Identification of a four copper folding intermediate in mammalian copper metallothionein by electrospray ionization mass spectrometry

 Mammalian metallothioneins (MT) are known to maximally bind 12 copper ions in two six-Cu(I) ion clusters. Using electrospray ionization mass spectrometry of MT at pH 4.5, a four-Cu(I) ion cluster was observed intermediate to a fully formed six Cu(I) in a single domain or a fully formed Cu₁₂MT species. The four-Cu(I) cluster was observed in both MT1 and MT3 isoforms. Addition of increasing amounts of Cu(I) to MT at pH 4.5 resulted in prominent ions whoses masses were consistent with apo-MT, Cu₄MT, Cu₆MT, and Cu₁₂MT. The cooperativity of cluster formation was reduced at pH 2.5. Addition of Cu(I) to apo-MT at a reduced pH resulted in a series of ions consistent with Cu₄ to Cu₁₂MT species. However, formation of the tetracopper MT species remained cooperative at low pH, suggesting that this species is very stable. To determine whether the tetracopper cluster was formed in either the α or β domain, domain peptides of MT3 were used. Addition of Cu(I) to the apo β domain resulted in a peak consistent with the formation of a four-Cu(I) cluster. This is consistent with reports that Cu(I) ions bind preferentially to the β domain of MTs. Received: 2 June 1998 / Accepted: 21 August 1998     

Decreased accumulation of superoxide dismutase 2 within mitochondria in the yeast model of Shwachman-Diamond syndrome

Mutations in the human SBDS gene is the most common cause of Shwachman-Diamond syndrome (SDS). The SBDS protein participates in ribosome biogenesis; however, effects beyond reduced translation efficiency are thought to be involved in SDS progression. Impaired mitochondrial function has been reported for cells lacking either SBDS or Sdo1p, the Saccharomyces cerevisiae SBDS ortholog. To better understand how the loss of SBDS/Sdo1p leads to mitochondria damage, we utilized the S. cerevisiae model of SDS. Yeast deleted for SDO1 show increased oxidative damage to mitochondrial proteins and a marked decrease in protein levels and activity of mitochondrial superoxide dismutase 2 (Sod2p), a key enzyme involved in defense against oxidants. Immature forms of Sod2p are observed in sdo1∆ cells suggesting a defect in proteolysis of the presequence. Yeast deleted for CYM1, encoding a presequence protease, display a similar reduction in Sod2p activity as sdo1∆ cells, as well as elevated oxidative damage, to mitochondrial proteins. Sod2p protein levels and activity are largely restored in a por1∆ sdo1∆ strain, lacking the major mitochondrial voltage-dependent anion channel. Together these results indicate that mitochondrial insufficiency in sdo1∆ cells may be linked to the accumulation of immature presequence containing proteins and this effect is a consequence, at least in part, from loss of counter-regulation of Por1p by Sdo1p.     

Improper protein trafficking contributes to artemisinin sensitivity in cells lacking the KDAC Rpd3p

Lysine deacetylases (KDACs) inhibitors may have therapeutic value in anti-malarial combination therapies with artemisinin. To evaluate connections between KDACs and artemisinin, Saccharomyces cerevisiae deletion mutants in KDAC genes were assayed. Deletion of RPD3, but not other KDAC genes, resulted in strong sensitivity to artemisinin, which was also observed in sit4Δ mutants with impaired endoplasmic reticulum (ER) to Golgi protein trafficking. Decreased accumulation of the transporters Pdr5p, Fur4p, and Tat2p was observed in rpd3Δ and sit4Δ cells. The unfolded protein response is induced in rpd3Δ cells consistent with retention of proteins in the ER. Disruption of protein trafficking appears to sensitize cells to artemisinin and targeting these pathways may be useful as part of artemisinin based anti-malarial therapy.     

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.     

Activation of Cu,Zn-Superoxide Dismutase in the Absence of Oxygen and the Copper Chaperone CCS

Eukaryotic Cu,Zn-superoxide dismutases (SOD1s) are generally thought to acquire the essential copper cofactor and intramolecular disulfide bond through the action of the CCS copper chaperone. However, several metazoan SOD1s have been shown to acquire activity in vivo in the absence of CCS, and the Cu,Zn-SOD from Caenorhabditis elegans has evolved complete independence from CCS. To investigate SOD1 activation in the absence of CCS, we compared and contrasted the CCS-independent activation of C. elegans and human SOD1 to the strict CCS-dependent activation of Saccharomyces cerevisiae SOD1. Using a yeast expression system, both pathways were seen to acquire copper derived from cell surface transporters and compete for the same intracellular pool of copper. Like CCS, CCS-independent activation occurs rapidly with a preexisting pool of apo-SOD1 without the need for new protein synthesis. The two pathways, however, strongly diverge when assayed for the SOD1 disulfide. SOD1 molecules that are activated without CCS exhibit disulfide oxidation in vivo without oxygen and under copper-depleted conditions. The strict requirement for copper, oxygen, and CCS in disulfide bond oxidation appears exclusive to yeast SOD1, and we find that a unique proline at position 144 in yeast SOD1 is responsible for this disulfide effect. CCS-dependent and -independent pathways also exhibit differential requirements for molecular oxygen. CCS activation of SOD1 requires oxygen, whereas the CCS-independent pathway is able to activate SOD1s even under anaerobic conditions. In this manner, Cu,Zn-SOD from metazoans may retain activity over a wide range of physiological oxygen tensions.Oxygen is essential for aerobic respiration, but reactive byproducts of oxygen metabolism, such as the superoxide anion, can damage cellular molecules, including proteins, DNA, and lipids (1–3). SOD1s (copper- and zinc-containing superoxide dismutases) provide the primary defense against superoxide damage by catalytically removing it through a disproportionation reaction (4). This reaction involves redox cycling at the copper active site (5). SOD1s require several post-translational modifications to form an active molecule. Copper and zinc are bound by the enzyme, and an intramolecular disulfide bond is formed between two conserved cysteine residues. Although the zinc ion and disulfide bond are not directly involved in the disproportionation reaction, these modifications are required for proper stability and formation of the active site (6–10). The presence of an intramolecular disulfide bond is intriguing, given the fact that the cytosol favors reduced thiols.The activity of SOD1s in vivo is largely controlled through the aforementioned post-translational modifications. Most of what is currently known about activation of SOD1 in vivo has emerged through studies of the bakers'' yeast Saccharomyces cerevisiae SOD1. Here insertion of the catalytic copper requires the action of the copper chaperone for SOD³ (CCS) (11). CCS physically interacts with SOD1 to deliver the copper ion and catalyze the disulfide bond formation in an oxygen-dependent manner (12–15). In fact, S. cerevisiae SOD1 (ySOD1) is completely dependent on CCS for insertion of the catalytic copper and oxidation of the disulfide bond (11, 15, 16).Although ySOD1 is dependent on CCS for activity, other eukaryotic SOD1s are not. Mouse and human SOD1 (hSOD1), when expressed in CCS^−/− mouse fibroblasts and in ccs1Δ yeast, still retain some SOD1 activity (17–19). Moreover, the genome for the nematode Caenorhabditis elegans does not contain a CCS-like gene, yet harbors several Cu,Zn-SODs. Previous studies with C. elegans SOD-1 (wSOD-1) have shown that this SOD is activated completely independently of CCS (20). Together, these studies present a strong case for a second SOD1 activation mechanism independent of CCS.There must be inherent differences in SOD1 sequences that dictate whether the enzyme uses CCS or the CCS-independent pathway or both. Through targeted mutagenesis, sequences near the C terminus have been previously identified as being important (19). Yeast SOD1 contains dual prolines at positions 142 and 144, which when mutated in combination allow for CCS-independent activation. Conversely, hSOD1 and wSOD-1 contain non-proline residues at these positions, and if dual prolines are introduced, then CSS-independent activation is blocked (19, 20). How this pair of prolines influences SOD1 activation is not understood.It is interesting that nature has developed two activation mechanisms for such a key enzyme in oxidative stress protection, and these are not likely to be redundant. It was previously predicted that the two pathways draw upon distinct sources of copper (19), since the addition of the catalytic copper ion is limiting for enzyme activation. However, since disulfide oxidation is also limiting for enzyme activity, it is possible that the two pathways diverge at this level. In the current study, we investigate the requirements and regulation of the CCS-dependent and -independent SOD1 activation pathways. Our results strongly indicate that the two pathways do not diverge at the level of upstream copper transporter sources or the kinetics of copper incorporation into SOD1 but rather at the level of disulfide bond formation. Copper is required for CCS-mediated disulfide bond oxidation in yeast SOD1, whereas SOD1s that can be activated without CCS show no such requirement for copper in disulfide oxidation. Moreover, oxygen is required for enzyme activation through CCS, but the CCS-independent pathway is able to bypass the need for molecular oxygen. This allows for significant SOD1 activity to be found at a variety of oxygen concentrations by utilizing two activation pathways.     

Modelling Marine Predator Habitat Using the Abundance of Its Pelagic Prey in the Tropical South-Western Pacific

Ecosystems - Understanding the ecological mechanisms underpinning distribution patterns is vital in managing populations of mobile marine species. This study is a first step towards an integrated...     

Activation of CuZn superoxide dismutases from Caenorhabditis elegans does not require the copper chaperone CCS

Reactive oxygen species are produced as the direct result of aerobic metabolism and can cause damage to DNA, proteins, and lipids. A principal defense against reactive oxygen species involves the superoxide dismutases (SOD) that act to detoxify superoxide anions. Activation of CuZn-SODs in eukaryotic cells occurs post-translationally and is generally dependent on the copper chaperone for SOD1 (CCS), which inserts the catalytic copper cofactor and catalyzes the oxidation of a conserved disulfide bond that is essential for activity. In contrast to other eukaryotes, the nematode Caenorhabditis elegans does not contain an obvious CCS homologue, and we have found that the C. elegans intracellular CuZn-SODs (wSOD-1 and wSOD-5) are not dependent on CCS for activation when expressed in Saccharomyces cerevisiae. CCS-independent activation of CuZn-SODs is not unique to C. elegans; however, this is the first organism identified that appears to exclusively use this alternative pathway. As was found for mammalian SOD1, wSOD-1 exhibits a requirement for reduced glutathione in CCS-independent activation. Unexpectedly, wSOD-1 was inactive even in the presence of CCS when glutathione was depleted. Our investigation of the cysteine residues that form the disulfide bond in wSOD-1 suggests that the ability of wSODs to readily form this disulfide bond may be the key to obtaining high levels of activation through the CCS-independent pathway. Overall, these studies demonstrate that the CuZn-SODs of C. elegans have uniquely evolved to acquire copper without the copper chaperone and this may reflect the lifestyle of this organism.