Myosin phosphatase autoinhibition in gastrointestinal smooth muscle contraction

胃肠平滑肌收缩中的肌球蛋白磷酸酶自抑制

• 批准号: 8079574
• 负责人: MASUMI ETO
• 金额: $ 38.49万
• 依托单位: UNIVERSITY OF VIRGINIA
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-07-01 至 2013-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8079574
• 关键词: Active Sites; Address; Agonist; Amino Acid Substitution; Asthma; Binding; Biochemistry; Biological Assay; Biophysics; Biosensor; Calorimetry; Cerebrovascular Spasm; Cleaved cell; Collaborations; Complex; Computer Simulation; Confocal Microscopy; Coronary Artery Vasospasm; Cyclic AMP; Cyclic AMP Receptor Protein; Cyclic AMP-Dependent Protein Kinases; Cyclic GMP; Cyclic Nucleotides; Data; Development; Disease; Down-Regulation; Dyspepsia; Erectile dysfunction; Fluorescence; Fluorescence Resonance Energy Transfer; Freezing; Functional disorder; Fundus; GTP-Binding Proteins; Gastrointestinal Diseases; Gastrointestinal tract structure; Generations; Goals; Guanine Nucleotide Exchange Factors; Guanosine Triphosphate; Guanosine Triphosphate Phosphohydrolases; Hypertension; In Vitro; Intestines; Kinetics; Knock-out; Knockout Mice; Laboratories; Lead; Left; Life; Ligation; Mediating; Mediator of activation protein; Modeling; Molecular; Molecular Models; Monitor; Mus; Muscle Contraction; Muscle Tonus; Muscle relaxation phase; Myography; Myosin ATPase; Myosin Regulatory Light Chains; NMR Spectroscopy; Neoplasm Metastasis; Nucleotides; Operative Surgical Procedures; Organ Culture Techniques; Outcome; Pathology; Pathway interactions; Phosphorylation; Phosphorylation Site; Phosphotransferases; Physiological; Physiology; Play; Proteins; Recombinant Proteins; Recombinants; Regulation; Relative (related person); Relaxation; Research; Resolution; Rho-associated kinase; Role; Signal Pathway; Signal Transduction; Smooth Muscle; Smooth Muscle Myocytes; Smooth Muscle Myosins; Sphincter; Stomach; Structure; Structure-Activity Relationship; Surface Plasmon Resonance; Technology; Testing; Time; Tissues; Translating; alpha helix; attenuation; cell motility; flash photolysis; gastric fundus; gastrointestinal; ileum; in vivo; insight; interdisciplinary approach; migration; molecular modeling; myosin phosphatase; novel; photolysis; prevent; public health relevance; research study; simulation; small hairpin RNA; telokin; tumor

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.

描述（申请人提供）：平滑肌（SM）收缩状态的改变在胃肠道疾病中起着关键作用，例如胃、肠和括约肌功能障碍、运动异常和其他病理。 SM 受到复杂的信号通路网络的严格调节，这些信号通路通过肌球蛋白的磷酸化来调节收缩性。肌球蛋白轻链磷酸酶（MLCP）是这些信号通路的主要下游靶标，但对其抑制和激活的分子机制知之甚少，这是我们建议的重点。 RhoA GTPase、Rho 激酶 (ROCK) 和其他激酶的效应器在 Thr696 和 Thr853 磷酸化 MLCP 靶向亚基 (MYPT1) 并抑制 MLCP，而 cAMP/cGMP 信号和环核苷酸靶点 telokin 逆转抑制作用，导致 GI SM 松弛。我们的合作为 MYPT1 磷酸化抑制 MLCP 活性的机制建立了一个新模型。在该模型中，包含 Thr696 或 Thr853 处磷酸化 MYPT1 的片段直接与 MLCP 的活性位点结合，导致 MLCP 的自身抑制。在目标 1 中，我们将确定 MYPT1 自抑制 (AI) 域（包括 Thr696 (AI-1) 或 Thr853 (AI-2)）在调节肠道 SM 音调中的结构/功能关系，以便现在严格测试该模型。该模型将使用 FRET 生物传感器在活体眼底 SM 细胞中进行验证。第二个目标将解决 cAMP/cGMP 信号如何改变 MLCP 活性，以消除 RhoA 介导的 Ca2+ 敏化，从而导致松弛。环核苷酸被认为是 GI SM 中生理上重要的松弛介质。显示激活 MLCP 的主要环核苷酸靶标是 telokin，它在 GI SM 中表达最高。与野生型相比，telokin 敲除小鼠的 pCa 力关系左移。在目标 2a 中，我们将确定 telokin 诱导的 MLCP 活性激活的分子机制。表面等离子共振、等温量热法、GST-pull down、邻近连接测定（PLA）和 FRET 生物传感器将用于测试 telokin 诱导的 MLCP 激活的两种分子模型。功能测定将利用 telokin -/- 和 WT 小鼠的 GI SM。在目标 2b 中，我们将检验以下假设：环核苷酸诱导的不同胃肠道平滑肌中 MLCP 的去自抑制是由多种途径介导的，但主要由不同的途径来放松 Ca2+ 敏化力。我们将使用笼状核苷酸的光解来确定非telokin介导的环核苷酸自抑制的减弱是否是通过Rap1的Epac激活、MYPT1的Ser695的磷酸化或RhoA的抑制性磷酸化来下调RhoA活性而发生的。使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贡献的大小和层次，并得出 GI SM 中环核苷酸诱导的松弛的新机械计算模型。我们预计眼底和回肠 SM 将由不同的通路主导，反映它们不同的功能作用。研究结果将为靶向治疗带来新的见解。 公众健康相关性：由平滑肌组织异常收缩和舒张引起的疾病，胃、肠和括约肌功能障碍、消化不良、肠道疾病、手术引起的肠道动力下降、高血压、脑和冠状血管痉挛、勃起功能障碍和支气管哮喘等疾病都是由异常引起的。 平滑肌组织的收缩和舒张。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。这种收缩机制还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情况。研究结果应转化为针对这些疾病的新疗法。