First, we compared the presentation of model proteins undergoing RQC degradation or steady-state turnover, using model genes without and with a stop codon (non-stop proteins, resulting in ribosome stalling or stop proteins, normal translation termination, respectively). The employed model proteins contained a destabilization domain whose folding state can be controlled by the addition of a ligand. The results show that degradation/presentation of non-stop proteins is independent of their folding potential, implying that RQC could be exploited for the sampling of folding-efficient proteins, a common trait of viral proteins. Moreover, in the presence of folding ligand, non-stop proteins have a markedly faster presentation than their stop counterparts, which could be highly beneficial for early detection of infections.
Next, we evaluated the contribution of RQC degradation on global MHC-I antigen presentation by comparing the immunopeptidome of human cells lacking the Listerin E3 ligase, responsible for the ubiquitination of stalled nascent chains, with their parental WT cell line. Immunoprecipitation of MHC-I with bound immunopeptides and mass-spectrometric analysis resulted in the identification of 3658 presented peptides. From those, 103 peptides had significantly higher intensity in WT than Listerin knock-out cells, and therefore represent RQC targets. In accordance, proteome quantification of the corresponding samples indicated that for the majority of cases the observed immunopeptide changes are due to alterations in presentation and not in expression. Gene ontology analysis of the targets did not reveal a significant enrichment for a specific molecular function, cellular component or biological process, highlighting the capacity of RQC to target a wide variety of proteins. In conclusion, our results reveal a co-opted function of RQC in sampling functionally diverse proteins that are otherwise difficult to present, indicating that RQC has great potential for helping the immune system to identify virus-infected cells. Importantly, our immunopeptidome analysis also provided the first dataset of endogenous RQC substrates in human cells. This, in combination with our in-depth proteome analysis, brought us a first glimpse into the largely uncharacterized natural causes for aberrant translation and RQC recruitment.
The work developed in the Hartl laboratory was presented to the broader public in the Open Day of the Max Planck Institutes in Martinsried on November 17th, 2018. Approximate 4500 visitors were welcomed to our institute with lectures and guided tours through the laboratories. The specific results of the IMMUNO-RQC project were presented in the EMBO conference “Protein quality control: From mechanisms to disease” on April/May 2019 in the form of a poster. The presentation was received with great interest by the scientific community, and was awarded a poster prize. The corresponding manuscript is in the final stages of preparation, and will be submitted soon to a broad-interest journal with the open access option.
