Enzyme activity is regulated by several mechanisms, including phosphorylation. Phosphorylation is a key signal transduction process in all eukaryotic cells and is thus crucial for virtually all cellular processes. In addition to its direct effect on protein structure, phosphorylation also affects protein-protein interactions, such as binding to scaffolding 14-3-3 proteins, which selectively recognize phosphorylated motifs. These interactions then modulate the catalytic activity, cellular localisation and interactions of phosphorylated enzymes through different mechanisms. The aim of this mini-review is to highlight several examples of 14-3-3 protein-dependent mechanisms of enzyme regulation previously studied in our laboratory over the past decade. More specifically, we address here the regulation of the human enzymes ubiquitin ligase Nedd4-2, procaspase-2, calcium-calmodulin dependent kinases CaMKK1/2, and death-associated protein kinase 2 (DAPK2) and yeast neutral trehalase Nth1.

• MeSH
• fosforylace MeSH
• lidé MeSH
• proteiny 14-3-3 * metabolismus   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH
• Názvy látek
• proteiny 14-3-3 * MeSH

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.

• MeSH
• alosterická regulace fyziologie   MeSH
• proteiny 14-3-3 chemie   metabolismus   MeSH
• Saccharomyces cerevisiae MeSH
• sekundární struktura proteinů MeSH
• trehalasa chemie   metabolismus   MeSH
• vápník chemie   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH
• Názvy látek
• proteiny 14-3-3 MeSH
• trehalasa MeSH
• vápník MeSH

Many aspects of protein function regulation require specific protein-protein interactions to carry out the exact biochemical and cellular functions. The highly conserved members of the 14-3-3 protein family mediate such interactions and through binding to hundreds of other proteins provide multitude of regulatory functions, thus playing key roles in many cellular processes. The 14-3-3 protein binding can affect the function of the target protein in many ways including the modulation of its enzyme activity, its subcellular localization, its structure and stability, or its molecular interactions. In this minireview, we focus on mechanisms of the 14-3-3 protein-dependent regulation of three important 14-3-3 binding partners: yeast neutral trehalase Nth1, regulator of G-protein signaling 3 (RGS3), and phosducin.

• MeSH
• DNA-glykosylasy chemie   ultrastruktura   MeSH
• DNA-lyasa (apurinová nebo apyrimidinová) chemie   ultrastruktura   MeSH
• fosfoproteiny chemie   ultrastruktura   MeSH
• konformace proteinů MeSH
• lidé MeSH
• molekulární sekvence - údaje MeSH
• multienzymové komplexy chemie   ultrastruktura   MeSH
• oční proteiny chemie   ultrastruktura   MeSH
• proteiny 14-3-3 chemie   ultrastruktura   MeSH
• proteiny RGS chemie   ultrastruktura   MeSH
• proteiny vázající GTP - regulátory chemie   ultrastruktura   MeSH
• Schizosaccharomyces pombe - proteiny chemie   ultrastruktura   MeSH
• sekvence aminokyselin MeSH
• vazba proteinů MeSH
• vazebná místa MeSH
• vztahy mezi strukturou a aktivitou MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH
• Názvy látek
• DNA-glykosylasy MeSH
• DNA-lyasa (apurinová nebo apyrimidinová) MeSH
• fosfoproteiny MeSH
• multienzymové komplexy MeSH
• Nth1 protein, S pombe MeSH Prohlížeč
• oční proteiny MeSH
• phosducin MeSH Prohlížeč
• proteiny 14-3-3 MeSH
• proteiny RGS MeSH
• proteiny vázající GTP - regulátory MeSH
• RGS3 protein, human MeSH Prohlížeč
• Schizosaccharomyces pombe - proteiny MeSH

Mammalian P2X receptors contain 10 conserved cysteine residues in their ectodomains, which form five disulfide bonds (SS1-5). Here, we analyzed the relevance of these disulfide pairs in rat P2X4 receptor function by replacing one or both cysteines with alanine or threonine, expressing receptors in HEK293 cells and studying their responsiveness to ATP in the absence and presence of ivermectin, an allostenic modulator of these channels. Response to ATP was not altered when both cysteines forming the SS3 bond (C132-C159) were replaced with threonines. Replacement of SS1 (C116-C165), SS2 (C126-C149) and SS4 (C217-C227), but not SS5 (C261-C270), cysteine pairs with threonines resulted in decreased sensitivity to ATP and faster deactivation times. The maximum current amplitude was reduced in SS2, SS4 and SS5 double mutants and could be partially rescued by ivermectin in SS2 and SS5 double mutants. This response pattern was also observed in numerous single residue mutants, but receptor function was not affected when the 217 cysteine was replaced with threonine or arginine or when the 261 cysteine was replaced with alanine. These results suggest that the SS1, SS2 and SS4 bonds contribute substantially to the structure of the ligand binding pocket, while the SS5 bond located towards the transmembrane domain contributes to receptor gating.

• MeSH
• cystein chemie   genetika   MeSH
• gating iontového kanálu fyziologie   MeSH
• HEK293 buňky MeSH
• konzervovaná sekvence MeSH
• krysa rodu Rattus MeSH
• lidé MeSH
• purinergní receptory P2X4 chemie   genetika   metabolismus   MeSH
• vazebná místa MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Intramural MeSH
• Názvy látek
• cystein MeSH
• purinergní receptory P2X4 MeSH

The 14-3-3 proteins are a family of acidic regulatory molecules found in all eukaryotes. 14-3-3 proteins function as molecular scaffolds by modulating the conformation of their binding partners. Through the functional modulation of a wide range of binding partners, 14-3-3 proteins are involved in many processes, including cell cycle regulation, metabolism control, apoptosis, and control of gene transcription. This minireview includes a short overview of 14-3-3 proteins and then focuses on their role in the regulation of two important binding partners: FOXO forkhead transcription factors and an enzyme tyrosine hydroxylase.

• MeSH
• forkhead transkripční faktory metabolismus   MeSH
• konformace proteinů MeSH
• konzervovaná sekvence MeSH
• kvarterní struktura proteinů MeSH
• lidé MeSH
• molekulární modely MeSH
• molekulární sekvence - údaje MeSH
• protein - isoformy MeSH
• proteiny 14-3-3 chemie   genetika   metabolismus   MeSH
• sekvence aminokyselin MeSH
• tyrosin-3-monooxygenasa metabolismus   MeSH
• vazba proteinů MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH
• Názvy látek
• forkhead transkripční faktory MeSH
• protein - isoformy MeSH
• proteiny 14-3-3 MeSH
• tyrosin-3-monooxygenasa MeSH

The FOXO subgroup of forkhead transcription factors plays a central role in cell-cycle control, differentiation, metabolism control, stress response and apoptosis. Therefore, the function of these important molecules is tightly controlled by a wide range of protein-protein interactions and posttranslational modifications including phosphorylation, acetylation and ubiquitination. The mechanisms by which these processes regulate FOXO activity are mostly elusive. This review focuses on recent advances in structural studies of forkhead transcription factors and the insights they provide into the mechanism of DNA recognition. On the basis of these data, we discuss structural aspects of protein-protein interactions and posttranslational modifications that target the forkhead domain and the nuclear localization signal of FOXO proteins.

• MeSH
• acetylace MeSH
• forkhead transkripční faktory chemie   fyziologie   MeSH
• fosforylace MeSH
• konformace proteinů MeSH
• lidé MeSH
• posttranslační úpravy proteinů MeSH
• ubikvitin metabolismus   MeSH
• vztahy mezi strukturou a aktivitou MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH
• Názvy látek
• forkhead transkripční faktory MeSH
• ubikvitin MeSH

Beside of the protein crystallography or NMR, another attractive option in protein structure analysis has recently appeared: computer modeling of the protein structure based on homology and similarity with proteins of already known structures. We have used the combination of computer modeling with spectroscopic techniques, such as steady-state or time-resolved fluorescence spectroscopy, and with molecular biology techniques. This method could provide useful structural information in the cases where crystal or NMR structure is not available. Molecular modeling of the ATP site within the H4-H5-loop revealed eight amino acids residues, namely besides the previously reported amino acids Asp443, Lys480, Lys501, Gly502 and Arg544, also Glu446, Phe475 and Gln482, which form the complete ATP recognition site. Moreover, we have proved that a hydrogen bond between Arg423 and Glu472 supports the connection of two opposite halves of the ATP-binding pocket. Similarly, the conserved residue Pro489 is important for the proper interaction of the third and fourth beta-strands, which both contain residues that take part in the ATP-binding. Alternatively, molecular dynamics simulation combined with dynamic fluorescence spectroscopy revealed that 14-3-3 zeta C-terminal stretch is directly involved in the interaction of 14-3-3 protein with the ligand. Phosphorylation at Thr232 induces a conformational change of the C-terminus, which is presumably responsible for observed inhibition of binding abilities. Phosphorylation at Thr232 induces more extended conformation of 14-3-3zeta C-terminal stretch and changes its interaction with the rest of the 14-3-3 molecule. This could explain negative regulatory effect of phosphorylation at Thr232 on 14-3-3 binding properties.

• MeSH
• adenosintrifosfát chemie   metabolismus   MeSH
• alkoholoxidoreduktasy MeSH
• DNA vazebné proteiny fyziologie   MeSH
• fluorescenční spektrometrie metody   MeSH
• fosfoproteiny fyziologie   MeSH
• fosforylace MeSH
• konformace proteinů * MeSH
• konzervovaná sekvence MeSH
• molekulární modely * MeSH
• počítačová simulace * MeSH
• proteiny 14-3-3 chemie   metabolismus   MeSH
• sekvence aminokyselin MeSH
• sekvenční homologie aminokyselin MeSH
• sodíko-draslíková ATPasa chemie   fyziologie   MeSH
• threonin fyziologie   MeSH
• vazebná místa MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• adenosintrifosfát MeSH
• alkoholoxidoreduktasy MeSH
• C-terminal binding protein MeSH Prohlížeč
• DNA vazebné proteiny MeSH
• fosfoproteiny MeSH
• proteiny 14-3-3 MeSH
• sodíko-draslíková ATPasa MeSH
• threonin MeSH

Lifetime analysis of tryptophan fluorescence of the mitochondrial processing peptidase (MPP) from Saccharomyces cerevisiae clearly proved that substrate binding evoked a conformational change of the alpha-subunit while presence of substrate influenced neither the lifetime components nor the average lifetime of the tryptophan excited state of the beta-MPP subunit. Interestingly, lifetime analysis of tryptophan fluorescence decay of the alpha-MPP subunit revealed about 11% of steady-state fractional intensity due to the long-lived lifetime component, indicating that at least one tryptophan residue is partly buried at the hydrophobic microenvironment. Computer modeling, however, predicted none of three tryptophans, which the alpha-subunit contains, as deeply buried in the protein matrix. We conclude this as a consequence of a possible dimeric (oligomeric) structure.

• MeSH
• dimerizace MeSH
• Escherichia coli genetika   MeSH
• fluorescence MeSH
• konformace proteinů MeSH
• krysa rodu Rattus MeSH
• metaloendopeptidasy chemie   genetika   metabolismus   MeSH
• molekulární modely MeSH
• MPP peptidasa MeSH
• počítačová simulace MeSH
• podjednotky proteinů MeSH
• proteinové prekurzory metabolismus   MeSH
• protony MeSH
• rekombinantní proteiny chemie   metabolismus   MeSH
• renaturace proteinů MeSH
• Saccharomyces cerevisiae enzymologie   genetika   MeSH
• sbalování proteinů MeSH
• sekvence aminokyselin MeSH
• synchrotrony MeSH
• techniky dvojhybridového systému MeSH
• tryptofan metabolismus   MeSH
• vazba proteinů MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• metaloendopeptidasy MeSH
• podjednotky proteinů MeSH
• proteinové prekurzory MeSH
• protony MeSH
• rekombinantní proteiny MeSH
• tryptofan MeSH

In the present work, we studied the interactions of recombinant alpha1 and alpha2 integrin I domains with cations Tb(3+), Mn(2+), Mg(2+) and Ca(2+). We observed that alpha1 and alpha2 I domains bind these cations with significantly different characteristics. The binding of Mg(2+) by the alpha1 I domain was accompanied by significant changes of tryptophan fluorescence which could be interpreted as a conformational change. Comparison of the alpha1 integrin I domain structure obtained by comparative modeling with a known structure of the alpha2 integrin I domain shows distinct differences in the metal ion binding sites which could explain the differences in cation binding.

• MeSH
• CD antigeny chemie   metabolismus   MeSH
• fluorescence MeSH
• hořčík metabolismus   MeSH
• integrin alfa1 MeSH
• integrin alfa2 MeSH
• kationty metabolismus   MeSH
• konformace proteinů MeSH
• molekulární modely MeSH
• molekulární sekvence - údaje MeSH
• rekombinantní proteiny chemie   metabolismus   MeSH
• sekvence aminokyselin MeSH
• terbium metabolismus   MeSH
• tryptofan chemie   MeSH
• vazebná místa MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• srovnávací studie MeSH
• Názvy látek
• CD antigeny MeSH
• hořčík MeSH
• integrin alfa1 MeSH
• integrin alfa2 MeSH
• kationty MeSH
• rekombinantní proteiny MeSH
• terbium MeSH
• tryptofan MeSH

The effect of aminophospholipid glycation on lipid order and lipid bilayer hydration was investigated using time-resolved fluorescence spectroscopy. The changes of lipid bilayer hydration were estimated both from its effect on the fluorescence lifetime of The 1-[4-(trimethylammonium)-phenyl]-6-phenylhexa-1,3,5-triene (TMA-DPH) and 1,6-diphenylhexa-1,3,5-triene (DPH) and using solvatochromic shift studies with 1-anilinonaphthalene-8-sulfonic acid. The head-group and acyl chain order were determined from time-resolved fluorescence anisotropy measurements of the TMA-DPH and DPH. The suspensions of small unilamellar vesicles (with phosphatidylethanolamine/phosphatidylcholine molar ratio 1:2.33) were incubated with glyceraldehyde and it was found that aminophospholipids react with glyceraldehyde to form products with the absorbance and the fluorescence properties typical for protein advanced glycation end products. The lipid glycation was accompanied by the progressive oxidative modification of unsaturated fatty acid residues. It was found that aminophospholipid glycation increased the head-group hydration and lipid order in both regions of the membrane. The lipid oxidation accompanying the lipid glycation affected mainly the lipid order, while the effect on the lipid hydration was small. The increase in the lipid order was presumably the result of two effects: (1) the modification of head-groups of phosphatidylethanolamine by glycation; and (2) the degradation of unsaturated fatty acid residues by oxidation.

• Publikační typ
• časopisecké články MeSH