重组p300组蛋白乙酰转移酶结构域的表达及应用

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.     

p300/CBP及其相关因子PCAF与转录调控

p300/CBP及相关因子PCAF具有乙酰转移酶活性,能通过乙酰化组蛋白和非组蛋白的方式参与基因的转录调控.同时,它们能在转录因子和基本转录复合物之间起到桥梁作用,而且也能为整合多种转录因子提供支架,是一种典型的转录辅激活子. p300/CBP与细胞周期调控、细胞凋亡以及癌症的发生等过程之间有着直接的联系。本文概括了p300/CBP与PCAF的基本特性,并简要介绍它们与其他蛋白之间的相互作用,特别是E1A的最新研究进展。     

组蛋白乙酰转移酶PCAF通过与PML蛋白的作用而定位于细胞核ND10结构

PCAF(P300/CBP associated factor,P300/CBP相关因子)是一种重要的组蛋白乙酰转移酶,主要通过使核小体组蛋白发生乙酰化而参与基因转录的调控。P300/CBP是一种能够与PCAF相互作用的宿主细胞蛋白,定位于细胞核ND10结构(nuclear dot10)。ND10是由70余种蛋白质分子组成的、以分散的斑点或团块样方式存在于细胞核内的大分子复合物,与基因转录调控过程有着密切关系。早幼粒细胞白血病蛋白(promyelocytic leukemia protein,PML)是ND10的主要组成成分,对于维持ND10的正常结构与功能至关重要。在成功构建pGBK-PML3、pGAD-PCAF、pGEX-PCAF、pFlag-PCAF和pcDNA-PML3重组质粒的基础上,用酵母双杂交证明PCAF与PML3之间存在着相互作用;用GST-pull down技术证明GST-PCAF能够将PML3蛋白捕获并沉降下来,表明在体外条件下两者之间存在直接的相互作用;用免疫共沉淀和荧光共定位证明在体内PCAF能够与PML发生直接的相互作用而定位于ND10。这些结果为进一步深入研究PCAF的生物学功能和作用模式奠定了基础。     

可逆的组蛋白乙酰化调控p16的表达

p16^INK4a通过抑制CDK4/6的活性而在细胞周期进行中发挥重要的作用,研究发现,组蛋白乙酰转移酶p300能促进p16^INK4a启动子活性,而组蛋白去乙酰化酶HDAC3/4能够逆转由p300介导的p16^INK4a启动子活性的增加,HDAC3/4能够降低p16^INK4a mRNA和蛋白质的水平．染色质免疫沉淀(ChIP)实验结果表明转染p300表达质粒能够逆转由HDAC3/4介导的p16^INK4a启动子组蛋白的低乙酰化状态．此外,免疫荧光实验结果表明HDAC4的核质穿梭起着重要的作用．免疫印迹和染色质免疫沉淀实验证明HDAC的抑制剂丁酸钠盐(NaBu)能通过诱导组蛋白的高乙酰化而促进p16^INK4a的表达．基于这些实验结果,推测出可逆的组蛋白乙酰化参与p16^INK4a基因转录调控的模型．     

CBP/P300介导的PTIP乙酰化可能促进下游肿瘤相关基因CCDC121和GPR75的转录

PTIP (Pax transactivation domain-interacting protein,PAXIP1)蛋白参与MLL3 (lysine methyltransferase 2C,KMT2C)/MLL4(lysine methyltransferase 2B,KMT2B)组蛋白H3K4甲基转移酶复合体,促进基因的转录激活。但关于PTIP蛋白的乙酰化修饰及人宫颈癌细胞中PTIP转录激活靶基因的研究尚无报道。本研究以稳定表达SFB-PTIP蛋白的293T细胞株为对象,通过免疫沉淀和Western印迹法发现,PTIP蛋白能够发生乙酰化修饰,乙酰转移酶CBP/P300介导PTIP蛋白的乙酰化过程,CBP是主要乙酰转移酶。去乙酰化酶HDAC1/2/3介导PTIP蛋白的去乙酰化过程。选择性HDAC1-3抑制剂MS-275促进PTIP蛋白乙酰化水平上升,且呈剂量和时间依赖性。以人宫颈癌HeLa细胞为研究对象,RNA-seq和RT-qPCR证明,CCDC121 (coiled-coil domain containing121)和G蛋白偶联受体75 (G protein-coupled receptor 75,GPR75)是PTIP的转录激活靶基因(P<0. 01)。和对照相比,MS-275介导的蛋白质乙酰化水平上升,促进基因CCDC121和GPR75的mRNA转录呈现不同程度的剂量依赖性上升(P<0. 01,P<0. 05)。由此推测,MS-275有可能是通过促进PTIP蛋白的乙酰化水平,促进CCDC121和GPR75的转录。但其详细机制有待进一步研究。本研究对今后深入探讨PTIP乙酰化修饰对下游肿瘤相关基因转录的调节和功能,如肿瘤发生、进展等方面的调控机制提供了基础。     

乙酰转移酶基因NAA10的生物学功能及其在肿瘤中的作用

N端乙酰转移酶A（N-acetyltransferase A, NatA）复合体是真核生物最主要的N端氨基酸α位乙酰转移酶,N端α位乙酰转移酶10基因（N-α-acetyltransferase 10, NAA10）编码的N端α位乙酰转移酶10蛋白（N-α-acetyltransferase 10 protein, Naa10p）是NatA的催化亚基. Naa10p具备新生蛋白质N端氨基酸α位乙酰化活性、成熟蛋白质Lys残基ε位乙酰化活性以及对部分转录因子的协同调节作用. Naa10p能够通过调节细胞周期促进细胞增殖,通过调节雷帕霉素靶蛋白（mechanistic target of rapamycin, mTOR）通路促进细胞自噬,并通过多种不同的分子信号通路抑制细胞运动能力.根据乙酰化底物的不同,Naa10p还在细胞凋亡的调控中起双重作用. Naa10p参与的生物学过程还涉及血管生成和神经发育等. Naa10p在多种癌症组织中呈过表达,但其预后意义随肿瘤不同而有较大差别.对Naa10p的生理生化研究必将使我们对细胞的生理病理学过程及其机制的了解更加深入全面.本文将从蛋白质结构、机制功能及临床意义等不同角度系统地阐述NAA10的研究现状与进展.     

p300乙酰化在卡介苗感染中的作用

研究p300乙酰化在卡介苗(bacillus Calmette Guérin,BCG)感染中的作用。构建THP-1巨噬细胞模型,比较BCG感染前后p300蛋白表达水平和组蛋白H3乙酰化水平的改变,加入p300特异性抑制剂Delphinidin,观察细胞内组蛋白H3乙酰化水平的变化。结果表明,在分化成熟的THP-1细胞系中,BCG感染能够上调p300蛋白表达水平和组蛋白H3乙酰化水平,加入p300特异性抑制剂Delphinidin后,组蛋白H3乙酰化水平降低。BCG感染通过p300途径导致蛋白质乙酰化水平发生改变。     

腺苷酸活化蛋白激酶活化对单核细胞-内皮细胞黏附的影响及其机制北大核心CSCD

本研究旨在探讨腺苷酸活化蛋白激酶（AMP-activated protein kinase,AMPK）活化对单核细胞与内皮细胞黏附的影响及其分子机制。用不同剂量的AMPK激动剂5-氨基咪唑-4-甲酰胺核糖核苷酸（AICAR,0~2 mmol/L）或AMPK抑制剂compound C（10 mmol/L）处理肿瘤坏死因子α（tumor necrosis factorα,TNFα,10 ng/m L）诱导的人主动脉内皮细胞（human aortic endothelial cells,HAECs）,用TNFα诱导过表达活性型或显性抑制型AMPK蛋白的HAECs。用荧光染色法观察AMPK对荧光标记的单核THP-1细胞与HAECs黏附的影响。用荧光定量PCR检测血管细胞黏附分子1（vascular cell adhesion molecule-1,VCAM-1）和细胞间黏附分子1（intercellular cell adhesion molecule-1,ICAM-1）m RNA表达水平,用ELISA法检测二者的蛋白分泌量;用Western blot检测核因子-kappa B（nuclear factor-kappa B,NF-κB）p65的211位点赖氨酸乙酰化水平,用ELISA法检测NF-κB p65DNA结合活性,并用试剂盒检测p300乙酰转移酶活性。通过小干扰RNA抑制HAECs组蛋白乙酰转移酶p300蛋白表达后,检测TNFα对NF-κB p65 DNA结合活性、黏附分子ICAM-1、VCAM-1的表达及单核细胞黏附率的影响。结果显示,AICAR显著抑制TNFα诱导的单核细胞与HAECs的黏附,在HAECs中下调TNFα诱导的ICAM-1、VCAM-1的m RNA水平上调和蛋白分泌。AICAR的效应可以被AMPK抑制剂compound C完全阻断。转染活性型AMPKα显著抑制TNFα诱导的ICAM-1、VCAM-1m RNA表达和分泌,以及单核细胞-内皮细胞黏附,而转染显性抑制型AMPKα则无明显影响。RNAi干预抑制p300活性显著抑制TNFα诱导的黏附分子表达和单核-内皮细胞黏附。AMPK激活可抑制TNFα诱导的p300乙酰转移酶活性,抑制NF-κB p65的211位赖氨酸的乙酰化,降低NF-κB p65 DNA结合活性。以上结果提示,AMPK激活抑制单核细胞-内皮细胞黏附,作用机制可能与其降低p300酶活性,下调NF-κB p65转录活性密切相关。     

乙酰转移酶MYST家族的生物学功能

组蛋白乙酰化是表观遗传修饰的重要方式,主要受到组蛋白乙酰转移酶（histone acetyltransferases, HATs)和组蛋白去乙酰化酶（histone deacetylase, HDACs）催化. MYST是人类HATs的4大家族之一,包括MOF(males absent on the first),TIP60 (tat interacting protein 60 kD),结合ORC1的组蛋白乙酰转移酶（histone acetyltransferase binding to ORC1, HBO1),单核细胞白血病锌指蛋白(monocytic leukemia zinc finger protein, MOZ)和MOZ相关蛋白（MOZ related factor, MORF）等,均具有典型的MYST结构域.MYST介导的乙酰化是重要的翻译后修饰,其催化底物包括组蛋白和非组蛋白,如组蛋白H3, H4, H2A, H2A突变体,以及许多参与DNA代谢、细胞增殖和发育调控的蛋白因子. MYST蛋白家族参与许多细胞的生理过程,本文主要综述其在调节基因转录、DNA损伤修复和肿瘤发生发展等方面的生物学功能.     

p300/CBP-associated factor histone acetyltransferase processing of a peptide substrate. Kinetic analysis of the catalytic mechanism

p300/CBP-associated factor (PCAF) is a histone acetyltransferase that plays an important role in the remodeling of chromatin and the regulation of gene expression. It has been shown to catalyze preferentially acetylation of the epsilon-amino group of lysine 14 in histone H3. In this study, the kinetic mechanism of PCAF was evaluated with a 20-amino acid peptide substrate derived from the amino terminus of histone H3 (H3-20) and recombinant bacterially expressed PCAF catalytic domain (PCAF(cat)). The enzymologic behavior of full-length PCAF and PCAF(cat) were shown to be similar. PCAF-catalyzed acetylation of the substrate H3-20 was shown to be specific for Lys-14, analogous to its behavior with the full-length histone H3 protein. Two-substrate kinetic analysis displayed an intersecting line pattern, consistent with a ternary complex mechanism for PCAF. The dead-end inhibitor analog desulfo-CoA was competitive versus acetyl-CoA and noncompetitive versus H3-20. The dead-end analog inhibitor H3-20 K14A was competitive versus H3-20 and uncompetitive versus acetyl-CoA. The potent bisubstrate analog inhibitor H3-CoA-20 was competitive versus acetyl-CoA and noncompetitive versus H3-20. Taken together, these inhibition patterns support an ordered BiBi kinetic mechanism for PCAF in which acetyl-CoA binding precedes H3-20 binding. Viscosity experiments suggest that diffusional release of product is not rate-determining for PCAF catalysis. These results provide a mechanistic framework for understanding the detailed catalytic behavior of an important subset of the histone acetyltransferases and have significant implications for molecular regulation of and inhibitor design for these enzymes.     

CREB-binding protein/p300 activates MyoD by acetylation

The myogenic protein MyoD requires two nuclear histone acetyltransferases, CREB-binding protein (CBP)/p300 and PCAF, to transactivate muscle promoters. MyoD is acetylated by PCAF in vitro, which seems to increase its affinity for DNA. We here show that MyoD is constitutively acetylated in muscle cells. In vitro, MyoD is acetylated both by CBP/p300 and by PCAF on two lysines located at the boundary of the DNA binding domain. MyoD acetylation by CBP/p300 (as well as by PCAF) increases its activity on a muscle-specific promoter, as assessed by microinjection experiments. MyoD mutants that cannot be acetylated in vitro are not activated in the functional assay. Our results provide direct evidence that MyoD acetylation functionally activates the protein and show that both PCAF and CBP/p300 are candidate enzymes for MyoD acetylation in vivo.     

Histone acetyltransferase p300/CBP‐associated factor is an effective suppressor of secretory immunoglobulin synthesis in immature B cells

The histone acetyltransferase p300/CBP‐associated factor (PCAF) catalyzes acetylation of core histones and plays important roles in epigenetics by altering the chromatin structure in vertebrates. In this study, PCAF‐deficient DT40 mutants were analyzed and it was found that PCAF participates in regulation of secretory IgM heavy chain (H‐chain) synthesis. Remarkably, PCAF‐deficiency causes an increase in the amount of secretory IgM H‐chain mRNA, but not in that of IgM light chain and membrane‐bound IgM H‐chain mRNAs, resulting in dramatic up‐regulation of the amount of secretory IgM protein. These findings suggest that PCAF regulates soluble antibody production and is thus an effective suppressor of secretory IgM H‐chain synthesis.