DISRUPTION AND EXPRESSION OF MAST CELL PROTEASE GENES

描述（改编自申请人的摘要）：小鼠肥大细胞（MC）表达 至少12种丝氨酸蛋白酶（命名为小鼠MC 蛋白酶（mMCP）1至10、组织蛋白酶G和颗粒酶B）和外肽酶 （命名为小鼠MC羧肽酶A（mMC-CPA），其具有酶活性 尽管这些颗粒蛋白酶及其人类同源物具有 对于鉴定组织中不同的MC群体以及 了解MC的发展，大部分的生物基质， 蛋白酶还有待确定。据推测， 每个MC表达的蛋白酶与其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活化。虽然许多 MCP彼此高度同源，每种蛋白酶具有独特组 在其底物结合裂缝中的氨基酸。cDNA的克隆和 编码各种小鼠MC蛋白酶及其人类同源物的基因现在 允许使用补充方法来解决其功能， 新陈代谢.在特定目标1中，17号染色体复合体，其中小鼠MC 类胰蛋白酶基因的存在，将被映射和测序，以确定其余的 复合体中的蛋白酶基因。将产生转基因小鼠， 这些类胰蛋白酶基因中的某些被破坏， 基因对心脏、肺、子宫中MC的发育和功能的影响， 其他器官也将接受评估。例如，功能效果将 在肺中通过确定是否MC细胞活化来评估， 特定的类胰蛋白酶无效小鼠使气道对增强的应答准备就绪， 特异性激动剂。在特定目标2中，重组小鼠和人胰蛋白酶将 以评估其底物特异性，并确定 它们在体内失活和代谢的机制。最后 重组胰蛋白酶将用于获得低分子量抑制剂 对每种蛋白酶都有特异性。

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.