Mitochondrial Hsp60, Resistance to Oxidative Stress, and the Labile Iron Pool Are Closely Connected in Saccharomyces cerevisiae

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195

168

Oxidative modifications of cellular components have been described as one of the main contributions to aged phenotype. In Saccharomyces cerevisiae, two distinct life spans can be considered, replicative and chronological. The relationship between both aging models is still not clear despite suggestions that these phenomena may be related. In this work, we show that replicative and chronological-aged yeast cells are affected by an oxidative stress situation demonstrated by increased protein carbonylation when compared with young cells. The data on the identification of these oxidatively modified proteins gives clues to better understand cellular dysfunction that occurs during aging. Strikingly, although in both aging models metabolic differences are important, major targets are almost the same. Common targets include stress resistance proteins (Hsp60 and Hsp70) and enzymes involved in glucose metabolism such as enolase, glyceraldehydes-3-P dehydrogenase, fructose-1,6-biphosphate aldolase, pyruvate decarboxylase, and alcohol dehydrogenase. In both aging models, calorie restriction results in decreased damage to these proteins. In addition, chronological-aged cells grown under glucose restriction displayed lowered levels of lipid peroxidation product lipofuscin. Intracellular iron concentration is kept almost unchanged, whereas in non-restricted cells, the values increase up 4 -5 times. The pro-oxidant effects of such increased iron concentration would account for the damage observed. Also, calorie-restricted cells show undamaged catalase, which clearly appears carbonylated in cells grown at a high glucose concentration. These results may explain lengthening of the viability of chronological-aged cells and could have an important role in replicative life span extension by calorie restriction.

Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Oxidative Damage to Specific Proteins in Replicative and Chronological-aged Saccharomyces cerevisiae

Reverter-Branchat

Cabiscol

Tamarit

et al. 2004

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195

168

“…Evaluation of Oxidative Stress after High pH Exposure-Exponentially growing wild type DBY746 cells (A 660 0.5) in YPD medium were incubated for 1 h with dihydrorhodamine 123 (D1054, Sigma) at 2.5 g/ml essentially as described previously (27). Aliquots were taken and made 30 mM KOH (which raised the pH to 8.1) or 2 mM hydrogen peroxide and further incubated with shaking for 30 and 90 min, respectively.…”

Section: Strains and Growthmentioning

confidence: 99%

Characterization of the Calcium-mediated Response to Alkaline Stress in Saccharomyces cerevisiae

Viladevall¹,

Serrano²,

Ruiz³

et al. 2004

189

212

Exposure of the yeast Saccharomyces cerevisiae to alkaline stress resulted in adaptive changes that involved remodeling the gene expression. Recent evidence suggested that the calcium-activated protein phosphatase calcineurin could play a role in alkaline stress signaling. By using an aequorin luminescence reporter, we showed that alkaline stress resulted in a sharp and transient rise in cytoplasmic calcium. This increase was largely abolished by addition of EGTA to the medium or in cells lacking Mid1 or Cch1, components of the high affinity cell membrane calcium channel. Under these circumstances, the alkaline response of different calcineurin-sensitive transcriptional promoters was also blocked. Therefore, exposure to alkali resulted in entry of calcium from the external medium, and this triggered a calcineurin-mediated response. The involvement of calcineurin and Crz1/Tcn1, the transcription factor activated by the phosphatase, in the transcriptional response triggered by alkalinization has been globally assessed by DNA microarray analysis in a time course experiment using calcineurin-deficient (cnb1) and crz1 mutants. We found that exposure to pH 8.0 increased at least 2-fold the mRNA levels of 266 genes. In many cases (60%) the response was rather early (peak after 10 min). The transcriptional response of 27 induced genes (10%) was reduced or fully abolished in cnb1 cells. In general, the response of crz1 mutants was similar to that of calcineurin-deficient cells. By analysis of a systematic deletion library, we found 48 genes whose mutation resulted in increased sensitivity to the calcineurin inhibitor FK506. Twenty of these mutations (42%) also provoked alkaline pH sensitivity. In conclusion, our results demonstrated that calcium signaling and calcineurin activation represented a significant component of the yeast response to environmental alkalinization.Calcium-mediated signaling mechanisms are used by virtually every eukaryotic cell to regulate a wide variety or cellular processes, including gene expression. Transient increases in cytosolic calcium results in activation of diverse enzymes, such as the protein phosphatase calcineurin. Calcineurin is a heterodimer of catalytic subunit and regulatory subunits. In the yeast Saccharomyces cerevisiae, the catalytic subunit is encoded by two genes, CNA1 and CNA2 (1), whereas a single gene, CNB1, encodes the regulatory subunit (2). Cells lacking the catalytic subunits, or the regulatory subunit, are deficient in calcineurin activity.Exposure of yeast cells to a number of signals, such as ␣-factor (3, 4), glucose (5), sphingosine (6), and certain stress conditions (7-9), triggers a rise in cytoplasmic calcium. This increase in calcium can be a consequence of external calcium influx or release from internal stores, such as the vacuole, and results in activation of calcineurin. For instance, hyperosmotic shock has been reported to provoke calcium release from vacuolar stores (8) through Yvc1, a member of the transient receptor potential channel family, and to trig...

“…Hsp60 proteins play an important role in the cellular protection against oxidative stress (4,5). Studies with mutant strains of Saccharomyces cerevisiae exposed to various oxidants show that a decrease in Hsp60 expression results in an increased sensitivity toward oxidative stress (reduced cell viability) and increased levels of oxidized mitochondrial proteins, including Hsp60 itself (5).…”

mentioning

confidence: 99%

Potential Role of Methionine Sulfoxide in the Inactivation of the Chaperone GroEL by Hypochlorous Acid (HOCl) and Peroxynitrite (ONOO–)

Khor

Fisher

Schöneich

2004

GroEL is an Escherichia coli molecular chaperone that functions in vivo to fold newly synthesized polypeptides as well as to bind and refold denatured proteins during stress. This protein is a suitable model for its eukaryotic homolog, heat shock protein 60 (Hsp60), due to the high number of conserved amino acid sequences and similar function. Here, we will provide evidence that GroEL is rather insensitive to oxidants produced endogenously during metabolism, such as nitric oxide ( ⅐ NO) or hydrogen peroxide (H 2 O 2 ), but is efficiently modified and inactivated by reactive species generated by phagocytes, such as peroxynitrite (ONOO ؊ ) and hypochlorous acid (HOCl). For the exposure of 17.5 M GroEL to 100 -250 M HOCl, the major pathway of inactivation was through the oxidation of methionine to methionine sulfoxide, established through mass spectrometric detection of methionine sulfoxide and the reactivation of a significant fraction of inactivated GroEL by the enzyme methionine sulfoxide reductase B/A (MsrB/A). In addition to the oxidation of methionine, HOCl caused the conversion of cysteine to cysteic acid and this product may account for the remainder of inactivated GroEL not recoverable through MsrB/A. In contrast, HOCl produced only negligible yields of 3-chlorotyrosine. A remarkable finding was the conversion of Met 111 and Met 114 to Met sulfone, which suggests a rather low reduction potential of these 2 residues in GroEL. The high sensitivity of GroEL toward HOCl and ONOO ؊ suggests that this protein may be a target for bacterial killing by phagocytes.GroEL and its eukaryotic analog, heat shock protein 60 (Hsp60), 1 are highly sequence-related members of the Group I subclass of chaperonin 60 (Cpn60) (1). These proteins assist the folding of newly synthesized polypeptides (GroEL) or translocated preproteins (mitochondrial Hsp60). The functional unit of GroEL (and of most Cpn60 proteins) is a sandwich of two heptameric rings, which are stacked end to end. Depending on the protein substrate, different ligands such as K ϩ , Mg 2ϩ , ATP, and the cofactor GroES (or Hsp10) may be required for proper folding. Following the trapping of an unfolded or misfolded protein substrate in the hydrophobic interior of GroEL, the binding of ATP and GroES causes a conformational transition, which changes the interior surface properties from hydrophobic to hydrophilic, thus triggering protein folding (1, 2). Mammalian Hsp60 differs from GroEL in that it forms stable and functional heptameric rings in the absence of ATP and its cofactor Hsp10 (3). Our rationale for investigating the oxidative inactivation of GroEL is 2-fold: (i) its potential involvement in bacterial killing by phagocytes and (ii) a potential role for its analog, Hsp60, in an inflammatory and proapoptotic response during cardiovascular disease, as described below.Hsp60 proteins play an important role in the cellular protection against oxidative stress (4, 5). Studies with mutant strains of Saccharomyces cerevisiae exposed to various oxidants show that a decre...