ENDOGENOUS INHIBITORS OF ADAMS/MDC PROTEINASES

ADAMS/MDC 蛋白酶的内源抑制剂

• 批准号: 6097405
• 负责人: Jay William FOX
• 金额: $ 11.1万
• 依托单位: UNIVERSITY OF VIRGINIA
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-09-01 至 2002-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6097405
• 关键词: affinity chromatography; genetic library; human tissue; mass spectrometry; melanoma; metalloendopeptidases; protease inhibitor; serum; surface plasmon resonance; tissue /cell culture; yeast two hybrid system

A new group of proteins termed "ADAMS" (A Disintegrin And Metal Metalloproteinase protein" or "MDC" (Metalloproteinase Disintegrin Cysteine-rich protein) have recently been identified. This group of proteins has been implicated in several important biological processes including fertilization, development and cell surface shedding. Additional physiological roles will likely be identified for the various members of the group. ADAMS are type-1 integral membrane proteins having a multiple domain structure comprised of metalloproteinase, disintegrin-like, cysteine-rich, EGF-like, transmembrane and cytoplasmic domains. Most proteolytic enzyme systems have endogenous inhibitors that regulate the proteinases' biological regulation. Given the extensive distribution of the ADAMS, we hypothesize that there is a non-TIMP endogenous inhibitor/proteinase system for the metalloproteinase domain of the ADAMS. This hypothesis is supported by the identification of endogenous serum inhibitors of the snake venom metalloproteinases (homologs of the ADAMS) in opossum, woodrat, mongoose and certain snakes. We have preliminary data that supports the presence of a non-TIMP inhibitor(s) in human serum that inhibits ADAM 9, an ADAM family member we have cloned and sequenced from a human metastatic melanoma. We propose to isolate the ADAM proteinase inhibitor(s) from human serum and conditioned cell culture medium using conventional and affinity chromatography. The inhibitor will be partially sequenced by a combination of Edman and mass spectrometric techniques and the data used to clone and sequence its cDNA. The mechanism by which the inhibitor binds to ADAM 9 and blocks proteolytic activity and the kinetics of inhibition will be studied using surface plasmon resonance techniques. The structural requirements for proteinase inhibition by the inhibitor will be probed using reduced and alkylated inhibitor. Additionally, the inhibitor will be subjected to limited proteolysis and the fragments tested for inhibitory activity in order to identify regions of the inhibitor involved in proteinase interaction. Recombinant proteins representing the inhibitor and its domains will be expressed for functional studies. A second approach to identify ADAM 9 inhibitors will be to use a yeast two-hybrid screen. Characterization of ADAM binding proteins thus identified will be performed as described above. The discovery and characterization of endogenous inhibitors of ADAMs will mark a significant contribution to the understanding of the biology and biochemistry of the ADAMs. The broad-ranging biological attributes of members of the ADAMs family make it likely that pathologies associated with these proteins will be discovered in the near future. Isolation and characterization of these inhibits could therefore be of future importance in developing novel pharmacological agents for the regulation of ADAMs proteins' functions in pathological states.

一组新的蛋白质称为“亚当斯”（一种去整合素和金属金属蛋白酶蛋白质）或“MDC”（金属蛋白酶去整合素富含半胱氨酸蛋白质）最近已被鉴定。这组蛋白质涉及几个重要的生物过程，包括受精，发育和细胞表面脱落。 其他生理角色可能会被确定为该组的各个成员。 亚当斯是1型整合膜蛋白，具有由金属蛋白酶、去整合素样、富含半胱氨酸、EGF样、跨膜和胞质结构域组成的多结构域结构。大多数蛋白水解酶系统都有调节蛋白酶生物调节的内源性抑制剂。 鉴于亚当斯的广泛分布，我们假设亚当斯的金属蛋白酶结构域存在非TIMP内源性抑制剂/蛋白酶系统。 这一假设是支持的内源性血清抑制剂的蛇毒金属蛋白酶（同源物的亚当斯）负鼠，林鼠，猫鼬和某些蛇的鉴定。 我们有初步的数据支持在人血清中存在抑制ADAM 9的非TIMP抑制剂，ADAM 9是我们从人转移性黑素瘤中克隆和测序的ADAM家族成员。我们建议使用常规层析和亲和层析从人血清和条件细胞培养基中分离ADAM蛋白酶抑制剂。 将通过Edman和质谱技术的组合对抑制剂进行部分测序，并将数据用于克隆和测序其cDNA。 将使用表面等离子体共振技术研究抑制剂与ADAM 9结合并阻断蛋白水解活性和抑制动力学的机制。 将使用还原和烷基化抑制剂探索抑制剂对蛋白酶抑制的结构要求。此外，将对抑制剂进行有限的蛋白水解，并检测片段的抑制活性，以鉴定参与蛋白酶相互作用的抑制剂区域。 将表达代表抑制剂及其结构域的重组蛋白用于功能研究。 鉴定ADAM 9抑制剂的第二种方法是使用酵母双杂交筛选。 如上所述进行由此鉴定的ADAM结合蛋白的表征。 亚当斯内源性抑制剂的发现和表征将标志着对亚当斯生物学和生物化学的理解的重大贡献。 亚当斯家族成员的广泛生物学属性使得与这些蛋白质相关的病理学可能在不久的将来被发现。 因此，这些抑制剂的分离和表征在开发用于调节病理状态下的亚当斯蛋白功能的新型药理学试剂中可能具有未来的重要性。