Apurinic/apyrimidinic Endodeoxyribonuclease 1 Research Articles

Apurinic/apyrimidinic endodeoxyribonuclease 1 (APE1) promotes stress granule formation via YBX1 phosphorylation in ovarian cancer

APE1 is an essential gene involved in DNA damage repair, the redox regulation of transcriptional factors (TFs) and RNA processing. APE1 overexpression is common in cancers and correlates with poor patient survival. Stress granules (SGs) are phase-separated cytoplasmic assemblies that cells form in response to environmental stresses. Precise regulation of SGs is pivotal to cell survival, whereas their dysregulation is increasingly linked to diseases. Whether APE1 engages in modulating SG dynamics is worthy of investigation. In this study, we demonstrate that APE1 colocalizes with SGs and promotes their formation. Through phosphoproteome profiling, we discover that APE1 significantly alters the phosphorylation landscape of ovarian cancer cells, particularly the phosphoprofile of SG proteins. Notably, APE1 promotes the phosphorylation of Y-Box binding protein 1 (YBX1) at S174 and S176, leading to enhanced SG formation and cell survival. Moreover, expression of the phosphomutant YBX1 S174/176E mimicking hyperphosphorylation in APE1-knockdown cells recovered the impaired SG formation. These findings shed light on the functional importance of APE1 in SG regulation and highlight the importance of YBX1 phosphorylation in SG dynamics.

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 GW4869 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.

Development of a DNAzyme Walker for the Detection of APE1 in Living Cancer Cells.

DNAzyme walker technology is a compelling option for bioanalytical and drug delivery applications. While nucleic acid and protein targets have been used to activate DNAzyme walkers, investigations into enzyme-triggered DNAzyme walkers in living cells are still in their early stages. The base excision repair (BER) pathway presents an array of enzymes that are overexpressed in cancer cells. Here, we introduce a DNAzyme walker system that sensitively and specifically detects the BER enzyme apurinic/apyrimidinic endodeoxyribonuclease 1 (APE1). We constructed the DNAzyme walker on the surface of 20 nm-diameter gold nanoparticles. We achieved a detection limit of 160 fM of APE1 in a buffer and in whole cell lysate equivalent to the amount of APE1 in a single HeLa cell in a sample volume of 100 μL. Confocal imaging of the DNAzyme walking reveals a cytoplasmic distribution of APE1 in HeLa cells. Walking activity is tunable to exogenous Mn2+ concentrations and the uptake of the DNAzyme walker system does not require transfection assistance. We demonstrate the investigative potential of the DNAzyme walker for up-regulated or overactive enzyme biomarkers of the BER pathway in cancer cells.

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

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. 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. 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. APE1 regulates specific miRNAs having prognostic value in HCC.

Co-delivery of siAPE1 and melatonin by 125I-loaded PSMA-targeted nanoparticles for the treatment of prostate cancer

Background: Both apurinic/apyrimidinic endodeoxyribonuclease 1 (APE1) inhibition and melatonin suppress prostate cancer (PCa) growth. This study evaluated the therapeutic efficiency of self-assembled and prostate-specific membrane antigen (PSMA)-targeted nanocarrier loading 125I radioactive particles and encapsulating siRNA targeting APE1 (siAPE1) and melatonin for PCa. Methods: The linear polyarginine R12 polypeptide was prepared using Fmoc-Arg-Pbf-OH. The PSMA-targeted polymer was synthesized by conjugating azide-modified R12 peptide to PSMA monoclonal antibody (mAb). Before experiments, the PSMA-R12 nanocarrier was installed with melatonin and siAPE1, which were subsequently labeled by 125I radioactive particles. In vitro biocompatibility and cytotoxicity of nanocomposites were examined in LNCaP cells and in vivo biodistribution and pharmacokinetics were determined using PCa tumor-bearing mice. Results: PSMA-R12 nanocarrier was ~120 nm in size and was increased to ~150 nm by melatonin encapsulation. PSMA-R12 nanoparticles had efficient loading capacities of siAPE1, melatonin, and 125I particles. The co-delivery of melatonin and siAPE1 by PSMA-R12-125I showed synergistic effects on suppressing LNCaP cell proliferation and Bcl-2 expression and promoting cell apoptosis and caspase-3 expression. Pharmacokinetics analysis showed that Mel@PSMA-R12-125I particles had high uptake activity in the liver, spleen, kidney, intestine, and tumor, and were accumulated in the tumor sites within the first 8 h p.i., but was rapidly cleared from all the tested organs at 24 h p.i. Administration of nanoparticles to PCa tumors in vivo showed that Mel@PSMA-R12-125I/siAPE1 had high efficiency in suppressing PCa tumor growth. Conclusion: The PSMA-targeted nanocarrier encapsulating siAPE1 and melatonin is a promising therapeutic strategy for PCa.

AUF1 recognizes 8-oxo-guanosine embedded in DNA and stimulates APE1 endoribonuclease activity.

The existence of modified rNMPs 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 including ours. Although the activity of APE1, a key enzyme of the base excision repair pathway, in the repair of rAP sites results as efficient as the canonical deoxyribose monophosphate abasic sites (dAP), its incision repair 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 on this lesion and ii) characterize the molecular and biological relevance of this interaction using human cancer cell lines. By using an unbiased proteomic approach, we discovered that 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; iii) both proteins are involved in modulating the formation of DNA:RNA hybrids. These data 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. By showing a novel function of AUF1, our findings shed new light on the process of maintenance of genome stability in mammalian cells towards oxidative stress-related damages.

Essential oil of lemon myrtle (Backhousia citriodora) induces S-phase cell cycle arrest and apoptosis in HepG2 cells

Ethnopharmacological relevanceLemon myrtle (Backhousia citriodora F.Muell.) leaves, whether fresh or dried, are used traditionally in folk medicine to treat wounds, cancers, skin infections, and other infectious conditions. However, the targets and mechanisms related to anti-cancer effect of lemon myrtle are unavailable. In our study, we found that the essential oil of lemon myrtle (LMEO) showed anti-cancer activity in vitro, and we initially explored its mechanism of action. Materials and methodsWe analyzed the chemical compositions of LMEO by GC-MS. We tested the cytotoxicity of LMEO on various cancer cell lines using the MTT assay. Network pharmacology was used also to analyze the targets of LMEO. Moreover, the mechanisms of LMEO were investigated through scratch assay, flow cytometry analysis, and western blot in the HepG2 liver cancer cell line. ResultsLMEO showed cytotoxicity on various cancer cell lines with values of IC50 40.90 ± 2.23 (liver cancer HepG2 cell line), 58.60 ± 6.76 (human neuroblastoma SH-SY5Y cell line), 68.91 ± 4.62 (human colon cancer HT-29 cell line) and 57.57 ± 7.61 μg/mL (human non-small cell lung cancer A549 cell line), respectively. The major cytotoxic chemical constituent in LMEO was identified as citrals, which accounted for 74.9% of the content. Network pharmacological analysis suggested that apurinic/apyrimidinic endodeoxyribonuclease 1 (APEX1), androgen receptor (AR), cyclin-dependent kinases 1 (CDK1), nuclear factor erythroid 2-related factor 2 (Nrf-2), fatty acid synthase (FASN), epithelial growth factor receptor (EGFR), estrogen receptor 1 (ERα) and cyclin-dependent kinases 4 (CDK4) are potential cytotoxic targets of LMEO. These targets are closely related to cell migration, cycle and apoptosis. Notley, the p53 protein had the highest confidence to co-associate with the eight common targets, which was further confirmed by scratch assay, flow cytometry analysis, and western blot in the HepG2 liver cancer cell line. LMEO significantly inhibited the migration of HepG2 cells in time-dependent and dose-dependent manner. Moreover, LMEO caused a S-phase blocking on HepG2 cells and promoted apoptosis in the meanwhile. Western blot results indicated that p53 protein, Cyclin A2 and Bax proteins were up-regulated, while Cyclin E1 and Bcl-2 proteins were down-regulated. ConclusionLMEO showed cytotoxicity in various cancer cell lines in vitro. Pharmacological networks showed LMEO to have multi-component and multi-targeting effects that are related to inhibit migration of HepG2 cells, and affect cell cycle S-phase arrest and apoptosis through modulation of p53 protein.

SIRT1/APE1 promotes the viability of gastric cancer cells by inhibiting p53 to suppress ferroptosis.

Gastric cancer (GC) is a common cancer worldwide with high mortality. Sirtuin 1 (SIRT1) and apurinic/apyrimidinic endodeoxyribonuclease 1 (APE1) are abnormally expressed in GC cells and related to p53, which is involved in ferroptosis. Thus, we explore the mechanism via which SIRT1, APE1, and p53 impact ferroptosis in GC cells. Specifically, GC cells were transfected with small-interfering RNA for SIRT1 (SiSIRT1) or small-interfering RNA for APE1 (SiAPE1) or with short-hairpin RNA for p53, and the cell viability, Fe2+, malondialdehyde (MDA), and glutathione (GSH) contents were detected by cell counting kit-8 assay and enzyme-linked immunosorbent assay. Western blot, immunofluorescence, and quantitative real-time polymerase chain reaction were conducted to quantify SIRT1, APE1, p53, solute carrier family 7 member 11 (SLC7A11), and glutathione peroxidase 4 (GPX4) levels in GC cells. Silencing of SIRT1 decreased viability, GSH content, and expressions of GPX4 and SLC7A11, while increased Fe2+, MDA content, and p53 expression in GC cells. Such aforementioned effects were reversed by APE1 overexpression. Also, SiAPE1 generated the same effects as SiSIRT1 on the above aspects, which was offset by p53 silencing. In short, SIRT1/APE1 promotes the growth of GC cells by targeting p53 to inhibit ferroptosis.

Low level of BRCA2 in peripheral blood lymphocytes is associated with an increased risk for head and neck squamous cell carcinoma (HNSCC) in a population of North-East India: a case-control study.

We investigated the role of DNA repair proteins breast cancer susceptibility 2 (BRCA2), xeroderma pigmentosum group D (XPD) and apurinic/apyrimidinic endodeoxyribonuclease 1 (APE1) in determining the risk for head and neck squamous cell cancer (HNSCC) in a case-control study from North-East India. Expression of BRCA2, XPD and APE1 genes in the matched tumour, normal adjacent tissue (NAT) and blood of 12 HNSCC patients and blood of 8 age- and gender-matched controls was determined by quantitative real-time PCR. Results were validated by expression analysis of the corresponding proteins in the peripheral blood lymphocytes (PBLs) of 228 subjects (106 patients and 122 controls) by a slot-blot immunoassay. Expression of the BRCA2 and XPD genes in tumour tissue of HNSCC patients declined progressively as the cancer stage advanced, was reverse that of the NAT, but was mirrored by the expression in the blood. BRCA2 and XPD proteins were significantly (p < 0.0001) downregulated in the PBL of HNSCC patients to 71% and 77% the levels in controls, showing significant negative correlation with HNSCC stage (Spearman correlation coefficient (r s) of -0.9060, p < 0.0001 for BRCA2; r s of -0.8008, p < 0.01 for XPD). On the contrary, APE1 was significantly upregulated in PBL of HNSCC patients to 1.47 fold the level in controls, showing significant positive correlation with HNSCC stage (r s of 0.7023, p < 0.01). Classification and regression tree analyses predicted low levels of BRCA2 protein in PBL as the single most important risk factor for HNSCC, independent of gender. Smokers above 36 years of age with low level of BRCA2 appeared to exhibit a 1.78-fold increased risk for HNSCC (with a 1.78-fold increased risk for HNSCC (OR = 1.78, 95% confidence interval (CI) = 0.33-9.52) though this risk was not significant statistically. Similarly, low levels of BRCA2 appeared to indicate a moderate, but non-significant risk for HNSCC in non-smokers aged between 36 and 56 years (OR = 1.15, 95% CI = 0.21-6.37). Low level of BRCA2 protein in the peripheral blood indicates increased risk for HNSCC.

NEDD4 attenuates oxidized low‑density lipoprotein‑induced inflammation and dysfunction in vascular endothelial cells via regulating APEX1 expression.

Atherosclerosis chiefly results from inflammation as well as vascular endothelial cell dysfunction. Methylation levels of neuronally expressed developmentally downregulated 4 (NEDD4) were found to be fortified in atherosclerosis patients and NEDD4 deficiency enhanced vascular calcification. However, the exact function of NEDD4 in inflammation and vascular endothelial dysfunction remains to be elucidated. In the present study, CCK-8 assay was used to estimate cell viability. Reverse transcription-quantitative PCR was adopted to examine the expression of NEDD4, inflammation-associated enzymes and apurinic/apyrimidinic endodeoxyribonuclease 1 (APEX1). Western blotting was used to test NEDD4, endothelial nitric oxide synthase, inducible nitric oxide synthase and APEX1 protein levels. Cytotoxicity was detected by a lactate dehydrogenase (LDH) kit. Reactive oxygen species level was tested by a corresponding kit. Vascular cell adhesion molecule 1 and intercellular adhesion molecule 1 contents were examined with ELISA. Cell adhesion assays evaluated the adhesion of endothelial cells. Co-immunoprecipitation assay was used to test the relationship between NEDD4 and APEX1. The data revealed that NEDD4 expression rapidly declined in oxidized low density lipoprotein (ox-LDL)-induced human umbilical vein endothelial cells (HUVECs). Following NEDD4 overexpression, the active damage, inflammatory release and endothelial cell dysfunction in ox-LDL-induced HUVECs were attenuated. After co-transfection of APEX1 interference plasmids and NEDD4 overexpression plasmids, cell damage, inflammatory release and endothelial cell dysfunction in ox-LDL-induced HUVECs were improved again. Taken together, NEDD4 attenuated ox-LDL-induced inflammation and endothelial dysfunction by regulating APEX1 expression.

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 mammaliancells 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.

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.

Silver nanoparticles induced testicular damage targeting NQO1 and APE1 dysregulation, apoptosis via Bax/Bcl-2 pathway, fibrosis via TGF-β/α-SMA upregulation in rats

In medicine, silver nanoparticles (AgNPs) are employed often. They do, however, have negative impacts, particularly on the reproductive organs. This research aimed to assess AgNP impact on the testis and the possible intracellular mechanisms to induce testicular deteriorations in rats at various concentrations and different time intervals. Sprague Dawley rats (n = 40) were allocated into four equal groups: the control one, and three other groups injected intra-peritoneally with AgNP solution 0.25, 0.5, and 1 mg/kg b.w. respectively for 15 and 30 days. Our findings revealed that AgNPs reduced body and testicular weights, estradiol (E2) and testosterone (T) hormone levels, and sperm parameters while elevating the nitric oxide and malondialdehyde levels with inhibition of reduced glutathione contents in testicular tissue. Interestingly, AgNPs significantly upregulated the testicular inducible nitric oxide synthase, B cell lymphoma 2 (Bcl-2)-associated X, transforming growth factor, and alpha-smooth muscle actin (α-SMA) expression levels. However, apurinic/apyrimidinic endo deoxyribonuclease 1 (APE1), NAD (P) H quinone dehydrogenase 1 (NQO1), and Bcl-2 expression levels were all downregulated indicating exhaustion of body antioxidant and repairing defense mechanisms in testicles in comparison with the control rats. Various histological alterations were also detected which dramatically increased in rats sacrificed after 30 days such as loss of the lining cells of seminiferous tubules with no spermatozoa and tubular irregularities associated with thickening of their basement membranes. Immunolabeling implicated in the apoptotic pathway revealed a negative expression of Bcl-2 and marked immunoreactivity for caspase-3 after 30 days of AgNP treatment in comparison to the control rats. To our knowledge, there have been no previous publications on the role of the α-SMA, APE1, and NQO1 genes in the molecular pathogenesis of AgNP testicular cytotoxicity following AgNP acute and chronic exposure.Graphical

Space microgravity improves proliferation of human iPSC-derived cardiomyocytes.

SummaryIn microgravity, cells undergo profound changes in their properties. However, how human cardiac progenitors respond to space microgravity is unknown. In this study, we evaluated the effect of space microgravity on differentiation of human induced pluripotent stem cell (hiPSC)-derived cardiac progenitors compared with 1G cultures on the International Space Station (ISS). Cryopreserved 3D cardiac progenitors were cultured for 3 weeks on the ISS. Compared with 1G cultures, the microgravity cultures had 3-fold larger sphere sizes, 20-fold higher counts of nuclei, and increased expression of proliferation markers. Highly enriched cardiomyocytes generated in space microgravity showed improved Ca2+ handling and increased expression of contraction-associated genes. Short-term exposure (3 days) of cardiac progenitors to space microgravity upregulated genes involved in cell proliferation, survival, cardiac differentiation, and contraction, consistent with improved microgravity cultures at the late stage. These results indicate that space microgravity increased proliferation of hiPSC-cardiomyocytes, which had appropriate structure and function.

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.

Nuclear translocation of RIG-I promotes cellular apoptosis

Cell death is important in the elimination of damaged cells such as virus-infected cells and also is closely involved in the pathogenesis of autoimmune diseases such as systemic lupus erythematosus (SLE). The retinoic acid-inducible gene-I (RIG-I), one cytosolic RNA innate sensor, can trigger antiviral innate response by inducing production of type I interferons (IFN-I). However, the function of RIG-I, once translocated from cytoplasm to nucleus at the late stage of viral infection when IFN-I production is almost terminated, remains poorly understood. Here, we reported that RIG-I is accumulated in the nucleus of macrophages and fibroblasts after virus infection, and nuclear RIG-I is present in peripheral blood mononuclear cells (PBMCs) from SLE patients. We found that nuclear RIG-I interacts with the first 20 amino acids of apurinic/apyrimidinic endodeoxyribonuclease 1 (APEX1) and attenuates the anti-apoptotic properties of APEX1, therefore promoting apoptosis of virus-infected cells to suppress viral infection through an IFN-I-independent way at the late stage of viral infection. Together, our findings reveal a non-canonical role of nuclear RIG-I in the induction of cellular apoptosis, besides its activation of IFN-I expression as the cytosolic innate sensor. This study provides new insight to the regulation of infection, IFN-I and autoimmune diseases by nuclear RIG-I-APEX1 interaction.

Potential Role of APEX1 During Ferroptosis.

Ferroptosis is a recently discovered category of programmed cell death. It is much different from other types of cell death such as apoptosis, necrosis and autophagy. The main pathological feature of ferroptosis is the accumulation of iron-dependent lipid peroxidation. The typical changes in the morphological features of ferroptosis include cell volume shrinkage and increased mitochondrial membrane area. The mechanisms of ferroptosis may be mainly related to lipid peroxidation accumulation, imbalance in amino acid antioxidant system, and disturbance of iron metabolism. Besides, hypoxia-inducible factor (HIF), nuclear factor-E2-related factor 2 (Nrf2), and p53 pathway have been demonstrated to be involved in ferroptosis. At present, the molecular mechanisms of ferroptosis pathway are still unmapped. In this review, an outlook has been put forward about the crucial role of apurinic/apyrimidinic endodeoxyribonuclease 1 (APEX1) in the regulation of ferroptosis. APEX1 plays an important role in the regulation of intracellular redox balance and can be used as a potential inhibitor of ferroptotic cell death. Bioinformatics analysis indicated that the mRNA level of APEX1 is decreased in cases of ferroptosis triggered by erastin. Besides, it was found that there was a significant correlation between APEX1 and genes in the ferroptosis pathway. We have discussed the possibility to employ APEX1 inducers or inhibitors in the regulation of ferroptosis as a new strategy for the treatment of various human diseases.

Selenoprotein T Protects Endothelial Cells against Lipopolysaccharide-Induced Activation and Apoptosis.

Sepsis is an exaggerated immune response upon infection with lipopolysaccharide (LPS) as the main causative agent. LPS-induced activation and apoptosis of endothelial cells (EC) can lead to organ dysfunction and finally organ failure. We previously demonstrated that the first twenty amino acids of the Apurinic/Apyrimidinic Endodeoxyribonuclease 1 (APEX1) are sufficient to inhibit EC apoptosis. To identify genes whose regulation by LPS is affected by this N-terminal APEX1 peptide, EC were transduced with an expression vector for the APEX1 peptide or an empty control vector and treated with LPS. Following RNA deep sequencing, genes upregulated in LPS-treated EC expressing the APEX1 peptide were identified bioinformatically. Selected candidates were validated by semi-quantitative real time PCR, a promising one was Selenoprotein T (SELENOT). For functional analyses, an expression vector for SELENOT was generated. To study the effect of SELENOT expression on LPS-induced EC activation and apoptosis, the SELENOT vector was transfected in EC. Immunostaining showed that SELENOT was expressed and localized in the ER. EC transfected with the SELENOT plasmid showed no activation and reduced apoptosis induced by LPS. SELENOT as well as APEX1(1-20) can protect EC against activation and apoptosis and could provide new therapeutic approaches in the treatment of sepsis.

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.

Short DNA/RNA heteroduplex oligonucleotide interacting proteins are key regulators of target gene silencing.

Antisense oligonucleotide (ASO)-based therapy is one of the next-generation therapy, especially targeting neurological disorders. Many cases of ASO-dependent gene expression suppression have been reported. Recently, we developed a tocopherol conjugated DNA/RNA heteroduplex oligonucleotide (Toc-HDO) as a new type of drug. Toc-HDO is more potent, stable, and efficiently taken up by the target tissues compared to the parental ASO. However, the detailed mechanisms of Toc-HDO, including its binding proteins, are unknown. Here, we developed native gel shift assays with fluorescence-labeled nucleic acids samples extracted from mice livers. These assays revealed two Toc-HDO binding proteins, annexin A5 (ANXA5) and carbonic anhydrase 8 (CA8). Later, we identified two more proteins, apurinic/apyrimidinic endodeoxyribonuclease 1 (APEX1) and flap structure-specific endonuclease 1 (FEN1) by data mining. shRNA knockdown studies demonstrated that all four proteins regulated Toc-HDO activity in Hepa1–6, mouse hepatocellular cells. In vitro binding assays and fluorescence polarization assays with purified recombinant proteins characterized the identified proteins and pull-down assays with cell lysates demonstrated the protein binding to the Toc-HDO and ASO in a biological environment. Taken together, our findings provide a brand new molecular biological insight as well as future directions for HDO-based disease therapy.