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）： 项目摘要：候选人的直接职业计划是助理项目科学家的进步，并获得了多年的项目工作，以获得转基因小鼠方法/遗传学的技能。长期目标：通过心血管生物学独立研究获得独立的RO1资金和教职员工。该建议旨在确定促进内皮屏障完整性的信号分子。内皮细胞 - 细胞连接破坏加收缩应力纤维的形成增加容器的渗透性。内皮屏障功能的失调有助于心血管病理中的血管泄漏。 RAP1 GTPase稳定内皮连接。 KRIT1和CCM2突变会产生“脑海绵状畸形”疾病，其特征是内皮异常的血管病变。初步工作将KRIT1鉴定为具有β-整合蛋白结合位点的RAP1效应子，并证明了与稳定的皮质肌动蛋白相关的内皮连接剂的KRIT1和CCM2靶向，并降低了渗透性。假设：KRIT1是一种RAP1效应子募集ICAP1，调节β-整合蛋白和/或CCM2，通过Rho-GTPases调节F-肌动蛋白介导的屏障功能。具体目的是：1）测试KRIT1定位和函数是否由ICAP1和CCM2结合介导。 2）在调节连接稳定性方面，评估KRIT1 PERS域与β-积聚蛋白的相互作用。 3）在调节与屏障功能相关的肌动蛋白细胞骨架时，检查与KRIT1的RAC和RHO-GTPase相互作用。培养细胞中的分子相互作用将通过体外蛋白结合测定和共免疫沉淀进行测试，并使用突变结合结构域蛋白的siRNA耗竭和异位表达。使用β-整合蛋白，KRIT1和CCM2突变小鼠，将在体内测试渗透率。并在tran膨胀过滤器中使用动物培养的内皮细胞进行体外体外。分子定位将使用共聚焦免疫荧光显微镜进行成像，并将实时微型成像以及可视化标记的异位蛋白和荧光Rho-GTPase生物传感器的动力学定位。相关性：该项目将增加对一般的血管通透性的理解，尤其是CCM疾病。它可能会鉴定出靶标分子，以用于病理泄漏的药理学治疗，以及对CCM2疾病的遗传治疗。 （抽象的结尾）