CHARACTERIZATION OF TWO NOVEL BCL-2-ASSCOCIATED PROTEINS

超过85%的人滤泡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Bcl-2已被证明可以阻断细胞凋亡， 多种造血和神经细胞类型，并可能有助于 滤泡性淋巴瘤通过这种类型的机制。试图设计 基于阻断Bcl-2功能的治疗受到阻碍， 关于Bcl-2的分子机制知之甚少， 行动上 我们广泛的长期目标是确定Bcl-2的机制， 2行动，通过表征Bcl-2与其他细胞的相互作用， proteins. 在初步研究中，我们克隆了两个编码新基因的cDNA， BAP 1和BAP 2蛋白在体外与Bcl-2结合。BAP 1和BAP 2是 来自不同的，但相关的基因，并定义了一个新的家庭， Bcl-2相关蛋白。BAP 2蛋白在一个细胞中的强制表达 白细胞介素-3（IL-3）依赖性细胞系导致加速凋亡 在IL-3退出后。因此，BAP蛋白可能发挥重要作用 在细胞凋亡中的作用。这项建议的具体目标是：1） 表征正常小鼠中BAP 1和BAP 2基因的表达 组织和确定的造血细胞系; 2）研究物理 Bcl-2与BAP 1和BAP 2蛋白之间的相互作用 造血细胞系; 3）研究BAP 1和 BAP 2阻断Bcl-2功能;以及4）研究物理相互作用 BAP 1和BAP 2蛋白与Bcl-2蛋白的其他成员之间的相互作用 Bcl-XL、Bcl-XS和Mcl-1。这些研究可能 为旨在阻断Bcl-2功能的新疗法提供基础， vivo. BAP 1和BAP 2蛋白在正常小鼠组织中的表达， 将通过免疫组织化学研究确定的造血细胞系 和使用针对该蛋白产生的多克隆抗血清进行蛋白质印迹 重组蛋白将进行北方印迹和原位杂交。 用于研究mRNA表达。 研究细胞内 蛋白质-蛋白质相互作用，我们将共表达Bcl-2与BAP 1或BAP 2， IL-3依赖性细胞系，并确定Bcl-2是否可以特异性 与BAP 1或BAP 2共免疫沉淀。与此同时，我们将调查 BAP 1或BAP 2的强制表达对Bcl-2表达能力的影响， 抑制IL-3撤除后的细胞凋亡。调查 BAP蛋白和Bcl-2蛋白的其他成员之间的相互作用 蛋白质家族将使用纯化的重组体在体外进行 proteins.

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.