Synonymous codon usage regulates translation initiation.

Nonoptimal synonymous codons repress gene expression, but the underlying mechanisms are poorly understood. We and others have previously shown that nonoptimal codons slow translation elongation speeds and thereby trigger messenger RNA (mRNA) degradation. Nevertheless, transcript levels are often insufficient to explain protein levels, suggesting additional mechanisms by which codon usage regulates gene expression. Using reporters in human and Drosophila cells, we find that transcript levels account for less than half of the variation in protein abundance due to codon usage. This discrepancy is explained by translational differences whereby nonoptimal codons repress translation initiation. Nonoptimal transcripts are also less bound by the translation initiation factors eIF4E and eIF4G1, providing a mechanistic explanation for their reduced initiation rates. Importantly, translational repression can occur without mRNA decay and deadenylation, and it does not depend on the known nonoptimality sensor, CNOT3. Our results reveal a potent mechanism of regulation by codon usage where nonoptimal codons repress further rounds of translation.

Fatty acid desaturase activity in mature red blood cells and implications for blood storage quality.

BACKGROUND: Increases in the red blood cell (RBC) degree of fatty acid desaturation are reported in response to exercise, aging, or diseases associated with systemic oxidant stress. However, no studies have focused on the presence and activity of fatty acid desaturases (FADS) in the mature RBC. STUDY DESIGN AND METHODS: Steady state metabolomics and isotope-labeled tracing experiments, immunofluorescence approaches, and pharmacological interventions were used to determine the degree of fatty acid unsaturation, FADS activity as a function of storage, oxidant stress, and G6PD deficiency in human and mouse RBCs. RESULTS: In 250 blood units from the REDS III RBC Omics recalled donor population, we report a storage-dependent accumulation of free mono-, poly-(PUFAs), and highly unsaturated fatty acids (HUFAs), which occur at a faster rate than saturated fatty acid accumulation. Through a combination of immunofluorescence, pharmacological inhibition, tracing experiments with stable isotope-labeled fatty acids, and oxidant challenge with hydrogen peroxide, we demonstrate the presence and redox-sensitive activity of FADS2, FADS1, and FADS5 in the mature RBC. Increases in PUFAs and HUFAs in human and mouse RBCs correlate negatively with storage hemolysis and positively with posttransfusion recovery. Inhibition of these enzymes decreases accumulation of free PUFAs and HUFAs in stored RBCs, concomitant to increases in pyruvate/lactate ratios. Alterations of this ratio in G6PD deficient patients or units supplemented with pyruvate-rich rejuvenation solutions corresponded to decreased PUFA and HUFA accumulation. CONCLUSION: Fatty acid desaturases are present and active in mature RBCs. Their activity is sensitive to oxidant stress, storage duration, and alterations of the pyruvate/lactate ratio.

Protein-L-isoaspartate O-methyltransferase is required for in vivo control of oxidative damage in red blood cells.

Red blood cells have the special challenge of a large amount of reactive oxygen species (from their substantial iron load and Fenton reactions) combined with the inability to synthesize new gene products. Considerable progress has been made in elucidating the multiple pathways by which red blood cells neutralize reactive oxygen species via NADPH driven redox reactions. However, far less is known about how red blood cells repair the inevitable damage that does occur when reactive oxygen species break through anti-oxidant defenses. When structural and functional proteins become oxidized, the only remedy available to red blood cells is direct repair of the damaged molecules, as red blood cells cannot synthesize new proteins. Amongst the most common amino acid targets of oxidative damage is the conversion of asparagine and aspartate side chains into a succinimidyl group through deamidation or dehydration, respectively. Red blood cells express an L-Isoaspartyl methyltransferase (PIMT, gene name PCMT1) that can convert succinimidyl groups back to an aspartate. Herein, we report that deletion of PCMT1 significantly alters red blood cell metabolism in a healthy state, but does not impair the circulatory lifespan of red blood cells. Through a combination of genetic ablation, bone marrow transplantation and oxidant stimulation with phenylhydrazine in vivo or blood storage ex vivo, we use omics approaches to show that, when animals are exposed to oxidative stress, red blood cells from PCMT1 knockout undergo significant metabolic reprogramming and increased hemolysis. This is the first report of an essential role of PCMT1 for normal RBC circulation during oxidative stress.

ZOOMICS: Comparative Metabolomics of Red Blood Cells From Old World Monkeys and Humans.

As part of the ZOOMICS project, we set out to investigate common and diverging metabolic traits in the blood metabolome across various species by taking advantage of recent developments in high-throughput metabolomics. Here we provide the first comparative metabolomics analysis of fresh and stored human (n = 21, 10 males, 11 females), olive baboon (n = 20), and rhesus macaque (n = 20) red blood cells at baseline and upon 42 days of storage under blood bank conditions. The results indicated similarities and differences across species, which ultimately resulted in a differential propensity to undergo morphological alterations and lyse as a function of the duration of refrigerated storage. Focusing on purine oxidation, carboxylic acid, fatty acid, and arginine metabolism further highlighted species-specific metabolic wiring. For example, through a combination of steady state measurements and 13C6 15N4-arginine tracing experiments, we report an increase in arginine catabolism into ornithine in humans, suggestive of species-specific arginase 1 activity and nitric oxide synthesis-an observation that may impact the translatability of cardiovascular disease studies carried out in non-human primates (NHPs). Finally, we correlated metabolic measurements to storage-induced morphological alterations via scanning electron microscopy and hemolysis, which were significantly lower in human red cells compared to both NHPs.

Impact of taurine on red blood cell metabolism and implications for blood storage.

BACKGROUND: Taurine is an antioxidant that is abundant in some common energy drinks. Here we hypothesized that the antioxidant activity of taurine in red blood cells (RBCs) could be leveraged to counteract storage-induced oxidant stress. STUDY DESIGN AND METHODS: Metabolomics analyses were performed on plasma and RBCs from healthy volunteers (n = 4) at baseline and after consumption of a whole can of a common, taurine-rich (1000 mg/serving) energy drink. Reductionistic studies were also performed by incubating human RBCs with taurine ex vivo (unlabeled or 13 C15 N-labeled) at increasing doses (0, 100, 500, and 1000 µmol/L) at 37°C for up to 16 hours, with and without oxidant stress challenge with hydrogen peroxide (0.1% or 0.5%). Finally, we stored human and murine RBCs under blood bank conditions in additives supplemented with 500 µmol/L taurine, before metabolomics and posttransfusion recovery studies. RESULTS: Consumption of energy drinks increased plasma and RBC levels of taurine, which was paralleled by increases in glycolysis and glutathione (GSH) metabolism in the RBC. These observations were recapitulated ex vivo after incubation with taurine and hydrogen peroxide. Taurine levels in the RBCs from the REDS-III RBC-Omics donor biobank were directly proportional to the total levels of GSH and glutathionylated metabolites and inversely correlated to oxidative hemolysis measurements. Storage of human RBCs in the presence of taurine improved energy and redox markers of storage quality and increased posttransfusion recoveries in FVB mice. CONCLUSION: Taurine modulates RBC antioxidant metabolism in vivo and ex vivo, an observation of potential relevance to transfusion medicine.

Parabiosis Incompletely Reverses Aging-Induced Metabolic Changes and Oxidant Stress in Mouse Red Blood Cells.

Mature red blood cells (RBCs) not only account for ~83% of the total host cells in the human body, but they are also exposed to all body tissues during their circulation in the bloodstream. In addition, RBCs are devoid of de novo protein synthesis capacity and, as such, they represent a perfect model to investigate system-wide alterations of cellular metabolism in the context of aging and age-related oxidant stress without the confounding factor of gene expression. In the present study, we employed ultra-high-pressure liquid chromatography coupled with mass spectrometry (UHPLC-MS)-based metabolomics and proteomics to investigate RBC metabolism across age in male mice (6, 15, and 25 months old). We report that RBCs from aging mice face a progressive decline in the capacity to cope with oxidant stress through the glutathione/NADPH-dependent antioxidant systems. Oxidant stress to tryptophan and purines was accompanied by declines in late glycolysis and methyl-group donors, a potential compensatory mechanism to repair oxidatively damaged proteins. Moreover, heterochronic parabiosis experiments demonstrated that the young environment only partially rescued the alterations in one-carbon metabolism in old mice, although it had minimal to no impact on glutathione homeostasis, the pentose phosphate pathway, and oxidation of purines and tryptophan, which were instead aggravated in old heterochronic parabionts.