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Impact of 3-week citrulline supplementation on postprandial protein metabolism in malnourished older patients: The Ciproage randomized controlled trial.
Bouillanne, O, Melchior, JC, Faure, C, Paul, M, Canouï-Poitrine, F, Boirie, Y, Chevenne, D, Forasassi, C, Guery, E, Herbaud, S, et al
Clinical nutrition (Edinburgh, Scotland). 2019;(2):564-574
Abstract
BACKGROUND Citrulline (CIT), is not extracted by the splanchnic area, can stimulate muscle protein synthesis and could potentially find clinical applications in conditions involving low amino acid (AA) intake, such as in malnourished older subjects. OBJECTIVE Our purpose was to research the effects of CIT supplementation on protein metabolism in particular on non-oxidative leucine disposal (NOLD, primary endpoint), and splanchnic extraction of amino acids in malnourished older patients. DESIGN This prospective randomized multicenter study determined whole-body and liver protein synthesis, splanchnic protein metabolism and appendicular skeletal muscle mass (ASMM) in 24 malnourished older patients [80-92 years; 18 women and 6 men] in inpatient rehabilitation units. All received an oral dose of 10 g of CIT or an equimolar mixture of six non-essential amino acids (NEAAs), as isonitrogenous placebo, for 3 weeks. RESULTS NOLD and albumin fractional synthesis rates were not different between the NEAA and CIT groups. Splanchnic extraction of dietary amino acid tended to decrease (p = 0.09) in the CIT group (45.2%) compared with the NEAA group (60.3%). Total differences in AA and NEAA area under the curves between fed-state and postabsorptive-state were significantly higher in the CIT than in the NEAA group. There were no significant differences for body mass index, fat mass (FM), lean mass (LM) or ASMM in the whole population except for a tendential decrease in FM for the citrulline group (p = 0.089). Compared with Day 1, lean mass and ASMM significantly increased (respectively p = 0.016 and p = 0.018) at Day 20 in CIT-treated women (mean respective increase of 1.7 kg and 1.1 kg), and fat mass significantly decreased (p = 0.001) at Day 20 in CIT-group women (mean decrease of 1.3 kg). CONCLUSIONS Our results demonstrate that CIT supplementation has no effect on whole-body protein synthesis or liver protein synthesis in malnourished older subjects. However, CIT supplementation was associated with a higher systemic AA availability. In the subgroup of women, CIT supplementation increased LM and ASMM, and decreased FM.
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The problem of genetic code misreading during protein synthesis.
Joshi, K, Cao, L, Farabaugh, PJ
Yeast (Chichester, England). 2019;(1):35-42
Abstract
Saccharomyces cerevisiae has been an important model for determining the frequency of translational misreading events, those in which a tRNA pairs incorrectly to the mRNA and inserts an amino acid not specified by the codon in the mRNA. Misreading errors have been quantified in vivo using reporter protein systems or mass spectrometry with both approaches converging on a simple model for most misreading. The available data show that misreading tRNAs must form stereotypical base mismatches that correspond to those that can mimic Watson-Crick base pairs when formed in the ribosomal A site. Errors involving other mismatches occur significantly less frequently. This work debunks the idea of an average misreading frequency of 5 × 10-4 per codon that extends across the genetic code. Instead, errors come in two distinct classes-high frequency and low frequency events-with most errors being of the low frequency type. A comparison of misreading errors in S. cerevisiae and Escherichia coli suggests the existence of a mechanism that reduces misreading frequency in yeast; this mechanism may operate in eukaryotes generally.
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3.
Phosphorylation and Signal Transduction Pathways in Translational Control.
Proud, CG
Cold Spring Harbor perspectives in biology. 2019;(7)
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Abstract
Protein synthesis, including the translation of specific messenger RNAs (mRNAs), is regulated by extracellular stimuli such as hormones and by the levels of certain nutrients within cells. This control involves several well-understood signaling pathways and protein kinases, which regulate the phosphorylation of proteins that control the translational machinery. These pathways include the mechanistic target of rapamycin complex 1 (mTORC1), its downstream effectors, and the mitogen-activated protein (MAP) kinase (extracellular ligand-regulated kinase [ERK]) signaling pathway. This review describes the regulatory mechanisms that control translation initiation and elongation factors, in particular the effects of phosphorylation on their interactions or activities. It also discusses current knowledge concerning the impact of these control systems on the translation of specific mRNAs or subsets of mRNAs, both in physiological processes and in diseases such as cancer.
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Translational regulation and deregulation in erythropoiesis.
Vatikioti, A, Karkoulia, E, Ioannou, M, Strouboulis, J
Experimental hematology. 2019;:11-20
Abstract
Translational regulation plays a critical role in erythropoiesis, as it reflects the translational needs of enucleated mature erythroid cells in the absence of transcription and the large translational demands of balanced globin chain synthesis during erythroid maturation. In addition, red blood cells need to respond quickly to changes in their environment and the demands of the organism. Translational regulation occurs at several levels in erythroid cells, including the differential utilization of upstream open reading frames during differentiation and in response to signaling and the employment of RNA-binding proteins in an erythroid cell-specific fashion. Translation initiation is a critical juncture for translational regulation in response to environmental signals such as heme and iron availability, whereas regulatory mechanisms for ribosome recycling are consistent with recent observations highlighting the importance of maintaining adequate ribosome levels in differentiating erythroid cells. Translational deregulation in erythroid cells leads to disease associated with ineffective erythropoiesis, further highlighting the pivotal role translational regulation in erythropoiesis plays in human physiology and homeostasis. Overall, erythropoiesis has served as a unique model that has provided invaluable insight into translational regulation.
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Cycloheximide can distort measurements of mRNA levels and translation efficiency.
Santos, DA, Shi, L, Tu, BP, Weissman, JS
Nucleic acids research. 2019;(10):4974-4985
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Abstract
Regulation of the efficiency with which an mRNA is translated into proteins represents a key mechanism for controlling gene expression. Such regulation impacts the number of actively translating ribosomes per mRNA molecule, referred to as translation efficiency (TE), which can be monitored using ribosome profiling and RNA-seq, or by evaluating the position of an mRNA in a polysome gradient. Here we show that in budding yeast, under nutrient limiting conditions, the commonly used translation inhibitor cycloheximide induces rapid transcriptional upregulation of hundreds of genes involved in ribosome biogenesis. Cycloheximide also prevents translation of these newly transcribed messages, leading to an apparent drop in TE of these genes under conditions that include key transitions during the yeast metabolic cycle, meiosis, and amino acid starvation; however, this effect is abolished when cycloheximide pretreatment is omitted. This response requires TORC1 signaling, and is modulated by the genetic background as well as the vehicle used to deliver the drug. The present work highlights an important caveat to the use of translation inhibitors when measuring TE or mRNA levels, and will hopefully aid in future experimental design as well as interpretation of prior results.
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Chiral checkpoints during protein biosynthesis.
Kuncha, SK, Kruparani, SP, Sankaranarayanan, R
The Journal of biological chemistry. 2019;(45):16535-16548
Abstract
Protein chains contain only l-amino acids, with the exception of the achiral glycine, making the chains homochiral. This homochirality is a prerequisite for proper protein folding and, hence, normal cellular function. The importance of d-amino acids as a component of the bacterial cell wall and their roles in neurotransmission in higher eukaryotes are well-established. However, the wider presence and the corresponding physiological roles of these specific amino acid stereoisomers have been appreciated only recently. Therefore, it is expected that enantiomeric fidelity has to be a key component of all of the steps in translation. Cells employ various molecular mechanisms for keeping d-amino acids away from the synthesis of nascent polypeptide chains. The major factors involved in this exclusion are aminoacyl-tRNA synthetases (aaRSs), elongation factor thermo-unstable (EF-Tu), the ribosome, and d-aminoacyl-tRNA deacylase (DTD). aaRS, EF-Tu, and the ribosome act as "chiral checkpoints" by preferentially binding to l-amino acids or l-aminoacyl-tRNAs, thereby excluding d-amino acids. Interestingly, DTD, which is conserved across all life forms, performs "chiral proofreading," as it removes d-amino acids erroneously added to tRNA. Here, we comprehensively review d-amino acids with respect to their occurrence and physiological roles, implications for chiral checkpoints required for translation fidelity, and potential use in synthetic biology.
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Effects of acute oral feeding on protein metabolism and muscle protein synthesis in individuals with cancer.
van der Meij, BS, De Groot, LM, Deutz, NEP, Engelen, MPKJ
Nutrition (Burbank, Los Angeles County, Calif.). 2019;:110531
Abstract
Weight loss and muscle loss are common in individuals living with cancer, with ≤50% experiencing involuntary weight loss at any time point in their cancer journey, and between 11% and 74% having sarcopenia or significant muscle loss. These changes in body composition are related to poor outcomes such as increased treatment toxicity, impaired quality of life, and reduced survival duration. Poor outcomes are not restricted to those who are underweight with severe weight loss; sarcopenia alone has been shown to be a prognostic marker across all body mass index categories, ranging from underweight to obesity To understand the mechanism of nutrition interventions in cancer and to develop effective future interventions, it is necessary to look at the acute effects of feeding on the response of the body and the ability to reach an anabolic response. The aim of this study was to explore and summarize the emerging evidence on metabolic effects of acute oral interventions on whole body protein kinetics and muscle protein synthesis in individuals with cancer.
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It's not just about protein turnover: the role of ribosomal biogenesis and satellite cells in the regulation of skeletal muscle hypertrophy.
Brook, MS, Wilkinson, DJ, Smith, K, Atherton, PJ
European journal of sport science. 2019;(7):952-963
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Abstract
Skeletal muscle has indispensable roles in regulating whole body health (e.g. glycemic control, energy consumption) and, in being central in locomotion, is crucial in maintaining quality-of-life. Therefore, understanding the regulation of muscle mass is of significant importance. Resistance exercise training (RET) combined with supportive nutrition is an effective strategy to achieve muscle hypertrophy by driving chronic elevations in muscle protein synthesis (MPS). The regulation of muscle protein synthesis is a coordinated process, in requiring ribosomes to translate mRNA and sufficient myonuclei density to provide the platform for ribosome and mRNA transcription; as such MPS is determined by both translational efficiency (ribosome activity) and translational capacity (ribosome number). Moreover, as the muscle protein pool expands during hypertrophy, translation capacity (i.e. ribosomes and myonuclei content) could theoretically become rate-limiting such that an inability to expand these pools through ribosomal biogenesis and satellite cell (SC) mediated myonuclear addition could limit growth potential. Simple measures of RNA (ribosome content) and DNA (SC/Myonuclei number) concentrations reveal that these pools do increase with hypertrophy; yet whether these adaptations are a pre-requisite or a limiting factor for hypertrophy is unresolved and highly debated. This is primarily due to methodological limitations and many assumptions being made on static measures or correlative associations. However recent advances within the field using stable isotope tracers shows promise in resolving these questions in muscle adaptation.
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Substrate-assisted mechanism of catalytic hydrolysis of misaminoacylated tRNA required for protein synthesis fidelity.
Ilchenko, MM, Rybak, MY, Rayevsky, AV, Kovalenko, OP, Dubey, IY, Tukalo, MA
The Biochemical journal. 2019;(4):719-732
Abstract
d-aminoacyl-tRNA-deacylase (DTD) prevents the incorporation of d-amino acids into proteins during translation by hydrolyzing the ester bond between mistakenly attached amino acids and tRNAs. Despite extensive study of this proofreading enzyme, the precise catalytic mechanism remains unknown. Here, a combination of biochemical and computational investigations has enabled the discovery of a new substrate-assisted mechanism of d-Tyr-tRNATyr hydrolysis by Thermus thermophilus DTD. Several functional elements of the substrate, misacylated tRNA, participate in the catalysis. During the hydrolytic reaction, the 2'-OH group of the А76 residue of d-Tyr-tRNATyr forms a hydrogen bond with a carbonyl group of the tyrosine residue, stabilizing the transition-state intermediate. Two water molecules participate in this reaction, attacking and assisting ones, resulting in a significant decrease in the activation energy of the rate-limiting step. The amino group of the d-Tyr aminoacyl moiety is unprotonated and serves as a general base, abstracting the proton from the assisting water molecule and forming a more nucleophilic ester-attacking species. Quantum chemical methodology was used to investigate the mechanism of hydrolysis. The DFT-calculated deacylation reaction is in full agreement with the experimental data. The Gibbs activation energies for the first and second steps were 10.52 and 1.05 kcal/mol, respectively, highlighting that the first step of the hydrolysis process is the rate-limiting step. Several amino acid residues of the enzyme participate in the coordination of the substrate and water molecules. Thus, the present work provides new insights into the proofreading details of misacylated tRNAs and can be extended to other systems important for translation fidelity.
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Determinants of skeletal muscle protein turnover following severe burn trauma in children.
Malagaris, I, Herndon, DN, Polychronopoulou, E, Rontoyanni, VG, Andersen, CR, Suman, OE, Porter, C, Sidossis, LS
Clinical nutrition (Edinburgh, Scotland). 2019;(3):1348-1354
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Abstract
BACKGROUND & AIMS Burns remain the fifth cause of non-fatal pediatric injuries globally, with muscle cachexia being a hallmark of the stress response to burns. Burn-induced muscle wasting is associated with morbidity, yet the determinants of muscle protein catabolism in response to burn trauma remains unclear. Our objective was to determine the effect of patient and injury characteristics on muscle protein kinetics in burn patients. METHODS This retrospective, observational study was performed using protein kinetic data from pediatric patients who had severe burns (>30% of the total body surface area burned) and underwent cross-limb stable isotope infusions between 1999 and 2008 as part of prospective clinical trials. Mixed multiple regression models were used to assess associations between patient/injury characteristics and muscle protein fractional synthesis rate (FSR), net balance (NB), and rates of phenylalanine appearance (Ra; index of protein breakdown) and disappearance (Rd; index of protein synthesis) across the leg. RESULTS A total of 268 patients who underwent 499 studies were analyzed. Increasing time post injury was associated with greater FSR (p < 0.001) and NB (p = 0.01). Males were more catabolic than females (as indicated by lower NB, p = 0.04 and greater Ra, p = 0.008), a consequence of higher protein breakdown rather than lower synthesis. Increasing burn size was associated with higher protein synthesis rate (as indicated by higher FSR, p = 0.019) and higher protein breakdown rates (as indicated by greater Ra, p = 0.001). FSR was negatively associated with age (p < 0.001). CONCLUSIONS Data from this large patient cohort show that injury severity, sex, and time post injury influence skeletal muscle wasting in burned children. These findings suggest that individual patient characteristics should be considered when devising therapies to improve the acute care and rehabilitation of burn survivors.