O akci
14-3-3 is a family of highly conserved and ubiquitously expressed eukaryotic proteins. 14-3-3 proteins have regulatory functions and act in multiple cellular processes such as cell cycle, signal transduction, or cell death. For the proper function of this rigid and highly helical protein, its dimeric state is essential [1]. Mammalian 14-3-3 proteins have 7 known isoforms named β, γ, ε, ζ, η, θ, and σ, which can form both homo- and heterodimers. These isoforms differ in expression levels in various tissues, thus in their total concentrations in these tissues [2-4].
Most studies of 14-3-3 proteins to date have been focused on homodimers and not heterodimers. However, in a mixture of multiple isoforms in addition to homodimers, heterodimers are inevitably formed. Therefore, accurate quantification of the thermodynamic and the kinetic parameters of individual dimerization and thus the ability to quantify the populations of homo- and heterodimers for different tissues with known expression levels of individual 14-3-3 isoforms.
In previous years our group designed an FRET assay to quantitatively determine 14-3-3 dimerization parameters, namely rate and dissociation equilibrium constants (koff, KD) [5]. It allowed to reveal that phosphorylation of ζ at Ser58 increases its dimerization KD by six orders of magnitude from (5.5 ± 0.8) nM (ζ/ζ) to (4.3 ± 0.3) mM (pζ/pζ). In addition to hetero-dimers between different 14-3-3 isoforms, we were able to reveal another relevant type of heterodimers between phosphorylated and not phosphorylated 14-3-3 isoforms (pζ/ζ), not considered before [5-7]. The heterodimerization Kd of pζ/ζ was determined as (2.5 ± 0.3) mM implying that at typical expression levels of phosphorylated 14-3-3ζ (pζ), about 85% exist as monomers and approximately 15% exist in the heterodimeric state (pζ/ζ).
We also propose the usage of mathematical models allowing us to predict the time dependence as a consequence of changes in expression levels of individual isoforms (e.g. at stress response). Our model also predicts the expected concentrations of all possible variants of 14-3-3 homo-, hetero-dimers, and monomers in a mixture.
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- G. Gogl, K. V. Tugaeva, P. Eberling, C. Kostmann, G. Trave, N. N. Sluchanko, Nat Commun, 12, (2021), 1677
- K. Gardino, S. J. Smerdon, and M. B. Yaffe, “Structural determinants of 14-3-3 binding specificities and regulation of subcellular localization of 14-3-3-ligand complexes: A comparison of the X-ray crystal structures of all human 14-3-3 isoforms,” Jun 2006. doi: 10.1016/j.semcancer.2006.03.007.
- X. Yang et al., “Structural basis for protein-protein interactions in the 14-3-3 protein family,” 2006. [Online]. Available: www.pnas.orgcgidoi10.1073pnas.0605779103
- Z. Trošanová, P. Louša, A. Kozeleková, T. Brom, N. Gašparik, J. Tungli, V. Weisová, E. Župa, G. Žoldák, J. Hritz, J. Mol. Biol., 434, (2022), 167479
- Z. Jandová, Z. Trošanová, V. Weisová, C. Oostenbrink, J. Hritz, Biochim Biophys Acta Proteins Proteom, 1866, (2018), 442-450
- Kozeleková, A. Náplavová, T. Brom, N. Gašparik, J. Šimek, J. Houser, J. Hritz, Front. Chem., 10, (2022), 1-17
This project has received funding from the European Union’s Horizon Europe program under the grant agreement No. 101087124. We acknowledge CF Biomolecular Interactions and Crystallography of CIISB, Instruct-CZ Centre, supported by MEYS CR (LM2023042) and European Regional Development Fund-Project „UP CIISB“(No. CZ.02.1.01/0.0/0.0/18_046/0015974). We acknowledge CEITEC Proteomics Core Facility of CIISB, Instruct-CZ Centre, supported by MEYS CR (LM2023042, e-INFRA CZ (ID:90254))
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