P20-Mediated Suppression of Vascular Force

描述(由申请人提供)：血管平滑肌松弛通常被认为是激活的逆转，即肌浆[Ca~(2+)]肌球蛋白调节轻链(MRLC)去磷酸化减少。我们称这种松弛形式为“失活”，因为它是激活机制的逆转。有些刺激会产生一种不涉及“去激活”机制的松弛。尽管MRLC的磷酸化水平持续升高，但[cGMP]或[cAMP]的升高会降低平滑肌张力。我们将这一过程称为力抑制，以将其与通过减少MRCL磷酸化来减小力的失活机制区分开来。在这项提议中，我们提出的证据表明，导致力抑制的机制涉及磷酸化p20(也称为热休克蛋白20或HSP20)与平滑肌细丝的结合。我们已经有初步数据表明，重组的磷酸化p20抑制了皮肤颈动脉的作用力。我们也有初步数据表明，力抑制与缩短速度的降低无关。这表明，力抑制是在交叉桥水平上调节的。为了进一步研究这一现象，我们建议研究重组突变体p20在剥皮的颈动脉中的作用，并对完整的颈动脉进行详细的肌肉力学研究。目的：1.验证磷酸化p20通过p20肽(110-121)中的肌动蛋白结合域特异性抑制皮肤平滑肌力的假说。这个p20结构域与肌钙蛋白1中的一个称为抑制肽的结构域具有很高的序列同源性。目的2.验证力抑制受p20丝氨酸16磷酸化调控的假说。目的3.验证力抑制是在交叉桥水平介导的假说。在发达国家，力抑制的不适当反应与大多数发病率和死亡率有关。目前的许多治疗方法都是以平滑肌肉为目标来降低肌张力。以力抑制通路(即p20)为靶点可能会提供类似的好处。

DESCRIPTION (provided by the applicant): Vascular smooth muscle relaxation is typically hypothesized to be the reversal of activation, i.e. reduction of myoplasmic [Ca2+] myosin regulatory light chain (MRLC) dephosphorylation. We term this form of relaxation as "deactivation" because it is the reversal of activation mechanisms. Some stimuli induce a relaxation that dose not involve "deactivation" mechanisms. Elevations in [cGMP] or [cAMP] can reduce smooth muscle tone despite persistent elevations in MRLC phosphorylation levels. We term this process 'force suppression' to separate it from deactivation mechanisms which reduce force by reducing MRCL phosphorylation.In this proposal, we present evidence suggesting that the mechanism responsible for 'force suppression' involves binding of phosphorylate p20 (also known as heat shock protein 20 or HSP20) to smooth muscle thin filaments. We have preliminary data that recombinant phosphorylated p20 suppresses force in skinned carotid artery. We also have preliminary data that force suppression is not associated with reductions in shortening velocity. This suggests that force suppression is mediated at the crossbridge level. To further study this phenomenon, we propose to study recombinant mutant p20 in skinned carotid artery and to perform detailed muscle mechanics in intact carotid artery. Thee aims are:Aim 1. Test the hypothesis that phosphorylated p20 specifically suppresses skinned smooth muscle force via the actin binding domain contained in the peptide p20 (110-121). This p20 domain has a high sequence homology with a domain in troponin 1 called the inhibitory peptide.Aim 2. Test the hypothesis that force suppression is regulated by p20 phosphorylation at serine 16.Aim 3. Test the hypothesis that force suppression is mediated at the crossbridge level.Inappropriate responses of smooth muscle are involved in most morbidity and mortality in the developed world. Many current treatments target smooth muscle to reduce tone. It is possible that targeting the force suppression pathway (i.e. p20) might provide comparable benefits.