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性羧基肽酶在生理和病理过程中的重要作用。我们将强调膜结合的羧基肽酶(CP)M通过细胞因子调节的B1激动素受体作为信号调节器的作用如下。假设1：CPM通过产生B1受体激动剂并有效地将其运送到膜上的受体，是B1激肽系统的重要调节因子。具体目的1：通过确定CPM和B1受体是否共定位于细胞表面，阐明CPM在产生Des-Arg激动素以激活B1受体中的作用；(Ii)CPM产生Des-Arg激动素B1激动剂并将其传递到受体并刺激相应信号的能力；(Iii)B1反应与CPM水解细胞表面B2受体激动剂的能力的关系。假设2：CPM的C末端反式甲状腺素样结构域包含一个独特的二硫键，该键连接酶，使其能够改变对细胞表面底物的访问，并允许更有效地将产物输送到膜受体。具体目标2：确定CPM的C-末端结构域在调节其在细胞膜上的多肽底物的水解、定位和脱落中的作用。我们将：(I)表达重组形式的CPM，其中C-末端的结构域被灵活的系链取代，两个C-末端的半胱氨酸已经突变，或者与血管紧张素转换酶的C-末端跨膜区形成嵌合形式；(Ii)对于每种形式的CPM，研究：大小从2到53个氨基酸的多肽底物的动力学参数、抑制剂的亲和力、最适pH和稳定性；(Iii)确定膜的释放速度和方式；(Iv)确定对CPM膜定位的影响。具体目标3：通过以下方法确定CPM C-末端结构域对其产生和运送Des-Arg-kinins到B1激动素受体能力的功能重要性：(I)研究C-末端结构域突变对CPM和B1受体在膜上共定位的影响；(Ii)通过测量细胞内钙离子、花生四烯酸释放和一氧化氮产生的增加来确定突变对其转化B2激动剂和产生B1受体反应能力的影响。这些研究将为参与生理和病理生理过程的CPM的结构和功能提供新的信息。例如，CPM调节缓激肽的活性，控制肾脏中的盐和水的排泄，并产生B1受体激动剂，后者被炎性细胞因子上调，导致受体信号的延长和重要心血管介质如一氧化氮的产生。

DESCRIPTION (provided by applicant): Our long-term objective is to understand the important roles of regulatory carboxypeptidases in physiological and pathological processes. We will emphasize the role of membrane-bound carboxypeptidase (CP) M as a regulator of signaling through cytokine-regulated B1 kinin receptor as follows. Hypothesis 1: CPM is an important regulator of the B1 kinin system via its ability to generate the B1 receptor agonist and efficiently deliver it to the receptor on the membrane. Specific Aim 1: Elucidate the role of CPM in generating des-Arg kinins for activation of B1 receptors by determining: (i) whether CPM and the B1 receptor are co-localized on the cell surface; (ii) the ability of CPM to generate and deliver the des-Arg kinin B1 agonists to the receptor and stimulate a corresponding signal; (iii) the relationship of the B1 response to the ability of CPM to hydrolyze B2 receptor agonists on the cell surface. Hypothesis 2: The C-terminal transthyretin-like domain of CPM contains a unique disulfide bond that tethers the enzyme, orienting it to alter access to substrates on the cell surface and allow more efficient delivery of products to membrane receptors. Specific Aim 2: Determine the role of the C-terminal domain of CPM in regulating its hydrolysis of peptide substrates on the cell membrane, its localization and shedding. We will: (i) express recombinant forms of CPM in which the C-terminal domain is replaced with a flexible tether, the two C-terminal cysteines have been mutated or a chimeric form with the C-terminal transmembrane region of angiotensin I converting enzyme; (ii) investigate, for each form of CPM: kinetic parameters for peptide substrates ranging in size from 2 to 53 amino acids, inhibitor affinity, pH optimum and stability; (iii) determine the rate and mode of release from the membrane; (iv) determine the effect on CPM's membrane localization. Specific Aim 3: Determine the functional importance of the C-terminal domain of CPM on its ability to generate and deliver des-Arg-kinins to the B1 kinin receptor by: (i) investigating the effect of C-terminal domain mutations on the co-localization of CPM and B1 receptor on the membrane; (ii) determining the effect of mutations on its ability to convert B2 agonists and generate the B1 receptor responses by measuring increases in intracellular calcium, arachidonic acid release and nitric oxide production. These studies will provide novel information on the structure and function of CPM that can be involved in physiological and pathophysiological processes. For example, CPM regulation of bradykinin activity, which controls salt and water excretion in the kidney, and generation of agonists for the B1 receptor, which is upregulated by inflammatory cytokines, resulting in prolonged receptor signaling and production of important cardiovascular mediators such as nitric oxide.