Hydrogen/deuterium exchange (HDX) is a well-established analytical technique that enables monitoring of protein dynamics and interactions by probing the isotope exchange of backbone amides. It has virtually no limitations in terms of protein size, flexibility, or reaction conditions and can thus be performed in solution at different pH values and temperatures under controlled redox conditions. Thanks to its coupling with mass spectrometry (MS), it is also straightforward to perform and has relatively high throughput, making it an excellent complement to the high-resolution methods of structural biology. Given the recent expansion of artificial intelligence-aided protein structure modeling, there is considerable demand for techniques allowing fast and unambiguous validation of in silico predictions; HDX-MS is well-placed to meet this demand. Here we present a protocol for HDX-MS and illustrate its use in characterizing the dynamics and structural changes of a dimeric heme-containing oxygen sensor protein as it responds to changes in its coordination and redox state. This allowed us to propose a mechanism by which the signal (oxygen binding to the heme iron in the sensing domain) is transduced to the protein's functional domain.
The nonradioactive method, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) in the presence of Phos-tag (Phos-tag electrophoresis), is used to evaluate a kinase autophosphorylation and/or phosphotransfer reaction from a kinase/ATP to its protein substrate. This method outperforms radioisotope methods using [32P]ATP for detecting trace amounts of phosphorylated protein in fresh protein preparations. Phos-tag electrophoresis has been used to perform detailed analyses of the kinase activity of a heme-based oxygen sensor-specifically, a globin-coupled histidine kinase from the soil bacterium Anaeromyxobacter sp. Fw109-5 (AfGcHK).
The iron atom possesses unique properties, being manifested in the heme molecule. In addition to the involvement of heme in many processes (such as oxygen or electron transport and catalysis of enzyme reaction), it can also regulate key pathways critical for health. Heme-containing sensor proteins mediate the heme regulation role. The review aims to underline the main characteristics of the heme-containing sensor proteins group, either sensing heme or sensing a gas molecule through a heme-binding site, on the example of specific representatives from each sensor subgroup.
- MeSH
- hem * chemie fyziologie MeSH
- hemoproteiny * chemie fyziologie MeSH
- lidé MeSH
- Check Tag
- lidé MeSH
The heme-based oxygen sensor protein AfGcHK is a globin-coupled histidine kinase in the soil bacterium Anaeromyxobacter sp. Fw109-5. Its C-terminal functional domain exhibits autophosphorylation activity induced by oxygen binding to the heme-Fe(II) complex located in the oxygen-sensing N-terminal globin domain. A detailed understanding of the signal transduction mechanisms in heme-containing sensor proteins remains elusive. Here, we investigated the role of the globin domain's dimerization interface in signal transduction in AfGcHK. We present a crystal structure of a monomeric imidazole-bound AfGcHK globin domain at 1.8 Å resolution, revealing that the helices of the WT globin dimer are under tension and suggesting that Tyr-15 plays a role in both this tension and the globin domain's dimerization. Biophysical experiments revealed that whereas the isolated WT globin domain is dimeric in solution, the Y15A and Y15G variants in which Tyr-15 is replaced with Ala or Gly, respectively, are monomeric. Additionally, we found that although the dimerization of the full-length protein is preserved via the kinase domain dimerization interface in all variants, full-length AfGcHK variants bearing the Y15A or Y15G substitutions lack enzymatic activity. The combined structural and biophysical results presented here indicate that Tyr-15 plays a key role in the dimerization of the globin domain of AfGcHK and that globin domain dimerization is essential for internal signal transduction and autophosphorylation in this protein. These findings provide critical insights into the signal transduction mechanism of the histidine kinase AfGcHK from Anaeromyxobacter.
- MeSH
- bakteriální proteiny chemie metabolismus MeSH
- fosforylace MeSH
- globiny chemie metabolismus MeSH
- histidinkinasa chemie metabolismus MeSH
- konformace proteinů, alfa-helix MeSH
- konformace proteinů MeSH
- krystalografie rentgenová MeSH
- molekulární modely MeSH
- multimerizace proteinu MeSH
- Myxococcales chemie metabolismus MeSH
- proteinové domény MeSH
- signální transdukce MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Publikační typ
- abstrakt z konference MeSH
- Publikační typ
- abstrakt z konference MeSH
- Publikační typ
- abstrakt z konference MeSH
Heme-based oxygen sensors allow bacteria to regulate their activity based on local oxygen levels. YddV, a globin-coupled oxygen sensor with diguanylate cyclase activity from Escherichia coli, regulates cyclic-di-GMP synthesis based on oxygen availability. Stable and active samples of the full-length YddV protein were prepared by attaching it to maltose binding protein (MBP). To better understand the full-length protein's structure, the interactions between its domains were examined by performing a kinetic analysis. The diguanylate cyclase reaction catalyzed by YddV-MBP exhibited Michaelis-Menten kinetics. Its pH optimum was 8.5-9.0, and catalysis required either Mg2+ or Mn2+; other divalent metal ions gave no activity. The most active form of YddV-MBP had a 5-coordinate Fe(III) heme complex; its kinetic parameters were KmGTP 84 ± 21 μM and kcat 1.2 min-1. YddV-MBP with heme Fe(II), heme Fe(II)-O2, and heme Fe(II)-CO complexes had kcat values of 0.3 min-1, 0.95 min-1, and 0.3 min-1, respectively, suggesting that catalysis is regulated by the heme iron's redox state and axial ligand binding. The kcat values for heme Fe(III) complexes of L65G, L65Q, and Y43A YddV-MBP mutants bearing heme distal amino acid replacements were 0.15 min-1, 0.26 min-1 and 0.54 min-1, respectively, implying that heme distal residues play key regulatory roles by mediating signal transduction between the sensing and functional domains. Ultracentrifugation and size exclusion chromatography experiments showed that YddV-MBP is primarily dimeric in solution, with a sedimentation coefficient around 8. The inactive heme-free H93A mutant is primarily octameric, suggesting that catalytically active dimer formation requires heme binding.
- MeSH
- hem chemie MeSH
- katalytická doména MeSH
- kinetika MeSH
- ligandy MeSH
- lyasy štěpící vazby P-O chemie genetika metabolismus MeSH
- oxidace-redukce MeSH
- proteiny z Escherichia coli chemie genetika metabolismus MeSH
- substituce aminokyselin MeSH
- vazba proteinů MeSH
- železo chemie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Although ellipticine (Elli) is an efficient anticancer agent, it exerts several adverse effects. One approach to decrease the adverse effects of drugs is their encapsulation inside a suitable nanocarrier, allowing targeted delivery to tumour tissue whereas avoiding healthy cells. We constructed a nanocarrier from apoferritin (Apo) bearing ellipticine, ApoElli, and subsequently characterized. The nanocarrier exhibits a narrow size distribution suggesting its suitability for entrapping the hydrophobic ellipticine molecule. Ellipticine was released from ApoElli into the water environment under pH 6.5, but only less than 20% was released at pH 7.4. The interaction of ApoElli with microsomal membrane particles containing cytochrome P450 (CYP) biotransformation enzymes accelerated the release of ellipticine from this nanocarrier making it possible to be transferred into this membrane system even at pH 7.4 and facilitating CYP-mediated metabolism. Reactive metabolites were formed not only from free ellipticine, but also from ApoElli, and both generated covalent DNA adducts. ApoElli was toxic in UKF-NB-4 neuroblastoma cells, but showed significantly lower cytotoxicity in non-malignant fibroblast HDFn cells. Ellipticine either free or released from ApoElli was concentrated in the nuclei of neuroblastoma cells, concentrations of which being significantly higher in nuclei of UKF-NB-4 than in HDFn cells. In HDFn the higher amounts of ellipticine were sequestrated in lysosomes. The extent of ApoElli entering the nuclei in UKF-NB-4 cells was lower than that of free ellipticine and correlated with the formation of ellipticine-derived DNA adducts. Our study indicates that the ApoElli form of ellipticine seems to be a promising tool for neuroblastoma treatment.
- MeSH
- adukty DNA genetika metabolismus MeSH
- antitumorózní látky chemie farmakologie MeSH
- apoferritiny chemie farmakologie MeSH
- cytochrom P-450 CYP3A metabolismus MeSH
- elipticiny chemie farmakologie MeSH
- fosforylace MeSH
- histony metabolismus MeSH
- lidé MeSH
- nádorové buněčné linie MeSH
- nanočástice * MeSH
- neuroblastom farmakoterapie enzymologie genetika patologie MeSH
- nosiče léků * MeSH
- příprava léků MeSH
- uvolňování léčiv MeSH
- viabilita buněk účinky léků MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Endocrine disruptors (EDs) are compounds that interfere with the balance of the endocrine system by mimicking or antagonising the effects of endogenous hormones, by altering the synthesis and metabolism of natural hormones, or by modifying hormone receptor levels. The synthetic estrogen 17α-ethinylestradiol (EE2) and the environmental carcinogen benzo[a]pyrene (BaP) are exogenous EDs whereas the estrogenic hormone 17β-estradiol is a natural endogenous ED. Although the biological effects of these individual EDs have partially been studied previously, their toxicity when acting in combination has not yet been investigated. Here we treated Wistar rats with BaP, EE2 and estradiol alone or in combination and studied the influence of EE2 and estradiol on: (i) the expression of cytochrome P450 (CYP) 1A1 and 1B1 in rat liver on the transcriptional and translational levels; (ii) the inducibility of these CYP enzymes by BaP in this rat organ; (iii) the formation of BaP-DNA adducts in rat liver in vivo; and (iv) the generation of BaP-induced DNA adducts after activation of BaP with hepatic microsomes of rats exposed to BaP, EE2 and estradiol and with recombinant rat CYP1A1 in vitro. BaP acted as a strong and moderate inducer of CYP1A1 and 1B1 in rat liver, respectively, whereas EE2 or estradiol alone had no effect on the expression of these enzymes. However, when EE2 was administered to rats together with BaP, it significantly decreased the potency of BaP to induce CYP1A1 and 1B1 gene expression. For EE2, but not estradiol, this also correlated with a reduction of BaP-induced CYP1A1 enzyme activity in rat hepatic microsomes. Further, while EE2 and estradiol did not form covalent adducts with DNA, they affected BaP-derived DNA adduct formations in vivo and in vitro. The observed decrease in BaP-DNA adduct levels in rat liver in vivo resulted from the inhibition of CYP1A1-mediated BaP bioactivation by EE2 and estradiol. Our results indicate that BaP genotoxicity mediated through its activation by CYP1A1 in rats in vivo is modulated by estradiol and its synthetic derivative EE2.
- MeSH
- benzopyren toxicita MeSH
- cytochrom P-450 CYP1A1 biosyntéza genetika MeSH
- endokrinní disruptory toxicita MeSH
- estradiol toxicita MeSH
- ethinylestradiol toxicita MeSH
- jaterní mikrozomy účinky léků enzymologie MeSH
- krysa rodu rattus MeSH
- potkani Wistar MeSH
- regulace genové exprese enzymů * účinky léků MeSH
- synergismus léků MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- mužské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
