Cu,Zn superoxide dismutase maturation and activity are regulated by COMMD1.
ABSTRACT: The maturation and activation of the anti-oxidant Cu,Zn superoxide dismutase (SOD1) are highly regulated processes that require several post-translational modifications. The maturation of SOD1 is initiated by incorporation of zinc and copper ions followed by disulfide oxidation leading to the formation of enzymatically active homodimers. Our present data indicate that homodimer formation is a regulated final step in SOD1 maturation and implicate the recently characterized copper homeostasis protein COMMD1 in this process. COMMD1 interacts with SOD1, and this interaction requires CCS-mediated copper incorporation into SOD1. COMMD1 does not regulate disulfide oxidation of SOD1 but reduces the level of SOD1 homodimers. RNAi-mediated knockdown of COMMD1 expression results in a significant induction of SOD1 activity and a consequent decrease in superoxide anion concentrations, whereas overexpression of COMMD1 exerts exactly the opposite effects. Here, we identify COMMD1 as a novel protein regulating SOD1 activation and associate COMMD1 function with the production of free radicals.
Project description:The Copper Metabolism MURR1 domain protein 1 (COMMD1) is a protein involved in multiple cellular pathways, including copper homeostasis, NF-?B and hypoxia signalling. Acting as a scaffold protein, COMMD1 mediates the levels, stability and proteolysis of its substrates (e.g. the copper-transporters ATP7B and ATP7A, RELA and HIF-1?). Recently, we established an interaction between the Cu/Zn superoxide dismutase 1 (SOD1) and COMMD1, resulting in a decreased maturation and activation of SOD1. Mutations in SOD1, associated with the progressive neurodegenerative disorder Amyotrophic Lateral Sclerosis (ALS), cause misfolding and aggregation of the mutant SOD1 (mSOD1) protein. Here, we identify COMMD1 as a novel regulator of misfolded protein aggregation as it enhances the formation of mSOD1 aggregates upon binding. Interestingly, COMMD1 co-localizes to the sites of mSOD1 inclusions and forms high molecular weight complexes in the presence of mSOD1. The effect of COMMD1 on protein aggregation is client-specific as, in contrast to mSOD1, COMMD1 decreases the abundance of mutant Parkin inclusions, associated with Parkinson's disease. Aggregation of a polyglutamine-expanded Huntingtin, causative of Huntington's disease, appears unaltered by COMMD1. Altogether, this study offers new research directions to expand our current knowledge on the mechanisms underlying aggregation disease pathologies.
Project description:Metallochaperones are a diverse family of trafficking molecules that provide metal ions to protein targets for use as cofactors. The copper chaperone for superoxide dismutase (Ccs1) activates immature copper-zinc superoxide dismutase (Sod1) by delivering copper and facilitating the oxidation of the Sod1 intramolecular disulfide bond. Here, we present structural, spectroscopic, and cell-based data supporting a novel copper-induced mechanism for Sod1 activation. Ccs1 binding exposes an electropositive cavity and proposed "entry site" for copper ion delivery on immature Sod1. Copper-mediated sulfenylation leads to a sulfenic acid intermediate that eventually resolves to form the Sod1 disulfide bond with concomitant release of copper into the Sod1 active site. Sod1 is the predominant disulfide bond-requiring enzyme in the cytoplasm, and this copper-induced mechanism of disulfide bond formation obviates the need for a thiol/disulfide oxidoreductase in that compartment.
Project description:The CCS copper chaperone is critical for maturation of Cu, Zn-superoxide dismutase (SOD1) through insertion of the copper co-factor and oxidization of an intra-subunit disulfide. The disulfide helps stabilize the SOD1 polypeptide, which can be particularly important in cases of amyotrophic lateral sclerosis (ALS) linked to misfolding of mutant SOD1. Surprisingly, however, over-expressed CCS was recently shown to greatly accelerate disease in a G93A SOD1 mouse model for ALS. Herein we show that disease in these G93A/CCS mice correlates with incomplete oxidation of the SOD1 disulfide. In the brain and spinal cord, CCS over-expression failed to enhance oxidation of the G93A SOD1 disulfide and if anything, effected some accumulation of disulfide-reduced SOD1. This effect was mirrored in culture with a C244,246S mutant of CCS that has the capacity to interact with SOD1 but can neither insert copper nor oxidize the disulfide. In spite of disulfide effects, there was no evidence for increased SOD1 aggregation. If anything, CCS over-expression prevented SOD1 misfolding in culture as monitored by detergent insolubility. This protection against SOD1 misfolding does not require SOD1 enzyme activation as the same effect was obtained with the C244,246S allele of CCS. In the G93A SOD1 mouse, CCS over-expression was likewise associated with a lack of obvious SOD1 misfolding marked by detergent insolubility. CCS over-expression accelerates SOD1-linked disease without the hallmarks of misfolding and aggregation seen in other mutant SOD1 models. These studies are the first to indicate biological effects of CCS in the absence of SOD1 enzymatic activation.
Project description:We use NMR directly in live human cells to describe the complete post-translational maturation process of human superoxide dismutase 1 (SOD1). We follow, at atomic resolution, zinc binding, homodimer formation and copper uptake, and discover that copper chaperone for SOD1 oxidizes the SOD1 intrasubunit disulfide bond through both copper-dependent and copper-independent mechanisms. Our approach represents a new strategy for structural investigation of endogenously expressed proteins in a physiological (cellular) environment.
Project description: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.
Project description: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.
Project description:Dominant mutations in Cu,Zn-superoxide dismutase (SOD1) cause a familial form of amyotrophic lateral sclerosis (fALS), and aggregation of mutant SOD1 has been proposed to play a role in neurodegeneration. A growing body of evidence suggests that fALS-causing mutations destabilize the native structure of SOD1, leading to aberrant protein interactions for aggregation. SOD1 becomes stabilized and enzymatically active after copper and zinc binding and intramolecular disulfide formation, but it remains unknown which step(s) in the SOD1 maturation process is important in the pathological aggregation. In this study we have shown that apoSOD1 without disulfide is the most facile state for formation of amyloid-like fibrillar aggregates. fALS mutations impair either zinc binding, disulfide formation, or both, leading to accumulation of the aggregation-prone, apo, and disulfide-reduced SOD1. Moreover, we have found that the copper chaperone for SOD1 (CCS) facilitates maturation of SOD1 and that CCS overexpression ameliorates intracellular aggregation of mutant SOD1 in vivo. Based on our in vivo and in vitro results, we propose that facilitation of post-translational modifications is a promising strategy to reduce SOD1 aggregation in the cell.
Project description:Canine copper toxicosis is an autosomal recessive disorder characterized by hepatic copper accumulation resulting in liver fibrosis and eventually cirrhosis. We have identified COMMD1 as the gene underlying copper toxicosis in Bedlington terriers. Although recent studies suggest that COMMD1 regulates hepatic copper export via an interaction with the Wilson disease protein ATP7B, its importance in hepatic copper homeostasis is ill-defined. In this study, we aimed to assess the effect of Commd1 deficiency on hepatic copper metabolism in mice. Liver-specific Commd1 knockout mice (Commd1(?hep)) were generated and fed either a standard or a copper-enriched diet. Copper homeostasis and liver function were determined in Commd1(?hep) mice by biochemical and histological analyses, and compared to wild-type littermates. Commd1(?hep) mice were viable and did not develop an overt phenotype. At six weeks, the liver copper contents was increased up to a 3-fold upon Commd1 deficiency, but declined with age to concentrations similar to those seen in controls. Interestingly, Commd1(?hep) mice fed a copper-enriched diet progressively accumulated copper in the liver up to a 20-fold increase compared to controls. These copper levels did not result in significant induction of the copper-responsive genes metallothionein I and II, neither was there evidence of biochemical liver injury nor overt liver pathology. The biosynthesis of ceruloplasmin was clearly augmented with age in Commd1(?hep) mice. Although COMMD1 expression is associated with changes in ATP7B protein stability, no clear correlation between Atp7b levels and copper accumulation in Commd1(?hep) mice could be detected. Despite the absence of hepatocellular toxicity in Commd1(?hep) mice, the changes in liver copper displayed several parallels with copper toxicosis in Bedlington terriers. Thus, these results provide the first genetic evidence for COMMD1 to play an essential role in hepatic copper homeostasis and present a valuable mouse model for further understanding of the molecular mechanisms underlying hepatic copper homeostasis.
Project description:Superoxide dismutase 1 (Sod1) is an important antioxidative enzyme that converts superoxide anions to hydrogen peroxide and water. Active Sod1 is a homodimer containing one zinc ion, one copper ion, and one disulfide bond per subunit. Maturation of Sod1 depends on its copper chaperone (Ccs1). Sod1 and Ccs1 are dually localized proteins that reside in the cytosol and in the intermembrane space of mitochondria. The import of Ccs1 into mitochondria depends on the mitochondrial disulfide relay system. However, the exact mechanism of this import process has been unclear. In this study we detail the import and folding pathway of Ccs1 and characterize its interaction with the oxidoreductase of the mitochondrial disulfide relay Mia40. We identify cysteines at positions 27 and 64 in domain I of Ccs1 as critical for mitochondrial import and interaction with Mia40. On interaction with Mia40, these cysteines form a structural disulfide bond that stabilizes the overall fold of domain I. Although the cysteines are essential for the accumulation of functional Ccs1 in mitochondria, they are dispensable for the enzymatic activity of cytosolic Ccs1. We propose a model in which the Mia40-mediated oxidative folding of domain I controls the cellular distribution of Ccs1 and, consequently, active Sod1.
Project description:Copper-responsive intracellular ATP7B trafficking is crucial for maintaining the copper balance in mammalian hepatocytes and thus copper levels in organs. The copper metabolism domain-containing protein 1 (COMMD1) binds both the ATP7B copper transporter and phosphatidylinositol (4,5)-bisphosphate [PtdIns(4,5)P2], whereas COMMD1 loss causes hepatocyte copper accumulation. Although it is clear that COMMD1 is localized to endocytic trafficking complexes, a direct function for COMMD1 in ATP7B trafficking has not yet been defined. In this study, experiments using quantitative colocalization analysis reveal that COMMD1 modulates copper-responsive ATP7B trafficking through recruitment to PtdIns(4,5)P2 Decreased COMMD1 abundance results in loss of ATP7B from lysosomes and the trans-Golgi network (TGN) in high copper conditions, although excess expression of COMMD1 also disrupts ATP7B trafficking and TGN structure. Overexpression of COMMD1 mutated to inhibit PtdIns(4,5)P2 binding has little impact on ATP7B trafficking. A mechanistic PtdIns(4,5)P2-mediated function for COMMD1 is proposed that is consistent with decreased cellular copper export as a result of disruption of the ATP7B trafficking itinerary and early endosome accumulation when COMMD1 is depleted. PtdIns(4,5)P2 interaction with COMMD1 as well as COMMD1 abundance could both be important in maintenance of specific membrane protein trafficking pathways.