Function and regulation of constitutive protein translation in platelets

项目摘要 本研究的目的是：1)阐明蛋白质翻译起始的分子信号转导机制。 循环的人类血小板和表征血浆生长因子或激素驱动的途径，绘制 再生人血小板蛋白质组，并确定构型翻译在血小板功能中的作用 反应性；2)测定ArGONAUTE2(小鼠的AGO2、AGO2)在血小板mRNA、microRNA中的作用 (MiRNA)和蛋白的表达、血小板的反应性以及由miRNA介导的血小板的特殊作用 在止血和血栓形成潜力方面抑制翻译。这项提案中的研究将阐明 血小板功能的分子生理学的基本概念。此外，这些研究还将具有深远的意义 对两个具有广泛临床意义的领域的影响：生长因子/激素的变化 动态平衡及药物翻译抑制剂在疾病中的应用和发展 治疗。令人惊讶的是，翻译在所谓的静息血小板中的作用几乎没有引起人们的注意。 发行量，遵循翻译重要性有限的总体假设。在先前的研究中 “静息”的血小板转化，体外血小板从其天然血浆环境中移除，以及 翻译逐渐停顿下来。我们对这些先前的研究的解释是，相反，血小板会经历 在血浆存在的情况下，例如，在循环中的结构性翻译，并且这种分子的支出 资源和细胞能量学对血小板蛋白的动态平衡和功能至关重要。我们的强者 初步数据支持这一假设，我们提出了一系列创新的方法来研究这一点 对血小板生物学及其功能结果知之甚少。我们第一次证明了 由于翻译的启动，循环中的血小板经历了一个强大的蛋白质组的结构性翻译 由血浆携带的生长因子和激素驱动的信号，这一过程受血小板调节 MiRNAs和AGO2，以及构成的miRNA在血小板中调节的翻译调节血小板的功能。 我们将采用一系列创新的方法来检验以下假设：目标1-1)“静息”血小板 在血液循环中，由于血浆生长因子和 激素；2)血浆生长因子驱动的结构性翻译维持血小板蛋白稳态 对止血功能是必需的；目标2-1)血小板中丰富的miRNAs和Ago2介导的RNA抑制 调节血浆生长因子和激素驱动的结构性翻译；2)Ago2/miRNA介导 抑制使血小板保持在必要的反应状态，以支持止血，同时限制血栓形成。 总之，这些研究的结果将极大地扩展我们对血小板控制的理解 功能、止血和血栓形成以及对血小板反应性和生长因子功能的影响 或疾病中的激素失衡，以及临床翻译抑制剂对这些过程的潜在影响。

Project Summary The goals of this proposal are: 1) to elucidate the molecular signaling of protein translation initiation in circulating human platelets and characterize the plasma growth factor- or hormone-driven pathways, map the regenerating human platelet proteome, and determine the roles of constitutive translation in platelet functional reactivity; 2) to determine the roles of ARGONAUTE2 (AGO2, Ago2 in mice) in platelet mRNA, microRNA (miRNA) and protein expression, platelet reactivity, and the specific roles of platelet miRNA-mediated suppression of translation in hemostasis and thrombotic potential. The studies in this proposal will elucidate essential concepts of the molecular physiology of platelet function. In addition, these studies will have profound implications for two areas of widespread clinical significance: alterations in growth factor/hormone homeostasis, and current use and development of pharmacological inhibitors of translation in disease treatment. Surprisingly little attention has been paid to roles of translation in so-called “resting” platelets in circulation, following the overall presumption that translation is of limited importance. In prior studies of “resting” platelet translation, ex vivo platelets were removed from their native plasma environment, and translation steadily came to a halt. We interpret these prior studies to suggest instead that platelets undergo translation constitutively in the presence of plasma, e.g., in circulation, and that this expenditure of molecular resources and cellular energetics is critical for platelet protein homeostasis and functionality. Our strong preliminary data support this hypothesis and we propose a series of innovative approaches to investigate this poorly understood aspect of platelet biology and its functional outcomes. We have shown for the first time that platelets in circulation undergo constitutive translation of a robust proteome due to translation initiation signaling driven by plasma-borne growth factors and hormones, that this process is modulated by platelet miRNAs and AGO2, and that constitutive miRNA-modulated translation in platelets regulates platelet function. We will pursue an array of innovative approaches to test the following hypotheses: Aim 1- 1) “resting” platelets in circulation are translationally active due to ongoing signaling initiated by plasma growth factors and hormones; 2) constitutive translation driven by plasma growth factors maintains platelet protein homeostasis necessary for hemostatic function; Aim 2 - 1) abundant miRNAs and Ago2-mediated RNA inhibition in platelets modulate constitutive translation driven by plasma growth factors and hormones; 2) Ago2/miRNA-mediated suppression maintains platelets in a reactive state necessary to support hemostasis while limiting thrombosis. Together, the outcomes of these studies will significantly expand our understanding of control of platelet function, hemostasis and thrombosis, as well as implications for platelet reactivity and function in growth factor or hormone imbalance in disease, and potential impacts of clinical translation inhibitors on these processes.