Structure and Function of Carboxypeptidases

羧肽酶的结构和功能

• 批准号: 6775417
• 负责人: Randal A Skidgel
• 金额: $ 32.97万
• 依托单位: UNIVERSITY OF ILLINOIS AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-07-01 至 2009-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6775417
• 关键词: CHO cells; MDCK cell; arachidonate; bradykinin; calcium ion; carboxypeptidase; cell membrane; cell surface receptors; chemical kinetics; chimeric proteins; enzyme mechanism; enzyme structure; hydrolysis; nitric oxide; plasminogen; protein localization; protein sequence; protein structure function; protein transport; recombinant proteins; stimulant /agonist; tissue /cell culture

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.

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重要作用。我们将强调膜结合羧基肽酶（CP） M作为细胞因子调节的B1激肽受体信号的调节剂的作用如下。假设1:CPM通过产生B1受体激动剂并有效地将其传递到膜上的受体，是B1激肽系统的重要调节剂。具体目的1：通过确定(i) CPM和B1受体是否在细胞表面共定位，阐明CPM在产生去精氨酸激肽激活B1受体中的作用；（ii） CPM产生和传递des-Arg kinin B1激动剂到受体并刺激相应信号的能力；（iii） B1反应与CPM在细胞表面水解B2受体激动剂能力的关系。假设2:CPM的c端转甲状腺素样结构域包含一个独特的二硫键，该键连接酶，定向其改变对细胞表面底物的访问，并允许更有效地将产物递送到膜受体。具体目标2：确定CPM的c端结构域在调节其对细胞膜上肽底物的水解、定位和脱落中的作用。我们将：(i)表达重组形式的CPM，其中c端结构域被柔性系链取代，两个c端半胱氨酸已经突变或与血管紧张素i转换酶的c端跨膜区嵌合形式；（ii）研究每种形式的CPM：从2到53个氨基酸大小的肽底物的动力学参数，抑制剂亲和力，最佳pH值和稳定性；（iii）确定从膜上释放的速率和方式；（iv）确定对CPM膜定位的影响。具体目标3：确定CPM的c端结构域对其产生和传递去精氨酸激肽到B1激肽受体的能力的功能重要性：(i)研究c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、花生四烯酸释放和一氧化氮生成的增加，确定突变对其转化B2激动剂和产生B1受体反应的能力的影响。这些研究将为CPM的结构和功能提供新的信息，CPM可能参与生理和病理生理过程。例如，CPM调节缓激肽活性，它控制肾脏中的盐和水的排泄，以及B1受体的激动剂的产生，B1受体被炎症细胞因子上调，导致受体信号传导延长，并产生重要的心血管介质，如一氧化氮。