BACKGROUND: Cystathionine β-synthase (CBS)-deficient homocystinuria (HCU) is an inherited disorder of sulfur amino acid metabolism with varying severity and organ complications, and a limited knowledge about underlying pathophysiological processes. Here we aimed at getting an in-depth insight into disease mechanisms using a transgenic mouse model of HCU (I278T). METHODS: We assessed metabolic, proteomic and sphingolipidomic changes, and mitochondrial function in tissues and body fluids of I278T mice and WT controls. Furthermore, we evaluated the efficacy of methionine-restricted diet (MRD) in I278T mice. RESULTS: In WT mice, we observed a distinct tissue/body fluid compartmentalization of metabolites with up to six-orders of magnitude differences in concentrations among various organs. The I278T mice exhibited the anticipated metabolic imbalance with signs of an increased production of hydrogen sulfide and disturbed persulfidation of free aminothiols. HCU resulted in a significant dysregulation of liver proteome affecting biological oxidations, conjugation of compounds, and metabolism of amino acids, vitamins, cofactors and lipids. Liver sphingolipidomics indicated upregulation of the pro-proliferative sphingosine-1-phosphate signaling pathway. Liver mitochondrial function of HCU mice did not seem to be impaired compared to controls. MRD in I278T mice improved metabolic balance in all tissues and substantially reduced dysregulation of liver proteome. CONCLUSION: The study highlights distinct tissue compartmentalization of sulfur-related metabolites in normal mice, extensive metabolome, proteome and sphingolipidome disruptions in I278T mice, and the efficacy of MRD to alleviate some of the HCU-related biochemical abnormalities.

• Keywords
• Cystathionine beta-synthase, Homocystinuria, Metabolomics, Methionine restriction, Proteomics,
• MeSH
• Cystathionine beta-Synthase * metabolism   deficiency   genetics   MeSH
• Homocystinuria * metabolism   genetics   MeSH
• Liver * metabolism   MeSH
• Lipidomics methods   MeSH
• Metabolomics * methods   MeSH
• Mitochondria metabolism   MeSH
• Disease Models, Animal * MeSH
• Mice, Transgenic * MeSH
• Mice MeSH
• Proteome metabolism   MeSH
• Proteomics * methods   MeSH
• Sphingolipids * metabolism   MeSH
• Animals MeSH
• Check Tag
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Cystathionine beta-Synthase * MeSH
• Proteome MeSH
• Sphingolipids * MeSH

BACKGROUND: In animals, dietary sulfur amino acid restriction (SAAR) improves metabolic health, possibly mediated by altering sulfur amino acid metabolism and enhanced anti-obesogenic processes in adipose tissue. AIM: To assess the effects of SAAR over time on the plasma and urine SAA-related metabolites (sulfurome) in humans with overweight and obesity, and explore whether such changes were associated with body weight, body fat and adipose tissue gene expression. METHODS: Fifty-nine subjects were randomly allocated to SAAR (∼2 g SAA, n = 31) or a control diet (∼5.6 g SAA, n = 28) consisting of plant-based whole-foods and supplemented with capsules to titrate contents of SAA. Sulfurome metabolites in plasma and urine at baseline, 4 and 8 weeks were measured using HPLC and LC-MS/MS. mRNA-sequencing of subcutaneous white adipose tissue (scWAT) was performed to assess changes in gene expression. Data were analyzed with mixed model regression. Principal component analyses (PCA) were performed on the sulfurome data to identify potential signatures characterizing the response to SAAR. RESULTS: SAAR led to marked decrease of the main urinary excretion product sulfate (p < 0.001) and plasma and/or 24-h urine concentrations of cystathionine, sulfite, thiosulfate, H2S, hypotaurine and taurine. PCA revealed a distinct metabolic signature related to decreased transsulfuration and H2S catabolism that predicted greater weight loss and android fat mass loss in SAAR vs. controls (all pinteraction < 0.05). This signature correlated positively with scWAT expression of genes in the tricarboxylic acid cycle, electron transport and β-oxidation (FDR = 0.02). CONCLUSION: SAAR leads to distinct alterations of the plasma and urine sulfurome in humans, and predicted increased loss of weight and android fat mass, and adipose tissue lipolytic gene expression in scWAT. Our data suggest that SAA are linked to obesogenic processes and that SAAR may be useful for obesity and related disorders. TRIAL IDENTIFIER: https://clinicaltrials.gov/study/NCT04701346.

• Keywords
• Adipose tissue, Cysteine, Gene expression, Hydrogen sulfide, Methionine, Randomized controlled trial, Sulfur amino acid restriction, Transsulfuration,
• MeSH
• Amino Acids, Sulfur * metabolism   blood   MeSH
• Adult MeSH
• Middle Aged MeSH
• Humans MeSH
• Metabolome MeSH
• Overweight * metabolism   genetics   MeSH
• Obesity * metabolism   genetics   MeSH
• Gene Expression Regulation MeSH
• Adipose Tissue * metabolism   MeSH
• Check Tag
• Adult MeSH
• Middle Aged MeSH
• Humans MeSH
• Male MeSH
• Female MeSH
• Publication type
• Journal Article MeSH
• Randomized Controlled Trial MeSH
• Names of Substances
• Amino Acids, Sulfur * MeSH

Classical homocystinuria (HCU) is the most common inherited disorder of sulfur amino acid metabolism caused by deficiency in cystathionine beta-synthase (CBS) activity and characterized by severe elevation of homocysteine in blood and tissues. Treatment with dietary methionine restriction is not optimal, and poor compliance leads to serious complications. We developed an enzyme replacement therapy (ERT) and studied its efficacy in a severe form of HCU in mouse (the I278T model). Treatment was initiated before or after the onset of clinical symptoms in an effort to prevent or reverse the phenotype. ERT substantially reduced and sustained plasma homocysteine concentration at around 100 μM and normalized plasma cysteine for up to 9 months of treatment. Biochemical balance was also restored in the liver, kidney, and brain. Furthermore, ERT corrected liver glucose and lipid metabolism. The treatment prevented or reversed facial alopecia, fragile and lean phenotype, and low bone mass. In addition, structurally defective ciliary zonules in the eyes of I278T mice contained low density and/or broken fibers, while administration of ERT from birth partially rescued the ocular phenotype. In conclusion, ERT maintained an improved metabolic pattern and ameliorated many of the clinical complications in the I278T mouse model of HCU.

• Keywords
• PEGylation, alopecia, bone density, cystathionine beta-synthase, enzyme replacement, eye defect, homocysteine, inborn error of metabolism, metabolomics, preclinical studies,
• MeSH
• Amino Acids, Sulfur blood   metabolism   MeSH
• Cystathionine beta-Synthase administration & dosage   chemistry   MeSH
• Enzyme Replacement Therapy * MeSH
• Phenotype * MeSH
• Glucose metabolism   MeSH
• Homocystinuria diagnosis   metabolism   therapy   MeSH
• Liver drug effects   metabolism   MeSH
• Lipid Metabolism MeSH
• Disease Models, Animal MeSH
• Mice MeSH
• Oxidative Stress MeSH
• Polyethylene Glycols chemistry   MeSH
• Drug Evaluation, Preclinical MeSH
• Animals MeSH
• Check Tag
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, N.I.H., Extramural MeSH
• Names of Substances
• Amino Acids, Sulfur MeSH
• Cystathionine beta-Synthase MeSH
• Glucose MeSH
• Polyethylene Glycols MeSH

Classical homocystinuria (HCU) is an inborn error of sulfur amino acid metabolism caused by deficient activity of cystathionine β-synthase (CBS), resulting in an accumulation of homocysteine and a concomitant decrease of cystathionine and cysteine in blood and tissues. In mice, the complete lack of CBS is neonatally lethal. In this study, newborn CBS-knockout (KO) mice were treated with recombinant polyethyleneglycolylated human truncated CBS (PEG-CBS). Full survival of the treated KO mice, along with a positive impact on metabolite levels in plasma, liver, brain, and kidneys, was observed. The PEG-CBS treatment prevented an otherwise fatal liver disease characterized by steatosis, death of hepatocytes, and ultrastructural abnormalities of endoplasmic reticulum and mitochondria. Furthermore, treatment of the KO mice for 5 mo maintained the plasma metabolite balance and completely prevented osteoporosis and changes in body composition that characterize both the KO model and human patients. These findings argue that early treatment of patients with HCU with PEG-CBS may prevent clinical symptoms of the disease possibly without the need of dietary protein restriction.-Majtan, T., Hůlková, H., Park, I., Krijt, J., Kožich, V., Bublil, E. M., Kraus, J. P. Enzyme replacement prevents neonatal death, liver damage, and osteoporosis in murine homocystinuria.

• Keywords
• PEGylation, cystathionine β-synthase, homocysteine, preclinical drug development, rare inherited disease,
• MeSH
• Cystathionine beta-Synthase genetics   metabolism   therapeutic use   MeSH
• Homocystinuria drug therapy   enzymology   metabolism   pathology   MeSH
• Liver drug effects   enzymology   metabolism   pathology   MeSH
• Disease Models, Animal MeSH
• Mice, Knockout MeSH
• Mice MeSH
• Liver Diseases enzymology   prevention & control   MeSH
• Osteoporosis prevention & control   MeSH
• Recombinant Proteins therapeutic use   MeSH
• Body Composition drug effects   MeSH
• Fatty Liver enzymology   prevention & control   MeSH
• Animals MeSH
• Check Tag
• Male MeSH
• Mice MeSH
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, N.I.H., Extramural MeSH
• Names of Substances
• Cystathionine beta-Synthase MeSH
• Recombinant Proteins MeSH

Misfolding and aggregation of mutant enzymes have been proposed to play role in the pathogenesis of homocystinuria due to cystathionine β-synthase (CBS) deficiency. Chemical chaperones have been recently shown to facilitate proper assembly of several CBS mutants. To asses the number of patients that may respond to chaperone therapy, we examined the effect of selected CBS ligands and osmolytes on assembly and activity of 27 CBS mutants that represent 70% of known CBS alleles. The mutant enzymes were expressed in a bacterial system, and their properties were assessed by native Western blotting and sensitive liquid chromatography tandem mass spectrometry (LC-MS/MS) assay, respectively. We studied the chaperoning activity of δ-aminolevulinic acid (δ-ALA)-a heme precursor-and of three osmolytes betaine, 2-aminoethanesulfonic acid (taurine), and glycerol. Fourteen mutants responded by at least 30% increase in the amount of correctly assembled tetramers and enzymatic activity to the coexpressional presence of either 0.5 mM δ-ALA, 100 mM betaine, and/or 750 mM glycerol. Eight of these mutants (p.R266K, p.P49L, p.R125Q, p.K102N, p.R369C, p.V180A, p.P78R, p.S466L) were rescuable by all of these three substances. Four mutants showed increased formation of tetramers that was not accompanied by changes in activity. Topology of mutations appeared to determine the chaperone responsiveness, as 11 of 14 solvent-exposed mutations were substantially more responsive than three of 13 buried mutations. This study identified chaperone-responsive mutants that represent 56 of 713 known patient-derived CBS alleles and may serve as a basis for exploring pharmacological approaches aimed at correcting misfolding in homocystinuria.

• MeSH
• Alleles MeSH
• Betaine pharmacology   therapeutic use   MeSH
• Cystathionine beta-Synthase chemistry   drug effects   genetics   metabolism   MeSH
• Escherichia coli metabolism   MeSH
• Glycerol pharmacology   MeSH
• Homocystinuria drug therapy   genetics   metabolism   MeSH
• Polymorphism, Single Nucleotide physiology   MeSH
• Protein Conformation drug effects   MeSH
• Aminolevulinic Acid pharmacology   therapeutic use   MeSH
• Humans MeSH
• Ligands * MeSH
• Molecular Chaperones pharmacology   therapeutic use   MeSH
• Protein Multimerization drug effects   MeSH
• Mutant Proteins chemistry   drug effects   metabolism   MeSH
• Protein Folding drug effects   MeSH
• Taurine pharmacology   therapeutic use   MeSH
• Protein Binding MeSH
• Structure-Activity Relationship MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Evaluation Study MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Betaine MeSH
• Cystathionine beta-Synthase MeSH
• Glycerol MeSH
• Aminolevulinic Acid MeSH
• Ligands * MeSH
• Molecular Chaperones MeSH
• Mutant Proteins MeSH
• Taurine MeSH