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

与发育障碍有关的两种溶酶体转运蛋白的结构和功能研究

• 批准号: 10703221
• 负责人: Philip Schmiege
• 金额: $ 1.81万
• 依托单位: UT SOUTHWESTERN MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-08-10 至 2023-12-04
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10703221
• 关键词: 3-Dimensional; Adult; Affect; Antibodies; Aspartate; Automobile Driving; Binding; Binding Proteins; Binding Sites; Biochemical; Biological Assay; Cell physiology; Central Nervous System; Cessation of life; Complex; Cryoelectron Microscopy; Crystal Formation; Cysteine; Cystine; Cystinosis; Cytosol; Data; Developmental Delay Disorders; Disease; Electrophysiology (science); Elements; Environment; Finnish Type Sialic Acid Storage Disease; Future; Genetic Diseases; Glutamates; Human; Infant; Infantile Form Sialuria; Inherited; Investigation; Kidney; Knowledge; Length; Lysosomal Storage Diseases; Lysosomes; Mediating; Membrane; Membrane Potentials; Membrane Proteins; Membrane Transport Proteins; Methods; Molecular; Molecular Conformation; Monoclonal Antibodies; Mutagenesis; Mutation; Neurologic; Neurotransmitters; Organ; Pathway interactions; Play; Polysaccharides; Process; Proteins; Protons; Radiolabeled; Rare Diseases; Recycling; Research; Resolution; Role; Sampling; Sialic Acids; Structure; Symptoms; Testing; Tissues; Translational Research; Vision; Work; autosome; body system; design; developmental disease; dimer; disease-causing mutation; insight; lysosomal proteins; mutant; neurotransmitter transport; protein function; protein purification; protein transport; proteoliposomes; reconstruction; screening; sialic acid permease; symporter; therapeutic development; translational therapeutics; uptake

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.

项目概要 溶酶体贮积病 (LSD) 包括 70 多种常染色体隐性遗传病， 是由溶酶体蛋白质故障引起的，导致溶酶体中特定底物的积聚。 这些相对罕见的疾病往往出现在婴儿身上，尽管成人形式也存在。胱氨酸病和婴儿病 唾液酸贮积病 (ISSD) 是两种较常见的 LSD。胱氨酸中毒的症状包括肾 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。胱氨酸病是由突变引起的 胱氨酸蛋白，一种溶酶体 H+：胱氨酸同向转运体，而 ISSD 是由唾液酸蛋白突变引起的， 溶酶体 H+：唾液酸同向转运蛋白和神经递质单向转运蛋白。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。在我的 初步研究中，我生成了一种结构特异性的单克隆抗体，可以识别腔域 胱氨酸，Fab3H5。使用这种抗体，我解析了人全长胱氨酸的三种结构： pH 7.5 时，面向 apo 构象的分辨率为 3.4-Å；pH 7.5 时，面向内的 apo 构象的分辨率为 3.2-Å pH 5.0，胱氨酸结合构象，分辨率为 3.4-Å。这些结构揭示了所涉及的残基 在介导其底物胱氨酸运输的细胞质和管腔门中，以及残基 与胱氨酸结合口袋中的胱氨酸相互作用。该蛋白质的初步电生理数据 进一步鉴定了突变 D205N 和 D305N（消除了转运）以及突变 Q284A 和 D346N， 其保持与野生型蛋白质相似的转运活性。为了完整地定义分子 胱氨酸转运机制，我建议解决这些胱氨酸突变体的结构并进行测试 他们通过电生理学分析，建立了更完整的结合和运输机制的图景。 参考Sialin和ISSD，我的前期工作已经得到了单克隆抗体；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。所以我首先会优化 抗体筛选过程。这将使我能够分离出结构特异性的单克隆抗体 可用于结构研究。然后我将使用与以下相同的通用方法来解决 Siialin 的结构 胱氨酸已取得成功。由于Sialin也是H+：唾液酸同向转运体，不同的pH环境会影响 用于捕获不同的构象。唾液酸蛋白还结合和运输各种底物。因此，我将 还解析了Sialin的底物结合结构，从而提供了有关结合口袋的更多信息 的基板。然后将通过电生理学测试重要的机械和底物结合残基 （针对唾液酸）和放射性标记底物摄取测定（针对神经递质）。这些拟议的研究将 揭示 LSD Cystinosis 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。