Rap1 - Krit1 Regulation of Endothelial Permeability

Rap1 - Krit1 内皮渗透性调节

• 批准号: 7474157
• 负责人: REBECCA A STOCKTON
• 金额: $ 11.34万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-08-01 至 2013-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7474157
• 关键词: Actin-Binding Protein; Actins; Acute; Adhesions; Animals; Arterial Fatty Streak; Atherosclerosis; Binding; Binding Sites; Biological Assay; Biology; Biosensor; Blood; Blood Vessels; CCM1 gene; Cardiovascular Pathology; Cardiovascular system; Cavernous Malformation; Cell Adhesion; Cell Line; Cell-Cell Adhesion; Cells; Cerebrum; Co-Immunoprecipitations; Cultured Cells; Cytoplasmic Tail; Cytoskeleton; Disease; Disruption; Ectopic Expression; Endothelial Cells; Endothelium; Extracellular Matrix; Faculty; Family; Focal Adhesions; Funding; Genetic; Goals; Guanosine Triphosphate Phosphohydrolases; Hemostatic function; Image; Immunofluorescence Microscopy; In Vitro; Inflammation; Inflammatory Response; Integrin Binding; Integrin beta Chains; Integrins; Intercellular Junctions; Kinetics; Label; Lesion; Leukocytes; Link; Liquid substance; Lung; Mediating; Mentors; Methodology; Molecular; Monomeric GTP-Binding Proteins; Movement; Mutant Strains Mice; Mutate; Mutation; Nuclear; Nuclear Translocation; PTB Domain; Pathology; Peptide Signal Sequences; Permeability; Pharmacological Treatment; Phosphotransferases; Physiology; Play; Positioning Attribute; Principal Investigator; Process; Protein Binding; Protein Binding Domain; Proteins; Recruitment Activity; Regulation; Reperfusion Injury; Research; Role; Scientist; Signal Transduction; Signaling Molecule; Site; Small Interfering RNA; Stress Fibers; Structure; Swelling; Talin; Tertiary Protein Structure; Testing; Thrombin; Time; Transgenic Mice; Vascular Permeabilities; Work; abstracting; angiogenesis; career; cerebral cavernous malformations; cytarabine/methotrexate protocol; ezrin; in vivo; interstitial; macromolecule; migration; moesin; novel; programs; protein function; radixin protein; receptor; response; rho; rho GTP-Binding Proteins; scaffold; skills; therapeutic target

DESCRIPTION (provided by applicant): PROJECT SUMMARY: Candidate's immediate career plans are advancement to Asst Project Scientist with several years' mentored project work to gain skills in transgenic mouse methodology/genetics. Long-term goal: acquire independent RO1 funding and a faculty position with independent research in cardiovascular biology. This proposal aims to identify signaling molecules promoting endothelial barrier integrity. Endothelial cell-cell junction disruption plus contractile stress fiber formation increase vessel permeability. Dysregulation of endothelial barrier function contributes to vessel leak in cardiovascular pathology. Rap1 GTPase stabilizes endothelial junctions. KRIT1 and CCM2 mutations produce "Cerebral Cavernous Malformations" disease characterized by vascular lesions with abnormal endothelium. Preliminary work identified KRIT1 as a Rap1 effectors having beta-integrin binding site, and demonstrated KRIT1 and CCM2 targeting to endothelial junctions that was associated with stabilized cortical actin and decreased permeability. HYPOTHESIS: KRIT1 is a Rap1 effector recruiting ICAP1 modulating beta-integrins and/or CCM2, regulating f-actin mediated barrier function through Rho-GTPases. SPECIFIC AIMS are to: 1) test whether KRIT1 localization and function are mediated by ICAP1 and CCM2 binding. 2) Evaluate KRIT1 PERM domain interactions with beta-integrins in regulating junctional stability. 3) Examine Rac- and Rho-GTPase interactions with KRIT1 in modulating actin cytoskeleton associated with barrier function. Molecular interactions in cultured cells will be tested with in vitro protein binding assays and co-immunoprecipitations, using siRNA depletion and ectopic expression of mutated binding domain proteins. Permeability will be tested in vivo with pulmonary vascular leak assays using beta-integrin, KRIT1, and CCM2 mutant mice; and in vitro using the animal's cultured endothelial cells in Tran swell filters. Molecular localization will be imaged using confocal immunofluorescence microscopy, plus real-time micro imaging to visualize kinetic localization of labeled ectopic proteins and fluorescent Rho-GTPase biosensors. RELEVANCE: This project will increase understanding of vascular permeability in general and CCM disease in particular. It will potentially identify target molecules for pharmacological treatment of pathological leak, and genetic treatment of CCM2 disease. (End of Abstract)

描述（由申请人提供）： 项目概要：候选人的近期职业计划是晋升为助理项目科学家，并通过几年的指导项目工作获得转基因小鼠方法学/遗传学方面的技能。长期目标：获得独立的RO 1资金和心血管生物学独立研究的教师职位。该提议旨在鉴定促进内皮屏障完整性的信号分子。内皮细胞-细胞连接破坏加上收缩应力纤维形成增加血管通透性。内皮屏障功能失调导致心血管病理学中的血管渗漏。Rap 1 GTdR稳定内皮连接。KRIT 1和CCM 2突变产生“脑海绵状血管畸形”疾病，其特征是血管病变伴异常内皮。初步工作确定KRIT 1作为Rap 1效应器具有β-整联蛋白结合位点，并证明KRIT 1和CCM 2靶向内皮连接，这与稳定的皮质肌动蛋白和降低的渗透性有关。假设：KRIT 1是一种Rap 1效应子，募集ICAP 1调节β-整联蛋白和/或CCM 2，通过Rho-GTP酶调节f-肌动蛋白介导的屏障功能。具体目的是：1）测试KRIT 1定位和功能是否由ICAP 1和CCM 2结合介导。2)评估KRIT 1 PERM结构域与β-整联蛋白在调节连接稳定性中的相互作用。3)检查Rac和Rho-GTdR与KRIT 1在调节与屏障功能相关的肌动蛋白细胞骨架中的相互作用。培养细胞中的分子相互作用将通过体外蛋白质结合试验和免疫共沉淀试验，使用突变结合结构域蛋白的siRNA耗竭和异位表达进行检测。将使用β-整联蛋白、KRIT 1和CCM 2突变小鼠通过肺血管渗漏试验在体内检测渗透性;并使用Tran swell滤器中的动物培养内皮细胞在体外检测渗透性。分子定位将使用共聚焦免疫荧光显微镜成像，加上实时显微成像，以可视化标记的异位蛋白和荧光Rho-GTdR生物传感器的动力学定位。相关性：该项目将增加对血管通透性的理解，特别是CCM疾病。它将潜在地识别用于病理性渗漏的药物治疗和CCM 2疾病的基因治疗的靶分子。(End摘要）