University of Cambridge
Characterization of endocytic escape pathway and its role in adaptive immunity
Abstract
dc:description.abstractIn dendritic cells, endocytic escape appears critical for the induction of cytotoxic T-cell responses against viruses and tumors, in a process termed cross-presentation. During cross-presentation, dendritic cells present exogenous antigens on MHC class I, and the predominant molecular model for it is one where exogenous antigens are first released from endosomes into the cytosol, and further processed like endogenous proteins for presentation on MHC class I. Endocytic escape of antigens appears to be the rate-limiting step of cross-presentation, yet we know very little about the molecular mechanisms involved. To identify the machinery involved in endocytic escape of proteins, the Kozik lab has established a genetic screening strategy where escape of a ribosome-inactivating protein, called saporin, is being followed. When saporin enters the cytosol, it causes translational arrest quantified using puromycin labelling of newly synthesized polypeptides. Using the saporin assay, the Kozik lab performed CRISPR/Cas9 genetic screen targeting 200 candidate genes. The screen yielded one strong hit: pore-forming protein, perforin-2 (*Mpeg1*; Macrophage-expressed gene 1) and we proposed perforin-2 forms pores in endolysosomal compartments, allowing for endocytic escape of saporin. The main aim of my PhD was to extend our understanding of mechanisms governing endocytic escape through perforin-2, to identify other members of endocytic escape pathway, and to study their role in cross-presentation. We demonstrated that perforin-2 activity might be regulated by proteolytic cleavage and set out to identify the protease involved. The cleavage depends on low pH and can be stimulated by a range of pathogen-associated molecular patterns. To identify the new players of the endocytic escape pathway, we employed a genome-wide CRISPR/Cas9 genetic screening approach, where we followed endocytic escape of either saporin or of another translation inhibitor, a small peptide AGAAMSH (here named Pep). The screen confirmed that *Mpeg1* is a main regulator of endocytic escape of saporin in MutuDCs, and we identified and validated multiple potential *Mpeg1* regulators, including proteins involved in trafficking and posttranslational modifications. Interestingly, the screen with use of Pep as a translation inhibitor identified largely different hits. Many of the negative regulation hits were mitochondrial proteins, especially complex I related, which might indicate the involvement of mitochondrial ROS in endocytic escape of Pep. Finally, we went to address whether any of the validated hits indeed play a role in cross-presentation. Using the B3Z T cell hybridoma-based cross-presentation assay, we did not observe defects in cross-presentation in *Mpeg1* knockouts, however, knockout of a negative regulator of saporin escape, *Sppl2a*, seemed to increase cross-presentation of soluble ovalbumin, whereas knockout of a negative regulator of Pep escape, *Chchd5*, might enhance cross-presentation of immune complexes of ovalbumin. We hope that further characterization and cross-presentation studies will shed more light on the role of validated hits in the endocytic escape pathways, and on how different endocytic escape pathways contribute to adaptive immunity.
Degree
thesis:*- Name dc:type.qualificationname
- Doctor of Philosophy (PhD)
- Level dc:type.qualificationlevel
- Doctoral
- Grantor dc:publisher.institution
- University of Cambridge
- Year dc:date.issued
- 2023
Author and committee
dc:creator, dc:contributor.*- Author dc:creator
-
- Krawczyk, Patrycja Anna
- Advisor dc:contributor.advisor
-
- Kozik, Patrycja
Subjects
dc:subject × 5Rights
dc:rightsIdentifiers
dc:identifier.*- DOI dc:identifier.doi
- https://doi.org/10.17863/CAM.106152
- OAI identifier oai:identifier
- oai:www.repository.cam.ac.uk:1810/364520