Structure and Function of Carboxypeptidases

描述（由申请人提供）：我们的长期目标是了解调节羧肽酶在生理和病理过程中的作用。膜结合羧肽酶（CP）M是激肽释放酶-激肽系统的重要调节剂，通过其产生G蛋白偶联的激肽B1受体（B1 R）的脱精氨酸-激肽激动剂的能力。激肽是肾和心血管功能以及炎症过程的重要调节剂。主要目的是研究一种新的变构蛋白质-蛋白质之间的相互作用CPM和B1 R，增强受体信号。具体目的1：探索CPM和B1 R在膜上的相互作用，并确定对它们的相互作用重要的残基。假设：CPM外部的带正电荷的残基与膜磷脂结合，以使CPM在膜上定向，并且与B1 R的基础相互作用由其C-末端结构域介导，而变构调节由催化结构域附近的表面介导。我们将：（i）确定CPM和B1 R是否直接相互作用。(ii)研究CPM上的外部正电荷对于正确的膜取向和与B1 R结合的重要性。(ii)确定B1 R上介导其与CPM结合的位点。(iii)确定CPM上与B1 R相互作用的位点。(iv)产生肽干扰CPM/B1 R相互作用。具体目标二：阐明CPM和B1受体之间的相互作用导致受体活化的变构调节的特征。假设：CPM通过与变构位点结合来增强B1 R功能，并且CPM与底物结合导致B1 R构象变化和增强的受体活化。我们将确定：（i）CPM特异性和肽底物亲和力对B1 R活化的变构调节的作用和（ii）CPM变构调节对B1 R构象的影响。具体目标3：确定CPM介导的B1 R活化变构调节在G蛋白偶联、-arrestin相互作用和下游信号传导中的作用，并确定CPM/B1 R相互作用在调节内皮屏障功能中的重要性。我们的假设是，CPM与B1 R的相互作用增强了所有B1 R信号通过基础的相互作用，但进一步增强Gq耦合的反应变构调制。此外，CPM/B1 R相互作用的破坏将减弱B2 R激动剂对B1 R介导的内皮屏障损伤。我们将确定：（i）CPM与B1 R相互作用介导的G蛋白偶联vs.激动剂结合。(ii)B1受体激动剂激活与CPM介导的变构增强中β-arrestin-2募集的参与。(iii)B1 R通过Gq和Gi介导的途径响应激动剂或CPM相互作用的下游信号输出。(iv)CPM在调节内皮/血管通透性的B1 R信号变构调节中的作用。这些研究将阐明一种新的机制，变构增强B1 R信号的CPM，也产生其配体。了解如何调节这一过程可能会导致治疗肾脏和心血管疾病的新药的开发。 公共卫生相关性：我们正在研究两种蛋白质，羧肽酶M和B1受体，在细胞表面上的相互作用，导致影响细胞和器官功能的调节分子的产生增加。已知B1受体调节肾脏和心血管功能，特别是在感染或炎症的情况下。了解这些蛋白质的相互作用如何被增强或阻断可能会导致治疗肾脏和心血管疾病的新药的开发。

DESCRIPTION (provided by applicant): Our long term objective is to understand the roles of regulatory carboxypeptidases in physiological and pathological processes. Membrane-bound carboxypeptidase (CP) M is an important regulator of the kallikrein- kinin system via its ability to generate des-Arg-kinin agonists of the G-protein coupled kinin B1 receptor (B1R). Kinins are important regulators of renal and cardiovascular function and inflammatory processes. The major objective is to investigate a novel allosteric protein-protein interaction between CPM and B1R that enhances receptor signaling. Specific Aim 1: To explore the interaction of CPM and B1R on the membrane and determine the residues important for their interaction. Hypothesis: positively charged residues on CPM's exterior bind to membrane phospholipids to orient CPM on the membrane and basal interaction with the B1R is mediated by its C-terminal domain whereas allosteric modulation is mediated by a surface near the catalytic domain. We will: (i) determine whether CPM and B1R directly interact. (ii) investigate the importance of the exterior positive charges on CPM for proper membrane orientation and binding to the B1R. (ii) determine the site(s) on the B1R that mediates its binding to CPM. (iii) determine the site(s) on CPM that interacts with the B1R. (iv) generate peptides to interfere with CPM/B1R interactions. Specific Aim 2: To elucidate the characteristics of the interaction between CPM and the B1R that results in allosteric modulation of receptor activation. Hypothesis: CPM enhances B1R function by binding to an allosteric site and substrate binding by CPM results in B1R conformational change and enhanced receptor activation. We will determine: (i) the role of CPM specificity and peptide substrate affinity on allosteric modulation of B1R activation and (ii) the effect of CPM allosteric modulation on B1R conformation. Specific Aim 3: To determine the role of CPM-mediated allosteric modulation of B1R activation in G-protein coupling, -arrestin interaction and downstream signaling and to determine the importance of CPM/B1R interaction in regulating endothelial barrier function. Our hypothesis is that CPM interaction with the B1R enhances all B1R signaling via basal interaction but further enhances Gq-coupled responses by allosteric modulation. Furthermore, disruption of CPM/B1R interaction will attenuate B1R-mediated endothelial barrier damage in response to B2R agonist. We will determine: (i) G-protein coupling mediated by CPM interaction with the B1R vs. agonist binding. (ii) the involvement of -arrestin-2 recruitment in B1R agonist activation vs. CPM mediated allosteric enhancement . (iii) the downstream signaling output from the B1R via Gq and Gi mediated pathways in response to agonist or CPM interaction. (iv) the role of CPM in allosteric modulation of B1R signaling in regulating endothelial/vascular permeability. These studies will elucidate a novel mechanism for allosteric enhancement of B1R signaling by CPM which also generates its ligand. Understanding how this process can be regulated could lead to the development of novel drugs to treat renal and cardiovascular diseases. PUBLIC HEALTH RELEVANCE: We are investigating the interaction of two proteins, carboxypeptidase M and B1 receptor, on the cell surface that results in increased production of regulatory molecules that affect cell and organ function. The B1 receptor is known to regulate kidney and cardiovascular function, especially under conditions of infection or inflammation. Understanding how the interaction of these proteins can be enhanced or blocked could lead to the development of novel drugs to treat renal and cardiovascular diseases.