FUNCTIONS OF PPRB-SPECIFIC PROTEASES IN APPOTOSIS

这是一个香农奖提供部分支持的研究 项目不属于指定的研究所的资金范围，但 都处于优秀的边缘。 香农奖旨在 提供支持，以测试该方法的可行性;进一步发展 测试和完善研究技术;进行二次分析 可用的数据集;或进行离散项目， PI的研究能力或为已经存在的 有价值的申请。 下面的摘要摘自原文 主要研究者提交的文件。 磷酸化不足的（p110/pRB），但不是磷酸化的 (p120/ppRB），视网膜母细胞瘤蛋白（RB）的形式作为内源性的 细胞增殖、分化和衰老抑制剂。 最 最近有报道称Rb基因缺失也与 在透镜纤维中存在不适当的细胞凋亡（或程序性细胞死亡） 细胞 然而，RB蛋白在细胞凋亡中的作用仍然是未知的。 未知 我们发现，在细胞凋亡过程中， 各种抗癌剂（例如，VP-16）在人早幼粒细胞白血病中的作用 HL 60细胞中p110/pRB表达水平高，无明显变化。 与此相反，p120/ppRB在开始时迅速降低， 化疗诱导的细胞凋亡，随后首先诱导 RB的另一种磷酸化形式，p115/ppRB，然后通过几种 较小的磷酸化RB相关蛋白，包括p68和p48。 我们 已经发展了一种假说，即ppRB特异性蛋白酶（ppRB-PTS） 在发病时由多种抗癌药物诱导 细胞凋亡，以及ppRB-PTS的激活和随后的降解 p120/ppRB的表达可能在细胞凋亡的调控中起积极作用。 因此，我们广泛的长期目标是确定 ppRB-PTS在细胞凋亡中的作用。 该提案的具体目标如下。 (1)建立检测ppRB-PTS活性的体外方法。 药物诱导的ppRB-PTS活性将在体外通过 孵育由以下物质制备的底物蛋白p115/ppRB 代谢[32 P]正磷酸标记的免疫沉淀物，VP-16- 处理的HL 60细胞，用来自未标记的VP-16- 处理的细胞，然后检测[32 P]-标记的p68和 第48页。 (2)纯化ppRB-PTS蛋白并克隆其基因。 ppRB-PTS 将通过使用FPLC、RB抗体的层析柱进行纯化 和含有切割位点的合成寡肽，然后用凝胶 洗脱 将纯化的ppRB-PTS进行测序，并将其cDNA探针和 将产生抗体。 将克隆编码ppRB-PTS的基因 利用其cDNA探针和/或抗体。 (3)对一个主要的ppRB进行测序- PTS切割位点位于RB上。 p68和其他ppRB片段将被 使用RB抗体亲和柱和凝胶洗脱纯化。 序列 将纯化的片段与真实的人的片段进行比较。 RB蛋白和至少一个ppRB-PTS切割位点。 (4)研究p120/ppRB降解在化疗中的作用。 诱导凋亡。 将ppRB-PTS基因转染并表达 在确定的细胞系中，对细胞凋亡的影响将是 研究了 此外，将测试ppRB-PTS切割是否 含有位点的寡肽可用作细胞凋亡的抑制剂， 凋亡，以及bcl-2癌蛋白的过表达是否可以阻断细胞凋亡。 中毒相关ppRB降解。 这些研究将提供 深入了解ppRB降解的分子机制， 为开发新的治疗方法提供基础， ppRB-PTS在体内的功能。

This is a Shannon Award providing partial support for the research projects that fall short of the assigned institute's funding range but are in the margin of excellence. The Shannon Award is intended to provide support to test the feasibility of the approach; develop further tests and refine research techniques; perform secondary analysis of available data sets; or conduct discrete projects that can demonstrate the PI's research capabilities or lend additional weight to an already meritorious application. The abstract below is taken from the original document submitted by the principal investigator. The underphosphorylated (p110/pRB), but not the phosphorylated (p120/ppRB), form of retinoblastoma protein (RB) serves as an endogenous inhibitor of cell proliferation, differentiation and senescence. Most recently, it has been reported that loss of Rb gene is also associated with inappropriate apoptosis (or programmed cell death) in lens fibre cells. However, how RB protein functions during apoptosis is still unknown. We have found that during the process of apoptosis induced by various anticancer agents (e.g., VP-16) in human promyelocytic leukemia HL60 cells, the level of p110/pRB was high and not substantially changed. In contrast, p120/ppRB was rapidly reduced just at the onset of chemotherapy-induced apoptosis, which were followed first by induction of another phosphorylated form of RB,p115/ppRB, and then by of several smaller phosphorylated, RB-related proteins, including p68 and p48. We have developed a hypothesis that a ppRB-specific protease(s) (ppRB-PTS) is/are induced by a variety of anticancer drugs at the onset of apoptosis, and that the activation of ppRB-PTS and consequent degradation of p120/ppRB may play an active role in regulating cellular apoptosis. Therefore, our broad, long-term objective is to define the function of ppRB-PTS in apoptosis. The Specific Aims of this proposal are following. (1) To develop an in vitro assay for detection of the ppRB-PTS activity. The drug-induced ppRB-PTS activity will be detected in vitro by incubating a substrate protein, p115/ppRB, prepared from immunoprecipitates of metabolically [32P]orthophosphate-labeled, VP-16- treated HL60 cells, with a whole cell extract from unlabeled, VP-16- treated cells, followed by detecting production of [32P]-labeled p68 and p48. (2) To purify the ppRB-PTS protein and clone its gene. ppRB-PTS will be purified by using chromatographic columns of FPLC, RB antibody and the cleavage site-containing synthetic oligopeptides, followed by gel elution. The purified ppRB-PTS will be sequenced, and its cDNA probe and antibody will be generated. The gene encoding ppRB-PTS will be cloned by using its cDNA probe and/or antibody. (3) To sequence a major ppRB- PTS cleavage site located on RB. p68 and other ppRB fragments will be purified using RB antibody affinity columns and gel elution. Sequences of the purified fragments will be compared to that of the authentic human RB protein, and at least one ppRB-PTS cleavage site will be determined. (4) To study functions of the p120/ppRB degradation in chemotherapy- induced apoptosis. The ppRB-PTS gene will be transfected and expressed in a defined cell line, and the effects to cellular apoptosis will be studied. In addition, it will be tested whether the ppRB-PTS cleavage site-containing oligopeptides can be used as an inhibitor of cellular apoptosis, and whether overexpression of bcl-2 oncoprotein can block the apoptosis-associated ppRB degradation. These studies should provide insight into the molecular mechanisms of the ppRB degradation, and may provide a basis for the development of novel therapies aimed t activating the ppRB-PTS function in vivo.