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Biochimie Dec 2020One hypothesis regarding the cause of diabetic complications is that advanced glycation end products (AGEs) bind to the AGE receptor and induce changes in gene...
One hypothesis regarding the cause of diabetic complications is that advanced glycation end products (AGEs) bind to the AGE receptor and induce changes in gene expression. However, what AGEs exist in vivo and how individual AGEs are produced and impact body metabolic process to cause diabetes complications are not understood. We developed a new precise method to measure AGEs using LC-MS/MS with a new column and measured 7 free AGEs, including N(6)-carboxymethyllysine (CML), N(6)-(1-carboxyethyl)-l-lysine (CEL) and N5-(5-hydro-5-methyl-4-imidazolon-2-yl)L-ornithine (MG-H1), in human blood components. Blood was obtained from 9 people, and free AGEs were measured in individual blood components with LC-MS/MS before and after a meal. Free CML and CEL were abundant in erythrocytes, with 92% of free CML and 85% of free CEL localized in erythrocytes. In contrast, 60% of free MG-H1 was distributed in the serum. After the meal, free serum MG-H1 increased, but CML and CEL did not. CML and CEL are mainly distributed in erythrocytes and were not affected by the meal, indicating that they are produced in vivo. However, the main source of MG-H1 is the meal. The effect of genetic polymorphisms on AGEs was also investigated. Low activity type aldehyde dehydrogenase 2 (ALDH2) increased the CML concentration in the blood. This is the first observation that shows that the metabolic process of CML and CEL is different from that of MG-H1 and the effect of ALDH2 SNPs on CML.
Topics: Adult; Alcohol Dehydrogenase; Aldehyde Dehydrogenase, Mitochondrial; Chromatography, High Pressure Liquid; Erythrocytes; Female; Glycation End Products, Advanced; Healthy Volunteers; Humans; Lysine; Male; Meals; Middle Aged; Ornithine; Polymorphism, Single Nucleotide; Tandem Mass Spectrometry; Young Adult
PubMed: 32946992
DOI: 10.1016/j.biochi.2020.09.010 -
Biomolecules Jul 2021There is a pressing need for molecular targets and biomarkers in gastric cancer (GC). We aimed at identifying aberrations in L-arginine metabolism with therapeutic and...
There is a pressing need for molecular targets and biomarkers in gastric cancer (GC). We aimed at identifying aberrations in L-arginine metabolism with therapeutic and diagnostic potential. Systemic metabolites were quantified using mass spectrometry in 293 individuals and enzymes' gene expression was quantified in 29 paired tumor-normal samples using qPCR and referred to cancer pathology and molecular landscape. Patients with cancer or benign disorders had reduced systemic arginine, citrulline, and ornithine and elevated symmetric dimethylarginine and dimethylamine. Citrulline and ornithine depletion was accentuated in metastasizing cancers. Metabolite diagnostic panel had 91% accuracy in detecting cancer and 70% accuracy in differentiating cancer from benign disorders. Gastric tumors had upregulated and downregulated , , , and expression. upregulation was less and downregulation was more pronounced in metastatic cancers. Tumor and expression was inversely related to local advancement. Enzyme up- or downregulation was greater or significant solely in cardia subtype. Metabolic reprogramming in GC includes aberrant L-arginine metabolism, reflecting GC subtype and pathology, and is manifested by altered interplay of its intermediates and enzymes. Exploiting L-arginine metabolic pathways for diagnostic and therapeutic purposes is warranted. Functional studies on , , and in GC are needed.
Topics: Aged; Arginine; Argininosuccinate Lyase; Cell Differentiation; Citrulline; DNA, Complementary; Female; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Humans; Intracellular Signaling Peptides and Proteins; Male; Mass Spectrometry; Metabolomics; Middle Aged; Mitochondrial Membrane Transport Proteins; Neoplasm Metastasis; Nitric Oxide Synthase Type II; Ornithine; Polymerase Chain Reaction; Protein-Arginine N-Methyltransferases; Reproducibility of Results; Stomach Neoplasms; Transcriptome
PubMed: 34439753
DOI: 10.3390/biom11081086 -
Molecules (Basel, Switzerland) Jan 2023Ornithine aminotransferase (OAT) is overexpressed in hepatocellular carcinoma (HCC), and we previously showed that inactivation of OAT inhibits the growth of HCC....
Ornithine aminotransferase (OAT) is overexpressed in hepatocellular carcinoma (HCC), and we previously showed that inactivation of OAT inhibits the growth of HCC. Recently, we found that (3,4)-3-amino-4-fluorocyclopentenecarboxylic acid () was a potent inactivator of γ-aminobutyric acid aminotransferase (GABA-AT), proceeding by an enamine mechanism. Here we describe our investigations into the activity and mechanism of as an inactivator of human OAT. We have found that exhibits 10-fold less inactivation efficiency (/) against OAT than GABA-AT. A comprehensive mechanistic study was carried out to understand its inactivation mechanism with OAT. p and electrostatic potential calculations were performed to further support the notion that the α,β-unsaturated alkene of is critical for enhancing acidity and nucleophilicity of the corresponding intermediates and ultimately responsible for the improved inactivation efficiency of over the corresponding saturated analogue (). Intact protein mass spectrometry and the crystal structure complex with OAT provide evidence to conclude that mainly inactivates OAT through noncovalent interactions, and that, unlike with GABA-AT, covalent binding with OAT is a minor component of the total inhibition which is unique relative to other monofluoro-substituted derivatives. Furthermore, based on the results of transient-state measurements and free energy calculations, it is suggested that the α,β-unsaturated carboxylate group of PLP-bound may be directly involved in the inactivation cascade by forming an enolate intermediate. Overall, compound exhibits unusual structural conversions which are catalyzed by specific residues within OAT, ultimately leading to an enamine mechanism-based inactivation of OAT through noncovalent interactions and covalent modification.
Topics: Humans; Amino Acids; Carcinoma, Hepatocellular; Enzyme Inhibitors; Ornithine-Oxo-Acid Transaminase; Liver Neoplasms; gamma-Aminobutyric Acid; Carboxylic Acids; Ornithine
PubMed: 36770800
DOI: 10.3390/molecules28031133 -
Journal of the American Chemical Society Mar 2023A deaminative reductive coupling of amino acid pyridinium salts with aryl bromides has been developed to enable efficient synthesis of noncanonical amino acids and...
A deaminative reductive coupling of amino acid pyridinium salts with aryl bromides has been developed to enable efficient synthesis of noncanonical amino acids and diversification of peptides. This method transforms natural, commercially available lysine, ornithine, diaminobutanoic acid, and diaminopropanoic acid to aryl alanines and homologated derivatives with varying chain lengths. Attractive features include ability to transverse scales, tolerance of pharma-relevant (hetero)aryls and biorthogonal functional groups, and the applicability beyond monomeric amino acids to short and macrocyclic peptide substrates. The success of this work relied on high-throughput experimentation to identify complementary reaction conditions that proved critical for achieving the coupling of a broad scope of aryl bromides with a range of amino acid and peptide substrates including macrocyclic peptides.
Topics: Amino Acids; Bromides; Amines; Peptides; Ornithine
PubMed: 36853652
DOI: 10.1021/jacs.2c11451 -
Archives of Biochemistry and Biophysics Nov 2022Tyrosine-430 of d-ornithine/d-lysine decarboxylase (DOKDC) is located in the active site, and was suggested to be responsible for the D-stereospecificity of the enzyme....
Tyrosine-430 of d-ornithine/d-lysine decarboxylase (DOKDC) is located in the active site, and was suggested to be responsible for the D-stereospecificity of the enzyme. We have prepared the Y430F mutant form of Salmonella enterica serovar typhimurium DOKDC and evaluated its catalytic activity with D- and l-lysine and ornithine. The kinetic results show that the Y430F mutant has measurable decarboxylase activity with both D- and l-lysine and ornithine, which wild type DOKDC does not. Spectroscopic experiments show that these amino acids bind to form external aldimine complexes with the pyridoxal-5'-phosphate with λ = 425 nm. In addition, we have obtained crystal structures of Y430F DOKDC bound to HEPES, putrescine, d-ornithine, d-lysine, and d-arginine. The d-amino acids bind in the crystals to form equilibrium mixtures of gem-diamine and external aldimine complexes. Furthermore, the crystal structures reveal an unexpected allosteric product activator site for putrescine located on the 2-fold axis between the two active sites. Putrescine binds by donating hydrogen bonds from the ammonium groups to Asp-361 and Gln-358 in the specificity helix of both chains. Addition of 0.1-1 mM putrescine eliminates the lag in steady state kinetics and abolishes the sigmoid kinetics. The catalytic loop was modeled with AlphaFold2, and the model shows that Glu-181 can form additional hydrogen bonds with the bound putrescine, likely stabilizing the catalytic closed conformation.
Topics: Ornithine; Putrescine; Ornithine Decarboxylase; Lysine; Allosteric Regulation; Mutagenesis, Site-Directed; Carboxy-Lyases; Pyridoxal Phosphate; Kinetics; Salmonella
PubMed: 36265649
DOI: 10.1016/j.abb.2022.109429 -
Environmental Microbiology Oct 2022Pseudomonas aeruginosa is an important Gram-negative pathogen with intrinsic resistance to many clinically used antibiotics. It is particularly troublesome in nosocomial... (Review)
Review
Pseudomonas aeruginosa is an important Gram-negative pathogen with intrinsic resistance to many clinically used antibiotics. It is particularly troublesome in nosocomial infections, immunocompromised patients, and individuals with cystic fibrosis. Antimicrobial resistance (AMR) is a huge threat to global health, with a predicted 10 million people dying from resistant infections by 2050. A promising therapy for combatting AMR infections is phage therapy. However, more research is required to investigate mechanisms that may influence the efficacy of phage therapy. An important overlooked aspect is the impact of membrane lipid remodelling on phage binding ability. P. aeruginosa undergoes changes in membrane lipids when it encounters phosphorus stress, an environmental perturbation that is likely to occur during infection. Lipid changes include the substitution of glycerophospholipids with surrogate glycolipids and the over-production of ornithine-containing aminolipids. Given that membrane lipids are known to influence the structure and function of membrane proteins, we propose that changes in the composition of membrane lipids during infection may alter phage binding and subsequent phage infection dynamics. Consideration of such effects needs to be urgently prioritised in order to develop the most effective phage therapy strategies for P. aeruginosa infections.
Topics: Anti-Bacterial Agents; Bacteriophages; Glycerophospholipids; Glycolipids; Humans; Membrane Lipids; Membrane Proteins; Ornithine; Phage Therapy; Phosphorus; Pseudomonas Infections; Pseudomonas aeruginosa
PubMed: 35837865
DOI: 10.1111/1462-2920.16136 -
Journal of Animal Science Apr 2022In livestock species, the enterocytes of the small intestine are responsible for the synthesis of citrulline and arginine from glutamine and proline. At present, little...
In livestock species, the enterocytes of the small intestine are responsible for the synthesis of citrulline and arginine from glutamine and proline. At present, little is known about de novo synthesis of citrulline and arginine in horses. To test the hypothesis that horses of different age groups can utilize glutamine and proline for the de novo synthesis of citrulline and arginine, jejunal enterocytes from 19 horses of three different age groups: neonates (n = 4; 7.54 ± 2.36 d of age), adults (n = 9; 6.4 ± 0.35 yr), and aged (n = 6; 22.9 ± 1.0 yr) with healthy gastrointestinal tracts were used in the present study. Enterocytes were isolated from the jejunum and incubated at 37 °C for 30 min in oxygenated (95% O2/5% CO2) Krebs bicarbonate buffer (pH 7.4) containing 5 mM D-glucose and 0 mM, 2-mM L-[U-14C]glutamine, or 2 mM L-[U-14C]proline plus 2 mM L-glutamine. Concentrations of arginine, citrulline, and ornithine in cells plus medium were determined using high-performance liquid chromatography. Results indicate that the rate of oxidation of glutamine to CO2 was high in enterocytes from neonatal horses, but low in cells from adult and aged horses. Enterocytes from all age groups of horses did not degrade proline into CO2. Regardless of age, equine enterocytes formed ornithine from glutamine and proline, but failed to convert ornithine into citrulline and arginine. Because arginine is an essential substrate for the synthesis of not only proteins, but also nitrogenous metabolites (e.g., nitric oxide, polyamines, and creatine), our novel findings have important implications for the nutrition, performance, and health of horses.
Topics: Animals; Arginine; Carbon Dioxide; Citrulline; Enterocytes; Glutamine; Horses; Ornithine; Proline
PubMed: 35275603
DOI: 10.1093/jas/skac077 -
Chinese Medical Journal Aug 2022Hypertrophic cardiomyopathy (HCM) is an underdiagnosed genetic heart disease worldwide. The management and prognosis of obstructive HCM (HOCM) and non-obstructive HCM...
BACKGROUND
Hypertrophic cardiomyopathy (HCM) is an underdiagnosed genetic heart disease worldwide. The management and prognosis of obstructive HCM (HOCM) and non-obstructive HCM (HNCM) are quite different, but it also remains challenging to discriminate these two subtypes. HCM is characterized by dysmetabolism, and myocardial amino acid (AA) metabolism is robustly changed. The present study aimed to delineate plasma AA and derivatives profiles, and identify potential biomarkers for HCM.
METHODS
Plasma samples from 166 participants, including 57 cases of HOCM, 52 cases of HNCM, and 57 normal controls (NCs), who first visited the International Cooperation Center for HCM, Xijing Hospital between December 2019 and September 2020, were collected and analyzed by high-performance liquid chromatography-mass spectrometry based on targeted AA metabolomics. Three separate classification algorithms, including random forest, support vector machine, and logistic regression, were applied for the identification of specific AA and derivatives compositions for HCM and the development of screening models to discriminate HCM from NC as well as HOCM from HNCM.
RESULTS
The univariate analysis showed that the serine, glycine, proline, citrulline, glutamine, cystine, creatinine, cysteine, choline, and aminoadipic acid levels in the HCM group were significantly different from those in the NC group. Four AAs and derivatives (Panel A; proline, glycine, cysteine, and choline) were screened out by multiple feature selection algorithms for discriminating HCM patients from NCs. The receiver operating characteristic (ROC) analysis in Panel A yielded an area under the ROC curve (AUC) of 0.83 (0.75-0.91) in the training set and 0.79 (0.65-0.94) in the validation set. Moreover, among 10 AAs and derivatives (arginine, phenylalanine, tyrosine, proline, alanine, asparagine, creatine, tryptophan, ornithine, and choline) with statistical significance between HOCM and HNCM, 3 AAs (Panel B; arginine, proline, and ornithine) were selected to differentiate the two subgroups. The AUC values in the training and validation sets for Panel B were 0.83 (0.74-0.93) and 0.82 (0.66-0.98), respectively.
CONCLUSIONS
The plasma AA and derivatives profiles were distinct between the HCM and NC groups. Based on the differential profiles, the two established screening models have potential value in assisting HCM screening and identifying whether it is obstructive.
Topics: Humans; Amino Acids; Cysteine; Cardiomyopathy, Hypertrophic; Biomarkers; Proline; Arginine; Ornithine; Glycine; Choline
PubMed: 36156511
DOI: 10.1097/CM9.0000000000002279 -
Metabolomics : Official Journal of the... Sep 2021Vitiligo pathogenesis is complicated, and several possibilities were suggested. However, it is well-known that the metabolism of pigments plays a significant role in the...
INTRODUCTION
Vitiligo pathogenesis is complicated, and several possibilities were suggested. However, it is well-known that the metabolism of pigments plays a significant role in the pathogenicity of the disease.
OBJECTIVES
We explored the role of amino acids in vitiligo using targeted metabolomics.
METHODS
The amino acid profile was studied in plasma using liquid chromatography. First, 22 amino acids were derivatized and precisely determined. Next, the concentrations of the amino acids and the molar ratios were calculated in 31 patients and 34 healthy individuals.
RESULTS
The differential concentrations of amino acids were analyzed and eight amino acids, i.e., cysteine, arginine, lysine, ornithine, proline, glutamic acid, histidine, and glycine were observed differentially. The ratios of cysteine, glutamic acid, and proline increased significantly in Vitiligo patients, whereas arginine, lysine, ornithine, glycine, and histidine decreased significantly compared to healthy individuals. Considering the percentage of skin area, we also showed that glutamic acid significantly has a higher amount in patients with less than 25% involvement compared to others. Finally, cysteine and lysine are considered promising candidates for diagnosing and developing the disorder with high accuracy (0.96).
CONCLUSION
The findings are consistent with the previously illustrated mechanism of Vitiligo, such as production deficiency in melanin and an increase in immune activity and oxidative stress. Furthermore, new evidence was provided by using amino acids profile toward the pathogenicity of the disorder.
Topics: Amino Acids; Arginine; Cysteine; Glutamates; Glycine; Histidine; Humans; Lysine; Metabolomics; Ornithine; Proline; Vitiligo
PubMed: 34562159
DOI: 10.1007/s11306-021-01843-x -
Nature Microbiology Apr 2020Polyamines are essential metabolites that play an important role in cell growth, stress adaptation and microbial virulence. To survive and multiply within a human host,...
Polyamines are essential metabolites that play an important role in cell growth, stress adaptation and microbial virulence. To survive and multiply within a human host, pathogenic bacteria adjust the expression and activity of polyamine biosynthetic enzymes in response to different environmental stresses and metabolic cues. Here, we show that ornithine capture by the ribosome and the nascent peptide SpeFL controls polyamine synthesis in γ-proteobacteria by inducing the expression of the ornithine decarboxylase SpeF, via a mechanism involving ribosome stalling and transcription antitermination. In addition, we present the cryogenic electron microscopy structure of an Escherichia coli ribosome stalled during translation of speFL in the presence of ornithine. The structure shows how the ribosome and the SpeFL sensor domain form a highly selective binding pocket that accommodates a single ornithine molecule but excludes near-cognate ligands. Ornithine pre-associates with the ribosome and is then held in place by the sensor domain, leading to the compaction of the SpeFL effector domain and blocking the action of release factor 1. Thus, our study not only reveals basic strategies by which nascent peptides assist the ribosome in detecting a specific metabolite, but also provides a framework for assessing how ornithine promotes virulence in several human pathogens.
Topics: Amino Acid Sequence; Bacterial Proteins; Base Sequence; Binding Sites; Enterococcus faecalis; Escherichia coli; Models, Molecular; Ornithine; Ornithine Decarboxylase; Peptide Termination Factors; Phylogeny; Polyamines; Protein Binding; Protein Biosynthesis; Protein Interaction Domains and Motifs; RNA, Transfer; Ribosomes; Salmonella typhimurium; Sequence Alignment; Sequence Homology, Amino Acid; Thermus thermophilus; Virulence
PubMed: 32094585
DOI: 10.1038/s41564-020-0669-1