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。血管异常收缩，如血管痉挛，是血管疾病引起猝死的主要原因。我们发现鞘氨酰磷胆碱（SPC）是一种新的介质，可引起rho激酶介导的血管异常收缩。在本研究中，我们试图鉴定SPC的下游分子及其调控机制，得到了以下结果：1。我们发现spc诱导的血管平滑肌异常收缩依赖于胆固醇，而富含胆固醇的膜结构域膜筏在血管平滑肌异常收缩中起关键作用。这些结果作为非常高影响力的发现被介绍到Circulation research编辑部。我们获得了突变Fyn的重组蛋白，即野生型Fyn、组成型活性Fyn和显性阴性Fyn。通过细胞膜渗透，我们首次证实了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实验中证明了肌动球蛋白的Ca^<2+>-不依赖滑动。我们成功地制作了脂筏模型膜作为杂交脂质体，并发现SPC选择性地与膜筏结合。我们成功地纯化了人血管平滑肌的膜筏组分，并利用串联质谱技术鉴定了膜筏中定位的几个新的蛋白。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.