亚热带山区土壤未培养微生物L-赖氨酸脱羧酶Ldc1E中关键氨基酸残基的功能鉴定

本研究旨在探讨L-赖氨酸脱羧酶Ldc1E关键氨基酸在底物识别和催化过程中的作用;通过生物信息学方法选择突变位点,并利用直接定点突变技术,完成了6个关键氨基酸残基突变和功能鉴定研究。突变酶D692N最适温度和pH值分别为40℃和6.5。突变酶D692N比野生型Ldc1E对高温具有更强的耐受性,在40℃～55℃温浴1 h后剩余酶活力达到35%以上,在60℃温浴1 h后仍然保留20%的酶活力;而野生型酶Ldc1E在50℃以上温浴1 h后几乎失活。此外,50 mmol/L DMSO、5 mmol/L Al~(3+)和Ca~(2+)对突变酶的酶活力有激活作用,而Al3+对野生型酶Ldc1E具有明显抑制作用。突变酶D692N的分子动力学常数K_m升高了1.78倍,k_(cat)下降了20.2倍。突变酶S221A、H245A、D330A、H366A、F607Y经检测酶催化活性丧失。研究结果表明氨基酸残基位点D692对酶与底物的结合具有重要影响;而S221、H245、D330、H366、F607是Ldc1E酶活性能够体现的关键氨基酸位点,不可替换。本研究为探究L-赖氨酸脱羧酶的结构与功能关系提供理论参考。     

β-葡萄糖苷酶的糖耐受和促活性质研究

【目的】以葡萄糖耐受并促活的β-葡萄糖苷酶Bgl2A为出发材料,寻找与β-葡萄糖苷酶的葡萄糖耐受和促活性质相关的重要氨基酸残基位点并对其进行突变;对突变酶性质进行检测,结合分子对接,探究突变对酶的糖耐受和促活性质的影响及机制;进而对葡萄糖不耐受的Bgl3A (Bgl2A:A22S/V224S)进行分子改造,以获得应用潜能更好的突变酶。【方法】通过序列和结构比对、统计耦联分析和结构分析,选取Bgl2A底物通道口、蛋白质表面以及活性中心附近可能间接影响葡萄糖耐受和促活性质的残基作为突变位点,构建了多个突变酶,并对其酶学性质进行检测。【结果】以Bgl2A为出发酶,D322I、W325A、W126Y、F172N、C173I和N226V的糖耐受和促活性质显著提升。分子对接提示,这些突变可能是通过变构效应影响活性中心与葡萄糖结合的自由能,从而改变酶葡萄糖耐受和促活性质。据此,在Bgl3A分子上对应构建多个突变体,筛选获得了较出发酶在糖耐受和促活性质提升的同时保持较高酶活和稳定性的突变酶N226V和F172N。【结论】除了酶与葡萄糖直接结合的位点,不与葡萄糖直接相互作用的位点也可通过远程作用间接影响...     

链霉菌GXT6 β-葡萄糖苷酶的酶学性质及葡萄糖耐受性分子改造

【目的】从经过全基因组测序的链霉菌GXT6中克隆、表达一个编码糖基水解酶家族3的新β-葡萄糖苷酶基因,研究重组酶的酶学性质并进行相关葡萄糖耐受性的氨基酸残基的分子改造,提高其对葡萄糖的耐受性。【方法】根据链霉菌GXT6的全基因测序结果,对其中一个注释为糖基水解酶的基因设计引物,PCR扩增目的基因,以p SE380为表达载体构建重组质粒,转化至大肠杆菌中诱导表达;采用镍亲和层析技术纯化重组蛋白质,对目的蛋白质进行酶学性质研究;采用定点饱和突变的方法对重组酶进行相关氨基酸残基的分子改造。【结果】从链霉菌GXT6中克隆到一个编码糖基水解酶家族3的新β-葡萄糖苷酶基因,并在大肠杆菌中表达。酶学性质研究结果表明该β-葡萄糖苷酶的最适温度为40°C,最适p H为6.0,Km值为(0.4712±0.0180) mmol/L,Vmax值为(128.000±1.741)μmol/(min·mg),葡萄糖抑制常数Ki值为(1.8880±0.1307)mmol/L。该BGL3-GXT6能够水解黄豆苷、染料木苷、甜茶苷、虎杖苷、淫羊藿苷。还对BGL3-GXT6中与葡萄糖耐受性可能相关的氨基酸残基位点81-Trp和233-Trp进行了定点饱和突变,获得了25个具有酶活的突变酶并对其进行酶学性质研究。其中W233位点饱和突变后获得的突变酶的Km和葡萄糖抑制常数Ki值与重组酶BGL3-GXT6相比均发生明显变化,葡萄糖耐受性有不同程度的提高,最高的提高了209倍。【结论】本研究获得的BGL3-GXT6对天然底物甜茶苷、黄豆苷、染料木苷、虎杖苷和淫羊藿苷具有水解功能,这些特性表明该β-葡萄糖苷酶在理论研究及在工业中有一定的应用价值。     

7-木糖紫杉烷糖基水解酶LXYL-P1-1和LXYL-P1-2活性中心预测及初步的功能分析

7-木糖紫杉烷糖基水解酶LXYL-P1-1和LXYL-P1-2是克隆自真菌香菇的两个双功能酶(序列一致性97%),具有β-木糖苷酶/β-葡萄糖苷酶双重活性,能特异性地水解移除7-木糖-10-去乙酰紫杉醇等紫杉烷上的木糖基。采用生物信息学方法对两个酶蛋白进行酶活性中心预测,初步确定Asp300和Glu529分别为亲核试剂和一般酸/碱催化剂,而Asn172-Gly173-Arg174和Lys207-His208为底物结合结构域。以LXYL-P1-2为研究对象,以毕赤酵母细胞为表达宿主,应用定点突变技术获得了N172A、G173A、R174A、K207A、H208A、D300N和E529Q突变体,并进行了酶活性分析。结果显示:在分别以PNP-Xyl、PNP-Glc和7-木糖-10-去乙酰紫杉醇为底物时,N172A、G173A、R174A、K207A、D300N和E529Q的β-木糖苷酶与β-葡萄糖苷酶活性大幅度下降甚至完全消失;H208A的β-木糖苷酶活性也显著下降,但仍保持98%的β-葡萄糖苷酶活性。其结果初步验证了对上述两个酶蛋白的活性中心的预测,为进一步揭示7-木糖紫杉烷糖基水解酶结构与功能的关系提供了实验依据。     

长双歧杆菌N-乙酰氨基己糖1-位激酶的活性位点

【目的】研究长双歧杆菌(Bifidobacterium longum)JCM1217的N-乙酰氨基己糖1-位激酶(Nacetylhexosamine 1-kinase,Nah K)中对催化活性有影响的位点。【方法】利用点突变试剂盒,获得Nah K的4个位点的共10种单点突变体表达菌株。诱导表达并纯化野生型和突变体酶,用DNS法和NADH偶联的微孔板分光光度法检测野生型及突变体酶的最适p H和最适Mg~(2+)浓度,并测定酶促反应动力学参数。【结果】D208A、D208N、D208E和I24A四种突变体的催化活性几乎丧失。突变体H31A、H31V、F247A和I24V的最适p H由野生型的7.5变为7.0,突变体H31A和F247A的最适Mg~(2+)浓度由野生型的5 mmol/L变为10 mmol/L。反应动力学参数测定结果表明,突变体F247Y对底物Glc NAc/Gal NAc及ATP的催化活性均高于野生型。【结论】通过定点突变,确定了对Nah K催化活性有影响的4个位点,并且获得了一个催化效率提高的突变体(F247Y),为进一步对Nah K进行分子改造奠定了一定基础。     

定向进化提高嗜热拟青霉J18耐热β-1,3-1,4-葡聚糖酶在酸性条件下的催化能力

应用定向进化技术提高了嗜热拟青霉Paecilomyces thermophila J18耐热β-1,3-1,4-葡聚糖酶(PtLic16A)在酸性条件下的催化能力.结合易错PCR和DNA改组的方法,构建了β-葡聚糖酶的突变体文库;利用刚果红染色法建立了阳性克隆的高通量筛选体系.筛选得到的突变酶PtLic 16AM1的反应最适pH由7.0变化至5.5,且保持了原有的耐热性和比酶活.突变酶的DNA序列中有4个点位发生突变,引发了4处氨基酸替换,分别是T58S、Y110N、G195E和D221G.结构模拟结果显示,发生突变的4个氨基酸位点中,Y110N位置靠近酶活性中心,而T58S、G195E和D221G则离酶活性中心较远,其中T58S、G195E可能对酶最适pH的变化起到了关键作用.     

嗜甲基菌DM11菌株二氯甲烷脱卤素酶基因的定点诱变

为了研究嗜甲基菌（Methylophilus）DM11菌株二氯甲烷脱卤素酶的不同氨基酸残基在底物结合、谷胱甘肽（GSH）亲和以及催化活力中的作用,对编码该酶的基因进行了定点诱变研究。将保守的103位色氨酸（W）分别用苯丙氨酸（F）、缬氨酸（V）或天冬酞胺（N）替换,109位精氨酸（R）用亮氨酸（L）替换,117位色氨酸用酪氨酸（Y）或苯丙氨酸替换,得到6种突变酶。其中3种突变酶具有较低的活力,另外3种突变酶没有活力。突变酶W117Y的性质与野生型酶明显不同。     

定点突变对酰胺水解酶DamH可溶性表达和酶活的影响

摘要:【目的】DamH是一种具有酯酶活性的酰胺水解酶,其非活性中心氨基酸残基的突变对重组酶可溶性表达和比酶活产生一定的影响。拟探索DamH的活性中心氨基酸残基构成,并对其非活性中心氨基酸残基突变对可溶性表达和比酶活的影响进行研究。【方法】通过重叠延伸的方法对DamH可能的活性中心氨基酸S149、E244和H274以及非活性中心氨基酸D165及N192进行定点突变,通过静息细胞测活验证了S149、E244和H274 在催化2-氯-N-(2’-甲基-6’-乙基苯基)乙酰胺(CMEPA)水解反应中的作用,通过Ni2+- NTA亲和层析对D165及N192突变子进行纯化,对突变株和野生型比酶活进行比较。【结果】研究表明S149A使DamH的CMEPA 水解酶活性下降为野生型的5%,E244A和H274A突变导致其失去活性;D165P和N192P突变影响到DamH的可溶性表达,表达量分别为野生型的28.2%和20.8%,突变子N192P、D165P比酶活分别为野生型比酶活的55.5%和49.7%。【结论】DamH催化酯类底物和芳基酰胺类底物可能共用同一活性中心S149、E244和H274,其两个α螺旋的转角处氨基酸侧链极性和刚性结构的改变对可溶性表达以及活性有很大的影响。     

E92A是HIV-1整合酶耐药突变N155S的活性回复突变

HIV-1整合酶是目前抗艾滋病药物研发的重要靶点之一,整合酶的耐药突变是导致整合酶抑制剂类药物治疗失败的主要原因,但突变产生耐药性的机理仍不清楚.本工作通过人工构建突变型整合酶,测试其活性和耐药性,对整合酶的耐药机理进行初步探索.构建整合酶的突变型包括E92A、N155S两种单突变及E92A/N155S双突变.通过基因工程操作引入突变、构建质粒、表达纯化得到整合酶蛋白.用基于磁珠的整合酶链转移ELISA测试整合酶的链转移活性,用S-1360和Raltegravir两种抑制剂测试整合酶的耐药性.另外,用Autodock软件做了S-1360和整合酶核心区(包括野生型和突变型)的分子对接.结果表明,N155S突变使整合酶链转移活性下降约80%,而E92A/N155S双突变仅使活性下降约42%,这表明N155S突变基础上的E92A突变可使整合酶的活性大幅回复.E92A和E92A/N155S对不同的抑制剂可产生不同的耐药性,它们对Raltegravir的耐药性强于对S-1360.突变对整合酶活性和耐药性的影响主要是通过改变整合酶活性中心结构实现的,E92A突变可能导致其与周围残基静电相互作用减弱,间接影响到D64和D116残基,产生活性回复作用.     

Role of the ASK1-SEK1-JNK1-HIPK1 signal in Daxx trafficking and ASK1 oligomerization

Overexpression of JNK binding domain inhibited glucose deprivation-induced JNK1 activation, relocalization of Daxx from the nucleus to the cytoplasm, and apoptosis signal-regulating kinase 1 (ASK1) oligomerization in human prostate adenocarcinoma DU-145 cells. However, SB203580, a p38 inhibitor, did not prevent relocalization of Daxx and oligomerization of ASK1 during glucose deprivation. Studies from in vivo labeling and immune complex kinase assay demonstrated that phosphorylation of Daxx occurred during glucose deprivation, and its phosphorylation was mediated through the ASK1-SEK1-JNK1-HIPK1 signal transduction pathway. Data from immunofluorescence staining and protein interaction assay suggest that phosphorylated Daxx may be translocated to the cytoplasm, bind to ASK1, and subsequently lead to ASK1 oligomerization. Mutation of Daxx Ser667 to Ala results in suppression of Daxx relocalization during glucose deprivation, suggesting that Ser667 residue plays an important role in the relocalization of Daxx. Unlike wild-type Daxx, a Daxx deletion mutant (amino acids 501-625) mainly localized to the cytoplasm, where it associated with ASK1, activated JNK1, and induced ASK1 oligomerization without glucose deprivation. Taken together, these results show that glucose deprivation activates the ASK1-SEK1-JNK1-HIPK1 pathway, and the activated HIPK1 is probably involved in the relocalization of Daxx from the nucleus to the cytoplasm. The relocalized Daxx may play an important role in glucose deprivation-induced ASK1 oligomerization.     

DEC1 and MIC-1

Comment on: Qian Y, et al. Proc Natl Acad Sci USA 2012; 109:11300-5.     

LINE-1编码蛋白L1-ORF1的原核表达纯化和多克隆抗体制备

目的: 制备具有肿瘤组织特异性表达的L1-ORF1蛋白多克隆抗体并进行初步应用研究。方法:采取基因工程表达方法制备L1-ORF1蛋白,免疫家兔制备多克隆抗体,间接ELISA检测抗体效价,Western blot和细胞免疫荧光方法检测抗体特异性,免疫检测验证其识别肿瘤细胞内L1-ORF1蛋白的特异性。结果:制备的抗L1-ORF1蛋白多克隆抗体具有很高的敏感性与特异性,免疫学检测表明该抗体不仅能检测出正常细胞中瞬时表达的L1-ORF1蛋白,而且可检测出肿瘤细胞中天然表达的L1-ORF1蛋白。结论:制备的多克隆抗体具有较高的敏感性与特异性,为以后该抗体的进一步应用奠定了基础。     

Antagonism between Cry1Ac1 and Cyt1A1 toxins of bacillus thuringiensis

Most strains of the insecticidal bacterium Bacillus thuringiensis have a combination of different protoxins in their parasporal crystals. Some of the combinations clearly interact synergistically, like the toxins present in B. thuringiensis subsp. israelensis. In this paper we describe a novel joint activity of toxins from different strains of B. thuringiensis. In vitro bioassays in which we used pure, trypsin-activated Cry1Ac1 proteins from B. thuringiensis subsp. kurstaki, Cyt1A1 from B. thuringiensis subsp. israelensis, and Trichoplusia ni BTI-Tn5B1-4 cells revealed contrasting susceptibility characteristics. The 50% lethal concentrations (LC50s) were estimated to be 4,967 of Cry1Ac1 per ml of medium and 11.69 ng of Cyt1A1 per ml of medium. When mixtures of these toxins in different proportions were assayed, eight different LC50s were obtained. All of these LC50s were significantly higher than the expected LC50s of the mixtures. In addition, a series of bioassays were performed with late first-instar larvae of the cabbage looper and pure Cry1Ac1 and Cyt1A1 crystals, as well as two different combinations of the two toxins. The estimated mean LC50 of Cry1Ac1 was 2.46 ng/cm2 of diet, while Cyt1A1 crystals exhibited no toxicity, even at very high concentrations. The estimated mean LC50s of Cry1Ac1 crystals were 15.69 and 19.05 ng per cm2 of diet when these crystals were mixed with 100 and 1,000 ng of Cyt1A1 crystals per cm2 of diet, respectively. These results indicate that there is clear antagonism between the two toxins both in vitro and in vivo. Other joint-action analyses corroborated these results. Although this is the second report of antagonism between B. thuringiensis toxins, our evidence is the first evidence of antagonism between toxins from different subspecies of B. thuringiensis (B. thuringiensis subsp. kurstaki and B. thuringiensis subsp. israelensis) detected both in vivo and in vitro. Some possible explanations for this relationship are discussed.     

Regulation of PCTAIRE1 protein stability by AKT1, LKB1 and BRCA1

PCTAIRE1, also known as CDK16, is a cyclin-dependent kinase that is regulated by cyclin Y. It is a member of the serine-threonine family of kinases and its functions have primarily been implicated in cellular processes like vesicular transport, neuronal growth and development, myogenesis, spermatogenesis and cell proliferation. However, as extensive studies on PCTAIRE1 have not yet been conducted, the signaling pathways for this kinase involved in governing many cellular processes are yet to be elucidated in detail. Here, we report the association of PCTAIRE1 with important cellular proteins involved in major cell signaling pathways, especially cell proliferation. In particular, here we show that PCTAIRE1 interacts with AKT1, a key player of the PI3K signaling pathway that is responsible for promoting cell survival and proliferation. Our studies show that PCTAIRE1 is a substrate of AKT1 that gets stabilized by it. Further, we show that PCTAIRE1 also interacts with and is degraded by LKB1, a kinase that is known to suppress cellular proliferation and also regulate cellular energy metabolism. Moreover, our results show that PCTAIRE1 is also degraded by BRCA1, a well-known tumor suppressor. Together, our studies highlight the regulation of PCTAIRE1 by key players of the major cell signaling pathways involved in regulating cell proliferation, and therefore, provide crucial links that could be explored further to elucidate the mechanistic role of PCTAIRE1 in cell proliferation and tumorigenesis.     

Antagonism between Cry1Ac1 and Cyt1A1 Toxins of Bacillus thuringiensis

Most strains of the insecticidal bacterium Bacillus thuringiensis have a combination of different protoxins in their parasporal crystals. Some of the combinations clearly interact synergistically, like the toxins present in B. thuringiensis subsp. israelensis. In this paper we describe a novel joint activity of toxins from different strains of B. thuringiensis. In vitro bioassays in which we used pure, trypsin-activated Cry1Ac1 proteins from B. thuringiensis subsp. kurstaki, Cyt1A1 from B. thuringiensis subsp. israelensis, and Trichoplusia ni BTI-Tn5B1-4 cells revealed contrasting susceptibility characteristics. The 50% lethal concentrations (LC₅₀s) were estimated to be 4,967 of Cry1Ac1 per ml of medium and 11.69 ng of Cyt1A1 per ml of medium. When mixtures of these toxins in different proportions were assayed, eight different LC₅₀s were obtained. All of these LC₅₀s were significantly higher than the expected LC₅₀s of the mixtures. In addition, a series of bioassays were performed with late first-instar larvae of the cabbage looper and pure Cry1Ac1 and Cyt1A1 crystals, as well as two different combinations of the two toxins. The estimated mean LC₅₀ of Cry1Ac1 was 2.46 ng/cm² of diet, while Cyt1A1 crystals exhibited no toxicity, even at very high concentrations. The estimated mean LC₅₀s of Cry1Ac1 crystals were 15.69 and 19.05 ng per cm² of diet when these crystals were mixed with 100 and 1,000 ng of Cyt1A1 crystals per cm² of diet, respectively. These results indicate that there is clear antagonism between the two toxins both in vitro and in vivo. Other joint-action analyses corroborated these results. Although this is the second report of antagonism between B. thuringiensis toxins, our evidence is the first evidence of antagonism between toxins from different subspecies of B. thuringiensis (B. thuringiensis subsp. kurstaki and B. thuringiensis subsp. israelensis) detected both in vivo and in vitro. Some possible explanations for this relationship are discussed.     

Isolation and Characterization of Ubiquitin-activating Enzyme E1-domain Containing 1, UBE1DC1

Ubiquitin and other ubiquitin-like proteins play important roles in post-translational modification. They are phylogenetically well-conserved in eukaryotes. Activated by other proteins, ubiquitin and ubiquitin-like proteins can covalently modify target proteins. The enzymes responsible for the activation of this modification have been known to include UBA1, SAE2, UBA3, SAE1 and ULA1. Here we report a new ubiquitin activating enzyme like cDNA, named ubiquitin activating enzyme E1-domain containing 1 (UBE1DC1), whose cDNA is 2654 base pairs in length and contains an open reading frame encoding 404 amino acids. The UBE1DC1 gene consists of 12 exons and is located at human chromosome 3q22. The result of RT-PCR showed that UBE1DC1 is expressed in most of human tissues. These two authors contributed equally to this paper. The nucleotide sequence reported in this paper has been submitted to GenBank under accession number AY253672.