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.

Basic information

Original name

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

Authors

TROŠANOVÁ, Zuzana (703 Slovakia, belonging to the institution), Petr LOUŠA (203 Czech Republic, belonging to the institution), Aneta KOZELEKOVÁ (203 Czech Republic, belonging to the institution), Tomáš BROM (203 Czech Republic, belonging to the institution), Norbert GAŠPARIK (703 Slovakia, belonging to the institution), Ján TUNGLI (703 Slovakia, belonging to the institution), Veronika WEISOVÁ (703 Slovakia, belonging to the institution), Erik ŽUPA (703 Slovakia, belonging to the institution), Gabriel ŽOLDÁK and Jozef HRITZ (703 Slovakia, guarantor, belonging to the institution)

Edition

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

Other information

Language

English

Type of outcome

Článek v odborném periodiku

Field of Study

10608 Biochemistry and molecular biology

Country of publisher

United Kingdom of Great Britain and Northern Ireland

Confidentiality degree

není předmětem státního či obchodního tajemství

References:

Impact factor

Impact factor: 5.600

RIV identification code

RIV/00216224:14740/22:00125485

Organization unit

Central European Institute of Technology

UT WoS

000791750700015

Keywords in English

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

Tags

International impact, Reviewed
Změněno: 9/10/2024 13:16, Ing. Martina Blahová

Abstract

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.

Links

GF20-05789L, research and development project
Name: Charakterizace přirozeně neuspořádaných proteinů
Investor: Czech Science Foundation, Partner Agency (Austria)
LTAUSA18168, research and development project
Name: Selektivní NMR značení jako nástroj pro charakterizaci proteinových komplexů zapojených do neurodegenerativních onemocnění
Investor: Ministry of Education, Youth and Sports of the CR, Selective NMR labelling as a tool for characterization of protein complexes involved in neurodegenerative diseases., INTER-ACTION
7AMB16DE005, research and development project
Name: Navržení fluorescenčních analytických metod pro kvantifikaci protein-proteinových interakcí v rámci rodiny proteinu 14-3-3.
Investor: Ministry of Education, Youth and Sports of the CR
90043, large research infrastructures
Name: CIISB