Paratrimastix pyriformis is a free-living flagellate thriving in low-oxygen freshwater sediments. It belongs to the group Metamonada along with human parasites, such as Giardia and Trichomonas. Like other metamonads, P. pyriformis has a mitochondrion-related organelle (MRO) which in this protist is primarily involved in one-carbon folate metabolism. The MRO contains four members of the solute carrier family 25 (SLC25) responsible for the exchange of metabolites across the mitochondrial inner membrane. Here, we characterise the function of the adenine nucleotide carrier PpMC1 by thermostability shift and transport assays. We show that it transports ATP, ADP and, to a lesser extent, AMP, but not phosphate. The carrier is distinct in function and origin from both ADP/ATP carriers and ATP-Mg/phosphate carriers, and it most likely represents a distinct class of adenine nucleotide carriers.

• MeSH
• adenosinmonofosfát metabolismus   MeSH
• adenosintrifosfát metabolismus   MeSH
• lidé MeSH
• mitochondriální membrány metabolismus   MeSH
• mitochondrie metabolismus   MeSH
• paraziti * metabolismus   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

5-aminolevulinic acid (5-ALA) is the first precursor of the heme biosynthesis pathway, accumulated in acute intermittent porphyria (AIP), an inherited metabolic disease characterized by porphobilinogen deaminase deficiency. An increased incidence of hepatocellular carcinoma (HCC) has been reported as a long-term manifestation in symptomatic AIP patients. 5-ALA is an α-aminoketone prone to oxidation, yielding reactive oxygen species and 4,5-dioxovaleric acid. A high concentration of 5-ALA presents deleterious pro-oxidant potential. It can induce apoptosis, DNA damage, mitochondrial dysfunction, and altered expression of carcinogenesis-related proteins. Several hypotheses of the increased risk of HCC rely on the harmful effect of elevated 5-ALA in the liver of AIP patients, which could promote a pro-carcinogenic environment. We investigated the global transcriptional changes and perturbed molecular pathways in HepG2 cells following exposure to 5-ALA 25 mM for 2 h and 24 h using DNA microarray. Distinct transcriptome profiles were observed. 5-ALA '25 mM-2h' upregulated 10 genes associated with oxidative stress response and carcinogenesis. Enrichment analysis of differentially expressed genes by KEGG, Reactome, MetaCoreTM, and Gene Ontology, showed that 5-ALA '25 mM-24h' enriched pathways involved in drug detoxification, oxidative stress, DNA damage, cell death/survival, cell cycle, and mitochondria dysfunction corroborating the pro-oxidant properties of 5-ALA. Furthermore, our results disclosed other possible processes such as senescence, immune responses, endoplasmic reticulum stress, and also some putative effectors, such as sequestosome, osteopontin, and lon peptidase 1. This study provided additional knowledge about molecular mechanisms of 5-ALA toxicity which is essential to a deeper understanding of AIP and HCC pathophysiology. Furthermore, our findings can contribute to improving the efficacy of current therapies and the development of novel biomarkers and targets for diagnosis, prognosis, and therapeutic strategies for AHP/AIP and associated HCC.

• MeSH
• akutní intermitentní porfyrie * komplikace   genetika   metabolismus   MeSH
• hepatocelulární karcinom * genetika   metabolismus   MeSH
• karcinogeneze MeSH
• kyselina aminolevulová metabolismus   farmakologie   MeSH
• lidé MeSH
• nádory jater * genetika   MeSH
• reaktivní formy kyslíku metabolismus   MeSH
• transkriptom MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

Protein phosphorylation is a common phenomenon in human flavoproteins although the functional consequences of this site-specific modification are largely unknown. Here, we evaluated the effects of site-specific phosphorylation (using phosphomimetic mutations at sites S40, S82 and T128) on multiple functional aspects as well as in the structural stability of the antioxidant and disease-associated human flavoprotein NQO1 using biophysical and biochemical methods. In vitro biophysical studies revealed effects of phosphorylation at different sites such as decreased binding affinity for FAD and structural stability of its binding site (S82), conformational stability (S40 and S82) and reduced catalytic efficiency and functional cooperativity (T128). Local stability measurements by H/D exchange in different ligation states provided structural insight into these effects. Transfection of eukaryotic cells showed that phosphorylation at sites S40 and S82 may reduce steady-levels of NQO1 protein by enhanced proteasome-induced degradation. We show that site-specific phosphorylation of human NQO1 may cause pleiotropic and counterintuitive effects on this multifunctional protein with potential implications for its relationships with human disease. Our approach allows to establish relationships between site-specific phosphorylation, functional and structural stability effects in vitro and inside cells paving the way for more detailed analyses of phosphorylation at the flavoproteome scale.

• MeSH
• antioxidancia metabolismus   MeSH
• flavinadenindinukleotid chemie   MeSH
• flavoproteiny metabolismus   MeSH
• fosforylace MeSH
• lidé MeSH
• mutace MeSH
• NAD(P)H dehydrogenasa (chinon) * metabolismus   MeSH
• nádory * genetika   MeSH
• proteasomový endopeptidasový komplex metabolismus   MeSH
• vazba proteinů MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

Bilirubin is among the most potent of the endogenous antioxidants. Data developed over the last three decades have convincingly demonstrated the protective effects of mildly elevated serum bilirubin concentrations; whereas lower levels of it have been associated with an increased risk of various diseases of civilization, commonly accompanied with increased oxidative stress. Even tiny, micromolar changes of serum bilirubin concentrations have been associated with substantial modulation for the risk of these diseases. However, clinical data published in the current literature are influenced by many confounding factors that have not been properly controlled for. These include the use of improper reference intervals, which are mostly used as common intervals without any partitioning for gender, ethnicity, age, or other important factors (such as smoking). The clinical chemistry methods used for bilirubin determination have not been standardized; in fact, these methods are known to be among the least reliable of any used in clinical chemistry labs. As a result, the data from epidemiological studies are not always comparable. Therefore, it is highly recommended to conduct properly-designed large epidemiological studies. Based on this data, the establishment of decision limits is highly warranted, especially for the lower concentration values of serum bilirubin.

• MeSH
• bilirubin analýza   MeSH
• biologické markery analýza   MeSH
• diagnóza MeSH
• lidé MeSH
• mladý dospělý MeSH
• pravidla klinického rozhodování MeSH
• referenční hodnoty MeSH
• senioři nad 80 let MeSH
• senioři MeSH
• Check Tag
• lidé MeSH
• mladý dospělý MeSH
• mužské pohlaví MeSH
• senioři nad 80 let MeSH
• senioři MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH

P38alpha kinase plays an important role in the regulation of both cell stress response and cell fate. In this study, we report that p38alpha kinase-deficient embryonic stem cells exhibit a higher production of reactive oxygen species (ROS) in contrast to their wild-type counterpart. Analysis of the expressions of NADPH oxidases (NOXs) and dual oxidases, crucial enzymes involved in intracellular ROS formation, shows NOX2/gp91phox is over-expressed in p38alpha deficient cells. The particular increase in superoxide formation was confirmed by the specific detection of hydroethidine derivate 2-hydroxyethidium. ROS formation decreased when the level of NOX2 was silenced by siRNA in p38alpha deficient cells. These data suggest the importance of p38alpha kinase in the regulation of ROS metabolism in embryonic stem cells and the significance of the observed phenomena of cancer cell-like phenotypes, which is discussed.

• MeSH
• buněčná diferenciace fyziologie   MeSH
• genový knockdown MeSH
• genový knockout MeSH
• kultivované buňky MeSH
• membránový potenciál mitochondrií fyziologie   MeSH
• mitochondrie metabolismus   MeSH
• mitogenem aktivovaná proteinkinasa 14 genetika   metabolismus   MeSH
• myší embryonální kmenové buňky metabolismus   MeSH
• myši MeSH
• NADPH-oxidasa 2 genetika   metabolismus   MeSH
• superoxidy metabolismus   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

α-synuclein amyloid fibrils are found in surviving neurons of Parkinson's disease affected patients, but the role they play in the disease development is still under debate. A growing number of evidences points to soluble oligomers as the major cytotoxic species, while insoluble fibrillar aggregates could even play a protection role. In this work, we investigate α-synuclein fibrils dissociation induced at high pressure by means of Small Angle X-ray Scattering and Fourier Transform Infrared Spectroscopy. Fibrils were produced from wild type α-synuclein and two familial mutants, A30P and A53T. Our results enlighten the different reversible nature of α-synuclein fibrils fragmentation at high pressure and suggest water excluded volumes presence in the fibrils core. Wild type and A30P species stabilized at high pressure are highly amyloidogenic and quickly re-associate into fibrils upon decompression, while A53T species shows a partial reversibility of the process likely due to the presence of an intermediate oligomeric state stabilized at high pressure. The amyloid fibrils dissociation process is here suggested to be associated to a negative activation volume, supporting the notion that α-synuclein fibrils are in a high-volume and high-compressibility state and hinting at the presence of a hydration-mediated activated state from which dissociation occurs.

• MeSH
• alfa-synuklein chemie   genetika   metabolismus   MeSH
• amyloid chemie   genetika   metabolismus   MeSH
• bodová mutace MeSH
• difrakce rentgenového záření MeSH
• lidé MeSH
• maloúhlový rozptyl MeSH
• Parkinsonova nemoc genetika   metabolismus   MeSH
• rozpustnost MeSH
• spektroskopie infračervená s Fourierovou transformací MeSH
• tlak MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

A detailed kinetic study based on steady-state and pre-steady-state measurements is described for the highly enantioselective epoxide hydrolase Kau2. The enzyme, which is a member of the α/β-hydrolase fold family, preferentially reacts with the (S,S)-enantiomer of trans-stilbene oxide (TSO) with an E value of ∼200. The enzyme follows a classical two-step catalytic mechanism with formation of an alkyl-enzyme intermediate in the first step and hydrolysis of this intermediate in a rate-limiting second step. Tryptophan fluorescence quenching during TSO conversion appears to correlate with alkylation of the enzyme. The steady-state data are consistent with (S,S) and (R,R)-TSO being two competing substrates with marked differences in k(cat) and K(M) values. The high enantiopreference of the epoxide hydrolase is best explained by pronounced differences in the second-order alkylation rate constant (k2/K(S)) and the alkyl-enzyme hydrolysis rate k3 between the (S,S) and (R,R)-enantiomers of TSO. Our data suggest that during conversion of (S,S)-TSO the two active site tyrosines, Tyr(157) and Tyr(259), serve mainly as electrophilic catalysts in the alkylation half-reaction, polarizing the oxirane oxygen of the bound epoxide through hydrogen bond formation, however, without fully donating their hydrogens to the forming alkyl-enzyme intermediate.

• MeSH
• aktivace enzymů MeSH
• chemické modely * MeSH
• epoxid hydrolasy chemie   ultrastruktura   MeSH
• katalýza MeSH
• kinetika MeSH
• konformace proteinů MeSH
• simulace molekulového dockingu * MeSH
• stabilita enzymů MeSH
• stilbeny chemie   MeSH
• substrátová specifita MeSH
• vazba proteinů MeSH
• vazebná místa MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Understanding the interaction between graphene oxide (GO) and the biomolecules is fundamentally essential, especially for disease- and drug-related peptides and proteins. In this study, the interaction between GO and albumins (bovine serum albumin, human serum albumin, and bovine alpha-lactalbumin) has been performed by fluorescence and UV-Vis spectroscopic techniques. The fluorescence quenching mechanism between GO and aromatic acids residues with intrinsic fluorescence was determined as mainly static quenching in combination with dynamic quenching. The optimal conditions for the most effective affinity between albumins and GO have been estimated at neutral pH and room temperature. The strong impact of the size of graphene oxide on the interaction between proteins and graphene oxide has been confirmed, as well. The interaction between GO and albumins has been examined as electrostatic and hydrophobic. The electrostatic interaction was confirmed by pH effect, while the hydrophobic interaction was proved by the presence of Poloxamer188. The CD spectra of albumins exhibit decreasing helicity in the secondary structure of albumins upon the addition of GO. However, no significant changes in position and shape of characteristic negative bands have been noted. Mentioned changes indicate the successful interaction between GO and proteins, the predominantly α-helical structure of albumins has been preserved.

• MeSH
• cirkulární dichroismus MeSH
• grafit chemie   MeSH
• koncentrace vodíkových iontů MeSH
• laktalbumin chemie   MeSH
• lidé středního věku MeSH
• lidé MeSH
• oxidy chemie   MeSH
• sérový albumin hovězí chemie   MeSH
• sérový albumin chemie   MeSH
• skot MeSH
• teplota MeSH
• velikost částic MeSH
• zvířata MeSH
• Check Tag
• lidé středního věku MeSH
• lidé MeSH
• skot MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

HIV integrase (IN) catalyzes the insertion of proviral DNA into the host cell chromosome. While IN has strict sequence requirements for the viral cDNA ends, the integration site preference has been shown to be very diverse. Here, we mapped the HIV IN strand transfer reaction requirements using various short oligonucleotides (ON) that mimic the target DNA. Most double stranded DNA dodecamers served as excellent IN targets with variable integration efficiency depending mostly on the ON sequences. The preferred integration was lost with any changes in the geometry of the DNA double helical structures. Various hairpin-loop-forming ONs also served as efficient integration targets. Similar integration preferences were also observed for ONs, in which the nucleotide hairpin loop was replaced with a flexible aliphatic linker. The integration biases with all target DNA structures tested were significantly influenced by changes in the resulting secondary ON structures.

• MeSH
• dimerizace MeSH
• DNA virů genetika   MeSH
• HIV-1 genetika   MeSH
• HIV-integrasa genetika   izolace a purifikace   MeSH
• integrace viru genetika   MeSH
• katalýza MeSH
• konformace nukleové kyseliny MeSH
• molekulární sekvence - údaje MeSH
• oligonukleotidy genetika   MeSH
• sekvence nukleotidů MeSH
• substrátová specifita genetika   MeSH
• vztahy mezi strukturou a aktivitou MeSH
• Publikační typ
• práce podpořená grantem MeSH

We investigated hydrogen peroxide production in mitochondria with low (liver, heart, brain) and high (brown adipose tissue, BAT) content of glycerophosphate dehydrogenase (mGPDH). ROS production at state 4 due to electron backflow from mGPDH was low, but after inhibition of electron transport with antimycin A high rates of mGPDH-dependent ROS production were observed in liver, heart and brain mitochondria. When this ROS production was related to activity of mGPDH, many-fold higher ROS production was found in contrast to succinate- (39-, 28-, 3-fold) or pyruvate plus malate-dependent ROS production (32-, 96-, 5-fold). This specific rate of mGPDH-dependent ROS production was also exceedingly higher (28-, 66-, 22-fold) compared to that in BAT. mGPDH-dependent ROS production was localized to the dehydrogenase+CoQ and complex III, the latter being the highest in all mitochondria but BAT. Our results demonstrate high efficiency of mGPDH-dependent ROS production in mammalian mitochondria with a low content of mGPDH and suggest its endogenous inhibition in BAT.

• MeSH
• antimycin A farmakologie   MeSH
• financování organizované MeSH
• glycerolfosfátdehydrogenasa metabolismus   MeSH
• hnědá tuková tkáň metabolismus   MeSH
• jaterní mitochondrie metabolismus   účinky léků   MeSH
• křečci praví MeSH
• krysa rodu rattus MeSH
• kyselina jantarová metabolismus   MeSH
• kyselina pyrohroznová metabolismus   MeSH
• mitochondrie metabolismus   účinky léků   MeSH
• mozek metabolismus   MeSH
• peroxid vodíku metabolismus   MeSH
• potkani Wistar MeSH
• reaktivní formy kyslíku metabolismus   MeSH
• respirační komplex III metabolismus   MeSH
• srdeční mitochondrie metabolismus   účinky léků   MeSH
• transport elektronů MeSH
• zvířata MeSH
• Check Tag
• křečci praví MeSH
• krysa rodu rattus MeSH
• mužské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• techniky in vitro MeSH