Proteomic Approach to Further Understanding the Role of*

描述(由申请人提供)： 骨髓增生异常综合征(MDS)是一组异质性的克隆性疾病，其特征是进行性外周血细胞减少，骨髓细胞增多或正常，红系和巨核系形态异常，并有进展为急性白血病的倾向。尽管与这些疾病相关的发病率和死亡率很高，但其发病和进展的分子机制仍不完全清楚。最近在大量MDS病例中发现了涉及转录因子RUNX-1的突变，特别是那些进展为白血病的病例。这项建议使用RUNX-1的S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DNA外，还参与重要的蛋白质-蛋白质相互作用，从而调节它们的活性。凝胶过滤层析的初步研究表明，在小鼠巨核细胞系中，RUNX-1至少参与了大约150-300 kDa和&gt；669 kDa的两个稳定的多蛋白复合体。这一建议利用新的强大的蛋白质组学技术来鉴定这些复合体的成分，并探讨它们的功能意义。它利用了我们开发的一项技术，用于在哺乳动物细胞中对重组蛋白进行代谢生物素标记。该高亲和力标签与标志表位相结合，用于天然条件下对复合体的串联亲和纯化。然后使用全通道LC/MS/MS质谱仪，以无偏见的方式全面鉴定相关蛋白质。在验证结果后，探讨了所识别的蛋白质的功能意义，并检查了与MDS相关的RUNX-1突变对它们相互作用的影响。我们的初步结果支持最近关于RUNX-1和GATA-1之间的物理相互作用的报道，GATA-1是红系和巨核细胞末端成熟的中心锌指转录因子。这些分子的相互作用区域将被进一步定义，并在酵母特异性突变筛选中识别破坏结合的突变。然后，这些突变体将被用来检验这一假设，即RUNX-1：GATA-1相互作用在正常的巨核细胞生成中具有重要的功能，并可能被MDS相关突变破坏。这些研究应该提供重要的见解，促进MDS的新疗法的设计。

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.