Structure and Function of Carboxypeptidases

羧肽酶的结构和功能

• 批准号: 6850824
• 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/6850824
• 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性羧肽酶在生理和病理过程中的重要作用。我们将强调的作用，膜结合羧肽酶（CP）M作为一个调节器的信号转导通过甘氨酸调节B1激肽受体如下。假设1：CPM是B1激肽系统的重要调节剂，它能够产生B1受体激动剂并有效地将其递送到膜上的受体。具体目标1：通过测定以下参数阐明CPM在产生des-Arg激肽以激活B1受体中的作用：（i）CPM和B1受体是否共定位于细胞表面;（ii）CPM产生des-Arg激肽B1激动剂并将其递送至受体并刺激相应信号的能力;（iii）B1应答与CPM水解细胞表面上的B2受体激动剂的能力的关系。假设二：CPM的C-末端甲状腺素运载蛋白样结构域含有一个独特的二硫键，该二硫键束缚酶，使其定向以改变对细胞表面底物的接近，并允许更有效地将产物递送至膜受体。具体目标二：确定CPM的C-末端结构域在调节其水解细胞膜上的肽底物、其定位和脱落中的作用。我们将：（i）表达CPM的重组形式，其中C-末端结构域被柔性系链取代，两个C-末端半胱氨酸已经突变，或者是与血管紧张素I转化酶的C-末端跨膜区的嵌合形式;（ii）对于每种形式的CPM，研究：大小为2至53个氨基酸的肽底物的动力学参数、抑制剂亲和力、最适pH和稳定性;（iii）确定从膜释放的速率和模式;（iv）确定对CPM的膜定位的影响。具体目标3：通过以下方法确定CPM的C-末端结构域对其产生des-Arg-激肽并将其递送至B1激肽受体的能力的功能重要性：（i）研究C-末端结构域突变对CPM和B1受体在膜上的共定位的影响;（二）通过测量B2受体激动剂和B1受体应答的增加来确定突变对其转化B2受体激动剂和产生B1受体应答的能力的影响，细胞内钙、花生四烯酸释放和一氧化氮产生。这些研究将提供新的信息CPM的结构和功能，可以参与生理和病理生理过程。例如，CPM调节缓激肽活性，其控制肾脏中的盐和水排泄，以及B1受体激动剂的产生，其被炎性细胞因子上调，导致延长的受体信号传导和重要的心血管介质如一氧化氮的产生。