RESUMEN
Ubiquitin-specific protease 7 (USP7) has been implicated in cancer progression and neurodevelopment. However, its molecular targets remain poorly characterized. We combined quantitative proteomics, transcriptomics, and epigenomics to define the core USP7 network. Our multi-omics analysis reveals USP7 as a control hub that links genome regulation, tumor suppression, and histone H2A ubiquitylation (H2AK119ub1) by noncanonical Polycomb-repressive complexes (ncPRC1s). USP7 strongly stabilizes ncPRC1.6 and, to a lesser extent, ncPRC1.1. Moreover, USP7 represses expression of AUTS2, which suppresses H2A ubiquitylation by ncPRC1.3/5. Collectively, these USP7 activities promote the genomic deposition of H2AK119ub1 by ncPRC1, especially at transcriptionally repressed loci. Notably, USP7-dependent changes in H2AK119ub1 levels are uncoupled from H3K27me3. Even complete loss of the PRC1 catalytic core and H2AK119ub1 has only a limited effect on H3K27me3. Besides defining the USP7 regulome, our results reveal that H2AK119ub1 dosage is largely disconnected from H3K27me3.
RESUMEN
Background & Aims: Antigen-specific immunotherapy is a promising strategy to treat HBV infection and hepatocellular carcinoma (HCC). To facilitate killing of malignant and/or infected hepatocytes, it is vital to know which T cell targets are presented by human leucocyte antigen (HLA)-I complexes on patient-derived hepatocytes. Here, we aimed to reveal the hepatocyte-specific HLA-I peptidome with emphasis on peptides derived from HBV proteins and tumour-associated antigens (TAA) to guide development of antigen-specific immunotherapy. Methods: Primary human hepatocytes were isolated with high purity from (HBV-infected) non-tumour and HCC tissues using a newly designed perfusion-free procedure. Hepatocyte-derived HLA-bound peptides were identified by unbiased mass spectrometry (MS), after which source proteins were subjected to Gene Ontology and pathway analysis. HBV antigen and TAA-derived HLA peptides were searched for using targeted MS, and a selection of peptides was tested for immunogenicity. Results: Using unbiased data-dependent acquisition (DDA), we acquired a high-quality HLA-I peptidome of 2 × 105 peptides that contained 8 HBV-derived peptides and 14 peptides from 8 known HCC-associated TAA that were exclusive to tumours. Of these, 3 HBV- and 12 TAA-derived HLA peptides were detected by targeted MS in the sample they were originally identified in by DDA. Moreover, 2 HBV- and 2 TAA-derived HLA peptides were detected in samples in which no identification was made using unbiased MS. Finally, immunogenicity was demonstrated for 5 HBV-derived and 3 TAA-derived peptides. Conclusions: We present a first HLA-I immunopeptidome of isolated primary human hepatocytes, devoid of immune cells. Identified HBV-derived and TAA-derived peptides directly aid development of antigen-specific immunotherapy for chronic HBV infection and HCC. The described methodology can also be applied to personalise immunotherapeutic treatment of liver diseases in general. Lay summary: Immunotherapy that aims to induce immune responses against a virus or tumour is a promising novel treatment option to treat chronic HBV infection and liver cancer. For the design of successful therapy, it is essential to know which fragments (i.e. peptides) of virus-derived and tumour-specific proteins are presented to the T cells of the immune system by diseased liver cells and are thus good targets for immunotherapy. Here, we have isolated liver cells from patients who have chronic HBV infection and/or liver cancer, analysed what peptides are presented by these cells, and assessed which peptides are able to drive immune responses.
RESUMEN
The rapid, sensitive and specific detection of SARS-CoV-2 is critical in responding to the current COVID-19 outbreak. In this proof-of-concept study, we explored the potential of targeted mass spectrometry (MS) based proteomics for the detection of SARS-CoV-2 proteins in both research samples and clinical specimens. First, we assessed the limit of detection for several SARS-CoV-2 proteins by parallel reaction monitoring (PRM) MS in infected Vero E6 cells. For tryptic peptides of Nucleocapsid protein, the limit of detection was estimated to be in the mid-attomole range (9E-13 g). Next, this PRM methodology was applied to the detection of viral proteins in various COVID-19 patient clinical specimens, such as sputum and nasopharyngeal swabs. SARS-CoV-2 proteins were detected in these samples with high sensitivity in all specimens with PCR Ct values <24 and in several samples with higher CT values. A clear relationship was observed between summed MS peak intensities for SARS-CoV-2 proteins and Ct values reflecting the abundance of viral RNA. Taken together, these results suggest that targeted MS based proteomics may have the potential to be used as an additional tool in COVID-19 diagnostics.