GART Functions as a Novel Methyltransferase in the RUVBL1/ß-Catenin Signaling Pathway to Promote Tumor Stemness in Colorectal Cancer.

Tumor stemness is associated with the recurrence and incurability of colorectal cancer (CRC), which lacks effective therapeutic targets and drugs. Glycinamide ribonucleotide transformylase (GART) fulfills an important role in numerous types of malignancies. The present study aims to identify the underlying mechanism through which GART may promote CRC stemness, as to developing novel therapeutic methods. An elevated level of GART is associated with poor outcomes in CRC patients and promotes the proliferation and migration of CRC cells. CD133+ cells with increased GART expression possess higher tumorigenic and proliferative capabilities both in vitro and in vivo. GART is identified to have a novel methyltransferase function, whose enzymatic activity center is located at the E948 site. GART also enhances the stability of RuvB-like AAA ATPase 1 (RUVBL1) through methylating its K7 site, which consequently aberrantly activates the Wnt/ß-catenin signaling pathway to induce tumor stemness. Pemetrexed (PEM), a compound targeting GART, combined with other chemotherapy drugs greatly suppresses tumor growth both in a PDX model and in CRC patients. The present study demonstrates a novel methyltransferase function of GART and the role of the GART/RUVBL1/ß-catenin signaling axis in promoting CRC stemness. PEM may be a promising therapeutic agent for the treatment of CRC.

MiR-199a-5p-containing macrophage-derived extracellular vesicles inhibit SMARCA4 and alleviate atherosclerosis by reducing endothelial cell pyroptosis.

BACKGROUND: Endothelial cell disturbance underpins a role in pathogenesis of atherosclerosis. Notably, accumulating studies indicate the substantial role of microRNAs (miRs) in atherosclerosis, and miR-199a-5p dysregulation has been associated with atherosclerosis and other cardiovascular disorders. However, the effect of miR-199a-5p on the phenotypes of endothelial cells and atherosclerosis remains largely unknown. METHODS: ApoE-/- male mice were fed with high-fat diet for detection of inflammation and aorta plaque area. Extracellular vesicles (EVs) were separated from THP-1-derived macrophage (THP-1-DM) that was treated by oxidized low-density lipoprotein, followed by co-culture with human aortic endothelial cells (HAECs). Ectopic expression and downregulation of miR-199a-5p were done in THP-1-DM-derived EVs to assess pyroptosis and lactate dehydrogenase (LDH) of HAECs. Binding relationship between miR-199a-5p and SMARCA4 was evaluated by luciferase activity assay. RESULTS: EVs derived from ox-LDL-induced THP-1-DM expedited inflammation and aorta plaque area in atherosclerotic mice. Besides, miR-199a-5p expression was reduced in EVs from ox-LDL-induced THP-1-DM, and miR-199a-5p inhibition facilitated HAEC pyroptosis and LDH activity. Moreover, miR-199a-5p targeted and restricted SMARCA4, and then SMARCA4 activated the NF-κB pathway by increasing PODXL expression in HAECs. CONCLUSION: EV-packaged inhibited miR-199a-5p from macrophages expedites endothelial cell pyroptosis and further accelerates atherosclerosis through the SMARCA4/PODXL/NF-κB axis, providing promising targets and strategies for the prevention and treatment of atherosclerosis.

Antiviral activity and underlying mechanisms of baicalin against avian infectious bronchitis virus in vitro.

Baicalin, a flavonoid compound extracted from the dry root of Scutellaria baicalensis Georgi, has been shown to have anti-inflammation, anti-viral, anti-bacterial, and immunomodulatory activity. However, the effect of baicalin against avian infectious bronchitis virus (IBV) remains unknown. The purpose of this study was to investigate the anti-IBV activity and underlying mechanism of baicalin in vitro. The results showed that baicalin has a direct virucidal effect but no prophylactic effect on IBV infection. The mRNA and protein of IBV N were decreased significantly when IBV-infected cells were treated with baicalin during the multiple stages of the virus replication cycle, including viral adsorption, invasion, internalization, and release. Stress granule (SG) formation resulted from the increase of G3BP1 and the phosphorylation of the PKR/eIF2α due to the treatment of IBV-infected cells with baicalin. The inhibitory activity of baicalin on IBV replication was increased when G3BP1 expression was inhibited, and the down-regulation of G3BP1 expression occurred when the expression of PKR and eIF2α was inhibited. These findings revealed that baicalin activates phosphorylation of the PKR/eIF2α pathway and induces SG formation by targeting G3BP1, initiating the antiviral response to suppress IBV replication in Vero cells. The results suggest that baicalin is a promising candidate drug to treat or prevent IBV infection.RESEARCH HIGHLIGHTS Baicalin inhibits IBV replication by reducing IBV N protein and mRNA.Baicalin disturbs multiple stages of the IBV life cycle.Baicalin activates PKR/eIF2α pathway and induces stress granule formation to exert anti-IBV response.

Phillygenin activates PKR/eIF2α pathway and induces stress granule to exert anti-avian infectious bronchitis virus.

The prevalence of avian infectious bronchitis virus (IBV) is still one of causes inducing severe losses of production in the poultry industry worldwide. Vaccination does not completely prevent IBV infection and spread due to immune failure and viral mutations. ForsythiaeFructus and its compounds have been widely used in a lot of prescriptions of the traditional Chinese medicine for a long history, and it is well-known as safety and efficiency in heat-clearing and detoxifying. This study aims to investigate the anti-IBV activity and mechanism of phillygenin. The results showed that phillygenin inhibited IBV replication by disturbing multiple stages of the virus life cycle, including viral adsorption, invasion, internalization, and release in Vero cells. After being treated with 100, 125 and 150 µg/mL phillygenin, the expression of G3BP1 was significantly increased and the phosphorylation of PKR/eIF2α was activated, which increased stress granule, thereby triggering the antiviral response in Vero cells. The anti-virus activity of PHI was decreased when G3BP1 was interfered by si-RNA, and G3BP1 was down-regulated when PKR/eIF2α was interfered by si-RNA. In conclusion, our findings indicate that phillygenin activates PKR/eIF2α pathway and induces stress granule formation to exert anti-IBV, which holds promise to develop into a novel anti-IBV drug. Further study in vivo is needed to explore phillygenin as a potential and effective drug to prevent IB in poultry.

Joint effects of antibiotics and quorum sensing inhibitors on resistance development in bacteria.

Quorum sensing inhibitors (QSIs) are promising alternatives to antibiotics. While QSIs have great application potential in a variety of fields, their joint effects with antibiotics on bacteria, especially on antibiotic resistance mutations, remain largely unexplored. Herein, we report the joint effects of four commonly used antibiotics and two QSIs on bacterial growth and resistance mutations in E. coli. It was found that QSIs presented antagonistic or additive effects with antibiotics on bacterial growth, and more importantly, QSIs exhibited an attenuating effect on antibiotic-induced resistance mutations. Further analysis demonstrated that antibiotics might enhance resistance mutations by promoting the expressions of rpoS, lexA and recA, while QSIs attenuated the mutations by promoting the expressions of mutS and uvrD. The present research provides a comprehensive understanding of the joint effects of antibiotics and QSIs on bacteria, which may benefit the risk assessment of their combined exposure.

RUVBL1, a novel C-RAF-binding protein, activates the RAF/MEK/ERK pathway to promote lung cancer tumorigenesis.

Lung cancer remains the leading cause of cancer-related deaths in the world. The RAF/MEK/ERK pathway controls many fundamental cellular functions and plays key roles in lung carcinogenesis. However, the proteins that regulate this pathway remain largely unknown. Here, we identified a novel C-RAF-binding protein, RUVBL1, which activates the RAF/MEK/ERK pathway by inhibiting phosphorylation of the C-RAF protein at serine 259. RUVBL1 expression was elevated in lung adenocarcinoma tissues. In addition, knocking out RUVBL1 effectively inhibited the proliferation and invasion of A549 cells. In vivo experiments, RUVBL1 deficiency significantly decreased the tumorigensis of lung cancer. In conclusion, we have shown that RUVBL1 could activate the RAF/MEK/ERK pathway by inhibiting phosphorylation of the C-RAF protein at serine 259, to promote lung cancer progression. Therefore, RUVBL1 could represent a novel therapeutic target for lung cancer treatment.

Functional amyloids as inhibitors of plasmid DNA replication.

DNA replication is tightly regulated to constrain the genetic material within strict spatiotemporal boundaries and copy numbers. Bacterial plasmids are autonomously replicating DNA molecules of much clinical, environmental and biotechnological interest. A mechanism used by plasmids to prevent over-replication is 'handcuffing', i.e. inactivating the replication origins in two DNA molecules by holding them together through a bridge built by a plasmid-encoded initiator protein (Rep). Besides being involved in handcuffing, the WH1 domain in the RepA protein assembles as amyloid fibres upon binding to DNA in vitro. The amyloid state in proteins is linked to specific human diseases, but determines selectable and epigenetically transmissible phenotypes in microorganisms. Here we have explored the connection between handcuffing and amyloidogenesis of full-length RepA. Using a monoclonal antibody specific for an amyloidogenic conformation of RepA-WH1, we have found that the handcuffed RepA assemblies, either reconstructed in vitro or in plasmids clustering at the bacterial nucleoid, are amyloidogenic. The replication-inhibitory RepA handcuff assembly is, to our knowledge, the first protein amyloid directly dealing with DNA. Built on an amyloid scaffold, bacterial plasmid handcuffs can bring a novel molecular solution to the universal problem of keeping control on DNA replication initiation.

Overexpression of Twinkle-helicase protects cardiomyocytes from genotoxic stress caused by reactive oxygen species.

Mitochondrial DNA (mtDNA) in adult human heart is characterized by complex molecular forms held together by junctional molecules of unknown biological significance. These junctions are not present in mouse hearts and emerge in humans during postnatal development, concomitant with increased demand for oxidative metabolism. To analyze the role of mtDNA organization during oxidative stress in cardiomyocytes, we used a mouse model, which recapitulates the complex mtDNA organization of human hearts by overexpression of the mitochondrial helicase, TWINKLE. Overexpression of TWINKLE rescued the oxidative damage induced replication stalling of mtDNA, reduced mtDNA point mutation load, and modified mtDNA rearrangements in heterozygous mitochondrial superoxide dismutase knockout hearts, as well as ameliorated cardiomyopathy in mice superoxide dismutase knockout in a p21-dependent manner. We conclude that mtDNA integrity influences cell survival and reason that tissue specific modes of mtDNA maintenance represent an adaptation to oxidative stress.

Influence of retinoblastoma-related gene silencing on the initiation of DNA replication by African cassava mosaic virus Rep in cells of mature leaves in Nicotiana benthamiana plants.

BACKGROUND: Geminiviruses mainly infect terminally differentiated tissues and cells in plants. They need to reprogramme host cellular machinery for DNA replication. This process is thought to be mediated by inactivation of cell-cycle repressor proteins and by induction of host DNA synthesis protein expression through actions of the geminviral replication initiator protein (Rep). FINDINGS: Exploiting a Nicotiana benthamiana pOri2 line, which is transformed with a transgene consisting of a direct repeat of the African cassava mosaic virus (ACMV)-replication origin (Ori) flanking a non-viral DNA region, and virus-induced RNA silencing (VIGS), the impact of host gene expression on replication of the ACMV-derived replicon was investigated. The ACMV Rep trans-replicated the viral episomal replicon in leaves of young but not older pOri2 plants. Upon VIGS-mediated down-regulation of N. benthamiana NbRBR1, the retinoblastoma-related protein gene coding for a negative cell-cycle suppressor, recovered the ability of ACMV Rep for trans DNA replication, whereas the silencing of NbPCNA coding for the sliding clamp of DNA polymerase had no effect. CONCLUSIONS: These results suggest that the cellular machinery for DNA replication in differentiated tissues of older leaves cannot be reprogrammed by Rep alone but may need other uncharacterised viral and plant factors.

Lack of effect of RuvB-like proteins on DNA damage signaling activation.

Ataxia telangiectasia mutated (ATM) kinase is a central player in cellular response to DNA damage. Phosphorylation of the histone H2AX by ATM is required for the accumulation of repair proteins at the sites of double-strand breaks. Recently, it was reported that the histone acetyltransferase Tat interactive protein-60 (IPP60) is required to acetylate ATM prior to its activation. The RuvB-like proteins TIP48 and TIP49 are known to be necessary for the assembly and functional activity of the TIP60 acetyltransferase complex. In the present communication, we investigated the requirements of IIP48 and IIP49 for ATM activation by monitoring the cell cycle distribution and H2AX phosphorylation after irradiation of IIP48- and IIP49-depleted cells. We found that neither the cell cycle norgammay-H2AX were affected in IIP48- and IIP49-silenced cells, suggesting that the IIP60 chromatin modification complex is not engaged in DNA damage signaling upstream of ATM.

UvrD303, a hyperhelicase mutant that antagonizes RecA-dependent SOS expression by a mechanism that depends on its C terminus.

Genomic integrity is critical for an organism's survival and ability to reproduce. In Escherichia coli, the UvrD helicase has roles in nucleotide excision repair and methyl-directed mismatch repair and can limit reactions by RecA under certain circumstances. UvrD303 (D403A D404A) is a hyperhelicase mutant, and when expressed from a multicopy plasmid, it results in UV sensitivity (UV(s)), recombination deficiency, and antimutability. In order to understand the molecular mechanism underlying the UV(s) phenotype of uvrD303 cells, this mutation was transferred to the E. coli chromosome and studied in single copy. It is shown here that uvrD303 mutants are UV sensitive, recombination deficient, and antimutable and additionally have a moderate defect in inducing the SOS response after UV treatment. The UV-sensitive phenotype is epistatic with recA and additive with uvrA and is partially suppressed by removing the LexA repressor. Furthermore, uvrD303 is able to inhibit constitutive SOS expression caused by the recA730 mutation. The ability of UvrD303 to antagonize SOS expression was dependent on its 40 C-terminal amino acids. It is proposed that UvrD303, via its C terminus, can decrease the levels of RecA activity in the cell.

Helicases as antiviral drug targets.

1. We have demonstrated for the first time that the helicase of a ribonucleic acid virus, the SARS coronavirus (SARS-CoV), is a valid target for drug development. 2. Using high throughput screen and chemical synthesis, several lead compounds targeting the SARS-CoV helicase have been identified. We have shown that these compounds can inhibit SARS-CoV helicase activity and viral growth in cell culture systems. These compounds can potentially be used to target other viruses.

Prohibitin and the SWI/SNF ATPase subunit BRG1 are required for effective androgen antagonist-mediated transcriptional repression of androgen receptor-regulated genes.

Androgen antagonists or androgen deprivation are the primary therapeutic modalities for the treatment of prostate cancer. Invariably, however, the disease becomes progressive and unresponsive to androgen ablation therapy (hormone refractory). The molecular mechanisms by which androgen antagonists inhibit prostate cancer proliferation are not fully defined. In this study, we identify two molecules which are required for effective prostate cancer cell responsiveness to androgen antagonists. We establish that androgen receptor (AR)-dependent transcriptional suppression by androgen antagonists requires the tumor suppressor prohibitin. This requirement for prohibitin was demonstrated using structurally-distinct androgen antagonists, stable and transient knockdown of prohibitin and transfected and endogenous AR-responsive genes. The SWI-SNF complex core ATPase BRG1, but not its closely-related counterpart ATPase BRM, is required for this repressive action of prohibitin on AR-responsive promoters. Androgen antagonists induce recruitment of prohibitin and BRG1 to endogenous AR-responsive promoters and induce a physical association between AR and prohibitin and BRG1. The recruitment of prohibitin to endogenous AR-responsive promoters is dependent upon antagonist-bound AR. Prohibitin binding in the prostate-specific antigen (PSA) promoter results in the recruitment of BRG1 and the dissociation of p300 from the PSA promoter. These findings suggest that prohibitin may function through BRG1-mediated local chromatin remodeling activity and the removal of p300-mediated acetylation to produce androgen antagonist-mediated transcriptional repression. Furthermore, in addition to its necessary role in AR-mediated transcriptional repression, we demonstrate that prohibitin is required for full and efficient androgen antagonist-mediated growth suppression of prostate cancer cells.

The fission yeast pfh1(+) gene encodes an essential 5' to 3' DNA helicase required for the completion of S-phase.

The Cdc24 protein plays an essential role in chromosomal DNA replication in the fission yeast Schizosaccharomyces pombe, most likely via its direct interaction with Dna2, a conserved endonuclease-helicase protein required for Okazaki fragment processing. To gain insights into Cdc24 function, we isolated cold-sensitive chromosomal suppressors of the temperature-sensitive cdc24-M38 allele. One of the complementation groups of such suppressors defined a novel gene, pfh1(+), encoding an 805 amino acid nuclear protein highly homologous to the Saccharomyces cerevisiae Pif1p and Rrm3p DNA helicase family proteins. The purified Pfh1 protein displayed single-stranded DNA-dependent ATPase activity as well as 5' to 3' DNA helicase activity in vitro. Reverse genetic analysis in S.pombe showed that helicase activity was essential for the function of the Pfh1 protein in vivo. Schizosaccharomyces pombe cells carrying the cold-sensitive pfh1-R20 allele underwent cell cycle arrest in late S/G2-phase of the cell cycle when shifted to the restrictive temperature. This arrest was dependent upon the presence of a functional late S/G2 DNA damage checkpoint, suggesting that Pfh1 is required for the completion of DNA replication. Furthermore, at their permissive temperature pfh1-R20 cells were highly sensitive to the DNA-alkylating agent methyl methanesulphonate, implying a further role for Pfh1 in the repair of DNA damage.

The Bloom's syndrome helicase stimulates the activity of human topoisomerase IIIalpha.

Bloom's syndrome (BS) is a disorder associated with chromosomal instability and a predisposition to the development of cancer. The BS gene product, BLM, is a DNA helicase of the RecQ family that forms a complex in vitro and in vivo with topoisomerase IIIalpha. Here, we show that BLM stimulates the ability of topoisomerase IIIalpha to relax negatively supercoiled DNA. Moreover, DNA binding analyses indicate that BLM recruits topoisomerase IIIalpha to its DNA substrate. Consistent with this, a mutant form of BLM that retains helicase activity, but is unable to bind topoisomerase IIIalpha, fails to stimulate topoisomerase activity. These results indicate that a physical association between BLM and topoisomerase IIIalpha is a prerequisite for their functional biochemical interaction.

XPD codon 751 polymorphism, metabolism genes, smoking, and bladder cancer risk.

Cigarette smoking is the main risk factor for bladder cancer, accounting for at least 50% of bladder cancer in men. Cigarette smoke is a rich source of arylamines, which are detoxified by the NAT2 enzyme and activated by the NAT1 enzyme to highly reactive species that can form bulky adducts on DNA. DNA damage from such adducts is mainly repaired by the nucleotide excision repair pathway, in which the XPD protein functions in opening the DNA helix. We hypothesized that an XPD codon 751 polymorphism (Lys-to-Gln amino acid change) could affect the repair of smoking-induced DNA damage and could be associated with bladder-cancer risk. We also hypothesized that allelic variants of the NAT1 and NAT2 genes might modify the effect of the XPD codon 751 polymorphism on smoking-associated bladder-cancer risk. We determined the XPD codon 751 genotype for 228 bladder-cancer cases and 210 controls who were frequency-matched to cases by age, sex, and ethnicity, and we used our previously published data on the NAT1 and NAT2 genotypes for these same individuals (J. A. Taylor et al., Cancer Res., 58: 3603-3610, 1998). We found a slight decrease in risk for the XPD codon 751 Gln/Gln genotype (adjusted odds ratio: 0.8; 95% confidence interval: 0.4-1.3) compared with subjects with the Lys/Lys or Lys/Gln genotypes. The analysis with smoking showed that smokers with the Lys/Lys or Lys/Gln genotypes were twice as likely to have bladder cancer than smokers with the Gln/Gln genotype (test of interaction P = 0.03). The combined presence of the NAT1/NAT2 high-risk genotype and the XPD Lys/Lys or Lys/Gln genotypes ignoring smoking had an odds ratio that was only slightly higher than expected, assuming no genotype-genotype interaction (P = 0.52). We found little evidence for a gene-gene-exposure, three-way interaction among the XPD codon 751 genotype, smoking, and the NAT1/NAT2 genotype.

1-Methyl-4-phenylpyridinium ion, a toxin that can cause parkinsonism, alters branched structures of DNA.

During replication, human mitochondrial DNA (mtDNA) takes on a triple-stranded structure known as a D-loop, which is implicated in replication and transcription. 1-Methyl-4-phenylpyridinium ion (MPP+), a toxin inducing parkinsonism, inhibits mtDNA replication, possibly by resolving the D-loops. For initiation of mtDNA replication, mitochondria are thought to have another triple-stranded structure, an R-loop. The R-loop, which is resolved by a bacterial junction-specific helicase, RecG, is also resolved by MPP+. Because mitochondrial D-loops are likewise resolved by RecG, the D- and R-loops may share a similar branched structure. MPP+ resolves cruciform DNA in supercoiled DNA. MPP+ converts a stacked conformation to an extended conformation in a synthetic Holliday junction. This conversion is reversed by 1 mM Mg(2+), as is the resolution of the D-loops or cruciform DNA. These observations suggest that the junction structure of mitochondrial D- and R-loops is affected by MPP+.

Genetic evidence that the elevated levels of Escherichia coli helicase II antagonize recombinational DNA repair.

Some phages survive irradiation much better upon multiple than upon single infection, a phenomenon known as multiplicity reactivation (MR). Long ago MR of UV-irradiated lambda red phage in E. coli cells was shown to be a manifestation of recA-dependent recombinational DNA repair. We used this experimental model to assess the influence of helicase II on the type of recombinational repair responsible for MR. Since helicase II is encoded by the SOS-inducible uvrD gene, SOS-inducing treatments such as irradiating recA(+) or heating recA441 cells were used. We found: i) that MR was abolished by the SOS-inducing treatments; ii) that in uvrD background these treatments did not affect MR; and iii) that the presence of a high-copy plasmid vector carrying the uvrD(+) allele together with its natural promoter region was sufficient to block MR. From these results we infer that helicase II is able to antagonize the type of recA-dependent recombinational repair acting on multiple copies of UV-damaged lambda DNA and that its anti-recombinogenic activity is operative at elevated levels only.

Virulence gene regulation by peptides in staphylococci and other Gram-positive bacteria.

In staphylococci, autoinducing peptides activate agr. a global regulator of the expression of genes encoding virulence factors and other exoproteins. During the past year, there have been major advances in the structure-function analysis of these peptides and the regulation of a virulence factor by an autoinducing peptide in pneumococci has been demonstrated.

[Specific control of gene expression by oligonucleotides: from chemistry to clinical aspects]. / Le contrôle spécifique de l'expression des gènes par les oligonucléotides: de la chimie à la clinique.

Oligonucleotides can be synthesized for specific regulation of gene expression within the living cell. The oligonucleotide acts either on messenger RNA (nonsense and ribozyme strategies) or on DNA (triple helix or antigene strategy). This pharmacological approache requires chemically modified oligonucleotides and appropriate vectors. Applications in gene therapy can also be developed using a DNA vector to produce regulator RNA (nonsense, ribozyme, antigene) within the same cells. Early clinical trials are being conducted to determine the therapeutic efficacy of these two approaches.