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Dive into the research topics where Hana Engstová is active.

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Featured researches published by Hana Engstová.


Biochimica et Biophysica Acta | 1998

Fatty acid cycling mechanism and mitochondrial uncoupling proteins

Petr Ježek; Hana Engstová; Markéta Žáčková; Anibal E. Vercesi; Alexandre D. T. Costa; Paulo Arruda; Keith D. Garlid

We hypothesize that fatty acid-induced uncoupling serves in bioenergetic systems to set the optimum efficiency and tune the degree of coupling of oxidative phosphorylation. Uncoupling results from fatty acid cycling, enabled by several phylogenetically specialized proteins and, to a lesser extent, by other mitochondrial carriers. It is suggested that the regulated uncoupling in mammalian mitochondria is provided by uncoupling proteins UCP-1, UCP-2 and UCP-3, whereas in plant mitochondria by PUMP and StUCP, all belonging to the gene family of mitochondrial carriers. UCP-1, and hypothetically UCP-3, serve mostly to provide nonshivering thermogenesis in brown adipose tissue and skeletal muscle, respectively. Fatty acid cycling was documented for UCP-1, PUMP and ADP/ATP carrier, and is predicted also for UCP-2 and UCP-3. UCP-1 mediates a purine nucleotide-sensitive uniport of monovalent unipolar anions, including anionic fatty acids. The return of protonated fatty acid leads to H+ uniport and uncoupling. UCP-2 is probably involved in the regulation of body weight and energy balance, in fever, and defense against generation of reactive oxygen species. PUMP has been discovered in potato tubers and immunologically detected in fruits and corn, whereas StUCP has been cloned and sequenced froma a potato gene library. PUMP is supposed to act in the termination of synthetic processes in mature fruits and during the climacteric respiratory rise.


Journal of Biological Chemistry | 2001

Natural and Azido Fatty Acids Inhibit Phosphate Transport and Activate Fatty Acid Anion Uniport Mediated by the Mitochondrial Phosphate Carrier

Hana Engstová; Markéta Z̆ác̆ková; Michal Růz̆ic̆ka; Andrea Meinhardt; Jan Hanuš; Reinhard Krämer; Petr Jez̆ek

The electroneutral Pi uptake via the phosphate carrier (PIC) in rat liver and heart mitochondria is inhibited by fatty acids (FAs), by 12-(4-azido-2-nitrophenylamino)dodecanoic acid (AzDA) and heptylbenzoic acid (∼1 μm doses) and by lauric, palmitic, or 12-azidododecanoic acids (∼0.1 mmdoses). In turn, reconstituted E. coli-expressed yeast PIC mediated anionic FA uniport with a similar pattern leading to FA cycling and H+ uniport. The kinetics of Pi/Pi exchange on recombinant PIC in the presence of AzDA better corresponded to a competitive inhibition mechanism. Methanephosphonate was identified as a new PIC substrate. Decanephosphonate, butanephosphonate, 4-nitrophenylphosphate, and other Pi analogs were not translocated and did not inhibit Pi transport. However, methylenediphosphonate and iminodi(methylenephosphonate) inhibited both electroneutral Pi uptake and FA cycling via PIC. AzDA analog 16-(4-azido-2-nitrophenylamino)-[3H4]-hexadecanoic acid (3H-AzHA) bound upon photoactivation to several mitochondrial proteins, including the 30- and 34-kDa bands. The latter was ascribed to PIC due to its specific elution pattern on Blue Sepharose and Affi-Gel. 3H-AzHA photolabeling of recombinant PIC was prevented by methanephosphonate and diphosphonates and after premodification with 4-azido-2-nitrophenylphosphate. Hence, the demonstrated PIC interaction with monovalent long-chain FA anions, but with divalent phosphonates of short chain only, indicates a pattern distinct from that valid for the mitochondrial uncoupling protein-1.


ACS Applied Materials & Interfaces | 2016

RGDS- and TAT-Conjugated Upconversion of NaYF4:Yb3+/Er3+&SiO2 Nanoparticles: In Vitro Human Epithelioid Cervix Carcinoma Cellular Uptake, Imaging, and Targeting

Uliana Kostiv; Ilya Kotelnikov; Miroslav Šlouf; Jan Kucka; Hana Engstová; Petr Ježek; Daniel Horák

Starting NaYF4:Yb(3+)/Er(3+) nanoparticles with size tuned from 24 to 33 nm were prepared by high-temperature coprecipitation of lanthanide chlorides in high-boiling organic solvents. To enhance colloidal stability in aqueous medium, an aminosilica shell was introduced on the surface by hydrolysis and condensation of tetramethyl orthosilicate and (3-aminopropyl)trimethoxysilane using a reverse microemulsion technique; to form alkyne groups, reaction with 4-pentynoic acid followed. Finally, the cell adhesive and cell penetrating azidopentanoyl-GGGRGDSGGGY-NH2 (RGDS) and azidopentanoyl-GGGRKKRRQRRR-NH2 (TAT) peptides were conjugated to the upconversion particles via Cu(I)-catalyzed alkyne-azide cycloaddition. The concentrations of the peptides bound to the nanoparticle surfaces and amount of adsorbed residual Cu(I) catalyst were determined using an (125)I-radiolabeled RGDS peptide and a (64)Cu(I)-doped catalyst, respectively. Targeting and uptake of the RGDS- and TAT-conjugated NaYF4:Yb(3+)/Er(3+)&SiO2 nanoparticles by human cervix carcinoma HeLa cells were monitored by confocal microscopy. RGDS-conjugated nanoparticle probes were mainly localized on the cell plasma membrane due to specific binding of the peptide to the corresponding integrins. In contrast, the TAT-conjugated nanoparticles were able to cross the cell membrane and accumulate in the cell cytoplasm. Thus, this new peptide bioconjugation approach supported both extra- and intracellular nanoparticle uptake, enabling targeting and imaging of the specific tumor phenotypes.


The FASEB Journal | 2016

Hypoxic HepG2 cell adaptation decreases ATP synthase dimers and ATP production in inflated cristae by mitofilin down-regulation concomitant to MICOS clustering

Lydie Plecitá-Hlavatá; Hana Engstová; Lukáš Alán; Tomáš Špaček; Andrea Dlasková; Katarína Smolková; Jitka Špačková; Jan Tauber; Vendula Stradalova; Jan Malínský; Mark Lessard; Joerg Bewersdorf; Petr Ježek

The relationship of the inner mitochondrial membrane (IMM) cristae structure and intracristal space (ICS) to oxidative phosphorylation (oxphos) is not well understood. Mitofilin (subunit Mic60) of the mitochondrial contact site and cristae organizing system (MICOS) IMM complex is attached to the outer membrane (OMM) via the sorting and assembly machinery/topogenesis of mitochondrial outer membrane β‐barrel proteins (SAM/TOB) complex and controls the shape of the cristae. ATP synthase dimers determine sharp cristae edges, whereas trimeric OPA1 tightens ICS outlets. Metabolism is altered during hypoxia, and we therefore studied cristae morphology in HepG2 cells adapted to 5% oxygen for 72 h. Three dimensional (3D), super‐resolution biplane fluorescence photoactivation localization microscopy with Eos‐conjugated, ICS‐located lactamase‐β indicated hypoxic ICS expansion with an unchanged OMM (visualized by Eos‐mitochondrial fission protein‐1). 3D direct stochastic optical reconstruction microscopy immunocytochemistry revealed foci of clustered mitofilin (but not MIC OS subunit Mic19) in contrast to its even normoxic distribution. Mitofilin mRNA and protein decreased by ~20%. ATP synthase dimers vs. monomers and state‐3/state‐4 respiration ratios were lower during hypoxia. Electron microscopy confirmed ICS expansion (maximum in glycolytic cells), which was absent in reduced or OMM‐detached cristae of OPA1‐ and mitofilin‐silenced cells, respectively. Hypoxic adaptation is reported as rounding sharp cristae edges and expanding cristae width (ICS) by partial mitofilin/Mic60 down‐regulation. Mitofilin‐depleted MICOS detaches from SAM while remaining MICOS with mitofilin redistributes toward higher interdistances. This phenomenon causes partial oxphos dormancy in glycolytic cells via disruption of ATP synthase dimers.—Plecitá‐Hlavatá, L., Engstová, H., Alán, L., Špaček, T., Dlasková, A., Smolková, K., Špačková, J., Tauber, J., Strádalová, V., Malínský, J., Lessard, M., Bewersdorf, J., Ježek, P. Hypoxic HepG2 cell adaptation decreases ATP synthase dimers and ATP production in inflated cristae by mitofilin down‐regulation concomitant to MICOS clustering. FASEB J. 30, 1941–1957 (2016). www.fasebj.org


Beilstein Journal of Nanotechnology | 2015

Silica-coated upconversion lanthanide nanoparticles: The effect of crystal design on morphology, structure and optical properties

Uliana Kostiv; Miroslav Šlouf; Hana Macková; Alexander Zhigunov; Hana Engstová; Katarína Smolková; Petr Ježek; Daniel Horák

Summary NaYF4:Yb3+/Er3+ nanoparticles were synthesized by thermal decomposition of lanthanide trifluoroacetates using oleylamine (OM) as both solvent and surface binding ligand. The effect of reaction temperature and time on the properties of the particles was investigated. The nanoparticles were characterized by transmission electron microscopy (TEM), electron diffraction (ED), energy dispersive spectroscopy (EDX), dynamic light scattering (DLS), thermogravimetric analysis (TGA), elemental analysis and X-ray diffraction (XRD) to determine morphology, size, polydispersity, crystal structure and elemental composition of the nanocrystals. TEM microscopy revealed that the morphology of the nanoparticles could be fine-tuned by modifying of the synthetic conditions. A cubic-to-hexagonal phase transition of the NaYF4:Yb3+/Er3+ nanoparticles at temperatures above 300 °C was confirmed by both ED and XRD. Upconversion luminescence under excitation at 980 nm was observed in the luminescence spectra of OM–NaYF4:Yb3+/Er3+ nanoparticles. Finally, the OM–NaYF4:Yb3+/Er3+ nanoparticles were coated with a silica shell to enable further functionalization and increase biocompatibility and stability in aqueous media, preventing particle aggregation.


Biochimica et Biophysica Acta | 2017

Antioxidant mechanism of mitochondria-targeted plastoquinone SkQ1 is suppressed in aglycemic HepG2 cells dependent on oxidative phosphorylation

Jan Ježek; Hana Engstová; Petr Ježek

Previously suggested antioxidant mechanisms for mitochondria-targeted plastoquinone SkQ1 included: i) ion-pairing of cationic SkQ1+ with free fatty acid anions resulting in uncoupling; ii) SkQ1H2 ability to interact with lipoperoxyl radical; iii) interference with electron flow at the inner ubiquinone (Q) binding site of Complex III (Qi), involving the reduction of SkQ1 to SkQ1H2 by ubiquinol. We elucidated SkQ1 antioxidant properties by confocal fluorescence semi-quantification of mitochondrial superoxide (Jm) and cytosolic H2O2 (Jc) release rates in HepG2 cells. Only in glycolytic cells, SkQ1 prevented the rotenone-induced enhancement of Jm and Jc but not basal releases without rotenone. The effect ceased in glutaminolytic aglycemic cells, in which the redox parameter NAD(P)H/FAD increased after rotenone in contrast to its decrease in glycolytic cells. Autofluorescence decay indicated decreased NADPH/NADH ratios with rotenone in both metabolic modes. SkQ1 did not increase cell respiration and diminished Jm established high by antimycin or myxothiazol but not by stigmatellin. The revealed SkQ1 antioxidant modes reflect its reduction to SkQ1H2 at Complex I IQ or Complex III Qi site. Both reductions diminish electron diversions to oxygen thus attenuating superoxide formation. Resulting SkQ1H2 oxidizes back to SkQ1at the second (flavin) Complex I site, previously indicated for MitoQ10. Regeneration proceeds only at lower NAD(P)H/FAD in glycolytic cells. In contrast, cyclic SkQ1 reduction/SkQ1H2 oxidation does not substantiate antioxidant activity in intact cells in the absence of oxidative stress (neither pro-oxidant activity, representing a great advantage). A targeted delivery to oxidative-stressed tissues is suggested for the effective antioxidant therapy based on SkQ1.


Scientific Reports | 2017

Nkx6.1 decline accompanies mitochondrial DNA reduction but subtle nucleoid size decrease in pancreatic islet β-cells of diabetic Goto Kakizaki rats

Tomáš Špaček; Vojtěch Pavluch; Lukáš Alán; Nikola Capková; Hana Engstová; Andrea Dlasková; Zuzana Berková; Frantisek Saudek; Petr Ježek

Hypertrophic pancreatic islets (PI) of Goto Kakizaki (GK) diabetic rats contain a lower number of β-cells vs. non-diabetic Wistar rat PI. Remaining β-cells contain reduced mitochondrial (mt) DNA per nucleus (copy number), probably due to declining mtDNA replication machinery, decreased mt biogenesis or enhanced mitophagy. We confirmed mtDNA copy number decrease down to <30% in PI of one-year-old GK rats. Studying relations to mt nucleoids sizes, we employed 3D superresolution fluorescent photoactivable localization microscopy (FPALM) with lentivirally transduced Eos conjugate of mt single-stranded-DNA-binding protein (mtSSB) or transcription factor TFAM; or by 3D immunocytochemistry. mtSSB (binding transcription or replication nucleoids) contoured “nucleoids” which were smaller by 25% (less diameters >150 nm) in GK β-cells. Eos-TFAM-visualized nucleoids, composed of 72% localized TFAM, were smaller by 10% (immunochemically by 3%). A theoretical ~70% decrease in cell nucleoid number (spatial density) was not observed, rejecting model of single mtDNA per nucleoid. The β-cell maintenance factor Nkx6.1 mRNA and protein were declining with age (>12-fold, 10 months) and decreasing with fasting hyperglycemia in GK rats, probably predetermining the impaired mtDNA replication (copy number decrease), while spatial expansion of mtDNA kept nucleoids with only smaller sizes than those containing much higher mtDNA in non-diabetic β-cells.


Biochimica et Biophysica Acta | 2018

3D super-resolution microscopy reflects mitochondrial cristae alternations and mtDNA nucleoid size and distribution

Andrea Dlasková; Hana Engstová; Tomáš Špaček; Anežka Kahancová; Vojtěch Pavluch; Katarína Smolková; Jitka Špačková; Martin Bartoš; Lydie Hlavatá; Petr Ježek

3D super-resolution microscopy based on the direct stochastic optical reconstruction microscopy (dSTORM) with primary Alexa-Fluor-647-conjugated antibodies is a powerful method for accessing changes of objects that could be normally resolved only by electron microscopy. Despite the fact that mitochondrial cristae yet to become resolved, we have indicated changes in cristae width and/or morphology by dSTORM of ATP-synthase F1 subunit α (F1α). Obtained 3D images were analyzed with the help of Ripleys K-function modeling spatial patterns or transferring them into distance distribution function. Resulting histograms of distances frequency distribution provide most frequent distances (MFD) between the localized single antibody molecules. In fasting state of model pancreatic β-cells, INS-1E, MFD between F1α were ~80 nm at 0 and 3 mM glucose, whereas decreased to 61 nm and 57 nm upon glucose-stimulated insulin secretion (GSIS) at 11 mM and 20 mM glucose, respectively. Shorter F1α interdistances reflected cristae width decrease upon GSIS, since such repositioning of F1α correlated to average 20 nm and 15 nm cristae width at 0 and 3 mM glucose, and 9 nm or 8 nm after higher glucose simulating GSIS (11, 20 mM glucose, respectively). Also, submitochondrial entities such as nucleoids of mtDNA were resolved e.g. after bromo-deoxyuridine (BrDU) pretreatment using anti-BrDU dSTORM. MFD in distances distribution histograms reflected an average nucleoid diameter (<100 nm) and average distances between nucleoids (~1000 nm). Double channel PALM/dSTORM with Eos-lactamase-β plus anti-TFAM dSTORM confirmed the latter average inter-nucleoid distance. In conclusion, 3D single molecule (dSTORM) microscopy is a reasonable tool for studying mitochondrion.


RSC Advances | 2017

Correction: Biodistribution of upconversion/magnetic silica-coated NaGdF4:Yb3+/Er3+ nanoparticles in mouse models

Uliana Kostiv; Lenka Rajsiglova; Dominika Luptáková; Tomáš Pluháček; Luca Vannucci; Vladimír Havlíček; Hana Engstová; Daniel Jirák; Miroslav Šlouf; Peter Makovicky; Radislav Sedlacek; Daniel Horák

Correction for ‘Biodistribution of upconversion/magnetic silica-coated NaGdF4:Yb3+/Er3+ nanoparticles in mouse models’ by Uliana Kostiv et al., RSC Adv., 2017, 7, 45997–46006.


Free Radical Biology and Medicine | 2017

Superoxide Generation, Bioenergetics Parameters, and Mitochondrial Morphology in Insulinoma INS-1E Cells upon Glucose Addition and ATPase Inhibitory Factor (IF1) Knockdown

Andrea Dlasková; Anežka Kahancová; Lydie Plecitá-Hlavatá; Hana Engstová; Jan auber; Tomáš Špaček; Jan Ježek; Katarína Smolková; Petr Ježek

We studied mitochondrial superoxide formation under conditions when the respiration substrates suddenly increase upon glucose addition, hence one might expect the elevated superoxide formation. We aimed to study this situation also while determining the concomitant bioenergetics responses. We have clearly demonstrated that mitochondria as the glucose sensor in pancreatic β-cells do decrease superoxide release to the matrix upon GSIS. To correlate resulting changes in surplus superoxide release to the matrix with bioenergetics parameters, we also inspected respiration and mitochondrial membrane potential ΔΨm. The employed 14 mM incremental glucose in cells cultivated with 11 mM glucose induced a small respiration elevation resulting in a slight non-significant increase in ratio of state 3/state 4 respiration. We further confirmed presence of the ATPase Inhibitory Factor 1, IF1, in pancreatic beta cells and studied its role in superoxide generation, regulation of bioenergetics parameters and mitochondrial architecture in INS1E cells.

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Petr Ježek

Academy of Sciences of the Czech Republic

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Andrea Dlasková

Academy of Sciences of the Czech Republic

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Katarína Smolková

Academy of Sciences of the Czech Republic

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Lydie Plecitá-Hlavatá

Academy of Sciences of the Czech Republic

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Tomáš Špaček

Academy of Sciences of the Czech Republic

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Lukáš Alán

Academy of Sciences of the Czech Republic

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Daniel Horák

Academy of Sciences of the Czech Republic

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Miroslav Šlouf

Academy of Sciences of the Czech Republic

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Uliana Kostiv

Academy of Sciences of the Czech Republic

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Jan Ježek

Academy of Sciences of the Czech Republic

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