Flavoprotein oxidoreductases are members of a large protein family of specialized dehydrogenases, which include type II NADH dehydrogenase, pyridine nucleotide-disulphide oxidoreductases, ferredoxin-NAD+ reductases, NADH oxidases, and NADH peroxidases, playing a crucial role in the metabolism of several prokaryotes and eukaryotes. Although several studies have been performed on single members or protein subgroups of flavoprotein oxidoreductases, a comprehensive analysis on structure–function relationships among the different members and subgroups of this great dehydrogenase family is still missing. Here, we present a structural comparative analysis showing that the investigated flavoprotein oxidoreductases have a highly similar overall structure, although the investigated dehydrogenases are quite different in functional annotations and global amino acid composition. The different functional annotation is ascribed to their participation in species-specific metabolic pathways based on the same biochemical reaction, i.e., the oxidation of specific cofactors, like NADH and FADH₂. Notably, the performed comparative analysis sheds light on conserved sequence features that reflect very similar oxidation mechanisms, conserved among flavoprotein oxidoreductases belonging to phylogenetically distant species, as the bacterial type II NADH dehydrogenases and the mammalian apoptosis-inducing factor protein, until now retained as unique protein entities in Bacteria/Fungi or Animals, respectively. Furthermore, the presented computational analyses will allow consideration of FAD/NADH oxidoreductases as a possible target of new small molecules to be used as modulators of mitochondrial respiration for patients affected by rare diseases or cancer showing mitochondrial dysfunction, or antibiotics for treating bacterial/fungal/protista infections.

Glutaminolysis is known to correlate with ovarian cancer aggressiveness and invasion. However, how this affects the tumor microenvironment is elusive. Here, we show that ovarian cancer cells become addicted to extracellular glutamine when silenced for glutamine synthetase (GS), similar to naturally occurring GS-low, glutaminolysis-high ovarian cancer cells. Glutamine addiction elicits a crosstalk mechanism whereby cancer cells release N -acetylaspartate (NAA) which, through the inhibition of the NMDA receptor, and synergistically with IL-10, enforces GS expression in macrophages. In turn, GS-high macrophages acquire M2-like, tumorigenic features. Supporting this in␣vitro model, in silico data and the analysis of ascitic fluid isolated from ovarian cancer patients prove that an M2-like macrophage phenotype, IL-10 release, and NAA levels positively correlate with disease stage. Our study uncovers the unprecedented role of glutamine metabolism in modulating macrophage polarization in highly invasive ovarian cancer and highlights the anti-inflammatory, protumoral function of NAA.

Mitochondrial diseases are a plethora of inherited neuromuscular disorders sharing defects in mitochondrial respiration, but largely different from one another for genetic basis and pathogenic mechanism. Whole exome sequencing was performed in a familiar trio (trio-WES) with a child affected by severe epileptic encephalopathy associated with respiratory complex I deficiency and mitochondrial DNA depletion in skeletal muscle. By trio-WES we identified biallelic mutations in SLC25A10, a nuclear gene encoding a member of the mitochondrial carrier family. Genetic and functional analyses conducted on patient fibroblasts showed that SLC25A10 mutations are associated with reduction in RNA quantity and aberrant RNA splicing, and to absence of SLC25A10 protein and its transporting function. The yeast SLC25A10 ortholog knockout strain showed defects in mitochondrial respiration and mitochondrial DNA content, similarly to what observed in the patient skeletal muscle, and growth susceptibility to oxidative stress. Albeit patient fibroblasts were depleted in the main antioxidant molecules NADPH and glutathione, transport assays demonstrated that SLC25A10 is unable to transport glutathione. Here, we report the first recessive mutations of SLC25A10 associated to an inherited severe mitochondrial neurodegenerative disorder. We propose that SLC25A10 loss-of-function causes pathological disarrangements in respiratory-demanding conditions and oxidative stress vulnerability.

ADP/ATP carriers (AACs) are mitochondrial transport proteins playing a strategic role in maintaining the respiratory chain activity, fueling the cell with ATP, and also regulating mitochondrial apoptosis. To understand if AACs might represent a new molecular target for cancer treatment, we evaluated AAC expression levels in cancer/normal tissue pairs available on the Tissue Cancer Genome Atlas database (TCGA), observing that AACs are dysregulated in most of the available samples. It was observed that at least two AACs showed a significant differential expression in all the available kidney cancer/normal tissue pairs. Thus, we investigated AAC expression in the corresponding kidney non-cancer (HK2)/cancer (RCC-Shaw and CaKi-1) cell lines, grown in complete medium or serum starvation, for investigating how metabolic alteration induced by different growth conditions might influence AAC expression and resistance to mitochondrial apoptosis initiators, such as “staurosporine” or the AAC highly selective inhibitor “carboxyatractyloside”. Our analyses showed that AAC2 and AAC3 transcripts are more expressed than AAC1 in all the investigated kidney cell lines grown in complete medium, whereas serum starvation causes an increase of at least two AAC transcripts in kidney cancer cell lines compared to non-cancer cells. However, the total AAC protein content is decreased in the investigated cancer cell lines, above all in the serum-free medium. The observed decrease in AAC protein content might be responsible for the decrease of OXPHOS activity and for the observed lowered sensitivity to mitochondrial apoptosis induced by staurosporine or carboxyatractyloside. Notably, the cumulative probability of the survival of kidney cancer patients seriously decreases with the decrease of AAC1 expression in KIRC and KIRP tissues making AAC1 a possible new biomarker of metabolic remodeling and survival in kidney cancers.

The transcription factors ADR1 or CAT8 are required for RTG pathway activation and evasion from yeast acetic acid-induced programmed cell death in raffinose –

Carnitine O-acetyltransferase (CRAT) is a key mitochondrial enzyme involved in maintaining metabolic homeostasis by mediating the reversible transfer of acetyl groups between acetyl-CoA and carnitine. This enzymatic activity ensures the optimal functioning of mitochondrial carbon flux by preventing acetyl-CoA accumulation, buffering metabolic flexibility, and regulating the balance between fatty acid and glucose oxidation. CRAT’s interplay with the mitochondrial carnitine shuttle, involving carnitine palmitoyltransferases (CPT1 and CPT2) and the carnitine carrier (SLC25A20), underscores its critical role in energy metabolism. Emerging evidence highlights the structural and functional diversity of CRAT and structurally related acetyltransferases across cellular compartments, illustrating their coordinated role in lipid metabolism, amino acid catabolism, and mitochondrial bioenergetics. Moreover, the structural insights into CRAT have paved the way for understanding its regulation and identifying potential modulators with therapeutic applications for diseases such as diabetes, mitochondrial disorders, and cancer. This review examines CRAT’s structural and functional aspects, its relationships with carnitine shuttle members and other carnitine acyltransferases, and its broader role in metabolic health and disease. The potential for targeting CRAT and its associated pathways offers promising avenues for therapeutic interventions aimed at restoring metabolic equilibrium and addressing metabolic dysfunction in disease states.