Figure 1.
Enhanced interaction of COMMD1 with ALS-associated SOD1 mutant proteins relative to SOD1 wild-type.
A. 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 [27]. 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. 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. C. 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 CuCl2, 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.
Figure 2.
COMMD1 mediates aggregation of ALS-associated SOD1 mutants.
A. SOD1-Flag WT and mutant (A4V, G37R, G85R, D90A, G93A, E100G) constructs were co-transfected with either EV (–) or HA-COMMD1 in HEK293T cells prior to detection of SOD1 aggregates by means of filter trap analysis on cellulose acetate membrane as described in Experimental procedures section (40 μg protein; upper panel). Aggregates were analysed on the same membranes and visualized by IB using antibodies directed against the Flag-tag. Input samples (15 μg protein; lower panel), corresponding to the filter trap analysis, represent total cell lysates of each condition, and were visualized using antibodies directed against the Flag- and HA-tag, and α-Tubulin, respectively. Input samples were analysed on separated membranes. Graph represents the relative COMMD1-mediated induction in SOD1 aggregates compared to EV conditions (representative for at least 3 independent experiments). EV, SOD1 WT and mSOD1 D90A values were considered as 0 as these proteins do not form aggregates. * indicates significantly increase in mSOD1 aggregation mediated by COMMD1 (** p<0.005, *** p<0.001, **** p<0.0001). n.s. = non-significant. B. Filter trap analysis of Neuro2A cells transiently transfected with SOD1-Flag mutants G85R and G93A in combination with either EV (–) or HA-COMMD1. Detection of aggregates and input samples was performed as described in Figure 2A. Graph represents the relative COMMD1-mediated induction in mSOD1 aggregates compared to EV conditions (representative for at least 3 independent experiments). * indicates significantly increase in mSOD1 aggregation mediated by COMMD1 (* p<0.05). C. Filter trap analysis performed in HEK293T cells stably transfected with shControl or shCOMMD1 and co-transfected with SOD1-Flag WT and mutants (A4V, G37R, G85R, D90A, G93A, E100G). Aggregates were analysed on the same membranes. Analyses were performed as described in Figure 2A. Input samples were analysed on separated membranes. Indicated values represent the relative COMMD1-mediated reduction in mSOD1 aggregates compared to EV conditions (mSOD1 aggregation upon EV co-transfection was set a 1.0; representative for at least 3 independent experiments). EV, SOD1 WT and mSOD1 D90A values were considered as 0 as these proteins do not form aggregates. Graph represents the relative COMMD1-mediated reduction in SOD1 aggregates compared to EV conditions (representative for at least 3 independent experiments). EV, SOD1 WT and mSOD1 D90A values were considered as 0 as these proteins do not form aggregates. * indicates significantly decrease in mSOD1 aggregation upon COMMD1 knock-down (* p<0.05, ** p<0.005, **** p<0.0001). n.s. = non-significant.
Figure 3.
Formation of mSOD1 high molecular mass species is regulated by COMMD1.
A. 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. 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. C. 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.
Figure 4.
COMMD1 is in complex with aggregated mSOD1 proteins.
A. Neuro2A cells were transient transfected with eYFP-tagged SOD1 WT, or mSOD1 G85R and G93A, with or without mCherry-COMMD1, as indicated. 72 h post-transfection, cells were fixed and imaged using a confocal microscope. B. HEK293T cells were transient transfected with either EV (–) or HA-COMMD1 in combination with EV, or Flag-tagged mSOD1 G85R or G93A constructs prior to lysis and detection of SOD1 multimeric complexes using blue native PAGE (30 μg). Immunoblotting was performed using antibodies directed against the Flag-tag and HA-COMMD1 proteins. Total protein levels were analysed in presence of β-ME on a SDS-PAGE gel (30 μg). Photoshop analysis was used to generate overlay, using a marked region on the membrane as focus point.
Figure 5.
COMMD1-mediated induction of mSOD1 aggregation is affected by a SOD1 C6S substitution.
A. 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 Figure 1A. 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 Figure 1A. C. 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 Figure 2A.
Figure 6.
COMMD1 diminishes Parkin C289G aggregation.
A. 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. 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 Figure 1A. 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.
Figure 7.
Aggregation of polyglutamine proteins is unaffected by COMMD1.
A. HEK293T cells were transfected with eYFP-HttQ74 and eYFP-HttQ119 alone, or in co-expression with HA-COMMD1 or V5-DNAJB6 prior to lysis and detection of polyQ HMW complexes using SDS-PAGE. HMW aggregating complexes were trapped and detected in the stacking gel. IB was performed using indicated antibodies. Samples were analysed on the same membranes. Graph represents the relative fraction of insoluble HttQ74 and Q119 HMW species under EV conditions or upon co-expression of COMMD1 or DNAJB6. Insoluble Htt HMW species present in EV conditions were set at 1. B. Filter trap analysis of HEK293T cells transfected with the polyQ proteins eYFP-HttQ74 and eYFP-HttQ119, in co-expression with EV or HA-COMMD1. Samples were prepared as described under Experimental procedures and diluted as indicated. Immunoblotting was performed antibodies directed against the eYFP-tag. Densitometric quantification of insoluble fraction of HttQ74 and HttQ119, in absence or presence of exogenous COMMD1 (5x dilution). EV conditions were set at 1. p = 0.0425, indicates a significant decrease in insoluble HttQ74 aggregates mediated by COMMD1. n.s. = non-significant.