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

在日本，血管疾病是一种死亡率很高的危及生命的疾病。血管收缩异常，如血管痉挛，被认为是血管疾病导致猝死的主要原因。鞘氨醇磷脂酰胆碱(SPC)是一种由Rho-K介导的引起血管异常收缩的新介质。在本研究中，我们试图鉴定SPC的下游分子及其调控机制，并获得了以下结果。我们发现，SPC诱导的异常收缩依赖于胆固醇，而富含胆固醇的膜域，即膜筏，在人血管平滑肌异常收缩中起着关键作用。这些结果在《循环研究》的编辑部被作为非常高的影响发现进行了介绍。我们获得了突变的Fyn重组蛋白，即野生型Fyn、成分活性Fyn和显性负性Fyn。通过膜通透性将这些突变的Fyn蛋白应用于胞浆，我们首次证明了Fyn酪氨酸激酶在SPC和Rho-kinase诱导的血管平滑肌异常收缩中起重要作用。在高纯度收缩蛋白的体外运动分析中，我们首次证明了肌动球蛋白的钙非依赖性滑动。我们成功地制备了脂筏模型膜作为杂化脂质体，发现SPC选择性地结合到膜筏上。我们成功地从人血管平滑肌中分离纯化了膜FRAFT组分，并利用串联质谱仪的功能蛋白质组学方法鉴定了几种新的定位于膜FRAFT的蛋白质。我们发现了几个可以选择性抑制血管异常收缩信号转导的分子，这一点在本研究中得到了阐明。

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.