Nedd4-2 E3 ligase regulates Na+ homeostasis by ubiquitinating various channels and membrane transporters, including the epithelial sodium channel ENaC. In turn, Nedd4-2 dysregulation leads to various conditions, including electrolytic imbalance, respiratory distress, hypertension, and kidney diseases. However, Nedd4-2 regulation remains mostly unclear. The present study aims at elucidating Nedd4-2 regulation by structurally characterizing Nedd4-2 and its complexes using several biophysical techniques. Our cryo-EM reconstruction shows that the C2 domain blocks the E2-binding surface of the HECT domain. This blockage, ubiquitin-binding exosite masking by the WW1 domain, catalytic C922 blockage and HECT domain stabilization provide the structural basis for Nedd4-2 autoinhibition. Furthermore, Ca2+-dependent C2 membrane binding disrupts C2/HECT interactions, but not Ca2+ alone, whereas 14-3-3 protein binds to a flexible region of Nedd4-2 containing the WW2 and WW3 domains, thereby inhibiting its catalytic activity and membrane binding. Overall, our data provide key mechanistic insights into Nedd4-2 regulation toward fostering the development of strategies targeting Nedd4-2 function.

• MeSH
• elektronová kryomikroskopie MeSH
• HEK293 buňky MeSH
• lidé MeSH
• molekulární modely MeSH
• proteinové domény MeSH
• proteiny 14-3-3 * metabolismus   chemie   MeSH
• ubikvitinace MeSH
• ubikvitinligasy Nedd4 * metabolismus   chemie   genetika   ultrastruktura   MeSH
• vápník * metabolismus   MeSH
• vazba proteinů MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• Nedd4 protein, human MeSH Prohlížeč
• Nedd4L protein, human MeSH Prohlížeč
• proteiny 14-3-3 * MeSH
• ubikvitinligasy Nedd4 * MeSH
• vápník * MeSH

Cell signaling regulates several physiological processes by receiving, processing, and transmitting signals between the extracellular and intracellular environments. In signal transduction, phosphorylation is a crucial effector as the most common posttranslational modification. Selectively recognizing specific phosphorylated motifs of target proteins and modulating their functions through binding interactions, the yeast 14-3-3 proteins Bmh1 and Bmh2 are involved in catabolite repression, carbon metabolism, endocytosis, and mitochondrial retrograde signaling, among other key cellular processes. These conserved scaffolding molecules also mediate crosstalk between ubiquitination and phosphorylation, the spatiotemporal control of meiosis, and the activity of ion transporters Trk1 and Nha1. In humans, deregulation of analogous processes triggers the development of serious diseases, such as diabetes, cancer, viral infections, microbial conditions and neuronal and age-related diseases. Accordingly, the aim of this review article is to provide a brief overview of the latest findings on the functions of yeast 14-3-3 proteins, focusing on their role in modulating the aforementioned processes.

• Klíčová slova
• 14-3-3 proteins, adaptor protein, molecular mechanism, phosphorylation, protein-protein interaction, scaffolding, yeast,
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH

Ca2+ /CaM-dependent protein kinase kinases 1 and 2 (CaMKK1 and CaMKK2) phosphorylate and enhance the catalytic activity of downstream kinases CaMKI, CaMKIV, and protein kinase B. Accordingly, CaMKK1 and CaMKK2 regulate key physiological and pathological processes, such as tumorigenesis, neuronal morphogenesis, synaptic plasticity, transcription factor activation, and cellular energy homeostasis, and promote cell survival. Both CaMKKs are partly inhibited by phosphorylation, which in turn triggers adaptor and scaffolding protein 14-3-3 binding. However, 14-3-3 binding only significantly affects CaMKK1 function. CaMKK2 activity remains almost unchanged after complex formation for reasons still unclear. Here, we aim at structurally characterizing CaMKK1:14-3-3 and CaMKK2:14-3-3 complexes by SAXS, H/D exchange coupled to MS, and fluorescence spectroscopy. The results revealed that complex formation suppresses the interaction of both phosphorylated CaMKKs with Ca2+ /CaM and affects the structure of their kinase domains and autoinhibitory segments. But these effects are much stronger on CaMKK1 than on CaMKK2 because the CaMKK1:14-3-3γ complex has a more compact and rigid structure in which the active site of the kinase domain directly interacts with the last two C-terminal helices of the 14-3-3γ protein, thereby inhibiting CaMKK1. In contrast, the CaMKK2:14-3-3 complex has a looser and more flexible structure, so 14-3-3 binding only negligibly affects the catalytic activity of CaMKK2. Therefore, Ca2+ /CaM binding suppression and the interaction of the kinase active site of CaMKK1 with the last two C-terminal helices of 14-3-3γ protein provide the structural basis for 14-3-3-mediated CaMKK1 inhibition.

• Klíčová slova
• 14-3-3 proteins, CaMKK, SAXS, calcium/calmodulin-dependent protein kinase, fluorescence spectroscopy, hydrogen/deuterium exchange coupled to MS, protein-protein interaction,
• MeSH
• difrakce rentgenového záření MeSH
• fosforylace MeSH
• katalytická doména MeSH
• kinasa proteinkinasy závislé na vápníku a kalmodulinu * chemie   metabolismus   MeSH
• maloúhlový rozptyl MeSH
• proteiny 14-3-3 * metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• kinasa proteinkinasy závislé na vápníku a kalmodulinu * MeSH
• proteiny 14-3-3 * MeSH

Covalent DNA interstrand crosslinks are toxic DNA damage lesions that block the replication machinery that can cause a genomic instability. Ubiquitous abasic DNA sites are particularly susceptible to spontaneous cross-linking with a base from the opposite DNA strand. Detection of a crosslink induces the DNA helicase ubiquitination that recruits NEIL3, a DNA glycosylase responsible for the lesion removal. NEIL3 utilizes several zinc finger domains indispensable for its catalytic NEI domain repairing activity. They recruit NEIL3 to the repair site and bind the single-stranded DNA. However, the molecular mechanism underlying their roles in the repair process is unknown. Here, we report the structure of the tandem zinc-finger GRF domain of NEIL3 and reveal the molecular details of its interaction with DNA. Our biochemical data indicate the preferential binding of the GRF domain to the replication fork. In addition, we obtained a structure for the catalytic NEI domain in complex with the DNA reaction intermediate that allowed us to construct and validate a model for the interplay between the NEI and GRF domains in the recognition of an interstrand cross-link. Our results suggest a mechanism for recognition of the DNA replication X-structure by NEIL3, a key step in the interstrand cross-link repair.

• MeSH
• DNA-glykosylasy metabolismus   MeSH
• DNA-helikasy metabolismus   MeSH
• DNA chemie   MeSH
• endodeoxyribonukleasy metabolismus   MeSH
• jednovláknová DNA MeSH
• oprava DNA * MeSH
• poškození DNA MeSH
• zinek MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• DNA-glykosylasy MeSH
• DNA-helikasy MeSH
• DNA MeSH
• endodeoxyribonukleasy MeSH
• jednovláknová DNA MeSH
• NEIL3 protein, mouse MeSH Prohlížeč
• zinek MeSH

Signal transduction cascades efficiently transmit chemical and/or physical signals from the extracellular environment to intracellular compartments, thereby eliciting an appropriate cellular response. Most often, these signaling processes are mediated by specific protein-protein interactions involving hundreds of different receptors, enzymes, transcription factors, and signaling, adaptor and scaffolding proteins. Among them, 14-3-3 proteins are a family of highly conserved scaffolding molecules expressed in all eukaryotes, where they modulate the function of other proteins, primarily in a phosphorylation-dependent manner. Through these binding interactions, 14-3-3 proteins participate in key cellular processes, such as cell-cycle control, apoptosis, signal transduction, energy metabolism, and protein trafficking. To date, several hundreds of 14-3-3 binding partners have been identified, including protein kinases, phosphatases, receptors and transcription factors, which have been implicated in the onset of various diseases. As such, 14-3-3 proteins are promising targets for pharmaceutical interventions. However, despite intensive research into their protein-protein interactions, our understanding of the molecular mechanisms whereby 14-3-3 proteins regulate the functions of their binding partners remains insufficient. This review article provides an overview of the current state of the art of the molecular mechanisms whereby 14-3-3 proteins regulate their binding partners, focusing on recent structural studies of 14-3-3 protein complexes.

• Klíčová slova
• 14-3-3 proteins, adaptor protein, molecular mechanism, phosphorylation, protein-protein interactions, scaffolding,
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH

Neural precursor cells expressed developmentally downregulated protein 4-2 (Nedd4-2), a homologous to the E6-AP carboxyl terminus (HECT) ubiquitin ligase, triggers the endocytosis and degradation of its downstream target molecules by regulating signal transduction through interactions with other targets, including 14-3-3 proteins. In our previous study, we found that 14-3-3 binding induces a structural rearrangement of Nedd4-2 by inhibiting interactions between its structured domains. Here, we used time-resolved fluorescence intensity and anisotropy decay measurements, together with fluorescence quenching and mass spectrometry, to further characterize interactions between Nedd4-2 and 14-3-3 proteins. The results showed that 14-3-3 binding affects the emission properties of AEDANS-labeled WW3, WW4, and, to a lesser extent, WW2 domains, and reduces their mobility, but not those of the WW1 domain, which remains mobile. In contrast, 14-3-3 binding has the opposite effect on the active site of the HECT domain, which is more solvent exposed and mobile in the complexed form than in the apo form of Nedd4-2. Overall, our results suggest that steric hindrance of the WW3 and WW4 domains combined with conformational changes in the catalytic domain may account for the 14-3-3 binding-mediated regulation of Nedd4-2.

• MeSH
• endozomální třídící komplexy pro transport * metabolismus   MeSH
• katalytická doména MeSH
• nervové kmenové buňky * metabolismus   MeSH
• proteiny 14-3-3 metabolismus   MeSH
• ubikvitinligasy Nedd4 metabolismus   MeSH
• ubikvitinligasy metabolismus   MeSH
• vazba proteinů MeSH
• WW domény MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• endozomální třídící komplexy pro transport * MeSH
• proteiny 14-3-3 MeSH
• ubikvitinligasy Nedd4 MeSH
• ubikvitinligasy MeSH

Neural precursor cell expressed developmentally down-regulated 4 ligase (Nedd4-2) is an E3 ubiquitin ligase that targets proteins for ubiquitination and endocytosis, thereby regulating numerous ion channels, membrane receptors and tumor suppressors. Nedd4-2 activity is regulated by autoinhibition, calcium binding, oxidative stress, substrate binding, phosphorylation and 14-3-3 protein binding. However, the structural basis of 14-3-3-mediated Nedd4-2 regulation remains poorly understood. Here, we combined several techniques of integrative structural biology to characterize Nedd4-2 and its complex with 14-3-3. We demonstrate that phosphorylated Ser342 and Ser448 are the key residues that facilitate 14-3-3 protein binding to Nedd4-2 and that 14-3-3 protein binding induces a structural rearrangement of Nedd4-2 by inhibiting interactions between its structured domains. Overall, our findings provide the structural glimpse into the 14-3-3-mediated Nedd4-2 regulation and highlight the potential of the Nedd4-2:14-3-3 complex as a pharmacological target for Nedd4-2-associated diseases such as hypertension, epilepsy, kidney disease and cancer.

• MeSH
• down regulace MeSH
• fosforylace MeSH
• myši genetika   metabolismus   MeSH
• proteiny 14-3-3 genetika   metabolismus   MeSH
• ubikvitinace MeSH
• ubikvitinligasy Nedd4 genetika   metabolismus   MeSH
• vazba proteinů MeSH
• WW domény * MeSH
• zvířata MeSH
• Check Tag
• myši genetika   metabolismus   MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• Nedd4l protein, mouse MeSH Prohlížeč
• proteiny 14-3-3 MeSH
• Sfn protein, mouse MeSH Prohlížeč
• ubikvitinligasy Nedd4 MeSH

Phosducin (Pdc) is a conserved phosphoprotein that, when unphosphorylated, binds with high affinity to the complex of βγ-subunits of G protein transducin (Gtβγ). The ability of Pdc to bind to Gtβγ is inhibited through its phosphorylation at S54 and S73 within the N-terminal domain (Pdc-ND) followed by association with the scaffolding protein 14-3-3. However, the molecular basis for the 14-3-3-dependent inhibition of Pdc binding to Gtβγ is unclear. By using small-angle x-ray scattering, high-resolution NMR spectroscopy, and limited proteolysis coupled with mass spectrometry, we show that phosphorylated Pdc and 14-3-3 form a complex in which the Pdc-ND region 45-80, which forms a part of Pdc's Gtβγ binding surface and contains both phosphorylation sites, is restrained within the central channel of the 14-3-3 dimer, with both 14-3-3 binding motifs simultaneously participating in protein association. The N-terminal part of Pdc-ND is likely located outside the central channel of the 14-3-3 dimer, but Pdc residues 20-30, which are also involved in Gtβγ binding, are positioned close to the surface of the 14-3-3 dimer. The C-terminal domain of Pdc is located outside the central channel and its structure is unaffected by the complex formation. These results indicate that the 14-3-3 protein-mediated inhibition of Pdc binding to Gtβγ is based on steric occlusion of Pdc's Gtβγ binding surface.

• MeSH
• difrakce rentgenového záření MeSH
• fosfoproteiny antagonisté a inhibitory   chemie   MeSH
• fosforylace MeSH
• krysa rodu Rattus MeSH
• maloúhlový rozptyl MeSH
• oční proteiny antagonisté a inhibitory   chemie   MeSH
• proteinové domény MeSH
• proteiny 14-3-3 chemie   metabolismus   MeSH
• proteiny vázající GTP - regulátory antagonisté a inhibitory   chemie   MeSH
• proteolýza MeSH
• protonová magnetická rezonanční spektroskopie MeSH
• sekundární struktura proteinů MeSH
• vazba proteinů MeSH
• vztahy mezi strukturou a aktivitou MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• fosfoproteiny MeSH
• oční proteiny MeSH
• phosducin MeSH Prohlížeč
• proteiny 14-3-3 MeSH
• proteiny vázající GTP - regulátory MeSH

Phosducin (Pdc), a highly conserved phosphoprotein involved in the regulation of retinal phototransduction cascade, transcriptional control, and modulation of blood pressure, is controlled in a phosphorylation-dependent manner, including the binding to the 14-3-3 protein. However, the molecular mechanism of this regulation is largely unknown. Here, the solution structure of Pdc and its interaction with the 14-3-3 protein were investigated using small angle x-ray scattering, time-resolved fluorescence spectroscopy, and hydrogen-deuterium exchange coupled to mass spectrometry. The 14-3-3 protein dimer interacts with Pdc using surfaces both inside and outside its central channel. The N-terminal domain of Pdc, where both phosphorylation sites and the 14-3-3-binding motifs are located, is an intrinsically disordered protein that reduces its flexibility in several regions without undergoing dramatic disorder-to-order transition upon binding to 14-3-3. Our data also indicate that the C-terminal domain of Pdc interacts with the outside surface of the 14-3-3 dimer through the region involved in Gtβγ binding. In conclusion, we show that the 14-3-3 protein interacts with and sterically occludes both the N- and C-terminal Gtβγ binding interfaces of phosphorylated Pdc, thus providing a mechanistic explanation for the 14-3-3-dependent inhibition of Pdc function.

• Klíčová slova
• 14-3-3 protein, fluorescence, hydrogen-deuterium exchange, phosducin, protein complex, protein phosphorylation, small-angle x-ray scattering (SAXS),
• MeSH
• fosfoproteiny chemie   genetika   metabolismus   MeSH
• fosforylace MeSH
• krysa rodu Rattus MeSH
• lidé MeSH
• molekulární modely MeSH
• oční proteiny chemie   genetika   metabolismus   MeSH
• proteiny 14-3-3 chemie   genetika   metabolismus   MeSH
• proteiny vázající GTP - regulátory chemie   genetika   metabolismus   MeSH
• sekvence aminokyselin MeSH
• terciární struktura proteinů MeSH
• vazba proteinů MeSH
• vazebná místa MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• fosfoproteiny MeSH
• oční proteiny MeSH
• phosducin MeSH Prohlížeč
• proteiny 14-3-3 MeSH
• proteiny vázající GTP - regulátory MeSH
• YWHAZ protein, human MeSH Prohlížeč

Protein-protein interaction was investigated using a protein nanoprobe capable of photo-initiated cross-linking in combination with high-resolution and tandem mass spectrometry. This emerging experimental approach introduces photo-analogs of amino acids within a protein sequence during its recombinant expression, preserves native protein structure and is suitable for mapping the contact between two proteins. The contact surface regions involved in the well-characterized interaction between two molecules of human 14-3-3ζ regulatory protein were used as a model. The employed photo-initiated cross-linking techniques extend the number of residues shown to be within interaction distance in the contact surface of the 14-3-3ζ dimer (Gln8-Met78). The results of this study are in agreement with our previously published data from molecular dynamic calculations based on high-resolution chemical cross-linking data and Hydrogen/Deuterium exchange mass spectrometry. The observed contact is also in accord with the 14-3-3ζ X-ray crystal structure (PDB 3dhr). The results of the present work are relevant to the structural biology of transient interaction in the 14-3-3ζ protein, and demonstrate the ability of the chosen methodology (the combination of photo-initiated cross-linking protein nanoprobes and mass spectrometry analysis) to map the protein-protein interface or regions with a flexible structure.

• MeSH
• fotochemické procesy MeSH
• lidé MeSH
• mapování interakce mezi proteiny metody   MeSH
• molekulární modely MeSH
• multimerizace proteinu MeSH
• proteiny 14-3-3 chemie   metabolismus   MeSH
• sekvence aminokyselin MeSH
• spektrometrie hmotnostní - ionizace laserem za účasti matrice metody   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• proteiny 14-3-3 MeSH