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Novel Roles for Phosphoinositide Signaling in alpha-Granule Biogenesis

磷酸肌醇信号传导在 α 颗粒生物发生中的新作用

基本信息

批准号：
10656287
负责人：
CHARLES S. ABRAMS
金额：
$ 50.45万
依托单位：
UNIVERSITY OF PENNSYLVANIA
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-05-10 至 2025-04-30
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10656287
关键词：
Address Affect Alpha Granule Binding Binding Proteins Biochemical Biogenesis Biology Blood Blood Cells Blood Platelets Blood coagulation Bone Marrow Cell membrane Cells Collaborations Cytoplasmic Granules Data Defect Development Environment GOLPH3 gene Golgi Apparatus Growth Factor Hematopoiesis Hematopoietic stem cells Human Impairment Indiana Individual Inflammation Knock-out Left Link Maintenance Mediating Megakaryocytes Megakaryocytopoieses Membrane Morphology Multivesicular Body Mus Mutate Mutation Myocardial Infarction Neurons Pathologic Patients Phenotype Phosphatidylinositol Transfer Protein Phosphatidylinositols Phospholipids Phosphorylation Physiological Platelet Activation Play Process Production Property Protein Family Protein Isoforms Proteins Publications Publishing Regulation Research Role Second Messenger Systems Signal Pathway Signal Transduction Stroke Syndrome Testing Thrombosis Transforming Growth Factor beta Universities cytokine human disease in vivo member monomer novel novel therapeutic intervention protein transport recruit trafficking trans-Golgi Network

项目摘要

Phosphorylated phosphatidylinositols (phosphoinositides) are a type of membrane bound phospholipid that impact multiple diverse processes required for megakaryopoiesis and the activation of platelets. We have recently published in Developmental Cell that phosphoinositides in neuronal cells initiate intracellular trafficking by recruiting effector proteins such as GOLPH3 that are involved in vesicular fusion and budding of plasma membranes during Golgi biogenesis. Since megakaryocyte α-granules are derived from the trans-Golgi network and Multi-Vesicular Bodies, I hypothesize that phosphoinositide signaling is necessary for the intracellular trafficking required for the biogenesis of α-granules. PhosphatidylInositol Transfer Proteins (PITPs) are members of a small protein family that bind and transfer phosphoinositide monomers from one cellular compartment to another and thereby enable phosphoinositide synthesis. We have made the unexpected observation that the two predominant PITP isoforms found within megakaryocytes, PITPα and PITPβ play previously unrecognized but essential roles in the trafficking of cargo from the Multi-Vesicular Body to α-granules. Loss of PITP-mediated phosphoinositide synthesis produces morphologic defects similar to what is seen in humans with Gray Platelet Syndrome. The overall hypothesis of this Proposal is that phosphoinositide signaling mediated by PITPs is necessary for the membrane dynamics and protein trafficking required for the biogenesis and maintenance of megakaryocyte α-granules. In Aim 1 of the Project, we will rigorously analyze the discrete biochemical properties of individual PITP isoforms in megakaryocytes. Our preliminary data shows that the two PITP isoforms control phosphoinositide signaling through biochemically distinct mechanisms. In Aim 2, we will determine how phosphoinositide signaling contributes to alpha granule biogenesis and function. In conjunction with Project 2, we will test the hypothesis that phosphoinositide synthesis within discrete microdomains of megakaryocytes and platelets regulates effector proteins such as NBEAL2 (the mutated protein responsible for the Gray Platelet Syndrome). This signaling cascade modulates NBEAL2’s ability to mediate membrane dynamics and protein trafficking. We will also analyze in detail the functional roles of α- granules with ex vivo rheologic and ultramicroscopy studies that will be performed with Project 2, in vivo thrombosis studies with Project 3, and in vivo inflammation studies with Project 4.

磷酸化磷脂酰肌醇（磷酸肌醇）是一种膜结合蛋白磷脂影响巨核细胞生成和巨核细胞生成所需的多种不同过程血小板的活化。我们最近在《发育细胞》上发表了磷酸肌醇在神经元细胞中，通过招募 GOLPH3 等效应蛋白来启动细胞内运输在高尔基体生物发生过程中参与囊泡融合和质膜出芽。由于巨核细胞 α 颗粒源自跨高尔基体网络和多囊泡身体，我假设磷酸肌醇信号对于细胞内运输是必需的 α-颗粒的生物发生所必需的。磷脂酰肌醇转移蛋白 (PITP) 是一个小蛋白质家族的成员，可结合并转移磷酸肌醇单体细胞区室相互连接，从而实现磷酸肌醇的合成。我们已经做了意外的观察发现，两种主要的 PITP 亚型在巨核细胞、PITPα 和 PITPβ 在将货物从多囊泡体贩运到 α 颗粒。 PITP介导的丧失磷酸肌醇合成会产生与人类中所见相似的形态缺陷灰血小板综合症。该提案的总体假设是磷酸肌醇信号传导 PITP 介导的膜动力学和蛋白质运输是必要的巨核细胞α颗粒的生物发生和维持。在该项目的目标 1 中，我们将严格分析单个 PITP 异构体的离散生化特性巨核细胞。我们的初步数据表明，两种 PITP 亚型控制磷酸肌醇通过不同的生化机制发出信号。在目标 2 中，我们将确定如何磷酸肌醇信号传导有助于α颗粒的生物发生和功能。结合在项目 2 中，我们将测试以下假设：离散的磷酸肌醇合成巨核细胞和血小板的微结构域调节效应蛋白，例如 NBEAL2（导致灰血小板综合症的突变蛋白）。该信号级联调节 NBEAL2 介导膜动力学和蛋白质运输的能力。我们还将分析在通过离体流变学和超显微镜研究详细说明 α-颗粒的功能作用将与项目 2 一起进行，体内血栓形成研究与项目 3 一起进行，以及体内炎症研究研究项目 4。