In the genome of Drosophila melanogaster, four genes coding for aldehyde oxidases (AOX1-4) were identified on chromosome 3. Phylogenetic analysis showed that the AOX gene cluster evolved via independent duplication events in the vertebrate and invertebrate lineages. The functional role and the substrate specificity of the distinct Drosophila AOX enzymes is unknown. Two loss-of-function mutant alleles in this gene region, low pyridoxal oxidase (Po lpo ) and aldehyde oxidase-1 (Aldox-1 n1 ) are associated with a phenotype characterized by undetectable AOX enzymatic activity. However, the genes involved and the corresponding mutations have not yet been identified. In this study we characterized the activities, substrate specificities and expression profiles of the four AOX enzymes in D. melanogaster. We show that the Po lpo -associated phenotype is the consequence of a structural alteration of the AOX1 gene. We identified an 11-bp deletion in the Po lpo allele, resulting in a frameshift event, which removes the molybdenum cofactor domain of the encoded enzyme. Furthermore, we show that AOX2 activity is detectable only during metamorphosis and characterize a Minos-AOX2 insertion in this developmental gene that disrupts its activity. We demonstrate that the Aldox-1 n1 phenotype maps to the AOX3 gene and AOX4 activity is not detectable in our assays.
Adaptation to nutrient scarcity involves an orchestrated response of metabolic and signaling pathways to maintain homeostasis. We find that in the fat body of fasting Drosophila , lysosomal export of cystine coordinates remobilization of internal nutrient stores with reactivation of the growth regulator target of rapamycin complex 1 (TORC1). Mechanistically, cystine was reduced to cysteine and metabolized to acetyl-coenzyme A (acetyl-CoA) by promoting CoA metabolism. In turn, acetyl-CoA retained carbons from alternative amino acids in the form of tricarboxylic acid cycle intermediates and restricted the availability of building blocks required for growth. This process limited TORC1 reactivation to maintain autophagy and allowed animals to cope with starvation periods. We propose that cysteine metabolism mediates a communication between lysosomes and mitochondria, highlighting how changes in diet divert the fate of an amino acid into a growth suppressive program.
Regulatory in vitro genotoxicity testing exhibits shortcomings in specificity and mode of action (MoA) information. Thus, the aim of this work was to evaluate the performance of the novel MultiFlow V R assay composed of mechanistic biomarkers quantified in TK6 cells after treatment (4 and 24 hr): gH2AX (DNA double strand breaks), phosphorylated H3 (mitotic cells), translocated p53 (genotoxicity), and cleaved PARP1 (apoptosis). A reference dataset of 31 compounds with well-established MoA was studied using the MicroFlow V R micronucleus assay. A positive call was raised following the earlier published criteria from Litron Laboratories. In the light of our data, these evaluation criteria should probably be adjusted since only 8/11 (73%) nongenotoxicants and 18/20 (90%) genotoxicants were correctly identified. Moreover, there is a need for new in vitro tools to delineate the predominant MoA as in the MicroFlow V R assay only 5/9 (56%) aneugens and 4/11 (36%) clastogens were correctly classified. In contrast, the MultiFlow V R assay provides more in-depth information about the MoA and therefore reliably discriminates clastogens, aneugens, and nongenotoxicants. By using a labspecific, practical threshold for the aforementioned biomarkers, 10/11 (91%) nongenotoxicants and 19/20 genotoxicants (95%), 9/11 (82%) clastogens, and 8/9 (89%) aneugens were correctly categorized, suggesting a clear improvement over the MicroFlow V R . Furthermore, the MultiFlow markers were benchmarked against established methods to assess the validity of the data. Altogether, these findings demonstrated good agreement between the MultiFlow V R assay and the benchmarking methods. Finally, p21 may improve class discrimination given the correct identification of 4/4 (100%) aneugens and 2/5 (40%) clastogens.
The highly regulated process of adapting to cellular nutritional status depends on lysosomal mTORC1 (mechanistic Target of Rapamycin Complex 1), that integrates nutrients availability via the sensing of amino acids to promote growth and anabolism 1 . Nutrient restriction inhibits mTORC1 activity, which in turn induces autophagy, a crucial adaptive process that recycles internal nutrient stores to promote survival 2 . However, as successful amino acid recycling through autophagic degradation reactivates mTORC1 signaling over time 3,4 , it is unclear how autophagy can be maintained during prolonged starvation. Here, we show that one particular amino acid, cysteine, acts in a feedback loop to limit mTORC1 reactivation in vivo. We provide evidence that lysosomal export of cystine through the cystine transporter cystinosin fuels a metabolic pathway that suppresses mTORC1 signaling and maintains autophagy during starvation. This pathway involves reduction to cysteine, cysteine catabolism to acetyl-CoA and subsequent fueling of the TCA cycle. Accordingly, the starvation sensitivity phenotype of animals lacking cystinosin is rescued by dietary supplementation with cysteine and TCA cycle components as well as by reducing mTORC1 activity. We propose that cysteine mediates a communication between lysosomes and mitochondria to control mTORC1 signaling under prolonged starvation, highlighting how changes in nutrient availability divert the fate of an amino acid into a growth suppressive program to maintain the balance between nutrient supply and consumption.Organisms constantly cope with variations in nutrient supply by adjusting metabolism, a process controlled by the major growth regulator mTORC1. Nutrient scarcity inhibits mTORC1 to limit growth and promote catabolic programs, including autophagy. Autophagy involves the sequestration of cytosolic material into autophagosomes that fuse with lysosomes for cargo degradation and recycling. Efficient lysosomal degradation generates new amino acids that in turn reactivate mTORC1 3-6 . However, how mTORC1 inhibition and activation is balanced over the course of starvation remains unclear.
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