Myosin phosphatase autoinhibition in gastrointestinal smooth muscle contraction

描述(申请人提供)：平滑肌收缩状态的改变在胃、肠和括约肌功能障碍、运动异常和其他病理的胃肠道疾病中起着关键作用。SM受到一个复杂的信号通路网络的关键调节，该网络通过肌球蛋白的磷酸化来调节收缩能力。肌球蛋白轻链磷酸酶(MLCP)是这些信号通路的主要下游靶点，但其抑制和激活的分子机制尚不清楚，这是我们建议的重点。RhoA GTPase、Rho-Kinase(ROCK)等酶的效应物，在Thr696和Thr853处磷酸化MLCP靶向亚基(MYPT1)，抑制MLCP，而cAMP/cGMP信号和环核苷酸靶标telokin逆转抑制效应，导致GI SM松弛。我们的合作为MLCP活性抑制MYPT1磷酸化的机制带来了一个新的模型。在该模型中，包括Thr696或Thr853处磷酸化MYPT1的片段直接与MLCP的活性部位结合，导致MLCP的自身抑制。在目标1中，我们将确定MYPT1自身抑制(AI)结构域，包括Thr696(AI-1)或Thr853(AI-2)在肠道SM音调调节中的结构/功能关系，以严格检验这一模型。该模型将在活体眼底SM细胞中使用FRET生物传感器进行验证。第二个目标将研究cAMP/cGMP信号如何改变MLCP活性，以消除RhoA介导的钙敏感性，从而导致松弛。环核苷酸被公认为胃肠粘膜松弛的重要生理介质。激活MLCP的一个主要环核苷酸靶点是telokin，它在GI SM中表达最高。与野生型相比，telokin基因敲除小鼠的PCA-force关系左移。在目标2a中，我们将确定telokin诱导MLCP活性激活的分子机制(S)。表面等离子体共振、等温量热法、GST-下拉、邻近连接分析和FRET生物传感器将被用来测试两种端粒素诱导MLCP激活的分子模型。功能分析将利用Telokin-/-和WT小鼠的GI SM。在目标2b中，我们将检验这一假说，即环核苷酸诱导的MLCP在不同的胃肠道平滑肌中的去自身抑制是由多种途径介导的，但以不同的途径为主，以松弛钙敏感力。我们将利用笼中核苷酸的光解来确定非端粒素介导的环核苷酸是否通过EPAC激活Rap1下调RhoA活性，通过磷酸化MYPT1的Ser695或抑制RhoA的磷酸化来减弱环核苷酸的自抑制作用。利用我们的体内和体外数据进行模拟和拟合，我们期望建立这些信号通路的贡献的大小和等级，并得出新的环核苷酸诱导GI SM松弛的机制计算模型。我们预计，眼底和回肠SM将由不同的通路主导，反映出它们不同的功能作用。这些发现应该会为靶向治疗带来新的见解。 与公共卫生相关：由平滑肌组织的异常收缩和松弛引起的疾病，如胃、肠和括约肌功能障碍，消化不良，肠道疾病，手术导致的肠道动力减退，高血压，脑和冠状动脉血管痉挛，勃起功能障碍，以及支气管哮喘等疾病，以及其他疾病，都是由平滑肌组织的异常收缩和松弛引起的。我们正在研究特定蛋白质的作用，这些蛋白质通过复杂的信号通路调节胃肠道平滑肌细胞的收缩机制。这种收缩机制也在细胞迁移中发挥作用，例如在胃肠道发育和肿瘤转移过程中发生的细胞迁移。这项研究的结果应该转化为针对这些疾病的新疗法。

DESCRIPTION (provided by applicant): Alterations in the contractile state of smooth muscle (SM) plays a key role in gastrointestinal diseases such as gastric, intestinal and sphincter dysfunction, abnormal motility and other pathologies. SM is critically modulated by a complex network of signaling pathways that regulate contractility through phosphorylation of myosin. Myosin light chain phosphatase (MLCP) is a major downstream target of these signaling pathways yet the molecular mechanisms responsible for its inhibition and activation are poorly understood and this is the focus of our proposal. The effector of RhoA GTPase, Rho-kinase (ROCK) and other kinases, phosphorylate MLCP targeting subunit (MYPT1), at Thr696 and Thr853 and inhibit MLCP, while cAMP/cGMP signals and the cyclic nucleotide target, telokin, reverse the inhibitory effect, causing GI SM relaxation. Our collaboration has lead to a novel model for the mechanism underlying the inhibition of MLCP activity upon MYPT1 phosphorylation. In the model, the segment including the phosphorylated MYPT1 at Thr696 or Thr853 directly binds to the active site of MLCP, resulting in an autoinhibition of MLCP. In Aim 1 we will determine the structure / function relationship of MYPT1 autoinhibitory (AI) domains including Thr696 (AI-1) or Thr853 (AI-2) in the regulation of gut SM tone to now rigorously test this model. The model will be validated in live fundus SM cells using FRET biosensors. The second aim will address how cAMP/cGMP signals alter MLCP activity to eliminate RhoA-mediated Ca2+ sensitization resulting in relaxation. Cyclic nucleotides are well established as physiologically important mediators of relaxation in GI SM. A major cyclic nucleotide target shown to activate MLCP is telokin, which is most highly expressed in GI SM. The pCa-force relationship is left shifted in telokin knockout mice compared to wild type. In Aim 2a we will determine the molecular mechanism(s) of telokin- induced activation of MLCP activity. Surface plasmon resonance, isothermal calorimetry, GST-pull down, proximity ligation assays (PLA) and a FRET biosensor will be used to test two molecular models of telokin- induced activation of MLCP. Functional assays will utilize GI SM from telokin -/- and WT mice. In Aim 2b we will test the hypothesis that cyclic nucleotide-induced de-autoinhibition of MLCP in different gastrointestinal smooth muscles is mediated by multiple, but dominated by different pathways to relax Ca2+ sensitized force. We will determine using photolysis of caged nucleotides, whether non-telokin mediated attenuation of the autoinhibition by cyclic nucleotides occurs through down regulation of RhoA activity by Epac activation of Rap1, through phosphorylation of Ser695 of MYPT1 or inhibitory phosphorylation of RhoA. Using our in vivo and in vitro data for simulations and fitting we expect to establish the magnitudes and hierarchy of the contribution of these signaling pathways and arrive at new mechanistic computational models of cyclic nucleotide-induced relaxation in GI SM. We expect that fundus and ileum SM will be dominated by different pathways, reflecting their different functional roles. Findings should lead to new insights for targeting therapies. PUBLIC HEALTH RELEVANCE: Diseases such as g by abnormal contraction and relaxation of smooth muscle tissues gastric, intestinal and sphincter dysfunction, dyspepsia, intestinal bowel disease, surgery- induced decreased gut motility, hypertension, cerebral and coronary vasospasm, erectile dysfunction, and bronchial asthma, among other diseases are caused by abnormal contraction and relaxation of smooth muscle tissues. We are studying the role of specific proteins, which through complex signaling pathways regulate the contractile machinery in gastrointestinal smooth muscle cells. This contractile machinery also functions in cell migration, such as occurs during development of the gastrointestinal tract and in tumor metastasis. The results of the research should translate into novel treatments for targeting these diseases.