THE BIOCHEMISTRY AND FUNCTION OF HRK

HRK 的生物化学和功能

• 批准号: 6103381
• 负责人: Gabriel Nunez
• 金额: $ 19.84万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-02-01 至 2000-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6103381
• 关键词: Adenoviridae; BCL2 gene /protein; HeLa cells; apoptosis; cell population study; confocal scanning microscopy; gene expression; gene interaction; genetically modified animals; immunocytochemistry; immunoprecipitation; in situ hybridization; laboratory mouse; microinjections; neoplastic cell; polymerase chain reaction; protein structure function; regulatory gene; site directed mutagenesis; transcription factor; western blottings; yeast two hybrid system

Programmed cell death (PCD) often occurs via apoptosis, a physiological form of cellular demise common during embryogenesis, turnover of tissues and selection of cell populations. Several genes have been recently identified that activate, execute or inhibit the cell death pathway. The bcl-2 family of apoptosis-regulatory genes modulates cell death induced by a wide array of signals. Two members of the family, bcl-2 and bcl-x are expressed in many tissues and their importance is underscored by the finding that mutant mice deficient in bcl-2 or bcl-x exhibit lethal phenotypes due to abnormal apoptosis. Moreover, deregulated expression of Bcl-2 and Bcl-x proteins play an important role in cancer development and resistance of tumor cells to therapy- induced apoptosis. Yet, the biochemical mechanism by which Bcl-2 family members regulate apoptosis is poorly understood. We have identified a novel gene, harakiri, that functions as a regulator of Bcl-2 and Bcl-X/L and apoptotic cell death in mammalian cells. The harakiri product (HRK) does not exhibit significant homology to Bcl-2 or Bcl-X/L and lacks conserved BH1 and BH2 domains which are shared by BCL-2 family members. HRK contains an 8 amino acid BH3 motif which is found in proteins of the BCL-2 family. Significantly, HRK physically interacts with anti- apoptosis proteins Bcl-2 and Bcl-X/L but not with death-promoting Bcl-2 members such as Bax and Bak. Expression of HRK induces rapid onset of cell death in mammalian cells. Importantly the death-promoting activity of HRK is repressed by Bcl-2 and Bcl-X/L suggesting that HRK is a common target for the anti-apoptosis proteins Bcl-2 and Bcl-XL. In this proposal we propose experiments (i) to further characterize the molecular interaction between HRK and Bcl-2/Bclk-X/L and identify additional targets of HRK; (ii) to examine the expression, regulation, subcellular localization; (iii) develop recombinant adenoviruses to study cellular targets of HRK and its ability to activate cell death in normal and cancer cells, and (iv) to generate mutant mice deficient in HRK to determine its physiological role in vivo. The studies outlined in this proposal should provide novel sight into the apoptosis pathway which could lead to alternative therapeutic strategies for treatment of cancer.

程序性细胞死亡（PCD）通常通过细胞凋亡发生，这是一种生理性的细胞凋亡。 在胚胎发育过程中常见的细胞死亡形式，组织的更替 和细胞群体的选择。 最近有几个基因 识别激活，执行或抑制细胞死亡途径。 凋亡调控基因bcl-2家族调节细胞死亡 由一系列信号引起的。 家族的两个成员bcl-2 和bcl-x在许多组织中表达， bcl-2或bcl-x缺陷的突变小鼠 由于异常凋亡而表现出致命的表型。 此外，委员会认为， Bcl-2和Bcl-x蛋白的表达失调在肿瘤的发生发展中起重要作用。 在癌症发展和肿瘤细胞对治疗的抗性中的作用- 诱导凋亡。 然而，Bcl-2家族的生化机制 成员调控细胞凋亡的机制知之甚少。 我们已经确定了一 一种新的基因，harakiri，作为Bcl-2和Bcl-X/L的调节因子发挥作用 和哺乳动物细胞中的凋亡性细胞死亡。 Harakiri产品（HRK） 与Bcl-2或Bcl-X/L没有显著的同源性， BCL-2家族成员共有的保守的BH 1和BH 2结构域。 HRK含有一个8个氨基酸的BH 3基序，它存在于哺乳动物的蛋白质中。 BCL-2家族 值得注意的是，HRK与抗- 凋亡蛋白Bcl-2和Bcl-X/L，但不含促死蛋白Bcl-2 成员如Bax和巴克。 HRK的表达可诱导细胞凋亡的快速发生， 哺乳动物细胞的死亡。 重要的是， 的HRK被Bcl-2和Bcl-X/L抑制，表明HRK是一种常见的 抗凋亡蛋白Bcl-2和Bcl-XL的靶点。 在这 建议我们提出实验（i）进一步表征 HRK与Bcl-2/Bclk-X/L的分子相互作用及鉴定 HRK的其他靶点;（ii）检查HRK的表达，调节， 亚细胞定位;（iii）开发重组腺病毒， 研究HRK的细胞靶点及其激活细胞死亡的能力， 正常细胞和癌细胞，以及（iv）产生缺乏以下的突变小鼠： HRK以确定其在体内的生理作用。 研究概述 这一建议将为细胞凋亡途径提供新的视角， 这可能会导致替代的治疗策略， 癌