Search

Sprouting can enhance the bioavailability and stimulate the production of health-promoting compounds. This research explored the potential health benefits of wheat sprouting, focusing on underexplored areas in existing literature such as alterations in phenylalanine ammonia-lyase (PAL) activity and glutathione levels during wheat sprouting. Furthermore, special attention was directed toward asparagine (Asn), the main precursor of acrylamide formation, as regulatory agencies are actively seeking to impose limitations on the presence of acrylamide in baked products. The results demonstrate elevated levels of PAL (4.5-fold at 48 h of sprouting), antioxidants, and total phenolics (1.32 mg gallic acid equivalent/g dry matter at 72 h of sprouting), coupled with a reduction in Asn (i.e. 11-fold at 48 h of sprouting) and glutathione concentrations, after wheat sprouting. These findings suggest that sprouting can unlock health-promoting properties in wheat. Optimizing the sprouting process to harness these benefits, however, may have implications for the techno-functionality of wheat flour in food processing., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Ltd.)

Acrylamide (AA) is a neoformed compound in heated foods, mainly produced between asparagine (Asn) and glucose (Glc) during the Maillard reaction. Galacturonic acid (GalA), the major component of pectin, exhibits high activity in AA formation. This study investigated the pathway for AA formation between GalA and Asn. Three possible pathways were proposed: 1) The carbonyl group of GalA directly interacts with Asn to produce AA; 2) GalA undergoes an oxidative cleavage reaction to release α-dicarbonyl compounds, which subsequently leads to AA production; 3) 5-formyl-2-furancarboxylic acid, the thermal degradation product of GalA, reacts with Asn to generate AA. Structural analysis revealed that the COOH group in GalA accelerated intramolecular protonation and electron transfer processes, thereby increasing the formation of AA precursors such as decarboxylated Schiff base and α-dicarbonyl compounds, promoting AA formation. This study provides a theoretical basis and new insights into the formation and control of AA., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Peptides are receiving significant attention in pharmaceutical sciences due to their applications as anti-inflammatory drugs; however, many aspects of their interactions and mechanisms at the molecular level are not well-known. This work explores the molecular structure of two peptides-(i) cysteine (Cys)-asparagine (Asn)-serine (Ser) (CNS) as a molecule in the gas phase and solvated in water in zwitterion form, and (ii) the crystal structure of the dipeptide serine-asparagine (SN), a reliable peptide indication whose experimental cell parameters are well known. A search was performed by means of atomistic calculations based on density functional theory (DFT). These calculations matched the experimental crystal structure of SN, validating the CNS results and useful for assignments of our experimental spectroscopic IR bands. Our calculations also explore the intercalation of CNS into the interlayer space of montmorillonite (MNT). Our quantum mechanical calculations show that the conformations of these peptides change significantly during intercalation into the confined interlayer space of MNT. This intercalation is energetically favorable, indicating that this process can be a useful preparation for therapeutic anti-inflammatory applications and showing high stability and controlled release processes.

SIRT5, one of the mammalian sirtuins, specifically recognizes succinyl-lysine residues on proteins and catalyzes the desuccinylation reaction. In this study, we characterized SIRT5 mutants with hydrophobic amino acid substitutions at Q140 and N141, in addition to the catalytic residue H158, known as an active site residue, by the Michaelis-Menten analysis and X-ray crystallography. Kinetic analysis showed that the catalytic efficiency (k cat /K m ) of the Q140L and N141V mutants decreased to 0.02 times and 0.0038 times that of the wild-type SIRT5, respectively, with the activity of the N141V mutant becoming comparable to that of the H158M mutant. Our findings indicate that N141 contributes significantly to the desuccinylation reaction., (© 2024 Federation of European Biochemical Societies.)

This study presents a novel and selective method for the determination of l-asparagine in diverse potato varieties under various storage conditions. L-asparagine levels serve as a crucial indicator for acrylamide formation, a hazardous substance in processed potato products. The fluorometric method utilized blue-emitting CDs (B-CDs), orange-emitting CDs (O-CDs), and the enzyme L-asparaginase for ratiometric detection of L-asparagine. Upon enzymatic hydrolysis of L-asparagine by L-asparaginase, liberated ammonia induced a pH increase in the reaction medium. This pH shift enhanced the fluorescence of B-CDs while simultaneously decreasing that of O-CDs, enabling sensitive and selective L-asparagine quantification. Comprehensive characterization of the CDs was performed using various spectroscopic techniques and transmission electron microscopy. The method demonstrated excellent sensitivity (LOD = 0.31 μM) and a wide linear range (1.0-50.0 μM). When the method was applied to potato samples, high recovery values (98.00-100.33 %) with low relative standard deviations (RSDs) were achieved, confirming the accuracy and precision of the method. The approach was employed to determine L-asparagine levels in three potato varieties (Lady Rosetta, Spunta, and Nicola) under different storage temperatures and durations. This method provides a valuable tool for monitoring L-asparagine content in potatoes, potentially aiding in the mitigation of acrylamide formation during processing. The robust performance and simplicity of the proposed technique make it suitable for routine analysis in both research and industrial applications within the potato industry., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

A novel fluorometric method for the determination of L-asparaginase, an enzyme crucial in cancer therapy and food industry applications, is presented. This sensitive and selective approach utilizes L-asparagine and two pH-sensitive carbon dots (blue-N-CDs and red-N-CDs) as probes. The interaction between L-asparagine and L-asparaginase liberates ammonia, causing an increase in pH. This pH change simultaneously decreases the fluorescence of blue-N-CDs while enhancing the emission of red-N-CDs, enabling ratiometric detection of L-asparaginase. Comprehensive characterization of both carbon dots and investigation of their response mechanism towards L-asparaginase were conducted using ultraviolet-visible spectrophotometry, fluorescence spectroscopy, and transmission electron microscopy (TEM) imaging techniques. The designed approach demonstrates outstanding linearity (20 to 2000 U L -1 ) and a low detection limit (6.95 U L -1 ) for L-asparaginase quantification. Moreover, when tested to human serum samples, the detection system exhibits outstanding selectivity and high recovery rates (96.15% to 99.75%) with low standard deviation, underscoring its suitability for practical implementation in clinical diagnostics., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Therapeutic antibodies have predominantly been IgG-based. However, the ongoing clinical trial of MOv18 IgE has highlighted the potential of using IgE antibodies in cancer therapy. While extensive studies targeting IgG glycosylation resulted in a rational basis for the development of enhanced biotherapeutics, IgE glycosylation remains an area with limited analyses. Previous studies on the role of IgE glycosylation present conflicting data with one study emphasizing the importance of N275 and T277 residues for FcεRI binding whereas another asserts the nonsignificance of IgE glycosylation in receptor interaction. While existing literature underscores the significance of glycans at the N275 position for binding to FcεR1 receptor and initiation of anaphylaxis, the role of other IgE glycosylation sites in folding or receptor binding remains elusive. This study systematically investigates the functional significance of N-linked glycosylation sites in the heavy chain of IgE which validates the pivotal role of N275 residue in IgE secretion and stability. Replacement of this asparagine to non-amine group moieties does not affect IgE function in vitro, yet substitution with aspartic acid compromises antibody yield. The deglycosylated IgE variant exhibits superior efficacy, challenging the conventional importance of glycosylation for effector function. In summary, our study unveils an intricate relationship between N-glycosylation sites and the structural-functional dynamics of IgE antibodies. Furthermore, it offers novel insights into the IgE scaffold, paving the way for the development of more effective and stable IgE-based therapeutics., (© 2024 Wiley Periodicals LLC.)

Heliorhodopsin (HeR) is a new rhodopsin family discovered in 2018 through functional metagenomic analysis. Similar to microbial rhodopsins, HeR has an all-trans retinal chromophore, and its photoisomerization to the 13-cis form triggers a relatively slow photocycle with sequential intermediate states (K, M, and O intermediates). The O intermediate has a relatively long lifetime and is a putative active state for transferring signals or regulating enzymatic reactions. Although the first discovered HeR, 48C12, was found in bacteria and the second HeR (TaHeR) was found in archaea, their key amino acid residues and molecular architectures have been recognized to be well conserved. Nevertheless, the rise and decay kinetics of the O intermediate are faster in 48C12 than in TaHeR. Here, using a new infrared spectroscopic technique with quantum cascade lasers, we clarified that the hydrogen bond between transmembrane helices (TM) 3 and 4 is essential for the altered O kinetics (Ser112 and Asn138 in 48C12). Interconverting mutants of 48C12 and TaHeR clearly revealed that the hydrogen bond is important for regulating the dynamics of the O intermediate. Overall, our study sheds light on the importance of the hydrogen bond between TM3 and TM4 in heliorhodopsins, similar to the DC gate in channelrhodopsins., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Cancer is a leading concern and important cause of death worldwide. Cancer is a non-communicable illness defined as uncontrolled division of cells. It can develop into metastatic cancer when tumor cells migrate to other organs. In recent years evidence has emerged that the bioavailability of Asn play a crucial role in cancer metastasis. Asn is a non-essential amino acid formed from an ATP dependent catalyzed reaction by the enzyme asparagine synthetase (ASNS), where Asp and Gln are converted to Asn and Glu, respectively. The human ASNS enzyme consist of 561 amino acids, with a molecular weight of 64 KDa. ASNS governs the activation of transcriptional factors that regulate the process of metastasis. In this work the 3D model of ASNS in E. coli (AS-B) and the human ASNS docked with its different ligands have been used to study the 3D mechanism of the conversion of Asp and Gln to Asn and Glu, in human ASNS. The stability evaluation of the docked complexes was checked by molecular dynamic simulation through the bioinformatic tool Desmond. The binding residues and their interactions can be exploited for the development of inhibitors, as well as for finding new drug molecules against ASNS and prevention of metastatic cancer., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Riaz et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Deamidation frequently is invoked as an important driver of crystallin aggregation and cataract formation. Here, we characterized the structural and biophysical consequences of cumulative Asn to Asp changes in γD-crystallin. Using NMR spectroscopy, we demonstrate that N- or C-terminal domain-confined or fully Asn to Asp changed γD-crystallin exhibits essentially the same 1 H- 15 N HSQC spectrum as the wild-type protein, implying that the overall structure is retained. Only a very small thermodynamic destabilization for the overall Asn to Asp γD-crystallin variants was noted by chaotropic unfolding, and assessment of the colloidal stability, by measuring diffusion interaction parameters, yielded no substantive differences in association propensities. Furthermore, using molecular dynamics simulations, no significant changes in dynamics for proteins with Asn to Asp or iso-Asp changes were detected. Our combined results demonstrate that substitution of all Asn by Asp residues, reflecting an extreme case of deamidation, did not affect the structure and biophysical properties of γD-crystallin. This suggests that these changes alone cannot be the major determinant in driving cataract formation., (© 2024 The Author(s). Protein Science published by Wiley Periodicals LLC on behalf of The Protein Society.)

Site-specific modifications of aspartate residues spontaneously occur in crystallin, the major protein in the lens. One of the primary modification sites is Asp151 in αA-crystallin. Isomerization and racemization alter the crystallin backbone structure, reducing its stability by inducing abnormal crystallin-crystallin interactions and ultimately leading to the insolubilization of crystallin complexes. These changes are considered significant factors in the formation of senile cataracts. However, the mechanisms driving spontaneous isomerization and racemization have not been experimentally demonstrated. In this study, we generated αA-crystallins with different homo-oligomeric sizes and/or containing an asparagine residue at position 151, which is more prone to isomerization and racemization. We characterized their structure, hydrophobicity, chaperone-like function, and heat stability, and examined their propensity for isomerization and racemization. The results show that the two differently sized αA-crystallin variants possessed similar secondary structures but exhibited different chaperone-like functions depending on their oligomeric sizes. The rate of isomerization and racemization of Asp151, as assessed by the deamidation of Asn151, was also found to depend on the oligomeric sizes of αA-crystallin. The predominant isomerization product via deamidation of Asn151 in the different-sized αA-crystallin variants was L-β-Asp in vitro, while various modifications occurred around Asp151 in vivo. The disparity between the findings of this in vitro study and in vivo studies suggests that the isomerization of Asp151 in vivo may be more complex than what occurs in vitro., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Sun et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

The ninhydrin reaction is commonly used for the detection of amino acids. However, in the literature, different conditions with respect to the buffer system, its pH and concentration, type of organic solvent, incubation time, and temperature, as well as the concentrations of the reagents, are described. To identify the most suitable conditions, colour development with reagents of varying compositions and different reaction temperatures and times were investigated using asparagine as a model amino acid. Asparagine was selected since it is one of the most abundant free amino acids in many types of samples. The optimal reaction mixture consisted of 0.8 mol L -1 potassium acetate, 1.6 mol L -1 acetic acid, 20 mg mL -1 ninhydrin and 0.8 mg mL -1 hydrindantin in DMSO/acetate buffer 40/60 ( v / v ) (final concentrations). The best reaction condition was heating the samples in 1.5 mL reaction tubes to 90 °C for 45 min. Afterwards, the samples were diluted with 2-propanol/water 50/50 ( v / v ) and the absorbance was measured at 570 nm. The proteinogenic amino acids showed a similar response except for cysteine and proline. The method was highly sensitive and showed excellent linearity as well as intra-day and inter-day reproducibility.

High-mannose-type glycan structure of N-glycoproteins plays important roles in the proper folding of proteins in sorting glycoprotein secretion and degradation of misfolded proteins in the endoplasmic reticulum (ER). The Glc 1 Man 9 GlcNAc 2 (G1M9)-type N-glycan is one of the most important signaling molecules in the ER. However, current chemical synthesis strategies are laborious, warranting more practical approaches for G1M9-glycopeptide development. Wang et al. reported the procedure to give G1M9-Asn-Fmoc through chemical modifications and purifications from 40 chicken eggs, but only 3.3 mg of G1M9-glycopeptide was obtained. Therefore, better methods are needed to obtain more than 10 mg of G1M9-glycopeptide. In this study, we report the preparation of G1M9-glycopeptide (13.2 mg) linking Asn-Gly-Thr triad as consensus sequence from 40 chicken eggs. In this procedure, λ-carrageenan treatment followed by papain treatment was used to separate the Fc region of IgY antibody that harbors high-mannose glycans. Moreover, cotton hydrophilic interaction liquid chromatography was adapted for easy purification. The resulting G1M9-Asn(Fmoc)-Gly-Thr was identified by nuclear magnetic resonance and mass spectroscopy. G1M9-Asn(Fmoc)-Gly, G1M9-Asn(Fmoc), and G1M9-OH were also detected by mass spectroscopy. Here, our developed G1M9-tripeptide might be useful for the elucidation of glycoprotein functions as well as the specific roles of the consensus sequence., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Yuto Nakamachi, Shinji Tanaka, Taiki Kuribara, Kiichiro Totani has patent #P7261366 issued to KH Neochem Co. Ltd. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Maillard reaction (MR) plays a pivotal role in the food flavor industry, including a cascade of reactions starting with the reaction between amino compounds and reducing sugars, and thus provides various colors and flavors. A new group of volatile compounds called pyrazinones found in MR are now getting more attention. In this study, eight volatile pyrazinones were found in the asparagine MR systems, in which 3,5-dimethyl- and 3,6-dimethyl-2(1 H )-pyrazinones were reported for the first time. The major formation pathways were the reactions between asparagine and α-dicarbonyls, with decarboxylation as a critical step. Besides, novel alternative pathways involving alanine amidation and successive reactions with α-dicarbonyls were explored and successfully formed eight pyrazinones. The major differences between alanine-amidated pathways and decarboxylation pathways are the amidation step and absence of the decarboxylation step. For the alanine-amidated pathways, the higher the temperature, the better the amidation effect. The optimal amidation temperature was 200 °C in this study. The reaction between the alanine amide and α-dicarbonyls after amidation can happen at low temperatures, such as 35 and 50 °C, proposing the possibility of pyrazinone formation in real food systems. Further investigations should be conducted to investigate volatile pyrazinones in various food systems as well as the biological effects and kinetic formation differences of the volatile pyrazinones.

Background: In wheat-derived bakery products, the quantity of free asparagine (fAsn) has been identified as a key factor in acrylamide (AA) formation. Based on this assumption, four varieties of common wheat (Triticum aestivum L.), Stromboli, Montecarlo, Sothys and Cosmic, selected for their different fAsn content inside the grain, were studied to evaluate their potential in the production of pizza with reduced AA levels. To this purpose, wholemeal and refined flours were obtained from each variety., Results: The fAsn content ranged from 0.25 to 3.30 mmol kg -1 , with higher values for wholemeal flours which also showed greater amount of ash, fibre and damaged starch than refined wheat flours. All types of flours were separately used to produce wood oven baked pizza base, according to the Traditional Speciality Guaranteed EU Regulation (97/2010). AA reduction in the range 47-68% was found for all the selected wheat cultivars, compared with a commercial flour, with significantly lower values registered when refined flour was used. Moreover, refined leavened dough samples showed decreased levels of fAsn and reducing sugars due to the fermentation activity of yeasts. Furthermore, it was confirmed that pizza made with wholemeal flours exhibited lower rapidly digestible starch (RDS) and rapidly available glucose (RAG) values compared to that prepared with the refined flour., Conclusion: This study clearly shows that a reduced asparagine content in wheat flour is a key factor in the mitigation of AA formation in pizza base. Unfortunately, at the same time, it is highlighted how it is necessary to sacrifice the beneficial effects of fibre intake, such as lowering the glycaemic index, in order to reduce AA. © 2024 Society of Chemical Industry., (© 2024 Society of Chemical Industry.)

Synergistic cross-linkers could improve the taste acceptability of ready-to-eat sea cucumber (RSC). Besides, the hardness of RSC was increased by 331.00% and 266.87% after synergistic cross-linking. Synergistic cross-linking treatment could ameliorate the non-enzymatic degradation of RSC collagen and polysaccharides. Gaussian calculations results showed that dipeptides containing asparagine residues may have different reaction pathways. The main cleavage pathways of CH 3 CO-Asn-Gly-NHCH 3 (NG) might be water-assisted side chain cyclization, stepwise cyclamide hydrolysis via a Gemdiol Intermediate, deamination, and peptide bond breakage. The relative free energy of cyclamide hydrolysis process of NG was increased by 8.2 kcal/mol after synergistic cross-linking. The mass spectrometry results showed that typical peptides could cleavage at NG, CH 3 CO-Asn-Lys-NHCH 3 (NK) and CH 3 CO-Asn-Leu-NHCH 3 (NL) sites after heating, which justified the breakage pattern of peptides in Gaussian calculations. It can offer a comprehensive theoretical basis for the processing of the ready-to-eat sea cucumber with storage stability., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Amadori rearrangement products of asparagine with glucose (Asn-Glc-ARP) were first prepared through Maillard model reactions and identified via liquid chromatography-mass spectroscopy. With the study on the effect of the reaction temperature, pH values, and reaction time, the ideal reaction condition for accumulation of Asn-Glc-ARP was determined at 100 °C for 40 min under pH 7. Asparagine (Asn) was prone to degrade from Asn-Glc-ARP in alkaline pH values within a lower temperature range, while in an acidic environment with high temperatures, deamidation of Asn-Glc-ARP to Asp-Glc-ARP (Amadori rearrangement products of aspartic acid with glucose) was displayed as the dominant pathway. The deamidation reaction on the side chain of the amide group took place at Asn-Glc-ARP and transferred it into the hydroxyl group, forming Asp-Glc-ARP at the end. Considering that lyophilization as pretreatment led to limited water activity, a single aspartic acid was not deamidated from Asn directly nor did it degrade from Asp-Glc-ARP even at 120 °C. The degradation of Asn-Glc-ARP through tandem mass spectrometry (MS/MS) analysis showed the obvious fragment ion at m / z 211, indicating that the stable oxonium ion formed during fragmentation. The structure of Asn-Glc-ARP was proposed as 1-deoxy-1-l-asparagino-d-fructose after separation and purification. Also, the content of Asn-Glc-ARP within dry jujube fruit (HeTianYuZao) was quantitated as high as 8.1 ± 0.5 mg/g.

Acrylamide is a probable carcinogen in humans and is formed when reducing sugars react with free asparagine (Asn) during thermal processing of food. Although breeding for low reducing sugars worked well in potatoes, it is less successful in cereals. However, reducing free Asn in cereals has great potential for reducing acrylamide formation, despite the role that Asn plays in nitrogen transport and amino acid biosynthesis. In this perspective, we summarize the efforts aimed at reducing free Asn in cereal grains and discuss the potentials and challenges associated with targeting this essential amino acid, especially in a seed-specific manner.

Aspartate/asparagine-β-hydroxylase (AspH) is a transmembrane 2-oxoglutarate (2OG)-dependent oxygenase that catalyzes the post-translational hydroxylation of aspartate- and asparagine-residues in epidermal growth factor-like domains (EGFDs) of its substrate proteins. Upregulation of ASPH and translocation of AspH from the endoplasmic reticulum membrane to the surface membrane of cancer cells is associated with enhanced cell motility and worsened clinical prognosis. AspH is thus a potential therapeutic and diagnostic target for cancer. This chapter describes methods for the production and purification of soluble constructs of recombinant human AspH suitable for biochemical and crystallographic studies. The chapter also describes efficient methods for performing turnover and inhibition assays which monitor catalysis of isolated recombinant human AspH in vitro using solid phase extraction coupled to mass spectrometry (SPE-MS). The SPE-MS assays employ synthetic disulfide- or thioether-bridged macrocyclic oligopeptides as substrates; a macrocycle is an apparently essential requirement for productive AspH catalysis and mimics an EGFD disulfide isomer that is not typically observed in crystal and NMR structures. SPE-MS assays can be used to monitor catalysis of 2OG oxygenases other than AspH; the methods described herein are representative for 2OG oxygenase SPE-MS assays useful for performing kinetic and/or inhibition studies., (Copyright © 2024. Published by Elsevier Inc.)

Thermal processing techniques can lead to the formation of heat-induced toxic substances. Acrylamide is one contaminant that has received much scientific attention in recent years, and it is formed essentially during the Maillard reaction when foods rich in carbohydrates, particularly reducing sugars (glucose, fructose), and certain free amino acids, especially asparagine (ASN), are processed at high temperatures (>120°C). The highly variable free ASN concentration in raw materials makes it challenging for food businesses to keep acrylamide content below the European Commission benchmark levels, while avoiding flavor, color, and texture impacts on their products. Free ASN concentrations in crops are affected by environment, genotype, and soil fertilization, which can also influence protein content and amino acid composition. This review aims to provide an overview of free ASN and acrylamide quantification methods and mitigation strategies for acrylamide formation in foods, focusing on adding pulse flours to cereal-based snacks and bakery products. Overall, this review emphasizes the importance of these mitigation strategies in minimizing acrylamide formation in plant-based products and ensuring safer and healthier food options., (© 2023 The Authors. Comprehensive Reviews in Food Science and Food Safety published by Wiley Periodicals LLC on behalf of Institute of Food Technologists.)

Asparagine (Asn) deamidation and aspartic acid (Asp) isomerization are common degradation pathways that affect the stability of therapeutic antibodies. These modifications can pose a significant challenge in the development of biopharmaceuticals. As such, the early engineering and selection of chemically stable monoclonal antibodies (mAbs) can substantially mitigate the risk of subsequent failure. In this study, we introduce a novel in silico approach for predicting deamidation and isomerization sites in therapeutic antibodies by analyzing the structural environment surrounding asparagine and aspartate residues. The resulting quantitative structure-activity relationship (QSAR) model was trained using previously published forced degradation data from 57 clinical-stage mAbs. The predictive accuracy of the model was evaluated for four different states of the protein structure: (1) static homology models, (2) enhancing low-frequency vibrational modes during short molecular dynamics (MD) runs, (3) a combination of (2) with a protonation state reassignment, and (4) conventional full-atomistic MD simulations. The most effective QSAR model considered the accessible surface area (ASA) of the residue, the pKa value of the backbone amide, and the root mean square deviations of both the alpha carbon and the side chain. The accuracy was further enhanced by incorporating the QSAR model into a decision tree, which also includes empirical information about the sequential successor and the position in the protein. The resulting model has been implemented as a plugin named "Forecasting Reactivity of Isomerization and Deamidation in Antibodies" in MOE software, completed with a user-friendly graphical interface to facilitate its use.

L-asparaginase from Escherichia coli (EcA) has been used for the treatment of acute lymphoid leukemia (ALL) since the 1970s. Nevertheless, the enzyme has a second specificity that results in glutaminase breakdown, resulting in depletion from the patient's body, causing severe adverse effects. Despite the huge interest in the use of this enzyme, the exact process of glutamine depletion is still unknown and there is no consensus regarding L-asparagine hydrolysis. Here, we investigate the role of T12, Y25, and T89 in asparaginase and glutaminase activities. We obtained individual clones containing mutations in the T12, Y25 or T89 residues. After the recombinant production of wild-type and mutated EcA, The purified samples were subjected to structural analysis using Nano Differential Scanning Fluorimetry, which revealed that all samples contained thermostable molecules in their active structural conformation, the homotetramer conformation. The quaternary conformation was confirmed by DLS and SEC. The activity enzymatic assay combined with molecular dynamics simulation identified the contribution of T12, Y25, and T89 residues in EcA glutaminase and asparaginase activities. Our results mapped the enzymatic behavior paving the way for the designing of improved EcA enzymes, which is important in the treatment of ALL., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier B.V.)

Acrylamide (AA) is a toxic substance formed in many carbohydrate-rich food products, whose formation can be reduced by adding some additives. Furthermore, the type of food consumed determines the AA intake. According to the compiled information, the first route causing AA formation is the Maillard reaction. Some interventions, such as reducing AA precursors in raw materials, (i.e., asparagine), reducing sugars, or decreasing temperature and processing time can be applied to limit AA formation in food products. The L-asparaginase is more widely used in potato products. Also, coatings loaded with proteins, enzymes, and phenolic compounds are new techniques for reducing AA content. Enzymes have a reducing effect on AA formation by acting on asparagine; proteins by competing with amino acids to participate in Maillard, and phenolic compounds through their radical scavenging activity. On the other hand, some synthetic and natural additives increase the formation of AA. Due to the high exposure to AA and its toxic effects, it is essential to recognize suitable food additives to reduce the health risks for consumers. In this sense, this study focuses on different additives that are proven to be effective in the reduction or formation of AA in food products.

The present study reports the structural and functional characterization of a new glutaminase-free recombinant L-asparaginase (PrASNase) from Pseudomonas resinovorans IGS-131. PrASNase showed substrate specificity to L-asparagine, and its kinetic parameters, K m , V max , and k cat were 9.49 × 10 -3  M, 25.13 IUmL -1  min -1 , and 3.01 × 10 3  s -1 , respectively. The CD spectra showed that PrASNase consisted of 18.5 % helix, 21.5 % antiparallel sheets, 4.2 % parallel sheets, 14 % turns, and rest other structures. FTIR was used for the functional characterization, and molecular docking predicted that the substrate interacts with serine, alanine, and glutamine in the binding pocket of PrASNase. Differing from known asparaginases, structural characterization by small-angle X-ray scattering (SAXS) and analytical ultracentrifugation (AUC) unambiguously revealed PrASNase to exist as a monomer in solution at low temperatures and oligomerized to a higher state with temperature rise. Through SAXS studies and enzyme assay, PrASNase was found to be mostly monomer and catalytically active at 37 °C. Furthermore, this glutaminase-free PrASNase showed killing effects against WIL2-S and TF-1.28 cells with IC 50 of 7.4 μg.mL -1 and 5.6 μg.mL -1 , respectively. This is probably the first report with significant findings of fully active L-asparaginase in monomeric form using SAXS and AUC and demonstrated the potential of PrASNase in inhibiting cancerous cells, making it a potential therapeutic candidate., Competing Interests: Declaration of competing interest The authors have declared that they have no conflict of interest related to this work., (Copyright © 2023. Published by Elsevier B.V.)

Coffee is a relevant source of dietary exposure for neoformed furan, alkyl furans and acrylamide. In this study, different statistical methods (hierarchical cluster analysis, correlation analysis, partial least squares regression analysis) were used for characterizing the formation of these process contaminants in green coffee beans roasted under the same standardized conditions. The results displayed a strong correlation between sucrose levels and furans in relation to the other sugars analyzed, while acrylamide formation was strongly related to the free asparagine. The data suggest that a sufficiently large amino acid pool in green coffee favors Maillard-induced acrylamide formation from asparagine, while reactions amongst the carbonyl-containing sugar fragmentation products leading to furan formation are suppressed. If the pool of free amino acids is small, it is depleted faster during roasting, thus favoring the formation of furans by caramelization, basically a sugar degradation process in which reactive carbonyl substances are generated and react together., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Crystallin aggregation in the eye lens is involved in the pathogenesis of cataracts. The aggregation is considered to be promoted by non-enzymatic post-translational modifications, such as the deamidation and stereoinversion of amino acid residues. Although in a previous study, the deamidated asparagine residues were detected in γS-crystallin in vivo, it is unclear which deamidated residues have the most impact on the aggregation under physiological conditions. In this study, we investigated the deamidation impacts of all Asn residues in γS-crystallin for the structural and aggregation properties utilizing deamidation mimetic mutants (N14D, N37D, N53D, N76D, and N143D). The structural impacts were investigated using circular dichroism analysis and molecular dynamics simulations, and the aggregation properties were analyzed by gel filtration chromatography and spectrophotometric methods. No significant structural impacts of all mutations were detected. However, the N37D mutation decreased thermal stability and changed some intermolecular hydrogen-bond formations. Aggregation analysis indicated that the superiority of the aggregation rate in each mutant varied with temperature. Deamidation at any Asn residues promoted γS-crystallin aggregation, and the deamidation at Asn37, Asn53, and Asn76 were suggested to be the most impactful in the formation of insoluble aggregations.

This study unveiled the effect of the suspected precursors of acrylamide (asparagine, glutamine) combined/separated with different formulations of glucose, fructose, and sucrose. To better understand the interaction between acrylamide precursors, cooking technique (deep vs air frying), and temperature (170 °C vs 190 °C), seven potato models from starch, sugars, amino acids, water and hydrocolloids (alginate and agar) were formulated. In line with previous findings, the present results showed that asparagine, glucose and fructose played an important role in acrylamide formation in these synthetic potato models. Furthermore, glutamine and sodium alginate might have an inhibitory effect on acrylamide formation. A significant impact of frying technique was also revealed. On the other hand, GC-FID analysis detected acrylamide in only these three models, (glucose-fructose, sucrose and asparagine-glucose/fructose/sucrose models > LOD 333.33 µg.kg -1 )., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Maillard reaction products obtained from the model system of binary amino acids (asparagine and proline) with glucose were first studied. GC-MS results showed that proline-specific aromatic compounds, 2,3-dihydro-1 H -pyrrolizines and cyclopent[ b ]azepin-8(1 H )-ones, were dominant among overall products, followed by pyrazines at different temperatures. Aspartic acid was first applied to model reactions as the precise control of asparagine deamidation, and lysine was further introduced into model systems for improving pyrazine formation. Quantitative results of model reaction products demonstrated that pyrazines were not significantly increased in deamidated states (Asn-Asp-Pro and Asp-Pro) while proline-specific compounds had a rapid enhancement at the same time. With excellent ability to form pyrazines, lysine did help to increase the formation of pyrazines, but still far fewer than pyrrolizines and azepines. It was assumed that proline would preferentially react with α-dicarbonyl compounds in Maillard reaction cascades with lower activation energies.

Fragmentation studies of cationized amino acids and small peptides as studied using guided ion beam tandem mass spectrometry (GIBMS) are reviewed. After a brief examination of the key attributes of the GIBMS approach, results for a variety of systems are examined, compared, and contrasted. Cationization of amino acids, diglycine, and triglycine with alkali cations generally leads to dissociations in which the intact biomolecule is lost. Exceptions include most lithiated species as well as a few examples for sodiated and one example for potassiated species. Like the lithiated species, cationization by protons leads to numerous dissociation channels. Results for protonated glycine, cysteine, asparagine, diglycine, and a series of tripeptides are reviewed, along with the thermodynamic consequences that can be gleaned. Finally, the important physiological process of the deamidation of asparagine (Asn) residues is explored by the comparison of five dipeptides in which the C-terminal partner (AsnXxx) is altered. The GIBMS thermochemistry is shown to correlate well with kinetic results from solution phase studies., (© 2021 John Wiley & Sons Ltd.)

Recombinant adeno-associated viral (AAV) vectors have taken center stage as gene delivery vehicles for gene therapy. Asparagine deamidation of AAV capsid proteins has been reported to reduce vector stability and potency of AAV gene therapy products. Deamidation of asparagine residue is a common post-translational modification of proteins that is detected and quantified by liquid chromatography-tandem mass spectrometry (LC-MS)-based peptide mapping. However, artificial deamidation can be spontaneously induced during sample preparation for peptide mapping prior to LC-MS analysis. We have developed an optimized sample preparation method to reduce and minimize deamidation artifacts induced during sample preparation for peptide mapping, which typically takes several hours to complete. To shorten turnaround time of deamidation results and to avoid artificial deamidation, we developed orthogonal RPLC-MS and RPLC-fluorescence detection methods for direct deamidation analysis at the intact AAV9 capsid protein level to routinely support downstream purification, formulation development, and stability testing. Similar trends of increasing deamidation of AAV9 capsid proteins in stability samples were observed at the intact protein level and peptide level, indicating that the developed direct deamidation analysis of intact AAV9 capsid proteins is comparable to the peptide mapping-based deamidation analysis and both methods are suitable for deamidation monitoring of AAV9 capsid proteins., Competing Interests: Declarations of competing interest None, (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Context: The inhibitory effect of asparagine (Asn) and its derivatives on iron (Fe) corrosion was studied by performing density functional theory (DFT) calculations. In this paper, the global and local reactivity descriptors of Asn in the protonated and neutral forms were evaluated. Also, the changes in reactivity were investigated when dipeptides were combined with Asn. Due to the increase in the reaction centers within their molecular structure, there was an enhancement in the inhibitory effect of these dipeptides. Moreover, the adsorption energies (E ads ) and the adsorption configurations of Asn and small peptides (SPs) with most stability were determined on the surface of Fe(111). It was found that dipeptides had a chemical adsorption on these substrates. In the protonated forms, there was an enhancement in the absolute values of E ads between the inhibitors and the Fe(111) surfaces. Peptides were more likely to be adsorbed on the Fe surfaces, showing the great inhibitory effect of these moieties. The results of the current research demonstrate the possibility of utilizing SPs as efficient "green" corrosion inhibitors., Methods: DFT computations were undertaken by employing the BIOVIA Material Studio with B3LYP-D3 functional and 6-31 + G* basis set. The theoretical evaluation of the inhibitory effect of asparagine (Asn) dipeptides, and the potential analysis of small peptides to protect against the corrosion of Fe, was done., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Background: Acrylamide (AA) is a potential carcinogen formed in food rich in carbohydrate during heating. Recently, AA has been found in several fruit products, such as prune juice, sugarcane molasses and canned black olives. This study focused on the role of galacturonic acid (GalA), the main acid hydrolysis product of fruit pectin, in AA formation in three model systems - asparagine (Asn)/glucose (Glc), Asn/GalA, and Asn/Glc/GalA - during heating under different pH values (pH 3.8-7.8), Glc concentration (0-0.1 mol L -1 ), molar ratio of substrates (Asn/Glc = 1:1, 0.025-0.5 mol L -1 ) and temperature (120-180 °C) for 30 min, respectively., Results: The results suggested that the addition of 0.1 mol L -1 GalA strongly accelerated AA formation in a manner dependent on pH value and temperature (P < 0.05). AA concentration under different Glc concentration and molar ratio of substrates suggested that GalA was more reactive than Glc when reacted with Asn. Furthermore, the Amadori rearrangement product/Schiff base/oxazolidine-5-one were identified as the intermediates formed in the Asn/GalA model system using ultra-performance liquid chromatography-quadrupole-time-of-flight-mass spectrometry., Conclusion: The results suggested that Maillard reaction between Asn and GalA might contribute to AA formation. This study is significant in elucidating the contribution of interaction between components for AA formation in fruit products. © 2022 Society of Chemical Industry., (© 2022 Society of Chemical Industry.)

Protein deamidation is a degradation mechanism that significantly impacts both pharmaceutical and physiological proteins. Deamidation impacts two amino acids, Asn and Gln, where the net neutral residues are converted into their acidic forms. While there are multiple similarities between the reaction mechanisms of the two residues, the impact of Gln deamidation has been noted to be most significant on physiological proteins while Asn deamidation has been linked to both pharmaceutical and physiological proteins. For this purpose, we sought to analyze the thermochemical and kinetic properties of the different reactions of Gln deamidation relative to Asn deamidation. In this study, we mapped the deamidation of Gln-X dipeptides into Glu-X dipeptides using density functional theory (DFT). Full network mapping facilitated the prediction of reaction selectivity between the two primary pathways, as well as between the two products of Gln-X deamidation as a function of solvent dielectric. To achieve this analysis, we studied a total of 77 dipeptide reactions per solvent dielectric (308 total reactions). Modeled at a neutral pH and using quantum chemical and statistical thermodynamic methods, we computed the following values: enthalpy of reaction (Δ H RXN ), entropy (Δ S RXN ), Gibbs free energy of reaction (Δ G RXN ), activation energy ( E A ), and the Arrhenius preexponential factor (log(A)) for each dipeptide. Additionally, using chemical reaction principles, we generated a database of computed rate coefficients for all possible N-terminus Gln-X deamidation reactions at a neutral pH, predicted the most likely deamidation reaction mechanism for each dipeptide reaction, analyzed our results against our prior study on Asn-X deamidation, and matched our results against qualitative trends previously noted by experimental literature.

The l-asparaginase (l-ASNase) enzyme catalyzes the conversion of the non-essential amino acid l-asparagine into l-aspartic acid and ammonia. Importantly, the l-ASNases are used as a key part of the treatment of acute lymphoblastic leukemia (ALL); however, despite their benefits, they trigger severe side effects because they have their origin in bacterial species ( Escherichia coli and Erwinia chrysanthemi ). Therefore, one way to solve these side effects is the use of l-ASNases with characteristics similar to those of bacterial types, but from different sources. In this sense, Cavia porcellus l-ASNase (CpA) of mammalian origin is a promising enzyme because it possesses similarities with bacterial species. In this work, the hydrolysis reaction for C. porcellus l-asparaginase was studied from an atomistic point of view. The QM/MM methodology was employed to describe the reaction, from which it was found that the conversion mechanism of l-asparagine into l-aspartic acid occurs in four steps. It was identified that the nucleophilic attack and release of the ammonia group is the rate-limiting step of the reaction. In this step, the nucleophile (Thr 19 ) attacks the substrate (ASN) leading to the formation of a covalent intermediate and release of the leaving group (ammonia). The calculated energy barrier is 18.9 kcal mol -1 , at the M06-2X+D3(0)/6-311+G(2d,2p)//CHARMM36 level of theory, which is in agreement with the kinetic data available in the literature, 15.9 kcal mol -1 (derived from the k cat value of 38.6 s -1 ). These catalytic aspects will hopefully pave the way toward enhanced forms of CpA. Finally, our work emphasizes that computational calculations may enhance the rational design of mutations to improve the catalytic properties of the CpA enzyme.

Many membrane-resident and secreted proteins, including growth factors and their receptors are N-glycosylated. The initial N-glycan structure is synthesized in the endoplasmic reticulum (ER) as a branched structure on a lipid anchor (dolicholpyrophosphate) and then co-translationally, "en bloc" transferred and linked via N-acetylglucosamine to asparagine within a specific N-glycosylation acceptor sequence of the nascent recipient protein. In the ER and then the Golgi apparatus, the N-linked glycan structure is modified by hydrolytic removal of sugar residues ("trimming") followed by re-glycosylation with additional sugar residues ("processing") such as galactose, fucose or sialic acid to form complex N-glycoproteins. While the sequence of the reactions leading to biosynthesis, "en bloc" transfer and processing of N-glycans is well investigated, it is still not completely understood how N-glycans affect the biological fate and function of N-glycoproteins. This review will discuss the biology of N-glycoprotein synthesis, processing and function with specific reference to the physiology and pathophysiology of the immune and nervous system, as well as infectious diseases such as Covid-19., (© 2023. The Author(s), under exclusive license to Springer Nature Switzerland AG.)

In silico prediction methods were developed to predict protein asparagine (Asn) deamidation. The method is based on understanding deamidation mechanism on structural level with machine learning. Our structure-based method is more accurate than the sequence-based method which is still widely used in protein engineering process. In addition, molecular dynamics simulation was applied to study the time occupancy of nucleophilic attack distance, which is hypothesized as the most important step toward the rate-limiting succinimide intermediate formation. A more accurate prediction method for distinguishing potentially liable amino acid residues would allow their elimination or reduction as early as possible in the drug discovery process. It is possible that such quantitative protein structure-property relationship tools can also be applied to other protein hotspot predictions., (© 2023. Springer Science+Business Media, LLC, part of Springer Nature.)

Antinutrients (ANs) interact with proteins changing its behavior and may affect Maillard reaction (MR). This work aimed to study the effect of phytic acid, tannic acid, and saponin on asparagine-glucose MR. The effect of AN concentration (0-1 mM) and reaction time (3-30 min at 150 °C) on the formation of melanoidins and acrylamide was determined. Other MR compounds were analyzed by gas chromatography and nuclear magnetic resonance. The ANs effect on asparagine-glucose thermal behavior was studied by differential scanning calorimetry. Results showed that ANs increase the melanoidins formation. Acrylamide content increased in saponin and phytic acid presence. The volatile profile was similar among the samples and formed mainly by pyrazines (>50%). ANs affect glucose's melting point, however, only phytic acid and saponin affect asparagine and glucose thermal behavior. The results presented in this work are important for food science and the industry to control MR in processed foods., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

The ubiquitin E3 ligase substrate adapter cereblon (CRBN) is a target of thalidomide and lenalidomide 1 , therapeutic agents used in the treatment of haematopoietic malignancies 2-4 and as ligands for targeted protein degradation 5-7 . These agents are proposed to mimic a naturally occurring degron; however, the structural motif recognized by the thalidomide-binding domain of CRBN remains unknown. Here we report that C-terminal cyclic imides, post-translational modifications that arise from intramolecular cyclization of glutamine or asparagine residues, are physiological degrons on substrates for CRBN. Dipeptides bearing the C-terminal cyclic imide degron substitute for thalidomide when embedded within bifunctional chemical degraders. Addition of the degron to the C terminus of proteins induces CRBN-dependent ubiquitination and degradation in vitro and in cells. C-terminal cyclic imides form adventitiously on physiologically relevant timescales throughout the human proteome to afford a degron that is endogenously recognized and removed by CRBN. The discovery of the C-terminal cyclic imide degron defines a regulatory process that may affect the physiological function and therapeutic engagement of CRBN., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)

Protein deamidation can severely alter the physicochemical characteristics and biological functions of protein therapeutics. Cobratide is a non-addictive analgesic with wide clinical acceptance. However, the Asn residue at position 48 from the N-terminus of the cobratide amino acid sequence (N48) tends to degrade during purification, storage, and transport. This characteristic could severely affect the drug safety and clinical efficacy of cobratide. Traditional methods for quantitating deamidation reported in previous research are characterised by low efficiency and accuracy; the quality control of cobratide via this method is limited. Herein, we developed an improved 18O-labelling method based on the detection of a unique peptide (i.e., the protein fragment of cobratide containing the N48 deamidation hotspot after enzymolysis) using an Orbitrap high-resolution mass spectrometer to quantify deamidated cobratide. The limits of detection and quantification of this method reached 0.02 and 0.025 μM, respectively, and inter- and intra-day precision values of the method were <3%. The accuracy of the 18O-labelling strategy was validated by using samples containing synthesised peptides with a known ratio of deamidation impurities and also by comparing the final total deamidation results with our previously developed capillary electrophoresis method. The recoveries for deamidation (Asp), deamidation isomerisation (iso-Asp), and total deamidation were 101.52 ± 1.17, 102.42 ± 1.82, and 103.55 ± 1.07, respectively. The robustness of the method was confirmed by verifying the chromatographic parameters. Our results demonstrate the applicability of the 18O-labelling strategy for detecting protein deamidation and lay a robust foundation for protein therapeutics studies and drug quality consistency evaluations.

Adeno-associated viral capsid proteins (AAV VP) are the major components that determine the tissue specificity and immunogenicity, and in vivo transduction performance of the vector. It was reported that asparagine deamidation of AAV capsid proteins leads to charge variants/heterogeneity and altered vector function, reduction of stability, and potency of AAV gene therapy products. Deamidation of asparagine residue is a common post-translational modification of proteins and is mostly detected and quantified by liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based peptide mapping. However, deamidation can be spontaneously introduced during sample preparation before LC-MS/MS analysis. So far, no optimal sample preparations, instead, traditional sample preparation has been used for AAV VP peptide mapping, resulting in exaggerating the original deamidation levels. It is important to accurately monitor and provide true value of asparagine deamidation for development of AAV gene therapy products. In this study, we evaluated denaturation temperatures, digestion durations, and digestion temperatures using three different sample preparation formats for LC-MS/MS-based assessment of deamidation of AAV9 capsid proteins. The results demonstrated that the optimal sample preparation method for AAV9 VP peptide mapping minimized asparagine deamidation artifacts. Although AAV9 was used for method optimization, this study may also provide a guidance on how to control deamidation artifacts for other AAV serotypes.

Ammonia loss from L -asparaginyls is a nonenzymatic reaction spontaneously occurring in all proteins and eventually resulting in damaging isoaspartate residues that hamper protein function and induce proteinopathy related to aging. Here, we discuss theoretical considerations supporting the possibility of a full repair reaction and present the first experimental evidence of its existence. If confirmed, the true repair of L -asparaginyl deamidation could open new avenues for preventing aging and neurodegenerative diseases.

A current method to locate sites of N -linked glycosylation on a protein involves the identification of deamidated sites after releasing the glycans with peptide- N -glycosidase F (PNGase F). PNGase F deglycosylation converts glycosylated Asn residues into Asp. The 1-Da mass tag created by this process is observable by liquid chromatography-tandem mass spectrometry analysis. A potential interference to this method of N -glycosylation site mapping is the chemical deamidation of Asn residues, which occurs spontaneously and can result in false positives. Deamidation is a pH-dependent process that results in the formation of iso-Asp (i-Asp) and native Asp (n-Asp) by a succinimide intermediate, whereas PNGase F deglycosylation results in the conversion of the glycosylation Asn residue into n-Asp. N -linked glycosylation sites can thus be identified by the presence of a single chromatographic peak corresponding to an n-Asp residue within the consensus sequence Asn-X-Ser/Thr, whereas sites of deamidation led to 2 chromatographic peaks resulting from the presence of n-Asp and i-Asp. The intent of this study is to alert investigators in the field to the potential and unexpected errors resulting from this phenomenon and to suggest a strategy to overcome this pitfall and limit the number of false-positive identifications., Competing Interests: Conflict of Interest Disclosures: The authors declare no conflicts of interest., (Copyright © 2022 Association of Biomolecular Resource Facilities. All rights reserved.)

Models of asparagine-containing dipeptides specifically designed to favor intrinsic folding into an Asx turn were characterized both theoretically, by using quantum chemistry, and experimentally, by using laser spectroscopy in the gas phase. Both approaches provided evidence for the spontaneous folding of both the Asn-Ala and Asn-Gly dipeptide models into the most stable Asx turn, a conformation stabilized by a C10 H-bond that was very similar to a type II' β-turn. In parallel, analysis of Asx turns implicating asparagine in crystallized protein structures in the Protein Data Bank revealed a sequence-dependent behavior. In Asn-Ala sequences, the Asx turn was found in conjunction with a type I β-turn for which the first of the four defining residues was Asn. The observation that the Asx turn in these structures is mostly of type II' (i. e., its most stable innate structure) suggests that this motif might foster the formation and/or enhance the stability of the backbone β-turn. In contrast, the Asx turns observed in Asn-Gly sequences extensively adopted a type II Asx-turn structure, thus suggesting that their formation should be ascribed to other factors, such as hydration. The fact that the Asx turn in a Asn-Gly sequence is also often found in combination with a hydrated β-bulge supports the premise that a Asn-Gly sequence might efficiently promote the formation of the β-bulge secondary structure., (© 2022 Wiley-VCH GmbH.)

The asparaginase II pathway consists of an asparagine transaminase [l-asparagine + α-keto acid ⇆ α-ketosuccinamate + l-amino acid] coupled to ω-amidase [α-ketosuccinamate + H 2 O → oxaloacetate + NH 4 + ]. The net reaction is: l-asparagine + α-keto acid + H 2 O → oxaloacetate + l-amino acid + NH 4 + . Thus, in the presence of a suitable α-keto acid substrate, the asparaginase II pathway generates anaplerotic oxaloacetate at the expense of readily dispensable asparagine. Several studies have shown that the asparaginase II pathway is important in photorespiration in plants. However, since its discovery in rat tissues in the 1950s, this pathway has been almost completely ignored as a conduit for asparagine metabolism in mammals. Several mammalian transaminases can catalyze transamination of asparagine, one of which - alanine-glyoxylate aminotransferase type 1 (AGT1) - is important in glyoxylate metabolism. Glyoxylate is a precursor of oxalate which, in the form of its calcium salt, is a major contributor to the formation of kidney stones. Thus, transamination of glyoxylate with asparagine may be physiologically important for the removal of potentially toxic glyoxylate. Asparaginase has been the mainstay treatment for certain childhood leukemias. We suggest that an inhibitor of ω-amidase may potentiate the therapeutic benefits of asparaginase treatment., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Deamidation of asparagine and glutamine alters protein structures and affects the chemical and biological properties of proteins. Protein deamidation has been demonstrated to be associated with protein folding, enzymatic activity, and degradation, as well as aging, cancer, and neurodegenerative diseases. To gain a better understanding on the biological roles of protein deamidation in aging and diseases, mass spectrometry (MS) has been employed in the identification of deamidated protein species and comprehensive characterization of deamidation sites. Three main MS approaches, top-down, middle-down, and bottom-up have been applied in the study of protein deamidation with high sensitivity, throughput, and accuracy. In this review, we discuss the application of top-down and middle-down MS in the study of protein deamidation, including sample preparation methods, separation strategies, MS and MS/MS techniques and data analysis. The advantages and drawbacks of these two approaches are also discussed and compared with those of the bottom-up method. The development of top-down and middle-down MS methods provides new strategies for protein deamidation analysis and gives new insights into the biological significance of protein deamidation in diseases., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Deamidation is a spontaneous degenerative protein modification (DPM) that disrupts the structure and function of both endogenous proteins and various therapeutic agents. While deamidation has long been recognized as a critical event in human aging and multiple degenerative diseases, research progress in this field has been restricted by the technical challenges associated with studying this DPM in complex biological samples. Asparagine (Asn) deamidation generates L-aspartic acid (L-Asp), D-aspartic acid (D-Asp), L-isoaspartic acid (L-isoAsp) or D-isoaspartic acid (D-isoAsp) residues at the same position of Asn in the affected protein, but each of these amino acids displays similar hydrophobicity and cannot be effectively separated by reverse phase liquid chromatography. The Asp and isoAsp isoforms are also difficult to resolve using mass spectrometry since they have the same mass and fragmentation pattern in MS/MS. Moreover, the 13 C peaks of the amidated peptide are often misassigned as monoisotopic peaks of the corresponding deamidated peptides in protein database searches. Furthermore, typical protein isolation and proteomic sample preparation methods induce artificial deamidation that cannot be distinguished from the physiological forms. To better understand the role of deamidation in biological aging and degenerative pathologies, new technologies are now being developed to address these analytical challenges, including mixed mode electrostatic-interaction modified hydrophilic interaction liquid chromatography (emHILIC). When coupled to high resolution, high accuracy tandem mass spectrometry this technology enables unprecedented, proteome-wide study of the 'deamidome' of complex samples. The current article therefore reviews recent advances in sample preparation methods, emHILIC-MS/MS technology, and MS instrumentation / data processing approaches to achieving accurate and reliable characterization of protein deamidation in complex biological and clinical samples., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Asparagines in proteins deamidate spontaneously, which changes the chemical structure of a protein and often affects its function. Current prediction algorithms for asparagine deamidation require a structure as an input or are too slow to be applied at a proteomic scale. We present NGOME-Lite, a new version of our sequence-based predictor for spontaneous asparagine deamidation that is faster by over two orders of magnitude at a similar degree of accuracy. The algorithm takes into account intrinsic sequence propensities and slowing down of deamidation by local structure. NGOME-Lite can run in a proteomic analysis mode that provides the half-time of the intact form of each protein, predicted by taking into account sequence propensities and structural protection or sequence propensities only, and a structure protection factor. The detailed analysis mode also provides graphical output for all Asn residues in the query sequence. We applied NGOME-Lite to over 257,000 sequences in 38 proteomes and found that different taxa differ in their predicted deamidation dynamics. Spontaneous protein deamidation is faster in Eukarya than in Bacteria because of a higher degree of structural protection in the latter. Predicted protein deamidation half-lifes correlate with protein turnover in human, mouse, rat, C. elegans and budding yeast but not in two plants and two bacteria. NGOME-Lite is implemented in a docker container available at https://ngome.proteinphysiologylab.org., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Asparagine is one of the precursors of acrylamide and toxic fungal secondary metabolites, both carcinogenic compounds. In the present study, the optimal conditions to deplete asparagine by Aureobasidium pullulans (L1 and L8) from potato and wheat flour matrices were investigated. Through a colorimetric plate-assay with phenol red as indicator dye, both strains demonstrated to be able to produce L-asparaginase from 20 ∘C to 30 ∘C for L1 and only at 20 ∘C for L8 strain starting from 48 h of incubation. The ability of both yeasts to reduce asparagine content in potato and wheat flour was studied by in vitro spectrophotometric assay. Both strains showed a great ability to totally reduce asparagine at 20 ∘C after 15 min of incubation in potato homogenate, conversely in wheat flour, the highest reduction was detected after a longer exposition time (60 min). As known, L1 and L8 diamine asparaginase to aspartic acid. For this reason, both amino acids were tested to verify the antifungal effect against Rhizoctonia solani (Rs1) and Fusarium graminearum (F3) mycelial growth . Asparagine (120 mg/L) increased Rs1 and F3 mycelial growth respectively by 4.4% and 18.9%; conversely, aspartic acid significantly inhibited both respectively by 8.2% and 12.0%., Competing Interests: The author declares no conflict of interest., (© 2022 The Author(s). Published by IMR Press.)

Deamidation of asparagine (Asn) and isomerization of aspartic acid (Asp) residues are among the most commonly observed spontaneous post-translational modifications (PTMs) in proteins. Understanding and predicting a protein sequence's propensity for such PTMs can help expedite protein therapeutic discovery and development. In this study, we used proton-affinity calculations with semi-empirical quantum mechanics and microsecond long equilibrium molecular dynamics simulations to investigate mechanistic roles of structural conformation and chemical environment in dictating spontaneous degradation of Asn and Asp residues in 131 clinical-stage therapeutic antibodies. Backbone secondary structure, side-chain rotamer conformation and solvent accessibility were found to be key molecular indicators of Asp isomerization and Asn deamidation. Comparative analysis of backbone dihedral angles along with N-H proton affinity calculations provides a mechanistic explanation for the strong influence of the identity of the n + 1 residue on the rate of Asn/Asp degradation. With these findings, we propose a minimalistic physics-based classification model that can be leveraged to predict deamidation and isomerization propensity of proteins.

The conformational preferences of three azadipeptides Ac-Pro-azaXaa-NHMe [Xaa = Asn (1), Asp (2), Ala (3)] have been carried out in gas phase and solution (water) using the density functional method B3LYP/6-311 +  + G(d,p) to explore the effect of the change of side chain of azaamino acids at the i + 2 position on the stability of these components. The most stable conformations of compounds (1), (2), and (3) have an amid bond oriented trans, trans, and cis, respectively, in gas phase, whereas the orientation of amid bond in water solvent of compounds (2) and (3) has changed to cis and trans, respectively. We have also noticed the importance of backbone-side chain hydrogen bonds in the stabilization of the β turn motif in gas phase since this motif is more stable in the case of compounds (1) and (2) and less stable in the case of compound (3) in which these hydrogen bonds are absent. Furthermore, the βII(βII') turn structure is more stable than βI turn for all conformations of the three compounds in gas phase, while it is not true in the case of some conformations in solution. Moreover, the stability of β turn increases from azaAsn to azaAsp which could be due to the side chain's basic nature of azaAsn. In general, hydrogen bonds were found to play a key role in the stabilization of these compounds since most of conformers are lower in energy when they have more than two hydrogen bond interactions while conformations with one or no hydrogen bonds are higher in energy and thus less stable., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)