scholarly journals Hepatitis C Virus Downregulates Ubiquitin-Conjugating Enzyme E2S Expression To Prevent Proteasomal Degradation of NS5A, Leading to Host Cells More Sensitive to DNA Damage

2018 ◽  
Vol 93 (2) ◽  
Author(s):  
Hang T. Pham ◽  
Tram T. T. Nguyen ◽  
Lap P. Nguyen ◽  
Sang-Seop Han ◽  
Yun-Sook Lim ◽  
...  
Keyword(s):  
Dna Damage ◽  
Antiviral Activity ◽  
Host Cells ◽  
Protein Homeostasis ◽  
Viral Propagation ◽  
Ns5a Protein ◽  
Ubiquitin Conjugating Enzyme ◽  
Infected Cells ◽  
Dependent Pathway ◽  
Conjugating Enzyme

ABSTRACT Hepatitis C virus (HCV) infection may cause chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma. HCV exploits cellular machineries to establish persistent infection. We demonstrate here that ubiquitin-conjugating enzyme E2S (UBE2S), a member of the ubiquitin-conjugating enzyme family (E2s), was downregulated by endoplasmic reticulum stress caused by HCV in Huh7 cells. UBE2S interacted with domain I of HCV NS5A and degraded NS5A protein through the Lys11-linked proteasome-dependent pathway. Overexpression of UBE2S suppressed viral propagation, while depletion of UBE2S expression increased viral infectivity. Enzymatically inactive UBE2S C95A mutant exerted no antiviral activity, suggesting that ubiquitin-conjugating enzymatic activity was required for the suppressive role of UBE2S. Chromatin ubiquitination plays a crucial role in the DNA damage response. We showed that the levels of UBE2S and Lys11 chains bound to the chromatin were markedly decreased in the context of HCV replication, rendering HCV-infected cells more sensitive to DNA damage. These data suggest that HCV counteracts antiviral activity of UBE2S to optimize viral propagation and may contribute to HCV-induced liver pathogenesis. IMPORTANCE Protein homeostasis is essential to normal cell function. HCV infection disturbs the protein homeostasis in the host cells. Therefore, host cells exert an anti-HCV activity in order to maintain normal cellular metabolism. We showed that UBE2S interacted with HCV NS5A and degraded NS5A protein through the Lys11-linked proteasome-dependent pathway. However, HCV has evolved to overcome host antiviral activity. We demonstrated that the UBE2S expression level was suppressed in HCV-infected cells. Since UBE2S is an ubiquitin-conjugating enzyme and this enzyme activity is involved in DNA damage repair, HCV-infected cells are more sensitive to DNA damage, and thus UBE2S may contribute to viral oncogenesis.

DNA Damage-Induced Foci of E2 Ubiquitin-Conjugating Enzyme are Detectable upon Co-transfection with an Interacting E3 Ubiquitin Ligase

2015 ◽  
Vol 54 (2) ◽  
pp. 147-157 ◽  
Author(s):  
Degui Wang ◽  
Yingxia Tian ◽  
Dong Wei ◽  
Yuhong Jing ◽  
Haitao Niu ◽  
...  
Keyword(s):  
Dna Damage ◽  
Ubiquitin Ligase ◽  
E3 Ubiquitin Ligase ◽  
Ubiquitin Conjugating Enzyme ◽  
Conjugating Enzyme

scholarly journals DNA double-strand breaks in the Toxoplasma gondii-infected cells by the action of reactive oxygen species

2020 ◽  
Vol 13 (1) ◽  
Author(s):  
Haohan Zhuang ◽  
Chaoqun Yao ◽  
Xianfeng Zhao ◽  
Xueqiu Chen ◽  
Yimin Yang ◽  
...  
Keyword(s):  
Dna Damage ◽  
Toxoplasma Gondii ◽  
Double Strand Breaks ◽  
Host Cells ◽  
Oxygen Species ◽  
Dna Double Strand Breaks ◽  
Strand Breaks ◽  
Damage Response ◽  
Infected Cells

Abstract Background Toxoplasma gondii is an obligate parasite of all warm-blooded animals around the globe. Once infecting a cell, it manipulates the host’s DNA damage response that is yet to be elucidated. The objectives of the present study were three-fold: (i) to assess DNA damages in T. gondii-infected cells in vitro; (ii) to ascertain causes of DNA damage in T. gondii-infected cells; and (iii) to investigate activation of DNA damage responses during T. gondii infection. Methods HeLa, Vero and HEK293 cells were infected with T. gondii at a multiplicity of infection (MOI) of 10:1. Infected cells were analyzed for a biomarker of DNA double-strand breaks (DSBs) γH2AX at 10 h, 20 h or 30 h post-infection using both western blot and immunofluorescence assay. Reactive oxygen species (ROS) levels were measured using 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA), and ROS-induced DNA damage was inhibited by a ROS inhibitor N-acetylcysteine (NAC). Lastly, DNA damage responses were evaluated by detecting the active form of ataxia telangiectasia mutated/checkpoint kinase 2 (ATM/CHK2) by western blot. Results γH2AX levels in the infected HeLa cells were significantly increased over time during T. gondii infection compared to uninfected cells. NAC treatment greatly reduced ROS and concomitantly diminished γH2AX in host cells. The phosphorylated ATM/CHK2 were elevated in T. gondii-infected cells. Conclusions Toxoplasma gondii infection triggered DNA DSBs with ROS as a major player in host cells in vitro. It also activated DNA damage response pathway ATM/CHK2. Toxoplasma gondii manages to keep a balance between survival and apoptosis of its host cells for the benefit of its own survival.

scholarly journals SARS-CoV-2 triggers DNA damage response in Vero E6 cells

2021 ◽  
Author(s):  
Joshua Victor ◽  
Jamie Deutsch ◽  
Annalis Whitaker ◽  
Erica N. Lamkin ◽  
Anthony March ◽  
...  
Keyword(s):  
Dna Damage ◽  
Dna Damage Response ◽  
Host Cells ◽  
Kidney Cells ◽  
The Novel ◽  
African Green Monkey Kidney ◽  
Downstream Effector ◽  
Damage Response ◽  
Green Monkey ◽  
Infected Cells

AbstractThe novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus responsible for the current COVID-19 pandemic and has now infected more than 200 million people with more than 4 million deaths globally. Recent data suggest that symptoms and general malaise may continue long after the infection has ended in recovered patients, suggesting that SARS-CoV-2 infection has profound consequences in the host cells. Here we report that SARS-CoV-2 infection can trigger a DNA damage response (DDR) in African green monkey kidney cells (Vero E6). We observed a transcriptional upregulation of the Ataxia telangiectasia and Rad3 related protein (ATR) in infected cells. In addition, we observed enhanced phosphorylation of CHK1, a downstream effector of the ATR DNA damage response, as well as H2AX. Strikingly, SARS-CoV-2 infection lowered the expression of TRF2 shelterin-protein complex, and reduced telomere lengths in infected Vero E6 cells. Thus, our observations suggest SARS-CoV-2 may have pathological consequences to host cells beyond evoking an immunopathogenic immune response.

Bortezomib Impairs Myeloma Cells (MM) Homologous Recombination Through Inhibition of the E2-Ubiquitin-Conjugating Enzyme UBC13

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1834-1834
Author(s):  
Paola Neri ◽  
Li Ren ◽  
Jordan Johnson ◽  
Kathy J Gratton ◽  
Erin Stebner ◽  
...  
Keyword(s):  
Dna Damage ◽  
Dna Damage Repair ◽  
Proteasome Inhibition ◽  
Nuclear Accumulation ◽  
Transcriptional Level ◽  
Damage Repair ◽  
Dna Dsbs ◽  
Ubiquitin Conjugating Enzyme ◽  
Repair Genes ◽  
Conjugating Enzyme

Abstract Abstract 1834 Background: Chromosomal instability is a defining feature of clonal MM plasma cells resulting in the perpetual accumulation of genomic aberrations. In addition to its role in protein homeostasis the ubiquitin-proteasome system is also involved in the regulation of DNA damage repair proteins. We have recently reported that proteasome inhibition induces a BRCAness state in MM cells (MM) resulting in a contextual synthetic lethality when combined with PARP inhibitors. We now report on the mechanisms by which bortezomib impairs homologous recombination (HR) mediated repair of DNA double stranded breaks (DNA-DSBs). Methods and results: Using the DR-GFP/SceI reporter assay in which cells are transfected with a plasmid tandemly expressing mutated GFP genes (5'-Sce/GFP – truncated IGFP-3') and followed by infection with an adenovirus (AdNGUS24i) expressing the yeast Sce endonuclease, we have confirmed that treatment with bortezomib does severely impair HR in MM cells. At the transcriptional level, proteasome inhibition transiently (6–12 hours post treatment) reduced the mRNA expression of several HR and DNA damage repair genes (BRCA1, BRCA2, RAD51 and FANCD2) followed by recovery to baseline levels by 24 hours. This transient downregulation of these DNA damage repair genes was also confirmed in a promoter luciferase reporter screen. At the post-trancriptional level, examining the dynamics and kinetics of DNA-DSBs repair induced by radiation therapy or PARP inhibition, we have demonstrated that bortezomib does not alter the initial phase of DNA damage sensing (MRN complex recruitment, ATM and H2AX phosphorylation and MDC1 activation) but does impair the Lys63-poly-ubiquitylation of histones γH2AX and H2A. This histone ubiquitylation is a required modification for the second wave of DNA repair proteins recruitment and retention of BRCA1 and RAD51 at the sites of DNA-DSBs. Furthermore, we have shown that bortezomib treatment depleted the nuclear pools of ubiquitin and abrogated the poly-ubiquitylation (lack of co-localization poly-Ub FK2 foci with γH2AX), but not the phosphorylation and foci formation of histone H2AX. Therefore, bortezomib appears to impair histones (H2AX and H2A) poly-ubiquitylation, a process that is dependent on the activation and recruitment of the E3 ubiquitin-protein ligases RNF8 and RNF-168 as well as the ubiquitin-E2 conjugating enzyme UBC13 to the sites of DNA-DSBs. Western blot analysis of the nuclear and cytoplasmic fractions of cellular extracts, revealed that bortezomib dramatically reduced the nuclear accumulation of UBC13 in response to DNA-DSBs as well as the formation of UBC13-Ubiquitin thiolester bonds formation and hence impaired the transfer of ubiquitin to nucleosomal histones. Conclusion: Our studies demonstrate that proteasome inhibition with bortezomib severely impairs homology-mediated repair of DNA breaks in MM cells at the post-transcriptional level by altering the nuclear accumulation and function of the E2-ubiquitin conjugating enzyme UBC13. These results explain the observed clinical synergy between bortezomib and several DNA damaging agents and support further clinical investigation of the combination of these class of drugs in MM patients. Disclosures: Neri: Celgene: Honoraria, Research Funding. Bahlis:Celgene: Honoraria, Speakers Bureau.

scholarly journals Bacterial DNA damage effectors in host cells

2018 ◽  
Vol 16 (3) ◽  
pp. 26-36
Author(s):  
Vladimir G. Druzhinin ◽  
Elizaveta D. Baranova ◽  
Vladislav Yu. Buslaev ◽  
Lyudmila V. Matskova ◽  
Alina V. Tolstikova
Keyword(s):  
Dna Damage ◽  
Bacterial Species ◽  
Host Cells ◽  
Host Organism ◽  
Mutation Process ◽  
Bacterial Dna ◽  
Bacterial Microbiota ◽  
Repair Efficiency ◽  
Infected Cells ◽  
Genotoxic Action

The microbiota has a significant, and sometimes decisive, effect on the host's homeostasis. The results of recent metagenomic studies confirm the importance of microbiota in maintaining health or its impact on the development of acute, chronic and neoplastic diseases. One of the important aspects of microbiota exposure is the ability of many bacterial species to induce mutations or modulate a mutation process in the cells of the host organism. This review summarizes the main experimental data revealing various mechanisms of genotoxic action of a bacterial microbiota, including direct damage to the DNA structure, induction of oxidative stress, delay in replication, and a decrease in repair efficiency. It is emphasized that bacteria use different strategies to ensure their own survival and replication, including. by suppressing the repair of host cell DNA, by promoting the survival of infected cells, despite the presence of DNA damage therein.

scholarly journals Virucidal Activity of Moringa a From Moringa Oleifera Seeds Against Influenza A Viruses by Regulating TFEB

2020 ◽  
Author(s):  
Yongai Xiong ◽  
Muhammad Shahid Riaz Rajoka ◽  
MengXun Zhang ◽  
Ning Liang ◽  
Zhendan He
Keyword(s):  
Antiviral Activity ◽  
Inflammatory Cytokines ◽  
Influenza A ◽  
Moringa Oleifera ◽  
Cytopathic Effect ◽  
Cellular Protein ◽  
Host Cells ◽  
Influenza A Viruses ◽  
Different Types ◽  
Infected Cells

Abstract BackgroundInfluenza A viruses (IAVs) are highly contagious pathogens infecting human and numerous animals. The viruses cause millions of infection cases and thousands of deaths every year, thus making IAVs a continual threat to global health. MethodsMoringa A was isolated from Moringa oleifera seeds and tested for antiviral activity against H1N1. The antiviral activity of Moringa A was tested by checking their effect on hemagglutination and PFU activities of the studied virus, and the cytopathic effect was observed too. Additionally, the different types of treatment experiments were performed to complement the analysis of the antiviral activity of Moringa A, and the contents of inflammatory cytokines and the expression of TFEB were detected.ResultsMoringa A inhibits virus replication in host cells, and it protects infected cells from cytopathic effect induced by IAVs. The EC50 and EC90 value of Moringa A for IAVs were 1.27 and 5.30 μM, respectively. The different types of treatment experiments revealed that Moringa A has a significant inhibitory effect on the IAVs both before and after drug addition. What’s more, Moringa A was observed to decrease the levels of inflammatory cytokines TNF-α, IL-6, IL-1β and IFN-β in H1N1 infected RAW264.7 cells. Finaly, Moringa A was found to inhibit the expression and nuclear transfer of the cellular protein transcription factor EB (TFEB), and weaken the autophagy in infected cells, which could be an important antiviral mechnism of Moringa A. ConclusionsMoringa A has potent antiviral activity against IVAs, which could be due to the autophagy inhibition property.

scholarly journals Regulation of p53 Localization and Activity by Ubc13

2006 ◽  
Vol 26 (23) ◽  
pp. 8901-8913 ◽  
Author(s):  
Aaron Laine ◽  
Ivan Topisirovic ◽  
Dayong Zhai ◽  
John C. Reed ◽  
Katherine L. B. Borden ◽  
...  
Keyword(s):  
Dna Damage ◽  
Transcriptional Activity ◽  
Ubiquitin Ligases ◽  
Wild Type ◽  
Terminal Domain ◽  
Ubiquitin Conjugating Enzyme ◽  
P53 Stability ◽  
Conjugating Enzyme

ABSTRACT The abundance and activity of p53 are regulated largely by ubiquitin ligases. Here we demonstrate a previously undisclosed regulation of p53 localization and activity by Ubc13, an E2 ubiquitin-conjugating enzyme. While increasing p53 stability, Ubc13 decreases p53 transcriptional activity and increases its localization to the cytoplasm, changes that require its ubiquitin-conjugating activity. Ubc13 elicits K63-dependent ubiquitination of p53, which attenuates Hdm2-induced polyubiquitination of p53. Ubc13 association with p53 requires an intact C-terminal domain of p53 and is markedly stronger with a p53 mutant that cannot tetramerize. Expression of Ubc13 in vivo increases the pool of monomeric p53, indicating that Ubc13 affects tetramerization of p53. Significantly, wild-type but not mutant Ubc13 is associated with polysomes and enriches p53 within this fraction. In response to DNA damage, Ubc13 is no longer capable of facilitating p53 monomerization, in part due to a decrease in its own levels which is p53 dependent. Our findings point to a newly discerned mechanism important in the regulation of p53 organization, localization, and activity by Ubc13.

scholarly journals Characterizing Cellular Responses During Oncolytic Maraba Virus Infection

2019 ◽  
Vol 20 (3) ◽  
pp. 580 ◽  
Author(s):  
Golnoush Hassanzadeh ◽  
Thet Naing ◽  
Tyson Graber ◽  
Seyed Jafarnejad ◽  
David Stojdl ◽  
...  
Keyword(s):  
High Selectivity ◽  
Cellular Responses ◽  
Oncolytic Viruses ◽  
Host Cells ◽  
End Joining ◽  
Viral Propagation ◽  
Infected Cells ◽  
Translational Arrest ◽  
Low Cytotoxicity

The rising demand for powerful oncolytic virotherapy agents has led to the identification of Maraba virus, one of the most potent oncolytic viruses from Rhabdoviridae family which displays high selectivity for killing malignant cells and low cytotoxicity in normal cells. Although the virus is readied to be used for clinical trials, the interactions between the virus and the host cells is still unclear. Using a newly developed interferon-sensitive mutant Maraba virus (MG1), we have identified two key regulators of global translation (4E-BP1 and eIF2α) as being involved in the regulation of protein synthesis in the infected cells. Despite the translational arrest upon viral stress, we showed an up-regulation of anti-apoptotic Bcl-xL protein that provides a survival benefit for the host cell, yet facilitates effective viral propagation. Given the fact that eIF5B canonically regulates 60S ribosome subunit end joining and is able to replace the role of eIF2 in delivering initiator tRNA to the 40S ribosome subunit upon the phosphorylation of eIF2α we have tested whether eIF5B mediates the translation of target mRNAs during MG1 infection. Our results show that the inhibition of eIF5B significantly down-regulates the level of Bcl-xL steady-state mRNA, thus indirectly attenuates viral propagation.

scholarly journals DNA Double Strand Breaks in the Toxoplasma Gondii-Infected Cells by the Action of Reactive Oxygen Species

2020 ◽  
Author(s):  
Haohan Zhuang ◽  
Chaoqun Yao ◽  
Xianfeng Zhao ◽  
Yi Yang ◽  
Xueqiu Chen ◽  
...  
Keyword(s):  
Dna Damage ◽  
Dna Damage Response ◽  
Double Strand Breaks ◽  
Host Cells ◽  
Oxygen Species ◽  
Dna Double Strand Breaks ◽  
Strand Breaks ◽  
Damage Response ◽  
Infected Cells

Abstract Background: Toxoplasma gondii (T. gondii) is an obligate parasite of the warm-blooded animals with a worldwide distribution. Once having entered a host cell, it manipulates host’s DNA damage response that is yet to be investigated. The objectives of the present study were three-fold: 1) to assess DNA damages in T. gondii-infected cells in vitro; 2) to ascertain sources causing DNA damage in T. gondii-infected cells; 3) to investigate activation of DNA damage response during T. gondii infection.Methods: HeLa, Vero and HEK293 cells were infected with T. gondii at multiplicity of infection (MOI) of 10:1. Infected cells at 10 h, 20 h or 30 h post infection were analyzed for a DNA double strand breaks (DSBs) biomarker γH2AX using Western blot and immunofluorescence assay. Reactive oxygen species (ROS) levels were examined using 2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA), and the impact of ROS on DNA damage was assessed by inhibition using a ROS inhibitor N-acetylcysteine (NAC). Lastly, DNA damage response in these T. gondii-infected cells was evaluated by detecting the expression of active form of ataxia telangiectasia mutated/checkpoint kinase 2 (ATM/CHK2) with Western blot. Results: Compared to uninfected cells, γH2AX expression in the infected HeLa cells at 10 h, 20 h, and 30 h was increased over time during T. gondii infection. NAC treatment reduced ROS level in host cells and significantly decreased the expression of γH2AX. Expression of phosphorylated ATM/CHK2 was elevated in T. gondii-infected cells.Conclusion: T. gondii infection triggered DNA DSBs with ROS as a major player in host cells in vitro. It also concomitantly activated DNA damage response pathway ATM/CHK2. T. gondii struggles a balance between survival and apoptosis of its host cells for the benefit of its own survival.

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