A Gain of Superoxide Dismutase (SOD) Activity Obtained with CCS, the Copper Metallochaperone for SOD1

Paul J Schmidt,Minerva Ramos-Gomez,Valeria Cizewski Culotta

doi:10.1074/jbc.274.52.36952

Paul J Schmidt, Minerva Ramos-Gomez + Show 1 more

Open Access

https://doi.org/10.1074/jbc.274.52.36952

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Abstract

The incorporation of copper ions into the cytosolic superoxide dismutase (SOD1) is accomplished in vivo by the action of the copper metallochaperone CCS (copper chaperone for SOD1). Mammalian CCS is comprised of three distinct protein domains, with a central region exhibiting remarkable homology (approximately 50% identity) to SOD1 itself. Conserved in CCS are all the SOD1 zinc binding ligands and three of four histidine copper binding ligands. In CCS the fourth histidine is replaced by an aspartate (Asp(200)). Despite this conservation of sequence between SOD1 and CCS, CCS exhibited no detectable SOD activity. Surprisingly, however, a single D200H mutation, targeting the fourth potential copper ligand in CCS, granted significant superoxide scavenging activity to this metallochaperone that was readily detected with CCS expressed in yeast. This mutation did not inhibit the metallochaperone capacity of CCS, and in fact, D200H CCS appears to represent a bifunctional SOD that can self-activate itself with copper. The aspartate at CCS position 200 is well conserved among mammalian CCS molecules, and we propose that this residue has evolved to preclude deleterious reactions involving copper bound to CCS.

Highlights

In eukaryotic cells, copper is delivered to specific protein targets via the action of a family of copper carrier proteins termed “metallochaperones” [1]
All four of the zinc binding ligands of superoxide dismutase 1 (SOD1) and three of four histidine copper binding ligands are present in CCS; the fourth histidine is replaced by an aspartate residue, which is a possible ligand for copper [35] (Fig. 1A)
Based on sequence analysis alone it would seem plausible that mammalian CCS should possess SOD activity

Summary

EXPERIMENTAL PROCEDURES

Yeast Strains and Plasmids—The isogenic wild type SY1699 and lys null SY2950 strains were previously described [27], as was the sod1⌬ sod2⌬ strain KS100 [28]. The vector for expression of human CCS in yeast cells is pCCS-HIS, containing the human CCS coding sequence under the control of the S. cerevisiae PGK1 (phosphoglycerol kinase) promoter. Human SOD1 was expressed in yeast by the pLC1 vector (URA3 2␮) where the SOD1 cDNA was placed under the control of the PGK1 promoter as described [31]. PSM703 is the PGK1 promoter-containing vector (URA3 2␮) that served as the parent plasmid for construction of all human SOD1 and CCS expression plasmids. For immunoprecipitation of human CCS and yeast SOD1, 50 –100 ␮g of cell lysate protein was reacted with either anti-CCS (1:100 dilution), anti-yeast SOD1 (1:500 dilution; kind gift of Dan Kosman, SUNY, Buffalo), or preimmune serum (1:100 dilution) in a buffer containing 50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 150 mM NaCl, and 0.1% Nonidet P-40, as needed. The mixture was subjected to microcentrifugation for 1 min at 4 °C, and the resulting supernatants were concentrated on Microcon-10 Microconcentrator (Amicon) columns

RESULTS

A Copper Chaperone with SOD Activity

DISCUSSION