MOLECULAR BASIS OF GLYCOSPHINGOLIPID BINDING SPECIFICITY

描述(由申请人提供)：富含糖鞘糖脂(GSL)的‘筏’和小窝是膜微域，被认为是参与肿瘤发生的信号蛋白的侧向组织部位。由于小窝蛋白，即小窝蛋白-1的过表达与肿瘤细胞的存活、侵袭和转移潜能有关，靶向富含GSL的小窝可能被证明是一种有效的新疗法，用于转移、肿瘤发生和肿瘤进展的功能干扰。GSL富集区的形成和维持过程还没有很好的定义，但预计涉及到特定的蛋白质，可以在细胞之间和细胞内结合和转移GSL。我们的目标是利用Dinshaw Patel(斯隆-凯特琳研究所，NY)的结构专业知识和Rhoderick Brown(Hormel Institute，MN)实验室的分子生物学和糖鞘脂专业知识，阐明人类糖脂转移蛋白(GLTP)和相关同源物的结构，并利用结构、动力学和突变方法鉴定与糖脂连接选择性有关的折叠结构域。这项研究的基本原理是，解决GLTP及相关同源物在apo和糖脂连接状态下的结构将使绘制GLTP功能所涉及的蛋白质结构域和相关关键氨基酸残基成为可能。获得这一知识将为未来开发能够在显示异常GLTP活性的致癌细胞中特异性靶向GLTP的药理学药物提供基础。这项拟议的工作具有创新性，因为它利用了对人类GLTP的首次结构性洞察。GLTP的新的两层、全α螺旋拓扑结构与其他已知的脂结合/转移蛋白的折叠拓扑结构明显不同，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。我们期望，拟议的人类GLTP及其相关同源物的结构-动力学-突变研究将为这个新兴的新蛋白质家族的功能工作提供无与伦比的见解。这一新知识有望为以新的和创新的方式使用GLTP提供基础，例如将特定的GSL抗原引入癌细胞，以帮助通过免疫治疗手段实现对患病细胞的定向破坏。

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.