Nikolai N. Sluchanko

14-3-3 Proteins and regulation of cytoskeleton

The proteins of the 14-3-3 family are universal adapters participating in multiple processes running in the cell. We describe the structure, isoform composition, and distribution of 14-3-3 proteins in different tissues. Different elements of 14-3-3 structure important for dimer formation and recognition of protein targets are analyzed in detail. Special attention is paid to analysis of posttranslational modifications playing important roles in regulation of 14-3-3 function. The data of the literature concerning participation of 14-3-3 in regulation of intercellular contacts and different elements of cytoskeleton formed by microfilaments are analyzed. We also describe participation of 14-3-3 in regulation of small G-proteins and protein kinases important for proper functioning of cytoskeleton. The data on the interaction of 14-3-3 with different components of microtubules are presented, and the probable role of 14-3-3 in developing of certain neurodegenerative diseases is discussed. The data of the literature concerning the role of 14-3-3 in formation and normal functioning of intermediate filaments are also reviewed. It is concluded that due to its adapter properties 14-3-3 plays an important role in cytoskeleton regulation. The cytoskeletal proteins that are abundant in the cell might compete with the other protein targets of 14-3-3 and therefore can indirectly regulate many intracellular processes that are dependent on 14-3-3.

Oligomeric structure of 14-3-3 protein: What do we know about monomers?

14‐3‐3s predominantly form homo‐/heterodimers that are in equilibrium with corresponding monomers. Dimer/monomer equilibrium depends on the nature and phosphorylation of Ser58 of certain 14‐3‐3 isoforms. The structure and properties of 14‐3‐3 dimers are well characterized, whereas 14‐3‐3 monomers are less investigated. Therefore design and analysis of dimer‐incapable mutants of 14‐3‐3 are important. Truncated or heavily mutated proteins are not ideal since their structure may be distorted. Phosphomimicking mutations, such as S58(D/E), induce incomplete dimer dissociation. A recently characterized monomeric 14‐3‐3 contains few mutations and retains the original secondary structure. Monomeric 14‐3‐3 interacts with phosphorylated target proteins and has higher chaperone‐like activity than dimeric 14‐3‐3. Further investigation of the properties of monomeric 14‐3‐3 is important for understanding its yet poorly characterized role in different cellular processes.

The purple Trp288Ala mutant of Synechocystis OCP persistently quenches phycobilisome fluorescence and tightly interacts with FRP.

In Cyanobacteria, the Orange Carotenoid Protein (OCP) and Fluorescence Recovery Protein (FRP) are central to the photoprotective mechanism consisting in regulated quenching of phycobilisome (PBs) fluorescence. Due to a transient and flexible nature of the light-activated red quenching form, OCPR, which is obtained from the stable dark-adapted orange form, OCPO, by photoconversion, the detailed mechanism of photoprotection remains unclear. Here we demonstrate that our recently described W288A mutant of the Synechocystis OCP (hereinafter called OCPW288A) is a fully functional analogue of the OCPR form which is capable of constitutive PBs fluorescence quenching in vitro with no need of photoactivation. This PBs quenching effect is abolished in the presence of FRP, which interacts with OCPW288A with micromolar affinity and an apparent stoichiometry of 1:1, unexpectedly, implying dissociation of the FRP dimers. This establishes OCPW288A as a robust model system providing novel insights into the interplay between OCP and FRP to regulate photoprotection in cyanobacteria.

Phosphomimicking mutations of human 14-3-3ζ affect its interaction with tau protein and small heat shock protein HspB6.

Effect of phosphomimicking mutations of 14-3-3ζ on its interaction with phosphorylated shortest isoform of human tau protein and phosphorylated human small heat shock protein HspB6 (Hsp20) was analyzed. Chemical crosslinking and native gel electrophoresis indicate that mutations S184E and T232E weakly affect interaction of 14-3-3 with phosphorylated tau protein, whereas mutations S58E and S58E/S184E/T232E significantly impair interaction of 14-3-3 and tau. Size-exclusion chromatography, chemical crosslinking and immunoprecipitation revealed that phosphomimicking mutations S58E and S58E/S184E/T232E strongly decrease, mutation T232E weakly affects and mutation S184E improves interaction of 14-3-3 with phosphorylated HspB6. Thus, mutation mimicking phosphorylation of Ser58 dramatically decreases interaction of 14-3-3 with two target proteins and this effect might be due to destabilization of the dimeric structure of 14-3-3 and/or conformational changes of the target-binding site. The mutation mimicking phosphorylation of Thr232 weakly affects interaction of 14-3-3 with both proteins. The mutation mimicking phosphorylation of Ser184 does not markedly affect interaction with tau protein and improves the interaction of 14-3-3 with HspB6. Thus, effect of 14-3-3 phosphorylation depends on the nature of the target protein and therefore, phosphorylation of 14-3-3 might affect its target specificity.

Phosphorylation of more than one site is required for tight interaction of human tau protein with 14-3-3ζ

MINT‐7233285, MINT‐7233297, MINT‐7233310: 14‐3‐3 zeta (uniprotkb:P63104) and Tau 3 (uniprotkb:P10636‐3) bind (MI:0407) by comigration in non‐denaturing gel electrophoresis (MI:0404)

Effect of mutations mimicking phosphorylation on the structure and properties of human 14-3-3ζ

Effect of mutations mimicking phosphorylation on the structure of human 14-3-3zeta protein was analyzed by different methods. Mutation S58E increased intrinsic Trp fluorescence and binding of bis-ANS to 14-3-3. At low protein concentration mutation S58E increased the probability of dissociation of dimeric 14-3-3 and its susceptibility to proteolysis. Mutation S184E slightly increased Stokes radius and thermal stability of 14-3-3. Mutation T232E induced only small increase of Stokes radius and sedimentation coefficient that probably reflect the changes in the size or shape of 14-3-3. At low protein concentration the triple mutant S58E/S184E/T232E tended to dissociate, whereas at high concentration its properties were comparable with those of the wild type protein. The triple mutant was highly susceptible to proteolysis. Thus, mutation mimicking phosphorylation of Ser58 destabilized, whereas mutation of Ser184 induced stabilization of 14-3-3zeta structure.

Moonlighting chaperone-like activity of the universal regulatory 14-3-3 proteins

The ubiquitous eukaryotic 14‐3‐3 proteins coordinate multiple cellular processes due to their well‐known regulatory function, which is based on specific recognition of phosphorylated motifs in their partners. In this context, 14‐3‐3 proteins have been called ‘chaperones’. Although in the classical meaning this is not fully correct, recent studies have revealed that they can indeed be an integral part of the protein quality control system, as they (a) display ATP‐independent anti‐aggregation (‘holdase’) activity, similar to that of the unrelated small heat shock proteins, (b) assist in clearing misfolded proteins by directing them to proteasomes or aggresomes, (c) cooperate with classical chaperones for substrate refolding, and also (d) are associated with neurodegenerative disorders by affecting aggregation of tau, prion protein, α‐synuclein, huntingtin, etc. Importantly, these activities are usually independent of substrate phosphorylation and therefore should be considered as distinct, ‘moonlighting’ functions of 14‐3‐3 proteins that mimic and complement the functions of dedicated molecular chaperones. Although the precise mechanism of this activity is still unknown, it has been shown that it is not dependent on the unstructured C‐terminal region or the amphipathic phosphopeptide‐binding groove. However, since disassembly of 14‐3‐3 dimers significantly increases their chaperone‐like activity, the dimer interface, located in the N terminus, possessing a high disorder propensity and pronounced hydrophobicity, is likely to be involved. Various factors affecting the oligomeric status of 14‐3‐3 proteins can thus regulate the balance between regulatory phosphomotif binding and genuine chaperone‐like activity. Understanding the latter mode of 14‐3‐3 functioning is fundamental to defining the underlying molecular mechanisms for a range of human disorders.

Probable Participation of 14-3-3 in Tau Protein Oligomerization and Aggregation

Aggregation of tau proteins followed by formation of paired helical filaments and neurofibrillary tangles is considered as a hallmark of certain neurodegenerative disorders such as different tauopathies and Alzheimers disease (AD). Tau aggregation is dependent on the presence of polyanions, cellular redox state, limited proteolysis, and different posttranslational modifications among which tau phosphorylation plays a particularly important role. Although it is still debatable whether tau aggregation is harmful or protective for the cell, detailed analysis of molecular mechanisms underlying this process seems to be of great importance for understanding AD pathogenesis. This review is focused on universal adapter proteins 14-3-3 that seem to be significant partners to tau protein in neurons. 14-3-3 interacts with nonphosphorylated tau and promotes its interaction with and phosphorylation by a number of protein kinases. 14-3-3 induces aggregation of nonphosphorylated tau and does not affect aggregation of tau phosphorylated at specific sites. Due to its high concentration in neurons, 14-3-3 can compete with tubulin for interaction with tau. Binding to phosphorylated tau, 14-3-3 might inhibit its dephosphorylation by protein phosphatases and by this means indirectly affect interaction of tau with microtubules and tau aggregation. Finally, 14-3-3 might promote sequestration of dangerous small tau oligomers and stabilize tau aggregates. We propose that 14-3-3 should be considered an important participant of the complex process of tau aggregation and as a potential therapeutic target in treating AD.

Effect of phosphorylation on interaction of human tau protein with 14-3-3ζ

Interaction of the shortest isoform of tau protein (tau3) with human 14-3-3zeta was analyzed by means of native gel electrophoresis, chemical crosslinking and size-exclusion chromatography. Phosphorylation by cAMP-dependent protein kinase (up to 2 mole of phosphate per mole of tau3) strongly enhanced interaction of tau3 with 14-3-3. Apparent K(D) of the complexes formed by phosphorylated tau3 and 14-3-3 was close to 2 microM, whereas the corresponding constant for unphosphorylated tau3 was at least 10 times higher. The stoichiometry of the complexes formed by phosphorylated tau3 and 14-3-3 was variable and was different from 1:1. 14-3-3 decreased the probability of formation of chemically crosslinked large homooligomers of phosphorylated tau3 and at the same time induced formation of crosslinked heterooligomeric complexes of tau3 and 14-3-3 with an apparent molecular mass of 120-140 kDa.

Properties of the monomeric form of human 14-3-3ζ protein and its interaction with tau and HspB6.

Dimers formed by seven isoforms of the human 14-3-3 protein participate in multiple cellular processes. The dimeric form has been extensively characterized; however, little is known about the structure and properties of the monomeric form of 14-3-3. The monomeric form is involved in the assembly of homo- and heterodimers, which could partially dissociate back into monomers in response to phosphorylation at Ser58. To obtain monomeric forms of human 14-3-3ζ, we produced four protein constructs with different combinations of mutated (M) or wild-type (W) segments E(5), (12)LAE(14), and (82)YREKIE(87). Under a wide range of expression conditions in Escherichia coli, the MMM and WMM mutants were insoluble, whereas WMW and MMW mutants were soluble, highly expressed, and purified to homogeneity. WMW and MMW mutants remained monomeric over a wide range of concentrations while retaining the α-helical structure characteristic of wild-type 14-3-3. However, WMW and MMW mutants were highly susceptible to proteolysis and had much lower thermal stabilities than the wild-type protein. Using WMW and MMW mutants, we show that the monomeric form interacts with the tau protein and with the HspB6 protein, in both cases forming complexes with a 1:1 stoichiometry, in contrast to the 2:1 and/or 2:2 complexes formed by wild-type 14-3-3. Significantly, this interaction requires phosphorylation of tau protein and HspB6. Because of minimal changes in structure, MMW and especially WMW mutant proteins are promising candidates for analyzing the effect of monomerization on the physiologically important properties of 14-3-3ζ.