【作者】 李强；

【导师】 朱力；

【作者基本信息】 苏州大学 ， 细胞生物学， 2014， 硕士

【摘要】 神经导向因子Semaphorin4D (Sema4D; CD100)已经证明在血小板上表达,并且通过接触依赖性方式加强胶原诱导的糖蛋白VI (GPVI)/FcR γ复合体信号通路中脾酪氨酸激酶(Syk)的活化来促进血栓形成。我们之前的研究已经证明血小板Sema4D胞外段可以被金属蛋白酶ADAM17切割,产生一个120kDa的具有生物学活性的大片段。并且,我们也鉴定到Sema4D胞内段部分序列与钙调蛋白结合,调节血小板Sema4D的胞外切割。但我们的进一步研究发现,还有其他的调控机制存在。首先,我们探索了蛋白激酶A(PKA)是否参与Sema4D胞外切割的调控。PKA是环腺苷酸(cAMP)依赖的蛋白激酶,PKA在血小板内起着负反馈作用,PKA的活化能够抑制刺激剂诱导的血小板活化,维持血小板在静息状态从而自由循环。抑制PKA的活性能够诱导金属蛋白酶介导的血小板GPIbα的切割。为了研究PKA是否也参与Sema4D胞外段切割,我们使用PKA抑制剂H89和PKA活化剂forskolin来进行一系列实验,结果表明抑制PKA能够诱导金属蛋白酶介导的Sema4D胞外切割,活化PKA能够抑制刺激剂诱导的Sema4D胞外切割。进一步研究发现抑制PKA导致的Sema4D胞外段切割是通过提高ADAM17酶活性引起的,而不是通过使钙调蛋白从Sema4D解离造成的。同时,我们注意到Sema4D也发生胞内段的切割,主要表现为在某些情况下,血小板Sema4D发生胞外切割后残余的28kDa胞内小片段会消失。但是,Sema4D的胞内水解切割的详细机制还不清楚。之前的研究已经证明钙调蛋白能够和Sema4D胞内段结合,并且与钙调蛋白结合的分子往往都是钙蛋白酶(calpain)的底物。这给我们带来新的启示：Sema4D胞内结构域可能被胞内钙离子依赖的半胱氨酸蛋白酶calpain切割。因此,我们对calpain调节Sema4D切割的作用进行了研究。结果表明：1)血小板Sema4D胞内切割主要是由非生理性刺激剂包括W7、A23187和dibucaine所介导。2)W7、A23187和dibucaine能够显著地提高calpain的活性,使calpain能够水解切割Sema4D胞内段。3)calpain参与调节Sema4D胞内段的切割并不依赖于ADAM17。4) calpain介导的血小板Sema4D胞内切割与血小板活化和凋亡有关。因此,我们的结果揭示了调节Sema4D胞内外切割的新机制,为研究与Sema4D切割相关的胞内蛋白和信号通路以及相关疾病提供了新的思路。更多还原

【Abstract】 Axon guidance molecule Sema4D (Semaphoring4D) has been proved to be expressed on platelets, and supports thrombus formation by amplifying Syk activation, an early step in collagen signaling via the glycoprotein VI (GPVI)/FcRy complex, where in a contact-dependent manner. We have shown that Sema4D exodomain can be cleaved by the metalloprotease ADAM17and produces a120kDa exodomain fragment that retains biological activity. And we also identified a calmodulin-binding domain in Sema4D that regulates its exodomain shedding in platelets. But our further study in here found that there are other regulatory mechanisms exist.First, we explored whether protein kinase A (PKA) is involved in the regulation of Sema4D exodomain shedding. PKA is a cAMP-dependent protein kinase. Activation of PKA has a negative feedback on agonists induced platelet activation and maintain platelets in a resting state to allow free circulation. Inhibition of PKA could results in metalloproteinase-dependent platelet GPIba shedding. To explore whether PKA involved in Sema4D exodomain shedding, we used PKA inhibitor H89and PKA activator forskolin to perform a series of experiments, and the results showed that inhibition of PKA induces metalloproteinase-dependent Sema4D exodomain shedding, activation of PKA could inhibits agonists induced Sema4D exodomain shedding. Further studies showed that inhibition of PKA induced Sema4D exodomain shedding was caused by enhancing ADAM17activity without dissociation of calmodulin from Sema4D.At the same time, we noticed that Sema4D undergoes intracellular domain cleavage as well since the28-kDa fragment disappears in some cases. However, the detail mechanism of the intracellular proteolytic cleavage of Sema4D has not been defined. As the ability of a protein to bind calmodulin confers a strong likelihood that this protein is a substrate for calpain. So, calmodulin binding to Sema4D give us an inspiration that Sema4D intracellular domain may be cleaved by an intracellular Ca2+-dependent cysteine protease calpain. Here we investigated the potential role of calpain in regulating Sema4D intracellular proteolytic cleavage. Our results showed that1) the intracellular cleavage of Sema4D was mediated by non-physiological agonists, such as W7, A23187and dibucaine;2) W7, A23187and dibucaine significantly enhanced calpain activity, which enable calpain to cleave Sema4D intracellular domain proteolytically;3) calpain in regulation of Sema4D intracellular cleavage may occur independently of ADAM17;4) Sema4D intracellular cleavage also involved in platelet activation and apoptosis induced by A23187.Therefore, our data reveal some new regulation mechanisms for platelet Sema4D extracellular and intracellular cleavage and that may replenish Sema4D shedding related intracellular protein, signaling pathways and related diseases.更多还原