Mechanisms for activating Cu- and Zn-containing superoxide dismutase in the absence of the CCS Cu chaperone - PubMed (original) (raw)

Mechanisms for activating Cu- and Zn-containing superoxide dismutase in the absence of the CCS Cu chaperone

Mark C Carroll et al. Proc Natl Acad Sci U S A. 2004.

Erratum in

Abstract

The Cu- and Zn-containing superoxide dismutase 1 (SOD1) largely obtains Cu in vivo by means of the action of the Cu chaperone CCS. Yet, in the case of mammalian SOD1, a secondary pathway of activation is apparent. Specifically, when human SOD1 is expressed in either yeast or mammalian cells that are null for CCS, the SOD1 enzyme retains a certain degree of activity. This CCS-independent activity is evident with both wild-type and mutant variants of SOD1 that have been associated with familial amyotrophic lateral sclerosis. We demonstrate here that the CCS-independent activation of mammalian SOD1 involves glutathione, particularly the reduced form, or GSH. A role for glutathione in CCS-independent activation was seen with human SOD1 molecules that were expressed in either yeast cells or immortalized fibroblasts. Compared with mammalian SOD1, the Saccharomyces cerevisiae enzyme cannot obtain Cu without CCS in vivo, and this total dependence on CCS involves the presence of dual prolines near the C terminus of the SOD1 polypeptide. Indeed, the insertion of such prolines into human SOD1 rendered this molecule refractory to CCS-independent activation. The possible implications of multiple pathways for SOD1 activation are discussed in the context of SOD1 evolutionary biology and familial amyotrophic lateral sclerosis.

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Figures

Fig. 3.

Fig. 3.

Distinct concentrations of glutathione are required to support cell growth and to stimulate hSOD1 activity in vivo. The indicated strains expressing hSOD1 from pLC1 were grown in SD medium supplemented with the designated concentrations of glutathione (GSHout). After five to six doublings, an aliquot of cells were harvested and assayed for hSOD1 activity by the native gel assay (Top), for SOD1 polypeptide levels by immunoblotting (Middle), and for intracellular glutathione levels (GSHin). Glutathione measurements represent the average of duplicate samples, in which range was <10%. To help correct for the increased levels of SOD1 protein in _gsh1_Δ cells¶, only approximately one-third as much total cellular protein was assayed in the native gel and immunoblot in the case of untreated GSH– cells. Cells that were not harvested were diluted to OD600 = 0.075 and cultured for an additional 14 h under the same glutathione-treatment conditions, and total cell growth was determined by absorbance at 600 nm (Bottom). The following strains were used: GSH1+, PS131 (_lys7_Δ); and GSH1–, PS132 (_lys7_Δ _gsh1_Δ).

Fig. 2.

Fig. 2.

Glutathione depletion inhibits CCS-independent activation of human SOD1. The indicated yeast strains expressing wild-type hSOD1 from pLC1 were tested either for SOD activity and hSOD1 polypeptide levels, as shown in Fig. 1 (A); lysine independent growth in air (B); or effect of metal chelators during gel electrophoresis (C). The following strains were used: CCS+ GSH1+, KS107 (_sod1_Δ); CCS+ GSH1–, MC107 (_sod1_Δ _gsh1_Δ); CCS– GSH1+, PS131 (_lys7_Δ); CCS– GSH1–, PS132 (_lys7_Δ _gsh1_Δ); CCS– GLR1+, 614 (_lys7_Δ); and CCS– GLR1–, MC112 (_lys7_Δ _glr1_Δ). (A) Cells were grown for five to six doublings in SD medium before preparation of cell lysates. We assayed 40 μg of cellular protein, except for in lane 6, which contained 15 μg to help normalize hSOD1 polypeptide levels. (B) We spotted 2 × 104 and 2 × 103 cells of the indicated strains onto SD medium lacking lysine and allowed them to grow for 2 days either in air (+O2) or in anaerobic culture jars (–O2). (C) Native gel electrophoresis and nitro blue tetrazolium staining for SOD1 activity was carried out as described for A except that 8% polyacrylamide gels were poured that contained 0.1 mM EDTA where indicated. (All other native gels of this study used 12% precast gels that were not treated with EDTA.) Large arrow indicates position of major hSOD1 activity species, whereas smaller arrows indicate hSOD1 that was apparently acquired Cu during electrophoresis (see Discussion).

Fig. 1.

Fig. 1.

Activity of human wild-type and ALS mutant SOD1 in yeast cells lacking CCS. The indicated yeast strains were grown in SD medium to confluency over night. Forty micrograms of lysate protein was subject to either native gel electrophoresis and nitro blue tetrazolium staining for SOD activity (A Upper and B Upper) or to denaturing SDS gel electrophoresis and immunoblotting using an antibody directed against human SOD1 (A Lower and B Lower). (A) Strains were transformed with either pLC1 expressing human wild-type SOD1 (hSOD1, +) or pSM703 empty vector control (hSOD1, –). The following strains were used: yCCS+ ySOD1–, KS107 (_sod1_Δ); yCCS– ySOD1–, LS101 (_sod1_Δ _lys7_Δ); and yCCS– ySOD1+, PS131 (_lys7_Δ). Arrow indicates position of hSOD1. The activity band of slower migration represents S. cerevisiae SOD2. (B) Strains were transformed with either pLC1 (WT) or with the YEp351-hSOD1-based plasmids (21) for expression of the indicated FALS mutant alleles of SOD1. The following strains were used: CCS+, KS107; and CCS–, LS101. The YEp351-hSOD1-based plasmids generally yield higher overall expression levels than those of pLC1. The effects of point mutations on hSOD1 mobility on both native and denaturing gels are not unusual (30).

Fig. 5.

Fig. 5.

The role of C-terminal prolines in determining the dependence of SOD1 on CCS. (A) An alignment of the C-terminal region of S. cerevisiae and human SOD1. Black and gray residues represent amino acid identity and similarity, respectively. White boxes mark positions where amino acid substitutions were introduced. Gray box indicates conserved disulfide cysteine. (B) The indicated strains expressing wild-type (WT) or the mutant variants of yeast SOD1 were subject to the native gel assay for SOD1 activity (Upper) and immunoblot analysis for ySOD1 using an antibody directed against yeast SOD1 (Lower). To help correct for the higher SOD1 polypeptide levels seen in gsh1_Δ mutants¶,1/3 as much total cellular protein was assayed in lane 11 compared with lanes 9 and 10. (C) Activity and polypeptide levels of human wild-type (WT) and mutant hSOD1 expressed in yeast was assayed as in Fig. 1. (D) The indicated strains transformed with empty pSM703 vector (V) or plasmids expressing wild-type (WT) or the indicated mutant hSOD1 were assayed for lysine-independent aerobic growth, as described for Fig. 2_B. The following strains were used in B, lanes 1–8, C, and D: CCS+, KS107 (_sod1_Δ); and CCS–, LS101 (_sod1_Δ _lys7_Δ). The following strains were used in B, lanes 9–11: CCS+ GSH1+, KS107 (_sod1_Δ); CCS– GSH1+, LS101 (_sod1_Δ _lys7_Δ); and CCS– GSH1–, PS132 (_lys7_Δ _gsh1_Δ).

Fig. 4.

Fig. 4.

Glutathione depletion inhibits CCS-independent activation of mammalian SOD1 in fibroblasts. Immortalized fibroblasts from CCS+/+ and CCS–/– mice that were either nontransgenic for hSOD1 (Ntg) or transgenic for G37R hSOD1 (line 42, ref. 17) were cultured in the presence or absence of 50 μM BSO to deplete intracellular glutathione. We subjected 20 μg of lysate protein to SOD1 activity analysis by the native gel, as described for Fig. 1, and to immunoblot analysis to monitor SOD1 polypeptide levels by using an antibody that recognizes both human and mouse SOD1 (lanes 1–4) or is specific to hSOD1 (lanes 5–8). GSHin, total intracellular glutathione levels, where numbers represent the averages of at least two independent experimental trials; range is indicated below the blots.

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