Adenosine undergoes ATP-dependent phosphorylation catalyzed by adenosine kinase (ADK). In plants, ADK also phosphorylates cytokinin ribosides, transport forms of the hormone. Here, we investigated the substrate preferences, oligomeric states, and structures of ADKs from moss (Physcomitrella patens) and maize (Zea mays) alongside metabolomic and phenotypic analyses. We showed that dexamethasone-inducible ZmADK overexpressor lines in Arabidopsis can benefit from a higher number of lateral roots and larger root areas under nitrogen starvation. We discovered that maize and moss enzymes can form dimers upon increasing protein concentration, setting them apart from the monomeric human and protozoal ADKs. Structural and kinetic analyses revealed a catalytically inactive unique dimer. Within the dimer, both active sites are mutually blocked. The activity of moss ADKs, exhibiting a higher propensity to dimerize, was 10-fold lower compared with maize ADKs. Two monomeric structures in a ternary complex highlight the characteristic transition from an open to a closed state upon substrate binding. This suggests that the oligomeric state switch can modulate the activity of moss ADKs and probably other plant ADKs. Moreover, dimer association represents a novel negative feedback mechanism, helping to maintain steady levels of adenosine and AMP.

• Keywords
• Physcomitrella patens, Zea mays, Adenosine kinase, SnRK, crystal structure, cytokinin, overexpression, purine, riboside,
• MeSH
• Adenosine Kinase * metabolism   genetics   chemistry   MeSH
• Arabidopsis genetics   enzymology   metabolism   MeSH
• Zea mays * enzymology   genetics   metabolism   MeSH
• Bryopsida * enzymology   genetics   metabolism   MeSH
• Protein Multimerization MeSH
• Plant Proteins * metabolism   genetics   chemistry   MeSH
• Substrate Specificity MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Adenosine Kinase * MeSH
• Plant Proteins * MeSH

MOTIVATION: The increasing use of big data and optimized prediction methods in metabolomics requires techniques aligned with biological assumptions to improve early symptom diagnosis. One major challenge in predictive data analysis is handling confounding factors-variables influencing predictions but not directly included in the analysis. RESULTS: Detecting and correcting confounding factors enhances prediction accuracy, reducing false negatives that contribute to diagnostic errors. This study reviews concept drift detection methods in metabolomic predictions and selects the most appropriate ones. We introduce a new implementation of concept drift analysis in predictive classifiers using metabolomics data. Known confounding factors were confirmed, validating our approach and aligning it with conventional methods. Additionally, we identified potential confounding factors that may influence biomarker analysis, which could introduce bias and impact model performance. AVAILABILITY AND IMPLEMENTATION: Based on biological assumptions supported by detected concept drift, these confounding factors were incorporated into correction of prediction algorithms to enhance their accuracy. The proposed methodology has been implemented in Semi-Automated Pipeline using Concept Drift Analysis for improving Metabolomic Predictions (SAPCDAMP), an open-source workflow available at https://github.com/JanaSchwarzerova/SAPCDAMP.

• Publication type
• Journal Article MeSH

Glutamate carboxypeptidase II (GCPII, also known as PSMA or FOLH1) is responsible for the cleavage of N-acetyl-aspartyl-glutamate (NAAG) to N-acetyl-aspartate and glutamate in the central nervous system and facilitates the intestinal absorption of folate by processing dietary folyl-poly-γ-glutamate in the small intestine. The physiological function of GCPII in other organs like kidneys is still not known. GCPII inhibitors are neuroprotective in various conditions (e.g., ischemic brain injury) in vivo; however, their utilization as potential drug candidates has not been investigated in regard to not yet known GCPII activities. To explore the GCPII role and possible side effects of GCPII inhibitors, we performed parallel metabolomic and lipidomic analysis of the cerebrospinal fluid (CSF), urine, plasma, and brain tissue of mice with varying degrees of GCPII deficiency (fully deficient in Folh1, -/-; one allele deficient in Folh1, +/-; and wild type, +/+). Multivariate analysis of metabolites showed no significant differences between wild-type and GCPII-deficient mice (except for NAAG), although changes were observed between the sex and age. NAAG levels were statistically significantly increased in the CSF, urine, and plasma of GCPII-deficient mice. However, no difference in NAAG concentrations was found in the whole brain lysate likely because GCPII, as an extracellular enzyme, can affect only extracellular and not intracellular NAAG concentrations. Regarding the lipidome, the most pronounced genotype-linked changes were found in the brain tissue. In brains of GCPII-deficient mice, we observed statistically significant enrichment in phosphatidylcholine-based lipids and reduction of sphingolipids and phosphatidylethanolamine plasmalogens. We hypothesize that the alteration of the NAA-NAAG axis by absent GCPII activity affected myelin composition. In summary, the absence of GCPII and thus similarly its inhibition do not have detrimental effects on metabolism, with just minor changes in the brain lipidome.

• Keywords
• FOLH1, N-acetyl-aspartyl-glutamate, folyl-poly-γ-glutamyl hydrolase I, glutamate carboxypeptidase II, lipidomics, metabolomics,
• MeSH
• Dipeptides metabolism   MeSH
• Glutamate Carboxypeptidase II * genetics   metabolism   MeSH
• Glutamic Acid MeSH
• Lipidomics * MeSH
• Lipids chemistry   MeSH
• Metabolomics * MeSH
• Brain metabolism   MeSH
• Mice MeSH
• Animals MeSH
• Check Tag
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Dipeptides MeSH
• Folh1 protein, mouse MeSH Browser
• Glutamate Carboxypeptidase II * MeSH
• isospaglumic acid MeSH Browser
• Glutamic Acid MeSH
• Lipids MeSH

Three genetically determined enzyme defects of purine de novo synthesis (PDNS) have been identified so far in humans: adenylosuccinate lyase (ADSL) deficiency, 5-amino-4-imidazole carboxamide-ribosiduria (AICA-ribosiduria), and deficiency in bifunctional enzyme phosphoribosylaminoimidazole carboxylase and phosphoribosylaminoimidazolesuccinocarboxamide synthase (PAICS). Clinical signs of these defects are mainly neurological, such as seizures, psychomotor retardation, epilepsy, autistic features, etc. This work aims to describe the metabolic changes of CRISPR-Cas9 genome-edited HeLa cells deficient in the individual steps of PDNS to better understand known and potential defects of the pathway in humans. High-performance liquid chromatography coupled with mass spectrometry was used for both targeted and untargeted metabolomic analyses. The statistically significant features from the untargeted study were identified by fragmentation analysis. Data from the targeted analysis were processed in Cytoscape software to visualize the most affected metabolic pathways. Statistical significance of PDNS intermediates preceding deficient enzymes was the highest (p-values 10 × 10-7-10 × 10-15) in comparison with the metabolites from other pathways (p-values of up to 10 × 10-7). Disturbed PDNS resulted in an altered pool of adenine and guanine nucleotides. However, the adenylate energy charge was not different from controls. Different profiles of acylcarnitines observed among deficient cell lines might be associated with a specific enzyme deficiency rather than global changes related to the PDNS pathway. Changes detected in one-carbon metabolism might reduce the methylation activity of the deficient cells, thus affecting the modification state of DNA, RNA, and proteins.

• Keywords
• HeLa cells, mass spectrometry, metabolomics, purine de novo synthesis, rare metabolic disorders,
• Publication type
• Journal Article MeSH

BACKGROUND: Tauopathies represent heterogeneous groups of neurodegenerative diseases that are characterised by abnormal deposition of the microtubule-associated protein tau. Alzheimer's disease is the most prevalent tauopathy, affecting more than 35 million people worldwide. In this study we investigated changes in metabolic pathways associated with tau-induced neurodegeneration. METHODS: Cerebrospinal fluid (CSF), plasma and brain tissue were collected from a transgenic rat model for tauopathies and from age-matched control animals. The samples were analysed by targeted and untargeted metabolomic methods using high-performance liquid chromatography coupled to mass spectrometry. Unsupervised and supervised statistical analysis revealed biochemical changes associated with the tauopathy process. RESULTS: Energy deprivation and potentially neural apoptosis were reflected in increased purine nucleotide catabolism and decreased levels of citric acid cycle intermediates and glucose. However, in CSF, increased levels of citrate and aconitate that can be attributed to glial activation were observed. Other significant changes were found in arginine and phosphatidylcholine metabolism. CONCLUSIONS: Despite an enormous effort invested in development of biomarkers for tauopathies during the last 20 years, there is no clinically used biomarker or assay on the market. One of the most promising strategies is to create a panel of markers (e.g., small molecules, proteins) that will be continuously monitored and correlated with patients' clinical outcome. In this study, we identified several metabolic changes that are affected during the tauopathy process and may be considered as potential markers of tauopathies in humans.

• Keywords
• Metabolomics, Tau protein, Tauopathy, Transgenic rat model,
• MeSH
• Apoptosis genetics   MeSH
• Biomarkers metabolism   MeSH
• Rats MeSH
• Humans MeSH
• Metabolomics MeSH
• Disease Models, Animal MeSH
• Brain metabolism   pathology   MeSH
• Mutation genetics   MeSH
• Rats, Inbred SHR MeSH
• Rats, Transgenic MeSH
• tau Proteins genetics   metabolism   MeSH
• Tauopathies cerebrospinal fluid   diagnosis   genetics   MeSH
• Animals MeSH
• Check Tag
• Rats MeSH
• Humans MeSH
• Male MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Biomarkers MeSH
• tau Proteins MeSH