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Journal of Proteomics May 2024Optimization of broiler chicken breast muscle protein accretion is key for the efficient production of poultry meat, whose demand is steadily increasing. In a context...
Optimization of broiler chicken breast muscle protein accretion is key for the efficient production of poultry meat, whose demand is steadily increasing. In a context where antimicrobial growth promoters use is being restricted, it is important to find alternatives as well as to characterize the effect of immunological stress on broiler chicken's growth. Despite its importance, research on broiler chicken muscle protein dynamics has mostly been limited to the study of mixed protein turnover. The present study aims to characterize the effect of a bacterial challenge and the feed supplementation of citrus and cucumber extracts on broiler chicken individual breast muscle proteins fractional synthesis rates (FSR) using a recently developed dynamic proteomics pipeline. Twenty-one day-old broiler chickens were administered a single HO dose before being culled at different timepoints. A total of 60 breast muscle protein extracts from five experimental groups (Unchallenged, Challenged, Control Diet, Diet 1 and Diet 2) were analysed using a DDA proteomics approach. Proteomics data was filtered in order to reliably calculate multiple proteins FSR making use of a newly developed bioinformatics pipeline. Broiler breast muscle proteins FSR uniformly decreased following a bacterial challenge, this change was judged significant for 15 individual proteins, the two major functional clusters identified as well as for mixed breast muscle protein. Citrus or cucumber extract feed supplementation did not show any effect on the breast muscle protein FSR of immunologically challenged broilers. The present study has identified potential predictive markers of breast muscle growth and provided new information on broiler chicken breast muscle protein synthesis which could be essential for improving the efficiency of broiler chicken meat production. SIGNIFICANCE: The present study constitutes the first dynamic proteomics study conducted in a farm animal species which has characterized FSR in a large number of proteins, establishing a precedent for biomarker discovery and assessment of health and growth status. Moreover, it has been evidenced that the decrease in broiler chicken breast muscle protein following an immune challenge is a coordinated event which seems to be the main cause of the decreased growth observed in these animals.
Topics: Animals; Chickens; Muscle Proteins; Dietary Supplements; Diet; Muscles; Animal Feed; Meat
PubMed: 38484873
DOI: 10.1016/j.jprot.2024.105158 -
Science Advances Mar 2024Cancer diagnosis by metabolic MRI proposes to follow the fate of glycolytic precursors such as pyruvate or glucose, and their in vivo conversion into lactate. This study...
Cancer diagnosis by metabolic MRI proposes to follow the fate of glycolytic precursors such as pyruvate or glucose, and their in vivo conversion into lactate. This study compares the H MRI outlooks afforded by these metabolites when targeting a pancreatic cancer model. Exogenously injected [3,3',3″-H]-pyruvate was visible only briefly; it generated a deuterated lactate signal throughout the body that faded after ~5 min, showing a minor concentration bias at the rims of the tumors. [6,6'-H]-glucose by contrast originated a lactate signal that localized clearly within the tumors, persisting for over an hour. Investigations alternating deuterated and nondeuterated glucose injections revealed correlations between the lactate generation and the glucose available at the tumor, evidencing a continuous and avid glucose consumption generating well-localized lactate signatures as driven by the Warburg effect. This is by contrast to the transient and more promiscuous pyruvate-to-lactate transformation, which seemed subject to transporter and kinetics effects. The consequences of these observations within metabolic MRI are briefly discussed.
Topics: Humans; Pyruvic Acid; Deuterium; Magnetic Resonance Spectroscopy; Glucose; Magnetic Resonance Imaging; Pancreatic Neoplasms; Lactic Acid; Molecular Imaging
PubMed: 38478615
DOI: 10.1126/sciadv.adm8600 -
International Journal of Molecular... Mar 2024Liquid chromatography with mass spectrometry (LC-MS)-based metabolomics detects thousands of molecular features (retention time-/ pairs) in biological samples per...
Liquid chromatography with mass spectrometry (LC-MS)-based metabolomics detects thousands of molecular features (retention time-/ pairs) in biological samples per analysis, yet the metabolite annotation rate remains low, with 90% of signals classified as unknowns. To enhance the metabolite annotation rates, researchers employ tandem mass spectral libraries and challenging in silico fragmentation software. Hydrogen/deuterium exchange mass spectrometry (HDX-MS) may offer an additional layer of structural information in untargeted metabolomics, especially for identifying specific unidentified metabolites that are revealed to be statistically significant. Here, we investigate the potential of hydrophilic interaction liquid chromatography (HILIC)-HDX-MS in untargeted metabolomics. Specifically, we evaluate the effectiveness of two approaches using hypothetical targets: the post-column addition of deuterium oxide (DO) and the on-column HILIC-HDX-MS method. To illustrate the practical application of HILIC-HDX-MS, we apply this methodology using the in silico fragmentation software MS-FINDER to an unknown compound detected in various biological samples, including plasma, serum, tissues, and feces during HILIC-MS profiling, subsequently identified as -acetylspermidine.
Topics: Deuterium; Chromatography, Liquid; Metabolomics; Hydrogen Deuterium Exchange-Mass Spectrometry; Hydrophobic and Hydrophilic Interactions
PubMed: 38474147
DOI: 10.3390/ijms25052899 -
European Radiology Experimental Mar 2024Magnetic resonance (MR) imaging of deuterated glucose, termed deuterium metabolic imaging (DMI), is emerging as a biomarker of pathway-specific glucose metabolism in...
BACKGROUND
Magnetic resonance (MR) imaging of deuterated glucose, termed deuterium metabolic imaging (DMI), is emerging as a biomarker of pathway-specific glucose metabolism in tumors. DMI is being studied as a useful marker of treatment response in a scan-rescan scenario. This study aims to evaluate the repeatability of brain DMI.
METHODS
A repeatability study was performed in healthy volunteers from December 2022 to March 2023. The participants consumed 75 g of [6,6'-H]glucose. The delivery of H-glucose to the brain and its conversion to H-glutamine + glutamate, H-lactate, and H-water DMI was imaged at baseline and at 30, 70, and 120 min. DMI was performed using MR spectroscopic imaging on a 3-T system equipped with a H/H-tuned head coil. Coefficients of variation (CoV) were computed for estimation of repeatability and between-subject variability. In a set of exploratory analyses, the variability effects of region, processing, and normalization were estimated.
RESULTS
Six male participants were recruited, aged 34 ± 6.5 years (mean ± standard deviation). There was 42 ± 2.7 days between sessions. Whole-brain levels of glutamine + glutamate, lactate, and glucose increased to 3.22 ± 0.4 mM, 1.55 ± 0.3 mM, and 3 ± 0.7 mM, respectively. The best signal-to-noise ratio and repeatability was obtained at the 120-min timepoint. Here, the within-subject whole-brain CoVs were -10% for all metabolites, while the between-subject CoVs were -20%.
CONCLUSIONS
DMI of glucose and its downstream metabolites is feasible and repeatable on a clinical 3 T system.
TRIAL REGISTRATION
ClinicalTrials.gov, NCT05402566 , registered the 25th of May 2022.
RELEVANCE STATEMENT
Brain deuterium metabolic imaging of healthy volunteers is repeatable and feasible at clinical field strengths, enabling the study of shifts in tumor metabolism associated with treatment response.
KEY POINTS
• Deuterium metabolic imaging is an emerging tumor biomarker with unknown repeatability. • The repeatability of deuterium metabolic imaging is on par with FDG-PET. • The study of deuterium metabolic imaging in clinical populations is feasible.
Topics: Humans; Male; Deuterium; Glucose; Glutamates; Glutamine; Healthy Volunteers; Lactates; Adult
PubMed: 38472611
DOI: 10.1186/s41747-024-00426-4 -
Nature Communications Mar 2024We present a hydrogen/deuterium exchange workflow coupled to tandem mass spectrometry (HX-MS) that supports the acquisition of peptide fragment ions alongside their...
We present a hydrogen/deuterium exchange workflow coupled to tandem mass spectrometry (HX-MS) that supports the acquisition of peptide fragment ions alongside their peptide precursors. The approach enables true auto-curation of HX data by mining a rich set of deuterated fragments, generated by collisional-induced dissociation (CID), to simultaneously confirm the peptide ID and authenticate MS-based deuteration calculations. The high redundancy provided by the fragments supports a confidence assessment of deuterium calculations using a combinatorial strategy. The approach requires data-independent acquisition (DIA) methods that are available on most MS platforms, making the switch to HX-MS straightforward. Importantly, we find that HX-DIA enables a proteomics-grade approach and wide-spread applications. Considerable time is saved through auto-curation and complex samples can now be characterized and at higher throughput. We illustrate these advantages in a drug binding analysis of the ultra-large protein kinase DNA-PKcs, isolated directly from mammalian cells.
Topics: Animals; Deuterium; Deuterium Exchange Measurement; Hydrogen; Tandem Mass Spectrometry; Peptides; Mammals
PubMed: 38467655
DOI: 10.1038/s41467-024-46610-3 -
RSC Advances Mar 2024The incorporation of deuterium into peptides and proteins holds broad applications across various fields, such as drug development and structural characterization....
The incorporation of deuterium into peptides and proteins holds broad applications across various fields, such as drug development and structural characterization. Nevertheless, current methods for peptide/protein deuteration often target exchangeable labile sites or require harsh conditions for stable modification. In this study, we present a late-stage approach utilizing an alkaline phosphate solution to achieve deuteration of non-exchangeable backbone sites of peptides and proteins. The specific deuteration regions are identified through ultraviolet photodissociation (UVPD) and mass spectrometry analysis. This deuteration strategy demonstrates site and structure selectivity, with a notable affinity for labeling the α-helix regions of myoglobin. The deuterium method is particularly suitable for peptides and proteins that remain stable under high pH conditions.
PubMed: 38464689
DOI: 10.1039/d3ra08636d -
BioRxiv : the Preprint Server For... Feb 2024The G protein-coupled metabotropic glutamate receptors form homodimers and heterodimers with highly diverse responses to glutamate and varying physiological function....
The G protein-coupled metabotropic glutamate receptors form homodimers and heterodimers with highly diverse responses to glutamate and varying physiological function. The molecular basis for this diversity remains poorly delineated. We employ molecular dynamics, single-molecule spectroscopy, and hydrogen-deuterium exchange to dissect the pathway of activation triggered by glutamate. We find that activation entails multiple loosely coupled steps and identify a novel pre-active intermediate whose transition to the active state forms dimer interactions that set signaling efficacy. Such subunit interactions generate functional diversity that differs across homodimers and heterodimers. The agonist-bound receptor is remarkably dynamic, with low occupancy of G protein-coupling conformations, providing considerable headroom for modulation of the landscape by allosteric ligands. Sites of sequence diversity within the dimerization interface and diverse coupling between activation rearrangements may contribute to precise decoding of glutamate signals and transients over broad spatial and temporal scales.
PubMed: 38464305
DOI: 10.1101/2024.02.28.582567 -
BioRxiv : the Preprint Server For... Feb 2024Magnesium (Mg) uptake systems are present in all domains of life given the vital role of this ion. Bacteria acquire Mg via conserved Mg channels and transporters. The...
Magnesium (Mg) uptake systems are present in all domains of life given the vital role of this ion. Bacteria acquire Mg via conserved Mg channels and transporters. The transporters are required for growth when Mg is limiting or during bacterial pathogenesis, but, despite their significance, there are no known structures for these transporters. Here we report the first structure of the Mg transporter MgtA solved by single particle cryo-electron microscopy (cryo-EM). Using mild membrane extraction, we obtained high resolution structures of both a homodimeric form (2.9 Å), the first for a P-type ATPase, and a monomeric form (3.6 Å). Each monomer unit of MgtA displays a structural architecture that is similar to other P-type ATPases with a transmembrane domain and two soluble domains. The dimer interface consists of contacts between residues in adjacent soluble nucleotide binding and phosphotransfer regions of the haloacid dehalogenase (HAD) domain. We suggest oligomerization is a conserved structural feature of the diverse family of P-type ATPase transporters. The ATP binding site and conformational dynamics upon nucleotide binding to MgtA were characterized using a combination of cryo-EM, molecular dynamics simulations, hydrogen-deuterium exchange mass spectrometry, and mutagenesis. Our structure also revealed a Mg ion in the transmembrane segments, which, when combined with sequence conservation and mutagenesis studies, allowed us to propose a model for Mg transport across the lipid bilayer. Finally, our work revealed the N-terminal domain structure and cytoplasmic Mg binding sites, which have implications for related P-type ATPases defective in human disease.
PubMed: 38464158
DOI: 10.1101/2024.02.28.582502 -
Biophysical Journal Apr 2024COG0523 proteins, also known as nucleotide-dependent metallochaperones, are a poorly understood class of small P-loop G3E GTPases. Multiple family members play critical...
COG0523 proteins, also known as nucleotide-dependent metallochaperones, are a poorly understood class of small P-loop G3E GTPases. Multiple family members play critical roles in bacterial pathogen survival during an infection as part of the adaptive response to host-mediated "nutritional immunity." Our understanding of the structure, dynamics, and molecular-level function of COG0523 proteins, apart from the eukaryotic homolog, Zng1, remains in its infancy. Here, we use X-ray absorption spectroscopy to establish that Acinetobacter baumannii (Ab) ZigA coordinates Zn using all three cysteines derived from the invariant CXCC motif to form an S(N/O) coordination complex, a feature inconsistent with the Zn-bound crystal structure of a distantly related COG0523 protein of unknown function from Escherichia coli, EcYjiA. The binding of Zn and guanine nucleotides is thermodynamically linked in AbZigA, and this linkage is more favorable for the substrate GTP relative to the product GDP. Part of this coupling originates with nucleotide-induced stabilization of the G-domain tertiary structure as revealed by global thermodynamics measurements and hydrogen-deuterium exchange mass spectrometry (HDX-MS). HDX-MS also reveals that the HDX behavior of the G2 (switch 1) loop is highly sensitive to nucleotide status and becomes more exchange labile in the GDP (product)-bound state. Significant long-range perturbation of local stability in both the G-domain and the C-terminal domain define a candidate binding pocket for a client protein that appears sensitive to nucleotide status (GDP versus GTP). We place these new insights into the structure, dynamics, and energetics of intermolecular metal transfer into the context of a model for AbZigA metallochaperone function.
Topics: Humans; Zinc; Acinetobacter baumannii; Nucleotides; Bacteria; Guanosine Triphosphate; Protein Binding; Guanosine Diphosphate
PubMed: 38459695
DOI: 10.1016/j.bpj.2024.03.010 -
ELife Mar 2024The physical basis of phase separation is thought to consist of the same types of bonds that specify conventional macromolecular interactions yet is unsatisfyingly often...
The physical basis of phase separation is thought to consist of the same types of bonds that specify conventional macromolecular interactions yet is unsatisfyingly often referred to as 'fuzzy'. Gaining clarity on the biogenesis of membraneless cellular compartments is one of the most demanding challenges in biology. Here, we focus on the chromosome passenger complex (CPC), that forms a chromatin body that regulates chromosome segregation in mitosis. Within the three regulatory subunits of the CPC implicated in phase separation - a heterotrimer of INCENP, Survivin, and Borealin - we identify the contact regions formed upon droplet formation using hydrogen/deuterium exchange mass spectrometry (HXMS). These contact regions correspond to some of the interfaces seen between individual heterotrimers within the crystal lattice they form. A major contribution comes from specific electrostatic interactions that can be broken and reversed through initial and compensatory mutagenesis, respectively. Our findings reveal structural insight for interactions driving liquid-liquid demixing of the CPC. Moreover, we establish HXMS as an approach to define the structural basis for phase separation.
Topics: Phase Separation; Cell Cycle Proteins; Chromosomes; Mitosis; Cytoskeleton; Chromosome Segregation; Aurora Kinase B
PubMed: 38456462
DOI: 10.7554/eLife.92709