MOLECULAR BASIS OF GLYCOSPHINGOLIPID BINDING SPECIFICITY

描述（由申请人提供）：鞘糖脂（GSL）富集的“筏”和小泡是膜微结构域，被认为是参与肿瘤发生的信号蛋白的横向组织位点。由于选择的小泡蛋白，即小泡蛋白-1的过表达与肿瘤细胞的存活、侵袭和转移潜力有关，因此靶向富含gsl的小泡可能被证明是一种新的治疗方法，可用于转移、肿瘤发生和肿瘤进展的功能破坏。富含gsl的结构域形成和维持的过程还没有很好的定义，但预计涉及到可以在细胞之间和细胞内结合和转移gsl的特定蛋白质。我们的目标是利用Dinshaw Patel （Sloan-Kettering研究所，NY）的结构专业知识和Rhoderick Brown （Hormel研究所，MN）实验室的分子生物学和糖鞘脂专业知识，利用结构、动力学和突变方法，阐明人类糖脂转移蛋白（GLTP）和相关同源物的结构，并鉴定/表征负责糖脂配体选择性的折叠结构域。这项研究的基本原理是，解决GLTP及其相关同源物在载脂蛋白和糖脂配体状态下的结构，将能够绘制与GLTP功能相关的蛋白质结构域和相关关键氨基酸残基。获得这一知识将为追求未来的药物开发提供基础，这些药物可以专门针对显示异常GLTP活性的致癌细胞中的GLTP。这项提议的工作是创新的，因为它利用了有史以来第一次对人类GLTP的结构见解。这种新型的双层全α螺旋拓扑结构与其他已知的脂质结合/转移蛋白的折叠拓扑结构明显不同，这表明GLTP折叠基序定义了一个新的蛋白质家族。这些研究将利用我们最近在GLTP和相关点突变体的分子克隆和表达方面取得的成功。我们期望对人类gltp及其相关同源物的结构-动力学-突变研究将为这一新兴蛋白质家族的功能运作提供无与伦比的见解。这一新发现有望为GLTP的创新应用奠定基础，例如将特异性GSL抗原引入癌细胞，从而通过免疫治疗手段实现病变细胞的靶向破坏。

DESCRIPTION (provided by applicant): Glycosphingolipids (GSL)-enriched 'rafts' and caveolae are membrane microdomains that putatively function as lateral organizing sites for signaling proteins involved in oncogenesis. Because the overexpression of select caveolar proteins, i.e. caveolin-1, is associated with tumor cell survival, aggression and metastatic potential, targeting GSL-enriched caveolae may prove useful as a new therapy for the functional disruption of metastatis, tumorigenesis, and tumor progression. The processes by which GSL-enriched domains are formed and maintained are not well defined but are expected to involve specific proteins that can bind and transfer GSLs between and within cells. Our objective is to elucidate the structure of human glycolipid transfer protein (GLTP) and related orthologs and to identify/-characterize folding domains responsible for glycolipid liganding selectivity using structural, dynamical, and mutational approaches by taking advantage of the structural expertise of the Dinshaw Patel (Sloan-Kettering Institute, NY) and the molecular biological and glycosphingolipid expertise of the Rhoderick Brown (Hormel Institute, MN) laboratories. The rationale for the research is that, solving the structure of GLTP and related orthologs in their apo and glycolipid-liganded states will enable mapping of the protein domains and associated key amino acid residues involved in GLTP functionality. Acquiring this knowledge will provide a foundation for pursuing the future development of pharmacologic agents that can specifically target GLTP in oncogenic cells displaying aberrant GLTP activity. The proposed work is innovative because it capitalizes on the first-ever, structural insights into human GLTP. The novel two-layer, all alpha helical topology of GLTP differs distinctly from the folding topologies of other known lipid binding/transfer proteins, suggesting that the GLTP folding motif defines a novel family of proteins. The studies will take advantage of our recent successes in the molecular cloning and expression of GLTP and related point mutants. It is our expectation that the proposed structural-dynamics-mutational studies of human GLTPs and related orthologs will provide unparalleled insights into the functional workings of this emerging new protein family. This new knowledge is expected to be significant by providing a foundation for using GLTP in new and innovative ways, such as introducing specific GSL antigens into cancer cells to help achieve targeted destruction of diseased cells via immunotherapeutic means.