Membrane Binding and Imaging Applications of Annexins

膜联蛋白的膜结合和成像应用

• 批准号: 6773458
• 负责人: Jonathan F Tait
• 金额: $ 26.87万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-08-01 至 2008-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6773458
• 关键词: annexins; apoptosis; binding proteins; binding sites; bioengineering /biomedical engineering; bioimaging /biomedical imaging; biological signal transduction; gene mutation; intermolecular interaction; kidney metabolism; laboratory mouse; membrane; phospholipids; protein engineering; protein structure function; radionuclide imaging /scanning; site directed mutagenesis; technology /technique development

DESCRIPTION (provided by applicant): Annexins are a family of calcium-dependent phospholipid binding proteins with diverse physiological functions that generally involve membrane binding either within or outside the cell. Annexin V is also being developed for clinical use as an imaging agent for detection of organ transplant rejection, response to cancer chemotherapy, and myocardial infarction. Membrane binding is critical for the proposed functions and clinical uses of annexins, yet the structural requirements for membrane binding are still poorly understood. Furthermore, wild-type annexin V has certain properties that limit its utility as a nuclear-medicine imaging agent. This proposal will clarify the mechanism of annexin-membrane binding, and will evaluate the utility of site-directed annexin V mutants as potential imaging agents. The long-term goals are to understand the structure-function relationships of annexins, and to develop clinical applications of annexins as diagnostic and therapeutic agents. By better understanding the structural correlates of membrane binding, we will be able to design improved agents for diagnosis and treatment in thrombosis, organ transplantation, and cancer chemotherapy. The specific aims of this proposal are: 1) Develop a family of mutant annexin V molecules with a wide range of membrane binding affinities. Using newly developed methods, measure the binding of the mutant annexin V molecules to phospholipid vesicles and cell membranes in vitro under conditions of low membrane occupancy. 2) Develop a family of mutant annexin V molecules with different net charges. Measure in vivo biodistribution of Tc-labeled _mutant proteins in normal mice. Correlate in vitro properties (binding affinity and net charge) with in vivo organ uptake. 3) Develop a set of mutant annexin V proteins with a wide range of dissociation rates. Measure biodistribution and target uptake of selected mutants in mice with hepatic apoptosis. Determine which mutant proteins have lower renal uptake without loss of uptake in apoptotic target tissues. Serially measure target uptake and retention of wild-type annexin V and selected mutants in mice with apoptosis of liver. Determine which mutant proteins have faster removal from the target site, thus allowing for repeated measurement of PS expression over short periods of time (up to one day).

描述（由申请人提供）： 膜联蛋白是钙依赖性磷脂结合蛋白的家族，具有多种生理功能，通常涉及细胞内或外部的膜结合。还在开发膜联蛋白V作为临床用作作为成像剂，以检测器官移植排斥，对癌症化学疗法的反应和心肌梗塞。膜结合对于融合蛋白的拟议功能和临床用途至关重要，但是膜结合的结构要求仍然很少了解。此外，野生型膜联蛋白V具有某些特性，可以限制其作为核药物成像剂的效用。该提案将阐明膜联蛋白 - 膜结合的机制，并将评估位于定向的膜联蛋白V突变体作为潜在成像剂的效用。长期目标是了解膜联蛋白的结构功能关系，并开发膜毒素作为诊断和治疗剂的临床应用。通过更好地了解膜结合的结构相关性，我们将能够设计改进的药物，用于血栓形成，器官移植和癌症化疗中的诊断和治疗。该提案的具体目的是：1）开发具有各种膜结合亲和力的突变膜融合蛋白V分子家族。使用新开发的方法，在低膜占用率的条件下，在体外测量突变膜融合蛋白V分子与磷脂囊泡和细胞膜的结合。 2）开发一个具有不同净电荷的突变体膜联蛋白V分子家族。测量正常小鼠中TC标记_Mutant蛋白的体内生物分布。将体外特性（结合亲和力和净电荷）与体内器官摄取相关。 3）开发一组具有广泛解离速率的突变膜融合蛋白V蛋白。测量肝凋亡小鼠中所选突变体的靶向摄取。确定哪些突变蛋白具有较低的肾脏摄取，而凋亡靶组织中的摄取却损失了。在肝脏细胞凋亡的小鼠中，野生型膜联蛋白V和选定突变体的序列测量靶向摄取和保留。确定哪些突变蛋白从目标位点更快地去除，从而可以在短时间内重复测量PS表达（最多一天）。