Autophagy suppression in DNA damaged cells occurs through a newly identified p53-proteasome-LC3 axis.

Macroautophagy is thought to have a critical role in shaping and refining cellular proteostasis in eukaryotic cells recovering from DNA damage. Here, we report a mechanism by which autophagy is suppressed in cells exposed to bacterial toxin-, chemical-, or radiation-mediated sources of genotoxicity. Autophagy suppression is directly linked to cellular responses to DNA damage, and specifically the stabilization of the tumor suppressor p53, which is both required and sufficient for regulating the ubiquitination and proteasome-dependent reduction in cellular pools of microtubule-associated protein 1 light chain 3 (LC3A/B), a key precursor of autophagosome biogenesis and maturation, in both epithelial cells and an ex vivo organoid model. Our data indicate that suppression of autophagy, through a newly identified p53-proteasome-LC3 axis, is a conserved cellular response to multiple sources of genotoxicity. Such a mechanism could potentially be important for realigning proteostasis in cells undergoing DNA damage repair.

Topoisomerase 2ß Induces DNA Breaks To Regulate Human Papillomavirus Replication.

Topoisomerases regulate higher-order chromatin structures through the transient breaking and religating of one or both strands of the phosphodiester backbone of duplex DNA. TOP2ß is a type II topoisomerase that induces double-strand DNA breaks at topologically associated domains (TADS) to relieve torsional stress arising during transcription or replication. TADS are anchored by CCCTC-binding factor (CTCF) and SMC1 cohesin proteins in complexes with TOP2ß. Upon DNA cleavage, a covalent intermediate DNA-TOP2ß (TOP2ßcc) is transiently generated to allow for strand passage. The tyrosyl-DNA phosphodiesterase TDP2 can resolve TOP2ßcc, but failure to do so quickly can lead to long-lasting DNA breaks. Given the role of CTCF/SMC1 proteins in the human papillomavirus (HPV) life cycle, we investigated whether TOP2ß proteins contribute to HPV pathogenesis. Our studies demonstrated that levels of both TOP2ß and TDP2 were substantially increased in cells with high-risk HPV genomes, and this correlated with large amounts of DNA breaks. Knockdown of TOP2ß with short hairpin RNAs (shRNAs) reduced DNA breaks by over 50% as determined through COMET assays. Furthermore, this correlated with substantially reduced formation of repair foci such as phosphorylated H2AX (γH2AX), phosphorylated CHK1 (pCHK1), and phosphorylated SMC1 (pSMC1) indicative of impaired activation of DNA damage repair pathways. Importantly, knockdown of TOP2ß also blocked HPV genome replication. Our previous studies demonstrated that CTCF/SMC1 factors associate with HPV genomes at sites in the late regions of HPV31, and these correspond to regions that also bind TOP2ß. This study identifies TOP2ß as responsible for enhanced levels of DNA breaks in HPV-positive cells and as a regulator of viral replication.IMPORTANCE High-risk human papillomaviruses (HPVs) infect epithelial cells and induce viral genome amplification upon differentiation. HPV proteins activate DNA damage repair pathways by inducing high numbers of DNA breaks in both viral and cellular DNAs. This activation is required for HPV genome replication. TOP2ß is a type II topoisomerase that induces double-strand DNA breaks at topologically associated domains (TADS) to relieve torsional stress arising during transcription or replication. Our studies demonstrate that TOP2ß levels are increased in HPV-positive cells and that this is required for HPV replication. Importantly, our studies further show that knockdown of TOP2ß reduces the number of breaks by over 50% in HPV-positive cells and that this correlates with substantially impaired activation of DNA repair pathways. This study identifies a critical mechanism by which HPV replication is regulated by the topoisomerase TOP2ß through DNA break formation.

Pathogenesis of Human Papillomaviruses Requires the ATR/p62 Autophagy-Related Pathway.

High-risk human papillomaviruses (HPVs) constitutively activate the ataxia telangiectasia and Rad3-related (ATR) DNA damage response pathway, and this is required for viral replication. In fibroblasts, activated ATR regulates transcription of inflammatory genes through its negative effects on the autophagosome cargo protein p62. In addition, suppression of p62 results in increased levels of the transcription factor GATA4, leading to cellular senescence. In contrast, in HPV-positive keratinocytes, we observed that activation of ATR resulted in increased levels of phosphorylated p62, which in turn lead to reduced levels of GATA4. Knockdown of ATR in HPV-positive cells resulted in decreased p62 phosphorylation and increased GATA4 levels. Transcriptome sequencing (RNA-seq) analysis of HPV-positive cells identified inflammatory genes and interferon factors as negative transcriptional targets of ATR. Furthermore, knockdown of p62 or overexpression of GATA4 in HPV-positive cells leads to inhibition of viral replication. These findings identify a novel role of the ATR/p62 signaling pathway in HPV-positive cells.IMPORTANCE High-risk human papillomaviruses (HPVs) infect epithelial cells and induce viral genome amplification upon differentiation. HPV proteins activate the ATR DNA damage repair pathway, and this is required for HPV genome amplification. In the present study, we show that HPV-induced ATR activation also leads to suppression of expression of inflammatory response genes. This suppression results from HPV-induced phosphorylation of the autophagosome cargo protein p62 which regulates the levels of the transcription factor GATA4. Activation of p62 in normal fibroblasts results in senescence, but this is not seen in HPV-positive keratinocytes. Importantly, knockdown of p62 or overexpression of GATA4 in HPV-positive cells abrogates viral replication. This study demonstrates that activation of ATR in HPV-positive cells triggers a p62-directed pathway inducing suppression of inflammatory gene expression independent of DNA repair and facilitating HPV replication.

Topoisomerase IIß-binding protein 1 activates expression of E2F1 and p73 in HPV-positive cells for genome amplification upon epithelial differentiation.

High-risk human papillomaviruses (HPVs) constitutively activate ataxia telangiectasia mutated (ATM) and ataxia telangiectasia- and Rad3-related (ATR) DNA damage repair pathways for viral genome amplification. HPVs activate these pathways through the immune regulator STAT-5. For the ATR pathway, STAT-5 increases expression of the topoisomerase IIß-binding protein 1 (TopBP1), a scaffold protein that binds ATR and recruits it to sites of DNA damage. TopBP1 also acts as a transcriptional regulator, and we investigated how this activity influenced the HPV life cycle. We determined that TopBP1 levels are increased in cervical intraepithelial neoplasias as well as cervical carcinomas, consistent with studies in HPV-positive cell lines. Suppression of TopBP1 by shRNAs impairs HPV genome amplification and activation of the ATR pathway but does not affect the total levels of ATR and CHK1. In contrast, knockdown reduces the expression of other DNA damage factors such as RAD51 and Mre11 but not BRCA2 or NBS1. Interestingly, TopBP1 positively regulates the expression of E2F1, a TopBP1-binding partner, and p73 in HPV-positive cells in contrast to its effects in other cell types. TopBP1 transcriptional activity is regulated by AKT, and treatment with AKT inhibitors suppresses expression of E2F1 and p73 without interfering with ATR signaling. Importantly, the levels of p73 are elevated in HPV-positive cells and its knockdown impairs HPV genome amplification. This demonstrates that p73, like p63 and p53, is an important regulator of the HPV life cycle that is controlled by the transcriptional activating properties of the multifunctional TopBP1 protein.

Relationship among pregnancy associated plasma protein-A levels, clinical characteristics, and coronary artery disease extent in patients with chronic stable angina pectoris.

AIMS: To assess, in chronic stable angina (CSA) patients, the relationship among clinical characteristics and cardiovascular risk factors, extent of coronary artery disease (CAD), and pregnancy-associated plasma protein-A (PAPP-A) levels. METHODS AND RESULTS: We studied 643 CSA patients (63+/-10 years, 482 men) undergoing diagnostic coronary angiography; 97 with angiographically normal coronary arteries or <50% stenosis, 127 with single vessel disease (VD), and 419 with multi-VD. Patients' age, gender, cardiovascular risk factors, body mass index, history of previous myocardial infarction, angina class, left ventricular ejection fraction (LVEF), and treatment were assessed at study entry. PAPP-A levels (mIU/L) were higher in men than in women (6.2+/-2.4 vs. 5.2+/-1.8; P<0.001) and in hypertensive vs. normotensive patients (6.4+/-2.8 vs. 5.8+/-2.1; P=0.01). PAPP-A correlated directly with age (r=0.19, P<0.001) and inversely with LVEF (r=-0.11, P=0.01). Patients with multivessel disease (VD) had higher PAPP-A levels (6.45+/-2.58) than those with single-VD (5.49+/-1.54, P<0.001) or normal coronaries (4.62+/-1.17, P<0.001). Male gender, age, history of a previous MI, hypercholesterolaemia, and PAPP-A levels were independent predictors for the presence of CAD. CONCLUSION: In CSA patients PAPP-A levels correlate with age, male gender, hypertension, and CAD extent. In the present study, PAPP-A was an independent predictor for the presence and extent of CAD.

Pregnancy-associated plasma protein A and its endogenous inhibitor, the proform of eosinophil major basic protein (proMBP), are related to complex stenosis morphology in patients with stable angina pectoris.

BACKGROUND: The metalloproteinase pregnancy-associated plasma protein-A (PAPP-A) has been implicated in coronary plaque disruption. Its endogenous inhibitor, the proform of eosinophil major basic protein (proMBP), may also play a role in this process. Atheromatous plaque disruption often presents as complex angiographic lesions. We sought to assess whether PAPP-A, proMBP, and PAPP-A/ProMBP ratio are markers of angiographic plaque complexity in patients with chronic stable angina. METHODS AND RESULTS: We studied 396 stable angina patients (age 63+/-10 years, 230 men) of whom 289 had angiographically documented coronary artery disease (> or =75% stenosis). All coronary stenoses > or =30% diameter reduction (n =531 in 322 patients) were assessed and classified as complex (n =228) or smooth (n =303) by previously validated criteria. PAPP-A, proMBP, and C-reactive protein (hs-CRP) serum levels were measured by ELISA. Patients with complex coronary stenoses had a significantly (P<0.001) higher PAPP-A/proMBP ratio (3.1+/-1.2 versus 2.7+/-0.8x10(-3)) and PAPP-A levels (5.9+/-1.6 versus 5.1+/-1.4 mIU/L) than those without. On univariate analysis, male gender (P<0.001), age (P<0.001), previous history of myocardial infarction (P=0.013), reduced ejection fraction (P<0.001), severe coronary artery disease (P<0.001), aspirin treatment (P<0.001), PAPP-A levels (P<0.001), and PAPP-A/proMBP ratio (P<0.001) were correlated with the number of complex stenoses. Multiple regression analysis showed that male gender, age, severe coronary artery disease, and PAPP-A/proMBP ratio were independent predictors of the number of angiographically complex stenoses. CONCLUSIONS: In patients with stable angina, PAPP-A and PAPP-A/proMBP ratio are associated with angiographic plaque complexity.