Towards prediction of maturation-dependent kokumi taste in cheese by comprehensive high throughput quantitation of glutamyl dipeptides.

The study focuses on the comprehensive analysis of glutamyl dipeptides in cheese, particularly their formation during the cheese ripening process and the influence of various factors, such as origin, the use of various mold cultures, and cheese types. For the first time, all three subgroups of glutamyl dipeptides, namely α-Glu-X, X-Glu, and γ-Glu-X, are covered in a comprehensive analytical LC-MS/MS method offering robust quantitation of all 56 glutamyl dipeptides. The workflow includes a simplified extraction protocol and an optimized separation of the analytes on the stationary phase. Validation experiments demonstrate the method's reliability, including repeatability, detection limits, and recovery. The comprehensive analysis of all glutamyl dipeptides in 122 cheese samples with ripening times between 2 weeks and 15 years shows a strong increase in all peptide classes with prolonged ripening and particularly in the presence of mold.

Metabolomics Reveals the Regulatory Mechanisms of Antioxidant Dipeptides Enhancing the Tolerance of Lager Yeast against Ethanol Stress.

The antioxidant dipeptides (Ala-His, AH; Thr-Tyr, TY; and Phe-Cys, FC) significantly enhanced the lager yeast tolerance of ethanol stress. The enhancement mechanisms were further elucidated through physiological responses and metabolomics analysis. The results indicated that antioxidant dipeptides significantly increased the lager yeast biomass and budding rate. The primary mechanisms by which antioxidant dipeptides improved lager yeast tolerance involved decreasing intracellular reactive oxygen species (ROS) levels and increasing energy metabolism. Specifically, the addition of FC resulted in a 27.44% reduction in intracellular ROS content and a 26.14% increase in the ATP level compared to the control. Metabolomics analysis further explored the potential mechanisms underlying the protective effects of FC, identifying 63 upregulated and 103 downregulated metabolites. The analysis revealed that FC altered intracellular metabolites related to glutathione metabolism, purine metabolism, starch and sucrose metabolism, and ABC transporters, thereby enhancing yeast stress tolerance. The results suggest that FC is an effective enhancer for improving lager yeast tolerance to ethanol stress.

Cyclic homodimer formation by singlet oxygen-mediated oxidation of carnosine.

Although carnosine (ß-Ala-L-His) is one of physiological protectants against in vivo damages caused by reactive oxygen species (ROS), its reactivity against singlet oxygen (1O2), an ROS, is still unclear at the molecular level. Theoretically, the reaction consists of two steps: i) oxygenation of the His side chain to form an electrophilic endoperoxide and ii) nucleophilic addition to the endoperoxide. In this study, the end product of 1O2-mediated carnosine oxidation was evaluated using 2D-NMR and other analytical methods both in the presence and absence of external nucleophiles. Interestingly, as the end product without external nucleophile, a cyclic homodimer was confirmed under our particular conditions. The reaction was also replicated in pork specimens.

Efficient Solution-Phase Dipeptide Synthesis Using Titanium Tetrachloride and Microwave Heating.

Microwaves have been successfully employed in the Lewis acid titanium tetrachloride-assisted synthesis of peptide systems. Dipeptide systems with their amino function differently protected with urethane protecting groups have been synthesized in short periods of time and with high yields. The formation of the peptide bond between the two reacting amino acids was achieved in pyridine by using titanium tetrachloride as a condensing agent and heating the reaction mixture with a microwave reactor. The reaction conditions are compatible with amino acids featuring various side chains and different protecting groups on both the amino function and side chains. Additionally, the substrates retain their chiral integrity after reaction.

A Deep Mining Strategy for Peptide Rapid Identification in Lactobacillus reuteri Based on LC-MS/MS Integrated with FBMN and De Novo Sequencing.

Lactobacillus reuteri (L. reuteri) is widely recognized as a probiotic that produces prebiotics. However, studies on bioactive peptides or amino acid (AA) derivatives produced by L. reuteri are still lacking, whereas many bioactive peptides and AA derivatives have been found in other Lactobacillus species. In addition, rapid identification of peptides is challenged by the large amount of data and is limited by the coverage of protein databases. In this study, we performed a rapid and thorough profile of peptides in L. reuteri incorporating Global Natural Products Social Molecular Networking (GNPS) platform database searching, de novo sequencing, and deep mining, based on feature-based molecular networking (FBMN). According to FBMN, it was found that peptides containing identical or similar AA compositions were grouped into the same clusters, especially cyclic dipeptides (CDPs). Therefore, the grouping characteristics of clusters, differences in precursor ions, and characteristic fragment ions were utilized for the mining of deeply unknown compounds. Through this strategy, a total of 192 compounds, including 184 peptides, were rapidly identified. Among them, 53 CDPs, including four novel ones, were found for the first time in L. reuteri. Then, one of the novel CDPs, cyclo(5-OMe-Glu-4-OH-Pro), was isolated and characterized, which was consistent with the identification results. Moreover, some of the identified peptides exhibited considerable interactions with seven anti-inflammatory-related target proteins through molecular docking. According to the binding energies of peptides with different AA consistencies, it was considered that the existence of unnatural AAs in CDPs might contribute to their anti-inflammatory activity. These results provide a valuable strategy for the rapid identification of peptides, including CDPs. This study also reveals the substance basis for the potential anti-inflammatory effects exerted by L. reuteri.

Development of QSARs for cysteine-containing di- and tripeptides with antioxidant activity:influence of the cysteine position.

Antioxidants agents play an essential role in the food industry for improving the oxidative stability of food products. In the last years, the search for new natural antioxidants has increased due to the potential high toxicity of chemical additives. Therefore, the synthesis and evaluation of the antioxidant activity in peptides is a field of current research. In this study, we performed a Quantitative Structure Activity Relationship analysis (QSAR) of cysteine-containing 19 dipeptides and 19 tripeptides. The main objective is to bring information on the relationship between the structure of peptides and their antioxidant activity. For this purpose, 1D and 2D molecular descriptors were calculated using the PaDEL software, which provides information about the structure, shape, size, charge, polarity, solubility and other aspects of the compounds. Different QSAR model for di- and tripeptides were developed. The statistic parameters for di-peptides model (R2train = 0.947 and R2test = 0.804) and for tripeptide models (R2train = 0.923 and R2test = 0.847) indicate that the generated models have high predictive capacity. Then, the influence of the cysteine position was analyzed predicting the antioxidant activity for new di- and tripeptides, and comparing them with glutathione. In dipeptides, excepting SC, TC and VC, the activity increases when cysteine is at the N-terminal position. For tripeptides, we observed a notable increase in activity when cysteine is placed in the N-terminal position.

Anti-bacterial cyclic dipeptides from Hormonema dematioides, an endophytic fungus of Juniperus communis.

Hormonema sp., an endophytic fungi found in the medicinal plant of Juniperus communis leaves, was reported to possess antimicrobial compounds from its unidentified species. In this study, 21 cyclic dipeptides (1-21) were isolated and identified from H. dematioides. All the 21 isolated cyclic dipeptides were reported for the first time from the genus Hormonema. The antimicrobial activity by the disc diffusion method showed that five compounds including cyclo(Pro-Gly) (9), cyclo(Phe-Ile) (11), cyclo(Ile-Val) (12), cyclo(Val-Ala) (17), and cyclo(Ala-Phe) (20) inhibited the growth of Staphylococcus aureus with inhibition zone diameters ranging from 12 to 30 mm. Further bioassay demonstrated that four cyclic dipeptides (9, 12, 17, and 20) showed significant antibacterial activity against S. aureus with MIC values of 0.04, 0.39, 0.01, and 0.10 µg/mL, respectively, as compared to the positive control (ciprofloxacin, MIC = 0.08 µg/mL). However, none of these cyclic dipeptides showed obvious anti-fungal activity against Candida albicans.

Targeting prolidase. A survey of the literature data to depict a structure-activity relationship frame and to address future studies for drug development.

Prolidase (EC.3.4.13.9) is a Mn+2-dependent dipeptidase that is well known to play a crucial role in several physiological and pathological processes affecting humans. More in particular, this enzyme is involved in the cleavage of proline- and hydroxyproline-containing dipeptides (imidodipeptides), providing a fine regulation of the homeostasis of these two amino acids. Hyperactivity or deficiency of prolidase have been clearly associated to the development and progress of several acute and chronic syndromes (e.g. chronic liver fibrosis, viral and acute hepatitis, cancer, neurological disorders, inflammation, skin diseases, intellectual disability, respiratory infection). Thus, targeting prolidase and modulating its activity is an intriguing field of research with a great therapeutic potential for the next future and for the design of specific and selective drugs. Prolidase can be exploited in two essential ways: as an activator of proline containing prodrugs and by direct interaction. In this latter case, few specific ligands for the title enzyme have been described, but with no reports about their structure-activity relationship. The aim of this comprehensive review is to gather all available information on prolidase targeting so far reported in the literature, to rationalize the observed data and effect into a preliminary structure-relationship picture, to comment about the effectiveness of each reported ligands, and to address future research activities providing new potential and putative natural, semisynthetic, and purely synthetic molecules able to trigger prolidase as the main biological target.

Molecular self-assembled helix peptide nanotubes based on some amino acid molecules and their dipeptides: molecular modeling studies.

CONTEXT: The paper considers the features of the structure and dipole moments of several amino acids and their dipeptides which play an important role in the formation of the peptide nanotubes based on them. The influence of the features of their chirality (left L and right D) and the alpha-helix conformations of amino acids are taken into account. In particular, amino acids with aromatic rings, such as phenylalanine (Phe/F), and branched-chain amino acids (BCAAs)-leucine (Leu/L) and isoleucine (Ile/I)-as well as corresponding dipeptides (diphenylalanine (FF), dileucine (LL), and diisoleucine (II)) are considered. The main features and properties of these dipeptide structures and peptide nanotubes (PNTs), based on them, are investigated using computational molecular modeling and quantum-chemical semi-empirical calculations. Their polar, piezoelectric, and photoelectronic properties and features are studied in detail. The results of calculations of dipole moments and polarization, as well as piezoelectric coefficients and band gap width, for different types of helical peptide nanotubes are presented. The calculated values of the chirality indices of various nanotubes are given, depending on the chirality of the initial dipeptides-the results obtained are consistent with the law of changes in the type of chirality as the hierarchy of molecular structures becomes more complex. The influence of water molecules in the internal cavity of nanotubes on their physical properties is estimated. A comparison of the results of these calculations by various computational methods with the available experimental data is presented and discussed. METHOD: The main tool for molecular modeling of all studied nanostructures in this work was the HyperChem 8.01 software package. The main approach used here is the Hartree-Fock (HF) self-consistent field (SCF) with various quantum-chemical semi-empirical methods (AM1, PM3, RM1) in the restricted Hartree-Fock (RHF) and in the unrestricted Hartree-Fock (UHF) approximations. Optimization of molecular systems and the search for their optimal geometry is carried out in this work using the Polak-Ribeire algorithm (conjugate gradient method), which determines the optimized geometry at the point of their minimum total energy. For such optimized structures, dipole moments D and electronic energy levels (such as EHOMO and ELUMO), as well as the band gap Eg = ELUMO - EHOMO, were then calculated. For each optimized molecular structure, the volume was calculated using the QSAR program implemented also in the HyperChem software package.

Selection of adjunct cultures for the ripening of plant cheese analogues.

Fermentation contributes to the taste and odor of plant cheeses. The selection of functional cultures for the fermentation of plant cheeses, however, is in its infancy. This study aimed to select lactic acid bacteria for ripening of soy and lupin cheese analogues. Bacillus velezensis and B. amyloliquefaciens were used for germination of seeds to produce proteolytic enzymes; Lactococcus lactis and Lactiplantibacillus plantarum served as primary acidifying cultures. Levilactobacillus hammesii, Furfurilactobacillus milii, or Lentilactobacillus buchneri were assessed as adjunct cultures for the ripening of plant cheese. Growth of bacilli was inhibited at low pH. Both Lc. lactis and Lp. plantarum were inactived during plant cheese ripening. Cell counts of Lv. hammesii remained stable over 45 d of ripening while Ff. milii and Lt. buchneri grew slowly. Sequencing of full length 16S rRNA genes confirmed that the inocula the plant cheeses accounted for more than 98% of the bacterial communities. HPLC analysis revealed that Lt. buchneri metabolized lactate to acetate and 1,2-propanediol during ripening. Bacilli enhanced proteolysis as measured by quantification of free amino nitrogen, and the release of glutamate. LC-MS/MS analysis quantified kokumi-active dipeptides. The concentrations of γ-Glu-Leu, γ-Glu-Ile, and γ-Glu-Ala, γ-Glu-Cys in unripened cheeses were increased by seed germination but γ-Glu-Phe was degraded. Lt. buchneri but not Lv. hammesii or Ff. milii accumulated γ-Glu-Val, γ-Glu-Ile or γ-Glu-Leu during ripening, indicating strain-specific differences. In conclusion, a consortium of bacilli, acidification cultures and adjunct cultures accumulates taste- and kokumi-active compounds during ripening of plant cheeses.

The high-throughput solid-phase extraction of cis-cyclo(L-Leu-L-Pro) and cis-cyclo(L-Phe-L-Pro) from Lactobacillus plantarum demonstrates efficacy against multidrug-resistant bacteria and influenza A (H3N2) virus.

Introduction: 2,5-diketopiperazines are the simplest forms of cyclic dipeptides (CDPs) and have diverse frameworks with chiral side chains that are useful for drug development. Previous research has investigated the antimicrobial properties of proline-linked CDPs and their combinations in the culture filtrate (CF) of Lactobacillus plantarum LBP-K10 using anion exchange chromatography (AEC). However, the quantity of CDPs showcasing notable anti-influenza virus activity derived from AECs was generally lower than those originating from Lactobacillus CF. Methods: To address this issue, the study aims to propose a more efficient method for isolating CDPs and to introduce the antiviral combinations of CDPs obtained using a new method. The study employed a novel technique entailing high-throughput C18-based solid-phase extraction with a methanol gradient (MeSPE). The MeSPE method involved increasing the methanol concentration from 5% to 50% in 5% increments. Results: The methanol SPE fractions (MeSPEfs) eluted with methanol concentrations between 35% and 45% evinced substantial efficacy in inhibiting the influenza A/H3N2 virus via plaque-forming assay. MeSPEf-45, the 45% MeSPEf, exhibited exceptional efficacy in preventing viral infections in Madin-Darby kidney cells, surpassing both individual CDPs and the entire set of MeSPEfs. To identify the specific antiviral components of MeSPEf-45, all MeSPEfs were further fractionated through preparative high-performance liquid chromatography (prep-HPLC). MeSPEf-45 fractions S8 and S11 presented the highest activity against multidrug-resistant bacteria and influenza A/H3N2 virus among all MeSPEfs, with 11 common fractions. Antiviral fractions S8 and S11 were identified as proline-based CDPs, specifically cis-cyclo(L-Leu-L-Pro) and cis-cyclo(L-Phe-L-Pro), using gas chromatography-mass spectrometry. The combination of MeSPEf-45 fractions S8 and S11 displayed superior antibacterial and anti-influenza virus effects compared to the individual fractions S8 and S11. Discussion: High-throughput MeSPE-derived MeSPEfs and subsequent HPLC-fractionated fractions presents an innovative approach to selectively purify large amounts of potent antimicrobial CDPs from bacterial CF. The findings also show the effectiveness of physiologically bioactive combinations that utilize fractions not containing CDP. This study provides the initial evidence demonstrating the antimicrobial properties of CDPs acquired through high-throughput SPE techniques.

Chiral zwitterionic stationary phases based on Cinchona alkaloids and dipeptides: Application in chiral separation of dipeptides under reversed phase conditions.

Chromatographic behavior of novel chiral stationary phases with bonded selectors based on Cinchona alkaloids modified with dipeptides was studied using dipeptides as probe molecules. Buffer-free and salt containing hydro-organic solutions were used as the mobile phases. The selectors exhibit pseudoenantiomeric behavior with respect to the L/D or LL/DD enantiomers and do not behave so with respect to the LD/DL enantiomers. The alkaloid part of the selectors is the driver of enantioselectivity, while the dipeptide substituent plays a modulating role. The quinidine-based selectors demonstrate stronger adsorption affinity and higher enantioselectivity as compared to the quinine-based selectors. The dipeptide analytes containing a glycyl fragment are weaker retained and their enantiomers are worse separated comparing to dipeptides with both units being larger amino acids. Moreover, a phenyl group in the structure of a dipeptide analyte facilitates enantioseparation. The effect of the mobile phase composition on retention depends on the hydrophobicity of an analyte. Hydrophobic dipeptides are better eluted by methanol-rich solvents, hydrophilic dipeptides are better eluted with water-rich solvents, and dipeptides with an intermediate hydrophobicity demonstrate a U-shaped or more complicated dependence of the retention factor on the percentage of methanol. Even a small buffer addition to the mobile phase decreases retention, but the ion-exchange mechanism was not confirmed. The effect of an electrolyte is rather due to the shielding of the charged groups of the selector reducing thereby electrostatic interaction between the selector and analyte. Efficiency of the novel columns is comparable to that of other brush-type chiral columns, the highest achieved number of the theoretical plates per 1 m varying between 30,000 and 40,000.

Antibacterial and antibiofilm properties of two cyclic dipeptides produced by a new desert Streptomyces sp. HG-17 strain against multidrug-resistant pathogenic bacteria.

INTRODUCTION: The emergence of multidrug-resistant bacteria and biofilms requires discovering new antimicrobial agents from unexplored environments. OBJECTIVES: This study aims to isolate and characterize a new actinobacterial strain from the Hoggar Mountains in southern Algeria and evaluate its ability to produce bioactive molecules with potential antibacterial and antibiofilm activities. METHODS: A novel halotolerant actinobacterial strain, designated HG-17, was isolated from the Hoggar Mountains, and identified based on phenotypic characterizations, 16S rDNA sequence analysis, and phylogenetic analysis. The antibacterial and antibiofilm activities of the strain were assessed, and the presence of biosynthetic genes (PKS-I and NRPS) was confirmed. Two active compounds, HG-7 and HG-9, were extracted butanol solvent, purified by HPLC, and their chemical structures were elucidated using ESI mass spectrometry and NMR spectroscopy. RESULTS: The strain HG-17 was identified as Streptomyces purpureus NBRC with 98.8% similarity. It exhibited strong activity against multidrug-resistant and biofilm-forming bacteria. The two purified active compounds, HG-7 and HG-9, were identified as cyclo-(d-cis-hydroxyproline-l-phenylalanine) and cyclo-(l-prolone-l-tyrosine), respectively. The minimum inhibitory concentrations (MICs) of HG-7 and HG-9 ranged from 3 to 15 µg/mL, comparable to the MICs of tetracycline (8 to 15 µg/mL). Their minimum biofilm inhibitory concentration (MBIC 50%) showed good inhibition from 48.0 to 52.0% at concentrations of 1 to 7 µg/mL against the tested bacteria. CONCLUSION: This is the first report of cyclo-(d-cis-hydroxyproline-l-phenylalanine) and cyclo-(l-prolone-l-tyrosine) antibiotics from S. purpureus and their anti-multi-drug-resistant and biofilm-forming bacteria. These results indicate that both antibiotics could be used as effective therapeutics to control infections associated with multidrug-resistant bacteria.

The protective role of commensal gut microbes and their metabolites against bacterial pathogens.

Multidrug-resistant microorganisms have become a major public health concern around the world. The gut microbiome is a gold mine for bioactive compounds that protect the human body from pathogens. We used a multi-omics approach that integrated whole-genome sequencing (WGS) of 74 commensal gut microbiome isolates with metabolome analysis to discover their metabolic interaction with Salmonella and other antibiotic-resistant pathogens. We evaluated differences in the functional potential of these selected isolates based on WGS annotation profiles. Furthermore, the top altered metabolites in co-culture supernatants of selected commensal gut microbiome isolates were identified including a series of dipeptides and examined for their ability to prevent the growth of various antibiotic-resistant bacteria. Our results provide compelling evidence that the gut microbiome produces metabolites, including the compound class of dipeptides that can potentially be applied for anti-infection medication, especially against antibiotic-resistant pathogens. Our established pipeline for the discovery and validation of bioactive metabolites from the gut microbiome as novel candidates for multidrug-resistant infections represents a new avenue for the discovery of antimicrobial lead structures.

Peptide Transporter 1-Mediated Dipeptide Transport Promotes Hepatocellular Carcinoma Metastasis by Activating MAP4K4/G3BP2 Signaling Axis.

Cancer metastasis is the leading cause of mortality in patients with hepatocellular carcinoma (HCC). To meet the rapid malignant growth and transformation, tumor cells dramatically increase the consumption of nutrients, such as amino acids. Peptide transporter 1 (PEPT1), a key transporter for small peptides, has been found to be an effective and energy-saving intracellular source of amino acids that are required for the growth of tumor cells. Here, the role of PEPT1 in HCC metastasis and its underlying mechanisms is explored. PEPT1 is upregulated in HCC cells and tissues, and high PEPT1 expression is associated with poor prognosis in patients with HCC. PEPT1 overexpression dramatically promoted HCC cell migration, invasion, and lung metastasis, whereas its knockdown abolished these effects both in vitro and in vivo. Mechanistic analysis revealed that high PEPT1 expression increased cellular dipeptides in HCC cells that are responsible for activating the MAP4K4/G3BP2 signaling pathway, ultimately facilitating the phosphorylation of G3BP2 at Thr227 and enhancing HCC metastasis. Taken together, these findings suggest that PEPT1 acts as an oncogene in promoting HCC metastasis through dipeptide-induced MAP4K4/G3BP2 signaling and that the PEPT1/MAP4K4/G3BP2 axis can serve as a promising therapeutic target for metastatic HCC.

Gold and Silver Nanoparticles as Biosensors: Characterization of Surface and Changes in the Adsorption of Leucine Dipeptide under the Influence of Substituent Changes.

Early detection of diseases can increase the chances of successful treatment and survival. Therefore, it is necessary to develop a method for detecting or sensing biomolecules that cause trouble in living organisms. Disease sensors should possess specific properties, such as selectivity, reproducibility, stability, sensitivity, and morphology, for their routine application in medical diagnosis and treatment. This work focuses on biosensors in the form of surface-functionalized gold (AuNPs) and silver nanoparticles (AgNPs) prepared using a less-time-consuming, inexpensive, and efficient synthesis route. This allows for the production of highly pure and stable (non-aggregating without stabilizers) nanoparticles with a well-defined spherical shape, a desired diameter, and a monodisperse distribution in an aqueous environment, as confirmed by transmission electron microscopy with energy-dispersive X-ray spectroscopy (TEM-EDS), X-ray diffraction (XRD), photoelectron spectroscopy (XPS), ultraviolet-visible (UV-VIS) spectroscopy, and dynamic light scattering (DLS). Thus, these nanoparticles can be used routinely as biomarker sensors and drug-delivery platforms for precision medicine treatment. The NPs' surface was coated with phosphonate dipeptides of L-leucine (Leu; l-Leu-C(R1)(R2)PO3H2), and their adsorption was monitored using SERS. Reproducible spectra were analyzed to determine the orientation of the dipeptides (coating layers) on the nanoparticles' surface. The appropriate R2 side chain of the dipeptide can be selected to control the arrangement of these dipeptides. This allows for the proper formation of a layer covering the nanoparticles while also simultaneously interacting with the surrounding biological environment, such as cells, tissues, and biological fluids.

pH-sensitive release of nitric oxide gas using peptide-graphene co-assembled hybrid nanosheets.

Nitric oxide (NO) donating drugs such as organic nitrates have been used to treat cardiovascular diseases for more than a century. These donors primarily produce NO systemically. It is however sometimes desirable to control the amount, location, and time of NO delivery. We present the design of a novel pH-sensitive NO release system that is achieved by the synthesis of dipeptide diphenylalanine (FF) and graphene oxide (GO) co-assembled hybrid nanosheets (termed as FF@GO) through weak molecular interactions. These hybrid nanosheets were characterised by using X-ray diffraction, Raman spectroscopy, Fourier transform infrared spectroscopy, zeta potential measurements, X-ray photoelectron spectroscopy, scanning and transmission electron microscopies. The weak molecular interactions, which include electrostatic, hydrogen bonding and π-π stacking, are pH sensitive due to the presence of carboxylic acid and amine functionalities on GO and the dipeptide building blocks. Herein, we demonstrate that this formulation can be loaded with NO gas with the dipeptide acting as an arresting agent to inhibit NO burst release at neutral pH; however, at acidic pH it is capable of releasing NO at the rate of up to 0.6 µM per minute, comparable to the amount of NO produced by healthy endothelium. In conclusion, the innovative conjugation of dipeptide with graphene can store and release NO gas under physiologically relevant concentrations in a pH-responsive manner. pH responsive NO-releasing organic-inorganic nanohybrids may prove useful for the treatment of cardiovascular diseases and other pathologies.

Elucidating uptake and metabolic fate of dipeptides in CHO cell cultures using 13C labeling experiments and kinetic modeling.

The rapidly growing market of biologics including monoclonal antibodies has stimulated the need to improve biomanufacturing processes including mammalian host systems such as Chinese Hamster Ovary (CHO) cells. Cell culture media formulations continue to be enhanced to enable intensified cell culture processes and optimize cell culture performance. Amino acids, major components of cell culture media, are consumed in large amounts by CHO cells. Due to their low solubility and poor stability, certain amino acids including tyrosine, leucine, and phenylalanine can pose major challenges leading to suboptimal bioprocess performance. Dipeptides have the potential to replace amino acids in culture media. However, very little is known about the cleavage, uptake, and utilization kinetics of dipeptides in CHO cell cultures. In this study, replacing amino acids, including leucine and tyrosine by their respective dipeptides including but not limited to Ala-Leu and Gly-Tyr, supported similar cell growth, antibody production, and lactate profiles. Using 13C labeling techniques and spent media studies, dipeptides were shown to undergo both intracellular and extracellular cleavage in cultures. Extracellular cleavage increased with the culture duration, indicating cleavage by host cell proteins that are likely secreted and accumulate in cell culture over time. A kinetic model was built and for the first time, integrated with 13C labeling experiments to estimate dipeptide utilization rates, in CHO cell cultures. Dipeptides with alanine at the N-terminus had a higher utilization rate than dipeptides with alanine at the C-terminus and dipeptides with glycine instead of alanine at N-terminus. Simultaneous supplementation of more than one dipeptide in culture led to reduction in individual dipeptide utilization rates indicating that dipeptides compete for the same cleavage enzymes, transporters, or both. Dipeptide utilization rates in culture and cleavage rates in cell-free experiments appeared to follow Michaelis-Menten kinetics, reaching a maximum at higher dipeptide concentrations. Dipeptide utilization behavior was found to be similar in cell-free and cell culture environments, paving the way for future testing approaches for dipeptides in cell-free environments prior to use in large-scale bioreactors. Thus, this study provides a deeper understanding of the fate of dipeptides in CHO cell cultures through an integration of cell culture, 13C labeling, and kinetic modeling approaches providing insights in how to best use dipeptides in media formulations for robust and optimal mammalian cell culture performance.

Contribution of γ-Glutamyl-Cysteine Ligases of Limosilactobacillus reuteri to the Formation of Kokumi-Active γ-Glutamyl Dipeptides in Sourdough Bread.

Kokumi-active γ-glutamyl dipeptides accumulate during sourdough fermentation. γ-Glutamylcysteine ligases (Gcls) of Limosilactobacillus reuteri synthesize γ-glutamyl dipeptides during growth in sourdough. This study aimed to evaluate the contribution of Gcls from strains of L. reuteri in the formation of kokumi-active γ-glutamyl dipeptides in sourdough bread. Among 12 acceptor amino acids, the three Gcls of L. reuteri were the most active to Cys. With the acceptor amino acids Ile, Leu, and Phe, Gcl1 was more active than Gcl2 and Gcl3. Accordingly, Gcl1 contributed to the γ-Glu-Ile synthesis in sourdough fermentation. Proofing and baking strongly influenced the concentration of γ-glutamyl dipeptides in bread. The addition of 10% sourdough increased the content of γ-Glu-Leu and γ-Glu-Phe but not of other γ-glutamyl dipeptides in bread. In conclusion, the accumulation of kokumi γ-glutamyl dipeptides in sourdoughs was attributed to the combined activity of cereal enzymes, γ-glutamyl-cysteine ligases, and other microbial enzymes.

Orphan lysosomal solute carrier MFSD1 facilitates highly selective dipeptide transport.

Orphan solute carrier (SLC) represents a group of membrane transporters whose exact functions and substrate specificities are not known. Elucidating the function and regulation of orphan SLC transporters is not only crucial for advancing our knowledge of cellular and molecular biology but can potentially lead to the development of new therapeutic strategies. Here, we provide evidence for the biological function of a ubiquitous orphan lysosomal SLC, the Major Facilitator Superfamily Domain-containing Protein 1 (MFSD1), which has remained phylogenetically unassigned. Targeted metabolomics revealed that dipeptides containing either lysine or arginine residues accumulate in lysosomes of cells lacking MFSD1. Whole-cell patch-clamp electrophysiological recordings of HEK293-cells expressing MFSD1 on the cell surface displayed transport affinities for positively charged dipeptides in the lower mM range, while dipeptides that carry a negative net charge were not transported. This was also true for single amino acids and tripeptides, which MFSD1 failed to transport. Our results identify MFSD1 as a highly selective lysosomal lysine/arginine/histidine-containing dipeptide exporter, which functions as a uniporter.