Investigating cellular function and biochemical mechanism for STK24-CCM3 complex

研究 STK24-CCM3 复合物的细胞功能和生化机制

基本信息

批准号：
9020243
负责人：
Titus Jonathon Boggon
金额：
$ 31.64万
依托单位：
YALE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2014
资助国家：
美国
起止时间：
2014-05-01 至 2018-02-28
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9020243
关键词：
Acute Address Affect Arthritis Binding Biochemical Biological Biological Models Blood Vessels Cell physiology Cells Cellular biology Cerebral hemisphere hemorrhage Chronic Complex Cytoplasmic Granules Data Disease Docking Dysplasia Endothelial Cells Exocytosis Family G Protein-Coupled Receptor Signaling G-Protein-Coupled Receptors Gene Expression Genes Goals Grant Hand Health Host Defense Hypersensitivity Immune response Infectious Agent Inflammation Inflammatory Ischemia Knockout Mice Lead Ligands Lipid Binding Lipids Mass Spectrum Analysis Matrix Metalloproteinases Mediating Membrane Microscopic Modeling Molecular Mus Natural Immunity Neuraxis Outcome Pattern Peroxidases Phosphotransferases Process Protein-Serine-Threonine Kinases Proteins Proteomics Regulation Reperfusion Therapy Research Rest Role Seizures Sepsis Small Interfering RNA Solid Sterility Stimulus Stroke Structure Surface System Testing Therapeutic Tissues Transplantation Vascular Diseases base cell motility cerebral cavernous malformations directional cell experience extracellular follow-up genetic regulatory protein germinal center kinases human disease insight migration neurovascular neutrophil new therapeutic target protein complex response success

项目摘要

DESCRIPTION (provided by applicant): One of our research emphases is to understand GPCR (G protein-coupled receptor) signaling mechanisms and functions. In a siRNA screen, we unexpectedly discovered that Serine/Threonine Kinase (STK) 24 and its binding partner Cerebral Cavernous Malformation (CCM) 3 in regulation of neutrophil degranulation. STK24, together with MST4 and STK25, belongs to the germinal center kinase (GCK) III sub-family of Sterile-20 kinases. These kinases are known to interact with CCM3. CCM3 has been directly, whereas the GCK III kinases indirectly, implicated in the CCM disease. CCM is a vascular pathological condition that affects the vasculature of the central nervous system and results in stroke, seizure and cerebral hemorrhage. Despite the potential importance of the STK-CCM3 complex, their cellular functions and underlying biochemical mechanisms remain obscure. Following our discovery of their role in degranulation, we identified CCM3 and STK24 interaction with an exocytosis regulatory protein, UNC13D, through the mass spectrometry (MS) analysis. Furthermore, we have gathered solid preliminary results to help us understand how STK24 and CCM3 mechanistically regulate the degranulation and how extracellular stimuli, including GPCR ligands, regulate the release of STK24/CCM3-controlled granule pools via Ca2+. Thus, our findings provide the first insights into a cellular function of the STK24-CCM3 complex with well- defined biochemical basis. Moreover, our present study has unraveled previously unknown roles of STK24 and CCM3 in the regulation of neutrophil degranulation, a process important for acute innate immune responses and tissue damage that occurs in chronic and systemic inflammation and ischemia-reperfusion, but understudied. Furthermore, our new mechanism for regulation of degranulation would facilitate the general understanding of ligand-stimulated exocytosis, which degranulation is a form of and occurs in many cells including endothelial cells. Thus our study may pave the way for the eventual understanding of the pathogenic basis for the CCM disease and exert potentially high impact on a number of fields (basic cell biology, innate immunity, and CCM disease). In the present study, we will focus on neutrophil degranulation by testing the hypothesis that the STK24 and CCM3 complex is a key negative regulator of neutrophil degranulation. We will investigate the biochemical and molecular mechanisms by which CCM3 and STK24 regulate degranulation.

描述（由申请人提供）：我们的研究重点之一是了解GPCR（G蛋白偶联受体）信号传导机制和功能。在 siRNA 筛选中，我们意外地发现丝氨酸/苏氨酸激酶 (STK) 24 及其结合伙伴脑海绵状血管瘤 (CCM) 3 参与中性粒细胞脱颗粒的调节。 STK24 与 MST4 和 STK25 一起属于 Sterile-20 激酶的生发中心激酶 (GCK) III 亚家族。已知这些激酶与 CCM3 相互作用。 CCM3 直接与 CCM 疾病有关，而 GCK III 激酶则间接与 CCM 疾病有关。 CCM 是一种血管病理性疾病，影响中枢神经系统的脉管系统，导致中风、癫痫发作和脑出血。尽管 STK-CCM3 复合物具有潜在的重要性，但它们的细胞功能和潜在的生化机制仍然不清楚。在发现 CCM3 和 STK24 在脱颗粒中的作用后，我们通过质谱 (MS) 分析确定了 CCM3 和 STK24 与胞吐调节蛋白 UNC13D 的相互作用。此外，我们还收集了可靠的初步结果，以帮助我们了解 STK24 和 CCM3 如何机械调节脱颗粒，以及细胞外刺激（包括 GPCR 配体）如何通过 Ca2+ 调节 STK24/CCM3 控制的颗粒池的释放。因此，我们的研究结果首次深入了解了 STK24-CCM3 复合物的细胞功能，并具有明确的生化基础。此外，我们目前的研究揭示了 STK24 和 CCM3 在中性粒细胞脱粒调节中以前未知的作用，这是一个对于慢性和全身炎症和缺血再灌注中发生的急性先天免疫反应和组织损伤很重要的过程，但尚未得到充分研究。此外，我们的脱颗粒调节新机制将有助于对配体刺激的胞吐作用的一般理解，脱颗粒是包括内皮细胞在内的许多细胞的一种形式并发生在许多细胞中。因此，我们的研究可能为最终了解 CCM 疾病的致病基础铺平道路，并对许多领域（基础细胞生物学、先天免疫和 CCM 疾病）产生潜在的重大影响。在本研究中，我们将通过测试 STK24 和 CCM3 复合物是中性粒细胞脱颗粒的关键负调节因子这一假设来关注中性粒细胞脱颗粒。我们将研究 CCM3 和 STK24 调节脱颗粒的生化和分子机制。