植物冷激蛋白的研究进展

冷激蛋白是存在于细菌、植物与动物中的一类高度保守的核酸结合蛋白,其通过RNA分子伴侣活性参与转录、翻译及生长发育和逆境胁迫应答等细胞生理活动。本文主要从植物冷激蛋白的结构、表达模式、生物学功能以及应用前景等几个方面介绍了植物冷激蛋白的研究进展。     

Selective mRNA degradation by polynucleotide phosphorylase in cold shock adaptation in Escherichia coli

Upon cold shock, Escherichia coli cell growth transiently stops. During this acclimation phase, specific cold shock proteins (CSPs) are highly induced. At the end of the acclimation phase, their synthesis is reduced to new basal levels, while the non-cold shock protein synthesis is resumed, resulting in cell growth reinitiation. Here, we report that polynucleotide phosphorylase (PNPase) is required to repress CSP production at the end of the acclimation phase. A pnp mutant, upon cold shock, maintained a high level of CSPs even after 24 h. PNPase was found to be essential for selective degradation of CSP mRNAs at 15 degrees C. In a poly(A) polymerase mutant and a CsdA RNA helicase mutant, CSP expression upon cold shock was significantly prolonged, indicating that PNPase in concert with poly(A) polymerase and CsdA RNA helicase plays a critical role in cold shock adaptation.     

A family of cold shock proteins in Bacillus subtilis is essential for cellular growth and for efficient protein synthesis at optimal and low temperatures

Like other bacteria, Bacillus subtilis possesses a family of homologous small acidic proteins (CspB, CspC and CspD, identity > 70%) that are strongly induced in response to cold shock. We show that deletion of cspC or cspD genes did not result in a detectable phenotype; in contrast, csp double mutants exhibited severe reduction in cellular growth at 15°C as well as at 37°C, including impairment of survival during the stationary phase. Two-dimensional gel analysis showed that protein synthesis was deregulated in csp double mutants and that the loss of one or two CSPs led to an increase in the synthesis of the remaining CSP(s) at 37°C and after cold shock, suggesting that CSPs down-regulate production of members from this protein family. A cspB/C/D triple mutant (64BCDbt) could only be generated in the presence of cspB in trans on a plasmid that was not lost, in spite of lack of antibiotic pressure, indicating that a minimum of one csp gene is essential for viability of B . subtilis . After cold shock, synthesis of CspB in 64BCDbt was drastically lower than in wild-type cells accompanied by cessation in growth and strong reduction in general protein synthesis. As CspB, CspC and CspD are shown to bind to RNA in a co-operative and interactive manner, CSPs are suggested to function as RNA chaperones facilitating the initiation of translation under optimal and low temperatures.     

Binding preferential of chickpea cold shock protein during nucleic acid interactions

Cold shock proteins (CSPs) have a widespread occurrence from prokaryotes to eukaryotes including plants. These proteins are known to possess nucleic acid binding properties. CSPs have a single cold shock domain in prokaryotes while N-terminal and C-terminal flanking regions are present in eukaryotic CSPs. The objective of this study was to investigate nucleic acid binding preferential for the chickpea CSP. Full cDNA of chickpea CSP was cloned and sequenced. The sequence was submitted to GenBank (accession no. KM036036) at NCBI. Multiple sequence alignment and phylogenetic analysis further revealed that the inferred amino acid sequence belongs to CSP family. Molecular docking was performed between the CSP and variety of nucleic acids entities. These results suggest that CSPs of chickpea possess preferential binding affinity for single stranded nucleic acids. Docking results suggest that homo-polymer entities of RNA polyU RNA (20mer) form most stable complex.     

Single-stranded nucleic acid binding in Arabidopsis thaliana cold shock protein is cold shock domain dependent

Cold shock proteins (CSPs) are ancient nucleic acid-binding proteins and well conserved from bacteria to animals as well as plants. In prokaryotes, CSPs possess a single cold shock domain (CSD) while animal CSPs, flanked by N- and C-terminal domains, are commonly named Y-box proteins. Interestingly, the plants CSPs contain auxiliary C-terminal domains in addition to their N-terminal CSD. The CSPs have been shown to play important role in development and stress adaptation in various plant species. The objective of this study was to find out the possible nucleic acid-binding affinities of whole CSP as well as independent domains, so that role of each individual domain may be revealed in Arabidopsis thaliana, the model plant species. The structure of CSP 3 protein from A. thaliana was modeled by homology-based approach and docking was done with different nucleic acid types.     

微生物产生的冷休克蛋白研究进展

冷休克蛋白(cold shock protein,Csp)首先在大肠杆菌中发现,它与微生物对冷环境的适应及多种细胞功能有关。冷休克蛋白基因是一段编码70个左右氨基酸的DNA序列,在这段序列中有5′非翻译区(5′UTR)、冷盒及下游盒等特征。冷休克蛋白作为DNA或RNA结合蛋白在基因表达调控过程中起重要作用。冷休克蛋白在转录、mRNA稳定性及翻译等几个水平上被严格调控。     

过表达海洋微生物宏基因组MbCSP提高转基因拟南芥的抗旱和耐寒性

干旱和低温是影响农作物生长发育的重要因素,培育转基因作物是解决此问题的有效途径。冷激蛋白（Cold Shock Proteins,CSPs）是一类高度保守的核酸结合蛋白,参与非生物胁迫应答等细胞生理活动,转CSP基因可增强作物抗逆能力。以海洋微生物宏基因组DNA为模板,采用锚定PCR的方法克隆得到了MbCSP基因,其ORF为216 bp,编码一个由71个氨基酸构成的蛋白;对其进行同源性分析,显示该氨基酸序列与EcCSPG、EcCSPA（大肠杆菌 Escherichia coli）,BsCspB（枯草芽孢杆菌Bacillus subtilis）和BcCspA（蜡样芽孢杆菌 Bacillus cereus）等冷休克蛋白氨基酸序列同源性在60%~90%;对该氨基酸序列进行多重序列比对和系统发育树分析,结果发现MbCSP蛋白包含RNP1（KGFGFI）和RNP2（VFVHF）等CSP蛋白经典的保守结构域,其与EcCspG（大肠杆菌）和CmCspG、CmCspB（堆肥宏基因组）等生物的冷休克蛋白亲缘关系较近。为进一步探讨冷休克蛋白MbCSP的功能,构建了植物表达载体pTF101-MbCSP,采用花序浸染法转化拟南芥,经过除草剂筛选和PCR检测,获得转基因植株。进行半定量RT-PCR分析,选择表达量最高的阳性株系进行后续的生理检测。结果表明：在干旱胁迫及低温胁迫下,转基因拟南芥的生长状况明显优于野生型,其生物量显著高于野生型植株;转基因拟南芥的叶片相对含水量、叶绿素含量和超氧化物歧化酶活性均高于野生型拟南芥,而丙二醛含量则低于野生型拟南芥。上述结果表明,过表达海洋微生物宏基因组MbCSP能够提高转基因拟南芥的抗旱和耐寒能力,为培育转基因作物新品种奠定了基础。     

The effects of ionic strength on protein stability: the cold shock protein family

Continuum electrostatic models are used to examine in detail the mechanism of protein stabilization and destabilization due to salt near physiological concentrations. Three wild-type cold shock proteins taken from mesophilic, thermophilic, and hyperthermophilic bacteria are studied using these methods. The model is validated by comparison with experimental data collected for these proteins. In addition, a number of single point mutants and three designed sequences are examined. The results from this study demonstrate that the sensitivity of protein stability toward salt is correlated with thermostability in the cold shock protein family. The calculations indicate that the mesophile is stabilized by the presence of salt while the thermophile and hyperthermophile are destabilized. A decomposition of the salt influence at a residue level permits identification of regions of the protein sequences that contribute toward the observed salt-dependent stability. This model is used to rationalize the effect of various point mutations with regard to sensitivity toward salt. Finally, it is demonstrated that designed cold shock protein variants exhibit electrostatic properties similar to the natural thermophilic and hyperthermophilic proteins.     

The role of cold-shock proteins in low-temperature adaptation of food-related bacteria

There is a considerable interest in the cold adaptation of food-related bacteria, including starter cultures for industrial food fermentations, food spoilage bacteria and food-borne pathogens. Mechanisms that permit low-temperature growth involve cellular modifications for maintaining membrane fluidity, the uptake or synthesis of compatible solutes, the maintenance of the structural integrity of macromolecules and macromolecule assemblies, such as ribosomes and other components that affect gene expression. A specific cold response that is shared by nearly all food-related bacteria is the induction of the synthesis so-called cold-shock proteins (CSPs), which are small (7 kDa) proteins that are involved in mRNA folding, protein synthesis and/or freeze protection. In addition, CSPs are able to bind RNA and it is believed that these proteins act as RNA chaperones, thereby reducing the increased secondary folding of RNA at low temperatures. In this review established and novel aspects concerning the structure, function and control of these CSPs are discussed. A model for bacterial cold adaptation, with a central role for ribosomal functioning, and possible mechanisms for low-temperature sensing are discussed.     

Differentiation between cold shock proteins and cold acclimation proteins in a mesophilic gram-positive bacterium, Enterococcus faecalis JH2-2.

Transfer of Enterococcus faecalis to a cold temperature (8 degrees C for 4 to 30 h) led to increased expression of 11 cold shock proteins (CSPs). Furthermore, this mesophilic prokaryote synthesized 10 cold acclimation proteins, five of them distinct from CSPs, during continuous growth (4 days) at the same temperature (8 degrees C).     

A cold-regulated nucleic acid-binding protein of winter wheat shares a domain with bacterial cold shock proteins

The molecular mechanisms of cold acclimation are still largely unknown; however, it has been established that overwintering plants such as winter wheat increases freeze tolerance during cold treatments. In prokaryotes, cold shock proteins are induced by temperature downshifts and have been proposed to function as RNA chaperones. A wheat cDNA encoding a putative nucleic acid-binding protein, WCSP1, was isolated and found to be homologous to the predominant CspA of Escherichia coli. The putative WCSP1 protein contains a three-domain structure consisting of an N-terminal cold shock domain with two internal conserved consensus RNA binding domains and an internal glycine-rich region, which is interspersed with three C-terminal CX(2)CX(4)HX(4)C (CCHC) zinc fingers. Each domain has been described independently within several nucleotide-binding proteins. Northern and Western blot analyses showed that WCSP1 mRNA and protein levels steadily increased during cold acclimation, respectively. WCSP1 induction was cold-specific because neither abscisic acid treatment, drought, salinity, nor heat stress induced WCSP1 expression. Nucleotide binding assays determined that WCSP1 binds ssDNA, dsDNA, and RNA homopolymers. The capacity to bind dsDNA was nearly eliminated in a mutant protein lacking C-terminal zinc fingers. Structural and expression similarities to E. coli CspA suggest that WCSP1 may be involved in gene regulation during cold acclimation.