Quantitation of Human 14-3-3ζ Dimerization and the Effect of
Phosphorylation on Dimer-monomer Equilibria

J 2022

Quantitation of Human 14-3-3ζ Dimerization and the Effect of Phosphorylation on Dimer-monomer Equilibria

TROŠANOVÁ, Zuzana, Petr LOUŠA, Aneta KOZELEKOVÁ, Tomáš BROM, Norbert GAŠPARIK et. al.

Základní údaje

Originální název

Quantitation of Human 14-3-3ζ Dimerization and the Effect of Phosphorylation on Dimer-monomer Equilibria

Autoři

TROŠANOVÁ, Zuzana (703 Slovensko, domácí), Petr LOUŠA (203 Česká republika, domácí), Aneta KOZELEKOVÁ (203 Česká republika, domácí), Tomáš BROM (203 Česká republika, domácí), Norbert GAŠPARIK (703 Slovensko, domácí), Ján TUNGLI (703 Slovensko, domácí), Veronika WEISOVÁ (703 Slovensko, domácí), Erik ŽUPA (703 Slovensko, domácí), Gabriel ŽOLDÁK a Jozef HRITZ (703 Slovensko, garant, domácí)

Vydání

Journal of Molecular Biology, Elsevier, 2022, 0022-2836

Další údaje

Jazyk

angličtina

Typ výsledku

Článek v odborném periodiku

Obor

10608 Biochemistry and molecular biology

Stát vydavatele

Velká Británie a Severní Irsko

Utajení

není předmětem státního či obchodního tajemství

Odkazy

URL

Impakt faktor

Impact factor: 5.600

Kód RIV

RIV/00216224:14740/22:00125485

Organizační jednotka

Středoevropský technologický institut

DOI

http://dx.doi.org/10.1016/j.jmb.2022.167479

UT WoS

000791750700015

Klíčová slova anglicky

14-3-3; phosphorylation; dimerization; dissociation constant; FRET; NMR

Štítky

CF BIC, CF PROT, rivok

Příznaky

Mezinárodní význam, Recenzováno

Změněno: 9. 10. 2024 13:16, Ing. Martina Blahová

Anotace

V originále

14-3-3 proteins are universal regulatory proteins and their function depends on their oligomeric form which may alter between the monomeric, homodimeric and heterodimeric states. The populations of individual oligomeric forms are controlled by Kd values of the dimer–monomer equilibria between the involved isoforms. This complex picture is extended by post-translational modifications, e.g. phosphorylation. In this work, we describe the equilibria between monomers, homo- and heterodimers of the 14-3-3ζ isoform in the unmodified and phosphorylated form. To cover a wide range of dimerization affinities, we combined solution NMR, microscale thermophoresis, native PAGE, and a set of novel fluorescence assays. Using a FRET based assay, we also determined the kinetic parameters of dimerization. We found that phosphorylation of 14-3-3ζ at Ser58 increases its homodimeric Kd value by 6 orders of magnitude. The presented assays allow to efficiently monitor 14-3-3ζ dimerization as a function of external factors, such as temperature, salt concentration, and client protein binding. For instance, we obtained values of both transient and equilibrium thermodynamic constants for the dimerization, and observed a substantial decrease of 14-3-3ζ dimer dissociation rate upon binding to the doubly phosphorylated regulatory domain of tyrosine hydroxylase. In summary, our work provides a conceptual framework to characterise the isoform exchanges of homo- and heterodimers which can significantly deepen our knowledge about the regulatory function of 14-3-3 proteins.

Návaznosti

GF20-05789L, projekt VaV

Název: Charakterizace přirozeně neuspořádaných proteinů

Investor: Grantová agentura ČR, Characterization of intrinsically disordered proteins, Partnerská agentura (Rakousko)

LTAUSA18168, projekt VaV

Název: Selektivní NMR značení jako nástroj pro charakterizaci proteinových komplexů zapojených do neurodegenerativních onemocnění

Investor: Ministerstvo školství, mládeže a tělovýchovy ČR, Selektivní NMR značení jako nástroj pro charakterizaci proteinových komplexů zapojených do neurodegenerativních onemocnění, INTER-ACTION

7AMB16DE005, projekt VaV

Název: Navržení fluorescenčních analytických metod pro kvantifikaci protein-proteinových interakcí v rámci rodiny proteinu 14-3-3.

Investor: Ministerstvo školství, mládeže a tělovýchovy ČR, Design of fluorescence-based analytical tool for quantification of the protein-protein interaction within the 14-3-3 family

90043, velká výzkumná infrastruktura

Název: CIISB

Citovat

TROŠANOVÁ, Zuzana, Petr LOUŠA, Aneta KOZELEKOVÁ, Tomáš BROM, Norbert GAŠPARIK, Ján TUNGLI, Veronika WEISOVÁ, Erik ŽUPA, Gabriel ŽOLDÁK a Jozef HRITZ. Quantitation of Human 14-3-3ζ Dimerization and the Effect of Phosphorylation on Dimer-monomer Equilibria. Journal of Molecular Biology. Elsevier, 2022, roč. 434, č. 7, s. 1-13. ISSN 0022-2836. Dostupné z: https://dx.doi.org/10.1016/j.jmb.2022.167479.

@article{1839678,
   author = {Trošanová, Zuzana and Louša, Petr and Kozeleková, Aneta and Brom, Tomáš and Gašparik, Norbert and Tungli, Ján and Weisová, Veronika and Župa, Erik and Žoldák, Gabriel and Hritz, Jozef},
   article_number = {7},
   doi = {http://dx.doi.org/10.1016/j.jmb.2022.167479},
   keywords = {14-3-3; phosphorylation; dimerization; dissociation constant; FRET; NMR},
   language = {eng},
   issn = {0022-2836},
   journal = {Journal of Molecular Biology},
   title = {Quantitation of Human 14-3-3ζ Dimerization and the Effect of Phosphorylation on Dimer-monomer Equilibria},
   url = {https://www.sciencedirect.com/science/article/pii/S0022283622000481},
   volume = {434},
   year = {2022}
}

TY  - JOUR
ID  - 1839678
AU  - Trošanová, Zuzana - Louša, Petr - Kozeleková, Aneta - Brom, Tomáš - Gašparik, Norbert - Tungli, Ján - Weisová, Veronika - Župa, Erik - Žoldák, Gabriel - Hritz, Jozef
PY  - 2022
TI  - Quantitation of Human 14-3-3ζ Dimerization and the Effect of Phosphorylation on Dimer-monomer Equilibria
JF  - Journal of Molecular Biology
VL  - 434
IS  - 7
SP  - 1-13
EP  - 1-13
PB  - Elsevier
SN  - 00222836
KW  - 14-3-3
KW  - phosphorylation
KW  - dimerization
KW  - dissociation constant
KW  - FRET
KW  - NMR
UR  - https://www.sciencedirect.com/science/article/pii/S0022283622000481
N2  - 14-3-3 proteins are universal regulatory proteins and their function depends on their oligomeric form which may alter between the monomeric, homodimeric and heterodimeric states. The populations of individual oligomeric forms are controlled by Kd values of the dimer–monomer equilibria between the involved isoforms. This complex picture is extended by post-translational modifications, e.g. phosphorylation. In this work, we describe the equilibria between monomers, homo- and heterodimers of the 14-3-3ζ isoform in the unmodified and phosphorylated form. To cover a wide range of dimerization affinities, we combined solution NMR, microscale thermophoresis, native PAGE, and a set of novel fluorescence assays. Using a FRET based assay, we also determined the kinetic parameters of dimerization. We found that phosphorylation of 14-3-3ζ at Ser58 increases its homodimeric Kd value by 6 orders of magnitude. The presented assays allow to efficiently monitor 14-3-3ζ dimerization as a function of external factors, such as temperature, salt concentration, and client protein binding. For instance, we obtained values of both transient and equilibrium thermodynamic constants for the dimerization, and observed a substantial decrease of 14-3-3ζ dimer dissociation rate upon binding to the doubly phosphorylated regulatory domain of tyrosine hydroxylase. In summary, our work provides a conceptual framework to characterise the isoform exchanges of homo- and heterodimers which can significantly deepen our knowledge about the regulatory function of 14-3-3 proteins.
ER  -

TROŠANOVÁ, Zuzana, Petr LOUŠA, Aneta KOZELEKOVÁ, Tomáš BROM, Norbert GAŠPARIK, Ján TUNGLI, Veronika WEISOVÁ, Erik ŽUPA, Gabriel ŽOLDÁK a Jozef HRITZ. Quantitation of Human 14-3-3ζ Dimerization and the Effect of Phosphorylation on Dimer-monomer Equilibria. \textit{Journal of Molecular Biology}. Elsevier, 2022, roč.~434, č.~7, s.~1-13. ISSN~0022-2836. Dostupné z: https://dx.doi.org/10.1016/j.jmb.2022.167479.

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