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Epidermal growth factor-like domains (EGFDs) have important functions in cell-cell signaling. Both secreted and cell surface human EGFDs are subject to extensive modifications, including aspartate and asparagine residue C3-hydroxylations catalyzed by the 2-oxoglutarate oxygenase aspartate/asparagine-β-hydroxylase (AspH). Although genetic studies show AspH is important in human biology, studies on its physiological roles have been limited by incomplete knowledge of its substrates. Here, we redefine the consensus sequence requirements for AspH-catalyzed EGFD hydroxylation based on combined analysis of proteomic mass spectrometric data and mass spectrometry-based assays with isolated AspH and peptide substrates. We provide cellular and biochemical evidence that the preferred site of EGFD hydroxylation is embedded within a disulfide-bridged macrocycle formed of 10 amino acid residues. This definition enabled the identification of previously unassigned hydroxylation sites in three EGFDs of human fibulins as AspH substrates. A non-EGFD containing protein, lymphocyte antigen-6/plasminogen activator urokinase receptor domain containing protein 6B (LYPD6B) was shown to be a substrate for isolated AspH, but we did not observe evidence for LYPD6B hydroxylation in cells. AspH-catalyzed hydroxylation of fibulins is of particular interest given their important roles in extracellular matrix dynamics. In conclusion, these results lead to a revision of the consensus substrate requirements for AspH and expand the range of observed and potential AspH-catalyzed hydroxylation in cells, which will enable future study of the biological roles of AspH.

Amino acids are essential measurements for the potential growth stage because of connecting to protein structures and functions. The objective of this paper was to analyze chromosomes feature at plastid region of rice represented by nucleotide, synonymous codon, and amino acid usage to predict gene expression through codon usage pattern. The results showed that the values of the codon adaption index ranged from 0.733 in chromosome 9 to 0.631 in chromosome 8 with full length of these two chromosomes were 3738 and 1635 respectively. The higher value of guanine and cytosine content was 60% in chromosomes 9 while the lower values was 37% in chromosomes 11. Eight chromosomes (ch1, ch2, ch3, ch5, ch7, ch8, ch10, and ch12) were greater value of modified relative codon bias than threshold (threshold: 0.66) especially in cysteine for ch1, ch2, ch5, ch10, and ch12. While other remaining chromosomes were less than the threshold. Relative synonymous codon usage found that the over-represented of amino acids were asparagine, aspartate, cysteine, glutamate, and phenylalanine across all 12 chromosomes. These results would establish a platform for more and further projects concerning rice breeding and genetics and codon optimization in the amino acids for developing varieties. These results also will help breeders to select desirable genes through the genome for improve target traits.

L-Asparaginase is an antileukemic agent that depletes L-asparagine “an important nutrient for cancer cells” through the hydrolysis of L-asparagine into L-aspartic acid and ammonia leading to leukemia cell starvation and apoptosis in susceptible leukemic cell populations. Moreover currently, bacterial L-asparaginase has been limited by problems of lower productivity, stability, selectivity and a number of toxicities along with the resistance towards bacterial L-asparaginase. Then the current work aimed to provide pure L-asparaginase with in-vitro efficacy against various human carcinomas without adverse effects related to current L-asparaginase formulations. Submerged fermentation (SMF) bioprocess was applied and improved to maximize L-asparaginase production from Fusarium equiseti AHMF4 as alternative sources of bacteria. The enzyme production in SMF was maximized to reach 40.78 U mL−1 at the 7th day of fermentation with initial pH 7.0, incubation temperature 30 °C, 1.0% glucose as carbon source, 0.2% asparagine as nitrogen source, 0.1% alanine as amino acid supplement and 0.1% KH2PO 4 . The purification of AHMF4 L-asparaginase yielded 2.67-fold purification and 48% recovery with final specific activity of 488.1 U mg−1 of protein. Purified L-asparaginase was characterized as serine protease enzyme with molecular weight of 45.7 kDa beside stability at neutral pH and between 20 and 40 °C. Interestingly, purified L-asparaginase showed promising DPPH radical scavenging activity (IC50 69.12 μg mL−1) and anti-proliferative activity against cervical epitheloid carcinoma (Hela), epidermoid larynx carcinoma (Hep-2), hepatocellular carcinoma (HepG-2), Colorectal carcinoma (HCT-116), and breast adenocarcinoma (MCF-7) with IC50 equal to 2.0, 5.0, 12.40, 8.26 and 22.8 μg mL−1, respectively. The enzyme showed higher activity, selectivity and anti-proliferative activity against cancerous cells along with tiny cytotoxicity toward normal cells (WI-38) which indicates that it has selective toxicity and it could be applied as a less toxic alternative to the current formulations.

Graphical abstract, Sweet cherry fruit cracking is a complex physiological disorder that causes significant economic losses. Despite many years of research there is a lack of understanding of the mechanisms involved in cracking. Here, skin and flesh tissue from the cracking susceptible 'Early Bigi’ and the cracking tolerant ‘Regina’ cultivars were sampled prior and just after water dipping treatment to identify water-affected metabolic networks that putatively involved in fruit cracking. Primary metabolites, most strongly those involved in sugars and amino acid metabolism, such as glucose and asparagine, shifted in 'Early Bigi’ compared with ‘Regina’ tissues following water exposure. Comparisons between cultivars, tissues and dipping points identified significant differentially expressed genes. Particularly, genes related to abscisic acid, ethylene biosynthesis, pectin metabolism, expansins and aquaporins were altered in water-exposed tissues. To further characterize the role of these genes in cracking, their single nucleotide variants of the coding regions was studied in another eight sweet cherry cultivars, which differ in their sensitivity to cracking, revealing a strong link mainly between pectin metabolism-related genes and cracking-phenotypes. Integrated metabolomic and transcriptomic profiling uncovered genotypic- and tissue-specific metabolic pathways, including tricarboxylic acid cycle, cell enlargement, lipid and ethanol biosynthesis, and plant defense that putatively are involved in fruit cracking. Based on these results, a model which describes the skin and flesh metabolic reprogramming during water-induced fruit cracking in the susceptible 'Early Bigi’ cultivar is presented. Τhis study can help to explore novel candidate genes and metabolic pathways for cracking tolerance in sweet cherry.

Asparagine is an efficient nitrogen transport and storage carrier. Asparagine synthesis occurs by the amination of aspartate which is catalyzed by asparagine synthetase (ASN) in plants. Complete genome-wide analysis and classifications of the ASN gene family have recently been reported in different plants. However, systematic analysis and expression profiles of these genes have not been performed in apple (Malus domestica). Here, a comprehensive bioinformatics approach was applied to identify MdASNs in apple. Then, plant phylogenetic tree, chromosome location, conserved protein motif, gene structure, and expression pattern of MdASNs were analyzed. Five members were identified and distributed on 4 chromosomes with conserved GATase-7 and ASN domains. Expression analysis indicated that all MdASNs mRNA accumulated at the highest level in reproductive organs, namely flowers or fruits, which may be associated with the redistribution of free amino acids in plant metabolic organs and reservoirs. Additionally, most of MdASNs were dramatically up-regulated under various nitrogen supplies, especially in the aboveground part. Taken together, MdASNs may be assigned to be responsible for the nitrogen metabolism and asparagine synthesis in apple.

l -Asparaginases catalyze the conversion of l -asparagine to aspartate. Their applications as antineoplastic drug against different types of cancers, as tool for biosensing asparagine levels in blood and food items, and as processing aid for acrylamide mitigation have created special interest in these enzymes. Some of these applications require thermostable l -asparaginases and hyperthermophiles are a good source of such enzymes. A search for homologs of l -asparaginases in the genomes of hyperthermophilic archaea revealed that most of them contain more than one type of l -asparaginase. In fact, two types of l -asparaginases have been characterized from members of Euryarchaeota and Crenarchaeota. This chapter describes l -asparaginases from hyperthermophilic archaea and their potential applications.

Protein content in the inferior grains (IG) has a great impact on eating quality of indica-japonica rice hybrids. However, the mechanisms of the difference in protein accumulation between IG and superior grains (SG) remains unclear. A field experiment was carried out using an indica-japonica hybrid Yongyou17 and two nitrogen levels. The result showed that prolamin concentration was significantly higher in IG than SG, while there was no significant difference in glutelin concentration between the two types of grains. Totally, 465 metabolites were identified, and 26 “core metabolites” differentially accumulated in IG could be divided into three types. Asparagine and 1-aminocyclopropanecarboxylic acid were markedly increased in IG, while flavonoid compounds were obviously reduced, in comparison with those in SG. Weighted gene co-expression network analysis indicated that bisque4 and darkorange2 modules for type 1 and type 3 of the “core metabolites” were closely associated with the synthesis of ribosomal protein and stress response, respectively. Eight hub genes, i.e. rpL32_8.1, novel.907, RPS15a and RPL13a.4 in bisque4 and OsSAP5, OsHSP20, PRX12 and DUF3741 were identified in darkorange2. In addition, genes WDR, R2R3-MYB and bHLHs were significantly down-regulated in IG, companied by reduced synthesis of flavonoid compounds. IG had higher ethylene and N levels, resulting more protein accumulation. The current study provides a new insight into the regulation network of protein synthesis in IG of indica-japonica rice hybrids.

L-asparaginase is an enzyme that catalyzes the degradation of asparagine and successfully used in the treatment of acute lymphoblastic leukemia. L-asparaginase toxicity is either related to hypersensitivity to the foreign protein or to a secondary L-glutaminase activity that causes inhibition of protein synthesis. PEGylated versions have been incorporated into the treatment protocols to reduce immunogenicity and an alternative L-asparaginase derived from Dickeya chrysanthemi is used in patients with anaphylactic reactions to the E. coli L-asparaginase. Alternative approaches commonly explore new sources of the enzyme as well as the use of protein engineering techniques to create less immunogenic, more stable variants with lower L-glutaminase activity. This article reviews the main strategies used to overcome L-asparaginase shortcomings and introduces recent tools that can be used to create therapeutic enzymes with improved features.

The β subunit of bovine luteinizing hormone (LH) was crystallized and its structure solved to 3.15 ​Å resolution by molecular replacement using human chorionic gonadotropin (hCG) β subunit as search model. The asymmetric unit contains two copies of the β subunit that are related by a non-crystallographic symmetry (NCS) two-fold axis, both copies of which contain proteolytic cleavages after amino acid 100. It is noteworthy that the oligosaccharide moieties covalently attached at asparagine 13 were particularly pronounced in the electron density, allowing seven sugar residues to be defined. The α subunit of LH, which is common to all glycosylated gonadotropin hormones, was placed by superposition of hCG on the LH beta subunits, thereby yielding a model for the intact hormone., Graphical abstract A model of luteinizing hormone based on the known α subunit and the β subunit determined here by X-ray crystallography.Image 1, Highlights • The X-ray crystallographic structure of the beta subunit of bovine luteinizing hormone (LH) has been determined at 3.15 A resolution • A model for the entire LH has been proposed based on the beta subunit structure reported here and the known structure if the common alpha subunit • A large amount (7 sugar residues on one molecule in the asymmetric unit, 5 on the other) of the oligosaccharide attached at asn 13 was visible in electron density maps • Each of the two molecules in the asymmetric unit, which contains an NCS twofold axis, is associated with two molecules of beta octylglucoside • The beta subunit has a proteolytic cleavage between amino acids 100 and 101, which releases the “seatbelt” peptide, though that peptide remains associated through an S–S bridge to the core.

SUMMARY Artificial glycan holes on recombinant Env-based vaccines occur when a potential N-linked glycosylation site (PNGS) is under-occupied, but not on their viral counterparts. Native-like SOSIP trimers, including clinical candidates, contain such holes in the glycan shield that induce strain-specific neutralizing antibodies (NAbs) or non-NAbs. To eliminate glycan holes and mimic the glycosylation of native BG505 Env, we replace all 12 NxS sequons on BG505 SOSIP with NxT. All PNGS, except N133 and N160, are nearly fully occupied. Occupancy of the N133 site is increased by changing N133 to NxS, whereas occupancy of the N160 site is restored by reverting the nearby N156 sequon to NxS. Hence, PNGS in close proximity, such as in the N133-N137 and N156-N160 pairs, affect each other’s occupancy. We further apply this approach to improve the occupancy of several Env strains. Increasing glycan occupancy should reduce off-target immune responses to vaccine antigens., Graphical Abstract, In brief Derking et al. present a strategy to enhance glycan occupancy on recombinant HIV-1 envelope trimers to mimic that of the virus. This strategy eliminates artificial glycan holes on these trimers that induce non-neutralizing antibodies. The induction of these antibodies could be distractive for immuno-focusing strategies.

Hind-limb unloaded (HU) mouse is a well-recognized model of muscle atrophy; however, the molecular changes in the skeletal muscle during unloading are poorly characterized. We have used Raman spectroscopy to evaluate the structure and behavior of signature molecules involved in regulating muscle structural and functional health. The Raman spectroscopic analysis of gastrocnemius muscles was compared between 16-18 weeks old HU c57Bl/6J mice and ground-based controls. The spectra showed that the signals for asparagine and glutamine were reduced in HU mice, possibly indicating increased catabolism. The peaks for hydroxyproline and proline were split, pointing towards molecular breakdown and reduced tendon repair. We also report a consistently increased intensity in> 1300 cm-1 range in the Raman spectra along with a shift towards higher frequencies in the HU mice, indicating activation of sarcoplasmic reticulum (SR) stress during HU., Highlights • A thorough understanding of molecular changes underlying muscle detriment in disuse partly remains elusive. • The Raman spectroscopy is applied to characterize the molecular changes in the skeletal muscle of hindlimb unloaded mice. • The conformational changes and the breakdown of the molecular bonds in the skeletal muscle dictates loss of muscle mass. • The therapeutic drugs targeting specific molecules can be designed by identifying the changes in the skeletal muscles.

The spontaneous conversion of asparagine residues to aspartic acid or iso-aspartic acid, via deamidation, is a major pathway of protein degradation and is often seriously disruptive to biological systems. Deamidation has been shown to negatively affect both in vitro stability and in vivo biological function of diverse classes of proteins. During protein therapeutics development, deamidation liabilities that are overlooked necessitate expensive and time-consuming remediation strategies, sometimes leading to termination of the project. In this paper, we apply machine learning to a large (n = 776) liquid chromatography-tandem mass spectrometry (LC-MS/MS) dataset of monoclonal antibody peptides to create computational models for the post-translational modification asparagine deamidation, using the random decision forest method. We show that our categorical model predicts antibody deamidation with nearly 5% increased accuracy and 0.2 MCC over the best currently available models. Surprisingly, our model also paces or outperforms advanced and conventional models on an independent non-antibody dataset. In addition to deamidation probability, we are able to accurately predict deamidation rate (R2 = 0.963 and Q2 = 0.822), a capability with no peer in current models. This method should enable significant improvement in protein candidate selection, especially in biopharmaceutical development, and can be applied with similar accuracy to enzymes, monoclonal antibodies, next-generation formats, vaccine component antigens, and gene therapy vectors such as adeno-associated virus. Keywords: deamidation, machine learning, prediction, developability, stability, drug development, therapeutic protein, antibody, mab, IgG

The composition of Serra da Estrela PDO cheeses (total fat, total protein, salt and free amino acids) was assessed using NIR spectrophotometry and UPLC-DAD-MS/MS. In total, 24 cheeses were acquired from 6 certified cheesemakers located in 5 different municipalities within the delimited PDO geographical region. Cheeses were produced from raw ewe milk of two autochthonous Portuguese sheep breeds, between November 2017 and March 2018, and were acquired after 45 days of maturation. The data include the mean (and respective standard deviations) levels of moisture (%), total fat (%), total protein (%) and salt (%), obtained by NIR spectrophotometry. As well the mean (and respective standard deviations) of free amino acids contents (mg/100 g of cheese, in wet basis) evaluated using a UPLC-DAD-MS/MS method are also shown. The latter data include information regarding 8 essential free amino acids (histidine, leucine-isoleucine, lysine, methionine, phenylalanine, threonine, tryptophan and valine) and 9 non-essential free amino acids (arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, proline, tyrosine and serine). Leucine and isoleucine, being isomers, were quantified together. Leucine-isoleucine, phenylalanine and serine were the most abundant essential free amino acids and cysteine, proline and asparagine were the most abundant non-essential free amino acids. Free amino acids contents depended on the cheese producer as well as on the production time-period., The authors are grateful to the Foundation for Science and Technology (FCT, Portugal) and FEDER under Programme PT2020 for financial support to CIMO - UID/AGR/00690/2019, to Associate Laboratory LSRE-LCM - UID/EQU/50020/2019, to the strategic funding CEB - UID/BIO/04469/2019 and BioTecNorte operation (NORTE-01-0145-FEDER-000004). The authors would also like to acknowledge the funding provided by the approved Project, with reference 02/SAICT/2016/23290, entitled "Characterization and Valorization of QSE PDO and its ability for health promotion (QCLASSE)", financed by FCT. S. Falcão thanks National funding by FCT- Foundation for Science and Technology, P.I., through the institutional scientific employment program-contract., info:eu-repo/semantics/publishedVersion

11 pags., 6 figs., 4 tabs., The aim of this work was to determine the effect of temperature on the formation of acrylamide in cocoa beans during drying treatment by an experimental and computational study, in order to assess the presence of this neoformed compound from postharvest stage. The computational study was conducted on the reaction between fructose, glyoxal from glucose, and on asparagine at the M06-2X/6-31+G(d,p) level, under cocoa bean drying conditions at 323.15 to 343.15 K. The proposed reaction for acrylamide formation consisted of seven steps, which required to progress a via cyclic transition state of the four members. In addition, step III (decarboxylation) was considered to be the rate-determining step. Glucose followed an E1-like elimination and fructose exhibited an E1cb-like elimination. Computational model showed that the reaction of acrylamide formation was favored by fructose rather than glucose. The content of reducing sugars, asparagine and acrylamide in fermented and dried cocoa from two subregions of Antioquia-Colombia, as well as roasted cocoa, were evaluated by UHPLC-C-CAD and UHPLC-QqQ. The concentrations of monosaccharides measured at the end of the fermentation and drying process of cocoa nibs showed greater decreases in the levels of fructose as compared to glucose, supporting the main model hypothesis. Acrylamide formation only occurred in Bajo Cauca due to the presence of both precursors and fast drying time (72 h). Finally, it was possible to find the conditions to which acrylamide can be formed from the drying process and not only from roasting, information that can be used for future control strategies., This work was supported by Government of Antioquia through Science and Technology Fund (General System of Royalties), Antioquia-Colombia, contract 4600003895. It was also supported by Colciencias-Colfuturo and Universidad Nacional de Colombia, scholarship No. 647-2015.

Acrylamide is a process contaminant formed in bread and other baked products during baking. It forms because of a reaction between reducing sugars and the amino acid asparagine via the Maillard reaction. Since the discovery of acrylamide in food in 2002 and the related public health concerns, much effort has been and continues to be put into reducing the levels in food. Bread may not appear to contain high levels of acrylamide when compared to other cereal or potato products but because of its high consumption rate it is one of the big contributors to daily acrylamide intake in many countries and hence the importance of acrylamide reduction in breads. This chapter reviews the background to acrylamide in food, gives an overview of the mitigation techniques, looks at guidelines and legislation, and gives suggestions on how bakers can use this information to reduce acrylamide levels in their products.

Proliferation of acute lymphoblastic leukemic cells is nutritionally dependent on the external supply of asparagine. l-asparaginase, an enzyme hydrolyzing l-asparagine in blood, is used for treatment of acute lymphoblastic leukemic and other related blood cancers. Although previous studies demonstrated that l-asparaginase suppresses the proliferation of cultured solid tumor cells, it remains unclear whether this enzyme prevents the growth of solid tumors in vivo. In this study, we demonstrated the importance of optimizing dosing schedules for the anti-tumor activity of l-asparaginase in 4T1 breast tumor-bearing mice. Cultures of several types of murine solid tumor cells were dependent on the external supply of asparagine. Among them, we selected murine 4T1 breast cancer cells and implanted them into BALB/c female mice kept under standardized light/dark cycle conditions. The growth of 4T1 tumor cells implanted in mice was significantly suppressed by intravenous administration of l-asparaginase during the light phase, whereas its administration during the dark phase failed to show significant anti-tumor activity. Decreases in plasma asparagine levels due to the administration of l-asparaginase were closely related to the dosing time-dependency of its anti-tumor effects. These results suggest that the anti-tumor efficacy of l-asparaginase in breast tumor-bearing mice is improved by optimizing the dosing schedule. Keywords: l-asparaginase, Asparagine, Solid tumor, Chrono-pharmacotherapy

A newly isolated salt-tolerant alkaliphilic actinomycete, Nocardiopsis dassonvillei strain OK-18 grows on mineral salts medium with glucose as carbon source. It also grows and produces protease with amino acids as sole carbon source. The synthesis of extracellular alkaline protease parallel to growth was repressible by substrate concentrations. The absolute production of the protease was delinked with growth under nutritional stress, as protease production was high, despite poor growth. When amino acids served as the sole source of carbon and nitrogen, the enzyme production was significantly controlled by the number of amino acids. Maximal protease production was achieved with proline, asparagine, tyrosine, alanine, methionine and valine as sole source of carbon and nitrogen in minimal medium. With the increasing number of different amino acids in the presence and absence of glucose, the protease production was synergistically lower as compared to complex medium.

Microbacterium oleivorans is a predominant member of hydrocarbon-contaminated environments. We here report on the genomic analysis of M. oleivorans strain Wellendorf that was isolated from an indoor door handle. The partial genome of M. oleivorans strain Wellendorf consists of 2,916,870bp of DNA with 2831 protein-coding genes and 49 RNA genes. The organism appears to be a versatile mesophilic heterotroph potentially capable of hydrolysis a suite of carbohydrates and amino acids. Genomic analysis revealed metabolic versatility with genes involved in the metabolism and transport of glucose, fructose, rhamnose, galactose, xylose, arabinose, alanine, aspartate, asparagine, glutamate, serine, glycine, threonine and cysteine. This is the first detailed analysis of a Microbacterium oleivorans genome.

BackgroundTau cleavage by different proteolytic enzymes generates short, aggregation-prone fragments that have been implicated in the pathogenesis of Alzheimer’s disease (AD). Asparagine endopeptidase (AEP) activity in particular has been associated with tau dysfunction and aggregation, and the activity of the protease is increased in both aging and AD.Methods and ResultsUsing a mass spectrometry approach we identified a novel tau cleavage site at N167 and confirmed its processing by AEP. In combination with the previously known site at N368, we show that AEP cleavage yields a tau fragment that is present in both control and AD brains at similar levels. AEP is a lysosomal enzyme, and our data suggest that it is expressed in microglia rather than in neurons. Accordingly, we observe tau cleavage at N167 and N368 after endocytotic uptake into microglia, but not neurons. However, tau168-368 does not accumulate in microglia and we thus conclude that the fragment is part of a proteolytic cascade leading to tau degradation.ConclusionsWhile we confirm previous studies showing increased overall AEP activity in AD brains, our data suggests that AEP-mediated cleavage of tau is a physiological event occurring during microglial degradation of the secreted neuronal protein. The disease-associated increase in active AEP may thus be related to pro-inflammatory conditions in AD brains, and our findings argue against AEP inhibition as a therapeutic approach in AD.

In recent years, there has been a consensus that functional foods play a key role in reducing disease and improving health. The development of these food products using biotechnology processes has become a promising area of research in academia and industry. Protein sources and associated by-products can be used to produce bioactive hydrolysates and peptides with potential use as functional food ingredients. Resistant starches that improve colonic health and microbiota can be produced by enzymatic hydrolysis of starch sources. Lipases can be used for the production of certain fatty acids as flavoring and antioxidant esters. Enzymes such as tannase, phytase, and l -asparaginase have certain specificities for the production of functional foods. Tannase is used for bioconversion of tannin-rich materials into value-added products. l -Asparaginase is an enzyme that catalyzes the hydrolysis of l -asparagine, not allowing the reaction of reducing sugars with this amino acid for the generation of acrylamide. Phytase has been found increasingly interesting for use in the processing and manufacturing of food, particularly because the decline in food phytate results in enhancement of mineral bioavailability. Accordingly, this chapter will present a review of recent studies from the technical literature that explored different enzymes for the production of functional foods.

The glycation of hemoglobin to form stable Amadori products and advanced glycation end products causes structural perturbation in the protein. We have applied Fourier-transform infrared (FTIR) spectroscopy and a coupled HPLC-ATR-FTIR system toward the characterization of the reaction products (Rt = 5.5–6.5 min) at pH 8 in the T = 25–140°C range of the Glucose/Asparagine model system. The initial reaction species of the Asn-Gluc reaction were characterized by detection of the vibrational marker bands of the CAsn N CGluc moiety of the Schiff base. The UV-vis spectrum of the reaction product of Hb-Schiff base showed the formation of a 423 nm species and the FTIR spectrum modifications in the AmideIvibration. The FTIR band observed at 1611 cm− 1 indicates the formation of beta-sheet structures. Taken together, these observations suggest the formation of a stable six-coordinated low-spin His64-Fe(III)-His93 species, which is known as hemichrome in which the distal His64 (F6) is coordinated to the heme Fe, thereby blocking irreversibly the O2 binding site.

The allergenicity of gluten proteins in foods made with wheat flour, including bread, is a serious public health concern. The deamidation of gluten proteins, a reaction that converts glutamine and asparagine residues to glutamic-acid and aspartic-acid residue, respectively, is an effective method for reducing allergenicity and improving physicochemical functions, including the solubility and surface properties of gluten proteins. The deamidation of gluten proteins following acid-, alkaline-, heat-, enzyme-, or cation-exchange-resin treatment yields increased solubility in aqueous solution and improved emulsifying, foaming, and gelation properties compared with untreated proteins. In addition, the deamidation of gliadin, a major allergenic protein in gluten, using carboxylated cation-exchange resin is reportedly effective at reducing its reactivity with the sera of patients that exhibit a positive radioallergosorbent test to wheat and oral allergenicity, including intestinal permeability, increased serum allergen, or allergen-specific immunoglobulin E (IgE) levels, FcɛRI expression on mast cell surfaces, and increased serum and intestinal histamine levels.

Acrylamide is a processing contaminant that forms from free asparagine and reducing sugars, such as glucose, fructose and maltose, during high-temperature cooking and processing. Fried, baked and roasted potato, cereal and coffee products are the major sources of dietary intake. The Maillard reaction, which produces acrylamide, also gives rise to the colors, flavors and aromas associated with fried, baked and roasted foods. Acrylamide is a probable (Group 2a) human carcinogen and the European Food Safety Authority has expressed its concern regarding the possible neoplastic effects of dietary intake. This led the European Commission to propose tighter regulations on the presence of acrylamide in food. There is evidence that the most effective measures to reduce acrylamide formation in food by modifying processes and improving quality control have already been implemented, and further substantial reductions may not be achievable without a step change in the acrylamide-forming potential of crop products. This chapter describes the work that has been done to identify low acrylamide-forming varieties of potato, wheat and rye, define the crop management measures that can be taken to mitigate the problem, and elucidate the genetic control of acrylamide-forming potential. It also describes the progress that has been made in reducing the acrylamide-forming potential of potato by genetic modification, and the potential for further improvement in all affected crops using modern plant breeding and biotechnology techniques.

A number of clinical trials have shown that KRAS mutations of colorectal cancer (CRC) can predict a lack of responses to anti-epidermal growth factor receptor–based therapy. Recently, there have been several studies to elucidate metabolism reprogramming in cancer. However, it remains to be investigated how mutated KRAS can coordinate the metabolic shift to sustain CRC tumor growth. In this study, we found that KRAS mutation in CRC caused alteration in amino acid metabolism. KRAS mutation causes a marked decrease in aspartate level and an increase in asparagine level in CRC. Using several human CRC cell lines and clinical specimens of primary CRC, we demonstrated that the expression of asparagine synthetase (ASNS), an enzyme that synthesizes asparagine from aspartate, was upregulated by mutated KRAS and that ASNS expression was induced by KRAS-activated signaling pathway, in particular PI3K-AKT-mTOR pathway. Importantly, we demonstrated that KRAS-mutant CRC cells could become adaptive to glutamine depletion through asparagine biosynthesis by ASNS and that asparagine addition could rescue the inhibited growth and viability of cells grown under the glutamine-free condition in vitro. Notably, a pronounced growth suppression of KRAS-mutant CRC was observed upon ASNS knockdown in vivo. Furthermore, combination of L-asparaginase plus rapamycin markedly suppressed the growth of KRAS-mutant CRC xenografts in vivo, whereas either L-asparaginase or rapamycin alone was not effective. These results indicate ASNS might be a novel therapeutic target against CRCs with mutated KRAS.

Micrococcus luteus is a predominant member of skin microbiome. We here report on the genomic analysis of Micrococcus luteus strain O'Kane that was isolated from an elevator. The partial genome assembly of Micrococcus luteus strain O'Kane is 2.5Mb with 2256 protein-coding genes and 62 RNA genes. Genomic analysis revealed metabolic versatility with genes involved in the metabolism and transport of glucose, galactose, fructose, mannose, alanine, aspartate, asparagine, glutamate, glutamine, glycine, serine, cysteine, methionine, arginine, proline, histidine, phenylalanine, and fatty acids. Genomic comparison to other M. luteus representatives identified the potential to degrade polyhydroxybutyrates, as well as several antibiotic resistance genes absent from other genomes.

Gram-negative bacteria possess specialized biogenesis machineries that facilitate the export of amyloid subunits, the fibers of which are key components of their biofilm matrix. The secretion of bacterial functional amyloid requires a specialized outer-membrane protein channel through which unfolded amyloid substrates are translocated. We previously reported the crystal structure of the membrane-spanning domain of the amyloid subunit transporter FapF from Pseudomonas. However, the structure of the periplasmic domain, which is essential for amyloid transport, is yet to be determined. Here, we present the crystal structure of the N-terminal periplasmic domain at 1.8-Å resolution. This domain forms a novel asymmetric trimeric coiled coil that possesses a single buried tyrosine residue as well as an extensive hydrogen-bonding network within a glutamine layer. This new structural insight allows us to understand this newly described functional amyloid secretion system in greater detail.

Axenic culture of Leishmania is generally performed in rich, serum-supplemented media which sustain robust growth over multiple passages. The use of such undefined media, however, obscures proteomic analyses and confounds the study of metabolism. We have established a simple, defined culture medium that supports the sustained growth of promastigotes over multiple passages and which yields parasites that have similar infectivity to macrophages to parasites grown in a conventional semi-defined medium. We have exploited this medium to investigate the amino acid requirements of promastigotes in culture and have found that phenylalanine, tryptophan, arginine, leucine, lysine and valine are essential for viability in culture. Most of the 20 proteogenic amino acids promote growth of Leishmania promastigotes, with the exception of alanine, asparagine, and glycine. This defined medium will be useful for further studies of promastigote substrate requirements, and will facilitate future proteomic and metabolomic analyses.

l -Asparaginase II, which is widely accepted as the therapeutic enzyme for the treatment of leukemia and lymphoma, depletes the l -asparagine in the blood; hence, it hinders the protein synthetic machinery of cancerous tissue. It has a wide distribution in nature and each source has its own advantages. Through the intervention of recombinant DNA technology, the production of the enzyme could be manipulated in desirable ways. There are many obstacles to overcome for the effective use of this enzyme such as its immunogenicity, low plasma half-life, protease action, etc. Advances in the therapeutic field are helping to find solutions for these hindrances. At the same time, directed evolution and mutagenesis studies are ongoing to increase its properties such as thermostability and storage stability. The route of delivery is equally important to the development of the drug. Nanoparticle coating and PEGylation are the techniques being used nowadays. This chapter deals with the importance of asparaginase, its sources and method of production, and also the methods used to manipulate the strain for drug improvement to increase clinical efficiency.