Functional roles of membrane domain and its localized component proteins in abnormal vascular contraction

血管疾病是危及生命的疾病，在日本死亡率很高。血管收缩异常，如血管痉挛，被认为是由于血管疾病引起的猝死的主要原因。我们发现鞘氨醇磷酸胆碱（SPC）是一种新型介质，可导致Rho激酶介导的血管异常收缩。本研究试图对SPC下游分子及其调控机制进行鉴定，并获得以下结果.我们发现SPC引起的异常收缩依赖于胆固醇，而富含胆固醇的膜结构域膜筏在人血管平滑肌的异常收缩中起着关键作用。这些结果被《流通研究》编辑部作为非常高影响力的发现介绍。我们获得了突变Fyn的重组蛋白，即野生型Fyn、组成型活性Fyn和显性失活Fyn。通过膜透化技术将这些重组突变Fyn蛋白应用于细胞质中，我们首次证明了Fyn酪氨酸激酶在SPC和Rho激酶诱导的血管平滑肌异常收缩中起重要作用。我们首次在高纯度收缩蛋白的体外运动试验中发现肌动球蛋白的非Ca ^<2 +>依赖性滑动.我们成功地将脂筏模型膜制成杂化脂质体，发现SPC选择性地与膜筏结合.我们成功地从人血管平滑肌中纯化了膜筏组分，并利用功能蛋白质组学技术和串联质谱技术鉴定了几个定位于膜筏中的新蛋白.我们发现了几种分子，可以选择性地抑制异常血管收缩的信号转导，这是由本研究澄清。

Vascular diseases are life-threatening diseases with high mortality in Japan. Abnormal vascular contraction, such as vasospasm, is known as major cause of sudden death due to vascular diseases. We identified sphingosylphosphorylcholine (SPC) as a novel mediator which causes abnormal vascular contraction mediated by Rho-kinase. In this study, we attempted to identify downstream molecules of SPC and their regulatory mechanisms, and obtained the following results.1. We found that SPC-induced abnormal contraction depends on cholesterol, and cholesterol-enriched membrane domain, membrane raft, plays a pivotal role in abnormal contraction of human vascular smooth muscle. These results were introduced as very high impact discovery in the Editorial Section of Circulation Research.2. We obtained recombinant proteins of mutated Fyn, namely, wild type of Fyn, constitutively active Fyn, and dominant negative Fyn. Using cytosolic application of these recombinant mutated Fyn proteins by membrane permeabilization, we demonstrated the first direst evidence that Fyn tyrosine kinase plays an important role in abnormal contraction of vascular smooth muscle induced by SPC and Rho-kinase.3. We showed for the first time that Ca^<2+>-independent sliding of actomyosin in an in vitro motility assay of highly purified contractile proteins.4. We succeeded to make lipid raft model membrane as a hybrid liposome and found that SPC selectively binds to membrane raft.5. We succeeded to purify membrane raft fraction from human vascular smooth muscle, and identified several novel proteins localized in membrane raft, using functional proteomics by tandem mass spectrometer.6. We identified several molecules which can selectively inhibit the signal transduction of abnormal vascular contraction, which was clarified by this study.