Translational stalling events that result in ribosome collisions induce Ribosome-associated Quality Control (RQC) in order to degrade potentially toxic truncated nascent proteins. For RQC induction, the collided ribosomes are first marked by the Hel2/ZNF598 E3 ubiquitin ligase to recruit the RQT complex for subunit dissociation. In yeast, uS10 is polyubiquitinated by Hel2, whereas eS10 is preferentially monoubiquitinated by ZNF598 in human cells for an unknown reason. Here, we characterize the ubiquitination activity of ZNF598 and its importance for human RQT-mediated subunit dissociation using the endogenous XBP1u and poly(A) translation stallers. Cryo-EM analysis of a human collided disome reveals a distinct composite interface, with substantial differences to yeast collided disomes. Biochemical analysis of collided ribosomes shows that ZNF598 forms K63-linked polyubiquitin chains on uS10, which are decisive for mammalian RQC initiation. The human RQT (hRQT) complex composed only of ASCC3, ASCC2 and TRIP4 dissociates collided ribosomes dependent on the ATPase activity of ASCC3 and the ubiquitin-binding capacity of ASCC2. The hRQT-mediated subunit dissociation requires the K63-linked polyubiquitination of uS10, while monoubiquitination of eS10 or uS10 is not sufficient. Therefore, we conclude that ZNF598 functionally marks collided mammalian ribosomes by K63-linked polyubiquitination of uS10 for the trimeric hRQT complex-mediated subunit dissociation.
- MeSH
- DNA-helikasy metabolismus MeSH
- lidé MeSH
- proteosyntéza MeSH
- ribozomy metabolismus MeSH
- Saccharomyces cerevisiae - proteiny * genetika metabolismus MeSH
- Saccharomyces cerevisiae * genetika metabolismus MeSH
- transkripční faktory metabolismus MeSH
- transportní proteiny * metabolismus MeSH
- ubikvitinace MeSH
- ubikvitinligasy metabolismus MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Tuberculosis (TB) remains one of the major health concerns worldwide. Mycobacterium tuberculosis (Mtb), the causative agent of TB, can flexibly change its metabolic processes during different life stages. Regulation of key metabolic enzyme activities by intracellular conditions, allosteric inhibition or feedback control can effectively contribute to Mtb survival under different conditions. Phosphofructokinase (Pfk) is one of the key enzymes regulating glycolysis. Mtb encodes two Pfk isoenzymes, Pfk A/Rv3010c and Pfk B/Rv2029c, which are differently expressed upon transition to the hypoxia-induced non-replicating state of the bacteria. While pfkB gene and protein expression are upregulated under hypoxic conditions, Pfk A levels decrease. Here, we present biochemical characterization of both Pfk isoenzymes, revealing that Pfk A and Pfk B display different kinetic properties. Although the glycolytic activity of Pfk A is higher than that of Pfk B, it is markedly inhibited by an excess of both substrates (fructose-6-phosphate and ATP), reaction products (fructose-1,6-bisphosphate and ADP) and common metabolic allosteric regulators. In contrast, synthesis of fructose-1,6-bisphosphatase catalyzed by Pfk B is not regulated by higher levels of substrates, and metabolites. Importantly, we found that only Pfk B can catalyze the reverse gluconeogenic reaction. Pfk B thus can support glycolysis under conditions inhibiting Pfk A function.
- MeSH
- adenosindifosfát metabolismus farmakologie MeSH
- adenosintrifosfát metabolismus farmakologie MeSH
- alosterická regulace MeSH
- bakteriální proteiny antagonisté a inhibitory metabolismus MeSH
- enzymová indukce MeSH
- fosfofruktokinasy antagonisté a inhibitory metabolismus MeSH
- fruktosadifosfáty biosyntéza farmakologie MeSH
- fruktosafosfáty metabolismus farmakologie MeSH
- glukoneogeneze MeSH
- glykolýza MeSH
- hexosafosfáty metabolismus MeSH
- izoenzymy antagonisté a inhibitory metabolismus MeSH
- katalýza MeSH
- kinetika MeSH
- kyslík farmakologie MeSH
- L-laktátdehydrogenasa metabolismus MeSH
- Mycobacterium tuberculosis účinky léků enzymologie MeSH
- pyruvátkinasa metabolismus MeSH
- rekombinantní proteiny metabolismus MeSH
- substrátová specifita MeSH
- zpětná vazba fyziologická MeSH
- Publikační typ
- časopisecké články MeSH
- srovnávací studie MeSH
Colorectal cancer (CRC) ranks among the most common cancers worldwide. Surgical removal remains the best strategy for treatment of resectable tumors. An important part of caring for patients after surgery is monitoring for early detection of a possible relapse of the disease. Efforts are being made to improve the sensitivity and specificity of routinely used carcinoembryonic antigen (CEA) with the use of additional biomarkers such as microRNAs. The aim of our study was to evaluate the prognostic potential of microRNAs and their use as markers of disease recurrence. The quantitative estimation of CEA, CA19-9, and 22 selected microRNAs (TaqMan Advanced miRNA Assays) was performed in 85 paired (preoperative and postoperative) blood plasma samples of CRC patients and in samples taken during the follow-up period. We have revealed a statistically significant decrease in plasma levels for miR-20a, miR-23a, miR-210, and miR-223a (p = 0.0093, p = 0.0013, p = 0.0392, and p = 0.0214, respectively) after surgical removal of the tumor tissue. A statistically significant relation to prognosis (overall survival; OS) was recorded for preoperative plasma levels of miR-20a, miR-21, and miR-23a (p = 0.0236, p = 0.0316, and p =0.0271, respectively) in a subgroup of patients who underwent palliative surgery. The best discrimination between patients with favorable and unfavorable outcomes was achieved by a combination of CEA, CA19-9 with miR-21, miR-20a, and miR-23a (p < 0.0001). The use of these microRNAs for early disease recurrence detection was affected by a low specificity in comparison with CEA and CA19-9. CEA and CA19-9 had high specificity but low sensitivity. Our results show the benefit of combining currently used standard biomarkers and microRNAs for precise prognosis estimation.
- Publikační typ
- časopisecké články MeSH
Cílem této studie je analyzovat reliabilitu a predikční validitu testové sestavy motorických testů nej- častěji využívaných v kontrolním testování při tréninku běhu na 100 m překážek žen. Bylo analyzováno sedm testů používaných pro predikci sportovní výkonnosti překážkářek. Testování se zúčastnily nejlepší překážkářky z oddílů Ostrava (Vítkovice, Poruba), Třinec, Opava a Olomouc. Na základě výsledků regresní krokové analýzy bylo zjištěno, že klíčovými testy pro predikci budoucí překážkářské výkonosti jsou testy běh na 120 m a skok do dálky z místa.
The aim of this study is to analyse the reliability and prediction validity of a battery of motor tests used in 100m Hurdles woman. Seven control tests were analyzed and used for a prediction of performance and training improvement and to evaluate their importance for a future hurdles performance. Best athletes (woman, 100m hurdles runners) from Ostrava (Vítkovice, Poruba), Třinec, Opava and Olomouc have been asked to undergo the testing. Based on the results of stepwise method we can conclude that key tests in the whole battery are 120m run and standing long jump. The result of the multi-regress analyzes shows that only these two tests can predict the performance on women´s 100m hurdles.
- Klíčová slova
- motorické testování,
- MeSH
- běh * MeSH
- lidé MeSH
- motorické dovednosti MeSH
- reprodukovatelnost výsledků MeSH
- sportovní výkon MeSH
- výkonnost * MeSH
- Check Tag
- lidé MeSH
- ženské pohlaví MeSH
Mycobacterium tuberculosis (MTb), the causative agent of tuberculosis, can persist in macrophages for decades, maintaining its basic metabolic activities. Phosphoenolpyruvate carboxykinase (Pck; EC 4.1.1.32) is a key player in central carbon metabolism regulation. In replicating MTb, Pck is associated with gluconeogenesis, but in non-replicating MTb, it also catalyzes the reverse anaplerotic reaction. Here, we explored the role of selected cysteine residues in function of MTb Pck under different redox conditions. Using mass spectrometry analysis we confirmed formation of S-S bridge between cysteines C391 and C397 localized in the C-terminal subdomain. Molecular dynamics simulations of C391-C397 bridged model indicated local conformation changes needed for formation of the disulfide. Further, we used circular dichroism and Raman spectroscopy to analyze the influence of C391 and C397 mutations on Pck secondary and tertiary structures, and on enzyme activity and specificity. We demonstrate the regulatory role of C391 and C397 that form the S-S bridge and in the reduced form stabilize Pck tertiary structure and conformation for gluconeogenic and anaplerotic reactions.
- MeSH
- aminokyselinové motivy MeSH
- biokatalýza * MeSH
- cystein metabolismus MeSH
- disulfidy metabolismus MeSH
- fosfoenolpyruvátkarboxykinasa (závislá na ATP) chemie metabolismus MeSH
- kinetika MeSH
- molekulární modely MeSH
- mutace genetika MeSH
- mutageneze cílená MeSH
- mutantní proteiny chemie MeSH
- Mycobacterium tuberculosis enzymologie MeSH
- sekundární struktura proteinů MeSH
- sekvence aminokyselin MeSH
- stabilita enzymů MeSH
- substrátová specifita MeSH
- tandemová hmotnostní spektrometrie MeSH
- terciární struktura proteinů MeSH
- vztahy mezi strukturou a aktivitou MeSH
- Publikační typ
- časopisecké články MeSH
Mycobacterium tuberculosis (MTb) je lidský intracelulární patogen, jehož flexibilní metabolismus mu umožňuje přežívat v hostiteli ve formě tzv. latentní infekce bez symptomů onemocnění po dlouhé období. K reaktivaci MTb s následným rozvojem akutní tuberkulózy může dojít kdykoliv. Více ohroženi jsou především lidé s oslabeným imunitním systémem, jako jsou HIV pozitivní pacienti, lidé trpící podvýživou, postižení diabetem nebo rakovinou.
Mycobacterium tuberculosis (MTb) is a human intracellular pathogen, which can survive in the host due to its flexible metabolism in a form of latent infection without any symptoms of illness for a long period. Reactivating tuberculosis (post‑primarily) can occur any time. Especially people with compromised immune systems, such as people living with HIV, malnutrition, diabetes or cancer, have a much higher risk of falling ill with MTb or reactivation of latent infection into acute tuberculosis.
- MeSH
- AIDS MeSH
- diabetes mellitus MeSH
- HIV infekce MeSH
- lidé MeSH
- multirezistentní tuberkulóza imunologie MeSH
- nádory MeSH
- podvýživa komplikace MeSH
- přenos infekční nemoci MeSH
- rizikové faktory MeSH
- tuberkulóza * epidemiologie imunologie MeSH
- vakcíny proti tuberkulóze MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- práce podpořená grantem MeSH
Tuberculosis, the second leading infectious disease killer after HIV, remains a top public health priority. The causative agent of tuberculosis, Mycobacterium tuberculosis (Mtb), which can cause both acute and clinically latent infections, reprograms metabolism in response to the host niche. Phosphoenolpyruvate carboxykinase (Pck) is the enzyme at the center of the phosphoenolpyruvate-pyruvate-oxaloacetate node, which is involved in regulating the carbon flow distribution to catabolism, anabolism, or respiration in different states of Mtb infection. Under standard growth conditions, Mtb Pck is associated with gluconeogenesis and catalyzes the metal-dependent formation of phosphoenolpyruvate. In non-replicating Mtb, Pck can catalyze anaplerotic biosynthesis of oxaloacetate. Here, we present insights into the regulation of Mtb Pck activity by divalent cations. Through analysis of the X-ray structure of Pck-GDP and Pck-GDP-Mn2+ complexes, mutational analysis of the GDP binding site, and quantum mechanical (QM)-based analysis, we explored the structural determinants of efficient Mtb Pck catalysis. We demonstrate that Mtb Pck requires presence of Mn2+ and Mg2+ cations for efficient catalysis of gluconeogenic and anaplerotic reactions. The anaplerotic reaction, which preferably functions in reducing conditions that are characteristic for slowed or stopped Mtb replication, is also effectively activated by Fe2+ in the presence of Mn2+ or Mg2+ cations. In contrast, simultaneous presence of Fe2+ and Mn2+ or Mg2+ inhibits the gluconeogenic reaction. These results suggest that inorganic ions can contribute to regulation of central carbon metabolism by influencing the activity of Pck. Furthermore, the X-ray structure determination, biochemical characterization, and QM analysis of Pck mutants confirmed the important role of the Phe triad for proper binding of the GDP-Mn2+ complex in the nucleotide binding site and efficient catalysis of the anaplerotic reaction.
- MeSH
- aktivace enzymů MeSH
- fosfoenolpyruvátkarboxykinasa (závislá na ATP) chemie genetika metabolismus MeSH
- glukoneogeneze MeSH
- katalýza MeSH
- kationty dvojmocné MeSH
- kineze MeSH
- konformace proteinů MeSH
- lidé MeSH
- molekulární modely MeSH
- molekulární sekvence - údaje MeSH
- multimerizace proteinu MeSH
- mutace MeSH
- Mycobacterium tuberculosis enzymologie genetika MeSH
- nukleotidy metabolismus MeSH
- sekvence aminokyselin MeSH
- sekvenční seřazení MeSH
- vazebná místa MeSH
- vodíková vazba MeSH
- vztahy mezi strukturou a aktivitou MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Tuberculosis remains a major health concern worldwide. Eradication of its causative agent, the bacterial pathogen Mycobacterium tuberculosis, is particularly challenging due to a vast reservoir of latent carriers of the disease. Despite the misleading terminology of a so-called dormant state associated with latent infections, the bacteria have to maintain basic metabolic activities. Hypoxic conditions have been widely used as an in vitro system to study this dormancy. Such studies identified a rearrangement of central carbon metabolism to exploit fermentative processes caused by the lack of oxygen. Phosphoenolpyruvate carboxykinase (Pck; EC 4.1.1.32) is the enzyme at the center of these metabolic rearrangements. Although Pck is associated with gluconeogenesis under standard growth conditions, the enzyme can catalyze the reverse reaction, supporting anaplerosis of the tricarboxylic acid cycle, under conditions leading to slowed or stopped bacterial replication. To study the mechanisms that regulate the switch between two Pck functions, we systematically investigated factors influencing the gluconeogenic and anaplerotic reaction kinetics. We demonstrate that a reducing environment, as found under hypoxia-triggered non-replicating conditions, accelerates the reaction in the anaplerotic direction. Furthermore, we identified proteins that interact with Pck. The interaction between Pck and the reduced form of mycobacterial thioredoxin, gene expression of which is increased under hypoxic conditions, also increased the Pck anaplerotic activity. We thus propose that a reducing environment and the protein-protein interaction with thioredoxin in particular enable the Pck anaplerotic function under fermentative growth conditions.
- MeSH
- bakteriální proteiny genetika metabolismus MeSH
- citrátový cyklus fyziologie MeSH
- fosfoenolpyruvátkarboxykinasa (závislá na ATP) genetika metabolismus MeSH
- Mycobacterium tuberculosis enzymologie genetika MeSH
- oxidace-redukce MeSH
- regulace genové exprese enzymů fyziologie MeSH
- regulace genové exprese u bakterií fyziologie MeSH
- thioredoxiny genetika metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Tuberkulóza je jednou z nejstarších nemocí, a přestože je k dispozici řada léků, je v současnosti nejrozšířenější bakteriální infekcí ve světě a společně s HIV je největším zabijákem. Přispívá k tomu vysoký výskyt MTb kmenů s resistencí k současně používaným lékům a zvýšená citlivost HIV positivních pacientů k nákaze MTb. Více než třetina světové populace je nakažena MTb, které přežívá v latentní podobě u lidí bez příznaků a může být kdykoliv reaktivována snížením imunity, či ko-infekcí dalšími patogeny. Proto je v současnosti urgentní potřeba porozumět mechanismům, kterými se MTb brání imunitnímu systému člověka a které umožňují této baktérii dlouhodobě přežívat v hostiteli. Tyto poznatky umožní vývoj léků s novým mechanismem účinku.
Tuberculosis (TB), one of the oldest human diseases, is still one of the biggest killers, despite the availability of drugs. Increased prevalence of multidrug-resistant MTb strains and dramatically increasing host susceptibility to MTb in HIV-1 positive patients contribute to high prevalence of this bacterial disease. Almost one-third of the world's population is infected with MTb. In the majority of these cases, MTb persists in macrophages in a latent form and can be re-activated any time by immunosuppression or co-infection with other pathogens. Thus, there is an urgent need for better understanding of MTb metabolism, its adaptation to latency, and for discovery of new anti-tuberculosis drugs.
- MeSH
- antituberkulotika terapeutické užití MeSH
- BCG vakcína imunologie škodlivé účinky terapeutické užití MeSH
- dítě MeSH
- dospělí MeSH
- extenzivně rezistentní tuberkulóza MeSH
- lidé MeSH
- multirezistentní tuberkulóza MeSH
- Mycobacterium tuberculosis * metabolismus patogenita účinky léků MeSH
- Check Tag
- dítě MeSH
- dospělí MeSH
- lidé MeSH
- Publikační typ
- práce podpořená grantem MeSH
