Protein radical labeling, like fast photochemical oxidation of proteins (FPOP), coupled to a top-down mass spectrometry (MS) analysis offers an alternative analytical method for probing protein structure or protein interaction with other biomolecules, for instance, proteins and DNA. However, with the increasing mass of studied analytes, the MS/MS spectra become complex and exhibit a low signal-to-noise ratio. Nevertheless, these difficulties may be overcome by protein isotope depletion. Thus, we aimed to use protein isotope depletion to analyze FPOP-oxidized samples by top-down MS analysis. For this purpose, we prepared isotopically natural (IN) and depleted (ID) forms of the FOXO4 DNA binding domain (FOXO4-DBD) and studied the protein-DNA interaction interface with double-stranded DNA, the insulin response element (IRE), after exposing the complex to hydroxyl radicals. As shown by comparing tandem mass spectra of natural and depleted proteins, the ID form increased the signal-to-noise ratio of useful fragment ions, thereby enhancing the sequence coverage by more than 19%. This improvement in the detection of fragment ions enabled us to detect 22 more oxidized residues in the ID samples than in the IN sample. Moreover, less common modifications were detected in the ID sample, including the formation of ketones and lysine carbonylation. Given the higher quality of ID top-down MSMS data set, these results provide more detailed information on the complex formation between transcription factors and DNA-response elements. Therefore, our study highlights the benefits of isotopic depletion for quantitative top-down proteomics. Data are available via ProteomeXchange with the identifier PXD044447.

• MeSH
• DNA MeSH
• Ions MeSH
• Isotopes MeSH
• Proteins * analysis   MeSH
• Tandem Mass Spectrometry * MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• DNA MeSH
• Ions MeSH
• Isotopes MeSH
• Proteins * 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.

• Keywords
• 14-3-3 proteins, adaptor protein, molecular mechanism, phosphorylation, protein-protein interactions, scaffolding,
• Publication type
• Journal Article MeSH
• Review MeSH

14-3-3 proteins are important dimeric scaffolds that regulate the function of hundreds of proteins in a phosphorylation-dependent manner. The SARS-CoV-2 nucleocapsid (N) protein forms a complex with human 14-3-3 proteins upon phosphorylation, which has also been described for other coronaviruses. Here, we report a high-resolution crystal structure of 14-3-3 bound to an N phosphopeptide bearing the phosphoserine 197 in the middle. The structure revealed two copies of the N phosphopeptide bound, each in the central binding groove of each 14-3-3 monomer. A complex network of hydrogen bonds and water bridges between the peptide and 14-3-3 was observed explaining the high affinity of the N protein for 14-3-3 proteins.

• MeSH
• COVID-19 MeSH
• Phosphopeptides chemistry   MeSH
• Phosphoproteins chemistry   MeSH
• Coronavirus Nucleocapsid Proteins * chemistry   MeSH
• Humans MeSH
• 14-3-3 Proteins * chemistry   MeSH
• SARS-CoV-2 * MeSH
• Protein Binding MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Phosphopeptides MeSH
• Phosphoproteins MeSH
• Coronavirus Nucleocapsid Proteins * MeSH
• nucleocapsid phosphoprotein, SARS-CoV-2 MeSH Browser
• 14-3-3 Proteins * MeSH

FOXO transcription factors regulate cellular homeostasis, longevity and response to stress. FOXO1 (also known as FKHR) is a key regulator of hepatic glucose production and lipid metabolism, and its specific inhibition may have beneficial effects on diabetic hyperglycemia by reducing hepatic glucose production. Moreover, all FOXO proteins are considered potential drug targets for drug resistance prevention in cancer therapy. However, the development of specific FOXO inhibitors requires a detailed understanding of structural differences between individual FOXO DNA-binding domains. The high-resolution structure of the DNA-binding domain of FOXO1 reported in this study and its comparison with structures of other FOXO proteins revealed differences in both their conformation and flexibility. These differences are encoded by variations in protein sequences and account for the distinct functions of FOXO proteins. In particular, the positions of the helices H1, H2 and H3, whose interface form the hydrophobic core of the Forkhead domain, and the interactions between hydrophobic residues located on the interface between the N-terminal segment, the H2-H3 loop, and the recognition helix H3 differ among apo FOXO1, FOXO3 and FOXO4 proteins. Therefore, the availability of apo structures of DNA-binding domains of all three major FOXO proteins will support the development of FOXO-type-specific inhibitors.

• Keywords
• DNA-binding domain, FOXO1, Forkhead domain, nuclear magnetic resonance, structure,
• MeSH
• Forkhead Box Protein O1 chemistry   genetics   metabolism   MeSH
• Forkhead Transcription Factors chemistry   genetics   metabolism   MeSH
• Hydrophobic and Hydrophilic Interactions MeSH
• Humans MeSH
• Magnetic Resonance Spectroscopy MeSH
• Models, Molecular MeSH
• Mice MeSH
• Forkhead Box Protein O3 chemistry   genetics   metabolism   MeSH
• Protein Domains MeSH
• Protein Structure, Secondary MeSH
• Sequence Analysis, Protein MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Forkhead Box Protein O1 MeSH
• Forkhead Transcription Factors MeSH
• Forkhead Box Protein O3 MeSH

Phosphatidylinositol 4-kinase IIIβ (PI4KB) is a key enzyme of the Golgi system because it produces its lipid hallmark - the phosphatidylinositol 4-phosphate (PI4P). It is recruited to Golgi by the Golgi resident ACBD3 protein, regulated by 14-3-3 proteins and it also serves as an adaptor because it recruits the small GTPase Rab11. Here, we analyzed the protein complexes formed by PI4KB in vitro using small angle x-ray scattering (SAXS) and we discovered that these protein complexes are highly flexible. The 14-3-3:PI4KB:Rab11 protein complex has 2:1:1 stoichiometry and its different conformations are rather compact, however, the ACBD3:PI4KB protein complex has both, very compact and very extended conformations. Furthermore, in vitro reconstitution revealed that the membrane is necessary for the formation of ACBD3:PI4KB:Rab11 protein complex at physiological (nanomolar) concentrations.

• MeSH
• Adaptor Proteins, Signal Transducing metabolism   MeSH
• Phosphotransferases (Alcohol Group Acceptor) metabolism   MeSH
• Intracellular Membranes metabolism   MeSH
• Scattering, Small Angle MeSH
• Membrane Proteins metabolism   MeSH
• Protein Multimerization * MeSH
• 14-3-3 Proteins metabolism   MeSH
• rab GTP-Binding Proteins metabolism   MeSH
• Recombinant Proteins metabolism   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• ACBD3 protein, human MeSH Browser
• Adaptor Proteins, Signal Transducing MeSH
• Phosphotransferases (Alcohol Group Acceptor) MeSH
• Membrane Proteins MeSH
• phosphatidylinositol 4-kinase IIIbeta, human MeSH Browser
• 14-3-3 Proteins MeSH
• rab GTP-Binding Proteins MeSH
• rab11 protein MeSH Browser
• Recombinant Proteins MeSH

14-3-3 proteins bind phosphorylated binding partners to regulate several of their properties, including enzymatic activity, stability and subcellular localization. Here, two crystal structures are presented: the crystal structures of the 14-3-3 protein (also known as Bmh1) from the yeast Lachancea thermotolerans in the unliganded form and bound to a phosphopeptide derived from human PI4KB (phosphatidylinositol 4-kinase B). The structures demonstrate the high evolutionary conservation of ligand recognition by 14-3-3 proteins. The structural analysis suggests that ligand recognition by 14-3-3 proteins evolved very early in the evolution of eukaryotes and remained conserved, underlying the importance of 14-3-3 proteins in physiology.

• Keywords
• 14-3-3 proteins, Bmh1, Bmh2, Lachancea thermotolerans, PI4KB, crystal structure, phosphopeptide,
• MeSH
• 1-Phosphatidylinositol 4-Kinase chemistry   genetics   metabolism   MeSH
• Escherichia coli genetics   metabolism   MeSH
• Gene Expression MeSH
• Phosphoproteins chemistry   genetics   metabolism   MeSH
• Fungal Proteins chemistry   genetics   metabolism   MeSH
• Cloning, Molecular MeSH
• Protein Conformation, alpha-Helical MeSH
• Conserved Sequence MeSH
• Crystallography, X-Ray MeSH
• Humans MeSH
• Ligands MeSH
• Evolution, Molecular MeSH
• Models, Molecular MeSH
• Plasmids chemistry   metabolism   MeSH
• Protein Isoforms chemistry   genetics   metabolism   MeSH
• 14-3-3 Proteins chemistry   genetics   metabolism   MeSH
• Recombinant Proteins chemistry   genetics   metabolism   MeSH
• Saccharomycetales chemistry   metabolism   MeSH
• Amino Acid Sequence MeSH
• Sequence Alignment MeSH
• Structural Homology, Protein MeSH
• Protein Binding MeSH
• Binding Sites MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• 1-Phosphatidylinositol 4-Kinase MeSH
• Phosphoproteins MeSH
• Fungal Proteins MeSH
• Ligands MeSH
• Protein Isoforms MeSH
• 14-3-3 Proteins MeSH
• Recombinant Proteins MeSH

Phosducin (Pdc), a highly conserved phosphoprotein, plays an important role in the regulation of G protein signaling, transcriptional control, and modulation of blood pressure. Pdc is negatively regulated by phosphorylation followed by binding to the 14-3-3 protein, whose role is still unclear. To gain insight into the role of 14-3-3 in the regulation of Pdc function, we studied structural changes of Pdc induced by phosphorylation and 14-3-3 protein binding using time-resolved fluorescence spectroscopy. Our data show that the phosphorylation of the N-terminal domain of Pdc at Ser-54 and Ser-73 affects the structure of the whole Pdc molecule. Complex formation with 14-3-3 reduces the flexibility of both the N- and C-terminal domains of phosphorylated Pdc, as determined by time-resolved tryptophan and dansyl fluorescence. Therefore, our data suggest that phosphorylated Pdc undergoes a conformational change when binding to 14-3-3. These changes involve the G(t)βγ binding surface within the N-terminal domain of Pdc, and thus could explain the inhibitory effect of 14-3-3 on Pdc function.

• MeSH
• Spectrometry, Fluorescence MeSH
• Phosphatidylcholines MeSH
• Phosphoproteins chemistry   metabolism   MeSH
• Phosphorylation MeSH
• Rats MeSH
• Humans MeSH
• Molecular Sequence Data MeSH
• Eye Proteins chemistry   metabolism   MeSH
• 14-3-3 Proteins metabolism   MeSH
• GTP-Binding Protein Regulators chemistry   metabolism   MeSH
• Amino Acid Sequence MeSH
• Serine metabolism   MeSH
• Protein Structure, Tertiary MeSH
• Tryptophan MeSH
• Protein Binding MeSH
• Animals MeSH
• Check Tag
• Rats MeSH
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• 1-myristoyl-2-(12-((5-dimethylamino-1-naphthalenesulfonyl)amino)dodecanoyl)-sn-glycero-3-phosphocholine MeSH Browser
• Phosphatidylcholines MeSH
• Phosphoproteins MeSH
• Eye Proteins MeSH
• phosducin MeSH Browser
• 14-3-3 Proteins MeSH
• GTP-Binding Protein Regulators MeSH
• Serine MeSH
• Tryptophan MeSH
• YWHAZ protein, human MeSH Browser

Regulator of G protein signaling (RGS) proteins function as GTPase-activating proteins for the α-subunit of heterotrimeric G proteins. The function of certain RGS proteins is negatively regulated by 14-3-3 proteins, a family of highly conserved regulatory molecules expressed in all eukaryotes. In this study, we provide a structural mechanism for 14-3-3-dependent inhibition of RGS3-Gα interaction. We have used small angle x-ray scattering, hydrogen/deuterium exchange kinetics, and Förster resonance energy transfer measurements to determine the low-resolution solution structure of the 14-3-3ζ·RGS3 complex. The structure shows the RGS domain of RGS3 bound to the 14-3-3ζ dimer in an as-yet-unrecognized manner interacting with less conserved regions on the outer surface of the 14-3-3 dimer outside its central channel. Our results suggest that the 14-3-3 protein binding affects the structure of the Gα interaction portion of RGS3 as well as sterically blocks the interaction between the RGS domain and the Gα subunit of heterotrimeric G proteins.

• MeSH
• Circular Dichroism MeSH
• Phosphorylation MeSH
• Mass Spectrometry MeSH
• Humans MeSH
• Scattering, Small Angle MeSH
• 14-3-3 Proteins chemistry   genetics   metabolism   MeSH
• GTPase-Activating Proteins chemistry   genetics   metabolism   MeSH
• RGS Proteins MeSH
• GTP-Binding Proteins chemistry   genetics   metabolism   MeSH
• Fluorescence Resonance Energy Transfer MeSH
• Protein Structure, Secondary MeSH
• Signal Transduction MeSH
• Protein Structure, Tertiary MeSH
• Protein Binding MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• 14-3-3 Proteins MeSH
• GTPase-Activating Proteins MeSH
• RGS Proteins MeSH
• GTP-Binding Proteins MeSH
• RGS3 protein, human MeSH Browser