Figure 3.
Disease-associated defective proteins were captured by TanGIBLE. (A) TanGIBLE interacts with the translocation-defective prepro-form of insulin mutants. HeLa cells expressing C-terminally T7-tagged insulin WT or R6C and Flag-tagged TanGIBLE were treated with 10 μM MG-132, as indicated. At 4 h after MG-132 treatment, the cells were lysed and immunoprecipitated (IP) with anti-Flag M2 agarose beads. Precipitates were blotted using the indicated antibodies. (B) Schematic of the 10S TanGIBLE developed as a negative control in this study. (a) Reported structure model of the UBL domain of BAG6 (PDB: 4EEW). The side chains of the Ile60, Val65, and Val81 residues in the BAG6 UBL domain are exposed on the surface of the domain and make a closely associated hydrophobic cluster. Note that Ile60 of BAG6 UBL corresponds to the ubiquitin Ile44 critical for ubiquitin-mediated protein–protein interactions. Except for Ile60, the hydrophobicity of these residues (Ile32, Val65, and Val81) is highly conserved in BAG6 orthologs from various vertebrates but is not conserved in ubiquitin and other UBL proteins, suggesting that the surface-exposed hydrophobicity is BAG6 UBL-specific. (b and c) Since the tandemly aligned N200-6S fragment was found to show non-negligible binding ability with model defective proteins, we further introduced mutations in the UBL domain. Four conserved hydrophobic residues in the UBL domain (correspond to Ile32, Ile60, Val65, and Val81 of human BAG6, indicated in red characters), as well as six hydrophobic residues in the BUILD domain (identical to the N200-6S mutations, see Fig. 2 B), were substituted simultaneously with hydrophilic serine residues. The tandemly aligned fragment with these mutated domains was designated 10S TanGIBLE and verified as a negative control. (C and D) HeLa cells expressing C-terminally T7-tagged R6C insulin, Flag-tagged TanGIBLE, and its 10S negative control fragment were treated with 10 μM MG-132, and Flag-precipitates were blotted using the indicated antibodies. TanGIBLE co-precipitated R6C insulin, while 10S mutated TanGIBLE did not. Prolonged exposure of the same blot showed ladder-like modifications of prepro-insulin exclusively in the TanGIBLE precipitates (D, lane 3). Yellow and black asterisks indicate non-specific and immunoglobulin signals, respectively. (E) HeLa cells expressing Flag-tagged TanGIBLE, T7-tagged N3a-PrP, and Prl-PrP were treated with 10 μM MG-132, and Flag-precipitates were blotted using anti-PrP antibody. Note that N3a-PrP was co-precipitated with TanGIBLE with multiple ladder-like signals (indicated with red arrowheads), whereas very little glycosyl-modified Prl-PrP co-precipitated with TanGIBLE (indicated by blue lines). Unmodified PrP species are indicated with black arrowheads. (F) TanGIBLE co-precipitated N3a-PrP in the presence of MG-132, while 10S mutated TanGIBLE did not precipitate N3a-PrP, even with MG-132. Asterisk indicates signal of immunoglobulin. Source data are available for this figure: SourceData F3.

Disease-associated defective proteins were captured by TanGIBLE. (A) TanGIBLE interacts with the translocation-defective prepro-form of insulin mutants. HeLa cells expressing C-terminally T7-tagged insulin WT or R6C and Flag-tagged TanGIBLE were treated with 10 μM MG-132, as indicated. At 4 h after MG-132 treatment, the cells were lysed and immunoprecipitated (IP) with anti-Flag M2 agarose beads. Precipitates were blotted using the indicated antibodies. (B) Schematic of the 10S TanGIBLE developed as a negative control in this study. (a) Reported structure model of the UBL domain of BAG6 (PDB: 4EEW). The side chains of the Ile60, Val65, and Val81 residues in the BAG6 UBL domain are exposed on the surface of the domain and make a closely associated hydrophobic cluster. Note that Ile60 of BAG6 UBL corresponds to the ubiquitin Ile44 critical for ubiquitin-mediated protein–protein interactions. Except for Ile60, the hydrophobicity of these residues (Ile32, Val65, and Val81) is highly conserved in BAG6 orthologs from various vertebrates but is not conserved in ubiquitin and other UBL proteins, suggesting that the surface-exposed hydrophobicity is BAG6 UBL-specific. (b and c) Since the tandemly aligned N200-6S fragment was found to show non-negligible binding ability with model defective proteins, we further introduced mutations in the UBL domain. Four conserved hydrophobic residues in the UBL domain (correspond to Ile32, Ile60, Val65, and Val81 of human BAG6, indicated in red characters), as well as six hydrophobic residues in the BUILD domain (identical to the N200-6S mutations, see Fig. 2 B), were substituted simultaneously with hydrophilic serine residues. The tandemly aligned fragment with these mutated domains was designated 10S TanGIBLE and verified as a negative control. (C and D) HeLa cells expressing C-terminally T7-tagged R6C insulin, Flag-tagged TanGIBLE, and its 10S negative control fragment were treated with 10 μM MG-132, and Flag-precipitates were blotted using the indicated antibodies. TanGIBLE co-precipitated R6C insulin, while 10S mutated TanGIBLE did not. Prolonged exposure of the same blot showed ladder-like modifications of prepro-insulin exclusively in the TanGIBLE precipitates (D, lane 3). Yellow and black asterisks indicate non-specific and immunoglobulin signals, respectively. (E) HeLa cells expressing Flag-tagged TanGIBLE, T7-tagged N3a-PrP, and Prl-PrP were treated with 10 μM MG-132, and Flag-precipitates were blotted using anti-PrP antibody. Note that N3a-PrP was co-precipitated with TanGIBLE with multiple ladder-like signals (indicated with red arrowheads), whereas very little glycosyl-modified Prl-PrP co-precipitated with TanGIBLE (indicated by blue lines). Unmodified PrP species are indicated with black arrowheads. (F) TanGIBLE co-precipitated N3a-PrP in the presence of MG-132, while 10S mutated TanGIBLE did not precipitate N3a-PrP, even with MG-132. Asterisk indicates signal of immunoglobulin. Source data are available for this figure: SourceData F3.

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