STRUCTURE/FUNCTION OF CARBOXYPEPTIDASES

总体目标是通过强调三个广泛的研究领域来阐明调节羧基肽酶（CPs）在生理和病理过程中的作用：1)结构，2)细胞靶向和3)功能。假设1:CPM的结构包含调节羧基肽酶家族独有的特征。具体目的1：结晶重组羧肽酶（CP） M并确定其三维结构。假设2:CPD的三个羧基肽酶同源结构域是独特的：结构域1和2是具有独特特异性的活性CPs，而结构域3是无活性的，但可以结合底物并影响结构域1和2的动力学。具体目标2：通过(i)表达含有1、2或3个活性位点结构域的CPD重组形式，确定CPD的CP同源结构域的特征及其在完整分子中的相互作用；（ii）研究每种形式的CPD：底物特异性、合成和天然肽的动力学参数、抑制剂亲和性、金属离子激活/抑制、最佳pH值和稳定性；（iii）确定肽底物结合结构域3的特异性。假设3:CPM在其胞外区域含有独特的基底外侧靶向信号。特异性目的3：通过转染不同CPM缺失突变体和融合构建体的MDCK细胞，确定CPM的细胞外结构域是否介导了极化的Madin Darby犬肾（MDCK）细胞中CPM异常的基底外侧分选，然后研究CPM向根尖和基底外侧分选。假设4：调节性羧肽酶抑制纤溶酶原活化；CPN与纤溶酶原或tPA直接相互作用，细胞CPM和CPD通过下调纤溶酶原与细胞的结合。具体目标4：研究CPN对溶液中纤溶酶原活化的抑制作用，以及CPM和CPD对细胞表面纤溶酶原活化的调节作用，通过(i)阐明CPN的50 kDa和83 kDa亚基的作用及其作用机制（即结合或水解）；（ii）测定表达不同水平CPM和CPD的细胞表面纤溶酶原的结合和活化。这些研究将提供有关可参与多种生理和病理生理过程的调节性cp的结构和功能的新信息，例如：(i)调节缓激肽活性，控制肾脏中的盐和水排泄；（ii）产生B1受体的激动剂（例如，des-Arg9-bradykinin），该受体被炎症细胞因子上调；（iii）调节纤溶酶原的激活，这对纤维蛋白凝块的血管内溶解、伤口愈合、血管生成、组织重塑和肿瘤细胞的转移至关重要。

The overall objective is to elucidate the roles of regulatory carboxypeptidases (CPs) in physiological and pathological processes by emphasizing three broad areas of investigation: l) Structure, 2) Cellular targeting and 3) Functions. Hypothesis 1: The structure of CPM contains features that are unique to the regulatory carboxypeptidase family. Specific Aim 1: Crystallize recombinant carboxypeptidase (CP) M and determine its three dimensional structure. Hypothesis 2: The three carboxypeptidase homology domains of CPD are unique: Domains 1 and 2 are active CPs with unique specificities whereas domain 3 is inactive, but binds substrates and influences the kinetics of domains 1 and 2. Specific Aim 2: Determine the characteristics of the CP homology domains of CPD and their interactions in the intact molecule by (i) expressing recombinant forms of CPD containing 1, 2 or 3 of the active site domains; (ii) investigating, for each form of CPD: substrate specificity, kinetic parameters for synthetic and naturally occurring peptides, inhibitor affmity, metal ion activation/inhibition, pH optimum and stability; (iii) determining the specificity of binding of peptide substrates for domain 3. Hypothesis 3: CPM contains a unique basolateral targeting signal in its extracellular domain. Specific Aim 3: Determine whether the extracellular domain of CPM mediates its unusual basolateral sorting in polarized Madin Darby canine kidney (MDCK) cells by transfecting MDCK cells with various CPM deletion mutants and fusion constructs and then investigate their sorting to the apical and basolateral domains. Hypothesis 4: Regulatory carboxypeptidases inhibit plasminogen activation; CPN by direct interaction with plasminogen or tPA and cellular CPM and CPD by downregulating plasminogen binding to cells. Specific Aim 4: Investigate CPN inhibition of plasminogen activation in solution and CPM and CPD regulation of plasminogen activation on the cell surface by (i) elucidating the role of the 50 kDa and 83 kDa subunits of CPN and the mechanism(s) by which they act (i.e., binding or hydrolysis); (ii) measuring plasminogen binding and activation on the surface of cells expressing different levels of CPM and CPD. These studies will provide novel information regarding the structure and function of regulatory CPs that can be involved in a variety of physiological and pathophysiological processes such as: (i) regulation of bradykinin activity which controls salt and water excretion in the kidney; (ii) generation of agonists (e.g., des-Arg9-bradykinin) for the B1 receptor which is upregulated by inflammatory cytokines; (iii) regulation of plasminogen activation which is critical to the intravascular dissolution of fibrin clots, wound healing, angiogenesis, tissue remodeling and metastasis of neoplastic cells.