Neutral trehalase 1 (Nth1) from Saccharomyces cerevisiae catalyzes disaccharide trehalose hydrolysis and helps yeast to survive adverse conditions, such as heat shock, starvation or oxidative stress. 14-3-3 proteins, master regulators of hundreds of partner proteins, participate in many key cellular processes. Nth1 is activated by phosphorylation followed by 14-3-3 protein (Bmh) binding. The activation mechanism is also potentiated by Ca(2+) binding within the EF-hand-like motif. This review summarizes the current knowledge about trehalases and the molecular and structural basis of Nth1 activation. The crystal structure of fully active Nth1 bound to 14-3-3 protein provided the first high-resolution view of a trehalase from a eukaryotic organism and showed 14-3-3 proteins as structural modulators and allosteric effectors of multi-domain binding partners.
The 14-3-3 proteins, a family of highly conserved scaffolding proteins ubiquitously expressed in all eukaryotic cells, interact with and regulate the function of several hundreds of partner proteins. Yeast neutral trehalases (Nth), enzymes responsible for the hydrolysis of trehalose to glucose, compared with trehalases from other organisms, possess distinct structure and regulation involving phosphorylation at multiple sites followed by binding to the 14-3-3 protein. Here we report the crystal structures of yeast Nth1 and its complex with Bmh1 (yeast 14-3-3 isoform), which, together with mutational and fluorescence studies, indicate that the binding of Nth1 by 14-3-3 triggers Nth1's activity by enabling the proper 3D configuration of Nth1's catalytic and calcium-binding domains relative to each other, thus stabilizing the flexible part of the active site required for catalysis. The presented structure of the Bmh1:Nth1 complex highlights the ability of 14-3-3 to modulate the structure of a multidomain binding partner and to function as an allosteric effector. Furthermore, comparison of the Bmh1:Nth1 complex structure with those of 14-3-3:serotonin N-acetyltransferase and 14-3-3:heat shock protein beta-6 complexes revealed similarities in the 3D structures of bound partner proteins, suggesting the highly conserved nature of 14-3-3 affects the structures of many client proteins.
- MeSH
- arylalkylamin-N-acetyltransferasa metabolismus MeSH
- chemické databáze * MeSH
- fosforylace MeSH
- glukosa metabolismus MeSH
- katalytická doména MeSH
- konformace proteinů MeSH
- krystalografie rentgenová MeSH
- molekulární modely MeSH
- proteinové domény MeSH
- proteiny 14-3-3 genetika metabolismus MeSH
- proteiny tepelného šoku chemie metabolismus MeSH
- Saccharomyces cerevisiae - proteiny chemie metabolismus MeSH
- Saccharomyces cerevisiae enzymologie genetika metabolismus MeSH
- trehalasa chemie metabolismus MeSH
- trehalosa metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
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- MeSH
- celiakie diagnóza dietoterapie klasifikace MeSH
- diferenciální diagnóza MeSH
- disacharidasy nedostatek MeSH
- idiopatické střevní záněty diagnóza MeSH
- intestinální absorpce fyziologie MeSH
- laktasa nedostatek MeSH
- lidé MeSH
- malabsorpční syndromy * diagnostické zobrazování diagnóza etiologie MeSH
- metabolismus sacharidů fyziologie MeSH
- nesnášenlivost laktózy diagnóza dietoterapie MeSH
- podvýživa etiologie MeSH
- proteiny metabolismus MeSH
- syndrom krátkého střeva diagnóza etiologie MeSH
- syndrom slepé kličky diagnóza etiologie MeSH
- tenké střevo fyziologie patofyziologie MeSH
- trávení fyziologie MeSH
- tropická sprue diagnóza farmakoterapie MeSH
- tuky metabolismus MeSH
- Whippleova nemoc diagnóza terapie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- přehledy MeSH
Trehalases hydrolyze the non-reducing disaccharide trehalose amassed by cells as a universal protectant and storage carbohydrate. Recently, it has been shown that the activity of neutral trehalase Nth1 from Saccharomyces cerevisiae is mediated by the 14-3-3 protein binding that modulates the structure of both the catalytic domain and the region containing the EF-hand-like motif, whose role in the activation of Nth1 is unclear. In this work, the structure of the Nth1·14-3-3 complex and the importance of the EF-hand-like motif were investigated using site-directed mutagenesis, hydrogen/deuterium exchange coupled to mass spectrometry, chemical cross-linking, and small angle x-ray scattering. The low resolution structural views of Nth1 alone and the Nth1·14-3-3 complex show that the 14-3-3 protein binding induces a significant structural rearrangement of the whole Nth1 molecule. The EF-hand-like motif-containing region forms a separate domain that interacts with both the 14-3-3 protein and the catalytic trehalase domain. The structural integrity of the EF-hand like motif is essential for the 14-3-3 protein-mediated activation of Nth1, and calcium binding, although not required for the activation, facilitates this process by affecting its structure. Our data suggest that the EF-hand like motif-containing domain functions as the intermediary through which the 14-3-3 protein modulates the function of the catalytic domain of Nth1.
- MeSH
- aktivace enzymů MeSH
- katalytická doména MeSH
- molekulární modely MeSH
- motivy EF-ruky * MeSH
- proteiny 14-3-3 chemie metabolismus MeSH
- Saccharomyces cerevisiae - proteiny chemie metabolismus MeSH
- Saccharomyces cerevisiae enzymologie MeSH
- sekvence aminokyselin MeSH
- trehalasa chemie metabolismus MeSH
- vápník metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
BACKGROUND: Trehalases are highly conserved enzymes catalyzing the hydrolysis of trehalose in a wide range of organisms. The activity of yeast neutral trehalase Nth1 is regulated in a 14-3-3- and a calcium-dependent manner. The Bmh proteins (the yeast 14-3-3 isoforms) recognize phosphorylated Nth1 and enhance its enzymatic activity through an unknown mechanism. METHODS: To investigate the structural basis of interaction between Nth1 and Bmh1, we used hydrogen/deuterium exchange coupled to mass spectrometry, circular dichroism spectroscopy and homology modeling to identify structural changes occurring upon the complex formation. RESULTS: Our results show that the Bmh1 protein binding affects structural properties of several regions of phosphorylated Nth1: the N-terminal segment containing phosphorylation sites responsible for Nth1 binding to Bmh, the region containing the calcium binding domain, and segments surrounding the active site of the catalytic trehalase domain. The complex formation between Bmh1 and phosphorylated Nth1, however, is not accompanied by the change in the secondary structure composition but rather the change in the tertiary structure. CONCLUSIONS: The 14-3-3 protein-dependent activation of Nth1 is based on the structural change of both the calcium binding domain and the catalytic trehalase domain. These changes likely increase the accessibility of the active site, thus resulting in Nth1 activation. GENERAL SIGNIFICANCE: The results presented here provide a structural view of the 14-3-3 protein-dependent activation of yeast neutral trehalase Nth1, which might be relevant to understand the process of Nth1 activity regulation as well as the role of the 14-3-3 proteins in the regulation of other enzymes.
- MeSH
- aktivace enzymů MeSH
- cirkulární dichroismus MeSH
- hmotnostní spektrometrie MeSH
- konformace proteinů MeSH
- molekulární modely MeSH
- proteiny 14-3-3 metabolismus MeSH
- Saccharomyces cerevisiae enzymologie MeSH
- trehalasa metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Trehalases are important highly conserved enzymes found in a wide variety of organisms and are responsible for the hydrolysis of trehalose that serves as a carbon and energy source as well as a universal stress protectant. Emerging evidence indicates that the enzymatic activity of the neutral trehalase Nth1 in yeast is enhanced by 14-3-3 protein binding in a phosphorylation-dependent manner through an unknown mechanism. In the present study, we investigated in detail the interaction between Saccharomyces cerevisiae Nth1 and 14-3-3 protein isoforms Bmh1 and Bmh2. We determined four residues that are phosphorylated by PKA (protein kinase A) in vitro within the disordered N-terminal segment of Nth1. Sedimentation analysis and enzyme kinetics measurements show that both yeast 14-3-3 isoforms form a stable complex with phosphorylated Nth1 and significantly enhance its enzymatic activity. The 14-3-3-dependent activation of Nth1 is significantly more potent compared with Ca2+-dependent activation. Limited proteolysis confirmed that the 14-3-3 proteins interact with the N-terminal segment of Nth1 where all phosphorylation sites are located. Site-directed mutagenesis in conjunction with the enzyme activity measurements in vitro and the activation studies of mutant forms in vivo suggest that Ser60 and Ser83 are sites primarily responsible for PKA-dependent and 14-3-3-mediated activation of Nth1.
- MeSH
- aktivace enzymů MeSH
- fosforylace MeSH
- mutace MeSH
- proteiny 14-3-3 metabolismus MeSH
- proteolýza MeSH
- Saccharomyces cerevisiae enzymologie MeSH
- spektrometrie hmotnostní - ionizace laserem za účasti matrice MeSH
- trehalasa genetika metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- MeSH
- aplikace orální MeSH
- benzamidy MeSH
- biguanidy dějiny MeSH
- diabetes mellitus 2. typu farmakoterapie MeSH
- disacharidasy MeSH
- hypoglykemika dějiny MeSH
- inhibitory dipeptidylpeptidasy 4 MeSH
- inkretiny MeSH
- krevní glukóza účinky léků MeSH
- lidé MeSH
- sulfonylmočovinové sloučeniny MeSH
- thiazolidindiony MeSH
- Check Tag
- lidé MeSH
BACKGROUND: Combination of platinum derivatives with paclitaxel is currently the standard front line regimen for patients with epithelial ovarian carcinoma, and represents also an active regimen in patients with metastatic breast or unknown primary carcinomas. Measurement of intestinal permeability represents one of the potential methods of noninvasive laboratory assessment of gastrointestinal mucositis induced by chemotherapy, but little is known about intestinal permeability in patients treated with paclitaxel or platinum. METHODS: Intestinal permeability was assessed in 36 breast and ovarian cancer patients treated with paclitaxel/platinum combination by measuring, using capillary gas chromatography, urinary sucrose, lactulose, xylose and mannitol after oral challenge. The significance of differences during the therapy compared to pre-treatment values was studied by Wilcoxon paired test. The differences between groups of patient were studied by Mann-Whitney U test. Fisher exact test was used to compare the frequency in different subgroups. RESULTS: After administration of the first dose, a significant (p < 0.05) decrease in xylose absorption and increased lactulose/mannitol, sucrose/mannitol, lactulose/xylose and sucrose/xylose ratios were observed, but these parameters returned subsequently to pre-treatment levels. Patients who experienced serious (grade 3 or 4) toxicity had at baseline significantly lower percentages of xylose, mannitol and sucrose, and higher lactulose/mannitol ratio. Nine of 13 (69%) patients with baseline lactulose/mannitol ratio 0.070 or above experienced serious toxicity compared to 4 out of 23 patients (17%) with the ratio below 0.070 (p = 0.002). Post-treatment lactulose, lactulose/mannitol, sucrose/mannitol and lactulose/xylose ratios were significantly increased in patients with serious toxicity. CONCLUSION: A transient significant increase in lactulose/monosaccharide and sucrose/monosaccharide ratios was observed in ovarian and breast cancer patients treated with paclitaxel and platinum. Increased lactulose absorption, lactulose/mannitol, sucrose/mannitol and lactulose/xylose ratios were evident in patients with grade 3 or 4 toxicity, and increased baseline lactulose/mannitol ratio predicted serious toxicity.
- MeSH
- adenokarcinom farmakoterapie metabolismus patologie MeSH
- cisplatina aplikace a dávkování MeSH
- dospělí MeSH
- financování organizované MeSH
- gastrointestinální trakt metabolismus MeSH
- intestinální absorpce účinky léků MeSH
- karboplatina aplikace a dávkování MeSH
- laktulosa metabolismus MeSH
- lidé středního věku MeSH
- lidé MeSH
- mannitol metabolismus MeSH
- monoklonální protilátky aplikace a dávkování MeSH
- nádory glandulární a epitelové farmakoterapie metabolismus patologie MeSH
- nádory prsu farmakoterapie metabolismus patologie MeSH
- nádory vaječníků farmakoterapie metabolismus patologie MeSH
- paclitaxel aplikace a dávkování MeSH
- permeabilita buněčné membrány účinky léků MeSH
- protokoly antitumorózní kombinované chemoterapie terapeutické užití MeSH
- sacharasa metabolismus MeSH
- senioři MeSH
- xylosa metabolismus MeSH
- Check Tag
- dospělí MeSH
- lidé středního věku MeSH
- lidé MeSH
- senioři MeSH
- ženské pohlaví MeSH
The increase in ammonia and ethanol in the exhaled breath stream following mouthwashes by aqueous solutions of urea and sugar (sucrose), respectively, has been investigated by analysing exhaled breath in real time using selected ion flow tube mass spectrometry, SIFT-MS. It is shown that the measured levels of these compounds in the stream of exhaled breath can be much greater than the endogenous levels originating at the alveolar boundary. Thus, it is concluded that without careful preparation, mouth production of these compounds, and other compounds as yet unidentified, can seriously compromise the quantification of truly endogenous trace compounds present in blood and in the alveolar breath, as required for clinical diagnosis, and can probably introduce additional compounds into the breath stream that could seriously mislead breath analysis. The concentrations of both the urea and sucrose solutions used to enhance the ammonia and ethanol levels were larger than normally present in food and drinks and so in most situations such severe enhancements will not occur.
