QPP: Protease that Prevents Apoptosis in Quiescent Cells

超出提供的空间。QPP(静止细胞脯氨酸二肽酶)(DPP7)是一种控制处于细胞周期GO期的淋巴细胞和神经细胞存活的蛋白酶，因为QPP抑制剂可以诱导这些细胞的凋亡。一、qpp突变小鼠的构建为确定qpp在体内的功能意义提供了直接的途径。将使用四个系统，以便对QPP在不同细胞类型和发育阶段中的作用进行广泛分析：(A)将分析传统的QPP-/-小鼠是否存在发育缺陷。(B)如果QPP-/-基因在胚胎上是致命的，将通过将胎儿肝脏移植到RAG-2-/-小鼠身上来测试淋巴发育。(C)如果qpp-/-基因型的致死性发生在淋巴干细胞发育之前，将使用RAG-2缺陷的囊胚互补系统产生嵌合体小鼠，该系统仅允许纯合qpp突变在淋巴系中表达。(D)为了确定QPP在成熟细胞和单个器官中的作用，将利用Cre-loxP重组系统对条件QPP Ko小鼠进行工程改造，该系统可控制纯合子突变的表达。I1.体外抑制QPP的表达，以确定QPP在人淋巴细胞和神经细胞中的作用。将采用四个系统：(A)由于QPP蛋白酶活性需要二聚化，因此将筛选酶活性部位突变的显性负活性。(B)RNA干扰(RNAi)将用于沉默QPP转录本。(C)将测试反义寡核苷酸以阻断原代淋巴细胞和神经细胞中QPP蛋白的表达。(D)将重组腺病毒中的全长反义QPP基因导入人和小鼠细胞系和原代细胞，以阻断QPPo II1的表达。我们将对QPP底物(S)进行表征，以了解QPP诱导的GO细胞存活的机制和/或途径。(A)根据工作假设，LKLF和神经营养因子以及新的候选物质将被筛选为QPP的潜在底物。(B)为了确认QPP在体外对候选底物的切割在功能上是重要的，将进行共定位实验。总而言之，这些研究将有助于更好地理解体内和体外真核细胞中构成GO的生存程序。此外，它们还将为分析静止细胞中默认的PCD途径提供一个工具。表演网站========================================Section End===========================================

EXCEED THE SPACE PROVIDED. QPP (Quiescent cell Proline di-Peptidase) (DPP7) is a protease hypothesized to control the survival of lymphocytes and neuronal cells that are in the GO stage of cell cycle, because QPP inhibitors induce apoptosis in these cells. I. The construction of QPP mutant mice provides a direct approach for defining the functional significance of QPP in vivo. Four systems will be used which will allow a broad analysis of the role of QPP in various cell types and stages of development: (a) Conventional QPP-/- mice will be analyzed for defects in development. (b) If the QPP-/- genotype is embryonically lethal, lymphoid development will be tested by fetal liver transfer into RAG-2-/- mice. (c) If lethality of the QPP-/- genotype occurs before lymphoid stern cell development, chimeric mice will be generated, using the RAG-2-deficient blastocyst complementation system that allows expression of the homozygous QPP mutation in the lymphoid lineage only. (d) To define the role of QPP in mature cells and individual organs, conditional QPP ko mice will be engineered, using the Cre-loxP recombination system that provides control of expression of the homozygous mutation. I1. Inhibition of QPP expression in vitro will be carried out to define the role of QPP in human lymphocytes and neuronal cells. Four systems will be employed: (a) Since the QPP protease activity requires dimerization, enzyme active site mutants will be screened for dominant negative activity. (b) RNA interference (RNAi) will be used for silencing QPP transcripts. (c) Antisense oligos will be tested for blocking QPP protein expression in primary lymphocytes and neuronal cells. (d) A full-length antisense QPP cDNA in a recombinant adenovirus will be introduced into human and mouse cell lines and primary cells to block expression of QPPo II1. Characterization of QPP substrate(s) will be carried out to understand the mechanism and/or pathway of QPP-rnediated survival of GO cells. (a) Based on the working hypotheses, LKLF and neurotrophins, as well as novel candidates, will be screened as potential substrates of QPP. (b) To confirm that cleavage of a candidate substrate by QPP in vitro is functionally significant, co-localization experiments will be carried out. Collectively, these studies will lead to a better understanding of the constitutive GO survival program in eukaryotic cells in vivo and in vitro. In addition, they will provide a tool for the analysis of the default PCD pathway in quiescent cells. PERFORMANCE SITE ========================================Section End===========================================