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
Tumor markers known also as cancer or oncological markers are substances produced either by tumor or normal cells as a response to the presence of cancer . The markers are used for the detection, diagnosis, prognosis and control of treatment. There is no absolutely specific or universal cancer marker; therefore different markers for different cancer types are used in clinical practice. A problematic feature of cancer markers is their falsely positive results under physiological and non-cancer conditions.
In this study the levels of auto-antibodies formed against the activation peptide of procathepsin D were determined by the ELISA method using a synthetic multiantigen peptide containing the N-terminal amino acid sequence of the activation peptide of procathepsin D as a coating antigen. Based on the results auto-antibodies cannot be considered suitable cancer markers.
Metody PCR se úspěšně využívají při určování původních surovin v masných, mlékárenských a rybích produktech. Obecně se všechny zmíněné metody dají aplikovat pro odhalení použití levnější suroviny místo dražší, nebo odhalit kvalitu přísad, ale důležitá je i interpretace výsledků. Prozatím neexistuje univerzální přesná metoda, která by jednoznačně dokázala určit druhový původ surovin v potravinách. V tomto článku jsou ríastíněny současné výzkumnétrendy v oblasti DNA technologií pro určováním pravosti potravin.
PCR methods have been successfully used in identifying the main ingredients of meat, dairy and fish products. Generally, all the described methods can be applied to detect the substitution of expensive raw materials for by cheaper ones, or reveal the quality of ingredients, but it is the correct interpretation of results. So far there is no universal exact method, which could clearly identify the origin of the ingredients in foods. This article outlines the current research trends in the field of DNA technology for the determination of food authenticity.
