H4组蛋白相互作用伙伴的蛋白质组学分析
Analysis of interaction partners of H4 histone by a new proteomics approach
摘要：
我们描述了一种改进的TAP方法，用于纯化和分析多蛋白复合物，这里称为DEF-TAP（串联亲和纯化后的差异洗脱分级）。其基本的新功能是用于最后的纯化步骤的6XHis-Ni++相互作用，这是耐各种苛刻的洗涤条件，包括高离子强度和存在的有机溶剂。这使我们能够在蛋白酶消化之前使用各种分级方案，这有望提高所分析的蛋白质混合物的覆盖率，并且还提供了对纯化的大分子复合物的结构和所涉及的蛋白质相互作用的性质的额外了解。我们通过分析从HeLa细胞中纯化的含组蛋白H4的可溶性核复合物来说明我们的新方法。特别是，我们观察到不同的分馏模式的HAT 1和RbAp 46蛋白相比，RbAp 48蛋白，所有确定为H4组蛋白的相互作用的合作伙伴。此外，我们报告了许可的MCM 2 -7复合物的所有组分和可溶性H4的相互作用伙伴之间的糖尿病相关DAXX蛋白。最后，我们表明，HAT 1需要N-末端尾部的H4稳定协会与此组蛋白。
英文摘要：
We describe a modification of the TAP method for purification and analysis of multiprotein complexes, termed here DEF-TAP (for Differential Elution Fractionation after Tandem Affinity Purification). Its essential new feature is the use for last purification step of 6XHis-Ni++ interaction, which is resistant to a variety of harsh washing conditions, including high ionic strength and presence of organic solvents. This allows us to use various fractionation schemes before the protease digestion, which is expected to improve the coverage of the analysed protein mixture and also to provide an additional insight into the structure of the purified macromolecular complex and the nature of protein-protein interactions involved. We illustrate our new approach by analysis of soluble nuclear complexes containing histone H4 purified from HeLa cells. In particular, we observed different fractionation patterns of HAT1 and RbAp46 proteins as compared to RbAp48 protein, all identified as interaction partners of H4 histone. In addition, we report all components of the licensing MCM2-7 complex and the apoptosis-related DAXX protein among the interaction partners of the soluble H4. Finally, we show that HAT1 requires N-terminal tail of H4 for its stable association with this histone.
机构、总结、中文摘要均由AI分析而来，可能存在错误，请以论文正文为准。
