HDAC6 ZnF-UBP domain interacts with CFTR-ΔF508 aggregates and ubiquitin C terminus. A, protein-protein interaction assay of ubiquitin with either His-tagged wild-type HDAC6 ZnF-UBP domain, a R1155A/Y1184A mutant, or wild-type USP5 UBP domain. His-tagged proteins were incubated with or without ubiquitin at room temperature for 30 min before Ni-NTA was added. The resin was washed extensively prior to elution with SDS-PAGE loading buffer. The input and eluted proteins were resolved by SDS-PAGE and visualized by Coomassie staining. B, both HDAC6 ZnF-UBP and USP5 UBP, but not HDAC6 ZnF-UBP RY mutant, interact with GFP-CFTR-ΔF508 protein aggregates. Cultured 293T cells were transfected with GFP-CFTR-ΔF508 and treated with either MG132 or Me₂SO, as indicated. The lysates were incubated with HDAC6 ZnF-UBP (top panel) or HDAC6 ZnF-UBP RY mutant (bottom panel). CFTR-ΔF508 aggregates were immunoprecipitated with an anti-GFP antibody, resolved by SDS-PAGE, and visualized by Coomassie staining. C, both HDAC6 ZnF-UBP and USP5-UBP interact with ataxin-1-82Q-GFP aggregates. Cultured 293T cells were transfected with human ataxin-1-82Q-GFP and treated with either MG132 or Me₂SO, as indicated. The lysates were incubated with HDAC6 ZnF-UBP, HDAC6 ZnF-UBP RY mutant, or USP5-UBP. Ataxin-1-82Q-GFP aggregates were immunoprecipitated with an anti-GFP antibody, resolved by SDS-PAGE, and visualized by Coomassie staining. D, GFP overexpression did not cause aggresome formation. The cells were transfected with pLenti-GFP construct and treated with either Me₂SO or MG132 in Me₂SO. The cell lysates were collected 2 days after transfection; incubated with either USP5 UBP, HDAC6 ZnF-UBP, or HDAC6 ZnF-UBP-RY mutant proteins; and immunoprecipitated by anti-GFP antibody. Input and precipitated samples were resolved by SDS-PAGE and visualized by Coomassie staining. E, protein-protein interaction assays of His-tagged HDAC6 ZnF-UBP domain with ubiquitin and ubiquitin-AFC. The His-tagged HDAC6 ZnF-UBP domain was incubated with ubiquitin or ubiquitin-AFC before applied to a Talon (cobalt) column. The input and eluted proteins were resolved by SDS-PAGE and visualized by Coomassie staining. F, protein-protein interaction assays of N-terminal His-tagged ubiquitin and ubiquitin mutants with HDAC6 ZnF-UBP domain. N-terminal His-tagged ubiquitin and ubiquitin mutant proteins were incubated with HDAC6 ZnF-UBP domain before applied to a Talon (cobalt) column. The input and eluted proteins were resolved by SDS-PAGE and visualized by Coomassie staining. MW, molecular mass.

CFTR-ΔF508 protein aggregates contain unanchored ubiquitin generated by deubiquitinase ataxin-3. A–C, the specificity of the C-terminal ubiquitin anti-Ubi^C antibody. A and B, AFC-conjugated ubiquitin (lane 1), pure monoubiquitin (lane 2), and tetraubiquitin (lane 3) peptides were resolved by SDS-PAGE and detected in Western analysis by either anti-Ubi^C antibody (A) or anti-Ubi^N antibody (B). C, ubiquitin C-terminal truncation mutants Ub71 (lane 1), Ub74 (lane 2), and Ub75 (lane 3) and point mutant G75A/G76A were resolved by SDS-PAGE and detected in Western analysis by either anti-Ubi^N antibody (top panel) or anti-Ubi^C antibody (bottom panel). D–H, cultured 293T cells were transfected with GFP-CFTRΔF508 and either scrambled (lanes 1–6) or ataxin-3 siRNA (lanes 7–12) and treated with either Me₂SO (lanes 1–3 and 7–9) or MG132 (lanes 4–6 and 10–12). Cell lysates were collected 2 days after transfection. GFP-CFTRΔF508 aggregates were immunoprecipitated with GFP antibody and resolved by SDS-PAGE and probed for GFP (D), ubiquitin C termini by anti-Ubi^C antibody (E), ubiquitin N termini by anti-Ubi^N antibody (F), HDAC6 (G), and ataxin-3 (H). IB, immunoblot; Ub, ubiquitin.

A schematic representation of aggresome formation. Under normal conditions, polyubiquitinated misfolded proteins are efficiently degraded by ubiquitin proteasome. When ubiquitin proteasome is disrupted or overwhelmed, polyubiquitinated misfolded proteins form aggregates. Deubiquitinase ataxin-3 interacts with polyubiquitinated protein aggregates, generating unanchored ubiquitin or ubiquitin chains. HDAC6 recognizes and binds these unanchored ubiquitin C-terminal tails in protein aggregates and recruits them to dynein motor complexes that subsequently transport the aggregated cargo to the aggresomes.

The structures of HDAC6 ZnF-UBP domain and its complexes with ubiquitin and RLRGG peptide. A, ribbon diagram of HDAC6 ZnF-UBP domain. The residues in the ubiquitin-binding site are shown in cyan. B, overlay of structures of HDAC6 ZnF-UBP domain (shown in ribbon representation) in complex with ubiquitin (yellow sticks) and RLRGG peptide (cyan sticks). C, structures of HDAC6 ZnF-UBP domain and its complex with RLRGG peptide, showing the ubiquitin binding pocket of HDAC6 ZnF-UBP domain. The last three residues of the peptide (RGG) are shown as sticks. D, overlay of the ubiquitin-binding site of HDAC6 ZnF-UBP (green) and its complex with RLRGG peptide (cyan). The peptide is shown in gray. The different conformations of Arg-1155 and Tyr-1156 can be clearly observed. E, the complex structure of HDAC6-UBP and RLRGG peptide, showing the hydrogen bond network. The peptide is colored by B-factors. The water molecules are shown in red. F, overlay of structures of HDAC6 ZnF-UBP (cyan) and its complex with RLRGG peptide (green), showing that minimal change occurred in the overall protein fold upon peptide binding. The protein is shown in ribbon representation, and the peptide is shown as sticks (yellow).

Unanchored ubiquitin was co-localized with ataxin-3 in CFTR-ΔF508 aggregates. A549 cells expressing GFP-CFTR-ΔF508 were transfected with either scrambled RNAi (A, B, and D) or ataxin-3 RNAi (C and E) and treated with either Me₂SO (DMSO, A) or MG132 (B–E). The cells were labeled for GFP (green), ataxin-3 (red), C-terminal ubiquitin (Ub C-term, magenta in A–C), HDAC6 (magenta in D and E), and DAPI (blue). Perinuclear areas in B1 and D1 (white boxes) are shown in B′ and D′, respectively. The ataxin-3 siRNA-mediated knockdown efficiency varied among cells, because we observed that some cells have efficient knockdown (arrow in E), whereas other cells have significant residual levels of ataxin-3 (arrowhead in E). Scale bars, 10 μm.