Molybdenum cofactor (Moco) is an essential prosthetic group that mediates the activity of 4 animal oxidases and is required for viability. Humans with mutations in the genes encoding Moco-biosynthetic enzymes suffer from Moco deficiency, a neonatal lethal inborn error of metabolism. Caenorhabditis elegans has recently emerged as a useful and tractable genetic discovery engine for Moco biology. Here, we identify and characterize K10D2.7/ moc-6 , the C. elegans ortholog of human MOCS2A, a sulfur-carrier protein essential for Moco synthesis. Using CRISPR/Cas9 gene editing, we generate 3 null mutations in K10D2.7/ moc-6 and with these alleles genetically demonstrate that K10D2.7/ moc-6 is necessary for endogenous Moco synthesis in C. elegans.

The amino acid cysteine is critical for many aspects of life, yet excess cysteine is toxic. Therefore, animals require pathways to maintain cysteine homeostasis. In mammals, high cysteine activates cysteine dioxygenase, a key enzyme in cysteine catabolism. The mechanism by which cysteine dioxygenase is regulated remains largely unknown. We discovered that C. elegans cysteine dioxygenase (cdo-1) is transcriptionally activated by high cysteine and the hypoxia inducible transcription factor (hif-1). hif-1- dependent activation of cdo-1 occurs downstream of an H2S-sensing pathway that includes rhy-1, cysl-1, and egl-9. cdo-1 transcription is primarily activated in the hypodermis where it is sufficient to drive sulfur amino acid metabolism. EGL-9 and HIF-1 are core members of the cellular hypoxia response. However, we demonstrate that the mechanism of HIF-1-mediated induction of cdo-1 functions largely independent of EGL-9 prolyl hydroxylation and the von Hippel-Lindau E3 ubiquitin ligase; classical hypoxia signaling pathway components. We propose that the intersection of hif-1 and cdo-1 reveals a negative feedback loop for maintaining cysteine homeostasis. High cysteine stimulates the production of an H2S signal. H2S then activates the rhy-1/cysl-1/egl-9 signaling pathway, increasing HIF-1-mediated transcription of cdo-1, promoting degradation of cysteine via CDO-1.

No abstract

Molybdenum cofactor (Moco) is a prosthetic group necessary for the activity of 4 unique enzymes, including the essential sulfite oxidase (SUOX-1). Moco is required for life; humans with inactivating mutations in the genes encoding Moco-biosynthetic enzymes display Moco deficiency, a rare and lethal inborn error of metabolism. Despite its importance to human health, little is known about how Moco moves among and between cells, tissues, and organisms. The prevailing view is that cells that require Moco must synthesize Moco de novo. Although, the nematode Caenorhabditis elegans appears to be an exception to this rule and has emerged as a valuable system for understanding fundamental Moco biology. C. elegans has the seemingly unique capacity to both synthesize its own Moco as well as acquire Moco from its microbial diet. However, the relative contribution of Moco from the diet or endogenous synthesis has not been rigorously evaluated or quantified biochemically. We genetically removed dietary or endogenous Moco sources in C. elegans and biochemically determined their impact on animal Moco content and SUOX-1 activity. We demonstrate that dietary Moco deficiency dramatically reduces both animal Moco content and SUOX-1 activity. Furthermore, these biochemical deficiencies have physiological consequences; we show that dietary Moco deficiency alone causes sensitivity to sulfite, the toxic substrate of SUOX-1. This work establishes the biochemical consequences of depleting dietary Moco or endogenous Moco synthesis in C. elegans and quantifies the surprising contribution of the diet to maintaining Moco homeostasis in C. elegans.

The amino acid cysteine is critical for many aspects of life, yet excess cysteine is toxic. Therefore, animals require pathways to maintain cysteine homeostasis. In mammals, high cysteine activates cysteine dioxygenase, a key enzyme in cysteine catabolism. The mechanism by which cysteine dioxygenase is regulated remains largely unknown. We discovered that C. elegans cysteine dioxygenase (cdo-1) is transcriptionally activated by high cysteine and the hypoxia inducible transcription factor (hif-1). hif-1- dependent activation of cdo-1 occurs downstream of an H2S-sensing pathway that includes rhy-1, cysl-1, and egl-9. cdo-1 transcription is primarily activated in the hypodermis where it is sufficient to drive sulfur amino acid metabolism. EGL-9 and HIF-1 are core members of the cellular hypoxia response. However, we demonstrate that the mechanism of HIF-1-mediated induction of cdo-1 functions largely independent of EGL-9 prolyl hydroxylation and the von Hippel-Lindau E3 ubiquitin ligase; classical hypoxia signaling pathway components. We propose that the intersection of hif-1 and cdo-1 reveals a negative feedback loop for maintaining cysteine homeostasis. High cysteine stimulates the production of an H2S signal. H2S then activates the rhy-1/cysl-1/egl-9 signaling pathway, increasing HIF-1-mediated transcription of cdo-1, promoting degradation of cysteine via CDO-1.