Structure and Function of Protein C

描述（申请人提供）：该竞争性更新申请集中于活化蛋白C（APC）的功能性蛋白质组学研究，APC是一种发挥两种主要的不同活性的酶-（1）通过在需要APC辅因子的反应中靶向凝血因子Va和VIIIa的抗凝活性，和（2）通过靶向两种APC受体对细胞的直接抗凋亡和抗炎作用，蛋白酶激活受体1（PAR 1）和内皮蛋白C受体（EPCR）。我们已发表的工作和未发表的初步数据直接导致拟议的研究，并为我们的假设提供强有力的支持。我们假设APC对细胞的EPCR依赖性作用至少部分来自APC在PAR 1中Arg 41处的裂解，该裂解反应涉及APC上不同于因子Va外切位点的PAR 1特异性外切位点。我们假设细胞上的PAR 1通过细胞外蛋白环和/或跨膜螺旋-螺旋相互作用直接与EPCR相互作用。我们假设有额外的辅助因子或衔接子影响APC对细胞的直接作用。我们假设APC的抗凋亡活性需要APC内吞作用，并且内吞的APC直接在细胞中发挥细胞内抗凋亡活性。我们推测APC通过下调caspase 3的产生发挥抗凋亡活性，APC可能作用于线粒体释放cyt c的上游。在测试这些假设，我们建议：1）扩大突变框架理解APC的酶靶向其关键底物，2）表征高密度脂蛋白（HDL）和因子V作为APC辅因子，3）澄清机制负责APC的直接影响内皮细胞，和4）评估APC的新的细胞内抗凋亡活性。在相关的翻译研究中，我们建议评估血栓性疾病和APC受体，PAR 1和EPCR之间的关系，以及血栓性疾病和APC脂质辅因子，葡萄糖神经酰胺的血浆水平之间的关系。这些研究将提高我们理解、诊断和治疗血栓性疾病的能力。

DESCRIPTION (provided by applicant): This competing renewal application is focused on functional proteomics studies of activated protein C (APC), an enzyme that exerts two major, distinct activities - (1) anticoagulant activity by targeting coagulation factors Va and Vllla in reactions requiring APC cofactors and (2) direct anti-apoptotic and anti-inflammatory effects on cells by targeting two APC receptors, protease activated receptor 1 (PAR1) and Endothelial Protein C Receptor (EPCR). Our published work and unpublished preliminary data lead directly to the proposed studies and provide strong support for our hypotheses. We hypothesize that EPCR-dependent effects of APC on cells derive, at least in part, from APC's cleavage at Arg41 in PAR1 in a reaction involving PAR1-specific exosites on APC that differ from factor Va exosites. We hypothesize that PAR1 on cells interacts directly with EPCR via extracellular protein loops and/or via transmembrane helix-helix interactions. We hypothesize that there are additional cofactors or adaptors that influence APC's direct effects on cells. We hypothesize that APC endocytosis is required for APC's anti-apoptotic activity and that endocytosed APC directly exerts intracellular anti-apoptotic activity in cells. We hypothesize that APC exerts antiapoptotic activity by downregulating caspase 3 generation and that APC might act upstream of mitochondrial release of cyt c. In testing these hypotheses, we propose: 1) to expand the mutational framework for understanding APC's enzymatic targeting of its key substrates, 2) to characterize high density lipoprotein (HDL) and factor V as APC cofactors, 3) to clarify mechanisms responsible for APC's direct effects on endothelial cells, and 4) to evaluate APC's novel intracellular anti-apoptotic activity. In related translational research, we propose to assess relationships between thrombotic disease and the APC receptors, PAR1 and EPCR, and between thrombotic disease and plasma levels of the APC lipid cofactor, glucosylceramide. These studies will improve our ability to understand, diagnose and treat thrombotic diseases.