Proteomic Approach to Further Understanding the Role of*

进一步了解*作用的蛋白质组学方法

• 批准号: 7128102
• 负责人: ALAN B. CANTOR
• 金额: $ 33.01万
• 依托单位: BOSTON CHILDREN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-09-30 至 2009-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7128102
• 关键词: affinity chromatography; affinity labeling; bone marrow disorder; gene induction /repression; gene mutation; gene rearrangement; hematopoiesis; laboratory mouse; mass spectrometry; protein binding; protein protein interaction; protein purification; proteomics; recombinant proteins; transcription factor; yeast two hybrid system

DESCRIPTION (provided by applicant): Myelodysplastic syndromes (MDS) are a heterogeneous group of clonal disorders characterized by progressive peripheral blood cytopenias, hypercellular or normocellular bone marrow, morphologic abnormalities of the erythroid and megkaryocytic lineages, and propensity to progress to acute leukemia. Despite the high morbidity and mortality associated with these disorders, the molecular mechanisms underlying their pathogenesis and progression remain incompletely understood. Mutations involving the transcription factor Runx-1 have recently been identified in a large number of MDS cases, especially those that progress to leukemia. This proposal uses Runx-1's recently discovered role in normal megakaryopoiesis as a model to better understand the mechanisms by which it may be perturbed by MDS associated mutations. Many transcription factors, in addition to binding DMA, make important protein-protein interactions that modulate their activity. Preliminary studies using gel filtration chromatography indicate that Runx-1 participates in at least two stable multiprotein complexes of approximately 150-300 kDa and >669 kDa in a murine megakaryoblastic cell line. This proposal utilizes new powerful proteomic technology to identify components of these complexes and probe their functional significance. It takes advantage of a technique we developed for metabolic biotin tagging of recombinant proteins in mammalian cells. This high affinity tag is combined with a FLAG epitope for tandem affinity purification of complexes under native conditions. Whole lane LC/MS/MS mass spectrometry is then used to comprehensively identify associated proteins in a non-biased manner. After validation of results, the functional significance of identified proteins is probed, and the effect of MDS-associated Runx-1 mutations on their interactions examined. Our preliminary results support recent reports of a physical interaction between Runx-1 and GATA-1, a zinc finger transcription factor central to erythroid and megakaryocyte terminal maturation. The interacting domains of these molecules will be further defined, and mutations that disrupt binding identified in a yeast altered specificity mutant screen. These mutants will then be used to test the hypothesis that Runx-1 :GATA-1 interactions are functionally important in normal megakaryopoiesis, and may be disrupted by MDS-associated mutations. These studies should provide important insights that will facilitate the design of novel therapies for MDS.

描述（由申请人提供）： 骨髓增生异常综合征（MDS）是一组异质性克隆性疾病，其特征在于进行性外周血细胞减少、骨髓细胞过多或正常、红细胞和巨核细胞谱系的形态异常以及进展为急性白血病的倾向。 尽管与这些疾病相关的高发病率和死亡率，但其发病机制和进展的分子机制仍不完全清楚。涉及转录因子Runx-1的突变最近在大量MDS病例中被鉴定，特别是进展为白血病的那些。该提案使用Runx-1最近发现的在正常巨核细胞生成中的作用作为模型，以更好地理解其可能被MDS相关突变干扰的机制。许多转录因子，除了结合DMA，使重要的蛋白质-蛋白质相互作用，调节其活动。使用凝胶过滤色谱法的初步研究表明，Runx-1在鼠巨核细胞系中参与至少两种稳定的多蛋白复合物，约150 - 300 kDa和> 669 kDa。该建议利用新的强大的蛋白质组学技术，以确定这些复合物的组成部分，并探讨其功能意义。它利用了我们开发的用于哺乳动物细胞中重组蛋白的代谢生物素标记的技术。这种高亲和力标签与FLAG表位组合，用于在天然条件下串联亲和纯化复合物。 然后使用全泳道LC/MS/MS质谱法以非偏倚方式全面鉴定相关蛋白质。在验证结果后，探测鉴定的蛋白质的功能意义，并检查MDS相关的Runx-1突变对其相互作用的影响。我们的初步结果支持最近的报告Runx-1和加塔-1，中央红细胞和巨核细胞终末成熟的锌指转录因子之间的物理相互作用。这些分子的相互作用结构域将被进一步定义，并且在酵母特异性改变突变体筛选中鉴定出破坏结合的突变。然后，这些突变体将用于检验Runx-1：加塔-1相互作用在正常巨核细胞生成中具有重要功能，并且可能被MDS相关突变破坏的假设。 这些研究应该提供重要的见解，这将有助于设计新的治疗MDS。