Final Report Summary - REDOXDYNAMICS (In vivo dynamics of nitrosylation and glutathionylation in Chlamydomonas reinhardtii and Saccharomyces cerevisiae)
ROS/RNS mainly act as signaling molecules through a set of post-translational modifications of thiol residues on proteins which are mainly controlled by thioredoxins (TRXs) and glutaredoxins (GRXs). Three reversible redox PTMs, disulfide bonds (SS), nitrosylation (SNO) and glutathionylation (SSG), have recently emerged as important mechanisms of cell regulation and signaling. These modifications can affect the function of numerous proteins. They appear to play a major role in numerous fundamental cell processes and are implicated in a broad spectrum of human diseases. TRXs are small thiol oxydoreductases involved in many cellular processes. TRXs play an essential role in the control of redox PTMs.
One of our main objectives was to analyze from a global point of view, using proteomics and mass spectrometry, the redox proteome of Saccharomyces cerevisiae under diverse physiological growth conditions in order to elucidate not only the proteins putatively subjected to a redox control but also the cellular processes regulated by redox signaling.
By using our proteomic approach, more than 1000 proteins have been identified as putative TRX targets when Saccharomyces cells were grown under fermentative or respiratory conditions. The final list of this global analysis is in progress, but the first data show that the identified proteins are involved in numerous cellular processes such as protein degradation, translation, redox homeostasis or stress responses. We have found proteins already known to be redox regulated such as 2-Cys peroxiredoxin or the transcription factor YAP1, validating our method, but also proteins whose redox regulation remain unknown. Therefore, these results considerably broaden the importance of Trx-dependent redox regulation and signaling in eukaryotic cell.
Interestingly, the autophagy proteins Atg4 and Atg8 have been also found in our study. This finding prompted us to further investigate the connection between autophagy and redox balance in yeasts. Autophagy is a major catabolic process by which eukaryotic cells degrade intracellular material. Autophagy is characterized by the formation of double membrane vesicles, named autophagosomes, where the internal material is included to be degraded. The autophagy machinery is composed of Atg proteins that mediate the formation and regulation of the autophagosome. In the last years, mounting evidence suggests that ROS may play a role in the control of autophagy. Atg4 is a cysteine protease with a dual function involved in the Atg8 maduration and, consequently, in autophagosome formation. At present, Atg4 is the only Atg protein whose activity has been shown to be redox regulated; however, the molecular mechanism involved in this redox regulation has not been elucidated.
Based on the proteomic results and also on our knowledge and experience in redox biology and autophagy fields, we decided to analyze the redox regulation of Atg4 activity from Saccharomyces cerevisiae. By using a combination of biochemical assays, redox titrations and site-directed mutagenesis, we have shown that Atg4 is regulated by oxidoreduction of a single disulfide bond between Cys338 and Cys394. This disulfide has a low redox potential and is very efficiently reduced by thioredoxin, suggesting that this oxidoreductase plays an important role in Atg4 regulation. Accordingly, we found that autophagy activation by rapamycin was more pronounced in a thioredoxin mutant compared to wild-type cells. Moreover, in vivo studies indicated that Cys338 and Cys394 are required for the proper regulation of autophagosome biogenesis, since mutation of these cysteines resulted in increased recruitment of Atg8 to the phagophore assembly site. Thus, we have proposed that the fine tuning of Atg4 activity depending on the intracellular redox state may regulate autophagosome formation. In our opinion, this study will be very useful for both the Redox Biology and Autophagy communities since it has demonstrated the importance of the redox regulation of autophagy, not only in yeasts, but also in any eukaryotic cell.
Another of our objectives was to revise a pioneering TRX targets identification work in the group to elucidate the redox-regulated proteins and also the cellular pathways subjected to redox control in photosynthetic organisms such as Chlamydomonas reinhardtii. Taking advantage of the recent improvements in mass spectrometry and also in Chlamydomonas genome annotation, we have identified more than one thousand putative targets by using the mutant version of the cytosolic thioredoxin 1 grafted on an affinity column. We have found proteins involved in many cellular processes such as stress response or carbon metabolism, suggesting that all these processes may be subjected to redox regulation. For instance, we have identified all Calvin-Benson Cycle enzymes in this study, and their redox regulation will be biochemically analyzed in the laboratory. In addition, we have identified around 200 putative modified cysteine sites, pointing out the exact residue that may be subjected to the redox regulation. Furthermore, we have performed a comparative analysis of the different proteomes found to be redox regulated by glutathionylation, nitrosylation or by thioredoxin, suggesting that redox post-translational modifications are interconnected and they should not be analyzed independently.
Another objective has been to elucidate the role of ROS in the control of autophagy in photosynthetic organisms. We have analyzed, in a collaboration with another laboratory, the role of gluthatione on autophagy induced by endoplasmic reticulum stress.
One of our main objectives was to analyze from a global point of view, using proteomics and mass spectrometry, the redox proteome of Saccharomyces cerevisiae under diverse physiological growth conditions in order to elucidate not only the proteins putatively subjected to a redox control but also the cellular processes regulated by redox signaling.
By using our proteomic approach, more than 1000 proteins have been identified as putative TRX targets when Saccharomyces cells were grown under fermentative or respiratory conditions. The final list of this global analysis is in progress, but the first data show that the identified proteins are involved in numerous cellular processes such as protein degradation, translation, redox homeostasis or stress responses. We have found proteins already known to be redox regulated such as 2-Cys peroxiredoxin or the transcription factor YAP1, validating our method, but also proteins whose redox regulation remain unknown. Therefore, these results considerably broaden the importance of Trx-dependent redox regulation and signaling in eukaryotic cell.
Interestingly, the autophagy proteins Atg4 and Atg8 have been also found in our study. This finding prompted us to further investigate the connection between autophagy and redox balance in yeasts. Autophagy is a major catabolic process by which eukaryotic cells degrade intracellular material. Autophagy is characterized by the formation of double membrane vesicles, named autophagosomes, where the internal material is included to be degraded. The autophagy machinery is composed of Atg proteins that mediate the formation and regulation of the autophagosome. In the last years, mounting evidence suggests that ROS may play a role in the control of autophagy. Atg4 is a cysteine protease with a dual function involved in the Atg8 maduration and, consequently, in autophagosome formation. At present, Atg4 is the only Atg protein whose activity has been shown to be redox regulated; however, the molecular mechanism involved in this redox regulation has not been elucidated.
Based on the proteomic results and also on our knowledge and experience in redox biology and autophagy fields, we decided to analyze the redox regulation of Atg4 activity from Saccharomyces cerevisiae. By using a combination of biochemical assays, redox titrations and site-directed mutagenesis, we have shown that Atg4 is regulated by oxidoreduction of a single disulfide bond between Cys338 and Cys394. This disulfide has a low redox potential and is very efficiently reduced by thioredoxin, suggesting that this oxidoreductase plays an important role in Atg4 regulation. Accordingly, we found that autophagy activation by rapamycin was more pronounced in a thioredoxin mutant compared to wild-type cells. Moreover, in vivo studies indicated that Cys338 and Cys394 are required for the proper regulation of autophagosome biogenesis, since mutation of these cysteines resulted in increased recruitment of Atg8 to the phagophore assembly site. Thus, we have proposed that the fine tuning of Atg4 activity depending on the intracellular redox state may regulate autophagosome formation. In our opinion, this study will be very useful for both the Redox Biology and Autophagy communities since it has demonstrated the importance of the redox regulation of autophagy, not only in yeasts, but also in any eukaryotic cell.
Another of our objectives was to revise a pioneering TRX targets identification work in the group to elucidate the redox-regulated proteins and also the cellular pathways subjected to redox control in photosynthetic organisms such as Chlamydomonas reinhardtii. Taking advantage of the recent improvements in mass spectrometry and also in Chlamydomonas genome annotation, we have identified more than one thousand putative targets by using the mutant version of the cytosolic thioredoxin 1 grafted on an affinity column. We have found proteins involved in many cellular processes such as stress response or carbon metabolism, suggesting that all these processes may be subjected to redox regulation. For instance, we have identified all Calvin-Benson Cycle enzymes in this study, and their redox regulation will be biochemically analyzed in the laboratory. In addition, we have identified around 200 putative modified cysteine sites, pointing out the exact residue that may be subjected to the redox regulation. Furthermore, we have performed a comparative analysis of the different proteomes found to be redox regulated by glutathionylation, nitrosylation or by thioredoxin, suggesting that redox post-translational modifications are interconnected and they should not be analyzed independently.
Another objective has been to elucidate the role of ROS in the control of autophagy in photosynthetic organisms. We have analyzed, in a collaboration with another laboratory, the role of gluthatione on autophagy induced by endoplasmic reticulum stress.