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Evidence for a role for the putative Drosophila hGRX1 orthologue in copper homeostasis

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Abstract

Glutaredoxins are a family of small molecular weight proteins that have a central role in cellular redox regulation. Human GRX1 (hGRX1) has also been shown to play an integral role in copper homeostasis by regulating the redox activity of the metalated sites of copper chaperones such as ATOX1 and SOD1, and the copper efflux proteins ATP7A and ATP7B. To further elucidate the role of hGRX1 in copper homeostasis, we examined the impact of RNA interference-mediated knockdown of CG6852, a putative Drosophila orthologue of hGRX1. CG6852 shares ~41 % amino acid identity with hGRX1 and key functional domains including the metal-binding CXXC motif are conserved between the two proteins. Knockdown of CG6852 in the adult midline caused a thoracic cleft and reduced scutellum, phenotypes that were exacerbated by additional knockdown of copper uptake transporters Ctr1A and Ctr1B. Knockdown of CG6852 in the adult eye enhanced a copper-deficiency phenotype caused by Ctr1A knockdown while ubiquitous knockdown of CG6852 resulted a mild systemic copper deficiency. Therefore we conclude that CG6852 is a putative orthologue of hGRX1 and may play an important role in Drosophila copper homeostasis.

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Abbreviations

SOD1:

Superoxide dismutase 1

Cu:

Copper

BCS:

Bathocuproine disulfonate

PBS:

Phosphate buffered saline

COX:

Cytochrome c oxidase

PNR:

Pannier

GSH:

Glutathione

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Acknowledgments

This work was supported by Project Grant funding from the National Health and Medical Research Council of Australia (RB, Project Grant #606609). The Australian Drosophila Biomedical Research Support Facility assisted in the import and quarantine of fly strains used in this research. All transgenic Drosophila experiments carried out in this research were performed with the approval of the Monash University Institutional Biosafety Committee.

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Authors and Affiliations

  1. School of Biological Sciences, Monash University, Melbourne, VIC, Australia

    Stephen W. Mercer & Richard Burke

  2. Department of Pathology, University of Melbourne, Melbourne, VIC, Australia

    Stephen W. Mercer

Authors
  1. Stephen W. Mercer
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  2. Richard Burke
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Correspondence to Stephen W. Mercer.

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10534_2016_9946_MOESM1_ESM.tif

Figure S1. CG6852 RNAi line effectively decreases CG6852 transcript levels. Weak ubiquitous RNAi-mediated suppression of CG6852 using DA-GAL4 (DA>CG6852RNAi) significantly decreased CG6852 expression levels compared with wild type (DA>w1118). n≥3, *** p-value <0.001. 1-3: DA>CG6852RNAi, CG6852 primer pair. 4-6: DA>w1118, CG6852 primer pair. 7: DA>CG6852RNAi, RP49 primer pair. 8: DA>w1118, RP49 primer pair. (TIFF 8180 kb)

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Mercer, S.W., Burke, R. Evidence for a role for the putative Drosophila hGRX1 orthologue in copper homeostasis. Biometals 29, 705–713 (2016). https://doi.org/10.1007/s10534-016-9946-0

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