PROTEASE THAT PREVENTS APOPTOSIS IN QUIESCENT CELLS

防止静止细胞凋亡的蛋白酶

• 批准号: 6362360
• 负责人: Brigitte T. Huber
• 金额: $ 29.31万
• 依托单位: TUFTS UNIVERSITY BOSTON
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-03-01 至 2003-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6362360
• 关键词: RNA splicing; apoptosis; cell cycle; cysteine endopeptidases; enzyme activity; enzyme substrate; gene expression; gene mutation; genetic library; genetically modified animals; human subject; interleukin 2; laboratory mouse; lymphocyte; nucleic acid sequence; polymerase chain reaction; posttranslational modifications; protease inhibitor; radiotracer; serine proteinases; tissue /cell culture; yeast two hybrid system

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.

一种新的抗肿瘤基因的功能鉴定、生化纯化和克隆 新的胞质丝氨酸蛋白酶，QPP（静止脯氨酸二肽酶）， 阻止静止的淋巴细胞进行细胞程序化 死亡（PCD）形成了工作假设的基础，即细胞凋亡是 通过主动机制在静息淋巴细胞中被阻断。QPP似乎是 对于静息T和B细胞的存活至关重要，因为特异性 这种酶的抑制导致细胞半胱天冬酶的激活， PCD。这项提案的目标是证实这些有趣的 观察并确定G/o淋巴细胞中的凋亡途径。I. 直接分析QPP在淋巴细胞中的作用， (DN)变体将通过突变其催化位点来构建， 它仍然结合它的特定底物，但不再切割它。 野生型和DN QPP构建体将在细胞系中体外表达 和原代T细胞，以及通过使用RAG-2 囊胚ES互补系统。二.了解功能 QPP在保护淋巴细胞免于PCD中的意义， 必须识别生理底物。两种方法将是 使用：i）rDN QPP蛋白作为亲和基质以提取底物 来自淋巴细胞裂解物;和ii）具有DN的酵母双杂交系统 QPP作为诱饵筛选淋巴细胞cDNA文库。的蛋白质 将通过以下方法验证这些方法鉴定的物质是否为QPP的底物： 它们对野生型酶切割的敏感性，然后是N- 末端序列分析三.初步数据显示， 通过阻断QPP在静止淋巴细胞中启动caspase级联反应 与其他表征良好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 标记的zVADfb 3，一种不可逆的半胱天冬酶抑制剂，将被用作 活性定点亲和试剂，结合2D凝胶 分析，以鉴定所涉及的半胱天冬酶。四.所有测试的细胞， 在其印迹中还含有类似于QPP的蛋白酶活性，即， 在所有组织中表达的1.7 kB条带和2.5 kB条带 主要见于淋巴细胞和胰腺。而且 QPP EST测序显示含有内部序列的克隆 差距。因此，将确定这种酶是否与 剪接和/或以组织特异性方式差异修饰。 总之，这些研究将提供新的见解， 维持免疫系统中静息淋巴细胞的稳态。