A Point Mutation in the Exon Junction Complex Factor Y14 Disrupts Its Function in mRNA Cap Binding and Translation Enhancement.

PubMed

Chuang, Tzu-Wei; Lee, Kuo-Ming; Lou, Yuan-Chao; Lu, Chia-Chen; Tarn, Woan-Yuh

2016-04-15

Eukaryotic mRNA biogenesis involves a series of interconnected steps mediated by RNA-binding proteins. The exon junction complex core protein Y14 is required for nonsense-mediated mRNA decay (NMD) and promotes translation. Moreover, Y14 binds the cap structure of mRNAs and inhibits the activity of the decapping enzyme Dcp2. In this report, we show that an evolutionarily conserved tryptophan residue (Trp-73) of Y14 is critical for its binding to the mRNA cap structure. A Trp-73 mutant (W73V) bound weakly to mRNAs and failed to protect them from degradation. However, this mutant could still interact with the NMD and mRNA degradation factors and retained partial NMD activity. In addition, we found that the W73V mutant could not interact with translation initiation factors. Overexpression of W73V suppressed reporter mRNA translation in vitro and in vivo and reduced the level of a set of nascent proteins. These results reveal a residue of Y14 that confers cap-binding activity and is essential for Y14-mediated enhancement of translation. Finally, we demonstrated that Y14 may selectively and differentially modulate protein biosynthesis. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Biological role and structural mechanism of twinfilin–capping protein interaction

PubMed Central

Falck, Sandra; Paavilainen, Ville O; Wear, Martin A; Grossmann, J Günter; Cooper, John A; Lappalainen, Pekka

2004-01-01

Twinfilin and capping protein (CP) are highly conserved actin-binding proteins that regulate cytoskeletal dynamics in organisms from yeast to mammals. Twinfilin binds actin monomer, while CP binds the barbed end of the actin filament. Remarkably, twinfilin and CP also bind directly to each other, but the mechanism and role of this interaction in actin dynamics are not defined. Here, we found that the binding of twinfilin to CP does not affect the binding of either protein to actin. Furthermore, site-directed mutagenesis studies revealed that the CP-binding site resides in the conserved C-terminal tail region of twinfilin. The solution structure of the twinfilin–CP complex supports these conclusions. In vivo, twinfilin's binding to both CP and actin monomer was found to be necessary for twinfilin's role in actin assembly dynamics, based on genetic studies with mutants that have defined biochemical functions. Our results support a novel model for how sequential interactions between actin monomers, twinfilin, CP, and actin filaments promote cytoskeletal dynamics. PMID:15282541

Atomic-resolution structure of the CAP-Gly domain of dynactin on polymeric microtubules determined by magic angle spinning NMR spectroscopy.

PubMed

Yan, Si; Guo, Changmiao; Hou, Guangjin; Zhang, Huilan; Lu, Xingyu; Williams, John Charles; Polenova, Tatyana

2015-11-24

Microtubules and their associated proteins perform a broad array of essential physiological functions, including mitosis, polarization and differentiation, cell migration, and vesicle and organelle transport. As such, they have been extensively studied at multiple levels of resolution (e.g., from structural biology to cell biology). Despite these efforts, there remain significant gaps in our knowledge concerning how microtubule-binding proteins bind to microtubules, how dynamics connect different conformational states, and how these interactions and dynamics affect cellular processes. Structures of microtubule-associated proteins assembled on polymeric microtubules are not known at atomic resolution. Here, we report a structure of the cytoskeleton-associated protein glycine-rich (CAP-Gly) domain of dynactin motor on polymeric microtubules, solved by magic angle spinning NMR spectroscopy. We present the intermolecular interface of CAP-Gly with microtubules, derived by recording direct dipolar contacts between CAP-Gly and tubulin using double rotational echo double resonance (dREDOR)-filtered experiments. Our results indicate that the structure adopted by CAP-Gly varies, particularly around its loop regions, permitting its interaction with multiple binding partners and with the microtubules. To our knowledge, this study reports the first atomic-resolution structure of a microtubule-associated protein on polymeric microtubules. Our approach lays the foundation for atomic-resolution structural analysis of other microtubule-associated motors.

Structure of the antiviral assembly inhibitor CAP-1 complex with the HIV-1 CA protein.

PubMed

Kelly, Brian N; Kyere, Sampson; Kinde, Isaac; Tang, Chun; Howard, Bruce R; Robinson, Howard; Sundquist, Wesley I; Summers, Michael F; Hill, Christopher P

2007-10-19

The CA domain of the human immunodeficiency virus type 1 (HIV-1) Gag polyprotein plays critical roles in both the early and late phases of viral replication and is therefore an attractive antiviral target. Compounds with antiviral activity were recently identified that bind to the N-terminal domain of CA (CA N) and inhibit capsid assembly during viral maturation. We have determined the structure of the complex between CA N and the antiviral assembly inhibitor N-(3-chloro-4-methylphenyl)-N'-{2-[({5-[(dimethylamino)-methyl]-2-furyl}-methyl)-sulfanyl]ethyl}-urea) (CAP-1) using a combination of NMR spectroscopy and X-ray crystallography. The protein undergoes a remarkable conformational change upon CAP-1 binding, in which Phe32 is displaced from its buried position in the protein core to open a deep hydrophobic cavity that serves as the ligand binding site. The aromatic ring of CAP-1 inserts into the cavity, with the urea NH groups forming hydrogen bonds with the backbone oxygen of Val59 and the dimethylamonium group interacting with the side-chains of Glu28 and Glu29. Elements that could be exploited to improve binding affinity are apparent in the structure. The displacement of Phe32 by CAP-1 appears to be facilitated by a strained main-chain conformation, which suggests a potential role for a Phe32 conformational switch during normal capsid assembly.

Remodeling of the pioneer translation initiation complex involves translation and the karyopherin importin β

PubMed Central

Sato, Hanae; Maquat, Lynne E.

2009-01-01

Mammalian mRNAs lose and acquire proteins throughout their life span while undergoing processing, transport, translation, and decay. How translation affects messenger RNA (mRNA)–protein interactions is largely unknown. The pioneer round of translation uses newly synthesized mRNA that is bound by cap-binding protein 80 (CBP80)–CBP20 (also known as the cap-binding complex [CBC]) at the cap, poly(A)-binding protein N1 (PABPN1) and PABPC1 at the poly(A) tail, and, provided biogenesis involves pre-mRNA splicing, exon junction complexes (EJCs) at exon–exon junctions. Subsequent rounds of translation engage mRNA that is bound by eukaryotic translation initiation factor 4E (eIF4E) at the cap and PABPC1 at the poly(A) tail, but that lacks detectable EJCs and PABPN1. Using the level of intracellular iron to regulate the translation of specific mRNAs, we show that translation promotes not only removal of EJC constituents, including the eIF4AIII anchor, but also replacement of PABPN1 by PABPC1. Remarkably, translation does not affect replacement of CBC by eIF4E. Instead, replacement of CBC by eIF4E is promoted by importin β (IMPβ): Inhibiting the binding of IMPβ to the complex of CBC–IMPα at an mRNA cap using the IMPα IBB (IMPβ-binding) domain or a RAN variant increases the amount of CBC-bound mRNA and decreases the amount of eIF4E-bound mRNA. Our studies uncover a previously unappreciated role for IMPβ and a novel paradigm for how newly synthesized messenger ribonucleoproteins (mRNPs) are matured. PMID:19884259

Translation initiation mediated by nuclear cap-binding protein complex.

PubMed

Ryu, Incheol; Kim, Yoon Ki

2017-04-01

In mammals, cap-dependent translation of mRNAs is initiated by two distinct mechanisms: cap-binding complex (CBC; a heterodimer of CBP80 and 20)-dependent translation (CT) and eIF4E-dependent translation (ET). Both translation initiation mechanisms share common features in driving cap- dependent translation; nevertheless, they can be distinguished from each other based on their molecular features and biological roles. CT is largely associated with mRNA surveillance such as nonsense-mediated mRNA decay (NMD), whereas ET is predominantly involved in the bulk of protein synthesis. However, several recent studies have demonstrated that CT and ET have similar roles in protein synthesis and mRNA surveillance. In a subset of mRNAs, CT preferentially drives the cap-dependent translation, as ET does, and ET is responsible for mRNA surveillance, as CT does. In this review, we summarize and compare the molecular features of CT and ET with a focus on the emerging roles of CT in translation. [BMB Reports 2017; 50(4): 186-193].

Inhibition of Mitogen-activated Protein Kinase (MAPK)-interacting Kinase (MNK) Preferentially Affects Translation of mRNAs Containing Both a 5'-Terminal Cap and Hairpin.

PubMed

Korneeva, Nadejda L; Song, Anren; Gram, Hermann; Edens, Mary Ann; Rhoads, Robert E

2016-02-12

The MAPK-interacting kinases 1 and 2 (MNK1 and MNK2) are activated by extracellular signal-regulated kinases 1 and 2 (ERK1/2) or p38 in response to cellular stress and extracellular stimuli that include growth factors, cytokines, and hormones. Modulation of MNK activity affects translation of mRNAs involved in the cell cycle, cancer progression, and cell survival. However, the mechanism by which MNK selectively affects translation of these mRNAs is not understood. MNK binds eukaryotic translation initiation factor 4G (eIF4G) and phosphorylates the cap-binding protein eIF4E. Using a cell-free translation system from rabbit reticulocytes programmed with mRNAs containing different 5'-ends, we show that an MNK inhibitor, CGP57380, affects translation of only those mRNAs that contain both a cap and a hairpin in the 5'-UTR. Similarly, a C-terminal fragment of human eIF4G-1, eIF4G(1357-1600), which prevents binding of MNK to intact eIF4G, reduces eIF4E phosphorylation and inhibits translation of only capped and hairpin-containing mRNAs. Analysis of proteins bound to m(7)GTP-Sepharose reveals that both CGP and eIF4G(1357-1600) decrease binding of eIF4E to eIF4G. These data suggest that MNK stimulates translation only of mRNAs containing both a cap and 5'-terminal RNA duplex via eIF4E phosphorylation, thereby enhancing the coupled cap-binding and RNA-unwinding activities of eIF4F. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Protein phosphatase PPM1G regulates protein translation and cell growth by dephosphorylating 4E binding protein 1 (4E-BP1).

PubMed

Liu, Jianyu; Stevens, Payton D; Eshleman, Nichole E; Gao, Tianyan

2013-08-09

Protein translation initiation is a tightly controlled process responding to nutrient availability and mitogen stimulation. Serving as one of the most important negative regulators of protein translation, 4E binding protein 1 (4E-BP1) binds to translation initiation factor 4E and inhibits cap-dependent translation in a phosphorylation-dependent manner. Although it has been demonstrated previously that the phosphorylation of 4E-BP1 is controlled by mammalian target of rapamycin in the mammalian target of rapamycin complex 1, the mechanism underlying the dephosphorylation of 4E-BP1 remains elusive. Here, we report the identification of PPM1G as the phosphatase of 4E-BP1. A coimmunoprecipitation experiment reveals that PPM1G binds to 4E-BP1 in cells and that purified PPM1G dephosphorylates 4E-BP1 in vitro. Knockdown of PPM1G in 293E and colon cancer HCT116 cells results in an increase in the phosphorylation of 4E-BP1 at both the Thr-37/46 and Ser-65 sites. Furthermore, the time course of 4E-BP1 dephosphorylation induced by amino acid starvation or mammalian target of rapamycin inhibition is slowed down significantly in PPM1G knockdown cells. Functionally, the amount of 4E-BP1 bound to the cap-dependent translation initiation complex is decreased when the expression of PPM1G is depleted. As a result, the rate of cap-dependent translation, cell size, and protein content are increased in PPM1G knockdown cells. Taken together, our study has identified protein phosphatase PPM1G as a novel regulator of cap-dependent protein translation by negatively controlling the phosphorylation of 4E-BP1.

Structure of catabolite activator protein with cobalt(II) and sulfate

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

Rao, Ramya R.; Lawson, Catherine L., E-mail: cathy.lawson@rutgers.edu

2014-04-15

The crystal structure of E. coli catabolite activator protein with bound cobalt(II) and sulfate ions at 1.97 Å resolution is reported. The crystal structure of cyclic AMP–catabolite activator protein (CAP) from Escherichia coli containing cobalt(II) chloride and ammonium sulfate is reported at 1.97 Å resolution. Each of the two CAP subunits in the asymmetric unit binds one cobalt(II) ion, in each case coordinated by N-terminal domain residues His19, His21 and Glu96 plus an additional acidic residue contributed via a crystal contact. The three identified N-terminal domain cobalt-binding residues are part of a region of CAP that is important for transcriptionmore » activation at class II CAP-dependent promoters. Sulfate anions mediate additional crystal lattice contacts and occupy sites corresponding to DNA backbone phosphate positions in CAP–DNA complex structures.« less

Allosteric Coupling of CARMIL and V-1 Binding to Capping Protein Revealed by Hydrogen-Deuterium Exchange.

PubMed

Johnson, Britney; McConnell, Patrick; Kozlov, Alex G; Mekel, Marlene; Lohman, Timothy M; Gross, Michael L; Amarasinghe, Gaya K; Cooper, John A

2018-05-29

Actin assembly is important for cell motility. The ability of actin subunits to join or leave filaments via the barbed end is critical to actin dynamics. Capping protein (CP) binds to barbed ends to prevent subunit gain and loss and is regulated by proteins that include V-1 and CARMIL. V-1 inhibits CP by sterically blocking one binding site for actin. CARMILs bind at a distal site and decrease the affinity of CP for actin, suggested to be caused by conformational changes. We used hydrogen-deuterium exchange with mass spectrometry (HDX-MS) to probe changes in structural dynamics induced by V-1 and CARMIL binding to CP. V-1 and CARMIL induce changes in both proteins' binding sites on the surface of CP, along with a set of internal residues. Both also affect the conformation of CP's ββ subunit "tentacle," a second distal actin-binding site. Concerted regulation of actin assembly by CP occurs through allosteric couplings between CP modulator and actin binding sites. Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.

The function of yeast CAP family proteins in lipid export, mating, and pathogen defense.

PubMed

Darwiche, Rabih; El Atab, Ola; Cottier, Stéphanie; Schneiter, Roger

2018-04-01

In their natural habitat, yeast cells are constantly challenged by changing environmental conditions and a fierce competition for limiting resources. To thrive under such conditions, cells need to adapt and divide quickly, and be able to neutralize the toxic compounds secreted by their neighbors. Proteins like the pathogen-related yeast, Pry proteins, which belong to the large CAP/SCP/TAPS superfamily, may have an important role in this function. CAP proteins are conserved from yeast to man and are characterized by a unique αβα sandwich fold. They are mostly secreted glycoproteins and have been implicated in many different physiological processes including pathogen defense, virulence, venom toxicity, and sperm maturation. Yeast members of this family bind and export sterols as well as fatty acids, and they render cells resistant to eugenol, an antimicrobial compound present in clove oil. CAP family members might thus exert their various physiological functions through binding, sequestration, and neutralization of such small hydrophobic compounds. © 2017 Federation of European Biochemical Societies.

Coupling ligand recognition to protein folding in an engineered variant of rabbit ileal lipid binding protein.

PubMed

Kouvatsos, Nikolaos; Meldrum, Jill K; Searle, Mark S; Thomas, Neil R

2006-11-28

We have engineered a variant of the beta-clam shell protein ILBP which lacks the alpha-helical motif that caps the central binding cavity; the mutant protein is sufficiently destabilised that it is unfolded under physiological conditions, however, it unexpectedly binds its natural bile acid substrates with high affinity forming a native-like beta-sheet rich structure and demonstrating strong thermodynamic coupling between ligand binding and protein folding.

Reconstitution of high affinity. cap alpha. /sub 2/ adrenergic agonist binding by fusion with a pertussis toxin substrate

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

Kim, M.H.; Neubig, R.R.

1986-03-05

High affinity ..cap alpha../sub 2/ adrenergic agonist binding is thought to occur via a coupling of the ..cap alpha../sub 2/ receptor with N/sub i/, the inhibitory guanyl nucleotide binding protein. Human platelet membranes pretreated at pH 11.5 exhibit a selective inactivation of agonist binding and N/sub i/. To further study the mechanism of agonist binding, alkali treated membranes (ATM) were mixed with membranes pretreated with 10 ..mu..M phenoxybenzamine to block ..cap alpha../sub 2/ receptors (POB-M). The combined membrane pellet was incubated in 50% polyethylene glycol (PEG) to promote membrane-membrane fusion and assayed for binding to the ..cap alpha../sub 2/ agonistmore » (/sup 3/H)UK 14,304 (UK) and the antagonist (/sup 3/H) yohimbine. PEG treatment resulted in a 2-4 fold enhancement of UK binding whereas yohimbine binding was unchanged. No enhancement of UK binding was observed in the absence of PEG treatment. The reconstitution was dependent on the addition of POB-M. They found that a 1:1 ratio of POB-M:ATM was optimal. Reconstituted binding was inhibited by GppNHp. Fusion of rat C6 glioma cell membranes, which do not contain ..cap alpha../sub 2/ receptors, also enhanced agonist binding to ATM. Fusion of C6 membranes from cells treated with pertussis toxin did not enhance (/sup 3/H) UK binding. These data show that a pertussis toxin sensitive membrane component, possibly N/sub i/, can reconstitute high affinity ..cap alpha../sub 2/ agonist binding.« less

Tropomodulins: pointed-end capping proteins that regulate actin filament architecture in diverse cell types

PubMed Central

Yamashiro, Sawako; Gokhin, David S.; Kimura, Sumiko; Nowak, Roberta B.; Fowler, Velia M.

2012-01-01

Tropomodulins are a family of four proteins (Tmods 1–4) that cap the pointed ends of actin filaments in actin cytoskeletal structures in a developmentally regulated and tissue-specific manner. Unique among capping proteins, Tmods also bind tropomyosins (TMs), which greatly enhance the actin filament pointed-end capping activity of Tmods. Tmods are defined by a tropomyosin (TM)-regulated/Pointed-End Actin Capping (TM-Cap) domain in their unstructured N-terminal portion, followed by a compact, folded Leucine-Rich Repeat/Pointed-End Actin Capping (LRR-Cap) domain. By inhibiting actin monomer association and dissociation from pointed ends, Tmods regulate regulate actin dynamics and turnover, stabilizing actin filament lengths and cytoskeletal architecture. In this review, we summarize the genes, structural features, molecular and biochemical properties, actin regulatory mechanisms, expression patterns, and cell and tissue functions of Tmods. By understanding Tmods’ functions in the context of their molecular structure, actin regulation, binding partners, and related variants (leiomodins 1–3), we can draw broad conclusions that can explain the diverse morphological and functional phenotypes that arise from Tmod perturbation experiments in vitro and in vivo. Tmod-based stabilization and organization of intracellular actin filament networks provide key insights into how the emergent properties of the actin cytoskeleton drive tissue morphogenesis and physiology. PMID:22488942

Role of a guanine nucleotide-binding protein in. cap alpha. /sub 1/-adrenergic receptor-mediated Ca/sup 2 +/ mobilization in DDT/sub 1/ MF-2 cells

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

Cornett, L.E.; Norris, J.S.

1987-11-01

In this study the mechanisms involved in ..cap alpha../sub 1/-adrenergic receptor-mediated Ca/sup 2 +/ mobilization at the level of the plasma membrane were investigated. Stimulation of /sup 45/Ca/sup 2 +/ efflux from saponin-permeabilized DDT/sub 1/ MF-2 cells was observed with the addition of either the ..cap alpha../sub 1/-adrenergic agonist phenylephrine and guanosine-5'-triphosphate or the nonhydrolyzable guanine nucleotide guanylyl-imidodiphosphate. In the presence of (/sup 32/P) NAD, pertussis toxin was found to catalyze ADP-ribosylation of a M/sub r/ = 40,500 (n = 8) peptide in membranes prepared from DDT/sub 1/, MF-2 cells, possibly the ..cap alpha..-subunit of N/sub i/. However, stimulation ofmore » unidirectional /sup 45/Ca/sup 2 +/ efflux by phenylephrine was not affected by previous treatment of cells with 100 ng/ml pertussis toxin. These data suggest that the putative guanine nucleotide-binding protein which couples the ..cap alpha../sub 1/-adrenergic receptor to Ca/sup 2 +/ mobilization in DDT/sub 1/ MF-2 cells is not a pertussis toxin substrate and may possibly be an additional member of guanine nucleotide binding protein family.« less

Sensitivity of genera Porphyromonas and Prevotella to the bactericidal action of C-terminal domain of human CAP18 and its analogues.

PubMed

Isogai, E; Isogai, H; Matuo, K; Hirose, K; Kowashi, Y; Okumuara, K; Hirata, M

2003-10-01

This paper reports the effect of the synthesized 27-amino acid sequence in the C-terminal domain of human CAP18 (hCAP18), a human cationic antibacterial protein or cathelicidin, on certain strains belonging to the genera Porophyromonas and Prevotella. The domain binds lipopolysaccharides (LPS) from Porophyromonas gingivalis and Porophyromonas circumdentaria as well as enterobacterial LPS. Two analogues of hCAP18, designated LL/CAP18 and FF/CAP18, were also tested to determine whether additional activity was obtained. The analogue peptides replaced with hydrophobic and cationic amino acid residues showed more potent bactericidal and LPS-binding activities than the original one.

ENTRAPMENT OF PROTEINS IN GLYCOGEN-CAPPED AND HYDRAZIDE-ACTIVATED SUPPORTS

PubMed Central

Jackson, Abby J.; Xuan, Hai; Hage, David S.

2010-01-01

A method is described for the entrapment of proteins in hydrazide-activated supports using oxidized glycogen as a capping agent. This approach is demonstrated using human serum albumin (HSA) as a model binding agent. After optimization of this method, a protein content of 43 (± 1) mg HSA/g support was obtained for porous silica. The entrapped HSA supports could retain a low mass drug (S-warfarin) and had activities and equilibrium constants comparable to those for soluble HSA. It was also found that this approach could be used with other proteins and binding agents that had masses between 5.8 and 150 kDa. PMID:20470745

Specific high-affinity binding sites for a synthetic gliadin heptapeptide of human peripheral blood lymphocytes

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

Payan, D.G.; Horvath, K.; Graf, L.

1987-03-23

The synthetic peptide containing residues 43-49 of ..cap alpha..-gliadin, the major protein component of gluten, has previously been shown to inhibit the production of lymphokine activities by mononuclear leukocytes. The authors demonstrate using radiolabeled ..cap alpha..-gliadin(43-49) that human peripheral blood lymphocytes express approximately 20,000-25,000 surface receptors for this peptide, with a dissociation constant (K/sub D/) of 20 nM. In addition, binding is inhibited by naloxone and an enkephalin analog, thus confirming the functional correlate which demonstrates inhibition by these agents of ..cap alpha..-gliadin(43-49) functional effects. Furthermore, B-lymphocytes bind specifically a greater amount of (/sup 125/I)..cap alpha..-gliadin(43-49) than T-lymphocytes. The lymphocytemore » ..cap alpha..-gliadin(43-49) receptor may play an important role in mediating the immunological response to ..cap alpha..-gliadin. 16 references, 4 figures.« less

Identification of Actin-Binding Proteins from Maize Pollen

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

Staiger, C.J.

Specific Aims--The goal of this project was to gain an understanding of how actin filament organization and dynamics are controlled in flowering plants. Specifically, we proposed to identify unique proteins with novel functions by investigating biochemical strategies for the isolation and characterization of actin-binding proteins (ABPs). In particular, our hunt was designed to identify capping proteins and nucleation factors. The specific aims included: (1) to use F-actin affinity chromatography (FAAC) as a general strategy to isolate pollen ABPs (2) to produce polyclonal antisera and perform subcellular localization in pollen tubes (3) to isolate cDNA clones for the most promising ABPsmore » (4) to further purify and characterize ABP interactions with actin in vitro. Summary of Progress By employing affinity chromatography on F-actin or DNase I columns, we have identified at least two novel ABPs from pollen, PrABP80 (gelsolin-like) and ZmABP30, We have also cloned and expressed recombinant protein, as well as generated polyclonal antisera, for 6 interesting ABPs from Arabidopsis (fimbrin AtFIM1, capping protein a/b (AtCP), adenylyl cyclase-associated protein (AtCAP), AtCapG & AtVLN1). We performed quantitative analyses of the biochemical properties for two of these previously uncharacterized ABPs (fimbrin and capping protein). Our studies provide the first evidence for fimbrin activity in plants, demonstrate the existence of barbed-end capping factors and a gelsolin-like severing activity, and provide the quantitative data necessary to establish and test models of F-actin organization and dynamics in plant cells.« less

Sequestration by IFIT1 Impairs Translation of 2′O-unmethylated Capped RNA

PubMed Central

Lacerda, Livia; Benda, Christian; Holze, Cathleen; Eberl, Christian H.; Mann, Angelika; Kindler, Eveline; Gil-Cruz, Cristina; Ziebuhr, John; Thiel, Volker; Pichlmair, Andreas

2013-01-01

Viruses that generate capped RNA lacking 2′O methylation on the first ribose are severely affected by the antiviral activity of Type I interferons. We used proteome-wide affinity purification coupled to mass spectrometry to identify human and mouse proteins specifically binding to capped RNA with different methylation states. This analysis, complemented with functional validation experiments, revealed that IFIT1 is the sole interferon-induced protein displaying higher affinity for unmethylated than for methylated capped RNA. IFIT1 tethers a species-specific protein complex consisting of other IFITs to RNA. Pulsed stable isotope labelling with amino acids in cell culture coupled to mass spectrometry as well as in vitro competition assays indicate that IFIT1 sequesters 2′O-unmethylated capped RNA and thereby impairs binding of eukaryotic translation initiation factors to 2′O-unmethylated RNA template, which results in inhibition of translation. The specificity of IFIT1 for 2′O-unmethylated RNA serves as potent antiviral mechanism against viruses lacking 2′O-methyltransferase activity and at the same time allows unperturbed progression of the antiviral program in infected cells. PMID:24098121

Structural basis for LeishIF4E-1 modulation by an interacting protein in the human parasite Leishmania major.

PubMed

Meleppattu, Shimi; Arthanari, Haribabu; Zinoviev, Alexandra; Boeszoermenyi, Andras; Wagner, Gerhard; Shapira, Michal; Léger-Abraham, Mélissa

2018-03-19

Leishmania parasites are unicellular pathogens that are transmitted to humans through the bite of infected sandflies. Most of the regulation of their gene expression occurs post-transcriptionally, and the different patterns of gene expression required throughout the parasites' life cycle are regulated at the level of translation. Here, we report the X-ray crystal structure of the Leishmania cap-binding isoform 1, LeishIF4E-1, bound to a protein fragment of previously unknown function, Leish4E-IP1, that binds tightly to LeishIF4E-1. The molecular structure, coupled to NMR spectroscopy experiments and in vitro cap-binding assays, reveal that Leish4E-IP1 allosterically destabilizes the binding of LeishIF4E-1 to the 5' mRNA cap. We propose mechanisms through which Leish4E-IP1-mediated LeishIF4E-1 inhibition could regulate translation initiation in the human parasite.

Structure of apo-CAP reveals that large conformational changes are necessary for DNA binding

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

Sharma, Hitesh; Yu, Shaoning; Kong, Jilie

2009-10-21

The binding of cAMP to the Escherichia coli catabolite gene activator protein (CAP) produces a conformational change that enables it to bind specific DNA sequences and regulate transcription, which it cannot do in the absence of the nucleotide. The crystal structures of the unliganded CAP containing a D138L mutation and the unliganded WT CAP were determined at 2.3 and 3.6 {angstrom} resolution, respectively, and reveal that the two DNA binding domains have dimerized into one rigid body and their two DNA recognition helices become buried. The WT structure shows multiple orientations of this rigid body relative to the nucleotide bindingmore » domain supporting earlier biochemical data suggesting that the inactive form exists in an equilibrium among different conformations. Comparison of the structures of the liganded and unliganded CAP suggests that cAMP stabilizes the active DNA binding conformation of CAP through the interactions that the N{sup 6} of the adenosine makes with the C-helices. These interactions are associated with the reorientation and elongation of the C-helices that precludes the formation of the inactive structure.« less

Modulation of translation-initiation in CHO-K1 cells by rapamycin-induced heterodimerization of engineered eIF4G fusion proteins.

PubMed

Schlatter, Stefan; Senn, Claudia; Fussenegger, Martin

2003-07-20

Translation-initiation is a predominant checkpoint in mammalian cells which controls protein synthesis and fine-tunes the flow of information from gene to protein. In eukaryotes, translation-initiation is typically initiated at a 7-methyl-guanylic acid cap posttranscriptionally linked to the 5' end of mRNAs. Alternative cap-independent translation-initiation involves 5' untranslated regions (UTR) known as internal ribosome entry sites, which adopt a particular secondary structure. Translation-initiating ribosome assembly at cap or IRES elements is mediated by a multiprotein complex of which the initiation factor 4F (eIF4F) consisting of eIF4A (helicase), eIF4E (cap-binding protein), and eIF4G is a major constituent. eIF4G is a key target of picornaviral protease 2A, which cleaves this initiation factor into eIF4G(Delta) and (Delta)eIF4G to redirect the cellular translation machinery exclusively to its own IRES-containing transcripts. We have designed a novel translation control system (TCS) for conditional as well as adjustable translation of cap- and IRES-dependent transgene mRNAs in mammalian cells. eIF4G(Delta) and (Delta)eIF4G were fused C- and N-terminally to the FK506-binding protein (FKBP) and the FKBP-rapamycin-binding domain (FRB) of the human FKBP-rapamycin-associated protein (FRAP), respectively. Rapamycin-induced heterodimerization of eIF4G(Delta)-FKBP and FRB-(Delta)eIF4G fusion proteins reconstituted a functional chimeric elongation factor 4G in a dose-dependent manner. Rigorous quantitative expression analysis of cap- and IRES-dependent SEAP- (human placental secreted alkaline phosphatase) and luc- (Photinus pyralis luciferase) encoding reporter constructs confirmed adjustable translation control and revealed increased production of desired proteins in response to dimerization-induced heterologous eIF4G in Chinese hamster ovary (CHO-K1) cells. Copyright 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 83: 210-225, 2003.

Inhibition of Poly(A)-binding protein with a synthetic RNA mimic reduces pain sensitization in mice.

PubMed

Barragán-Iglesias, Paulino; Lou, Tzu-Fang; Bhat, Vandita D; Megat, Salim; Burton, Michael D; Price, Theodore J; Campbell, Zachary T

2018-01-02

Nociceptors rely on cap-dependent translation to rapidly induce protein synthesis in response to pro-inflammatory signals. Comparatively little is known regarding the role of the regulatory factors bound to the 3' end of mRNA in nociceptor sensitization. Poly(A)-binding protein (PABP) stimulates translation initiation by bridging the Poly(A) tail to the eukaryotic initiation factor 4F complex associated with the mRNA cap. Here, we use unbiased assessment of PABP binding specificity to generate a chemically modified RNA-based competitive inhibitor of PABP. The resulting RNA mimic, which we designated as the Poly(A) SPOT-ON, is more stable than unmodified RNA and binds PABP with high affinity and selectivity in vitro. We show that injection of the Poly(A) SPOT-ON at the site of an injury can attenuate behavioral response to pain. Collectively, these results suggest that PABP is integral for nociceptive plasticity. The general strategy described here provides a broad new source of mechanism-based inhibitors for RNA-binding proteins and is applicable for in vivo studies.

Steady-state EB cap size fluctuations are determined by stochastic microtubule growth and maturation

PubMed Central

Rickman, Jamie; Duellberg, Christian; Cade, Nicholas I.; Griffin, Lewis D.; Surrey, Thomas

2017-01-01

Growing microtubules are protected from depolymerization by the presence of a GTP or GDP/Pi cap. End-binding proteins of the EB1 family bind to the stabilizing cap, allowing monitoring of its size in real time. The cap size has been shown to correlate with instantaneous microtubule stability. Here we have quantitatively characterized the properties of cap size fluctuations during steady-state growth and have developed a theory predicting their timescale and amplitude from the kinetics of microtubule growth and cap maturation. In contrast to growth speed fluctuations, cap size fluctuations show a characteristic timescale, which is defined by the lifetime of the cap sites. Growth fluctuations affect the amplitude of cap size fluctuations; however, cap size does not affect growth speed, indicating that microtubules are far from instability during most of their time of growth. Our theory provides the basis for a quantitative understanding of microtubule stability fluctuations during steady-state growth. PMID:28280102

Control of eIF4E cellular localization by eIF4E-binding proteins, 4E-BPs.

PubMed

Rong, Liwei; Livingstone, Mark; Sukarieh, Rami; Petroulakis, Emmanuel; Gingras, Anne-Claude; Crosby, Katherine; Smith, Bradley; Polakiewicz, Roberto D; Pelletier, Jerry; Ferraiuolo, Maria A; Sonenberg, Nahum

2008-07-01

Eukaryotic initiation factor (eIF) 4E, the mRNA 5'-cap-binding protein, mediates the association of eIF4F with the mRNA 5'-cap structure to stimulate cap-dependent translation initiation in the cytoplasm. The assembly of eIF4E into the eIF4F complex is negatively regulated through a family of repressor proteins, called the eIF4E-binding proteins (4E-BPs). eIF4E is also present in the nucleus, where it is thought to stimulate nuclear-cytoplasmic transport of certain mRNAs. eIF4E is transported to the nucleus via its interaction with 4E-T (4E-transporter), but it is unclear how it is retained in the nucleus. Here we show that a sizable fraction (approximately 30%) of 4E-BP1 is localized to the nucleus, where it binds eIF4E. In mouse embryo fibroblasts (MEFs) subjected to serum starvation and/or rapamycin treatment, nuclear 4E-BPs sequester eIF4E in the nucleus. A dramatic loss of nuclear 4E-BP1 occurs in c-Ha-Ras-expressing MEFs, which fail to show starvation-induced nuclear accumulation of eIF4E. Therefore, 4E-BP1 is a regulator of eIF4E cellular localization.

Control of eIF4E cellular localization by eIF4E-binding proteins, 4E-BPs

PubMed Central

Rong, Liwei; Livingstone, Mark; Sukarieh, Rami; Petroulakis, Emmanuel; Gingras, Anne-Claude; Crosby, Katherine; Smith, Bradley; Polakiewicz, Roberto D.; Pelletier, Jerry; Ferraiuolo, Maria A.; Sonenberg, Nahum

2008-01-01

Eukaryotic initiation factor (eIF) 4E, the mRNA 5′-cap-binding protein, mediates the association of eIF4F with the mRNA 5′-cap structure to stimulate cap-dependent translation initiation in the cytoplasm. The assembly of eIF4E into the eIF4F complex is negatively regulated through a family of repressor proteins, called the eIF4E-binding proteins (4E-BPs). eIF4E is also present in the nucleus, where it is thought to stimulate nuclear-cytoplasmic transport of certain mRNAs. eIF4E is transported to the nucleus via its interaction with 4E-T (4E-transporter), but it is unclear how it is retained in the nucleus. Here we show that a sizable fraction (∼30%) of 4E-BP1 is localized to the nucleus, where it binds eIF4E. In mouse embryo fibroblasts (MEFs) subjected to serum starvation and/or rapamycin treatment, nuclear 4E-BPs sequester eIF4E in the nucleus. A dramatic loss of nuclear 4E-BP1 occurs in c-Ha-Ras–expressing MEFs, which fail to show starvation-induced nuclear accumulation of eIF4E. Therefore, 4E-BP1 is a regulator of eIF4E cellular localization. PMID:18515545

Elucidation of the pharmacokinetics of prednisone and prednisolone: elimination and the effect of estrogen

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

Gustavson, L.E.

Several aspects of the pharmacokinetics of the interconvertible glucocorticoids prednisone and prednisolone have been studied. The pharmacokinetics of prednisolone were examined in postmenopausal women taking conjugated estrogens and age-matched control women. The subjects received iv bolus doses of 0.14 and 0.55 mg/kg prednisolone. Expected increases in clearance and volume of distribution with increasing dose were observed for total prednisolone in all subjects. At both doses, significant decreases in total and unbound prednisolone clearance were observed in the women taking estrogen compared to the controls. Volume of distribution was unchanged. The decreases in clearance are smaller than those observed in youngmore » women taking oral contraceptives indicating that factors other than estrogen administration may influence prednisolone clearance in oral contraceptive users. While the protein binding of prednisolone is well characterized, little is known about the protein binding of prednisone. Equilibrium dialysis employing (/sup 3/H)prednisone was used to study the binding of prednisone in human plasma containing endogenous hydrocortisone. Plasma was obtained from volunteers with normal and elevated transcortin binding capacities (CAP/sub T/). Prednisolone binding exhibits marked concentration dependence and sensitivity to CAP/sub T/. In contrast, prednisone binding is independent of concentration and CAP/sub T/.« less

Formin and capping protein together embrace the actin filament in a ménage à trois

PubMed Central

Shekhar, Shashank; Kerleau, Mikael; Kühn, Sonja; Pernier, Julien; Romet-Lemonne, Guillaume; Jégou, Antoine; Carlier, Marie-France

2015-01-01

Proteins targeting actin filament barbed ends play a pivotal role in motile processes. While formins enhance filament assembly, capping protein (CP) blocks polymerization. On their own, they both bind barbed ends with high affinity and very slow dissociation. Their barbed-end binding is thought to be mutually exclusive. CP has recently been shown to be present in filopodia and controls their morphology and dynamics. Here we explore how CP and formins may functionally coregulate filament barbed-end assembly. We show, using kinetic analysis of individual filaments by microfluidics-assisted fluorescence microscopy, that CP and mDia1 formin are able to simultaneously bind barbed ends. This is further confirmed using single-molecule imaging. Their mutually weakened binding enables rapid displacement of one by the other. We show that formin FMNL2 behaves similarly, thus suggesting that this is a general property of formins. Implications in filopodia regulation and barbed-end structural regulation are discussed. PMID:26564775

Eukaryotic Initiation Factor (eIF) 4F Binding to Barley Yellow Dwarf Virus (BYDV) 3′-Untranslated Region Correlates with Translation Efficiency*

PubMed Central

Banerjee, Bidisha; Goss, Dixie J.

2014-01-01

Eukaryotic initiation factor (eIF) 4F binding to mRNA is the first committed step in cap-dependent protein synthesis. Barley yellow dwarf virus (BYDV) employs a cap-independent mechanism of translation initiation that is mediated by a structural BYDV translation element (BTE) located in the 3′-UTR of its mRNA. eIF4F bound the BTE and a translationally inactive mutant with high affinity, thus questioning the role of eIF4F in translation of BYDV. To examine the effects of eIF4F in BYDV translation initiation, BTE mutants with widely different in vitro translation efficiencies ranging from 5 to 164% compared with WT were studied. Using fluorescence anisotropy to obtain quantitative data, we show 1) the equilibrium binding affinity (complex stability) correlated well with translation efficiency, whereas the “on” rate of binding did not; 2) other unidentified proteins or small molecules in wheat germ extract prevented eIF4F binding to mutant BTE but not WT BTE; 3) BTE mutant-eIF4F interactions were found to be both enthalpically and entropically favorable with an enthalpic contribution of 52–90% to ΔG° at 25 °C, suggesting that hydrogen bonding contributes to stability; and 4) in contrast to cap-dependent and tobacco etch virus internal ribosome entry site interaction with eIF4F, poly(A)-binding protein did not increase eIF4F binding. Further, the eIF4F bound to the 3′ BTE with higher affinity than for either m7G cap or tobacco etch virus internal ribosome entry site, suggesting that the 3′ BTE may play a role in sequestering host cell initiation factors and possibly regulating the switch from replication to translation. PMID:24379412

Mutational Analysis of Plant Cap-Binding Protein eIF4E Reveals Key Amino Acids Involved in Biochemical Functions and Potyvirus Infection▿

PubMed Central

German-Retana, Sylvie; Walter, Jocelyne; Doublet, Bénédicte; Roudet-Tavert, Geneviève; Nicaise, Valérie; Lecampion, Cécile; Houvenaghel, Marie-Christine; Robaglia, Christophe; Michon, Thierry; Le Gall, Olivier

2008-01-01

The eukaryotic translation initiation factor 4E (eIF4E) (the cap-binding protein) is involved in natural resistance against several potyviruses in plants. In lettuce, the recessive resistance genes mo11 and mo12 against Lettuce mosaic virus (LMV) are alleles coding for forms of eIF4E unable, or less effective, to support virus accumulation. A recombinant LMV expressing the eIF4E of a susceptible lettuce variety from its genome was able to produce symptoms in mo11 or mo12 varieties. In order to identify the eIF4E amino acid residues necessary for viral infection, we constructed recombinant LMV expressing eIF4E with point mutations affecting various amino acids and compared the abilities of these eIF4E mutants to complement LMV infection in resistant plants. Three types of mutations were produced in order to affect different biochemical functions of eIF4E: cap binding, eIF4G binding, and putative interaction with other virus or host proteins. Several mutations severely reduced the ability of eIF4E to complement LMV accumulation in a resistant host and impeded essential eIF4E functions in yeast. However, the ability of eIF4E to bind a cap analogue or to fully interact with eIF4G appeared unlinked to LMV infection. In addition to providing a functional mutational map of a plant eIF4E, this suggests that the role of eIF4E in the LMV cycle might be distinct from its physiological function in cellular mRNA translation. PMID:18480444

A novel type of light-harvesting antenna protein of red algal origin in algae with secondary plastids.

PubMed

Sturm, Sabine; Engelken, Johannes; Gruber, Ansgar; Vugrinec, Sascha; Kroth, Peter G; Adamska, Iwona; Lavaud, Johann

2013-07-30

Light, the driving force of photosynthesis, can be harmful when present in excess; therefore, any light harvesting system requires photoprotection. Members of the extended light-harvesting complex (LHC) protein superfamily are involved in light harvesting as well as in photoprotection and are found in the red and green plant lineages, with a complex distribution pattern of subfamilies in the different algal lineages. Here, we demonstrate that the recently discovered "red lineage chlorophyll a/b-binding-like proteins" (RedCAPs) form a monophyletic family within this protein superfamily. The occurrence of RedCAPs was found to be restricted to the red algal lineage, including red algae (with primary plastids) as well as cryptophytes, haptophytes and heterokontophytes (with secondary plastids of red algal origin). Expression of a full-length RedCAP:GFP fusion construct in the diatom Phaeodactylum tricornutum confirmed the predicted plastid localisation of RedCAPs. Furthermore, we observed that similarly to the fucoxanthin chlorophyll a/c-binding light-harvesting antenna proteins also RedCAP transcripts in diatoms were regulated in a diurnal way at standard light conditions and strongly repressed at high light intensities. The absence of RedCAPs from the green lineage implies that RedCAPs evolved in the red lineage after separation from the the green lineage. During the evolution of secondary plastids, RedCAP genes therefore must have been transferred from the nucleus of the endocytobiotic alga to the nucleus of the host cell, a process that involved complementation with pre-sequences allowing import of the gene product into the secondary plastid bound by four membranes. Based on light-dependent transcription and on localisation data, we propose that RedCAPs might participate in the light (intensity and quality)-dependent structural or functional reorganisation of the light-harvesting antennae of the photosystems upon dark to light shifts as regularly experienced by diatoms in nature. Remarkably, in plastids of the red lineage as well as in green lineage plastids, the phycobilisome based cyanobacterial light harvesting system has been replaced by light harvesting systems that are based on members of the extended LHC protein superfamily, either for one of the photosystems (PS I of red algae) or for both (diatoms). In their proposed function, the RedCAP protein family may thus have played a role in the evolutionary structural remodelling of light-harvesting antennae in the red lineage.

Adenylyl cyclase-associated protein 1 in metastasis of squamous cell carcinoma of the head and neck and non-small cell lung cancer

NASA Astrophysics Data System (ADS)

Kakurina, G. V.; Kolegova, E. S.; Cheremisina, O. V.; Zavyalov, A. A.; Shishkin, D. A.; Kondakova, I. V.; Choinzonov, E. L.

2016-08-01

Progression of tumors and metastasis in particular is one of the main reasons of the high mortality rate among cancer patients. The primary role in developing metastases plays cell locomotion which requires remodeling of the actin cytoskeleton. Form, dynamics, localization and mechanical properties of the actin cytoskeleton are regulated by a variety of actin-binding proteins, which include the adenylyl cyclase-associated protein 1 (CAP1). The study is devoted to the investigation of CAP1 level depending on the presence or absence of metastases in patients with squamous cell carcinoma of the head and neck (SCCHN) and non-small cell lung cancer (NSCLC). The results show the contribution of CAP1 to SCCHN and NSCLC progression. We detected the connection between the tissue protein CAP1 level and the stage of NSCLC and SCCHN disease. Also the levels of the CAP1 protein in tissues of primary tumors and metastases in lung cancer were different. Our data showed that CAP is important in the development of metastases, which suggests further perspectives in the study of this protein for projecting metastasis of NSCLC and SCCHN.

Characterization of rat leydig cell gonadotropin receptor structure by affinity cross-linking

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

Zhang, Q.Y.; Hwang, J.; Menon, K.M.J.

1986-05-01

The gonadotropin receptor from rat leydig cell has been characterized with respect to binding kinetics and physiological regulation. The present study was intended to examine the structure of the receptor. Leydig cell suspension was prepared by either collagenase digestion or by mechanical disruption of the testis. The cells were incubated with /sup 125/I-hCG and the unreacted hCG was removed by centrifugation. The /sup 125/I-hCG was then covalently linked to the cell surface receptor using cleavable (dithiobis (succinimidyl propionate)) and non-cleavable (disuccinimidyl suberate) cross-linking reagents. The extracted cross-linked membrane proteins were resolved on SDS-polyacrylamide gels under reducing and non-reducing conditions andmore » subjected to autoradiographic analysis. Under non-reducing conditions, two labeled species with M/sub r/ = 87,000 and 120,000 were detected. However, only one labeled band was detected under reducing conditions with M/sub r/ = 64,000. The binding of /sup 125/I-hCG to the receptor was inhibited by hCG and LH, but not by a number of peptides and proteins. The data suggest that hCG receptor in leydig cell is an oligomeric complex consisting of four subunits, ..cap alpha cap alpha beta gamma... The ..beta.. and ..gamma.. subunits are each linked to an ..cap alpha.. subunit through disulfide linkage and the hormone binds to each ..cap alpha.. subunit. The two dimers formed (..cap alpha beta cap alpha gamma..) are associated by noncovalent interactions.« less

The Reconstruction of Condition-Specific Transcriptional Modules Provides New Insights in the Evolution of Yeast AP-1 Proteins

PubMed Central

Goudot, Christel; Etchebest, Catherine

2011-01-01

AP-1 proteins are transcription factors (TFs) that belong to the basic leucine zipper family, one of the largest families of TFs in eukaryotic cells. Despite high homology between their DNA binding domains, these proteins are able to recognize diverse DNA motifs. In yeasts, these motifs are referred as YRE (Yap Response Element) and are either seven (YRE-Overlap) or eight (YRE-Adjacent) base pair long. It has been proposed that the AP-1 DNA binding motif preference relies on a single change in the amino acid sequence of the yeast AP-1 TFs (an arginine in the YRE-O binding factors being replaced by a lysine in the YRE-A binding Yaps). We developed a computational approach to infer condition-specific transcriptional modules associated to the orthologous AP-1 protein Yap1p, Cgap1p and Cap1p, in three yeast species: the model yeast Saccharomyces cerevisiae and two pathogenic species Candida glabrata and Candida albicans. Exploitation of these modules in terms of predictions of the protein/DNA regulatory interactions changed our vision of AP-1 protein evolution. Cis-regulatory motif analyses revealed the presence of a conserved adenine in 5′ position of the canonical YRE sites. While Yap1p, Cgap1p and Cap1p shared a remarkably low number of target genes, an impressive conservation was observed in the YRE sequences identified by Yap1p and Cap1p. In Candida glabrata, we found that Cgap1p, unlike Yap1p and Cap1p, recognizes YRE-O and YRE-A motifs. These findings were supported by structural data available for the transcription factor Pap1p (Schizosaccharomyces pombe). Thus, whereas arginine and lysine substitutions in Cgap1p and Yap1p proteins were reported as responsible for a specific YRE-O or YRE-A preference, our analyses rather suggest that the ancestral yeast AP-1 protein could recognize both YRE-O and YRE-A motifs and that the arginine/lysine exchange is not the only determinant of the specialization of modern Yaps for one motif or another. PMID:21695268

Synthesis, properties, and biological activity of boranophosphate analogs of the mRNA cap: versatile tools for manipulation of therapeutically relevant cap-dependent processes

PubMed Central

Kowalska, Joanna; Wypijewska del Nogal, Anna; Darzynkiewicz, Zbigniew M.; Buck, Janina; Nicola, Corina; Kuhn, Andreas N.; Lukaszewicz, Maciej; Zuberek, Joanna; Strenkowska, Malwina; Ziemniak, Marcin; Maciejczyk, Maciej; Bojarska, Elzbieta; Rhoads, Robert E.; Darzynkiewicz, Edward; Sahin, Ugur; Jemielity, Jacek

2014-01-01

Modified mRNA cap analogs aid in the study of mRNA-related processes and may enable creation of novel therapeutic interventions. We report the synthesis and properties of 11 dinucleotide cap analogs bearing a single boranophosphate modification at either the α-, β- or γ-position of the 5′,5′-triphosphate chain. The compounds can potentially serve either as inhibitors of translation in cancer cells or reagents for increasing expression of therapeutic proteins in vivo from exogenous mRNAs. The BH3-analogs were tested as substrates and binding partners for two major cytoplasmic cap-binding proteins, DcpS, a decapping pyrophosphatase, and eIF4E, a translation initiation factor. The susceptibility to DcpS was different between BH3-analogs and the corresponding analogs containing S instead of BH3 (S-analogs). Depending on its placement, the boranophosphate group weakened the interaction with DcpS but stabilized the interaction with eIF4E. The first of the properties makes the BH3-analogs more stable and the second, more potent as inhibitors of protein biosynthesis. Protein expression in dendritic cells was 2.2- and 1.7-fold higher for mRNAs capped with m27,2′-OGppBH3pG D1 and m27,2′-OGppBH3pG D2, respectively, than for in vitro transcribed mRNA capped with m27,3′-OGpppG. Higher expression of cancer antigens would make mRNAs containing m27,2′-OGppBH3pG D1 and m27,2′-OGppBH3pG D2 favorable for anticancer immunization. PMID:25150148

Structural determination of importin alpha in complex with beak and feather disease virus capsid nuclear localization signal

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

Patterson, Edward I.; EH Graham Centre for Agricultural Innovation; Dombrovski, Andrew K.

2013-09-06

Highlights: •Circovirus capsid proteins contain large nuclear localization signals (NLS). •A method of nuclear import has not been elucidated. •Beak and feather disease virus (BFDV) capsid NLS was crystallized with importin α. •The structure showed BFDV NLS binding to the major site of importin α. •Result shows implications for mechanism of nuclear transport for all circoviruses. -- Abstract: Circoviruses represent a rapidly increasing genus of viruses that infect a variety of vertebrates. Replication requires shuttling viral molecules into the host cell nucleus, a process facilitated by capsid-associated protein (Cap). Whilst a nuclear localization signal (NLS) has been shown to mediatemore » nuclear translocation, the mode of nuclear transport remains to be elucidated. To better understand this process, beak and feather disease virus (BFDV) Cap NLS was crystallized with nuclear import receptor importin-α (Impα). Diffraction yielded structural data to 2.9 Å resolution, and the binding site on both Impα and BFDV Cap NLS were well resolved. The binding mechanism for the major site is likely conserved across circoviruses as supported by the similarity of NLSs in circovirus Caps. This finding illuminates a crucial step for infection of host cells by this viral family, and provides a platform for rational drug design against the binding interface.« less

A new MIF4G domain-containing protein, CTIF, directs nuclear cap-binding protein CBP80/20-dependent translation

PubMed Central

Kim, Kyoung Mi; Cho, Hana; Choi, Kobong; Kim, Jaedong; Kim, Bong-Woo; Ko, Young-Gyu; Jang, Sung Key; Kim, Yoon Ki

2009-01-01

During or right after mRNA export via the nuclear pore complex (NPC) in mammalian cells, mRNAs undergo translation mediated by nuclear cap-binding proteins 80 and 20 (CBP80/20). After CBP80/20-dependent translation, CBP80/20 is replaced by cytoplasmic cap-binding protein eIF4E, which directs steady-state translation. Nonsense-mediated mRNA decay (NMD), one of the best-characterized mRNA surveillance mechanisms, has been shown to occur on CBP80/20-bound mRNAs. However, despite the tight link between CBP80/20-dependent translation and NMD, the underlying molecular mechanism and cellular factors that mediate CBP80/20-dependent translation remain obscure. Here, we identify a new MIF4G domain-containing protein, CTIF (CBP80/20-dependent translation initiation factor). CTIF interacts directly with CBP80 and is part of the CBP80/20-dependent translation initiation complex. Depletion of endogenous CTIF from an in vitro translation system selectively blocks the translation of CBP80-bound mRNAs, while addition of purified CTIF restores it. Accordingly, down-regulation of endogenous CTIF abrogates NMD. Confocal microscopy shows that CTIF is localized to the perinuclear region. Our observations demonstrate the existence of CBP80/20-dependent translation and support the idea that CBP80/20-dependent translation is mechanistically different from steady-state translation through identification of a specific cellular protein, CTIF. PMID:19648179

IFIT3 and IFIT2/3 promote IFIT1-mediated translation inhibition by enhancing binding to non-self RNA.

PubMed

Fleith, Renata C; Mears, Harriet V; Leong, Xin Yun; Sanford, Thomas J; Emmott, Edward; Graham, Stephen C; Mansur, Daniel S; Sweeney, Trevor R

2018-06-01

Interferon-induced proteins with tetratricopeptide repeats (IFITs) are highly expressed during the cell-intrinsic immune response to viral infection. IFIT1 inhibits translation by binding directly to the 5' end of foreign RNAs, particularly those with non-self cap structures, precluding the recruitment of the cap-binding eukaryotic translation initiation factor 4F and ribosome recruitment. The presence of IFIT1 imposes a requirement on viruses that replicate in the cytoplasm to maintain mechanisms to avoid its restrictive effects. Interaction of different IFIT family members is well described, but little is known of the molecular basis of IFIT association or its impact on function. Here, we reconstituted different complexes of IFIT1, IFIT2 and IFIT3 in vitro, which enabled us to reveal critical aspects of IFIT complex assembly. IFIT1 and IFIT3 interact via a YxxxL motif present in the C-terminus of each protein. IFIT2 and IFIT3 homodimers dissociate to form a more stable heterodimer that also associates with IFIT1. We show for the first time that IFIT3 stabilizes IFIT1 protein expression, promotes IFIT1 binding to a cap0 Zika virus reporter mRNA and enhances IFIT1 translation inhibition. This work reveals molecular aspects of IFIT interaction and provides an important missing link between IFIT assembly and function.

Functional properties of an isolated. cap alpha beta. heterodimeric human placenta insulin-like growth factor 1 receptor complex

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

Feltz, S.M.; Swanson, M.L.; Wemmie, J.A.

1988-05-03

Treatment of human placenta membranes at pH 8.5 in the presence of 2.0 mM dithiothreitol (DTT) for 5 min, followed by the simultaneous removal of the DTT and pH adjustment of pH 7.6, resulted in the formation of a functional ..cap alpha beta.. heterodimeric insulin-like growth factor 1 (IGF-1) receptor complex from the native ..cap alpha../sub 2/..beta../sub 2/ heterotetrameric disulfide-linked state. The membrane-bound ..cap alpha beta.. heterodimeric complex displayed similar curvilinear /sup 125/I-IGF-1 equilibrium binding compared to the ..cap alpha../sub 2/..beta../sub 2/ heterotetrameric complex. /sup 125/I-IGF-1 binding to both the isolated ..cap alpha../sub 2/..beta../sub 2/ heterotetrameric and ..cap alpha beta..more » heterodimeric complexes demonstrated a marked straightening of the Scatchard plots, compared to the placenta membrane-bound IGF-1 receptors, with a 2-fold increase in the high-affinity binding component. IGF-1 stimulation of IGF-1 receptor autophosphorylation indicated that the ligand-dependent activation of ..cap alpha beta.. heterodimeric protein kinase activity occurred concomitant with the reassociation into a covalent ..cap alpha../sub 2/..beta../sub 2/ heterotetrameric state. These data demonstrate that (i) a combination of alkaline pH and DTT treatment of human placenta membranes results in the formation of an ..cap alpha beta.. heterodimeric IGF-1 receptor complex, (ii) unlike the insulin receptor, high-affinity homogeneous IGF-1 binding occurs in both the ..cap alpha../sub 2/..beta../sub 2/ heterotetrameric and ..cap alpha beta.. heterodimeric complexes, and (iii) IGF-1-dependent autophosphorylation of the ..cap alpha beta.. heterodimeric IGF-1 receptor complex correlates wit an IGF-1 dependent covalent reassociation into an ..cap alpha../sub 2/..beta../sub 2/ heterotetrameric disulfide-linked state.« less

Prostate Cancer Risk in Relation to IGF-1 and its Genetic Determinants: A Case Control Study Within the Cancer Prostate Sweden Project (CAPS)

DTIC Science & Technology

2006-05-01

and the GHRH receptor (GHRHR). Ghrelin (GHRL), a recently identified new peptide hormone produced by endocrine cells in the stomach, also stimulates...GHRL Ghrelin GHSR Growth hormone secretagogue receptor IGFALS IGF binding protein, acid labile subunit IGFBP1 - 6 IGF-binding proteins 1 to 6

The human insulin mRNA is partly translated via a cap- and eIF4A-independent mechanism

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

Fred, Rikard G., E-mail: Rikard.Fred@mcb.uu.se; Sandberg, Monica; Pelletier, Jerry

Highlights: {yields} The polypyrimidine tract binding protein binds to the 5'-UTR of the insulin mRNA. {yields} Insulin mRNA can be translated via a cap-independent mechanism. {yields} The fraction cap-independent insulin synthesis increases during conditions of stress. {yields} The {beta}-cell is able to uphold basal insulin biosynthesis under conditions of stress. -- Abstract: The aim of this study was to investigate whether cap-independent insulin mRNA translation occurs in human pancreatic islets at basal conditions, during stimulation at a high glucose concentration and at conditions of nitrosative stress. We also aimed at correlating cap-independent insulin mRNA translation with binding of the IRESmore » trans-acting factor polypyrimidine tract binding protein (PTB) to the 5'-UTR of insulin mRNA. For this purpose, human islets were incubated for 2 h in the presence of low (1.67 mM) or high glucose (16.7 mM). Nitrosative stress was induced by addition of 1 mM DETA/NO and cap-dependent mRNA translation was inhibited with hippuristanol. Insulin biosynthesis rates were determined by radioactive labeling and immunoprecipitation. PTB affinity to insulin mRNA 5'-UTR was assessed by a magnetic micro bead pull-down procedure. We observed that in the presence of 1.67 mM glucose, approximately 70% of the insulin mRNA translation was inhibited by hippuristanol. Corresponding value from islets incubated at 16.7 mM glucose was 93%. DETA/NO treatment significantly decreased the translation of insulin by 85% in high glucose incubated islets, and by 50% at a low glucose concentration. The lowered insulin biosynthesis rates of DETA/NO-exposed islets were further suppressed by hippuristanol with 55% at 16.7 mM glucose but not at 1.67 mM glucose. Thus, hippuristanol-induced inhibition of insulin biosynthesis was less pronounced in DETA/NO-treated islets as compared to control islets. We observed also that PTB bound specifically to the insulin mRNA 5'-UTR in vitro, and that this binding corresponded well with rates of cap-independent insulin biosynthesis at the different conditions. In conclusion, our studies show that insulin biosynthesis is mainly cap-dependent at a high glucose concentration, but that the cap-independent biosynthesis of insulin can constitute as much as 40-100% of all insulin biosynthesis during conditions of nitrosative stress. These data suggest that the pancreatic {beta}-cell is able to uphold basal insulin synthesis at conditions of starvation and stress via a cap- and eIF4A-independent mechanism, possibly mediated by the binding of PTB to the 5'-UTR of the human insulin mRNA.« less

The human fatty acid-binding protein family: Evolutionary divergences and functions

PubMed Central

2011-01-01

Fatty acid-binding proteins (FABPs) are members of the intracellular lipid-binding protein (iLBP) family and are involved in reversibly binding intracellular hydrophobic ligands and trafficking them throughout cellular compartments, including the peroxisomes, mitochondria, endoplasmic reticulum and nucleus. FABPs are small, structurally conserved cytosolic proteins consisting of a water-filled, interior-binding pocket surrounded by ten anti-parallel beta sheets, forming a beta barrel. At the superior surface, two alpha-helices cap the pocket and are thought to regulate binding. FABPs have broad specificity, including the ability to bind long-chain (C16-C20) fatty acids, eicosanoids, bile salts and peroxisome proliferators. FABPs demonstrate strong evolutionary conservation and are present in a spectrum of species including Drosophila melanogaster, Caenorhabditis elegans, mouse and human. The human genome consists of nine putatively functional protein-coding FABP genes. The most recently identified family member, FABP12, has been less studied. PMID:21504868

CAPS drives trans-SNARE complex formation and membrane fusion through syntaxin interactions.

PubMed

James, Declan J; Kowalchyk, Judith; Daily, Neil; Petrie, Matt; Martin, Thomas F J

2009-10-13

Ca(2+)-dependent activator protein for secretion (CAPS) is an essential factor for regulated vesicle exocytosis that functions in priming reactions before Ca(2+)-triggered fusion of vesicles with the plasma membrane. However, the precise events that CAPS regulates to promote vesicle fusion are unclear. In the current work, we reconstituted CAPS function in a SNARE-dependent liposome fusion assay using VAMP2-containing donor and syntaxin-1/SNAP-25-containing acceptor liposomes. The CAPS stimulation of fusion required PI(4,5)P(2) in acceptor liposomes and was independent of Ca(2+), but Ca(2+) dependence was restored by inclusion of synaptotagmin. CAPS stimulated trans-SNARE complex formation concomitant with the stimulation of full membrane fusion at physiological SNARE densities. CAPS bound syntaxin-1, and CAPS truncations that competitively inhibited syntaxin-1 binding also inhibited CAPS-dependent fusion. The results revealed an unexpected activity of a priming protein to accelerate fusion by efficiently promoting trans-SNARE complex formation. CAPS may function in priming by organizing SNARE complexes on the plasma membrane.

Activity and subcellular compartmentalization of peroxisome proliferator-activated receptor alpha are altered by the centrosome-associated protein CAP350.

PubMed

Patel, Hansa; Truant, Ray; Rachubinski, Richard A; Capone, John P

2005-01-01

Peroxisome proliferator-activated nuclear hormone receptors (PPAR) are ligand-activated transcription factors that play pivotal roles in governing metabolic homeostasis and cell growth. PPARs are primarily in the nucleus but, under certain circumstances, can be found in the cytoplasm. We show here that PPAR(alpha) interacts with the centrosome-associated protein CAP350. CAP350 also interacts with PPAR(delta), PPAR(gamma) and liver-X-receptor alpha, but not with the 9-cis retinoic acid receptor, RXR(alpha). Immunofluorescence analysis indicated that PPAR(alpha) is diffusely distributed in the nucleus and excluded from the cytoplasm. However, in the presence of coexpressed CAP350, PPAR(alpha) colocalizes with CAP350 to discrete nuclear foci and to the centrosome, perinuclear region and intermediate filaments. In contrast, the subcellular distribution of RXR(alpha) or of thyroid hormone receptor alpha was not altered by coexpression of CAP350. An amino-terminal fragment of CAP350 was localized exclusively to nuclear foci and was sufficient to recruit PPAR(alpha) to these sites. Mutation of the single putative nuclear hormone receptor interacting signature motif LXXLL present in this fragment had no effect on its subnuclear localization but abrogated recruitment of PPAR(alpha) to nuclear foci. Surprisingly, mutation of the LXXLL motif in this CAP350 subfragment did not prevent its binding to PPAR(alpha) in vitro, suggesting that this motif serves some function other than PPAR(alpha) binding in recruiting PPAR(alpha) to nuclear spots. CAP350 inhibited PPAR(alpha)-mediated transactivation in an LXXLL-dependent manner, suggesting that CAP350 represses PPAR(alpha) function. Our findings implicate CAP350 in a dynamic process that recruits PPAR(alpha) to discrete nuclear and cytoplasmic compartments and suggest that altered intracellular compartmentalization represents a regulatory process that modulates PPAR function.

Novel single-stranded DNA binding protein-assisted fluorescence aptamer switch based on FRET for homogeneous detection of antibiotics.

PubMed

Wang, Ye; Gan, Ning; Zhou, You; Li, Tianhua; Cao, Yuting; Chen, Yinji

2017-01-15

Herein, a smart single-stranded DNA binding protein (SSB)-assisted fluorescence aptamer switch based on fluorescence resonance energy transfer (FRET) was designed. The FRET switch was synthesized by connecting SSB labeled quantum dots (QDs@SSB) as donor with aptamer (apt) labeled gold nanoparticles (AuNPs@apt) as acceptor, and it was employed for detecting chloramphenicol (CAP) in a homogenous solution. In the assay, the interaction between core-shell QDs@SSB and AuNPs@apt leads to a dramatic quenching (turning off). After adding CAP in the detection system, AuNPs@apt can bind the target specifically then separate QDs@SSB with AuNPs@apt-target, resulting in restoring the fluorescence intensity of QDs (turning on). Consequently, the fluorescence intensity recovers and the recovery extent can be used for detection of CAP in homogenous phase via optical responses. Under optimal conditions, the fluorescence intensity increased linearly with increasing concentrations of CAP from 0.005 to 100ngmL -1 . The limit of this fluorescence aptamer switch was around 3pgmL -1 for CAP detection. When the analyte is changed, the assay can be applied to detect other targets only by changing relative aptamer in AuNPs@apt probe. Furthermore, it has potential to be served as a simple, sensitive and portable platform for antibiotic contaminants detection in biological and environmental samples. Copyright © 2016 Elsevier B.V. All rights reserved.

Structure of human IFIT1 with capped RNA reveals adaptable mRNA binding and mechanisms for sensing N1 and N2 ribose 2′-O methylations

PubMed Central

Laudenbach, Beatrice Theres; Martínez-Montero, Saúl; Cencic, Regina; Habjan, Matthias; Pichlmair, Andreas; Damha, Masad J.; Pelletier, Jerry; Nagar, Bhushan

2017-01-01

IFIT1 (IFN-induced protein with tetratricopeptide repeats-1) is an effector of the host innate immune antiviral response that prevents propagation of virus infection by selectively inhibiting translation of viral mRNA. It relies on its ability to compete with the translation initiation factor eIF4F to specifically recognize foreign capped mRNAs, while remaining inactive against host mRNAs marked by ribose 2′-O methylation at the first cap-proximal nucleotide (N1). We report here several crystal structures of RNA-bound human IFIT1, including a 1.6-Å complex with capped RNA. IFIT1 forms a water-filled, positively charged RNA-binding tunnel with a separate hydrophobic extension that unexpectedly engages the cap in multiple conformations (syn and anti) giving rise to a relatively plastic and nonspecific mode of binding, in stark contrast to eIF4E. Cap-proximal nucleotides encircled by the tunnel provide affinity to compete with eIF4F while allowing IFIT1 to select against N1 methylated mRNA. Gel-shift binding assays confirm that N1 methylation interferes with IFIT1 binding, but in an RNA-dependent manner, whereas translation assays reveal that N1 methylation alone is not sufficient to prevent mRNA recognition at high IFIT1 concentrations. Structural and functional analysis show that 2′-O methylation at N2, another abundant mRNA modification, is also detrimental for RNA binding, thus revealing a potentially synergistic role for it in self- versus nonself-mRNA discernment. Finally, structure-guided mutational analysis confirms the importance of RNA binding for IFIT1 restriction of a human coronavirus mutant lacking viral N1 methylation. Our structural and biochemical analysis sheds new light on the molecular basis for IFIT1 translational inhibition of capped viral RNA. PMID:28251928

RPA and POT1: friends or foes at telomeres?

PubMed

Flynn, Rachel Litman; Chang, Sandy; Zou, Lee

2012-02-15

Telomere maintenance in cycling cells relies on both DNA replication and capping by the protein complex shelterin. Two single-stranded DNA (ssDNA)-binding proteins, replication protein A (RPA) and protection of telomere 1 (POT1) play critical roles in DNA replication and telomere capping, respectively. While RPA binds to ssDNA in a non-sequence-specific manner, POT1 specifically recognizes singlestranded TTAGGG telomeric repeats. Loss of POT1 leads to aberrant accumulation of RPA at telomeres and activation of the ataxia telangiectasia and Rad3-related kinase (ATR)-mediated checkpoint response, suggesting that POT1 antagonizes RPA binding to telomeric ssDNA. The requirement for both POT1 and RPA in telomere maintenance and the antagonism between the two proteins raises the important question of how they function in concert on telomeric ssDNA. Two interesting models were proposed by recent studies to explain the regulation of POT1 and RPA at telomeres. Here, we discuss how these models help unravel the coordination, and also the antagonism, between POT1 and RPA during the cell cycle.

Aptamer-based SERRS Sensor for Thrombin Detection

PubMed Central

Cho, Hansang; Baker, Brian R.; Wachsmann-Hogiu, Sebastian; Pagba, Cynthia V.; Laurence, Ted A.; Lane, Stephen M.; Lee, Luke P.; Tok, Jeffrey B.-H.

2012-01-01

We describe an aptamer-based Surface Enhanced Resonance Raman Scattering (SERRS) sensor with high sensitivity, specificity, and stability for the detection of a coagulation protein, human α-thrombin. The sensor achieves high sensitivity and a limit of detection of 100 pM by monitoring the SERRS signal change upon the single step of thrombin binding to immobilized thrombin binding aptamer. The selectivity of the sensor is demonstrated by the specific discrimination of thrombin from other protein analytes. The specific recognition and binding of thrombin by the thrombin binding aptamer is essential to the mechanism of the aptamer-based sensor, as shown through measurements using negative control oligonucleotides. In addition, the sensor can detect 1 nM thrombin in the presence of complex biofluids, such as 10% fetal calf serum, demonstrating that the immobilized, 5'-capped, 3'-capped aptamer is sufficiently robust for clinical diagnostic applications. Furthermore, the proposed sensor may be implemented for multiplexed detection using different aptamer-Raman probe complexes. PMID:19367849

Dissecting the telomere-inner nuclear membrane interface formed in meiosis.

PubMed

Pendlebury, Devon F; Fujiwara, Yasuhiro; Tesmer, Valerie M; Smith, Eric M; Shibuya, Hiroki; Watanabe, Yoshinori; Nandakumar, Jayakrishnan

2017-12-01

Tethering telomeres to the inner nuclear membrane (INM) allows homologous chromosome pairing during meiosis. The meiosis-specific protein TERB1 binds the telomeric protein TRF1 to establish telomere-INM connectivity and is essential for mouse fertility. Here we solve the structure of the human TRF1-TERB1 interface to reveal the structural basis for telomere-INM linkage. Disruption of this interface abrogates binding and compromises telomere-INM attachment in mice. An embedded CDK-phosphorylation site within the TRF1-binding region of TERB1 provides a mechanism for cap exchange, a late-pachytene phenomenon involving the dissociation of the TRF1-TERB1 complex. Indeed, further strengthening this interaction interferes with cap exchange. Finally, our biochemical analysis implicates distinct complexes for telomere-INM tethering and chromosome-end protection during meiosis. Our studies unravel the structure, stoichiometry, and physiological implications underlying telomere-INM tethering, thereby providing unprecedented insights into the unique function of telomeres in meiosis.

Immunogenicity and immunoprotection of porcine circovirus type 2 (PCV2) Cap protein displayed by Lactococcus lactis.

PubMed

Li, Peng-Cheng; Qiao, Xu-Wen; Zheng, Qi-Sheng; Hou, Ji-Bo

2016-01-27

The capsid (Cap) protein, an important immunoprotective protein of porcine circovirus type 2 (PCV2), was expressed on the cell surface of the Gram-positive food-grade bacterium, Lactococcus lactis. Cap protein was fused to the peptidoglycan binding domain (known as the protein anchor domain, PA) of the lactococcal AcmA cell-wall hydrolase. The Cap protein fusion was non-covalently rebound to the surface of non-genetically modified, non-living high-binder L. lactis cells (designated Gram-positive enhancer matrix (GEM) particles). Expression of the recombinant GEM-displaying capsid protein (GEM-PA-Cap) was verified by Western blotting and immunofluorescence and transmission electron microscopy assays. To evaluate the immunogenicity of the recombinant Cap protein (rCap), 20 PCV2-seronegative piglets were immunized with the GEM-PA-Cap subunit vaccine, GEM alone, or phosphate-buffered saline (PBS, challenge control and empty control). Each group consisted of five piglets. The results showed that the level of PCV2-specific antibodies in piglets immunized with the GEM-PA-Cap subunit vaccine was significantly higher than that of the piglets immunized with GEM alone or the control group at all the time points post-vaccination (P<0.01). After challenge with the PCV2 wild-type strain, piglets that received the GEM-PA-Cap subunit vaccine showed significantly higher average daily weight gain (DWG) and shorter fever duration than the other two groups (P<0.001). Furthermore, a significant reduction in the gross lung lesion scores and lymph node lesion scores was noted in the GEM-PA-Cap-immunized group compared with the scores of the GEM or PBS-treated group (P<0.01). The results suggest that recombinant rCap displayed by L. lactis GEM particles provided the piglets with significant immunoprotection from PCV2-associated disease. Thus, the novel GEM-PA-Cap subunit vaccine has potential to be considered an effective and safe candidate vaccine against PCV2 infection in piglets. Copyright © 2015. Published by Elsevier Ltd.

Condensin II Regulates Interphase Chromatin Organization Through the Mrg-Binding Motif of Cap-H2

PubMed Central

Wallace, Heather A.; Klebba, Joseph E.; Kusch, Thomas; Rogers, Gregory C.; Bosco, Giovanni

2015-01-01

The spatial organization of the genome within the eukaryotic nucleus is a dynamic process that plays a central role in cellular processes such as gene expression, DNA replication, and chromosome segregation. Condensins are conserved multi-subunit protein complexes that contribute to chromosome organization by regulating chromosome compaction and homolog pairing. Previous work in our laboratory has shown that the Cap-H2 subunit of condensin II physically and genetically interacts with the Drosophila homolog of human MORF4-related gene on chromosome 15 (MRG15). Like Cap-H2, Mrg15 is required for interphase chromosome compaction and homolog pairing. However, the mechanism by which Mrg15 and Cap-H2 cooperate to maintain interphase chromatin organization remains unclear. Here, we show that Cap-H2 localizes to interband regions on polytene chromosomes and co-localizes with Mrg15 at regions of active transcription across the genome. We show that co-localization of Cap-H2 on polytene chromosomes is partially dependent on Mrg15. We have identified a binding motif within Cap-H2 that is essential for its interaction with Mrg15, and have found that mutation of this motif results in loss of localization of Cap-H2 on polytene chromosomes and results in partial suppression of Cap-H2-mediated compaction and homolog unpairing. Our data are consistent with a model in which Mrg15 acts as a loading factor to facilitate Cap-H2 binding to chromatin and mediate changes in chromatin organization. PMID:25758823

Fusion of a Novel Genetically Engineered Chitosan Affinity Protein and Green Fluorescent Protein for Specific Detection of Chitosan In Vitro and In Situ

PubMed Central

Nampally, Malathi; Moerschbacher, Bruno Maria

2012-01-01

Chitin is the second most abundant polysaccharide, present, e.g., in insect and arthropod exoskeletons and fungal cell walls. In some species or under specific conditions, chitin appears to be enzymatically de-N-acetylated to chitosan—e.g., when pathogenic fungi invade their host tissues. Here, the deacetylation of chitin is assumed to represent a pathogenicity mechanism protecting the fungus from the host's chitin-driven immune response. While highly specific chitin binding lectins are well known and easily available, this is not the case for chitosan-specific probes. This is partly due to the poor antigenicity of chitosan so that producing high-affinity, specific antibodies is difficult. Also, lectins with specificity to chitosan have been described but are not commercially available, and our attempts to reproduce the findings were not successful. We have, therefore, generated a fusion protein between a chitosanase inactivated by site-directed mutagenesis, the green fluorescent protein (GFP), and StrepII, as well as His6 tags for purification and detection. The recombinant chitosan affinity protein (CAP) expressed in Escherichia coli was shown to specifically bind to chitosan, but not to chitin, and the affinity increased with decreasing degree of acetylation. In vitro, CAP detection was possible either based on GFP fluorescence or using Strep-Tactin conjugates or anti-His5 antibodies. CAP fluorescence microscopy revealed binding to the chitosan exposing endophytic infection structures of the wheat stem rust fungus, but not the chitin exposing ectophytic infection structures, verifying its suitability for in situ chitosan staining. PMID:22367086

Protein Translation and Signaling in Human Eosinophils

PubMed Central

Esnault, Stephane; Shen, Zhong-Jian; Malter, James S.

2017-01-01

We have recently reported that, unlike IL-5 and GM-CSF, IL-3 induces increased translation of a subset of mRNAs. In addition, we have demonstrated that Pin1 controls the activity of mRNA binding proteins, leading to enhanced mRNA stability, GM-CSF protein production and prolonged eosinophil (EOS) survival. In this review, discussion will include an overview of cap-dependent protein translation and its regulation by intracellular signaling pathways. We will address the more general process of mRNA post-transcriptional regulation, especially regarding mRNA binding proteins, which are critical effectors of protein translation. Furthermore, we will focus on (1) the roles of IL-3-driven sustained signaling on enhanced protein translation in EOS, (2) the mechanisms regulating mRNA binding proteins activity in EOS, and (3) the potential targeting of IL-3 signaling and the signaling leading to mRNA binding activity changes to identify therapeutic targets to treat EOS-associated diseases. PMID:28971096

Thermodynamic and Kinetics Analysis of Peptides Derived from CapZ, NDR, p53, HDM2, and HDM4 Binding to Human S100B

PubMed Central

Wafer, Lucas N.; Streicher, Werner W.; McCallum, Scott A.; Makhatadze, George I.

2012-01-01

S100B is a member of the S100 subfamily of EF-hand proteins that has been implicated in malignant melanoma and neurodegenerative conditions such as Alzheimer's and Parkinson's disease. Calcium-induced conformational changes expose a hydrophobic binding cleft, facilitating interactions with a wide variety of nuclear, cytoplasmic, and extracellular target proteins. Previously, peptides derived from CapZ, p53, NDR, HDM2 and HDM4 have been shown to interact with S100B in a calcium-dependent manner. However, the thermodynamic and kinetic basis of these interactions remains largely unknown. To gain further insight, these peptides were screened against the S100B protein using isothermal titration calorimetry and nuclear magnetic resonance. All peptides were found to have binding affinities in the low micromolar to nanomolar range. Binding-induced changes in the line shapes of S100B backbone 1H and 15N were monitored to obtain the dissociation constants and the kinetic binding parameters. The large microscopic Kon rate constants observed in this study, Kon ≥1×107 M-1s-1, suggest that S100B utilizes a “fly casting mechanism” in the recognition of these peptide targets. PMID:22913742

How to find the optimal partner--studies of snurportin 1 interactions with U snRNA 5' TMG-cap analogues containing modified 2-amino group of 7-methylguanosine.

PubMed

Piecyk, Karolina; Niedzwiecka, Anna; Ferenc-Mrozek, Aleksandra; Lukaszewicz, Maciej; Darzynkiewicz, Edward; Jankowska-Anyszka, Marzena

2015-08-01

Snurportin 1 is an adaptor protein that mediates the active nuclear import of uridine-rich small nuclear RNAs (U snRNA) by the importin-β receptor pathway. Its cellular activity influences the overall transport yield of small ribonucleoprotein complexes containing N(2),N(2),7-trimethylguanosine (TMG) capped U snRNA. So far little is still known about structural requirements related to molecular recognition of the trimethylguanosine moiety by snurportin in solution. Since these interactions are of a great biomedical importance, we synthesized a series of new 7-methylguanosine cap analogues with extended substituents at the exocyclic 2-amino group to gain a deeper insight into how the TMG-cap is adapted into the snurportin cap-binding pocket. Prepared chemical tools were applied in binding assays using emission spectroscopy. Surprisingly, our results revealed strict selectivity of snurportin towards the TMG-cap structure that relied mainly on its structural stiffness and compactness. Copyright © 2015 Elsevier Ltd. All rights reserved.

The mRNA cap-binding protein Cbc1 is required for high and timely expression of genes by promoting the accumulation of gene-specific activators at promoters.

PubMed

Li, Tianlu; De Clercq, Nikki; Medina, Daniel A; Garre, Elena; Sunnerhagen, Per; Pérez-Ortín, José E; Alepuz, Paula

2016-02-01

The highly conserved Saccharomyces cerevisiae cap-binding protein Cbc1/Sto1 binds mRNA co-transcriptionally and acts as a key coordinator of mRNA fate. Recently, Cbc1 has also been implicated in transcription elongation and pre-initiation complex (PIC) formation. Previously, we described Cbc1 to be required for cell growth under osmotic stress and to mediate osmostress-induced translation reprogramming. Here, we observe delayed global transcription kinetics in cbc1Δ during osmotic stress that correlates with delayed recruitment of TBP and RNA polymerase II to osmo-induced promoters. Interestingly, we detect an interaction between Cbc1 and the MAPK Hog1, which controls most gene expression changes during osmostress, and observe that deletion of CBC1 delays the accumulation of the activator complex Hot1-Hog1 at osmostress promoters. Additionally, CBC1 deletion specifically reduces transcription rates of highly transcribed genes under non-stress conditions, such as ribosomal protein (RP) genes, while having low impact on transcription of weakly expressed genes. For RP genes, we show that recruitment of the specific activator Rap1, and subsequently TBP, to promoters is Cbc1-dependent. Altogether, our results indicate that binding of Cbc1 to the capped mRNAs is necessary for the accumulation of specific activators as well as PIC components at the promoters of genes whose expression requires high and rapid transcription. Copyright © 2016 Elsevier B.V. All rights reserved.

Functional reconstitution of prostaglandin E receptor from bovine adrenal medulla with guanine nucleotide binding proteins

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

Negishi, M.; Ito, S.; Yokohama, H.

1988-05-15

Prostaglandin E/sub 2/ (PEG/sub 2/) was found to bind specifically to a 100,000 x g pellet prepared from bovine adrenal medulla. The PGE receptor was associated with a GTP-binding protein (G-protein) and could be covalently cross-linked with this G-protein by dithiobis(succinimidyl propionate) in the 100,000 x g pellet. In order to characterize the G-protein associated with the PGE receptor and reconstitute these proteins in phospholipid vesicles, the authors purified the G-protein to apparent homogeneity from the 100,000 x g pellet. The G-protein served as a substrate of pertussis toxin but differed in its ..cap alpha.. subunit from two known pertussismore » toxin substrate G-proteins (G/sub i/ and G/sub 0/) purified from bovine brain. The molecular weight of the ..cap alpha.. subunit was 40,000, which is between those of G/sub i/ and G/sub 0/. The purified protein was also distinguished immunologically from G/sub i/ and G/sub 0/ and was referred to as G/sub am/. Reconstitution of the PGE receptor with pure C/sub am/, G/sub i/, or G/sub 0/ in phospholipid vesicles resulted in a remarkable restoration of (/sup 3/H)PGE/sub 2/ binding activity in a GTP-dependent manner. The efficiency of these three G-proteins in this capacity was roughly equal. When pertussis toxin- or N-ethylmaleimide-treated G-proteins, instead of the native ones, were reconstituted into vesicles, the restoration of binding activity was no longer observed. These results indicate that the PGE receptor can couple functionally with G/sub am/, G/sub i/, or G/sub 0/ in phospholipid vesicles and suggest that G/sub am/ may be involved in signal transduction of the PGE receptor in bovine adrenal medulla.« less

Binding Linkage in a Telomere DNA–Protein Complex at the Ends of Oxytricha nova Chromosomes

PubMed Central

Buczek, Pawel; Orr, Rochelle S.; Pyper, Sean R.; Shum, Mili; Ota, Emily Kimmel Irene; Gerum, Shawn E.; Horvath, Martin P.

2005-01-01

Alpha and beta protein subunits of the telomere end binding protein from Oxytricha nova (OnTEBP) combine with telomere single strand DNA to form a protective cap at the ends of chromosomes. We tested how protein–protein interactions seen in the co-crystal structure relate to DNA binding through use of fusion proteins engineered as different combinations of domains and subunits derived from OnTEBP. Joining alpha and beta resulted in a protein that bound single strand telomere DNA with high affinity (KD-DNA=1.4 nM). Another fusion protein, constructed without the C-terminal protein–protein interaction domain of alpha, bound DNA with 200-fold diminished affinity (KD-DNA=290 nM) even though the DNA-binding domains of alpha and beta were joined through a peptide linker. Adding back the alpha C-terminal domain as a separate protein restored high-affinity DNA binding. The binding behaviors of these fusion proteins and the native protein subunits are consistent with cooperative linkage between protein-association and DNA-binding equilibria. Linking DNA–protein stability to protein–protein contacts at a remote site may provide a trigger point for DNA–protein disassembly during telomere replication when the single strand telomere DNA must exchange between a very stable OnTEBP complex and telomerase. PMID:15967465

Nuclear assortment of eIF4E coincides with shut-off of host protein synthesis upon poliovirus infection.

PubMed

Sukarieh, R; Sonenberg, N; Pelletier, J

2010-05-01

Eukaryotic initiation factor (eIF) 4E is a subunit of the cap-binding protein complex, eIF4F, which recognizes the cap structure of cellular mRNAs to facilitate translation initiation. eIF4E is assembled into the eIF4F complex via its interaction with eIF4G, an event that is under Akt/mTOR regulation. The eIF4E-eIF4G interaction is regulated by the eIF4E binding partners, eIF4E-binding proteins and eIF4E-transporter. Cleavage of eIF4G occurs upon poliovirus infection and is responsible for the shut-off of host-cell protein synthesis observed early in infection. Here, we document that relocalization of eIF4E to the nucleus occurs concomitantly with cleavage of eIF4G upon poliovirus infection. This event is not dependent upon virus replication, but is dependent on eIF4G cleavage. We postulate that eIF4E nuclear relocalization may contribute to the shut-off of host protein synthesis that is a hallmark of poliovirus infection by perturbing the circular status of actively translating mRNAs.

Towards novel efficient and stable nuclear import signals: synthesis and properties of trimethylguanosine cap analogs modified within the 5',5'-triphosphate bridge.

PubMed

Zytek, Malgorzata; Kowalska, Joanna; Lukaszewicz, Maciej; Wojtczak, Blazej A; Zuberek, Joanna; Ferenc-Mrozek, Aleksandra; Darzynkiewicz, Edward; Niedzwiecka, Anna; Jemielity, Jacek

2014-12-07

A trimethylguanosine (TMG) cap is present at the 5' end of several small nuclear and nucleolar RNAs. Recently, it has been reported that the TMG cap is a potential nuclear import signal for nucleus-targeting therapeutic nucleic acids and proteins. The import is mediated by recognition of the TMG cap by the snRNA transporting protein, snurportin1. This work describes the synthesis and properties of a series of dinucleotide TMG cap (m3(2,2,7)GpppG) analogs modified in the 5',5'-triphosphate bridge as tools to study TMG cap-dependent biological processes. The bridge was altered at different positions by introducing either bridging (imidodiphosphate, O to NH and methylenebisphosphonate, O to CH2) or non-bridging (phosphorothioate, O to S and boranophosphate, O to BH3) modifications, or by elongation to tetraphosphate. The stability of novel analogs in blood serum was studied to reveal that the α,β-bridging O to NH substitution (m3(2,2,7)GppNHpG) confers the highest resistance. Short RNAs capped with analogs containing α,β-bridging (m3(2,2,7)GppNHpG) or β-non-bridging (m3(2,2,7)GppSpG D2) modifications were resistant to decapping pyrophosphatase, hNudt16. Preliminary studies on binding by human snurportin1 revealed that both O to NH and O to S substitutions support this binding. Due to favorable properties in all three assays, m3(2,2,7)GppNHpG was selected as a promising candidate for further studies on the efficiency of the TMG cap as a nuclear import signal.

La-related protein 1 (LARP1) repression of TOP mRNA translation is mediated through its cap-binding domain and controlled by an adjacent regulatory region

PubMed Central

Philippe, Lucas; Vasseur, Jean-Jacques; Debart, Françoise

2018-01-01

Abstract Cell growth is a complex process shaped by extensive and coordinated changes in gene expression. Among these is the tightly regulated translation of a family of growth-related mRNAs defined by a 5′ terminal oligopyrimidine (TOP) motif. TOP mRNA translation is partly controlled via the eukaryotic initiation factor 4F (eIF4F), a translation factor that recognizes the mRNA 5′ cap structure. Recent studies have also implicated La-related protein 1 (LARP1), which competes with eIF4F for binding to mRNA 5′ ends. However, it has remained controversial whether LARP1 represses TOP mRNA translation directly and, if so, what features define its mRNA targets. Here, we show that the C-terminal half of LARP1 is necessary and sufficient to control TOP mRNA translation in cells. This fragment contains the DM15 cap-binding domain as well as an adjacent regulatory region that we identified. We further demonstrate that purified LARP1 represses TOP mRNA translation in vitro through the combined recognition of both the TOP sequence and cap structure, and that its intrinsic repressive activity and affinity for these features are subject to regulation. These results support a model whereby the translation of TOP mRNAs is controlled by a growth-regulated competition between eIF4F and LARP1 for their 5′ ends. PMID:29244122

Dissecting the telomere–inner nuclear membrane interface formed in meiosis

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

Pendlebury, Devon F.; Fujiwara, Yasuhiro; Tesmer, Valerie M.

Tethering telomeres to the inner nuclear membrane (INM) allows homologous chromosome pairing during meiosis. The meiosis-specific protein TERB1 binds the telomeric protein TRF1 to establish telomere–INM connectivity and is essential for mouse fertility. Here we solve the structure of the human TRF1–TERB1 interface to reveal the structural basis for telomere–INM linkage. Disruption of this interface abrogates binding and compromises telomere–INM attachment in mice. An embedded CDK-phosphorylation site within the TRF1-binding region of TERB1 provides a mechanism for cap exchange, a late-pachytene phenomenon involving the dissociation of the TRF1–TERB1 complex. Indeed, further strengthening this interaction interferes with cap exchange. Finally, ourmore » biochemical analysis implicates distinct complexes for telomere–INM tethering and chromosome-end protection during meiosis. Our studies unravel the structure, stoichiometry, and physiological implications underlying telomere–INM tethering, thereby providing unprecedented insights into the unique function of telomeres in meiosis.« less

Alpha-catenin-dependent recruitment of the centrosomal protein CAP350 to adherens junctions allows epithelial cells to acquire a columnar shape.

PubMed

Gavilan, Maria P; Arjona, Marina; Zurbano, Angel; Formstecher, Etienne; Martinez-Morales, Juan R; Bornens, Michel; Rios, Rosa M

2015-03-01

Epithelial morphogenesis involves a dramatic reorganisation of the microtubule cytoskeleton. How this complex process is controlled at the molecular level is still largely unknown. Here, we report that the centrosomal microtubule (MT)-binding protein CAP350 localises at adherens junctions in epithelial cells. By two-hybrid screening, we identified a direct interaction of CAP350 with the adhesion protein α-catenin that was further confirmed by co-immunoprecipitation experiments. Block of epithelial cadherin (E-cadherin)-mediated cell-cell adhesion or α-catenin depletion prevented CAP350 localisation at cell-cell junctions. Knocking down junction-located CAP350 inhibited the establishment of an apico-basal array of microtubules and impaired the acquisition of columnar shape in Madin-Darby canine kidney II (MDCKII) cells grown as polarised epithelia. Furthermore, MDCKII cystogenesis was also defective in junctional CAP350-depleted cells. CAP350-depleted MDCKII cysts were smaller and contained either multiple lumens or no lumen. Membrane polarity was not affected, but cortical microtubule bundles did not properly form. Our results indicate that CAP350 may act as an adaptor between adherens junctions and microtubules, thus regulating epithelial differentiation and contributing to the definition of cell architecture. We also uncover a central role of α-catenin in global cytoskeleton remodelling, in which it acts not only on actin but also on MT reorganisation during epithelial morphogenesis.

Alpha-catenin-Dependent Recruitment of the Centrosomal Protein CAP350 to Adherens Junctions Allows Epithelial Cells to Acquire a Columnar Shape

PubMed Central

Zurbano, Angel; Formstecher, Etienne; Martinez-Morales, Juan R.; Bornens, Michel; Rios, Rosa M.

2015-01-01

Epithelial morphogenesis involves a dramatic reorganisation of the microtubule cytoskeleton. How this complex process is controlled at the molecular level is still largely unknown. Here, we report that the centrosomal microtubule (MT)-binding protein CAP350 localises at adherens junctions in epithelial cells. By two-hybrid screening, we identified a direct interaction of CAP350 with the adhesion protein α-catenin that was further confirmed by co-immunoprecipitation experiments. Block of epithelial cadherin (E-cadherin)-mediated cell-cell adhesion or α-catenin depletion prevented CAP350 localisation at cell-cell junctions. Knocking down junction-located CAP350 inhibited the establishment of an apico-basal array of microtubules and impaired the acquisition of columnar shape in Madin-Darby canine kidney II (MDCKII) cells grown as polarised epithelia. Furthermore, MDCKII cystogenesis was also defective in junctional CAP350-depleted cells. CAP350-depleted MDCKII cysts were smaller and contained either multiple lumens or no lumen. Membrane polarity was not affected, but cortical microtubule bundles did not properly form. Our results indicate that CAP350 may act as an adaptor between adherens junctions and microtubules, thus regulating epithelial differentiation and contributing to the definition of cell architecture. We also uncover a central role of α-catenin in global cytoskeleton remodelling, in which it acts not only on actin but also on MT reorganisation during epithelial morphogenesis. PMID:25764135

Mextli proteins use both canonical bipartite and novel tripartite binding modes to form eIF4E complexes that display differential sensitivity to 4E-BP regulation

PubMed Central

Peter, Daniel; Weber, Ramona; Köne, Carolin; Chung, Min-Yi; Ebertsch, Linda; Truffault, Vincent; Weichenrieder, Oliver; Igreja, Cátia; Izaurralde, Elisa

2015-01-01

The eIF4E-binding proteins (4E-BPs) are a diverse class of translation regulators that share a canonical eIF4E-binding motif (4E-BM) with eIF4G. Consequently, they compete with eIF4G for binding to eIF4E, thereby inhibiting translation initiation. Mextli (Mxt) is an unusual 4E-BP that promotes translation by also interacting with eIF3. Here we present the crystal structures of the eIF4E-binding regions of the Drosophila melanogaster (Dm) and Caenorhabditis elegans (Ce) Mxt proteins in complex with eIF4E in the cap-bound and cap-free states. The structures reveal unexpected evolutionary plasticity in the eIF4E-binding mode, with a classical bipartite interface for Ce Mxt and a novel tripartite interface for Dm Mxt. Both interfaces comprise a canonical helix and a noncanonical helix that engage the dorsal and lateral surfaces of eIF4E, respectively. Remarkably, Dm Mxt contains a C-terminal auxiliary helix that lies anti-parallel to the canonical helix on the eIF4E dorsal surface. In contrast to the eIF4G and Ce Mxt complexes, the Dm eIF4E–Mxt complexes are resistant to competition by bipartite 4E-BPs, suggesting that Dm Mxt can bind eIF4E when eIF4G binding is inhibited. Our results uncovered unexpected diversity in the binding modes of 4E-BPs, resulting in eIF4E complexes that display differential sensitivity to 4E-BP regulation. PMID:26294658

Single-molecule visualization of a formin-capping protein ‘decision complex' at the actin filament barbed end

PubMed Central

Bombardier, Jeffrey P.; Eskin, Julian A.; Jaiswal, Richa; Corrêa, Ivan R.; Xu, Ming-Qun; Goode, Bruce L.; Gelles, Jeff

2015-01-01

Precise control of actin filament length is essential to many cellular processes. Formins processively elongate filaments, whereas capping protein (CP) binds to barbed ends and arrests polymerization. While genetic and biochemical evidence has indicated that these two proteins function antagonistically, the mechanism underlying the antagonism has remained unresolved. Here we use multi-wavelength single-molecule fluorescence microscopy to observe the fully reversible formation of a long-lived ‘decision complex' in which a CP dimer and a dimer of the formin mDia1 simultaneously bind the barbed end. Further, mDia1 displaced from the barbed end by CP can randomly slide along the filament and later return to the barbed end to re-form the complex. Quantitative kinetic analysis reveals that the CP-mDia1 antagonism that we observe in vitro occurs through the decision complex. Our observations suggest new molecular mechanisms for the control of actin filament length and for the capture of filament barbed ends in cells. PMID:26566078

Mapping the binding site of snurportin 1 on native U1 snRNP by cross-linking and mass spectrometry

PubMed Central

Kühn-Hölsken, Eva; Lenz, Christof; Dickmanns, Achim; Hsiao, He-Hsuan; Richter, Florian M.; Kastner, Berthold; Ficner, Ralf; Urlaub, Henning

2010-01-01

Mass spectrometry allows the elucidation of molecular details of the interaction domains of the individual components in macromolecular complexes subsequent to cross-linking of the individual components. Here, we applied chemical and UV cross-linking combined with tandem mass-spectrometric analysis to identify contact sites of the nuclear import adaptor snurportin 1 to the small ribonucleoprotein particle U1 snRNP in addition to the known interaction of m3G cap and snurportin 1. We were able to define previously unknown sites of protein–protein and protein–RNA interactions on the molecular level within U1 snRNP. We show that snurportin 1 interacts with its central m3G-cap-binding domain with Sm proteins and with its extreme C-terminus with stem-loop III of U1 snRNA. The crosslinking data support the idea of a larger interaction area between snurportin 1 and U snRNPs and the contact sites identified prove useful for modeling the spatial arrangement of snurportin 1 domains when bound to U1 snRNP. Moreover, this suggests a functional nuclear import complex that assembles around the m3G cap and the Sm proteins only when the Sm proteins are bound and arranged in the proper orientation to the cognate Sm site in U snRNA. PMID:20421206

Effects of composite antimicrobial peptides in weanling piglets challenged with deoxynivalenol: II. Intestinal morphology and function.

PubMed

Xiao, H; Tan, B E; Wu, M M; Yin, Y L; Li, T J; Yuan, D X; Li, L

2013-10-01

Deoxynivalenol (DON) affects animal and human health and targets the gastrointestinal tract. The objective of this study was to evaluate the ability of composite antimicrobial peptides (CAP) to repair intestinal injury in piglets challenged with DON. A total of 28 piglets (Duroc × Landrace × Large Yorkshire) weaned at 28 d of age were randomly assigned to receive 1 of 4 treatments (7 pigs/treatment): negative control, basal diet (NC), basal diet + 0.4% composite antimicrobial peptide (CAP), basal diet + 4 mg/kg DON (DON), and basal diet + 4 mg/kg DON + 0.4% CAP (DON + CAP). After an adaptation period of 7 d, blood samples were collected on d 15 and 30 after the initiation of treatment for determinations of the concentrations of D-lactate and diamine oxidase. At the end of the study, all piglets were slaughtered to obtain small intestines for the determination of intestinal morphology, epithelial cell proliferation, and protein expression in the mammalian target of rapamycin (mTOR) signaling pathway. The results showed that DON increased serum concentrations of D-lactate and diamine oxidase, and these values in the CAP and DON + CAP treatments were less than those in the NC and DON treatments, respectively (P < 0.05). The villous height/crypt depth in the jejunum and ileum and the goblet cell number in the ileum in the CAP and DON + CAP treatments were greater than those in the NC and DON treatments (P < 0.05). The proliferating cell nuclear antigen (PCNA) labeling indexes for the jejunum and ileum in the DON + CAP treatment were greater than those in the DON treatment (P < 0.05). The DON decreased (P < 0.05) the relative protein expression of phosphorylated Akt (Protein Kinase B) and mTOR in the jejunal and ileal mucosa and of phosphorylated 4E-binding protein 1 (p-4EBP1) in the jejunal mucosa, whereas CAP increased (P < 0.05) the protein expression of p-4EBP1 in the jejunum. These findings showed that DON could enhance intestinal permeability, damage villi, cause epithelial cell apoptosis, and inhibit protein synthesis, whereas CAP improved intestinal morphology and promoted intestinal epithelial cell proliferation and protein synthesis, indicating that CAP may repair the intestinal injury induced by DON.

Myofibril growth during cardiac hypertrophy is regulated through dual phosphorylation and acetylation of the actin capping protein CapZ

PubMed Central

Lin, Ying-Hsi; Warren, Chad M.; Li, Jieli; McKinsey, Timothy A.; Russell, Brenda

2016-01-01

The mechanotransduction signaling pathways initiated in heart muscle by increased mechanical loading are known to lead to long-term transcriptional changes and hypertrophy, but the rapid events for adaptation at the sarcomeric level are not fully understood. The goal of this study was to test the hypothesis that actin filament assembly during cardiomyocyte growth is regulated by post-translational modifications (PTMs) of CapZβ1. In rapidly hypertrophying neonatal rat ventricular myocytes (NRVMs) stimulated by phenylephrine (PE), two-dimensional gel electrophoresis (2DGE) of CapZβ1 revealed a shift toward more negative charge. Consistent with this, mass spectrometry identified CapZβ1 phosphorylation on serine-204 and acetylation on lysine-199, two residues which are near the actin binding surface of CapZβ1. Ectopic expression of dominant negative PKCε (dnPKCε) in NRVMs blunted the PE-induced increase in CapZ dynamics, as evidenced by the kinetic constant (Kfrap) of fluorescence recovery after photobleaching (FRAP), and concomitantly reduced phosphorylation and acetylation of CapZβ1. Furthermore, inhibition of class I histone deacetylases (HDACs) increased lysine-199 acetylation on CapZβ1, which increased Kfrap of CapZ and stimulated actin dynamics. Finally, we show that PE treatment of NRVMs results in decreased binding of HDAC3 to myofibrils, suggesting a signal-dependent mechanism for the regulation of sarcomere-associated CapZβ1 acetylation. Taken together, this dual regulation through phosphorylation and acetylation of CapZβ1 provides a novel model for the regulation of myofibril growth during cardiac hypertrophy. PMID:27185186

Analysis of Carbohydrate-Carbohydrate Interactions Using Sugar-Functionalized Silicon Nanoparticles for Cell Imaging.

PubMed

Lai, Chian-Hui; Hütter, Julia; Hsu, Chien-Wei; Tanaka, Hidenori; Varela-Aramburu, Silvia; De Cola, Luisa; Lepenies, Bernd; Seeberger, Peter H

2016-01-13

Protein-carbohydrate binding depends on multivalent ligand display that is even more important for low affinity carbohydrate-carbohydrate interactions. Detection and analysis of these low affinity multivalent binding events are technically challenging. We describe the synthesis of dual-fluorescent sugar-capped silicon nanoparticles that proved to be an attractive tool for the analysis of low affinity interactions. These ultrasmall NPs with sizes of around 4 nm can be used for NMR quantification of coupled sugars. The silicon nanoparticles are employed to measure the interaction between the cancer-associated glycosphingolipids GM3 and Gg3 and the associated kD value by surface plasmon resonance experiments. Cell binding studies, to investigate the biological relevance of these carbohydrate-carbohydrate interactions, also benefit from these fluorescent sugar-capped nanoparticles.

Selection and identification of single-domain antibody fragment against capsid protein of porcine circovirus type 2 (PCV2) from C. bactrianus.

PubMed

Yang, Shunli; Shang, Youjun; Yin, Shuanghui; Tian, Hong; Chen, Yan; Sun, Shiqi; Jin, Ye; Liu, Xiangtao

2014-07-15

Single-domain variable heavy chain (VHH) antibody fragments are derived from heavy-chain antibodies of Camelids. Their comparatively small size, solubility, high affinity and specificity to the targets antigen make them suitable for many biotechnological applications. In this study, a VHH library was constructed from porcine circovirus type 2 (PCV2) vaccine immunized C. bactrianus and three VHH fragments specific to the capsid protein of PCV2 (PCV2 Cap) were selected and characterized. The selected VHH clones (VHH-c1/c3/c4) were stably expressed as soluble protein in E. coli, and were specific to PCV2 Cap except VHH-c3 which shows binding activity with both PCV1 and PCV2 Cap by ELISA. All the VHH-cs show high association rate constant and dissociation rate constant, which was 1.84 × 10(5)M(-1)s(-1), 9.00 × 10(-3)s(-1) for VHH-c1, 5.49 × 10(4)M(-1)s(-1), 9.91 × 10(-3)s(-1) and 1.46 × 10(5)M(-1)s(-1), 1.18 × 10(-3)s(-1) for VHH-c3 and VHH-c4 assessed by surface plasmon resonance (SPR). Additionally, the selected three VHH-cs can bind to different epitopes of PCV2 Cap that was determined by additive ELISA. Our study confirmed that VHHs with high affinity and specificity to PCV2 Cap can be selected from an immune VHH library, and have the potential application for effective and fast diagnostic development of PCV2. Copyright © 2014 Elsevier B.V. All rights reserved.

Guanosine 5'-triphosphate binding protein (G/sub i/) and two additional pertussis toxin substrates associated with muscarinic receptors in rat heart myocytes: characterization and age dependency

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

Moscona-Amir, E.; Henis, Y.I.; Sokolovsky, M.

1988-07-12

The coupling of muscarinic receptors with G-proteins was investigated in cultured myocytes prepared from the hearts of newborn rats. The coupling was investigated in both young (5 days after plating) and aged (14 days after plating) cultures, in view of the completely different effects of 5'-guanylyl imidodiphosphate (Gpp(NH)p) on muscarinic agonist binding to homogenates from young vs aged cultures. Pretreatment of cultures from both ages by Bordetella pertussis toxin (IAP) was found to eliminate any Gpp(NH)p effect on carbamylcholine binding. IAP by itself induced a rightward shift in the carbamylcholine competition curve in homogenates from aged cultures, but no suchmore » effect was observed in homogenates from young cultures. IAP-catalyzed (/sup 32/P)ADP-ribosylation of membrane preparations from young and aged cultures revealed major differences between them. Young cultures exhibited a major IAP substrate at 40 kDa, which was also recognized by anti-..cap alpha../sub i/ antibodies, and two novel IAP substrates at 28 and 42 kDa, which were weakly ADP-ribosylated by the toxin and were not recognized with either anti-..cap alpha../sub i/ or anti-..cap alpha../sub 0/ antibodies. In aged cultures, only the 40-kDa band (ribosylated to a lower degree) was detected. The parallel age-dependent changes in the three IAP substrates (28, 40, and 42 kDa) and in the interactions of the G-protein(s) with the muscarinic receptors strongly suggest close association between the two phenomena. All of these age-dependent changes in the G-protein related parameters were prevented by phosphatidylcholine-liposome treatment of the aged cultures. The role of the membrane lipid composition in these phenomena is discussed.« less

Role of the C-terminal Extension of Formin 2 in Its Activation by Spire Protein and Processive Assembly of Actin Filaments*

PubMed Central

Montaville, Pierre; Kühn, Sonja; Compper, Christel; Carlier, Marie-France

2016-01-01

Formin 2 (Fmn2), a member of the FMN family of formins, plays an important role in early development. This formin cooperates with profilin and Spire, a WASP homology domain 2 (WH2) repeat protein, to stimulate assembly of a dynamic cytoplasmic actin meshwork that facilitates translocation of the meiotic spindle in asymmetric division of mouse oocytes. The kinase-like non-catalytic domain (KIND) of Spire directly interacts with the C-terminal extension of the formin homology domain 2 (FH2) domain of Fmn2, called FSI. This direct interaction is required for the synergy between the two proteins in actin assembly. We have recently demonstrated how Spire, which caps barbed ends via its WH2 domains, activates Fmn2. Fmn2 by itself associates very poorly to filament barbed ends but is rapidly recruited to Spire-capped barbed ends via the KIND domain, and it subsequently displaces Spire from the barbed end to elicit rapid processive assembly from profilin·actin. Here, we address the mechanism by which Spire and Fmn2 compete at barbed ends and the role of FSI in orchestrating this competition as well as in the processivity of Fmn2. We have combined microcalorimetric, fluorescence, and hydrodynamic binding assays, as well as bulk solution and single filament measurements of actin assembly, to show that removal of FSI converts Fmn2 into a Capping Protein. This activity is mimicked by association of KIND to Fmn2. In addition, FSI binds actin at filament barbed ends as a weak capper and plays a role in displacing the WH2 domains of Spire from actin, thus allowing the association of actin-binding regions of FH2 to the barbed end. PMID:26668326

Time-resolved spectroscopy of dye-labeled photoactive yellow protein suggests a pathway of light-induced structural changes in the N-terminal cap.

PubMed

Hoersch, Daniel; Otto, Harald; Cusanovich, Michael A; Heyn, Maarten P

2009-07-14

The photoreceptor PYP responds to light activation with global conformational changes. These changes are mainly located in the N-terminal cap of the protein, which is approximately 20 A away from the chromophore binding pocket and separated from it by the central beta-sheet. The question of the propagation of the structural change across the central beta-sheet is of general interest for the superfamily of PAS domain proteins, for which PYP is the structural prototype. Here we measured the kinetics of the structural changes in the N-terminal cap by transient absorption spectroscopy on the ns to second timescale. For this purpose the cysteine mutants A5C and N13C were prepared and labeled with thiol reactive 5-iodoacetamidofluorescein (IAF). A5 is located close to the N-terminus, while N13 is part of helix alpha1 near the functionally important salt bridge E12-K110 between the N-terminal cap and the central anti-parallel beta-sheet. The absorption spectrum of the dye is sensitive to its environment, and serves as a sensor for conformational changes near the labeling site. In both labeled mutants light activation results in a transient red-shift of the fluorescein absorption spectrum. To correlate the conformational changes with the photocycle intermediates of the protein, we compared the kinetics of the transient absorption signal of the dye with that of the p-hydroxycinnamoyl chromophore. While the structural change near A5 is synchronized with the rise of the I(2) intermediate, which is formed in approximately 200 mus, the change near N13 is delayed and rises with the next intermediate I(2)', which forms in approximately 2 ms. This indicates that different parts of the N-terminal cap respond to light activation with different kinetics. For the signaling pathway of photoactive yellow protein we propose a model in which the structural signal propagates from the chromophore binding pocket across the central beta-sheet via the N-terminal region to helix alpha1, resulting in a large change in the protein conformation.

Streamlined Synthesis and Assembly of a Hybrid Sensing Architecture with Solid Binding Proteins and Click Chemistry.

PubMed

Swift, Brian J F; Shadish, Jared A; DeForest, Cole A; Baneyx, François

2017-03-22

Combining bioorthogonal chemistry with the use of proteins engineered with adhesive and morphogenetic solid-binding peptides is a promising route for synthesizing hybrid materials with the economy and efficiency of living systems. Using optical sensing of chloramphenicol as a proof of concept, we show here that a GFP variant engineered with zinc sulfide and silica-binding peptides on opposite sides of its β-barrel supports the fabrication of protein-capped ZnS:Mn nanocrystals that exhibit the combined emission signatures of organic and inorganic fluorophores. Conjugation of a chloramphenicol-specific DNA aptamer to the protein shell through strain-promoted azide-alkyne cycloaddition and spontaneous concentration of the resulting nanostructures onto SiO 2 particles mediated by the silica-binding sequence enables visual detection of environmentally and clinically relevant concentrations of chloramphenicol through analyte-mediated inner filtering of sub-330 nm excitation light.

Capped RNA primer binding to influenza polymerase and implications for the mechanism of cap-binding inhibitors

PubMed Central

Pflug, Alexander; Gaudon, Stephanie; Resa-Infante, Patricia; Lethier, Mathilde; Reich, Stefan; Schulze, Wiebke M

2018-01-01

Abstract Influenza polymerase uses short capped primers snatched from nascent Pol II transcripts to initiate transcription of viral mRNAs. Here we describe crystal structures of influenza A and B polymerase bound to a capped primer in a configuration consistent with transcription initiation (’priming state’) and show by functional assays that conserved residues from both the PB2 midlink and cap-binding domains are important for positioning the capped RNA. In particular, mutation of PB2 Arg264, which interacts with the triphosphate linkage in the cap, significantly and specifically decreases cap-dependent transcription. We also compare the configuration of the midlink and cap-binding domains in the priming state with their very different relative arrangement (called the ‘apo’ state) in structures where the potent cap-binding inhibitor VX-787, or a close analogue, is bound. In the ‘apo’ state the inhibitor makes additional interactions to the midlink domain that increases its affinity beyond that to the cap-binding domain alone. The comparison suggests that the mechanism of resistance of certain mutations that allow virus to escape from VX-787, notably PB2 N510T, can only be rationalized if VX-787 has a dual mode of action, direct inhibition of capped RNA binding as well as stabilization of the transcriptionally inactive ‘apo’ state. PMID:29202182

Probing the Energetics of Dynactin Filament Assembly and the Binding of Cargo Adaptor Proteins Using Molecular Dynamics Simulation and Electrostatics-Based Structural Modeling.

PubMed

Zheng, Wenjun

2017-01-10

Dynactin, a large multiprotein complex, binds with the cytoplasmic dynein-1 motor and various adaptor proteins to allow recruitment and transportation of cellular cargoes toward the minus end of microtubules. The structure of the dynactin complex is built around an actin-like minifilament with a defined length, which has been visualized in a high-resolution structure of the dynactin filament determined by cryo-electron microscopy (cryo-EM). To understand the energetic basis of dynactin filament assembly, we used molecular dynamics simulation to probe the intersubunit interactions among the actin-like proteins, various capping proteins, and four extended regions of the dynactin shoulder. Our simulations revealed stronger intersubunit interactions at the barbed and pointed ends of the filament and involving the extended regions (compared with the interactions within the filament), which may energetically drive filament termination by the capping proteins and recruitment of the actin-like proteins by the extended regions, two key features of the dynactin filament assembly process. Next, we modeled the unknown binding configuration among dynactin, dynein tails, and a number of coiled-coil adaptor proteins (including several Bicaudal-D and related proteins and three HOOK proteins), and predicted a key set of charged residues involved in their electrostatic interactions. Our modeling is consistent with previous findings of conserved regions, functional sites, and disease mutations in the adaptor proteins and will provide a structural framework for future functional and mutational studies of these adaptor proteins. In sum, this study yielded rich structural and energetic information about dynactin and associated adaptor proteins that cannot be directly obtained from the cryo-EM structures with limited resolutions.

Reconfiguring the AR-TIF2 Protein–Protein Interaction HCS Assay in Prostate Cancer Cells and Characterizing the Hits from a LOPAC Screen

PubMed Central

Fancher, Ashley T.; Hua, Yun; Camarco, Daniel P.; Close, David A.; Strock, Christopher J.

2016-01-01

Abstract The continued activation of androgen receptor (AR) transcription and elevated expression of AR and transcriptional intermediary factor 2 (TIF2) coactivator observed in prostate cancer (CaP) recurrence and the development of castration-resistant CaP (CRPC) support a screening strategy for small-molecule inhibitors of AR-TIF2 protein–protein interactions (PPIs) to find new drug candidates. Small molecules can elicit tissue selective effects, because the cells of distinct tissues express different levels and cohorts of coregulatory proteins. We reconfigured the AR-TIF2 PPI biosensor (PPIB) assay in the PC-3 CaP cell line to determine whether AR modulators and hits from an AR-TIF2 PPIB screen conducted in U-2 OS cells would behave differently in the CaP cell background. Although we did not observe any significant differences in the compound responses between the assay performed in osteosarcoma and CaP cells, the U-2 OS AR-TIF2 PPIB assay would be more amenable to screening, because both the virus and cell culture demands are lower. We implemented a testing paradigm of counter-screens and secondary hit characterization assays that allowed us to identify and deprioritize hits that inhibited/disrupted AR-TIF2 PPIs and AR transcriptional activation (AR-TA) through antagonism of AR ligand binding or by non-specifically blocking nuclear receptor trafficking. Since AR-TIF2 PPI inhibitor/disruptor molecules act distally to AR ligand binding, they have the potential to modulate AR-TA in a cell-specific manner that is distinct from existing anti-androgen drugs, and to overcome the development of resistance to AR antagonism. We anticipate that the application of this testing paradigm to characterize the hits from an AR-TIF2 PPI high-content screening campaign will enable us to prioritize the AR-TIF2 PPI inhibitor/disruptor leads that have potential to be developed into novel therapeutics for CaP and CRPC. PMID:27606620

Hypoxia induces IGFBP3 in esophageal squamous cancer cells through HIF-1α-mediated mRNA transcription and continuous protein synthesis

PubMed Central

Natsuizaka, Mitsuteru; Naganuma, Seiji; Kagawa, Shingo; Ohashi, Shinya; Ahmadi, Azal; Subramanian, Harry; Chang, Sanders; Nakagawa, Kei J.; Ji, Xinjun; Liebhaber, Stephen A.; Klein-Szanto, Andres J.; Nakagawa, Hiroshi

2012-01-01

Insulin-like growth factor binding protein (IGFBP)-3 regulates cell proliferation and apoptosis in esophageal squamous cell carcinoma (ESCC) cells. We have investigated how the hypoxic tumor microenvironment in ESCC fosters the induction of IGFBP3. RNA interference experiments revealed that hypoxia-inducible factor (HIF)-1α, but not HIF-2α, regulates IGFBP3 mRNA induction. By chromatin immunoprecipitation and transfection assays, HIF-1α was found to transactivate IGFBP3 through a novel hypoxia responsive element (HRE) located at 57 kb upstream from the transcription start site. Metabolic labeling experiments demonstrated hypoxia-mediated inhibition of global protein synthesis. 7-Methyl GTP-cap binding assays suggested that hypoxia suppresses cap-dependent translation. Experiments using pharmacological inhibitors for mammalian target of rapamycin (mTOR) suggested that a relatively weak mTOR activity may be sufficient for cap-dependent translation of IGFBP3 under hypoxic conditions. Bicistronic RNA reporter transfection assays did not validate the possibility of an internal ribosome entry site as a potential mechanism for cap-independent translation for IGFBP3 mRNA. Finally, IGFBP3 mRNA was found enriched to the polysomes. In aggregate, our study establishes IGFBP3 as a direct HIF-1α target gene and that polysome enrichment of IGFBP3 mRNA may permit continuous translation under hypoxic conditions.—Natsuizaka, M., Naganuma, S., Kagawa, S., Ohashi, S., Ahmadi, A., Subramanian, H., Chang, S., Nakagawa, K. J., Ji, X., Liebhaber, S. A., Klein-Szanto, A. J., Nakagawa, H. Hypoxia induces IGFBP3 in esophageal squamous cancer cells through HIF-1α-mediated mRNA transcription and continuous protein synthesis. PMID:22415309

Structure of the human M2 muscarinic acetylcholine receptor bound to an antagonist

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

Haga, Kazuko; Kruse, Andrew C.; Asada, Hidetsugu

2012-03-15

The parasympathetic branch of the autonomic nervous system regulates the activity of multiple organ systems. Muscarinic receptors are G-protein-coupled receptors that mediate the response to acetylcholine released from parasympathetic nerves. Their role in the unconscious regulation of organ and central nervous system function makes them potential therapeutic targets for a broad spectrum of diseases. The M2 muscarinic acetylcholine receptor (M2 receptor) is essential for the physiological control of cardiovascular function through activation of G-protein-coupled inwardly rectifying potassium channels, and is of particular interest because of its extensive pharmacological characterization with both orthosteric and allosteric ligands. Here we report the structuremore » of the antagonist-bound human M2 receptor, the first human acetylcholine receptor to be characterized structurally, to our knowledge. The antagonist 3-quinuclidinyl-benzilate binds in the middle of a long aqueous channel extending approximately two-thirds through the membrane. The orthosteric binding pocket is formed by amino acids that are identical in all five muscarinic receptor subtypes, and shares structural homology with other functionally unrelated acetylcholine binding proteins from different species. A layer of tyrosine residues forms an aromatic cap restricting dissociation of the bound ligand. A binding site for allosteric ligands has been mapped to residues at the entrance to the binding pocket near this aromatic cap. The structure of the M2 receptor provides insights into the challenges of developing subtype-selective ligands for muscarinic receptors and their propensity for allosteric regulation.« less

The ELAV RNA-stability factor HuR binds the 5′-untranslated region of the human IGF-IR transcript and differentially represses cap-dependent and IRES-mediated translation

PubMed Central

Meng, Zheng; King, Peter H.; Nabors, L. Burt; Jackson, Nateka L.; Chen, Ching-Yi; Emanuel, Peter D.; Blume, Scott W.

2005-01-01

The type I insulin-like growth factor receptor (IGF-IR) is an integral component in the control of cell proliferation, differentiation and apoptosis. The IGF-IR mRNA contains an extraordinarily long (1038 nt) 5′-untranslated region (5′-UTR), and we have characterized a diverse series of proteins interacting with this RNA sequence which may provide for intricate regulation of IGF-IR gene expression at the translational level. Here, we report the purification and identification of one of these IGF-IR 5′-UTR-binding proteins as HuR, using a novel RNA crosslinking/RNase elution strategy. Because HuR has been predominantly characterized as a 3′-UTR-binding protein, enhancing mRNA stability and generally increasing gene expression, we sought to determine whether HuR might serve a different function in the context of its binding the IGF-IR 5′-UTR. We found that HuR consistently repressed translation initiation through the IGF-IR 5′-UTR. The inhibition of translation by HuR was concentration dependent, and could be reversed in trans by addition of a fragment of the IGF-IR 5′-UTR containing the HuR binding sites as a specific competitor, or abrogated by deletion of the third RNA recognition motif of HuR. We determined that HuR repressed translation initiation through the IGF-IR 5′-UTR in cells as well, and that siRNA knockdown of HuR markedly increased IGF-IR protein levels. Interestingly, we also found that HuR potently inhibited IGF-IR translation mediated through internal ribosome entry. Kinetic assays were performed to investigate the mechanism of translation repression by HuR and the dynamic interplay between HuR and the translation apparatus. We found that HuR, occupying a cap-distal position, significantly delayed translation initiation mediated by cap-dependent scanning, but was eventually displaced from its binding site, directly or indirectly, as a consequence of ribosomal scanning. However, HuR perpetually blocked the activity of the IGF-IR IRES, apparently arresting the IRES-associated translation pre-initiation complex in an inactive state. This function of HuR as a 5′-UTR-binding protein and dual-purpose translation repressor may be critical for the precise regulation of IGF-IR expression essential to normal cellular homeostasis. PMID:15914670

The Cyclase-Associated Protein Cap1 Is Important for Proper Regulation of Infection-Related Morphogenesis in Magnaporthe oryzae

PubMed Central

Zhou, Xiaoying; Zhang, Haifeng; Li, Guotian; Shaw, Brian; Xu, Jin-Rong

2012-01-01

Surface recognition and penetration are critical steps in the infection cycle of many plant pathogenic fungi. In Magnaporthe oryzae, cAMP signaling is involved in surface recognition and pathogenesis. Deletion of the MAC1 adenylate cyclase gene affected appressorium formation and plant infection. In this study, we used the affinity purification approach to identify proteins that are associated with Mac1 in vivo. One of the Mac1-interacting proteins is the adenylate cyclase-associated protein named Cap1. CAP genes are well-conserved in phytopathogenic fungi but none of them have been functionally characterized. Deletion of CAP1 blocked the effects of a dominant RAS2 allele and resulted in defects in invasive growth and a reduced intracellular cAMP level. The Δcap1 mutant was defective in germ tube growth, appressorium formation, and formation of typical blast lesions. Cap1-GFP had an actin-like localization pattern, localizing to the apical regions in vegetative hyphae, at the periphery of developing appressoria, and in circular structures at the base of mature appressoria. Interestingly, Cap1, similar to LifeAct, did not localize to the apical regions in invasive hyphae, suggesting that the apical actin cytoskeleton differs between vegetative and invasive hyphae. Domain deletion analysis indicated that the proline-rich region P2 but not the actin-binding domain (AB) of Cap1 was responsible for its subcellular localization. Nevertheless, the AB domain of Cap1 must be important for its function because CAP1 ΔAB only partially rescued the Δcap1 mutant. Furthermore, exogenous cAMP induced the formation of appressorium-like structures in non-germinated conidia in CAP1 ΔAB transformants. This novel observation suggested that AB domain deletion may result in overstimulation of appressorium formation by cAMP treatment. Overall, our results indicated that CAP1 is important for the activation of adenylate cyclase, appressorium morphogenesis, and plant infection in M. oryzae. CAP1 may also play a role in feedback inhibition of Ras2 signaling when Pmk1 is activated. PMID:22969430

Localization of TFIIB binding regions using serial analysis of chromatin occupancy

PubMed Central

Yochum, Gregory S; Rajaraman, Veena; Cleland, Ryan; McWeeney, Shannon

2007-01-01

Background: RNA Polymerase II (RNAP II) is recruited to core promoters by the pre-initiation complex (PIC) of general transcription factors. Within the PIC, transcription factor for RNA polymerase IIB (TFIIB) determines the start site of transcription. TFIIB binding has not been localized, genome-wide, in metazoans. Serial analysis of chromatin occupancy (SACO) is an unbiased methodology used to empirically identify transcription factor binding regions. In this report, we use TFIIB and SACO to localize TFIIB binding regions across the rat genome. Results: A sample of the TFIIB SACO library was sequenced and 12,968 TFIIB genomic signature tags (GSTs) were assigned to the rat genome. GSTs are 20–22 base pair fragments that are derived from TFIIB bound chromatin. TFIIB localized to both non-protein coding and protein-coding loci. For 21% of the 1783 protein-coding genes in this sample of the SACO library, TFIIB binding mapped near the characterized 5' promoter that is upstream of the transcription start site (TSS). However, internal TFIIB binding positions were identified in 57% of the 1783 protein-coding genes. Internal positions are defined as those within an inclusive region greater than 2.5 kb downstream from the 5' TSS and 2.5 kb upstream from the transcription stop. We demonstrate that both TFIIB and TFIID (an additional component of PICs) bound to internal regions using chromatin immunoprecipitation (ChIP). The 5' cap of transcripts associated with internal TFIIB binding positions were identified using a cap-trapping assay. The 5' TSSs for internal transcripts were confirmed by primer extension. Additionally, an analysis of the functional annotation of mouse 3 (FANTOM3) databases indicates that internally initiated transcripts identified by TFIIB SACO in rat are conserved in mouse. Conclusion: Our findings that TFIIB binding is not restricted to the 5' upstream region indicates that the propensity for PIC to contribute to transcript diversity is far greater than previously appreciated. PMID:17997859

The human papillomavirus type 16 E6 oncoprotein activates mTORC1 signaling and increases protein synthesis.

PubMed

Spangle, Jennifer M; Münger, Karl

2010-09-01

The mammalian target of rapamycin (mTOR) kinase acts as a cellular rheostat that integrates signals from a variety of cellular signal transduction pathways that sense growth factor and nutrient availability as well as intracellular energy status. It was previously reported that the human papillomavirus type 16 (HPV16) E6 oncoprotein may activate the S6 protein kinase (S6K) through binding and E6AP-mediated degradation of the mTOR inhibitor tuberous sclerosis complex 2 (TSC2) (Z. Lu, X. Hu, Y. Li, L. Zheng, Y. Zhou, H. Jiang, T. Ning, Z. Basang, C. Zhang, and Y. Ke, J. Biol. Chem. 279:35664-35670, 2004; L. Zheng, H. Ding, Z. Lu, Y. Li, Y. Pan, T. Ning, and Y. Ke, Genes Cells 13:285-294, 2008). Our results confirmed that HPV16 E6 expression causes an increase in mTORC1 activity through enhanced phosphorylation of mTOR and activation of downstream signaling pathways S6K and eukaryotic initiation factor binding protein 1 (4E-BP1). However, we did not detect a decrease in TSC2 levels in HPV16 E6-expressing cells. We discovered, however, that HPV16 E6 expression causes AKT activation through the upstream kinases PDK1 and mTORC2 under conditions of nutrient deprivation. We show that HPV16 E6 expression causes an increase in protein synthesis by enhancing translation initiation complex assembly at the 5' mRNA cap and an increase in cap-dependent translation. The increase in cap-dependent translation likely results from HPV16 E6-induced AKT/mTORC1 activation, as the assembly of the translation initiation complex and cap-dependent translation are rapamycin sensitive. Lastly, coexpression of the HPV16 E6 and E7 oncoproteins does not affect HPV16 E6-induced activation of mTORC1 and cap-dependent translation. HPV16 E6-mediated activation of mTORC1 signaling and cap-dependent translation may be a mechanism to promote viral replication under conditions of limited nutrient supply in differentiated, HPV oncoprotein-expressing proliferating cells.

Efficient Cleavage of Ribosome-Associated Poly(A)-Binding Protein by Enterovirus 3C Protease

PubMed Central

Kuyumcu-Martinez, N. Muge; Joachims, Michelle; Lloyd, Richard E.

2002-01-01

Poliovirus (PV) causes a rapid and drastic inhibition of host cell cap-dependent protein synthesis during infection while preferentially allowing cap-independent translation of its own genomic RNA via an internal ribosome entry site element. Inhibition of cap-dependent translation is partly mediated by cleavage of an essential translation initiation factor, eIF4GI, during PV infection. In addition to cleavage of eIF4GI, cleavage of eIF4GII and poly(A)-binding protein (PABP) has been recently proposed to contribute to complete host translation shutoff; however, the relative importance of eIF4GII and PABP cleavage has not been determined. At times when cap-dependent translation is first blocked during infection, only 25 to 35% of the total cellular PABP is cleaved; therefore, we hypothesized that the pool of PABP associated with polysomes may be preferentially targeted by viral proteases. We have investigated what cleavage products of PABP are produced in vivo and the substrate determinants for cleavage of PABP by 2A protease (2Apro) or 3C protease (3Cpro). Our results show that PABP in ribosome-enriched fractions is preferentially cleaved in vitro and in vivo compared to PABP in other fractions. Furthermore, we have identified four N-terminal PABP cleavage products produced during PV infection and have shown that viral 3C protease generates three of the four cleavage products. Also, 3Cpro is more efficient in cleaving PABP in ribosome-enriched fractions than 2Apro in vitro. In addition, binding of PABP to poly(A) RNA stimulates 3Cpro-mediated cleavage and inhibits 2Apro-mediated cleavage. These results suggest that 3Cpro plays a major role in processing PABP during virus infection and that the interaction of PABP with translation initiation factors, ribosomes, or poly(A) RNA may promote its cleavage by viral 2A and 3C proteases. PMID:11836384

Porcine circovirus type 2 activates PI3K/Akt and p38 MAPK pathways to promote interleukin-10 production in macrophages via Cap interaction of gC1qR

PubMed Central

Wang, Tongtong; Zhang, Xiujuan; Chen, Yu; Cui, Beibei; Li, Delong; Zhao, Xiaomin; Zhang, Wenlong; Chang, Lingling; Tong, Dewen

2016-01-01

Porcine circovirus type 2 (PCV2) infection caused PCV2-associated diseases (PCVAD) is one of the major emerging immunosuppression diseases in pig industry. In this study, we investigated how PCV2 inoculation increases interleukin (IL)-10 expression in porcine alveolar macrophages (PAMs). PCV2 inoculation significantly upregulated IL-10 expression compared with PCV1. Upon initial PCV2 inoculation, PI3K/Akt cooperated with NF-κB pathways to promote IL-10 transcription via p50, CREB and Ap1 transcription factors, whereas inhibition of PI3K/Akt activation blocked Ap1 and CREB binding to the il10 promoter, and decreased the binding level of NF-κB1 p50 with il10 promoter, leading to great reduction in early IL-10 transcription. In the later phase of inoculation, PCV2 further activated p38 MAPK and ERK pathways to enhance IL-10 production by promoting Sp1 binding to the il10 promoter. For PCV2-induced IL-10 production in macrophages, PCV2 capsid protein Cap, but not the replicase Rep or ORF3, was the critical component. Cap activated PI3K/Akt, p38 MAPK, and ERK signaling pathways to enhance IL-10 expression. In the whole process, gC1qR mediated PCV2-induced PI3K/Akt and p38 MAPK activation to enhance IL-10 induction by interaction with Cap. Depletion of gC1qR blocked PI3K/Akt and p38 MAPK activation, resulting in significant decrease in IL-10 production in PCV2-inoculated cells. Thus, gC1qR might be a critical functional receptor for PCV2-induced IL-10 production. Taken together, these data demonstrated that Cap protein binding with host gC1qR induction of PI3K/Akt and p38 MAPK signalings activation is a critical process in enhancing PCV2-induced IL-10 production in porcine alveolar macrophages. PMID:26883107

Nutritional stress affects an atypical cap-binding protein in Leishmania.

PubMed

Zinoviev, Alexandra; Manor, Shachar; Shapira, Michal

2012-12-01

Many eukaryotes encode multiple isoforms of the cap-binding translation initiation factor (eIF4E). Leishmanias and other trypanosomatids encode four paralogs of this protein, but none can complement the eIF4E function in a yeast mutant. A low conservation is observed between the four paralogs, suggesting they assist these organisms survive a multitude of conditions encountered throughout the life cycle. Earlier attempts to decipher their function led to identification of LeishIF4E-4 as the canonical translation initiation factor. LeishIF4E-1 appears to function during thermal stress, via a mechanism not yet understood. LeishIF4E-3 hardly binds cap-4 and is, therefore, less likely to serve as a typical initiation factor. Although it interacts with an eIF4G homolog, LeishIF4G-4, the two polypeptides do not co-migrate on sucrose gradients. While LeishIF4E-3 enters large particles that increase in size during nutritional stress, LeishIF4G-4 is found only in the top fractions. Confocal microscopy localized LeishIF4E-3 (but not LeishIF4G-4) within nutritional stress-induced granules. Accordingly, interaction between the two proteins reduced upon starvation. We therefore propose that under normal conditions, LeishIF4G-4 sequesters LeishIF4E-3 in the cytoplasm. During a nutritional stress, LeishIF4E-3 is modified and released from LeishIF4G-4 to enter stress granules, where inactive mRNAs are stored. Binding of LeishIF4G-4 to LeishIF4E-3 requires a short peptide within the LeishIF4G-4 N-terminus, which bears no similarity to the consensus 4E-binding peptide, YXXXXLΦ. Mutational analysis combined with structure prediction indicates that this interaction is based on an obligatory, conserved α helix in LeishIF4G-4. These features further highlight the uniqueness of LeishIF4E-3 and how it interacts with its binding partners.

Essential motions and energetic contributions of individual residues in a peptide bound to an SH3 domain.

PubMed Central

Kolafa, J; Perram, J W; Bywater, R P

2000-01-01

We have studied protein-ligand interactions by molecular dynamics simulations using software designed to exploit parallel computing architectures. The trajectories were analyzed to extract the essential motions and to estimate the individual contributions of fragments of the ligand to overall binding enthalpy. Two forms of the bound ligand are compared, one with the termini blocked by covalent derivatization, and one in the underivatized, zwitterionic form. The ends of the peptide tend to bind more loosely in the capped form. We can observe significant motions in the bound ligand and distinguish between motions of the peptide backbone and of the side chains. This could be useful in designing ligands, which fit optimally to the binding protein. We show that it is possible to determine the different contributions of each residue in a peptide to the enthalpy of binding. Proline is a major net contributor to binding enthalpy, in keeping with the known propensity for this family of proteins to bind proline-rich peptides. PMID:10919999

Mechanism and Regulation of Protein Synthesis in Saccharomyces cerevisiae

PubMed Central

Dever, Thomas E.; Kinzy, Terri Goss; Pavitt, Graham D.

2016-01-01

In this review, we provide an overview of protein synthesis in the yeast Saccharomyces cerevisiae. The mechanism of protein synthesis is well conserved between yeast and other eukaryotes, and molecular genetic studies in budding yeast have provided critical insights into the fundamental process of translation as well as its regulation. The review focuses on the initiation and elongation phases of protein synthesis with descriptions of the roles of translation initiation and elongation factors that assist the ribosome in binding the messenger RNA (mRNA), selecting the start codon, and synthesizing the polypeptide. We also examine mechanisms of translational control highlighting the mRNA cap-binding proteins and the regulation of GCN4 and CPA1 mRNAs. PMID:27183566

4EBP-Dependent Signaling Supports West Nile Virus Growth and Protein Expression

PubMed Central

Shives, Katherine D.; Massey, Aaron R.; May, Nicholas A.; Morrison, Thomas E.; Beckham, J. David

2016-01-01

West Nile virus (WNV) is a (+) sense, single-stranded RNA virus in the Flavivirus genus. WNV RNA possesses an m7GpppNm 5′ cap with 2′-O-methylation that mimics host mRNAs preventing innate immune detection and allowing the virus to translate its RNA genome through the utilization of cap-dependent translation initiation effectors in a wide variety of host species. Our prior work established the requirement of the host mammalian target of rapamycin complex 1 (mTORC1) for optimal WNV growth and protein expression; yet, the roles of the downstream effectors of mTORC1 in WNV translation are unknown. In this study, we utilize gene deletion mutants in the ribosomal protein kinase called S6 kinase (S6K) and eukaryotic translation initiation factor 4E-binding protein (4EBP) pathways downstream of mTORC1 to define the role of mTOR-dependent translation initiation signals in WNV gene expression and growth. We now show that WNV growth and protein expression are dependent on mTORC1 mediated-regulation of the eukaryotic translation initiation factor 4E-binding protein/eukaryotic translation initiation factor 4E-binding protein (4EBP/eIF4E) interaction and eukaryotic initiation factor 4F (eIF4F) complex formation to support viral growth and viral protein expression. We also show that the canonical signals of mTORC1 activation including ribosomal protein s6 (rpS6) and S6K phosphorylation are not required for WNV growth in these same conditions. Our data suggest that the mTORC1/4EBP/eIF4E signaling axis is activated to support the translation of the WNV genome. PMID:27763553

Mutations in STAMBP, encoding a deubiquitinating enzyme, cause Microcephaly-Capillary Malformation syndrome

PubMed Central

McDonell, Laura M.; Mirzaa, Ghayda M.; Alcantara, Diana; Schwartzentruber, Jeremy; Carter, Melissa T.; Lee, Leo J.; Clericuzio, Carol L.; Graham, John M.; Morris-Rosendahl, Deborah J.; Polster, Tilman; Acsadi, Gyula; Townshend, Sharron; Williams, Simon; Halbert, Anne; Isidor, Bertrand; Smyser, Christopher D.; Paciorkowski, Alex R.; Willing, Marcia; Woulfe, John; Das, Soma; Beaulieu, Chandree L.; Marcadier, Janet; Geraghty, Michael T.; Frey, Brendan J.; Majewski, Jacek; Bulman, Dennis E.; Dobyns, William B.; O’Driscoll, Mark; Boycott, Kym M.

2014-01-01

Microcephaly-capillary malformation (MIC-CAP) syndrome exhibits severe microcephaly with progressive cortical atrophy, intractable epilepsy, profound developmental delay and multiple small capillary malformations on the skin. We employed whole-exome sequencing of five patients with MIC-CAP syndrome and identified novel recessive mutations in STAMBP, a gene encoding the deubiquitinating (DUB) isopeptidase STAMBP (STAM-binding protein)/AMSH (Associated Molecule with the SH3 domain of STAM), that plays a key role in cell surface receptor-mediated endocytosis and sorting. Patient cell lines showed reduced STAMBP expression associated with accumulation of ubiquitin-conjugated protein aggregates, elevated apoptosis and insensitive activation of the RAS-MAPK and PI3K-AKT-mTOR pathways. The latter cellular phenotype is significant considering the established connection between these pathways and their association with vascular and capillary malformations. Furthermore, our findings of a congenital human disorder caused by a defective DUB protein that functions in endocytosis, implicates ubiquitin-conjugate aggregation and elevated apoptosis as factors potentially influencing the progressive neuronal loss underlying MIC-CAP. PMID:23542699

A Genome-Wide RNAi Screen Identifies FOXO4 as a Metastasis-Suppressor through Counteracting PI3K/AKT Signal Pathway in Prostate Cancer

PubMed Central

Su, Bing; Gao, Lingqiu; Baranowski, Catherine; Gillard, Bryan; Wang, Jianmin; Ransom, Ryan; Ko, Hyun-Kyung; Gelman, Irwin H.

2014-01-01

Activation of the PI3K/AKT signal pathway is a known driving force for the progression to castration-recurrent prostate cancer (CR-CaP), which constitutes the major lethal phenotype of CaP. Here, we identify using a genomic shRNA screen the PI3K/AKT-inactivating downstream target, FOXO4, as a potential CaP metastasis suppressor. FOXO4 protein levels inversely correlate with the invasive potential of a panel of human CaP cell lines, with decreased mRNA levels correlating with increased incidence of clinical metastasis. Knockdown (KD) of FOXO4 in human LNCaP cells causes increased invasion in vitro and lymph node (LN) metastasis in vivo without affecting indices of proliferation or apoptosis. Increased Matrigel invasiveness was found by KD of FOXO1 but not FOXO3. Comparison of differentially expressed genes affected by FOXO4-KD in LNCaP cells in culture, in primary tumors and in LN metastases identified a panel of upregulated genes, including PIP, CAMK2N1, PLA2G16 and PGC, which, if knocked down by siRNA, could decrease the increased invasiveness associated with FOXO4 deficiency. Although only some of these genes encode FOXO promoter binding sites, they are all RUNX2-inducible, and RUNX2 binding to the PIP promoter is increased in FOXO4-KD cells. Indeed, the forced expression of FOXO4 reversed the increased invasiveness of LNCaP/shFOXO4 cells; the forced expression of FOXO4 did not alter RUNX2 protein levels, yet it decreased RUNX2 binding to the PIP promoter, resulting in PIP downregulation. Finally, there was a correlation between FOXO4, but not FOXO1 or FOXO3, downregulation and decreased metastasis-free survival in human CaP patients. Our data strongly suggest that increased PI3K/AKT-mediated metastatic invasiveness in CaP is associated with FOXO4 loss, and that mechanisms to induce FOXO4 re-expression might suppress CaP metastatic aggressiveness. PMID:24983969

The eIF4E-binding proteins are modifiers of cytoplasmic eIF4E relocalization during the heat shock response.

PubMed

Sukarieh, R; Sonenberg, N; Pelletier, J

2009-05-01

Stress granules (SGs) arise as a consequence of cellular stress, contain stalled translation preinitiation complexes, and are associated with cell survival during environmental insults. SGs are dynamic entities with proteins relocating into and out of them during stress. Among the repertoire of proteins present in SGs is eukaryotic initiation factor 4E (eIF4E), a translation factor required for cap-dependent translation and that regulates a rate-limiting step for protein synthesis. Herein, we demonstrate that localization of eIF4E to SGs is dependent on the presence of a family of repressor proteins, eIF4E-binding proteins (4E-BPs). Our results demonstrate that 4E-BPs regulate the SG localization of eIF4E.

V-1 regulates capping protein activity in vivo

PubMed Central

Jung, Goeh; Wu, Xufeng S.; Piszczek, Grzegorz; Chen, Bi-Chang; Betzig, Eric; Hammer, John A.

2016-01-01

Capping Protein (CP) plays a central role in the creation of the Arp2/3-generated branched actin networks comprising lamellipodia and pseudopodia by virtue of its ability to cap the actin filament barbed end, which promotes Arp2/3-dependent filament nucleation and optimal branching. The highly conserved protein V-1/Myotrophin binds CP tightly in vitro to render it incapable of binding the barbed end. Here we addressed the physiological significance of this CP antagonist in Dictyostelium, which expresses a V-1 homolog that we show is very similar biochemically to mouse V-1. Consistent with previous studies of CP knockdown, overexpression of V-1 in Dictyostelium reduced the size of pseudopodia and the cortical content of Arp2/3 and induced the formation of filopodia. Importantly, these effects scaled positively with the degree of V-1 overexpression and were not seen with a V-1 mutant that cannot bind CP. V-1 is present in molar excess over CP, suggesting that it suppresses CP activity in the cytoplasm at steady state. Consistently, cells devoid of V-1, like cells overexpressing CP described previously, exhibited a significant decrease in cellular F-actin content. Moreover, V-1–null cells exhibited pronounced defects in macropinocytosis and chemotactic aggregation that were rescued by V-1, but not by the V-1 mutant. Together, these observations demonstrate that V-1 exerts significant influence in vivo on major actin-based processes via its ability to sequester CP. Finally, we present evidence that V-1’s ability to sequester CP is regulated by phosphorylation, suggesting that cells may manipulate the level of active CP to tune their “actin phenotype.” PMID:27791032

V-1 regulates capping protein activity in vivo.

PubMed

Jung, Goeh; Alexander, Christopher J; Wu, Xufeng S; Piszczek, Grzegorz; Chen, Bi-Chang; Betzig, Eric; Hammer, John A

2016-10-25

Capping Protein (CP) plays a central role in the creation of the Arp2/3-generated branched actin networks comprising lamellipodia and pseudopodia by virtue of its ability to cap the actin filament barbed end, which promotes Arp2/3-dependent filament nucleation and optimal branching. The highly conserved protein V-1/Myotrophin binds CP tightly in vitro to render it incapable of binding the barbed end. Here we addressed the physiological significance of this CP antagonist in Dictyostelium, which expresses a V-1 homolog that we show is very similar biochemically to mouse V-1. Consistent with previous studies of CP knockdown, overexpression of V-1 in Dictyostelium reduced the size of pseudopodia and the cortical content of Arp2/3 and induced the formation of filopodia. Importantly, these effects scaled positively with the degree of V-1 overexpression and were not seen with a V-1 mutant that cannot bind CP. V-1 is present in molar excess over CP, suggesting that it suppresses CP activity in the cytoplasm at steady state. Consistently, cells devoid of V-1, like cells overexpressing CP described previously, exhibited a significant decrease in cellular F-actin content. Moreover, V-1-null cells exhibited pronounced defects in macropinocytosis and chemotactic aggregation that were rescued by V-1, but not by the V-1 mutant. Together, these observations demonstrate that V-1 exerts significant influence in vivo on major actin-based processes via its ability to sequester CP. Finally, we present evidence that V-1's ability to sequester CP is regulated by phosphorylation, suggesting that cells may manipulate the level of active CP to tune their "actin phenotype."

Relationship between helix stability and binding affinities: molecular dynamics simulations of Bfl-1/A1-binding pro-apoptotic BH3 peptide helices in explicit solvent.

PubMed

Modi, Vivek; Lama, Dilraj; Sankararamakrishnan, Ramasubbu

2013-01-01

The anti-apoptotic protein Bfl-1, also known as A1, belongs to the Bcl-2 family of proteins and interacts with pro-apoptotic Bcl-2 counterparts to regulate programmed cell death. As demonstrated for other anti-apoptotic Bcl-2 proteins, Bfl-1/A1 has also been shown to be overexpressed in various human cancers and hence they are attractive targets for anticancer drugs. Peptides derived from the BH3 region of pro-apoptotic Bcl-2 proteins have been shown to elicit similar biological response as that of parent proteins. BH3 peptides from different pro-apoptotic proteins have wide range of affinities for Bfl-1/A1. Experimentally determined complex structures show that the hydrophobic side of amphipathic BH3 peptides binds to the hydrophobic groove formed by the α-helical bundle of Bfl-1/A1 protein. Apart from the length and amino acid composition, a BH3 peptide's ability to form a stable helical structure has been suggested to be important for its high binding affinity. Molecular dynamics simulations of three BH3 peptides derived from the pro-apoptotic proteins Bak, Bid, and Bmf were carried out each for a period of at least 100 ns after 2 ns equilibration run. The length of simulated BH3 peptides varied from 22 to 24 residues and their binding affinities for Bfl-1/A1 varied from 1 to 180 nM. Our results show that the hydrophobic residues from the hydrophobic face of BH3 peptides tend to cluster together quickly to avoid being exposed to the solvent. This resulted in either reduction of helix length or complete loss of helical character. Bak and Bid BH3 peptides with high affinities for Bf1-1/A1 have stable helical segments in the N-terminal region. The highly conserved Leu residue lies just outside the helical region at the C-terminal end. Capping interactions arising out of N-cap residues seem to be extremely important to maintain the helical stability. Favorable hydrophilic interactions between residues also give further stability to the helix fragment and at least one of the interacting residues resides within the helical region. Bmf BH3 peptide with a weaker binding affinity for Bmf-1/A1 completely lost its helical character at the end of 100 ns production run and a further 50 ns simulation showed that the Bmf peptide continues to remain in random conformation. The present study clearly establishes a link between a BH3 peptide's ability to form a stable helical segment and its high binding affinity for an anti-apoptotic protein. To further test this hypothesis, we simulated a mutant Bmf peptide for 100 ns in which two residues R129 and H146 were substituted by Asn in silico in the wild-type peptide. Introduction of N-terminal Asn clearly enabled the formation of capping interactions at the N-terminus and resulted in a stable N-terminal helical segment. This demonstrates that the knowledge of interactions that help to maintain stable helical segments in a high-affinity BH3 peptide will help in designing highly specific peptide-based drugs/inhibitors. Such molecules will have the ability to bind a particular anti-apoptotic protein with high affinity.

Cross reactivity between European hornet and yellow jacket venoms.

PubMed

Severino, M G; Caruso, B; Bonadonna, P; Labardi, D; Macchia, D; Campi, P; Passalacqua, G

2010-08-01

Cross-reactions between venoms may be responsible for multiple diagnostic positivities in hymenoptera allergy. There is limited data on the cross-reactivity between Vespula spp and Vespa crabro, which is an important cause of severe reactions in some parts of Europe. We studied by CAP-inhibition assays and immunoblotting the cross-reactivity between the two venoms. Sera from patients with non discriminative skin/CAP positivity to both Vespula and Vespa crabro were collected for the analyses. Inhibition assays were carried out with a CAP method, incubating the sera separately with both venoms and subsequently measuring the specific IgE to venoms themselves. Immunoblotting was performed on sera with ambiguous results at the CAP-inhibition. Seventeen patients had a severe reaction after Vespa crabro sting and proved skin and CAP positive also to vespula. In 11/17 patients, Vespula venom completely inhibited IgE binding to VC venom, whereas VC venom inhibited binding to Vespula venom only partially (<75%). In 6 subjects the CAP-inhibition provided inconclusive results and their sera were analysed by immunoblotting. The SDS-PAGE identified hyaluronidase, phospholipase A1 and antigen 5 as the main proteins of the venoms. In 5 sera the levels of IgE against antigen 5 of Vespa crabro were higher than IgE against Vespula germanica, thus indicating a true sensitisation to crabro. In the case of multiple positivities to Vespa crabro and Vespula spp the CAP inhibition is helpful in detecting the cross-reactivities.

Intramolecular Dynamics within the N-Cap-SH3-SH2 Regulatory Unit of the c-Abl Tyrosine Kinase Reveal Targeting to the Cellular Membrane*♦

PubMed Central

de Oliveira, Guilherme A. P.; Pereira, Elen G.; Ferretti, Giulia D. S.; Valente, Ana Paula; Cordeiro, Yraima; Silva, Jerson L.

2013-01-01

c-Abl is a key regulator of cell signaling and is under strict control via intramolecular interactions. In this study, we address changes in the intramolecular dynamics coupling within the c-Abl regulatory unit by presenting its N-terminal segment (N-Cap) with an alternative function in the cell as c-Abl becomes activated. Using small angle x-ray scattering, nuclear magnetic resonance, and confocal microscopy, we demonstrate that the N-Cap and the Src homology (SH) 3 domain acquire μs-ms motions upon N-Cap association with the SH2-L domain, revealing a stabilizing synergy between these segments. The N-Cap-myristoyl tether likely triggers the protein to anchor to the membrane because of these flip-flop dynamics, which occur in the μs-ms time range. This segment not only presents the myristate during c-Abl inhibition but may also trigger protein localization inside the cell in a functional and stability-dependent mechanism that is lost in Bcr-Abl+ cells, which underlie chronic myeloid leukemia. This loss of intramolecular dynamics and binding to the cellular membrane is a potential therapeutic target. PMID:23928308

Intramolecular dynamics within the N-Cap-SH3-SH2 regulatory unit of the c-Abl tyrosine kinase reveal targeting to the cellular membrane.

PubMed

de Oliveira, Guilherme A P; Pereira, Elen G; Ferretti, Giulia D S; Valente, Ana Paula; Cordeiro, Yraima; Silva, Jerson L

2013-09-27

c-Abl is a key regulator of cell signaling and is under strict control via intramolecular interactions. In this study, we address changes in the intramolecular dynamics coupling within the c-Abl regulatory unit by presenting its N-terminal segment (N-Cap) with an alternative function in the cell as c-Abl becomes activated. Using small angle x-ray scattering, nuclear magnetic resonance, and confocal microscopy, we demonstrate that the N-Cap and the Src homology (SH) 3 domain acquire μs-ms motions upon N-Cap association with the SH2-L domain, revealing a stabilizing synergy between these segments. The N-Cap-myristoyl tether likely triggers the protein to anchor to the membrane because of these flip-flop dynamics, which occur in the μs-ms time range. This segment not only presents the myristate during c-Abl inhibition but may also trigger protein localization inside the cell in a functional and stability-dependent mechanism that is lost in Bcr-Abl(+) cells, which underlie chronic myeloid leukemia. This loss of intramolecular dynamics and binding to the cellular membrane is a potential therapeutic target.

Role of the C-terminal Extension of Formin 2 in Its Activation by Spire Protein and Processive Assembly of Actin Filaments.

PubMed

Montaville, Pierre; Kühn, Sonja; Compper, Christel; Carlier, Marie-France

2016-02-12

Formin 2 (Fmn2), a member of the FMN family of formins, plays an important role in early development. This formin cooperates with profilin and Spire, a WASP homology domain 2 (WH2) repeat protein, to stimulate assembly of a dynamic cytoplasmic actin meshwork that facilitates translocation of the meiotic spindle in asymmetric division of mouse oocytes. The kinase-like non-catalytic domain (KIND) of Spire directly interacts with the C-terminal extension of the formin homology domain 2 (FH2) domain of Fmn2, called FSI. This direct interaction is required for the synergy between the two proteins in actin assembly. We have recently demonstrated how Spire, which caps barbed ends via its WH2 domains, activates Fmn2. Fmn2 by itself associates very poorly to filament barbed ends but is rapidly recruited to Spire-capped barbed ends via the KIND domain, and it subsequently displaces Spire from the barbed end to elicit rapid processive assembly from profilin·actin. Here, we address the mechanism by which Spire and Fmn2 compete at barbed ends and the role of FSI in orchestrating this competition as well as in the processivity of Fmn2. We have combined microcalorimetric, fluorescence, and hydrodynamic binding assays, as well as bulk solution and single filament measurements of actin assembly, to show that removal of FSI converts Fmn2 into a Capping Protein. This activity is mimicked by association of KIND to Fmn2. In addition, FSI binds actin at filament barbed ends as a weak capper and plays a role in displacing the WH2 domains of Spire from actin, thus allowing the association of actin-binding regions of FH2 to the barbed end. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

PI(4,5)P2-binding effector proteins for vesicle exocytosis

PubMed Central

Martin, Thomas F. J.

2014-01-01

PI(4,5)P2 participates directly in priming and possibly fusion steps of Ca2+-triggered vesicle exocytosis. High concentration nanodomains of PI(4,5)P2 reside on the plasma membrane of neuroendocrine cells. A subset of vesicles that co-localize with PI(4,5)P2 domains appear to undergo preferential exocytosis in stimulated cells. PI(4,5)P2 directly regulates vesicle exocytosis by recruiting and activating PI(4,5)P2-binding proteins that regulate SNARE protein function including CAPS, Munc13-1/2, synaptotagmin-1, and other C2 domain-containing proteins. These PI(4,5)P2 effector proteins are coincidence detectors that engage in multiple interactions at vesicle exocytic sites. The SNARE protein syntaxin-1 also binds to PI(4,5)P2, which promotes clustering, but an activating role for PI(4,5)P2 in syntaxin-1 function remains to be fully characterized. Similar principles underlie polarized constitutive vesicle fusion mediated in part by the PI(4,5)P2-binding subunits of the exocyst complex (Sec3, Exo70). Overall, focal vesicle exocytosis occurs at sites landmarked by PI(4,5)P2, which serves to recruit and/or activate multifunctional PI(4,5)P2-binding proteins. PMID:25280637

Systematic identification of non-coding RNA 2,2,7-trimethylguanosine cap structures in Caenorhabditis elegans

PubMed Central

Jia, Dong; Cai, Lun; He, Housheng; Skogerbø, Geir; Li, Tiantian; Aftab, Muhammad Nauman; Chen, Runsheng

2007-01-01

Background The 2,2,7-trimethylguanosine (TMG) cap structure is an important functional characteristic of ncRNAs with critical cellular roles, such as some snRNAs. Here we used immunoprecipitation with both K121 and R1131 anti-TMG antibodies to systematically identify the TMG cap structures for all presently characterized ncRNAs in C. elegans. Results The two anti-TMG antibodies precipitated a similar group of the C. elegans ncRNAs. All snRNAs known to have a TMG cap structure were found in the precipitate, indicating that our identification system was efficient. Other ncRNA families related to splicing, such as SL RNAs and Sm Y RNAs, were also found in the precipitate, as were 7 C/D box snoRNAs. Further analysis showed that the SL RNAs and the Sm Y RNAs shared a very similar Sm binding site element (AAU4–5GGA), which sequence composition differed somewhat from those of other U snRNAs. There were also 16 ncRNAs without an Sm binding site element in the precipitate, suggesting that for these ncRNAs, TMG formation may occur independently of Sm proteins. Conclusion Our results showed that most ncRNAs predicted to be transcribed by RNA polymerase II had a TMG cap, while those predicted to be transcribed by RNA plymerase III or located in introns did not have a TMG cap structure. Compared to ncRNAs without a TMG cap, TMG-capped ncRNAs tended to have higher expression levels. Five functionally non-annotated ncRNAs also have a TMG cap structure, which might be helpful for identifying the cellular roles of these ncRNAs. PMID:17903271

Systematic identification of non-coding RNA 2,2,7-trimethylguanosine cap structures in Caenorhabditis elegans.

PubMed

Jia, Dong; Cai, Lun; He, Housheng; Skogerbø, Geir; Li, Tiantian; Aftab, Muhammad Nauman; Chen, Runsheng

2007-09-29

The 2,2,7-trimethylguanosine (TMG) cap structure is an important functional characteristic of ncRNAs with critical cellular roles, such as some snRNAs. Here we used immunoprecipitation with both K121 and R1131 anti-TMG antibodies to systematically identify the TMG cap structures for all presently characterized ncRNAs in C. elegans. The two anti-TMG antibodies precipitated a similar group of the C. elegans ncRNAs. All snRNAs known to have a TMG cap structure were found in the precipitate, indicating that our identification system was efficient. Other ncRNA families related to splicing, such as SL RNAs and Sm Y RNAs, were also found in the precipitate, as were 7 C/D box snoRNAs. Further analysis showed that the SL RNAs and the Sm Y RNAs shared a very similar Sm binding site element (AAU4-5GGA), which sequence composition differed somewhat from those of other U snRNAs. There were also 16 ncRNAs without an Sm binding site element in the precipitate, suggesting that for these ncRNAs, TMG formation may occur independently of Sm proteins. Our results showed that most ncRNAs predicted to be transcribed by RNA polymerase II had a TMG cap, while those predicted to be transcribed by RNA plymerase III or located in introns did not have a TMG cap structure. Compared to ncRNAs without a TMG cap, TMG-capped ncRNAs tended to have higher expression levels. Five functionally non-annotated ncRNAs also have a TMG cap structure, which might be helpful for identifying the cellular roles of these ncRNAs.

The eIF4E-binding proteins are modifiers of cytoplasmic eIF4E relocalization during the heat shock response

PubMed Central

Sukarieh, R.; Sonenberg, N.; Pelletier, J.

2009-01-01

Stress granules (SGs) arise as a consequence of cellular stress, contain stalled translation preinitiation complexes, and are associated with cell survival during environmental insults. SGs are dynamic entities with proteins relocating into and out of them during stress. Among the repertoire of proteins present in SGs is eukaryotic initiation factor 4E (eIF4E), a translation factor required for cap-dependent translation and that regulates a rate-limiting step for protein synthesis. Herein, we demonstrate that localization of eIF4E to SGs is dependent on the presence of a family of repressor proteins, eIF4E-binding proteins (4E-BPs). Our results demonstrate that 4E-BPs regulate the SG localization of eIF4E. PMID:19244480

DARPin-Based Crystallization Chaperones Exploit Molecular Geometry as a Screening Dimension in Protein Crystallography.

PubMed

Batyuk, Alexander; Wu, Yufan; Honegger, Annemarie; Heberling, Matthew M; Plückthun, Andreas

2016-04-24

DARPin libraries, based on a Designed Ankyrin Repeat Protein consensus framework, are a rich source of binding partners for a wide variety of proteins. Their modular structure, stability, ease of in vitro selection and high production yields make DARPins an ideal starting point for further engineering. The X-ray structures of around 30 different DARPin complexes demonstrate their ability to facilitate crystallization of their target proteins by restricting flexibility and preventing undesired interactions of the target molecule. However, their small size (18 kDa), very hydrophilic surface and repetitive structure can limit the DARPins' ability to provide essential crystal contacts and their usefulness as a search model for addressing the crystallographic phase problem in molecular replacement. To optimize DARPins for their application as crystallization chaperones, rigid domain-domain fusions of the DARPins to larger proteins, proven to yield high-resolution crystal structures, were generated. These fusions were designed in such a way that they affect only one of the terminal capping repeats of the DARPin and do not interfere with residues involved in target binding, allowing to exchange at will the binding specificities of the DARPin in the fusion construct. As a proof of principle, we designed rigid fusions of a stabilized version of Escherichia coli TEM-1 β-lactamase to the C-terminal capping repeat of various DARPins in six different relative domain orientations. Five crystal structures representing four different fusion constructs, alone or in complex with the cognate target, show the predicted relative domain orientations and prove the validity of the concept. Copyright © 2016 Elsevier Ltd. All rights reserved.

Using the Stylophora pistillata genome and cell cultures to understand the mechanism of aragonite precipitation in corals

NASA Astrophysics Data System (ADS)

Mass, T.; Drake, J.; Haramaty, L.; Zelzion, U.; Bhattacharya, D.; Rosenthal, Y.; Falkowski, P. G.

2012-12-01

Atmospheric CO 2 levels are rising rapidly, resulting in a decrease in both oceanic pH, and the carbonate saturation state (Ω). It has been hypothesized that calcifying marine organisms, including reef-building corals, will be affected by the decline of the carbonate saturation state. However, it is still unclear how corals will respond to these changes, as their skeletal formation is biologically mediated and occurs in isolated space rather than directly from seawater. In corals new skeletal material is precipitated in the subcalicoblastic space between the skeleton and the calicoblastic epithelium which, does not exceed a few nanometers and contains the ''calcifying fluid''. The goal of our project is to understand how these fluids respond to changes in the surrounding seawater and in turn affects the biologically mediated calcification mechanisms at the molecular, cellular and tissue levels. While it is generally thought that an organic matrix, which contain a suite of proteins, lipids and poly-saccharides, take part in calcification process, the specific mechanism by which the mineral is precipitated is unknown. The organic matrix composed of two fractions: the soluble organic matrix (SOM) and the insoluble organic matrix (IOM). It is suggested that the IOM plays a role as structural proteins forming a framework for crystal growth whereas the SOM plays a role in nucleation and crystal growth. To address this question we have investigated both the structural framework proteins (Drake et al abstract submitted to the AGU fall meeting) the role of proteins in nucleation and crystal growth (this work). Here, we established cell cultures and sequenced the 458-megabase genome of the stony coral, Stylophora pistillata, using next-generation sequencing technology. This genome contains 21,678 predicted protein-coding genes. Many of the known protein components of invertebrate skeletal matrices are acidic and/or contain repeated sequences. We searched for genes encoding proteins with the following characteristics: (1) high content (>35%) of acidic amino acids (aspartate and glutamate), (2) at least 150 residues, and (3) a signal peptide. This approach revealed eight coral acidic proteins (CAPs). We confirmed the sequence of four candidates (CAP1-3 and 8). A search for similarity in the UniProt database and published coral's genomes and ESTs reveals high similarity of CAPs 2, 3, and 8 to both invertebrate and vertebrate acidic rich proteins. CAP1, however, has no significant matches in any of the databases. We expressed and purified these four proteins to examine their role in coral bio-mineralization. We show that the pure CAPs bind Ca+2, and furthermore, individual CAPs can precipitate calcium carbonate in vitro in artificial seawater. These results strongly suggest that aragonite precipitation in the calcifying region of corals is promoted by a highly conserved set of acidic proteins. Based purely on thermodynamic grounds, the predicted change in surface ocean pH should not affect the binding ability of these acidic proteins. To the extent these proteins are responsible for calcification in this and other corals, we suggest that corals will continue to calcify at pH changes predicted to occur in this century.

LIM-domain proteins, LIMD1, Ajuba, and WTIP are required for microRNA-mediated gene silencing

PubMed Central

James, Victoria; Zhang, Yining; Foxler, Daniel E.; de Moor, Cornelia H.; Kong, Yi Wen; Webb, Thomas M.; Self, Tim J.; Feng, Yungfeng; Lagos, Dimitrios; Chu, Chia-Ying; Rana, Tariq M.; Morley, Simon J.; Longmore, Gregory D.; Bushell, Martin; Sharp, Tyson V.

2010-01-01

In recent years there have been major advances with respect to the identification of the protein components and mechanisms of microRNA (miRNA) mediated silencing. However, the complete and precise repertoire of components and mechanism(s) of action remain to be fully elucidated. Herein we reveal the identification of a family of three LIM domain-containing proteins, LIMD1, Ajuba and WTIP (Ajuba LIM proteins) as novel mammalian processing body (P-body) components, which highlight a novel mechanism of miRNA-mediated gene silencing. Furthermore, we reveal that LIMD1, Ajuba, and WTIP bind to Ago1/2, RCK, Dcp2, and eIF4E in vivo, that they are required for miRNA-mediated, but not siRNA-mediated gene silencing and that all three proteins bind to the mRNA 5′ m7GTP cap–protein complex. Mechanistically, we propose the Ajuba LIM proteins interact with the m7GTP cap structure via a specific interaction with eIF4E that prevents 4EBP1 and eIF4G interaction. In addition, these LIM-domain proteins facilitate miRNA-mediated gene silencing by acting as an essential molecular link between the translationally inhibited eIF4E-m7GTP-5′cap and Ago1/2 within the miRISC complex attached to the 3′-UTR of mRNA, creating an inhibitory closed-loop complex. PMID:20616046

Ybp1 and Gpx3 signaling in Candida albicans govern hydrogen peroxide-induced oxidation of the Cap1 transcription factor and macrophage escape.

PubMed

Patterson, Miranda J; McKenzie, Christopher G; Smith, Deborah A; da Silva Dantas, Alessandra; Sherston, Sam; Veal, Elizabeth A; Morgan, Brian A; MacCallum, Donna M; Erwig, Lars-Peter; Quinn, Janet

2013-12-20

As Candida albicans is the major fungal pathogen of humans, there is an urgent need to understand how this pathogen evades toxic reactive oxygen species (ROS) generated by the host immune system. A key regulator of antioxidant gene expression, and thus ROS resistance, in C. albicans is the AP-1-like transcription factor Cap1. Despite this, little is known regarding the intracellular signaling mechanisms that underlie the oxidation and activation of Cap1. Therefore, the aims of this study were; (i) to identify the regulatory proteins that govern Cap1 oxidation, and (ii) to investigate the importance of Cap1 oxidation in C. albicans pathogenesis. In response to hydrogen peroxide (H2O2), but not glutathione-depleting/modifying oxidants, Cap1 oxidation, nuclear accumulation, phosphorylation, and Cap1-dependent gene expression, is mediated by a glutathione peroxidase-like enzyme, which we name Gpx3, and an orthologue of the Saccharomyces cerevisiae Yap1 binding protein, Ybp1. In addition, Ybp1 also functions to stabilise Cap1 and this novel function is conserved in S. cerevisiae. C. albicans cells lacking Cap1, Ybp1, or Gpx3, are unable to filament and thus, escape from murine macrophages after phagocytosis, and also display defective virulence in the Galleria mellonella infection model. Ybp1 is required to promote the stability of fungal AP-1-like transcription factors, and Ybp1 and Gpx3 mediated Cap1-dependent oxidative stress responses are essential for the effective killing of macrophages by C. albicans. Activation of Cap1, specifically by H2O2, is a prerequisite for the subsequent filamentation and escape of this fungal pathogen from the macrophage.

New structural insights into the molecular deciphering of mycobacterial lipoglycan binding to C-type lectins: lipoarabinomannan glycoform characterization and quantification by capillary electrophoresis at the subnanomole level.

PubMed

Nigou, J; Vercellone, A; Puzo, G

2000-06-23

Lipoarabinomannans are key molecules of the mycobacterial envelopes involved in many steps of tuberculosis immunopathogenesis. Several of the biological activities of lipoarabinomannans are mediated by their ability to bind human C-type lectins, such as the macrophage mannose receptor, the mannose-binding protein and the surfactant proteins A and D. The lipoarabinomannan mannooligosaccharide caps have been demonstrated to be involved in the binding to the lectin carbohydrate recognition domains. We report an original analytical approach, based on capillary electrophoresis monitored by laser-induced fluorescence, allowing the absolute quantification, in nanomole quantities of lipoarabinomannan, of the number of mannooligosaccharide units per lipoarabinomannan molecule. Moreover, this analytical approach was successful for the glycosidic linkage determination of the mannooligosaccharide motifs and has been applied to the comparative analysis of parietal and cellular lipoarabinomannans of Mycobacterium bovis BCG and Mycobacterium tuberculosis H37Rv, H37Ra and Erdman strains. Significant differences were observed in the amounts of the various mannooligosaccharide units between lipoarabinomannans of different strains and between parietal and cellular lipoarabinomannans of the same strain. Nevertheless, no relationship was found between the number of mannooligosaccharide caps and the virulence of the corresponding strain. The results of the present study should help us to gain more understanding of the molecular basis of lipoarabinomannan discrimination in the process of binding to C-type lectins. Copyright 2000 Academic Press.

The Drosophila telomere-capping protein Verrocchio binds single-stranded DNA and protects telomeres from DNA damage response

PubMed Central

Cicconi, Alessandro; Micheli, Emanuela; Vernì, Fiammetta; Jackson, Alison; Gradilla, Ana Citlali; Cipressa, Francesca; Raimondo, Domenico; Bosso, Giuseppe; Wakefield, James G.; Ciapponi, Laura; Cenci, Giovanni; Gatti, Maurizio

2017-01-01

Abstract Drosophila telomeres are sequence-independent structures maintained by transposition to chromosome ends of three specialized retroelements rather than by telomerase activity. Fly telomeres are protected by the terminin complex that includes the HOAP, HipHop, Moi and Ver proteins. These are fast evolving, non-conserved proteins that localize and function exclusively at telomeres, protecting them from fusion events. We have previously suggested that terminin is the functional analogue of shelterin, the multi-protein complex that protects human telomeres. Here, we use electrophoretic mobility shift assay (EMSA) and atomic force microscopy (AFM) to show that Ver preferentially binds single-stranded DNA (ssDNA) with no sequence specificity. We also show that Moi and Ver form a complex in vivo. Although these two proteins are mutually dependent for their localization at telomeres, Moi neither binds ssDNA nor facilitates Ver binding to ssDNA. Consistent with these results, we found that Ver-depleted telomeres form RPA and γH2AX foci, like the human telomeres lacking the ssDNA-binding POT1 protein. Collectively, our findings suggest that Drosophila telomeres possess a ssDNA overhang like the other eukaryotes, and that the terminin complex is architecturally and functionally similar to shelterin. PMID:27940556

Innate immune restriction and antagonism of viral RNA lacking 2'-O methylation

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

Hyde, Jennifer L.; Diamond, Michael S., E-mail: diamond@borcim.wustl.edu; Molecular Microbiology, Washington University School of Medicine, St Louis., MO 63110

N-7 and 2′-O methylation of host cell mRNA occurs in the nucleus and results in the generation of cap structures (cap 0, m{sup 7}GpppN; cap 1, m{sup 7}GpppNm) that control gene expression by modulating nuclear export, splicing, turnover, and protein synthesis. Remarkably, RNA cap modification also contributes to mammalian cell host defense as viral RNA lacking 2′-O methylation is sensed and inhibited by IFIT1, an interferon (IFN) stimulated gene (ISG). Accordingly, pathogenic viruses that replicate in the cytoplasm have evolved mechanisms to circumvent IFIT1 restriction and facilitate infection of mammalian cells. These include: (a) generating cap 1 structures on theirmore » RNA through cap-snatching or virally-encoded 2′-O methyltransferases, (b) using cap-independent means of translation, or (c) using RNA secondary structural motifs to antagonize IFIT1 binding. This review will discuss new insights as to how specific modifications at the 5′-end of viral RNA modulate host pathogen recognition responses to promote infection and disease.« less

Construction of plasmid, bacterial expression, purification, and assay of dengue virus type 2 NS5 methyltransferase.

PubMed

Boonyasuppayakorn, Siwaporn; Padmanabhan, Radhakrishnan

2014-01-01

Dengue virus (DENV), a member of mosquito-borne flavivirus, causes self-limiting dengue fever as well as life-threatening dengue hemorrhagic fever and dengue shock syndrome. Its positive sense RNA genome has a cap at the 5'-end and no poly(A) tail at the 3'-end. The viral RNA encodes a single polyprotein, C-prM-E-NS1-NS2A-NS2B-NS3-NS4A-NS4B-NS5. The polyprotein is processed into 3 structural proteins (C, prM, and E) and 7 nonstructural (NS) proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, NS5). NS3 and NS5 are multifunctional enzymes performing various tasks in viral life cycle. The N-terminal domain of NS5 has distinct GTP and S-adenosylmethionine (SAM) binding sites. The role of GTP binding site is implicated in guanylyltransferase (GTase) activity of NS5. The SAM binding site is involved in both N-7 and 2'-O-methyltransferase (MTase) activities involved in formation of type I cap. The C-terminal domain of NS5 catalyzes RNA-dependent RNA polymerase (RdRp) activity involved in RNA synthesis. We describe the construction of the MTase domain of NS5 in an E. coli expression vector, purification of the enzyme, and conditions for enzymatic assays of N7- and 2'O-methyltransferase activities that yield the final type I 5'-capped RNA ((7Me)GpppA2'OMe-RNA).

TERRA and hnRNPA1 orchestrate an RPA-to-POT1 switch on telomeric single-stranded DNA.

PubMed

Flynn, Rachel Litman; Centore, Richard C; O'Sullivan, Roderick J; Rai, Rekha; Tse, Alice; Songyang, Zhou; Chang, Sandy; Karlseder, Jan; Zou, Lee

2011-03-24

Maintenance of telomeres requires both DNA replication and telomere 'capping' by shelterin. These two processes use two single-stranded DNA (ssDNA)-binding proteins, replication protein A (RPA) and protection of telomeres 1 (POT1). Although RPA and POT1 each have a critical role at telomeres, how they function in concert is not clear. POT1 ablation leads to activation of the ataxia telangiectasia and Rad3-related (ATR) checkpoint kinase at telomeres, suggesting that POT1 antagonizes RPA binding to telomeric ssDNA. Unexpectedly, we found that purified POT1 and its functional partner TPP1 are unable to prevent RPA binding to telomeric ssDNA efficiently. In cell extracts, we identified a novel activity that specifically displaces RPA, but not POT1, from telomeric ssDNA. Using purified protein, here we show that the heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1) recapitulates the RPA displacing activity. The RPA displacing activity is inhibited by the telomeric repeat-containing RNA (TERRA) in early S phase, but is then unleashed in late S phase when TERRA levels decline at telomeres. Interestingly, TERRA also promotes POT1 binding to telomeric ssDNA by removing hnRNPA1, suggesting that the re-accumulation of TERRA after S phase helps to complete the RPA-to-POT1 switch on telomeric ssDNA. Together, our data suggest that hnRNPA1, TERRA and POT1 act in concert to displace RPA from telomeric ssDNA after DNA replication, and promote telomere capping to preserve genomic integrity.

Molecular requirements for actin-based lamella formation in Drosophila S2 cells

PubMed Central

Rogers, Stephen L.; Wiedemann, Ursula; Stuurman, Nico; Vale, Ronald D.

2003-01-01

Cell migration occurs through the protrusion of the actin-enriched lamella. Here, we investigated the effects of RNAi depletion of ∼90 proteins implicated in actin function on lamella formation in Drosophila S2 cells. Similar to in vitro reconstitution studies of actin-based Listeria movement, we find that lamellae formation requires a relatively small set of proteins that participate in actin nucleation (Arp2/3 and SCAR), barbed end capping (capping protein), filament depolymerization (cofilin and Aip1), and actin monomer binding (profilin and cyclase-associated protein). Lamellae are initiated by parallel and partially redundant signaling pathways involving Rac GTPases and the adaptor protein Nck, which stimulate SCAR, an Arp2/3 activator. We also show that RNAi of three proteins (kette, Abi, and Sra-1) known to copurify with and inhibit SCAR in vitro leads to SCAR degradation, revealing a novel function of this protein complex in SCAR stability. Our results have identified an essential set of proteins involved in actin dynamics during lamella formation in Drosophila S2 cells. PMID:12975351

Phospholipid arrays on porous polymer coatings generated by micro-contact spotting

PubMed Central

de Freitas, Monica; Tröster, Lea-Marie; Jochum, Tobias; Levkin, Pavel A; Hirtz, Michael; Fuchs, Harald

2017-01-01

Nanoporous poly(2-hydroxyethyl methacrylate-co-ethylene dimethacrylate) (HEMA-EDMA) is used as a 3D mesh for spotting lipid arrays. Its porous structure is an ideal matrix for lipid ink to infiltrate, resulting in higher fluorescent signal intensity as compared to similar arrays on strictly 2D substrates like glass. The embedded lipid arrays show high stability against washing steps, while still being accessible for protein and antibody binding. To characterize binding to polymer-embedded lipids we have applied Streptavidin as well as biologically important biotinylated androgen receptor binding onto 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(cap biotinyl) (Biotinyl Cap PE) and anti-DNP IgE recognition of 2,4-dinitrophenyl[1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[6-[(2,4-dinitrophenyl)amino]hexanoyl] (DNP)] antigen. This approach adds lipid arrays to the range of HEMA polymer applications and makes this solid substrate a very attractive platform for a variety of bio-applications. PMID:28487815

Clinical characterisation of pneumonia caused by atypical pathogens combining classic and novel predictors.

PubMed

Masiá, M; Gutiérrez, F; Padilla, S; Soldán, B; Mirete, C; Shum, C; Hernández, I; Royo, G; Martin-Hidalgo, A

2007-02-01

The aim of this study was to characterise community-acquired pneumonia (CAP) caused by atypical pathogens by combining distinctive clinical and epidemiological features and novel biological markers. A population-based prospective study of consecutive patients with CAP included investigation of biomarkers of bacterial infection, e.g., procalcitonin, C-reactive protein and lipopolysaccharide-binding protein (LBP) levels. Clinical, radiological and laboratory data for patients with CAP caused by atypical pathogens were compared by univariate and multivariate analysis with data for patients with typical pathogens and patients from whom no organisms were identified. Two predictive scoring models were developed with the most discriminatory variables from multivariate analysis. Of 493 patients, 94 had CAP caused by atypical pathogens. According to multivariate analysis, patients with atypical pneumonia were more likely to have normal white blood cell counts, have repetitive air-conditioning exposure, be aged <65 years, have elevated aspartate aminotransferase levels, have been exposed to birds, and have lower serum levels of LBP. Two different scoring systems were developed that predicted atypical pathogens with sensitivities of 35.2% and 48.8%, and specificities of 93% and 91%, respectively. The combination of selected patient characteristics and laboratory data identified up to half of the cases of atypical pneumonia with high specificity, which should help clinicians to optimise initial empirical therapy for CAP.

Stk1-mediated phosphorylation stimulates the DNA-binding properties of the Staphylococcus aureus SpoVG transcriptional factor.

PubMed

Bischoff, Markus; Brelle, Solène; Minatelli, Sabrina; Molle, Virginie

2016-05-13

The stage V sporulation protein G (SpoVG) homolog of Staphylococcus aureus is a modulator of virulence factor synthesis and antibiotic resistance in this clinically important gram-positive pathogen. Here we demonstrate that SpoVG can be phosphorylated by the staphylococcal Ser/Thr protein kinase Stk1 and that phosphorylation positively affects its DNA-binding properties. Mass spectrometric analyses and site directed mutagenesis identified Thr4, Thr13, Thr24 and Ser41 as phospho-acceptors. Stk1-mediated phosphorylation markedly enhanced the DNA binding activity of SpoVG towards the promoter regions of target genes such as capA, lip, and nuc1. Similarly, trans-complementation of the S. aureus ΔyabJ-spoVG mutant SM148 with a SpoVG derivative that mimics constitutive phosphorylation, SpoVG_Asp, exhibited capA, lip, and nuc1 transcript levels that were comparable to the levels seen with the wild-type, whereas trans-complementation with a phosphoablative variant of SpoVG (SpoVG_Ala) produced transcript levels similar to the ones seen in SM148. Our data suggest that the expression/activity of this transcription factor is tightly controlled in S. aureus by transcriptional, post-transcriptional and post-translational mechanisms. Copyright © 2016 Elsevier Inc. All rights reserved.

Dynamin1 is a novel target for IRSp53 protein and works with mammalian enabled (Mena) protein and Eps8 to regulate filopodial dynamics.

PubMed

Chou, Ai Mei; Sem, Kai Ping; Wright, Graham Daniel; Sudhaharan, Thankiah; Ahmed, Sohail

2014-08-29

Filopodia are dynamic actin-based structures that play roles in processes such as cell migration, wound healing, and axonal guidance. Cdc42 induces filopodial formation through IRSp53, an Inverse-Bin-Amphiphysins-Rvs (I-BAR) domain protein. Previous work from a number of laboratories has shown that IRSp53 generates filopodia by coupling membrane protrusion with actin dynamics through its Src homology 3 domain binding partners. Here, we show that dynamin1 (Dyn1), the large guanosine triphosphatase, is an interacting partner of IRSp53 through pulldown and Förster resonance energy transfer analysis, and we explore its role in filopodial formation. In neuroblastoma cells, Dyn1 localizes to filopodia, associated tip complexes, and the leading edge just behind the anti-capping protein mammalian enabled (Mena). Dyn1 knockdown reduces filopodial formation, which can be rescued by overexpressing wild-type Dyn1 but not the GTPase mutant Dyn1-K44A and the loss-of-function actin binding domain mutant Dyn1-K/E. Interestingly, dynasore, an inhibitor of Dyn GTPase, also reduced filopodial number and increased their lifetime. Using rapid time-lapse total internal reflection fluorescence microscopy, we show that Dyn1 and Mena localize to filopodia only during initiation and assembly. Dyn1 actin binding domain mutant inhibits filopodial formation, suggesting a role in actin elongation. In contrast, Eps8, an actin capping protein, is seen most strongly at filopodial tips during disassembly. Taken together, the results suggest IRSp53 partners with Dyn1, Mena, and Eps8 to regulate filopodial dynamics. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Use of entrapment and high-performance affinity chromatography to compare the binding of drugs and site-specific probes with normal and glycated human serum albumin

PubMed Central

Jackson, Abby J.; Anguizola, Jeanethe; Pfaunmiller, Erika L.; Hage, David S.

2013-01-01

Protein entrapment and high-performance affinity chromatography were used with zonal elution to examine the changes in binding that occurred for site-specific probes and various sulfonylurea drugs with normal and glycated forms of human serum albumin (HSA). Samples of this protein in a soluble form were physically entrapped within porous silica particles by using glycogen-capped hydrazide-activated silica; these supports were then placed into 1.0 cm × 2.1 mm inner diameter columns. Initial zonal elution studies were performed using (R)-warfarin and L-tryptophan as probes for Sudlow sites I and II (i.e., the major drug binding sites of HSA), giving quantitative measures of binding affinities in good agreement with literature values. It was also found for solutes with multisite binding to the same proteins, such as many sulfonylurea drugs, that this method could be used to estimate the global affinity of the solute for the entrapped protein. This entrapment and zonal approach provided retention information with precisions of ±0.1–3.3% (± one standard deviation) and elution within 0.50–3.00 min for solutes with binding affinities of 1 × 104–3 × 105 M−1. Each entrapped-protein column was used for many binding studies, which decreased the cost and amount of protein needed per injection (e.g., the equivalent of only 125–145 pmol of immobilized HSA or glycated HSA per injection over 60 sample application cycles). This method can be adapted for use with other proteins and solutes and should be valuable in high-throughput screening or quantitative studies of drug–protein binding or related biointeractions. PMID:23657448

The PHD Finger Protein MMD1/DUET Ensures the Progression of Male Meiotic Chromosome Condensation and Directly Regulates the Expression of the Condensin Gene CAP-D3.

PubMed

Wang, Jun; Niu, Baixiao; Huang, Jiyue; Wang, Hongkuan; Yang, Xiaohui; Dong, Aiwu; Makaroff, Christopher; Ma, Hong; Wang, Yingxiang

2016-08-01

Chromosome condensation, a process mediated by the condensin complex, is essential for proper chromosome segregation during cell division. Unlike rapid mitotic chromosome condensation, meiotic chromosome condensation occurs over a relatively long prophase I and is unusually complex due to the coordination with chromosome axis formation and homolog interaction. The molecular mechanisms that regulate meiotic chromosome condensation progression from prophase I to metaphase I are unclear. Here, we show that the Arabidopsis thaliana meiotic PHD-finger protein MMD1/DUET is required for progressive compaction of prophase I chromosomes to metaphase I bivalents. The MMD1 PHD domain is required for its function in chromosome condensation and binds to methylated histone tails. Transcriptome analysis and qRT-PCR showed that several condensin genes exhibit significantly reduced expression in mmd1 meiocytes. Furthermore, MMD1 specifically binds to the promoter region of the condensin subunit gene CAP-D3 to enhance its expression. Moreover, cap-d3 mutants exhibit similar chromosome condensation defects, revealing an MMD1-dependent mechanism for regulating meiotic chromosome condensation, which functions in part by promoting condensin gene expression. Together, these discoveries provide strong evidence that the histone reader MMD1/DUET defines an important step for regulating the progression of meiotic prophase I chromosome condensation. © 2016 American Society of Plant Biologists. All rights reserved.

The PHD Finger Protein MMD1/DUET Ensures the Progression of Male Meiotic Chromosome Condensation and Directly Regulates the Expression of the Condensin Gene CAP-D3[OPEN

PubMed Central

Wang, Jun; Niu, Baixiao; Huang, Jiyue; Wang, Hongkuan; Yang, Xiaohui; Dong, Aiwu

2016-01-01

Chromosome condensation, a process mediated by the condensin complex, is essential for proper chromosome segregation during cell division. Unlike rapid mitotic chromosome condensation, meiotic chromosome condensation occurs over a relatively long prophase I and is unusually complex due to the coordination with chromosome axis formation and homolog interaction. The molecular mechanisms that regulate meiotic chromosome condensation progression from prophase I to metaphase I are unclear. Here, we show that the Arabidopsis thaliana meiotic PHD-finger protein MMD1/DUET is required for progressive compaction of prophase I chromosomes to metaphase I bivalents. The MMD1 PHD domain is required for its function in chromosome condensation and binds to methylated histone tails. Transcriptome analysis and qRT-PCR showed that several condensin genes exhibit significantly reduced expression in mmd1 meiocytes. Furthermore, MMD1 specifically binds to the promoter region of the condensin subunit gene CAP-D3 to enhance its expression. Moreover, cap-d3 mutants exhibit similar chromosome condensation defects, revealing an MMD1-dependent mechanism for regulating meiotic chromosome condensation, which functions in part by promoting condensin gene expression. Together, these discoveries provide strong evidence that the histone reader MMD1/DUET defines an important step for regulating the progression of meiotic prophase I chromosome condensation. PMID:27385818

Cap-proximal nucleotides via differential eIF4E binding and alternative promoter usage mediate translational response to energy stress.

PubMed

Tamarkin-Ben-Harush, Ana; Vasseur, Jean-Jacques; Debart, Françoise; Ulitsky, Igor; Dikstein, Rivka

2017-02-08

Transcription start-site (TSS) selection and alternative promoter (AP) usage contribute to gene expression complexity but little is known about their impact on translation. Here we performed TSS mapping of the translatome following energy stress. Assessing the contribution of cap-proximal TSS nucleotides, we found dramatic effect on translation only upon stress. As eIF4E levels were reduced, we determined its binding to capped-RNAs with different initiating nucleotides and found the lowest affinity to 5'cytidine in correlation with the translational stress-response. In addition, the number of differentially translated APs was elevated following stress. These include novel glucose starvation-induced downstream transcripts for the translation regulators eIF4A and Pabp, which are also translationally-induced despite general translational inhibition. The resultant eIF4A protein is N-terminally truncated and acts as eIF4A inhibitor. The induced Pabp isoform has shorter 5'UTR removing an auto-inhibitory element. Our findings uncovered several levels of coordination of transcription and translation responses to energy stress.

The protein network surrounding the human telomere repeat binding factors TRF1, TRF2, and POT1

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

Giannone, Richard J; McDonald, W Hayes; Hurst, Gregory

Telomere integrity (including telomere length and capping) is critical in overall genomic stability. Telomere repeat binding factors and their associated proteins play vital roles in telomere length regulation and end protection. In this study, we explore the protein network surrounding telomere repeat binding factors, TRF1, TRF2, and POT1 using dual-tag affinity purification in combination with multidimensional protein identification technology liquid chromatography - tandem mass spectrometry (MudPIT LC-MS/MS). After control subtraction and data filtering, we found that TRF2 and POT1 co-purified all six members of the telomere protein complex, while TRF1 identified five of six components at frequencies that lend evidencemore » towards the currently accepted telomere architecture. Many of the known TRF1 or TRF2 interacting proteins were also identified. Moreover, putative associating partners identified for each of the three core components fell into functional categories such as DNA damage repair, ubiquitination, chromosome cohesion, chromatin modification/remodeling, DNA replication, cell cycle and transcription regulation, nucleotide metabolism, RNA processing, and nuclear transport. These putative protein-protein associations may participate in different biological processes at telomeres or, intriguingly, outside telomeres.« less

Stimulation of translation by human Unr requires cold shock domains 2 and 4, and correlates with poly(A) binding protein interaction.

PubMed

Ray, Swagat; Anderson, Emma C

2016-03-03

The RNA binding protein Unr, which contains five cold shock domains, has several specific roles in post-transcriptional control of gene expression. It can act as an activator or inhibitor of translation initiation, promote mRNA turnover, or stabilise mRNA. Its role depends on the mRNA and other proteins to which it binds, which includes cytoplasmic poly(A) binding protein 1 (PABP1). Since PABP1 binds to all polyadenylated mRNAs, and is involved in translation initiation by interaction with eukaryotic translation initiation factor 4G (eIF4G), we investigated whether Unr has a general role in translational control. We found that Unr strongly stimulates translation in vitro, and mutation of cold shock domains 2 or 4 inhibited its translation activity. The ability of Unr and its mutants to stimulate translation correlated with its ability to bind RNA, and to interact with PABP1. We found that Unr stimulated the binding of PABP1 to mRNA, and that Unr was required for the stable interaction of PABP1 and eIF4G in cells. siRNA-mediated knockdown of Unr reduced the overall level of cellular translation in cells, as well as that of cap-dependent and IRES-dependent reporters. These data describe a novel role for Unr in regulating cellular gene expression.

An ice-binding and tandem beta-sandwich domain-containing protein in Shewanella frigidimarina is a potential new type of ice adhesin.

PubMed

Vance, Tyler D R; Graham, Laurie A; Davies, Peter L

2018-04-01

Out of the dozen different ice-binding protein (IBP) structures known, the DUF3494 domain is the most widespread, having been passed many times between prokaryotic and eukaryotic microorganisms by horizontal gene transfer. This ~25-kDa β-solenoid domain with an adjacent parallel α-helix is most commonly associated with an N-terminal secretory signal peptide. However, examples of the DUF3494 domain preceded by tandem Bacterial Immunoglobulin-like (BIg) domains are sometimes found, though uncharacterized. Here, we present one such protein (SfIBP_1) from the Antarctic bacterium Shewanella frigidimarina. We have confirmed and characterized the ice-binding activity of its ice-binding domain using thermal hysteresis measurements, fluorescent ice plane affinity analysis, and ice recrystallization inhibition assays. X-ray crystallography was used to solve the structure of the SfIBP_1 ice-binding domain, to further characterize its ice-binding surface and unique method of stabilizing or 'capping' the ends of the solenoid structure. The latter is formed from the interaction of two loops mediated by a combination of tandem prolines and electrostatic interactions. Furthermore, given their domain architecture and membrane association, we propose that these BIg-containing DUF3494 IBPs serve as ice-binding adhesion proteins that are capable of adsorbing their host bacterium onto ice. Submitted new structure to the Protein Data Bank (PDB: 6BG8). © 2018 Federation of European Biochemical Societies.

PRMT5 is essential for the eIF4E-mediated 5′-cap dependent translation

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

Lim, Ji-Hong; Lee, Yoon-Mi; Lee, Gibok

2014-10-03

Highlights: • PRMT5 participates in syntheses of HIF-1α, c-Myc and cyclin D1 proteins. • PRMT5 promotes the 5′-cap dependent translation. • PRMT5 is required for eIF4E binding to mRNA 5′-cap. • PRMT5 is essential for eIF4E-dependent cell proliferation. - Abstract: It is becoming clear that PRMT5 plays essential roles in cell cycle progression, survival, and responses to external stresses. However, the precise mechanisms underlying such roles of PRMT5 have not been clearly understood. Previously, we have demonstrated that PRMT5 participates in cellular adaptation to hypoxia by ensuring 5′-cap dependent translation of HIF-1α. Given that c-Myc and cyclin D1 expressions aremore » also tightly regulated in 5′-cap dependent manner, we here tested the possibility that PRMT5 promotes cell proliferation by increasing de novo syntheses of the oncoproteins. c-Myc and cyclin D1 were found to be noticeably downregulated by PRMT5 knock-down. A RNA immunoprecipitation analysis, which can identify RNA–protein interactions, showed that PRMT5 is required for the interaction among eIF4E and 5′-UTRs of HIF-1α, c-Myc and cyclin D1 mRNAs. In addition, PRMT5 knock-down inhibited cell proliferation by inducing cell cycle arrest at the G1 phase. More importantly, ectopic expression of eIF4E significantly rescued the cell cycle progression and cell proliferation even in PRMT5-deficeint condition. Based on these results, we propose that PRMT5 determines cell fate by regulating 5′-cap dependent translation of proteins essential for proliferation and survival.« less

Crystal Structure of Serine Racemase that Produces Neurotransmitter d-Serine for Stimulation of the NMDA Receptor

NASA Astrophysics Data System (ADS)

Goto, Masaru

d-Serine is an endogenous coagonist for the N-methyl-d-aspartate receptor and is involved in excitatory neurotransmission in the brain. Mammalian pyridoxal 5’-phosphate-dependent serine racemase, which is localized in the mammalian brain, catalyzes the racemization of l-serine to yield d-serine and vice versa. We have determined the structures of three forms of the mammalian enzyme homolog from Schizosaccharomyces pombe. Lys57 and Ser82 located on the protein and solvent sides, respectively, with respect to the cofactor plane, are acid-base catalysts that shuttle protons to the substrate. The modified enzyme, which has a unique lysino-d-alanyl residue at the active site, also binds the substrate serine in the active site, suggesting that the lysino-d-alanyl residue acts as a catalytic base in the same manner as Lys57 of the wild type enzyme.

Structural studies of human glioma pathogenesis-related protein 1

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

Asojo, Oluwatoyin A., E-mail: oasojo@unmc.edu; Koski, Raymond A.; Bonafé, Nathalie

2011-10-01

Structural analysis of a truncated soluble domain of human glioma pathogenesis-related protein 1, a membrane protein implicated in the proliferation of aggressive brain cancer, is presented. Human glioma pathogenesis-related protein 1 (GLIPR1) is a membrane protein that is highly upregulated in brain cancers but is barely detectable in normal brain tissue. GLIPR1 is composed of a signal peptide that directs its secretion, a conserved cysteine-rich CAP (cysteine-rich secretory proteins, antigen 5 and pathogenesis-related 1 proteins) domain and a transmembrane domain. GLIPR1 is currently being investigated as a candidate for prostate cancer gene therapy and for glioblastoma targeted therapy. Crystal structuresmore » of a truncated soluble domain of the human GLIPR1 protein (sGLIPR1) solved by molecular replacement using a truncated polyalanine search model of the CAP domain of stecrisp, a snake-venom cysteine-rich secretory protein (CRISP), are presented. The correct molecular-replacement solution could only be obtained by removing all loops from the search model. The native structure was refined to 1.85 Å resolution and that of a Zn{sup 2+} complex was refined to 2.2 Å resolution. The latter structure revealed that the putative binding cavity coordinates Zn{sup 2+} similarly to snake-venom CRISPs, which are involved in Zn{sup 2+}-dependent mechanisms of inflammatory modulation. Both sGLIPR1 structures have extensive flexible loop/turn regions and unique charge distributions that were not observed in any of the previously reported CAP protein structures. A model is also proposed for the structure of full-length membrane-bound GLIPR1.« less

Human La binds mRNAs through contacts to the poly(A) tail.

PubMed

Vinayak, Jyotsna; Marrella, Stefano A; Hussain, Rawaa H; Rozenfeld, Leonid; Solomon, Karine; Bayfield, Mark A

2018-05-04

In addition to a role in the processing of nascent RNA polymerase III transcripts, La proteins are also associated with promoting cap-independent translation from the internal ribosome entry sites of numerous cellular and viral coding RNAs. La binding to RNA polymerase III transcripts via their common UUU-3'OH motif is well characterized, but the mechanism of La binding to coding RNAs is poorly understood. Using electromobility shift assays and cross-linking immunoprecipitation, we show that in addition to a sequence specific UUU-3'OH binding mode, human La exhibits a sequence specific and length dependent poly(A) binding mode. We demonstrate that this poly(A) binding mode uses the canonical nucleic acid interaction winged helix face of the eponymous La motif, previously shown to be vacant during uridylate binding. We also show that cytoplasmic, but not nuclear La, engages poly(A) RNA in human cells, that La entry into polysomes utilizes the poly(A) binding mode, and that La promotion of translation from the cyclin D1 internal ribosome entry site occurs in competition with cytoplasmic poly(A) binding protein (PABP). Our data are consistent with human La functioning in translation through contacts to the poly(A) tail.

Structural basis for the ligand-binding specificity of fatty acid-binding proteins (pFABP4 and pFABP5) in gentoo penguin.

PubMed

Lee, Chang Woo; Kim, Jung Eun; Do, Hackwon; Kim, Ryeo-Ok; Lee, Sung Gu; Park, Hyun Ho; Chang, Jeong Ho; Yim, Joung Han; Park, Hyun; Kim, Il-Chan; Lee, Jun Hyuck

2015-09-11

Fatty acid-binding proteins (FABPs) are involved in transporting hydrophobic fatty acids between various aqueous compartments of the cell by directly binding ligands inside their β-barrel cavities. Here, we report the crystal structures of ligand-unbound pFABP4, linoleate-bound pFABP4, and palmitate-bound pFABP5, obtained from gentoo penguin (Pygoscelis papua), at a resolution of 2.1 Å, 2.2 Å, and 2.3 Å, respectively. The pFABP4 and pFABP5 proteins have a canonical β-barrel structure with two short α-helices that form a cap region and fatty acid ligand binding sites in the hydrophobic cavity within the β-barrel structure. Linoleate-bound pFABP4 and palmitate-bound pFABP5 possess different ligand-binding modes and a unique ligand-binding pocket due to several sequence dissimilarities (A76/L78, T30/M32, underlining indicates pFABP4 residues) between the two proteins. Structural comparison revealed significantly different conformational changes in the β3-β4 loop region (residues 57-62) as well as the flipped Phe60 residue of pFABP5 than that in pFABP4 (the corresponding residue is Phe58). A ligand-binding study using fluorophore displacement assays shows that pFABP4 has a relatively strong affinity for linoleate as compared to pFABP5. In contrast, pFABP5 exhibits higher affinity for palmitate than that for pFABP4. In conclusion, our high-resolution structures and ligand-binding studies provide useful insights into the ligand-binding preferences of pFABPs based on key protein-ligand interactions. Copyright © 2015 Elsevier Inc. All rights reserved.

Flagellar Cap Protein FliD Mediates Adherence of Atypical Enteropathogenic Escherichia coli to Enterocyte Microvilli

PubMed Central

Sampaio, Suely C. F.; Luiz, Wilson B.; Vieira, Mônica A. M.; Ferreira, Rita C. C.; Garcia, Bruna G.; Sinigaglia-Coimbra, Rita; Sampaio, Jorge L. M.; Ferreira, Luís C. S.

2016-01-01

The expression of flagella correlates with different aspects of bacterial pathogenicity, ranging from adherence to host cells to activation of inflammatory responses by the innate immune system. In the present study, we investigated the role of flagella in the adherence of an atypical enteropathogenic Escherichia coli (aEPEC) strain (serotype O51:H40) to human enterocytes. Accordingly, isogenic mutants deficient in flagellin (FliC), the flagellar structural subunit; the flagellar cap protein (FliD); or the MotAB proteins, involved in the control of flagellar motion, were generated and tested for binding to differentiated Caco-2 cells. Binding of the aEPEC strain to enterocytes was significantly impaired in strains with the fliC and fliD genes deleted, both of which could not form flagella on the bacterial surface. A nonmotile but flagellated MotAB mutant also showed impaired adhesion to Caco-2 cells. In accordance with these observations, adhesion of aEPEC strain 1711-4 to Caco-2 cells was drastically reduced after the treatment of Caco-2 cells with purified FliD. In addition, incubation of aEPEC bacteria with specific anti-FliD serum impaired binding to Caco-2 cells. Finally, incubation of Caco-2 cells with purified FliD, followed by immunolabeling, showed that the protein was specifically bound to the microvillus tips of differentiated Caco-2 cells. The aEPEC FliD or anti-FliD serum also reduced the adherence of prototype typical enteropathogenic, enterohemorrhagic, and enterotoxigenic E. coli strains to Caco-2 cells. In conclusion, our findings further strengthened the role of flagella in the adherence of aEPEC to human enterocytes and disclosed the relevant structural and functional involvement of FliD in the adhesion process. PMID:26831466

Exploring Step‐by‐Step Assembly of Nanoparticle:Cytochrome Biohybrid Photoanodes

PubMed Central

Hwang, Ee Taek; Orchard, Katherine L.; Hojo, Daisuke; Beton, Joseph; Lockwood, Colin W. J.; Adschiri, Tadafumi

2017-01-01

Abstract Coupling light‐harvesting semiconducting nanoparticles (NPs) with redox enzymes has been shown to create artificial photosynthetic systems that hold promise for the synthesis of solar fuels. High quantum yields require efficient electron transfer from the nanoparticle to the redox protein, a property that can be difficult to control. Here, we have compared binding and electron transfer between dye‐sensitized TiO2 nanocrystals or CdS quantum dots and two decaheme cytochromes on photoanodes. The effect of NP surface chemistry was assessed by preparing NPs capped with amine or carboxylic acid functionalities. For the TiO2 nanocrystals, binding to the cytochromes was optimal when capped with a carboxylic acid ligand, whereas for the CdS QDs, better adhesion was observed for amine capped ligand shells. When using TiO2 nanocrystals, dye‐sensitized with a phosphonated bipyridine Ru(II) dye, photocurrents are observed that are dependent on the redox state of the decaheme, confirming that electrons are transferred from the TiO2 nanocrystals to the surface via the decaheme conduit. In contrast, when CdS NPs are used, photocurrents are not dependent on the redox state of the decaheme, consistent with a model in which electron transfer from CdS to the photoanode bypasses the decaheme protein. These results illustrate that although the organic shell of NPs nanoparticles crucially affects coupling with proteinaceous material, the coupling can be difficult to predict or engineer. PMID:28920010

Structural Basis for Ligand Regulation of the Fatty Acid-binding Protein 5, Peroxisome Proliferator-activated Receptor β/δ (FABP5-PPARβ/δ) Signaling Pathway*

PubMed Central

Armstrong, Eric H.; Goswami, Devrishi; Griffin, Patrick R.; Noy, Noa; Ortlund, Eric A.

2014-01-01

Fatty acid-binding proteins (FABPs) are a widely expressed group of calycins that play a well established role in solubilizing cellular fatty acids. Recent studies, however, have recast FABPs as active participants in vital lipid-signaling pathways. FABP5, like its family members, displays a promiscuous ligand binding profile, capable of interacting with numerous long chain fatty acids of varying degrees of saturation. Certain “activating” fatty acids induce the protein's cytoplasmic to nuclear translocation, stimulating PPARβ/δ transactivation; however, the rules that govern this process remain unknown. Using a range of structural and biochemical techniques, we show that both linoleic and arachidonic acid elicit FABP5's translocation by permitting allosteric communication between the ligand-sensing β2 loop and a tertiary nuclear localization signal within the α-helical cap of the protein. Furthermore, we show that more saturated, nonactivating fatty acids inhibit nuclear localization signal formation by destabilizing this activation loop, thus implicating FABP5 specifically in cis-bonded, polyunsaturated fatty acid signaling. PMID:24692551

Specificity in Transition State Binding: The Pauling Model Revisited

PubMed Central

Amyes, Tina L.; Richard, John P.

2013-01-01

Linus Pauling proposed that the large rate accelerations for enzymes are due to the high specificity of the protein catalyst for binding the reaction transition state. The observation that stable analogs of the transition states for enzymatic reactions often act as tight-binding binding inhibitors provided early support for this simple and elegant proposal. We review experimental results which support the proposal that Pauling’s model provides a satisfactory explanation for the rate accelerations for many heterolytic enzymatic reactions through high energy reaction intermediates, such as proton transfer and decarboxylation. Specificity in transition state binding is obtained when the total intrinsic binding energy of the substrate is significantly larger than the binding energy observed at the Michaelis complex. The results of recent studies to characterize the specificity in binding of the enolate oxygen at the transition state for the 1,3-isomerization reaction catalyzed by ketosteroid isomerase are reviewed. Interactions between pig heart succinyl-CoA:3-oxoacid coenzyme A transferase (SCOT) and the nonreacting portions of CoA are responsible for a rate increase of 3 × 1012-fold, which is close to the estimated total 5 × 1013-fold enzymatic rate acceleration. Studies that partition the interactions between SCOT and CoA into their contributing parts are reviewed. Interactions of the protein with the substrate phosphodianion group provide a ca. 12 kcal/mol stabilization of the transition state for the reactions catalyzed by triosephosphate isomerase, orotidine 5′-monophosphate decarboxylase and α-glycerol phosphate dehydrogenase. The interactions of these enzymes with the substrate piece phosphite dianion provide a 6 – 8 kcal/mol stabilization of the transition state for reaction of the appropriate truncated substrate. Enzyme activation by phosphite dianion reflects the higher dianion affinity for binding to the enzyme-transition state complex compared with the free enzyme. Evidence is presented that supports a model in which the binding energy of the phosphite dianion piece, or the phosphodianion group of the whole substrate, is utilized to drive an enzyme conformational change from an inactive open form EO to an active closed form EC, by closure of a phosphodianion gripper loop. Members of the enolase and haloalkanoic acid dehalogenase superfamilies use variable capping domains to interact with nonreacting portions of the substrate and sequester the substrate from interaction with bulk solvent. Interactions of this capping domain with the phenyl group of mandelate have been shown to activate mandelate racemase for catalysis of deprotonation of α-carbonyl carbon. We propose that an important function of these capping domains is to utilize the binding interactions with nonreacting portions of the substrate to activate the enzyme for catalysis. PMID:23327224

In vitro chloramphenicol detection in a Haemophilus influenza model using an aptamer-polymer based electrochemical biosensor.

PubMed

Yadav, Saurabh K; Agrawal, Bharati; Chandra, Pranjal; Goyal, Rajendra N

2014-05-15

A sensitive and selective electrochemical biosensor is developed for the determination of chloramphenicol (CAP) exploring its direct electron transfer processes in in-vitro model and pharmaceutical samples. This biosensor exploits a selective binding of CAP with aptamer, immobilized onto the poly-(4-amino-3-hydroxynapthalene sulfonic acid) (p-AHNSA) modified edge plane pyrolytic graphite. The electrochemical reduction of CAP was observed in a well-defined peak. A quartz crystal microbalance (QCM) study is performed to confirm the interaction between the polymer film and the aptamer. Cyclic voltammetry (CV) and square wave voltammetry (SWV) were used to detect CAP. The in-vitro CAP detection is performed using the bacterial strain of Haemophilus influenza. A significant accumulation of CAP by the drug sensitive H. influenza strain is observed for the first time in this study using a biosensor. Various parameters affecting the CAP detection in standard solution and in in vitro detection are optimized. The detection of CAP is linear in the range of 0.1-2500 nM with the detection limit and sensitivity of 0.02 nM and 0.102 µA/nM, respectively. CAP is also detected in the presence of other common antibiotics and proteins present in the real sample matrix, and negligible interference is observed. Copyright © 2013 Elsevier B.V. All rights reserved.

Pheromone induction of agglutination in Saccharomyces cerevisiae a cells

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

Terrance, K.; Lipke, P.N.

1987-10-01

a-Agglutinin, the cell surface sexual agglutinin of yeast a cells, was assayed by its ability to bind its complementary agglutinin, ..cap alpha..-agglutinin. The specific binding of /sup 125/I-..cap alpha..-agglutinin to a cells treated with the sex pheromone ..cap alpha..-factor was 2 to 2.5 times that of binding to a cells not treated with ..cap alpha..-factor. Competition with unlabeled ..cap alpha..-agglutinin revealed that the increased binding was due to increased cell surface expression of a-agglutinin, with no apparent change in the binding constant. The increase in site number was similar to the increase in cellular agglutinability. Increased expression of a-agglutinin followedmore » the same kinetics as the increase in cellular agglutinability, with a 10-min lag followed by a 15- to 20-min response time. Induction kinetics were similar in cells in phases G1 and G2 of the cell cycle. Maximal expression levels were similar in cells treated with excess pheromone and in cells exposed to pheromone after destruction of constitutively expressed a-agglutinin.« less

Selective inhibition by chloramphenicol of pregnenolone-16. cap alpha. -carbonitrile-inducible rat liver cytochrome P-450 isozymes

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

Graves, P.E.; Kaminsky, L.S.; Halpert, J.

Pregnenolone-16 ..cap alpha..-carbonitrile (PCN) has been shown to induce, in male rats, cytochrome P-450 isozymes responsible for the formation of R-10-hydroxywarfarin and R-dehydrowarfarin. Antibodies to the major PCN-inducible isozyme (PB/PCN-E) inhibit both activities in microsomal preparations. Recently the authors have shown that PCN treatment of female rats also induces the formation of both R-warfarin metabolites. However, in both sexes chloramphenicol (CAP) treatment selectively inhibits only the rate of formation of the R-dehydrowarfarin. A decrease in microsomal P-450 content occurs after in vivo administration of CAP to PCN-treated rats of both sexes. This is in contrast to the lack of effectmore » of CAP on P-450 levels in phenobarbital-treated rats. Covalent binding of /sup 14/C-CAP to microsomal protein in vitro was increased 3 to 4-fold following PCN treatment. Chromatographic evidences suggests the presence of at least two PCN-induced isozymes of similar molecular weights in both male and female rat liver microsomes. These data are consistent with the multiplicity of PCN-inducible P-450 in rat liver.« less

GIGYF1/2 proteins use auxiliary sequences to selectively bind to 4EHP and repress target mRNA expression

PubMed Central

Peter, Daniel; Weber, Ramona; Sandmeir, Felix; Wohlbold, Lara; Helms, Sigrun; Bawankar, Praveen; Valkov, Eugene; Igreja, Cátia; Izaurralde, Elisa

2017-01-01

The eIF4E homologous protein (4EHP) is thought to repress translation by competing with eIF4E for binding to the 5′ cap structure of specific mRNAs to which it is recruited through interactions with various proteins, including the GRB10-interacting GYF (glycine–tyrosine–phenylalanine domain) proteins 1 and 2 (GIGYF1/2). Despite its similarity to eIF4E, 4EHP does not interact with eIF4G and therefore fails to initiate translation. In contrast to eIF4G, GIGYF1/2 bind selectively to 4EHP but not eIF4E. Here, we present crystal structures of the 4EHP-binding regions of GIGYF1 and GIGYF2 in complex with 4EHP, which reveal the molecular basis for the selectivity of the GIGYF1/2 proteins for 4EHP. Complementation assays in a GIGYF1/2-null cell line using structure-based mutants indicate that 4EHP requires interactions with GIGYF1/2 to down-regulate target mRNA expression. Our studies provide structural insights into the assembly of 4EHP–GIGYF1/2 repressor complexes and reveal that rather than merely facilitating 4EHP recruitment to transcripts, GIGYF1/2 proteins are required for repressive activity. PMID:28698298

Detection of ovomucoid-specific low-affinity IgE in infants and its relationship to eczema.

PubMed

Kawamoto, Norio; Kamemura, Norio; Kido, Hiroshi; Fukao, Toshiyuki

2017-06-01

Allergen-specific low-affinity IgE was previously detected in cord blood by a highly sensitive densely carboxylated protein (DCP) chip, but not by ImmunoCAP. Here, we investigated the presence of low-affinity IgE during the early life of infants and observed its relationship with eczema. We conducted a birth cohort study, collecting sera at birth and 6 and 14 months of age (n = 110). We monitored the ovomucoid (OM)- and egg white (EW)-specific IgE (sIgE) by ImmunoCAP or DCP chip and analyzed the antigen affinity of sIgE by binding inhibition assays in the presence or absence of a mild chaotropic agent, diethyl amine (DEA). The low- and high-affinity OM-sIgEs and sensitization risk factors were analyzed by a multivariate logistic analysis. The OM-sIgE measured by DCP chip significantly correlated with that measured by ImmunoCAP, but some samples assessed as OM-sIgE positive by DCP chip were considered OM-sIgE negative by ImmunoCAP. Binding inhibition analysis after DEA treatment was performed for participants judged as OM-sIgE positive by DCP chip at 14 M. The group assessed as negative for OM- and EW-sIgE by ImmunoCAP at 6 and 14 months showed a larger binding inhibition curve shift after DEA treatment than did the group assessed as positive at these times, indicating the presence of low-affinity sIgE antibodies at 14 months. The logistic regression analysis found that persistent eczema from 6 to 14 months is a significant risk factor for developing high-affinity, but not low-affinity, sIgE. Human infant peripheral blood contains allergen-specific low-affinity sIgE. Persistent eczema is related to the development of high-affinity, but not low-affinity, IgE. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Identification of small molecule inhibitors of the Chikungunya virus nsP1 RNA capping enzyme.

PubMed

Feibelman, Kristen M; Fuller, Benjamin P; Li, Linfeng; LaBarbera, Daniel V; Geiss, Brian J

2018-06-01

Chikungunya virus (CHIKV) is an arthropod-borne alphavirus. Alphaviruses are positive strand RNA viruses that require a 5' cap structure to direct translation of the viral polyprotein and prevent degradation of the viral RNA genome by host cell nucleases. Formation of the 5' RNA cap is orchestrated by the viral protein nsP1, which binds GTP and provides the N-7 methyltransferase and guanylyltransferase activities that are necessary for cap formation. Viruses with aberrant nsP1 activity are unable to replicate effectively suggesting that nsP1 is a promising target for antiviral drug discovery. Given the absence of commercially available antiviral therapies for CHIKV, it is imperative to identify compounds that could be developed as potential therapeutics. This study details a high-throughput screen of 3051 compounds from libraries containing FDA-approved drugs, natural products, and known bioactives against CHIKV nsP1 using a fluorescence polarization-based GTP competition assay. Several small molecule hits from this screen were able to compete with GTP for the CHIKV nsP1 GTP binding site at low molar concentrations. Compounds were also evaluated with an orthogonal assay that measured the ability of nsP1 to perform the guanylation step of the capping reaction in the presence of inhibitor. In addition, live virus assays with CHIKV and closely related alphavirus, Sindbis virus, were used in conjunction with cell toxicity assays to determine the antiviral activity of compounds in cell culture. The naturally derived compound lobaric acid was found to inhibit CHIKV nsP1 GTP binding and guanylation as well as attenuate viral growth in vitro at both 24 hpi and 48 hpi in hamster BHK21 and human Huh 7 cell lines. These data indicate that development of lobaric acid and further exploration of CHIKV nsP1 as a drug target may aid in the progress of anti-alphaviral drug development strategies. Copyright © 2018. Published by Elsevier B.V.

Specificity in transition state binding: the Pauling model revisited.

PubMed

Amyes, Tina L; Richard, John P

2013-03-26

Linus Pauling proposed that the large rate accelerations for enzymes are caused by the high specificity of the protein catalyst for binding the reaction transition state. The observation that stable analogues of the transition states for enzymatic reactions often act as tight-binding inhibitors provided early support for this simple and elegant proposal. We review experimental results that support the proposal that Pauling's model provides a satisfactory explanation for the rate accelerations for many heterolytic enzymatic reactions through high-energy reaction intermediates, such as proton transfer and decarboxylation. Specificity in transition state binding is obtained when the total intrinsic binding energy of the substrate is significantly larger than the binding energy observed at the Michaelis complex. The results of recent studies that aimed to characterize the specificity in binding of the enolate oxygen at the transition state for the 1,3-isomerization reaction catalyzed by ketosteroid isomerase are reviewed. Interactions between pig heart succinyl-coenzyme A:3-oxoacid coenzyme A transferase (SCOT) and the nonreacting portions of coenzyme A (CoA) are responsible for a rate increase of 3 × 10(12)-fold, which is close to the estimated total 5 × 10(13)-fold enzymatic rate acceleration. Studies that partition the interactions between SCOT and CoA into their contributing parts are reviewed. Interactions of the protein with the substrate phosphodianion group provide an ~12 kcal/mol stabilization of the transition state for the reactions catalyzed by triosephosphate isomerase, orotidine 5'-monophosphate decarboxylase, and α-glycerol phosphate dehydrogenase. The interactions of these enzymes with the substrate piece phosphite dianion provide a 6-8 kcal/mol stabilization of the transition state for reaction of the appropriate truncated substrate. Enzyme activation by phosphite dianion reflects the higher dianion affinity for binding to the enzyme-transition state complex compared with that of the free enzyme. Evidence is presented that supports a model in which the binding energy of the phosphite dianion piece, or the phosphodianion group of the whole substrate, is utilized to drive an enzyme conformational change from an inactive open form E(O) to an active closed form E(C), by closure of a phosphodianion gripper loop. Members of the enolase and haloalkanoic acid dehalogenase superfamilies use variable capping domains to interact with nonreacting portions of the substrate and sequester the substrate from interaction with bulk solvent. Interactions of this capping domain with the phenyl group of mandelate have been shown to activate mandelate racemase for catalysis of deprotonation of α-carbonyl carbon. We propose that an important function of these capping domains is to utilize the binding interactions with nonreacting portions of the substrate to activate the enzyme for catalysis.

Structure and function of flavivirus NS5 methyltransferase.

PubMed

Zhou, Yangsheng; Ray, Debashish; Zhao, Yiwei; Dong, Hongping; Ren, Suping; Li, Zhong; Guo, Yi; Bernard, Kristen A; Shi, Pei-Yong; Li, Hongmin

2007-04-01

The plus-strand RNA genome of flavivirus contains a 5' terminal cap 1 structure (m7GpppAmG). The flaviviruses encode one methyltransferase, located at the N-terminal portion of the NS5 protein, to catalyze both guanine N-7 and ribose 2'-OH methylations during viral cap formation. Representative flavivirus methyltransferases from dengue, yellow fever, and West Nile virus (WNV) sequentially generate GpppA-->m7GpppA-->m7GpppAm. The 2'-O methylation can be uncoupled from the N-7 methylation, since m7GpppA-RNA can be readily methylated to m7GpppAm-RNA. Despite exhibiting two distinct methylation activities, the crystal structure of WNV methyltransferase at 2.8 A resolution showed a single binding site for S-adenosyl-L-methionine (SAM), the methyl donor. Therefore, substrate GpppA-RNA should be repositioned to accept the N-7 and 2'-O methyl groups from SAM during the sequential reactions. Electrostatic analysis of the WNV methyltransferase structure showed that, adjacent to the SAM-binding pocket, is a highly positively charged surface that could serve as an RNA binding site during cap methylations. Biochemical and mutagenesis analyses show that the N-7 and 2'-O cap methylations require distinct buffer conditions and different side chains within the K61-D146-K182-E218 motif, suggesting that the two reactions use different mechanisms. In the context of complete virus, defects in both methylations are lethal to WNV; however, viruses defective solely in 2'-O methylation are attenuated and can protect mice from later wild-type WNV challenge. The results demonstrate that the N-7 methylation activity is essential for the WNV life cycle and, thus, methyltransferase represents a novel target for flavivirus therapy.

Human La binds mRNAs through contacts to the poly(A) tail

PubMed Central

Vinayak, Jyotsna; Marrella, Stefano A; Hussain, Rawaa H; Rozenfeld, Leonid; Solomon, Karine; Bayfield, Mark A

2018-01-01

Abstract In addition to a role in the processing of nascent RNA polymerase III transcripts, La proteins are also associated with promoting cap-independent translation from the internal ribosome entry sites of numerous cellular and viral coding RNAs. La binding to RNA polymerase III transcripts via their common UUU-3’OH motif is well characterized, but the mechanism of La binding to coding RNAs is poorly understood. Using electromobility shift assays and cross-linking immunoprecipitation, we show that in addition to a sequence specific UUU-3’OH binding mode, human La exhibits a sequence specific and length dependent poly(A) binding mode. We demonstrate that this poly(A) binding mode uses the canonical nucleic acid interaction winged helix face of the eponymous La motif, previously shown to be vacant during uridylate binding. We also show that cytoplasmic, but not nuclear La, engages poly(A) RNA in human cells, that La entry into polysomes utilizes the poly(A) binding mode, and that La promotion of translation from the cyclin D1 internal ribosome entry site occurs in competition with cytoplasmic poly(A) binding protein (PABP). Our data are consistent with human La functioning in translation through contacts to the poly(A) tail. PMID:29447394

FY2017 Defense Appropriations Fact Sheet: Selected Highlights of H.R. 5293 and S. 3000

DTIC Science & Technology

2016-06-17

legally binding caps on discretionary spending for defense programs and for non-defense programs, which were established by P.L. 114-74, the Bipartisan...funding for FY2017 that (1) exceeds the relevant BBA cap ; and (2) is also exempt from that spending cap because it is classified as funding for so-called...Overseas Contingency Operations (OCO). The 2015 BBA increased binding caps on defense and non-defense discretionary appropriations for FY2016 and

Fact Sheet: Selected Highlights of the FY2017 National Defense Authorization Act (H.R. 4909)

DTIC Science & Technology

2016-05-12

shaped by the legally binding caps on discretionary spending for defense programs and for non-defense programs, which were established by P.L. 114-74...of Defense (DOD) funding for FY2017 that (1) exceeds the relevant BBA cap ; and (2) is exempt from that spending cap because it is classified as...funding for so- called Overseas Contingency Operations (OCO). The 2015 BBA increased binding caps on defense and non-defense discretionary appropriations

SPArking Interest in the Long Noncoding RNA World: A New Class of 5' SnoRNA-Stabilized LncRNA that Influences Alternative Splicing.

PubMed

Li, Ruohan; Fox, Archa H

2016-11-03

Delving deeply into the locus deleted in Prader-Willi syndrome, in this issue of Molecular Cell, Wu et al. (2016) identify SPA RNAs, a new class of 5' snoRNA-capped lncRNAs that sequester RNA binding proteins and influence alternative splicing. Copyright © 2016 Elsevier Inc. All rights reserved.

An integrated catch-and-hold mechanism activates nicotinic acetylcholine receptors.

PubMed

Jadey, Snehal; Auerbach, Anthony

2012-07-01

In neuromuscular acetylcholine (ACh) receptor channels (AChRs), agonist molecules bind with a low affinity (LA) to two sites that can switch to high affinity (HA) and increase the probability of channel opening. We measured (by using single-channel kinetic analysis) the rate and equilibrium constants for LA binding and channel gating for several different agonists of adult-type mouse AChRs. Almost all of the variation in the equilibrium constants for LA binding was from differences in the association rate constants. These were consistently below the limit set by diffusion and were substantially different even though the agonists had similar sizes and the same charge. This suggests that binding to resting receptors is not by diffusion alone and, hence, that each binding site can undergo two conformational changes ("catch" and "hold") that connect three different structures (apo-, LA-bound, and HA-bound). Analyses of ACh-binding protein structures suggest that this binding site, too, may adopt three discrete structures having different degrees of loop C displacement ("capping"). For the agonists we tested, the logarithms of the equilibrium constants for LA binding and LA↔HA gating were correlated. Although agonist binding and channel gating have long been considered to be separate processes in the activation of ligand-gated ion channels, this correlation implies that the catch-and-hold conformational changes are energetically linked and together comprise an integrated process having a common structural basis. We propose that loop C capping mainly reflects agonist binding, with its two stages corresponding to the formation of the LA and HA complexes. The catch-and-hold reaction coordinate is discussed in terms of preopening states and thermodynamic cycles of activation.

Capping of the N-terminus of PSD-95 by calmodulin triggers its postsynaptic release

PubMed Central

Zhang, Yonghong; Matt, Lucas; Patriarchi, Tommaso; Malik, Zulfiqar A; Chowdhury, Dhrubajyoti; Park, Deborah K; Renieri, Alessandra; Ames, James B; Hell, Johannes W

2014-01-01

Postsynaptic density protein-95 (PSD-95) is a central element of the postsynaptic architecture of glutamatergic synapses. PSD-95 mediates postsynaptic localization of AMPA receptors and NMDA receptors and plays an important role in synaptic plasticity. PSD-95 is released from postsynaptic membranes in response to Ca2+ influx via NMDA receptors. Here, we show that Ca2+/calmodulin (CaM) binds at the N-terminus of PSD-95. Our NMR structure reveals that both lobes of CaM collapse onto a helical structure of PSD-95 formed at its N-terminus (residues 1–16). This N-terminal capping of PSD-95 by CaM blocks palmitoylation of C3 and C5, which is required for postsynaptic PSD-95 targeting and the binding of CDKL5, a kinase important for synapse stability. CaM forms extensive hydrophobic contacts with Y12 of PSD-95. The PSD-95 mutant Y12E strongly impairs binding to CaM and Ca2+-induced release of PSD-95 from the postsynaptic membrane in dendritic spines. Our data indicate that CaM binding to PSD-95 serves to block palmitoylation of PSD-95, which in turn promotes Ca2+-induced dissociation of PSD-95 from the postsynaptic membrane. PMID:24705785

Capping of the N-terminus of PSD-95 by calmodulin triggers its postsynaptic release.

PubMed

Zhang, Yonghong; Matt, Lucas; Patriarchi, Tommaso; Malik, Zulfiqar A; Chowdhury, Dhrubajyoti; Park, Deborah K; Renieri, Alessandra; Ames, James B; Hell, Johannes W

2014-06-17

Postsynaptic density protein-95 (PSD-95) is a central element of the postsynaptic architecture of glutamatergic synapses. PSD-95 mediates postsynaptic localization of AMPA receptors and NMDA receptors and plays an important role in synaptic plasticity. PSD-95 is released from postsynaptic membranes in response to Ca(2+) influx via NMDA receptors. Here, we show that Ca(2+)/calmodulin (CaM) binds at the N-terminus of PSD-95. Our NMR structure reveals that both lobes of CaM collapse onto a helical structure of PSD-95 formed at its N-terminus (residues 1-16). This N-terminal capping of PSD-95 by CaM blocks palmitoylation of C3 and C5, which is required for postsynaptic PSD-95 targeting and the binding of CDKL5, a kinase important for synapse stability. CaM forms extensive hydrophobic contacts with Y12 of PSD-95. The PSD-95 mutant Y12E strongly impairs binding to CaM and Ca(2+)-induced release of PSD-95 from the postsynaptic membrane in dendritic spines. Our data indicate that CaM binding to PSD-95 serves to block palmitoylation of PSD-95, which in turn promotes Ca(2+)-induced dissociation of PSD-95 from the postsynaptic membrane. © 2014 The Authors.

Structural and thermodynamic characterization of the recognition of the S100-binding peptides TRTK12 and p53 by calmodulin

PubMed Central

Wafer, Lucas N; Tzul, Franco O; Pandharipande, Pranav P; McCallum, Scott A; Makhatadze, George I

2014-01-01

Calmodulin (CaM) is a multifunctional messenger protein that activates a wide variety of signaling pathways in eukaryotic cells in a calcium-dependent manner. CaM has been proposed to be functionally distinct from the S100 proteins, a related family of eukaryotic calcium-binding proteins. Previously, it was demonstrated that peptides derived from the actin-capping protein, TRTK12, and the tumor-suppressor protein, p53, interact with multiple members of the S100 proteins. To test the specificity of these peptides, they were screened using isothermal titration calorimetry against 16 members of the human S100 protein family, as well as CaM, which served as a negative control. Interestingly, both the TRTK12 and p53 peptides were found to interact with CaM. These interactions were further confirmed by both fluorescence and nuclear magnetic resonance spectroscopies. These peptides have distinct sequences from the known CaM target sequences. The TRTK12 peptide was found to independently interact with both CaM domains and bind with a stoichiometry of 2:1 and dissociations constants Kd,C-term = 2 ± 1 µM and Kd,N-term = 14 ± 1 µM. In contrast, the p53 peptide was found to interact only with the C-terminal domain of CaM, Kd,C-term =2 ± 1 µM, 25°C. Using NMR spectroscopy, the locations of the peptide binding sites were mapped onto the structure of CaM. The binding sites for both peptides were found to overlap with the binding interface for previously identified targets on both domains of CaM. This study demonstrates the plasticity of CaM in target binding and may suggest a possible overlap in target specificity between CaM and the S100 proteins. PMID:24947426

Diversity and cold adaptation of microorganisms isolated from the Schirmacher Oasis, Antarctica

NASA Astrophysics Data System (ADS)

Mojib, Nazia; Bej, Asim K.; Hoover, Richard

2008-08-01

We have investigated the feasibility of the PCR amplification of the 16S rRNA genes from eubacteria and Archea on samples collected on Whatman FTA filters from Schirmacher Oasis for the study of culture-independent analysis of the microbial diversity. Both conventional PCR and real-time TaqmaTM PCR successfully amplified the targeted genes. A number of diverse groups of psychrotolerant microorganisms with various pigments have been isolated when cultured on agar medium. 16S rRNA gene analysis of these isolates helped us to identify closest taxonomic genus Pseudomonas, Frigoribacterium, Arthrobacter, Flavobacterium, and Janthinobacterium. It is possible that the pigments play protective role from solar UV radiation, which is prevalent in Antarctic continent especially during Austral summer months. Study of the expression of cold adaptive protein CapB and ice-binding protein IBP using western blots showed positive detection of both or either of these proteins in 6 out of 8 isolates. Since the CapB and IBP protein structure greatly varies in microorganisms, it is possible that the 2 isolates with negative results could have a different class of these proteins. The expression of the CapB and the IBP in these isolates suggest that these proteins are essential for the survival in the Antarctic cold and subzero temperatures and protect themselves from freeze-damage. The current study provided sufficient data to further investigate the rich and diverse biota of psychrotolerant extremophiles in the Antarctic Schirmacher Oasis using both culture-independent and culture-based approaches; and understand the mechanisms of cold tolerance.

Binding of /sup 3/H-acetylcholine to cholinergic receptors in bovine cerebral arteries

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

Shimohama, S.; Tsukahara, T.; Taniguchi, T.

Cholinergic receptor sites in bovine cerebral arteries were analyzed using radioligand binding techniques with the cholinergic agonist, /sup 3/H-acetylcholine (ACh), as the ligand. Specific binding of /sup 3/H-ACh to membrane preparations of bovine cerebral arteries was saturable, of two binding sites, with dissociation constant (K/sub D/) values of 0.32 and 23.7 nM, and maximum binding capacity (Bmax) values of 67 and 252 fmol/mg protein, respectively. Specific binding of /sup 3/H-ACh was displaced effectively by muscarinic cholinergic agents and less effectively by nicotinic cholinergic agents. IC/sub 50/ values of cholinergic drugs for /sup 3/H-ACh binding were as follows: atropine, 38.5 nM;more » ACh, 59.8 nM; oxotremorine, 293 nM; scopolamine 474 nM; carbamylcholine, 990 nM. IC/sub 50/ values of nicotinic cholinergic agents such as nicotine, cytisine and ..cap alpha..-bungarotoxin exceeded 50 ..mu..M. Choline acetyltransferase activity was 1.09 nmol/mg protein/hour in the cerebral arteries. These findings suggest that the cholinergic nerves innervate the bovine cerebral arteries and that there are at least two classes of ACh binding sites of different affinities on muscarinic reporters in these arteries. 18 references, 2 figures, 2 tables.« less

Molecular Probing of the HPV-16 E6 Protein Alpha Helix Binding Groove with Small Molecule Inhibitors

PubMed Central

Rietz, Anne; Petrov, Dino P.; Bartolowits, Matthew; DeSmet, Marsha; Davisson, V. Jo; Androphy, Elliot J.

2016-01-01

The human papillomavirus (HPV) HPV E6 protein has emerged as a central oncoprotein in HPV-associated cancers in which sustained expression is required for tumor progression. A majority of the E6 protein interactions within the human proteome use an alpha-helix groove interface for binding. The UBE3A/E6AP HECT domain ubiquitin ligase binds E6 at this helix-groove interface. This enables formation of a trimeric complex with p53, resulting in destruction of this tumor suppressor. While recent x-ray crystal structures are useful, examples of small molecule probes that can modulate protein interactions at this interface are limited. To develop insights useful for potential structure-based design of ligands for HPV E6, a series of 2,6-disubstituted benzopyranones were prepared and tested as competitive antagonists of E6-E6AP helix-groove interactions. These small molecule probes were used in both binding and functional assays to evaluate recognition features of the E6 protein. Evidence for an ionic functional group interaction within the helix groove was implicated by the structure-activity among the highest affinity ligands. The molecular topographies of these protein-ligand interactions were evaluated by comparing the binding and activities of single amino acid E6 mutants with the results of molecular dynamic simulations. A group of arginine residues that form a rim-cap over the E6 helix groove offer compensatory roles in binding and recognition of the small molecule probes. The flexibility and impact on the overall helix-groove shape dictated by these residues offer new insights for structure-based targeting of HPV E6. PMID:26915086

Entrapment of alpha1-acid glycoprotein in high-performance affinity columns for drug-protein binding studies.

PubMed

Bi, Cong; Jackson, Abby; Vargas-Badilla, John; Li, Rong; Rada, Giana; Anguizola, Jeanethe; Pfaunmiller, Erika; Hage, David S

2016-05-15

A slurry-based method was developed for the entrapment of alpha1-acid glycoprotein (AGP) for use in high-performance affinity chromatography to study drug interactions with this serum protein. Entrapment was achieved based on the physical containment of AGP in hydrazide-activated porous silica supports and by using mildly oxidized glycogen as a capping agent. The conditions needed for this process were examined and optimized. When this type of AGP column was used in binding studies, the association equilibrium constant (Ka) measured by frontal analysis at pH 7.4 and 37°C for carbamazepine with AGP was found to be 1.0 (±0.5)×10(5)M(-1), which agreed with a previously reported value of 1.0 (±0.1)×10(5)M(-1). Binding studies based on zonal elution were conducted for several other drugs with such columns, giving equilibrium constants that were consistent with literature values. An entrapped AGP column was also used in combination with a column containing entrapped HSA in a screening assay format to compare the binding of various drugs to AGP and HSA. These results also agreed with previous data that have been reported in literature for both of these proteins. The same entrapment method could be extended to other proteins and to the investigation of additional types of drug-protein interactions. Potential applications include the rapid quantitative analysis of biological interactions and the high-throughput screening of drug candidates for their binding to a given protein. Copyright © 2015 Elsevier B.V. All rights reserved.

Entrapment of Alpha1-Acid Glycoprotein in High-Performance Affinity Columns for Drug-Protein Binding Studies

PubMed Central

Bi, Cong; Jackson, Abby; Vargas-Badilla, John; Li, Rong; Rada, Giana; Anguizola, Jeanethe; Pfaunmiller, Erika; Hage, David S.

2015-01-01

A slurry-based method was developed for the entrapment of alpha1-acid glycoprotein (AGP) for use in high-performance affinity chromatography to study drug interactions with this serum protein. Entrapment was achieved based on the physical containment of AGP in hydrazide-activated porous silica supports and by using mildly oxidized glycogen as a capping agent. The conditions needed for this process were examined and optimized. When this type of AGP column was used in binding studies, the association equilibrium constant (Ka) measured by frontal analysis at pH 7.4 and 37°C for carbamazepine with AGP was found to be 1.0 (± 0.5) × 105 M−1, which agreed with a previously reported value of 1.0 (± 0.1) × 105 M−1. Binding studies based on zonal elution were conducted for several other drugs with such columns, giving equilibrium constants that were consistent with literature values. An entrapped AGP column was also used in combination with a column containing entrapped HSA in a screening assay format to compare the binding of various drugs to AGP and HSA. These results also agreed with previous data that have been reported in literature for both of these proteins. The same entrapment method could be extended to other proteins and to the investigation of additional types of drug-protein interactions. Potential applications include the rapid quantitative analysis of biological interactions and the high-throughput screening of drug candidates for their binding to a given protein. PMID:26627938

Crystal Structure of MpPR-1i, a SCP/TAPS protein from Moniliophthora perniciosa, the fungus that causes Witches’ Broom Disease of Cacao

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

Baroni, Renata M.; Luo, Zhipu; Darwiche, Rabih

The pathogenic fungi Moniliophthora perniciosa causes Witches’ Broom Disease (WBD) of cacao. The structure of MpPR-1i, a protein expressed by M. perniciosa when it infects cacao, are presented. This is the first reported de novo structure determined by single-wavelength anomalous dispersion phasing upon soaking with selenourea. Each monomer has flexible loop regions linking the core alpha-beta-alpha sandwich topology that comprise ~50% of the structure, making it difficult to generate an accurate homology model of the protein. MpPR-1i is monomeric in solution but is packed as a high ~70% solvent content, crystallographic heptamer. The greatest conformational flexibility between monomers is foundmore » in loops exposed to the solvent channel that connect the two longest strands. MpPR-1i lacks the conserved CAP tetrad and is incapable of binding divalent cations. MpPR-1i has the ability to bind lipids, which may have roles in its infection of cacao. These lipids likely bind in the palmitate binding cavity as observed in tablysin-15, since MpPR-1i binds palmitate with comparable affinity as tablysin-15. Further studies are required to clarify the possible roles and underlying mechanisms of neutral lipid binding, as well as their effects on the pathogenesis of M. perniciosa so as to develop new interventions for WBD.« less

The nuclear cap-binding complex interacts with the U4/U6·U5 tri-snRNP and promotes spliceosome assembly in mammalian cells

PubMed Central

Pabis, Marta; Neufeld, Noa; Steiner, Michaela C.; Bojic, Teodora; Shav-Tal, Yaron; Neugebauer, Karla M.

2013-01-01

The nuclear cap-binding complex (CBC) binds to the 7-methyl guanosine cap present on every RNA polymerase II transcript. CBC has been implicated in many aspects of RNA biogenesis; in addition to roles in miRNA biogenesis, nonsense-mediated decay, 3′-end formation, and snRNA export from the nucleus, CBC promotes pre-mRNA splicing. An unresolved question is how CBC participates in splicing. To investigate CBC’s role in splicing, we used mass spectrometry to identify proteins that copurify with mammalian CBC. Numerous components of spliceosomal snRNPs were specifically detected. Among these, three U4/U6·U5 snRNP proteins (hBrr2, hPrp4, and hPrp31) copurified with CBC in an RNA-independent fashion, suggesting that a significant fraction of CBC forms a complex with the U4/U6·U5 snRNP and that the activity of CBC might be associated with snRNP recruitment to pre-mRNA. To test this possibility, CBC was depleted from HeLa cells by RNAi. Chromatin immunoprecipitation and live-cell imaging assays revealed decreased cotranscriptional accumulation of U4/U6·U5 snRNPs on active transcription units, consistent with a requirement for CBC in cotranscriptional spliceosome assembly. Surprisingly, recruitment of U1 and U2 snRNPs was also affected, indicating that RNA-mediated interactions between CBC and snRNPs contribute to splicing. On the other hand, CBC depletion did not impair snRNP biogenesis, ruling out the possibility that decreased snRNP recruitment was due to changes in nuclear snRNP concentration. Taken together, the data support a model whereby CBC promotes pre-mRNA splicing through a network of interactions with and among spliceosomal snRNPs during cotranscriptional spliceosome assembly. PMID:23793891

Conservation of an ATP-binding domain among recA proteins from Proteus vulgaris, erwinia carotovora, Shigella flexneri, and Escherichia coli K-12 and B/r

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

Knight, K.L.; Hess, R.M.; McEntee, K.

1988-06-01

The purified RecA proteins encoded by the cloned genes from Proteus vulgaris, Erwinia carotovora, Shigella flexneri, and Escherichia coli B/r were compared with the RecA protein from E. coli K-12. Each of the proteins hydrolyzed ATP in the presence of single-stranded DNA, and each was covalently modified with the photoaffinity ATP analog 8-azidoadenosine 5'-triphosphate (8N/sub 3/ATP). Two-dimensional tryptic maps of the four heterologous RecA proteins demonstrated considerable structural conservation among these bacterial genera. Moreover, when the (..cap alpha..-/sup 32/P)8N/sub 3/ATP-modified proteins were digested with trypsin and analyzed by high-performance liquid chromatography, a single peak of radioactivity was detected in eachmore » of the digests and these peptides eluted identically with the tryptic peptide T/sub 31/ of the E. coli K-12 RecA protein, which was the unique site of 8N/sub 3/ATP photolabeling. Each of the heterologous recA genes hybridized to oligonucleotide probes derived from the ATP-binding domain sequence of the E. coli K-12 gene. These last results demonstrate that the ATP-binding domain of the RecA protein has been strongly conserved for greater than 10/sup 7/ years.« less

Purification and immunolocalization of an annexin-like protein in pea seedlings

NASA Technical Reports Server (NTRS)

Clark, G. B.; Dauwalder, M.; Roux, S. J.

1992-01-01

As part of a study to identify potential targets of calcium action in plant cells, a 35-kDa, annexin-like protein was purified from pea (Pisum sativum L.) plumules by a method used to purify animal annexins. This protein, called p35, binds to a phosphatidylserine affinity column in a calcium-dependent manner and binds 45Ca2+ in a dot-blot assay. Preliminary sequence data confirm a relationship for p35 with the annexin family of proteins. Polyclonal antibodies have been raised which recognize p35 in Western and dot blots. Immunofluorescence and immunogold techniques were used to study the distribution and subcellular localization of p35 in pea plumules and roots. The highest levels of immunostain were found in young developing vascular cells producing wall thickenings and in peripheral root-cap cells releasing slime. This localization in cells which are actively involved in secretion is of interest because one function suggested for the animal annexins is involvement in the mediation of exocytosis.

No Escaping the Rat Race: Simulated Night Shift Work Alters the Time-of-Day Variation in BMAL1 Translational Activity in the Prefrontal Cortex.

PubMed

Marti, Andrea R; Patil, Sudarshan; Mrdalj, Jelena; Meerlo, Peter; Skrede, Silje; Pallesen, Ståle; Pedersen, Torhild T; Bramham, Clive R; Grønli, Janne

2017-01-01

Millions of people worldwide work during the night, resulting in disturbed circadian rhythms and sleep loss. This may cause deficits in cognitive functions, impaired alertness and increased risk of errors and accidents. Disturbed circadian rhythmicity resulting from night shift work could impair brain function and cognition through disrupted synthesis of proteins involved in synaptic plasticity and neuronal function. Recently, the circadian transcription factor brain-and-muscle arnt-like protein 1 (BMAL1) has been identified as a promoter of mRNA translation initiation, the most highly regulated step in protein synthesis, through binding to the mRNA "cap". In this study we investigated the effects of simulated shift work on protein synthesis markers. Male rats ( n = 40) were exposed to forced activity, either in their rest phase (simulated night shift work) or in their active phase (simulated day shift work) for 3 days. Following the third work shift, experimental animals and time-matched undisturbed controls were euthanized (rest work at ZT12; active work at ZT0). Tissue lysates from two brain regions (prefrontal cortex, PFC and hippocampus) implicated in cognition and sleep loss, were analyzed with m 7 GTP (cap) pull-down to examine time-of-day variation and effects of simulated shift work on cap-bound protein translation. The results show time-of-day variation of protein synthesis markers in PFC, with increased protein synthesis at ZT12. In the hippocampus there was little difference between ZT0 and ZT12. Active phase work did not induce statistically significant changes in protein synthesis markers at ZT0 compared to time-matched undisturbed controls. Rest work, however, resulted in distinct brain-region specific changes of protein synthesis markers compared to time-matched controls at ZT12. While no changes were observed in the hippocampus, phosphorylation of cap-bound BMAL1 and its regulator S6 kinase beta-1 (S6K1) was significantly reduced in the PFC, together with significant reduction in the synaptic plasticity associated protein activity-regulatedcytoskeleton-associated protein (Arc). Our results indicate considerable time-of-day and brain-region specific variation in cap-dependent translation initiation. We concludethat simulated night shift work in rats disrupts the pathways regulating the circadian component of the translation of mRNA in the PFC, and that this may partly explain impaired waking function during night shift work.

Structural basis for ligand regulation of the fatty acid-binding protein 5, peroxisome proliferator-activated receptor β/δ (FABP5-PPARβ/δ) signaling pathway.

PubMed

Armstrong, Eric H; Goswami, Devrishi; Griffin, Patrick R; Noy, Noa; Ortlund, Eric A

2014-05-23

Fatty acid-binding proteins (FABPs) are a widely expressed group of calycins that play a well established role in solubilizing cellular fatty acids. Recent studies, however, have recast FABPs as active participants in vital lipid-signaling pathways. FABP5, like its family members, displays a promiscuous ligand binding profile, capable of interacting with numerous long chain fatty acids of varying degrees of saturation. Certain "activating" fatty acids induce the protein's cytoplasmic to nuclear translocation, stimulating PPARβ/δ transactivation; however, the rules that govern this process remain unknown. Using a range of structural and biochemical techniques, we show that both linoleic and arachidonic acid elicit FABP5's translocation by permitting allosteric communication between the ligand-sensing β2 loop and a tertiary nuclear localization signal within the α-helical cap of the protein. Furthermore, we show that more saturated, nonactivating fatty acids inhibit nuclear localization signal formation by destabilizing this activation loop, thus implicating FABP5 specifically in cis-bonded, polyunsaturated fatty acid signaling. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

A Conserved Interaction between a C-Terminal Motif in Norovirus VPg and the HEAT-1 Domain of eIF4G Is Essential for Translation Initiation

PubMed Central

Leen, Eoin N.; Sorgeloos, Frédéric; Correia, Samantha; Chaudhry, Yasmin; Cannac, Fabien; Pastore, Chiara; Xu, Yingqi; Graham, Stephen C.; Matthews, Stephen J.; Goodfellow, Ian G.; Curry, Stephen

2016-01-01

Translation initiation is a critical early step in the replication cycle of the positive-sense, single-stranded RNA genome of noroviruses, a major cause of gastroenteritis in humans. Norovirus RNA, which has neither a 5´ m7G cap nor an internal ribosome entry site (IRES), adopts an unusual mechanism to initiate protein synthesis that relies on interactions between the VPg protein covalently attached to the 5´-end of the viral RNA and eukaryotic initiation factors (eIFs) in the host cell. For murine norovirus (MNV) we previously showed that VPg binds to the middle fragment of eIF4G (4GM; residues 652–1132). Here we have used pull-down assays, fluorescence anisotropy, and isothermal titration calorimetry (ITC) to demonstrate that a stretch of ~20 amino acids at the C terminus of MNV VPg mediates direct and specific binding to the HEAT-1 domain within the 4GM fragment of eIF4G. Our analysis further reveals that the MNV C terminus binds to eIF4G HEAT-1 via a motif that is conserved in all known noroviruses. Fine mutagenic mapping suggests that the MNV VPg C terminus may interact with eIF4G in a helical conformation. NMR spectroscopy was used to define the VPg binding site on eIF4G HEAT-1, which was confirmed by mutagenesis and binding assays. We have found that this site is non-overlapping with the binding site for eIF4A on eIF4G HEAT-1 by demonstrating that norovirus VPg can form ternary VPg-eIF4G-eIF4A complexes. The functional significance of the VPg-eIF4G interaction was shown by the ability of fusion proteins containing the C-terminal peptide of MNV VPg to inhibit in vitro translation of norovirus RNA but not cap- or IRES-dependent translation. These observations define important structural details of a functional interaction between norovirus VPg and eIF4G and reveal a binding interface that might be exploited as a target for antiviral therapy. PMID:26734730

Changes in root cap pH are required for the gravity response of the Arabidopsis root

NASA Technical Reports Server (NTRS)

Fasano, J. M.; Swanson, S. J.; Blancaflor, E. B.; Dowd, P. E.; Kao, T. H.; Gilroy, S.

2001-01-01

Although the columella cells of the root cap have been identified as the site of gravity perception, the cellular events that mediate gravity signaling remain poorly understood. To determine if cytoplasmic and/or wall pH mediates the initial stages of root gravitropism, we combined a novel cell wall pH sensor (a cellulose binding domain peptide-Oregon green conjugate) and a cytoplasmic pH sensor (plants expressing pH-sensitive green fluorescent protein) to monitor pH dynamics throughout the graviresponding Arabidopsis root. The root cap apoplast acidified from pH 5.5 to 4.5 within 2 min of gravistimulation. Concomitantly, cytoplasmic pH increased in columella cells from 7.2 to 7.6 but was unchanged elsewhere in the root. These changes in cap pH preceded detectable tropic growth or growth-related pH changes in the elongation zone cell wall by 10 min. Altering the gravity-related columella cytoplasmic pH shift with caged protons delayed the gravitropic response. Together, these results suggest that alterations in root cap pH likely are involved in the initial events that mediate root gravity perception or signal transduction.

Structure-Function Relationship of the Bik1-Bim1 Complex.

PubMed

Stangier, Marcel M; Kumar, Anil; Chen, Xiuzhen; Farcas, Ana-Maria; Barral, Yves; Steinmetz, Michel O

2018-04-03

In budding yeast, the microtubule plus-end tracking proteins Bik1 (CLIP-170) and Bim1 (EB1) form a complex that interacts with partners involved in spindle positioning, including Stu2 and Kar9. Here, we show that the CAP-Gly and coiled-coil domains of Bik1 interact with the C-terminal ETF peptide of Bim1 and the C-terminal tail region of Stu2, respectively. The crystal structures of the CAP-Gly domain of Bik1 (Bik1CG) alone and in complex with an ETF peptide revealed unique, functionally relevant CAP-Gly elements, establishing Bik1CG as a specific C-terminal phenylalanine recognition domain. Unlike the mammalian CLIP-170-EB1 complex, Bik1-Bim1 forms ternary complexes with the EB1-binding motifs SxIP and LxxPTPh, which are present in diverse proteins, including Kar9. Perturbation of the Bik1-Bim1 interaction in vivo affected Bik1 localization and astral microtubule length. Our results provide insight into the role of the Bik1-Bim1 interaction for cell division, and demonstrate that the CLIP-170-EB1 module is evolutionarily flexible. Copyright © 2018 Elsevier Ltd. All rights reserved.

Nuclear localization signal regulates porcine circovirus type 2 capsid protein nuclear export through phosphorylation.

PubMed

Hou, Qiang; Hou, Shaohua; Chen, Qing; Jia, Hong; Xin, Ting; Jiang, Yitong; Guo, Xiaoyu; Zhu, Hongfei

2018-02-15

The open reading frame 2 (ORF2) of Porcine circovirus type 2 (PCV2) encodes the major Capsid (Cap) protein, which self-assembles into virus-like particle (VLP) of similar morphology to the PCV2 virion and accumulates in the nucleus through the N-terminal arginine-rich nuclear localization signal (NLS). In this study, PCV2 Cap protein and its derivates were expressed via the baculovirus expression system, and the cellular localization of the recombinant proteins were investigated using anti-Cap mAb by imaging flow cytometry. Analysis of subcellular localization of Cap protein and its variants demonstrated that NLS mediated Cap protein nuclear export as well as nuclear import, and a phosphorylation site (S17) was identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS) in the NLS domain to regulate Cap protein nuclear export. Phosphorylation of NLS regulating the PCV2 Cap protein nuclear export was also demonstrated in PK15 cells by fluorescence microscopy. Moreover, the influence of Rep and Rep' protein on Cap protein subcellular localization was investigated in PK15 cells. Phosphorylation of NLS regulating Cap protein nuclear export provides more detailed knowledge of the PCV2 viral life cycle. Copyright © 2018 Elsevier B.V. All rights reserved.

Serum-deprivation stimulates cap-binding by PARN at the expense of eIF4E, consistent with the observed decrease in mRNA stability

PubMed Central

Seal, Ruth; Temperley, Richard; Wilusz, Jeffrey; Lightowlers, Robert N.; Chrzanowska-Lightowlers, Zofia M. A.

2005-01-01

PARN, a poly(A)-specific ribonuclease, binds the 5′ cap-structure of mRNA and initiates deadenylation-dependent decay. Eukaryotic initiation factor 4E (eIF4E) also binds to the cap structure, an interaction that is critical for initiating cap-dependent translation. The stability of various mRNA transcripts in human cell lines is reduced under conditions of serum starvation as determined by both functional and chemical half-lives. Serum starvation also leads to enhanced cap association by PARN. In contrast, the 5′ cap occupancy by eIF4E decreases under serum-deprivation, as does the translation of reporter transcripts. Further, we show that PARN is a phosphoprotein and that this modification can be modulated by serum status. Taken together, these data are consistent with a natural competition existing at the 5′ cap structure between PARN and eIF4E that may be regulated by changes in post-translational modifications. These phosphorylation-induced changes in the interplay of PARN and eIF4E may determine whether the mRNA is translated or decayed. PMID:15653638

The X-ray Crystallographic Structure and Activity Analysis of a Pseudomonas-Specific Subfamily of the HAD Enzyme Superfamily Evidences a Novel Biochemical Function

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

Peisach,E.; Wang, L.; Burroughs, A.

2008-01-01

The haloacid dehalogenase (HAD) superfamily is a large family of proteins dominated by phosphotransferases. Thirty-three sequence families within the HAD superfamily (HADSF) have been identified to assist in function assignment. One such family includes the enzyme phosphoacetaldehyde hydrolase (phosphonatase). Phosphonatase possesses the conserved Rossmanniod core domain and a C1-type cap domain. Other members of this family do not possess a cap domain and because the cap domain of phosphonatase plays an important role in active site desolvation and catalysis, the function of the capless family members must be unique. A representative of the capless subfamily, PSPTO{_}2114, from the plant pathogenmore » Pseudomonas syringae, was targeted for catalytic activity and structure analyses. The X-ray structure of PSPTO{_}2114 reveals a capless homodimer that conserves some but not all of the intersubunit contacts contributed by the core domains of the phosphonatase homodimer. The region of the PSPTO{_}2114 that corresponds to the catalytic scaffold of phosphonatase (and other HAD phosphotransfereases) positions amino acid residues that are ill suited for Mg+2 cofactor binding and mediation of phosphoryl group transfer between donor and acceptor substrates. The absence of phosphotransferase activity in PSPTO{_}2114 was confirmed by kinetic assays. To explore PSPTO{_}2114 function, the conservation of sequence motifs extending outside of the HADSF catalytic scaffold was examined. The stringently conserved residues among PSPTO{_}2114 homologs were mapped onto the PSPTO{_}2114 three-dimensional structure to identify a surface region unique to the family members that do not possess a cap domain. The hypothesis that this region is used in protein-protein recognition is explored to define, for the first time, HADSF proteins which have acquired a function other than that of a catalyst. Proteins 2008.« less

Crystal Structure of the GRAS Domain of SCARECROW-LIKE7 in Oryza sativa

PubMed Central

Li, Shengping; Zhao, Yanhe; Zhao, Zheng; Wu, Xiuling; Sun, Lifang; Liu, Qingsong; Wu, Yunkun

2016-01-01

GRAS proteins belong to a plant-specific protein family with many members and play essential roles in plant growth and development, functioning primarily in transcriptional regulation. Proteins in the family are minimally defined as containing the conserved GRAS domain. Here, we determined the structure of the GRAS domain of Os-SCL7 from rice (Oryza sativa) to 1.82 Å. The structure includes cap and core subdomains and elucidates the features of the conserved GRAS LRI, VHIID, LRII, PFYRE, and SAW motifs. The structure is a dimer, with a clear groove to accommodate double-stranded DNA. Docking a DNA segment into the groove to generate an Os-SCL7/DNA complex provides insight into the DNA binding mechanism of GRAS proteins. Furthermore, the in vitro DNA binding property of Os-SCL7 and model-defined recognition residues are assessed by electrophoretic mobility shift analysis and mutagenesis assays. These studies reveal the structure and preliminary DNA interaction mechanisms of GRAS proteins and open the door to in-depth investigation and understanding of the individual pathways in which they play important roles. PMID:27081181

Gamma Interferon-Induced Guanylate Binding Protein 1 Is a Novel Actin Cytoskeleton Remodeling Factor

PubMed Central

Ostler, Nicole; Britzen-Laurent, Nathalie; Liebl, Andrea; Naschberger, Elisabeth; Lochnit, Günter; Ostler, Markus; Forster, Florian; Kunzelmann, Peter; Ince, Semra; Supper, Verena; Praefcke, Gerrit J. K.; Schubert, Dirk W.; Stockinger, Hannes; Herrmann, Christian

2014-01-01

Gamma interferon (IFN-γ) regulates immune defenses against viruses, intracellular pathogens, and tumors by modulating cell proliferation, migration, invasion, and vesicle trafficking processes. The large GTPase guanylate binding protein 1 (GBP-1) is among the cellular proteins that is the most abundantly induced by IFN-γ and mediates its cell biologic effects. As yet, the molecular mechanisms of action of GBP-1 remain unknown. Applying an interaction proteomics approach, we identified actin as a strong and specific binding partner of GBP-1. Furthermore, GBP-1 colocalized with actin at the subcellular level and was both necessary and sufficient for the extensive remodeling of the fibrous actin structure observed in IFN-γ-exposed cells. These effects were dependent on the oligomerization and the GTPase activity of GBP-1. Purified GBP-1 and actin bound to each other, and this interaction was sufficient to impair the formation of actin filaments in vitro, as demonstrated by atomic force microscopy, dynamic light scattering, and fluorescence-monitored polymerization. Cosedimentation and band shift analyses demonstrated that GBP-1 binds robustly to globular actin and slightly to filamentous actin. This indicated that GBP-1 may induce actin remodeling via globular actin sequestering and/or filament capping. These results establish GBP-1 as a novel member within the family of actin-remodeling proteins specifically mediating IFN-γ-dependent defense strategies. PMID:24190970

Gamma interferon-induced guanylate binding protein 1 is a novel actin cytoskeleton remodeling factor.

PubMed

Ostler, Nicole; Britzen-Laurent, Nathalie; Liebl, Andrea; Naschberger, Elisabeth; Lochnit, Günter; Ostler, Markus; Forster, Florian; Kunzelmann, Peter; Ince, Semra; Supper, Verena; Praefcke, Gerrit J K; Schubert, Dirk W; Stockinger, Hannes; Herrmann, Christian; Stürzl, Michael

2014-01-01

Gamma interferon (IFN-γ) regulates immune defenses against viruses, intracellular pathogens, and tumors by modulating cell proliferation, migration, invasion, and vesicle trafficking processes. The large GTPase guanylate binding protein 1 (GBP-1) is among the cellular proteins that is the most abundantly induced by IFN-γ and mediates its cell biologic effects. As yet, the molecular mechanisms of action of GBP-1 remain unknown. Applying an interaction proteomics approach, we identified actin as a strong and specific binding partner of GBP-1. Furthermore, GBP-1 colocalized with actin at the subcellular level and was both necessary and sufficient for the extensive remodeling of the fibrous actin structure observed in IFN-γ-exposed cells. These effects were dependent on the oligomerization and the GTPase activity of GBP-1. Purified GBP-1 and actin bound to each other, and this interaction was sufficient to impair the formation of actin filaments in vitro, as demonstrated by atomic force microscopy, dynamic light scattering, and fluorescence-monitored polymerization. Cosedimentation and band shift analyses demonstrated that GBP-1 binds robustly to globular actin and slightly to filamentous actin. This indicated that GBP-1 may induce actin remodeling via globular actin sequestering and/or filament capping. These results establish GBP-1 as a novel member within the family of actin-remodeling proteins specifically mediating IFN-γ-dependent defense strategies.

Regulation of Cdc42/Rac Signaling in the Establishment of Cell Polarity and Control of Cell Motility

DTIC Science & Technology

2004-08-01

Irazoqui Breast Cancer Predoctoral Traineeship Final Report Introduction Cdc42p, together with other polarity proteins, becomes polarized to a cap... Cancer Biology, Certificate in Cell and Molecular Biology. "* Awarded the Jane Coffin Childs Postdoctoral Fellowship for continuing research in the...Bemlp binds directly to both the Cdc42p-directed GEF Department of Pharmacology and Cancer Biology Duke University Medical Center, Durham, NC 27710

A Family of Helminth Molecules that Modulate Innate Cell Responses via Molecular Mimicry of Host Antimicrobial Peptides

PubMed Central

Hutchinson, Andrew T.; To, Joyce; Taylor, Nicole L.; Norton, Raymond S.; Perugini, Matthew A.

2011-01-01

Over the last decade a significant number of studies have highlighted the central role of host antimicrobial (or defence) peptides in modulating the response of innate immune cells to pathogen-associated ligands. In humans, the most widely studied antimicrobial peptide is LL-37, a 37-residue peptide containing an amphipathic helix that is released via proteolytic cleavage of the precursor protein CAP18. Owing to its ability to protect against lethal endotoxaemia and clinically-relevant bacterial infections, LL-37 and its derivatives are seen as attractive candidates for anti-sepsis therapies. We have identified a novel family of molecules secreted by parasitic helminths (helminth defence molecules; HDMs) that exhibit similar biochemical and functional characteristics to human defence peptides, particularly CAP18. The HDM secreted by Fasciola hepatica (FhHDM-1) adopts a predominantly α-helical structure in solution. Processing of FhHDM-1 by F. hepatica cathepsin L1 releases a 34-residue C-terminal fragment containing a conserved amphipathic helix. This is analogous to the proteolytic processing of CAP18 to release LL-37, which modulates innate cell activation by classical toll-like receptor (TLR) ligands such as lipopolysaccharide (LPS). We show that full-length recombinant FhHDM-1 and a peptide analogue of the amphipathic C-terminus bind directly to LPS in a concentration-dependent manner, reducing its interaction with both LPS-binding protein (LBP) and the surface of macrophages. Furthermore, FhHDM-1 and the amphipathic C-terminal peptide protect mice against LPS-induced inflammation by significantly reducing the release of inflammatory mediators from macrophages. We propose that HDMs, by mimicking the function of host defence peptides, represent a novel family of innate cell modulators with therapeutic potential in anti-sepsis treatments and prevention of inflammation. PMID:21589904

Capsaicinoids improve consequences of physical activity.

PubMed

Sahin, Kazim; Orhan, Cemal; Tuzcu, Mehmet; Sahin, Nurhan; Erten, Fusun; Juturu, Vijaya

2018-01-01

The purpose of this study was to investigate the effects of capsaicinoids (CAPs) on lipid metabolism, inflammation, antioxidant status and the changes in gene products involved in these metabolic functions in exercised rats. A total of 28 male Wistar albino rats were randomly divided into four groups (n = 7) (i) No exercise and no CAPs, (ii) No exercise + CAPs (iii) Regular exercise, (iv) Regular exercise + CAPs. Rats were administered as 0.2 mg capsaicinoids from 10 mg/kg BW/day Capsimax ® daily for 8 weeks. A significant decrease in lactate and malondialdehyde (MDA) levels and increase in activities of antioxidant enzymes were observed in the combination of regular exercise and CAPs group ( P < 0.0001). Regular exercise + CAPs treated rats had greater nuclear factor-E2-related factor-2 (Nrf2) and heme oxygenase-1 (HO-1) levels in muscle than regular exercise and no exercise rats ( P < 0.001). Nevertheless, regular exercise + CAPs treated had lower nuclear factor kappa B (NF-κB) and IL-10 levels in muscle than regular exercise and control rats ( P < 0.001). Muscle sterol regulatory element-binding protein 1c (SREBP-1c), liver X receptors (LXR), ATP citrate lyase (ACLY) and fatty acid synthase (FAS) levels in the regular exercise + CAPs group were lower than all groups ( P < 0.05). However, muscle PPAR-γ level was higher in the regular exercise and CAPs alone than the no exercise rats. These results suggest CAPs with regular exercise may enhance lipid metabolism by regulation of gene products involved in lipid and antioxidant metabolism including SREBP-1c, PPAR-γ, and Nrf2 pathways in rats.

Utilization of phage display to identify antigenic regions in the PCV2 capsid protein for the evaluation of serological responses in mice and pigs.

PubMed

Santos, Marcus Rebouças; Assao, Viviane Sisdelli; Santos, Fabiana de Almeida Araújo; Salgado, Rafael Locatelli; Carneiro, Ana Paula; Fietto, Juliana Lopes Rangel; Bressan, Gustavo Costa; de Almeida, Márcia Rogéria; Lobato, Zelia Inês Portela; Ueira-Veira, Carlos; Goulart, Luíz Ricardo; Silva-Júnior, Abelardo

2018-07-01

Porcine circovirus 2 (PCV2) is associated with a series of swine diseases. There is a great interest in improving our understanding of the immunology of PCV2, especially the properties of the viral capsid protein Cap-PCV2 and how they relate to the immunogenicity of the virus and the subsequent development of vaccines. Phage display screening has been widely used to study binding affinities for target proteins. The aim of this study was to use phage display screening to identify antigenic peptides in the PCV2 capsid protein. After the selection of peptides, five of them presented similarity to sequences found in cap-PCV2, and four peptides were synthesized and used for immunization in mice: 51-CTFGYTIKRTVT-62 (PS14), 127-CDNFVTKATALTY-138 (PS34), 164-CKPVLDSTIDY-173 (PC12), and 79-CFLPPGGGSNT-88 (PF1). Inoculation with the PC12 peptide led to the highest production of antibodies. Furthermore, we used the PC12 peptide as an antigen to examine the humoral response of swine serum by ELISA. The sensitivity and specificity of this assay was 88.9% and 92.85%, respectively. Altogether, characterization of immunogenic epitopes in the capsid protein of PCV2 may contribute to the improvement of vaccines and diagnostics.

Structure and Function of Flavivirus NS5 Methyltransferase

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

Zhou,Y.; Ray, D.; Zhao, Y.

2007-01-01

The plus-strand RNA genome of flavivirus contains a 5' terminal cap 1 structure (m{sup 7}GpppAmG). The flaviviruses encode one methyltransferase, located at the N-terminal portion of the NS5 protein, to catalyze both guanine N-7 and ribose 2'-OH methylations during viral cap formation. Representative flavivirus methyltransferases from dengue, yellow fever, and West Nile virus (WNV) sequentially generate GpppA {yields} m{sup 7}GpppA {yields} m{sup 7}GpppAm. The 2'-O methylation can be uncoupled from the N-7 methylation, since m{sup 7}GpppA-RNA can be readily methylated to m{sup 7}GpppAm-RNA. Despite exhibiting two distinct methylation activities, the crystal structure of WNV methyltransferase at 2.8 {angstrom} resolution showedmore » a single binding site for S-adenosyl-L-methionine (SAM), the methyl donor. Therefore, substrate GpppA-RNA should be repositioned to accept the N-7 and 2'-O methyl groups from SAM during the sequential reactions. Electrostatic analysis of the WNV methyltransferase structure showed that, adjacent to the SAM-binding pocket, is a highly positively charged surface that could serve as an RNA binding site during cap methylations. Biochemical and mutagenesis analyses show that the N-7 and 2'-O cap methylations require distinct buffer conditions and different side chains within the K{sub 61}-D{sub 146}-K{sub 182}-E{sub 218} motif, suggesting that the two reactions use different mechanisms. In the context of complete virus, defects in both methylations are lethal to WNV; however, viruses defective solely in 2'-O methylation are attenuated and can protect mice from later wild-type WNV challenge. The results demonstrate that the N-7 methylation activity is essential for the WNV life cycle and, thus, methyltransferase represents a novel target for flavivirus therapy.« less

A sequence of basic residues in the porcine circovirus type 2 capsid protein is crucial for its co-expression and co-localization with the replication protein.

PubMed

Huang, Liping; Van Renne, Nicolaas; Liu, Changming; Nauwynck, Hans J

2015-12-01

Porcine circovirus type 2 (PCV2) encodes two major proteins: the replication protein (Rep) and the capsid protein (Cap). Cap displays a conserved stretch of basic residues situated on the inside of the capsid, whose role is so far unknown. We used a reverse-genetics approach to investigate its function and found that mutations in these amino acids hindered Cap mRNA translation and hampered Cap/Rep co-localization, yielding unfit viruses. Intriguingly, co-transfection with a WT PCV2 of a different genotype partially rescued mutant Cap expression, showing the importance of this basic pattern for efficient translation of Cap mRNA into protein. Our results show that Cap and Rep are expressed independently of each other, and that this amino acid sequence of Cap is vital for virus propagation. This study provides a method for studying unfit PCV2 virions and offers new insights into the intracellular modus vivendi of PCV2.

In silico analysis of surface structure variation of PCV2 capsid resulting from loop mutations of its capsid protein (Cap)

PubMed Central

Wang, Aibing; Zhang, Lijie; Khayat, Reza

2016-01-01

Outbreaks of porcine circovirus (PCV) type 2 (PCV2)-associated diseases have caused substantial economic losses worldwide in the last 20 years. The PCV capsid protein (Cap) is the sole structural protein and main antigenic determinant of this virus. In this study, not only were phylogenetic trees reconstructed, but variations of surface structure of the PCV capsid were analysed in the course of evolution. Unique surface patterns of the icosahedral fivefold axes of the PCV2 capsid were identified and characterized, all of which were absent in PCV type 1 (PCV1). Icosahedral fivefold axes, decorated with Loops BC, HI and DE, were distinctly different between PCV2 and PCV1. Loops BC, determining the outermost surface around the fivefold axes of PCV capsids, had limited homology between Caps of PCV1 and PCV2. A conserved tyrosine phosphorylation motif in Loop HI that might be recognized by non-receptor tyrosine kinase(s) in vivo was present only in PCV2. Particularly, the concurrent presence of 60 pairs of the conserved tyrosine and a canonical PXXP motif on the PCV2 capsid surface could be a mechanism for PXXP motif binding to and activation of an SH3-domain-containing tyrosine kinase in host cells. Additionally, a conserved cysteine in Loop DE of the PCV2 Cap was substituted by an arginine in PCV1, indicating potentially distinct assembly mechanisms of the capsid in vitro between PCV1 and PCV2. Therefore, these unique patterns on the PCV2 capsid surface, absent in PCV1 isolates, might be related to cell entry, virus function and pathogenesis. PMID:27902320

In silico analysis of surface structure variation of PCV2 capsid resulting from loop mutations of its capsid protein (Cap).

PubMed

Wang, Naidong; Zhan, Yang; Wang, Aibing; Zhang, Lijie; Khayat, Reza; Yang, Yi

2016-12-01

Outbreaks of porcine circovirus (PCV) type 2 (PCV2)-associated diseases have caused substantial economic losses worldwide in the last 20 years. The PCV capsid protein (Cap) is the sole structural protein and main antigenic determinant of this virus. In this study, not only were phylogenetic trees reconstructed, but variations of surface structure of the PCV capsid were analysed in the course of evolution. Unique surface patterns of the icosahedral fivefold axes of the PCV2 capsid were identified and characterized, all of which were absent in PCV type 1 (PCV1). Icosahedral fivefold axes, decorated with Loops BC, HI and DE, were distinctly different between PCV2 and PCV1. Loops BC, determining the outermost surface around the fivefold axes of PCV capsids, had limited homology between Caps of PCV1 and PCV2. A conserved tyrosine phosphorylation motif in Loop HI that might be recognized by non-receptor tyrosine kinase(s) in vivo was present only in PCV2. Particularly, the concurrent presence of 60 pairs of the conserved tyrosine and a canonical PXXP motif on the PCV2 capsid surface could be a mechanism for PXXP motif binding to and activation of an SH3-domain-containing tyrosine kinase in host cells. Additionally, a conserved cysteine in Loop DE of the PCV2 Cap was substituted by an arginine in PCV1, indicating potentially distinct assembly mechanisms of the capsid in vitro between PCV1 and PCV2. Therefore, these unique patterns on the PCV2 capsid surface, absent in PCV1 isolates, might be related to cell entry, virus function and pathogenesis.

Capped mRNAs with reduced secondary structure can function in extracts from poliovirus-infected cells

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

Sonenberg, N.; Guertin, D.; Lee, K.A.W.

1982-12-01

Extracts form poliovirus-infected HeLa cells were used to study ribosome binding of native and denatured reovirus mRNAs and translation of capped mRNAs with different degrees of secondary structure. Here, the authors demonstrate that ribosomes in extracts from poliovirus-infected cells could form initiation complexes with denatured reovirus mRNA, in contrast to their inability to bind native reovirus mRNA. Furthermore, the capped alfalfa mosiac virus 4 RNA, which is most probable devoid of stable secondary structure at its 5' end, could be translated at much higher efficiency than could other capped mRNAs in extracts from poliovirus-infected cells.

Glycation of whey protein with dextrans of different molar mass: Effect on immunoglobulin E-binding capacity with blood sera obtained from patients with cow milk protein allergy.

PubMed

Xu, Lei; Gong, Yuansheng; Gern, James E; Ikeda, Shinya; Lucey, John A

2018-05-16

A growing concern around the world is the number of people who are suffering from food protein allergies. One potential approach to decrease protein allergenicity is to block IgE-binding epitopes of the protein allergen by attachment of polysaccharides via the Maillard reaction (i.e., glycation). Protein glycation has been extensively studied to modify various functional properties. We wanted to examine whether glycates could reduce IgE binding in patients with cow milk protein allergy and to explore how the size (molar mass; M W ) of the polysaccharide affects this IgE-binding capacity. Glycation was performed using the initial step of the Maillard reaction performed in aqueous solutions. The specific goal of this study was to reduce the IgE-binding capacity of whey protein isolate (WPI) through glycation with dextran (DX). Blood sera were obtained from 8 patients who had been diagnosed with cow milk protein allergy, and a composite sera sample was used for IgE-binding analysis by the ImmunoCap (Phadia, Uppsala, Sweden) method. The WPI was glycated with DX of M W ranging from 1 to 2,000 kDa, and the M W of purified glycates was determined using size-exclusion chromatography coupled with multiangle laser light scattering. The WPI to DX molar ratios in the glycates made from DX that had M W values of 1, 3.5, 10 (G10), 150, 500, and 2,000 kDa were 1:4, 1:3, 1:2, 1:1.5, 1:1, and 1:1, respectively. With the increase in the M W of DX, there was an increase in the M W values of the corresponding glycates but a decrease in the number of bound DX. The WPI-DX glycates had lower whey protein IgE-binding capacity than native WPI, with the lowest IgE-binding capacity obtained in the G10 glycate. The DX binding ratios and morphology results from atomic force microscopy images suggested that glycation of WPI with small-M W DX resulted in extensive protein surface coverage, probably due to the attachment of up to 4 DX molecules per whey protein. The lower IgE binding of the G10 glycate was likely due to greater steric hindrance (or a physical barrier) at the surface of the protein. In summary, our results demonstrate that glycating WPI with DX via Maillard reaction can potentially be used to decrease the allergenicity of whey protein. Copyright © 2018 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Tankyrase 2 Poly(ADP-Ribose) Polymerase Domain-Deleted Mice Exhibit Growth Defects but Have Normal Telomere Length and Capping

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

Hsiao, Susan J; Poitras, Marc; Cook, Brandoch

Regulation of telomere length maintenance and capping are a critical cell functions in both normal and tumor cells. Tankyrase 2 (Tnks2) is a poly(ADP-ribose) polymerase (PARP) that has been shown to modify itself and TRF1, a telomere-binding protein. We show here by overexpression studies that tankyrase 2, like its closely related homolog tankyrase 1, can function as a positive regulator of telomere length in human cells, dependent on its catalytic PARP activity. To study the role of Tnks2 in vivo, we generated mice with the Tnks2 PARP domain deleted. These mice are viable and fertile but display a growth retardationmore » phenotype. Telomere analysis by quantitative fluorescence in situ hybridization (FISH), flow-FISH, and restriction fragment analysis showed no change in telomere length or telomere capping in these mice. To determine the requirement foTnks2 in long-term maintenance of telomeres, we generated embryonic stem cells with the Tnks2 PARP domain deleted and observed no change, even upon prolonged growth, in telomere length or telomere capping. Together these results suggest that Tnkjs2 has a role in normal growth and development but is not essential for telomere length maintenance or telomere capping in mice.« less

How capping protein enhances actin filament growth and nucleation on biomimetic beads.

PubMed

Wang, Ruizhe; Carlsson, Anders E

2015-11-25

Capping protein (CP), which caps the growing ends of actin filaments, accelerates actin-based motility. Recent experiments on biomimetic beads have shown that CP also enhances the rate of actin filament nucleation. Proposed explanations for these phenomena include (i) the actin funneling hypothesis (AFH), in which the presence of CP increases the free-actin concentration, and (ii) the monomer gating model, in which CP binding to actin filament barbed ends makes more monomers available for filament nucleation. To establish how CP increases the rates of filament elongation and nucleation on biomimetic beads, we perform a quantitative modeling analysis of actin polymerization, using rate equations that include actin filament nucleation, polymerization and capping, as modified by monomer depletion near the surface of the bead. With one adjustable parameter, our simulation results match previously measured time courses of polymerized actin and filament number. The results support a version of the AFH where CP increases the local actin monomer concentration at the bead surface, but leaves the global free-actin concentration nearly constant. Because the rate of filament nucleation increases with the monomer concentration, the increased local monomer concentration enhances actin filament nucleation. We derive a closed-form formula for the characteristic CP concentration where the local free-actin concentration reaches half the bulk value, and find it to be comparable to the global Arp2/3 complex concentration. We also propose an experimental protocol for distinguishing branching nucleation of filaments from spontaneous nucleation.

Importance of the lid and cap domains for the catalytic activity of gastric lipases.

PubMed

Miled, N; Bussetta, C; De caro, A; Rivière, M; Berti, L; Canaan, S

2003-09-01

Human gastric lipase (HGL) is an enzyme secreted by the stomach, which is stable and active despite the highly acidic environment. It has been clearly established that this enzyme is responsible for 30% of the fat digestion processes occurring in human. This globular protein belongs to the alpha/beta hydrolase fold family and its catalytic serine is deeply buried under a domain called the extrusion domain, which is composed of a 'cap' domain and a segment consisting of 58 residues, which can be defined as a lid. The exact roles played by the cap and the lid domains during the catalytic step have not yet been elucidated. We have recently solved the crystal structure of the open form of the dog gastric lipase in complex with a covalent inhibitor. The detergent molecule and the inhibitor were mimicking a triglyceride substrate that would interact with residues belonging to both the cap and the lid domains. In this study, we have investigated the role of the cap and the lid domains, using site-directed mutagenesis procedures. We have produced truncated mutants lacking the lid and the cap. After expressing these mutants and purifying them, their activity was found to have decreased drastically in comparison with the wild type HGL. The lid and the cap domains play an important role in the catalytic reaction mechanism. Based on these results and the structural data (open form of DGL), we have pointed out the cap and the lid residues involved in the binding with the lipidic substrate.

Dysregulation of mTOR signaling in fragile X syndrome.

PubMed

Sharma, Ali; Hoeffer, Charles A; Takayasu, Yukihiro; Miyawaki, Takahiro; McBride, Sean M; Klann, Eric; Zukin, R Suzanne

2010-01-13

Fragile X syndrome, the most common form of inherited mental retardation and leading genetic cause of autism, is caused by transcriptional silencing of the Fmr1 gene. The fragile X mental retardation protein (FMRP), the gene product of Fmr1, is an RNA binding protein that negatively regulates translation in neurons. The Fmr1 knock-out mouse, a model of fragile X syndrome, exhibits cognitive deficits and exaggerated metabotropic glutamate receptor (mGluR)-dependent long-term depression at CA1 synapses. However, the molecular mechanisms that link loss of function of FMRP to aberrant synaptic plasticity remain unclear. The mammalian target of rapamycin (mTOR) signaling cascade controls initiation of cap-dependent translation and is under control of mGluRs. Here we show that mTOR phosphorylation and activity are elevated in hippocampus of juvenile Fmr1 knock-out mice by four functional readouts: (1) association of mTOR with regulatory associated protein of mTOR; (2) mTOR kinase activity; (3) phosphorylation of mTOR downstream targets S6 kinase and 4E-binding protein; and (4) formation of eukaryotic initiation factor complex 4F, a critical first step in cap-dependent translation. Consistent with this, mGluR long-term depression at CA1 synapses of FMRP-deficient mice is exaggerated and rapamycin insensitive. We further show that the p110 subunit of the upstream kinase phosphatidylinositol 3-kinase (PI3K) and its upstream activator PI3K enhancer PIKE, predicted targets of FMRP, are upregulated in knock-out mice. Elevated mTOR signaling may provide a functional link between overactivation of group I mGluRs and aberrant synaptic plasticity in the fragile X mouse, mechanisms relevant to impaired cognition in fragile X syndrome.

Maintenance of dendritic spine morphology by partitioning-defective 1b through regulation of microtubule growth.

PubMed

Hayashi, Kenji; Suzuki, Atsushi; Hirai, Syu-ichi; Kurihara, Yasuyuki; Hoogenraad, Casper C; Ohno, Shigeo

2011-08-24

Dendritic spines are postsynaptic structures that receive excitatory synaptic input from presynaptic terminals. Actin and its regulatory proteins play a central role in morphogenesis of dendritic spines. In addition, recent studies have revealed that microtubules are indispensable for the maintenance of mature dendritic spine morphology by stochastically invading dendritic spines and regulating dendritic localization of p140Cap, which is required for actin reorganization. However, the regulatory mechanisms of microtubule dynamics remain poorly understood. Partitioning-defective 1b (PAR1b), a cell polarity-regulating serine/threonine protein kinase, is thought to regulate microtubule dynamics by inhibiting microtubule binding of microtubule-associated proteins. Results from the present study demonstrated that PAR1b participates in the maintenance of mature dendritic spine morphology in mouse hippocampal neurons. Immunofluorescent analysis revealed PAR1b localization in the dendrites, which was concentrated in dendritic spines of mature neurons. PAR1b knock-down cells exhibited decreased mushroom-like dendritic spines, as well as increased filopodia-like dendritic protrusions, with no effect on the number of protrusions. Live imaging of microtubule plus-end tracking proteins directly revealed decreases in distance and duration of microtubule growth following PAR1b knockdown in a neuroblastoma cell line and in dendrites of hippocampal neurons. In addition, reduced accumulation of GFP-p140Cap in dendritic protrusions was confirmed in PAR1b knock-down neurons. In conclusion, the present results suggested a novel function for PAR1b in the maintenance of mature dendritic spine morphology by regulating microtubule growth and the accumulation of p140Cap in dendritic spines.

Structure of an N276-Dependent HIV-1 Neutralizing Antibody Targeting a Rare V5 Glycan Hole Adjacent to the CD4 Binding Site.

PubMed

Wibmer, Constantinos Kurt; Gorman, Jason; Anthony, Colin S; Mkhize, Nonhlanhla N; Druz, Aliaksandr; York, Talita; Schmidt, Stephen D; Labuschagne, Phillip; Louder, Mark K; Bailer, Robert T; Abdool Karim, Salim S; Mascola, John R; Williamson, Carolyn; Moore, Penny L; Kwong, Peter D; Morris, Lynn

2016-11-15

All HIV-1-infected individuals develop strain-specific neutralizing antibodies to their infecting virus, which in some cases mature into broadly neutralizing antibodies. Defining the epitopes of strain-specific antibodies that overlap conserved sites of vulnerability might provide mechanistic insights into how broadly neutralizing antibodies arise. We previously described an HIV-1 clade C-infected donor, CAP257, who developed broadly neutralizing plasma antibodies targeting an N276 glycan-dependent epitope in the CD4 binding site. The initial CD4 binding site response potently neutralized the heterologous tier 2 clade B viral strain RHPA, which was used to design resurfaced gp120 antigens for single-B-cell sorting. Here we report the isolation and structural characterization of CAP257-RH1, an N276 glycan-dependent CD4 binding site antibody representative of the early CD4 binding site plasma response in donor CAP257. The cocrystal structure of CAP257-RH1 bound to RHPA gp120 revealed critical interactions with the N276 glycan, loop D, and V5, but not with aspartic acid 368, similarly to HJ16 and 179NC75. The CAP257-RH1 monoclonal antibody was derived from the immunoglobulin-variable IGHV3-33 and IGLV3-10 genes and neutralized RHPA but not the transmitted/founder virus from donor CAP257. Its narrow neutralization breadth was attributed to a binding angle that was incompatible with glycosylated V5 loops present in almost all HIV-1 strains, including the CAP257 transmitted/founder virus. Deep sequencing of autologous CAP257 viruses, however, revealed minority variants early in infection that lacked V5 glycans. These glycan-free V5 loops are unusual holes in the glycan shield that may have been necessary for initiating this N276 glycan-dependent CD4 binding site B-cell lineage. The conserved CD4 binding site on gp120 is a major target for HIV-1 vaccine design, but key events in the elicitation and maturation of different antibody lineages to this site remain elusive. Studies have shown that strain-specific antibodies can evolve into broadly neutralizing antibodies or in some cases act as helper lineages. Therefore, characterizing the epitopes of strain-specific antibodies may help to inform the design of HIV-1 immunogens to elicit broadly neutralizing antibodies. In this study, we isolate a narrowly neutralizing N276 glycan-dependent antibody and use X-ray crystallography and viral deep sequencing to describe how gp120 lacking glycans in V5 might have elicited these early glycan-dependent CD4 binding site antibodies. These data highlight how glycan holes can play a role in the elicitation of B-cell lineages targeting the CD4 binding site. Copyright © 2016 Wibmer et al.

Structure of an N276-Dependent HIV-1 Neutralizing Antibody Targeting a Rare V5 Glycan Hole Adjacent to the CD4 Binding Site

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

Wibmer, Constantinos Kurt; Gorman, Jason; Anthony, Colin S.

ABSTRACT All HIV-1-infected individuals develop strain-specific neutralizing antibodies to their infecting virus, which in some cases mature into broadly neutralizing antibodies. Defining the epitopes of strain-specific antibodies that overlap conserved sites of vulnerability might provide mechanistic insights into how broadly neutralizing antibodies arise. We previously described an HIV-1 clade C-infected donor, CAP257, who developed broadly neutralizing plasma antibodies targeting an N276 glycan-dependent epitope in the CD4 binding site. The initial CD4 binding site response potently neutralized the heterologous tier 2 clade B viral strain RHPA, which was used to design resurfaced gp120 antigens for single-B-cell sorting. Here we report themore » isolation and structural characterization of CAP257-RH1, an N276 glycan-dependent CD4 binding site antibody representative of the early CD4 binding site plasma response in donor CAP257. The cocrystal structure of CAP257-RH1 bound to RHPA gp120 revealed critical interactions with the N276 glycan, loop D, and V5, but not with aspartic acid 368, similarly to HJ16 and 179NC75. The CAP257-RH1 monoclonal antibody was derived from the immunoglobulin-variable IGHV3-33 and IGLV3-10 genes and neutralized RHPA but not the transmitted/founder virus from donor CAP257. Its narrow neutralization breadth was attributed to a binding angle that was incompatible with glycosylated V5 loops present in almost all HIV-1 strains, including the CAP257 transmitted/founder virus. Deep sequencing of autologous CAP257 viruses, however, revealed minority variants early in infection that lacked V5 glycans. These glycan-free V5 loops are unusual holes in the glycan shield that may have been necessary for initiating this N276 glycan-dependent CD4 binding site B-cell lineage. IMPORTANCEThe conserved CD4 binding site on gp120 is a major target for HIV-1 vaccine design, but key events in the elicitation and maturation of different antibody lineages to this site remain elusive. Studies have shown that strain-specific antibodies can evolve into broadly neutralizing antibodies or in some cases act as helper lineages. Therefore, characterizing the epitopes of strain-specific antibodies may help to inform the design of HIV-1 immunogens to elicit broadly neutralizing antibodies. In this study, we isolate a narrowly neutralizing N276 glycan-dependent antibody and use X-ray crystallography and viral deep sequencing to describe how gp120 lacking glycans in V5 might have elicited these early glycan-dependent CD4 binding site antibodies. These data highlight how glycan holes can play a role in the elicitation of B-cell lineages targeting the CD4 binding site.« less

Structure of an N276-Dependent HIV-1 Neutralizing Antibody Targeting a Rare V5 Glycan Hole Adjacent to the CD4 Binding Site

PubMed Central

Wibmer, Constantinos Kurt; Gorman, Jason; Anthony, Colin S.; Mkhize, Nonhlanhla N.; Druz, Aliaksandr; York, Talita; Schmidt, Stephen D.; Labuschagne, Phillip; Louder, Mark K.; Bailer, Robert T.; Abdool Karim, Salim S.; Mascola, John R.; Williamson, Carolyn; Moore, Penny L.

2016-01-01

ABSTRACT All HIV-1-infected individuals develop strain-specific neutralizing antibodies to their infecting virus, which in some cases mature into broadly neutralizing antibodies. Defining the epitopes of strain-specific antibodies that overlap conserved sites of vulnerability might provide mechanistic insights into how broadly neutralizing antibodies arise. We previously described an HIV-1 clade C-infected donor, CAP257, who developed broadly neutralizing plasma antibodies targeting an N276 glycan-dependent epitope in the CD4 binding site. The initial CD4 binding site response potently neutralized the heterologous tier 2 clade B viral strain RHPA, which was used to design resurfaced gp120 antigens for single-B-cell sorting. Here we report the isolation and structural characterization of CAP257-RH1, an N276 glycan-dependent CD4 binding site antibody representative of the early CD4 binding site plasma response in donor CAP257. The cocrystal structure of CAP257-RH1 bound to RHPA gp120 revealed critical interactions with the N276 glycan, loop D, and V5, but not with aspartic acid 368, similarly to HJ16 and 179NC75. The CAP257-RH1 monoclonal antibody was derived from the immunoglobulin-variable IGHV3-33 and IGLV3-10 genes and neutralized RHPA but not the transmitted/founder virus from donor CAP257. Its narrow neutralization breadth was attributed to a binding angle that was incompatible with glycosylated V5 loops present in almost all HIV-1 strains, including the CAP257 transmitted/founder virus. Deep sequencing of autologous CAP257 viruses, however, revealed minority variants early in infection that lacked V5 glycans. These glycan-free V5 loops are unusual holes in the glycan shield that may have been necessary for initiating this N276 glycan-dependent CD4 binding site B-cell lineage. IMPORTANCE The conserved CD4 binding site on gp120 is a major target for HIV-1 vaccine design, but key events in the elicitation and maturation of different antibody lineages to this site remain elusive. Studies have shown that strain-specific antibodies can evolve into broadly neutralizing antibodies or in some cases act as helper lineages. Therefore, characterizing the epitopes of strain-specific antibodies may help to inform the design of HIV-1 immunogens to elicit broadly neutralizing antibodies. In this study, we isolate a narrowly neutralizing N276 glycan-dependent antibody and use X-ray crystallography and viral deep sequencing to describe how gp120 lacking glycans in V5 might have elicited these early glycan-dependent CD4 binding site antibodies. These data highlight how glycan holes can play a role in the elicitation of B-cell lineages targeting the CD4 binding site. PMID:27581986

Hormonal regulation of hepatic glycogenolysis in the carp, Cyprinus carpio

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

Janssens, P.A.; Lowrey, P.

1987-04-01

Carp (Cyprinus carpio) liver maintained normal glycogen content and enzyme complement for several days in organ culture. Epinephrine-stimulated glycogenolysis, phosphorylase activation, and cyclic AMP (cAMP) accumulation in a concentration-dependent manner with EC/sub 50/s of 100, 100, and 500 nM, respectively. These actions were blocked by the ..beta..-adrenergic antagonist, propranolol, but not by the ..cap alpha..-adrenergic antagonist phentolamine. Glycogenolysis and tissue cAMP were uninfluenced by 10/sup -6/ M arginine vasotocin, arginine vasopressin, lysine vasotocin, lysine vasopressin, mesotocin, or oxytocin, but were slightly increased by 10/sup -5/ M isotocin and slightly decreased by 10/sup -6/ M angiotensin II. (/sup 125/I)-iodocyanopindolol (ICP), amore » ..beta..-adrenergic ligand, bound to isolated carp liver membranes with a K/sub D/ of 83 pM. Maximum binding of 45 fmol/mg protein was at 600 pM. Propranolol, isoprenaline, epinephrine, phenylephrine, norepinephrine, and phenoxybenzamine displaced ICP with K/sub D/s of 100 nM, 2, 20, 20, 60, and 200 ..mu..M, respectively. The ..cap alpha..-adrenergic antagonists, yohimbine and prazosin, showed no specific binding. These data provide evidence that catecholamines act via ..beta..-adrenergic receptors in carp liver and that ..cap alpha..-adrenergic receptors are not present. Vasoactive peptides play no significant role in regulation of carp liver glycogenolysis.« less

A Sensitive and Robust High-Throughput Screening Assay for Inhibitors of the Chikungunya Virus nsP1 Capping Enzyme.

PubMed

Bullard-Feibelman, Kristen M; Fuller, Benjamin P; Geiss, Brian J

2016-01-01

Chikungunya virus (CHIKV) is a mosquito-borne Alphavirus that causes severe and debilitating disease symptoms. Alarmingly, transmission rates of CHIKV have increased dramatically over the last decade resulting in 1.7 million suspected cases in the Western hemisphere alone. There are currently no antivirals for treatment of CHIKV infection and novel anti-alphaviral compounds are badly needed. nsP1 is the alphavirus protein responsible for the methyltransferase and guanylyltransferase activities necessary for formation of the 5' type 0 cap structure added to newly formed viral RNA. Formation of this cap depends on nsP1 binding GTP and transferring a methylated GMP to nascent viral RNA. We have developed a fluorescence polarization-based assay that monitors displacement of a fluorescently-labeled GTP analog in real time. Determining the relative affinities of 15 GTP analogs for nsP1 GTP revealed important structural aspects of GTP that will inform identification of inhibitors able to outcompete GTP for the nsP1 binding site. Validation of the assay for HTS was completed and a secondary orthogonal assay that measures guanylation activity was developed in order to evaluate hits from future drug screens. This platform provides an avenue for identification of potent nsP1 inhibitors, which would potentially provide compounds capable of treating disease caused by CHIKV infection.

Molecular characterization of emaraviruses associated with Pigeonpea sterility mosaic disease.

PubMed

Kumar, Surender; Subbarao, B L; Hallan, Vipin

2017-09-19

Sterility Mosaic Disease (SMD) of pigeonpea (Cajanus cajan (L.) Millspaugh) is a complex disease due to various factors including the presence of a mixed infection. Comparison of dsRNA profile and small RNA (sRNA) deep sequencing analysis of samples from three locations revealed the presence of Pigeonpea sterility mosaic virus-I and II (PPSMV-I and II) from Chevella and only PPSMV-II from Bengaluru and Coimbatore. PPSMV-I genome consisted of four while PPSMV-II encompassed six RNAs. The two viruses have modest sequence homology between their corresponding RNA 1-4 encoding RdRp, glycoprotein precursor, nucleocapsid and movement proteins and the corresponding orthologs of other emaraviruses. However, PPSMV-II is more related to Fig mosaic virus (FMV) than to PPSMV-I. ELISA based detection methodology was standardized to identify these two viruses, uniquely. Mite inoculation of sub-isolate Chevella sometimes resulted in few- to- many pigeonpea plants containing PPSMV-I alone. The study shows that (i) the N-terminal region of RdRp (SRD-1) of both the viruses contain "cap-snatching" endonuclease domain and a 13 AA cap binding site at the C-terminal, essential for viral cap-dependent transcription similar to the members of Bunyaviridae family and (ii) P4 is the movement protein and may belong to '30 K superfamily' of MPs.

Comparative Plasmodium gene overexpression reveals distinct perturbation of sporozoite transmission by profilin

PubMed Central

Sato, Yuko; Hliscs, Marion; Dunst, Josefine; Goosmann, Christian; Brinkmann, Volker; Montagna, Georgina N.; Matuschewski, Kai

2016-01-01

Plasmodium relies on actin-based motility to migrate from the site of infection and invade target cells. Using a substrate-dependent gliding locomotion, sporozoites are able to move at fast speed (1–3 μm/s). This motility relies on a minimal set of actin regulatory proteins and occurs in the absence of detectable filamentous actin (F-actin). Here we report an overexpression strategy to investigate whether perturbations of F-actin steady-state levels affect gliding locomotion and host invasion. We selected two vital Plasmodium berghei G-actin–binding proteins, C-CAP and profilin, in combination with three stage-specific promoters and mapped the phenotypes afforded by overexpression in all three extracellular motile stages. We show that in merozoites and ookinetes, additional expression does not impair life cycle progression. In marked contrast, overexpression of C-CAP and profilin in sporozoites impairs circular gliding motility and salivary gland invasion. The propensity for productive motility correlates with actin accumulation at the parasite tip, as revealed by combinations of an actin-stabilizing drug and transgenic parasites. Strong expression of profilin, but not C-CAP, resulted in complete life cycle arrest. Comparative overexpression is an alternative experimental genetic strategy to study essential genes and reveals effects of regulatory imbalances that are not uncovered from deletion-mutant phenotyping. PMID:27226484

Cbp80 is needed for the expression of piRNA components and piRNAs

PubMed Central

Colombo, Martino; Hernandez, Greco; Beuchle, Dirk; Berger, Fabienne; Peischl, Stephan; Bruggmann, Rémy

2017-01-01

Cap binding protein 80 (Cbp80) is the larger subunit of the nuclear cap-binding complex (nCBC), which is known to play important roles in nuclear mRNA processing, export, stability and quality control events. Reducing Cbp80 mRNA levels in the female germline revealed that Cbp80 is also involved in defending the germline against transposable elements. Combining such knockdown experiments with large scale sequencing of small RNAs further showed that Cbp80 is involved in the initial biogenesis of piRNAs as well as in the secondary biogenesis pathway, the ping-pong amplification cycle. We further found that Cbp80 knockdown not only led to the upregulation of transposons, but also to delocalization of Piwi, Aub and Ago3, key factors in the piRNA biosynthesis pathway. Furthermore, compared to controls, levels of Piwi and Aub were also reduced upon knock down of Cbp80. On the other hand, with the same treatment we could not detect significant changes in levels or subcellular distribution (nuage localization) of piRNA precursor transcripts. This shows that Cbp80 plays an important role in the production and localization of the protein components of the piRNA pathway and it seems to be less important for the production and export of the piRNA precursor transcripts. PMID:28746365

Gel-sol transition of the cytoplasm and its regulation

NASA Astrophysics Data System (ADS)

Janmey, Paul A.

1991-05-01

The cytoplasm of motile cells contains a dynamic system of filamentous protein polymers that endow the cell with elasticity permitting it to maintain its shape in the presence of mechanical forces encountered in vivo. Part of this cytoskeleton is composed of filaments of polymerized actin. Remodeling of this network is required for cell motility and cytoplasmic restructuring, and the reversible polymerization of actin per se has been suggested to cause morphologic changes such as cell ruffling and pseudopd extension. Changes in the degree of polymerization of acting and in the association of actin filaments into supramolecular structures are often associated with cell activation. Such activation is initiated by extracellular signals that bind to receptors which are often coupled by G-proteins to the production of intracellular second messangers. Cytoplasmic gel-sol transitions therefore can occur by formation and dissolution of actin networks, mediated by a variety of actin-binding proteins which are regulated by intracellular signalling molecules such as Ca2+ and polyphosphoinositides. The effects of three actin binding proteins: profilin, gelsolin and ABP (Tilamin) on the polymerization of actin and the viscoelasticity of the resulting networks measured in vitro suggest possible roles of these proteins in vivo. In particular, gelsolin, which activated by Ca2+ to sever and cap actin filaments, and released from filament ends by PIP2, appears to be a likely candidate for regulation of gel-sol transitions in response to cell activation. Recent results demonstrate that the hydrolysis of ATP that occurs following actin polymerization also influences the structure of the resulting filament. In addition being regulated by acting-binding proteins, the viscoelasticity of actin networks is also affected by the presence of the other two classes of cytoplasmic protein polymers, microtubules and intermediate filaments.

CARD8 is a negative regulator for NLRP3 inflammasome, but mutant NLRP3 in cryopyrin-associated periodic syndromes escapes the restriction

PubMed Central

2014-01-01

Introduction NLRP3 plays a role in sensing various pathogen components or stresses in the innate immune system. Once activated, NLRP3 associates with apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) and procaspase-1 to form a large protein complex termed inflammasome. Although some investigators have proposed a model of NLRP3-inflammasome containing an adaptor protein caspase recruitment domain-containing protein 8 (CARD8), the role of this molecule remains obscure. This study aimed to clarify the interaction between CARD8 and wild-type NLRP3 as well as mutant forms of NLRP3 linked with cryopyrin-associated periodic syndromes (CAPS). Methods In here HEK293 expression system, cells were transfected with the cDNAs for inflammasome components. Also used were peripheral blood mononuclear cells (PBMCs) and human monocyte-derived macrophages (HMDMs) from healthy volunteers. The interaction of CARD8 and NLRP3 was studied by immunoprecipitation. The effect of CARD8 expression on IL-1β secretion was assessed by ELISA. CARD8 knockdown experiments were carried out by transfection of the specific siRNA into HMDMs. Results In HEK293 cells, CARD8 interacted with wild-type NLRP3, but not with CAPS-associated mutant NLRP3. CARD8 significantly reduced IL-1β secretion from cells transfected with wild-type NLRP3, but not if they were transfected with mutant NLRP3. In addition, association of endogenously expressed CARD8 with NLRP3 was confirmed in resting PBMCs, and CARD8 knockdown resulted in higher amount of IL-1β secretion from HMDMs. Conclusions Until specific stimuli activate NLRP3, CARD8 holds NLRP3, and is supposed to prevent activation by subtle stimuli. However, CAPS-associated mutant NLRP3 is unable to bind with CARD8, which might be relevant to the pathogenesis of CAPS. PMID:24517500

CARD8 is a negative regulator for NLRP3 inflammasome, but mutant NLRP3 in cryopyrin-associated periodic syndromes escapes the restriction.

PubMed

Ito, Sayaka; Hara, Yukichi; Kubota, Tetsuo

2014-02-12

NLRP3 plays a role in sensing various pathogen components or stresses in the innate immune system. Once activated, NLRP3 associates with apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) and procaspase-1 to form a large protein complex termed inflammasome. Although some investigators have proposed a model of NLRP3-inflammasome containing an adaptor protein caspase recruitment domain-containing protein 8 (CARD8), the role of this molecule remains obscure. This study aimed to clarify the interaction between CARD8 and wild-type NLRP3 as well as mutant forms of NLRP3 linked with cryopyrin-associated periodic syndromes (CAPS). In here HEK293 expression system, cells were transfected with the cDNAs for inflammasome components. Also used were peripheral blood mononuclear cells (PBMCs) and human monocyte-derived macrophages (HMDMs) from healthy volunteers. The interaction of CARD8 and NLRP3 was studied by immunoprecipitation. The effect of CARD8 expression on IL-1β secretion was assessed by ELISA. CARD8 knockdown experiments were carried out by transfection of the specific siRNA into HMDMs. In HEK293 cells, CARD8 interacted with wild-type NLRP3, but not with CAPS-associated mutant NLRP3. CARD8 significantly reduced IL-1β secretion from cells transfected with wild-type NLRP3, but not if they were transfected with mutant NLRP3. In addition, association of endogenously expressed CARD8 with NLRP3 was confirmed in resting PBMCs, and CARD8 knockdown resulted in higher amount of IL-1β secretion from HMDMs. Until specific stimuli activate NLRP3, CARD8 holds NLRP3, and is supposed to prevent activation by subtle stimuli. However, CAPS-associated mutant NLRP3 is unable to bind with CARD8, which might be relevant to the pathogenesis of CAPS.

Translation initiation on mRNAs bound by nuclear cap-binding protein complex CBP80/20 requires interaction between CBP80/20-dependent translation initiation factor and eukaryotic translation initiation factor 3g.

PubMed

Choe, Junho; Oh, Nara; Park, Sungjin; Lee, Ye Kyung; Song, Ok-Kyu; Locker, Nicolas; Chi, Sung-Gil; Kim, Yoon Ki

2012-05-25

In the cytoplasm of mammalian cells, either cap-binding proteins 80 and 20 (CBP80/20) or eukaryotic translation initiation factor (eIF) 4E can direct the initiation of translation. Although the recruitment of ribosomes to mRNAs during eIF4E-dependent translation (ET) is well characterized, the molecular mechanism for CBP80/20-dependent translation (CT) remains obscure. Here, we show that CBP80/20-dependent translation initiation factor (CTIF), which has been shown to be preferentially involved in CT but not ET, specifically interacts with eIF3g, a component of the eIF3 complex involved in ribosome recruitment. By interacting with eIF3g, CTIF serves as an adaptor protein to bridge the CBP80/20 and the eIF3 complex, leading to efficient ribosome recruitment during CT. Accordingly, down-regulation of CTIF using a small interfering RNA causes a redistribution of CBP80 from polysome fractions to subpolysome fractions, without significant consequence to eIF4E distribution. In addition, down-regulation of eIF3g inhibits the efficiency of nonsense-mediated mRNA decay, which is tightly coupled to CT but not to ET. Moreover, the artificial tethering of CTIF to an intercistronic region of dicistronic mRNA results in translation of the downstream cistron in an eIF3-dependent manner. These findings support the idea that CT mechanistically differs from ET.

[Cytoskeletal actin and its associated proteins. Some examples in Protista].

PubMed

Guillén, N; Carlier, M F; Brugerolle, G; Tardieux, I; Ausseil, J

1998-06-01

Many processes, cell motility being an example, require cells to remodel the actin cytoskeleton in response to both intracellular and extracellular signals. Reorganization of the actin cytoskeleton involves the rapid disassembly and reassembly of actin filaments, a phenomenon regulated by the action of particular actin-binding proteins. In recent years, an interest in studying actin regulation in unicellular organisms has arisen. Parasitic protozoan are among these organisms and studies of the cytoskeleton functions of these protozoan are relevant related to either cell biology or pathogenicity. To discuss recent data in this field, a symposium concerning "Actin and actin-binding proteins in protists" was held on May 8-11 in Paris, France, during the XXXV meeting of the French Society of Protistology. As a brief summary of the symposium we report here findings concerning the in vitro actin dynamic assembly, as well as the characterization of several actin-binding proteins from the parasitic protozoan Entamoeba histolytica, Trichomonas vaginalis and Plasmodium knowlesi. In addition, localization of actin in non-pathogen protists such as Prorocentrum micans and Crypthecodinium cohnii is also presented. The data show that some actin-binding proteins facilitate organization of filaments into higher order structures as pseudopods, while others have regulatory functions, indicating very particular roles for actin-binding proteins. One of the proteins discussed during the symposium, the actin depolymerizing factor ADF, was shown to enhance the treadmilling rate of actin filaments. In vitro, ADF binds to the ADP-bound forms of G-actin and F-actin, thereby participating in and changing the rate of actin assembly. Biochemical approaches allowed the identification of a protein complex formed by HSP/C70-cap32-34 which might also be involved in depolymerization of F-actin in P. knowlesi. Molecular and cellular approaches were used to identify proteins such as ABP-120 and myosin IB at the leading edge of E. histolytica. ABP-120 organizes F-actin in a network and myosin IB participates in the pseudopod formation. Similar approaches using T. vaginalis resulted in the discovery of an actin-binding protein that participate in the F-actin reorganization during adhesion of parasites to target cells. This protein is homologous to alpha-actinin from other eukaryotic cells. Finally, by using cell biology approaches, F-actin was observed in the cytoplasm as well as in the nucleus of Dinoflagellates. The recent developments in the molecular genetics of protozoa will provide new insights to understand the roles of actin-binding proteins during cytoskeleton activities.

Enhanced protective immune response to PCV2 subunit vaccine by co-administration of recombinant porcine IFN-γ in mice.

PubMed

Wang, Yi-Ping; Liu, Dan; Guo, Long-Jun; Tang, Qing-Hai; Wei, Yan-Wu; Wu, Hong-Li; Liu, Jian-Bo; Li, Sheng-Bin; Huang, Li-Ping; Liu, Chang-Ming

2013-01-21

The capsid (Cap) protein of PCV2 is the major immunogenic protein that is crucial to induce PCV2-specific neutralizing antibodies and protective immunity; thus, it is a suitable target antigen for the research and development of genetically engineered vaccines against PCV2 infection. IFN-γ has exhibited potential efficacy as an immune adjuvant that enhances the immunogenicity of certain vaccines in experimental animal models. In this study, three recombinant proteins: PCV2-Cap protein, porcine IFN-γ (PoIFN-γ), and the fusion protein (Cap-PoIFN-γ) of PCV2-Cap protein and PoIFN-γ were respectively expressed in the baculovirus system, and analyzed by Western blot and indirect ELISA. Additionally, we evaluated the enhancement of the protective immune response to the Cap protein-based PCV2 subunit vaccine elicited by co-administration of PoIFN-γ in mice. Vaccination of mice with the PCV2-Cap+PoIFN-γ vaccine elicited significantly higher levels of PCV2-specific IPMA antibodies, neutralizing antibodies, and lymphocyte proliferative responses compared to the Cap-PoIFN-γ vaccine, the PCV2-Cap vaccine, and LG-strain. Following virulent PCV2 challenge, no viraemia was detected in all immunized groups, and the viral loads in lungs of the PCV2-Cap+PoIFN-γ group were significantly lower compared to the Cap-PoIFN-γ group, the LG-strain group, and the mock group, but slightly lower compared to the PCV2-Cap group. These findings suggested that PoIFN-γ substantially enhanced the protective immune response to the Cap protein-based PCV2 subunit vaccine, and that the PCV2-Cap+PoIFN-γ subunit vaccine potentially serves as an attractive candidate vaccine for the prevention and control of PCV2-associated diseases. Copyright © 2012 Elsevier Ltd. All rights reserved.

Structural, kinetic, and thermodynamic studies of specificity designed HIV-1 protease.

PubMed

Alvizo, Oscar; Mittal, Seema; Mayo, Stephen L; Schiffer, Celia A

2012-07-01

HIV-1 protease recognizes and cleaves more than 12 different substrates leading to viral maturation. While these substrates share no conserved motif, they are specifically selected for and cleaved by protease during viral life cycle. Drug resistant mutations evolve within the protease that compromise inhibitor binding but allow the continued recognition of all these substrates. While the substrate envelope defines a general shape for substrate recognition, successfully predicting the determinants of substrate binding specificity would provide additional insights into the mechanism of altered molecular recognition in resistant proteases. We designed a variant of HIV protease with altered specificity using positive computational design methods and validated the design using X-ray crystallography and enzyme biochemistry. The engineered variant, Pr3 (A28S/D30F/G48R), was designed to preferentially bind to one out of three of HIV protease's natural substrates; RT-RH over p2-NC and CA-p2. In kinetic assays, RT-RH binding specificity for Pr3 increased threefold compared to the wild-type (WT), which was further confirmed by isothermal titration calorimetry. Crystal structures of WT protease and the designed variant in complex with RT-RH, CA-p2, and p2-NC were determined. Structural analysis of the designed complexes revealed that one of the engineered substitutions (G48R) potentially stabilized heterogeneous flap conformations, thereby facilitating alternate modes of substrate binding. Our results demonstrate that while substrate specificity could be engineered in HIV protease, the structural pliability of protease restricted the propagation of interactions as predicted. These results offer new insights into the plasticity and structural determinants of substrate binding specificity of the HIV-1 protease. Copyright © 2012 The Protein Society.

PERK Signal-Modulated Protein Translation Promotes the Survivability of Dengue 2 Virus-Infected Mosquito Cells and Extends Viral Replication.

PubMed

Hou, Jiun-Nan; Chen, Tien-Huang; Chiang, Yi-Hsuan; Peng, Jing-Yun; Yang, Tsong-Han; Cheng, Chih-Chieh; Sofiyatun, Eny; Chiu, Cheng-Hsun; Chiang-Ni, Chuan; Chen, Wei-June

2017-09-20

Survival of mosquitoes from dengue virus (DENV) infection is a prerequisite of viral transmission to the host. This study aimed to see how mosquito cells can survive the infection during prosperous replication of the virus. In C6/36 cells, global protein translation was shut down after infection by DENV type 2 (DENV2). However, it returned to a normal level when infected cells were treated with an inhibitor of the protein kinase RNA (PKR)-like ER kinase (PERK) signaling pathway. Based on a 7-Methylguanosine 5'-triphosphate (m7GTP) pull-down assay, the eukaryotic translation initiation factor 4F (eIF4F) complex was also identified in DENV2-infected cells. This suggests that most mosquito proteins are synthesized via canonical cap-dependent translation. When the PERK signal pathway was inhibited, both accumulation of reactive oxygen species and changes in the mitochondrial membrane potential increased. This suggested that ER stress response was alleviated through the PERK-mediated shutdown of global proteins in DENV2-infected C6/36 cells. In the meantime, the activities of caspases-9 and -3 and the apoptosis-related cell death rate increased in C6/36 cells with PERK inhibition. This reflected that the PERK-signaling pathway is involved in determining cell survival, presumably by reducing DENV2-induced ER stress. Looking at the PERK downstream target, α-subunit of eukaryotic initiation factor 2 (eIF2α), an increased phosphorylation status was only shown in infected C6/36 cells. This indicated that recruitment of ribosome binding to the mRNA 5'-cap structure could have been impaired in cap-dependent translation. It turned out that shutdown of cellular protein translation resulted in a pro-survival effect on mosquito cells in response to DENV2 infection. As synthesis of viral proteins was not affected by the PERK signal pathway, an alternate mode other than cap-dependent translation may be utilized. This finding provides insights into elucidating how the PERK signal pathway modulates dynamic translation of proteins and helps mosquito cells survive continuous replication of the DENV2. It was ecologically important for virus amplification in mosquitoes and transmission to humans.

PERK Signal-Modulated Protein Translation Promotes the Survivability of Dengue 2 Virus-Infected Mosquito Cells and Extends Viral Replication

PubMed Central

Hou, Jiun-Nan; Chen, Tien-Huang; Chiang, Yi-Hsuan; Peng, Jing-Yun; Yang, Tsong-Han; Cheng, Chih-Chieh; Sofiyatun, Eny; Chiu, Cheng-Hsun; Chiang-Ni, Chuan; Chen, Wei-June

2017-01-01

Survival of mosquitoes from dengue virus (DENV) infection is a prerequisite of viral transmission to the host. This study aimed to see how mosquito cells can survive the infection during prosperous replication of the virus. In C6/36 cells, global protein translation was shut down after infection by DENV type 2 (DENV2). However, it returned to a normal level when infected cells were treated with an inhibitor of the protein kinase RNA (PKR)-like ER kinase (PERK) signaling pathway. Based on a 7-Methylguanosine 5′-triphosphate (m7GTP) pull-down assay, the eukaryotic translation initiation factor 4F (eIF4F) complex was also identified in DENV2-infected cells. This suggests that most mosquito proteins are synthesized via canonical cap-dependent translation. When the PERK signal pathway was inhibited, both accumulation of reactive oxygen species and changes in the mitochondrial membrane potential increased. This suggested that ER stress response was alleviated through the PERK-mediated shutdown of global proteins in DENV2-infected C6/36 cells. In the meantime, the activities of caspases-9 and -3 and the apoptosis-related cell death rate increased in C6/36 cells with PERK inhibition. This reflected that the PERK-signaling pathway is involved in determining cell survival, presumably by reducing DENV2-induced ER stress. Looking at the PERK downstream target, α-subunit of eukaryotic initiation factor 2 (eIF2α), an increased phosphorylation status was only shown in infected C6/36 cells. This indicated that recruitment of ribosome binding to the mRNA 5′-cap structure could have been impaired in cap-dependent translation. It turned out that shutdown of cellular protein translation resulted in a pro-survival effect on mosquito cells in response to DENV2 infection. As synthesis of viral proteins was not affected by the PERK signal pathway, an alternate mode other than cap-dependent translation may be utilized. This finding provides insights into elucidating how the PERK signal pathway modulates dynamic translation of proteins and helps mosquito cells survive continuous replication of the DENV2. It was ecologically important for virus amplification in mosquitoes and transmission to humans. PMID:28930151

Global Low Frequency Protein Motions in Long-Range Allosteric Signaling

NASA Astrophysics Data System (ADS)

McLeish, Tom; Rogers, Thomas; Townsend, Philip; Burnell, David; Pohl, Ehmke; Wilson, Mark; Cann, Martin; Richards, Shane; Jones, Matthew

2015-03-01

We present a foundational theory for how allostery can occur as a function of low frequency dynamics without a change in protein structure. Elastic inhomogeneities allow entropic ``signalling at a distance.'' Remarkably, many globular proteins display just this class of elastic structure, in particular those that support allosteric binding of substrates (long-range co-operative effects between the binding sites of small molecules). Through multi-scale modelling of global normal modes we demonstrate negative co-operativity between the two cAMP ligands without change to the mean structure. Crucially, the value of the co-operativity is itself controlled by the interactions around a set of third allosteric ``control sites.'' The theory makes key experimental predictions, validated by analysis of variant proteins by a combination of structural biology and isothermal calorimetry. A quantitative description of allostery as a free energy landscape revealed a protein ``design space'' that identified the key inter- and intramolecular regulatory parameters that frame CRP/FNR family allostery. Furthermore, by analyzing naturally occurring CAP variants from diverse species, we demonstrate an evolutionary selection pressure to conserve residues crucial for allosteric control. The methodology establishes the means to engineer allosteric mechanisms that are driven by low frequency dynamics.

Wise, a context-dependent activator and inhibitor of Wnt signalling.

PubMed

Itasaki, Nobue; Jones, C Michael; Mercurio, Sara; Rowe, Alison; Domingos, Pedro M; Smith, James C; Krumlauf, Robb

2003-09-01

We have isolated a novel secreted molecule, Wise, by a functional screen for activities that alter the anteroposterior character of neuralised Xenopus animal caps. Wise encodes a secreted protein capable of inducing posterior neural markers at a distance. Phenotypes arising from ectopic expression or depletion of Wise resemble those obtained when Wnt signalling is altered. In animal cap assays, posterior neural markers can be induced by Wnt family members, and induction of these markers by Wise requires components of the canonical Wnt pathway. This indicates that in this context Wise activates the Wnt signalling cascade by mimicking some of the effects of Wnt ligands. Activation of the pathway was further confirmed by nuclear accumulation of beta-catenin driven by Wise. By contrast, in an assay for secondary axis induction, extracellularly Wise antagonises the axis-inducing ability of Wnt8. Thus, Wise can activate or inhibit Wnt signalling in a context-dependent manner. The Wise protein physically interacts with the Wnt co-receptor, lipoprotein receptor-related protein 6 (LRP6), and is able to compete with Wnt8 for binding to LRP6. These activities of Wise provide a new mechanism for integrating inputs through the Wnt coreceptor complex to modulate the balance of Wnt signalling.

Insights into Structural and Mechanistic Features of Viral IRES Elements

PubMed Central

Martinez-Salas, Encarnacion; Francisco-Velilla, Rosario; Fernandez-Chamorro, Javier; Embarek, Azman M.

2018-01-01

Internal ribosome entry site (IRES) elements are cis-acting RNA regions that promote internal initiation of protein synthesis using cap-independent mechanisms. However, distinct types of IRES elements present in the genome of various RNA viruses perform the same function despite lacking conservation of sequence and secondary RNA structure. Likewise, IRES elements differ in host factor requirement to recruit the ribosomal subunits. In spite of this diversity, evolutionarily conserved motifs in each family of RNA viruses preserve sequences impacting on RNA structure and RNA–protein interactions important for IRES activity. Indeed, IRES elements adopting remarkable different structural organizations contain RNA structural motifs that play an essential role in recruiting ribosomes, initiation factors and/or RNA-binding proteins using different mechanisms. Therefore, given that a universal IRES motif remains elusive, it is critical to understand how diverse structural motifs deliver functions relevant for IRES activity. This will be useful for understanding the molecular mechanisms beyond cap-independent translation, as well as the evolutionary history of these regulatory elements. Moreover, it could improve the accuracy to predict IRES-like motifs hidden in genome sequences. This review summarizes recent advances on the diversity and biological relevance of RNA structural motifs for viral IRES elements. PMID:29354113

Multiple receptors mobilize calcium through a pertussis toxin (PT) sensitive GTP-binding protein in human neutrophils (PMN's)

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

Lad, P.M.; Olson, C.V.; Grewal, I.S.

1986-03-05

Treatment of PMN's with PT causes an abolition of chemotaxis, enzyme release, superoxide generation and aggregation caused by f-met-leu-phe (FMLP),C5a and platelet activating factor (PAF). Lectin (Con-A) induced capping and receptor induced shape change are abolished, but phagocytosis is unaltered. In whole cells, calcium mobilization induced by FMLP, PAF and Con-A inhibited by PT although the FMLP-mediated effect is more susceptible to PT's effects. Treatment of PMN's with phorbol 12-myristate 13 acetate (PMA) causes an abolition of calcium mobilization by all agents in a range which also inhibits cap formation. Investigation of calcium uptake reveals PT sensitive and insensitive components.more » Reciprocal interactions between Ns and Ni proteins are also observed since pretreatment with FMLP and PAF causes a stimulation of Ns-mediated cyclic AMP enhancement while pretreatment with Ns linked receptors (PGE/sub 1/ and beta receptor agonists) inhibits calcium mobilization. Comparative peptide mapping studies indicate substantial similarity between Ni proteins in PMN's, platelets and human erythrocytes. The authors results suggest that the Ni linked calcium mobilization sensitive to PMA is important to the regulation of the human neutrophil.« less

The unique Leishmania EIF4E4 N-terminus is a target for multiple phosphorylation events and participates in critical interactions required for translation initiation.

PubMed

de Melo Neto, Osvaldo P; da Costa Lima, Tamara D C; Xavier, Camila C; Nascimento, Larissa M; Romão, Tatiany P; Assis, Ludmila A; Pereira, Mariana M C; Reis, Christian R S; Papadopoulou, Barbara

2015-01-01

The eukaryotic initiation factor 4E (eIF4E) recognizes the mRNA cap structure and, together with eIF4G and eIF4A, form the eIF4F complex that regulates translation initiation in eukaryotes. In trypanosomatids, 2 eIF4E homologues (EIF4E3 and EIF4E4) have been shown to be part of eIF4F-like complexes with presumed roles in translation initiation. Both proteins possess unique N-terminal extensions, which can be targeted for phosphorylation. Here, we provide novel insights on the Leishmania infantum EIF4E4 function and regulation. We show that EIF4E4 is constitutively expressed throughout the parasite development but is preferentially phosphorylated in exponentially grown promastigote and amastigote life stages, hence correlating with high levels of translation. Phosphorylation targets multiple serine-proline or threonine-proline residues within the N-terminal extension of EIF4E4 but does not require binding to the EIF4E4's partner, EIF4G3, or to the cap structure. We also report that EIF4E4 interacts with PABP1 through 3 conserved boxes at the EIF4E4 N-terminus and that this interaction is a prerequisite for efficient EIF4E4 phosphorylation. EIF4E4 is essential for Leishmania growth and an EIF4E4 null mutant was only obtained in the presence of an ectopically provided wild type gene. Complementation for the loss of EIF4E4 with several EIF4E4 mutant proteins affecting either phosphorylation or binding to mRNA or to EIF4E4 protein partners revealed that, in contrast to other eukaryotes, only the EIF4E4-PABP1 interaction but neither the binding to EIF4G3 nor phosphorylation is essential for translation. These studies also demonstrated that the lack of both EIF4E4 phosphorylation and EIF4G3 binding leads to a non-functional protein. Altogether, these findings further highlight the unique features of the translation initiation process in trypanosomatid protozoa.

The unique Leishmania EIF4E4 N-terminus is a target for multiple phosphorylation events and participates in critical interactions required for translation initiation

PubMed Central

de Melo Neto, Osvaldo P; da Costa Lima, Tamara D C; Xavier, Camila C; Nascimento, Larissa M; Romão, Tatiany P; Assis, Ludmila A; Pereira, Mariana M C; Reis, Christian R S; Papadopoulou, Barbara

2015-01-01

The eukaryotic initiation factor 4E (eIF4E) recognizes the mRNA cap structure and, together with eIF4G and eIF4A, form the eIF4F complex that regulates translation initiation in eukaryotes. In trypanosomatids, 2 eIF4E homologues (EIF4E3 and EIF4E4) have been shown to be part of eIF4F-like complexes with presumed roles in translation initiation. Both proteins possess unique N-terminal extensions, which can be targeted for phosphorylation. Here, we provide novel insights on the Leishmania infantum EIF4E4 function and regulation. We show that EIF4E4 is constitutively expressed throughout the parasite development but is preferentially phosphorylated in exponentially grown promastigote and amastigote life stages, hence correlating with high levels of translation. Phosphorylation targets multiple serine-proline or threonine-proline residues within the N-terminal extension of EIF4E4 but does not require binding to the EIF4E4's partner, EIF4G3, or to the cap structure. We also report that EIF4E4 interacts with PABP1 through 3 conserved boxes at the EIF4E4 N-terminus and that this interaction is a prerequisite for efficient EIF4E4 phosphorylation. EIF4E4 is essential for Leishmania growth and an EIF4E4 null mutant was only obtained in the presence of an ectopically provided wild type gene. Complementation for the loss of EIF4E4 with several EIF4E4 mutant proteins affecting either phosphorylation or binding to mRNA or to EIF4E4 protein partners revealed that, in contrast to other eukaryotes, only the EIF4E4-PABP1 interaction but neither the binding to EIF4G3 nor phosphorylation is essential for translation. These studies also demonstrated that the lack of both EIF4E4 phosphorylation and EIF4G3 binding leads to a non-functional protein. Altogether, these findings further highlight the unique features of the translation initiation process in trypanosomatid protozoa. PMID:26338184

Nuclear TRIM25 Specifically Targets Influenza Virus Ribonucleoproteins to Block the Onset of RNA Chain Elongation.

PubMed

Meyerson, Nicholas R; Zhou, Ligang; Guo, Yusong R; Zhao, Chen; Tao, Yizhi J; Krug, Robert M; Sawyer, Sara L

2017-11-08

TRIM25 is an E3 ubiquitin ligase that activates RIG-I to promote the antiviral interferon response. The NS1 protein from all strains of influenza A virus binds TRIM25, although not all virus strains block the interferon response, suggesting alternative mechanisms for TRIM25 action. Here we present a nuclear role for TRIM25 in specifically restricting influenza A virus replication. TRIM25 inhibits viral RNA synthesis through a direct mechanism that is independent of its ubiquitin ligase activity and the interferon pathway. This activity can be inhibited by the viral NS1 protein. TRIM25 inhibition of viral RNA synthesis results from its binding to viral ribonucleoproteins (vRNPs), the structures containing individual viral RNA segments, the viral polymerase, and multiple viral nucleoproteins. TRIM25 binding does not inhibit initiation of capped-RNA-primed viral mRNA synthesis by the viral polymerase. Rather, the onset of RNA chain elongation is inhibited because TRIM25 prohibits the movement of RNA into the polymerase complex. Copyright © 2017 Elsevier Inc. All rights reserved.

Small Maf proteins (MafF, MafG, MafK): History, structure and function.

PubMed

Katsuoka, Fumiki; Yamamoto, Masayuki

2016-07-25

The small Maf proteins (sMafs) are basic region leucine zipper (bZIP)-type transcription factors. The basic region of the Maf family is unique among the bZIP factors, and it contributes to the distinct DNA-binding mode of this class of proteins. MafF, MafG and MafK are the three vertebrate sMafs, and no functional differences have been observed among them in terms of their bZIP structures. sMafs form homodimers by themselves, and they form heterodimers with cap 'n' collar (CNC) proteins (p45 NF-E2, Nrf1, Nrf2, and Nrf3) and also with Bach proteins (Bach1 and Bach2). Because CNC and Bach proteins cannot bind to DNA as monomers, sMafs are indispensable partners that are required by CNC and Bach proteins to exert their functions. sMafs lack the transcriptional activation domain; hence, their homodimers act as transcriptional repressors. In contrast, sMafs participate in transcriptional activation or repression depending on their heterodimeric partner molecules and context. Mouse genetic analyses have revealed that various biological pathways are under the regulation of CNC-sMaf heterodimers. In this review, we summarize the history and current progress of sMaf studies in relation to their partners. Copyright © 2016 Elsevier B.V. All rights reserved.

Munc13-4 reconstitutes calcium-dependent SNARE-mediated membrane fusion

PubMed Central

Boswell, Kristin L.; James, Declan J.; Esquibel, Joseph M.; Bruinsma, Stephen; Shirakawa, Ryutaro; Horiuchi, Hisanori

2012-01-01

Munc13-4 is a widely expressed member of the CAPS/Munc13 protein family proposed to function in priming secretory granules for exocytosis. Munc13-4 contains N- and C-terminal C2 domains (C2A and C2B) predicted to bind Ca2+, but Ca2+-dependent regulation of Munc13-4 activity has not been described. The C2 domains bracket a predicted SNARE-binding domain, but whether Munc13-4 interacts with SNARE proteins is unknown. We report that Munc13-4 bound Ca2+ and restored Ca2+-dependent granule exocytosis to permeable cells (platelets, mast, and neuroendocrine cells) dependent on putative Ca2+-binding residues in C2A and C2B. Munc13-4 exhibited Ca2+-stimulated SNARE interactions dependent on C2A and Ca2+-dependent membrane binding dependent on C2B. In an apparent coupling of membrane and SNARE binding, Munc13-4 stimulated SNARE-dependent liposome fusion dependent on putative Ca2+-binding residues in both C2A and C2B domains. Munc13-4 is the first priming factor shown to promote Ca2+-dependent SNARE complex formation and SNARE-mediated liposome fusion. These properties of Munc13-4 suggest its function as a Ca2+ sensor at rate-limiting priming steps in granule exocytosis. PMID:22508512

Munc13-4 reconstitutes calcium-dependent SNARE-mediated membrane fusion.

PubMed

Boswell, Kristin L; James, Declan J; Esquibel, Joseph M; Bruinsma, Stephen; Shirakawa, Ryutaro; Horiuchi, Hisanori; Martin, Thomas F J

2012-04-16

Munc13-4 is a widely expressed member of the CAPS/Munc13 protein family proposed to function in priming secretory granules for exocytosis. Munc13-4 contains N- and C-terminal C2 domains (C2A and C2B) predicted to bind Ca(2+), but Ca(2+)-dependent regulation of Munc13-4 activity has not been described. The C2 domains bracket a predicted SNARE-binding domain, but whether Munc13-4 interacts with SNARE proteins is unknown. We report that Munc13-4 bound Ca(2+) and restored Ca(2+)-dependent granule exocytosis to permeable cells (platelets, mast, and neuroendocrine cells) dependent on putative Ca(2+)-binding residues in C2A and C2B. Munc13-4 exhibited Ca(2+)-stimulated SNARE interactions dependent on C2A and Ca(2+)-dependent membrane binding dependent on C2B. In an apparent coupling of membrane and SNARE binding, Munc13-4 stimulated SNARE-dependent liposome fusion dependent on putative Ca(2+)-binding residues in both C2A and C2B domains. Munc13-4 is the first priming factor shown to promote Ca(2+)-dependent SNARE complex formation and SNARE-mediated liposome fusion. These properties of Munc13-4 suggest its function as a Ca(2+) sensor at rate-limiting priming steps in granule exocytosis.

L-selectin-carbohydrate interactions: relevant modifications of the Lewis x trisaccharide.

PubMed

Sanders, W J; Katsumoto, T R; Bertozzi, C R; Rosen, S D; Kiessling, L L

1996-11-26

Protein-carbohydrate interactions are known to mediate cell-cell recognition and adhesion events. Specifically, three carbohydrate binding proteins termed selectins (E-, P-, and L-selectin) have been shown to be essential for leukocyte rolling along the vascular endothelium, the first step in the recruitment of leukocytes from the blood into inflammatory sites or into secondary lymphoid organs. Although this phenomenon is well-established, little is known about the molecular-level interactions on which it depends. All three selectins recognize sulfated and sialylated derivatives of the Lewis x [Le(x):Gal beta 1-->4(Fuc alpha 1-->3)GlcNAc] and Lewis a [Le(a): Gal beta 1-->3(Fuc alpha 1-->4)GlcNAc] trisaccharide cores with affinities in the millimolar range, and it is believed that variants of these structures are the carbohydrate determinants of selectin recognition. Recently it was shown that the mucin GlyCAM-1, a secreted physiological ligand for L-selectin, is capped with sulfated derivatives of sialyl Lewis x [sLe(x): Sia alpha 2-->3Gal beta 1-->4(Fuc alpha 1-->3)GlcNAc] and that sulfation is required for the high-affinity interaction between GlyCAM-1 and L-selectin. To elucidate the important sites of sulfation on Le(x) with respect to L-selectin recognition, we have synthesized six sulfated Le(x) analogs and determined their abilities to block binding of a recombinant L-selectin-Ig chimera to immobilized GlyCAM-1. Our results suggest that 6-sulfo sLe(x) binds to L-selectin with higher affinity than does sLe(x) or 6'-sulfo sLe(x) and that sulfation of sLe(x) capping groups on GlyCAM-1 at the 6-position is important for L-selectin recognition.

Prostate Cancer Risk in Relation to IGF-1 and its Genetic Determinants: A Case Control Study Within the Cancer Prostate Sweden Project (CAPS)

DTIC Science & Technology

2007-05-01

releasing hormone (GHRH), and the GHRH receptor (GHRHR). Ghrelin (GHRL), a recently identified new peptide hormone produced by endocrine cells in...synthesis IGF1R IGF-I receptor GHRL Ghrelin GHSR Growth hormone secretagogue receptor IGFALS IGF binding protein, acid labile subunit IGFBP1 - 6...Hasinoff MJ, Fischer M, et al. Genetic linkage and association of the growth hormone secretagogue receptor ( ghrelin receptor) gene in human obesity. Diabetes

Human cytomegalovirus TRS1 protein associates with the 7-methylguanosine mRNA cap and facilitates translation.

PubMed

Ziehr, Benjamin; Lenarcic, Erik; Vincent, Heather A; Cecil, Chad; Garcia, Benjamin; Shenk, Thomas; Moorman, Nathaniel J

2015-06-01

Viruses rely on the host translation machinery for the synthesis of viral proteins. Human cells have evolved sensors that recognize viral RNAs and inhibit mRNA translation in order to limit virus replication. Understanding how viruses manipulate the host translation machinery to gain access to ribosomes and disable the antiviral response is therefore a critical aspect of the host/pathogen interface. In this study, we used a proteomics approach to identify human cytomegalovirus (HCMV) proteins that might contribute to viral mRNA translation. The HCMV TRS1 protein (pTRS1) associated with the 7-methylguanosine mRNA cap, increased the total level of protein synthesis, and colocalized with mRNAs undergoing translation initiation during infection. pTRS1 stimulated translation of a nonviral reporter gene and increased the translation of a reporter containing an HCMV 5' untranslated region (5'UTR) to a greater extent. The preferential effect of pTRS1 on translation of an mRNA containing a viral 5'UTR required the pTRS1 RNA and double-stranded RNA-dependent protein kinase (PKR)-binding domains, and was likely the result of PKR inhibition. However, pTRS1 also stimulated the total level of protein synthesis and translation directed by an HCMV 5'UTR in cells lacking PKR. Thus our results demonstrate that pTRS1 stimulates translation through both PKR-dependent and PKR-independent mechanisms. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

5'-Phosphorothiolate Dinucleotide Cap Analogues: Reagents for Messenger RNA Modification and Potent Small-Molecular Inhibitors of Decapping Enzymes.

PubMed

Wojtczak, Blazej A; Sikorski, Pawel J; Fac-Dabrowska, Kaja; Nowicka, Anna; Warminski, Marcin; Kubacka, Dorota; Nowak, Elzbieta; Nowotny, Marcin; Kowalska, Joanna; Jemielity, Jacek

2018-05-09

The 5' cap consists of 7-methylguanosine (m 7 G) linked by a 5'-5'-triphosphate bridge to messenger RNA (mRNA) and acts as the master regulator of mRNA turnover and translation initiation in eukaryotes. Cap analogues that influence mRNA translation and turnover (either as small molecules or as part of an RNA transcript) are valuable tools for studying gene expression, which is often also of therapeutic relevance. Here, we synthesized a series of 15 dinucleotide cap (m 7 GpppG) analogues containing a 5'-phosphorothiolate (5'-PSL) moiety (i.e., an O-to-S substitution within the 5'-phosphoester) and studied their biological properties in the context of three major cap-binding proteins: translation initiation factor 4E (eIF4E) and two decapping enzymes, DcpS and Dcp2. While the 5'-PSL moiety was neutral or slightly stabilizing for cap interactions with eIF4E, it significantly influenced susceptibility to decapping. Replacing the γ-phosphoester with the 5'-PSL moiety (γ-PSL) prevented β-γ-pyrophosphate bond cleavage by DcpS and conferred strong inhibitory properties. Combining the γ-PSL moiety with α-PSL and β-phosphorothioate (PS) moiety afforded first cap-derived hDcpS inhibitor with low nanomolar potency. Susceptibility to Dcp2 and translational properties were studied after incorporation of the new analogues into mRNA transcripts by RNA polymerase. Transcripts containing the γ-PSL moiety were resistant to cleavage by Dcp2. Surprisingly, superior translational properties were observed for mRNAs containing the α-PSL moiety, which were Dcp2-susceptible. The overall protein expression measured in HeLa cells for this mRNA was comparable to mRNA capped with the translation augmenting β-PS analogue reported previously. Overall, our study highlights 5'-PSL as a synthetically accessible cap modification, which, depending on the substitution site, can either reduce susceptibility to decapping or confer superior translational properties on the mRNA. The 5'-PSL-analogues may find application as reagents for the preparation of efficiently expressed mRNA or for investigation of the role of decapping enzymes in mRNA processing or neuromuscular disorders associated with decapping.

5'-Phospho-RNA Acceptor Specificity of GDP Polyribonucleotidyltransferase of Vesicular Stomatitis Virus in mRNA Capping.

PubMed

Ogino, Minako; Ogino, Tomoaki

2017-03-15

The GDP polyribonucleotidyltransferase (PRNTase) domain of the multifunctional L protein of rhabdoviruses, such as vesicular stomatitis virus (VSV) and rabies virus, catalyzes the transfer of 5'-phospho-RNA (pRNA) from 5'-triphospho-RNA (pppRNA) to GDP via a covalent enzyme-pRNA intermediate to generate a 5'-cap structure (GpppA). Here, using an improved oligo-RNA capping assay with the VSV L protein, we showed that the Michaelis constants for GDP and pppAACAG (VSV mRNA-start sequence) are 0.03 and 0.4 μM, respectively. A competition assay between GDP and GDP analogues in the GpppA formation and pRNA transfer assay using GDP analogues as pRNA acceptors indicated that the PRNTase domain recognizes the C-2-amino group, but not the C-6-oxo group, N-1-hydrogen, or N-7-nitrogen, of GDP for the cap formation. 2,6-Diaminopurine-riboside (DAP), 7-deazaguanosine (7-deaza-G), and 7-methylguanosine (m 7 G) diphosphates efficiently accepted pRNA, resulting in the formation of DAPpppA, 7-deaza-GpppA, and m 7 GpppA (cap 0), respectively. Furthermore, either the 2'- or 3'-hydroxyl group of GDP was found to be required for efficient pRNA transfer. A 5'-diphosphate form of antiviral ribavirin weakly inhibited the GpppA formation but did not act as a pRNA acceptor. These results indicate that the PRNTase domain has a unique guanosine-binding mode different from that of eukaryotic mRNA capping enzyme, guanylyltransferase. IMPORTANCE mRNAs of nonsegmented negative-strand (NNS) RNA viruses, such as VSV, possess a fully methylated cap structure, which is required for mRNA stability, efficient translation, and evasion of antiviral innate immunity in host cells. GDP polyribonucleotidyltransferase (PRNTase) is an unconventional mRNA capping enzyme of NNS RNA viruses that is distinct from the eukaryotic mRNA capping enzyme, guanylyltransferase. In this study, we studied the pRNA acceptor specificity of VSV PRNTase using various GDP analogues and identified chemical groups of GDP as essential for the substrate activity. The findings presented here are useful not only for understanding the mechanism of the substrate recognition with PRNTase but also for designing antiviral agents targeting this enzyme. Copyright © 2017 American Society for Microbiology.

5′-Phospho-RNA Acceptor Specificity of GDP Polyribonucleotidyltransferase of Vesicular Stomatitis Virus in mRNA Capping

PubMed Central

Ogino, Minako

2017-01-01

ABSTRACT The GDP polyribonucleotidyltransferase (PRNTase) domain of the multifunctional L protein of rhabdoviruses, such as vesicular stomatitis virus (VSV) and rabies virus, catalyzes the transfer of 5′-phospho-RNA (pRNA) from 5′-triphospho-RNA (pppRNA) to GDP via a covalent enzyme-pRNA intermediate to generate a 5′-cap structure (GpppA). Here, using an improved oligo-RNA capping assay with the VSV L protein, we showed that the Michaelis constants for GDP and pppAACAG (VSV mRNA-start sequence) are 0.03 and 0.4 μM, respectively. A competition assay between GDP and GDP analogues in the GpppA formation and pRNA transfer assay using GDP analogues as pRNA acceptors indicated that the PRNTase domain recognizes the C-2-amino group, but not the C-6-oxo group, N-1-hydrogen, or N-7-nitrogen, of GDP for the cap formation. 2,6-Diaminopurine-riboside (DAP), 7-deazaguanosine (7-deaza-G), and 7-methylguanosine (m7G) diphosphates efficiently accepted pRNA, resulting in the formation of DAPpppA, 7-deaza-GpppA, and m7GpppA (cap 0), respectively. Furthermore, either the 2′- or 3′-hydroxyl group of GDP was found to be required for efficient pRNA transfer. A 5′-diphosphate form of antiviral ribavirin weakly inhibited the GpppA formation but did not act as a pRNA acceptor. These results indicate that the PRNTase domain has a unique guanosine-binding mode different from that of eukaryotic mRNA capping enzyme, guanylyltransferase. IMPORTANCE mRNAs of nonsegmented negative-strand (NNS) RNA viruses, such as VSV, possess a fully methylated cap structure, which is required for mRNA stability, efficient translation, and evasion of antiviral innate immunity in host cells. GDP polyribonucleotidyltransferase (PRNTase) is an unconventional mRNA capping enzyme of NNS RNA viruses that is distinct from the eukaryotic mRNA capping enzyme, guanylyltransferase. In this study, we studied the pRNA acceptor specificity of VSV PRNTase using various GDP analogues and identified chemical groups of GDP as essential for the substrate activity. The findings presented here are useful not only for understanding the mechanism of the substrate recognition with PRNTase but also for designing antiviral agents targeting this enzyme. PMID:28053102

Aptamer-mediated colorimetric method for rapid and sensitive detection of chloramphenicol in food.

PubMed

Yan, Chao; Zhang, Jing; Yao, Li; Xue, Feng; Lu, Jianfeng; Li, Baoguang; Chen, Wei

2018-09-15

We report an aptamer-mediated colorimetric method for sensitive detection of chloramphenicol (CAP). The aptamer of CAP is immobilized by the hybridization with pre-immobilized capture probe in the microtiter plate. The horseradish peroxidase (HRP) is covalently attached to the aptamer by the biotin-streptavidin system for signal production. CAP will preferably bind with aptamer due to the high binding affinity, which attributes to the release of aptamer and HRP and thus, affects the optical signal intensity. Quantitative determination of CAP is successfully achieved in the wide range from 0.001 to 1000 ng/mL with detection limit of 0.0031 ng/mL, which is more sensitive than traditional immunoassays. This method is further validated by measuring the recovery of CAP spiked in two different food matrices (honey and fish). The aptamer-mediated colorimetric method can be a useful protocol for rapid and sensitive screening of CAP, and may be used as an alternative means for traditional immunoassays. Copyright © 2018 Elsevier Ltd. All rights reserved.

Transgenic Brassica rapa plants over-expressing eIF(iso)4E variants show broad-spectrum Turnip mosaic virus (TuMV) resistance.

PubMed

Kim, Jinhee; Kang, Won-Hee; Hwang, Jeena; Yang, Hee-Bum; Dosun, Kim; Oh, Chang-Sik; Kang, Byoung-Cheorl

2014-08-01

The protein-protein interaction between VPg (viral protein genome-linked) of potyviruses and eIF4E (eukaryotic initiation factor 4E) or eIF(iso)4E of their host plants is a critical step in determining viral virulence. In this study, we evaluated the approach of engineering broad-spectrum resistance in Chinese cabbage (Brassica rapa) to Turnip mosaic virus (TuMV), which is one of the most important potyviruses, by a systematic knowledge-based approach to interrupt the interaction between TuMV VPg and B. rapa eIF(iso)4E. The seven amino acids in the cap-binding pocket of eIF(iso)4E were selected on the basis of other previous results and comparison of protein models of cap-binding pockets, and mutated. Yeast two-hybrid assay and co-immunoprecipitation analysis demonstrated that W95L, K150L and W95L/K150E amino acid mutations of B. rapa eIF(iso)4E interrupted its interaction with TuMV VPg. All eIF(iso)4E mutants were able to complement an eIF4E-knockout yeast strain, indicating that the mutated eIF(iso)4E proteins retained their function as a translational initiation factor. To determine whether these mutations could confer resistance, eIF(iso)4E W95L, W95L/K150E and eIF(iso)4E wild-type were over-expressed in a susceptible Chinese cabbage cultivar. Evaluation of the TuMV resistance of T1 and T2 transformants demonstrated that the over-expression of the eIF(iso)4E mutant forms can confer resistance to multiple TuMV strains. These data demonstrate the utility of knowledge-based approaches for the engineering of broad-spectrum resistance in Chinese cabbage. © 2014 BSPP AND JOHN WILEY & SONS LTD.

Structure of RNA polymerase complex and genome within a dsRNA virus provides insights into the mechanisms of transcription and assembly.

PubMed

Wang, Xurong; Zhang, Fuxian; Su, Rui; Li, Xiaowu; Chen, Wenyuan; Chen, Qingxiu; Yang, Tao; Wang, Jiawei; Liu, Hongrong; Fang, Qin; Cheng, Lingpeng

2018-06-25

Most double-stranded RNA (dsRNA) viruses transcribe RNA plus strands within a common innermost capsid shell. This process requires coordinated efforts by RNA-dependent RNA polymerase (RdRp) together with other capsid proteins and genomic RNA. Here we report the near-atomic resolution structure of the RdRp protein VP2 in complex with its cofactor protein VP4 and genomic RNA within an aquareovirus capsid using 200-kV cryoelectron microscopy and symmetry-mismatch reconstruction. The structure of these capsid proteins enabled us to observe the elaborate nonicosahedral structure within the double-layered icosahedral capsid. Our structure shows that the RdRp complex is anchored at the inner surface of the capsid shell and interacts with genomic dsRNA and four of the five asymmetrically arranged N termini of the capsid shell proteins under the fivefold axis, implying roles for these N termini in virus assembly. The binding site of the RNA end at VP2 is different from the RNA cap binding site identified in the crystal structure of orthoreovirus RdRp λ3, although the structures of VP2 and λ3 are almost identical. A loop, which was thought to separate the RNA template and transcript, interacts with an apical domain of the capsid shell protein, suggesting a mechanism for regulating RdRp replication and transcription. A conserved nucleoside triphosphate binding site was localized in our RdRp cofactor protein VP4 structure, and interactions between the VP4 and the genomic RNA were identified.

Highly selective colorimetric bacteria sensing based on protein-capped nanoparticles.

PubMed

Qiu, Suyan; Lin, Zhenyu; Zhou, Yaomin; Wang, Donggen; Yuan, Lijuan; Wei, Yihua; Dai, Tingcan; Luo, Linguang; Chen, Guonan

2015-02-21

A rapid and cost-effective colorimetric sensor has been developed for the detection of bacteria (Bacillus subtilis was selected as an example). The sensor was designed to rely on lysozyme-capped AuNPs with the advantages of effective amplification and high specificity. In the sensing system, lysozyme was able to bind strongly to Bacillus subtilis, which effectively induced a color change of the solution from light purple to purplish red. The lowest concentration of Bacillus subtilis detectable by the naked eye was 4.5 × 10(3) colony-forming units (CFU) mL(-1). Similar results were discernable from UV-Vis absorption measurements. A good specificity was observed through a statistical analysis method using the SPSS software (version 17.0). This simple colorimetric sensor may therefore be a rapid and specific method for a bacterial detection assay in complex samples.

Induction of viral, 7-methyl-guanosine cap-independent translation and oncolysis by mitogen-activated protein kinase-interacting kinase-mediated effects on the serine/arginine-rich protein kinase.

PubMed

Brown, Michael C; Bryant, Jeffrey D; Dobrikova, Elena Y; Shveygert, Mayya; Bradrick, Shelton S; Chandramohan, Vidyalakshmi; Bigner, Darell D; Gromeier, Matthias

2014-11-01

Protein synthesis, the most energy-consuming process in cells, responds to changing physiologic priorities, e.g., upon mitogen- or stress-induced adaptations signaled through the mitogen-activated protein kinases (MAPKs). The prevailing status of protein synthesis machinery is a viral pathogenesis factor, particularly for plus-strand RNA viruses, where immediate translation of incoming viral RNAs shapes host-virus interactions. In this study, we unraveled signaling pathways centered on the ERK1/2 and p38α MAPK-interacting kinases MNK1/2 and their role in controlling 7-methyl-guanosine (m(7)G) "cap"-independent translation at enterovirus type 1 internal ribosomal entry sites (IRESs). Activation of Raf-MEK-ERK1/2 signals induced viral IRES-mediated translation in a manner dependent on MNK1/2. This effect was not due to MNK's known functions as eukaryotic initiation factor (eIF) 4G binding partner or eIF4E(S209) kinase. Rather, MNK catalytic activity enabled viral IRES-mediated translation/host cell cytotoxicity through negative regulation of the Ser/Arg (SR)-rich protein kinase (SRPK). Our investigations suggest that SRPK activity is a major determinant of type 1 IRES competency, host cell cytotoxicity, and viral proliferation in infected cells. We are targeting unfettered enterovirus IRES activity in cancer with PVSRIPO, the type 1 live-attenuated poliovirus (PV) (Sabin) vaccine containing a human rhinovirus type 2 (HRV2) IRES. A phase I clinical trial of PVSRIPO with intratumoral inoculation in patients with recurrent glioblastoma (GBM) is showing early promise. Viral translation proficiency in infected GBM cells is a core requirement for the antineoplastic efficacy of PVSRIPO. Therefore, it is critically important to understand the mechanisms controlling viral cap-independent translation in infected host cells. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Porcine circovirus-2 capsid protein induces cell death in PK15 cells

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

Walia, Rupali; Dardari, Rkia, E-mail: rdardari@ucalgary.ca; Chaiyakul, Mark

Studies have shown that Porcine circovirus (PCV)-2 induces apoptosis in PK15 cells. Here we report that cell death is induced in PCV2b-infected PK15 cells that express Capsid (Cap) protein and this effect is enhanced in interferon gamma (IFN-γ)-treated cells. We further show that transient PCV2a and 2b-Cap protein expression induces cell death in PK15 cells at rate similar to PCV2 infection, regardless of Cap protein localization. These data suggest that Cap protein may have the capacity to trigger different signaling pathways involved in cell death. Although further investigation is needed to gain deeper insights into the nature of the pathwaysmore » involved in Cap-induced cell death, this study provides evidence that PCV2-induced cell death in kidney epithelial PK15 cells can be mapped to the Cap protein and establishes the need for future research regarding the role of Cap-induced cell death in PCV2 pathogenesis. - Highlights: • IFN-γ enhances PCV2 replication that leads to cell death in PK15 cells. • IFN-γ enhances nuclear localization of the PCV2 Capsid protein. • Transient PCV2a and 2b-Capsid protein expression induces cell death. • Cell death is not dictated by specific Capsid protein sub-localization.« less

Tropomyosin modulates erythrocyte membrane stability

PubMed Central

An, Xiuli; Salomao, Marcela; Guo, Xinhua; Gratzer, Walter; Mohandas, Narla

2007-01-01

The ternary complex of spectrin, actin, and 4.1R (human erythrocyte protein 4.1) defines the nodes of the erythrocyte membrane skeletal network and is inseparable from membrane stability under mechanical stress. These junctions also contain tropomyosin (TM) and the other actin-binding proteins, adducin, protein 4.9, tropomodulin, and a small proportion of capZ, the functions of which are poorly defined. Here, we have examined the consequences of selective elimination of TM from the membrane. We have shown that the mechanical stability of the membranes of resealed ghosts devoid of TM is grossly, but reversibly, impaired. That the decreased membrane stability of TM-depleted membranes is the result of destabilization of the ternary complex of the network junctions is demonstrated by the strongly facilitated entry into the junctions in situ of a β-spectrin peptide, containing the actin- and 4.1R-binding sites, after extraction of the TM. The stabilizing effect of TM is highly specific, in that it is only the endogenous isotype, and not the slightly longer muscle TM that can bind to the depleted membranes and restore their mechanical stability. These findings have enabled us identify a function for TM in elevating the mechanical stability of erythrocyte membranes by stabilizing the spectrin-actin-4.1R junctional complex. PMID:17008534

Structure of 2C-Methyl-D-erythritol-2,4-cyclodiphosphate Synthase from Shewanella oneidensis at 1.6 angstrom: Identification of Farnesyl pyrophosphate Trapped in a Hydrophobic Cavity

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

Ni, Shuisong; Robinson, Howard; Marsing, Gregory C.

2004-11-01

1. Introduction Enzymes in the non-mevalonate pathway for isoprenoid synthesis have gained recent attention because of their potential value as targets for antibiotic drug development. 2C-methyl-D-erythritol-2,4 cyclophosphate (MECDP) synthase is the fifth enzyme in the seven enzyme non-mevalonate pathway for synthesis of isopentenyl diphosphate. Four groups have published structures of MECDP synthase at resolutions varying from 1.6Å to 2.8Å, either in the presence or absence of substrate from Escherichia coli (Richard et al., 2002; Kemp et al., 2002; Steinbacher et al., 2002) or from Thermus thermophilus (Kishida et al., 2003). Among these structures, the protein always exists as a homotrimermore » either with a crystallographic or a non-crystallographic three-fold symmetry axis and an active site formed in a cleft between adjacent monomers. While the overall shape of the proteins is highly similar among these structures, each of the four reported structures contain different combinations of metal ions in the active site including a Zn2+ ion only (Steinbacher et al., 2002), a Mn2+ ion only (Richard et al., 2002), Zn2+ and Mn2+ ions (Kemp et al., 2002) or two Mg2+ ions (Kishida et al., 2003). Furthermore, two of the structures are reported to contain a hydrophobic channel along the three-fold symmetry axis that is capped by a cluster of three arginine side chains (one from each monomer) at one end of the cavity and a cluster of three glutamic acid side chains (one from each monomer) at the other side of the cavity. In a 1.8Å resolution structure, Kemp et al. (2002) reported a sulfate ion coordinated to the arginine cap and solvent trapped in a hydrophobic cavity. In a lower 2.8Å resolution structure, Richard et al. (2002) concluded that geranyl diphosphate, GPP, was most likely trapped by the arginine cap and hydrophobic cavity (Richard et al., 2002), however, the low resolution of the data together with the presence of the crystallographic symmetry axis prohibited a definitive analysis of the identity and mode of binding of the bound molecule. Kishida et al. (2003) reported that no cavity existed in a 1.6Å structure of the SO3437 homolog from Thermus thermophilus, presumably due to tighter packing of the protein from the thermophilic organism. Steinbacher et al. (2002) make no description of a hydrophobic cavity in a lower resolution (2.5-3.2Å) of the Escherichia coli protein. Here, we report a high-resolution (1.6Å) structure of MECDP synthase from Shewanella oneidensis in the absence of substrate in the active site. We provide unambiguous data that confirms the presence of Zn2+ in one of the metal binding sites and observe what appears to be farnesyl diphosphate (FPP) bound in the hydrophobic cavity along the non-crystallographic three-fold symmetry axis of the homotrimer. The high-resolution structure clarifies the mode of binding of the pyrophosphate of FPP in the arginine cluster that caps the hydrophobic cavity.« less

Novel Broad Spectrum Inhibitors Targeting the Flavivirus Methyltransferase

PubMed Central

Liu, Binbin; Banavali, Nilesh K.; Jones, Susan A.; Zhang, Jing; Li, Zhong; Kramer, Laura D.; Li, Hongmin

2015-01-01

The flavivirus methyltransferase (MTase) is an essential enzyme that sequentially methylates the N7 and 2’-O positions of the viral RNA cap, using S-adenosyl-L-methionine (SAM) as a methyl donor. We report here that small molecule compounds, which putatively bind to the SAM-binding site of flavivirus MTase and inhibit its function, were identified by using virtual screening. In vitro methylation experiments demonstrated significant MTase inhibition by 13 of these compounds, with the most potent compound displaying sub-micromolar inhibitory activity. The most active compounds showed broad spectrum activity against the MTase proteins of multiple flaviviruses. Two of these compounds also exhibited low cytotoxicity and effectively inhibited viral replication in cell-based assays, providing further structural insight into flavivirus MTase inhibition. PMID:26098995

Flightless I interacts with NMMIIA to promote cell extension formation, which enables collagen remodeling

PubMed Central

Arora, Pamma D.; Wang, Yongqiang; Bresnick, Anne; Janmey, Paul A.; McCulloch, Christopher A.

2015-01-01

We examined the role of the actin-capping protein flightless I (FliI) in collagen remodeling by mouse fibroblasts. FliI-overexpressing cells exhibited reduced spreading on collagen but formed elongated protrusions that stained for myosin10 and fascin and penetrated pores of collagen-coated membranes. Inhibition of Cdc42 blocked formation of cell protrusions. In FliI-knockdown cells, transfection with constitutively active Cdc42 did not enable protrusion formation. FliI-overexpressing cells displayed increased uptake and degradation of exogenous collagen and strongly compacted collagen fibrils, which was blocked by blebbistatin. Mass spectrometry analysis of FliI immunoprecipitates showed that FliI associated with nonmuscle myosin IIA (NMMIIA), which was confirmed by immunoprecipitation. GFP-FliI colocalized with NMMIIA at cell protrusions. Purified FliI containing gelsolin-like domains (GLDs) 1–6 capped actin filaments efficiently, whereas FliI GLD 2–6 did not. Binding assays showed strong interaction of purified FliI protein (GLD 1–6) with the rod domain of NMMIIA (kD = 0.146 μM), whereas FliI GLD 2–6 showed lower binding affinity (kD = 0.8584 μM). Cells expressing FliI GLD 2–6 exhibited fewer cell extensions, did not colocalize with NMMIIA, and showed reduced collagen uptake compared with cells expressing FliI GLD 1–6. We conclude that FliI interacts with NMMIIA to promote cell extension formation, which enables collagen remodeling in fibroblasts. PMID:25877872

Human cytomegalovirus TRS1 protein associates with the 7-methylguanosine mRNA cap and facilitates translation

PubMed Central

Ziehr, Benjamin; Lenarcic, Erik; Vincent, Heather A.; Cecil, Chad; Garcia, Benjamin; Shenk, Thomas; Moorman, Nathaniel J.

2015-01-01

Viruses rely on the host translation machinery for the synthesis of viral proteins. Human cells have evolved sensors that recognize viral RNAs and inhibit mRNA translation in order to limit virus replication. Understanding how viruses manipulate the host translation machinery to gain access to ribosomes and disable the antiviral response is therefore a critical aspect of the host:pathogen interface. In this study we used a proteomics approach to identify human cytomegalovirus (HCMV) proteins that might contribute to viral mRNA translation. The HCMV TRS1 protein (pTRS1) associated with the 7-methylguanosine (m7G) mRNA cap, increased the total level of protein synthesis, and co-localized with mRNAs undergoing translation initiation during infection. pTRS1 stimulated translation of a non-viral reporter gene and increased the translation of a reporter containing an HCMV 5’ untranslated region (5’UTR) to a greater extent. The preferential effect of pTRS1 on translation of an mRNA containing a viral 5’UTR required the pTRS1 RNA and PKR binding domains, and was likely the result of PKR inhibition. However pTRS1 also stimulated the total level of protein synthesis and translation directed by an HCMV 5’UTR in cells lacking PKR. Thus our results demonstrate that pTRS1 stimulates translation through both PKR-dependent and PKR-independent mechanisms. PMID:25894605

Iodine-131-labeled, transferrin-capped polypyrrole nanoparticles for tumor-targeted synergistic photothermal-radioisotope therapy.

PubMed

Song, Xuejiao; Liang, Chao; Feng, Liangzhu; Yang, Kai; Liu, Zhuang

2017-08-22

Combining different therapeutic functions within single tumor-targeted nanoscale delivery systems is promising to overcome the limitations of conventional cancer therapies. Herein, transferrin that recognizes transferrin receptors up-regulated on tumor cells is pre-labeled with iodine-131 ( 131 I) and then utilized as the stabilizer in the fabrication of polypyrrole (PPy) nanoparticles. The obtained transferrin-capped PPy@Tf- 131 I nanoparticles could be used for tumor-targeted radioisotope therapy (RIT) and photothermal therapy (PTT), by employing beta-emission from 131 I and the intrinsic high near-infrared (NIR) absorbance of PPy, respectively. Owing to the transferrin-mediated tumor targeting, PPy@Tf- 131 I nanoparticles exhibit obviously enhanced in vitro cancer cell binding and in vivo tumor uptake compared to its non-targeting counterpart. The combined RIT and PTT based on PPy@Tf- 131 I nanoparticles is then conducted, achieving a remarkable synergistic therapeutic effect. This work thus demonstrates a rather simple one-step approach to fabricate tumor-targeting nanoparticles based on protein-capped conjugated polymers, promising for combination cancer therapy with great efficacy and high safety.

A short conserved motif in ALYREF directs cap- and EJC-dependent assembly of export complexes on spliced mRNAs

PubMed Central

Gromadzka, Agnieszka M.; Steckelberg, Anna-Lena; Singh, Kusum K.; Hofmann, Kay; Gehring, Niels H.

2016-01-01

The export of messenger RNAs (mRNAs) is the final of several nuclear posttranscriptional steps of gene expression. The formation of export-competent mRNPs involves the recruitment of export factors that are assumed to facilitate transport of the mature mRNAs. Using in vitro splicing assays, we show that a core set of export factors, including ALYREF, UAP56 and DDX39, readily associate with the spliced RNAs in an EJC (exon junction complex)- and cap-dependent manner. In order to elucidate how ALYREF and other export adaptors mediate mRNA export, we conducted a computational analysis and discovered four short, conserved, linear motifs present in RNA-binding proteins. We show that mutation in one of the new motifs (WxHD) in an unstructured region of ALYREF reduced RNA binding and abolished the interaction with eIF4A3 and CBP80. Additionally, the mutation impaired proper localization to nuclear speckles and export of a spliced reporter mRNA. Our results reveal important details of the orchestrated recruitment of export factors during the formation of export competent mRNPs. PMID:26773052

Use of antibodies specific to defined regions of scorpion. cap alpha. -toxin to study its interaction with its receptor site on the sodium channel

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

Ayeb, M.E.; Bahraoui, E.M.; Granier, C.

1986-10-21

Five antibody populations selected by immunoaffinity chromatography for the specificity toward various regions of toxin II of the scorpion Androctonus australis Hector were used to probe the interaction of this protein with its receptor site on the sodium channel. These studies indicate that two antigenic sites, one located around the disulfide bridge 12-63 and one encompassing residues 50-59, are involved in the molecular mechanisms of toxicity neutralization. Fab fragments specific to the region around disulfide bridge 12-63 inhibit binding of the /sup 125/I-labeled toxin to its receptor site. Also, these two antigenic regions are inaccessible to the antibodies when themore » toxin is bound to its receptor site. In contrast, the two other antigenic sites encompassing the only ..cap alpha..-helix region (residues 23-32) and a ..beta..-turn structure (residues 32-35) are accessible to the respective antibodies when the toxin is bound to its receptor. Together, these data support the recent proposal that a region made of residues that are conserved in the scorpion toxin family is involved in the binding of the toxin to the receptor.« less

p130Cas-associated Protein (p140Cap) as a New Tyrosine-phosphorylated Protein Involved in Cell Spreading

PubMed Central

Di Stefano, Paola; Cabodi, Sara; Erba, Elisabetta Boeri; Margaria, Valentina; Bergatto, Elena; Giuffrida, Maria Gabriella; Silengo, Lorenzo; Tarone, Guido; Turco, Emilia; Defilippi, Paola

2004-01-01

Integrin-mediated cell adhesion stimulates a cascade of signaling pathways that control cell proliferation, migration, and survival, mostly through tyrosine phosphorylation of signaling molecules. p130Cas, originally identified as a major substrate of v-Src, is a scaffold molecule that interacts with several proteins and mediates multiple cellular events after cell adhesion and mitogen treatment. Here, we describe a novel p130Cas-associated protein named p140Cap (Cas-associated protein) as a new tyrosine phosphorylated molecule involved in integrin- and epidermal growth factor (EGF)-dependent signaling. By affinity chromatography of human ECV304 cell extracts on a MBP-p130Cas column followed by mass spectrometry matrix-assisted laser desorption ionization/time of flight analysis, we identified p140Cap as a protein migrating at 140 kDa. We detected its expression in human, mouse, and rat cells and in different mouse tissues. Endogenous and transfected p140Cap proteins coimmunoprecipitate with p130Cas in ECV304 and in human embryonic kidney 293 cells and associate with p130Cas through their carboxy-terminal region. By immunofluorescence analysis, we demonstrated that in ECV304 cells plated on fibronectin, the endogenous p140Cap colocalizes with p130Cas in the perinuclear region as well as in lamellipodia. In addition p140Cap codistributes with cortical actin and actin stress fibers but not with focal adhesions. We also show that p140Cap is tyrosine phosphorylated within 15 min of cell adhesion to integrin ligands. p140Cap tyrosine phosphorylation is also induced in response to EGF through an EGF receptor dependent-mechanism. Interestingly expression of p140Cap in NIH3T3 and in ECV304 cells delays the onset of cell spreading in the early phases of cell adhesion to fibronectin. Therefore, p140Cap is a novel protein associated with p130Cas and actin cytoskeletal structures. Its tyrosine phosphorylation by integrin-mediated adhesion and EGF stimulation and its involvement in cell spreading on matrix proteins suggest that p140Cap plays a role in controlling actin cytoskeleton organization in response to adhesive and growth factor signaling. PMID:14657239

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

Schellenberg, Matthew J; Appel, C Denise; Adhikari, Sanjay

The topoisomerase II (topo II) DNA incision-and-ligation cycle can be poisoned (for example following treatment with cancer chemotherapeutics) to generate cytotoxic DNA double-strand breaks (DSBs) with topo II covalently conjugated to DNA. Tyrosyl-DNA phosphodiesterase 2 (Tdp2) protects genomic integrity by reversing 5'-phosphotyrosyl–linked topo II–DNA adducts. Here, X-ray structures of mouse Tdp2–DNA complexes reveal that Tdp2 β–2-helix–β DNA damage–binding 'grasp', helical 'cap' and DNA lesion–binding elements fuse to form an elongated protein-DNA conjugate substrate-interaction groove. The Tdp2 DNA-binding surface is highly tailored for engagement of 5'-adducted single-stranded DNA ends and restricts nonspecific endonucleolytic or exonucleolytic processing. Structural, mutational and functional analysesmore » support a single–metal ion catalytic mechanism for the exonuclease-endonuclease-phosphatase (EEP) nuclease superfamily and establish a molecular framework for targeted small-molecule blockade of Tdp2-mediated resistance to anticancer topoisomerase drugs.« less

Effects of light on protein patterns in gravitropically stimulated root caps of corn

NASA Technical Reports Server (NTRS)

Feldman, L. J.; Gildow, V.

1984-01-01

In certain cultivars of corn (Zea mays var. Merit), light stimulates gravitropic bending of the root by influencing events in the root cap. In this paper, we report on changes in root cap proteins which occur as a result of the light treatment and single out specific proteins as potentially having a role in mediating the gravitropic response. For this work, we have used root caps maintained aseptically in culture media supplemented with auxin. If auxin is deleted from the culture medium, the protein profiles observed following illumination differ from that seen in caps provided light while in auxin-supplemented media. We also report that several of the proteins for which synthesis is stimulated by light appear to turn over rapidly, usually within 0.5 hour of formation.

The Rabies Virus L Protein Catalyzes mRNA Capping with GDP Polyribonucleotidyltransferase Activity.

PubMed

Ogino, Minako; Ito, Naoto; Sugiyama, Makoto; Ogino, Tomoaki

2016-05-21

The large (L) protein of rabies virus (RABV) plays multiple enzymatic roles in viral RNA synthesis and processing. However, none of its putative enzymatic activities have been directly demonstrated in vitro. In this study, we expressed and purified a recombinant form of the RABV L protein and verified its guanosine 5'-triphosphatase and GDP polyribonucleotidyltransferase (PRNTase) activities, which are essential for viral mRNA cap formation by the unconventional mechanism. The RABV L protein capped 5'-triphosphorylated but not 5'-diphosphorylated RABV mRNA-start sequences, 5'-AACA(C/U), with GDP to generate the 5'-terminal cap structure G(5')ppp(5')A. The 5'-AAC sequence in the substrate RNAs was found to be strictly essential for RNA capping with the RABV L protein. Furthermore, site-directed mutagenesis showed that some conserved amino acid residues (G1112, T1170, W1201, H1241, R1242, F1285, and Q1286) in the PRNTase motifs A to E of the RABV L protein are required for cap formation. These findings suggest that the putative PRNTase domain in the RABV L protein catalyzes the rhabdovirus-specific capping reaction involving covalent catalysis of the pRNA transfer to GDP, thus offering this domain as a target for developing anti-viral agents.

High-Pressure NMR and SAXS Reveals How Capping Modulates Folding Cooperativity of the pp32 Leucine-rich Repeat Protein.

PubMed

Zhang, Yi; Berghaus, Melanie; Klein, Sean; Jenkins, Kelly; Zhang, Siwen; McCallum, Scott A; Morgan, Joel E; Winter, Roland; Barrick, Doug; Royer, Catherine A

2018-04-27

Many repeat proteins contain capping motifs, which serve to shield the hydrophobic core from solvent and maintain structural integrity. While the role of capping motifs in enhancing the stability and structural integrity of repeat proteins is well documented, their contribution to folding cooperativity is not. Here we examined the role of capping motifs in defining the folding cooperativity of the leucine-rich repeat protein, pp32, by monitoring the pressure- and urea-induced unfolding of an N-terminal capping motif (N-cap) deletion mutant, pp32-∆N-cap, and a C-terminal capping motif destabilization mutant pp32-Y131F/D146L, using residue-specific NMR and small-angle X-ray scattering. Destabilization of the C-terminal capping motif resulted in higher cooperativity for the unfolding transition compared to wild-type pp32, as these mutations render the stability of the C-terminus similar to that of the rest of the protein. In contrast, deletion of the N-cap led to strong deviation from two-state unfolding. In both urea- and pressure-induced unfolding, residues in repeats 1-3 of pp32-ΔN-cap lost their native structure first, while the C-terminal half was more stable. The residue-specific free energy changes in all regions of pp32-ΔN-cap were larger in urea compared to high pressure, indicating a less cooperative destabilization by pressure. Moreover, in contrast to complete structural disruption of pp32-ΔN-cap at high urea concentration, its pressure unfolded state remained compact. The contrasting effects of the capping motifs on folding cooperativity arise from the differential local stabilities of pp32, whereas the contrasting effects of pressure and urea on the pp32-ΔN-cap variant arise from their distinct mechanisms of action. Copyright © 2018 Elsevier Ltd. All rights reserved.

A region rich in aspartic acid, arginine, tyrosine, and glycine (DRYG) mediates eukaryotic initiation factor 4B (eIF4B) self-association and interaction with eIF3.

PubMed Central

Méthot, N; Song, M S; Sonenberg, N

1996-01-01

The binding of mRNA to the ribosome is mediated by eukaryotic initiation factors eukaryotic initiation factor 4F (eIF4F), eIF4B, eIF4A, and eIF3, eIF4F binds to the mRNA cap structure and, in combination with eIF4B, is believed to unwind the secondary structure in the 5' untranslated region to facilitate ribosome binding. eIF3 associates with the 40S ribosomal subunit prior to mRNA binding. eIF4B copurifies with eIF3 and eIF4F through several purification steps, suggesting the involvement of a multisubunit complex during translation initiation. To understand the mechanism by which eIF4B promotes 40S ribosome binding to the mRNA, we studied its interactions with partner proteins by using a filter overlay (protein-protein [far Western]) assay and the two-hybrid system. In this report, we show that eIF4B self-associates and also interacts directly with the p170 subunit of eIF3. A region rich in aspartic acid, arginine, tyrosine, and glycine, termed the DRYG domain, is sufficient for self-association of eIF4B, both in vitro and in vivo, and for interaction with the p170 subunit of eIF3. These experiments suggest that eIF4B participates in mRNA-ribosome binding by acting as an intermediary between the mRNA and eIF3, via a direct interaction with the p170 subunit of eIF3. PMID:8816444

Structural and Biophysical Characterization of the Mycobacterium tuberculosis Protein Rv0577, a Protein Associated with Neutral Red Staining of Virulent Tuberculosis Strains and Homologue of the Streptomyces coelicolor Protein KbpA [Structural and Biophysical Characterization of Rv0577, a Protein Associated with Neutral Red Staining of Virulent Strains of Mycobacterium tuberculosis and homolog of the Streptomyces coelicolor protein KbpA

DOE PAGES

Buchko, Garry W.; Echols, Nathaniel; Flynn, E. Megan; ...

2017-07-10

Mycobacterium tuberculosis protein Rv0577 is a prominent antigen in tuberculosis patients, the component responsible for neutral red staining of virulent strains of M. tuberculosis, a putative component in a methylglyoxal detoxification pathway, and an agonist of toll-like receptor 2. It also has an amino acid sequence that is 36% identical to that of Streptomyces coelicolor AfsK-binding protein A (KbpA), a component in the complex secondary metabolite pathways in the Streptomyces genus. To gain insight into the biological function of Rv0577 and the family of KpbA kinase regulators, the crystal structure for Rv0577 was determined to a resolution of 1.75 Å,more » binding properties with neutral red and deoxyadenosine were surveyed, backbone dynamics were measured, and thermal stability was assayed by circular dichroism spectroscopy. The protein is composed of four approximate repeats with a βαβββ topology arranged radially in consecutive pairs to form two continuous eight-strand β-sheets capped on both ends with an α-helix. The two β-sheets intersect in the center at roughly a right angle and form two asymmetric deep “saddles” that may serve to bind ligands. Nuclear magnetic resonance chemical shift perturbation experiments show that neutral red and deoxyadenosine bind to Rv0577. Binding to deoxyadenosine is weaker with an estimated dissociation constants of 4.1 ± 0.3 mM for saddle 1. Heteronuclear steady-state { 1H}– 15N nuclear Overhauser effect, T 1, and T 2 values were generally uniform throughout the sequence with only a few modest pockets of differences. As a result, circular dichroism spectroscopy characterization of the thermal stability of Rv0577 indicated irreversible unfolding upon heating with an estimated melting temperature of 56 °C.« less

Structural and Biophysical Characterization of the Mycobacterium tuberculosis Protein Rv0577, a Protein Associated with Neutral Red Staining of Virulent Tuberculosis Strains and Homologue of the Streptomyces coelicolor Protein KbpA [Structural and Biophysical Characterization of Rv0577, a Protein Associated with Neutral Red Staining of Virulent Strains of Mycobacterium tuberculosis and homolog of the Streptomyces coelicolor protein KbpA

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

Buchko, Garry W.; Echols, Nathaniel; Flynn, E. Megan

Mycobacterium tuberculosis protein Rv0577 is a prominent antigen in tuberculosis patients, the component responsible for neutral red staining of virulent strains of M. tuberculosis, a putative component in a methylglyoxal detoxification pathway, and an agonist of toll-like receptor 2. It also has an amino acid sequence that is 36% identical to that of Streptomyces coelicolor AfsK-binding protein A (KbpA), a component in the complex secondary metabolite pathways in the Streptomyces genus. To gain insight into the biological function of Rv0577 and the family of KpbA kinase regulators, the crystal structure for Rv0577 was determined to a resolution of 1.75 Å,more » binding properties with neutral red and deoxyadenosine were surveyed, backbone dynamics were measured, and thermal stability was assayed by circular dichroism spectroscopy. The protein is composed of four approximate repeats with a βαβββ topology arranged radially in consecutive pairs to form two continuous eight-strand β-sheets capped on both ends with an α-helix. The two β-sheets intersect in the center at roughly a right angle and form two asymmetric deep “saddles” that may serve to bind ligands. Nuclear magnetic resonance chemical shift perturbation experiments show that neutral red and deoxyadenosine bind to Rv0577. Binding to deoxyadenosine is weaker with an estimated dissociation constants of 4.1 ± 0.3 mM for saddle 1. Heteronuclear steady-state { 1H}– 15N nuclear Overhauser effect, T 1, and T 2 values were generally uniform throughout the sequence with only a few modest pockets of differences. As a result, circular dichroism spectroscopy characterization of the thermal stability of Rv0577 indicated irreversible unfolding upon heating with an estimated melting temperature of 56 °C.« less

Design, production and molecular structure of a new family of artificial alpha-helicoidal repeat proteins (αRep) based on thermostable HEAT-like repeats.

PubMed

Urvoas, Agathe; Guellouz, Asma; Valerio-Lepiniec, Marie; Graille, Marc; Durand, Dominique; Desravines, Danielle C; van Tilbeurgh, Herman; Desmadril, Michel; Minard, Philippe

2010-11-26

Repeat proteins have a modular organization and a regular architecture that make them attractive models for design and directed evolution experiments. HEAT repeat proteins, although very common, have not been used as a scaffold for artificial proteins, probably because they are made of long and irregular repeats. Here, we present and validate a consensus sequence for artificial HEAT repeat proteins. The sequence was defined from the structure-based sequence analysis of a thermostable HEAT-like repeat protein. Appropriate sequences were identified for the N- and C-caps. A library of genes coding for artificial proteins based on this sequence design, named αRep, was assembled using new and versatile methodology based on circular amplification. Proteins picked randomly from this library are expressed as soluble proteins. The biophysical properties of proteins with different numbers of repeats and different combinations of side chains in hypervariable positions were characterized. Circular dichroism and differential scanning calorimetry experiments showed that all these proteins are folded cooperatively and are very stable (T(m) >70 °C). Stability of these proteins increases with the number of repeats. Detailed gel filtration and small-angle X-ray scattering studies showed that the purified proteins form either monomers or dimers. The X-ray structure of a stable dimeric variant structure was solved. The protein is folded with a highly regular topology and the repeat structure is organized, as expected, as pairs of alpha helices. In this protein variant, the dimerization interface results directly from the variable surface enriched in aromatic residues located in the randomized positions of the repeats. The dimer was crystallized both in an apo and in a PEG-bound form, revealing a very well defined binding crevice and some structure flexibility at the interface. This fortuitous binding site could later prove to be a useful binding site for other low molecular mass partners. Copyright © 2010 Elsevier Ltd. All rights reserved.

The N-terminal tropomyosin- and actin-binding sites are important for leiomodin 2's function.

PubMed

Ly, Thu; Moroz, Natalia; Pappas, Christopher T; Novak, Stefanie M; Tolkatchev, Dmitri; Wooldridge, Dayton; Mayfield, Rachel M; Helms, Gregory; Gregorio, Carol C; Kostyukova, Alla S

2016-08-15

Leiomodin is a potent actin nucleator related to tropomodulin, a capping protein localized at the pointed end of the thin filaments. Mutations in leiomodin-3 are associated with lethal nemaline myopathy in humans, and leiomodin-2-knockout mice present with dilated cardiomyopathy. The arrangement of the N-terminal actin- and tropomyosin-binding sites in leiomodin is contradictory and functionally not well understood. Using one-dimensional nuclear magnetic resonance and the pointed-end actin polymerization assay, we find that leiomodin-2, a major cardiac isoform, has an N-terminal actin-binding site located within residues 43-90. Moreover, for the first time, we obtain evidence that there are additional interactions with actin within residues 124-201. Here we establish that leiomodin interacts with only one tropomyosin molecule, and this is the only site of interaction between leiomodin and tropomyosin. Introduction of mutations in both actin- and tropomyosin-binding sites of leiomodin affected its localization at the pointed ends of the thin filaments in cardiomyocytes. On the basis of our new findings, we propose a model in which leiomodin regulates actin poly-merization dynamics in myocytes by acting as a leaky cap at thin filament pointed ends. © 2016 Ly, Moroz, et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

Recombinant Flagellin-Porcine Circovirus Type 2 Cap Fusion Protein Promotes Protective Immune Responses in Mice

PubMed Central

Zhang, Chunyan; Zhu, Shanshan; Wei, Li; Yan, Xu; Wang, Jing; Quan, Rong; She, Ruiping; Hu, Fengjiao; Liu, Jue

2015-01-01

The Cap protein of porcine circovirus type 2 (PCV2) that serves as a major host-protective immunogen was used to develop recombinant vaccines for control of PCV2-associated diseases. Growing research data have demonstrated the high effectiveness of flagellin as an adjuvant for humoral and cellular immune responses. Here, a recombinant protein was designed by fusing a modified version of bacterial flagellin to PCV2 Cap protein and expressed in a baculovirus system. When administered without adjuvant to BALB/c mice, the flagellin-Cap fusion protein elicited stronger PCV2-specific IgG antibody response, higher neutralizing antibody levels, milder histopathological changes and lower viremia, as well as higher secretion of cytokines such as TNF-α and IFN-γ that conferred better protection against virus challenge than those in the recombinant Cap alone-inoculated mice. These results suggest that the recombinant Cap protein when fused to flagellin could elicit better humoral and cellular immune responses against PCV2 infection in a mouse model, thereby acting as an attractive candidate vaccine for control of the PCV2-associated diseases. PMID:26070075

Kinetics of antibody-induced modulation of respiratory syncytial virus antigens in a human epithelial cell line

PubMed Central

Sarmiento, Rosa E; Tirado, Rocio G; Valverde, Laura E; Gómez-Garcia, Beatriz

2007-01-01

Background The binding of viral-specific antibodies to cell-surface antigens usually results in down modulation of the antigen through redistribution of antigens into patches that subsequently may be internalized by endocytosis or may form caps that can be expelled to the extracellular space. Here, by use of confocal-laser-scanning microscopy we investigated the kinetics of the modulation of respiratory syncytial virus (RSV) antigen by RSV-specific IgG. RSV-infected human epithelial cells (HEp-2) were incubated with anti-RSV polyclonal IgG and, at various incubation times, the RSV-cell-surface-antigen-antibody complexes (RSV Ag-Abs) and intracellular viral proteins were detected by indirect immunoflourescence. Results Interaction of anti-RSV polyclonal IgG with RSV HEp-2 infected cells induced relocalization and aggregation of viral glycoproteins in the plasma membrane formed patches that subsequently produced caps or were internalized through clathrin-mediated endocytosis participation. Moreover, the concentration of cell surface RSV Ag-Abs and intracellular viral proteins showed a time dependent cyclic variation and that anti-RSV IgG protected HEp-2 cells from viral-induced death. Conclusion The results from this study indicate that interaction between RSV cell surface proteins and specific viral antibodies alter the expression of viral antigens expressed on the cells surface and intracellular viral proteins; furthermore, interfere with viral induced destruction of the cell. PMID:17608950

Phosphatidylinositol 4,5-bisphosphate optical uncaging potentiates exocytosis

PubMed Central

Wierda, Keimpe DB; Pinheiro, Paulo S; Nadler, André; McCarthy, Anthony W; Ziomkiewicz, Iwona; Kruse, Martin; Reither, Gregor; Rettig, Jens; Lehmann, Martin; Haucke, Volker; Hille, Bertil

2017-01-01

Phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2] is essential for exocytosis. Classical ways of manipulating PI(4,5)P2 levels are slower than its metabolism, making it difficult to distinguish effects of PI(4,5)P2 from those of its metabolites. We developed a membrane-permeant, photoactivatable PI(4,5)P2, which is loaded into cells in an inactive form and activated by light, allowing sub-second increases in PI(4,5)P2 levels. By combining this compound with electrophysiological measurements in mouse adrenal chromaffin cells, we show that PI(4,5)P2 uncaging potentiates exocytosis and identify synaptotagmin-1 (the Ca2+ sensor for exocytosis) and Munc13-2 (a vesicle priming protein) as the relevant effector proteins. PI(4,5)P2 activation of exocytosis did not depend on the PI(4,5)P2-binding CAPS-proteins, suggesting that PI(4,5)P2 uncaging may bypass CAPS-function. Finally, PI(4,5)P2 uncaging triggered the rapid fusion of a subset of readily-releasable vesicles, revealing a rapid role of PI(4,5)P2 in fusion triggering. Thus, optical uncaging of signaling lipids can uncover their rapid effects on cellular processes and identify lipid effectors. PMID:29068313

Antibodies from a Human Survivor Define Sites of Vulnerability for Broad Protection Against Ebolaviruses

DTIC Science & Technology

2017-03-31

GP proteins from EBOV, BDBV, and SUDV by ELISA . We found that 72% of the mAbs were also able to recognize BDBV GP, whereas only 11% demonstrated...349 mAbs reacted with all three ebolavirus glycoproteins by ELISA (Figure S1B). Within this subset, 16 mAbs belonged to the glycan cap epitope group...Miller et al., 2012; Wang et al., 2016), we first examined the capacity of the GP base-binding NAbs to inhibit it in a competitive ELISA (Figure 5A

Characterisation of a DNA sequence element that directs Dictyostelium stalk cell-specific gene expression.

PubMed

Ceccarelli, A; Zhukovskaya, N; Kawata, T; Bozzaro, S; Williams, J

2000-12-01

The ecmB gene of Dictyostelium is expressed at culmination both in the prestalk cells that enter the stalk tube and in ancillary stalk cell structures such as the basal disc. Stalk tube-specific expression is regulated by sequence elements within the cap-site proximal part of the promoter, the stalk tube (ST) promoter region. Dd-STATa, a member of the STAT transcription factor family, binds to elements present in the ST promoter-region and represses transcription prior to entry into the stalk tube. We have characterised an activatory DNA sequence element, that lies distal to the repressor elements and that is both necessary and sufficient for expression within the stalk tube. We have mapped this activator to a 28 nucleotide region (the 28-mer) within which we have identified a GA-containing sequence element that is required for efficient gene transcription. The Dd-STATa protein binds to the 28-mer in an in vitro binding assay, and binding is dependent upon the GA-containing sequence. However, the ecmB gene is expressed in a Dd-STATa null mutant, therefore Dd-STATa cannot be responsible for activating the 28-mer in vivo. Instead, we identified a distinct 28-mer binding activity in nuclear extracts from the Dd-STATa null mutant, the activity of this GA binding activity being largely masked in wild type extracts by the high affinity binding of the Dd-STATa protein. We suggest, that in addition to the long range repression exerted by binding to the two known repressor sites, Dd-STATa inhibits transcription by direct competition with this putative activator for binding to the GA sequence.

Importance of α–helix N–capping motif in stabilization of ββα fold

PubMed Central

Koscielska-Kasprzak, Katarzyna; Cierpicki, Tomasz; Otlewski, Jacek

2003-01-01

FSD-1 (full sequence design 1) is a protein folded in a ββα motif, designed on the basis of the second zinc finger domain of Zif268 by a substitution of its metal coordination site with a hydrophobic core. In this work, we analyzed the possibility of introducing the DNA recognition motif of the template zinc finger (S13RSDH17) into FSD-1 sequence in order to obtain a small DNA-binding module devoid of cross-link(s) or metal cofactors. The hybrid protein was unfolded, as judged by CD and NMR criteria. To reveal the role of each of the five amino acids, which form the N-capping motif of the α-helix, we analyzed conformational and stability properties of eight FSD-1 mutants. We used a shielded methyl group of Leu 18 and a CD signal at 215 nm as a convenient measure of the folded state. Glu 17→His substitution at the N3 in S13NEKE17 variant decreased the folded structure content from 90% to 25% (equivalent to 1.8 kcal • mole−1 destabilization) by disruption of N-capping interactions, and had the most significant effect among single mutants studied here. The Ncap Asn 14 substitution with Arg considerably decreased stability, reducing structure content from 90% to 40% (1.4 kcal • mole−1 destabilization) by disruption of a helix-capping hydrogen bond and destabilization of a helix macrodipole. The N1 Glu 15→Ser mutation also produced a considerable effect (1.0 kcal • mole−1 destabilization), again emphasizing the significance of electrostatic interactions in α-helix stabilization. PMID:12761399

Role of p70S6K1-mediated phosphorylation of eIF4B and PDCD4 proteins in the regulation of protein synthesis.

PubMed

Dennis, Michael D; Jefferson, Leonard S; Kimball, Scot R

2012-12-14

Modulation of mRNA binding to the 40 S ribosomal subunit during translation initiation controls not only global rates of protein synthesis but also regulates the pattern of protein expression by allowing for selective inclusion, or exclusion, of mRNAs encoding particular proteins from polysomes. The mRNA binding step is modulated by signaling through a protein kinase known as the mechanistic target of rapamycin complex 1 (mTORC1). mTORC1 directly phosphorylates the translational repressors eIF4E binding proteins (4E-BP) 1 and 2, releasing them from the mRNA cap binding protein eIF4E, thereby promoting assembly of the eIF4E·eIF4G complex. mTORC1 also phosphorylates the 70-kDa ribosomal protein S6 kinase 1 (p70S6K1), which subsequently phosphorylates eIF4B, and programmed cell death 4 (PDCD4), which sequesters eIF4A from the eIF4E·eIF4G complex, resulting in repressed translation of mRNAs with highly structured 5'-untranslated regions. In the present study, we compared the role of the 4E-BPs in the regulation of global rates of protein synthesis to that of eIF4B and PDCD4. We found that maintenance of eIF4E interaction with eIF4G was not by itself sufficient to sustain global rates of protein synthesis in the absence of mTORC1 signaling to p70S6K1; phosphorylation of both eIF4B and PDCD4 was additionally required. We also found that the interaction of eIF4E with eIF4G was maintained in the liver of fasted rats as well as in serum-deprived mouse embryo fibroblasts lacking both 4E-BP1 and 4E-BP2, suggesting that the interaction of eIF4G with eIF4E is controlled primarily through the 4E-BPs.

HnRNP-like proteins as post-transcriptional regulators.

PubMed

Yeap, Wan-Chin; Namasivayam, Parameswari; Ho, Chai-Ling

2014-10-01

Plant cells contain a diverse repertoire of RNA-binding proteins (RBPs) that coordinate a network of post-transcriptional regulation. RBPs govern diverse developmental processes by modulating the gene expression of specific transcripts. Recent gene annotation and RNA sequencing clearly showed that heterogeneous nuclear ribonucleoprotein (hnRNP)-like proteins which form a family of RBPs, are also expressed in higher plants and serve specific plant functions. In addition to their involvement in post-transcriptional regulation from mRNA capping to translation, they are also involved in telomere regulation, gene silencing and regulation in chloroplast. Here, we review the involvement of plant hnRNP-like proteins in post-transcription regulation of RNA processes and their functional roles in control of plant developmental processes especially plant-specific functions including flowering, chloroplastic-specific mRNA regulation, long-distance phloem transportation and plant responses to environmental stresses. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

Patterns of gene expression in atrophying skeletal muscles: response to food deprivation

NASA Technical Reports Server (NTRS)

Jagoe, R. Thomas; Lecker, Stewart H.; Gomes, Marcelo; Goldberg, Alfred L.

2002-01-01

During fasting and many systemic diseases, muscle undergoes rapid loss of protein and functional capacity. To define the transcriptional changes triggering muscle atrophy and energy conservation in fasting, we used cDNA microarrays to compare mRNAs from muscles of control and food-deprived mice. Expression of >94% of genes did not change, but interesting patterns emerged among genes that were differentially expressed: 1) mRNAs encoding polyubiquitin, ubiquitin extension proteins, and many (but not all) proteasome subunits increased, which presumably contributes to accelerated protein breakdown; 2) a dramatic increase in mRNA for the ubiquitin ligase, atrogin-1, but not most E3s; 3) a significant suppression of mRNA for myosin binding protein H (but not other myofibrillar proteins) and IGF binding protein 5, which may favor cell protein loss; 4) decreases in mRNAs for several glycolytic enzymes and phosphorylase kinase subunits, and dramatic increases in mRNAs for pyruvate dehydrogenase kinase 4 and glutamine synthase, which should promote glucose sparing and gluconeogenesis. During fasting, metallothionein mRNA increased dramatically, mRNAs for extracellular matrix components fell, and mRNAs that may favor cap-independent mRNA translation rose. Significant changes occurred in mRNAs for many growth-related proteins and transcriptional regulators. These transcriptional changes indicate a complex adaptive program that should favor protein degradation and suppress glucose oxidation in muscle. Similar analysis of muscles atrophying for other causes is allowing us to identify a set of atrophy-specific changes in gene expression.

Entropy as a Driver of Selectivity for Inhibitor Binding to Histone Deacetylase 6.

PubMed

Porter, Nicholas J; Wagner, Florence F; Christianson, David W

2018-05-18

Among the metal-dependent histone deacetylases, the class IIb isozyme HDAC6 is remarkable because of its role in the regulation of microtubule dynamics in the cytosol. Selective inhibition of HDAC6 results in microtubule hyperacetylation, leading to cell cycle arrest and apoptosis, which is a validated strategy for cancer chemotherapy and the treatment of other disorders. HDAC6 inhibitors generally consist of a Zn 2+ -binding group such as a hydroxamate, a linker, and a capping group; the capping group is a critical determinant of isozyme selectivity. Surprisingly, however, even "capless" inhibitors exhibit appreciable HDAC6 selectivity. To probe the chemical basis for this selectivity, we now report high-resolution crystal structures of HDAC6 complexed with capless cycloalkyl hydroxamate inhibitors 1-4. Each inhibitor hydroxamate group coordinates to the catalytic Zn 2+ ion with canonical bidentate geometry. Additionally, the olefin moieties of compounds 2 and 4 bind in an aromatic crevice between the side chains of F583 and F643. Reasoning that similar binding could be achieved in the representative class I isozyme HDAC8, we employed isothermal titration calorimetry to study the thermodynamics of inhibitor binding. These measurements indicate that the entropy of inhibitor binding is generally positive for binding to HDAC6 and negative for binding to HDAC8, resulting in ≤313-fold selectivity for binding to HDAC6 relative to HDAC8. Thus, favorable binding entropy contributes to HDAC6 selectivity. Notably, cyclohexenyl hydroxamate 2 represents a promising lead for derivatization with capping groups that may further enhance its impressive 313-fold thermodynamic selectivity for HDAC6 inhibition.

CAPS and Munc13: CATCHRs that SNARE Vesicles.

PubMed

James, Declan J; Martin, Thomas F J

2013-12-04

CAPS (Calcium-dependent Activator Protein for Secretion, aka CADPS) and Munc13 (Mammalian Unc-13) proteins function to prime vesicles for Ca(2+)-triggered exocytosis in neurons and neuroendocrine cells. CAPS and Munc13 proteins contain conserved C-terminal domains that promote the assembly of SNARE complexes for vesicle priming. Similarities of the C-terminal domains of CAPS/Munc13 proteins with Complex Associated with Tethering Containing Helical Rods domains in multi-subunit tethering complexes (MTCs) have been reported. MTCs coordinate multiple interactions for SNARE complex assembly at constitutive membrane fusion steps. We review aspects of these diverse tethering and priming factors to identify common operating principles.

Characteristic of the Oxidative Stress in Blood of Patients in Dependence of Community-Acquired Pneumonia Severity.

PubMed

Muravlyova, Larissa; Molotov-Luchankiy, Vilen; Bakirova, Ryszhan; Klyuyev, Dmitriy; Demidchik, Ludmila; Lee, Valentina

2016-03-15

At the present time the alternation of the oxidative metabolism is considered as one of the leading pathogenic mechanisms in the development and progression of community-acquired pneumonia (CAP). However the nature and direction of the oxidative protein changes in CAP patient's blood had been almost unexplored. To define oxidative and modified proteins in erythrocytes and blood plasma of CAP patients. Blood plasma and erythrocytes obtained from: 42 patients with moderate severity pneumonia, 12 patients with grave severity pneumonia and 32 healthy volunteers. Content of advanced oxidation protein products, malondialdehyde and reactive carbonyl derivatives were estimated as indicators of the oxidative stress and oxidative damage of proteins. In patients with grave severity the level of oxidative proteins and MDA in erythrocytes exceeded both: control values and similar meanings in CAP patients with moderate severity. The further growth of MDA in this group patients' blood plasma was observed, but the level of oxidative proteins decreased in comparison with those in CAP patients with moderate severity. To sum up, our derived data show, that injury of erythrocytes' redox-status and blood plasma components plays an essential role in development and progression CAP.

Iron Homeostasis and Nutritional Iron Deficiency123

PubMed Central

Theil, Elizabeth C.

2011-01-01

Nonheme food ferritin (FTN) iron minerals, nonheme iron complexes, and heme iron contribute to the balance between food iron absorption and body iron homeostasis. Iron absorption depends on membrane transporter proteins DMT1, PCP/HCP1, ferroportin (FPN), TRF2, and matriptase 2. Mutations in DMT1 and matriptase-2 cause iron deficiency; mutations in FPN, HFE, and TRF2 cause iron excess. Intracellular iron homeostasis depends on coordinated regulation of iron trafficking and storage proteins encoded in iron responsive element (IRE)-mRNA. The noncoding IRE-mRNA structures bind protein repressors, IRP1 or 2, during iron deficiency. Integration of the IRE-RNA in translation regulators (near the cap) or turnover elements (after the coding region) increases iron uptake (DMT1/TRF1) or decreases iron storage/efflux (FTN/FPN) when IRP binds. An antioxidant response element in FTN DNA binds Bach1, a heme-sensitive transcription factor that coordinates expression among antioxidant response proteins like FTN, thioredoxin reductase, and quinone reductase. FTN, an antioxidant because Fe2+ and O2 (reactive oxygen species generators) are consumed to make iron mineral, is also a nutritional iron concentrate that is an efficiently absorbed, nonheme source of iron from whole legumes. FTN protein cages contain thousands of mineralized iron atoms and enter cells by receptor-mediated endocytosis, an absorption mechanism distinct from transport of nonheme iron salts (ferrous sulfate), iron chelators (ferric-EDTA), or heme. Recognition of 2 nutritional nonheme iron sources, small and large (FTN), will aid the solution of iron deficiency, a major public health problem, and the development of new policies on iron nutrition. PMID:21346101

Biofortification of soybean meal: immunological properties of the 27 kDa γ-zein.

PubMed

Krishnan, Hari B; Jang, Sungchan; Kim, Won-Seok; Kerley, Monty S; Oliver, Melvin J; Trick, Harold N

2011-02-23

Legumes, including soybeans ( Glycine max ), are deficient in sulfur-containing amino acids, which are required for the optimal growth of monogastric animals. This deficiency can be overcome by expressing heterologous proteins rich in sulfur-containing amino acids in soybean seeds. A maize 27 kDa γ-zein, a cysteine-rich protein, has been successfully expressed in several crops including soybean, barley, and alfalfa with the intent to biofortify these crops for animal feed. Previous work has shown that the maize 27 kDa zein can withstand digestion by pepsin and elicit an immunogenic response in young pigs. By use of sera from patients who tested positive by ImmunoCAP assay for elevated IgE to maize proteins, specific IgE binding to the 27 kDa γ-zein is demonstrated. Bioinformatic analysis using the full-length and 80 amino acid sliding window FASTA searches identified significant sequence homology of the 27 kDa γ-zein with several known allergens. Immunoblot analysis using human serum that cross-reacts with maize seed proteins also revealed specific IgE-binding to the 27 kDa γ-zein in soybean seed protein extracts containing the 27 kDa zein. This study demonstrates for the first time the allergenicity potential of the 27 kDa γ-zein and the potential that this protein has to limit livestock performance when used in soybeans that serve as a biofortified feed supplement.

An ultrastable conjugate of silver nanoparticles and protein formed through weak interactions

NASA Astrophysics Data System (ADS)

Brahmkhatri, Varsha P.; Chandra, Kousik; Dubey, Abhinav; Atreya, Hanudatta S.

2015-07-01

In recent years, silver nanoparticles (AgNPs) have attracted significant attention owing to their unique physicochemical, optical, conductive and antimicrobial properties. One of the properties of AgNPs which is crucial for all applications is their stability. In the present study we unravel a mechanism through which silver nanoparticles are rendered ultrastable in an aqueous solution in complex with the protein ubiquitin (Ubq). This involves a dynamic and reversible association and dissociation of ubiquitin from the surface of AgNP. The exchange occurs at a rate much greater than 25 s-1 implying a residence time of <40 ms for the protein. The AgNP-Ubq complex remains stable for months due to steric stabilization over a wide pH range compared to unconjugated AgNPs. NMR studies reveal that the protein molecules bind reversibly to AgNP with an approximate dissociation constant of 55 μM and undergo fast exchange. At pH > 4 the positively charged surface of the protein comes in contact with the citrate capped AgNP surface. Further, NMR relaxation-based experiments suggest that in addition to the dynamic exchange, a conformational rearrangement of the protein takes place upon binding to AgNP. The ultrastability of the AgNP-Ubq complex was found to be useful for its anti-microbial activity, which allowed the recycling of this complex multiple times without the loss of stability. Altogether, the study provides new insights into the mechanism of protein-silver nanoparticle interactions and opens up new avenues for its application in a wide range of systems.In recent years, silver nanoparticles (AgNPs) have attracted significant attention owing to their unique physicochemical, optical, conductive and antimicrobial properties. One of the properties of AgNPs which is crucial for all applications is their stability. In the present study we unravel a mechanism through which silver nanoparticles are rendered ultrastable in an aqueous solution in complex with the protein ubiquitin (Ubq). This involves a dynamic and reversible association and dissociation of ubiquitin from the surface of AgNP. The exchange occurs at a rate much greater than 25 s-1 implying a residence time of <40 ms for the protein. The AgNP-Ubq complex remains stable for months due to steric stabilization over a wide pH range compared to unconjugated AgNPs. NMR studies reveal that the protein molecules bind reversibly to AgNP with an approximate dissociation constant of 55 μM and undergo fast exchange. At pH > 4 the positively charged surface of the protein comes in contact with the citrate capped AgNP surface. Further, NMR relaxation-based experiments suggest that in addition to the dynamic exchange, a conformational rearrangement of the protein takes place upon binding to AgNP. The ultrastability of the AgNP-Ubq complex was found to be useful for its anti-microbial activity, which allowed the recycling of this complex multiple times without the loss of stability. Altogether, the study provides new insights into the mechanism of protein-silver nanoparticle interactions and opens up new avenues for its application in a wide range of systems. Electronic supplementary information (ESI) available. See DOI: 10.1039/c5nr03047a

Activity of calcium activated protease in skeletal muscles and its changes in atrophy and stretch

NASA Technical Reports Server (NTRS)

Ellis, S.; Nagainis, P. A.

1984-01-01

The reduction of protein content in skeletal muscle undergoing disuse-induced atrophy is correlated with accelerated rates of protein degradation and reduced rates of protein synthesis (Goldspink, 1977). It is not known in what manner myofibers are partially disassembled during disuse atrophy to fibers of smaller diameter; nor is it known which proteases are responsible for this morphological change in contractile protein mass. Dayton and colleagues (1975) have suggested that the Ca(2+)-activated protease (CaP) may initiate myofibril degradation. The discovery of a form of CaP that is activatable by nano-molar concentrations of Ca(2+) indicates that CaP activity may be regulated by physiological concentrations of Ca(2+) (Mellgren, 1980). The enhancement of proteolysis by the Ca(2+) ionophore A23187, reported by Etlinger (1979), is consistent with a significant role for CaP in protein degradation. It was of interest, therefore, to measure the levels of CaP activity and the CaP inhibitor in extracts obtained from skeletal muscles of rat and chicken limbs undergoing disuse atrophy or stretch hypertrophy, respectively.

Subunits of the Drosophila Actin-Capping Protein Heterodimer Regulate Each Other at Multiple Levels

PubMed Central

Amândio, Ana Rita; Gaspar, Pedro; Whited, Jessica L.; Janody, Florence

2014-01-01

The actin-Capping Protein heterodimer, composed of the α and β subunits, is a master F-actin regulator. In addition to its role in many cellular processes, Capping Protein acts as a main tumor suppressor module in Drosophila and in humans, in part, by restricting the activity of Yorkie/YAP/TAZ oncogenes. We aimed in this report to understand how both subunits regulate each other in vivo. We show that the levels and capping activities of both subunits must be tightly regulated to control F-actin levels and consequently growth of the Drosophila wing. Overexpressing capping protein α and β decreases both F-actin levels and tissue growth, while expressing forms of Capping Protein that have dominant negative effects on F-actin promote tissue growth. Both subunits regulate each other's protein levels. In addition, overexpressing one of the subunit in tissues knocked-down for the other increases the mRNA and protein levels of the subunit knocked-down and compensates for its loss. We propose that the ability of the α and β subunits to control each other's levels assures that a pool of functional heterodimer is produced in sufficient quantities to restrict the development of tumor but not in excess to sustain normal tissue growth. PMID:24788460

Tracing the Evolutionary History of the CAP Superfamily of Proteins Using Amino Acid Sequence Homology and Conservation of Splice Sites.

PubMed

Abraham, Anup; Chandler, Douglas E

2017-10-01

Proteins of the CAP superfamily play numerous roles in reproduction, innate immune responses, cancer biology, and venom toxicology. Here we document the breadth of the CAP (Cysteine-RIch Secretory Protein (CRISP), Antigen 5, and Pathogenesis-Related) protein superfamily and trace the major events in its evolution using amino acid sequence homology and the positions of exon/intron borders within their genes. Seldom acknowledged in the literature, we find that many of the CAP subfamilies present in mammals, where they were originally characterized, have distinct homologues in the invertebrate phyla. Early eukaryotic CAP genes contained only one exon inherited from prokaryotic predecessors and as evolution progressed an increasing number of introns were inserted, reaching 2-5 in the invertebrate world and 5-15 in the vertebrate world. Focusing on the CRISP subfamily, we propose that these proteins evolved in three major steps: (1) origination of the CAP/PR/SCP domain in bacteria, (2) addition of a small Hinge domain to produce the two-domain SCP-like proteins found in roundworms and anthropoids, and (3) addition of an Ion Channel Regulatory domain, borrowed from invertebrate peptide toxins, to produce full length, three-domain CRISP proteins, first seen in insects and later to diversify into multiple subtypes in the vertebrate world.

Tetracycline-inducible protein expression in pancreatic cancer cells: Effects of CapG overexpression

PubMed Central

Tonack, Sarah; Patel, Sabina; Jalali, Mehdi; Nedjadi, Taoufik; Jenkins, Rosalind E; Goldring, Christopher; Neoptolemos, John; Costello, Eithne

2011-01-01

AIM: To establish stable tetracycline-inducible pancreatic cancer cell lines. METHODS: Suit-2, MiaPaca-2, and Panc-1 cells were transfected with a second generation reverse tetracycline-controlled transactivator protein (rtTA2S-M2), under the control of either a cytomegalovirus (CMV) or a chicken β-actin promoter, and the resulting clones were characterised. RESULTS: Use of the chicken (β-actin) promoter proved superior for both the production and maintenance of doxycycline-inducible cell lines. The system proved versatile, enabling transient inducible expression of a variety of genes, including GST-P, CYP2E1, S100A6, and the actin capping protein, CapG. To determine the physiological utility of this system in pancreatic cancer cells, stable inducible CapG expressors were established. Overexpressed CapG was localised to the cytoplasm and the nuclear membrane, but was not observed in the nucleus. High CapG levels were associated with enhanced motility, but not with changes to the cell cycle, or cellular proliferation. In CapG-overexpressing cells, the levels and phosphorylation status of other actin-moduating proteins (Cofilin and Ezrin/Radixin) were not altered. However, preliminary analyses suggest that the levels of other cellular proteins, such as ornithine aminotransferase and enolase, are altered upon CapG induction. CONCLUSION: We have generated pancreatic-cancer derived cell lines in which gene expression is fully controllable. PMID:21528072

Molecular Basis for Impaired DNA Damage Response Function Associated with the RAP80 ΔE81 Defect*

PubMed Central

Anamika; Markin, Craig J.; Rout, Manoj K.; Spyracopoulos, Leo

2014-01-01

Signal transduction within the DNA damage response is driven by the flux of protein-protein interaction cascades that ultimately recruit repair complexes to sites of damage. The protein RAP80 plays a central role in the damage response by targeting BRCA1/BRCA2 tumor suppressors to DNA damage foci through multivalent binding of Lys-63-linked polyubiquitin chains. Mutations within the high penetrance BRCA1/BRCA2 genes account for ∼20% of familial breast cancers. The genetic basis for the remaining cancers remains unknown, but may involve defects in binding partners for BRCA1 and BRCA2 that lead to impaired targeting to foci and a concomitant role in the pathogenesis of cancer. Recently, an in-frame deletion mutation (ΔE81) in a conserved region from the first ubiquitin interaction motif of RAP80 has been linked to an increase in chromosomal abnormalities. Using NMR spectroscopy, we demonstrate that the N-cap motif within the α-helix of the first ubiquitin interaction motif from ΔE81 undergoes a structural frameshift that leads to abolishment of multivalent binding of polyubiquitin chains. Loss of this single glutamate residue disrupts favorable electrostatic interactions between RAP80 and ubiquitin, establishing a plausible molecular basis for a potential predisposition to cancer unrelated to mutations within BRCA1/BRCA2 genes. PMID:24627472

Identification of surface domain structure on enamel crystals using polyamidoamine dendrimer

NASA Astrophysics Data System (ADS)

Chen, Haifeng; Clarkson, Brian H.; Orr, Bradford; Majoros, Istvan; Banaszak Holl, Mark M.

2002-03-01

The control of hydroxyapatite crystal nucleation and crystal growth is central to the mineralization and remineralization of enamel and dentin of teeth. However, the precise biomolecular mechanisms involved remain obscure. The intimate association between the crystal's surface and extracellular protein components implies a modulating role for organic crystal interactions probably mediated via specific crystal surface domains. These include lattice defects and specific stereochemical arrays on associated organic molecules. The nature of protein-crystal interaction depends upon the physical forces of attraction / repulsion between specific biomolecular groups and crystal surface domains. The proposed study is to utilize specific polyamidoamine (PAMAM) dendrimers, also known as “artificial proteins”, acting as nanoprobe. These will be used to probe specific surface domain on the surface of the naturally derived crystals of hydroxyapatite and to determine how control of growth and dissolution may be affected at the biomolecular level. The hydroxyapatite crystals are extracted from the maturation stage enamel of rats. Three types of PAMAM dendrimers, respectively with amine-, carboxylic acid and methyl-capped surface, will be applied in the study. The dendrimer binding on the surface of the hydoxyapatite crystals will be characterized using atomic force microscopy (AFM). The different dendrimer binding on the crystals will disclose the specific surface domain structure on the crystals, which is assumed to be important in binding the extracellular protein.

Increased helix and protein stability through the introduction of a new tertiary hydrogen bond.

PubMed

Peterson, R W; Nicholson, E M; Thapar, R; Klevit, R E; Scholtz, J M

1999-03-12

In an effort to quantify the importance of hydrogen bonding and alpha-helix formation to protein stability, a capping box motif was introduced into the small phosphocarrier protein HPr. Previous studies had confirmed that Ser46, at the N-cap position of the short helix-B in HPr, serves as an N-cap in solution. Thus, only a single-site mutation was required to produce a canonical S-X-X-E capping box: Lys49 at the N3 position was substituted with a glutamic acid residue. Thermal and chemical denaturation studies on the resulting K49E HPr show that the designed variant is approximately 2 kcal mol-1 more stable than the wild-type protein. However, NMR studies indicate that the side-chain of Glu49 does not participate in the expected capping H-bond interaction, but instead forms a new tertiary H-bond that links helix-B to the four-stranded beta-sheet of HPr. Here, we demonstrate that a strategy in which new non-native H-bonds are introduced can generate proteins with increased stability. We discuss why the original capping box design failed, and compare the energetic consequences of the new tertiary side-chain to main-chain H-bond with a local (helix-capping) side-chain to main-chain H-bond on the protein's global stability. Copyright 1999 Academic Press.

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

Spliced leader RNA of trypanosomes: in vivo mutational analysis reveals extensive and distinct requirements for trans splicing and cap4 formation.

PubMed Central

Lücke, S; Xu, G L; Palfi, Z; Cross, M; Bellofatto, V; Bindereif, A

1996-01-01

In trypanosomes mRNAs are generated through trans splicing. The spliced leader (SL) RNA, which donates the 5'-terminal mini-exon to each of the protein coding exons, plays a central role in the trans splicing process. We have established in vivo assays to study in detail trans splicing, cap4 modification, and RNP assembly of the SL RNA in the trypanosomatid species Leptomonas seymouri. First, we found that extensive sequences within the mini-exon are required for SL RNA function in vivo, although a conserved length of 39 nt is not essential. In contrast, the intron sequence appears to be surprisingly tolerant to mutation; only the stem-loop II structure is indispensable. The asymmetry of the sequence requirements in the stem I region suggests that this domain may exist in different functional conformations. Second, distinct mini-exon sequences outside the modification site are important for efficient cap4 formation. Third, all SL RNA mutations tested allowed core RNP assembly, suggesting flexible requirements for core protein binding. In sum, the results of our mutational analysis provide evidence for a discrete domain structure of the SL RNA and help to explain the strong phylogenetic conservation of the mini-exon sequence and of the overall SL RNA secondary structure; they also suggest that there may be certain differences between trans splicing in nematodes and trypanosomes. This approach provides a basis for studying RNA-RNA interactions in the trans spliceosome. Images PMID:8861965

Model of turnover kinetics in the lamellipodium: implications of slow- and fast- diffusing capping protein and Arp2/3 complex

NASA Astrophysics Data System (ADS)

McMillen, Laura M.; Vavylonis, Dimitrios

2016-12-01

Cell protrusion through polymerization of actin filaments at the leading edge of motile cells may be influenced by spatial gradients of diffuse actin and regulators. Here we study the distribution of two of the most important regulators, capping protein and Arp2/3 complex, which regulate actin polymerization in the lamellipodium through capping and nucleation of free barbed ends. We modeled their kinetics using data from prior single molecule microscopy experiments on XTC cells. These experiments have provided evidence for a broad distribution of diffusion coefficients of both capping protein and Arp2/3 complex. The slowly diffusing proteins appear as extended ‘clouds’ while proteins bound to the actin filament network appear as speckles that undergo retrograde flow. Speckle appearance and disappearance events correspond to assembly and dissociation from the actin filament network and speckle lifetimes correspond to the dissociation rate. The slowly diffusing capping protein could represent severed capped actin filament fragments or membrane-bound capping protein. Prior evidence suggests that slowly diffusing Apr2/3 complex associates with the membrane. We use the measured rates and estimates of diffusion coefficients of capping protein and Arp2/3 complex in a Monte Carlo simulation that includes particles in association with a filament network and diffuse in the cytoplasm. We consider two separate pools of diffuse proteins, representing fast and slowly diffusing species. We find a steady state with concentration gradients involving a balance of diffusive flow of fast and slow species with retrograde flow. We show that simulations of FRAP are consistent with prior experiments performed on different cell types. We provide estimates for the ratio of bound to diffuse complexes and calculate conditions where Arp2/3 complex recycling by diffusion may become limiting. We discuss the implications of slowly diffusing populations and suggest experiments to distinguish among mechanisms that influence long range transport.

Inhibition of Poliovirus-Induced Cleavage of Cellular Protein PCBP2 Reduces the Levels of Viral RNA Replication

PubMed Central

Chase, Amanda J.; Daijogo, Sarah

2014-01-01

ABSTRACT Due to their small genome size, picornaviruses must utilize host proteins to mediate cap-independent translation and viral RNA replication. The host RNA-binding protein poly(rC) binding protein 2 (PCBP2) is involved in both processes in poliovirus infected cells. It has been shown that the viral proteinase 3CD cleaves PCBP2 and contributes to viral translation inhibition. However, cleaved PCBP2 remains active in viral RNA replication. This would suggest that both cleaved and intact forms of PCBP2 have a role in the viral RNA replication cycle. The picornavirus genome must act as a template for both translation and RNA replication. However, a template that is actively being translated cannot function as a template for RNA replication, suggesting that there is a switch in template usage from translation to RNA replication. We demonstrate that the cleavage of PCBP2 by the poliovirus 3CD proteinase is a necessary step for efficient viral RNA replication and, as such, may be important for mediating a switch in template usage from translation to RNA replication. IMPORTANCE Poliovirus, like all positive-strand RNA viruses that replicate in the cytoplasm of eukaryotic cells, uses its genomic RNA as a template for both viral protein synthesis and RNA replication. Given that these processes cannot occur simultaneously on the same template, poliovirus has evolved a mechanism(s) to facilitate the switch from using templates for translation to using them for RNA synthesis. This study explores one possible scenario for how the virus alters the functions of a host cell RNA binding protein to mediate, in part, this important transition. PMID:24371074

Cap analog and Potato virus A HC-Pro silencing suppressor improve GFP transient expression using an infectious virus vector in Nicotiana benthamiana.

PubMed

Tahmasebi, Amin-Alah; Afsharifar, Alireza

2017-06-01

Transient expression of proteins in plants has become a choice to facilitate recombinant protein production with its fast and easy application. On the other hand, host defensive mechanisms have been reported to reduce the efficiency of transient expression in plants. Hence, this study was designed to evaluate the effect of cap analog and Potato virus A helper component proteinase (PVA HC-Pro) on green fluorescent protein (GFP) expression efficiency. N . benthamiana leaves were inoculated with capped or un-capped RNA transcripts of a Turnip crinkle virus (TCV) construct containing a green fluorescent protein reporter gene (TCV-sGFP) in place of its coat protein (CP) ORF. PVA HC-Pro as a viral suppressor of RNA silencing was infiltrated in trans by Agrobacterium tumefaciens , increased the GFP foci diameter to six and even more cells in both capped and un capped treatments. The expression level of GFP in inoculated plants with TCV-sGFP transcript pre-infiltrated with PVA HC-Pro was 12.97-fold higher than the GFP accumulation level in pre-infiltrated leaves with empty plasmid (EP) control. Also, the yield of GFP in inoculated N. benthamiana plants with capped TCV-sGFP transcript pre-infiltrated with EP and PVA HC-Pro was 1.54 and 1.2-fold respectively, greater than the level of GFP expressed without cap analog application at 5 days post inoculation (dpi). In addition, the movement of TCV-sGFP was increased in some cells of inoculated leaves with capped transcripts. Results of this study indicated that PVA HC-Pro and mRNA capping can increase GFP expression and its cell to cell movement in N. benthamiana .

HIV-1 Rev protein specifies the viral RNA export pathway by suppressing TAP/NXF1 recruitment

PubMed Central

Taniguchi, Ichiro; Mabuchi, Naoto; Ohno, Mutsuhito

2014-01-01

Nuclear RNA export pathways in eukaryotes are often linked to the fate of a given RNA. Therefore, the choice of export pathway should be well-controlled to avoid an unfavorable effect on gene expression. Although some RNAs could be exported by more than one pathway, little is known about how the choice is regulated. This issue is highlighted when the human immunodeficiency virus type 1 (HIV-1) Rev protein induces the export of singly spliced and unspliced HIV-1 transcripts. How these RNAs are exported is not well understood because such transcripts should have the possibility of utilizing CRM1-dependent export via Rev or cellular TAP/NXF1-dependent export via the transcription/export (TREX) complex, or both. Here we found that Rev suppressed TAP/NXF1-dependent export of model RNA substrates that recapitulated viral transcripts. In this effect, Rev interacted with the cap-binding complex and inhibited the recruitment of the TREX complex. Thus, Rev controls the identity of the factor occupying the cap-proximal region that determines the RNA export pathway. This ribonucleoprotein remodeling activity of Rev may favor viral gene expression. PMID:24753416

Capping protein is essential for cell migration in vivo and for filopodial morphology and dynamics

PubMed Central

Sinnar, Shamim A.; Antoku, Susumu; Saffin, Jean-Michel; Cooper, Jon A.; Halpain, Shelley

2014-01-01

Capping protein (CP) binds to barbed ends of growing actin filaments and inhibits elongation. CP is essential for actin-based motility in cell-free systems and in Dictyostelium. Even though CP is believed to be critical for creating the lamellipodial actin structure necessary for protrusion and migration, CP's role in mammalian cell migration has not been directly tested. Moreover, recent studies have suggested that structures besides lamellipodia, including lamella and filopodia, may have unappreciated roles in cell migration. CP has been postulated to be absent from filopodia, and thus its role in filopodial activity has remained unexplored. We report that silencing CP in both cultured mammalian B16F10 cells and in neurons of developing neocortex impaired cell migration. Moreover, we unexpectedly observed that low levels of CP were detectable in the majority of filopodia. CP depletion decreased filopodial length, altered filopodial shape, and reduced filopodial dynamics. Our results support an expansion of the potential roles that CP plays in cell motility by implicating CP in filopodia as well as in lamellipodia, both of which are important for locomotion in many types of migrating cells. PMID:24829386

Influenza Polymerase Can Adopt an Alternative Configuration Involving a Radical Repacking of PB2 Domains.

PubMed

Thierry, Eric; Guilligay, Delphine; Kosinski, Jan; Bock, Thomas; Gaudon, Stephanie; Round, Adam; Pflug, Alexander; Hengrung, Narin; El Omari, Kamel; Baudin, Florence; Hart, Darren J; Beck, Martin; Cusack, Stephen

2016-01-07

Influenza virus polymerase transcribes or replicates the segmented RNA genome (vRNA) into respectively viral mRNA or full-length copies and initiates RNA synthesis by binding the conserved 3' and 5' vRNA ends (the promoter). In recent structures of promoter-bound polymerase, the cap-binding and endonuclease domains are configured for cap snatching, which generates capped transcription primers. Here, we present a FluB polymerase structure with a bound complementary cRNA 5' end that exhibits a major rearrangement of the subdomains within the C-terminal two-thirds of PB2 (PB2-C). Notably, the PB2 nuclear localization signal (NLS)-containing domain translocates ∼90 Å to bind to the endonuclease domain. FluA PB2-C alone and RNA-free FluC polymerase are similarly arranged. Biophysical and cap-dependent endonuclease assays show that in solution the polymerase explores different conformational distributions depending on which RNA is bound. The inherent flexibility of the polymerase allows it to adopt alternative conformations that are likely important during polymerase maturation into active progeny RNPs. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Nat1 promotes translation of specific proteins that induce differentiation of mouse embryonic stem cells.

PubMed

Sugiyama, Hayami; Takahashi, Kazutoshi; Yamamoto, Takuya; Iwasaki, Mio; Narita, Megumi; Nakamura, Masahiro; Rand, Tim A; Nakagawa, Masato; Watanabe, Akira; Yamanaka, Shinya

2017-01-10

Novel APOBEC1 target 1 (Nat1) (also known as "p97," "Dap5," and "Eif4g2") is a ubiquitously expressed cytoplasmic protein that is homologous to the C-terminal two thirds of eukaryotic translation initiation factor 4G (Eif4g1). We previously showed that Nat1-null mouse embryonic stem cells (mES cells) are resistant to differentiation. In the current study, we found that NAT1 and eIF4G1 share many binding proteins, such as the eukaryotic translation initiation factors eIF3 and eIF4A and ribosomal proteins. However, NAT1 did not bind to eIF4E or poly(A)-binding proteins, which are critical for cap-dependent translation initiation. In contrast, compared with eIF4G1, NAT1 preferentially interacted with eIF2, fragile X mental retardation proteins (FMR), and related proteins and especially with members of the proline-rich and coiled-coil-containing protein 2 (PRRC2) family. We also found that Nat1-null mES cells possess a transcriptional profile similar, although not identical, to the ground state, which is established in wild-type mES cells when treated with inhibitors of the ERK and glycogen synthase kinase 3 (GSK3) signaling pathways. In Nat1-null mES cells, the ERK pathway is suppressed even without inhibitors. Ribosome profiling revealed that translation of mitogen-activated protein kinase kinase kinase 3 (Map3k3) and son of sevenless homolog 1 (Sos1) is suppressed in the absence of Nat1 Forced expression of Map3k3 induced differentiation of Nat1-null mES cells. These data collectively show that Nat1 is involved in the translation of proteins that are required for cell differentiation.

African Swine Fever Virus NP868R Capping Enzyme Promotes Reovirus Rescue during Reverse Genetics by Promoting Reovirus Protein Expression, Virion Assembly, and RNA Incorporation into Infectious Virions

PubMed Central

Eaton, Heather E.; Kobayashi, Takeshi; Dermody, Terence S.; Johnston, Randal N.

2017-01-01

ABSTRACT Reoviruses, like many eukaryotic viruses, contain an inverted 7-methylguanosine (m7G) cap linked to the 5′ nucleotide of mRNA. The traditional functions of capping are to promote mRNA stability, protein translation, and concealment from cellular proteins that recognize foreign RNA. To address the role of mRNA capping during reovirus replication, we assessed the benefits of adding the African swine fever virus NP868R capping enzyme during reovirus rescue. C3P3, a fusion protein containing T7 RNA polymerase and NP868R, was found to increase protein expression 5- to 10-fold compared to T7 RNA polymerase alone while enhancing reovirus rescue from the current reverse genetics system by 100-fold. Surprisingly, RNA stability was not increased by C3P3, suggesting a direct effect on protein translation. A time course analysis revealed that C3P3 increased protein synthesis within the first 2 days of a reverse genetics transfection. This analysis also revealed that C3P3 enhanced processing of outer capsid μ1 protein to μ1C, a previously described hallmark of reovirus assembly. Finally, to determine the rate of infectious-RNA incorporation into new virions, we developed a new recombinant reovirus S1 gene that expressed the fluorescent protein UnaG. Following transfection of cells with UnaG and infection with wild-type virus, passage of UnaG through progeny was significantly enhanced by C3P3. These data suggest that capping provides nontraditional functions to reovirus, such as promoting assembly and infectious-RNA incorporation. IMPORTANCE Our findings expand our understanding of how viruses utilize capping, suggesting that capping provides nontraditional functions to reovirus, such as promoting assembly and infectious-RNA incorporation, in addition to enhancing protein translation. Beyond providing mechanistic insight into reovirus replication, our findings also show that reovirus reverse genetics rescue is enhanced 100-fold by the NP868R capping enzyme. Since reovirus shows promise as a cancer therapy, efficient reovirus reverse genetics rescue will accelerate production of recombinant reoviruses as candidates to enhance therapeutic potency. NP868R-assisted reovirus rescue will also expedite production of recombinant reovirus for mechanistic insights into reovirus protein function and structure. PMID:28298603

Cross-Linked Peptidoglycan Mediates Lysostaphin Binding to the Cell Wall Envelope of Staphylococcus aureus†

PubMed Central

Gründling, Angelika; Schneewind, Olaf

2006-01-01

Staphylococcus simulans bv. staphylolyticus secretes lysostaphin, a bacteriocin that cleaves pentaglycine cross bridges in the cell wall of Staphylococcus aureus. The C-terminal cell wall-targeting domain (CWT) of lysostaphin is required for selective binding of this bacteriocin to S. aureus cells; however, the molecular target for this was unknown. We used purified green fluorescent protein fused to CWT (GFP-CWT) to reveal species-specific association of the reporter with staphylococci. GFP-CWT bound S. aureus cells as well as purified peptidoglycan sacculi. The addition of cross-linked murein, disaccharides linked to interconnected wall peptides, blocked GFP-CWT binding to staphylococci, whereas murein monomers or lysostaphin-solubilized cell wall fragments did not. S. aureus strain Newman variants lacking the capacity for synthesizing polysaccharide capsule (capFO), poly-N-acetylglucosamine (icaAC), lipoprotein (lgt), cell wall-anchored proteins (srtA), or the glycolipid anchor of lipoteichoic acid (ypfP) bound GFP-CWT similar to wild-type staphylococci. A tagO mutant strain, defective in the synthesis of polyribitol wall teichoic acid attached to the cell wall envelope, displayed increased GFP-CWT binding. In contrast, a femAB mutation, reducing both the amount and the length of peptidoglycan cross-linking (monoglycine cross bridges), showed a dramatic reduction in GFP-CWT binding. Thus, the CWT domain of lysostaphin directs the bacteriocin to cross-linked peptidoglycan, which also serves as the substrate for its glycyl-glycine endopeptidase domain. PMID:16547033

Cross-linked peptidoglycan mediates lysostaphin binding to the cell wall envelope of Staphylococcus aureus.

PubMed

Gründling, Angelika; Schneewind, Olaf

2006-04-01

Staphylococcus simulans bv. staphylolyticus secretes lysostaphin, a bacteriocin that cleaves pentaglycine cross bridges in the cell wall of Staphylococcus aureus. The C-terminal cell wall-targeting domain (CWT) of lysostaphin is required for selective binding of this bacteriocin to S. aureus cells; however, the molecular target for this was unknown. We used purified green fluorescent protein fused to CWT (GFP-CWT) to reveal species-specific association of the reporter with staphylococci. GFP-CWT bound S. aureus cells as well as purified peptidoglycan sacculi. The addition of cross-linked murein, disaccharides linked to interconnected wall peptides, blocked GFP-CWT binding to staphylococci, whereas murein monomers or lysostaphin-solubilized cell wall fragments did not. S. aureus strain Newman variants lacking the capacity for synthesizing polysaccharide capsule (capFO), poly-N-acetylglucosamine (icaAC), lipoprotein (lgt), cell wall-anchored proteins (srtA), or the glycolipid anchor of lipoteichoic acid (ypfP) bound GFP-CWT similar to wild-type staphylococci. A tagO mutant strain, defective in the synthesis of polyribitol wall teichoic acid attached to the cell wall envelope, displayed increased GFP-CWT binding. In contrast, a femAB mutation, reducing both the amount and the length of peptidoglycan cross-linking (monoglycine cross bridges), showed a dramatic reduction in GFP-CWT binding. Thus, the CWT domain of lysostaphin directs the bacteriocin to cross-linked peptidoglycan, which also serves as the substrate for its glycyl-glycine endopeptidase domain.

Self-assembly of virus-like particles of porcine circovirus type 2 capsid protein expressed from Escherichia coli

PubMed Central

2010-01-01

Background Porcine circovirus 2 (PCV2) is a serious problem to the swine industry and can lead to significant negative impacts on profitability of pork production. Syndrome associated with PCV2 is known as porcine circovirus closely associated with post-weaning multisystemic wasting syndrome (PMWS). The capsid (Cap) protein of PCV2 is a major candidate antigen for development of recombinant vaccine and serological diagnostic method. The recombinant Cap protein has the ability to self-assemble into virus-like particles (VLPs) in vitro, it is particularly opportunity to develop the PV2 VLPs vaccine in Escherichia coli,(E.coli ), because where the cost of the vaccine must be weighed against the value of the vaccinated pig, when it was to extend use the VLPs vaccine of PCV2. Results In this report, a highly soluble Cap-tag protein expressed in E.coli was constructed with a p-SMK expression vector with a fusion tag of small ubiquitin-like modifiers (SUMO). The recombinant Cap was purified using Ni2+ affinity resins, whereas the tag was used to remove the SUMO protease. Simultaneously, the whole native Cap protein was able to self-assemble into VLPs in vitro when viewed under an electron microscope. The Cap-like particles had a size and shape that resembled the authentic Cap. The result could also be applied in the large-scale production of VLPs of PCV2 and could be used as a diagnostic antigen or a potential VLP vaccine against PCV2 infection in pigs. Conclusion we have, for the first time, utilized the SUMO fusion motif to successfully express the entire authentic Cap protein of PCV2 in E. coli. After the cleavage of the fusion motif, the nCap protein has the ability to self-assemble into VLPs, which can be used as as a potential vaccine to protect pigs from PCV2-infection. PMID:20646322

Mitotic protein kinase CDK1 phosphorylation of mRNA translation regulator 4E-BP1 Ser83 may contribute to cell transformation

DOE PAGES

Velasquez, Celestino; Cheng, Erdong; Shuda, Masahiro; ...

2016-07-11

mTOR-directed 4E-BP1 phosphorylation promotes cap-dependent translation and tumorigen-esis. During mitosis, CDK1 substitutes for mTOR and fully phosphorylates 4E-BP1 at canoni-cal as well a non-canonical S83 site resulting in a mitosis-specific hyperphosphorylated δ isoform. Colocalization studies with a phospho-S83 specific antibody indicate that 4E-BP1 S83 phosphorylation accumulates at centrosomes during prophase, peaks at metaphase, and decreases through telophase. While S83 phosphorylation of 4E-BP1 does not affect in vitro cap-dependent translation, nor eIF4G/4E-BP1 cap-binding, expression of an alanine substitution mutant 4E-BP1.S83A partially reverses rodent cell transformation induced by Merkel cell polyomavirus (MCV) small T (sT) antigen viral oncoprotein. In contrast to inhibitorymore » mTOR 4E-BP1 phosphorylation, these findings suggest that mitotic CDK1-directed phosphorylation of δ-4E-BP1 may yield a gain-of-function, distinct from translation regulation, that may be important in tumorigenesis and mitotic centrosome function.« less

Back to basics: the untreated rabbit reticulocyte lysate as a competitive system to recapitulate cap/poly(A) synergy and the selective advantage of IRES-driven translation.

PubMed

Soto Rifo, Ricardo; Ricci, Emiliano P; Décimo, Didier; Moncorgé, Olivier; Ohlmann, Théophile

2007-01-01

Translation of most eukaryotic mRNAs involves the synergistic action between the 5' cap structure and the 3' poly(A) tail at the initiation step. The poly(A) tail has also been shown to stimulate translation of picornavirus internal ribosome entry sites (IRES)-directed translation. These effects have been attributed principally to interactions between eIF4G and poly(A)-binding protein (PABP) but also to the participation of PABP in other steps during translation initiation. As the rabbit reticulocyte lysate (RRL) does not recapitulate this cap/poly(A) synergy, several systems based on cellular cell-free extracts have been developed to study the effects of poly(A) tail in vitro but they generally exhibit low translational efficiency. Here, we describe that the non-nuclease-treated RRL (untreated RRL) is able to recapitulate the effects of poly(A) tail on translation in vitro. In this system, translation of a capped/polyadenylated RNA was specifically inhibited by either Paip2 or poly(rA), whereas translation directed by HCV IRES remained unaffected. Moreover, cleavage of eIF4G by FMDV L protease strongly stimulated translation directed by the EMCV IRES, thus recapitulating the competitive advantage that the proteolytic processing of eIF4G confers to IRES-driven RNAs.

A short conserved motif in ALYREF directs cap- and EJC-dependent assembly of export complexes on spliced mRNAs.

PubMed

Gromadzka, Agnieszka M; Steckelberg, Anna-Lena; Singh, Kusum K; Hofmann, Kay; Gehring, Niels H

2016-03-18

The export of messenger RNAs (mRNAs) is the final of several nuclear posttranscriptional steps of gene expression. The formation of export-competent mRNPs involves the recruitment of export factors that are assumed to facilitate transport of the mature mRNAs. Using in vitro splicing assays, we show that a core set of export factors, including ALYREF, UAP56 and DDX39, readily associate with the spliced RNAs in an EJC (exon junction complex)- and cap-dependent manner. In order to elucidate how ALYREF and other export adaptors mediate mRNA export, we conducted a computational analysis and discovered four short, conserved, linear motifs present in RNA-binding proteins. We show that mutation in one of the new motifs (WxHD) in an unstructured region of ALYREF reduced RNA binding and abolished the interaction with eIF4A3 and CBP80. Additionally, the mutation impaired proper localization to nuclear speckles and export of a spliced reporter mRNA. Our results reveal important details of the orchestrated recruitment of export factors during the formation of export competent mRNPs. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

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

Kim, Kyoung Mi; Cho, Hana; Kim, Yoon Ki, E-mail: yk-kim@korea.ac.kr

Highlights: Black-Right-Pointing-Pointer CDKN1A mRNA is a bona fide NMD substrate. Black-Right-Pointing-Pointer The uORF of CDKN1A mRNA is efficiently translated. Black-Right-Pointing-Pointer Translation of downstream main ORF is negatively regulated by translation of uORF in CDKN1A mRNA. -- Abstract: The first round of translation occurs on mRNAs bound by nuclear cap-binding complex (CBC), which is composed of nuclear cap-binding protein 80 and 20 (CBP80/20). During this round of translation, aberrant mRNAs are recognized and downregulated in abundance by nonsense-mediated mRNA decay (NMD), which is one of the mRNA quality control mechanisms. Here, our microarray analysis reveals that the level of cyclin-dependent kinasemore » inhibitor 1A (CDKN1A; also known as Waf1/p21) mRNAs increases in cells depleted of cellular NMD factors. Intriguingly, CDKN1A mRNA contains an upstream open reading frame (uORF), which is a NMD-inducing feature. Using chimeric reporter constructs, we find that the uORF of CDKN1A mRNA negatively modulates translation of the main downstream ORF. These findings provide biological insights into the possible role of NMD in diverse biological pathways mediated by CDKN1A.« less

sST2 translation is regulated by FGF2 via an hnRNP A1-mediated IRES-dependent mechanism.

PubMed

Kunze, Michael M; Benz, Fabienne; Brauß, Thilo F; Lampe, Sebastian; Weigand, Julia E; Braun, Johannes; Richter, Florian M; Wittig, Ilka; Brüne, Bernhard; Schmid, Tobias

2016-07-01

Translation is an energy-intensive process and tightly regulated. Generally, translation is initiated in a cap-dependent manner. Under stress conditions, typically found within the tumor microenvironment in association with e.g. nutrient deprivation or hypoxia, cap-dependent translation decreases, and alternative modes of translation initiation become more important. Specifically, internal ribosome entry sites (IRES) facilitate translation of specific mRNAs under otherwise translation-inhibitory conditions. This mechanism is controlled by IRES trans-acting factors (ITAF), i.e. by RNA-binding proteins, which interact with and determine the activity of selected IRESs. We aimed at characterizing the translational regulation of the IL-33 decoy receptor sST2, which was enhanced by fibroblast growth factor 2 (FGF2). We identified and verified an IRES within the 5'UTR of sST2. Furthermore, we found that MEK/ERK signaling contributes to FGF2-induced, sST2-IRES activation and translation. Determination of the sST2-5'UTR structure by in-line probing followed by deletion analyses identified 23 nucleotides within the sST2-5'UTR to be required for optimal IRES activity. Finally, we show that the RNA-binding protein heterogeneous ribonucleoprotein A1 (hnRNP A1) binds to the sST2-5'UTR, acts as an ITAF, and thus controls the activity of the sST2-IRES and consequently sST2 translation. Specifically, FGF2 enhances nuclear-cytoplasmic translocation of hnRNP A1, which requires intact MEK/ERK activity. In summary, we provide evidence that the sST2-5'UTR contains an IRES element, which is activated by a MEK/ERK-dependent increase in cytoplasmic localization of hnRNP A1 in response to FGF2, enhancing the translation of sST2. Copyright © 2016 Elsevier B.V. All rights reserved.

Small-angle X-ray and neutron scattering demonstrates that cell-free expression produces properly formed disc-shaped nanolipoprotein particles

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

Cleveland, Thomas E.; He, Wei; Evans, Angela C.

Nanolipoprotein particles (NLPs), composed of membrane scaffold proteins and lipids, have been used to support membrane proteins in a native-like bilayer environment for biochemical and structural studies. Traditionally, these NLPs have been prepared by the controlled removal of detergent from a detergent-solubilized protein-lipid mixture. Recently, an alternative method has been developed using direct cell-free expression of the membrane scaffold protein in the presence of preformed lipid vesicles, which spontaneously produces NLPs without the need for detergent at any stage. Using SANS/SAXS, we show here that NLPs produced by this cell-free expression method are structurally indistinguishable from those produced using detergentmore » removal methodologies. This further supports the utility of single step cell-free methods for the production of lipid binding proteins. Lastly, in addition, detailed structural information describing these NLPs can be obtained by fitting a capped core-shell cylinder type model to all SANS/SAXS data simultaneously.« less

Small-angle X-ray and neutron scattering demonstrates that cell-free expression produces properly formed disc-shaped nanolipoprotein particles

DOE PAGES

Cleveland, Thomas E.; He, Wei; Evans, Angela C.; ...

2018-02-13

Nanolipoprotein particles (NLPs), composed of membrane scaffold proteins and lipids, have been used to support membrane proteins in a native-like bilayer environment for biochemical and structural studies. Traditionally, these NLPs have been prepared by the controlled removal of detergent from a detergent-solubilized protein-lipid mixture. Recently, an alternative method has been developed using direct cell-free expression of the membrane scaffold protein in the presence of preformed lipid vesicles, which spontaneously produces NLPs without the need for detergent at any stage. Using SANS/SAXS, we show here that NLPs produced by this cell-free expression method are structurally indistinguishable from those produced using detergentmore » removal methodologies. This further supports the utility of single step cell-free methods for the production of lipid binding proteins. Lastly, in addition, detailed structural information describing these NLPs can be obtained by fitting a capped core-shell cylinder type model to all SANS/SAXS data simultaneously.« less

Protein synthesis in geostimulated root caps

NASA Technical Reports Server (NTRS)

Feldman, L. J.

1982-01-01

A study is presented of the processes occurring in the root cap of corn which are requisite for the formation of root cap inhibitor and which can be triggered or modulated by both light and gravity. The results of this study indicate the importance of protein synthesis for light-induced gravitropic bending in roots. Root caps in which protein synthesis is prevented are unable to induce downward bending. This suggests that light acts by stimulating proteins which are necessary for the translation of the gravitropic stimulus into a growth response (downward bending). The turnover of protein with time was also examined in order to determine whether light acts by stimulating the synthesis of unique proteins required for downward growth. It is found that auxin in combination with light allows for the translation of the gravitropic stimulus into a growth response at least in part through the modification of protein synthesis. It is concluded that unique proteins are stimulated by light and are involved in promoting the downward growth in roots which are responding to gravity.

Analysis of the function of Spire in actin assembly and its synergy with formin and profilin.

PubMed

Bosch, Montserrat; Le, Kim Ho Diep; Bugyi, Beata; Correia, John J; Renault, Louis; Carlier, Marie-France

2007-11-30

The Spire protein, together with the formin Cappuccino and profilin, plays an important role in actin-based processes that establish oocyte polarity. Spire contains a cluster of four actin-binding WH2 domains. It has been shown to nucleate actin filaments and was proposed to remain bound to their pointed ends. Here we show that the multifunctional character of the WH2 domains allows Spire to sequester four G-actin subunits binding cooperatively in a tight SA(4) complex and to nucleate, sever, and cap filaments at their barbed ends. Binding of Spire to barbed ends does not affect the thermodynamics of actin assembly at barbed ends but blocks barbed end growth from profilin-actin. The resulting Spire-induced increase in profilin-actin concentration enhances processive filament assembly by formin. The synergy between Spire and formin is reconstituted in an in vitro motility assay, which provides a functional basis for the genetic interplay between Spire, formin, and profilin in oogenesis.

The human insulin receptor mRNA contains a functional internal ribosome entry segment

PubMed Central

Spriggs, Keith A.; Cobbold, Laura C.; Ridley, Simon H.; Coldwell, Mark; Bottley, Andrew; Bushell, Martin; Willis, Anne E.; Siddle, Kenneth

2009-01-01

Regulation of mRNA translation is an important mechanism determining the level of expression of proteins in eukaryotic cells. Translation is most commonly initiated by cap-dependent scanning, but many eukaryotic mRNAs contain internal ribosome entry segments (IRESs), providing an alternative means of initiation capable of independent regulation. Here, we show by using dicistronic luciferase reporter vectors that the 5′-UTR of the mRNA encoding human insulin receptor (hIR) contains a functional IRES. RNAi-mediated knockdown showed that the protein PTB was required for maximum IRES activity. Electrophoretic mobility shift assays confirmed that PTB1, PTB2 and nPTB, but not unr or PTB4, bound to hIR mRNA, and deletion mapping implicated a CCU motif 448 nt upstream of the initiator AUG in PTB binding. The IR-IRES was functional in a number of cell lines, and most active in cells of neuronal origin, as assessed by luciferase reporter assays. The IRES was more active in confluent than sub-confluent cells, but activity did not change during differentiation of 3T3-L1 fibroblasts to adipocytes. IRES activity was stimulated by insulin in sub-confluent cells. The IRES may function to maintain expression of IR protein in tissues such as the brain where mRNA translation by cap-dependent scanning is less effective. PMID:19654240

New candidate markers of head and neck squamous cell carcinoma progression

NASA Astrophysics Data System (ADS)

Kakurina, G. V.; Kolegova, E. S.; Cheremisina, O. V.; Kulbakin, D. E.; Choinzonov, E. L.

2017-09-01

The tumor progression in head and neck squamous cell carcinoma (HNSCC) is one of the main causes of high mortality of the patients with HNSCC. The tumor progression, particularly the metastasis, is characterized by the changes in the composition, functions and structure of different proteins. We have previously shown that serum of HNSCC patients contains the proteins which regulate various cellular processes—adenylyl cyclase associated protein 1 (CAP1), protein phosphatase 1 B (PPM1B), etc. The levels of CAP1 and PPM1B were determined using the enzyme immunoassay. The results of this study show that CAP1 and PPM1B take a part in the progression of HNSCC. The levels of CAP1 and PPM1B in the tumor and in morphologically normal tissue depended on the prevalence of the tumor process. The CAP1 and PPM1B levels were significantly higher in tumor tissue of the patients with regional metastasis. Our data allow assuming the potential possibility for predicting the outcome of the HNSCC measuring the level of tissue CAP1.

MoCAP proteins regulated by MoArk1-mediated phosphorylation coordinate endocytosis and actin dynamics to govern development and virulence of Magnaporthe oryzae

PubMed Central

Yang, Jun; Chen, Deng; Liu, Muxing; Zhang, Haifeng; Zheng, Xiaobo; Wang, Ping; Peng, Youliang; Zhang, Zhengguang

2017-01-01

Actin organization is a conserved cellular process that regulates the growth and development of eukaryotic cells. It also governs the virulence process of pathogenic fungi, such as the rice blast fungus Magnaporthe oryzae, with mechanisms not yet fully understood. In a previous study, we found that actin-regulating kinase MoArk1 displays conserved functions important in endocytosis and actin organization, and MoArk1 is required for maintaining the growth and full virulence of M. oryzae. To understand how MoArk1 might function, we identified capping protein homologs from M. oryzae (MoCAP) that interact with MoArk1 in vivo. MoCAP is heterodimer consisting of α and β subunits MoCapA and MoCapB. Single and double deletions of MoCAP subunits resulted in abnormal mycelial growth and conidia formation. The ΔMocap mutants also exhibited reduced appressorium penetration and invasive hyphal growth within host cells. Furthermore, the ΔMocap mutants exhibited delayed endocytosis and abnormal cytoskeleton assembly. Consistent with above findings, MoCAP proteins interacted with MoAct1, co-localized with actin during mycelial development, and participated in appressorial actin ring formation. Further analysis revealed that the S85 residue of MoCapA and the S285 residue of MoCapB were subject to phosphorylation by MoArk1 that negatively regulates MoCAP functions. Finally, the addition of exogenous phosphatidylinositol 4,5-bisphosphate (PIP2) failed to modulate actin ring formation in ΔMocap mutants, in contrast to the wild-type strain, suggesting that MoCAP may also mediate phospholipid signaling in the regulation of the actin organization. These results together demonstrate that MoCAP proteins whose functions are regulated by MoArk1 and PIP2 are important for endocytosis and actin dynamics that are directly linked to growth, conidiation and pathogenicity of M. oryzae. PMID:28542408

The Est3 protein associates with yeast telomerase through an OB-fold domain

PubMed Central

Lee, Jaesung S.; Mandell, Edward K.; Tucey, Timothy M.; Morris, Danna K.; Victoria, Lundblad

2009-01-01

The Est3 protein is a small regulatory subunit of yeast telomerase which is dispensable for enzyme catalysis but essential for telomere replication in vivo. Using structure prediction combined with in vivo characterization, we show here that Est3 consists of a predicted OB (oligo-saccharide/oligo-nucleotide binding) fold. Mutagenesis of predicted surface residues was used to generate a functional map of one surface of Est3, which identified a site that mediates association with the telomerase complex. Surprisingly, the predicted OB-fold of Est3 is structurally similar to the OB-fold of the mammalian TPP1 protein, despite the fact that Est3 and TPP1, as components of telomerase and a telomere capping complex, respectively, perform functionally distinct tasks at chromosome ends. The analysis performed on Est3 may be instructive in generating comparable missense mutations on the surface of the OB-fold domain of TPP1. PMID:19172754

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

Zhao, Baoyu; Yi, Guanghui; Du, Fenglei

The recent outbreak of Zika virus (ZIKV) has infected over 1 million people in over 30 countries. ZIKV replicates its RNA genome using virally encoded replication proteins. Nonstructural protein 5 (NS5) contains a methyltransferase for RNA capping and a polymerase for viral RNA synthesis. Here we report the crystal structures of full-length NS5 and its polymerase domain at 3.0 Å resolution. The NS5 structure has striking similarities to the NS5 protein of the related Japanese encephalitis virus. The methyltransferase contains in-line pockets for substrate binding and the active site. Key residues in the polymerase are located in similar positions tomore » those of the initiation complex for the hepatitis C virus polymerase. The polymerase conformation is affected by the methyltransferase, which enables a more efficiently elongation of RNA synthesis in vitro. Altogether, our results will contribute to future studies on ZIKV infection and the development of inhibitors of ZIKV replication.« less

NMR Model of PrgI-SipD Interaction and its Implications in the Needle-Tip Assembly of the Salmonella Type III Secretion System

PubMed Central

Rathinavelan, Thenmalarchelvi; Lara-Tejero, Maria; Lefebre, Matthew; Chatterjee, Srirupa; McShan, Andrew C.; Guo, Da-Chuan; Tang, Chun; Galan, Jorge E.; De Guzman, Roberto N.

2014-01-01

Salmonella and other pathogenic bacteria use the type III secretion system (T3SS) to inject virulence proteins into human cells to initiate infections. The structural component of the T3SS contains a needle and a needle tip. The needle is assembled from PrgI needle protomers and the needle tip is capped with several copies of the SipD tip protein. How a tip protein docks on the needle is unclear. A crystal structure of a PrgI-SipD fusion protein docked on the PrgI needle results in steric clash of SipD at the needle tip when modeled on the recent atomic structure of the needle. Thus, there is currently no good model of how SipD is docked on the PrgI needle tip. Previously, we showed by NMR paramagnetic relaxation enhancement (PRE) methods that a specific region in the SipD coiled-coil is the binding site for PrgI. Others have hypothesized that a domain of the tip protein – the N-terminal α-helical hairpin, has to swing away during the assembly of the needle apparatus. Here, we show by PRE methods that a truncated form of SipD lacking the α-helical hairpin domain binds more tightly to PrgI. Further, PRE-based structure calculations revealed multiple PrgI binding sites on the SipD coiled-coil. Our PRE results together with the recent NMR-derived atomic structure of the Salmonella needle suggest a possible model of how SipD might dock at the PrgI needle tip. SipD and PrgI are conserved in other bacterial T3SSs, thus our results have wider implication in understanding other needle-tip complexes. PMID:24951833

Identification of a negative element in the human vimentin promoter: modulation by the human T-cell leukemia virus type I Tax protein.

PubMed Central

Salvetti, A; Lilienbaum, A; Li, Z; Paulin, D; Gazzolo, L

1993-01-01

The vimentin gene is a member of the intermediate filament multigene family and encodes a protein expressed, in vivo, in all mesenchymal derivatives and, in vitro, in cell types of various origin. We have previously demonstrated that the expression of this growth-regulated gene could be trans activated by the 40-kDa Tax protein of HTLV-I (human T-cell leukemia virus type I) and that responsiveness to this viral protein was mediated by the presence of an NF-kappa B binding site located between -241 and -210 bp upstream of the mRNA cap site (A. Lilienbaum, M. Duc Dodon, C. Alexandre, L. Gazzolo, and D. Paulin, J. Virol. 64:256-263, 1990). These previous assays, performed with deletion mutants of the vimentin promoter linked to the chloramphenicol acetyltransferase gene, also revealed the presence of an upstream negative region between -529 and -241 bp. Interestingly, the inhibitory activity exerted by this negative region was overcome after cotransfection of a Tax-expressing plasmid. In this study, we further characterize the vimentin negative element and define the effect of the Tax protein on the inhibitory activity of this element. We first demonstrate that a 187-bp domain (-424 to -237 bp) behaves as a negative region when placed upstream either of the NF-kappa B binding site of vimentin or of a heterologous enhancer such as that present in the desmin gene promoter. The negative effect can be further assigned to a 32-bp element which is indeed shown to repress the basal or induced activity of the NF-kappa B binding site.(ABSTRACT TRUNCATED AT 250 WORDS) Images PMID:8417364

The X-ray Crystal Structures of Human {alpha}-Phosphomannomutase 1 Reveal the Structural Basis of Congenital Disorder of Glycosylation Type 1a

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

Silvaggi,N.; Zhang, C.; Lu, Z.

2006-01-01

Carbohydrate-deficient glycoprotein syndrome type 1a (CDG-1a) is a congenital disease characterized by severe defects in nervous system development. It is caused by mutations in alpha -phosphomannomutase (of which there are two isozymes, {alpha}-PMM1 and {alpha}-PPM2). Here we report the X-ray crystal structures of human {alpha}-PMM1 in the open conformation, with and without the bound substrate, {alpha}-D-mannose 1-phosphate. {alpha}-PMM1, like most Haloalkanoic Acid Dehalogenase Superfamily (HADSF) members, consists of two domains, the cap and core, which open to bind substrate and then close to provide a solvent exclusive environment for catalysis. The substrate phosphate group is observed at a positively chargedmore » site of the cap domain, rather than at the core domain phosphoryl-transfer site defined by the D19 nucleophile and Mg{sup 2+} cofactor. This suggests that substrate binds first to the cap and then is swept into the active site upon cap closure. The orientation of the acid/base residue D21 suggests that {alpha}-PMM uses a different method of protecting the aspartylphosphate from hydrolysis than the HADSF member {beta}-phosphoglucomutase. It is hypothesized that the electrostatic repulsion of positive charges at the interface of the cap and core domains stabilizes {alpha}-PMM1 in the open conformation, and that the negatively charged substrate binds to the cap, thereby facilitating its closure over the core domain. The two isozymes {alpha}-PMM1 and {alpha}-PMM2 are shown to have a conserved active-site structure and to display similar kinetic properties. Analysis of the known mutation sites in the context of the structures reveals the genotype-phenotype relationship underlying CDG-1a.« less

Goat's milk allergy without cow's milk allergy: suppression of non-cross-reactive epitopes on caprine β-casein.

PubMed

Hazebrouck, S; Ah-Leung, S; Bidat, E; Paty, E; Drumare, M-F; Tilleul, S; Adel-Patient, K; Wal, J-M; Bernard, H

2014-04-01

Goat's milk (GM) allergy associated with tolerance to cow's milk (CM) has been reported in patients without history of CM allergy and in CM-allergic children successfully treated with oral immunotherapy. The IgE antibodies from GM-allergic/CM-tolerant patients recognize caprine β-casein (βcap) without cross-reacting with bovine β-casein (βbov) despite a sequence identity of 91%. In this study, we investigated the non-cross-reactive IgE-binding epitopes of βcap. Recombinant βcap was genetically modified by substituting caprine domains with the bovine counterparts and by performing site-directed mutagenesis. We then evaluated the recognition of modified βcap by IgE antibodies from 11 GM-allergic/CM-tolerant patients and 11 CM-allergic patients or by monoclonal antibodies (mAb) raised against caprine caseins. The allergenic potency of modified βcap was finally assessed by degranulation tests of humanized rat basophil leukaemia (RBL)-SX38 cells. Non-cross-reactive epitopes of βcap were found in domains 44-88 and 130-178. The substitutions A55T/T63P/L75P and P148H/S152P induced the greatest decrease in IgE reactivity of GM-allergic/CM-tolerant patients towards βcap. The pivotal role of threonine 63 was particularly revealed as its substitution also impaired the recognition of βcap by specific mAb, which could discriminate between βcap and βbov. The modified βcap containing the five substitutions was then unable to trigger the degranulation of RBL-SX38 cells passively sensitized with IgE antibodies from GM-allergic/CM-tolerant patients. Although IgE-binding epitopes are spread all over βcap, a non-cross-linking version of βcap was generated with only five amino acid substitutions and could thus provide new insight for the design of hypoallergenic variants. © 2013 John Wiley & Sons Ltd.

Influenza A Virus Host Shutoff Disables Antiviral Stress-Induced Translation Arrest

PubMed Central

Khaperskyy, Denys A.; Emara, Mohamed M.; Johnston, Benjamin P.; Anderson, Paul; Hatchette, Todd F.; McCormick, Craig

2014-01-01

Influenza A virus (IAV) polymerase complexes function in the nucleus of infected cells, generating mRNAs that bear 5′ caps and poly(A) tails, and which are exported to the cytoplasm and translated by host machinery. Host antiviral defences include mechanisms that detect the stress of virus infection and arrest cap-dependent mRNA translation, which normally results in the formation of cytoplasmic aggregates of translationally stalled mRNA-protein complexes known as stress granules (SGs). It remains unclear how IAV ensures preferential translation of viral gene products while evading stress-induced translation arrest. Here, we demonstrate that at early stages of infection both viral and host mRNAs are sensitive to drug-induced translation arrest and SG formation. By contrast, at later stages of infection, IAV becomes partially resistant to stress-induced translation arrest, thereby maintaining ongoing translation of viral gene products. To this end, the virus deploys multiple proteins that block stress-induced SG formation: 1) non-structural protein 1 (NS1) inactivates the antiviral double-stranded RNA (dsRNA)-activated kinase PKR, thereby preventing eIF2α phosphorylation and SG formation; 2) nucleoprotein (NP) inhibits SG formation without affecting eIF2α phosphorylation; 3) host-shutoff protein polymerase-acidic protein-X (PA-X) strongly inhibits SG formation concomitant with dramatic depletion of cytoplasmic poly(A) RNA and nuclear accumulation of poly(A)-binding protein. Recombinant viruses with disrupted PA-X host shutoff function fail to effectively inhibit stress-induced SG formation. The existence of three distinct mechanisms of IAV-mediated SG blockade reveals the magnitude of the threat of stress-induced translation arrest during viral replication. PMID:25010204

A cyclodextrin-capped histone deacetylase inhibitor.

PubMed

Amin, Jahangir; Puglisi, Antonino; Clarke, James; Milton, John; Wang, Minghua; Paranal, Ronald M; Bradner, James E; Spencer, John

2013-06-01

We have synthesized a β-cyclodextrin (βCD)-capped histone deacetylase (HDAC) inhibitor 3 containing an alkyl linker and a zinc-binding hydroxamic acid motif. Biological evaluation (HDAC inhibition studies) of 3 enabled us to establish the effect of replacing an aryl cap (in SAHA (vorinostat,)) 1 by a large saccharidic scaffold "cap". HDAC inhibition was observed for 3, to a lesser extent than SAHA, and rationalized by molecular docking into the active site of HDAC8. However, compound 3 displayed no cellular activity. Copyright © 2013 Elsevier Ltd. All rights reserved.

Identification of liver protein targets modified by tienilic acid metabolites using a two-dimensional Western blot-mass spectrometry approach

NASA Astrophysics Data System (ADS)

Methogo, Ruth Menque; Dansette, Patrick M.; Klarskov, Klaus

2007-12-01

A combined approach based on two-dimensional electrophoresis-immuno-blotting and nanoliquid chromatography coupled on-line with electrospray ionization mass spectrometry (nLC-MS/MS) was used to identify proteins modified by a reactive intermediate of tienilic acid (TA). Liver homogenates from rats exposed to TA were fractionated using ultra centrifugation; four fractions were obtained and subjected to 2D electrophoresis. Following transfer to PVDF membranes, modified proteins were visualized after India ink staining, using an anti-serum raised against TA and ECL detection. Immuno-reactive spots were localized on the PVDF membrane by superposition of the ECL image, protein spots of interest were excised, digested on the membrane with trypsin followed by nLC-MS/MS analysis and protein identification. A total of 15 proteins were identified as likely targets modified by a TA reactive metabolite. These include selenium binding protein 2, senescence marker protein SMP-30, adenosine kinase, Acy1 protein, adenosylhomocysteinase, capping protein (actin filament), protein disulfide isomerase, fumarylacetoacetase, arginase chain A, ketohexokinase, proteasome endopeptidase complex, triosephosphate isomerase, superoxide dismutase, dna-type molecular chaperone hsc73 and malate dehydrogenase.

Fibroblast activation protein is induced by inflammation and degrades type I collagen in thin-cap fibroatheromata

PubMed Central

Brokopp, Chad E.; Schoenauer, Roman; Richards, Peter; Bauer, Stefan; Lohmann, Christine; Emmert, Maximilian Y.; Weber, Benedikt; Winnik, Stephan; Aikawa, Elena; Graves, Kirk; Genoni, Michele; Vogt, Peter; Lüscher, Thomas F.; Renner, Christoph; Hoerstrup, Simon P.; Matter, Christian M.

2011-01-01

Aims Collagen degradation in atherosclerotic plaques with thin fibrous caps renders them more prone to rupture. Fibroblast activation protein (FAP) plays a role in arthritis and tumour formation through its collagenase activity. However, the significance of FAP in thin-cap human fibroatheromata remains unknown. Methods and results We detected enhanced FAP expression in type IV–V human aortic atheromata (n = 12), compared with type II–III lesions (n = 9; P < 0.01) and healthy aortae (n = 8; P < 0.01) by immunostaining and western blot analyses. Fibroblast activation protein was also increased in thin-cap (<65 µm) vs. thick-cap (≥65 µm) human coronary fibroatheromata (n = 12; P < 0.01). Fibroblast activation protein was expressed by human aortic smooth muscle cells (HASMC) as shown by colocalization on immunofluorescent aortic plaque stainings (n = 10; P < 0.01) and by flow cytometry in cell culture. Although macrophages did not express FAP, macrophage burden in human aortic plaques correlated with FAP expression (n = 12; R2= 0.763; P < 0.05). Enzyme-linked immunosorbent assays showed a time- and dose-dependent up-regulation of FAP in response to human tumour necrosis factor α (TNFα) in HASMC (n = 6; P < 0.01). Moreover, supernatants from peripheral blood-derived macrophages induced FAP expression in cultured HASMC (n = 6; P < 0.01), an effect abolished by blocking TNFα (n = 6; P < 0.01). Fibroblast activation protein associated with collagen-poor regions in human coronary fibrous caps and digested type I collagen and gelatin in vitro (n = 6; P < 0.01). Zymography revealed that FAP-mediated collagenase activity was neutralized by an antibody directed against the FAP catalytic domain both in HASMC (n = 6; P < 0.01) and in fibrous caps of atherosclerotic plaques (n = 10; P < 0.01). Conclusion Fibroblast activation protein expression in HASMC is induced by macrophage-derived TNFα. Fibroblast activation protein associates with thin-cap human coronary fibroatheromata and contributes to type I collagen breakdown in fibrous caps. PMID:21292680

New insights into respirable protein powder preparation using a nano spray dryer.

PubMed

Bürki, K; Jeon, I; Arpagaus, C; Betz, G

2011-04-15

In this study the Nano Spray Dryer B-90 (BÜCHI Labortechnik AG, Flawil, Switzerland) was evaluated with regard to the drying of proteins and the preparation of respirable powders in the size range of 1-5 μm. β-galactosidase was chosen as a model protein and trehalose was added as a stabilizer. The influence of inlet temperature, hole size of the spray cap membrane and ethanol concentration in the spray solution was studied using a 3³ full factorial design. The investigated responses were enzyme activity, particle size, span, yield and shelf life. Furthermore, the particle morphology was examined. The inlet temperature as well as the interaction of inlet temperature and spray cap size significantly influenced the enzyme activity. Full activity was retained with the optimized process. The particle size was affected by the hole size of the spray cap membrane and the ethanol content. The smallest cap led to a monodisperse particle size distribution and the greatest yield of particles of respirable size. Higher product recovery was achieved with lower inlet temperatures, higher ethanol contents and smaller cap sizes. Particle morphology differed depending on the cap size. The protein exhibited higher storage stability when spray dried without ethanol and when a larger spray cap size was used. Copyright © 2011 Elsevier B.V. All rights reserved.

A pH-activatable nanoparticle with signal-amplification capabilities for non-invasive imaging of tumour malignancy.

PubMed

Mi, Peng; Kokuryo, Daisuke; Cabral, Horacio; Wu, Hailiang; Terada, Yasuko; Saga, Tsuneo; Aoki, Ichio; Nishiyama, Nobuhiro; Kataoka, Kazunori

2016-08-01

Engineered nanoparticles that respond to pathophysiological parameters, such as pH or redox potential, have been developed as contrast agents for the magnetic resonance imaging (MRI) of tumours. However, beyond anatomic assessment, contrast agents that can sense these pathological parameters and rapidly amplify their magnetic resonance signals are desirable because they could potentially be used to monitor the biological processes of tumours and improve cancer diagnosis. Here, we report an MRI contrast agent that rapidly amplifies magnetic resonance signals in response to pH. We confined Mn(2+) within pH-sensitive calcium phosphate (CaP) nanoparticles comprising a poly(ethylene glycol) shell. At a low pH, such as in solid tumours, the CaP disintegrates and releases Mn(2+) ions. Binding to proteins increases the relaxivity of Mn(2+) and enhances the contrast. We show that these nanoparticles could rapidly and selectively brighten solid tumours, identify hypoxic regions within the tumour mass and detect invisible millimetre-sized metastatic tumours in the liver.

Insights into Cdc13 Dependent Telomere Length Regulation

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

M Mason; E Skordalakes

Cdc13 is a single stranded telomere binding protein that specifically localizes to the telomere ends of budding yeasts and is essential for cell viability. It caps the ends of chromosomes thus preventing chromosome end-to-end fusions and exonucleolytic degradation, events that could lead to genomic instability and senescence, the hallmark of aging. Cdc13 is also involved in telomere length regulation by recruiting or preventing access of telomerase to the telomeric overhang. Recruitment of telomerase to the telomeres for G-strand extension is required for continuous cell division, while preventing its access to the telomeres through capping the chromosome ends prevents mitotic eventsmore » that could lead to cell immortality, the hall mark of carcinogenesis. Cdc13 and its putative homologues human CTC1 and POT1 are therefore key to many biological processes directly associated with life extension and cancer prevention and can be viewed as an ideal target for cancer and age related therapies.« less

A pH-activatable nanoparticle with signal-amplification capabilities for non-invasive imaging of tumour malignancy

NASA Astrophysics Data System (ADS)

Mi, Peng; Kokuryo, Daisuke; Cabral, Horacio; Wu, Hailiang; Terada, Yasuko; Saga, Tsuneo; Aoki, Ichio; Nishiyama, Nobuhiro; Kataoka, Kazunori

2016-08-01

Engineered nanoparticles that respond to pathophysiological parameters, such as pH or redox potential, have been developed as contrast agents for the magnetic resonance imaging (MRI) of tumours. However, beyond anatomic assessment, contrast agents that can sense these pathological parameters and rapidly amplify their magnetic resonance signals are desirable because they could potentially be used to monitor the biological processes of tumours and improve cancer diagnosis. Here, we report an MRI contrast agent that rapidly amplifies magnetic resonance signals in response to pH. We confined Mn2+ within pH-sensitive calcium phosphate (CaP) nanoparticles comprising a poly(ethylene glycol) shell. At a low pH, such as in solid tumours, the CaP disintegrates and releases Mn2+ ions. Binding to proteins increases the relaxivity of Mn2+ and enhances the contrast. We show that these nanoparticles could rapidly and selectively brighten solid tumours, identify hypoxic regions within the tumour mass and detect invisible millimetre-sized metastatic tumours in the liver.

Comparative Plasmodium gene overexpression reveals distinct perturbation of sporozoite transmission by profilin.

PubMed

Sato, Yuko; Hliscs, Marion; Dunst, Josefine; Goosmann, Christian; Brinkmann, Volker; Montagna, Georgina N; Matuschewski, Kai

2016-07-15

Plasmodium relies on actin-based motility to migrate from the site of infection and invade target cells. Using a substrate-dependent gliding locomotion, sporozoites are able to move at fast speed (1-3 μm/s). This motility relies on a minimal set of actin regulatory proteins and occurs in the absence of detectable filamentous actin (F-actin). Here we report an overexpression strategy to investigate whether perturbations of F-actin steady-state levels affect gliding locomotion and host invasion. We selected two vital Plasmodium berghei G-actin-binding proteins, C-CAP and profilin, in combination with three stage-specific promoters and mapped the phenotypes afforded by overexpression in all three extracellular motile stages. We show that in merozoites and ookinetes, additional expression does not impair life cycle progression. In marked contrast, overexpression of C-CAP and profilin in sporozoites impairs circular gliding motility and salivary gland invasion. The propensity for productive motility correlates with actin accumulation at the parasite tip, as revealed by combinations of an actin-stabilizing drug and transgenic parasites. Strong expression of profilin, but not C-CAP, resulted in complete life cycle arrest. Comparative overexpression is an alternative experimental genetic strategy to study essential genes and reveals effects of regulatory imbalances that are not uncovered from deletion-mutant phenotyping. © 2016 Sato et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

7-methylguanosine diphosphate (m(7)GDP) is not hydrolyzed but strongly bound by decapping scavenger (DcpS) enzymes and potently inhibits their activity.

PubMed

Wypijewska, Anna; Bojarska, Elzbieta; Lukaszewicz, Maciej; Stepinski, Janusz; Jemielity, Jacek; Davis, Richard E; Darzynkiewicz, Edward

2012-10-09

Decapping scavenger (DcpS) enzymes catalyze the cleavage of a residual cap structure following 3' → 5' mRNA decay. Some previous studies suggested that both m(7)GpppG and m(7)GDP were substrates for DcpS hydrolysis. Herein, we show that mononucleoside diphosphates, m(7)GDP (7-methylguanosine diphosphate) and m(3)(2,2,7)GDP (2,2,7-trimethylguanosine diphosphate), resulting from mRNA decapping by the Dcp1/2 complex in the 5' → 3' mRNA decay, are not degraded by recombinant DcpS proteins (human, nematode, and yeast). Furthermore, whereas mononucleoside diphosphates (m(7)GDP and m(3)(2,2,7)GDP) are not hydrolyzed by DcpS, mononucleoside triphosphates (m(7)GTP and m(3)(2,2,7)GTP) are, demonstrating the importance of a triphosphate chain for DcpS hydrolytic activity. m(7)GTP and m(3)(2,2,7)GTP are cleaved at a slower rate than their corresponding dinucleotides (m(7)GpppG and m(3)(2,2,7)GpppG, respectively), indicating an involvement of the second nucleoside for efficient DcpS-mediated digestion. Although DcpS enzymes cannot hydrolyze m(7)GDP, they have a high binding affinity for m(7)GDP and m(7)GDP potently inhibits DcpS hydrolysis of m(7)GpppG, suggesting that m(7)GDP may function as an efficient DcpS inhibitor. Our data have important implications for the regulatory role of m(7)GDP in mRNA metabolic pathways due to its possible interactions with different cap-binding proteins, such as DcpS or eIF4E.

Light-regulated protein and mRNA synthesis in root caps of maize

NASA Technical Reports Server (NTRS)

Feldman, L. J.; Piechulla, B.; Sun, P. S.

1988-01-01

Illumination of maize roots initiates changes in mRNA levels and in the activities of proteins within the root cap. Using Northern analysis we showed a 5-6 fold increase in the levels of three specific mRNAs and a 14-fold increase in plastid mRNA. This increase is rapid, occurring within 30 minutes of illumination. With prolonged periods of darkness following illumination, messages return to levels observed in dark, control caps. For two species of mRNA illumination results in a reduction in message levels. Light-stimulated increases in the levels of specific mRNAs are proportionally greater than are increases in the activities of corresponding proteins. We suggest that the light-stimulated increase in protein activity in root caps may be preceded by and occur as a consequence of enhanced levels of mRNA. Our work suggests that photomorphogenesis in roots could involve changes in the levels of a wide variety of mRNAs within the root cap.

Comparison of N-terminal modifications on neurotensin(8-13) analogues correlates peptide stability but not binding affinity with in vivo efficacy.

PubMed

Orwig, Kevin S; Lassetter, McKensie R; Hadden, M Kyle; Dix, Thomas A

2009-04-09

Neurotensin(8-13) and two related analogues were used as model systems to directly compare various N-terminal peptide modifications representing both commonly used and novel capping groups. Each N-terminal modification prevented aminopeptidase cleavage but surprisingly differed in its ability to inhibit cleavage at other sites, a phenomenon attributed to long-range conformational effects. None of the capping groups were inherently detrimental to human neurotensin receptor 1 (hNTR1) binding affinity or receptor agonism. Although the most stable peptides exhibited the lowest binding affinities and were the least potent receptor agonists, they produced the largest in vivo effects. Of the parameters studied only stability significantly correlated with in vivo efficacy, demonstrating that a reduction in binding affinity at NTR1 can be countered by increased in vivo stability.

Conformational changes induced in the eukaryotic translation initiation factor eIF4E by a clinically relevant inhibitor, ribavirin triphosphate

PubMed Central

Volpon, Laurent; Osborne, Michael J.; Zahreddine, Hiba; Romeo, Andrea A.; Borden, Katherine L.B.

2013-01-01

The eukaryotic translation initiation factor eIF4E is highly elevated in human cancers including acute myeloid leukemia (AML). A potential anticancer agent, ribavirin, targets eIF4E activity in AML patients corresponding to clinical responses. To date, ribavirin is the only direct inhibitor of eIF4E to reach clinical trials. We showed that ribavirin acts as a competitive inhibitor of the methyl 7-guanosine (m7G) cap, the natural ligand of eIF4E. Here we examine the conformational changes occurring in human eIF4E upon binding the active metabolite of ribavirin, ribavirin triphosphate (RTP). Our NMR data revealed an unexpected concentration dependence on RTP affinity for eIF4E. We observed NMR spectra characteristic of tight binding at low micromolar concentrations (2-5μM eIF4E) but much weaker affinity at more typical NMR concentrations (50-200μM). Comparison of chemical shift perturbation and line broadening suggest that the two eIF4E-RTP complexes differ in the precise positioning of RTP within the cap binding pocket, with the high affinity complex showing more extensive changes to the central β-sheet and dorsal surface of eIF4E, similar to m7G cap. The differences between high and low affinity complexes arise due to concentration dependent aggregation of eIF4E and RTP. Given the intracellular concentrations of eIF4E and RTP and the differential binding toward the W56A eIF4E mutant the high affinity complex is the most physiologically relevant. In summary, these findings demonstrate that RTP binds in the cap-binding site but also suggests new features of this pocket that should be considered in both drug design efforts and reveal new insights into ligand eIF4E recognition. PMID:23583375

Relative stability of the open and closed conformations of the active site loop of streptavidin

NASA Astrophysics Data System (ADS)

Ignacio J., General; Meirovitch, Hagai

2011-01-01

The eight-residue surface loop, 45-52 (Ser, Ala, Val, Gly, Asn, Ala, Glu, Ser), of the homotetrameric protein streptavidin has a "closed" conformation in the streptavidin-biotin complex, where the corresponding binding affinity is one of the strongest found in nature (ΔG ˜ -18 kcal/mol). However, in most of the crystal structures of apo (unbound) streptavidin, the loop conformation is "open" and typically exhibits partial disorder and high B-factors. Thus, it is plausible to assume that the loop structure is changed from open to closed upon binding of biotin, and the corresponding difference in free energy, ΔF = Fopen - Fclosed in the unbound protein, should therefore be considered in the total absolute free energy of binding. ΔF (which has generally been neglected) is calculated here using our "hypothetical scanning molecular-dynamics" (HSMD) method. We use a protein model in which only the atoms closest to the loop are considered (the "template") and they are fixed in the x-ray coordinates of the free protein; the x-ray conformation of the closed loop is attached to the same (unbound) template and both systems are capped with the same sphere of TIP3P water. Using the force field of the assisted model building with energy refinement (AMBER), we carry out two separate MD simulations (at temperature T = 300 K), starting from the open and closed conformations, where only the atoms of the loop and water are allowed to move (the template-water and template-loop interactions are considered). The absolute Fopen and Fclosed (of loop + water) are calculated from these trajectories, where the loop and water contributions are obtained by HSMD and a thermodynamic integration (TI) process, respectively. The combined HSMD-TI procedure leads to total (loop + water) ΔF = -27.1 ± 2.0 kcal/mol, where the entropy TΔS constitutes 34% of ΔF, meaning that the effect of S is significant and should not be ignored. Also, ΔS is positive, in accord with the high flexibility of the open loop observed in crystal structures, while the energy ΔE is unexpectedly negative, thus also adding to the stability of the open loop. The loop and the 250 capped water molecules are the largest system studied thus far, which constitutes a test for the efficiency of HSMD-TI; this efficiency and technical issues related to the implementation of the method are also discussed. Finally, the result for ΔF is a prediction that will be considered in the calculation of the absolute free energy of binding of biotin to streptavidin, which constitutes our next project.

FRET Studies Between CdTe Capped by Small-Molecule Ligands and Fluorescent Protein

NASA Astrophysics Data System (ADS)

Zhang, Yue; Zhou, Dejian; He, Junhui

2014-12-01

Water-soluble luminescent semiconductor nanocrystals also known as quantum dots (QDs) that have prominent photostability, wide absorption cross sections and tunable narrow emission, have been shown as promising probes in immunoassays. QDs are often used as donors in fluorescence resonance energy transfer (FRET) based sensors using organic dyes or fluorescent proteins as acceptors. Here, the FRET between a QD donor and fluorescent protein acceptors has been studied. The fluorescent protein (FP)mCherry appended with a hexa-histidine-tag could effectively self-assemble onto CdTe to produce small donor-acceptor distances and hence highly efficient FRET (efficiency > 80%) at relatively low FP:CdTe copy numbers (ca.1). Using the Förster dipole-dipole interaction formula, the Förster radius (R0) and respective donor-acceptor distances for the CdTe-FP FRET systems have been calculated. The binding constants (Kd) of the QD-FP systems have also been evaluated by the emission spectra.

Structure and function of the Zika virus full-length NS5 protein

DOE PAGES

Zhao, Baoyu; Yi, Guanghui; Du, Fenglei; ...

2017-03-27

The recent outbreak of Zika virus (ZIKV) has infected over 1 million people in over 30 countries. ZIKV replicates its RNA genome using virally encoded replication proteins. Nonstructural protein 5 (NS5) contains a methyltransferase for RNA capping and a polymerase for viral RNA synthesis. Here we report the crystal structures of full-length NS5 and its polymerase domain at 3.0 Å resolution. The NS5 structure has striking similarities to the NS5 protein of the related Japanese encephalitis virus. The methyltransferase contains in-line pockets for substrate binding and the active site. Key residues in the polymerase are located in similar positions tomore » those of the initiation complex for the hepatitis C virus polymerase. The polymerase conformation is affected by the methyltransferase, which enables a more efficiently elongation of RNA synthesis in vitro. Altogether, our results will contribute to future studies on ZIKV infection and the development of inhibitors of ZIKV replication.« less

Controllable synthesis of protein-conjugated lead sulfide nanocubes by using bovine hemoglobin as a capping agent

NASA Astrophysics Data System (ADS)

Yang, Guangrui; Qin, Dezhi; Zhang, Li

2014-06-01

A simple, convenient, and controllable strategy was reported in this contribution for protein-assisted synthesis BHb-conjugated PbS nanocubes. Powder X-ray diffraction, energy disperse X-ray spectroscopy, transmission electron microscopy, high-resolution transmission electron microscopy, and selected-area electron diffraction characterizations were used to determine the structure and morphology of BHb-conjugated PbS nanocubes. The prepared PbS nanocrystals with cubic rock salt structure were uniform and monodispersed with homogeneous size around 12 nm. The results of Fourier transform infrared and circular dichroism assay proved that Pb2+/PbS had coordination interaction with functional groups of BHb besides physical-binding effect, and the secondary structure of protein significantly changed with this interaction. Thermogravimetric analysis results confirmed the existence of BHb in PbS nanocrystals and indicated that the conjugate bonds existed between PbS and BHb. A clear perspective was shown here that special nanostructure could be created by using proteins as a mediating template at the inorganic-organic interface.

Toward the mechanism of eIF4F-mediated ribosomal attachment to mammalian capped mRNAs.

PubMed

Kumar, Parimal; Hellen, Christopher U T; Pestova, Tatyana V

2016-07-01

Ribosomal attachment to mammalian capped mRNAs is achieved through the cap-eukaryotic initiation factor 4E (eIF4E)-eIF4G-eIF3-40S chain of interactions, but the mechanism by which mRNA enters the mRNA-binding channel of the 40S subunit remains unknown. To investigate this process, we recapitulated initiation on capped mRNAs in vitro using a reconstituted translation system. Formation of initiation complexes at 5'-terminal AUGs was stimulated by the eIF4E-cap interaction and followed "the first AUG" rule, indicating that it did not occur by backward scanning. Initiation complexes formed even at the very 5' end of mRNA, implying that Met-tRNAi (Met) inspects mRNA from the first nucleotide and that initiation does not have a "blind spot." In assembled initiation complexes, the cap was no longer associated with eIF4E. Omission of eIF4A or disruption of eIF4E-eIF4G-eIF3 interactions converted eIF4E into a specific inhibitor of initiation on capped mRNAs. Taken together, these results are consistent with the model in which eIF4E-eIF4G-eIF3-40S interactions place eIF4E at the leading edge of the 40S subunit, and mRNA is threaded into the mRNA-binding channel such that Met-tRNAi (Met) can inspect it from the first nucleotide. Before entering, eIF4E likely dissociates from the cap to overcome steric hindrance. We also found that the m(7)G cap specifically interacts with eIF3l. © 2016 Kumar et al.; Published by Cold Spring Harbor Laboratory Press.

A systematic analysis of atomic protein-ligand interactions in the PDB.

PubMed

Ferreira de Freitas, Renato; Schapira, Matthieu

2017-10-01

As the protein databank (PDB) recently passed the cap of 123 456 structures, it stands more than ever as an important resource not only to analyze structural features of specific biological systems, but also to study the prevalence of structural patterns observed in a large body of unrelated structures, that may reflect rules governing protein folding or molecular recognition. Here, we compiled a list of 11 016 unique structures of small-molecule ligands bound to proteins - 6444 of which have experimental binding affinity - representing 750 873 protein-ligand atomic interactions, and analyzed the frequency, geometry and impact of each interaction type. We find that hydrophobic interactions are generally enriched in high-efficiency ligands, but polar interactions are over-represented in fragment inhibitors. While most observations extracted from the PDB will be familiar to seasoned medicinal chemists, less expected findings, such as the high number of C-H···O hydrogen bonds or the relatively frequent amide-π stacking between the backbone amide of proteins and aromatic rings of ligands, uncover underused ligand design strategies.

Understanding the self-assembly of proteins onto gold nanoparticles and quantum dots driven by metal-histidine coordination.

PubMed

Aldeek, Fadi; Safi, Malak; Zhan, Naiqian; Palui, Goutam; Mattoussi, Hedi

2013-11-26

Coupling of polyhistidine-appended biomolecules to inorganic nanocrystals driven by metal-affinity interactions is a greatly promising strategy to form hybrid bioconjugates. It is simple to implement and can take advantage of the fact that polyhistidine-appended proteins and peptides are routinely prepared using well established molecular engineering techniques. A few groups have shown its effectiveness for coupling proteins onto Zn- or Cd-rich semiconductor quantum dots (QDs). Expanding this conjugation scheme to other metal-rich nanoparticles (NPs) such as AuNPs would be of great interest to researchers actively seeking effective means for interfacing nanostructured materials with biology. In this report, we investigated the metal-affinity driven self-assembly between AuNPs and two engineered proteins, a His7-appended maltose binding protein (MBP-His) and a fluorescent His6-terminated mCherry protein. In particular, we investigated the influence of the capping ligand affinity to the nanoparticle surface, its density, and its lateral extension on the AuNP-protein self-assembly. Affinity gel chromatography was used to test the AuNP-MPB-His7 self-assembly, while NP-to-mCherry-His6 binding was evaluated using fluorescence measurements. We also assessed the kinetics of the self-assembly between AuNPs and proteins in solution, using time-dependent changes in the energy transfer quenching of mCherry fluorescent proteins as they immobilize onto the AuNP surface. This allowed determination of the dissociation rate constant, Kd(-1) ∼ 1-5 nM. Furthermore, a close comparison of the protein self-assembly onto AuNPs or QDs provided additional insights into which parameters control the interactions between imidazoles and metal ions in these systems.

The Role of RCAS1 and oxytocinase in immune tolerance during pregnancy.

PubMed

Wicherek, L; Dutsch-Wicherek, M; Mak, P; Klimek, M

2005-01-01

To determine and compare the level of RCAS1 (receptor-binding cancer antigen expressed in SiSo cells) in placentas at term as well as oxytocinase/cystine amino peptidase (CAP) serum level a few days before labor in order to evaluate their possible role in the regulation of maternal immune response during pregnancy and in initiation of labor. We estimated the RCAS1 content in 44 placental tissue samples, using Western blot method. We also assessed CAP serum level by its enzymatic activity, using L-cystine-di-beta-naphthylamide as a synthetic substrate. The statistical analysis was performed using Shapiro-Wilk procedure. Student's t test was applied to compare the differences between parametric data. A value of p < 0.05 was considered significant. RCAS1 was found in all placental tissue samples examined. The differences in the RCAS1 relative amount depended on the onset of labor, with the highest level in induced labor and the lowest in spontaneous labor. The differences were also observed in the CAP serum level with the highest level in pregnant women whose labor was induced. We have observed a link between the expression of the two proteins examined and the onset of the labor. Therefore, we posit that RCAS1 and CAP may play a role in the downregulation of the maternal immune response during pregnancy and may participate in the initiation of the labor. Copyright (c) 2005 S. Karger AG, Basel.

Human recombinant cementum attachment protein (hrPTPLa/CAP) promotes hydroxyapatite crystal formation in vitro and bone healing in vivo.

PubMed

Montoya, Gonzalo; Arenas, Jesús; Romo, Enrique; Zeichner-David, Margarita; Alvarez, Marco; Narayanan, A Sampath; Velázquez, Ulises; Mercado, Gabriela; Arzate, Higinio

2014-12-01

Cementum extracellular matrix is similar to other mineralized tissues; however, this unique tissue contains molecules only present in cementum. A cDNA of these molecules, cementum attachment protein (hrPTPLa/CAP) was cloned and expressed in a prokaryotic system. This molecule is an alternative splicing of protein tyrosine phosphatase-like A (PTPLa). In this study, we wanted to determine the structural and functional characteristics of this protein. Our results indicate that hrPTPLa/CAP contains a 43.2% α-helix, 8.9% β-sheet, 2% β-turn and 45.9% random coil secondary structure. Dynamic light scattering shows that this molecule has a size distribution of 4.8 nm and aggregates as an estimated mass of 137 kDa species. AFM characterization and FE-SEM studies indicate that this protein self-assembles into nanospheres with sizes ranging from 7.0 to 27 nm in diameter. Functional studies demonstrate that hrPTPLa/CAP promotes hydroxyapatite crystal nucleation: EDS analysis revealed that hrPTPLa/CAP-induced crystals had a 1.59 ± 0.06 Ca/P ratio. Further confirmation with MicroRaman spectrometry and TEM confirm the presence of hydroxyapatite. In vivo studies using critical-size defects in rat cranium showed that hrPTPLa/CAP promoted 73% ± 2.19% and 87% ± 1.97% new bone formation at 4 and 8 weeks respectively. Although originally identified in cementum, PTPLa/CAP is very effective at inducing bone repair and healing and therefore this novel molecule has a great potential to be used for mineralized tissue bioengineering and tissue regeneration. Copyright © 2014 Elsevier Inc. All rights reserved.

Resident CAPS on dense-core vesicles docks and primes vesicles for fusion

PubMed Central

Kabachinski, Greg; Kielar-Grevstad, D. Michelle; Zhang, Xingmin; James, Declan J.; Martin, Thomas F. J.

2016-01-01

The Ca2+-dependent exocytosis of dense-core vesicles in neuroendocrine cells requires a priming step during which SNARE protein complexes assemble. CAPS (aka CADPS) is one of several factors required for vesicle priming; however, the localization and dynamics of CAPS at sites of exocytosis in live neuroendocrine cells has not been determined. We imaged CAPS before, during, and after single-vesicle fusion events in PC12 cells by TIRF micro­scopy. In addition to being a resident on cytoplasmic dense-core vesicles, CAPS was present in clusters of approximately nine molecules near the plasma membrane that corresponded to docked/tethered vesicles. CAPS accompanied vesicles to the plasma membrane and was present at all vesicle exocytic events. The knockdown of CAPS by shRNA eliminated the VAMP-2–dependent docking and evoked exocytosis of fusion-competent vesicles. A CAPS(ΔC135) protein that does not localize to vesicles failed to rescue vesicle docking and evoked exocytosis in CAPS-depleted cells, showing that CAPS residence on vesicles is essential. Our results indicate that dense-core vesicles carry CAPS to sites of exocytosis, where CAPS promotes vesicle docking and fusion competence, probably by initiating SNARE complex assembly. PMID:26700319

Adenylyl cyclase-associated protein-1/CAP1 as a biological target substrate of gelatinase B/MMP-9

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

Cauwe, Benedicte; Martens, Erik; Van den Steen, Philippe E.

2008-09-10

Matrix metalloproteinases (MMPs) are classically associated with the turnover of secreted structural and functional proteins. Although MMPs have been shown to process also a kaleidoscope of membrane-associated substrates, little is known about the processing of intracellular proteins by MMPs. Physiological and pathological cell apoptosis, necrosis and tumor lysis by chemotherapy, radiotherapy or immunological cytotoxicity, are examples of conditions in which an overload of intracellular proteins becomes accessible to the action of MMPs. We used a model system of dying human myelomonocytic cells to study the processing of intracellular protein substrates by gelatinase B/MMP-9 in vitro. Adenylyl cyclase-associated protein-1 or CAP1more » was identified as a novel and most efficient substrate of gelatinase B/MMP-9. The presence of CAP1 in the extracellular milieu in vivo was documented by analysis of urine of patients with systemic autoimmune diseases. Whereas no active MMP-9 could be detected in urines of healthy controls, all urine samples of patients with clinical parameters of renal failure contained activated MMP-9 and/or MMP-2. In addition, in some of these patients indications of CAP1 cleavage are observed, implying CAP1 degradation in vivo. The high turnover rate of CAP1 by MMP-9, comparable to that of gelatin as the natural extracellular substrate of this enzyme, may be critical to prevent pathological conditions associated with considerable cytolysis.« less

Receptor clustering drives polarized assembly of ankyrin.

PubMed

Jefford, G; Dubreuil, R R

2000-09-08

Expression of the L1 family cell adhesion molecule neuroglian in Drosophila S2 cells leads to cell aggregation and polarized ankyrin accumulation at sites of cell-cell contact. Thus neuroglian adhesion generates a spatial cue for polarized assembly of ankyrin and the spectrin cytoskeleton. Here we characterized a chimera of the extracellular and transmembrane domains of rat CD2 fused to the cytoplasmic domain of neuroglian. The chimera was used to test the hypothesis that clustering of neuroglian at sites of adhesion generates the signal that activates ankyrin binding. Abundant expression of the chimera at the plasma membrane was not a sufficient cue to drive ankyrin assembly, since ankyrin remained diffusely distributed throughout the cytoplasm of CD2-neuroglian-expressing cells. However, ankyrin became highly enriched at sites of antibody-induced capping of CD2-neuroglian. Spectrin codistributed with ankyrin at capped sites. A green fluorescent protein-tagged ankyrin was used to monitor ankyrin distribution in living cells. Enhanced green fluorescent protein-ankyrin behaved identically to antibody-stained endogenous ankyrin, proving that the polarized accumulation of ankyrin was not an artifact of fixing and staining cells. We propose a model in which clustering of neuroglian induces a conformational change in the cytoplasmic domain that drives polarized assembly of the spectrin cytoskeleton.

Replication Protein A-1 Has a Preference for the Telomeric G-rich Sequence in Trypanosoma cruzi.

PubMed

Pavani, Raphael Souza; Vitarelli, Marcela O; Fernandes, Carlos A H; Mattioli, Fabio F; Morone, Mariana; Menezes, Milene C; Fontes, Marcos R M; Cano, Maria Isabel N; Elias, Maria Carolina

2018-05-01

Replication protein A (RPA), the major eukaryotic single-stranded binding protein, is a heterotrimeric complex formed by RPA-1, RPA-2, and RPA-3. RPA is a fundamental player in replication, repair, recombination, and checkpoint signaling. In addition, increasing evidences have been adding functions to RPA in telomere maintenance, such as interaction with telomerase to facilitate its activity and also involvement in telomere capping in some conditions. Trypanosoma cruzi, the etiological agent of Chagas disease is a protozoa parasite that appears early in the evolution of eukaryotes. Recently, we have showed that T. cruziRPA presents canonical functions being involved with DNA replication and DNA damage response. Here, we found by FISH/IF assays that T. cruziRPA localizes at telomeres even outside replication (S) phase. In vitro analysis showed that one telomeric repeat is sufficient to bind RPA-1. Telomeric DNA induces different secondary structural modifications on RPA-1 in comparison with other types of DNA. In addition, RPA-1 presents a higher affinity for telomeric sequence compared to randomic sequence, suggesting that RPA may play specific roles in T. cruzi telomeric region. © 2017 The Author(s) Journal of Eukaryotic Microbiology © 2017 International Society of Protistologists.

Transcriptome-wide studies uncover the diversity of modes of mRNA recruitment to eukaryotic ribosomes.

PubMed

Shatsky, Ivan N; Dmitriev, Sergey E; Andreev, Dmitri E; Terenin, Ilya M

2014-01-01

The conventional paradigm of translation initiation in eukaryotes states that the cap-binding protein complex eIF4F (consisting of eIF4E, eIF4G and eIF4A) plays a central role in the recruitment of capped mRNAs to ribosomes. However, a growing body of evidence indicates that this paradigm should be revised. This review summarizes the data which have been mostly accumulated in a post-genomic era owing to revolutionary techniques of transcriptome-wide analysis. Unexpectedly, these techniques have uncovered remarkable diversity in the recruitment of cellular mRNAs to eukaryotic ribosomes. These data enable a preliminary classification of mRNAs into several groups based on their requirement for particular components of eIF4F. They challenge the widely accepted concept which relates eIF4E-dependence to the extent of secondary structure in the 5' untranslated regions of mRNAs. Moreover, some mRNA species presumably recruit ribosomes to their 5' ends without the involvement of either the 5' m(7)G-cap or eIF4F but instead utilize eIF4G or eIF4G-like auxiliary factors. The long-standing concept of internal ribosome entry site (IRES)-elements in cellular mRNAs is also discussed.

Capsaicin modulates acetylcholine release at the myoneural junction.

PubMed

Thyagarajan, Baskaran; Potian, Joseph G; Baskaran, Padmamalini; McArdle, Joseph J

2014-12-05

Transient receptor potential (TRP) proteins are non-selective cation channel proteins that are expressed throughout the body. Previous studies demonstrated the expression of TRP Vanilloid 1 (TRPV1), capsaicin (CAP) receptor, in sensory neurons. Recently, we reported TRPV1 expression in mouse motor nerve terminals [MNTs; (Thyagarajan et al., 2009)], where we observed that CAP protected MNTs from botulinum neurotoxin A (BoNT/A). Phrenic nerve diaphragm nerve muscle preparations (NMP) isolated from isoflurane anesthetized adult mice were analyzed for twitch tension, spontaneous (mEPCs) and nerve stimulus evoked (EPCs) acetylcholine release. When acutely applied to isolated NMP, CAP produced a concentration-dependent decline of twitch tension and produced a significant decline in the amplitude of EPCs and quantal content without any effect on the mEPCs. The suppression of nerve stimulus evoked acetylcholine release by CAP was antagonized by capsazepine (CPZ), a TRPV1 antagonist. CAP did not suppress phrenic nerve stimulus evoked acetylcholine release in TRPV1 knockout mice. Also, CAP treatment, in vitro, interfered with the localization of adapter protein 2 in cholinergic Neuro 2a cells. Wortmannin, (WMN; non-selective phosphoinositol kinase inhibitor), mimicked the effects of CAP by inhibiting the acetylcholine exocytosis. Our data suggest that TRPV1 proteins expressed at the MNT are coupled to the exo-endocytic mechanisms to regulate neuromuscular functions. Copyright © 2014 Elsevier B.V. All rights reserved.

A homogeneous and "off-on" fluorescence aptamer-based assay for chloramphenicol using vesicle quantum dot-gold colloid composite probes.

PubMed

Miao, Yang-Bao; Ren, Hong-Xia; Gan, Ning; Zhou, You; Cao, Yuting; Li, Tianhua; Chen, Yinji

2016-07-27

In this work, a novel homogeneous and signal "off-on" aptamer based fluorescence assay was successfully developed to detect chloramphenicol (CAP) residues in food based on the fluorescence resonance energy transfer (FRET). The vesicle nanotracer was prepared through labeling single stranded DNA binding protein (SSB) on limposome-CdSe/ZnS quantum dot (SSB/L-QD) complexes. It was worth mentioning that the signal tracer (SSB/L-QD) with vesicle shape, which was fabricated being encapsulated with a number of quantum dots and SSB. The nanotracer has excellent signal amplification effects. The vesicle composite probe was formed by combining aptamer labeled nano-gold (Au-Apt) and SSB/L-QD. Which based on SSB's specific affinity towards aptamer. This probe can't emit fluoresce which is in "off" state because the signal from SSB/L-QD as donor can be quenched by the Au-aptas acceptor. When CAP was added in the composite probe solution, the aptamer on the Au-Apt can be preferentially bounded with CAP then release from the composite probe, which can turn the "off" signal of SSB/L-QD tracer into "on" state. The assay indicates excellent linear response to CAP from 0.001 nM to 10 nM and detection limit down to 0.3 pM. The vesicle probes with size of 88 nm have strong signal amplification. Because a larger number of QDs can be labeled inside the double phosphorus lipid membrane. Besides, it was employed to detect CAP residues in the milk samples with results being agreed well with those from ELISA, verifying its accuracy and reliability. Copyright © 2016 Elsevier B.V. All rights reserved.

Murine Adseverin (D5), a Novel Member of the Gelsolin Family, and Murine Adseverin Are Induced by Interleukin-9 in T-Helper Lymphocytes

PubMed Central

Robbens, Johan; Louahed, Jamila; De Pestel, Kathleen; Van Colen, Inge; Ampe, Christophe; Vandekerckhove, Joel; Renauld, Jean-Christophe

1998-01-01

We identified a number of upregulated genes by differential screening of interleukin-9-stimulated T-helper lymphocytes. Interestingly, two of these messengers encode proteins that are similar to proteins of the gelsolin family. The first displays a typical structure of six homologous domains and shows a high level of identity (90%) with bovine adseverin (or scinderin) and may therefore be considered the murine adseverin homolog. The second encodes a protein with only five segments. Sequence comparison shows that most of the fifth segment and a short amino-terminal part of the sixth segment (amino acids 528 to 628 of adseverin) are missing, and thus, this form may represent an alternatively spliced product derived from the same gene. The corresponding protein is called mouse adseverin (D5). We expressed both proteins in Escherichia coli and show that mouse adseverin displays the typical characteristics of all members of the gelsolin family with respect to actin binding (capping, severing, and nucleation) and its regulation by Ca2+. In contrast, mouse adseverin (D5) fails to nucleate actin polymerization, although like mouse adseverin and gelsolin, it severs and caps actin filaments in a Ca2+-dependent manner. Adseverin is present in all of the tissues and most of the cell lines tested, although at low concentrations. Mouse adseverin (D5) was found only in blood cells and in cell lines derived from T-helper lymphocytes and mast cells, where it is weakly expressed. In a gel filtration experiment, we demonstrated that mouse adseverin forms a 1:2 complex with G actin which is stable only in the presence of Ca2+, while no stable complex was observed for mouse adseverin (D5). PMID:9671468

Gene expression profiling in human skeletal muscle during recovery from eccentric exercise

PubMed Central

Mohoney, D. J.; Safdar, A.; Parise, G.; Melov, S.; Fu, Minghua; MacNeil, L.; Kaczor, J.; Payne, E. T.; Tarnopolsky, M. A.

2009-01-01

We used cDNA microarrays to screen for differentially expressed genes during recovery from exercise-induced muscle damage in humans. Male subjects (n = 4) performed 300 maximal eccentric contractions, and skeletal muscle biopsy samples were analyzed at 3 h and 48 h after exercise. In total, 113 genes increased 3 h postexercise, and 34 decreased. At 48 h postexercise, 59 genes increased and 29 decreased. On the basis of these data, we chose 19 gene changes and conducted secondary analyses using real-time RT-PCR from muscle biopsy samples taken from 11 additional subjects who performed an identical bout of exercise. Real-time RT-PCR analyses confirmed that exercise-induced muscle damage led to a rapid (3 h) increase in sterol response element binding protein 2 (SREBP-2), followed by a delayed (48 h) increase in the SREBP-2 gene targets Acyl CoA:cholesterol acyltransferase (ACAT)-2 and insulin-induced gene 1 (insig-1). The expression of the IL-1 receptor, a known regulator of SREBP-2, was also elevated after exercise. Taken together, these expression changes suggest a transcriptional program for increasing cholesterol and lipid synthesis and/or modification. Additionally, damaging exercise induced the expression of protein kinase H11, capping protein Z alpha (capZα), and modulatory calcineurin-interacting protein 1 (MCIP1), as well as cardiac ankryin repeat protein 1 (CARP1), DNAJB2, c-myc, and junD, each of which are likely involved in skeletal muscle growth, remodeling, and stress management. In summary, using DNA microarrays and RT-PCR, we have identified novel genes that respond to skeletal muscle damage, which, given the known biological functions, are likely involved in recovery from and/or adaptation to damaging exercise. PMID:18321953

The evolution of milk casein genes from tooth genes before the origin of mammals.

PubMed

Kawasaki, Kazuhiko; Lafont, Anne-Gaelle; Sire, Jean-Yves

2011-07-01

Caseins are among cardinal proteins that evolved in the lineage leading to mammals. In milk, caseins and calcium phosphate (CaP) form a huge complex called casein micelle. By forming the micelle, milk maintains high CaP concentrations, which help altricial mammalian neonates to grow bone and teeth. Two types of caseins are known. Ca-sensitive caseins (α(s)- and β-caseins) bind Ca but precipitate at high Ca concentrations, whereas Ca-insensitive casein (κ-casein) does not usually interact with Ca but instead stabilizes the micelle. Thus, it is thought that these two types of caseins are both necessary for stable micelle formation. Both types of caseins show high substitution rates, which make it difficult to elucidate the evolution of caseins. Yet, recent studies have revealed that all casein genes belong to the secretory calcium-binding phosphoprotein (SCPP) gene family that arose by gene duplication. In the present study, we investigated exon-intron structures and phylogenetic distributions of casein and other SCPP genes, particularly the odontogenic ameloblast-associated (ODAM) gene, the SCPP-Pro-Gln-rich 1 (SCPPPQ1) gene, and the follicular dendritic cell secreted peptide (FDCSP) gene. The results suggest that contemporary Ca-sensitive casein genes arose from a putative common ancestor, which we refer to as CSN1/2. The six putative exons comprising CSN1/2 are all found in SCPPPQ1, although ODAM also shares four of these exons. By contrast, the five exons of the Ca-insensitive casein gene are all reminiscent of FDCSP. The phylogenetic distribution of these genes suggests that both SCPPPQ1 and FDCSP arose from ODAM. We thus argue that all casein genes evolved from ODAM via two different pathways; Ca-sensitive casein genes likely originated directly from SCPPPQ1, whereas the Ca-insensitive casein genes directly differentiated from FDCSP. Further, expression of ODAM, SCPPPQ1, and FDCSP was detected in dental tissues, supporting the idea that both types of caseins evolved as Ca-binding proteins. Based on these findings, we propose two alternative hypotheses for micelle formation in primitive milk. The conserved biochemical characteristics in caseins and their immediate ancestors also suggest that many slight genetic modifications have created modern caseins, proteins vital to the sustained success of mammals.

cap alpha. /sub i/-3 cDNA encodes the. cap alpha. subunit of G/sub k/, the stimulatory G protein of receptor-regulated K/sup +/ channels

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

Codina, J.; Olate, J.; Abramowitz, J.

1988-05-15

cDNA cloning has identified the presence in the human genome of three genes encoding ..cap alpha.. subunits of pertussis toxin substrates, generically called G/sub i/. They are named ..cap alpha../sub i/-1, ..cap alpha../sub i/-2 and ..cap alpha../sub i/-3. However, none of these genes has been functionally identified with any of the ..cap alpha.. subunits of several possible G proteins, including pertussis toxin-sensitive G/sub p/'s, stimulatory to phospholipase C or A/sub 2/, G/sub i/, inhibitory to adenylyl cyclase, or G/sub k/, stimulatory to a type of K/sup +/ channels. The authors now report the nucleotide sequence and the complete predicted aminomore » acid sequence of human liver ..cap alpha../sub i/-3 and the partial amino acid sequence of proteolytic fragments of the ..cap alpha.. subunit of human erythrocyte G/sub k/. The amino acid sequence of the proteolytic fragment is uniquely encoded by the cDNA of ..cap alpha../sub i/-3, thus identifying it as ..cap alpha../sub k/. The probable identity of ..cap alpha../sub i/-1 with ..cap alpha../sub p/ and possible roles for ..cap alpha../sub i/-2, as well as additional roles for ..cap alpha../sub i/-1 and ..cap alpha../sub i/-3 (..cap alpha../sub k/) are discussed.« less

The antioxidative and hepatoprotective effects comparison of Chinese angelica polysaccharide(CAP)and selenizing CAP (sCAP) in CCl4 induced hepatic injury mice.

PubMed

Gao, Zhenzhen; Zhang, Chao; Tian, Weijun; Liu, Kuanhui; Hou, Ranran; Yue, Chanjuan; Wu, Yi; Wang, Deyun; Liu, Jiaguo; Hu, Yuanliang; Yang, Ying

2017-04-01

Chinese angelica polysaccharides (CAP) and selenizing CAP (sCAP) were prepared and identified through FTIR and SEM observation. Their antioxidant activities in vitro and hepatoprotective effects in vivo were compared by free radical-scavenging tests or with CCl 4 -induced hepatic injury model mice. The results showed that for DPPH radical, superoxide anion and hydroxyl radical, the scavenging capabilities of sCAP were significantly stronger than those of CAP . In hepatic injury model mice, sCAP could significantly reduce ALT, AST and ALP contents and raised TP content in serum, significantly reduce MDA and ROS contents and raised SOD and T-AOC activities in liver homogenate in comparison with CAP; obviously relieve the pathological changes of liver and significantly inhibit the expressions of p-ERK, p-JNK and p-p38 protein as compared with those in model control group. These results indicate that selenylation modification can enhance the antioxidant and hepatoprotective actions of Chinese angelica polysaccharide. A action mechanism of sCAP is suppressing the protein expression of MAPK signaling pathway. Copyright © 2017 Elsevier B.V. All rights reserved.

Fluorescence microscopic analysis of antifungal effects of cold atmospheric pressure plasma in Saccharomyces cerevisiae.

PubMed

Itooka, Koki; Takahashi, Kazuo; Izawa, Shingo

2016-11-01

Cold atmospheric pressure plasma (CAP) has potential to be utilized as an alternative method for sterilization in food industries without thermal damage or toxic residues. In contrast to the bactericidal effects of CAP, information regarding the efficacy of CAP against eukaryotic microorganisms is very limited. Therefore, herein we investigated the effects of CAP on the budding yeast Saccharomyces cerevisiae, with a focus on the cellular response to CAP. The CAP treatment caused oxidative stress responses including the nuclear accumulation of the oxidative stress responsive transcription factor Yap1, mitochondrial fragmentation, and enhanced intracellular oxidation. Yeast cells also induced the expression of heat shock protein (HSP) genes and formation of Hsp104 aggregates when treated with CAP, suggesting that CAP denatures proteins. As phenomena unique to eukaryotic cells, the formation of cytoplasmic mRNP granules such as processing bodies and stress granules and changes in the intracellular localization of Ire1 were caused by the treatment with CAP, indicating that translational repression and endoplasmic reticulum (ER) stress were induced by the CAP treatment. These results suggest that the fungicidal effects of CAP are attributed to the multiple severe stresses.

Identification of a factor in HeLa cells specific for an upstream transcriptional control sequence of an EIA-inducible adenovirus promoter and its relative abundance in infected and uninfected cells.

PubMed Central

SivaRaman, L; Subramanian, S; Thimmappaya, B

1986-01-01

Utilizing the gel electrophoresis/DNA binding assay, a factor specific for the upstream transcriptional control sequence of the EIA-inducible adenovirus EIIA-early promoter has been detected in HeLa cell nuclear extract. Analysis of linker-scanning mutants of the promoter by DNA binding assays and methylation-interference experiments show that the factor binds to the 17-nucleotide sequence 5' TGGAGATGACGTAGTTT 3' located between positions -66 and -82 upstream from the cap site. This sequence has been shown to be essential for transcription of this promoter. The EIIA-early-promoter specific factor was found to be present at comparable levels in uninfected HeLa cells and in cells infected with either wild-type adenovirus or the EIA-deletion mutant dl312 under conditions in which the EIA proteins are induced to high levels [7 or 20 hr after infection in the presence of arabinonucleoside (cytosine arabinoside)]. Based on the quantitation in DNA binding assays, it appears that the mechanism of EIA-activated transcription of the EIIA-early promoter does not involve a net change in the amounts of this factor. Images PMID:2942943

Gelsolin in Onychophora and Tardigrada with notes on its variability in the Ecdysozoa.

PubMed

Thiruketheeswaran, Prasath; Greven, Hartmut; D'Haese, Jochen

2017-01-01

Rearrangements of the filamentous actin network involve a broad range of actin binding proteins. Among these, the gelsolin proteins sever actin filaments, cap their fast growing end and nucleate actin assembly in a calcium-dependent manner. Here, we focus on the gelsolin of the onychophoran Peripatoides novaezealandiae and the eutardigrade Hypsibius dujardini. From the cDNA of P. novaezealandiae we obtained the complete coding sequence with an open reading frame of 2178bp. It encodes a protein of 726 amino acids with a calculated molecular mass of 82,610.9Da and a pI of 5.57. This sequence is comprised of six segments (S1-S6). However, analysis of data from TardiBase reveals that the gelsolin of the eutardigrade Hypsibius dujardini has only three segments (S1-S3). The coding sequence consist of 1119bp for 373 amino acids with a calculated molecular mass of 42,440.95Da and a pI of 6.17. The Peripatoides and Hypsibius gelsolin revealed both conserved binding motifs for G-actin, F-actin and phosphatidylinositol 4,5-bisphosphate (PIP 2 ), along with a full set of type-1 and type-2 Ca 2+ -binding sites which could result in the binding of eight and four calcium ions, respectively. Both gelsolin proteins lack a C-terminal latch-helix indicating a more rapid activation in the submicromolar Ca 2+ range. We suggest that a gelsolin with three segments was present in the last common ancestor of the ecdysozoan clade Panarthropoda (Onychophora, Tardigrada, Arthropoda), primarily because the gelsolin of all non-Ecdysozoa studied so far (except Chordata) reveals this number of segments. Mapping of our molecular data onto a well-established phylogeny revealed that the number of gelsolin segments does not correlate with the phylogenetic lineage but rather with particular functional demands to alter the kinetics of actin polymerization. Copyright © 2016 Elsevier Inc. All rights reserved.

Root Secreted Metabolites and Proteins Are Involved in the Early Events of Plant-Plant Recognition Prior to Competition

PubMed Central

Badri, Dayakar V.; De-la-Peña, Clelia; Lei, Zhentian; Manter, Daniel K.; Chaparro, Jacqueline M.; Guimarães, Rejane L.; Sumner, Lloyd W.; Vivanco, Jorge M.

2012-01-01

The mechanism whereby organisms interact and differentiate between others has been at the forefront of scientific inquiry, particularly in humans and certain animals. It is widely accepted that plants also interact, but the degree of this interaction has been constricted to competition for space, nutrients, water and light. Here, we analyzed the root secreted metabolites and proteins involved in early plant neighbor recognition by using Arabidopsis thaliana Col-0 ecotype (Col) as our focal plant co-cultured in vitro with different neighbors [A. thaliana Ler ecotype (Ler) or Capsella rubella (Cap)]. Principal component and cluster analyses revealed that both root secreted secondary metabolites and proteins clustered separately between the plants grown individually (Col-0, Ler and Cap grown alone) and the plants co-cultured with two homozygous individuals (Col-Col, Ler-Ler and Cap-Cap) or with different individuals (Col-Ler and Col-Cap). In particularly, we observed that a greater number of defense- and stress- related proteins were secreted when our control plant, Col, was grown alone as compared to when it was co-cultured with another homozygous individual (Col-Col) or with a different individual (Col-Ler and Col-Cap). However, the total amount of defense proteins in the exudates of the co-cultures was higher than in the plant alone. The opposite pattern of expression was identified for stress-related proteins. These data suggest that plants can sense and respond to the presence of different plant neighbors and that the level of relatedness is perceived upon initial interaction. Furthermore, the role of secondary metabolites and defense- and stress-related proteins widely involved in plant-microbe associations and abiotic responses warrants reassessment for plant-plant interactions. PMID:23056382

Purification and characterization of the glycoprotein hormone. cap alpha. -subunit-like material secreted by HeLa cells

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

Cox, G.S.; Rimerman, R.A.

1988-08-23

The protein secreted by HeLa cells that cross-reacts with antiserum developed against the ..cap alpha..-subunit of human chorionic gonadotropin (hCG) has been purified approximately 30,000-fold from concentrated culture medium by organic solvent fractionation followed by ion exchange, gel filtration, and lectin affinity chromatography. The final preparation had a specific activity (by RIA) of 6.8 x 10/sup 5/ ng of ..cap alpha../mg of protein and appeared homogeneous by electrophoresis on reducing/denaturing polyacrylamide gels (SDS-PAGE). Amino acid analysis indicated that HeLa-..cap alpha.. had a composition very similar to that of the urinary hCG ..cap alpha..-subunit. However, comparison of hCG-..cap alpha.. and HeLa-..capmore » alpha.. demonstrated that the tumor-associated subunit was not identical with its normal counterpart. The purified tumor protein had an apparent molecular weight greater than that of the urinary ..cap alpha..-subunit when analyzed by SDS-PAGE, and this difference was even greater when a partially purified preparation was examined by an immunoblot technique (Western). Isoelectric focusing of the HeLa and hCG subunits demonstrated that the tumor protein had a lower pI. Immunoprecipitation and electrophoresis of ..cap alpha..-subunit from HeLa cultures labeled with (/sup 3/H)fucose indicated that the tumor subunit was fucosylated, whereas analysis of hCG-..cap alpha.. hydrosylates by HPLC confirmed previous reports that the placental subunit does not contain fucose. The results indicate that, regardless of whether or not a single ..cap alpha..-subunit gene is being expressed in both normal and neoplastic tissues, posttranslational modifications lead to a highly altered subunit in the tumor. The differences observed may be useful in diagnosing neoplastic vs hyperplastic conditions and may lend insight into the mechanism of ectopic hormone production by tumors.« less

Selection of specific protein binders for pre-defined targets from an optimized library of artificial helicoidal repeat proteins (alphaRep).

PubMed

Guellouz, Asma; Valerio-Lepiniec, Marie; Urvoas, Agathe; Chevrel, Anne; Graille, Marc; Fourati-Kammoun, Zaineb; Desmadril, Michel; van Tilbeurgh, Herman; Minard, Philippe

2013-01-01

We previously designed a new family of artificial proteins named αRep based on a subgroup of thermostable helicoidal HEAT-like repeats. We have now assembled a large optimized αRep library. In this library, the side chains at each variable position are not fully randomized but instead encoded by a distribution of codons based on the natural frequency of side chains of the natural repeats family. The library construction is based on a polymerization of micro-genes and therefore results in a distribution of proteins with a variable number of repeats. We improved the library construction process using a "filtration" procedure to retain only fully coding modules that were recombined to recreate sequence diversity. The final library named Lib2.1 contains 1.7×10(9) independent clones. Here, we used phage display to select, from the previously described library or from the new library, new specific αRep proteins binding to four different non-related predefined protein targets. Specific binders were selected in each case. The results show that binders with various sizes are selected including relatively long sequences, with up to 7 repeats. ITC-measured affinities vary with Kd values ranging from micromolar to nanomolar ranges. The formation of complexes is associated with a significant thermal stabilization of the bound target protein. The crystal structures of two complexes between αRep and their cognate targets were solved and show that the new interfaces are established by the variable surfaces of the repeated modules, as well by the variable N-cap residues. These results suggest that αRep library is a new and versatile source of tight and specific binding proteins with favorable biophysical properties.

Selection of Specific Protein Binders for Pre-Defined Targets from an Optimized Library of Artificial Helicoidal Repeat Proteins (alphaRep)

PubMed Central

Chevrel, Anne; Graille, Marc; Fourati-Kammoun, Zaineb; Desmadril, Michel; van Tilbeurgh, Herman; Minard, Philippe

2013-01-01

We previously designed a new family of artificial proteins named αRep based on a subgroup of thermostable helicoidal HEAT-like repeats. We have now assembled a large optimized αRep library. In this library, the side chains at each variable position are not fully randomized but instead encoded by a distribution of codons based on the natural frequency of side chains of the natural repeats family. The library construction is based on a polymerization of micro-genes and therefore results in a distribution of proteins with a variable number of repeats. We improved the library construction process using a “filtration” procedure to retain only fully coding modules that were recombined to recreate sequence diversity. The final library named Lib2.1 contains 1.7×109 independent clones. Here, we used phage display to select, from the previously described library or from the new library, new specific αRep proteins binding to four different non-related predefined protein targets. Specific binders were selected in each case. The results show that binders with various sizes are selected including relatively long sequences, with up to 7 repeats. ITC-measured affinities vary with Kd values ranging from micromolar to nanomolar ranges. The formation of complexes is associated with a significant thermal stabilization of the bound target protein. The crystal structures of two complexes between αRep and their cognate targets were solved and show that the new interfaces are established by the variable surfaces of the repeated modules, as well by the variable N-cap residues. These results suggest that αRep library is a new and versatile source of tight and specific binding proteins with favorable biophysical properties. PMID:24014183

Peptide functionalized gold nanoparticles: the influence of pH on binding efficiency

NASA Astrophysics Data System (ADS)

Harrison, Emma; Hamilton, Jeremy W. J.; Macias-Montero, Manuel; Dixon, Dorian

2017-07-01

We report herein on the synthesis of mixed monolayer gold nanoparticles (AuNPs) capped with both polyethylene glycol (PEG) and one of three peptides. Either a receptor-mediated endocytosis peptide, an endosomal escape pathway (H5WYG) peptide or the Nrp-1 targeting RGD peptide (CRGDK) labeled with FITC. All three peptides have a thiol containing cysteine residue which can be used to bind the peptides to the AuNPs. In order to investigate the influence of pH on peptide attachment, PEGylated AuNPs were centrifuged, the supernatant removed, and the nanoparticles were then re-suspended in a range of pH buffer solutions above, below and at the respective isoelectric points of the peptides before co-functionalization. Peptide attachment was investigated using dynamic light scattering, Ultra-violet visible spectroscopy (UV/Vis), FTIR and photo luminescence spectroscopy. UV/Vis analysis coupled with protein assay results and photoluminescence of the FITC tagged RGD peptide concluded that a pH of ∼8 optimized the cysteine binding and stability, irrespective of the peptide used.

Structure of a short-chain dehydrogenase/reductase (SDR) within a genomic island from a clinical strain of Acinetobacter baumannii

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

Shah, Bhumika S., E-mail: bhumika.shah@mq.edu.au; Tetu, Sasha G.; Harrop, Stephen J.

2014-09-25

The structure of a short-chain dehydrogenase encoded within genomic islands of A. baumannii strains has been solved to 2.4 Å resolution. This classical SDR incorporates a flexible helical subdomain. The NADP-binding site and catalytic side chains are identified. Over 15% of the genome of an Australian clinical isolate of Acinetobacter baumannii occurs within genomic islands. An uncharacterized protein encoded within one island feature common to this and other International Clone II strains has been studied by X-ray crystallography. The 2.4 Å resolution structure of SDR-WM99c reveals it to be a new member of the classical short-chain dehydrogenase/reductase (SDR) superfamily. Themore » enzyme contains a nucleotide-binding domain and, like many other SDRs, is tetrameric in form. The active site contains a catalytic tetrad (Asn117, Ser146, Tyr159 and Lys163) and water molecules occupying the presumed NADP cofactor-binding pocket. An adjacent cleft is capped by a relatively mobile helical subdomain, which is well positioned to control substrate access.« less

Capsaicin-induced activation of ERK1/2 and its involvement in GAP-43 expression and CGRP depletion in organotypically cultured DRG neurons.

PubMed

Li, Yunfeng; Liu, Guixiang; Li, Hao; Xu, Youzheng; Zhang, Hong; Liu, Zhen

2013-04-01

Low concentrations of capsaicin (CAP) stimulate and high concentrations of CAP can be toxic to the primary sensory neurons of the dorsal root ganglion (DRG). CAP induces the phosphorylation of extracellular signal-regulated protein kinases 1/2 (ERK1/2) in DRG neurons. The effect of the activation of ERK1/2 by different concentrations of CAP on growth-associated protein 43 (GAP-43) expression and calcitonin gene-related peptide (CGRP) depletion in DRG neurons remains unknown. In the present study, organotypic embryonic 15-day-old rat DRG explants were used to determine the effect of different concentrations of CAP on GAP-43 expression and CGRP depletion. The results showed that, compared to unstimulated control cultures, GAP-43 and pERK1/2 protein levels increased at a low concentration (2 μmol/L) of CAP and decreased at a higher concentration (10 μmol/L). The number of CGRP-immunoreactive (IR) migrating neurons also decreased in CAP-treated cultures. The increase in GAP-43 levels and CGRP depletion could be blocked by the administration of ERK1/2 inhibitor PD98059. The results of the present study imply that CAP at different concentrations has different effects on GAP-43 expression and CGRP depletion. These effects were involved in the activation of ERK1/2 in organotypically cultured DRG neurons stimulated with CAP. These data may provide new insights for further development potential therapeutic applications of CAP with moderate dose on neurogenic inflammation.

Reflections on the history of pre-mRNA processing and highlights of current knowledge: A unified picture

PubMed Central

Darnell, James E.

2013-01-01

Several strong conclusions emerge concerning pre-mRNA processing from both old and newer experiments. The RNAPII complex is involved with pre-mRNA processing through binding of processing proteins to the CTD (carboxyl terminal domain) of the largest RNAPII subunit. These interactions are necessary for efficient processing, but whether factor binding to the CTD and delivery to splicing sites is obligatory or facilitatory is unsettled. Capping, addition of an m7Gppp residue (cap) to the initial transcribed residue of a pre-mRNA, occurs within seconds. Splicing of pre-mRNA by spliceosomes at particular sites is most likely committed during transcription by the binding of initiating processing factors and ∼50% of the time is completed in mammalian cells before completion of the primary transcript. This fact has led to an outpouring in the literature about “cotranscriptional splicing.” However splicing requires several minutes for completion and can take longer. The RNAPII complex moves through very long introns and also through regions dense with alternating exons and introns at an average rate of ∼3 kb per min and is, therefore, not likely detained at each splice site for more than a few seconds, if at all. Cleavage of the primary transcript at the 3′ end and polyadenylation occurs within 30 sec or less at recognized polyA sites, and the majority of newly polyadenylated pre-mRNA molecules are much larger than the average mRNA. Finally, it seems quite likely that the nascent RNA most often remains associated with the chromosomal locus being transcribed until processing is complete, possibly acquiring factors related to the transport of the new mRNA to the cytoplasm. PMID:23440351

Mini-thin filaments regulated by troponin–tropomyosin

PubMed Central

Gong, Huiyu; Hatch, Victoria; Ali, Laith; Lehman, William; Craig, Roger; Tobacman, Larry S.

2005-01-01

Striated muscle thin filaments contain hundreds of actin monomers and scores of troponins and tropomyosins. To study the cooperative mechanism of thin filaments, “mini-thin filaments” were generated by isolating particles nearly matching the minimal structural repeat of thin filaments: a double helix of actin subunits with each strand approximately seven actins long and spanned by a troponin–tropomyosin complex. One end of the particles was capped by a gelsolin (segment 1–3)–TnT fusion protein (substituting for normal TnT), and the other end was capped by tropomodulin. EM showed that the particles were 46 ± 9 nm long, with a knob-like mass attributable to gelsolin at one end. Average actin, tropomyosin, and gelsolin–troponin composition indicated one troponin–tropomyosin attached to each strand of the two-stranded actin filament. The minifilaments thus nearly represent single regulatory units of thin filaments. The myosin S1 MgATPase rate stimulated by the minifilaments was Ca2+-sensitive, indicating that single regulatory length particles are sufficient for regulation. Ca2+ bound cooperatively to cardiac TnC in conventional thin filaments but noncooperatively to cardiac TnC in minifilaments in the absence of myosin. This suggests that thin filament Ca2+-binding cooperativity reflects indirect troponin–troponin interactions along the long axis of conventional filaments, which do not occur in minifilaments. Despite noncooperative Ca2+ binding to minifilaments in the absence of myosin, Ca2+ cooperatively activated the myosin S1-particle ATPase rate. Two-stranded single regulatory units therefore may be sufficient for myosin-mediated Ca2+-binding cooperativity. Functional mini-thin filaments are well suited for biochemical and structural analysis of thin-filament regulation. PMID:15644437

Effects of mycoplasma contamination on phenotypic expression of mitochondrial mutants in human cells.

PubMed

Doersen, C J; Stanbridge, E J

1981-04-01

HeLa cells sensitive to the mitochondrial protein synthesis inhibitors erythromycin (ERY) and chloramphenicol (CAP) and HeLa variants resistant to the effects of these drugs were purposefully infected with drug-sensitive and -resistant mycoplasma strains. Mycoplasma hyorhinis and the ERY-resistant strain of Mycoplasma orale, MO-ERYr, did not influence the growth of HeLa and ERY-resistant ERY2301 cells in the presence or absence of ERY. M. hyorhinis also did not affect the growth of HeLa and CAP-resistant Cap-2 cells in the presence or absence of CAP. However, both HeLa and Cap-2 cells infected with the CAP-resistant strain of M. hyorhinis, MH-CAPr, were more sensitive to the cytotoxic effect of CAP. This may be due to the glucose dependence of the cells, which was compromised by the increased utilization of glucose by MH-CAPr in these infected cell cultures. In vitro protein synthesis by isolated mitochondria was significantly altered by mycoplasma infection of the various cell lines. A substantial number of mycoplasmas copurified with the mitochondria, resulting in up to a sevenfold increase in the incorporation of [3H]leucine into the trichloroacetic acid-insoluble material. More importantly, the apparent drug sensitivity or resistance of mitochondrial preparations from mycoplasma-infected cells reflected the drug sensitivity or resistance of the contaminating mycoplasmas. These results illustrate the hazards in interpreting mitochondrial protein synthesis data derived from mycoplasma-infected cell lines, particularly putative mitochondrially encoded mutants resistant to inhibitors of mitochondrial protein synthesis.

RStrucFam: a web server to associate structure and cognate RNA for RNA-binding proteins from sequence information.

PubMed

Ghosh, Pritha; Mathew, Oommen K; Sowdhamini, Ramanathan

2016-10-07

RNA-binding proteins (RBPs) interact with their cognate RNA(s) to form large biomolecular assemblies. They are versatile in their functionality and are involved in a myriad of processes inside the cell. RBPs with similar structural features and common biological functions are grouped together into families and superfamilies. It will be useful to obtain an early understanding and association of RNA-binding property of sequences of gene products. Here, we report a web server, RStrucFam, to predict the structure, type of cognate RNA(s) and function(s) of proteins, where possible, from mere sequence information. The web server employs Hidden Markov Model scan (hmmscan) to enable association to a back-end database of structural and sequence families. The database (HMMRBP) comprises of 437 HMMs of RBP families of known structure that have been generated using structure-based sequence alignments and 746 sequence-centric RBP family HMMs. The input protein sequence is associated with structural or sequence domain families, if structure or sequence signatures exist. In case of association of the protein with a family of known structures, output features like, multiple structure-based sequence alignment (MSSA) of the query with all others members of that family is provided. Further, cognate RNA partner(s) for that protein, Gene Ontology (GO) annotations, if any and a homology model of the protein can be obtained. The users can also browse through the database for details pertaining to each family, protein or RNA and their related information based on keyword search or RNA motif search. RStrucFam is a web server that exploits structurally conserved features of RBPs, derived from known family members and imprinted in mathematical profiles, to predict putative RBPs from sequence information. Proteins that fail to associate with such structure-centric families are further queried against the sequence-centric RBP family HMMs in the HMMRBP database. Further, all other essential information pertaining to an RBP, like overall function annotations, are provided. The web server can be accessed at the following link: http://caps.ncbs.res.in/rstrucfam .

Analysis of the promoter of the cudA gene reveals novel mechanisms of Dictyostelium cell type differentiation.

PubMed

Fukuzawa, M; Williams, J G

2000-06-01

The cudA gene encodes a nuclear protein that is essential for normal multicellular development. At the slug stage cudA is expressed in the prespore cells and in a sub-region of the prestalk zone. We show that cap site distal promoter sequences direct cudA expression in prespore cells, while proximal sequences direct expression in the prestalk sub-region. The promoter domain that directs prespore-specific transcription consists of a positively acting region, that has the potential to direct expression in all cells within the slug, and a negatively acting region that prevents expression in the prestalk cells. Dd-STATa is the STAT protein that regulates commitment to stalk cell gene expression, where it is known to function as a transcriptional repressor. We show that Dd-STATa binds in vitro to the positively acting part of the prespore domain of the cudA promoter. However, Dd-STATa cannot be utilised for this purpose in vivo, because analysis of a Dd-STATa null mutant strain shows that Dd-STATa is not necessary for cudA transcription in prespore cells. In contrast, the part of the cudA promoter that directs prestalk-specific expression contains a binding site for Dd-STATa that is essential for its biological activity. Dd-STATa appears therefore to serve as a direct activator of cudA transcription in prestalk cells, while a protein with a DNA binding specificity highly related to that of Dd-STATa is utilised to activate cudA transcription in prespore cells.

Francisella tularensis Live Vaccine Strain deficient in capB and overexpressing the fusion protein of IglA, IglB, and IglC from the bfr promoter induces improved protection against F. tularensis respiratory challenge.

PubMed

Jia, Qingmei; Bowen, Richard; Lee, Bai-Yu; Dillon, Barbara Jane; Masleša-Galić, Saša; Horwitz, Marcus A

2016-09-22

A safer and more effective vaccine than the unlicensed Francisella tularensis Live Vaccine Strain (LVS) is needed to protect against the biowarfare agent F. tularensis. Previously, we developed an LVS ΔcapB mutant that is significantly safer than LVS and provides potent protective immunity against F. tularensis respiratory challenge when administered intranasally but limited protection when administered intradermally unless as part of a prime-boost vaccination strategy. To improve the immunogenicity and efficacy of LVS ΔcapB, we developed recombinant LVS ΔcapB (rLVS ΔcapB) strains overexpressing various F. tularensis Francisella Pathogenicity Island (FPI) proteins - IglA, IglB and IglC, and a fusion protein (IglABC) comprising immunodominant epitopes of IglA, IglB, and IglC downstream of different Francisella promoters, including the bacterioferritin (bfr) promoter. We show that rLVS ΔcapB/bfr-iglA, iglB, iglC, and iglABC express more IglA, IglB, IglC or IglABC than parental LVS ΔcapB in broth and in human macrophages, and stably express FPI proteins in macrophages and mice absent antibiotic selection. In response to IglC and heat-inactivated LVS, spleen cells from mice immunized intradermally with rLVS ΔcapB/bfr-iglC or bfr-iglABC secrete greater amounts of interferon-gamma and/or interleukin-17 than those from mice immunized with LVS ΔcapB, comparable to those from LVS-immunized mice. Mice immunized with rLVS ΔcapB/bfr-iglA, iglB, iglC or iglABC produce serum antibodies at levels similar to LVS-immunized mice. Mice immunized intradermally with rLVS ΔcapB/bfr-iglABC and challenged intranasally with virulent F. tularensis Schu S4 survive longer than sham- and LVS ΔcapB-immunized mice. Mice immunized intranasally with rLVS ΔcapB/bfr-iglABC - but not with LVS - just before or after respiratory challenge with F. tularensis Schu S4 are partially protected; protection is correlated with induction of a strong innate immune response. Thus, rLVS ΔcapB/bfr-iglABC shows improved immunogenicity and protective efficacy compared with parental LVS ΔcapB and, in contrast to LVS, has partial efficacy as immediate pre- and post-exposure prophylaxis. Copyright © 2016 Elsevier Ltd. All rights reserved.

Role of tachykinins in ozone-induced acute lung injury in guinea pigs

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

Tepper, J.S.; Costa, D.L.; Fitzgerald, S.

To examine the hypothesis that the acute reversible changes caused by ozone (O3) exposure are mediated by tachykinin release, guinea pigs were depleted of tachykinins by use of repeated capsaicin (CAP) injections before O3 exposure in an attempt to prevent O3-induced functional changes. Unexpectedly, CAP pretreatment caused divergent results in the functional responses to O3. Ventilatory measurements obtained from CAP-pretreated O3-exposed (CAP-O3) animals were exacerbated rather than diminished compared with the effects of O3 alone. Similarly, lavage fluid protein accumulation was enhanced in the CAP-O3 group compared with the O3-exposed group. In better agreement with our initial hypothesis, the CAP-O3more » group was less responsive than the O3-exposed animals to histamine aerosol challenge. Additionally, Evans blue dye accumulation, a hallmark of tachykinin release, was increased in O3-exposed animals and was partially blocked in the CAP-O3 group. These data suggest that tachykinin-containing sensory fibers are unlikely to mediate the acute effects of O3 exposure on tidal breathing and lavage fluid protein accumulation but may play a role in causing post-O3 airway hyperreactivity and protein extravasation into the trachea.« less

Effect of arabinogalactan proteins from the root caps of pea and Brassica napus on Aphanomyces euteiches zoospore chemotaxis and germination.

PubMed

Cannesan, Marc Antoine; Durand, Caroline; Burel, Carole; Gangneux, Christophe; Lerouge, Patrice; Ishii, Tadashi; Laval, Karine; Follet-Gueye, Marie-Laure; Driouich, Azeddine; Vicré-Gibouin, Maïté

2012-08-01

Root tips of many plant species release a number of border, or border-like, cells that are thought to play a major role in the protection of root meristem. However, little is currently known on the structure and function of the cell wall components of such root cells. Here, we investigate the sugar composition of the cell wall of the root cap in two species: pea (Pisum sativum), which makes border cells, and Brassica napus, which makes border-like cells. We find that the cell walls are highly enriched in arabinose and galactose, two major residues of arabinogalactan proteins. We confirm the presence of arabinogalactan protein epitopes on root cap cell walls using immunofluorescence microscopy. We then focused on these proteoglycans by analyzing their carbohydrate moieties, linkages, and electrophoretic characteristics. The data reveal (1) significant structural differences between B. napus and pea root cap arabinogalactan proteins and (2) a cross-link between these proteoglycans and pectic polysaccharides. Finally, we assessed the impact of root cap arabinogalactan proteins on the behavior of zoospores of Aphanomyces euteiches, an oomycetous pathogen of pea roots. We find that although the arabinogalactan proteins of both species induce encystment and prevent germination, the effects of both species are similar. However, the arabinogalactan protein fraction from pea attracts zoospores far more effectively than that from B. napus. This suggests that root arabinogalactan proteins are involved in the control of early infection of roots and highlights a novel role for these proteoglycans in root-microbe interactions.

Effect of Arabinogalactan Proteins from the Root Caps of Pea and Brassica napus on Aphanomyces euteiches Zoospore Chemotaxis and Germination12[C][W

PubMed Central

Cannesan, Marc Antoine; Durand, Caroline; Burel, Carole; Gangneux, Christophe; Lerouge, Patrice; Ishii, Tadashi; Laval, Karine; Follet-Gueye, Marie-Laure; Driouich, Azeddine; Vicré-Gibouin, Maïté

2012-01-01

Root tips of many plant species release a number of border, or border-like, cells that are thought to play a major role in the protection of root meristem. However, little is currently known on the structure and function of the cell wall components of such root cells. Here, we investigate the sugar composition of the cell wall of the root cap in two species: pea (Pisum sativum), which makes border cells, and Brassica napus, which makes border-like cells. We find that the cell walls are highly enriched in arabinose and galactose, two major residues of arabinogalactan proteins. We confirm the presence of arabinogalactan protein epitopes on root cap cell walls using immunofluorescence microscopy. We then focused on these proteoglycans by analyzing their carbohydrate moieties, linkages, and electrophoretic characteristics. The data reveal (1) significant structural differences between B. napus and pea root cap arabinogalactan proteins and (2) a cross-link between these proteoglycans and pectic polysaccharides. Finally, we assessed the impact of root cap arabinogalactan proteins on the behavior of zoospores of Aphanomyces euteiches, an oomycetous pathogen of pea roots. We find that although the arabinogalactan proteins of both species induce encystment and prevent germination, the effects of both species are similar. However, the arabinogalactan protein fraction from pea attracts zoospores far more effectively than that from B. napus. This suggests that root arabinogalactan proteins are involved in the control of early infection of roots and highlights a novel role for these proteoglycans in root-microbe interactions. PMID:22645070

Two Routes to Genetic Suppression of RNA Trimethylguanosine Cap Deficiency via C-Terminal Truncation of U1 snRNP Subunit Snp1 or Overexpression of RNA Polymerase Subunit Rpo26.

PubMed

Qiu, Zhicheng R; Schwer, Beate; Shuman, Stewart

2015-04-24

The trimethylguanosine (TMG) caps of small nuclear (sn) RNAs are synthesized by the enzyme Tgs1 via sequential methyl additions to the N2 atom of the m(7)G cap. Whereas TMG caps are inessential for Saccharomyces cerevisiae vegetative growth at 25° to 37°, tgs1∆ cells that lack TMG caps fail to thrive at 18°. The cold-sensitive defect correlates with ectopic stoichiometric association of nuclear cap-binding complex (CBC) with the residual m(7)G cap of the U1 snRNA and is suppressed fully by Cbc2 mutations that weaken cap binding. Here, we show that normal growth of tgs1∆ cells at 18° is also restored by a C-terminal deletion of 77 amino acids from the Snp1 subunit of yeast U1 snRNP. These results underscore the U1 snRNP as a focal point for TMG cap function in vivo. Casting a broader net, we conducted a dosage suppressor screen for genes that allowed survival of tgs1∆ cells at 18°. We thereby recovered RPO26 (encoding a shared subunit of all three nuclear RNA polymerases) and RPO31 (encoding the largest subunit of RNA polymerase III) as moderate and weak suppressors of tgs1∆ cold sensitivity, respectively. A structure-guided mutagenesis of Rpo26, using rpo26∆ complementation and tgs1∆ suppression as activity readouts, defined Rpo26-(78-155) as a minimized functional domain. Alanine scanning identified Glu89, Glu124, Arg135, and Arg136 as essential for rpo26∆ complementation. The E124A and R135A alleles retained tgs1∆ suppressor activity, thereby establishing a separation-of-function. These results illuminate the structure activity profile of an essential RNA polymerase component. Copyright © 2015 Qiu et al.

Overexpression of adenylate cyclase-associated protein 2 is a novel prognostic marker in malignant melanoma.

PubMed

Masugi, Yohei; Tanese, Keiji; Emoto, Katsura; Yamazaki, Ken; Effendi, Kathryn; Funakoshi, Takeru; Mori, Mariko; Sakamoto, Michiie

2015-12-01

Malignant melanoma is one of the lethal malignant tumors worldwide. Previously we reported that adenylate cyclase-associated protein 2 (CAP2), which is a well-conserved actin regulator, was overexpressed in hepatocellular carcinoma; however, CAP2 expression in other clinical cancers remains unclear. The aim of the current study was to clarify the clinicopathological significance of CAP2 overexpression in malignant melanoma. Immunohistochemical analyses revealed that many melanoma cells exhibited diffuse cytoplasmic expression of CAP2, whereas no normal melanocytes showed detectable immunostaining for CAP2. A high level of CAP2 expression was seen in 14 of 50 melanomas and was significantly correlated with greater tumor thickness and nodular melanoma subtypes. In addition, a high level of CAP2 expression was associated with poor overall survival in univariate and multivariate analyses. For 13 patients, samples of primary and metastatic melanoma tissue were available: four patients exhibited higher levels of CAP2 expression in metastatic tumor compared to the primary site, whereas no patient showed lower levels of CAP2 expression in metastatic melanomas. Our findings show that CAP2 overexpression is a novel prognostic marker in malignant melanoma and that CAP2 expression seems to increase stepwise during tumor progression, suggesting the involvement of CAP2 in the aggressive behavior of malignant melanoma. © 2015 Japanese Society of Pathology and Wiley Publishing Asia Pty Ltd.

DARPP-32 interaction with adducin may mediate rapid environmental effects on striatal neurons.

PubMed

Engmann, Olivia; Giralt, Albert; Gervasi, Nicolas; Marion-Poll, Lucile; Gasmi, Laila; Filhol, Odile; Picciotto, Marina R; Gilligan, Diana; Greengard, Paul; Nairn, Angus C; Hervé, Denis; Girault, Jean-Antoine

2015-12-07

Environmental enrichment has multiple effects on behaviour, including modification of responses to psychostimulant drugs mediated by striatal neurons. However, the underlying molecular and cellular mechanisms are not known. Here we show that DARPP-32, a hub signalling protein in striatal neurons, interacts with adducins, which are cytoskeletal proteins that cap actin filaments' fast-growing ends and regulate synaptic stability. DARPP-32 binds to adducin MARCKS domain and this interaction is modulated by DARPP-32 Ser97 phosphorylation. Phospho-Thr75-DARPP-32 facilitates β-adducin Ser713 phosphorylation through inhibition of a cAMP-dependent protein kinase/phosphatase-2A cascade. Caffeine or 24-h exposure to a novel enriched environment increases adducin phosphorylation in WT, but not T75A mutant mice. This cascade is implicated in the effects of brief exposure to novel enriched environment on dendritic spines in nucleus accumbens and cocaine locomotor response. Our results suggest a molecular pathway by which environmental changes may rapidly alter responsiveness of striatal neurons involved in the reward system.

Abscisic acid and ethephon regulation of cellulase in the endosperm cap and radicle during lettuce seed germination.

PubMed

Chen, Bingxian; Ma, Jun; Xu, Zhenjiang; Wang, Xiaofeng

2016-10-01

The purpose of this study was to investigate the role of cellulase in endosperm cap weakening and radicle elongation during lettuce (Lactuca sativa L.) seed germination. The application of abscisic acid (ABA) or ethephon inhibits or promotes germination, respectively, by affecting endosperm cap weakening and radicle elongation. Cellulase activities, and related protein and transcript abundances of two lettuce cellulase genes, LsCEL1 and LsCEL2, increase in the endosperm cap and radicle prior to radicle protrusion following imbibition in water. ABA or ethephon reduce or elevate, respectively, cellulase activity, and related protein and transcript abundances in the endosperm cap. Taken together, these observations suggest that cellulase plays a role in endosperm cap weakening and radicle elongation during lettuce seed germination, and that the regulation of cellulase in the endosperm cap by ABA and ethephon play a role in endosperm cap weakening. However, the influence of ABA and ethephon on radicle elongation may not be through their effects on cellulase. © 2016 Institute of Botany, Chinese Academy of Sciences.

Autistic-like behavioral phenotypes in a mouse model with copy number variation of the CAPS2/CADPS2 gene.

PubMed

Sadakata, Tetsushi; Shinoda, Yo; Oka, Megumi; Sekine, Yukiko; Furuichi, Teiichi

2013-01-04

Ca²⁺-dependent activator protein for secretion 2 (CAPS2 or CADPS2) facilitates secretion and trafficking of dense-core vesicles. Recent genome-wide association studies of autism have identified several microdeletions due to copy number variation (CNV) in one of the chromosome 7q31.32 alleles on which the locus for CAPS2 is located in autistic patients. To evaluate the biological significance of reducing CAPS2 copy number, we analyzed CAPS2 heterozygous mice. Our present findings suggest that adequate levels of CAPS2 protein are critical for normal brain development and behavior, and that allelic changes due to CNV may contribute to autistic symptoms in combination with deficits in other autism-associated genes. Copyright © 2012 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Enhanced porcine circovirus Cap protein production by Pichia pastoris with a fuzzy logic DO control based methanol/sorbitol co-feeding induction strategy.

PubMed

Ding, Jian; Zhang, Chunling; Gao, Minjie; Hou, Guoli; Liang, Kexue; Li, Chunhua; Ni, Jianping; Li, Zhen; Shi, Zhongping

2014-05-10

Porcine circovirus Cap protein production by P. pastoris with strong AOX promoter suffered with the problems with traditional pure methanol induction: (1) inefficient methanol metabolism; (2) extensive oxygen supply load; (3) difficulty in stable DO control; (4) low protein titer. In this study, based on the difference of DO change patterns in response to methanol and sorbitol additions, a novel fuzzy control system was proposed to automatically regulate the co-feeding rates of methanol and sorbitol for efficient Cap protein induction. With aid of the proposed control system when setting DO control level at 10%, overall fermentation performance was significantly improved: (1) DO could be stably controlled under mild aeration condition; (2) methanol consumption rate could be restricted at moderate level and the major enzymes involved with methanol metabolism were largely activated; (3) Cap protein concentration reached a highest level of 198mg/L, which was about 64% increase over the best one using the pure methanol induction strategies. Copyright © 2014 Elsevier B.V. All rights reserved.

CdTe quantum dots: aqueous phase synthesis, stability studies and protein conjugation for development of biosensors

NASA Astrophysics Data System (ADS)

Borse, Vivek; Sadawana, Mayur; Srivastava, Rohit

2016-04-01

Synthesis of quantum dots (QDs) in aqueous medium is advantageous as compared to the organic solvent mediated synthesis, as the aqueous synthesis is less toxic, reagent effective, easily reproducible and importantly, synthesized QDs have biological compatibility. The QDs should be aqueous in nature for use in cell imaging, drug labeling, tracking and delivery. Structural modifications are necessary to enable their use in biosensing application. In this work, mercaptopropionic acid capped cadmium telluride QDs (MPA-CdTe QDs) were synthesized by hydrothermal method and characterized by various techniques. Water and various biochemical buffers were used to study the fluorescence intensity stability of the QDs at different physicochemical conditions. QDs stored in 4° C showed excellent stability of fluorescence intensity values as compared to the samples stored at room temperature. Staphylococcal protein A (SPA) was conjugated with the QDs (SPA-QDs) and characterized using UV and fluorescence spectroscopy, zeta potential, HRTEM, FTIR, and AFM. Blue shift was observed in the fluorescence emission spectra that may be due to reduction in the surface charge as carboxyl groups on QDs were replaced by amino groups of SPA. This SPA conjugated to QDs enables binding of the C-terminal of antibodies on its surface allowing N-terminal binding site remain free to bind with antigenic biomarkers. Thus, the biosensor i.e. antibody bound on SPA-QDs would bind to the antigenic biomarkers in sample and the detection system could be developed. As QDs have better fluorescence properties than organic dyes, this biosensor will provide high sensitivity and quantitative capability in diagnostics.

Normalizing translation through 4E-BP prevents mTOR-driven cortical mislamination and ameliorates aberrant neuron integration.

PubMed

Lin, Tiffany V; Hsieh, Lawrence; Kimura, Tomoki; Malone, Taylor J; Bordey, Angélique

2016-10-04

Hyperactive mammalian target of rapamycin complex 1 (mTORC1) is a shared molecular hallmark in several neurodevelopmental disorders characterized by abnormal brain cytoarchitecture. The mechanisms downstream of mTORC1 that are responsible for these defects remain unclear. We show that focally increasing mTORC1 activity during late corticogenesis leads to ectopic placement of upper-layer cortical neurons that does not require altered signaling in radial glia and is accompanied by changes in layer-specific molecular identity. Importantly, we found that decreasing cap-dependent translation by expressing a constitutively active mutant of the translational repressor eukaryotic initiation factor 4E-binding protein 1 (4E-BP1) prevents neuronal misplacement and soma enlargement, while partially rescuing dendritic hypertrophy induced by hyperactive mTORC1. Furthermore, overactivation of translation alone through knockdown of 4E-BP2 was sufficient to induce neuronal misplacement. These data show that many aspects of abnormal brain cytoarchitecture can be prevented by manipulating a single intracellular process downstream of mTORC1, cap-dependent translation.

Direct /sup 125/I-radioligand assays for serum progesterone compared with assays involving extraction of serum

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

Ratcliffe, W.A.; Corrie, J.E.T.; Dalziel, A.H.

1982-06-01

Two direct radioimmunoassays for progesterone in 50 ..mu..L of unextracted serum or plasma with assays involving extraction of serum were compared. The direct assays include the use of either danazol at pH 7.4 or 8-anilino-1-naphthalenesulfonic acid at pH 4.0 to displace progesterone from serum binding-proteins. Progesterone is then assayed by using an antiserum to a progesterone 11..cap alpha..-hemisuccinyl conjugate and the radioligand /sup 125/I-labeled progesterone 11..cap alpha..-glucuronyl tyramine, with separation by double-antibody techniques. Direct assays with either displacing agent gave good analytical recovery of progesterone added to human serum, and progesterone values for patients' specimens correlated well (r > 0.96)more » with results of assays involving extraction of serum. Precision was similar with each displacing agent over the working range 2.5-100 nmol/L and superior to that of extraction assays. We conclude that these direct assays of progesterone are analytically valid and more robust, precise, and technically convenient than many conventional methods involving extraction of serum.« less

VHL negatively regulates SARS coronavirus replication by modulating nsp16 ubiquitination and stability.

PubMed

Yu, Xiao; Chen, Shuliang; Hou, Panpan; Wang, Min; Chen, Yu; Guo, Deyin

2015-04-03

Eukaryotic cellular and most viral RNAs carry a 5'-terminal cap structure, a 5'-5' triphosphate linkage between the 5' end of the RNA and a guanosine nucleotide (cap-0). SARS coronavirus (SARS-CoV) nonstructural protein nsp16 functions as a methyltransferase, to methylate mRNA cap-0 structure at the ribose 2'-O position of the first nucleotide to form cap-1 structures. However, whether there is interplay between nsp16 and host proteins was not yet clear. In this report, we identified several potential cellular nsp16-interacting proteins from a human thymus cDNA library by yeast two-hybrid screening. VHL, one of these proteins, was proven to interact with nsp16 both in vitro and in vivo. Further studies showed that VHL can inhibit SARS-CoV replication by regulating nsp16 ubiquitination and promoting its degradation. Our results have revealed the role of cellular VHL in the regulation of SARS-CoV replication. Copyright © 2015 Elsevier Inc. All rights reserved.

DNA and Protein Requirements for Substrate Conformational Changes Necessary for Human Flap Endonuclease-1-catalyzed Reaction*

PubMed Central

Algasaier, Sana I.; Exell, Jack C.; Bennet, Ian A.; Thompson, Mark J.; Gotham, Victoria J. B.; Shaw, Steven J.; Craggs, Timothy D.; Finger, L. David; Grasby, Jane A.

2016-01-01

Human flap endonuclease-1 (hFEN1) catalyzes the essential removal of single-stranded flaps arising at DNA junctions during replication and repair processes. hFEN1 biological function must be precisely controlled, and consequently, the protein relies on a combination of protein and substrate conformational changes as a prerequisite for reaction. These include substrate bending at the duplex-duplex junction and transfer of unpaired reacting duplex end into the active site. When present, 5′-flaps are thought to thread under the helical cap, limiting reaction to flaps with free 5′-termini in vivo. Here we monitored DNA bending by FRET and DNA unpairing using 2-aminopurine exciton pair CD to determine the DNA and protein requirements for these substrate conformational changes. Binding of DNA to hFEN1 in a bent conformation occurred independently of 5′-flap accommodation and did not require active site metal ions or the presence of conserved active site residues. More stringent requirements exist for transfer of the substrate to the active site. Placement of the scissile phosphate diester in the active site required the presence of divalent metal ions, a free 5′-flap (if present), a Watson-Crick base pair at the terminus of the reacting duplex, and the intact secondary structure of the enzyme helical cap. Optimal positioning of the scissile phosphate additionally required active site conserved residues Tyr40, Asp181, and Arg100 and a reacting duplex 5′-phosphate. These studies suggest a FEN1 reaction mechanism where junctions are bound and 5′-flaps are threaded (when present), and finally the substrate is transferred onto active site metals initiating cleavage. PMID:26884332

Effects of mycoplasma contamination on phenotypic expression of mitochondrial mutants in human cells.

PubMed Central

Doersen, C J; Stanbridge, E J

1981-01-01

HeLa cells sensitive to the mitochondrial protein synthesis inhibitors erythromycin (ERY) and chloramphenicol (CAP) and HeLa variants resistant to the effects of these drugs were purposefully infected with drug-sensitive and -resistant mycoplasma strains. Mycoplasma hyorhinis and the ERY-resistant strain of Mycoplasma orale, MO-ERYr, did not influence the growth of HeLa and ERY-resistant ERY2301 cells in the presence or absence of ERY. M. hyorhinis also did not affect the growth of HeLa and CAP-resistant Cap-2 cells in the presence or absence of CAP. However, both HeLa and Cap-2 cells infected with the CAP-resistant strain of M. hyorhinis, MH-CAPr, were more sensitive to the cytotoxic effect of CAP. This may be due to the glucose dependence of the cells, which was compromised by the increased utilization of glucose by MH-CAPr in these infected cell cultures. In vitro protein synthesis by isolated mitochondria was significantly altered by mycoplasma infection of the various cell lines. A substantial number of mycoplasmas copurified with the mitochondria, resulting in up to a sevenfold increase in the incorporation of [3H]leucine into the trichloroacetic acid-insoluble material. More importantly, the apparent drug sensitivity or resistance of mitochondrial preparations from mycoplasma-infected cells reflected the drug sensitivity or resistance of the contaminating mycoplasmas. These results illustrate the hazards in interpreting mitochondrial protein synthesis data derived from mycoplasma-infected cell lines, particularly putative mitochondrially encoded mutants resistant to inhibitors of mitochondrial protein synthesis. PMID:6965101

A novel role for the condensin II complex in cellular senescence.

PubMed

Yokoyama, Yuhki; Zhu, Hengrui; Zhang, Rugang; Noma, Ken-ichi

2015-01-01

Although cellular senescence is accompanied by global alterations in genome architecture, how the genome is restructured during the senescent processes is not well understood. Here, we show that the hCAP-H2 subunit of the condensin II complex exists as either a full-length protein or an N-terminus truncated variant (ΔN). While the full-length hCAP-H2 associates with mitotic chromosomes, the ΔN variant exists as an insoluble nuclear structure. When overexpressed, both hCAP-H2 isoforms assemble this nuclear architecture and induce senescence-associated heterochromatic foci (SAHF). The hCAP-H2ΔN protein accumulates as cells approach senescence, and hCAP-H2 knockdown inhibits oncogene-induced senescence. This study identifies a novel mechanism whereby condensin drives senescence via nuclear/genomic reorganization.

Widespread Positive Selection Drives Differentiation of Centromeric Proteins in the Drosophila melanogaster subgroup.

PubMed

Beck, Emily A; Llopart, Ana

2015-11-25

Rapid evolution of centromeric satellite repeats is thought to cause compensatory amino acid evolution in interacting centromere-associated kinetochore proteins. Cid, a protein that mediates kinetochore/centromere interactions, displays particularly high amino acid turnover. Rapid evolution of both Cid and centromeric satellite repeats led us to hypothesize that the apparent compensatory evolution may extend to interacting partners in the Condensin I complex (i.e., SMC2, SMC4, Cap-H, Cap-D2, and Cap-G) and HP1s. Missense mutations in these proteins often result in improper centromere formation and aberrant chromosome segregation, thus selection for maintained function and coevolution among proteins of the complex is likely strong. Here, we report evidence of rapid evolution and recurrent positive selection in seven centromere-associated proteins in species of the Drosophila melanogaster subgroup, and further postulate that positive selection on these proteins could be a result of centromere drive and compensatory changes, with kinetochore proteins competing for optimal spindle attachment.

Fluorescence Polarization Screening Assays for Small Molecule Allosteric Modulators of ABL Kinase Function

PubMed Central

Grover, Prerna; Shi, Haibin; Baumgartner, Matthew; Camacho, Carlos J.; Smithgall, Thomas E.

2015-01-01

The ABL protein-tyrosine kinase regulates intracellular signaling pathways controlling diverse cellular processes and contributes to several forms of cancer. The kinase activity of ABL is repressed by intramolecular interactions involving its regulatory Ncap, SH3 and SH2 domains. Small molecules that allosterically regulate ABL kinase activity through its non-catalytic domains may represent selective probes of ABL function. Here we report a screening assay for chemical modulators of ABL kinase activity that target the regulatory interaction of the SH3 domain with the SH2-kinase linker. This fluorescence polarization (FP) assay is based on a purified recombinant ABL protein consisting of the N-cap, SH3 and SH2 domains plus the SH2-kinase linker (N32L protein) and a short fluorescein-labeled probe peptide that binds to the SH3 domain. In assay development experiments, we found that the probe peptide binds to the recombinant ABL N32L protein in vitro, producing a robust FP signal that can be competed with an excess of unlabeled peptide. The FP signal is not observed with control N32L proteins bearing either an inactivating mutation in the SH3 domain or enhanced SH3:linker interaction. A pilot screen of 1200 FDA-approved drugs identified four compounds that specifically reduced the FP signal by at least three standard deviations from the untreated controls. Secondary assays showed that one of these hit compounds, the antithrombotic drug dipyridamole, enhances ABL kinase activity in vitro to a greater extent than the previously described ABL agonist, DPH. Docking studies predicted that this compound binds to a pocket formed at the interface of the SH3 domain and the linker, suggesting that it activates ABL by disrupting this regulatory interaction. These results show that screening assays based on the non-catalytic domains of ABL can identify allosteric small molecule regulators of kinase function, providing a new approach to selective drug discovery for this important kinase system. PMID:26222440

Fluorescence Polarization Screening Assays for Small Molecule Allosteric Modulators of ABL Kinase Function.

PubMed

Grover, Prerna; Shi, Haibin; Baumgartner, Matthew; Camacho, Carlos J; Smithgall, Thomas E

2015-01-01

The ABL protein-tyrosine kinase regulates intracellular signaling pathways controlling diverse cellular processes and contributes to several forms of cancer. The kinase activity of ABL is repressed by intramolecular interactions involving its regulatory Ncap, SH3 and SH2 domains. Small molecules that allosterically regulate ABL kinase activity through its non-catalytic domains may represent selective probes of ABL function. Here we report a screening assay for chemical modulators of ABL kinase activity that target the regulatory interaction of the SH3 domain with the SH2-kinase linker. This fluorescence polarization (FP) assay is based on a purified recombinant ABL protein consisting of the N-cap, SH3 and SH2 domains plus the SH2-kinase linker (N32L protein) and a short fluorescein-labeled probe peptide that binds to the SH3 domain. In assay development experiments, we found that the probe peptide binds to the recombinant ABL N32L protein in vitro, producing a robust FP signal that can be competed with an excess of unlabeled peptide. The FP signal is not observed with control N32L proteins bearing either an inactivating mutation in the SH3 domain or enhanced SH3:linker interaction. A pilot screen of 1200 FDA-approved drugs identified four compounds that specifically reduced the FP signal by at least three standard deviations from the untreated controls. Secondary assays showed that one of these hit compounds, the antithrombotic drug dipyridamole, enhances ABL kinase activity in vitro to a greater extent than the previously described ABL agonist, DPH. Docking studies predicted that this compound binds to a pocket formed at the interface of the SH3 domain and the linker, suggesting that it activates ABL by disrupting this regulatory interaction. These results show that screening assays based on the non-catalytic domains of ABL can identify allosteric small molecule regulators of kinase function, providing a new approach to selective drug discovery for this important kinase system.

Murine Efficacy and Pharmacokinetic Evaluation of the Flaviviral NS5 Capping Enzyme 2-Thioxothiazolidin-4-One Inhibitor BG-323

PubMed Central

Bullard, Kristen M.; Gullberg, Rebekah C.; Soltani, Elnaz; Steel, J. Jordan; Geiss, Brian J.; Keenan, Susan M.

2015-01-01

Arthropod-borne flavivirus infection continues to cause significant morbidity and mortality worldwide. Identification of drug targets and novel antiflaviviral compounds to treat these diseases has become a global health imperative. A previous screen of 235,456 commercially available small molecules identified the 2-thioxothiazolidin-4-one family of compounds as inhibitors of the flaviviral NS5 capping enzyme, a promising target for antiviral drug development. Rational drug design methodologies enabled identification of lead compound BG-323 from this series. We have shown previously that BG-323 potently inhibits NS5 capping enzyme activity, displays antiviral effects in dengue virus replicon assays and inhibits growth of West Nile and yellow fever viruses with low cytotoxicity in vitro. In this study we further characterized BG-323’s antiviral activity in vitro and in vivo. We found that BG-323 was able to reduce replication of WNV (NY99) and Powassan viruses in culture, and we were unable to force resistance into WNV (Kunjin) in long-term culture experiments. We then evaluated the antiviral activity of BG-323 in a murine model. Mice were challenged with WNV NY99 and administered BG-323 or mock by IP inoculation immediately post challenge and twice daily thereafter. Mice were bled and viremia was quantified on day three. No significant differences in viremia were observed between BG-323-treated and control groups and clinical scores indicated both BG-323-treated and control mice developed signs of illness on approximately the same day post challenge. To determine whether differences in in vitro and in vivo efficacy were due to unfavorable pharmacokinetic properties of BG-323, we conducted a pharmacokinetic evaluation of this small molecule. Insights from pharmacokinetic studies indicate that BG-323 is cell permeable, has a low efflux ratio and does not significantly inhibit two common cytochrome P450 (CYP P450) isoforms thus suggesting this molecule may be less likely to cause adverse drug interactions. However, the T1/2 of BG-323 was suboptimal and the percent of drug bound to plasma binding proteins was high. Future studies with BG-323 will be aimed at increasing the T1/2 and determining strategies for mitigating the effects of high plasma protein binding, which likely contribute to low in vivo efficacy. PMID:26075394

Murine Efficacy and Pharmacokinetic Evaluation of the Flaviviral NS5 Capping Enzyme 2-Thioxothiazolidin-4-One Inhibitor BG-323.

PubMed

Bullard, Kristen M; Gullberg, Rebekah C; Soltani, Elnaz; Steel, J Jordan; Geiss, Brian J; Keenan, Susan M

2015-01-01

Arthropod-borne flavivirus infection continues to cause significant morbidity and mortality worldwide. Identification of drug targets and novel antiflaviviral compounds to treat these diseases has become a global health imperative. A previous screen of 235,456 commercially available small molecules identified the 2-thioxothiazolidin-4-one family of compounds as inhibitors of the flaviviral NS5 capping enzyme, a promising target for antiviral drug development. Rational drug design methodologies enabled identification of lead compound BG-323 from this series. We have shown previously that BG-323 potently inhibits NS5 capping enzyme activity, displays antiviral effects in dengue virus replicon assays and inhibits growth of West Nile and yellow fever viruses with low cytotoxicity in vitro. In this study we further characterized BG-323's antiviral activity in vitro and in vivo. We found that BG-323 was able to reduce replication of WNV (NY99) and Powassan viruses in culture, and we were unable to force resistance into WNV (Kunjin) in long-term culture experiments. We then evaluated the antiviral activity of BG-323 in a murine model. Mice were challenged with WNV NY99 and administered BG-323 or mock by IP inoculation immediately post challenge and twice daily thereafter. Mice were bled and viremia was quantified on day three. No significant differences in viremia were observed between BG-323-treated and control groups and clinical scores indicated both BG-323-treated and control mice developed signs of illness on approximately the same day post challenge. To determine whether differences in in vitro and in vivo efficacy were due to unfavorable pharmacokinetic properties of BG-323, we conducted a pharmacokinetic evaluation of this small molecule. Insights from pharmacokinetic studies indicate that BG-323 is cell permeable, has a low efflux ratio and does not significantly inhibit two common cytochrome P450 (CYP P450) isoforms thus suggesting this molecule may be less likely to cause adverse drug interactions. However, the T1/2 of BG-323 was suboptimal and the percent of drug bound to plasma binding proteins was high. Future studies with BG-323 will be aimed at increasing the T1/2 and determining strategies for mitigating the effects of high plasma protein binding, which likely contribute to low in vivo efficacy.

A search for structurally similar cellular internal ribosome entry sites

PubMed Central

Baird, Stephen D.; Lewis, Stephen M.; Turcotte, Marcel; Holcik, Martin

2007-01-01

Internal ribosome entry sites (IRES) allow ribosomes to be recruited to mRNA in a cap-independent manner. Some viruses that impair cap-dependent translation initiation utilize IRES to ensure that the viral RNA will efficiently compete for the translation machinery. IRES are also employed for the translation of a subset of cellular messages during conditions that inhibit cap-dependent translation initiation. IRES from viruses like Hepatitis C and Classical Swine Fever virus share a similar structure/function without sharing primary sequence similarity. Of the cellular IRES structures derived so far, none were shown to share an overall structural similarity. Therefore, we undertook a genome-wide search of human 5′UTRs (untranslated regions) with an empirically derived structure of the IRES from the key inhibitor of apoptosis, X-linked inhibitor of apoptosis protein (XIAP), to identify novel IRES that share structure/function similarity. Three of the top matches identified by this search that exhibit IRES activity are the 5′UTRs of Aquaporin 4, ELG1 and NF-kappaB repressing factor (NRF). The structures of AQP4 and ELG1 IRES have limited similarity to the XIAP IRES; however, they share trans-acting factors that bind the XIAP IRES. We therefore propose that cellular IRES are not defined by overall structure, as viral IRES, but are instead dependent upon short motifs and trans-acting factors for their function. PMID:17591613

Biomarkers in Pediatric Community-Acquired Pneumonia.

PubMed

Principi, Nicola; Esposito, Susanna

2017-02-19

Community-acquired pneumonia (CAP) is an infectious disease caused by bacteria, viruses, or a combination of these infectious agents. The severity of the clinical manifestations of CAP varies significantly. Consequently, both the differentiation of viral from bacterial CAP cases and the accurate assessment and prediction of disease severity are critical for effectively managing individuals with CAP. To solve questionable cases, several biomarkers indicating the etiology and severity of CAP have been studied. Unfortunately, only a few studies have examined the roles of these biomarkers in pediatric practice. The main aim of this paper is to detail current knowledge regarding the use of biomarkers to diagnose and treat CAP in children, analyzing the most recently published relevant studies. Despite several attempts, the etiologic diagnosis of pediatric CAP and the estimation of the potential outcome remain unsolved problems in most cases. Among traditional biomarkers, procalcitonin (PCT) appears to be the most effective for both selecting bacterial cases and evaluating the severity. However, a precise cut-off separating bacterial from viral and mild from severe cases has not been defined. The three-host protein assay based on C-reactive protein (CRP), tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), plasma interferon-γ protein-10 (IP-10), and micro-array-based whole genome expression arrays might offer more advantages in comparison with former biomarkers. However, further studies are needed before the routine use of those presently in development can be recommended.

Treatment of Ras-induced cancers by the F-actin cappers tensin and chaetoglobosin K, in combination with the caspase-1 inhibitor N1445.

PubMed

Tikoo, A; Cutler, H; Lo, S H; Chen, L B; Maruta, H

1999-01-01

For transforming normal fibroblasts to malignant cells, oncogenic Ras mutants such as v-Ha-ras require Rho family GTPases (Rho, Rac, and CDC42) that are responsible for controlling actin-cytoskeleton organization. Ras activates Rac through a PI-3 kinase-mediated pathway. Rac causes uncapping of actin filaments (F-actin) at the plus-ends, through phosphatidylinositol 4,5 bisphosphate (PIP2), and eventually induces membrane ruffling. Several distinct F-actin/PIP2-binding proteins, such as gelsolin, which severs and caps the plus-ends of actin filaments, or HS1, which cross-links actin filaments, have been shown to suppress v-Ha-Ras-induced malignant transformation when they are overexpressed. Interestingly, an F-actin cross-linking drug (photosensitizer) called MKT-077 suppresses Ras transformation. Thus, an F-actin capping/severing drug might also have an anticancer potential. This study was conducted to determine first whether Ras-induced malignant phenotype (anchorage-independent growth) is suppressed by overexpression of the gene encoding a large plus-end F-actin capping protein called tensin and second to test the anti-Ras potential of a unique fungal antibiotic (small compound) called chaetoglobosin K (CK) that also caps the plus-ends of actin filaments. DNA transfection with a retroviral vector carrying the tensin cDNA was used to overexpress tensin in v-Ha-Ras-transformed NIH 3T3 cells. All stable tensin transfectants rarely formed colonies in soft agar, indicating that tensin suppresses the anchorage-independent growth. The anti-Ras action of CK was determined by incubating the Ras-transformants in the presence of CK in soft agar. Two microM CK almost completely inhibited their colony formation, indicating that CK also suppresses the malignant phenotype. However, unlike tensin, CK causes an apoptosis of Ras-transformed NIH 3T3 cells and, less effectively, of normal NIH 3T3 cells, indicating that CK has an F-actin capping-independent side effect(s). CK-induced apoptosis is at least in part caused by CK-induced inhibition of the kinase PKB/AKT. However, a specific ICE/caspase-1 inhibitor called N1445 completely abolished the CK-induced apoptosis by reactivating PKB, but without affecting the CK-induced suppression of Ras transformation. Like the F-actin cross-linking drug MKT-077, the F-actin capping drug CK may be useful for the treatment of Ras-associated cancers if it is combined with the ICE inhibitor N1445, which abolishes the side effect of CK. Our observations that two distinct F-actin capping molecules (i.e., tensin and CK) suppress Ras-induced malignant phenotype strongly suggest, if not prove, that capping of actin filaments at the plus-ends alone is sufficient to block one of the Ras signaling pathways essential for its oncogenicity. This notion is compatible with the fact that Ras induces the uncapping of actin filaments at the plus-ends through the Rac/PIP2 pathway.

Removal of Protein Capping Enhances the Antibacterial Efficiency of Biosynthesized Silver Nanoparticles

PubMed Central

Jain, Navin; Bhargava, Arpit; Rathi, Mohit; Dilip, R. Venkataramana; Panwar, Jitendra

2015-01-01

The present study demonstrates an economical and environmental affable approach for the synthesis of “protein-capped” silver nanoparticles in aqueous solvent system. A variety of standard techniques viz. UV-visible spectroscopy, transmission electron microscopy (TEM), energy dispersive spectroscopy (EDS) and X-ray diffraction (XRD) measurements were employed to characterize the shape, size and composition of nanoparticles. The synthesized nanoparticles were found to be homogenous, spherical, mono-dispersed and covered with multi-layered protein shell. In order to prepare bare silver nanoparticles, the protein shell was removed from biogenic nanoparticles as confirmed by UV-visible spectroscopy, FTIR and photoluminescence analysis. Subsequently, the antibacterial efficacy of protein-capped and bare silver nanoparticles was compared by bacterial growth rate and minimum inhibitory concentration assay. The results revealed that bare nanoparticles were more effective as compared to the protein-capped silver nanoparticles with varying antibacterial potential against the tested Gram positive and negative bacterial species. Mechanistic studies based on ROS generation and membrane damage suggested that protein-capped and bare silver nanoparticles demonstrate distinct mode of action. These findings were strengthened by the TEM imaging along with silver ion release measurements using inductively coupled plasma atomic emission spectroscopy (ICP-AES). In conclusion, our results illustrate that presence of protein shell on silver nanoparticles can decrease their bactericidal effects. These findings open new avenues for surface modifications of nanoparticles to modulate and enhance their functional properties. PMID:26226385

An integrated catch-and-hold mechanism activates nicotinic acetylcholine receptors

PubMed Central

Jadey, Snehal

2012-01-01

In neuromuscular acetylcholine (ACh) receptor channels (AChRs), agonist molecules bind with a low affinity (LA) to two sites that can switch to high affinity (HA) and increase the probability of channel opening. We measured (by using single-channel kinetic analysis) the rate and equilibrium constants for LA binding and channel gating for several different agonists of adult-type mouse AChRs. Almost all of the variation in the equilibrium constants for LA binding was from differences in the association rate constants. These were consistently below the limit set by diffusion and were substantially different even though the agonists had similar sizes and the same charge. This suggests that binding to resting receptors is not by diffusion alone and, hence, that each binding site can undergo two conformational changes (“catch” and “hold”) that connect three different structures (apo-, LA-bound, and HA-bound). Analyses of ACh-binding protein structures suggest that this binding site, too, may adopt three discrete structures having different degrees of loop C displacement (“capping”). For the agonists we tested, the logarithms of the equilibrium constants for LA binding and LA↔HA gating were correlated. Although agonist binding and channel gating have long been considered to be separate processes in the activation of ligand-gated ion channels, this correlation implies that the catch-and-hold conformational changes are energetically linked and together comprise an integrated process having a common structural basis. We propose that loop C capping mainly reflects agonist binding, with its two stages corresponding to the formation of the LA and HA complexes. The catch-and-hold reaction coordinate is discussed in terms of preopening states and thermodynamic cycles of activation. PMID:22732309

Cap-independent protein synthesis is enhanced by betaine under hypertonic conditions.

PubMed

Carnicelli, Domenica; Arfilli, Valentina; Onofrillo, Carmine; Alfieri, Roberta R; Petronini, Pier Giorgio; Montanaro, Lorenzo; Brigotti, Maurizio

2017-02-12

Protein synthesis is one of the main cellular functions inhibited during hypertonic challenge. The subsequent accumulation of the compatible osmolyte betaine during the later adaptive response allows not only recovery of translation but also its stimulation. In this paper, we show that betaine modulates translation by enhancing the formation of cap-independent 48 S pre-initiation complexes, leaving cap-dependent 48 S pre-initiation complexes basically unchanged. In the presence of betaine, CrPV IRES- and sodium-dependent neutral amino acid transporter-2 (SNAT2) 5'-UTR-driven translation is 2- and 1.5-fold stimulated in MCF7 cells, respectively. Thus, betaine could provide an advantage in translation of messengers coding for proteins implicated in the response of cells to different stressors, which are often recognized by ribosomal 40 S subunit through simplified cap-independent mechanisms. Copyright © 2017 Elsevier Inc. All rights reserved.

Plasminogen associates with phosphatidylserine-exposing platelets and contributes to thrombus lysis under flow

PubMed Central

Whyte, Claire S.; Swieringa, Frauke; Mastenbroek, Tom G.; Lionikiene, Ausra S.; Lancé, Marcus D.; van der Meijden, Paola E. J.; Heemskerk, Johan W. M.

2015-01-01

The interaction of plasminogen with platelets and their localization during thrombus formation and fibrinolysis under flow are not defined. Using a novel model of whole blood thrombi, formed under flow, we examine dose-dependent fibrinolysis using fluorescence microscopy. Fibrinolysis was dependent upon flow and the balance between fibrin formation and plasminogen activation, with tissue plasminogen activator-mediated lysis being more efficient than urokinase plasminogen activator-mediated lysis. Fluorescently labeled plasminogen radiates from platelet aggregates at the base of thrombi, primarily in association with fibrin. Hirudin attenuates, but does not abolish plasminogen binding, denoting the importance of fibrin. Flow cytometry revealed that stimulation of platelets with thrombin/convulxin significantly increased the plasminogen signal associated with phosphatidylserine (PS)-exposing platelets. Binding was attenuated by tirofiban and Gly-Pro-Arg-Pro amide, confirming a role for fibrin in amplifying plasminogen binding to PS-exposing platelets. Confocal microscopy revealed direct binding of plasminogen and fibrinogen to different platelet subpopulations. Binding of plasminogen and fibrinogen co-localized with PAC-1 in the center of spread platelets. In contrast, PS-exposing platelets were PAC-1 negative, and bound plasminogen and fibrinogen in a protruding “cap.” These data show that different subpopulations of platelets harbor plasminogen by diverse mechanisms and provide an essential scaffold for the accumulation of fibrinolytic proteins that mediate fibrinolysis under flow. PMID:25712989

The Double Face of Mucin-Type O-Glycans in Lectin-Mediated Infection and Immunity.

PubMed

Morozov, Vasily; Borkowski, Julia; Hanisch, Franz-Georg

2018-05-11

Epithelial human blood group antigens (HBGAs) on O-glycans play roles in pathogen binding and the initiation of infection, while similar structures on secretory mucins exert protective functions. These double-faced features of O-glycans in infection and innate immunity are reviewed based on two instructive examples of bacterial and viral pathogens. Helicobacter pylori represents a class 1 carcinogen in the human stomach. By expressing blood group antigen-binding adhesin ( BabA ) and LabA adhesins that bind to Lewis-b and LacdiNAc, respectively, H. pylori colocalizes with the mucin MUC5AC in gastric surface epithelia, but not with MUC6, which is cosecreted with trefoil factor family 2 ( TFF2 ) by deep gastric glands. Both components of the glandular secretome are concertedly up-regulated upon infection. While MUC6 expresses GlcNAc-capped glycans as natural antibiotics for H. pylori growth control, TFF2 may function as a probiotic lectin. In viral infection human noroviruses of the GII genogroup interact with HBGAs via their major capsid protein, VP1. HBGAs on human milk oligosaccharides (HMOs) may exert protective functions by binding to the P2 domain pocket on the capsid. We discuss structural details of the P2 carbohydrate-binding pocket in interaction with blood group H/Lewis-b HMOs and fucoidan-derived oligofucoses as effective interactors for the most prevalent norovirus strains, GII.4 and GII.17.

Molecular and functional characterization of the first tick CAP2b (periviscerokinin) receptor from Rhipicephalus (Boophilus) microplus

USDA-ARS?s Scientific Manuscript database

The cDNA of the receptor for CAP2b/periviscerokinin (PVK) neuropeptides, designated Rhimi-CAP2b-R, was cloned from synganglia of tick Rhipicephalus (Boophilus) microplus. This receptor is the ortholog of the insect CAP2b/PVK receptor, as concluded from analyses of the predicted protein sequence, ph...

Ionization of isocitrate bound to pig hear NADP/sup +/-dependent isocitrate dehydrogenase: /sup 13/C NMR study of substrate binding

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

Ehrlich, R.S.; Colman, R.F.

1987-06-16

Isocitrate and ..cap alpha..-ketoglutarate have been synthesized with carbon-13 enrichment at specific positions. The /sup 13/C NMR spectra of these derivatives were measured as a function of pH. The magnitudes of the changes in chemical shifts with pH for free isocitrate and the magnesium-isocitrate complex suggest that the primary site of ionization at the ..beta..-carboxyl. In the presence of the enzyme NADP/sup +/-dependent isocitrate dehydrogenase and the activating metal magnesium, the carbon-13 resonances of all three carboxyls remain constant from pH 5.5 to pH 7.5. Thus, the carboxyls remain in the ionized form in the enzyme-isocitrate complex. The ..cap alpha..-hydroxylmore » carbon resonance could not be located in the enzyme-isocitrate complex, suggesting immobilization of this group. Magnesium produces a 2 ppm downfield shift of the ..beta..-carboxyl but does not change the resonances of the ..cap alpha..- and ..gamma..-carboxyls. This result is consistent with metal activation of both the dehydrogenation and decarboxylation reactions. The /sup 13/C NMR spectrum of ..cap alpha..-ketoglutarate remains unchanged in the presence of isocitrate dehydrogenase, implying the absence of alterations in geometry in the enzyme-bound form. Formation of the quaternary complex with Mg/sup 2 +/ and NADPH leads to loss of the ..cap alpha..-ketoglutarate resonances and the appearance of new resonances characteristic of ..cap alpha..-hydroxyglutarate. In addition, a broad peak ascribed to the enol form of ..cap alpha..-ketoglutarate is observed. The substantial change in the shift of the ..beta..-carboxyl of isocitrate and the lack of significant shifts in the other carboxyls of isocitrate or ..cap alpha..-ketoglutarate suggest that interaction of the ..beta..-carboxyl with the enzyme contributes to the tighter binding of isocitrate and may be significant for the oxidative decarboxylation function of isocitrate dehydrogenase.« less

Taxonomic distribution and origins of the extended LHC (light-harvesting complex) antenna protein superfamily

PubMed Central

2010-01-01

Background The extended light-harvesting complex (LHC) protein superfamily is a centerpiece of eukaryotic photosynthesis, comprising the LHC family and several families involved in photoprotection, like the LHC-like and the photosystem II subunit S (PSBS). The evolution of this complex superfamily has long remained elusive, partially due to previously missing families. Results In this study we present a meticulous search for LHC-like sequences in public genome and expressed sequence tag databases covering twelve representative photosynthetic eukaryotes from the three primary lineages of plants (Plantae): glaucophytes, red algae and green plants (Viridiplantae). By introducing a coherent classification of the different protein families based on both, hidden Markov model analyses and structural predictions, numerous new LHC-like sequences were identified and several new families were described, including the red lineage chlorophyll a/b-binding-like protein (RedCAP) family from red algae and diatoms. The test of alternative topologies of sequences of the highly conserved chlorophyll-binding core structure of LHC and PSBS proteins significantly supports the independent origins of LHC and PSBS families via two unrelated internal gene duplication events. This result was confirmed by the application of cluster likelihood mapping. Conclusions The independent evolution of LHC and PSBS families is supported by strong phylogenetic evidence. In addition, a possible origin of LHC and PSBS families from different homologous members of the stress-enhanced protein subfamily, a diverse and anciently paralogous group of two-helix proteins, seems likely. The new hypothesis for the evolution of the extended LHC protein superfamily proposed here is in agreement with the character evolution analysis that incorporates the distribution of families and subfamilies across taxonomic lineages. Intriguingly, stress-enhanced proteins, which are universally found in the genomes of green plants, red algae, glaucophytes and in diatoms with complex plastids, could represent an important and previously missing link in the evolution of the extended LHC protein superfamily. PMID:20673336

Telomere Capping Proteins are Structurally Related to RPA with an additional Telomere-Specific Domain

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

Gelinas, A.; Paschini, M; Reyes, F

Telomeres must be capped to preserve chromosomal stability. The conserved Stn1 and Ten1 proteins are required for proper capping of the telomere, although the mechanistic details of how they contribute to telomere maintenance are unclear. Here, we report the crystal structures of the C-terminal domain of the Saccharomyces cerevisiae Stn1 and the Schizosaccharomyces pombe Ten1 proteins. These structures reveal striking similarities to corresponding subunits in the replication protein A complex, further supporting an evolutionary link between telomere maintenance proteins and DNA repair complexes. Our structural and in vivo data of Stn1 identify a new domain that has evolved to supportmore » a telomere-specific role in chromosome maintenance. These findings endorse a model of an evolutionarily conserved mechanism of DNA maintenance that has developed as a result of increased chromosomal structural complexity.« less

Structural and Biophysical Characterization of the Mycobacterium tuberculosis Protein Rv0577, a Protein Associated with Neutral Red Staining of Virulent Tuberculosis Strains and Homologue of the Streptomyces coelicolor Protein KbpA

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

Buchko, Garry W.; Echols, Nathaniel; Flynn, E. Megan

The 261-residue Mycobacterium tuberculosis protein Rv0577 is a prominent antigen in tuberculosis patients, the responsible component for neutral red staining of virulent strains of M. tuberculosis, a putative component in a methylglyoxal detoxification pathway, and an agonist of toll-like receptor 2. It also has 36% sequence identity to AfsK-binding protein A (KbpA), a component in the complex secondary metabolite pathways in the Streptomycetes genus from which many commercial antibiotics are derived. To gain insight into the biological function of Rv0577 and the family of KpbA kinase regulators, the crystal structure for Rv0577 was determined to a resolution of 1.75 Åmore » (3OXH), binding properties with neutral red and deoxyadenosine (Ado) surveyed, backbone dynamics measured, and thermal stability assayed by CD spectroscopy. The protein is composed of four approximate repeats with an topology arranged radially in consecutive pairs to form two continuous eight-strand -sheets capped on both ends with an -helix. The two -sheets intersect in the center at roughly a right angle and form an asymmetric deep “saddle” on both sides of the protein, saddle one (P11 to A129) and saddle two (L143 to A258), that may serve to bind ligands. NMR chemical shift perturbation experiments show that neutral red binds to Rv0577, further cementing the role of Rv0577 in the neutral red staining of virulent strains of M. tuberculosis. Similar experiments show that adenosine also bind to Rv0577, although less tightly, with estimated dissociation constants of 4.1 ± 0.3 mM for saddle one and > 1 M for saddle two. Heteronuclear steady-state {1H}-15N NOE, T1, and T2 values were generally uniform through-out the sequence with only a few modest pockets of differences suggestive of slightly different motion in loops between -strands in saddle 1. Circular dichroism spectroscopy characterization of the thermal stability of Rv0577 indicated irreversible unfolding upon heating with an estimated melting temperature of 56 °C. While it is not known if Rv0577 has a kinase regulatory role similar to its Streptomyces homolog KbpA, protein kinase and phosphatase signaling help M. tuberculosis adapt to the hostile host environment during infections. Consequently, new anti-tuberculosis drugs targeting Rv0577 may act by interfering with multiple mechanisms; a potential signaling machinery as well as toll-like receptor 2 activation and the methylglyoxal detoxification pathway.« less

The natural absence of RPA1N domain did not impair Leishmania amazonensis RPA-1 participation in DNA damage response and telomere protection.

PubMed

Da Silveira, Rita De Cássia Viveiros; Da Silva, Marcelo Santos; Nunes, Vinícius Santana; Perez, Arina Marina; Cano, Maria Isabel Nogueira

2013-04-01

We have previously shown that the subunit 1 of Leishmania amazonensis RPA (LaRPA-1) alone binds the G-rich telomeric strand and is structurally different from other RPA-1. It is analogous to telomere end-binding proteins described in model eukaryotes whose homologues were not identified in the protozoan´s genome. Here we show that LaRPA-1 is involved with damage response and telomere protection although it lacks the RPA1N domain involved with the binding with multiple checkpoint proteins. We induced DNA double-strand breaks (DSBs) in Leishmania using phleomycin. Damage was confirmed by TUNEL-positive nuclei and triggered a G1/S cell cycle arrest that was accompanied by nuclear accumulation of LaRPA-1 and RAD51 in the S phase of hydroxyurea-synchronized parasites. DSBs also increased the levels of RAD51 in non-synchronized parasites and of LaRPA-1 and RAD51 in the S phase of synchronized cells. More LaRPA-1 appeared immunoprecipitating telomeres in vivo and associated in a complex containing RAD51, although this interaction needs more investigation. RAD51 apparently co-localized with few telomeric clusters but it did not immunoprecipitate telomeric DNA. These findings suggest that LaRPA-1 and RAD51 work together in response to DNA DSBs and at telomeres, upon damage, LaRPA-1 works probably to prevent loss of single-stranded DNA and to assume a capping function.

Biomarkers in systemic juvenile idiopathic arthritis: a comparison with biomarkers in cryopyrin-associated periodic syndromes.

PubMed

Nirmala, Nanguneri; Grom, Alexei; Gram, Hermann

2014-09-01

This review summarizes biomarkers in systemic juvenile idiopathic arthritis (sJIA). Broadly, the markers are classified under protein, cellular, gene expression and genetic markers. We also compare the biomarkers in sJIA to biomarkers in cryopyrin-associated periodic syndrome (CAPS). Recent publications showing the similarity of clinical response of sJIA and CAPS to anti-interleukin 1 therapies prompted a comparison at the biomarker level. sJIA traditionally is classified under the umbrella of juvenile idiopathic arthritis. At the clinical phenotypic level, sJIA has several features that are more similar to those seen in CAPS. In this review, we summarize biomarkers in sJIA and CAPS and draw upon the various similarities and differences between the two families of diseases. The main differences between sJIA and CAPS biomarkers are genetic markers, with CAPS being a family of monogenic diseases with mutations in NLRP3. There have been a small number of publications describing cellular biomarkers in sJIA with no such studies described for CAPS. Many of the protein marker's characteristics of sJIA are also seen to characterize CAPS. The gene expression data in both sJIA and CAPS show a strong upregulation of innate immunity pathways. In addition, we describe a strong similarity between sJIA and CAPS at the gene expression level in which several genes that form a part of the erythropoiesis signature are upregulated in both sJIA and CAPS.

Biomarkers in Systemic Juvenile Idiopathic Arthritis: A comparison with biomarkers in Cryopyrin Associated Periodic Syndromes

PubMed Central

Nirmala, Nanguneri; Grom, Alexei; Gram, Hermann

2015-01-01

Purpose of review This review summarizes biomarkers in Systemic Juvenile Idiopathic Arthritis (sJIA). Broadly, the markers are classified under protein, cellular, gene expression and genetic markers. We also compare the biomarkers in sJIA to biomarkers in cryopyrin associated periodic syndromes (CAPS). Recent findings Recent publications showing the similarity of clinical response of sJIA and CAPS to anti IL1 therapies prompted a comparison at the biomarker level. Summary sJIA traditionally is classified under the umbrella of juvenile idiopathic arthritis. At the clinical phenotypic level, sJIA has several features that are more similar to those seen in Cryopyrin Associated Periodic Syndromes (CAPS). In this review, we summarize biomarkers in sJIA and CAPS and draw upon the various similarities and differences between the two families of diseases. The main difference between sJIA and CAPS biomarkers are genetic markers with CAPS being a family of monogenic diseases with mutations in NLRP3. There have been a small number of publications describing cellular biomarkers in sJIA with no such studies described for CAPS. Many of the protein markers characteristic of sJIA are also seen to characterize CAPS. The gene expression data in both sJIA and CAPS show a strong upregulation of innate immunity pathways. In addition, we describe a strong similarity between sJIA and CAPS at the gene expression level where several genes that form a part of the erythropoiesis signature are upregulated in both sJIA and CAPS. PMID:25050926

COLLAPSED ABNORMAL POLLEN1 Gene Encoding the Arabinokinase-Like Protein Is Involved in Pollen Development in Rice1[C][W][OA

PubMed Central

Ueda, Kenji; Yoshimura, Fumiaki; Miyao, Akio; Hirochika, Hirohiko; Nonomura, Ken-Ichi; Wabiko, Hiroetsu

2013-01-01

We isolated a pollen-defective mutant, collapsed abnormal pollen1 (cap1), from Tos17 insertional mutant lines of rice (Oryza sativa). The cap1 heterozygous plant produced equal numbers of normal and collapsed abnormal grains. The abnormal pollen grains lacked almost all cytoplasmic materials, nuclei, and intine cell walls and did not germinate. Genetic analysis of crosses revealed that the cap1 mutation did not affect female reproduction or vegetative growth. CAP1 encodes a protein consisting of 996 amino acids that showed high similarity to Arabidopsis (Arabidopsis thaliana) l-arabinokinase, which catalyzes the conversion of l-arabinose to l-arabinose 1-phosphate. A wild-type genomic DNA segment containing CAP1 restored mutants to normal pollen grains. During rice pollen development, CAP1 was preferentially expressed in anthers at the bicellular pollen stage, and the effects of the cap1 mutation were mainly detected at this stage. Based on the metabolic pathway of l-arabinose, cap1 pollen phenotype may have been caused by toxic accumulation of l-arabinose or by inhibition of cell wall metabolism due to the lack of UDP-l-arabinose derived from l-arabinose 1-phosphate. The expression pattern of CAP1 was very similar to that of another Arabidopsis homolog that showed 71% amino acid identity with CAP1. Our results suggested that CAP1 and related genes are critical for pollen development in both monocotyledonous and dicotyledonous plants. PMID:23629836

Resident CAPS on dense-core vesicles docks and primes vesicles for fusion.

PubMed

Kabachinski, Greg; Kielar-Grevstad, D Michelle; Zhang, Xingmin; James, Declan J; Martin, Thomas F J

2016-02-15

The Ca(2+)-dependent exocytosis of dense-core vesicles in neuroendocrine cells requires a priming step during which SNARE protein complexes assemble. CAPS (aka CADPS) is one of several factors required for vesicle priming; however, the localization and dynamics of CAPS at sites of exocytosis in live neuroendocrine cells has not been determined. We imaged CAPS before, during, and after single-vesicle fusion events in PC12 cells by TIRF micro-scopy. In addition to being a resident on cytoplasmic dense-core vesicles, CAPS was present in clusters of approximately nine molecules near the plasma membrane that corresponded to docked/tethered vesicles. CAPS accompanied vesicles to the plasma membrane and was present at all vesicle exocytic events. The knockdown of CAPS by shRNA eliminated the VAMP-2-dependent docking and evoked exocytosis of fusion-competent vesicles. A CAPS(ΔC135) protein that does not localize to vesicles failed to rescue vesicle docking and evoked exocytosis in CAPS-depleted cells, showing that CAPS residence on vesicles is essential. Our results indicate that dense-core vesicles carry CAPS to sites of exocytosis, where CAPS promotes vesicle docking and fusion competence, probably by initiating SNARE complex assembly. © 2016 Kabachinski, Kielar-Grevstad, et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

An Aromatic Cap Seals the Substrate Binding Site in an ECF-Type S Subunit for Riboflavin

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

Karpowich, Nathan K.; Song, Jinmei; Wang, Da-Neng

2016-06-13

ECF transporters are a family of active membrane transporters for essential micronutrients, such as vitamins and trace metals. Found exclusively in archaea and bacteria, these transporters are composed of four subunits: an integral membrane substrate-binding subunit (EcfS), a transmembrane coupling subunit (EcfT), and two ATP-binding cassette ATPases (EcfA and EcfA'). We have characterized the structural basis of substrate binding by the EcfS subunit for riboflavin from Thermotoga maritima, TmRibU. TmRibU binds riboflavin with high affinity, and the protein–substrate complex is exceptionally stable in solution. The crystal structure of riboflavin-bound TmRibU reveals an electronegative binding pocket at the extracellular surface inmore » which the substrate is completely buried. Analysis of the intermolecular contacts indicates that nearly every available substrate hydrogen bond is satisfied. A conserved aromatic residue at the extracellular end of TM5, Tyr130, caps the binding site to generate a substrate-bound, occluded state, and non-conservative mutation of Tyr130 reduces the stability of this conformation. Using a novel fluorescence binding assay, we find that an aromatic residue at this position is essential for high-affinity substrate binding. Comparison with other S subunit structures suggests that TM5 and Loop5-6 contain a dynamic, conserved motif that plays a key role in gating substrate entry and release by S subunits of ECF transporters.« less

Bonding in the first-row diatomic molecules within the local spin-density approximation

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

Painter, G.S.; Averill, F.W.

1982-08-15

The Hohenberg-Kohn-Sham density-functional equations in the local spin-density approximation (LSDA) have been solved with essentially no loss of accuracy for dimers of the first row of the Periodic Table with the use of a fully-self-consistent spin-polarized Gaussian-orbital approach. Spectroscopic constants (binding energies, equilibrium separations, and ground-state vibrational frequencies) have been derived from the calculated potential-energy curves. Intercomparison of results obtained using the exchange-correlation functionals of Slater (scaled exchange or X..cap alpha..), Gunnarsson and Lundqvist (GL), and Vosko, Wilk, and Nusair (VWN) permits assessment of the relative merits of each and serves to identify general shortcomings in the LSDA. Basic trendsmore » are similar for each functional, but the treatment of the spin dependence of the exchange-correlation energy in the GL and VWN functionals yields a variation of the binding energy across the series which is more systematic than that in the X..cap alpha.. approximation. Agreement between the present results and those of Dunlap, Connolly, and Sabin in the X..cap alpha.., approximation confirms the accuracy of the variational charge-density-fit procedure used in the latter work. The refinements in correlation treatment within the VWN functional are reflected in improvements in binding energies which are only slight for most dimers in the series. This behavior is attributed to the error remaining in the exchange channel within the LSDA and demonstrates the necessity for self-interaction corrections for more accurate binding-energy determinations. Within the current LSDA, absolute accuracies of the VWN functional for the first-row dimers are within 2.3 eV for binding energies, 0.07 a.u. for bond lengths, and approx.200 cm/sup -1/ for vibrational frequencies.« less

Injury downregulates the expression of the human cathelicidin protein hCAP18/LL-37 in atopic dermatitis.

PubMed

Mallbris, Lotus; Carlén, Lina; Wei, Tianling; Heilborn, Johan; Nilsson, Margareta Frohm; Granath, Fredrik; Ståhle, Mona

2010-05-01

Reduced production of antimicrobial peptides was proposed to contribute to susceptibility for skin infections in atopic dermatitis (AD). Focusing on the human cathelicidin protein, hCAP18, the aim of the present study was to explore whether reduced hCAP18 expression is a constitutive trait in AD and if established inducers affect the expression of hCAP18 in the skin of AD. First, we compared levels of hCAP18 mRNA between lesional skin in AD and psoriasis and verified significantly lower expression of hCAP18 mRNA in AD. In non-lesional skin, however, there was no difference between AD, psoriasis and healthy, indicating that there is no constitutive defect in the production of hCAP18 in AD patients. In healthy skin, hCAP18 was reported to be rapidly induced following wounding and here we verified this pattern in healthy controls and in psoriasis. In AD lesions, however, the expression of hCAP18 mRNA was markedly suppressed following wounding. Obviously, the inflammation in AD lesions neutralizes the expected induction of hCAP18 and even induces suppression. Notably, the mechanism to upregulate hCAP18 following vitamin D treatment was functional in lesional as well as in non-lesional AD indicating that the CAMP gene is normally regulated in this respect. In addition, cultured primary keratinocytes from non-lesional skin of psoriasis, AD and healthy skin, upregulated hCAP18mRNA following treatment with vitamin D. Itching is a hallmark of AD and scratching inevitably injures the skin. Failure to upregulate hCAP18 in eczema following injury is likely to affect antimicrobial protection and tissue repair in AD.

Cryopyrin-associated periodic syndromes: background and therapeutics.

PubMed

Kubota, Tetsuo; Koike, Ryuji

2010-06-01

Cryopyrin-associated periodic syndromes (CAPS) are caused by mutations of the gene encoding the NLR family protein NLRP3, which together with caspase-1 and adaptor proteins constitutes a protein complex termed the inflammasome. In innate immune reactions, a variety of stimuli activate the NLRP3 inflammasome, triggering caspase-1 to process proIL-1 and thus to produce mature IL-1. Excessive production of IL-1 by monocytes/macrophages is the central pathophysiology of CAPS, and daily injection of the IL-1 receptor antagonist anakinra rapidly ameliorates the inflammatory symptoms in most cases. Furthermore, double-blind, placebo-controlled clinical trials have recently confirmed the efficacy and safety of rilonacept, a fusion protein of the IL-1 receptor and IgG Fc, and canakinumab, a human anti-IL-1 monoclonal antibody, as novel long-lasting agents for treating CAPS.

Hydrolytic properties and substrate specificity of the foot-and-mouth disease leader protease.

PubMed

Santos, Jorge A N; Gouvea, Iuri E; Júdice, Wagner A S; Izidoro, Mario A; Alves, Fabiana M; Melo, Robson L; Juliano, Maria A; Skern, Tim; Juliano, Luiz

2009-08-25

Foot-and-mouth disease virus, a global animal pathogen, possesses a single-stranded RNA genome that, on release into the infected cell, is immediately translated into a single polyprotein. This polyprotein product is cleaved during synthesis by proteinases contained within it into the mature viral proteins. The first cleavage is performed by the leader protease (Lb(pro)) between its own C-terminus and the N-terminus of VP4. Lb(pro) also specifically cleaves the two homologues of cellular eukaryotic initiation factor (eIF) 4G, preventing translation of capped mRNA. Viral protein synthesis is initiated internally and is thus unaffected. We used a panel of specifically designed FRET peptides to examine the effects of pH and ionic strength on Lb(pro) activity and investigate the size of the substrate binding site and substrate specificity. Compared to the class prototypes, papain and the cathepsins, Lb(pro) possesses several unusual characteristics, including a high sensitivity to salt and a very specific substrate binding site extending up to P(7). Indeed, almost all substitutions investigated were detrimental to Lb(pro) activity. Analysis of structural data showed that Lb(pro) binds residues P(1)-P(3) in an extended conformation, whereas residues P(4)-P(7) are bound in a short 3(10) helix. The specificity of Lb(pro) as revealed by the substituted peptides could be explained for all positions except P(5). Strikingly, Lb(pro) residues L178 and L143 contribute to the architecture of more than one substrate binding pocket. The diverse functions of these two Lb(pro) residues explain why Lb(pro) is one of the smallest, but simultaneously most specific, papain-like enzymes.

Two related trypanosomatid eIF4G homologues have functional differences compatible with distinct roles during translation initiation

PubMed Central

Moura, Danielle MN; Reis, Christian RS; Xavier, Camila C; da Costa Lima, Tamara D; Lima, Rodrigo P; Carrington, Mark; de Melo Neto, Osvaldo P

2015-01-01

In higher eukaryotes, eIF4A, eIF4E and eIF4G homologues interact to enable mRNA recruitment to the ribosome. eIF4G acts as a scaffold for these interactions and also interacts with other proteins of the translational machinery. Trypanosomatid protozoa have multiple homologues of eIF4E and eIF4G and the precise function of each remains unclear. Here, 2 previously described eIF4G homologues, EIF4G3 and EIF4G4, were further investigated. In vitro, both homologues bound EIF4AI, but with different interaction properties. Binding to distinct eIF4Es was also confirmed; EIF4G3 bound EIF4E4 while EIF4G4 bound EIF4E3, both these interactions required similar binding motifs. EIF4G3, but not EIF4G4, interacted with PABP1, a poly-A binding protein homolog. Work in vivo with Trypanosoma brucei showed that both EIF4G3 and EIF4G4 are cytoplasmic and essential for viability. Depletion of EIF4G3 caused a rapid reduction in total translation while EIF4G4 depletion led to changes in morphology but no substantial inhibition of translation. Site-directed mutagenesis was used to disrupt interactions of the eIF4Gs with either eIF4E or eIF4A, causing different levels of growth inhibition. Overall the results show that only EIF4G3, with its cap binding partner EIF4E4, plays a major role in translational initiation. PMID:25826663

An immunoblotting analysis of cross-reactivity between melon, and plantago and grass pollens.

PubMed

García Ortiz, J C; Ventas, P; Cosmes, P; López-Asunsolo, A

1996-01-01

It is known that most patients with type I allergy to pollens also suffer intolerance to fruits. Recently, an epidemiological and CAP-inhibition study has shown a new clustering of allergy between melon and Plantago and grass pollens. The aim of the present study was to confirm these results by immunoblotting analysis and inhibition of immunoblotting. Sera from 3 patients with confirmed allergy to melon, and Dactylis glomerata and Plantago lanceolata pollens were used for the in vitro studies. SDS-PAGE and immunoblotting analysis with a pool of sera revealed that several distinct protein bands were shared by the three extracts at 14, 31, and a spectrum between 40 and 70 kDa, approximately. Immunoblotting inhibition experiments, performed with extracts of melon, Plantago and Dactylis, showed that all allergens of melon blotting were almost completely inhibited by grass and Plantago pollen extracts. Inversely, the melon extract was capable of inhibiting IgE-binding to various allergens of Dactylis at high mol mass and partially to the band at 14 kDa. Moreover, the melon almost totally inhibited the IgE-binding capacity to the proteins of Plantago extract. Taken together, the results support the presence of structurally similar allergens in melon, Plantago and grass pollens, and that all allergenic epitopes of the melon are present in these pollens.

Coactosin accelerates cell dynamism by promoting actin polymerization.

PubMed

Hou, Xubin; Katahira, Tatsuya; Ohashi, Kazumasa; Mizuno, Kensaku; Sugiyama, Sayaka; Nakamura, Harukazu

2013-07-01

During development, cells dynamically move or extend their processes, which are achieved by actin dynamics. In the present study, we paid attention to Coactosin, an actin binding protein, and studied its role in actin dynamics. Coactosin was associated with actin and Capping protein in neural crest cells and N1E-115 neuroblastoma cells. Accumulation of Coactosin to cellular processes and its association with actin filaments prompted us to reveal the effect of Coactosin on cell migration. Coactosin overexpression induced cellular processes in cultured neural crest cells. In contrast, knock-down of Coactosin resulted in disruption of actin polymerization and of neural crest cell migration. Importantly, Coactosin was recruited to lamellipodia and filopodia in response to Rac signaling, and mutated Coactosin that cannot bind to F-actin did not react to Rac signaling, nor support neural crest cell migration. It was also shown that deprivation of Rac signaling from neural crest cells by dominant negative Rac1 (DN-Rac1) interfered with neural crest cell migration, and that co-transfection of DN-Rac1 and Coactosin restored neural crest cell migration. From these results we have concluded that Coactosin functions downstream of Rac signaling and that it is involved in neurite extension and neural crest cell migration by actively participating in actin polymerization. Copyright © 2013 Elsevier Inc. All rights reserved.

Alpha3, a transposable element that promotes host sexual reproduction.

PubMed

Barsoum, Emad; Martinez, Paula; Aström, Stefan U

2010-01-01

Theoretical models predict that selfish DNA elements require host sex to persist in a population. Therefore, a transposon that induces sex would strongly favor its own spread. We demonstrate that a protein homologous to transposases, called alpha3, was essential for mating type switch in Kluyveromyces lactis. Mutational analysis showed that amino acids conserved among transposases were essential for its function. During switching, sequences in the 5' and 3' flanking regions of the alpha3 gene were joined, forming a DNA circle, showing that alpha3 mobilized from the genome. The sequences encompassing the alpha3 gene circle junctions in the mating type alpha (MATalpha) locus were essential for switching from MATalpha to MATa, suggesting that alpha3 mobilization was a coupled event. Switching also required a DNA-binding protein, Mating type switch 1 (Mts1), whose binding sites in MATalpha were important. Expression of Mts1 was repressed in MATa/MATalpha diploids and by nutrients, limiting switching to haploids in low-nutrient conditions. A hairpin-capped DNA double-strand break (DSB) was observed in the MATa locus in mre11 mutant strains, indicating that mating type switch was induced by MAT-specific DSBs. This study provides empirical evidence for selfish DNA promoting host sexual reproduction by mediating mating type switch.

Drosophila cell cycle under arrest: uncapped telomeres plead guilty.

PubMed

Cenci, Giovanni

2009-04-01

Telomeres are specialized structures that protect chromosome ends from degradation and fusion events. In most organisms, telomeres consist of short, repetitive G-rich sequences added to chromosome ends by a reverse transcriptase with an internal RNA template, called telomerase. Specific DNA-binding protein complexes associate with telomeric sequences preventing chromosome ends from being recognized as DNA double strand breaks (DSBs). Telomeres that lose their cap activate the DNA damage response (DDR) likewise DSBs and, if inappropriately repaired, generate telomeric fusions, which eventually lead to genome instability. In Drosophila there is not telomerase, and telomere length is maintained by transposition of three specialized retroelements. However, fly telomeres are protected by multi protein complexes like their yeast and vertebrate counterparts; these complexes bind chromosome ends in a sequence-independent fashion and are required to prevent checkpoint activation and end-to-end fusion. Uncapped Drosophila telomeres elicit a DDR just as dysfunctional human telomeres. Most interestingly, uncapped Drosophila telomeres also activate the spindle assembly checkpoint (SAC) by recruiting the SAC kinase BubR1. BubR1 accumulations at chromosome ends trigger the SAC that inhibits the metaphase-to-anaphase transition. These findings, reviewed here, highlight an intriguing and unsuspected connection between telomeres and cell cycle regulation, providing a clue to understand human telomere function.

An Electrochemical Impedimetric Aptasensing Platform for Sensitive and Selective Detection of Small Molecules Such as Chloramphenicol

PubMed Central

Pilehvar, Sanaz; Dierckx, Tarryn; Blust, Ronny; Breugelmans, Tom; De Wael, Karolien

2014-01-01

We report on the aptadetection of chloramphenicol (CAP) using electrochemical impedance spectroscopy. The detection principle is based on the changes of the interfacial properties of the electrode after the interaction of the ssDNA aptamers with the target molecules. The electrode surface is partially blocked due to the formation of the aptamer-CAP complex, resulting in an increase of the interfacial electron-transfer resistance of the redox probe detected by electrochemical impedance spectroscopy or cyclic voltammetry. We observed that the ratio of polarization resistance had a linear relationship with the concentrations of CAP in the range of 1.76–127 nM, and a detection limit of 1.76 nM was obtained. The covalent binding of CAP-aptamer on the electrode surface combined with the unique properties of aptamers and impedimetric transduction leads to the development of a stable and sensitive electrochemical aptasensor for CAP. PMID:25004156

Capped antigenomic RNA transcript facilitates rescue of a plant rhabdovirus.

PubMed

Qian, Shasha; Chen, Xiaolan; Sun, Kai; Zhang, Yang; Li, Zhenghe

2017-06-13

Recovery of recombinant negative-stranded RNA viruses from cloned cDNAs is an inefficient process as multiple viral components need to be delivered into cells for reconstitution of infectious entities. Previously studies have shown that authentic viral RNA termini are essential for efficient virus rescue. However, little is known about the activity of viral RNAs processed by different strategies in supporting recovery of plant negative-stranded RNA virus. In this study, we used several versions of hammerhead ribozymes and a truncated cauliflower mosaic virus 35S promoter to generate precise 5' termini of sonchus yellow net rhabdovirus (SYNV) antigenomic RNA (agRNA) derivatives. These agRNAs were co-expressed with the SYNV core proteins in Nicotiana benthamiana leaves to evaluate their efficiency in supporting fluorescent reporter gene expression from an SYNV minireplicon (MR) and rescue of full-length virus. Optimization of hammerhead ribozyme cleavage activities led to improved SYNV MR reporter gene expression. Although the MR agRNA processed by the most active hammerhead variants is comparable to the capped, precisely transcribed agRNA in supporting MR activity, efficient recovery of recombinant SYNV was only achieved with capped agRNA. Further studies showed that the capped SYNV agRNA permitted transient expression of the nucleocapsid (N) protein, and an agRNA derivatives unable to express the N protein in cis exhibited dramatically reduced rescue efficiency. Our study reveals superior activity of precisely transcribed, capped SYNV agRNAs to uncapped, hammerhead ribozyme-processed agRNAs, and suggests a cis-acting function for the N protein expressed from the capped agRNA during recovery of SYNV from plasmids.

Capping protein regulatory cycle driven by CARMIL and V-1 may promote actin network assembly at protruding edges

PubMed Central

Fujiwara, Ikuko; Remmert, Kirsten; Piszczek, Grzegorz; Hammer, John A.

2014-01-01

Although capping protein (CP) terminates actin filament elongation, it promotes Arp2/3-dependent actin network assembly and accelerates actin-based motility both in vitro and in vivo. In vitro, capping protein Arp2/3 myosin I linker (CARMIL) antagonizes CP by reducing its affinity for the barbed end and by uncapping CP-capped filaments, whereas the protein V-1/myotrophin sequesters CP in an inactive complex. Previous work showed that CARMIL can readily retrieve CP from the CP:V-1 complex, thereby converting inactive CP into a version with moderate affinity for the barbed end. Here we further clarify the mechanism of this exchange reaction, and we demonstrate that the CP:CARMIL complex created by complex exchange slows the rate of barbed-end elongation by rapidly associating with, and dissociating from, the barbed end. Importantly, the cellular concentrations of V-1 and CP determined here argue that most CP is sequestered by V-1 at steady state in vivo. Finally, we show that CARMIL is recruited to the plasma membrane and only at cell edges undergoing active protrusion. Assuming that CARMIL is active only at this location, our data argue that a large pool of freely diffusing, inactive CP (CP:V-1) feeds, via CARMIL-driven complex exchange, the formation of weak-capping complexes (CP:CARMIL) at the plasma membrane of protruding edges. In vivo, therefore, CARMIL should promote Arp2/3-dependent actin network assembly at the leading edge by promoting barbed-end capping there. PMID:24778263

eIF4A RNA Helicase Associates with Cyclin-Dependent Protein Kinase A in Proliferating Cells and Is Modulated by Phosphorylation1[OPEN

PubMed Central

Bush, Maxwell S.; Pierrat, Olivier; Nibau, Candida; Mikitova, Veronika; Zheng, Tao; Corke, Fiona M. K.; Mayberry, Laura K.; Browning, Karen S.

2016-01-01

Eukaryotic initiation factor 4A (eIF4A) is a highly conserved RNA-stimulated ATPase and helicase involved in the initiation of messenger RNA translation. Previously, we found that eIF4A interacts with cyclin-dependent kinase A (CDKA), the plant ortholog of mammalian CDK1. Here, we show that this interaction occurs only in proliferating cells where the two proteins coassociate with 5′-cap-binding protein complexes, eIF4F or the plant-specific eIFiso4F. CDKA phosphorylates eIF4A on a conserved threonine residue (threonine-164) within the RNA-binding motif 1b TPGR. In vivo, a phospho-null (APGR) variant of the Arabidopsis (Arabidopsis thaliana) eIF4A1 protein retains the ability to functionally complement a mutant (eif4a1) plant line lacking eIF4A1, whereas a phosphomimetic (EPGR) variant fails to complement. The phospho-null variant (APGR) rescues the slow growth rate of roots and rosettes, together with the ovule-abortion and late-flowering phenotypes. In vitro, wild-type recombinant eIF4A1 and its phospho-null variant both support translation in cell-free wheat germ extracts dependent upon eIF4A, but the phosphomimetic variant does not support translation and also was deficient in ATP hydrolysis and helicase activity. These observations suggest a mechanism whereby CDK phosphorylation has the potential to down-regulate eIF4A activity and thereby affect translation. PMID:27388680

A Panoptic Uncovering of the Dynamical Evolution of the Zika Virus NS5 Methyltransferase Binding Site Loops- Zeroing in on the Molecular Landscape.

PubMed

Devnarain, Nikita; Soliman, Mahmoud E S

2018-06-20

The global threat of the Zika virus to humanity is real. Innovative and potent anti-Zika virus drugs are still at large, due to the lack of anti-Zika virus drugs that have passed phase 1 trials. Experimental research has revealed novel inhibitors of Zika virus NS5 methyltransferase enzyme. This study has taken a step further to provide insight into the molecular dynamics of Zika virus and inhibitor binding, which have not been established experimentally. Movements of the methyltransferase binding site loops have a large role to play in the methylation of the viral mRNA cap, which is essential for Zika virus replication. Here we pinpoint the binding interactions between each potential inhibitor and the methyltransferase, residues that are responsible for binding, as well as which inhibitor-bound complex renders the methyltransferase more stable. We also highlight the conformational changes that occur within the methyltransferase to accommodate binding of inhibitors and consequences of those changes upon the RNA- and cap-binding sites in the methyltransferase. This research will improve the understanding of the Zika virus NS5 methyltransferase enzyme, and will be beneficial in driving the development of anti-Zika virus drugs. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

TCTEX1D4, a novel protein phosphatase 1 interactor: connecting the phosphatase to the microtubule network

PubMed Central

Korrodi-Gregório, Luís; Vieira, Sandra I.; Esteves, Sara L. C.; Silva, Joana V.; Freitas, Maria João; Brauns, Ann-Kristin; Luers, Georg; Abrantes, Joana; Esteves, Pedro J.; da Cruz e Silva, Odete A. B.; Fardilha, Margarida; da Cruz e Silva, Edgar F.

2013-01-01

Summary Reversible phosphorylation plays an important role as a mechanism of intracellular control in eukaryotes. PPP1, a major eukaryotic Ser/Thr-protein phosphatase, acquires its specificity by interacting with different protein regulators, also known as PPP1 interacting proteins (PIPs). In the present work we characterized a physiologically relevant PIP in testis. Using a yeast two-hybrid screen with a human testis cDNA library, we identified a novel PIP of PPP1CC2 isoform, the T-complex testis expressed protein 1 domain containing 4 (TCTEX1D4) that has recently been described as a Tctex1 dynein light chain family member. The overlay assays confirm that TCTEX1D4 interacts with the different spliced isoforms of PPP1CC. Also, the binding domain occurs in the N-terminus, where a consensus PPP1 binding motif (PPP1BM) RVSF is present. The distribution of TCTEX1D4 in testis suggests its involvement in distinct functions, such as TGFβ signaling at the blood–testis barrier and acrosome cap formation. Immunofluorescence in human ejaculated sperm shows that TCTEX1D4 is present in the flagellum and in the acrosome region of the head. Moreover, TCTEX1D4 and PPP1 co-localize in the microtubule organizing center (MTOC) and microtubules in cell cultures. Importantly, the TCTEX1D4 PPP1BM seems to be relevant for complex formation, for PPP1 retention in the MTOC and movement along microtubules. These novel results open new avenues to possible roles of this dynein, together with PPP1. In essence TCTEX1D4/PPP1C complex appears to be involved in microtubule dynamics, sperm motility, acrosome reaction and in the regulation of the blood–testis barrier. PMID:23789093

Structural basis for the substrate selectivity of a HAD phosphatase from Thermococcus onnurineus NA1.

PubMed

Ngo, Tri Duc; Van Le, Binh; Subramani, Vinod Kumar; Thi Nguyen, Chi My; Lee, Hyun Sook; Cho, Yona; Kim, Kyeong Kyu; Hwang, Hye-Yeon

2015-05-22

Proteins in the haloalkaloic acid dehalogenase (HAD) superfamily, which is one of the largest enzyme families, is generally composed of a catalytic core domain and a cap domain. Although proteins in this family show broad substrate specificities, the mechanisms of their substrate recognition are not well understood. In this study, we identified a new substrate binding motif of HAD proteins from structural and functional analyses, and propose that this motif might be crucial for interacting with hydrophobic rings of substrates. The crystal structure of TON_0338, one of the 17 putative HAD proteins identified in a hyperthermophilic archaeon, Thermococcus onnurineus NA1, was determined as an apo-form at 2.0 Å resolution. In addition, we determined the crystal structure TON_0338 in complex with Mg(2+) or N-cyclohexyl-2-aminoethanesulfonic acid (CHES) at 1.7 Å resolution. Examination of the apo-form and CHES-bound structures revealed that CHES is sandwiched between Trp58 and Trp61, suggesting that this Trp sandwich might function as a substrate recognition motif. In the phosphatase assay, TON_0338 was shown to have high activity for flavin mononucleotide (FMN), and the docking analysis suggested that the flavin of FMN may interact with Trp58 and Trp61 in a way similar to that observed in the crystal structure. Moreover, the replacement of these tryptophan residues significantly reduced the phosphatase activity for FMN. Our results suggest that WxxW may function as a substrate binding motif in HAD proteins, and expand the diversity of their substrate recognition mode. Copyright © 2015 Elsevier Inc. All rights reserved.

Cytotoxicity Induced by a Redox-silent Analog of Tocotrienol in Human Mesothelioma H2452 Cell Line via Suppression of Cap-dependent Protein Translation.

PubMed

Sato, Ayami; Ueno, Haruka; Takase, Akari; Ando, Akira; Sekine, Yuko; Yano, Tomohiro

2016-04-01

De novo synthesis of proteins is regulated by cap-dependent protein translation. Aberrant activation of the translation is a hallmark of many cancer types including malignant mesothelioma (MM). We previously reported that a redox-silent analog of α-tocotrienol, 6-O-carboxypropyl-α-tocotrienol (T3E) induces potent cytotoxicity against human MM cells. However, the detailed mechanism of cytotoxicity of T3E remains unclear. In this study, we investigated if T3E induced potent cytotoxicity aganist MM cells. T3E reduced the formation of the cap-dependent translation complex and induced inactivation of oncogene from rat sarcoma virus (RAS). These events were associated with T3E cytotoxicity in MM cells. Furthermore, atorvastatin, an inhibitor of RAS function, had similar effects on MM cells. Moreover, 4EGI-1, a specific inhibitor of the cap-dependent translation complex, induced severe cytotoxicity in MM cells. Overall, T3E had a cytotoxic effect on MM cells via disruption of the activated cap-dependent translation complex through inactivation of RAS. Copyright© 2016 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

Differentiation to adipocytes in accompanied by an increase in the amounts of Gi- and Go-proteins in 3T3-L1 cells

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

Watkins, D.C.; Northup, J.K.; Malbon, C.C.

Treatment of cultures of 3T3-L1 cells with methylisobutyl-xanthine and dexamethasone has been shown to result in accumulation of lipid and conversion to the morphology of adipocytes in more than 90% of the cells. The status of the stimulatory (Gs), inhibitory (Gi) and Go-proteins during the course of 3T3-L1 differentiation was examined. The amount of alpha subunit of Gs (..cap alpha..Gs), assayed by radiolabeling in the presence of cholera toxin and (/sup 32/P)NAD/sup +/, increased upon differentiation as previously described by others. The amounts of ..cap alpha..Gi and ..cap alpha..Go assayed by radiolabeling in the presence of pertussis toxin and (/supmore » 32/P)NAD/sup +/ increased 3-fold upon differentiation. Immunoblots of cell membranes subjected to gel electrophoresis in sodium dodecyl sulfate were probed with two rabbit antisera raised against bovine brain ..cap alpha..Go and with one raised against the..beta..-subunit of the bovine rod-outer-segment G-protein, referred to as transducin. The immunoblotting data confirm the increase upon differentiation of ..cap alpha..Go and also demonstrate an increase in the amount of the ..beta..-subunit. Thus differentiation of 3T3-L1 cells is accompanied by dramatic changes in the complexion of G-proteins in the membranes.« less

Amine-capped ZnS-Mn2+ nanocrystals for fluorescence detection of trace TNT explosive.

PubMed

Tu, Renyong; Liu, Bianhua; Wang, Zhenyang; Gao, Daming; Wang, Feng; Fang, Qunling; Zhang, Zhongping

2008-05-01

Mn2+-doped ZnS nanocrystals with an amine-capping layer have been synthesized and used for the fluorescence detection of ultratrace 2,4,6-trinitrotoluene (TNT) by quenching the strong orange Mn2+ photoluminescence. The organic amine-capped nanocrystals can bind TNT species from solution and atmosphere by the acid-base pairing interaction between electron-rich amino ligands and electron-deficient aromatic rings. The resultant TNT anions bound onto the amino monolayer can efficiently quench the Mn2+ photoluminescence through the electron transfer from the conductive band of ZnS to the lowest unoccupied molecular orbital (LUMO) of TNT anions. The amino ligands provide an amplified response to the binding events of nitroaromatic compounds by the 2- to approximately 5-fold increase in quenching constants. Moreover, a large difference in quenching efficiency was observed for different types of nitroaromatic analytes, dependent on the affinity of nitro analytes to the amino monolayer and their electron-accepting abilities. The amine-capped nanocrystals can sensitively detect down to 1 nM TNT in solution or several parts-per-billion of TNT vapor in atmosphere. The ion-doped nanocrystal sensors reported here show a remarkable air/solution stability, high quantum yield, and strong analyte affinity and, therefore, are well-suited for detecting the ultratrace TNT and distinguishing different nitro compounds.

Microwave-Assisted Hydrothermal Rapid Synthesis of Calcium Phosphates: Structural Control and Application in Protein Adsorption

PubMed Central

Cai, Zhu-Yun; Peng, Fan; Zi, Yun-Peng; Chen, Feng; Qian, Qi-Rong

2015-01-01

Synthetic calcium phosphate (CaP)-based materials have attracted much attention in the biomedical field. In this study, we have investigated the effect of pH values on CaP nanostructures prepared using a microwave-assisted hydrothermal method. The hierarchical nanosheet-assembled hydroxyapatite (HAP) nanostructure was prepared under weak acidic conditions (pH 5), while the HAP nanorod was prepared under neutral (pH 7) and weak alkali (pH 9) condition. However, when the pH value increases to 11, a mixed product of HAP nanorod and tri-calcium phosphate nanoparticle was obtained. The results indicated that the pH value of the initial reaction solution played an important role in the phase and structure of the CaP. Furthermore, the protein adsorption and release performance of the as-prepared CaP nanostructures were investigated by using hemoglobin (Hb) as a model protein. The sample that was prepared at pH = 11 and consisted of mixed morphologies of nanorods and nanoprisms showed a higher Hb protein adsorption capacity than the sample prepared at pH 5, which could be explained by its smaller size and dispersed structure. The results revealed the relatively high protein adsorption capacity of the as-prepared CaP nanostructures, which show promise for applications in various biomedical fields such as drug delivery and protein adsorption. PMID:28347064

Microwave-Assisted Hydrothermal Rapid Synthesis of Calcium Phosphates: Structural Control and Application in Protein Adsorption.

PubMed

Cai, Zhu-Yun; Peng, Fan; Zi, Yun-Peng; Chen, Feng; Qian, Qi-Rong

2015-07-31

Synthetic calcium phosphate (CaP)-based materials have attracted much attention in the biomedical field. In this study, we have investigated the effect of pH values on CaP nanostructures prepared using a microwave-assisted hydrothermal method. The hierarchical nanosheet-assembled hydroxyapatite (HAP) nanostructure was prepared under weak acidic conditions (pH 5), while the HAP nanorod was prepared under neutral (pH 7) and weak alkali (pH 9) condition. However, when the pH value increases to 11, a mixed product of HAP nanorod and tri-calcium phosphate nanoparticle was obtained. The results indicated that the pH value of the initial reaction solution played an important role in the phase and structure of the CaP. Furthermore, the protein adsorption and release performance of the as-prepared CaP nanostructures were investigated by using hemoglobin (Hb) as a model protein. The sample that was prepared at pH = 11 and consisted of mixed morphologies of nanorods and nanoprisms showed a higher Hb protein adsorption capacity than the sample prepared at pH 5, which could be explained by its smaller size and dispersed structure. The results revealed the relatively high protein adsorption capacity of the as-prepared CaP nanostructures, which show promise for applications in various biomedical fields such as drug delivery and protein adsorption.

The Enigmatic Alphavirus Non-Structural Protein 3 (nsP3) Revealing Its Secrets at Last

PubMed Central

Götte, Benjamin; Liu, Lifeng

2018-01-01

Alphaviruses encode 4 non-structural proteins (nsPs), most of which have well-understood functions in capping and membrane association (nsP1), polyprotein processing and RNA helicase activity (nsP2) and as RNA-dependent RNA polymerase (nsP4). The function of nsP3 has been more difficult to pin down and it has long been referred to as the more enigmatic of the nsPs. The protein comprises three domains, an N-terminal macro domain, a central zinc-binding domain and a C-terminal hypervariable domain (HVD). In this article, we review old and new literature about the functions of the three domains. Much progress in recent years has contributed to a picture of nsP3, particularly through its HVD as a hub for interactions with host cell molecules, with multiple effects on the biology of the host cell at early points in infection. These and many future discoveries will provide targets for anti-viral therapies as well as strategies for modification of vectors for vaccine and oncolytic interventions. PMID:29495654

Leiomodin and tropomodulin in smooth muscle

NASA Technical Reports Server (NTRS)

Conley, C. A.

2001-01-01

Evidence is accumulating to suggest that actin filament remodeling is critical for smooth muscle contraction, which implicates actin filament ends as important sites for regulation of contraction. Tropomodulin (Tmod) and smooth muscle leiomodin (SM-Lmod) have been found in many tissues containing smooth muscle by protein immunoblot and immunofluorescence microscopy. Both proteins cofractionate with tropomyosin in the Triton-insoluble cytoskeleton of rabbit stomach smooth muscle and are solubilized by high salt. SM-Lmod binds muscle tropomyosin, a biochemical activity characteristic of Tmod proteins. SM-Lmod staining is present along the length of actin filaments in rat intestinal smooth muscle, while Tmod stains in a punctate pattern distinct from that of actin filaments or the dense body marker alpha-actinin. After smooth muscle is hypercontracted by treatment with 10 mM Ca(2+), both SM-Lmod and Tmod are found near alpha-actinin at the periphery of actin-rich contraction bands. These data suggest that SM-Lmod is a novel component of the smooth muscle actin cytoskeleton and, furthermore, that the pointed ends of actin filaments in smooth muscle may be capped by Tmod in localized clusters.

Meiotic recombination protein Rec12: functional conservation, crossover homeostasis and early crossover/non-crossover decision

PubMed Central

Kan, Fengling; Davidson, Mari K.; Wahls, Wayne P.

2011-01-01

In fission yeast and other eukaryotes, Rec12 (Spo11) is thought to catalyze the formation of dsDNA breaks (DSBs) that initiate homologous recombination in meiosis. Rec12 is orthologous to the catalytic subunit of topoisomerase VI (Top6A). Guided by the crystal structure of Top6A, we engineered the rec12 locus to encode Rec12 proteins each with a single amino acid substitution in a conserved residue. Of 21 substitutions, 10 significantly reduced or abolished meiotic DSBs, gene conversion, crossover recombination and the faithful segregation of chromosomes. Critical residues map within the metal ion-binding pocket toprim (E179A, D229A, D231A), catalytic region 5Y-CAP (R94A, D95A, Y98F) and the DNA-binding interface (K201A, G202E, R209A, K242A). A subset of substitutions reduced DSBs but maintained crossovers, demonstrating crossover homeostasis. Furthermore, a strong separation of function mutation (R304A) suggests that the crossover/non-crossover decision is established early by a protein–protein interaction surface of Rec12. Fission yeast has multiple crossovers per bivalent, and chromosome segregation was robust above a threshold of about one crossover per bivalent, below which non-disjunction occurred. These results support structural and functional conservation among Rec12/Spo11/Top6A family members for the catalysis of DSBs, and they reveal how Rec12 regulates other features of meiotic chromosome dynamics. PMID:21030440

Mammalian poly(A)-binding protein is a eukaryotic translation initiation factor, which acts via multiple mechanisms.

PubMed

Kahvejian, Avak; Svitkin, Yuri V; Sukarieh, Rami; M'Boutchou, Marie-Noël; Sonenberg, Nahum

2005-01-01

Translation initiation is a multistep process involving several canonical translation factors, which assemble at the 5'-end of the mRNA to promote the recruitment of the ribosome. Although the 3' poly(A) tail of eukaryotic mRNAs and its major bound protein, the poly(A)-binding protein (PABP), have been studied extensively, their mechanism of action in translation is not well understood and is confounded by differences between in vivo and in vitro systems. Here, we provide direct evidence for the involvement of PABP in key steps of the translation initiation pathway. Using a new technique to deplete PABP from mammalian cell extracts, we show that extracts lacking PABP exhibit dramatically reduced rates of translation, reduced efficiency of 48S and 80S ribosome initiation complex formation, and impaired interaction of eIF4E with the mRNA cap structure. Supplementing PABP-depleted extracts with wild-type PABP completely rectified these deficiencies, whereas a mutant of PABP, M161A, which is incapable of interacting with eIF4G, failed to restore translation. In addition, a stronger inhibition (approximately twofold) of 80S as compared to 48S ribosome complex formation (approximately 65% vs. approximately 35%, respectively) by PABP depletion suggests that PABP plays a direct role in 60S subunit joining. PABP can thus be considered a canonical translation initiation factor, integral to initiation complex formation at the 5'-end of mRNA.

Mammalian poly(A)-binding protein is a eukaryotic translation initiation factor, which acts via multiple mechanisms

PubMed Central

Kahvejian, Avak; Svitkin, Yuri V.; Sukarieh, Rami; M'Boutchou, Marie-Noël; Sonenberg, Nahum

2005-01-01

Translation initiation is a multistep process involving several canonical translation factors, which assemble at the 5′-end of the mRNA to promote the recruitment of the ribosome. Although the 3′ poly(A) tail of eukaryotic mRNAs and its major bound protein, the poly(A)-binding protein (PABP), have been studied extensively, their mechanism of action in translation is not well understood and is confounded by differences between in vivo and in vitro systems. Here, we provide direct evidence for the involvement of PABP in key steps of the translation initiation pathway. Using a new technique to deplete PABP from mammalian cell extracts, we show that extracts lacking PABP exhibit dramatically reduced rates of translation, reduced efficiency of 48S and 80S ribosome initiation complex formation, and impaired interaction of eIF4E with the mRNA cap structure. Supplementing PABP-depleted extracts with wild-type PABP completely rectified these deficiencies, whereas a mutant of PABP, M161A, which is incapable of interacting with eIF4G, failed to restore translation. In addition, a stronger inhibition (approximately twofold) of 80S as compared to 48S ribosome complex formation (∼65% vs. ∼35%, respectively) by PABP depletion suggests that PABP plays a direct role in 60S subunit joining. PABP can thus be considered a canonical translation initiation factor, integral to initiation complex formation at the 5′-end of mRNA. PMID:15630022

Synthesis and structure-activity relationship of histone deacetylase (HDAC) inhibitors with triazole-linked cap group.

PubMed

Chen, Po C; Patil, Vishal; Guerrant, William; Green, Patience; Oyelere, Adegboyega K

2008-05-01

Histone deacetylase (HDAC) inhibition is a recent, clinically validated therapeutic strategy for cancer treatment. Small molecule HDAC inhibitors identified so far fall in to three distinct structural motifs: the zinc-binding group (ZBG), a hydrophobic linker, and a recognition cap group. Here we report the suitability of a 1,2,3-triazole ring as a surface recognition cap group-linking moiety in suberoylanilide hydroxamic acid-like (SAHA-like) HDAC inhibitors. Using "click" chemistry (Huisgen cycloaddition reaction), several triazole-linked SAHA-like hydroxamates were synthesized. Structure-activity relationship revealed that the position of the triazole moiety as well as the identity of the cap group markedly affected the in vitro HDAC inhibition and cell growth inhibitory activities of this class of compounds.

Band 3 in aging and neurological disease.

PubMed

Kay, M M

1991-01-01

Senescent cell antigen appears on old cells and marks them for death by initiating the binding of IgG autoantibody and subsequent removal by phagocytes in mammals and other vertebrates. We have created a synthetic aging antigen that blocks binding of IgG to senescent cells in vitro. Synthetic senescent cell antigen might be effective in preventing cellular destruction in vivo in certain diseases, and can be used to manipulate cellular life span in situ. Senescent cell antigen is generated by the modification of an important structural and transport membrane molecule, protein band 3. Band 3 is present in cellular, nuclear, Golgi, and mitochondrial membranes as well as in cell membranes. Band 3 proteins in nucleated cells participate in cell surface patching and capping. Band 3 maintains acid-base balance by mediating the exchange of anions (e.g., chloride, bicarbonate), and is the binding site for glycolytic enzymes. It is responsible for CO2 exchange in all tissues and organs. Thus, it is the most heavily used anion transport system in the body. Band 3 is a major transmembrane structural protein which attaches the plasma membrane to the internal cell cytoskeleton by binding to band 2.1 (ankyrin). Oxidation generates senescent cell antigen in situ. Band 3 is present in the central nervous system, and differences have been described in band 3 between young and aging brain tissue. One autosomal recessive neurological disease, choreoacanthocytosis, is associated with band 3 abnormalities. The 150 residues of the carboxyl terminus segment of band 3 appear to be altered. In brains from Alzheimer's disease patients, antibodies to aged band 3 label the amyloid core of classical plaques and the microglial cells located in the middle of the plaque in tissue sections, and an abnormal band 3 in immunoblots. Band 3 protein(s) in mammalian brain performs the same functions as that of erythroid band 3. These functions is anion transport, ankyrin binding, and generation of senescent cell antigen, an aging antigen that terminates the life of cells. Structural similarity of brain and erythroid band 3 is suggested by the reaction of antibodies to synthetic peptides of erythroid band 3 with brain band 3, the inhibition of anion transport by the same inhibitors, and an equal degree of inhibition of brain and erythrocyte anion transport by synthetic peptides of erythroid band 3. One of these segments, pep-COOH, contains antigenic determinants of senescent cell antigen.(ABSTRACT TRUNCATED AT 400 WORDS)

Biomimetic assembly of polypeptide-stabilized CaCO(3) nanoparticles.

PubMed

Zhang, Zhongping; Gao, Daming; Zhao, Hui; Xie, Chenggen; Guan, Guijian; Wang, Dapeng; Yu, Shu-Hong

2006-05-04

In this paper, we report a simple polypeptide-directed strategy for fabricating large spherical assembly of CaCO(3) nanoparticles. Stepwise growth and assembly of a large number of nanoparticles have been observed, from the formation of an amorphous liquidlike CaCO(3)-polypeptide precursor, to the crystallization and stabilization of polypeptide-capped nanoparticles, and eventually, the spherical assembly of nanoparticles. The "soft" poly(aspartate)-capping layer binding on a nanoparticle surface resulted in the unusual soft nature of nanoparticle assembly, providing a reservoir of primary nanoparticles with a moderate mobility, which is the basis of a new strategy for reconstructing nanoparticle assembly into complex nanoparticle architectures. Moreover, the findings of the secondary assembly of nanoparticle microspheres and the morphology transformation of nanoparticle assembly demonstrate a flexible and controllable pathway for manipulating the shapes and structures of nanoparticle assembly. In addition, the combination of the polypeptide with a double hydrophilic block copolymer (DHBC) allows it to possibly further control the shape and complexity of the nanoparticle assembly. A clear perspective is shown here that more complex nanoparticle materials could be created by using "soft" biological proteins or peptides as a mediating template at the organic-inorganic interface.

An Exportin-1–dependent microRNA biogenesis pathway during human cell quiescence

PubMed Central

Martinez, Ivan; Hayes, Karen E.; Barr, Jamie A.; Harold, Abby D.; Xie, Mingyi; Bukhari, Syed I. A.; Vasudevan, Shobha; Steitz, Joan A.; DiMaio, Daniel

2017-01-01

The reversible state of proliferative arrest known as “cellular quiescence” plays an important role in tissue homeostasis and stem cell biology. By analyzing the expression of miRNAs and miRNA-processing factors during quiescence in primary human fibroblasts, we identified a group of miRNAs that are induced during quiescence despite markedly reduced expression of Exportin-5, a protein required for canonical miRNA biogenesis. The biogenesis of these quiescence-induced miRNAs is independent of Exportin-5 and depends instead on Exportin-1. Moreover, these quiescence-induced primary miRNAs (pri-miRNAs) are modified with a 2,2,7-trimethylguanosine (TMG)-cap, which is known to bind Exportin-1, and knockdown of Exportin-1 or trimethylguanosine synthase 1, responsible for (TMG)-capping, inhibits their biogenesis. Surprisingly, in quiescent cells Exportin-1–dependent pri-miR-34a is present in the cytoplasm together with a small isoform of Drosha, implying the existence of a different miRNA processing pathway in these cells. Our findings suggest that during quiescence the canonical miRNA biogenesis pathway is down-regulated and specific miRNAs are generated by an alternative pathway to regulate genes involved in cellular growth arrest. PMID:28584122

Direct Regulation of Androgen Receptor Activity by Potent CYP17 Inhibitors in Prostate Cancer Cells*

PubMed Central

Soifer, Harris S.; Souleimanian, Naira; Wu, Sijian; Voskresenskiy, Anatoliy M.; Kisaayak Collak, Filiz; Cinar, Bekir; Stein, Cy A.

2012-01-01

TOK-001 and abiraterone are potent 17-heteroarylsteroid (17-HAS) inhibitors of Cyp17, one of the rate-limiting enzymes in the biosynthesis of testosterone from cholesterol in prostate cancer cells. Nevertheless, the molecular mechanism underlying the prevention of prostate cell growth by 17-HASs still remains elusive. Here, we assess the effects of 17-HASs on androgen receptor (AR) activity in LNCaP and LAPC-4 cells. We demonstrate that both TOK-001 and abiraterone reduced AR protein and mRNA expression, and antagonized AR-dependent promoter activation induced by androgen. TOK-001, but not abiraterone, is an effective apparent competitor of the radioligand [3H]R1881 for binding to the wild type and various mutant AR (W741C, W741L) proteins. In agreement with these data, TOK-001 is a consistently superior inhibitor than abiraterone of R1881-induced transcriptional activity of both wild type and mutant AR. However, neither agent was able to trans-activate the AR in the absence of R1881. Our data demonstrate that phospho-4EBP1 levels are significantly reduced by TOK-001 and to a lesser extent by abiraterone alcohol, and suggest a mechanism by which cap-dependent translation is suppressed by blocking assembly of the eIF4F and eIF4G complex to the mRNA 5′ cap. Thus, the effects of these 17-HASs on AR signaling are complex, ranging from a decrease in testosterone production through the inhibition of Cyp17 as previously described, to directly reducing both AR protein expression and R1881-induced AR trans-activation. PMID:22174412

Mutations to the Formin Homology 2 Domain of INF2 Protein Have Unexpected Effects on Actin Polymerization and Severing*

PubMed Central

Ramabhadran, Vinay; Gurel, Pinar S.; Higgs, Henry N.

2012-01-01

INF2 (inverted formin 2) is a formin protein with unusual biochemical characteristics. As with other formins, the formin homology 2 (FH2) domain of INF2 accelerates actin filament assembly and remains at the barbed end, modulating elongation. The unique feature of INF2 is its ability to sever filaments and enhance depolymerization, which requires the C-terminal region. Physiologically, INF2 acts in the secretory pathway and is mutated in two human diseases, focal and segmental glomerulosclerosis and Charcot-Marie-Tooth disease. In this study, we investigate the effects of mutating two FH2 residues found to be key in other formins: Ile-643 and Lys-792. Surprisingly, neither mutation abolishes barbed end binding, as judged by pyrene-actin and total internal reflection (TIRF) microscopy elongation assays. The I643A mutation causes tight capping of a subset of filaments, whereas K792A causes slow elongation of all filaments. The I643A mutation has a minor inhibitory effect on polymerization activity but causes almost complete abolition of severing and depolymerization activity. The K792A mutation has relatively small effects on polymerization, severing, and depolymerization. In cells, the K792A mutant causes actin accumulation around the endoplasmic reticulum to a similar extent as wild type, whereas the I643A mutant causes no measurable polymerization. The inability of I643A to induce actin polymerization in cells is explained by its inability to promote robust actin polymerization in the presence of capping protein. These results highlight an important point: it is dangerous to assume that mutation of conserved FH2 residues will have equivalent effects in all formins. The work also suggests that both mutations have effects on the mechanism of processive elongation. PMID:22879592

Anaphylaxis to pork kidney is related to IgE antibodies specific for galactose-alpha-1,3-galactose.

PubMed

Morisset, M; Richard, C; Astier, C; Jacquenet, S; Croizier, A; Beaudouin, E; Cordebar, V; Morel-Codreanu, F; Petit, N; Moneret-Vautrin, D A; Kanny, G

2012-05-01

Carbohydrate-specific IgE antibodies present on nonprimate mammalian proteins were incriminated recently in delayed meat anaphylaxis. The aim of this study was to explore whether anaphylaxis to mammalian kidney is also associated with galactose-α-1,3-galactose (αGal)-specific IgE. Fourteen patients with anaphylaxis to pork or beef kidney underwent prick tests to meat and kidney. Some patients also underwent skin tests to Erbitux(®) (cetuximab). IgE antibodies to αGal, swine urine proteins, beef and pork meat, serum albumin proteins, cat, and rFel d 1 were measured by ImmunoCAP(®). The αGal levels were estimated in meats and kidney by ELISA inhibition assay. Cross-reactivity between αGal and pork kidney was studied with the ImmunoCAP(®) inhibition assay. Among the 14 patients, 12 presented with anaphylactic shock. Reactions occurred within 2 h from exposure in 67% of patients. Associated risk factors were observed in 10 cases, and alcohol was the main cofactor. Three patients underwent an oral challenge to pork kidney, and anaphylaxis occurred after ingestion of small quantities (1-2 g). Prick tests to kidney were positive in 54% of patients. All tested patients showed positive skin tests to Erbitux(®). All patients tested positive for IgE to αGal, with levels ranging from 0.4 to 294 kU/l. IgE binding to αGal was inhibited by raw pork kidney extract (mean, 77%; range, 55-87%), which showed a high amount of αGal determinants. Pork or beef kidney anaphylaxis is related to αGal IgE. Its peculiar severity could be due to an elevated content of αGal epitopes in kidney. © 2012 John Wiley & Sons A/S.

BAG3: a new player in the heart failure paradigm.

PubMed

Knezevic, Tijana; Myers, Valerie D; Gordon, Jennifer; Tilley, Douglas G; Sharp, Thomas E; Wang, JuFang; Khalili, Kamel; Cheung, Joseph Y; Feldman, Arthur M

2015-07-01

BAG3 is a cellular protein that is expressed predominantly in skeletal and cardiac muscle but can also be found in the brain and in the peripheral nervous system. BAG3 functions in the cell include: serving as a co-chaperone with members of the heat-shock protein family of proteins to facilitate the removal of misfolded and degraded proteins, inhibiting apoptosis by interacting with Bcl2 and maintaining the structural integrity of the Z-disk in muscle by binding with CapZ. The importance of BAG3 in the homeostasis of myocytes and its role in the development of heart failure was evidenced by the finding that single allelic mutations in BAG3 were associated with familial dilated cardiomyopathy. Furthermore, significant decreases in the level of BAG3 have been found in end-stage failing human heart and in animal models of heart failure including mice with heart failure secondary to trans-aortic banding and in pigs after myocardial infarction. Thus, it becomes relevant to understand the cellular biology and molecular regulation of BAG3 expression in order to design new therapies for the treatment of patients with both hereditary and non-hereditary forms of dilated cardiomyopathy.

Probing the effect of charge transfer enhancement in off resonance mode SERS via conjugation of the probe dye between silver nanoparticles and metal substrates.

PubMed

Selvakannan, Pr; Ramanathan, Rajesh; Plowman, Blake J; Sabri, Ylias M; Daima, Hemant K; O'Mullane, Anthony P; Bansal, Vipul; Bhargava, Suresh K

2013-08-21

The charge transfer-mediated surface enhanced Raman scattering (SERS) of crystal violet (CV) molecules that were chemically conjugated between partially polarized silver nanoparticles and optically smooth gold and silver substrates has been studied under off-resonant conditions. Tyrosine molecules were used as a reducing agent to convert silver ions into silver nanoparticles where oxidised tyrosine caps the silver nanoparticle surface with its semiquinone group. This binding through the quinone group facilitates charge transfer and results in partially oxidised silver. This establishes a chemical link between the silver nanoparticles and the CV molecules, where the positively charged central carbon of CV molecules can bind to the terminal carboxylate anion of the oxidised tyrosine molecules. After drop casting Ag nanoparticles bound with CV molecules it was found that the free terminal amine groups tend to bind with the underlying substrates. Significantly, only those CV molecules that were chemically conjugated between the partially polarised silver nanoparticles and the underlying gold or silver substrates were found to show SERS under off-resonant conditions. The importance of partial charge transfer at the nanoparticle/capping agent interface and the resultant conjugation of CV molecules to off resonant SERS effects was confirmed by using gold nanoparticles prepared in a similar manner. In this case the capping agent binds to the nanoparticle through the amine group which does not facilitate charge transfer from the gold nanoparticle and under these conditions SERS enhancement in the sandwich configuration was not observed.

Nucleostemin inhibits TRF1 dimerization and shortens its dynamic association with the telomere

PubMed Central

Meng, Lingjun; Hsu, Joseph K.; Zhu, Qubo; Lin, Tao; Tsai, Robert Y. L.

2011-01-01

TRF1 is a key component of the telomere-capping complex and binds double-strand telomeric DNA as homodimers. So far, it is not clear whether TRF1 dimerization coincides with its telomere binding or is actively controlled before it binds the telomere, and in the latter case, how this event might affect its telomere association. We previously found that TRF1 dimerization and its telomere binding can be increased by GNL3L, which is the vertebrate paralogue of nucleostemin (NS). Here, we show that NS and GNL3L bind TRF1 directly but competitively through two separate domains of TRF1. In contrast to GNL3L, NS prevents TRF1 dimerization through a mechanism not determined by its ability to displace TRF1-bound GNL3L. Furthermore, NS is capable of shortening the dynamic association of TRF1 with the telomere in normal and TRF2ΔBΔM-induced telomere-damaged cells without affecting the amount of telomere-bound TRF1 proteins in vivo. Importantly, NS displays a protective function against the formation of telomere-dysfunction-induced foci. This work demonstrates that TRF1 dimerization is actively and oppositely regulated by NS and GNL3L extrachromosomally. Changing the relative amount of TRF1 monomers versus dimers in the nucleoplasm might affect the dynamic association of TRF1 with the telomere and the repair of damaged telomeres. PMID:22045740

Drosophila Casein Kinase I Alpha Regulates Homolog Pairing and Genome Organization by Modulating Condensin II Subunit Cap-H2 Levels

PubMed Central

Nguyen, Huy Q.; Nye, Jonathan; Buster, Daniel W.; Klebba, Joseph E.; Rogers, Gregory C.; Bosco, Giovanni

2015-01-01

The spatial organization of chromosomes within interphase nuclei is important for gene expression and epigenetic inheritance. Although the extent of physical interaction between chromosomes and their degree of compaction varies during development and between different cell-types, it is unclear how regulation of chromosome interactions and compaction relate to spatial organization of genomes. Drosophila is an excellent model system for studying chromosomal interactions including homolog pairing. Recent work has shown that condensin II governs both interphase chromosome compaction and homolog pairing and condensin II activity is controlled by the turnover of its regulatory subunit Cap-H2. Specifically, Cap-H2 is a target of the SCFSlimb E3 ubiquitin-ligase which down-regulates Cap-H2 in order to maintain homologous chromosome pairing, chromosome length and proper nuclear organization. Here, we identify Casein Kinase I alpha (CK1α) as an additional negative-regulator of Cap-H2. CK1α-depletion stabilizes Cap-H2 protein and results in an accumulation of Cap-H2 on chromosomes. Similar to Slimb mutation, CK1α depletion in cultured cells, larval salivary gland, and nurse cells results in several condensin II-dependent phenotypes including dispersal of centromeres, interphase chromosome compaction, and chromosome unpairing. Moreover, CK1α loss-of-function mutations dominantly suppress condensin II mutant phenotypes in vivo. Thus, CK1α facilitates Cap-H2 destruction and modulates nuclear organization by attenuating chromatin localized Cap-H2 protein. PMID:25723539

A Receptor-Like Kinase Mediates Ammonium Homeostasis and Is Important for the Polar Growth of Root Hairs in Arabidopsis[W

PubMed Central

Bai, Ling; Ma, Xiaonan; Zhang, Guozeng; Song, Shufei; Zhou, Yun; Gao, Lijie; Miao, Yuchen; Song, Chun-Peng

2014-01-01

Ammonium (NH4+) is both a necessary nutrient and an important signal in plants, but can be toxic in excess. Ammonium sensing and regulatory mechanisms in plant cells have not been fully elucidated. To decipher the complex network of NH4+ signaling, we analyzed [Ca2+]cyt-associated protein kinase (CAP) genes, which encode signaling components that undergo marked changes in transcription levels in response to various stressors. We demonstrated that CAP1, a tonoplast-localized receptor-like kinase, regulates root hair tip growth by maintaining cytoplasmic Ca2+ gradients. A CAP1 knockout mutant (cap1-1) produced elevated levels of cytoplasmic NH4+. Furthermore, root hair growth of cap1-1 was inhibited on Murashige and Skoog medium, but NH4+ depletion reestablished the Ca2+ gradient necessary for normal growth. The lower net NH4+ influx across the vacuolar membrane and relatively alkaline cytosolic pH of cap1-1 root hairs implied that mutation of CAP1 increased NH4+ accumulation in the cytoplasm. Furthermore, CAP1 functionally complemented the npr1 (nitrogen permease reactivator protein) kinase yeast mutant, which is defective in high-affinity NH4+ uptake via MEP2 (methylammonium permease 2), distinguishing CAP1 as a cytosolic modulator of NH4+ levels that participates in NH4+ homeostasis-regulated root hair growth by modulating tip-focused cytoplasmic Ca2+ gradients. PMID:24769480

Spiral wound extraction cartridge

DOEpatents

Wisted, Eric E.; Lundquist, Susan H.

1999-01-01

A cartridge device for removing an analyte from a fluid comprises a hollow core, a sheet composite comprising a particulate-loaded porous membrane and optionally at least one reinforcing spacer sheet, the particulate being capable of binding the analyte, the sheet composite being formed into a spiral configuration about the core, wherein the sheet composite is wound around itself and wherein the windings of sheet composite are of sufficient tightness so that adjacent layers are essentially free of spaces therebetween, two end caps which are disposed over the core and the lateral ends of the spirally wound sheet composite, and means for securing the end caps to the core, the end caps also being secured to the lateral ends of the spirally wound sheet composite. A method for removing an analyte from a fluid comprises the steps of providing a spirally wound element of the invention and passing the fluid containing the analyte through the element essentially normal to a surface of the sheet composite so as to bind the analyte to the particulate of the particulate-loaded porous membrane, the method optionally including the step of eluting the bound analyte from the sheet composite.

3′ Cap-Independent Translation Enhancers of Plant Viruses

PubMed Central

Simon, Anne E.; Miller, W. Allen

2014-01-01

In the absence of a 5′ cap, plant positive-strand RNA viruses have evolved a number of different elements in their 3′ untranslated region (UTR) to attract initiation factors and/or ribosomes to their templates. These 3′ cap-independent translational enhancers (3′ CITEs) take different forms, such as I-shaped, Y-shaped, T-shaped, or pseudoknotted structures, or radiate multiple helices from a central hub. Common features of most 3′ CITEs include the ability to bind a component of the translation initiation factor eIF4F complex and to engage in an RNA-RNA kissing-loop interaction with a hairpin loop located at the 5′ end of the RNA. The two T-shaped structures can bind to ribosomes and ribosomal subunits, with one structure also able to engage in a simultaneous long-distance RNA-RNA interaction. Several of these 3′ CITEs are interchangeable and there is evidence that natural recombination allows exchange of modular CITE units, which may overcome genetic resistance or extend the virus’s host range. PMID:23682606

Genome-wide Control of Heterochromatin Replication by the Telomere Capping Protein TRF2.

PubMed

Mendez-Bermudez, Aaron; Lototska, Liudmyla; Bauwens, Serge; Giraud-Panis, Marie-Josèphe; Croce, Olivier; Jamet, Karine; Irizar, Agurtzane; Mowinckel, Macarena; Koundrioukoff, Stephane; Nottet, Nicolas; Almouzni, Genevieve; Teulade-Fichou, Mare-Paule; Schertzer, Michael; Perderiset, Mylène; Londoño-Vallejo, Arturo; Debatisse, Michelle; Gilson, Eric; Ye, Jing

2018-05-03

Hard-to-replicate regions of chromosomes (e.g., pericentromeres, centromeres, and telomeres) impede replication fork progression, eventually leading, in the event of replication stress, to chromosome fragility, aging, and cancer. Our knowledge of the mechanisms controlling the stability of these regions is essentially limited to telomeres, where fragility is counteracted by the shelterin proteins. Here we show that the shelterin subunit TRF2 ensures progression of the replication fork through pericentromeric heterochromatin, but not centromeric chromatin. In a process involving its N-terminal basic domain, TRF2 binds to pericentromeric Satellite III sequences during S phase, allowing the recruitment of the G-quadruplex-resolving helicase RTEL1 to facilitate fork progression. We also show that TRF2 is required for the stability of other heterochromatic regions localized throughout the genome, paving the way for future research on heterochromatic replication and its relationship with aging and cancer. Copyright © 2018 Elsevier Inc. All rights reserved.

Robust and specific ratiometric biosensing using a copper-free clicked quantum dot-DNA aptamer sensor

NASA Astrophysics Data System (ADS)

Zhang, Haiyan; Feng, Guoqiang; Guo, Yuan; Zhou, Dejian

2013-10-01

We report herein the successful preparation of a compact and functional CdSe-ZnS core-shell quantum dot (QD)-DNA conjugate via highly efficient copper-free ``click chemistry'' (CFCC) between a dihydro-lipoic acid-polyethylene glycol-azide (DHLA-PEG-N3) capped QD and a cyclooctyne modified DNA. This represents an excellent balance between the requirements of high sensitivity, robustness and specificity for the QD-FRET (Förster resonance energy transfer) based sensor as confirmed by a detailed FRET analysis on the QD-DNA conjugate, yielding a relatively short donor-acceptor distance of ~5.8 nm. We show that this CFCC clicked QD-DNA conjugate is not only able to retain the native fluorescence quantum yield (QY) of the parent DHLA-PEG-N3 capped QD, but also well-suited for robust and specific biosensing; it can directly quantitate, at the pM level, both labelled and unlabelled complementary DNA probes with a good SNP (single-nucleotide polymorphism) discrimination ability in complex media, e.g. 10% human serum via target-binding induced FRET changes between the QD donor and the dye acceptor. Furthermore, this sensor has also been successfully exploited for the detection, at the pM level, of a specific protein target (thrombin) via the encoded anti-thrombin aptamer sequence in the QD-DNA conjugate.We report herein the successful preparation of a compact and functional CdSe-ZnS core-shell quantum dot (QD)-DNA conjugate via highly efficient copper-free ``click chemistry'' (CFCC) between a dihydro-lipoic acid-polyethylene glycol-azide (DHLA-PEG-N3) capped QD and a cyclooctyne modified DNA. This represents an excellent balance between the requirements of high sensitivity, robustness and specificity for the QD-FRET (Förster resonance energy transfer) based sensor as confirmed by a detailed FRET analysis on the QD-DNA conjugate, yielding a relatively short donor-acceptor distance of ~5.8 nm. We show that this CFCC clicked QD-DNA conjugate is not only able to retain the native fluorescence quantum yield (QY) of the parent DHLA-PEG-N3 capped QD, but also well-suited for robust and specific biosensing; it can directly quantitate, at the pM level, both labelled and unlabelled complementary DNA probes with a good SNP (single-nucleotide polymorphism) discrimination ability in complex media, e.g. 10% human serum via target-binding induced FRET changes between the QD donor and the dye acceptor. Furthermore, this sensor has also been successfully exploited for the detection, at the pM level, of a specific protein target (thrombin) via the encoded anti-thrombin aptamer sequence in the QD-DNA conjugate. Electronic supplementary information (ESI) available: Details on the synthesis, purification and characterisation of the DHLA-PEG600-N3, cyclooctyne-DNA, and QD-TBA20 conjugates as well as all supporting figures and tables. See DOI: 10.1039/c3nr02897f

Canakinumab for treatment of cryopyrin-associated periodic syndrome.

PubMed

Feist, Eugen; Burmester, Gerd R

2010-11-01

Autoinflammatory syndromes such as cryopyrin-associated periodic syndromes (CAPS) place a heavy burden on affected individuals as well as on their families due to significant morbidity and increased mortality. The inflammatory response in CAPS is caused by an overwhelming activation of the pro-inflammatory cytokine IL-1, which was identified as a promising treatment target. This article focuses on the pathogenic background and different clinical manifestations in CAPS. Furthermore, the development program and characteristics of canakinumab, a recently approved fully human anti-IL-1β mAb for the treatment of CAPS, are described and compared to other available IL-1 blocking agents. Canakinumab targets selectively human IL-1ß with high affinity and prevents the cytokine from interaction to its receptor and, thus, effectively blocks the inflammatory response in CAPS. In all studies performed, canakinumab showed a rapid improvement of symptoms of CAPS and a complete clinical response was achieved in most patients. Inflammatory markers such as C-reactive protein and serum amyloid-A protein were reduced to normal levels within few days. In comparison to other IL-1 blockers, canakinumab provides a longer plasma half-life and less injection site reactions. Canakinumab offers the possibility of permanent disease control, almost symptom-free life, and hopefully less long-term morbidity and mortality in patients with CAPS.

Eukaryotic Translation Initiation Factor 4E Availability Controls the Switch between Cap-Dependent and Internal Ribosomal Entry Site-Mediated Translation†

PubMed Central

Svitkin, Yuri V.; Herdy, Barbara; Costa-Mattioli, Mauro; Gingras, Anne-Claude; Raught, Brian; Sonenberg, Nahum

2005-01-01

Translation of m7G-capped cellular mRNAs is initiated by recruitment of ribosomes to the 5′ end of mRNAs via eukaryotic translation initiation factor 4F (eIF4F), a heterotrimeric complex comprised of a cap-binding subunit (eIF4E) and an RNA helicase (eIF4A) bridged by a scaffolding molecule (eIF4G). Internal translation initiation bypasses the requirement for the cap and eIF4E and occurs on viral and cellular mRNAs containing internal ribosomal entry sites (IRESs). Here we demonstrate that eIF4E availability plays a critical role in the switch from cap-dependent to IRES-mediated translation in picornavirus-infected cells. When both capped and IRES-containing mRNAs are present (as in intact cells or in vitro translation extracts), a decrease in the amount of eIF4E associated with the eIF4F complex elicits a striking increase in IRES-mediated viral mRNA translation. This effect is not observed in translation extracts depleted of capped mRNAs, indicating that capped mRNAs compete with IRES-containing mRNAs for translation. These data explain numerous reported observations where viral mRNAs are preferentially translated during infection. PMID:16287867

Eukaryotic translation initiation factor 4E availability controls the switch between cap-dependent and internal ribosomal entry site-mediated translation.

PubMed

Svitkin, Yuri V; Herdy, Barbara; Costa-Mattioli, Mauro; Gingras, Anne-Claude; Raught, Brian; Sonenberg, Nahum

2005-12-01

Translation of m7G-capped cellular mRNAs is initiated by recruitment of ribosomes to the 5' end of mRNAs via eukaryotic translation initiation factor 4F (eIF4F), a heterotrimeric complex comprised of a cap-binding subunit (eIF4E) and an RNA helicase (eIF4A) bridged by a scaffolding molecule (eIF4G). Internal translation initiation bypasses the requirement for the cap and eIF4E and occurs on viral and cellular mRNAs containing internal ribosomal entry sites (IRESs). Here we demonstrate that eIF4E availability plays a critical role in the switch from cap-dependent to IRES-mediated translation in picornavirus-infected cells. When both capped and IRES-containing mRNAs are present (as in intact cells or in vitro translation extracts), a decrease in the amount of eIF4E associated with the eIF4F complex elicits a striking increase in IRES-mediated viral mRNA translation. This effect is not observed in translation extracts depleted of capped mRNAs, indicating that capped mRNAs compete with IRES-containing mRNAs for translation. These data explain numerous reported observations where viral mRNAs are preferentially translated during infection.

RNA Cap Methyltransferase Activity Assay

PubMed Central

Trotman, Jackson B.; Schoenberg, Daniel R.

2018-01-01

Methyltransferases that methylate the guanine-N7 position of the mRNA 5′ cap structure are ubiquitous among eukaryotes and commonly encoded by viruses. Here we provide a detailed protocol for the biochemical analysis of RNA cap methyltransferase activity of biological samples. This assay involves incubation of cap-methyltransferase-containing samples with a [32P]G-capped RNA substrate and S-adenosylmethionine (SAM) to produce RNAs with N7-methylated caps. The extent of cap methylation is then determined by P1 nuclease digestion, thin-layer chromatography (TLC), and phosphorimaging. The protocol described here includes additional steps for generating the [32P]G-capped RNA substrate and for preparing nuclear and cytoplasmic extracts from mammalian cells. This assay is also applicable to analyzing the cap methyltransferase activity of other biological samples, including recombinant protein preparations and fractions from analytical separations and immunoprecipitation/pulldown experiments. PMID:29644259

Class I β-1,3-Glucanase and Chitinase Are Expressed in the Micropylar Endosperm of Tomato Seeds Prior to Radicle Emergence1

PubMed Central

Wu, Chun-Ta; Leubner-Metzger, Gerhard; Meins, Frederick; Bradford, Kent J.

2001-01-01

β-1,3-Glucanase (EC 3.2.1.39) and chitinase (EC 3.2.1.14) mRNAs, proteins, and enzyme activities were expressed specifically in the micropylar tissues of imbibed tomato (Lycopersicon esculentum Mill.) seeds prior to radicle emergence. RNA hybridization and immunoblotting demonstrated that both enzymes were class I basic isoforms. β-1,3-Glucanase was expressed exclusively in the endosperm cap tissue, whereas chitinase localized to both endosperm cap and radicle tip tissues. β-1,3-Glucanase and chitinase appeared in the micropylar tissues of gibberellin-deficient gib-1 tomato seeds only when supplied with gibberellin. Accumulation of β-1,3-glucanase mRNA, protein and enzyme activity was reduced by 100 μM abscisic acid, which delayed or prevented radicle emergence but not endosperm cap weakening. In contrast, expression of chitinase mRNA, protein, and enzyme activity was not affected by abscisic acid. Neither of these enzymes significantly hydrolyzed isolated tomato endosperm cap cell walls. Although both β-1,3-glucanase and chitinase were expressed in tomato endosperm cap tissue prior to radicle emergence, we found no evidence that they were directly involved in cell wall modification or tissue weakening. Possible functions of these hydrolases during tomato seed germination are discussed. PMID:11457981

Reverse Micelle Mediated synthesis of Calcium Phosphate Nanocarriers for Controlled Release of Bovine Serum Albumin (BSA)

PubMed Central

Dasgupta, Sudip; Bandyopadhyay, Amit; Bose, Susmita

2010-01-01

Calcium phosphate (CaP) nanoparticle with calcium to phosphorus (Ca:P) molar ratio of 1.5:1 were synthesized using reverse micro emulsion. Ca(NO3)2.4H2O and H3PO4 were used as aqueous phase, cyclohexane as organic phase, and poly(oxyethylene)12 nonylphenol ether (NP-12) as surfactant. Depending on calcination temperature between 600 and 800 °C, CaP nanoparticle showed different phases calcium deficient hydroxyapatite (CDHA) and β-tricalcium phosphate (β-TCP), particle size between 48 and 69 nm, the BET specific average surface area between 73 m2/g and 57 m2/g. Bovine serum albumin (BSA) was used as a model protein to study loading and release behavior. Adsorptive property of BSA was investigated with the change in BET surface area of these nanoparticle and the pH of the suspension. At pH 7.5, maximum amount of BSA was adsorbed onto CaP nanoparticle. The release kinetics of BSA showed a gradual time dependent increase at pH 4.0 and 6.0 buffer solutions. However, the amount of released protein was significantly smaller at pH 7.2. BSA release rate also varied depending on the presence of different phases of CaPs in the system, β-TCP or CDHA. These results suggest that BSA protein release rate can be controlled by changing particle size, surface area and phase composition of CaP nanocarriers. PMID:19435617

Centrosomal protein Dzip1l binds Cby, promotes ciliary bud formation, and acts redundantly with Bromi to regulate ciliogenesis in the mouse.

PubMed

Wang, Chengbing; Li, Jia; Takemaru, Ken-Ichi; Jiang, Xiaogang; Xu, Guoqiang; Wang, Baolin

2018-03-15

The primary cilium is a microtubule-based organelle required for Hedgehog (Hh) signaling and consists of a basal body, a ciliary axoneme and a compartment between the first two structures, called the transition zone (TZ). The TZ serves as a gatekeeper to control protein composition in cilia, but less is known about its role in ciliary bud formation. Here, we show that centrosomal protein Dzip1l is required for Hh signaling between Smoothened and Sufu. Dzip1l colocalizes with basal body appendage proteins and Rpgrip1l, a TZ protein. Loss of Dzip1l results in reduced ciliogenesis and dysmorphic cilia in vivo Dzip1l interacts with, and acts upstream of, Cby, an appendage protein, in ciliogenesis. Dzip1l also has overlapping functions with Bromi (Tbc1d32) in ciliogenesis, cilia morphogenesis and neural tube patterning. Loss of Dzip1l arrests ciliogenesis at the stage of ciliary bud formation from the TZ. Consistent with this, Dzip1l mutant cells fail to remove the capping protein Cp110 (Ccp110) from the distal end of mother centrioles and to recruit Rpgrip1l to the TZ. Therefore, Dzip1l promotes ciliary bud formation and is required for the integrity of the TZ. © 2018. Published by The Company of Biologists Ltd.

Subcomponent self-assembly and guest-binding properties of face-capped Fe4L4(8+) capsules.

PubMed

Bilbeisi, Rana A; Clegg, Jack K; Elgrishi, Noémie; de Hatten, Xavier; Devillard, Marc; Breiner, Boris; Mal, Prasenjit; Nitschke, Jonathan R

2012-03-21

A general method for preparing Fe(4)L(4) face-capped tetrahedral cages through subcomponent self-assembly was developed and has been demonstrated using four different C(3)-symmetric triamines, 2-formylpyridine, and iron(II). Three of the triamines were shown also to form Fe(2)L(3) helicates when the appropriate stoichiometry of subcomponents was used. Two of the cages were observed to have nearly identical Fe-Fe distances in the solid state, which enabled their ligands to be coincorporated into a collection of mixed cages. Only one of the cages combined a sufficiently large cavity with the sufficiently small pores required for guest binding, taking up a wide variety of guest species in size- and shape-selective fashion.

LcrV Mutants That Abolish Yersinia Type III Injectisome Function

PubMed Central

Ligtenberg, Katherine Given; Miller, Nathan C.; Mitchell, Anthony; Plano, Gregory V.

2013-01-01

LcrV, the type III needle cap protein of pathogenic Yersinia, has been proposed to function as a tether between YscF, the needle protein, and YopB-YopD to constitute the injectisome, a conduit for the translocation of effector proteins into host cells. Further, insertion of LcrV-capped needles from a calcium-rich environment into host cells may trigger the low-calcium signal for effector translocation. Here, we used a genetic approach to test the hypothesis that the needle cap responds to the low-calcium signal by promoting injectisome assembly. Growth restriction of Yersinia pestis in the absence of calcium (low-calcium response [LCR+] phenotype) was exploited to isolate dominant negative lcrV alleles with missense mutations in its amber stop codon (lcrV*327). The addition of at least four amino acids or the eight-residue Strep tag to the C terminus was sufficient to generate an LCR− phenotype, with variant LcrV capping type III needles that cannot assemble the YopD injectisome component. The C-terminal Strep tag appears buried within the cap structure, blocking effector transport even in Y. pestis yscF variants that are otherwise calcium blind, a constitutive type III secretion phenotype. Thus, LcrV*327 mutants arrest the needle cap in a state in which it cannot respond to the low-calcium signal with either injectisome assembly or the activation of type III secretion. Insertion of the Strep tag at other positions of LcrV produced variants with wild-type LCR+, LCR−, or dominant negative LCR− phenotypes, thereby allowing us to identify discrete sites within LcrV as essential for its attributes as a secretion substrate, needle cap, and injectisome assembly factor. PMID:23222719

The ITO-capped WO3 nanowires biosensor based on field-effect transistor in label-free protein sensing

NASA Astrophysics Data System (ADS)

Shariati, Mohsen

2017-05-01

The fabrication of ITO-capped WO3 nanowires associated with their bio-sensing properties in field-effect transistor diagnostics basis as a biosensor has been reported. The bio-sensing property for manipulated nanowires elucidated that the grown nanostructures were very sensitive to protein. The ITO-capped WO3 nanowires biosensor showed an intensive bio-sensing activity against reliable protein. Polylysine strongly charged bio-molecule was applied as model system to demonstrate the implementation of materialized biosensor. The employed sensing mechanism was `label-free' and depended on bio-molecule's intrinsic charge. For nanowires synthesis, the vapor-liquid-solid mechanism was used. Nanowires were beyond a few hundred nanometers in lengths and around 15-20 nm in diameter, while the globe cap's size on the nanowires was around 15-25 nm. The indium tin oxide (ITO) played as catalyst in nanofabrication for WO3 nanowires growth and had outstanding role in bio-sensing especially for bio-molecule adherence. In applied electric field presence, the fabricated device showed the great potential to enhance medical diagnostics.

Exploring bis-(indolyl)methane moiety as an alternative and innovative CAP group in the design of histone deacetylase (HDAC) inhibitors.

PubMed

Giannini, Giuseppe; Marzi, Mauro; Marzo, Maria Di; Battistuzzi, Gianfranco; Pezzi, Riccardo; Brunetti, Tiziana; Cabri, Walter; Vesci, Loredana; Pisano, Claudio

2009-05-15

In order to gather further knowledge about the structural requirements on histone deacetylase inhibitors (HDACi), starting from the schematic model of the common pharmacophore that characterizes this class of molecules (surface recognition CAP group-connection unit-linker region-Zinc Binding Group), we designed and synthesized a series of hydroxamic acids containing a bis-(indolyl)methane moiety. HDAC inhibition profile and antiproliferative activity were evaluated.

Crystal structure of Bacillus anthracis transpeptidase enzyme CapD.

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

Wu, R.; Richter, S.; Zhang, R.

2009-09-04

Bacillus anthracis elaborates a poly-{gamma}-d-glutamic acid capsule that protects bacilli from phagocytic killing during infection. The enzyme CapD generates amide bonds with peptidoglycan cross-bridges to anchor capsular material within the cell wall envelope of B. anthracis. The capsular biosynthetic pathway is essential for virulence during anthrax infections and can be targeted for anti-infective inhibition with small molecules. Here, we present the crystal structures of the {gamma}-glutamyltranspeptidase CapD with and without {alpha}-l-Glu-l-Glu dipeptide, a non-hydrolyzable analog of poly-{gamma}-d-glutamic acid, in the active site. Purified CapD displays transpeptidation activity in vitro, and its structure reveals an active site broadly accessible for poly-{gamma}-glutamatemore » binding and processing. Using structural and biochemical information, we derive a mechanistic model for CapD catalysis whereby Pro{sup 427}, Gly{sup 428}, and Gly{sup 429} activate the catalytic residue of the enzyme, Thr{sup 352}, and stabilize an oxyanion hole via main chain amide hydrogen bonds.« less

Crimean-Congo Hemorrhagic Fever Virus Nucleocapsid Protein Augments mRNA Translation.

PubMed

Jeeva, Subbiah; Cheng, Erdong; Ganaie, Safder S; Mir, Mohammad A

2017-08-01

Crimean-Congo hemorrhagic fever virus (CCHFV) is a tick-borne Nairovirus of the Bunyaviridae family, causing severe illness with high mortality rates in humans. Here, we demonstrate that CCHFV nucleocapsid protein (CCHFV-NP) augments mRNA translation. CCHFV-NP binds to the viral mRNA 5' untranslated region (UTR) with high affinity. It facilitates the translation of reporter mRNA both in vivo and in vitro with the assistance of the viral mRNA 5' UTR. CCHFV-NP equally favors the translation of both capped and uncapped mRNAs, demonstrating the independence of this translation strategy on the 5' cap. Unlike the canonical host translation machinery, inhibition of eIF4F complex, an amalgam of three initiation factors, eIF4A, eIF4G, and eIF4E, by the chemical inhibitor 4E1RCat did not impact the CCHFV-NP-mediated translation mechanism. However, the proteolytic degradation of eIF4G alone by the human rhinovirus 2A protease abrogated this translation strategy. Our results demonstrate that eIF4F complex formation is not required but eIF4G plays a critical role in this translation mechanism. Our results suggest that CCHFV has adopted a unique translation mechanism to facilitate the translation of viral mRNAs in the host cell cytoplasm where cellular transcripts are competing for the same translation apparatus. IMPORTANCE Crimean-Congo hemorrhagic fever, a highly contagious viral disease endemic to more than 30 countries, has limited treatment options. Our results demonstrate that NP favors the translation of a reporter mRNA harboring the viral mRNA 5' UTR. It is highly likely that CCHFV uses an NP-mediated translation strategy for the rapid synthesis of viral proteins during the course of infection. Shutdown of this translation mechanism might selectively impact viral protein synthesis, suggesting that an NP-mediated translation strategy is a target for therapeutic intervention against this viral disease. Copyright © 2017 American Society for Microbiology.

Paced Reading in Semantic Dementia: Word Knowledge Contributes to Phoneme Binding in Rapid Speech Production

ERIC Educational Resources Information Center

Jefferies, Elizabeth; Grogan, John; Mapelli, Cristina; Isella, Valeria

2012-01-01

Patients with semantic dementia (SD) show deficits in phoneme binding in immediate serial recall: when attempting to reproduce a sequence of words that they no longer fully understand, they show frequent migrations of phonemes between items (e.g., cap, frog recalled as "frap, cog"). This suggests that verbal short-term memory emerges directly from…

The pattern of tyrosine phosphorylation in human sperm in response to binding to zona pellucida or hyaluronic acid.

PubMed

Sati, Leyla; Cayli, Sevil; Delpiano, Elena; Sakkas, Denny; Huszar, Gabor

2014-05-01

In mammalian species, acquisition of sperm fertilization competence is dependent on the phenomenon of sperm capacitation. One of the key elements of capacitation is protein tyrosine phosphorylation (TP) in various sperm membrane regions. In previous studies performed, the pattern of TP was examined in human sperm bound to zona pellucida of oocytes. In the present comparative study, TP patterns upon sperm binding to the zona pellucida or hyaluronic acid (HA) were investigated in spermatozoa arising from the same semen samples. Tyrosine phosphorylation, visualized by immunofluorescence, was localized within the acrosomal cap, equatorial head region, neck, and the principal piece. Tyrosine phosphorylation has increased in a time-related manner as capacitation progressed, and the phosphorylation pattern was identical within the principal piece and neck, regardless of the sperm bound to the zona pellucida or HA. Thus, the data demonstrated that the patterns of sperm activation-related TP were similar regardless of the spermatozoa bound to zona pellucida or HA. Further, sperm with incomplete development, as detected by excess cytoplasmic retention, failed to exhibit TP.

The Pattern of Tyrosine Phosphorylation in Human Sperm in Response to Binding to Zona Pellucida or Hyaluronic Acid

PubMed Central

Sati, Leyla; Cayli, Sevil; Delpiano, Elena; Sakkas, Denny

2014-01-01

In mammalian species, acquisition of sperm fertilization competence is dependent on the phenomenon of sperm capacitation. One of the key elements of capacitation is protein tyrosine phosphorylation (TP) in various sperm membrane regions. In previous studies performed, the pattern of TP was examined in human sperm bound to zona pellucida of oocytes. In the present comparative study, TP patterns upon sperm binding to the zona pellucida or hyaluronic acid (HA) were investigated in spermatozoa arising from the same semen samples. Tyrosine phosphorylation, visualized by immunofluorescence, was localized within the acrosomal cap, equatorial head region, neck, and the principal piece. Tyrosine phosphorylation has increased in a time-related manner as capacitation progressed, and the phosphorylation pattern was identical within the principal piece and neck, regardless of the sperm bound to the zona pellucida or HA. Thus, the data demonstrated that the patterns of sperm activation-related TP were similar regardless of the spermatozoa bound to zona pellucida or HA. Further, sperm with incomplete development, as detected by excess cytoplasmic retention, failed to exhibit TP. PMID:24077441

The N-terminus of IpaB provides a potential anchor to the Shigella type III secretion system tip complex protein IpaD

PubMed Central

Dickenson, Nicholas E.; Arizmendi, Olivia; Patil, Mrinalini K.; Toth, Ronald T.; Middaugh, C. Russell; Picking, William D.; Picking, Wendy L.

2014-01-01

The type III secretion system (T3SS) is an essential virulence factor for Shigella flexneri, providing a conduit through which host-altering effectors are injected directly into a host cell to promote uptake. The type III secretion apparatus (T3SA) is comprised of a basal body, external needle, and regulatory tip complex. The nascent needle is a polymer of MxiH capped by a pentamer of invasion plasmid antigen D (IpaD). Exposure to bile salts (e.g. deoxycholate) causes a conformational change in IpaD and promotes recruitment of IpaB to the needle tip. It has been proposed that IpaB senses contact with host cell membranes, recruiting IpaC and inducing full secretion of T3SS effectors. While the steps of T3SA maturation and their external triggers have been identified, details of specific protein interactions and mechanisms have remained difficult to study due to the hydrophobic nature of the IpaB and IpaC translocator proteins. Here we explored the ability for a series of soluble N-terminal IpaB peptides to interact with IpaD. We found that DOC is required for the interaction and that a region of IpaB between residues 11–27 is required for maximum binding, which was confirmed in vivo. Furthermore, intramolecular FRET measurements indicated that movement of the IpaD distal domain away from the protein core accompanied the binding of IpaB11-226. Together these new findings provide important new insight into the interactions and potential mechanisms that define the maturation of the Shigella T3SA needle tip complex and provide a foundation for further studies probing T3SS activation. PMID:24236510

mRNA-Selective Translation Induced by FSH in Primary Sertoli Cells

PubMed Central

Musnier, Astrid; León, Kelly; Morales, Julia; Reiter, Eric; Boulo, Thomas; Costache, Vlad; Vourc'h, Patrick; Heitzler, Domitille; Oulhen, Nathalie; Poupon, Anne; Boulben, Sandrine; Cormier, Patrick

2012-01-01

FSH is a key hormonal regulator of Sertoli cell secretory activity, required to optimize sperm production. To fulfil its biological function, FSH binds a G protein-coupled receptor, the FSH-R. The FSH-R-transduced signaling network ultimately leads to the transcription or down-regulation of numerous genes. In addition, recent evidence has suggested that FSH might also regulate protein translation. However, this point has never been demonstrated conclusively yet. Here we have addressed this issue in primary rat Sertoli cells endogenously expressing physiological levels of FSH-R. We observed that, within 90 min of stimulation, FSH not only enhanced overall protein synthesis in a mammalian target of rapamycin-dependent manner but also increased the recruitment of mRNA to polysomes. m7GTP pull-down experiments revealed the functional recruitment of mammalian target of rapamycin and p70 S6 kinase to the 5′cap, further supported by the enhanced phosphorylation of one of p70 S6 kinase targets, the eukaryotic initiation factor 4B. Importantly, the scaffolding eukaryotic initiation factor 4G was also recruited, whereas eukaryotic initiation factor 4E-binding protein, the eukaryotic initiation factor 4E generic inhibitor, appeared to play a minor role in translational regulations induced by FSH, in contrast to what is generally observed in response to anabolic factors. This particular regulation of the translational machinery by FSH stimulation might support mRNA-selective translation, as shown here by quantitative RT-PCR amplification of the c-fos and vascular endothelial growth factor mRNA but not of all FSH target mRNA, in polysomal fractions. These findings add a new level of complexity to FSH biological roles in its natural target cells, which has been underappreciated so far. PMID:22383463

O2 and CO binding to tetraaza-tripodal-capped iron(II) porphyrins.

PubMed

Ruzié, Christian; Even, Pascale; Ricard, David; Roisnel, Thierry; Boitrel, Bernard

2006-02-06

A series of tris(2-aminoethylamine) (tren) capped iron(II) porphyrins has been synthesized and characterized and their affinities for dioxygen and carbon monoxide measured. The X-ray structure of the basic scaffold with nickel inserted in the porphyrin is also reported. All the ligands differ by the nature of the group(s) attached to the secondary amine functions of the cap. These various substitutions were introduced to probe if a hydrogen bond with these secondary amine groups acting as the donor could rationalize the high affinity of these myoglobin models. This work clearly indicates that the cage structure of the tren predominates over all the other appended groups with the exception of p-nitrophenol.

Binding of purified and radioiodinated capsular polysaccharides from Cryptococcus neoformans serotype A strains to capsule-free mutants.

PubMed Central

Small, J M; Mitchell, T G

1986-01-01

Strains 6, 15, 98, 110, and 145 of Cryptococcus neoformans serotype A vary in capsule size, animal virulence, and susceptibility to in vitro phagocytosis. The isolated capsular polysaccharides (CPSs) differ in monosaccharide composition ratios and molecular size, as determined by gel filtration. The purpose of this investigation was to characterize the binding of CPSs to capsule-free mutants of C. neoformans and to examine CPSs from these strains for differences in their ability to bind, to determine whether such differences might explain the variation in the pathobiology of these strains. CPSs were partially periodate oxidized, tyraminated, iodinated with 125I, and used in binding studies with two capsule-free mutants of C. neoformans, strain 602 and Cap59. Binding was specific for yeast species and for polysaccharide and was saturable, which is consistent with a receptor-mediated mechanism of attachment. Binding occurred rapidly and was only slowly reversible. Binding was also independent of pH from pH 5.5 to 8, of cation concentrations, and of competition by sugars up to 1.0 M concentrations. Only a portion of CPS was capable of binding, and strains varied in the extent to which their CPS bound. CPS-15-IV (peak IV was the major polysaccharide peak on DEAE-cellulose chromatography of CPS from strain 15) had the highest proportion of binding (40%), followed by CPS from strains 98, 6, 145, 110, and 15-III (peak III was an earlier eluting fraction of CPS from strain 15). The CPSs differed similarly in their ability to competitively inhibit binding. Treatment of CPS, but not yeast cells, with proteinase XIV abolished binding without altering the CPS gross structure. Treatment of yeast cells with proteases, heat, or formaldehyde did not alter binding, and both strain 602 and Cap59 bound CPS similarly. Binding to encapsulated yeast cells was minimal. PMID:3536747

Binding of purified and radioiodinated capsular polysaccharides from Cryptococcus neoformans serotype A strains to capsule-free mutants

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

Small, J.M.; Mitchell, T.G.

Strains 6, 15, 98, 110, and 145 of Cryptococcus neoformans serotype A vary in capsule size, animal virulence, and susceptibility to in vitro phagocytosis. The isolated capsular polysaccharides (CPSs) differ in monosaccharide composition ratios and molecular size, as determined by gel filtration. The purpose of this investigation was to characterize the binding of CPSs to capsule-free mutants of C. neoformans and to examine CPSs from these strains for differences in their ability to bind, to determine whether such differences might explain the variation in the pathobiology of these strains. CPSs were partially periodate oxidized, tyraminated, iodinated with /sup 125/I, andmore » used in binding studies with two capsule-free mutants of C. neoformans, strain 602 and Cap59. Binding was specific for yeast species and for polysaccharide and was saturable, which is consistent with a receptor-mediated mechanism of attachment. Binding occurred rapidly and was only slowly reversible. Binding was also independent of pH from pH 5.5 to 8, of cation concentrations, and of competition by sugars up to 1.0 M concentrations. Only a portion of CPS was capable of binding, and strains varied in the extent to which their CPS bound. CPS-15-IV (peak IV was the major polysaccharide peak on DEAE-cellulose chromatography of CPS from strain 15) had the highest proportion of binding (40%), followed by CPS from strains 98, 6, 145, 110, and 15-III (peak III was an earlier eluting fraction of CPS from strain 15). The CPSs differed similarly in their ability to competitively inhibit binding. Treatment of CPS, but not yeast cells, with proteinase XIV abolished binding without altering the CPS gross structure. Treatment of yeast cells with proteases, heat, or formaldehyde did not alter binding, and both strain 602 and Cap59 bound CPS similarly. Binding to encapsulated yeast cells was minimal.« less

Inhibition of metabolism and DNA binding of polycyclic aromatic hydrocarbons (PAHs) by plant phenols in epidermis of SENCAR mice

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

Das, M.; Bik, D.P.; Bickers, D.R.

1986-03-05

Naturally occurring plant phenols such as tannic acid (TA), quercetin (QT), myricetin (MY) and anthraflavic acid (AA) have been shown to inhibit the mutagenicity of several bay-region diolepoxides of PAHs. Since skin is a target for PAH carcinogenesis, they investigated the effect of these plant phenols on epidermal aryl hydrocarbon hydroxylase (AHH) activity and the binding of PAHs to DNA in SENCAR mice. Each of the plant phenols tested was found to be an in vitro and in vivo inhibitor of epidermal AHH activity with I/sub 50/ values ranging from 4.4 x 10/sup -5/ - 12.4 x 10/sup -5/M inmore » control and 3-methylcholanthrene (MCA) pretreated skin. On an equimolar basis TA was the most potent inhibitor with a Ki of 81 ..mu..M. Incubation of TA, QT, MY and AA with epidermal microsomes resulted in varying degrees of inhibition of enzyme mediated covalent binding of benzo(a)pyrene (BP) to calf thymus DNA. TA (25 ..mu..M) showed maximum inhibition (64%). A single topical application (12 ..mu..mol) of TA, QT, MY and AA resulted in significant decrease in the binding of BP, BP-7,8-diol and 7,12-dimethylbenz(a)anthracene to epidermal DNA. The formation of (+)-7..beta..,8..cap alpha..-dihydroxy-9..cap alpha..,10..cap alpha..-epoxy-7,8,9,10-tetrahydro-BP-deoxyguanine adduct in epidermis was significantly reduced (62-86%) following topical application of the plant phenols. Their results suggest that some of these plant phenols have substantial though variable potential to modify the risk of PAHs induced skin carcinogenicity.« less

A New Framework for Understanding IRES-mediated Translation

PubMed Central

Komar, Anton A.; Mazumder, Barsanjit; Merrick, William C.

2012-01-01

Studies over the past 5 or so years have indicated that the traditional clustering of mechanisms for translation initiation in eukaryotes into cap-dependent and cap-independent (or IRES-mediated) is far too narrow. From individual studies of a number of mRNAs encoding proteins that are regulatory in nature (i.e. likely to be needed in small amounts such as transcription factors, protein kinases, etc.), it is now evident that mRNAs exist that blur these boundaries. This review seeks to set the basic ground rules for the analysis of different initiation pathways that are associated with these new mRNAs as well as related to the more traditional mechanisms, especially the cap-dependent translational process that is the major route of initiation of mRNAs for housekeeping proteins and thus, the bulk of protein synthesis in most cells. It will become apparent that a mixture of descriptions is likely to become the norm in the near future (i.e. m7G-assisted internal initiation). PMID:22555019

RNA-dependent RNA polymerase: Addressing Zika outbreak by a phylogeny-based drug target study.

PubMed

Stephen, Preyesh; Lin, Sheng-Xiang

2018-01-01

Since the first major outbreak of Zika virus (ZIKV) in 2007, ZIKV is spreading explosively through South and Central America, and recent reports in highly populated developing countries alarm the possibility of a more catastrophic outbreak. ZIKV infection in pregnant women leads to embryonic microcephaly and Guillain-Barré syndrome in adults. At present, there is limited understanding of the infectious mechanism, and no approved therapy has been reported. Despite the withdrawal of public health emergency, the WHO still considers the ZIKV as a highly significant and long-term public health challenge that the situation has to be addressed rapidly. Non-structural protein 5 is essential for capping and replication of viral RNA and comprises a methyltransferase and RNA-dependent RNA polymerase (RdRp) domain. We used molecular modeling to obtain the structure of ZIKV RdRp, and by molecular docking and phylogeny analysis, we here demonstrate the potential sites for drug screening. Two metal binding sites and an NS3-interacting region in ZIKV RdRp are demonstrated as potential drug screening sites. The docked structures reveal a remarkable degree of conservation at the substrate binding site and the potential drug screening sites. A phylogeny-based approach is provided for an emergency preparedness, where similar class of ligands could target phylogenetically related proteins. © 2017 John Wiley & Sons A/S.

A disulfide-stabilized conformer of methionine synthase reveals an unexpected role for the histidine ligand of the cobalamin cofactor

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

Datta, Supratim; Koutmos, Markos; Pattridge, Katherine A.

2008-07-08

B{sub 12}-dependent methionine synthase (MetH) from Escherichia coli is a large modular protein that is alternately methylated by methyltetrahydrofolate to form methylcobalamin and demethylated by homocysteine to form cob(I)alamin. Major domain rearrangements are required to allow cobalamin to react with three different substrates: homocysteine, methyltetrahydrofolate, and S-adenosyl-l-methionine (AdoMet). These same rearrangements appear to preclude crystallization of the wild-type enzyme. Disulfide cross-linking was used to lock a C-terminal fragment of the enzyme into a unique conformation. Cysteine point mutations were introduced at Ile-690 and Gly-743. These cysteine residues span the cap and the cobalamin-binding module and form a cross-link that reducesmore » the conformational space accessed by the enzyme, facilitating protein crystallization. Here, we describe an x-ray structure of the mutant fragment in the reactivation conformation; this conformation enables the transfer of a methyl group from AdoMet to the cobalamin cofactor. In the structure, the axial ligand to the cobalamin, His-759, dissociates from the cobalamin and forms intermodular contacts with residues in the AdoMet-binding module. This unanticipated intermodular interaction is expected to play a major role in controlling the distribution of conformers required for the catalytic and the reactivation cycles of the enzyme.« less

Citrus sinensis annotation project (CAP): a comprehensive database for sweet orange genome.

PubMed

Wang, Jia; Chen, Dijun; Lei, Yang; Chang, Ji-Wei; Hao, Bao-Hai; Xing, Feng; Li, Sen; Xu, Qiang; Deng, Xiu-Xin; Chen, Ling-Ling

2014-01-01

Citrus is one of the most important and widely grown fruit crop with global production ranking firstly among all the fruit crops in the world. Sweet orange accounts for more than half of the Citrus production both in fresh fruit and processed juice. We have sequenced the draft genome of a double-haploid sweet orange (C. sinensis cv. Valencia), and constructed the Citrus sinensis annotation project (CAP) to store and visualize the sequenced genomic and transcriptome data. CAP provides GBrowse-based organization of sweet orange genomic data, which integrates ab initio gene prediction, EST, RNA-seq and RNA-paired end tag (RNA-PET) evidence-based gene annotation. Furthermore, we provide a user-friendly web interface to show the predicted protein-protein interactions (PPIs) and metabolic pathways in sweet orange. CAP provides comprehensive information beneficial to the researchers of sweet orange and other woody plants, which is freely available at http://citrus.hzau.edu.cn/.

Differentially expressed proteins in nitric oxide-stimulated NIH/3T3 fibroblasts: implications for inhibiting cancer development.

PubMed

Shim, Dong Hwi; Lim, Joo Weon; Kim, Hyeyoung

2015-03-01

Recent evidence shows that nitric oxide (NO) may exhibit both pro-cancer and anti-cancer activities. The present study aimed to determine the differentially expressed proteins in NO-treated NIH/3T3 fibroblasts in order to investigate whether NO induces proteins with pro-cancer or anti-cancer effects. The cells were treated with 300 μM of an NO donor 3,3-bis-(aminoethyl)-1-hydroxy-2-oxo-1-triazene (NOC-18) for 12 h. The changed protein patterns, which were separated by two-dimensional electrophoresis using pH gradients of 4-7, were conclusively identified by matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) analysis of the peptide digests. Seventeen differentially expressed proteins were identified in NOC-18-treated cells. Nine proteins [vinculin protein, keratin 19, ubiquitous tropomodulin, F-actin capping protein (α1 subunit), tropomyosin 3, 26S proteasome-associated pad1 homolog, T-complex protein 1 (ε subunit) N(G)-dimethylarginine dimethylaminohydrolase, and heat shock protein 90] were increased and eight proteins (heat shock protein 70, glucosidase II, lamin B1, calreticulin, nucleophosmin 1, microtubule-associated protein retinitis pigmentosa/end binding family member 1, 150 kD oxygen-regulated protein precursor, and heat shock 70-related protein albino or pale green 2) were decreased by NOC-18 in the cells. Thirteen proteins are related to the suppression of cancer cell proliferation, invasion, and metastasis while two proteins (heat shock protein 90 and N(G)-dimethylarginine dimethylaminohydrolase) are related to carcinogenesis. The functions of 150 kD oxygen-regulated protein precursor and T-complex protein 1 (ε subunit) are unknown in relation to carcinogenesis. Most proteins differentially expressed by NOC-18 are involved in inhibiting cancer development.

Preparation of Curcumin-Piperazine Coamorphous Phase and Fluorescence Spectroscopic and Density Functional Theory Simulation Studies on the Interaction with Bovine Serum Albumin.

PubMed

Pang, Wenzhe; Lv, Jie; Du, Shuang; Wang, Jiaojiao; Wang, Jing; Zeng, Yanli

2017-09-05

In the present study, a new coamorphous phase (CAP) of bioactive herbal ingredient curcumin (CUR) with high solubilitythe was screened with pharmaceutically acceptable coformers. Besides, to provide basic information for the best practice of physiological and pharmaceutical preparations of CUR-based CAP, the interaction between CUR-based CAP and bovine serum albumin (BSA) was studied at the molecular level in this paper. CAP of CUR and piperazine with molar ratio of 1:2 was prepared by EtOH-assisted grinding. The as-prepared CAP was characterized by powder X-ray diffraction, modulated temperature differential scanning calorimetry, thermogravimetric analysis, Fourier-transform infrared, and solid-state 13 C nuclear magnetic resonance. The 1:2 CAP stoichioimetry was sustained by C═O···H hydrogen bonds between the N-H group of the piperazine and the C═O group of CUR; piperazine stabilized the diketo structure of CUR in CAP. The dissolution rate of CUR-piperazine CAP in 30% ethanol-water was faster than that of CUR; the t 50 values were 243.1 min for CUR and 4.378 min for CAP. Furthermore, interactions of CUR and CUR-piperazine CAP with BSA were investigated by fluorescence spectroscopy and density functional theory (DFT) calculation. The binding constants (K b ) of CUR and CUR-piperazine CAP with BSA were 10.0 and 9.1 × 10 3 L mol -1 at 298 K, respectively. Moreover, DFT simulation indicated that the interaction energy values of hydrogen-bonded interaction in the tryptophan-CUR and tryptophan-CUR-piperazine complex were -26.1 and -17.9 kJ mol -1 , respectively. In a conclusion, after formation of CUR-piperazine CAP, the interaction forces between CUR and BSA became weaker.

Plant villin, lily P-135-ABP, possesses G-actin binding activity and accelerates the polymerization and depolymerization of actin in a Ca2+-sensitive manner.

PubMed

Yokota, Etsuo; Tominaga, Motoki; Mabuchi, Issei; Tsuji, Yasunori; Staiger, Christopher J; Oiwa, Kazuhiro; Shimmen, Teruo

2005-10-01

From germinating pollen of lily, two types of villins, P-115-ABP and P-135-ABP, have been identified biochemically. Ca(2+)-CaM-dependent actin-filament binding and bundling activities have been demonstrated for both villins previously. Here, we examined the effects of lily villins on the polymerization and depolymerization of actin. P-115-ABP and P-135-ABP present in a crude protein extract prepared from germinating pollen bound to a DNase I affinity column in a Ca(2+)-dependent manner. Purified P-135-ABP reduced the lag period that precedes actin filament polymerization from monomers in the presence of either Ca(2+) or Ca(2+)-CaM. These results indicated that P-135-ABP can form a complex with G-actin in the presence of Ca(2+) and this complex acts as a nucleus for polymerization of actin filaments. However, the nucleation activity of P-135-ABP is probably not relevant in vivo because the assembly of G-actin saturated with profilin, a situation that mimics conditions found in pollen, was not accelerated in the presence of P-135-ABP. P-135-ABP also enhanced the depolymerization of actin filaments during dilution-mediated disassembly. Growth from filament barbed ends in the presence of Ca(2+)-CaM was also prevented, consistent with filament capping activity. These results suggested that lily villin is involved not only in the arrangement of actin filaments into bundles in the basal and shank region of the pollen tube, but also in regulating and modulating actin dynamics through its capping and depolymerization (or fragmentation) activities in the apical region of the pollen tube, where there is a relatively high concentration of Ca(2+).

Complex absorbing potential based Lorentzian fitting scheme and time dependent quantum transport.

PubMed

Xie, Hang; Kwok, Yanho; Jiang, Feng; Zheng, Xiao; Chen, GuanHua

2014-10-28

Based on the complex absorbing potential (CAP) method, a Lorentzian expansion scheme is developed to express the self-energy. The CAP-based Lorentzian expansion of self-energy is employed to solve efficiently the Liouville-von Neumann equation of one-electron density matrix. The resulting method is applicable for both tight-binding and first-principles models and is used to simulate the transient currents through graphene nanoribbons and a benzene molecule sandwiched between two carbon-atom chains.

Green synthesis of biogenic silver nanoparticles using Solanum tuberosum extract and their interaction with human serum albumin: Evidence of "corona" formation through a multi-spectroscopic and molecular docking analysis.

PubMed

Ali, Mohd Sajid; Altaf, Mohammad; Al-Lohedan, Hamad A

2017-08-01

Biogenic silver nanoparticles (AgNPs) have been synthesized by using Solanum tuberosum (potato) extract (PE) as a reducing as well as stabilizing agent which is reasonably cheaper, non-toxic and easily available material. The green synthesis of silver nanoparticles has been carried out by very simple method and the nanoparticles were characterized by surface plasmon band as well as TEM measurements. The PE-AgNPs were highly dispersed in the solution and found to be spherical with around 10nm in size. Interaction of these nanoparticles was studied with plasma protein HSA by means of various spectroscopies, such as, UV-visible, fluorescence, DLS, CD and FTIR spectroscopies. The HSA was found to form the protein "corona" around the starch-capped PE-AgNPs. Absorption spectroscopy revealed that the interaction between HSA and PE-AgNPs resulted in the ground state complex formation. Due to the strong absorption of PE-AgNPs, the inner filter effect was corrected for the fluorescence data. PE-AgNPs were found to quench the fluorescence of HSA with a small blue shift attributed to the increase in the hydrophobicity near tryptophan residue due to the presence of amylopectin and amylose units in the starch. The value of n, Hill's constant, was found to be >1 which determines the existence of a cooperative binding between nanoparticle and albumin. Several parameters such as Stern-Volmer and binding constants in addition to the thermodynamic parameters have been analyzed and discussed which established that the complex formation has taken place via static quenching mechanism and the corona formation between albumin and PE-AgNPs was entropy driven process. Binding of biogenic PE-AgNPs to the HSA slightly affected the secondary structure of latter with a small decrease in α-helical contents resulting in the partial unfolding of the protein, though the structural motif remained the same. Molecular docking simulations revealed various possible binding modes between PE-AgNPs and albumin. Copyright © 2017 Elsevier B.V. All rights reserved.

Reverse micelle-mediated synthesis of calcium phosphate nanocarriers for controlled release of bovine serum albumin.

PubMed

Dasgupta, Sudip; Bandyopadhyay, Amit; Bose, Susmita

2009-10-01

Calcium phosphate (CaP) nanoparticles with a calcium to phosphorus (Ca:P) molar ratio of 1.5:1 were synthesized using reverse microemulsion. Ca(NO(3))(2).4H(2)O and H(3)PO(4) were used as the aqueous phase, cyclohexane as the organic phase and poly(oxyethylene)(12) nonylphenol ether (NP-12) as the surfactant. Depending on the calcination temperature between 600 and 800 degrees C, CaP nanoparticle showed different phases of calcium-deficient hydroxyapatite (CDHA) and beta-tricalcium phosphate (beta-TCP), particle size between 48 and 69 nm, and a BET specific average surface area between 73 and 57 m(2)g(-1). Bovine serum albumin (BSA) was used as a model protein to study loading and release behavior. The adsorptive property of BSA was investigated by the change in BET surface area of these nanoparticles and the pH of the suspension. At pH 7.5, the maximum amount of BSA was adsorbed onto CaP nanoparticle. The release kinetics of BSA showed a gradual time-dependent increase in pH 4.0 and 6.0 buffer solutions. However, the amount of protein released was significantly smaller at pH 7.2. The BSA release rate also varied depending on the presence of different phases of CaPs in the system, beta-TCP or CDHA. These results suggest that the BSA protein release rate can be controlled by changing the particle size, surface area and phase composition of the CaP nanocarriers.

Cold argon-oxygen plasma species oxidize and disintegrate capsid protein of feline calicivirus

PubMed Central

Mor, Sunil K.; Higgins, LeeAnn; Armien, Anibal; Youssef, Mohammed M.; Bruggeman, Peter J.; Goyal, Sagar M.

2018-01-01

Possible mechanisms that lead to inactivation of feline calicivirus (FCV) by cold atmospheric-pressure plasma (CAP) generated in 99% argon-1% O2 admixture were studied. We evaluated the impact of CAP exposure on the FCV viral capsid protein and RNA employing several cultural, molecular, proteomic and morphologic characteristics techniques. In the case of long exposure (2 min) to CAP, the reactive species of CAP strongly oxidized the major domains of the viral capsid protein (VP1) leading to disintegration of a majority of viral capsids. In the case of short exposure (15 s), some of the virus particles retained their capsid structure undamaged but failed to infect the host cells in vitro. In the latter virus particles, CAP exposure led to the oxidation of specific amino acids located in functional peptide residues in the P2 subdomain of the protrusion (P) domain, the dimeric interface region of VP1 dimers, and the movable hinge region linking the S and P domains. These regions of the capsid are known to play an essential role in the attachment and entry of the virus to the host cell. These observations suggest that the oxidative effect of CAP species inactivates the virus by hindering virus attachment and entry into the host cell. Furthermore, we found that the oxidative impact of plasma species led to oxidation and damage of viral RNA once it becomes unpacked due to capsid destruction. The latter effect most likely plays a secondary role in virus inactivation since the intact FCV genome is infectious even after damage to the capsid. PMID:29566061

Immunoprecipitation of Tri-methylated Capped RNA.

PubMed

Hayes, Karen E; Barr, Jamie A; Xie, Mingyi; Steitz, Joan A; Martinez, Ivan

2018-02-05

Cellular quiescence (also known as G 0 arrest) is characterized by reduced DNA replication, increased autophagy, and increased expression of cyclin-dependent kinase p27 Kip1 . Quiescence is essential for wound healing, organ regeneration, and preventing neoplasia. Previous findings indicate that microRNAs (miRNAs) play an important role in regulating cellular quiescence. Our recent publication demonstrated the existence of an alternative miRNA biogenesis pathway in primary human foreskin fibroblast (HFF) cells during quiescence. Indeed, we have identified a group of pri-miRNAs (whose mature miRNAs were found induced during quiescence) modified with a 2,2,7-trimethylguanosine (TMG)-cap by the trimethylguanosine synthase 1 (TGS1) protein and transported to the cytoplasm by the Exportin-1 (XPO1) protein. We used an antibody against (TMG)-caps (which does not cross-react with the (m 7 G)-caps that most pri-miRNAs or mRNAs contain [Luhrmann et al ., 1982]) to perform RNA immunoprecipitations from total RNA extracts of proliferating or quiescent HFFs. The novelty of this assay is the specific isolation of pri-miRNAs as well as other non-coding RNAs containing a TMG-cap modification.

Luminescent Quantum Dot Bioconjugates in Fluorescence Resonance Energy Transfer (FRET) Assays

NASA Astrophysics Data System (ADS)

Clapp, Aaron; Medintz, Igor; Goldman, Ellen; Anderson, George; Mauro, J. Matthew; Mattoussi, Hedi

2003-03-01

Colloidal semiconductor quantum dots (QDs) such as those made of CdSe-ZnS core-shell nanocrystals offer a promising alternative to organic dyes in a variety of biological tagging applications. They exhibit high resistance to chemical and photo-degradations, are highly luminescent, and show unique size-specific optical and spectroscopic properties. We have previously demonstrated a useful method for attaching proteins to CdSe-ZnS QDs using dihydrolipoic acid (DHLA) surface capping groups and electrostatic self-assembly in aqueous environments. We have used this conjugation strategy to build solution-based QD-conjugate sensors based on fluorescence resonance energy transfer (FRET) between QD donors and dye-labeled protein acceptors. Specific binding between the QD-ligand donor and dye-labeled receptor was achieved. In another example, the dye receptor was grafted directly onto the protein, then immobilized onto the QD surface via an electrostatic self-assembly process. The QD-complexes were optically excited in a region where absorption of the dye is negligible compared to that of the nanocrystals. We observed a continuous decrease of the QD emission accompanied by a steady and pronounced increase of the acceptor emission as the ratio of dye to QD was increased. The results of these experiments suggest efficient resonance energy transfer between the QD donor and the dye acceptor upon ligand-receptor binding. We will present these data and discuss other aspects such as donor-acceptor separation distance, degree of overlap between absorption of the acceptor and emission of the QD, and reverse FRET (upon ligand-receptor release) in a reversible assay.

Simple and green synthesis of protein-conjugated CdS nanoparticles and spectroscopic study on the interaction between CdS and zein

NASA Astrophysics Data System (ADS)

Qin, Dezhi; Zhang, Li; Du, Xian; Wang, Yabo; Zhang, Qiuxia

2016-09-01

The present study demonstrates the role of zein molecules in synthesizing CdS nanoassemblies through protein-directed, green synthetic approach. Zein molecules can as capping ligand and stabilizing agent to regulate the nucleation and growth of CdS nanocrystals, and the obtained products are organic-inorganic nanocomposites. The analysis of surface charge and conductivity indicates that strong electrostatic force restricts mobility of ions, which creates a local supersaturation surrounding the binding sites of zein and reduces the activated energy of nucleation. The interaction between Cd2+/CdS and zein molecules was systematically investigated through spectroscopy techniques. Fourier transform infrared (FT-IR) spectra were used to envisage the binding of the functional groups of zein with the surface of CdS nanoparticles. Ultraviolet visible (UV-Vis) and photoluminescence (PL) spectra results show that Cd2+/CdS might interact with the aromatic amino acids of protein molecules and change its chemical microenvironment. The quantum-confined effect of nanocrystals is confirmed by optical absorption spectrum due to the small size (3-5 nm) of CdS particles. The data of circular dichroism (CD) spectra indicate that the formation of CdS nanocrystals could lead to the conformational change of zein molecules. Moreover, the possible mechanism of CdS nanocrystals growth in zein solution was also discussed. The weak interactions such as Van der Waals, hydrophobic forces and hydrogen bonds in zein molecules should play a crucial factor in the self-assembly of small nanoparticles.

The activities of the C-terminal regions of the formin protein disheveled-associated activator of morphogenesis (DAAM) in actin dynamics.

PubMed

Vig, Andrea Teréz; Földi, István; Szikora, Szilárd; Migh, Ede; Gombos, Rita; Tóth, Mónika Ágnes; Huber, Tamás; Pintér, Réka; Talián, Gábor Csaba; Mihály, József; Bugyi, Beáta

2017-08-18

Disheveled-associated activator of morphogenesis (DAAM) is a diaphanous-related formin protein essential for the regulation of actin cytoskeleton dynamics in diverse biological processes. The conserved formin homology 1 and 2 (FH1-FH2) domains of DAAM catalyze actin nucleation and processively mediate filament elongation. These activities are indirectly regulated by the N- and C-terminal regions flanking the FH1-FH2 domains. Recently, the C-terminal diaphanous-autoregulatory domain (DAD) and the C terminus (CT) of formins have also been shown to regulate actin assembly by directly interacting with actin. Here, to better understand the biological activities of DAAM, we studied the role of DAD-CT regions of Drosophila DAAM in its interaction with actin with in vitro biochemical and in vivo genetic approaches. We found that the DAD-CT region binds actin in vitro and that its main actin-binding element is the CT region, which does not influence actin dynamics on its own. However, we also found that it can tune the nucleating activity and the filament end-interaction properties of DAAM in an FH2 domain-dependent manner. We also demonstrate that DAD-CT makes the FH2 domain more efficient in antagonizing with capping protein. Consistently, in vivo data suggested that the CT region contributes to DAAM-mediated filopodia formation and dynamics in primary neurons. In conclusion, our results demonstrate that the CT region of DAAM plays an important role in actin assembly regulation in a biological context. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Characterization of muscarinic receptors on intact human neuroblastoma cells: coupling to phosphoinositide hydrolysis and phosphorylation by phorbol esters

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

Serra, M.; Watson, M.; Roeske, W.R.

Cloned human neuroblastoma cells (SH-SY5Y) were grown. High affinity binding of (/sup 3/H)(-)quinuclidinyl benzilate ((/sup 3/H)(-)QNB) and its quaternary derivative (/sup 3/H)(-)methyl QNB to muscarinic receptors (MR) on intact SH-SY5Y cells was studied. A 30 min rinse time gave a ratio of specific/total binding of 90% for both ligands. Association rates of (/sup 3/H)(-)QNB and (/sup 3/H)(-)methyl QNB were determined. Both ligands reached steady state by 60 min at 37/sup 0/C. Rates of dissociation for both radioligands were biphasic, although (/sup 3/H)(-)methyl QNB was faster. Saturation studies yielded K/sub d/ (dissociation constant) values of 16 and 260 pM and B/submore » max/ (maximal MR density) values of 172 and 134 fmoles/mg prot for (/sup 3/H)(-)QNB and (/sup 3/H)(-)methyl QNB, respectively. Activation of protein kinase C by phorbol esters produced increased phosphorylation of cellular proteins. Pretreatment with 100 nM of 4..beta..-phorbol 12..beta..-myristate 13..cap alpha..-acetate (PMA) induced a decrease in agonist affinity for MR, suggesting a PMA-promoted phosphorylation of the MR protein. Phosphoinositide (PhI) turnover was measured by MR agonist-induced accumulation of inositol-1-phosphate in the presence of Li/sup + +/ (10 mM). Only carbachol and acetylcholine elicited potent responses with oxotremorine (16%) pilocarpine (17%) and McN-A-343 (8%) appearing to be weak partial agonist of low efficacy.« less

Direct work function measurement by gas phase photoelectron spectroscopy and its application on PbS nanoparticles.

PubMed

Axnanda, Stephanus; Scheele, Marcus; Crumlin, Ethan; Mao, Baohua; Chang, Rui; Rani, Sana; Faiz, Mohamed; Wang, Suidong; Alivisatos, A Paul; Liu, Zhi

2013-01-01

Work function is a fundamental property of a material's surface. It is playing an ever more important role in engineering new energy materials and efficient energy devices, especially in the field of photovoltaic devices, catalysis, semiconductor heterojunctions, nanotechnology, and electrochemistry. Using ambient pressure X-ray photoelectron spectroscopy (APXPS), we have measured the binding energies of core level photoelectrons of Ar gas in the vicinity of several reference materials with known work functions (Au(111), Pt(111), graphite) and PbS nanoparticles. We demonstrate an unambiguously negative correlation between the work functions of reference samples and the binding energies of Ar 2p core level photoelectrons detected from the Ar gas near the sample surface region. Using this experimentally determined linear relationship between the surface work function and Ar gas core level photoelectron binding energy, we can measure the surface work function of different materials under different gas environments. To demonstrate the potential applications of this ambient pressure XPS technique in nanotechnology and solar energy research, we investigate the work functions of PbS nanoparticles with various capping ligands: methoxide, mercaptopropionic acid, and ethanedithiol. Significant Fermi level position changes are observed for PbS nanoparticles when the nanoparticle size and capping ligands are varied. The corresponding changes in the valence band maximum illustrate that an efficient quantum dot solar cell design has to take into account the electrochemical effect of the capping ligand as well.

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

Knox, Anna Sophia; Paller, Michael H.; Milliken, Charles E.

One challenge to all remedial approaches for contaminated sediments is the continued influx of contaminants from uncontrolled sources following remediation. We investigated the effects of ongoing contamination in mesocosms employing sediments remediated by different types of active and passive caps and in-situ treatment. Our hypothesis was that the sequestering agents used in active caps and in situ treatment will bind elements (arsenic, chromium, cadmium, cobalt, copper, nickel, lead, selenium, and zinc) from ongoing sources thereby reducing their bioavailability and protecting underlying remediated sediments from recontamination. Most element concentrations in surface water remained significantly lower in mesocosms with apatite and mixedmore » amendment caps than in mesocosms with passive caps (sand), uncapped sediment, and spike solution throughout the 2520 hour experiment. Element concentrations were significantly higher in Lumbriculus variegatus from untreated sediment than in Lumbriculus from most active caps. Moreover, Pearson correlations between element concentrations in Lumbriculus and metal concentrations in the top 2.5 cm of sediment or cap measured by diffusive gradient in thin films (DGT) sediment probes were generally strong (as high as 0.98) and significant (p<0.05) for almost all tested elements. Metal concentrations in both Lumbriculus and sediment/cap were lowest in apatite, mixed amendment, and activated carbon treatments. Finally, these findings show that some active caps can protect remediated sediments by reducing the bioavailable pool of metals/metalloids in ongoing sources of contamination.« less

Environmental impact of ongoing sources of metal contamination on remediated sediments

DOE PAGES

Knox, Anna Sophia; Paller, Michael H.; Milliken, Charles E.; ...

2016-04-29

One challenge to all remedial approaches for contaminated sediments is the continued influx of contaminants from uncontrolled sources following remediation. We investigated the effects of ongoing contamination in mesocosms employing sediments remediated by different types of active and passive caps and in-situ treatment. Our hypothesis was that the sequestering agents used in active caps and in situ treatment will bind elements (arsenic, chromium, cadmium, cobalt, copper, nickel, lead, selenium, and zinc) from ongoing sources thereby reducing their bioavailability and protecting underlying remediated sediments from recontamination. Most element concentrations in surface water remained significantly lower in mesocosms with apatite and mixedmore » amendment caps than in mesocosms with passive caps (sand), uncapped sediment, and spike solution throughout the 2520 hour experiment. Element concentrations were significantly higher in Lumbriculus variegatus from untreated sediment than in Lumbriculus from most active caps. Moreover, Pearson correlations between element concentrations in Lumbriculus and metal concentrations in the top 2.5 cm of sediment or cap measured by diffusive gradient in thin films (DGT) sediment probes were generally strong (as high as 0.98) and significant (p<0.05) for almost all tested elements. Metal concentrations in both Lumbriculus and sediment/cap were lowest in apatite, mixed amendment, and activated carbon treatments. Finally, these findings show that some active caps can protect remediated sediments by reducing the bioavailable pool of metals/metalloids in ongoing sources of contamination.« less

Integrated system for temperature-controlled fast protein liquid chromatography comprising improved copolymer modified beaded agarose adsorbents and a travelling cooling zone reactor arrangement.

PubMed

Müller, Tobias K H; Cao, Ping; Ewert, Stephanie; Wohlgemuth, Jonas; Liu, Haiyang; Willett, Thomas C; Theodosiou, Eirini; Thomas, Owen R T; Franzreb, Matthias

2013-04-12

An integrated approach to temperature-controlled chromatography, involving copolymer modified agarose adsorbents and a novel travelling cooling zone reactor (TCZR) arrangement, is described. Sepharose CL6B was transformed into a thermoresponsive cation exchange adsorbent (thermoCEX) in four synthetic steps: (i) epichlorohydrin activation; (ii) amine capping; (iii) 4,4'-azobis(4-cyanovaleric acid) immobilization; and 'graft from' polymerization of poly(N-isopropylacrylamide-co-N-tert-butylacrylamide-co-acrylic acid-co-N,N'-methylenebisacrylamide). FT-IR, (1)H NMR, gravimetry and chemical assays allowed precise determination of the adsorbent's copolymer composition and loading, and identified the initial epoxy activation step as a critical determinant of 'on-support' copolymer loading, and in turn, protein binding performance. In batch binding studies with lactoferrin, thermoCEX's binding affinity and maximum adsorption capacity rose smoothly with temperature increase from 20 to 50 °C. In temperature shifting chromatography experiments employing thermoCEX in thermally jacketed columns, 44-51% of the lactoferrin adsorbed at 42 °C could be desorbed under binding conditions by cooling the column to 22 °C, but the elution peaks exhibited strong tailing. To more fully exploit the potential of thermoresponsive chromatography adsorbents, a new column arrangement, the TCZR, was developed. In TCZR chromatography, a narrow discrete cooling zone (special assembly of copper blocks and Peltier elements) is moved along a bespoke fixed-bed separation columnfilled with stationary phase. In tests with thermoCEX, it was possible to recover 65% of the lactoferrin bound at 35 °C using 8 successive movements of the cooling zone at a velocity of 0.1mm/s; over half of the recovered protein was eluted in the first peak in more concentrated form than in the feed. Intra-particle diffusion of desorbed protein out of the support pores, and the ratio between the velocities of the cooling zone and mobile phase were identified as the main parameters affecting TCZR performance. In contrast to conventional systems, which rely on cooling the whole column to effect elution and permit only batch-wise operation, TCZR chromatography generates sharp concentrated elution peaks without tailing effects and appears ideally suited for continuous operation. Copyright © 2013 Elsevier B.V. All rights reserved.

Paracellular transport in the collecting duct

PubMed Central

Hou, Jianghui

2016-01-01

Purpose of review The paracellular pathway through the tight junction provides an important route for chloride reabsorption in the collecting duct of the kidney. This review describes recent findings of how defects in paracellular chloride permeation pathway may cause kidney diseases and how such a pathway may be regulated to maintain normal chloride homeostasis. Recent findings The tight junction in the collecting duct expresses two important claudin genes – claudin-4 and claudin-8. Transgenic knockout of either claudin gene causes hypotension, hypochloremia, and metabolic alkalosis in experimental animals. The claudin-4 mediated chloride permeability can be regulated by a protease endogenously expressed by the collecting duct cell – Cap1. Cap1 regulates the intercellular interaction of claudin-4 and its membrane stability. KLHL3, previously identified as a causal gene for Gordon’s syndrome, also known as pseudohypoaldosteronism II (PHA-II), directly interacts with claudin-8 and regulates its ubiquitination and degradation. The dominant PHA-II mutation (R528H) in KLHL3 abolishes claudin-8 binding, ubiquitination, and degradation. Summary The paracellular chloride permeation pathway in the kidney is an important but understudied area in nephrology. It plays vital roles in renal salt handling and regulation of extracellular fluid volume and blood pressure. Two claudin proteins – claudin-4 and claudin-8 contribute to the function of this paracellular pathway. Deletion of either claudin protein from the collecting duct causes renal chloride reabsorption defects and low blood pressure. Claudins can be regulated on post-translational levels by several mechanisms involving protease and ubiquitin ligase. Deregulation of claudins may cause human hypertension as exemplified in the Gordon’s syndrome. PMID:27490784

Sperm membrane proteins associated with the boar semen cryopreservation.

PubMed

Guimarães, Daianny B; Barros, Tatyane B; van Tilburg, Maurício F; Martins, Jorge A M; Moura, Arlindo A; Moreno, Frederico B; Monteiro-Moreira, Ana C; Moreira, Renato A; Toniolli, Ricardo

2017-08-01

This study aimed to define sperm membrane protein markers of semen freezability of boars with the aid of a proteomic approach. Semen from fourteen adult boars were subjected to slow freezing and rapid thawing. After thawing, sperm vigor and motility were analyzed, and based on these results, animals were separated into two groups: good (GFEs) and poor freezability (PFEs). Sperm membrane proteins were extracted and subjected to two-dimensional electrophoresis. Stained gels were analyzed by computerized resources to indicate differentially expressed protein spots, that were identified by mass spectrometry. Six animals showed good freezability with average sperm vigor and motility of 2.2±0.8 and 41.8±22.9, respectively, whereas eight boars showed poor freezability, with 1.9±0.6 and 26.8±17.5 of sperm vigor sperm motility, respectively. An average of 263±62.2 spots per gel and 234.2±54.6 of spots consistently present in all gels were detected. The intensities of five spots were significantly different between groups. Fc fragment of IgG binding protein and lactadherin were more intense in the PFE group, while Arylsulfatase A and F-actin capping protein subunit alpha 1 were more expressed in the GEF group. Based on their functions and interactions with other proteins, we conclude that these four sperm membrane proteins may act as potential markers of boar semen freezability. Copyright © 2017. Published by Elsevier B.V.

DNA 3' pp 5' G de-capping activity of aprataxin: effect of cap nucleoside analogs and structural basis for guanosine recognition

DOE PAGES

Chauleau, Mathieu; Jacewicz, Agata; Shuman, Stewart

2015-05-24

DNA 3' pp 5'G caps synthesized by the 3'-PO 4/5'-OH ligase RtcB have a strong impact on enzymatic reactions at DNA 3'-OH ends. Aprataxin, an enzyme that repairs A5'pp5'DNA ends formed during abortive ligation by classic 3'-OH/5'-PO 4 ligases, is also a DNA 3' de-capping enzyme, converting DNAppG to DNA 3'p and GMP. By taking advantage of RtcB's ability to utilize certain GTP analogs to synthesize DNAppN caps, we show that aprataxin hydrolyzes inosine and 6-O-methylguanosine caps, but is not adept at removing a deoxyguanosine cap. We report a 1.5 Å crystal structure of aprataxin in a complex with GMP,more » which reveals that: (i) GMP binds at the same position and in the same anti nucleoside conformation as AMP; and (ii) aprataxin makes more extensive nucleobase contacts with guanine than with adenine, via a hydrogen bonding network to the guanine O6, N1, N2 base edge. Alanine mutations of catalytic residues His147 and His149 abolish DNAppG de-capping activity, suggesting that the 3' de-guanylylation and 5' de-adenylylation reactions follow the same pathway of nucleotidyl transfer through a covalent aprataxin-(His147)–NMP intermediate. Alanine mutation of Asp63, which coordinates the guanosine ribose hydroxyls, impairs DNAppG de-capping.« less

Dissociation of the Tubulin-sequestering and Microtubule Catastrophe-promoting Activities of Oncoprotein 18/Stathmin

PubMed Central

Howell, Bonnie; Larsson, Niklas; Gullberg, Martin; Cassimeris, Lynne

1999-01-01

Oncoprotein 18/stathmin (Op18) has been identified recently as a protein that destabilizes microtubules, but the mechanism of destabilization is currently controversial. Based on in vitro microtubule assembly assays, evidence has been presented supporting conflicting destabilization models of either tubulin sequestration or promotion of microtubule catastrophes. We found that Op18 can destabilize microtubules by both of these mechanisms and that these activities can be dissociated by changing pH. At pH 6.8, Op18 slowed microtubule elongation and increased catastrophes at both plus and minus ends, consistent with a tubulin-sequestering activity. In contrast, at pH 7.5, Op18 promoted microtubule catastrophes, particularly at plus ends, with little effect on elongation rates at either microtubule end. Dissociation of tubulin-sequestering and catastrophe-promoting activities of Op18 was further demonstrated by analysis of truncated Op18 derivatives. Lack of a C-terminal region of Op18 (aa 100–147) resulted in a truncated protein that lost sequestering activity at pH 6.8 but retained catastrophe-promoting activity. In contrast, lack of an N-terminal region of Op18 (aa 5–25) resulted in a truncated protein that still sequestered tubulin at pH 6.8 but was unable to promote catastrophes at pH 7.5. At pH 6.8, both the full length and the N-terminal–truncated Op18 bound tubulin, whereas truncation at the C-terminus resulted in a pronounced decrease in tubulin binding. Based on these results, and a previous study documenting a pH-dependent change in binding affinity between Op18 and tubulin, it is likely that tubulin sequestering observed at lower pH resulted from the relatively tight interaction between Op18 and tubulin and that this tight binding requires the C-terminus of Op18; however, under conditions in which Op18 binds weakly to tubulin (pH 7.5), Op18 stimulated catastrophes without altering tubulin subunit association or dissociation rates, and Op18 did not depolymerize microtubules capped with guanylyl (α, β)-methylene diphosphonate–tubulin subunits. We hypothesize that weak binding between Op18 and tubulin results in free Op18, which is available to interact with microtubule ends and thereby promote catastrophes by a mechanism that likely involves GTP hydrolysis. PMID:9880330

Spiral wound extraction cartridge

DOEpatents

Wisted, E.E.; Lundquist, S.H.

1999-04-27

A cartridge device for removing an analyte from a fluid comprises a hollow core, a sheet composite comprising a particulate-loaded porous membrane and optionally at least one reinforcing spacer sheet, the particulate being capable of binding the analyte, the sheet composite being formed into a spiral configuration about the core, wherein the sheet composite is wound around itself and wherein the windings of sheet composite are of sufficient tightness so that adjacent layers are essentially free of spaces therebetween, two end caps which are disposed over the core and the lateral ends of the spirally wound sheet composite, and means for securing the end caps to the core, the end caps also being secured to the lateral ends of the spirally wound sheet composite. A method for removing an analyte from a fluid comprises the steps of providing a spirally wound element of the invention and passing the fluid containing the analyte through the element essentially normal to a surface of the sheet composite so as to bind the analyte to the particulate of the particulate-loaded porous membrane, the method optionally including the step of eluting the bound analyte from the sheet composite. 4 figs.

A biochemical framework for eIF4E-dependent mRNA export and nuclear recycling of the export machinery.

PubMed

Volpon, Laurent; Culjkovic-Kraljacic, Biljana; Sohn, Hye Seon; Blanchet-Cohen, Alexis; Osborne, Michael J; Borden, Katherine L B

2017-06-01

The eukaryotic translation initiation factor eIF4E acts in the nuclear export and translation of a subset of mRNAs. Both of these functions contribute to its oncogenic potential. While the biochemical mechanisms that underlie translation are relatively well understood, the molecular basis for eIF4E's role in mRNA export remains largely unexplored. To date, over 3000 transcripts, many encoding oncoproteins, were identified as potential nuclear eIF4E export targets. These target RNAs typically contain a ∼50-nucleotide eIF4E sensitivity element (4ESE) in the 3' UTR and a 7-methylguanosine cap on the 5' end. While eIF4E associates with the cap, an unknown factor recognizes the 4ESE element. We previously identified cofactors that functionally interacted with eIF4E in mammalian cell nuclei including the leucine-rich pentatricopeptide repeat protein LRPPRC and the export receptor CRM1/XPO1. LRPPRC simultaneously interacts with both eIF4E bound to the 5' mRNA cap and the 4ESE element in the 3' UTR. In this way, LRPPRC serves as a specificity factor to recruit 4ESE-containing RNAs within the nucleus. Further, we show that CRM1 directly binds LRPPRC likely acting as the export receptor for the LRPPRC-eIF4E-4ESE RNA complex. We also found that Importin 8, the nuclear importer for cap-free eIF4E, imports RNA-free LRPPRC, potentially providing both coordinated nuclear recycling of the export machinery and an important surveillance mechanism to prevent futile export cycles. Our studies provide the first biochemical framework for the eIF4E-dependent mRNA export pathway. © 2017 Volpon et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

Cryopreservation of bull semen is associated with carbonylation of sperm proteins.

PubMed

Mostek, Agnieszka; Dietrich, Mariola Aleksandra; Słowińska, Mariola; Ciereszko, Andrzej

2017-04-01

Artificial insemination with cryopreserved semen enables affordable, large-scale dissemination of gametes with superior genetics. However, cryopreservation can cause functional and structural damage to spermatozoa that is associated with reactive oxygen species (ROS) production, impairment of sperm motility and decreased fertilizing potential, but little attention has been paid to protein changes. The goal of this study was to investigate the oxidative modifications (measured as carbonylation level changes) of bull spermatozoa proteins triggered by the cryopreservation process. Flow cytometry and computer-assisted sperm analysis were used to evaluate changes in viability, ROS level and motility of spermatozoa. Western blotting, in conjunction with two-dimensional electrophoresis (2D-oxyblot) and matrix-assisted laser desorption/ionization time-of-flight/time-of-flight spectrometry, was employed to identify and quantify the specifically carbonylated spermatozoa proteins. Cryopreservation decreased motility and viability but increased the number of ROS-positive cells. We identified 11 proteins (ropporin-1, outer dense fiber protein 2, glutathione S-transferase, triosephosphate isomerase, capping protein beta 3 isoform, actin-related protein M1, actin-related protein T2, NADH dehydrogenase, isocitrate dehydrogenase, cilia- and flagella-associated protein 161, phosphatidylethanolamine-binding protein 4) showing differences in protein carbonylation in response to cryopreservation. The identified proteins are associated with cytoskeleton and flagella organization, detoxification and energy metabolism. Moreover, almost all of the identified carbonylated proteins are involved in capacitation. Our results indicate for the first time that cryopreservation induces oxidation of selected sperm proteins via carbonylation. We suggest that carbonylation of sperm proteins could be a direct result of oxidative stress and potentially lead to disturbances of capacitation-involved proteins or could indicate cryopreservation-induced premature capacitation. Copyright © 2017. Published by Elsevier Inc.

Proteomic analysis on the alteration of protein expression in the early-stage placental villous tissue of electromagnetic fields associated with cell phone exposure.

PubMed

Luo, Qiong; Jiang, Ying; Jin, Min; Xu, Jian; Huang, He-Feng

2013-09-01

To explore the possible adverse effects and search for cell phone electromagnetic field (EMF)-responsive proteins in human early reproduction, a proteomics approach was employed to investigate the changes in protein expression profile induced by cell phone EMF in human chorionic tissues of early pregnancy in vivo. Volunteer women about 50 days pregnant were exposed to EMF at the average absorption rate of 1.6 to 8.8 W/kg for 1 hour with the irradiation device placed 10 cm away from the umbilicus at the midline of the abdomen. The changes in protein profile were examined using 2-dimensional electrophoresis (2-DE). Up to 15 spots have yielded significant change at least 2- to 2.5-folds up or down compared to sham-exposed group. Twelve proteins were identified- procollagen-proline, eukaryotic translation elongation factor 1 delta, chain D crystal structure of human vitamin D-binding protein, thioredoxin-like 3, capping protein, isocitrate dehydrogenase 3 alpha, calumenin, Catechol-O-methyltransferase protein, proteinase inhibitor 6 (PI-6; SerpinB6) protein, 3,2-trans-enoyl-CoA isomerase protein, chain B human erythrocyte 2,3-bisphosphoglycerate mutase, and nucleoprotein. Cell phone EMF might alter the protein profile of chorionic tissue of early pregnancy, during the most sensitive stage of the embryos. The exposure to EMF may cause adverse effects on cell proliferation and development of nervous system in early embryos. Furthermore, 2-DE coupled with mass spectrometry is a promising approach to elucidate the effects and search for new biomarkers for environmental toxic effects.

Biomarkers in community-acquired pneumonia: a state-of-the-art review.

PubMed

Seligman, Renato; Ramos-Lima, Luis Francisco; Oliveira, Vivian do Amaral; Sanvicente, Carina; Pacheco, Elyara F; Dalla Rosa, Karoline

2012-11-01

Community-acquired pneumonia (CAP) exhibits mortality rates, between 20% and 50% in severe cases. Biomarkers are useful tools for searching for antibiotic therapy modifications and for CAP diagnosis, prognosis and follow-up treatment. This non-systematic state-of-the-art review presents the biological and clinical features of biomarkers in CAP patients, including procalcitonin, C-reactive protein, copeptin, pro-ANP (atrial natriuretic peptide), adrenomedullin, cortisol and D-dimers.

Protein and carotenoid synthesis and turnover in gravistimulated root caps

NASA Technical Reports Server (NTRS)

Feldman, L. J.

1984-01-01

In certain cultivars of corn gravitropic bending occurs only after the root cap, the site of gravity perception, is exposed to light. Light appears to trigger or to remove some block in the gravity translation process. Using light sensitive cultivars of corn, it was shown that light affects various processes in the cap. The roles of these light-induced processes in gravitropic bending in roots were studied.