Membrane Serine Protease Activities in Protease Activated Receptor Signaling

描述(由申请人提供)：蛋白酶激活受体(PARs)是一种独特的G蛋白偶联受体(GPCRs)，使细胞能够感知其环境中的特定蛋白酶。PARs正在成为几种疾病的有吸引力的治疗靶点，包括心血管疾病、关节炎、结肠炎、哮喘、神经退行性疾病和癌症。单个PAR能够激活屏障破坏、促炎信号和抗炎屏障保护性信号通路。这种选择性信号是如何介导的还知之甚少，这是开发有效的针对PARs的激动剂和拮抗剂用于治疗的重要障碍。更好地了解诱导差异PAR信号的蛋白酶环境的性质，以及参与PAR激活的分子机制是至关重要的。我们发现，两种膜锚定的丝氨酸蛋白酶，睾丸蛋白和基质金属丝氨酸酶，是PAR2的细胞特异性内源性激活因子，可能分别在内皮细胞和上皮细胞中调节PAR2的局部空间和时间信号。我们的数据表明，GPI锚定的丝氨酸蛋白酶，Tstisin，是一种促血管生成因子，可以激活微血管系统中的PAR2，促进对血管生成至关重要的毛细血管生长。我们还发现，跨膜丝氨酸蛋白酶，即Mattritase，可能支持PAR2依赖的信号通路，对维持肠上皮屏障至关重要。该研究计划的目标是确定这些激活PAR2的膜丝氨酸蛋白酶的活性，并开发一种新的前药策略来检测和靶向它们的活性。这项研究计划将利用体外培养和在小鼠体内的研究来测试以下目标：1)确定睾丸素在血管生成过程中对PAR2信号的贡献，2)确定PARS在调节前列腺素&GT酶介导的肠上皮屏障关闭中的调节作用，以及3)重组炭疽毒素以靶向细胞表面激活PAR2膜锚定的丝氨酸蛋白酶。

DESCRIPTION (provided by applicant): Protease-activated receptors (PARs) are distinct G protein-coupled receptors (GPCRs) that allow cells to sense specific proteases in their environment. PARs are emerging as attractive therapeutic targets for several diseases, including cardiovascular diseases, arthritis, colitis, asthma, neurodegenerative conditions and cancer. Individual PARs are able to activate pathways that confer both barrier disruptive, proinflammatory signaling, as well as anti-inflammatory barrier protective signaling pathways. How this selective signaling is mediated is poorly understood, and represents a significant impediment to the development of effective agonists and antagonists targeting PARs for therapeutic uses. A better understanding of the nature of the protease environment that induces differential PAR signaling, and the molecular mechanisms involved in protease activation of PARs are critical. We have discovered that two membrane-anchored serine proteases, Testisin and Matriptase, are cell-specific endogenous activators of PAR2, and likely modulate localized spatial and temporal signaling of PAR2 in endothelial and epithelial cells, respectively. Our data suggests that the GPI anchored serine protease, Testisin, is a proangiogenic factor that can activate PAR2 in the microvasculature, and facilitate capillary growth important for angiogenesis. We also find that the transmembrane serine protease, Matriptase, may sustain a PAR2 dependent signaling pathway important for maintaining the intestinal epithelial barrier. The goal of the research plan is to define the activities of these PAR2-activating membrane serine proteases and to develop a novel prodrug strategy for both detecting and targeting their activities. The research plan will utilize in vitro cultures in concert with in vivo studies in mic to test the following aims: 1) to determine the contribution of Testisin to PAR2 signaling during angiogenesis, 2) to determine the role of PARs in regulating in regulating Prostasin->Matriptase mediated intestinal epithelial barrier closure, and 3) to reengineer anthrax toxins to target PAR2-activating membrane-anchored serine proteases on the cell surface.