Just one position-independent lysine residue can direct MelanA into proteasomal degradation following N-terminal fusion of ubiquitin

Abstract

N-terminal stable in frame fusion of ubiquitin (Ub) has been shown to target the fusion protein for proteasomal degradation. This pathway, called the Ub fusion degradation (UFD), might also elevate MHC class I (MHC-I) antigen presentation of specific antigens. The UFD, mainly studied on cytosolic proteins, has been described to be mediated by polyubiquitination of specific lysine residues within the fused Ub moiety. Using the well characterized melanoma-specific antigen MelanA as a model protein, we analyzed the requirements of the UFD for ubiquitination and proteasomal degradation of a transmembrane protein. Here we show that fusion of the non-cleavable Ub(G76V) variant to the N-terminus of MelanA results in rapid proteasomal degradation via the endoplasmic reticulum-associated degradation (ERAD) pathway and, consequently, leads to an increased MHC-I antigen presentation. While lysine residues within Ub are dispensable for these effects, the presence of one single lysine residue, irrespectively of its location along the fusion protein, is sufficient to induce degradation of MelanA. These results show that the ubiquitination, ER to cytosol relocation and proteasomal degradation of a transmembrane protein can be increased by N-terminal fusion of Ub at the presence of at least one, position independent lysine residue. These findings are in contrast to the conventional wisdom concerning the UFD and indicate a new concept to target a protein into the ubiquitin-proteasome system (UPS) and thus for enhanced MHC-I antigen presentation, and might open up new possibilities in the development of tumor vaccines.

Figure 1. Ub^G76VMelanA-SL is rapidly degraded by the 26S proteasome and exhibits increased MHC-I antigen presentation.

(A) Schematic representation of MelanA-SL and Ub^G76VMelanA-SL proteins. (B) After transient transfection, HeLa cells expressing MelanA-SL or Ub^G76VMelanA-SL were treated with PIs (20 µM MG-132+5 µM LC) or DMSO as a solvent control for 4 h prior to lysis. Whole cell lysates were analyzed by Western blot using an anti-MelanA Ab. As a loading control, the Western blot was reprobed using an anti-β-actin Ab. (C) HeLa-K^b cells were transiently transfected with expression plasmids coding for MelanA, MelanA-SL or Ub^G76VMelanA-SL. H2-K^b-SL complexes presented on the surface of MelanA-positive cells were quantified by flow cytometry using the mAb 25D1.16 conjugated to allophycocyanin (25D1.16-APC). Following fixation and permeabilization, intracellular MelanA was detected by staining with anti-MelanA and a secondary Alexa488-conjugated anti-mouse Ab. A representative histogram plot is shown. (D) Depiction of the mean fluorescence intensity (MFI) of the 25D1.16 staining of 36 independent experiments. The statistical analyses were performed using the paired two-tailed student's t-test (_***p<0.0001). (E) HeLa-K^b cells were transiently transfected with expression plasmids coding for MelanA-SL or Ub^G76VMelanA-SL and qRT-PCR was performed from total RNA. The normalized mRNA amount of MelanA-SL was set to 1 and the mRNA relation of Ub^G76VMelanA-SL to MelanA-SL is depicted. Bars represent mean ± SD from three independent experiments.

Figure 2. Insertion of SL does not affect the subcellular localization of MelanA.

Following transient transfection HeLa cells expressing MelanA or MelanA-SL were treated with (A) DMSO as a solvent control or (B) PIs (20 µM MG-132+5 µM LC) for 4 h. Cells were stained for MelanA (green) with an anti-MelanA Ab and for TGN using an anti-TGN46 Ab (red). The nucleus was illuminated with DAPI (blue). (C) Following transient transfection HeLa cells expressing MelanA-SL or Ub^G76VMelanA-SL were treated with PIs or DMSO for 4 h. The plasma membrane was permeabilized using 20 µM digitonin followed by a cell fractionation (S = supernatant; P = pellet) and analyzed by Western blot. Staining for the ER-resident protein calnexin served as a control for the quality of the fractionation. As a loading control, the Western blot was reprobed using an anti-β-actin Ab. (D) After transient transfection HeLa cells expressing MelanA-SL or Ub^G76VMelanA-SL were treated with PIs or DMSO for 4 h. The ER fraction was isolated using an ER isolation kit and analyzed by Western blot. Staining for the ER-resident protein calnexin served as a positive control and Hsp90 as a negative control to assess the quality of the fractionation.

Figure 3. The enhanced proteasomal degradation and MHC-I antigen presentation of Ub^G76VMelanA-SL are independent of the Lys residues within the fused Ub^G76V.

(A) Following transient transfection HeLa cells expressing MelanA-SL, Ub^G76VMelanA-SL or Ub^G76V(K0)MelanA-SL were treated with PIs or DMSO for 4 h. Whole cell lysates were analyzed by Western blot. As a loading control, the Western blot was reprobed using an anti-β-actin Ab. (B) Immunoprecipitation (IP) of ubiquitinated MelanA-SL species. After transient transfection, MelanA-SL and variants thereof were coexpressed with HA- tagged Ub. MelanA was precipitated under denaturing conditions from whole cell lysates using an anti-MelanA Ab. Ubiquitinated MelanA was detected by an anti-HA Western blot. To demonstrate that equal amounts of MelanA and variants thereof are used in the IP, a part of the cell lysate was collected before the IP and stained with an anti-MelanA Ab. As a loading control, the Western blot was reprobed using an anti-β-actin Ab (upper panels). (C) Following transient transfection of HeLa-K^b cells with MelanA-SL, Ub^G76VMelanA-SL or Ub^G76V(K0)MelanA-SL, H2-K^b-SL complexes presented on the surface of MelanA-positive cells were quantified by flow cytometry using 25D1.16-APC. A representative histogram plot is shown. (D) Depiction of the mean fluorescence intensity (MFI) of the 25D1.16 staining of 36 independent experiments. The statistical analysis was performed using the paired two-tailed student's t-test (_*** p<0.0001).

Figure 4. Only one lysine residue, independently of its position, can target Ub^G76V(K0)MelanA-SL into the UPS.

(A) Schematic depiction of Ub^G76V(K0)MelanA-SL in the ER membrane. The Lys residues within MelanA-SL are marked in red. (B+C) HeLa cells were transiently transfected with expression plasmids coding for MelanA-SL, Ub^G76VMelanA-SL, Ub^G76V(K0)MelanA-SL or variants thereof harboring cumulative Lys to Arg mutations (B) from the C- to the N-terminus or (C) from the N- to the C-terminus. Cells were treated with PIs or DMSO for 4 h. Whole cell lysates were analyzed by Western blot. As a loading control, the Western blot was reprobed using an anti-β-actin Ab. (D) Depiction of the mean fluorescence intensity (MFI) of the 25D1.16 staining of six independent experiments using MelanA-SL and Ub^G76V(K0)MelanA-SL^K19only. The statistical analysis was performed using the paired two-tailed student's t-test (_***p<0.0001). (E) Following transient transfection, HeLa cells expressing MelanA-SL, Ub^G76VMelanA-SL, Ub^G76V(K0)MelanA-SL or Ub^G76V(K0)MelanA-SL variants harbouring only one of the lysine residues within MelanA were treated with PIs or DMSO for 4 h prior to lysis. Whole cell lysates were analyzed by Western blot. As a loading control, the Western blot was reprobed using an anti-β-actin Ab.

Figure 5. Mutation of all Lys residues within Ub^G76VMelanA-SL abrogates its entry into the UFD-pathway.

Following transient transfection, HeLa cells expressing MelanA-SL, Ub^G76VMelanA-SL, Ub^G76V(K0)MelanA-SL or Ub^G76V(K0)MelanA^(K0)-SL were treated with PIs or DMSO for 4 h prior to lysis. Whole cell lysates were analyzed by Western blot. As a loading control, the Western blot was reprobed using an anti-β-actin Ab.

Figure 6. Ub^G76VMelanA-SL and Ub^G76V(K0)MelanA-SL are degraded via the ERAD-pathway.

After transient transfection, HeLa cells expressing MelanA-SL, Ub^G76VMelanA-SL or Ub^G76V(K0)MelanA-SL (A) or GFP, Ub^G76VGFP or UbMGFP (B) were treated with PIs or DMSO in the presence or absence of the ERAD-inhibitor Eey1 for 4 h. Whole cell lysates were analyzed by Western blot. As a loading control, the Western blot was reprobed using an anti-β-actin Ab. (C) HeLa cells were transiently transfected with MelanA-SL or Ub^G76VMelanA-SL and treated with PIs or DMSO for 4 h or ExoA for 18 h. Whole cell lysates were analyzed by Western blot. As a loading control, the Western blot was reprobed using an anti-β-actin Ab. To document the uptake of ExoA the Western blot was stained with an anti-ExoA Ab. (D) 24 h after transient transfection of MelanA-SL or Ub^G76VMelanA-SL in HeLa-K^b cells, ExoA was added for 18 h. After acid wash a fraction of the cells was harvested (0 h). The other part was further incubated for 4 h in complete RPMI 1640 medium with or without ExoA. H2-K^b-SL complexes presented on the surface of MelanA-positive cells were quantified by flow cytometry using 25D1.16-APC. The mean fluorescence intensity (MFI) of the 25D1.16 staining of four independent experiments is depicted. Bars represent mean ± SD.