14-3-3ζ Plays a key role in the modulation of neuroplasticity underlying the antidepressant-like effects of Zhi-Zi-Chi-Tang.

BACKGROUND: Zhi-Zi-Chi-Tang (ZZCT) is an effective traditional Chinese medicinal formula. ZZCT has been used for the treatment of depression for centuries. Its clinical efficacy in relieving depression has been confirmed. However, the molecular mechanisms of ZZCT regarding neuroplasticity in the pathogenesis of depression have not yet been elucidated. PURPOSE: The present study aimed to examine the effects of ZZCT on neuroplasticity in mice exposed to chronic unpredictable mild stress (CUMS), and to explore the underlying molecular mechanisms. METHODS: For this purpose, a murine model of depression was established using the CUMS procedure. Following the intragastric administration of ZZCT or fluoxetine, classic behavioral experiments were performed to observe the efficacy of ZZCT as an antidepressant. Immunofluorescence was used to label and quantify microtubule-associated protein (MAP2) and postsynaptic density protein (PSD95) in the hippocampus. Golgi staining was applied to visualize the dendritic spine density of neurons in the hippocampi. Isolated hippocampal slices were prepared to induce long-term potentiation (LTP) in the CA1 area. The hippocampal protein expression levels of glycogen synthase kinase-3ß (GSK-3ß), p-GSK-3ß (Ser9), cAMP response element binding protein (CREB), p-CREB (Ser133), brain-derived neurotrophic factor (BDNF) and 14-3-3ζ were detected using western blot analysis. The interaction of 14-3-3ζ and p-GSK-3ß (Ser9) was examined using co-immunoprecipitation. LV-shRNA was used to knockdown 14-3-3ζ by an intracerebroventricular injection. RESULTS: ZZCT (6 g/kg) and fluoxetine (20 mg/kg) alleviated depressive-like behavior, restored hippocampal MAP2+ PSD95+ intensity, and reversed the dendritic spine density of hippocampal neurons and LTP in the CA1 region of mice exposed to CUMS. Both low and high doses of ZZCT (3 and 6 g/kg) significantly promoted the binding of 14-3-3ζ to p-GSK-3ß (Ser9) in the hippocampus, and ZZCT (6 g/kg) significantly promoted the phosphorylation of GSK-3ß Ser9 and CREB Ser133 in the hippocampus. ZZCT (3 and 6 g/kg) upregulated hippocampal BDNF expression in mice exposed to CUMS. LV-sh14-3-3ξ reduced the antidepressant effects of ZZCT. CONCLUSION: ZZCT exerted antidepressant effects against CUMS-stimulated depressive-like behavior mice. The knockdown of 14-3-3ζ using lentivirus confirmed that 14-3-3ζ was involved in the ZZCT-mediated antidepressant effects through GSK-3ß/CREB/BDNF signaling. On the whole, these results suggest that the antidepressant effects of ZZCT are attributed to restoring damage by neuroplasticity enhancement via the 14-3-3ζ/GSK-3ß/CREB/BDNF signaling pathway.

N-linked glycoproteomic profiling in esophageal squamous cell carcinoma.

BACKGROUND: Mass spectrometry-based proteomics and glycomics reveal post-translational modifications providing significant biological insights beyond the scope of genomic sequencing. AIM: To characterize the N-linked glycoproteomic profile in esophageal squamous cell carcinoma (ESCC) via two complementary approaches. METHODS: Using tandem multilectin affinity chromatography for enrichment of N-linked glycoproteins, we performed N-linked glycoproteomic profiling in ESCC tissues by two-dimensional gel electrophoresis (2-DE)-based and isobaric tags for relative and absolute quantification (iTRAQ) labeling-based mass spectrometry quantitation in parallel, followed by validation of candidate glycoprotein biomarkers by Western blot. RESULTS: 2-DE-based and iTRAQ labeling-based quantitation identified 24 and 402 differentially expressed N-linked glycoproteins, respectively, with 15 in common, demonstrating the outperformance of iTRAQ labeling-based quantitation over 2-DE and complementarity of these two approaches. Proteomaps showed the distinct compositions of functional categories between proteins and glycoproteins with differential expression associated with ESCC. Western blot analysis validated the up-regulation of total procathepsin D and high-mannose procathepsin D, and the down-regulation of total haptoglobin, high-mannose clusterin, and GlcNAc/sialic acid-containing fraction of 14-3-3ζ in ESCC tissues. The serum levels of glycosylated fractions of clusterin, proline-arginine-rich end leucine-rich repeat protein, and haptoglobin in patients with ESCC were remarkably higher than those in healthy controls. CONCLUSION: Our study provides insights into the aberrant N-linked glycoproteome associated with ESCC, which will be a valuable resource for future investigations.

[Gene cloning, induction, and prokaryotic expression of a Sm14-3-3 protein from Salvia miltiorrhiza].

14-3-3 proteins are important proteins in plants, as they regulate plant growth and development and the response to biotic or abiotic stresses. In this study, a 14-3-3 gene(GenBank accession: OM683281) was screened from the cDNA library of the medicinal species Salvia miltiorrhiza by yeast two-hybrid and cloned. The open reading frame(ORF) was 780 bp, encoding 259 amino a cids. Bioinformatics analysis predicted that the protein was a non-transmembrane protein with the molecular formula of C_(1287)H_(2046)N_(346)O_(422)S_9, relative molecular weight of 29.4 kDa, and no signal peptide. Homologous sequence alignment and phylogenetic tree analysis proved that the protein belonged to 14-3-3 family and had close genetic relationship with the 14-3-3 proteins from Arabidopsis thaliana, Oryza sativa, and Nicotiana tabacum. The 14-3-3 gene was ligated to the prokaryotic expression vector pGEX-4 T-1 and then transformed into Escherichia coli BL21 for the expression of recombinant protein. Real-time fluorescent quantitative PCR showed that the expression of this gene was different among roots, stems, leaves, and flowers of S. miltiorrhiza. To be specific, the highest expression was found in leaves, followed by stems, and the lowest expression was detected in flowers. S. miltiorrhiza plants were treated with 15% PEG(simulation of drought), and hormones salicylic acid, methyl jasmonate, and ethephon, respectively, and the expression of 14-3-3 gene peaked at the early stage of induction. Therefore, the gene can quickly respond to abiotic stresses such as drought and plant hormone treatments such as salicylic acid, jasmonic acid, and ethylene. This study lays the foundation for revealing the molecular mechanism of 14-3-3 protein regulating tanshinone biosynthesis and responding to biotic and abiotic stresses.

Crystal structure of potato 14-3-3 protein St14f revealed the importance of helix I in StFDL1 recognition.

In potato (Solanum tuberosum L.), 14-3-3 protein forms a protein complex with the FLOWERING LOCUS T (FT)-like protein StSP6A and the FD-like protein StFDL1 to activate potato tuber formation. Eleven 14-3-3 isoforms were reported in potato, designated as St14a-k. In this study, the crystal structure of the free form of St14f was determined at 2.5 Å resolution. Three chains were included in the asymmetric unit of the St14f free form crystal, and the structural deviation among the three chain structures was found on the C-terminal helix H and I. The St14f free form structure in solution was also investigated by nuclear magnetic resonance (NMR) residual dipolar coupling analysis, and the chain B in the crystal structure was consistent with NMR data. Compared to other crystal structures, St14f helix I exhibited a different conformation with larger B-factor values. Larger B-factor values on helix I were also found in the 14-3-3 free form structure with higher solvent contents. The mutation in St14f Helix I stabilized the complex with StFDL1. These data clearly showed that the flexibility of helix I of 14-3-3 protein plays an important role in the recognition of target protein.

pH modulates interaction of 14-3-3 proteins with pollen plasma membrane H+ ATPases independently from phosphorylation.

Pollen grains transport the sperm cells through the style tissue via a fast-growing pollen tube to the ovaries where fertilization takes place. Pollen tube growth requires a precisely regulated network of cellular as well as molecular events including the activity of the plasma membrane H+ ATPase, which is known to be regulated by reversible protein phosphorylation and subsequent binding of 14-3-3 isoforms. Immunodetection of the phosphorylated penultimate threonine residue of the pollen plasma membrane H+ ATPase (LilHA1) of Lilium longiflorum pollen revealed a sudden increase in phosphorylation with the start of pollen tube growth. In addition to phosphorylation, pH modulated the binding of 14-3-3 isoforms to the regulatory domain of the H+ ATPase, whereas metabolic components had only small effects on 14-3-3 binding, as tested with in vitro assays using recombinant 14-3-3 isoforms and phosphomimicking substitutions of the threonine residue. Consequently, local H+ influxes and effluxes as well as pH gradients in the pollen tube tip are generated by localized regulation of the H+ ATPase activity rather than by heterogeneous localized distribution in the plasma membrane.

14-3-3ζ: A suppressor of inflammatory arthritis.

Inflammatory arthritis (IA) is a common disease that affects millions of individuals worldwide. Proinflammatory events during IA pathogenesis are well studied; however, loss of protective immunity remains underexplored. Earlier, we reported that 14-3-3zeta (ζ) has a role in T-cell polarization and interleukin (IL)-17A signal transduction. Here, we demonstrate that 14-3-3ζ knockout (KO) rats develop early-onset severe arthritis in two independent models of IA, pristane-induced arthritis and collagen-induced arthritis. Arthritic 14-3-3ζ KO animals showed an increase in bone loss and immune cell infiltration in synovial joints. Induction of arthritis coincided with the loss of anti-14-3-3ζ antibodies; however, rescue experiments to supplement the 14-3-3ζ antibody by passive immunization did not suppress arthritis. Instead, 14-3-3ζ immunization during the presymptomatic phase resulted in significant suppression of arthritis in both wild-type and 14-3-3ζ KO animals. Mechanistically, 14-3-3ζ KO rats exhibited elevated inflammatory gene signatures at the messenger RNA and protein levels, particularly for IL-1ß. Furthermore, the immunization with recombinant 14-3-3ζ protein suppressed IL-1ß levels, significantly increased anti-14-3-3ζ antibody levels and collagen production, and preserved bone quality. The 14-3-3ζ protein increased collagen expression in primary rat mesenchymal cells. Together, our findings indicate that 14-3-3ζ causes immune suppression and extracellular remodeling, which lead to a previously unrecognized IA-suppressive function.

Rice G protein γ subunit qPE9-1 modulates root elongation for phosphorus uptake by involving 14-3-3 protein OsGF14b and plasma membrane H+ -ATPase.

Heterotrimeric G protein is involved in plant growth and development, while the role of rice (Oryza sativa) G protein γ subunit qPE9-1 in response to low-phosphorus (LP) conditions remains unclear. The gene expression of qPE9-1 was significantly induced in rice roots under LP conditions. Rice varieties carrying the qPE9-1 allele showed a stronger primary root response to LP than the varieties carrying the qpe9-1 allele (mutant of the qPE9-1 allele). Transgenic rice plants with the qPE9-1 allele had longer primary roots and higher P concentrations than those with the qpe9-1 allele under LP conditions. The plasma membrane (PM) H+ -ATPase was important for the qPE9-1-mediated response to LP. Furthermore, OsGF14b, a 14-3-3 protein that acts as a key component in activating PM H+ -ATPase for root elongation, is also involved in the qPE9-1 mediation. Moreover, the overexpression of OsGF14b in WYJ8 (carrying the qpe9-1 allele) partially increased primary root length under LP conditions. Experiments using R18 peptide (a 14-3-3 protein inhibitor) showed that qPE9-1 is important for primary root elongation and H+ efflux under LP conditions by involving the 14-3-3 protein. In addition, rhizosheath weight, total P content, and the rhizosheath soil Olsen-P concentration of qPE9-1 lines were higher than those of qpe9-1 lines under soil drying and LP conditions. These results suggest that the G protein γ subunit qPE9-1 in rice plants modulates root elongation for phosphorus uptake by involving the 14-3-3 protein OsGF14b and PM H+ -ATPase, which is required for rice P use.

CO2 -responsive CCT protein interacts with 14-3-3 proteins and controls the expression of starch synthesis-related genes.

CO2 -responsive CCT protein (CRCT) is a positive regulator of starch synthesis-related genes such as ADP-glucose pyrophosphorylase large subunit 1 and starch branching enzyme I particularly in the leaf sheath of rice (Oryza sativa L.). The promoter GUS analysis revealed that CRCT expressed exclusively in the vascular bundle, whereas starch synthesis-related genes were expressed in different sites such as mesophyll cell and starch storage parenchyma cell. However, the chromatin immunoprecipitation (ChIP) using a FLAG-CRCT overexpression line and subsequent qPCR analyses showed that the 5'-flanking regions of these starch synthesis-related genes tended to be enriched by ChIP, suggesting that CRCT can bind to the promoter regions of these genes. The monomer of CRCT is 34.2 kDa; however, CRCT was detected at 270 kDa via gel filtration chromatography, suggesting that CRCT forms a complex in vivo. Immunoprecipitation and subsequent MS analysis pulled down several 14-3-3-like proteins. A yeast two-hybrid analysis and bimolecular fluorescence complementation assays confirmed the interaction between CRCT and 14-3-3-like proteins. Although there is an inconsistency in the place of expression, this study provides important findings regarding the molecular function of CRCT to control the expression of key starch synthesis-related genes.

Proteomics-Guided Study on Buyang Huanwu Decoction for Its Neuroprotective and Neurogenic Mechanisms for Transient Ischemic Stroke: Involvements of EGFR/PI3K/Akt/Bad/14-3-3 and Jak2/Stat3/Cyclin D1 Signaling Cascades.

Buyang Huanwu Decoction (BHD), a classic traditional Chinese medicine (TCM) formula, has been used for recovering neurological dysfunctions and treating post-stroke disability in China for 200 years. In the present study, we investigated the effects of BHD on inhibiting neuronal apoptosis, promoting proliferation and differentiation of neural stem cells (NSCs) and neurite formation and enhancing learning and memory functional recovery in an experimental rat ischemic stroke model. BHD significantly reduced infarct volume and decreased cell apoptosis in the ischemic brain. BHD enhanced neuronal cell viability in vitro. BHD dose-dependently promoted the proliferation of NSCs in ischemic rat brains in vivo. Moreover, BHD promoted neuronal and astrocyte differentiation in primary cultured NSCs in vitro. Water maze test revealed that BHD promoted the recovery of learning function but not memory functions in the transient ischemic rats. We then investigated the changes of the cellular signaling molecules by using two-dimension (2D) gel electrophoresis and focused on the PI3K/Akt/Bad and Jak2/Stat3/cyclin D1signaling pathway to uncover its underlying mechanisms for its neuroprotective and neurogenetic effects. BHD significantly upregulated the expression of p-PI3K, p-Akt, and p-Bad as well as the expression of p-Jak, p-Stat3, and cyclin D1 in vitro and in vivo. In addition, BHD upregulated Hes1 and downregulated cav-1 in vitro and in vivo. Taken together, these results suggest that BHD has neuroprotective effects and neurogenesis-promoting effects via activating PI3K/Akt/Bad and Jak2/Stat3/Cyclin D1 signaling pathways. Graphical Abstract Buyang Huanwu Decoction (BHD) activates the PI3K-AKT-BAD pathway in the ischemic brain for neuroprotection. BHD also activates JAK2/STAT3/Cyclin D1 signaling cascades for promoting neurogenesis in the hippocampus of post-ischemic brains. Moreover, BHD inhibits the expression of caveolin-1 and increases the expression of HES1 for promoting neuronal differentiation. The neuroprotective and neurogenesis-promoting effects in the hippocampus of post-ischemic brains promote learning ability.

Tetramethylpyrazine Attenuates the Endotheliotoxicity and the Mitochondrial Dysfunction by Doxorubicin via 14-3-3γ/Bcl-2.

Doxorubicin (Dox) with cardiotoxicity and endotheliotoxicity limits its clinical application for cancer. The toxicitic mechanism involves excess ROS generation. 14-3-3s have the protective effects on various injured tissues and cells. Tetramethylpyrazine (TMP) is an alkaloid extracted from the rhizome of Ligusticum wallichii and has multiple bioactivities. We hypothesize that TMP has the protective effects on vascular endothelium by upregulating 14-3-3γ. To test the hypothesis, Dox-induced endotheliotoxicity was used to establish vascular endothelium injury models in mice and human umbilical vein endothelial cells. The effects of TMP were assessed by determining thoracic aortic strips' endothelium-dependent dilation (EDD), as well as LDH, CK, caspase-3, SOD, CAT, GSH-Px activities and MDA level in serum, apoptotic rate, and histopathological changes of vascular tissue (in vivo). Also, cell viability, LDH and caspase-3 activities, ROS generation, levels of NAD+/NADH and GSH/GSSG, MMP, mPTP opening, and apoptotic rate were evaluated (in vitro). The expression of 14-3-3γ and Bcl-2, as well as phosphorylation of Bad (S112), were determined by Western blot. Our results showed that Dox-induced injury to vascular endothelium was decreased by TMP via upregulating 14-3-3γ expression in total protein and Bcl-2 expression in mitochondria, activating Bad (S112) phosphorylation, maintaining EDD, reducing LDH, CK, and caspase-3 activities, thereby causing a reduction in apoptotic rate, and histopathological changes of vascular endothelium (in vivo). Furthermore, TMP increased cell viability and MMP levels, maintained NAD+/NADH, GSH/GSSG balance, decreased LDH and caspase-3 activities, ROS generation, mPTP opening, and apoptotic rate (in vitro). However, the protective effects to vascular endothelium of TMP were significantly canceled by pAD/14-3-3γ-shRNA, an adenovirus that caused knockdown 14-3-3γ expression, or ABT-737, a specific Bcl-2 inhibitor. In conclusion, this study is the first to demonstrate that TMP protects the vascular endothelium against Dox-induced injury via upregulating 14-3-3γ expression, promoting translocation of Bcl-2 to the mitochondria, closing mPTP, maintaining MMP, inhibiting RIRR mechanism, suppressing oxidative stress, improving mitochondrial function, and alleviating Dox-induced endotheliotoxicity.

Crosstalk between 14-3-3θ and AF4 enhances MLL-AF4 activity and promotes leukemia cell proliferation.

PURPOSE: The t(4;11)(q21;q23) translocation characterizes a form of acute lymphoblastic leukemia with a poor prognosis. It results in a fusion gene encoding a chimeric transcription factor, MLL-AF4, that deregulates gene expression through a variety of still controversial mechanisms. To provide new insights into these mechanisms, we examined the interaction between AF4, the most common MLL fusion partner, and the scaffold protein 14-3-3θ, in the context of t(4;11)-positive leukemia. METHODS: Protein-protein interactions were analyzed using immunoprecipitation and in vitro binding assays, and by fluorescence microscopy in t(4;11)-positive RS4;11 and MV4-11 leukemia cells and in HEK293 cells. Protein and mRNA expression levels were determined by Western blotting and RT-qPCR, respectively. A 5-bromo-2'-deoxyuridine assay and an annexin V/propidium iodide assay were used to assess proliferation and apoptosis rates, respectively, in t(4;11)-positive and control cells. Chromatin immunoprecipitation was performed to assess binding of 14-3-3θ and AF4 to a specific promoter element. RESULTS: We found that AF4 and 14-3-3θ are nuclear interactors, that 14-3-3θ binds Ser588 of AF4 and that 14-3-3θ forms a complex with MLL-AF4. In addition, we found that in t(4;11)-positive cells, 14-3-3θ knockdown decreased the expression of MLL-AF4 target genes, induced apoptosis and hampered cell proliferation. Moreover, we found that 14-3-3θ knockdown impaired the recruitment of AF4, but not of MLL-AF4, to target chromatin. Overall, our data indicate that the activity of the chimeric transcription factor MLL-AF4 depends on the cellular availability of 14-3-3θ, which triggers the transactivating function and subsequent degradation of AF4. CONCLUSIONS: From our data we conclude that the scaffold protein 14-3-3θ enhances the aberrant activity of the chimeric transcription factor MLL-AF4 and, therefore, represents a new player in the molecular pathogenesis of t(4;11)-positive leukemia and a new promising therapeutic target.

Site-Directed Fragment-Based Screening for the Discovery of Protein-Protein Interaction Stabilizers.

Modulation of protein-protein interactions (PPIs) by small molecules has emerged as a valuable approach in drug discovery. Compared to direct inhibition, PPI stabilization is vastly underexplored but has strong advantages, including the ability to gain selectivity by targeting an interface formed only upon association of proteins. Here, we present the application of a site-directed screening technique based on disulfide trapping (tethering) to select for fragments that enhance the affinity between protein partners. We target the phosphorylation-dependent interaction between the hub protein 14-3-3σ and a peptide derived from Estrogen Receptor α (ERα), an important breast cancer target that is negatively regulated by 14-3-3σ. We identify orthosteric stabilizers that increase 14-3-3/ERα affinity up to 40-fold and propose the mechanism of stabilization based on X-ray crystal structures. These fragments already display partial selectivity toward ERα-like motifs over other representative 14-3-3 clients. This first of its kind study illustrates the potential of the tethering approach to overcome the hurdles in systematic PPI stabilizer discovery.

Gastrodin Protects Cardiomyocytes from Anoxia/Reoxygenation Injury by 14-3-3η.

Gastrodin (GAS) is the major component isolated from the rhizome of the Chinese traditional medicinal herb "Tianma." Many clinical studies have found that GAS protects cardiomyocytes in cardiovascular diseases, although the effects and underlying mechanisms on cardiovascular anoxia/reoxygenation (A/R) injury remain unknown. This study is aimed at exploring the effect of gastrodin on cardiomyocytes in A/R injury. Our results suggested that the protective effect of GAS on cardiomyocytes is associated with upregulated 14-3-3η levels. Pretreatment with GAS could increase the cell viability and decrease the activities of creatine phosphokinase (CPK) and lactate dehydrogenase (LDH). GAS could also reduce reactive oxygen species (ROS) production, inhibit mitochondrial permeability transition pore (mPTP) opening, alter the maintenance of the mitochondrial membrane potential (∆Ψm), decrease the activation of caspase-3, and finally restrain cell apoptosis. Downregulating 14-3-3η levels by transfection with siRNA14-3-3η clearly attenuated the protective effect of GAS on cardiomyocytes in A/R injury.

WRINKLED1 as a novel 14-3-3 client: function of 14-3-3 proteins in plant lipid metabolism.

The conserved plant 14-3-3 proteins (14-3-3s) function by binding to phosphorylated client proteins to regulate their function. Previous studies indicate that 14-3-3s are involved in the regulation of plant primary metabolism; however, not much is known regarding the functions of 14-3-3s in plant oil biosynthesis. Our recent work shows that 14-3-3 plays a role in mediating plant oil biosynthesis through interacting with the transcription factor, WRINKLED1 (WRI1). WRI1 is critical for the transcriptional control of plant oil biosynthesis. Arabidopsis WRI1 physically interacts with 14-3-3s. Transient co-expression of AtWRI1 with 14-3-3s enhances plant oil biosynthesis in leaves of Nicotiana benthamiana. Transgenic plants overexpressing of a 14-3-3 show enhanced seed oil content. Co-expression of a 14-3-3 with AtWRI1 results in increased transcriptional activity and protein stability of AtWRI1. Our transcriptional regulation model supports a concept that interaction of a 14-3-3 with a transcription factor enhances the transcriptional activity through protein stabilization.

Tanshinone IIA Pretreatment Protects H9c2 Cells against Anoxia/Reoxygenation Injury: Involvement of the Translocation of Bcl-2 to Mitochondria Mediated by 14-3-3η.

Tanshinone IIA is an important component that is isolated from danshen (Salvia miltiorrhiza), which is known to be beneficial for cardiovascular health. In this study, we determined the effects of Tanshinone IIA and its underlying mechanisms of action in an anoxia/reoxygenation (A/R) cell line model. Prior to inducing A/R injury, rat cardiomyocyte-derived cell line H9c2 was stimulated with 8 µM of Tanshinone IIA for 48 hours. When compared with the A/R group, the Tanshinone IIA treatment significantly increased cell viability and decreased lactate dehydrogenase activity. Tanshinone IIA upregulated 14-3-3η expression and facilitated Bcl-2 translocation to the mitochondrial outer membrane, which bound with voltage-dependent anion channel 1. In addition, pretreatment with Tanshinone IIA reduced the generation of reactive oxygen species and cytochrome c release, inactivated caspase-3, prevented mitochondrial permeability transition pore opening, and reduced the percentage of apoptotic cells. Moreover, treatment with Tanshinone IIA reduced the level of malondialdehyde, thereby increasing the activity of superoxide dismutase and glutathione peroxidase. Silencing the expression of 14-3-3η by adenovirus blocked the above-mentioned results. These novel findings showed that pretreatment with Tanshinone IIA alleviated H9c2 cell damage against A/R injury and was associated with upregulation of 14-3-3η, thereby facilitating Bcl-2 translocation to the mitochondrial outer membrane and preventing mitochondrial permeability transition pore opening, decreasing cytochrome c release, preventing caspase-3 activation, and restraining apoptosis.

(-)-Epigallocatechin-3-gallate stimulates myogenic differentiation through TAZ activation.

Muscle loss is a typical process of aging. Green tea consumption is known to slow down the progress of aging. Their underlying mechanisms, however, remain largely unknown. In this study, we investigated the effect of (-)-epigallocatechin-3-gallate (EGCG), a polyphenolic compound of green tea, on myogenic differentiation and found that EGCG significantly increases myogenic differentiation. After EGCG treatment, the expression of myogenic marker genes, such as myosin heavy chain, are increased through activation of TAZ, a transcriptional coactivator with a PDZ-binding motif. TAZ-knockdown does not stimulate EGCG-induced myogenic differentiation. EGCG facilitates the interaction between TAZ and MyoD, which stimulates MyoD-mediated gene transcription. EGCG induces nuclear localization of TAZ through the dephosphorylation of TAZ at its Ser89 residue, which relieves 14-3-3 binding in the cytosol. Interestingly, inactivation of Lats kinase is observed after EGCG treatment, which is responsible for the production of dephosphorylated TAZ. Together, these results suggest that EGCG induces myogenic differentiation through TAZ, suggesting that TAZ plays an important role in EGCG induced muscle regeneration.

Costunolide and dehydrocostuslactone combination treatment inhibit breast cancer by inducing cell cycle arrest and apoptosis through c-Myc/p53 and AKT/14-3-3 pathway.

Our previous studies demonstrated that volatile oil from saussurea lappa root (VOSL), rich in two natural sesquiterpene lactones, costunolide (Cos) and dehydrocostuslactone (Dehy), exerts better anti-breast cancer efficacy and lower side effects than Cos or Dehy alone in vivo, however, their anti-cancer molecular mechanisms were still unknown. In this study, we investigated the underlying mechanisms of Cos and Dehy combination treatment (CD) on breast cancer cells through proteomics technology coupled with Western blot validation. Ingenuity Pathways Analysis (IPA) results based on the differentially expressed proteins revealed that both VOSL and CD affect the 14-3-3-mediated signaling, c-Myc mediated apoptosis signaling and protein kinase A (PKA) signaling. Western blot coupled with cell cycle and apoptosis analysis validated the results of proteomics analysis. Cell cycle arrest and apoptosis were induced in a dose-dependent manner, and the expressions of p53 and p-14-3-3 were significantly up-regulated, whereas the expressions of c-Myc, p-AKT, p-BID were significantly down-regulated, furthermore, the ratio of BAX/BCL-2 were significantly increased in breast cancer cells after CD and VOSL treatment. The findings indicated that VOSL and CD could induce breast cancer cell cycle arrest and apoptosis through c-Myc/p53 and AKT/14-3-3 signaling pathways and may be novel effective candidates for breast cancer treatment.

Anthocyanins from roselle extract arrest cell cycle G2/M phase transition via ATM/Chk pathway in p53-deficient leukemia HL-60 cells.

Cell cycle regulation is an important issue in cancer therapy. Delphinidin and cyanidin are two major anthocyanins of the roselle plant (Hibiscus sabdariffa). In the present study, we investigated the effect of Hibiscus anthocyanins (HAs) on cell cycle arrest in human leukemia cell line HL-60 and the analyzed the underlying molecular mechanisms. HAs extracted from roselle calyces (purity 90%) markedly induced G2/M arrest evaluated with flow cytometry analysis. Western blot analyses revealed that HAs (0.1-0.7 mg mL-1 ) induced G2/M arrest via increasing Tyr15 phosphorylation of Cdc2, and inducing Cdk inhibitors p27 and p21. HAs also induced phosphorylation of upstream signals related to G2/M arrest such as phosphorylation of Cdc25C tyrosine phosphatase at Ser216, increasing the binding of pCdc25C with 14-3-3 protein. HAs-induced phosphorylation of Cdc25C could be activated by ATM checkpoint kinases, Chk1, and Chk2. We first time confirmed that ATM-Chk1/2-Cdc25C pathway as a critical mechanism for G2/M arrest in HAs-induced leukemia cell cycle arrest, indicating that this compound could be a promising anticancer candidate or chemopreventive agents for further investigation. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 1290-1304, 2017.

Potato Tuber Induction is Regulated by Interactions Between Components of a Tuberigen Complex.

Photoperiod-regulated flowering and potato tuber formation involve leaf-produced mobile signals, florigen and tuberigen, respectively. The major protein component of florigen has been identified as the FLOWERING LOCUS T (FT) protein. In rice, an FT-like protein, Heading date 3a (Hd3a), induces flowering by making the florigen activation complex (FAC) through interactions with 14-3-3 and OsFD1, a rice FD-like protein. In potato, StSP6A, an FT-like protein, was identified as a major component of tuberigen. However, the molecular mechanism of how StSP6A triggers tuber formation remains elusive. Here we analyzed the significance of the formation of a complex including StSP6A, 14-3-3 and FD-like proteins in tuberization. Yeast two-hybrid, bimolecular fluorescence complementation and in vitro pull-down assays showed that StSP6A and StFDL1, a potato FD-like protein, interact with St14-3-3s. StSP6A overexpression induced early tuberization in a 14-3-3-dependent manner, and suppression of StFDL1 delayed tuberization. These results strongly suggest that an FAC-like complex, the tuberigen activation complex (TAC), comprised of StSP6A, St14-3-3s and StFDL1, regulates potato tuber formation.

14-3-3 protein mediates plant seed oil biosynthesis through interaction with AtWRI1.

Plant 14-3-3 proteins are phosphopeptide-binding proteins, belonging to a large family of proteins involved in numerous physiological processes including primary metabolism, although knowledge about the function of 14-3-3s in plant lipid metabolism is sparse. WRINKLED1 (WRI1) is a key transcription factor that governs plant oil biosynthesis. At present, AtWRI1-interacting partners remain largely unknown. Here, we show that 14-3-3 proteins are able to interact with AtWRI1, both in yeast and plant cells. Transient co-expression of 14-3-3- and AtWRI1-encoding cDNAs led to increased oil biosynthesis in Nicotiana benthamiana leaves. Stable transgenic plants overproducing a 14-3-3 protein also displayed increased seed oil content. Co-production of a 14-3-3 protein with AtWRI1 enhanced the transcriptional activity of AtWRI1. The 14-3-3 protein was found to increase the stability of AtWRI1. A possible 14-3-3 binding motif was identified in one of the two AP2 domains of AtWRI1, which was also found to be critical for the interaction of AtWRI1 with an E3 ligase linker protein. Thus, we hypothesize a regulatory mechanism by which the binding of 14-3-3 to AtWRI1 interferes with the interaction of AtWRI1 and the E3 ligase, thereby protecting AtWRI1 from degradation. Taken together, our studies identified AtWRI1 as a client of 14-3-3 proteins and provide insights into a role of 14-3-3 in mediating plant oil biosynthesis.