A Biotin Switch-Based Proteomics Approach Identifies 14-3-3ζ as a Target of Sirt1 in the Metabolic Regulation of Caspase-2

PubMed Central

Andersen, Joshua L.; Thompson, J. Will; Lindblom, Kelly R.; Johnson, Erika S.; Yang, Chih-Sheng; Lilley, Lauren R.; Freel, Christopher D.; Moseley, M. Arthur; Kornbluth, Sally

2011-01-01

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 novel 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 promotes 14-3-3ζ dissociation from caspase-2 in both egg extract and human cell lines. These data reveal a role for Sirt1 in modulating apoptotic sensitivity, in response to metabolic changes, by antagonizing 14-3-3ζ acetylation. PMID:21884983

Molecular tweezers modulate 14-3-3 protein-protein interactions

NASA Astrophysics Data System (ADS)

Bier, David; Rose, Rolf; Bravo-Rodriguez, Kenny; Bartel, Maria; Ramirez-Anguita, Juan Manuel; Dutt, Som; Wilch, Constanze; Klärner, Frank-Gerrit; Sanchez-Garcia, Elsa; Schrader, Thomas; Ottmann, Christian

2013-03-01

Supramolecular chemistry has recently emerged as a promising way to modulate protein functions, but devising molecules that will interact with a protein in the desired manner is difficult as many competing interactions exist in a biological environment (with solvents, salts or different sites for the target biomolecule). We now show that lysine-specific molecular tweezers bind to a 14-3-3 adapter protein and modulate its interaction with partner proteins. The tweezers inhibit binding between the 14-3-3 protein and two partner proteins—a phosphorylated (C-Raf) protein and an unphosphorylated one (ExoS)—in a concentration-dependent manner. Protein crystallography shows that this effect arises from the binding of the tweezers to a single surface-exposed lysine (Lys214) of the 14-3-3 protein in the proximity of its central channel, which normally binds the partner proteins. A combination of structural analysis and computer simulations provides rules for the tweezers' binding preferences, thus allowing us to predict their influence on this type of protein-protein interactions.

Hormone-induced 14-3-3γ Adaptor Protein Regulates Steroidogenic Acute Regulatory Protein Activity and Steroid Biosynthesis in MA-10 Leydig Cells*

PubMed Central

Aghazadeh, Yasaman; Rone, Malena B.; Blonder, Josip; Ye, Xiaoying; Veenstra, Timothy D.; Hales, D. Buck; Culty, Martine; Papadopoulos, Vassilios

2012-01-01

Cholesterol is the sole precursor of steroid hormones in the body. The import of cholesterol to the inner mitochondrial membrane, the rate-limiting step in steroid biosynthesis, relies on the formation of a protein complex that assembles at the outer mitochondrial membrane called the transduceosome. The transduceosome contains several mitochondrial and cytosolic components, including the steroidogenic acute regulatory protein (STAR). Human chorionic gonadotropin (hCG) induces de novo synthesis of STAR, a process shown to parallel maximal steroid production. In the hCG-dependent steroidogenic MA-10 mouse Leydig cell line, the 14-3-3γ protein was identified in native mitochondrial complexes by mass spectrometry and immunoblotting, and its levels increased in response to hCG treatment. The 14-3-3 proteins bind and regulate the activity of many proteins, acting via target protein activation, modification and localization. In MA-10 cells, cAMP induces 14-3-3γ expression parallel to STAR expression. Silencing of 14-3-3γ expression potentiates hormone-induced steroidogenesis. Binding motifs of 14-3-3γ were identified in components of the transduceosome, including STAR. Immunoprecipitation studies demonstrate a hormone-dependent interaction between 14-3-3γ and STAR that coincides with reduced 14-3-3γ homodimerization. The binding site of 14-3-3γ on STAR was identified to be Ser-194 in the STAR-related sterol binding lipid transfer (START) domain, the site phosphorylated in response to hCG. Taken together, these results demonstrate that 14-3-3γ negatively regulates steroidogenesis by binding to Ser-194 of STAR, thus keeping STAR in an unfolded state, unable to induce maximal steroidogenesis. Over time 14-3-3γ homodimerizes and dissociates from STAR, allowing this protein to induce maximal mitochondrial steroid formation. PMID:22427666

Structural Characterization of Phosducin and Its Complex with the 14-3-3 Protein*

PubMed Central

Kacirova, Miroslava; Kosek, Dalibor; Kadek, Alan; Man, Petr; Vecer, Jaroslav; Herman, Petr; Obsilova, Veronika; Obsil, Tomas

2015-01-01

Phosducin (Pdc), a highly conserved phosphoprotein involved in the regulation of retinal phototransduction cascade, transcriptional control, and modulation of blood pressure, is controlled in a phosphorylation-dependent manner, including the binding to the 14-3-3 protein. However, the molecular mechanism of this regulation is largely unknown. Here, the solution structure of Pdc and its interaction with the 14-3-3 protein were investigated using small angle x-ray scattering, time-resolved fluorescence spectroscopy, and hydrogen-deuterium exchange coupled to mass spectrometry. The 14-3-3 protein dimer interacts with Pdc using surfaces both inside and outside its central channel. The N-terminal domain of Pdc, where both phosphorylation sites and the 14-3-3-binding motifs are located, is an intrinsically disordered protein that reduces its flexibility in several regions without undergoing dramatic disorder-to-order transition upon binding to 14-3-3. Our data also indicate that the C-terminal domain of Pdc interacts with the outside surface of the 14-3-3 dimer through the region involved in Gtβγ binding. In conclusion, we show that the 14-3-3 protein interacts with and sterically occludes both the N- and C-terminal Gtβγ binding interfaces of phosphorylated Pdc, thus providing a mechanistic explanation for the 14-3-3-dependent inhibition of Pdc function. PMID:25971962

Bacterial co-expression of human Tau protein with protein kinase A and 14-3-3 for studies of 14-3-3/phospho-Tau interaction

PubMed Central

Tugaeva, Kristina V.; Tsvetkov, Philipp O.

2017-01-01

Abundant regulatory 14-3-3 proteins have an extremely wide interactome and coordinate multiple cellular events via interaction with specifically phosphorylated partner proteins. Notwithstanding the key role of 14-3-3/phosphotarget interactions in many physiological and pathological processes, they are dramatically underexplored. Here, we focused on the 14-3-3 interaction with human Tau protein associated with the development of several neurodegenerative disorders, including Alzheimer’s and Parkinson’s diseases. Among many known phosphorylation sites within Tau, protein kinase A (PKA) phosphorylates several key residues of Tau and induces its tight interaction with 14-3-3 proteins. However, the stoichiometry and mechanism of 14-3-3 interaction with phosphorylated Tau (pTau) are not clearly elucidated. In this work, we describe a simple bacterial co-expression system aimed to facilitate biochemical and structural studies on the 14-3-3/pTau interaction. We show that dual co-expression of human fetal Tau with PKA in Escherichia coli results in multisite Tau phosphorylation including also naturally occurring sites which were not previously considered in the context of 14-3-3 binding. Tau protein co-expressed with PKA displays tight functional interaction with 14-3-3 isoforms of a different type. Upon triple co-expression with 14-3-3 and PKA, Tau protein could be co-purified with 14-3-3 and demonstrates complex which is similar to that formed in vitro between individual 14-3-3 and pTau obtained from dual co-expression. Although used in this study for the specific case of the previously known 14-3-3/pTau interaction, our co-expression system may be useful to study of other selected 14-3-3/phosphotarget interactions and for validations of 14-3-3 complexes identified by other methods. PMID:28575131

Exercise-induced TBC1D1 Ser237 phosphorylation and 14-3-3 protein binding capacity in human skeletal muscle.

PubMed

Frøsig, Christian; Pehmøller, Christian; Birk, Jesper B; Richter, Erik A; Wojtaszewski, Jørgen F P

2010-11-15

TBC1D1 is a Rab-GTPase activating protein involved in regulation of GLUT4 translocation in skeletal muscle. We here evaluated exercise-induced regulation of TBC1D1 Ser237 phosphorylation and 14-3-3 protein binding capacity in human skeletal muscle. In separate experiments healthy men performed all-out cycle exercise lasting either 30 s, 2  min or 20  min. After all exercise protocols, TBC1D1 Ser237 phosphorylation increased (∼70-230%, P < 0.005), with the greatest response observed after 20  min of cycling. Interestingly, capacity of TBC1D1 to bind 14-3-3 protein showed a similar pattern of regulation, increasing 60-250% (P < 0.001). Furthermore, recombinant 5AMP-activated protein kinase (AMPK) induced both Ser237 phosphorylation and 14-3-3 binding properties on human TBC1D1 when evaluated in vitro. To further characterize the role of AMPK as an upstream kinase regulating TBC1D1, extensor digitorum longus muscle (EDL) from whole body α1 or α2 AMPK knock-out and wild-type mice were stimulated to contract in vitro. In wild-type and α1 knock-out mice, contractions resulted in a similar ∼100% increase (P < 0.001) in Ser237 phosphorylation. Interestingly, muscle of α2 knock-out mice were characterized by reduced protein content of TBC1D1 (∼50%, P < 0.001) as well as in basal and contraction-stimulated (∼60%, P < 0.001) Ser237 phosphorylation, even after correction for the reduced TBC1D1 protein content. This study shows that TBC1D1 is Ser237 phosphorylated and 14-3-3 protein binding capacity is increased in response to exercise in human skeletal muscle. Furthermore, we show that the catalytic α2 AMPK subunit is the main (but probably not the only) donor of AMPK activity regulating TBC1D1 Ser237 phosphorylation in mouse EDL muscle.

Modulation of 14-3-3 protein interactions with target polypeptides by physical and metabolic effectors.

PubMed

Athwal, G S; Lombardo, C R; Huber, J L; Masters, S C; Fu, H; Huber, S C

2000-04-01

The proteins commonly referred to as 14-3-3s have recently come to prominence in the study of protein:protein interactions, having been shown to act as allosteric or steric regulators and possibly scaffolds. The binding of 14-3-3 proteins to the regulatory phosphorylation site of nitrate reductase (NR) was studied in real-time by surface plasmon resonance, using primarily an immobilized synthetic phosphopeptide based on spinach NR-Ser543. Both plant and yeast 14-3-3 proteins were shown to bind the immobilized peptide ligand in a Mg2+-stimulated manner. Stimulation resulted from a reduction in KD and an increase in steady-state binding level (Req). As shown previously for plant 14-3-3s, fluorescent probes also indicated that yeast BMH2 interacted directly with cations, which bind and affect surface hydrophobicity. Binding of 14-3-3s to the phosphopeptide ligand occurred in the absence of divalent cations when the pH was reduced below neutral, and the basis for enhanced binding was a reduction in K(D). At pH 7.5 (+Mg2+), AMP inhibited binding of plant 14-3-3s to the NR based peptide ligand. The binding of AMP to 14-3-3s was directly demonstrated by equilibrium dialysis (plant), and from the observation that recombinant plant 14-3-3s have a low, but detectable, AMP phosphatase activity.

14-3-3ζ: A numbers game in adipocyte function?

PubMed Central

Lim, Gareth E.; Johnson, James D.

2016-01-01

ABSTRACT Molecular scaffolds are often viewed as passive signaling molecules that facilitate protein-protein interactions. However, new evidence gained from the use of loss-of-function or gain-of-function models is dispelling this notion. Our own recent discovery of 14-3-3ζ as an essential regulator of adipogenesis highlights the complex roles of this member of the 14-3-3 protein family. Depletion of the 14-3-3ζ isoform affected parallel pathways that drive adipocyte development, including pathways controlling the stability of key adipogenic transcription factors and cell cycle progression. Going beyond adipocyte differentiation, this study opens new avenues of research in the context of metabolism, as 14-3-3ζ binds to a variety of well-established metabolic proteins that harbor its canonical phosphorylation binding motifs. This suggests that 14-3-3ζ may contribute to key metabolic signaling pathways, such as those that facilitate glucose uptake and fatty acid metabolism. Herein, we discuss these novel areas of research, which will undoubtedly shed light onto novel roles of 14-3-3ζ, and perhaps its related family members, on glucose homeostasis. PMID:27386155

Phosphorylation Dependence and Stoichiometry of the Complex Formed by Tyrosine Hydroxylase and 14-3-3γ*

PubMed Central

Kleppe, Rune; Rosati, Sara; Jorge-Finnigan, Ana; Alvira, Sara; Ghorbani, Sadaf; Haavik, Jan; Valpuesta, José María; Heck, Albert J. R.; Martinez, Aurora

2014-01-01

Phosphorylated tyrosine hydroxylase (TH) can form complexes with 14-3-3 proteins, resulting in enzyme activation and stabilization. Although TH was among the first binding partners identified for these ubiquitous regulatory proteins, the binding stoichiometry and the activation mechanism remain unknown. To address this, we performed native mass spectrometry analyses of human TH (nonphosphorylated or phosphorylated on Ser19 (TH-pS19), Ser40 (TH-pS40), or Ser19 and Ser40 (TH-pS19pS40)) alone and together with 14-3-3γ. Tetrameric TH-pS19 (224 kDa) bound 14-3-3γ (58.3 kDa) with high affinity (Kd = 3.2 nM), generating complexes containing either one (282.4 kDa) or two (340.8 kDa) dimers of 14-3-3. Electron microscopy also revealed one major population of an asymmetric complex, consistent with one TH tetramer and one 14-3-3 dimer, and a minor population of a symmetric complex of one TH tetramer with two 14-3-3 dimers. Lower phosphorylation stoichiometries (0.15–0.54 phosphate/monomer) produced moderate changes in binding kinetics, but native MS detected much less of the symmetric TH:14-3-3γ complex. Interestingly, dephosphorylation of [32P]-TH-pS19 was mono-exponential for low phosphorylation stoichiometries (0.18–0.52), and addition of phosphatase accelerated the dissociation of the TH-pS19:14-3-3γ complex 3- to 4-fold. All together this is consistent with a model in which the pS19 residues in the TH tetramer contribute differently in the association to 14-3-3γ. Complex formation between TH-pS40 and 14-3-3γ was not detected via native MS, and surface plasmon resonance showed that the interaction was very weak. Furthermore, TH-pS19pS40 behaved similarly to TH-pS19 in terms of binding stoichiometry and affinity (Kd = 2.1 nM). However, we found that 14-3-3γ inhibited the phosphorylation rate of TH-pS19 by PKA (3.5-fold) on Ser40. We therefore conclude that Ser40 does not significantly contribute to the binding of 14-3-3γ, and rather has reduced accessibility in

The polyadenosine RNA-binding protein ZC3H14 interacts with the THO complex and coordinately regulates the processing of neuronal transcripts.

PubMed

Morris, Kevin J; Corbett, Anita H

2018-06-15

The polyadenosine RNA-binding protein ZC3H14 is important in RNA processing. Although ZC3H14 is ubiquitously expressed, mutation of the ZC3H14 gene causes a non-syndromic form of intellectual disability. Here, we examine the function of ZC3H14 in the brain by identifying ZC3H14-interacting proteins using unbiased mass spectrometry. Through this analysis, we identified physical interactions between ZC3H14 and multiple RNA processing factors. Notably, proteins that comprise the THO complex were amongst the most enriched proteins. We demonstrate that ZC3H14 physically interacts with THO components and that these proteins are required for proper RNA processing, as loss of ZC3H14 or THO components leads to extended bulk poly(A) tail length. Furthermore, we identified the transcripts Atp5g1 and Psd95 as shared RNA targets of ZC3H14 and the THO complex. Our data suggest that ZC3H14 and the THO complex are important for proper processing of Atp5g1 and Psd95 RNA, as depletion of ZC3H14 or THO components leads to decreased steady-state levels of each mature transcript accompanied by accumulation of Atp5g1 and Psd95 pre-mRNA in the cytoplasm. Taken together, this work provides the first unbiased identification of nuclear ZC3H14-interacting proteins from the brain and links the functions of ZC3H14 and the THO complex in the processing of RNA.

Modulation of 14-3-3/Phosphotarget Interaction by Physiological Concentrations of Phosphate and Glycerophosphates

PubMed Central

Sluchanko, Nikolai N.; Chebotareva, Natalia A.; Gusev, Nikolai B.

2013-01-01

Molecular mechanisms governing selective binding of a huge number of various phosphorylated protein partners to 14-3-3 remain obscure. Phosphate can bind to 14-3-3 and therefore being present at high intracellular concentration, which undergoes significant changes under physiological conditions, phosphate can theoretically regulate interaction of 14-3-3 with phosphorylated targets. In order to check this hypothesis we analyzed effect of phosphate and other natural abundant anions on interaction of 14-3-3 with phosphorylated human small heat shock protein HspB6 (Hsp20) participating in regulation of different intracellular processes. Inorganic phosphate, glycerol-1-phosphate and glycerol-2-phosphate at physiologically relevant concentrations (5-15 mM) significantly destabilized complexes formed by 14-3-3ζ and phosphorylated HspB6 (pHspB6), presumably, via direct interaction with the substrate-binding site of 14-3-3. Phosphate also destabilized complexes between pHspB6 and 14-3-3γ or the monomeric mutant form of 14-3-3ζ. Inorganic sulfate and pyrophosphate were less effective in modulation of 14-3-3 interaction with its target protein. The inhibitory effect of all anions on pHspB6/14-3-3 interaction was concentration-dependent. It is hypothesized that physiological changes in phosphate anions concentration can modulate affinity and specificity of interaction of 14-3-3 with its multiple targets and therefore the actual phosphointeractome of 14-3-3. PMID:23977325

Modulation of 14-3-3/phosphotarget interaction by physiological concentrations of phosphate and glycerophosphates.

PubMed

Sluchanko, Nikolai N; Chebotareva, Natalia A; Gusev, Nikolai B

2013-01-01

Molecular mechanisms governing selective binding of a huge number of various phosphorylated protein partners to 14-3-3 remain obscure. Phosphate can bind to 14-3-3 and therefore being present at high intracellular concentration, which undergoes significant changes under physiological conditions, phosphate can theoretically regulate interaction of 14-3-3 with phosphorylated targets. In order to check this hypothesis we analyzed effect of phosphate and other natural abundant anions on interaction of 14-3-3 with phosphorylated human small heat shock protein HspB6 (Hsp20) participating in regulation of different intracellular processes. Inorganic phosphate, glycerol-1-phosphate and glycerol-2-phosphate at physiologically relevant concentrations (5-15 mM) significantly destabilized complexes formed by 14-3-3ζ and phosphorylated HspB6 (pHspB6), presumably, via direct interaction with the substrate-binding site of 14-3-3. Phosphate also destabilized complexes between pHspB6 and 14-3-3γ or the monomeric mutant form of 14-3-3ζ. Inorganic sulfate and pyrophosphate were less effective in modulation of 14-3-3 interaction with its target protein. The inhibitory effect of all anions on pHspB6/14-3-3 interaction was concentration-dependent. It is hypothesized that physiological changes in phosphate anions concentration can modulate affinity and specificity of interaction of 14-3-3 with its multiple targets and therefore the actual phosphointeractome of 14-3-3.

Arsenite Stress Down-regulates Phosphorylation and 14-3-3 Binding of Leucine-rich Repeat Kinase 2 (LRRK2), Promoting Self-association and Cellular Redistribution*

PubMed Central

Mamais, Adamantios; Chia, Ruth; Beilina, Alexandra; Hauser, David N.; Hall, Christine; Lewis, Patrick A.; Cookson, Mark R.; Bandopadhyay, Rina

2014-01-01

Mutations in the gene encoding leucine-rich repeat kinase 2 (LRRK2) are a common genetic cause of Parkinson disease, but the mechanisms whereby LRRK2 is regulated are unknown. Phosphorylation of LRRK2 at Ser910/Ser935 mediates interaction with 14-3-3. Pharmacological inhibition of its kinase activity abolishes Ser910/Ser935 phosphorylation and 14-3-3 binding, and this effect is also mimicked by pathogenic mutations. However, physiological situations where dephosphorylation occurs have not been defined. Here, we show that arsenite or H2O2-induced stresses promote loss of Ser910/Ser935 phosphorylation, which is reversed by phosphatase inhibition. Arsenite-induced dephosphorylation is accompanied by loss of 14-3-3 binding and is observed in wild type, G2019S, and kinase-dead D2017A LRRK2. Arsenite stress stimulates LRRK2 self-association and association with protein phosphatase 1α, decreases kinase activity and GTP binding in vitro, and induces translocation of LRRK2 to centrosomes. Our data indicate that signaling events induced by arsenite and oxidative stress may regulate LRRK2 function. PMID:24942733

The Polyadenosine RNA-binding Protein, Zinc Finger Cys3His Protein 14 (ZC3H14), Regulates the Pre-mRNA Processing of a Key ATP Synthase Subunit mRNA*

PubMed Central

Wigington, Callie P.; Morris, Kevin J.; Newman, Laura E.; Corbett, Anita H.

2016-01-01

Polyadenosine RNA-binding proteins (Pabs) regulate multiple steps in gene expression. This protein family includes the well studied Pabs, PABPN1 and PABPC1, as well as the newly characterized Pab, zinc finger CCCH-type containing protein 14 (ZC3H14). Mutations in ZC3H14 are linked to a form of intellectual disability. To probe the function of ZC3H14, we performed a transcriptome-wide analysis of cells depleted of either ZC3H14 or the control Pab, PABPN1. Depletion of PABPN1 affected ∼17% of expressed transcripts, whereas ZC3H14 affected only ∼1% of expressed transcripts. To assess the function of ZC3H14 in modulating target mRNAs, we selected the gene encoding the ATP synthase F0 subunit C (ATP5G1) transcript. Knockdown of ZC3H14 significantly reduced ATP5G1 steady-state mRNA levels. Consistent with results suggesting that ATP5G1 turnover increases upon depletion of ZC3H14, double knockdown of ZC3H14 and the nonsense-mediated decay factor, UPF1, rescues ATP5G1 transcript levels. Furthermore, fractionation reveals an increase in the amount of ATP5G1 pre-mRNA that reaches the cytoplasm when ZC3H14 is depleted and that ZC3H14 binds to ATP5G1 pre-mRNA in the nucleus. These data support a role for ZC3H14 in ensuring proper nuclear processing and retention of ATP5G1 pre-mRNA. Consistent with the observation that ATP5G1 is a rate-limiting component for ATP synthase activity, knockdown of ZC3H14 decreases cellular ATP levels and causes mitochondrial fragmentation. These data suggest that ZC3H14 modulates pre-mRNA processing of select mRNA transcripts and plays a critical role in regulating cellular energy levels, observations that have broad implications for proper neuronal function. PMID:27563065

The Fn14 cytoplasmic tail binds tumour-necrosis-factor-receptor-associated factors 1, 2, 3 and 5 and mediates nuclear factor-kappaB activation.

PubMed Central

Brown, Sharron A N; Richards, Christine M; Hanscom, Heather N; Feng, Sheau-Line Y; Winkles, Jeffrey A

2003-01-01

Fn14 is a growth-factor-inducible immediate-early-response gene encoding a 102-amino-acid type I transmembrane protein. The human Fn14 protein was recently identified as a cell-surface receptor for the tumour necrosis factor (TNF) superfamily member named TWEAK (TNF-like weak inducer of apoptosis). In the present paper, we report that the human TWEAK extracellular domain can also bind the murine Fn14 protein. Furthermore, site-specific mutagenesis and directed yeast two-hybrid interaction assays revealed that the TNFR-associated factor (TRAF) 1, 2, 3 and 5 adaptor molecules bind the murine Fn14 cytoplasmic tail at an overlapping, but non-identical, amino acid sequence motif. We also found that TWEAK treatment of quiescent NIH 3T3 cells stimulates inhibitory kappaBalpha phosphorylation and transcriptional activation of a nuclear factor-kappaB (NF-kappaB) enhancer/luciferase reporter construct. Fn14 overexpression in transiently transfected NIH 3T3 cells also promotes NF-kappaB activation, and this cellular response requires an intact TRAF binding site. These results indicate that Fn14 is a functional TWEAK receptor that can associate with four distinct TRAF family members and stimulate the NF-kappaB transcription factor signalling pathway. PMID:12529173

14-3-3 proteins mediate inhibitory effects of cAMP on salt-inducible kinases (SIKs).

PubMed

Sonntag, Tim; Vaughan, Joan M; Montminy, Marc

2018-02-01

The salt-inducible kinase (SIK) family regulates cellular gene expression via the phosphorylation of cAMP-regulated transcriptional coactivators (CRTCs) and class IIA histone deacetylases, which are sequestered in the cytoplasm by phosphorylation-dependent 14-3-3 interactions. SIK activity toward these substrates is inhibited by increases in cAMP signaling, although the underlying mechanism is unclear. Here, we show that the protein kinase A (PKA)-dependent phosphorylation of SIKs inhibits their catalytic activity by inducing 14-3-3 protein binding. SIK1 and SIK3 contain two functional PKA/14-3-3 sites, while SIK2 has four. In keeping with the dimeric nature of 14-3-3s, the presence of multiple binding sites within target proteins dramatically increases binding affinity. As a result, loss of a single 14-3-3-binding site in SIK1 and SIK3 abolished 14-3-3 association and rendered them insensitive to cAMP. In contrast, mutation of three sites in SIK2 was necessary to fully block cAMP regulation. Superimposed on the effects of PKA phosphorylation and 14-3-3 association, an evolutionary conserved domain in SIK1 and SIK2 (the so called RK-rich region; 595-624 in hSIK2) is also required for the inhibition of SIK2 activity. Collectively, these results point to a dual role for 14-3-3 proteins in repressing a family of Ser/Thr kinases as well as their substrates. © 2017 Federation of European Biochemical Societies.

Comparative interactomics: analysis of arabidopsis 14-3-3 complexes reveals highly conserved 14-3-3 interactions between humans and plants.

PubMed

Paul, Anna-Lisa; Liu, Li; McClung, Scott; Laughner, Beth; Chen, Sixue; Ferl, Robert J

2009-04-01

As a first step in the broad characterization of plant 14-3-3 multiprotein complexes in vivo, stringent and specific antibody affinity purification was used to capture 14-3-3s together with their interacting proteins from extracts of Arabidopsis cell suspension cultures. Approximately 120 proteins were identified as potential in vivo 14-3-3 interacting proteins by mass spectrometry of the recovered complexes. Comparison of the proteins in this data set with the 14-3-3 interacting proteins from a similar study in human embryonic kidney cell cultures revealed eight interacting proteins that likely represent reasonably abundant, fundamental 14-3-3 interaction complexes that are highly conserved across all eukaryotes. The Arabidopsis 14-3-3 interaction data set was also compared to a yeast in vivo 14-3-3 interaction data set. Four 14-3-3 interacting proteins are conserved in yeast, humans, and Arabidopsis. Comparisons of the data sets based on biochemical function revealed many additional similarities in the human and Arabidopsis data sets that represent conserved functional interactions, while also leaving many proteins uniquely identified in either Arabidopsis or human cells. In particular, the Arabidopsis interaction data set is enriched for proteins involved in metabolism.

Characterization of 14-3-3 isoforms expressed in the Echinococcus granulosus pathogenic larval stage.

PubMed

Teichmann, Aline; Vargas, Daiani M; Monteiro, Karina M; Meneghetti, Bruna V; Dutra, Cristine S; Paredes, Rodolfo; Galanti, Norbel; Zaha, Arnaldo; Ferreira, Henrique B

2015-04-03

The 14-3-3 protein family of eukaryotic regulators was studied in Echinococcus granulosus, the causative agent of cystic hydatid disease. These proteins mediate important cellular processes in eukaryotes and are expected to play important roles in parasite biology. Six isoforms of E. granulosus 14-3-3 genes and proteins (Eg14-3-3.1-6) were analyzed, and their phylogenetic relationships were established with bona fide 14-3-3 orthologous proteins from eukaryotic species. Eg14-3-3 isoforms with previous evidence of expression (Eg14-3-3.1-4) in E. granulosus pathogenic larval stage (metacestode) were cloned, and recombinant proteins were used for functional studies. These protein isoforms were detected in different components of E. granulosus metacestode, including interface components with the host. The roles that are played by Eg14-3-3 proteins in parasite biology were inferred from the repertoires of interacting proteins with each isoform, as assessed by gel overlay, cross-linking, and affinity chromatography assays. A total of 95 Eg14-3-3 protein ligands were identified by mass spectrometry. Eg14-3-3 isoforms have shared partners (44 proteins), indicating some overlapping functions; however, they also bind exclusive partners (51 proteins), suggesting Eg14-3-3 functional specialization. These ligand repertoires indicate the involvement of Eg14-3-3 proteins in multiple biochemical pathways in the E. granulosus metacestode and note some degree of isoform specialization.

Neural precursor cell-expressed developmentally down-regulated protein 4-2 (Nedd4-2) regulation by 14-3-3 protein binding at canonical serum and glucocorticoid kinase 1 (SGK1) phosphorylation sites.

PubMed

Chandran, Sindhu; Li, Hui; Dong, Wuxing; Krasinska, Karolina; Adams, Chris; Alexandrova, Ludmila; Chien, Allis; Hallows, Kenneth R; Bhalla, Vivek

2011-10-28

Regulation of epithelial Na(+) channel (ENaC)-mediated transport in the distal nephron is a critical determinant of blood pressure in humans. Aldosterone via serum and glucocorticoid kinase 1 (SGK1) stimulates ENaC by phosphorylation of the E3 ubiquitin ligase Nedd4-2, which induces interaction with 14-3-3 proteins. However, the mechanisms of SGK1- and 14-3-3-mediated regulation of Nedd4-2 are unclear. There are three canonical SGK1 target sites on Nedd4-2 that overlap phosphorylation-dependent 14-3-3 interaction motifs. Two of these are termed "minor," and one is termed "major," based on weak or strong binding to 14-3-3 proteins, respectively. By mass spectrometry, we found that aldosterone significantly stimulates phosphorylation of a minor, relative to the major, 14-3-3 binding site on Nedd4-2. Phosphorylation-deficient minor site Nedd4-2 mutants bound less 14-3-3 than did wild-type (WT) Nedd4-2, and minor site Nedd4-2 mutations were sufficient to inhibit SGK1 stimulation of ENaC cell surface expression. As measured by pulse-chase and cycloheximide chase assays, a major binding site Nedd4-2 mutant had a shorter cellular half-life than WT Nedd4-2, but this property was not dependent on binding to 14-3-3. Additionally, a dimerization-deficient 14-3-3ε mutant failed to bind Nedd4-2. We conclude that whereas phosphorylation at the Nedd4-2 major site is important for interaction with 14-3-3 dimers, minor site phosphorylation by SGK1 may be the relevant molecular switch that stabilizes Nedd4-2 interaction with 14-3-3 and thus promotes ENaC cell surface expression. We also propose that major site phosphorylation promotes cellular Nedd4-2 protein stability, which potentially represents a novel form of regulation for turnover of E3 ubiquitin ligases.

The phosphatidylinositol transfer protein RdgBβ binds 14-3-3 via its unstructured C-terminus, whereas its lipid-binding domain interacts with the integral membrane protein ATRAP (angiotensin II type I receptor-associated protein).

PubMed

Garner, Kathryn; Li, Michelle; Ugwuanya, Natalie; Cockcroft, Shamshad

2011-10-01

PITPs [PI (phosphatidylinositol) transfer proteins] bind and transfer PI between intracellular membranes and participate in many cellular processes including signalling, lipid metabolism and membrane traffic. The largely uncharacterized PITP RdgBβ (PITPNC1; retinal degeneration type B β), contains a long C-terminal disordered region following its defining N-terminal PITP domain. In the present study we report that the C-terminus contains two tandem phosphorylated binding sites (Ser(274) and Ser(299)) for 14-3-3. The C-terminus also contains PEST sequences which are shielded by 14-3-3 binding. Like many proteins containing PEST sequences, the levels of RdgBβ are regulated by proteolysis. RdgBβ is degraded with a half-life of 4 h following ubiquitination via the proteasome. A mutant RdgBβ which is unable to bind 14-3-3 is degraded even faster with a half-life of 2 h. In vitro, RdgBβ is 100-fold less active than PITPα for PI transfer, and RdgBβ proteins (wild-type and a mutant that cannot bind 14-3-3) expressed in COS-7 cells or endogenous proteins from heart cytosol do not exhibit transfer activity. When cells are treated with PMA, the PITP domain of RdgBβ interacts with the integral membrane protein ATRAP (angiotensin II type I receptor-associated protein; also known as AGTRAP) causing membrane recruitment. We suggest that RdgBβ executes its function following recruitment to membranes via its PITP domain and the C-terminal end of the protein could regulate entry to the hydrophobic cavity.

14-3-3ε Boosts Bleomycin-induced DNA Damage Response by Inhibiting the Drug-Resistant Activity of MVP

PubMed Central

Tang, Siwei; Bai, Chen; Yang, Pengyuan; Chen, Xian

2013-01-01

Major vault protein (MVP) is the predominant constituent of the vault particle, the largest known ribonuclear protein complex. Although emerging evidences have been establishing the links between MVP (vault) and multidrug resistance (MDR), little is known regarding exactly how the MDR activity of MVP is modulated during cellular response to drug-induced DNA damage (DDR). Bleomycin (BLM), an anti-cancer drug, induces DNA double-stranded breaks (DSBs) and consequently triggers the cellular DDR. Due to its physiological implications in hepatocellular carcinoma (HCC) and cell fate decision, 14-3-3ε was chosen as the pathway-specific bait protein to identify the critical target(s) responsible for HCC MDR. By using LC-MS/MS-based proteomic approach, MVP was first identified in the BLM-induced 14-3-3ε interactome formed in HCC cells. Biological characterization revealed that MVP possesses specific activity to promote the resistance to the BLM-induced DDR. On the other hand, 14-3-3ε enhances BLM-induced DDR by interacting with MVP. Mechanistic investigation further revealed that 14-3-3ε, in a phosphorylation-dependent manner, binds to the phosphorylated sites at both Thr52 and Ser864 of the monomer of MVP. Consequently, the phosphorylation-dependent binding between 14-3-3ε and MVP inhibits the drug-resistant activity of MVP for an enhanced DDR to BLM treatment. Our findings provide an insight into the mechanism underlying how the BLM-induced interaction between 14-3-3ε and MVP modulates MDR, implicating novel strategy to overcome the chemotherapeutic resistance through interfering specific protein-protein interactions. PMID:23590642

Kank regulates RhoA-dependent formation of actin stress fibers and cell migration via 14-3-3 in PI3K-Akt signaling.

PubMed

Kakinuma, Naoto; Roy, Badal Chandra; Zhu, Yun; Wang, Yong; Kiyama, Ryoiti

2008-05-05

Phosphoinositide-3 kinase (PI3K)/Akt signaling is activated by growth factors such as insulin and epidermal growth factor (EGF) and regulates several functions such as cell cycling, apoptosis, cell growth, and cell migration. Here, we find that Kank is an Akt substrate located downstream of PI3K and a 14-3-3-binding protein. The interaction between Kank and 14-3-3 is regulated by insulin and EGF and is mediated through phosphorylation of Kank by Akt. In NIH3T3 cells expressing Kank, the amount of actin stress fibers is reduced, and the coexpression of 14-3-3 disrupted this effect. Kank also inhibits insulin-induced cell migration via 14-3-3 binding. Furthermore, Kank inhibits insulin and active Akt-dependent activation of RhoA through binding to 14-3-3. Based on these findings, we hypothesize that Kank negatively regulates the formation of actin stress fibers and cell migration through the inhibition of RhoA activity, which is controlled by binding of Kank to 14-3-3 in PI3K-Akt signaling.

14-3-3 Proteins Interact with a Hybrid Prenyl-Phosphorylation Motif to Inhibit G Proteins

PubMed Central

Riou, Philippe; Kjær, Svend; Garg, Ritu; Purkiss, Andrew; George, Roger; Cain, Robert J.; Bineva, Ganka; Reymond, Nicolas; McColl, Brad; Thompson, Andrew J.; O’Reilly, Nicola; McDonald, Neil Q.; Parker, Peter J.; Ridley, Anne J.

2013-01-01

Summary Signaling through G proteins normally involves conformational switching between GTP- and GDP-bound states. Several Rho GTPases are also regulated by RhoGDI binding and sequestering in the cytosol. Rnd proteins are atypical constitutively GTP-bound Rho proteins, whose regulation remains elusive. Here, we report a high-affinity 14-3-3-binding site at the C terminus of Rnd3 consisting of both the Cys241-farnesyl moiety and a Rho-associated coiled coil containing protein kinase (ROCK)-dependent Ser240 phosphorylation site. 14-3-3 binding to Rnd3 also involves phosphorylation of Ser218 by ROCK and/or Ser210 by protein kinase C (PKC). The crystal structure of a phosphorylated, farnesylated Rnd3 peptide with 14-3-3 reveals a hydrophobic groove in 14-3-3 proteins accommodating the farnesyl moiety. Functionally, 14-3-3 inhibits Rnd3-induced cell rounding by translocating it from the plasma membrane to the cytosol. Rnd1, Rnd2, and geranylgeranylated Rap1A interact similarly with 14-3-3. In contrast to the canonical GTP/GDP switch that regulates most Ras superfamily members, our results reveal an unprecedented mechanism for G protein inhibition by 14-3-3 proteins. PMID:23622247

Direct Interaction between Scaffolding Proteins RACK1 and 14-3-3ζ Regulates Brain-derived Neurotrophic Factor (BDNF) Transcription*

PubMed Central

Neasta, Jérémie; Kiely, Patrick A.; He, Dao-Yao; Adams, David R.; O'Connor, Rosemary; Ron, Dorit

2012-01-01

RACK1 is a scaffolding protein that spatially and temporally regulates numerous signaling cascades. We previously found that activation of the cAMP signaling pathway induces the translocation of RACK1 to the nucleus. We further showed that nuclear RACK1 is required to promote the transcription of the brain-derived neurotrophic factor (BDNF). Here, we set out to elucidate the mechanism underlying cAMP-dependent RACK1 nuclear translocation and BDNF transcription. We identified the scaffolding protein 14-3-3ζ as a direct binding partner of RACK1. Moreover, we found that 14-3-3ζ was necessary for the cAMP-dependent translocation of RACK1 to the nucleus. We further observed that the disruption of RACK1/14-3-3ζ interaction with a peptide derived from the RACK1/14-3-3ζ binding site or shRNA-mediated 14-3-3ζ knockdown inhibited cAMP induction of BDNF transcription. Together, these data reveal that the function of nuclear RACK1 is mediated through its interaction with 14-3-3ζ. As RACK1 and 14-3-3ζ are two multifunctional scaffolding proteins that coordinate a wide variety of signaling events, their interaction is likely to regulate other essential cellular functions. PMID:22069327

Cannabinoid receptor activation inhibits cell cycle progression by modulating 14-3-3β.

PubMed

Jung, Hye-Won; Park, Inae; Ghil, Sungho

2014-09-01

Cannabinoids display various pharmacological activities, including tumor regression, anti-inflammatory and neuroprotective effects. To investigate the molecular mechanisms underlying the pharmacological effects of cannabinoids, we used a yeast two-hybrid system to screen a mouse brain cDNA library for proteins interacting with type 1 cannabinoid receptor (CB1R). Using the intracellular loop 3 of CB1R as bait, we identified 14-3-3β as an interacting partner of CB1R and confirmed their interaction using affinity-binding assays. 14-3-3β has been reported to induce a cell cycle delay at the G2/M phase. We tested the effects of cannabinoids on cell cycle progression in HeLa cells synchronized using a double-thymidine block-and-release protocol and found an increase in the population of G2/M phase cells. We further found that CB1R activation augmented the interaction of 14-3-3β with Wee1 and Cdc25B, and promoted phosphorylation of Cdc2 at Tyr-15. These results suggest that cannabinoids induce cell cycle delay at the G2/M phase by activating 14-3-3β.

The human RNA-binding protein and E3 ligase MEX-3C binds the MEX-3-recognition element (MRE) motif with high affinity.

PubMed

Yang, Lingna; Wang, Chongyuan; Li, Fudong; Zhang, Jiahai; Nayab, Anam; Wu, Jihui; Shi, Yunyu; Gong, Qingguo

2017-09-29

MEX-3 is a K-homology (KH) domain-containing RNA-binding protein first identified as a translational repressor in Caenorhabditis elegans , and its four orthologs (MEX-3A-D) in human and mouse were subsequently found to have E3 ubiquitin ligase activity mediated by a RING domain and critical for RNA degradation. Current evidence implicates human MEX-3C in many essential biological processes and suggests a strong connection with immune diseases and carcinogenesis. The highly conserved dual KH domains in MEX-3 proteins enable RNA binding and are essential for the recognition of the 3'-UTR and post-transcriptional regulation of MEX-3 target transcripts. However, the molecular mechanisms of translational repression and the consensus RNA sequence recognized by the MEX-3C KH domain are unknown. Here, using X-ray crystallography and isothermal titration calorimetry, we investigated the RNA-binding activity and selectivity of human MEX-3C dual KH domains. Our high-resolution crystal structures of individual KH domains complexed with a noncanonical U-rich and a GA-rich RNA sequence revealed that the KH1/2 domains of human MEX-3C bound MRE10, a 10-mer RNA (5'-CAGAGUUUAG-3') consisting of an eight-nucleotide MEX-3-recognition element (MRE) motif, with high affinity. Of note, we also identified a consensus RNA motif recognized by human MEX-3C. The potential RNA-binding sites in the 3'-UTR of the human leukocyte antigen serotype ( HLA-A2 ) mRNA were mapped with this RNA-binding motif and further confirmed by fluorescence polarization. The binding motif identified here will provide valuable information for future investigations of the functional pathways controlled by human MEX-3C and for predicting potential mRNAs regulated by this enzyme. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Phosphorylation of Arabidopsis ubiquitin ligase ATL31 is critical for plant carbon/nitrogen nutrient balance response and controls the stability of 14-3-3 proteins.

PubMed

Yasuda, Shigetaka; Sato, Takeo; Maekawa, Shugo; Aoyama, Shoki; Fukao, Yoichiro; Yamaguchi, Junji

2014-05-30

Ubiquitin ligase plays a fundamental role in regulating multiple cellular events in eukaryotes by fine-tuning the stability and activity of specific target proteins. We have previously shown that ubiquitin ligase ATL31 regulates plant growth in response to nutrient balance between carbon and nitrogen (C/N) in Arabidopsis. Subsequent study demonstrated that ATL31 targets 14-3-3 proteins for ubiquitination and modulates the protein abundance in response to C/N-nutrient status. However, the underlying mechanism for the targeting of ATL31 to 14-3-3 proteins remains unclear. Here, we show that ATL31 interacts with 14-3-3 proteins in a phosphorylation-dependent manner. We identified Thr(209), Ser(247), Ser(270), and Ser(303) as putative 14-3-3 binding sites on ATL31 by motif analysis. Mutation of these Ser/Thr residues to Ala in ATL31 inhibited the interaction with 14-3-3 proteins, as demonstrated by yeast two-hybrid and co-immunoprecipitation analyses. Additionally, we identified in vivo phosphorylation of Thr(209) and Ser(247) on ATL31 by MS analysis. A peptide competition assay showed that the application of synthetic phospho-Thr(209) peptide, but not the corresponding unphosphorylated peptide, suppresses the interaction between ATL31 and 14-3-3 proteins. Moreover, Arabidopsis plants overexpressing mutated ATL31, which could not bind to 14-3-3 proteins, showed accumulation of 14-3-3 proteins and growth arrest in disrupted C/N-nutrient conditions similar to wild-type plants, although overexpression of intact ATL31 resulted in repression of 14-3-3 accumulation and tolerance to the conditions. Together, these results demonstrate that the physiological role of phosphorylation at 14-3-3 binding sites on ATL31 is to modulate the binding ability and stability of 14-3-3 proteins to control plant C/N-nutrient response. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

The mitochondrial targeting chaperone 14-3-3ε regulates a RIG-I translocon that mediates membrane-association and innate antiviral immunity

PubMed Central

Liu, Helene Minyi; Loo, Yueh-Ming; Horner, Stacy M.; Zornetzer, Gregory A.; Katze, Michael G.; Gale, Michael

2012-01-01

Summary RIG-I is a cytosolic pathogen recognition receptor that initiates immune responses against RNA viruses. Upon viral RNA recognition, anti-viral signalling requires RIG-I redistribution from the cytosol to membranes where it binds the adaptor protein, MAVS. Here we identify the mitochondrial targeting chaperone protein, 14-3-3ε, as a RIG-I-binding partner and essential component of a translocation complex or “translocon” containing RIG-I, 14-3-3ε, and the TRIM25 ubiquitin ligase. The RIG-I translocon directs RIG-I redistribution from the cytosol to membranes where it mediates MAVS-dependent innate immune signalling during acute RNA virus infection. 14-3-3ε is essential for the stable interaction of RIG-I with TRIM25, which facilitates RIG-I ubiquitination and initiation of innate immunity against hepatitis C virus and other pathogenic RNA viruses. Our results define 14-3-3ε as a key component of a RIG-I translocon required for innate antiviral immunity. PMID:22607805

The mitochondrial targeting chaperone 14-3-3ε regulates a RIG-I translocon that mediates membrane association and innate antiviral immunity.

PubMed

Liu, Helene Minyi; Loo, Yueh-Ming; Horner, Stacy M; Zornetzer, Gregory A; Katze, Michael G; Gale, Michael

2012-05-17

RIG-I is a cytosolic pathogen recognition receptor that initiates immune responses against RNA viruses. Upon viral RNA recognition, antiviral signaling requires RIG-I redistribution from the cytosol to membranes where it binds the adaptor protein, MAVS. Here we identify the mitochondrial targeting chaperone protein, 14-3-3ε, as a RIG-I-binding partner and essential component of a translocation complex or "translocon" containing RIG-I, 14-3-3ε, and the TRIM25 ubiquitin ligase. The RIG-I translocon directs RIG-I redistribution from the cytosol to membranes where it mediates MAVS-dependent innate immune signaling during acute RNA virus infection. 14-3-3ε is essential for the stable interaction of RIG-I with TRIM25, which facilitates RIG-I ubiquitination and initiation of innate immunity against hepatitis C virus and other pathogenic RNA viruses. Our results define 14-3-3ε as a key component of a RIG-I translocon required for innate antiviral immunity. Copyright © 2012 Elsevier Inc. All rights reserved.

Scaffold Functions of 14-3-3 Adaptors in B Cell Immunoglobulin Class Switch DNA Recombination

PubMed Central

White, Clayton A.; Li, Guideng; Pone, Egest J.; Xu, Zhenming; Casali, Paolo

2013-01-01

Class switch DNA recombination (CSR) of the immunoglobulin heavy chain (IgH) locus crucially diversifies antibody biological effector functions. CSR involves the induction of activation-induced cytidine deaminase (AID) expression and AID targeting to switch (S) regions by 14-3-3 adaptors. 14-3-3 adaptors specifically bind to 5′-AGCT-3′ repeats, which make up for the core of all IgH locus S regions. They selectively target the upstream and downstream S regions that are set to undergo S–S DNA recombination. We hypothesized that 14-3-3 adaptors function as scaffolds to stabilize CSR enzymatic elements on S regions. Here we demonstrate that all seven 14-3-3β, 14-3-3ε, 14-3-3γ, 14-3-3η, 14-3-3σ, 14-3-3τ and 14-3-3ζ adaptors directly interacted with AID, PKA-Cα (catalytic subunit) and PKA-RIα (regulatory inhibitory subunit) and uracil DNA glycosylase (Ung). 14-3-3 adaptors, however, did not interact with AID C-terminal truncation mutant AIDΔ(180–198) or AIDF193A and AIDL196A point-mutants (which have been shown not to bind to S region DNA and fail to mediate CSR). 14-3-3 adaptors colocalized with AID and replication protein A (RPA) in B cells undergoing CSR. 14-3-3 and AID binding to S region DNA was disrupted by viral protein R (Vpr), an accessory protein of human immunodeficiency virus type-1 (HIV-1), which inhibited CSR without altering AID expression or germline IH-CH transcription. Accordingly, we demonstrated that 14-3-3 directly interact with Vpr, which in turn, also interact with AID, PKA-Cα and Ung. Altogether, our findings suggest that 14-3-3 adaptors play important scaffold functions and nucleate the assembly of multiple CSR factors on S regions. They also show that such assembly can be disrupted by a viral protein, thereby allowing us to hypothesize that small molecule compounds that specifically block 14-3-3 interactions with AID, PKA and/or Ung can be used to inhibit unwanted CSR. PMID:24282540

Peroxisome Proliferator-Activated Receptors Protect against Apoptosis via 14-3-3

PubMed Central

Wu, Kenneth K.

2010-01-01

Peroxisome proliferator-activated receptors (PPARs) were reported to prevent cells from stress-induced apoptosis and protect tissues against ischemia-reperfusion injury. The underlying transcriptional mechanism is unclear. Recent reports indicate that the antiapoptotic actions of ligand-activated PPARδ and PPARγ are mediated through enhanced binding of PPAR to the promoter of 14-3-3ε and upregulation of 14-3-3ε expression. We propose that ligand-activated PPARα exerts its anti-apoptotic actions via the identical pathway. The PPAR to 14-3-3 transcriptional axis plays an important role in protection of cell and tissue integrity and is a target for drug discovery. PMID:20862376

Novel soluble, high-affinity gastrin-releasing peptide binding proteins in Swiss 3T3 fibroblasts.

PubMed

Kane, M A; Portanova, L B; Kelley, K; Holley, M; Ross, S E; Boose, D; Escobedo-Morse, A; Alvarado, B

1994-01-01

Swiss 3T3 cells contained substantial amounts of soluble and specific [125I]GRP binders. Like the membrane-associated GRP receptor, they were of high affinity, saturable, bound to GRP(14-27) affinity gels, and exhibited specificity for GRP(14-27) binding. They differed in that acid or freezing destroyed specific binding, specific binding exhibited different time and temperature effects, no detergent was required for their solubilization, ammonium sulfate fractionation yielded different profiles, the M(rs) were lower, GRP(1-16) also blocked binding, and a polyclonal anti-GRP receptor antiserum did not bind on Western blots. The isolated, soluble GRP binding protein(s) rapidly degraded [125I]GRP. These soluble GRP binding proteins may play a role in the regulation of the mitogenic effects of GRP on these cells.

Identification of Novel 14-3-3 Residues That Are Critical for Isoform-specific Interaction with GluN2C to Regulate N-Methyl-d-aspartate (NMDA) Receptor Trafficking*

PubMed Central

Chung, Connie; Wu, Wei-Hua; Chen, Bo-Shiun

2015-01-01

The 14-3-3 family of proteins is widely distributed in the CNS where they are major regulators of essential neuronal functions. There are seven known mammalian 14-3-3 isoforms (ζ,, τ, ϵ, η, β, and σ), which generally function as adaptor proteins. Previously, we have demonstrated that 14-3-3ϵ isoform dynamically regulates forward trafficking of GluN2C-containing NMDA receptors (NMDARs) in cerebellar granule neurons, that when expressed on the surface, promotes neuronal survival following NMDA-induced excitotoxicity. Here, we report 14-3-3 isoform-specific binding and functional regulation of GluN2C. In particular, we show that GluN2C C-terminal domain (CTD) binds to all 14-3-3 isoforms except 14-3-3σ, and binding is dependent on GluN2C serine 1096 phosphorylation. Co-expression of 14-3-3 (ζ and ϵ) and GluN1/GluN2C promotes the forward delivery of receptors to the cell surface. We further identify novel residues serine 145, tyrosine 178, and cysteine 189 on α-helices 6, 7, and 8, respectively, within ζ-isoform as part of the GluN2C binding motif and independent of the canonical peptide binding groove. Mutation of these conserved residues abolishes GluN2C binding and has no functional effect on GluN2C trafficking. Reciprocal mutation of alanine 145, histidine 180, and isoleucine 191 on 14-3-3σ isoform promotes GluN2C binding and surface expression. Moreover, inhibiting endogenous 14-3-3 using a high-affinity peptide inhibitor, difopein, greatly diminishes GluN2C surface expression. Together, these findings highlight the isoform-specific structural and functional differences within the 14-3-3 family of proteins, which determine GluN2C binding and its essential role in targeting the receptor to the cell surface to facilitate glutamatergic neurotransmission. PMID:26229101

Cotton (Gossypium hirsutum) 14-3-3 proteins participate in regulation of fibre initiation and elongation by modulating brassinosteroid signalling.

PubMed

Zhou, Ying; Zhang, Ze-Ting; Li, Mo; Wei, Xin-Zheng; Li, Xiao-Jie; Li, Bing-Ying; Li, Xue-Bao

2015-02-01

Cotton (Gossypium hirsutum) fibre is an important natural raw material for textile industry in the world. Understanding the molecular mechanism of fibre development is important for the development of future cotton varieties with superior fibre quality. In this study, overexpression of Gh14-3-3L in cotton promoted fibre elongation, leading to an increase in mature fibre length. In contrast, suppression of expression of Gh14-3-3L, Gh14-3-3e and Gh14-3-3h in cotton slowed down fibre initiation and elongation. As a result, the mature fibres of the Gh14-3-3 RNAi transgenic plants were significantly shorter than those of wild type. This 'short fibre' phenotype of the 14-3-3 RNAi cotton could be partially rescued by application of 2,4-epibrassinolide (BL). Expression levels of the BR-related and fibre-related genes were altered in the Gh14-3-3 transgenic fibres. Furthermore, we identified Gh14-3-3 interacting proteins (including GhBZR1) in cotton. Site mutation assay revealed that Ser163 in GhBZR1 and Lys51/56/53 in Gh14-3-3L/e/h were required for Gh14-3-3-GhBZR1 interaction. Nuclear localization of GhBZR1 protein was induced by BR, and phosphorylation of GhBZR1 by GhBIN2 kinase was helpful for its binding to Gh14-3-3 proteins. Additionally, 14-3-3-regulated GhBZR1 protein may directly bind to GhXTH1 and GhEXP promoters to regulate gene expression for responding rapid fibre elongation. These results suggested that Gh14-3-3 proteins may be involved in regulating fibre initiation and elongation through their interacting with GhBZR1 to modulate BR signalling. Thus, our study provides the candidate intrinsic genes for improving fibre yield and quality by genetic manipulation. © 2014 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd.

14-3-3γ Prevents Centrosome Amplification and Neoplastic Progression.

PubMed

Mukhopadhyay, Amitabha; Sehgal, Lalit; Bose, Arunabha; Gulvady, Anushree; Senapati, Parijat; Thorat, Rahul; Basu, Srikanta; Bhatt, Khyati; Hosing, Amol S; Balyan, Renu; Borde, Lalit; Kundu, Tapas K; Dalal, Sorab N

2016-06-02

More than 80% of malignant tumors show centrosome amplification and clustering. Centrosome amplification results from aberrations in the centrosome duplication cycle, which is strictly coordinated with DNA-replication-cycle. However, the relationship between cell-cycle regulators and centrosome duplicating factors is not well understood. This report demonstrates that 14-3-3γ localizes to the centrosome and 14-3-3γ loss leads to centrosome amplification. Loss of 14-3-3γ results in the phosphorylation of NPM1 at Thr-199, causing early centriole disjunction and centrosome hyper-duplication. The centrosome amplification led to aneuploidy and increased tumor formation in mice. Importantly, an increase in passage of the 14-3-3γ-knockdown cells led to an increase in the number of cells containing clustered centrosomes leading to the generation of pseudo-bipolar spindles. The increase in pseudo-bipolar spindles was reversed and an increase in the number of multi-polar spindles was observed upon expression of a constitutively active 14-3-3-binding-defective-mutant of cdc25C (S216A) in the 14-3-3γ knockdown cells. The increase in multi-polar spindle formation was associated with decreased cell viability and a decrease in tumor growth. Our findings uncover the molecular basis of regulation of centrosome duplication by 14-3-3γ and inhibition of tumor growth by premature activation of the mitotic program and the disruption of centrosome clustering.

Dynamic interactions between 14-3-3 proteins and phosphoproteins regulate diverse cellular processes

PubMed Central

2004-01-01

14-3-3 proteins exert an extraordinarily widespread influence on cellular processes in all eukaryotes. They operate by binding to specific phosphorylated sites on diverse target proteins, thereby forcing conformational changes or influencing interactions between their targets and other molecules. In these ways, 14-3-3s ‘finish the job’ when phosphorylation alone lacks the power to drive changes in the activities of intracellular proteins. By interacting dynamically with phosphorylated proteins, 14-3-3s often trigger events that promote cell survival – in situations from preventing metabolic imbalances caused by sudden darkness in leaves to mammalian cell-survival responses to growth factors. Recent work linking specific 14-3-3 isoforms to genetic disorders and cancers, and the cellular effects of 14-3-3 agonists and antagonists, indicate that the cellular complement of 14-3-3 proteins may integrate the specificity and strength of signalling through to different cellular responses. PMID:15167810

Pulmonary microRNA profiles identify involvement of Creb1 and Sec14l3 in bronchial epithelial changes in allergic asthma

PubMed Central

Bartel, Sabine; Schulz, Nikola; Alessandrini, Francesca; Schamberger, Andrea C.; Pagel, Philipp; Theis, Fabian J.; Milger, Katrin; Noessner, Elfriede; Stick, Stephen M.; Kicic, Anthony; Eickelberg, Oliver; Freishtat, Robert J.; Krauss-Etschmann, Susanne

2017-01-01

Asthma is highly prevalent, but current therapies cannot influence the chronic course of the disease. It is thus important to understand underlying early molecular events. In this study, we aimed to use microRNAs (miRNAs) - which are critical regulators of signaling cascades - to identify so far uncharacterized asthma pathogenesis pathways. Therefore, deregulation of miRNAs was assessed in whole lungs from mice with ovalbumin (OVA)-induced allergic airway inflammation (AAI). In silico predicted target genes were confirmed in reporter assays and in house-dust-mite (HDM) induced AAI and primary human bronchial epithelial cells (NHBE) cultured at the air-liquid interface. We identified and validated the transcription factor cAMP-responsive element binding protein (Creb1) and its transcriptional co-activators (Crtc1-3) as targets of miR-17, miR-144, and miR-21. Sec14-like 3 (Sec14l3) - a putative target of Creb1 - was down-regulated in both asthma models and in NHBE cells upon IL13 treatment, while it’s expression correlated with ciliated cell development and decreased along with increasing goblet cell metaplasia. Finally, we propose that Creb1/Crtc1-3 and Sec14l3 could be important for early responses of the bronchial epithelium to Th2-stimuli. This study shows that miRNA profiles can be used to identify novel targets that would be overlooked in mRNA based strategies. PMID:28383034

Pulmonary microRNA profiles identify involvement of Creb1 and Sec14l3 in bronchial epithelial changes in allergic asthma.

PubMed

Bartel, Sabine; Schulz, Nikola; Alessandrini, Francesca; Schamberger, Andrea C; Pagel, Philipp; Theis, Fabian J; Milger, Katrin; Noessner, Elfriede; Stick, Stephen M; Kicic, Anthony; Eickelberg, Oliver; Freishtat, Robert J; Krauss-Etschmann, Susanne

2017-04-06

Asthma is highly prevalent, but current therapies cannot influence the chronic course of the disease. It is thus important to understand underlying early molecular events. In this study, we aimed to use microRNAs (miRNAs) - which are critical regulators of signaling cascades - to identify so far uncharacterized asthma pathogenesis pathways. Therefore, deregulation of miRNAs was assessed in whole lungs from mice with ovalbumin (OVA)-induced allergic airway inflammation (AAI). In silico predicted target genes were confirmed in reporter assays and in house-dust-mite (HDM) induced AAI and primary human bronchial epithelial cells (NHBE) cultured at the air-liquid interface. We identified and validated the transcription factor cAMP-responsive element binding protein (Creb1) and its transcriptional co-activators (Crtc1-3) as targets of miR-17, miR-144, and miR-21. Sec14-like 3 (Sec14l3) - a putative target of Creb1 - was down-regulated in both asthma models and in NHBE cells upon IL13 treatment, while it's expression correlated with ciliated cell development and decreased along with increasing goblet cell metaplasia. Finally, we propose that Creb1/Crtc1-3 and Sec14l3 could be important for early responses of the bronchial epithelium to Th2-stimuli. This study shows that miRNA profiles can be used to identify novel targets that would be overlooked in mRNA based strategies.

Dynamic interaction between 14-3-3zeta and bax during TNF-α-induced apoptosis in living cells

NASA Astrophysics Data System (ADS)

Gao, Xuejuan; Xing, Da; Chen, Tongsheng

2006-09-01

Bax, a proapoptotic member of the Bcl-2 family, localizes largely in the cytoplasm but redistributes to mitochondria and undergoes oligomerization to induce the release of apoptogenic factors such as cytochrome c in response to apoptotic stimuli. Cytoplasmic protein 14-3-3zeta binds to Bax and, upon apoptotic stimulation, releases Bax by a caspase-independent mechanism. However, the direct interaction of the cytoplasmic 14-3-3zeta and Bax in living cells has not been observed. In present study, to monitor the dynamic interaction between 14-3-3zeta and Bax in living cells in real time during apoptosis induced by tumor necrosis factor (TNF-α), DsRed-14-3-3zeta plasmid is constructed. By cotransfecting DsRed- 14-3-3zeta and GFP-Bax plasmids into human lung adenocarcinoma cells (ASTC-a-1), we observe the dynamic interaction between Bax and 14-3-3zeta using fluorescence resonance energy transfer (FRET) technique on laser scanning confocal microscope. The results show that 14-3-3zeta remains in the cytoplasm but GFP-Bax translocates to mitochondria completely after TNF-α stimulation. These results reveal that 14-3-3zeta binds directly to Bax in healthy cells, and that 14-3-3zeta negatively regulates Bax translocation to mitochondria during TNF-α-induced apoptosis.

Multiple elements regulate nuclear/cytoplasmic shuttling of FOXO1: characterization of phosphorylation- and 14-3-3-dependent and -independent mechanisms.

PubMed Central

Zhao, Xiangshan; Gan, Lixia; Pan, Haiyun; Kan, Donghui; Majeski, Michael; Adam, Stephen A; Unterman, Terry G

2004-01-01

FOXO1, a Forkhead transcription factor, is an important target of insulin and growth factor action. Phosphorylation of Thr-24, Ser-256 and Ser-319 promotes nuclear exclusion of FOXO1, yet the mechanisms regulating nuclear/cytoplasmic shuttling of FOXO1 are poorly understood. Previous studies have identified an NLS (nuclear localization signal) in the C-terminal basic region of the DBD (DNA-binding domain), and a leucine-rich, leptomycin-B sensitive NES (nuclear export signal) located further downstream. Here, we find that other elements in the DBD also contribute to nuclear localization, and that multiple mechanisms contribute to nuclear exclusion of FOXO1. Phosphorylation of Ser-319 and a cluster of nearby residues (Ser-322, Ser-325 and Ser-329) functions co-operatively with the nearby NES to promote nuclear exclusion. The N-terminal region of FOXO1 (amino acids 1-149) also is sufficient to promote nuclear exclusion, and does so through multiple mechanisms. Amino acids 1-50 are sufficient to promote nuclear exclusion of green fluorescent protein fusion proteins, and the phosphorylation of Thr-24 is required for this effect. A leucine-rich, leptomycin B-sensitive export signal is also present nearby. Phosphorylated FOXO1 binds 14-3-3 proteins, and co-precipitation studies with tagged proteins indicate that 14-3-3 binding involves co-operative interactions with both Thr-24 and Ser-256. Ser-256 is located in the C-terminal region of the DBD, where 14-3-3 proteins may interfere both with DNA-binding and with nuclear-localization functions. Together, these studies demonstrate that multiple elements contribute to nuclear/cytoplasmic shuttling of FOXO1, and that phosphorylation and 14-3-3 binding regulate the cellular distribution and function of FOXO1 through multiple mechanisms. The presence of these redundant mechanisms supports the concept that the regulation of FOXO1 function plays a critical role in insulin and growth factor action. PMID:14664696

Chemical Genetics of 14-3-3 Regulation and Role in Tumor Development

DTIC Science & Technology

2005-11-01

inhibitors , our group had identified a series of inhibitory compounds. When tested one of these, TK10, shows an inhibitory effect on 14-3-3 sigma nuclear...potential regulators of 14-3-3 sigma function. 5 BODY Determine the biological activity of the newly identified inhibitor of 14-3- &T nuclear export TKI0 I...have previously shown that an inhibitor of FOXOla nuclear export, TK10, inhibits the export of 14- 3-3 from the nucleus while TK10 does not affect

14-3-3η Amplifies Androgen Receptor Actions in Prostate Cancer

PubMed Central

Titus, Mark A.; Tan, Jiann-an; Gregory, Christopher W.; Ford, O. Harris; Subramanian, Romesh R.; Fu, Haian; Wilson, Elizabeth M.; Mohler, James L.; French, Frank S.

2009-01-01

Purpose Androgen receptor (AR) abundance and AR-regulated gene expression in castration-recurrent prostate cancer (CaP) are indicative of AR activation in the absence of testicular androgen. AR transactivation of target genes in castration-recurrent CaP occurs in part through mitogen signaling that amplifies the actions of AR and its coregulators. Herein we report on the role of 14-3-3η in AR action. Experimental Design and Results AR and 14-3-3η co-localized in COS cell nuclei with and without androgen and 14-3-3η promoted AR nuclear localization in the absence of androgen. 14-3-3η interacted with AR in cell-free binding and coimmunoprecipitation assays. In the recurrent human CaP cell line, CWR-R1, native endogenous AR transcriptional activation was stimulated by 14-3-3η at low DHT concentrations and was increased by EGF. Moreover, the DHT and EGF dependent increase in AR transactivation was inhibited by a dominant negative 14-3-3η. In the CWR22 CaP xenograft model, 14-3-3η expression was increased by androgen, suggesting a feed-forward mechanism that potentiates both 14-3-3η and AR actions. 14-3-3η mRNA and protein decreased following castration of tumor bearing mice and increased in tumors of castrate mice after treatment with testosterone. CWR22 tumors that recurred 5 months after castration contained 14-3-3η levels similar to the androgen-stimulated tumors removed before castration. In a human prostate tissue microarray of clinical specimens, 14-3-3η localized with AR in nuclei and the similar amounts expressed in castration-recurrent CaP, androgen-stimulated CaP and benign prostatic hyperplasia were consistent with AR activation in recurrent CaP. Conclusion 14-3-3η enhances androgen and mitogen induced AR transcriptional activity in castration-recurrent CaP. PMID:19996220

Induction of AID-targeting adaptor 14-3-3γ is mediated by NF-κB-dependent recruitment of CFP1 to the 5′-CpG-3′-rich 14-3-3γ promoter and is sustained by E2A

PubMed Central

Mai, Thach; Pone, Egest J.; Li, Guideng; Lam, Tonika S.; Moehlman, J’aime; Xu, Zhenming; Casali, Paolo

2013-01-01

Class switch DNA recombination (CSR) crucially diversifies antibody biological effectors functions. 14-3-3γ specifically binds to the 5′-AGCT-3′ repeats in the IgH locus switch (S) regions. By directly interacting with the C-terminal region of activation-induced cytidine deaminase (AID), 14-3-3γ targets this enzyme to S regions to mediate CSR. Here, we showed that 14-3-3γ was expressed in germinal center B cells in vivo and induced in B cells by T-dependent and T-independent primary CSR-inducing stimuli in vitro in humans and mice. Induction of 14-3-3γ was rapid, peaking within 3 h of stimulation by lipopolysaccharides (LPS), and sustained over the course of AID and CSR induction. It was dependent on recruitment of NF-κB to the 14-3-3γ gene promoter. The NF-κB recruitment enhanced the occupancy of the CpG island within the 14-3-3γ promoter by CFP1, a component of the COMPASS histone methyltransferase complex, and promoter-specific enrichment of histone 3 lysine 4 trimethylation (H3K4me3), which is indicative of open chromatin state and marks transcription-competent promoters. NF-κB also potentiated the binding of B cell lineage-specific factor E2A to an E-box motif located immediately downstream of the two closely-spaced transcription start sites (TSSs) for sustained 14-3-3γ expression and CSR induction. Thus, 14-3-3γ induction in CSR is enabled by the CFP1-mediated H3K4me3 enrichment in the promoter, dependent on NF-κB and sustained by E2A. PMID:23851690

14-3-3ζ and aPKC-ι synergistically facilitate epithelial-mesenchymal transition of cholangiocarcinoma via GSK-3β/Snail signaling pathway.

PubMed

Yang, Yan; Liu, Yan; He, Jun-Chuang; Wang, Jian-Ming; Schemmer, Peter; Ma, Chao-Qun; Qian, Ya-Wei; Yao, Wei; Zhang, Jian; Qi, Wei-Peng; Fu, Yang; Feng, Wei; Yang, Tao

2016-08-23

Cholangiocarcinoma (CCA) invasion and metastasis are the primary causes of poor survival rates in patients. The epithelial-mesenchymal transition (EMT) is a crucial step in cancer invasion and metastasis. However, it is still unclear of the molecular mechanism. In this study, the expression of 14-3-3ζ and atypical protein kinase C-ι (aPKC-ι) was further detected in CCA tissues and cell lines. Meanwhile, we established the EMT model of CCA cells and investigated 14-3-3ζ and aPKC-ι co-regulatory effect on the EMT in vitro and in vivo. Further, we identified the downstream molecular glycogen synthase kinase 3 beta (GSK-3β)/Snail signalling pathway that contribute to regulating the EMT. Our data showed that the expression of 14-3-3ζ and aPKC-ι was synergistically increased in CCA tissues compared with adjacent noncancerous tissues and was intimately associated with differentiation and the tumour-node-metastasis (TNM) stage. Multivariate Cox regression analysis indicated that high 14-3-3ζ and aPKC-ι expression separately predicted a poor prognosis and were independent prognostic indicators in patients with CCA. The CO-IP experiment confirmed that the mutual binding relationship between 14-3-3ζ and aPKC-ι. Small interfering RNAs and siRNA rescue experiment demonstrated that 14-3-3ζ and aPKC-ι regulated each other. In addition, 14-3-3ζ and aPKC-ι pretreatment by si-RNA inhibit the phosphorylated GSK-3β and Snail expression during EMT. Meanwhile, silence of 14-3-3ζ or aPKC-ι suppressed CCA cells migration, metastasis and proliferation in vitro and in vivo. Our study demonstrates that 14-3-3ζ and aPKC-ι synergistically facilitate EMT of CCA via GSK-3β/Snail signalling pathway, and may be potential therapeutic target for CCA.

14-3-3ζ and aPKC-ι synergistically facilitate epithelial-mesenchymal transition of cholangiocarcinoma via GSK-3β/snail signaling pathway

PubMed Central

He, Jun-chuang; Wang, Jian-ming; Schemmer, Peter; Ma, Chao-qun; Qian, Ya-wei; Yao, Wei; Zhang, Jian; Qi, Wei-peng; Fu, Yang; Feng, Wei; Yang, Tao

2016-01-01

Cholangiocarcinoma (CCA) invasion and metastasis are the primary causes of poor survival rates in patients. The epithelial-mesenchymal transition (EMT) is a crucial step in cancer invasion and metastasis. However, it is still unclear of the molecular mechanism. In this study, the expression of 14-3-3ζ and atypical protein kinase C-ι (aPKC-ι) was further detected in CCA tissues and cell lines. Meanwhile, we established the EMT model of CCA cells and investigated 14-3-3ζ and aPKC-ι co-regulatory effect on the EMT in vitro and in vivo. Further, we identified the downstream molecular glycogen synthase kinase 3 beta (GSK-3β)/Snail signalling pathway that contribute to regulating the EMT. Our data showed that the expression of 14-3-3ζ and aPKC-ι was synergistically increased in CCA tissues compared with adjacent noncancerous tissues and was intimately associated with differentiation and the tumour-node-metastasis (TNM) stage. Multivariate Cox regression analysis indicated that high 14-3-3ζ and aPKC-ι expression separately predicted a poor prognosis and were independent prognostic indicators in patients with CCA. The CO-IP experiment confirmed that the mutual binding relationship between 14-3-3ζ and aPKC-ι. Small interfering RNAs and siRNA rescue experiment demonstrated that 14-3-3ζ and aPKC-ι regulated each other. In addition, 14-3-3ζ and aPKC-ι pretreatment by si-RNA inhibit the phosphorylated GSK-3β and Snail expression during EMT. Meanwhile, silence of 14-3-3ζ or aPKC-ι suppressed CCA cells migration, metastasis and proliferation in vitro and in vivo. Our study demonstrates that 14-3-3ζ and aPKC-ι synergistically facilitate EMT of CCA via GSK-3β/Snail signalling pathway, and may be potential therapeutic target for CCA. PMID:27409422

Functional identification of a novel 14-3-3 epsilon splicing variant suggests dimerization is not necessary for 14-3-3 epsilon to inhibit UV-induced apoptosis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Han, Dingding; Ye, Guangming; Liu, Tingting

2010-05-28

14-3-3 proteins function as a dimer and have been identified to involve in diverse signaling pathways. Here we reported the identification of a novel splicing variant of human 14-3-3 epsilon (14-3-3 epsilon sv), which is derived from a novel exon 1' insertion. The insertion contains a stop codon and leads to a truncated splicing variant of 14-3-3 epsilon. The splicing variant is translated from the exon 2 and results in the deletion of an N-terminal {alpha}-helix which is crucial for the dimerization. Therefore, the 14-3-3 epsilon sv could not form a dimer with 14-3-3 zeta. However, after UV irradiation 14-3-3more » epsilon sv could also support cell survival, suggesting monomer of 14-3-3 epsilon is sufficient to protect cell from apoptosis.« less

The RNA-binding protein, ZC3H14, is required for proper poly(A) tail length control, expression of synaptic proteins, and brain function in mice.

PubMed

Rha, Jennifer; Jones, Stephanie K; Fidler, Jonathan; Banerjee, Ayan; Leung, Sara W; Morris, Kevin J; Wong, Jennifer C; Inglis, George Andrew S; Shapiro, Lindsey; Deng, Qiudong; Cutler, Alicia A; Hanif, Adam M; Pardue, Machelle T; Schaffer, Ashleigh; Seyfried, Nicholas T; Moberg, Kenneth H; Bassell, Gary J; Escayg, Andrew; García, Paul S; Corbett, Anita H

2017-10-01

A number of mutations in genes that encode ubiquitously expressed RNA-binding proteins cause tissue specific disease. Many of these diseases are neurological in nature revealing critical roles for this class of proteins in the brain. We recently identified mutations in a gene that encodes a ubiquitously expressed polyadenosine RNA-binding protein, ZC3H14 (Zinc finger CysCysCysHis domain-containing protein 14), that cause a nonsyndromic, autosomal recessive form of intellectual disability. This finding reveals the molecular basis for disease and provides evidence that ZC3H14 is essential for proper brain function. To investigate the role of ZC3H14 in the mammalian brain, we generated a mouse in which the first common exon of the ZC3H14 gene, exon 13 is removed (Zc3h14Δex13/Δex13) leading to a truncated ZC3H14 protein. We report here that, as in the patients, Zc3h14 is not essential in mice. Utilizing these Zc3h14Δex13/Δex13mice, we provide the first in vivo functional characterization of ZC3H14 as a regulator of RNA poly(A) tail length. The Zc3h14Δex13/Δex13 mice show enlarged lateral ventricles in the brain as well as impaired working memory. Proteomic analysis comparing the hippocampi of Zc3h14+/+ and Zc3h14Δex13/Δex13 mice reveals dysregulation of several pathways that are important for proper brain function and thus sheds light onto which pathways are most affected by the loss of ZC3H14. Among the proteins increased in the hippocampi of Zc3h14Δex13/Δex13 mice compared to control are key synaptic proteins including CaMK2a. This newly generated mouse serves as a tool to study the function of ZC3H14 in vivo. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Dual phosphorylation of Btk by Akt/protein kinase b provides docking for 14-3-3ζ, regulates shuttling, and attenuates both tonic and induced signaling in B cells.

PubMed

Mohammad, Dara K; Nore, Beston F; Hussain, Alamdar; Gustafsson, Manuela O; Mohamed, Abdalla J; Smith, C I Edvard

2013-08-01

Bruton's tyrosine kinase (Btk) is crucial for B-lymphocyte activation and development. Mutations in the Btk gene cause X-linked agammaglobulinemia (XLA) in humans and X-linked immunodeficiency (Xid) in mice. Using tandem mass spectrometry, 14-3-3ζ was identified as a new binding partner and negative regulator of Btk in both B-cell lines and primary B lymphocytes. The activated serine/threonine kinase Akt/protein kinase B (PKB) phosphorylated Btk on two sites prior to 14-3-3ζ binding. The interaction sites were mapped to phosphoserine pS51 in the pleckstrin homology domain and phosphothreonine pT495 in the kinase domain. The double-alanine, S51A/T495A, replacement mutant failed to bind 14-3-3ζ, while phosphomimetic aspartate substitutions, S51D/T495D, caused enhanced interaction. The phosphatidylinositol 3-kinase (PI3-kinase) inhibitor LY294002 abrogated S51/T495 phosphorylation and binding. A newly characterized 14-3-3 inhibitor, BV02, reduced binding, as did the Btk inhibitor PCI-32765 (ibrutinib). Interestingly, in the presence of BV02, phosphorylation of Btk, phospholipase Cγ2, and NF-κB increased strongly, suggesting that 14-3-3 also regulates B-cell receptor (BCR)-mediated tonic signaling. Furthermore, downregulation of 14-3-3ζ elevated nuclear translocation of Btk. The loss-of-function mutant S51A/T495A showed reduced tyrosine phosphorylation and ubiquitination. Conversely, the gain-of-function mutant S51D/T495D exhibited intense tyrosine phosphorylation, associated with Btk ubiquitination and degradation, likely contributing to the termination of BCR signaling. Collectively, this suggests that Btk could become an important new candidate for the general study of 14-3-3-mediated regulation.

Dual Phosphorylation of Btk by Akt/Protein Kinase B Provides Docking for 14-3-3ζ, Regulates Shuttling, and Attenuates both Tonic and Induced Signaling in B Cells

PubMed Central

Nore, Beston F.; Hussain, Alamdar; Gustafsson, Manuela O.; Mohamed, Abdalla J.

2013-01-01

Bruton's tyrosine kinase (Btk) is crucial for B-lymphocyte activation and development. Mutations in the Btk gene cause X-linked agammaglobulinemia (XLA) in humans and X-linked immunodeficiency (Xid) in mice. Using tandem mass spectrometry, 14-3-3ζ was identified as a new binding partner and negative regulator of Btk in both B-cell lines and primary B lymphocytes. The activated serine/threonine kinase Akt/protein kinase B (PKB) phosphorylated Btk on two sites prior to 14-3-3ζ binding. The interaction sites were mapped to phosphoserine pS51 in the pleckstrin homology domain and phosphothreonine pT495 in the kinase domain. The double-alanine, S51A/T495A, replacement mutant failed to bind 14-3-3ζ, while phosphomimetic aspartate substitutions, S51D/T495D, caused enhanced interaction. The phosphatidylinositol 3-kinase (PI3-kinase) inhibitor LY294002 abrogated S51/T495 phosphorylation and binding. A newly characterized 14-3-3 inhibitor, BV02, reduced binding, as did the Btk inhibitor PCI-32765 (ibrutinib). Interestingly, in the presence of BV02, phosphorylation of Btk, phospholipase Cγ2, and NF-κB increased strongly, suggesting that 14-3-3 also regulates B-cell receptor (BCR)-mediated tonic signaling. Furthermore, downregulation of 14-3-3ζ elevated nuclear translocation of Btk. The loss-of-function mutant S51A/T495A showed reduced tyrosine phosphorylation and ubiquitination. Conversely, the gain-of-function mutant S51D/T495D exhibited intense tyrosine phosphorylation, associated with Btk ubiquitination and degradation, likely contributing to the termination of BCR signaling. Collectively, this suggests that Btk could become an important new candidate for the general study of 14-3-3-mediated regulation. PMID:23754751

Subcellular localization of full-length human myeloid leukemia factor 1 (MLF1) is independent of 14-3-3 proteins.

PubMed

Molzan, Manuela; Ottmann, Christian

2013-03-01

Myeloid leukemia factor 1 (MLF1) is associated with the development of leukemic diseases such as acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS). However, information on the physiological function of MLF1 is limited and mostly derived from studies identifying MLF1 interaction partners like CSN3, MLF1IP, MADM, Manp and the 14-3-3 proteins. The 14-3-3-binding site surrounding S34 is one of the only known functional features of the MLF1 sequence, along with one nuclear export sequence (NES) and two nuclear localization sequences (NLS). It was recently shown that the subcellular localization of mouse MLF1 is dependent on 14-3-3 proteins. Based on these findings, we investigated whether the subcellular localization of human MLF1 was also directly 14-3-3-dependent. Live cell imaging with GFP-fused human MLF1 was used to study the effects of mutations and deletions on its subcellular localization. Surprisingly, we found that the subcellular localization of full-length human MLF1 is 14-3-3-independent, and is probably regulated by other as-yet-unknown proteins.

Identification of a host 14-3-3 Protein that Interacts with Xanthomonas effector AvrRxv

PubMed Central

Whalen, Maureen; Richter, Todd; Zakhareyvich, Kseniya; Yoshikawa, Masayasu; Al-Azzeh, Dana; Adefioye, Adeshola; Spicer, Greg; Mendoza, Laura L.; Morales, Christine Q.; Klassen, Vicki; Perez-Baron, Gina; Toebe, Carole S.; Tzovolous, Ageliki; Gerstman, Emily; Evans, Erika; Thompson, Cheryl; Lopez, Mary; Ronald, Pamela C.

2009-01-01

AvrRxv is a member of a family of pathogen effectors present in pathogens of both plant and mammalian species. Xanthomonas campestris pv. vesicatoria strains carrying AvrRxv induce a hypersensitive response (HR) in the tomato cultivar Hawaii 7998. Using a yeast two-hybrid screen, we identified a 14-3-3 protein from tomato that interacts with AvrRxv called AvrRxv Interactor 1 (ARI1). The interaction was confirmed in vitro with affinity chromatography. Using mutagenesis, we identified a 14-3-3-binding domain in AvrRxv and demonstrated that a mutant in that domain showed concomitant loss of interaction with ARI1 and HR-inducing activity in tomato. These results demonstrate that the AvrRxv bacterial effector recruits 14-3-3 proteins for its function within host cells. AvrRxv homologues YopP and YopJ from Yersinia do not have AvrRxv-specific HR-inducing activity when delivered into tomato host cells by Agrobacterium. Although YopP itself cannot induce HR, its C-terminal domain containing the catalytic residues can replace that of AvrRxv in an AvrRxv-YopP chimera for HR-inducing activity. Phylogenetic analysis indicates that the sequences encoding the C-termini of family members are evolving independently from those encoding the N-termini. Our results support a model in which there are three functional domains in proteins of the family, translocation, interaction, and catalytic. PMID:21796232

Differential endothelial transcriptomics identifies semaphorin 3G as a vascular class 3 semaphorin.

PubMed

Kutschera, Simone; Weber, Holger; Weick, Anja; De Smet, Frederik; Genove, Guillem; Takemoto, Minoru; Prahst, Claudia; Riedel, Maria; Mikelis, Constantinos; Baulande, Sylvain; Champseix, Catherine; Kummerer, Petra; Conseiller, Emmanuel; Multon, Marie-Christine; Heroult, Melanie; Bicknell, Roy; Carmeliet, Peter; Betsholtz, Christer; Augustin, Hellmut G

2011-01-01

To characterize the role of a vascular-expressed class 3 semaphorin (semaphorin 3G [Sema3G]). Semaphorins have been identified as axon guidance molecules. Yet, they have more recently also been characterized as attractive and repulsive regulators of angiogenesis. Through a transcriptomic screen, we identified Sema3G as a molecule of angiogenic endothelial cells. Sema3G-deficient mice are viable and exhibit no overt vascular phenotype. Yet, LacZ expression in the Sema3G locus revealed intense arterial vascular staining in the angiogenic vasculature, starting at E9.5, which was detectable throughout adolescence and downregulated in adult vasculature. Sema3G is expressed as a full-length 100-kDa secreted molecule that is processed by furin proteases to yield 95- and a 65-kDa Sema domain-containing subunits. Full-length Sema3G binds to NP2, whereas processed Sema3G binds to NP1 and NP2. Expression profiling and cellular experiments identified autocrine effects of Sema3G on endothelial cells and paracrine effects on smooth muscle cells. Although the mouse knockout phenotype suggests compensatory mechanisms, the experiments identify Sema3G as a primarily endothelial cell-expressed class 3 semaphorin that controls endothelial and smooth muscle cell functions in autocrine and paracrine manners, respectively.

Site-specific regulatory interaction between spinach leaf sucrose-phosphate synthase and 14-3-3 proteins

NASA Technical Reports Server (NTRS)

Toroser, D.; Athwal, G. S.; Huber, S. C.; Davies, E. (Principal Investigator)

1998-01-01

We report an Mg2+-dependent interaction between spinach leaf sucrose-phosphate synthase (SPS) and endogenous 14-3-3 proteins, as evidenced by co-elution during gel filtration and co-immunoprecipitation. The content of 14-3-3s associated with an SPS immunoprecipitate was inversely related to activity, and was specifically reduced when tissue was pretreated with 5-aminoimidazole-4-carboxamide riboside, suggesting metabolite control in vivo. A synthetic phosphopeptide based on Ser-229 was shown by surface plasmon resonance to bind a recombinant plant 14-3-3, and addition of the phosphorylated SPS-229 peptide was found to stimulate the SPS activity of an SPS:14-3-3 complex. Taken together, the results suggest a regulatory interaction of 14-3-3 proteins with Ser-229 of SPS.

14-3-3 Proteins Modulate the ETS Transcription Factor ETV1 in Prostate Cancer

PubMed Central

Oh, Sangphil; Shin, Sook; Lightfoot, Stan A.; Janknecht, Ralf

2013-01-01

Overexpression of the ETS-related transcription factor ETV1 can initiate neoplastic transformation of the prostate. ETV1 activity is highly regulated by phosphorylation, but the underlying mechanisms are unknown. Here we report that all 14-3-3 proteins, with the exception of the tumor suppressor 14-3-3σ, can bind to ETV1 in a condition manner dictated by its prominent phosphorylation site S216. All non-σ 14-3-3 proteins synergized with ETV1 to activate transcription of its target genes MMP-1 and MMP-7, which regulate extracellular matrix in the prostate tumor microenvironment. S216 mutation or 14-3-3τ downregulation was sufficient to reduce ETV1 protein levels in prostate cancer cells, indicating that non-σ 14-3-3 proteins protect ETV1 from degradation. Notably, S216 mutation also decreased ETV1-dependent migration and invasion in benign prostate cells. Downregulation of 14-3-3τ reduced prostate cancer cell invasion and growth in the same manner as ETV1 attenuation. Lastly, we showed that 14-3-3τ and 14-3-3ε were overexpressed in human prostate tumors. Taken together, our results demonstrated that non-σ 14-3-3 proteins are important modulators of ETV1 function that promote prostate tumorigenesis. PMID:23774214

14-3-3 α and 14-3-3 ζ contribute to immune responses in planarian Dugesia japonica.

PubMed

Lu, Qingqing; Wu, Suge; Zhen, Hui; Deng, Hongkuan; Song, Qian; Ma, Kaifu; Cao, Zhonghong; Pang, Qiuxiang; Zhao, Bosheng

2017-06-05

14-3-3 proteins are a family of highly conserved acidic proteins that regulate cellular processes. They act as a kind of important signaling molecules taking part in many crucial decisions throughout the development process. We have isolated and characterized two members of the 14-3-3 family, namely, Dj14-3-3 α and Dj14-3-3 ζ in the planarian Dugesia japonica. The Dj14-3-3 α and ζ genes encode polypeptides of 260 and 255 amino acids respectively. We have proved that the Dj14-3-3 α and ζ genes were especially expressed in the pharynx in adult and regenerating planarians by in situ hybridization and they were not involved in regeneration process. Besides, Dj14-3-3 α and ζ genes can compensate each other in planarians by RNA interference. The Dj14-3-3 α and ζ were significantly up-regulated expression when planarians were stimulated with the pathogen-associated molecular patterns including lipopolysaccharide (LPS), peptidoglycan (PGN), β-Glu and Poly (I:C), indicating that the Dj14-3-3 α and ζ may be involved in the immune responses. Copyright © 2017 Elsevier B.V. All rights reserved.

Plant 14-3-3 proteins assist ion channels and pumps.

PubMed

de Boer, A H

2002-08-01

Turgor pressure is a cellular parameter, important for a range of physiological processes in plants, like cell elongation, gas exchange and gravitropic/phototropic bending. Regulation of turgor pressure involves ion and water transport at the expense of metabolic energy (ATP). The primary pump in the plasma membrane (the H(+)-ATPase) is a key player in turgor regulation since it provides the driving force for ion uptake, followed by water influx through osmosis. Using the phytotoxin fusicoccin (a well-known activator of the ATPase) as a tool, 14-3-3 proteins were identified as regulators of the H(+)-ATPase. Since fusicoccin has a dramatic effect on K(+) accumulation and cellular respiration as well, we studied whether 14-3-3 proteins play a role in the regulation of the mitochondrial F(0)F(1)-ATP synthase and ion channels in the vacuolar and plasma membranes. Besides the plasma membrane H(+)-ATPase, we have identified thus far at least four other transport proteins that are regulated by 14-3-3 proteins. The mechanism of regulation will be described and the possibility that 14-3-3 proteins act as coordinators of ion transporters with varied but interdependent functions will be discussed.

Differential interaction and aggregation of 3-repeat and 4-repeat tau isoforms with 14-3-3{zeta} protein

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sadik, Golam; Tanaka, Toshihisa, E-mail: tanaka@psy.med.osaka-u.ac.jp; Kato, Kiyoko

2009-05-22

Tau isoforms, 3-repeat (3R) and 4-repeat tau (4R), are differentially involved in neuronal development and in several tauopathies. 14-3-3 protein binds to tau and 14-3-3/tau association has been found both in the development and in tauopathies. To understand the role of 14-3-3 in the differential regulation of tau isoforms, we have performed studies on the interaction and aggregation of 3R-tau and 4R-tau, either phosphorylated or unphosphorylated, with 14-3-3{zeta}. We show by surface plasmon resonance studies that the interaction between unphosphorylated 3R-tau and 14-3-3{zeta} is {approx}3-folds higher than that between unphosphorylated 4R-tau and 14-3-3{zeta}. Phosphorylation of tau by protein kinase Amore » (PKA) increases the affinity of both 3R- and 4R-tau for 14-3-3{zeta} to a similar level. An in vitro aggregation assay employing both transmission electron microscopy and fluorescence spectroscopy revealed the aggregation of unphosphorylated 4R-tau to be significantly higher than that of unphosphorylated 3R-tau following the induction of 14-3-3{zeta}. The filaments formed from 3R- and 4R-tau were almost similar in morphology. In contrast, the aggregation of both 3R- and 4R-tau was reduced to a similar low level after phosphorylation with PKA. Taken together, these results suggest that 14-3-3{zeta} exhibits a similar role for tau isoforms after PKA-phosphorylation, but a differential role for unphosphorylated tau. The significant aggregation of 4R-tau by 14-3-3{zeta} suggests that 14-3-3 may act as an inducer in the generation of 4R-tau-predominant neurofibrillary tangles in tauopathies.« less

14-3-3 proteins regulate desmosomal adhesion via plakophilins.

PubMed

Rietscher, Katrin; Keil, René; Jordan, Annemarie; Hatzfeld, Mechthild

2018-05-22

Desmosomes are essential for strong intercellular adhesion and are abundant in tissues exposed to mechanical strain. At the same time, desmosomes need to be dynamic to allow for remodeling of epithelia during differentiation or wound healing. Phosphorylation of desmosomal plaque proteins appears to be essential for desmosome dynamics. However, the mechanisms of how context-dependent post-translational modifications regulate desmosome formation, dynamics or stability are incompletely understood. Here, we show that growth factor signaling regulates the phosphorylation-dependent association of plakophilins 1 and 3 (PKP1 and PKP3) with 14-3-3 protein isoforms, and uncover unique and partially antagonistic functions of members of the 14-3-3 family in the regulation of desmosomes. 14-3-3γ associated primarily with cytoplasmic PKP1 phosphorylated at S155 and destabilized intercellular cohesion of keratinocytes by reducing its incorporation into desmosomes. In contrast, 14-3-3σ (also known as stratifin, encoded by SFN ) interacted preferentially with S285-phosphorylated PKP3 to promote its accumulation at tricellular contact sites, leading to stable desmosomes. Taken together, our study identifies a new layer of regulation of intercellular adhesion by 14-3-3 proteins. © 2018. Published by The Company of Biologists Ltd.

Analysis of 14-3-3 Family Member Function in Xenopus Embryos by Microinjection of Antisense Morpholino Oligos

NASA Astrophysics Data System (ADS)

Lau, Jeffrey M. C.; Muslin, Anthony J.

The 14-3-3 intracellular phosphoserine/threonine-binding proteins are adapter molecules that regulate signal transduction, cell cycle, nutrient sensing, apoptotic, and cytoskeletal pathways. There are seven 14-3-3 family members, encoded by separate genes, in vertebrate organisms. To evaluate the role of individual 14-3-3 proteins in vertebrate embryonic development, we utilized an antisense morpholino oligo microinjection technique in Xenopus laevis embryos. By use of this method, we showed that embryos lacking specific 14-3-3 proteins displayed unique phenotypic abnormalities. Specifically, embryos lacking 14-3-3 τ exhibited gastrulation and axial patterning defects, but embryos lacking 14-3-3 γ exhibited eye defects without other abnormalities, and embryos lacking 14-3-3 ζ appeared completely normal. These and other results demonstrate the power and specificity of the morpholino antisense oligo microinjection technique.

Proteomics Profiling Reveals Carbohydrate Metabolic Enzymes and 14-3-3 Proteins Play Important Roles for Starch Accumulation during Cassava Root Tuberization.

PubMed

Wang, Xuchu; Chang, Lili; Tong, Zheng; Wang, Dongyang; Yin, Qi; Wang, Dan; Jin, Xiang; Yang, Qian; Wang, Liming; Sun, Yong; Huang, Qixing; Guo, Anping; Peng, Ming

2016-01-21

Cassava is one of the most important root crops as a reliable source of food and carbohydrates. Carbohydrate metabolism and starch accumulation in cassava storage root is a cascade process that includes large amounts of proteins and cofactors. Here, comparative proteomics were conducted in cassava root at nine developmental stages. A total of 154 identified proteins were found to be differentially expressed during starch accumulation and root tuberization. Many enzymes involved in starch and sucrose metabolism were significantly up-regulated, and functional classification of the differentially expressed proteins demonstrated that the majority were binding-related enzymes. Many proteins were took part in carbohydrate metabolism to produce energy. Among them, three 14-3-3 isoforms were induced to be clearly phosphorylated during storage root enlargement. Overexpression of a cassava 14-3-3 gene in Arabidopsis thaliana confirmed that the older leaves of these transgenic plants contained higher sugar and starch contents than the wild-type leaves. The 14-3-3 proteins and their binding enzymes may play important roles in carbohydrate metabolism and starch accumulation during cassava root tuberization. These results not only deepened our understanding of the tuberous root proteome, but also uncovered new insights into carbohydrate metabolism and starch accumulation during cassava root enlargement.

Proteomics Profiling Reveals Carbohydrate Metabolic Enzymes and 14-3-3 Proteins Play Important Roles for Starch Accumulation during Cassava Root Tuberization

PubMed Central

Wang, Xuchu; Chang, Lili; Tong, Zheng; Wang, Dongyang; Yin, Qi; Wang, Dan; Jin, Xiang; Yang, Qian; Wang, Liming; Sun, Yong; Huang, Qixing; Guo, Anping; Peng, Ming

2016-01-01

Cassava is one of the most important root crops as a reliable source of food and carbohydrates. Carbohydrate metabolism and starch accumulation in cassava storage root is a cascade process that includes large amounts of proteins and cofactors. Here, comparative proteomics were conducted in cassava root at nine developmental stages. A total of 154 identified proteins were found to be differentially expressed during starch accumulation and root tuberization. Many enzymes involved in starch and sucrose metabolism were significantly up-regulated, and functional classification of the differentially expressed proteins demonstrated that the majority were binding-related enzymes. Many proteins were took part in carbohydrate metabolism to produce energy. Among them, three 14-3-3 isoforms were induced to be clearly phosphorylated during storage root enlargement. Overexpression of a cassava 14-3-3 gene in Arabidopsis thaliana confirmed that the older leaves of these transgenic plants contained higher sugar and starch contents than the wild-type leaves. The 14-3-3 proteins and their binding enzymes may play important roles in carbohydrate metabolism and starch accumulation during cassava root tuberization. These results not only deepened our understanding of the tuberous root proteome, but also uncovered new insights into carbohydrate metabolism and starch accumulation during cassava root enlargement. PMID:26791570

Modulators of 14-3-3 Protein–Protein Interactions

PubMed Central

2017-01-01

Direct interactions between proteins are essential for the regulation of their functions in biological pathways. Targeting the complex network of protein–protein interactions (PPIs) has now been widely recognized as an attractive means to therapeutically intervene in disease states. Even though this is a challenging endeavor and PPIs have long been regarded as “undruggable” targets, the last two decades have seen an increasing number of successful examples of PPI modulators, resulting in growing interest in this field. PPI modulation requires novel approaches and the integrated efforts of multiple disciplines to be a fruitful strategy. This perspective focuses on the hub-protein 14-3-3, which has several hundred identified protein interaction partners, and is therefore involved in a wide range of cellular processes and diseases. Here, we aim to provide an integrated overview of the approaches explored for the modulation of 14-3-3 PPIs and review the examples resulting from these efforts in both inhibiting and stabilizing specific 14-3-3 protein complexes by small molecules, peptide mimetics, and natural products. PMID:28968506

Participation of 14-3-3ε and 14-3-3ζ proteins in the phagocytosis, component of cellular immune response, in Aedes mosquito cell lines.

PubMed

Trujillo-Ocampo, Abel; Cázares-Raga, Febe Elena; Del Angel, Rosa María; Medina-Ramírez, Fernando; Santos-Argumedo, Leopoldo; Rodríguez, Mario H; Hernández-Hernández, Fidel de la Cruz

2017-08-01

Better knowledge of the innate immune system of insects will improve our understanding of mosquitoes as potential vectors of diverse pathogens. The ubiquitously expressed 14-3-3 protein family is evolutionarily conserved from yeast to mammals, and at least two isoforms of 14-3-3, the ε and ζ, have been identified in insects. These proteins have been shown to participate in both humoral and cellular immune responses in Drosophila. As mosquitoes of the genus Aedes are the primary vectors for arboviruses, causing several diseases such as dengue fever, yellow fever, Zika and chikungunya fevers, cell lines derived from these mosquitoes, Aag-2 from Aedes aegypti and C6/36 HT from Aedes albopictus, are currently used to study the insect immune system. Here, we investigated the role of 14-3-3 proteins (ε and ζ isoform) in phagocytosis, the main cellular immune responses executed by the insects, using Aedes spp. cell lines. We evaluated the mRNA and protein expression of 14-3-3ε and 14-3-3ζ in C6/36 HT and Aag-2 cells, and demonstrated that both proteins were localised in the cytoplasm. Further, in C6/36 HT cells treated with a 14-3-3 specific inhibitor we observed a notable modification of cell morphology with filopodia-like structure caused through cytoskeleton reorganisation (co-localization of 14-3-3 proteins with F-actin), more importantly the decrease in Salmonella typhimurium, Staphylococcus aureus and E. coli phagocytosis and reduction in phagolysosome formation. Additionally, silencing of 14-3-3ε and 14-3-3ζ expression by mean of specific DsiRNA confirmed the decreased phagocytosis and phagolysosome formation of pHrodo labelled E. coli and S. aureus bacteria by Aag-2 cells. The 14-3-3ε and 14-3-3ζ proteins modulate cytoskeletal remodelling, and are essential for phagocytosis of Gram-positive and Gram-negative bacteria in Aedes spp. cell lines.

14-3-3 protein is a regulator of the mitochondrial and chloroplast ATP synthase.

PubMed

Bunney, T D; van Walraven, H S; de Boer, A H

2001-03-27

Mitochondrial and chloroplast ATP synthases are key enzymes in plant metabolism, providing cells with ATP, the universal energy currency. ATP synthases use a transmembrane electrochemical proton gradient to drive synthesis of ATP. The enzyme complexes function as miniature rotary engines, ensuring energy coupling with very high efficiency. Although our understanding of the structure and functioning of the synthase has made enormous progress in recent years, our understanding of regulatory mechanisms is still rather preliminary. Here we report a role for 14-3-3 proteins in the regulation of ATP synthases. These 14-3-3 proteins are highly conserved phosphoserine/phosphothreonine-binding proteins that regulate a wide range of enzymes in plants, animals, and yeast. Recently, the presence of 14-3-3 proteins in chloroplasts was illustrated, and we show here that plant mitochondria harbor 14-3-3s within the inner mitochondrial-membrane compartment. There, the 14-3-3 proteins were found to be associated with the ATP synthases, in a phosphorylation-dependent manner, through direct interaction with the F(1) beta-subunit. The activity of the ATP synthases in both organelles is drastically reduced by recombinant 14-3-3. The rapid reduction in chloroplast ATPase activity during dark adaptation was prevented by a phosphopeptide containing the 14-3-3 interaction motif, demonstrating a role for endogenous 14-3-3 in the down-regulation of the CF(o)F(1) activity. We conclude that regulation of the ATP synthases by 14-3-3 represents a mechanism for plant adaptation to environmental changes such as light/dark transitions, anoxia in roots, and fluctuations in nutrient supply.

Cytochrome bo(3) from Escherichia coli: the binding and turnover of nitric oxide.

PubMed

Butler, Clive; Forte, Elena; Maria Scandurra, Francesca; Arese, Marzia; Giuffré, Alessandro; Greenwood, Colin; Sarti, Paolo

2002-09-06

The reaction of nitric oxide (NO) with fast and reduced cytochrome bo(3)(cyt bo(3)) from Escherichia coli has been investigated. The stoichiometry of NO binding to cyt bo(3) was determined using an NO electrode in the [NO] range 1-14 microM. Under reducing conditions, the initial decrease in [NO] following the addition of cyt bo(3) corresponded to binding of 1 NO molecule per cyt bo(3) functional unit. After this "rapid" NO binding phase, there was a slow, but significant rate of NO consumption ( approximately 0.3molNOmol bo(3)(-1)min(-1)), indicating that cyt bo(3) possesses a low level of NO reductase activity. The binding of NO to fast pulsed enzyme was also investigated. The results show that in the [NO] range used (1-14 microM) both fast and pulsed oxidised cyt bo(3) bind NO with a stoichiometry of 1:1 with an observed dissociation constant of K(d)=5.6+/-0.6 microM and that NO binding was inhibited by the presence of Cl(-). The binding of nitrite to the binuclear centre causes spectral changes similar to those observed upon NO binding to fast cyt bo(3). These results are discussed in relation to the model proposed by Wilson and co-workers [FEBS Lett. 414 (1997) 281] where the binding of NO to Cu(B)(II) results in the formation of the nitrosonium (Cu(B)(I)-NO(+)) complex. NO(+) then reacts with OH(-), a Cu(B) ligand, to form nitrite, which can bind at the binuclear centre. This work suggests for the first time that the binding of NO to oxidised cyt bo(3) does result in the reduction of Cu(B).

Capacity for cooperative binding of thyroid hormone (T3) receptor dimers defines wild type T3 response elements.

PubMed

Brent, G A; Williams, G R; Harney, J W; Forman, B M; Samuels, H H; Moore, D D; Larsen, P R

1992-04-01

Thyroid hormone response elements (T3REs) have been identified in a variety of promoters including those directing expression of rat GH (rGH), alpha-myosin heavy chain (rMHC), and malic enzyme (rME). A detailed biochemical and genetic analysis of the rGH element has shown that it consists of three hexamers related to the consensus [(A/G)GGT(C/A)A]. We have extended this analysis to the rMHC and rME elements. Binding of highly purified thyroid hormone receptor (T3R) to T3REs was determined using the gel shift assay, and thyroid hormone (T3) induction was measured in transient tranfections. We show that the wild type version of each of the three elements binds T3R dimers cooperatively. Mutational analysis of the rMHC and rME elements identified domains important for binding T3R dimers and allowed a direct determination of the relationship between T3R binding and function. In each element two hexamers are required for dimer binding, and mutations that interfere with dimer formation significantly reduce T3 induction. Similar to the rGH element, the rMHC T3RE contains three hexameric domains arranged as a direct repeat followed by an inverted copy, although the third domain is weaker than in rGH. All three are required for full function and T3R binding. The rME T3RE is a two-hexamer direct repeat T3RE, which also binds T3R monomer and dimer. Across a series of mutant elements, there was a strong correlation between dimer binding in vitro and function in vivo for rMHC (r = 0.99, P less than 0.01) and rME (r = 0.67, P less than 0.05) T3REs. Our results demonstrate a similar pattern of T3R dimer binding to a diverse array of hexameric sequences and arrangements in three wild type T3REs. Addition of nuclear protein enhanced T3R binding but did not alter the specificity of binding to wild type or mutant elements. Binding of purified T3R to T3REs was highly correlated with function, both with and without the addition of nuclear protein. T3R dimer formation is the common

Tunicate cytostatic factor TC14-3 induces a polycomb group gene and histone modification through Ca2+ binding and protein dimerization

PubMed Central

2012-01-01

Background As many invertebrate species have multipotent cells that undergo cell growth and differentiation during regeneration and budding, many unique and interesting homeostatic factors are expected to exist in those animals. However, our understanding of such factors and global mechanisms remains very poor. Single zooids of the tunicate, Polyandrocarpa misakiensis, can give off as many as 40 buds during the life span. Bud development proceeds by means of transdifferentiation of very limited number of cells and tissues. TC14-3 is one of several different but closely related polypeptides isolated from P. misakiensis. It acts as a cytostatic factor that regulates proliferation, adhesion, and differentiation of multipotent cells, although the molecular mechanism remains uncertain. The Polycomb group (PcG) genes are involved in epigenetic control of genomic activity in mammals. In invertebrates except Drosophila, PcG and histone methylation have not been studied so extensively, and genome-wide gene regulation is poorly understood. Results When Phe65 of TC14-3 was mutated to an acidic amino acid, the resultant mutant protein failed to dimerize. The replacement of Thr69 with Arg69 made dimers unstable. When Glu106 was changed to Gly106, the resultant mutant protein completely lost Ca2+ binding. All these mutant proteins lacked cytostatic activity, indicating the requirement of protein dimerization and calcium for the activity. Polyandrocarpa Eed, a component of PcG, is highly expressed during budding, like TC14-3. When wild-type and mutant TC14-3s were applied in vivo and in vitro to Polyandrocarpa cells, only wild-type TC14-3 could induce Eed without affecting histone methyltransferase gene expression. Eed-expressing cells underwent trimethylation of histone H3 lysine27. PmEed knockdown by RNA interference rescued cultured cells from the growth-inhibitory effects of TC14-3. Conclusion These results show that in P. misakiensis, the cytostatic activity of TC14-3 is

Covalent binding of cyclohexene, 1,3-cyclohexadiene and 1,4-cyclohexadiene on Si(1 1 1)-7 × 7

NASA Astrophysics Data System (ADS)

Tao, Feng; Wang, Zhong Hai; Xu, Guo Qin

2003-05-01

The adsorption reactions and binding configurations of cyclohexene, 1,3-cyclohexadiene and 1,4-cyclohexadiene on Si(1 1 1)-7 × 7 were studied using high-resolution electron energy loss spectroscopy (HREELS), ultraviolet photoelectron spectroscopy (UPS), X-ray photoelectron spectroscopy (XPS) and DFT calculation. The covalent attachments of these unsaturated hydrocarbons to Si(1 1 1)-7 × 7 through the formation of Si-C linkages are clearly demonstrated by the observation of the Si-C stretching mode at 450-500 cm -1 in their HREELS spectra. For chemisorbed cyclohexene, the involvement of π CC in binding is further supported by the absence of CC stretching modes and the disappearance of the π CC photoemission. The chemisorption of both 1,3-cyclohexadiene and 1,4-cyclohexadiene leads to the formation of cyclohexene-like intermediates through di-σ bonding. The existence of one π CC bond in their chemisorbed states is confirmed by the observation of the CC and (sp 2)CH stretching modes and the UPS and XPS results. DFT calculations show that [4 + 2]-like cycloaddition is thermodynamically preferred for 1,3-cyclohexadiene on Si(1 1 1)-7 × 7, but a [2 + 2]-like reaction mechanism is proposed for the covalent attachment of cyclohexene and 1,4-cyclohexadiene.

Characterization of little skate (Leucoraja erinacea) recombinant transthyretin: Zinc-dependent 3,3',5-triiodo-l-thyronine binding.

PubMed

Suzuki, Shunsuke; Kasai, Kentaro; Yamauchi, Kiyoshi

2015-01-01

Transthyretin (TTR) diverged from an ancestral 5-hydroxyisourate hydrolase (HIUHase) by gene duplication at some early stage of chordate evolution. To clarify how TTR had participated in the thyroid system as an extracellular thyroid hormone (TH) binding protein, TH binding properties of recombinant little skate Leucoraja erinacea TTR was investigated. At the amino acid level, skate TTR showed 37-46% identities with the other vertebrate TTRs. Because the skate TTR had a unique histidine-rich segment in the N-terminal region, it could be purified by Ni-affinity chromatography. The skate TTR was a 46-kDa homotetramer of 14.5kDa subunits, and had one order of magnitude higher affinity for 3,3',5-triiodo-l-thyronine (T3) and some halogenated phenols than for l-thyroxine. However, the skate TTR had no HIUHase activity. Ethylenediaminetetraacetic acid (EDTA) treatment inhibited [(125)I]T3 binding activity whereas the addition of Zn(2+) to the EDTA-treated TTR recovered [(125)I]T3 binding activity in a Zn(2+) concentration-dependent manner. Scatchard analysis revealed the presence of two classes of binding site for T3, with dissociation constants of 0.24 and 17nM. However, the high-affinity sites were completely abolished with 1mM EDTA, whereas the remaining low-affinity sites decreased binding capacity. The number of zinc per TTR was quantified to be 4.5-6.3. Our results suggest that skate TTR has tight Zn(2+)-binding sites, which are essential for T3 binding to at least the high-affinity sites. Zn(2+) binding to the N-terminal histidine-rich segment may play an important role in acquisition or reinforcement of TH binding ability during early evolution of TTR. Copyright © 2015 Elsevier Inc. All rights reserved.

Recognition of xyloglucan by the crystalline cellulose-binding site of a family 3a carbohydrate-binding module

PubMed Central

Hernandez-Gomez, Mercedes C.; Rydahl, Maja G.; Rogowski, Artur; Morland, Carl; Cartmell, Alan; Crouch, Lucy; Labourel, Aurore; Fontes, Carlos M. G. A.; Willats, William G. T.; Gilbert, Harry J; Knox, J. Paul

2018-01-01

Type A non-catalytic carbohydrate-binding modules (CBMs), exemplified by CtCBM3acipA, are widely believed to specifically target crystalline cellulose through entropic forces. Here we have tested the hypothesis that type A CBMs can also bind to xyloglucan, a soluble β-1,4-glucan containing α-1,6-xylose side chains. CtCBM3acipA bound to xyloglucan in cell walls and arrayed on solid surfaces. Xyloglucan and cellulose were shown to bind to the same planar surface on CBM3acipA. A range of type A CBMs from different families were shown to bind to xyloglucan in solution with ligand binding driven by enthalpic changes. The nature of CBM-polysaccharide interactions is discussed. PMID:26193423

Comparative and evolutionary analysis of the 14-3-3 family genes in eleven fishes.

PubMed

Cao, Jun; Tan, Xiaona

2018-07-01

14-3-3 proteins are a type of highly conserved acidic proteins, which are distributed over a wide variety of organisms and are involved in multiple cellular processes. While the comparative and evolutionary analysis of this gene family is unavailable in various fish species. In this study, we identified 101 putative 14-3-3 genes in 11 fish species and divided them into 5 groups via phylogenetic analysis. Synteny analysis implied conserved and dynamic evolution characteristics near the 14-3-3 gene loci in some vertebrates. We also found that some recombination events have accelerated the evolution of this gene family. Moreover, a positive selection site was also identified, and mutation of this site could reduce the 14-3-3 stability. Divergent expression profiles of the zebrafish 14-3-3 genes were further investigated under organophosphorus stress, suggesting that they may be involved in the different osmoregulation and immune response. The results will serve as a foundation for the further functional investigation into the 14-3-3 genes in fishes. Copyright © 2018 Elsevier B.V. All rights reserved.

Human CRISP-3 binds serum alpha(1)B-glycoprotein across species.

PubMed

Udby, Lene; Johnsen, Anders H; Borregaard, Niels

2010-04-01

CRISP-3 was previously shown to be bound to alpha(1)B-glycoprotein (A1BG) in human serum/plasma. All mammalian sera are supposed to contain A1BG, although its presence in rodent sera is not well-documented. Since animal sera are often used to supplement buffers in experiments, in particular such that involve cell cultures, binding proteins present in sera might interfere in the experiments. We examined sera from five different animal species for CRISP-3 binding proteins using gel filtration and ligand blotting. We developed a rapid method for isolation of proteins that bind to human CRISP-3 and identified the isolated proteins by mass spectrometry and N-terminal sequencing. We identified A1BG as a CRISP-3 binding protein in sera from cow, horse and rabbit. CRISP-3 bound kininogen 1 in mouse serum, whereas rat serum showed no CRISP-3 binding activity. In equine serum, we furthermore detected a possible CRISP, already bound to A1BG. It seems to be a common mechanism that A1BGs bind CRISPs, also across species. Apart from the possible physiological implications hereof, complex binding of CRISPs by A1BG (and other proteins) may interfere with the detection and function of CRISPs, when these are studied in the presence of animal sera. Copyright 2009 Elsevier B.V. All rights reserved.

The Crystal Structure of Giardia duodenalis 14-3-3 in the Apo Form: When Protein Post-Translational Modifications Make the Difference

PubMed Central

Fiorillo, Annarita; di Marino, Daniele; Bertuccini, Lucia; Via, Allegra; Pozio, Edoardo; Camerini, Serena; Ilari, Andrea; Lalle, Marco

2014-01-01

The 14-3-3s are a family of dimeric evolutionary conserved pSer/pThr binding proteins that play a key role in multiple biological processes by interacting with a plethora of client proteins. Giardia duodenalis is a flagellated protozoan that affects millions of people worldwide causing an acute and chronic diarrheal disease. The single giardial 14-3-3 isoform (g14-3-3), unique in the 14-3-3 family, needs the constitutive phosphorylation of Thr214 and the polyglycylation of its C-terminus to be fully functional in vivo. Alteration of the phosphorylation and polyglycylation status affects the parasite differentiation into the cyst stage. To further investigate the role of these post-translational modifications, the crystal structure of the g14-3-3 was solved in the unmodified apo form. Oligomers of g14-3-3 were observed due to domain swapping events at the protein C-terminus. The formation of filaments was supported by TEM. Mutational analysis, in combination with native PAGE and chemical cross-linking, proved that polyglycylation prevents oligomerization. In silico phosphorylation and molecular dynamics simulations supported a structural role for the phosphorylation of Thr214 in promoting target binding. Our findings highlight unique structural features of g14-3-3 opening novel perspectives on the evolutionary history of this protein family and envisaging the possibility to develop anti-giardial drugs targeting g14-3-3. PMID:24658679

[3H]-nitrendipine binding in membranes obtained from hypoxic and reoxygenated heart.

PubMed

Matucci, R; Bennardini, F; Sciammarella, M L; Baccaro, C; Stendardi, I; Franconi, F; Giotti, A

1987-04-01

We compared the binding properties of [3H]-nitrendipine in heart membranes from normal guinea-pig heart and from hypoxic or hypoxic and reoxygenated heart. The [3H]-nitrendipine binds a single class of high capacity (Bmax 667.2 +/- 105.2) with high affinity (KD 0.14 +/- 0.02) binding sites. By contrast, in membranes of hypoxic and reoxygenated heart the Bmax decreases significantly while it remains unaffected during hypoxia. Xanthinoxidase activity is increased in hypoxic-reoxygenated hearts.

14-3-3η Autoantibodies: Diagnostic Use in Early Rheumatoid Arthritis.

PubMed

Maksymowych, Walter P; Boire, Gilles; van Schaardenburg, Dirkjan; Wichuk, Stephanie; Turk, Samina; Boers, Maarten; Siminovitch, Katherine A; Bykerk, Vivian; Keystone, Ed; Tak, Paul Peter; van Kuijk, Arno W; Landewé, Robert; van der Heijde, Desiree; Murphy, Mairead; Marotta, Anthony

2015-09-01

To describe the expression and diagnostic use of 14-3-3η autoantibodies in early rheumatoid arthritis (RA). 14-3-3η autoantibody levels were measured using an electrochemiluminescent multiplexed assay in 500 subjects (114 disease-modifying antirheumatic drug-naive patients with early RA, 135 with established RA, 55 healthy, 70 autoimmune, and 126 other non-RA arthropathy controls). 14-3-3η protein levels were determined in an earlier analysis. Two-tailed Student t tests and Mann-Whitney U tests compared differences among groups. Receiver-operator characteristic (ROC) curves were generated and diagnostic performance was estimated by area under the curve (AUC), as well as specificity, sensitivity, and likelihood ratios (LR) for optimal cutoffs. Median serum 14-3-3η autoantibody concentrations were significantly higher (p < 0.0001) in patients with early RA (525 U/ml) when compared with healthy controls (235 U/ml), disease controls (274 U/ml), autoimmune disease controls (274 U/ml), patients with osteoarthritis (259 U/ml), and all controls (265 U/ml). ROC curve analysis comparing early RA with healthy controls demonstrated a significant (p < 0.0001) AUC of 0.90 (95% CI 0.85-0.95). At an optimal cutoff of ≥ 380 U/ml, the ROC curve yielded a sensitivity of 73%, a specificity of 91%, and a positive LR of 8.0. Adding 14-3-3η autoantibodies to 14-3-3η protein positivity enhanced the identification of patients with early RA from 59% to 90%; addition of 14-3-3η autoantibodies to anticitrullinated protein antibodies (ACPA) and/or rheumatoid factor (RF) increased identification from 72% to 92%. Seventy-two percent of RF- and ACPA-seronegative patients were positive for 14-3-3η autoantibodies. 14-3-3η autoantibodies, alone and in combination with the 14-3-3η protein, RF, and/or ACPA identified most patients with early RA.

Immunological characterization of eristostatin and echistatin binding sites on alpha IIb beta 3 and alpha V beta 3 integrins.

PubMed Central

Marcinkiewicz, C; Rosenthal, L A; Mosser, D M; Kunicki, T J; Niewiarowski, S

1996-01-01

Two disintegrins with a high degree of amino acid sequence similarity, echistatin and eristostatin, showed a low level of interaction with Chinese hamster ovary (CHO) cells, but they bound to CHO cells transfected with alpha IIb beta 3 genes (A5 cells) and to CHO cells transfected with alpha v beta 3 genes (VNRC3 cells) in a reversible and saturable manner. Scatchard analysis revealed that eristostatin bound to 816000 sites per A5 cell (Kd 28 nM) and to 200000 sites (Kd 14 nM) per VNRC3 cell respectively. However, VNRC3 cells did not bind to immobilized eristostatin. Echistatin bound to 495000 sites (Kd 53 nM) per A5 cell and to 443000 sites (Kd 20 nM) per VNRC3 cell. As determined by flow cytometry, radiobinding assay and adhesion studies, binding of both disintegrins to A5 cells and resting platelets and binding of echistatin to VNRC3 cells resulted in the expression of ligand-induced binding sites (LIBS) on the beta 3 subunit. Eristostatin inhibited, more strongly than echistatin, the binding of three monoclonal antibodies: OPG2 (RGD motif dependent), A2A9 (alpha IIb beta 3 complex dependent) and 7E3 (alpha IIb beta 3 and alpha v beta 3 complex dependent) to A5 cells, to resting and to activated platelets and to purified alpha IIb beta 3. Experiments in which echistatin and eristostatin were used alone or in combination to inhibit the binding of 7E3 and OPG2 antibodies to resting platelets suggested that these two disintegrins bind to different but overlapping sites on alpha IIb beta 3 integrin. Monoclonal antibody LM 609 and echistatin seemed to bind to different sites on alpha v beta 3 integrin. However, echistatin inhibited binding of 7E3 antibody to VNRC3 cells and to purified alpha v beta 3 suggesting that alpha v beta 3 and alpha IIb beta 3 might share the same epitope to which both echistatin and 7E3 bind. Eristostatin had no effect in these systems, providing further evidence that it binds to a different epitope on alpha v beta 3. PMID:8760368

On the role of residue phosphorylation in 14-3-3 partners: AANAT as a case study

PubMed Central

Masone, Diego; Uhart, Marina; Bustos, Diego M.

2017-01-01

Twenty years ago, a novel concept in protein structural biology was discovered: the intrinsically disordered regions (IDRs). These regions remain largely unstructured under native conditions and the more are studied, more properties are attributed to them. Possibly, one of the most important is their ability to conform a new type of protein-protein interaction. Besides the classical domain-to-domain interactions, IDRs follow a ‘fly-casting’ model including ‘induced folding’. Unfortunately, it is only possible to experimentally explore initial and final states. However, the complete movie of conformational changes of protein regions and their characterization can be addressed by in silico experiments. Here, we simulate the binding of two proteins to describe how the phosphorylation of a single residue modulates the entire process. 14-3-3 protein family is considered a master regulator of phosphorylated proteins and from a modern point-of-view, protein phosphorylation is a three component system, with writers (kinases), erasers (phosphatases) and readers. This later biological role is attributed to the 14-3-3 protein family. Our molecular dynamics results show that phosphorylation of the key residue Thr31 in a partner of 14-3-3, the aralkylamine N-acetyltransferase, releases the fly-casting mechanism during binding. On the other hand, the non-phosphorylation of the same residue traps the proteins, systematically and repeatedly driving the simulations into wrong protein-protein conformations. PMID:28387381

Genome-wide association analysis in East Asians identifies breast cancer susceptibility loci at 1q32.1, 5q14.3 and 15q26.1.

PubMed

Cai, Qiuyin; Zhang, Ben; Sung, Hyuna; Low, Siew-Kee; Kweon, Sun-Seog; Lu, Wei; Shi, Jiajun; Long, Jirong; Wen, Wanqing; Choi, Ji-Yeob; Noh, Dong-Young; Shen, Chen-Yang; Matsuo, Keitaro; Teo, Soo-Hwang; Kim, Mi Kyung; Khoo, Ui Soon; Iwasaki, Motoki; Hartman, Mikael; Takahashi, Atsushi; Ashikawa, Kyota; Matsuda, Koichi; Shin, Min-Ho; Park, Min Ho; Zheng, Ying; Xiang, Yong-Bing; Ji, Bu-Tian; Park, Sue K; Wu, Pei-Ei; Hsiung, Chia-Ni; Ito, Hidemi; Kasuga, Yoshio; Kang, Peter; Mariapun, Shivaani; Ahn, Sei Hyun; Kang, Han Sung; Chan, Kelvin Y K; Man, Ellen P S; Iwata, Hiroji; Tsugane, Shoichiro; Miao, Hui; Liao, Jiemin; Nakamura, Yusuke; Kubo, Michiaki; Delahanty, Ryan J; Zhang, Yanfeng; Li, Bingshan; Li, Chun; Gao, Yu-Tang; Shu, Xiao-Ou; Kang, Daehee; Zheng, Wei

2014-08-01

In a three-stage genome-wide association study among East Asian women including 22,780 cases and 24,181 controls, we identified 3 genetic loci newly associated with breast cancer risk, including rs4951011 at 1q32.1 (in intron 2 of the ZC3H11A gene; P=8.82×10(-9)), rs10474352 at 5q14.3 (near the ARRDC3 gene; P=1.67×10(-9)) and rs2290203 at 15q26.1 (in intron 14 of the PRC1 gene; P=4.25×10(-8)). We replicated these associations in 16,003 cases and 41,335 controls of European ancestry (P=0.030, 0.004 and 0.010, respectively). Data from the ENCODE Project suggest that variants rs4951011 and rs10474352 might be located in an enhancer region and transcription factor binding sites, respectively. This study provides additional insights into the genetics and biology of breast cancer.

Characterization of chlorophyll binding to LIL3.

PubMed

Mork-Jansson, Astrid Elisabeth; Eichacker, Lutz Andreas

2018-01-01

The light harvesting like protein 3 (LIL 3) from higher plants, has been linked to functions in chlorophyll and tocopherol biosynthesis, photo-protection and chlorophyll transfer. However, the binding of chlorophyll to LIL3 is unclear. We present a reconstitution protocol for chlorophyll binding to LIL3 in DDM micelles. It is shown in the absence of lipids and carotenoids that reconstitution of chlorophyll binding to in vitro expressed LIL3 requires pre-incubation of reaction partners at room temperature. We show chlorophyll a but not chlorophyll b binding to LIL3 at a molar ratio of 1:1. Neither dynamic light scattering nor native PAGE, enabled a discrimination between binding of chlorophyll a and/or b to LIL3.

Characterization of chlorophyll binding to LIL3

PubMed Central

Mork-Jansson, Astrid Elisabeth

2018-01-01

The light harvesting like protein 3 (LIL 3) from higher plants, has been linked to functions in chlorophyll and tocopherol biosynthesis, photo-protection and chlorophyll transfer. However, the binding of chlorophyll to LIL3 is unclear. We present a reconstitution protocol for chlorophyll binding to LIL3 in DDM micelles. It is shown in the absence of lipids and carotenoids that reconstitution of chlorophyll binding to in vitro expressed LIL3 requires pre-incubation of reaction partners at room temperature. We show chlorophyll a but not chlorophyll b binding to LIL3 at a molar ratio of 1:1. Neither dynamic light scattering nor native PAGE, enabled a discrimination between binding of chlorophyll a and/or b to LIL3. PMID:29390011

The Silencing of a 14-3-3ɛ Homolog in Tenebrio molitor Leads to Increased Antimicrobial Activity in Hemocyte and Reduces Larval Survivability.

PubMed

Seo, Gi Won; Jo, Yong Hun; Seong, Jeong Hwan; Park, Ki Beom; Patnaik, Bharat Bhusan; Tindwa, Hamisi; Kim, Sun-Am; Lee, Yong Seok; Kim, Yu Jung; Han, Yeon Soo

2016-08-20

The 14-3-3 family of phosphorylated serine-binding proteins acts as signaling molecules in biological processes such as metabolism, division, differentiation, autophagy, and apoptosis. Herein, we report the requirement of 14-3-3ɛ isoform from Tenebrio molitor (Tm14-3-3ɛ) in the hemocyte antimicrobial activity. The Tm14-3-3ɛ transcript is 771 nucleotides in length and encodes a polypeptide of 256 amino acid residues. The protein has the typical 14-3-3 domain, the nuclear export signal (NES) sequence, and the peptide binding residues. The Tm14-3-3ɛ transcript shows a significant three-fold expression in the hemocyte of T. molitor larvae when infected with Escherichia coli Tm14-3-3ɛ silenced larvae show significantly lower survival rates when infected with E. coli. Under Tm14-3-3ɛ silenced condition, a strong antimicrobial activity is elicited in the hemocyte of the host inoculated with E. coli. This suggests impaired secretion of antimicrobial peptides (AMP) into the hemolymph. Furthermore, a reduction in AMP secretion under Tm14-3-3ɛ silenced condition would be responsible for loss in the capacity to kill bacteria and might explain the reduced survivability of the larvae upon E. coli challenge. This shows that Tm14-3-3ɛ is required to maintain innate immunity in T. molitor by enabling antimicrobial secretion into the hemolymph and explains the functional specialization of the isoform.

The Silencing of a 14-3-3ɛ Homolog in Tenebrio molitor Leads to Increased Antimicrobial Activity in Hemocyte and Reduces Larval Survivability

PubMed Central

Seo, Gi Won; Jo, Yong Hun; Seong, Jeong Hwan; Park, Ki Beom; Patnaik, Bharat Bhusan; Tindwa, Hamisi; Kim, Sun-Am; Lee, Yong Seok; Kim, Yu Jung; Han, Yeon Soo

2016-01-01

The 14-3-3 family of phosphorylated serine-binding proteins acts as signaling molecules in biological processes such as metabolism, division, differentiation, autophagy, and apoptosis. Herein, we report the requirement of 14-3-3ɛ isoform from Tenebrio molitor (Tm14-3-3ɛ) in the hemocyte antimicrobial activity. The Tm14-3-3ɛ transcript is 771 nucleotides in length and encodes a polypeptide of 256 amino acid residues. The protein has the typical 14-3-3 domain, the nuclear export signal (NES) sequence, and the peptide binding residues. The Tm14-3-3ɛ transcript shows a significant three-fold expression in the hemocyte of T. molitor larvae when infected with Escherichia coli Tm14-3-3ɛ silenced larvae show significantly lower survival rates when infected with E. coli. Under Tm14-3-3ɛ silenced condition, a strong antimicrobial activity is elicited in the hemocyte of the host inoculated with E. coli. This suggests impaired secretion of antimicrobial peptides (AMP) into the hemolymph. Furthermore, a reduction in AMP secretion under Tm14-3-3ɛ silenced condition would be responsible for loss in the capacity to kill bacteria and might explain the reduced survivability of the larvae upon E. coli challenge. This shows that Tm14-3-3ɛ is required to maintain innate immunity in T. molitor by enabling antimicrobial secretion into the hemolymph and explains the functional specialization of the isoform. PMID:27556493

The Fibroblast Growth Factor 14·Voltage-gated Sodium Channel Complex Is a New Target of Glycogen Synthase Kinase 3 (GSK3)*

PubMed Central

Shavkunov, Alexander S.; Wildburger, Norelle C.; Nenov, Miroslav N.; James, Thomas F.; Buzhdygan, Tetyana P.; Panova-Elektronova, Neli I.; Green, Thomas A.; Veselenak, Ronald L.; Bourne, Nigel; Laezza, Fernanda

2013-01-01

The FGF14 protein controls biophysical properties and subcellular distribution of neuronal voltage-gated Na+ (Nav) channels through direct binding to the channel C terminus. To gain insights into the dynamic regulation of this protein/protein interaction complex, we employed the split luciferase complementation assay to screen a small molecule library of kinase inhibitors against the FGF14·Nav1.6 channel complex and identified inhibitors of GSK3 as hits. Through a combination of a luminescence-based counter-screening, co-immunoprecipitation, patch clamp electrophysiology, and quantitative confocal immunofluorescence, we demonstrate that inhibition of GSK3 reduces the assembly of the FGF14·Nav channel complex, modifies FGF14-dependent regulation of Na+ currents, and induces dissociation and subcellular redistribution of the native FGF14·Nav channel complex in hippocampal neurons. These results further emphasize the role of FGF14 as a critical component of the Nav channel macromolecular complex, providing evidence for a novel GSK3-dependent signaling pathway that might control excitability through specific protein/protein interactions. PMID:23640885

Identification of the heparin binding site on adeno-associated virus serotype 3B (AAV-3B)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lerch, Thomas F.; Chapman, Michael S., E-mail: chapmami@ohsu.edu

2012-02-05

Adeno-associated virus is a promising vector for gene therapy. In the current study, the binding site on AAV serotype 3B for the heparan sulfate proteoglycan (HSPG) receptor has been characterized. X-ray diffraction identified a disaccharide binding site at the most positively charged region on the virus surface. The contributions of basic amino acids at this and other sites were characterized using site-directed mutagenesis. Both heparin and cell binding are correlated to positive charge at the disaccharide binding site, and transduction is significantly decreased in AAV-3B vectors mutated at this site to reduce heparin binding. While the receptor attachment sites ofmore » AAV-3B and AAV-2 are both in the general vicinity of the viral spikes, the exact amino acids that participate in electrostatic interactions are distinct. Diversity in the mechanisms of cell attachment by AAV serotypes will be an important consideration for the rational design of improved gene therapy vectors.« less

Identification of the heparin binding site on adeno-associated virus serotype 3B (AAV-3B)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lerch, Thomas F.; Chapman, Michael S.

2012-05-24

Adeno-associated virus is a promising vector for gene therapy. In the current study, the binding site on AAV serotype 3B for the heparan sulfate proteoglycan (HSPG) receptor has been characterized. X-ray diffraction identified a disaccharide binding site at the most positively charged region on the virus surface. The contributions of basic amino acids at this and other sites were characterized using site-directed mutagenesis. Both heparin and cell binding are correlated to positive charge at the disaccharide binding site, and transduction is significantly decreased in AAV-3B vectors mutated at this site to reduce heparin binding. While the receptor attachment sites ofmore » AAV-3B and AAV-2 are both in the general vicinity of the viral spikes, the exact amino acids that participate in electrostatic interactions are distinct. Diversity in the mechanisms of cell attachment by AAV serotypes will be an important consideration for the rational design of improved gene therapy vectors.« less

Binding of (/sup 3/H)forskolin to solubilized preparations of adenylate cyclase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nelson, C.A.; Seamon, K.B.

1988-01-01

The binding of (/sup 3/H)forskolin to proteins solubilized from bovine brain membranes was studied by precipitating proteins with polyethylene glycol and separating (/sup 3/H)forskolin bound to protein from free (/sup 3/H)forskolin by rapid filtration. The K/sub d/ for (/sup 3/H)forskolin binding to solubilized proteins was 14 nM which was similar to that for (/sup 3/H)forskolin binding sites in membranes from rat brain and human platelets. Forskolin analogs competed for (/sup 3/H)forskolin binding sites with the same rank potency in both brain membranes and in proteins solubilized from brain membranes. (/sup 3/H)forskolin bound to proteins solubilized from membranes with a Bmaxmore » of 38 fmolmg protein which increased to 94 fmolmg protein when GppNHp was included in the binding assay. In contrast, GppNHp had no effect on (/sup 3/H)forskolin binding to proteins solubilized from membranes preactivated with GppNHp. Solubilized adenylate cyclase from non-preactivated membranes had a basal activity of 130 pmolmgmin which was increased 7-fold by GppNHp. In contrast, adenylate cyclase from preactivated membranes had a basal activity of 850 pmolmgmin which was not stimulated by GppNHp or forskolin« less

Elucidation of the 14-3-3ζ interactome reveals critical roles of RNA-splicing factors during adipogenesis.

PubMed

Mugabo, Yves; Sadeghi, Mina; Fang, Nancy N; Mayor, Thibault; Lim, Gareth E

2018-05-04

Adipogenesis involves a complex signaling network requiring strict temporal and spatial organization of effector molecules. Molecular scaffolds, such as 14-3-3 proteins, facilitate such organization, and we have previously identified 14-3-3ζ as an essential scaffold in adipocyte differentiation. The interactome of 14-3-3ζ is large and diverse, and it is possible that novel adipogenic factors may be present within it, but this possibility has not yet been tested. Herein, we generated mouse embryonic fibroblasts from mice overexpressing a tandem affinity purification (TAP) epitope-tagged 14-3-3ζ molecule. After inducing adipogenesis, TAP-14-3-3ζ complexes were purified, followed by MS analysis to determine the 14-3-3ζ interactome. We observed more than 100 proteins that were unique to adipocyte differentiation, 56 of which were novel interacting partners. Among these, we were able to identify previously established regulators of adipogenesis ( i.e. Ptrf/Cavin1) within the 14-3-3ζ interactome, confirming the utility of this approach to detect adipogenic factors. We found that proteins related to RNA metabolism, processing, and splicing were enriched in the interactome. Analysis of transcriptomic data revealed that 14-3-3ζ depletion in 3T3-L1 cells affected alternative splicing of mRNA during adipocyte differentiation. siRNA-mediated depletion of RNA-splicing factors within the 14-3-3ζ interactome, that is, of Hnrpf, Hnrpk, Ddx6, and Sfpq, revealed that they have essential roles in adipogenesis and in the alternative splicing of Pparg and the adipogenesis-associated gene Lpin1 In summary, we have identified novel adipogenic factors within the 14-3-3ζ interactome. Further characterization of additional proteins within the 14-3-3ζ interactome may help identify novel targets to block obesity-associated expansion of adipose tissues. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

Identification of the 14-3-3 gene family in Rafflesia cantleyi

NASA Astrophysics Data System (ADS)

Rosli, Khadijah; Wan, Kiew-Lian

2018-04-01

Rafflesia is known to be the largest flower in the world. Due to its size and appearance, it is considered to be very unique. Little is known about the molecular biology of this rare parasitic flowering plant as it is very difficult to locate and has a short life-span as a flower. Physiological activities in plants are regulated by signalling regulators such as the members of the 14-3-3 gene family. The number of members of this gene family varies in plants and there are thirteen known members in Arabidopsis thaliana. Their role is to bind to phosphorylated targets to complete signal transduction processes. Sequence comparison using BLAST of transcriptome data from three different Rafflesia cantleyi floral bud stages against the Swissprot database revealed 27 transcripts annotated as members of this gene family. All of the transcripts were expressed during floral bud stage 1 (S1) while 14 and four transcripts were expressed during floral bud stages 2 (S2) and 3 (S3), respectively. Significant downregulation was recorded for six and nine transcripts at S1 vs. S2 and S2 vs. S3 respectively. This gene family may play a critical role as signalling regulators during the development of Rafflesia floral bud.

UVB-induced nuclear translocation of TC-PTP by AKT/14-3-3σ axis inhibits keratinocyte survival and proliferation.

PubMed

Kim, Mihwa; Morales, Liza D; Baek, Minwoo; Slaga, Thomas J; DiGiovanni, John; Kim, Dae Joon

2017-10-31

Understanding protein subcellular localization is important to determining the functional role of specific proteins. T-cell protein tyrosine phosphatase (TC-PTP) contains bipartite nuclear localization signals (NLSI and NLSII) in its C-terminus. We previously have demonstrated that the nuclear form of TC-PTP (TC45) is mainly localized to the cytoplasm in keratinocytes and it is translocated to the nucleus following UVB irradiation. Here, we report that TC45 is translocated by an AKT/14-3-3σ-mediated mechanism in response to UVB exposure, resulting in increased apoptosis and decreased keratinocyte proliferation. We demonstrate that UVB irradiation increased phosphorylation of AKT and induced nuclear translocation of 14-3-3σ and TC45. However, inhibition of AKT blocked nuclear translocation of TC45 and 14-3-3σ. Site-directed mutagenesis of 14-3-3σ binding sites within TC45 showed that a substitution at Threonine 179 (TC45/T179A) effectively blocked UVB-induced nuclear translocation of ectopic TC45 due to the disruption of the direct binding between TC45 and 14-3-3σ. Overexpression of TC45/T179A in keratinocytes resulted in a decrease of UVB-induced apoptosis which corresponded to an increase in nuclear phosphorylated STAT3, and cell proliferation was higher in TC45/T179A-overexpressing keratinocytes compared to control keratinocytes following UVB irradiation. Furthermore, deletion of TC45 NLSII blocked its UVB-induced nuclear translocation, indicating that both T179 and NLSII are required. Taken together, our findings suggest that AKT and 14-3-3σ cooperatively regulate TC45 nuclear translocation in a critical step of an early protective mechanism against UVB exposure that signals the deactivation of STAT3 in order to promote keratinocyte cell death and inhibit keratinocyte proliferation.

14-3-3 sigma and 14-3-3 zeta plays an opposite role in cell growth inhibition mediated by transforming growth factor-beta 1.

PubMed

Hong, Hye-Young; Jeon, Woo-Kwang; Bae, Eun-Jin; Kim, Shin-Tae; Lee, Ho-Jae; Kim, Seong-Jin; Kim, Byung-Chul

2010-03-01

The expression of 14-3-3 proteins is dysregulated in various types of cancer. This study was undertaken to investigate the effects of 14-3-3 zeta and 14-3-3 sigma on cell growth inhibition mediated by transforming growth factor-beta 1 (TGF-beta1). Mouse mammary epithelial cells (Eph4) that are transformed with oncogenic c-H-Ras (EpRas) and no longer sensitive to TGF-beta1-mediated growth inhibition displayed increased expression of 14-3-3 zeta and decreased expression of 14-3-3 sigma compared with parental Eph4 cells. Using small interfering RNA-mediated knockdown and overexpression of 14-3-3 sigma or 14-3-3 zeta, we showed that 14-3-3 sigma is required for TGF-beta1-mediated growth inhibition whereas 14-3-3 zeta negatively modulates this growth inhibitory response. Notably, overexpression of 14-3-3 zeta increased the level of Smad3 protein that is phosphorylated at linker regions and cannot mediate the TGF-beta1 growth inhibitory response. Consistent with this finding, mutation of the 14-3-3 zeta phosphorylation sites in Smad3 markedly reduced the 14-3-3 zeta-mediated inhibition of TGF-beta1-induced p15 promoter-reporter activity and cell cycle arrest, suggesting that these residues are critical targets of 14-3-3 zeta in the suppression of TGF-beta1-mediated growth. Taken together, our findings indicate that dysregulation of 14-3-3 sigma or 14-3-3 zeta contributes to TGF-beta1 resistance in cancer cells.

A novel pathway down-modulating T cell activation involves HPK-1-dependent recruitment of 14-3-3 proteins on SLP-76.

PubMed

Di Bartolo, Vincenzo; Montagne, Benjamin; Salek, Mogjiborahman; Jungwirth, Britta; Carrette, Florent; Fourtane, Julien; Sol-Foulon, Nathalie; Michel, Frédérique; Schwartz, Olivier; Lehmann, Wolf D; Acuto, Oreste

2007-03-19

The SH2 domain-containing leukocyte protein of 76 kD (SLP-76) is a pivotal element of the signaling machinery controlling T cell receptor (TCR)-mediated activation. Here, we identify 14-3-3epsilon and zeta proteins as SLP-76 binding partners. This interaction was induced by TCR ligation and required phosphorylation of SLP-76 at serine 376. Ribonucleic acid interference and in vitro phosphorylation experiments showed that serine 376 is the target of the hematopoietic progenitor kinase 1 (HPK-1). Interestingly, either S376A mutation or HPK-1 knockdown resulted in increased TCR-induced tyrosine phosphorylation of SLP-76 and phospholipase C-gamma1. Moreover, an SLP-76-S376A mutant induced higher interleukin 2 gene transcription than wild-type SLP-76. These data reveal a novel negative feedback loop involving HPK-1-dependent serine phosphorylation of SLP-76 and 14-3-3 protein recruitment, which tunes T cell activation.

Genome-wide association analysis in East Asians identifies breast cancer susceptibility loci at 1q32.1, 5q14.3 and 15q26.1

PubMed Central

Cai, Qiuyin; Zhang, Ben; Sung, Hyuna; Low, Siew-Kee; Kweon, Sun-Seog; Lu, Wei; Shi, Jiajun; Long, Jirong; Wen, Wanqing; Choi, Ji-Yeob; Noh, Dong-Young; Shen, Chen-Yang; Matsuo, Keitaro; Teo, Soo-Hwang; Kim, Mi Kyung; Khoo, Ui Soon; Iwasaki, Motoki; Hartman, Mikael; Takahashi, Atsushi; Ashikawa, Kyota; Matsuda, Koichi; Shin, Min-Ho; Park, Min Ho; Zheng, Ying; Xiang, Yong-Bing; Ji, Bu-Tian; Park, Sue K.; Wu, Pei-Ei; Hsiung, Chia-Ni; Ito, Hidemi; Kasuga, Yoshio; Kang, Peter; Mariapun, Shivaani; Ahn, Sei Hyun; Kang, Han Sung; Chan, Kelvin Y. K.; Man, Ellen P. S.; Iwata, Hiroji; Tsugane, Shoichiro; Miao, Hui; Liao, Jiemin; Nakamura, Yusuke; Kubo, Michiaki; Delahanty, Ryan J.; Zhang, Yanfeng; Li, Bingshan; Li, Chun; Gao, Yu-Tang; Shu, Xiao-Ou; Kang, Daehee; Zheng, Wei

2014-01-01

In a three-stage genome-wide association study among East Asian women including 22,780 cases and 24,181 controls, we identified three novel genetic loci associated with breast cancer risk, including rs4951011 at 1q32.1 (in intron 2 of the ZC3H11A gene, P = 8.82 × 10−9), rs10474352 at 5q14.3 (near the ARRDC3 gene, P = 1.67 × 10−9), and rs2290203 at 15q26.1 (in intron 14 of the PRC1 gene, P = 4.25 × 10−8). These associations were replicated in European-ancestry populations including 16,003 cases and 41,335 controls (P = 0.030, 0.004, and 0.010, respectively). Data from the ENCODE project suggest that variants rs4951011 and rs10474352 may be located in an enhancer region and transcription factor binding sites, respectively. This study provides additional insights into the genetics and biology of breast cancer. PMID:25038754

The 14-3-3 protein PAR-5 regulates the asymmetric localization of the LET-99 spindle positioning protein.

PubMed

Wu, Jui-Ching; Espiritu, Eugenel B; Rose, Lesilee S

2016-04-15

PAR proteins play important roles in establishing cytoplasmic polarity as well as regulating spindle positioning during asymmetric division. However, the molecular mechanisms by which the PAR proteins generate asymmetry in different cell types are still being elucidated. Previous studies in Caenorhabditis elegans revealed that PAR-3 and PAR-1 regulate the asymmetric localization of LET-99, which in turn controls spindle positioning by affecting the distribution of the conserved force generating complex. In wild-type embryos, LET-99 is localized in a lateral cortical band pattern, via inhibition at the anterior by PAR-3 and at the posterior by PAR-1. In this report, we show that the 14-3-3 protein PAR-5 is also required for cortical LET-99 asymmetry. PAR-5 associated with LET-99 in pull-down assays, and two PAR-5 binding sites were identified in LET-99 using the yeast two-hybrid assay. Mutation of these sites abolished binding in yeast and altered LET-99 localization in vivo: LET-99 was present at the highest levels at the posterior pole of the embryo instead of a band in par-5 embryos. Together the results indicate that PAR-5 acts in a mechanism with PAR-1 to regulate LET-99 cortical localization. Copyright © 2016 Elsevier Inc. All rights reserved.

The 14-3-3 Protein PAR-5 Regulates the Asymmetric localization of the LET-99 Spindle Positioning Protein

PubMed Central

Rose, Lesilee S.

2016-01-01

PAR proteins play important roles in establishing cytoplasmic polarity as well as regulating spindle positioning during asymmetric division. However, the molecular mechanisms by which the PAR proteins generate asymmetry in different cell types are still being elucidated. Previous studies in C. elegans revealed that PAR-3 and PAR-1 regulate the asymmetric localization of LET-99, which in turn controls spindle positioning by affecting the distribution of the conserved force generating complex. In wild-type embryos, LET-99 is localized in a lateral cortical band pattern, via inhibition at the anterior by PAR-3 and at the posterior by PAR-1. In this report, we show that the 14-3-3 protein PAR-5 is also required for cortical LET-99 asymmetry. PAR-5 associated with LET-99 in pull-down assays, and two PAR-5 binding sites were identified in LET-99 using the yeast two-hybrid assay. Mutation of these sites abolished binding in yeast and altered LET-99 localization in vivo: LET-99 was present at the highest levels at the posterior pole of the embryo instead of a band in par-5 embryos. Together the results indicate that PAR-5 acts in a mechanism with PAR-1 to regulate LET-99 cortical localization. PMID:26921457

Light modulated activity of root alkaline/neutral invertase involves the interaction with 14-3-3 proteins.

PubMed

Gao, Jing; van Kleeff, Paula J M; Oecking, Claudia; Li, Ka Wan; Erban, Alexander; Kopka, Joachim; Hincha, Dirk K; de Boer, Albertus H

2014-12-01

Alkaline/neutral invertases (A/N-Invs) are now recognized as essential proteins in plant life. They catalyze the irreversible breakdown of sucrose into glucose and fructose and thus supply the cells with energy as well as signaling molecules. In this study we report on a mechanism that affects the activity of the cytosolic invertase AtCINV1 (At-A/N-InvG or AT1G35580). We demonstrate that Ser547 at the extreme C-terminus of the AtCINV1 protein is a substrate of calcium-dependent kinases (CPK3 and 21) and that phosphorylation creates a high-affinity binding site for 14-3-3 proteins. The invertase as such has basal activity, but we provide evidence that interaction with 14-3-3 proteins enhances its activity. The analysis of three quadruple 14-3-3 mutants generated from six T-DNA insertion mutants of the non-epsilon family shows both specificity as well as redundancy for this function of 14-3-3 proteins. The strong reduction in hexose levels in the roots of one 14-3-3 quadruple mutant plant is in line with the activating function of 14-3-3 proteins. The physiological relevance of this mechanism that affects A/N-invertase activity is underscored by the light-induced activation and is another example of the central role of 14-3-3 proteins in mediating dark/light signaling. The nature of the light-induced signal that travels from the shoot to root and the question whether this signal is transmitted via cytosolic Ca(++) changes that activate calcium-dependent kinases, await further study. © 2014 The Authors The Plant Journal © 2014 John Wiley & Sons Ltd.

The GCKIII Kinase Sps1 and the 14-3-3 Isoforms, Bmh1 and Bmh2, Cooperate to Ensure Proper Sporulation in Saccharomyces cerevisiae

PubMed Central

Slubowski, Christian J.; Paulissen, Scott M.; Huang, Linda S.

2014-01-01

Sporulation in the budding yeast Saccharomyces cerevisiae is a developmental program initiated in response to nutritional deprivation. Sps1, a serine/threonine kinase, is required for sporulation, but relatively little is known about the molecular mechanisms through which it regulates this process. Here we show that SPS1 encodes a bona-fide member of the GCKIII subfamily of STE20 kinases, both through phylogenetic analysis of the kinase domain and examination of its C-terminal regulatory domain. Within the regulatory domain, we find Sps1 contains an invariant ExxxPG region conserved from plant to human GCKIIIs that we call the EPG motif; we show this EPG motif is important for SPS1 function. We also find that Sps1 is phosphorylated near its N-terminus on Threonine 12, and that this phosphorylation is required for the efficient production of spores. In Sps1, Threonine 12 lies within a 14-3-3 consensus binding sequence, and we show that the S. cerevisiae 14-3-3 proteins Bmh1 and Bmh2 bind Sps1 in a Threonine 12-dependent fashion. This interaction is significant, as BMH1 and BMH2 are required during sporulation and genetically interact with SPS1 in sporulating cells. Finally, we observe that Sps1, Bmh1 and Bmh2 are present in both the nucleus and cytoplasm during sporulation. We identify a nuclear localization sequence in Sps1 at amino acids 411–415, and show that this sequence is necessary and sufficient for nuclear localization. Taken together, these data identify regions within Sps1 critical for its function and indicate that SPS1 and 14-3-3s act together to promote proper sporulation in S. cerevisiae. PMID:25409301

The chromatin-binding protein HMGN3 stimulates histone acetylation and transcription across the Glyt1 gene

PubMed Central

Barkess, Gráinne; Postnikov, Yuri; Campos, Chrisanne D.; Mishra, Shivam; Mohan, Gokula; Verma, Sakshi; Bustin, Michael; West, Katherine L.

2013-01-01

HMGNs are nucleosome-binding proteins that alter the pattern of histone modifications and modulate the binding of linker histones to chromatin. The HMGN3 family member exists as two splice forms, HMGN3a which is full-length and HMGN3b which lacks the C-terminal RD (regulatory domain). In the present study, we have used the Glyt1 (glycine transporter 1) gene as a model system to investigate where HMGN proteins are bound across the locus in vivo, and to study how the two HMGN3 splice variants affect histone modifications and gene expression. We demonstrate that HMGN1, HMGN2, HMGN3a and HMGN3b are bound across the Glyt1 gene locus and surrounding regions, and are not enriched more highly at the promoter or putative enhancer. We conclude that the peaks of H3K4me3 (trimethylated Lys4 of histone H3) and H3K9ac (acetylated Lys9 of histone H3) at the active Glyt1a promoter do not play a major role in recruiting HMGN proteins. HMGN3a/b binding leads to increased H3K14 (Lys14 of histone H3) acetylation and stimulates Glyt1a expression, but does not alter the levels of H3K4me3 or H3K9ac enrichment. Acetylation assays show that HMGN3a stimulates the ability of PCAF [p300/CREB (cAMP-response-element-binding protein)-binding protein-associated factor] to acetylate nucleosomal H3 in vitro, whereas HMGN3b does not. We propose a model where HMGN3a/b-stimulated H3K14 acetylation across the bodies of large genes such as Glyt1 can lead to more efficient transcription elongation and increased mRNA production. PMID:22150271

Differential binding of /sup 3/H-imipramine and /sup 3/H-mianserin in rat cerebral cortex

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dumbrille-Ross, A.; Tang, S.W.; Coscina, D.V.

1981-11-16

Drug competition profiles, effect of raphe lesion, and sodium dependency of the binding of two antidepressant drugs /sup 3/H-imipramine and /sup 3/H-mianserin to rat cerebral cortex homogenate were compared to examine whether the drugs bound to a common ''antidepressant receptor.'' Of the neurotransmitters tested, only serotonin displaced binding of both /sup 3/H-imipramine and /sup 3/H-mianserin. /sup 3/H-Mianserin binding was potently displaced by serotonin S/sub 2/ antagonists and exhibited a profile similar to that of /sup 3/H-spiperone binding. In the presence of the serotonin S/sub 2/ antagonist spiperone, antihistamines (H/sub 1/) potently displaced /sup 3/H-mianserin binding. /sup 3/H-Imipramine binding was displacedmore » potently by serotonin uptake inhibitors. The order of potency of serotonergic drugs in displacing /sup 3/H-imipramine binding was not similar to their order in displacing /sup 3/H-spiperone or -3H-serotonin binding. Prior midbrain raphe lesions greatly decreased the binding of /sup 3/H-imipramine but did not alter binding of /sup 3/H-mianserin. Binding of /sup 3/H-imipramine but not /sup 3/H-mianserin was sodium dependent. These results show that /sup 3/H-imipramine and /sup 3/H-mianserin bind to different receptors. /sup 3/H-Imipramine binds to a presynaptic serotonin receptor which is probably related to a serotonin uptake recognition site, the binding of which is sodium dependent. /sup 3/H-Mianserin binds to postsynaptic receptors, possibly both serotonin S/sub 2/ and histamine H/sub 1/ receptors, the binding of which is sodium independent.« less

Characterization of hyaluronate binding proteins isolated from 3T3 and murine sarcoma virus transformed 3T3 cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Turley, E.A.; Moore, D.; Hayden, L.J.

1987-06-02

A hyaluronic acid binding fraction was purified from the supernatant media of both 3T3 and murine sarcoma virus (MSV) transformed 3T3 cultures by hyaluronate and immunoaffinity chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis resolved the hyaluronate affinity-purified fraction into three major protein bands of estimated molecular weight (M/sub r,e/) 70K, 66K, and 56K which contained hyaluronate binding activity and which were termed hyaluronate binding proteins (HABP). Hyaluronate affinity chromatography combined with immunoaffinity chromatography, using antibody directed against the larger HABP, allowed a 20-fold purification of HABP. Fractions isolated from 3T3 supernatant medium also contained additional binding molecules in the molecular weightmore » range of 20K. This material was present in vanishingly small amounts and was not detected with a silver stain or with (/sup 35/S)methionine label. The three protein species isolated by hyaluronate affinity chromatography (M/sub r,e/ 70K, 66K, and 56K) were related to one another since they shared antigenic determinants and exhibited similar pI values. In isocratic conditions, HABP occurred as aggregates of up to 580 kilodaltons. Their glycoprotein nature was indicated by their incorporation of /sup 3/H-sugars. Enzyme-linked immunoadsorbent assay showed they were antigenically distinct from other hyaluronate binding proteins such as fibronectin, cartilage link protein, and the hyaluronate binding region of chondroitin sulfate proteoglycan. The results are discussed with regard both to the functional significance of hyaluronate-cell surface interactions in transformed as well as normal cells and to the relationship of HABP to other reported hyaluronate binding proteins.« less

14-3-3 coordinates microtubules, Rac, and myosin II to control cell mechanics and cytokinesis

PubMed Central

Zhou, Qiongqiong; Kee, Yee-Seir; Poirier, Christopher C.; Jelinek, Christine; Osborne, Jonathan; Divi, Srikanth; Surcel, Alexandra; Will, Marie E.; Eggert, Ulrike S.; Müller-Taubenberger, Annette; Iglesias, Pablo A.; Cotter, Robert J.; Robinson, Douglas N.

2010-01-01

Summary Background During cytokinesis, regulatory signals are presumed to emanate from the mitotic spindle. However, what these signals are and how they lead to the spatiotemporal changes in the cortex structure, mechanics, and regional contractility are not well understood in any system. Results To investigate pathways that link the microtubule network to the cortical changes that promote cytokinesis, we used chemical genetics in Dictyostelium to identify genetic suppressors of nocodazole, a microtubule depolymerizer. We identified 14-3-3 and found that it is enriched in the cortex, helps maintain steady state microtubule length, contributes to normal cortical tension, modulates actin wave formation, and controls the symmetry and kinetics of cleavage furrow contractility during cytokinesis. Furthermore, 14-3-3 acts downstream of a Rac small GTPase (RacE), associates with myosin II heavy chain and is needed to promote myosin II bipolar thick filament remodeling. Conclusion 14-3-3 connects microtubules, Rac and myosin II to control several aspects of cortical dynamics, mechanics, and cytokinesis cell shape change. Further, 14-3-3 interacts directly with myosin II heavy chain to promote bipolar thick filament remodeling and distribution. Overall, 14-3-3 appears to integrate several critical cytoskeletal elements that drive two important processes cytokinesis shape change and cell mechanics. PMID:20951045

Proteomic profiling of tandem affinity purified 14-3-3 protein complexes in Arabidopsis thaliana

PubMed Central

Chang, Ing-Feng; Curran, Amy; Woolsey, Rebekah; Quilici, David; Cushman, John; Mittler, Ron; Harmon, Alice; Harper, Jeffrey

2014-01-01

In eukaryotes, 14-3-3 dimers regulate hundreds of functionally diverse proteins (clients), typically in phosphorylation-dependent interactions. To uncover new clients, a 14-3-3 omega (At1g78300) from Arabidopsis was engineered with a “tandem affinity purification” (TAP) tag and expressed in transgenic plants. Purified complexes were analyzed by tandem mass spectrometry. Results indicate that 14-3-3 omega can dimerize with at least 10 of the 12 14-3-3 isoforms expressed in Arabidopsis. The identification here of 121 putative clients provides support for in vivo 14-3-3 interactions with a diverse array of proteins, including those involved in: (1) Ion transport, such as a K+ channel (GORK), a Cl− channel (CLCg), Ca2+ channels belonging to the glutamate receptor family (GLRs 1.2, 2.1, 2.9, 3.4, 3.7); (2) hormone signaling, such as ACC synthase (isoforms ACS-6, 7 and 8 involved in ethylene synthesis) and the brassinolide receptors BRI1 and BAK1; (3) transcription, such as 7 WRKY family transcription factors; (4) metabolism, such as phosphoenol pyruvate (PEP) carboxylase; and (5) lipid signaling, such as phospholipase D (β, and γ). More than 80% (101) of these putative clients represent previously unidentified 14-3-3 interactors. These results raise the number of putative 14-3-3 clients identified in plants to over 300. PMID:19452453

Proteomic profiling of tandem affinity purified 14-3-3 protein complexes in Arabidopsis thaliana.

PubMed

Chang, Ing-Feng; Curran, Amy; Woolsey, Rebekah; Quilici, David; Cushman, John C; Mittler, Ron; Harmon, Alice; Harper, Jeffrey F

2009-06-01

In eukaryotes, 14-3-3 dimers regulate hundreds of functionally diverse proteins (clients), typically in phosphorylation-dependent interactions. To uncover new clients, 14-3-3 omega (At1g78300) from Arabidopsis was engineered with a "tandem affinity purification" tag and expressed in transgenic plants. Purified complexes were analyzed by tandem MS. Results indicate that 14-3-3 omega can dimerize with at least 10 of the 12 14-3-3 isoforms expressed in Arabidopsis. The identification here of 121 putative clients provides support for in vivo 14-3-3 interactions with a diverse array of proteins, including those involved in: (i) Ion transport, such as a K(+) channel (GORK), a Cl(-) channel (CLCg), Ca(2+) channels belonging to the glutamate receptor family (1.2, 2.1, 2.9, 3.4, 3.7); (ii) hormone signaling, such as ACC synthase (isoforms ACS-6, -7 and -8 involved in ethylene synthesis) and the brassinolide receptors BRI1 and BAK1; (iii) transcription, such as 7 WRKY family transcription factors; (iv) metabolism, such as phosphoenol pyruvate carboxylase; and (v) lipid signaling, such as phospholipase D (beta and gamma). More than 80% (101) of these putative clients represent previously unidentified 14-3-3 interactors. These results raise the number of putative 14-3-3 clients identified in plants to over 300.

Endothelin-1 Inhibits the Epithelial Na+ Channel through βPix/14-3-3/Nedd4-2

PubMed Central

Pavlov, Tengis S.; Chahdi, Ahmed; Ilatovskaya, Daria V.; Levchenko, Vladislav; Vandewalle, Alain; Pochynyuk, Oleh

2010-01-01

Epithelial Na+ channels (ENaCs) mediate sodium reabsorption in the cortical collecting duct (CCD), but the regulatory pathways that modulate the activity of these channels are incompletely understood. Here, we observed that endothelin-1 (ET-1) attenuates ENaC activity acutely by reducing the channel's open probability and chronically by decreasing the number of channels in the plasma membrane. To investigate whether β1Pix, a signaling protein activated by ET-1, mediates ENaC activity, we reconstituted ENaC in CHO cells with or without coexpressed β1Pix and found that β1Pix negatively regulates ENaC. Knockdown of βPix in native principal cells abolished the ET-1-induced decrease in ENaC channel number. Furthermore, we found that βPix does not decrease ENaC activity through its guanine nucleotide exchange factor (GEF) activity for Rac1 and Cdc42. Instead, coexpression of β1Pix mutant constructs revealed that β1Pix affects ENaC activity through binding 14-3-3 proteins. Coimmunoprecipitation experiments supported a physical interaction between β1Pix and 14-3-3β in cultured principal cells. Coexpression of 14-3-3β increased ENaC activity in CHO cells, but concomitant expression of β1Pix attenuated this increase. Recruitment of 14-3-3β by β1Pix impaired the interaction of 14-3-3β with the ubiquitin ligase Nedd4-2, thereby promoting ubiquitination and degradation of ENaC. Taken together, these results suggest that the inhibitory effects of chronic ET-1 on ENaC result from βPix interacting with the 14-3-3/Nedd4-2 pathway. PMID:20338996

14-3-3 proteins tune non-muscle myosin II assembly.

PubMed

West-Foyle, Hoku; Kothari, Priyanka; Osborne, Jonathan; Robinson, Douglas N

2018-05-04

The 14-3-3 family comprises a group of small proteins that are essential, ubiquitous, and highly conserved across eukaryotes. Overexpression of the 14-3-3 proteins σ, ϵ, ζ, and η correlates with high metastatic potential in multiple cancer types. In Dictyostelium , 14-3-3 promotes myosin II turnover in the cell cortex and modulates cortical tension, cell shape, and cytokinesis. In light of the important roles of 14-3-3 proteins across a broad range of eukaryotic species, we sought to determine how 14-3-3 proteins interact with myosin II. Here, conducting in vitro and in vivo studies of both Dictyostelium (one 14-3-3 and one myosin II) and human proteins (seven 14-3-3s and three nonmuscle myosin IIs), we investigated the mechanism by which 14-3-3 proteins regulate myosin II assembly. Using in vitro assembly assays with purified myosin II tail fragments and 14-3-3, we demonstrate that this interaction is direct and phosphorylation-independent. All seven human 14-3-3 proteins also altered assembly of at least one paralog of myosin II. Our findings indicate a mechanism of myosin II assembly regulation that is mechanistically conserved across a billion years of evolution from amebas to humans. We predict that altered 14-3-3 expression in humans inhibits the tumor suppressor myosin II, contributing to the changes in cell mechanics observed in many metastatic cancers. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

Inhibition of (/sup 3/H)nitrendipine binding by phospholipase A/sub 2/

DOE Office of Scientific and Technical Information (OSTI.GOV)

Goldman, M.E.; Pisano, J.J.

Phospholipase A/sub 2/ from several sources inhibited (/sup 3/H)nitrendipine binding to membranes from brain, heart and ileal longitudinal muscle. The enzymes from bee venom and Russell's viper venom were most potent, having IC/sub 50/ values of approximately 5 and 14 ng/ml, respectively, in all three membrane preparations. Inhibition of binding by bee venom phospholipase A/sub 2/ was time- and dose-dependent. Mastoparan, a known facilitator of phospholipase A/sub 2/ enzymatic activity, shifted the bee venom phospholipase A/sub 2/ dose-response curve to the left. Pretreatment of brain membranes with bee venom phospholipase A/sub 2/ (10 ng/ml) for 15 min caused a 2-foldmore » increase in the K/sub d/ without changing the B/sub max/ compared with untreated membranes. Extension of the preincubation period to 30 min caused no further increase in the K/sub d/ but significantly decreased the B/sub max/ to 71% the value for untreated membranes. (/sup 3/H)Nitrendipine, preincubated with bee venom phospholipase A/sub 2/, was recovered and found to be fully active, indicating that the phospholipase A/sub 2/ did not modify the ligand. It is concluded that phospholipase A/sub 2/ acts on the membrane at or near the (/sup 3/H)nitrendipine binding site and that phospholipids play a key role in the interactions of 1,4 dihydropyridine calcium channel antagonists with the dihydropyridine binding site. 33 references, 3 figures, 1 table.« less

Lipopolysaccharide (LPS)-binding protein stimulates CD14-dependent Toll-like receptor 4 internalization and LPS-induced TBK1-IKKϵ-IRF3 axis activation.

PubMed

Tsukamoto, Hiroki; Takeuchi, Shino; Kubota, Kanae; Kobayashi, Yohei; Kozakai, Sao; Ukai, Ippo; Shichiku, Ayumi; Okubo, Misaki; Numasaki, Muneo; Kanemitsu, Yoshitomi; Matsumoto, Yotaro; Nochi, Tomonori; Watanabe, Kouichi; Aso, Hisashi; Tomioka, Yoshihisa

2018-05-14

Toll-like receptor 4 (TLR4) is an indispensable immune receptor for lipopolysaccharide (LPS), a major component of the Gram-negative bacterial cell wall. Following LPS stimulation, TLR4 transmits the signal from the cell surface and becomes internalized in an endosome. However, the spatial regulation of TLR4 signaling is not fully understood. Here, we investigated the mechanisms of LPS-induced TLR4 internalization and clarified the roles of the extracellular LPS-binding molecules, LPS-binding protein (LBP), and glycerophosphatidylinositol-anchored protein (CD14). LPS stimulation of CD14-expressing cells induced TLR4 internalization in the presence of serum, and an inhibitory anti-LBP mAb blocked its internalization. Addition of LBP to serum-free cultures restored LPS-induced TLR4 internalization to comparable levels of serum. The secretory form of the CD14 (sCD14) induced internalization but required a much higher concentration than LBP. An inhibitory anti-sCD14 mAb was ineffective for serum-mediated internalization. LBP lacking the domain for LPS transfer to CD14 and a CD14 mutant with reduced LPS binding both attenuated TLR4 internalization. Accordingly, LBP is an essential serum molecule for TLR4 internalization, and its LPS transfer to membrane-anchored CD14 (mCD14) is a prerequisite. LBP induced the LPS-stimulated phosphorylation of TBK1, IKKϵ, and IRF3, leading to IFN-β expression. However, LPS-stimulated late activation of NFκB or necroptosis were not affected. Collectively, our results indicate that LBP controls LPS-induced TLR4 internalization, which induces TLR adaptor molecule 1 (TRIF)-dependent activation of the TBK1-IKKϵ-IRF3-IFN-β pathway. In summary, we showed that LBP-mediated LPS transfer to mCD14 is required for serum-dependent TLR4 internalization and activation of the TRIF pathway. Copyright © 2018, The American Society for Biochemistry and Molecular Biology.

Potential antimicrobial agents from triazole-functionalized 2H-benzo[b][1,4]oxazin-3(4H)-ones.

PubMed

Bollu, Rajitha; Banu, Saleha; Bantu, Rajashaker; Reddy, A Gopi; Nagarapu, Lingaiah; Sirisha, K; Kumar, C Ganesh; Gunda, Shravan Kumar; Shaik, Kamal

2017-12-01

A series of substituted triazole functionalized 2H-benzo[b][1,4]oxazin-3(4H)-ones were synthesized by employing click chemistry and further characterized based on 1 H NMR, 13 C NMR, IR and mass spectral studies. All the synthesized derivatives were screened for their in vitro antimicrobial activities. Further, molecular docking studies were accomplished to explore the binding interactions between 1,2,3-triazol-4-yl-2H-benzo[b][1,4]oxazin-3(4H)-one and the active site of Staphylococcus aureus (CrtM) dehydrosqualene synthase (PDB ID: 2ZCS). These docking studies revealed that the synthesized derivatives showed high binding energies and strong H-bond interactions with the dehydrosqualene synthase validating the observed antimicrobial activity data. Based on antimicrobial activity and docking studies, the compounds 9c, 9d and 9e were identified as promising antimicrobial leads. Copyright © 2017 Elsevier Ltd. All rights reserved.

Exploring the DNA binding/cleavage, cellular accumulation and topoisomerase inhibition of 2-hydroxy-3-(aminomethyl)-1,4-naphthoquinone Mannich bases and their platinum(II) complexes.

PubMed

Neves, Amanda P; Pereira, Michelle X G; Peterson, Erica J; Kipping, Ralph; Vargas, Maria D; Silva, Floriano P; Carneiro, J Walkimar M; Farrell, Nicholas P

2013-02-01

Several chlorido and amino Pt(2+) complexes of 2-hydroxy-3-(aminomethyl)-1,4-naphthoquinone Mannich bases HL exhibiting moderate to high cytotoxicity against cancer cell lines were studied in order to investigate their modes of DNA binding, in vitro DNA strand breaks, mechanism of topoisomerase (Topo I) inhibition and cellular accumulation. DNA model base studies have shown that complex 1a [Pt(HL1)Cl(2)] was capable of binding covalently to 9-ethylguanine (9-EtG) and 5'-GMP. (1)H NMR and mass spectrometry studies have shown that both chlorides were substituted by 9-EtG ligands, whereas 5'-GMP was able to replace only one chlorido ligand, due to steric hindrance. The chlorido Pt(2+) complexes [Pt(HL)Cl(2)] highly accumulate in prostate (PC-3) and melanoma (MDA-MB-435) cell lines, being able to induce DNA strand breaks in vitro and inhibit Topo I by a catalytic mode. On the other hand, the free 2-hydroxy-3-(aminomethyl)-1,4-naphthoquinones HL and the amino Pt(2+) complexes [Pt(L(-))(NH(3))(2)]NO(3) neither cause DNA strand breakage nor exhibit strong DNA interaction, nevertheless the latter were also found to be catalytic inhibitors of Topo I at 100μM. Thus, coordination of the Mannich bases HL to the "PtCl(2)" fragment substantially affects the chemical and biophysical properties of the pro-ligands, leading to an improvement of their DNA binding properties and generating compounds that cleave DNA and catalytically inhibit Topo I. Finally, the high cytotoxicity exhibited by the free (uncomplexed) 2-hydroxy-3-(aminomethyl)-1,4-naphthoquinones might be associated with their decomposition in solution, which is not observed for the Pt(2+) complexes. Copyright © 2012 Elsevier Inc. All rights reserved.

H3K9me2/3 Binding of the MBT Domain Protein LIN-61 Is Essential for Caenorhabditis elegans Vulva Development

PubMed Central

Koester-Eiserfunke, Nora; Fischle, Wolfgang

2011-01-01

MBT domain proteins are involved in developmental processes and tumorigenesis. In vitro binding and mutagenesis studies have shown that individual MBT domains within clustered MBT repeat regions bind mono- and dimethylated histone lysine residues with little to no sequence specificity but discriminate against the tri- and unmethylated states. However, the exact function of promiscuous histone methyl-lysine binding in the biology of MBT domain proteins has not been elucidated. Here, we show that the Caenorhabditis elegans four MBT domain protein LIN-61, in contrast to other MBT repeat factors, specifically interacts with histone H3 when methylated on lysine 9, displaying a strong preference for di- and trimethylated states (H3K9me2/3). Although the fourth MBT repeat is implicated in this interaction, H3K9me2/3 binding minimally requires MBT repeats two to four. Further, mutagenesis of residues conserved with other methyl-lysine binding MBT regions in the fourth MBT repeat does not abolish interaction, implicating a distinct binding mode. In vivo, H3K9me2/3 interaction of LIN-61 is required for C. elegans vulva development within the synMuvB pathway. Mutant LIN-61 proteins deficient in H3K9me2/3 binding fail to rescue lin-61 synMuvB function. Also, previously identified point mutant synMuvB alleles are deficient in H3K9me2/3 interaction although these target residues that are outside of the fourth MBT repeat. Interestingly, lin-61 genetically interacts with two other synMuvB genes, hpl-2, an HP1 homologous H3K9me2/3 binding factor, and met-2, a SETDB1 homologous H3K9 methyl transferase (H3K9MT), in determining C. elegans vulva development and fertility. Besides identifying the first sequence specific and di-/trimethylation binding MBT domain protein, our studies imply complex multi-domain regulation of ligand interaction of MBT domains. Our results also introduce a mechanistic link between LIN-61 function and biology, and they establish interplay of the H3K9me2/3

14-3-3 zeta is a molecular target in guggulsterone induced apoptosis in head and neck cancer cells.

PubMed

Macha, Muzafar A; Matta, Ajay; Chauhan, Ss; Siu, Kw Michael; Ralhan, Ranju

2010-11-30

The five-year survival rates for head and neck squamous cell carcinoma (HNSCC) patients are less than 50%, and the prognosis has not improved, despite advancements in standard multi-modality therapies. Hence major emphasis is being laid on identification of novel molecular targets and development of multi-targeted therapies. 14-3-3 zeta, a multifunctional phospho-serine/phospho-threonine binding protein, is emerging as an effector of pro-survival signaling by binding to several proteins involved in apoptosis (Bad, FKHRL1 and ASK1) and may serve as an appropriate target for head and neck cancer therapy. Herein, we determined effect of guggulsterone (GS), a farnesoid X receptor antagonist, on 14-3-3 zeta associated molecular pathways for abrogation of apoptosis in head and neck cancer cells. Head and neck cancer cells were treated with guggulsterone (GS). Effect of GS-treatment was evaluated using cell viability (MTT) assay and apoptosis was verified by annexin V, DNA fragmentation and M30 CytoDeath antibody assay. Mechanism of GS-induced apoptosis was determined by western blotting and co-IP assays using specific antibodies. Using in vitro models of head and neck cancer, we showed 14-3-3 zeta as a key player regulating apoptosis in GS treated SCC4 cells. Treatment with GS releases BAD from the inhibitory action of 14-3-3 zeta in proliferating HNSCC cells by activating protein phosphatase 2A (PP2A). These events initiate the intrinsic mitochondrial pathway of apoptosis, as revealed by increased levels of cytochrome c in cytoplasmic extracts of GS-treated SCC4 cells. In addition, GS treatment significantly reduced the expression of anti-apoptotic proteins, Bcl-2, xIAP, Mcl1, survivin, cyclin D1 and c-myc, thus committing cells to apoptosis. These events were followed by activation of caspase 9, caspase 8 and caspase 3 leading to cleavage of its downstream target, poly-ADP-ribose phosphate (PARP). GS targets 14-3-3 zeta associated cellular pathways for reducing

IL-3 specifically inhibits GM-CSF binding to the higher affinity receptor

DOE Office of Scientific and Technical Information (OSTI.GOV)

Taketazu, F.; Chiba, S.; Shibuya, K.

1991-02-01

The inhibition of binding between human granulocyte-macrophage colony-stimulating factor (GM-CSF) and its receptor by human interleukin-3 (IL-3) was observed in myelogenous leukemia cell line KG-1 which bore the receptors both for GM-CSF and IL-3. In contrast, this phenomenon was not observed in histiocytic lymphoma cell line U-937 or in gastric carcinoma cell line KATO III, both of which have apparent GM-CSF receptor but an undetectable IL-3 receptor. In KG-1 cells, the cross-inhibition was preferentially observed when the binding of GM-CSF was performed under the high-affinity binding condition; i.e., a low concentration of 125I-GM-CSF was incubated. Scatchard analysis of 125I-GM-CSF bindingmore » to KG-1 cells in the absence and in the presence of unlabeled IL-3 demonstrated that IL-3 inhibited GM-CSF binding to the higher-affinity component of GM-CSF receptor on KG-1 cells. Moreover, a chemical cross-linking study has revealed that the cross-inhibition of the GM-CSF binding observed in KG-1 cells is specific for the beta-chain, Mr 135,000 binding protein which has been identified as a component forming the high-affinity GM-CSF receptor existing specifically on hemopoietic cells.« less

[3H]MK-801 binding sites in post-mortem human frontal cortex.

PubMed

Kornhuber, J; Mack-Burkhardt, F; Kornhuber, M E; Riederer, P

1989-03-29

The binding of [3H]MK-801 ((+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine maleate) was investigated in extensively washed homogenates of post-mortem human frontal cortex. The association of [3H]MK-801 proceeded slowly (t1/2 = 553 min) and reached equilibrium only after a prolonged incubation (greater than 24 h). The dissociation of [3H]MK-801 from the binding site was also slow (t1/2 = 244 min). Glutamate, glycine and magnesium markedly increased the rate of association (t1/2 = 14.8 min) and dissociation (t1/2 = 36.5 min). At equilibrium, the binding was not altered by these substances. Specific binding was linear with protein concentration, was saturable, reversible, stereoselective, heat-labile and was nearly absent in the white matter. Scatchard analysis of the saturation curves obtained at equilibrium indicated that there was a high-affinity (Kd1 1.39 +/- 0.21 nM, Bmax1 0.483 +/- 0.084 pmol/mg protein) and a low-affinity (Kd2 116.25 +/- 50.79 nM, Bmax2 3.251 +/- 0.991 pmol/mg protein) binding site. All competition curves obtained with (+)-MK-801, (-)-MK-801, phencyclidine and ketamine had Hill coefficients of less than unity and were best explained by a two-site model. Thus, our results demonstrate the presence of binding sites for MK-801 in post-mortem human brains and provide evidence for binding site heterogeneity. Furthermore, glutamate, glycine and magnesium accelerate the association and dissociation of [3H]MK-801 to and from its binding sites. The results add support to the hypothesis that MK-801, glutamate, glycine and magnesium all bind to different sites on the NMDA receptor-ion channel complex.

14 CFR 73.3 - Special use airspace.

Code of Federal Regulations, 2010 CFR

2010-01-01

... 14 Aeronautics and Space 2 2010-01-01 2010-01-01 false Special use airspace. 73.3 Section 73.3 Aeronautics and Space FEDERAL AVIATION ADMINISTRATION, DEPARTMENT OF TRANSPORTATION (CONTINUED) AIRSPACE... defined dimensions identified by an area on the surface of the earth wherein activities must be confined...

Histone H3 Lysine 14 (H3K14) Acetylation Facilitates DNA Repair in a Positioned Nucleosome by Stabilizing the Binding of the Chromatin Remodeler RSC (Remodels Structure of Chromatin)*

PubMed Central

Duan, Ming-Rui; Smerdon, Michael J.

2014-01-01

Histone H3 acetylation is induced by UV damage in yeast and may play an important role in regulating the repair of UV photolesions in nucleosome-loaded genomic loci. However, it remains elusive how H3 acetylation facilitates repair. We generated a strongly positioned nucleosome containing homogeneously acetylated H3 at Lys-14 (H3K14ac) and investigated possible mechanisms by which H3K14 acetylation modulates repair. We show that H3K14ac does not alter nucleosome unfolding dynamics or enhance the repair of UV-induced cyclobutane pyrimidine dimers by UV photolyase. Importantly, however, nucleosomes with H3K14ac have a higher affinity for purified chromatin remodeling complex RSC (Remodels the Structure of Chromatin) and show greater cyclobutane pyrimidine dimer repair compared with unacetylated nucleosomes. Our study indicates that, by anchoring RSC, H3K14 acetylation plays an important role in the unfolding of strongly positioned nucleosomes during repair of UV damage. PMID:24515106

The ShcA SH2 domain engages a 14-3-3/PI3'K signaling complex and promotes breast cancer cell survival.

PubMed

Ursini-Siegel, J; Hardy, W R; Zheng, Y; Ling, C; Zuo, D; Zhang, C; Podmore, L; Pawson, T; Muller, W J

2012-11-29

The ShcA adapter protein transmits activating signals downstream of receptor and cytoplasmic tyrosine kinases through the establishment of phosphotyrosine-dependent complexes. In this regard, ShcA possesses both a phosphotyrosine-binding domain (PTB) and Src homology 2 domain (SH2), which bind phosphotyrosine residues in a sequence-specific manner. Although the majority of receptor tyrosine kinases expressed in breast cancer cells bind the PTB domain, very little is known regarding the biological importance of SH2-driven ShcA signaling during mammary tumorigenesis. To address this, we employed transgenic mice expressing a mutant ShcA allele harboring a non-functional SH2 domain (ShcR397K) under the transcriptional control of the endogenous ShcA promoter. Using transplantation approaches, we demonstrate that SH2-dependent ShcA signaling within the mammary epithelial compartment is essential for breast tumor outgrowth, survival and the development of lung metastases. We further show that the ShcA SH2 domain activates the AKT pathway, potentially through a novel SH2-mediated complex between ShcA, 14-3-3ζ and the p85 regulatory subunit of phosphatidylinositol 3 (PI3') kinase. This study is the first to demonstrate that the SH2 domain of ShcA is critical for tumor survival during mammary tumorigenesis.

Unraveling 14-3-3 proteins in C4 panicoids with emphasis on model plant Setaria italica reveals phosphorylation-dependent subcellular localization of RS splicing factor.

PubMed

Kumar, Karunesh; Muthamilarasan, Mehanathan; Bonthala, Venkata Suresh; Roy, Riti; Prasad, Manoj

2015-01-01

14-3-3 proteins are a large multigenic family of regulatory proteins ubiquitously found in eukaryotes. In plants, 14-3-3 proteins are reported to play significant role in both development and response to stress stimuli. Therefore, considering their importance, genome-wide analyses have been performed in many plants including Arabidopsis, rice and soybean. But, till date, no comprehensive investigation has been conducted in any C4 panicoid crops. In view of this, the present study was performed to identify 8, 5 and 26 potential 14-3-3 gene family members in foxtail millet (Si14-3-3), sorghum (Sb14-3-3) and maize (Zm14-3-3), respectively. In silico characterization revealed large variations in their gene structures; segmental and tandem duplications have played a major role in expansion of these genes in foxtail millet and maize. Gene ontology annotation showed the participation of 14-3-3 proteins in diverse biological processes and molecular functions, and in silico expression profiling indicated their higher expression in all the investigated tissues. Comparative mapping was performed to derive the orthologous relationships between 14-3-3 genes of foxtail millet and other Poaceae members, which showed a higher, as well as similar percentage of orthology among these crops. Expression profiling of Si14-3-3 genes during different time-points of abiotic stress and hormonal treatments showed a differential expression pattern of these genes, and sub-cellular localization studies revealed the site of action of Si14-3-3 proteins within the cells. Further downstream characterization indicated the interaction of Si14-3-3 with a nucleocytoplasmic shuttling phosphoprotein (SiRSZ21A) in a phosphorylation-dependent manner, and this demonstrates that Si14-3-3 might regulate the splicing events by binding with phosphorylated SiRSZ21A. Taken together, the present study is a comprehensive analysis of 14-3-3 gene family members in foxtail millet, sorghum and maize, which provides

Identification of C3b-binding Small Molecule Complement Inhibitors Using Cheminformatics

PubMed Central

Garcia, Brandon L.; Skaff, D. Andrew; Chatterjee, Arindam; Hanning, Anders; Walker, John K.; Wyckoff, Gerald J.; Geisbrecht, Brian V.

2017-01-01

The complement system is an elegantly regulated biochemical cascade formed by the collective molecular recognition properties and proteolytic activities of over two dozen membrane-bound or serum proteins. Complement plays diverse roles in human physiology which include acting as a sentry against invading microorganisms, priming of the adaptive immune response, and removal of immune complexes. However, dysregulation of complement can serve as a trigger for a wide range of human diseases which include autoimmune, inflammatory, and degenerative conditions. Despite several potential advantages of modulating complement with small molecule inhibitors, small molecule drugs are highly underrepresented in the current complement-directed therapeutics pipeline. In this study we have employed a cheminformatics drug discovery approach based on the extensive structural and functional knowledge available for the central proteolytic fragment of the cascade, C3b. Using parallel in silico screening methodologies we identified 45 small molecules which putatively bind C3b near ligand-guided functional hot-spots. Surface plasmon resonance experiments resulted in the validation of seven dose-dependent C3b-binding compounds. Competition-based biochemical assays demonstrated the ability of several C3b-binding compounds to interfere with binding of the original C3b ligand which guided their discovery. In vitro assays of complement function identified a single complement inhibitory compound, termed cmp-5, and mechanistic studies of the cmp-5 inhibitory mode revealed it acts at the level of C5 activation. This study has led to the identification of a promising new class of C3b-binding small molecule complement inhibitors, and to our knowledge, provides the first demonstration of cheminformatics-based complement-directed drug discovery. PMID:28298523

Comparison of (/sup 3/H)nicotine and (/sup 3/H)acetylcholine binding in mouse brain: regional distribution

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sershen, H.; Reith, M.E.; Hashim, A.

1985-06-01

In a continuing study of nicotine binding sites, the authors determined the relative amount of nicotine binding and acetylcholine binding in various brain regions of C57/BL and of DBA mice. Although midbrain showed the highest and cerebellum the lowest binding for both (/sup 3/H)nicotine and (/sup 3/H)acetylcholine, the ratio of nicotine to acetylcholine binding showed a three-fold regional variation. Acetylcholine inhibition of (/sup 3/H)nicotine binding indicated that a portion of nicotine binding was not inhibited by acetylcholine. These results indicate important differences between the binding of (+/-)-(/sup 3/H)nicotine and that of (/sup 3/H)acetylcholine.

Many Routes to an Antibody Heavy-Chain CDR3: Necessary, Yet Insufficient, for Specific Binding

DOE PAGES

D'Angelo, Sara; Ferrara, Fortunato; Naranjo, Leslie; ...

2018-03-08

Because of its great potential for diversity, the immunoglobulin heavy-chain complementarity-determining region 3 (HCDR3) is taken as an antibody molecule’s most important component in conferring binding activity and specificity. For this reason, HCDR3s have been used as unique identifiers to investigate adaptive immune responses in vivo and to characterize in vitro selection outputs where display systems were employed. Here, we show that many different HCDR3s can be identified within a target-specific antibody population after in vitro selection. For each identified HCDR3, a number of different antibodies bearing differences elsewhere can be found. In such selected populations, all antibodies with themore » same HCDR3 recognize the target, albeit at different affinities. In contrast, within unselected populations, the majority of antibodies with the same HCDR3 sequence do not bind the target. In one HCDR3 examined in depth, all target-specific antibodies were derived from the same VDJ rearrangement, while non-binding antibodies with the same HCDR3 were derived from many different V and D gene rearrangements. Careful examination of previously published in vivo datasets reveals that HCDR3s shared between, and within, different individuals can also originate from rearrangements of different V and D genes, with up to 26 different rearrangements yielding the same identical HCDR3 sequence. On the basis of these observations, we conclude that the same HCDR3 can be generated by many different rearrangements, but that specific target binding is an outcome of unique rearrangements and VL pairing: the HCDR3 is necessary, albeit insufficient, for specific antibody binding.« less

Many Routes to an Antibody Heavy-Chain CDR3: Necessary, Yet Insufficient, for Specific Binding

DOE Office of Scientific and Technical Information (OSTI.GOV)

D'Angelo, Sara; Ferrara, Fortunato; Naranjo, Leslie

Because of its great potential for diversity, the immunoglobulin heavy-chain complementarity-determining region 3 (HCDR3) is taken as an antibody molecule’s most important component in conferring binding activity and specificity. For this reason, HCDR3s have been used as unique identifiers to investigate adaptive immune responses in vivo and to characterize in vitro selection outputs where display systems were employed. Here, we show that many different HCDR3s can be identified within a target-specific antibody population after in vitro selection. For each identified HCDR3, a number of different antibodies bearing differences elsewhere can be found. In such selected populations, all antibodies with themore » same HCDR3 recognize the target, albeit at different affinities. In contrast, within unselected populations, the majority of antibodies with the same HCDR3 sequence do not bind the target. In one HCDR3 examined in depth, all target-specific antibodies were derived from the same VDJ rearrangement, while non-binding antibodies with the same HCDR3 were derived from many different V and D gene rearrangements. Careful examination of previously published in vivo datasets reveals that HCDR3s shared between, and within, different individuals can also originate from rearrangements of different V and D genes, with up to 26 different rearrangements yielding the same identical HCDR3 sequence. On the basis of these observations, we conclude that the same HCDR3 can be generated by many different rearrangements, but that specific target binding is an outcome of unique rearrangements and VL pairing: the HCDR3 is necessary, albeit insufficient, for specific antibody binding.« less

Nucleocytoplasmic Shuttling of the Golgi Phosphatidylinositol 4-Kinase Pik1 Is Regulated by 14-3-3 Proteins and Coordinates Golgi Function with Cell Growth

PubMed Central

Demmel, Lars; Beck, Mike; Klose, Christian; Schlaitz, Anne-Lore; Gloor, Yvonne; Hsu, Peggy P.; Havlis, Jan; Shevchenko, Andrej; Krause, Eberhard; Kalaidzidis, Yannis

2008-01-01

The yeast phosphatidylinositol 4-kinase Pik1p is essential for proliferation, and it controls Golgi homeostasis and transport of newly synthesized proteins from this compartment. At the Golgi, phosphatidylinositol 4-phosphate recruits multiple cytosolic effectors involved in formation of post-Golgi transport vesicles. A second pool of catalytically active Pik1p localizes to the nucleus. The physiological significance and regulation of this dual localization of the lipid kinase remains unknown. Here, we show that Pik1p binds to the redundant 14-3-3 proteins Bmh1p and Bmh2p. We provide evidence that nucleocytoplasmic shuttling of Pik1p involves phosphorylation and that 14-3-3 proteins bind Pik1p in the cytoplasm. Nutrient deprivation results in relocation of Pik1p from the Golgi to the nucleus and increases the amount of Pik1p–14-3-3 complex, a process reversed upon restored nutrient supply. These data suggest a role of Pik1p nucleocytoplasmic shuttling in coordination of biosynthetic transport from the Golgi with nutrient signaling. PMID:18172025

Somatomedin-1 binding protein-3: insulin-like growth factor-1 binding protein-3, insulin-like growth factor-1 carrier protein.

PubMed

2003-01-01

Somatomedin-1 binding protein-3 [insulin-like growth factor-1 binding protein-3, SomatoKine] is a recombinant complex of insulin-like growth factor-1 (rhIGF-1) and binding protein-3 (IGFBP-3), which is the major circulating somatomedin (insulin-like growth factor) binding protein; binding protein-3 regulates the delivery of somatomedin-1 to target tissues. Somatomedin-1 binding protein-3 has potential as replacement therapy for somatomedin-1 which may become depleted in indications such as major surgery, organ damage/failure and traumatic injury, resulting in catabolism. It also has potential for the treatment of osteoporosis; diseases associated with protein wasting including chronic renal failure, cachexia and severe trauma; and to attenuate cardiac dysfunction in a variety of disease states, including after severe burn trauma. Combined therapy with somatomedin-1 and somatomedin-1 binding protein-3 would prolong the duration of action of somatomedin-1 and would reduce or eliminate some of the undesirable effects associated with somatomedin-1 monotherapy. Somatomedin-1 is usually linked to binding protein-3 in the normal state of the body, and particular proteases clip them apart in response to stresses and release somatomedin-1 as needed. Therefore, somatomedin-1 binding protein-3 is a self-dosing system and SomatoKine would augment the natural supply of these linked compounds. Somatomedin-1 binding protein-3 was developed by Celtrix using its proprietary recombinant protein production technology. Subsequently, Celtrix was acquired by Insmed Pharmaceuticals on June 1 2000. Insmed and Avecia, UK, have signed an agreement for the manufacturing of SomatoKine and its components, IGF-1 and binding protein-3. CGMP clinical production of SomatoKine and its components will be done in Avecia's Advanced Biologics Centre, Billingham, UK, which manufactures recombinant-based medicines and vaccines with a capacity of up to 1000 litres. In 2003, manufacturing of SomatoKine is

Binding selectivity of vitamin K3 based chemosensors towards nickel(II) and copper(II) metal ions

NASA Astrophysics Data System (ADS)

Patil, Amit; Lande, Dipali N.; Nalkar, Archana; Gejji, Shridhar P.; Chakrovorty, Debamitra; Gonnade, Rajesh; Moniz, Tânia; Rangel, Maria; Pereira, Eulália; Salunke-Gawali, Sunita

2017-09-01

The vitamin K3 derivatives 2-methyl-3-[(pyridin-2-ylmethyl)-amino]-1,4-naphthoquinone (M-1), 2-methyl-3-[(pyridin-2-ylethyl)-amino]-1,4-naphthoquinone (M-2), 2-methyl-3-((2-(thiophen-2-yl)methyl)amino)naphthalene-1,4-dione (M-3) and 2-methyl-3-((2-(thiophen-2-yl)ethyl)amino)naphthalene-1,4-dione (M-4) have been synthesized, characterized and studied for their chemosensor abilities towards transition metal ions. Crystal structures of M-1 to M-4 revealed a variety of Nsbnd H⋯O, Csbnd H⋯O, Csbnd H⋯π and π⋯π interactions. Minor variations in such interactions by chemical stimuli such as metal ions, results in change in color that can be visualized by naked eyes. It has been shown that electronic structure and 1H NMR, vibrational as well as electronic spectra from the density functional theory agree well with the experiments. The metal ion binding in ethanol, ethanol-water and in mild base triethylamine brings forth recognizing ability of M-1 toward Ni2+ whereas M-2 exhibits large sensing ability for Cu2+ ion. Interestingly M-1 display varying metal ion binding specificity in different solvents with the association constant in ethanol being 11,786 M-1 for Ni2+ compared to 9462 M-1 for the Cu2+. A reversal in preferential binding of M-2 with the respective association constants being 4190 M-1 and 6370 M-1 is discernible.

Plasma Membrane CRPK1-Mediated Phosphorylation of 14-3-3 Proteins Induces Their Nuclear Import to Fine-Tune CBF Signaling during Cold Response.

PubMed

Liu, Ziyan; Jia, Yuxin; Ding, Yanglin; Shi, Yiting; Li, Zhen; Guo, Yan; Gong, Zhizhong; Yang, Shuhua

2017-04-06

In plant cells, changes in fluidity of the plasma membrane may serve as the primary sensor of cold stress; however, the precise mechanism and how the cell transduces and fine-tunes cold signals remain elusive. Here we show that the cold-activated plasma membrane protein cold-responsive protein kinase 1 (CRPK1) phosphorylates 14-3-3 proteins. The phosphorylated 14-3-3 proteins shuttle from the cytosol to the nucleus, where they interact with and destabilize the key cold-responsive C-repeat-binding factor (CBF) proteins. Consistent with this, the crpk1 and 14-3-3κλ mutants show enhanced freezing tolerance, and transgenic plants overexpressing 14-3-3λ show reduced freezing tolerance. Further study shows that CRPK1 is essential for the nuclear translocation of 14-3-3 proteins and for 14-3-3 function in freezing tolerance. Thus, our study reveals that the CRPK1-14-3-3 module transduces the cold signal from the plasma membrane to the nucleus to modulate CBF stability, which ensures a faithfully adjusted response to cold stress of plants. Copyright © 2017 Elsevier Inc. All rights reserved.

16 CFR 3.14 - Intervention.

Code of Federal Regulations, 2010 CFR

2010-01-01

... 16 Commercial Practices 1 2010-01-01 2010-01-01 false Intervention. 3.14 Section 3.14 Commercial... ADJUDICATIVE PROCEEDINGS Pleadings § 3.14 Intervention. (a) Any individual, partnership, unincorporated.... The Administrative Law Judge or the Commission may by order permit the intervention to such extent and...

16 CFR 3.14 - Intervention.

Code of Federal Regulations, 2014 CFR

2014-01-01

... 16 Commercial Practices 1 2014-01-01 2014-01-01 false Intervention. 3.14 Section 3.14 Commercial... ADJUDICATIVE PROCEEDINGS Pleadings § 3.14 Intervention. (a) Any individual, partnership, unincorporated.... The Administrative Law Judge or the Commission may by order permit the intervention to such extent and...

16 CFR 3.14 - Intervention.

Code of Federal Regulations, 2012 CFR

2012-01-01

... 16 Commercial Practices 1 2012-01-01 2012-01-01 false Intervention. 3.14 Section 3.14 Commercial... ADJUDICATIVE PROCEEDINGS Pleadings § 3.14 Intervention. (a) Any individual, partnership, unincorporated.... The Administrative Law Judge or the Commission may by order permit the intervention to such extent and...

16 CFR 3.14 - Intervention.

Code of Federal Regulations, 2011 CFR

2011-01-01

... 16 Commercial Practices 1 2011-01-01 2011-01-01 false Intervention. 3.14 Section 3.14 Commercial... ADJUDICATIVE PROCEEDINGS Pleadings § 3.14 Intervention. (a) Any individual, partnership, unincorporated.... The Administrative Law Judge or the Commission may by order permit the intervention to such extent and...

16 CFR 3.14 - Intervention.

Code of Federal Regulations, 2013 CFR

2013-01-01

... 16 Commercial Practices 1 2013-01-01 2013-01-01 false Intervention. 3.14 Section 3.14 Commercial... ADJUDICATIVE PROCEEDINGS Pleadings § 3.14 Intervention. (a) Any individual, partnership, unincorporated.... The Administrative Law Judge or the Commission may by order permit the intervention to such extent and...

The inhibitor protein of phosphorylated nitrate reductase from spinach (Spinacia oleracea) leaves is a 14-3-3 protein.

PubMed

Bachmann, M; Huber, J L; Liao, P C; Gage, D A; Huber, S C

1996-06-03

The inhibitor protein (IP) that inactivates spinach leaf NADH:nitrate reductase (NR) has been identified for the first time as a member of the eukaryotic 14-3-3 protein family based on three lines of evidence. First, the sequence of an eight amino acid tryptic peptide, obtained from immunopurified IP, matched that of a highly conserved region of the 14-3-3 proteins. Second, an authentic member of the 14-3-3 family, recombinant Arabidopsis GF14omega, caused inactivation of phospho-NR in a magnesium-dependent manner identical to IP. Third, an anti-GF14 monoclonal antibody cross-reacted with IP and anti-IP monoclonal antibodies cross-reacted with GF14omega.

17 CFR 14.3 - Hearings.

Code of Federal Regulations, 2012 CFR

2012-04-01

... 17 Commodity and Securities Exchanges 1 2012-04-01 2012-04-01 false Hearings. 14.3 Section 14.3 Commodity and Securities Exchanges COMMODITY FUTURES TRADING COMMISSION RULES RELATING TO SUSPENSION OR DISBARMENT FROM APPEARANCE AND PRACTICE § 14.3 Hearings. Hearings required or permitted to be held under...

The Rho ADP-ribosylating C3 exoenzyme binds cells via an Arg-Gly-Asp motif.

PubMed

Rohrbeck, Astrid; Höltje, Markus; Adolf, Andrej; Oms, Elisabeth; Hagemann, Sandra; Ahnert-Hilger, Gudrun; Just, Ingo

2017-10-27

The Rho ADP-ribosylating C3 exoenzyme (C3bot) is a bacterial protein toxin devoid of a cell-binding or -translocation domain. Nevertheless, C3 can efficiently enter intact cells, including neurons, but the mechanism of C3 binding and uptake is not yet understood. Previously, we identified the intermediate filament vimentin as an extracellular membranous interaction partner of C3. However, uptake of C3 into cells still occurs (although reduced) in the absence of vimentin, indicating involvement of an additional host cell receptor. C3 harbors an Arg-Gly-Asp (RGD) motif, which is the major integrin-binding site, present in a variety of integrin ligands. To check whether the RGD motif of C3 is involved in binding to cells, we performed a competition assay with C3 and RGD peptide or with a monoclonal antibody binding to β1-integrin subunit and binding assays in different cell lines, primary neurons, and synaptosomes with C3-RGD mutants. Here, we report that preincubation of cells with the GRGDNP peptide strongly reduced C3 binding to cells. Moreover, mutation of the RGD motif reduced C3 binding to intact cells and also to recombinant vimentin. Anti-integrin antibodies also lowered the C3 binding to cells. Our results indicate that the RGD motif of C3 is at least one essential C3 motif for binding to host cells and that integrin is an additional receptor for C3 besides vimentin. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Long non-coding RNA CRYBG3 blocks cytokinesis by directly binding G-actin.

PubMed

Pei, Hailong; Hu, Wentao; Guo, Ziyang; Chen, Huaiyuan; Ma, Ji; Mao, Weidong; Li, Bingyan; Wang, Aiqing; Wan, Jianmei; Zhang, Jian; Nie, Jing; Zhou, Guangming; Hei, Tom K

2018-06-22

The dynamic interchange between monomeric globular actin (G-actin) and polymeric filamentous actin filaments (F-actin) is fundamental and essential to many cellular processes including cytokinesis and maintenance of genomic stability. Here we report that the long non-coding RNA LNC CRYBG3 directly binds G-actin to inhibit its polymerization and formation of contractile rings, resulting in M-Phase cell arrest. Knockdown of LNC CRYBG3 in tumor cells enhanced their malignant phenotypes. Nucleotide sequence 228-237 of the full-length LNC CRYBG3 and the ser14 domain of beta-actin are essential for their interaction, and mutation of either of these sites abrogated binding of LNC CRYBG3 to G-actin. Binding of LNC CRYBG3 to G-actin blocked nuclear localization of MAL, which consequently kept serum response factor (SRF) away from the promoter region of several immediate early genes, including JUNB and Arp3, which are necessary for cellular proliferation, tumor growth, adhesion, movement, and metastasis. These findings reveal a novel lncRNA-actin-MAL-SRF pathway and highlight LNC CRYBG3 as a means to block cytokinesis and treat cancer by targeting the actin cytoskeleton. Copyright ©2018, American Association for Cancer Research.

Acetaminophen structure-toxicity studies: In vivo covalent binding of a nonhepatotoxic analog, 3-hydroxyacetanilide

DOE Office of Scientific and Technical Information (OSTI.GOV)

Roberts, S.A.; Price, V.F.; Jollow, D.J.

1990-09-01

High doses of 3-hydroxyacetanilide (3HAA), a structural isomer of acetaminophen, do not produce hepatocellular necrosis in normal male hamsters or in those sensitized to acetaminophen-induced liver damage by pretreatment with a combination of 3-methylcholanthrene, borneol, and diethyl maleate. Although 3HAA was not hepatotoxic, the administration of acetyl-labeled (3H or 14C)3HAA (400 mg/kg, ip) produced levels of covalently bound radiolabel that were similar to those observed after an equimolar, hepatotoxic dose of (G-3H)acetaminophen. The covalent nature of 3HAA binding was demonstrated by retention of the binding after repetitive organic solvent extraction following protease digestion. Hepatic and renal covalent binding after 3HAAmore » was approximately linear with both dose and time. In addition, 3HAA produced only a modest depletion of hepatic glutathione, suggesting the lack of a glutathione threshold. 3-Methylcholanthrene pretreatment increased and pretreatment with cobalt chloride and piperonyl butoxide decreased the hepatic covalent binding of 3HAA, indicating the involvement of cytochrome P450 in the formation of the 3HAA reactive metabolite. The administration of multiple doses or a single dose of (ring-3H)3HAA to hamsters pretreated with a combination of 3-methylcholanthrene, borneol, and diethyl maleate produced hepatic levels of 3HAA covalent binding that were in excess of those observed after a single, hepatotoxic acetaminophen dose. These data suggest that the nature and/or the intracellular processing of the reactive metabolites of acetaminophen and 3HAA are different. These data also demonstrate that absolute levels of covalently bound xenobiotic metabolites cannot be utilized as absolute predictors of cytotoxic potential.« less

Comparison of (/sup 3/H)pirenzepine and (/sup 3/H)quinuclidinylbenzilate binding to muscarinic cholinergic receptors in rat brain

DOE Office of Scientific and Technical Information (OSTI.GOV)

Luthin, G.R.; Wolfe, B.B.

The properties of (/sup 3/H)quinuclidinylbenzilate ( (/sup 3/H)QNB) binding and (/sup 3/H)pirenzepine ( (/sup 3/H)PZ) binding to various regions of rat brain were compared. (/sup 3/H)PZ appeared to bind with high affinity to a single site, with a Kd value of approximately 15 nM in the cerebral cortex. The rank order of potencies of muscarinic drugs to inhibit binding of either (/sup 3/H)QNB or (/sup 3/H)PZ was QNB greater than atropine . scopolamine greater than pirenzepine greater than oxotremorine greater than bethanechol. Muscarinic antagonists (except PZ) inhibited both (/sup 3/H)PZ and (/sup 3/H)QNB binding with Hill coefficients of approximately 1.more » PZ inhibited (/sup 3/H)QNB binding in cortex with a Hill coefficient of 0.7, but inhibited (/sup 3/H)PZ binding with a Hill coefficient of 1.0. Hill coefficients for agonists were less than 1. The density of (/sup 3/H)PZ binding sites was approximately half the density of (/sup 3/H)QNB binding sites in cortex, striatum and hippocampus. In pons-medulla and cerebellum, the densities of (/sup 3/H)PZ binding sites were 20 and 0%, respectively, relative to the densities of (/sup 3/H)QNB binding sites. When unlabeled PZ was used to compete for (/sup 3/H)QNB binding, the relative number of high-affinity PZ binding sites in cortex, pons and cerebellum agreed with the relative number of (/sup 3/H)PZ binding sites in those regions. The binding of (/sup 3/H)PZ and (/sup 3/H)QNB was nonadditive in cortex. GTP inhibited high-affinity oxotremorine binding, but not PZ binding. Together, these data suggest that (/sup 3/H)PZ binds to a subset of (/sup 3/H)QNB binding sites. Whether this subset reflects the existence of subtypes of muscarinic receptors or is a consequence of coupling to another membrane protein remains to be seen.« less

Phosphorylation of Krüppel-like factor 3 (KLF3/BKLF) and C-terminal binding protein 2 (CtBP2) by homeodomain-interacting protein kinase 2 (HIPK2) modulates KLF3 DNA binding and activity.

PubMed

Dewi, Vitri; Kwok, Alister; Lee, Stella; Lee, Ming Min; Tan, Yee Mun; Nicholas, Hannah R; Isono, Kyo-ichi; Wienert, Beeke; Mak, Ka Sin; Knights, Alexander J; Quinlan, Kate G R; Cordwell, Stuart J; Funnell, Alister P W; Pearson, Richard C M; Crossley, Merlin

2015-03-27

Krüppel-like factor 3 (KLF3/BKLF), a member of the Krüppel-like factor (KLF) family of transcription factors, is a widely expressed transcriptional repressor with diverse biological roles. Although there is considerable understanding of the molecular mechanisms that allow KLF3 to silence the activity of its target genes, less is known about the signal transduction pathways and post-translational modifications that modulate KLF3 activity in response to physiological stimuli. We observed that KLF3 is modified in a range of different tissues and found that the serine/threonine kinase homeodomain-interacting protein kinase 2 (HIPK2) can both bind and phosphorylate KLF3. Mass spectrometry identified serine 249 as the primary phosphorylation site. Mutation of this site reduces the ability of KLF3 to bind DNA and repress transcription. Furthermore, we also determined that HIPK2 can phosphorylate the KLF3 co-repressor C-terminal binding protein 2 (CtBP2) at serine 428. Finally, we found that phosphorylation of KLF3 and CtBP2 by HIPK2 strengthens the interaction between these two factors and increases transcriptional repression by KLF3. Taken together, our results indicate that HIPK2 potentiates the activity of KLF3. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Lil3 dimerization and chlorophyll binding in Arabidopsis thaliana.

PubMed

Mork-Jansson, Astrid Elisabeth; Gargano, Daniela; Kmiec, Karol; Furnes, Clemens; Shevela, Dmitriy; Eichacker, Lutz Andreas

2015-10-07

The two-helix light harvesting like (Lil) protein Lil3 belongs to the family of chlorophyll binding light harvesting proteins of photosynthetic membranes. A function in tetrapyrrol synthesis and stabilization of geranylgeraniol reductase has been shown. Lil proteins contain the chlorophyll a/b-binding motif; however, binding of chlorophyll has not been demonstrated. We find that Lil3.2 from Arabidopsis thaliana forms heterodimers with Lil3.1 and binds chlorophyll. Lil3.2 heterodimerization (25±7.8 nM) is favored relative to homodimerization (431±59 nM). Interaction of Lil3.2 with chlorophyll a (231±49 nM) suggests that heterodimerization precedes binding of chlorophyll in Arabidopsis thaliana. Copyright © 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Identification of C3b-Binding Small-Molecule Complement Inhibitors Using Cheminformatics.

PubMed

Garcia, Brandon L; Skaff, D Andrew; Chatterjee, Arindam; Hanning, Anders; Walker, John K; Wyckoff, Gerald J; Geisbrecht, Brian V

2017-05-01

The complement system is an elegantly regulated biochemical cascade formed by the collective molecular recognition properties and proteolytic activities of more than two dozen membrane-bound or serum proteins. Complement plays diverse roles in human physiology, such as acting as a sentry against invading microorganisms, priming of the adaptive immune response, and removal of immune complexes. However, dysregulation of complement can serve as a trigger for a wide range of human diseases, which include autoimmune, inflammatory, and degenerative conditions. Despite several potential advantages of modulating complement with small-molecule inhibitors, small-molecule drugs are highly underrepresented in the current complement-directed therapeutics pipeline. In this study, we have employed a cheminformatics drug discovery approach based on the extensive structural and functional knowledge available for the central proteolytic fragment of the cascade, C3b. Using parallel in silico screening methodologies, we identified 45 small molecules that putatively bind C3b near ligand-guided functional hot spots. Surface plasmon resonance experiments resulted in the validation of seven dose-dependent C3b-binding compounds. Competition-based biochemical assays demonstrated the ability of several C3b-binding compounds to interfere with binding of the original C3b ligand that guided their discovery. In vitro assays of complement function identified a single complement inhibitory compound, termed cmp-5, and mechanistic studies of the cmp-5 inhibitory mode revealed it acts at the level of C5 activation. This study has led to the identification of a promising new class of C3b-binding small-molecule complement inhibitors and, to our knowledge, provides the first demonstration of cheminformatics-based, complement-directed drug discovery. Copyright © 2017 by The American Association of Immunologists, Inc.

The Prognostic Value of 14-3-3 Isoforms in Vulvar Squamous Cell Carcinoma Cases: 14-3-3β and ε Are Independent Prognostic Factors for These Tumors

PubMed Central

Wang, Zhihui; Nesland, Jahn M.; Suo, Zhenhe; Trope, Claes G.; Holm, Ruth

2011-01-01

Background The 14-3-3 family is comprised of highly conserved proteins that are functionally important in the maintenance of homeostasis. Their involvement with the cell cycle, their association with proto-oncogenes and oncogenes, and their abnormal expression in various tumors has linked this family of proteins to the etiology of human cancer. Mounting evidence now indicates that 14-3-3σ is a cancer suppressor gene but the roles of the other 14-3-3 isoforms and their interactions in tumorigenesis have not yet been elucidated. In our current study, we examined the expression of 14-3-3β, γ, ε, ζ, η and τ in a large series of vulvar squamous cell carcinomas to evaluate any clinical significance. Methods Tumor biopsies from 298 vulvar carcinomas were examined by immunohistochemistry for the expression of 14-3-3β, γ, ε, ζ, η and τ. Statistical analyses were employed to validate any associations between the expression of any 14-3-3 isoform and clinicopathologic variables for this disease. Results High cytoplasmic levels of 14-3-3β, γ, ζ, ε and η were observed in 79%, 58%, 50%, 86% and 54% of the vulvar carcinomas analyzed, respectively, whereas a low nuclear expression of 14-3-3τ was present in 80% of these cases. The elevated cytoplasmic expression of 14-3-3β, γ, ε, ζ and η was further found to be associated with advanced disease and aggressive features of these cancers. The overexpression of cytoplasmic 14-3-3β and ε significantly correlated with a poor disease-specific survival by univariate analysis (P = 0.007 and P = 0.04, respectively). The independent prognostic significance of these factors was confirmed by multivariate analysis (P = 0.007 and P = 0.009, respectively). Conclusions We reveal for the first time that the 14-3-3β, γ, ε, ζ, η and τ isoforms may be involved in the progression of vulvar carcinomas. Furthermore, our analyses show that high cytoplasmic levels of 14-3-3β and ε independently correlate with

A 14-3-3 Family Protein from Wild Soybean (Glycine Soja) Regulates ABA Sensitivity in Arabidopsis

PubMed Central

Sun, Xiaoli; Sun, Mingzhe; Jia, Bowei; Chen, Chao; Qin, Zhiwei; Yang, Kejun; Shen, Yang; Meiping, Zhang; Mingyang, Cong; Zhu, Yanming

2015-01-01

It is widely accepted that the 14-3-3 family proteins are key regulators of multiple stress signal transduction cascades. By conducting genome-wide analysis, researchers have identified the soybean 14-3-3 family proteins; however, until now, there is still no direct genetic evidence showing the involvement of soybean 14-3-3s in ABA responses. Hence, in this study, based on the latest Glycine max genome on Phytozome v10.3, we initially analyzed the evolutionary relationship, genome organization, gene structure and duplication, and three-dimensional structure of soybean 14-3-3 family proteins systematically. Our results suggested that soybean 14-3-3 family was highly evolutionary conserved and possessed segmental duplication in evolution. Then, based on our previous functional characterization of a Glycine soja 14-3-3 protein GsGF14o in drought stress responses, we further investigated the expression characteristics of GsGF14o in detail, and demonstrated its positive roles in ABA sensitivity. Quantitative real-time PCR analyses in Glycine soja seedlings and GUS activity assays in PGsGF14O:GUS transgenic Arabidopsis showed that GsGF14o expression was moderately and rapidly induced by ABA treatment. As expected, GsGF14o overexpression in Arabidopsis augmented the ABA inhibition of seed germination and seedling growth, promoted the ABA induced stomata closure, and up-regulated the expression levels of ABA induced genes. Moreover, through yeast two hybrid analyses, we further demonstrated that GsGF14o physically interacted with the AREB/ABF transcription factors in yeast cells. Taken together, results presented in this study strongly suggested that GsGF14o played an important role in regulation of ABA sensitivity in Arabidopsis. PMID:26717241

12 CFR 3.14 - Remedies.

Code of Federal Regulations, 2011 CFR

2011-01-01

... 12 Banks and Banking 1 2011-01-01 2011-01-01 false Remedies. 3.14 Section 3.14 Banks and Banking COMPTROLLER OF THE CURRENCY, DEPARTMENT OF THE TREASURY MINIMUM CAPITAL RATIOS; ISSUANCE OF DIRECTIVES Enforcement § 3.14 Remedies. A bank that does not have or maintain the minimum capital ratios applicable to it...

12 CFR 3.14 - Remedies.

Code of Federal Regulations, 2010 CFR

2010-01-01

... 12 Banks and Banking 1 2010-01-01 2010-01-01 false Remedies. 3.14 Section 3.14 Banks and Banking COMPTROLLER OF THE CURRENCY, DEPARTMENT OF THE TREASURY MINIMUM CAPITAL RATIOS; ISSUANCE OF DIRECTIVES Enforcement § 3.14 Remedies. A bank that does not have or maintain the minimum capital ratios applicable to it...

Multiple sup 3 H-oxytocin binding sites in rat myometrial plasma membranes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Crankshaw, D.; Gaspar, V.; Pliska, V.

1990-01-01

The affinity spectrum method has been used to analyse binding isotherms for {sup 3}H-oxytocin to rat myometrial plasma membranes. Three populations of binding sites with dissociation constants (Kd) of 0.6-1.5 x 10(-9), 0.4-1.0 x 10(-7) and 7 x 10(-6) mol/l were identified and their existence verified by cluster analysis based on similarities between Kd, binding capacity and Hill coefficient. When experimental values were compared to theoretical curves constructed using the estimated binding parameters, good fits were obtained. Binding parameters obtained by this method were not influenced by the presence of GTP gamma S (guanosine-5'-O-3-thiotriphosphate) in the incubation medium. The bindingmore » parameters agree reasonably well with those found in uterine cells, they support the existence of a medium affinity site and may allow for an explanation of some of the discrepancies between binding and response in this system.« less

A structured proteomic approach identifies 14-3-3Sigma as a novel and reliable protein biomarker in panel based differential diagnostics of liver tumors.

PubMed

Reis, Henning; Pütter, Carolin; Megger, Dominik A; Bracht, Thilo; Weber, Frank; Hoffmann, Andreas-C; Bertram, Stefanie; Wohlschläger, Jeremias; Hagemann, Sascha; Eisenacher, Martin; Scherag, André; Schlaak, Jörg F; Canbay, Ali; Meyer, Helmut E; Sitek, Barbara; Baba, Hideo A

2015-06-01

Hepatocellular carcinoma (HCC) is a major lethal cancer worldwide. Despite sophisticated diagnostic algorithms, the differential diagnosis of small liver nodules still is difficult. While imaging techniques have advanced, adjuvant protein-biomarkers as glypican3 (GPC3), glutamine-synthetase (GS) and heat-shock protein 70 (HSP70) have enhanced diagnostic accuracy. The aim was to further detect useful protein-biomarkers of HCC with a structured systematic approach using differential proteome techniques, bring the results to practical application and compare the diagnostic accuracy of the candidates with the established biomarkers. After label-free and gel-based proteomics (n=18 HCC/corresponding non-tumorous liver tissue (NTLT)) biomarker candidates were tested for diagnostic accuracy in immunohistochemical analyses (n=14 HCC/NTLT). Suitable candidates were further tested for consistency in comparison to known protein-biomarkers in HCC (n=78), hepatocellular adenoma (n=25; HCA), focal nodular hyperplasia (n=28; FNH) and cirrhosis (n=28). Of all protein-biomarkers, 14-3-3Sigma (14-3-3S) exhibited the most pronounced up-regulation (58.8×) in proteomics and superior diagnostic accuracy (73.0%) in the differentiation of HCC from non-tumorous hepatocytes also compared to established biomarkers as GPC3 (64.7%) and GS (45.4%). 14-3-3S was part of the best diagnostic three-biomarker panel (GPC3, HSP70, 14-3-3S) for the differentiation of HCC and HCA which is of most important significance. Exclusion of GS and inclusion of 14-3-3S in the panel (>1 marker positive) resulted in a profound increase in specificity (+44.0%) and accuracy (+11.0%) while sensitivity remained stable (96.0%). 14-3-3S is an interesting protein biomarker with the potential to further improve the accuracy of differential diagnostic process of hepatocellular tumors. This article is part of a Special Issue entitled: Medical Proteomics. Copyright © 2014 Elsevier B.V. All rights reserved.

MMPP Attenuates Non-Small Cell Lung Cancer Growth by Inhibiting the STAT3 DNA-Binding Activity via Direct Binding to the STAT3 DNA-Binding Domain.

PubMed

Son, Dong Ju; Zheng, Jie; Jung, Yu Yeon; Hwang, Chul Ju; Lee, Hee Pom; Woo, Ju Rang; Baek, Song Yi; Ham, Young Wan; Kang, Min Woong; Shong, Minho; Kweon, Gi Ryang; Song, Min Jong; Jung, Jae Kyung; Han, Sang-Bae; Kim, Bo Yeon; Yoon, Do Young; Choi, Bu Young; Hong, Jin Tae

2017-01-01

Rationale: Signal transducer and activator of transcription-3 (STAT3) plays a pivotal role in cancer biology. Many small-molecule inhibitors that target STAT3 have been developed as potential anticancer drugs. While designing small-molecule inhibitors that target the SH2 domain of STAT3 remains the leading focus for drug discovery, there has been a growing interest in targeting the DNA-binding domain (DBD) of the protein. Methods: We demonstrated the potential antitumor activity of a novel, small-molecule (E)-2-methoxy-4-(3-(4-methoxyphenyl)prop-1-en-1-yl)phenol (MMPP) that directly binds to the DBD of STAT3, in patient-derived non-small cell lung cancer (NSCLC) xenograft model as well as in NCI-H460 cell xenograft model in nude mice. Results: MMPP effectively inhibited the phosphorylation of STAT3 and its DNA binding activity in vitro and in vivo . It induced G1-phase cell cycle arrest and apoptosis through the regulation of cell cycle- and apoptosis-regulating genes by directly binding to the hydroxyl residue of threonine 456 in the DBD of STAT3. Furthermore, MMPP showed a similar or better antitumor activity than that of docetaxel or cisplatin. Conclusion: MMPP is suggested to be a potential candidate for further development as an anticancer drug that targets the DBD of STAT3.

GA binding protein augments autophagy via transcriptional activation of BECN1-PIK3C3 complex genes

PubMed Central

Zhu, Wan; Swaminathan, Gayathri; Plowey, Edward D

2014-01-01

Macroautophagy is a vesicular catabolic trafficking pathway that is thought to protect cells from diverse stressors and to promote longevity. Recent studies have revealed that transcription factors play important roles in the regulation of autophagy. In this study, we have identified GA binding protein (GABP) as a transcriptional regulator of the combinatorial expression of BECN1-PIK3C3 complex genes involved in autophagosome initiation. We performed bioinformatics analyses that demonstrated highly conserved putative GABP sites in genes that encode BECN1/Beclin 1, several BECN1 interacting proteins, and downstream autophagy proteins including the ATG12–ATG5-ATG16L1 complex. We demonstrate that GABP binds to the promoter regions of BECN1-PIK3C3 complex genes and activates their transcriptional activities. Knockdown of GABP reduced BECN1-PIK3C3 complex transcripts, BECN1-PIK3C3 complex protein levels and autophagy in cultured cells. Conversely, overexpression of GABP increased autophagy. Nutrient starvation increased GABP-dependent transcriptional activity of BECN1-PIK3C3 complex gene promoters and increased the recruitment of GABP to the BECN1 promoter. Our data reveal a novel function of GABP in the regulation of autophagy via transcriptional activation of the BECN1-PIK3C3 complex. PMID:25046113

Arachidonic acid depletion extends survival of cold-stored platelets by interfering with the [glycoprotein Ibα – 14-3-3ζ] association

PubMed Central

van der Wal, Dianne E.; Gitz, Eelo; Du, Vivian X.; Lo, Kimberly S.L.; Koekman, Cornelis A.; Versteeg, Sabine; Akkerman, Jan Willem N.

2012-01-01

Background Cold storage of platelets reduces bacterial growth and preserves their hemostatic properties better than current procedures do. However, storage at 0°C induces [14-3-3ζ-glycoprotein Ibα] association, 14-3-3ζ release from phospho-Bad, Bad activation and apoptosis. Design and Methods We investigated whether arachidonic acid, which also binds 14-3-3ζ, contributes to coldinduced apoptosis. Results Cold storage activated P38-mitogen-activated protein kinase and released arachidonic acid, which accumulated due to cold inactivation of cyclooxygenase-1/thromboxane synthase. Accumulated arachidonic acid released 14-3-3ζ from phospho-Bad and decreased the mitochondrial membrane potential, which are steps in the induction of apoptosis. Addition of arachidonic acid did the same and its depletion made platelets resistant to cold-induced apoptosis. Incubation with biotin-arachidonic acid revealed formation of an [arachidonic acid-14-3-3ζ-glycoprotein Ibα] complex. Indomethacin promoted complex formation by accumulating arachidonic acid and released 14-3-3ζ from cyclo-oxygenase-1. Arachidonic acid depletion prevented the cold-induced reduction of platelet survival in mice. Conclusions We conclude that cold storage induced apoptosis through an [arachidonic acid-14-3-3ζ-glycoprotein Ibα] complex, which released 14-3-3ζ from Bad in an arachidonic acid-dependent manner. Although arachidonic acid depletion reduced agonist-induced thromboxane A2 formation and aggregation, arachidonic acid repletion restored these functions, opening ways to reduce apoptosis during storage without compromising hemostatic functions post-transfusion. PMID:22371179

Cationic Peptides and Peptidomimetics Bind Glycosaminoglycans as Potential Sema3A Pathway Inhibitors

PubMed Central

Corredor, Miriam; Bonet, Roman; Moure, Alejandra; Domingo, Cecilia; Bujons, Jordi; Alfonso, Ignacio; Pérez, Yolanda; Messeguer, Àngel

2016-01-01

Semaphorin3A (Sema3A) is a vertebrate-secreted protein that was initially characterized as a repulsive-guidance cue. Semaphorins have crucial roles in several diseases; therefore, the development of Sema3A inhibitors is of therapeutic interest. Sema3A interacts with glycosaminoglycans (GAGs), presumably through its C-terminal basic region. We used different biophysical techniques (i.e., NMR, surface plasmon resonance, isothermal titration calorimetry, fluorescence, and UV-visible spectroscopy) to characterize the binding of two Sema3A C-terminus-derived basic peptides (FS2 and NFS3) to heparin and chondroitin sulfate A. We found that these peptides bind to both GAGs with affinities in the low-micromolar range. On the other hand, a peptoid named SICHI (semaphorin-induced chemorepulsion inhibitor), which is positively charged at physiological pH, was first identified by our group as being able to block Sema3A chemorepulsion and growth-cone collapse in axons at the extracellular level. To elucidate the direct target for the reported SICHI inhibitory effect in the Sema3A signaling pathway, we looked first to the protein-protein interaction between secreted Sema3A and the Nrp1 receptor. However, our results show that SICHI does not bind directly to the Sema3A sema domain or to Nrp1 extracellular domains. We evaluated a new, to our knowledge, hypothesis, according to which SICHI binds to GAGs, thereby perturbing the Sema3A-GAG interaction. By using the above-mentioned techniques, we observed that SICHI binds to GAGs and competes with Sema3A C-terminus-derived basic peptides for binding to GAGs. These data support the ability of SICHI to block the biologically relevant interaction between Sema3A and GAGs, thus revealing SICHI as a new, to our knowledge, class of inhibitors that target the GAG-protein interaction. PMID:27028639

14-3-3 eta isoform colocalizes TDP-43 on the coarse granules in the anterior horn cells of patients with sporadic amyotrophic lateral sclerosis.

PubMed

Umahara, Takahiko; Uchihara, Toshiki; Shibata, Noriyuki; Nakamura, Ayako; Hanyu, Haruo

2016-09-01

The immunolocalization of the 14-3-3 eta isoform in the anterior horn cells (AHCs) of patients with sporadic amyotrophic lateral sclerosis (ALS) and controls was examined. Compared with the immunolocalization of other 14-3-3 isoforms, the immunolocalization of the 14-3-3 eta isoform was either synaptic at the periphery of AHCs, spindle-shaped in neurites, or granular in the cytoplasm. By double labeling with phosphorylated (p-)TDP-43, the transactivation response DNA binding protein of 43kDa (TDP-43) demonstrated frequent colocalization of the 14-3-3 eta isoform in granular structures (90%) and spindle-shaped structures (85.4%), but not in p-TDP-43-positive round inclusions. It is speculated that the 14-3-3 eta isoform is associated with not only a synaptic pathology of ALS but also TDP-positive small lesions in the cytoplasm and neurites. The absence of eta-like immunoreactivity in p-TDP-43-positive large inclusions suggests the restricted relevance of the 14-3-3 eta isoform during ALS pathogenesis to some phases of the p-TDP pathology. Copyright © 2016 Elsevier B.V. All rights reserved.

Penicillin binding protein 3 of Staphylococcus aureus NCTC 8325-4 binds and activates human plasminogen.

PubMed

Kylväjä, Riikka; Ojalehto, Tuomas; Kainulainen, Veera; Virkola, Ritva; Westerlund-Wikström, Benita

2016-08-04

Staphylococcus aureus is a versatile pathogen expressing a number of virulence-associated adhesive molecules. In a previous study, we generated in a secretion-competent Escherichia coli strain a library of random FLAG-tag positive (FTP) polypeptides of S. aureus. To identify adhesive proteins and gain additional knowledge on putative virulence factors of S. aureus, we here screened the FTP library against human serum proteins. Staphylococcus aureus NCTC 8325-4, origin of the FTP library, adhered to immobilized plasminogen in vitro. In an enzyme-linked immunoassay a C-terminal part of penicillin binding protein 3 (PBP3), included in the FTP library, bound to immobilized plasminogen. We expressed and purified full-length PBP3 and its C-terminal fragments as recombinant proteins. In a time-resolved fluorometry-based assay the PBP3 polypeptides bound to immobilized plasminogen. The polypeptides enhanced formation of plasmin from plasminogen as analyzed by cleavage of a chromogenic plasmin substrate. The present findings, although preliminary, demonstrate reliably that S. aureus NCTC 8325-4 adheres to immobilized plasminogen in vitro and that the adhesion may be mediated by a C-terminal fragment of the PBP3 protein. The full length PBP3 and the penicillin binding C-terminal domain of PBP3 expressed as recombinant proteins bound plasminogen and activated plasminogen to plasmin. These phenomena were inhibited by the lysine analogue ε-aminocaproic acid suggesting that the binding is mediated by lysine residues. A detailed molecular description of surface molecules enhancing the virulence of S. aureus will aid in understanding of its pathogenicity and help in design of antibacterial drugs in the future.

Crystal structure of importin-α3 bound to the nuclear localization signal of Ran-binding protein 3.

PubMed

Koyama, Masako; Matsuura, Yoshiyuki

2017-09-23

Ran-binding protein 3 (RanBP3) is a primarily nuclear Ran-binding protein that functions as an accessory factor in the Ran GTPase system. RanBP3 associates with Ran-specific nucleotide exchange factor RCC1 and enhances its catalytic activity towards Ran. RanBP3 also promotes CRM1-mediated nuclear export as well as CRM1-independent nuclear export of β-catenin, Smad2, and Smad3. Nuclear import of RanBP3 is dependent on the nuclear import adaptor protein importin-α and, RanBP3 is imported more efficiently by importin-α3 than by other members of the importin-α family. Protein kinase signaling pathways control nucleocytoplasmic transport through phosphorylation of RanBP3 at Ser58, immediately C-terminal to the nuclear localization signal (NLS) in the N-terminal region of RanBP3. Here we report the crystal structure of human importin-α3 bound to an N-terminal fragment of human RanBP3 containing the NLS sequence that is necessary and sufficient for nuclear import. The structure reveals that RanBP3 binds to importin-α3 residues that are strictly conserved in all seven isoforms of human importin-α at the major NLS-binding site, indicating that the region of importin-α outside the NLS-binding site, possibly the autoinhibotory importin-β1-binding domain, may be the key determinant for the preferential binding of RanBP3 to importin-α3. Computational docking simulation indicates that phosphorylation of RanBP3 at Ser58 could potentially stabilize the association of RanBP3 with importin-α through interactions between the phosphate moiety of phospho-Ser58 of RanBP3 and a cluster of basic residues (Arg96 and Lys97 in importin-α3) on armadillo repeat 1 of importin-α. Copyright © 2017 Elsevier Inc. All rights reserved.

Characterization of high affinity (/sup 3/H)triazolam binding in rat brain

DOE Office of Scientific and Technical Information (OSTI.GOV)

Earle, M.; Concas, A.; Yamamura, H.I.

1986-03-01

The hypnotic Triazolam (TZ), a triazolo (1,4)-benzodiazepine, displays a short physiological half life and has been used for the treatment of insomnia related to anxiety states. Specific binding properties of this recently tritiated TZ were characterized. The authors major objectives were the direct measurement of the temperature dependence and the GABA effect on (/sup 3/H)TZ binding. Saturation studies showed a shift to lower affinity at 37/sup 0/C (K/sub d/ = 0.25 +/- 0.01 nM at O/sup 0/C; K/sub d/ = 1.46 +/- 0.03 nM at 37/sup 0/C) while the B/sub max/ values remained unchanged (1003 +/- 37 fmoles/mg prot. atmore » 0/sup 0/C and 1001 +/- 43 fmoles/mg prot. at 37/sup 0/C). Inhibition studies showed that (/sup 3/H)TZ binding displayed no GABA shift at 0/sup 0/C(K/sub i/ 0.37 +/- 0.03 nM/- GABA and K/sub i/ = 0.55 +/- 0.13 nM/+GABA) but a nearly two-fold shift was apparent at 37/sup 0/C (K/sub i/ = 2.92 +/- 0.2 nM/-GABA; K/sub i/ = 1.37 +/- 0.11 mM/+GABA). These results were also confirmed by saturation studies in the presence or absence of GABA showing a shift to higher affinity in the presence of GABA only at 37/sup 0/C. In Ro 15-1788/(/sup 3/H)TZ competition experiments the presence of GABA did not affect the inhibitory potency of Ro 15-1788 on (/sup 3/H)TZ binding at both temperatures. In conclusion (/sup 3/H)TZ binding showed an extremely high affinity for benzodiazepine receptors. In contrast to reported literature, the findings suggest that TZ interacts with benzodiazepine receptors similar to other benzodiazepine agonists.« less

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

PubMed Central

Liang, Shu-Man; Chen, Shyh-Chang; Wang, John; Hsu, Chiun; Wu, Yao-Ming; Liou, Jun-Yang

2013-01-01

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. PMID:23483955

Photoaffinity labelling of the cardiac calcium channel. (-)-[3H]azidopine labels a 165 kDa polypeptide, and evidence against a [3H]-1,4-dihydropyridine-isothiocyanate being a calcium-channel-specific affinity ligand.

PubMed

Ferry, D R; Goll, A; Glossmann, H

1987-04-01

The arylazide 1,4-dihydropyridine (-)-[3H]azidopine binds to a saturable population of sites in guinea-pig heart membranes with a dissociation constant (KD) of 30 +/- 7 pM and a density (Bmax.) of 670 +/- 97 fmol/mg of protein. This high-affinity binding site is assumed to reside on voltage-operated calcium channels because reversible binding is blocked stereoselectively by 1,4-dihydropyridine channel blockers and by the enantiomers of Bay K 8644. A low-affinity (KD 25 +/- 7 nM) high-capacity (Bmax. 21.6 +/- 9 pmol/mg of protein) site does not bind (-)- or (+)-Bay K 8644, but is blocked by high concentrations (greater than 500 nM) of dihydro-2,6-dimethyl-4-(2-isothiocyanatophenyl)-3,5-pyridinedicarboxy lic acid dimethyl ester (1,4-DHP-isothiocyanate) or, e.g., (+/-)-nicardipine. (-)-[3H]Azidopine was photoincorporated covalently into bands of 165 +/- 8, 39 +/- 2 and 35 +/- 3 kDa, as determined by SDS/polyacrylamide-gel electrophoresis. Labelling of the 165 kDa band is protected stereoselectively by 1,4-dihydropyridine enantiomers at low (nM) concentrations and by (-)- and (+)-Bay K 8644, whereas the lower-Mr bands are not. Thus, only the 165 kDa band is the calcium-channel-linked 1,4-dihydropyridine receptor. Photolabelling of the 39 or 35 kDa bands was only blocked by 10 microM-1,4-DHP-isothiocyanate or 50 microM-(+/-)-nicardipine but not by 10 microM-(-)-Bay K 8644. [3H]-1,4-DHP-isothiocyanate binds to guinea-pig heart membranes with a KD of 0.35 nM and dissociates with a k-1 of 0.2 min-1 at 30 degrees C. [3H]-1,4 DHP-isothiocyanate irreversibly labels bands of 39 and 35 kDa which are protected by greater than 10 microM-(+/-)-nicardipine or unlabelled ligand but not by 10 microM-(-)-Bay K 8644. Thus, [3H]-1,4-DHP-isothiocyanate is not an affinity probe for the calcium channel.

Functional analysis of Paracoccidioides brasiliensis 14-3-3 adhesin expressed in Saccharomyces cerevisiae.

PubMed

Assato, Patricia Akemi; da Silva, Julhiany de Fátima; de Oliveira, Haroldo Cesar; Marcos, Caroline Maria; Rossi, Danuza; Valentini, Sandro Roberto; Mendes-Giannini, Maria José Soares; Zanelli, Cleslei Fernando; Fusco-Almeida, Ana Marisa

2015-11-04

14-3-3 proteins comprise a family of eukaryotic multifunctional proteins involved in several cellular processes. The Pb14-3-3 of Paracoccidioides brasiliensis seems to play an important role in the Paracoccidioides-host interaction. Paracoccidioides brasiliensis is an etiological agent of paracoccidioidomycosis, which is a systemic mycosis that is endemic in Latin America. In the initial steps of the infection, Paracoccidioides spp. synthetizes adhesins that allow it to adhere and invade host cells. Therefore, the aim of this work was to perform a functional analysis of Pb14-3-3 using Saccharomyces cerevisiae as a model. The functional analysis of Pb14-3-3 was performed in S. cerevisiae, and it was found that Pb14-3-3 partially complemented S. cerevisiae proteins Bmh1p and Bmh2p, which are recognized as two yeast 14-3-3 homologues. When we evaluated the adhesion profile of S. cerevisiae transformants, Pb14-3-3 acted as an adhesin in S. cerevisiae; however, Bmh1p did not show this function. The influence of Pb14-3-3 in S. cerevisiae ergosterol pathway was also evaluated and our results showed that Pb14-3-3 up-regulates genes involved in ergosterol biosynthesis. Our data showed that Pb14-3-3 was able to partially complement Bmh1p and Bmh2p proteins in S. cerevisiae; however, we suggest that Pb14-3-3 has a differential role as an adhesin. In addition, Pb-14-3-3 may be involved in Paracoccidioides spp. ergosterol biosynthesis which makes it an interest as a therapeutic target.

Autoradiographic localization of sigma receptor binding sites in guinea pig and rat central nervous system with (+)3H-3-(3-hydroxyphenyl)-N-(1-propyl)piperidine

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gundlach, A.L.; Largent, B.L.; Snyder, S.H.

1986-06-01

(+)3H-3-PPP ((+)3H-3-(3-Hydroxyphenyl)-N-(1-propyl)-piperidine) binds with high affinity to brain membranes with a pharmacological profile consistent with that of sigma receptors. The distribution of (+)3H-3-PPP binding sites in brain and spinal cord of both guinea pig and rat has been determined by in vitro autoradiography with binding densities quantitated by computer-assisted densitometry. (+)3H-3-PPP binding to slide-mounted brain sections is saturable and displays high affinity and a pharmacological specificity very similar to sites labeled in homogenates. (+)3H-3-PPP binding sites are heterogeneously distributed. Highest concentrations of binding sites occur in spinal cord, particularly the ventral horn and dorsal root ganglia; the pons-medulla, associated withmore » the cranial nerve and pontine nuclei and throughout the brain stem reticular formation; the cerebellum, over the Purkinje cell layer; the midbrain, particularly the central gray and red nucleus; and hippocampus, over the pyramidal cell layer. Lowest levels are seen in the basal ganglia and parts of the thalamus, while all other areas, including hypothalamus and cerebral cortex, exhibit moderate grain densities. Quinolinic acid-induced lesions of the hippocampus indicate that (+)3H-3-PPP labels hippocampal pyramidal cells and granule cells in the dentate gyrus. Intrastriatal injection of ibotenic acid dramatically reduces (+)3H-3-PPP binding in this area, while injection of 6-hydroxydopamine produces a relatively slight decrease. The distribution of (+)3H-3-PPP binding sites does not correlate with the receptor distribution of any recognized neurotransmitter or neuropeptide, including dopamine. However, there is a notable similarity between the distribution of (+)3H-3-PPP sites and high-affinity binding sites for psychotomimetic opioids, such as the benzomorphan (+)SKF 10,047.« less

SOX10 Regulates Expression of the SH3-Domain Kinase Binding Protein 1 (Sh3kbp1) locus in Schwann Cells via an Alternative Promoter

PubMed Central

Hodonsky, Chani J.; Kleinbrink, Erica L.; Charney, Kira N.; Prasad, Megana; Bessling, Seneca L.; Jones, Erin A.; Srinivasan, Rajini; Svaren, John; McCallion, Andrew S.; Antonellis, Anthony

2011-01-01

The transcription factor SOX10 has essential roles in neural crest-derived cell populations, including myelinating Schwann cells—specialized glial cells responsible for ensheathing axons in the peripheral nervous system. Importantly, SOX10 directly regulates the expression of genes essential for proper myelin function. To date, only a handful of SOX10 target loci have been characterized in Schwann cells. Addressing this lack of knowledge will provide a better understanding of Schwann cell biology and candidate loci for relevant diseases such as demyelinating peripheral neuropathies. We have identified a highly-conserved SOX10 binding site within an alternative promoter at the SH3-domain kinase binding protein 1 (Sh3kbp1) locus. The genomic segment identified at Sh3kbp1 binds to SOX10 and displays strong promoter activity in Schwann cells in vitro and in vivo. Mutation of the SOX10 binding site ablates promoter activity, and ectopic expression of SOX10 in SOX10-negative cells promotes the expression of endogenous Sh3kbp1. Combined, these data reveal Sh3kbp1 as a novel target of SOX10 and raise important questions regarding the function of SH3KBP1 isoforms in Schwann cells. PMID:22037207

Interaction between cardiac myosin-binding protein C and formin Fhod3.

PubMed

Matsuyama, Sho; Kage, Yohko; Fujimoto, Noriko; Ushijima, Tomoki; Tsuruda, Toshihiro; Kitamura, Kazuo; Shiose, Akira; Asada, Yujiro; Sumimoto, Hideki; Takeya, Ryu

2018-05-08

Mutations in cardiac myosin-binding protein C (cMyBP-C) are a major cause of familial hypertrophic cardiomyopathy. Although cMyBP-C has been considered to regulate the cardiac function via cross-bridge arrangement at the C-zone of the myosin-containing A-band, the mechanism by which cMyBP-C functions remains unclear. We identified formin Fhod3, an actin organizer essential for the formation and maintenance of cardiac sarcomeres, as a cMyBP-C-binding protein. The cardiac-specific N-terminal Ig-like domain of cMyBP-C directly interacts with the cardiac-specific N-terminal region of Fhod3. The interaction seems to direct the localization of Fhod3 to the C-zone, since a noncardiac Fhod3 variant lacking the cMyBP-C-binding region failed to localize to the C-zone. Conversely, the cardiac variant of Fhod3 failed to localize to the C-zone in the cMyBP-C-null mice, which display a phenotype of hypertrophic cardiomyopathy. The cardiomyopathic phenotype of cMyBP-C-null mice was further exacerbated by Fhod3 overexpression with a defect of sarcomere integrity, whereas that was partially ameliorated by a reduction in the Fhod3 protein levels, suggesting that Fhod3 has a deleterious effect on cardiac function under cMyBP-C-null conditions where Fhod3 is aberrantly mislocalized. Together, these findings suggest the possibility that Fhod3 contributes to the pathogenesis of cMyBP-C-related cardiomyopathy and that Fhod3 is critically involved in cMyBP-C-mediated regulation of cardiac function via direct interaction.

Use of (2-/sup 14/C)mevalonate and saponin-bound (/sup 14/C)-3-hydroxy-3-methylglutaric acid for the biosynthesis of terpenoids in leaves of Dioscorea deltoidea

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gurielidze, K.G.; Paseshnichenko, V.A.; Vasil'eva, I.S.

After the introduction of (2-/sup 14/C)acetate into leaves of Dioscorea deltoidea, a radioactive furonanalog of deltafolin - protodeltofolin, containing two-thirds of the label in the 3-hydroxy-3-methylglutaryl portion - was isolated from them. Radioactive ..beta..-careotene and sterols were isolated from cut young leaves of Dioscorea 24 h after the introduction of (/sup 14/C) protodeltofolin into them, using chromatography on a column of silica gel and precipitation of sterols in the form of digitonins for this purpose. The incorporation of radioactivity from (/sup 14/C)-3-hydroxy-3-methyl-glutaric acid, bound in the form of a saponin, and ..beta..-carotene came to 0.18-0.80%, while incorporation into sterols camemore » to 0.07-2.86% of the radioactivity of the alcohol extract. Thereby it was shown that 3-hydroxyl-3-methylglutaric acid, bound in the form of the saponin, can be used to form terpenoids in Dioscorea leaves. It was suggested that the binding of hydroxymethylglutaric acid to saponin represents one of the mechanisms of regulation of the rate of terpenoid biosynthesis in Dioscorea leaves.« less

Localization of 14-3-3 proteins in the nuclei of arabidopsis and maize.

PubMed

Bihn, E A; Paul, A L; Wang, S W; Erdos, G W; Ferl, R J

1997-12-01

It has been demonstrated that 14-3-3 proteins are present in the nuclei of Arabidopsis thaliana and Zea mays cells using laser scanning confocal microscopy and immunocytochemistry with monoclonal antibodies against plant 14-3-3 proteins. Confirmation of nuclear localization provides insight into the range of functions normally attributed to 14-3-3 proteins, especially since the association of 14-3-3s with transcription factors is (thus far) a phenomenon unique to plants, and since 14-3-3 proteins do not possess a recognizable nuclear targeting sequence.

High Affinity Binding of Epibatidine to Serotonin Type 3 Receptors*

PubMed Central

Drisdel, Renaldo C.; Sharp, Douglas; Henderson, Tricia; Hales, Tim G.; Green, William N.

2008-01-01

Epibatidine and mecamylamine are ligands used widely in the study of nicotinic acetylcholine receptors (nAChRs) in the central and peripheral nervous systems. In the present study, we find that nicotine blocks only 75% of 125I-epibatidine binding to rat brain membranes, whereas ligands specific for serotonin type 3 receptors (5-HT3Rs) block the remaining 25%. 125I-Epibatidine binds with a high affinity to native 5-HT3Rs of N1E-115 cells and to receptors composed of only 5-HT3A subunits expressed in HEK cells. In these cells, serotonin, the 5-HT3R-specific antagonist MDL72222, and the 5-HT3R agonist chlorophenylbiguanide readily competed with 125I-epibatidine binding to 5-HT3Rs. Nicotine was a poor competitor for 125I-epibatidine binding to 5-HT3Rs. However, the noncompetitive nAChR antagonist mecamylamine acted as a potent competitive inhibitor of 125I-epibatidine binding to 5-HT3Rs. Epibatidine inhibited serotonin-induced currents mediated by endogenous 5-HT3Rs in neuroblastoma cell lines and 5-HT3ARs expressed in HEK cells in a competitive manner. Our results demonstrate that 5-HT3Rs are previously uncharacterized high affinity epibatidine binding sites in the brain and indicate that epibatidine and mecamylamine act as 5-HT3R antagonists. Previous studies that depended on epibatidine and mecamylamine as nAChR-specific ligands, in particular studies of analgesic properties of epibatidine, may need to be reinterpreted with respect to the potential role of 5-HT3Rs. PMID:17702741

3H[2-(2-benzofuranyl)-2-imidazoline] (BFI) binding in human platelets: modulation by tranylcypromine.

PubMed

Wiest, S A; Steinberg, M I

1999-08-01

2-(2-Benzofuranyl)-2-imidazoline (BFI) is a highly selective ligand for imidazoline-type 2 (I2) binding sites that are known to be associated with monoamine oxidase (MAO). Recently we demonstrated a potentiation of 3H-BFI binding in human but not in rat brain by the nonselective MAO inhibitor tranylcypromine. In the present studies, we evaluated the effect of tranylcypromine on the binding of 3H-BFI to human platelet inner membranes. Membranes were incubated with 3H-BFI at 22 degrees C in 50 mM Tris, 1.5 mM EDTA, pH 7.5. Saturation experiments with 3H-BFI (0.5-80 nM) were analyzed using non-linear curve fitting. Addition of tranylcypromine (0.1 mM) increased the number of 3H-BFI binding sites (Bmax=0.35+/-0.06 vs. 1.87+/-0.15 pmol/mg protein for vehicle and tranylcypromine, respectively) and increased 3H-BFI affinity slightly (KD =16.0+/-4.1 vs. 6.5+/-0.3 nM for vehicle and tranylcypromine, respectively). In competitive binding experiments using the less selective I2 ligand, 3H-idazoxan, tranylcypromine only weakly inhibited binding. Preincubation of platelet membranes with tranylcypromine (1 nM-10 microM) enhanced the Bmax of 3H-BFI binding in a concentration-dependent manner peaking at 1 microM (13 x control) and returning to near baseline at 100 microM. 3H-BFI binding was displaced monophasically (in order of decreasing potency) by BFI > or = 2-(4,5-dihydroimidazol-2-yl)quinoline (BU224) > or = cirazoline >idazoxan >(1,4-benzodioxan-2-methoxy-2-yl)-2-imidazoline (RX821002)= moxonidine. Amiloride, clorgyline, guanabenz and clonidine displayed biphasic curves with nanomolar high affinity components. Tranylcypromine altered the competition curves for all ligands (except BFI) by increasing the affinities for clonidine and RX821002 and decreasing affinities for BU224, cirazoline, guanabenz, idazoxan, clorgyline, moxonidine, and amiloride. Thus, in human platelets tranylcypromine exposes a high capacity 3H-BFI binding site distinct from previously described I2 sites

Mutations in protein-binding hot-spots on the hub protein Smad3 differentially affect its protein interactions and Smad3-regulated gene expression.

PubMed

Schiro, Michelle M; Stauber, Sara E; Peterson, Tami L; Krueger, Chateen; Darnell, Steven J; Satyshur, Kenneth A; Drinkwater, Norman R; Newton, Michael A; Hoffmann, F Michael

2011-01-01

Hub proteins are connected through binding interactions to many other proteins. Smad3, a mediator of signal transduction induced by transforming growth factor beta (TGF-β), serves as a hub protein for over 50 protein-protein interactions. Different cellular responses mediated by Smad3 are the product of cell-type and context dependent Smad3-nucleated protein complexes acting in concert. Our hypothesis is that perturbation of this spectrum of protein complexes by mutation of single protein-binding hot-spots on Smad3 will have distinct consequences on Smad3-mediated responses. We mutated 28 amino acids on the surface of the Smad3 MH2 domain and identified 22 Smad3 variants with reduced binding to subsets of 17 Smad3-binding proteins including Smad4, SARA, Ski, Smurf2 and SIP1. Mutations defective in binding to Smad4, e.g., D408H, or defective in nucleocytoplasmic shuttling, e.g., W406A, were compromised in modulating the expression levels of a Smad3-dependent reporter gene or six endogenous Smad3-responsive genes: Mmp9, IL11, Tnfaip6, Fermt1, Olfm2 and Wnt11. However, the Smad3 mutants Y226A, Y297A, W326A, K341A, and E267A had distinct differences on TGF-β signaling. For example, K341A and Y226A both reduced the Smad3-mediated activation of the reporter gene by ∼50% but K341A only reduced the TGF-β inducibilty of Olfm2 in contrast to Y226A which reduced the TGF-β inducibility of all six endogenous genes as severely as the W406A mutation. E267A had increased protein binding but reduced TGF-β inducibility because it caused higher basal levels of expression. Y297A had increased TGF-β inducibility because it caused lower Smad3-induced basal levels of gene expression. Mutations in protein binding hot-spots on Smad3 reduced the binding to different subsets of interacting proteins and caused a range of quantitative changes in the expression of genes induced by Smad3. This approach should be useful for unraveling which Smad3 protein complexes are critical for

Collagenase-3 binds to a specific receptor and requires the low density lipoprotein receptor-related protein for internalization

NASA Technical Reports Server (NTRS)

Barmina, O. Y.; Walling, H. W.; Fiacco, G. J.; Freije, J. M.; Lopez-Otin, C.; Jeffrey, J. J.; Partridge, N. C.

1999-01-01

We have previously identified a specific receptor for collagenase-3 that mediates the binding, internalization, and degradation of this ligand in UMR 106-01 rat osteoblastic osteosarcoma cells. In the present study, we show that collagenase-3 binding is calcium-dependent and occurs in a variety of cell types, including osteoblastic and fibroblastic cells. We also present evidence supporting a two-step mechanism of collagenase-3 binding and internalization involving both a specific collagenase-3 receptor and the low density lipoprotein receptor-related protein. Ligand blot analysis shows that (125)I-collagenase-3 binds specifically to two proteins ( approximately 170 kDa and approximately 600 kDa) present in UMR 106-01 cells. Western blotting identified the 600-kDa protein as the low density lipoprotein receptor-related protein. Our data suggest that the 170-kDa protein is a specific collagenase-3 receptor. Low density lipoprotein receptor-related protein-null mouse embryo fibroblasts bind but fail to internalize collagenase-3, whereas UMR 106-01 and wild-type mouse embryo fibroblasts bind and internalize collagenase-3. Internalization, but not binding, is inhibited by the 39-kDa receptor-associated protein. We conclude that the internalization of collagenase-3 requires the participation of the low density lipoprotein receptor-related protein and propose a model in which the cell surface interaction of this ligand requires a sequential contribution from two receptors, with the collagenase-3 receptor acting as a high affinity primary binding site and the low density lipoprotein receptor-related protein mediating internalization.

Docking, molecular dynamics, binding energy-MM-PBSA studies of naphthofuran derivatives to identify potential dual inhibitors against BACE-1 and GSK-3β.

PubMed

Kumar, Akhil; Srivastava, Gaurava; Negi, Arvind S; Sharma, Ashok

2018-01-19

BACE-1 and GSK-3β both are potential therapeutic drug targets for Alzheimer's disease. Recently, both these targets received attention for designing dual inhibitors. Till now only two scaffolds (triazinone and curcumin) derivatives have been reported as BACE-1 and GSK-3β dual inhibitors. In our previous work, we have reported first in class dual inhibitor for BACE-1 and GSK-3β. In this study, we have explored other naphthofuran derivatives for their potential to inhibit BACE-1 and GSK-3β through docking, molecular dynamics, binding energy (MM-PBSA). These computational methods were performed to estimate the binding affinity of naphthofuran derivatives towards the BACE-1 and GSK-3β. In the docking results, two derivatives (NS7 and NS9) showed better binding affinity as compared to previously reported inhibitors. Hydrogen bond occupancy of NS7 and NS9 generated from MD trajectories showed good interaction with the flap residues Gln73, Thr72 of BACE-1 and Arg141, Thr138 residues of GSK-3β. MM-PBSA and energy decomposition per residue revealed different components of binding energy and relative importance of amino acid involved in binding. The results showed that the binding of inhibitors was majorly governed by the hydrophobic interactions and suggesting that hydrophobic interactions might be the key to design dual inhibitors for BACE1-1 and GSK-3β. Distance between important pair of amino acid residues indicated that BACE-1 and GSK-3β adopt closed conformation and become inactive after ligand binding. The results suggested that naphthofuran derivatives might act as dual inhibitor against BACE-1 and GSK-3β.

NF-kappaB binds to a polymorphic repressor element in the MMP-3 promoter.

PubMed

Borghaei, Ruth C; Rawlings, P Lyle; Javadi, Masoud; Woloshin, Joanna

2004-03-26

A 5T/6T polymorphic site in the matrix metalloproteinase-3 (MMP-3) promoter has been identified as a repressor element involved in inhibiting induction of MMP-3 transcription by interleukin 1; and the 6T allele has been associated with decreased expression of MMP-3 as compared to the 5T allele. Zinc-binding protein-89 (ZBP-89) was cloned from a yeast one-hybrid assay via its ability to interact with this site, but when the protein was over-expressed, it resulted in activation of the MMP-3 promoter rather than repression. Here we show that in nuclear extracts isolated from human gingival fibroblasts stimulated with IL-1, this site is bound by p50 and p65 components of NF-kappaB in addition to ZBP-89, and that recombinant p50 binds preferentially to the 6T binding site. These results are consistent with a role for NF-kappaB in limiting the cytokine induced expression of MMP-3.

The RNA-Binding Site of Poliovirus 3C Protein Doubles as a Phosphoinositide-Binding Domain.

PubMed

Shengjuler, Djoshkun; Chan, Yan Mei; Sun, Simou; Moustafa, Ibrahim M; Li, Zhen-Lu; Gohara, David W; Buck, Matthias; Cremer, Paul S; Boehr, David D; Cameron, Craig E

2017-12-05

Some viruses use phosphatidylinositol phosphate (PIP) to mark membranes used for genome replication or virion assembly. PIP-binding motifs of cellular proteins do not exist in viral proteins. Molecular-docking simulations revealed a putative site of PIP binding to poliovirus (PV) 3C protein that was validated using nuclear magnetic resonance spectroscopy. The PIP-binding site was located on a highly dynamic α helix, which also functions in RNA binding. Broad PIP-binding activity was observed in solution using a fluorescence polarization assay or in the context of a lipid bilayer using an on-chip, fluorescence assay. All-atom molecular dynamics simulations of the 3C protein-membrane interface revealed PIP clustering and perhaps PIP-dependent conformations. PIP clustering was mediated by interaction with residues that interact with the RNA phosphodiester backbone. We conclude that 3C binding to membranes will be determined by PIP abundance. We suggest that the duality of function observed for 3C may extend to RNA-binding proteins of other viruses. Copyright © 2017 Elsevier Ltd. All rights reserved.

Distinct peptide binding specificities of Src homology 3 (SH3) protein domains can be determined by modulation of local energetics across the binding interface.

PubMed

Gorelik, Maryna; Davidson, Alan R

2012-03-16

The yeast Nbp2p SH3 and Bem1p SH3b domains bind certain target peptides with similar high affinities, yet display vastly different affinities for other targets. To investigate this unusual behavior, we have solved the structure of the Nbp2p SH3-Ste20 peptide complex and compared it with the previously determined structure of the Bem1p SH3b bound to the same peptide. Although the Ste20 peptide interacts with both domains in a structurally similar manner, extensive in vitro studies with domain and peptide mutants revealed large variations in interaction strength across the binding interface of the two complexes. Whereas the Nbp2p SH3 made stronger contacts with the peptide core RXXPXXP motif, the Bem1p SH3b domain made stronger contacts with residues flanking the core motif. Remarkably, this modulation of local binding energetics can explain the distinct and highly nuanced binding specificities of these two domains.

Dronerarone acts as a selective inhibitor of 3,5,3'-triiodothyronine binding to thyroid hormone receptor-alpha1: in vitro and in vivo evidence.

PubMed

Van Beeren, H C; Jong, W M C; Kaptein, E; Visser, T J; Bakker, O; Wiersinga, W M

2003-02-01

Dronedarone (Dron), without iodine, was developed as an alternative to the iodine-containing antiarrhythmic drug amiodarone (AM). AM acts, via its major metabolite desethylamiodarone, in vitro and in vivo as a thyroid hormone receptor alpha(1) (TRalpha(1)) and TRbeta(1) antagonist. Here we investigate whether Dron and/or its metabolite debutyldronedarone inhibit T(3) binding to TRalpha(1) and TRbeta(1) in vitro and whether dronedarone behaves similarly to amiodarone in vivo. In vitro, Dron had a inhibitory effect of 14% on the binding of T(3) to TRalpha(1), but not on TRbeta(1). Desethylamiodarone inhibited T(3) binding to TRalpha(1) and TRbeta(1) equally. Debutyldronedarone inhibited T(3) binding to TRalpha(1) by 77%, but to TRbeta(1) by only 25%. In vivo, AM increased plasma TSH and rT(3), and decreased T(3). Dron decreased T(4) and T(3), rT(3) did not change, and TSH fell slightly. Plasma total cholesterol was increased by AM, but remained unchanged in Dron-treated animals. TRbeta(1)-dependent liver low density lipoprotein receptor protein and type 1 deiodinase activities decreased in AM-treated, but not in Dron-treated, animals. TRalpha(1)-mediated lengthening of the QTc interval was present in both AM- and Dron-treated animals. The in vitro and in vivo findings suggest that dronedarone via its metabolite debutyldronedarone acts as a TRalpha(1)-selective inhibitor.

(Z)-2-(3-Chlorobenzylidene)-3,4-dihydro-N-(2-methoxyethyl)-3-oxo-2H-benzo[b][1,4]oxazine-6-carboxamide as GSK-3β inhibitor: Identification by virtual screening and its validation in enzyme- and cell-based assay.

PubMed

Joshi, Prashant; Gupta, Mehak; Vishwakarma, Ram A; Kumar, Ajay; Bharate, Sandip B

2017-06-01

Glycogen synthase kinase 3β (GSK-3β) is a widely investigated molecular target for numerous diseases including Alzheimer's disease, cancer, and diabetes mellitus. The present study was aimed to discover new scaffolds for GSK-3β inhibition, through protein structure-guided virtual screening approach. With the availability of large number of GSK-3β crystal structures with varying degree of RMSD in protein backbone and RMSF in side chain geometry, herein appropriate crystal structures were selected based on the characteristic ROC curve and percentage enrichment of actives. The validated docking protocol was employed to screen a library of 50,000 small molecules using molecular docking and binding affinity calculations. Based on the GLIDE docking score, Prime MMGB/SA binding affinity, and interaction pattern analysis, the top 50 ligands were selected for GSK-3β inhibition. (Z)-2-(3-chlorobenzylidene)-3,4-dihydro-N-(2-methoxyethyl)-3-oxo-2H-benzo[b][1,4]oxazine-6-carboxamide (F389-0663, 7) was identified as a potent inhibitor of GSK-3β with an IC 50 value of 1.6 μm. Further, GSK-3β inhibition activity was then investigated in cell-based assay. The treatment of neuroblastoma N2a cells with 12.5 μm of F389-0663 resulted in the significant increase in GSK-3β Ser9 levels, which is indicative of the GSK-3β inhibitory activity of a compound. The molecular dynamic simulations were carried out to understand the interactions of F389-0663 with GSK-3β protein. © 2016 John Wiley & Sons A/S.

The MTA family proteins as novel histone H3 binding proteins.

PubMed

Wu, Meng; Wang, Lina; Li, Qian; Li, Jiwen; Qin, Jun; Wong, Jiemin

2013-01-03

The nucleosome remodeling and histone deacetylase complex (Mi2/NRD/NuRD/NURD) has a broad role in regulation of transcription, DNA repair and cell cycle. Previous studies have revealed a specific interaction between NURD and histone H3N-terminal tail in vitro that is not observed for another HDAC1/2-containing complex, Sin3A. However, the subunit(s) responsible for specific binding of H3 by NURD has not been defined. In this study, we show among several class I HDAC-containing corepressor complexes only NURD exhibits a substantial H3 tail-binding activity in vitro. We present the evidence that the MTA family proteins within the NURD complex interact directly with H3 tail. Extensive in vitro binding assays mapped the H3 tail-binding domain to the C-terminal region of MTA1 and MTA2. Significantly, although the MTA1 and MTA2 mutant proteins with deletion of the C-terminal H3 tail binding domain were assembled into the endogenous NURD complex when expressed in mammalian cells, the resulting NURD complexes were deficient in binding H3 tail in vitro, indicating that the MTA family proteins are required for the observed specific binding of H3 tail peptide by NURD in vitro. However, chromatin fractionation experiments show that the NURD complexes with impaired MTA1/2-H3 tail binding activity remained to be associated with chromatin in cells. Together our study reveals a novel histone H3-binding activity for the MTA family proteins and provides evidence that the MTA family proteins mediate the in vitro specific binding of H3 tail peptide by NURD complex. However, multiple mechanisms are likely to contribute to the chromatin association of NURD complex in cells. Our finding also raises the possibility that the MTA family proteins may exert their diverse biological functions at least in part through their direct interaction with H3 tail.

The MTA family proteins as novel histone H3 binding proteins

PubMed Central

2013-01-01

Background The nucleosome remodeling and histone deacetylase complex (Mi2/NRD/NuRD/NURD) has a broad role in regulation of transcription, DNA repair and cell cycle. Previous studies have revealed a specific interaction between NURD and histone H3N-terminal tail in vitro that is not observed for another HDAC1/2-containing complex, Sin3A. However, the subunit(s) responsible for specific binding of H3 by NURD has not been defined. Results In this study, we show among several class I HDAC-containing corepressor complexes only NURD exhibits a substantial H3 tail-binding activity in vitro. We present the evidence that the MTA family proteins within the NURD complex interact directly with H3 tail. Extensive in vitro binding assays mapped the H3 tail-binding domain to the C-terminal region of MTA1 and MTA2. Significantly, although the MTA1 and MTA2 mutant proteins with deletion of the C-terminal H3 tail binding domain were assembled into the endogenous NURD complex when expressed in mammalian cells, the resulting NURD complexes were deficient in binding H3 tail in vitro, indicating that the MTA family proteins are required for the observed specific binding of H3 tail peptide by NURD in vitro. However, chromatin fractionation experiments show that the NURD complexes with impaired MTA1/2-H3 tail binding activity remained to be associated with chromatin in cells. Conclusions Together our study reveals a novel histone H3-binding activity for the MTA family proteins and provides evidence that the MTA family proteins mediate the in vitro specific binding of H3 tail peptide by NURD complex. However, multiple mechanisms are likely to contribute to the chromatin association of NURD complex in cells. Our finding also raises the possibility that the MTA family proteins may exert their diverse biological functions at least in part through their direct interaction with H3 tail. PMID:23286669

Protective immunity against Eimeria maxima induced by vaccines of Em14-3-3 antigen.

PubMed

Liu, Tingqi; Huang, Jingwei; Ehsan, Muhammad; Wang, Shuai; Fei, Hong; Zhou, Zhouyang; Song, Xiaokai; Yan, Ruofeng; Xu, Lixin; Li, Xiangrui

2018-04-15

Eimeria maxima 14-3-3 (Em14-3-3) open reading frame (ORF) which consisted of 861 bp encoding a protein of 286 amino acids was successfully amplified and sequenced. Subsequently, the Em14-3-3 ORF was subcloned into pET-32a (+) and pVAX1, respectively. RT-PCR and immunoblot analyses confirmed that the target gene was successfully transcribed and expressed in vivo. Immunofluorescence analysis showed that Em14-3-3 was expressed in both the sporozoites and merozoites. The animal experiments demonstrated that both rEm14-3-3 and pVAX1-14-3-3 could clearly alleviate jejunum lesions and body weight loss. The Em14-3-3 vaccines could increase oocyst decrease ratio, as well as produce an anticoccidial index of more than 165. The percentages of CD4 + in both the Em14-3-3 immunized groups were much higher, when compared with those of PBS, pET32a (+), and pVAX1 controls (P < 0.05). Similarly, the anti-Em14-3-3 antibody titers of both rEm14-3-3 and pVAX1-14-3-3 immunized groups showed higher levels compared with those of PBS, pET32a (+), and pVAX1 controls (P < 0.05). The IFN-γ and tumor growth factor-β (TGF-β) levels showed significant increments in the rEm14-3-3 and pVAX1-14-3-3 immunized groups, when compared with those in the negative controls (P < 0.05). These results demonstrated that Em14-3-3 could be used as a promising antigen candidate for developing vaccines against E. maxima. Copyright © 2018 Elsevier B.V. All rights reserved.

14 CFR 420.3 - Applicability.

Code of Federal Regulations, 2010 CFR

2010-01-01

... 14 Aeronautics and Space 4 2010-01-01 2010-01-01 false Applicability. 420.3 Section 420.3 Aeronautics and Space COMMERCIAL SPACE TRANSPORTATION, FEDERAL AVIATION ADMINISTRATION, DEPARTMENT OF... operating a site that only supports amateur rocket activities as defined in 14 CFR 1.1, does not need a...

14 CFR 420.3 - Applicability.

Code of Federal Regulations, 2011 CFR

2011-01-01

... 14 Aeronautics and Space 4 2011-01-01 2011-01-01 false Applicability. 420.3 Section 420.3 Aeronautics and Space COMMERCIAL SPACE TRANSPORTATION, FEDERAL AVIATION ADMINISTRATION, DEPARTMENT OF... operating a site that only supports amateur rocket activities as defined in 14 CFR 1.1, does not need a...

DNA binding by the ribosomal DNA transcription factor rrn3 is essential for ribosomal DNA transcription.

PubMed

Stepanchick, Ann; Zhi, Huijun; Cavanaugh, Alice H; Rothblum, Katrina; Schneider, David A; Rothblum, Lawrence I

2013-03-29

The human homologue of yeast Rrn3 is an RNA polymerase I-associated transcription factor that is essential for ribosomal DNA (rDNA) transcription. The generally accepted model is that Rrn3 functions as a bridge between RNA polymerase I and the transcription factors bound to the committed template. In this model Rrn3 would mediate an interaction between the mammalian Rrn3-polymerase I complex and SL1, the rDNA transcription factor that binds to the core promoter element of the rDNA. In the course of studying the role of Rrn3 in recruitment, we found that Rrn3 was in fact a DNA-binding protein. Analysis of the sequence of Rrn3 identified a domain with sequence similarity to the DNA binding domain of heat shock transcription factor 2. Randomization, or deletion, of the amino acids in this region in Rrn3, amino acids 382-400, abrogated its ability to bind DNA, indicating that this domain was an important contributor to DNA binding by Rrn3. Control experiments demonstrated that these mutant Rrn3 constructs were capable of interacting with both rpa43 and SL1, two other activities demonstrated to be essential for Rrn3 function. However, neither of these Rrn3 mutants was capable of functioning in transcription in vitro. Moreover, although wild-type human Rrn3 complemented a yeast rrn3-ts mutant, the DNA-binding site mutant did not. These results demonstrate that DNA binding by Rrn3 is essential for transcription by RNA polymerase I.

DNA Binding by the Ribosomal DNA Transcription Factor Rrn3 Is Essential for Ribosomal DNA Transcription*

PubMed Central

Stepanchick, Ann; Zhi, Huijun; Cavanaugh, Alice H.; Rothblum, Katrina; Schneider, David A.; Rothblum, Lawrence I.

2013-01-01

The human homologue of yeast Rrn3 is an RNA polymerase I-associated transcription factor that is essential for ribosomal DNA (rDNA) transcription. The generally accepted model is that Rrn3 functions as a bridge between RNA polymerase I and the transcription factors bound to the committed template. In this model Rrn3 would mediate an interaction between the mammalian Rrn3-polymerase I complex and SL1, the rDNA transcription factor that binds to the core promoter element of the rDNA. In the course of studying the role of Rrn3 in recruitment, we found that Rrn3 was in fact a DNA-binding protein. Analysis of the sequence of Rrn3 identified a domain with sequence similarity to the DNA binding domain of heat shock transcription factor 2. Randomization, or deletion, of the amino acids in this region in Rrn3, amino acids 382–400, abrogated its ability to bind DNA, indicating that this domain was an important contributor to DNA binding by Rrn3. Control experiments demonstrated that these mutant Rrn3 constructs were capable of interacting with both rpa43 and SL1, two other activities demonstrated to be essential for Rrn3 function. However, neither of these Rrn3 mutants was capable of functioning in transcription in vitro. Moreover, although wild-type human Rrn3 complemented a yeast rrn3-ts mutant, the DNA-binding site mutant did not. These results demonstrate that DNA binding by Rrn3 is essential for transcription by RNA polymerase I. PMID:23393135

Regulation of the Water Channel Aquaporin-2 via 14-3-3θ and -ζ*

PubMed Central

Moeller, Hanne B.; Slengerik-Hansen, Joachim; Aroankins, Takwa; Assentoft, Mette; MacAulay, Nanna; Moestrup, Soeren K.; Bhalla, Vivek; Fenton, Robert A.

2016-01-01

The 14-3-3 family of proteins are multifunctional proteins that interact with many of their cellular targets in a phosphorylation-dependent manner. Here, we determined that 14-3-3 proteins interact with phosphorylated forms of the water channel aquaporin-2 (AQP2) and modulate its function. With the exception of σ, all 14-3-3 isoforms were abundantly expressed in mouse kidney and mouse kidney collecting duct cells (mpkCCD14). Long-term treatment of mpkCCD14 cells with the type 2 vasopressin receptor agonist dDAVP increased mRNA and protein levels of AQP2 alongside 14-3-3β and -ζ, whereas levels of 14-3-3η and -θ were decreased. Co-immunoprecipitation (co-IP) studies in mpkCCD14 cells uncovered an AQP2/14-3-3 interaction that was modulated by acute dDAVP treatment. Additional co-IP studies in HEK293 cells determined that AQP2 interacts selectively with 14-3-3ζ and -θ. Use of phosphatase inhibitors in mpkCCD14 cells, co-IP with phosphorylation deficient forms of AQP2 expressed in HEK293 cells, or surface plasmon resonance studies determined that the AQP2/14-3-3 interaction was modulated by phosphorylation of AQP2 at various sites in its carboxyl terminus, with Ser-256 phosphorylation critical for the interactions. shRNA-mediated knockdown of 14-3-3ζ in mpkCCD14 cells resulted in increased AQP2 ubiquitylation, decreased AQP2 protein half-life, and reduced AQP2 levels. In contrast, knockdown of 14-3-3θ resulted in increased AQP2 half-life and increased AQP2 levels. In conclusion, this study demonstrates phosphorylation-dependent interactions of AQP2 with 14-3-3θ and -ζ. These interactions play divergent roles in modulating AQP2 trafficking, phosphorylation, ubiquitylation, and degradation. PMID:26645691

Commercial valerian interactions with [3H]Flunitrazepam and [3H]MK-801 binding to rat synaptic membranes.

PubMed

Ortiz, José G; Rassi, Nicole; Maldonado, Patricia M; González-Cabrera, Silvia; Ramos, Igmeris

2006-09-01

Valeriana officinalis extracts are used in folkloric medicine for their sedative, hypnotic and tranquilizer effects. Using [3H]flunitrazepam binding as an indicator, the interactions of commercial Valerian extracts with GABA(A) receptors were examined. There was considerable fluctuation among the different extracts, some mildly enhanced [3H]flunitrazepam binding, others had no effect and others had inhibitory effects, independent of standardization by valerenic acid. Central depression can also be accomplished by a reduction of excitatory transmission. Valerian extracts had modest inhibitory effects on [3H]MK-801 binding, an indicator of NMDA-Valerian interactions. Spectral analyses (UV region) did not show marked differences among the different extracts. The inhibitory effects of one of the extracts on [3H]flunitrazepam binding was somewhat stable, while on [3H]MK-801 binding the inhibitory effects were lost within months. These results suggest that particular care should be taken in analysing and interpreting results from commercial Valerian preparations. Copyright (c) 2006 John Wiley & Sons, Ltd.

Identification of a New Functional Domain in Angiopoietin-like 3 (ANGPTL3) and Angiopoietin-like 4 (ANGPTL4) Involved in Binding and Inhibition of Lipoprotein Lipase (LPL)S⃞

PubMed Central

Lee, E-Chiang; Desai, Urvi; Gololobov, Gennady; Hong, Seokjoo; Feng, Xiao; Yu, Xuan-Chuan; Gay, Jason; Wilganowski, Nat; Gao, Cuihua; Du, Ling-Ling; Chen, Joan; Hu, Yi; Zhao, Sharon; Kirkpatrick, Laura; Schneider, Matthias; Zambrowicz, Brian P.; Landes, Greg; Powell, David R.; Sonnenburg, William K.

2009-01-01

Angiopoietin-like 3 (ANGPTL3) and angiopoietin-like 4 (ANGPTL4) are secreted proteins that regulate triglyceride (TG) metabolism in part by inhibiting lipoprotein lipase (LPL). Recently, we showed that treatment of wild-type mice with monoclonal antibody (mAb) 14D12, specific for ANGPTL4, recapitulated the Angptl4 knock-out (-/-) mouse phenotype of reduced serum TG levels. In the present study, we mapped the region of mouse ANGPTL4 recognized by mAb 14D12 to amino acids Gln29–His53, which we designate as specific epitope 1 (SE1). The 14D12 mAb prevented binding of ANGPTL4 with LPL, consistent with its ability to neutralize the LPL-inhibitory activity of ANGPTL4. Alignment of all angiopoietin family members revealed that a sequence similar to ANGPTL4 SE1 was present only in ANGPTL3, corresponding to amino acids Glu32–His55. We produced a mouse mAb against this SE1-like region in ANGPTL3. This mAb, designated 5.50.3, inhibited the binding of ANGPTL3 to LPL and neutralized ANGPTL3-mediated inhibition of LPL activity in vitro. Treatment of wild-type as well as hyperlipidemic mice with mAb 5.50.3 resulted in reduced serum TG levels, recapitulating the lipid phenotype found in Angptl3-/- mice. These results show that the SE1 region of ANGPTL3 and ANGPTL4 functions as a domain important for binding LPL and inhibiting its activity in vitro and in vivo. Moreover, these results demonstrate that therapeutic antibodies that neutralize ANGPTL4 and ANGPTL3 may be useful for treatment of some forms of hyperlipidemia. PMID:19318355

Two nucleotide binding sites modulate ( sup 3 H) glyburide binding to rat cortex membranes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Johnson, D.E.; Gopalakrishnan, M.; Triggle, D.J.

1991-03-11

The effects of nucleotides on the binding of the ATP-dependent K{sup +}-channel antagonist ({sup 3}H)glyburide (GLB) to rat cortex membranes were examined. Nucleotide triphosphates (NTPs) and nucleotide diphosphate (NDPs) inhibited the binding of GLB. This effect was dependent on the presence of dithiothreitol (DTT). Inhibition of binding by NTPs, with the exception of ATP{gamma}S, was dependent on the presence of Mg{sup 2+}. GLB binding showed a biphasic response to ADP: up to 3 mM, ADP inhibited binding, and above this concentration GLB binding increased rapidly, and was restored to normal levels by 10 mM ADP. In the presence of Mg{supmore » 2+}, ADP did not stimulate binding. Saturation analysis in the presence of Mg{sup 2+} and increasing concentrations of ADP showed that ADP results primarily in a change of the B{sub max} for GLB binding. The differential effects of NTPS and NDPs indicate that two nucleotide binding sites regulate GLB binding.« less

A gene variation of 14-3-3 zeta isoform in rat hippocampus.

PubMed

Murakami, K; Situ, S Y; Eshete, F

1996-11-14

A variant form of 14-3-3 zeta was isolated from the rat hippocampal cDNA library. The cloned cDNA is 1687 bp in length and it contains an entire ORF (nt = 63-797) with 245 amino acids that is characteristic to 14-3-3 zeta subtype. By comparing with reported sequences of 14-3-3 zeta, we found three nucleotide substitutions within the coding sequence in our clone; C<-->T transition at nt = 325 and G<-->C transversions at nt = 387 and 388. Both are missense mutations, leading ACG (Thr) to ATG (Met) and CGT (Arg) to GCT (Ala) conversions at residue 88 and 109, respectively. Our results show that at least three different genetic variants of 14-3-3 zeta are present in rat species which results in protein variations. Such mutation in the amino acid sequence is an important indication of the diverse functions of this protein and may also contribute to the recent contradictory observations regarding the role of the 14-3-3 zeta subtype.

Bak Conformational Changes Induced by Ligand Binding: Insight into BH3 Domain Binding and Bak Homo-Oligomerization

PubMed Central

Pang, Yuan-Ping; Dai, Haiming; Smith, Alyson; Meng, X. Wei; Schneider, Paula A.; Kaufmann, Scott H.

2012-01-01

Recently we reported that the BH3-only proteins Bim and Noxa bind tightly but transiently to the BH3-binding groove of Bak to initiate Bak homo-oligomerization. However, it is unclear how such tight binding can induce Bak homo-oligomerization. Here we report the ligand-induced Bak conformational changes observed in 3D models of Noxa·Bak and Bim·Bak refined by molecular dynamics simulations. In particular, upon binding to the BH3-binding groove, Bim and Noxa induce a large conformational change of the loop between helices 1 and 2 and in turn partially expose a remote groove between helices 1 and 6 in Bak. These observations, coupled with the reported experimental data, suggest formation of a pore-forming Bak octamer, in which the BH3-binding groove is at the interface on one side of each monomer and the groove between helices 1 and 6 is at the interface on the opposite side, initiated by ligand binding to the BH3-binding groove. PMID:22355769

Biopanning and characterization of peptides with Fe3O4 nanoparticles-binding capability via phage display random peptide library technique.

PubMed

You, Fei; Yin, Guangfu; Pu, Ximing; Li, Yucan; Hu, Yang; Huang, Zhongbin; Liao, Xiaoming; Yao, Yadong; Chen, Xianchun

2016-05-01

Functionalization of inorganic nanoparticles (NPs) play an important role in biomedical applications. A proper functionalization of NPs can improve biocompatibility, avoid a loss of bioactivity, and further endow NPs with unique performances. Modification with vairous specific binding biomolecules from random biological libraries has been explored. In this work, two 7-mer peptides with sequences of HYIDFRW and TVNFKLY were selected from a phage display random peptide library by using ferromagnetic NPs as targets, and were verified to display strong binding affinity to Fe3O4 NPs. Fourier transform infrared spectrometry, fluorescence microscopy, thermal analysis and X-ray photoelectron spectroscopy confirmed the presence of peptides on the surface of Fe3O4 NPs. Sequence analyses revealed that the probable binding mechanism between the peptide and Fe3O4 NPs might be driven by Pearson hard acid-hard base specific interaction and hydrogen bonds, accompanied with hydrophilic interactions and non-specific electrostatic attractions. The cell viability assay indicated a good cytocompatibility of peptide-bound Fe3O4 NPs. Furthermore, TVNFKLY peptide and an ovarian tumor cell A2780 specific binding peptide (QQTNWSL) were conjugated to afford a liner 14-mer peptide (QQTNWSLTVNFKLY). The binding and targeting studies showed that 14-mer peptide was able to retain both the strong binding ability to Fe3O4 NPs and the specific binding ability to A2780 cells. The results suggested that the Fe3O4-binding peptides would be of great potential in the functionalization of Fe3O4 NPs for the tumor-targeted drug delivery and magnetic hyperthermia. Copyright © 2016 Elsevier B.V. All rights reserved.

The Role and Regulatory Mechanism of 14-3-3 Sigma in Human Breast Cancer

PubMed Central

Ko, SeungSang; Kim, Ji Young; Jeong, Joon; Lee, Jong Eun; Yang, Woo Ick

2014-01-01

Purpose 14-3-3 sigma (σ) is considered to be an important tumor suppressor and decreased expression of the same has been reported in many malignant tumors by hypermethylation at its promoter or ubiquitin-mediated proteolysis by estrogen-responsive ring finger protein (Efp). In this study, we investigated the significance of 14-3-3 σ expression in human breast cancer and its regulatory mechanism. Methods Efp was silenced using small interfering RNA (siRNA) in the MCF-7 breast cancer cell line in order to examine its influence on the level of 14-3-3 σ protein. The methylation status of the 14-3-3 σ promoter was also evaluated by methylation-specific polymerase chain reaction (PCR). The expression of Efp and 14-3-3 σ in 220 human breast carcinoma tissues was assessed by immunohistochemistry. Other clinicopathological parameters were also evaluated. Results Silencing Efp in the MCF-7 breast cancer cell line resulted in increased expression of 14-3-3 σ. The Efp-positive human breast cancers were more frequently 14-3-3 σ-negative (60.5% vs. 39.5%). Hypermethylation of 14-3-3 σ was common (64.9%) and had an inverse association with 14-3-3 σ positivity (p=0.072). Positive 14-3-3 σ expression was significantly correlated with poor prognosis: disease-free survival (p=0.008) and disease-specific survival (p=0.009). Conclusion Our data suggests that in human breast cancer, the regulation of 14-3-3 σ may involve two mechanisms: ubiquitin-mediated proteolysis by Efp and downregulation by hypermethylation. However, the inactivation of 14-3-3 σ is probably achieved mainly by hypermethylation. Interestingly, 14-3-3 σ turned out to be a very significant poor prognostic indicator, which is in contrast to its previously known function as a tumor suppressor, suggesting a different role of 14-3-3 σ in breast cancer. PMID:25320618

From Binding-Induced Dynamic Effects in SH3 Structures to Evolutionary Conserved Sectors.

PubMed

Zafra Ruano, Ana; Cilia, Elisa; Couceiro, José R; Ruiz Sanz, Javier; Schymkowitz, Joost; Rousseau, Frederic; Luque, Irene; Lenaerts, Tom

2016-05-01

Src Homology 3 domains are ubiquitous small interaction modules known to act as docking sites and regulatory elements in a wide range of proteins. Prior experimental NMR work on the SH3 domain of Src showed that ligand binding induces long-range dynamic changes consistent with an induced fit mechanism. The identification of the residues that participate in this mechanism produces a chart that allows for the exploration of the regulatory role of such domains in the activity of the encompassing protein. Here we show that a computational approach focusing on the changes in side chain dynamics through ligand binding identifies equivalent long-range effects in the Src SH3 domain. Mutation of a subset of the predicted residues elicits long-range effects on the binding energetics, emphasizing the relevance of these positions in the definition of intramolecular cooperative networks of signal transduction in this domain. We find further support for this mechanism through the analysis of seven other publically available SH3 domain structures of which the sequences represent diverse SH3 classes. By comparing the eight predictions, we find that, in addition to a dynamic pathway that is relatively conserved throughout all SH3 domains, there are dynamic aspects specific to each domain and homologous subgroups. Our work shows for the first time from a structural perspective, which transduction mechanisms are common between a subset of closely related and distal SH3 domains, while at the same time highlighting the differences in signal transduction that make each family member unique. These results resolve the missing link between structural predictions of dynamic changes and the domain sectors recently identified for SH3 domains through sequence analysis.

From Binding-Induced Dynamic Effects in SH3 Structures to Evolutionary Conserved Sectors

PubMed Central

Ruiz Sanz, Javier; Schymkowitz, Joost; Rousseau, Frederic

2016-01-01

Src Homology 3 domains are ubiquitous small interaction modules known to act as docking sites and regulatory elements in a wide range of proteins. Prior experimental NMR work on the SH3 domain of Src showed that ligand binding induces long-range dynamic changes consistent with an induced fit mechanism. The identification of the residues that participate in this mechanism produces a chart that allows for the exploration of the regulatory role of such domains in the activity of the encompassing protein. Here we show that a computational approach focusing on the changes in side chain dynamics through ligand binding identifies equivalent long-range effects in the Src SH3 domain. Mutation of a subset of the predicted residues elicits long-range effects on the binding energetics, emphasizing the relevance of these positions in the definition of intramolecular cooperative networks of signal transduction in this domain. We find further support for this mechanism through the analysis of seven other publically available SH3 domain structures of which the sequences represent diverse SH3 classes. By comparing the eight predictions, we find that, in addition to a dynamic pathway that is relatively conserved throughout all SH3 domains, there are dynamic aspects specific to each domain and homologous subgroups. Our work shows for the first time from a structural perspective, which transduction mechanisms are common between a subset of closely related and distal SH3 domains, while at the same time highlighting the differences in signal transduction that make each family member unique. These results resolve the missing link between structural predictions of dynamic changes and the domain sectors recently identified for SH3 domains through sequence analysis. PMID:27213566

Mutation of CD2AP and SH3KBP1 Binding Motif in Alphavirus nsP3 Hypervariable Domain Results in Attenuated Virus.

PubMed

Mutso, Margit; Morro, Ainhoa Moliner; Smedberg, Cecilia; Kasvandik, Sergo; Aquilimeba, Muriel; Teppor, Mona; Tarve, Liisi; Lulla, Aleksei; Lulla, Valeria; Saul, Sirle; Thaa, Bastian; McInerney, Gerald M; Merits, Andres; Varjak, Margus

2018-04-27

Infection by Chikungunya virus (CHIKV) of the Old World alphaviruses (family Togaviridae) in humans can cause arthritis and arthralgia. The virus encodes four non-structural proteins (nsP) (nsP1, nsp2, nsP3 and nsP4) that act as subunits of the virus replicase. These proteins also interact with numerous host proteins and some crucial interactions are mediated by the unstructured C-terminal hypervariable domain (HVD) of nsP3. In this study, a human cell line expressing EGFP tagged with CHIKV nsP3 HVD was established. Using quantitative proteomics, it was found that CHIKV nsP3 HVD can bind cytoskeletal proteins, including CD2AP, SH3KBP1, CAPZA1, CAPZA2 and CAPZB. The interaction with CD2AP was found to be most evident; its binding site was mapped to the second SH3 ligand-like element in nsP3 HVD. Further assessment indicated that CD2AP can bind to nsP3 HVDs of many different New and Old World alphaviruses. Mutation of the short binding element hampered the ability of the virus to establish infection. The mutation also abolished ability of CD2AP to co-localise with nsP3 and replication complexes of CHIKV; the same was observed for Semliki Forest virus (SFV) harbouring a similar mutation. Similar to CD2AP, its homolog SH3KBP1 also bound the identified motif in CHIKV and SFV nsP3.

Mutations in Protein-Binding Hot-Spots on the Hub Protein Smad3 Differentially Affect Its Protein Interactions and Smad3-Regulated Gene Expression

PubMed Central

Schiro, Michelle M.; Stauber, Sara E.; Peterson, Tami L.; Krueger, Chateen; Darnell, Steven J.; Satyshur, Kenneth A.; Drinkwater, Norman R.; Newton, Michael A.; Hoffmann, F. Michael

2011-01-01

Background Hub proteins are connected through binding interactions to many other proteins. Smad3, a mediator of signal transduction induced by transforming growth factor beta (TGF-β), serves as a hub protein for over 50 protein-protein interactions. Different cellular responses mediated by Smad3 are the product of cell-type and context dependent Smad3-nucleated protein complexes acting in concert. Our hypothesis is that perturbation of this spectrum of protein complexes by mutation of single protein-binding hot-spots on Smad3 will have distinct consequences on Smad3-mediated responses. Methodology/Principal Findings We mutated 28 amino acids on the surface of the Smad3 MH2 domain and identified 22 Smad3 variants with reduced binding to subsets of 17 Smad3-binding proteins including Smad4, SARA, Ski, Smurf2 and SIP1. Mutations defective in binding to Smad4, e.g., D408H, or defective in nucleocytoplasmic shuttling, e.g., W406A, were compromised in modulating the expression levels of a Smad3-dependent reporter gene or six endogenous Smad3-responsive genes: Mmp9, IL11, Tnfaip6, Fermt1, Olfm2 and Wnt11. However, the Smad3 mutants Y226A, Y297A, W326A, K341A, and E267A had distinct differences on TGF-β signaling. For example, K341A and Y226A both reduced the Smad3-mediated activation of the reporter gene by ∼50% but K341A only reduced the TGF-β inducibilty of Olfm2 in contrast to Y226A which reduced the TGF-β inducibility of all six endogenous genes as severely as the W406A mutation. E267A had increased protein binding but reduced TGF-β inducibility because it caused higher basal levels of expression. Y297A had increased TGF-β inducibility because it caused lower Smad3-induced basal levels of gene expression. Conclusions/Significance Mutations in protein binding hot-spots on Smad3 reduced the binding to different subsets of interacting proteins and caused a range of quantitative changes in the expression of genes induced by Smad3. This approach should be useful

Dynamic imaging of interaction between protein 14-3-3 and Bid in living cells

NASA Astrophysics Data System (ADS)

Chen, Tongsheng; Xing, Da; Wang, Jinjun

2006-02-01

The 14-3-3 proteins are known to sequester certain pro-apoptotic members of this family. BH3- interacting domain death agonist (Bid) may contribute to tumor necrosis factor α(TNF-α)-induced neuronal death, although regulation by 14-3-3 has not been reported. In this study we examined whether 14-3-3 proteins interact with Bid/tBid during TNF-α-induced cell death. The TNF-αtriggered Bid cleavage and tBid translocated to mitochondria. Human lung adenocarcinoma cells were co-transfected with both CFP-Bid and 14-3-3-YFP plasmids, and the dynamical interaction between the Bid/tBid and 14-3-3 were performed on laser confocal fluorescence microscope in single living cell during TNF-α-induced cell apoptosis. The Bid distribute equally only in the cytoplasm of healthy cells, and the 14-3-3 protein distribute not only in the cytoplasm but also in the nucleus of healthy cells. Our data showed that the tBid aggregate, but the 14-3-3 protein does not aggregate as the tBid, and the 14-3-3 protein separate from the aggregated tBid, implying that the 14-3-3 proteins do not interact with the aggregated tBid after TNF-αtreatment.

[3H]aniracetam binds to specific recognition sites in brain membranes.

PubMed

Fallarino, F; Genazzani, A A; Silla, S; L'Episcopo, M R; Camici, O; Corazzi, L; Nicoletti, F; Fioretti, M C

1995-08-01

[3H]Aniracetam bound to specific and saturable recognition sites in membranes prepared from discrete regions of rat brain. In crude membrane preparation from rat cerebral cortex, specific binding was Na+ independent, was still largely detectable at low temperature (4 degrees C), and underwent rapid dissociation. Scatchard analysis of [3H]aniracetam binding revealed a single population of sites with an apparent KD value of approximately 70 nM and a maximal density of 3.5 pmol/mg of protein. Specifically bound [3H]aniracetam was not displaced by various metabolites of aniracetam, nor by other pyrrolidinone-containing nootropic drugs such as piracetam or oxiracetam. Subcellular distribution studies showed that a high percentage of specific [3H]aniracetam binding was present in purified synaptosomes or mitochondria, whereas specific binding was low in the myelin fraction. The possibility that at least some [3H]aniracetam binding sites are associated with glutamate receptors is supported by the evidence that specific binding was abolished when membranes were preincubated at 37 degrees C under fast shaking (a procedure that substantially reduced the amount of glutamate trapped in the membranes) and could be restored after addition of either glutamate or alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) but not kainate. The action of AMPA was antagonized by DNQX, which also reduced specific [3H]aniracetam binding in unwashed membranes. High levels of [3H]aniracetam binding were detected in hippocampal, cortical, or cerebellar membranes, which contain a high density of excitatory amino acid receptors.(ABSTRACT TRUNCATED AT 250 WORDS)

GNL3L Inhibits Estrogen Receptor-Related Protein Activities by Competing for Coactivator Binding

PubMed Central

Yasumoto, Hiroaki; Meng, Lingjun; Lin, Tao; Zhu, Qubo; Tsai, Robert Y.L.

2010-01-01

Summary Guanine-nucleotide binding protein 3-like (GNL3L) is the closest homologue of a stem cell-enriched factor nucleostemin in vertebrates. They share the same yeast orthologue, Grn1p, but only GNL3L can rescue the growth-deficient phenotype in Grn1p-null yeasts. To determine the unique function of GNL3L, we identified estrogen receptor-related protein-γ (ERRγ) as a GNL3L-specific binding protein. GNL3L and ERRγ are coexpressed in the eye, kidney and muscle, and co-reside in the nucleoplasm. The interaction between GNL3L and ERRγ requires the intermediate domain of GNL3L and the AF2-domain of ERRγ. Gain- and loss-of-function experiments show that GNL3L can inhibit the transcriptional activities of ERR genes in a cell-based reporter system, which does not require the nucleolar localization of GNL3L. We further demonstrate that GNL3L is able to reduce the steroid receptor coactivator (SRC) binding and the SRC-mediated transcriptional coactivation of ERRγ. This work reveals a novel mechanism that negatively regulates the transcriptional function of ERRγ by GNL3L through coactivator competition. PMID:17623774

Interactions of L-3,5,3'-Triiodothyronine, Allopregnanolone, and Ivermectin with the GABAA Receptor: Evidence for Overlapping Intersubunit Binding Modes

PubMed Central

Westergard, Thomas; Salari, Reza; Martin, Joseph V.; Brannigan, Grace

2015-01-01

Structural mechanisms of modulation of γ-aminobutyric acid (GABA) type A receptors by neurosteroids and hormones remain unclear. The thyroid hormone L-3,5,3’-triiodothyronine (T3) inhibits GABAA receptors at micromolar concentrations and has common features with neurosteroids such as allopregnanolone (ALLOP). Here we use functional experiments on α2β1γ2 GABAA receptors expressed in Xenopus oocytes to detect competitive interactions between T3 and an agonist (ivermectin, IVM) with a crystallographically determined binding site at subunit interfaces in the transmembrane domain of a homologous receptor (glutamate-gated chloride channel, GluCl). T3 and ALLOP also show competitive effects, supporting the presence of both a T3 and ALLOP binding site at one or more subunit interfaces. Molecular dynamics (MD) simulations over 200 ns are used to investigate the dynamics and energetics of T3 in the identified intersubunit sites. In these simulations, T3 molecules occupying all intersubunit sites (with the exception of the α-β interface) display numerous energetically favorable conformations with multiple hydrogen bonding partners, including previously implicated polar/acidic sidechains and a structurally conserved deformation in the M1 backbone. PMID:26421724

Clinical and treatment effects on /sup 3/H-clonidine and /sup 3/H-imipramine binding in elderly depressed patients

DOE Office of Scientific and Technical Information (OSTI.GOV)

Georgotas, A.; Schweitzer, J.; McCue, R.E.

1987-06-01

/sup 3/H-clonidine and /sup 3/H-imipramine binding were measured in depressed patients, 55 years and older. There was no significant difference in either /sup 3/H-clonidine or /sup 3/H-imipramine binding between depressed patients and age- and sex-matched controls. There was no significant correlation between /sup 3/H-clonidine or /sup 3/H-imipramine binding and severity of depression before treatment. There was a significant negative correlation between the K/sub D/ of /sup 3/H-imipramine binding sites and Hamilton score over seven weeks of antidepressant treatment. There was no significant difference between receptor data of responders and nonresponders to antidepressant treatment. 19 references, 2 tables.

Characterization of ( sup 3 H)alprazolam binding to central benzodiazepine receptors

DOE Office of Scientific and Technical Information (OSTI.GOV)

McCabe, R.T.; Mahan, D.R.; Smith, R.B.

1990-10-01

The binding of the triazolobenzodiazepine ({sup 3}H)alprazolam was studied to characterize the in vitro interactions with benzodiazepine receptors in membrane preparations of rat brain. Studies using nonequilibrium and equilibrium binding conditions for ({sup 3}H)alprazolam resulted in high specific to nonspecific (signal to noise) binding ratios. The binding of ({sup 3}H)alprazolam was saturable and specific with a low nanomolar affinity for benzodiazepine receptors in the rat brain. The Kd was 4.6 nM and the Bmax was 2.6 pmol/mg protein. GABA enhanced ({sup 3}H)alprazolam binding while several benzodiazepine receptor ligands were competitive inhibitors of this drug. Compounds that bind to other receptormore » sites had a very weak or negligible effect on ({sup 3}H)alprazolam binding. Alprazolam, an agent used as an anxiolytic and in the treatment of depression, acts in vitro as a selective and specific ligand for benzodiazepine receptors in the rat brain. The biochemical binding profile does not appear to account for the unique therapeutic properties which distinguish this compound from the other benzodiazepines in its class.« less

Functional characterization of transcription factor binding sites for HNF1-alpha, HNF3-beta (FOXA2), HNF4-alpha, Sp1 and Sp3 in the human prothrombin gene enhancer.

PubMed

Ceelie, H; Spaargaren-Van Riel, C C; De Jong, M; Bertina, R M; Vos, H L

2003-08-01

Prothrombin is a key component in blood coagulation. Overexpression of prothrombin leads to an increased risk of venous thrombosis. Therefore, the study of the transcriptional regulation of the prothrombin gene may help to identify mechanisms of overexpression. The aim of our study was to localize the regions within the prothrombin enhancer responsible for its activity, to identify the proteins binding to these regions, and to establish their functional importance. We constructed a set of prothrombin promoter 5' deletion constructs containing the firefly luciferase reporter gene, which were transiently transfected in HepG2, HuH7 and HeLa cells. Putative transcription factor (TF) binding sites were evaluated by electrophoretic mobility shift assays. The functional importance of each TF binding site was evaluated by site directed mutagenesis and transient transfection of the mutant constructs. We confirmed the major contribution of the enhancer region to the transcriptional activity of the prothrombin promoter. Analysis of this region revealed putative binding sites for hepatocyte nuclear factor HNF4, HNF3-beta and specificity protein(Sp)1. We identified six different TFs binding to three evolutionary conserved sites in the enhancer: HNF4-alpha (site 1), HNF1-alpha, HNF3-beta and an as yet unidentified TF (site 2) and the ubiquitously expressed TFs Sp1 and Sp3 (site 3). Mutagenesis studies showed that loss of binding of HNF3-beta resulted in a considerable decrease of enhancer activity, whereas loss of HNF4-alpha or Sp1/Sp3 resulted in milder reductions. The prothrombin enhancer plays a major role in regulation of prothrombin expression. Six different TFs are able to bind to this region. At least three of these TFs, HNF4-alpha, HNF3-beta and Sp1/Sp3, are important in regulation of prothrombin expression.

Crystal structure of the Candida albicans Kar3 kinesin motor domain fused to maltose-binding protein

DOE Office of Scientific and Technical Information (OSTI.GOV)

Delorme, Caroline; Joshi, Monika; Allingham, John S., E-mail: allinghj@queensu.ca

2012-11-30

Highlights: Black-Right-Pointing-Pointer The Candida albicans Kar3 motor domain structure was solved as a maltose-binding protein fusion. Black-Right-Pointing-Pointer The electrostatic surface and part of the ATPase pocket of the motor domain differs markedly from other kinesins. Black-Right-Pointing-Pointer The MBP-Kar3 interface highlights a new site for intramolecular or intermolecular interactions. -- Abstract: In the human fungal pathogen Candida albicans, the Kinesin-14 motor protein Kar3 (CaKar3) is critical for normal mitotic division, nuclear fusion during mating, and morphogenic transition from the commensal yeast form to the virulent hyphal form. As a first step towards detailed characterization of this motor of potential medical significance,more » we have crystallized and determined the X-ray structure of the motor domain of CaKar3 as a maltose-binding protein (MBP) fusion. The structure shows strong conservation of overall motor domain topology to other Kar3 kinesins, but with some prominent differences in one of the motifs that compose the nucleotide-binding pocket and the surface charge distribution. The MBP and Kar3 modules are arranged such that MBP interacts with the Kar3 motor domain core at the same site where the neck linker of conventional kinesins docks during the 'ATP state' of the mechanochemical cycle. This site differs from the Kar3 neck-core interface in the recent structure of the ScKar3Vik1 heterodimer. The position of MBP is also completely distinct from the Vik1 subunit in this complex. This may suggest that the site of MBP interaction on the CaKar3 motor domain provides an interface for the neck, or perhaps a partner subunit, at an intermediate state of its motile cycle that has not yet been observed for Kinesin-14 motors.« less

Analysis of the interactions between GMF and Arp2/3 complex in two binding sites by molecular dynamics simulation.

PubMed

Popinako, A; Antonov, M; Dibrova, D; Chemeris, A; Sokolova, O S

2018-02-05

The Arp2/3 complex plays a key role in nucleating actin filaments branching. The glia maturation factor (GMF) competes with activators for interacting with the Arp2/3 complex and initiates the debranching of actin filaments. In this study, we performed a comparative analysis of interactions between GMF and the Arp2/3 complex and identified new amino acid residues involved in GMF binding to the Arp2/3 complex at two separate sites, revealed by X-ray and single particle EM techniques. Using molecular dynamics simulations we demonstrated the quantitative and qualitative changes in hydrogen bonds upon binding with GMF. We identified the specific amino acid residues in GMF and Arp2/3 complex that stabilize the interactions and estimated the mean force profile for the GMF using umbrella sampling. Phylogenetic and structural analyses of the recently defined GMF binding site on the Arp3 subunit indicate a new mechanism for Arp2/3 complex inactivation that involves interactions between the Arp2/3 complex and GMF at two binding sites. Copyright © 2018 Elsevier Inc. All rights reserved.

Juvenile hormone-binding proteins of Melanoplus bivittatus identified by EFDA photoaffinity labeling

DOE Office of Scientific and Technical Information (OSTI.GOV)

Winder, B.S.

1988-01-01

Proteins that bind juvenile hormone in the hemolymph and fat body of the grasshopper, Melanoplus bivittatus were identified by photoaffinity labeling with radiolabeled epoxyfarnesyl diazoacetate ({sup 3}H-EFDA), and were characterized by electrophoretic analysis. A protocol was developed which allowed detection of {sup 3}H-EFDA that was covalently linked to proteins upon exposure to ultraviolet light at 254 nm. Quantification of protein-linked {sup 3}H-EFDA by liquid scintillation spectrometry took advantage of the differential solubility of unlinked {sup 3}H-EFDA in toluene alone, and of the protein-linked {sup 3}H-EFDA in toluene plus the detergent, Triton X-100. Competition between EFDA and juvenile hormone (JH) formore » binding to JH-specific binding sites was measured by hydroxyapatite protein binding assays in the presence of radiolabeled JH or EFDA and competing non-radiolabeled hormone. The protein-linked EFDA was detected on fluorograms of SDS or nondenaturing polyacrylamide gels (PAGE), and by liquid scintillation spectrometry of membranes to which the proteins had been electrophoretically transferred. Proteins which specifically bound JH were identified by photolabeling proteins in the presence and absence of nonlabeled JH-III.« less

Chemokine CCR3 ligands-binding peptides derived from a random phage-epitope library.

PubMed

Houimel, Mehdi; Mazzucchelli, Luca

2013-01-01

Eosinophils are major effectors cells implicated in a number of chronic inflammatory diseases in humans, particularly bronchial asthma and allergic rhinitis. The human chemokine receptor C-C receptor 3 (hCCR3) provides a mechanism for the recruitment of eosinophils into tissue and thus has recently become an attractive biological target for therapeutic intervention. In order to develop peptides antagonists of hCCR3-hCCL11 (human eotaxin) interactions, a random bacteriophage hexapeptide library was used to map structural features of hCCR3 by determining the epitopes of neutralizing anti-hCCR3 mAb 7B11. This mAb t is selective for hCCR3 and exhibit potent antagonist activity in receptor binding and functional assays. After three rounds of biopanning, four mAb7B11-binding peptides were identified from a 6-mer linear peptide library. The phage bearing the peptides showed specific binding to immobilized mAb 7B11 with over 94% of phages bound being competitively inhibited by free synthetic peptides. In FACScan analysis all selected phage peptides were able to strongly inhibit the binding of mAb 7B11 to hCCR3-transfected preB-300-19 murine cells. Furthermore, synthetic peptides of the corresponding phage epitopes were effective in blocking the antibody-hCCR3 interactions and to inhibit the binding of hCCL11 to hCCR3 transfectants. Chemically synthesized peptides CKGERF, FERKGK, SSMKVK and RHVSSQ, effectively competed for (125)I-hCCL11 binding to hCCR3 with IC(50) ranging from 3.5 to 9.7μM. Calcium release and chemotaxis of hCCR3 transfectants or human eosinophils were inhibited by all peptides in a dose-dependent manner. Furthermore, they showed inhibitory effects on chemotaxis of human eosinophils induced by hCCL11, hCCL5, hCCL7, hCCL8, and hCCL24. Specificities of all selected peptides were assessed with hCXCR1, hCXCR2, hCXCR3, and hCCR5 receptors. Peptides CKGERF and FERKGK showed inhibitory effects on eosinophil chemotaxis in a murine model of mCCL11-induced

H3.Y discriminates between HIRA and DAXX chaperone complexes and reveals unexpected insights into human DAXX-H3.3-H4 binding and deposition requirements

PubMed Central

Zink, Lisa-Maria; Delbarre, Erwan; Eberl, H. Christian; Keilhauer, Eva C.; Bönisch, Clemens; Pünzeler, Sebastian; Bartkuhn, Marek; Collas, Philippe; Mann, Matthias

2017-01-01

Abstract Histone chaperones prevent promiscuous histone interactions before chromatin assembly. They guarantee faithful deposition of canonical histones and functionally specialized histone variants into chromatin in a spatial- and temporally-restricted manner. Here, we identify the binding partners of the primate-specific and H3.3-related histone variant H3.Y using several quantitative mass spectrometry approaches, and biochemical and cell biological assays. We find the HIRA, but not the DAXX/ATRX, complex to recognize H3.Y, explaining its presence in transcriptionally active euchromatic regions. Accordingly, H3.Y nucleosomes are enriched in the transcription-promoting FACT complex and depleted of repressive post-translational histone modifications. H3.Y mutational gain-of-function screens reveal an unexpected combinatorial amino acid sequence requirement for histone H3.3 interaction with DAXX but not HIRA, and for H3.3 recruitment to PML nuclear bodies. We demonstrate the importance and necessity of specific H3.3 core and C-terminal amino acids in discriminating between distinct chaperone complexes. Further, chromatin immunoprecipitation sequencing experiments reveal that in contrast to euchromatic HIRA-dependent deposition sites, human DAXX/ATRX-dependent regions of histone H3 variant incorporation are enriched in heterochromatic H3K9me3 and simple repeat sequences. These data demonstrate that H3.Y's unique amino acids allow a functional distinction between HIRA and DAXX binding and its consequent deposition into open chromatin. PMID:28334823

EV71 3D Protein Binds with NLRP3 and Enhances the Assembly of Inflammasome Complex

PubMed Central

Wan, Pin; Pan, Pan; Zhang, Yecheng; Wu, Kailang; Liu, Yingle; Wu, Jianguo

2017-01-01

Activation of NLRP3 inflammasome is important for effective host defense against invading pathogen. Together with apoptosis-associated speck-like protein containing CARD domain (ASC), NLRP3 induces the cleavage of caspase-1 to facilitate the maturation of interleukin-1beta (IL-1β), an important pro-inflammatory cytokine. IL-1β subsequently plays critical roles in inflammatory responses by activating immune cells and inducing many secondary pro-inflammatory cytokines. Although the role of NLRP3 inflammasome in immune response is well defined, the mechanism underlying its assembly modulated by pathogen infection remains largely unknown. Here, we identified a novel mechanism by which enterovirus 71 (EV71) facilitates the assembly of NLRP3 inflammasome. Our results show that EV71 induces production and secretion of IL-1β in macrophages and peripheral blood mononuclear cells (PBMCs) through activation of NLRP3 inflammasome. EV71 replication and protein synthesis are required for NLRP3-mediated activation of IL-1β. Interestingly, EV71 3D protein, a RNA-dependent RNA polymerase (RdRp) was found to stimulate the activation of NLRP3 inflammasome, the cleavage of pro-caspase-1, and the release of IL-1β through direct binding to NLRP3. More importantly, 3D interacts with NLRP3 to facilitate the assembly of inflammasome complex by forming a 3D-NLRP3-ASC ring-like structure, resulting in the activation of IL-1β. These findings demonstrate a new role of 3D as an important player in the activation of inflammatory response, and identify a novel mechanism underlying the modulation of inflammasome assembly and function induced by pathogen invasion. PMID:28060938

Cone arrestin binding to JNK3 and Mdm2: conformational preference and localization of interaction sites

PubMed Central

Song, Xiufeng; Gurevich, Eugenia V.; Gurevich, Vsevolod V.

2008-01-01

Arrestins are multi-functional regulators of G protein-coupled receptors. Receptor-bound arrestins interact with >30 remarkably diverse proteins and redirect the signaling to G protein-independent pathways. The functions of free arrestins are poorly understood, and the interaction sites of the non-receptor arrestin partners are largely unknown. In this study, we show that cone arrestin, the least studied member of the family, binds c-Jun N-terminal kinase (JNK3) and Mdm2 and regulates their subcellular distribution. Using arrestin mutants with increased or reduced structural flexibility, we demonstrate that arrestin in all conformations binds JNK3 comparably, whereas Mdm2 preferentially binds cone arrestin ‘frozen’ in the basal state. To localize the interaction sites, we expressed separate N- and C-domains of cone and rod arrestins and found that individual domains bind JNK3 and remove it from the nucleus as efficiently as full-length proteins. Thus, the arrestin binding site for JNK3 includes elements in both domains with the affinity of partial sites on individual domains sufficient for JNK3 relocalization. N-domain of rod arrestin binds Mdm2, which localizes its main interaction site to this region. Comparable binding of JNK3 and Mdm2 to four arrestin subtypes allowed us to identify conserved residues likely involved in these interactions. PMID:17680991

A genome-wide association study identifies colorectal cancer susceptibility loci on chromosomes 10p14 and 8q23.3.

PubMed

Tomlinson, Ian P M; Webb, Emily; Carvajal-Carmona, Luis; Broderick, Peter; Howarth, Kimberley; Pittman, Alan M; Spain, Sarah; Lubbe, Steven; Walther, Axel; Sullivan, Kate; Jaeger, Emma; Fielding, Sarah; Rowan, Andrew; Vijayakrishnan, Jayaram; Domingo, Enric; Chandler, Ian; Kemp, Zoe; Qureshi, Mobshra; Farrington, Susan M; Tenesa, Albert; Prendergast, James G D; Barnetson, Rebecca A; Penegar, Steven; Barclay, Ella; Wood, Wendy; Martin, Lynn; Gorman, Maggie; Thomas, Huw; Peto, Julian; Bishop, D Timothy; Gray, Richard; Maher, Eamonn R; Lucassen, Anneke; Kerr, David; Evans, D Gareth R; Schafmayer, Clemens; Buch, Stephan; Völzke, Henry; Hampe, Jochen; Schreiber, Stefan; John, Ulrich; Koessler, Thibaud; Pharoah, Paul; van Wezel, Tom; Morreau, Hans; Wijnen, Juul T; Hopper, John L; Southey, Melissa C; Giles, Graham G; Severi, Gianluca; Castellví-Bel, Sergi; Ruiz-Ponte, Clara; Carracedo, Angel; Castells, Antoni; Försti, Asta; Hemminki, Kari; Vodicka, Pavel; Naccarati, Alessio; Lipton, Lara; Ho, Judy W C; Cheng, K K; Sham, Pak C; Luk, J; Agúndez, Jose A G; Ladero, Jose M; de la Hoya, Miguel; Caldés, Trinidad; Niittymäki, Iina; Tuupanen, Sari; Karhu, Auli; Aaltonen, Lauri; Cazier, Jean-Baptiste; Campbell, Harry; Dunlop, Malcolm G; Houlston, Richard S

2008-05-01

To identify colorectal cancer (CRC) susceptibility alleles, we conducted a genome-wide association study. In phase 1, we genotyped 550,163 tagSNPs in 940 familial colorectal tumor cases (627 CRC, 313 high-risk adenoma) and 965 controls. In phase 2, we genotyped 42,708 selected SNPs in 2,873 CRC cases and 2,871 controls. In phase 3, we evaluated 11 SNPs showing association at P < 10(-4) in a joint analysis of phases 1 and 2 in 4,287 CRC cases and 3,743 controls. Two SNPs were taken forward to phase 4 genotyping (10,731 CRC cases and 10,961 controls from eight centers). In addition to the previously reported 8q24, 15q13 and 18q21 CRC risk loci, we identified two previously unreported associations: rs10795668, located at 10p14 (P = 2.5 x 10(-13) overall; P = 6.9 x 10(-12) replication), and rs16892766, at 8q23.3 (P = 3.3 x 10(-18) overall; P = 9.6 x 10(-17) replication), which tags a plausible causative gene, EIF3H. These data provide further evidence for the 'common-disease common-variant' model of CRC predisposition.

Structure, Subunit Topology, and Actin-binding Activity of the Arp2/3 Complex from Acanthamoeba

PubMed Central

Mullins, R. Dyche; Stafford, Walter F.; Pollard, Thomas D.

1997-01-01

The Arp2/3 complex, first isolated from Acanthamoeba castellani by affinity chromatography on profilin, consists of seven polypeptides; two actinrelated proteins, Arp2 and Arp3; and five apparently novel proteins, p40, p35, p19, p18, and p14 (Machesky et al., 1994). The complex is homogeneous by hydrodynamic criteria with a Stokes' radius of 5.3 nm by gel filtration, sedimentation coefficient of 8.7 S, and molecular mass of 197 kD by analytical ultracentrifugation. The stoichiometry of the subunits is 1:1:1:1:1:1:1, indicating the purified complex contains one copy each of seven polypeptides. In electron micrographs, the complex has a bilobed or horseshoe shape with outer dimensions of ∼13 × 10 nm, and mathematical models of such a shape and size are consistent with the measured hydrodynamic properties. Chemical cross-linking with a battery of cross-linkers of different spacer arm lengths and chemical reactivities identify the following nearest neighbors within the complex: Arp2 and p40; Arp2 and p35; Arp3 and p35; Arp3 and either p18 or p19; and p19 and p14. By fluorescent antibody staining with anti-p40 and -p35, the complex is concentrated in the cortex of the ameba, especially in linear structures, possibly actin filament bundles, that lie perpendicular to the leading edge. Purified Arp2/3 complex binds actin filaments with a K d of 2.3 μM and a stoichiometry of approximately one complex molecule per actin monomer. In electron micrographs of negatively stained samples, Arp2/3 complex decorates the sides of actin filaments. EDC/NHS cross-links actin to Arp3, p35, and a low molecular weight subunit, p19, p18, or p14. We propose structural and topological models for the Arp2/3 complex and suggest that affinity for actin filaments accounts for the localization of complex subunits to actinrich regions of Acanthamoeba. PMID:9015304

14 CFR 158.3 - Definitions.

Code of Federal Regulations, 2012 CFR

2012-01-01

... 14 Aeronautics and Space 3 2012-01-01 2012-01-01 false Definitions. 158.3 Section 158.3 Aeronautics and Space FEDERAL AVIATION ADMINISTRATION, DEPARTMENT OF TRANSPORTATION (CONTINUED) AIRPORTS... airport to support aeronautical operations and related activities. Baggage tugs, belt loaders, cargo...

14 CFR 158.3 - Definitions.

Code of Federal Regulations, 2014 CFR

2014-01-01

... 14 Aeronautics and Space 3 2014-01-01 2014-01-01 false Definitions. 158.3 Section 158.3 Aeronautics and Space FEDERAL AVIATION ADMINISTRATION, DEPARTMENT OF TRANSPORTATION (CONTINUED) AIRPORTS... airport to support aeronautical operations and related activities. Baggage tugs, belt loaders, cargo...

STD-NMR experiments identify a structural motif with novel second-site activity against West Nile virus NS2B-NS3 protease.

PubMed

Schöne, Tobias; Grimm, Lena Lisbeth; Sakai, Naoki; Zhang, Linlin; Hilgenfeld, Rolf; Peters, Thomas

2017-10-01

West Nile virus (WNV) belongs to the genus Flavivirus of the family Flaviviridae. This mosquito-borne virus that is highly pathogenic to humans has been evolving into a global threat during the past two decades. Despite many efforts, neither antiviral drugs nor vaccines are available. The viral protease NS2B-NS3 pro is essential for viral replication, and therefore it is considered a prime drug target. However, success in the development of specific NS2B-NS3 pro inhibitors had been moderate so far. In the search for new structural motifs with binding affinity for NS2B-NS3 pro , we have screened a fragment library, the Maybridge Ro5 library, employing saturation transfer difference (STD) NMR experiments as readout. About 30% of 429 fragments showed binding to NS2B-NS3 pro . Subsequent STD-NMR competition experiments using the known active site fragment A as reporter ligand yielded 14 competitively binding fragments, and 22 fragments not competing with A. In a fluorophore-based protease assay, all of these fragments showed inhibition in the micromolar range. Interestingly, 10 of these 22 fragments showed a notable increase of STD intensities in the presence of compound A suggesting cooperative binding. The most promising non-competitive inhibitors 1 and 2 (IC 50 ∼ 500 μM) share a structural motif that may guide the development of novel second-site (potentially allosteric) inhibitors of NS2B-NS3 pro . To identify the matching protein binding site, chemical shift perturbation studies employing 1 H, 15 N-TROSY-HSQC experiments with uniformly 2 H, 15 N-labeled protease were performed in the presence of 1, and in the concomitant absence or presence of A. The data suggest that 1 interacts with Met 52* of NS2B, identifying a secondary site adjacent to the binding site of A. Therefore, our study paves the way for the synthesis of novel bidentate NS2B-NS3 pro inhibitors. Copyright © 2017 Elsevier B.V. All rights reserved.

38 CFR 3.14 - Validity of enlistments.

Code of Federal Regulations, 2013 CFR

2013-07-01

... 38 Pensions, Bonuses, and Veterans' Relief 1 2013-07-01 2013-07-01 false Validity of enlistments. 3.14 Section 3.14 Pensions, Bonuses, and Veterans' Relief DEPARTMENT OF VETERANS AFFAIRS ADJUDICATION Pension, Compensation, and Dependency and Indemnity Compensation General § 3.14 Validity of...

38 CFR 3.14 - Validity of enlistments.

Code of Federal Regulations, 2012 CFR

2012-07-01

... 38 Pensions, Bonuses, and Veterans' Relief 1 2012-07-01 2012-07-01 false Validity of enlistments. 3.14 Section 3.14 Pensions, Bonuses, and Veterans' Relief DEPARTMENT OF VETERANS AFFAIRS ADJUDICATION Pension, Compensation, and Dependency and Indemnity Compensation General § 3.14 Validity of...

38 CFR 3.14 - Validity of enlistments.

Code of Federal Regulations, 2011 CFR

2011-07-01

... 38 Pensions, Bonuses, and Veterans' Relief 1 2011-07-01 2011-07-01 false Validity of enlistments. 3.14 Section 3.14 Pensions, Bonuses, and Veterans' Relief DEPARTMENT OF VETERANS AFFAIRS ADJUDICATION Pension, Compensation, and Dependency and Indemnity Compensation General § 3.14 Validity of...

38 CFR 3.14 - Validity of enlistments.

Code of Federal Regulations, 2014 CFR

2014-07-01

... 38 Pensions, Bonuses, and Veterans' Relief 1 2014-07-01 2014-07-01 false Validity of enlistments. 3.14 Section 3.14 Pensions, Bonuses, and Veterans' Relief DEPARTMENT OF VETERANS AFFAIRS ADJUDICATION Pension, Compensation, and Dependency and Indemnity Compensation General § 3.14 Validity of...

38 CFR 3.14 - Validity of enlistments.

Code of Federal Regulations, 2010 CFR

2010-07-01

... 38 Pensions, Bonuses, and Veterans' Relief 1 2010-07-01 2010-07-01 false Validity of enlistments. 3.14 Section 3.14 Pensions, Bonuses, and Veterans' Relief DEPARTMENT OF VETERANS AFFAIRS ADJUDICATION Pension, Compensation, and Dependency and Indemnity Compensation General § 3.14 Validity of...

Hyperglycemia decreases expression of 14-3-3 proteins in an animal model of stroke.

PubMed

Jeon, Seong-Jun; Sung, Jin-Hee; Koh, Phil-Ok

2016-07-28

Diabetes is a severe metabolic disorder and a major risk factor for stroke. Stroke severity is worse in patients with diabetes compared to the non-diabetic population. The 14-3-3 proteins are a family of conserved acidic proteins that are ubiquitously expressed in cells and tissues. These proteins are involved in many cellular processes including metabolic pathways, signal transduction, protein trafficking, protein synthesis, and cell cycle control. This study investigated 14-3-3 proteins expression in the cerebral cortex of animals with diabetes, cerebral ischemic injury and a combination of both diabetes and cerebral ischemic injury. Diabetes was induced by intraperitoneal injection of streptozotocin (40mg/kg) in adult male rats. After 4 weeks of treatment, middle cerebral artery occlusion (MCAO) was performed for the induction of focal cerebral ischemia and cerebral cortex tissue was collected 24h after MCAO. We confirmed that diabetes increases infarct volume following MCAO compared to non-diabetic animals. In diabetic animals with MCAO injury, reduction of 14-3-3 β/α, 14-3-3 ζ/δ, 14-3-3 γ, and 14-3-3 ε isoforms was detected. The expression of these proteins was significantly decreased in diabetic animals with MCAO injury compared to diabetic-only and MCAO-only animals. Moreover, Western blot analysis ascertained the decreased expression of 14-3-3 family proteins in diabetic animals with MCAO injury, including β/α, ζ/δ, γ, ε, τ, and η isoforms. These results show the changes of 14-3-3 proteins expression in streptozotocin-induced diabetic animals with MCAO injury. Thus, these findings suggest that decreases in 14-3-3 proteins might be involved in the regulation of 14-3-3 proteins under the presence of diabetes following MCAO. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Generation of mice deficient in RNA-binding motif protein 3 (RBM3) and characterization of its role in innate immune responses and cell growth

DOE Office of Scientific and Technical Information (OSTI.GOV)

Matsuda, Atsushi; Core Research for Evolution Science and Technology, Japan Science and Technology Agency, Chiyoda-ku, Tokyo 102-0075; Ogawa, Masahiro

Highlights: {yields} We identified RNA-binding motif protein 3 (RBM3) as CpG-B DNA-binding protein. {yields} RBM3 translocates from the nucleus to the cytoplasm and co-localized with CpG-B DNA. {yields} We newly generated Rbm3-deficient (Rbm3{sup -/-}) mice. {yields} DNA-mediated cytokine gene induction was normally occured in Rbm3{sup -/-} cells. {yields}Rbm3{sup -/-} MEFs showed poorer proliferation rate and increased number of G2-phase cells. -- Abstract: The activation of innate immune responses is critical to host defense against microbial infections, wherein nucleic acid-sensing pattern recognition receptors recognize DNA or RNA from viruses or bacteria and activate downstream signaling pathways. In a search for newmore » DNA-sensing molecules that regulate innate immune responses, we identified RNA-binding motif protein 3 (RBM3), whose role has been implicated in the regulation of cell growth. In this study, we generated Rbm3-deficient (Rbm3{sup -/-}) mice to study the role of RBM3 in immune responses and cell growth. Despite evidence for its interaction with immunogenic DNA in a cell, no overt phenotypic abnormalities were found in cells from Rbm3{sup -/-} mice for the DNA-mediated induction of cytokine genes. Interestingly, however, Rbm3{sup -/-} mouse embryonic fibroblasts (MEFs) showed poorer proliferation rates as compared to control MEFs. Further cell cycle analysis revealed that Rbm3{sup -/-} MEFs have markedly increased number of G2-phase cells, suggesting a hitherto unknown role of RBM3 in the G2-phase control. Thus, these mutant mice and cells may provide new tools with which to study the mechanisms underlying the regulation of cell cycle and oncogenesis.« less

On the inhibitor effects of bergamot juice flavonoids binding to the 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) enzyme.

PubMed

Leopoldini, Monica; Malaj, Naim; Toscano, Marirosa; Sindona, Giovanni; Russo, Nino

2010-10-13

Density functional theory was applied to study the binding mode of new flavonoids as possible inhibitors of the 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR), an enzyme that catalyzes the four-electron reduction of HMGCoA to mevalonate, the committed step in the biosynthesis of sterols. The investigated flavonoid conjugates brutieridin and melitidin were recently quantified in the bergamot fruit extracts and identified to be structural analogues of statins, lipids concentration lowering drugs that inhibit HMGR. Computations allowed us to perform a detailed analysis of the geometrical and electronic features affecting the binding of these compounds, as well as that of the excellent simvastatin drug, to the active site of the enzyme and to give better insight into the inhibition process.

Genome-Wide Identification, Phylogeny, and Expression Analyses of the 14-3-3 Family Reveal Their Involvement in the Development, Ripening, and Abiotic Stress Response in Banana

PubMed Central

Li, Meiying; Ren, Licheng; Xu, Biyu; Yang, Xiaoliang; Xia, Qiyu; He, Pingping; Xiao, Susheng; Guo, Anping; Hu, Wei; Jin, Zhiqiang

2016-01-01

Plant 14-3-3 proteins act as critical components of various cellular signaling processes and play an important role in regulating multiple physiological processes. However, less information is known about the 14-3-3 gene family in banana. In this study, 25 14-3-3 genes were identified from the banana genome. Based on the evolutionary analysis, banana 14-3-3 proteins were clustered into ε and non-ε groups. Conserved motif analysis showed that all identified banana 14-3-3 genes had the typical 14-3-3 motif. The gene structure of banana 14-3-3 genes showed distinct class-specific divergence between the ε group and the non-ε group. Most banana 14-3-3 genes showed strong transcript accumulation changes during fruit development and postharvest ripening in two banana varieties, indicating that they might be involved in regulating fruit development and ripening. Moreover, some 14-3-3 genes also showed great changes after osmotic, cold, and salt treatments in two banana varieties, suggested their potential role in regulating banana response to abiotic stress. Taken together, this systemic analysis reveals the involvement of banana 14-3-3 genes in fruit development, postharvest ripening, and response to abiotic stress and provides useful information for understanding the functions of 14-3-3 genes in banana. PMID:27713761

The 14-3-3σ gene promoter is methylated in both human melanocytes and melanoma

PubMed Central

2009-01-01

Background Recent evidence demonstrates that 14-3-3σ acts as a tumor suppressor gene inactivated by methylation of its 5' CpG islands in epithelial tumor cells, while remaining un-methylated in normal human epithelia. The methylation analysis of 14-3-3σ has been largely overlooked in melanoma. Methods The methylation status of 14-3-3σ CpG island in melanocytes and melanoma cells was analyzed by methylation-specific sequencing (MSS) and quantitative methylation-specific PCR (Q-MSP). 14-3-3σ mRNA and protein expression in cell lines was detected by real-time RT-PCR and western blot. Melanoma cells were also treated by 5-aza-2'-deoxycytidine (DAC), a demethylating agent, and/or histone deacetylase inhibitor, Trichostatin A (TSA), to evaluate their effects on 14-3-3σ gene expression. Results 14-3-3σ is hypermethylated in both human melanocytes and most melanoma cells in a lineage-specific manner, resulting in the silencing of 14-3-3σ gene expression and the active induction of 14-3-3σ mRNA and protein expression following treatment with DAC. We also observed a synergistic effect upon gene expression when DAC was combined with TSA. The promoter methylation status of 14-3-3σ was analyzed utilizing Q-MSP in 20 melanoma tissue samples and 10 cell lines derived from these samples, showing that the majority of melanoma samples maintain their hypermethylation status of the 14-3-3σ gene. Conclusion 14-3-3σ is hypermethylated in human melanoma in a cell-linage specific manner. Spontaneous demethylation and re-expression of 14-3-3σ is a rare event in melanoma, indicating 14-3-3σ might have a tentative role in the pathogenesis of melanoma. PMID:19473536

The RPAP3-Cterminal domain identifies R2TP-like quaternary chaperones.

PubMed

Maurizy, Chloé; Quinternet, Marc; Abel, Yoann; Verheggen, Céline; Santo, Paulo E; Bourguet, Maxime; C F Paiva, Ana; Bragantini, Benoît; Chagot, Marie-Eve; Robert, Marie-Cécile; Abeza, Claire; Fabre, Philippe; Fort, Philippe; Vandermoere, Franck; M F Sousa, Pedro; Rain, Jean-Christophe; Charpentier, Bruno; Cianférani, Sarah; Bandeiras, Tiago M; Pradet-Balade, Bérengère; Manival, Xavier; Bertrand, Edouard

2018-05-29

R2TP is an HSP90 co-chaperone that assembles important macro-molecular machineries. It is composed of an RPAP3-PIH1D1 heterodimer, which binds the two essential AAA+ATPases RUVBL1/RUVBL2. Here, we resolve the structure of the conserved C-terminal domain of RPAP3, and we show that it directly binds RUVBL1/RUVBL2 hexamers. The human genome encodes two other proteins bearing RPAP3-C-terminal-like domains and three containing PIH-like domains. Systematic interaction analyses show that one RPAP3-like protein, SPAG1, binds PIH1D2 and RUVBL1/2 to form an R2TP-like complex termed R2SP. This co-chaperone is enriched in testis and among 68 of the potential clients identified, some are expressed in testis and others are ubiquitous. One substrate is liprin-α2, which organizes large signaling complexes. Remarkably, R2SP is required for liprin-α2 expression and for the assembly of liprin-α2 complexes, indicating that R2SP functions in quaternary protein folding. Effects are stronger at 32 °C, suggesting that R2SP could help compensating the lower temperate of testis.

2,3-DPG-Hb complex: a hypothesis for an asymmetric binding.

PubMed

Pomponi, M; Bertonati, C; Fuglei, E; Wiig, O; Derocher, A E

2000-05-15

This study was undertaken to test the symmetry of 2,3-diphosphoglycerate (2,3-DPG) binding site in hemoglobin (Hb). From Arnone's study [A. Arnone, Nature (London) 237 (1972) 146] the 2,3-DPG binding site is located at the top of the cavity, that runs through the center of the deoxy-Hb molecule. However, it is possible that this symmetry reported by Arnone, for crystals of 2,3-DPG-Hb complex, might not be conserved in solution. In this paper, we report the 31P nuclear magnetic resonances of the 2,3-DPG interaction with Hb. The 2,3-DPG chemical shifts of the P2 and P3 resonance are both pH- and hemoglobin-dependent [protein from man, polar bear (Ursus maritimus), Arctic fox (Alopex lagopus) and bovine]. 2,3-DPG binds tightly to deoxyhemoglobin and weakly, nevertheless significantly, to oxyhemoglobin. In particular, our results suggest similar spatial position of the binding site of 2,3-DPG in both forms of Hb in solutions. However, the most unexpected result was the apparent loss of symmetry in the binding site, which might correlate with the ability of the hemoglobin to modulate its functional behavior. The different interactions of the phosphate groups indicate small differences in the quaternary structure of the different deoxy forms of hemoglobin. Given the above structural perturbation an asymmetric binding in the complex could justify, at least in part, different physiological properties of Hb. Regardless, functionally relevant effects of 2,3-DPG seem to be measured and best elucidated through solution studies.

14 CFR 60.3 - Definitions.

Code of Federal Regulations, 2014 CFR

2014-01-01

... 14 Aeronautics and Space 2 2014-01-01 2014-01-01 false Definitions. 60.3 Section 60.3 Aeronautics and Space FEDERAL AVIATION ADMINISTRATION, DEPARTMENT OF TRANSPORTATION (CONTINUED) AIRMEN FLIGHT SIMULATION TRAINING DEVICE INITIAL AND CONTINUING QUALIFICATION AND USE § 60.3 Definitions. In addition to...

14 CFR 60.3 - Definitions.

Code of Federal Regulations, 2012 CFR

2012-01-01

... 14 Aeronautics and Space 2 2012-01-01 2012-01-01 false Definitions. 60.3 Section 60.3 Aeronautics and Space FEDERAL AVIATION ADMINISTRATION, DEPARTMENT OF TRANSPORTATION (CONTINUED) AIRMEN FLIGHT SIMULATION TRAINING DEVICE INITIAL AND CONTINUING QUALIFICATION AND USE § 60.3 Definitions. In addition to...

14 CFR 60.3 - Definitions.

Code of Federal Regulations, 2011 CFR

2011-01-01

... 14 Aeronautics and Space 2 2011-01-01 2011-01-01 false Definitions. 60.3 Section 60.3 Aeronautics and Space FEDERAL AVIATION ADMINISTRATION, DEPARTMENT OF TRANSPORTATION (CONTINUED) AIRMEN FLIGHT SIMULATION TRAINING DEVICE INITIAL AND CONTINUING QUALIFICATION AND USE § 60.3 Definitions. In addition to...

14 CFR 60.3 - Definitions.

Code of Federal Regulations, 2010 CFR

2010-01-01

... 14 Aeronautics and Space 2 2010-01-01 2010-01-01 false Definitions. 60.3 Section 60.3 Aeronautics and Space FEDERAL AVIATION ADMINISTRATION, DEPARTMENT OF TRANSPORTATION (CONTINUED) AIRMEN FLIGHT SIMULATION TRAINING DEVICE INITIAL AND CONTINUING QUALIFICATION AND USE § 60.3 Definitions. In addition to...

14 CFR 60.3 - Definitions.

Code of Federal Regulations, 2013 CFR

2013-01-01

... 14 Aeronautics and Space 2 2013-01-01 2013-01-01 false Definitions. 60.3 Section 60.3 Aeronautics and Space FEDERAL AVIATION ADMINISTRATION, DEPARTMENT OF TRANSPORTATION (CONTINUED) AIRMEN FLIGHT SIMULATION TRAINING DEVICE INITIAL AND CONTINUING QUALIFICATION AND USE § 60.3 Definitions. In addition to...

14 CFR 33.3 - General.

Code of Federal Regulations, 2010 CFR

2010-01-01

... 14 Aeronautics and Space 1 2010-01-01 2010-01-01 false General. 33.3 Section 33.3 Aeronautics and Space FEDERAL AVIATION ADMINISTRATION, DEPARTMENT OF TRANSPORTATION AIRCRAFT AIRWORTHINESS STANDARDS: AIRCRAFT ENGINES General § 33.3 General. Each applicant must show that the aircraft engine concerned meets...

14 CFR 33.3 - General.

Code of Federal Regulations, 2011 CFR

2011-01-01

... 14 Aeronautics and Space 1 2011-01-01 2011-01-01 false General. 33.3 Section 33.3 Aeronautics and Space FEDERAL AVIATION ADMINISTRATION, DEPARTMENT OF TRANSPORTATION AIRCRAFT AIRWORTHINESS STANDARDS: AIRCRAFT ENGINES General § 33.3 General. Each applicant must show that the aircraft engine concerned meets...

14 CFR 234.3 - Applicability.

Code of Federal Regulations, 2010 CFR

2010-01-01

... 14 Aeronautics and Space 4 2010-01-01 2010-01-01 false Applicability. 234.3 Section 234.3 Aeronautics and Space OFFICE OF THE SECRETARY, DEPARTMENT OF TRANSPORTATION (AVIATION PROCEEDINGS) ECONOMIC REGULATIONS AIRLINE SERVICE QUALITY PERFORMANCE REPORTS § 234.3 Applicability. This part applies to certain...

14 CFR 101.3 - Waivers.

Code of Federal Regulations, 2011 CFR

2011-01-01

... 14 Aeronautics and Space 2 2011-01-01 2011-01-01 false Waivers. 101.3 Section 101.3 Aeronautics and Space FEDERAL AVIATION ADMINISTRATION, DEPARTMENT OF TRANSPORTATION (CONTINUED) AIR TRAFFIC AND GENERAL OPERATING RULES MOORED BALLOONS, KITES, AMATEUR ROCKETS AND UNMANNED FREE BALLOONS General § 101.3...

14 CFR 101.3 - Waivers.

Code of Federal Regulations, 2010 CFR

2010-01-01

... 14 Aeronautics and Space 2 2010-01-01 2010-01-01 false Waivers. 101.3 Section 101.3 Aeronautics and Space FEDERAL AVIATION ADMINISTRATION, DEPARTMENT OF TRANSPORTATION (CONTINUED) AIR TRAFFIC AND GENERAL OPERATING RULES MOORED BALLOONS, KITES, AMATEUR ROCKETS AND UNMANNED FREE BALLOONS General § 101.3...

14 CFR 101.3 - Waivers.

Code of Federal Regulations, 2012 CFR

2012-01-01

... 14 Aeronautics and Space 2 2012-01-01 2012-01-01 false Waivers. 101.3 Section 101.3 Aeronautics and Space FEDERAL AVIATION ADMINISTRATION, DEPARTMENT OF TRANSPORTATION (CONTINUED) AIR TRAFFIC AND GENERAL OPERATING RULES MOORED BALLOONS, KITES, AMATEUR ROCKETS AND UNMANNED FREE BALLOONS General § 101.3...

14 CFR 101.3 - Waivers.

Code of Federal Regulations, 2014 CFR

2014-01-01

... 14 Aeronautics and Space 2 2014-01-01 2014-01-01 false Waivers. 101.3 Section 101.3 Aeronautics and Space FEDERAL AVIATION ADMINISTRATION, DEPARTMENT OF TRANSPORTATION (CONTINUED) AIR TRAFFIC AND GENERAL OPERATING RULES MOORED BALLOONS, KITES, AMATEUR ROCKETS AND UNMANNED FREE BALLOONS General § 101.3...

14 CFR 101.3 - Waivers.

Code of Federal Regulations, 2013 CFR

2013-01-01

... 14 Aeronautics and Space 2 2013-01-01 2013-01-01 false Waivers. 101.3 Section 101.3 Aeronautics and Space FEDERAL AVIATION ADMINISTRATION, DEPARTMENT OF TRANSPORTATION (CONTINUED) AIR TRAFFIC AND GENERAL OPERATING RULES MOORED BALLOONS, KITES, AMATEUR ROCKETS AND UNMANNED FREE BALLOONS General § 101.3...

14 CFR 77.3 - Standards.

Code of Federal Regulations, 2010 CFR

2010-01-01

... 14 Aeronautics and Space 2 2010-01-01 2010-01-01 false Standards. 77.3 Section 77.3 Aeronautics and Space FEDERAL AVIATION ADMINISTRATION, DEPARTMENT OF TRANSPORTATION (CONTINUED) AIRSPACE OBJECTS... section 16 of the Federal Airport Act; (3) Developing technical standards and guidance in the design and...

Interactions of L-3,5,3'-Triiodothyronine [corrected], Allopregnanolone, and Ivermectin with the GABAA Receptor: Evidence for Overlapping Intersubunit Binding Modes.

PubMed

Westergard, Thomas; Salari, Reza; Martin, Joseph V; Brannigan, Grace

2015-01-01

Structural mechanisms of modulation of γ-aminobutyric acid (GABA) type A receptors by neurosteroids and hormones remain unclear. The thyroid hormone L-3,5,3'-triiodothyronine (T3) inhibits GABAA receptors at micromolar concentrations and has common features with neurosteroids such as allopregnanolone (ALLOP). Here we use functional experiments on α2β1γ2 GABAA receptors expressed in Xenopus oocytes to detect competitive interactions between T3 and an agonist (ivermectin, IVM) with a crystallographically determined binding site at subunit interfaces in the transmembrane domain of a homologous receptor (glutamate-gated chloride channel, GluCl). T3 and ALLOP also show competitive effects, supporting the presence of both a T3 and ALLOP binding site at one or more subunit interfaces. Molecular dynamics (MD) simulations over 200 ns are used to investigate the dynamics and energetics of T3 in the identified intersubunit sites. In these simulations, T3 molecules occupying all intersubunit sites (with the exception of the α-β interface) display numerous energetically favorable conformations with multiple hydrogen bonding partners, including previously implicated polar/acidic sidechains and a structurally conserved deformation in the M1 backbone.

Identification of 14-3-3β in human gastric cancer cells and its potency as a diagnostic and prognostic biomarker.

PubMed

Tseng, Chien-Wei; Yang, Jyh-Chin; Chen, Chiung-Nien; Huang, Hsuan-Cheng; Chuang, Kai-Neng; Lin, Chen-Ching; Lai, Hong-Shiee; Lee, Po-Huang; Chang, King-Jen; Juan, Hsueh-Fen

2011-06-01

Gastric cancer is the second most common cause of cancer deaths worldwide and due to its poor prognosis, it is important that specific biomarkers are identified to enable its early detection. Through 2-D gel electrophoresis and MALDI-TOF-TOF-based proteomics approaches, we found that 14-3-3β, which was one of the proteins that were differentially expressed by 5-fluorouracil-treated gastric cancer SC-M1 cells, was upregulated in gastric cancer cells. 14-3-3β levels in tissues and serum were further validated in gastric cancer patients and controls. The results showed that 14-3-3β levels were elevated in tumor tissues (n=40) in comparison to normal tissues (n=40; p<0.01), and serum 14-3-3β levels in cancer patients (n=145) were also significantly higher than those in controls (n=63; p<0.0001). Elevated serum 14-3-3β levels highly correlated with the number of lymph node metastases, tumor size and a reduced survival rate. Moreover, overexpression of 14-3-3β enhanced the growth, invasiveness and migratory activities of tumor cells. Twenty-eight proteins involved in anti-apoptosis and tumor progression were also found to be differentially expressed in 14-3-3β-overexpressing gastric cancer cells. Overall, these results highlight the significance of 14-3-3β in gastric cancer cell progression and suggest that it has the potential to be used as a diagnostic and prognostic biomarker in gastric cancer. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

SMAD3 augments FoxO3-induced MuRF-1 promoter activity in a DNA-binding-dependent manner

PubMed Central

Bollinger, Lance M.; Witczak, Carol A.; Houmard, Joseph A.

2014-01-01

Muscle-specific RING finger-1 (MuRF-1), a ubiquitin ligase and key regulator of proteasome-dependent protein degradation, is highly expressed during skeletal muscle atrophy. The transcription factor forkhead box O3 (FoxO3) induces MuRF-1 expression, but the direct role of other major atrophy-related transcription factors, such as SMAD3, is largely unknown. The goal of this study was to determine whether SMAD3 individually regulates, or with FoxO3 coordinately regulates, MuRF-1 expression. In cultured myotubes or human embryonic kidney cells, MuRF-1 mRNA content and promoter activity were increased by FoxO3 but not by SMAD3 overexpression. However, FoxO3 and SMAD3 coexpression synergistically increased MuRF-1 mRNA and promoter activity. Mutation of the SMAD-binding element (SBE) in the proximal MuRF-1 promoter or overexpression of a SMAD3 DNA-binding mutant attenuated FoxO3-dependent MuRF-1 promoter activation, showing that SMAD binding to DNA is required for optimal activation of FoxO3-induced transcription of MuRF-1. Using chromatin immunoprecipitation, SMAD3 DNA binding increased FoxO3 abundance and SBE mutation reduced FoxO3 abundance on the MuRF-1 promoter. Furthermore, SMAD3 overexpression dose-dependently increased FoxO3 protein content, and coexpression of FoxO3 and SMAD3 synergistically increased FoxO-dependent gene transcription [assessed with a FoxO response element (FRE)-driven reporter]. Collectively, these results show that SMAD3 regulates transcription of MuRF-1 by increasing FoxO3 binding at a conserved FRE-SBE motif within the proximal promoter region, and by increasing FoxO3 protein content and transcriptional activity. These data are the first to indicate that two major transcription factors regulating protein degradation, FoxO3 and SMAD3, converge to coordinately and directly regulate transcription of MuRF-1. PMID:24920680

(CAG)(n)-hairpin DNA binds to Msh2-Msh3 and changes properties of mismatch recognition.

PubMed

Owen, Barbara A L; Yang, Zungyoon; Lai, Maoyi; Gajec, Maciej; Gajek, Maciez; Badger, John D; Hayes, Jeffrey J; Edelmann, Winfried; Kucherlapati, Raju; Wilson, Teresa M; McMurray, Cynthia T

2005-08-01

Cells have evolved sophisticated DNA repair systems to correct damaged DNA. However, the human DNA mismatch repair protein Msh2-Msh3 is involved in the process of trinucleotide (CNG) DNA expansion rather than repair. Using purified protein and synthetic DNA substrates, we show that Msh2-Msh3 binds to CAG-hairpin DNA, a prime candidate for an expansion intermediate. CAG-hairpin binding inhibits the ATPase activity of Msh2-Msh3 and alters both nucleotide (ADP and ATP) affinity and binding interfaces between protein and DNA. These changes in Msh2-Msh3 function depend on the presence of A.A mispaired bases in the stem of the hairpin and on the hairpin DNA structure per se. These studies identify critical functional defects in the Msh2-Msh3-CAG hairpin complex that could misdirect the DNA repair process.

14 CFR 67.3 - Issue.

Code of Federal Regulations, 2014 CFR

2014-01-01

... 14 Aeronautics and Space 2 2014-01-01 2014-01-01 false Issue. 67.3 Section 67.3 Aeronautics and Space FEDERAL AVIATION ADMINISTRATION, DEPARTMENT OF TRANSPORTATION (CONTINUED) AIRMEN MEDICAL STANDARDS AND CERTIFICATION General § 67.3 Issue. A person who meets the medical standards prescribed in this...

14 CFR 67.3 - Issue.

Code of Federal Regulations, 2010 CFR

2010-01-01

... 14 Aeronautics and Space 2 2010-01-01 2010-01-01 false Issue. 67.3 Section 67.3 Aeronautics and Space FEDERAL AVIATION ADMINISTRATION, DEPARTMENT OF TRANSPORTATION (CONTINUED) AIRMEN MEDICAL STANDARDS AND CERTIFICATION General § 67.3 Issue. A person who meets the medical standards prescribed in this...

14 CFR 67.3 - Issue.

Code of Federal Regulations, 2013 CFR

2013-01-01

... 14 Aeronautics and Space 2 2013-01-01 2013-01-01 false Issue. 67.3 Section 67.3 Aeronautics and Space FEDERAL AVIATION ADMINISTRATION, DEPARTMENT OF TRANSPORTATION (CONTINUED) AIRMEN MEDICAL STANDARDS AND CERTIFICATION General § 67.3 Issue. A person who meets the medical standards prescribed in this...

14 CFR 67.3 - Issue.

Code of Federal Regulations, 2012 CFR

2012-01-01

... 14 Aeronautics and Space 2 2012-01-01 2012-01-01 false Issue. 67.3 Section 67.3 Aeronautics and Space FEDERAL AVIATION ADMINISTRATION, DEPARTMENT OF TRANSPORTATION (CONTINUED) AIRMEN MEDICAL STANDARDS AND CERTIFICATION General § 67.3 Issue. A person who meets the medical standards prescribed in this...

14 CFR 67.3 - Issue.

Code of Federal Regulations, 2011 CFR

2011-01-01

... 14 Aeronautics and Space 2 2011-01-01 2011-01-01 false Issue. 67.3 Section 67.3 Aeronautics and Space FEDERAL AVIATION ADMINISTRATION, DEPARTMENT OF TRANSPORTATION (CONTINUED) AIRMEN MEDICAL STANDARDS AND CERTIFICATION General § 67.3 Issue. A person who meets the medical standards prescribed in this...

14 CFR 255.3 - Definitions.

Code of Federal Regulations, 2013 CFR

2013-01-01

... REGULATIONS AIRLINE COMPUTER RESERVATIONS SYSTEMS § 255.3 Definitions. Availability means information provided... 14 Aeronautics and Space 4 2013-01-01 2013-01-01 false Definitions. 255.3 Section 255.3... presentation of carrier schedules, fares, rules or availability to a subscriber by means of a computer terminal...

14 CFR 255.3 - Definitions.

Code of Federal Regulations, 2010 CFR

2010-01-01

... REGULATIONS AIRLINE COMPUTER RESERVATIONS SYSTEMS § 255.3 Definitions. Availability means information provided... 14 Aeronautics and Space 4 2010-01-01 2010-01-01 false Definitions. 255.3 Section 255.3... presentation of carrier schedules, fares, rules or availability to a subscriber by means of a computer terminal...

14 CFR 255.3 - Definitions.

Code of Federal Regulations, 2014 CFR

2014-01-01

... REGULATIONS AIRLINE COMPUTER RESERVATIONS SYSTEMS § 255.3 Definitions. Availability means information provided... 14 Aeronautics and Space 4 2014-01-01 2014-01-01 false Definitions. 255.3 Section 255.3... presentation of carrier schedules, fares, rules or availability to a subscriber by means of a computer terminal...

14 CFR 255.3 - Definitions.

Code of Federal Regulations, 2012 CFR

2012-01-01

... REGULATIONS AIRLINE COMPUTER RESERVATIONS SYSTEMS § 255.3 Definitions. Availability means information provided... 14 Aeronautics and Space 4 2012-01-01 2012-01-01 false Definitions. 255.3 Section 255.3... presentation of carrier schedules, fares, rules or availability to a subscriber by means of a computer terminal...

14 CFR 255.3 - Definitions.

Code of Federal Regulations, 2011 CFR

2011-01-01

... REGULATIONS AIRLINE COMPUTER RESERVATIONS SYSTEMS § 255.3 Definitions. Availability means information provided... 14 Aeronautics and Space 4 2011-01-01 2011-01-01 false Definitions. 255.3 Section 255.3... presentation of carrier schedules, fares, rules or availability to a subscriber by means of a computer terminal...

Structural basis of nSH2 regulation and lipid binding in PI3Kα.

PubMed

Miller, Michelle S; Schmidt-Kittler, Oleg; Bolduc, David M; Brower, Evan T; Chaves-Moreira, Daniele; Allaire, Marc; Kinzler, Kenneth W; Jennings, Ian G; Thompson, Philip E; Cole, Philip A; Amzel, L Mario; Vogelstein, Bert; Gabelli, Sandra B

2014-07-30

We report two crystal structures of the wild-type phosphatidylinositol 3-kinase α (PI3Kα) heterodimer refined to 2.9 Å and 3.4 Å resolution: the first as the free enzyme, the second in complex with the lipid substrate, diC4-PIP₂, respectively. The first structure shows key interactions of the N-terminal SH2 domain (nSH2) and iSH2 with the activation loop that suggest a mechanism by which the enzyme is inhibited in its basal state. In the second structure, the lipid substrate binds in a positively charged pocket adjacent to the ATP-binding site, bordered by the P-loop, the activation loop and the iSH2 domain. An additional lipid-binding site was identified at the interface of the ABD, iSH2 and kinase domains. The ability of PI3Kα to bind an additional PIP₂ molecule was confirmed in vitro by fluorescence quenching experiments. The crystal structures reveal key differences in the way the nSH2 domain interacts with wild-type p110α and with the oncogenic mutant p110αH1047R. Increased buried surface area and two unique salt-bridges observed only in the wild-type structure suggest tighter inhibition in the wild-type PI3Kα than in the oncogenic mutant. These differences may be partially responsible for the increased basal lipid kinase activity and increased membrane binding of the oncogenic mutant.

Characterization of (/sup 3/H)forskolin binding sites in the iris-ciliary body of the albino rabbit

DOE Office of Scientific and Technical Information (OSTI.GOV)

Goldman, M.E.; Mallorga, P.; Pettibone, D.J.

1988-01-01

(/sup 3/H)forskolin binding sites were identified using membranes prepared from the iris-ciliary body of adult, albino rabbits. Scatchard analysis of saturation binding experiments demonstrated that (/sup 3/H)forskolin bound to a single population of high affinity sites. The K/sub d/ and B/sub max/ values were 8.7 +- 0.9 nM and 119.0 +- 30.9 fmolmg prot. using membranes prepared from frozen tissue and 17.0 +- 6.2 nM and 184.4 +- 47.2 fmolmg prot. using fresh tissue. The binding of (/sup 3/H)forskolin was magnesium-dependent. The B/sub max/ was enhanced by sodium fluoride and Gpp(NH)p, a nonhydrolyzable guanine nucleotide analog. Forskolin was the mostmore » potent inhibitor of (/sup 3/H)forskolin binding; two commercially-available analogs were weaker inhibitors. In an adenylate cyclase assay, there was the same rank order of potency to enhance enzyme activity. Based upon binding affinities, magnesium-dependence, sensitivity to sodium fluoride and Gpp(NH)p, rank order of potencies of analogs and correlation of binding with adenylate cyclase activity, these studies suggest that the (/sup 3/H)forskolin binding site in the iris-ciliary body is similar to the binding site in other tissues« less

Changes in Brain 14-3-3 Proteins in Response to Insulin Resistance Induced by a High Palatable Diet.

PubMed

Bock, Hugo; Zimmer, Aline Rigon; Zimmer, Eduardo Rigon; de Souza, Diogo Onofre Gomes; Portela, Luis Valmor Cruz; Saraiva-Pereira, Maria Luiza

2015-08-01

The 14-3-3 protein family takes part in a wide range of cellular processes and is expressed in all eukaryotic organisms. In mammals, seven isoforms (β, ε, η, γ, τ, ζ, and σ) have been identified. 14-3-3 proteins are suggested to modulate the insulin-signaling cascade in the brain. The aim of this study was to investigate whether insulin resistance state induced by high palatable diet modulates expression of the 14-3-3 proteins in brain. Wistar male rats (n = 8) were divided into two experimental groups: insulin resistant (IR), induced by high palatable diet, and control (CO) group. Biochemical parameters (glucose tolerance test and plasma lipid profile) were evaluated after 130 days. Brain structures (cortex and hippocampus) were dissected for evaluation of messenger RNA (mRNA) and protein levels of different 14-3-3 proteins. Statistical analyses included Student t test and Pearson correlation. Significant decrease was observed in Ywhah and in Ywahq mRNA levels in the cortex of IR group, while no changes were observed in the hippocampus. Significant increase of θ isoform was observed in hippocampus IR group by immunodetection, while no differences were detected in the remaining isoforms. Inverse correlation was observed between blood glucose levels in cortex IR group and both Ywhah and Ywhaq mRNA levels. Protein levels of Creb and phosphatidylinositide 3-kinases (PI3K) showed to be increased in the hippocampus. These alterations may be due to a compensatory effect of impaired insulin signaling. We demonstrated differential expression of 14-3-3 isoforms throughout brain regions of rats with IR. As a whole, our results indicate that brain 14-3-3 levels are influenced by different diets.

14 CFR 95.3 - Symbols.

Code of Federal Regulations, 2013 CFR

2013-01-01

... 14 Aeronautics and Space 2 2013-01-01 2013-01-01 false Symbols. 95.3 Section 95.3 Aeronautics and Space FEDERAL AVIATION ADMINISTRATION, DEPARTMENT OF TRANSPORTATION (CONTINUED) AIR TRAFFIC AND GENERAL OPERATING RULES IFR ALTITUDES General § 95.3 Symbols. For the purposes of this part— (a) COP means...

14 CFR 95.3 - Symbols.

Code of Federal Regulations, 2012 CFR

2012-01-01

... 14 Aeronautics and Space 2 2012-01-01 2012-01-01 false Symbols. 95.3 Section 95.3 Aeronautics and Space FEDERAL AVIATION ADMINISTRATION, DEPARTMENT OF TRANSPORTATION (CONTINUED) AIR TRAFFIC AND GENERAL OPERATING RULES IFR ALTITUDES General § 95.3 Symbols. For the purposes of this part— (a) COP means...

14 CFR 95.3 - Symbols.

Code of Federal Regulations, 2014 CFR

2014-01-01

... 14 Aeronautics and Space 2 2014-01-01 2014-01-01 false Symbols. 95.3 Section 95.3 Aeronautics and Space FEDERAL AVIATION ADMINISTRATION, DEPARTMENT OF TRANSPORTATION (CONTINUED) AIR TRAFFIC AND GENERAL OPERATING RULES IFR ALTITUDES General § 95.3 Symbols. For the purposes of this part— (a) COP means...

14 CFR 95.3 - Symbols.

Code of Federal Regulations, 2011 CFR

2011-01-01

... 14 Aeronautics and Space 2 2011-01-01 2011-01-01 false Symbols. 95.3 Section 95.3 Aeronautics and Space FEDERAL AVIATION ADMINISTRATION, DEPARTMENT OF TRANSPORTATION (CONTINUED) AIR TRAFFIC AND GENERAL OPERATING RULES IFR ALTITUDES General § 95.3 Symbols. For the purposes of this part— (a) COP means...

14 CFR 95.3 - Symbols.

Code of Federal Regulations, 2010 CFR

2010-01-01

... 14 Aeronautics and Space 2 2010-01-01 2010-01-01 false Symbols. 95.3 Section 95.3 Aeronautics and Space FEDERAL AVIATION ADMINISTRATION, DEPARTMENT OF TRANSPORTATION (CONTINUED) AIR TRAFFIC AND GENERAL OPERATING RULES IFR ALTITUDES General § 95.3 Symbols. For the purposes of this part— (a) COP means...

14 CFR 298.3 - Classification.

Code of Federal Regulations, 2013 CFR

2013-01-01

... 14 Aeronautics and Space 4 2013-01-01 2013-01-01 false Classification. 298.3 Section 298.3... REGULATIONS EXEMPTIONS FOR AIR TAXI AND COMMUTER AIR CARRIER OPERATIONS General § 298.3 Classification. (a) There is hereby established a classification of air carriers, designated as “air taxi operators,” which...

14 CFR 298.3 - Classification.

Code of Federal Regulations, 2012 CFR

2012-01-01

... 14 Aeronautics and Space 4 2012-01-01 2012-01-01 false Classification. 298.3 Section 298.3... REGULATIONS EXEMPTIONS FOR AIR TAXI AND COMMUTER AIR CARRIER OPERATIONS General § 298.3 Classification. (a) There is hereby established a classification of air carriers, designated as “air taxi operators,” which...

14 CFR 298.3 - Classification.

Code of Federal Regulations, 2014 CFR

2014-01-01

... 14 Aeronautics and Space 4 2014-01-01 2014-01-01 false Classification. 298.3 Section 298.3... REGULATIONS EXEMPTIONS FOR AIR TAXI AND COMMUTER AIR CARRIER OPERATIONS General § 298.3 Classification. (a) There is hereby established a classification of air carriers, designated as “air taxi operators,” which...

14 CFR 298.3 - Classification.

Code of Federal Regulations, 2010 CFR

2010-01-01

... 14 Aeronautics and Space 4 2010-01-01 2010-01-01 false Classification. 298.3 Section 298.3... REGULATIONS EXEMPTIONS FOR AIR TAXI AND COMMUTER AIR CARRIER OPERATIONS General § 298.3 Classification. (a) There is hereby established a classification of air carriers, designated as “air taxi operators,” which...

14 CFR 298.3 - Classification.

Code of Federal Regulations, 2011 CFR

2011-01-01

... 14 Aeronautics and Space 4 2011-01-01 2011-01-01 false Classification. 298.3 Section 298.3... REGULATIONS EXEMPTIONS FOR AIR TAXI AND COMMUTER AIR CARRIER OPERATIONS General § 298.3 Classification. (a) There is hereby established a classification of air carriers, designated as “air taxi operators,” which...

Combining peptide recognition specificity and context information for the prediction of the 14-3-3-mediated interactome in S. cerevisiae and H. sapiens.

PubMed

Panni, Simona; Montecchi-Palazzi, Luisa; Kiemer, Lars; Cabibbo, Andrea; Paoluzi, Serena; Santonico, Elena; Landgraf, Christiane; Volkmer-Engert, Rudolf; Bachi, Angela; Castagnoli, Luisa; Cesareni, Gianni

2011-01-01

Large-scale interaction studies contribute the largest fraction of protein interactions information in databases. However, co-purification of non-specific or indirect ligands, often results in data sets that are affected by a considerable number of false positives. For the fraction of interactions mediated by short linear peptides, we present here a combined experimental and computational strategy for ranking the reliability of the inferred partners. We apply this strategy to the family of 14-3-3 domains. We have first characterized the recognition specificity of this domain family, largely confirming the results of previous analyses, while revealing new features of the preferred sequence context of 14-3-3 phospho-peptide partners. Notably, a proline next to the carboxy side of the phospho-amino acid functions as a potent inhibitor of 14-3-3 binding. The position-specific information about residue preference was encoded in a scoring matrix and two regular expressions. The integration of these three features in a single predictive model outperforms publicly available prediction tools. Next we have combined, by a naïve Bayesian approach, these "peptide features" with "protein features", such as protein co-expression and co-localization. Our approach provides an orthogonal reliability assessment and maps with high confidence the 14-3-3 peptide target on the partner proteins. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Investigation of naphthofuran moiety as potential dual inhibitor against BACE-1 and GSK-3β: molecular dynamics simulations, binding energy, and network analysis to identify first-in-class dual inhibitors against Alzheimer's disease.

PubMed

Kumar, Akhil; Srivastava, Gaurava; Srivastava, Swati; Verma, Seema; Negi, Arvind S; Sharma, Ashok

2017-08-01

BACE-1 and GSK-3β are potential therapeutic drug targets for Alzheimer's disease. Recently, both the targets received attention for designing dual inhibitors for Alzheimer's disease. Until now, only two-scaffold triazinone and curcumin have been reported as BACE-1 and GSK-3β dual inhibitors. Docking, molecular dynamics, clustering, binding energy, and network analysis of triazinone derivatives with BACE-1 and GSK-3β was performed to get molecular insight into the first reported dual inhibitor. Further, we designed and evaluated a naphthofuran series for its ability to inhibit BACE-1 and GSK-3β with the computational approaches. Docking study of naphthofuran series showed a good binding affinity towards both the targets. Molecular dynamics, binding energy, and network analysis were performed to compare their binding with the targets and amino acids responsible for binding. Naphthofuran series derivatives showed good interaction within the active site residues of both of the targets. Hydrogen bond occupancy and binding energy suggested strong binding with the targets. Dual-inhibitor binding was mostly governed by the hydrophobic interactions for both of the targets. Per residue energy decomposition and network analysis identified the key residues involved in the binding and inhibiting BACE-1 and GSK-3β. The results indicated that naphthofuran series derivative 11 may be a promising first-in-class dual inhibitor against BACE-1 and GSK-3β. This naphthofuran series may be further explored to design better dual inhibitors. Graphical abstract Naphthofuran derivative as a dual inhibitor for BACE-1 and GSK-3β.

The diversity of H3 loops determines the antigen-binding tendencies of antibody CDR loops.

PubMed

Tsuchiya, Yuko; Mizuguchi, Kenji

2016-04-01

Of the complementarity-determining regions (CDRs) of antibodies, H3 loops, with varying amino acid sequences and loop lengths, adopt particularly diverse loop conformations. The diversity of H3 conformations produces an array of antigen recognition patterns involving all the CDRs, in which the residue positions actually in contact with the antigen vary considerably. Therefore, for a deeper understanding of antigen recognition, it is necessary to relate the sequence and structural properties of each residue position in each CDR loop to its ability to bind antigens. In this study, we proposed a new method for characterizing the structural features of the CDR loops and obtained the antigen-binding ability of each residue position in each CDR loop. This analysis led to a simple set of rules for identifying probable antigen-binding residues. We also found that the diversity of H3 loop lengths and conformations affects the antigen-binding tendencies of all the CDR loops. © 2016 The Protein Society.

Proteomic analysis identifies a novel function for galectin-3 in the cell entry of parvovirus.

PubMed

Garcin, Pierre; Cohen, Sarah; Terpstra, Sanne; Kelly, Isabelle; Foster, Leonard J; Panté, Nelly

2013-02-21

Cellular factors associated with the parvovirus minute virus of mice (MVM) during infection are thought to play important roles in the MVM life cycle but only a few of these have been identified. Here we used a proteomic-based approach in order to identify host-binding partners of MVM. Using purified MVM as bait for immunoprecipitation assays, a total of 150 proteins were identified in MVM immunoprecipitates by quantitative liquid chromatography-tandem mass spectrometry. Galectin-3 was one of six proteins showing a statistically significant enrichment across replicates. Small interfering RNA depletion studies revealed an important role for galectin-3 in MVM endocytosis and infectivity in LA9 mouse fibroblast cells. Galectin-3-depleted cells were less susceptible to MVM infection than control cells and showed a significant reduction of MVM cellular uptake, but not of MVM binding to the cell surface. Our results indicate an important role for galectin-3 in the cellular uptake of MVM. We propose that galectin-3 facilitates the access of MVM to its receptor(s) at the plasma membrane and in this way promotes MVM endocytosis. Copyright © 2012 Elsevier B.V. All rights reserved.

Increased /sup 3/H-spiperone binding sites in mesolimbic area related to methamphetamine-induced behavioral hypersensitivity

DOE Office of Scientific and Technical Information (OSTI.GOV)

Akiyama, K.; Sato, M.; Otsuki, S.

1982-02-01

The specific /sup 3/H-spiperone binding to membrane homogenates of the striatum, mesolimbic area, and frontal cortex was examined in two groups of rats pretreated once daily with saline or 4 mg/kg of methamphetamine (MAP) for 14 days. At 7 days following cessation of chronic pretreatment, all rats received an injection of 4 mg/kg of MAP and were decapitated 1 hr after the injection. In the chronic saline-pretreatment group, the single administration of MAP induced significant changes in the number (Bmax) of specific /sup 3/H-spiperone binding sites (a decrease in the striatum and an increase in the mesolimbic area and frontalmore » cortex), but no significant changes in the affinity (KD) in any brain area. The chronic MAP pretreatment markedly augmented the changes in Bmax in the striatum and mesolimbic area. The increase in specific /sup 3/H-spiperone binding sites in the mesolimbic area is discussed in relation to MAP-induced behavioral hypersensitivity.« less

Pretreatment 14-3-3 epsilon level is predictive for advanced extranodal NK/T cell lymphoma therapeutic response to asparaginase-based chemotherapy.

PubMed

Qiu, Yajuan; Zhou, Zhiyuan; Li, Zhaoming; Lu, Lisha; Li, Ling; Li, Xin; Wang, Xinhua; Zhang, Mingzhi

2017-03-01

The aim of the present study was to identify the potential relevant biomarkers to predict the therapeutic response of advanced extranodal natural killer/T cell lymphoma(ENKTL) treated with asparaginase-based treatment. Proteomic technology is used to identify differentially expressed proteins between chemotherapy-resistant and chemotherapy-sensitive patients. Then enzyme-linked immunosorbent assay is used to validate the predictive value of selective biomarkers. A total of 61 upregulated and 22 downregulated proteins are identified in chemotherapy-resistant patients compared with chemotherapy-sensitive patients. Furthermore, they validated that pretreatment high level 14-3-3 epsilon(ε)(≥61.95 ng/mL, 84.0 and 95.2% for sensitivity and specificity, respectively) is associated with poor 2-year overall survival (OS) (5.3 vs 68.8%, p<0.0001) and PFS (4.5 vs 76.9%, p<0.0001). In multivariate survival analysis, pretreatment high level 14-3-3 epsilon significantly is correlated with both inferior OS (p = 0.033) and PFS (p = 0.005). These findings indicate that pretreatment high level 14-3-3 epsilon is an independent predictor of chemotherapy-resistance and poor prognosis for patients with advanced ENKTL in the era of asparaginase. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Structure and Calcium Binding Properties of a Neuronal Calcium-Myristoyl Switch Protein, Visinin-Like Protein 3.

PubMed

Li, Congmin; Lim, Sunghyuk; Braunewell, Karl H; Ames, James B

2016-01-01

Visinin-like protein 3 (VILIP-3) belongs to a family of Ca2+-myristoyl switch proteins that regulate signal transduction in the brain and retina. Here we analyze Ca2+ binding, characterize Ca2+-induced conformational changes, and determine the NMR structure of myristoylated VILIP-3. Three Ca2+ bind cooperatively to VILIP-3 at EF2, EF3 and EF4 (KD = 0.52 μM and Hill slope of 1.8). NMR assignments, mutagenesis and structural analysis indicate that the covalently attached myristoyl group is solvent exposed in Ca2+-bound VILIP-3, whereas Ca2+-free VILIP-3 contains a sequestered myristoyl group that interacts with protein residues (E26, Y64, V68), which are distinct from myristate contacts seen in other Ca2+-myristoyl switch proteins. The myristoyl group in VILIP-3 forms an unusual L-shaped structure that places the C14 methyl group inside a shallow protein groove, in contrast to the much deeper myristoyl binding pockets observed for recoverin, NCS-1 and GCAP1. Thus, the myristoylated VILIP-3 protein structure determined in this study is quite different from those of other known myristoyl switch proteins (recoverin, NCS-1, and GCAP1). We propose that myristoylation serves to fine tune the three-dimensional structures of neuronal calcium sensor proteins as a means of generating functional diversity.

Cadherin-related family member 3, a childhood asthma susceptibility gene product, mediates rhinovirus C binding and replication.

PubMed

Bochkov, Yury A; Watters, Kelly; Ashraf, Shamaila; Griggs, Theodor F; Devries, Mark K; Jackson, Daniel J; Palmenberg, Ann C; Gern, James E

2015-04-28

Members of rhinovirus C (RV-C) species are more likely to cause wheezing illnesses and asthma exacerbations compared with other rhinoviruses. The cellular receptor for these viruses was heretofore unknown. We report here that expression of human cadherin-related family member 3 (CDHR3) enables the cells normally unsusceptible to RV-C infection to support both virus binding and replication. A coding single nucleotide polymorphism (rs6967330, C529Y) was previously linked to greater cell-surface expression of CDHR3 protein, and an increased risk of wheezing illnesses and hospitalizations for childhood asthma. Compared with wild-type CDHR3, cells transfected with the CDHR3-Y529 variant had about 10-fold increases in RV-C binding and progeny yields. We developed a transduced HeLa cell line (HeLa-E8) stably expressing CDHR3-Y529 that supports RV-C propagation in vitro. Modeling of CDHR3 structure identified potential binding sites that could impact the virus surface in regions that are highly conserved among all RV-C types. Our findings identify that the asthma susceptibility gene product CDHR3 mediates RV-C entry into host cells, and suggest that rs6967330 mutation could be a risk factor for RV-C wheezing illnesses.

Investigating the clinical potential for 14-3-3 zeta protein to serve as a biomarker for epithelial ovarian cancer

PubMed Central

2013-01-01

Objective Recently, 14-3-3 zeta protein was identified as a potential serum biomarker of epithelial ovarian cancer (EOC). The goal of this study was to investigate the clinical potential of 14-3-3 zeta protein for monitoring EOC progression compared with CA-125 and HE4. Design Prospective follow-up study. Setting University of Pecs Medical Center Department of Obstetrics and Gynecology/Oncology (Pecs, Hungary). Population Thirteen EOC patients with advanced stage (FIGO IIb-IIIc) epithelial ovarian cancer that underwent radical surgery and received six consecutive cycles of first line chemotherapy (paclitaxel, carboplatin) in 21-day intervals. Methods Pre- and post-chemotherapy computed tomography (CT) scans were performed. Serum levels of CA-125, HE4, and 14-3-3 zeta protein were detected by enzyme-linked immunosorbent assay (ELISA) and quantitative electrochemiluminescence assay (ECLIA). Main outcome measures Serum levels of CA-125, HE4, and 14-3-3 zeta protein, as well as lesion size according to pre- and post-chemotherapy CT scans. Results Serum levels of CA-125 and HE4 were found to significantly decrease following chemotherapy, and this was consistent with the decrease in lesion size detected post-chemotherapy. In contrast, 14-3-3 zeta protein levels did not significantly differ in healthy postmenopausal patients versus EOC patients. Conclusions Determination of CA-125 and HE4 serum levels for the determination of the risk of ovarian malignancy algorithm (ROMA) represents a useful tool for the prediction of chemotherapy efficacy for EOC patients. However, levels of 14-3-3 zeta protein were not found to vary significantly as a consequence of treatment. Therefore we question if 14-3-3 zeta protein is a reliable biomarker, which correlates with the clinical behavior of EOC. PMID:24238270

Experimental and Computational Studies of Binding of Dinitrogen, Nitriles, Azides, Diazoalkanes, Pyridine and Pyrazines to M(PR3)2(CO)3 (M=Mo, W; R=Me, iPr)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hoff, C.D.; Muckerman, J.; Achord, P.

2009-08-17

The enthalpies of binding of a number of N-donor ligands to the complex Mo(P{sup i}Pr{sub 3}){sub 2}(CO){sub 3} in toluene have been determined by solution calorimetry and equilibrium measurements. The measured binding enthalpies span a range of {approx}10 kcal mol{sup -1}: {Delta}H{sub binding} = -8.8 {+-} 1.2 (N{sub 2}-Mo(P{sup i}Pr{sub 3}){sub 2}(CO){sub 3}); -10.3 {+-} 0.8 (N{sub 2}); -11.2 {+-} 0.4 (AdN{sub 3} (Ad = 1-adamantyl)); -13.8 {+-} 0.5 (N{sub 2}CHSiMe{sub 3}); -14.9 {+-} 0.9 (pyrazine = pz); -14.8 {+-} 0.6 (2,6-Me{sub 2}pz); -15.5 {+-} 1.8 (Me{sub 2}NCN); -16.6 {+-} 0.4 (CH{sub 3}CN); -17.0 {+-} 0.4 (pyridine); -17.5 {+-} 0.8more » ([4-CH{sub 3}pz][PF{sub 6}] (in tetrahydrofuran)); -17.6 {+-} 0.4 (C{sub 6}H{sub 5}CN); -18.6 {+-} 1.8 (N{sub 2}CHC(=O)OEt); and -19.3 {+-} 2.5 kcal mol{sup -1} (pz)Mo(P{sup i}Pr{sub 3}){sub 2}(CO){sub 3}). The value for the isonitrile AdNC (-29.0 {+-} 0.3) is 12.3 kcal mol{sup -1} more exothermic than that of the nitrile AdCN (-16.7 {+-} 0.6 kcal mol{sup -1}). The enthalpies of binding of a range of arene nitrile ligands were also studied, and remarkably, most nitrile complexes were clustered within a 1 kcal mol{sup -1} range despite dramatic color changes and variation of v{sub CN}. Computed structural and spectroscopic parameters for the complexes Mo(P{sup i}Pr{sub 3}){sub 2}(CO){sub 3}L are in good agreement with experimental data. Computed binding enthalpies for Mo(P{sup i}Pr{sub 3}){sub 2}(CO){sub 3}L exhibit considerable scatter and are generally smaller compared to the experimental values, but relative agreement is reasonable. Computed enthalpies of binding using a larger basis set for Mo(PMe{sub 3}){sub 2}(CO){sub 3}L show a better fit to experimental data than that for Mo(P{sup i}Pr{sub 3}){sub 2}(CO){sub 3}L using a smaller basis set. Crystal structures of Mo(P{sup i}Pr{sub 3}){sub 2}(CO){sub 3}(AdCN), W(P{sup i}Pr{sub 3}){sub 2}(CO){sub 3}(Me{sub 2}NCN), W(P{sup i}Pr{sub 3}){sub 2}(CO){sub 3}(2,6-F{sub 2}C

Inhibition of /sup 3/H-leukotriene D4 binding to guinea pig lung receptors by the novel leukotriene antagonist ICI 198,615

DOE Office of Scientific and Technical Information (OSTI.GOV)

Aharony, D.; Falcone, R.C.; Krell, R.D.

1987-12-01

The specific binding of (/sup 3/H)5(S)hydroxy-6(R)-S-cysteinylglycyl -7(E),9(E),11(Z),14(Z)-eicosatetraenoic acid ((/sup 3/H)LTD4) to receptors on guinea pig lung parenchymal membranes and its inhibition by ICI 198,615, a representative example of a new class of leukotriene antagonists, was characterized by a receptor-ligand binding assay. (/sup 3/H)LTD4 bound specifically and rapidly (Kon = 0.29 +/- 0.6 nM-1.min-1) reaching equilibrium within 15 min. The rate of binding was greatly inhibited in the presence of ICI 198,615. Excess LTD4 or ICI 198,615 slowly (t1/2 = 20 min) dissociated about 70% of the receptor-bound (/sup 3/H)LTD4, whereas in combination with GTP analogs, both induced a rapid (t1/2more » less than 5 min) and full dissociation. Equilibrium saturation analysis of (/sup 3/H)LTD4 binding demonstrated a saturable (Bmax = 1014 +/- 174 fmol/mg) and high affinity (Kd = 0.43 +/- 0.09 nM) binding site. A high degree of stereoselectivity was demonstrated with inhibition of binding by the stereoisomers of LTD4: S,R much greater than R,R greater than R,S much greater than S,S. The rank order for inhibition of binding by peptide leukotriene was: LTD4 greater than 5(S)-hydroxy-6(R)-S-cysteinyl-7(E),9(E),11(Z),14(Z)-eicosatetraenoic acid much greater than 5(S)hydroxy-6(R)-S-glutathionyl-7(E),9(E),11(Z),14(Z)-eicosatetraenoic acid (potency ratios were: 1:4:590). In competition assays, ICI 198,615 competitively inhibited binding of (/sup 3/H)LTD4 (Ki = 0.27 +/- 0.16 nM) and was 2300-fold and 3100-fold more potent than LY171883 or FPL55712. These data, together with results obtained previously in functional receptor assays, illustrate that this new class of leukotriene antagonists are the most potent and selective competitive antagonists of LTD4 receptors yet described.« less

Binding efficiency of recombinant collagen-binding basic fibroblast growth factors (CBD-bFGFs) and their promotion for NIH-3T3 cell proliferation.

PubMed

Wu, Zhenxu; Zhou, Yulai; Chen, Li; Hu, Mingxin; Wang, Yu; Li, Linlong; Wang, Zongliang; Zhang, Peibiao

2018-03-01

The recombinant basic fibroblast growth factor (bFGF) containing collagen-binding domain (CBD) has been found to be a potential therapeutic factor in tissue regeneration. However, its binding efficiency and quantification remain uncertain. In this research, massive recombinant bFGFs with good bioactivity for enhancing the proliferation of NIH-3T3 cells were achieved. An ELISA-based quantitative method was set up to investigate the binding efficiency of CBD-bFGFs on collagen films. It indicated that the CBDs significantly increased the collagen-binding ability of bFGF (P < .05), with the optimum binding condition first determined to be in the pH range of 7.5-9.5 (P < .05). Then, the relevant equations to calculate the binding density of bFGF, C-bFGF, and V-bFGF were acquired. Analysis confirmed that the bioactivity of immobilized bFGFs was well correlated with the density of growth factor on collagen films. Based on this research, the density of growth factor is a logical and applicable dosage unit for quantification of binding efficiency of growth factors, rather than traditional concentration of soluble growth factors in tissue engineering applications. © 2018 Wiley Periodicals, Inc.

Design and synthesis of highly potent benzodiazepine gamma-secretase inhibitors: preparation of (2S,3R)-3-(3,4-difluorophenyl)-2-(4-fluorophenyl)-4- hydroxy-N-((3S)-1-methyl-2-oxo-5- phenyl-2,3-dihydro-1H-benzo[e][1,4]-diazepin-3-yl)butyramide by use of an asymmetric Ireland-Claisen rearrangement.

PubMed

Churcher, Ian; Williams, Susie; Kerrad, Sonia; Harrison, Timothy; Castro, José L; Shearman, Mark S; Lewis, Huw D; Clarke, Earl E; Wrigley, Jonathan D J; Beher, Dirk; Tang, Yui S; Liu, Wensheng

2003-06-05

Novel benzodiazepine-containing gamma-secretase inhibitors for potential use in Alzheimer's disease have been designed that incorporate a substituted hydrocinnamide C-3 side chain. A syn combination of alpha-alkyl or aryl and beta-hydroxy or hydroxymethyl substituents was shown to give highly potent compounds. In particular, (2S,3R)-3-(3,4-difluorophenyl)-2-(4-fluorophenyl)-4-hydroxy-N-((3S)-2-oxo-5-phenyl-2,3-dihydro-1H-benzo[e][1,4]diazepin-3-yl)butyramide (34) demonstrated excellent in vitro potency (IC(50) = 0.06 nM). 34 could also be selectively methylated to give [(3)H]-28, which is of use in radioligand binding assays.

Design and Synthesis of 1-(3-(dimethylamino)propyl)-1-(4-fluorophenyl)-1,3-dihydroisobenzofuran-5-carbonitrile (Citalopram) Analogues as Novel Probes for the Serotonin Transporter S1 and S2 Binding Sites

PubMed Central

Banala, Ashwini K.; Zhang, Peng; Plenge, Per; Cyriac, George; Kopajtic, Theresa; Katz, Jonathan L.; Loland, Claus Juul; Newman, Amy Hauck

2013-01-01

The serotonin transporter (SERT) is the primary target for antidepressant drugs. The existence of a high affinity primary orthosteric binding site (S1) and a low affinity secondary site (S2) has been described and their relation to antidepressant pharmacology has been debated. Herein, structural modifications to the N-, 4, 5, and 4’-positions of (±)citalopram (1) are reported. All of the analogues were SERT-selective and demonstrated that steric bulk was tolerated at the SERT S1 site, including two dimeric ligands (15 and 51.) In addition, 8 analogues were identified with similar potencies to S-1 for decreasing the dissociation of [3H]S-1 from the S1 site, via allosteric modulation at S2. Both dimeric compounds had similar affinities for the SERT S1 site (Ki=19.7 and 30.2 nM, respectively), whereas only the N-substituted analogue, 51, was as effective as S-1 in allosterically modulating the binding of [3H]S-1 via S2. PMID:24237160

PtdIns(3,4,5)P3 binding is necessary for WAVE2-induced formation of lamellipodia.

PubMed

Oikawa, Tsukasa; Yamaguchi, Hideki; Itoh, Toshiki; Kato, Masayoshi; Ijuin, Takeshi; Yamazaki, Daisuke; Suetsugu, Shiro; Takenawa, Tadaomi

2004-05-01

Polarized cell movement is triggered by the development of a PtdIns(3,4,5)P(3) gradient at the membrane, which is followed by rearrangement of the actin cytoskeleton. The WASP family verprolin homologous protein (WAVE) is essential for lamellipodium formation at the leading edge by activating the Arp2/3 complex downstream of Rac GTPase. Here, we report that WAVE2 binds to PtdIns(3,4,5)P(3) through its basic domain. The amino-terminal portion of WAVE2, which includes the PtdIns(3,4,5)P(3)-binding sequence, was localized at the leading edge of lamellipodia induced by an active form of Rac (RacDA) or by treatment with platelet-derived growth factor (PDGF). Production of PtdIns(3,4,5)P(3) at the cell membrane by myristoylated phosphatidylinositol-3-OH kinase (PI(3)K) is sufficient to recruit WAVE2 in the presence of dominant-negative Rac and latrunculin, demonstrating that PtdIns(3,4,5)P(3) alone is able to recruit WAVE2. Expression of a full-length mutant of WAVE2 that lacks the lipid-binding activity inhibited proper formation of lamellipodia induced by RacDA. These results suggest that one of the products of PI(3)K, PtdIns(3,4,5)P(3), recruits WAVE2 to the polarized membrane and that this recruitment is essential for lamellipodium formation at the leading edge.

Identification and Characterization of a Secondary Sodium-Binding Site and the Main Selectivity Determinants in the Human Concentrative Nucleoside Transporter 3.

PubMed

Arimany-Nardi, C; Claudio-Montero, A; Viel-Oliva, A; Schmidtke, P; Estarellas, C; Barril, X; Bidon-Chanal, A; Pastor-Anglada, M

2017-06-05

The family of concentrative Na + /nucleoside cotransporters in humans is constituted by three subtypes, namely, hCNT1, hCNT2, and hCNT3. Besides their different nucleoside selectivity, hCNT1 and hCNT2 have a Na + /nucleoside stoichiometry of 1:1, while for hCNT3 it is 2:1. This distinct stoichiometry of subtype 3 might hint the existence of a secondary sodium-binding site that is not present in the other two subtypes, but to date their three-dimensional structures remain unknown and the residues implicated in Na + binding are unclear. In this work, we have identified and characterized the Na + binding sites of hCNT3 by combining molecular modeling and mutagenesis studies. A model of the transporter was obtained by homology modeling, and key residues of two sodium-binding sites were identified and verified with a mutagenesis strategy. The structural model explains the altered sodium-binding properties of the hCNT3C602R polymorphic variant and supports previously generated data identifying the determinant residues of nucleoside selectivity, paving the way to understand how drugs can target this plasma membrane transporter.

Photofrin binds to procaspase-3 and mediates photodynamic treatment-triggered methionine oxidation and inactivation of procaspase-3

PubMed Central

Hsieh, Y-J; Chien, K-Y; Lin, S-Y; Sabu, S; Hsu, R-M; Chi, L-M; Lyu, P-C; Yu, J-S

2012-01-01

Diverse death phenotypes of cancer cells can be induced by Photofrin-mediated photodynamic therapy (PDT), which has a decisive role in eliciting a tumor-specific immunity for long-term tumor control. However, the mechanism(s) underlying this diversity remain elusive. Caspase-3 is a critical factor in determining cell death phenotypes in many physiological settings. Here, we report that Photofrin-PDT can modify and inactivate procaspase-3 in cancer cells. In cells exposed to an external apoptotic trigger, high-dose Photofrin-PDT pretreatment blocked the proteolytic activation of procaspase-3 by its upstream caspase. We generated and purified recombinant procaspase-3-D3A (a mutant without autolysis/autoactivation activity) to explore the underlying mechanism(s). Photofrin could bind directly to procaspase-3-D3A, and Photofrin-PDT-triggered inactivation and modification of procaspase-3-D3A was seen in vitro. Mass spectrometry-based quantitative analysis for post-translational modifications using both 16O/18O- and 14N/15N-labeling strategies revealed that Photofrin-PDT triggered a significant oxidation of procaspase-3-D3A (mainly on Met-27, -39 and -44) in a Photofrin dose-dependent manner, whereas the active site Cys-163 remained largely unmodified. Site-directed mutagenesis experiments further showed that Met-44 has an important role in procaspase-3 activation. Collectively, our results reveal that Met oxidation is a novel mechanism for the Photofrin-PDT-mediated inactivation of procaspase-3, potentially explaining at least some of the complicated cell death phenotypes triggered by PDT. PMID:22785533

Interaction of Tl +3 with mononucleotides: metal ion binding and sugar conformation

NASA Astrophysics Data System (ADS)

Nafisi, Sh.; Mohajerani, N.; Hadjiakhoondi, A.; Monajemi, M.; Garib, F.

2001-05-01

The interaction of Tl 3+ with sodium salts of adenosine-5'-monophosphate (5'-AMP), guanosine-5'-monophosphate (5'-GMP), cytidine-5'-monophosphate (5'-CMP), thymidine 5'-monophosphate (5'-dTMP) in ratios 1 and 2 have been studied in neutral pH. The solid complexes were isolated and characterized by Fourier transform infrared (FTIR) and 1H NMR spectroscopy. In the Tl 2(AMP) 3, Tl 3+ binds directly to N-7 and indirectly to the N-1 position of the pyrimidine ring and phosphate group with sugar moiety in C2'-endoanti. The crystalline salt of Tl 2(GMP) 3 show direct Tl-N-7 and Tl-PO 3(inner-sphere) binding. The conformation of ribose moiety in Tl 2(GMP) 3 is C3'-endoanti. In the Tl 2(CMP) 3, Tl 3+ bind directly to N-3 and PO32- (inner-sphere). The conformation of ribose moiety in Tl 2(CMP) 3 is C2'-endoanti. In the Tl 2(dTMP) 3, Tl 3+ bind indirectly to carbonyl group. The sugar moiety in Tl 2(dTMP) 3 is C3'-endoanti.

The clinicopathological and prognostic impact of 14-3-3 sigma expression on vulvar squamous cell carcinomas

PubMed Central

Wang, Zhihui; Tropè, Claes G; Suo, Zhenhe; Trøen, Gunhild; Yang, Guanrui; Nesland, Jahn M; Holm, Ruth

2008-01-01

Background 14-3-3 sigma (σ) promotes G2/M cell cycle arrest by sequestering cyclin B1-CDC2 complex in cytoplasm. Down-regulation of 14-3-3σ, which has been demonstrated in various carcinomas, may contribute to malignant transformation. However, the exact role of 14-3-3σ in the pathogenesis of vulvar carcinoma is not fully characterized, and the prognostic impact of 14-3-3σ protein expression is still unknown. Methods We investigated the 14-3-3σ expression in a series of 302 vulvar squamous cell carcinomas using immunohistochemistry and its associations with clinicopathological factors and clinical outcome. Results In cytoplasm, nucleus and cytoplasm/nucleus of vulvar carcinomas high 14-3-3σ protein expression was found in 72%, 59% and 75% of the carcinomas, respectively, and low levels in 28%, 41% and 25% of the cases, respectively. High level of 14-3-3σ in cytoplasm, nucleus and cytoplasm/nucleus was significantly correlated to large tumor diameter (p = 0.001, p = 0.002 and p = 0.001, respectively) and deep invasion (p = 0.01, p = 0.001 and p = 0.007, respectively). Variations of 14-3-3σ protein expression were not associated to disease-specific survival. Conclusion Our results indicate that 14-3-3σ may be involved in the development of a subset of vulvar squamous cell carcinomas by down-regulation of 14-3-3σ protein. Neither cytoplasmic nor nuclear level of 14-3-3σ expression was associated with prognosis. PMID:18950492

Structural model of the p14/SF3b155 · branch duplex complex.

PubMed

Schellenberg, Matthew J; Dul, Erin L; MacMillan, Andrew M

2011-01-01

Human p14 (SF3b14), a component of the spliceosomal U2 snRNP, interacts directly with the pre-mRNA branch adenosine within the context of the bulged duplex formed between the pre-mRNA branch region and U2 snRNA. This association occurs early in spliceosome assembly and persists within the fully assembled spliceosome. Analysis of the crystal structure of a complex containing p14 and a peptide derived from p14-associated SF3b155 combined with the results of cross-linking studies has suggested that the branch nucleotide interacts with a pocket on a non-canonical RNA binding surface formed by the complex. Here we report a structural model of the p14 · bulged duplex interaction based on a combination of X-ray crystallography of an adenine p14/SF3b155 peptide complex, biochemical comparison of a panel of disulfide cross-linked protein-RNA complexes, and small-angle X-ray scattering (SAXS). These studies reveal specific recognition of the branch adenosine within the p14 pocket and establish the orientation of the bulged duplex RNA bound on the protein surface. The intimate association of one surface of the bulged duplex with the p14/SF3b155 peptide complex described by this model buries the branch nucleotide at the interface and suggests that p14 · duplex interaction must be disrupted before the first step of splicing.

Structural model of the p14/SF3b155·branch duplex complex

PubMed Central

Schellenberg, Matthew J.; Dul, Erin L.; MacMillan, Andrew M.

2011-01-01

Human p14 (SF3b14), a component of the spliceosomal U2 snRNP, interacts directly with the pre-mRNA branch adenosine within the context of the bulged duplex formed between the pre-mRNA branch region and U2 snRNA. This association occurs early in spliceosome assembly and persists within the fully assembled spliceosome. Analysis of the crystal structure of a complex containing p14 and a peptide derived from p14-associated SF3b155 combined with the results of cross-linking studies has suggested that the branch nucleotide interacts with a pocket on a non-canonical RNA binding surface formed by the complex. Here we report a structural model of the p14•bulged duplex interaction based on a combination of X-ray crystallography of an adenine p14/SF3b155 peptide complex, biochemical comparison of a panel of disulfide cross-linked protein–RNA complexes, and small-angle X-ray scattering (SAXS). These studies reveal specific recognition of the branch adenosine within the p14 pocket and establish the orientation of the bulged duplex RNA bound on the protein surface. The intimate association of one surface of the bulged duplex with the p14/SF3b155 peptide complex described by this model buries the branch nucleotide at the interface and suggests that p14•duplex interaction must be disrupted before the first step of splicing. PMID:21062891

14 CFR 440.3 - Definitions.

Code of Federal Regulations, 2010 CFR

2010-01-01

... 14 Aeronautics and Space 4 2010-01-01 2010-01-01 false Definitions. 440.3 Section 440.3 Aeronautics and Space COMMERCIAL SPACE TRANSPORTATION, FEDERAL AVIATION ADMINISTRATION, DEPARTMENT OF... space flight participant, for the purposes of this part, is not a customer. Federal range facility means...

14 CFR 440.3 - Definitions.

Code of Federal Regulations, 2011 CFR

2011-01-01

... 14 Aeronautics and Space 4 2011-01-01 2011-01-01 false Definitions. 440.3 Section 440.3 Aeronautics and Space COMMERCIAL SPACE TRANSPORTATION, FEDERAL AVIATION ADMINISTRATION, DEPARTMENT OF... space flight participant, for the purposes of this part, is not a customer. Federal range facility means...

14 CFR 3.1 - Applicability.

Code of Federal Regulations, 2012 CFR

2012-01-01

... 14 Aeronautics and Space 1 2012-01-01 2012-01-01 false Applicability. 3.1 Section 3.1 Aeronautics and Space FEDERAL AVIATION ADMINISTRATION, DEPARTMENT OF TRANSPORTATION DEFINITIONS GENERAL...-certificated product, or (2) A product, part, appliance or material that may be used on a type-certificated...

14 CFR 3.1 - Applicability.

Code of Federal Regulations, 2010 CFR

2010-01-01

... 14 Aeronautics and Space 1 2010-01-01 2010-01-01 false Applicability. 3.1 Section 3.1 Aeronautics and Space FEDERAL AVIATION ADMINISTRATION, DEPARTMENT OF TRANSPORTATION DEFINITIONS GENERAL...-certificated product, or (2) A product, part, appliance or material that may be used on a type-certificated...

14 CFR 3.1 - Applicability.

Code of Federal Regulations, 2011 CFR

2011-01-01

... 14 Aeronautics and Space 1 2011-01-01 2011-01-01 false Applicability. 3.1 Section 3.1 Aeronautics and Space FEDERAL AVIATION ADMINISTRATION, DEPARTMENT OF TRANSPORTATION DEFINITIONS GENERAL...-certificated product, or (2) A product, part, appliance or material that may be used on a type-certificated...

14 CFR 3.1 - Applicability.

Code of Federal Regulations, 2013 CFR

2013-01-01

... 14 Aeronautics and Space 1 2013-01-01 2013-01-01 false Applicability. 3.1 Section 3.1 Aeronautics and Space FEDERAL AVIATION ADMINISTRATION, DEPARTMENT OF TRANSPORTATION DEFINITIONS GENERAL...-certificated product, or (2) A product, part, appliance or material that may be used on a type-certificated...

14 CFR 3.1 - Applicability.

Code of Federal Regulations, 2014 CFR

2014-01-01

... 14 Aeronautics and Space 1 2014-01-01 2014-01-01 false Applicability. 3.1 Section 3.1 Aeronautics and Space FEDERAL AVIATION ADMINISTRATION, DEPARTMENT OF TRANSPORTATION DEFINITIONS GENERAL...-certificated product, or (2) A product, part, appliance or material that may be used on a type-certificated...

Distinct requirements within the Msh3 nucleotide binding pocket for mismatch and double-strand break repair.

PubMed

Kumar, Charanya; Williams, Gregory M; Havens, Brett; Dinicola, Michelle K; Surtees, Jennifer A

2013-06-12

In Saccharomyces cerevisiae, repair of insertion/deletion loops is carried out by Msh2-Msh3-mediated mismatch repair (MMR). Msh2-Msh3 is also required for 3' non-homologous tail removal (3' NHTR) in double-strand break repair. In both pathways, Msh2-Msh3 binds double-strand/single-strand junctions and initiates repair in an ATP-dependent manner. However, the kinetics of the two processes appear different; MMR is likely rapid in order to coordinate with the replication fork, whereas 3' NHTR has been shown to be a slower process. To understand the molecular requirements in both repair pathways, we performed an in vivo analysis of well-conserved residues in Msh3 that are hypothesized to be required for MMR and/or 3' NHTR. These residues are predicted to be involved in either communication between the DNA-binding and ATPase domains within the complex or nucleotide binding and/or exchange within Msh2-Msh3. We identified a set of aromatic residues within the FLY motif of the predicted Msh3 nucleotide binding pocket that are essential for Msh2-Msh3-mediated MMR but are largely dispensable for 3' NHTR. In contrast, mutations in other regions gave similar phenotypes in both assays. Based on these results, we suggest that the two pathways have distinct requirements with respect to the position of the bound ATP within Msh3. We propose that the differences are related, at least in part, to the kinetics of each pathway. Proper binding and positioning of ATP is required to induce rapid conformational changes at the replication fork, but is less important when more time is available for repair, as in 3' NHTR. Copyright © 2013 Elsevier Ltd. All rights reserved.

Distinct requirements within the Msh3 nucleotide binding pocket for mismatch and double-strand break repair

PubMed Central

Kumar, Charanya; Williams, Gregory M.; Havens, Brett; Dinicola, Michelle; Surtees, Jennifer A.

2013-01-01

In Saccharomyces cerevisiae, repair of insertion/deletion loops is carried out by Msh2-Msh3-mediated mismatch repair (MMR). Msh2-Msh3 is also required for 3’ non-homologous tail removal (3’NHTR) in double-strand break repair. In both pathways, Msh2-Msh3 binds double-strand/single-strand junctions and initiates repair in an ATP-dependent manner. However, the kinetics of the two processes appear different; MMR is likely rapid in order to coordinate with the replication fork, whereas 3’ NHTR has been shown to be a slower process. To understand the molecular requirements in both repair pathways, we performed an in vivo analysis of well conserved residues in Msh3 that are hypothesized to be required for MMR and/or 3’NHTR. These residues are predicted to be involved in either communication between the DNA-binding and ATPase domains within the complex or nucleotide binding and/or exchange within Msh2-Msh3. We identified a set of aromatic residues within the FLY motif of the predicted Msh3 nucleotide binding pocket that are essential for Msh2-Msh3-mediated MMR but are largely dispensable for 3’NHTR. In contrast, mutations in other regions gave similar phenotypes in both assays. Based on these results, we suggest the two pathways have distinct requirements with respect to the position of the bound ATP within Msh3. We propose that the differences are related, at least in part, to the kinetics of each pathway. Proper binding and positioning of ATP is required to induce rapid conformational changes at the replication fork, but is less important when more time is available for repair, as in 3’ NHTR. PMID:23458407

Studies on molecular structure, vibrational spectra and molecular docking analysis of 3-Methyl-1,4-dioxo-1,4-dihydronaphthalen-2-yl 4-aminobenzoate

NASA Astrophysics Data System (ADS)

Suresh, D. M.; Amalanathan, M.; Hubert Joe, I.; Bena Jothy, V.; Diao, Yun-Peng

2014-09-01

The molecular structure, vibrational analysis and molecular docking analysis of the 3-Methyl-1,4-dioxo-1,4-dihydronaphthalen-2-yl 4-aminobenzoate (MDDNAB) molecule have been carried out using FT-IR and FT-Raman spectroscopic techniques and DFT method. The equilibrium geometry, harmonic vibrational wave numbers, various bonding features have been computed using density functional method. The calculated molecular geometry has been compared with experimental data. The detailed interpretation of the vibrational spectra has been carried out by using VEDA program. The hyper-conjugative interactions and charge delocalization have been analyzed using natural bond orbital (NBO) analysis. The simulated FT-IR and FT-Raman spectra satisfactorily coincide with the experimental spectra. The PES and charge analysis have been made. The molecular docking was done to identify the binding energy and the Hydrogen bonding with the cancer protein molecule.

Ethanol intake and sup 3 H-serotonin uptake II: A study in alcoholic patients using platelets sup 3 H-paroxetine binding

DOE Office of Scientific and Technical Information (OSTI.GOV)

Daoust, M.; Boucly, P.; Ernouf, D.

1991-01-01

The kinetic parameters of {sup 3}H-paroxetine binding and {sup 3}H-serotonin uptake were studied in platelets of alcoholic patients. There was no difference between alcoholic and non alcoholic subjects in {sup 3}H-paroxetine binding. When binding and {sup 3}H-serotonin uptake were studied, in the same plasma of the same subjects, the Vmax of serotonin uptake was increased in alcoholics. The data confirm the involvement of serotonin uptake system in alcohol dependance and suggest that serotonin uptake and paroxetine binding sites may be regulated independently in this pathology.

Identification of StARD3 as a Lutein-binding Protein in the Macula of the Primate Retina†

PubMed Central

Li, Binxing; Vachali, Preejith; Frederick, Jeanne M.; Bernstein, Paul S.

2011-01-01

Lutein, zeaxanthin and their metabolites are the xanthophyll carotenoids that form the macular pigment of the human retina. Epidemiological evidence suggests that high levels of these carotenoids in the diet, serum and macula are associated with decreased risk of age-related macular degeneration (AMD), and the AREDS2 study is prospectively testing this hypothesis. Understanding the biochemical mechanisms underlying the selective uptakes of lutein and zeaxanthin into the human macula may provide important insights into the physiology of the human macula in health and disease. GSTP1 is the macular zeaxanthin-binding protein, but the identity of the human macular lutein-binding protein has remained elusive. Prior identification of the silkworm lutein-binding protein (CBP) as a member of the steroidogenic acute regulatory domain (StARD) protein family, and selective labeling of monkey photoreceptor inner segments by anti-CBP antibody provided an important clue toward identifying the primate retina lutein-binding protein. Homology of CBP to all 15 human StARD proteins was analyzed using database searches, western blotting and immunohistochemistry, and we here provide evidence to identify StARD3 (also known as MLN64) as a human retinal lutein-binding protein. Further, recombinant StARD3 selectively binds lutein with high affinity (KD = 0.45 micromolar) when assessed by surface plasmon resonance (SPR) binding assays. Our results demonstrate previously unrecognized, specific interactions of StARD3 with lutein and provide novel avenues to explore its roles in human macular physiology and disease. PMID:21322544

Purification of L-( sup 3 H) Nicotine eliminates low affinity binding

DOE Office of Scientific and Technical Information (OSTI.GOV)

Romm, E.; Marks, M.J.; Collins, A.C.

1990-01-01

Some studies of L-({sup 3}H) nicotine binding to rodent and human brain tissue have detected two binding sites as evidenced by nonlinear Scatchard plots. Evidence presented here indicated that the low affinity binding site is not stereospecific, is not inhibited by low concentrations of cholinergic agonists and is probably due to breakdown products of nicotine since purification of the L-({sup 3}H)nicotine eliminates the low affinity site.

The Interaction of Integrin αIIbβ3 with Fibrin Occurs through Multiple Binding Sites in the αIIb β-Propeller Domain*

PubMed Central

Podolnikova, Nataly P.; Yakovlev, Sergiy; Yakubenko, Valentin P.; Wang, Xu; Gorkun, Oleg V.; Ugarova, Tatiana P.

2014-01-01

The currently available antithrombotic agents target the interaction of platelet integrin αIIbβ3 (GPIIb-IIIa) with fibrinogen during platelet aggregation. Platelets also bind fibrin formed early during thrombus growth. It was proposed that inhibition of platelet-fibrin interactions may be a necessary and important property of αIIbβ3 antagonists; however, the mechanisms by which αIIbβ3 binds fibrin are uncertain. We have previously identified the γ370–381 sequence (P3) in the γC domain of fibrinogen as the fibrin-specific binding site for αIIbβ3 involved in platelet adhesion and platelet-mediated fibrin clot retraction. In the present study, we have demonstrated that P3 can bind to several discontinuous segments within the αIIb β-propeller domain of αIIbβ3 enriched with negatively charged and aromatic residues. By screening peptide libraries spanning the sequence of the αIIb β-propeller, several sequences were identified as candidate contact sites for P3. Synthetic peptides duplicating these segments inhibited platelet adhesion and clot retraction but not platelet aggregation, supporting the role of these regions in fibrin recognition. Mutant αIIbβ3 receptors in which residues identified as critical for P3 binding were substituted for homologous residues in the I-less integrin αMβ2 exhibited reduced cell adhesion and clot retraction. These residues are different from those that are involved in the coordination of the fibrinogen γ404–411 sequence and from auxiliary sites implicated in binding of soluble fibrinogen. These results map the binding of fibrin to multiple sites in the αIIb β-propeller and further indicate that recognition specificity of αIIbβ3 for fibrin differs from that for soluble fibrinogen. PMID:24338009

Arabidopsis Histone Reader EMSY-LIKE 1 Binds H3K36 and Suppresses Geminivirus Infection.

PubMed

Coursey, Tami; Milutinovic, Milica; Regedanz, Elizabeth; Brkljacic, Jelena; Bisaro, David M

2018-06-06

Histone post-translational modifications (PTMs) impart information that regulates chromatin structure and activity. Their effects are mediated by histone reader proteins that bind specific PTMs to modify chromatin and/or recruit appropriate effectors to alter the chromatin landscape. Despite their crucial juxtaposition between information and functional outcome, relatively few plant histone readers have been identified, and nothing is known about their impact on viral chromatin and pathogenesis. We used the geminivirus Cabbage leaf curl virus (CaLCuV) as a model to functionally characterize two recently identified reader proteins, EMSY-LIKE 1 and 3 (EML1 and EML3), which contain Tudor-like Agenet domains predictive of histone PTM binding function. Here, we show that mutant Arabidopsis plants exhibit contrasting hypersusceptible ( eml1 ) and tolerant ( eml3 ) responses to CaLCuV infection, and that EML1 deficiency correlates with RNA polymerase II (Pol II) enrichment on viral chromatin and upregulated viral gene expression. Consistent with reader activity, EML1 and EML3 associate with nucleosomes and with CaLCuV chromatin, suggesting a direct impact on pathogenesis. We also demonstrate that EML1 and EML3 bind peptides containing histone H3 lysine 36 (H3K36), a PTM usually associated with active gene expression. The interaction encompasses multiple H3K36 PTMs, including methylation and acetylation, suggesting nuanced regulation. Further, EML1 and EML3 associate with similar regions of viral chromatin, implying possible competition between the two readers. Regions of EML1 and EML3 association correlate with sites of trimethylated H3K36 (H3K36me3) enrichment, consistent with regulation of geminivirus chromatin by direct EML targeting. IMPORTANCE Histone PTMs convey information that regulates chromatin compaction and DNA accessibility. Histone reader proteins bind specific PTMs and translate their effects by modifying chromatin and/or by recruiting effectors that alter

Identification of a 3rd Na+ Binding Site of the Glycine Transporter, GlyT2.

PubMed

Subramanian, Nandhitha; Scopelitti, Amanda J; Carland, Jane E; Ryan, Renae M; O'Mara, Megan L; Vandenberg, Robert J

2016-01-01

The Na+/Cl- dependent glycine transporters GlyT1 and GlyT2 regulate synaptic glycine concentrations. Glycine transport by GlyT2 is coupled to the co-transport of three Na+ ions, whereas transport by GlyT1 is coupled to the co-transport of only two Na+ ions. These differences in ion-flux coupling determine their respective concentrating capacities and have a direct bearing on their functional roles in synaptic transmission. The crystal structures of the closely related bacterial Na+-dependent leucine transporter, LeuTAa, and the Drosophila dopamine transporter, dDAT, have allowed prediction of two Na+ binding sites in GlyT2, but the physical location of the third Na+ site in GlyT2 is unknown. A bacterial betaine transporter, BetP, has also been crystallized and shows structural similarity to LeuTAa. Although betaine transport by BetP is coupled to the co-transport of two Na+ ions, the first Na+ site is not conserved between BetP and LeuTAa, the so called Na1' site. We hypothesized that the third Na+ binding site (Na3 site) of GlyT2 corresponds to the BetP Na1' binding site. To identify the Na3 binding site of GlyT2, we performed molecular dynamics (MD) simulations. Surprisingly, a Na+ placed at the location consistent with the Na1' site of BetP spontaneously dissociated from its initial location and bound instead to a novel Na3 site. Using a combination of MD simulations of a comparative model of GlyT2 together with an analysis of the functional properties of wild type and mutant GlyTs we have identified an electrostatically favorable novel third Na+ binding site in GlyT2 formed by Trp263 and Met276 in TM3, Ala481 in TM6 and Glu648 in TM10.

Allosteric regulation of tryptophan synthase channeling: the internal aldimine probed by trans-3-indole-3'-acrylate binding.

PubMed

Casino, Patricia; Niks, Dimitri; Ngo, Huu; Pan, Peng; Brzovic, Peter; Blumenstein, Lars; Barends, Thomas Reinier; Schlichting, Ilme; Dunn, Michael F

2007-07-03

Substrate channeling in the tryptophan synthase bienzyme complex from Salmonella typhimurium is regulated by allosteric interactions triggered by binding of ligand to the alpha-site and covalent reaction at the beta-site. These interactions switch the enzyme between low-activity forms with open conformations and high-activity forms with closed conformations. Previously, allosteric interactions have been demonstrated between the alpha-site and the external aldimine, alpha-aminoacrylate, and quinonoid forms of the beta-site. Here we employ the chromophoric l-Trp analogue, trans-3-indole-3'-acrylate (IA), and noncleavable alpha-site ligands (ASLs) to probe the allosteric properties of the internal aldimine, E(Ain). The ASLs studied are alpha-d,l-glycerol phosphate (GP) and d-glyceraldehyde 3-phosphate (G3P), and examples of two new classes of high-affinity alpha-site ligands, N-(4'-trifluoromethoxybenzoyl)-2-aminoethyl phosphate (F6) and N-(4'-trifluoromethoxybenzenesulfonyl)-2-aminoethyl phosphate (F9), that were previously shown to bind to the alpha-site by optical spectroscopy and X-ray crystal structures [Ngo, H., Harris, R., Kimmich, N., Casino, P., Niks, D., Blumenstein, L., Barends, T. R., Kulik, V., Weyand, M., Schlichting, I., and Dunn, M. F. (2007) Synthesis and characterization of allosteric probes of substrate channeling in the tryptophan synthase bienzyme complex, Biochemistry 46, 7713-7727]. The binding of IA to the beta-site is stimulated by the binding of GP, G3P, F6, or F9 to the alpha-site. The binding of ASLs was found to increase the affinity of the beta-site of E(Ain) for IA by 4-5-fold, demonstrating for the first time that the beta-subunit of the E(Ain) species undergoes a switching between low- and high-affinity states in response to the binding of ASLs.

17 CFR 260.7a-3 - Number of copies; filing; signatures; binding.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 17 Commodity and Securities Exchanges 3 2010-04-01 2010-04-01 false Number of copies; filing; signatures; binding. 260.7a-3 Section 260.7a-3 Commodity and Securities Exchanges SECURITIES AND EXCHANGE... § 260.7a-3 Number of copies; filing; signatures; binding. (a) Three copies of the complete application...

17 CFR 260.5a-3 - Number of copies; filing; signatures; binding.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 17 Commodity and Securities Exchanges 3 2010-04-01 2010-04-01 false Number of copies; filing; signatures; binding. 260.5a-3 Section 260.5a-3 Commodity and Securities Exchanges SECURITIES AND EXCHANGE... § 260.5a-3 Number of copies; filing; signatures; binding. (a) Three copies of each statement of...

14 CFR 155.3 - Applicable law.

Code of Federal Regulations, 2010 CFR

2010-01-01

... 14 Aeronautics and Space 3 2010-01-01 2010-01-01 false Applicable law. 155.3 Section 155.3... RELEASE OF AIRPORT PROPERTY FROM SURPLUS PROPERTY DISPOSAL RESTRICTIONS § 155.3 Applicable law. (a... transfer to the requirements of applicable law. Based on the laws cited in this paragraph, the...

17 CFR 260.4c-3 - Number of copies; filing; signatures; binding.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 17 Commodity and Securities Exchanges 3 2010-04-01 2010-04-01 false Number of copies; filing; signatures; binding. 260.4c-3 Section 260.4c-3 Commodity and Securities Exchanges SECURITIES AND EXCHANGE... § 260.4c-3 Number of copies; filing; signatures; binding. (a) Three copies of every application and of...

Epigenetic priors for identifying active transcription factor binding sites.

PubMed

Cuellar-Partida, Gabriel; Buske, Fabian A; McLeay, Robert C; Whitington, Tom; Noble, William Stafford; Bailey, Timothy L

2012-01-01

Accurate knowledge of the genome-wide binding of transcription factors in a particular cell type or under a particular condition is necessary for understanding transcriptional regulation. Using epigenetic data such as histone modification and DNase I, accessibility data has been shown to improve motif-based in silico methods for predicting such binding, but this approach has not yet been fully explored. We describe a probabilistic method for combining one or more tracks of epigenetic data with a standard DNA sequence motif model to improve our ability to identify active transcription factor binding sites (TFBSs). We convert each data type into a position-specific probabilistic prior and combine these priors with a traditional probabilistic motif model to compute a log-posterior odds score. Our experiments, using histone modifications H3K4me1, H3K4me3, H3K9ac and H3K27ac, as well as DNase I sensitivity, show conclusively that the log-posterior odds score consistently outperforms a simple binary filter based on the same data. We also show that our approach performs competitively with a more complex method, CENTIPEDE, and suggest that the relative simplicity of the log-posterior odds scoring method makes it an appealing and very general method for identifying functional TFBSs on the basis of DNA and epigenetic evidence. FIMO, part of the MEME Suite software toolkit, now supports log-posterior odds scoring using position-specific priors for motif search. A web server and source code are available at http://meme.nbcr.net. Utilities for creating priors are at http://research.imb.uq.edu.au/t.bailey/SD/Cuellar2011. t.bailey@uq.edu.au Supplementary data are available at Bioinformatics online.

The SARS Coronavirus 3a protein binds calcium in its cytoplasmic domain.

PubMed

Minakshi, Rinki; Padhan, Kartika; Rehman, Safikur; Hassan, Md Imtaiyaz; Ahmad, Faizan

2014-10-13

The Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) is a positive stranded RNA virus with ∼30kb genome. Among all open reading frames (orfs) of this virus, the orf3a is the largest, and encodes a protein of 274 amino acids, named as 3a protein. Sequence analysis suggests that the orf3a aligned to one calcium pump present in Plasmodium falciparum and the enzyme glutamine synthetase found in Leptospira interrogans. This sequence similarity was found to be limited only to amino acid residues 209-264 which form the cytoplasmic domain of the orf3a. Furthermore, this region was predicted to be involved in the calcium binding. Owing to this hypothesis, we were driven to establish its calcium binding property in vitro. Here, we expressed and purified the cytoplasmic domain of the 3a protein, called Cyto3a, as a recombinant His-tagged protein in the E. coli. The calcium binding nature was established by performing various staining methods such as ruthenium red and stains-all. (45)Ca overlay method was also done to further support the data. Since the 3a protein forms ion channels, we were interested to see any conformational changes occurring in the Cyot3a upon calcium binding, using fluorescence spectroscopy and circular dichroism. These studies clearly indicate a significant change in the conformation of the Cyto3a protein after binding with calcium. Our results strongly suggest that the cytoplasmic domain of the 3a protein of SARS-CoV binds calcium in vitro, causing a change in protein conformation. Copyright © 2014 Elsevier B.V. All rights reserved.