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.

Perturbations affecting the homoeostasis of endoplasmic reticulum (ER) activate an adaptive signaling known as the unfolded protein response or UPR. Many studies have reported the association between neurological disorders and ER stress. Decreasing ER stress may therefore aid in therapeutic control of neuronal diseases. Sodium 4-phenylbutyrate (4-PBA), a small molecule, has been shown to alleviate ER stress and various neurological diseases, but the mechanistic basis of its action is not well understood. Using an iTRAQ based LC-MS technique we have delineated the effect of 4-PBA on the proteome of human neuroblastoma cells (SK-N-SH) during Tunicamycin-induced ER stress. The proteomic profile of 4-PBA-treated cells revealed that 4-PBA does not alter the cellular proteome to adapt towards ER stress. However, it can alleviate both the toxicity and proteomic alterations, induced by an ER stress inducer. Hence, the therapeutic effect of 4-PBA is primarily due to its ability to resolve ER stress rather than its ability to alter the expression of proteins required for maintaining ER proteostasis. Thus, we posit here that 4-PBA acts as an authentic chemical chaperone by aiding protein folding in the ER.

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Imbalance in protein homeostasis in specific subcellular organelles is alleviated through organelle-specific stress response pathways. As a canonical example of stress activated pathway, accumulation of misfolded proteins in ER activates unfolded protein response (UPR) in almost all eukaryotic organisms. However, very little is known about the involvement of proteins of other organelles that help to maintain the cellular protein homeostasis during ER stress. In this study, using iTRAQ-based LC-MS approach, we identified organelle enriched proteins that are differentially expressed in yeast (Saccharomyces cerevisiae) during ER stress in the absence of UPR sensor Ire1p. We have identified about 750 proteins from enriched organelle fraction in three independent iTRAQ experiments. Induction of ER stress resulted in the differential expression of 93 proteins in WT strains, 40 of which were found to be dependent on IRE1. Our study reveals a cross-talk between ER- and mitochondrial proteostasis exemplified by an Ire1p-dependent induction of Hsp60p, a mitochondrial chaperone. Thus, in this study, we show changes in protein levels in various organelles in response to ER stress. A large fraction of these changes were dependent on canonical UPR signalling through Ire1, highlighting the importance of interorganellar cross-talk during stress.

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Summary Serum and amniotic fluid samples from five pregnant women were analyzed for different proteinase inhibitors by ion‐exchange chromatography on DEAEcellulose. Alpha‐2 macroglobulin was found to be absent in amniotic fluid. Even though α‐1 antichymotrypsin was identified in amniotic fluid, its concentration gradient between serum and amniotic fluid (20–60) was much higher than that of α‐1 antitrypsin (8–20). Two forms of α‐1 antitrypsin were identified in both the fluids. In pregnancy, the ratio of the two forms was altered in favour of the less anionic form compared to normal sera. The contribution of the heat stable proteinase inhibitor for total antitryptic activity in amniotic fluid was more than in serum. The observation that urine samples from immature infants had higher heat stable inhibitory activity than urine samples from normal infants suggests that the heat stable inhibitor in amniotic fluid may arise from fetal kidney.