Heat shock induces peroxidase activity in Neurospora crassa and confers tolerance toward oxidative stress

Heat shock treatment of 14-h-old Neurospora crassa mycelium, for 1 h at 48 degrees C, led to the induction of high levels of peroxidase (EC. 1.11.1.7) activity. No significant change was observed in the superoxide dismutase content. Colonies formed by plating conidial suspensions on sorbose-medium also exhibited high peroxidase activity following exposure to hyperthermia and were found to be resistant to normally toxic doses of H2O2. Thus one of the heat shock proteins of N. crassa has the function of protection against oxidative stress.     

hsp80 of Neurospora crassa: cDNA cloning, gene mapping, and studies of mRNA accumulation under stress.

Using mRNA isolated from Neurospora crassa mycelium, grown for 14 h at normal growth temperature of 28 degrees C, and heat shocked for 1 h at 48 degrees C, a cDNA library was prepared in the expression vector lambda gt11. Following immunoscreening of this library with a polyclonal antiserum raised against a 80-kilodalton heat-shock protein (HSP80), cDNA clones containing 1.1- and 1.4-kilobase inserts were selected. Analysis of the partial nucleotide sequence and the deduced amino acid sequence of the cDNA clones revealed a remarkable extent of homology with other eukaryotic stress-90 family proteins; 85% identity of the amino acid sequence with that of yeast HSP90(82) was seen. The C-terminal end of the sequence contained the MEEVD motif, characteristic of eukaryotic stress proteins with a predominantly cytosolic localization. The gene for N. crassa HSP80 was mapped to the right arm of linkage group V, using restriction fragment length polymorphism mapping. Its expression during heat shock and recovery was monitored by probing Northern blots of RNA isolated from mycelium grown under various stress conditions.     

Global whole-cell FTICR mass spectrometric proteomics analysis of the heat shock response in the radioresistant bacterium Deinococcus radiodurans

The results of previous studies indicated that D. radiodurans mounts a regulated protective response to heat shock, and that expression of more than 130 genes, including classical chaperones such as the groESL and dnaKJ operons and proteases such as clpB are induced in response to elevated temperature. In addition, previous qualitative whole-cell mass spectrometric studies conducted under heat shock conditions indicated global changes in the D. radiodurans proteome. To enable the discovery of novel heat shock inducible proteins as well as gain greater biological insight into the classical heat shock response at the protein level, we undertook the global whole-cell FTICR mass spectrometric proteomics study reported here. We have greatly increased the power of this approach by conducting a large number of replicate experiments in addition to taking a semiquantitative approach to data analysis, finding good reproducibility between replicates. Through this analysis, we have identified with high confidence a core set of classical heat shock proteins whose expression increases dramatically and reproducibly in response to elevated temperature. In addition, we have found that the heat shock proteome includes a large number of induced proteins that have not been identified previously as heat responsive, and have therefore been designated as candidate responders. Finally, our results are consistent with the hypothesis that elevated temperature stress could lead to cross-protection against other related stresses.     

Ethanol and carbon-source starvation enhance the accumulation of HSP80 in Neurospora crassa

In Neurospora crassa, heat shock results in the induction of 9 to 11 heat shock proteins (HSP), of which HSP80 is the most abundant and the first to be synthesized. The induction of HSP80 was investigated during normal growth (2% sucrose) and under sucrose starvation. Transfer of mycelium to a medium supplemented with ethanol stimulated the synthesis of HSP80, even at the normal growth temperature of 28 degrees C. It was also synthesized under carbon starvation conditions, where the medium was supplemented with 0.02% sucrose, 0.3% acetate, 0.2% lactate, or ethanol. A 30-35 kilodalton polypeptide was induced by heat shock in carbon-sufficient media, but in 0.02% sucrose and 0.3% acetate containing media it was synthesized at normal temperatures. While the overall heat shock response remained unaltered in these cultures, the abundance of HSP90 and HSP70, relative to HSP80, was greater. HSP80 appears to be controlled by carbon-catabolite repression as well as heat shock. Another high molecular mass protein (tentatively designated alc'80') was observed to be induced by heat shock, provided carbon starvation conditions prevailed concurrently.     

Evidence for copurification of micronuclei in sucrose density gradient-enriched plasma membranes from cell lines

Sucrose density gradient-enriched membrane preparations and membrane fraction enrichment through affinity purification techniques are commonly used in proteomic analysis. However, published proteomic profiles characterized by the above methods show the presence of nuclear proteins in addition to membrane proteins. While shuttling of nuclear proteins across cellular compartments and their transient residency at membrane interfaces could explain some of these observations, the presence of nuclear proteins in proteomic profiles generated with crude and enriched membranes could be the result of nonspecific contamination of nuclear debris during cell fractionation procedures. We hypothesized that micronuclei arising from the genomic instability inherent to cancer cells may copurify with plasma membrane fractions on sucrose gradients. Using sucrose gradient-enriched plasma membranes from breast cancer cell lines derived from the MCF-7 cell line, we provide experimental evidence to indicate that micronuclei are present in fresh preparations of plasma membranes. The origin of these micronuclei was traced to budding of nuclei in intact cells. Furthermore, mass spectrometric analysis confirmed the presence of nuclear proteins as well as membrane and associated signaling proteins in sucrose gradient-enriched preparations.     

Some aspects of the regulation of pyruvate kinase levels in Neurospora crassa

Pyruvate kinase levels were monitored in Neurospora crassa mycelium (grown on different carbon sources for varying time intervals) by immunoprecipitation using polyclonal antibodies raised against a purified enzyme preparation. Pyruvate kinase specific mRNA was demonstrated by hybridization of Northern and dot blots of total RNA with a N. crassa pyruvate kinase gene fragment. Two pyruvate kinase specific mRNA species were detected in mycelia of all ages examined. An age-dependent and carbon source dependent variation in the pyruvate kinase protein and mRNA levels was encountered: both registered an increase for up to about 20 h and a subsequent decline; growth on acetate and sucrose resulted in significantly higher yields of both, relative to that on medium containing ethanol and alanine. Stress caused by heat shock depressed the pyruvate kinase mRNA levels.     

Evidence for three differentially regulated catalase genes in Neurospora crassa: effects of oxidative stress, heat shock, and development.

Genetic and biochemical studies demonstrated that Neurospora crassa possesses three catalases encoded by three separate structural genes. The specific activities of the three enzymes varied in response to superoxide-mediated stress, heat shock, and development. The three loci, which we designated cat-1, cat-2, and cat-3, map to the right arms of chromosomes III, VII, and III, respectively. The cat-1-encoded enzyme (designated Cat-1; estimated molecular weight, 315,000; pI 5.2) was the predominant catalase in rapid-growth mycelium, and its activity was substantially increased in paraquat-treated and heat-shocked mycelium. Cat-2 (Mw, 165,000; pI 5.4) was absent from rapid-growth mycelium but present at low levels in conidia and stationary-phase mycelium. It was the predominant catalase in extracts derived from mycelium that had been heat shocked for 2 h. Cat-3 (Mw, 340,000; pI 5.5) was the predominant catalase in extracts from mature conidia.     

Reassembling proteins and chaperones in human nuclear matrix protein fractions.

To detect putative filament forming components, nuclear matrix proteins were searched for proteins extensively reassembling from urea solution. Eight proteins, ubiquitously occurring in various human cell types, but not apparent in the cytosol, were registered by means of two-dimensional gel electrophoresis. They consisted of a protein exhibiting a novel amino acid sequence; of nuclear lamin B2, RbAp46, and RbAp48; and of four as yet unknown proteins. Furthermore, partial sequencing, mass spectrometry, and immunodetection of proteins demonstrated the presence of molecular chaperones and protein folding catalysts in the nuclear matrix fractions. In addition to a TCP-1-related protein, certain members of the heat shock, PDI, and calreticulin family of proteins were detected. On the basis of the absence of several other heat shock proteins in the nuclear matrix fraction, a general contamination by cytoplasmic chaperones appears unlikely.     

Specificity of commercial anti-spectrin antibody in the study of fungal and Oomycete spectrin: cross-reaction with proteins other than spectrin

Spectrin was first described in erythrocytes where it forms a filamentous network in the cytoplasmic face of the plasma membrane and participates in the membrane's structural integrity in addition to controlling the lateral mobility of integral membrane proteins. In fungi, spectrin-like proteins have been described in the plasma membrane, concentrated mainly in the region of maximum apical expansion. This localization led to the idea of a spectrin based membrane skeleton in fungi participating in mechanical integrity of the plasma membrane, generating and maintaining cell polarity. The occurrence of spectrin-like proteins in filamentous fungi, yeasts and Oomycetes, however, is questionable since the presence of such proteins has only been demonstrated with immunochemical methods using antibodies whose specificity is unclear. There is no evidence of a gene coding for the high molecular weight alphabeta-spectrin in the genome of these organisms. Mass spectrometric analysis of the anti alphabeta-spectrin immunoreacting peptides from Neurospora crassa and Phytophthora infestans identified them as elongation factor 2 (NCU07700.4) and Hsp70 (PITG_13237.1), respectively. An attempt was made to correlate the reactivity of anti-spectrin antibody to a common feature of these three proteins i.e., spectrin, elongation factor 2 and heat shock protein 70, in that they all have a hydrophobic region implicated in chaperon activity.     

Heat shock inhibits and activates different protein degradation pathways and proteinase activities in Neurospora crassa

Abstract In Neurospora crassa , heat shock treatment inhibits proteolytic activity. ATP-independent proteinases were analysed after polyacrylamide gel electrophoresis using renaturing gelatine gels. Proteinases of 24, 29, and 130 kDa were shown to be inhibited by heat shock and were further characterized as to their properties. A major part of the heat shock-induced inhibition is probably due to suppression of de novo synthesis of proteinases as deduced from experiments with cycloheximide. During several hours of recovery from heat shock, the inhibition of overall protein degradation and ATP-independent proteinases is reversed. Azocasein assays as well as pulse-chase experiments further showed that ATP-dependent protein degradation is only slightly affected by heat shock. Two ATP-binding proteinases of about 60 and 160 kDa even show an increased activity after heat shock. The degradation rate of heat shock proteins is inhibited by heat shock treatment, indicating that they are degraded by ATP-independent proteinases. Western blot analysis of a ∼40-kDa degradation product of HSP70 containing its amino terminal portion revealed a reduction in the amount of this peptide after heat shock.     

Characterization of a low molecular mass autophosphorylating protein in cultured sugarcane cells and its identification as a nucleoside diphosphate kinase.

A low molecular mass (18 kD) phosphoprotein (pp18) was characterized and purified from cultured sugarcane (Saccharum officinarum L.) cell line H50-7209. Autophosphorylation assays were used to detect pp18 after separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Only pp18 was detected by a brief in situ phosphorylation method, whereas additional putative protein kinases were detected by an extended method. pp18 was present in both microsomal membrane and soluble fractions and exhibited anomalous turnover of 32P label during in vitro phosphorylation experiments with highest levels present at shorter incubation times. Two major isoforms of the protein were identified in two-dimensional isoelectric focusing/SDS-PAGE of crude extracts and microsomal fractions. The levels of pp18 were enhanced approximately 4-fold by heat shock at 36 degrees C and the elevated pp18 decayed after heat shock was discontinued. pp18 was purified to apparent homogeneity, could be phosphorylated on serine residues, and also exhibited kinase-like activity toward histone H1. The amino acid sequence obtained from a cyanogen bromide digest was greater than 80% identical to nucleoside diphosphate (NDP) kinases from a variety of organisms. Biochemical analysis of the purified protein confirmed the identity as NDP kinase. Thus, NDP kinase appears to be modulated by heat shock in plants.     

LEDGF binds to heat shock and stress-related element to activate the expression of stress-related genes

We have investigated the mechanism by which LEDGF protects cells against environmental stress. Our earlier report showed that a low level of LEDGF was present in the nucleus of most cell types and significant elevation of LEDGF level was induced by heat and oxidative stress. The cells overexpressing LEDGF-activated expression of heat shock proteins and enhanced survival of many cell types. Here we show that LEDGF binds to heat shock element (HSE) and stress-related regulatory element (STRE) to activate the expression of stress-related genes (Hsp27 and alphaB-crystallin). Apparently, HSE and STRE are present in promoters of many stress-related genes. Elevation of many stress-related proteins (STRPs) induced by LEDGF may protect cells against environmental stress. In yeast, it has been demonstrated that a single stress can activate the expression of multiple STRPs. This is known as "cross-protection," and now similar mechanism has been found in mammalian cells and LEDGF plays a vital role in it.     

Proteomic identification of protein markers of stages of heart formation in humans

On the basis of the results of proteomic analysis and mass spectrometric identification of human myocardium proteins exhibiting pronounced quantitative changes in the dynamics of prenatal cardiogenesis, changes in the expression level of proteins of three families (mitochondrial, contractile, and heat shock) have been identified. The complex of human myocardium mitochondrial proteins (for example, α and β isoforms of ATP synthase, aconitase 2, creatine phosphokinase M-subunit, and 60-kDa heat shock protein) largely finishes its development according to the adult type by developmental week 24. The formation of the protein composition of human myocardium contractile structures (for example, desmin, myosin regulatory light chain 2, fetal ventricular essential isoform 1, canonical α-tropomyosin, and fetal isoform 6) reflects the initial stage of myofibril development until developmental week 8 (replacement of fetal isoforms of contractile proteins with adult ones with the involvement of the phosphorylated isoform of 27-kDa heat shock protein), the stage of their qualitative and quantitative structuring by developmental weeks 20–24, and the final formation of the adult phenotype of contractile structures by 2 years of life.     

Glucose metabolism in Neurospora is altered by heat shock and by disruption of HSP30

We compared the metabolism of [1-13C]glucose by wild type cells of Neurospora crassa at normal growth temperature and at heat shock temperatures, using nuclear magnetic resonance analysis of cell extracts. High temperature led to increased incorporation of 13C into trehalose, relative to all other metabolites, and there was undetectable synthesis of glycerol, which was a prominent metabolite of glucose at normal temperature (30 degrees C). Heat shock strongly reduced formation of tricarboxylic acid cycle intermediates, approximately 10-fold, and mannitol synthesis was severely depressed at 46 degrees C, but only moderately reduced at 45 degrees C. A mutant strain of N. crassa that lacks the small alpha-crystallin-related heat shock protein, Hsp30, shows poor survival during heat shock on a nutrient medium with restricted glucose. An analysis of glucose metabolism of this strain showed that, unlike the wild type strain, Hsp30-deficient cells may accumulate unphosphorylated glucose at high temperature. This suggestion that glucose-phosphorylating hexokinase activity might be depressed in mutant cells led us to compare hexokinase activity in the two strains at high temperature. Hexokinase was reduced more than 35% in the mutant cell extracts, relative to wild type extracts. alpha-Crystallin and an Hsp30-enriched preparation protected purified hexokinase from thermal inactivation in vitro, supporting the proposal that Hsp30 may directly stabilize hexokinase in vivo during heat shock.