Disrupted zinc-binding sites in structures of pathogenic SOD1 variants D124V and H80R.

Mutations in human copper-zinc superoxide dismutase (SOD1) cause an inherited form of the fatal neurodegenerative disease amyotrophic lateral sclerosis (ALS). Here, we present structures of the pathogenic SOD1 variants D124V and H80R, both of which demonstrate compromised zinc-binding sites. The disruption of the zinc-binding sites in H80R SOD1 leads to conformational changes in loop elements, permitting non-native SOD1-SOD1 interactions that mediate the assembly of these proteins into higher-order filamentous arrays. Analytical ultracentrifugation sedimentation velocity experiments indicate that these SOD1 variants are more prone to monomerization than the wild-type enzyme. Although D124V and H80R SOD1 proteins appear to have fully functional copper-binding sites, inductively coupled plasma mass spectrometery (ICP-MS) and anomalous scattering X-ray diffraction analyses reveal that zinc (not copper) occupies the copper-binding sites in these variants. The absence of copper in these proteins, together with the results of covalent thiol modification experiments in yeast strains with and without the gene encoding the copper chaperone for SOD1 (CCS), suggests that CCS may not fully act on newly translated forms of these polypeptides. Overall, these findings lend support to the hypothesis that immature mutant SOD1 species contribute to toxicity in SOD1-linked ALS.

Structural and biophysical properties of the pathogenic SOD1 variant H46R/H48Q.

Over 100 mutations in the gene encoding human copper-zinc superoxide dismutase (SOD1) cause an inherited form of the fatal neurodegenerative disease amyotrophic lateral sclerosis (ALS). Two pathogenic SOD1 mutations, His46Arg (H46R) and His48Gln (H48Q), affect residues that act as copper ligands in the wild type enzyme. Transgenic mice expressing a human SOD1 variant containing both mutations develop paralytic disease akin to ALS. Here we show that H46R/H48Q SOD1 possesses multiple characteristics that distinguish it from the wild type. These properties include the following: (1) an ablated copper-binding site, (2) a substantially weakened affinity for zinc, (3) a binding site for a calcium ion, (4) the ability to form stable heterocomplexes with the copper chaperone for SOD1 (CCS), and (5) compromised CCS-mediated oxidation of the intrasubunit disulfide bond in vivo. The results presented here, together with data on pathogenic SOD1 proteins coming from cell culture and transgenic mice, suggest that incomplete posttranslational modification of nascent SOD1 polypeptides via CCS may be a characteristic shared by familial ALS SOD1 mutants, leading to a population of destabilized, off-pathway folding intermediates that are toxic to motor neurons.

Down-regulation of a manganese transporter in the face of metal toxicity.

The yeast Smf1p Nramp manganese transporter is posttranslationally regulated by environmental manganese. Smf1p is stabilized at the cell surface with manganese starvation, but is largely degraded in the vacuole with physiological manganese through a mechanism involving the Rsp5p adaptor complex Bsd2p/Tre1p/Tre2p. We now describe an additional level of Smf1p regulation that occurs with toxicity from manganese, but not other essential metals. This regulation is largely Smf1p-specific. As with physiological manganese, toxic manganese triggers vacuolar degradation of Smf1p by trafficking through the multivesicular body. However, regulation by toxic manganese does not involve Bsd2p/Tre1p/Tre2p. Toxic manganese triggers both endocytosis of cell surface Smf1p and vacuolar targeting of intracellular Smf1p through the exocytic pathway. Notably, the kinetics of vacuolar targeting for Smf1p are relatively slow with toxic manganese and require prolonged exposures to the metal. Down-regulation of Smf1p by toxic manganese does not require transport activity of Smf1p, whereas such transport activity is needed for Smf1p regulation by manganese starvation. Furthermore, the responses to manganese starvation and manganese toxicity involve separate cellular compartments. We provide evidence that manganese starvation is sensed within the lumen of the secretory pathway, whereas manganese toxicity is sensed within an extra-Golgi/cytosolic compartment of the cell.

The effects of glutaredoxin and copper activation pathways on the disulfide and stability of Cu,Zn superoxide dismutase.

Mutations in Cu,Zn superoxide dismutase (SOD1) can cause amyotrophic lateral sclerosis (ALS) through mechanisms proposed to involve SOD1 misfolding, but the intracellular factors that modulate folding and stability of SOD1 are largely unknown. By using yeast and mammalian expression systems, we demonstrate here that SOD1 stability is governed by post-translational modification factors that target the SOD1 disulfide. Oxidation of the human SOD1 disulfide in vivo was found to involve both the copper chaperone for SOD1 (CCS) and the CCS-independent pathway for copper activation. When both copper pathways were blocked, wild type SOD1 stably accumulated in yeast cells with a reduced disulfide, whereas ALS SOD1 mutants A4V, G93A, and G37R were degraded. We describe here an unprecedented role for the thiol oxidoreductase glutaredoxin in reducing the SOD1 disulfide and destabilizing ALS mutants. Specifically, the major cytosolic glutaredoxin of yeast was seen to reduce the intramolecular disulfide of ALS SOD1 mutant A4V SOD1 in vivo and in vitro. By comparison, glutaredoxin was less reactive toward the disulfide of wild type SOD1. The apo-form of A4V SOD1 was highly reactive with glutaredoxin but not SOD1 containing both copper and zinc. Glutaredoxin therefore preferentially targets the immature form of ALS mutant SOD1 lacking metal co-factors. Overall, these studies implicate a critical balance between cellular reductants such as glutaredoxin and copper activation pathways in controlling the disulfide and stability of SOD1 in vivo.

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

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.