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Membrane Binding Properties of the GM2 Activator Protein

GM2 激活蛋白的膜结合特性

基本信息

批准号：
7821481
负责人：
GAIL E FANUCCI
金额：
$ 23.72万
依托单位：
UNIVERSITY OF FLORIDA
依托单位国家：
美国
项目类别：
财政年份：
2006
资助国家：
美国
起止时间：
2006-05-05 至 2012-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7821481
关键词：
AB Variant Tay-Sachs Disease Address Affect Arts Baculovirus Expression System Behavior Binding CD1 Antigens Calorimetry Catabolism Cell Death Cell membrane Cells Clinical Complex Data Defect Development Digestion Disease Electron Spin Resonance Spectroscopy Electrostatics Endocytosis Event Fluorescence Fluorescence Spectroscopy G(M2) Activator Protein G(M2) Ganglioside Gangliosides Gangliosidoses Gangliosidoses GM2 Glycosphingolipids Head Hex A In Vitro Inborn Genetic Diseases Insecta Label Lead Ligands Lipid Bilayers Lipid Binding Lipids Location Lysosomal Storage Diseases Lysosomes Maps Measurement Membrane Membrane Fluidity Metabolism Methodology Methods Micelles Modeling Molecular Molecular Conformation Mutation Neurons Oligosaccharides Pathway interactions Pharmaceutical Preparations Phase Phospholipase D Platelet Activating Factor Platelet Factor 4 Play Property Protein Binding Proteins Reaction Reporting Research Research Personnel Resolution Role Sandhoff Disease Scheme Shapes Signal Transduction Site Solutions Spin Labels Structure Surface Syndrome System Tay-Sachs Disease Techniques Temperature Testing Titrations Vesicle Work X-Ray Crystallography Yeasts base beta-n-acetylhexosaminidase bis(monoacylglyceryl)phosphate gene therapy glycosylation in vivo late endosome lipid metabolism physical property research study simulation

项目摘要

DESCRIPTION (provided by applicant): The GM2 Activator Protein (GM2AP) plays an essential role in the catabolism of GM2 in lysosomes. GM2 is a complex glycosphingolipid (GSL) that is a degradation intermediate in the breakdown of GM1 which is found in relatively high concentrations in the outer surface of plasma membranes in neuronal cells. GM2AP acts as a substrate specific co-factor by solubilizing GM2 from the intralysozomal vesicles and presenting the oligosaccharide moiety to beta-hexosaminidase A (HexA) for hydrolytic cleavage. Mutations in either HexA or GM2AP lead to the storage of GM2 within lysozomes and cell death. A well known example of this type of lysosomal storage disease is Tay-Sachs syndrome. The mechanism for how the GM2AP accessory protein regulates enzymatic degradation of GM2 as the first step in the GM2 lipid metabolism remains unclear. The proposed work will focus on elucidating the molecular details of this mechanism through site-directed spin labeling anomposition of late endosomes and intralysosomal vesicles are believed to alter the bilayer physical properties allowing GM2AP to extract GM2 from membranes. The current hypothesis is that in membranes of high curvature of neuronal lysozome composition, the binding is a transient event. Fluorescence based binding measurements will be used to test this hypothesis. Recently, crystal structures of the GM2AP bound to GM2, POPG and PAF were reported. The model of conformational changes predicted by X-ray crystallography data for specific and non-specific substrates will be tested by SDSL EPR experiments of GM2AP in solution where GM2 is extracted from micelles, as well as from lipid bilayers. Currently, the GM2AP has been expressed in E.coli, insect cells and in yeast. The effects of glycosylation on the membrane binding properties will also be examined. The general relevance of this research is that congenital mutations in GM2AP or HexA result in lysozomal storage diseases. The GM2-gangliosidoses (including Tay-Sachs disease) are a group of inherited disorders that result from defects in the catabolism of GM2. In fact, a variety of lysosomal storage diseases (Pompe, Fabry and the gangliosidoses) result from aberrations in lipid metabolism. A more detailed understanding of this critical first step in GM2 catabolism may lead to developments in drug or gene therapies for these fatal diseases.

描述（由申请人提供）：GM2激活蛋白（GM2AP）在溶酶体中GM2的分解代谢中发挥重要作用。 GM2 是一种复杂的鞘糖脂 (GSL)，是 GM1 分解过程中的降解中间体，GM1 在神经元细胞质膜外表面的浓度相对较高。 GM2AP 通过溶解溶菌体内囊泡中的 GM2 并将寡糖部分呈递给 β-己糖胺酶 A (HexA) 进行水解裂解，充当底物特异性辅因子。 HexA 或 GM2AP 的突变会导致 GM2 在溶酶体中储存并导致细胞死亡。这种溶酶体贮积症的一个众所周知的例子是泰-萨克斯综合征。 GM2AP 辅助蛋白如何调节 GM2 酶促降解作为 GM2 脂质代谢的第一步，其机制仍不清楚。拟议的工作将侧重于通过晚期内涵体和溶酶体内囊泡的定点自旋标记反位来阐明该机制的分子细节，据信可以改变双层物理特性，从而使 GM2AP 从膜中提取 GM2。目前的假设是，在神经元溶酶体成分的高曲率膜中，结合是一个短暂的事件。基于荧光的结合测量将用于检验这一假设。最近，报道了与 GM2、POPG 和 PAF 结合的 GM2AP 的晶体结构。由特定和非特定底物的 X 射线晶体学数据预测的构象变化模型将通过溶液中 GM2AP 的 SDSL EPR 实验进行测试，其中 GM2 从胶束以及脂质双层中提取。目前，GM2AP已在大肠杆菌、昆虫细胞和酵母中表达。还将检查糖基化对膜结合特性的影响。这项研究的总体意义在于，GM2AP 或 HexA 的先天性突变会导致溶菌体贮积病。 GM2 神经节苷脂沉积症（包括泰萨克斯病）是一组由 GM2 分解代谢缺陷引起的遗传性疾病。事实上，多种溶酶体贮积症（庞贝病、法布里病和神经节苷脂病）都是由脂质代谢异常引起的。对 GM2 分解代谢这一关键第一步的更详细了解可能会导致针对这些致命疾病的药物或基因疗法的发展。