Elucidating the in vivo mechanism(s) of peroxiredoxins in oxidative stress protection and aging – PrxAGE

A CONTEXT
We address the mechanisms of oxidative stress and aging by focusing on the moonlighting enzymes peroxiredoxins (Prx). Prxs were initially described as degrading H2O2 by use of a reactive cysteine (CP), then shown to operate in H2O2 signaling, and later to undergo hyperoxidation of CP by H2O2, which inactivate their H2O2 scavenging and signaling function and turn them into a redox-activated chaperone holdase. Sulfiredoxin (Srx) catalyzes reduction of hyperoxidized CP, thereby switching enzyme functions.
We recently showed that increased gene dosage of TSA1, which encodes the major S. cerevisiae Prx, increases yeast replicative lifespan by means of a novel protein quality control (PQC) pathway, together with the Hsp70 chaperones. We also observed that yeast adaptation to extreme H2O2 levels is uniquely dependent of Tsa1 peroxidase activity. TSA1, and similarly other Prxs have multiple phenotypes, but except the two above examples, unambiguously linking them to a given Prx molecular function is impossible. The structure-function relationship of the peroxidase activity of Prxs is relatively well understood, but the PQC mechanism is largely unknown, and its approach is difficult, as the two functions are molecularly intertwined.

B. GENERAL AIM
We aim at elucidating the mechanisms by which Prxs operate their in vivo functions by systematically linking enzyme activity to phenotypes during oxidative stress and aging, which entails gaining knowledge of the still unknown Prxs PQC molecular function. We also aim exploring our hypothesis that Prxs mediate some of the H2O2 stress protective effects of H2S.

C STRATEGIC APPROACH
1. We propose generating mutations unambiguously separating Prx-moonlighting activities, as today the best strategy of exploring Prx functions and physiological scope, by:
- Structure-function mutations design, based on large Prx structure knowledge,
- Selection of random TSA1 mutations using growth conditions providing TSA1 genetically tractable phenotypes, presumably linked to one or the other Tsa1 moonlighting activities, and our sophisticated yeast single cell biology microfluidic device, which allow monitoring H2O2 adaptation and replicative lifespan.
2. Extensively characterize mutants
- in vitro by biophysical-biochemical approaches
- in vivo, by cell biological and biochemical approaches
3. Explore the effects of H2S during aging and H2O2 stress and their modulation by Prxs
We propose establishing a yeast strain conditionally overproducing H2S to explore the effects of this gas on H2O2 tolerance, signaling, and replicative lifespan.
Using this strain, we propose testing our hypothesis that Prxs mediate some of the effects of H2S by operating as a new type of H2S oxidase.
4. Link enzymatic activities and phenotypes, under calibrated conditions, those used in the genetic selections, and others also requiring TSA1, but for which the unknown reason of this requirement will be revealed.

D THE CONSORTIUM
The project relies on the high complementarity and synergy between three research teams that have generated the project preliminary data and conceived overall strategy. M. Toledano (CEA-Saclay, coordinator), expert in yeast genetics and biochemistry of oxidative stress and redox enzymes, together with G. Charvin (CNRS-IGBMC, Strasbourg), expert in quantitative system biology who invented the microfluidic device will conduct the genetic selection and appended molecular biology, and S. Rahuel-Clermont (CRNS-Nancy), expert in molecular redox enzymology will design mutants together with M. Toledano, and conduct the in vitro analyses of mutants.

E EXPECTED RESULTS
This project should provide unprecedented insights regarding the structure-function of 2-Cys Prxs, their physiological scope, and reciprocally push the limits of our knowledge of the molecular processes underlying oxidative stress and aging, opening novel research directions in the approach to age-linked neurodegenerative diseases and cancer.