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.     

The binding of 14-3-3γ to membranes studied by intrinsic fluorescence spectroscopy

Human 14-3-3 proteins contain two conserved tryptophan residues in each monomer, Trp60 and Trp233 in isoform γ. 14-3-3γ binds to negatively charged membranes and here we show that membrane binding can be monitored by steady-state intrinsic fluorescence spectroscopy. Measurements with W60F and W233F 14-3-3γ mutants revealed that Trp60 is the major contributor to the emission fluorescence, whereas the fluorescence of Trp233, which π-stacks with Tyr184, is quenched. The fluorescence is reduced and red-shifted upon specific binding of a phosphate ligand, and further red-shifted upon binding of 14-3-3γ to the membrane, compatible with solvent exposure of Trp60. Moreover, our results support that membrane binding involves the non-conserved, convex area of 14-3-3γ, and that Trp residues do not intercalate in the bilayer.     

Revisiting PPARγ as a target for the treatment of metabolic disorders

As the prevalence of obesity has increased explosively over the last several decades, associated metabolic disorders, including type 2 diabetes, dyslipidemia, hypertension, and cardiovascular diseases, have been also increased. Thus, new strategies for preventing and treating them are needed. The nuclear peroxisome proliferator-activated receptors (PPARs) are involved fundamentally in regulating energy homeostasis; thus, they have been considered attractive drug targets for addressing metabolic disorders. Among the PPARs, PPARγ is a master regulator of gene expression for metabolism, inflammation, and other pathways in many cell types, especially adipocytes. It is a physiological receptor of the potent anti-diabetic drugs of the thiazolidinediones (TZDs) class, including rosiglitazone (Avandia). However, TZDs have undesirable and severe side effects, such as weight gain, fluid retention, and cardiovascular dysfunction. Recently, many reports have suggested that PPARγ could be modulated by post-translational modifications (PTMs), and modulation of PTM has been considered as novel approaches for treating metabolic disorders with fewer side effects than the TZDs. In this review, we discuss how PTM of PPARγ may be regulated and issues to be considered in making novel anti-diabetic drugs that can modulate the PTM of PPARγ. [BMB Reports 2014; 47(11): 599-608]     

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.     

Minireview: PPARγ as the target of obesogens

The peroxisome proliferator-activated receptor gamma (PPARγ) is a key regulator of adipogenesis and is medically important for its connections to obesity and the treatment of type II diabetes. Activation of this receptor by certain natural or xenobiotic compounds has been shown to stimulate adipogenesis in vitro and in vivo. Obesogens are chemicals that ultimately increase obesity through a variety of potential mechanisms, including activation of PPARγ. The first obesogen for which a definitive mechanism of action has been elucidated is the PPARγ and RXR activator tributyltin; however, not all chemicals that activate PPARγ are adipogenic or correlated with obesity in humans. There are multiple mechanisms through which obesogens can target PPARγ that may not involve direct activation of the receptor. Ligand-independent mechanisms could act through obesogen-mediated post-translational modification of PPARγ which cause receptor de-repression or activation. PPARγ is active in multipotent stem cells committing to the adipocyte fate during fat cell development. By modifying chromatin structure early in development, obesogens have the opportunity to influence the promoter activity of PPARγ, or the ability of PPARγ to bind to its target genes, ultimately biasing the progenitor pool towards the fat lineage. Obesogens that act by directly or indirectly activating PPARγ, by increasing the levels of PPARγ protein, or enhancing its recruitment to promoters of key genes in the adipogenic pathway may ultimately play an important role in adipogenesis and obesity.     

Re-emergence of an orphan therapeutic target for the treatment of resistant prostate cancer – a thorough conformational and binding analysis for ROR-γ protein

Recent studies have linked a deadly form of prostate cancer known as metastatic castration-resistant prostate cancer to retinoic acid-related orphan-receptor gamma (ROR-γ). Most of these studies continued to place ROR-γ as orphan because of unidentifiable inhibitor. Recently identified inhibitors of ROR-γ and their therapeutic potential were evaluated, among which inhibitor XY018 was the potent. However, molecular understanding of the conformational features of XY018-ROR-γ complex is still elusive. Herein, molecular dynamics simulations were conducted on HC9-ROR-γ and XY018-ROR-γ complexes to understand their conformational features at molecular level and the influence of XY018 binding on the dynamics of ROR-γ with the aid of post-dynamic analytical tools. These include; principal component analysis, radius of gyration, binding free energy calculation (MM/GBSA), per-residue fluctuation and hydrogen bond occupancy. Findings from this study revealed that (1) hydrophobic packing contributes significantly to binding free energy, (2) Ile136 and Leu60 exhibited high hydrogen-bond occupancy in XY018-ROR-γ and HC9-ROR-γ, respectively, (3) XY018-ROR-γ displayed a relatively high loop region residue fluctuation compared to HC9-ROR-γ, (4) electrostatic interactions are a potential binding force in XY018-ROR-γ complex compared to HC9-ROR-γ, (5) XY018-ROR-γ assumes a rigid conformation which is highlighted by a decrease in residual fluctuation, (6) XY018 could potentially induce pseudoporphyria, nephritis and interstitial nephritis but potentially safe in renal failure. This study could serve as a base line for the design of new potential ROR-γ inhibitors.     

A biotin switch-based proteomics approach identifies 14-3-3ζ as a target of Sirt1 in the metabolic regulation of caspase-2

While lysine acetylation in the nucleus is well characterized, comparatively little is known about its significance in cytoplasmic signaling. Here we show that inhibition of the Sirt1 deacetylase, which is primarily cytoplasmic in cancer cell lines, sensitizes these cells to caspase-2-dependent death. To identify relevant Sirt1 substrates, we developed a proteomics strategy, enabling the identification of a range of putative substrates, including 14-3-3ζ, a known direct regulator of caspase-2. We show here that inhibition of Sirtuin activity accelerates caspase activation and overrides caspase-2 suppression by nutrient abundance. Furthermore, 14-3-3ζ is acetylated prior to caspase activation, and supplementation of Xenopus egg extract with glucose-6-phosphate, which promotes caspase-2/14-3-3ζ binding, enhances 14-3-3ζ-directed Sirtuin activity. Conversely, inhibiting Sirtuin activity promotes14-3-3ζ dissociation from caspase-2 in both egg extract and human cultured cells. These data reveal a role for Sirt1 in modulating apoptotic sensitivity, in response to metabolic changes, by antagonizing 14-3-3ζ acetylation.     

DNA polymerase β as a novel target for chemotherapeutic intervention of colorectal cancer

Chemoprevention presents a major strategy for the medical management of colorectal cancer. Most drugs used for colorectal cancer therapy induce DNA-alkylation damage, which is primarily repaired by the base excision repair (BER) pathway. Thus, blockade of BER pathway is an attractive option to inhibit the spread of colorectal cancer. Using an in silico approach, we performed a structure-based screen by docking small-molecules onto DNA polymerase β (Pol-β) and identified a potent anti-Pol-β compound, NSC-124854. Our goal was to examine whether NSC-124854 could enhance the therapeutic efficacy of DNA-alkylating agent, Temozolomide (TMZ), by blocking BER. First, we determined the specificity of NSC-124854 for Pol-β by examining in vitro activities of APE1, Fen1, DNA ligase I, and Pol-β-directed single nucleotide (SN)- and long-patch (LP)-BER. Second, we investigated the effect of NSC-124854 on the efficacy of TMZ to inhibit the growth of mismatch repair (MMR)-deficient and MMR-proficient colon cancer cell lines using in vitro clonogenic assays. Third, we explored the effect of NSC-124854 on TMZ-induced in vivo tumor growth inhibition of MMR-deficient and MMR-proficient colonic xenografts implanted in female homozygous SCID mice. Our data showed that NSC-124854 has high specificity to Pol-β and blocked Pol-β-directed SN- and LP-BER activities in in vitro reconstituted system. Furthermore, NSC-124854 effectively induced the sensitivity of TMZ to MMR-deficient and MMR-proficient colon cancer cells both in vitro cell culture and in vivo xenograft models. Our findings suggest a potential novel strategy for the development of highly specific structure-based inhibitor for the prevention of colonic tumor progression.     

Overexpression of the 14-3-3γ protein in uterine leiomyoma cells results in growth retardation and increased apoptosis

Protein 14-3-3γ was significantly reduced in human uterine leiomyoma compared to the adjacent normal myometrium tissue. To investigate the possible link between the reduced 14-3-3γ expression and uterine leiomyoma growth, we have overexpressed 14-3-3γ protein in uterine leiomyomal cells and its effects on cell proliferation and apoptosis were analyzed. Over-expression of 14-3-3γ was achieved by transducing into two types of uterine leiomyoma cells (primary culture cells and immortal stem cells) with a 14-3-3γ expressing adenovirus vector. Differentially expressed proteins were screened by the proteomics tool (TMT-LCTMS), followed by PANTHER database analysis to single out specifically modified signaling pathway proteins, which were confirmed by Phospho-MAPK Antibody Array and Western blots analysis. The results showed that increase in 14-3-3γ expression in both two types of human uterine leiomyoma cells inhibited cell proliferation and induced apoptosis. Proteomic screening has found 42 proteins, among 5846, that were significantly affected. PANTHER database and GeneMANIA analysis of the differentially expressed proteins have found that proteins involved in apoptosis signaling and cytoskeletal/adhesion were among the ones affected the most. Further analysis of the key signaling pathways have found that over-expression of 14-3-3γ resulted in reductions in the phosphorylations of multiple signaling molecules, including AKT, pan, ERK1/2, GSK-3 α/β, MEK1/2, Foxo1 and Vimentin. In conclusion, the loss of 14-3-3γ may have causal effects on the growth of uterine leiomyoma, which may function through modifying multiple signaling pathways, including AKT-Foxo and/or MEK1/2-ERK1/2.     

Design of novel lead molecules against RhoG protein as cancer target – a computational study

Cancer is a class of diseases characterized by uncontrolled cell growth. Every year more than 2 million people are affected by the disease. Rho family proteins are actively involved in cytoskeleton regulation. Over-expression of Rho family proteins show oncogenic activity and promote cancer progression. In the present work RhoG protein is considered as novel target of cancer. It is a member of Rho family and Rac subfamily protein, which plays pivotal role in regulation of microtubule formation, cell migration and contributes in cancer progression. In order to understand the binding interaction between RhoG protein and the DH domain of Ephexin-4 protein, the 3D structure of RhoG was evaluated and Molecular Dynamic Simulations was performed to stabilize the structure. The 3D structure of RhoG protein was validated and active site identified using standard computational protocols. Protein–protein docking of RhoG with Ephexin-4 was done to understand binding interactions and the active site structure. Virtual screening was carried out with ligand databases against the active site of RhoG protein. The efficiency of virtual screening is analysed with enrichment factor and area under curve values. The binding free energy of docked complexes was calculated using prime MM-GBSA module. The SASA, FOSA, FISA, PISA and PSA values of ligands were carried out. New ligands with high docking score, glide energy and acceptable ADME properties were prioritized as potential inhibitors of RhoG protein.     

Identification of 14-3-3β Gene as a Novel miR-152 Target Using a Proteome-based Approach

Recent studies demonstrated that miR-152 overexpression down-regulates the nonclassical human leukocyte antigen (HLA) class I molecule HLA-G in human tumors thereby contributing to their immune surveillance. Using two-dimensional gel electrophoresis followed by MALDI-TOF mass spectrometry, the protein expression profile of HLA-G⁺, miR-152^low cells, and their miR-152-overexpressing (miR^high) counterparts was compared leading to the identification of 24 differentially expressed proteins. These were categorized according to their function and localization demonstrating for most of them an important role in the initiation and progression of tumors. The novel miR-152 target 14-3-3 protein β/α/YWHAB (14-3-3β) is down-regulated upon miR-152 overexpression, although its overexpression was often found in tumors of distinct origin. The miR-152-mediated reduction of the 14-3-3β expression was accompanied by an up-regulation of BAX protein expression resulting in a pro-apoptotic phenotype. In contrast, the reconstitution of 14-3-3β expression in miR-152^high cells increased the expression of the anti-apoptotic BCL2 gene, enhances the proliferative activity in the presence of the cytostatic drug paclitaxel, and causes resistance to apoptosis induced by this drug. By correlating clinical microarray data with the patients'' outcome, a link between 14-3-3β and HLA-G expression was found, which could be associated with poor prognosis and overall survival of patients with tumors. Because miR-152 controls both the expression of 14-3-3β and HLA-G, it exerts a dual role in tumor cells by both altering the immunogenicity and the tumorigenicity.     

Amygdala 14-3-3ζ as a novel modulator of escalating alcohol intake in mice

Alcoholism is a devastating brain disorder that affects millions of people worldwide. The development of alcoholism is caused by alcohol-induced maladaptive changes in neural circuits involved in emotions, motivation, and decision-making. Because of its involvement in these processes, the amygdala is thought to be a key neural structure involved in alcohol addiction. However, the molecular mechanisms that govern the development of alcoholism are incompletely understood. We have previously shown that in a limited access choice paradigm, C57BL/6J mice progressively escalate their alcohol intake and display important behavioral characteristic of alcohol addiction, in that they become insensitive to quinine-induced adulteration of alcohol. This study used the limited access choice paradigm to study gene expression changes in the amygdala during the escalation to high alcohol consumption in C57BL/6J mice. Microarray analysis revealed that changes in gene expression occurred predominantly after one week, i.e. during the initial escalation of alcohol intake. One gene that stood out from our analysis was the adapter protein 14-3-3ζ, which was up-regulated during the transition from low to high alcohol intake. Independent qPCR analysis confirmed the up-regulation of amygdala 14-3-3ζ during the escalation of alcohol intake. Subsequently, we found that local knockdown of 14-3-3ζ in the amygdala, using RNA interference, dramatically augmented alcohol intake. In addition, knockdown of amygdala 14-3-3ζ promoted the development of inflexible alcohol drinking, as apparent from insensitivity to quinine adulteration of alcohol. This study identifies amygdala 14-3-3ζ as a novel key modulator that is engaged during escalation of alcohol use.     

PPAR-γ as a therapeutic target in cardiovascular disease: evidence and uncertainty

Peroxisome proliferator-activated receptor γ (PPAR-γ) is a key regulator of fatty acid metabolism, promoting its storage in adipose tissue and reducing circulating concentrations of free fatty acids. Activation of PPAR-γ has favorable effects on measures of adipocyte function, insulin sensitivity, lipoprotein metabolism, and vascular structure and function. Despite these effects, clinical trials of thiazolidinedione PPAR-γ activators have not provided conclusive evidence that they reduce cardiovascular morbidity and mortality. The apparent disparity between effects on laboratory measurements and clinical outcomes may be related to limitations of clinical trials, adverse effects of PPAR-γ activation, or off-target effects of thiazolidinedione agents. This review addresses these issues from a clinician's perspective and highlights several ongoing clinical trials that may help to clarify the therapeutic role of PPAR-γ activators in cardiovascular disease.     

Interacting domains of P14-3-3 and actin involved in protein–protein interactions of living cells

14-3-3 proteins are conserved regulatory proteins present in all eukaryotic cells that control numerous cellular activities via targeted protein interactions. To elucidate the interaction between P14-3-3 from Physarum polycephalum and actin in living cells, PCR and DNA recombination were used to generate various P14-3-3 and actin constructs. Yeast two-hybrid assay and FRET were employed to characterize the interaction between P14-3-3 and actin. The two-hybrid assay indicated that P14-3-3 N-terminal 76–108 amino acids and the C-terminal 207–216 amino acids played an important role in mediating interactions with actin, and the actin N-terminal 1–54 amino acids and the C-terminal 326–376 amino acids are also crucial in the interactions with the mPa, a P14-3-3 with mutations at Ser62 (Ser62 → Gly62). Mutations to potential phosphorylation sites did not affect interactions between P14-3-3 and actin. FRET results demonstrated that P14-3-3 co-localized with actin with a FRET efficiency of 22.2% and a distance of 7.4 nm and that P14-3-3 N-terminal 76–108 and C-terminal 207–216 amino acids were important in mediating this interaction, the truncated actin peptides without either the N-terminal 1–54 or C-terminal 326–376 amino acids interacted with P14-3-3, consistent with the results obtained from the yeast two-hybrid assay. Based on data obtained, we identified critical actin and P14-3-3 contact regions.     

Estimation of Förster's distance between two ends of Dps protein from mycobacteria: distance heterogeneity as a function of oligomerization and DNA binding

Dps protein (DNA binding Protein from Starved Cells) from Mycobacterium smegmatis (Ms-Dps) is known to undergo an in vitro irreversible oligomeric transition from trimer to dodecamer. This transition helps the protein to provide for bimodal protection to the bacterial DNA from the free radical and Fenton mediated damages in the stationary state. The protein exists as a stable trimer, when purified from E. coli cells transformed with an over-expression plasmid. Both trimer as well as dodecamer are known to exhibit ferroxidation activity, thus removing toxic hydroxyl radicals in vivo, whereas iron accumulation and non-sequence specific DNA binding activity are found in dodecamer only. This seems to be aided by the positively charged long C-terminal tail of the protein. We used frequency domain phase-modulation fluorescence spectroscopy and Förster Resonance Energy Transfer (FRET) to monitor this oligomeric switch from a trimer to a dodecamer and to elucidate the structure of DNA–Dps dodecamer complex. As Ms-Dps is devoid of any Cysteine residues, a Serine is mutated to Cysteine (S169C) at a position adjacent to the putative DNA binding domain. This Cysteine is subsequently labeled with fluorescent probe and another probe is placed at the N-terminus, as crystal structure of the protein reveals several side-chain interactions between these two termini, and both are exposed towards the surface of the protein. Here, we report the Förster's distance distribution in the trimer and the dodecamer in the presence and absence of DNA. Through discrete lifetime analysis of the probes tagged at the respective regions in the macromolecule, coupled with Maximum Entropy Method (MEM) analysis, we show that the dodecamer, upon DNA binding shows conformational heterogeneity in overall structure, perhaps mediated by a non-specific DNA–protein interaction. On the other hand, the nature of DNA–Dps interaction is not known and several models exist in literature. We show here with the help of fluorescence anisotropy measurements of labeled DNA having different length and unlabeled native dodecameric protein that tandem occupation of DNA binding sites by a series of Dps molecules perhaps guide the tight packing of Dps over DNA backbone.     

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.