Small heat shock proteins in the amphibian Pelophylax bergeri: Cloning and characterization of Hsp27 and Hsp30 cDNAs and their expression analysis in ex vivo skin exposed to abiotic stresses.

Small Heat Shock Proteins (sHSP) are molecular chaperones that play an essential role in maintaining protein homeostasis and promoting cell survival. In this work, for the first time, multiple cDNAs encoding for small Hsp27 and Hsp30, designed, respectively, as PbHsp27-(1-2) and PbHsp30-(1-5), were cloned and characterized in the amphibian Pelophylax bergeri, which is a suitable model for studying biological responses to environmental perturbations. Domain architecture analysis showed that PbHsp27 and PbHsp30 cDNAs displayed the typical signature motifs of the sHSP family such as the conserved α-crystallin domain flanked by variable N-terminal and C-terminal regions. Phylogenetic analysis revealed that PbHsp27 and PbHsp30 clustered, respectively, with Hsp27 and Hsp30 members of other vertebrates, but more closely with amphibians. Overall PbHsp27 and PbHsp30 transcriptional activity, analyzed by qRT-PCR, evidenced that, in ex vivo skin exposed to thermal shock and cadmium treatment, PbHsp27 and PbHsp30 mRNAs were inducible and regulated differently. This study provides the basis for future research on the potential use of PbHsp27 and PbHsp30 as biomarkers of proteotoxic stress in amphibians.

TORC1-signalling is down-regulated in Saccharomyces cerevisiae hsp30Δ cells by SNF1-dependent mechanisms.

Hsp30 is a plasma membrane localized heat shock protein in Saccharomyces cerevisiae whose expression is induced by numerous environmental stressors. Elucidation of its mechanism of action has remained elusive primarily because hsp30Δ cells do not show a strong phenotype. To identify cellular functions associated with Hsp30, we thus compared the transcriptome of BY4741hsp30Δ with that of its wild type counterpart. Our studies indicate down-regulation of the target of rapamycin complex 1 (TORC1)-dependent gene-expression programme in hsp30Δ cells. We further show that TORC1-signalling through its effectors (Sch9 and Tap42) was down-regulated in the deletion strain. Specifically, (a) phosphorylation levels of Sch9 were lower and nuclear exclusion of Rim15 (Sch9-downstream function) was overridden in hsp30Δ cells, (b) membrane association of Tor1 and Tap42 was lower in hsp30Δ cells, and (c) Tap42-downstream functions were abrogated in the deletion strain. Furthermore, transcription factors Rtg1, Rtg3, Gat1, and Gln3 were localized in the nucleus of the hsp30Δ as observed upon inactivation of TORC1. Studies aimed at determining how TORC1-signalling is down-regulated in hsp30Δ cells indicated that total reducing sugar levels were lower and ADP:ATP ratio was higher in hsp30Δ cells -conditions known to activate the Snf1 kinase and consequently to the inactivation of TORC1. We thus determined if TORC1-signalling could be restored in hsp30Δ cells upon the deletion of SNF1. Sch9 phosphorylation levels (TORC1-signalling) was restored to wild type levels in hsp30Δsnf1Δ cells. TORC1-signalling is thus down-regulated in hsp30Δ cells by SNF1-dependent mechanisms. A probable role for Hsp30 is discussed.

Effect of hemin, baicalein and heme oxygenase-1 (HO-1) enzyme activity inhibitors on Cd-induced accumulation of HO-1, HSPs and aggresome-like structures in Xenopus kidney epithelial cells.

Cadmium is a highly toxic environmental pollutant that can cause many adverse effects including cancer, neurological disease and kidney damage. Aquatic amphibians are particularly susceptible to this toxicant as it was shown to cause developmental abnormalities and genotoxic effects. In mammalian cells, the accumulation of heme oxygenase-1 (HO-1), which catalyzes the breakdown of heme into CO, free iron and biliverdin, was reported to protect cells against potentially lethal concentrations of CdCl2. In the present study, CdCl2 treatment of A6 kidney epithelial cells, derived from the frog, Xenopus laevis, induced the accumulation of HO-1, heat shock protein 70 (HSP70) and HSP30 as well as an increase in the production of aggregated protein and aggresome-like structures. Treatment of cells with inhibitors of HO-1 enzyme activity, tin protoporphyrin (SnPP) and zinc protoporphyrin (ZnPP), enhanced CdCl2-induced actin cytoskeletal disorganization and the accumulation of HO-1, HSP70, aggregated protein and aggresome-like structures. Treatment of cells with hemin and baicalein, which were previously shown to provide cytoprotection against various stresses, induced HO-1 accumulation in a concentration-dependent manner. Also, treatment of cells with hemin and baicalein suppressed CdCl2-induced actin dysregulation and the accumulation of aggregated protein and aggresome-like structures. This cytoprotective effect was inhibited by SnPP. These results suggest that HO-1-mediated protection against CdCl2 toxicity includes the maintenance of actin cytoskeletal and microtubular structure and the suppression of aggregated protein and aggresome-like structures.

Effect of isothiocyanates, BITC and PEITC, on stress protein accumulation, protein aggregation and aggresome-like structure formation in Xenopus A6 kidney epithelial cells.

Numerous studies have elucidated the health benefits of organosulfur compounds, known as isothiocyanates (ITCs), derived from cruciferous vegetables. As electrophiles, ITCs have the ability to directly bind and modify thiol-containing compounds such as glutathione and cellular protein, including tubulin. While the biochemical effects of ITCs have been well characterized, less information is available regarding their effects on the accumulation of stress-inducible heme oxygenase-1 (HO-1), heat shock proteins (HSPs) and the possible formation of aggregated protein due to thiol modification. The present study has examined the effect of the ITCs, benzyl isothiocyanate (BITC) and phenethyl isothiocyanate (PEITC), on the accumulation of HO-1, HSP70 and HSP30 in Xenopus laevis A6 kidney epithelial cells. Immunoblot analysis revealed that both BITC and PEITC induced the accumulation of HO-1 and HSP70 whereas HSP30 levels were enhanced only in cells treated with BITC. Immunocytochemistry determined that ITC treatment induced F-actin disorganization and membrane ruffling and enhanced accumulation of HO-1 in the cytoplasm. Additionally, BITC induced enhanced levels of ubiquitinated protein, aggregated protein, and the collapse and fragmentation of microtubules. In comparison, treatment of cells with the proteasomal inhibitor, MG132, induced the accumulation of all three stress proteins, aggregated protein and aggresome-like structures. Finally, cells pretreated with BITC inhibited the formation of MG132-induced aggresome-like structures in the perinuclear region. This latter finding suggests that BITC-induced microtubule fragmentation may impede the movement of aggregated protein via microtubules and their subsequent coalescence into aggresome-like structures in the perinuclear region.

The expression and function of hsp30-like small heat shock protein genes in amphibians, birds, fish, and reptiles.

Small heat shock proteins (sHSPs) are a superfamily of molecular chaperones with important roles in protein homeostasis and other cellular functions. Amphibians, reptiles, fish and birds have a shsp gene called hsp30, which was also referred to as hspb11 or hsp25 in some fish and bird species. Hsp30 genes, which are not found in mammals, are transcribed in response to heat shock or other stresses by means of the heat shock factor that is activated in response to an accumulation of unfolded protein. Amino acid sequence analysis revealed that representative HSP30s from different classes of non-mammalian vertebrates were distinct from other sHSPs including HSPB1/HSP27. Studies with amphibian and fish recombinant HSP30 determined that they were molecular chaperones since they inhibited heat- or chemically-induced aggregation of unfolded protein. During non-mammalian vertebrate development, hsp30 genes were differentially expressed in selected tissues. Also, heat shock-induced stage-specific expression of hsp30 genes in frog embryos was regulated at the level of chromatin structure. In adults and/or tissue culture cells, hsp30 gene expression was induced by heat shock, arsenite, cadmium or proteasomal inhibitors, all of which enhanced the production of unfolded/damaged protein. Finally, immunocytochemical analysis of frog and chicken tissue culture cells revealed that proteotoxic stress-induced HSP30 accumulation co-localized with aggresome-like inclusion bodies. The congregation of damaged protein in aggresomes minimizes the toxic effect of aggregated protein dispersed throughout the cell. The current availability of probes to detect the presence of hsp30 mRNA or encoded protein has resulted in the increased use of hsp30 gene expression as a marker of proteotoxic stress in non-mammalian vertebrates.

The CgHaa1-Regulon Mediates Response and Tolerance to Acetic Acid Stress in the Human Pathogen Candida glabrata.

To thrive in the acidic vaginal tract, Candida glabrata has to cope with high concentrations of acetic acid. The mechanisms underlying C. glabrata tolerance to acetic acid at low pH remain largely uncharacterized. In this work, the essential role of the CgHaa1 transcription factor (encoded by ORF CAGL0L09339g) in the response and tolerance of C. glabrata to acetic acid is demonstrated. Transcriptomic analysis showed that CgHaa1 regulates, directly or indirectly, the expression of about 75% of the genes activated under acetic acid stress. CgHaa1-activated targets are involved in multiple physiological functions including membrane transport, metabolism of carbohydrates and amino acids, regulation of the activity of the plasma membrane H+-ATPase, and adhesion. Under acetic acid stress, CgHaa1 increased the activity and the expression of the CgPma1 proton pump and contributed to increased colonization of vaginal epithelial cells by C. glabrata CgHAA1, and two identified CgHaa1-activated targets, CgTPO3 and CgHSP30, are herein demonstrated to be determinants of C. glabrata tolerance to acetic acid. The protective effect of CgTpo3 and of CgHaa1 was linked to a role of these proteins in reducing the accumulation of acetic acid inside C. glabrata cells. In response to acetic acid stress, marked differences were found in the regulons controlled by CgHaa1 and by its S. cerevisiae ScHaa1 ortholog, demonstrating a clear divergent evolution of the two regulatory networks. The results gathered in this study significantly advance the understanding of the molecular mechanisms underlying the success of C. glabrata as a vaginal colonizer.

The small heat shock protein, HSP30, is associated with aggresome-like inclusion bodies in proteasomal inhibitor-, arsenite-, and cadmium-treated Xenopus kidney cells.

In the present study, treatment of Xenopus laevis A6 kidney epithelial cells with the proteasomal inhibitor, MG132, or the environmental toxicants, sodium arsenite or cadmium chloride, induced the accumulation of the small heat shock protein, HSP30, in total and in both soluble and insoluble protein fractions. Immunocytochemical analysis revealed the presence of relatively large HSP30 structures primarily in the perinuclear region of the cytoplasm. All three of the stressors promoted the formation of aggresome-like inclusion bodies as determined by immunocytochemistry and laser scanning confocal microscopy using a ProteoStat aggresome dye and additional aggresomal markers, namely, anti-γ-tubulin and anti-vimentin antibodies. Further analysis revealed that HSP30 co-localized with these aggresome-like inclusion bodies. In most cells, HSP30 was found to envelope or occur within these structures. Finally, we show that treatment of cells with withaferin A, a steroidal lactone with anti-inflammatory, anti-tumor, and proteasomal inhibitor properties, also induced HSP30 accumulation that co-localized with aggresome-like inclusion bodies. It is possible that proteasomal inhibitor or metal/metalloid-induced formation of aggresome-like inclusion bodies may sequester toxic protein aggregates until they can be degraded. While the role of HSP30 in these aggresome-like structures is not known, it is possible that they may be involved in various aspects of aggresome-like inclusion body formation or transport.

Accumulation of heme oxygenase-1 (HSP32) in Xenopus laevis A6 kidney epithelial cells treated with sodium arsenite, cadmium chloride or proteasomal inhibitors.

The present study examined the effect of sodium arsenite, cadmium chloride, heat shock and the proteasomal inhibitors MG132, withaferin A and celastrol on heme oxygenase-1 (HO-1; also known as HSP32) accumulation in Xenopus laevis A6 kidney epithelial cells. Immunoblot analysis revealed that HO-1 accumulation was not induced by heat shock but was enhanced by sodium arsenite and cadmium chloride in a dose- and time-dependent fashion. Immunocytochemistry revealed that these metals induced HO-1 accumulation in a granular pattern primarily in the cytoplasm. Additionally, in 20% of the cells arsenite induced the formation of large HO-1-containing perinuclear structures. In cells recovering from sodium arsenite or cadmium chloride treatment, HO-1 accumulation initially increased to a maximum at 12h followed by a 50% reduction at 48 h. This initial increase in HO-1 levels was likely the result of new synthesis as it was inhibited by cycloheximide. Interestingly, treatment of cells with a mild heat shock enhanced HO-1 accumulation induced by low concentrations of sodium arsenite and cadmium chloride. Finally, we determined that HO-1 accumulation was induced in A6 cells by the proteasomal inhibitors, MG132, withaferin A and celastrol. An examination of heavy metal and proteasomal inhibitor-induced HO-1 accumulation in amphibians is of importance given the presence of toxic heavy metals in aquatic habitats.

Distinct patterns of HSP30 and HSP70 degradation in Xenopus laevis A6 cells recovering from thermal stress.

Heat shock proteins (HSPs) are molecular chaperones that assist in protein synthesis, folding and degradation and prevent stress-induced protein aggregation. In this study, we examined the pattern of accumulation of HSP30 and HSP70 in Xenopus laevis A6 kidney epithelial cells recovering from heat shock. Immunoblot analysis revealed the presence of elevated levels of HSP30 after 72h of recovery. However, the relative levels of HSP70 declined to near control levels after 24h. The relative levels of both hsp30 and hsp70 mRNA were reduced to low levels after 24h of recovery from heat shock. Pretreatment of cells with cycloheximide, a translational inhibitor, produced a rapid decline in HSP70 but not HSP30. The cycloheximide-associated decline of HSP70 was blocked by the proteasomal inhibitor, MG132, but had little effect on the relative level of HSP30. Also, treatment of cells with the phosphorylation inhibitor, SB203580, in addition to cycloheximide treatment enhanced the stability of HSP30 compared to cycloheximide alone. Immunocytochemical studies detected the presence of HSP30 accumulation in a granular pattern in the cytoplasm of recovering cells and its association with aggresome-like structures, which was enhanced in the presence of SB203580. This study has shown that the relative levels of heat shock-induced HSP30 persist during recovery in contrast to HSP70. While HSP70 is degraded by the ubiquitin-proteasome system, it is likely that the presence of HSP30 multimeric complexes that are known to associate with unfolded protein as well as its association with aggresome-like structures may delay its degradation.

Synteny analysis provides a route to design genus-specific PCR primers for rapid identification of all Saccharomyces species.

The genus Saccharomyces comprises seven single-genome species (S. arboricola, S. cerevisiae, S. eubayanus, S. kudriavzevii, S. mikatae, S. paradoxus and S. uvarum) and two hybrid species - S. pastorianus (S. cerevisiae plus S. eubayanus) and S. bayanus (mostly S. uvarum plus S. eubayanus). Species-specific primers have already been developed for the identification of each of the single-genome species, and these primers can usually detect both genomes in hybrids. It would be advantageous if a single reaction could detect any member of the clade. We have investigated three potentially generic approaches to design genus-specific primers. Two methods that both use sequence alignment differences for primer design were only partly successful. A third method used synteny data to identify 136 target genes that are potentially present only in all species of the Saccharomyces clade. HSP30 (YCR021C) was fully successful; different primer pairs were developed with high G+C content for use at 63 °C. In < 3 h, using a robust colony-PCR followed by gel electrophoresis, the method can reliably detect any member of the genus. This novel approach still uses conventional sequence alignment mismatches but relies principally on the presence of the target gene only within the genus Saccharomyces.

Enhanced HSP30 and HSP70 accumulation in Xenopus cells subjected to concurrent sodium arsenite and cadmium chloride stress.

Heat shock proteins (HSPs) are molecular chaperones that aid in protein folding, translocation and in preventing stress-induced protein aggregation. The present study examined the effect of simultaneous sodium arsenite and cadmium chloride treatment on the pattern of HSP30 and HSP70 accumulation in A6 kidney epithelial cells of the frog, Xenopus laevis. Immunoblot analysis revealed that HSP30 and HSP70 accumulation in concurrent stressor treatments were significantly higher than the sum of HSP30 or HSP70 accumulation in individual treatments. This finding suggested a synergistic action between sodium arsenite and cadmium chloride. KNK437 inhibitor studies indicated that the combined stressor-induced accumulation of HSPs may be regulated, at least in part, at the level of transcription. Immunocytochemistry revealed that simultaneous treatment of cells with the two stressors induced HSP30 accumulation primarily in the cytoplasm in a punctate pattern with some dysregulation of F-actin structure. Increased ubiquitinated protein accumulation was observed with combined sodium arsenite and cadmium chloride treatment compared to individual stressors suggesting an impairment of the ubiquitin proteasome degradation system. The addition of a mild heat shock further enhanced the accumulation of HSP30 and HSP70 in response to relatively low concentrations of sodium arsenite plus cadmium chloride.

Structural alteration of Escherichia coli Hsp31 by thermal unfolding increases chaperone activity.

Escherichia coli Hsp31, encoded by hchA, is a heat-inducible molecular chaperone. We found that Hsp31 undergoes a conformational change via temperature-induced unfolding, generating a high molecular weight (HMW) form with enhanced chaperone activity. Although it has previously been reported that some subunits of the Hsp31 crystal structure show structural heterogeneity with increased hydrophobic surfaces, Hsp31 basically forms a dimer. We found that a C-terminal deletion (CΔ19) of Hsp31 exhibited structurally and functionally similar characteristics to that of the HMW form. Both the CΔ19 and HMW forms achieved a structure with considerably more ß-sheets and less α-helices than the native dimeric form, exposing a portion of its hydrophobic surfaces. The structural alterations were determined from its spectral changes in circular dichroism, intrinsic fluorescence of tryptophan residues, and fluorescence of bis-ANS binding to a hydrophobic surface. Interestingly, during thermal transition, the dimeric Hsp31 undergoes a conformational change to the HMW species via the CΔ19 structure, as monitored with near-UV CD spectrum, implying that the CΔ19 resembles an intermediate state between the dimer and the HMW form. From these results, we propose that Hsp31 transforms itself into a fully functional chaperone by altering its tertiary and quaternary structures.

Withaferin A induces proteasome inhibition, endoplasmic reticulum stress, the heat shock response and acquisition of thermotolerance.

In the present study, withaferin A (WA), a steroidal lactone with anti-inflammatory and anti-tumor properties, inhibited proteasome activity and induced endoplasmic reticulum (ER) and cytoplasmic HSP accumulation in Xenopus laevis A6 kidney epithelial cells. Proteasomal inhibition by WA was indicated by an accumulation of ubiquitinated protein and a decrease in chymotrypsin-like activity. Additionally, immunoblot analysis revealed that treatment of cells with WA induced the accumulation of HSPs including ER chaperones, BiP and GRP94, as well as cytoplasmic/nuclear HSPs, HSP70 and HSP30. Furthermore, WA-induced an increase in the relative levels of the protein kinase, Akt, while the levels of actin were unchanged compared to control. Northern blot experiments determined that WA induced an accumulation in bip, hsp70 and hsp30 mRNA but not eIF-1α mRNA. Interestingly, WA acted synergistically with mild heat shock to enhance HSP70 and HSP30 accumulation to a greater extent than the sum of both stressors individually. This latter phenomenon was not observed with BiP or GRP94. Immunocytochemical analysis indicated that WA-induced BiP accumulation occurred mainly in the perinuclear region in a punctate pattern, while HSP30 accumulation occurred primarily in a granular pattern in the cytoplasm with some staining in the nucleus. Prolonged exposure to WA resulted in disorganization of the F-actin cytoskeleton as well as the production of relatively large HSP30 staining structures that co-localized with F-actin. Finally, prior exposure of cells to WA treatment, which induced the accumulation of HSPs conferred a state of thermal protection since it protected the F-actin cytoskeleton against a subsequent cytotoxic thermal challenge.

Temperature dependent N-glycosylation of plasma membrane heat shock protein Hsp30p in Saccharomyces cerevisiae.

The HSP30 gene of the budding yeast Saccharomyces cerevisiae encodes a seven-transmembrane heat shock protein expressed in response to various types of stress including heat shock. Although Hsp30p contains a potential N-glycosylation consensus sequence (Asn(2)-Asp(3)-Thr(4)), whether it is actually N-glycosylated has not been verified. Here we demonstrate that N-glycosylation is induced at Asn(2) of Hsp30p by severe heat shock, ethanol stress, and acetic acid stress. Mild heat shock and glucose depletion induced the expression but not N-glycosylation of Hsp30p, indicating the N-glycosylation to be dependent on temperature and environmental conditions. N-glycosylation did not affect on the intracellular localization of Hsp30p but its physiological role under severe heat shock conditions. Since limited information is available on stress-responsive or condition-induced N-glycosylation, our findings provide new insight into the regulation of cellular stress response in yeast.

Sodium arsenite and cadmium chloride induction of proteasomal inhibition and HSP accumulation in Xenopus laevis A6 kidney epithelial cells.

Sodium arsenite (NA) and cadmium chloride (CdCl(2)) are relatively abundant environmental toxicants that have multiple toxic effects including carcinogenesis, dysfunction of gene regulation and DNA and protein damage. In the present study, treatment of Xenopus laevis A6 kidney epithelial cells with concentrations of NA (20-30 µM) or CdCl(2) (100-200 µM) that induced HSP30 and HSP70 accumulation also produced an increase in the relative levels of ubiquitinated protein. Actin protein levels were unchanged in these experiments. In time course experiments, the levels of ubiquitinated protein and HSPs increased over a 24h exposure to NA or CdCl(2). Furthermore, treatment of cells with NA or CdCl(2) reduced the relative levels of proteasome chymotrypsin (CT)-like activity compared to control. Interestingly, pretreatment of cells with the HSP accumulation inhibitor, KNK437, prior to NA or CdCl(2) exposure decreased the relative levels of ubiquitinated protein as well as HSP30 and HSP70. A similar finding was made with ubiquitinated protein induced by proteasomal inhibitors, MG132 and celastrol, known to induce HSP accumulation in A6 cells. However, the NA- or CdCl(2)-induced decrease in proteasome CT-like activity was not altered by KNK437 pretreatment. This study has shown for the first time in poikilothermic vertebrates that NA and CdCl(2) can inhibit proteasomal activity and that there is a possible association between HSP accumulation and the mechanism of protein ubiquitination.

Transcription of the Hsp30, Hsp70, and Hsp90 heat shock protein genes is modulated by the PalA protein in response to acid pH-sensing in the fungus Aspergillus nidulans.

Heat shock proteins are molecular chaperones linked to a myriad of physiological functions in both prokaryotes and eukaryotes. In this study, we show that the Aspergillus nidulans hsp30 (ANID_03555.1), hsp70 (ANID_05129.1), and hsp90 (ANID_08269.1) genes are preferentially expressed in an acidic milieu, whose expression is dependent on the palA (+) background under optimal temperature for fungal growth. Heat shock induction of these three hsp genes showed different patterns in response to extracellular pH changes in the palA(+) background. However, their accumulation upon heating for 2 h was almost unaffected by ambient pH changes in the palA (-) background. The PalA protein is a member of a conserved signaling cascade that is involved in the pH-mediated regulation of gene expression. Moreover, we identified several genes whose expression at pH 5.0 is also dependent on the palA (+) background. These results reveal novel aspects of the heat- and pH-sensing networks of A. nidulans.

Effects of triclocarban, triclosan, and methyl triclosan on thyroid hormone action and stress in frog and mammalian culture systems.

Triclosan (TCS) and triclocarban (TCC) are widely used broad spectrum bactericides that are common pollutants of waterways and soils. Methyl triclosan (mTCS) is the predominant bacterial TCS metabolite. Previous studies have shown that TCS disrupts thyroid hormone (TH) action; however, the effects of mTCS or TCC are not known. The present study uses the cultured frog tadpole tail fin biopsy (C-fin) assay and the TH-responsive rat pituitary GH3 cell line to assess the effects of these three chemicals (1-1000 nM) on TH signaling and cellular stress within 48 h. mRNA abundance of TH receptor ß, Rana larval keratin type I (TH-response), heat shock protein 30, and catalase (stress-response) was measured using quantitative real-time polymerase chain reaction in the C-fin assay. The TH-responsive gene transcripts encoding growth hormone, deiodinase I, and prolactin were measured in GH3 cells with the heat shock protein 70 transcript acting as a cellular stress indicator. We found alteration of stress indicators at a wide range of concentrations of TCS, mTCS, and TCC in both test systems. mTCS and TCC affected TH-responsive gene transcripts at the highest concentration in mammalian cells, whereas a modest effect included lower concentrations in the C-fin assay. In contrast, TCS did not affect TH-responsive transcripts. These results identify nontarget biological effects of these bacteriocides on amphibian and mammalian cells and suggest the TH-disrupting effects observed for TCS could be mediated through its metabolite.

SIR2 and other genes are abundantly expressed in long-lived natural segregants for replicative aging of the budding yeast Saccharomyces cerevisiae.

We investigated the mechanism underlying the natural variation in longevity within natural populations using the model budding yeast, Saccharomyces cerevisiae. We analyzed whole-genome gene expression in four progeny of a natural S. cerevisiae strain that display differential replicative aging. Genes with different expression levels in short- and long-lived strains were classified disproportionately into metabolism, transport, development, transcription or cell cycle, and organelle organization (mitochondrial, chromosomal, and cytoskeletal). With several independent validating experiments, we detected 15 genes with consistent differential expression levels between the long- and the short-lived progeny. Among those 15, SIR2, HSP30, and TIM17 were upregulated in long-lived strains, which is consistent with the known effects of gene silencing, stress response, and mitochondrial function on aging. The link between SIR2 and yeast natural life span variation offers some intriguing ties to the allelic association of the human homolog SIRT1 to visceral obesity and metabolic response to lifestyle intervention.

Curcumin-induced inhibition of proteasomal activity, enhanced HSP accumulation and the acquisition of thermotolerance in Xenopus laevis A6 cells.

In the present study, curcumin, a phenolic compound with anti-inflammatory, anti-tumor and anti-amyloid properties, inhibited proteasomal activity and induced the accumulation of HSPs in the frog model system, Xenopus laevis. Treatment of A6 kidney epithelial cells with curcumin enhanced ubiquitinated protein levels and inhibited chymotrypsin-like activity. Furthermore, exposure of cells to 10-50 µM curcumin for 24h induced HSP30 and HSP70 accumulation. This phenomenon was controlled at the transcriptional level since pre-treatment of cells with KNK437, a heat shock factor 1 (HSF1) inhibitor, repressed HSP accumulation. Additionally, elevation of the incubation temperature from 22 to 30 °C greatly enhanced the curcumin-induced accumulation of HSP30 and HSP70. Immunocytochemical analysis revealed that curcumin-induced HSP30 was detectable primarily in the cytoplasm in a punctate pattern with minimal detrimental effects on the actin cytoskeleton. Finally, prior exposure of cells to curcumin conferred a state of thermotolerance since it protected them against a subsequent cytotoxic thermal challenge. These findings are of importance given the interest in identifying agents that can upregulate HSP levels with minimal effects on cell structure or function as a therapeutic treatment of protein folding diseases.

The forkhead transcription factor Hcm1 promotes mitochondrial biogenesis and stress resistance in yeast.

In Saccharomyces cerevisiae, the forkhead transcription factor Hcm1 is involved in chromosome segregation, spindle pole dynamics, and budding. We found that Hcm1 interacts with the histone deacetylase Sir2 and shifts from cytoplasm to the nucleus in the G(1)/S phase or in response to oxidative stress stimuli. The nuclear localization of Hcm1 depends on the activity of Sir2 as revealed by activators and inhibitors of the sirtuins and the Δsir2 mutant. Hcm1-overexpressing cells display more mitochondria that can be attributed to increased amounts of Abf2, a protein involved in mitochondrial biogenesis. These cells also show higher rates of oxygen consumption and improved resistance to oxidative stress that would be explained by increased catalase and Sod2 activities and molecular chaperones such as Hsp26, Hsp30, and members of Hsp70 family. Microarray analyses also reveal increased expression of genes involved in mitochondrial energy pathways and those allowing the transition from the exponential to the stationary phase. Taken together, these results describe a new and relevant role of Hcm1 for mitochondrial functions, suggesting that this transcription factor would participate in the adaptation of cells from fermentative to respiratory metabolism.