Biology subjects

Singh, R. K.

Publications and source records attributed to Singh, R. K..

46 records · Page 3Linked to original sources

bioRxiv2020

Septins disruption controls tumor growth and enhances efficacy of Herceptin

Septin expressions are altered in cancer cells and exhibit poor prognoses in malignancies. As the first approach to develop a septin filament targeting agent, we optimized the structure of Forchlorfenuron (FCF), a known plant cytokinin to generate UR214-9, which contrary to FCF, causes septin-2/9 filamental structural catastrophe in cancer cells without altering cellular septin protein levels. In-silico docking using septin-2/septin-2 dimer complex showed that UR214-9 displaced the guanine carbonyl oxygen from the GDP binding domain and showed increased binding energy than FCF(-8.59vs-7.21). UR214-9 reduced cancer cell growth, downregulated HER2/STAT-3 axis and controlled growth of HER2+ pancreatic, breast and ovarian cancer xenografts in NSG mice and enhanced response of Herceptin against HER2+breast cancer xenograft. Transcriptome analysis of UR214-9 exposed cells demonstrated significant perturbation of <20 genes compared to afatinib which impacted >1200 genes in JIMT-1 breast cancer cells indicating target specificity and non-transcriptional functions of UR214-9. In summary, disrupting septins via UR214-9 is a new approach to control the growth of HER2+ malignancies.

cancer biology↗

bioRxiv2020

Semecarpus anacardium Linn. leaf extract exhibits activities against breast cancer and prolongs the survival of tumor bearing mice

Semecarpus anacardium Linn. is commonly used in various traditional medicines from ancient times. The nuts have been described in Ayurveda medication systems to treat numerous clinical ailments. However, isolating phytochemical constituents from nuts remains challenging and exhibits cytotoxic effects on other cells. In this study, we have standardized procedures for isolating phytochemicals from the leaf extract. The ethyl acetate leaf extract selectively affects cancer cells in a dose-dependent manner (IC50: 0.57 g/ml in MCF-7 cells) in various cancer cell lines. Next, we examined if the extract incubation could induce cell cycle arrest and suppress cell migration in the cell culture model. Consistent with this idea, the leaf extract could potentially affect the aggressive migration nature of cancer cells. Moreover, oral administration of extract significantly restored tumor growth in mice. Together, these observations suggest the anti-cancer activities of S. anacardium leaf potential for both in vitro and in vivo models. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=90 SRC="FIGDIR/small/898940v3_ufig1.gif" ALT="Figure 1"> View larger version (45K): org.highwire.dtl.DTLVardef@3fefborg.highwire.dtl.DTLVardef@594510org.highwire.dtl.DTLVardef@c525d2org.highwire.dtl.DTLVardef@1362978_HPS_FORMAT_FIGEXP M_FIG C_FIG

pharmacology and toxicology↗

bioRxiv2020

Virtaul screening of MAP-Tau protein inhibitors from Semecarpus anacardium Linn. leaf extract for cancer prevention

Semecarpus anacardium is a well known Indian medicinal plant with various medicinal properties like hypoglycemic, antioxidant, anticancer, anti-inflammatory, anti-geriatric, antimicrobial and hair growth promoter, etc. The molecular mechanism of metabolites from fruiting bodies of S. anacardium against cancer has been described but anticancerous properties in its leaves are still unknown. The leaves were extracted in petroleum ether, ethyl acetate and methanol and assayed for anticancer activity using MTT assay. The active extract was evaluated for mode of cell death induction using EtBr-AO double staining and analyzed for phytochemical constituents using GC-MS, followed by molecular docking studies for exploration of possibility for anticancer agents and Drugability. In this study, ethyl acetate extract of leaf was found potent cytotoxic in MCF-7 cells and also induced apoptosis. It has also found the SLE is safe for normal cells. The molecular docking studies were done to explore the probable mechanism of action of the extract which showed 9 compounds are targeting the microtubule-associated protein tau (MAPT). MAPT promotes assembling and prevents disassembling to arrest the cell cycle. The overexpression of MAPT induces chemoresistance to cancerous cells against conventional drugs like paclitaxel. We have identified 17 compounds from ethyl acetate extract of S. anacardium leaves and drawn its chemical structure by using chembiodraw software to transform into pdb format. Further, the compounds have been subjected for molecular docking study to investigate its interactive efficiency with MAPT protein. The compound 13 had higher interactive potential to MAPT with binding energy -31.75 kcal/mol and lowest binding energy (-15.44 kcal/mol) was observed in compound 6. The present study suggested that the compounds from leaves of S. anacardium could be alternative approach of conventional drug for cancer treatment with cost effective and less side effect.

bioinformatics↗

bioRxiv2019

Molecular characterization and differential expression reveal functional divergence of stress-responsive enzymes in C4 panicoid models, Setaria italica and Setaria viridis

Stress-responsive genes regulate the morpho-physiological as well as molecular responses of plants to environmental cues. In addition to known genes, there are several unknown genes underlying stress-responsive machinery. One such machinery is the sophisticated biochemical carbon-concentrating mechanism of the C4 photosynthetic pathway that enables the plants to survive in high temperatures, high light intensities and drought conditions. Despite the importance of C4 photosynthesis, no comprehensive study has been performed to identify and characterize the key enzymes involved in this process among sequenced Poaceae genomes. In the present study, five major classes of enzymes that are reported to play roles in C4 biochemical carbon-concentrating mechanism were identified in sequenced Poaceae genomes with emphasis on the model crops, Setaria italica and S. viridis. Further analysis revealed that segmental and tandem duplications have contributed to the expansion of these gene families. Comparative genome mapping and molecular dating provided insights into their duplication and divergence in the course of evolution. Expression profiling of candidate genes in contrasting S. italica cultivars subjected to abiotic stresses and hormone treatments showed distinct stress-specific upregulation of SiCaH1, Si{beta}CaH5, SiPEPC2, SiPPDK2, SiMDH8 and SiNADP-ME5 in the tolerant cultivar. Altogether, the study highlights key stress-responsive genes that could serve as potential candidates for elucidating their precise roles in stress tolerance. Key messageComprehensive analysis of stress-responsive gene families in C4 model plants, Setaria italica and S. viridis identified SiCaH1, SiPEPC2, SiPPDK2, SiMDH8 and SiNADP-ME5 as potential candidates for engineering abiotic stress tolerance.

plant biology↗

bioRxiv2019

SRSF2 regulation of MDM2 reveals splicing as a therapeutic vulnerability of the p53 pathway

MDM2 is an oncogene and critical negative regulator of tumor suppressor p53. Genotoxic stress causes alternative splicing of MDM2 transcripts, which leads to alterations in p53 activity and contributes to tumorigenesis. MDM2-ALT1 is one of transcripts predominantly produced in response to genotoxic stress and is comprised of terminal coding exons 3 and 12. Previously, we found that SRSF1 induces MDM2-ALT1 by promoting MDM2 exon 11 skipping. Here we report that splicing regulator SRSF2 antagonizes the regulation of SRSF1 by facilitating the inclusion of exon 11 through binding at two conserved exonic splicing enhancers. Overexpression of SRSF2 reduced the generation of MDM2-ALT1 in genotoxic stress condition, whereas knockdown induces the expression of MDM2-ALT1 in absence of genotoxic stress. Consistently, blocking the exon 11 SRSF2 binding sites using oligonucleotides promotes MDM2-ALT1. The regulation of MDM2 splicing by SRSF2 is also conserved in mouse as mutation of one SRSF2 binding site in Mdm2 exon 11, using CRISPR-Cas9, increases the expression MDM2-ALT1 homolog Mdm2-MS2 and proliferation of NIH 3T3 cells. Taken together, these findings underscore the relevance of MDM2 alternative splicing in cancer and suggest that p53 levels can be modulated by artificially regulating MDM2 splicing.

cancer biology↗

bioRxiv2019

Detection and characterization of a novel copper-dependent intermediate in a lytic polysaccharide monooxygenase

Lytic polysaccharide monooxygenases (LPMOs) are copper-containing enzymes capable of oxidizing crystalline cellulose and the enzyme has large practical application in the process of refining biomass. The LPMO catalytic mechanism still remains debated despite several proposed reaction mechanisms. Here, we report a long-lived intermediate (t[1/2] = 6 - 8 minutes) observed in an LPMO from Thermoascus aurantiacus (TaLPMO9A). The intermediate with a strong absorption around 420 nm is formed when reduced LPMO-Cu(I) reacts with H2O2. UV-vis absorption spectroscopy, electron paramagnetic resonance (EPR), and stopped-flow spectroscopy indicate that the observed long-lived intermediate involves the copper center and a nearby tyrosine (Tyr175). We propose that the reaction with H2O2 first forms a highly reactive short-lived Cu(III)-intermediate which is subsequently transformed into the observed long-lived copper-dependent intermediate. Since sub-equimolar amount of H2O2 to LPMO boosts oxidation of phosphoric acid swollen cellulose (PASC) suggests that the long-lived copper-dependent intermediate is part of the catalytic mechanism for LPMOs. The proposed mechanism offers new perspectives in the oxidative reaction mechanism of copper enzymes and hence for the biomass oxidation and the reactivity of copper in biological systems.

biochemistry↗

bioRxiv2019

TLR4-dependent signaling drives extracellular catabolism of low-density lipoprotein aggregates

ObjectiveAggregation and modification of low-density lipoproteins (LDL) promotes their retention and accumulation in the arteries. This is a critical initiating factor during atherosclerosis. Macrophage catabolism of aggregated LDL (agLDL) occurs using a specialized extracellular, hydrolytic compartment, the lysosomal synapse (LS). Compartment formation by local actin polymerization and delivery of lysosomal contents by exocytosis promotes acidification of the compartment and degradation of agLDL. Internalization of metabolites such as cholesterol promotes foam cell formation, a process that drives atherogenesis. Further, there is accumulating evidence for the involvement of TLR4 and its adaptor protein MyD88 in atherosclerosis. Here, we investigated the role of TLR4 in catabolism of agLDL using the LS and foam cell formation.\n\nApproach and ResultsUsing bone marrow-derived macrophages (BMMs) from knockout mice, we find that TLR4 and MyD88 regulate compartment formation, lysosome exocytosis, acidification of the compartment and foam cell formation. Using siRNA, pharmacological inhibition and knockout BMMs, we implicate SYK, PI3 kinase and Akt in agLDL catabolism using the LS. Using bone marrow transplantation of LDL receptor knockout mice with TLR4KO bone marrow, we show that deficiency of TLR4 protects macrophages from lipid accumulation during atherosclerosis. Finally, we demonstrate that macrophages in vivo form an extracellular compartment and exocytose lysosome contents similar to that observed in vitro for degradation of agLDL.\n\nConclusionsWe present a mechanism in which interaction of macrophages with agLDL initiates a TLR4 signaling pathway, resulting in formation of the LS, catabolism of agLDL and lipid accumulation in vitro and in vivo.

cell biology↗

bioRxiv2019

Overcoming ibrutinib resistance by targeting phosphatidylinositol-3-kinase signaling in diffuse large B-cell lymphoma

Diffuse large B-cell lymphoma is the most common subtype of non-Hodgkin lymphoma; 40% of patients relapse following a complete response or are refractory to therapy. The activated subtype of diffuse large B-cell lymphoma relies upon B-cell receptor signaling for survival; this signaling can be modulated by the activity of Brutons tyrosine kinase. Targeting that kinase with its inhibitor ibrutinib provides a potential therapeutic approach for the activated B-cell subtype of diffuse large B-cell lymphoma. However, non-Hodgkin lymphoma is often resistant to ibrutinib or soon develops resistance after exposure to it. In this study, we explored the development of acquired ibrutinib resistance. After generating three isogenic ibrutinib-resistant diffuse large B-cell lymphoma cell lines, we investigated the deregulated pathways that are associated with colony formation, growth rates, and tumorigenic properties. We found that reduced levels of Brutons tyrosine kinase and enhanced phosphatidylinositol 3-kinase/AKT signaling were hallmarks of these ibrutinib-resistant cells. Upregulation of phosphatidylinositol-3-kinase-beta expression in those cells drove resistance and was reversed by the blocking activity of phosphatidylinositol-3-kinase-beta/delta. Treatment with the selective phosphatidylinositol-3-kinase-beta/delta dual inhibitor KA2237 reduced both tumorigenic properties and survival-based phosphatidylinositol-3-kinase/AKT/mTOR signaling of these ibrutinib-resistant cells. Additionally, combining KA2237 with currently available chemotherapeutic agents synergistically inhibited the metabolic growth of these ibrutinib-resistant cells. This study elucidates the compensatory upregulated phosphatidylinositol-3-kinase/AKT axis that emerges in ibrutinib-resistant cells.

cancer biology↗

bioRxiv2018

Transient kinetic analysis of SWR1C-catalyzed H2A.Z deposition unravels the impact of nucleosome dynamics and the asymmetry of stepwise histone exchange

The SWR1C chromatin remodeling enzyme catalyzes an ATP-dependent replacement of nucleosomal H2A with the H2A.Z variant, regulating key DNA-mediated processes, such as transcription and DNA repair. Here we investigate the transient kinetic mechanism of the histone exchange reaction employing ensemble FRET, fluorescence correlation spectroscopy (FCS), and the steady state kinetics of ATP hydrolysis. Our studies indicate that SWR1C modulates nucleosome dynamics on both the millisecond and microsecond timescales, poising the nucleosome for the dimer exchange reaction. The transient kinetic analysis of the remodeling reaction performed under single turnover conditions unraveled a striking asymmetry in the ATP-dependent replacement of nucleosomal dimers, promoted by localized DNA translocation. Taken together, our transient kinetic studies identify new intermediates and provide crucial insights into the SWR1C-catalyzed dimer exchange reaction, as well as shedding light on how the mechanics of H2A.Z deposition might contribute to transcriptional regulation in vivo.

biochemistry↗

bioRxiv2018

Rbfox splicing factors maintain skeletal muscle mass by regulating calpain3 and proteostasis

Alternative splicing promotes proteomic diversity important for cellular differentiation and cell fate determination. Here, we show that deletion of the highly conserved Rbfox1 and Rbfox2 alternative splicing regulators in adult mouse skeletal muscle causes rapid, severe loss of muscle mass. Homeostasis of skeletal muscle tissue requires a dynamic balance between protein synthesis and degradation (proteostasis) but the mechanisms that regulate this balance are not well understood. Rbfox deletion did not cause reduced global protein synthesis, but resulted in reduced autophagy flux and altered splicing of hundreds of transcripts including Capn3, which produced an active form of calpain3 protease. The results indicate Rbfox proteins regulate proteostasis in skeletal muscle tissue by control of calpain and autophagy-lysosome pathways.\n\nHighlightsO_LIProteostasis in adult skeletal muscle is post-transcriptionally regulated, in part by alternative splicing via Rbfox1/2\nC_LIO_LIRbfox1/2 regulate hundreds of targets in skeletal muscle, including Calpn3, to maintain muscle mass in adult mice\nC_LIO_LIAutophagy flux is markedly decreased in muscle lacking Rbfox1/2\nC_LIO_LIAs for neurons, altered proteostasis is detrimental to adult muscle\nC_LI

molecular biology↗