蛋白质的氧化重折叠

经过近几十年来广泛而深入的研究,蛋白质氧化重折叠的机制已得到相当详细的阐明。1在已研究过的蛋白质中,大多数蛋白质都是沿着多途径而非单一、特定的途径进行氧化重折叠,这与折叠能量景观学说是一致的。2正是氨基酸残基间的天然相互作用而不是非天然的相互作用控制蛋白质的折叠过程。这一结论与含非天然二硫键的折叠中间体在牛胰蛋白酶抑制剂（BPTI）折叠中所起的重要作用并非相互排斥,因为后者仅仅是进行链内二硫键重排的化学反应所必需,与控制肽链折叠无直接关系。3根据对BPTI的研究,二硫键曾被认为仅仅具有稳定蛋白质天然结构的作用,既不决定折叠途径也不决定其三维构象。这一观点不适用于其它蛋白质。对凝乳酶原的研究表明,天然二硫键的形成是恢复天然构象的前提。天然二硫键的形成与肽键的正确折叠相辅相成,更具有普遍意义。4在氧化重折叠的早期,二硫键的形成基本上是一个随机过程,随着肽链的折叠二硫键的形成越来越受折叠中间体构象的限制。提高重组蛋白质的复性产率是生物技术领域中的一个巨大的挑战。除了分子聚集外,在折叠过程中所形成的二硫键错配分子是导致低复性率的另一个主要原因。氧化重折叠机制的阐明为解决此问题提供了有益的启示。如上所述,在折叠的后期,二硫键的形成决定于折叠中间体的构象,类天然、有柔性的结构有利于天然二硫键形成和正确折叠,具有这类结构的分子为有效的折叠中间体,最终都能转变为天然产物;而无效折叠中间体往往具有稳定的结构,使巯基、二硫键内埋妨碍二硫键重排,并因能垒的障碍不利于进一步折叠。因此,降低无效折叠中间体的稳定性使之转变为有效折叠中间体是提高含二硫键蛋白质复性率的一条基本原则,实验证明,碱性pH、低温、降低蛋白质稳定性的试剂、蛋白质二硫键异构酶、改变蛋白质一级结构是实现这一原则的有效手段。此外,这里还就氧化重折叠的基础和应用研究的前景进行了讨论。     

植物氧化胁迫信号应答的研究进展

干旱、盐害以及极端温度等非生物胁迫是影响植物生长发育的重要因子。植物在遭受胁迫时,活性氧的快速积累导致胞内氧化还原稳态被打破,进一步诱导产生次级氧化胁迫损伤。除了初级非生物胁迫胁迫信号外,植物细胞也需要产生一系列的次级氧化胁迫信号。氧化还原信号的感知与传递在植物氧化胁迫应答过程中发挥重要的作用,其生物化学基础是功能蛋白质发生的氧化还原翻译后修饰,分别又由多种具有氧化还原活性的小分子介导。本文综述了近年来植物氧化还原信号的研究进展,展望了未来的研究方向,以期为研究植物氧化胁迫应答及氧化还原信号转导提供参考。     

镉暴露对黑斑蛙精巢ROS的诱导及其蛋白质氧化损伤作用机理

重金属镉对精巢发育、呼吸及神经系统信号转导等途径均有不良影响,被认为是造成两栖动物种群数量急剧下降的重要原因之一。然而,有关镉对精巢损伤的分子机理还不清楚。通过对镉暴露后的黑斑蛙精巢活性氧自由基(ROS)、蛋白质羰基(PCO)以及DNA蛋白质交联(DPC)等指标的系统分析,探讨了镉对精巢毒害的分子作用机理。随镉浓度的增加,黑斑蛙精巢细胞线粒体ROS随镉暴露浓度的增加而升高,0.5、1.0 mg/L镉染毒组与对照组比较有显著性差异(P<0.05);精巢组织PCO和DPC也随镉暴露浓度的增加而逐渐上升,且均呈明显的浓度-效应关系。结果表明:镉诱导机体产生ROS,进而导致蛋白质氧化损伤以及DNA损伤,说明精巢组织ROS的产生是镉致雄性生殖毒效应机制的重要因素之一。     

球孢白僵菌响应氧化胁迫的分子机制研究进展

球孢白僵菌作为模式丝状真菌,以分生孢子、菌丝体、虫菌体等多种形态存在,在真菌孢子发育、寄主与宿主互作的研究中具有重要意义。同时,球孢白僵菌又是一类广泛应用的真菌杀虫剂,对森林防护和农业生产具有实际应用价值。球孢白僵菌的相关基因被敲除后,突变体响应氧化胁迫,孢子发育和毒力会发生改变。本文综述了近年来球孢白僵菌在响应氧化胁迫方面的研究进展,为丝状真菌氧化胁迫信号途径的研究提供参考。     

蛋白质巯基亚硝基化———一种典型氧化还原依赖的蛋白质翻译后修饰

综述了蛋白质巯基亚硝基化修饰的特点、检测方法、功能研究、相关疾病和发展态势.蛋白质巯基亚硝基化(S-nitrosation)是指一氧化氮(nitricoxide,NO)及其衍生物修饰蛋白质半胱氨酸(cysteine,Cys)巯基—SH生成—SNO,其是一种典型的氧化还原依赖的蛋白质翻译后修饰,也是一氧化氮发挥其广泛信号转导作用的新的重要途径.     

tRNA介导蛋白质工程

在遗传信息从DNA到蛋白质流动的过程中,tRNA携带特异的氨基酸参与蛋白质合成,对于维持蛋白质翻译的忠实性起着非常重要的作用,生物体内共有20种氨酰tRNA合成酶,每一种均对尖于一种氨基酸和一个tRNA类型,但是这种翻译过程仅仅限于20种天然氨基酸,因此在进行传统的蛋白质工程研究时常常受到限制,事实上,在蛋白质工程中借助于校正 tRNA定点掺入非天然氨基酸可以提供蛋白质的结构信息,改进蛋白质检测与分离的方法,甚至赋予蛋白质某些新的特性,随着生物技术的发展和完善,tRNA介导蛋白质工程将不仅在蛋白质工程中发挥潜能,而且在研制新型生物材料和疾病诊断及药物治疗方面起到推动作用。     

氧化还原稳态调控机制与肿瘤治疗

肿瘤往往处于高氧化状态,随着研究的不断深入,人们逐渐发现细胞内氧化还原状态与肿瘤的关系高度复杂,其促瘤和抑瘤效应均有报道。明确氧化还原稳态调控机制,有望为精准靶向该机制,提高肿瘤治疗效果带来新的契机。该文将对氧化还原稳态调控机制及其在肿瘤治疗中应用的最新进展作一综述。     

Caspase-1介导的蛋白质分泌机制新进展

此前多数观点认为内质网一高尔基体运输是经典的分泌型蛋白质分泌出胞的途径。它们要先翻译出一段信号肽,这段信号肽将mRNA连同核糖体转移到内质网上并使合成的蛋白质保存于内质网内,而后通过高尔基体分泌出胞。     

tRNA介导蛋白质工程

在遗传信息从DNA到蛋白质流动的过程中,tRNA携带特异的氨基酸参与蛋白质合成,对于维持蛋白质翻译的忠实性起着非常重要的作用。生物体内共有20种氨酰tRNA合成酶,每一种均对应于一种氨基酸和一个tRNA类型。但是这种翻译过程仅仅限于20种天然氨基酸,因此在进行传统的蛋白质工程研究时常常受到限制。事实上,在蛋白质工程中借助于校正tRNA定点掺入非天然氨基酸可以提供蛋白质的结构信息,改进蛋白质检测与分离的方法,甚至赋予蛋白质某些新的特性。随着生物技术的发展和完善,tRNA介导蛋白质工程将不仅在蛋白质工程中发挥潜能,而且在研制新型生物材料和疾病诊断及药物治疗方面起到推动作用。     

Protein Thermostability: Mechanism and Control Through Protein Engineering

Chemical modification and protein engineering especially are now the useful tools for thermo-stabilizing proteins, and also for elucidating the mechanism of protein stability. The information on the mechanism so far accumulated indicate that a single or few amino acid replacement(s) in a protein is/are sufficient to enhance protein thermostability. Salt bridges inside protein molecule or decrease of internal or external hydrophobicity, respectively, may contribute to increased thermostability. However, generalized molecular reasons for protein thermostability and generalized methods for protein stabilization have not yet been proposed. Some of typical examples of the application of protein engineering to stabilize proteins are presented. They are based on information concerning the tertiary structure of the proteins or their related proteins. Even if such structural information is unavailable, one can replace amino acid(s) in a protein by mutagenesis of the gene coding for the protein via the application of chemicals to the gene (or the plasmid harbouring the gene) or organism. A promising strategy involving transfer of the identified gene into a thermophile and subsequent growth at higher temperatures (thermal adaptation) is described.     

Molecular and Biochemical Characterization of Short Duration Quality Protein Maize

Fifty microsatellite or simple sequence repeat (SSR) markers, spread across the maize genome were used for analyzing a set of 19 elite Quality Protein Maize (QPM) lines, including seventeen lines developed in India and two at CIMMYT, Mexico. Polymorphic profiles for 47 SSR loci have aided in differentiating the QPM inbred lines. The polymorphism information content (PIC) values among the inbreds ranged from 0.06 (umc2229) to 0.70 (umc1071) with an average of 0.45 per primer-pair. The genetic relationships as indicated by the cluster analysis of SSR data were largely in congruence with the known pedigree of the QPM lines. The study resulted in identification of two SSR markers, umc1071 and umc1063 with higher PIC values of 0.70 and 0.64, respectively. The tryptophan content among the genotypes was found to vary considerably. Two genotypes viz., VOL 2 and VOL 8 were found to differ significantly for tryptophan content (0.51% and 0.94%, respectively). Both these QPM genotypes being derived from the same non-QPM parent CM 145, makes them ideal for mapping of modifiers for tryptophan content.     

Biochemical Evidence for a Timing Mechanism in Prochlorococcus

Organisms coordinate biological activities into daily cycles using an internal circadian clock. The circadian oscillator proteins KaiA, KaiB, and KaiC are widely believed to underlie 24-h oscillations of gene expression in cyanobacteria. However, a group of very abundant cyanobacteria, namely, marine Prochlorococcus species, lost the third oscillator component, KaiA, during evolution. We demonstrate here that the remaining Kai proteins fulfill their known biochemical functions, although KaiC is hyperphosphorylated by default in this system. These data provide biochemical support for the observed evolutionary reduction of the clock locus in Prochlorococcus and are consistent with a model in which a mechanism that is less robust than the well-characterized KaiABC protein clock of Synechococcus is sufficient for biological timing in the very stable environment that Prochlorococcus inhabits.Cyanobacteria are photosynthetic prokaryotes that are known to possess a true circadian clock. Gene expression and other biological activities follow rhythmic cycles with a circa 24-h period. Rhythmic behavior is maintained even in the absence of environmental stimuli such as light and temperature. The underlying core oscillator consisting of the clock proteins KaiA, KaiB, and KaiC is the only characterized prokaryotic circadian oscillator. It was previously demonstrated that these three proteins, together with ATP, can produce 24-h oscillations of KaiC phosphorylation in vitro (17). The essential roles of KaiA and KaiB in oppositely influencing KaiC phosphorylation are well documented for the oscillator of “Synechococcus elongatus” PCC 7942 (hereafter S. elongatus), the species for which most bacterial circadian research has been conducted. Thus, it is puzzling that marine cyanobacteria of the genus Prochlorococcus, probably the most abundant photosynthetic organisms on Earth (5, 28), contain homologs of only two of these clock proteins, KaiC and KaiB (3, 11, 20). Laboratory cultures (10) as well as natural Prochlorococcus populations (24) display a rhythmic cell cycle together with a daily periodicity of gene expression that can be explained by the functioning of a circadian clock. Alternatively, these rhythms could be controlled directly by the daylight (10). The functional role of the Kai proteins from Prochlorococcus has remained entirely unclear and has not been experimentally addressed thus far.In the well-studied protein clock of S. elongatus, KaiC hexamers are at the center of the circadian oscillator, combining three intrinsic enzymatic activities: autokinase, autophosphatase, and ATPase. KaiA and KaiB modulate KaiC''s activities in opposite manners. KaiA seems to be essential for the shift between autophosphatase and autokinase, and for generating KaiC phosphorylation rhythms, by stabilizing C-terminal residues of KaiC, the A-loops (12). Thus, the absence of KaiA should have consequences for the enzymatic activities of the remaining Kai proteins of Prochlorococcus. In this study, the previously unknown functions of the Prochlorococcus sp. strain MED4 protein KaiB (ProKaiB) and ProKaiC are examined. In our in vitro experiments, we analyzed the recombinant proteins ProKaiB and ProKaiC in direct comparison to the core oscillator of S. elongatus, which consists of S. elongatus KaiA (SynKaiA), SynKaiB, and SynKaiC. We show here that both clock proteins from Prochlorococcus sp. strain MED4 independently exhibit their known biochemical functions, although the influence of ProKaiB on ProKaiC dephosphorylation is different certainly due to the absence of KaiA, the third protein of the oscillator. For ProKaiC, we demonstrate ATPase activity as well as the phosphorylation of serine 427 (S427) and threonine 428 (T428) using mass spectrometry and high-resolution sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE). Moreover, we suggest that the deletion of kaiA is compensated by the enhanced autophosphorylation activity of ProKaiC. Our results might have further implications for the analysis of a possible timing mechanism in other bacterial species, such as purple bacteria that encode KaiB and KaiC homologs but that lack the KaiA component.     

Compatible solutes reduce ROS-induced potassium efflux in Arabidopsis roots

Reactive oxygen species (ROS) are known to be primarily responsible for the impairment of cellular function under numerous abiotic and biotic stress conditions. In this paper, using non-invasive microelectrode ion flux measuring (MIFE) system, we show that the application of a hydroxyl radical (OH*)-generating Cu2+/ascorbate (Cu/a) mixture to Arabidopsis roots results in a massive, dose-dependent efflux of K+ from epidermal cells in the elongation zone. Pharmacological experiments suggest that both outward-rectifying K+ channels and non-selective cation channels (NSCCs) mediate such effluxes. Low (5 mM) concentrations of compatible solutes (glycine betaine, proline, mannitol, trehalose or myo-inositol) significantly reduces OH*-induced K+ efflux, similar to our previous reports for NaCl-induced K+ efflux. Importantly, a significant reduction in K+ efflux was found using osmolytes with no reported free radical scavenging activity, as well as those for which a role in free radical scavenging has been demonstrated. This indicates that compatible solutes must play other (regulatory) roles, in addition to free radical scavenging, in mitigating the damaging effects of oxidative stress.     

冷敏感植物的低温光抑制及其生化保护机制

阐述了冷敏感植物光抑制的发光机理、光与低温在光抑制中的关系及冷敏感植物在低温光抑制过程中的生理化变化,并结合冷锻炼对植物的影响,介绍了植物防制光破坏的生化机制。     

The Mechanism of N-Terminal Acetylation of Protein

Abstract

This article summarizes all experimental facts concerning the cold denaturation of single-domain, multi-domain, and multimeric globular proteins in aqueous solutions with and without urea and guanidine hydrochloride. The facts obtained by various experimental techniques are analyzed thermodynamically and it is shown that the cold denaturation is a general phenomenon caused by the very specific and strongly termperature-dependent interaction of protein nonpolar groups with water. Hydration of these groups, in contrast to expectations, is favorable thermodynamically, i.e., the Gibbs energy of hydration is negative and increases in magnitude at a temperature decrease. As a result, the polypeptide chain, tightly packed in a compact native structure, unfolds at a sufficiently low temperature, exposing internal nonpolar groups to water. The reev-aluation of the hydration effect on the base of direct calorimetric studies of protein denaturation and of transfer of non-polar compounds into water leads to revision of the conventional conception on the mechanism of hydrophobic interaction. The last appears to be a complex effect in which the positive contributor is van der Waals interactions between the nonpolar groups and not the hydration of these groups as it was usually supposed.