Intraluminal vesicle trafficking is involved in the secretion of base excision repair protein APE1.

The apurinic/apyrimidinic endodeoxyribonuclease 1 (APE1) is an essential enzyme of the base excision repair pathway of non-distorting DNA lesions. In response to genotoxic treatments, APE1 is highly secreted (sAPE1) in association with small-extracellular vesicles (EVs). Interestingly, its presence in the serum of patients with hepatocellular or non-small-cell-lung cancers may represent a prognostic biomarker. The mechanism driving APE1 to associate with EVs is unknown, but is of paramount importance in better understanding the biological roles of sAPE1. Because APE1 lacks an endoplasmic reticulum-targeting signal peptide, it can be secreted through an unconventional protein secretion endoplasmic reticulum-Golgi-independent pathway, which includes an endosome-based secretion of intraluminal vesicles, mediated by multivesicular bodies (MVBs). Using HeLa and A549 cell lines, we investigated the role of endosomal sorting complex required for transport protein pathways (either-dependent or -independent) in the constitutive or trichostatin A-induced secretion of sAPE1, by means of manumycin A and GW 4869 treatments. Through an in-depth biochemical analysis of late-endosomes (LEs) and early-endosomes (EEs), we observed that the distribution of APE1 on density gradient corresponded to that of LE-CD63, LE-Rab7, EE-EEA1 and EE-Rab 5. Interestingly, the secretion of sAPE1, induced by cisplatin genotoxic stress, involved an autophagy-based unconventional secretion requiring MVBs. The present study enlightens the central role played by MVBs in the secretion of sAPE1 under various stimuli, and offers new perspectives in understanding the biological relevance of sAPE1 in cancer cells.

Light-strand bias and enriched zones of embedded ribonucleotides are associated with DNA replication and transcription in the human-mitochondrial genome.

Abundant ribonucleoside-triphosphate (rNTP) incorporation into DNA by DNA polymerases in the form of ribonucleoside monophosphates (rNMPs) is a widespread phenomenon in nature, resulting in DNA-structural change and genome instability. The rNMP distribution, characteristics, hotspots and association with DNA metabolic processes in human mitochondrial DNA (hmtDNA) remain mostly unknown. Here, we utilize the ribose-seq technique to capture embedded rNMPs in hmtDNA of six different cell types. In most cell types, the rNMPs are preferentially embedded on the light strand of hmtDNA with a strong bias towards rCMPs; while in the liver-tissue cells, the rNMPs are predominately found on the heavy strand. We uncover common rNMP hotspots and conserved rNMP-enriched zones across the entire hmtDNA, including in the control region, which links the rNMP presence to the frequent hmtDNA replication-failure events. We show a strong correlation between coding-sequence size and rNMP-embedment frequency per nucleotide on the non-template, light strand in all cell types, supporting the presence of transient RNA-DNA hybrids preceding light-strand replication. Moreover, we detect rNMP-embedment patterns that are only partly conserved across the different cell types and are distinct from those found in yeast mtDNA. The study opens new research directions to understand the biology of hmtDNA and genomic rNMPs.

Clinical Significance of Apurinic/Apyrimidinic Endodeoxyribonuclease 1 and MicroRNA Axis in Hepatocellular Carcinoma.

Background and Aims: Identification of prognostic factors for hepatocellular carcinoma (HCC) opens new perspectives for therapy. Circulating and cellular onco-miRNAs are noncoding RNAs which can control the expression of genes involved in oncogenesis through post-transcriptional mechanisms. These microRNAs (miRNAs) are considered novel prognostic and predictive factors in HCC. The apurinic/apyrimidinic endodeoxyribonuclease 1 (APE1) contributes to the quality control and processing of specific onco-miRNAs and is a negative prognostic factor in several tumors. The present work aims to: a) define APE1 prognostic value in HCC; b) identify miRNAs regulated by APE1 and their relative target genes and c) study their prognostic value. Methods: We used The Cancer Genome Atlas (commonly known as TCGA) data analysis to evaluate the expression of APE1 in HCC. To identify differentially-expressed miRNAs (DEmiRNAs) upon APE1 depletion through specific small interfering RNA, we used NGS and nanostring approaches in the JHH-6 HCC tumor cell line. Bioinformatics analyses were performed to identify signaling pathways involving APE1-regulated miRNAs. Microarray analysis was performed to identify miRNAs correlating with serum APE1 expression. Results: APE1 is considerably overexpressed in HCC tissues compared to normal liver, according to the TCGA-liver HCC (known as LIHC) dataset. Enrichment analyses showed that APE1-regulated miRNAs are implicated in signaling and metabolic pathways linked to cell proliferation, transformation, and angiogenesis, identifying Cyclin Dependent Kinase 6 and Lysosomal Associated Membrane Protein 2 as targets. miR-33a-5p, miR-769, and miR-877 are related to lower overall survival in HCC patients. Through array profiling, we identified eight circulating DE-miRNAs associated with APE1 overexpression. A training phase identified positive association between sAPE1 and miR-3180-3p and miR-769. Conclusions: APE1 regulates specific miRNAs having prognostic value in HCC.

In Vitro Assay to Measure APE1 Enzymatic Activity on Ribose Monophosphate Abasic Site.

APE1 (apurinic/apyrimidinic endodeoxyribonuclease 1) is a central enzyme of the base excision repair (BER) pathway playing a pivotal role in protecting mammalian cells against genotoxins and in safeguarding genome stability. Recently, we demonstrated the APE1 ability to process abasic ribonucleotides embedded in DNA. Here, we provide a pipeline of protocols to quantify endodeoxyribonuclease activity by APE1 on these substrates, by using recombinant protein and whole-cell extracts. The repair capacity is measured by using fluorescent oligonucleotide substrates, which are then separated by polyacrylamide gel electrophoresis and detected by imaging scanning. The specificity of APE1 action is demonstrated using specific APE1 enzymatic inhibitors.

Revisiting Two Decades of Research Focused on Targeting APE1 for Cancer Therapy: The Pros and Cons.

APE1 is an essential endodeoxyribonuclease of the base excision repair pathway that maintains genome stability. It was identified as a pivotal factor favoring tumor progression and chemoresistance through the control of gene expression by a redox-based mechanism. APE1 is overexpressed and serum-secreted in different cancers, representing a prognostic and predictive factor and a promising non-invasive biomarker. Strategies directly targeting APE1 functions led to the identification of inhibitors showing potential therapeutic value, some of which are currently in clinical trials. Interestingly, evidence indicates novel roles of APE1 in RNA metabolism that are still not fully understood, including its activity in processing damaged RNA in chemoresistant phenotypes, regulating onco-miRNA maturation, and oxidized RNA decay. Recent data point out a control role for APE1 in the expression and sorting of onco-miRNAs within secreted extracellular vesicles. This review is focused on giving a portrait of the pros and cons of the last two decades of research aiming at the identification of inhibitors of the redox or DNA-repair functions of APE1 for the definition of novel targeted therapies for cancer. We will discuss the new perspectives in cancer therapy emerging from the unexpected finding of the APE1 role in miRNA processing for personalized therapy.

Design and advanced characterization of quercetin-loaded nano-liposomes prepared by high-pressure homogenization.

Quercetin-loaded nano-liposomes were prepared by high-pressure homogenization (HPH) at different pressures (up to 150 MPa) and number of passes (up to 3) to define the best processing conditions allowing the lowest particle size and the highest encapsulation efficiency (EE). The process at 150 MPa for 1 pass was the best, producing quercetin-loaded liposomes with the lowest particle size and 42% EE. Advanced techniques (multi-detector asymmetrical-flow field flow fractionation and analytical ultracentrifugation combined with transmission electron microscopy) were further used for the characterization of the liposomes which were oblong in shape (ca. 30 nm). Results highlight the need for several techniques to study nano-sized, polydisperse samples. The potential of quercetin-loaded liposomes against colon cancer cells was demonstrated. Results prove that HPH is an efficient and sustainable method for liposome preparation and highlight the remarkable role of process optimisation as well as the powerfulness of advanced methodologies for the characterisation of nano-structures.

Role of condensates in modulating DNA repair pathways and its implication for chemoresistance.

For cells, it is important to repair DNA damage, such as double-strand and single-strand DNA breaks, because unrepaired DNA can compromise genetic integrity, potentially leading to cell death or cancer. Cells have multiple DNA damage repair pathways that have been the subject of detailed genetic, biochemical, and structural studies. Recently, the scientific community has started to gain evidence that the repair of DNA double-strand breaks may occur within biomolecular condensates and that condensates may also contribute to DNA damage through concentrating genotoxic agents used to treat various cancers. Here, we summarize key features of biomolecular condensates and note where they have been implicated in the repair of DNA double-strand breaks. We also describe evidence suggesting that condensates may be involved in the repair of other types of DNA damage, including single-strand DNA breaks, nucleotide modifications (e.g., mismatch and oxidized bases), and bulky lesions, among others. Finally, we discuss old and new mysteries that could now be addressed considering the properties of condensates, including chemoresistance mechanisms.

AUF1 Recognizes 8-Oxo-Guanosine Embedded in DNA and Stimulates APE1 Endoribonuclease Activity.

Aims: The existence of modified ribonucleotide monophosphates embedded in genomic DNA, as a consequence of oxidative stress conditions, including 8-oxo-guanosine and ribose monophosphate abasic site (rAP), has been recently highlighted by several works and associated with oxidative stress conditions. Although human apurinic-apyrimidinic endodeoxyribonuclease 1 (APE1), a key enzyme of the base-excision repair pathway, repairs rAP sites and canonical deoxyribose monophosphate abasic sites with similar efficiency, its incision-repairing activity on 8-oxo-guanosine is very weak. The aims of this work were to: (i) identify proteins able to specifically bind 8-oxo-guanosine embedded in DNA and promote APE1 endoribonuclease activity on this lesion, and (ii) characterize the molecular and biological relevance of this interaction using human cancer cell lines. Results: By using an unbiased proteomic approach, we discovered that the AU-rich element RNA-binding protein 1 (AUF1) actively recognizes 8-oxo-guanosine and stimulates the APE1 enzymatic activity on this DNA lesion. By using orthogonal approaches, we found that: (i) the interaction between AUF1 and APE1 is modulated by H2O2-treatment; (ii) depletion of APE1 and AUF1 causes the accumulation of single- and double- strand breaks; and (iii) both proteins are involved in modulating the formation of DNA:RNA hybrids. Innovation: These results establish unexpected functions of AUF1 in modulating genome stability and improve our knowledge of APE1 biology with respect to 8-oxo-guanosine embedded in DNA. Conclusion: By showing a novel function of AUF1, our findings shed new light on the process of genome stability in mammalian cells toward oxidative stress-related damages. Antioxid. Redox Signal. 39, 411-431.

APE1 interacts with the nuclear exosome complex protein MTR4 and is involved in cisplatin- and 5-fluorouracil-induced RNA damage response.

The nuclear RNA surveillance mechanism is essential for cancer cell survival and is ensured by the RNA nuclear exosome including some co-factors, such as the RNA helicase MTR4. Recent studies suggest an involvement of DNA repair proteins such as apurinic/apyrimidinic (AP) endodeoxyribonuclease 1 (APE1), a major endodeoxyribonuclease of Base Excision Repair (BER), in RNA metabolism and RNA decay of oxidized and abasic RNA. Cisplatin (CDDP) and 5-fluorouracil (5-FU) are commonly used for the treatment of solid tumours. Whether APE1 is involved in the elimination of CDDP- or 5-FU-damaged RNA is unknown, as is its possible interaction with the nuclear exosome complex. Here, by using different human cancer cell models, we demonstrated that: (a) APE1 is involved in the elimination of damaged-RNA, upon CDDP- and 5-FU-treatments, in a MTR4-independent manner; (b) the interaction between APE1 and MTR4 is stimulated by CDDP- and 5-FU-treatments through lysine residues in the APE1 N-terminal region and is, in part, mediated by nucleic acids and (c) APE1- and MTR4-depletion lead to the generation of R-loop formation causing the activation of the DNA damage response (DDR) pathway through the ATM-p53-p21 axis. Our data demonstrate a role of MTR4 in DDR underpinning the function of APE1 in controlling the RNA quality upon genotoxic treatments with possible implications in chemoresistance.

A regulatory network comprising let-7 miRNA and SMUG1 is associated with good prognosis in ER+ breast tumours.

Single-strand selective uracil-DNA glycosylase 1 (SMUG1) initiates base excision repair (BER) of uracil and oxidized pyrimidines. SMUG1 status has been associated with cancer risk and therapeutic response in breast carcinomas and other cancer types. However, SMUG1 is a multifunctional protein involved, not only, in BER but also in RNA quality control, and its function in cancer cells is unclear. Here we identify several novel SMUG1 interaction partners that functions in many biological processes relevant for cancer development and treatment response. Based on this, we hypothesized that the dominating function of SMUG1 in cancer might be ascribed to functions other than BER. We define a bad prognosis signature for SMUG1 by mapping out the SMUG1 interaction network and found that high expression of genes in the bad prognosis network correlated with lower survival probability in ER+ breast cancer. Interestingly, we identified hsa-let-7b-5p microRNA as an upstream regulator of the SMUG1 interactome. Expression of SMUG1 and hsa-let-7b-5p were negatively correlated in breast cancer and we found an inhibitory auto-regulatory loop between SMUG1 and hsa-let-7b-5p in the MCF7 breast cancer cells. We conclude that SMUG1 functions in a gene regulatory network that influence the survival and treatment response in several cancers.

Combining Deep Phenotyping of Serum Proteomics and Clinical Data via Machine Learning for COVID-19 Biomarker Discovery.

The persistence of long-term coronavirus-induced disease 2019 (COVID-19) sequelae demands better insights into its natural history. Therefore, it is crucial to discover the biomarkers of disease outcome to improve clinical practice. In this study, 160 COVID-19 patients were enrolled, of whom 80 had a "non-severe" and 80 had a "severe" outcome. Sera were analyzed by proximity extension assay (PEA) to assess 274 unique proteins associated with inflammation, cardiometabolic, and neurologic diseases. The main clinical and hematochemical data associated with disease outcome were grouped with serological data to form a dataset for the supervised machine learning techniques. We identified nine proteins (i.e., CD200R1, MCP1, MCP3, IL6, LTBP2, MATN3, TRANCE, α2-MRAP, and KIT) that contributed to the correct classification of COVID-19 disease severity when combined with relative neutrophil and lymphocyte counts. By analyzing PEA, clinical and hematochemical data with statistical methods that were able to handle many variables in the presence of a relatively small sample size, we identified nine potential serum biomarkers of a "severe" outcome. Most of these were confirmed by literature data. Importantly, we found three biomarkers associated with central nervous system pathologies and protective factors, which were downregulated in the most severe cases.

APE1 controls DICER1 expression in NSCLC through miR-33a and miR-130b.

Increasing evidence suggests different, not completely understood roles of microRNA biogenesis in the development and progression of lung cancer. The overexpression of the DNA repair protein apurinic/apyrimidinic endodeoxyribonuclease 1 (APE1) is an important cause of poor chemotherapeutic response in lung cancer and its involvement in onco-miRNAs biogenesis has been recently described. Whether APE1 regulates miRNAs acting as prognostic biomarkers of lung cancer has not been investigated, yet. In this study, we analyzed miRNAs differential expression upon APE1 depletion in the A549 lung cancer cell line using high-throughput methods. We defined a signature of 13 miRNAs that strongly correlate with APE1 expression in human lung cancer: miR-1246, miR-4488, miR-24, miR-183, miR-660, miR-130b, miR-543, miR-200c, miR-376c, miR-218, miR-146a, miR-92b and miR-33a. Functional enrichment analysis of this signature revealed its biological relevance in cancer cell proliferation and survival. We validated DICER1 as a direct functional target of the APE1-regulated miRNA-33a-5p and miR-130b-3p. Importantly, IHC analyses of different human tumors confirmed a negative correlation existing between APE1 and Dicer1 protein levels. DICER1 downregulation represents a prognostic marker of cancer development but the mechanisms at the basis of this phenomenon are still completely unknown. Our findings, suggesting that APE1 modulates DICER1 expression via miR-33a and miR-130b, reveal new mechanistic insights on DICER1 regulation, which are of relevance in lung cancer chemoresistance and cancer invasiveness.

Brain Exosomes: Friend or Foe in Alzheimer's Disease?

Alzheimer's disease (AD) is the most common neurodegenerative disease. It is known to be a multifactorial disease and several causes are associated with its occurrence as well as progression. However, the accumulation of amyloid beta (Aß) is widely considered its major pathogenic hallmark. Additionally, neurofibrillary tangles (NFT), mitochondrial dysfunction, oxidative stress, and aging (cellular senescence) are considered as additional hits affecting the disease pathology. Several studies are now suggesting important role of inflammation in AD, which shifts our thought towards the brain's resident immune cells, microglia, and astrocytes; how they interact with neurons; and how these interactions are affected by intra and extracellular stressful factors. These interactions can be modulated by different mechanisms and pathways, in which exosomes could play an important role. Exosomes are multivesicular bodies secreted by nearly all types of cells. The exosomes secreted by glial cells or neurons affect the interactions and thus the physiology of these cells by transmitting miRNAs, proteins, and lipids. Exosomes can serve as a friend or foe to the neuron function, depending upon the carried signals. Exosomes, from the healthy microenvironment, may assist neuron function and health, whereas, from the stressed microenvironment, they carry oxidative and inflammatory signals to the neurons and thus prove detrimental to the neuronal function. Furthermore, exosomes can cross the blood-brain barrier (BBB), and from the blood plasma they can enter the brain cells and activate microglia and astrocytes. Exosomes can transport Aß or Tau, cytokines, miRNAs between the cells, and alter the physiology of recipient cells. They can also assist in Aß clearance and regulation of synaptic activity. The exosomes derived from different cells play different roles, and this field is still in its infancy stage. This review advocates exosomes' role as a friend or foe in neurodegenerative diseases, especially in the case of Alzheimer's disease.

Integrated multi-omics analyses on patient-derived CRC organoids highlight altered molecular pathways in colorectal cancer progression involving PTEN.

BACKGROUND: Colorectal cancer (CRC) represents the fourth leading cause of cancer-related deaths. The heterogeneity of CRC identity limits the usage of cell lines to study this type of tumor because of the limited representation of multiple features of the original malignancy. Patient-derived colon organoids (PDCOs) are a promising 3D-cell model to study tumor identity for personalized medicine, although this approach still lacks detailed characterization regarding molecular stability during culturing conditions. Correlation analysis that considers genomic, transcriptomic, and proteomic data, as well as thawing, timing, and culturing conditions, is missing. METHODS: Through integrated multi-omics strategies, we characterized PDCOs under different growing and timing conditions, to define their ability to recapitulate the original tumor. RESULTS: Whole Exome Sequencing allowed detecting temporal acquisition of somatic variants, in a patient-specific manner, having deleterious effects on driver genes CRC-associated. Moreover, the targeted NGS approach confirmed that organoids faithfully recapitulated patients' tumor tissue. Using RNA-seq experiments, we identified 5125 differentially expressed transcripts in tumor versus normal organoids at different time points, in which the PTEN pathway resulted of particular interest, as also confirmed by further phospho-proteomics analysis. Interestingly, we identified the PTEN c.806_817dup (NM_000314) mutation, which has never been reported previously and is predicted to be deleterious according to the American College of Medical Genetics and Genomics (ACMG) classification. CONCLUSION: The crosstalk of genomic, transcriptomic and phosphoproteomic data allowed to observe that PDCOs recapitulate, at the molecular level, the tumor of origin, accumulating mutations over time that potentially mimic the evolution of the patient's tumor, underlining relevant potentialities of this 3D model.

Coping with RNA damage with a focus on APE1, a BER enzyme at the crossroad between DNA damage repair and RNA processing/decay.

Interest in RNA damage as a novel threat associated with several human pathologies is rapidly increasing. Knowledge on damaged RNA recognition, repair, processing and decay is still scanty. Interestingly, in the last few years, more and more evidence put a bridge between DNA damage repair enzymes and the RNA world. The Apurinic/apyrimidinic endodeoxyribonuclease 1 (APE1) was firstly identified as a crucial enzyme of the base excision repair (BER) pathway preserving genome stability toward non-distorting DNA lesion-induced damages. Later, an unsuspected role of APE1 in controlling gene expression was discovered and its pivotal involvement in several human pathologies, ranging from tumor progression to neurodegenerative diseases, has emerged. Recent novel findings indicate a role of APE1 in RNA metabolism, particularly in processing activities of damaged (abasic and oxidized) RNA and in the regulation of oncogenic microRNAs (miRNAs). Even though the role of miRNAs in human pathologies is well-known, the mechanisms underlying their quality control are still totally unexplored. A detailed knowledge of damaged RNA decay processes in human cells is crucial in order to understand the molecular processes involved in multiple pathologies. This cutting-edge perspective article will highlight these emerging aspects of damaged RNA processing and decay, focusing the attention on the involvement of APE1 in RNA world.

Enzymatically active apurinic/apyrimidinic endodeoxyribonuclease 1 is released by mammalian cells through exosomes.

The apurinic/apyrimidinic endodeoxyribonuclease 1 (APE1), the main AP-endonuclease of the DNA base excision repair pathway, is a key molecule of interest to researchers due to its unsuspected roles in different nonrepair activities, such as: i) adaptive cell response to genotoxic stress, ii) regulation of gene expression, and iii) processing of microRNAs, which make it an excellent drug target for cancer treatment. We and others recently demonstrated that APE1 can be secreted in the extracellular environment and that serum APE1 may represent a novel prognostic biomarker in hepatocellular and non-small-cell lung cancers. However, the mechanism by which APE1 is released extracellularly was not described before. Here, using three different approaches for exosomes isolation: commercial kit, nickel-based isolation, and ultracentrifugation methods and various mammalian cell lines, we elucidated the mechanisms responsible for APE1 secretion. We demonstrated that APE1 p37 and p33 forms are actively secreted through extracellular vesicles (EVs), including exosomes from different mammalian cell lines. We then observed that APE1 p33 form is generated by proteasomal-mediated degradation and is enzymatically active in EVs. Finally, we revealed that the p33 form of APE1 accumulates in EVs upon genotoxic treatment by cisplatin and doxorubicin, compounds commonly found in chemotherapy pharmacological treatments. Taken together, these findings provide for the first time evidence that a functional Base Excision Repair protein is delivered through exosomes in response to genotoxic stresses, shedding new light into the complex noncanonical biological functions of APE1 and opening new intriguing perspectives on its role in cancer biology.

Differential capacity of CD90+ cells in autophagy activation following chemotherapy in hepatocellular carcinoma.

INTRODUCTION AND OBJECTIVES: Analysis of cancer biomarkers is an important tool in developing targeted-therapy and in modulating chemoresistance. Here, we analyze the relevance of CD90, a marker of cancer stem cells (CSC) in hepatocellular carcinoma (HCC) and its correlation with autophagy. MATERIALS AND METHODS: For in vivo study, 86 specimens were collected from 43 patients undergoing liver resections. In each patient, HCC nodule (HCC) and surrounding non-tumor (SNT) were collected. For in vitro study, HCC cells JHH6 subpopulations expressing CD90+ and CD90- were isolated using magnetic-sorter and confirmed by flow-cytometry. Upon doxorubicin treatment, autophagy turn-over was analyzed by RTqPCR for mRNA expression, Western blot for protein expression, and autophagosome staining for autophagy-flux. Cytotoxicity test was performed by MTT assay. Gene and protein analysis were performed in clinical samples together with immunohistostaining. RESULTS: CD90 mRNA expression was higher in HCC than in SNT for 8-fold (p < 0.001). LC3-II protein was up-regulated in the HCC in comparison with the SNT (p < 0.05). In vitro model showed that CD90+ and CD90- cells had diverse expressions of autophagy-related genes. Upon doxorubicin treatment, autophagy was activated in both cells by increasing LC3-II protein expression, autophagic vacuoles, and dysregulation of autophagy-related mRNAs. A differential autophagic capacity was noticed between two subpopulations and it was correlated with cellular toxicity assay. CONCLUSIONS: We demonstrated the relevance of differential autophagy capacity of CD90+ cells in HCC. Autophagy was involved in cancer-defense mechanism against doxorubicin. Cancer promoting function of autophagy in CD90+ cells was also related to cancer environment.

Cleavage of the APE1 N-Terminal Domain in Acute Myeloid Leukemia Cells Is Associated with Proteasomal Activity.

Apurinic/apyrimidinic endonuclease 1 (APE1), the main mammalian AP-endonuclease for the resolution of DNA damages through the base excision repair (BER) pathway, acts as a multifunctional protein in different key cellular processes. The signals to ensure temporo-spatial regulation of APE1 towards a specific function are still a matter of debate. Several studies have suggested that post-translational modifications (PTMs) act as dynamic molecular mechanisms for controlling APE1 functionality. Interestingly, the N-terminal region of APE1 is a disordered portion functioning as an interface for protein binding, as an acceptor site for PTMs and as a target of proteolytic cleavage. We previously demonstrated a cytoplasmic accumulation of truncated APE1 in acute myeloid leukemia (AML) cells in association with a mutated form of nucleophosmin having aberrant cytoplasmic localization (NPM1c+). Here, we mapped the proteolytic sites of APE1 in AML cells at Lys31 and Lys32 and showed that substitution of Lys27, 31, 32 and 35 with alanine impairs proteolysis. We found that the loss of the APE1 N-terminal domain in AML cells is dependent on the proteasome, but not on granzyme A/K as described previously. The present work identified the proteasome as a contributing machinery involved in APE1 cleavage in AML cells, suggesting that acetylation can modulate this process.

Architecture of The Human Ape1 Interactome Defines Novel Cancers Signatures.

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.

Role of phase partitioning in coordinating DNA damage response: focus on the Apurinic Apyrimidinic Endonuclease 1 interactome.

Liquid-liquid phase separation (LLPS) is a way to concentrate biochemical reactions while excluding noninteracting components. Disordered domains of proteins, as well as interaction with RNA, favor condensation but are not mandatory for modulating this process. Recent insights about phase-separation mechanisms pointed to new fascinating models that could explain how cells could cope with DNA damage responses, conferring both spatial and temporal fine regulation. APE1 is a multifunctional protein belonging to the Base Excision Repair (BER) pathway, bearing additional 'non-canonical' DNA-repair functions associated with processes like RNA metabolism. Recently, it has been highlighted that several DNA repair enzymes, such as 53BP1 and APE1, are endowed with RNA binding abilities. In this work, after reviewing the recent literature supporting a role of LLPS in DDR, we analyze, as a proof of principle, the interactome of APE1 using a bioinformatics approach to look for clues of LLPS in BER. Some of the APE1 interactors are associated with cellular processes in which LLPS has been either proved or proposed and are involved in different pathogenic events. This work might represent a paradigmatical pipeline for evaluating the relevance of LLPS in DDR.