Pro-inflammatory Cytokines Alter the Immunopeptidome Landscape by Modulation of HLA-B Expression

Antigen presentation on HLA molecules is a major mechanism by which the immune system monitors self and non-self-recognition. Importantly, HLA-I presentation has gained much attention through its role in eliciting anti-tumor immunity. Several determinants controlling the peptides presented on HLA have been uncovered, mainly through the study of model substrates and large-scale immunopeptidome analyses. These determinants include the relative abundances of proteins in the cell, the stability or turnover rate of these proteins and the binding affinities of a given peptide to the HLA haplotypes found in a cell. However, the regulatory principles involved in selection and regulation of specific antigens in response to tumor pro-inflammatory signals remain largely unknown. Here, we chose to examine the effect that TNFα and IFNγ stimulation may exert on the immunopeptidome landscape of lung cancer cells. We show that the expression of many of the proteins involved in the class I antigen presentation pathway are changed by pro-inflammatory cytokines. Further, we could show that increased expression of the HLA-B allomorph drives a significant change in HLA-bound antigens, independently of the significant changes observed in the cellular proteome. Finally, we observed increased HLA-B levels in correlation with tumor infiltration across the TCGA lung cancer cohorts. Taken together, our results suggest that the immunopeptidome landscape should be examined in the context of anti-tumor immunity whereby signals in the microenvironment may be critical in shaping and modulating this important aspect of host-tumor interactions

ND10-MEDIATED ROLES OF IFI16 IN SENSING AND SUPPRESSING HERPES SIMPLEX VIRUS-1

The antiviral factor interferon inducible protein 16 (IFI16) is a sensor of foreign DNA in the nucleus. A critical member of innate and intrinsic immunity processes, IFI16 binds herpesvirus DNA in the nucleus during infection. In response to DNA binding, IFI16 stimulates the production of interferon and cytokines through the STING-TBK1-IRF3 pathway. IFI16 has also been shown to have other critical roles during infection, serving as a suppressor of viral transcription. During Herpes Simplex Virus 1 (HSV-1) infection, these roles of IFI16 require it to be recruited to pre-replication sites in the nucleus where the viral genome is deposited. Despite all that is known about the function of IFI16 during infection, many of the cofactors responsible for binding HSV-1 genomes and mediating IFN upregulation through STING remain unknown. Using a combination of proteomics and virology - based methods, I characterized many of the interactions of IFI16 during HSV-1 infection. The newly identified interactions of IFI16 lend insight into the roles it plays in binding to and suppressing the replication of HSV-1 genomes. Through immunopurification and mass spectrometry analysis, IFI16 was found to interact with proteins from the sub-nuclear Nuclear Domain 10 (ND10). This complex has also been shown to associate with HSV-1 genomes and is recruited to pre-replication sites during infection, thereby playing a role in antiviral cellular defense. In addition, IFI16 was shown to associate with many HSV-1 proteins involved in HSV-1 genome transcription. I demonstrated that the ND10 component PML and the HIN200 domain of IFI16 are necessary for the recruitment of IFI16 to HSV-1 pre-replication sites. Finally, I examined the role of IFI16 and PML in IFN-induced apoptotic induction during HSV-1 infection

Interactions of the Antiviral Factor Interferon Gamma-Inducible Protein 16 (IFI16) Mediate Immune Signaling and Herpes Simplex Virus-1 Immunosuppression

The interferon-inducible protein IFI16 has emerged as a critical antiviral factor and sensor of viral DNA. IFI16 binds nuclear viral DNA, triggering expression of antivi-ral cytokines during infection with herpesviruses. The knowledge of the mechanisms and protein interactions through which IFI16 exerts its antiviral functions re-mains limited. Here, we provide the first characteriza-tion of endogenous IFI16 interactions following infection with the prominent human pathogen herpes simplex vi-rus 1 (HSV-1). By integrating proteomics and virology approaches, we identified and validated IFI16 interac-tions with both viral and host proteins that are involved in HSV-1 immunosuppressive mechanisms and host an-tiviral responses. We discover that during early HSV-1 infection, IFI16 is recruited to sub-nuclear puncta an

Altered Protein Abundance and Localization Inferred from Sites of Alternative Modification by Ubiquitin and SUMO

Protein modification by ubiquitin or SUMO can alter the function, stability or activity of target proteins. Previous studies have identified thousands of substrates that were modified by ubiquitin or SUMO on the same lysine residue. However, it remains unclear whether such overlap could result from a mere higher solvent accessibility, whether proteins containing those sites are associated with specific functional traits, and whether selectively perturbing their modification by ubiquitin or SUMO could result in different phenotypic outcomes. Here, we mapped reported lysine modification sites across the human proteome and found an enrichment of sites reported to be modified by both ubiquitin and SUMO. Our analysis uncovered thousands of proteins containing such sites, which we term Sites of Alternative Modification (SAMs). Among more than 36,000 sites reported to be modified by SUMO, 51.8% have also been reported to be modified by ubiquitin. SAM-containing proteins are associated with diverse biological functions including cell cycle, DNA damage, and transcriptional regulation. As such, our analysis highlights numerous proteins and pathways as putative targets for further elucidating the crosstalk between ubiquitin and SUMO. Comparing the biological and biochemical properties of SAMs versus other non-overlapping modification sites revealed that these sites were associated with altered cellular localization or abundance of their host proteins. Lastly, using S. cerevisiae as model, we show that mutating the SAM motif in a protein can influence its ubiquitination as well as its localization and abundance.</p

The proteasome regulator PSME4 modulates proteasome activity and antigen diversity to abrogate antitumor immunity in NSCLC

Immunotherapy revolutionized treatment options in cancer, yet the mechanisms underlying resistance in many patients remain poorly understood. Cellular proteasomes have been implicated in modulating antitumor immunity by regulating antigen processing, antigen presentation, inflammatory signaling and immune cell activation. However, whether and how proteasome complex heterogeneity may affect tumor progression and the response to immunotherapy has not been systematically examined. Here, we show that proteasome complex composition varies substantially across cancers and impacts tumor–immune interactions and the tumor microenvironment. Through profiling of the degradation landscape of patient-derived non-small-cell lung carcinoma samples, we find that the proteasome regulator PSME4 is upregulated in tumors, alters proteasome activity, attenuates presented antigenic diversity and associates with lack of response to immunotherapy. Collectively, our approach affords a paradigm by which proteasome composition heterogeneity and function should be examined across cancer types and targeted in the context of precision oncology