Mitochondrial targets in hyperammonemia: Addressing urea cycle function to improve drug therapies.

The urea cycle (UC) is a critically important metabolic process for the disposal of nitrogen (ammonia) produced by amino acids catabolism. The impairment of this liver-specific pathway induced either by primary genetic defects or by secondary causes, namely those associated with hepatic disease or drug administration, may result in serious clinical consequences. Urea cycle disorders (UCD) and certain organic acidurias are the major groups of inherited rare diseases manifested with hyperammonemia (HA) with UC dysregulation. Importantly, several commonly prescribed drugs, including antiepileptics in monotherapy or polytherapy from carbamazepine to valproic acid or specific antineoplastic agents such as asparaginase or 5-fluorouracil may be associated with HA by mechanisms not fully elucidated. HA, disclosing an imbalance between ammoniagenesis and ammonia disposal via the UC, can evolve to encephalopathy which may lead to significant morbidity and central nervous system damage. This review will focus on biochemical mechanisms related with HA emphasizing some poorly understood perspectives behind the disruption of the UC and mitochondrial energy metabolism, namely: i) changes in acetyl-CoA or NAD+ levels in subcellular compartments; ii) post-translational modifications of key UC-related enzymes, namely acetylation, potentially affecting their catalytic activity; iii) the mitochondrial sirtuins-mediated role in ureagenesis. Moreover, the main UCD associated with HA will be summarized to highlight the relevance of investigating possible genetic mutations to account for unexpected HA during certain pharmacological therapies. The ammonia-induced effects should be avoided or overcome as part of safer therapeutic strategies to protect patients under treatment with drugs that may be potentially associated with HA.

Pathogenic SLC25A26 variants impair SAH transport activity causing mitochondrial disease.

The SLC25A26 gene encodes a mitochondrial inner membrane carrier that transports S-adenosylmethionine (SAM) into the mitochondrial matrix in exchange for S-adenosylhomocysteine (SAH). SAM is the predominant methyl-group donor for most cellular methylation processes, of which SAH is produced as a by-product. Pathogenic, biallelic SLC25A26 variants are a recognized cause of mitochondrial disease in children, with a severe neonatal onset caused by decreased SAM transport activity. Here, we describe two, unrelated adult cases, one of whom presented with recurrent episodes of severe abdominal pain and metabolic decompensation with lactic acidosis. Both patients had exercise intolerance and mitochondrial myopathy associated with biallelic variants in SLC25A26, which led to marked respiratory chain deficiencies and mitochondrial histopathological abnormalities in skeletal muscle that are comparable to those previously described in early-onset cases. We demonstrate using both mouse and fruit fly models that impairment of SAH, rather than SAM, transport across the mitochondrial membrane is likely the cause of this milder, late-onset phenotype. Our findings associate a novel pathomechanism with a known disease-causing protein and highlight the quests of precision medicine in optimizing diagnosis, therapeutic intervention and prognosis.

Stable Isotope Labeling of Amino Acids in Flies (SILAF) Reveals Differential Phosphorylation of Mitochondrial Proteins Upon Loss of OXPHOS Subunits.

Drosophila melanogaster has been a workhorse of genetics and cell biology for more than a century. However, proteomic-based methods have been limited due to the complexity and dynamic range of the fly proteome and the lack of efficient labeling methods. Here, we advanced a chemically defined food source into direct stable-isotope labeling of amino acids in flies (SILAF). It allows for the rapid and cost-efficient generation of a large number of larvae or flies, with full incorporation of lysine-[13C6] after six labeling days. SILAF followed by fractionation and enrichment gave proteomic insights at a depth of 7196 proteins and 8451 phosphorylation sites, which substantiated metabolic regulation on enzymatic level. We applied SILAF to quantify the mitochondrial phosphoproteome of an early-stage leucine-rich PPR motif-containing protein (LRPPRC)-knockdown fly model of mitochondrial disease that almost exclusively affects protein levels of the oxidative phosphorylation (OXPHOS) system. While the mitochondrial compartment was hypo-phosphorylated, two conserved phosphosites on OXPHOS subunits NDUFB10 and NDUFA4 were significantly upregulated upon impaired OXPHOS function. The ease and versatility of the method actuate the fruit fly as an appealing model in proteomic and posttranslational modification studies, and it enlarges potential metabolic applications based on heavy amino acid diets.

The one-carbon pool controls mitochondrial energy metabolism via complex I and iron-sulfur clusters.

Induction of the one-carbon cycle is an early hallmark of mitochondrial dysfunction and cancer metabolism. Vital intermediary steps are localized to mitochondria, but it remains unclear how one-carbon availability connects to mitochondrial function. Here, we show that the one-carbon metabolite and methyl group donor S-adenosylmethionine (SAM) is pivotal for energy metabolism. A gradual decline in mitochondrial SAM (mitoSAM) causes hierarchical defects in fly and mouse, comprising loss of mitoSAM-dependent metabolites and impaired assembly of the oxidative phosphorylation system. Complex I stability and iron-sulfur cluster biosynthesis are directly controlled by mitoSAM levels, while other protein targets are predominantly methylated outside of the organelle before import. The mitoSAM pool follows its cytosolic production, establishing mitochondria as responsive receivers of one-carbon units. Thus, we demonstrate that cellular methylation potential is required for energy metabolism, with direct relevance for pathophysiology, aging, and cancer.

Severe congenital lactic acidosis and hypertrophic cardiomyopathy caused by an intronic variant in NDUFB7.

Mutations in structural subunits and assembly factors of complex I of the oxidative phosphorylation system constitute the most common cause of mitochondrial respiratory chain defects. Such mutations can present a wide range of clinical manifestations, varying from mild deficiencies to severe, lethal disorders. We describe a patient presenting intrauterine growth restriction and anemia, which displayed postpartum hypertrophic cardiomyopathy, lactic acidosis, encephalopathy, and a severe complex I defect with fatal outcome. Whole genome sequencing revealed an intronic biallelic mutation in the NDUFB7 gene (c.113-10C>G) and splicing pattern alterations in NDUFB7 messenger RNA were confirmed by RNA Sequencing. The detected variant resulted in a significant reduction of the NDUFB7 protein and reduced complex I activity. Complementation studies with expression of wild-type NDUFB7 in patient fibroblasts normalized complex I function. Here we report a case with a primary complex I defect due to a homozygous mutation in an intron region of the NDUFB7 gene.