RETICULOCYTE-SPECIFIC UBIQUITINATING ENZYMES

网状细胞特异性泛素化酶

• 批准号: 3245253
• 负责人: Cecile M. Pickart
• 金额: $ 18.03万
• 依托单位: STATE UNIVERSITY OF NEW YORK AT BUFFALO
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-02-01 至 1996-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3245253
• 关键词: cell differentiation; chemical conjugate; complementary DNA; enzyme mechanism; enzyme substrate; gene deletion mutation; genetic models; immunochemistry; immunoprecipitation; laboratory mouse; molecular cloning; northern blottings; nuclear runoff assay; nucleic acid sequence; posttranslational modifications; protein degradation; protein metabolism; reticulocytes; tissue /cell culture; ubiquitin; western blottings

Protein modification by ubiquitin plays important roles in such processes as cell cycle control, DNA repair, ribosome biogenesis, the heat shock response, and intracellular protein degradation. Substrates include cytoplasmic, nuclear, and integral membrane proteins. The diverse functions of ubiquitin generally involve the activity of distinct ubiquitin-conjugating enzymes. We have found that the conjugating enzymes E2-20K and E2-230K appear to be expressed only in the erythroid lineage. The discovery of ubiquitin-conjugating enzymes expressed in a tissue-specific fashion suggests that the functions of ubiquitination involve not only general cellular processes (above), but extend to specific differentiative events as well. The differentiation of erythroid cells is a global remodelling process whose mechanism is poorly understood. Because the levels E2-20K and E2-230K are high in the reticulocyte rather than the erythrocyte, it is likely that these enzymes represent a novel class of erythroid proteins which do not function in the mature erythrocyte but rather in the differentiative process that gives rise to it. It is proposed that E2-20K and E2-230K provide functions critical for erythroid differentiation. This hypothesis will be tested as follows. The genes for E2-20k and E2-230K will be cloned, sequenced, and used to generate murine erythroleukemia (MEL) cell derivatives in which these genes have been deleted by homologous recombination. We will then test whether such mutations affect the process of erythroid differentiation as represented in this model system. Potential conjugative and degradative targets of these E2's will be identified as specific proteins whose ubiquitination or turnover drifters between mutant and parental cells. A long-term goal is to relate the observed differentiative phenotypes to the nature of the observed substrates. The results are expected to be highly novel in providing the first insights into roles of ubiquitin in development. In addition, the experiments are expected to yield major insights into the mechanism of erythroid differentiation, with possible applications in the understanding of congenital diserythropoietic anemias.

泛素的蛋白质修饰在这一过程中起着重要的作用。 如细胞周期控制、DNA修复、核糖体生物发生、热休克 反应和细胞内蛋白质的降解。衬底包括 细胞质、核和完整的膜蛋白。不同的 泛素的功能一般涉及不同的活性。 泛素偶联酶。我们已经发现，结合酶 E2-20K和E2-230K似乎只在红系中表达。 泛素结合酶的发现 组织特异性时尚表明泛素化的功能 不仅涉及一般的细胞过程(如上所述)，而且扩展到特定的 差异化的事件也是如此。 红系细胞的分化是一个全球性的重塑过程 其机制还知之甚少。因为E2-20K和 E2-230K在网织红细胞中含量较高，而不是在红细胞中 很可能这些酶代表了一类新的红系蛋白 它们不在成熟的红细胞中起作用，而是在 产生它的差异化过程。建议将E2-20K 和E2-230K提供了红系分化的关键功能。这 假设将被检验如下。E2-20K和E2-230K基因 将被克隆、测序并用于产生小鼠红白血病 这些基因已被同源基因删除的(MEL)细胞衍生品 重组。然后我们将测试这些突变是否会影响这一过程 如该模型系统中所示的红系分化。 这些E2‘S的潜在结合和降解靶点将是 鉴定为泛素化或周转漂移的特定蛋白质 突变细胞和亲本细胞之间。一个长期目标是将 观察到的与观察到的性质相区别的表型 底物。预计结果将是非常新颖的，为 首次洞察泛素在发育中的作用。此外， 预计实验将对这一机制产生重大见解 红系辨证及其在认识中的可能应用 先天性失血性贫血。