DNA Binding Activity Research Articles

Acetylation of xenogeneic silencer H-NS regulates biofilm development through the nitrogen homeostasis regulator in Shewanella.

Adjusting intracellular metabolic pathways and adopting suitable live state such as biofilms, are crucial for bacteria to survive environmental changes. Although substantial progress has been made in understanding how the histone-like nucleoid-structuring (H-NS) protein modulates the expression of the genes involved in biofilm formation, the precise modification that the H-NS protein undergoes to alter its DNA binding activity is still largely uncharacterized. This study revealed that acetylation of H-NS at Lys19 inhibits biofilm development in Shewanella oneidensis MR-1 by downregulating the expression of glutamine synthetase, a critical enzyme in glutamine synthesis. We further found that nitrogen starvation, a likely condition in biofilm development, induces deacetylation of H-NS and the trimerization of nitrogen assimilation regulator GlnB. The acetylated H-NS strain exhibits significantly lower cellular glutamine concentration, emphasizing the requirement of H-NS deacetylation in Shewanella biofilm development. Moreover, we discovered in vivo that the activation of glutamine biosynthesis pathway and the concurrent suppression of the arginine synthesis pathway during both pellicle and attached biofilms development, further suggesting the importance of fine tune nitrogen assimilation by H-NS acetylation in Shewanella. In summary, posttranslational modification of H-NS endows Shewanella with the ability to respond to environmental needs by adjusting the intracellular metabolism pathways.

Chemical Engineering of Artificial Transcription Factors by Orthogonal Palladium(II)‐Mediated S‐Arylation Reactions

AbstractSite‐selective functionalization strategies are in high demand to prepare well‐defined homogeneous proteins for basic research and biomedical applications. In this regard, cysteine‐based reactions have enabled a broad set of transformations to produce modified proteins for various applications. However, these approaches were mainly employed to modify a single reactive site with a specific transformation. Achieving site selectivity or multiple transformations, essential for preparing complex biomolecules, remains challenging. Herein we demonstrate the power of combining palladium(II)‐mediated C−S bond formation and C−S bond cleavage reactions to selectively edit desired cysteine sites in complex and uniquely modified proteins. We developed an orthogonal palladium(II) strategy for rapid and effective diversification of multiple cysteine sites (3–6 residues) with various transformations. Importantly, we employed our approach to prepare 10 complex analogues, including modified, stapled, and multimeric proteins on a milligram scale. Furthermore, we also synthesized a focused library of stabilized artificial transcription factors that displayed enhanced stability and potent DNA binding activity. Our approach enables rapid and effective protein editing and opens new avenues to engineer new biomolecules for fundamental research and therapeutic applications.

Chemical Engineering of Artificial Transcription Factors by Orthogonal Palladium(II)-Mediated S-Arylation Reactions.

Site-selective functionalization strategies are in high demand to prepare well-defined homogeneous proteins for basic research and biomedical applications. In this regard, cysteine-based reactions have enabled a broad set of transformations to produce modified proteins for various applications. However, these approaches were mainly employed to modify a single reactive site with a specific transformation. Achieving site selectivity or multiple transformations, essential for preparing complex biomolecules, remains challenging. Herein we demonstrate the power of combining palladium(II)-mediated C-S bond formation and C-S bond cleavage reactions to selectively edit desired cysteine sites in complex and uniquely modified proteins. We developed an orthogonal palladium(II) strategy for rapid and effective diversification of multiple cysteine sites (3-6 residues) with various transformations. Importantly, we employed our approach to prepare 10 complex analogues, including modified, stapled, and multimeric proteins on a milligram scale. Furthermore, we also synthesized a focused library of stabilized artificial transcription factors that displayed enhanced stability and potent DNA binding activity. Our approach enables rapid and effective protein editing and opens new avenues to engineer new biomolecules for fundamental research and therapeutic applications.

Targeted mutagenesis of the β-strand DNA binding region of African swine fever virus histone-like protein (pA104R) impairs DNA-binding activity and antibody recognition

African Swine Fever (ASF) is a highly contagious disease caused by a double-stranded DNA virus (ASFV). Despite significant advances made over the last decade, issues such as residual virulence and absence of differentiating infected from vaccinated animals (DIVA) capacity remain an obstacle in the development of live attenuated vaccines (LAVs) against ASFV. It is, therefore, necessary to identify novel strategies to improve vaccine safety, by rational mutagenesis of virulence associated genes and generation of DIVA markers. ASFV encodes a HU (histone-like protein from E. coli strain U93) homolog protein, pA104R, which is involved in viral genome assembly and host immune recognition. A phylogenetic analysis revealed that pA104R is highly conserved among ASFV isolates, suggesting that it can be a good target for vaccine design. Thus, we selectively mutated the β-strand DNA binding region (BDR) of pA104R to attenuate its enzymatic activity, and identified and mutated several B-cell epitopes present in pA104R to generate a negative marker. Residues K64, K66, and R69 in the BDR were identified as relevant for pA104R activity, with double mutation of the first two showing additive attenuation. pA104R-reactive IgM and IgG epitopes were also identified in the bottom of the BDR, with selective mutagenesis drastically reducing antibody recognition and, when combined with mutations in the arm of the BDR, leading to a further reduction of DNA-binding activity. Interestingly, the immunodominant pA104R-reactive IgG epitope was mainly recognized by IgG1 suggesting that pA104R induces a dominant Th2 response. In sum, the rational mutagenesis can reduce pA104R-DNA binding activity and immune reactivity, providing a rationale for the development of an ASFV pA104R-based DIVA vaccine.

AQP1 as a novel biomarker to predict prognosis and tumor immunity in glioma patients

Abstract Background Glioma is a kind of nervous system cancer with a low overall survival rate. Aquaporin 1 (AQP1) is linked to a number of cancers. Its prognostic relevance and immunological consequences in gliomas, however, are unclear. Objectives Our objective was to thoroughly examine the modified expression of AQP1, its prognostic significance, and its correlation with immune cells and markers to discover innovative molecular immunotherapy strategies for glioma patients. Methods RNA sequencing data from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) databases were used. In addition, we used real-time reverse transcription polymerase chain reaction (RT-PCR) and Western Blot methods to monitor AQP1 expression in glioma tissues. Results AQP1 expression was greater in gliomas than in traumatized brain tissues. The increased AQP1 expression in gliomas was additionally confirmed through immunohistochemical analysis in the Human Protein Atlas (HPA) repository. An elevated level of AQP1 expression was identified as a separate determinant of the overall survival (OS) and prognosis of individuals with glioma. AQP1 expression was shown to be tightly linked to the tumor immune milieu, immune checkpoint blockade (ICB) and temozolomide drug reaction. In conclusion, the 50 genes that show coexpression with AQP1 indicate that the predominant functions and pathways are related to anterior pattern specification, pattern specification, regionalization, high-density lipoprotein particles, protein–lipid complexes, glycosaminoglycan binding, DNA-binding transcription repressor specific activation to RNA polymerase II, DNA-binding transcription repressor activity, nitrogen metabolism, alpha-linolenic acid metabolism, and fat digestion and absorption. Conclusions The results indicate that AQP1 could serve as both a predictive marker and a potential treatment target in glioma.

Metformin protects retinal pigment epithelium cells against H2O2-induced oxidative stress and inflammation via the Nrf2 signaling cascade.

Dysfunctions of retinal pigment epithelium (RPE) attributed to oxidative stress and inflammation are implicated with age-related macular degeneration (AMD). A debate on the curative role of metformin in AMD has been raised, though several recent clinical studies support the lower odds by using metformin. This study aimed to determine whether metformin could exert cytoprotection against RPE oxidative damages and the potential mechanisms. A cellular AMD model was established by treating ARPE-19 cells with hydrogen peroxide (H2O2) for 24h. The reactive oxygen species (ROS) generation, expression of antioxidant enzymes, and levels of pro-inflammatory cytokines were monitored under administrations with H2O2 with/without metformin. The expression and DNA-binding activity of transcription factor erythroid-related factor 2 (Nrf2) were determined by western blot, immunofluorescence, and electrophoretic mobility shift assay. Knockout of Nrf2 was conducted by CRISPR/Cas9 gene deletion system. Metformin pretreatment significantly improved the H2O2-induced low viability of ARPE-19 cells, reduced ROS production, and increased contents of antioxidative molecules. Concurrently, metformin also suppressed levels of pro-inflammatory cytokines caused by H2O2. The metformin-augmented nuclear translocation and DNA-binding activity of Nrf2 were further verified by the increased expression of its downstream targets. Genetic deletion of Nrf2 blocked the cytoprotective role of metformin. Metformin possesses antioxidative and anti-inflammatory properties in ARPE-19 cells by activating the Nrf2 signaling. It supports the potential use for the control and prevention of AMD.

Artemisinin protects dopaminergic neurons against 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced neurotoxicity in a mouse model of Parkinson’s disease

Artemisinin is an antimalarial drug that has been used for almost half a century. However, the anti-Parkinson's disease (PD) effects of artemisinin with respect to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced oxidative stress have not yet been investigated while focusing on NF-E2–related factor 2 (Nrf2) signaling. Thus, we sought to assess the behavioral and oxidative mechanistic effects of artemisinin on MPTP-induced toxicity via the Nrf2 signaling pathway. We explored this through immunohistochemical assays, ELISA, in differentiated PC12 cells treated with siRNA, and with a PD mouse model. Artemisinin increased Nrf2 DNA-binding activity and HO-1 and NQO1 expression. Artemisinin treatment protected cells against MPP+ -induced neuronal death signaling, including NADH dehydrogenase activity, reactive oxygen species, mitochondrial membrane potential, and cleaved caspase-3. Moreover, it protected cells against MPTP-induced behavioral impairments and significantly reduced dopaminergic neuronal loss. Additionally, Nrf2 pre-inhibition using ML385 neutralized the inhibitory effects of artemisinin on dopaminergic neuronal damage and behavioral impairments induced by MPTP. Our results suggest that artemisinin inhibits MPTP-induced behavioral and neurotoxic effects in mice. This provides a foundation for further research to evaluate artemisinin as a potential therapeutic agent for PD.

BZIP transcription factor FabR: Redox-dependent mechanism controlling docosahexaenoic acid biosynthesis and H2O2 stress response in Schizochytrium sp.

Schizochytrium sp. is an important industrial strain for commercial production of docosahexaenoic acid (DHA), which plays essential physiological roles in infant development and human health. The regulatory network for DHA biosynthesis and lipid accumulation in Schizochytrium remains poorly understood. FabR (fatty acid biosynthesis repressor), a basic leucine zipper (bZIP) transcription factor, was transcriptionally downregulated under low-nitrogen condition. Deletion of fabR gene (mutant ΔfabR) increased production of total lipids and DHA by 30.1% and 46.5%, respectively. ΔfabR displayed H2O2 stress resistance higher than that of parental strain or complementation strain CfabR. FabR bound specifically to 7-bp pseudo-palindromic sequence 5′-ATTSAAT-3′ in upstream regions and repressed transcription of fatty acid biosynthesis genes (acl, fas, pfa) and antioxidant defense genes (cat, sod1, sod2, gpx). DNA binding activity of FabR was regulated in a redox-dependent manner. Under oxidative condition, FabR forms intermolecular disulfide bonds between two Cys46 residues of dimers; its DNA binding activity is thereby lost, and the transcription of its target genes is enhanced through derepression. Our findings clarify the redox-dependent mechanism that modulates FabR activity governing lipid and DHA biosynthesis and H2O2 stress response in Schizochytrium.

PI(3,4,5)P3 allosteric regulation of repressor activator protein 1 controls antigenic variation in trypanosomes

African trypanosomes evade host immune clearance by antigenic variation, causing persistent infections in humans and animals. These parasites express a homogeneous surface coat of variant surface glycoproteins (VSGs). They transcribe one out of hundreds of VSG genes at a time from telomeric expression sites (ESs) and periodically change the VSG expressed by transcriptional switching or recombination. The mechanisms underlying the control of VSG switching and its developmental silencing remain elusive. We report that telomeric ES activation and silencing entail an on/off genetic switch controlled by a nuclear phosphoinositide signaling system. This system includes a nuclear phosphatidylinositol 5-phosphatase (PIP5Pase), its substrate PI(3,4,5)P3, and the repressor-activator protein 1 (RAP1). RAP1 binds to ES sequences flanking VSG genes via its DNA binding domains and represses VSG transcription. In contrast, PI(3,4,5)P3 binds to the N-terminus of RAP1 and controls its DNA binding activity. Transient inactivation of PIP5Pase results in the accumulation of nuclear PI(3,4,5)P3, which binds RAP1 and displaces it from ESs, activating transcription of silent ESs and VSG switching. The system is also required for the developmental silencing of VSG genes. The data provides a mechanism controlling reversible telomere silencing essential for the periodic switching in VSG expression and its developmental regulation.

PI(3,4,5)P3 allosteric regulation of repressor activator protein 1 controls antigenic variation in trypanosomes.

African trypanosomes evade host immune clearance by antigenic variation, causing persistent infections in humans and animals. These parasites express a homogeneous surface coat of variant surface glycoproteins (VSGs). They transcribe one out of hundreds of VSG genes at a time from telomeric expression sites (ESs) and periodically change the VSG expressed by transcriptional switching or recombination. The mechanisms underlying the control of VSG switching and its developmental silencing remain elusive. We report that telomeric ES activation and silencing entail an on/off genetic switch controlled by a nuclear phosphoinositide signaling system. This system includes a nuclear phosphatidylinositol 5-phosphatase (PIP5Pase), its substrate PI(3,4,5)P3, and the repressor-activator protein 1 (RAP1). RAP1 binds to ES sequences flanking VSG genes via its DNA binding domains and represses VSG transcription. In contrast, PI(3,4,5)P3 binds to the N-terminus of RAP1 and controls its DNA binding activity. Transient inactivation of PIP5Pase results in the accumulation of nuclear PI(3,4,5)P3, which binds RAP1 and displaces it from ESs, activating transcription of silent ESs and VSG switching. The system is also required for the developmental silencing of VSG genes. The data provides a mechanism controlling reversible telomere silencing essential for the periodic switching in VSG expression and its developmental regulation.

Transcription factor network analysis of the Cf-19-mediated resistance response in tomato infected by Cladosporium fulvum

Tomato leaf mold, caused by Cladosporium fulvum, is a common disease in tomato cultivation. The Cf-19 gene is an effective resistance gene to C. fulvum. Current studies have shown that the Cf-19-mediated resistance response pathway is regulated by a variety of TFs. In this study, in order to better understand the role of TFs in the Cf-19-mediated immune response of tomato against C. fulvum, differentially expressed TFs in different response stages were identified based on RNA-seq data. The results showed that a total of 37 families with 171 TF members were identified. These differentially expressed TFs were enriched in “nucleus” in the component category, “DNA-binding transcription factor activity” in the function category and “Regulation of transcription” in the process category in the Gene Ontology (GO) analysis results. Four pathways, including plant hormone signal transduction, MAPK signaling, plant-pathogen interaction and circadian rhythm, were enriched in the KEGG analysis results. Further analysis showed that 45 TFs were involved in the above four major pathways, and some TFs in different pathways had interaction relationships, which might be the key nodes mediating cross-talk between different pathways. EREB was predicted to be the core of multigroup interactions, and its interaction with WRKY80 is the node connecting the MAPK pathway and plant-pathogen interaction pathway. The function of the EREB gene was analyzed by VIGS, and it was found that the downregulated expression of this gene decreased the expression of WRKY80 and the disease resistance mediated by Cf-19.

Altered m6A RNA methylation governs denervation-induced muscle atrophy by regulating ubiquitin proteasome pathway

BackgroundDenervation-induced muscle atrophy is complex disease involving multiple biological processes with unknown mechanisms. N6-methyladenosine (m6A) participates in skeletal muscle physiology by regulating multiple levels of RNA metabolism, but its impact on denervation-induced muscle atrophy is still unclear. Here, we aimed to explore the changes, functions, and molecular mechanisms of m6A RNA methylation during denervation-induced muscle atrophy.MethodsDuring denervation-induced muscle atrophy, the m6A immunoprecipitation sequencing (MeRIP-seq) as well as enzyme-linked immunosorbent assay analysis were used to detect the changes of m6A modified RNAs and the involved biological processes. 3-deazidenosine (Daa) and R-2-hydroxyglutarate (R-2HG) were used to verify the roles of m6A RNA methylation. Through bioinformatics analysis combined with experimental verification, the regulatory roles and mechanisms of m6A RNA methylation had been explored.ResultsThere were many m6A modified RNAs with differences during denervation-induced muscle atrophy, and overall, they were mainly downregulated. After 72 h of denervation, the biological processes involved in the altered mRNA with m6A modification were mainly related to zinc ion binding, ubiquitin protein ligase activity, ATP binding and sequence-specific DNA binding and transcription coactivator activity. Daa reduced overall m6A levels in healthy skeletal muscles, which reduced skeletal muscle mass. On the contrary, the increase in m6A levels mediated by R-2HG alleviated denervation induced muscle atrophy. The m6A RNA methylation regulated skeletal muscle mass through ubiquitin–proteasome pathway.ConclusionThis study indicated that decrease in m6A RNA methylation was a new symptom of denervation-induced muscle atrophy, and confirmed that targeting m6A alleviated denervation-induced muscle atrophy.

Irisin inhibits CCK-8-induced TNF-α production via integrin αVβ5-NF-κB signaling pathways in Nile tilapia (Oreochromis niloticus)

Irisin, a secreted myokine generated by fibronectin type III domain-containing protein 5, has recently shown the potential to alleviate inflammation. Cholecystokinin-octapeptide (CCK-8) is closely associated with the inflammatory factor TNF-α, a central cytokine in inflammatory reactions. However, the interactions between irisin and CCK-8 in regulating TNF-α production and the underlying mechanism have not yet been elucidated. In the present study, irisin treatment inhibited the basal and the CCK-8-induced TNF-α production in vivo. Additionally, neutralizing circulating irisin using an irisin antiserum significantly augmented the CCK-8-induced stimulation of TNF-α levels. Moreover, the incubation of head kidney cells with irisin or CCK-8 has opposite effects on TNF-α secretion. Notably, irisin treatment inhibited basal and CCK-8-stimulated TNF-α release and gene transcription in head kidney cells. Mechanistically, the inhibitory actions of irisin on basal and CCK-8-induced TNF-α production could be negated by co-administered with the selective integrin αVβ5 inhibitor cilengitide. In addition, the inhibitory effect of irisin on basal and CCK-8-triggered TNF-α production could be abolished by the inhibition of the nuclear factor-kappa B (NF-κB) signaling pathway. Furthermore, irisin impeded CCK-8-induced phosphorylation and degradation of IκBα, simultaneously inhibiting NF-κB phosphorylation, preventing its translocation into the nucleus, and suppressing its DNA-binding activity induced by CCK-8. Collectively, these results suggest that the inhibitory effect of irisin on TNF-α production caused by CCK-8 is mediated via the integrin αVβ5-NF-κB signaling pathways in tilapia.

Bilobalide attenuates lipopolysaccharide‑induced HepG2 cell injury by inhibiting TLR4‑NF‑κB signaling via the PI3K/Akt pathway.

Inflammation is involved in the pathological process underlying a number of liver diseases. Bilobalide (BB) is a natural compound from Ginkgo biloba leaves that was recently demonstrated to exert hepatoprotective effects by inhibiting oxidative stress in the liver cancer cell line HepG2. The anti-inflammatory activity of BB has been reported in recent studies. The major objective of the present study was to investigate whether BB could attenuate inflammation-associated cell damage. HepG2 cells were cultured with lipopolysaccharide (LPS) and BB, and cell damage was evaluated by measuring cell viability using MTT assay. The activity of the NF-κB signaling pathway was assessed by measuring the levels of IκBα, NF-κB p65, phosphorylated (p)-IκBα, p-p65, p65 DNA-binding activity and inflammatory cytokines IL-1β, IL-6 and TNF-α. A toll-like receptor (TLR)4 inhibitor (CLI-095) was used to detect the involvement of TLR4 in cell injury caused by LPS. In addition, the PI3K/Akt inhibitor LY294002 was applied to explore the involvement of the PI3K/Akt axis in mediating the effects of BB. The results demonstrated that LPS induced HepG2 cell injury. LPS also elevated the levels of p-IκBα, p-p65, p65 DNA-binding activity and inflammatory cytokines. However, CLI-095 significantly attenuated the LPS-induced cell damage and inhibited the activation of NF-κB signaling. BB also dose-dependently attenuated the LPS-induced cell damage, activation of NF-κB signaling and TLR4 overexpression. Furthermore, it was observed that LY294002 diminished the cytoprotective effects of BB on cell injury, TLR4 expression and NF-κB activation. These findings indicated that BB could attenuate LPS-induced inflammatory injury to HepG2 cells by regulating TLR4-NF-κB signaling.

Synthesis, crystal structure, DNA binding, and anticancer activity of the cobalt(II), nickel(II), and copper(II) complexes of 9-benzothiazolanthrahydrazone

In the present study, we synthesized and extensively characterized a novel ligand, 9-THA, based on 9-benzothiazolanthrahydrazone, along with its three corresponding transition metal complexes: 9-THA-Co (1), 9-THA-Ni (2), and 9-THA-Cu (3). The crystallographic analysis revealed distinct coordination geometries - six-coordinate for 9-THA-Co and 9-THA-Ni, binding three and two 9-THA ligands, respectively, and four-coordinate for 9-THA-Cu, binding one 9-THA ligand. In vitro anticancer screening demonstrated that 9-THA-Cu exhibited significant growth inhibition in HeLa-229, NCI-H460, and SK-OV-3 cancer cells with IC50 values of 12.10 ± 0.54, 12.12 ± 0.58, and 17.65 ± 0.66 μM, respectively. Moreover, it displayed comparable cytotoxicity to cisplatin on HL-7702 normal liver cells. In contrast, 9-THA and 9-THA-Co/9-THA-Ni did not exhibit significant growth inhibition in the tested cancer cells, suggesting that Cu(II) played a pivotal role in its anticancer mechanism. Further investigation revealed the intercalative DNA binding mode of 9-THA-Cu by UV–Vis and fluorescent spectral analyses. It also exhibited competitive binding with two typical DNA intercalators, Gel-red (GR) and ethidium bromide (EB). Cell cycle and cell apoptosis experiments indicated that 9-THA-Cu at a concentration of 22 μM could induce cell cycle arrest in the S phase in 40 % of HeLa-229 cancer cells. Simultaneously, it induced 33.2 % apoptosis in HeLa-229 cells, suggesting that 9-THA-Cu may employ multiple mechanisms to exert its anticancer effects.

C-di-GMP inhibits the DNA binding activity of H-NS in Salmonella

Cyclic di-GMP (c-di-GMP) is a second messenger that transduces extracellular stimuli into cellular responses and regulates various biological processes in bacteria. H-NS is a global regulatory protein that represses expression of many genes, but how H-NS activity is modulated by environmental signals remains largely unclear. Here, we show that high intracellular c-di-GMP levels, induced by environmental cues, relieve H-NS-mediated transcriptional silencing in Salmonella enterica serovar Typhimurium. We find that c-di-GMP binds to the H-NS protein to inhibit its binding to DNA, thus derepressing genes silenced by H-NS. However, c-di-GMP is unable to displace H-NS from DNA. In addition, a K107A mutation in H-NS abolishes response to c-di-GMP but leaves its DNA binding activity unaffected in vivo. Our results thus suggest a mechanism by which H-NS acts as an environment-sensing regulator in Gram-negative bacteria.

The Whole-Exome Sequencing of a Cohort of 19 Families with Adolescent Idiopathic Scoliosis (AIS): Candidate Pathways.

A significant genetic involvement has been known for decades to exist in adolescent idiopathic scoliosis (AIS), a spine deformity affecting 1-3% of the world population. However, though biomechanical and endocrinological theories have emerged, no clear pathophysiological explanation has been found. Data from the whole-exome sequencing performed on 113 individuals in 19 multi-generational families with AIS have been filtered and analyzed via interaction pathways and functional category analysis (Varaft, Bingo and Panther). The subsequent list of 2566 variants has been compared to the variants already described in the literature, with an 18% match rate. The familial analysis in two families reveals mutations in the BICD2 gene, supporting the involvement of the muscular system in AIS etiology. The cellular component analysis revealed significant enrichment in myosin-related and neuronal activity-related categories. All together, these results reinforce the suspected role of the neuronal and muscular systems, highlighting the calmodulin pathway and suggesting a role of DNA-binding activities in AIS physiopathology.

STEM-09. EXPLORING THE ROLE OF TP53 IN REDOX REGULATION AND RESPONSE OF GLIOBLASTOMA STEM CELLS TO ROS-INDUCING AGENTS

Abstract Glioblastoma, the most malignant primary tumor of the central nervous system in adults presents a major therapeutic challenge due to the presence of chemo-radioresistant glioblastoma stem cells (GSCs). These GSCs adapt the thioredoxin (Trx) and glutathione (GSH) antioxidant systems to counteract treatment-induced reactive oxygen species (ROS) and promote their survival. The tumor suppressor gene wild-type (wt)TP53 regulates the expression of pro-oxidant or antioxidant genes in response to chemoradiotherapy based on p53 DNA-binding activity in a reducing microenvironment. Given the importance of p53 as a redox-sensitive protein, we hypothesized that p53 might protect GSCs against ROS-inducing agents. Auranofin (Au), a FDA-approved gold(I)-containing compound inhibits thioredoxin reductase 1 (TrxR1), a key antioxidant protein. We investigated the effects of Au on different GSCs including wtp53 GSC line and its counterpart p53-knockdown. We assessed viability, neurosphere formation, ROS levels and expression of p53 and TrXR1 to evaluate the cytotoxicity of Au, alone and in combination with L-buthionine sulfoximine (L-BSO), a GSH-depleting agent. We conducted TCGA analysis to explore the correlation between TrxR1 and p53. Our findings revealed a positive significant correlation between p53 and TrxR1 expression, consistent with lower levels of TrxR1 in p53-knockdown GSCs compared to their wt-p53 counterparts. P53-knockdown GSCs displayed a higher basal level of ROS, which further increased upon treatment with Au. P53-knockdown significantly increased the sensitivity of cells to Au, with pronounced effects for Au/L-BSO combination. Our findings reveal that expression levels of wtp53 affect the role of p53 in TrxR1-redox regulation and sensitivity to ROS-inducing agents in GSCs.

Validation of a robust and rapid liquid chromatography tandem mass spectrometric method for the quantitative analysis of VK-2019, a selective EBNA1 inhibitor.

EBNA1 is an Epstein Barr virus (EBV) protein expressed in all EBV-associated cancers. EBNA1 plays a critical role in the replication and maintenance of EBV episomes in latently infected cells. VK-2019 was developed as a highly specific inhibitor of EBNA1 DNA binding activity and is currently in phase 1 development as a treatment for EBV-associated carcinomas. A sensitive and reliable method was developed to quantify VK-2019 in human plasma using liquid chromatography with tandem mass spectrometry to perform detailed pharmacokinetic studies. VK-2019 was extracted from plasma using protein precipitation with acetonitrile. Separation of VK-2019, two purported metabolites, and the internal standard, VK-2019-d6, was achieved with a Zorbax XDB C18 column using a gradient flow over 6 min. VK-2019 was detected using a SCIEX 4500 triple quadrupole mass spectrometer operating in positive electrospray ionization mode. The assay range was 0.5-500 ng/mL and proved to be accurate and precise. Dilutions of 1:10 were accurately quantified. VK-2019 was stable in plasma at -70°C for approximately 18 months. The method was applied to assess the total plasma concentrations of VK-2019 in a patient who received a single and multiple oral daily doses of 120 mg.

Abstract 15636: NFATC1 Modulates Atrial Excitability and Arrhythmogenesis

Transcription factors have emerged as important loci for atrial fibrillation (AF) susceptibility. We identified a novel mutation (M527L) in a conserved site of the transcription factor NFATC1 in a multigenerational family with an autosomal dominant young onset AF (<40 years old) phenotype. M527L reduces NFATC1 nuclear translocation (nuclear/total cellular ratio: M527L 0.50±0.02; WT 0.64±0.04, p<1E-5; n=10/each), DNA binding (M527L 0.69±0.02; WT 0.83±0.01, p=0.02; n=3/ each), and transcriptional activity (M527L 0.0003±0.00005; WT 0.0009±0.0002, p=0.01; n=5/each) in expression systems, underscoring the functional consequences of this mutation. To study the broader role of NFATC1 in atrial excitability we employed a CRISPR/Cas9 strategy to create an nfatc1 null (KO) zebrafish. KO fish exhibited reduced survival, with ~80% of juvenile fish succumbing to sudden death (log rank test p=0.0001), with no observable change in body or heart gross morphology. Explanted juvenile atria displayed spontaneous tachyarrhythmias or irregular rhythm (29 /40 KO atria vs 11/28 WT atria; Chi-Squared, p=0.0013) accompanied by irregular Ca 2+ handling (10/11 KO atria vs 5/16 WT atria). Furthermore, we found an abbreviated action potential duration in KO atrium (80±1 ms in KO (n=607) vs 91±1 ms in WT (n=441), p=0.0001), but not ventricle (186±3 ms in KO (n=109) vs 191±2 ms in WT (n=160), p=0.8). Taken together, our data suggests that NFATC1 is a modulator of atrial excitability and plays a role in AF susceptibility. Experiments are ongoing to further define the mechanisms underlying arrhythmia susceptibility and to identify the gene networks downstream of NFATC1 in the atria.