ENDOGENOUS INHIBITORS OF ADAMS/MDC PROTEINASES

最近发现了一组新的蛋白质，称为ADAMS(去整合素和金属金属蛋白酶蛋白)或MDC(金属蛋白酶去整合素半胱氨酸富含蛋白)。这组蛋白质参与了几个重要的生物学过程，包括受精、发育和细胞表面脱落。可能会为小组的不同成员确定额外的生理角色。ADAMS是由金属蛋白酶、去整合素样区、富含半胱氨酸、EGF样区、跨膜区和胞浆区组成的I型整合膜蛋白。大多数蛋白水解酶系统都有内源抑制物来调节蛋白水解酶的生物调节。鉴于ADAMS的广泛分布，我们假设ADAMS的金属蛋白酶结构域存在一个非TIMP内源性抑制物/蛋白酶系统。在负鼠、猫鼬、猫鼬和某些蛇中发现了蛇毒金属蛋白酶的内源性血清抑制物(亚当斯的同源物)，这支持了这一假说。我们有初步数据支持在人血清中存在抑制ADAM 9的非TIMP抑制物(S)，ADAM 9是我们从人类转移性黑色素瘤中克隆并测序的ADAM家族成员。我们建议用常规层析和亲和层析从人血清和条件细胞培养液中分离ADAM蛋白水解酶抑制剂(S)。该抑制物将通过结合EDMAN和质谱学技术以及用于克隆和测序其cDNA的数据进行部分测序。该抑制剂与ADAM 9结合并阻断蛋白分解活性的机制和抑制的动力学将用表面等离子体共振技术进行研究。将使用还原和烷基化的抑制剂来探讨该抑制剂抑制蛋白水解酶的结构要求。此外，将对抑制物进行有限的蛋白分解，并对片段进行抑制活性测试，以确定参与蛋白酶相互作用的抑制物区域。代表该抑制物及其结构域的重组蛋白将被表达用于功能研究。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，将进行这样鉴定的ADAM结合蛋白的鉴定。ADAMS内源性抑制物的发现和表征将标志着对ADAMS生物学和生物化学的理解做出了重大贡献。亚当斯家族成员广泛的生物学特性使得与这些蛋白质相关的病理学很有可能在不久的将来被发现。因此，分离和鉴定这些抑制物对于开发新的药理药物以调节ADAMS蛋白在病理状态下的功能具有重要意义。

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.