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

项目总结 溶酶体储存疾病(LSD)包括一组70多种遗传性常染色体隐性遗传性疾病 是由溶酶体蛋白中的故障引起的，导致溶酶体中特定底物的堆积。 这些相对罕见的疾病往往出现在婴儿身上，尽管成人也存在。胱氨酸病与婴幼儿 唾液酸贮积症(ISSD)是两种较常见的LSD。膀胱炎的症状包括肾脏 和视力问题，而ISSD的症状包括中枢神经系统问题和过早死亡。 这两种遗传性疾病也会导致严重的发育迟缓。胱氨酸病是由基因突变引起的 蛋白Cystinosin是一种溶酶体H+：Cystine共转运蛋白，而ISSD是由蛋白Sialin的突变引起的， 溶酶体H+：唾液酸转运体和神经递质单转运体。结构和功能数据将有助于摆脱 疾病突变影响这些蛋白质功能的确切分子机制。在我的 初步研究，我产生了一种识别管腔结构域的结构特异性单抗 Cystinosin，Fab3H5。使用这种抗体，我解决了人类全长半胱氨酸氨基转移酶的三种结构：一种外向结构-- 在pH 7.5时，面对3.4？分辨率的载脂蛋白构象；在3.2？分辨率时，向内的载脂蛋白构象 PH 5.0，3.4°分辨率的半胱氨酸构象。这些结构揭示了所涉及的残基 在胞液和管腔门中，介导其底物胱氨酸的运输，以及 在胱氨球蛋白的结合口袋中与胱氨酸相互作用。该蛋白的初步电生理数据 进一步鉴定了取消转运的突变D205N和D305N，以及突变Q284A和D346N， 它保持了与野生型蛋白相似的运输活性。为了充分定义分子 半胱氨酸氨基转移的机制，我建议解决这些半胱氨酸氨基突变体的结构并进行测试 通过电生理检测，构建了更完整的结合和运输机制的图景。 参考Sialin和ISSD，我的初步工作已经产生了一种单抗；然而，3D 用这种抗体重建Sialin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性的单抗，它是 可用于结构研究。然后，我将使用相同的通用方法来解决Sialin的结构问题 成功地使用了半胱氨酸氨基转移酶。由于Sialin也是H+：唾液酸转运体，不同的pH环境将 用来捕捉不同的构象。西拉林还结合和运输各种底物。因此，我会 还解决了Sialin的底物结合结构，从而提供了有关结合口袋的更多信息(S) 在底物上。重要的机械和底物结合残基将通过电生理学进行测试 (唾液酸)和放射性标记底物摄取分析(神经递质)。这些拟议的研究将 揭示LSD膀胱和ISSD背后的潜在分子机制，为进一步研究铺平道路 翻译研究和治疗设计。

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.