丝氨酸/苏氨酸蛋白激酶突变体MEKK3~(KI)的构建及功能鉴定

有丝分裂原活化蛋白激酶激酶激酶MEKK3(mitogen-activated protein kinase/extracellular signal-regulated kinase kinase3)是一种丝氨酸/苏氨酸蛋白激酶,属于MAP3K(mitogen-activated protein kinase kinase kinase)家族,其在细胞增殖、凋亡、炎症反应、肿瘤的发生中发挥了重要的作用。利用分子克隆手段构建激酶功能失活(kinase inactive)型突变体MEKK3KI(391位赖氨酸突变为甲硫氨酸,K391→M391)的真核表达载体,并通过检测MEKK3KI蛋白对NudC的磷酸化作用及对细胞周期的影响以鉴定其生物活性。根据GenBank提供的人源MEKK3的cNA序列(NM_203351),扩增出野生型MEKK3(MEKK3~(WT))的目的基因,用重叠延伸PCR定点突变技术扩增激酶功能失活型MEKK3KI目的基因,并将其分别克隆至带有FLAG标签的真核表达载体p CMV-tag-2c和带有GFP标签的真核表达载体pEGFP-C1中。DNA序列分析结果显示,MEKK3KI基因序列成功将391位点的赖氨酸(K)突变为甲硫氨酸(M),表明突变体构建成功;免疫印迹分析显示,MEKK3~(WT)、MEKK3KI重组体均在He La细胞中高效表达;体外模拟磷酸化实验结果显示,野生型MEKK3~(WT)在体外可以磷酸化NudC,而激酶功能失活型突变体MEKK3KI无法将其磷酸化;激光共聚焦显微镜观察发现,野生型MEKK3在HeLa细胞中过表达,细胞核形态异常,与凋亡细胞核形态相似,而失活型MEKK3过表达不会导致细胞核明显变化。总之,本实验成功构建了野生型、失活型MEKK3的真核表达载体,同时发现其可以磷酸化NudC,促进细胞凋亡,为进一步揭示MEKK3生物学功能奠定了基础。     

丝氨酸/苏氨酸蛋白激酶突变体MEKK3的构建及功能鉴定

有丝分裂原活化蛋白激酶激酶激酶MEKK3(mitogen-activated protein kinase/extracellular signal-regulated kinase kinase 3)是一种丝氨酸/苏氨酸蛋白激酶,属于MAP3K(Mitogen-activated protein kinase kinase kinase)家族,其在细胞增殖、凋亡、炎症反应、肿瘤的发生中发挥了重要的作用。本文利用分子克隆手段构建激酶功能失活(kinase inactive)型突变体MEKK3KI(391位赖氨酸突变为甲硫氨酸,K391→M391)的真核表达载体,并通过检测MEKK3KI对NudC的磷酸化作用及对细胞周期的影响以鉴定其激酶活性。根据GenBank提供的人源MEKK3的cDNA序列(NM_203351),扩增出野生型MEKK3(MEKK3WT)的目的基因,用重叠延伸PCR定点突变技术扩增激酶功能失活型MEKK3KI目的基因,并将其分别克隆至带有FLAG标签的真核表达载体pCMV-tag-2c和带有GFP标签的真核表达载体pEGFP-C1中。DNA序列分析结果显示,MEKK3KI基因序列成功将391位点的赖氨酸(K)突变为甲硫氨酸(M),表明突变体构建成功;免疫印迹分析显示,MEKK3WT、MEKK3KI重组体均在HeLa细胞中高效表达;体外模拟磷酸化实验结果显示,野生型MEKK3WT在体外可以磷酸化NudC,而激酶功能失活型突变体MEKK3KI无法将其磷酸化;激光共聚焦显微镜观察发现,野生型MEKK3转染HeLa细胞,细胞核形态异常,与凋亡细胞核形态相似,而失活型MEKK3过量表达不会导致细胞核明显变化。总之,本实验成功构建了野生型、失活型MEKK3的真核表达载体,为进一步揭示MEKK3生物学功能奠定了基础。     

水稻β-1,3-葡聚糖酶疏水簇特性的研究

研究了41个水稻β-1,3葡聚糖酶的疏水簇(HC)的结构特点、HC的疏水氨基酸的突变倾向、密码子使用类型与偏好、为同种氨基酸二连体组合编码的密码子二连体组合使用类型与偏好.研究结果显示,水稻β-1,3葡聚糖酶至少有20个保守性较高的基本的HC,HC内至少含有≥1个疏水核心或有≥2个高度保守的疏水氨基酸位点.HC内似乎有I易向L、V、F突变,M易向I、V突变,W易向F突变,而反向突变较难的倾向.HC中的疏水氨基酸位点和为丙氨酸（A）编码的密码子使用频率与水稻基因组的统计数据出现明显偏差,为部分同种氨基酸二连     

c-Jun氨基末端激酶3结构域突变质粒的构建、鉴定与转染

为构建c-Jun氨基末端激酶3 (c-Jun N-terminal kinase3,JNK3)的p VP16-eGFP-Myc-JNK3活化环(activation-loop,A-loop)结构域突变型重组质粒,通过A-loop突变型JNK3的c DNA序列以及p VP16-eGFP-Myc的酶切位点设计引物,PCR扩增目的基因,使用限制性内切酶Mlu I与Xba I将基因克隆到p VP16-eGFP-Myc真核表达载体中,构建p VP16-eGFP-Myc-JNK3(A-loop)结构域突变型重组质粒。转化后,通过双酶切鉴定与DNA测序判断是否成功构建重组质粒,使用Trans IntroTMEL Transfection Reagent转染人胚肾(human emborynic kidney,HEK) 293T细胞,通过荧光显微镜下观察融合蛋白表达情况。结果表明:构建p VP16-eGFP-Myc-JNK3 (A-loop)结构域突变型重组质粒,双酶切鉴定和测序结果显示构建成功; p VP16-eGFP-MycJNK3 (A-loop)成功转染HEK293T细胞且表达。鼠源JNK3结构域突变型质粒构建成功,对进一步研究JNK3结构域在相关疾病的作用和机制提供实验工具。     

大肠杆菌ATCase抗反馈抑制突变体的构建及其对胞苷积累的影响

为解决胞苷生物合成途径中天冬氨酸氨甲酰转移酶受胞苷三磷酸反馈抑制调节的问题,通过对其碱基序列和蛋白质结构分析,利用基因定点突变的方法构建了大肠杆菌的ATCase突变酶,得到三个突变体:M1(H20L)、M2(K60E)、M3(K94E),并在E.coli DH5α中对融合蛋白进行了表达.酶活测定表明,M1、M2、M3的ATCase酶相对活性都比野生型M0的高,分别为野生型M0的1.10、1.22和1.37倍,且比活力都有不同程度提高.与含野生型pyrBI基因的M0相比,含突变型基因的M1、M2和M3均对15,mmol/L的CTP具有强的抗反馈抑制作用,且M1、M2和M3的抗CTP反馈抑制作用分别是M0的5.4、6.0和8.5倍.最后将各突变质粒转入到E.coli Cyt10(Δcdd)中进行发酵培养,结果表明,与未含突变基因菌株相比,各含突变基因菌株的胞苷积累量均有不同程度的提高,说明ATCase定点突变使胞苷的合成积累途径得到了不同程度的强化.     

新型冠状病毒膜蛋白的生物信息学分析

为了研究新型冠状病毒膜蛋白(SARS-CoV-2 M蛋白)结构及性质.基于生物信息学分析M蛋白质基因结构、二级结构和三级结构、翻译后的修饰和进化历程.结果表明,M蛋白为疏水性蛋白,其基因编码区长度为669bp,编码222个氨基酸;M蛋白启动子区内不存在甲基化位点,存在17潜在的转录因子结合位点;其二级结构以无规则卷曲和...     

利用酵母功能检测技术筛选肝癌组织p53基因显性负抑制效应突变体

p5 3突变体的显性负抑制效应(dominant negative effect)是指突变型p5 3蛋白抑制细胞内野生型p5 3蛋白发挥正常功能的一种特性,是肿瘤发生的重要原因之一.利用酵母中分离的等位基因功能分析技术(functional analysis of separated alleles in yeast,FASAY)建立了一种简便稳定的p5 3突变体显性负抑制效应检测和筛选方法,并应用于检测2 8例原发性肝癌组织(HCC)样本的p5 3单点突变体,发现了1 2例为显性负抑制效应突变体,其中突变位点Met2 4 6Val为首次报道具有显性负抑制效应.进一步分析这些突变p5 3蛋白的结构,发现这些具有显性负抑制效应的突变位点大多位于p5 3蛋白表面与DNA特异性序列相互作用的区域.     

多种肌红蛋白突变体与Cu(Ⅱ)相互作用机制光谱法研究

运用多种光谱法研究PCR定点突变获得的肌红蛋白突变体(D44K、D60K、K56D)与Cu(Ⅱ)的相互作用.结果表明:Cu(Ⅱ)对突变体的猝灭机理与野生型相同,均为静态猝灭,但结合常数、结合位点数、热力学常数、结合距离以及三维构象方面发生了一些变化.在相同温度下,蛋白与Cu(Ⅱ)的结合能力顺序为Mb(WT)相似文献   

德国小蠊致敏原Bla g 2的Glu 233突变的分子对接研究

为探讨Bla g 2的E233A突变对抗原抗体相互作用的影响,从RCSB数据库下载德国小蠊致敏原Bla g 2野生型及其单克隆抗体4C3的NMR结构,运用Swiss PDB Viewer将Bla g 2野生型(E233-93Q)构建为突变型(E233A-93Q)三维结构;并将Bla g 2野生型和突变型分别与其单克隆抗体4C3进行分子动力学模拟和蛋白-蛋白对接.结果表明:突变体E233A与单克隆抗体4C3结合能力下降,进而增加了与Ig E的结合力,可能加重过敏反应的发生.     

人锰超氧化物歧化酶hSOD2-Q46K突变体的表达与功能分析

目的:构建人锰超氧化物歧化酶(hSOD2)-Q46K突变体,简化获得hSOD2突变体的步骤,探讨hSOD2结构与功能的关系.方法:通过聚合酶链式反应(PCR)体外定点突变技术将hSOD2的46位Q氨基酸突变为K氨基酸,经测序鉴定正确后将p ET15b-hSOD2-Q46K突变体重组质粒转化Rosetta-gami大肠杆菌,通过异丙基-β-D-硫代半乳糖苷(IPTG)诱导目的蛋白表达.利用SWISS-MODEL Workspace对hSOD2-Q46K突变体进行同源建模,利用Swiss-Pdb-Viewer软件对建模结果进行分析.利用Ni-NTA亲和层析纯化目的蛋白后用超氧化物歧化酶(SOD)检测试剂盒检测目的蛋白的SOD活力.利用浓度为0.1 mmol/L百草枯(PQ)压力下生长情况测定表达hSOD2-Q46K和hSOD2蛋白的Rosetta-gami大肠杆菌的抗氧化能力.结果:测序结果表明46位密码子已由CAG突变为AAG.经过IPTG诱导后野生型和突变体均能表达出大小约为25 k Da的蛋白.SOD活性检测表明hSOD2-Q46K突变体的活力为899 U/mg,比野生型蛋白活性下降了65.4%.三维结构分析显示,突变点周围氨基酸间氢键被部分打破.结论:成功构建hSOD2-Q46K突变体并成功得到了纯化的突变体蛋白,hSOD2-Q46K的SOD活力比野生型降低了65.4%,且百草枯压力下表达hSOD2-Q46K蛋白的大肠杆菌A600达到0.84,而野生型蛋白的大肠杆菌A600达到1.14.这可能是因为突变点与周围氨基酸间氢键打破,突变点周围失去了原来的稳定结构,底物通道入口处氨基酸排列疏散,影响电子在氨基酸间的传递,影响静电导向机制,从而阻碍氧自由基在底物通道的通行,使hSOD2的催化活力下降.     

百合无症病毒衣壳蛋白基因克隆和蛋白分析

根据已报道的LSV CP基因序列合成两条寡聚核苷酸引物,模板为感染LSV的百合叶片的总RNA,通过反转录-聚合酶链式反应(RT-PCR)扩增出大小为876bp的LSV CP基因,经测序后,对该基因编码区全长序列及相应的氨基酸序列用生物信息学软件系统进行序列分析及结构功能预测.结果表明:该基因由876个核苷酸组成,编码291个氨基酸;与GeneBank公布的其他LSV分离物的基因序列同源性为93.4%～99.0%,氨基酸同源性为84.8%～99.5%;它含有一个卷曲螺旋结构和多个磷酸化位点,平均疏水值为-0.432;含有Carlaviruses完整的衣壳蛋白保守结构域,二级结构以α-螺旋和无规则卷曲为主.     

Modulation of an RNA-binding protein by abscisic-acid-activated protein kinase

Protein kinases are involved in stress signalling in both plant and animal systems. The hormone abscisic acid mediates the responses of plants to stresses such as drought, salinity and cold. Abscisic-acid-activated protein kinase (AAPK -- found in guard cells, which control stomatal pores -- has been shown to regulate plasma membrane ion channels. Here we show that AAPK-interacting protein 1 (AKIP1), with sequence homology to heterogeneous nuclear RNA-binding protein A/B, is a substrate of AAPK. AAPK-dependent phosphorylation is required for the interaction of AKIP1 with messenger RNA that encodes dehydrin, a protein implicated in cell protection under stress conditions. AAPK and AKIP1 are present in the guard-cell nucleus, and in vivo treatment of such cells with abscisic acid enhances the partitioning of AKIP1 into subnuclear foci which are reminiscent of nuclear speckles. These results show that phosphorylation-regulated RNA target discrimination by heterogeneous nuclear RNA-binding proteins may be a general phenomenon in eukaryotes, and implicate a plant hormone in the regulation of protein dynamics during rapid subnuclear reorganization.     

Structural differences between a ras oncogene protein and the normal protein

One of the most commonly found transforming ras oncogenes in human tumours has a valine codon replacing the glycine codon at position 12 of the normal c-Ha-ras gene. To understand the structural reasons behind cell transformation arising from this single amino acid substitution, we have determined the crystal structure of the GDP-bound form of the mutant protein, p21(Val-12), encoded by this oncogene. We report here the overall structure of p21(Val-12) at 2.2 A resolution and compare it with the structure of the normal c-Ha-ras protein. One of the major differences is that the loop of the transforming ras protein that binds the beta-phosphate of the guanine nucleotide is enlarged. Such a change in the 'catalytic site' conformation could explain the reduced GTPase activity of the mutant, which keeps the protein in the GTP bound 'signal on' state for a prolonged period time, ultimately causing cell transformation.     

A keratin cytoskeletal protein regulates protein synthesis and epithelial cell growth

Cell growth, an increase in mass and size, is a highly regulated cellular event. The Akt/mTOR (mammalian target of rapamycin) signalling pathway has a central role in the control of protein synthesis and thus the growth of cells, tissues and organisms. A striking example of a physiological context requiring rapid cell growth is tissue repair in response to injury. Here we show that keratin 17, an intermediate filament protein rapidly induced in wounded stratified epithelia, regulates cell growth through binding to the adaptor protein 14-3-3sigma. Mouse skin keratinocytes lacking keratin 17 (ref. 4) show depressed protein translation and are of smaller size, correlating with decreased Akt/mTOR signalling activity. Other signalling kinases have normal activity, pointing to the specificity of this defect. Two amino acid residues located in the amino-terminal head domain of keratin 17 are required for the serum-dependent relocalization of 14-3-3sigma from the nucleus to the cytoplasm, and for the concomitant stimulation of mTOR activity and cell growth. These findings reveal a new and unexpected role for the intermediate filament cytoskeleton in influencing cell growth and size by regulating protein synthesis.     

The LDL-receptor-related protein, LRP, is an apolipoprotein E-binding protein

The low-density-lipoprotein (LDL) receptor is a cell-surface protein that plays an important part in the metabolism of cholesterol by mediating the uptake of LDL from plasma into cells. Although LDL particles bind to the LDL receptor through their apolipoprotein B (apo B) and apolipoprotein E (apo E) moieties, other apo E-containing particles, like chylomicron remnants, are not dependent on the LDL receptor for uptake into cells. Chylomicrons formed in the intestinal mucosa during the absorption of the products of digestion, are processed by the peripheral circulation by lipoprotein lipase, which catalyses the breakdown of triglycerides in chylomicrons to free fatty acids and glycerol. The resulting chylomicron remnants, which are cholesterol-rich lipoproteins, are subsequently taken up in the liver. A second distinct protein that binds to apo E-containing lipoproteins, but not to LDL, has been proposed to be the receptor mediating the clearance of chylomicron remnants from the plasma. This protein has a relative molecular mass (Mr) of 56,000 (56K). More recent studies have failed, however, to establish whether this protein is a cell-surface receptor. Here we describe crosslinking experiments in which apo E liposomes were found to bind specifically to the cell surface of hepG2 cells and to human liver membranes. The size and immunological cross-reactivity of the protein to which the liposomes bound was indistinguishable from that of the recently cloned and sequenced LDL-receptor-related protein, LRP. We therefore conclude that the LRP might function as an apo E receptor.