ATAD2 suppression enhances the combinatorial effect of gemcitabine and radiation in pancreatic cancer cells.

Amalgamation of target-based approach along with chemo and/or radiotherapy could be an effective strategy to combat pancreatic cancer (PC). ATAD2 (ATPase family AAA domain containing 2) is a potential oncoprotein and a poor prognostic factor in PC. ATAD2 inhibition sensitizes pancreatic cancer cells (PCCs) to gemcitabine (GEM). ATAD2 silencing also enhances radio-sensitivity in lung cancer cells. We therefore hypothesise that ATAD2 suppression along with application of GEM and radiation could show additive/synergistic response in reducing PC progression. At first, immunofluorescence and western blot analysis are carried out to investigate ATAD2 expression upon irradiation in PCCs (BxPC3, and PANC1). Colony forming assay is performed to analyse the radio-sensitization effect of ATAD2 suppressed PCCs in absence and presence of GEM. To evaluate the consequences of irradiation, expression of markers for DNA damage and repair as well as apoptosis and autophagy are also assessed. Results indicate that ATAD2 is elevated in irradiated PCCs. Silencing ATAD2 sensitizes PCCs to radiation. Survival fraction analysis of PCCs shows that combination treatment of GEM and radiation (with minimal doses) is more effective when ATAD2 is suppressed. Further, when GEM-radiation combination is applied to ATAD2 silenced PCCs, expression of DNA damage marker like γH2AX is induced, whereas DNA damage repair proteins (pChk1, and pChk2) are downregulated. Moreover, ATAD2 suppressed PCCs are more susceptible to apoptosis and autophagy in presence of radiation and GEM. Collectively, these findings support our hypothesis and demonstrate that targeting ATAD2 enhances efficacy of GEM-radiation treatment in PCCs.

Effects of Probiotics at the Interface of Metabolism and Immunity to Prevent Colorectal Cancer-Associated Gut Inflammation: A Systematic Network and Meta-Analysis With Molecular Docking Studies.

Background: Dysbiosis/imbalance in the gut microbial composition triggers chronic inflammation and promotes colorectal cancer (CRC). Modulation of the gut microbiome by the administration of probiotics is a promising strategy to reduce carcinogenic inflammation. However, the mechanism remains unclear. Methods: In this study, we presented a systematic network, meta-analysis, and molecular docking studies to determine the plausible mechanism of probiotic intervention in diminishing CRC-causing inflammations. Results: We selected 77 clinical, preclinical, in vitro, and in vivo articles (PRISMA guidelines) and identified 36 probiotics and 135 training genes connected to patients with CRC with probiotic application. The meta-analysis rationalizes the application of probiotics in the prevention and treatment of CRC. An association network is generated with 540 nodes and 1,423 edges. MCODE cluster analysis identifies 43 densely interconnected modules from the network. Gene ontology (GO) and pathway enrichment analysis of the top scoring and functionally significant modules reveal stress-induced metabolic pathways (JNK, MAPK), immunomodulatory pathways, intrinsic apoptotic pathways, and autophagy as contributors for CRC where probiotics could offer major benefits. Based on the enrichment analyses, 23 CRC-associated proteins and 7 probiotic-derived bacteriocins were selected for molecular docking studies. Results indicate that the key CRC-associated proteins (e.g., COX-2, CASP9, PI3K, and IL18R) significantly interact with the probiotic-derived bacteriocins (e.g., plantaricin JLA-9, lactococcin A, and lactococcin mmfii). Finally, a model for probiotic intervention to reduce CRC-associated inflammation has been proposed. Conclusion: Probiotics and/or probiotic-derived bacteriocins could directly interact with CRC-promoting COX2. They could modulate inflammatory NLRP3 and NFkB pathways to reduce CRC-associated inflammation. Probiotics could also activate autophagy and apoptosis by regulating PI3K/AKT and caspase pathways in CRC. In summary, the potential mechanisms of probiotic-mediated CRC prevention include multiple signaling cascades, yet pathways related to metabolism and immunity are the crucial ones.

MicroRNA-217 modulates pancreatic cancer progression via targeting ATAD2.

AIMS: Pancreatic cancer is a fatal disease across the world with 5 years survival rate less than 10%. ATAD2, a valid cancer drug-target, is overexpressed in pancreatic malignancy with high oncogenic potential. However, the mechanism of the upregulated expression of ATAD2 in pancreatic cancer is unknown. Since microRNAs (miRNAs) could potentially control target mRNA expressions, and are involved in cancer as tumor-suppressors, oncomiR or both, we examine the possibility of miRNA-mediated regulation of ATAD2 in pancreatic cancer cells (PCCs). MAIN METHODS: Our in-silico approach first identifies hsa-miR-217 as a candidate regulator for ATAD2 expression. For further validation, luciferase reporter assay is performed. We overexpress hsa-miRNA-217 and assess cellular viability, migration, apoptosis and cell cycle progression in three different PCCs (BxPC3, PANC1, and MiaPaCa2). KEY FINDINGS: We find hsa-miRNA-217 has potential binding site at the 3'UTR of ATAD2. Luciferase assay confirms that ATAD2 is a direct target of hsa-miR-217. Overexpression of hsa-miR-217 drastically downregulates ATAD2 expression in PCCs, thus, corroborating binding studies. The elevated expression of hsa-miRNA-217 diminishes cell proliferation and migration as well as induces apoptosis and cell cycle arrest in PCCs. Finally, siRNA mediated ATAD2 knockdown or overexpression of hsa-miRNA-217 in PCCs showed inactivation of the AKT signaling pathway. Therefore, hsa-miR-217 abrogates pancreatic cancer progression through inactivation of the AKT signaling pathway and this might be partly due to miR-217 mediated suppression of ATAD2 expression. SIGNIFICANCE: The application of hsa-miR-217 mimic could be a promising therapeutic strategy for the treatment of pancreatic cancer patients in near future.

Oncogenic potential of ATAD2 in stomach cancer and insights into the protein-protein interactions at its AAA + ATPase domain and bromodomain.

ATAD2 has recently been shown to promote stomach cancer. However, nothing is known about the functional network of ATAD2 in stomach carcinogenesis. This study illustrates the oncogenic potential of ATAD2 and the participation of its ATPase and bromodomain in stomach malignancy. Expression of ATAD2 in stomach cancer is analyzed by in silico and in vitro techniques including western blot and immunofluorescence microscopy of stomach cancer cells (SCCs) and tissues. The oncogenic potential of ATAD2 is examined thoroughly using genetic alterations, driver gene prediction, survival analysis, identification of interacting partners, and analysis of canonical pathways. To understand the protein-protein interactions (PPI) at residue level, molecular docking and molecular dynamics simulations (1200 ns) are performed. Enhanced expression of ATAD2 is observed in H. pylori-infected SCCs, patient biopsy tissues, and all stages and grades of stomach cancer. High expression of ATAD2 is found to be negatively correlated with the survival of stomach cancer patients. ATAD2 is a cancer driver gene with 37 mutational sites and a predictable factor for stomach cancer prognosis with high accuracy. The top canonical pathways of ATAD2 indicate its participation in stomach malignancy. The ATAD2-PPI in stomach cancer identify top-ranked partners; ESR1, SUMO2, SPTN2, and MYC show preference for the bromodomain whereas NCOA3 and HDA11 have preference for the ATPase domain of ATAD2. The oncogenic characterization of ATAD2 provides strong evidence to consider ATAD2 as a stomach cancer biomarker. These studies offer an insight for the first time into the ATAD2-PPI interface presenting a novel target for cancer therapeutics. Communicated by Ramaswamy H. Sarma.

Emerging oncogene ATAD2: Signaling cascades and therapeutic initiatives.

ATAD2 is a promising oncoprotein with tumor-promoting functions in many cancers. It is a valid cancer drug-target and a potential cancer-biomarker for multiple malignancies. As a cancer/testis antigen (CTA), ATAD2 could also be a probable candidate for immunotherapy. It is a unique CTA that belongs to both AAA+ ATPase and bromodomain family proteins. Since 2007, several research groups have been reported on the pleiotropic oncogenic functions of ATAD2 in diverse signaling pathways, including Rb/E2F-cMyc pathway, steroid hormone signaling pathway, p53 and p38-MAPK-mediated apoptotic pathway, AKT pathway, hedgehog signaling pathway, HIF1α signaling pathway, and Epithelial to Mesenchymal Transition (EMT) pathway in various cancers. In all these pathways, ATAD2 participates in chromatin dynamics, DNA replication, and gene transcription, demonstrating its role as an epigenetic reader and transcription factor or coactivator to promote tumorigenesis. However, despite the progress, an overall mechanism of ATAD2-mediated oncogenesis in diverse origin is elusive. In this review, we summarize the accumulated evidence to envision the overall ATAD2 signaling networks during carcinogenesis and highlight the area where missing links await further research. Besides, the structure-function aspect of ATAD2 is also discussed. Since the efforts have already been initiated to explore targeted drug molecules and RNA-based therapeutic alternatives against ATAD2, their potency and prospects have been elucidated. Together, we believe this is a well-rounded review on ATAD2, facilitating a new drift in ATAD2 research, essential for its clinical implication as a biomarker and/or cancer drug-target.

Systematic Network and Meta-analysis on the Antiviral Mechanisms of Probiotics: A Preventive and Treatment Strategy to Mitigate SARS-CoV-2 Infection.

With the alarming rise of infected cases and deaths, COVID-19 is a pandemic, affecting 220 countries worldwide. Until now, no specific treatment is available against SARS-CoV-2. The causal virus SARS-CoV-2 primarily infects lung cells, leading to respiratory illness ranging in severity from the common cold to deadly pneumonia. This, with comorbidities, worsens the clinical outcome, particularly for immunosuppressed individuals with COVID-19. Interestingly, the commensal gut microbiota has been shown to improve lung infections by modulating the immune system. Therefore, fine-tuning of the gut microbiome with probiotics could be an alternative strategy for boosting immunity and treating COVID-19. Here, we present a systematic biological network and meta-analysis to provide a rationale for the implementation of probiotics in preventing and/or treating COVID-19. We have identified 90 training genes from the literature analysis (according to PRISMA guidelines) and generated an association network concerning the candidate genes linked with COVID-19 and probiotic treatment. The functional modules and pathway enrichment analysis of the association network clearly show that the application of probiotics could have therapeutic effects on ACE2-mediated virus entry, activation of the systemic immune response, nlrp3-mediated immunomodulatory pathways, immune cell migration resulting in lung tissue damage and cardiovascular difficulties, and altered glucose/lipid metabolic pathways in the disease prognosis. We also demonstrate the potential mechanistic domains as molecular targets for probiotic applications to combat the viral infection. Our study, therefore, offers probiotics-mediated novel preventive and therapeutic strategies for COVID-19 warfare.

HIF1α-dependent upregulation of ATAD2 promotes proliferation and migration of stomach cancer cells in response to hypoxia.

Stomach cancer is a difficult-to-treat disease. Lack of detection markers and limited understanding of the disease mechanisms contribute to the aggressive nature of stomach cancer cells (SCCs). Recently, an ATPase, ATAD2 has been found to be highly expressed in stomach cancer contributing to increased malignancy. However, nothing is known about the mechanism of ATAD2 upregulation and its involvement in stomach carcinogenesis. Since hypoxic microenvironment plays a crucial role in the progression of solid tumors like stomach cancer; we have examined the regulation and function of ATAD2 expression in hypoxic SCCs. ATAD2 is induced in hypoxia-treated SCCs. Stomach adenocarcinoma and metastatic tissues with high HIF1α level also show enhanced ATAD2 expression. In the absence of hypoxia-inducible factor HIF1α, ATAD2 protein level is found to be less indicating towards a potential correlation between them. We identify the presence of HIF1α-binding site (HBS) and HIF1α ancillary site (HAS) in the ATAD2 promoter. Using both in vitro and in vivo binding studies, we confirm that HIF1α binds with the ATAD2 promoter in hypoxic condition. ATAD2 upregulation promotes proliferation and migration of SCCs exposed to hypoxia. Thus, we identify ATAD2 as a hypoxia-responsive and HIF1α-regulated gene and elucidate that upregulated expression of ATAD2 enhances tumor-promoting functions in hypoxic SCCs. Therefore, we propose ATAD2 as a promising therapeutic target for stomach cancer.

Mechanisms of interactions of the nucleotide cofactor with the RepA protein of plasmid RSF1010. Binding dynamics studied using the fluorescence stopped-flow method.

The dynamics of the nucleotide binding to a single, noninteracting nucleotide-binding site of the hexameric helicase RepA protein of plasmid RSF1010 has been examined, using the fluorescence stopped-flow method. The experiments have been performed with fluorescent analogues of ATP and ADP, TNP-ATP and TNP-ADP, respectively. In the presence of Mg(2+), the association of the cofactors proceeds as a sequential three-step process [Formula: see text] The sequential nature of the mechanism indicates the lack of significant conformational equilibria of the helicase prior to nucleotide binding. The major conformational change of the RepA helicase-nucleotide complex occurs in the formation of (H-N)(2), which is characterized by a very high value of the partial equilibrium constant and large positive changes in the apparent enthalpy and entropy. Strong stabilizing interactions between subunits of the RepA hexamer contribute to the observed dynamics and energetics of the internal transitions of the formed complexes. Magnesium cations mediate the efficient and fast conformational transitions of the protein, in a manner independent of the structure of the cofactor phosphate group. The ssDNA bound to the enzyme preferentially selects a single intermediate of the RepA-ATP analogue complex, (H-N)(2), while the DNA has no effect on the intermediates of the RepA-ADP complex. Allosteric interactions between the nucleotide- and DNA-binding site are established in the initial stages of formation of the complex. Moreover, in the presence of the single-stranded DNA, all the transitions in the nucleotide binding to the helicase become sensitive to the structure of the phosphate group of the cofactor.

Interactions of the Escherichia coli DnaB-DnaC protein complex with nucleotide cofactors. 1. Allosteric conformational transitions of the complex.

Interactions of nucleotide cofactors with both protein components of the Escherichia coli DnaB helicase complex with the replication factor, the DnaC protein, have been examined using MANT-nucleotide analogues. At saturation, in all examined stationary complexes, including the binary, DnaB-DnaC, and tertiary, DnaB-DnaC-ssDNA, complexes, the helicase binds six cofactor molecules. Thus, protein-protein and protein-DNA interactions do not affect the maximum stoichiometry of the helicase-nucleotide interactions. The single-stranded DNA dramatically increases the ATP analogue affinity, while it has little effect on the affinity of the NDP analogues, indicating that stationary complexes reflect allosteric interactions between the DNA- and NTP-binding site prior to the cofactor hydrolysis step and subsequent to product release. In the binary complex, the DnaC protein diminishes the intrinsic affinity and increases the negative cooperativity in the cofactor binding to the helicase; an opposite effect of the protein on the cofactor-helicase interactions occurs in the tertiary complex. The DnaC protein retains its nucleotide binding capability in the binary and tertiary complexes with the helicase. Surprisingly, the DnaC protein-nucleotide interactions, in the binary and tertiary complexes, are characterized by positive cooperativity. The DnaC assembles on the helicase as a hexamer, which exists in two conformational states and undergoes an allosteric transition, induced by the cofactor. Cooperativity of the allosteric transition depends on the structure of the phosphate group of the nucleotide. The significance of the results for the DnaB-DnaC complex activities is discussed.

Mechanism of NTP hydrolysis by the Escherichia coli primary replicative helicase DnaB protein. 2. Nucleotide and nucleic acid specificities.

The kinetic mechanism of NTP binding and hydrolysis by the Escherichia coli replicative helicase, the DnaB protein, in the absence and presence of the single-stranded DNA (ssDNA), has been quantitatively examined using the rapid quench-flow technique, under single-turnover conditions. In the case of both the free helicase and the enzyme-ssDNA complexes, the mechanism is independent of the type of base of the cofactor or the DNA; the bimolecular association is followed by the reversible chemical hydrolysis and subsequent conformational transition of the enzyme-product complex. The NTP hydrolysis step is significantly faster for the purine than for the pyrimidine cofactor, both in the absence and in the presence of the DNA. The temperature effect indicates that the nature of intermediates of the purine nucleotide, ATP, is different from the nature of the analogous intermediates of the pyrimidine nucleotide, CTP. Nevertheless, both types of cofactors seem to approach a similar "exit" state at the end of the reaction. The effect of ssDNA on the kinetics of NTP hydrolysis depends on the type of nucleotide cofactor and the base composition of the DNA and is centered at the hydrolysis step. Homoadenosine ssDNA oligomers are particularly effective in increasing the hydrolysis rate. The allosteric signal from the DNA, which activates the NTP hydrolysis, comes predominantly from the strong DNA-binding subsite. The role of the weak DNA-binding subsite is to modulate the allosteric effect of the strong subsite. The significance of these results for the mechanism of the free energy transduction by the DnaB helicase is discussed.

Escherichia coli DnaB helicase-DnaC protein complex: allosteric effects of the nucleotides on the nucleic acid binding and the kinetic mechanism of NTP hydrolysis. 3.

Allosteric interactions between the DNA- and NTP-binding sites of the Escherichia coli DnaB helicase engaged in the DnaB-DnaC complex and the mechanism of NTP hydrolysis by the complex have been examined using the fluorescence titration, analytical ultracentrifugation, and rapid quench-flow technique. Surprisingly, the ssDNA affinity of the DnaB-DnaC complex is independent of the structure of the phosphate group of the cofactor bound to the helicase. Thus, the DnaC protein eliminates the antagonistic allosteric effect of NTP and NDP on the ssDNA affinity of the enzyme. The protein changes the engagement of the DNA-binding subsites of the helicase in interactions with the nucleic acid, depending on the structure of the phosphate group of the present nucleotide cofactor and profoundly affects the structure of the bound DNA. Moreover, the ssDNA affinity of the helicase in the DnaB-DnaC complex is under the control of the nucleotide-binding site of the DnaC protein. The protein does not affect the NTP hydrolysis mechanism of the helicase. Nevertheless, the rate of the chemical step is diminished in the DnaB-DnaC complex. In the tertiary DnaB-DnaC-ssDNA complex, the ssDNA changes the internal dynamics between intermediates of the pyrimidine cofactor, in a manner independent of the base composition of the DNA, while the hydrolysis step of the purine cofactor is specifically stimulated by the homoadenosine ssDNA. The significance of these results for functional activities of the DnaB-DnaC complex is discussed.

Accumulation of p-hydroxybenzoic acid in hairy roots of Daucus carota.

We report here the accumulation of p-hydroxybenzoic acid in Agrobacterium rhizogenes-induced hairy root cultures of Daucus carota. This phenolic acid finds application in food, pharmaceutical and polymer industries. Metabolic profiling of phenolics by HPLC/ESI-MS from these hairy roots showed a considerable amount of p-hydroxybenzoic acid accumulation both in cytosol and in the cell wall. Analyses of HCl and NaOH treated soluble phenolic fractions resulted in the elution of peaks with same retention time and similar UV-absorption spectra as observed with p-hydroxybenzoic acid standard. This suggests that p-hydroxybenzoic acid is present in the cytosol as free-form (unconjugated). A correlation has been drawn between the accumulation of soluble and wall-bound phenolic acids on a time-course basis. An apparent absence of any p-hydroxybenzoic acid-glucoside supports this observation, which in turn encourages the idea of its incorporation in the cell wall in an alkaline-labile form.

Kinetic mechanisms of the nucleotide cofactor binding to the strong and weak nucleotide-binding site of the Escherichia coli PriA helicase. 2.

Kinetics of the nucleotide binding to the strong (S) and weak (W) nucleotide-binding site of the Escherichia coli PriA helicase have been studied using the fluorescence stopped-flow technique. Experiments were performed with TNP-ADP and TNP-ATP analogues. Binding of the ADP analogue to the strong binding site is a four-step sequential reaction: (PriA)S + D (k1)<-->(k(-1)) + (S)1 (k2)<-->(k(-2)) (S)2 (k3)<-->(k(-3)) (S)3 (k4)<-->(k(-4)) (S)4. Association of TNP-ATP proceeds through an analogous three-step mechanism. The first two steps and intermediates are similar for both cofactors. However, the (S)3 intermediate is dramatically different for ADP and ATP analogues. Its emission is close to the emission of the free TNP-ADP, while it is by a factor of approximately 16 larger than the free TNP-ATP fluorescence. Thus, only the ADP analogue passes through an intermediate where it leaves the hydrophobic cleft of the site. This behavior corroborates with the fact that ADP leaves the ATPase site without undergoing a chemical change. The fast bimolecular step and the sequential mechanism indicate that the site is easily accessible to the cofactor, and it does not undergo an adjustment prior to binding. The subsequent step is also fast and stabilizes the complex. Magnesium profoundly affects the population of intermediates. The data indicate that the dominant (S)2 species is a part of the ATP catalytic cycle. ADP analogue binding to the weak nucleotide-binding site proceeds in a simpler two-step mechanism: (PriA)W + D (k1)<-->(k(-1)) (W)1 (k2)<-->(k(-2)) (W)2 with (W)1 being a dominant intermediate both in the presence and in the absence of Mg2+. The results indicate that the weak site is an allosteric control site in the functioning of the PriA helicase.