FUNCTIONS OF RB PROTEASE(S) IN APOPTOSIS

描述（改编自研究者摘要）： 磷酸化（p110/pRB）形式，但非磷酸化（pI 2 O/ppRB）形式 视网膜母细胞瘤蛋白（RB）作为一种内源性抑制剂， 增殖、分化和衰老。 最近， 据报道，Rb基因的缺失也与不适当的 透镜纤维细胞的凋亡（或程序性细胞死亡）。 但如何 RB蛋白在细胞凋亡过程中的作用尚不清楚。 的 研究人员发现，在诱导细胞凋亡的过程中， 通过各种抗癌剂（例如，VP-16）在人早幼粒细胞 白血病HL 60细胞p110/pRB表达增高， 实质性改变。 与此相反，p120/ppRB迅速减少， 在化疗诱导的细胞凋亡开始时， 首先通过诱导RB的另一种磷酸化形式p115/ppRB， 然后是几种较小的磷酸化的RB相关蛋白质， 包括p68和p48。 他们提出了一个假设，即ppRB- 特异性蛋白酶（ppRB-PTS）由多种 抗癌药物在细胞凋亡开始时， ppRB-PTS和随后的p12 O/ppRB降解可能起积极作用 调节细胞凋亡。 因此，广泛、长期 目的探讨ppRB-PTS在细胞凋亡中的作用。 的 这项建议的具体目标如下：1）制定一项 体外测定用于检测ppRB-PTS活性。 所述药物诱导的 ppRB-PTS活性将通过孵育底物在体外检测 蛋白质，p115/ppRB，从代谢性 [32 P]正磷酸盐标记的VP-16处理的HL 60细胞，含全细胞 从未标记的VP-16处理的细胞中提取，然后检测 生产[32 P]-标记的p68和p48; 2）纯化ppRB-PTS 并克隆其基因。 ppRB-PTS将通过使用 FPLC、RB抗体和切割位点的色谱柱- 含有合成的寡肽，然后进行凝胶洗脱。 的 将纯化的ppRB-PTS进行测序，并将其cDNA探针和抗体 将被生成。 编码ppRB-PTS的基因将用 其cDNA探针和/或抗体; 3）对主要ppRB-PTS切割进行测序 地点在RB。 p68和其他ppRB片段将使用 RB抗体亲和柱和凝胶洗脱。 纯化的多肽的序列 将片段与真实的人RB蛋白的片段进行比较，并且 将确定至少一个ppRB-PTS切割位点; 4）研究 p120/ppRB降解在化疗诱导的细胞凋亡中的作用。 ppRB-PTS基因将在确定的细胞中转染和表达 line，并研究其对细胞凋亡的影响。 在 此外，将测试含有ppRB-PTS切割位点的 寡肽可用作细胞凋亡的抑制剂， bcl-2癌蛋白过表达是否能阻断细胞凋亡， 相关的ppRB降解。 这些研究应该提供洞察力， ppRB降解的分子机制，并可能提供 为开发旨在激活的新疗法奠定了基础 ppRB-PTS在体内的功能。

DESCRIPTION (adapted from investigator's abstract): The under- phosphorylated (p110/pRB), but not the phosphorylated (pI2O/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. The investigators 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 one of several smaller phosphorylated, RB-related proteins, including p68 and p48. They 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 p12O/ppRB may play an active role in regulating cellular apoptosis. Therefore, the broad, long-term objective is to define the function of ppRB-PTS in apoptosis. The Specific Aims of this proposal are the 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 at activating the ppRB-PTS function in vivo.