蛋白质的功能障碍引起的，导致特定底物在溶酶体中的积累。 这些相对罕见的疾病往往表现在婴儿身上，尽管成人形式也存在。胱氨酸病与婴儿 唾液酸储存病（ISSD）是两种常见的LSD。胱氨酸病的症状包括肾 和视力问题，而ISSD的症状包括中枢神经系统问题和早逝。 这两种遗传疾病也会导致严重的发育迟缓。胱氨酸病是由突变引起的， 蛋白质胱氨酸蛋白酶，一种溶酶体H+：胱氨酸同向转运体，而ISSD是由蛋白质唾液酸蛋白的突变引起的， 溶酶体H+：唾液酸同向转运体和神经递质单向转运体。结构和功能数据将有助于摆脱 阐明疾病突变影响这些蛋白质功能的确切分子机制。在我 在初步研究中，我产生了一种结构特异性单克隆抗体， 胱氨酸蛋白酶Fab 3 H5使用这种抗体，我解决了人类全长胱氨酸蛋白酶的三种结构：一个外向的- 在pH 7.5时，在3.4-μ m分辨率下的面向apo构象，在3.2-μ m分辨率下的面向apo构象， pH 5.0，和胱氨酸结合构象在3.4- 100分辨率。这些结构揭示了所涉及的残基 在介导其底物胱氨酸转运的胞质和腔门中，以及 在胱氨酸蛋白酶的结合口袋中与胱氨酸相互作用。这种蛋白质的初步电生理数据 进一步鉴定了破坏转运的突变D205 N和D305 N，以及突变Q284 A和D346 N， 其保持与野生型蛋白相似的转运活性。为了完全确定分子 为了研究胱氨酸蛋白酶体的转运机制，我建议解决这些胱氨酸蛋白酶体突变体的结构，并测试 他们与电生理分析，建立一个更完整的图片的结合和运输机制。 关于Sialin和ISSD，我的初步工作已经产生了单克隆抗体;然而，3D 用该抗体重建唾液酸蛋白没有产生可见的二级结构。因此，我将首先优化 抗体筛选过程。这将使我能够分离出一种结构特异的单克隆抗体， 可用于结构研究。然后，我将使用相同的一般方法解决Sialin的结构， 用胱抑素成功了由于唾液酸蛋白也是H+：唾液酸同向转运体，因此不同的pH环境将影响唾液酸的转运。 用于捕获不同的构象。唾液酸蛋白还结合和运输各种底物。所以我会 还解决了Sialin的底物结合结构，从而提供了更多关于结合口袋的信息 的基板。然后通过电生理学检测重要的机制和底物结合残基 (for唾液酸）和放射性标记的底物摄取测定（用于神经递质）。这些拟议的研究将 揭示LSD胱氨酸病和ISSD背后的潜在分子机制，为进一步研究铺平道路。 转化研究和治疗设计。

PROJECT SUMMARY Lysosomal storage diseases (LSDs) comprise a group of over 70 inherited autosomal recessive disorders that are caused by malfunctions in lysosomal proteins, leading to a buildup of specific substrates in the lysosome. These relatively rare disorders tend to manifest in infants, although adult forms also exist. Cystinosis and Infantile Sialic acid Storage Disease (ISSD) are two of the more common LSDs. Symptoms of Cystinosis include renal and vision problems, while symptoms of ISSD include problems with the central nervous system and early death. Both of these genetic disorders also cause severe developmental delay. Cystinosis is caused by mutations in the protein Cystinosin, a lysosomal H+:cystine symporter, while ISSD is caused by mutations in the protein Sialin, a lysosomal H+:sialic acid symporter and neurotransmitter uniporter. Structural and functional data will help shed light on the exact molecular mechanism by which disease mutations affect the function of these proteins. In my preliminary studies, I generated a structurally specific monoclonal antibody that recognizes the luminal domain of Cystinosin, Fab3H5. Using this antibody, I solved three structures of human full-length Cystinosin: an outward- facing apo conformation at 3.4-Å resolution at pH 7.5, an inward-facing apo conformation at 3.2-Å resolution at pH 5.0, and a cystine-bound conformation at 3.4-Å resolution. These structures revealed the residues involved in the cytosolic and luminal gates that mediate the transport of its substrate cystine, as well as the residues that interact with cystine in the binding pocket of Cystinosin. Preliminary electrophysiological data for this protein further identified mutations D205N and D305N, which abolished transport, and mutations Q284A and D346N, which maintained a transport activity similar to the wild-type protein. In order to fully define the molecular mechanism of transport for Cystinosin, I propose to solve the structure of these Cystinosin mutants and test them with electrophysiological assays, building a more complete picture of the binding and transport mechanism. With reference to Sialin and ISSD, my preliminary work has yielded a monoclonal antibody; however, the 3D reconstruction of Sialin with this antibody did not yield visible secondary structures. Therefore, I will first optimize the antibody screening process. This will allow me to isolate a structurally specific monoclonal antibody that I can use for structural studies. I will then solve the structure of Sialin using the same general approach that has been successful with Cystinosin. Since Sialin is also a H+:sialic acid symporter, different pH environments will be used to capture different conformations. Sialin also binds and transports various substrates. Therefore, I will also solve the substrate-bound structures of Sialin, thus providing more information about the binding pocket(s) of the substrates. Important mechanistic and substrate binding residues will then be tested via electrophysiology (for sialic acid) and radiolabeled substrate uptake assays (for neurotransmitters). These proposed studies will reveal the underlying molecular mechanisms behind the LSDs Cystinosis and ISSD, paving the way for further translational research and therapeutic design.