Department of Protein Structures - Institute of Physiology AS CR, v.v.i.

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The study of structure and function of 14-3-3 proteins and their complexes

Our research team has been studying the 14-3-3 proteins which are highly conserved regulatory molecules found in all eukaryotes. 14-3-3 proteins have the ability of binding the functionally different signal proteins, including kinases, fosfatases and transmembrane receptors by changing their function. Through the functional modulation of a wide range of binding partners, 14-3-3 proteins are involved in many processes, including cell cycle regulation, metabolism control, apoptosis, and control of gene transcription. More than 300 proteins have been described as binding partners till now. We employ both biophysical (fluorescence spectroscopy, analytical ultracentrifugation, SAXS, mass spectrometry, isothermal titration calorimetry, X-ray crystallography, protein structure modeling, etc.) and biochemical (recombinant protein expression, site-directed mutagenesis, enzyme kinetics) approaches to understand the details of how the activity and function of protein-protein complexes are regulated.

Research Projects

The 14-3-3 proteins are a family of regulatory molecules, which specifically bind to phosphoserine (or phosphothreonine)- containing motifs (pSer/pThr) in a sequence-specific manner. Through these binding reactions, the 14-3-3 proteins play key regulatory roles in signal transduction, cell cycle control, metabolism control and apoptosis. More than 200 14-3-3 binding partners have been reported so far and some of them play prominent roles in cancer development (e.g. transcription factors p53 and FOXO), neurodegeneration (e.g. Tau protein, ASK1 kinase), cardiovascular diseases (e.g. RGS proteins, phosducin) or inflammation (e.g. NFkB, ASK1 kinase). However, the detailed mechanisms of the14-3-3 protein-mediated regulations are mostly elusive, mainly due to the lack of structural data.

Main goal of our research is a mechanistic understanding of the 14-3-3 protein function in the regulation of selected 14-3-3 protein binding partners. In recent years we have been studying the 14-3-3 protein-mediated regulation of forkhead transcription factor FOXO4, tyrosine hydroxylase, or regulator of G-protein signaling RGS3. Our current projects are focused on regulation of neutral trehalase Nth1, protein kinase ASK1 and phosducin.

Neutral trehalase Nth1 is an enzyme that catalyzes the hydrolysis of trehalose (non-reducing sugar found in a wide variety of organisms) in yeast S. cerevisiae and its enzymatic activity is regulated in the 14-3-3 protein-dependent manner. This project is funded by Czech Science Foundation (Project P207/11/0455).

Protein kinase ASK1, a member of the mitogen-activated protein kinase kinase kinase family, activates JNK and p38 MAP kinase signaling pathways in response to various stress stimuli, including oxidative stress, endoplasmic reticulum stress, and calcium ion influx . ASK1 plays a key role in the pathogenesis of multiple diseases including cancer, neurodegeneration and cardiovascular diseases and is considered as a promising therapeutic target. The activity of ASK1 is regulated by several other proteins including thioredoxin and the 14-3-3 protein that both function as physiological inhibitors of ASK1. This project is funded by Czech Science Foundation (Project 14-10061S).

Phosducin (Pdc) is a highly conserved acidic phosphoprotein that regulates visual signal transduction by modulating the amount of transducin Gtαβγ heterotrimer through competitionwith the Gta subunit for binding to the Gtβγ complex. Pdc was also shown to regulate the cardiovascular system by modulating sympathetic activity and blood pressure. Phosducin function is regulated through the phosphorylation and the binding to the 14-3-3 protein. This project is funded by Czech Science Foundation (Project P305/11/0708).

Transient receptor potential (TRP) channels are a wide family of non-selective ion channels responsible for monovalent and divalent cation influx into the cells. Members of this family are involved in many sensory processes such as invertebrate vision and hearing, mammalian temperature-, mechano- and chemo-sensation. Both the N- and C-terminal intracellular domains are comprised of many different domains that are responsible for binding different compounds that can regulate the channels. Our goal is to provide the structural insight into the interactions of TRP channels with ATP, calmodulin and PIP. This project is funded by Czech Science Foundation (Project P301/10/1159).

Gallery

SAXS low-resolution structure of the pNth1:Bmh1 complex (B). (Kopecka et. al., JBC 2014, 289:13948-13961)

Proposed mechanism for the 14-3-3 protein-dependent activation of Nth1. The active site (AS) of Nth1 in the absence of Bmh (yeast 14-3-3 isoform) is buried within the structure of the trehalase domain and the enzyme is catalytically inactive. Phosphorylated Nth1 is recognized by Bmh protein and its binding to the N-terminal pSer60 and pSer83 induces conformational change within both the Ca²⁺-binding domain (shown in orange) and the catalytic domain in close proximity to the active site. This structural change enables substrate and product entry and departure, respectively, hence the enzyme activation. (Kopecka et. al., JBC 2014, 289:13948-13961; Macakova et. al., BBA-Gen. Subjects 2013, 1830:4491-4499)

HD-MS reveals the binding surface of pNth1. Graphs represent HDX kinetics for selected pNth1 regions that show slower exchange kinetics upon Bmh1 binding. A. Regions from unstructured N-terminal part. B. Regions from the catalytic trehalase domain mapped on its homology structural model. (Macakova et. al., BBA-Gen. Subjects 2013, 1830:4491-4499)

Specific trehalase activity of pNth1 WT and mutants upon the Bmh1 activation in vitro.(Veisova et. al., Biochemical Journal 2012, 443:663-670)

Structure of human 14-3-3 protein (zeta isoform) with the modeled C-terminal segment. (Veisova et. al., Biochemistry 2010, 49(18):3853-61)

Continuous distribution of sedimentation coefficients, c(s), for Bmh1 and Nth1 alone and in the complex. (Veisova et. al., Biochemical Journal 2012, 443:663-670)