Reductive stress leads to the loss of disulfide bond formation and induces the unfolded protein response of the endoplasmic reticulum (UPR(ER)), necessary to regain proteostasis in the compartment. Here we show that peroxide accumulation during reductive stress attenuates UPR(ER) amplitude by altering translation without any discernible effect on transcription. Through a comprehensive genetic screen in Saccharomyces cerevisiae, we identify modulators of reductive stress-induced UPR(ER) and demonstrate that oxidative quality control (OQC) genes modulate this cellular response in the presence of chronic but not acute reductive stress. Using a combination of microarray and relative quantitative proteomics, we uncover a non-canonical translation attenuation mechanism that acts in a bipartite manner to selectively downregulate highly expressed proteins, decoupling the cell's transcriptional and translational response during reductive ER stress. Finally, we demonstrate that PERK, a canonical translation attenuator in higher eukaryotes, helps in bypassing a ROS-dependent, non-canonical mode of translation attenuation.

Quantitative proteomics using LC-MS has emerged as an essential tool for addressing different biological questions. Various labelling methods have been effectively employed for quantitative proteomics studies. However, these are fraught with several challenges, including reproducibility and the number of samples that can be analysed at a given time. To this end, unlabelled proteomics is a promising field, and the recently developed sequential window acquisition of all theoretical fragment ion spectra (SWATH-MS) method aims to address these limitations. In this study, we compared SWATH-MS to isobaric tag for relative and absolute quantitation (iTRAQ), a widely used labelled method for relative quantitation. For this, we used yeast, Saccharomyces cerevisiae, since almost all its proteins are identified. More importantly, the abundance of each protein is well documented. We found that although a similar number of proteins could be quantitated using the two techniques, SWATH had the advantage of quantifying a larger percentage of low abundance proteins (below 60 ppm). Thus, based on our analysis, we believe that these two techniques are complementary and can synergistically improve the number of quantifiable proteins. SWATH's ability to quantify low abundant proteins could be an asset in biomarker discovery studies.

Mycobacterium tuberculosis modulates expression of various metabolism-related genes to adapt in the adverse host environment. The gene coding for M. tuberculosis S-adenosylhomocysteine hydrolase (Mtb-SahH) is essential for optimal growth and the protein product is involved in intermediary metabolism. However, the relevance of SahH in mycobacterial physiology is unknown. In this study, we analyze the role of Mtb-SahH in regulating homocysteine concentration in surrogate host Mycobacterium smegmatis. Mtb-SahH catalyzes reversible hydrolysis of S-adenosylhomocysteine to homocysteine and adenosine and we demonstrate that the conserved His363 residue is critical for bi-directional catalysis. Mtb-SahH is regulated by serine/threonine phosphorylation of multiple residues by M. tuberculosis PknB. Major phosphorylation events occur at contiguous residues Thr219, Thr220 and Thr221, which make pivotal contacts with cofactor NAD+. Consequently, phosphorylation negatively modulates affinity of enzyme towards NAD+ as well as SAH-synthesis. Thr219, Thr220 and Thr221 are essential for enzyme activity, and therefore, responsible for SahH-mediated regulation of homocysteine.