Proteomics and Phospho-Proteomics Profiling of the Co-Formulation of Type I and II Interferons, HeberFERON, in the Glioblastoma-Derived Cell Line U-87 MG

Abstract

HeberFERON, a co-formulation of Interferon (IFN)-α2b and IFN-γ, has effects on skin cancer and other solid tumors. It has antiproliferative effects over glioblastoma multiform (GBM) clones and cultured cell lines, including U-87 MG. Here, we report the first label-free quantitative proteomic and phospho-proteomic analyses to evaluate changes induced by HeberFERON after 72 h incubation of U-87 MG that can explain the effect on cellular proliferation. LC-MS/MS, functional enrichment and networking analysis were performed. We identified 7627 proteins; 122 and 211 were down- and up-regulated by HeberFERON (fold change > 2; p < 0.05), respectively. We identified 23,549 peptides (5692 proteins) and 8900 phospho-peptides; 523 of these phospho-peptides (359 proteins) were differentially modified. Proteomic enrichment showed IFN signaling and its control, direct and indirect antiviral mechanisms were the main modulated processes. Phospho-proteome enrichment displayed the cell cycle as one of the most commonly targeted events together with cytoskeleton organization; translation/RNA splicing, autophagy and DNA repair, as represented biological processes. There is a high interconnection of phosphoproteins in a molecular network; mTOR occupies a centric hub with interactions with translation machinery, cytoskeleton and autophagy components. Novel phosphosites and others with unknown biological functionality in key players in the aforementioned processes were regulated by HeberFERON and involved CDK and ERK kinases. These findings open new experimental hypotheses regarding HeberFERON action. The results obtained contribute to a better understanding of HeberFERON effector mechanisms in the context of GBM treatment.

An Atlas of Protein Phosphorylation Dynamics During Interferon Signaling

“…This led to the identification of >700 total IFN- regulated phosphorylation sites in >500 proteins, vastly increasing our current understanding of factors associated with these pathways. The results we present differentiate themselves from other phosphoproteomic efforts in this area (for example (Vázquez-Blomquist et al , 2022; Viengkhou et al , 2021)) due to the short IFN stimulation periods that we used to capture rapid and transient events relevant to the known timings of JAK/STAT signaling, as well as the comprehensive nature of our approach using all three IFN types. Furthermore, the work adds a complementary additional layer of knowledge to recent studies that have investigated the common and unique specificities of different IFNs with regards to their antiviral activities, or their transcriptome and proteome re-wiring capabilities (Guo et al , 2020; Lum et al , 2024; Matos et al , 2019; Megger et al , 2017; Schuhenn et al , 2022; Yan et al , 2004).…”