Abstract
Copper plays key roles in catalytic and regulatory biochemical reactions essential for normal growth, development, and health. Dietary copper deficiencies or mutations in copper homeostasis genes can lead to abnormal musculoskeletal development, cognitive disorders, and poor growth. In yeast and mammals, copper is acquired through the activities of the CTR1 family of high-affinity copper transporters. However, the mechanisms of systemic responses to dietary or tissue-specific copper deficiency remain unclear. Here, taking advantage of the animal model Caenorhabditis elegans for studying whole-body copper homeostasis, we investigated the role of a C. elegans CTR1 homolog, CHCA-1, in copper acquisition and in worm growth, development, and behavior. Using sequence homology searches, we identified 10 potential orthologs to mammalian CTR1 Among these genes, we found that chca-1, which is transcriptionally up-regulated in the intestine and hypodermis of C. elegans during copper deficiency, is required for normal growth, reproduction, and maintenance of systemic copper balance under copper deprivation. The intestinal copper transporter CUA-1 normally traffics to endosomes to sequester excess copper, and we found here that loss of chca-1 caused CUA-1 to mislocalize to the basolateral membrane under copper overload conditions. Moreover, animals lacking chca-1 exhibited significantly reduced copper avoidance behavior in response to toxic copper conditions compared with WT worms. These results establish that CHCA-1-mediated copper acquisition in C. elegans is crucial for normal growth, development, and copper-sensing behavior.
Highlights
Copper plays key roles in catalytic and regulatory biochemical reactions essential for normal growth, development, and health
We found that chca-1, which is transcriptionally up-regulated in the intestine and hypodermis of C. elegans during copper deficiency, is required for normal growth, reproduction, and maintenance of systemic copper balance under copper deprivation
Worm copper transporter 1 (CTR1) candidate proteins were further analyzed based on the conserved features of copper transporter gene (CTR) proteins, such as number of transmembrane domains and copper-transporting motifs at the N and C termini, and the second predicted TMD
Summary
To identify potential genes for copper acquisition in C. elegans, a Basic Local Alignment Search Tool (BLAST) search was performed using the human CTR1 protein sequence as a probe. Values with asterisk are significantly different from vector under the same culture condition (two-way ANOVA, Sidak post hoc test, ****, p Ͻ 0.0001.) Error bars in this figure represent mean Ϯ S.E. other CTR candidate genes did not change CUA-1.1 localization in the intestine (Fig. S4), suggesting the CHCA-1 is a major player in intestinal copper homeostasis. At least 100 synchronized L4 CHCA-1::GFP-expressing worms were used following 2.5 days of copper or BCS-supplemented cultures in each condition (one-way ANOVA, Dunnett post hoc test, ns, not significant). Transgenic worms expressing WT CHCA-1 in the intestine in the mutant background significantly rescued tm6506 growth during copper deficiency (Fig. 6D) These results suggest a role for vesicular CHCA-1 in mediating copper acquisition in the worm intestine, and that CHCA-1 protein abundance, in contrast with mammalian CTR1, is not regulated by copper status. Our results suggest that copper-sensing and/or corresponding behavior of C. elegans is associated with the copperdependent neuropeptide maturation in neurons that requires CHCA-1 activity and balanced body copper levels
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