Role of FA protein complexes in hematopoiesis

FA 蛋白复合物在造血中的作用

• 批准号: 6984197
• 负责人: QISHEN PANG
• 金额: $ 37.38万
• 依托单位: CINCINNATI CHILDRENS HOSP MED CTR
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-07-05 至 2009-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6984197
• 关键词: DNA binding protein; DNA damage; apoptosis; cell proliferation; clinical research; congenital aplastic anemia; cytotoxicity; enzyme activity; gene mutation; genetically modified animals; heat shock proteins; hematopoiesis; hematopoietic stem cells; human subject; immunocytochemistry; immunoprecipitation; interferon gamma; laboratory mouse; mass spectrometry; mitomycin C; protein protein interaction; protein structure function; serine threonine protein kinase; tumor necrosis factor alpha

DESCRIPTION (provided by applicant): Hematopoietic cells from patients with Fanconi anemia (FA) exhibit both hypersensitive to cross-linking agents such as mitomycin C and exaggerated apoptotic responses to a variety of extracellular cues for mitogenic inhibition and apoptosis. One of the FA proteins, FANCC, functions to protect cells from cytotoxicity induced by combinations of either interferon gamma (IFN-gamma) and tumor necrosis factor alpha (TNF-alpha) or double-stranded RNA (dsRNA) and IFN-gamma. Our studies have demonstrated that the pro-apoptotic protein kinase PKR is required for cytokine hypersensitivity of FA cells. More recently, we showed that FANCC forms a ternary complex with the PKR kinase and molecular chaperone Hsp70, and mutations in 3 major FA genes, FANCA, FANCC and FANCG, affect the structural integrity of the complex. This suggests that 1 or more FA proteins participate in the formation of a multi-subunit protein complex, which we term the PKR-FA protein complex. We hypothesize that the FA proteins modulate stress responses by forming the PKR-FA protein complex and that a proper control of the activity of the stress kinase PKR is one consequence of structural and functional integrity of the complex. The long-term goals of our study are to characterize the functional assembly of the PKR-FA protein complex and to ultimately define the role of this newly identified FA protein-containing complex in hematopoiesis. We plan to build on our preliminary structural and biochemical characterizations of the PKR-FANCC-Hsp70 ternary complex to explore in molecular details the roles that FA proteins and other components play in the formation and function of the PKR-FA protein complex, interactions between components of the complex and functional significance of these interactions, and the roles of individual components in the structural and functional integrity of the complex. We will also investigate the in vivo role of the PKR-FA complex in proliferation and apoptotic response (to inhibitory cytokines IFN-gamma and TNF-alpha or DNA damaging agents such as mitomycin C) of hematopoietic progenitor cells using bone marrow cells from FA patients and FA knockout mice. Finally, we will examine the assembly of the FA protein-containing complexes in a living cell exposed to specified cellular stresses. The expectation is that the proposed study will provide insight into the biological function of the PKR-FA protein complex with regard to hematopoiesis and the relationship between the PKR-FA protein complex and the FA nuclear complex identified for DNA damage response.

描述（由申请人提供）：来自范可尼贫血 (FA) 患者的造血细胞表现出对交联剂（如丝裂霉素 C）过敏，并且对各种有丝分裂抑制和细胞凋亡的细胞外信号表现出过度的细胞凋亡反应。 FA 蛋白之一 FANCC 的作用是保护细胞免受干扰素 γ (IFN-γ) 和肿瘤坏死因子 α (TNF-α) 或双链 RNA (dsRNA) 和 IFN-γ 组合诱导的细胞毒性。我们的研究表明，促凋亡蛋白激酶 PKR 是 FA 细胞细胞因子超敏反应所必需的。最近，我们发现 FANCC 与 PKR 激酶和分子伴侣 Hsp70 形成三元复合物，并且 3 个主要 FA 基因 FANCA、FANCC 和 FANCG 的突变会影响复合物的结构完整性。这表明 1 个或多个 FA 蛋白参与多亚基蛋白复合物的形成，我们将其称为 PKR-FA 蛋白复合物。我们假设 FA 蛋白通过形成 PKR-FA 蛋白复合物来调节应激反应，并且对应激激酶 PKR 活性的适当控制是该复合物结构和功能完整性的结果之一。我们研究的长期目标是表征 PKR-FA 蛋白复合物的功能组装，并最终确定这种新鉴定的含 FA 蛋白复合物在造血过程中的作用。我们计划在 PKR-FANCC-Hsp70 三元复合物的初步结构和生化表征的基础上，在分子细节上探索 FA 蛋白和其他成分在 PKR-FA 蛋白复合物的形成和功能中所起的作用，成分之间的相互作用这些相互作用的复杂性和功能意义，以及各个成分在复杂的结构和功能完整性中的作用。我们还将使用来自 FA 患者的骨髓细胞，研究 PKR-FA 复合物在造血祖细胞增殖和凋亡反应（对抑制性细胞因子 IFN-γ 和 TNF-α 或 DNA 损伤剂，如丝裂霉素 C）中的体内作用和 FA 敲除小鼠。最后，我们将检查暴露于特定细胞应激的活细胞中含有 FA 蛋白的复合物的组装情况。期望所提出的研究将深入了解 PKR-FA 蛋白复合物在造血方面的生物学功能，以及 PKR-FA 蛋白复合物与用于 DNA 损伤反应的 FA 核复合物之间的关系。