14-3-3ε augments OGT stability by binding with S20-phosphorylated OGT.

The relationship between O-linked N-acetylglucosamine (O-GlcNAc) transferase (OGT) and mitosis is intertwined. Besides the numerous mitotic OGT substrates that have been identified, OGT itself is also a target of the mitotic machinery. Previously, our investigations have shown that Checkpoint kinase 1 (Chk1) phosphorylates OGT at Ser-20 to increase OGT levels during cytokinesis, suggesting that OGT levels oscillate as mitosis progresses. Herein we studied its underlying mechanism. We set out from an R17C mutation of OGT, which is a uterine carcinoma mutation in The Cancer Genome Atlas. We found that R17C abolishes the S20 phosphorylation of OGT, as it lies in the Chk1 phosphorylating consensus motif. Consistent with our previous report that pSer-20 is essential for OGT level increases during cytokinesis, we further demonstrate that the R17C mutation renders OGT less stable, decreases vimentin phosphorylation levels and results in cytokinesis defects. Based on bioinformatic predictions, pSer-20 renders OGT more likely to interact with 14-3-3 proteins, the phospho-binding signal adaptor/scaffold protein family. By screening the seven isoforms of 14-3-3 family, we show that 14-3-3ε specifically associates with Ser-20-phosphorylated OGT. Moreover, we studied the R17C and S20A mutations in xenograft models and demonstrated that they both inhibit uterine carcinoma compared to wild-type OGT, probably due to less cellular reproduction. Our work is a sequel of our previous report on pS20 of OGT and is in line with the notion that OGT is intricately regulated by the mitotic network.

Positive 14-3-3 protein in cerebrospinal fluid followed by poppy-induced delayed post-hypoxic leukoencephalopathy: A case report.

Background: Delayed post-hypoxic leukoencephalopathy (DPHL) is characterized by a biphasic clinical course, with complete recovery from coma to a fully conscious state lasting one to four weeks (lucid interval), followed by abrupt neurological deterioration as an indirect consequence of hypoxic events like carbon monoxide poisoning and narcotic drug overdose. To our best knowledge, there are no documented cases in literature of choreoathetosis and dementia following poppy-induced DPHL with 14-3-3 protein in cerebrospinal fluid (CSF). Case presentation: We report the case of a 70-year-old female who underwent cardiopulmonary resuscitation (CPR) due to overdose of homemade refined opium poppy paste two weeks prior to presentation. She presented a progressive cognitive decline, along with the development of apraxia and choreic movement affecting her tongue and bilateral upper and lower extremities. During the symptomatic phase, brain magnetic resonance imaging (MRI) showed bilateral symmetrical hyperintense signals mostly in central frontal, temporal, and parieto-occipital lobes in the diffusion weighted imaging (DWI) and fluid-attenuated inversion recovery (FLAIR) sequences which are the characteristic findings of DPHL. CSF routine analysis, as well as toxicology screening, autoimmune and paraneoplastic encephalitis panels were negative, but the presence of 14-3-3 protein in the CSF was detected. With steroid therapy, hyperbaric oxygen therapy and symptomatic treatment, she experienced gradual improvement in cognition, motivation, and psychomotor function. Conclusion: DPHL represents a distinct form of encephalopathy characterized by unique clinical course and imaging features. It is the first report of DPHL with positive 14-3-3 protein in CSF. The potential of 14-3-3 protein as a biomarker for diagnosing DPHL and its ability to predict disease severity and prognosis warrants further research.

Unraveling the Protective Role of Oleocanthal and Its Oxidation Product, Oleocanthalic Acid, against Neuroinflammation.

Neuroinflammation is a critical aspect of various neurodegenerative diseases, such as Alzheimer's and Parkinson's diseases. This study investigates the anti-neuroinflammatory properties of oleocanthal and its oxidation product, oleocanthalic acid, using the BV-2 cell line activated with lipopolysaccharide. Our findings revealed that oleocanthal significantly inhibited the production of pro-inflammatory cytokines and reduced the expression of inflammatory genes, counteracted oxidative stress induced by lipopolysaccharide, and increased cell phagocytic activity. Conversely, oleocanthalic acid was not able to counteract lipopolysaccharide-induced activation. The docking analysis revealed a plausible interaction of oleocanthal, with both CD14 and MD-2 leading to a potential interference with TLR4 signaling. Since our data show that oleocanthal only partially reduces the lipopolysaccharide-induced activation of NF-kB, its action as a TLR4 antagonist alone cannot explain its remarkable effect against neuroinflammation. Proteomic analysis revealed that oleocanthal counteracts the LPS modulation of 31 proteins, including significant targets such as gelsolin, clathrin, ACOD1, and four different isoforms of 14-3-3 protein, indicating new potential molecular targets of the compound. In conclusion, oleocanthal, but not oleocanthalic acid, mitigates neuroinflammation through multiple mechanisms, highlighting a pleiotropic action that is particularly important in the context of neurodegeneration.

14-3-3σ restricts YY1 to the cytoplasm, promoting therapy resistance, and tumor progression in colorectal cancer.

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.

The clinicopathological significance and prognostic impact of 14-3-3σ/stratifin expression on patients with surgically resectable intrahepatic cholangiocarcinoma.

INTRODUCTION: Intrahepatic cholangiocarcinoma (iCCA) is the second most common primary liver cancer after hepatocellular carcinoma. Through data mining of publicly available iCCA transcriptomic datasets from the Gene Expression Omnibus, we identified SFN as the most significantly up-regulated gene in iCCA compared to normal tissue, focusing on the Gene Ontology term "cell proliferation" (GO:0008283). SFN encodes the 14-3-3σ protein, also known as stratifin, which plays crucial roles in various cellular processes. MATERIALS AND METHODS: Immunohistochemistry was used to assess stratifin expression in 182 patients with localized iCCAs undergoing surgical resection. Patients were divided into low and high expression groups, and the association between stratifin expression and clinicopathological features was analyzed. Univariate and multivariate survival analyses were performed to assess overall survival (OS), disease-specific survival (DSS), local recurrence-free survival (LRFS), and metastasis-free survival (MeFS). RESULTS: Elevated stratifin expression in iCCAs was significantly associated with the absence of hepatitis, positive surgical margins, advanced primary tumor stages, and higher histological grades (all p ≤ 0.011). Survival analyses demonstrated a significant negative association between stratifin expression and all prognostic indicators, including OS, DSS, LRFS, and MeFS (all p ≤ 0.0004). Multivariate analysis revealed that stratifin overexpression was significantly correlated with poorer outcomes in terms of DSS, LRFS, and MeFS (all p < 0.001). CONCLUSIONS: These findings suggest that stratifin may play a crucial role in iCCA oncogenesis and tumor progression, serving as a potential novel prognostic biomarker.

Canine ovarian epithelial tumours: histopathological and immunohistochemical evaluation with proposed histopathological classification system.

Canine ovarian epithelial tumours (OETs) are currently divided into ovarian adenomas and carcinomas, which are further inconsistently subclassified as papillary or cystic, whereas in human medicine, OETs are subdivided into several subtypes. This study aimed to establish clear morphological features enabling more consistent distinction between benign OETs and ovarian carcinomas (OvCas) as well as defining different histopathological patterns of canine OvCas. Analysis revealed a mitotic count threshold of >2 as a potential criterion for differentiating OvCas from benign OETs. Alongside ovarian adenomas, ovarian borderline tumours were introduced as a distinct category among benign OETs. OvCas exhibited five different histopathological patterns, namely papillary, solid with tubular differentiation, micropapillary, cystic and sarcomatous. Since some OvCas can morphologically overlap with other ovarian tumours, the expression of cytokeratin 7, a cytokeratin expressed in ovarian epithelium, was assessed and proved helpful, although it was not expressed in all cases. Furthermore, we investigated the expression of 14-3-3σ and cyclooxygenase 2 (COX-2). Based on the frequent expression of 14-3-3σ, this marker appears to have a role in canine OETs since it is not expressed in normal canine ovaries. The infrequent expression of COX-2 suggests that it is a poor candidate as a potential therapeutic target in canine OvCas.

Regulation of Caenorhabditis elegans HLH-30 subcellular localization dynamics: Evidence for a redox-dependent mechanism.

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.

The fusicoccin story revisited.

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.

RAS and SHOC2 Roles in RAF Activation and Therapeutic Considerations.

Mutations in RAS proteins play a pivotal role in the development of human cancers, driving persistent RAF activation and deregulating the Mitogen-Activated Protein Kinase (MAPK) signaling pathway. While progress has been made in targeting specific oncogenic RAS proteins, effective drug-based therapies for the majority of RAS mutations remain limited. Recent investigations on RAS-RAF complexes and the SHOC2-MRAS-PP1C holoenzyme complex have provided crucial insights into the structural and functional aspects of RAF activation within the MAPK signaling pathway. Moreover, these studies have also unveiled new blueprints for developing inhibitors allowing us to think beyond the current RAS and MEK inhibitors. In this review, we explore the roles of RAS and SHOC2 in activating RAF and discuss potential therapeutic strategies to target these proteins. A comprehensive understanding of the molecular interactions involved in RAF activation and their therapeutic implications holds the potential to drive innovative approaches in combating RAS/RAF-driven cancers.

Succinylation of 14-3-3 theta by CPT1A promotes survival and paclitaxel resistance in nasal type extranodal natural killer/T-cell lymphoma.

BACKGROUND: The aggressive and refractory extranodal natural killer/T-cell lymphoma, nasal type (ENKTL-NT) is a subtype of non-Hodgkin's lymphoma. Succinylation promotes progression in a variety of tumors, but its mechanism in ENKTL-NT is unclear. METHODS: Bioinformatic analysis was performed to screen differentially expressed genes in the ENKTL dataset. Cell transfection techniques were used for knockdown and overexpression of genes. The mRNA and protein expression were detected using RT-qPCR and western blot, respectively. Immunohistochemical staining was used to assess protein expression in situ. For the detection of cell proliferation activity, CCK-8, clonal formation, and EDU staining assays were used. Flow cytometry was employed to detect apoptosis. Co-immunoprecipitation was utilized for the identification of protein interactions and succinylation modifications. RESULTS: Succinyltransferase CPT1A was highly elevated in ENKTL-NT and was associated with a dismal prognosis. CPT1A knockdown suppressed SNK-6 cells' proliferation and induced apoptosis, while these effects were reversed by the overexpression of 14-3-3theta. Co-immunoprecipitation results showed that CPT1A caused succinylation of 14-3-3theta at site of K85, thereby enhancing the protein stability. Suppression of CPT1A-induced succinylation of 14-3-3theta by ST1326 resulted in the inhibition of SNK-6 cell proliferation and increased apoptosis. Paclitaxel combined with knockdown of CPT1A significantly inhibited the proliferation of ENKTL-NT compared to paclitaxel alone. CONCLUSION: CPT1A induces succinylation of 14-3-3theta at the K85 site, promoting ENKTL-NT proliferation. The anti-ENKTL activity of paclitaxel was improved when combined with CPT1A knockdown.

14-3-3σ downregulation sensitizes pancreatic cancer to carbon ions by suppressing the homologous recombination repair pathway.

This study explored the role of 14-3-3σ in carbon ion-irradiated pancreatic adenocarcinoma (PAAD) cells and xenografts and clarified the underlying mechanism. The clinical significance of 14-3-3σ in patients with PAAD was explored using publicly available databases. 14-3-3σ was silenced or overexpressed and combined with carbon ions to measure cell proliferation, cell cycle, and DNA damage repair. Immunoblotting and immunofluorescence (IF) assays were used to determine the underlying mechanisms of 14-3-3σ toward carbon ion radioresistance. We used the BALB/c mice to evaluate the biological behavior of 14-3-3σ in combination with carbon ions. Bioinformatic analysis revealed that PAAD expressed higher 14-3-3σ than normal pancreatic tissues; its overexpression was related to invasive clinicopathological features and a worse prognosis. Knockdown or overexpression of 14-3-3σ demonstrated that 14-3-3σ promoted the survival of PAAD cells after carbon ion irradiation. And 14-3-3σ was upregulated in PAAD cells during DNA damage (carbon ion irradiation, DNA damaging agent) and promotes cell recovery. We found that 14-3-3σ resulted in carbon ion radioresistance by promoting RPA2 and RAD51 accumulation in the nucleus in PAAD cells, thereby increasing homologous recombination repair (HRR) efficiency. Blocking the HR pathway consistently reduced 14-3-3σ overexpression-induced carbon ion radioresistance in PAAD cells. The enhanced radiosensitivity of 14-3-3σ depletion on carbon ion irradiation was also demonstrated in vivo. Altogether, 14-3-3σ functions in tumor progression and can be a potential target for developing biomarkers and treatment strategies for PAAD along with incorporating carbon ion irradiation.

The 14-3-3 protein nt GF14e interacts with CIPK2 and increases low potassium stress in tobacco.

Potassium (K+) plays a role in enzyme activation, membrane transport, and osmotic regulation processes. An increase in potassium content can significantly improve the elasticity and combustibility of tobacco and reduce the content of harmful substances. Here, we report that the expression analysis of Nt GF14e, a 14-3-3 gene, increased markedly after low-potassium treatment (LK). Then, chlorophyll content, POD activity and potassium content, were significantly increased in overexpression of Nt GF14e transgenic tobacco lines compared with those in the wild type plants. The net K+ efflux rates were severely lower in the transgenic plants than in the wild type under LK stress. Furthermore, transcriptome analysis identified 5708 upregulated genes and 2787 downregulated genes between Nt GF14e overexpressing transgenic tobacco plants. The expression levels of some potassium-related genes were increased, such as CBL-interacting protein kinase 2 (CIPK2), Nt CIPK23, Nt CIPK25, H+-ATPase isoform 2 a (AHA2a), Nt AHA4a, Stelar K+ outward rectifier 1(SKOR1), and high affinity K+ transporter 5 (HAK5). The result of yeast two-hybrid and luciferase complementation imaging experiments suggested Nt GF14e could interact with CIPK2. Overall, these findings indicate that NtGF14e plays a vital roles in improving tobacco LK tolerance and enhancing potassium nutrition signaling pathways in tobacco plants.

14-3-3ζ suppresses RANKL signaling by destabilizing TRAF6.

Macrophages are essential regulators of inflammation and bone loss. Receptor activator of nuclear factor-κß ligand (RANKL), a pro-inflammatory cytokine, is responsible for macrophage differentiation to osteoclasts and bone loss. We recently showed that 14-3-3ζ-knockout (YwhazKO) rats exhibit increased bone loss in the inflammatory arthritis model. 14-3-3ζ is a cytosolic adaptor protein that actively participates in many signaling transductions. However, the role of 14-3-3ζ in RANKL signaling or bone remodeling is unknown. We investigated how 14-3-3ζ affects osteoclast activity by evaluating its role in RANKL signaling. We utilized 14-3-3ζ-deficient primary bone marrow-derived macrophages obtained from wildtype and YwhazKO animals and RAW264.7 cells generated using CRISPR-Cas9. Our results showed that 14-3-3ζ-deficient macrophages, upon RANKL stimulation, have bigger and stronger tartrate-resistant acid phosphatase-positive multinucleated cells and increased bone resorption activity. The presence of 14-3-3ζ suppressed RANKL-induced MAPK and AKT phosphorylation, transcription factors (NFATC1 and p65) nuclear translocation, and subsequently, gene induction (Rank, Acp5, and Ctsk). Mechanistically, 14-3-3ζ interacts with TRAF6, an essential component of the RANKL receptor complex. Upon RANKL stimulation, 14-3-3ζ-TRAF6 interaction was increased, while RANK-TRAF6 interaction was decreased. Importantly, 14-3-3ζ supported TRAF6 ubiquitination and degradation by the proteasomal pathway, thus dampening the downstream RANKL signaling. Together, we show that 14-3-3ζ regulates TRAF6 levels to suppress inflammatory RANKL signaling and osteoclast activity. To the best of our knowledge, this is the first report on 14-3-3ζ regulation of RANKL signaling and osteoclast activation.

A 14-3-3 Protein Ca16R Acts Positively in Pepper Immunity against Ralstonia solanacearum by Interacting with CaASR1.

Although 14-3-3 proteins have been implicated in plant growth, development, and stress response, their roles in pepper immunity against R. solanacearum remain poorly understood. In this study, a 14-3-3-encoding gene in pepper, Ca16R, was found to be upregulated by R. solanacearum inoculation (RSI), its silencing significantly reduced the resistance of pepper plants to RSI, and its overexpression significantly enhanced the resistance of Nicotiana benthamiana to RSI. Consistently, its transient overexpression in pepper leaves triggered HR cell death, indicating that it acts positively in pepper immunity against RSI, and it was further found to act positively in pepper immunity against RSI by promoting SA but repressing JA signaling. Ca16R was also found to interact with CaASR1, originally using pull-down combined with a spectrum assay, and then confirmed using bimolecular fluorescence complementation (BiFC) and a pull-down assay. Furthermore, we found that CaASR1 transient overexpression induced HR cell death and SA-dependent immunity while repressing JA signaling, although this induction and repression was blocked by Ca16R silencing. All these data indicate that Ca16R acts positively in pepper immunity against RSI by interacting with CaASR1, thereby promoting SA-mediated immunity while repressing JA signaling. These results provide new insight into mechanisms underlying pepper immunity against RSI.

AGR2-mediated unconventional secretion of 14-3-3ε and α-actinin-4, responsive to ER stress and autophagy, drives chemotaxis in canine mammary tumor cells.

BACKGROUND: Canine mammary tumors (CMTs) in intact female dogs provide a natural model for investigating metastatic human cancers. Our prior research identified elevated expression of Anterior Gradient 2 (AGR2), a protein disulfide isomerase (PDI) primarily found in the endoplasmic reticulum (ER), in CMT tissues, highly associated with CMT progression. We further demonstrated that increased AGR2 expression actively influences the extracellular microenvironment, promoting chemotaxis in CMT cells. Unraveling the underlying mechanisms is crucial for assessing the potential of therapeutically targeting AGR2 as a strategy to inhibit a pro-metastatic microenvironment and impede tumor metastasis. METHODS: To identify the AGR2-modulated secretome, we employed proteomics analysis of the conditioned media (CM) from two CMT cell lines ectopically expressing AGR2, compared with corresponding vector-expressing controls. AGR2-regulated release of 14-3-3ε (gene: YWHAE) and α-actinin 4 (gene: ACTN4) was validated through ectopic expression, knockdown, and knockout of the AGR2 gene in CMT cells. Extracellular vesicles derived from CMT cells were isolated using either differential ultracentrifugation or size exclusion chromatography. The roles of 14-3-3ε and α-actinin 4 in the chemotaxis driven by the AGR2-modulated CM were investigated through gene knockdown, antibody-mediated interference, and recombinant protein supplement. Furthermore, the clinical relevance of the release of 14-3-3ε and α-actinin 4 was assessed using CMT tissue-immersed saline and sera from CMT-afflicted dogs. RESULTS: Proteomics analysis of the AGR2-modulated secretome revealed increased abundance in 14-3-3ε and α-actinin 4. Ectopic expression of AGR2 significantly increased the release of 14-3-3ε and α-actinin 4 in the CM. Conversely, knockdown or knockout of AGR2 expression remarkably reduced their release. Silencing 14-3-3ε or α-actinin 4 expression diminished the chemotaxis driven by AGR2-modulated CM. Furthermore, AGR2 controls the release of 14-3-3ε and α-actinin 4 primarily via non-vesicular routes, responding to the endoplasmic reticulum (ER) stress and autophagy activation. Knockout of AGR2 resulted in increased α-actinin 4 accumulation and impaired 14-3-3ε translocation in autophagosomes. Depletion of extracellular 14-3-3ε or α-actinin 4 reduced the chemotaxis driven by AGR2-modulated CM, whereas supplement with recombinant 14-3-3ε in the CM enhanced the CM-driven chemotaxis. Notably, elevated levels of 14-3-3ε or α-actinin 4 were observed in CMT tissue-immersed saline compared with paired non-tumor samples and in the sera of CMT dogs compared with healthy dogs. CONCLUSION: This study elucidates AGR2's pivotal role in orchestrating unconventional secretion of 14-3-3ε and α-actinin 4 from CMT cells, thereby contributing to paracrine-mediated chemotaxis. The insight into the intricate interplay between AGR2-involved ER stress, autophagy, and unconventional secretion provides a foundation for refining strategies aimed at impeding metastasis in both canine mammary tumors and potentially human cancers.

Adaptor protein 14-3-3zeta promotes corneal wound healing via regulating cell homeostasis, a potential novel therapy for corneal injury.

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.

Development of a NanoBRET assay for evaluation of 14-3-3σ molecular glues.

We report the development of a 384-well formatted NanoBRET assay to characterize molecular glues of 14-3-3/client interactions in living cells. The seven isoforms of 14-3-3 are dimeric hub proteins with diverse roles including transcription factor regulation and signal transduction. 14-3-3 interacts with hundreds of client proteins to regulate their function and is therefore an ideal therapeutic target when client selectivity can be achieved. We have developed the NanoBRET system for three 14-3-3σ client proteins CRAF, TAZ, and estrogen receptor α (ERα), which represent three specific binding modes. We have measured stabilization of 14-3-3σ/client complexes by molecular glues with EC50 values between 100 nM and 1 µM in cells, which align with the EC50 values calculated by fluorescence anisotropy in vitro. Developing this NanoBRET system for the hub protein 14-3-3σ allows for a streamlined approach, bypassing multiple optimization steps in the assay development process for other 14-3-3σ clients. The NanoBRET system allows for an assessment of PPI stabilization in a more physiologically relevant, cell-based environment using full-length proteins. The method is applicable to diverse protein-protein interactions (PPIs) and offers a robust platform to explore libraries of compounds for both PPI stabilizers and inhibitors.

14-3-3ε/YWHAE regulates the transcriptional expression of cardiac sodium channel NaV1.5.

BACKGROUND: Cardiac voltage-gated sodium channel alpha subunit 5 (NaV1.5) encoded by SCN5A is associated with arrhythmia disorders. However, the molecular mechanism underlying NaV1.5 expression remains to be fully elucidated. Previous studies have reported that the 14-3-3 family acts as an adaptor involved in regulating kinetic characteristics of cardiac ion channels. OBJECTIVE: The purpose of this study was to establish 14-3-3ε/YWHAE, a member of the 14-3-3 family, as a crucial regulator of NaV1.5 and to explore the potential role of 14-3-3ε in the heart. METHODS: Western blotting, patch clamping, real-time reverse transcription-polymerase chain reaction, RNA immunoprecipitation, electrocardiogram recording, echocardiography, and histologic analysis were performed. RESULTS: YWHAE overexpression significantly reduced the expression level of SCN5A mRNA and sodium current density, whereas YWHAE knockdown significantly increased SCN5A mRNA expression and sodium current density in HEK293/NaV1.5 and H9c2 cells. Similar results were observed in mice injected with adeno-associated virus serotype 9-mediated YWHAE knockdown. The effect of 14-3-3ε on NaV1.5 expression was abrogated by knockdown of TBX5, a transcription factor of NaV1.5. An interaction between 14-3-3ε protein and TBX5 mRNA was identified, and YWHAE overexpression significantly decreased TBX5 mRNA stability without affecting SCN5A mRNA stability. In addition, mice subjected to adeno-associated virus serotype 9-mediated YWHAE knockdown exhibited shorter R-R intervals and higher prevalence of premature ventricular contractions. CONCLUSION: Our data unveil a novel regulatory mechanism of NaV1.5 by 14-3-3ε and highlight the significance of 14-3-3ε in transcriptional regulation of NaV1.5 expression and cardiac arrhythmias.

The Development of CDC25A-Derived Phosphoseryl Peptides That Bind 14-3-3ε with High Affinities.

Overexpression of the 14-3-3ε protein is associated with suppression of apoptosis in cutaneous squamous cell carcinoma (cSCC). This antiapoptotic activity of 14-3-3ε is dependent on its binding to CDC25A; thus, inhibiting 14-3-3ε - CDC25A interaction is an attractive therapeutic approach to promote apoptosis in cSCC. In this regard, designing peptide inhibitors of 14-3-3ε - CDC25A interactions is of great interest. This work reports the rational design of peptide analogs of pS, a CDC25A-derived peptide that has been shown to inhibit 14-3-3ε-CDC25A interaction and promote apoptosis in cSCC with micromolar IC50. We designed new peptide analogs in silico by shortening the parent pS peptide from 14 to 9 amino acid residues; then, based on binding motifs of 14-3-3 proteins, we introduced modifications in the pS(174-182) peptide. We studied the binding of the peptides using conventional molecular dynamics (MD) and steered MD simulations, as well as biophysical methods. Our results showed that shortening the pS peptide from 14 to 9 amino acids reduced the affinity of the peptide. However, substituting Gln176 with either Phe or Tyr amino acids rescued the binding of the peptide. The optimized peptides obtained in this work can be candidates for inhibition of 14-3-3ε - CDC25A interactions in cSCC.

Structural Features and Physiological Associations of Human 14-3-3ζ Pseudogenes.

There are about 14,000 pseudogenes that are mutated or truncated sequences resembling functional parent genes. About two-thirds of pseudogenes are processed, while others are duplicated. Although initially thought dead, emerging studies indicate they have functional and regulatory roles. We study 14-3-3ζ, an adaptor protein that regulates cytokine signaling and inflammatory diseases, including rheumatoid arthritis, cancer, and neurological disorders. To understand how 14-3-3ζ (gene symbol YWHAZ) performs diverse functions, we examined the human genome and identified nine YWHAZ pseudogenes spread across many chromosomes. Unlike the 32 kb exon-to-exon sequence in YWHAZ, all pseudogenes are much shorter and lack introns. Out of six, four YWHAZ exons are highly conserved, but the untranslated region (UTR) shows significant diversity. The putative amino acid sequence of pseudogenes is 78-97% homologous, resulting in striking structural similarities with the parent protein. The OMIM and Decipher database searches revealed chromosomal loci containing pseudogenes are associated with human diseases that overlap with the parent gene. To the best of our knowledge, this is the first report on pseudogenes of the 14-3-3 family protein and their implications for human health. This bioinformatics-based study introduces a new insight into the complexity of 14-3-3ζ's functions in biology.