The shortened replicative life span of prohibitin mutants of yeast appears to be due to defective mitochondrial segregation in old mother cells

Prohibitin proteins have been implicated in cell proliferation, aging, respiratory chain assembly and the maintenance of mitochondrial integrity. The prohibitins of Saccharomyces cerevisiae, Phb1 and Phb2, have strong sequence similarity with their human counterparts prohibitin and BAP37, making yeast a good model organism in which to study prohibitin function. Both yeast and mammalian prohibitins form high-molecular-weight complexes (Phb1/2 or prohibitin/BAP37, respectively) in the inner mitochondrial membrane. Expression of prohibitins declines with senescence, both in mammalian fibroblasts and in yeast. With a total loss of prohibitins, the replicative (budding) life span of yeast is reduced, whilst the chronological life span (the survival of stationary cells over time) is relatively unaffected. This effect of prohibitin loss on the replicative life span is still apparent in the absence of an assembled respiratory chain. It also does not reflect the production of extrachromosomal ribosomal DNA circles (ERCs), a genetic instability thought to be a major cause of replicative senescence in yeast. Examination of cells containing a mitochondrially targeted green fluorescent protein indicates this shortened life span is a reflection of defective mitochondrial segregation from the mother to the daughter in the old mother cells of phb mutant strains. Old mother phb mutant cells display highly aberrant mitochondrial morphology and, frequently, a delayed segregation of mitochondria to the daughter. They often arrest growth with their last bud strongly attached and with the mitochondria adjacent to the septum between the mother and the daughter cell.     

A mother's sacrifice: what is she keeping for herself?

Individual cells of the budding yeast, Saccharomyces cerevisiae, have a limited life span and undergo a form of senescence termed replicative aging. Replicative life span is defined as the number of daughter cells produced by a yeast mother cell before she ceases dividing. Replicative aging is asymmetric: a mother cell ages but the age of her daughter cells is 'reset' to zero. Thus, one or more senescence factors have been proposed to accumulate asymmetrically between mother and daughter yeast cells and lead to mother-specific replicative senescence once a crucial threshold has been reached. Here we evaluate potential candidates for senescence factors and age-associated phenotypes and discuss potential mechanisms underlying the asymmetry of replicative aging in budding yeast.     

Telomere length is independently associated with age,oxidative biomarkers,and sport training in skeletal muscle of healthy adult males

In skeletal muscle, which mainly contains postmitotic myonuclei, it has been suggested that telomere length remains roughly constant throughout adult life, or shortens in response to physiopathological conditions in muscle diseases or in the elderly. However, telomere length results from both the replicative history of a specific tissue and the exposure to environmental, DNA damage-related factors, therefore the predictive biological significance of telomere measures should combine the analysis of the various interactive factors. In the present study, we analysed any relationship between telomere length [mean and minimum terminal restriction fragment (TRF) length] chronological age, oxidative damage (4-HNE, protein carbonyls), catalase activity, and heat shock proteins expression (αB-crystallin, Hsp27, Hsp90) in semitendinous muscle biopsies of 26 healthy adult males between 20 and 50 years of age, also exploring the influence of regular exercise participation. The multiple linear regression analysis identified age, 4-HNE, catalase, and training status as significant independent variables associated with telomere length and jointly accounting for ～30–36% of interindividual variation in mean and/or minimum TRF length. No association has been identified between telomere length and protein carbonyl, αB-crystallin, Hsp27, and Hsp90, as well as between age and the variables related to stress response. Our results showed that skeletal muscle from healthy adults displays an age-dependent telomere attrition and that oxidised environment plays an age-independent contribution, partially influenced by exercise training.     

Higher respiratory activity decreases mitochondrial reactive oxygen release and increases life span in Saccharomyces cerevisiae

Increased replicative longevity in Saccharomyces cerevisiae because of calorie restriction has been linked to enhanced mitochondrial respiratory activity. Here we have further investigated how mitochondrial respiration affects yeast life span. We found that calorie restriction by growth in low glucose increased respiration but decreased mitochondrial reactive oxygen species production relative to oxygen consumption. Calorie restriction also enhanced chronological life span. The beneficial effects of calorie restriction on mitochondrial respiration, reactive oxygen species release, and replicative and chronological life span could be mimicked by uncoupling agents such as dinitrophenol. Conversely, chronological life span decreased in cells treated with antimycin (which strongly increases mitochondrial reactive oxygen species generation) or in yeast mutants null for mitochondrial superoxide dismutase (which removes superoxide radicals) and for RTG2 (which participates in retrograde feedback signaling between mitochondria and the nucleus). These results suggest that yeast aging is linked to changes in mitochondrial metabolism and oxidative stress and that mild mitochondrial uncoupling can increase both chronological and replicative life span.     

The Hsp90 chaperone complex is both a facilitator and a repressor of the dsRNA-dependent kinase PKR.

PKR, a member of the eukaryotic initiation-factor 2alpha (eIF-2alpha) kinase family, mediates the host antiviral response and is implicated in tumor suppression and apoptosis. Here we show that PKR is regulated by the heat shock protein 90 (Hsp90) molecular chaperone complex. Mammalian PKR expressed in budding yeast depends on several components of the Hsp90 complex for accumulation and activity. In mammalian cells, inhibition of Hsp90 function with geldanamycin (GA) during de novo synthesis of PKR also interferes with its accumulation and activity. Hsp90 and its co-chaperone p23 bind to PKR through its N-terminal double-stranded (ds) RNA binding region as well as through its kinase domain. Both dsRNA and GA induce the rapid dissociation of Hsp90 and p23 from mature PKR, activate PKR both in vivo and in vitro and within minutes trigger the phosphorylation of the PKR substrate eIF-2alpha. A short-term exposure of cells to the Hsp90 inhibitors GA or radicicol not only derepresses PKR, but also activates the Raf-MAPK pathway. This suggests that the Hsp90 complex may more generally assist the regulatory domains of kinases and other Hsp90 substrates.     

Induction of Hsp104 synthesis in Saccharomyces cerevisiae in the stationary growth phase is inhibited by the petite mutation

The elevation of Hsp104 (heat shock protein) content under heat stress plays a key role in the development of thermotolerance in yeast (Saccharomyces cerevisiae) cells. Hsp104 synthesis is increased under heat stress and in the stationary growth phase. The loss of mitochondrial DNA (petite mutation) was shown to inhibit the induction of Hsp104 synthesis under heat stress (39°C) and during the transition to the stationary growth phase. Also, the petite mutation suppressed the increase in activity of antioxidant enzymes in the stationary phase, which accompanied by decrease in thermotolerance. At the same time, mutation inhibited production of reactive oxygen species and prevented cell death under heat shock in the logarithmic growth phase. The results of this study suggest that disruption of the mitochondrial functional state suppresses the expression of yeast nuclear genes upon upon entry into the stationary growth phase.     

酿酒酵母衰老机制研究进展

酿酒酵母衰老机制的研究对解析高等真核生物衰老的分子机制具有重要意义。酿酒酵母有两种衰老形式：时序衰老（chronologicalaging）和复制衰老（replicative aging）。酿酒酵母衰老研究中通常使用的寿命定义有两种：世代寿命和时序寿命。前者是指单个酿酒酵母细胞在死亡之前的分裂次数;后者是指一定数量的酵母细胞在后二次生长和稳定期的存活时间。本文分别综述了这两种衰老形式的分子机制及两者的相同点和不同点。     

Sensitivity to Hsp90-targeting drugs can arise with mutation to the Hsp90 chaperone, cochaperones and plasma membrane ATP binding cassette transporters of yeast.

The Hsp90 molecular chaperone catalyses the final activation step of many of the most important regulatory proteins of eukaryotic cells. The antibiotics geldanamycin and radicicol act as highly selective inhibitors of in vivo Hsp90 function through their ability to bind within the ADP/ATP binding pocket of the chaperone. Drugs based on these compounds are now being developed as anticancer agents, their administration having the potential to inactivate simultaneously several of the targets critical for counteracting multistep carcinogenesis. This investigation used yeast to show that cells can be rendered hypersensitive to Hsp90 inhibitors by mutation to Hsp90 itself (within the Hsp82 isoform of yeast Hsp90, the point mutations T101I and A587T); with certain cochaperone defects and through the loss of specific plasma membrane ATP binding cassette transporters (Pdr5p, and to a lesser extent, Snq2p). The T101I hsp82 and A587T hsp82 mutations do not cause higher drug affinity for purified Hsp90 but may render the in vivo chaperone cycle more sensitive to drug inhibition. It is shown that these mutations render at least one Hsp90-dependent process (deactivation of heat-induced heat shock factor activity) more sensitive to drug inhibition in vivo.     

HSP90 controls SIR2 mediated gene silencing

In recent years, Hsp90 is found to interact with several telomeric proteins at various phases of cell cycle. The Hsp90 chaperone system controls assembly and disassembly of telomere structures and thus maintains the dynamic state of telomere. Here, for the first time we report that the activity of another telomeric protein Sir2p is modulated by Hsp82, the ortholog of Hsp90 from budding yeast (Saccharomyces cerevisiae). In a temperature sensitive Hsp90 deficient yeast strain (iG170Dhsp82), less abundant Sir2p is observed, resulting in de-repression of telomere silencing and a complete loss of mating type silencing. Intriguingly, over expression of Hsp90, either by exposing cells to heat shock or by introducing HSP82 overexpression plasmid also yields reduced level of Sir2p, with a consequential loss of telomere silencing. Thus, Hsp90 homeostasis maintains the cellular pool of Sir2p and thereby controls the reversible nature of telomere silencing. Interestingly, such regulation is independent of one of its major co-chaperones Sba1 (human ortholog of p23).     

Expression of a unique drug-resistant Hsp90 ortholog by the nematode Caenorhabditis elegans

In all species studied to date, the function of heat shock protein 90 (Hsp90), a ubiquitous and evolutionarily conserved molecular chaperone, is inhibited selectively by the natural product drugs geldanamycin (GA) and radicicol. Crystal structures of the N-terminal region of yeast and human Hsp90 have revealed that these compounds interact with the chaperone in a Bergerat-type adenine nucleotide-binding fold shared throughout the gyrase, Hsp90, histidine kinase mutL (GHKL) superfamily of adenosine triphosphatases. To better understand the consequences of disrupting Hsp90 function in a genetically tractable multicellular organism, we exposed the soil-dwelling nematode Caenorhabditis elegans to GA under a variety of conditions designed to optimize drug uptake. Mutations in the gene encoding C elegans Hsp90 affect larval viability, dauer development, fertility, and life span. However, exposure of worms to GA produced no discernable phenotypes, although the amino acid sequence of worm Hsp90 is 85% homologous to that of human Hsp90. Consistent with this observation, we found that solid phase-immobilized GA failed to bind worm Hsp90 from worm protein extracts or when translated in a rabbit reticulocyte lysate system. Further, affinity precipitation studies using chimeric worm-vertebrate fusion proteins or worm C-terminal truncations expressed in reticulocyte lysate revealed that the conserved nucleotide-binding fold of worm Hsp90 exhibits the novel ability to bind adenosine triphosphate but not GA. Despite its unusual GA resistance, worm Hsp90 appeared fully functional when expressed in a vertebrate background. It heterodimerized with its vertebrate counterpart and showed no evidence of compromising its essential cellular functions. Heterologous expression of worm Hsp90 in tumor cells, however, did not render them GA resistant. These findings provide new insights into the nature of unusual N-terminal nucleotide-binding fold of Hsp90 and suggest that target-related drug resistance is unlikely to emerge in patients receiving GA-like chemotherapeutic agents.     

NMR structure of hsp12, a protein induced by and required for dietary restriction-induced lifespan extension in yeast

Dietary restriction (DR) extends lifespan in yeast, worms, flies and mammals, suggesting that it may act via conserved processes. However, the downstream mechanisms by which DR increases lifespan remain unclear. We used a gel based proteomic strategy to identify proteins whose expression was induced by DR in yeast and thus may correlate with longevity. One protein up-regulated by DR was Hsp12, a small heat shock protein induced by various manipulations known to retard ageing. Lifespan extension by growth on 0.5% glucose (DR) was abolished in an hsp12Δ strain, indicating that Hsp12 is essential for the longevity effect of DR. In contrast, deletion of HSP12 had no effect on growth under DR conditions or a variety of environmental stresses, indicating that the effect of Hsp12 on lifespan is not due to increased general stress resistance. Unlike other small heat shock proteins, recombinant Hsp12 displayed negligible in vitro molecular chaperone activity, suggesting that its cellular function does not involve preventing protein aggregation. NMR analysis indicated that Hsp12 is monomeric and intrinsically unfolded in solution, but switches to a 4-helical conformation upon binding to membrane-mimetic SDS micelles. The structure of micelle-bound Hsp12 reported here is consistent with its recently proposed function as a membrane-stabilising 'lipid chaperone'. Taken together, our data suggest that DR-induced Hsp12 expression contributes to lifespan extension, possibly via membrane alterations.     

Inactivation of dual-specificity phosphatases is involved in the regulation of extracellular signal-regulated kinases by heat shock and hsp72

Extracellular signal-regulated kinase 1 (ERK1) and ERK2 (ERK1/2) dramatically enhance survival of cells exposed to heat shock. Using Cos-7 cells and primary human fibroblasts (IMR90 cells), we demonstrated that heat shock activates ERKs via two distinct mechanisms: stimulation of the ERK-activating kinases, MEK1/2, and inhibition of ERK dephosphorylation. Under milder heat shock conditions, activation of ERKs proceeded mainly through stimulation of MEK1/2, whereas under more severe heat shock MEK1/2 could no longer be activated and the inhibition of ERK phosphatases became critical. In Cos-7 cells, nontoxic heat shock caused rapid inactivation of the major ERK phosphatase, MKP-3, by promoting its aggregation, so that in cells exposed to 45 degrees C for 20 min, 90% of MKP-3 became insoluble. MKP-3 aggregation was reversible and, 1 h after heat shock, MKP-3 partially resolubilized. The redistribution of MKP-3 correlated with an increased rate of ERK dephosphorylation. Similar heat-induced aggregation, followed by partial resolubilization, was found with a distinct dual-specificity phosphatase MKP-1 but not with MKP-2. Therefore, MKP-3 and MKP-1 appeared to be critical heat-labile phosphatases involved in the activation of ERKs by heat shock. Expression of the major heat shock protein Hsp72 inhibited activation of MEK1/2 and prevented inactivation of MKP-3 and MKP-1. Hsp72DeltaEEVD mutant lacking a chaperone activity was unable to protect MKP-3 from heat inactivation but interfered with MEK1/2 activation similar to normal Hsp72. Hence, Hsp72 suppressed ERK activation by both protecting dual-specificity phosphatases, which was dependent on the chaperone activity, and suppressing MEK1/2, which was independent of the chaperone activity.     

Overexpressed Sod1p acts either to reduce or to increase the lifespans and stress resistance of yeast, depending on whether it is Cu(2+)-deficient or an active Cu,Zn-superoxide dismutase

Yeast overexpressing SOD1, the gene for Cu,Zn-superoxide dismutase (Cu,Zn-Sod), was used to determine how Sod1p overexpression influences the chronological lifespan [the survival of non-dividing stationary (G0) phase cells over time], the replicative lifespan (the number of buds produced by actively dividing yeast cells) and stress resistance. Increasing the level of active Cu,Zn-Sod in yeast was found to require either growth in the presence of high copper, or the simultaneous overexpression of both SOD1 and CCS1 (the latter being the gene that encodes the chaperone dedicated to Cu(2+)-loading of Sod1p in vivo). Dual SOD1 + CCS1 overexpression elevated the levels of Cu,Zn-Sod activity six- to eight-fold in vegetative cultures. It also increased the optimized survival of stationary cells up to two-fold, showing this chronological lifespan is ultimately limited by oxidative stress. In contrast, several detrimental effects resulted when the SOD1 gene was overexpressed in the absence of either high copper or a simultaneous overexpression of CCS1. Both the chronological and the replicative lifespans were shortened; the cells displayed an abnormally high level of endogenous oxidative stress, resulting in a high rate of spontaneous mutation. Such harmful effects were all reversed through the overexpression of CCS1. It is apparent therefore that they relate to the incomplete Cu(2+)-loading of the overexpressed Sod1p, most probably accumulation of a Cu(2+)-deficient Sod1p to appreciable levels in vivo. The same events may generate the detrimental effects that are frequently, though not universally, observed when Cu,Zn-Sod overexpression is attempted in metazoans.     

Hsp90 is involved in the formation of P-bodies and stress granules

Previously, we found that treatment of cells with the Hsp90 inhibitor geldanamycin (GA) leads to a substantial reduction in the number of processing bodies (P-bodies), and also alters the size and subcellular localization of stress granules. These findings imply that the chaperone activity of Hsp90 is involved in the formation of P-bodies and stress granules. To verify these observations, we examined whether another Hsp90 inhibitor radicicol (RA) affected P-bodies and stress granules. Treatment with RA reduced the level of the Hsp90 client protein Argonaute 2 and the number of P-bodies. Although stress granules still assembled in RA-treated cells upon heat shock, they were smaller and more dispersed in the cytoplasm than those in untreated cells. Furthermore eIF4E and eIF4E-transporter were dissociated selectively from stress granules in RA-treated cells. These observations were comparable to those obtained upon treatment with GA in our previous work. Thus, we conclude that abrogation of the chaperone activity of Hsp90 affects P-body formation and the integrity of stress granules.     

In vivo chaperone activity of heat shock protein 70 and thermotolerance

Heat shock protein 70 (Hsp70) is thought to play a critical role in the thermotolerance of mammalian cells, presumably due to its chaperone activity. We examined the chaperone activity and cellular heat resistance of a clonal cell line in which overexpression of Hsp70 was transiently induced by means of the tetracycline-regulated gene expression system. This single-cell-line approach circumvents problems associated with clonal variation and indirect effects resulting from constitutive overexpression of Hsp70. The in vivo chaperone function of Hsp70 was quantitatively investigated by using firefly luciferase as a reporter protein. Chaperone activity was found to strictly correlate to the level of Hsp70 expression. In addition, we observed an Hsp70 concentration dependent increase in the cellular heat resistance. In order to study the contribution of the Hsp70 chaperone activity, heat resistance of cells that expressed tetracycline-regulated Hsp70 was compared to thermotolerant cells expressing the same level of Hsp70 plus all of the other heat shock proteins. Overexpression of Hsp70 alone was sufficient to induce a similar recovery of cytoplasmic luciferase activity, as does expression of all Hsps in thermotolerant cells. However, when the luciferase reporter protein was directed to the nucleus, expression of Hsp70 alone was not sufficient to yield the level of recovery observed in thermotolerant cells. In addition, cells expressing the same level of Hsp70 found in heat-induced thermotolerant cells containing additional Hsps showed increased resistance to thermal killing but were more sensitive than thermotolerant cells. These results suggest that the inducible form of Hsp70 contributes to the stress-tolerant state by increasing the chaperone activity in the cytoplasm. However, its expression alone is apparently insufficient for protection of other subcellular compartments to yield clonal heat resistance to the level observed in thermotolerant cells.