A high content imaging assay for identification of specific inhibitors of native Plasmodium liver stage protein synthesis.

Plasmodium parasite resistance to antimalarial drugs is a serious threat to public health in malaria-endemic areas. Compounds that target core cellular processes like translation are highly desirable, as they should be capable of killing parasites in their liver and blood stage forms, regardless of molecular target or mechanism. Assays that can identify these compounds are thus needed. Recently, specific quantification of native Plasmodium berghei liver stage protein synthesis, as well as that of the hepatoma cells supporting parasite growth, was achieved via automated confocal feedback microscopy of the o-propargyl puromycin (OPP)-labeled nascent proteome, but this imaging modality is limited in throughput. Here, we developed and validated a miniaturized high content imaging (HCI) version of the OPP assay that increases throughput, before deploying this approach to screen the Pathogen Box. We identified only two hits; both of which are parasite-specific quinoline-4-carboxamides, and analogs of the clinical candidate and known inhibitor of blood and liver stage protein synthesis, DDD107498/cabamiquine. We further show that these compounds have strikingly distinct relationships between their antiplasmodial and translation inhibition efficacies. These results demonstrate the utility and reliability of the P. berghei liver stage OPP HCI assay for the specific, single-well quantification of Plasmodium and human protein synthesis in the native cellular context, allowing the identification of selective Plasmodium translation inhibitors with the highest potential for multistage activity.

Suspension Bead Loading (SBL): An Economical Protein Delivery Platform to Study URM1's Behavior in Live Cells.

Uniquely modified synthetic proteins are difficult to produce in large quantities, which could limit their use in various in vitro settings and in cellular studies. In this study, we developed a method named "suspension bead loading" (SBL), to deliver protein molecules into suspended living cells using glass beads, which significantly reduces the amount of protein required for effective delivery. We investigated the delivery efficiency of functionally different proteins and evaluated the cytotoxic effect of our method and the chemical and functional integrity of the delivered protein. We utilized SBL to address questions related to ubiquitin-related modifier 1 (URM1). Employing minimal protein quantities, SBL has enabled us to study its behavior within live cells under different redox conditions, including subcellular localization and conjugation patterns. We demonstrate that oxidative stress alters both the localization and conjugation pattern of URM1 in cells, highlighting its possible role in cellular response to such extreme conditions.

The crosstalk between metabolism and translation.

Metabolism and mRNA translation represent critical steps involved in modulating gene expression and cellular physiology. Being the most energy-consuming process in the cell, mRNA translation is strictly linked to cellular metabolism and in synchrony with it. Indeed, several mRNAs for metabolic pathways are regulated at the translational level, resulting in translation being a coordinator of metabolism. On the other hand, there is a growing appreciation for how metabolism impacts several aspects of RNA biology. For example, metabolic pathways and metabolites directly control the selectivity and efficiency of the translational machinery, as well as post-transcriptional modifications of RNA to fine-tune protein synthesis. Consistently, alterations in the intricate interplay between translational control and cellular metabolism have emerged as a critical axis underlying human diseases. A better understanding of such events will foresee innovative therapeutic strategies in human disease states.

Structural analysis of noncanonical translation initiation complexes.

Translation initiation is a highly regulated, multi-step process which is critical for efficient and accurate protein synthesis. In bacteria, initiation begins when mRNA, initiation factors, and a dedicated initiator fMet-tRNAfMet bind the small (30S) ribosomal subunit. Specific binding of fMet-tRNAfMet in the peptidyl (P) site is mediated by the inspection of the fMet moiety by initiation factor IF2 and of three conserved G-C base pairs in the tRNA anticodon stem by the 30S head domain. Tandem A-minor interactions form between 16S ribosomal RNA nucleotides A1339 and G1338 and tRNA base pairs G30-C40 and G29-C41, respectively. Swapping the G30-C40 pair of tRNAfMet with C-G reduces discrimination against the noncanonical start codon CUG in vitro, suggesting crosstalk between gripping of the anticodon stem and recognition of the start codon. Here, we solved electron cryomicroscopy structures of E. coli 70S initiation complexes containing an fMet-tRNAfMet G30-C40 variant paired to noncanonical CUG start codon, in the presence or absence of IF2 and the non-hydrolyzable GTP analog GDPCP, alongside structures of 70S initiation complexes containing this tRNAfMet variant paired to the canonical bacterial start codons AUG, GUG, and UUG. We find that the M1 mutation weakens A-minor interactions between tRNAfMet and 16S nucleotides A1339 and G1338, with IF2 strengthening the interaction of G1338 with the tRNA minor groove. These structures suggest how even slight changes to the recognition of the fMet-tRNAfMet anticodon stem by the ribosome can impact start codon selection.

The Anabolic Response to a Ground Beef Patty and Soy-Based Meat Alternative: A Randomized Controlled Trial.

BACKGROUND: Soy-based meat alternatives (SBMA) are becoming increasingly popular, but it is unclear if they have the same anabolic effect on skeletal muscle as animal meat. OBJECTIVE: We aimed to compare the stimulation of skeletal muscle protein synthesis by consumption of one or two 4 oz patties of SBMA with 4 oz (80%protein/20%fat) beef. METHODS: The study design was a randomized controlled trial. Participants were aged 18 to 40 years of age and in good general health with a BMI between 20 and 32 kg/m2. Stable isotope tracer methods were used (L-[ring-2H5] phenylalanine, [U-13C9-15N]- tyrosine and L-[ring-2H4] tyrosine) to quantify the response of muscle protein fractional synthetic rate to consumption of a single beef (4 oz), single SBMA (4 oz), or two 4 oz SBMA patties (8 oz). Whole-body rates of protein synthesis, breakdown and net balance, as well as plasma essential amino acid (EAA) concentrations, were also measured. RESULTS: The increase above basal in muscle protein FSR following consumption of the 4 oz beef patty (0.020 ± 0.016%/hour) was significantly greater than the increase following consumption of 4 oz SBMA (p = 0.021; 0.003 ± 0.010%/hour) but not 8 oz SBMA (p = 0.454; 0.013 ± 0.016%/hour). The maximal EAA concentration was significantly correlated (p = 0.046; r = 0.411) with the change in muscle FSR from the basal to postprandial period. In addition, the change in muscle FSR from the basal to postprandial period was significantly correlated (p = 0.046; r = 0.412) with the corresponding change in whole-body protein synthesis. CONCLUSION: Consumption of a 4 oz beef patty stimulates muscle and whole -body protein synthesis more than a 4 oz SBMA patty and similarly to 8 oz of SBMA. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT05197140.

Unacylated Ghrelin Protects Against Age-Related Loss of Muscle Mass and Contractile Dysfunction in Skeletal Muscle.

Sarcopenia, the progressive loss of muscle mass and function, universally affects older adults and is closely associated with frailty and reduced quality of life. Despite the inevitable consequences of sarcopenia and its relevance to healthspan, no pharmacological therapies are currently available. Ghrelin is a gut-released hormone that increases appetite and body weight through acylation. Acylated ghrelin activates its receptor, growth hormone secretagogue receptor 1a (GHSR1a), in the brain by binding to it. Studies have demonstrated that acyl and unacylated ghrelin (UnAG) both have protective effects against acute pathological conditions independent of receptor activation. Here, we investigated the long-term effects of UnAG in age-associated muscle atrophy and contractile dysfunction in mice. Four-month-old and 18-month-old mice were subjected to either UnAG or control treatment for 10 months. UnAG did not affect food consumption or body weight. Gastrocnemius and quadriceps muscle weights were reduced by 20%-30% with age, which was partially protected against by UnAG. Specific force, force per cross-sectional area, measured in isolated extensor digitorum longus muscle was diminished by 30% in old mice; however, UnAG prevented the loss of specific force. UnAG also protected from decreases in mitochondrial respiration and increases in hydrogen peroxide generation of skeletal muscle of old mice. Results of bulk mRNA-seq analysis and our contractile function data show that UnAG reversed neuromuscular junction impairment that occurs with age. Collectively, our data revealed the direct role of UnAG in mitigating sarcopenia in mice, independent of food consumption or body weight, implicating UnAG treatment as a potential therapy against sarcopenia.

Facile incorporation of non-canonical heme ligands in myoglobin through chemical protein synthesis.

The incorporation of non-canonical amino acids (ncAAs) into the metal coordination environments of proteins has endowed metalloproteins with enhanced properties and novel activities, particularly in hemoproteins. In this work, we disclose a scalable synthetic strategy that enables the production of myoglobin (Mb) variants with non-canonical heme ligands, i.e., HoCys and f4Tyr. The ncAA-containing Mb* variants (with H64V/V68A mutations) were obtained through two consecutive native chemical ligations and a subsequent desulfurization step, with overall isolated yield up to 28.6 % in over 10-milligram scales. After refolding and heme b cofactor reconstitution, the synthetic Mb* variants showed typical electronic absorption bands. When subjected to the catalysis of the cyclopropanation of styrene, both synthetic variants, however, were not as competent as the His-ligated Mb*. We envisioned that the synthetic method reported herein would be useful for incorporating a variety of ncAAs with diverse structures and properties into Mb for varied purposes.

The central role of transfer RNAs in mistranslation.

Transfer RNAs (tRNA) are essential small non-coding RNAs that enable the translation of genomic information into proteins in all life forms. The principal function of tRNAs is to bring amino acid building blocks to the ribosomes for protein synthesis. In the ribosome, tRNAs interact with messenger RNA (mRNA) to mediate the incorporation of amino acids into a growing polypeptide chain following the rules of the genetic code. Accurate interpretation of the genetic code requires tRNAs to carry amino acids matching their anticodon identity and decode the correct codon on mRNAs. Errors in these steps cause the translation of codons with the wrong amino acids (mistranslation), compromising the accurate flow of information from DNA to proteins. Accumulation of mutant proteins due to mistranslation jeopardizes proteostasis and cellular viability. However, the concept of mistranslation is evolving, with increasing evidence indicating that mistranslation can be used as a mechanism for survival and acclimatization to environmental conditions. In this review, we discuss the central role of tRNAs in modulating translational fidelity through their dynamic and complex interplay with translation factors. We summarize recent discoveries of mistranslating tRNAs and describe the underlying molecular mechanisms and the specific conditions and environments that enable and promote mistranslation.

The role of tRNA identity elements in aminoacyl-tRNA editing.

The rules of the genetic code are implemented by the unique features that define the amino acid identity of each transfer RNA (tRNA). These features, known as "identity elements," mark tRNAs for recognition by aminoacyl-tRNA synthetases (ARSs), the enzymes responsible for ligating amino acids to tRNAs. While tRNA identity elements enable stringent substrate selectivity of ARSs, these enzymes are prone to errors during amino acid selection, leading to the synthesis of incorrect aminoacyl-tRNAs that jeopardize the fidelity of protein synthesis. Many error-prone ARSs have evolved specialized domains that hydrolyze incorrectly synthesized aminoacyl-tRNAs. These domains, known as editing domains, also exist as free-standing enzymes and, together with ARSs, safeguard protein synthesis fidelity. Here, we discuss how the same identity elements that define tRNA aminoacylation play an integral role in aminoacyl-tRNA editing, synergistically ensuring the correct translation of genetic information into proteins. Moreover, we review the distinct strategies of tRNA selection used by editing enzymes and ARSs to avoid undesired hydrolysis of correctly aminoacylated tRNAs.

Uncovering a Latent Bioactive Interleukin-6 Glycoform.

A bioinspired semisynthesis of human-interleukin-6 bearing N-glycan at Asn143 (143glycosyl-IL-6) was performed by intentional glycosylation effects and protein folding chemistry for regioselective peptide-backbone activation. 143Glycosyl-IL-6 is a genetically coded cytokine, but isolation was difficult owing to a tiny amount. IL6-polypeptide (1-141-position) with an intentionally inserted cysteine at 142-position was expressed in E. coli. The expressed polypeptide was treated with a chemical folding process to make a specific helices bundle conformation through native two-disulfide bonds (43-49 and 72-82). Utilizing the successfully formed free-142-cysteine, sequential conversions using cyanation of 142-cysteine, hydrazinolysis, and thioesterification created a long polypeptide (1-141)-thioester. However, the resultant polypeptide-thioester caused considerable aggregation owing to a highly hydrophobic peptide sequence. After the reduction of two-disulfide bonds of polypeptide (1-141)-thioester, an unprecedented hydrophilic N-glycan tag was inserted at the resultant cysteine thiols. The N-glycan tags greatly stabilized polypeptide-thioester. The subsequent native chemical ligation and desulfurization successfully gave a whole 143glycosyl-IL-6 polypeptide (183-amino acids). Removal of four N-glycan tags and immediate one-pot in vitro folding protocol efficiently produced the folded 143glycosyl-IL-6. The folded 143glycosyl-IL-6 exhibited potent cell proliferation activity. The combined studies with molecular dynamics simulation, semisynthesis, and bioassays predict the bioactive conformation of latent 143glycosyl-IL-6.

Efficiently Substituting Dietary Fish Meal with Terrestrial Compound Protein Enhances Growth, Health, and Protein Synthesis in Largemouth Bass.

Inappropriate substitution of dietary fishmeal (FM) can adversely affect the growth, health, and metabolism of carnivorous fish species. To effectively reduce the amount of dietary FM in carnivorous largemouth bass (Micropterus salmoides), a terrestrial compound protein (Cpro) with chicken meal, bone meal, and black soldier fly protein was used to formulate four isoproteic (52%) and isolipidic (12%) diets, namely T1 (36% FM), T2 (30% FM), T3 (24% FM), and T4 (18% FM), for feeding juveniles (initial weight: ~12 g) for 81 days. Results indicated that the growth performance, feed efficiency, and morphological indicators, as well as muscle texture and edible quality of fish, did not differ significantly among the four groups. However, the muscle protein contents and ATP/AMP ratio of fish in the T4 group were significantly increased in comparison with those of fish in the T1 group, while the opposite was true for muscle glycogen. Compared with the T1 group, high serum total amino acid and MDA contents, as well as low AST activities, were observed in the T3 and T4 groups, and relatively high intestinal trypsin and lipase activities were found in the T2-T4 groups. The transcripts of intestinal proinflammatory cytokines (il-1ß, il-6, and tnf-α) were downregulated in the T2-T4 groups compared with T1 group, while the expression of anti-inflammatory cytokines (il-10) and tight junction (zo-1 and occludin) showed the reverse trend. The mRNA expression of positive regulators related to protein synthesis (sirt1, pgc1-α, pi3k, and akt) were significantly upregulated in the muscle of fish fed diets T3 and T4, while their negative regulators (4e-bp1) mRNA levels were downregulated. The results indicate that the dietary FM of largemouth bass could be effectively reduced to at least 18% by the Cpro, which is beneficial to health, digestion, and protein synthesis for maintaining accelerated growth.

Selective Activation of Peptide-Thioester Precursors for Templated Native Chemical Ligations.

Chemical protein synthesis enables access to proteins that would otherwise be difficult or impossible to obtain with traditional means such as recombinant expression. Chemoselective ligations provide the ability to join peptide segments prepared by solid-phase peptide synthesis. While native chemical ligation (NCL) is widely used, it is limited by the need for C-terminal thioesters with suitable reaction kinetics, properly placed native Cys or thiolated derivatives, and peptide segment solubility at low mM concentrations. Moreover, repetitive purifications to isolate ligated products are often yield-sapping, hampering efficiency and progress. In this work, we demonstrate the use of Controlled Activation of Peptides for Templated NCL (CAPTN). This traceless multi-segment templated NCL approach permits the one-pot synthesis of proteins by harnessing selective thioester activation and orthogonal conjugation chemistries to favor formation of full-length ligated product while minimizing side reactions. Importantly, CAPTN provides kinetic enhancements allowing ligations at sterically hindered junctions and low peptide concentrations. Additionally, this one-pot approach removes the need for intermediate purification. We report the synthesis of two E.coli ribosomal subunits S16 and S17 enabled by the chemical tools described herein. We anticipate that CAPTN will expedite the synthesis of valuable proteins and expand on templated approaches for chemical protein synthesis.

Conserved 5-methyluridine tRNA modification modulates ribosome translocation.

While the centrality of posttranscriptional modifications to RNA biology has long been acknowledged, the function of the vast majority of modified sites remains to be discovered. Illustrative of this, there is not yet a discrete biological role assigned for one of the most highly conserved modifications, 5-methyluridine at position 54 in tRNAs (m5U54). Here, we uncover contributions of m5U54 to both tRNA maturation and protein synthesis. Our mass spectrometry analyses demonstrate that cells lacking the enzyme that installs m5U in the T-loop (TrmA in Escherichia coli, Trm2 in Saccharomyces cerevisiae) exhibit altered tRNA modification patterns. Furthermore, m5U54-deficient tRNAs are desensitized to small molecules that prevent translocation in vitro. This finding is consistent with our observations that relative to wild-type cells, trm2Δ cell growth and transcriptome-wide gene expression are less perturbed by translocation inhibitors. Together our data suggest a model in which m5U54 acts as an important modulator of tRNA maturation and translocation of the ribosome during protein synthesis.

Mechanical Loading Modulates AMPK and mTOR Signaling in Muscle Cells.

Skeletal muscle adaptation to exercise involves various phenotypic changes that enhance the metabolic and contractile functions. One key regulator of these adaptive responses is the activation of AMPK, which is influenced by exercise intensity. However, the mechanistic understanding of AMPK activation during exercise remains incomplete. In this study, we utilized an in vitro model to investigate the effects of mechanical loading on AMPK activation and its interaction with the mTOR signaling pathway. Proteomic analysis of muscle cells subjected to static loading (SL) revealed distinct quantitative protein alterations associated with RNA metabolism, with 10% SL inducing the most pronounced response compared to lower intensities of 5% and 2% as well as the control. Additionally, 10% SL suppressed RNA and protein synthesis while activating AMPK and inhibiting the mTOR pathway. We also found that SRSF2, necessary for pre-mRNA splicing, is regulated by AMPK and mTOR signaling, which, in turn, is regulated in an intensity-dependent manner by SL with the highest expression in 2% SL. Further examination showed that the ADP/ATP ratio was increased after 10% SL compared to the control and that SL induced changes in mitochondrial biogenesis. Furthermore, Seahorse assay results indicate that 10% SL enhances mitochondrial respiration. These findings provide novel insights into the cellular responses to mechanical loading and shed light on the intricate AMPK-mTOR regulatory network in muscle cells.

Effect of Oral Skim Milk Administration on Skeletal Muscle Protein Synthesis after Total Gastrectomy in Rat.

Leucine is a branched-chain amino acid that is present in protein, and it is an essential factor in activating the mechanistic target of the rapamycin complex 1 signaling pathway and increasing muscle protein synthesis. However, the loss of digestive function after total gastrectomy leads to impaired protein absorption, potentially failing to stimulate muscle protein synthesis. Therefore, this study aimed to investigate whether muscle protein synthesis is enhanced by oral skim milk administration after total gastrectomy. Male Sprague Dawley rats were divided into total gastrectomy (TG) and sham surgery (S) groups. After five weeks postoperatively, we orally administered skim milk to achieve 3.1 g protein/kg body weight and collected blood and gastrocnemius muscle. The gastrocnemius muscle weight was significantly lower in the TG group than in the S group (p < 0.05). The increase in plasma leucine concentration was significantly lower in the TG group than in the S group (p < 0.05). The skeletal muscle protein synthesis and the phosphorylation of p70S6K and 4E-BP1 showed a similar increase in both groups. Even after TG, muscle protein synthesis was stimulated by consuming skim milk, accompanied by a sufficient rise in plasma leucine concentration.

Importance of Energy, Dietary Protein Sources, and Amino Acid Composition in the Regulation of Metabolism: An Indissoluble Dynamic Combination for Life.

PURPOSE: This paper aims to present a unique perspective that emphasizes the intricate interplay between energy, dietary proteins, and amino acid composition, underscoring their mutual dependence for health-related considerations. Energy and protein synthesis are fundamental to biological processes, crucial for the sustenance of life and the growth of organisms. METHODS AND RESULTS: We explore the intricate relationship between energy metabolism, protein synthesis, regulatory mechanisms, protein sources, amino acid availability, and autophagy in order to elucidate how these elements collectively maintain cellular homeostasis. We underscore the vital role this dynamic interplay has in preserving cell life. CONCLUSIONS: A deeper understanding of the link between energy and protein synthesis is essential to comprehend fundamental cellular processes. This insight could have a wide-ranging impact in several medical fields, such as nutrition, metabolism, and disease management.

Liriodendrin stimulates proliferation and milk protein synthesis of mammary epithelial cells via the PI3K-DDX18 signaling.

Liriodendrin is a lignan compound that is involved in a wide variety of physiological functions, however it is unknown whether liriodendrin plays an important role in milk production in the mammary glands. In this study, we explored the role and molecular mechanism of Liriodendrin in milk synthesis of mammary epithelial cells (MECs). Bovine MECs were treated with liriodendrin (0, 0.45, 0.9, 1.35, 1.8, and 2.25 mM) for 24 h. Liriodendrin dose-dependently increased cell number, cell cycle transition, and milk protein synthesis, as well as Cyclin D1 and mTOR phosphorylation, with the maximal effects observed at a dose of 1.35 mM. Liriodendrin increased the expression of DDX18, which mediated liriodendrin stimulation of Cyclin D1 and mTOR mRNA expression. PI3K inhibition and DDX18 knockdown experiments further confirmed that liriodendrin regulates the mRNA expression of Cyclin D1 and mTOR via the PI3K-DDX18 signaling. Mouse feeding experiment showed that liriodendrin dose-dependently promotes ß-casein and DDX18 expression in mouse mammary gland. In this study, DDX18 was found to be a novel positive regulator that plays a role in cell proliferation and synthesis of milk protein. These findings reveal that liriodendrin stimulates proliferation and milk protein synthesis of MECs via the PI3K-DDX18 signaling.

The Effect of Plant-Based Protein Ingestion on Athletic Ability in Healthy People-A Bayesian Meta-Analysis with Systematic Review of Randomized Controlled Trials.

Plant-based protein supplements are increasingly popular, yet their efficacy in enhancing athletic performance compared to animal protein, insect protein, or other protein types remains under investigation. This study aimed to assess the effectiveness of plant-based protein on athletic abilities such as muscle strength, endurance performance, and muscle protein synthesis (MPS) rate and compare it to no- or low-protein ingestion and non-plant protein sources. Randomized controlled trials (RCTs) evaluating the beneficial and harmful effects of plant-based protein ingestion on athletic ability in healthy individuals were considered. A systematic search of six databases yielded 2152 studies, which were screened using the Covidence systematic review tool. Thirty-one studies were included for meta-analysis after independent selection, data extraction, and risk of bias assessment by two reviewers. The meta-analysis employed a Bayesian approach using the Markov chain Monte Carlo (MCMC) method through a random-effects model. The results demonstrated that plant-based protein supplements provided greater benefits for athletic performance in healthy individuals compared to the no- or low-protein ingestion group [µ(SMD): 0.281, 95% CI: 0.159 to 0.412; heterogeneity τ: 0.18, 95% CI: 0.017 to 0.362]. However, when compared to other types of protein, plant-based protein ingestion was less effective in enhancing athletic ability [µ(SMD): -0.119, 95% CI: -0.209 to -0.028; heterogeneity τ: 0.076, 95% CI: 0.003 to 0.192]. A subgroup analysis indicated significant improvements in muscle strength and endurance performance in both young and older individuals consuming plant-based protein compared to those with no- or low-protein ingestion. Nonetheless, other protein types showed greater benefits in muscle strength compared to plant-based protein [µ(SMD): -0.133, 95% CI: -0.235 to -0.034; heterogeneity τ: 0.086, 95% CI: 0.004 to 0.214]. In conclusion, while plant-based protein ingestion demonstrates superior efficacy compared to low- or no-protein ingestion, it is not as effective as other protein types such as whey, beef, or milk protein in enhancing athletic performance in healthy individuals. Registration: Registered at the International Prospective Register of Systematic Reviews (PROSPERO) (identification code CRD42024555804).