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This study evaluated the effects of chemical modification, including ethanol, acetic acid, and natural deep eutectic solvents (NADES), on the secondary and tertiary structures, hydrophobicity, free amine content, protein-protein interactions, and functional properties of zein. The NADES used included choline chloride: oxalic acid, choline chloride: urea, choline chloride: glycerol, and glucose: citric acid. The results reveal that the NADES system significantly altered zein's structures, as evidenced by Fourier transform infrared spectroscopy, fluorescence, and Ultraviolet–Visible Spectroscopy analysis. Circular dichroism spectroscopy analysis indicated significant conformational change in modified zein, with decreased α-helix and increased random coil content. Notably, the NADES system leads to greater disruption of hydrogen bonds and facilitates the exposure of hydrophobic regions compared to water, ethanol, and acetic acid systems. This resulted in enhanced solubility, surface hydrophobicity, and free amine content in zein, indicating a more significant change in protein structure. In contrast, water and acetic acid solvents maintained more stable disulfide bonds within zein, which correlates with lower solubility and less unfolding. The NADES system promoted interactions between zein and its solvent components, improving emulsifying properties. Water, ethanol, and acetic acid systems had higher solubility in urea, thiourea, and dithiothreitol than the NADES system, revealing disruption of both covalent and noncovalent bonds in zein modified by NADES. Overall, this study highlights the superior ability of the NADES system to modify zein's structure and functionality compared to conventional solvents, suggesting its potential for enhancing protein applications in the industrial production of foods.

Hempseed protein isolate (HPI), a novel plant protein, possesses advantages as an alternative food protein from a nutritional and sustainable perspective. This study investigated HPI modification by examining the effects of high-pressure homogenization combined with high-intensity ultrasound (HPH + HIU) on the physicochemical and functionality of HPI. Firstly, the optimal homogenization pressure (180 MPa) was selected based on the solubility and particle size of HPI. Then, the effect of ultrasonic treatment time (2, 5, and 10 min) was studied at the optimal homogenization pressure. The results showed increased solubility of HPI after all treatments. Particularly, the HPH + HIU2min treatment had a synergistic effect that maximumly increased the solubility of HPI from 6.88 % to 22.89 % at neutral pH. This treatment significantly decreased the HPI’s particle size, β-sheet and total sulfhydryl contents while maximizing the random coil level, intrinsic fluorescence intensity and surface hydrophobicity compared to the single HPH or HIU2min treatments. The protein structure was modified and unfolded, enhancing the water-protein and oil-protein interactions, as reflected in the increase in water and oil absorption, foaming and emulsifying properties. However, extending the ultrasonic time to 5 min for the HPH + HIU treatment increased protein particle size and weakened the functional properties of HPI. Further prolonging the ultrasonic time to 10 min partially loosened the protein aggregates and restored the functional properties of HPI to some extent. The findings indicate a promising application of HPH + HIU as an efficient way for HPI modification to facilitate its broader application in the food industry.

Acrolein (ACR) is an α,β-unsaturated aldehyde with high reactivity towards nucleophiles in proteins. In this study, a typical phenolic compound phloretin (Phl) was employed to counteract protein modification induced by ACR (1 mM) in whey protein isolate (WPI, 10 mg/mL). The addition of Phl (2 mM) significantly reduced ACR-induced surge of protein carbonyls (from 1.65 to 0.65 μmol/mg protein) and loss of protein total sulfhydryl content (from 0.28 to 0.24 μmol/mg protein) whilst contributing to further reductions in protein surface hydrophobicity and intrinsic fluorescence. The incorporation of ACR into WPI was effectively interrupted by Phl as visualized by Western blot. Only 2.87 % of ACR remained in the presence of 2 mM Phl with the generation of Phl-ACR adducts, suggesting Phl could partially alleviate protein modification by scavenging of ACR. These findings could have important implications for employment of natural phenolic nucleophiles against the adverse effects of ACR towards dietary proteins.

Faba bean proteins are currently viewed as promising animal protein alternatives. However, certain functional properties e.g. relatively low solubility compared to whey protein isolates, can limit the application of faba bean protein isolates (FPIs) in certain food products. Therefore, it may be desirable to use modification approaches such as the application of ultrasound to alter such limiting physicochemical properties. In this study, Faba Bean Protein Isolates (FPIs) were treated by ultrasound with different frequencies (20 kHz, 40 kHz and 20 + 40 kHz) prior to hydration (1 %) at different pH levels (3, 7, and 9). Then the structure and physicochemical properties (i.e. particle size, ζ-potential, surface hydrophobicity, thermal behavior, and solubility) of control and untreated FPIs were investigated. Ultrasound treatment had no obvious effect on the molecular weight of FPIs, whereas it changed the secondary structure of FPIs from a more ordered structure to a more disordered structure. The applied treatment resulted in an increase in surface hydrophobicity across all treatment levels and pHs. It also decreased the particle size of FPI at pH 3, while it increased the particle size at pH 7 and 9, compared to the untreated FPI. In addition, the solubility and thermal properties of FPI were modified through the ultrasound treatment. The higher solubility of FPI could improve its potential to be used as a functional ingredient for many food applications. Ultrasound treatment at 20 kHz and 20 + 40 kHz had more effects on the physiochemical properties of FPI compared to that at 40 kHz. Overall, ultrasound treatment with different frequencies (20 kHz, 40 kHz, and 20 + 40 kHz) modified the structure and physiochemical properties of FPI to different degrees and may be beneficial for the development of FPI for certain food applications.

为促进花生蛋白的深加工和更广泛的应用，采用高静压联合碱性蛋白酶酶法改性花生蛋白。通过单因素试验考察了静压力、pH、酶添加量、酶解时间和酶解温度对花生蛋白溶解度的影响，在此基础上，采用正交试验优化花生蛋白联合改性工艺条件，并测定了联合改性花生蛋白的起泡性和泡沫稳定性、巯基和二硫键含量以及总还原能力。结果表明：花生蛋白联合改性最佳工艺条件为静压力300 MPa、1 g/100 mL碱性蛋白酶（20万U/g）添加量3.0 mL（100 mL质量分数5%的花生蛋白溶液）、酶解时间60 min、pH 10、酶解温度50 ℃，在此条件下联合改性花生蛋白溶解度为（82.87±0.51）%；联合改性花生蛋白的起泡性、泡沫稳定性、巯基含量、总还原能力显著提高，二硫键含量显著下降。综上，高静压联合酶法改性改善了花生蛋白的理化性质及功能特性，有利于其深加工及更广泛的应用。In order to promote the deep processing and broader application of peanut protein, peanut protein was modified by high hydrostatic pressure combined with alkaline protease hydrolysis. The effects of hydrostatic pressure, pH, enzyme addition amount, enzymatic time and enzymatic temperature on the solubility of peanut protein were investigated by single factor experiment, then orthogonal experiment was used to optimize the modification process of peanut protein. The foaming property and foam stability, sulfhydryl and disulfide bond contents, and total reducing capacity of the co-modified peanut protein were determined. The results showed that the optimal co-modification conditions for peanut protein were hydrostatic pressure 300 MPa, 1 g/100 mL alkaline protease(200 000 U/g) addition amount 3.0 mL (based on 100 mL peanut protein solution with mass fraction of 5%), pH 10, enzymatic temperature 50 ℃, and enzymatic time 60 min, and the protein solubility was (82.87±0.51)% under these conditions. The foaming property and foam stability, sulfhydryl group content, and total reducing capacity of co-modified peanut protein improved, while the disulfide bond content decreased. In conclusion, the combination of high hydrostatic pressure and enzymatic method can improve the physicochemical properties and functional properties of peanut protein, which facilitates the deep processing and broader application of peanut protein.

This study aimed to modify plant protein mixture to improve their functionality and digestibility by limited hydrolysis. Soy protein isolate and corn zein were mixed at the ratio of 5:1 (w/w), followed by limited hydrolysis using papain from 15 to 30 min. The structural characteristics, in vitro digestibility, and functional properties were evaluated. Also, DPPH radical scavenging activity was determined. The results indicated that the molecular weight of different modified samples was largely reduced by limited hydrolysis, and the proportion of random coil was significantly increased. Furthermore, the solubility, foaming, emulsifying and water-holding capacity of hydrolyzed protein mixture were significantly improved, which were close to those of whey protein isolate. In vitro digestibility after 30-min limited hydrolysis was remarkably elevated. In addition, the hydrolyzed protein mixture exhibited a higher antioxidant activity than those of untreated proteins. Overall, limited hydrolysis of protein mixture led to improved digestibility, functionality and antioxidant activity.

Oxidative stress is widely recognized as a key mechanism in the development of hypertension. Under pathological conditions, such as in hypertension, oxidative stress leads to irreversible posttranslational modifications of proteins, which result in loss of protein function and cellular damage. We have previously documented physiological and morphological changes across various blood and bone marrow cell lineages, all of which exhibit elevated oxidative stress. While cytoskeletal changes in erythrocytes have been well characterized in hereditary diseases, this is the first study, to our knowledge, to investigate cytoskeletal reorganization in erythrocytes from hypertensive patients. To this end, we compared the expression patterns and subcellular distribution of key cytoskeletal proteins in erythrocytes from hypertensive individuals with those from normotensive subjects using Western blot, flow cytometry, and confocal microscopy. Our results revealed the presence of three erythrocyte subpopulations with differential expression of glycophorin A. The persistent oxidative environment in hypertensive patients causes dysregulation in the expression of glycophorin A, Band 3 protein, protein 4.1, and ankyrin, as well as the reorganization of spectrin. These alterations in protein expression and distribution suggest that oxidative stress in hypertensive individuals may induce structural modifications, ultimately impairing erythrocyte membrane elasticity and function.

The functionalization of protein sidechains with highly water-soluble chlorotriazines (or derivatives thereof) using nucleophilic aromatic substitution reactions has been commonly employed to install various functional groups, including poly(ethylene glycol) tags or fluorogenic labels. Here, a poorly soluble dichlorotriazine with an appended indole is shown to react with a construct containing the disordered domain of BRCA1. Subsequently, this construct can undergo proteolytic cleavage to remove the SUMO-tag: the N-terminal poly(His) tag is still effective for purification. Steady-state fluorescence, circular dichroism spectroscopy, and isothermal titration calorimetry with the binding partner of BRCA1, PALB2, are used to characterize the indole-labeled BRCA1. Neither the reaction conditions nor the indole-tag appreciably alter the structure of the BRCA1. Mass spectrometry confirms that the target is modified once, although the location of modification cannot be determined by tandem mass spectrometry with collision-induced dissociation due to disadvantageous fragmentation patterns.

Covalent modification of proteins at specific, predetermined sites is essential for advancing biological and biopharmaceutical applications. Site-selective labeling techniques for protein modification allow us to effectively track biological function, intracellular dynamics, and localization. Despite numerous reports on modifying target proteins with functional chemical probes, unique organic reactions that achieve site-selective integration without compromising native functional properties remain a significant challenge. In this review, we delve into site-selective protein modification using synthetic probes, highlighting both chemical and computational methodologies for chemo- and regioselective modifications of naturally occurring amino acids, as well as proximity-driven protein-selective chemical modifications. We also underline recent traceless affinity labeling strategies that involve exchange/cleavage reactions and catalyst tethering modifications. The rapid development of computational infrastructure and methods has made the bioconjugation of proteins more accessible, enabling precise predictions of structural changes due to protein modifications. Hence, we discuss bioconjugational computational approaches, including molecular dynamics and artificial intelligence, underscoring their potential applications in enhancing our understanding of cellular biology and addressing current challenges in the field.

Auxenochlorella pyrenoidosa is a microalga that stands out due to its high protein content. The objective of this work was to study the effect of high-pressure homogenization (HPH) on the recovery of proteins from A. pyrenoidosa and their application as functional emulsifiers. Untreated and HPH-treated (400–800 bar, 1 and 4 passes) aqueous cellular suspensions were incubated at 40 °C for 6 h. The aqueous extracts were collected, the proteins were precipitated at pH 3, and the Auxenochlorella pyrenoidosa protein concentrates (APPC) were lyophilized. Increasing HPH pressure and number of passes (400–800 bar, 1 and 4 passes) improved protein recovery yield up to 57%. Higher HPH pressures also reduced α-helix and β-sheet structures, exposing the hydrophobic protein core. This protein modification led to APPCs with increased oil-holding capacity (2.83 g oil/g APPC). The surface tension of APPC solutions reached a minimum value of 28.6 mN/m at an APPC concentration of 2% w/w. The APPCs from untreated and HPH-treated biomass were used to stabilize nanoemulsions (2–6% sunflower oil), comparing one-step homogenization (high-speed homogenization) with the two-step homogenization method (combining high-speed and high-pressure homogenization). The two-step method led to significantly smaller oil droplets with narrow size distribution, leading to stable nanoemulsions with improved resistance to centrifugation and heating–cooling cycles. Due to APPC’s great emulsifying properties, A. pyrenoidosa proteins have a promising potential for various applications such as delivery systems stabilization. Additionally, the low energy requirements, continuous processing capability, and scalability of HPH make it a suitable process for industrial applications.

SUMO (small ubiquitin-like modifier) conjugation or SUMOylation, a post-translational modification, is a crucial regulator of protein function and cellular processes. In the context of neural stem cells (NSCs), SUMOylation has emerged as a key player, affecting their proliferation, differentiation, and survival. By modifying transcription factors, such as SOX1, SOX2, SOX3, SOX6, Bmi1, and Nanog, SUMOylation can either enhance or impair their transcriptional activity, thus impacting on NSCs self-renewal. Moreover, SUMOylation regulates neurogenesis and neuronal differentiation by modulating key proteins, such as Foxp1, Mecp2, MEF2A, and SOX10. SUMOylation is also crucial for the survival and proliferation of NSCs in both developing and adult brains. By regulating the activity of transcription factors, coactivators, and corepressors, SUMOylation acts as a molecular switch, inducing cofactor recruitment and function during development. Importantly, dysregulation of NSCs SUMOylation has been implicated in various disorders, including embryonic defects, ischemic cerebrovascular disease, glioma, and the harmful effects of benzophenone-3 exposure. Here we review the main findings on SUMOylation-mediated regulation of NSCs self-renewal, differentiation and survival. Better understanding NSCs SUMOylation mechanisms and its functional consequences might provide new strategies to promote neuronal differentiation that could contribute for the development of novel therapies targeting neurodegenerative diseases.

Introduction The escalating global changes have fostered conditions for the expansion and transmission of diverse biological factors, leading to the rise of emerging and reemerging infectious diseases. Complex viral infections, such as COVID-19, influenza, HIV, and Ebola, continue to surface, necessitating the development of effective vaccine technologies. Areas covered This review article highlights recent advancements in molecular biology, virology, and genomics that have propelled the design and development of innovative molecular tools. These tools have promoted new vaccine research platforms and directly improved vaccine efficacy. The review summarizes the cutting-edge molecular engineering tools used in creating novel vaccines and explores the rapidly expanding molecular tools landscape and potential directions for future vaccine development. Expert opinion The strategic application of advanced molecular engineering tools can address conventional vaccine limitations, enhance the overall efficacy of vaccine products, promote diversification in vaccine platforms, and form the foundation for future vaccine development. Prioritizing safety considerations of these novel molecular tools during vaccine development is crucial.

Streptomycetes possess numerous gene clusters and the potential to produce a large amount of natural products. Histone deacetylase (HDAC) inhibitors play an important role in the regulation of histone modifications in fungi, but their roles in prokaryotes remain poorly understood. Here, we investigated the global effects of the HDAC inhibitor, sodium butyrate (SB), on marine-derived Streptomyces olivaceus FXJ 8.021, particularly focusing on the activation of secondary metabolite biosynthesis. The antiSMASH analysis revealed 33 secondary metabolite biosynthetic gene clusters (BGCs) in strain FXJ 8.021, among which the silent lobophorin BGC was activated by SB. Transcriptomic data showed that the expression of genes involved in lobophorin biosynthesis (ge00097–ge00139) and CoA-ester formation (e.g., ge02824), as well as the glycolysis/gluconeogenesis pathway (e.g., ge01661), was significantly up-regulated in the presence of SB. Intracellular CoA-ester analysis confirmed that SB triggered the biosynthesis of CoA-ester, thereby increasing the precursor supply for lobophorin biosynthesis. Further acetylomic analysis revealed that the acetylation levels on 218 sites of 190 proteins were up-regulated and those on 411 sites of 310 proteins were down-regulated. These acetylated proteins were particularly enriched in transcriptional and translational machinery components (e.g., elongation factor GE04399), and their correlations with the proteins involved in lobophorin biosynthesis were established by protein–protein interaction network analysis, suggesting that SB might function via a complex hierarchical regulation to activate the expression of lobophorin BGC. These findings provide solid evidence that acetylated proteins triggered by SB could affect the expression of genes involved in the biosynthesis of primary and secondary metabolites in prokaryotes.

The functional properties of pumpkin seed proteins remain unutilized in numerous food and industrial applications. Several current approaches aim to improve the functional properties of pumpkin seed proteins, allowing their innovative potential to develop and modify significantly. Several strategies can be implemented to alter the functional properties of proteins isolated from pumpkin seeds. The first is enzymatic hydrolysis, regardless of whether, proteases may free peptide binding and profoundly impact the protein structure and functionality. Thermal treatment can include heating and cooling to replace protein conformation and increase solubility, emulsification, and gelation properties. Chemical modification techniques, including acylation and glycation, can also be used to improve stability, viscosity, and foaming ability. Functional properties and, where possible, ingredients with many applications may include exceptional possibilities for proteins modified in food preparations, such as dairy replacements, plant-based meat analogues, and free gluten that have an outstanding aspect, satisfactory quality, and nutritional profiles. As multiple different proteins act as precursors of active peptides, they can also be used to generate bio-specific foods. This review briefly provides information about various types of protein extraction techniques and functional properties that are modified by different types of processing technologies.

Abstract A method was developed that enables the magnesium chloride (MgCl2)‐activated S‐acetamidomethyl cysteine sulfoxide (Cys(Acm)(O)) to induce the sp2(C−H) sulfenylation of the indole of Trp residues. The reaction operates under mild acidic conditions using acetic acid or an ionic liquid to give the Trp‐sulfenylated products. Other than Trp, all other proteinogenic amino acids are unreactive to the sulfenylation conditions. We demonstrated the successful application of this reaction to various peptides, including lysozyme. Furthermore, we achieved the Trp‐modification of a monoclonal antibody by a MgCl2‐mediated reaction in an acidic ionic liquid. The resulting antibody exhibited antibody performance comparable to the parent protein. The amide moiety in the Acm group contributes to the difference in chemical behaviors between S‐Acm and S‐p‐methoxybenzyl (MBzl)‐protected cysteine sulfoxides. This is because the S‐Acm sulfoxide is converted to S‐chlorocysteine responsible for Trp‐sulfenylation under less acidic conditions than those required for the reaction of S‐MBzl sulfoxide. Based on this rationale, we prepared a linker possessing S‐Acm and S‐MBzl oxide moieties and subjected the linker to heterodimerization of DNA‐binding MYC and MAX peptides containing a Trp handle. The one‐pot/stepwise Cys‐Trp conjugation between the linker and DNA‐binding peptides allowed the generation of a heterodimeric MYC/MAX DNA binder.

Glycosylation is a crucial post-translational modification process where sugar molecules (glycans) are covalently linked to proteins, lipids, or other biomolecules. In this highly regulated and complex process, a series of enzymes are involved in adding, modifying, or removing sugar residues. This process plays a pivotal role in various biological functions, influencing the structure, stability, and functionality of the modified molecules. Glycosylation is essential in numerous biological processes, including cell adhesion, signal transduction, immune response, and biomolecular recognition. Dysregulation of glycosylation is associated with various diseases. Glycation, a post-translational modification characterized by the non-enzymatic attachment of sugar molecules to proteins, has also emerged as a crucial factor in various diseases. This review comprehensively explores the multifaceted role of glycation in disease pathogenesis, with a specific focus on its implications in osteoarthritis (OA). Glycosylation and glycation alterations wield a profound influence on OA pathogenesis, intertwining with disease onset and progression. Diverse studies underscore the multifaceted role of aberrant glycosylation in OA, particularly emphasizing its intricate relationship with joint tissue degradation and inflammatory cascades. Distinct glycosylation patterns, including N-glycans and O-glycans, showcase correlations with inflammatory cytokines, matrix metalloproteinases, and cellular senescence pathways, amplifying the degenerative processes within cartilage. Furthermore, the impact of advanced glycation end-products (AGEs) formation in OA pathophysiology unveils critical insights into glycosylation-driven chondrocyte behavior and extracellular matrix remodeling. These findings illuminate potential therapeutic targets and diagnostic markers, signaling a promising avenue for targeted interventions in OA management. In this comprehensive review, we aim to thoroughly examine the significant impact of glycosylation or AGEs in OA and explore its varied effects on other related conditions, such as liver-related diseases, immune system disorders, and cancers, among others. By emphasizing glycosylation's role beyond OA and its implications in other diseases, we uncover insights that extend beyond the immediate focus on OA, potentially revealing novel perspectives for diagnosing and treating OA.

Bioconjugation reactions are critical to the modification of peptides and proteins, permitting the introduction of biophysical probes onto proteins as well as drugs for use in antibody-targeted medicines. A diverse set of chemical reagents can be employed in these circumstances to covalently label protein side chains, such as the amine moiety in the side chain of lysine and the thiol functionality in cysteine residues, two of the more frequently employed sites for modification. To provide researchers with a thermodynamic survey of the reaction of these residues with frequently employed chemical modification reagents as well as reactive cellular intermediates also known to modify proteins non-enzymatically, a theoretical investigation of the overall thermodynamics of models of these reactions was undertaken at the T1 and G3(MP2) thermochemical recipe levels (gas phase), the M06-2X/6-311+G(2df,2p)/B3LYP/6-31G(d) (gas and water phase), and the M06-2X/cc-PVTZ(-f)++ density functional levels of theory (water phase). Discussions of the relationship between the reagent structure and the overall thermodynamics of amine or thiol modification are presented. Of additional interest are the observations that routine cellular intermediates such as certain thioesters, acyl phosphates, and acetyl-L-carnitine can contribute to non-enzymatic protein modifications. These reactions and representative click chemistry reactions were also investigated. The computational survey presented herein (>320 reaction computations were undertaken) should serve as a valuable resource for researchers undertaking protein bioconjugation. A concluding section addresses the ability of computation to provide predictions as to the potential for protein modification by new chemical entities, with a cautionary note on protein modification side reactions that may occur when employing synthetic substrates to measure enzyme kinetic activities.

This review presents a comprehensive analysis of plant-based proteins from soybeans, pulses, cereals, and pseudo-cereals by examining their structural properties, modification techniques, bioactivities, and applicability in food systems. It addresses the critical need for a proper utilization strategy of proteins from various plant sources amidst the rising environmental footprint of animal protein production. The inherent composition diversity among plant proteins, their nutritional profiles, digestibility, environmental impacts, and consumer acceptance are compared. The innovative modification techniques to enhance the functional properties of plant proteins are also discussed. The review also investigates the bioactive properties of plant proteins, including their antioxidant, antimicrobial, and antitumoral activities, and their role in developing meat analogs, dairy alternatives, baked goods, and 3D-printed foods. It underscores the consideration parameters of using plant proteins as sustainable, nutritious, and functional ingredients and advocates for research to overcome sensory and functional challenges for improved consumer acceptance and marketability.

The present study demonstrates the effects of pH-shifting treatments and magnetic field-assisted pH-shifting treatments on the properties of myofibrillar protein (MP) in frozen meat. The solubility results indicate that the pH-shifting treatments increased the solubility of MP from 16.8% to a maximum of 21.0% (pH 9). The values of surface hydrophobicity and protein particle size distribution indicate that the pH-shifting treatment effectively inhibited protein aggregation through electrostatic interactions. However, under higher pH conditions (pH 10, 11), the treatments assisted by the magnetic field increased the degree of aggregation. The total thiol content and SDS-PAGE results further suggest that the magnetic field-assisted pH-shifting treatment accelerated the formation of covalent bonds among MPs under the alkaline environment. The results of the Differential Scanning Calorimetry (DSC) and protein secondary structure analysis indicate that the magnetic field promoted the unfolding of protein structures in an alkaline environment, markedly reducing the effective pH levels of pH-shifting. Electron paramagnetic resonance (EPR) data indicate that the phenomenon might be associated with the increased concentration of free radicals caused by the magnetic field treatment. In summary, the application of magnetic field-assisted pH-shifting treatments could emerge as a potent and promising strategy to improve the protein properties in frozen meat.

Huauzontle (Chenopodium nuttalliae Saff.) is an underutilized pseudocereal native from Mexico, with high-quality protein content. This work subjected huauzontle protein isolate to ultrasonication, pH-shifting, or heat treatments, evaluating the effect on structural features, emulsifying, and foaming properties. FTIR spectra showed that the intensity of the amide I bands changed significantly, with mainly β-sheet and β-turn structures decreasing, and α-helix structure increasing. The latter structure is associated with increased surface activity and stable interfacial stability. Both, the emulsifying and the foaming properties were enhanced by protein modification. This work confirms that huauzontle protein treated by ultrasonication, pH-shifting, and heat induces changes in the protein's structure and physicochemical properties, contributing to expand its techno-functional applications as in the design of novel foods.

Bovine α-lactalbumin (BLA) induced severe cow's milk allergy. In this study, a novel strategy combining ultrasonication, performed before glycation, and phosphorylation was proposed to reduce BLA allergenicity. Result showed that IgE- and IgG-binding capacities and the release rates of histamine and interleukin-6 from RBL-2H3 were reduced. Moreover, intrinsic fluorescence intensity and surface hydrophobicity were decreased, whereas glycated sites (R10, N44, K79, K108, N102 and K114) and phosphorylated sites (Y36 and S112) of BLA were increased. Minimum allergenicity was detected during BLA treatment after ultrasonic prior to glycation and subsequent phosphorylation because of considerable increase in glycated and phosphorylated sites. Therefore, the decrease in allergenicity of BLA, the effect correlated well with the shielding effect of glycated sites combined with phosphorylated sites and the conformational changes. This study provides important theoretical foundations for improving and using the ultrasonic technology combined with protein modification in allergenic protein processing.

Protein–lipid conjugation is a widespread modification involved in many biological processes. Various lipids, including fatty acids, isoprenoids, sterols, glycosylphosphatidylinositol, sphingolipids, and phospholipids, are covalently linked with proteins. These modifications direct proteins to intracellular membranes through the hydrophobic nature of lipids. Some of these membrane-binding processes are reversible through delipidation or by reducing the affinity to membranes. Many signaling molecules undergo lipid modification, and their membrane binding is important for proper signal transduction. The conjugation of proteins to lipids also influences the dynamics and function of organellar membranes. Dysregulation of lipidation has been associated with diseases such as neurodegenerative diseases. In this review, we first provide an overview of diverse forms of protein–lipid conjugation and then summarize the catalytic mechanisms, regulation, and roles of these modifications. Key words: lipid, lipidation, membrane, organelle, protein modification

With the rising nutritional requirements of food and the advancement of technology in the food industry, protein modification has become one of the food industry's top concerns. Glycosylation products are safer than other chemical modifications ones and have a significant deal of potential to enhance food's sensory qualities. This article summarizes the progression of casein glycosylation interactions, including covalently bonded and non-covalently linked reactions, and analyses the effect of glycosylation on the functional properties of casein. Glycosylation is a promising and socially significant protein modification strategy that can improve the functional qualities of casein in a number of ways, potentially helping the dairy processing industry and even food innovation research.

Bioactive peptides can be obtained from plant, animal or marine sources. Bioactive peptides possess diverse biological activities such as antihypertension, antioxidant, antithrombosis, cholesterol-lowering, blood pressure-lowering, antibacterial, immune regulation, cell regulation, and mineral-binding properties. How to improve the conversion rate of proteins to peptides is always one of the difficulties in the research field of peptide. Ultra-high pressure technology is a novel non-thermal physical processing technology suitable for large-scale production, which has been widely used to the structural and functional characteristics of proteins, as well as the extraction and production efficiency of bioactive peptides. This review paper mainly focuses on the application of ultra-high pressure technology in protein modification and peptide preparation. The types, sources and biological activities of bioactive peptides prepared by ultra-high pressure technology are also summarized.

Post-translational modifications refer to the chemical alterations of proteins following their biosynthesis, leading to changes in protein properties. These modifications, which encompass acetylation, phosphorylation, methylation, SUMOylation, ubiquitination, and others, are pivotal in a myriad of cellular functions. Macroautophagy, also known as autophagy, is a major degradation of intracellular components to cope with stress conditions and strictly regulated by nutrient depletion, insulin signaling, and energy production in mammals. Intriguingly, in insects, 20-hydroxyecdysone signaling predominantly stimulates the expression of most autophagy-related genes while concurrently inhibiting mTOR activity, thereby initiating autophagy. In this review, we will outline post-translational modification-regulated autophagy in insects, including Bombyx mori and Drosophila melanogaster, in brief. A more profound understanding of the biological significance of post-translational modifications in autophagy machinery not only unveils novel opportunities for autophagy intervention strategies but also illuminates their potential roles in development, cell differentiation, and the process of learning and memory processes in both insects and mammals.

Abstract Central dogma is the most fundamental hypothesis in the field of molecular biology and explains the genetic information flow from DNA to protein. Beyond residue‐by‐residue transmission of sequential information, chemical modifications of DNA, RNA, and protein are also relayed in the course of gene expression. Here, this work presents recent evidence supporting bidirectional interplay between chromatin modifications and RNA modifications. Furthermore, several RNA modifications likely affect chemical modifications of proteins. The relay of chemical modifications occurs co‐transcriptionally or co‐translationally, ensuring crosstalk among chemical modifications at the DNA, RNA, and protein levels. Overall, this work proposes a hypothetical framework that represents transmission of chemical modification information among chromatin, RNA, and proteins.

The BTB (broad-complex, tramtrack, and bric-à-brac) domain, also known as the POZ (POX virus and zinc finger) domain, is a conserved protein–protein interaction domain present in various organisms. In this study, we conducted a genome-wide search to identify and characterize BTB genes in Solanum tuberosum. A total of 57 StBTBs were identified and analyzed for their physicochemical properties, chromosomal distribution, gene structure, conserved motifs, phylogenetic relationships, tissue-specific expression patterns, and responses to hormonal and stress treatments. We found that StBTBs were unevenly distributed across potato chromosomes and exhibited diverse gene structures and conserved motifs. Tissue-specific expression analysis revealed differential expression patterns across various potato tissues, implying their roles in plant growth and development. Furthermore, differential expression analysis under hormonal and stress treatments indicated the involvement of StBTBs in abiotic and biotic stress responses and hormone signaling pathways. Protein–protein interaction analysis identified potential interactions with ribosomal proteins, suggesting roles in translational regulation. Additionally, microRNA target site analysis revealed regulatory relationships between StBTBs and miRNAs. Our study provides a comprehensive understanding of the StBTB gene family in potato, laying the groundwork for further functional characterization and manipulation of these genes to improve stress tolerance and agricultural productivity in potato and related plant species.

The self-assembled structures of coix seeds affected the enzymatic efficiency and doesn‘t facilitate the release of more active peptides. The influence of heating combined with ultrasound pretreatment (HT + US) on the structure, enzymatic properties and hydrolysates (CHPs) of coix seed prolamin was investigated. Results showed that the structural of coix seed prolamins has changed after HT + US, including increased surface hydrophobicity, reduced α-helix and random coil content, and a decrease in particle size. So that, leads to changes in thermodynamic parameters such as an increase in the reaction rate constant and a decrease in activation energy, enthalpy and enthalpy. The fractions of

Delivering bioactive proteins into cells without carriers presents significant challenges in biomedical applications due to limited cell membrane permeability and the need for targeted delivery. Here, we introduce a novel carrier-free method that addresses these challenges by chemically modifying proteins with an acid-responsive cell-penetrating peptide (CPP) for selective intracellular delivery within tumours. Cytochrome C, a protein known for inducing apoptosis, served as a model for intracellular delivery of therapeutic proteins for cancer treatment. The CPP was protected with 2,3-dimethyl maleic anhydride (DMA) and chemically conjugated onto the protein surface, creating an acid-responsive protein delivery system. In the acidic tumour microenvironment, DMA deprotects and exposes the positively charged CPP, enabling membrane penetration. Both in vitro and in vivo assays validated the pH-dependent shielding mechanism, demonstrating the modified cytochrome C could induce apoptosis in cancer cells in a pH-selective manner. These findings provide a promising new approach for carrier-free and tumour-targeted intracellular delivery of therapeutic proteins for a wide range of potential applications.

Influenza A virus (IAV) is one of the most circulated human pathogens, and influenza disease, commonly known as the flu, remains one of the most recurring and prevalent infectious human diseases globally. IAV continues to challenge existing vaccines and antiviral drugs via its ability to evolve constantly. It is critical to identify the molecular determinants of IAV pathogenesis to understand the basis of flu severity in different populations and design improved antiviral strategies. In recent years, acetylation has been identified as one of the determinants of IAV pathogenesis. Acetylation was originally discovered as an epigenetic protein modification of histones. But, it is now known to be one of the ubiquitous protein modifications of both histones and non-histone proteins and a determinant of proteome complexity. Since our first observation in 2007, significant progress has been made in understanding the role of acetylation during IAV infection. Now, it is becoming clearer that acetylation plays a pro-IAV function via at least three mechanisms: (1) by reducing the host’s sensing of IAV infection, (2) by dampening the host’s innate antiviral response against IAV, and (3) by aiding the stability and function of viral and host proteins during IAV infection. In turn, IAV antagonizes the host deacetylases, which erase acetylation, to facilitate its replication. This review provides an overview of the research progress made on this subject so far and outlines research prospects for the significance of IAV-acetylation interplay.

Neutrophil and airway epithelial cell interactions are critical in the inflammatory response to viral infections including respiratory syncytial virus, Sendai virus, and SARS-CoV-2. Airway epithelial cell dysfunction during viral infections is likely mediated by the interaction of virus and recruited neutrophils at the airway epithelial barrier. Neutrophils are key early responders to viral infection. Neutrophil myeloperoxidase catalyzes the conversion of hydrogen peroxide to hypochlorous acid (HOCl). Previous studies have shown HOCl targets host neutrophil and endothelial cell plasmalogen lipids, resulting in the production of the chlorinated lipid, 2-chlorofatty aldehyde (2-ClFALD). We have previously shown that the oxidation product of 2-ClFALD, 2-chlorofatty acid (2-ClFA) is present in bronchoalveolar lavage fluid of Sendai virus-infected mice, which likely results from the attack of the epithelial plasmalogen by neutrophil-derived HOCl. Herein, we demonstrate small airway epithelial cells contain plasmalogens enriched with oleic acid at the sn-2 position unlike endothelial cells which contain arachidonic acid enrichment at the sn-2 position of plasmalogen. We also show neutrophil-derived HOCl targets epithelial cell plasmalogens to produce 2-ClFALD. Further, proteomics and over-representation analysis using the ω-alkyne analog of the 2-ClFALD molecular species, 2-chlorohexadecanal (2-ClHDyA) showed cell adhesion molecule binding and cell-cell junction enriched categories similar to that observed previously in endothelial cells. However, in contrast to endothelial cells, proteins in distinct metabolic pathways were enriched with 2-ClFALD modification, particularly pyruvate metabolism was enriched in epithelial cells and mitochondrial pyruvate respiration was reduced. Collectively, these studies demonstrate, for the first time, a novel plasmalogen molecular species distribution in airway epithelial cells that are targeted by myeloperoxidase-derived hypochlorous acid resulting in electrophilic 2-ClFALD, which potentially modifies epithelial physiology by modifying proteins.

Quinoa (Chenopodium quinoa Willd.) is a pseudocereal plant that originally came from South America. The trend of consuming quinoa is propelled by its well‒balanced amino acid profile compared to that of other plants. In addition, its gluten‒free nature makes quinoa a promising diet option for celiac disease patients. Protein accounts for approximately 17% of the quinoa seed composition and quinoa protein possesses excellent quality. Quinoa protein is mainly composed of 11S globulins (37%) and 2S albumins (35%), both of which are stabilized by disulfide bonds. To date, the alkaline extraction method is the most commonly used method to extract quinoa protein. The functional properties and digestibility of quinoa protein can be improved with the help of various modification methods, and as a result, the application of quinoa protein will be extended. In this review, the extraction method, modification of functional properties and digestibility of quinoa protein are thoroughly discussed, providing insights into the application of quinoa protein in plant‒based foods.

The chemoenzymatic synthesis of a series of dual N- and C-terminal–functionalized cholera toxin B subunit (CTB) glycoconjugates is described. Mucin 1 peptides bearing different levels of Tn antigen glycosylation [MUC1(Tn)] were prepared via solid-phase peptide synthesis. Using sortase-mediated ligation, the MUC1(Tn) epitopes were conjugated to the C-terminus of CTB in a well-defined manner allowing for high-density display of the MUC1(Tn) epitopes. This work explores the challenges of using sortase-mediated ligation in combination with glycopeptides and the practical considerations to obtain high levels of conjugation. Furthermore, we describe methods to combine two orthogonal labeling methodologies, oxime- and sortase-mediated ligation, to expand the biochemical toolkit and produce dual N- and C-terminal–labeled conjugates.

The purpose of this study was to investigate the oxidative stability and physicochemical properties of pork emulsion sausages with whey protein–tannic acid conjugate and native whey protein. Over the course of 21 days, the thiobarbituric acid reactive substances (TBARS) of sausages containing a whey protein–tannic acid conjugate were lower than those of sausages with regular whey protein (p < 0.05). Kinetically, sausage containing the whey protein–tannic acid conjugate (k = 0.0242 day−1) appeared to last longer than sausage containing regular whey protein (k = 0.0667 day−1). The addition of the whey protein–tannic acid conjugate had no effect on product texture because there was no difference in hardness, springiness, cohesiveness, or water-holding capacity between the control and treated samples at Day 0 (p > 0.05). Scanning electron microscopy revealed that, at Day 21, the control sausage exhibited emulsion coalescence, as evidenced by an increase in the number of oil droplets and large voids, but not the whey protein–tannic acid conjugate-added sausage. There was no variation in the L*, a*, and b* values of the sausages when the whey protein–tannic acid conjugate was added (p > 0.05). However, there was a little increase in ΔE value in the treated sample. Thus, the whey-protein–tannic acid conjugate appeared to stabilize the lipid and physicochemical properties of the sausages by lowering the rate of TBARS production, retaining texture, water-holding capacity, and color, as well as by minimizing lipid coalescence during refrigerated storage.

Most human proteins are modified by enzymes that act on the α-amino group of a newly synthesized polypeptide. Methionine aminopeptidases can remove the initiator methionine and expose the second amino acid for further modification by enzymes responsible for myristoylation, acetylation, methylation, or other chemical reactions. Specific acetyltransferases can also modify the initiator methionine and sometimes the acetylated methionine can be removed, followed by further modifications. These modifications at the protein N-termini play critical roles in cellular protein localization, protein-protein interaction, protein-DNA interaction, and protein stability. Consequently, the dysregulation of these modifications could significantly change the development and progression status of certain human diseases. The focus of this review is to highlight recent progress in our understanding of the roles of these modifications in regulating protein functions and how these enzymes have been used as potential novel therapeutic targets for various human diseases.

Advances in single particle cryo-EM data collection and processing have seen a significant rise in its use. However, the influences of the environment generated through grid preparation, by for example interactions of proteins with the air-water interface are poorly understood and can be a major hurdle in structure determination by cryo-EM. Initial interactions of proteins with the air-water interface occur quickly and proteins can adopt preferred orientation or partially unfold within hundreds of milliseconds. It has also been shown previously that thin-film layers create hydroxyl radicals. To investigate the potential this might have in cryo-EM sample preparation, we studied two proteins, HSPD1, and beta-galactosidase, and show that cysteine residues are modified in a time-dependent manner. In the case of both HSPD1 and beta-galactosidase, this putative oxidation is linked to partial protein unfolding, as well as more subtle structural changes. We show these modifications can be alleviated through increasing the speed of grid preparation, the addition of DTT, or by sequestering away from the AWI using continuous support films. We speculate that the modification is oxidation by reactive oxygen species which are formed and act at the air-water interface. Finally, we show grid preparation on a millisecond timescale outruns cysteine modification, showing that the reaction timescale is in the range of 100s to 1,000s milliseconds and offering an alternative approach to prevent spontaneous cysteine modification and its consequences during cryo-EM grid preparation.

Fibronectin (FN) enhances K+ channel activity by integrin-mediated mechanisms. As vascular smooth muscle (VSM) K+ channels mediate vasodilation, we hypothesized that modification of fibronectin, via advanced non-enzymatic glycation, would alter signaling of this extracellular matrix protein through these channels. Bovine FN (1 mg/ml) was glycated (gFN) for 5 days using methylglyoxal (50 mM), and albumin was similarly glycated as a non-matrix protein control. VSM cells were isolated from rat cerebral arteries for measurement of macroscopic K+ channel activity using whole cell patch clamp methodology. Pharmacological inhibitors, iberiotoxin (0.1 μM) and 4-aminopyridine (0.1 mM), were used to identify contributions of large-conductance, Ca2+-activated, K+ channels and voltage-gated K+ channels, respectively. Compared with baseline, native FN enhanced whole cell K+ current in a concentration-dependent manner, whereas gFN inhibited basal current. Furthermore, native albumin did not enhance basal K+ current, but the glycated form (gAlb) caused inhibition. gFN was shown to impair both the Kv and BKCa components of total macroscopic K+ current. Anti-integrin α5 and β1 antibodies attenuated the effects of both FN and gFN on macroscopic K+ current at +70 mV. Consistent with an action on BKCa activity, FN increased, whereas gFN decreased the frequency of spontaneous transient outward current (STOCs). In contrast, gAlb inhibited whole cell K+ current predominantly through Kv, showing little effect on STOCs. A function-blocking, anti-RAGE antibody partially reversed the inhibitory effects of gFN, suggesting involvement of this receptor. Further, gFN caused production of reactive oxygen species (ROS) by isolated VSMCs as revealed by the fluorescent indicator, DHE. Evoked ROS production was attenuated by the RAGE blocking antibody. Collectively, these studies identify ion channel-related mechanisms (integrin and ROS-mediated) by which protein glycation may modify VSMC function.

Kai Wang,1,* Jinqiu Rao,1,* Tingting Zhang,1 Qing Gao,2 Jichao Zhang,3 Chenxi Guang,3 Liqin Ding,3 Feng Qiu1,3 1School of Chinese Materia Medica, Tianjin University of Traditional Chinese Medicine, Tianjin 301617, People’s Republic of China; 2School of Integrative Medicine, Tianjin University of Traditional Chinese Medicine, Tianjin 301617, People’s Republic of China; 3State Key Laboratory of Component-based Chinese Medicine,Tianjin University of Traditional Chinese Medicine, Tianjin, 301617, People’s Republic of China*These authors contributed equally to this workCorrespondence: Feng QiuSchool of Chinese Materia Medica, Tianjin University of Traditional Chinese Medicine, 10 Poyanghu Road, West Area, Tuanbo New Town, Jinghai District, Tianjin 301617, People’s Republic of ChinaTel/ Fax +86-022-59596223Email fengqiu20070118@163.comIntroduction: Berberrubine (BRB), an isoquinoline alkaloid, is a major constituent of medicinal plants Coptis chinensis Franch or Phellodendron chinense Schneid. BRB exhibits various pharmacological activities, whereas exposure to BRB may cause toxicity in experimental animals.Methods: In this study, we thoroughly investigated the liver injury induced by BRB in mice and rats. To explore the underlying mechanism, a study of the metabolic activation of BRB was conducted. Furthermore, covalent modifications of cysteine residues of proteins were observed in liver homogenate samples of animals after exposure to BRB, by application of an exhaustive proteolytic digestion method.Results: It was demonstrated that BRB-induced hepatotoxicities in a time- and dose-dependent manner, based on the biochemical parameters ALT and AST. H&E stained histopathological examination showed the occurrence of obvious edema in liver of mice after intraperitoneal (i.p.) administration of BRB at a single dose of 100 mg/kg. Slight hepatotoxicity was also observed in rats given the same doses of BRB after six weeks of gavage. As a result, four GSH adducts derived from reactive metabolites of BRB were detected in microsomal incubations with BRB fortified with GSH as a trapping agent. Moreover, four cys-based adducts derived from reaction of electrophilic metabolites of BBR with proteins were found in livers.Conclusion: These results suggested that the formation of protein adducts originating from metabolic activation of BRB could be a crucial factor of the mechanism of BRB-induced toxicities.Keywords: berberrubine, hepatotoxicity, metabolic activation, protein modification

Enzymetic modification of proteins in order to break down specific peptide bonds and protein modification is widely used in the food industry. In this research, protein of faba bean seeds was hydrolyzed using alcalase and trypsin enzymes at three concentrations (1, 2 and 3%) and reaction times of 1-6 h at optimal temperature and pH of enzymes (50 and 37 °C, pH 8.5 and 7, respectively). Hydrolysis degree, 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity, and iron chelating activity of hydrolyzed proteins were investigated. The results showed that the degree of hydrolysis increased with increasing reaction time and concentration of alcalase and trypsin enzymes. The time of hydrolysis and type of enzyme has a significant effect on the degree of hydrolysis, the antioxidant and chelating activity of the faba bean protein hydrolysates (p < /em>

The omics approaches allow the scientific community to successfully identify genomic regions associated with traits of interest for marker-assisted breeding. Agronomic traits such as seed color, yield, growth habit, and stress tolerance have been the targets for soybean molecular breeding. Genes governing these traits often undergo post-transcriptional modifications, which should be taken into consideration when choosing elite genes for molecular breeding. Post-transcriptional regulations of genes include transcript regulations, protein modifications, and even the regulation of the translational machinery. Transcript regulations involve elements such as microRNAs (miRNAs) and long non-coding RNAs (lncRNAs) for the maintenance of transcript stability or regulation of translation efficiency. Protein modifications involve molecular modifications of target proteins and the alterations of their interacting partners. Regulations of the translational machinery include those on translation factors and the ribosomal protein complex. Post-transcriptional regulations usually involve a set of genes instead of a single gene. Such a property may facilitate molecular breeding. In this review, we will discuss the post-transcriptional modifications of genes related to favorable agronomic traits such as stress tolerance, growth, and nutrient uptake, using examples from soybean as well as other crops. The examples from other crops may guide the selection of genes for marker-assisted breeding in soybean.

Cells employ post-translational modifications (PTMs) as key mechanisms to expand proteome diversity beyond the inherent limitations of a concise genome. The ability to incorporate post-translationally modified amino acids into protein targets via chemical ligation of peptide fragments has enabled the access to homogeneous proteins bearing discrete PTM patterns and empowered functional elucidation of individual modification sites. Native chemical ligation (NCL) represents a powerful and robust means for convergent assembly of two homogeneous, unprotected peptides bearing an N-terminal cysteine residue and a C-terminal thioester, respectively. The subsequent discovery that protein cysteine residues can be chemoselectively desulfurized to alanine has ignited tremendous interest in preparing unnatural thiol-derived variants of proteogenic amino acids for chemical protein synthesis following the ligation-desulfurization logic. Recently, the 21st amino acid selenocysteine, together with other selenyl derivatives of amino acids, have been shown to facilitate ultrafast ligation with peptidyl selenoesters, while the advancement in deselenization chemistry has provided reliable bio-orthogonality to PTMs and other amino acids. The combination of these ligation techniques and desulfurization/deselenization chemistries has led to streamlined synthesis of multiple structurally-complex, post-translationally modified proteins. In this review, we aim to summarize the latest chemical synthesis of thiolated and selenylated amino-acid building blocks and exemplify their important roles in conquering challenging protein targets with distinct PTM patterns.

Phosphorylation is a widespread posttranslational modification that regulates numerous biological processes. Viruses can alter the physiological activities of host cells to promote virus particle replication, and manipulating phosphorylation is one of the mechanisms. Senecavirus A (SVA) is the causative agent of porcine idiopathic vesicular disease. Although numerous studies on SVA have been performed, comprehensive phosphoproteomics analysis of SVA infection is lacking. The present study performed a quantitative mass spectrometry-based phosphoproteomics survey of SVA infection in Instituto Biologico-Rim Suino-2 (IBRS-2) cells. Three parallel experiments were performed, and 4,520 phosphosites were quantified on 2,084 proteins. Gene Ontology (GO) functional enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses showed that many phosphorylated proteins were involved in apoptosis and spliceosome pathways, and subcellular structure localization analysis revealed that more than half were located in the nucleus. Motif analysis of proteins with differentially regulated phosphosites showed that proline, aspartic acid, and glutamic acid were the most abundant residues in the serine motif, while proline and arginine were the most abundant in the threonine motif. Forty phosphosites on 27 proteins were validated by parallel reaction monitoring (PRM) phosphoproteomics, and 30 phosphosites in 21 proteins were verified. Nine proteins with significantly altered phosphosites were further discussed, and eight [SRRM2, CDK13, DDX20, DDX21, BAD, ELAVL1, PDZ-binding kinase (PBK), and STAT3] may play a role in SVA infection. Finally, kinase activity prediction showed 10 kinases’ activity was reversed following SVA infection. It is the first phosphoproteomics analysis of SVA infection of IBRS-2 cells, and the results greatly expand our knowledge of SVA infection. The findings provide a basis for studying the interactions of other picornaviruses and their mammalian host cells.

Protein modification with non-canonical amino acids (ncAAs) represents a useful technology to afford homogenous samples of bioconjugates with site-specific modification. This technique can be directly applied to the detection of aberrant SUMOylation patterns, which are often indicative of disease states. Modified SUMO-trapping proteins, consisting of a catalytically inactive ULP1 fragment (UTAG) fused to the maltose-binding protein MBP, are useful reagents for the binding and labeling of SUMOylated proteins. Mutation of this UTAG fusion protein to facilitate amber suppression technologies for the genetic incorporation of ncAAs was assessed to provide a functional handle for modification. Ultimately, two sites in the maltose-binding protein (MBP) fusion were identified as ideal for incorporation and bioconjugation without perturbation to the SUMO-trapping ability of the UTAG protein. This functionality was then employed to label SUMOylated proteins in HeLa cells and demonstrate their enrichment in the nucleus. This modified UTAG-MBP-ncAA protein has far-reaching applications for both diagnostics and therapeutics.

In this study, the effect of solid-state fermentation (SSF) with Lactobacillus sakei MI401 and Pediococcus acidilactici PA-2 strains on functional properties of extruded (130 °C; 25 rpm) corn-milling by-products (CMB) and their albumin, globulin, and prolamin fractions was evaluated in order to produce stabilized and functionalized food/feed stock. Extrusion resulted in a considerable reduction of microbial contamination of CMB by five log cycles, increased damaged starch, water-absorption capacity, and lowered protein and fat contents by 12.4% and 37%, respectively. The application of SSF for the extruded CMB have been shown to improve the water absorption, foaming, and emulsifying capacity of albumins and globulins and also increased the digestibility and free radical scavenging activity of prolamins. The essential amino acid content (EAA) in CMB and antioxidant activity of prolamins was lowered after extrusion but significantly increased after SSF. The combination of the abovementioned treatments can be confirmed as a prospective functionalization of CMB, capable of potentially enhancing its safety and improving nutritional, biochemical, and technological properties of proteins.

Lipid oxidation is still a major problem complicating the development of food emulsions. In this study, an antioxidant Pickering emulsion stabilized by resveratrol-grafted zein (Z-R) conjugates and pectin (P) complex particles was prepared. The hydrophilic pectin successfully adjusted the wettability of Z-R; when the mass ratio of Z-R to P was 2:1 (Z-R/P2:1), the three-phase contact angle was 90.68°, and the wettability of the particles was close to neutral. Rheological analysis showed that the emulsion formed an elastic gel structure. FTIR spectra indicated that there was a hydrogen bond and electrostatic interaction between Z-R and P. The disappearance of characteristic infrared peaks of corn oil was due to a dense protective film formed on the surface of oil drops by Z-R/P2:1 particles, which was confirmed by confocal laser scanning microscopy. The emulsion stabilized by Z-R/P2:1 had excellent physical stability at a wide range of pH values (4–9), salt ion concentrations (0.04–0.15 mol·L−1) and storage times (0–30 days). The anti-lipid oxidation ability of the emulsion was outstanding; after storage for 14 days at room temperature, the MDA content in the emulsion was only 123.85 μmol/kg oil. In conclusion, the Z-R/P2:1 particles prepared in this study can effectively stabilize a Pickering emulsion and expand the usability of the method for constructing antioxidant Pickering emulsions.

Acetaminophen (APAP) is a mild analgesic and antipyretic used commonly worldwide. Although considered a safe and effective over-the-counter medication, it is also the leading cause of drug-induced acute liver failure. Its hepatotoxicity has been linked to the covalent binding of its reactive metabolite, N-acetyl p-benzoquinone imine (NAPQI), to proteins. The aim of this study was to identify APAP-protein targets in both rat and mouse liver, and to compare the results from both species, using bottom-up proteomics with data-dependent high resolution mass spectrometry and targeted multiple reaction monitoring (MRM) experiments. Livers from rats and mice, treated with APAP, were homogenized and digested by trypsin. Digests were then fractionated by mixed-mode solid-phase extraction prior to liquid chromatography-tandem mass spectrometry (LC-MS/MS). Targeted LC-MRM assays were optimized based on high-resolution MS/MS data from information-dependent acquisition (IDA) using control liver homogenates treated with a custom alkylating reagent yielding an isomeric modification to APAP on cysteine residues, to build a modified peptide database. A list of putative in vivo targets of APAP were screened from data-dependent high-resolution MS/MS analyses of liver digests, previous in vitro studies, as well as selected proteins from the target protein database (TPDB), an online resource compiling previous reports of APAP targets. Multiple protein targets in each species were found, while confirming modification sites. Several proteins were modified in both species, including ATP-citrate synthase, betaine-homocysteine S-methyltransferase 1, cytochrome P450 2C6/29, mitochondrial glutamine amidotransferase-like protein/ES1 protein homolog, glutamine synthetase, microsomal glutathione S-transferase 1, mitochondrial-processing peptidase, methanethiol oxidase, protein/nucleic acid deglycase DJ-1, triosephosphate isomerase and thioredoxin. The targeted method afforded better reproducibility for analysing these low-abundant modified peptides in highly complex samples compared to traditional data-dependent experiments.

Amino-terminal acetylation is catalyzed by a set of N-terminal acetyltransferases (NATs). The NatA complex (including X-linked Naa10 and Naa15) is the major acetyltransferase, with 40–50% of all mammalian proteins being potential substrates. However, the overall role of amino-terminal acetylation on a whole-organism level is poorly understood, particularly in mammals. Male mice lacking Naa10 show no globally apparent in vivo amino-terminal acetylation impairment and do not exhibit complete embryonic lethality. Rather Naa10 nulls display increased neonatal lethality, and the majority of surviving undersized mutants exhibit a combination of hydrocephaly, cardiac defects, homeotic anterior transformation, piebaldism, and urogenital anomalies. Naa12 is a previously unannotated Naa10-like paralog with NAT activity that genetically compensates for Naa10. Mice deficient for Naa12 have no apparent phenotype, whereas mice deficient for Naa10 and Naa12 display embryonic lethality. The discovery of Naa12 adds to the currently known machinery involved in amino-terminal acetylation in mice.

The ability to radiolabel proteins with [18F]fluoride enables the use of positron emission tomography (PET) for the early detection, staging and diagnosis of disease. The direct fluorination of native proteins through C-F bond formation is, however, a difficult task. The aqueous environments required by proteins severely hampers fluorination yields while the dry, organic solvents that promote nucleophilic fluorination can denature proteins. To circumvent these issues, indirect fluorination methods making use of prosthetic groups that are first fluorinated and then conjugated to a protein have become commonplace. But, when it comes to the radiofluorination of proteins, these indirect methods are not always suited to the short half-life of the fluorine-18 radionuclide (110 min). This review explores radiofluorination through bond formation with fluoride at boron, metal complexes, silicon, phosphorus and sulfur. The potential for these techniques to be used for the direct, aqueous radiolabeling of proteins with [18F]fluoride is discussed.