In this work, we have found that several proteins implicated mainly in glycolysis and fermentation become specifically carbonylated throughout industrial yeast biomass propagation, which affects their corresponding enzyme activities and lowers biomass fermentative capacity. This phenomenon can be directly related with the high oxidative stress which occurs at critical time points during the industrial process [2, 3]. Under these conditions, we have previously observed improved biomass yield and fermentative capacity as a result of TRX2 gene overexpression . We have demonstrated that TRX2 gene overexpression is able to reduce oxidative damages, global protein carbonylation and lipid peroxidation levels, and to increase total glutathione and antioxidant enzyme activities. The aim of the present study was to identify those proteins that present increased carbonylation after industrial biomass propagation and to find putative Trx2p protection targets that could highlight the improved phenotype of the TTRX2 strain .
From the western blot anti-DNP of 2D gels experiments using the T73 strain, we were able to identify selective protein carbonylation during the biomass propagation process. The main heat shock proteins Mif4p (Hsp60p), Ssa1p (Hsp70p) and Sse1p (Hsp90) become specifically carbonylated during the diauxic shift (15 h), as do the proteins relating with ATP metabolism. We obtained similar results for the oxidative stress-induced experiments by H2O2  and by using chronologically aged cells . These results suggest that oxidation of molecular chaperones may be due to their role as a proteins shield for ROS generated during the diauxic shift. Besides, another interesting oxidatively damaged protein at 15 h of growth is thioredoxin peroxidase Ahp1p, which uses thioredoxins as an electron donor and is highly implicated in the oxidative stress response . By the end of the process, the carbonyl content of them all had lowered, but their protein levels either increased or did not vary . Since carbonylation has been described as an irreversible process , the lower carbonyl content of these proteins can be explained by an effect of culture dilution as a consequence of biomass growth.
At the end of the propagation process, we observed that several proteins had been greatly damaged if compared to the initial growth steps. In this case, the proteins involved in glycolysis and fermentation, mitochondrial proteins (Cor5p and Ilv5p), and the proteins related to tricarboxylic acid cycle (Aco1p, Mls1p and Ach1p), all showed high carbonylation levels. We have previously demonstrated that cells grown under respiratory conditions (YPG medium) are better prepared to cope with oxidative stress than those grown under fermenting conditions (YPD medium), by reducing protein carbonyl content . However, this work reveals that glycolytic enzymes undergo vast damage in the cells grown under respiratory conditions for a long period (40 h). Furthermore, there have been reports that prolonged cultivation of S. cerevisiae in aerobic, glucose-limited chemostat cultures progressively lowers glycolytic enzyme activities . Among the most carbonylated glycolytic enzymes, we found Tdh3p (GAPDH), Eno1p and Adh1p, which are key enzymes in alcoholic fermentation. Two of them (Tdh3p and Adh1p) catalyze oxidative reactions by using NADH. These enzymes' high carbonylation levels could be the main reason to explain the fermentative capacity detriment previously observed in the T73 strain [2, 3, 36]. In fact, GAPDH has been identified as a target of oxidative modification in many different cellular systems, which suggests its possible regulatory role as a sensor of oxidative stress conditions . Krobitsch and colleagues  provided the first direct evidence for the oxidative inhibition of Tdh3p, and other glycolytic enzymes , in a controlled response that enables cells to redirect their carbohydrate flux from glycolysis to the pentose phosphate pathway, generating NADPH under stressful conditions. In addition, we have previously shown that ADH1 gene overexpression increases the NAD+/NADH ratio, and increases chronological and replicative lifespan extension in S. cerevisiae by diminishing oxidative stress . Thus, the Adh1p oxidative carbonylation and lowered ADH activity observed in the yeast propagation process may contribute to unbalance the NAD+/NADH ratio by enhancing oxidative stress.
It is interesting to note that most of the damaged proteins identified in our experiments undergo other oxidative modifications, especially at their cysteine residues . Le Moan and colleagues  demonstrated that after H2O2 addition, the proteome of oxidized protein thiols of several stress chaperons and glycolytic enzymes depends exclusively on the thioredoxin functions controlling the activity of oxidized proteins. Besides, many of these proteins are also targets of S-glutathionylation, even in algae and mammal cells, suggesting an important role of the redox state in the regulation of the protein function for several organisms [42, 43].
The TRX2-overexpressing strain displays lower carbonylation levels for the majority of damaged proteins observed in wild-type strain, especially at the end of the industrial process. Most of these proteins also present cysteine residues close to the active site or important Cys disulfide bonds for their structure and catalytic function . The increased Trx2p dosage (additional file 5) seems to stabilize the redox state of these cysteine residues by avoiding oxidative damage. As a result of less protein oxidative damage in the main glycolytic enzymes, TTRX2 strain exhibits greater GAPDH and ADH enzyme activities, suggesting a direct correlation between low carbonylation levels and great enzyme activity. The fact that the TTRX2 strain exhibits high levels of total glutathione during biomass propagation , and that several important proteins have lower carbonyl content, suggests that S-glutathionylation could protect against carbonylation. In addition, Tdh3p and Adh1p are susceptible to being modified by S-thiolation under H2O2 induced oxidative stress, which diminishes enzyme activity by at least 70% . Besides, it has been described that thioredoxins catalyze deglutathionylation in yeast by playing a key role in regulating the modification of proteins by the glutathione system . The fact that the TTRX2 strain shows greater ADH and PDC activity after dehydration may also be associated with the fact that, under fermentative conditions, the deglutathionylation of both enzymes is higher than in the control strain as a result of TRX2 overexpression.
The improved fermentative capacity previously observed for the TTRX2 strain may be chiefly due to less oxidative damage in the aforementioned enzymes. In addition, increased alcohol dehydrogenase activity could also help reduce oxidative stress in this strain, and may be a reason for biomass yield improvement since ADH1 gene overexpression increases yeast replicative and chronological lifespan [3, 40].
The comparison made between the TTRX2 and trx2 strains allowed us to identify new targets of Trx2p protection. Most of these proteins are related to mitochondria, even Fba1p, which is involved in glycolysis, but localizes to the mitochondrial surface upon oxidative stress . This suggests an implication of cytosolic thioredoxins in mitochondrial protein protection  under high endogenous oxidative stress conditions, as in the biomass production process.
One remarkable finding from this work is that Trx2p overexpression reduces Adh1p oligomers by the crosslinking caused by oxidative carbonylation. Although mild oxidation of a protein increases its degradation by proteasome 20S , excessive oxidation and cross-linking of proteins render them resistant to proteolytic degradation by the proteasome . In E. coli, more than 95% of total carbonylated proteins are insoluble proteins mainly detectable in an aggregate state . These authors proposed that some carbonylated proteins escape degradation in vivo by forming carbonylated protein aggregates, thus becoming non degradable which contributes to senescence. In several human neurodegenerative diseases it has been described how the proteins related to glycolysis and energy metabolism, cytoskeleton, chaperoning, cellular stress responses and members of the ubiquitin-proteasome system become aggregated as a result of oxidative damage . According to these data, we suggest that Trx2p is able to diminish protein carbonylation damage, thus preventing protein aggregates and oxidative damage expansion. The molecular mechanisms of Trx2p protein protection against oxidative carbonylation could be associated with other oxidative modifications, especially at protein cysteine residues, and this phenomenon deserves further investigation.