PROTEASE THAT PREVENTS APOPTOSIS IN QUIESCENT CELLS

A基因的功能鉴定、生化纯化及克隆 新的胞浆丝氨酸蛋白酶QPP(静止的脯氨酸二肽酶)， 阻止静止的淋巴细胞经历程序化的细胞 死亡(PCD)构成了细胞凋亡是 被一种活跃的机制阻断在静止的淋巴细胞中。QPP似乎是 对于静止的T和B细胞的生存至关重要，因为特定的 抑制该酶会激活细胞内的半胱氨酸氨基转移酶 PCD。这项提案的目标是证实这些耐人寻味的 观察并明确G/O淋巴细胞的凋亡途径。我。 直接分析QPP在显性负性淋巴细胞中的作用 (DN)变异体将通过突变其催化位点(S)来构建 它仍然结合其特定的底物，但不再切割它。 野生型和dN QPP构建物将在体外细胞系中表达 和原代T细胞，以及通过使用RAG-2在体内 胚泡发育补充系统。二、了解功能 QPP在PCD、ITS淋巴细胞保护中的意义 生理底物(S)必须确定。两种方法将是 使用：i)RDNQPP蛋白作为亲和基质提取底物 来自淋巴细胞裂解物；以及ii)具有DN的酵母-双杂交系统 以QPP为诱饵筛选淋巴细胞cDNA文库。这些蛋白质 这些方法确定的底物将被验证为QPP的底物 它们对野生型酶的敏感性，其次是N- 末端序列分析。三、初步数据显示， 阻断QPP在静止淋巴细胞中启动caspase级联反应 与其他特征明确的凋亡途径显著不同 在这些细胞中，如辐射或Fas诱导的PCD。放射性的 标记的zVADfmk，一种不可逆转的caspase抑制剂，将被用作 活性位点导向亲和剂与2D凝胶联合使用 分析，确定涉及的半胱氨酸天冬氨酸酶(S)。IV.所有电池都是这样测试的 Far含有一种在其印迹中类似于QPP的蛋白酶活性，即， 在所有组织中表达的1.7kB的条带和2.5kB的条带 这主要见于淋巴样细胞和胰腺。此外， 对QPP EST进行测序发现含有内部序列的克隆 差距。因此，将确定该酶是否存在差异 以组织特有的方式进行剪接和/或差异化修饰。 综上所述，这些研究将为 维持免疫系统中静止淋巴细胞的动态平衡。

The functional identification, biochemical purification and cloning of a novel cytosolic serine protease, QPP (Quiescent Proline di-Peptidase), which prevents quiescent lymphocytes from undergoing programmed cell death (PCD) forms the basis for the working hypothesis that apoptosis is blocked in resting lymphocytes by an active mechanism. QPP seems to be essential for the survival of resting T and B cells, because specific inhibition of this enzyme leads to activation of cellular caspases and PCD. The goals of this proposal are to substantiate these intriguing observations and to define the apoptosis pathway in G/o lymphocytes. I. to directly analyze the role of QPP in lymphocyte, a dominant negative (DN) variant will be constructed by mutating its catalytic site(s) such that it still binds its specific substrate, but no longer cleaves it. Wild type and DN QPP constructs will be expressed in vitro in cell lines and primary T cells, as well as in vivo by the use of the RAG-2 blastocyst ES complementation system. II. To understand the functional significance of QPP in the protection of lymphocytes from PCD, its physiological substrate(s) has to be identified. Two approaches will be used: i) rDN QPP protein as affinity matrix to extract the substrate from lymphocyte lysate; and ii) the yeast-two hybrid system with the DN QPP as bait for screening a lymphocyte cDNA library. The proteins identified by these methods will be verified as substrates of QPP by their susceptibility to cleavage by wild type enzyme, followed by N- terminal sequence analysis. III. Preliminary data indicate that the caspase cascade initiated in quiescent lymphocytes by blocking QPP differs significantly from other well characterized apoptotic pathways in these cells, such as irradiation- or Fas-induced PCD. Radioactively labeled zVADfmk, an irreversible caspase inhibitor, will be used as an active site-directed affinity reagent, in conjunction with 2D gel analysis, to identify the caspase(s) involved. IV. All cells tested so far contain a protease activity that resembles QPP in its blots, namely, a band of 1.7 kB that is expressed in all tissues and a band of 2.5 kB that is seen mainly in lymphoid cells and pancreas. Furthermore, the sequencing of QPP ESTs revealed clones that contain internal sequence gaps. Thus, it will be determined whether this enzyme is differentially spliced and/of differentially modified in a tissue specific manner. Taken together, these studies will provide new insights into the maintenance of homeostasis of resting lymphocytes in the immune system.