ENDOGENOUS INHIBITORS OF ADAMS/MDC PROTEINASES

最近发现了一组新的蛋白质，称为“ADAMS”（A Disintegrin And Metal Metalloproteinase protein）或“MDC”（Metalloproteinase Disintegrin半胱氨酸富蛋白）。这组蛋白参与了几个重要的生物过程，包括受精、发育和细胞表面脱落。额外的生理作用可能会被确定为群体的不同成员。ADAMS是1型整体膜蛋白，具有多结构域结构，包括金属蛋白酶、崩解素样结构域、富含半胱氨酸、egf样结构域、跨膜结构域和细胞质结构域。大多数蛋白水解酶系统都有内源性抑制剂来调节蛋白酶的生物学调节。鉴于ADAMS的广泛分布，我们假设ADAMS的金属蛋白酶结构域存在非timp内源性抑制剂/蛋白酶系统。在负鼠、木鼠、猫鼬和某些蛇类中发现了内源性蛇毒金属蛋白酶（ADAMS的同源物）的血清抑制剂，支持了这一假设。我们有初步数据支持在人血清中存在抑制ADAM 9的非timp抑制剂，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。我们提出用常规和亲和色谱法从人血清和条件细胞培养基中分离ADAM蛋白酶抑制剂。该抑制剂将通过结合Edman和质谱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adam9结合并阻断蛋白水解活性的机制和抑制动力学将使用表面等离子体共振技术进行研究。用还原性和烷基化抑制剂来探索抑制剂对蛋白酶抑制的结构要求。此外，抑制剂将进行有限的蛋白水解，并测试片段的抑制活性，以确定抑制剂参与蛋白酶相互作用的区域。代表该抑制剂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。因此鉴定的ADAM结合蛋白的表征将按照上述方法进行。ADAMs内源性抑制剂的发现和表征将对ADAMs生物学和生物化学的理解做出重大贡献。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在不久的将来可能会被发现。因此，这些抑制物的分离和表征可能对未来开发用于调节病理状态下ADAMs蛋白功能的新型药理药物具有重要意义。

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.