Structural Analysis of the Mannose 6-Phosphate Receptors

甘露糖 6-磷酸受体的结构分析

基本信息

批准号：
10327288
负责人：
Nancy M. Dahms
金额：
$ 60.95万
依托单位：
MEDICAL COLLEGE OF WISCONSIN
依托单位国家：
美国
项目类别：
财政年份：
1992
资助国家：
美国
起止时间：
1992-03-01 至 2023-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10327288
关键词：
Address Alpha-galactosidase Animal Model Binding Binding Sites Biochemical Biodistribution Biogenesis Biological Assay Catabolism Cell Death Cell Survival Cell physiology Cells Cellular Assay Child Complex Crystallization Data Data Reporting Differentiation and Growth Disease Dissociation Electron Microscopy Enzymes Excision FDA approved Fabry Disease Family Characteristics Functional disorder Genetic Diseases Glycoside Hydrolases Glycosphingolipids Goals Homeostasis Housing Human Hydrolase IGF Type 2 Receptor IGF2R gene Incidence Inherited Insulin-Like Growth Factor II Intravenous infusion procedures Kinetics Knowledge Ligand Binding Ligands Link Longevity Lysosomal Storage Diseases Lysosomes Maintenance Mass Spectrum Analysis Measures Mediating Mendelian disorder Metabolism Modeling Molecular Molecular Conformation Mus Mutagenesis Mutation N-terminal NMR Spectroscopy Nature Neonatal Screening Nutrient Organelles Organism Pathogenicity Patients Peptide Hydrolases Phagocytes Physiological Processes Plasma Plasminogen Play Polysaccharides Population Heterogeneity Positioning Attribute Process Production Property Proteins Rattus Receptor Gene Recombinants Recycling Resolution Roentgen Rays Role Specificity Structure System Techniques Testing Therapeutic Tissues Tumor Suppressor Proteins Urokinase Plasminogen Activator Receptor Variant X-Ray Crystallography antigen processing autosomal recessive trait base biophysical analysis biophysical techniques body system cell growth cell growth regulation cell motility design disease-causing mutation enzyme replacement therapy extracellular human disease improved inhibitor insight lysosomal proteins mannose 6 phosphate milligram novel particle protein aggregation receptor receptor binding receptor function treatment strategy

项目摘要

Lysosomes perform degradative metabolism critical to many endocytic, phagocytic, and autophagic processes. Cation-independent mannose 6-phosphate receptor (CI-MPR) plays a vital role in the biogenesis of lysosomes by delivering ~60 different newly synthesized hydrolytic enzymes with mannose 6-phosphate (M6P) on their N-glycans to lysosomes. Lysosomal storage diseases (LSDs) are caused by mutations in lysosomal proteins, mainly enzymes, that result in defective catabolism and substrate accumulation. Characteristic of the family of ~70 LSDs is their progressive and debilitating nature due to their impact on multiple organ systems. Treatment is symptomatic for most LSDs, with only 11 having FDA-approved therapies. CI-MPR's ability to internalize recombinant M6P- containing enzymes delivered to patients by bi-weekly intravenous infusion forms the basis of enzyme replacement therapy (ERT) for 9 of these therapies. However, structural knowledge of the interaction between CI-MPR and its cargo of ~60 different lysosomal enzymes is lacking. CI-MPR also binds a diverse set of extracellular non-M6P-containing ligands that mediate CI-MPR's tumor suppressor role and regulation of cell growth and differentiation. CI-MPR regulates plasma insulin-like growth factor 2 (IGF2) levels, and binds three components of the plasminogen activation system, plasminogen, urokinase-type plasminogen activator receptor (uPAR), and tissue-type PA (tPA). However, limited information is available concerning the molecular basis for CI-MPR's interaction with plasminogen and uPAR. Here we will expand upon our preliminary data that: 1) revealed the crystal structure of CI- MPR's N-terminal region that houses several ligand binding sites, 2) showed the first structural view of CI-MPR's entire extracellular region by high-resolution electron microscopy (EM), and 3) identified conformational changes elicited by pH and ligand binding. The overall structure of CI-MPR's 2300- residue extracellular region comprised of 15 domains will be determined alone and bound to ligands using an integrated approach combining single particle EM, mass spectrometry, X-ray crystallography and NMR spectroscopy. The first structure of a complex between CI-MPR and a lysosomal enzyme (Aim 1), plasminogen (Aim 2), and uPAR (Aim 2) will be elucidated. Kinetic analyses and cell-based assays will evaluate allosteric effects of each of CI-MPR's ligands (Aim 2). The mechanism of acidic pH-dependent ligand dissociation, which is essential to the functioning of CI-MPR and other endocytic receptors, will be probed using mutagenesis studies and NMR techniques (Aim 3). Our new rat model of the LSD Fabry disease will be used to test novel lysosomal enzyme-IGF2 fusion constructs for their ability to reduce substrate accumulation (Aim 4). These studies will provide insight for the design of improved therapeutics for the treatment of LSDs, and novel inhibitors of plasminogen activation.

溶酶体执行对许多内吞、吞噬和自噬至关重要的降解代谢流程。阳离子非依赖性甘露糖 6-磷酸受体 (CI-MPR) 在通过传递约 60 种不同的新合成的水解酶来进行溶酶体的生物合成甘露糖 6-磷酸 (M6P) 将其 N-聚糖转移至溶酶体。溶酶体贮积病（LSD）由溶酶体蛋白（主要是酶）的突变引起，导致分解代谢缺陷和底物积累。约 70 个 LSD 家族的特点是其进步和由于它们对多个器官系统的影响而使人衰弱。大多数情况下治疗都是针对症状的 LSD，其中只有 11 种药物具有 FDA 批准的疗法。 CI-MPR 内化重组 M6P- 的能力通过每两周静脉输注向患者输送含有酶的酶，形成酶的基础其中 9 种疗法的替代疗法 (ERT)。然而，相互作用的结构知识 CI-MPR 及其约 60 种不同溶酶体酶的货物之间缺乏联系。 CI-MPR 还结合介导 CI-MPR 肿瘤抑制作用的多种细胞外不含 M6P 的配体以及细胞生长和分化的调节。 CI-MPR 调节血浆胰岛素样生长因子 2 (IGF2) 水平，并结合纤溶酶原激活系统的三种成分：纤溶酶原，尿激酶型纤溶酶原激活剂受体 (uPAR) 和组织型 PA (tPA)。然而，有限有关 CI-MPR 与纤溶酶原相互作用的分子基础的信息是可用的 uPAR。在这里，我们将扩展我们的初步数据：1）揭示了 CI- 的晶体结构 MPR 的 N 末端区域包含多个配体结合位点，2) 显示了第一个结构视图 CI-MPR的整个细胞外区域通过高分辨率电子显微镜（EM）和3）鉴定 pH 值和配体结合引起的构象变化。 CI-MPR的2300-整体结构由 15 个结构域组成的残基胞外区将单独确定并与配体结合采用结合单粒子 EM、质谱、X 射线晶体学的综合方法和核磁共振波谱。 CI-MPR 和溶酶体酶复合物的第一个结构（目标 1）、纤溶酶原（目标 2）和 uPAR（目标 2）将被阐明。动力学分析和基于细胞的测定将评估每个 CI-MPR 配体的变构效应（目标 2）。酸性的作用机制 pH 依赖性配体解离，这对于 CI-MPR 和其他内吞作用的功能至关重要受体，将使用诱变研究和核磁共振技术进行探测（目标 3）。我们的新老鼠模型 LSD 法布里病的研究将用于测试新型溶酶体酶-IGF2 融合构建体的效果减少底物积累的能力（目标 4）。这些研究将为设计提供见解改进的 LSD 治疗方法以及新型纤溶酶原激活抑制剂。