The STIM/Orai interface and novel tools to control vascular smooth muscle phenotype

项目摘要 血管平滑肌细胞(VSMCs)对于维持血管壁、血管的完整性是至关重要的 音调，和收缩。它们有惊人的潜力在机械损伤或炎症时去分化。 从静止状态到增殖(合成)状态。这种高度的VSMC可塑性在伤口中至关重要 反应和愈合，但也使细胞易于在血管管腔附近不利地形成新的内膜。 新生内膜形成使动脉壁变紧，限制血流，是闭塞的致病基础。 心血管疾病，如动脉粥样硬化、再狭窄和系统性高血压。要解决的治疗方法 新生内膜形成罕见且非选择性。了解和确定目标的机制 人工合成的VSMCs具有最重要的临床意义。 在静止的和合成的VSMC之间的过渡中观察到的是一种表型 参与钙信号转导的蛋白质的调节。钙是一种重要的第二信使，参与了几乎 从增殖到凋亡的每一个细胞过程。当VSMC从收缩静止状态改变时 在增殖状态下，它们对电压门控钙通道的依赖较少，而对Orai1钙的依赖较多 频道。ORAI1在钙稳态过程中是必不可少的，这一过程被称为储存操作钙进入(SOCE)。 内质网(ER)中的钙存储是由一种被称为STIM的蛋白质家族感知的。vt.在.的基础上 储存耗尽，STIM蛋白寡聚，经历构象变化，从内质网迁移到内质网血浆 膜(PM)结。STIM的展开揭示了一个强大的互动网站，称为STIM-Orai 激活区(SOAR)，能够结合和激活高度钙选择性的PM Orai通道。 阐明和开发STIM-Orai结合界面是一种令人兴奋的新疗法 心血管疾病专用道。 Orai与STIM是如何结合在一起的，这是一个有很多猜测的话题。以前的研究表明，这两个人中的任何一个 ORAI胞浆区域被称为N-末端和跨膜延伸(TM4ext)，或 相邻的TM4exts需要形成二聚化结合口袋。为了解开STIM之谜- Orai偶联接口，我开发了一组新颖的PM系链多肽，它可以独占地询问 Orai1-3三种主要哺乳动物Orai亚型的TM4ext。我最近的工作呈现了一个令人兴奋的新 STIM-Orai相互作用的范例，认为TM4ext作为唯一结合区域是必要和充分的。 此外，我发现Orai3TM4ext在更多的时间内与STIM有巨大的结合作用 无处不在的Orai1。 这项F31提案建立在这些令人兴奋的发展之上，并代表着 詹姆斯·巴拉尼亚克的训练。这项提案的持续工作将询问STIM-Orai如何使用这些 新的多肽，以及它们如何被用作控制VSMC增殖和迁移的有力工具。

PROJECT ABSTRACT Vascular smooth muscle cells (VSMCs) are crucial in maintaining the integrity of vessel walls, vascular tone, and contraction. They have the amazing potential to dedifferentiate upon mechanical injury or inflammation from a quiescent state to a proliferative (synthetic) state. This high degree of VSMC plasticity is crucial in wound response and healing, but also predisposes the cell to adverse formation of neointima near the vascular lumen. Neointimal formation tightens the arterial wall, restricting blood flow and is the pathogenic base for occlusive cardiovascular diseases such as atherosclerosis, restenosis, and systemic hypertension. Therapies to address neointimal formation are scarce and non-selective. Understanding and targeting mechanisms utilized by synthetic VSMCs is of upmost clinical importance. What has been observed in the transition between quiescent and synthetic VSMCs, is a phenotypic modulation of proteins involved in calcium signaling. Calcium is a vital second messenger involved in nearly every cellular process from proliferation to apoptosis. As VSMCs change from their contractile quiescent state to a proliferative state, they rely less on voltage-gated calcium channels and depend more on the Orai1 calcium channel. Orai1 is essential in the calcium homeostatic process known as store operated calcium entry (SOCE). Calcium stores within the endoplasmic reticulum (ER) are sensed by a family of proteins known as STIM. Upon store depletion, STIM proteins oligomerize, undergo conformational change, and migrate from ER to ER-plasma membrane (PM) junctions. Unfolding of the STIM reveals a powerful interacting site known as the STIM-Orai activating region (SOAR), which is able to bind and activate the highly calcium selective PM Orai channels. Elucidation and exploitation of the STIM-Orai binding interface presents an exciting new therapeutic avenue for cardiovascular disease. How Orai couples with STIM is a topic under much speculation. Previous studies propose that either two Orai cytosolic regions are involved, known as the N-terminus and transmembrane extension (TM4ext), or that adjacent TM4exts are required to form a dimerized binding pocket. In order to solve the mystery of the STIM- Orai coupling interface, I developed a novel set of PM tethering peptides that can exclusively interrogate the TM4ext of any of the three major mammalian Orai subtypes, Orai1-3. My recent work presents an exciting new paradigm for STIM-Orai interacting, that the TM4ext is necessary and sufficient in acting as a sole binding region. Furthermore, I found out that the Orai3 TM4ext ahs tremendous binding interaction with STIM over the more ubiquitous Orai1. This F31 proposal builds upon these exciting developments and represents a huge integral part for the training of James Baraniak. Continual work on this proposal will interrogate how STIM-Orai interacts using these novel peptides, and how they may be used as a powerful tool in controlling VSMC proliferation and migration.