CHARACTERIZATION OF TWO NOVEL BCL-2-ASSCOCIATED PROTEINS

超过85%的人滤泡性B细胞淋巴瘤表现为 T(14；18)染色体易位，导致表达升高 Bcl-2癌蛋白的表达。BCL-2已被证明可以阻断细胞的凋亡 多种造血细胞和神经细胞类型，可能有助于 滤泡性淋巴瘤是由这种机制引起的。试图设计出 基于阻断Bcl-2功能的治疗受到以下因素的阻碍 目前对Bcl2的分子机制知之甚少 行动。我们广泛的、长期的目标是确定Bc l-1的机制。 2作用，通过表征Bcl-2与其他细胞的相互作用 蛋白质。 在初步研究中，我们克隆了两个编码新基因的cDNA BAP1和BAP2，在体外与Bcl2结合。BAP1和BAP2是 来自不同的，但相关的基因，并定义了一个新的家族 BCL-2相关蛋白。BAP2蛋白在沙门氏菌中的强制表达 白介素3(IL-3)依赖的细胞株加速细胞凋亡 停用IL-3后。因此，BAP蛋白可能扮演着重要的角色 在细胞凋亡中的作用。这项建议的具体目的是：1) BAP1和BAP2基因在正常小鼠中的表达特征 组织和已定义的造血细胞系；2)研究物理 Bcl-2与BAP1、BAP2蛋白相互作用的研究 3)研究BAP1、BAP1和BAP1的功能。 BAP2以阻断Bcl-2功能；以及4)研究物理相互作用 BAP1和BAP2蛋白与Bcl2蛋白其他成员之间的关系 家族，包括Bax、BclXL、BclXS和Mcl-1。这些研究可能 为靶向阻断Bcl2功能的新疗法提供基础 活着。 BAP1和BAP2蛋白在小鼠正常组织和正常组织中的表达 已确定的造血细胞系将通过免疫组织化学进行研究 用产生的多克隆抗血清进行Western blotting 重组蛋白。Northern印迹和原位杂交将 用于研究mRNA的表达。调查潜在的细胞内 蛋白质之间的相互作用，我们将在BAP1或BAP2中共表达Bcl2 IL-3依赖的细胞株，并确定Bcl2是否可以特异性地 与BAP1或BAP2免疫共沉淀。同时，我们将调查 强迫表达BAP1或BAP2对Bcl2表达能力的影响 抑制IL-3停用后的细胞凋亡。调查 BAP蛋白与Bcl2其他成员的相互作用 蛋白质家族将在体外进行，使用纯化的重组 蛋白质。

Greater than 85% percent of human follicular B cell lymphomas exhibit a t(14;18) chromosomal translocation, which results in elevated expression of the Bcl-2 oncoprotein. Bcl-2 has been shown to block apoptosis in a variety of hematopoietic and neuronal cell types and may contribute to follicular lymphomas by this type of mechanism. Attempts to devise therapies that are based on blocking Bcl-2 function, have been hindered by the fact that little is known about the molecular mechanism of Bcl-2 action. Our broad, long-term objective is to define the mechanism of Bcl- 2 action, by characterizing the interactions of Bcl-2 with other cellular proteins. In preliminary studies, we have cloned two cDNAs which code for novel proteins, BAP1 and BAP2, that bind to Bcl-2 in vitro. BAP1 and BAP2 are derived from distinct, yet related, genes, and define a novel family of Bcl-2-associated proteins. Forced expression of the BAP2 protein in an interleukin-3 (IL-3)-dependent cell line resulted in accelerated apoptosis following IL-3 withdrawal. Thus, the BAP proteins likely play important roles in cellular apoptosis. The Specific Aims of this proposal are: 1) to characterize expression of the BAP1 and BAP2 genes in normal murine tissues and defined hematopoietic cell lines; 2) to investigate physical interactions between Bcl-2 and the BAP1 and BAP2 proteins in defined hematopoietic cell lines; 3) to investigate the capabilities of BAP1 and BAP2 to block Bcl-2 function; and 4) to investigate physical interactions between the BAP1 and BAP2 proteins and other members of the Bcl-2 protein family, including Bax, Bcl-XL, Bcl-XS, and Mcl-1. These studies may provide a basis for novel therapies aimed at blocking Bcl-2 function in vivo. Expression of the BAP1 and BAP2 proteins in normal murine tissues and defined hematopoietic cell lines will be studied by immunohistochemistry and Western blotting using polyclonal antisera generated against the recombinant proteins. Northern blotting and in situ hybridization will be used to study mRNA expression. To investigate potential intracellular protein-protein interactions, we will coexpress Bcl-2 with BAP1 or BAP2 in IL-3-dependent cell lines, and determine whether Bcl-2 can be specifically coimmunoprecipitated with BAP1 or BAP2. In parallel, we will investigate the effects of forced BAP1 or BAP2 expression on the ability of Bcl-2 to suppress apoptosis following IL-3 withdrawal. Investigations of interactions between the BAP proteins and other members of the Bcl-2 protein family will be performed in vitro, using purified recombinant proteins.