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

AbstractSerine residues phosphorylated by protein kinase A (PKA) in the shortest isoform of human tau protein (τ3) were sequentially replaced by alanine and interaction of phosphorylated τ3 and its mutants with 14-3-3 was investigated. Mutation S156A slightly decreased interaction of phosphorylated τ3 with 14-3-3. Double mutations S156A/S267A and especially S156A/S235A, strongly inhibited interaction of phosphorylated τ3 with 14-3-3. Thus, two sites located in the Pro-rich region and in the pseudo repeats of τ3 are involved in phosphorylation-dependent interaction of τ3 with 14-3-3. The state of τ3 phosphorylation affects the mode of 14-3-3 binding and by this means might modify tau filament formation.Structured summaryMINT-7233358, MINT-7233372, MINT-7233384: 14-3-3 zeta (uniprotkb:P63104) and Tau 3 (uniprotkb:P10636-3) bind (MI:0407) by molecular sieving (MI:0071)MINT-7233323, MINT-7233334, MINT-7233346: Tau 3 (uniprotkb:P10636-3) and 14-3-3 zeta (uniprotkb:P63104) bind (MI:0407) by crosslinking studies (MI:0030)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

Structural basis for the interaction of a human small heat shock protein with the 14-3-3 universal signaling regulator

By interacting with hundreds of protein partners, 14-3-3 proteins coordinate vital cellular processes. Phosphorylation of the small heat shock protein, HSPB6, within its intrinsically disordered N-terminal domain activates its interaction with 14-3-3, ultimately triggering smooth muscle relaxation. After analyzing the binding of an HSPB6-derived phosphopeptide to 14-3-3 using isothermal calorimetry and X-ray crystallography, we have determined the crystal structure of the complete assembly consisting of the 14-3-3 dimer and full-length HSPB6 dimer and further characterized this complex in solution using fluorescence spectroscopy, small-angle X-ray scattering, and limited proteolysis. We show that selected intrinsically disordered regions of HSPB6 are transformed into well-defined conformations upon the interaction, whereby an unexpectedly asymmetric structure is formed. This structure provides the first atomic resolution snapshot of a human small HSP in functional state, explains how 14-3-3 proteins sequester their regulatory partners, and can inform the design of small-molecule interaction modifiers to be used as myorelaxants

The mechanism of SARS-CoV-2 nucleocapsid protein recognition by the human 14-3-3 proteins

The coronavirus nucleocapsid protein (N) controls viral genome packaging and contains numerous phosphorylation sites located within unstructured regions. Binding of phosphorylated SARS-CoV N to the host 14-3-3 protein in the cytoplasm was reported to regulate nucleocytoplasmic N shuttling. All seven isoforms of the human 14-3-3 are abundantly present in tissues vulnerable to SARS-CoV-2, where N can constitute up to ∼1% of expressed proteins during infection. Although the association between 14-3-3 and SARS-CoV-2 N proteins can represent one of the key host-pathogen interactions, its molecular mechanism and the specific critical phosphosites are unknown. Here, we show that phosphorylated SARS-CoV-2 N protein (pN) dimers, reconstituted via bacterial co-expression with protein kinase A, directly associate, in a phosphorylation-dependent manner, with the dimeric 14-3-3 protein, but not with its monomeric mutant. We demonstrate that pN is recognized by all seven human 14-3-3 isoforms with various efficiencies and deduce the apparent KD to selected isoforms, showing that these are in a low micromolar range. Serial truncations pinpointed a critical phosphorylation site to Ser197, which is conserved among related zoonotic coronaviruses and located within the functionally important, SR-rich region of N. The relatively tight 14-3-3/pN association could regulate nucleocytoplasmic shuttling and other functions of N via occlusion of the SR-rich region, and could also hijack cellular pathways by 14-3-3 sequestration. As such, the assembly may represent a valuable target for therapeutic intervention

Soluble Cyanobacterial Carotenoprotein as a Robust Antioxidant Nanocarrier and Delivery Module

To counteract oxidative stress, antioxidants including carotenoids are highly promising, yet their exploitation is drastically limited by the poor bioavailability and fast photodestruction, whereas current delivery systems are far from being efficient. Here we demonstrate that the recently discovered nanometer-sized water-soluble carotenoprotein from Anabaena sp. PCC 7120 (termed AnaCTDH) transiently interacts with liposomes to efficiently extract carotenoids via carotenoid-mediated homodimerization, yielding violet–purple protein samples. We characterize the spectroscopic properties of the obtained pigment–protein complexes and the thermodynamics of liposome–protein carotenoid transfer and demonstrate the delivery of carotenoid echinenone from AnaCTDH into liposomes with an efficiency of up to 70 ± 3%. Most importantly, we show efficient carotenoid delivery to membranes of mammalian cells, which provides protection from reactive oxygen species (ROS). Incubation of neuroblastoma cell line Tet21N in the presence of 1 μM AnaCTDH binding echinenone decreased antimycin A ROS production by 25% (p < 0.05). The described carotenoprotein may be considered as part of modular systems for the targeted antioxidant delivery.BMBF, 01DJ15007, Carotenoidbindende photoschaltbare Proteine: Lichtinduzierte Dynamik und Anwendungen in modernen mikroskopischen Verfahre

Hidden Disorder Propensity of the N-terminal Segment of Universal Adapter Protein 14-3-3 is Manifested in Its Monomeric Form: Novel Insights into Protein Dimerization and Multifunctionality

The multiplicity of functions of 14-3-3 proteins, integrated into many cellular interactions and signaling networks, is primarily based upon their dimeric α-helical structure that is capable of binding phosphorylated protein partners as well as displaying a “moonlighting” chaperone-like activity. The structure and functions of 14-3-3 proteins are regulated in different ways, including Ser58 phosphorylation in the interface, which shifts equilibrium towards the formation of protein monomers whose role is poorly understood. While modification of Ser58 induced only partial dissociation, the engineered triple mutation of human 14-3-3ζ located in the first α-helix deeply monomerized the protein, allowing for a structural analysis of the monomeric form. Dimer-incapable 14-3-3 proteins retained binding capacity and specificity towards some phosphopartners, and also demonstrated increased chaperone-like activity on various substrates. Here, we found a substantial propensity of the N-terminal segment (~ 40 residues) of 14-3-3 proteins to intrinsic disorder, showing remarkable conservation across different isoforms and organisms. We hypothesized that this intrinsic disorder propensity, hidden in the α-helical 14-3-3 dimer, can be manifested upon its dissociation and interrogated novel monomeric 14-3-3ζ carrying both monomerizing and S58E mutations (14-3-3ζmS58E). CD spectroscopy showed that, at physiological temperatures, this protein has ~ 10–15% reduced helicity relative to the wild type protein, corresponding to roughly 40 residues. Along with the known flexibility of C-terminus, SAXS-based modeling of the 14-3-3ζmS58E structure strongly suggested pliability of its N-terminus. The unraveled disorder propensity of the N-terminal tails of 14-3-3 proteins provides new clues for better understanding of the molecular mechanisms of dimerization and multifunctionality of these universal adapter proteins

Modulation of 14-3-3/phosphotarget interaction by physiological concentrations of phosphate and glycerophosphates.

Molecular mechanisms governing selective binding of a huge number of various phosphorylated protein partners to 14-3-3 remain obscure. Phosphate can bind to 14-3-3 and therefore being present at high intracellular concentration, which undergoes significant changes under physiological conditions, phosphate can theoretically regulate interaction of 14-3-3 with phosphorylated targets. In order to check this hypothesis we analyzed effect of phosphate and other natural abundant anions on interaction of 14-3-3 with phosphorylated human small heat shock protein HspB6 (Hsp20) participating in regulation of different intracellular processes. Inorganic phosphate, glycerol-1-phosphate and glycerol-2-phosphate at physiologically relevant concentrations (5-15 mM) significantly destabilized complexes formed by 14-3-3ζ and phosphorylated HspB6 (pHspB6), presumably, via direct interaction with the substrate-binding site of 14-3-3. Phosphate also destabilized complexes between pHspB6 and 14-3-3γ or the monomeric mutant form of 14-3-3ζ. Inorganic sulfate and pyrophosphate were less effective in modulation of 14-3-3 interaction with its target protein. The inhibitory effect of all anions on pHspB6/14-3-3 interaction was concentration-dependent. It is hypothesized that physiological changes in phosphate anions concentration can modulate affinity and specificity of interaction of 14-3-3 with its multiple targets and therefore the actual phosphointeractome of 14-3-3

Bacterial co-expression of human Tau protein with protein kinase A and 14-3-3 for studies of 14-3-3/phospho-Tau interaction.

Abundant regulatory 14-3-3 proteins have an extremely wide interactome and coordinate multiple cellular events via interaction with specifically phosphorylated partner proteins. Notwithstanding the key role of 14-3-3/phosphotarget interactions in many physiological and pathological processes, they are dramatically underexplored. Here, we focused on the 14-3-3 interaction with human Tau protein associated with the development of several neurodegenerative disorders, including Alzheimer's and Parkinson's diseases. Among many known phosphorylation sites within Tau, protein kinase A (PKA) phosphorylates several key residues of Tau and induces its tight interaction with 14-3-3 proteins. However, the stoichiometry and mechanism of 14-3-3 interaction with phosphorylated Tau (pTau) are not clearly elucidated. In this work, we describe a simple bacterial co-expression system aimed to facilitate biochemical and structural studies on the 14-3-3/pTau interaction. We show that dual co-expression of human fetal Tau with PKA in Escherichia coli results in multisite Tau phosphorylation including also naturally occurring sites which were not previously considered in the context of 14-3-3 binding. Tau protein co-expressed with PKA displays tight functional interaction with 14-3-3 isoforms of a different type. Upon triple co-expression with 14-3-3 and PKA, Tau protein could be co-purified with 14-3-3 and demonstrates complex which is similar to that formed in vitro between individual 14-3-3 and pTau obtained from dual co-expression. Although used in this study for the specific case of the previously known 14-3-3/pTau interaction, our co-expression system may be useful to study of other selected 14-3-3/phosphotarget interactions and for validations of 14-3-3 complexes identified by other methods

Solution structure of human steroidogenic acute regulatory protein STARD1 studied by small-angle X-ray scattering

Intracellular cholesterol transfer to mitochondria, a bottleneck of adrenal and gonadal steroidogenesis, relies on the functioning of the steroidogenic acute regulatory protein (StAR, STARD1), for which many disease-associated mutations have been described. Despite significant progress in the field, the exact mechanism of cholesterol binding and transfer by STARD1 still remains debatable and often considers significant structural rearrangements to achieve ligand binding. The crystal structure of STARD1, obtained recently at medium resolution, suggests that this protein has the same fold as other members of the START family. However, hydrodynamic properties and solution conformation of STARD1 are insufficiently characterized, partially due to poor solubility of this protein. Here, we used our recent protocol to obtain stable and soluble STARD1 and analyzed its hydrodynamic properties and solution conformation using a previously inapplicable small-angle X-ray scattering (SAXS). The SAXS data obtained exclusively from a monodisperse fraction of the monomeric protein suggest that, apart from movements of the flexible Ω1-loop, STARD1 unlikely undergoes significant spontaneous rearrangements proposed earlier as a gating mechanism for cholesterol binding. The consistency with the previously reported solution NMR structure of STARD6 suggests similarity of hydrodynamic behavior of other STARD-containing proteins

Effect of phosphate (P_i) on the interaction of pHspB6 with the wild type 14-3-3ζ, 14-3-3γ or with the 14-3-3ζ mutant being unable to form dimers (14-3-3ζ_m) studied by SEC.

<p>14-3-3ζ (39 µM) (<b>A</b>, <b>C</b>), 14-3-3γ (39 µM) (<b>B</b>, <b>D</b>), 14-3-3ζ_m (13 µM) (<b>E</b>, <b>F</b>) alone (black lines) or pHspB6 (21 µM) alone (orange lines) or the mixtures of pHspB6 and different 14-3-3 proteins (red lines) were pre-incubated and subjected to SEC in the absence (<b>A</b>, <b>B</b>, <b>E</b>) or in the presence (<b>C</b>, <b>D</b>, <b>F</b>) of 75 mM phosphate in elution buffer. Blue dashed lines represent algebraic sum of elution profiles of isolated 14-3-3 and isolated pHspB6 and indicate lack of 14-3-3/pB6 interaction. Representative results of three independent experiments are presented.</p