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Computational and Structural... 2022Synthetic lethality (SL) is an emerging therapeutic paradigm in cancer. We introduced a different approach to prioritize SL gene pairs through literature mining and...
Synthetic lethality (SL) is an emerging therapeutic paradigm in cancer. We introduced a different approach to prioritize SL gene pairs through literature mining and -mutant high-throughput screening (HTS) data. We matched essential genes from text-mining and mutant genes from the COSMIC and CCLE HTS datasets to build a prediction model of SL gene pairs. CCLE gene expression data were used to enrich the essential-mutant SL gene pairs using Spearman's correlation coefficient and literature mining. In total, 223 essential trigger terms were extracted and ranked. The threshold of the essential gene score ( ) was set to 10. We identified 586 genes essential for the SL prediction model of colon cancer. Seven essential -mutant SL gene pairs were identified in our model, including -/----/ and - gene pairs. Using -mutant HTS data validation, we identified two potential SL gene pairs, including the (essential gene)- (mutant gene) pair and - pair in the DLD-1 colon cancer cell line (Spearman's correlation values = 0.004786 and 0.00249, respectively). Based on further annotations by PubChem, we observed that digitonin targeted the complex comprising , especially in -mutated HCT116 cancer cells. Moreover, we experimentally demonstrated that exhibited selective vulnerability in -mutant colorectal cancer. We used literature mining and HTS data to identify candidates for SL targets for mutant colon cancer.
PubMed: 36212540
DOI: 10.1016/j.csbj.2022.09.025 -
PLoS Neglected Tropical Diseases Feb 2021In Trypanosoma brucei, there are fourteen enzymatic biotransformations that collectively convert glucose into five essential nucleotide sugars: UDP-Glc, UDP-Gal,...
In Trypanosoma brucei, there are fourteen enzymatic biotransformations that collectively convert glucose into five essential nucleotide sugars: UDP-Glc, UDP-Gal, UDP-GlcNAc, GDP-Man and GDP-Fuc. These biotransformations are catalyzed by thirteen discrete enzymes, five of which possess putative peroxisome targeting sequences. Published experimental analyses using immunofluorescence microscopy and/or digitonin latency and/or subcellular fractionation and/or organelle proteomics have localized eight and six of these enzymes to the glycosomes of bloodstream form and procyclic form T. brucei, respectively. Here we increase these glycosome localizations to eleven in both lifecycle stages while noting that one, phospho-N-acetylglucosamine mutase, also localizes to the cytoplasm. In the course of these studies, the heterogeneity of glycosome contents was also noted. These data suggest that, unlike other eukaryotes, all of nucleotide sugar biosynthesis in T. brucei is compartmentalized to the glycosomes in both lifecycle stages. The implications are discussed.
Topics: Life Cycle Stages; Microbodies; Nucleotides; Sugars; Trypanosoma brucei brucei
PubMed: 33592041
DOI: 10.1371/journal.pntd.0009132 -
Science Bulletin Mar 2024Silk is one of the toughest fibrous materials known despite spun at ambient temperature and pressure with water as a solvent. It is a great challenge to reproduce...
Silk is one of the toughest fibrous materials known despite spun at ambient temperature and pressure with water as a solvent. It is a great challenge to reproduce high-performance artificial fibers comparable to natural silk by bionic for the incomplete understanding of silkworm spinning in vivo. Here, we found that amphipol and digitonin stabilized the structure of natural silk fibroin (NSF) by a large-scale screening in vitro, and then studied the close-to-native ultrastructure and hierarchical assembly of NSF in the silk gland lumen. Our study showed that NSF formed reversible flexible nanofibrils mainly composed of random coils with a sedimentation coefficient of 5.8 S and a diameter of about 4 nm, rather than a micellar or rod-like structure assembled by the aggregation of globular NSF molecules. Metal ions were required for NSF nanofibril formation. The successive pH decrease from posterior silk gland (PSG) to anterior silk gland (ASG) resulted in a gradual increase in NSF hydrophobicity, thus inducing the sol-gelation transition of NSF nanofibrils. NSF nanofibrils were randomly dispersed from PSG to ASG-1, and self-assembled into anisotropic herringbone patterns at ASG-2 near the spinneret ready for silkworm spinning. Our findings reveal the controlled self-assembly mechanism of the multi-scale hierarchical architecture of NSF from nanofibrils to herringbone patterns programmed by metal ions and pH gradient, which provides novel insights into the spinning mechanism of silk-secreting animals and bioinspired design of high-performance fibers.
Topics: Animals; Bombyx; Silk; Fibroins; Solvents; Metals; Hydrogen-Ion Concentration
PubMed: 38245448
DOI: 10.1016/j.scib.2023.12.050 -
Cells Apr 2024The assessment of nanoparticle cytotoxicity is challenging due to the lack of customized and standardized guidelines for nanoparticle testing. Nanoparticles, with their...
The assessment of nanoparticle cytotoxicity is challenging due to the lack of customized and standardized guidelines for nanoparticle testing. Nanoparticles, with their unique properties, can interfere with biochemical test methods, so multiple tests are required to fully assess their cellular effects. For a more reliable and comprehensive assessment, it is therefore imperative to include methods in nanoparticle testing routines that are not affected by particles and allow for the efficient integration of additional molecular techniques into the workflow. Digital holographic microscopy (DHM), an interferometric variant of quantitative phase imaging (QPI), has been demonstrated as a promising method for the label-free assessment of the cytotoxic potential of nanoparticles. Due to minimal interactions with the sample, DHM allows for further downstream analyses. In this study, we investigated the capabilities of DHM in a multimodal approach to assess cytotoxicity by directly comparing DHM-detected effects on the same cell population with two downstream biochemical assays. Therefore, the dry mass increase in RAW 264.7 macrophages and NIH-3T3 fibroblast populations measured by quantitative DHM phase contrast after incubation with poly(alkyl cyanoacrylate) nanoparticles for 24 h was compared to the cytotoxic control digitonin, and cell culture medium control. Viability was then determined using a metabolic activity assay (WST-8). Moreover, to determine cell death, supernatants were analyzed for the release of the enzyme lactate dehydrogenase (LDH assay). In a comparative analysis, in which the average half-maximal effective concentration (EC) of the nanocarriers on the cells was determined, DHM was more sensitive to the effect of the nanoparticles on the used cell lines compared to the biochemical assays.
Topics: Animals; Mice; NIH 3T3 Cells; Nanoparticles; RAW 264.7 Cells; Cell Survival; Holography; Quantitative Phase Imaging
PubMed: 38667312
DOI: 10.3390/cells13080697 -
MethodsX 2021We describe here a simple method to enrich mitochondrial fractions from mammalian cells for downstream analyses in the lab. Mitochondria purification involves cell lysis...
We describe here a simple method to enrich mitochondrial fractions from mammalian cells for downstream analyses in the lab. Mitochondria purification involves cell lysis followed by separation of the organelles from the rest of the cellular components. Here, we use detergent to rupture the cell membrane of mammalian cells followed by differential centrifugation to enrich the organelles. Optimum conditions with respect to detergent concentration, time, sample size, and yield are discussed. The method's utility in downstream analyses and ease of processing multiple samples simultaneously is also described. All the reagents in this method can be assembled in-house, are economical, and are comparable, if not superior, to commercially available kits in terms of mitochondrial yield and integrity. • Rapid enrichment of mitochondria from mammalian cells using commonly available reagents. • Multiple samples can be processed simultaneously. • Works over a wide range of sample size (1 million to 100 million cells).
PubMed: 34434723
DOI: 10.1016/j.mex.2020.101197 -
Frontiers in Plant Science 2019Mitochondria play a central role in the energy metabolism of plants. At the same time, they provide energy for plant stress responses. We here report a first view on the...
Mitochondria play a central role in the energy metabolism of plants. At the same time, they provide energy for plant stress responses. We here report a first view on the mitochondrial Oxidative Phosphorylation (OXPHOS) system of the halophile (salt tolerant) plant . Mitochondria were purified from suspension cultures of and for comparison of , a closely related glycophyte (salt sensitive) plant. Mitochondria were treated with digitonin and solubilized protein complexes were analyzed by 2D Blue native/SDS polyacrylamide gel electrophoresis. The OXPHOS systems of the two compared plants exhibit some distinct differences. mitochondria include a very abundant respiratory supercomplex composed of monomeric complex I and dimeric complex III. At the same time the complexes II and IV are of reduced abundance. The stability of the OXPHOS complexes was investigated by combined salt and temperature treatments of isolated mitochondria. ATP synthase (complex V) is of increased stability in . Also, the I + III supercomplex is present in high abundance during stress treatments. These results give insights into the mitochondrial contribution to the plant salt stress response.
PubMed: 31456813
DOI: 10.3389/fpls.2019.01010 -
Proceedings of the National Academy of... Oct 2019Cytochrome oxidase (CcO), a membrane enzyme in the respiratory chain, catalyzes oxygen reduction by coupling electron and proton transfer through the enzyme with a...
Cytochrome oxidase (CcO), a membrane enzyme in the respiratory chain, catalyzes oxygen reduction by coupling electron and proton transfer through the enzyme with a proton pump across the membrane. In all crystals reported to date, bovine CcO exists as a dimer with the same intermonomer contacts, whereas CcOs and related enzymes from prokaryotes exist as monomers. Recent structural analyses of the mitochondrial respiratory supercomplex revealed that CcO monomer associates with complex I and complex III, indicating that the monomeric state is functionally important. In this study, we prepared monomeric and dimeric bovine CcO, stabilized using amphipol, and showed that the monomer had high activity. In addition, using a newly synthesized detergent, we determined the oxidized and reduced structures of monomer with resolutions of 1.85 and 1.95 Å, respectively. Structural comparison of the monomer and dimer revealed that a hydrogen bond network of water molecules is formed at the entry surface of the proton transfer pathway, termed the K-pathway, in monomeric CcO, whereas this network is altered in dimeric CcO. Based on these results, we propose that the monomer is the activated form, whereas the dimer can be regarded as a physiological standby form in the mitochondrial membrane. We also determined phospholipid structures based on electron density together with the anomalous scattering effect of phosphorus atoms. Two cardiolipins are found at the interface region of the supercomplex. We discuss formation of the monomeric CcO, dimeric CcO, and supercomplex, as well as their role in regulation of CcO activity.
Topics: Animals; Cardiolipins; Cattle; Crystallography, X-Ray; Digitonin; Electron Transport; Electron Transport Complex I; Electron Transport Complex IV; Hydrogen Bonding; Hydrogen-Ion Concentration; Mitochondria, Heart; Mitochondrial Membranes; Molecular Conformation; Oxidation-Reduction; Oxygen; Phospholipids; Phosphorus; Protein Binding; Protein Conformation; Protein Multimerization
PubMed: 31533957
DOI: 10.1073/pnas.1907183116 -
Identification of Novel Natural Products as Effective and Broad-Spectrum Anti-Zika Virus Inhibitors.Viruses Nov 2019Zika virus (ZIKV) infection during pregnancy leads to severe congenital Zika syndrome, which includes microcephaly and other neurological malformations. No therapeutic...
Zika virus (ZIKV) infection during pregnancy leads to severe congenital Zika syndrome, which includes microcephaly and other neurological malformations. No therapeutic agents have, so far, been approved for the treatment of ZIKV infection in humans; as such, there is a need for a continuous effort to develop effective and safe antiviral drugs to treat ZIKV-caused diseases. After screening a natural product library, we have herein identified four natural products with anti-ZIKV activity in Vero E6 cells, including gossypol, curcumin, digitonin, and conessine. Except for curcumin, the other three natural products have not been reported before to have anti-ZIKV activity. Among them, gossypol exhibited the strongest inhibitory activity against almost all 10 ZIKV strains tested, including six recent epidemic human strains. The mechanistic study indicated that gossypol could neutralize ZIKV infection by targeting the envelope protein domain III (EDIII) of ZIKV. In contrast, the other natural products inhibited ZIKV infection by targeting the host cell or cell-associated entry and replication stages of ZIKV. A combination of gossypol with any of the three natural products identified in this study, as well as with bortezomib, a previously reported anti-ZIKV compound, exhibited significant combinatorial inhibitory effects against three ZIKV human strains tested. Importantly, gossypol also demonstrated marked potency against all four serotypes of dengue virus (DENV) human strains in vitro. Taken together, this study indicates the potential for further development of these natural products, particularly gossypol, as the lead compound or broad-spectrum inhibitors against ZIKV and other flaviviruses, such as DENV.
Topics: Alkaloids; Animals; Antiviral Agents; Biological Products; Cell Survival; Chlorocebus aethiops; Curcumin; Dengue Virus; Digitonin; Drug Synergism; Gossypol; Humans; Molecular Structure; Vero Cells; Zika Virus; Zika Virus Infection
PubMed: 31684080
DOI: 10.3390/v11111019 -
The Journal of Biological Chemistry Mar 2023The nitric oxide synthase interacting protein (NOSIP), an E3-ubiquitin ligase, is involved in various processes like neuronal development, craniofacial development,...
The nitric oxide synthase interacting protein (NOSIP), an E3-ubiquitin ligase, is involved in various processes like neuronal development, craniofacial development, granulopoiesis, mitogenic signaling, apoptosis, and cell proliferation. The best-characterized function of NOSIP is the regulation of endothelial nitric oxide synthase activity by translocating the membrane-bound enzyme to the cytoskeleton, specifically in the G2 phase of the cell cycle. For this, NOSIP itself has to be translocated from its prominent localization, the nucleus, to the cytoplasm. Nuclear import of NOSIP was suggested to be mediated by the canonical transport receptors importin α/β. Recently, we found NOSIP in a proteomic screen as a potential importin 13 cargo. Here, we describe the nuclear shuttling characteristics of NOSIP in living cells and in vitro and show that it does not interact directly with importin α. Instead, it formed stable complexes with several importins (-β, -7, -β/7, -13, and transportin 1) and was also imported into the nucleus in digitonin-permeabilized cells by these factors. In living HeLa cells, transportin 1 seems to be the major nuclear import receptor for NOSIP. A detailed analysis of the NOSIP-transportin 1 interaction revealed a high affinity and an unusual binding mode, involving the N-terminal half of transportin 1. In contrast to nuclear import, nuclear export of NOSIP seems to occur mostly by passive diffusion. Thus, our results uncover additional layers in the larger process of endothelial nitric oxide synthase regulation.
Topics: Active Transport, Cell Nucleus; HeLa Cells; Humans; Protein Binding; Nitric Oxide Synthase Type III; Proteome; Ubiquitin-Protein Ligases; beta Karyopherins
PubMed: 36690276
DOI: 10.1016/j.jbc.2023.102932 -
Biochimica Et Biophysica Acta.... Oct 2022Triterpene glycosides are a diverse group of plant secondary metabolites, consisting of a sterol-like aglycon and one or several sugar groups. A number of triterpene...
Triterpene glycosides are a diverse group of plant secondary metabolites, consisting of a sterol-like aglycon and one or several sugar groups. A number of triterpene glycosides show membranolytic activity, and, therefore, are considered to be promising antimicrobial drugs. However, the interrelation between their structure, biological activities, and target membrane lipid composition remains elusive. Here we studied the antifungal effects of four Panax triterpene glycosides (ginsenosides) with sugar moieties at the C-3 (ginsenosides Rg3, Rh2), C-20 (compound K), and both (ginsenoside F2) positions in Saccharomyces cerevisiae mutants with altered sterol plasma membrane composition. We observed reduced cytostatic activity of the Rg3 and compound K in the UPC2-1 strain with high membrane sterol content. Moreover, LAM gene deletion reduced yeast resistance to Rg3 and digitonin, another saponin with glycosylated aglycon in the C-3 position. LAM genes encode plasma membrane-anchored StARkin superfamily-member sterol transporters. We also showed that the deletion of the ERG6 gene that inhibits ergosterol biosynthesis at the stage of zymosterol increased the cytostatic effects of Rg3 and Rh2, but not the other two tested ginsenosides. At the same time, in silico simulation revealed that the substitution of ergosterol with zymosterol in the membrane changes the spatial orientation of Rg3 and Rh2 in the membranes. These results imply that the plasma membrane sterol composition defines its interaction with triterpene glycoside depending on their glycoside group position. Our results also suggest that the biological role of membrane-anchored StARkin family protein is to protect eukaryotic cells from triterpenes glycosylated at the C-3 position.
Topics: Cytostatic Agents; Ergosterol; Ginsenosides; Saccharomyces cerevisiae; Sterols; Sugars; Triterpenes
PubMed: 35724740
DOI: 10.1016/j.bbamem.2022.183993