异柠檬酸脱氢酶在植物抗氧化胁迫中的作用

异柠檬酸脱氢酶(ICDH)是连接C-N代谢酶的关键,它催化三羧酸循环(TCA)中的异柠檬酸氧化脱羧形成α-酮戊二酸的可逆反应。ICDH的另一个重要功能是产生细胞质中的NADPH,NADPH在细胞中无处不在,介导许多氧化还原反应,并影响到几乎所有的代谢途径,涉及植物体内的活性氧(ROS)的生产和消耗,因此是传递胁迫信号的中转站。综述异柠檬酸脱氢酶在植物抗氧化胁迫中的作用,便于更好地理解ICDH在氧化胁迫中的信号传递和化学反应中所起的作用,为抗逆分子育种服务。     

脱落酸及其类似物对玉米线粒体异柠檬酸脱氢酶活性的影响

用（＋）ＡＢＡ及其两种ＲＣＡ系列类似物（ＲＣＡ．７ａ,ＲＣＡ．７ｂ）处理主米黄化芽提取的离体线粒体,三者均能促进线粒体上异柠檬酸脱氢酶（ＩＣＤＨ）的活性,另外,（＋）ＡＢＡ、ＲＣＡ．７Ａ及ＲＣＡ．７ｂ分子都有一个环己烯酮环（ｃｙｃｌｏｈｅｘｅｎｏｎｅ ｒｉｎｇ）,差别仅在侧链的不同,这提示,此环己烯酮环是ＡＢＡ表现此种促进作用所必需的。（＋）ＡＢＡ处理离体线粒体之前,先经抗ＡＢＡ结合蛋白的抗体（ａ     

异柠檬酸脱氢酶1 R132H突变型胶质细胞瘤及其维持端粒的代偿机制

异柠檬酸脱氢酶1（isocitrate dehydrogenase 1,IDH1）R132H是II-III级胶质瘤和少突胶质细胞瘤中最常见的突变基因。绝大多数IDH1^R132H突变型胶质细胞瘤并没有通过端粒酶的激活（在端粒酶逆转录酶TERT的介导下以RNA为模板延伸端粒长度）作为其端粒维持机制,而是通过一种依赖于同源重组（homologous recombination,HR）的代偿机制来维持端粒长度,该机制被称为端粒延长替代（alterative lengthening of telomere,ALT）,目前关于ALT形成的机制尚不完全清楚。最近的研究表明,端粒Shelterin复合物组分RAP1和非同源DNA末端连接（non-homologous end joining,NHEJ）修复因子XRCC1的表达在IDH1^R132H突变的胶质细胞瘤中均一致下调,导致端粒功能障碍并促进HR。同时,IDH1^R132H突变通过下调去甲基化酶KDM4B的活性水平,与α地中海贫血伴智力低下综合征X连锁（alpha thalassemia/mental retardation syndrome X-linked,ATRX）基因缺失协同作用促进ALT途径。基于这些研究,本文就突变IDH1^R132H的表达如何引发端粒功能障碍并改变端粒处的DNA修复途径偏好,进而与ATRX丢失协同作用促进ALT发生的机制进行综述。为临床靶向治疗IDH1^R132H突变型胶质细胞瘤提供参考。     

异柠檬酸脱氢酶突变对骨巨细胞瘤增殖水平的影响

[目的]探讨异柠檬酸脱氢酶(IDH)132、140、172位精氨酸突变对骨巨细胞瘤细胞增殖水平的影响。[方法]纳入2020年1月-2022年1月骨巨细胞瘤患者76例,检测骨巨细胞瘤组织和血清中IDH1和IDH2的基因序列和IDH底物2-羟基戊二酸(2HG)的水平。构建IDH1和IDH2敲除的骨巨细胞瘤细胞GCT-404细胞系,转染IDH野生型或突变型后检测细胞的增值水平。根据转染的IDH1或IDH2的基因型进行实验分组：(1)IDH1野生型组;(2)IDH2野生型组;(3)IDH1 R132H组;(4)IDH2 R140Q组;(5)IDH2 R172S组。[结果]骨巨细胞瘤患者中IDH突变型占比32.89%,野生型占比67.11%。IDH野生型患者肿瘤组织和血清中2HG的水平低于IDH突变型患者[(0.22±0.07)nmol/mg vs(2.58±0.24)nmol/mg,(1.73±0.29)μmol/L vs(9.89±1.08)μmol/L,P<0.05]。相比于IDH野生型,IDH1 R132H、IDH2 R140Q或IDH2 R172S显著促进GCT-404细胞的增殖(2.20±0.09 vs 4.25±0.12,P<0.05)和2HG水平(31.34±5.33 vs 56.32±8.32,P<0.05)。敲除IDH1或IDH2后,GCT-404细胞中2HG的水平显著下降(25.78±4.56 vs 10.22±2.04,P<0.05)。2HG处理后,GCT-404细胞的增殖水平均显著上升(1.56±0.08 vs 2.41±0.10,P<0.05)。相比于IDH野生型,IDH1 R132H、IDH2 R140Q或IDH2 R172S显著增加GCT-404细胞中HIF1A的表达。敲低HIF1A后GCT-404细胞的增殖水平显著下降(1.50±0.04 vs 0.94±0.03,P<0.05)。[结论]IDH1 R132H、IDH2 R140Q或IDH2 R172S通过调控2HG水平和HIF1A水平能显著增强骨巨细胞瘤细胞的增殖水平。     

植物NADP^＋-依赖型异柠檬酸脱氢酶的分子进化及功能研究进展

高等植物NADP^＋-依赖型异柠檬酸脱氢酶（ICDHs）定位于细胞质、线粒体、叶绿体和过氧化物酶体等植物细胞的不同部位,由不同的基因编码,属于一个高度保守的多同工酶蛋白家族。对近年来关于植物NADP^＋-依赖型异柠檬酸脱氢酶的分子进化及功能研究进行综述,同时提出了未来植物ICDHs的研究重点和方向。最新的分子系统学分析显示,植物中不同细胞定位的ICDH同工酶聚在各自相应的进化枝上,动物或植物中不同细胞器的ICDH同工酶均来源于各自祖先ICDH基因的独立倍增。最新的功能研究表明,ICDHs催化合成的α-酮戊二酸可为植物细胞对氨的吸收同化提供碳骨架,而NADPH可以维系细胞内的氧化还原平衡,帮助植物抵御氧化胁迫。     

巨噬细胞浸润在低级别胶质瘤预后预测中的研究

目的检测肿瘤相关性巨噬细胞（TAM）在低级别胶质瘤（LGG）分子亚型中的浸润程度和功能状态,分析TAM相关指标在LGG患者预后预测中的价值。 方法获取癌症基因组图谱（TCGA）中529例LGG数据,比较TAM浸润指标在IDH?1/2野生型低级别胶质瘤（IDH?1?/?2-wt LGG）和IDH?1?/?2突变型低级别胶质瘤（IDH?1/2-mu LGG）中的表达水平,分析CD163和IL-10在LGG患者预后预测中的价值。利用免疫组化法检测本中心17例IDH?1/2-?wt和19例IDH?1/2-mu LGG临床样本中TAM的浸润数量。使用Mann-Whitney U检验,COX回归和Kaplan-Meier法进行统计学分析。 结果TCGA数据分析表明,IDH?1/2-?wt LGG中巨噬细胞和TAM标记物CD68（Mann-Whitney U?=?19?425,P = 0.01）、CD11b（Mann-?Whitney U?=?15?836,P < 0.01）、IBA1（Mann-Whitney U?=?17?758,P < 0.01）、CD163（Mann-Whitney U?=?18?112,P < 0.01）、CD204（Mann-Whitney U?=?12?676,P < 0.01）以及TAM趋化因子CCL2（Mann-Whitney U?=?15?841,P < 0.01）、CCL5（Mann-Whitney U?=?11?326,P < 0.01）、POSTN（Mann-Whitney U?=?8?893,P < 0.01）、VEGF-A（Mann-Whitney U?=?14?433,P < 0.01）、Neurotensin（Mann-Whitney U?=?15?556,P < 0.01）的表达水平高于IDH?1/2-mu LGG。本中心免疫组化提示TAM在IDH?1/2-wt组中的浸润数量高于IDH?1/2-mu组（Mann-Whitney U?=?51,P < 0.01;Mann-Whitney U?=?35,P < 0.01;Mann-Whitney U?=?20,P < 0.01）。IDH?1/2-?wt LGG较IDH?1/2-mu LGG表达更多的免疫抑制因子TGF-β（Mann-Whitney U?=?15?459,P < 0.01）和IL-10（Mann-Whitney U?=?17?334,P < 0.01）。生存分析结果表明,CD163^highIDH-?wt LGG患者的预后较CD163^lowIDH-wt LGG患者差（P = 0.01）,IL-10^highIDH-mu LGG患者预后较IL-?10^lowIDH-?mu LGG患者差（P < 0.05）。 结论IDH?1/2-wt LGG和IDH?1/2-mu LGG中TAM的浸润数量及免疫抑制状态存在差异;TAM标志物CD163表达水平与IDH?1/2-wt LGG患者预后负相关,TAM相关免疫抑制因子IL-10表达水平与IDH?1/2-mu LGG患者的预后负相关。     

枯草杆菌及大肠杆菌异柠檬酸脱氢酶的酶学性质研究

对枯草杆菌异柠檬酸脱氢酶（BsIDH）、大肠杆菌异柠檬酸脱氢酶（EcIDH）和大肠杆菌异柠檬酸脱氢酶的突变体酶（EmIDH）进行了纯化和酶学性质鉴定。BsIDH和EcIDH对辅酶NADP^＋的特异性与NAD^＋相比,分别是NAD^＋的1330倍和3890倍。而EmIDH对NAD^＋的特异性与NADP^＋相比,是NADP^＋的122倍。因此BsIDH和EcIDH是NADP^＋依赖性异柠檬酸脱氢酶,而EmIDH的辅酶特异性已转换为NAD^＋依赖性。EcIDH、BsIDH和EmIDH对底物异柠檬酸的Km值分别为67.4 μmol/L、60.6 μmol/L和105.6 μmol/L。BsIDH和EcIDH的最适反应pH分别为8.2和8.0,EmIDH的最适pH为7.0。BsIDH和EmIDH的最适反应温度是45℃,EcIDH的最适温度为43℃。三种IDH的活性依赖于不同的二价金属离子的存在,Mn^2＋ 、Mg^2＋存在时酶活性最强,Cu^2＋ 、Ca^2＋ 、Zn^2＋和Ni2＋强烈抑制酶的活性。系统的酶学性质研究为深入认识IDH的催化与调节机制提供了更多依据。     

人脑胶质瘤中的IDH1/2突变

胶质母细胞瘤的基因组突变分析中发现的异柠檬酸脱氢酶(isocitrate dehydrogenase,IDH1)突变对胶质瘤的认识具有突破性意义。随后,在胶质瘤中发现了IDH1的R132碱基和IDH2的R172碱基突变。IDH1突变较多的发生在WHOII-III级胶质瘤和继发胶质母细胞瘤中。这种突变改变了异柠檬酸脱氢酶的结构,从而使将异柠檬酸转化为a-酮戊二酸的能力丧失,而获得将a-酮戊二酸转化为2-羟基戊二酸这一新的酶活性。在临床中,IDH1和IDH2突变已经显示对胶质瘤患者有诊断和预后意义。同时,现今也发展了一些检测方法。     

NAD+-依赖型异柠檬酸脱氢酶的结构和功能研究进展

NAD^ —依赖型异柠檬酸脱氢酶是一个核编码线粒体酶,参与三羧酸循环,负责催化异柠檬酸氧化脱羧成α-酮戊二酸,是循环路径中的限速酶。目前在酶学性质、亚基组成、基因克隆、蛋白组装与转运,以及功能等方面开展了许多研究。本文就这些方面的新进展进行综述。     

Primary Orthotopic Glioma Xenografts Recapitulate Infiltrative Growth and Isocitrate Dehydrogenase I Mutation

Malignant gliomas constitute a heterogeneous group of highly infiltrative glial neoplasms with distinct clinical and molecular features. Primary orthotopic xenografts recapitulate the histopathological and molecular features of malignant glioma subtypes in preclinical animal models. To model WHO grades III and IV malignant gliomas in transplantation assays, human tumor cells are xenografted into an orthotopic site, the brain, of immunocompromised mice. In contrast to secondary xenografts that utilize cultured tumor cells, human glioma cells are dissociated from resected specimens and transplanted without prior passage in tissue culture to generate primary xenografts. The procedure in this report details tumor sample preparation, intracranial transplantation into immunocompromised mice, monitoring for tumor engraftment and tumor harvesting for subsequent passage into recipient animals or analysis. Tumor cell preparation requires 2 hr and surgical procedure requires 20 min/animal.     

变铅青链霉菌异柠檬酸脱氢酶的异源表达及序列分析

通过同源性引物成功扩增和克隆了变铅青链霉菌(Streptomyces lividans)TK54的异柠檬酸脱氢酶(isocitrate dehydrogenase, IDH) (简称SlIDH)基因icd (GenBank登录号为EU661252).icd的起始密码子为GTG,GC含量为69.55 %,显示了链霉菌基因的高GC含量特征,实现了SlIDH在E.coli中的异源高效表达.0.5 mmol/L的IPTG为最佳诱导条件.SlIDH的分子量约为80 kD.在Mn~(2+)或Mg~(2+)条件下,SlIDH以NADP~+为辅酶时的活性分别为7.94 U/mg及4.00 U/mg,以NAD~+为辅酶时的活性分别为0.58 U/mg及0.27 U/mg,SlIDH更偏爱以NADP~+为辅酶.与不同种属单体IDH的氨基酸序列比对显示,SlIDH与单体IDH的序列一致性均在60 %以上.因此本工作首次以实验性证据初步鉴定了SlIDH为NADP-依赖型单体IDH.本工作为进一步探索单体IDH的结构与功能以及单体IDH与同源二聚体IDH的进化关系奠定了基础.     

Characterizing Lysine Acetylation of Isocitrate Dehydrogenase in Escherichia coli

The Escherichia coli isocitrate dehydrogenase (ICDH) is one of the tricarboxylic acid cycle enzymes, playing key roles in energy production and carbon flux regulation. E. coli ICDH was the first bacterial enzyme shown to be regulated by reversible phosphorylation. However, the effect of lysine acetylation on E. coli ICDH, which has no sequence similarity with its counterparts in eukaryotes, is still unclear. Based on previous studies of E. coli acetylome and ICDH crystal structures, eight lysine residues were selected for mutational and kinetic analyses. They were replaced with acetyllysine by the genetic code expansion strategy or substituted with glutamine as a classic approach. Although acetylation decreased the overall ICDH activity, its effects were different site by site. Deacetylation tests demonstrated that the CobB deacetylase could deacetylate ICDH both in vivo and in vitro, but CobB was only specific for lysine residues at the protein surface. On the other hand, ICDH could be acetylated by acetyl-phosphate chemically in vitro. And in vivo acetylation tests indicated that the acetylation level of ICDH was correlated with the amounts of intracellular acetyl-phosphate. This study nicely complements previous proteomic studies to provide direct biochemical evidence for ICDH acetylation.     

Shifted Cytosolic NADP+-Dependent Isocitrate Dehydrogenase on 2-D Gel in the Brain of Genetically Epileptic El Mice

We observed a spot on two-dimensional (2-D) gel in the epileptic mutant strain El mice with a similar molecular weight but with a different isoelectric point of approximately 0.2, compared with its mother strain ddY mice. The collected protein from the El mice was identified as cytosolic NADP⁺-dependent isocitrate dehydrogenase by internal amino acid sequencing. The enzyme is known to be maximally active during the development of the brain and to play an important role in NADPH production for fatty acids and cholesterol synthesis. In addition, alterations in cholesterol synthesis early in the development of the mammalian brain have been reported to lead to chronic epilepsy. The results in the present study therefore suggest that cytosolic NADP⁺-dependent isocitrate dehydrogenase might be involved in the epileptogenesis of the El mouse.     

Complete Reversal of Coenzyme Specificity of Isocitrate Dehydrogenase from <Emphasis Type="Italic">Haloferax volcanii</Emphasis>

Haloferax volcanii Ds-threo-isocitrate dehydrogenase (ICDH) was highly expressed in bacteria as inclusion bodies. The recombinant enzyme was refolded, purified and characterized, and was found to be NADP-dependent like the wild-type protein. Sequence alignment of several isocitrate dehydrogenases from evolutionarily divergent organisms including H. volcanii revealed that the amino acid residues involved in coenzyme specificity are highly conserved. Our objective was to switch the coenzyme specificity of halophilic ICDH by altering these conserved amino acids. We were able to switch coenzyme specificity from NADP⁺ to NAD⁺ by changing five amino acids by site-directed mutagenesis (Arg291, Lys343, Tyr344, Val350 and Tyr390). The five mutants of ICDH were overexpressed in Escherichia coli as inclusion bodies and each recombinant ICDH protein was refolded and purified, and its kinetic parameters were determined. Coenzyme specificity did not switch until all five amino acids were substituted.     

甘蔗绵蚜不同地理种群异柠檬酸脱氢酶（IDH）的多样性

用等位酶分析方法对三个用药背景不同的甘蔗绵蚜地理种群在9种酶（EST,G3PD,HEX,IDH,LDH,MDH,ME,PGI和PCM）上的遗传组成进行检测。结果显示：甘蔗绵蚜在9种酶共检测到9个等位酶位点,仅IDH位点具有多态性。在多态性的IDH位点共检测到3个等位基因,其中连续两年未曾用药的两院种群和用药较少的木棠种群均具有三个等位基因（a,b和c）,而用药次数最多的临高种群仅存在两个等位基因（a和b）。等位基因a的频率从两院种群到临高种群逐渐升高,而等位基因b的频率却逐渐降低。说明IDH在甘蔗绵蚜的种群遗传进化过程中起着重要作用,杀虫剂的选择压力可能对甘蔗绵蚜地理种群的遗传结构具有分化作用,同时也说明IDH在甘蔗绵蚜对杀虫剂的抗性产生中具有重要作用。IDH-a频率的升高,可能导致甘蔗绵蚜对杀虫剂产生抗性,可通过检测IDH位点等位基因频率的变化来监测甘蔗绵蚜对杀虫剂的抗性。     

Isocitrate dehydrogenase from the hyperthermophile Aeropyrum pernix: X-ray structure analysis of a ternary enzyme-substrate complex and thermal stability

Isocitrate dehydrogenase from Aeropyrum pernix (ApIDH) is a homodimeric enzyme that belongs to the beta-decarboxylating dehydrogenase family and is the most thermostable IDH identified. It catalyzes the NADP+ and metal-dependent oxidative decarboxylation of isocitrate to alpha-ketoglutarate. We have solved the crystal structures of a native ApIDH at 2.2 A, a pseudo-native ApIDH at 2.1 A, and of ApIDH in complex with NADP+, Ca2+ and d-isocitrate at 2.3 A. The pseudo-native ApIDH is in complex with etheno-NADP+ which was located at the surface instead of in the active site revealing a novel adenine-nucleotide binding site in ApIDH. The native and the pseudo-native ApIDHs were found in an open conformation, whereas one of the subunits of the ternary complex was closed upon substrate binding. The closed subunit showed a domain rotation of 19 degrees compared to the open subunit. The binding of isocitrate in the closed subunit was identical with that of the binary complex of porcine mitochondrial IDH, whereas the binding of NADP+ was similar to that of the ternary complex of IDH from Escherichiacoli. The reaction mechanism is likely to be conserved in the different IDHs. A proton relay chain involving at least five solvent molecules, the 5'-phosphate group of the nicotinamide-ribose and a coupled lysine-tyrosine pair in the active site, is postulated as essential in both the initial and the final steps of the catalytic reaction of IDH. ApIDH was found to be highly homologous to the mesophilic IDHs and was subjected to a comparative analysis in order to find differences that could explain the large difference in thermostability. Mutational studies revealed that a disulfide bond at the N terminus and a seven-membered inter-domain ionic network at the surface are major determinants for the higher thermostability of ApIDH compared to EcIDH. Furthermore, the total number of ion pairs was dramatically higher in ApIDH compared to the mesophilic IDHs if a cutoff of 4.2 A was used. A calculated net charge of only +1 compared to -19 and -25 in EcIDH and BsIDH, respectively, suggested a high degree of electrostatic optimization, which is known to be an important determinant for increased thermostability.