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

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.

• Klíčová slova
• 14-3-3 protein, allostery, crystal structure, enzyme, trehalase,
• 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 teplotního 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
• Názvy látek
• arylalkylamin-N-acetyltransferasa MeSH
• glukosa MeSH
• NTH1 protein, S cerevisiae MeSH Prohlížeč
• proteiny 14-3-3 MeSH
• proteiny teplotního šoku MeSH
• Saccharomyces cerevisiae - proteiny MeSH
• trehalasa MeSH
• trehalosa 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.

• Klíčová slova
• 14–3-3, Bmh, Calcium, Enzyme Mechanisms, H/D Exchange, Mass Spectrometry (MS), Neutral Trehalase, Protein Cross-linking, Protein Structure, SAXS,
• 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
• Názvy látek
• BMH1 protein, S cerevisiae MeSH Prohlížeč
• proteiny 14-3-3 MeSH
• Saccharomyces cerevisiae - proteiny MeSH
• trehalasa MeSH
• vápník MeSH