Fig 1.
Dislocation of ERAD substrates over the ER membrane is followed by deamidation.
A. In this study, we introduce a glycosylation consensus sequence in proinsulin to follow epitope trafficking. Schematic representation of the degradation route of PI-C31N. PI-C31N molecules are co-translationally translocated into the ER where N-linked glycosylation takes place. Dislocation of Δss-PPI-C31N molecules is accompanied by the removal of the N-linked glycan, leaving the protein deamidated. Dislocated insulin molecules are then targeted to the proteasome and the resulting proinsulin epitopes are imported into the ER by TAP, loaded onto HLA class I molecules and presented at the cell surface. B. Deamidation of an asparagine residue results in an aspartate residue.
Fig 2.
Expression of proinsulin mutants in K562 cells.
A. Schematic overview of constructs that were used in the experiments in this figure and mass-spectrometry analysis in Fig 6. B. Expression of mutant proinsulin constructs in cells detected by Western blot. Shorter exposure for C96N mutant is shown because of high protein levels. Fragments of the original image were spliced together (see S4 Fig for raw scans) C. Cell lysates were treated with Endo H before Western blot analysis of the removal of N-linked sugar groups from glycosylation proinsulin mutants. Fragments of the original image were spliced together (see S4 Fig for raw scans) D. Detection of expression of Δss-PPI-C31N mRNA species by RT-PCR.
Fig 3.
Presentation of the PPIB5-14 epitope requires dislocation of proinsulin over the ER membrane.
A. Representation of mass-spectrometry results of HLA-bound peptides that were eluded from affinity-purified MHC molecules derived from K562 HLA-A2 cells expressing the indicated mutant proinsulin constructs (see S3 Fig for spectra of eluted and synthetic peptides) B. Affinity of peptides that result from processing of mutant proinsulin constructs for HLA-A*02:01 molecules as predicted with NetMHC 3.4 (http://www.cbs.dtu.dk/services/NetMHC/).
Fig 4.
HRD1 catalytic activity is involved in proinsulin degradation.
A. K562 cells stably expressing HLA-A*02:01 and PPI were transduced with a lentiviral CRISPR-Cas9 vector containing a gRNA sequence directed against the 5’ region of the HRD1 gene and selected with puromycin. Monoclonal knockouts were generated by limited dilution and analyzed for HRD1 expression. Genomic DNA was isolated and sequenced for the presence of insertions of deletions (indels) within the gRNA target region. Both HRD1 alleles were aligned to a reference sequence (NCBI gene entry 84447). B. K562 cells from (A) were re-transduced with an empty cDNA vector (KO) or a cDNA vector encoding HRD1 (WT), or a catalytically inactive mutant (C1A). Cells were sorted based on mAmetrin expression to obtain pure populations. Next, Expression of HRD1 and proinsulin was assessed in corresponding cell lysates by immunoblotting. Human transferrin receptor was used as a loading control. C. K562 cells from (B) were treated with 200ug/mL CHX for the indicated times, followed by WB analysis of total proinsulin levels. A representative blot from three independent experiments is shown here. D. Results from 5 independent experiments of (C) were quantified, corrected for actin levels and normalized to t = 0 (Error bars = SD, paired Students’ T-test; *p<0,05; ** p<0,01; *** p<0,001, ns = non-significant). Data points are shown in S2 Table. E. The HLA-eluted peptidome from (rescued) HRD1 K.O. clones from (B) or parental cells was analyzed by mass spectrometry (N = 1) and quantified from MASCOT output. Abundance of specific peptides is shown as a percentage of their presentation in HRD1 K.O. + HRD1 WT rescued cells, in a white-red gradient-coded table. For raw data, see S3 Table.
Fig 5.
An E2-specific CRISPR knockout screen identifies UBE2G2 to be involved in PI-GFP degradation.
A. An arrayed CRISPR library targeting every known human E2-conjugating enzyme with three different gRNAs per gene [22] was transduced into K562 cells expressing PPI-GFP. Cells were selected with puromycin and GFP levels were assessed by flow cytometry 9 days post-infection. Results were quantified and compared to GFP levels in empty vector-expressing cells. For sample IDs, see S1 Table. B. Histograms of cells transduced with gRNAs targeting UBE2g2 or UBE2g1 (grey) or an empty vector control (red) from the screen shown in (A). PI-GFP levels were evaluated using flow cytometry. Raw values and calculations are shown in S2 Table.
Fig 6.
UBE2G2 catalytic activity is involved in proinsulin degradation.
A. K562 cells stably expressing HLA-A*02:01 and PPI were transduced with a CRISPR-Cas9 vector containing a gRNA sequence directed against the N-terminal region of the UBE2g2 gene and selected with puromycin. Monoclonal knockouts were generated by limited dilution and analyzed for UBE2g2 expression. Genomic DNA was isolated and sequenced for the presence of insertions of deletions (indels) within the gRNA target region. Both UBE2g2 alleles were aligned to a reference sequence (NCBI gene entry 7327). B. K562 cells from (C) were retransduced with an empty cDNA vector (KO) or a gRNA-resistant cDNA vector encoding wt HA-UBE2G2 (WT), or a catalytically inactive mutant HA-UBE2G2 (C89S). Cells were sorted for mAmetrin to enrich for gRNA-positive cells. Next, cell lysates were analyzed by WB for expression of UBE2G2 and proinsulin. Human transferrin receptor (TfR) was used as a loading control. C. K562 cell lines from (B) were treated with 200ug/mL CHX for the indicated times, followed by WB analysis of total proinsulin levels. A representative blot from three independent experiments is shown. D. Results from 6 independent experiments of (C) were quantified, corrected for actin levels and normalized to t = 0 (Error bars = SD, paired Students’ T-test; *p<0,05; **p<0,005, ns = non-significant) Data points are shown in S2 Table. E. The HLA-eluted peptidome from (rescued) UBE2G2 K.O. clones from (B) and parental cells was analyzed by mass spectrometry (N = 1) and quantified from MASCOT output. Abundance of specific peptides and total (>35 ion scored) peptides is shown as a percentage of their presentation in UBE2G2 K.O. + WT UBE2G2 cells in a white-red gradient-coded table. See S3 Table.