Architecture of The Human Ape1 Interactome Defines Novel Cancers Signatures

Abstract

APE1 is essential in cancer cells due to its central role in the Base Excision Repair pathway of DNA lesions and in the transcriptional regulation of genes involved in tumor progression/chemoresistance. Indeed, APE1 overexpression correlates with chemoresistance in more aggressive cancers, and APE1 protein-protein interactions (PPIs) specifically modulate different protein functions in cancer cells. Although important, a detailed investigation on the nature and function of protein interactors regulating APE1 role in tumor progression and chemoresistance is still lacking. The present work was aimed at analyzing the APE1-PPI network with the goal of defining bad prognosis signatures through systematic bioinformatics analysis. By using a well-characterized HeLa cell model stably expressing a flagged APE1 form, which was subjected to extensive proteomics analyses for immunocaptured complexes from different subcellular compartments, we here demonstrate that APE1 is a central hub connecting different subnetworks largely composed of proteins belonging to cancer-associated communities and/or involved in RNA-and DNA-metabolism. When we performed survival analysis in real cancer datasets, we observed that more than 80% of these APE1-PPI network elements is associated with bad prognosis. Our findings, which are hypothesis generating, strongly support the possibility to infer APE1-interactomic signatures associated with bad prognosis of different cancers; they will be of general interest for the future definition of novel predictive disease biomarkers. Future studies will be needed to assess the function of APE1 in the protein complexes we discovered. Data are available via ProteomeXchange with identifier PXD013368.Alteration of DNA repair mechanisms is an important hallmark of cancer cells, and plays a role both in the onset of an initial cancerous phenotype and in tumor progression. Tumor cells can develop drug resistance through repair mechanisms that counteract the DNA damage induced by chemotherapy or radiotherapy 1,2 . Thus, specific DNA repair inhibitors are often combined with DNA-damaging agents to improve therapy efficacy. Emerging evidences in tumor biology suggest that: i) protein-protein interactions (PPIs) specifically modulate both canonical and non-canonical roles of DNA repair enzymes; ii) RNA processing pathways participate in DNA-Damage Response (DDR); iii) defects in the above-mentioned regulatory mechanisms are associated with cancer genomic instability 3 . Very recent studies clearly show that many DNA repair proteins are associated with those involved in RNA metabolism, proving a role of their interactome network in undertaking non-canonical functions affecting gene expression in tumors. In addition, novel studies have shown that interaction of DDR components and miRNA biogenesis process is linked to cancer development 2 . In the context of these emerging lines, we already demonstrated the crucial role that enzymes belonging to the base excision DNA repair (BER) pathway play 4 . In particular, we showe...

Overexpression of SP6 Correlates with Osteosarcoma Metastasis and Poor Prognosis

“…In addition, a large number of DNA BER proteins have been investigated as a potential biomarker and therapeutic target [29][30][31].…”

Identification of RNA-binding proteins that partner with Lin28a to regulate Dnmt3a expression

“…After staining with colloidal Coomassie blue, whole gel lanes were cut into 12 slices, minced and washed with water. Corresponding proteins were separately in-gel reduced, S -alkylated with iodoacetamide and digested with trypsin, as previously reported 59 . Individual protein digests were then analyzed with a nanoLC-ESI-Q-Orbitrap-MS/MS platform consisting of an UltiMate 3000 HPLC RSLC nano system (Thermo Fisher Scientific) coupled to a Q-ExactivePlus mass spectrometer through a Nanoflex ion source (Thermo Fisher Scientific) 59 .…”

“…Corresponding proteins were separately in-gel reduced, S -alkylated with iodoacetamide and digested with trypsin, as previously reported 59 . Individual protein digests were then analyzed with a nanoLC-ESI-Q-Orbitrap-MS/MS platform consisting of an UltiMate 3000 HPLC RSLC nano system (Thermo Fisher Scientific) coupled to a Q-ExactivePlus mass spectrometer through a Nanoflex ion source (Thermo Fisher Scientific) 59 . Peptides were loaded on an Acclaim PepMap RSLC C18 column (150 mm × 75 μm ID, 2 μm particles, 100 Å pore size) (Thermo Fisher Scientific), and eluted with a gradient of solvent B (19.92/80/0.08 v/v/v water/acetonitrile/formic acid) in solvent A (99.9/0.1 v/v water/formic acid), at a flow rate of 300 nl/min 59 .…”

“…Individual protein digests were then analyzed with a nanoLC-ESI-Q-Orbitrap-MS/MS platform consisting of an UltiMate 3000 HPLC RSLC nano system (Thermo Fisher Scientific) coupled to a Q-ExactivePlus mass spectrometer through a Nanoflex ion source (Thermo Fisher Scientific) 59 . Peptides were loaded on an Acclaim PepMap RSLC C18 column (150 mm × 75 μm ID, 2 μm particles, 100 Å pore size) (Thermo Fisher Scientific), and eluted with a gradient of solvent B (19.92/80/0.08 v/v/v water/acetonitrile/formic acid) in solvent A (99.9/0.1 v/v water/formic acid), at a flow rate of 300 nl/min 59 . The gradient of solvent B started at 3%, increased to 40% over 40 min, raised to 80% over 5 min, remained at 80% for 4 min, and finally returned to 3% in 1 min, with a column equilibrating step of 30 min before the subsequent chromatographic run.…”

Mass spectrometry-based protein-protein interaction techniques and their applications in studies of DNA damage repair

“…DNA-(apurinic or apyrimidinic site) lyase (APEX1) functions as the AP endonuclease in the BER pathway. With the same co-IP strategy, an APEX1 protein‒protein interaction network was built, which revealed that the signature of APEX1 interactome is associated with poor prognosis in several types of cancers ( Ayyildiz et al, 2020 ).…”