Molecular cloning of cDNA and analysis of protein secondary structure of Candida albicans enolase, an abundant, immunodominant glycolytic enzyme.

We isolated and sequenced a clone for Candida albicans enolase from a C. albicans cDNA library by using molecular genetic techniques. The 1.4-kbp cDNA encoded one long open reading frame of 440 amino acids which was 87 and 75% similar to predicted enolases of Saccharomyces cerevisiae and enolases from other organisms, respectively. The cDNA included the entire coding region and predicted a protein of molecular weight 47,178. The codon usage was highly biased and similar to that found for the highly expressed EF-1 alpha proteins of C. albicans. Northern (RNA) blot analysis showed that the enolase cDNA hybridized to an abundant C. albicans mRNA of 1.5 kb present in both yeast and hyphal growth forms. The polypeptide product of the cloned cDNA, which was purified as a recombinant protein fused to glutathione S-transferase, had enolase enzymatic activity and inhibited radioimmunoprecipitation of a single C. albicans protein of molecular weight 47,000. Analysis of the predicted C. albicans enolase showed strong conservation in regions of alpha helices, beta sheets, and beta turns, as determined by comparison with the crystal structure of apo-enolase A of S. cerevisiae. The lack of cysteine residues and a two-amino-acid insertion in the main domain differentiated C. albicans enolase from S. cerevisiae enolase. Immunofluorescence of whole C. albicans cells by using a mouse antiserum generated against the purified fusion protein showed that enolase is not located on the surface of C. albicans. Recombinant C. albicans enolase will be useful in understanding the pathogenesis and host immune response in disseminated candidiasis, since enolase is an immunodominant antigen which circulates during disseminated infections.     

Differential expression of specific cellular defense proteins in rat hypothalamus under simulated microgravity induced conditions: Comparative proteomics

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.     

Neuronal expression of constitutive heat shock proteins: implications for neurodegenerative diseases

Neurodegenerative disorders such as Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis have been termed "protein misfolding disorders." These diseases differ widely in frequency and impact different classes of neurons. Heat shock proteins provide a line of defense against misfolded, aggregation-prone proteins and are among the most potent suppressors of neurodegeneration in animal models. Analysis of constitutively expressed heat shock proteins revealed variable levels of Hsc70 and Hsp27 in different classes of neurons in the adult rat brain. The differing levels of these constitutively expressed heat shock proteins in neuronal cell populations correlated with the relative frequencies of the previously mentioned neurodegenerative diseases.     

Protein expression during heat stress in thermo-intolerant and thermo-tolerant diatoms

Diatoms (Chrysophyta) are photosynthetic microorganisms that are abundant in the natural environment and often associated with specific habitat and water quality conditions. Their significance as bioindicators and as exploitable sources of fine chemicals makes them desirable candidates for the study of stress responses. The protein expression of a thermo-intolerant (Phaeodactylum tricornutum) and thermo-tolerant (Chaetoceros muelleri) diatom following exposure to elevated temperature was investigated using one- and two-dimensional gel electrophoresis and Western blot analysis. It was determined using SDS PAGE with ³⁵S-methionine labeled proteins and Western blot analysis using pea HSP70 antisera that higher temperatures and longer duration treatment were required to cause a noticeable stress response in C. muelleri compared to P. tricornutum. This may be explained by C. muelleri possessing higher amounts of constitutively expressed heat shock proteins, which allows these cells to rapidly adjust to temperature increases. Two-dimensional gel electrophoresis revealed that putative small heat shock proteins (smHSPs) may appear to play a role during heat stress in both diatoms, which is similar to the response in plants. SDS PAGE data are also presented characterizing the recovery of P. tricornutum after heat shock. These results suggest that there is a lag period between heat shock and stress protein synthesis in these thermo-intolerant cells. This supports the hypothesis that cells without higher amounts of constitutively expressed stress proteins have a greater sensitivity to increased temperature. Work is underway to identify particular stress proteins responsible for conveying thermo-tolerance and to determine if overexpression of these genes in thermo-intolerant diatoms affects their temperature sensitivity.     

Analysis of the heat-shock response displayed by two Chaetomium species originating from different thermal environments.

Three features of the heat shock response, reorganization of protein expression, intracellular accumulation of trehalose, and alteration in unsaturation degree of fatty acids were investigated in the thermophilic fungus Chaetomium thermophile and compared to the response displayed by a closely related mesophilic species, C. brasiliense. Thermophilic heat shock response paralleled the mesophilic response in many respects like (i) the temperature difference observed between normothermia and the upper limit of translational activity, (ii) the transient nature of the heat shock response at the level of protein expression including both the induction of heat shock proteins (HSPs) as well as the repression of housekeeping proteins, (iii) the presence of representatives of high-molecular-weight HSPs families, (iv) intracellular accumulation of trehalose, and finally (v) modifications in fatty acid composition. On the other hand, a great variability between the two organisms was observed for the proteins expressed during stress, in particular a protein of the HSP60 family that was only observed in C. thermophile. This peptide was also present constitutively at normal temperature and may thus fulfil thermophilic functions. It is shown that accumulation of trehalose does not play a part in thermophily but is only a stress response. C. thermophile contains less polyunsaturated fatty acids at normal temperature than C. brasiliense, a fact that can be directly related to thermophily. When subjected to heat stress, both organisms tended to accumulate shorter and less unsaturated fatty acids.     

荠菜LOS2基因的克隆与分析

利用RACE-PCR方法从荠菜(Capsella bursa-pastoris)中克隆到了新的LOS2全长基因(Cblos2)。序列分析表明,该基因的全长cDNA为1694bp,拥有一个由444个氨基酸组成的开放读码框,预测的CbLOS2蛋白包括一个烯醇酶N-端结构域、烯醇酶结构域以及与拟南芥的LOS2高度保守的DNA结合域和基因功能抑制域。生物信息学分析表明,Cblos2与LOS2极为相似。冷胁迫适应实验表明,Cblos2基因在荠菜中组成性表达,且其表达与胁迫适应过程密切相关。该研究表明Cblos2是具双重功能的植物烯醇酶基因家族的新成员。     

Heat shock response of Saccharomyces cerevisiae mutants altered in cyclic AMP-dependent protein phosphorylation.

When Saccharomyces cerevisiae cells grown at 23 degrees C were transferred to 36 degrees C, they initiated synthesis of heat shock proteins, acquired thermotolerance to a lethal heat treatment given after the temperature shift, and arrested their growth transiently at the G1 phase of the cell division cycle. The bcy1 mutant which resulted in production of cyclic AMP (cAMP)-independent protein kinase did not synthesize the three heat shock proteins hsp72A, hsp72B, and hsp41 after the temperature shift. The bcy1 cells failed to acquire thermotolerance to the lethal heat treatment and were not arrested at the G1 phase after the temperature shift. In contrast, the cyr1-2 mutant, which produced a low level of cAMP, constitutively produced three heat shock proteins and four other proteins without the temperature shift and was resistant to the lethal heat treatment. The results suggest that a decrease in the level of cAMP-dependent protein phosphorylation results in the heat shock response, including elevated synthesis of three heat shock proteins, acquisition of thermotolerance, and transient arrest of the cell cycle.     

Analysis of the Heat-Shock Response Displayed by Two Chaetomium Species Originating from Different Thermal Environments

Three features of the heat shock response, reorganization of protein expression, intracellular accumulation of trehalose, and alteration in unsaturation degree of fatty acids were investigated in the thermophilic fungus Chaetomium thermophile and compared to the response displayed by a closely related mesophilic species, C. brasiliense. Thermophilic heat shock response paralleled the mesophilic response in many respects like (i) the temperature difference observed between normothermia and the upper limit of translational activity, (ii) the transient nature of the heat shock response at the level of protein expression including both the induction of heat shock proteins (HSPs) as well as the repression of housekeeping proteins, (iii) the presence of representatives of high-molecular-weight HSPs families, (iv) intracellular accumulation of trehalose, and finally (v) modifications in fatty acid composition. On the other hand, a great variability between the two organisms was observed for the proteins expressed during stress, in particular a protein of the HSP60 family that was only observed in C. thermophile. This peptide was also present constitutively at normal temperature and may thus fulfil thermophilic functions. It is shown that accumulation of trehalose does not play a part in thermophily but is only a stress response. C. thermophile contains less polyunsaturated fatty acids at normal temperature than C. brasiliense, a fact that can be directly related to thermophily. When subjected to heat stress, both organisms tended to accumulate shorter and less unsaturated fatty acids.     

Structural organization of the spinach endoplasmic reticulum-luminal 70-kilodalton heat-shock cognate gene and expression of 70-kilodalton heat-shock genes during cold acclimation.

The 70-kD heat-shock proteins (HSP70s) are encoded by a multigene family in eukaryotes. In plants, the 70-kD heat-shock cognate (HSC70) proteins are located in organellar and cytosolic compartments of cells in most tissues. Previous work has indicated that HSC70 proteins of spinach (Spinacia oleracea) are actively synthesized during cold-acclimating conditions. We have isolated, sequenced, and characterized cDNA and genomic clones for the endoplasmic reticulum (ER) luminal HSC70 protein (immunoglobulin heavy chain-binding protein; BiP) of spinach. The spinach ER-luminal HSC70 is a constitutively expressed gene consisting of eight exons. Spinach BiP mRNA appears to be up-regulated during cold acclimation but is not expressed during water stress or heat shock. In contrast to the differential regulation of mRNA, the ER-luminal HSC70 protein levels remain constant in response to various environmental stresses. Two other members of the spinach 70-kD heat-shock (HS70) multigene family also show differential expression in response to a variety of environmental stresses. A constitutively expressed cytosolic HSC70 protein in spinach appears also to be up-regulated in response to both cold-acclimating and heat-shock treatments. Spinach also contains a cold-shock-induced HS70 gene that is not expressed during heat shock or water stress. Since HSP70s are considered to be involved with the chaperoning and folding of proteins, the data further support the concept that they may be important for maintaining cellular homeostasis and proper protein biogenesis during cold acclimation of spinach.     

Induction of heat shock protein 27 in rat embryos exposed to hyperthermia

Previously we reported that eight proteins were reproducibly induced in postimplantation rat embryos exposed to a brief heat shock (43°C, 15 min). The major heat-inducible rat embryo protein has now been identified as heat shock protein 72 (Hsp 72). In addition, the induction of Hsp 72 is temporally correlated with induction of thermotolerance. One of the other rat embryo proteins previously shown to be induced by elevated temperature is a heat shock protein of approximately 27 kilodaltons (Hsp 27). In this report we show that this protein is recognized by an antibody directed against a conserved peptide sequence of Hsp 27. Unlike Hsp 72, Hsp 27 is constitutively expressed in the rat embryo in the absence of any thermal stress; however, the level of Hsp 27 is increased approximately 2–3-fold after thermal stress (43°C, 10 min). Immunohistochemical analysis revealed that the constitutively expressed Hsp 27 is localized primarily to cells of the heart, cells that are uniquely resistant to the cytotoxic effects of hyperthermia. After thermal stress, Hsp 27 is expressed in all tissues of the embryo. Finally, our data show that Hsp 27 exists in the rat embryo as three major isoforms indicative of different phosphorylation states. Furthermore, most Hsp 27 in the heart is phosphorylated, whereas in the rest of the embryo, nonphosphorylated Hsp 27 predominates. After thermal stress, levels of phosphorylated isoforms increase dramatically in nonheart tissues of the embryo. Together, these results suggest that Hsp 27 may play a role in the development of thermotolerance in the postimplantation mammalian embryo. © 1996 Wiley-Liss, Inc.     

Lamin B is a prompt heat shock protein

The cellular response to hyperthermia involves the increased synthesis of heat shock proteins (HSPs) within several hours after treatment. In addition, a subset of proteins has been shown to be increased immediately after heating. These “prompt” HSPs are predominantly found in the nuclear matrix–intermediate filament fraction and are not present or detectable in unheated cells. Since the nuclear matrix has been suggested to be a target for heat-induced cell killing, prompt HSPs may play a prominent role in the heat shock response. Using Western blotting and flow cytometry, we found that an increase in the synthesis of lamin B, one of the major proteins of the nuclear lamina, is induced during heating at 45.5°C but not during heating at 42°C. Since it is an abundant protein which is constitutively expressed in mammalian cells, lamin B appears to be a unique member of the prompt HSP family. The kinetics of induction of lamin B during 45.5°C heating did not correlate with the dose-dependent reduction in cell survival. While increased levels of lamin B during 45.5°C heating do not appear to confer a survival advantage directly, a possible role for lamin B in cellular recovery after heat shock cannot be discounted. J Cell Physiol 178:28–34, 1999. © 1999 Wiley-Liss, Inc.     

Molecular characterization of specific heat shock proteins inBacillus subtilis

Heat shock inBacillus subtilis may induce as many as 66 proteins after temperature upshift from 37° to 48°C. Four induced proteins were analyzed by microsequencing techniques. These were identified as the homologues for GroEL, DnaK, enolase, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which are heat shock proteins in other systems. The identities of GroEL and DnaK were confirmed additionally by Western blot analysis. As a control, a protein whose synthesis was repressed approximately threefold by heat shock was identified by microsequencing as flagellin.     

Expression of heat shock proteins in response to high and low temperature extremes in diapausing pharate larvae of the gypsy moth,Lymantria dispar

Diapausing pharate first instars of the gypsy moth, Lymantria dispar, respond to high temperature (37–41°C) by suppressing normal protein synthesis and synthesizing a set of seven heat shock proteins with M_rs of 90,000, 75,000, 73,000, 60,000, 42,000, 29,000, and 22,000 as determined by SDS-PAGE. During recovery at 25°C from heat shock, synthesis of the heat shock proteins gradually decreases over a period of 6 h, while normal protein synthesis is restored. A subset of these same heat shock proteins is also expressed during recovery at 4°C or 25°C from brief exposures to low temperature (-10 to 20°C), and its expression is more intense with increased severity of cold exposure. During recovery at 4°C after 24 h at ?20°C, both 90,000 and 75,000 M_r heat shock proteins are expressed for more than 96 h. While normal protein synthesis is suppressed during heat shock and recovery from heat shock, normal protein synthesis coincides with synthesis of the heat shock proteins during recovery from low temperatures, thus implying that expression of the heat shock proteins is not invariably linked to suppression of normal protein synthesis. Western transfer, using a monoclonal antibody that recognizes the inducible form of the human 70,000 M_r heat shock protein, demonstrates that immunologically related proteins in the gypsy moth are expressed at 4°C and during recovery from cold and heat shock.     

Proteome analysis on differentially expressed proteins of the fat body of two silkworm breeds, Bombyx mori, exposed to heat shock exposure

Proteomes of heat tolerant (multivoltine) and heat susceptible (bivoltine) silkworms (Bombyx mori) in response to heat shock were studied. Detected proteins from fat body were identified by using MALDI-TOF/TOF spectrometer, MS/MS, and MS analysis. Eight proteins, including small heat shock proteins (sHSPs) and HSP70, were expressed similarly in both breeds, while 4 protein spots were expressed specifically in the bivoltine breed and 12 protein spots were expressed specifically in the multivoltine breed. In the present proteomics approach, 5 separate spots of sHSP proteins (HSP19.9, HSP20.1, HSP20.4, HSP20.8, and HSP21.4) were identified. Protein spot intensity of sHSPs was lower in the multivoltine breed than in the bivoltine breed after the 45°C heat shock treatment, while the difference between two breeds was not significant after the 41°C heat shock treatment. These results indicated that some other mechanisms might be engaged in thermal tolerance of multivotine breed except for the expression of sHSP and HSP70. There were visible differences in the intensity of heat shock protein expression between male and female, however, differences were not statistically significant.     

PROTEIN EXPRESSION DURING HEAT STRESS IN THERMO-INTOLERANT AND THERMO-TOLERANT DIATOMS

To better understand how diatoms are capable of responding to environmental stress, protein expression during heat treatment of a thermo-intolerant ( Phaeodactylum tricornutum ) and thermo-tolerant ( Chaetoceros muelleri ) diatom (Chrysophyta) was investigated. The stress response is a universal and conserved mechanism of cell survival to unfavorable conditions. Typically, a 10 to 15° C temperature elevation above cell growth optimal causes constitutively expressed proteins to decrease and heat shock proteins (HSPs) to increase. HSPs are categorized by molecular weight among five classes with each apparently specialized for a particular function that enhances cell survival. One-dimensional SDS-PAGE of diatoms subjected to heat treatment revealed that P. tricornutum exhibited a typical stress response, but C. muelleri did not exhibit a characteristic response even at a greatly elevated temperature (50° C). This result was confirmed by total soluble protein assays. Chaetoceros muelleri may contain higher basal levels of HSPs than P. tricornutum allowing C. muelleri to better tolerate elevated temperatures. Western blot analysis using pea HSP70 (70 kDa) antisera of heat-treated P. tricornutum and C. muelleri validated the hypothesis that thermo-tolerant cells contain higher levels of constitutively expressed HSPs. Two-dimensional gel electrophoresis of heat-treated cells indicate that the small HSPs (17–30 kDa) played a role in the stress response similar to that found in vascular plants. Ongoing work is focused on the manipulation of the stress response through over-expression of key hsp genes.     

TorsinA and heat shock proteins act as molecular chaperones: suppression of alpha-synuclein aggregation

TorsinA, a protein with homology to yeast heat shock protein104, has previously been demonstrated to colocalize with alpha-synuclein in Lewy bodies, the pathological hallmark of Parkinson's disease. Heat shock proteins are a family of chaperones that are both constitutively expressed and induced by stressors, and that serve essential functions for protein refolding and/or degradation. Here, we demonstrate that, like torsinA, specific molecular chaperone heat shock proteins colocalize with alpha-synuclein in Lewy bodies. In addition, using a cellular model of alpha-synuclein aggregation, we demonstrate that torsinA and specific heat shock protein molecular chaperones colocalize with alpha-synuclein immunopositive inclusions. Further, overexpression of torsinA and specific heat shock proteins suppress alpha-synuclein aggregation in this cellular model, whereas mutant torsinA has no effect. These data suggest that torsinA has chaperone-like activity and that the disease-associated GAG deletion mutant has a loss-of-function phenotype. Moreover, these data support a role for chaperone proteins, including torsinA and heat shock proteins, in cellular responses to neurodegenerative inclusions.