Rate Of Protein Synthesis Research Articles

Human hematopoietic stem cell vulnerability to ferroptosis.

Hematopoietic stem cells (HSCs) have a number of unique physiologic adaptations that enable lifelong maintenance of blood cell production, including a highly regulated rate of protein synthesis. Yet, the precise vulnerabilities that arise from such adaptations have not been fully characterized. Here, inspired by a bone marrow failure disorder due to the loss of the histone deubiquitinase MYSM1, characterized by selectively disadvantaged HSCs, we show how reduced protein synthesis in HSCs results in increased ferroptosis. HSC maintenance can be fully rescued by blocking ferroptosis, despite no alteration in protein synthesis rates. Importantly, this selective vulnerability to ferroptosis not only underlies HSC loss in MYSM1 deficiency but also characterizes a broader liability of human HSCs. Increasing protein synthesis rates via MYSM1 overexpression makes HSCs less susceptible to ferroptosis, more broadly illustrating the selective vulnerabilities that arise in somatic stem cell populations as a result of physiologic adaptations.

Modeling SILAC Data to Assess Protein Turnover in a Cellular Model of Diabetic Nephropathy.

Protein turnover rate is finely regulated through intracellular mechanisms and signals that are still incompletely understood but that are essential for the correct function of cellular processes. Indeed, a dysfunctional proteostasis often impacts the cell's ability to remove unfolded, misfolded, degraded, non-functional, or damaged proteins. Thus, altered cellular mechanisms controlling protein turnover impinge on the pathophysiology of many diseases, making the study of protein synthesis and degradation rates an important step for a more comprehensive understanding of these pathologies. In this manuscript, we describe the application of a dynamic-SILAC approach to study the turnover rate and the abundance of proteins in a cellular model of diabetic nephropathy. We estimated protein half-lives and relative abundance for thousands of proteins, several of which are characterized by either an altered turnover rate or altered abundance between diabetic nephropathic subjects and diabetic controls. Many of these proteins were previously shown to be related to diabetic complications and represent therefore, possible biomarkers or therapeutic targets. Beside the aspects strictly related to the pathological condition, our data also represent a consistent compendium of protein half-lives in human fibroblasts and a rich source of important information related to basic cell biology.

Comparison of the effects of commercial whey protein and native whey protein on muscle strength and muscle protein synthesis in rats.

Commercial whey protein (CWP) is generally produced in the cheese making process with heat treatment. Recently, native whey protein (NWP) can be obtained through microfiltration without heat treatment. The difference in physicochemical properties of CWP and NWP was confirmed in previous studies; however, in vivo research on the effect on muscle strength and protein synthesis is still lacking. In this study, rats were orally administered 1.56g protein/kg body weight of lyophilized beverages containing CWP and NWP for 8weeks. The biological value and net protein utilization in the NWP were significantly higher than in the CWP. Moreover, NWP increased muscle mass and grip strength compared to CWP. NWP also increased the phosphorylation of the mammalian target of rapamycin and ribosomal protein S6 kinase, pivotal proteins for muscle protein synthesis. These results suggest that NWP enhance muscle strength and protein synthesis more effectively than CWP.

Glycine Regulates Protein Turnover by Activating Protein Kinase B/Mammalian Target of Rapamycin and by Inhibiting MuRF1 and Atrogin-1 Gene Expression in C2C12 Myoblasts

Background: The regulation of protein turnover in skeletal muscle is essential for the maintenance of integrity, growth, and function of this tissue. We recently reported that glycine enhances skeletal muscle growth in young pigs. However, the underlying mechanisms remain unknown.Objective: This study was conducted with a mouse myoblast cell line, C2C12, to test the hypothesis that glycine activates protein kinase B/mammalian target of rapamycin (Akt/mTOR), as well as inhibits 5′-adenosine monophosphate-activated protein kinase (AMPK) and the expression of genes for proteolysis.Methods: C2C12 myoblasts were cultured with 0, 0.25 (physiologic concentration in mouse plasma), 0.5, or 1.0 mmol glycine/L. Cell proliferation, activation of mammalian target of rapamycin complex 1 (mTORC1), AMPK signaling, mRNA levels of atrogin-1 and muscle-specific ring finger protein 1 (MuRF1), and protein synthesis and degradation were measured in the absence or presence of an Akt inhibitor, LY294002, or an AMPK activator, 5-aminoimidazole-4-carboxyamide ribonucleoside (AICAR).Results: Compared with control cells, 0.25–1.0 mmol glycine/L enhanced cell growth (by 12–15%) after 24 h (P < 0.05). Glycine treatment led to increased DNA replication (by 70–80%) while enhancing mTORC1 activation by upregulating Akt and inhibiting AMPK signaling (P < 0.05). Accordingly, glycine exposure increased (P < 0.05) the rate of protein synthesis (by 20–80%) and inhibited (P < 0.05) the rate of protein degradation (by 15–30%) in a concentration-dependent manner in C2C12 cells. These observations were validated by the use of an Akt inhibitor, LY294002, or an AMPK activator, AICAR. Moreover, glycine addition resulted in decreased mRNA levels for atrogin-1 and MuRF1 (by 20–40% and 30–50%, respectively; P < 0.05). The repressing effect of glycine on the expression of MuRF1, instead of atrogin-1, was abolished by LY294002 (P < 0.05).Conclusions: These findings indicate that glycine plays a previously unrecognized role in enhancing protein synthesis and inhibiting protein degradation in C2C12 cells. Glycine regulates protein turnover by activating mTORC1 and by inhibiting the expression of genes for proteolysis. Our results indicate that glycine is a functional amino acid that improves muscle cell growth.

A dynamical stochastic model of yeast translation across the cell cycle

Translation is a central step in gene expression, however its quantitative and time-resolved regulation is poorly understood. We developed a discrete, stochastic model for protein translation in S. cerevisiae in a whole-transcriptome, single-cell context. A “base case” scenario representing an average cell highlights translation initiation rates as the main co-translational regulatory parameters. Codon usage bias emerges as a secondary regulatory mechanism through ribosome stalling. Demand for anticodons with low abundancy is shown to cause above-average ribosome dwelling times. Codon usage bias correlates strongly both with protein synthesis rates and elongation rates. Applying the model to a time-resolved transcriptome estimated by combining data from FISH and RNA-Seq experiments, it could be shown that increased total transcript abundance during the cell cycle decreases translation efficiency at single transcript level. Translation efficiency grouped by gene function shows highest values for ribosomal and glycolytic genes. Ribosomal proteins peak in S phase while glycolytic proteins rank highest in later cell cycle phases.

Measurement of protein in vivo turnover rate with metabolic labeling using LC-MS.

Understanding the protein dynamics of a drug target is important for pharmaceutical research because it provides insight into drug design, target engagement, pharmacodynamics and drug efficacy. Nonradioactive isotope labeling has been the method of choice for protein turnover measurement thanks to the advancement of high-resolution mass spectrometry. While the changes in proteome in cell cultures can be monitored precisely, as the culture media can be completely replaced with 2 H-, 15 N- or 13 C-labeled essential amino acids, quantifying rates of protein synthesis in vivo is more challenging. The amount of isotope tracer that can be administered into the body is relatively small compared with the existing protein, thus requiring more sensitive detection, and the precursor-product labeling relationship is more complicated to interpret. The purpose of this review is to provide an overview of the principles of in vivo protein turnover studies using deuterium water (2 H2 O) with an emphasis on targeted protein analysis by hybrid LC-MS assay platforms. The pursuit of these opportunities will facilitate drug discovery and research in preclinical and clinical stages.

Impact of silencing eEF2K expression on the malignant properties of chordoma.

Eukaryotic elongation factor 2 kinase (eukaryotic elongation factor 2 kinase, eEF2K) is a calcium calmodulin dependent protein kinase that keeps the highest energy consuming cellular process of protein synthesis under check through negative regulation. eEF2K pauses global protein synthesis rates at the translational elongation step by phosphorylating its only kown substrate elongation factor 2 (eEF2), a unique translocase activity in ekaryotic cells enabling the polypeptide chain elongation. Therefore, eEF2K is thought to preserve cellular energy pools particularly upon acute development of cellular stress conditions such as nutrient deprivation, hypoxia, or infections. Recently, high expression of this enzyme has been associated with poor prognosis in an array of solid tumor types. Therefore, in a growing number of studies tremendous effort is being directed to the development of treatment methods aiming to suppress eEF2K as a novel therapeutic approach in the fight against cancer. In our study, we aimed to investigate the changes in the tumorigenicity of chordoma cells in presence of gene silencing for eEF2K. Taking a transient gene silencing approach using siRNA particles, eEF2K gene expression was suppressed in chordoma cells. Silencing eEF2K expression was associated with a slight increase in cellular proliferation and a decrease in death rates. Furthermore, no alteration in the sensitivity of chordoma cells to chemotherapy was detected in response to the decrease in eEF2K expression which intriguingly promoted suppression of cell migratory and invasion related properties. Our findings indicate that the loss of eEF2K expression in chordoma cell lines results in the reduction of metastatic capacity.

Vicia faba Peptide Network Supplementation Does Not Differ From Milk Protein in Modulating Changes in Muscle Size During Short-Term Immobilization and Subsequent Remobilization, but Increases Muscle Protein Synthesis Rates During Remobilization in Healthy Young Men

BackgroundMuscle mass and strength decrease during short periods of immobilization and slowly recover during remobilization. Recent artificial intelligence applications have identified peptides that appear to possess anabolic properties in in vitro assays and murine models. ObjectivesThis study aimed to compare the impact of Vicia faba peptide network compared with milk protein supplementation on muscle mass and strength loss during limb immobilization and regain during remobilization. MethodsThirty young (24 ± 5 y) men were subjected to 7 d of one-legged knee immobilization followed by 14 d of ambulant recovery. Participants were randomly allocated to ingest either 10 g of the Vicia faba peptide network (NPN_1; n = 15) or an isonitrogenous control (milk protein concentrate; MPC; n = 15) twice daily throughout the study. Single-slice computed tomography scans were performed to assess quadriceps cross-sectional area (CSA). Deuterium oxide ingestion and muscle biopsy sampling were applied to measure myofibrillar protein synthesis rates. ResultsLeg immobilization decreased quadriceps CSA (primary outcome) from 81.9 ± 10.6 to 76.5 ± 9.2 cm2 and from 74.8 ± 10.6 to 71.5 ± 9.8 cm2 in the NPN_1 and MPC groups, respectively (P < 0.001). Remobilization partially recovered quadriceps CSA (77.3 ± 9.3 and 72.6 ± 10.0 cm2, respectively; P = 0.009), with no differences between the groups (P > 0.05). During immobilization, myofibrillar protein synthesis rates (secondary outcome) were lower in the immobilized leg (1.07% ± 0.24% and 1.10% ± 0.24%/d, respectively) than in the non-immobilized leg (1.55% ± 0.27% and 1.52% ± 0.20%/d, respectively; P < 0.001), with no differences between the groups (P > 0.05). During remobilization, myofibrillar protein synthesis rates in the immobilized leg were greater with NPN_1 than those with MPC (1.53% ± 0.38% vs. 1.23% ± 0.36%/d, respectively; P = 0.027). ConclusionNPN_1 supplementation does not differ from milk protein in modulating the loss of muscle size during short-term immobilization and the regain during remobilization in young men. NPN_1 supplementation does not differ from milk protein supplementation in modulating the myofibrillar protein synthesis rates during immobilization but further increases myofibrillar protein synthesis rates during remobilization.

Endurance training changes the expression of miR-1 and miR-133 and predicted genes in slow and fast twitch muscles

Purpose of the researchEndurance training can modify signaling and gene expression pathways that play a pivotal role in determining the phenotype of the fibers. The present study aimed to investigate the effects of endurance training on the expression of some myomiRs and related genes in slow and fast twitch muscles. MethodsTwenty healthy male adult Wistar rats (281 ± 14 g) were randomized to either control (n = 10) or treated (n = 10). The treated group performed an endurance program for eight weeks (running on a treadmill for eight weeks, 50 min, 23 m/min). After the end of the training protocol, the slow (soleus) and fast (EDL) twitch muscles were removed to assess the miR-1, miR-133 expression, and hdac4, mef2c genes, and protein by real-time PCR and western blot, respectively. ResultsThe soleus muscle miR-1 expression and mef2c gene in the treated group were significantly lower compared control (p = 0.0001). In contrast, miR-133 and hdac4 gene expression of the soleus muscle of the treated group increased significantly (p = 0001), and the EDL miR-133 and mef2c expression of the treated group increased in the compared control group (p = 0.0001). The EDL MEF2c protein expression in the treated group significantly decreased compared to the control group, although the expression of EDL HDAC4 protein significantly increased (p = 0.0001). ConclusionsEndurance training changes the expression of the miR-1, miR-133, and their predicted genes in slow and fast twitch muscles. Also, the rate of HDAC4 and MEF2c protein synthesis, which are upstream and downstream of these myomiRs, was affected by endurance training.

Prematurity blunts protein synthesis in skeletal muscle independently of body weight in neonatal pigs

BackgroundPostnatal growth failure in premature infants is associated with reduced lean mass accretion. Prematurity impairs the feeding-induced stimulation of translation initiation and protein synthesis in the skeletal muscle of neonatal pigs. The objective was to determine whether body weight independently contributes to the blunted postprandial protein synthesis.MethodsPreterm and term pigs that were either fasted or fed were stratified into quartiles according to birth weight to yield preterm and term groups of similar body weight; first and second quartiles of preterm pigs and third and fourth quartiles of term pigs were compared (preterm-fasted, n = 23; preterm-fed, n = 25; term-fasted, n = 21; term-fed, n = 21). Protein synthesis rates and mechanistic target of rapamycin complex 1 (mTORC1) activation in skeletal muscle were determined.ResultsRelative body weight gain was lower in preterm compared to term pigs. Prematurity attenuated the feeding-induced increase in mTORC1 activation in longissimus dorsi and gastrocnemius muscles (P < 0.05). Protein synthesis in gastrocnemius (P < 0.01), but not in longissimus dorsi muscle, was blunted by preterm birth.ConclusionA lower capacity of skeletal muscle to respond adequately to feeding may contribute to reduced body weight gain and lean mass accretion in preterm infants.ImpactThis study has shown that the feeding-induced increase in protein synthesis of skeletal and cardiac muscle is blunted in neonatal pigs born preterm compared to pigs born at term independently of birth weight.These findings support the notion that preterm birth, and not low birth weight, impairs the capacity of skeletal and cardiac muscle to upregulate mechanistic target of rapamycin-dependent anabolic signaling pathways and protein synthesis in response to the postprandial increase in insulin and amino acids.These observations suggest that a blunted anabolic response to feeding contributes to reduced lean mass accretion and altered body composition in preterm infants.

Increased interaction between endoplasmic reticulum and mitochondria following sleep deprivation

BackgroundProlonged cellular activity may overload cell function, leading to high rates of protein synthesis and accumulation of misfolded or unassembled proteins, which cause endoplasmic reticulum (ER) stress and activate the unfolded protein response (UPR) to re-establish normal protein homeostasis. Previous molecular work has demonstrated that sleep deprivation (SD) leads to ER stress in neurons, with a number of ER-specific proteins being upregulated to maintain optimal cellular proteostasis. It is still not clear which cellular processes activated by sleep deprivation lead to ER- stress, but increased cellular metabolism, higher request for protein synthesis, and over production of oxygen radicals have been proposed as potential contributing factors. Here, we investigate the transcriptional and ultrastructural ER and mitochondrial modifications induced by sleep loss.ResultsWe used gene expression analysis in mouse forebrains to show that SD was associated with significant transcriptional modifications of genes involved in ER stress but also in ER-mitochondria interaction, calcium homeostasis, and mitochondrial respiratory activity. Using electron microscopy, we also showed that SD was associated with a general increase in the density of ER cisternae in pyramidal neurons of the motor cortex. Moreover, ER cisternae established new contact sites with mitochondria, the so-called mitochondria associated membranes (MAMs), important hubs for molecule shuttling, such as calcium and lipids, and for the modulation of ATP production and redox state. Finally, we demonstrated that Drosophila male mutant flies (elav > linker), in which the number of MAMs had been genetically increased, showed a reduction in the amount and consolidation of sleep without alterations in the homeostatic sleep response to SD.ConclusionsWe provide evidence that sleep loss induces ER stress characterized by increased crosstalk between ER and mitochondria. MAMs formation associated with SD could represent a key phenomenon for the modulation of multiple cellular processes that ensure appropriate responses to increased cell metabolism. In addition, MAMs establishment may play a role in the regulation of sleep under baseline conditions.

Nitrogen and lysine utilization efficiencies, protein turnover, and blood urea concentrations in crossbred grower pigs at marginal dietary lysine concentration.

Nitrogen utilization efficiency (NUE) and lysine utilization efficiency (LUE) are key indicators of sustainable pork production and vary depending on nutritional and non-nutritional factors. The objective was to study NUE and LUE together with concentrations of blood urea nitrogen (BUN) and other metabolites in growing pigs fed diets with marginal Lys concentrations at 11-13 wk (40.5 kg mean BW) and 14 to 16 wk (60.2 kg mean BW). The cereal grain-soybean meal-based diets contained 10.6 and 7.9 g Lys/kg DM in periods 1 and 2, respectively. Feed intake and BW were measured for 508 individually penned pigs, and blood samples were collected 5 h after morning feeding at weeks 13 and 16. A subgroup of 48 barrows was used in a nitrogen (N) metabolism trial at weeks 13 and 16. In this subgroup, the mean N retention of pigs (27.3 g N/d) and mean LUE (70%) were not different between the periods, but NUE was higher in period 1 (47%) than in period 2 (43%) (P < 0.001). After administration of a single dose of 15N labeled glycine and measurement of 15N recovery in urine, the calculated whole-body protein turnover did not differ between the periods. The rate of protein synthesis was positively correlated with NUE (P < 0.001), but protein degradation was not. Excretion of urea-N in urine accounted for 80% of the total urinary N and was positively correlated with BUN. The N retention of all 508 pigs was estimated using an equation that was derived from the N metabolism data. N retention was on average 31.4 g/d, equal in both periods, and higher in barrows than in gilts in period 2, but not in period 1 (P = 0.003). The calculated NUE was, on average, 47% and was lower in barrows than in gilts (P < 0.001) and higher in period 1 than in period 2 (P < 0.001). The calculated LUE was, on average, 71%, and was lower in barrows than in gilts in period 2, but not in period 1 (P < 0.001). The BUN concentration was higher in barrows than in gilts (P < 0.001) and higher in period 1 than in period 2 (P < 0.001). BUN concentration was negatively correlated with NUE in Periods 1 (r = -0.50) and 2 (r = -0.15) (P < 0.05). We concluded that the maximum LUE was in the range of 70-72% under the conditions of this study, and only small differences between the periods and sexes existed. Protein synthesis, rather than degradation, appears to affect NUE. BUN concentration may be useful for estimating NUE in a large group of animals fed a diet with a marginal Lys concentration.

A simple geometrical model of the electrostatic environment around the catalytic center of the ribosome and its significance for the elongation cycle kinetics

The central function of the large subunit of the ribosome is to catalyze peptide bond formation. This biochemical reaction is conducted at the peptidyl transferase center (PTC). Experimental evidence shows that the catalytic activity is affected by the electrostatic environment around the peptidyl transferase center. Here, we set up a minimal geometrical model fitting the available x-ray solved structures of the ribonucleic cavity around the catalytic center of the large subunit of the ribosome. The purpose of this phenomenological model is to estimate quantitatively the electrostatic potential and electric field that are experienced during the peptidyl transfer reaction. At least two reasons motivate the need for developing this quantification. First, we inquire whether the electric field in this particular catalytic environment, made only of nucleic acids, is of the same order of magnitude as the one prevailing in catalytic centers of the proteic enzymes counterparts. Second, the protein synthesis rate is dependent on the nature of the amino acid sequentially incorporated in the nascent chain. The activation energy of the catalytic reaction and its detailed kinetics are shown to be dependent on the mechanical work exerted on the amino acids by the electric field, especially when one of the four charged amino acid residues (R, K, E, D) has previously been incorporated at the carboxy-terminal end of the peptidyl-tRNA. Physical values of the electric field provide quantitative knowledge of mechanical work, activation energy and rate of the peptide bond formation catalyzed by the ribosome. We show that our theoretical calculations are consistent with two independent sets of previously published experimental results. Experimental results for E.coli in the minimal case of the dipeptide bond formation when puromycin is used as the final amino acid acceptor strongly support our theoretically derived reaction time courses. Experimental Ribo-Seq results on E. coli and S. cerevisiae comparing the residence time distribution of ribosomes upon specific codons are also well accounted for by our theoretical calculations. The statistical queueing time theory was used to model the ribosome residence time per codon during nascent protein elongation and applied for the interpretation of the Ribo-Seq data. The hypo-exponential distribution fits the residence time observed distribution of the ribosome on a codon. An educated deconvolution of this distribution is used to estimate the rates of each elongation step in a codon specific manner. Our interpretation of all these results sheds light on the functional role of the electrostatic profile around the PTC and its impact on the ribosome elongation cycle.

Resveratrol directly suppresses proteolysis possibly via PKA/CREB signaling in denervated rat skeletal muscle.

Although there are reports that polyphenol resveratrol (Rsv) may cause muscle hypertrophy in basal conditions and attenuate muscle wasting in catabolic situations, its mechanism of action is still unclear. Our study evaluated the ex vivo effects of Rsv on protein metabolism and intracellular signaling in innervated (sham-operated; Sham) and 3-day sciatic denervated (Den) rat skeletal muscles. Rsv (10-4 M) reduced total proteolysis (40%) in sham muscles. Den increased total proteolysis (~40%) in muscle, which was accompanied by an increase in the activities of ubiquitin-proteasome (~3-fold) and lysosomal (100%) proteolytic systems. Rsv reduced total proteolysis (59%) in Den muscles by inhibiting the hyperactivation of ubiquitin-proteasome (50%) and lysosomal (~70%) systems. Neither Rsv nor Den altered calcium-dependent proteolysis in muscles. Mechanistically, Rsv stimulated PKA/CREB signaling in Den muscles, and PKA blockage by H89 (50μM) abolished the antiproteolytic action of the polyphenol. Rsv reduced FoxO4 phosphorylation (~60%) in both Sham and Den muscles and Akt phosphorylation (36%) in Den muscles. Rsv also caused a homeostatic effect in Den muscles by returning their protein synthesis rates to levels similar to Sham muscles. These data indicate that Rsv directly inhibits the proteolytic activity of lysosomal and ubiquitin-proteasome systems, mainly in Den muscles through, at least in part, the activation of PKA/CREB signaling.

Resilience and phenotypic plasticity of Arctic char (Salvelinus alpinus) facing cyclic hypoxia: insights into growth, energy stores and hepatic metabolism.

Arctic char (Salvelinus alpinus) is facing the decline of its southernmost populations due to several factors including rising temperatures and eutrophication. These conditions are also conducive to episodes of cyclic hypoxia, another possible threat to this species. In fact, lack of oxygen and reoxygenation can both have serious consequences on fish as a result of altered ATP balance and an elevated risk of oxidative burst. Thus, fish must adjust their phenotype to survive and equilibrate their energetic budget. However, their energy allocation strategy could imply a reduction in growth which could be deleterious for their fitness. Although the impact of cyclic hypoxia is a major issue for ecosystems and fisheries worldwide, our knowledge on how salmonid deal with high oxygen fluctuations remains limited. Our objective was to characterize the effects of cyclic hypoxia on growth and metabolism in Arctic char. We monitored growth parameters (specific growth rate, condition factor), hepatosomatic and visceral indexes, relative heart mass and hematocrit of Arctic char exposed to 30days of cyclic hypoxia. We also measured the hepatic protein synthesis rate, hepatic triglycerides as well as muscle glucose, glycogen and lactate and quantified hepatic metabolites during this treatment. The first days of cyclic hypoxia slightly reduce growth performance with a downward trend in specific growth rate in mass and condition factor variation compared to the control group. This acute exposure also induced a profound metabolome reorganization in the liver with an alteration of amino acid, carbohydrate and lipid metabolisms. However, fish rebalanced their metabolic activities and successfully maintained their growth and energetic reserves after 1month of cyclic hypoxia. These results demonstrate the impressive ability of Arctic char to cope with its changing environment but also highlight a certain vulnerability of this species during the first days of a cyclic hypoxia event.

Chronic Fetal Leucine Infusion Increases Rate of Leucine Oxidation but Not of Protein Synthesis in Late Gestation Fetal Sheep

BackgroundLeucine increases protein synthesis rates in postnatal animals and adults. Whether supplemental leucine has similar effects in the fetus has not been determined. ObjectiveTo determine the effect of a chronic leucine infusion on whole-body leucine oxidation and protein metabolic rates, muscle mass, and regulators of muscle protein synthesis in late gestation fetal sheep. MethodsCatheterized fetal sheep at ∼126 d of gestation (term = 147 d) received infusions of saline (CON, n = 11) or leucine (LEU; n = 9) adjusted to increase fetal plasma leucine concentrations by 50%–100% for 9 d. Umbilical substrate net uptake rates and protein metabolic rates were determined using a 1-13C leucine tracer. Myofiber myosin heavy chain (MHC) type and area, expression of amino acid transporters, and abundance of protein synthesis regulators were measured in fetal skeletal muscle. Groups were compared using unpaired t tests. ResultsPlasma leucine concentrations were 75% higher in LEU fetuses compared with CON by the end of the infusion period (P < 0.0001). Umbilical blood flow and uptake rates of most amino acids, lactate, and oxygen were similar between groups. Fetal whole-body leucine oxidation was 90% higher in LEU (P < 0.0005) but protein synthesis and breakdown rates were similar. Fetal and muscle weights and myofiber areas were similar between groups, however, there were fewer MHC type IIa fibers (P < 0.05), greater mRNA expression levels of amino acid transporters (P < 0.01), and a higher abundance of signaling proteins that regulate protein synthesis (P < 0.05) in muscle from LEU fetuses. ConclusionsA direct leucine infusion for 9 d in late gestation fetal sheep does not increase protein synthesis rates but results in higher leucine oxidation rates and fewer glycolytic myofibers. Increasing leucine concentrations in the fetus stimulates its own oxidation but also increases amino acid transporter expression and primes protein synthetic pathways in skeletal muscle.

New temperature-switchable acyl homoserine lactone-regulated expression vector.

Bacterial expression systems play an indispensable role in the biosynthesis of recombinant proteins. Different proteins and the tasks associated with them may require different systems. The purpose of this work is to make an expression vector that allows switching on and off the expression of the target gene during cell incubation. Several expression vectors for use in Escherichiacoli cells were developed using elements of the luxR/luxI type quorum sensing system of psychrophilic bacterium Aliivibrio logei. These vectors contain A.logei luxR2 and (optionally) luxI genes and LuxR2-regulated promoter, under the control of which a target gene is intended to be inserted. The synthesis of the target protein depends directly on the temperature: gene expression starts when the temperature drops to 22°C and stops when it rises to 37°C, which makes it possible to fix the desired amount of the target protein in the cell. At the same time, the expression of the target gene at a low temperature depends on the concentration of the autoinducer (L-homoserine N-(3-oxohexanoyl)-lactone, AI) in the culture medium in a wide range from 1nM to 10μM, which makes it possible to smoothly regulate the rate of target protein synthesis. Presence of luxI in the vector provides the possibility of autoinduction. Constructed expression vectors were tested with gfp, ardA, and ardB genes. At maximum, we obtained the target protein in an amount of up to 33% of the total cellular protein. KEY POINTS: • A.logei quorum sensing system elements were applied in new expression vectors • Expression of target gene is inducible at 22°C and it is switched off at 37°C • Target gene expression at 22°C is tunable by use different AI concentrations.

Effects of desiccation rate and inhibition of protein synthesis and the violaxanthin cycle on the rewetting recovery of Porella platyphylla

Following 5 different rates of 1-week desiccation to which Porella platyphylla (a desiccation-tolerant leafy liverwort) was subjected, physiological recovery upon rehydration (after 1 h, 24 h, 48 h) in light was monitored by cytological, photosynthetic (e-transport rate, photoprotective mechanisms), and other metabolic parameters using nuclear- and chloroplast-encoded protein synthesis inhibitors and a violaxanthin cycle inhibitor (DTT: dithiothreitol). Desiccation tolerance is mainly constitutive in P. platyphylla, allowing survival of rapid drying, and employs an active rehydration-induced repair and recovery mechanism. Following 24 hours of rehydration after the one-week dehydration in laboratory air (∼35% relative humidity /RH/), the chloroplasts had an irregular round shape. The recovery within 1 h of the thylakoid-function-related photosynthetic processes was extremely fast and independent of protein synthesis, while the overall recovery (except plants dried up at 76% RH, which depended on protein synthesis only to a limited extent) required de novo protein synthesis during rewetting. During the course of rehydration protein synthesis affects rehydrin expression, osmolite and general carbohydrate metabolism, the main repair and antioxidant mechanisms. Recovery was the best in samples dried up at 76% RH. Membrane permeability increased upon rehydration following 1-week dehydration. Normal membrane function recovered after 24 h (max. 48 h), except in plants that suffered drastic dehydration. Total protein content of plants dried under different conditions and of samples taken during recovery was generally lower than in the control plants at full turgor. The xanthophyll cycle has a great importance during the recovery in light; we confirmed the existence of a greater zeaxanthin-dependent and a smaller zeaxanthin-insensitive NPQ (nonphotochemical quenching). Plants dried under more favorable conditions had better light protection. The highest values of the osmotic potential belonged to the plants subjected to intense dehydration. Total soluble carbohydrate content was well-balanced and seemed to be unchanged following the different treatments. There was a significant increase in proline, while total fructan showed lower values at different degrees of 1-week desiccation. The cytoplasmic protein synthesis is likely to be involved in the change of the fructan content during rehydration.

Regulation of eIF4E guides a unique translational program to control erythroid maturation.

Translation control is essential in balancing hematopoietic precursors and differentiation; however, the mechanisms underlying this program are poorly understood. We found that the activity of the major cap-binding protein eIF4E is unexpectedly regulated in a dynamic manner throughout erythropoiesis that is uncoupled from global protein synthesis rates. Moreover, eIF4E activity directs erythroid maturation, and increased eIF4E expression maintains cells in an early erythroid state associated with a translation program driving the expression of PTPN6 and Igf2bp1. A cytosine-enriched motif in the 5' untranslated region is important for eIF4E-mediated translation specificity. Therefore, selective translation of key target genes necessary for the maintenance of early erythroid states by eIF4E highlights a unique mechanism used by hematopoietic precursors to rapidly elicit erythropoietic maturation upon need.

Monitoring the 5′UTR landscape reveals isoform switches to drive translational efficiencies in cancer

Transcriptional and translational control are key determinants of gene expression, however, to what extent these two processes can be collectively coordinated is still poorly understood. Here, we use Nanopore long-read sequencing and cap analysis of gene expression (CAGE-seq) to document the landscape of 5′ and 3′ untranslated region (UTR) isoforms and transcription start sites of epidermal stem cells, wild-type keratinocytes and squamous cell carcinomas. Focusing on squamous cell carcinomas, we show that a small cohort of genes with alternative 5′UTR isoforms exhibit overall increased translational efficiencies and are enriched in ribosomal proteins and splicing factors. By combining polysome fractionations and CAGE-seq, we further characterize two of these UTR isoform genes with identical coding sequences and demonstrate that the underlying transcription start site heterogeneity frequently results in 5′ terminal oligopyrimidine (TOP) and pyrimidine-rich translational element (PRTE) motif switches to drive mTORC1-dependent translation of the mRNA. Genome-wide, we show that highly translated squamous cell carcinoma transcripts switch towards increased use of 5′TOP and PRTE motifs, have generally shorter 5′UTRs and expose decreased RNA secondary structures. Notably, we found that the two 5′TOP motif-containing, but not the TOP-less, RPL21 transcript isoforms strongly correlated with overall survival in human head and neck squamous cell carcinoma patients. Our findings warrant isoform-specific analyses in human cancer datasets and suggest that switching between 5′UTR isoforms is an elegant and simple way to alter protein synthesis rates, set their sensitivity to the mTORC1-dependent nutrient-sensing pathway and direct the translational potential of an mRNA by the precise 5′UTR sequence.