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Wemekamp-Kamphuis HH Karatzas AK Wouters JA Abee T 《Applied and environmental microbiology》2002,68(2):456-463
Listeria monocytogenes is a psychrotrophic food-borne pathogen that is problematic for the food industry because of its ubiquitous distribution in nature and its ability to grow at low temperatures and in the presence of high salt concentrations. Here we demonstrate that the process of adaptation to low temperature after cold shock includes elevated levels of cold shock proteins (CSPs) and that the levels of CSPs are also elevated after treatment with high hydrostatic pressure (HHP). Two-dimensional gel electrophoresis combined with Western blotting performed with anti-CspB of Bacillus subtilis was used to identify four 7-kDa proteins, designated Csp1, Csp2, Csp3, and Csp4. In addition, Southern blotting revealed four chromosomal DNA fragments that reacted with a csp probe, which also indicated that a CSP family is present in L. monocytogenes LO28. After a cold shock in which the temperature was decreased from 37 degrees C to 10 degrees C the levels of Csp1 and Csp3 increased 10- and 3.5-fold, respectively, but the levels of Csp2 and Csp4 were not elevated. Pressurization of L. monocytogenes LO28 cells resulted in 3.5- and 2-fold increases in the levels of Csp1 and Csp2, respectively. Strikingly, the level of survival after pressurization of cold-shocked cells was 100-fold higher than that of cells growing exponentially at 37 degrees C. These findings imply that cold-shocked cells are protected from HHP treatment, which may affect the efficiency of combined preservation techniques. 相似文献
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In vivo radiolabeling of chloroplast proteins in grain sorghum (Sorghum bicolor L. cv. Texas 610) leaves and their separation by one-dimensional electrophoresis revealed at least 6 heat shock proteins (HSPs) between 24 and 94 kDa. of which the 24 kDa protein was the most prominent. All of these chloroplast heat shock proteins were found exclusively in the stroma. The 24 kDa heat shock protein, upon closer examination using two-dimensional electrophoresis proved to be two similarly-sized heat shock polypeptides with identical molecular masses and level of radiolahel incorporation, hut slightly different in isoeiectric points, suggesting isomers. Separation of stromal heat shock proteins synthesised in two other C4 monocotyledons ( Punicum miliaceum L. and Umchloa panictrides L.) revealed similar putative isomers. each of 24 kDa. Several other, previously unidentified, heat shock proteins between 22 and 38 kDa were also observed in all three species. In P. miliaceum. the most prominent HSP was the pair of 24 kDa proteins, whereas in U. panicoides. it was a group of 35 to 38 kDa HSPs that was most abundant. In vivo chlorophyll fluorescence measurements showed that no sustained impairment to photosynthetic efficiency had occurred for each species after the heat stress regime. However, when cytoplasmic protein synthesis was inhibited during the high temperature treatment, a dramatic decrease was observed in photosynthetic efficiency, suggesting a possible protective role for chloroplast heat shock proteins. It was also shown that a single chloroplast HSP complex of around 380 kDa was observed in the stroma of both 5. bicolor and P. miliaceum leaves in vivo. This was in contrast to the smaller HSP complex (200–265 kDa) observed in previous studies on chloroplast heat shock proteins in Cj species. 相似文献
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Cold shock proteins (Csps) play an important role in cold shock response of a diverse number of organisms ranging from bacteria to humans. Numerous studies of the Csp from various species showed that a two-state folding mechanism is conserved and the transition state (TS) appears to be very compact. However, the atomic details of the folding mechanism of Csp remain unclear. This study presents the folding mechanism of Csp in atomic detail using an all-atom Go model-based simulations. Our simulations predict that there may exist an en route intermediate, in which β strands 1-2-3 are well ordered and the contacts between β1 and β4 are almost developed. Such an intermediate might be too unstable to be detected in the previous fluorescence energy transfer experiments. The transition state ensemble has been determined from the P(fold) analysis and the TS appears even more compact than the intermediate state. 相似文献
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Enhanced Levels of Cold Shock Proteins in Listeria monocytogenes LO28 upon Exposure to Low Temperature and High Hydrostatic Pressure 总被引:2,自引:0,他引:2
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Henrike H. Wemekamp-Kamphuis Andreas K. Karatzas Jeroen A. Wouters Tjakko Abee 《Applied microbiology》2002,68(2):456-463
Listeria monocytogenes is a psychrotrophic food-borne pathogen that is problematic for the food industry because of its ubiquitous distribution in nature and its ability to grow at low temperatures and in the presence of high salt concentrations. Here we demonstrate that the process of adaptation to low temperature after cold shock includes elevated levels of cold shock proteins (CSPs) and that the levels of CSPs are also elevated after treatment with high hydrostatic pressure (HHP). Two-dimensional gel electrophoresis combined with Western blotting performed with anti-CspB of Bacillus subtilis was used to identify four 7-kDa proteins, designated Csp1, Csp2, Csp3, and Csp4. In addition, Southern blotting revealed four chromosomal DNA fragments that reacted with a csp probe, which also indicated that a CSP family is present in L. monocytogenes LO28. After a cold shock in which the temperature was decreased from 37°C to 10°C the levels of Csp1 and Csp3 increased 10- and 3.5-fold, respectively, but the levels of Csp2 and Csp4 were not elevated. Pressurization of L. monocytogenes LO28 cells resulted in 3.5- and 2-fold increases in the levels of Csp1 and Csp2, respectively. Strikingly, the level of survival after pressurization of cold-shocked cells was 100-fold higher than that of cells growing exponentially at 37°C. These findings imply that cold-shocked cells are protected from HHP treatment, which may affect the efficiency of combined preservation techniques. 相似文献
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微生物产生的冷休克蛋白研究进展 总被引:1,自引:0,他引:1
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。 相似文献
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The variable C-terminus of cysteine string proteins modulates exocytosis and protein-protein interactions 总被引:2,自引:0,他引:2
Cysteine string proteins (Csps) are vesicle proteins involved in neurotransmission and hormone exocytosis. They are composed of distinct domains: a variable N-terminus, a J-domain followed by a linker region, a cysteine-rich string, and a C-terminus which diverges among isoforms. Their precise function and interactions are not fully understood. Using insulin exocytosis as a model, we show that the linker region and the C-terminus, but not the variable N-terminus, regulate overall secretion. Moreover, endogenous Csp1 binds in a calcium-dependent manner to monomeric VAMP2, and this interaction requires the C-terminus of Csp. The interaction is isoform specific as recombinant Csp1 binds VAMP1 and VAMP7, but not VAMP3. Cross-linking in permeabilized clonal beta-cells revealed homodimerization of Csp which is stimulated by Ca(2+) and again modulated by the variant C-terminus. Our data suggest that both interactions of Csp occur during exocytosis and may explain the effect of the variant C-terminus of this chaperon protein on peptide hormone secretion. 相似文献
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Barley heat shock proteins have been cloned, characterized by hybrid release translation and sequenced. Clones coding for proteins of 17, 18, 30, 32 and 70 kDa have been obtained. Out of these the 32 and 30 kDa proteins have been characterized as precursors to plastidic proteins of 26 kDa by posttranslational transport and by cDNA sequencing. The coding regions of these two transcribed genes are highly homologous. Accumulation of the plastid HSP as well as of HSP 70 as well as their corresponding mRNAs has been studied in 2- to 6-day old seedlings and in the 7-day old barley leaf. The mRNA for all investigated proteins were only found after a heat shock; the mRNA levels increase towards the tip of the leaf and with development. Furthermore, under the conditions used the mRNAs for all investigated heat shock proteins accumulate in parallel. Unexpectedly, both proteins, HSP 70 and HSP 26, are found by western blotting in the 2-day old control plants in the absence of any inducing heat shock. At later stages of development and in the leaf gradient only immunoreactivity with HSP 70 was observed. In contrast to the levels of their mRNAs the highest levels of HSP 30–26 and 70 have been observed in the basal segments indicating that translational control plays a role during HSP expression. Under severe heat shock a protein of 30 kDa is induced whose identity is not known but which reacts with the antibody to HSP 30–26 and might represent the accumulating precursors of the plastidic proteins. 相似文献
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The induction of proteins after a 25 to 5 degrees C cold shock in the psychrotrophic Acinetobacter HH1-l was examined using two-dimensional polyacrylamide gel electrophoresis. In addition, effects of various carbon sources (acetate, Tween 80, and olive oil) on protein synthesis after cold shock were assessed. HH1-1 responded to cold shock by synthesizing both cold shock proteins (csps) and cold acclimation proteins (caps). The synthesis of two csps (89 and 18) was increased 2 h after cold shock by the cells, regardless of the carbon source provided. An additional csp (csp 12), with an estimated molecular mass of 12 kDa, was observed in cells grown in olive oil only. Csp 12 was also synthesized when cells were incubated at 30 degrees C, suggesting that this protein may serve as a general stress protein. In addition to csps, caps were observed post cold shock at 72 h in acetate-grown cells and at 140 h in cells grown in Tween 80 and olive oil. Induction of cold-acclimated periplasmic proteins was observed for cells grown in olive oil only, suggesting cells grown in olive oil may be stressed by low temperatures to a greater extent than cells grown in either acetate or Tween 80. 相似文献
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Biochemical characterization of the mammalian stress proteins and identification of two stress proteins as glucose- and Ca2+-ionophore-regulated proteins 总被引:37,自引:0,他引:37
W J Welch J I Garrels G P Thomas J J Lin J R Feramisco 《The Journal of biological chemistry》1983,258(11):7102-7111
Biochemical properties of the heat shock or stress proteins of mammalian cells have been investigated using two-dimensional gel electrophoresis and immunological techniques. Of the major mammalian stress proteins (Mr = 72,000, 73,000, and 90,000) and minor stress proteins (Mr = 80,000, 100,000, and 110,000), the 80- and 90-kDa proteins were found to be phosphoproteins in all cell types examined. The 100-kDa protein was found to incorporate phosphate in only some cell types examined. In studies of the metabolic incorporation of mannose into the stress proteins, only the 100-kDa protein was found to be a glycoprotein. Two of the stress proteins, the 80- and 100-kDa species, were found to be identical with the proteins induced in cells grown in the absence of glucose (i.e. the "glucose-regulated proteins"). These same two proteins also were induced in cells treated with the calcium ionophore A23187. To begin examining the intracellular location of these multiregulated proteins, immunofluorescence microscopy studies were carried out using a monoclonal antibody against the 100-kDa stress protein. The antigen was localized primarily with the Golgi apparatus and less prominently with the plasma membrane and nucleus. Heat shock treatment resulted in an increased number of the cells exhibiting a nuclear location of 100 kDa. 相似文献
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Studies of the small heat shock proteins of Caenorhabditis elegans using anti-peptide antibodies 总被引:1,自引:0,他引:1
Peptides corresponding to selected regions of the 16 kDa small heat shock proteins (hsps) of the nematode C. elegans were synthesized and used to elicit polyclonal antibodies. It was found that these antibodies reacted predominantly with either the 16 kDa or the 18 kDa proteins, suggesting a close structural similarity between these hsps. Western blots of two-dimensional gels revealed extensive heterogeneity in these proteins, probably resulting from post-synthetic modifications. The native structures of both size classes of hsps were found to consist of large complexes of 4-5 x 10(5) Da. 相似文献
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Putative 65 kDa protein of beet yellows closterovirus is a homologue of HSP70 heat shock proteins 总被引:7,自引:0,他引:7
A A Agranovsky V P Boyko A V Karasev E V Koonin V V Dolja 《Journal of molecular biology》1991,217(4):603-610
A portion of the RNA genome of beet yellows closterovirus (BYV) has been sequenced encompassing a complete long open reading frame (ORF) potentially encoding a 65 kDa protein. The sequence of this putative protein was strikingly similar to those of HSP70-related heat shock proteins. The counterparts of all the eight segments strongly conserved in HSP70s could be confidently identified in the BYV 65 kDa protein. It is suggested that some of these segments might be the ATP-binding site(s) and that, similarly to the heat shock proteins, the 65 kDa is probably ATP-binding. Generally, however, the divergence between the 65 kDa sequence and the sequences of the HSP70s was much more pronounced than that between any two members of the latter family, allowing a clearer delineation of clusters of conserved residues that might be crucial for protein function. It is suggested that these observations will be helpful in functional dissection of the proteins of the HSP70 family. Analysis of the sequence of a portion of the ORF found upstream from the 65 kDa ORF showed that the C-terminal domain of the encoded protein could be an RNA-dependent RNA polymerase closely related to those of tricornaviruses, a family of RNA plant viruses with three component genomes. 相似文献
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On the basis of acquired thermotolerance and cryotolerance, the optimal heat shock and cold shock temperatures have been determined
for Deinococcus radiodurans. A heat shock at 42°C maximized survival at the lethal temperature of 52°C and a cold shock at 20°C maximized survival after
repeated freeze-thawing. Enhanced survival from heat shock was found to be strongly dependent on growth stage, with its greatest
effect shortly after phase. Increased synthesis of a total of 67 proteins during heat shock and 42 proteins during cold shock
were observed by two-dimensional polyacrylamide gel electrophoresis (2D PAGE) and autoradiography. Eight of the most highly
induced heat shock proteins shown by 2D PAGE were identified by MALDI-MS as Hsp20, GroEL, DnaK, SodA, Csp, Protease I and
two proteins of unknown function. 相似文献
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Temperature-dependent binding to the thylakoid membranes of nuclear-coded chloroplast heat-shock proteins 总被引:5,自引:0,他引:5
Two nuclear-coded heat-shock proteins (HSP) of pea (Pisum sativum) are synthesized as larger precursors of 26 kDa and 30 kDa in vitro. They are transported post-translationally into isolated, homologous chloroplasts where they are processed to mature proteins of 22 kDa and 25 kDa, respectively. When the chloroplasts used for the transport are isolated from control plants grown at 25 degrees C the 22-kDa and 25-kDa HSPs are located in the stroma of the chloroplasts. However, when chloroplasts are prepared from heat-shocked plants both proteins are found bound to the thylakoid membranes. The transition of the non-binding to the binding status is comparatively sharp and occurs between 36 degrees C and 40 degrees C in the variety 'Rosa Krone'. The transition temperature has been determined at 38 degrees C for 'Rosa Krone' and at 40 degrees C for the variety 'Golf'. At 42 degrees C, 15-min treatment of the plants is sufficient to induce membrane binding, which persists for at least 4-6 h (but not for 24 h) after return to the ambient temperature. Once lost, membrane binding can be reinduced by a second heat-shock treatment in vivo. High light intensities during the heat shock interfere with the binding capacity for heat-shock proteins. 相似文献
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Low temperature is one of the major environmental challenges that Antarctic bacteria must face. Detailed studies of cold shock responses of cold-adapted microorganisms are still insufficient. Here, we cloned three cold shock protein (CSP) genes (Csp1137, Csp2039, and Csp2531) in the Antarctic bacterium Psychrobacter sp. G and their regulatory sequences were identified. The three CSPs were highly conserved with other known CspAs. qRT-PCR was performed to evaluate their expression characteristics under stress conditions, and the potential influence of regulatory sequences also was analyzed. The expression of Csp1137 was enhanced both by low (0, 10?°C) and high temperature (30?°C). The expression of Csp2039 was enhanced by low temperature (0?°C), but was lower than that of Csp1137. This can be explained by the absence in Csp2039 of the AT-rich UP element. Different from Csp1137, the expression of Csp2531 was inhibited by low temperature (0?°C), even with the presence of AT-rich UP element, and it was not sensitive to high temperature (30?°C). The expression of Csp1137 was enhanced by high salinity (90, 120), whereas that of Csp2531was enhanced by low salinity (0, 15). At 0?°C and a salinity of 15, the expression of Csp1137 was repressed initially, but then it increased greatly during the next 10?h. The expressions of Csp2039 and Csp2531 were repressed significantly under four different combinations of stress conditions. Our results showed that the role of the upstream regulation sequences were much more complex than previously thought. Also, gene expressions were also affected by the environmental salinity. These are helpful in further clarification of the adaptation mechanism of Psychrobacter sp. G. 相似文献
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Javed Iqbal Wang Li Murtaza Hasan Kefu Liu Umer Awan Yasmeen Saeed Yongqian Zhang Arif Muhammad Khan Amin Shah Hong Qing Yulin Deng 《Proteomics》2014,14(11):1424-1433
Microgravity severely halts the structural and functional cerebral capacity of astronauts especially affecting their brains due to the stress produced by cephalic fluid shift. We employed a rat tail suspension model to substantiate simulated microgravity (SM) in brain. In this study, comparative mass spectrometry was applied in order to demonstrate the differential expression of 17 specific cellular defense proteins. Gamma‐enolase, peptidyl‐prolyl cis‐trans isomerase A, glial fibrillary acidic protein, heat shock protein HSP 90‐alpha, 10 kDa heat shock protein, mitochondrial, heat shock cognate 71 kDa protein, superoxide dismutase 1 and dihydropyrimidinase‐related protein 2 were found to be upregulated by HPLC/ESI‐TOF. Furthermore, five differentially expressed proteins including 60 kDa heat shock protein, mitochondrial, heat shock protein HSP 90‐beta, peroxiredoxin‐2, stress‐induced‐phosphoprotein, and UCHL‐1 were found to be upregulated by HPLC/ESI‐Q‐TOF MS. In addition, downregulated proteins include cytochrome C, superoxide dismutase 2, somatic, and excitatory amino acid transporter 1 and protein DJ‐1. Validity of MS results was successfully performed by Western blot analysis of DJ‐1 protein. This study will not only help to understand the neurochemical responses produced under microgravity but also will give future direction to cure the proteomic losses and their after effects in astronauts. 相似文献
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The major heat shock protein (hsp) of Hydra vulgaris has recently been found to be a 60 kDa protein. Since in all organisms studied so far, the major heat shock protein is a 70 kDa protein, we have analyzed the relationship of hydra hsp60 to the highly conserved 70 kDa heat shock protein family. Genes and proteins related to the 70 kDa class of stress proteins are present in hydra. However, antibodies known to cross-react with hsp70 proteins in several different organisms do not cross-react with hydra hsp60 suggesting that hsp60 is not related to the conserved hsp70 proteins. 相似文献
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Dupont A Chwastyniak M Beseme O Guihot AL Drobecq H Amouyel P Pinet F 《Journal of proteome research》2008,7(8):3572-3582
Macrophages are believed to play a crucial role in atherogenesis and atherosclerotic plaque progression, mainly through their role in the accumulation of large amounts of cholesteryl ester and foam cell formation after the uptake into the arterial intima of oxidized LDL (oxLDL) particles known to be proatherogenic. The aim of this study was to use a differential proteomic approach to identify the response of human monocyte-derived macrophages after treatment with oxLDL for 24 h. Mass spectrometry analysis (MALDI-TOF) of 2D-DIGE gels made it possible to identify 9 intracellular and 3 secreted proteins that were up-regulated, 11 intracellular and 1 secreted proteins that were down-regulated, and 2 secreted proteins that were induced. This methodological approach not only confirmed the differential expression levels of proteins known to be regulated by oxLDL in macrophages, such as catalase and pyruvate kinase, but also identified oxLDL modulation of other proteins for the first time, including heat shock proteins (HSP) and Actin cytoskeletal proteins. Semiquantitative Western blot confirmed their role. The HSPs identified included heat shock cognate 71 kDa protein (Hsc70), 75 kDa glucose-regulated protein (GRP75), heat shock 70 kDa protein (Hsp70), and 60 kDa (Hsp60) proteins. These highly conserved intracellular protein chaperones, commonly seen in atherosclerotic plaques, appear to participate in protection against cellular stress. Interestingly, oxLDL also modulated several F-Actin capping proteins involved in Actin polymerization and motility: gelsolin, CapG, and CapZ. In conclusion, we have demonstrated the effects of oxLDL in the modulation of several proteins in human macrophages and established a functional profile of the human macrophage during the atherosclerotic process. 相似文献