Periodic Reporting for period 1 - IMMUNO-RQC (Role of Listerin ubiquitin ligase in MHC-I antigen presentation) Reporting period: 2017-05-01 to 2019-04-30 Summary of the context and overall objectives of the project Mammalian cells present their proteome content to the adaptive immune system through the display of peptides on MHC-I complexes. This allows for CD8+ T cells to selectively recognize and kill cells that present non-self antigens, for example due to viral infection or cancer transformation. The MHC-I peptides originate from protein degradation by the ubiquitin proteasome system, implicating that optimally-folding, long-lived proteins could be difficult to monitor in a timely fashion. To explain the kinetic discrepancy between protein half-life and MHC-I presentation, Defective Ribosomal Products (DRiPs) have been proposed to act as a significant source of self and viral peptides. Accordingly, cells exploit DRiP degradation for efficient processing of antigenic peptides. However, the underlying protein quality control pathways responsible for the efficient presentation of newly synthesized proteins remain elusive. The main goal of this project was to investigate the role of the Ribosomal Quality Control (RQC) pathway in sampling peptides for antigen presentation. The project therefore addresses a fundamental question in the field of immunology: how do cells ensure timely presentation of newly synthesized proteins. Understanding how this basic process works could potentially aid the development of new therapies, for example, for improving the immune-response to viral infections and cancers, or for attenuating auto-immune diseases. In the course of the action we showed that the RQC machinery generates antigenic peptides from nascent chains encoded by defective mRNA molecules, and that this is independent of folding efficiency. Furthermore, we demonstrated that RQC degradation substantially contributes to the composition of the immunopeptidome. The RQC contribution is especially pronounced for folding-efficient proteins where sampling might be difficult, as is expected to be the case for many viral proteins. Our analysis also provides the first dataset of natural RQC degradation substrates in human cells, providing new insights into this still largely uncharacterized degradation pathway. Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far 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. Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far) This is the first study to evaluate the contribution of a defined pathway to the global repertoire of antigens. We believe this represents a concrete step towards mechanistically understanding the 20-year old principles proposed by the DRiP hypothesis. Moreover, in collaboration with world-leading experts in the mass spectrometry field, we made use of pioneering technology to obtain quantitative immunopeptidome profiles of outstanding depth. With these results we could not only assess the RQC contribution to antigen presentation, but also identify endogenous RQC substrates. We thereby obtained the first dataset of RQC targets under normal growth conditions. Some of the obtained observations might help us to understand in the future why RQC-deficient mice have a neurodegenerative phenotype. In conclusion, the project made conceptual advances in the fields of immunology and protein quality control. Although the results are at present not directly applicable to the medical field, it advances on an interdisciplinary subject that is relevant not only for the development of anti-viral and anti-tumor immunotherapies, but also for the understanding of neurodegenerative diseases. The ribosomal quality cotrol pathway can generate peptides for MHC-I antigen presentation.