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. 2009 Nov;111(4):1062-73.
doi: 10.1111/j.1471-4159.2009.06388.x. Epub 2009 Sep 18.

Sequestosome 1/p62 links familial ALS mutant SOD1 to LC3 via an ubiquitin-independent mechanism

Jozsef Gal  1 , Anna-Lena Ström, David M Kwinter, Renée Kilty, Jiayu Zhang, Ping Shi, Weisi Fu, Marie W Wooten, Haining Zhu
Affiliations

Sequestosome 1/p62 links familial ALS mutant SOD1 to LC3 via an ubiquitin-independent mechanism

Jozsef Gal et al. J Neurochem. 2009 Nov.

Abstract

The p62/sequestosome 1 protein has been identified as a component of pathological protein inclusions in neurodegenerative diseases including amyotrophic lateral sclerosis (ALS). P62 has also been implicated in autophagy, a process of mass degradation of intracellular proteins and organelles. Autophagy is a critical pathway for degrading misfolded and/or damaged proteins, including the copper-zinc superoxide dismutase (SOD1) mutants linked to familial ALS. We previously reported that p62 interacted with ALS mutants of SOD1 and that the ubiquitin-association domain of p62 was dispensable for the interaction. In this study, we identified two distinct regions of p62 that were essential to its binding to mutant SOD1: the N-terminal Phox and Bem1 (PB1) domain (residues 1-104) and a separate internal region (residues 178-224) termed here as SOD1 mutant interaction region (SMIR). The PB1 domain is required for appropriate oligomeric status of p62 and the SMIR is the actual region interacting with mutant SOD1. Within the SMIR, the conserved W184, H190 and positively charged R183, R186, K187, and K189 residues are critical to the p62-mutant SOD1 interaction as substitution of these residues with alanine resulted in significantly abolished binding. In addition, SMIR and the p62 sequence responsible for the interaction with LC3, a protein essential for autophagy activation, are independent of each other. In cells lacking p62, the existence of mutant SOD1 in acidic autolysosomes decreased, suggesting that p62 can function as an adaptor between mutant SOD1 and the autophagy machinery. This study provides a novel molecular mechanism by which mutant SOD1 can be recognized by p62 in an ubiquitin-independent fashion and targeted for the autophagy-lysosome degradation pathway.

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Figures

Figure 1
Figure 1. The interaction and co-localization between p62 and the ALS-linked SOD1 mutants
(A) The interaction between endogenous p62 and mutant SOD1 in G93A SOD1 transgenic mouse spinal cord. Protein extracts were prepared from spinal cords dissected from 125-days old WT and G93A SOD1 transgenic mice and subjected to p62 immunoprecipitation followed by Western blotting using specified antibodies. (B). Co-localization of p62 and mutant SOD1 in primary mouse motor neurons. Primary motor neurons were prepared, cultured, transfected with DsRedM-p62 and WT or A4V mutant SOD1-EGFP, and subjected to confocal microscopic analysis. The scale bars are 10 μm.
Figure 2
Figure 2. Mapping the p62 domains necessary for the interaction with A4V SOD1
(A) The domain structure of the p62 protein and the exon structure of the mouse p62 gene. (B). The DsRedM-tagged full-length and domain deletion p62 mutants were co-transfected with HA-tagged A4V mutant SOD1. HA immunoprecipitation followed by Western blotting was performed to test which domains were required for the interaction. Deletion of the PB1 domain, exon 4 or exon 5 resulted in impaired binding of A4V SOD1 to p62. * denotes the antibody light chain in the immunoprecipitation samples. (C). HA-SOD1 immunoprecipitation was similarly carried out as in (B). The PB1 domain alone (lane 1) or PB1 and ZZ-type zinc finger together (lane 2) were not co-precipitated with A4V SOD1. The construct containing both PB1 domain and exon 4 (lane 3) was the minimal sequence required for interaction. (D). The interaction was tested by reciprocal FLAG-p62 immunoprecipitation. The protein expression levels in the cell lysates were examined in all experiments. The antibody used is specified beside each panel.
Figure 3
Figure 3. The role of the PB1 domain in the interaction
(A) The indicated p62 constructs, tagged with either the monomeric (DsRedM) or the tetrameric (DsRedT) DsRed were co-transfected with HA-tagged A4V SOD1. The cell extracts were subjected to HA immunoprecipitation followed by Western blotting. (B). The DsRedM tagged WT, K7A/D69A or R21A/D69A p62 construct was co-transfected with HA-tagged A4V SOD1. The cell extracts were subjected to HA immunoprecipitation followed by Western blotting. (C). Demonstration of the oligomerization status of selected DsRed-p62 constructs by native gel electrophoresis followed by Western blotting. A slower band (*) and a faster band (●) in native gel electrophoresis indicate an oligomeric and a monomeric form, respectively. The panels of the DsRed blot of the native gel were obtained with 5 sec (short) and 40 sec (long) exposition times of the same membrane. The protein expression levels in extracts were assessed in all experiments. The antibody used in each experiment is specified beside each panel.
Figure 4
Figure 4. Identification of the SOD1 mutant interaction region (SMIR)
(A) The HA-tagged A4V SOD1 interacted with the segment of p62 encoded by exons 4 and 5 only when it was tagged with the tetrameric DsRedT, but not the monomeric DsRedM. The indicated DsRed-tagged p62 constructs were co-transfected with HA-tagged A4V SOD1, and cellular extracts were subjected to HA immunoprecipitation. (B). The HA-tagged A4V SOD1 interacted with the DsRedT-exon 4, but not DsRedT-exon 5. The sequence encoded by exon 4 was termed as the SOD1 mutant interaction region (SMIR). (C). The DsRedT-tagged SMIR was co-immunoprecipitated with multiple ALS-linked SOD1 mutants (A4V, G37R, G85R and G93A) tagged with HA, but not with WT SOD1.
Figure 5
Figure 5. Critical residues in SMIR crucial for p62-mutant SOD1 interaction
(A) The alignment of the SMIR sequence of p62 from different organisms. The conserved cluster of positively charged residues (R183, R186, K187, K189) were indicated by “+” and the conserved W184 and H190 residues were also noted. (B). DsRedM-tagged p62 (WT, W184A, H190A or R183A/R186A/K187A/K189A) was co-transfected with HA-tagged A4V SOD1, and the extracts were subjected to HA immunoprecipitation.
Figure 6
Figure 6. The effect of the SMIR and LIR deletion on mutant SOD1 sequestration into p62 inclusions
DsRedM-p62, DsRedM-p62-ΔSMIR, DsRedM-p62-ΔLIR or DsRedM vector and GFP-tagged A4V mutant SOD1 were co-transfected into NSC34 cells. The inclusion formation was measured by simultaneously monitoring the GFP-positive SOD1 inclusions and DsRed-positive p62 inclusions under fluorescence microscope. Cells containing DsRed, GFP, and both inclusions were counted in 10 random viewfields. The percentage of cells containing inclusions out of the transfected cells was calculated and the average values from three independent experiments are shown.
Figure 7
Figure 7. p62 as an adaptor between mutant SOD1 and autophagy
(A) The p62-LC3 and p62-mutant SOD1 interactions are independent. HA-A4V SOD1, GFP-LC3 and FLAG-tagged FL p62, ΔSMIR-p62, ΔLIR-p62 or FLAG vector were co-transfected, and cellular extracts were subjected to FLAG-p62 immunoprecipitation followed by Western blotting using specified antibodies. (B). The acidification of mCherry-GFP-A4V SOD1 inclusions is impaired in p62 KO MEF cells. The mCherry-GFP-A4V SOD1 plasmid was transfected into WT or p62 KO MEF cells. Live cell imaging was performed 24 hours post transfection. The boxed areas of the entire cell images are enlarged to the right. Scare bars are 10 μm. (C). Quantification of the overlap rate of green and red puncta in the transfected WT and p62 KO MEF cells. A higher overlap rate in the p62 KO cells (p < 0.05) indicated less amount of mutant SOD1 in acidic autolysosomes.
Figure 8
Figure 8. Proposed model of how p62 may target mutant SOD1 aggregates to autophagy
LC3-II represents the phosphatidylethanolamine-conjugated form of LC3 and is associated with the autophagosomal membrane. Misfolded mutant SOD1 is recognized by p62, sequestered to form inclusions, and targeted to autophagy.
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