嗜酸氧化亚铁硫杆菌细胞色素C家族afe_0378基因转录表达差异及生物信息学挖掘

嗜酸氧化亚铁硫杆菌是一种极为重要的浸矿微生物,具有氧化各种还原性含硫物及亚铁等功能。采用实时荧光定量PCR技术及生物信息学等手段对该菌一个涉及铁硫氧化由afe_0378基因编码的细胞色素C家族蛋白CycA2进行了研究。结果表明,afe_0378基因在单质硫培养条件下转录水平是亚铁培养条件下的2.67倍。生物信息学分析表明afe_0378编码蛋白CycA2是分子质量为22.959 ku,pI为9.75的结合内膜的周质蛋白,二级结构为全α-螺旋,无β-折叠,包含2个相似结构域及N端一段跨膜疏水螺旋,属于细胞色素C4型蛋白家族。CycA2蛋白分子三维结构模建表明,双血红素与CycA2蛋白序列片段域C48-X-X-C51-H52及C149-X-X-C152-H153中的半胱氨酸共价连接。结合该菌硫代谢背景知识,推测CycA2蛋白的功能是介导细胞色素bc1复合体及终端氧化酶细胞色素aa3复合体之间的电子传递而参与硫代谢。     

Bcl-2家族蛋白调控线粒体膜通透性和细胞色素C释放的新机制

Bcl-2家族蛋白在调控线粒体功能和细胞色素C释放中起重要作用。最近发现Bcl-2分子通过与其他促凋亡分子相互作用调控线粒体外膜通透性,其具体分子机制尚不完全清楚。本课题组采用化学生物学方法,在研究Bax/Bak非依赖的细胞凋亡途径中,发现了一些小分子化合物能够诱导Bim表达量急剧升高,Bim能转位到线粒体上,与Bcl-2相互作用增强,并直接促进Bcl-2构象变化。有意义的是,Bim可以诱导Bcl-2功能发生转换并能够形成大的复合体通道来介导细胞色素C释放。研究结果提示Bcl-2分子可变成促凋亡分子,参与Bax/Bak非依赖的细胞色素C释放和细胞凋亡。     

线粒体与细胞凋亡调控

细胞凋亡是一个受到一系列相关基因严格调控的细胞死亡过程。线粒体是细胞凋亡调控的活动中心。在凋亡因子的刺激下,线粒体释放出不同促凋亡因子如细胞色素C、Smac／Diablo等,激活细胞内凋亡蛋白酶Caspase。我们发现,活化后的Caspase可以反过来作用于线粒体,引发更大量线粒体细胞色素c的释放,构成细胞色素c释放的正反馈调节机制,从而导致电子传递链的中断、膜电势的丧失、胞内ROS的升高以及线粒体产生ATP功能的完全丧失。Bcl-2家族蛋白在细胞色素C释放和细胞凋亡调控中起关键作用。     

心磷脂引起细胞色素C的氧化

心磷脂—细胞色素C—细胞色素C氧化酶体系吸收光谱的研究发现:心磷脂与氧化态细胞色素C结合产生230nm吸收峰;心磷脂与还原态细胞色素C作用,230nm吸收值上升,550nm吸收值下降,表明心磷脂可以引起细胞色素C的氧化。     

细胞色素b5家族蛋白序列分析

以牛肝微粒体细胞色素b5(CYB5-BOVIN)为切入点,利用生物学信息学方法获得一系列细胞色素b5家族的成员蛋白,同时对蛋白序列进行多重对齐分析及进化分析,借此为细胞色素b5蛋白的分子设计与构建提供指导意义。     

生物分离法制备细胞色素C质量的影响因素

细胞色素C为临床上应用广泛的生化药品。为了提高细胞色素C的质量,对制备过程中影响细胞色素C质量的因素进行考察,采用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳法(SDS-PAGE)对不同工艺制备的细胞色素C进行纯度分析,并对SDS-PAGE图谱中约2.5×10~4的蛋白质进行液相色谱-串联质谱法(LC-MS/MS)分析与鉴定。结果表明:在SDS-PAGE电泳图中,使用三氯乙酸(TCA)沉淀剂的传统工艺制备的细胞色素C样品除了有1.22×10~4的细胞色素C,还包括1个约2.5×10~4的蛋白分子;而不使用三氯乙酸沉淀剂的新工艺制备的细胞色素C样品,仅有1.22×10~4的细胞色素C,而未发现约2.5×10~4的蛋白。SDS-PAGE图谱中分子量2.5×10~4的蛋白分子的LC-MS/MS分析与鉴定结果表明其与猪源细胞色素C覆盖率最大(达到76%),结合其分子量大小,可以证明该蛋白为细胞色素C二聚体。细胞色素C制备过程中三氯乙酸沉淀剂易导致细胞色素C形成二聚体,故三氯乙酸是影响细胞色素C质量的关键因素之一。     

缺氧肺动脉平滑肌细胞中线粒体ATP敏感钾通道开放对细胞色素C的分布及细胞增殖的作用

为了探讨线粒体ATP敏感钾通道（mitochondrial ATP-sensitive K^＋channel,mito KATP）和线粒体膜电位（△ψm）在细胞缺氧信号转导中的作用以及对缺氧肺动脉平滑肌细胞中细胞色素C在细胞内的分布及细胞增殖的影响,本实验将人肺动脉平滑肌细胞进行常氧或24h缺氧培养,并将标本分为六组：（1）对照组;（2）mito KATP,开放剂diazoxide组;（3）mito KATP阻断剂5-HD组;（4）24h缺氧组;（5）24h缺氧＋diazoxide组;（6）24h缺氧＋5-HD组。利用激光共聚焦显微镜成像法检测△、ψm;线粒体／胞浆成分分离试剂盒（Bio Vision）分离线粒体和胞浆成分后,Western blot检测两者细胞色素C;Western blot检测细胞中caspase-9的蛋白表达量;MTT法及PI染色后流式细胞仪检测细胞增殖情况。结果显示：（1）diazoxide作用24h后,R-123荧光明显增强,胞浆细胞色素C与线粒体细胞色素C的比值明显降低,caspase-9的蛋白表达显著减少,细胞增殖明显增多、凋亡减少,与正常对照组相比较,均P〈0．05;而5-HD作用24h与正常对照组比较,上述指标无明显变化（P〉0．05）。（2）缺氧24h组,结果与diazoxide组相似,R-123荧光明显增强,胞浆细胞色素C与线粒体细胞色素C的比值明显降低,caspase-9的蛋白表达显著减少,细胞增殖明显增多、凋亡减少,与正常对照组相比较,均P〈0．05;24h缺氧＋diazoxide组与缺氧组相比较,R-123荧光明显增强,胞浆细胞色素C与线粒体细胞色素C的比值明显降低,caspase-9的蛋白表达显著减少,细胞增殖明显增多、凋亡减少（P〈0．05）;而24h缺氧＋5-HD组与缺氧组比较,R-123荧光明显降低,胞浆细胞色素C与线粒体细胞色素C的比值明显升高,caspase-9的蛋白表达显著增加,细胞增殖明显减少、凋亡增多（P〈0．05）。上述实验结果提示,缺氧可以引起mito KATP,的开放以及△ψm的去极化,并进而抑制细胞色素C从线粒体释放到胞浆,抑制线粒体凋亡途径,从而参与并影响肺动脉高压的发生、发展。     

青蒿素诱导人白血病细胞K562凋亡的线粒体机制

目的:探讨青蒿素诱导人白血病细胞K562凋亡的线粒体机制.方法:用青蒿素处理K562细胞.通过MTT比色法检别细胞增殖抑制的效果;荧光显微镜观察细胞的凋亡;流式细胞术(flow cytometry,FCM)进行细胞周期分析;Western-blotting测定药物作用前后线粒体、细胞浆细胞色素C的表达.结果:青蒿素抑制K562细胞的增殖,IC5D为1.5× 10-5mol·L-1;Hoechst33342/PI双荧光染色可观察到明显的核浓缩、凝集等细胞凋亡表现;流式细胞仪检测G2期细胞比例增高,S期减少;Western-blotting检测药物处理细胞后线粒体细胞色素C表达水平下调,细胞浆出现明显细胞色素C蛋白条带.结论:青蒿素可能通过线粒体细胞色素C途径诱导K562细胞凋亡.     

甘蔗细胞色素C基因的电子克隆与分析

以甘蔗类似细胞色素C的EST序列CF576943.1为探针,通过电子克隆技术获得了甘蔗细胞色素C基因（Cytochrome C,Cyt C）的一条cDNA全长序列,命名为ScCyt C.用生物信息学方法对该基因氨基酸序列与组成、亚细胞定位、跨膜区与信号肽、疏水性/亲水性、蛋白质二、三级结构以及功能等进行分析与预测.结果表明：Cyt C基因全长1 073 bp,编码112个氨基酸,该基因位于细胞质,为非分泌型蛋白,无规卷曲为主要二级结构原件,含有1个保守功能域,主要功能为翻译并且在不同植物中具有高度保守性.电子表达分析结果显示,该基因在甘蔗各个组织均有表达,其中在茎和根中的表达量比其他组织类型中表达量高,此外,该基因的表达可能受到低温的调控.为甘蔗细胞色素C基因的结构及其功能的研究奠定了一定的基础.     

Bcl—2家族蛋白与细胞凋亡

Bcl 2家族蛋白是在细胞凋亡过程中起关键性作用的一类蛋白质。在线粒体上 ,Bcl 2家族蛋白通过与其他凋亡蛋白的协同作用 ,调控线粒体结构与功能的稳定性 ,发挥着细胞凋亡“主开关”的作用。Bcl 2家族包括两类蛋白质 :一类是抗凋亡蛋白 ,另一类是促凋亡蛋白。在细胞凋亡时 ,Bcl 2家族中的促凋亡蛋白成员发生蛋白质的加工修饰 ,易位到线粒体的外膜上 ,引起细胞色素c、凋亡诱导因子等其他促凋亡因子的释放 ,导致细胞凋亡 ;而平时被隔离在线粒体等细胞器内的该家族的抗凋亡蛋白成员则抑制细胞色素c和凋亡诱导因子等促凋亡因子的释放 ,具有抑制细胞凋亡的功能。但一旦这类抗凋亡蛋白成员与激活的促凋亡蛋白发生相互作用后 ,便丧失了对细胞凋亡的抑制作用 ,造成线粒体等细胞器的功能丧失和细胞器内促凋亡因子的释放 ,导致细胞凋亡。现以Bcl 2家族调控细胞凋亡的最新研究进展为基础 ,对Bcl 2家族成员及其蛋白质结构、分布和调控细胞凋亡的分子机制进行综述。     

Human cytochrome P-450 PB-1: A multigene family involved in mephenytoin and steroid oxidations that maps to chromosome 10

The cytochrome P-450 monooxygenase system possesses catalytic activity toward many exogenous compounds (e.g., drugs, insecticides, and polycyclic aromatic hydrocarbons) and endogenous compounds (e.g., steroids, fatty acids, and prostaglandins). Multiple forms of cytochrome P-450 with different substrate specificities have been isolated. In the present paper we report the isolation and sequence of a cDNA clone for the human hepatic cytochrome P-450 responsible for mephenytoin (an anticonvulsant) oxidation. The mephenytoin cytochrome P-450 is analogous to the rat cytochrome P-450 form termed PB-1 (family P450C2C). We also report that human PB-1 is encoded by one of a small family of related genes all of which map to human chromosome 10q24.1-10q24.3. The endogenous role of this enzyme appears to be in steroid oxidations. This cytochrome P-450 family does not correspond to any of the hepatic cytochrome P-450 gene families previously mapped in humans.     

肝细胞线粒体凋亡途径及保护机制研究进展

线粒体在能量代谢、自由基产生、衰老、细胞凋亡中起重要作用。线粒体的基因突变,呼吸链缺陷,线粒体膜的改变等因素均会影响整个细胞的正常功能,从而导致病变。凋亡发生时,线粒体通透性转换孔开放,使得线粒体膜电位降低,呼吸链电子传递障碍,细胞ATP合成障碍,生成大量活性氧簇,线粒体发生水肿,线粒体外膜破裂,膜间隙释放大量促凋亡因子如细胞色素C。Bcl-2家族对线粒体的功能有调控作用,介导细胞色素C的释放,Caspase酶原的激活等。病毒性肝炎、酒精性肝病,梗阻性黄疸、肝癌、毒素和药物介导的肝损伤等疾病中都伴随着肝细胞凋亡的发生,目前保肝药物对肝细胞线粒体功能的保护机制主要体现在稳定线粒体膜功能,减轻氧化损伤等方面,针对临床疾病的治疗有很好的指导作用。     

肝细胞线粒体凋亡途径及保护机制研究进展

线粒体在能量代谢、自由基产生、衰老、细胞凋亡中起重要作用。线粒体的基因突变,呼吸链缺陷,线粒体膜的改变等因素均会影响整个细胞的正常功能,从而导致病变。凋亡发生时,线粒体通透性转换孔开放,使得线粒体膜电位降低,呼吸链电子传递障碍,细胞ATP合成障碍,生成大量活性氧簇,线粒体发生水肿,线粒体外膜破裂,膜间隙释放大量促凋亡因子如细胞色素C。Bcl-2家族对线粒体的功能有调控作用,介导细胞色素C的释放,Caspase酶原的激活等。病毒性肝炎、酒精性肝病,梗阻陛黄疸、肝癌、毒素和药物介导的肝损伤等疾病中都伴随着肝细胞凋亡的发生,目前保肝药物对肝细胞线粒体功能的保护机制主要体现在稳定线粒体膜功能,减轻氧化损伤等方面,针对临床疾病的治疗有很好的指导作用。     

Regulation of Angiotensin II–induced Neuromodulation by MARCKS in Brain Neurons

Bcl-2 family members either promote or repress programmed cell death. Bax, a death-promoting member, is a pore-forming, mitochondria-associated protein whose mechanism of action is still unknown. During apoptosis, cytochrome C is released from the mitochondria into the cytosol where it binds to APAF-1, a mammalian homologue of Ced-4, and participates in the activation of caspases. The release of cytochrome C has been postulated to be a consequence of the opening of the mitochondrial permeability transition pore (PTP). We now report that Bax is sufficient to trigger the release of cytochrome C from isolated mitochondria. This pathway is distinct from the previously described calcium-inducible, cyclosporin A–sensitive PTP. Rather, the cytochrome C release induced by Bax is facilitated by Mg²⁺ and cannot be blocked by PTP inhibitors. These results strongly suggest the existence of two distinct mechanisms leading to cytochrome C release: one stimulated by calcium and inhibited by cyclosporin A, the other Bax dependent, Mg²⁺ sensitive but cyclosporin insensitive.     

Tetrabiphenylporphyrin-based receptors for protein surfaces show sub-nanomolar affinity and enhance unfolding

A family of tetrabiphenylporphyrin-based receptors has been synthesized. Receptor 7 showed sub-nanomolar affinity (K(d)=0.67 nM) in binding to the surface of cytochrome c. In addition, a stoichiometric amount of the receptor 7 caused a lowering in the T(m) of cytochrome c from 85 to 35 degrees C.     

Expression, purification, and characterization of the CuA-cytochrome c domain from subunit II of the Bacillus subtilis cytochrome caa3 complex in Escherichia coli

Cytochrome caa3 from Bacillus subtilis is a member of the heme-copper oxidase family of integral membrane enzymes that includes mitochondrial cytochrome c oxidase. Subunit II of cytochrome caa3 has an extra 100 amino acids at its C-terminus, relative to its mitochondrial counterpart, and this extension encodes a heme C binding domain. Cytochrome caa3 has many of the properties of the complex formed between mitochondrial cytochrome c and mitochondrial cytochrome c oxidase. To examine more closely the interaction between cytochrome c and the oxidase we have cloned and expressed the Cu(A)-cytochrome c portion of subunit II from the cytochrome caa3 complex of B. subtilis. We are able to express about 2000 nmol, equivalent to 65 mg, of the Cu(A)-cytochrome c protein per litre of Escherichia coli culture. About 500 nmol is correctly targeted to the periplasmic space and we purify 50% of that by a combination of affinity chromatography and ammonium sulfate fractionation. The cytochrome c containing sub-domain is well-folded with a stable environment around the heme C center, as its mid-point potential and rates of reduction are indistinguishable from values for the cytochrome c domain of the holo-enzyme. However, the Cu(A) site lacks copper leading to an inherent instability in this sub-domain. Expression of B. subtilis cytochrome c, as exemplified by the Cu(A)-cytochrome c protein, can be achieved in E. coli, and we conclude that the cytochrome c and Cu(A) sub-domains behave independently despite their close physical and functional association.     

Neuronal Apoptosis: BH3-Only Proteins the Real Killers?

At present there is a poor understanding of the events that lead up to neuronal apoptosis that occurs in neurodegenerative diseases and following acute ischemic episodes. Apoptosis is critical for the elimination of unwanted neurons within the developing nervous system. The Bcl-2 family of proteins contains pro- and anti-apoptotic proteins that regulate the mitochondrial pathway of apoptosis. There is increasing interest in a subfamily of the Bcl-2 family, the BH3-only proteins, and their pro-apoptotic effects within neurons. Recently ischemic and seizure-induced neuronal injury has been shown to result in the activation of the BH3-only protein, Bid. This protein is cleaved and the truncated protein (tBid) translocates to the mitochondria. The translocation of tBid to the mitochondria is associated with the activation of outer mitochondrial membrane proteins Bax/Bak and the release of cytochrome C from the mitochondria. ER stress also has been implicated as a factor for the induction of apoptosis in ischemic neuronal injury. The induction of ER stress in hippocampal neurons has been shown to activate expression of bb3/PUMA, a member of the BH3-only gene family. Activation of PUMA is associated with the activation and clustering of the pro-apoptotic Bcl-2 family member Bax and the loss of cytochrome C from the mitochondria.     

Identification and location of alpha-helices in mammalian cytochromes P450

A model of the alpha-helical structure of mammalian cytochromes P450 is proposed. The location and sequence of alpha-helices in mammalian cytochromes P450 were predicted from their homology with those of cytochrome P450cam, and these sequences were generally confirmed as helical in nature by using a secondary structure prediction method. These analyses were applied to 26 sequences in 6 gene families of cytochrome P450. Mammalian cytochromes P450 consist of approximately 100 amino acid residues more than cytochrome P450cam. This difference was accounted for by three major areas of insertion: (1) at the N-terminus, (2) between helices C and D and between helices D and E, and (3) between helices J and K. Insertion 1 has been suggested by others as a membrane anchoring sequence, but the apparent insertions at 2 and 3 are novel observations; it is suggested that they may be involved in the binding of cytochrome P450 reductase. Only the mitochondrial cytochrome P450 family appeared to show a major variation from this pattern, as insertion 2 was absent, replaced by an insertion between helices G and H and between helices H and I. This may reflect the difference in electron donor proteins that bind to members of this cytochrome P450 family. Other than these differences the model of mammalian cytochromes P450 proposed maintains the general structure of cytochrome P450cam as determined by its alpha-helical composition.     

The role of soluble cytochrome c-551 in cyclic electron flow-driven active transport in Chromatium vinosum

Spheroplasts have been prepared from the photosynthetic purple sulfur bacterium Chromatium vinosum by lysozyme plus ethylenediaminetetraacetic acid treatment. These spheroplasts are able to take up alanine in the light, but light-dependent alanine uptake is lost upon subsequent washing of the spheroplasts. The observations that alanine uptake driven by a potassium plus valinomycin-induced membrane potential (outside positive) is not affected by washing and that light-dependent alanine uptake can be restored by addition of the supernatant from washing suggest that a soluble electron carrier is lost during washing. Light-dependent alanine uptake in washed spheroplasts could be restored by addition of C. vinosum cytochrome c-551. Other soluble electron carriers from C. vinosum (high-potential iron protein, cytochrome ‘f’, cytochrome c′ and the flavocytochrome c-552) did not restore alanine uptake nor did a variety of other soluble electron carrier proteins from other organisms. These results suggest that cytochrome c-551 functions as an electron carrier in the cyclic electron transfer chain of C. vinosum. Mitochondrial cytochrome c (equine heart) and cytochrome c-551 from Pseudomonas aeruginosa were highly effective in restoring light-dependent alanine uptake in washed spheroplasts, making it likely that C. vinosum cytochrome c-551 is related by evolution to the same cytochrome c family as these other two c cytochromes.