【摘要】 组蛋白乙酰化修饰是基因起始转录的关键步骤.p300等组蛋白乙酰转移酶(HATs)催化组蛋白和非组蛋白的乙酰化.HATs具有多种细胞功能,而且乙酰化对底物蛋白的功能改变也具有重要功能.组蛋白乙酰转移酶p300可乙酰化多种细胞内蛋白,某些病毒蛋白与p300有相互作用并促进病毒复制.因此,p300是细胞内具有广泛功能的转录激活因子.组蛋白乙酰转移酶结构域(HAT区)是p300乙酰化酶活性的最小中心功能域,在p300乙酰化底物中具有重要功能.本文重组表达了对应p300 HAT区的GST-p300 HAT蛋白,对其乙酰化酶的活性进行检测.结果证实,p300 HAT蛋白在体外可高效乙酰化组蛋白H3.随后,对体外乙酰化反应的条件进行优化.总之,本文构建了一种简单高效、非放射性体外乙酰化体系,适用于对潜在底物蛋白的乙酰化水平和机制进行分析,以及乙酰化蛋白的相关功能的研究.更多还原

【Abstract】 Acetylation of histones is believed to be the key step in transcription initiation.Histone acetyltransferases(HATs) such as p300 catalysed the acetylation of histones and a number of non-histone proteins.HATs appear to be capable of contributing to transcriptional activation,cell cycle progression,gene silencing,DNA repair and other cellular functions.Moreover,acetylation of proteins play a role in nuclear import,protein-protein interactions,protein stability and DNA binding affinity.The histone acetyltransferase p300 acetylates a variety of substrates including transcription factors,signaling regulators and cytoskeletal proteins.Besides,there are also reports concerning the potential of viral proteins to interact with p300 to favor virus replication.Therefore,p300 has been shown to be a versatile transcriptional co-activator in cells.The histone acetyltransferase domain of p300(HAT) is the minimal and central functional domain that bear intrinsic acetyltransferase activity to exert p300-dependent acetylation.In the present study,a recombinant protein corresponding to the HAT coding sequence of p300 was expressed in E.coli BL21(DE3) as a fusion protein with GST(GST-p300 HAT).Histone H3 was incubated with purified p300 HAT domain in the presence of Ac-CoA and detected for acetylation with antibodies against acetylated lysine to assess enzymatic activity.The results suggested that p300HAT domain efficiently acetylated histone H3 in vitro.We further optimize the reaction conditions of in vitro acetylation assay such as reaction buffer,quantity of Ac-CoA and p300 HAT used,and incubation time.In conclusion,we have developed a simple,robust,and non-radioactive in vitro assay for acetylation of histone and non-histone proteins.This assay can be modified easily to acetylate other candidate substrates for investigation of the degree and mechanism of acetylation,as well as the study on the functional advantage of these acetylated proteins.更多还原