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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 -
Methods in Molecular Biology (Clifton,... 2021Three-dimensional (3D) in vitro systems closely resemble tissue microenvironments and provide predictive models for studying cytotoxic drug responses. The ability to...
Three-dimensional (3D) in vitro systems closely resemble tissue microenvironments and provide predictive models for studying cytotoxic drug responses. The ability to capture the kinetic profiles of such responses in a dynamic and noninvasive way can further advance the utility of 3D cell cultures. Here, we describe the use of a luminescent lactate dehydrogenase (LDH) toxicity assay for monitoring time- and dose-dependent effects of drug treatment in 3D cancer spheroids. HCT116 spheroids formed in 96-well ultralow attachment plates were treated with increasing drug concentrations. Medium samples were collected at different timepoints, frozen, stored, and analyzed at the end of experiments using the luminescent LDH-Glo™ Assay. High assay sensitivity and low volume sampling enabled drug-induced toxicity profiling in a time- and dose-dependent manner.
Topics: Antineoplastic Agents; Digitonin; Dose-Response Relationship, Drug; Drug Screening Assays, Antitumor; Humans; Indicators and Reagents; L-Lactate Dehydrogenase; Luminescent Measurements; Neoplasms; Spheroids, Cellular; Time Factors; Toxicity Tests; Tumor Cells, Cultured
PubMed: 34033096
DOI: 10.1007/978-1-0716-1162-3_8 -
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 -
Experimental Parasitology Aug 2023Per-ARNT-Sim (PAS) domains constitute a family of domains present in a wide variety of prokaryotic and eukaryotic organisms. They form part of the structure of various...
Per-ARNT-Sim (PAS) domains constitute a family of domains present in a wide variety of prokaryotic and eukaryotic organisms. They form part of the structure of various proteins involved in diverse cellular processes. Regulation of enzymatic activity and adaptation to environmental conditions, by binding small ligands, are the main functions attributed to PAS-containing proteins. Recently, genes for a diverse set of proteins with a PAS domain were identified in the genomes of several protists belonging to the group of kinetoplastids, however, until now few of these proteins have been characterized. In this work, we characterize a phosphoglycerate kinase containing a PAS domain present in Trypanosoma cruzi (TcPAS-PGK). This PGK isoform is an active enzyme of 58 kDa with a PAS domain located at its N-terminal end. We identified the protein's localization within glycosomes of the epimastigote form of the parasite by differential centrifugation and selective permeabilization of its membranes with digitonin, as well as in an enriched mitochondrial fraction. Heterologous expression systems were developed for the protein with the N-terminal PAS domain (PAS-PGKc) and without it (PAS-PGKt), and the substrate affinities of both forms of the protein were determined. The enzyme does not exhibit standard Michaelis-Menten kinetics. When evaluating the dependence of the specific activity of the recombinant PAS-PGK on the concentration of its substrates 3-phosphoglycerate (3PGA) and ATP, two peaks of maximal activity were found for the complete enzyme with the PAS domain and a single peak for the enzyme without the domain. Km values measured for 3PGA were 219 ± 26 and 8.8 ± 1.3 μM, and for ATP 291 ± 15 and 38 ± 2.2 μM, for the first peak of PAS-PGKc and for PAS-PGKt, respectively, whereas for the second PAS-PGKc peak values of approximately 1.1-1.2 mM were estimated for both substrates. Both recombinant proteins show inhibition by high concentrations of their substrates, ATP and 3PGA. The presence of hemin and FAD exerts a stimulatory effect on PAS-PGKc, increasing the specific activity by up to 55%. This stimulation is not observed in the absence of the PAS domain. It strongly suggests that the PAS domain has an important function in vivo in T. cruzi in the modulation of the catalytic activity of this PGK isoform. In addition, the PAS-PGK through its PAS and PGK domains could act as a sensor for intracellular conditions in the parasite to adjust its intermediary metabolism.
Topics: Humans; Trypanosoma cruzi; Phosphoglycerate Kinase; Protein Isoforms; Chagas Disease; Adenosine Triphosphate
PubMed: 37353138
DOI: 10.1016/j.exppara.2023.108574 -
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 -
Methods in Molecular Biology (Clifton,... 2023Insect-transmitted trypanosomatid parasite infections cause life-threatening neglected tropical diseases (NTDs), including African sleeping sickness, Chagas disease and...
Insect-transmitted trypanosomatid parasite infections cause life-threatening neglected tropical diseases (NTDs), including African sleeping sickness, Chagas disease and leishmaniasis. In these parasites, glycosomes are unique organelles that are essential for the parasite survival. Proper biogenesis of glycosomes is crucial to ensure correct compartmentation of the glycosomal metabolism. Genetic or chemical disruption of the glycosome biogenesis leads to a mislocalization of the glycosomal enzymes into the cytosol, which results in toxicity to the parasites. Here, we describe a detailed protocol for biochemical fractionation of Trypanosoma brucei parasites to detect mislocalization of glycosomal proteins to the cytosol. This approach utilizes increasing concentrations of digitonin that first permeabilizes the plasma membrane, followed by permeabilization of other organelles, depending on their cholesterol content. Fractionated samples can be further analyzed using immunoblotting for specific marker proteins or quantified by the specific enzyme activities.
Topics: Animals; Trypanosoma; Microbodies; Trypanosoma brucei brucei; Protein Transport; Trypanosomiasis, African; Protozoan Proteins
PubMed: 36952205
DOI: 10.1007/978-1-0716-3048-8_32 -
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 -
Methods in Molecular Biology (Clifton,... 2023The protozoan parasite, Trypanosoma brucei, offers a simple system to study the growth and duplication of the Golgi. Cell lines stably expressing a photoactivatable GFP...
The protozoan parasite, Trypanosoma brucei, offers a simple system to study the growth and duplication of the Golgi. Cell lines stably expressing a photoactivatable GFP attached to an endogenous Golgi protein are permeabilized using digitonin. Photoactivation followed by imaging can then be used to follow the formation of the new Golgi.
Topics: Animals; Trypanosoma brucei brucei; Golgi Apparatus; Digitonin; Parasites; Protozoan Proteins
PubMed: 36512209
DOI: 10.1007/978-1-0716-2639-9_5 -
European Journal of Medicinal Chemistry Nov 2021Pathway activating mutations of the transcription factor NRF2 and its negative regulator KEAP1 are strongly correlative with poor clinical outcome with...
Pathway activating mutations of the transcription factor NRF2 and its negative regulator KEAP1 are strongly correlative with poor clinical outcome with pemetrexed/carbo(cis)platin/pembrolizumab (PCP) chemo-immunotherapy in lung cancer. Despite the strong genetic support and therapeutic potential for a NRF2 transcriptional inhibitor, currently there are no known direct inhibitors of the NRF2 protein or its complexes with MAF and/or DNA. Herein we describe the design of a novel and high-confidence homology model to guide a medicinal chemistry effort that resulted in the discovery of a series of peptides that demonstrate high affinity, selective binding to the Antioxidant Response Element (ARE) DNA and thereby displace NRF2-MAFG from its promoter, which is an inhibitory mechanism that to our knowledge has not been previously described. In addition to their activity in electrophoretic mobility shift (EMSA) and TR-FRET-based assays, we show significant dose-dependent ternary complex disruption of NRF2-MAFG binding to DNA by SPR, as well as cellular target engagement by thermal destabilization of HiBiT-tagged NRF2 in the NCI-H1944 NSCLC cell line upon digitonin permeabilization, and SAR studies leading to improved cellular stability. We report the characterization and unique profile of lead peptide 18, which we believe to be a useful in vitro tool to probe NRF2 biology in cancer cell lines and models, while also serving as an excellent starting point for additional in vivo optimization toward inhibition of NRF2-driven transcription to address a significant unmet medical need in non-small cell lung cancer (NSCLC).
Topics: Antioxidant Response Elements; DNA; Drug Design; Drug Stability; Electrophoretic Mobility Shift Assay; Half-Life; HeLa Cells; Humans; MafG Transcription Factor; NF-E2-Related Factor 2; Neoplasms; Peptides; Structure-Activity Relationship
PubMed: 34303079
DOI: 10.1016/j.ejmech.2021.113686