The Copper Metabolism MURR1 Domain Protein 1 (COMMD1) Modulates the Aggregation of Misfolded Protein Species in a Client-Specific Manner

The Copper Metabolism MURR1 domain protein 1 (COMMD1) is a protein involved in multiple cellular pathways, including copper homeostasis, NF-kappa 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 alpha). 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.</p

Liver-Specific Commd1 Knockout Mice Are Susceptible to Hepatic Copper Accumulation

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 (Commd1Δhep) were generated and fed either a standard or a copper-enriched diet. Copper homeostasis and liver function were determined in Commd1Δhep mice by biochemical and histological analyses, and compared to wild-type littermates. Commd1Δhep 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, Commd1Δhep 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 Commd1Δhep mice. Although COMMD1 expression is associated with changes in ATP7B protein stability, no clear correlation between Atp7b levels and copper accumulation in Commd1Δhep mice could be detected. Despite the absence of hepatocellular toxicity in Commd1Δhep 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

Delayed emergence of subdiffraction-sized mutant huntingtin fibrils following inclusion body formation

Aberrant aggregation of improperly folded proteins is the hallmark of several human neurodegenerative disorders, including Huntington’s Disease (HD) with autosomal-dominant inheritance. In HD, expansion of the CAG-repeat-encoded polyglutamine (polyQ) stretch beyond ~40 glutamines in huntingtin (Htt) and its N-terminal fragments leads to the formation of large (up to several μm) globular neuronal inclusion bodies (IBs) over time. We report direct observations of aggregating Htt exon 1 in living and fixed cells at enhanced spatial resolution by stimulated emission depletion (STED) microscopy and single-molecule super-resolution optical imaging. Fibrils of Htt exon 1 arise abundantly across the cytosolic compartment and also in neuritic processes only after nucleation and aggregation into a fairly advanced stage of growth of the prominent IB have taken place. Structural characterizations of fibrils by STED show a distinct length cutoff at ~1·5 μm and reveal subsequent coalescence (bundling/piling). Cytosolic fibrils are observed even at late stages in the process, side-by-side with the mature IB. Htt sequestration into the IB, which in neurons has been argued to be a cell-protective phenomenon, thus appears to saturate and over-power the cellular degradation systems and leaves cells vulnerable to further aggregation producing much smaller, potentially toxic, conformational protein species of which the fibrils may be comprised. We further found that exogenous delivery of the apical domain of the chaperonin subunit CCT1 to the cells via the cell medium reduced the aggregation propensity of mutant Htt exon 1 in general, and strongly reduced the occurrence of such late-stage fibrils in particular

Formation of mSOD1 high molecular mass species is regulated by COMMD1.

<p><b>A.</b> HEK293T cells were co-transfected with SOD1-Flag mutants (A4V, G37R, G85R, D90A, G93A, E100G) in combination with EV (–) or HA-COMMD1 prior to lysis and detection of SOD1 multimeric complexes using SDS-PAGE under non-reducing conditions (– β-ME; 40 μg). Immunoblotting was performed using antibodies directed against the Flag-tag and HA-COMMD1 proteins. Input samples were analysed in presence of β-ME (+ β-ME; 30 μg). Samples were analysed on separated membranes. <b>B.</b> HEK293T cells were co-transfected with SOD1-Flag WT in combination with EV (–) or HA-COMMD1 prior to lysis and analysed as described in Figure 3A. <b>C.</b> Visualization of SOD1 HMW complexes in Neuro2A cells transfected with SOD1-Flag mutants G85R and G93A in combination with EV (–) or HA-COMMD1 as described in Figure 3A. Samples were analysed on separated membranes.</p

COMMD1 diminishes Parkin C289G aggregation.

<p><b>A.</b> HEK293T cells were transfected with Parkin WT and C289G constructs alone, or in combination with HA-COMMD1. Supernatant (Sup) and pellet fractions were prepared as described in Experimental procedures. Immunoblotting was performed using indicated antibodies. Graphs represent relative fraction of insoluble Parkin proteins in absence or presence of exogenous COMMD1 (fractions quantified from both Parkin as well as Flag immunoblots). <b>B.</b> HEK293T cells were transient transfected with GST or COMMD1-GST alone in combination with Flag-Parkin constructs (WT and C289G). Cells were lysed, and GST fusion proteins were precipitated as described under <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0092408#pone-0092408-g001" target="_blank">Figure 1A</a>. Immunoblotting was performed using indicated antibodies. Densitometric quantification of interaction strength between COMMD1 and Parkin WT versus C289G mutant, normalized for total Parkin expression (input). Binding of COMMD1 to Parkin WT was set at 1.</p

COMMD1-mediated induction of mSOD1 aggregation is affected by a SOD1 C6S substitution.

<p><b>A.</b> HEK293T cells were transient transfected with either EV (–) or HA-COMMD1 in combination with GST only or SOD1-GST constructs (WT, C6S, C111S). GST PD was performed as described under <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0092408#pone-0092408-g001" target="_blank">Figure 1A</a>. <b>B.</b> HEK293T cells were transient transfected with EV (–) or HA-COMMD1 in combination with GST only or SOD1-GST constructs (WT, C6S, G93A, C6S/G93A, G85R, C6S/G85R). GST PD was performed as described under <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0092408#pone-0092408-g001" target="_blank">Figure 1A</a>. <b>C.</b> Filter trap analysis of HEK293T cells transiently transfected with SOD1-Flag mutants (C6S, G93A, C6S/G93A) in combination with either EV (–) or HA-COMMD1. Detection of aggregates was performed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0092408#pone-0092408-g002" target="_blank">Figure 2A</a>.</p

Enhanced interaction of COMMD1 with ALS-associated SOD1 mutant proteins relative to SOD1 wild-type.

<p><b>A.</b> HEK293T cells were transient transfected with empty vector (EV), HA-COMMD1 alone or in combination with SOD1-GST constructs (WT, A4V, G37R, G85R, D90A, G93A, E100G). GST-proteins were precipitated by means of GSH-sepharose beads prior to detection of their interaction with COMMD1 as visualized by immunoblotting for HA-COMMD1 (GST PD; upper panel), as described previously <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0092408#pone.0092408-Matsumoto1" target="_blank">[27]</a>. 30 μg of protein lysates were used for detection of WT and mutant SOD1-GST and HA-COMMD1 in total cell lysates (Input; lower panel) using antibodies directed against the HA- or GST-fusion proteins. Tubulin was used as loading control. <b>B.</b> Densitometric quantification of interaction strength between COMMD1 and SOD1 WT versus SOD1 mutants (A4V, G37R, G85R, D90A, G93A, E100G; GST PD), normalized for total SOD1 expression (input). Binding of COMMD1 to SOD1 WT was set at 1. * indicates significantly increased binding of COMMD1 to mSOD1 compared to SOD1 WT – COMMD1 (* p<0.05, ** p<0.005, *** p<0.0001). n.s.  =  non-significant. <b>C.</b> HEK293T cells were transient transfected with HA-COMMD1 alone or in combination with SOD1-GST constructs (WT and G93A). Cells were incubated overnight under basal conditions or with 150 μM CuCl_2, lysed, and GST fusion proteins were precipitated by means of GSH-sepharose beads prior to detection of their interaction with COMMD1 as visualized by immunoblotting for HA-COMMD1 (GST PD; upper panel). 30 μg of protein lysates were used for detection of SOD1-GST WT and G93A and HA-COMMD1 in total cell lysates (Input; lower panel) using antibodies directed against the HA- or GST-tags.</p