共查询到20条相似文献,搜索用时 31 毫秒
1.
Gloria Terrados Jean-Pascal Capp Yvan Canitrot Miguel García-Díaz Katarzyna Bebenek Tomas Kirchhoff Alberto Villanueva Fran?ois Boudsocq Valérie Bergoglio Christophe Cazaux Thomas A. Kunkel Jean-Sébastien Hoffmann Luis Blanco 《PloS one》2009,4(10)
Background
DNA polymerase lambda (Polλ) is a DNA repair polymerase, which likely plays a role in base excision repair (BER) and in non-homologous end joining (NHEJ) of DNA double-strand breaks (DSB).Principal Findings
Here, we described a novel natural allelic variant of human Polλ (hPolλ) characterized by a single nucleotide polymorphism (SNP), C/T variation in the first base of codon 438, resulting in the amino acid change Arg to Trp. In vitro enzyme activity assays of the purified W438 Polλ variant revealed that it retained both DNA polymerization and deoxyribose phosphate (dRP) lyase activities, but had reduced base substitution fidelity. Ectopic expression of the W438 hPolλ variant in mammalian cells increases mutation frequency, affects the DSB repair NHEJ pathway, and generates chromosome aberrations. All these phenotypes are dependent upon the catalytic activity of the W438 hPolλ.Conclusions
The expression of a cancer-related natural variant of one specialized DNA polymerase can be associated to generic instability at the cromosomal level, probably due a defective NHEJ. These results establish that chromosomal aberrations can result from mutations in specialized DNA repair polymerases. 相似文献2.
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Background
Glioblastoma Multiforme (GBM) is the most lethal form of brain tumor. Efficient DNA repair and anti-apoptotic mechanisms are making glioma treatment difficult. Proteases such as MMP9, cathepsin B and urokinase plasminogen activator receptor (uPAR) are over expressed in gliomas and contribute to enhanced cancer cell proliferation. Non-homologous end joining (NHEJ) repair mechanism plays a major role in double strand break (DSB) repair in mammalian cells.Methodology/Principal Findings
Here we show that silencing MMP9 in combination with uPAR/cathepsin B effects NHEJ repair machinery. Expression of DNA PKcs and Ku70/80 at both mRNA and protein levels in MMP9-uPAR (pMU) and MMP9-cathepsin B (pMC) shRNA-treated glioma xenograft cells were reduced. FACS analysis showed an increase in apoptotic peak and proliferation assays revealed a significant reduction in the cell population in pMU- and pMC-treated cells compared to untreated cells. We hypothesized that reduced NHEJ repair led to DSBs accumulation in pMU- and pMC-treated cells, thereby initiating cell death. This hypothesis was confirmed by reduced Ku70/Ku80 protein binding to DSB, increased comet tail length and elevated γH2AX expression in treated cells compared to control. Immunoprecipitation analysis showed that EGFR-mediated lowered DNA PK activity in treated cells compared to controls. Treatment with pMU and pMC shRNA reduced the expression of DNA PKcs and ATM, and elevated γH2AX levels in xenograft implanted nude mice. Glioma cells exposed to hypoxia and irradiation showed DSB accumulation and apoptosis after pMU and pMC treatments compared to respective controls.Conclusion/Significance
Our results suggest that pMU and pMC shRNA reduce glioma proliferation by DSB accumulation and increase apoptosis under normoxia, hypoxia and in combination with irradiation. Considering the radio- and chemo-resistant cancers favored by hypoxia, our study provides important therapeutic potential of MMP9, uPAR and cathepsin B shRNA in the treatment of glioma from clinical stand point. 相似文献4.
Background
Meiotic recombination between homologous chromosomes provides natural combinations of genetic variations and is a main driving force of evolution. It is initiated via programmed DNA double-strand breaks (DSB) and involves a specific axial chromosomal structure. So far, recombination regions have been mainly determined by experiments, both expensive and time-consuming.Results
SPoRE is a mathematical model that describes the non-uniform localisation of DSB and axis proteins sites, and distinguishes high versus low protein density. It is based on a combination of genomic signals, based on what is known from wet-lab experiments, whose contribution is precisely quantified. It models axis proteins accumulation at gene 5’-ends with a discrete approximation of their diffusion and convection along genes. It models DSB accumulation at approximated gene promoter positions with intergenic region length and GC-content. SPoRE can be used for prediction and it is parameterised in an obvious way that makes it easy to understand from a biological viewpoint.Conclusions
When compared to Saccharomyces cerevisiae experimental data, SPoRE predicts axis protein and DSB positions with high sensitivity and precision, axis protein density with an average local correlation r=0.63 and DSB density with an average local correlation r=0.62. SPoRE outbreaks previous DSB predictors, which are based on nucleotide patterning, and it reaches 85% of success rate in DSB prediction compared to 54% obtained by available tools on a benchmarked dataset.SPoRE is available at the address http://www.lcqb.upmc.fr/SPoRE/.Electronic supplementary material
The online version of this article (doi:10.1186/s12859-014-0391-1) contains supplementary material, which is available to authorized users. 相似文献5.
Jeffrey J. W. Verschuren Stella Trompet Joris Deelen David J. Stott Naveed Sattar Brendan M. Buckley Ian Ford Bastiaan T. Heijmans Henk-Jan Guchelaar Jeanine J. Houwing-Duistermaat P. Eline Slagboom J. Wouter Jukema 《PloS one》2013,8(2)
Purpose
DNA repair deficiencies have been postulated to play a role in the development and progression of cardiovascular disease (CVD). The hypothesis is that DNA damage accumulating with age may induce cell death, which promotes formation of unstable plaques. Defects in DNA repair mechanisms may therefore increase the risk of CVD events. We examined whether the joints effect of common genetic variants in 5 DNA repair pathways may influence the risk of CVD events.Methods
The PLINK set-based test was used to examine the association to myocardial infarction (MI) of the DNA repair pathway in GWAS data of 866 subjects of the GENetic DEterminants of Restenosis (GENDER) study and 5,244 subjects of the PROspective Study of Pravastatin in the Elderly at Risk (PROSPER) study. We included the main DNA repair pathways (base excision repair, nucleotide excision repair, mismatch repair, homologous recombination and non-homologous end-joining (NHEJ)) in the analysis.Results
The NHEJ pathway was associated with the occurrence of MI in both GENDER (P = 0.0083) and PROSPER (P = 0.014). This association was mainly driven by genetic variation in the MRE11A gene (PGENDER = 0.0001 and PPROSPER = 0.002). The homologous recombination pathway was associated with MI in GENDER only (P = 0.011), for the other pathways no associations were observed.Conclusion
This is the first study analyzing the joint effect of common genetic variation in DNA repair pathways and the risk of CVD events, demonstrating an association between the NHEJ pathway and MI in 2 different cohorts. 相似文献6.
White RR Miyata S Papa E Spooner E Gounaris K Selkirk ME Artavanis-Tsakonas K 《PLoS neglected tropical diseases》2011,5(10):e1340
Background
Trichinella spiralis is a zoonotic parasitic nematode that causes trichinellosis, a disease that has been identified on all continents except Antarctica. During chronic infection, T. spiralis larvae infect skeletal myofibres, severely disrupting their differentiation state.Methodology and Results
An activity-based probe, HA-Ub-VME, was used to identify deubiquitinating enzyme (DUB) activity in lysate of T. spiralis L1 larvae. Results were analysed by immuno-blot and immuno-precipitation, identifying a number of potential DUBs. Immuno-precipitated proteins were subjected to LC/MS/MS, yielding peptides with sequence homology to 5 conserved human DUBs: UCH-L5, UCH-L3, HAUSP, OTU 6B and Ataxin-3. The predicted gene encoding the putative UCH-L5 homologue, TsUCH37, was cloned and recombinant protein was expressed and purified. The deubiquitinating activity of this enzyme was verified by Ub-AMC assay. Co-precipitation of recombinant TsUCH37 showed that the protein associates with putative T. spiralis proteasome components, including the yeast Rpn13 homologue ADRM1. In addition, the UCH inhibitor LDN-57444 exhibited specific inhibition of recombinant TsUCH37 and reduced the viability of cultured L1 larvae.Conclusions
This study reports the identification of the first T. spiralis DUB, a cysteine protease that is putatively orthologous to the human protein, hUCH-L5. Results suggest that the interaction of this protein with the proteasome has been conserved throughout evolution. We show potential for the use of inhibitor compounds to elucidate the role of UCH enzymes in T. spiralis infection and their investigation as therapeutic targets for trichinellosis. 相似文献7.
Dan Yan Wooi Loon Ng Xiangming Zhang Ping Wang Zhaobin Zhang Yin-Yuan Mo Hui Mao Chunhai Hao Jeffrey J. Olson Walter J. Curran Ya Wang 《PloS one》2010,5(7)
Background
Radiotherapy kills tumor-cells by inducing DNA double strand breaks (DSBs). However, the efficient repair of tumors frequently prevents successful treatment. Therefore, identifying new practical sensitizers is an essential step towards successful radiotherapy. In this study, we tested the new hypothesis: identifying the miRNAs to target DNA DSB repair genes could be a new way for sensitizing tumors to ionizing radiation.Principal Findings
Here, we chose two genes: DNA-PKcs (an essential factor for non-homologous end-joining repair) and ATM (an important checkpoint regulator for promoting homologous recombination repair) as the targets to search their regulating miRNAs. By combining the database search and the bench work, we picked out miR-101. We identified that miR-101 could efficiently target DNA-PKcs and ATM via binding to the 3′- UTR of DNA-PKcs or ATM mRNA. Up-regulating miR-101 efficiently reduced the protein levels of DNA-PKcs and ATM in these tumor cells and most importantly, sensitized the tumor cells to radiation in vitro and in vivo.Conclusions
These data demonstrate for the first time that miRNAs could be used to target DNA repair genes and thus sensitize tumors to radiation. These results provide a new way for improving tumor radiotherapy. 相似文献8.
Carol HY Wu Julie YH Chan Jimmy Li-Jer Chou Samuel HH Chan Alice YW Chang 《Journal of biomedical science》2012,19(1):48
Background
Whereas brain death is a vitally important clinical phenomenon, our contemporary understanding on its underlying cellular mechanisms remains elusive. This study evaluated whether the ubiquitin-proteasome system (UPS) in the rostral ventrolateral medulla (RVLM), a neural substrate that our laboratory identified previously to be intimately related to brain death, is engaged in this fatal process.Methods
We performed proteomics, Western Blot, real-time PCR, ELISA and pharmacological experiments in conjunction with a clinically relevant experimental endotoxemia model of brain death based on intravenous administration of Escherichia coli lipopolysaccharide in adult male Sprague–Dawley rats.Results
Proteomics, Western blot and enzyme activity analyses demonstrated that polyubiquitination was preserved and de-ubiquitination by ubiquitin C-terminal hydrolase isozyme-L1 (UCH-L1) was sustained, alongside increased monoubiquitin availability or proteasome activity in RVLM over the course of experimental endotoxemia. However, real-time PCR revealed no significant alteration in proteasome subunit alpha type-1, ubiquitin or UCH-L1 at mRNA level. Functionally, whereas microinjection into the bilateral RVLM of proteasome inhibitors (lactacystin or proteasome inhibitor II) potentiated survival, an inhibitor of ubiquitin-recycling (ubiquitin aldehyde) or an UCH-L1 inhibitor exacerbated mortality.Conclusions
We proposed previously that the progression towards brain death entails a tug-of-war between pro-death and pro-life programs in RVLM. It is conceivable that ubiquitination or de-ubiquitination in RVLM participate in brain death by regulating the degradation of the proteins involved in those programs. 相似文献9.
Background
Bacillus anthracis is the bacterium responsible for causing anthrax. The ability of B. anthracis to cause disease is dependent on a secreted virulence factor, lethal toxin, that promotes survival of the bacteria in the host by impairing the immune response. A well-studied effect of lethal toxin is the killing of macrophages, although the molecular mechanisms involved have not been fully characterized.Methodology/Principal Findings
Here, we demonstrate that celastrol, a quinone methide triterpene derived from a plant extract used in herbal medicine, inhibits lethal toxin-induced death of RAW264.7 murine macrophages. Celastrol did not prevent cleavage of mitogen activated protein kinase kinase 1, a cytosolic target of the toxin, indicating that it did not inhibit the uptake or catalytic activity of lethal toxin. Surprisingly, celastrol conferred almost complete protection when it was added up to 1.5 h after intoxication, indicating that it could rescue cells in the late stages of intoxication. Since the activity of the proteasome has been implicated in intoxication using other pharmacological agents, we tested whether celastrol blocked proteasome activity. We found that celastrol inhibited the proteasome-dependent degradation of proteins in RAW264.7 cells, but only slightly inhibited proteasome-mediated cleavage of fluorogenic substrates in vitro. Furthermore, celastrol blocked stimulation of IL-18 processing, indicating that celastrol acted upstream of inflammasome activation.Conclusions/Significance
This work identifies celastrol as an inhibitor of lethal toxin-mediated macrophage lysis and suggests an inhibitory mechanism involving inhibition of the proteasome pathway. 相似文献10.
Stephanie Smith Jennifer Fox Marco Mejia Wanvipa Ruangpradit Alihossein Saberi Sunmi Kim Yongjun Choi Sehyun Oh Yucai Wang Kyungho Choi Lei Li Eric A. Hendrickson Shunichi Takeda Mark Muller Kyungjae Myung 《PloS one》2014,9(1)
Background
We have previously used the ATAD5-luciferase high-throughput screening assay to identify genotoxic compounds with potential chemotherapeutic capabilities. The successful identification of known genotoxic agents, including the histone deacetylase inhibitor (HDACi) trichostatin A (TSA), confirmed the specificity of the screen since TSA has been widely studied for its ability to cause apoptosis in cancer cells. Because many cancers have acquired mutations in DNA damage checkpoints or repair pathways, we hypothesized that these cancers may be susceptible to treatments that target compensatory pathways. Here, we used a panel of isogenic chicken DT40 B lymphocyte mutant and human cell lines to investigate the ability of TSA to define selective pathways that promote HDACi toxicity.Results
HDACi induced a DNA damage response and reduced viability in all repair deficient DT40 mutants although ATM-nulls were least affected. The most dramatic sensitivity was observed in mutants lacking the homology dependent repair (HDR) factor BLM or the non-homologous end-joining (NHEJ) and HDR factors, KU/RAD54, suggesting an involvement of either HDR or NHEJ in HDACi-induced cell death. To extend these findings, we measured the frequencies of HDR and NHEJ after HDACi treatment and monitored viability in human cell lines comparably deficient in HDR or NHEJ. Although no difference in HDR frequency was observed between HDACi treated and untreated cells, HDR-defective human cell lines were clearly more sensitive than wild type. Unexpectedly, cells treated with HDACis showed a significantly elevated NHEJ frequency.Conclusions
HDACi targeting drugs induced significant increases in NHEJ activity in human cell lines but did not alter HDR frequency. Moreover, HDR is required for cellular resistance to HDACi therapy; therefore, NHEJ does not appear to be a critical axis for HDACi resistance. Rather, HDACi compounds induced DNA damage, most likely double strand breaks (DSBs), and HDR proficiency is correlated with cell survival. 相似文献11.
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Marrocco K Criqui MC Zervudacki J Schott G Eisler H Parnet A Dunoyer P Genschik P 《PloS one》2012,7(4):e35173
Background
Selective protein degradation via the ubiquitin-26S proteasome is a major mechanism underlying DNA replication and cell division in all Eukaryotes. In particular, the APC/C (Anaphase Promoting Complex or Cyclosome) is a master ubiquitin protein ligase (E3) that targets regulatory proteins for degradation allowing sister chromatid separation and exit from mitosis. Interestingly, recent work also indicates that the APC/C remains active in differentiated animal and plant cells. However, its role in post-mitotic cells remains elusive and only a few substrates have been characterized.Methodology/Principal Findings
In order to identify novel APC/C substrates, we performed a yeast two-hybrid screen using as the bait Arabidopsis APC10/DOC1, one core subunit of the APC/C, which is required for substrate recruitment. This screen identified DRB4, a double-stranded RNA binding protein involved in the biogenesis of different classes of small RNA (sRNA). This protein interaction was further confirmed in vitro and in plant cells. Moreover, APC10 interacts with DRB4 through the second dsRNA binding motif (dsRBD2) of DRB4, which is also required for its homodimerization and binding to its Dicer partner DCL4. We further showed that DRB4 protein accumulates when the proteasome is inactivated and, most importantly, we found that DRB4 stability depends on APC/C activity. Hence, depletion of Arabidopsis APC/C activity by RNAi leads to a strong accumulation of endogenous DRB4, far beyond its normal level of accumulation. However, we could not detect any defects in sRNA production in lines where DRB4 was overexpressed.Conclusions/Significance
Our work identified a first plant substrate of the APC/C, which is not a regulator of the cell cycle. Though we cannot exclude that APC/C-dependent degradation of DRB4 has some regulatory roles under specific growth conditions, our work rather points to a housekeeping function of APC/C in maintaining precise cellular-protein concentrations and homeostasis of DRB4. 相似文献14.
Alison M Connor Nizar Mahomed Rajiv Gandhi Edward C Keystone Stuart A Berger 《Arthritis research & therapy》2012,14(2):R62-19
Introduction
Rheumatoid arthritis (RA) is a chronic inflammatory and destructive disease of the joint. The synovial lining consists of two main types of cells: synovial fibroblasts and macrophages. The macrophage-derived cytokine TNFα stimulates RA synovial fibroblasts to proliferate and produce growth factors, chemokines, proteinases and adhesion molecules, making them key players in the RA disease process. If proteins are not correctly folded, cellular stress occurs that can be relieved in part by increased degradation of the aberrant proteins by the proteasome or autophagy. We hypothesized that the activity of the protein degradation pathways would be increased in response to TNFα stimulation in RA synovial fibroblasts compared with control fibroblasts.Methods
Endoplasmic reticulum (ER) stress markers were examined in synovial fibroblasts by immunoblotting and PCR. Use of the autophagy and proteasome protein degradation pathways in response to TNFα stimulation was determined using a combination of experiments involving chemical inhibition of the autophagy or proteasome pathways followed by immunoblotting for the autophagy marker LC3, measurement of proteasome activity and long-lived protein degradation, and determination of cellular viability.Results
RA synovial fibroblasts are under acute ER stress, and the stress is increased in the presence of TNFα. Autophagy is the main pathway used to relieve the ER stress in unstimulated fibroblasts, and both autophagy and the proteasome are more active in RA synovial fibroblasts compared with control fibroblasts. In response to TNFα, the autophagy pathway but not the proteasome is consistently stimulated, yet there is an increased dependence on the proteasome for cell viability. If autophagy is blocked in the presence of TNFα, an increase in proteasome activity occurs in RA synovial fibroblasts but not in control cells.Conclusions
TNFα stimulation of synovial fibroblasts results in increased expression of ER stress markers. Survival of synovial fibroblasts is dependent on continuous removal of proteins by both the lysosome/autophagy and ubiquitin/proteasome protein degradation pathways. Both pathways are more active in RA synovial fibroblasts compared with control fibroblasts. These results may provide a better understanding of the mechanism of TNFα on prolonging the survival of synovial fibroblasts in RA tissue. 相似文献15.
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Background
The proteasome is a multi-subunit protein machine that is the final destination for cellular proteins that have been marked for degradation via an ubiquitin (Ub) chain appendage. These ubiquitylated proteins either bind directly to the intrinsic proteasome ubiqutin chain receptors Rpn10, Rpn13, or Rpt5, or are shuttled to the proteasome by Rad23, Dsk2, or Ddi1. The latter proteins share an Ub association domain (UBA) for binding poly-Ub chains and an Ub-like-domain (UBL) for binding to the proteasome. It has been proposed that shuttling receptors dock on the proteasome via Rpn1, but the precise nature of the docking site remains poorly defined.Results
To shed light on the recruitment of shuttling receptors to the proteasome, we performed both site-directed mutagenesis and genetic screening to identify mutations in Rpn1 that disrupt its binding to UBA-UBL proteins. Here we demonstrate that delivery of Ub conjugates and docking of Ddi1 (and to a lesser extent Dsk2) to the proteasome are strongly impaired by an aspartic acid to alanine point mutation in the highly-conserved D517 residue of Rpn1. Moreover, degradation of the Ddi1-dependent proteasome substrate, Ufo1, is blocked in rpn1-D517A yeast cells. By contrast, Rad23 recruitment to the proteasome is not affected by rpn1-D517A.Conclusions
These studies provide insight into the mechanism by which the UBA-UBL protein Ddi1 is recruited to the proteasome to enable Ub-dependent degradation of its ligands. Our studies suggest that different UBA-UBL proteins are recruited to the proteasome by distinct mechanisms. 相似文献18.
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Background
Existing in Permanent-wave solutions (PWS), thioglycolic acid (TGA) is widely used in hairdressing industry for its contribution to hair styling. However, the toxicity of TGA, especially its reproductive toxicity, gradually calls the attention of more and more researchers.Method
In this work, xenopus oocytes were pretreated with different concentration of TGA, and then activated by calcium ionophore A23187. During culture, the oocytes activation rates were taken note at different time after adding calcium ionophore A23187. At the end of the culture period, the nuclear status was detected under confocal microscope. In addition, some other samples were collected for Western-Blotting analysis.Result
TGA significantly inhibited the oocytes activation rate and pronuclear formation. It may be resulted from the inhibition of the degradation of p-ERK1, Mos and CyclinB2.Conclusion
TGA inhibits in vitro parthenogenetic activation of xenopus oocytes with inhibited the degradation of proteins involved in mitogenic-activated protein kinase (MAPK) and maturation-promoting factor (MPF) pathways. 相似文献20.