QPP: Protease that Prevents Apoptosis in Quiescent Cells

说明(申请人提供)：QPP(静止细胞脯氨酸二肽酶)，也被称为DPP2/7，是一种囊泡性蛋白酶，能将二肽从蛋白质的N端裂解，从而影响它们的半衰期和/或受体特异性。虽然这种酶的完整功能还远未被了解，但体外实验表明，QPP是原代淋巴细胞和神经细胞G0静止程序的主要调节因子。这一更新应用的主要目的是分析QPP介导的静息淋巴细胞存活的机制。目的I：确定QPP功能的最直接方法是产生QPP缺陷的突变小鼠。由于先前获得常规QP-/-小鼠的尝试受到胚胎致死性的限制，我们提出了一种新的替代方案来产生突变小鼠，利用Cre/IoxP重组酶系统，允许有条件的和可诱导的基因组改变。目的II：抑制QPP使静止的淋巴细胞进入细胞周期，导致c-Myc和P53表达上调，最终诱导细胞凋亡。因此，推测QPP酶活性是维持GO程序所必需的。为了验证这一假说，我们将描述QPP的表达调控和作用机制。将特别强调LKLF和STAT5，这两个转录因子分别参与QPP表达的正向和负向调控。此外，还将分析c-Myc和P53在细胞凋亡途径中的作用。目的III：为了全面理解真核细胞中的G0生存程序，必须确定QPP的生理底物(S)。由于这是最具雄心的计划，将采用各种方法来实现这一目标，包括筛选组合二肽底物文库，人和小鼠QPP与DPP4的结构比较，以及通过亲和矩阵层析分离生理底物(S)。总而言之，这些研究将增强我们对真核细胞中构成GO生存计划的有限理解。

DESCRIPTION (provided by applicant): QPP (Quiescent cell Proline di-Peptidase), also termed DPP2/7, is a vesicular protease that cleaves dipeptides from the N-terminus of proteins, thereby impacting their half-life and/or receptor specificity. While the full functional profile of this enzyme is far from understood, in vitro experiments demonstrate that QPP is a major regulator of the G0 quiescence program in primary lymphocytes and neuronal cells. The primary objective of this renewal application is to analyze the mechanism of QPP-mediated survival in resting lymphocytes. Aim I: The most direct approach to define the functional role of QPP is to generate a mutant mouse deficient in QPP. Since previous attempts to derive conventional QP-/- mice were curbed by embryonic lethality, we propose a novel alternative to generate the mutant mouse, utilizing the Cre/IoxP recombinase system that allows conditional and inducible genome alterations. Aim II: Inhibition of QPP advances quiescent lymphocytes into cell cycle, leading to upregulation of c- Myc and p53 and finally apoptosis induction. Thus, it is postulated that QPP enzyme activity is required for maintaining the Go program. To test this hypothesis, the control of expression and mechanism of action of QPP will be delineated. Special emphasis will be given to LKLF and STAT5, two transcription factors that are implicated in positive and negative regulation of QPP expression, respectively. Furthermore, the role of c-Myc and p53 in the apoptosis pathway will be analyzed. Aim III: To fully understand the G0 survival program in eukaryotic cells, it is essential to define the physiological substrate(s) of QPP. Since this is the most ambitious project, various approaches will be used to reach this goal, including the screen of combinatorial dipeptide substrate libraries, the structural comparison of human and murine QPP with that of DPP4, and the isolation of a physiological substrate(s) by affinity matrix chromatography. Collectively, these studies will augment our limited understanding of the constitutive Go survival program in eukaryotic cells.