Rate Of Protein Synthesis Research Articles

Growth and B-Phycoerythrin Production of Red Microalga Porphyridium purpureum (Porphyridiales, Rhodophyta) under Different Carbon Supply.

Red microalga Porphyridium purpureum (Bory) Drew is a well-known object of biotechnology due to its unique ability to synthesize a wide range of biologically active compounds. Enough minerals in an accessible form in a medium are a prerequisite for maintaining a high growth rate of P. purpureum. Carbon is the main element of microalgal biomass and is a component of all organic compounds. The work aimed to study the morphological features of cells and the accumulation and production of B-phycoerythrin and total protein in P. purpureum biomass in different ways of supplying CO2 into the culture. In Variant 1, CO2 was directly injected into a gas-air mixture (2-3 percent v/v) used for culture bubbling via capillary. In Variant 2, the air was supplied to the culture through the aquarium sparger. Variant 3 was like the first one but without the additional introduction of carbon dioxide. The application of the method for sparging atmospheric air led to a significant increase in both the productivity of the P. purpureum and the rate of protein and B-phycoerythrin synthesis in comparison with growing it using the air without spraying (two-and-a-half times, five times, and more than eight times, respectively). Moreover, there were significant changes in the morphological structure of P. purpureum cells, which were visualized both by microscopy and by changes in the color of the culture. Based on the experimental data obtained, the variants for the carbon supply experiment were ranked as follows: Variant 1 is better than Variant 2 and Variant 3. The use of atomization as a technological method made it possible to speed up the transfer of carbon dioxide from the air to the medium, which helped to keep the growth rate of P. purpureum biomass and B-phycoerythrin accumulation high.

Periostin- and podoplanin-positive cancer-associated fibroblast subtypes cooperate to shape the inflamed tumor microenvironment in aggressive pancreatic adenocarcinoma.

Cancer-associated fibroblasts (CAFs) are orchestrators of the pancreatic ductal adenocarcinoma (PDAC) microenvironment. Previously we described four CAF subtypes with specific molecular and functional features. Here, we have refined our CAF subtype signatures using RNAseq and immunostaining with the goal of defining bioinformatically the phenotypic stromal and tumor epithelial states associated with CAF diversity. We used primary CAF cultures grown from patient PDAC tumors, human data sets (in-house and public, including single-cell analyses), genetically engineered mouse PDAC tissues, and patient-derived xenografts (PDX) grown in mice. We found that CAF subtype RNAseq signatures correlated with immunostaining. Tumors rich in periostin-positive CAFs were significantly associated with shorter overall survival of patients. Periostin-positive CAFs were characterized by high proliferation and protein synthesis rates and low α-smooth muscle actin expression and were found in peri-/pre-tumoral areas. They were associated with highly cellular tumors and with macrophage infiltrates. Podoplanin-positive CAFs were associated with immune-related signatures and recruitment of dendritic cells. Importantly, we showed that the combination of periostin-positive CAFs and podoplanin-positive CAFs was associated with specific tumor microenvironment features in terms of stromal abundance and immune cell infiltrates. Podoplanin-positive CAFs identified an inflammatory CAF (iCAF)-like subset, whereas periostin-positive CAFs were not correlated with the published myofibroblastic CAF (myCAF)/iCAF classification. Taken together, these results suggest that a periostin-positive CAF is an early, activated CAF, associated with aggressive tumors, whereas a podoplanin-positive CAF is associated with an immune-related phenotype. These two subpopulations cooperate to define specific tumor microenvironment and patient prognosis and are of putative interest for future therapeutic stratification of patients. © 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Lipid hydroperoxides and oxylipins are mediators of denervation induced muscle atrophy

Loss of innervation is a key driver of age associated muscle atrophy and weakness (sarcopenia). Our laboratory has previously shown that denervation induced atrophy is associated with the generation of mitochondrial hydroperoxides and lipid mediators produced downstream of cPLA2 and 12/15 lipoxygenase (12/15-LOX). To define the pathological impact of lipid hydroperoxides generated in denervation-induced atrophy in vivo, we treated mice with liproxstatin-1, a lipid hydroperoxide scavenger. We treated adult male mice with 5 mg/kg liproxstain-1 or vehicle one day prior to sciatic nerve transection and daily for 7 days post-denervation before tissue analysis. Liproxstatin-1 treatment protected gastrocnemius mass and fiber cross sectional area (∼40% less atrophy post-denervation in treated versus untreated mice). Mitochondrial hydroperoxide generation was reduced 80% in vitro and by over 65% in vivo by liproxstatin-1 treatment in denervated permeabilized muscle fibers and decreased the content of 4-HNE by ∼25% post-denervation. Lipidomic analysis revealed detectable levels of 25 oxylipins in denervated gastrocnemius muscle and significantly increased levels for eight oxylipins that are generated by metabolism of fatty acids through 12/15-LOX. Liproxstatin-1 treatment reduced the level of three of the eight denervation-induced oxylipins, specifically 15-HEPE, 13-HOTrE and 17-HDOHE. Denervation elevated protein degradation rates in muscle and treatment with liproxstatin-1 reduced rates of protein breakdown in denervated muscle. In contrast, protein synthesis rates were unchanged by denervation. Targeted proteomics revealed a number of proteins with altered expression after denervation but no effect of liproxstain-1. Transcriptomic analysis revealed 203 differentially expressed genes in denervated muscle from vehicle or liproxstatin-1 treated mice, including ER stress, nitric oxide signaling, Gαi signaling, glucocorticoid receptor signaling, and other pathways. Overall, these data suggest lipid hydroperoxides and oxylipins are key drivers of increased protein breakdown and muscle loss associated with denervation induced atrophy and a potential target for sarcopenia intervention.

Protein synthesis inhibition and loss of homeostatic functions in astrocytes from an Alzheimer’s disease mouse model: a role for ER-mitochondria interaction

Deregulation of protein synthesis and ER stress/unfolded protein response (ER stress/UPR) have been reported in astrocytes. However, the relationships between protein synthesis deregulation and ER stress/UPR, as well as their role in the altered homeostatic support of Alzheimer’s disease (AD) astrocytes remain poorly understood. Previously, we reported that in astrocytic cell lines from 3xTg-AD mice (3Tg-iAstro) protein synthesis was impaired and ER-mitochondria distance was reduced. Here we show that impaired protein synthesis in 3Tg-iAstro is associated with an increase of p-eIF2α and downregulation of GADD34. Although mRNA levels of ER stress/UPR markers were increased two-three-fold, we found neither activation of PERK nor downstream induction of ATF4 protein. Strikingly, the overexpression of a synthetic ER-mitochondrial linker (EML) resulted in a reduced protein synthesis and augmented p-eIF2α without any effect on ER stress/UPR marker genes. In vivo, in hippocampi of 3xTg-AD mice, reduced protein synthesis, increased p-eIF2α and downregulated GADD34 protein were found, while no increase of p-PERK or ATF4 proteins was observed, suggesting that in AD astrocytes, both in vitro and in vivo, phosphorylation of eIF2α and impairment of protein synthesis are PERK-independent. Next, we investigated the ability of 3xTg-AD astrocytes to support metabolism and function of other cells of the central nervous system. Astrocyte-conditioned medium (ACM) from 3Tg-iAstro cells significantly reduced protein synthesis rate in primary hippocampal neurons. When added as a part of pericyte/endothelial cell (EC)/astrocyte 3D co-culture, 3Tg-iAstro, but not WT-iAstro, severely impaired formation and ramification of tubules, the effect, replicated by EML overexpression in WT-iAstro cells. Finally, a chemical chaperone 4-phenylbutyric acid (4-PBA) rescued protein synthesis, p-eIF2α levels in 3Tg-iAstro cells and tubulogenesis in pericyte/EC/3Tg-iAstro co-culture. Collectively, our results suggest that a PERK-independent, p-eIF2α-associated impairment of protein synthesis compromises astrocytic homeostatic functions, and this may be caused by the altered ER-mitochondria interaction.

INRI-seq enables global cell-free analysis of translation initiation and off-target effects of antisense inhibitors.

Ribosome profiling (Ribo-seq) is a powerful method for the transcriptome-wide assessment of protein synthesis rates and the study of translational control mechanisms. Yet, Ribo-seq also has limitations. These include difficulties with the analysis of translation-modulating molecules such as antibiotics, which are often toxic or challenging to deliver into living cells. Here, we have developed in vitro Ribo-seq (INRI-seq), a cell-free method to analyze the translational landscape of a fully customizable synthetic transcriptome. Using Escherichia coli as an example, we show how INRI-seq can be used to analyze the translation initiation sites of a transcriptome of interest. We also study the global impact of direct translation inhibition by antisense peptide nucleic acid (PNA) to analyze PNA off-target effects. Overall, INRI-seq presents a scalable, sensitive method to study translation initiation in a transcriptome-wide manner without the potentially confounding effects of extracting ribosomes from living cells.

Metabolic labeling unveils alterations in the turnover of HDL-associated proteins during diabetes progression in mice.

Several aspects of diabetes pathophysiology and complications result from hyperglycemia-induced alterations in the structure and function of plasma proteins. Furthermore, insulin has a significant influence on protein metabolism by affecting both the synthesis and degradation of proteins in various tissues. To understand the role of progressive hyperglycemia on plasma proteins, in this study, we measured the turnover rates of high-density lipoprotein (HDL)-associated proteins in control (chow diet), prediabetic [a high-fat diet (HFD) for 8 wk] or diabetic [HFD for 8 wk with low-dose streptozotocin (HFD + STZ) in weeks 5-8 of HFD] C57BL/6J mice using heavy water (2H2O)-based metabolic labeling approach. Compared with control mice, HFD and HFD + STZ mice showed elevations of fasting plasma glucose levels in the prediabetic and diabetic range, respectively. Furthermore, the HFD and HFD + STZ mice showed increased hepatic triglyceride (TG) levels, total plasma cholesterol, and plasma TGs. The kinetics of 40 proteins were quantified using the proteome dynamics method, which revealed an increase in the fractional synthesis rate (FSR) of HDL-associated proteins in the prediabetic mice compared with control mice, and a decrease in FSR in the diabetic mice. The pathway analysis revealed that proteins with altered turnover rates were involved in acute-phase response, lipid metabolism, and coagulation. In conclusion, prediabetes and diabetes have distinct effects on the turnover rates of HDL proteins. These findings suggest that an early dysregulation of the HDL proteome dynamics can provide mechanistic insights into the changes in protein levels in these conditions.NEW & NOTEWORTHY This study is the first to examine the role of gradual hyperglycemia during diabetes disease progression on HDL-associated protein dynamics in the prediabetes and diabetic mice. Our results show that the fractional synthesis rate of HDL-associated proteins increased in the prediabetic mice whereas it decreased in the diabetic mice compared with control mice. These kinetic changes can help to elucidate the mechanism of altered protein levels and HDL dysfunction during diabetes disease progression.

Multi-scale mechanobiological model for skeletal muscle hypertrophy.

Skeletal muscle adaptation is correlated to training exercise by triggering different signaling pathways that target many functions; in particular, the IGF1-AKT pathway controls protein synthesis and degradation. These two functions regulate the adaptation in size and strength of muscles. Computational models for muscle adaptation have focused on: the biochemical description of signaling pathways or the mechanical description of muscle function at organ scale; however, an interrelation between these two models should be considered to understand how an adaptation in muscle size affects the protein synthesis rate. In this research, a dynamical model for the IGF1-AKT signaling pathway is linked to a continuum-mechanical model describing the active and passive mechanical response of a muscle; this model is used to study the impact of the adaptive muscle geometry on the protein synthesis at the fiber scale. This new computational model links the signaling pathway to the mechanical response by introducing a growth tensor, and links the mechanical response to the signaling pathway through the evolution of the protein synthesis rate. The predicted increase in cross sectional area (CSA) due to an 8 weeks training protocol excellently agreed with experimental data. Further, our results show that muscle growth rate decreases, if the correlation between protein synthesis and CSA is negative. The outcome of this study suggests that multi-scale models coupling continuum mechanical properties and molecular functions may improve muscular therapies and training protocols.

Effect of caffeine on mitochondrial biogenesis in the skeletal muscle - A narrative review.

Caffeine is one of the most widely used substances as recreational drug for performance-enhancement in sport, underpinned by a strong evidence base. Although the effects of caffeine are widely investigated within the scope of performance physiology, the molecular effects of caffeine within skeletal muscle remain unclear. Evidence from invitro and invivo models suggest that caffeine regulates the glucose metabolism in the skeletal muscle. Moreover, caffeine seems to stimulate CaMKII, PPARδ/β, AMPK and PGC1α, classical markers of exercise-adaptations, including mitochondrial biogenesis and mitochondrial content. This review summarizes evidence to suggest caffeine-effects within skeletal muscle fibers, focusing on the putative role of caffeine on mitochondrial biogenesis to explore whether caffeine supplementation might be a strategy to enhance mitochondrial biogenesis.

The histone methyltransferase SETD2 negatively regulates cell size.

Cell size varies between cell types but is tightly regulated by cell intrinsic and extrinsic mechanisms. Cell size control is important for cell function, and changes in cell size are frequently observed in cancer. Here, we uncover a role for SETD2 in regulating cell size. SETD2 is a lysine methyltransferase and a tumor suppressor protein involved in transcription, RNA processing and DNA repair. At the molecular level, SETD2 is best known for associating with RNA polymerase II through its Set2-Rbp1 interacting (SRI) domain and methylating histone H3 on lysine 36 (H3K36) during transcription. Using multiple independent perturbation strategies, we identify SETD2 as a negative regulator of global protein synthesis rates and cell size. We provide evidence that overexpression of the H3K36 demethylase KDM4A or the oncohistone H3.3K36M also increase cell size. In addition, ectopic overexpression of a decoy SRI domain increased cell size, suggesting that the relevant substrate is engaged by SETD2 via its SRI domain. These data add a central role of SETD2 in regulating cellular physiology and warrant further studies on separating the different functions of SETD2 in cancer development.

Mycoprotein ingestion within or without its wholefood matrix results in equivalent stimulation of myofibrillar protein synthesis rates in resting and exercised muscle of young men.

Ingestion of mycoprotein stimulates skeletal muscle protein synthesis (MPS) rates to a greater extent than concentrated milk protein when matched for leucine content, potentially attributable to the wholefood nature of mycoprotein. We hypothesised that bolus ingestion of mycoprotein as part of its wholefood matrix would stimulate MPS rates to a greater extent compared with a leucine-matched bolus of protein concentrated from mycoprotein. Twenty-four healthy young (age, 21 ± 2 years; BMI, 24 ± 3 kg.m2) males received primed, continuous infusions of L-[ring-2H5]phenylalanine and completed a bout of unilateral resistance leg exercise before ingesting either 70 g mycoprotein (MYC; 31·4 g protein, 2·5 g leucine; n 12) or 38·2 g of a protein concentrate obtained from mycoprotein (PCM; 28·0 g protein, 2·5 g leucine; n 12). Blood and muscle samples (vastus lateralis) were taken pre- and (4 h) post-exercise/protein ingestion to assess postabsorptive and postprandial myofibrillar protein fractional synthetic rates (FSR) in resting and exercised muscle. Protein ingestion increased plasma essential amino acid and leucine concentrations (P < 0·0001), but more rapidly (both 60 v. 90 min; P < 0·0001) and to greater magnitudes (1367 v. 1346 μmol·l-1 and 298 v. 283 μmol·l-1, respectively; P < 0·0001) in PCM compared with MYC. Protein ingestion increased myofibrillar FSR (P < 0·0001) in both rested (MYC, Δ0·031 ± 0·007 %·h-1 and PCM, Δ0·020 ± 0·008 %·h-1) and exercised (MYC, Δ0·057 ± 0·011 %·h-1 and PCM, Δ0·058 ± 0·012 %·h-1) muscle, with no differences between conditions (P > 0·05). Mycoprotein ingestion results in equivalent postprandial stimulation of resting and post-exercise myofibrillar protein synthesis rates irrespective of whether it is consumed within or without its wholefood matrix.

Automated radiosynthesis and preclinical in vivo evaluation of [18F]Fluoroethylpuromycin as a potential radiotracer for imaging protein synthesis with PET

PurposeFrom a series of fluorinated analogues of puromycin, we recently identified [18F]fluoroethylpuromycin (FEPURO) as a potential candidate for imaging the rate of protein synthesis in vivo. Herein, we describe the automation of the radiosynthesis, and evaluation of [18F]FEPURO in vivo. Procedures[18F]FEPURO was radiosynthesised in an automated module. PET imaging was conducted in Wistar rats under control and blocking conditions using the protein synthesis inhibitor cycloheximide. Biodistribution and metabolite studies at 30, 60 and 120 min were conducted in healthy rats. ResultsAutomation of the radiosynthesis resulted in reduction of the synthesis time by half from the manual method. A steady increase in the SUV was observed in the time-activity curves for the whole brain as expected for a protein synthesis marker. However, rapid in vivo metabolism of [18F]FEPURO within 15 min in plasma as well as the brain (4 % of parent 30 min p.i.) indicated formation of the [18F]FET radio-metabolite in >90 % thus suggesting that observed increase in the brain uptake was due to the radiometabolite. Conclusions[18F]FEPURO is not a suitable PET radiotracer for imaging protein synthesis rates in brain in vivo due to its rapid metabolism. Further structural modifications to prevent in vivo metabolism are underway.

203 Estradiol Improves Proliferation and Protein Synthesis Rates of Primary Bovine Satellite Cells by Altering Mrna Abundance Related to Amino Acid and Micronutrient Metabolism

Abstract Approximately 90% of beef cattle on feed in the U.S. receive an anabolic implant to improve production. The mechanisms implants operate through to improve growth have not been deciphered. Estradiol (E2) is one of the primary hormones found in anabolic implants. The objective of this study was to determine the effects of E2 on mRNA abundance of genes associated with skeletal muscle growth in proliferating and fused bovine satellite cells (BSC). To accomplish this, BSC were isolated from six backgrounded steers and cultured. Proliferation experiments used BSC cultures grown to 75% confluency, followed by treatment with 1% fetal bovine serum (FBS) or 10 nM E2 in 1% FBS. Protein synthesis experiments used cultures grown to 80% confluency, at which point cultures were induced to differentiate and treated 48-h later with serum free media (SFM) or10 nM E2 in SFM. Isolation of RNA occurred 30-min and 12-h post-treatment in both experiments. The mRNA abundance of 94 genes associated with skeletal muscle growth were examined. Treatment with E2 increased (P&amp;lt; 0.05) both proliferation and protein synthesis rates compared with controls. In proliferating BSC, 30-min post-treatment four genes had abundance altered (P&amp;lt; 0.10) by E2, while 12-h post treatment two genes had abundance altered (P&amp;lt; 0.10). Fused BSC, 30-min post-treatment, E2 altered (P&amp;lt; 0.10) abundance of five genes, while 12-h post treatment eight genes were altered (P&amp;lt; 0.10). A subset of significant genes include Zip8 abundance increasing (P=0.03) 30-min post-treatment in E2 treated proliferating BSC, arginase-2 abundance increasing (P=0.03) 30-min post-treatment in E2 treated fused cultures, Retinoid X Receptor-gamma abundance increasing (P=0.05) 12-h post-treatment in E2 treated proliferating BSC, and superoxide dismutase-2 abundance increasing (P=0.05) 12-h post-treatment in E2 treated fused BSC. These results indicate that E2 may improve proliferation and protein synthesis rates of BSC by altering mRNA abundance related to amino acid and micronutrient metabolism.

PSII-A-4 Effects of Beef Cattle Breed Type and Steroid Hormones on Proliferation Rates of Bovine Satellite Cells

Abstract As the population increases, it is necessary to improve the sustainability of beef production. Bos indicus (BI) cattle are better adapted to nutritional stress, consume less water, and are better able to withstand thermal stress when compared to Bos taurus (BT) cattle. Additionally, anabolic implants, which typically contain the steroid hormones trenbolone acetate (TBA) and/or estradiol (E2), are commonly administered to beef cattle in the U.S. to improve production. The objective of this study was to determine the effects of cattle breed type and steroid hormones on proliferation of primary bovine satellite cells (BSC). Primary BSC were isolated from the Semimembranosus muscle of Angus (AN; n=2; 100% BT) and Santa Gertrudis (SG; n=2; 19% BI, 81% BT) yearling backgrounded steers and cultured. After the cells reached 70% confluency, cultures were treated with 1% fetal bovine serum (control), 10 nM TBA, 10 nM E2, or 10 nM TBA and E2 and proliferation rates were compared to control cultures. No treatment x breed interaction (P=0.17) was observed. Cultures from AN steers had increased (P=0.007) proliferation rates compared to cultures from SG steers. Steroid hormones increased (P=0.08) proliferation rates, regardless of breed. However, based on this preliminary data, trends are indicating that a breed*treatment interaction is present such that BSC isolated from AN steers are more responsive to treatment than BSC isolated from SG steers. Additional research needs to be completed to determine whether this interaction is still present once more replicates are completed, whether different hormone concentrations affect BSC from cattle of different breed types, and/or the effects of hormone treatment and breed type on BSC relative to differentiation and protein synthesis rate.

327 Parameter Updates in the Davis Growth Model for Growth, Intake, and Composition of Modern Angus-Cross Cattle

Abstract The Davis Growth Model is a dynamic model used to estimate body composition and growth in beef cattle by simulating total body DNA and protein turnover. Since model development and parametrization in the 1980s, the model has been re-structured to account for previous rate of protein accretion and prior nutrition. Original parameter estimates for alpha (maintenance constant, where net energy for maintenance is alpha x shrunk body weight0.75; 0.08406) and K2 (protein synthesis rate constant; 0.0444) may not fit modern, heavier, more productive cattle. Angus-cross steers (n = 24; initial body weight 402 ± 12 kg) were fed using automated feed weight stations in group pens (8 steers/pen). Steers were fed a high energy ration twice daily, at 10% greater than the intake from the previous day. Steers were measured for hip height, body weight (BW), and ultrasonic measurements for ribeye area and back fat every 28 d until harvest (d 128). These measurements were used to calculate empty BW, empty body fat, and empty percent body fat for alpha and K2 estimates. Parameter estimates (± SD) were 0.12661 (0.01587) and 0.04919 (0.00220) for alpha and K2, respectively. Parameter estimates in the current study reflect a large increase in apparent maintenance. Maintenance requirements were moderately correlated with back fat thickness and feed conversion efficiency. Protein turnover had very strong correlations with ribeye area and feed conversion efficiency. Average feed conversion efficiency in the current study was 0.17. To evaluate parameter estimates, the model was re-run using the new values for alpha and K2. On average, the model underpredicted final empty body weight by 0.08 kg and empty body fat percent by 0.01%-units. Considering the relatively large variation among observed alpha values and the magnitude of increase compared with those previously reported, further research is required to determine an accurate alpha.

Variability in Skeletal Muscle Protein Synthesis Rates in Critically Ill Patients.

(1) Background: Muscle protein synthesis in critically ill patients is, on average, normal despite dramatic muscle loss, but the variation is much larger than in controls. Here, we evaluate if this variation is due to 1) heterogeneity in synthesis rates, 2) morphological variation or infiltrating cells, or 3) heterogeneity in the synthesis of different protein fractions. (2) Methods: Muscle biopsies were taken from both legs of critically ill patients (n = 17). Mixed and mitochondrial protein synthesis rates and morphologies were evaluated in both legs. Synthesis rates of myosin and actin were determined in combined biopsies and compared with controls. (3) Results: Muscle protein synthesis rates had a large variability in the patients (1.4–10.8%/day). No differences in mixed and mitochondrial protein synthesis rates between both legs were observed. A microscopic examination revealed no morphological differences between the two legs or any infiltrating inflammatory cells. The synthesis rates for myosin were lower and for actin they were higher in the muscles of critically ill patients, compared with the controls. (4) Conclusions: The large variation in muscle protein synthesis rates in critically ill patients is not the result of heterogeneity in synthesis rates, nor due to infiltrating cells. There are differences in the synthesis rates of different proteins, but these do not explain the larger variations.

Mechanisms of action of cytoplasmic microRNAs. Part 2. MicroRNA-mediated post-translational silencing

The scientific review presents the mechanisms of action of cytoplasmic miRNAs, namely miRNA-mediated posttranslational silencing. To write the article, information was searched using Scopus, Web of Science, MedLine, PubMed, Google Scholar, EMBASE, Global Health, The Cochrane Library, CyberLeninka databases. It is stated that protein synthesis is a complex process which involved many regulators. It is known that the translation process consists of three main stages: initiation, elongation of the polypeptide chain and termination. It is presented that dozens of “basic” factors and numerous accessory proteins, both regulators and repressors of the process, take part in the translation initiation. The authors provide a kinetic model proposed by Christopher S. Fraser. According to this model, translation initiation is a ranked process. It is emphasized that subsequently the ribosome interacts with the beginning of the coding nucleotide sequence of mRNA. Modifications of nucleotides by elongation factors in the anticodon of tRNA regulate the dynamics of ribosome function and, thus, fine-tune the rate of protein synthesis. The authors state that translation termination is induced by the interaction of the decoding A-region of the ribosome with one of the three stop codons (UAA, UAG or UGA) of mRNA. “Termination factors” are also involved in the termination of translation. Scientists say that the main factors that regulate the functional activity of mRNA act on the cap and poly(A)tail, which protects mRNA from exonuclease action. Thus, various proteins surround mRNA molecule in the cell and support the existence and functional activity of mRNA. Each mRNA region interacts with a specific spectrum of RNA-binding proteins. The initiation of translation is a ranked process and is inextricably linked with mRNA degradation. It is widely believed that translation is largely controlled during the initiation period. The mechanism of silencing caused by mRNA degradation depends on the size of the complementary region.

Role of liver AMPK and GCN2 kinases in the control of postprandial protein metabolism in response to mid-term high or low protein intake in mice.

Protein synthesis and proteolysis are known to be controlled through mammalian target of rapamycin, AMP-activated kinase (AMPK) and general control non-derepressible 2 (GCN2) pathways, depending on the nutritional condition. This study aimed at investigating the contribution of liver AMPK and GCN2 on the adaptation to high variations in protein intake. To evaluate the answer of protein pathways to high- or low-protein diet, male wild-type mice and genetically modified mice from C57BL/6 background with liver-specific AMPK- or GCN2-knockout were fed from day 25 diets differing in their protein level as energy: LP (5%), NP (14%) and HP (54%). Two hours after a 1g test meal, protein synthesis rate was measured after a 13C valine flooding dose. The gene expression of key enzymes involved in proteolysis and GNC2 signaling pathway were quantified. The HP diet but not the LP diet was associated with a decrease in fractional synthesis rate by 29% in the liver compared to NP diet. The expression of mRNA encoding ubiquitin and Cathepsin D was not sensitive to the protein content. The deletion of AMPK or GCN2 in the liver did not affect nor protein synthesis rates and neither proteolysis markers in the liver or in the muscle, whatever the protein intake. In the postprandial state, protein level alters protein synthesis in the liver but not in the muscle. Taken together, these results suggest that liver AMPK and GCN2 are not involved in this adaptation to high- and low-protein diet observed in the postprandial period.

Increased rates of brain protein synthesis during [N1,N2] sleep: L-[1-11C]leucine PET studies in human subjects

During sleep, reduced brain energy demands provide an opportunity for biosynthetic processes like protein synthesis. Sleep is required for some forms of memory consolidation which requires de novo protein synthesis. We measured regional cerebral protein synthesis rates (rCPS) in human subjects to ascertain how rCPS is affected during sleep. Subjects underwent three consecutive L-[1-11C]leucine PET scans with simultaneous polysomnography: 1. rested awake, 2. sleep-deprived awake, 3. sleep. Measured rCPS were similar across the three conditions. Variations in sleep stage times during sleep scans were used to estimate rCPS in sleep stages under the assumption that measured rCPS is the weighted sum of rCPS in each stage, with weights reflecting time and availability of [11C]leucine in that stage. During sleep scans, subjects spent most of the time in N2, N3, and awake and very little time in N1 and REM; rCPS in N1 and REM could not be reliably estimated. When stages N1 and N2 were combined [N1,N2], estimates of rCPS were more robust. In selective regions, estimated rCPS were statistically significantly higher (30–39%) in [N1,N2] compared with N3; estimated rCPS in N3 were similar to values measured in sleep-deprived awake scans. Results indicate increased rates of protein synthesis linked to [N1,N2] sleep.

Delineation of proteome changes driven by cell size and growth rate.

Increasing cell size drives changes to the proteome, which affects cell physiology. As cell size increases, some proteins become more concentrated while others are diluted. As a result, the state of the cell changes continuously with increasing size. In addition to these proteomic changes, large cells have a lower growth rate (protein synthesis rate per unit volume). That both the cell’s proteome and growth rate change with cell size suggests they may be interdependent. To test this, we used quantitative mass spectrometry to measure how the proteome changes in response to the mTOR inhibitor rapamycin, which decreases the cellular growth rate and has only a minimal effect on cell size. We found that large cell size and mTOR inhibition, both of which lower the growth rate of a cell, remodel the proteome in similar ways. This suggests that many of the effects of cell size are mediated by the size-dependent slowdown of the cellular growth rate. For example, the previously reported size-dependent expression of some senescence markers could reflect a cell’s declining growth rate rather than its size per se. In contrast, histones and other chromatin components are diluted in large cells independently of the growth rate, likely so that they remain in proportion with the genome. Finally, size-dependent changes to the cell’s growth rate and proteome composition are still apparent in cells continually exposed to a saturating dose of rapamycin, which indicates that cell size can affect the proteome independently of mTORC1 signaling. Taken together, our results clarify the dependencies between cell size, growth, mTOR activity, and the proteome remodeling that ultimately controls many aspects of cell physiology.

Alaska Backcountry Expeditionary Hunting Promotes Exceptional Rates Of Muscle Protein Synthesis

Our Alaskan Backcountry Expeditionary Hunting (ABEH) paradigm represents an 8-14 day unscripted, remote expedition in the uninhabited Brooks Range. Despite negative energy balance (i.e., -9.7 ± 3.4 MJ/day) and significant weight loss (Δ-1.5 ± 0.7 kg) largely influenced by high levels of energy expenditure (i.e., 17.4 ± 2.6 MJ/day), participants retained skeletal muscle as determined by magnetic resonance imaging and lean tissue mass as indicated by dual energy x-ray absorptiometry. Preliminary evidence from these investigations demonstrated an increase in skeletal muscle follistatin-like protein 1, potentially enhancing the anabolism of skeletal muscle. PURPOSE: To determine integrated rates of skeletal muscle protein synthesis throughout the ABEH paradigm that may be relevant to skeletal muscle preservation. METHODS: The use of the virtual biopsy method was employed via 2H2O dosing and liquid chromatography/tandem mass spectrometry analysis to evaluate fractional synthetic rates (FSR) of muscle proteins in four participants (2 females [28 and 62 yr; 66.2 and 71.8 kg; 25.5 and 26.7 kg/m2] and 2 males [47 and 56 yr; 87.5 and 91.4 kg; 26.1 and 28.3 kg/m2]). RESULTS: As an indicator of skeletal muscle synthesis, mean FSR of serum carbonic anhydrase and creatine kinase M-type was 2.4% per day (range: 1.3-5.1% per day) and 4.0% per day (range: 1.4-8.2% per day), respectively. CONCLUSION: Our preliminary findings suggest that the ABEH-induced alterations in skeletal muscle FSR in our cohort are ~four-fold higher than reported in young individuals at baseline and ~ two-fold higher than after 3 weeks of exhaustive sprint interval training. Therefore, chronic mechanical overload may promote a sustained increase in the FSR of skeletal muscle despite limited nutritional provisions and negative energy balance. Research was supported by Pristine Ventures, Inc. Funding was also provided by an Institutional Development Award (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health under grant number 2P20GM103395 and Sloan Foundation Fellowship to Mr. Brandon Kowalski. The content is solely the responsibility of the authors and does not necessarily reflect the official views of the NIH and/or the Sloan Foundation.