DNA羟甲基化修饰在消化系统肿瘤中的研究进展

DNA羟甲基化修饰是基因组表观遗传学的重要调控方式,指5-甲基胞嘧啶(5-m C)在TET蛋白家族的催化作用下氧化生成5-羟甲基胞嘧啶(5-hm C),完成DNA胞嘧啶的去甲基化过程。基因组甲基化异常导致了多种肿瘤的发生,羟甲基化修饰作为去甲基化的一种,同样与肿瘤发生密不可分。在消化系统肿瘤发生发展过程中存在5-hm C含量的变化,其原因可能与TET蛋白家族、IDH突变等密切相关,提示DNA羟甲基化修饰参与了消化系统肿瘤的发生发展过程。本文围绕DNA羟甲基化修饰与消化系统肿瘤之间的关系进行综述,旨在为消化系统肿瘤羟甲基化修饰研究提供新方向。     

小鼠TET1蛋白核定位信号的预测与鉴定（英文）北大核心CSCD

TET(ten-eleven translocation)家族蛋白能够介导DNA的5-甲基胞嘧啶(5-methylcytosine,5m C)的氧化,产生5-羟甲基胞嘧啶(5-hydroxymethylcytosine,5hm C)。通过TET蛋白的催化,可以诱导特定靶基因的启动子区域Cp G岛的去甲基化,从而激活基因的转录。TET1蛋白是一个拥有2039个氨基酸的DNA去甲基化酶,通过预测,TET1拥有18个核定位信号(nuclear localization signals,NLSs),其中13个为单分型NLS,5个为双分型NLS。本文利用绿色荧光蛋白和各种突变体,首次确定了小鼠TET1蛋白的2个NLSs,分别存在于CXXC结构域和催化结构域,而且这2个NLSs对全长TET1的和定位都是必需的。我们的研究对深入理解TET1的蛋白结构与功能研究具有重要意义。     

DNA羟甲基化在小鼠胚胎干细胞中的研究进展

简要总结DNA羟甲基化在小鼠胚胎干细胞(mouse embryonic stem cells,mESC)中的最新研究进展.DNA甲基化(DNAmethylation)影响染色质的结构与功能,在发育与疾病发生过程中具有重要作用.2009年Tahiliani等发现TET1可以催化甲基化胞嘧啶(5-methylcytosine,5mC)氧化为羟甲基化胞嘧啶(5-hydroxymethylcytosine,5hmC).DNA羟甲基化(DNAhydroxymethylation)被认为是调节DNA甲基化的一种重要方式,成为了表观遗传学的研究热点之一.     

北京棒杆菌天冬氨酸激酶突变体Q316P的酶学性质

【目的】对北京棒杆菌(Corynebacterium pekinense)天冬氨酸激酶(Aspartate kinase,AK)进行改造,期望获得具有较高酶活力且酶活性质改善的高产天冬氨酸族氨基酸的优良突变株,并削弱甚至解除Thr对AK的反馈抑制作用。【方法】利用定点突变技术对Gln(Q)316位点进行突变,高通量筛选获得活力提高明显的突变体,并将其在大肠杆菌BL21中高效表达,对野生型(Wild type,WT)和突变体Q316P AK用镍柱纯化,进行酶动力学及酶学性质研究。【结果】获得突变体Q316P,并在大肠杆菌BL21中成功表达。与野生型相比,突变体Q316P的V_(max)提高8.53倍,n值由2.15降低为1.29,正协同性减弱;最适温度由25°C升高至30°C;最适p H由8.0降低至7.5,半衰期由3.8 h延长至5.0 h;且在实验范围浓度内,底物抑制剂苏氨酸对突变体Q316P表现出激活作用;Q316P AK对金属离子K+和有机溶剂甲醇表现出良好抗性。【结论】获得酶活力提高、酶学性质改善的突变体,并一定程度上解除苏氨酸对AK的反馈抑制,为构建高产天冬氨酸族氨基酸工程菌提供参考。     

变铅青链霉菌体内游离胞嘧啶的发现及检测

【背景】胞嘧啶(cytosine,C)是核酸分子4种基本碱基之一。胞嘧啶首先是以胞苷三磷酸 (cytosine triphosphate,CTP)的形式合成,在核酸基础代谢中没有形成游离胞嘧啶的特定途径。杀稻瘟菌素(blasticidin S,BS)和谷氏菌素生物合成途径均以游离的胞嘧啶为前体,前者的生物合成基因簇中包含一个能水解胞苷单磷酸(cytidine monophosphate,CMP)生成胞嘧啶的水解酶BlsM,后者的生物合成基因簇及其产生菌的基因组中均没有这个水解酶对应的同源蛋白。【目的】检测不同细菌中是否普遍存在游离的胞嘧啶,探究是否存在能产生游离胞嘧啶的同工酶或新途径。【方法】在BS异源表达菌株Streptomyces lividans WJ2中敲除blsM,高效液相色谱(HPLC)检测突变株和WJ2发酵产物;液相色谱-质谱联用(LC-MS)检测10个经过分级纯化的微生物细胞裂解液上清中是否存在游离的胞嘧啶。【结果】突变株WJ2?blsM菌株仍能合成杀稻瘟菌素,但各组分产量与WJ2菌株相比均明显降低;除了变铅青链霉菌,在10株被检测的菌株中,金黄色葡萄球菌(Staphylococcus aureus)、地中海拟无枝酸菌(Amycolatopsis mediterranei)、枯草芽孢杆菌(Bacillus subtilis)也检测到了含量较高的游离胞嘧啶。【结论】WJ2?blsM菌株仍具有产生BS的能力,说明野生型Streptomyces lividans菌株内存在一种未曾发现的游离胞嘧啶的产生方式,可以满足次级代谢的需求。另外,不同微生物中游离胞嘧啶的含量不同。     

DNA羟甲基化与表观遗传学调控

表观遗传学中的DNA甲基化与疾病的发生发展密不可分. DNA甲基化中的5-甲基胞嘧啶易发生氧化形成5 羟甲基胞嘧啶.此过程又称为羟甲基化修饰,已成为表观遗传学研究的一种新热点.羟甲基化与10-11易位家族蛋白（ten-eleven translocation,TET）的作用密切相关,它参与了基因的表达调控以及DNA去甲基化过程. 最近的羟甲基化研究主要集中在癌症和精神性疾病.针对日趋增多的相关研究,本文对DNA羟甲基化进行了全景式综述.     

组蛋白H3Thr3磷酸化在Sf9细胞有丝分裂中的动态定位

【目的】研究组蛋白H3Thr3磷酸化这一表观标记在Sf9细胞有丝分裂中的功能。【方法】通过固相法合成一段包含组蛋白H3第1-9位氨基酸的肽段[ARTKQTARKC],将第3位苏氨酸进行磷酸化修饰(P-Pep 226555)或者不修饰(NP-Pep 080472)后制备抗体,Western blot检测抗体的特异性。培养Sf9细胞,通过爬片制备细胞处于有丝分裂不同时期的玻片,以免疫荧光标记检测磷酸化H3Thr3(H3Thr3ph)抗体在Sf9细胞有丝分裂不同时期的定位特点。【结果】在Sf9细胞中,组蛋白H3Thr3的磷酸化发生在前期细胞染色体的特定位置;随着细胞周期的进行,磷酸化信号逐渐增强,中期达到最高水平,有丝分裂后期在形成子细胞的赤道板处均匀分布,末期仅在赤道板特定位置残留微弱的磷酸化信号。【结论】H3Thr3的磷酸化与Sf9细胞胞质有丝分裂相关。     

5－氮－2＇－脱氧胞苷（5－aza－CdR）对HL－60细胞分化和DNA甲基化酶的影响

以５－氮－２＇－脱氧胞苷（５－ａｚａ－ＣｄＲ）为诱导物,在０．５μｍｏｌ／Ｌ的最佳浓度下,可诱导ＨＬ－６０细胞分化达１５％左右。同时,用［￣３Ｈ］－ｍｅｔｈｙｌ－ｓ－ａｄｅｎｏｓｙｌｍｅｔｈｉｏｎｉｎｅ（￣３Ｈ－ＳＡＭ）为底物,通过同位素参入法,测定了不同浓度诱导物对ＨＬ－６０细胞ＤＮＡ甲基化酶活力的影响,发现在最佳诱导物浓度下,可使ＨＬ－６０细胞ＤＮＡ甲基化酶活力明显下降,此外,也比较了不同分化水平的ＨＬ－６０细胞中具有不同甲基化水平的ＤＮＡ在体外接受甲基的能力,从而证明５－ａｚａ－ＣｄＲ诱导ＨＬ－６０细胞分化与其ＤＮＡ甲基化状态密切相关。     

内消旋-二氨基庚二酸脱氢酶关键氨基酸位点的改造提高对烷基取代2-酮酸的催化活力

【背景】高效实现D-氨基酸的生物合成一直是人们关注的热点。内消旋-二氨基庚二酸脱氢酶(meso-diaminopimelate dehydrogenase,DAPDH)能够直接催化2-酮酸和氨合成D-氨基酸。【目的】提高DAPDH对烷基取代2-酮酸的催化活力,并解释其催化机制。【方法】以来源于嗜热共生杆菌(Symbiobacteriumthermophilum)的内消旋-二氨基庚二酸脱氢酶(StDAPDH)为模板,在前期结构分析结合被选择位点突变结果的基础上,确定对H227位进行定点饱和突变,并以D-丙氨酸、D-2-氨基丁酸、D-正缬氨酸、D-谷氨酸为底物进行筛选。【结果】获得突变体H227Q和H227N。突变体H227Q对丙酮酸、2-氧代丁酸、2-氧代戊酸、2-酮戊二酸的比活力比野生型分别提高了10.9、11.5、8.6和7.6倍。动力学参数表明,突变体H227Q同时提高了酶对底物的亲和力及催化常数,使其对丙酮酸的催化效率(k_(cat)/K_m)相较于野生型提高了9.4倍。利用分子模拟技术分析突变体H227Q与产物氨基酸之间的相互作用表明,227位的谷氨酰胺通过与氨基酸的羧酸形成氢键,使得氨基酸产物Cα上的氢和辅酶烟酰胺环C4原子之间的距离缩短。【结论】利用定向进化技术提高DAPDH对烷基取代2-酮酸的催化活力,有助于开发新型的高效生物催化剂,这些工作也为下一步继续进行更具挑战性的D-氨基酸研究提供了基础。     

代谢工程改造谷氨酸棒状杆菌合成及分泌途径生产L-缬氨酸

谷氨酸棒状杆菌是目前微生物发酵生产L-缬氨酸的主要工业菌株。文中首先在谷氨酸棒状杆菌VWB-1中敲除了alaT (丙氨酸氨基转移酶),获得突变菌株VWB-2,作为出发菌株。进而对L-缬氨酸合成途径关键酶——乙酰羟酸合酶 (ilvBN) 的调节亚基进行定点突变 (ilvBN1M13),解除L-缬氨酸对该酶的反馈抑制。然后辅助过量表达L-缬氨酸合成途径关键基因ilvBN1M13、乙酰羟酸异构酶 (ilvC)、二羟酸脱水酶 (ilvD)、支链氨基酸氨基转移酶 (ilvE),加强通往L-缬氨酸的碳代谢流,提高菌株的L-缬氨酸水平。最后,基于过量表达L-缬氨酸转运蛋白编码基因brnFE及其调控蛋白编码基因lrp1,提高细胞的L-缬氨酸转运能力。最终获得工程菌株VWB-2/pEC-XK99E-ilvBN1M13CE-lrp1-brnFE在5 L发酵罐中的L-缬氨酸产量达到461.4 mmol/L,糖酸转化率达到0.312 g/g葡萄糖。     

Analysis of mutagenesis induced by a thermolabile T4 phage deoxycytidylate hydroxymethylase suggests localized deoxyribonucleotide pool imbalance

Summary To understand the molecular basis of mutation stimulated by deoxyribonucleotide pool imbalance, we studied a temperature-sensitive T4 phage gene 42 mutant (LB3), which specifies a thermolabile deoxycytidylate hydroxymethylase. Analysis of rII mutations, revertible to wild type along either GC-to-AT or AT-to-GC transition pathways, showed 8- to 80-fold stimulation of GC-to-AT mutations at a semi-permissive temperature (34° C). One such marker, rII SN103, which showed the highest stimulation at 34° C, was sequenced after amplification of the template by polymerase chain reaction. The mutant site in rII SN103 was identified at nucleotide position 265 from the rII B translational start as an AT-to-GC transition, which changes TCA to CCA. Sequence analysis of revertants and pseudorevertants generated at 34° C showed that both cytosines within this triplet can undergo change to either thymine or adenine, consistent with the hypothesis that hydroxymethyldeoxycytidine triphosphate pools are depleted at replication sites. However, dNTP pool measurements in extracts of 34° C cultures showed no significant deviations from values obtained at 30° C, suggesting that pool imbalances occur only locally, close to replication forks. Our studies support the hypothesis that the imitator phenotype displayed by ts LB3 at semi-permissive temperature is a consequence of perturbation of the flow of nucleotide precursors into the DNA replication machinery. A putative localized depletion of hm-dCTP presumably enlarges effective dTTP/hm-dCTP and dATP/hm-dCTP pool ratios, resulting in the observed C-to-T transition and C-to-A transversion mutations.     

Synthesis of CDP-diglyceride from phosphatidylinositol and CMP

A reaction in which CDP-diglyceride and inositol are formed from 1-stearoyl, 2-arachidonoyl phosphatidylinositol and CMP occurs readily in dialyzed microsomal preparations from the mouse pancreas. The reaction is Mn²⁺-dependent, and it is inhibited by each of the two products, CDP-diglyceride and myoinositol. It is presumed to involve the back-reaction of CDP-diglyceride: inositol phosphatidyltransferase (phosphatidyl-inositol synthetase, EC.2.7.8.11.)     

CTP: CMP phosphotransferase activity in L1210 cells

Substrate converting enzymes interfering with the measurement of ribonucleotide reductase were assessed in cell-free extracts prepared from L1210 cells. Data show the presence of a myokinase-type enzyme activity (CTP:CMP phosphotransferase) which catalyzes the reaction: 2CDP in equilibrium CMP + CTP. This enzyme is not removed by passage of cell extracts over ATP-agarose columns. Monitoring of nucleoside diphosphate substrate level is, therefore, mandatory for obtaining accurate measurements of CDP reductase activity in crude cell extracts.     

Substrate-induced conformational changes in human UMP/CMP kinase

Human UMP/CMP kinase plays a crucial role in supplying precursors for nucleic acid synthesis by catalyzing the conversion of UMP, CMP, and dCMP into their diphosphate form. In addition, this kinase is an essential component of the activation cascade of medicinally relevant nucleoside analog prodrugs such as AraC, gemcitabine, and ddC. During the catalytic cycle the enzyme undergoes large conformational changes from open in the absence of substrates to closed in the presence of both phosphoryl donor and phosphoryl acceptor. Here we report the crystal structure of the substrate-free, open form of human UMP/CMP kinase. Comparison of the open structure with the closed state previously reported for the similar Dictyostelium discoideum UMP/CMP kinase reveals the conformational changes that occur upon substrate binding. We observe a classic example of induced fit where substrate-induced conformational changes in hinge residues result in rigid body movements of functional domains to form the catalytically competent state. In addition, a homology model of the human enzyme in the closed state based on the structure of D. discoideum UMP/CMP kinase aids to rationalize the substrate specificity of the human enzyme.     

CMP substitutions preferentially inhibit polysialic acid synthesis

It is widely reported that derivatives of sugar moieties can be used to metabolically label cell surface carbohydrates or inhibit a particular glycosylation. However, few studies address the effect of substitution of the cytidylmonophosphate (CMP) portion on sialyltransferase activities. Here we first synthesized 2'-O-methyl CMP and 5-methyl CMP and then asked if these CMP derivatives are recognized by alpha2,3-sialyltransferases (ST3Gal-III and ST3Gal-IV), alpha2,6-sialyltransferase (ST6Gal-I), and alpha2,8-sialyltransferase (ST8Sia-II, ST8Sia-III, and ST8Sia-IV). We found that ST3Gal-III and ST3Gal-IV but not ST6Gal-I was inhibited by 2'-O-methyl CMP as potently as by CMP, while ST3Gal-III, ST3Gal-IV, and ST6Gal-I were moderately inhibited by 5-methyl CMP. Previously, it was reported that polysialyltransferase ST8Sia-II but not ST8Sia-IV was inhibited by CMP N-butylneuraminic acid. We found that ST8Sia-IV as well as ST8Sia-II and ST8Sia-III are inhibited by 2'-O-methyl CMP as robustly as by CMP and moderately by 5-methyl CMP. Moreover, the addition of CMP, 2'-O-methyl CMP, and 5-methyl CMP to the culture medium resulted in the decrease of polysialic acid expression on the cell surface and NCAM of Chinese hamster ovary cells. These results suggest that 2'-O-methyl CMP and 5-methyl CMP can be used to preferentially inhibit sialyltransferases, in particular, polysialyltransferases in vitro and in vivo. Such inhibition may be useful to determine the function of a carbohydrate synthesized by a specific sialyltransferase such as polysialyltransferase.     

Effect of the nucleotides CMP and UMP on exhaustion in exercise rats

The aetiology of muscle fatigue has yet not been clearly established. Administration of two nucleotides, cytosine monophosphate (CMP) and uridine monophosphate (UMP), has been prescribed for the treatment of neuromuscular affections in humans. Patients treated with CMP/UMP recover from altered neurological functions and experience pain relief, thus the interest to investigate the possible effect of the drug on exhausting exercise. With such aim, we have determined, in exercised rats treated with CMP/UMP, exercise endurance, levels of lactate, glucose and glycogen, and the activity of several metabolic enzymes such as, creatine kinase (CK), lactate dehydrogenase (LDH), and aspartate aminotransferase (AST). Our results show that rats treated with CMP/UMP are able to endure longer periods of exercise (treadmill-run). Before exercise, muscle glucose level is significantly higher in treated rats, suggesting that the administration of CMP/UMP favours the entry of glucose in the muscle. Liver glycogen levels remains unaltered during exercise, suggesting that CMP/UMP may be implicated in maintaining the level of hepatic glycogen constant during exercise. Lactate dehydrogenase and aspartate aminotransferase activity is significantly lower in the liver of treated rats. These results suggest that administration of CMP/UMP enable rats to endure exercise by altering some metabolic parameters.