The phosphorylated redox proteome of Chlamydomonas reinhardtii: Revealing novel means for regulation of protein structure and function

Abstract

Post-translational modifications (PTMs) are covalent modifications to protein residues which may alter both conformation and activity, thereby modulating signaling and metabolic processes. While PTMs have been largely investigated independently, examination into how different modification interact, or crosstalk, will reveal a more complete understanding of the reciprocity of signaling cascades across numerous pathways. Combinatorial reversible thiol oxidation and phosphorylation in eukaryotes is largely recognized, but rigorous approaches for experimental discovery are underdeveloped. To begin meaningful interrogation of PTM crosstalk in systems biology research, knowledge of targeted proteins must be advanced. Herein, we demonstrate protein-level enrichment of reversibly oxidized proteoforms in Chlamydomonas reinhardtii with subsequent phosphopeptide analysis to determine the extent of phosphorylation in the redox thiol proteome. Label-free quantification was used to quantify 3353 oxidized Cys-sites on 1457 enriched proteins, where sequential phosphopeptide enrichment measured 1094 sites of phosphorylation on 720 proteins with 23% (172 proteins) also identified as reversibly oxidized. Proteins identified with both reversible oxidation and phosphorylation were involved in signaling transduction, ribosome and translation-related machinery, and metabolic pathways. Several redox-modified Calvin-Benson cycle proteins were found phosphorylated and many kinases/phosphatases involved in phosphorylation-dependent photosynthetic state transition and stress-response pathways had sites of reversible oxidation. Identification of redox proteins serves as a crucial element in understanding stress response in photosynthetic organisms and beyond, whereby knowing the ensemble of modifications co-occurring with oxidation highlights novel mechanisms for cellular control.

Photosynthetic Metabolism and Nitrogen Reshuffling Are Regulated by Reversible Cysteine Thiol Oxidation Following Nitrogen Deprivation in Chlamydomonas

“…Reversible thiol oxidation was enriched using an oxidized cysteine resin-assisted capture OxRAC) strategy, as described previously ( Figure 1 ) [ 21 , 33 ]. Briefly, 1 mg of protein lysates were incubated with 10 mM DTT on a Thermomixer in constant darkness at 25 °C and 1000 rpm for 1 h in order to reduce all reversibly oxidized cysteines.…”

“…The metabolic activity of C. reinhardtii is highly regulated by a portfolio of post-translational modifications (PTMs), including phosphorylation, glycosylation, and acylation [ 21 , 22 , 23 , 24 , 25 ]. Reversible oxidation is among the least understood PTM in phototrophic eukaryotes; although several studies have mapped the reversibly oxidized proteome of Chlamydomonas , there are still gaps differentiating the use of reversible oxidation for signaling and activity modulation and irreversible oxidation as markers for permanently damaged proteins [ 26 , 27 , 28 , 29 , 30 , 31 ].…”

Structural Basis for the Limited Response to Oxidative and Thiol-Conjugating Agents by Triosephosphate Isomerase From the Photosynthetic Bacteria Synechocystis

“…Post-translational modifications, such as reversible redox modifications, regulate protein activity during the Calvin-Benson cycle, both in vitro and in vivo (Zaffagnini et al, 2007 ; Michelet et al, 2008 , 2013 ; Hall et al, 2010 ; Friso and Van Wijk, 2015 ; McConnell et al, 2018 ). However, non-reversible modifications in response to reactive oxygen species (ROS) and reactive nitrogen species may lead to loss of enzymatic function or protein aggregation (Dahl et al, 2015 ; Hao et al, 2016 ).…”

Recent practical researches in the development of gluten-free breads

“…72 Redox regulation, control of reduction/oxidation of the disulfide bonds, as well as phosphorylation are the two major post-translational modifications of proteins. 73 Thioredoxin (Trx), 74 a small 12 -kDa protein, and glutathione, 75 a natural tripeptide, play central roles in the redox-dependent regulatory mechanisms.…”