Arachidonic Acid Binds 14-3-3ζ, Releases 14-3-3ζ from Phosphorylated BAD and Induces Aggregation of 14-3-3ζ

Polyunsaturated fatty acids, like arachidonic acid, can bind proteins and affect their function. The 14-3-3 proteins bind phosphorylated sites on a diverse array of client proteins and, in this way, are involved in many intracellular signaling pathways. In this study, we used a novel approach to discover that 14-3-3ζ is able to directly bind arachidonic acid. Furthermore, arachidonic acid, at physiological concentrations, reduced the binding of 14-3-3ζ to phosphorylated BAD, an interaction that is important in regulating apoptosis. In addition, high concentrations of arachidonic acid caused the polymerization of 14-3-3ζ, an event observed in neurodegenerative disorders. Taken together, these results indicate that arachidonic acid directly interacts with 14-3-3ζ and that this interaction may be important in both normal and pathological cellular events. If so, then factors that mediate the release, metabolism and reacylation of arachidonic acid into membranes represent key points of regulation.     

14-3-3 蛋白

介绍了14－3－3蛋白的基本结构和功能,并简要概述了14－3－3蛋白在信号转导,细胞周期调控以及前体蛋白的折叠与运输过程中的作用机理。     

Small-molecule modulators of 14-3-3 protein–protein interactions

14-3-3 Proteins are eukaryotic adapter proteins that regulate a plethora of physiological processes by binding to several hundred partner proteins. They play a role in biological activities as diverse as signal transduction, cell cycle regulation, apoptosis, host-pathogen interactions and metabolic control. As such, 14-3-3s are implicated in disease areas like cancer, neurodegeneration, diabetes, pulmonary disease, and obesity. Targeted modulation of 14-3-3 protein–protein interactions (PPIs) by small molecules is therefore an attractive concept for disease intervention. In recent years a number of examples of inhibitors and stabilizers of 14-3-3 PPIs have been reported promising a vivid future in chemical biology and drug development for this remarkable class of proteins.     

The down-regulated expression of 14-3-3σ may activate the other six 14-3-3 isoforms and they may take over the role of 14-3-3σ in the tumor genesis

Seven isoforms of 14-3-3 protein family have different functions in the cancer genesis and progress. It is found that six isoforms were up-regulated expression and inclined to sustain the cancer survival. Conversely, 14-3-3σ strongly promotes cancer apoptosis. Its down-regulated expression was found in many cancer tissues and thought to be an early event in the tumor genesis. Interestingly, no suggestions are made about the possible effect that the down-regulated expression of 14-3-3σ activated the other six 14-3-3 isoforms and they take over the role of 14-3-3σ in the tumor genesis. The inactivation of 14-3-3σ in the early stage of tumor genesis is a clue to trigger the other six 14-3-3 isoforms activation.     

The role of epigenetic inactivation of 14-3-3σ in human cancer

Cancer cells show characteristic alterations in DNA methylation patterns. Aberrant CpG methylation of specific promoters results in inactivation of tumor suppressor genes and therefore plays an important role in carcinogenesis. The p53-regulated gene 14-3-3σ undergoes frequent epigenetic silencing in several types of cancer, including carcinoma of the breast, prostate, and skin, suggesting that the loss of 14-3-3σ expression may be causally involved in tumor progression. Functional studies demonstrated that 14-3-3σ is involved in cell-cycle control and prevents the accumulation of chromosomal damage. The recent identification of novel 14-3-3if-associated proteins by a targeted proteomics approach implies that 14-3-3σ regulates diverse cellular processes, which may become deregulated after silencing of 14-3-3σ expression in cancer cells.     

14-3-3蛋白研究进展

14-3-3蛋白是高度保守的、所有真核生物细胞中都普遍存在的、在大多数生物物种中由一个基因家族编码的一类蛋白调控家族。它几乎参与生命体所有的生理反应过程,人们在各种组织细胞中发现了各种不同的14-3-3蛋白。作为与磷酸丝氨酸／苏氨酸结合的第一信号分子,14-3-3蛋白在细胞的信号转导中起着至关重要的作用,尤其是它直接参与调节蛋白激酶和蛋白磷酸化酶的活性,被称为蛋白质与蛋白质相互作用的”桥梁蛋白”;它可以与转录因子结合形成复合体,调节相关基因的表达。一些研究表明,14-3-3蛋白调控机制的紊乱可以直接导致疾病的发生,在临床上14-3-3蛋白常常可以作为诊断的标志物。     

Targeted proteomic analysis of 14-3-3σ in nasopharyngeal carcinoma

14-3-3σ is a potential tumor suppressor, and loss of 14-3-3σ expression plays an important role in carcinogenesis and metastasis. To explore the possible mechanism of 14-3-3σ in nasopharyngeal carcinoma (NPC) invasion and metastasis, targeted proteomic analysis was performed on 14-3-3σ-associated proteins from NPC cells. As the results, 112 proteins associated with 14-3-3σ were identified, and four 14-3-3σ-interacted proteins: keratin 8, epidermal growth factor receptor (EGFR), small GTP-binding protein RAB7, and p53 were confirmed by coimmunoprecipitation and Western blot analysis. The 14-3-3σ-associated proteins could be grouped into eight clusters based on their molecule functions. Protein–protein interaction (PPI) analysis indicated that 14-3-3σ/EGFR/keratin 8 interactions may be involved in the invasion and metastasis of NPC. 14-3-3σ/EGFR/keratin 8 could form complexes in NPC cells. 14-3-3σ downregulation in NPC may lead to the overexpression of EGFR and keratin 8, which increases the invasion ability of NPC cells possibly by activating the downstream signal molecules and reorganizing cytoskeleton. The data suggest that the biological functions of 14-3-3σ in NPC are diversified, and 14-3-3σ could inhibit the in vitro invasive ability of NPC cells possibly through 14-3-3σ/EGFR/keratin 8 interaction.     

14-3-3 η inhibits chondrogenic differentiation of ATDC5 cell

It was previously shown that 14-3-3η is overexpressed in the synovial fluid of patients with joint inflammation, which is often associated with growth failure. In this study, we investigated the role of 14-3-3η in chondrogenesis using ATDC5 cells. Upon treatment with TNF-α, cells overexpressed 14-3-3η with inhibition of chondrogenesis. Chondrogenesis was also inhibited by overexpression of 14-3-3η without TNF-α treatment, whereas silencing of 14-3-3η promoted chondrogenic differentiation. Further, G1 phase arrest was inhibited by overexpression of 14-3-3η. In summary, we suggest that 14-3-3η plays a regulatory role in chondrogenic differentiation.     

Effect of phosphorylation on interaction of human tau protein with 14-3-3ζ

Interaction of the shortest isoform of tau protein (τ3) with human 14-3-3ζ was analyzed by means of native gel electrophoresis, chemical crosslinking and size-exclusion chromatography. Phosphorylation by cAMP-dependent protein kinase (up to 2 mole of phosphate per mole of τ3) strongly enhanced interaction of τ3 with 14-3-3. Apparent K_D of the complexes formed by phosphorylated τ3 and 14-3-3 was close to 2 μM, whereas the corresponding constant for unphosphorylated τ3 was at least 10 times higher. The stoichiometry of the complexes formed by phosphorylated τ3 and 14-3-3 was variable and was different from 1:1. 14-3-3 decreased the probability of formation of chemically crosslinked large homooligomers of phosphorylated τ3 and at the same time induced formation of crosslinked heterooligomeric complexes of τ3 and 14-3-3 with an apparent molecular mass of 120–140 kDa.     

14-3-3ε Overexpression Contributes to Epithelial-Mesenchymal Transition of Hepatocellular Carcinoma

Background

14-3-3ε is implicated in regulating tumor progression, including hepatocellular carcinoma (HCC). Our earlier study indicated that elevated 14-3-3ε expression is significantly associated with higher risk of metastasis and lower survival rates of HCC patients. However, the molecular mechanisms of how 14-3-3ε regulates HCC tumor metastasis are still unclear.

Methodology and Principal Findings

In this study, we show that increased 14-3-3ε expression induces HCC cell migration and promotes epithelial-mesenchymal transition (EMT), which is determined by the reduction of E-cadherin expression and induction of N-cadherin and vimentin expression. Knockdown with specific siRNA abolished 14-3-3ε-induced cell migration and EMT. Furthermore, 14-3-3ε selectively induced Zeb-1 and Snail expression, and 14-3-3ε-induced cell migration was abrogated by Zeb-1 or Snail siRNA. In addition, the effect of 14-3-3ε-reduced E-cadherin was specifically restored by Zeb-1 siRNA. Positive 14-3-3ε expression was significantly correlated with negative E-cadherin expression, as determined by immunohistochemistry analysis in HCC tumors. Analysis of 14-3-3ε/E-cadherin expression associated with clinicopathological characteristics revealed that the combination of positive 14-3-3ε and negative E-cadherin expression is significantly correlated with higher incidence of HCC metastasis and poor 5-year overall survival. In contrast, patients with positive 14-3-3ε and positive E-cadherin expression had better prognostic outcomes than did those with negative E-cadherin expression.

Significance

Our findings show for the first time that E-cadherin is one of the downstream targets of 14-3-3ε in modulating HCC tumor progression. Thus, 14-3-3ε may act as an important regulator in modulating tumor metastasis by promoting EMT as well as cell migration, and it may serve as a novel prognostic biomarker or therapeutic target for HCC.     

植物14-3-3蛋白研究进展

14-3-3蛋白是真核生物中许多信号传导级联反应的主要调节分子,易于与具有磷酸化的丝氨酸和苏氨酸残基的靶蛋白互作进而调节碳氮代谢、三羧酸循环、莽草酸合成等多种生理过程中的多种酶活性。该文根据近年来国内外对14-3-3蛋白的研究进展,对植物中14-3-3蛋白的发现、基因鉴定、结构和功能以及14-3-3蛋白与其靶蛋白的互作机制进行综述,并对14-3-3蛋白的研究提出了进一步的展望。     

Histone Deacetylase 6 (HDAC6) Promotes the Pro-survival Activity of 14-3-3ζ via Deacetylation of Lysines within the 14-3-3ζ Binding Pocket

The phospho-binding protein 14-3-3ζ acts as a signaling hub controlling a network of interacting partners and oncogenic pathways. We show here that lysines within the 14-3-3ζ binding pocket and protein-protein interface can be modified by acetylation. The positive charge on two of these lysines, Lys⁴⁹ and Lys¹²⁰, is critical for coordinating 14-3-3ζ-phosphoprotein interactions. Through screening, we identified HDAC6 as the Lys⁴⁹/Lys¹²⁰ deacetylase. Inhibition of HDAC6 blocks 14-3-3ζ interactions with two well described interacting partners, Bad and AS160, which triggers their dephosphorylation at Ser¹¹² and Thr⁶⁴², respectively. Expression of an acetylation-refractory K49R/K120R mutant of 14-3-3ζ rescues both the HDAC6 inhibitor-induced loss of interaction and Ser¹¹²/Thr⁶⁴² phosphorylation. Furthermore, expression of the K49R/K120R mutant of 14-3-3ζ inhibits the cytotoxicity of HDAC6 inhibition. These data demonstrate a novel role for HDAC6 in controlling 14-3-3ζ binding activity.     

14-3-3 Proteins in the Regulation of Rotenone-Induced Neurotoxicity Might be via Its Isoform 14-3-3Epsilon’s Involvement in Autophagy

14-3-3 proteins have been confirmed to be involved in Parkinson’s disease. It has been reported that an increase of 14-3-3 (theta, epsilon, and gamma) expression has neuroprotective effect in response to rotenone and MPP⁺ in dopaminergic cell culture and transgenic C. elegans with alpha-synuclein overexpression. To further investigate the detail mechanism of 14-3-3 proteins in rotenone-induced dopamine neurotoxicity, we observed the expression of 14-3-3 isoforms, and the influence of 14-3-3epsilon knockdown on autophagic activity and cell function. The results showed that rotenone led to a decrease in expression of 14-3-3 protein and mRNA, and an increase in expression and aggregation of alpha-synuclein protein. Knockdown of 14-3-3epsilon expression in turn further aggravated PC12 cell damage, such as an enhancement of ROS formation, and a reduction of cell viability and ATP production. Further experiments confirmed that the autophagic activity was promoted with 14-3-3epsilon siRNA transfection, including an enhancement of autophagosome formation and the ratio of LC3-II/LC3-I. Therefore, we concluded that the regulation of 14-3-3 proteins in rotenone-induced neurotoxicity might be associated with its isoform 14-3-3epsilon’s involvement in autophagy, which might be considered a mechanism in addition to the currently known function of 14-3-3 proteins in neurodegenerative disease pathogenesis.     

Superhelical DNA as a preferential binding target of 14-3-3γ protein

The 14-3-3 protein family is a highly conserved and widely distributed group of proteins consisting of multiple isoforms in eukaryotes. Ubiquitously expressed, 14-3-3 proteins play key roles in DNA replication, cell cycle regulation, and apoptosis. The function of 14-3-3 proteins is mediated by interaction with a large number of other proteins and with DNA. It has been demonstrated that 14-3-3γ protein binds strongly to cruciform structures and is crucial for initiating replication. In this study, we analyzed DNA binding properties of the 14-3-3γ isoform to linear and supercoiled DNA. We demonstrate that 14-3-3γ protein binds strongly to long DNA targets, as evidenced by electrophoretic mobility shift assay on agarose gels. Binding of 14-3-3γ to DNA target results in the appearance of blurry, retarded DNA bands. Competition experiments with linear and supercoiled DNA on magnetic beads show very strong preference for supercoiled DNA. We also show by confocal microscopy that 14-3-3 protein in the HCT-116 cell line is co-localized with DNA cruciforms. This implies a role for the 14-3-3γ protein in its binding to local DNA structures which are stabilized by DNA supercoiling.     

Superhelical DNA as a preferential binding target of 14-3-3γ protein

The 14-3-3 protein family is a highly conserved and widely distributed group of proteins consisting of multiple isoforms in eukaryotes. Ubiquitously expressed, 14-3-3 proteins play key roles in DNA replication, cell cycle regulation, and apoptosis. The function of 14-3-3 proteins is mediated by interaction with a large number of other proteins and with DNA. It has been demonstrated that 14-3-3γ protein binds strongly to cruciform structures and is crucial for initiating replication. In this study, we analyzed DNA binding properties of the 14-3-3γ isoform to linear and supercoiled DNA. We demonstrate that 14-3-3γ protein binds strongly to long DNA targets, as evidenced by electrophoretic mobility shift assay on agarose gels. Binding of 14-3-3γ to DNA target results in the appearance of blurry, retarded DNA bands. Competition experiments with linear and supercoiled DNA on magnetic beads show very strong preference for supercoiled DNA. We also show by confocal microscopy that 14-3-3 protein in the HCT-116 cell line is co-localized with DNA cruciforms. This implies a role for the 14-3-3γ protein in its binding to local DNA structures which are stabilized by DNA supercoiling.     

Detection of 14-3-3ζ in cerebrospinal fluid following experimentally evoked seizures

Surrogate and peripheral (bio)markers of neuronal injury may be of value in assessing effects of seizures on the brain or epilepsy development following trauma. The presence of 14-3-3 isoforms in cerebrospinal fluid (CSF) is a diagnostic indicator of Creutzfeldt–Jakob disease but these proteins may also be present following acute neurological insults. Here, we examined neuronal and 14-3-3 proteins in CSF from rats after seizures. Seizures induced by intra-amygdala microinjection of 0.1 µg kainic acid (KA) caused damage which was mainly restricted to the ipsilateral CA3 subfield of the hippocampus. 14-3-3ζ was detected at significant levels in CSF sampled 4 h after seizures compared with near absence in control CSF. Neuron-specific nuclear protein (NeuN) was also elevated in CSF in seizure rats. CSF 14-3-3ζ levels were significantly lower in rats treated with 0.01 µg KA. These data suggest the presence of 14-3-3ζ within CSF may be a biomarker of acute seizure damage.     

Free energy calculations on the stability of the 14-3-3ζ protein

Mutations of cysteine are often introduced to e.g. avoid formation of non-physiological inter-molecular disulfide bridges in in-vitro experiments, or to maintain specificity in labeling experiments. Alanine or serine is typically preferred, which usually do not alter the overall protein stability, when the original cysteine was surface exposed. However, selecting the optimal mutation for cysteines in the hydrophobic core of the protein is more challenging. In this work, the stability of selected Cys mutants of 14-3-3ζ was predicted by free-energy calculations and the obtained data were compared with experimentally determined stabilities. Both the computational predictions as well as the experimental validation point at a significant destabilization of mutants C94A and C94S. This destabilization could be attributed to the formation of hydrophobic cavities and a polar solvation of a hydrophilic side chain. A L12E, M78K double mutant was further studied in terms of its reduced dimerization propensity. In contrast to naïve expectations, this double mutant did not lead to the formation of strong salt bridges, which was rationalized in terms of a preferred solvation of the ionic species. Again, experiments agreed with the calculations by confirming the monomerization of the double mutants. Overall, the simulation data is in good agreement with experiments and offers additional insight into the stability and dimerization of this important family of regulatory proteins.     

Metabolic-Stress-Induced Rearrangement of the 14-3-3ζ Interactome Promotes Autophagy via a ULK1- and AMPK-Regulated 14-3-3ζ Interaction with Phosphorylated Atg9

14-3-3ζ promotes cell survival via dynamic interactions with a vast network of binding partners, many of which are involved in stress regulation. We show here that hypoxia (low glucose and oxygen) triggers a rearrangement of the 14-3-3ζ interactome to favor an interaction with the core autophagy regulator Atg9A. Our data suggest that the localization of mammalian Atg9A to autophagosomes requires phosphorylation on the C terminus of Atg9A at S761, which creates a 14-3-3ζ docking site. Under basal conditions, this phosphorylation is maintained at a low level and is dependent on both ULK1 and AMPK. However, upon induction of hypoxic stress, activated AMPK bypasses the requirement for ULK1 and mediates S761 phosphorylation directly, resulting in an increase in 14-3-3ζ interactions, recruitment of Atg9A to LC3-positive autophagosomes, and enhanced autophagosome production. These data suggest a novel mechanism whereby the level of autophagy induction can be modulated by AMPK/ULK1-mediated phosphorylation of mammalian Atg9A.