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Bio-protocol May 2024Ribosomes are an archetypal ribonucleoprotein assembly. Due to ribosomal evolution and function, r-proteins share specific physicochemical similarities, making the...
Ribosomes are an archetypal ribonucleoprotein assembly. Due to ribosomal evolution and function, r-proteins share specific physicochemical similarities, making the riboproteome particularly suited for tailored proteome profiling methods. Moreover, the structural proteome of ribonucleoprotein assemblies reflects context-dependent functional features. Thus, characterizing the state of riboproteomes provides insights to uncover the context-dependent functionality of r-protein rearrangements, as they relate to what has been termed the ribosomal code, a concept that parallels that of the histone code, in which chromatin rearrangements influence gene expression. Compared to high-resolution ribosomal structures, omics methods lag when it comes to offering customized solutions to close the knowledge gap between structure and function that currently exists in riboproteomes. Purifying the riboproteome and subsequent shot-gun proteomics typically involves protein denaturation and digestion with proteases. The results are relative abundances of r-proteins at the ribosome population level. We have previously shown that, to gain insight into the stoichiometry of individual proteins, it is necessary to measure by proteomics bound r-proteins and normalize their intensities by the sum of r-protein abundances per ribosomal complex, i.e., 40S or 60S subunits. These calculations ensure that individual r-protein stoichiometries represent the fraction of each family/paralog relative to the complex, effectively revealing which r-proteins become substoichiometric in specific physiological scenarios. Here, we present an optimized method to profile the riboproteome of any organism as well as the synthesis rates of r-proteins determined by stable isotope-assisted mass spectrometry. Our method purifies the r-proteins in a reversibly denatured state, which offers the possibility for combined top-down and bottom-up proteomics. Our method offers a milder native denaturation of the r-proteome via a chaotropic GuHCl solution as compared with previous studies that use irreversible denaturation under highly acidic conditions to dissociate rRNA and r-proteins. As such, our method is better suited to conserve post-translational modifications (PTMs). Subsequently, our method carefully considers the amino acid composition of r-proteins to select an appropriate protease for digestion. We avoid non-specific protease cleavage by increasing the pH of our standardized r-proteome dilutions that enter the digestion pipeline and by using a digestion buffer that ensures an optimal pH for a reliable protease digestion process. Finally, we provide the R package ProtSynthesis to study the fractional synthesis rates of r-proteins. The package uses physiological parameters as input to determine peptide or protein fractional synthesis rates. Once the physiological parameters are measured, our equations allow a fair comparison between treatments that alter the biological equilibrium state of the system under study. Our equations correct peptide enrichment using enrichments in soluble amino acids, growth rates, and total protein accumulation. As a means of validation, our pipeline fails to find "false" enrichments in non-labeled samples while also filtering out proteins with multiple unique peptides that have different enrichment values, which are rare in our datasets. These two aspects reflect the accuracy of our tool. Our method offers the possibility of elucidating individual r-protein family/paralog abundances, PTM status, fractional synthesis rates, and dynamic assembly into ribosomal complexes if top-down and bottom-up proteomic approaches are used concomitantly, taking one step further into mapping the native and dynamic status of the r-proteome onto high-resolution ribosome structures. In addition, our method can be used to study the proteomes of all macromolecular assemblies that can be purified, although purification is the limiting step, and the efficacy and accuracy of the proteases may be limited depending on the digestion requirements. Key features • Efficient purification of the ribosomal proteome: streamlined procedure for the specific purification of the ribosomal proteome or complex Ome. • Accurate calculation of fractional synthesis rates: robust method for calculating fractional protein synthesis rates in macromolecular complexes under different physiological steady states. • Holistic ribosome methodology focused on plants: comprehensive approach that provides insights into the ribosomes and translational control of plants, demonstrated using cold acclimation [1]. • Tailored strategies for stable isotope labeling in plants: methodology focusing on materials and labeling considerations specific to free and proteinogenic amino acid analysis [2].
PubMed: 38737506
DOI: 10.21769/BioProtoc.4981 -
Structure (London, England : 1993) May 2024Immunogenetic studies have shown that specific HLA-B residues (67, 70, 97, and 156) mediate the impact of HLA class I on HIV infection, but the molecular basis is not...
Immunogenetic studies have shown that specific HLA-B residues (67, 70, 97, and 156) mediate the impact of HLA class I on HIV infection, but the molecular basis is not well understood. Here we evaluate the function of these residues within the protective HLA-B5701 allele. While mutation of Met67, Ser70, and Leu156 disrupt CD8 T cell recognition, substitution of Val97 had no significant impact. Thermal denaturation of HLA-B5701-peptide complexes revealed that Met67 and Leu156 maintain HLA-peptide stability, while Ser70 and Leu156 facilitate T cell receptor (TCR) interactions. Analyses of existing structures and structural models suggested that Val97 mediates HLA-peptide binding to inhibitory KIR3DL1 molecules, which was confirmed by experimental assays. These data thereby demonstrate that the genetic basis by which host immunity impacts HIV outcomes occurs by modulating HLA-B-peptide stability and conformation for interaction with TCR and killer immunoglobulin receptor (KIR) molecules. Moreover, they indicate a key role for epitope specificity and HLA-KIR interactions to HIV control.
PubMed: 38733995
DOI: 10.1016/j.str.2024.04.015 -
Bioorganic Chemistry Jul 2024Spectroscopic, biochemical, and computational modelling studies have been used to assess the binding capability of a set of minor groove binding (MGB) ligands against...
Spectroscopic, biochemical, and computational modelling studies have been used to assess the binding capability of a set of minor groove binding (MGB) ligands against the self-complementary DNA sequences 5'-d(CGCACTAGTGCG)-3' and 5'-d(CGCAGTACTGCG)-3'. The ligands were carefully designed to target the DNA response element, 5'-WGWWCW-3', the binding site for several nuclear receptors. Basic 1D H NMR spectra of the DNA samples prepared with three MGB ligands show subtle variations suggestive of how each ligand associates with the double helical structure of both DNA sequences. The variations among the investigated ligands were reflected in the line shape and intensity of 1D H and P-{H} NMR spectra. Rapid visual inspection of these 1D NMR spectra proves to be beneficial in providing valuable insights on MGB binding molecules. The NMR results were consistent with the findings from both UV DNA denaturation and molecular modelling studies. Both the NMR spectroscopic and computational analyses indicate that the investigated ligands bind to the minor grooves as antiparallel side-by-side dimers in a head-to-tail fashion. Moreover, comparisons with results from biochemical studies offered valuable insights into the mechanism of action, and antitumor activity of MGBs in relation to their structures, essential pre-requisites for future optimization of MGBs as therapeutic agents.
Topics: DNA; Ligands; Humans; Antineoplastic Agents; Molecular Structure; Nucleic Acid Conformation; Binding Sites; Structure-Activity Relationship; Models, Molecular; Dose-Response Relationship, Drug; Magnetic Resonance Spectroscopy; Cell Line, Tumor
PubMed: 38733748
DOI: 10.1016/j.bioorg.2024.107414 -
The Journal of Physical Chemistry. B May 2024The disaccharide trehalose is generally acknowledged as a superior stabilizer of proteins and other biomolecules in aqueous environments. Despite many theories aiming to... (Comparative Study)
Comparative Study
The disaccharide trehalose is generally acknowledged as a superior stabilizer of proteins and other biomolecules in aqueous environments. Despite many theories aiming to explain this, the stabilization mechanism is still far from being fully understood. This study compares the stabilizing properties of trehalose with those of the structurally similar disaccharide sucrose. The stability has been evaluated for the two proteins, lysozyme and myoglobin, at both low and high temperatures by determining the glass transition temperature, , and the denaturation temperature, . The results show that the sucrose-containing samples exhibit higher than the corresponding trehalose-containing samples, particularly at low water contents. The better stabilizing effect of sucrose at high temperatures may be explained by the fact that sucrose, to a greater extent, binds directly to the protein surface compared to trehalose. Both sugars show elevation with an increasing sugar-to-protein ratio, which allows for a more complete sugar shell around the protein molecules. Finally, no synergistic effects were found by combining trehalose and sucrose. Conclusively, the exact mechanism of protein stabilization may vary with the temperature, as influenced by temperature-dependent interactions between the protein, sugar, and water. This variability can make trehalose to a superior stabilizer under some conditions and sucrose under others.
Topics: Trehalose; Sucrose; Muramidase; Calorimetry, Differential Scanning; Myoglobin; Protein Stability; Animals; Temperature
PubMed: 38733344
DOI: 10.1021/acs.jpcb.4c00022 -
Polymers Apr 2024Meat quality and shelf life are important parameters affecting consumer perception and safety. Several factors contribute to the deterioration and spoilage of meat... (Review)
Review
Meat quality and shelf life are important parameters affecting consumer perception and safety. Several factors contribute to the deterioration and spoilage of meat products, including microbial growth, chemical reactions in the food's constituents, protein denaturation, lipid oxidation, and discoloration. This study reviewed the development of functional packaging biomaterials that interact with food and the environment to improve food's sensory properties and consumer safety. Bioactive packaging incorporates additive compounds such as essential oils, natural extracts, and chemical substances to produce composite polymers and polymer blends. The findings showed that the incorporation of additive compounds enhanced the packaging's functionality and improved the compatibility of the polymer-polymer matrices and that between the polymers and active compounds. Food preservatives are alternative substances for food packaging that prevent food spoilage and preserve quality. The safety of food contact materials, especially the flavor/odor contamination from the packaging to the food and the mass transfer from the food to the packaging, was also assessed. Flavor is a key factor in consumer purchasing decisions and also determines the quality and safety of meat products. Novel functional packaging can be used to preserve the quality and safety of packaged meat products.
PubMed: 38732702
DOI: 10.3390/polym16091232 -
International Journal of Molecular... May 2024Today, allergies have become a serious problem. PR-10 proteins are clinically relevant allergens that have the ability to bind hydrophobic ligands, which can...
Today, allergies have become a serious problem. PR-10 proteins are clinically relevant allergens that have the ability to bind hydrophobic ligands, which can significantly increase their allergenicity potential. It has been recently shown that not only the birch pollen allergen Bet v 1 but also the alder pollen allergen Aln g 1, might act as a true sensitizer of the immune system. The current investigation is aimed at the further study of the allergenic and structural features of Aln g 1. By using qPCR, we showed that Aln g 1 was able to upregulate alarmins in epithelial cells, playing an important role in sensitization. With the use of CD-spectroscopy and ELISA assays with the sera of allergic patients, we demonstrated that Aln g 1 did not completely restore its structure after thermal denaturation, which led to a decrease in its IgE-binding capacity. Using site-directed mutagenesis, we revealed that the replacement of two residues (Asp27 and Leu30) in the structure of Aln g 1 led to a decrease in its ability to bind to both IgE from sera of allergic patients and lipid ligands. The obtained data open a prospect for the development of hypoallergenic variants of the major alder allergen Aln g 1 for allergen-specific immunotherapy.
Topics: Humans; Pollen; Allergens; Antigens, Plant; Immunoglobulin E; Plant Proteins; Alnus
PubMed: 38732184
DOI: 10.3390/ijms25094965 -
International Journal of Molecular... Apr 2024Yamogenin is a steroidal saponin occurring in plant species such as , , , and sp. In this study, we evaluated in vitro cytotoxic, antioxidant, and antimicrobial...
An In Vitro Study on the Cytotoxic, Antioxidant, and Antimicrobial Properties of Yamogenin-A Plant Steroidal Saponin and Evaluation of Its Mechanism of Action in Gastric Cancer Cells.
Yamogenin is a steroidal saponin occurring in plant species such as , , , and sp. In this study, we evaluated in vitro cytotoxic, antioxidant, and antimicrobial properties of yamogenin. The cytotoxic activity was estimated on human colon cancer HCT116, gastric cancer AGS, squamous carcinoma UM-SCC-6 cells, and human normal fibroblasts with MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay. The amount of apoptotic and dead AGS cells after treatment with yamogenin was estimated with flow cytometry. Also, in yamogenin-treated AGS cells we investigated the reactive oxygen species (ROS) production, mitochondrial membrane depolarization, activity level of caspase-8 and -9, and gene expression at mRNA level with flow cytometry, luminometry, and RT-PCR, respectively. The antioxidant properties of yamogenin were assessed with DPPH (2,2-diphenyl-1-picrylhydrazyl) and ABTS (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) assays. The antimicrobial potential of the compound was estimated on , , , , , , , , , , and bacteria strains. Yamogenin showed the strongest cytotoxic effect on AGS cells (IC 18.50 ± 1.24 µg/mL) among the tested cell lines. This effect was significantly stronger in combinations of yamogenin with oxaliplatin or capecitabine than for the single compounds. Furthermore, yamogenin induced ROS production, depolarized mitochondrial membrane, and increased the activity level of caspase-8 and -9 in AGS cells. RT-PCR analysis revealed that this sapogenin strongly up-regulated expression at the mRNA level. These results indicate that yamogenin induced cell death via the extrinsic and intrinsic way of apoptosis. Antioxidant study showed that yamogenin had moderate in vitro potential (IC 704.7 ± 5.9 µg/mL in DPPH and 631.09 ± 3.51 µg/mL in ABTS assay) as well as the inhibition of protein denaturation properties (with IC 1421.92 ± 6.06 µg/mL). Antimicrobial test revealed a weak effect of yamogenin on bacteria strains, the strongest one being against (with MIC value of 350 µg/mL). In conclusion, yamogenin may be a potential candidate for the treatment and prevention of gastric cancers.
Topics: Humans; Antioxidants; Saponins; Stomach Neoplasms; Cell Line, Tumor; Apoptosis; Reactive Oxygen Species; Anti-Infective Agents; Membrane Potential, Mitochondrial; Plant Extracts; Antineoplastic Agents, Phytogenic
PubMed: 38731847
DOI: 10.3390/ijms25094627 -
International Journal of Molecular... Apr 2024leaves are rich sources of bioactive compounds with potential health benefits, including antioxidants and anti-inflammatory agents. Pressurized liquid extraction (PLE)...
leaves are rich sources of bioactive compounds with potential health benefits, including antioxidants and anti-inflammatory agents. Pressurized liquid extraction (PLE) stands out as a promising technique for effectively extracting valuable compounds from natural sources. In this study, we aimed to optimize PLE parameters, such as temperature, extraction duration, and pressure, to maximize bioactive compound (polyphenols, flavonoids, and ascorbic acid) yield from leaves and evaluate their antioxidant and anti-inflammatory activities. According to the outcomes of this research, the maximum achieved total polyphenol content was 24.10 mg gallic acid equivalents (GAE)/g of dry weight (dw), and the total flavonoid content was increased up to 19.89 mg rutin equivalents (RtE)/g dw. Moreover, after HPLC-DAD analysis, neochlorogenic and chlorogenic acids, catechin and epicatechin, rutin, and narirutin were identified and quantified. As far as the optimum ascorbic acid content is concerned, it was found to be 4.77 mg/g dw. The antioxidant activity was evaluated by three different methods: ferric reducing antioxidant power (FRAP), the DPPH method, and the anti-hydrogen peroxide activity (AHPA) method, resulting in 124.29 μmol ascorbic acid equivalent (AAE)/g dw, 131.28 μmol AAE/g dw, and 229.38 μmol AAE/g dw values, respectively. Lastly, the albumin denaturation inhibition was found to be 37.54%. These findings underscore the potential of PLE as an efficient extraction method for preparing extracts from leaves with the maximum content of bioactive compounds.
Topics: Moringa oleifera; Plant Leaves; Antioxidants; Plant Extracts; Flavonoids; Polyphenols; Ascorbic Acid; Anti-Inflammatory Agents; Chromatography, High Pressure Liquid; Pressure; Liquid-Liquid Extraction; Phytochemicals
PubMed: 38731845
DOI: 10.3390/ijms25094628 -
Foods (Basel, Switzerland) Apr 2024Antifreeze peptides have become effective antifreeze agents for frozen products, but their low quantity of active ingredients and high cost limit large-scale...
Antifreeze peptides have become effective antifreeze agents for frozen products, but their low quantity of active ingredients and high cost limit large-scale application. This study used the glycosylation of fish collagen peptides with glucosamine hydrochloride catalyzed by transglutaminase to obtain a transglutaminase-catalyzed glycosylation product (TGP) and investigate its antifreeze effect on tilapia. Compared with the blank group, the freshness (pH value of 6.31, TVB-N value of 21.7 mg/100 g, whiteness of 46.28), textural properties (especially hardness and elasticity), and rheological properties of the TGP groups were significantly improved. In addition, the protein structures of the samples were investigated using UV absorption and fluorescence spectroscopy. The results showed that the tertiary structure of the TGP groups changed to form a dense polymer. Therefore, this approach can reduce the denaturation and decomposition of muscle fibers and proteins in fish meat more effectively and has a better protective effect on muscle structure and protein aggregation, improving the stability of fish meat. This study reveals an innovative method for generating antifreeze peptides by enzymatic glycosylation, and glycosylated fish collagen peptide products can be used as new and effective green antifreeze agents in frozen foods.
PubMed: 38731690
DOI: 10.3390/foods13091319 -
BMC Bioinformatics May 2024Surveillance for genetic variation of microbial pathogens, both within and among species, plays an important role in informing research, diagnostic, prevention, and...
Surveillance for genetic variation of microbial pathogens, both within and among species, plays an important role in informing research, diagnostic, prevention, and treatment activities for disease control. However, large-scale systematic screening for novel genotypes remains challenging in part due to technological limitations. Towards addressing this challenge, we present an advancement in universal microbial high resolution melting (HRM) analysis that is capable of accomplishing both known genotype identification and novel genotype detection. Specifically, this novel surveillance functionality is achieved through time-series modeling of sequence-defined HRM curves, which is uniquely enabled by the large-scale melt curve datasets generated using our high-throughput digital HRM platform. Taking the detection of bacterial genotypes as a model application, we demonstrate that our algorithms accomplish an overall classification accuracy over 99.7% and perform novelty detection with a sensitivity of 0.96, specificity of 0.96 and Youden index of 0.92. Since HRM-based DNA profiling is an inexpensive and rapid technique, our results add support for the feasibility of its use in surveillance applications.
Topics: Machine Learning; Genotype; DNA, Bacterial; Algorithms; Nucleic Acid Denaturation
PubMed: 38730317
DOI: 10.1186/s12859-024-05747-0