Allosteric activation of yeast enzyme neutral trehalase by calcium and 14-3-3 protein

Alblova, Miroslava; Smidova, A.; Kalabova, Dana; Santo, Domenico Lentini; Obšil, Tomáš; Obšilová, Veronika

doi:10.33549/physiolres.933950

Cited by 6 publications

(4 citation statements)

References 104 publications

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“…The 13 residues (Pro 149 -Val 161 ) connecting both motifs and the last two residues (Asn 167 and Lys 168 ) were not visible in the electron density, likely due to their flexibility. Both pSer 139 and pSer 164 moieties are coordinated, as expected, by the side chains of 14-3-3c residues Lys 50 , Arg 57 , Arg 132 , and Tyr 133 , in agreement with previously published structures of 14-3-3 complexes [20,[31][32][33][34][35][36]. The comparison with the previously reported structure of an isolated pSer 164 motif bound to 14-3-3c [20] revealed similar but not identical mainchain conformation and interactions with 14-3-3c ( Figs 6C and 7A,C).…”

Section: Crystal Structure Of the 14-3-3:proc2 Ps139+ps164 Complexsupporting

confidence: 92%

14‐3‐3 protein binding blocks the dimerization interface of caspase‐2

et al. 2020

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Among all species, caspase-2 (C2) is the most evolutionarily conserved caspase required for effective initiation of apoptosis following death stimuli. C2 is activated through dimerization and autoproteolytic cleavage and inhibited through phosphorylation at Ser 139 and Ser 164 , within the linker between the caspase recruitment and p19 domains of the zymogen, followed by association with the adaptor protein 14-3-3, which maintains C2 in its immature form procaspase (proC2). However, the mechanism of 14-3-3-dependent inhibition of C2 activation remains unclear. Here, we report the structural characterization of the complex between proC2 and 14-3-3 by hydrogen/deuterium mass spectrometry and protein crystallography to determine the molecular basis for 14-3-3-mediated inhibition of C2 activation. Our data reveal that the 14-3-3 dimer interacts with proC2 not only through ligand-binding grooves but also through other regions outside the central channel, thus explaining the isoform-dependent specificity of 14-3-3 protein binding to proC2 and the substantially higher binding affinity of 14-3-3 protein to proC2 than to the doubly phosphorylated peptide. The formation of the complex between 14-3-3 protein and proC2 does not induce any large conformational change in proC2. Furthermore, 14-3-3 protein interacts with and masks both the nuclear localization sequence and the C-terminal region of the p12 domain of proC2 through transient interactions in which both the p19 and p12 domains of proC2 are not firmly docked onto the surface of 14-3-3. This masked region of p12 domain is involved in C2 dimerization. Therefore, 14-3-3 protein likely inhibits proC2 activation by blocking its dimerization surface. Databases Structural data are available in the Protein Data Bank under the accession numbers 6SAD and 6S9K. Abbreviations CARD, caspase recruitment domain; DSF, differential scanning fluorimetry; FP, fluorescence polarization assay; HDX-MS, hydrogen/ deuterium exchange mass spectrometry; NLS, nuclear localization sequence; PIDD, p53-induced protein with a death domain; proC2, caspase-2 residues 123-452 without the CARD domain; RAIDD, adaptor protein RIP-associated ICH-1/CAD-3 homologous protein with a death domain; SV-AUC, sedimentation velocity analytical ultracentrifugation.

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Section: Crystal Structure Of the 14-3-3:proc2 Ps139+ps164 Complexsupporting

confidence: 92%

14‐3‐3 protein binding blocks the dimerization interface of caspase‐2

et al. 2020

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“…In addition, PKA sites S20 and S21 regulate the dephosphorylation of Nth1 at the downstream Bmh1 site. Residues D478 and E674 serve as catalytic acids and bases, respectively, and the entire process is enhanced in the presence of Ca 2+ [72,73]. Therefore, the binding of Nth1 to 14-3-3 triggers the activity of Nth1 by stabilizing the three-dimensional structure of the catalytic domain and calcium-binding domain of Nth1, thereby stabilizing the active site required for catalysis.…”

Section: Gh37 Trehalasementioning

confidence: 99%

“…Metal ions may have a bidirectional effect on trehalases. Alblova et al demonstrated that the presence of Ca 2+ could enhance the activation of Nth1 of GH37 family [73]. Acidic trehalases remain unaffected by divalent cations.…”

Section: Effect Of Metal Ionsmentioning

confidence: 99%

Characterization, heterologous expression and engineering of trehalase for biotechnological applications

Gao

Gong

et al. 2022

Syst Microbiol and Biomanuf

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Trehalose is a non-reducing disaccharide connected by α-1,1-glycosidic bonds; it is widely distributed in bacteria, fungi, yeast, insects, and plant tissues and plays various roles. It can be hydrolyzed by trehalase into two glucose molecules. Trehalases from different sources have been expressed in Escherichia coli, Pichia pastoris, Saccharomyces cerevisiae, baculovirus-silkworm, and other expression systems; however, it is most common in E. coli. The structural characteristics of different glycoside hydrolase (GH) family trehalases and the sources of trehalase have been analyzed. The catalytic mechanism of GH37 trehalase has also been elucidated in detail. Moreover, the molecular modification of trehalase has mainly focused on directed evolution for improving enzyme activity. We comprehensively reviewed the current application status and adaptable transformations was comprehensively overviewed in the context of industrial performance. We suggest that the level of recombinant production is far from meeting industrial requirements, and the catalytic performance of trehalase needs to be improved urgently. Finally, we discuss developmental prospects and future trends.

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“…Here, we focus on the regulation of the final step in the trehalose pathway, the trehalase Nth1, which converts trehalose back to glucose (21). Nth1 has been extensively studied as a model for metabolic regulation because it has both an interesting regulatory mechanism and is experimentally well accessible (22) . We and others have shown that the catalytic activity of Nth1 is determined by both the cell cycle kinase CDK (cyclin dependent kinase) (16,19) and the metabolic kinase PKA (protein kinase A) (23,24).…”

Section: Introductionmentioning

confidence: 99%

Regulation of trehalase activity by multi-site phosphorylation and 14-3-3 interaction

Dengler

Örd

Schwab

et al. 2020

Preprint

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Protein phosphorylation enables a rapid adjustment of cellular activities to diverse intracellular and environmental stimuli. Many phosphoproteins are targeted on more than one site, which allows the integration of multiple signals and the implementation of complex responses. However, the hierarchy and interplay between multiple phospho-sites are often unknown. Here, we study multi-site phosphorylation using the yeast trehalase Nth1 and its activator, the 14-3-3 protein Bmh1, as a model. Nth1 is known to be phosphorylated by the metabolic kinase PKA on four serine residues and by the cell cycle kinase CDK on one residue. However, how these five phospho-sites adjust Nth1 activity remains unclear. Using a novel reporter construct, we investigated the contribution of the individual sites for the regulation of the trehalase and its 14-3-3 interactor. In contrast to the constitutively phosphorylated S20 and S83, the weaker sites S21 and S60 are only phosphorylated by increased PKA activity. For binding Bmh1, S83 functions as the high-affinity “gatekeeper” site, but successful binding of the Bmh1 dimer and thus Nth1 activation requires S60 as a secondary site. Under nutrient-poor conditions with low PKA activity, S60 is not efficiently phosphorylated and the cell cycle dependent phosphorylation of S66 by Cdk1 contributes to Nth1 activity, likely by providing an alternative Bmh1 binding site. Additionally, the PKA sites S20 and S21 modulate the dephosphorylation of Nth1 on downstream Bmh1 sites. In summary, our results expand our molecular understanding of Nth1 regulation and provide a new aspect of the interaction of 14-3-3 proteins with their targets.

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Allosteric activation of yeast enzyme neutral trehalase by calcium and 14-3-3 protein

Cited by 6 publications

References 104 publications

14‐3‐3 protein binding blocks the dimerization interface of caspase‐2

14‐3‐3 protein binding blocks the dimerization interface of caspase‐2

Characterization, heterologous expression and engineering of trehalase for biotechnological applications

Regulation of trehalase activity by multi-site phosphorylation and 14-3-3 interaction

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