MOLECULAR BASIS OF GLYCOSPHINGOLIPID BINDING SPECIFICITY

描述（由申请人提供）：富含鞘糖脂（GSL）的“筏”和小窝是膜微结构域，其作为参与肿瘤发生的信号蛋白的侧向组织位点发挥功能。由于选择小窝蛋白，即小窝蛋白-1的过表达与肿瘤细胞存活、侵袭和转移潜力相关，靶向富含GSL的小窝可能被证明可用作转移、肿瘤发生和肿瘤进展的功能性破坏的新疗法。GSL富集结构域形成和维持的过程尚未明确，但预计涉及可以在细胞之间和细胞内结合和转移GSL的特定蛋白质。我们的目标是阐明人糖脂转运蛋白（GLTP）和相关直向同源物的结构，并通过利用Dinshaw Patel的结构专业知识，使用结构，动力学和突变方法鉴定/表征负责糖脂配体选择性的折叠结构域。（Sloan-Kettering Institute，NY）和Rhoderick Brown（Hormel Institute，MN）实验室的分子生物学和鞘糖脂专业知识。该研究的基本原理是，解决GLTP和相关直系同源物在其载脂蛋白和糖脂配体状态下的结构，将使蛋白质结构域和参与GLTP功能的相关关键氨基酸残基的映射成为可能。获得这方面的知识将提供一个基础，追求未来的发展，药理学药物，可以特异性靶向GLTP在致癌细胞显示异常GLTP活性。拟议的工作是创新的，因为它利用了有史以来第一次对人类GLTP的结构性见解。新的两层，所有α螺旋拓扑结构的GLTP明显不同于其他已知的脂质结合/转移蛋白的折叠拓扑结构，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的成功。这是我们的期望，拟议的结构动力学突变的研究人类GLTP和相关的直系同源物将提供无与伦比的洞察这个新兴的新蛋白质家族的功能工作。这一新的知识预计将是重要的，通过提供一个基础，使用GLTP在新的和创新的方式，如引入特定的GSL抗原到癌细胞，以帮助实现有针对性的破坏病变细胞通过免疫手段。

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.