Creation and expression of SCRAP in EL4 cells
To study independently the generation of peptides from DRiPs versus retirees, we genetically fused the ProteoTuner destabilization domain to SIINFEKL followed by eGFP to create SCRAP (). We selected stable EL4 cell transfectants expressing SCRAP under the control of the CMV immediate early promoter. EL4/SCRAP cells exhibit minimal eGFP fluorescence detected above control EL4 autofluorescence levels (). Overnight treatment with Shield-1 greatly increases eGFP fluorescence in a dose-dependent manner (), with saturation attained at 2.5 µM. (Note that at lower concentrations, the effect of Shield-1 is transient; this is probably due to metabolism of the drug.) The stabilizing effect of Shield-1 is completely reversible; when EL4/SCRAP cells with a large pool of Shield-1–stabilized SCRAP were washed and cultured in the absence of Shield-1, GFP fluorescence decreased to background SCRAP levels (, right panel).
Although eGFP is a convenient tag for flow cytometric monitoring of protein expression and catabolism, not all SCRAP-gene products will fluoresce due to misfolding and mistranslation. We therefore examined SCRAP expression in the presence and absence of Shield-1 by immunoblotting with a GFP-specific mAb. This revealed a species with the predicted mobility of SCRAP (~42 kDa), whose levels increase with Shield-1 addition () and falls dramatically with Shield-1 removal. We next labeled EL4/SCRAP cells with [35
S]methionine for 10 min in the presence or absence of Shield-1 and chased for up to 130 min in the presence or absence of Shield-1. Total cell lysates were prepared using 95°C SDS-containing extraction buffer to maximize Ag recovery, and after neutralizing SDS denaturing activity, we analyzed species that interact with α-GFP Abs by SDS-PAGE. SCRAP levels rapidly decline in the absence of Shield-1 but remain virtually unchanged for up to 2 h in the presence of Shield-1 (). Another GFP-reactive species present in the immunoprecipitation, GFP 21, decayed with rapid half-life as previously reported (11
). Notably, the initial amount of GFP 21 detected was increased by Shield-1, demonstrating that it is derived from the folded fusion protein and is not the result of downstream initiation or other forms of DRiPs. This species likely results from autocatalytic cleavage of GFP during active site cyclization (15
displays decay curves of full-length SCRAP as determined by flow cytometry, immunoblotting, and pulse-chase analyses. SCRAP unfolded due to Shield-1 removal is degraded with a half-life of 30 min (as determined by immunoblotting and flow cytometry analyses), whereas nascent SCRAP is degraded twice as rapidly (τ = 16 min), demonstrating that nascent and retired SCRAP are handled in a distinct manner.
Taken together, these data indicate that by using Shield-1, we can control the stability of both nascent and mature, native SCRAP.
Presentation of SIINFEKL is only partially affected by protein stability
We next used Alexa 647-conjugated 25-D1.16 to examine the effect of Shield-1 on generation of Kb-SIINFEKL complexes from SCRAP via flow cytometry. To increase the resolution of detection, we briefly exposed cells to pH 3 to destroy preexisting Kb-SIINFEKL complexes. We then incubated cells at 37°C in growth medium and sampled every 60 min.
Kb-SIINFEKL complexes appear on the cell surface within 60 min after acid stripping and increase linearly over the next 5 h. Importantly, in the presence of a saturating Shield-1 concentration (5 µM), the generation of Kb-SIINFEKL complexes is reduced by as little as 30% in some experiments (), as deduced from rates determined by the slope of the linear regression curves. Over five experiments, the average reduction was 36% (3% SE) in five experiments. Complex generation requires both nascent protein synthesis and proteasome activity (), as it is completely blocked by treating cells with either cycloheximide (CHX) or MG132, which inhibit, respectively, protein synthesis and proteasome activity within seconds of their addition. As expected, Kb-SIINFEKL complex expression was completely blocked by brefeldin A (BFA), which prevents egress of complexes from the endoplasmic reticulum (ER) (). The specificity of Shield-1 treatment is clearly demonstrated by its lack of effect on overall cell surface Kb or Db molecules, whereas both MG132 and BFA, as expected, greatly impeded recovery of total MHC I after acid-washing (). The generation of Kb-SIINFEKL complexes in the presence of 5 µM Shield-1 cannot be attributed to residual proteasome degradation of protein detected by standard immunoblotting methodology, as levels of SCRAP rescued by Shield-1 and MG132 are indistinguishable (). These data indicate that the substrate responsible for DRiP presentation cannot be readily determined by standard biochemical means and likely represents a small fraction of the SCRAP synthesized.
FIGURE 2 MHC class I Ag presentation occurs in the presence or absence of stabilized substrates. A, EL4/SCRAP cells were washed briefly in cold citric acid buffer to remove existing Kb-SIINFEKL complexes from the cell surface. Cells were then cultured in the presence (more ...)
These data indicate that although decreasing nascent protein stability enhances peptide presentation, the effect is partial. This is due to the existence of a translation pool of DRiPs that are resistant to SCRAP stabilization and serve as the source of >50% of the antigenic peptides. As we failed to detect the DRiP pool by standard biochemical analysis, it suggests that this source of Ags constitutes a minor fraction of total SCRAP protein translated by the cell. In any event, the ability of Shield-1 to allow selectively peptide generation from DRiPs provides a tool for teasing out differences between the processing of DRiPs versus other sources of antigenic peptides.
Retirement is a much less efficient source of antigenic peptides
We next cultured acid-washed EL4/SCRAP cells with Shield-1 for 3.5 h to generate a native protein pool that we could “retire” by removing Shield-1. Cells were then rewashed in acid and resuspended in media lacking Shield-1 but containing CHX to prevent synthesis of new SCRAP protein. Degradation of SCRAP retirees resulted in a slight but statistically significant (p
< 0.05) increase in Kb
-SIINFEKL complexes compared with that of cells treated with ethanol vehicle alone or with MG132 to prevent SCRAP degradation (). Complexes presented in the presence of MG132 represent complexes present in the secretory pathway during acid treatment. Thus, complexes generated from the SCRAP retirement pool represent the difference between MG132-treated and -untreated cells. Extended CHX treatment may impact protein degradation (16
) and thus mask the true number of complexes generated by Shield-1 removal. We therefore also cultured cells in the absence of CHX after Shield-1 removal. An increase in the number of Kb
-SIINFEKL complexes similar in magnitude to cells treated with CHX was observed after retirement of SCRAP proteins () indicating that CHX treatment did not impact SCRAP degradation or Ag presentation.
FIGURE 3 Retired SCRAP protein minimally contributes to antigenic peptide presentation. EL4/SCRAP cells were washed in citric acid buffer and cultured with or without 5 µM Shield-1 for 3.5 h and then washed again in citric acid buffer followed by treatment (more ...)
The number of complexes generated from the SCRAP pool accumulated over 235 min is equivalent to the number of complexes generated during ~36 min of SCRAP synthesis in the presence of Shield-1. By this simple time-of-synthesis analysis, generating peptides from DRiPs is 6.5 times as efficient as generating them from retirees, providing a clear example of the importance of DRiPs in generating peptides from cellular gene products.
Presentation of SCRAP peptide expressed from rVV
Little is known about the differential contribution of processing pathways of a given gene product in different translational contexts. To address this issue, we inserted SCRAP into vaccinia virus under the control of the early/late p7.5 promoter. We infected DC2.4 cells with rVVs in the presence or absence of Shield-1 and measured GFP and Kb-SIINFEKL complexes over time.
As with transfected cells, the GFP signal remained low in the absence of Shield-1 in DC2.4 cells, increasing nearly linearly in the presence of Shield-1, consistent with a steady rate of translation and negligible degradation relative to the synthesis time window (). Complex formation required proper trafficking through the secretory pathway, de novo protein synthesis, and a functional proteasome, as the respective inhibitors prevented Ag presentation (). The rate of Kb-SIINFEKL complex generation was decreased 28% by Shield-1 (), remarkably similar to the findings with EL4 transfectants.
FIGURE 4 SCRAP expression by rVV results in rapid Ag presentation regardless of protein stabilization. DC2.4 cells were infected with rVV/SCRAP at a multiplicity of infection of 10 for 30 min and then cultured in 5 µM Shield-1 or equivalent amount of EtOH, (more ...)
These findings demonstrate that SCRAP Ag processing is highly similar when expressed by viral versus cellular translation machinery.
Eeyarestatin I selectively inhibits DRiP Ag presentation
Uniquely, our SCRAP system allows us to dissect processing of DRiPs versus other rapidly degraded polypeptides (RDPs) (11
) or retirees by the simple addition of Shield-1 to limit presentation to DRiPs. In screening drugs that selectivity modulate DRiP processing, we tested eeyarestatin I (Eer1), originally described as an inhibitor of ER-associated degradation (17
). We found that Eer1 reduces overall generation of Kb
-SIINFEKL by ~50%. In the presence of Shield-1, Eer1 reduced Kb
-SIINFEKL generation to nearly background levels, demonstrating that it inhibits processing of SIINFEKL from SCRAP DRiPs while sparing processing from Shield-1–responsive RDPs (). After acid stripping of class I molecules, Eer1 treatment reduced Kb
recovery over the next 100 or 300 min by ~20% (). This effect must be gauged by the partial effects of CHX and MG132 on recovery and is consistent with a significant effect of Eer1 on a sizeable pool of class I peptide ligands.
FIGURE 5 Eer1 inhibits Ag presentation of SCRAP DRiPs. A, EL4/SCRAP cells were prepared as in and cultured with ethanol or 5 µM Shield-1 combined with DMSO or 5 µM Eer1. At indicated times, cells were harvested and analyzed for Kb-SIINFEKL (more ...)
How does Eer1 interfere with Ag processing? We cultured EL4/SCRAP cells with Shield-1, removed Shield-1, and treated cells with either MG132 or Eer1. Eer1 had no significant effect on SCRAP accumulation or proteasome-mediated degradation (), which is consistent with its lack of effect on Shield-1–dependent presentation. This finding demonstrates that Eer1 does not inhibit DRiP Ag presentation by blocking protein synthesis or proteasome-mediated degradation.
To extend these results to other Ags, we infected DC2.4 cells with rVVs expressing a fusion protein consisting of influenza A virus nucleoprotein, SIINFEKL, and GFP (NP-S-GFP). Eer1 treatment of infected cells resulted in an ~50% decrease in Kb
-SIINFEKL levels (). By contrast, Eer1 had little effect on the generation of Kb
-SIINFEKL complexes from rVV-GFP-Ub-SIINFEKL, which expresses preprocessed cytosolic SIINFEKL liberated co-translationally by ubiquitin hydrolases from the fusion protein. (Note that we equalized the amount of Kb
-SIINFEKL complexes generated by the two viruses by partially inactivating the latter virus by UV irradiation, as otherwise it generates saturating amounts of SIINFEKL.) The failure of Eer1 to interfere with SIINFEKL presentation from GFP-Ub-SIINFEKL demonstrates that Eer1 does not block presentation by interfering with Kb
biogenesis per se, which is critical given the recent report that Eer1 can inhibit the import of some proteins into the ER (19
). Eer1 treatment of vaccinia virus-infected cells did not significantly affect expression of NP-S-GFP or GFP-Ub-S determined by immunoblotting with anti-GFP Abs (). Eer1 treatment of BMDCs infected with rVV-SCRAP in the presence or absence of Shield-1 resulted in a similar trend of inhibited DRiP Ag presentation ().
FIGURE 6 Eer1 partially inhibits DRiP Ag presentation but has a minimal effect on presentation of other forms of Ags during rVV infection. A, DC2.4 cells were infected with either NP-S-GFP, eGFP-Ub-S, or partially UV-inactivated eGFP-Ub-S expressing rVV for 30 (more ...)
Together, these data demonstrate that Eer1 inhibits DRiP presentation in a manner independent of protein synthesis, proteasome activity, TAP-mediated peptide transport into the ER, or peptide loading onto class I, therefore defining a novel step of the DRiP Ag presentation pathway.
Both Eer1 and Cl-PGA2 increase levels of polyubiquitylated proteins and inhibit DRiP presentation
Eer1 is believed to inhibit ER-associated degradation by binding to the AAA ATPase p97, leading to inhibition of substrate deubiquitylation by one or more p97-associated deubiquitinating (DUB) enzymes. Accordingly, Eer1 treatment is known to increase the levels of polyubiquitylated proteins (17
). We examined DUB participation in DRiP processing by treating EL4/SCRAP cells with another DUB inhibitor, 10-chloro-15-acetyl-1-methylester-PGA2
(hereafter termed Cl-PGA2) (20
). Cl-PGA2 behaved nearly identically to Eer1 in selectively inhibiting Ag presentation from the Shield-1–resistant SCRAP pool ().
FIGURE 7 Cl-PGA2 and Eer1 inhibit DRiP Ag presentation and increase levels of polyubiquitinated proteins in EL4/SCRAP cells. A, EL4/SCRAP cells were treated as in except Cl-PGA2 was used in place of Eer1. B, EL4/SCRAP cells were treated with either DMSO, (more ...)
Treating cells with either Eer1 or Cl-PGA2 increased cellular levels of polyubiquitylated proteins without inhibiting proteasome-mediated SCRAP degradation, consistent with the participation of DUBs in Ag processing ().
It is important to note that Eer1 and Cl-PGA2 have widely divergent chemical structures, greatly reducing the chance of their acting on a common non-DUB target. Rather, their highly similar effects on Ag processing strongly suggests that DUBs are necessary for the generation of peptides from SCRAP DRiPs whereas they are dispensable for generating peptides from Shield-1–responsive RDPs.