Replication of many viruses depends on phosphorylation of viral proteins by host protein kinases and subsequent recruitment of host protein partners. The nucleoprotein (N) of SARS-CoV-2 is heavily phosphorylated and recruits human phosphopeptide-binding 14-3-3 proteins early in infection, which is reversed prior to nucleocapsid assembly in new virions. Among the multiple phosphosites of N, which are particularly dense in the serine/arginine-rich interdomain region, phospho-Thr205 is highly relevant for 14-3-3 recruitment by SARS-CoV-2 N. The context of this site is mutated in most SARS-CoV-2 variants of concern. Among mutations that increase infectious virus titers, the S202R mutation (B.1.526 Iota) causes a striking replication boost (∼166-fold), although its molecular consequences have remained unclear. Here, we show that the S202R-mutated N phosphopeptide exhibits a 5-fold higher affinity for human 14-3-3ζ than the Wuhan variant and we rationalize this effect by solving a high-resolution crystal structure of the complex. The structure revealed an enhanced 14-3-3/N interface contributed by the Arg202 side chain that, in contrast to Ser202, formed multiple stabilizing contacts with 14-3-3, including water-mediated H-bonds and guanidinium pi-pi stacking. These findings provide a compelling link between the replicative fitness of SARS-CoV-2 and the N protein's affinity for host 14-3-3 proteins.

Macromolecular protein complexes carry out most cellular functions. Unfortunately, we lack the subunit composition for many human protein complexes. To address this gap we integrated >25,000 mass spectrometry experiments using a machine learning approach to identify >15,000 human protein complexes. We show our map of protein complexes is highly accurate and more comprehensive than previous maps, placing nearly 70% of human proteins into their physical contexts. We globally characterize our complexes using mass spectrometry based protein covariation data (ProteomeHD.2) and identify covarying complexes suggesting common functional associations. hu.MAP3.0 generates testable functional hypotheses for 472 uncharacterized proteins which we support using AlphaFold modeling. Additionally, we use AlphaFold modeling to identify 5871 mutually exclusive proteins in hu.MAP3.0 complexes suggesting complexes serve different functional roles depending on their subunit composition. We identify expression as the primary way cells and organisms relieve the conflict of mutually exclusive subunits. Finally, we import our complexes to EMBL-EBI's Complex Portal ( https://www.ebi.ac.uk/complexportal/home ) and provide complexes through our hu.MAP3.0 web interface ( https://humap3.proteincomplexes.org/ ). We expect our resource to be highly impactful to the broader research community.

Polo Like Kinase 1 (PLK1), a key regulator of mitosis whose overexpression is often associated with poor survival rates in cancer, continues to be widely investigated as an oncology drug target with clinical trials evaluating second and third generation inhibitors. In addition to the conserved N-terminal kinase domain (KD), a unique characteristic of the Polo-Like kinase family is the C-terminal polo-box domain (PBD). The PBD contains a phosphopeptide binding site that recognizes substrates primed by other kinases and furthermore is responsible for subcellular localization of PLK1 to specific sites in the nucleus including centrosomes and kinetochores. Another role of the PBD is its regulatory ability through domain-domain interactions with the KD to maintain an autoinhibited state of PLK1. Insights into post translational modifications and the PBD - KD domain-domain association have been obtained and show that key events in PLK1 regulation include phosphosubstrate binding, T210 phosphorylation and engagement with the Bora protein. These can induce an open and active conformation where the domain-domain inhibitory interactions no longer dominate. Further regulatory events recently described include the interchange between monomeric and dimeric forms, which can also serve to inhibit or activate PLK1 during the cell cycle. Different oligomeric forms of PLK1, existing as homodimers and heterodimers with PLK2, have been identified and likely play context dependent roles. This review provides an overview of recent information describing structural and mechanistic insights into inhibition of PLK1 and the temporal and spatial requirements of its activation and regulation. It also covers recent insights into the conformational regulation of other members of the Polo-Like kinase family. The implications of the conformational regulation of PLK1 with respect to cell cycle function and drug discovery are significant and are therefore discussed in detail.

14-3-3 proteins are highly conserved proteins that regulate numerous cellular processes mostly through phosphorylation-dependent protein-protein interactions. In the heart 14-3-3 proteins play critical roles in cardiac conduction pathways, excitation-contraction (EC) coupling, development and stress responses. This review summarizes the current understanding of cardiac 14-3-3 regulation and function, with particular emphasis on its role in ion channel regulation and β-adrenergic signalling. We discuss how 14-3-3 proteins act through three main mechanisms - masking, clamping, and scaffolding - to regulate target proteins, including Cx43, CaV1.2, NaV1.5, and various potassium channels. The seven mammalian 14-3-3 isoforms display distinct but overlapping functions, with tissue-specific expression patterns and isoform-specific regulation through phosphorylation and dimerization. Recent work has revealed 14-3-3's importance in cardiac development and stress responses, where it generally serves a cardioprotective role. However in some pathological contexts such as ischaemia-reperfusion injury, 14-3-3 can be detrimental. We highlight emerging themes in cardiac 14-3-3 biology, including its role in prolonging β-adrenergic signalling. Understanding the complex regulation of cardiac 14-3-3 and its numerous targets presents both opportunities and challenges for therapeutic development.

The main role of dimeric 14-3-3 proteins is to modulate the activity of several hundred binding partners by interacting with phosphorylated residues of the partner proteins, often located in disordered regions. The inherent flexibility or large size of 14-3-3 complexes hampers their structural characterization by X-ray crystallography, cryo-electron microscopy (EM) and traditional solution nuclear magnetic resonance (NMR) spectroscopy. Here, we employ solution 1D 19F-Trp NMR spectroscopy to characterize substrate binding and dimerization of 14-3-3 proteins, focusing on 14-3-3ζ - an abundant human isoform as an example. Both conserved Trp residues are located in distinct functionally important sites - the dimeric interface and the ligand-binding groove. We substituted them by 5F-Trp, thereby introducing a convenient NMR probe. Fluorination of the two Trp did not impact the stability and interaction properties of 14-3-3ζ in a substantive manner, permitting to carry out 19F NMR experiments to assess 14-3-3's structure and behavior. Importantly, 5F-Trp228 reports on binding of substrates in the amphipathic binding groove of 14-3-3ζ and permitted to distinguish distinct recognition modes. Thus, we established that 19F NMR is a powerful approach to evaluate the binding of partner proteins to 14-3-3 and to characterize the properties of the resulting complexes.

The accurate description and subsequent modeling of protein interactomes require quantification of their affinities at the proteome-wide scale. Here we develop and validate the Holdup Multiplex, a versatile assay with a mass spectrometry (MS) readout for profiling the affinities of a protein for large pools of peptides. The method can precisely quantify, in one single run, thousands of affinity constants over several orders of magnitude. The throughput, dynamic range, and sensitivity can be pushed to the performance limit of the MS readout. We applied the Holdup Multiplex to quantify in a few sample runs the affinities of the 14-3-3s, phosphoreader proteins highly abundant in humans, for 1000 different phosphopeptides. The seven human 14-3-3 isoforms were found to display similar specificities but staggered affinities, with 14-3-3γ being always the best binder and 14-3-3ε and σ being the weakest. Hundreds of new 14-3-3 binding sites were identified. We also identified dozens of 14-3-3 binding sites, some intervening in key signaling pathways, that were either stabilized or destabilized by the phytotoxin Fusicoccin-A. The results were corroborated by X-ray crystallography. Finally, we demonstrated the transferability of the Holdup Multiplex by quantifying the interactions of a PDZ domain for 5400 PBM peptides at once. The approach is applicable to any category of protein-binding ligands that can be quantifiable by mass spectrometry.

Molecular glues are small molecules that nucleate novel or stabilize natural, protein-protein interactions resulting in a ternary complex. Their success in targeting difficult to drug proteins of interest has led to ever-increasing interest in their use as therapeutics and research tools. While molecular glues and their targets vary in structure, inspection of diverse ternary complexes reveals commonalities. Whether of high or low molecular weight, molecular glues are often rigid and form direct hydrophobic interactions with their target protein. There is growing evidence that these hotspots can accommodate multiple ternary complex binding modes and enable targeting of traditionally undruggable targets. Advances in screening from the molecular glue degrader literature and insights in structure-based drug design, especially from the non-degrading tri-complex work, are likely intersectional.

Interferons (IFNs, types I-III) have pleiotropic functions in promoting antiviral and antitumor responses, as well as in modulating inflammation. Dissecting the signaling mechanisms elicited by different IFNs is therefore critical to understand their phenotypes. Here, we use mass spectrometry to investigate the early temporal dynamics of cellular protein phosphorylation in a human lung epithelial cell-line as it responds to stimulation with IFNα2, IFNβ, IFNω, IFNγ, or IFNλ1, representing all IFN types. We report an atlas of over 700 common or unique phosphorylation events reprogrammed by these different IFNs, revealing both previously known and uncharacterized modifications. While the proteins differentially phosphorylated following IFN stimulation have diverse roles, there is an enrichment of factors involved in chromatin remodeling, transcription, and RNA splicing. Functional screening and mechanistic studies identify that several proteins modified in response to IFNs contribute to host antiviral responses, either directly or by supporting IFN-stimulated gene or protein production. Among these, phosphorylation of PLEKHG3 at serine-1081 creates a phospho-regulated binding motif for the docking of 14-3-3 proteins, and together these factors contribute to coordinating efficient IFN-stimulated gene expression independent of early Janus kinase/signal transducer and activator of transcription signaling. Our findings map the global phosphorylation landscapes regulated by IFN types I, II, and III, and provide a key resource to explore their functional consequences.

Hub proteins interact with a host of client proteins and regulate multiple cellular functions. Dynamic hubs have a single binding interface for one client at a time resulting in competition among clients with the highest affinity. Dynamic dimeric hubs with two identical sites bind either two different client proteins or two chains of the same client to form homogenous complexes and could also form heterogeneous mixtures of interconverting complexes. Here, we review the interactions of the dimeric hubs 14-3-3 and LC8. 14-3-3 is a phosphoserine/threonine binding protein involved in structuring client proteins and regulating their phosphorylation. LC8 is involved in promoting the dimerization of client peptides and the rigidification of their disordered regions. Both 14-3-3 and LC8 are essential genes, with 14-3-3 playing a crucial role in apoptosis and cell cycle regulation, while LC8 is critical for the assembly of proteins involved in transport, DNA repair, and transcription. Interestingly, both protein dimers can dissociate by phosphorylation, which results in their interactome-wide changes. Their interactions are also regulated by the phosphorylation of their clients. Both form heterogeneous complexes with various functions including phase separation, signaling, and viral hijacking where they restrict the conformational heterogeneity of their dimeric clients that bind nucleic acids. This comparative analysis highlights the importance of dynamic protein-protein interactions in the diversity of functions of 14-3-3 and LC8 and how small differences in structures of interfaces explain why 14-3-3 is primarily involved in the regulation of phosphorylation states while LC8 is primarily involved in the regulation of assembly of large dynamic complexes.

Microtubule associated protein 2 (MAP2) interacts with the regulatory protein 14-3-3ζ in a cAMP-dependent protein kinase (PKA) phosphorylation dependent manner. Using selective phosphorylation, calorimetry, nuclear magnetic resonance, chemical crosslinking, and X-ray crystallography, we characterized interactions of 14-3-3ζ with various binding regions of MAP2c. Although PKA phosphorylation increases the affinity of MAP2c for 14-3-3ζ in the proline rich region and C-terminal domain, unphosphorylated MAP2c also binds the dimeric 14-3-3ζ via its microtubule binding domain and variable central domain. Monomerization of 14-3-3ζ leads to the loss of affinity for the unphosphorylated residues. In neuroblastoma cell extract, MAP2c is heavily phosphorylated by PKA and the proline kinase ERK2. Although 14-3-3ζ dimer or monomer do not interact with the residues phosphorylated by ERK2, ERK2 phosphorylation of MAP2c in the C-terminal domain reduces the binding of MAP2c to both oligomeric variants of 14-3-3ζ.

The 14-3-3 proteins are a highly conserved family of regulatory molecules that play crucial roles in various cellular processes. They are known for their ability to bind to phosphorylated serine and threonine residues on target proteins, which allows them to modulate their activity, localization, and stability. In mammals, there are seven known paralogs of 14-3-3 proteins, designated as β, ε, ζ, η, σ, τ, and γ. Each paralog has distinct biological functions and tissue distributions, which allow a diverse range of regulatory roles in cellular processes. The conformational plasticity of 14-3-3s regulates their interaction with protein partners but has not yet been thoroughly characterized. We investigated this topic by classical molecular dynamics simulations and observed how the γ, ε, and ζ paralogs exhibit different opening rates. A PCA analysis identified the main modes of these opening-conformational variations. Using correlation-based tools and simulations with single amino acid substitutions, we have recognized how the amphipathic 14-3-3 groove opening is triggered by a distally located aliphatic-π interaction. The identified residues form a partially conserved small cavity between helices H6, H7, and H8, representing a potential paralog-specific drug site.

The 14-3-3 proteins are highly conserved regulatory eukaryotic proteins, which are crucial in growth, development, and stress responses. However, systematic characterization of the 14-3-3 gene family in Brassicaceae species and their evolutionary relationships have not been comprehensively reported.

This study conducted genome-wide identification, structural characteristics, and comparative evolutionary analysis of 14-3-3 gene family members in Arabidopsis thaliana, A. lyrata, A. pumila, Camelina sativa, and Brassica oleracea using comparative genomics. Overall, a total of 108 14-3-3 genes, which were phylogenetically classified into ε and non-ε groups were identified in the five species, with the non-ε members exhibiting more similar exon-intron structures and conserved motif patterns. Collinearity analysis revealed that the Brassicaceae 14-3-3 gene family members underwent varying degrees of expansion following whole-genome duplication (WGD) events. Notably, the number of 14-3-3 gene family members between A. lyrata and A. thaliana remained similar despite the former having approximately 1.66-fold larger genome size. In contrast, the number of 14-3-3 gene family members in A. pumila and C. sativa increased in proportionately to their genome size, while gene members in the more distantly related species to A. thaliana, B. oleracea, showed irregular expansion patterns. Selection pressure analysis revealed that 14-3-3 homologs in all the five species underwent purifying selection, with the group ε members experiencing relatively weaker purifying selection. Cloning of ApGRF6-2 gene from A. pumila indicated that the ApGRF6-2 protein was localized in the cell membrane and cytoplasm, while ectopic overexpression of ApGRF6-2 in A. thaliana could promote early flowering by upregulating the expression of floral meristem identity genes.

This study provides a comprehensive and systematic identification of the 14-3-3 gene family members in five Brassicaceae species using updated genome sequences, and the results could form a basis for further validation of functional and molecular mechanisms of 14-3-3 genes in plant growth, development, abiotic stress responses, as well as flowering regulation.

Telomeres prevent ATM activation by sequestering chromosome termini within telomere loops (t-loops). Mitotic arrest promotes telomere linearity and a localized ATM-dependent telomere DNA damage response (DDR) through an unknown mechanism. Using unbiased interactomics, biochemical screening, molecular biology, and super-resolution imaging, we found that mitotic arrest-dependent (MAD) telomere deprotection requires the combined activities of the Chromosome passenger complex (CPC) on shelterin, and the BLM-TOP3A-RMI1/2 (BTR) complex on t-loops. During mitotic arrest, the CPC component Aurora Kinase B (AURKB) phosphorylated both the TRF1 hinge and TRF2 basic domains. Phosphorylation of the TRF1 hinge domain enhances CPC and TRF1 interaction through the CPC Survivin subunit. Meanwhile, phosphorylation of the TRF2 basic domain promotes telomere linearity, activates a telomere DDR dependent on BTR-mediated double Holliday junction dissolution, and leads to mitotic death. We identify that the TRF2 basic domain functions in mitosis-specific telomere protection and reveal a regulatory role for TRF1 in controlling a physiological ATM-dependent telomere DDR. The data demonstrate that MAD telomere deprotection is a sophisticated active mechanism that exposes telomere ends to signal mitotic stress.

Protein-protein interactions involving 14-3-3 proteins regulate various cellular activities in normal and pathological conditions. These interactions have mostly been reported to be phosphorylation-dependent, but the 14-3-3 proteins also interact with unphosphorylated proteins. In this work, we investigated whether phosphorylation is required, or, alternatively, whether negative charges are sufficient for 14-3-3ε binding. We substituted the pThr residue of pT(502-510) peptide by residues with a varying number of negative charges and investigated the binding of the peptides to 14-3-3ε using MD simulations and biophysical methods. We demonstrated that at least one negative charge is required for the peptides to bind 14-3-3ε, although phosphorylation is not necessary, and that two negative charges are preferable for high affinity binding. This discovery opens up new approaches for designing peptide-based 14-3-3 protein inhibitors.

Biomolecular condensates (BMCs) emerge as important players in RNA regulation. The RNA-binding protein Smaug forms cytosolic BMCs in mammals, insects and yeasts and affects mitochondrial function and/or responses to nutrient deprivation. Here, we found that the non-canonical activation of the Smoothened (SMO)-AMPK pathway, which is known to affect energy metabolism, triggers the immediate disassembly of BMCs formed by a number of human and rodent Smaug orthologs, whereas processing bodies remain rather unaltered. A non-phosphorylatable SMO mutant abrogated the effect, involving SMO phosphorylation in human (h)Smaug1 (also known as SAMD4A) BMCs regulation. Three mechanistically different SMO ligands, namely SAG, GSA-10 and cyclopamine, elicited a similar response, which was blocked upon AMPK pharmacological inhibition. Polysome disassembly by puromycin halted Smaug1 BMC dissolution, thus suggesting that unbound transcripts became translationally active. Single-molecule fluorescent in situ hybridization illustrated the release of UQCRC1 mRNA. Finally, Smaug1 is a phosphoprotein bound by 14-3-3 proteins, and the competitive inhibitor difopein blocked the response to non-canonical SMO stimulation. We propose that the regulated condensation and dispersion of Smaug1 BMCs generate translational changes that contribute to metabolic regulation downstream of the non-canonical SMO-AMPK axis.

Interaction of dimeric 14-3-3 proteins with phosphotargets regulates various physiological processes in plants, from flowering to transpiration and salt tolerance. Several genes express distinct 14-3-3 "isoforms," particularly numerous in plants, but these are unevenly studied even in model species. Here we systematically investigated twelve 14-3-3 isoforms from Arabidopsis thaliana. While all these proteins can homodimerize, four isoforms representing a supposedly more ancestral, epsilon phylogenetic group (iota, mu, omicron, epsilon), but not their eight non-epsilon counterparts (omega, phi, chi, psi, upsilon, nu, kappa, lambda), exhibit concentration-dependent monomerization, and pronounced surface hydrophobicity at physiologically relevant protein concentrations and under crowding conditions typical for the cell. We show that dramatically lowered thermodynamic stabilities entail aggregation of the epsilon group isoforms at near-physiological temperatures and accelerate their proteolytic degradation in vitro and in plant cell lysates. Mutations in 14-3-3 iota, inspired by structural analysis, helped us rescue non-epsilon behavior and pinpoint key positions responsible for the epsilon/non-epsilon demarcation. Combining two major demarcating positions (namely, 27th and 51st in omega) and differences in biochemical properties, we developed an epsilon/non-epsilon demarcation criterion that classified 89% of available 14-3-3 sequences from Dicots, Monocots, Gymnosperms, Ferns, and Lycophytes with 99.7% accuracy, and reliably predicted biochemical properties of a given 14-3-3 isoform, which we experimentally verified for distant 14-3-3 isoforms from Selaginella moellendorffii. The proven occurrence of isoforms of both groups in primitive plants refines the traditional phylogenetic, solely sequence-based analysis and provides intriguing insights into the evolutionary history of the epsilon phylogenetic group.

Prion diseases are fatal neurological disorders characterized by abnormal protein accumulation in the brain, leading to neurodegeneration, dementia, and ataxia. Sporadic Creutzfeldt-Jakob disease (sCJD), the most common form, accounts for 80-90% of cases and progresses rapidly, with most patients surviving <6 months to a year after symptom onset, indicating the importance of early diagnosis. The disease is classified into six subtypes based on PRNP gene polymorphisms, with differences in protein degradation patterns contributing to the diversity of clinical symptoms. However, diagnosis remains challenging because of the variability in clinical presentation and disease duration. Traditional diagnostic criteria established by the World Health Organization (WHO) rely on clinical findings, electroencephalogram, and cerebrospinal fluid tests, such as the 14-3-3 protein assay. However, these criteria require pathological confirmation, often delaying diagnosis. The recently proposed Hermann's criteria represent a significant advancement by incorporating newer biomarkers, including magnetic resonance imaging, real-time quaking-induced conversion assay, tau protein, and neurofilament light chain. These criteria improve diagnostic sensitivity and specificity but have a slightly higher risk of false positives. This review compares the effectiveness of these biomarkers with the WHO criteria and highlights the importance of early diagnosis for improving patient care.

Intrinsically disordered protein regions (IDRs) are structurally dynamic yet functional, often interacting with other proteins through short linear motifs (SLiMs). Proline residues in IDRs introduce conformational heterogeneity on a uniquely slow time scale arising from cis/trans isomerization of the Xaa-Pro peptide bond. Here, we explore the role of proline isomerization in the interaction between the prolactin receptor (PRLR) and 14-3-3. Using NMR spectroscopy, thermodynamic profiling, and molecular dynamics (MD) simulations, we uncover a unique proline isomer-dependent binding, with a cis conformation affinity 3 orders of magnitude higher than the trans. MD simulations identify structural constraints in the narrow 14-3-3 binding groove that provide an explanation for the observed isomer selectivity. The cis preference of WT PRLR introduces a slow kinetic component relevant to signal propagation and a steric component that impacts chain direction. Proline isomerization constitutes a previously unrecognized selective component relevant to the ubiquitous 14-3-3 interactome. Given the prevalence of prolines in IDRs and SLiMs, our study highlights the importance of considering the distinct properties of proline isomers in experimental design and data interpretation to fully comprehend IDR functionality.

14-3-3 proteins play a crucial role in the regulation of protein-protein interactions, impacting various cellular processes and disease mechanisms. Recent advancements have led to the development of stabilizers that enhance the binding of 14-3-3 proteins to clients, presenting promising therapeutic potentials. This perspective provides an updated overview of the latest developments in the field of 14-3-3 stabilizers, with a focus on their design, synthesis, and biological evaluation. We discuss the structural basis for the interaction between 14-3-3 proteins and their ligands, highlighting key modifications that enhance binding affinity and selectivity. Additionally, we explore the therapeutic applications of 14-3-3 stabilizers across major therapeutic areas such as cancer, metabolic disorders, and neurodegenerative diseases. By summarizing recent research findings and technological advancements, this perspective aims to shed light on the current state of 14-3-3 stabilizer developments and outline future directions for optimizing these compounds as effective therapeutic agents.

The L-type Ca2+ channel (CaV1.2) is essential for cardiac excitation-contraction coupling. To contribute to the inward Ca2+ flux that drives Ca2+-induced-Ca2+-release, CaV1.2 channels must be expressed on the sarcolemma; thus the regulatory mechanisms that tune CaV1.2 expression to meet contractile demand are an emerging area of research. A ubiquitously expressed protein called 14-3-3 has been proposed to affect Ca2+ channel trafficking in nonmyocytes; however, whether 14-3-3 has similar effects on CaV1.2 in cardiomyocytes is unknown. 14-3-3 preferentially binds phospho-serine/threonine residues to affect many cellular processes and is known to regulate cardiac ion channels including NaV1.5 and the human ether-à-go-go-related gene (hERG) potassium channel. Altered 14-3-3 expression and function have been implicated in cardiac pathologies including hypertrophy. Accordingly, we tested the hypothesis that 14-3-3 interacts with CaV1.2 in a phosphorylation-dependent manner and regulates cardiac CaV1.2 trafficking and recycling. Confocal imaging, proximity ligation assays, superresolution imaging, and coimmunoprecipitation revealed a population of 14-3-3 colocalized and closely associated with CaV1.2. The degree of 14-3-3/CaV1.2 colocalization increased upon stimulation of β-adrenergic receptors with isoproterenol. Notably, only the 14-3-3-associated CaV1.2 population displayed increased cluster size with isoproterenol, revealing a role for 14-3-3 as a nucleation factor that directs CaV1.2 superclustering. Isoproterenol-stimulated augmentation of sarcolemmal CaV1.2 expression, Ca2+ currents, and Ca2+ transients in ventricular myocytes were strengthened by 14-3-3 overexpression and attenuated by 14-3-3 inhibition. These data support a model where 14-3-3 interacts with CaV1.2 in a phosphorylation-dependent manner to promote enhanced trafficking/recycling, clustering, and activity during β-adrenergic stimulation.

14-3-3σ functions as an oncogene in colorectal cancer and is associated with therapy resistance. However, the mechanisms underlying these observations are not clear. The results in this report demonstrate that loss of 14-3-3σ in colorectal cancer cells leads to a decrease in tumor formation and increased sensitivity to chemotherapy. The increased sensitivity to chemotherapy is due to a decrease in the expression of UPR pathway genes in the absence of 14-3-3σ. 14-3-3σ promotes expression of the UPR pathway genes by binding to the transcription factor YY1 and preventing the nuclear localization of YY1. YY1, in the absence of 14-3-3σ, shows increased nuclear localization and binds to the promoter of the UPR pathway genes, resulting in decreased gene expression. Similarly, a YY1 mutant that cannot bind to 14-3-3σ also shows increased nuclear localization and is enriched on the promoter of the UPR pathway genes. Finally, inhibition of the UPR pathway with genetic or pharmacological approaches sensitizes colon cancer cells to chemotherapy. Our results identify a novel mechanism by which 14-3-3σ promotes tumor progression and therapy resistance in colorectal cancer by maintaining UPR gene expression.

Brassinosteroids (BRs) are vital plant steroid hormones sensed at the cell surface by a membrane signaling complex comprising the receptor kinase BRI1 and a SERK family co-receptor kinase. Activation of this complex lead to dissociation of the inhibitor protein BKI1 from the receptor and to differential phosphorylation of BZR1/BES1 transcription factors by the glycogen synthase kinase 3 protein BIN2. Many phosphoproteins of the BR signaling pathway, including BRI1, SERKs, BKI1 and BZR1/BES1 can associate with 14-3-3 proteins. In this study, we use quantitative ligand binding assays to define the minimal 14-3-3 binding sites in the N-terminal lobe of the BRI1 kinase domain, in BKI1, and in BZR1 from Arabidopsis thaliana. All three motifs require to be phosphorylated to specifically bind 14-3-3s with mid- to low-micromolar affinity. BR signaling components display minimal isoform preference within the 14-3-3 non-ε subgroup. 14-3-3λ and 14-3-3 ω isoform complex crystal structures reveal that BKI1 and BZR1 bind as canonical type II 14-3-3 linear motifs. Disruption of key amino acids in the phosphopeptide binding site through mutation impairs the interaction of 14-3-3λ with all three linear motifs. Notably, quadruple loss-of-function mutants from the non-ε group exhibit gain-of-function BR signaling phenotypes, suggesting a role for 14-3-3 proteins as overall negative regulators of the BR pathway. Collectively, our work provides further mechanistic and genetic evidence for the regulatory role of 14-3-3 proteins at various stages of the BR signaling cascade.

Protein phosphorylation is a crucial process in various cellular functions, and its irregularities have been implicated in several diseases, including cancer. Antibodies are commonly employed to detect protein phosphorylation in research. However, unlike the extensive studies on recognition mechanisms of the phosphate group by proteins such as kinases and phosphatases, only a few studies have explored antibody mechanisms. In this study, we produced and characterized two rabbit monoclonal antibodies that recognize a monophosphorylated Akt peptide. Through crystallography, thermodynamic mutational analyses, and molecular dynamics simulations, we investigated the unique recognition mechanism that enables higher binding affinity and selectivity of the antibodies compared to other generic proteins with lower binding affinity to phosphorylated epitopes. Our results demonstrate that molecular dynamics simulations provide novel insights into the dynamic aspects of molecular recognition of posttranslational modifications by proteins beyond static crystal structures, highlighting how specific atomic level interactions drive the exceptional affinity and selectivity of antibodies.

Protein-protein interactions involving 14-3-3 proteins regulate various cellular activities in normal and pathological conditions. These interactions have mostly been reported to be phosphorylation-dependent, but the 14-3-3 proteins also interact with unphosphorylated proteins. In this work, we investigated whether phosphorylation is required, or, alternatively, whether negative charges are sufficient for 14-3-3ε binding. We substituted the pThr residue of pT(502-510) peptide by residues with varying number of negative charges, and investigated binding of the peptides to 14-3-3ε using MD simulations and biophysical methods. We demonstrated that at least one negative charge is required for the peptides to bind 14-3-3ε while phosphorylation is not necessary, and that two negative charges are preferable for high affinity binding.

Here we describe labeling with barcodes and enrichment for biochemical analysis by sequencing (LABEL-seq), an assay for massively parallel profiling of pooled protein variants in human cells. By leveraging the intracellular self-assembly of an RNA-binding domain (RBD) with a stable, variant-encoding RNA barcode, LABEL-seq facilitates the direct measurement of protein properties and functions using simple affinity enrichments of RBD protein fusions, followed by high-throughput sequencing of co-enriched barcodes. Measurement of ~20,000 variant effects for ~1,600 BRaf variants revealed that variation at positions frequently mutated in cancer minimally impacted intracellular abundance but could dramatically alter activity, protein-protein interactions and druggability. Integrative analysis identified networks of positions with similar biochemical roles and enabled modeling of variant effects on cell proliferation and small molecule-promoted degradation. Thus, LABEL-seq enables direct measurement of multiple biochemical properties in a native cellular context, providing insights into protein function, disease mechanisms and druggability.

Phospholamban (PLN) is a sarco-endoplasmic reticulum (SER) membrane protein that regulates cardiac contraction/relaxation by reversibly inhibiting the SERCA2a Ca2+-reuptake pump. The R14Δ-PLN mutation causes severe cardiomyopathy that is resistant to conventional treatment. Protein complexes and higher-order supercomplexes such as intercalated disk components and Ca+2-cycling domains underlie many critical cardiac functions, a subset of which may be disrupted by R14Δ-PLN. Complexome profiling (CP) is a proteomics workflow for systematic analysis of high molecular weight (MW) protein complexes and supercomplexes. We hypothesize that R14Δ-PLN may alter a subset of these assemblies, and apply CP workflows to explore these changes in presymptomatic R14Δ/+ mice hearts. Ventricular tissues from presymptomatic 28wk-old WT and R14Δ/+ mice were homogenized under non-denaturing conditions, fractionated by size-exclusion chromatography (SEC) with a linear MW-range exceeding 5 MDa, and subjected to quantitative data-independent acquisition mass spectrometry (DIA-MS) analysis. Unfortunately, current workflows for the systematic analysis of CP data proved ill-suited for use in cardiac samples. Most rely upon curated protein complex databases to provide ground-truth for analysis; however, these are derived primarily from cancerous or immortalized cell lines and, consequently, cell-type specific complexes (including cardiac-specific machinery potentially affected in R14Δ-PLN hearts) are poorly covered. We thus developed PERCOM: a novel CP data-analysis strategy that does not rely upon these databases and can, furthermore, be implemented on widely available spreadsheet software. Applying PERCOM to our CP dataset resulted in the identification of 296 proteins with disrupted elution profiles. Hits were significantly enriched for mitochondrial and intercalated disk (ICD) supercomplex components. Changes to mitochondrial supercomplexes were associated with reduced expression of mitochondrial proteins and maximal oxygen consumption rate. The observed alterations to mitochondrial and ICD supercomplexes were replicated in a second cohort of "juvenile" 9wk-old mice. These early-stage changes to key cardiac machinery may contribute to R14Δ-PLN pathogenesis.

Macromolecular protein complexes carry out most functions in the cell including essential functions required for cell survival. Unfortunately, we lack the subunit composition for all human protein complexes. To address this gap we integrated >25,000 mass spectrometry experiments using a machine learning approach to identify > 15,000 human protein complexes. We show our map of protein complexes is highly accurate and more comprehensive than previous maps, placing ~75% of human proteins into their physical contexts. We globally characterize our complexes using protein co-variation data (ProteomeHD.2) and identify co-varying complexes suggesting common functional associations. Our map also generates testable functional hypotheses for 472 uncharacterized proteins which we support using AlphaFold modeling. Additionally, we use AlphaFold modeling to identify 511 mutually exclusive protein pairs in hu.MAP3.0 complexes suggesting complexes serve different functional roles depending on their subunit composition. We identify expression as the primary way cells and organisms relieve the conflict of mutually exclusive subunits. Finally, we import our complexes to EMBL-EBI's Complex Portal (https://www.ebi.ac.uk/complexportal/home) as well as provide complexes through our hu.MAP3.0 web interface (https://humap3.proteincomplexes.org/). We expect our resource to be highly impactful to the broader research community.

Bone and joint-related diseases, including osteoarthritis (OA), rheumatoid arthritis (RA), and bone tumors, pose significant health challenges due to their debilitating effects on the musculoskeletal system. 14-3-3 proteins, a family of conserved regulatory molecules, play a critical role in the pathology of these diseases. This review discusses the intricate structure and multifunctionality of 14-3-3 proteins, their regulation of signaling pathways, and their interactions with other proteins. We underscore the significance of 14-3-3 proteins in the regulation of osteoblasts, osteoclasts, chondrocytes, and bone remodeling, all key factors in the maintenance and dysfunction of bone and joint systems. Specific focus is directed toward elucidating the contribution of 14-3-3 proteins in the pathology of OA, RA, and bone malignancies, where dysregulated 14-3-3-mediated signaling cascades have been implicated in the disease processes. This review illuminates how the perturbation of 14-3-3 protein interactions can lead to the pathological manifestations observed in these disorders, including joint destruction and osteolytic activity. We highlight cutting-edge research that positions 14-3-3 proteins as potential biomarkers for disease progression and as innovative therapeutic targets, offering new avenues for disease intervention and management.

Fusicoccin-A (FC-A) is a diterpene glucoside produced by a pathogenic fungus. Since its discovery, FC-A has been widely recognized as a phytotoxin that induces stomatal opening and leaf wilting, eventually leading to plant death. In this study, we present the first evidence that FC-A enhances plant growth by stabilizing the protein-protein interaction between plasma membrane (PM) H+-ATPase and 14-3-3 in guard cells. Long-term treatment of Arabidopsis plants with FC-A resulted in ~ 30% growth enhancement. Structurally similar fusicoccin-J (FC-J) showed a similar degree of growth-promotion activity as FC-A, whereas the more hydrophilic fusicoccin-H (FC-H) exhibited no effect on plant growth, indicating that the enhancement of plant growth observed with FC-A and FC-J involves upregulation of the protein-protein interaction between PM H+-ATPase and 14-3-3 in guard cells, which promotes stomatal opening and photosynthesis.

Basic Helix-Loop-Helix (bHLH) transcription factors TFEB/TFE3 and HLH-30 are key regulators of autophagy induction and lysosomal biogenesis in mammals and C. elegans, respectively. While much is known about the regulation of TFEB/TFE3, how HLH-30 subcellular dynamics and transactivation are modulated are yet poorly understood. Thus, elucidating the regulation of C. elegans HLH-30 will provide evolutionary insight into the mechanisms governing the function of bHLH transcription factor family. We report here that HLH-30 is retained in the cytoplasm mainly through its conserved Ser201 residue and that HLH-30 physically interacts with the 14-3-3 protein FTT-2 in this location. The FoxO transcription factor DAF-16 is not required for HLH-30 nuclear translocation upon stress, despite that both proteins partner to form a complex that coordinately regulates several organismal responses. Similar as described for DAF-16, the importin IMB-2 assists HLH-30 nuclear translocation, but constitutive HLH-30 nuclear localization is not sufficient to trigger its distinctive transcriptional response. Furthermore, we identify FTT-2 as the target of diethyl maleate (DEM), a GSH depletor that causes a transient nuclear translocation of HLH-30. Together, our work demonstrates that the regulation of TFEB/TFE3 and HLH-30 family members is evolutionarily conserved and that, in addition to a direct redox regulation through its conserved single cysteine residue, HLH-30 can also be indirectly regulated by a redox-dependent mechanism, probably through FTT-2 oxidation.

Fusicoccin (FC) is one of the most studied fungal metabolites to date. The finding that the plasma membrane H+-ATPase in combination with 14-3-3 proteins acts as a high-affinity receptor for FC was a breakthrough in the field. Ever since, the binding of FC to the ATPase-14-3-3 receptor complex has taken center stage in explaining all FC-induced physiological effects. However, a more critical review shows that this is not evident for a number of FC-induced effects. This review challenges the notion that all FC-affected processes start with the binding to and activation of the plasma membrane ATPase, and raises the question of whether other proteins with a key role in the respective processes are directly targeted by FC. A second unresolved question is whether FC may be another example of a fungal molecule turning out to be a 'copy' of an as yet unknown plant molecule. In view of the evidence, albeit not conclusive, that plants indeed produce 'FC-like ligands', it is worthwhile making a renewed attempt with modern improved technology to answer this question; the answer might upgrade FC or its structural analogue(s) to the classification of plant hormone.

Enzyme activity is regulated by several mechanisms, including phosphorylation. Phosphorylation is a key signal transduction process in all eukaryotic cells and is thus crucial for virtually all cellular processes. In addition to its direct effect on protein structure, phosphorylation also affects protein-protein interactions, such as binding to scaffolding 14-3-3 proteins, which selectively recognize phosphorylated motifs. These interactions then modulate the catalytic activity, cellular localisation and interactions of phosphorylated enzymes through different mechanisms. The aim of this mini-review is to highlight several examples of 14-3-3 protein-dependent mechanisms of enzyme regulation previously studied in our laboratory over the past decade. More specifically, we address here the regulation of the human enzymes ubiquitin ligase Nedd4-2, procaspase-2, calcium-calmodulin dependent kinases CaMKK1/2, and death-associated protein kinase 2 (DAPK2) and yeast neutral trehalase Nth1.

The connection between aging and cancer is complex. Previous research has highlighted the association between the aging process of lung adenocarcinoma (LUAD) cells and the immune response, yet there remains a gap in confirming this through single-cell data validation. Here, we aim to develop a novel aging-related prognostic model for LUAD, and verify the alterations in the genome and immune microenvironment linked to cellular senescence.

We integrated a comprehensive collection of senescence genes from the GenAge and CellAge databases and employed the least absolute shrinkage and selection operator (LASSO) Cox analysis to construct and validate a novel prognostic model for LUAD. This model was then utilized to examine the relationship between aging, tumor somatic mutations, and immune cell infiltration. Additionally, we explored the heterogeneity of senescence and intercellular communication within the LUAD tumor microenvironment (TME) through single-cell transcriptomic data analysis.

By exploring the expression profiles of 586 cellular senescence-related genes in 428 LUAD patients, we constructed an aging-related genes (ARGs) risk model included 10 ARGs and validated it as an independent prognostic predictor for LUAD patients. Notably, patients with low aging scores (LAS group) exhibited better survival, lower tumor mutation burden (TMB), lower somatic mutation frequency, lower tumor proliferation rate, and an immune activated phenotype compared to patients with high aging scores (HAS group). While the HAS group was enriched in tumor cells and showed a lower infiltration of CD8-CCR7, CD8- CXCL13, CD8-GNLY, FCGR3A NK cells, XCL1 NK cells, plasma cell (PC) and other immune subsets. Furthermore, the SPP1 and TENASCIN pathways, associated with tumor immune escape and tumor progression, were also enriched in the HAS group. Additionally, our study also indicated that senescence levels were heterogeneous in the LUAD tumor microenvironment (TME), especially with tumor cells in the LAS group showing higher age scores compared to those in the HAS group.

Collectively, our findings underscore that ARRS through ARGs serves as a robust biomarker for the prognosis in LUAD.

The L-type Ca 2+ channel (Ca V 1.2) is essential for cardiac excitation-contraction coupling. To contribute to the inward Ca 2+ flux that drives Ca 2+ -induced-Ca 2+ -release, Ca V 1.2 channels must be expressed on the sarcolemma; thus the regulatory mechanisms that tune Ca V 1.2 expression to meet contractile demand are an emerging area of research. A ubiquitously expressed protein called 14-3-3 has been proposed to affect Ca 2+ channel trafficking in non-myocytes, however whether 14-3-3 has similar effects on Ca V 1.2 in cardiomyocytes is unknown. 14-3-3 preferentially binds phospho-serine/threonine residues to affect many cellular processes and is known to regulate cardiac ion channels including Na V 1.5 and hERG. Altered 14-3-3 expression and function have been implicated in cardiac pathologies including hypertrophy. Accordingly, we tested the hypothesis that 14-3-3 interacts with Ca V 1.2 in a phosphorylation-dependent manner and regulates cardiac Ca V 1.2 trafficking and recycling. Confocal imaging, proximity ligation assays, super-resolution imaging, and co-immunoprecipitation revealed a population of 14-3-3 colocalized and closely associated with Ca V 1.2. The degree of 14-3-3/Ca V 1.2 colocalization increased upon stimulation of β -adrenergic receptors with isoproterenol. Notably, only the 14-3-3-associated Ca V 1.2 population displayed increased cluster size with isoproterenol, revealing a role for 14-3-3 as a nucleation factor that directs Ca V 1.2 super-clustering. 14-3-3 overexpression increased basal Ca V 1.2 cluster size and Ca 2+ currents in ventricular myocytes, with maintained channel responsivity to isoproterenol. In contrast, isoproterenol-stimulated augmentation of sarcolemmal Ca V 1.2 expression and currents in ventricular myocytes were abrogated by 14-3-3 inhibition. These data support a model where 14-3-3 interacts with Ca V 1.2 in a phosphorylation-dependent manner to promote enhanced trafficking/recycling, clustering, and activity during β -adrenergic stimulation.

The L-type Ca 2+ channel, Ca V 1.2, plays an essential role in excitation-contraction coupling in the heart and in part regulates the overall strength of contraction during basal and fight- or-flight β -adrenergic signaling conditions. Proteins that modulate the trafficking and/or activity of Ca V 1.2 are interesting both from a physiological and pathological perspective, since alterations in Ca V 1.2 can impact action potential duration and cause arrythmias. A small protein called 14-3-3 regulates other ion channels in the heart and other Ca 2+ channels, but how it may interact with Ca V 1.2 in the heart has never been studied. Examining factors that affect Ca V 1.2 at rest and during β -adrenergic stimulation is crucial for our ability to understand and treat disease and aging conditions where these pathways are altered.

RAF family protein kinases are a key node in the RAS/RAF/MAP kinase pathway, the signaling cascade that controls cellular proliferation, differentiation, and survival in response to engagement of growth factor receptors on the cell surface. Over the past few years, structural and biochemical studies have provided new understanding of RAF autoregulation, RAF activation by RAS and the SHOC2 phosphatase complex, and RAF engagement with HSP90-CDC37 chaperone complexes. These studies have important implications for pharmacologic targeting of the pathway. They reveal RAF in distinct regulatory states and show that the functional RAF switch is an integrated complex of RAF with its substrate (MEK) and a 14-3-3 dimer. Here we review these advances, placing them in the context of decades of investigation of RAF regulation. We explore the insights they provide into aberrant activation of the pathway in cancer and RASopathies (developmental syndromes caused by germline mutations in components of the pathway).

Protein-protein interactions (PPIs) are of utmost importance for maintenance of cellular homeostasis. Herein, a central role can be found for 14-3-3 proteins. These hub-proteins are known to bind hundreds of interaction partners, thereby regulating their activity, localization, and/or stabilization. Due to their ability to bind a large variety of client proteins, studies of 14-3-3 protein complexes flourished over the last decades, aiming to gain greater molecular understanding of these complexes and their role in health and disease. Because of their crucial role within the cell, 14-3-3 protein complexes are recognized as highly interesting therapeutic targets, encouraging the discovery of small molecule modulators of these PPIs. We discuss various examples of 14-3-3-mediated regulation of its binding partners on a mechanistic level, highlighting the versatile and multi-functional role of 14-3-3 within the cell. Furthermore, an overview is given on the development of stabilizers of 14-3-3 protein complexes, from initially used natural products to fragment-based approaches. These studies show the potential of 14-3-3 PPI stabilizers as novel agents in drug discovery and as tool compounds to gain greater molecular understanding of the role of 14-3-3-based protein regulation.

While ultraviolet (UV) radiation damages DNA, eliciting the DNA damage response (DDR), it also damages RNA, triggering transcriptome-wide ribosomal collisions and eliciting a ribotoxic stress response (RSR). However, the relative contributions, timing, and regulation of these pathways in determining cell fate is unclear. Here we use time-resolved phosphoproteomic, chemical-genetic, single-cell imaging, and biochemical approaches to create a chronological atlas of signaling events activated in cells responding to UV damage. We discover that UV-induced apoptosis is mediated by the RSR kinase ZAK and not through the DDR. We identify two negative-feedback modules that regulate ZAK-mediated apoptosis: (1) GCN2 activation limits ribosomal collisions and attenuates ZAK-mediated RSR and (2) ZAK activity leads to phosphodegron autophosphorylation and its subsequent degradation. These events tune ZAK's activity to collision levels to establish regimes of homeostasis, tolerance, and death, revealing its key role as the cellular sentinel for nucleic acid damage.

Synthetic modulators of plant 14-3-3s are promising chemical tools both for understanding the 14-3-3-related signaling pathways and controlling plant physiology. Herein, we describe a novel small-molecule inhibitor for 14-3-3 proteins of Arabidopsis thaliana. The inhibitor was identified from unexpected products in a stock solution in dimethyl sulfoxide (DMSO) of an in-house chemical library. Mass spectroscopy, mutant-based analyses, fluorescence polarization assays, and thermal shift assays revealed that the inhibitor covalently binds to an allosteric site of 14-3-3 with isoform selectivity. Moreover, infiltration of the inhibitor to Arabidopsis leaves suppressed the stomatal aperture. The inhibitor should provide new insight into the design of potent and isoform-selective 14-3-3 modulators.

Severe corneal injury can lead to blindness even after prompt treatment. 14-3-3zeta, a member of an adaptor protein family, contributes to tissue repair by enhancing cellular viability and inhibiting fibrosis and inflammation in renal disease or arthritis. However, its role in corneal regeneration is less studied. In this study, filter disc of 2-mm diameter soaked in sodium hydroxide with a concentration of 0.5 N was placed at the center of the cornea for 30 s to establish a mouse model of corneal alkali injury. We found that 14-3-3zeta, which is mainly expressed in the epithelial layer, was upregulated following injury. Overexpression of 14-3-3zeta in ocular tissues via adeno-associated virus-mediated subconjunctival delivery promoted corneal wound healing, showing improved corneal structure and transparency. In vitro studies on human corneal epithelial cells showed that 14-3-3zeta was critical for cell proliferation and migration. mRNA-sequencing in conjunction with KEGG analysis and validation experiments revealed that 14-3-3zeta regulated the mRNA levels of ITGB1, PIK3R1, FGF5, PRKAA1 and the phosphorylation level of Akt, suggesting the involvement of the PI3K-Akt pathway in 14-3-3zeta-mediated tissue repair. 14-3-3zeta is a potential novel therapeutic candidate for treating severe corneal injury.