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DNA repair pathways are essential to maintain the integrity of the genome and prevent cell death and tumourigenesis. Here, we show that the Barrier-to-Autointegration Factor (Banf1) protein has a role in the repair of DNA double-strand breaks. Banf1 is characterized as a nuclear envelope protein and mutations in Banf1 are associated with the severe premature aging syndrome, Néstor–Guillermo Progeria Syndrome. We have previously shown that Banf1 directly regulates the activity of PARP1 in the repair of oxidative DNA lesions. Here, we show that Banf1 also has a role in modulating DNA double-strand break repair through regulation of the DNA-dependent Protein Kinase catalytic subunit, DNA-PKcs. Specifically, we demonstrate that Banf1 relocalizes from the nuclear envelope to sites of DNA double-strand breaks. We also show that Banf1 can bind to and directly inhibit the activity of DNA-PKcs. Supporting this, cellular depletion of Banf1 leads to an increase in non-homologous end-joining and a decrease in homologous recombination, which our data suggest is likely due to unrestrained DNA-PKcs activity. Overall, this study identifies how Banf1 regulates double-strand break repair pathway choice by modulating DNA-PKcs activity to control genome stability within the cell.

Platinum-based chemotherapy remains the cornerstone of treatment for most people with non-small cell lung cancer (NSCLC), either as adjuvant therapy in combination with a second cytotoxic agent or in combination with immunotherapy. Resistance to therapy, either in the form of primary refractory disease or evolutionary resistance, remains a significant issue in the treatment of NSCLC. Hence, predictive biomarkers and novel combinational strategies are required to improve the effectiveness and durability of treatment response 6for people with NSCLC. The aim of this study was to identify novel biomarkers and/or druggable proteins from deregulated protein networks within non-oncogene driven disease that are involved in the cellular response to cisplatin. Following exposure of NSCLC cells to cisplatin, in vitro quantitative mass spectrometry was applied to identify altered protein response networks. A total of 65 proteins were significantly deregulated following cisplatin exposure. These proteins were assessed to determine if they are druggable targets using novel machine learning approaches and to identify whether these proteins might serve as prognosticators of platinum therapy. Our data demonstrate novel candidates and drug-like molecules warranting further investigation to improve response to platinum agents in NSCLC.

Immunotherapy has revolutionised therapeutic approaches to fight cancer and, in certain diseases dramatically improves survival. Clinical responses to immune checkpoint blockade have in part been attributed to high mutational burden of tumours such as melanoma. High-risk acute myeloid leukaemia (AML) is defined by molecular and cytogenetic factors. AML has a low prevalence of somatic mutations and is predicted to have low immunogenicity. We aimed to determine how AMLs driven from different classes of oncogenes interact with endogenous anti-leukemic immune responses.

To assess the feasibility of a mass spectrometry (MS) based proteomics approach for identifying pathological differences associated with IgAN in clinical kidney biopsies.

This study investigated proteomic changes occurring in Anopheles gambiae and Anopheles stephensi during adult mosquito aging. These changes were evaluated using two-dimensional difference gel electrophoresis (2D-DIGE) and the identities of aging related proteins were determined using capillary high-pressure liquid chromatography (capHPLC) coupled with a linear ion-trap (LTQ)-Orbitrap XL hybrid mass spectrometry (MS). Here, we have described the techniques used to determine age associated proteomic changes occurring in heads and thoraces across three age groups; 1, 9 and 17 d old A. gambiae and 4 age groups; 1, 9, 17 and 34 d old A. stephensi. We have provided normalised spot volume raw data for all protein spots that were visible on 2D-DIGE images for both species and processed Orbitrap mass spectrometry data. For public access, mass spectrometry raw data are available via ProteomeXchange with identifier PXD002153. A detailed description of this study has been described elsewhere.

The receptor tyrosine kinase EphA2 is frequently upregulated in cancer, where it plays an intriguing dichotomous role that may lend itself to therapeutic intervention. However, much remains to be understood about how ephrin-mediated activation of EphA2 signaling can reverse its tumour-promoting ligand-independent effects and instead inhibit migration and invasion. We used quantitative phosphoproteomics to analyze EphA2 signaling in PC-3 prostate cancer cells with the aim to further elucidate its downstream signaling and pinpoint phosphorylation-dependent cellular processes that may explain its dichotomous role. Stable isotope labeling by amino acids in cell culture (SILAC) combined with liquid chromatography-coupled mass spectrometry (LC-MS/MS) was used to quantify changes in relative phosphosite abundance in response to EphA2 activation. We identified 113 significantly regulated phosphosites comprising 73 phosphoproteins, including many novel and several previously described downstream effector proteins of EphA2 signaling. More than two thirds of the regulated phosphosites showed reduced phosphorylation in response to EphA2 activation. This is consistent with EphA2 activation inhibiting oncogenic signaling pathways, including Akt and ERK-MAPK signaling. Protein association analysis using the STRING database showed that a large proportion of these differentially regulated proteins are downstream targets of Akt and ERK-MAPK, respectively. Gene ontology enrichment analysis found our dataset to be strongly enriched in proteins associated with the cytoskeleton and its regulation. Overall, our data suggest that diminished phosphorylation of cytoskeletal regulators downstream of Akt and ERK-MAPK signaling may be a major mechanism by which stimulation of EphA2 can alter cell morphology and inhibit cell motility.

Analysis of a tryptic digest of a recombinant D83A mutant of the murine dioxin receptor (D83A-DR) by CapHPLC-MALDI-TOF/TOF-MS/MS revealed a peptide corresponding to cleavage at a lysine that had either been mutated to an arginine or modified by dimethylation or formylation (Ser-Phe-Phe-Ala-Val-Ala-Leu-Me2K/FormK/Arg). High mass accuracy data were obtained for the pseudomolecular ions of synthetic peptides corresponding to the potential alternate structures of the variant D83A tryptic peptide and their CID products using an LTQ-Orbitrap-FTMS instrument. The high mass accuracies obtained by both FT-MS and FT-MS/MS were more than sufficient to enable differentiation between the synthetic peptides. The only potential fragmentation characteristic produced by CID was the formation of ions at −17 u relative to y ions of the Arg peptide. The variant D83A-DR tryptic peptide was also detected by CapHPLC-ESI-LTQ-Orbitrap-FT-MS and MS/MS with masses detected for the pseudomolecular and CID product ions being consistent with dimethylation of a C-terminal lysine. Fragmentation of the synthetic peptides by MALDI-TOF/TOF-MS/MS produced a range of fragmentation characteristics which provided a basis for distinguishing between peptides with C-terminal Me2K, FormK and Arg. These characteristics included specific immonium ions, apparent side chain fragmentation from the precursors of 28 or 30 u and satellite ions at −17 u relative to the y ions of the Arg peptide. Comparison of the fragmentation properties of the D83A-DR derived tryptic peptide produced by MALDI-TOF/TOF-MS/MS with those of the synthetic peptides corroborated the mass accuracy-based assignment of a dimethylated lysine at the C-terminus of the D83A-DR tryptic peptide. This represents the first documentation of any post-translational modification of DR, other than two previously described sites of phosphorylation