Mechanisms Driving Apoptosis Resistance in Pulmonary Hypertension

肺动脉高压中细胞凋亡抵抗的驱动机制

• 批准号: 10843723
• 负责人: Shannon Niedermeyer
• 金额: $ 8.08万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-08-01 至 2024-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10843723
• 关键词: AQP1 gene; Address; Animal Model; Apoptosis; Apoptotic; Automobile Driving; Binding Proteins; Biological Assay; Biotin; Biotinylation; Blood Vessels; CASP3 gene; Caspase; Cell Death; Cell Membrane Proteins; Cell Nucleus; Cell Separation; Cell physiology; Cells; Cessation of life; Co-Immunoprecipitations; Confocal Microscopy; Cytosol; Data; Development; Diagnosis; Disease; Disease Progression; Disease model; Educational process of instructing; Foundations; Fractionation; Funding; Future; Goals; Hypertrophy; Immunofluorescence Microscopy; Immunoprecipitation; In Situ Nick-End Labeling; Individual; Ischemia; K-Series Research Career Programs; Laboratories; Lesion; Ligase; Link; Lung; Maintenance; Malignant - descriptor; Malignant Neoplasms; Measures; Medial; Membrane; Membrane Proteins; Modeling; Molecular; Normal Cell; Nuclear; Outcome Measure; Oxidants; Pathway interactions; Patient Care; Peptide Hydrolases; Play; Pre-Clinical Model; Proliferating; Proteins; Proteomics; Pulmonary Hypertension; Pulmonary artery structure; Rattus; Regulation; Resistance; Resistant Hypertension; Role; Scientist; Site; Site-Directed Mutagenesis; Smooth Muscle Myocytes; Stains; Stimulus; System; Tail; Techniques; Testing; Toxin; Training; Water; Work; cancer type; career; design; experimental study; extracellular; gain of function; in silico; insight; interest; knock-down; loss of function; migration; mortality; new therapeutic target; novel; overexpression; prevent; protein expression; protein protein interaction; pulmonary arterial hypertension; pulmonary vascular cell proliferation; response; right ventricular failure; skills; water channel

PROJECT SUMMARY Pulmonary arterial hypertension (PAH) remains a fatal diagnosis despite available therapies. PAH is characterized by extensive remodeling of the pulmonary vasculature involving the formation of vaso-occlusive lesions and a thickened medial layer of the vascular wall, both of which contain pulmonary arterial smooth muscle cells (PASMCs). It has been demonstrated that PASMCs isolated from a well-established rat model of PAH are resistant to apoptosis under both basal and stimulated conditions. The cell membrane protein aquaporin 1 (AQP1) was initially described as a water transport channel, but more recently has been implicated in other cellular functions including migration and proliferation, and in several distinct cancer types, has been associated with apoptosis resistance. AQP1 is upregulated in PASMCs isolated from rat models of PAH suggesting a ‘quasi- malignant’ disease model, although AQP1’s exact role in apoptosis resistance is unclear. Exciting new data from an unpublished proteomics study using lung lysates demonstrates that AQP1 immunoprecipitates with total caspase-3, a enzymatic protein activated in apoptosis which is translocated to the nucleus where it initiates cell death. In proximity studies utilizing biotin ligase techniques, I have demonstrated that AQP1 and total caspase- 3 come within close proximity in live cells. Furthermore, in silico analysis of the AQP1 protein reveals 3 potential caspase-3 cleavage sites, which provide a mechanism for this protein-protein interaction. Together, these data suggest that AQP1 interacts with caspase-3, providing a novel relationship between AQP1, the caspase cascade, and resistance to apoptosis. This application serves to provide a training vehicle as I explore a potential mechanism by which AQP1 regulates apoptosis during PAH. Aim 1 is designed to determine whether the cytosolic caspase-3 cleavage site(s) on AQP1 are necessary for AQP1/caspase-3 interaction, Aim 2 serves to evaluate the impact of AQP1 on nuclear localization of capsase-3, and finally Aim 3 will establish if increased AQP1 is necessary and/or sufficient to confer apoptosis resistance. Techniques utilized to address these aims include but are not limited to protein expression and site directed mutagenesis, biotin ligase proximity assays, co-immunoprecipitation, animal models of PAH and primary cell isolation, immunofluorescence and confocal microscopy, nuclear/cytosolic fractionation, luminescent caspase-3/7 activity assay, Hoechst staining and TUNEL staining. Completion of this project will provide novel insight into the interaction between AQP1 and caspase-3 and the role for AQP1 in apoptosis resistance as well as provide a novel pathway for new therapeutic targets. The skills obtained in the design and execution of this study and the experimental results will provide the necessary foundation for a K award and an excellent platform on which to start a career as an independently funded clinician scientist focused on PAH. Insights gained from this work also could have implications beyond PAH to any diagnosis in which cellular resistance to apoptosis is essential to the development of disease.

项目摘要 肺动脉高压（PAH）仍然是致命的诊断目的地可用疗法。 pah是 其特征是涉及血管形成的肺脉管系统的大量重塑 病变和血管壁增厚的培养基层，两者都含有肺动脉平滑肌 细胞（PASMC）。已经证明，从公认的PAH大鼠模型中分离出的PASMC是 在碱性和刺激条件下对凋亡具有抗性。细胞膜蛋白水通道蛋白1 （AQP1）最初被描述为水传输通道，但最近与其他有关 包括迁移和增殖在内的细胞功能，以及在几种不同的癌症类型中，已经与 抗凋亡性。从PAH的大鼠模型中分离出的PASMC中，AQP1更新，这表明Quasi- 恶性疾病模型，尽管AQP1在抗凋亡耐药性中的确切作用尚不清楚。令人兴奋的新数据来自 一项未发表的蛋白质组学研究使用肺裂解液表明，AQP1免疫沉淀总计 caspase-3，一种在细胞凋亡中激活的酶促蛋白 死亡。在使用生物素连接酶技术的接近性研究中，我已经证明了AQP1和总caspase- 3在活细胞中近距离接近。此外，在对AQP1蛋白的计算机分析中显示了3个潜力 caspase-3裂解位点，为这种蛋白质蛋白质相互作用提供了一种机制。在一起，这些数据 建议AQP1与caspase-3相互作用，从而提供了AQP1之间的新型关系 级联反应和对凋亡的抗性。当我探索潜力时，该应用程序提供了训练工具 AQP1在PAH期间调节凋亡的机制。 AIM 1旨在确定是否 AQP1上的胞质caspase-3裂解位点对于AQP1/caspase-3相互作用是必需的，AIM 2用于 评估AQP1对Capsase-3的核定位的影响，最后AIM 3将确定是否增加 AQP1是必需的，并且足以赋予凋亡抗性。用于解决这些目标的技术 包括但不限于蛋白质表达和位置定向诱变，生物素连接酶接近测定， 共免疫沉淀，PAH的动物模型和原代细胞分离，免疫荧光和共焦 显微镜，核/胞质分馏，发光caspase-3/7活性测定，Hoechst染色和 TUNEL染色。该项目的完成将提供有关AQP1和 caspase-3和AQP1在凋亡耐药性中的作用，并为新疗法提供了新的途径 目标。在这项研究的设计和执行中获得的技能以及实验结果将提供 K奖的必要基础和一个出色的平台，以独立的职业生涯开始职业 资助的临床科学家专注于PAH。从这项工作中获得的见解也可能具有超越 PAH对任何诊断性的细胞抗凋亡对疾病的发展至关重要。