DISRUPTION AND EXPRESSION OF MAST CELL PROTEASE GENES

描述(改编自申请者摘要)：小鼠肥大细胞(MC) 至少12种丝氨酸蛋白酶的不同组合(指定为小鼠MC 蛋白酶(MMCP)1至10、组织蛋白酶G和颗粒酶B)和外肽酶 (指定为具有酶活性的小鼠MC羧肽酶A(MMC-CPA) 在中性pH条件下。尽管这些颗粒蛋白水解酶和它们的人类同源物 对于识别组织中不同的MC群体和 了解MC开发，大多数情况下的生物基质 蛋白水解酶还没有确定。据推测，病毒的数量和类型 每个MC所表达的蛋白与其所表达的蛋白质的数量和类型有关 必须在特定的组织环境中降解或激活。尽管许多人 MCP彼此高度同源，每个蛋白酶都有唯一的一组 底物结合缝隙中的氨基酸。CDNA和DNA的克隆 现在编码各种小鼠MC蛋白水解酶及其人类同源物的基因 允许使用互补的方法来解决其功能和 新陈代谢。在特定的目标1中，17号染色体复合体，其中小鼠MC 类胰蛋白酶基因驻留，将被绘制和测序，以确定剩余的 复合体中的蛋白酶基因。将产生的转基因小鼠具有 这些类胰蛋白酶基因中的某些基因被破坏，而去除这种基因的后果 心、肺、子宫中巨噬细胞发育和功能的基因， 和其他器官将被评估。例如，功能效果将是 通过确定MC细胞在肺中的激活情况来评估 特定的类胰蛋白酶缺失的小鼠为增强对 特定的激动剂。在特定目标2中，重组鼠和人类胰酶将 以评估它们的底物特性，并确定 它们在体内失活和代谢的机制。最后， 重组类胰蛋白酶将用于获得低分子量抑制剂 每种蛋白水解酶都是特定的。

DESCRIPTION (adapted from applicant's abstract): Mouse mast cells (MCs) express varied combinations of at least 12 serine proteases (designated mouse MC protease (mMCP) 1 to 10, cathepsin G, and granzyme B) and an exopeptidase (designated mouse MC carboxypeptidase A (mMC-CPA) that are enzymatically active at neutral pH. Although these granule proteases and their human homologues have been invaluable for identifying distinct populations of MCs in tissues and for understanding MC development, the biological substrates for most of the proteases have yet to be determined. Presumably, the number and type of proteases each MC expresses are related to the number and type of proteins it must degrade or activate in a particular tissue environment. Although many of the MCPs are highly homologous with one another, each protease has a unique set of amino acids in its substrate-binding cleft. The cloning of the cDNAs and genes that encode the varied mouse MC proteases and their human homologues now allows the use of complementary approaches to address their function and metabolism. In Specific Aim 1, the chromosome 17 complex, where the mouse MC tryptase genes reside, will be mapped and sequenced to identify the remaining protease genes in the complex. Transgenic mice will be generated that have certain of these tryptase genes disrupted and the consequences of ablating such genes on the development and function of the MCs in the heart, lung, uterus, and other organs will be assessed. For example, functional effects will be assessed in the lung by determining whether or not MC cell activation in a particular tryptase-null mouse primes the airways for augmented response to a specific agonist. In Specific Aim 2, recombinant mouse and human tryptases will be generated to evaluate their substrate specificities, and to determine the mechanisms by which they are inactivated and metabolized in vivo. Finally, the recombinant tryptases will be used to obtain low molecular weight inhibitors that are specific for each protease.