TNF receptor-associated factor 6 interacts with ALS-linked misfolded superoxide dismutase 1 and promotes aggregation

Abstract

Amyotrophic lateral sclerosis (ALS) is a fatal disease, characterized by the selective loss of motor neurons leading to paralysis. Mutations in the gene encoding superoxide dismutase 1 (SOD1) are the second most common cause of familial ALS, and considerable evidence suggests that these mutations result in an increase in toxicity due to protein misfolding. We previously demonstrated in the SOD1^G93A rat model that misfolded SOD1 exists as distinct conformers and forms deposits on mitochondrial subpopulations. Here, using SOD1^G93A rats and conformation-restricted antibodies specific for misfolded SOD1 (B8H10 and AMF7-63), we identified the interactomes of the mitochondrial pools of misfolded SOD1. This strategy identified binding proteins that uniquely interacted with either AMF7-63 or B8H10-reactive SOD1 conformers as well as a high proportion of interactors common to both conformers. Of this latter set, we identified the E3 ubiquitin ligase TNF receptor-associated factor 6 (TRAF6) as a SOD1 interactor, and we determined that exposure of the SOD1 functional loops facilitates this interaction. Of note, this conformational change was not universally fulfilled by all SOD1 variants and differentiated TRAF6 interacting from TRAF6 noninteracting SOD1 variants. Functionally, TRAF6 stimulated polyubiquitination and aggregation of the interacting SOD1 variants. TRAF6 E3 ubiquitin ligase activity was required for the former but was dispensable for the latter, indicating that TRAF6-mediated polyubiquitination and aggregation of the SOD1 variants are independent events. We propose that the interaction between misfolded SOD1 and TRAF6 may be relevant to the etiology of ALS.

Keywords: TNF receptor–associated factor (TRAF); amyotrophic lateral sclerosis (ALS) (Lou Gehrig disease); mitochondria; protein aggregation; protein misfolding; superoxide dismutase (SOD); ubiquitin.

Figure 1.

TRAF6 is a novel binding partner of B8H10 and AMF7-63-reactive misfolded SOD1 at the mitochondria. A, workflow for immunoprecipitation-based MS proteomics for the identification of binding partners of mitochondria-associated misfolded SOD1 in the SOD1^G93A rat model. Misfolded SOD1 was immunoprecipitated with the misfolded SOD1-specific conformation-restricted antibodies B8H10 and AMF7-63 from SOD1^G93A rat spinal cord mitochondria and binding partners identified by nanoLC-MS/MS. B, Venn diagram of identified misfolded SOD1-binding partners common to both B8H10 and AMF7-63 conformers or selective for either. C, novel binding-partner TRAF6 (UniProt ID: B5DF45) was identified based on six unique peptides covering 17% of the protein sequence. D and E, Flag-tagged SOD1 (WT or mutants) and Myc-tagged TRAF6 (WT or deletion mutants) were co-expressed in 293FT cells. Co-immunoprecipitations were performed on Flag–SOD1 or reciprocally on Myc–TRAF6, and co-precipitation of either binding partner was analyzed by immunoblotting. Immunoblotting for Flag–SOD1 (bait) is to demonstrate equal IP efficiency across conditions. Mock refers to transfection with an equivalent amount of empty vector. Note, the slower migrating band in TRAF6 lanes is auto-ubiquitinated TRAF6. Whole-cell lysates (WCL) were loaded to demonstrate equal plasmid expression. Actin serves as loading control. Data are representative of at least three independent experiments.

Figure 2.

C terminus of TRAF6 is necessary and sufficient for mutant SOD1 binding. A, schematic representation of generated Myc–TRAF6 deletion constructs. TRAF6^ΔC (aa. 1–288) comprises the N-terminal RING domain and zinc finger motifs. TRAF6^ΔN (aa. 289–522) comprises the central coiled-coil motif (CC) and the C-terminal MATH domain. B, protein structure models for TRAF6^WT, TRAF6^ΔC, and TRAF6^ΔN. C, Flag-tagged SOD1 (WT or mutants) and Myc-tagged TRAF6 (WT or deletion mutants) were co-expressed in 293FT cells. Co-immunoprecipitations were performed on Flag–SOD1, and Myc–TRAF6 co-precipitation was assessed by immunoblotting. Immunoblotting for Flag–SOD1 (bait) is to demonstrate equal IP efficiency across conditions. Mock refers to transfection with an equivalent amount of empty vector. WCL were loaded to demonstrate equal plasmid expression. Actin serves as loading control. Data are representative of at least three independent experiments.

Figure 3.

SOD1 mutation-dependent variation in the interaction with TRAF6 in cellulo. A–C, Flag-tagged SOD1 (WT or mutants) and Myc-tagged TRAF6 (WT or ΔN) were co-expressed in 293FT cells. Co-immunoprecipitations were performed on Flag–SOD1, and Myc–TRAF6 co-precipitation was analyzed by immunoblot. Immunoprecipitates were immunoblotted for Flag–SOD1 to demonstrate equal IP efficiency across conditions. Mock refers to transfection with an equivalent amount of empty vector. WCLs were loaded to demonstrate equal plasmid expression. Actin serves as loading control. B, densitometric analysis of the co-immunoprecipitated amount of Myc–TRAF6 relative to the amount of immunoprecipitated Flag–SOD1. Plotted values are the mean ± S.D. of four independent experiments. *, p < 0.05; **, p < 0.01; ***, p < 0.001; and ****, p < 0.0001.

Figure 4.

Exposure of epitopes associated with SOD1 misfolding discerns TRAF6-interacting from noninteracting mutants. A, schematic representation of Flag–SOD1 constructs, with the location of mutation sites relative to B8H10 (loop IV) and AMF7-63 (loop VII) antibody epitopes. SOD1^G127X is a truncation mutant with a nonnative C terminus and inherently lacks the AMF7-63 epitope. B, Flag-tagged SOD1 (WT or mutants) was expressed in 293FT cells. Misfolded SOD1 was immunoprecipitated with the B8H10 or AMF7-63 antibody, and precipitates were analyzed by Western blotting. Mock refers to transfection with an equivalent amount of empty vector. WCLs were loaded to demonstrate equal plasmid expression. Actin serves as loading control. Data are representative of three independent experiments.

Figure 5.

TRAF6-mediated polyubiquitination of mutant SOD1 depends on its ubiquitin ligase activity and is selective to TRAF6-interacting SOD1 variants. A, in cellulo ubiquitination assays with mutant SOD1 as substrate and TRAF6 as catalyzing E3 ubiquitin ligase. Flag–SOD1^A4V and SOD1^V148G (TRAF6-interacting) or Flag–SOD1^V148I (TRAF6-noninteracting) was co-expressed with Myc–TRAF6^WT or ubiquitin ligase-inactive TRAF6^C70A in the presence of HA-ubiquitin^WT in 293FT cells. Immunoprecipitations were performed on HA-ubiquitin, and immunoprecipitates were immunoblotted for Flag–SOD1 and HA-ubiquitin to demonstrate equal IP efficiency across conditions. B, in cellulo ubiquitin-linkage assays were performed similar to A, but in the presence of HA-ubiquitin^WT or single-lysine mutants. TRAF6 mediates mutant SOD1 polyubiquitination with primarily Lys-6, Lys-27, and Lys-29 linkages. Note that no polyubiquitinated mutant SOD1 was recovered in the 4% stacking gel (A) and therefore was removed prior to transfer in subsequent experiments. Mock refers to transfection with an equivalent amount of empty vector. WCLs were loaded to demonstrate plasmid expression. Actin serves as loading control. Data are representative of three independent experiments.

Figure 6.

TRAF6 modifies mutant SOD1 aggregation but independently of its ubiquitin ligase activity. A, C, and E, filter retardation assays in which protein aggregates ≥200 nm in diameter are captured on cellulose acetate membrane filters. Flag–SOD1 (WT or mutants) was either co-expressed with Myc–TRAF6^WT, TRAF6^ΔN, or ubiquitin ligase–inactive TRAF6^C70A, or Flag–SOD1 variants were expressed in 293FT cells that received siTRAF6 or siControl. Filter membranes were immunoblotted for Flag–SOD1. B, D, and F, Western blotting of cell lysates to demonstrate equal plasmid expression and to verify TRAF6 knockdown across conditions. Immunoblots also serve as loading controls for the filter membranes. Mock refers to transfection with an equivalent amount of empty vector. Actin serves as loading control. All data are representative of three independent experiments.

Figure 7.

TRAF6 is expressed in ALS disease–relevant cell types. A, Western blotting of whole-cell lysates from human iPSC-derived motor neurons differentiated for 14 days. Data are representative of two independent experiments. B and C, Western blotting of whole-cell lysates from mouse primary cortical astrocytes and neurons and quantification of the abundance of TRAF6 relative to total protein stained with Ponceau S. Data represent the mean ± S.D. from two independent cell preparations. TRAF6 is detected in all cell types as a 60-kDa immunoreactive band (corresponding to the reported size of TRAF6) and a faster-migrating band at about 48 kDa (unreported).