e1b 55-kilodalton protein: Topics by Science.gov

Sample records for e1b 55-kilodalton protein

Adenovirus E1B 19-Kilodalton Protein Modulates Innate Immunity through Apoptotic Mimicry

PubMed Central

Grigera, Fernando; Ucker, David S.; Cook, James L.

2014-01-01

ABSTRACT Cells that undergo apoptosis in response to chemical or physical stimuli repress inflammatory reactions, but cells that undergo nonapoptotic death in response to such stimuli lack this activity. Whether cells dying from viral infection exhibit a cell death-type modulatory effect on inflammatory reactions is unknown. We compared the effects on macrophage inflammatory responses of cells dying an apoptotic or a nonapoptotic death as a result of adenoviral infection. The results were exactly opposite to the predictions from the conventional paradigm. Cells dying by apoptosis induced by infection with an adenovirus type 5 (Ad5) E1B 19-kilodalton (E1B 19K) gene deletion mutant did not repress macrophage NF-κB activation or cytokine responses to proinflammatory stimuli, whereas cells dying a nonapoptotic death from infection with E1B 19K-competent, wild-type Ad5 repressed these macrophage inflammatory responses as well as cells undergoing classical apoptosis in response to chemical injury. The immunorepressive, E1B 19K-related cell death activity depended upon direct contact of the virally infected corpses with responder macrophages. Replacement of the viral E1B 19K gene with the mammalian Bcl-2 gene in cis restored the nonapoptotic, immunorepressive cell death activity of virally infected cells. These results define a novel function of the antiapoptotic, adenoviral E1B 19K protein that may limit local host innate immune inflammation during accumulation of virally infected cells at sites of infection and suggest that E1B 19K-deleted, replicating adenoviral vectors might induce greater inflammatory responses to virally infected cells than E1B 19K-positive vectors, because of the net effect of their loss-of-function mutation. IMPORTANCE We observed that cells dying a nonapoptotic cell death induced by adenovirus infection repressed macrophage proinflammatory responses while cells dying by apoptosis induced by infection with an E1B 19K deletion mutant virus did not
E4orf1 Limits the Oncolytic Potential of the E1B-55K Deletion Mutant Adenovirus▿

PubMed Central

Thomas, Michael A.; Broughton, Robin S.; Goodrum, Felicia D.; Ornelles, David A.

2009-01-01

Clinical trials have shown oncolytic adenoviruses to be tumor selective with minimal toxicity toward normal tissue. The virus ONYX-015, in which the gene encoding the early region 1B 55-kDa (E1B-55K) protein is deleted, has been most effective when used in combination with either chemotherapy or radiation therapy. Therefore, improving the oncolytic nature of tumor-selective adenoviruses remains an important objective for improving this form of cancer therapy. Cells infected during the G1 phase of the cell cycle with the E1B-55K deletion mutant virus exhibit a reduced rate of viral late protein synthesis, produce fewer viral progeny, and are less efficiently killed than cells infected during the S phase. Here we demonstrate that the G1 restriction imposed on the E1B-55K deletion mutant virus is due to the viral oncogene encoded by open reading frame 1 of early region 4 (E4orf1). E4orf1 has been reported to signal through the phosphatidylinositol 3′-kinase pathway leading to the activation of Akt, mTOR, and p70 S6K. Evidence presented here shows that E4orf1 may also induce the phosphorylation of Akt and p70 S6K in a manner that depends on Rac1 and its guanine nucleotide exchange factor Tiam1. Accordingly, agents that have been reported to disrupt the Tiam1-Rac1 interaction or to prevent phosphorylation of the ribosomal S6 kinase partially alleviated the E4orf1 restriction to late viral protein synthesis and enhanced tumor cell killing by the E1B-55K mutant virus. These results demonstrate that E4orf1 limits the oncolytic nature of a conditionally replicating adenovirus such as ONYX-015. The therapeutic value of similar oncolytic adenoviruses may be improved by abrogating E4orf1 function. PMID:19129452
E4orf1 limits the oncolytic potential of the E1B-55K deletion mutant adenovirus.

PubMed

Thomas, Michael A; Broughton, Robin S; Goodrum, Felicia D; Ornelles, David A

2009-03-01

Clinical trials have shown oncolytic adenoviruses to be tumor selective with minimal toxicity toward normal tissue. The virus ONYX-015, in which the gene encoding the early region 1B 55-kDa (E1B-55K) protein is deleted, has been most effective when used in combination with either chemotherapy or radiation therapy. Therefore, improving the oncolytic nature of tumor-selective adenoviruses remains an important objective for improving this form of cancer therapy. Cells infected during the G(1) phase of the cell cycle with the E1B-55K deletion mutant virus exhibit a reduced rate of viral late protein synthesis, produce fewer viral progeny, and are less efficiently killed than cells infected during the S phase. Here we demonstrate that the G(1) restriction imposed on the E1B-55K deletion mutant virus is due to the viral oncogene encoded by open reading frame 1 of early region 4 (E4orf1). E4orf1 has been reported to signal through the phosphatidylinositol 3'-kinase pathway leading to the activation of Akt, mTOR, and p70 S6K. Evidence presented here shows that E4orf1 may also induce the phosphorylation of Akt and p70 S6K in a manner that depends on Rac1 and its guanine nucleotide exchange factor Tiam1. Accordingly, agents that have been reported to disrupt the Tiam1-Rac1 interaction or to prevent phosphorylation of the ribosomal S6 kinase partially alleviated the E4orf1 restriction to late viral protein synthesis and enhanced tumor cell killing by the E1B-55K mutant virus. These results demonstrate that E4orf1 limits the oncolytic nature of a conditionally replicating adenovirus such as ONYX-015. The therapeutic value of similar oncolytic adenoviruses may be improved by abrogating E4orf1 function.
E1B-55K mediated regulation of RNF4 STUbL promotes HAdV gene expression.

PubMed

Müncheberg, Sarah; Hay, Ron T; Ip, Wing H; Meyer, Tina; Weiß, Christina; Brenke, Jara; Masser, Sawinee; Hadian, Kamyar; Dobner, Thomas; Schreiner, Sabrina

2018-04-25

HAdV E1B-55K is a multifunctional regulator of productive viral replication and oncogenic transformation in non-permissive mammalian cells. These functions depend on E1B-55K's posttranslational modification with the SUMO protein and its binding to HAdV E4orf6. Both early viral proteins recruit specific host factors to form an E3 Ubiquitin ligase complex that targets antiviral host substrates for proteasomal degradation. Recently, we reported that the PML-NB-associated factor Daxx represses efficient HAdV productive infection and is proteasomally degraded via a SUMO-E1B-55K-dependent, E4orf6-independent pathway, the details of which remained to be established.RNF4, a cellular SUMO-targeted Ubiquitin ligase (STUbL), induces ubiquitinylation of specific SUMOylated proteins and plays an essential role during DNA repair. Here, we show that E1B-55K recruits RNF4 to the insoluble nuclear matrix fraction of the infected cell to support RNF4/Daxx association, promoting Daxx PTM, and thus inhibiting this antiviral factor. Removing RNF4 from infected cells using RNAi resulted in blocking the proper establishment of viral replication centers and significantly diminished viral gene expression. These results provide a model for how HAdV antagonize the antiviral host responses by exploiting the functional capacity of cellular STUbLs. Thus, RNF4 and its STUbL function represent a positive factor during lytic infection and a novel candidate for future therapeutic antiviral intervention strategies. IMPORTANCE Daxx is a PML-NB-associated transcription factor, which was recently shown to repress efficient HAdV productive infection. To counteract this antiviral measurement during infection, Daxx is degraded via a novel pathway including viral E1B-55K and host proteasomes. This virus-mediated degradation is independent of the classical HAdV E3 Ubiquitin ligase complex, which is essential during viral infection to target other host antiviral substrates. To maintain productive viral life
Aggresome formation by the adenoviral protein E1B55K is not conserved among adenovirus species and is not required for efficient degradation of nuclear substrates.

PubMed

Blanchette, Paola; Wimmer, Peter; Dallaire, Frédéric; Cheng, Chi Ying; Branton, Philip E

2013-05-01

Much of the work on the basic molecular biology of human adenoviruses has been carried out on a very limited number of the more than 60 serotypes, primarily the highly related species C viruses adenovirus type 5 (Ad5) and Ad2 and, to some extent, Ad12 of species A. Until recently, it has been widely assumed that insights obtained with these model viruses were representative of all human adenoviruses. Recent studies on the E3 ubiquitin ligase formed by the viral E1B55K and E4orf6 proteins with a cellular Cullin-based complex indicated that although all species form such a functional complex, significant variations exist in terms of complex composition and the substrates that are degraded. In the present report we conducted a comprehensive analysis of the localization of E1B55K products from representatives of six of the seven adenovirus species in the presence and the absence of the corresponding E4orf6 protein. We found that although in some species E1B55K localized in aggresomes, such was not always the case, suggesting that these structures are not necessary for the efficient degradation of substrates. In addition, differences were evident in the localization of E1B55K, although all forms readily associated with PML. Finally, Ad5 E1B55K was seen to localize in close proximity to Rab11, a marker for the endosomal recycling compartment, and both focused at the microtubule organizing center. These findings suggest that E1B55K from some species may employ the transport system utilized by the membrane recycling pathway to assemble aggresomes and the possibility that this structure might then affect recycling of cell surface components.
Structure- and Modeling-based Identification of the Adenovirus E4orf4 Binding Site in the Protein Phosphatase 2A B55α Subunit*

PubMed Central

Horowitz, Ben; Sharf, Rakefet; Avital-Shacham, Meirav; Pechkovsky, Antonina; Kleinberger, Tamar

2013-01-01

The adenovirus E4orf4 protein regulates the progression of viral infection and when expressed outside the context of the virus it induces nonclassical, cancer cell-specific apoptosis. All E4orf4 functions known to date require an interaction between E4orf4 and protein phosphatase 2A (PP2A), which is mediated through PP2A regulatory B subunits. Specifically, an interaction with the B55α subunit is required for induction of cell death by E4orf4. To gain a better insight into the E4orf4-PP2A interaction, mapping of the E4orf4 interaction site in PP2A-B55α has been undertaken. To this end we used a combination of bioinformatics analyses of PP2A-B55α and of E4orf4, which led to the prediction of E4orf4 binding sites on the surface of PP2A-B55α. Mutation analysis, immunoprecipitation, and GST pulldown assays based on the theoretical predictions revealed that the E4orf4 binding site included the α1 and α2 helices described in the B55α structure and involved at least three residues located in these helices facing each other. Loss of E4orf4 binding was accompanied by reduced contribution of the B55α mutants to E4orf4-induced cell death. The identified E4orf4 binding domain lies above the previously described substrate binding site and does not overlap it, although its location could be consistent with direct or indirect effects on substrate binding. This work assigns for the first time a functional significance to the α1,α2 helices of B55α, and we suggest that the binding site defined by these helices could also contribute to interactions between PP2A and some of its cellular regulators. PMID:23530045
Characterization of a major 31-kilodalton peptidoglycan-bound protein of Legionella pneumophila

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

Butler, C.A.; Hoffman, P.S.

1990-05-01

A 31-kilodalton (kDa) protein was solubilized from the peptidoglycan (PG) fraction of Legionella pneumophila after treatment with either N-acetylmuramidase from the fungus Chalaropsis sp. or with mutanolysin from Streptomyces globisporus. The protein exhibited a ladderlike banding pattern by autoradiography when radiolabeled ((35S)cysteine or (35S)methionine) PG material was extensively treated with hen lysozyme. The banding patterns ranging between 31 and 45 kDa and between 55 and 60 kDa resolved as a single 31-kDa protein when the material was subsequently treated with N-acetylmuramidase. Analysis of the purified 31-kDa protein for diaminopimelic acid by gas chromatography revealed 1 mol of diaminopimelic acid permore » mol of protein. When outer membrane PG material containing the major outer membrane porin protein was treated with N-acetylmuramidase or mutanolysin, both the 28.5-kDa major outer membrane protein and the 31-kDa protein were solubilized from the PG material under reducing conditions. In the absence of 2-mercaptoethanol, a high-molecular-mass complex (100 kDa) was resolved. The results of this study indicate that a 31-kDa PG-bound protein is a major component of the cell wall of L. pneumophila whose function may be to anchor the major outer membrane protein to PG. Finally, a survey of other Legionella species and other serogroups of L. pneumophila suggested that PG-bound proteins may be a common feature of this genus.« less
E1B and E4 oncoproteins of adenovirus antagonize the effect of apoptosis inducing factor

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

Turner, Roberta L.; Wilkinson, John C., E-mail: john.wilkinson@ndsu.edu; Ornelles, David A., E-mail: ornelles@wakehealth.edu

2014-05-15

Adenovirus inundates the productively infected cell with linear, double-stranded DNA and an abundance of single-stranded DNA. The cellular response to this stimulus is antagonized by the adenoviral E1B and E4 early genes. A mutant group C adenovirus that fails to express the E1B-55K and E4ORF3 genes is unable to suppress the DNA-damage response. Cells infected with this double-mutant virus display significant morphological heterogeneity at late times of infection and frequently contain fragmented nuclei. Nuclear fragmentation was due to the translocation of apoptosis inducing factor (AIF) from the mitochondria into the nucleus. The release of AIF was dependent on active poly(ADP-ribose)more » polymerase-1 (PARP-1), which appeared to be activated by viral DNA replication. Nuclear fragmentation did not occur in AIF-deficient cells or in cells treated with a PARP-1 inhibitor. The E1B-55K or E4ORF3 proteins independently prevented nuclear fragmentation subsequent to PARP-1 activation, possibly by altering the intracellular distribution of PAR-modified proteins. - Highlights: • E1B-55K or E4orf3 prevents nuclear fragmentation. • Nuclear fragmentation requires AIF and PARP-1 activity. • Adenovirus DNA replication activates PARP-1. • E1B-55K or E4orf3 proteins alter the distribution of PAR.« less
High Efficiency CRISPR/Cas9-mediated Gene Editing in Primary Human T-cells Using Mutant Adenoviral E4orf6/E1b55k "Helper" Proteins.

PubMed

Gwiazda, Kamila S; Grier, Alexandra E; Sahni, Jaya; Burleigh, Stephen M; Martin, Unja; Yang, Julia G; Popp, Nicholas A; Krutein, Michelle C; Khan, Iram F; Jacoby, Kyle; Jensen, Michael C; Rawlings, David J; Scharenberg, Andrew M

2016-09-29

Many future therapeutic applications of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 and related RNA-guided nucleases are likely to require their use to promote gene targeting, thus necessitating development of methods that provide for delivery of three components-Cas9, guide RNAs and recombination templates-to primary cells rendered proficient for homology-directed repair. Here, we demonstrate an electroporation/transduction codelivery method that utilizes mRNA to express both Cas9 and mutant adenoviral E4orf6 and E1b55k helper proteins in association with adeno-associated virus (AAV) vectors expressing guide RNAs and recombination templates. By transiently enhancing target cell permissiveness to AAV transduction and gene editing efficiency, this novel approach promotes efficient gene disruption and/or gene targeting at multiple loci in primary human T-cells, illustrating its broad potential for application in translational gene editing.
Adenovirus Type 5 Early Region 1B 55K Oncoprotein-Dependent Degradation of Cellular Factor Daxx Is Required for Efficient Transformation of Primary Rodent Cells▿

PubMed Central

Schreiner, Sabrina; Wimmer, Peter; Groitl, Peter; Chen, Shuen-Yuan; Blanchette, Paola; Branton, Philip E.; Dobner, Thomas

2011-01-01

Early region 1B 55K (E1B-55K) from adenovirus type 5 (Ad5) is a multifunctional regulator of lytic infection and contributes in vitro to complete cell transformation of primary rodent cells in combination with Ad5 E1A. Inhibition of p53 activated transcription plays a key role in processes by which E1B-55K executes its oncogenic potential. Nevertheless, additional functions of E1B-55K or further protein interactions with cellular factors of DNA repair, transcription, and apoptosis, including Mre11, PML, and Daxx, may also contribute to the transformation process. In line with previous results, we performed mutational analysis to define a Daxx interaction motif within the E1B-55K polypeptide. The results from these studies showed that E1B-55K/Daxx binding is not required for inhibition of p53-mediated transactivation or binding and degradation of cellular factors (p53/Mre11). Surprisingly, these mutants lost the ability to degrade Daxx and showed reduced transforming potential in primary rodent cells. In addition, we observed that E1B-55K lacking the SUMO-1 conjugation site (SCS/K104R) was sufficient for Daxx interaction but no longer capable of E1B-55K-dependent proteasomal degradation of the cellular factor Daxx. These results, together with the observation that E1B-55K SUMOylation is required for efficient transformation, provides evidence for the idea that SUMO-1-conjugated E1B-55K-mediated degradation of Daxx plays a key role in adenoviral oncogenic transformation. We assume that the viral protein contributes to cell transformation through the modulation of Daxx-dependent pathways. This further substantiates the assumption that further mechanisms for efficient transformation of primary cells can be separated from functions required for the inhibition of p53-stimulated transcription. PMID:21697482
Isolation and characterization of a Treponema pallidum major 60-kilodalton protein resembling the groEL protein of Escherichia coli.

PubMed Central

Houston, L S; Cook, R G; Norris, S J

1990-01-01

A native structure containing the major 60-kilodalton common antigen polypeptide (designated TpN60) was isolated from Treponema pallidum subsp. pallidum (Nichols strain) through a combination of differential centrifugation and sucrose density gradient sedimentation. Gel filtration chromatography indicated that this structure is a high-molecular-weight homo-oligomer of TpN60. Antisera to TpN60 reacted with the groEL polypeptide of Escherichia coli, as determined by immunoperoxidase staining of two-dimensional electroblots. Electron microscopy of the isolated complex revealed a ringlike structure with a diameter of approximately 16 nm which was very similar in appearance to the groEL protein. Comparison of the N-terminal amino acid sequence of TpN60 with the deduced sequences of the E. coli groEL protein, related chaperonin proteins from mycobacteria and Coxiella burnetti, the hsp60 protein of Saccharomyces cerevisiae, the wheat ribulose bisphosphate carboxylase-oxygenase-subunit-binding protein (alpha subunit), and the human P1 mitochondrial protein indicated sequence identity at 8 of 22 to 10 of 22 residues (36 to 45% identity). We conclude that the oligomer of TpN60 is homologous to the groEL protein and related chaperonins found in a wide variety of procaryotes and eucaryotes and thus may represent a heat shock protein involved in protein folding and assembly. Images PMID:1971618
Self-regulation of 70-kilodalton heat shock proteins in Saccharomyces cerevisiae.

PubMed Central

Stone, D E; Craig, E A

1990-01-01

To determine whether the 70-kilodalton heat shock proteins of Saccharomyces cerevisiae play a role in regulating their own synthesis, we studied the effect of overexpressing the SSA1 protein on the activity of the SSA1 5'-regulatory region. The constitutive level of Ssa1p was increased by fusing the SSA1 structural gene to the GAL1 promoter. A reporter vector consisting of an SSA1-lacZ translational fusion was used to assess SSA1 promoter activity. In a strain producing approximately 10-fold the normal heat shock level of Ssa1p, induction of beta-galactosidase activity by heat shock was almost entirely blocked. Expression of a transcriptional fusion vector in which the CYC1 upstream activating sequence of a CYC1-lacZ chimera was replaced by a sequence containing a heat shock upstream activating sequence (heat shock element 2) from the 5'-regulatory region of SSA1 was inhibited by excess Ssa1p. The repression of an SSA1 upstream activating sequence by the SSA1 protein indicates that SSA1 self-regulation is at least partially mediated at the transcriptional level. The expression of another transcriptional fusion vector, containing heat shock element 2 and a lesser amount of flanking sequence, is not inhibited when Ssa1p is overexpressed. This suggests the existence of an element, proximal to or overlapping heat shock element 2, that confers sensitivity to the SSA1 protein. Images PMID:2181281
Activation of RNA polymerase III transcription of human Alu repetitive elements by adenovirus type 5: Requirement for the E1b 58-Kilodalton protein and the products of E4 open reading frames 3 and 6

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

Panning, B.; Smiley, J.R.

1993-06-01

Alu elements are the single most abundant class of dispersed repeated sequences in the human genome, comprising 5-10% of the mass of human DNA. This report demonstrates that Ad5 infection strongly stimulates Pol III transcription of human Alu elements in HeLa and 293 cells. In contrast to the cases of Ad5-induced Pol III transcriptional activation, this process requires the E1b 58-kDa protein and the products of E4 open reading frames (ORFs) 3 and 6 in addition to the E1a 289-residue product. These findings suggest novel regulatory properties of the Ad5 E1b and E4 proteins and raise the possibility that analogousmore » cellular trans-acting factors serve to modulate Alu expression in vivo.« less
Activation of RNA polymerase III transcription of human Alu repetitive elements by adenovirus type 5: requirement for the E1b 58-kilodalton protein and the products of E4 open reading frames 3 and 6.

PubMed Central

Panning, B; Smiley, J R

1993-01-01

We found that transcription of endogenous human Alu elements by RNA polymerase III was strongly stimulated following infection of HeLa cells with adenovirus type 5, leading to the accumulation of high levels of Alu transcripts initiated from Alu polymerase III promoters. In contrast to previously reported cases of adenovirus-induced activation of polymerase III transcription, induction required the E1b 58-kDa protein and the products of E4 open reading frames 3 and 6 in addition to the 289-residue E1a protein. In addition, E1a function was not required at high multiplicities of infection, suggesting that E1a plays an indirect role in Alu activation. These results suggest previously unsuspected regulatory properties of the adenovirus E1b and E4 gene products and provide a novel approach to the study of the biology of the most abundant class of dispersed repetitive DNA in the human genome. Images PMID:7684492
A tyrosine-phosphorylated 55-kilodalton motility-associated bovine sperm protein is regulated by cyclic adenosine 3',5'-monophosphates and calcium.

PubMed

Vijayaraghavan, S; Trautman, K D; Goueli, S A; Carr, D W

1997-06-01

Sperm motility is regulated by protein phosphorylation. We have recently shown that a serine/threonine phosphatase system is involved in motility regulation. Two of the components of the phosphatase system, GSK-3 and PP1gamma2, are regulated by tyrosine phosphorylation. During our investigation of sperm tyrosine-phosphorylated proteins we discovered a 55-kDa protein whose tyrosine phosphorylation correlates closely to the motility state of sperm. This protein is tyrosine phosphorylated to a much higher degree in motile caudal than in immotile caput epididymal sperm. Motility inhibition of caudal epididymal sperm by protein kinase A (PKA) anchoring inhibition or by ionomycin-induced calcium overload led to the virtual disappearance of tyrosine phosphorylation of the 55-kDa protein. Conversely, treatment of sperm with motility activators, isobutylmethylxanthine or 8-bromo-cAMP, resulted in increased tyrosine phosphorylation of the protein. The protein was present in the soluble 100 000 x g supernatants of sperm extracts and was heat labile. Chromatography through diethylaminoethyl-cellulose and Western blot analysis showed that this 55-kDa protein is not a regulatory subunit of PKA or alpha-tubulin. Our results represent the identification of a soluble protein whose tyrosine phosphorylation varies directly with motility and suggest that motility regulation may involve cross talk between PKA, calcium, and tyrosine kinase pathways.
Protein tyrosine phosphatase 1B (PTP1B) is dispensable for IgE-mediated cutaneous reaction in vivo.

PubMed

Yang, Ting; Xie, Zhongping; Li, Hua; Yue, Lei; Pang, Zheng; MacNeil, Adam J; Tremblay, Michel L; Tang, Jin-Tian; Lin, Tong-Jun

2016-01-01

Mast cells play a critical role in allergic reactions. The cross-linking of FcεRI-bound IgE with multivalent antigen initiates a cascade of signaling events leading to mast cell activation. It has been well-recognized that cross linking of FcεRI mediates tyrosine phosphorylation. However, the mechanism involved in tyrosine dephosphorylation in mast cells is less clear. Here we demonstrated that protein tyrosine phosphatase 1B (PTP1B)-deficient mast cells showed increased IgE-mediated phosphorylation of the signal transducer and activator of transcription 5 (STAT5) and enhanced production of CCL9 (MIP-1γ) and IL-6 in IgE-mediated mast cells activation in vitro. However, IgE-mediated calcium mobilization, β-hexaosaminidase release (degranulation), and phosphorylation of IκB and MAP kinases were not affected by PTP1B deficiency. Furthermore, PTP1B deficient mice showed normal IgE-dependent passive cutaneous anaphylaxis and late phase cutaneous reactions in vivo. Thus, PTP1B specifically regulates IgE-mediated STAT5 pathway, but is redundant in influencing mast cell function in vivo. Copyright © 2016 Elsevier Inc. All rights reserved.
Muscarinic agonists and phorbol esters increase tyrosine phosphorylation of a 40-kilodalton protein in hippocampal slices

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

Stratton, K.R.; Worley, P.F.; Huganir, R.L.

The authors have used the hippocampal slice preparation to investigate the regulation of protein tyrosine phosphorylation in brain. After pharmacological treatment of intact slices, proteins were separated by electrophoresis, and levels of protein tyrosine phosphorylation were assessed by immunoblotting with specific anti-phosphotyrosine antibodies. Phorbol esters, activators of the serine- and threonine-phosphorylating enzyme protein kinase C, selectively increase tyrosine phosphorylation of a soluble protein with an apparent molecular mass of approximately 40 kilodaltons. Muscarinic agonists such as carbachol and oxotremorine M that strongly activate the inositol phospholipid system also increase tyrosine phosphorylation of this protein. Neurotransmitter activation of the inositol phospholipidmore » system and protein kinase C appears to trigger a cascade leading to increased tyrosine phosphorylation.« less
Lower Growth Temperature Increases Alternative Pathway Capacity and Alternative Oxidase Protein in Tobacco 1

PubMed Central

Vanlerberghe, Greg C.; McIntosh, Lee

1992-01-01

Suspension cells of NT1 tobacco (Nicotiana tabacum L. cv bright yellow) have been used to study the effect of growth temperature on the CN-resistant, salicylhydroxamic acid-sensitive alternative pathway of respiration. Mitochondria isolated from cells maintained at 30°C had a low capacity to oxidize succinate via the alternative pathway, whereas mitochondria isolated from cells 24 h after transfer to 18°C displayed, on average, a 5-fold increase in this capacity (from 7 to 32 nanoatoms oxygen per milligram protein per minute). This represented an increase in alternative pathway capacity from 18 to 45% of the total capacity of electron transport. This increased capacity was lost upon transfer of cells back to 30°C. A monoclonal antibody to the terminal oxidase of the alternative pathway (the alternative oxidase) from Sauromatum guttatum (T.E. Elthon, R.L. Nickels, L. McIntosh [1989] Plant Physiology 89: 1311-1317) recognized a 35-kilodalton mitochondrial protein in tobacco. There was an excellent correlation between the capacity of the alternative path in isolated tobacco mitochondria and the levels of this 35-kilodalton alternative oxidase protein. Cycloheximide could inhibit both the increased level of the 35-kilodalton alternative oxidase protein and the increased alternative pathway capacity normally seen upon transfer to 18°C. We conclude that transfer of tobacco cells to the lower temperature increases the capacity of the alternative pathway due, at least in part, to de novo synthesis of the 35-kilodalton alternative oxidase protein. Images Figure 3 Figure 4 PMID:16652932
Acquired thermotolerance and heat shock in the extremely thermophilic archaebacterium Sulfolobus sp. strain B12

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

Trent, J.D.; Osipiuk, J.; Pinkau, T.

1990-03-01

The extreme thermophile Sulfolobus sp. strain B12 exhibits an acquired thermotolerance response. Thus, survival of cells from a 70{degrees}C culture at the lethal temperature of 92{degrees}C was enhanced by as much as 6 orders of magnitude over a 2-h period if the culture was preheated to 88{degrees}C for 60 min or longer before being exposed to the lethal temperature. In eubacteria and eucaryotes, acquired thermotolerance correlates with the induced synthesis of a dozen or so proteins known as heat shock proteins. In this Sulfolobus species, it correlates with the preferential synthesis of primarily one major protein (55 kilodaltons) and, tomore » a much lesser extent, two minor proteins (28 and 35 kilodaltons). Since the synthesis of all other proteins was radically reduced and these proteins were apparently not degraded or exported, their relative abundance within the cell increased during the time the cells were becoming thermotolerant. They could not yet be related to known heat shock proteins. In immunoassays, they were not cross-reactive with antibodies against heat shock proteins from Escherichia coli (DnaK and GroE), which are highly conserved between eubacteria and eucaryotes. However, it appears that if acquired thermotolerance depends on the synthesis of protective proteins, then in this extremely thermophilic archaebacterium it depends primarily on one protein.« less
Acquired thermotolerance and heat shock in the extremely thermophilic archaebacterium Sulfolobus sp. strain B12.

PubMed

Trent, J D; Osipiuk, J; Pinkau, T

1990-03-01

The extreme thermophile Sulfolobus sp. strain B12 exhibits an acquired thermotolerance response. Thus, survival of cells from a 70 degrees C culture at the lethal temperature of 92 degrees C was enhanced by as much as 6 orders of magnitude over a 2-h period if the culture was preheated to 88 degrees C for 60 min or longer before being exposed to the lethal temperature. In eubacteria and eucaryotes, acquired thermotolerance correlates with the induced synthesis of a dozen or so proteins known as heat shock proteins. In this Sulfolobus species, it correlates with the preferential synthesis of primarily one major protein (55 kilodaltons) and, to a much lesser extent, two minor proteins (28 and 35 kilodaltons). Since the synthesis of all other proteins was radically reduced and these proteins were apparently not degraded or exported, their relative abundance within the cell increased during the time the cells were becoming thermotolerant. They could not yet be related to known heat shock proteins. In immunoassays, they were not cross-reactive with antibodies against heat shock proteins from Escherichia coli (DnaK and GroE), which are highly conserved between eubacteria and eucaryotes. However, it appears that if acquired thermotolerance depends on the synthesis of protective proteins, then in this extremely thermophilic archaebacterium it depends primarily on one protein.

Characterization of a 29.4-kilodalton structural protein of Giardia lamblia and localization to the ventral disk [corrected

PubMed Central

Aggarwal, A; Adam, R D; Nash, T E

1989-01-01

The amino acid sequence of a 29.4-kilodalton [corrected] structural protein located in the ventral disk and axostyle of Giardia lamblia was determined. Clone lambda M16 from a mung bean expression library in lambda gt11 expressed a fusion protein recognized by three different isolate-specific antisera and sera from G. lamblia-infected gerbils. One of the three EcoRI fragments (M16; 1.26 kilobases) encoded the recognized protein. Sequence analysis revealed a single open reading frame of 813 base pairs. Two areas showed conservation of the positions of some amino acids. The abundance of arginine, glutamic acid, and threonine was increased. Two potential alpha-helical regions were deduced in the regions of repeats. Antisera to the M16 fusion protein reacted specifically with internal components of the ventral disk and axostyle, as well as Giardia fractions enriched for ventral disk structural proteins. An identical protein was recognized in different isolates by anti-M16, and a single identical band was recognized in Southern blots using the M16 1.26-kilobase fragment as a probe. Therefore, the 29.4-kilodaltion [corrected] protein appears to be highly conserved compared with variant surface proteins. Images PMID:2925253
Identification of the triazine receptor protein as a chloroplast gene product

PubMed Central

Steinback, Katherine E.; McIntosh, Lee; Bogorad, Lawrence; Arntzen, Charles J.

1981-01-01

The triazine herbicides inhibit photosynthesis by blocking electron transport at the second stable electron acceptor of photosystem II. This electron transport component of chloroplast thylakoid membranes is a protein-plastoquinone complex termed “B.” The polypeptide that is believed to be a component of the B complex has recently been identified as a 32- to 34-kilo-dalton polypeptide by using a photoaffinity labeling probe, azido-[14C]atrazine. A 34-kilodalton polypeptide of pea chloroplasts rapidly incorporates [35S]methionine in vivo and is also a rapidly labeled product of chloroplast-directed protein synthesis. Trypsin treatment of membranes tagged with azido-[14C]atrazine, [35S]methionine in vivo, or [35S]methionine in isolated intact chloroplasts results in identical, sequential alterations of the 34-kilo-dalton polypeptide to species of 32, then 18 and 16 kilodaltons. From the identical pattern of susceptibility to trypsin we conclude that the rapidly synthesized 34-kilodalton polypeptide that is a product of chloroplast-directed protein synthesis is identical to the triazine herbicide-binding protein of photosystem II. Chloroplasts of both triazine-susceptible and triazine-resistant biotypes of Amaranthus hybridus synthesize the 34-kilodalton polypeptide, but that of the resistant biotype does not bind the herbicide. Images PMID:16593133
Identification of the 64 kilodalton chloroplast stromal phosphoprotein as phosphoglucomutase. [Pisum sativum

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

Salvucci, M.E.; Drake, R.R.; Broadbent, K.P.

1990-05-01

Phosphorylation of the 64 kilodalton stromal phosphoprotein by incubation of pea (Pisum sativum) chloroplast extracts with ({gamma}-{sup 32}P)ATP decreased in the presence of Glc-6-P and Glc-1,6-P{sub 2}, but was stimulated by glucose. Two-dimensional gel electrophoresis following incubation of intact chloroplasts and stromal extracts with ({gamma}-{sup 32}P)ATP, or incubation of stromal extracts and partially purified phosphoglucomutase (EC 2.7.5.1) with ({sup 32}P)Glc-1-P showed that the identical 64 kilodalton polypeptide was labeled. A 62 kilodalton polypeptide was phosphorylated by incubation of tobacco (Nicotiana sylvestris) stromal extracts with either ({gamma}-{sup 32}P)ATP or ({sup 32}P)Glc-1-P. In contrast, an analogous polypeptide was not phosphorylated in extractsmore » from a tobacco mutant deficient in plastid phosphoglucomutase activity. The results indicate that the 64 (or 62) kilodalton chloroplast stromal phosphoprotein is phosphoglucomutase.« less
Bombyx mori nucleopolyhedrovirus nucleic acid binding proteins BRO-B and BRO-E associate with host T-cell intracellular antigen 1 homologue BmTRN-1 to influence protein synthesis during infection.

PubMed

Kotani, Eiji; Muto, Sayaka; Ijiri, Hiroshi; Mori, Hajime

2015-07-01

Previous reports have indicated that the Bombyx mori nucleopolyhedrovirus (BmNPV) nucleic acid binding proteins BRO-B and BRO-E are expressed during the early stage of infection and that the BRO family likely supports the regulation of mRNA; however, no study has directly examined the function of BRO family proteins in virus-permissive cells. Here, we show that BRO-B and BRO-E associate with cellular T-cell intracellular antigen 1 homologue (BmTRN-1), a translational regulator, and other cellular translation-related proteins in silkworm cells during viral infection. We created BM-N cells that expressed BRO-B/E to study molecular interactions between BmTRN-1 and BRO-B/E and how they influenced protein synthesis. Fluorescent microscopy revealed that BmTRN-1 was localized in cytoplasmic foci during BmNPV infection. Immunofluorescence studies confirmed that BmTRN-1 and BRO-B/E were colocalized in the amorphous conspicuous cytoplasmic foci. Reporter gene studies revealed that co-expression of BRO-B/E synergistically led to a significant decrease in protein synthesis from a designed transcript carrying the 5'untranslated region of a cellular mRNA with no significant change of transcript abundance. Additionally, RNA interference-mediated knockdown of BmTRN-1 resulted in a marked inhibition of the ability of BRO-B/E to regulate the transcript. These results suggested that the association of BmTRN-1 with BRO-B/E is responsible for the inhibitory regulation of certain mRNAs at the post-transcriptional level and add an additional mechanism for how baculoviruses control protein synthesis during infection.
Resistance of Adenoviral DNA Replication to Aphidicolin Is Dependent on the 72-Kilodalton DNA-Binding Protein

PubMed Central

Foster, David A.; Hantzopoulos, Petros; Zubay, Geoffrey

1982-01-01

Aphidicolin is a highly specific inhibitor of DNA polymerase α and has been most useful for assessing the role of this enzyme in various replication processes (J. A. Huberman, Cell 23:647-648, 1981). Both nuclear DNA replication and simian virus 40 DNA replication are highly sensitive to this drug (Krokan et al., Biochemistry 18:4431-4443, 1979), whereas mitochondrial DNA synthesis is completely insensitive (Zimmerman et al., J. Biol. Chem. 255:11847-11852, 1980). Adenovirus DNA replication is sensitive to aphidicolin, but only at much higher concentrations. These patterns of sensitivity are seen both in vivo and in vitro (Krokan et al., Biochemistry 18:4431-4443, 1979). A temperature-sensitive mutant of adenovirus type 5 known as H5ts125 is able to complete but not initiate new rounds of replication at nonpermissive temperatures (P. C. van der Vliet and J. S. Sussenbach, Virology 67:415-426, 1975). When cells infected with H5ts125 were shifted from permissive (33°C) to nonpermissive (41°C) conditions, the residual DNA synthesis (elongation) showed a striking increase in sensitivity to aphidicolin. The temperature-sensitive mutation of H5ts125 is in the gene for the 72-kilodalton single-stranded DNA-binding protein. This demonstrated that the increased resistance to aphidicolin shown by adenovirus DNA replication was dependent on that protein. It also supports an elongation role for both DNA polymerase α and the 72-kilodalton single-stranded DNA-binding protein in adenovirus DNA replication. Further support for an elongation role of DNA polymerase α came from experiments with permissive temperature conditions and inhibiting levels of aphidicolin in which it was shown that newly initiated strands failed to elongate to completion. Images PMID:6809958
Tumor-specific cytolysis caused by an E1B55K-attenuated adenovirus in nasopharyngeal carcinoma is augmented by cisplatin.

PubMed

Liu, Ran-Yi; Peng, Ji-Lin; Li, Yong-Qiang; Huang, Bi-Jun; Lin, Huan-Xin; Zhou, Ling; Luo, Hui-Ling; Huang, Wenlin

2013-12-01

An E1B55K-attenuated adenovirus, dl1520, has been shown to replicate selectively in and lyse tumor cells. In this study, the antitumor activities of dl1520, alone or in combination with the chemotherapeutic agent cisplatin, were investigated in nasopharyngeal carcinoma (NPC) cells. The results demonstrated that dl1520 replicated in and destroyed NPC cells, and induced apoptosis in vitro. In a nude mouse xenograft model, dl1520 significantly inhibited the growth of NPC cell xenografts, and the viral replication was associated with tumor regression. Importantly, the antitumor activity of dl1520 was augmented by the addition of cisplatin both in vitro and in vivo, showing that dl1520 and cisplatin have a synergistic anti-NPC effect. These data suggest that dl1520 exerts an efficient anti-NPC activity through oncolysis and the induction of apoptosis, which is enhanced synergistically by cisplatin. These findings indicate that oncolytic viral therapeutics using the E1B55K-attenuated adenovirus dl1520 could be promising in the comprehensive treatment of NPC, especially in combination with platinum-based chemotherapy. Copyright © 2013 Wiley Periodicals, Inc.
PDZ Protein Regulation of G Protein-Coupled Receptor Trafficking and Signaling Pathways.

PubMed

Dunn, Henry A; Ferguson, Stephen S G

2015-10-01

G protein-coupled receptors (GPCRs) contribute to the regulation of every aspect of human physiology and are therapeutic targets for the treatment of numerous diseases. As a consequence, understanding the myriad of mechanisms controlling GPCR signaling and trafficking is essential for the development of new pharmacological strategies for the treatment of human pathologies. Of the many GPCR-interacting proteins, postsynaptic density protein of 95 kilodaltons, disc large, zona occludens-1 (PDZ) domain-containing proteins appear most abundant and have similarly been implicated in disease mechanisms. PDZ proteins play an important role in regulating receptor and channel protein localization within synapses and tight junctions and function to scaffold intracellular signaling protein complexes. In the current study, we review the known functional interactions between PDZ domain-containing proteins and GPCRs and provide insight into the potential mechanisms of action. These PDZ domain-containing proteins include the membrane-associated guanylate-like kinases [postsynaptic density protein of 95 kilodaltons; synapse-associated protein of 97 kilodaltons; postsynaptic density protein of 93 kilodaltons; synapse-associated protein of 102 kilodaltons; discs, large homolog 5; caspase activation and recruitment domain and membrane-associated guanylate-like kinase domain-containing protein 3; membrane protein, palmitoylated 3; calcium/calmodulin-dependent serine protein kinase; membrane-associated guanylate kinase protein (MAGI)-1, MAGI-2, and MAGI-3], Na(+)/H(+) exchanger regulatory factor proteins (NHERFs) (NHERF1, NHERF2, PDZ domain-containing kidney protein 1, and PDZ domain-containing kidney protein 2), Golgi-associated PDZ proteins (Gα-binding protein interacting protein, C-terminus and CFTR-associated ligand), PDZ domain-containing guanine nucleotide exchange factors (GEFs) 1 and 2, regulator of G protein signaling (RGS)-homology-RhoGEFs (PDZ domain-containing RhoGEF and
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.
Human papillomavirus E6 protein enriches the CD55(+) population in cervical cancer cells, promoting radioresistance and cancer aggressiveness.

PubMed

Leung, Thomas Ho-Yin; Tang, Hermit Wai-Man; Siu, Michelle Kwan-Yee; Chan, David Wai; Chan, Karen Kar-Loen; Cheung, Annie Nga-Yin; Ngan, Hextan Yuen-Sheung

2018-02-01

Accumulating evidence indicates that the human papillomavirus (HPV) E6 protein plays a crucial role in the development of cervical cancer. Subpopulations of cells that reside within tumours are responsible for tumour resistance to cancer therapy and recurrence. However, the identity of such cells residing in cervical cancer and their relationship with the HPV-E6 protein have not been identified. Here, we isolated sphere-forming cells, which showed self-renewal ability, from primary cervical tumours. Gene expression profiling revealed that cluster of differentiation (CD) 55 was upregulated in primary cervical cancer sphere cells. Flow-cytometric analysis detected abundant CD55(+) populations among a panel of HPV-positive cervical cancer cell lines, whereas few CD55(+) cells were found in HPV-negative cervical cancer and normal cervical epithelial cell lines. The CD55(+) subpopulation isolated from the C33A cell line showed significant sphere-forming ability and enhanced tumourigenicity, cell migration, and radioresistance. In contrast, the suppression of CD55 in HPV-positive CaSki cells inhibited tumourigenicity both in vitro and in vivo, and sensitized cells to radiation treatment. In addition, ectopic expression of the HPV-E6 protein in HPV-negative cervical cancer cells dramatically enriched the CD55(+) subpopulation. CRISPR/Cas9 knockout of CD55 in an HPV-E6-overexpressing stable clone abolished the tumourigenic effects of the HPV-E6 protein. Taken together, our data suggest that HPV-E6 protein expression enriches the CD55(+) population, which contributes to tumourigenicity and radioresistance in cervical cancer cells. Targeting CD55 via CRISPR/Cas9 may represent a novel avenue for developing new strategies and effective therapies for the treatment of cervical cancer. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John
A 90-Kilodalton Endothelial Cell Molecule Mediating Lymphocyte Binding in Humans

NASA Astrophysics Data System (ADS)

Salmi, Marko; Jalkanen, Sirpa

1992-09-01

Interactions between leukocyte surface receptors and their ligands on vascular endothelial cells control lymphocyte traffic between the blood and various lymphoid organs, as well as extravasation of leukocytes into sites of inflammation. A heretofore undescribed 90-kilodalton human endothelial cell adhesion molecule (VAP-1) defined by a monoclonal antibody 1B2 is described. The expression pattern, molecular mass, functional properties, and an amino-terminal amino acid sequence define VAP-1 as an endothelial ligand for lymphocytes. VAP-1 helps to elucidate the complex heterotypic cell interactions that direct tissue-selective lymphocyte migration in man.
RNA Binding Protein RBM38 Regulates Expression of the 11-Kilodalton Protein of Parvovirus B19, Which Facilitates Viral DNA Replication.

PubMed

Ganaie, Safder S; Chen, Aaron Yun; Huang, Chun; Xu, Peng; Kleiboeker, Steve; Du, Aifang; Qiu, Jianming

2018-04-15

Human parvovirus B19 (B19V) expresses a single precursor mRNA (pre-mRNA), which undergoes alternative splicing and alternative polyadenylation to generate 12 viral mRNA transcripts that encode two structural proteins (VP1 and VP2) and three nonstructural proteins (NS1, 7.5-kDa protein, and 11-kDa protein). Splicing at the second 5' donor site (D2 site) of the B19V pre-mRNA is essential for the expression of VP2 and the 11-kDa protein. We previously identified that cis -acting intronic splicing enhancer 2 (ISE2) that lies immediately after the D2 site facilitates the recognition of the D2 donor for its efficient splicing. In this study, we report that ISE2 is critical for the expression of the 11-kDa viral nonstructural protein. We found that ISE2 harbors a consensus RNA binding motif protein 38 (RBM38) binding sequence, 5'-UGUGUG-3'. RBM38 is expressed during the middle stage of erythropoiesis. We first confirmed that RBM38 binds specifically with the ISE2 element in vitro The knockdown of RBM38 significantly decreases the level of spliced mRNA at D2 that encodes the 11-kDa protein but not that of the D2-spliced mRNA that encodes VP2. Importantly, we found that the 11-kDa protein enhances viral DNA replication and virion release. Accordingly, the knockdown of RBM38 decreases virus replication via downregulating 11-kDa protein expression. Taken together, these results suggest that the 11-kDa protein facilitates B19V DNA replication and that RBM38 is an essential host factor for B19V pre-mRNA splicing and for the expression of the 11-kDa protein. IMPORTANCE B19V is a human pathogen that can cause fifth disease, arthropathy, anemia in immunocompromised patients and sickle cell disease patients, myocarditis, and hydrops fetalis in pregnant women. Human erythroid progenitor cells (EPCs) are most susceptible to B19V infection and fully support viral DNA replication. The exclusive tropism of B19V for erythroid-lineage cells is dependent not only on the expression of viral
Adenovirus E1A and E1B-19K Proteins Protect Human Hepatoma Cells from Transforming Growth Factor β1-induced Apoptosis

PubMed Central

Tarakanova, Vera L.; Wold, William S. M.

2009-01-01

Primary and some transformed hepatocytes undergo apoptosis in response to transforming growth factor β1 (TGFβ). We report that infection with species C human adenovirus conferred resistance to TGFβ-induced apoptosis in human hepatocellular carcinoma cells (Huh-7). Protection against TGFβ-mediated cell death in adenovirus-infected cells correlated with the maintenance of normal nuclear morphology, lack of pro-caspases 8 and 3 processing, maintenance of the mitochondrial membrane potential, and lack of cellular DNA degradation. The TGFβ pro-apoptotic signaling pathway was blocked upstream of mitochondria in adenovirus-infected cells. Both the N-terminal sequences of the E1A proteins and the E1B-19K protein were necessary to protect infected cells against TGFβ-induced apoptosis. PMID:19854227
Insulin stimulates the tyrosine phosphorylation of a 61-kilodalton protein in rat adipocytes.

PubMed

Mooney, R A; Bordwell, K L

1992-03-01

Insulin stimulated the tyrosine phosphorylation of a 61-kilodalton (kDa) protein in rat adipocytes prelabeled for 2 h with [32P]orthophosphate. Tyrosine phosphorylation of this 61-kDa protein displayed very similar insulin concentration dependency to receptor autophosphorylation and tyrosine phosphorylation of a high molecular mass receptor substrate of 160 kDa. Phosphorylation of the 61-kDa protein was very rapid with maximum labeling attained at 30 sec, paralleling that of the other two proteins. Phosphoamino acid analysis revealed that each of the insulin-responsive phosphoproteins contained phosphoserine as well as phosphotyrosine, though the ratio of two phosphoamino acids recovered from each protein differed. The 61-kDa protein yielded relatively equal proportions of phosphoserine and phosphotyrosine. In contrast, the insulin receptor yielded relatively more label on phosphotyrosine than phosphoserine, whereas label incorporated into the 160-kDa protein was recovered primarily on phosphoserine. Cleveland peptide maps using either Staphylococcus aureus V8 proteinase or chymotrypsin revealed no similarities between the 61-kDa protein and the other tyrosine phosphorylated proteins. With subcellular fractionation, the 160-kDa protein was found in equal proportions in the high speed pellet (100,000 g) and supernatant. The 61-kDa protein had a similar distribution to that of the 160-kDa protein but was also detected in the low speed pellet (10,000 g). The insulin receptor was localized to the low speed pellet. In summary, rat adipocytes contain an insulin-dependent phosphotyrosyl protein of 61 kDa which is distinct from the more prominent high molecular mass receptor substrate. This 61-kDa protein has characteristics consistent with it being a substrate for the insulin receptor tyrosine kinase.
Identifying the substrate proteins of U-box E3s E4B and CHIP by orthogonal ubiquitin transfer.

PubMed

Bhuripanyo, Karan; Wang, Yiyang; Liu, Xianpeng; Zhou, Li; Liu, Ruochuan; Duong, Duc; Zhao, Bo; Bi, Yingtao; Zhou, Han; Chen, Geng; Seyfried, Nicholas T; Chazin, Walter J; Kiyokawa, Hiroaki; Yin, Jun

2018-01-01

E3 ubiquitin (UB) ligases E4B and carboxyl terminus of Hsc70-interacting protein (CHIP) use a common U-box motif to transfer UB from E1 and E2 enzymes to their substrate proteins and regulate diverse cellular processes. To profile their ubiquitination targets in the cell, we used phage display to engineer E2-E4B and E2-CHIP pairs that were free of cross-reactivity with the native UB transfer cascades. We then used the engineered E2-E3 pairs to construct "orthogonal UB transfer (OUT)" cascades so that a mutant UB (xUB) could be exclusively used by the engineered E4B or CHIP to label their substrate proteins. Purification of xUB-conjugated proteins followed by proteomics analysis enabled the identification of hundreds of potential substrates of E4B and CHIP in human embryonic kidney 293 cells. Kinase MAPK3 (mitogen-activated protein kinase 3), methyltransferase PRMT1 (protein arginine N -methyltransferase 1), and phosphatase PPP3CA (protein phosphatase 3 catalytic subunit alpha) were identified as the shared substrates of the two E3s. Phosphatase PGAM5 (phosphoglycerate mutase 5) and deubiquitinase OTUB1 (ovarian tumor domain containing ubiquitin aldehyde binding protein 1) were confirmed as E4B substrates, and β-catenin and CDK4 (cyclin-dependent kinase 4) were confirmed as CHIP substrates. On the basis of the CHIP-CDK4 circuit identified by OUT, we revealed that CHIP signals CDK4 degradation in response to endoplasmic reticulum stress.
Identifying the substrate proteins of U-box E3s E4B and CHIP by orthogonal ubiquitin transfer

PubMed Central

Bhuripanyo, Karan; Wang, Yiyang; Liu, Xianpeng; Zhou, Li; Liu, Ruochuan; Duong, Duc; Zhao, Bo; Bi, Yingtao; Zhou, Han; Chen, Geng; Seyfried, Nicholas T.; Chazin, Walter J.; Kiyokawa, Hiroaki; Yin, Jun

2018-01-01

E3 ubiquitin (UB) ligases E4B and carboxyl terminus of Hsc70-interacting protein (CHIP) use a common U-box motif to transfer UB from E1 and E2 enzymes to their substrate proteins and regulate diverse cellular processes. To profile their ubiquitination targets in the cell, we used phage display to engineer E2-E4B and E2-CHIP pairs that were free of cross-reactivity with the native UB transfer cascades. We then used the engineered E2-E3 pairs to construct “orthogonal UB transfer (OUT)” cascades so that a mutant UB (xUB) could be exclusively used by the engineered E4B or CHIP to label their substrate proteins. Purification of xUB-conjugated proteins followed by proteomics analysis enabled the identification of hundreds of potential substrates of E4B and CHIP in human embryonic kidney 293 cells. Kinase MAPK3 (mitogen-activated protein kinase 3), methyltransferase PRMT1 (protein arginine N-methyltransferase 1), and phosphatase PPP3CA (protein phosphatase 3 catalytic subunit alpha) were identified as the shared substrates of the two E3s. Phosphatase PGAM5 (phosphoglycerate mutase 5) and deubiquitinase OTUB1 (ovarian tumor domain containing ubiquitin aldehyde binding protein 1) were confirmed as E4B substrates, and β-catenin and CDK4 (cyclin-dependent kinase 4) were confirmed as CHIP substrates. On the basis of the CHIP-CDK4 circuit identified by OUT, we revealed that CHIP signals CDK4 degradation in response to endoplasmic reticulum stress. PMID:29326975
Mitotic MELK-eIF4B signaling controls protein synthesis and tumor cell survival

PubMed Central

Wang, Yubao; Begley, Michael; Li, Qing; Huang, Hai-Tsang; Lako, Ana; Eck, Michael J.; Gray, Nathanael S.; Mitchison, Timothy J.; Cantley, Lewis C.; Zhao, Jean J.

2016-01-01

The protein kinase maternal and embryonic leucine zipper kinase (MELK) is critical for mitotic progression of cancer cells; however, its mechanisms of action remain largely unknown. By combined approaches of immunoprecipitation/mass spectrometry and peptide library profiling, we identified the eukaryotic translation initiation factor 4B (eIF4B) as a MELK-interacting protein during mitosis and a bona fide substrate of MELK. MELK phosphorylates eIF4B at Ser406, a modification found to be most robust in the mitotic phase of the cell cycle. We further show that the MELK–eIF4B signaling axis regulates protein synthesis during mitosis. Specifically, synthesis of myeloid cell leukemia 1 (MCL1), an antiapoptotic protein known to play a role in cancer cell survival during cell division, depends on the function of MELK-elF4B. Inactivation of MELK or eIF4B results in reduced protein synthesis of MCL1, which, in turn, induces apoptotic cell death of cancer cells. Our study thus defines a MELK–eIF4B signaling axis that regulates protein synthesis during mitosis, and consequently influences cancer cell survival. PMID:27528663
A multicomponent complex is required for the AAUAAA-dependent cross-linking of a 64-kilodalton protein to polyadenylation substrates.

PubMed Central

Wilusz, J; Shenk, T; Takagaki, Y; Manley, J L

1990-01-01

A 64-kilodalton (kDa) polypeptide that is cross-linked by UV light specifically to polyadenylation substrate RNAs containing a functional AAUAAA element has been identified previously. Fractionated HeLa nuclear components that can be combined to regenerate efficient and accurate polyadenylation in vitro have now been screened for the presence of the 64-kDa protein. None of the individual components contained an activity which could generate the 64-kDa species upon UV cross-linking in the presence of substrate RNA. It was necessary to mix two components, cleavage stimulation factor and specificity factor, to reconstitute 64-kDa protein-RNA cross-linking. The addition of cleavage factors to this mixture very efficiently reconstituted the AAUAAA-specific 64-kDa protein-RNA interaction. The 64-kDa protein, therefore, is present in highly purified, reconstituted polyadenylation reactions. However, it is necessary to form a multicomponent complex to efficiently cross-link the protein to a substrate RNA. Images PMID:2304466
Cannabinoid Modulation of Eukaryotic Initiation Factors (eIF2α and eIF2B1) and Behavioral Cross-Sensitization to Cocaine in Adolescent Rats.

PubMed

Melas, Philippe A; Qvist, Johanna S; Deidda, Matteo; Upreti, Chirag; Wei, Ya Bin; Sanna, Fabrizio; Fratta, Walter; Scherma, Maria; Fadda, Paola; Kandel, Denise B; Kandel, Eric R

2018-03-13

Reduced eukaryotic Initiation Factor 2 (eIF2)α phosphorylation (p-eIF2α) enhances protein synthesis, memory formation, and addiction-like behaviors. However, p-eIF2α has not been examined with regard to psychoactive cannabinoids and cross-sensitization. Here, we find that a cannabinoid receptor agonist (WIN 55,212-2 mesylate [WIN]) reduced p-eIF2α in vitro by upregulating GADD34 (PPP1R15A), the recruiter of protein phosphatase 1 (PP1). The induction of GADD34 was linked to ERK/CREB signaling and to CREB-binding protein (CBP)-mediated histone hyperacetylation at the Gadd34 locus. In vitro, WIN also upregulated eIF2B1, an eIF2 activator subunit. We next found that WIN administration in vivo reduced p-eIF2α in the nucleus accumbens of adolescent, but not adult, rats. By contrast, WIN increased dorsal striatal levels of eIF2B1 and ΔFosB among both adolescents and adults. In addition, we found cross-sensitization between WIN and cocaine only among adolescents. These findings show that cannabinoids can modulate eukaryotic initiation factors, and they suggest a possible link between p-eIF2α and the gateway drug properties of psychoactive cannabinoids. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.
55. BOILER CHAMBER No. 1, LOOP B, STEAM DRUM AND ...

Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

55. BOILER CHAMBER No. 1, LOOP B, STEAM DRUM AND DOWNCOMERS LOOKING EAST (LOCATION LLL) - Shippingport Atomic Power Station, On Ohio River, 25 miles Northwest of Pittsburgh, Shippingport, Beaver County, PA
Biosynthesis of Nonspecific Lipid Transfer Proteins in Germinating Castor Bean Seeds 1

PubMed Central

Tsuboi, Shigeru; Watanabe, Shin-ichiro; Ozeki, Yoshihiro; Yamada, Mitsuhiro

1989-01-01

The biosynthesis of nonspecific lipid transfer proteins (ns-LTPs) in germinating castor bean (Ricinus communis L.) seeds were investigated. Lipid transfer activities of ns-LTPs in the cotyledons, axis, and endosperm increased with growth after germination. The activity increases were accompanied by increased amounts of ns-LTPs in each tissue, as measured by immunoblot using anti-ns-LTP serum. These results suggest that the ns-LTPs are synthesized de novo in each tissue after germination and not activated from inactive proteins synthesized before germination. Comparison of the immunoblot products in each tissue from 4-day-old seedlings indicate the occurrence of tissue-specific isoforms of ns-LTPs; 9 kilodaltons (major) and 7 kilodaltons (minor) in the cotyledons, and 7 kilodaltons (major) and 9 kilodaltons (minor) in the axis, whereas only the 8-kilodalton ns-LTP is present in the endosperm. In vitro translation from poly(A)+ RNAs from three tissues of castor bean seedlings and the detection of immunoprecipitated products indicate that translatable mRNAs for ns-LTPs exist in the three tissues a day before the synthesis of ns-LTPs; the translation products, which are 3.5 to 4.0 kilodaltons larger than ns-LTPs, were processed to the mature ns-LTPs. The production of mature ns-LTPs from translatable mRNAs without any delay suggests that gene expression of ns-LTPs in castor bean seedlings is controlled at a step before the formation of translatable mRNAs. Images Figure 3 Figure 4 Figure 5 PMID:16666886

Live-cell imaging RNAi screen identifies PP2A–B55α and importin-β1 as key mitotic exit regulators in human cells

PubMed Central

Schmitz, Michael H. A.; Held, Michael; Janssens, Veerle; Hutchins, James R. A.; Hudecz, Otto; Ivanova, Elitsa; Goris, Jozef; Trinkle-Mulcahy, Laura; Lamond, Angus I.; Poser, Ina; Hyman, Anthony A.; Mechtler, Karl; Peters, Jan-Michael; Gerlich, Daniel W.

2013-01-01

When vertebrate cells exit mitosis various cellular structures are re-organized to build functional interphase cells1. This depends on Cdk1 (cyclin dependent kinase 1) inactivation and subsequent dephosphorylation of its substrates2–4. Members of the protein phosphatase 1 and 2A (PP1 and PP2A) families can dephosphorylate Cdk1 substrates in biochemical extracts during mitotic exit5,6, but how this relates to postmitotic reassembly of interphase structures in intact cells is not known. Here, we use a live-cell imaging assay and RNAi knockdown to screen a genome-wide library of protein phosphatases for mitotic exit functions in human cells. We identify a trimeric PP2A–B55α complex as a key factor in mitotic spindle breakdown and postmitotic reassembly of the nuclear envelope, Golgi apparatus and decondensed chromatin. Using a chemically induced mitotic exit assay, we find that PP2A–B55α functions downstream of Cdk1 inactivation. PP2A–B55α isolated from mitotic cells had reduced phosphatase activity towards the Cdk1 substrate, histone H1, and was hyper-phosphorylated on all subunits. Mitotic PP2A complexes co-purified with the nuclear transport factor importin-β1, and RNAi depletion of importin-β1 delayed mitotic exit synergistically with PP2A–B55α. This demonstrates that PP2A–B55α and importin-β1 cooperate in the regulation of postmitotic assembly mechanisms in human cells. PMID:20711181
Sex pheromone receptor proteins. Visualization using a radiolabeled photoaffinity analog

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

Vogt, R.G.; Prestwich, G.D.; Riddiford, L.M.

1988-03-15

A tritium-labeled photoaffinity analog of a moth pheromone was used to covalently modify pheromone-selective binding proteins in the antennal sensillum lymph and sensory dendritic membranes of the male silk moth, Antheraea polyphemus. This analog, (E,Z)-6,11-(/sup 3/H)hexadecadienyl diazoacetate, allowed visualization of a 15-kilodalton soluble protein and a 69-kilodalton membrane protein in fluorescence autoradiograms of electrophoretically separated antennal proteins. Covalent modification of these proteins was specifically reduced when incubation and UV irradiation were conducted in the presence of excess unlabeled pheromone, (E,Z)-6,11-hexadecadienyl acetate. These experiments constitute the first direct evidence for a membrane protein of a chemosensory neuron interacting in a specificmore » fashion with a biologically relevant odorant.« less
The B55α Regulatory Subunit of Protein Phosphatase 2A Mediates Fibroblast Growth Factor-Induced p107 Dephosphorylation and Growth Arrest in Chondrocytes

PubMed Central

Daempfling, Lea

2013-01-01

Fibroblast growth factor (FGF)-induced growth arrest of chondrocytes is a unique cell type-specific response which contrasts with the proliferative response of most cell types and underlies several genetic skeletal disorders caused by activating FGF receptor (FGFR) mutations. We have shown that one of the earliest key events in FGF-induced growth arrest is dephosphorylation of the retinoblastoma protein (Rb) family member p107 by protein phosphatase 2A (PP2A), a ubiquitously expressed multisubunit phosphatase. In this report, we show that the PP2A-B55α holoenzyme (PP2A containing the B55α subunit) is responsible for this phenomenon. Only the B55α (55-kDa regulatory subunit, alpha isoform) regulatory subunit of PP2A was able to bind p107, and this interaction was induced by FGF in chondrocytes but not in other cell types. Small interfering RNA (siRNA)-mediated knockdown of B55α prevented p107 dephosphorylation and FGF-induced growth arrest of RCS (rat chondrosarcoma) chondrocytes. Importantly, the B55α subunit bound with higher affinity to dephosphorylated p107. Since the p107 region interacting with B55α is also the site of cyclin-dependent kinase (CDK) binding, B55α association may also prevent p107 phosphorylation by CDKs. FGF treatment induces dephosphorylation of the B55α subunit itself on several serine residues that drastically increases the affinity of B55α for the PP2A A/C dimer and p107. Together these observations suggest a novel mechanism of p107 dephosphorylation mediated by activation of PP2A through B55α dephosphorylation. This mechanism might be a general signal transduction pathway used by PP2A to initiate cell cycle arrest when required by external signals. PMID:23716589
Overproduction, purification, and ATPase activity of the Escherichia coli RuvB protein involved in DNA repair.

PubMed Central

Iwasaki, H; Shiba, T; Makino, K; Nakata, A; Shinagawa, H

1989-01-01

The ruvA and ruvB genes of Escherichia coli constitute an operon which belongs to the SOS regulon. Genetic evidence suggests that the products of the ruv operon are involved in DNA repair and recombination. To begin biochemical characterization of these proteins, we developed a plasmid system that overproduced RuvB protein to 20% of total cell protein. Starting from the overproducing system, we purified RuvB protein. The purified RuvB protein behaved like a monomer in gel filtration chromatography and had an apparent relative molecular mass of 38 kilodaltons in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, which agrees with the value predicted from the DNA sequence. The amino acid sequence of the amino-terminal region of the purified protein was analyzed, and the sequence agreed with the one deduced from the DNA sequence. Since the deduced sequence of RuvB protein contained the consensus sequence for ATP-binding proteins, we examined the ATP-binding and ATPase activities of the purified RuvB protein. RuvB protein had a stronger affinity to ADP than to ATP and weak ATPase activity. The results suggest that the weak ATPase activity of RuvB protein is at least partly due to end product inhibition by ADP. Images PMID:2529252
Calcium Homeostasis and Muscle Energy Metabolism Are Modified in HspB1-Null Mice.

PubMed

Picard, Brigitte; Kammoun, Malek; Gagaoua, Mohammed; Barboiron, Christiane; Meunier, Bruno; Chambon, Christophe; Cassar-Malek, Isabelle

2016-05-04

Hsp27-encoded by HspB1- is a member of the small heat shock proteins (sHsp, 12-43 kDa (kilodalton)) family. This protein is constitutively present in a wide variety of tissues and in many cell lines. The abundance of Hsp27 is highest in skeletal muscle, indicating a crucial role for muscle physiology. The protein identified as a beef tenderness biomarker was found at a crucial hub in a functional network involved in beef tenderness. The aim of this study was to analyze the proteins impacted by the targeted invalidation of HspB1 in the Tibialis anterior muscle of the mouse . Comparative proteomics using two-dimensional gel electrophoresis revealed 22 spots that were differentially abundant between HspB1 -null mice and their controls that could be identified by mass spectrometry. Eighteen spots were more abundant in the muscle of the mutant mice, and four were less abundant. The proteins impacted by the absence of Hsp27 belonged mainly to calcium homeostasis (Srl and Calsq1), contraction (TnnT3), energy metabolism (Tpi1, Mdh1, PdhB, Ckm, Pygm, ApoA1) and the Hsp proteins family (HspA9). These data suggest a crucial role for these proteins in meat tenderization. The information gained by this study could also be helpful to predict the side effects of Hsp27 depletion in muscle development and pathologies linked to small Hsps.
Calcium Homeostasis and Muscle Energy Metabolism Are Modified in HspB1-Null Mice

PubMed Central

Picard, Brigitte; Kammoun, Malek; Gagaoua, Mohammed; Barboiron, Christiane; Meunier, Bruno; Chambon, Christophe; Cassar-Malek, Isabelle

2016-01-01

Hsp27—encoded by HspB1—is a member of the small heat shock proteins (sHsp, 12–43 kDa (kilodalton)) family. This protein is constitutively present in a wide variety of tissues and in many cell lines. The abundance of Hsp27 is highest in skeletal muscle, indicating a crucial role for muscle physiology. The protein identified as a beef tenderness biomarker was found at a crucial hub in a functional network involved in beef tenderness. The aim of this study was to analyze the proteins impacted by the targeted invalidation of HspB1 in the Tibialis anterior muscle of the mouse. Comparative proteomics using two-dimensional gel electrophoresis revealed 22 spots that were differentially abundant between HspB1-null mice and their controls that could be identified by mass spectrometry. Eighteen spots were more abundant in the muscle of the mutant mice, and four were less abundant. The proteins impacted by the absence of Hsp27 belonged mainly to calcium homeostasis (Srl and Calsq1), contraction (TnnT3), energy metabolism (Tpi1, Mdh1, PdhB, Ckm, Pygm, ApoA1) and the Hsp proteins family (HspA9). These data suggest a crucial role for these proteins in meat tenderization. The information gained by this study could also be helpful to predict the side effects of Hsp27 depletion in muscle development and pathologies linked to small Hsps. PMID:28248227
Structure of a Protein Phosphatase 2A Holoenzyme: Insights into B55-Mediated Tau Dephosphorylation

PubMed Central

Xu, Yanhui; Chen, Yu; Zhang, Ping; Jeffrey, Philip D.; Shi, Yigong

2009-01-01

Summary Protein phosphatase 2A (PP2A) regulates many essential aspects of cellular physiology. Members of the regulatory B/B55/PR55 family are thought to play a key role in the dephosphorylation of Tau, whose hyperphosphorylation contributes to Alzheimer's disease. The underlying mechanisms of the PP2A-Tau connection remain largely enigmatic. Here, we report the complete reconstitution of a Tau dephosphorylation assay and the crystal structure of a heterotrimeric PP2A holoenzyme involving the regulatory subunit Bα. We show that Bα specifically and markedly facilitates dephosphorylation of the phosphorylated Tau in our reconstituted assay. The Bα subunit comprises a seven-bladed β propeller, with an acidic, substrate-binding groove located in the center of the propeller. The β propeller latches onto the ridge of the PP2A scaffold subunit with the help of a protruding β hairpin arm. Structure-guided mutagenesis studies revealed the underpinnings of PP2A-mediated dephosphorylation of Tau. PMID:18922469
CaMKII-MEDIATED PHOSPHORYLATION OF THE BOMBYX MORI LIPID STORAGE DROPLET PROTEIN-1 (BmLsd1), AN INSECT PAT FAMILY PROTEIN, IS ESSENTIAL FOR SILKMOTH SEX PHEROMONE BIOSYNTHESIS

USDA-ARS?s Scientific Manuscript database

The structurally-related members of the PAT family of proteins, which are so name based on similarity amongst perilipin, adipophilin/adipocyte differentiation-related protein (ADRP), and tail-interacting protein of 47 kilodaltons (TIP47), are cytoplasmic lipid droplet (LD)-associated proteins charac...
Identification of two Th1 cell epitopes on the Babesia bovis-encoded 77-kilodalton merozoite protein (Bb-1) by use of truncated recombinant fusion proteins.

PubMed Central

Brown, W C; Zhao, S; Woods, V M; Tripp, C A; Tetzlaff, C L; Heussler, V T; Dobbelaere, D A; Rice-Ficht, A C

1993-01-01

Previous studies have demonstrated the serologic and T-cell immunogenicity for cattle of a recombinant form of the apical complex-associated 77-kDa merozite protein of Babesia bovis, designated Bb-1. The present study characterizes the immunogenic epitopes of the Bb-1 protein. A series of recombinant truncated fusion proteins spanning the majority of the Bb-1 protein were expressed in Escherichia coli, and their reactivities with bovine peripheral blood mononuclear cells and T-cell clones derived from B. bovis-immune cattle and with rabbit antibodies were determined. Lymphocytes from two immune cattle were preferentially stimulated by the N-terminal half of the Bb-1 protein (amino acids 23 to 266, termed Bb-1A), localizing the T-cell epitopes to the Bb-1A portion of the molecule. CD4+ T-cell clones derived by stimulation with the intact Bb-1 fusion protein were used to identify two T-cell epitopes in the Bb-1A protein, consisting of amino acids SVVLLSAFSGN VWANEAEVSQVVK and FSDVDKTKSTEKT (residues 23 to 46 and 82 to 94). In contrast, rabbit antiserum raised against the intact fusion protein reacted only with the C-terminal half of the protein (amino acids 267 to 499, termed Bb-1B), which contained 28 tandem repeats of the tetrapeptide PAEK or PAET. Biological assays and Northern (RNA) blot analyses for cytokines revealed that following activation with concanavalin A, T-cell clones reactive against the two Bb-1A epitopes produced interleukin-2, gamma interferon, and tumor necrosis factors beta and alpha, but not interleukin-4, suggesting that the Bb-1 antigen preferentially stimulates the Th1 subset of CD4+ T cells in cattle. The studies described here report for the first time the characterization, by cytokine production, of the Th1 subset of bovine T cells and show that, as in mice, protozoal antigens can induce Th1 cells in ruminants. This first demonstration of B. bovis-encoded Th1 cell epitopes provides a rationale for incorporation of all or part of the Bb-1
Multiple transcriptional regulatory domains in the human immunodeficiency virus type 1 long terminal repeat are involved in basal and E1A/E1B-induced promoter activity.

PubMed Central

Kliewer, S; Garcia, J; Pearson, L; Soultanakis, E; Dasgupta, A; Gaynor, R

1989-01-01

The human immunodeficiency virus (HIV) type 1 long terminal repeat (LTR) is the site of activation of the HIV tat protein. However, additional transactivators, such as the adenovirus E1A and herpesvirus ICPO proteins, have also been shown to be capable of activating the HIV LTR. Analysis of adenovirus mutants indicated that complete transactivation of the HIV LTR was dependent on both the E1A and E1B proteins. To determine which regions of the HIV LTR were important for complete E1A/E1B activation, a variety of oligonucleotide-directed mutations in HIV transcriptional regulatory domains were assayed both in vivo and in vitro. S1 nuclease analysis of RNA prepared after transfection of these HIV constructs into HeLa cells infected with wild-type adenovirus indicated that the enhancer, SP1, TATA, and a portion of the transactivation-responsive element were each required for complete E1A/E1B-mediated activation of the HIV LTR. These same promoter elements were required for both basal and E1A/E1B-induced levels of transcription in in vitro transcription reactions performed with cellular extracts prepared from cells infected with dl434, an E1A/E1B deletion mutant, or wild-type adenovirus. No mutations were found that reduced only E1A/E1B-induced expression without proportionally reducing basal levels of transcription, suggesting that E1A/E1B-mediated induction of the HIV LTR requires multiple promoter elements which are also required for basal transcriptional levels. Unlike activation by the tat protein, there was not a rigid dependence on maintenance of the transactivation-responsive stem base pairing for E1A/E1B-mediated activation either in vivo or in vitro, indicating that activation occurs by a mechanism distinct from that of tat induction. Images PMID:2529378
Lower growth temperature increases alternative pathway capacity and alternative oxidase protein in tobacco.

PubMed

Vanlerberghe, G C; McIntosh, L

1992-09-01

Suspension cells of NT1 tobacco (Nicotiana tabacum L. cv bright yellow) have been used to study the effect of growth temperature on the CN-resistant, salicylhydroxamic acid-sensitive alternative pathway of respiration. Mitochondria isolated from cells maintained at 30 degrees C had a low capacity to oxidize succinate via the alternative pathway, whereas mitochondria isolated from cells 24 h after transfer to 18 degrees C displayed, on average, a 5-fold increase in this capacity (from 7 to 32 nanoatoms oxygen per milligram protein per minute). This represented an increase in alternative pathway capacity from 18 to 45% of the total capacity of electron transport. This increased capacity was lost upon transfer of cells back to 30 degrees C. A monoclonal antibody to the terminal oxidase of the alternative pathway (the alternative oxidase) from Sauromatum guttatum (T.E. Elthon, R.L. Nickels, L. McIntosh [1989] Plant Physiology 89: 1311-1317) recognized a 35-kilodalton mitochondrial protein in tobacco. There was an excellent correlation between the capacity of the alternative path in isolated tobacco mitochondria and the levels of this 35-kilodalton alternative oxidase protein. Cycloheximide could inhibit both the increased level of the 35-kilodalton alternative oxidase protein and the increased alternative pathway capacity normally seen upon transfer to 18 degrees C. We conclude that transfer of tobacco cells to the lower temperature increases the capacity of the alternative pathway due, at least in part, to de novo synthesis of the 35-kilodalton alternative oxidase protein.
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.
Oncogenic B-Raf(V600E) abrogates the AKT/B-Raf/Mps1 interaction in melanoma cells.

PubMed

Zhang, Ling; Shi, Ruyi; He, Chanting; Cheng, Caixia; Song, Bin; Cui, Heyang; Zhang, Yanyan; Zhao, Zhiping; Bi, Yanghui; Yang, Xiaofeng; Miao, Xiaoping; Guo, Jiansheng; Chen, Xing; Wang, Jinfen; Li, Yaoping; Cheng, Xiaolong; Liu, Jing; Cui, Yongping

2013-08-28

Activating B-Raf mutations that deregulate the mitogen-activated protein kinase (MAPK) pathway commonly occur in cancer. Although B-Raf(V600E) induces increased Mps1 protein contributing to centrosome amplification and chromosome instability, the regulatory mechanisms of Mps1 in melanoma cells is not fully understood. Here, we report that Mps1/AKT and B-Raf(WT)/ERK signaling form an auto-regulatory negative feedback loop in melanoma cells; notably, oncogenic B-Raf(V600E) abrogates the negative feedback loop, contributing the aberrant Mps1 functions and tumorigenesis. Our findings raise the possibility that targeting the oncogenic B-Raf and Mps1, especially when used in combination could potentially provide great therapeutic opportunities for cancer treatment. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.
Hormone signaling linked to silkmoth sex pheromone biosynthesis involves Ca2+/calmodulin-dependent protein kinase II-mediated phosphorylation of the insect PAT family protein Bombyx mori lipid storage droplet protein-1(BmLsd)

USDA-ARS?s Scientific Manuscript database

The structurally-related members of the PAT family of proteins, which are so name based on similarity amongst perilipin, adipophilin/adipocyte differentiation-related protein (ADRP), and tail-interacting protein of 47 kilodaltons (TIP47), are cytoplasmic lipid droplet (LD)-associated proteins charac...
The CCDC55 couples cannabinoid receptor CNR1 to a putative DISC1 schizophrenia pathway.

PubMed

Xie, J; Gizatullin, R; Vukojevic, V; Leopardi, R

2015-12-03

Our previous study suggested that the coiled coil domain-containing 55 gene (CCDC55), also named as NSRP1 (nuclear speckle splicing regulatory protein 1 (NSRP1)), was encompassed in a haplotype block spanning over the serotonin transporter (5-HTT) gene in patients with schizophrenia (SCZ). However, the neurobiological function of CCDC55 gene remains unknown. This study aims to uncover the potential role of CCDC55 in SCZ-associated molecular pathways. Using molecular cloning, sequencing and immune blotting to identify basic properties, yeast two-hybrid screening and glutathione S-transferase (GST) pull-down assay to test protein-protein interaction, and confocal laser scanning microscopy (CSLM) to show intracellular interaction of proteins. (i) CCDC55 is expressed as a nuclear protein in human neuronal cells; (ii) Protein-protein interaction analyses showed CCDC55 physically interacted with Ran binding protein 9 (RanBP9) and disrupted in schizophrenia 1 (DISC1); (iii) CCDC55 and RanBP9 co-localized in the nucleus of human neuronal cells; (iv) CCDC55 also interacted with the cannabinoid receptor 1 (CNR1), and with the brain cannabinoid receptor-interacting protein 1a (CNRIP1a); (v) CNR1 activation in differentiated human neuronal cells resulted in an altered RanBP9 localization. CCDC55 may be involved in a functional bridging between the CNR1 activation and the DISC1/RanBP9-associated pathways. Copyright © 2015 IBRO. Published by Elsevier Ltd. All rights reserved.
Myeloid protein tyrosine phosphatase 1B (PTP1B) deficiency protects against atherosclerotic plaque formation in the ApoE-/- mouse model of atherosclerosis with alterations in IL10/AMPKα pathway.

PubMed

Thompson, D; Morrice, N; Grant, L; Le Sommer, S; Ziegler, K; Whitfield, P; Mody, N; Wilson, H M; Delibegović, M

2017-08-01

Cardiovascular disease (CVD) is the most prevalent cause of mortality among patients with Type 1 or Type 2 diabetes, due to accelerated atherosclerosis. Recent evidence suggests a strong link between atherosclerosis and insulin resistance due to impaired insulin receptor (IR) signaling. Moreover, inflammatory cells, in particular macrophages, play a key role in pathogenesis of atherosclerosis and insulin resistance in humans. We hypothesized that inhibiting the activity of protein tyrosine phosphatase 1B (PTP1B), the major negative regulator of the IR, specifically in macrophages, would have beneficial anti-inflammatory effects and lead to protection against atherosclerosis and CVD. We generated novel macrophage-specific PTP1B knockout mice on atherogenic background (ApoE -/- /LysM-PTP1B). Mice were fed standard or pro-atherogenic diet, and body weight, adiposity (echoMRI), glucose homeostasis, atherosclerotic plaque development, and molecular, biochemical and targeted lipidomic eicosanoid analyses were performed. Myeloid-PTP1B knockout mice on atherogenic background (ApoE -/- /LysM-PTP1B) exhibited a striking improvement in glucose homeostasis, decreased circulating lipids and decreased atherosclerotic plaque lesions, in the absence of body weight/adiposity differences. This was associated with enhanced phosphorylation of aortic Akt, AMPKα and increased secretion of circulating anti-inflammatory cytokine interleukin-10 (IL-10) and prostaglandin E2 (PGE 2 ), without measurable alterations in IR phosphorylation, suggesting a direct beneficial effect of myeloid-PTP1B targeting. Here we demonstrate that inhibiting the activity of PTP1B specifically in myeloid lineage cells protects against atherosclerotic plaque formation, under atherogenic conditions, in an ApoE -/- mouse model of atherosclerosis. Our findings suggest for the first time that macrophage PTP1B targeting could be a therapeutic target for atherosclerosis treatment and reduction of CVD risk.
38 CFR 18b.55 - Depositions.

Code of Federal Regulations, 2010 CFR

2010-07-01

... 38 Pensions, Bonuses, and Veterans' Relief 2 2010-07-01 2010-07-01 false Depositions. 18b.55... Procedures § 18b.55 Depositions. Upon such terms as may be just, for the convenience of the parties or of the... be taken by deposition. ...
38 CFR 18b.55 - Depositions.

Code of Federal Regulations, 2011 CFR

2011-07-01

... 38 Pensions, Bonuses, and Veterans' Relief 2 2011-07-01 2011-07-01 false Depositions. 18b.55... Procedures § 18b.55 Depositions. Upon such terms as may be just, for the convenience of the parties or of the... be taken by deposition. ...
The Human Adenovirus Type 5 E4orf4 Protein Targets Two Phosphatase Regulators of the Hippo Signaling Pathway

PubMed Central

Mui, Melissa Z.; Zhou, Yiwang; Blanchette, Paola; Chughtai, Naila; Knight, Jennifer F.; Gruosso, Tina; Papadakis, Andreas I.; Huang, Sidong; Park, Morag; Gingras, Anne-Claude

2015-01-01

ABSTRACT When expressed alone at high levels, the human adenovirus E4orf4 protein exhibits tumor cell-specific p53-independent toxicity. A major E4orf4 target is the B55 class of PP2A regulatory subunits, and we have shown recently that binding of E4orf4 inhibits PP2AB55 phosphatase activity in a dose-dependent fashion by preventing access of substrates (M. Z. Mui et al., PLoS Pathog 9:e1003742, 2013, http://dx.doi.org/10.1371/journal.ppat.1003742). While interaction with B55 subunits is essential for toxicity, E4orf4 mutants exist that, despite binding B55 at high levels, are defective in cell killing, suggesting that other essential targets exist. In an attempt to identify additional targets, we undertook a proteomics approach to characterize E4orf4-interacting proteins. Our findings indicated that, in addition to PP2AB55 subunits, ASPP-PP1 complex subunits were found among the major E4orf4-binding species. Both the PP2A and ASPP-PP1 phosphatases are known to positively regulate effectors of the Hippo signaling pathway, which controls the expression of cell growth/survival genes by dephosphorylating the YAP transcriptional coactivator. We find here that expression of E4orf4 results in hyperphosphorylation of YAP, suggesting that Hippo signaling is affected by E4orf4 interactions with PP2AB55 and/or ASPP-PP1 phosphatases. Furthermore, knockdown of YAP1 expression was seen to enhance E4orf4 killing, again consistent with a link between E4orf4 toxicity and inhibition of the Hippo pathway. This effect may in fact contribute to the cancer cell specificity of E4orf4 toxicity, as many human cancer cells rely heavily on the Hippo pathway for their enhanced proliferation. IMPORTANCE The human adenovirus E4orf4 protein has been known for some time to induce tumor cell-specific death when expressed at high levels; thus, knowledge of its mode of action could be of importance for development of new cancer therapies. Although the B55 form of the phosphatase PP2A has long been
HTLV-1 bZIP factor protein targets the Rb/E2F-1 pathway to promote proliferation and apoptosis of primary CD4+ T cells

PubMed Central

Kawatsuki, A; Yasunaga, J-i; Mitobe, Y; Green, PL; Matsuoka, M

2016-01-01

Human T-cell leukemia virus type 1 (HTLV-1) is an oncogenic retrovirus that induces a fatal T-cell malignancy, adult T-cell leukemia (ATL). Among several regulatory/accessory genes in HTLV-1, HTLV-1 bZIP factor (HBZ) is the only viral gene constitutively expressed in infected cells. Our previous study showed that HBZ functions in two different molecular forms, HBZ protein and HBZ RNA. In this study, we show that HBZ protein targets retinoblastoma protein (Rb), which is a critical tumor suppressor in many types of cancers. HBZ protein interacts with the Rb/E2F-1 complex and activates the transcription of E2F-target genes associated with cell cycle progression and apoptosis. Mouse primary CD4+ T cells transduced with HBZ show accelerated G1/S transition and apoptosis, and importantly, T cells from HBZ transgenic (HBZ-Tg) mice also demonstrate enhanced cell proliferation and apoptosis. To evaluate the functions of HBZ protein alone in vivo, we generated a new transgenic mouse strain that expresses HBZ mRNA altered by silent mutations but encoding intact protein. In these mice, the numbers of effector/memory and Foxp3+ T cells were increased, and genes associated with proliferation and apoptosis were upregulated. This study shows that HBZ protein promotes cell proliferation and apoptosis in primary CD4+ T cells through activation of the Rb/E2F pathway, and that HBZ protein also confers onto CD4+ T-cell immunophenotype similar to those of ATL cells, suggesting that HBZ protein has important roles in dysregulation of CD4+ T cells infected with HTLV-1. PMID:26804169

Endothelial nitric-oxide synthase (eNOS) is activated through G-protein-coupled receptor kinase-interacting protein 1 (GIT1) tyrosine phosphorylation and Src protein.

PubMed

Liu, Songling; Premont, Richard T; Rockey, Don C

2014-06-27

Nitric oxide (NO) is a critical regulator of vascular tone and plays an especially prominent role in liver by controlling portal blood flow and pressure within liver sinusoids. Synthesis of NO in sinusoidal endothelial cells by endothelial nitric-oxide synthase (eNOS) is regulated in response to activation of endothelial cells by vasoactive signals such as endothelins. The endothelin B (ETB) receptor is a G-protein-coupled receptor, but the mechanisms by which it regulates eNOS activity in sinusoidal endothelial cells are not well understood. In this study, we built on two previous strands of work, the first showing that G-protein βγ subunits mediated activation of phosphatidylinositol 3-kinase and Akt to regulate eNOS and the second showing that eNOS directly bound to the G-protein-coupled receptor kinase-interacting protein 1 (GIT1) scaffold protein, and this association stimulated NO production. Here we investigated the mechanisms by which the GIT1-eNOS complex is formed and regulated. GIT1 was phosphorylated on tyrosine by Src, and Y293F and Y554F mutations reduced GIT1 phosphorylation as well as the ability of GIT1 to bind to and activate eNOS. Akt phosphorylation activated eNOS (at Ser(1177)), and Akt also regulated the ability of Src to phosphorylate GIT1 as well as GIT1-eNOS association. These pathways were activated by endothelin-1 through the ETB receptor; inhibiting receptor-activated G-protein βγ subunits blocked activation of Akt, GIT1 tyrosine phosphorylation, and ET-1-stimulated GIT1-eNOS association but did not affect Src activation. These data suggest a model in which Src and Akt cooperate to regulate association of eNOS with the GIT1 scaffold to facilitate NO production. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.
Regulation of ciliary retrograde protein trafficking by the Joubert syndrome proteins ARL13B and INPP5E.

PubMed

Nozaki, Shohei; Katoh, Yohei; Terada, Masaya; Michisaka, Saki; Funabashi, Teruki; Takahashi, Senye; Kontani, Kenji; Nakayama, Kazuhisa

2017-02-01

ARL13B (a small GTPase) and INPP5E (a phosphoinositide 5-phosphatase) are ciliary proteins encoded by causative genes of Joubert syndrome. We here showed, by taking advantage of a visible immunoprecipitation assay, that ARL13B interacts with the IFT46 -: IFT56 (IFT56 is also known as TTC26) dimer of the intraflagellar transport (IFT)-B complex, which mediates anterograde ciliary protein trafficking. However, the ciliary localization of ARL13B was found to be independent of its interaction with IFT-B, but dependent on the ciliary-targeting sequence RVEP in its C-terminal region. ARL13B-knockout cells had shorter cilia than control cells and exhibited aberrant localization of ciliary proteins, including INPP5E. In particular, in ARL13B-knockout cells, the IFT-A and IFT-B complexes accumulated at ciliary tips, and GPR161 (a negative regulator of Hedgehog signaling) could not exit cilia in response to stimulation with Smoothened agonist. This abnormal phenotype was rescued by the exogenous expression of wild-type ARL13B, as well as by its mutant defective in the interaction with IFT-B, but not by its mutants defective in INPP5E binding or in ciliary localization. Thus, ARL13B regulates IFT-A-mediated retrograde protein trafficking within cilia through its interaction with INPP5E. © 2017. Published by The Company of Biologists Ltd.
Isolation of human monoclonal antibodies to the envelope e2 protein of hepatitis C virus and their characterization.

PubMed

Shimizu, Yohko K; Hijikata, Minako; Oshima, Masamichi; Shimizu, Kazufumi; Alter, Harvey J; Purcell, Robert H; Yoshikura, Hiroshi; Hotta, Hak

2013-01-01

We isolated and characterized two human monoclonal antibodies to the envelope E2 protein of hepatitis C virus (HCV). Lymphoblastoid cell lines stably producing antibodies were obtained by immortalizing peripheral blood mononuclear cells of a patient with chronic hepatitis C using Epstein-Barr virus. Screening for antibody-positive clones was carried out by immunofluorescence with Huh7 cells expressing the E2 protein of HCV strain H (genotype 1a) isolated from the same patient. Isotype of resulting antibodies, #37 and #55, was IgG1/kappa and IgG1/lambda, respectively. Epitope mapping revealed that #37 and #55 recognize conformational epitopes spanning amino acids 429 to 652 and 508 to 607, respectively. By immunofluorescence using virus-infected Huh7.5 cells as targets both antibodies were reactive with all of the nine different HCV genotypes/subtypes tested. The antibodies showed a different pattern of immuno-staining; while #37 gave granular reactions mostly located in the periphery of the nucleus, #55 gave diffuse staining throughout the cytoplasm. Both antibodies were shown by immuno-gold electron microscopy to bind to intact viral particles. In a neutralization assay (focus-forming unit reduction using chimeric infectious HCV containing structural proteins derived from genotypes 1a, 1b, 2a, 2b, 3a, 4a, 5a, 6a, and 7a), #55 inhibited the infection of all HCV genotypes tested but genotype 7a to a lesser extent. #37 did not neutralize any of these viruses. As a broadly cross-neutralizing human antibody, #55 may be useful for passive immunotherapy of HCV infection.
Cloning and sequencing of a gene encoding a 21-kilodalton outer membrane protein from Bordetella avium and expression of the gene in Salmonella typhimurium.

PubMed Central

Gentry-Weeks, C R; Hultsch, A L; Kelly, S M; Keith, J M; Curtiss, R

1992-01-01

Three gene libraries of Bordetella avium 197 DNA were prepared in Escherichia coli LE392 by using the cosmid vectors pCP13 and pYA2329, a derivative of pCP13 specifying spectinomycin resistance. The cosmid libraries were screened with convalescent-phase anti-B. avium turkey sera and polyclonal rabbit antisera against B. avium 197 outer membrane proteins. One E. coli recombinant clone produced a 56-kDa protein which reacted with convalescent-phase serum from a turkey infected with B. avium 197. In addition, five E. coli recombinant clones were identified which produced B. avium outer membrane proteins with molecular masses of 21, 38, 40, 43, and 48 kDa. At least one of these E. coli clones, which encoded the 21-kDa protein, reacted with both convalescent-phase turkey sera and antibody against B. avium 197 outer membrane proteins. The gene for the 21-kDa outer membrane protein was localized by Tn5seq1 mutagenesis, and the nucleotide sequence was determined by dideoxy sequencing. DNA sequence analysis of the 21-kDa protein revealed an open reading frame of 582 bases that resulted in a predicted protein of 194 amino acids. Comparison of the predicted amino acid sequence of the gene encoding the 21-kDa outer membrane protein with protein sequences in the National Biomedical Research Foundation protein sequence data base indicated significant homology to the OmpA proteins of Shigella dysenteriae, Enterobacter aerogenes, E. coli, and Salmonella typhimurium and to Neisseria gonorrhoeae outer membrane protein III, Haemophilus influenzae protein P6, and Pseudomonas aeruginosa porin protein F. The gene (ompA) encoding the B. avium 21-kDa protein hybridized with 4.1-kb DNA fragments from EcoRI-digested, chromosomal DNA of Bordetella pertussis and Bordetella bronchiseptica and with 6.0- and 3.2-kb DNA fragments from EcoRI-digested, chromosomal DNA of B. avium and B. avium-like DNA, respectively. A 6.75-kb DNA fragment encoding the B. avium 21-kDa protein was subcloned into the
Characterization of the duck enteritis virus UL55 protein

PubMed Central

2011-01-01

Background Characteration of the newly identified duck enteritis virus UL55 gene product has not been reported yet. Knowledge of the protein UL55 can provide useful insights about its function. Results The newly identified duck enteritis virus UL55 gene was about 561 bp, it was amplified and digested for construction of a recombinant plasmid pET32a(+)/UL55 for expression in Escherichia coli. SDS-PAGE analysis revealed the recombinant protein UL55(pUL55) was overexpressed in Escherichia coli BL21 host cells after induction by 0.2 mM IPTG at 37°C for 4 h and aggregated as inclusion bodies. The denatured protein about 40 KDa named pUL55 was purified by washing five times, and used to immune rabbits for preparation of polyclonal antibody. The prepared polyclonal antibody against pUL55 was detected and determined by Agar immundiffusion and Neutralization test. The results of Wstern blotting assay and intracellular analysis revealed that pUL55 was expressed most abundantly during the late phase of replication and mainly distributed in cytoplasm in duck enteritis virus infected cells. Conclusions In this study, the duck enteritis virus UL55 protein was successfully expressed in prokaryotic expression system. Besides, we have prepared the polyclonal antibody against recombinant prtein UL55, and characterized some properties of the duck enteritis virus UL55 protein for the first time. The research will be useful for further functional analysis of this gene. PMID:21609474
Characterization of Three Novel Linear Neutralizing B-Cell Epitopes in the Capsid Protein of Swine Hepatitis E Virus.

PubMed

Chen, Yiyang; Liu, Baoyuan; Sun, Yani; Li, Huixia; Du, Taofeng; Nan, Yuchen; Hiscox, Julian A; Zhou, En-Min; Zhao, Qin

2018-07-01

Hepatitis E virus (HEV) causes liver disease in humans and is thought to be a zoonotic infection, with domestic animals, including swine and rabbits, being a reservoir. One of the proteins encoded by the virus is the capsid protein. This is likely the major immune-dominant protein and a target for vaccination. Four monoclonal antibodies (MAbs), three novel, 1E4, 2C7, and 2G9, and one previously characterized, 1B5, were evaluated for binding to the capsid protein from genotype 4 swine HEV. The results indicated that 625 DFCP 628 , 458 PSRPF 462 , and 407 EPTV 410 peptides on the capsid protein comprised minimal amino acid sequence motifs recognized by 1E4, 2C7, and 2G9, respectively. The data suggested that 2C7 and 2G9 epitopes were partially exposed on the surface of the capsid protein. Truncated genotype 4 swine HEV capsid protein (sp239, amino acids 368 to 606) can exist in multimeric forms. Preincubation of swine HEV with 2C7, 2G9, or 1B5 before addition to HepG2 cells partially blocked sp239 cell binding and inhibited swine HEV infection. The study indicated that 2C7, 2G9, and 1B5 partially blocked swine HEV infection of rabbits better than 1E4 or normal mouse IgG. The cross-reactivity of antibodies suggested that capsid epitopes recognized by 2C7 and 2G9 are common to HEV strains infecting most host species. Collectively, MAbs 2C7, 2G9, and 1B5 were shown to recognize three novel linear neutralizing B-cell epitopes of genotype 4 HEV capsid protein. These results enhance understanding of HEV capsid protein structure to guide vaccine and antiviral design. IMPORTANCE Genotype 3 and 4 HEVs are zoonotic viruses. Here, genotype 4 HEV was studied due to its prevalence in human populations and pig herds in China. To improve HEV disease diagnosis and prevention, a better understanding of the antigenic structure and neutralizing epitopes of HEV capsid protein are needed. In this study, the locations of three novel linear B-cell recognition epitopes within genotype 4
The tripartite leader sequence is required for ectopic expression of HAdV-B and HAdV-E E3 CR1 genes.

PubMed

Bair, Camden R; Kotha Lakshmi Narayan, Poornima; Kajon, Adriana E

2017-05-01

The unique repertoire of genes that characterizes the early region 3 (E3) of the different species of human adenovirus (HAdV) likely contributes to their distinct pathogenic traits. The function of many E3 CR1 proteins remains unknown possibly due to unidentified intrinsic properties that make them difficult to express ectopically. This study shows that the species HAdV-B- and HAdV-E-specific E3 CR1 genes can be expressed from vectors carrying the HAdV tripartite leader (TPL) sequence but not from traditional mammalian expression vectors. Insertion of the TPL sequence upstream of the HAdV-B and HAdV-E E3 CR1 open reading frames was sufficient to rescue protein expression from pCI-neo constructs in transfected 293T cells. The detection of higher levels of HAdV-B and HAdV-E E3 CR1 transcripts suggests that the TPL sequence may enhance gene expression at both the transcriptional and translational levels. Our findings will facilitate the characterization of additional AdV E3 proteins. Copyright © 2017 Elsevier Inc. All rights reserved.
gC1q-R/p32, a C1q-binding protein, is a receptor for the InlB invasion protein of Listeria monocytogenes.

PubMed

Braun, L; Ghebrehiwet, B; Cossart, P

2000-04-03

InlB is a Listeria monocytogenes protein that promotes entry of the bacterium into mammalian cells by stimulating tyrosine phosphorylation of the adaptor proteins Gab1, Cbl and Shc, and activation of phosphatidyl- inositol (PI) 3-kinase. Using affinity chromatography and enzyme-linked immunosorbent assay, we demonstrate a direct interaction between InlB and the mammalian protein gC1q-R, the receptor of the globular part of the complement component C1q. Soluble C1q or anti-gC1q-R antibodies impair InlB-mediated entry. Transient transfection of GPC16 cells, which are non-permissive to InlB-mediated entry, with a plasmid-expressing human gC1q-R promotes entry of InlB-coated beads. Furthermore, several experiments indicate that membrane recruitment and activation of PI 3-kinase involve an InlB-gC1q-R interaction and that gC1q-R associates with Gab1 upon stimulation of Vero cells with InlB. Thus, gC1q-R constitutes a cellular receptor involved in InlB-mediated activation of PI 3-kinase and tyrosine phosphorylation of the adaptor protein Gab1. After E-cadherin, the receptor for internalin, gC1q-R is the second identified mammalian receptor promoting entry of L. monocytogenes into mammalian cells.
Structures of Adenovirus Incomplete Particles Clarify Capsid Architecture and Show Maturation Changes of Packaging Protein L1 52/55k

PubMed Central

Condezo, Gabriela N.; Marabini, Roberto; Ayora, Silvia; Carazo, José M.; Alba, Raúl; Chillón, Miguel

2015-01-01

ABSTRACT Adenovirus is one of the most complex icosahedral, nonenveloped viruses. Even after its structure was solved at near-atomic resolution by both cryo-electron microscopy and X-ray crystallography, the location of minor coat proteins is still a subject of debate. The elaborated capsid architecture is the product of a correspondingly complex assembly process, about which many aspects remain unknown. Genome encapsidation involves the concerted action of five virus proteins, and proteolytic processing by the virus protease is needed to prime the virion for sequential uncoating. Protein L1 52/55k is required for packaging, and multiple cleavages by the maturation protease facilitate its release from the nascent virion. Light-density particles are routinely produced in adenovirus infections and are thought to represent assembly intermediates. Here, we present the molecular and structural characterization of two different types of human adenovirus light particles produced by a mutant with delayed packaging. We show that these particles lack core polypeptide V but do not lack the density corresponding to this protein in the X-ray structure, thereby adding support to the adenovirus cryo-electron microscopy model. The two types of light particles present different degrees of proteolytic processing. Their structures provide the first glimpse of the organization of L1 52/55k protein inside the capsid shell and of how this organization changes upon partial maturation. Immature, full-length L1 52/55k is poised beneath the vertices to engage the virus genome. Upon proteolytic processing, L1 52/55k disengages from the capsid shell, facilitating genome release during uncoating. IMPORTANCE Adenoviruses have been extensively characterized as experimental systems in molecular biology, as human pathogens, and as therapeutic vectors. However, a clear picture of many aspects of their basic biology is still lacking. Two of these aspects are the location of minor coat proteins in
Human Adenovirus Infection Causes Cellular E3 Ubiquitin Ligase MKRN1 Degradation Involving the Viral Core Protein pVII.

PubMed

Inturi, Raviteja; Mun, Kwangchol; Singethan, Katrin; Schreiner, Sabrina; Punga, Tanel

2018-02-01

Human adenoviruses (HAdVs) are common human pathogens encoding a highly abundant histone-like core protein, VII, which is involved in nuclear delivery and protection of viral DNA as well as in sequestering immune danger signals in infected cells. The molecular details of how protein VII acts as a multifunctional protein have remained to a large extent enigmatic. Here we report the identification of several cellular proteins interacting with the precursor pVII protein. We show that the cellular E3 ubiquitin ligase MKRN1 is a novel precursor pVII-interacting protein in HAdV-C5-infected cells. Surprisingly, the endogenous MKRN1 protein underwent proteasomal degradation during the late phase of HAdV-C5 infection in various human cell lines. MKRN1 protein degradation occurred independently of the HAdV E1B55K and E4orf6 proteins. We provide experimental evidence that the precursor pVII protein binding enhances MKRN1 self-ubiquitination, whereas the processed mature VII protein is deficient in this function. Based on these data, we propose that the pVII protein binding promotes MKRN1 self-ubiquitination, followed by proteasomal degradation of the MKRN1 protein, in HAdV-C5-infected cells. In addition, we show that measles virus and vesicular stomatitis virus infections reduce the MKRN1 protein accumulation in the recipient cells. Taken together, our results expand the functional repertoire of the HAdV-C5 precursor pVII protein in lytic virus infection and highlight MKRN1 as a potential common target during different virus infections. IMPORTANCE Human adenoviruses (HAdVs) are common pathogens causing a wide range of diseases. To achieve pathogenicity, HAdVs have to counteract a variety of host cell antiviral defense systems, which would otherwise hamper virus replication. In this study, we show that the HAdV-C5 histone-like core protein pVII binds to and promotes self-ubiquitination of a cellular E3 ubiquitin ligase named MKRN1. This mutual interaction between the pVII and
Role of the Acidic Tail of High Mobility Group Protein B1 (HMGB1) in Protein Stability and DNA Bending

PubMed Central

Belgrano, Fabricio S.; de Abreu da Silva, Isabel C.; Bastos de Oliveira, Francisco M.; Fantappié, Marcelo R.; Mohana-Borges, Ronaldo

2013-01-01

High mobility group box (HMGB) proteins are abundant nonhistone proteins found in all eukaryotic nuclei and are capable of binding/bending DNA. The human HMGB1 is composed of two binding motifs, known as Boxes A and B, are L-shaped alpha-helix structures, followed by a random-coil acidic tail that consists of 30 Asp and Glu residues. This work aimed at evaluating the role of the acidic tail of human HMGB1 in protein stability and DNA interactions. For this purpose, we cloned, expressed and purified HMGB1 and its tailless form, HMGB1ΔC, in E. coli strain. Tryptophan fluorescence spectroscopy and circular dichroism (CD) experiments clearly showed an increase in protein stability promoted by the acidic tail under different conditions, such as the presence of the chemical denaturant guanidine hydrochloride (Gdn.HCl), high temperature and low pH. Folding intermediates found at low pH for both proteins were denatured only in the presence of chemical denaturant, thus showing a relatively high stability. The acidic tail did not alter the DNA-binding properties of the protein, although it enhanced the DNA bending capability from 76° (HMGB1ΔC) to 91° (HMGB1), as measured using the fluorescence resonance energy transfer technique. A model of DNA bending in vivo was proposed, which might help to explain the interaction of HMGB1 with DNA and other proteins, i.e., histones, and the role of that protein in chromatin remodeling. PMID:24255708
PTP1B antisense oligonucleotide lowers PTP1B protein, normalizes blood glucose, and improves insulin sensitivity in diabetic mice.

PubMed

Zinker, Bradley A; Rondinone, Cristina M; Trevillyan, James M; Gum, Rebecca J; Clampit, Jill E; Waring, Jeffrey F; Xie, Nancy; Wilcox, Denise; Jacobson, Peer; Frost, Leigh; Kroeger, Paul E; Reilly, Regina M; Koterski, Sandra; Opgenorth, Terry J; Ulrich, Roger G; Crosby, Seth; Butler, Madeline; Murray, Susan F; McKay, Robert A; Bhanot, Sanjay; Monia, Brett P; Jirousek, Michael R

2002-08-20

The role of protein-tyrosine phosphatase 1B (PTP1B) in diabetes was investigated using an antisense oligonucleotide in ob/ob and db/db mice. PTP1B antisense oligonucleotide treatment normalized plasma glucose levels, postprandial glucose excursion, and HbA(1C). Hyperinsulinemia was also reduced with improved insulin sensitivity. PTP1B protein and mRNA were reduced in liver and fat with no effect in skeletal muscle. Insulin signaling proteins, insulin receptor substrate 2 and phosphatidylinositol 3 (PI3)-kinase regulatory subunit p50alpha, were increased and PI3-kinase p85alpha expression was decreased in liver and fat. These changes in protein expression correlated with increased insulin-stimulated protein kinase B phosphorylation. The expression of liver gluconeogenic enzymes, phosphoenolpyruvate carboxykinase, and fructose-1,6-bisphosphatase was also down-regulated. These findings suggest that PTP1B modulates insulin signaling in liver and fat, and that therapeutic modalities targeting PTP1B inhibition may have clinical benefit in type 2 diabetes.
Open Reading Frame E3-10.9K of Subspecies B1 Human Adenoviruses Encodes a Family of Late Orthologous Proteins That Vary in Their Predicted Structural Features and Subcellular Localization ▿

PubMed Central

Frietze, Kathryn M.; Campos, Samuel K.; Kajon, Adriana E.

2010-01-01

Subspecies B1 human adenoviruses (HAdV-B1s) are important causative agents of acute respiratory disease, but the molecular bases of their distinct pathobiology are still poorly understood. Marked differences in genetic content between HAdV-B1s and the well-characterized HAdV-Cs that may contribute to distinct pathogenic properties map to the E3 region. Between the highly conserved E3-19K and E3-10.4K/RIDα open reading frames (ORFs), and in the same location as the HAdV-C ADP/E3-11.6K ORF, HAdV-B1s carry ORFs E3-20.1K and E3-20.5K and a polymorphic third ORF, designated E3-10.9K, that varies in the size of its predicted product among HAdV-B1 serotypes and genomic variants. As an initial effort to define the function of the E3-10.9K ORF, we carried out a biochemical characterization of E3-10.9K-encoded orthologous proteins and investigated their expression in infected cells. Sequence-based predictions suggested that E3-10.9K orthologs with a hydrophobic domain are integral membrane proteins. Ectopically expressed, C-terminally tagged (with enhanced green fluorescent protein [EGFP]) E3-10.9K and E3-9K localized primarily to the plasma membrane, while E3-7.7K localized primarily to a juxtanuclear compartment that could not be identified. EGFP fusion proteins with a hydrophobic domain were N and O glycosylated. EGFP-tagged E3-4.8K, which lacked the hydrophobic domain, displayed diffuse cellular localization similar to that of the EGFP control. E3-10.9K transcripts from the major late promoter were detected at late time points postinfection. A C-terminally hemagglutinin-tagged version of E3-9K was detected by immunoprecipitation at late times postinfection in the membrane fraction of mutant virus-infected cells. These data suggest a role for ORF E3-10.9K-encoded proteins at late stages of HAdV-B1 replication, with potentially important functional implications for the documented ORF polymorphism. PMID:20739542
Identification of a Linked Set of Genes in Serpulina hyodysenteriae (B204) Predicted To Encode Closely Related 39-Kilodalton Extracytoplasmic Proteins

PubMed Central

Gabe, Jeffrey D.; Dragon, Elizabeth; Chang, Ray-Jen; McCaman, Michael T.

1998-01-01

A tandem pair of nearly identical genes from Serpulina hyodysenteriae (B204) were cloned and sequenced. The full open reading frame of one gene and the partial open reading frame of the neighboring gene appear to encode secreted proteins which are homologous to, yet distinct from, the 39-kDa extracytoplasmic protein purified from the membrane fraction of S. hyodysenteriae. We have designated these newly identified genes vspA and vspB (for variable surface protein). PMID:9440540
FBI-1 enhances transcription of the nuclear factor-kappaB (NF-kappaB)-responsive E-selectin gene by nuclear localization of the p65 subunit of NF-kappaB.

PubMed

Lee, Dong-Kee; Kang, Jae-Eun; Park, Hye-Jin; Kim, Myung-Hwa; Yim, Tae-Hee; Kim, Jung-Min; Heo, Min-Kyu; Kim, Kyu-Yeun; Kwon, Ho Jeong; Hur, Man-Wook

2005-07-29

The POZ domain is a highly conserved protein-protein interaction motif found in many regulatory proteins. Nuclear factor-kappaB (NF-kappaB) plays a key role in the expression of a variety of genes in response to infection, inflammation, and stressful conditions. We found that the POZ domain of FBI-1 (factor that binds to the inducer of short transcripts of human immunodeficiency virus-1) interacted with the Rel homology domain of the p65 subunit of NF-kappaB in both in vivo and in vitro protein-protein interaction assays. FBI-1 enhanced NF-kappaB-mediated transcription of E-selectin genes in HeLa cells upon phorbol 12-myristate 13-acetate stimulation and overcame gene repression by IkappaB alpha or IkappaB beta. In contrast, the POZ domain of FBI-1, which is a dominant-negative form of FBI-1, repressed NF-kappaB-mediated transcription, and the repression was cooperative with IkappaB alpha or IkappaB beta. In contrast, the POZ domain tagged with a nuclear localization sequence polypeptide of FBI-1 enhanced NF-kappaB-responsive gene transcription, suggesting that the molecular interaction between the POZ domain and the Rel homology domain of p65 and the nuclear localization by the nuclear localization sequence are important in the transcription enhancement mediated by FBI-1. Confocal microscopy showed that FBI-1 increased NF-kappaB movement into the nucleus and increased the stability of NF-kappaB in the nucleus, which enhanced NF-kappaB-mediated transcription of the E-selectin gene. FBI-1 also interacted with IkappaB alpha and IkappaB beta.
Avermectin B1

Integrated Risk Information System (IRIS)

Avermectin B1 ; CASRN 65195 - 55 - 3 Human health assessment information on a chemical substance is included in the IRIS database only after a comprehensive review of toxicity data , as outlined in the IRIS assessment development process . Sections I ( Health Hazard Assessments for Noncarcinogenic E
The 14.6 kd rubber elongation factor (Hev b 1) and 24 kd (Hev b 3) rubber particle proteins are recognized by IgE from patients with spina bifida and latex allergy.

PubMed

Yeang, H Y; Cheong, K F; Sunderasan, E; Hamzah, S; Chew, N P; Hamid, S; Hamilton, R G; Cardosa, M J

1996-09-01

Two major water-insoluble proteins are located on the surface of rubber particles in Hevea brasiliensis latex. A 14.6 kd protein (Hev b 1), found mainly on large rubber particles (> 350 mm in diameter), and a 24 kd protein (Hev b 3), found mainly on small rubber particles (average diameter, 70 nm), are recognized by IgE from patients with spina bifida and latex allergy. Although Hev b 1 (also called the rubber elongation factor [REF]) has previously been reported as a major latex allergen, this conclusion has been disputed on the basis of results from other studies. The allergenicity of Hev b 1 is verified in this study by testing the recombinant protein generated from its gene. Because allergenicity is confined to patients with spina bifida and not observed in adults sensitive to latex, it is not a major latex allergen. The identification of Hev b 3 as another allergen originating from rubber particles is confirmed by immunogold labeling and electron microscopy. Observations with the monoclonal antibody USM/RC2 developed against Hev b 3 show that the protein has a tendency to fragment into several polypeptides of lower molecular weight (from 24 kd to about 5 kd) when stored at -20 degrees C. There is also indication of protein aggregation from the appearance of proteins with molecular weights greater than 24 kd. Fragmentation of Hev b 3 is induced immediately on he addition of latex B-serum, which is normally compartmentalized in the lutoids in fresh latex. In the preparation of ammoniated latex (used for the manufacture of latex products), the lutoids are ruptured, and the released B-serum reacts with Hev b 3 on the rubber particles to give rise to an array of low molecular weight polypeptides that are allergenic to patients with spina bifida.
PTP1B antisense oligonucleotide lowers PTP1B protein, normalizes blood glucose, and improves insulin sensitivity in diabetic mice

PubMed Central

Zinker, Bradley A.; Rondinone, Cristina M.; Trevillyan, James M.; Gum, Rebecca J.; Clampit, Jill E.; Waring, Jeffrey F.; Xie, Nancy; Wilcox, Denise; Jacobson, Peer; Frost, Leigh; Kroeger, Paul E.; Reilly, Regina M.; Koterski, Sandra; Opgenorth, Terry J.; Ulrich, Roger G.; Crosby, Seth; Butler, Madeline; Murray, Susan F.; McKay, Robert A.; Bhanot, Sanjay; Monia, Brett P.; Jirousek, Michael R.

2002-01-01

The role of protein-tyrosine phosphatase 1B (PTP1B) in diabetes was investigated using an antisense oligonucleotide in ob/ob and db/db mice. PTP1B antisense oligonucleotide treatment normalized plasma glucose levels, postprandial glucose excursion, and HbA1C. Hyperinsulinemia was also reduced with improved insulin sensitivity. PTP1B protein and mRNA were reduced in liver and fat with no effect in skeletal muscle. Insulin signaling proteins, insulin receptor substrate 2 and phosphatidylinositol 3 (PI3)-kinase regulatory subunit p50α, were increased and PI3-kinase p85α expression was decreased in liver and fat. These changes in protein expression correlated with increased insulin-stimulated protein kinase B phosphorylation. The expression of liver gluconeogenic enzymes, phosphoenolpyruvate carboxykinase, and fructose-1,6-bisphosphatase was also down-regulated. These findings suggest that PTP1B modulates insulin signaling in liver and fat, and that therapeutic modalities targeting PTP1B inhibition may have clinical benefit in type 2 diabetes. PMID:12169659
Redox biology of Mycobacterium tuberculosis H37Rv: protein-protein interaction between GlgB and WhiB1 involves exchange of thiol-disulfide

PubMed Central

Garg, Saurabh; Alam, Md Suhail; Bajpai, Richa; Kishan, KV Radha; Agrawal, Pushpa

2009-01-01

Background Mycobacterium tuberculosis, an intracellular pathogen encounters redox stress throughout its life inside the host. In order to protect itself from the redox onslaughts of host immune system, M. tuberculosis appears to have developed accessory thioredoxin-like proteins which are represented by ORFs encoding WhiB-like proteins. We have earlier reported that WhiB1/Rv3219 is a thioredoxin like protein of M. tuberculosis and functions as a protein disulfide reductase. Generally thioredoxins have many substrate proteins. The current study aims to identify the substrate protein(s) of M. tuberculosis WhiB1. Results Using yeast two-hybrid screen, we identified alpha (1,4)-glucan branching enzyme (GlgB) of M. tuberculosis as a interaction partner of WhiB1. In vitro GST pull down assay confirmed the direct physical interaction between GlgB and WhiB1. Both mass spectrometry data of tryptic digests and in vitro labeling of cysteine residues with 4-acetamido-4' maleimidyl-stilbene-2, 2'-disulfonic acid showed that in GlgB, C95 and C658 are free but C193 and C617 form an intra-molecular disulfide bond. WhiB1 has a C37XXC40 motif thus a C40S mutation renders C37 to exist as a free thiol to form a hetero-disulfide bond with the cysteine residue of substrate protein. A disulfide mediated binary complex formation between GlgB and WhiB1C40S was shown by both in-solution protein-protein interaction and thioredoxin affinity chromatography. Finally, transfer of reducing equivalent from WhiB1 to GlgB disulfide was confirmed by 4-acetamido-4' maleimidyl-stilbene-2, 2'-disulfonic acid trapping by the reduced disulfide of GlgB. Two different thioredoxins, TrxB/Rv1471 and TrxC/Rv3914 of M. tuberculosis could not perform this reaction suggesting that the reduction of GlgB by WhiB1 is specific. Conclusion We conclude that M. tuberculosis GlgB has one intra-molecular disulfide bond which is formed between C193 and C617. WhiB1, a thioredoxin like protein interacts with GlgB and
Adenovirus type 5 E1A and E6 proteins of low-risk cutaneous beta-human papillomaviruses suppress cell transformation through interaction with FOXK1/K2 transcription factors.

PubMed

Komorek, Jessica; Kuppuswamy, Mohan; Subramanian, T; Vijayalingam, S; Lomonosova, Elena; Zhao, Ling-Jun; Mymryk, Joe S; Schmitt, Kimberly; Chinnadurai, G

2010-03-01

The adenovirus (Adv) oncoprotein E1A stimulates cell proliferation and inhibits differentiation. These activities are primarily linked to the N-terminal region (exon 1) of E1A, which interacts with multiple cellular protein complexes. The C terminus (exon 2) of E1A antagonizes these processes, mediated in part through interaction with C-terminal binding proteins 1 and 2 (CtBP1/2). To identify additional cellular E1A targets that are involved in the modulation of E1A C-terminus-mediated activities, we undertook tandem affinity purification of E1A-associated proteins. Through mass spectrometric analysis, we identified several known E1A-interacting proteins as well as novel E1A targets, such as the forkhead transcription factors, FOXK1/K2. We identified a Ser/Thr-containing sequence motif in E1A that mediated interaction with FOXK1/K2. We demonstrated that the E6 proteins of two beta-human papillomaviruses (HPV14 and HPV21) associated with epidermodysplasia verruciformis also interacted with FOXK1/K2 through a motif similar to that of E1A. The E1A mutants deficient in interaction with FOXK1/K2 induced enhanced cell proliferation and oncogenic transformation. The hypertransforming activity of the mutant E1A was suppressed by HPV21 E6. An E1A-E6 chimeric protein containing the Ser/Thr domain of the E6 protein in E1A interacted efficiently with FOXK1/K2 and inhibited cell transformation. Our results suggest that targeting FOXK1/K2 may be a common mechanism for certain beta-HPVs and Adv5. E1A exon 2 mutants deficient in interaction with the dual-specificity kinases DYRK1A/1B and their cofactor HAN11 also induced increased cell proliferation and transformation. Our results suggest that the E1A C-terminal region may suppress cell proliferation and oncogenic transformation through interaction with three different cellular protein complexes: FOXK1/K2, DYRK(1A/1B)/HAN11, and CtBP1/2.

Association of papillomavirus E6 proteins with either MAML1 or E6AP clusters E6 proteins by structure, function, and evolutionary relatedness

PubMed Central

Brimer, Nicole

2017-01-01

Papillomavirus E6 proteins bind to LXXLL peptide motifs displayed on targeted cellular proteins. Alpha genus HPV E6 proteins associate with the cellular ubiquitin ligase E6AP (UBE3A), by binding to an LXXLL peptide (ELTLQELLGEE) displayed by E6AP, thereby stimulating E6AP ubiquitin ligase activity. Beta, Gamma, and Delta genera E6 proteins bind a similar LXXLL peptide (WMSDLDDLLGS) on the cellular transcriptional co-activator MAML1 and thereby repress Notch signaling. We expressed 45 different animal and human E6 proteins from diverse papillomavirus genera to ascertain the overall preference of E6 proteins for E6AP or MAML1. E6 proteins from all HPV genera except Alpha preferentially interacted with MAML1 over E6AP. Among animal papillomaviruses, E6 proteins from certain ungulate (SsPV1 from pigs) and cetacean (porpoises and dolphins) hosts functionally resembled Alpha genus HPV by binding and targeting the degradation of E6AP. Beta genus HPV E6 proteins functionally clustered with Delta, Pi, Tau, Gamma, Chi, Mu, Lambda, Iota, Dyokappa, Rho, and Dyolambda E6 proteins to bind and repress MAML1. None of the tested E6 proteins physically and functionally interacted with both MAML1 and E6AP, indicating an evolutionary split. Further, interaction of an E6 protein was insufficient to activate degradation of E6AP, indicating that E6 proteins that target E6AP co-evolved to separately acquire both binding and triggering of ubiquitin ligase activation. E6 proteins with similar biological function clustered together in phylogenetic trees and shared structural features. This suggests that the divergence of E6 proteins from either MAML1 or E6AP binding preference is a major event in papillomavirus evolution. PMID:29281732
Escherichia coli F1Fo-ATP synthase with a b/δ fusion protein allows analysis of the function of the individual b subunits.

PubMed

Gajadeera, Chathurada S; Weber, Joachim

2013-09-13

The "stator stalk" of F1Fo-ATP synthase is essential for rotational catalysis as it connects the nonrotating portions of the enzyme. In Escherichia coli, the stator stalk consists of two (identical) b subunits and the δ subunit. In mycobacteria, one of the b subunits and the δ subunit are replaced by a b/δ fusion protein; the remaining b subunit is of the shorter b' type. In the present study, it is shown that it is possible to generate a functional E. coli ATP synthase containing a b/δ fusion protein. This construct allowed the analysis of the roles of the individual b subunits. The full-length b subunit (which in this case is covalently linked to δ in the fusion protein) is responsible for connecting the stalk to the catalytic F1 subcomplex. It is not required for interaction with the membrane-embedded Fo subcomplex, as its transmembrane helix can be removed. Attachment to Fo is the function of the other b subunit which in turn has only a minor (if any at all) role in binding to δ. Also in E. coli the second b subunit can be shortened to a b' type.
Natural products possessing protein tyrosine phosphatase 1B (PTP1B) inhibitory activity found in the last decades

PubMed Central

Jiang, Cheng-shi; Liang, Lin-fu; Guo, Yue-wei

2012-01-01

This article provides an overview of approximately 300 secondary metabolites with inhibitory activity against protein tyrosine phosphatase 1B (PTP1B), which were isolated from various natural sources or derived from synthetic process in the last decades. The structure-activity relationship and the selectivity of some compounds against other protein phosphatases were also discussed. Potential pharmaceutical applications of several PTP1B inhibitors were presented. PMID:22941286
[Solubilization Specificities Interferon beta-1b from Inclusion Bodies].

PubMed

Zhuravko, A S; Kononova, N V; Bobruskin, A I

2015-01-01

A new solubilization method of recombinant interferon beta-1b (IFNβ-1b) from the inclusion bodies was developed. This method allows to extract the target protein selectively in the solutions of different alcohols, such as ethanol, propanol and isopropanol. It was shown that the more effective IFNβ-1b solubilization was achieved in the 55% propanol solution. This method allowed to extract the target protein from inclusion bodies around 85-90%, and significantly reduced Escherichia coli content in the solubilizate, in comparison with standard methods.
Characterisation of Translation Elongation Factor eEF1B Subunit Expression in Mammalian Cells and Tissues and Co-Localisation with eEF1A2

PubMed Central

Janikiewicz, Justyna; Doig, Jennifer; Abbott, Catherine M.

2014-01-01

Translation elongation is the stage of protein synthesis in which the translation factor eEF1A plays a pivotal role that is dependent on GTP exchange. In vertebrates, eEF1A can exist as two separately encoded tissue-specific isoforms, eEF1A1, which is almost ubiquitously expressed, and eEF1A2, which is confined to neurons and muscle. The GTP exchange factor for eEF1A1 is a complex called eEF1B made up of subunits eEF1Bα, eEF1Bδ and eEF1Bγ. Previous studies have cast doubt on the ability of eEF1B to interact with eEF1A2, suggesting that this isoform might use a different GTP exchange factor. We show that eEF1B subunits are all widely expressed to varying degrees in different cell lines and tissues, and at different stages of development. We show that ablation of any of the subunits in human cell lines has a small but significant impact on cell viability and cycling. Finally, we show that both eEF1A1 and eEF1A2 colocalise with all eEF1B subunits, in such close proximity that they are highly likely to be in a complex. PMID:25436608
Solubilization of protein aggregates by the acid stress chaperones HdeA and HdeB.

PubMed

Malki, Abderrahim; Le, Hai-Tuong; Milles, Sigrid; Kern, Renée; Caldas, Teresa; Abdallah, Jad; Richarme, Gilbert

2008-05-16

The acid stress chaperones HdeA and HdeB of Escherichia coli prevent the aggregation of periplasmic proteins at acidic pH. We show in this report that they also form mixed aggregates with proteins that have failed to be solubilized at acidic pH and allow their subsequent solubilization at neutral pH. HdeA, HdeB, and HdeA and HdeB together display an increasing efficiency for the solubilization of protein aggregates at pH 3. They are less efficient for the solubilization of aggregates at pH 2, whereas HdeB is the most efficient. Increasing amounts of periplasmic proteins draw increasing amounts of chaperone into pellets, suggesting that chaperones co-aggregate with their substrate proteins. We observed a decrease in the size of protein aggregates in the presence of HdeA and HdeB, from very high molecular mass aggregates to 100-5000-kDa species. Moreover, a marked decrease in the exposed hydrophobicity of aggregated proteins in the presence of HdeA and HdeB was revealed by 1,1'-bis(4-anilino)naphtalene-5,5'-disulfonic acid binding experiments. In vivo, during the recovery at neutral pH of acid stressed bacterial cells, HdeA and HdeB allow the solubilization and renaturation of protein aggregates, including those formed by the maltose receptor MalE, the oligopeptide receptor OppA, and the histidine receptor HisJ. Thus, HdeA and HdeB not only help to maintain proteins in a soluble state during acid treatment, as previously reported, but also assist, both in vitro and in vivo, in the solubilization at neutral pH of mixed protein-chaperone aggregates formed at acidic pH, by decreasing the size of protein aggregates and the exposed hydrophobicity of aggregated proteins.
PRECLINICAL PHARMACOKINETIC ANALYSIS OF (E)-METHYL-4-ARYL-4-OXABUT-2-ENOATE, A NOVEL SER/THR PROTEIN KINASE B INIBITOR, IN RATS.

PubMed

Zhai, Qian-Qian; Pang, Jing; Li, Guo-Qing; Li, Cong-Ran; Wang, Yu-Cheng; Yu, Li-Yan; Li, Jian; YOUm, Xue-Fu

2017-01-01

(E)-Methyl-4-aryl-4-oxabut-2-enoate, designated YH-8, is a novel Serflhr protein kinase B (PknB) inhibitor, which is designed for the treatment of tuberculosis. The aim of this study was to investigate the pharmacokinetics, bioavailability, tissue distribution and excretion characteristics of YH-8 in rats and study its plasma protein binding in vitro. The pharmacokinetic properties were examined after intravenously injected YH-8 at 10 and 20 mg/kg and oral administrated YH-8 at 50, 100 and 200 mg/kg to rats. The concentrations of YH-8 in plasma were determined with LC-MS/MS, with a liquid-liquid extraction. The tissue distribution and urinary, fecal and -biliary excretion patterns of YH-8 were investigated following a single oral dosing of 100 mg/kg. The plasma protein binding rates of YH-8 were determined using ultra-filtration method. After intra- venous and oral administration, YH-8 showed dose-independent pharmacokinetic characteristics, with T(1/2) of approximately 5.5 h and 7.1 h, respectively. The oral absolute bioavailability of YH-8 was relatively low (about 12%). YH-8 was widely distributed in various tissues and showed substantial deposition in intestine, stomach, liver, lung and kidney. The drug was mainly eliminated via fecal excretion and its binding rate with plasma protein was concentration-dependent. In conclusion, this study as first provided the full pharmacokinetic characteristics of YH-8, which would be helpful for its further development and clinical application.
A selective antagonist reveals a potential role of G protein-coupled receptor 55 in platelet and endothelial cell function.

PubMed

Kargl, Julia; Brown, Andrew J; Andersen, Liisa; Dorn, Georg; Schicho, Rudolf; Waldhoer, Maria; Heinemann, Akos

2013-07-01

The G protein-coupled receptor 55 (GPR55) is a lysophosphatidylinositol (LPI) receptor that is also responsive to certain cannabinoids. Although GPR55 has been implicated in several (patho)physiologic functions, its role remains enigmatic owing mainly to the lack of selective GPR55 antagonists. Here we show that the compound CID16020046 ((4-[4-(3-hydroxyphenyl)-3-(4-methylphenyl)-6-oxo-1H,4H,5H,6H-pyrrolo[3,4-c]pyrazol-5-yl] benzoic acid) is a selective GPR55 antagonist. In yeast cells expressing human GPR55, CID16020046 antagonized agonist-induced receptor activation. In human embryonic kidney (HEK293) cells stably expressing human GPR55, the compound behaved as an antagonist on LPI-mediated Ca²⁺ release and extracellular signal-regulated kinases activation, but not in HEK293 cells expressing cannabinoid receptor 1 or 2 (CB₁ or CB₂). CID16020046 concentration dependently inhibited LPI-induced activation of nuclear factor of activated T-cells (NFAT), nuclear factor κ of activated B cells (NF-κB) and serum response element, translocation of NFAT and NF-κB, and GPR55 internalization. It reduced LPI-induced wound healing in primary human lung microvascular endothelial cells and reversed LPI-inhibited platelet aggregation, suggesting a novel role for GPR55 in platelet and endothelial cell function. CID16020046 is therefore a valuable tool to study GPR55-mediated mechanisms in primary cells and tissues.
Effective photoconductivity of exfoliated black phosphorus for optoelectronic switching under 1.55 μm optical excitation

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

Penillard, A., E-mail: anne.penillard@espci.fr; Tripon-Canseliet, C.; Maksimovic, I.

2016-01-14

We present a microwave photoconductive switch based on exfoliated black phosphorus and strongly responding to a 1.55 μm optical excitation. According to its number of atomic layers, exfoliated black phosphorus presents unique properties for optoelectronic applications, like a tunable direct bandgap from 0.3 eV to 2 eV, strong mobilities, and strong conductivities. The switch shows a maximum ON/OFF ratio of 17 dB at 1 GHz, and 2.2 dB at 20 GHz under 1.55-μm laser excitation at 50 mW, never achieved with bidimensional materials.
PTP1B Inhibitory and Anti-Inflammatory Effects of Secondary Metabolites Isolated from the Marine-Derived Fungus Penicillium sp. JF-55

PubMed Central

Lee, Dong-Sung; Jang, Jae-Hyuk; Ko, Wonmin; Kim, Kyoung-Su; Sohn, Jae Hak; Kang, Myeong-Suk; Ahn, Jong Seog; Kim, Youn-Chul; Oh, Hyuncheol

2013-01-01

Protein tyrosine phosphatase 1B (PTP1B) plays a major role in the negative regulation of insulin signaling, and is thus considered as an attractive therapeutic target for the treatment of diabetes. Bioassay-guided investigation of the methylethylketone extract of marine-derived fungus Penicillium sp. JF-55 cultures afforded a new PTP1B inhibitory styrylpyrone-type metabolite named penstyrylpyrone (1), and two known metabolites, anhydrofulvic acid (2) and citromycetin (3). Compounds 1 and 2 inhibited PTP1B activity in a dose-dependent manner, and kinetic analyses of PTP1B inhibition suggested that these compounds inhibited PTP1B activity in a competitive manner. In an effort to gain more biological potential of the isolated compounds, the anti-inflammatory effects of compounds 1–3 were also evaluated. Among the tested compounds, only compound 1 inhibited the production of NO and PGE2, due to the inhibition of the expression of iNOS and COX-2. Penstyrylpyrone (1) also reduced TNF-α and IL-1β production, and these anti-inflammatory effects were shown to be correlated with the suppression of the phosphorylation and degradation of IκB-α, NF-κB nuclear translocation, and NF-κB DNA binding activity. In addition, using inhibitor tin protoporphyrin (SnPP), an inhibitor of HO-1, it was verified that the inhibitory effects of penstyrylpyrone (1) on the pro-inflammatory mediators and NF-κB DNA binding activity were associated with the HO-1 expression. Therefore, these results suggest that penstyrylpyrone (1) suppresses PTP1B activity, as well as the production of pro-inflammatory mediators via NF-κB pathway, through expression of anti-inflammatory HO-1. PMID:23612372
Heat Shock Protein B1-Deficient Mice Display Impaired Wound Healing

PubMed Central

McNamee, Kay; Przybycien, Paulina M.; Lu, Xin; Williams, Richard O.; Bou-Gharios, George; Saklatvala, Jeremy; Dean, Jonathan L. E.

2013-01-01

There is large literature describing in vitro experiments on heat shock protein (hsp)B1 but understanding of its function in vivo is limited to studies in mice overexpressing human hspB1 protein. Experiments in cells have shown that hspB1 has chaperone activity, a cytoprotective role, regulates inflammatory gene expression, and drives cell proliferation. To investigate the function of the protein in vivo we generated hspB1-deficient mice. HspB1-deficient fibroblasts display increased expression of the pro-inflammatory cytokine, interleukin-6, compared to wild-type cells, but reduced proliferation. HspB1-deficient fibroblasts exhibit reduced entry into S phase and increased expression of cyclin-dependent kinase inhibitors p27kip1 and p21waf1. The expression of hspB1 protein and mRNA is also controlled by the cell cycle. To investigate the physiological function of hspB1 in regulating inflammation and cell proliferation we used an excisional cutaneous wound healing model. There was a significant impairment in the rate of healing of wounds in hspB1-deficient mice, characterised by reduced re-epithelialisation and collagen deposition but also increased inflammation. HspB1 deficiency augments neutrophil infiltration in wounds, driven by increased chemokine (C-X-C motif) ligand 1 expression. This appears to be a general mechanism as similar results were obtained in the air-pouch and peritonitis models of acute inflammation. PMID:24143227
Proteins bound to polyribosomal mRNA of the embryonic axis of kidney bean seeds

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

Pushkarev, V.M.; Galkin, A.P.

The protein composition of polyribosome-bound mRNAs from the embryonic axis of germinating kidney bean seeds, isolated by an improved procedure including treatment with EDTA, low concentrations of RNase T/sub 1/, and centrifugation in a linear 15-40% sucrose concentration gradient, was investigated. Electrophoretic analysis of the proteins of purified fragments of mRNP obtained by the method described showed the presence of two to three major polypeptides (molecular weights 50, 38, 32 kilodaltons) and a number of minor ones. It was established that one of the major proteins is associated with the poly(A) sequences of mRNA (50 kilodaltons). The similarity of themore » protein compositions of polyribosomal mRNAs of kidney bean embryos and other objects studied is discussed.« less
The B(E2;4^+1->2^+1) / B(E2;2^+1->0^+1) Ratio in Even-Even Nuclei

NASA Astrophysics Data System (ADS)

Loelius, C.; Sharon, Y. Y.; Zamick, L.; G"Urdal, G.

2009-10-01

We considered 207 even-even nuclei throughout the chart of nuclides for which the NNDC Tables had data on the energies and lifetimes of the 2^+1 and 4^+1 states. Using these data we calculated for each nucleus the electric quadrupole transition strengths B(E2;4^+1->2^+1) and B(E2;2^+1->0^+1), as well as their ratio. The internal conversion coefficients were obtained by using the NNDC HSICC calculator. For each nucleus we plotted the B(E2) ratio against A, N, and Z. We found that for close to 90% of the nuclei considered the ratio had values between 0.5 and 2.5. Most of the outliers had magic numbers of protons or neutrons. Our ratio results were compared with the theoretical predictions for this ratio by different models--10/7 in the rotational model and 2 in the simplest vibrational model. In the rotational regions (for 150 < A < 180 and A > 220) the ratios were indeed close to 10/7. For the few nuclei thought to be vibrational the ratios were usually less than 2. Otherwise, we got a wide scatter of ratio values. Hence other models, including the NpNn scheme, must be considered in interpreting these results.
Liver-Specific Deletion of Protein-Tyrosine Phosphatase 1B (PTP1B) Improves Metabolic Syndrome and Attenuates Diet-Induced Endoplasmic Reticulum Stress

PubMed Central

Delibegovic, Mirela; Zimmer, Derek; Kauffman, Caitlin; Rak, Kimberly; Hong, Eun-Gyoung; Cho, You-Ree; Kim, Jason K.; Kahn, Barbara B.; Neel, Benjamin G.; Bence, Kendra K.

2009-01-01

OBJECTIVE—The protein tyrosine phosphatase PTP1B is a negative regulator of insulin signaling; consequently, mice deficient in PTP1B are hypersensitive to insulin. Because PTP1B−/− mice have diminished fat stores, the extent to which PTP1B directly regulates glucose homeostasis is unclear. Previously, we showed that brain-specific PTP1B−/− mice are protected against high-fat diet–induced obesity and glucose intolerance, whereas muscle-specific PTP1B−/− mice have increased insulin sensitivity independent of changes in adiposity. Here we studied the role of liver PTP1B in glucose homeostasis and lipid metabolism. RESEARCH DESIGN AND METHODS—We analyzed body mass/adiposity, insulin sensitivity, glucose tolerance, and lipid metabolism in liver-specific PTP1B−/− and PTP1Bfl/fl control mice, fed a chow or high-fat diet. RESULTS—Compared with normal littermates, liver-specific PTP1B−/− mice exhibit improved glucose homeostasis and lipid profiles, independent of changes in adiposity. Liver-specific PTP1B−/− mice have increased hepatic insulin signaling, decreased expression of gluconeogenic genes PEPCK and G-6-Pase, enhanced insulin-induced suppression of hepatic glucose production, and improved glucose tolerance. Liver-specific PTP1B−/− mice exhibit decreased triglyceride and cholesterol levels and diminished expression of lipogenic genes SREBPs, FAS, and ACC. Liver-specific PTP1B deletion also protects against high-fat diet–induced endoplasmic reticulum stress response in vivo, as evidenced by decreased phosphorylation of p38MAPK, JNK, PERK, and eIF2α and lower expression of the transcription factors C/EBP homologous protein and spliced X box-binding protein 1. CONCLUSIONS—Liver PTP1B plays an important role in glucose and lipid metabolism, independent of alterations in adiposity. Inhibition of PTP1B in peripheral tissues may be useful for the treatment of metabolic syndrome and reduction of cardiovascular risk in addition to
A PP2A-B55 recognition signal controls substrate dephosphorylation kinetics during mitotic exit

PubMed Central

Cundell, Michael J.; Holder, James

2016-01-01

PP2A-B55 is one of the major phosphatases regulating cell division. Despite its importance for temporal control during mitotic exit, how B55 substrates are recognized and differentially dephosphorylated is unclear. Using phosphoproteomics combined with kinetic modeling to extract B55-dependent rate constants, we have systematically identified B55 substrates and assigned their temporal order in mitotic exit. These substrates share a bipartite polybasic recognition determinant (BPR) flanking a Cdk1 phosphorylation site. Experiments and modeling show that dephosphorylation rate is encoded into B55 substrates, including its inhibitor ENSA, by cooperative action of basic residues within the BPR. A complementary acidic surface on B55 decodes this signal, supporting a cooperative electrostatic mechanism for substrate selection. A further level of specificity is encoded into B55 substrates because B55 displays selectivity for phosphothreonine. These simple biochemical properties, combined with feedback control of B55 activity by the phosphoserine-containing substrate/inhibitor ENSA, can help explain the temporal sequence of events during exit from mitosis. PMID:27551054
Characterization of the Expression of the Petunia Glycine-Rich Protein-1 Gene Product 1

PubMed Central

Condit, Carol M.; McLean, B. Gail; Meagher, Richard B.

1990-01-01

We have examined the expression of the petunia (Petunia hybrida) glycine-rich protein-1 (ptGRP1) gene product using an antibody raised against a synthetic peptide comprising amino acids 22 through 36 of the mature ptGRP1 protein. This antibody recognizes a single protein of 23 kilodaltons. Cell fractionation studies showed that, as predicted (CM Condit, RB Meagher [1986] Nature 323: 178-181), ptGRP1 is most likely localized in the cell wall. In addition, it was found that (extractable) ptGRP1 is present in much higher abundance in unexpanded than in fully expanded tissue, with highest levels of accumulation in the bud. This same developmentally regulated pattern of protein expression was found in all varieties of petunia tested. In addition, tissue blots of petunia stem sections showed that ptGRP1 is localized to within the vascular tissue (to at least the phloem or cambium) and to either the epidermal cells or to a layer of collenchyma cells directly below the epidermis. Localization of ptGRP1 antigen in these cell types is shown to occur at different times in the overall development of the plant and at different quantitative levels. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 PMID:16667509
Sulfolobus chromatin proteins modulate strand displacement by DNA polymerase B1

PubMed Central

Sun, Fei; Huang, Li

2013-01-01

Strand displacement by a DNA polymerase serves a key role in Okazaki fragment maturation, which involves displacement of the RNA primer of the preexisting Okazaki fragment into a flap structure, and subsequent flap removal and fragment ligation. We investigated the role of Sulfolobus chromatin proteins Sso7d and Cren7 in strand displacement by DNA polymerase B1 (PolB1) from the hyperthermophilic archaeon Sulfolobus solfataricus. PolB1 showed a robust strand displacement activity and was capable of synthesizing thousands of nucleotides on a DNA-primed 72-nt single-stranded circular DNA template. This activity was inhibited by both Sso7d and Cren7, which limited the flap length to 3–4 nt at saturating concentrations. However, neither protein inhibited RNA displacement on an RNA-primed single-stranded DNA minicircle by PolB1. Strand displacement remained sensitive to modulation by the chromatin proteins when PolB1 was in association with proliferating cell nuclear antigen. Inhibition of DNA instead of RNA strand displacement by the chromatin proteins is consistent with the finding that double-stranded DNA was more efficiently bound and stabilized than an RNA:DNA duplex by these proteins. Our results suggest that Sulfolobus chromatin proteins modulate strand displacement by PolB1, permitting efficient removal of the RNA primer while inhibiting excessive displacement of the newly synthesized DNA strand during Okazaki fragment maturation. PMID:23821667
mTORC1 signalling and eIF4E/4E-BP1 translation initiation factor stoichiometry influence recombinant protein productivity from GS-CHOK1 cells.

PubMed

Jossé, Lyne; Xie, Jianling; Proud, Christopher G; Smales, C Mark

2016-12-15

Many protein-based biotherapeutics are produced in cultured Chinese hamster ovary (CHO) cell lines. Recent reports have demonstrated that translation of recombinant mRNAs and global control of the translation machinery via mammalian target of rapamycin (mTOR) signalling are important determinants of the amount and quality of recombinant protein such cells can produce. mTOR complex 1 (mTORC1) is a master regulator of cell growth/division, ribosome biogenesis and protein synthesis, but the relationship between mTORC1 signalling, cell growth and proliferation and recombinant protein yields from mammalian cells, and whether this master regulating signalling pathway can be manipulated to enhance cell biomass and recombinant protein production (rPP) are not well explored. We have investigated mTORC1 signalling and activity throughout batch culture of a panel of sister recombinant glutamine synthetase-CHO cell lines expressing different amounts of a model monoclonal IgG4, to evaluate the links between mTORC1 signalling and cell proliferation, autophagy, recombinant protein expression, global protein synthesis and mRNA translation initiation. We find that the expression of the mTORC1 substrate 4E-binding protein 1 (4E-BP1) fluctuates throughout the course of cell culture and, as expected, that the 4E-BP1 phosphorylation profiles change across the culture. Importantly, we find that the eIF4E/4E-BP1 stoichiometry positively correlates with cell productivity. Furthermore, eIF4E amounts appear to be co-regulated with 4E-BP1 amounts. This may reflect a sensing of either change at the mRNA level as opposed to the protein level or the fact that the phosphorylation status, as well as the amount of 4E-BP1 present, is important in the co-regulation of eIF4E and 4E-BP1. © 2016 The Author(s).
B1 Cell IgE Impedes Mast Cell-Mediated Enhancement of Parasite Expulsion through B2 IgE Blockade.

PubMed

Martin, Rebecca K; Damle, Sheela R; Valentine, Yolander A; Zellner, Matthew P; James, Briana N; Lownik, Joseph C; Luker, Andrea J; Davis, Elijah H; DeMeules, Martha M; Khandjian, Laura M; Finkelman, Fred D; Urban, Joseph F; Conrad, Daniel H

2018-02-13

Helminth infection is known for generating large amounts of poly-specific IgE. Here we demonstrate that innate-like B1 cells are responsible for this IgE production during infection with the nematode parasites Nippostrongylus brasiliensis and Heligmosomoides polygyrus bakeri. In vitro analysis of B1 cell immunoglobulin class switch recombination to IgE demonstrated a requirement for anti-CD40 and IL-4 that was further enhanced when IL-5 was added or when the B1 source was helminth infected mice. An IL-25-induced upregulation of IgE in B1 cells was also demonstrated. In T cell-reconstituted RAG1 -/- mice, N. brasiliensis clearance was enhanced with the addition of B2 cells in an IgE-dependent manner. This enhanced clearance was impeded by reconstitution with IgE sufficient B1 cells. Mucosal mast cells mediated the B2 cell enhancement of clearance in the absence of B1 cells. The data support B1 cell IgE secretion as a regulatory response exploited by the helminth. Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.
Comparative differences between T1a/b and T1e/m as substages in T1 urothelial carcinoma of the bladder.

PubMed

Turan, Turgay; Efiloğlu, Özgür; Günaydin, Bilal; Özkanli, Şeyma; Nikerel, Emrah; Atiş, Gökhan; Çaşkurlu, Turhan; Yildirim, Asif

2018-01-01

To evaluate the prognostic value of the depth of lamina propria invasion in patients with T1 bladder cancer and to display comparative differences between the T1a/b and T1e/m substaging systems. This study included 106 patients with primary stage T1 urothelial bladder tumours who underwent surgery between January 2009 and December 2014. Pathologic specimens were re-evaluated to confirm the diagnosis of T1 and substaging by the same pathologist using two systems: T1a and T1b, and T1m and T1e. Age, tumour size, multiplicity, associated carcinoma in situ, tumour grade, and T1 substaging system were investigated to detect the relation between disease progression and recurrence. The recurrence rate was 52% for T1a (n=42) vs. 76% for T1b (n=20) (p=0.028) and 55% for T1m (n=32) vs. 62% for T1e (n=30), respectively (p=0.446). There was no significant difference between the substaging groups for disease progression: T1a (n=12, 15%) vs. T1b (n=7, 27%), and T1m (n=8, 13.8%) vs. T1e (n=11, 23%) (p>0.05). In the multivariate analysis, tumour size >3 cm (p=0.008), multiplicity (p=0.049), and substaging T1b (p=0.043) were independent predictive factors for tumour recurrence. According to the Kaplan-Meier actuarial method, recurrence-free survival was significantly different in patients with pT1a tumours compared with those with pT1b tumours (p=0.033). Substaging T1 provides a prediction of disease recurrence. Regarding recurrence, T1a/b substaging can provide better knowledge of disease behaviour because it is predicted as more superior than T1 m/e, and it can help in determining the requirement for early cystectomy. Copyright® by the International Brazilian Journal of Urology.

IL-10 production by B cells expressing CD5 with the alternative exon 1B.

PubMed

Garaud, Soizic; Le Dantec, Christelle; de Mendoza, Agnès Revol; Mageed, Rizgar A; Youinou, Pierre; Renaudineau, Yves

2009-09-01

B lymphocytes are divided into two subpopulations, B1 and B2 cells based on expression of the T cell-associated protein CD5. Natural B1 cells are further divided into B1a cells that express CD5 on their membrane and B1b cells that do not but share most other biological characteristics of B1a cells. Recent studies from our laboratory have revealed, in humans, the existence of two alternative isoforms of the CD5 protein. A cell surface CD5 isoform which uses exon 1A (E1A) of the gene in B1a cells, and an intracellular isoform which uses exon 1B (E1B) mainly in human B1b cells. Indeed, the protein isoform encoded by transcripts containing E1B lack the leader peptide and is, thus, retained in the cytoplasm of B cells. The restriction of interleukin (IL)-10 to B1 lymphocytes in the mouse raises the possibility that the human CD5-E1B-expressing B cells produce IL-10. This prediction was confirmed in the CD5 negative Jok-1 B cells transfected with cDNA for either isoforms resulted in high level IL-10 production. Our data indicate that E1B-CD5-expressing B cells have the capacity to interfere with the immune response through their ability to produce high levels of IL-10.
Molecular cloning and immunoglobulin E reactivity of a natural rubber latex lecithinase homologue, the major allergenic component of Hev b 4.

PubMed

Sunderasan, E; Bahari, A; Arif, S A M; Zainal, Z; Hamilton, R G; Yeang, H Y

2005-11-01

Hev b 4 is an allergenic natural rubber latex (NRL) protein complex that is reactive in skin prick tests and in vitro immunoassays. On SDS-polyacrylamide gel electrophoresis (SDS-PAGE), Hev b 4 is discerned predominantly at 53-55 kDa together with a 57 kDa minor component previously identified as a cyanogenic glucosidase. Of the 13 NRL allergens recognized by the International Union of Immunological Societies, the 53-55 kDa Hev b 4 major protein is the only candidate that lacks complete cDNA and protein sequence information. We sought to clone the transcript encoding the Hev b 4 major protein, and characterize the native protein and its recombinant form in relation to IgE binding. The 5'/3' rapid amplification of cDNA ends method was employed to obtain the complete cDNA of the Hev b 4 major protein. A recombinant form of the protein was over-expressed in Escherichia coli. The native Hev b 4 major protein was deglycosylated by trifluoromethane sulphonic acid. Western immunoblots of the native, deglycosylated and recombinant proteins were performed using both polyclonal antibodies and sera from latex-allergic patients. The cDNA encoding the Hev b 4 major protein was cloned. Its open reading frame matched lecithinases in the conserved domain database and contained 10 predicted glycosylation sites. Detection of glycans on the Hev b 4 lecithinase homologue confirmed it to be a glycoprotein. The deglycosylated lecithinase homologue was discerned at 40 kDa on SDS-PAGE, this being comparable to the 38.53 kDa mass predicted by its cDNA. Deglycosylation of the lecithinase homologue resulted in the loss of IgE recognition, although reactivity to polyclonal rabbit anti-Hev b 4 was retained. IgE from latex-allergic patients also failed to recognize the non-glycosylated E. coli recombinant lecithinase homologue. The IgE epitopes of the Hev b 4 lecithinase homologue reside mainly in its carbohydrate moiety, which also account for the discrepancy between the observed molecular
TRAF2-binding BIR1 domain of c-IAP2/MALT1 fusion protein is essential for activation of NF-kappaB.

PubMed

Garrison, J B; Samuel, T; Reed, J C

2009-04-02

Marginal zone mucosa-associated lymphoid tissue (MALT) B-cell lymphoma is the most common extranodal non-Hodgkin lymphoma. The t(11;18)(q21;q21) translocation occurs frequently in MALT lymphomas and creates a chimeric NF-kappaB-activating protein containing the baculoviral IAP repeat (BIR) domains of c-IAP2 (inhibitor of apoptosis protein 2) fused with portions of the MALT1 protein. The BIR1 domain of c-IAP2 interacts directly with TRAF2 (TNFalpha-receptor-associated factor-2), but its role in NF-kappaB activation is still unclear. Here, we investigated the role of TRAF2 in c-IAP2/MALT1-induced NF-kappaB activation. We show the BIR1 domain of c-IAP2 is essential for NF-kappaB activation, whereas BIR2 and BIR3 domains are not. Studies of c-IAP2/MALT1 BIR1 mutant (E47A/R48A) that fails to activate NF-kappaB showed loss of TRAF2 binding, but retention of TRAF6 binding, suggesting that interaction of c-IAP2/MALT1 with TRAF6 is insufficient for NF-kappaB induction. In addition, a dominant-negative TRAF2 mutant or downregulation of TRAF2 achieved by small interfering RNA inhibited NF-kappaB activation by c-IAP2/MALT1 showing that TRAF2 is indispensable. Comparisons of the bioactivity of intact c-IAP2/MALT1 oncoprotein and BIR1 E47A/R48A c-IAP2/MALT1 mutant that cannot bind TRAF2 in a lymphoid cell line provided evidence that TRAF2 interaction is critical for c-IAP2/MALT1-mediated increases in the NF-kappaB activity, increased expression of endogenous NF-kappaB target genes (c-FLIP, TRAF1), and resistance to apoptosis.
Ki-1/57 and CGI-55 ectopic expression impact cellular pathways involved in proliferation and stress response regulation.

PubMed

Costa, Fernanda C; Saito, Angela; Gonçalves, Kaliandra A; Vidigal, Pedro M; Meirelles, Gabriela V; Bressan, Gustavo C; Kobarg, Jörg

2014-12-01

Ki-1/57 (HABP4) and CGI-55 (SERBP1) are regulatory proteins and paralogs with 40.7% amino acid sequence identity and 67.4% similarity. Functionally, they have been implicated in the regulation of gene expression on both the transcriptional and mRNA metabolism levels. A link with tumorigenesis is suggested, since both paralogs show altered expression levels in tumor cells and the Ki-1/57 gene is found in a region of chromosome 9q that represents a haplotype for familiar colon cancer. However, the target genes regulated by Ki-1/57 and CGI-55 are unknown. Here, we analyzed the alterations of the global transcriptome profile after Ki-1/57 or CGI-55 overexpression in HEK293T cells by DNA microchip technology. We were able to identify 363 or 190 down-regulated and 50 or 27 up-regulated genes for Ki-1/57 and CGI-55, respectively, of which 20 were shared between both proteins. Expression levels of selected genes were confirmed by qRT-PCR both after protein overexpression and siRNA knockdown. The majority of the genes with altered expression were associated to proliferation, apoptosis and cell cycle control processes, prompting us to further explore these contexts experimentally. We observed that overexpression of Ki-1/57 or CGI-55 results in reduced cell proliferation, mainly due to a G1 phase arrest, whereas siRNA knockdown of CGI-55 caused an increase in proliferation. In the case of Ki-1/57 overexpression, we found protection from apoptosis after treatment with the ER-stress inducer thapsigargin. Together, our data give important new insights that may help to explain these proteins putative involvement in tumorigenic events. Copyright © 2014 Elsevier B.V. All rights reserved.
Protein Phosphotyrosine Phosphatase 1B (PTP1B) in Calpain-dependent Feedback Regulation of Vascular Endothelial Growth Factor Receptor (VEGFR2) in Endothelial Cells

PubMed Central

Zhang, Yixuan; Li, Qiang; Youn, Ji Youn; Cai, Hua

2017-01-01

The VEGF/VEGFR2/Akt/eNOS/NO pathway is essential to VEGF-induced angiogenesis. We have previously discovered a novel role of calpain in mediating VEGF-induced PI3K/AMPK/Akt/eNOS activation through Ezrin. Here, we sought to identify possible feedback regulation of VEGFR2 by calpain via its substrate protein phosphotyrosine phosphatase 1B (PTP1B), and the relevance of this pathway to VEGF-induced angiogenesis, especially in diabetic wound healing. Overexpression of PTP1B inhibited VEGF-induced VEGFR2 and Akt phosphorylation in bovine aortic endothelial cells, while PTP1B siRNA increased both, implicating negative regulation of VEGFR2 by PTP1B. Calpain inhibitor ALLN induced VEGFR2 activation, which can be completely blocked by PTP1B overexpression. Calpain activation induced by overexpression or Ca/A23187 resulted in PTP1B cleavage, which can be blocked by ALLN. Moreover, calpain activation inhibited VEGF-induced VEGFR2 phosphorylation, which can be restored by PTP1B siRNA. These data implicate calpain/PTP1B negative feedback regulation of VEGFR2, in addition to the primary signaling pathway of VEGF/VEGFR2/calpain/PI3K/AMPK/Akt/eNOS. We next examined a potential role of PTP1B in VEGF-induced angiogenesis. Endothelial cells transfected with PTP1B siRNA showed faster wound closure in response to VEGF. Aortic discs isolated from PTP1B siRNA-transfected mice also had augmented endothelial outgrowth. Importantly, PTP1B inhibition and/or calpain overexpression significantly accelerated wound healing in STZ-induced diabetic mice. In conclusion, our data for the first time demonstrate a calpain/PTP1B/VEGFR2 negative feedback loop in the regulation of VEGF-induced angiogenesis. Modulation of local PTP1B and/or calpain activities may prove beneficial in the treatment of impaired wound healing in diabetes. PMID:27872190
Polyamine binding to proteins in oat and Petunia protoplasts

NASA Technical Reports Server (NTRS)

Mizrahi, Y.; Applewhite, P. B.; Galston, A. W.

1989-01-01

Previous work (A Apelbaum et al. [1988] Plant Physiol 88: 996-998) has demonstrated binding of labeled spermidine (Spd) to a developmentally regulated 18 kilodalton protein in tobacco tissue cultures derived from thin surface layer explants. To assess the general importance of such Spd-protein complexes, we attempted bulk isolation from protoplasts of Petunia and oat (Avena sativa). In Petunia, as in tobacco, fed radioactive Spd is bound to protein, but in oat, Spd is first converted to 1,3,-diaminopropane (DAP), probably by polyamine oxidase action. In oat, binding of DAP to protein depends on age of donor leaf and conditions of illumination and temperature, and the extraction of the DAP-protein complex depends upon buffer and pH. The yield of the DAP-protein complex was maximized by extraction of frozen-thawed protoplasts with a pH 8.8 carbonate buffer containing SDS. Its molecular size, based on Sephacryl column fractionation of ammonium sulfate precipitated material, exceeded 45 kilodaltons. Bound Spd or DAP can be released from their complexes by the action of Pronase, but not DNAse, RNAse, or strong salt solutions, indicating covalent attachment to protein.
Polyamine binding to proteins in oat and Petunia protoplasts.

PubMed

Mizrahi, Y; Applewhite, P B; Galston, A W

1989-01-01

Previous work (A Apelbaum et al. [1988] Plant Physiol 88: 996-998) has demonstrated binding of labeled spermidine (Spd) to a developmentally regulated 18 kilodalton protein in tobacco tissue cultures derived from thin surface layer explants. To assess the general importance of such Spd-protein complexes, we attempted bulk isolation from protoplasts of Petunia and oat (Avena sativa). In Petunia, as in tobacco, fed radioactive Spd is bound to protein, but in oat, Spd is first converted to 1,3,-diaminopropane (DAP), probably by polyamine oxidase action. In oat, binding of DAP to protein depends on age of donor leaf and conditions of illumination and temperature, and the extraction of the DAP-protein complex depends upon buffer and pH. The yield of the DAP-protein complex was maximized by extraction of frozen-thawed protoplasts with a pH 8.8 carbonate buffer containing SDS. Its molecular size, based on Sephacryl column fractionation of ammonium sulfate precipitated material, exceeded 45 kilodaltons. Bound Spd or DAP can be released from their complexes by the action of Pronase, but not DNAse, RNAse, or strong salt solutions, indicating covalent attachment to protein.
Expression of the 68-kilodalton neurofilament gene in aluminum intoxication

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

Muma, N.A.; Troncoso, J.C.; Hoffman, P.N.

1986-03-01

Intrathecal administration of aluminum salts induces accumulation of neurofilaments (NFs) in cell bodies and proximal axons of rabbit spinal motor neurons. Mechanisms leading to this pathological change are not well understood. Although impairments of NF transport have been demonstrated in this model, the hypothesis that NF accumulations are the result of an increase in NF synthesis needs to be explored. In rabbits, a large percentage of neurons develop accumulations of NFs following injections of aluminum lactate directly into the cisterna magna or into a reservoir placed in the lateral ventricle. To study levels of mRNA encoding cytoskeletal proteins, spinal cordmore » RNA was extracted, separated on a denaturing agarose gel, transferred to nitrocellulose paper, and hybridized to (/sup 32/P)-labeled cDNA clones encoding the mouse 68-kilodalton (kd) NF subunit and tubulin. Examining a constant amount of RNA, the radioactivity of labeled mRNA bands for the 68-kd NF subunit and for tubulin was decreased in spinal cords of aluminum-treated rabbits. These preliminary results will be followed up by in situ hybridization to determine levels of mRNA for tubulin and 68-kd NF subunit in affected and in normal spinal neurons. In conclusion, administration of aluminum decreased mRNA for the 608-kd NF protein in spinal neurons.« less
Mapping the B cell epitopes within the major capsid protein L1 of human papillomavirus type 16.

PubMed

Wang, Aiping; Li, Ning; Zhou, Jingming; Chen, Yumei; Jiang, Min; Qi, Yanhua; Liu, Hongliang; Liu, Yankai; Liu, Dongmin; Zhao, Jianguo; Wang, Yanwei; Zhang, Gaiping

2018-06-26

Persistent infection with human papillomavirus type16 (HPV16) has much association with the development of cervical cancer. L1 is the major capsid protein of HPV, it has been well investigated as a potential vaccine candidate. However, B cell epitopes present on L1 have not been well characterized. To identify the potential B-cell antigenic epitopes within HPV16 L1 protein, sixteen serial overlapping truncations (H1-H16) covering the whole region were expressed in E. coli and used in mice immunization. The mice antisera were tested in ELISA binding, IFA and HI assays. Finally, four fragments (H2, H4, H11, H12) were found to contain B cell epitopes of HPV16 L1 protein in ELISA and IFA assays, three fragments (H2, H3, H9) might contain neutralizing epitopes of HPV16 L1 protein in HI assay. Among them, H11 and H12 fragments contain B cell epitopes have never been reported before, and H3 was found as hemagglutination inhibition epitope for the first time. This work provides new insights to B cell epitopes on HPV16 L1 protein. Several new epitopes were identified and may provide some guidance for HPV16 subunit vaccine design. The results of this study might open new perspectives on the antibody-antigen reaction and have important implications for the development of epitopes-based protective HPV16 vaccines. Copyright © 2018. Published by Elsevier B.V.
Aldo-keto Reductase 1B15 (AKR1B15)

PubMed Central

Weber, Susanne; Salabei, Joshua K.; Möller, Gabriele; Kremmer, Elisabeth; Bhatnagar, Aruni; Adamski, Jerzy; Barski, Oleg A.

2015-01-01

Aldo-keto reductases (AKRs) comprise a superfamily of proteins involved in the reduction and oxidation of biogenic and xenobiotic carbonyls. In humans, at least 15 AKR superfamily members have been identified so far. One of these is a newly identified gene locus, AKR1B15, which clusters on chromosome 7 with the other human AKR1B subfamily members (i.e. AKR1B1 and AKR1B10). We show that alternative splicing of the AKR1B15 gene transcript gives rise to two protein isoforms with different N termini: AKR1B15.1 is a 316-amino acid protein with 91% amino acid identity to AKR1B10; AKR1B15.2 has a prolonged N terminus and consists of 344 amino acid residues. The two gene products differ in their expression level, subcellular localization, and activity. In contrast with other AKR enzymes, which are mostly cytosolic, AKR1B15.1 co-localizes with the mitochondria. Kinetic studies show that AKR1B15.1 is predominantly a reductive enzyme that catalyzes the reduction of androgens and estrogens with high positional selectivity (17β-hydroxysteroid dehydrogenase activity) as well as 3-keto-acyl-CoA conjugates and exhibits strong cofactor selectivity toward NADP(H). In accordance with its substrate spectrum, the enzyme is expressed at the highest levels in steroid-sensitive tissues, namely placenta, testis, and adipose tissue. Placental and adipose expression could be reproduced in the BeWo and SGBS cell lines, respectively. In contrast, AKR1B15.2 localizes to the cytosol and displays no enzymatic activity with the substrates tested. Collectively, these results demonstrate the existence of a novel catalytically active AKR, which is associated with mitochondria and expressed mainly in steroid-sensitive tissues. PMID:25577493
Cross-talk between Carboxypeptidase M and the Kinin B1 Receptor Mediates a New Mode of G Protein-coupled Receptor Signaling*

PubMed Central

Zhang, Xianming; Tan, Fulong; Brovkovych, Viktor; Zhang, Yongkang; Skidgel, Randal A.

2011-01-01

G protein-coupled receptor (GPCR) signaling is affected by formation of GPCR homo- or heterodimers, but GPCR regulation by other cell surface proteins is not well understood. We reported that the kinin B1 receptor (B1R) heterodimerizes with membrane carboxypeptidase M (CPM), facilitating receptor signaling via CPM-mediated conversion of bradykinin or kallidin to des-Arg kinin B1R agonists. Here, we found that a catalytically inactive CPM mutant that still binds substrate (CPM-E264Q) also facilitates efficient B1R signaling by B2 receptor agonists bradykinin or kallidin. This response required co-expression of B1R and CPM-E264Q in the same cell, was disrupted by antibody that dissociates CPM from B1R, and was not found with a CPM-E264Q-B1R fusion protein. An additional mutation that reduced the affinity of CPM for C-terminal Arg and increased the affinity for C-terminal Lys inhibited the B1R response to bradykinin (with C-terminal Arg) but generated a response to Lys9-bradykinin. CPM-E264Q-mediated activation of B1Rs by bradykinin resulted in increased intramolecular fluorescence resonance energy transfer (FRET) in a B1R FRET construct, similar to that generated directly by a B1R agonist. In cytokine-treated human lung microvascular endothelial cells, disruption of B1R-CPM heterodimers inhibited B1R-dependent NO production stimulated by bradykinin and blocked the increased endothelial permeability caused by treatment with bradykinin and pyrogallol (a superoxide generator). Thus, CPM and B1Rs on cell membranes form a critical complex that potentiates B1R signaling. Kinin peptide binding to CPM causes a conformational change in the B1R leading to intracellular signaling and reveals a new mode of GPCR activation by a cell surface peptidase. PMID:21454694
Human Parvovirus B19 NS1 Protein Aggravates Liver Injury in NZB/W F1 Mice

PubMed Central

Tsai, Chun-Chou; Chiu, Chun-Ching; Hsu, Jeng-Dong; Hsu, Huai-Sheng; Tzang, Bor-Show; Hsu, Tsai-Ching

2013-01-01

Human parvovirus B19 (B19) has been associated with a variety of diseases. However, the influence of B19 viral proteins on hepatic injury in SLE is still obscure. To elucidate the effects of B19 viral proteins on livers in SLE, recombinant B19 NS1, VP1u or VP2 proteins were injected subcutaneously into NZB/W F1 mice, respectively. Significant expressions of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) were detected in NZB/W F1 mice receiving B19 NS1 as compared to those mice receiving PBS. Markedly hepatocyte disarray and lymphocyte infiltration were observed in livers from NZB/WF 1 mice receiving B19 NS1 as compared to those mice receiving PBS. Additionally, significant increases of Tumor Necrosis Factor –α (TNF-α), TNF-α receptor, IκB kinase –α (IKK-α), nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor (IκB) and nuclear factor-kappa B (NF-κB) were detected in livers from NZB/W F1 mice receiving B19 NS1 as compared to those mice receiving PBS. Accordingly, significant increases of matrix metalloproteinase-9 (MMP9) and U-plasminogen activator (uPA) were also detected in livers from NZB/W F1 mice receiving B19 NS1 as compared to those mice receiving PBS. Contrarily, no significant variation on livers from NZB/W F1 mice receiving B19 VP1u or VP2 was observed as compared to those mice receiving PBS. These findings firstly demonstrated the aggravated effects of B19 NS1 but not VP1u or VP2 protein on hepatic injury and provide a clue in understanding the role of B19 NS1 on hepatic injury in SLE. PMID:23555760
Sperm Lysozyme-Like Protein 1 (SLLP1), an intra-acrosomal oolemmal-binding sperm protein, reveals filamentous organization in protein crystal form

PubMed Central

Zheng, Heping; Mandal, Arabinda; Shumilin, Igor A.; Chordia, Mahendra D.; Panneerdoss, Subbarayalu; Herr, John C.; Minor, Wladek

2016-01-01

Sperm Lysozyme-Like Protein 1 (SLLP1) is one of the lysozyme-like proteins predominantly expressed in mammalian testes that lacks bacteriolytic activity, localizes in the sperm acrosome, and exhibits high affinity for an oolemmal receptor, SAS1B. The crystal structure of mouse SLLP1 (mSLLP1) was determined at 2.15Å resolution. mSLLP1 monomer adopts a structural fold similar to that of chicken/mouse lysozymes retaining all four canonical disulfide bonds. mSLLP1 is distinct from c-lysozyme by substituting two essential catalytic residues (E35T/D52N), exhibiting different surface charge distribution, and by forming helical filaments approximately 75Å in diameter with a 25Å central pore comprised of six monomers per helix turn repeating every 33Å. Cross-species alignment of all reported SLLP1 sequences revealed a set of invariant surface regions comprising a characteristic fingerprint uniquely identifying SLLP1 from other c-lysozyme family members. The fingerprint surface regions reside around the lips of the putative glycan binding groove including three polar residues (Y33/E46/H113). A flexible salt bridge (E46-R61) was observed covering the glycan binding groove. The conservation of these regions may be linked to their involvement in oolemmal protein binding. Interaction between SLLP1 monomer and its oolemmal receptor SAS1B was modeled using protein-protein docking algorithms, utilizing the SLLP1 fingerprint regions along with the SAS1B conserved surface regions. This computational model revealed complementarity between the conserved SLLP1/SAS1B interacting surfaces supporting the experimentally-observed SLLP1/SAS1B interaction involved in fertilization. PMID:26198801
Sel1 repeat protein LpnE is a Legionella pneumophila virulence determinant that influences vacuolar trafficking.

PubMed

Newton, Hayley J; Sansom, Fiona M; Dao, Jenny; McAlister, Adrian D; Sloan, Joan; Cianciotto, Nicholas P; Hartland, Elizabeth L

2007-12-01

The environmental pathogen Legionella pneumophila possesses five proteins with Sel1 repeats (SLRs) from the tetratricopeptide repeat protein family. Three of these proteins, LpnE, EnhC, and LidL, have been implicated in the ability of L. pneumophila to efficiently establish infection and/or manipulate host cell trafficking events. Previously, we showed that LpnE is important for L. pneumophila entry into macrophages and epithelial cells. In further virulence studies here, we show that LpnE is also required for efficient infection of Acanthamoeba castellanii by L. pneumophila and for replication of L. pneumophila in the lungs of A/J mice. In addition, we found that the role of LpnE in host cell invasion is dependent on the eight SLR regions of the protein. A truncated form of LpnE lacking the two C-terminal SLR domains was unable to complement the invasion defect of an lpnE mutant of L. pneumophila 130b in both the A549 and THP-1 cell lines. The lpnE mutant displayed impaired avoidance of LAMP-1 association, suggesting that LpnE influenced trafficking of the L. pneumophila vacuole, similar to the case for EnhC and LidL. We also found that LpnE was present in L. pneumophila culture supernatants and that its export was independent of both the Lsp type II secretion system and the Dot/Icm type IV secretion system. The fact that LpnE was exported suggested that the protein may interact with a eukaryotic protein. Using LpnE as bait, we screened a HeLa cell cDNA library for interacting partners, using the yeast two-hybrid system. Examination of the protein-protein interaction between LpnE and a eukaryotic protein, obscurin-like protein 1, suggested that LpnE can interact with eukaryotic proteins containing immunoglobulin-like folds via the SLR regions. This investigation has further characterized the contribution of LpnE to L. pneumophila virulence and, more specifically, the importance of the SLR regions to LpnE function.
hnRNP A2/B1 interacts with influenza A viral protein NS1 and inhibits virus replication potentially through suppressing NS1 RNA/protein levels and NS1 mRNA nuclear export

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

Wang, Yimeng; Zhou, Jianhong; Du, Yuchun, E-mail: ydu@uark.edu

The NS1 protein of influenza viruses is a major virulence factor and exerts its function through interacting with viral/cellular RNAs and proteins. In this study, we identified heterogeneous nuclear ribonucleoprotein A2/B1 (hnRNP A2/B1) as an interacting partner of NS1 proteins by a proteomic method. Knockdown of hnRNP A2/B1 by small interfering RNA (siRNA) resulted in higher levels of NS vRNA, NS1 mRNA, and NS1 protein in the virus-infected cells. In addition, we demonstrated that hnRNP A2/B1 proteins are associated with NS1 and NS2 mRNAs and that knockdown of hnRNP A2/B1 promotes transport of NS1 mRNA from the nucleus to themore » cytoplasm in the infected cells. Lastly, we showed that knockdown of hnRNP A2/B1 leads to enhanced virus replication. Our results suggest that hnRNP A2/B1 plays an inhibitory role in the replication of influenza A virus in host cells potentially through suppressing NS1 RNA/protein levels and NS1 mRNA nucleocytoplasmic translocation. - Highlights: • Cellular protein hnRNP A2/B1 interacts with influenza viral protein NS1. • hnRNP A2/B1 suppresses the levels of NS1 protein, vRNA and mRNA in infected cells. • hnRNP A2/B1 protein is associated with NS1 and NS2 mRNAs. • hnRNP A2/B1 inhibits the nuclear export of NS1 mRNAs. • hnRNP A2/B1 inhibits influenza virus replication.« less
Compare the Difference of B-cell Epitopes of EgAgB1 and EgAgB3 Proteins Selected through Bioinformatic Analysis

NASA Astrophysics Data System (ADS)

An, Mengting; Zhang, Fengbo; Zhu, Yuejie; Zhao, Xiao; Ding, Jianbing

2018-01-01

Cystic echinococcosis, as a zoonosis, seriously endangers humans and animals, so early diagnosis of this disease is particularly important. Therefore, this study is to predict B-cell epitopes of EgAgB1 and EgAgB3 proteins by bioinformatics software. B-cell epitopes of EgAgB1 and EgAgB3 proteins are predicted using DNAStar and IEDB software. The results suggest that there are two potential B-cell epitopes in EgAgB1, which located in the 8-15 and 31-37 amino acid residue segments. And two potential B-cell epitopes in EgAgB2, located in the 20∼27 and 47∼53 amino acid residue segments. This study predicted the B-cell epitopes of EgAgB1 and EgAgB3 proteins, which laid the foundation for the early diagnosis of Cystic echinococcosis.
Asperentin B, a New Inhibitor of the Protein Tyrosine Phosphatase 1B.

PubMed

Wiese, Jutta; Aldemir, Hülya; Schmaljohann, Rolf; Gulder, Tobias A M; Imhoff, Johannes F

2017-06-21

In the frame of studies on secondary metabolites produced by fungi from deep-sea environments we have investigated inhibitors of enzymes playing key roles in signaling cascades of biochemical pathways relevant for the treatment of diseases. Here we report on a new inhibitor of the human protein tyrosine phosphatase 1B (PTP1B), a target in the signaling pathway of insulin. A new asperentin analog is produced by an Aspergillus sydowii strain isolated from the sediment of the deep Mediterranean Sea. Asperentin B ( 1 ) contains an additional phenolic hydroxy function at C-6 and exhibits an IC 50 value against PTP1B of 2 μM in vitro, which is six times stronger than the positive control, suramin. Interestingly, asperentin ( 2 ) did not show any inhibition of this enzymatic activity. Asperentin B ( 1 ) is discussed as possible therapeutic agents for type 2 diabetes and sleeping sickness.
Asperentin B, a New Inhibitor of the Protein Tyrosine Phosphatase 1B

PubMed Central

Wiese, Jutta; Aldemir, Hülya; Schmaljohann, Rolf; Gulder, Tobias A. M.; Imhoff, Johannes F.

2017-01-01

In the frame of studies on secondary metabolites produced by fungi from deep-sea environments we have investigated inhibitors of enzymes playing key roles in signaling cascades of biochemical pathways relevant for the treatment of diseases. Here we report on a new inhibitor of the human protein tyrosine phosphatase 1B (PTP1B), a target in the signaling pathway of insulin. A new asperentin analog is produced by an Aspergillus sydowii strain isolated from the sediment of the deep Mediterranean Sea. Asperentin B (1) contains an additional phenolic hydroxy function at C-6 and exhibits an IC50 value against PTP1B of 2 μM in vitro, which is six times stronger than the positive control, suramin. Interestingly, asperentin (2) did not show any inhibition of this enzymatic activity. Asperentin B (1) is discussed as possible therapeutic agents for type 2 diabetes and sleeping sickness. PMID:28635658
Protein tyrosine phosphatase 1B (PTP1B) is required for cardiac lineage differentiation of mouse embryonic stem cells.

PubMed

Eshkiki, Zahra Shokati; Ghahremani, Mohammad Hossein; Shabani, Parisa; Firuzjaee, Sattar Gorgani; Sadeghi, Asie; Ghanbarian, Hossein; Meshkani, Reza

2017-01-01

Protein tyrosine phosphatase 1B (PTP1B) has been shown to regulate multiple cellular events such as differentiation, cell growth, and proliferation; however, the role of PTP1B in differentiation of embryonic stem (ES) cells into cardiomyocytes remains unexplored. In the present study, we investigated the effects of PTP1B inhibition on differentiation of ES cells into cardiomyocytes. PTP1B mRNA and protein levels were increased during the differentiation of ES cells into cardiomyocytes. Accordingly, a stable ES cell line expressing PTP1B shRNA was established. In vitro, the number and size of spontaneously beating embryoid bodies were significantly decreased in PTP1B-knockdown cells, compared with the control cells. Decreased expression of cardiac-specific markers Nkx2-5, MHC-α, cTnT, and CX43, as assessed by real-time PCR analysis, was further confirmed by immunocytochemistry of the markers. The results also showed that PTP1B inhibition induced apoptosis in both differentiated and undifferentiated ES cells, as presented by increasing the level of cleaved caspase-3, cytochrome C, and cleaved PARP. Further analyses revealed that PTP1B inhibition did not change proliferation and pluripotency of undifferentiated ES cells. Taken together, the data presented here suggest that PTP1B is essential for proper differentiation of ES cells into cardiomyocytes.
RADIAL VELOCITY PLANETS DE-ALIASED: A NEW, SHORT PERIOD FOR SUPER-EARTH 55 Cnc e

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

Dawson, Rebekah I.; Fabrycky, Daniel C., E-mail: rdawson@cfa.harvard.ed, E-mail: daniel.fabrycky@gmail.co

2010-10-10

Radial velocity measurements of stellar reflex motion have revealed many extrasolar planets, but gaps in the observations produce aliases, spurious frequencies that are frequently confused with the planets' orbital frequencies. In the case of Gl 581 d, the distinction between an alias and the true frequency was the distinction between a frozen, dead planet and a planet possibly hospitable to life. To improve the characterization of planetary systems, we describe how aliases originate and present a new approach for distinguishing between orbital frequencies and their aliases. Our approach harnesses features in the spectral window function to compare the amplitude andmore » phase of predicted aliases with peaks present in the data. We apply it to confirm prior alias distinctions for the planets GJ 876 d and HD 75898 b. We find that the true periods of Gl 581 d and HD 73526 b/c remain ambiguous. We revise the periods of HD 156668 b and 55 Cnc e, which were afflicted by daily aliases. For HD 156668 b, the correct period is 1.2699 days and the minimum mass is (3.1 {+-} 0.4) M{sub +}. For 55 Cnc e, the correct period is 0.7365 days-the shortest of any known planet-and the minimum mass is (8.3 {+-} 0.3) M{sub +}. This revision produces a significantly improved five-planet Keplerian fit for 55 Cnc, and a self-consistent dynamical fit describes the data just as well. As radial velocity techniques push to ever-smaller planets, often found in systems of multiple planets, distinguishing true periods from aliases will become increasingly important.« less

Myelin protein zero gene mutated in Charcot-Marie-Tooth type 1B patients

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

Su, Ying; Li, Lanying; Lepercq, J.

1993-11-15

The autosomal dominant of Charcot-Marie-Tooth disease (CMT), whose gene is type 1B (CMT1B), has slow nerve conduction with demyelinated Schwann cells. In this study the abundant peripheral myelin protein zero (MPZ) gene, MPZ, was mapped 130 kb centromeric to the Fc receptor immunoglobulin gene cluster in band 1q22, and a major MPZ point mutation was found to cosegregate with CMT1B in one large CMT1B family. The MPZ point mutation in 18 of 18 related CMT1B pedigree 1 patients converts a positively charged lysine in codon 96 to a negatively charged glutamate. The same MPZ locus cosegregates with the CMT1B diseasemore » gene in a second CMT1B family [total multipoint logarithm of odds (lod) = 11.4 at [theta] = 0.00] with a splice junction mutation. Both mutations occur in MPZ protein regions otherwise conserved identically in human, rat, and cow since these species diverged 100 million years ago. MPZ protein, expressed exclusively in myelinated peripheral nerve Schwann cells, constitutes >50% of myelin protein. These mutations are anticipated to disrupt homophilic MPZ binding and result in CMT1B peripheral nerve demyelination.« less
Sperm Lysozyme-Like Protein 1 (SLLP1), an intra-acrosomal oolemmal-binding sperm protein, reveals filamentous organization in protein crystal form.

PubMed

Zheng, H; Mandal, A; Shumilin, I A; Chordia, M D; Panneerdoss, S; Herr, J C; Minor, W

2015-07-01

Sperm lysozyme-like protein 1 (SLLP1) is one of the lysozyme-like proteins predominantly expressed in mammalian testes that lacks bacteriolytic activity, localizes in the sperm acrosome, and exhibits high affinity for an oolemmal receptor, SAS1B. The crystal structure of mouse SLLP1 (mSLLP1) was determined at 2.15 Å resolution. mSLLP1 monomer adopts a structural fold similar to that of chicken/mouse lysozymes retaining all four canonical disulfide bonds. mSLLP1 is distinct from c-lysozyme by substituting two essential catalytic residues (E35T/D52N), exhibiting different surface charge distribution, and by forming helical filaments approximately 75 Å in diameter with a 25 Å central pore comprised of six monomers per helix turn repeating every 33 Å. Cross-species alignment of all reported SLLP1 sequences revealed a set of invariant surface regions comprising a characteristic fingerprint uniquely identifying SLLP1 from other c-lysozyme family members. The fingerprint surface regions reside around the lips of the putative glycan-binding groove including three polar residues (Y33/E46/H113). A flexible salt bridge (E46-R61) was observed covering the glycan-binding groove. The conservation of these regions may be linked to their involvement in oolemmal protein binding. Interaction between SLLP1 monomer and its oolemmal receptor SAS1B was modeled using protein-protein docking algorithms, utilizing the SLLP1 fingerprint regions along with the SAS1B conserved surface regions. This computational model revealed complementarity between the conserved SLLP1/SAS1B interacting surfaces supporting the experimentally observed SLLP1/SAS1B interaction involved in fertilization. © 2015 American Society of Andrology and European Academy of Andrology.
Protein tyrosine phosphatase 1B as a target for the treatment of impaired glucose tolerance and type II diabetes.

PubMed

Liu, Gang; Trevillyan, James M

2002-11-01

Protein tyrosine phosphatase 1B (PTP1B) is a negative regulator of the insulin signal transduction cascade, initiated when insulin binds to the insulin receptor. PTP1B-deficient mice are more sensitive to insulin, and have improved glycemic control and resistance to diet-induced obesity than wild-type control mice. Diabetic mice treated with PTP1B antisense oligonucleotides intraperitoneally have lower PTP1B protein levels in liver and fat, reduced plasma insulin, blood glucose and hemoglobin A1c (HbA1c) levels. These studies validate PTP1B as a promising drug discovery target for the treatment of insulin resistance, diabetes and obesity. Herein we review the recent advances in the structure-based design of potent and selective small molecule inhibitors of PTP1B, and discuss th e challenge of developing compounds with improved cell permeability and bioavailability.
Reduction of PTP1B induces differential expression of PI3-kinase (p85alpha) isoforms.

PubMed

Rondinone, Cristina M; Clampit, Jill; Gum, Rebecca J; Zinker, Bradley A; Jirousek, Michael R; Trevillyan, James M

2004-10-15

Protein tyrosine phosphatase 1B (PTP1B) inhibition increases insulin sensitivity and normalizes blood glucose levels in animals. The molecular events associated with PTP1B inhibition that increase insulin sensitivity remain controversial. Insulin resistant, diabetic ob/ob mice, dosed with PTP1B antisense for 3 weeks exhibited a decrease in PTP1B protein levels and a change in the expression level of p85alpha isoforms in liver, characterized by a reduction in p85alpha and an upregulation of the p50alpha and p55alpha isoforms. Transfection of mouse hepatocytes with PTP1B antisense caused a downregulation PTP1B and p85alpha protein levels. Furthermore, transfection of mouse hepatocytes with PTP1B siRNA downregulated p85alpha protein expression and enhanced insulin-induced PKB phosphorylation. Treatment of mouse hepatocytes with p85alpha antisense oligonucleotide caused a reduction of p85alpha and an increase in p50alpha and p55alpha isoforms and enhanced insulin-stimulated PKB activation. These results demonstrate that PTP1B inhibition causes a direct differential regulation of p85alpha isoforms of PI3-kinase in liver and that reduction of p85alpha may be one mechanism by which PTP1B inhibition improves insulin sensitivity and glucose metabolism in insulin-resistant states. Copyright 2004 Elsevier Inc.
75 FR 22508 - Airworthiness Directives; Eurocopter France Model AS350B, BA, B1, B2, B3, C, D, and D1; AS 355E...

Federal Register 2010, 2011, 2012, 2013, 2014

2010-04-29

... Airworthiness Directives; Eurocopter France Model AS350B, BA, B1, B2, B3, C, D, and D1; AS 355E, F, F1, F2, N... (b) None. Applicability (c) This AD applies to Model AS350B, BA, B1, B2, B3, C, D and D1; and AS 355E..., both dated November 16, 2005, is approved by the Director of the Federal Register as of May 14, 2010...
The B1 Protein Guides the Biosynthesis of a Lasso Peptide

NASA Astrophysics Data System (ADS)

Zhu, Shaozhou; Fage, Christopher D.; Hegemann, Julian D.; Mielcarek, Andreas; Yan, Dushan; Linne, Uwe; Marahiel, Mohamed A.

2016-10-01

Lasso peptides are a class of ribosomally synthesized and post-translationally modified peptides (RiPPs) with a unique lariat knot-like fold that endows them with extraordinary stability and biologically relevant activity. However, the biosynthetic mechanism of these fascinating molecules remains largely speculative. Generally, two enzymes (B for processing and C for cyclization) are required to assemble the unusual knot-like structure. Several subsets of lasso peptide gene clusters feature a “split” B protein on separate open reading frames (B1 and B2), suggesting distinct functions for the B protein in lasso peptide biosynthesis. Herein, we provide new insights into the role of the RiPP recognition element (RRE) PadeB1, characterizing its capacity to bind the paeninodin leader peptide and deliver its peptide substrate to PadeB2 for processing.
Intrinsically disordered chromatin protein NUPR1 binds to the C-terminal region of Polycomb RING1B

PubMed Central

Santofimia-Castaño, Patricia; Rizzuti, Bruno; Pey, Ángel L.; Soubeyran, Philippe; Vidal, Miguel; Urrutia, Raúl; Iovanna, Juan L.; Neira, José L.

2017-01-01

Intrinsically disordered proteins (IDPs) are ubiquitous in eukaryotes, and they are often associated with diseases in humans. The protein NUPR1 is a multifunctional IDP involved in chromatin remodeling and in the development and progression of pancreatic cancer; however, the details of such functions are unknown. Polycomb proteins are involved in specific transcriptional cascades and gene silencing. One of the proteins of the Polycomb complex is the Ring finger protein 1 (RING1). RING1 is related to aggressive tumor features in multiple cancer types. In this work we characterized the interaction between NUPR1 and the paralogue RING1B in vitro, in silico, and in cellulo. The interaction occurred through the C-terminal region of RING1B (C-RING1B), with an affinity in the low micromolar range (∼10 μM). The binding region of NUPR1, mapped by NMR, was a hydrophobic polypeptide patch at the 30s region of its sequence, as pinpointed by computational results and site-directed mutagenesis at Ala33. The association between C-RING1B and wild-type NUPR1 also occurred in cellulo as tested by protein ligation assays; this interaction is inhibited by trifluoperazine, a drug known to hamper binding of wild-type NUPR1 with other proteins. Furthermore, the Thr68Gln and Ala33Gln/Thr68Gln mutants had a reduction in the binding toward C-RING1B as shown by in vitro, in silico, and in cellulo studies. This is an example of a well-folded partner of NUPR1, because its other interacting proteins are also unfolded. We hypothesize that NUPR1 plays an active role in chromatin remodeling and carcinogenesis, together with Polycomb proteins. PMID:28720707
7 CFR 319.55-1 - Definitions.

Code of Federal Regulations, 2011 CFR

2011-01-01

..., DEPARTMENT OF AGRICULTURE FOREIGN QUARANTINE NOTICES Rice Rules and Regulations § 319.55-1 Definitions. (a) Seed or paddy rice. Unhusked rice in the form commonly used for seed purposes; the regulations in this subpart do not apply to husked or polished rice imported for food purposes. (b) Port of first arrival. The...
7 CFR 319.55-1 - Definitions.

Code of Federal Regulations, 2010 CFR

2010-01-01

..., DEPARTMENT OF AGRICULTURE FOREIGN QUARANTINE NOTICES Rice Rules and Regulations § 319.55-1 Definitions. (a) Seed or paddy rice. Unhusked rice in the form commonly used for seed purposes; the regulations in this subpart do not apply to husked or polished rice imported for food purposes. (b) Port of first arrival. The...
Enhanced expression of rat hepatic CYP2B1/2B2 and 2E1 by pyridine: differential induction kinetics and molecular basis of expression.

PubMed

Kim, H; Putt, D; Reddy, S; Hollenberg, P F; Novak, R F

1993-11-01

Expression of the cytochrome P450 (CYP) 2B subfamily in rat and rabbit hepatic tissues after pyridine (PY) treatment has been examined, and the molecular basis for enhanced 2B1/2B2 expression has been determined. P450 expression was monitored using metabolic activity, sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblot analyses, and the identity of the proteins was confirmed through N-terminus microsequence analysis. PY caused a dose-dependent elevation of hepatic CYP2B1/B2B levels in rats, which ranged from 4- to 22-fold over the dosing regimen of 100 to 400 mg PY/kg/day, for 3 days, respectively. PY at low dose failed to induce CYP2B in rabbit hepatic tissue, suggesting a species-dependent response in 2B expression. Anti-2B1 IgG addition to PY-induced microsomes inhibited benzphetamine N-demethylase activity by only approximately 15%, in sharp contrast to the approximately 73% inhibition observed for phenobarbital-induced microsomes, suggesting the induction of other form(s) of P450 having benzphetamine N-demethylase activity. Northern blot analysis revealed that PY treatment increased 2B1 and 2B2 poly(A)+ RNA levels approximately 69- and approximately 34-fold, respectively, whereas the 2E1 poly(A)+ RNA levels failed to increase. The results of this study show that PY induces CYP2B1/2B2 and that induction is species-dependent and kinetically distinguishable from 2E1 induction. Moreover, 2B1/2B2 induction occurs as a result of elevated mRNA levels associated with either transcriptional activation or mRNA stabilization, and it differs from the mechanism of hepatic 2E1 induction by PY.
Marburg Virus VP24 Protein Relieves Suppression of the NF-κB Pathway Through Interaction With Kelch-like ECH-Associated Protein 1.

PubMed

Edwards, Megan R; Basler, Christopher F

2015-10-01

Marburg virus (MARV) is an emerging zoonotic pathogen that causes hemorrhagic fever. MARV VP24 (mVP24) protein interacts with the host cell protein Kelch-like-ECH-associated protein 1 (Keap1). Keap1 interacts with and promotes the degradation of IκB kinase β (IKKβ), a component of the IκB kinase (IKK) complex that regulates nuclear factor-κB (NF-κB) activity. We studied whether mVP24 could relieve Keap1 repression of the NF-κB pathway. Coimmunoprecipitation assays were used to examine the interaction between Keap1 and IKKβ in the presence of wild-type mVP24 and mutants of mVP24 defective for binding to Keap1. Western blotting was used to determine levels of IKKβ expression in the presence of Keap1 and mVP24. NF-κB promoter-luciferase assays were used to determine the effect of mVP24 on Keap1-induced repression of activity. Expression of wild-type mVP24 disrupted the interaction of IKKβ and Keap1, whereas weakly interacting and noninteracting mVP24 mutants did not disrupt the interaction between Keap1 and IKKβ. The interaction of mVP24 with Keap1 enhanced IKKβ levels in the presence of Keap1. The interaction of mVP24 with Keap1 also relieved Keap1 repression of NF-κB reporter activity. mVP24 can relieve Keap1 repression of the NF-κB pathway through its interaction with Keap1. © The Author 2015. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.
Dual expression of Epstein-Barr virus, latent membrane protein-1 and human papillomavirus-16 E6 transform primary mouse embryonic fibroblasts through NF-κB signaling.

PubMed

Shimabuku, Tetsuya; Tamanaha, Ayumi; Kitamura, Bunta; Tanabe, Yasuka; Tawata, Natsumi; Ikehara, Fukino; Arakaki, Kazunari; Kinjo, Takao

2014-01-01

The prevalence of Epstein-Barr virus (EBV) and high-risk human papilloma virus (HPV) infections in patients with oral cancer in Okinawa, southwest islands of Japan, has led to the hypothesis that carcinogenesis is related to EBV and HPV co-infection. To explore the mechanisms of transformation induced by EBV and HPV co-infection, we analyzed the transformation of primary mouse embryonic fibroblasts (MEFs) expressing EBV and HPV-16 genes, alone or in combination. Expression of EBV latent membrane protein-1 (LMP-1) alone or in combination with HPV-16 E6 increased cell proliferation and decreased apoptosis, whereas single expression of EBV nuclear antigen-1 (EBNA-1), or HPV-16 E6 did not. Co-expression of LMP-1 and E6 induced anchorage-independent growth and tumor formation in nude mice, whereas expression of LMP-1 alone did not. Although the singular expression of these viral genes showed increased DNA damage and DNA damage response (DDR), co-expression of LMP-1 and E6 did not induce DDR, which is frequently seen in cancer cells. Furthermore, co-expression of LMP-1 with E6 increased NF-κB signaling, and the knockdown of LMP-1 or E6 in co-expressing cells decreased cell proliferation, anchorage independent growth, and NF-κB activation. These data suggested that expression of individual viral genes is insufficient for inducing transformation and that co-expression of LMP-1 and E6, which is associated with suppression of DDR and increased NF-κB activity, lead to transformation. Our findings demonstrate the synergistic effect by the interaction of oncogenes from different viruses on the transformation of primary MEFs.
The Role of a Novel Nucleolar Protein in Regulation of E2F1 in Breast Cancer

DTIC Science & Technology

2009-09-01

publication and successful defense of a PhD. 8 References 1. Paik JC, Wang B, Liu K, Lue J , Lin WC. Regulation of E2F1-induced apoptosis by...the nucleolar protein RRP1B. J Biol Chem. 2009 Dec 29. [E-pub ahead of print] 2. Hsieh SM, Look MP, Sieuwerts AM, Foekens JA, Hunter KW. Distinct...factor. J Biol Chem. 2009 Oct 16;284(42):28660-73. 4. Crawford NP, Walker RC, Lukes L, Officewala JS, Williams RW, Hunter KW. The Diasporin Pathway: a
Protein Tyrosine Phosphatase 1B (PTP1B): A Potential Target for Alzheimer's Therapy?

PubMed

Vieira, Marcelo N N; Lyra E Silva, Natalia M; Ferreira, Sergio T; De Felice, Fernanda G

2017-01-01

Despite significant advances in current understanding of mechanisms of pathogenesis in Alzheimer's disease (AD), attempts at drug development based on those discoveries have failed to translate into effective, disease-modifying therapies. AD is a complex and multifactorial disease comprising a range of aberrant cellular/molecular processes taking part in different cell types and brain regions. As a consequence, therapeutics for AD should be able to block or compensate multiple abnormal pathological events. Here, we examine recent evidence that inhibition of protein tyrosine phosphatase 1B (PTP1B) may represent a promising strategy to combat a variety of AD-related detrimental processes. Besides its well described role as a negative regulator of insulin and leptin signaling, PTB1B recently emerged as a modulator of various other processes in the central nervous system (CNS) that are also implicated in AD. These include signaling pathways germane to learning and memory, regulation of synapse dynamics, endoplasmic reticulum (ER) stress and microglia-mediated neuroinflammation. We propose that PTP1B inhibition may represent an attractive and yet unexplored therapeutic approach to correct aberrant signaling pathways linked to AD.
The family B1 GPCR: structural aspects and interaction with accessory proteins.

PubMed

Couvineau, Alain; Laburthe, Marc

2012-01-01

G protein coupled receptors (GPCRs) play a crucial role in physiology and pathophysiology in humans. Beside the large family A (rhodopsin-like receptors) and family C GPCR (metabotropic glutamate receptors), the small family B1 GPCR (secretin-like receptors) includes important receptors such as vasoactive intestinal peptide receptors (VPAC), pituitary adenylyl cyclase activating peptide receptor (PAC1R), secretin receptor (SECR), growth hormone releasing factor receptor (GRFR), glucagon receptor (GCGR), glucagon like-peptide 1 and 2 receptors (GLPR), gastric inhibitory peptide receptor (GIPR), parathyroid hormone receptors (PTHR), calcitonin receptors (CTR) and corticotropin-releasing factor receptors (CRFR). They represent very promising targets for the development of drugs having therapeutical impact on many diseases such as chronic inflammation, neurodegeneration, diabetes, stress and osteoporosis. Over the past decade, structure-function relationship studies have demonstrated that the N-terminal ectodomain (N-ted) of family B1 receptors plays a pivotal role in natural ligand recognition. Structural analysis of some family B1 GPCR N-teds revealed the existence of a Sushi domain fold consisting of two antiparallel β sheets stabilized by three disulfide bonds and a salt bridge. The family B1 GPCRs promote cellular responses through a signaling pathway including predominantly the Gsadenylyl cyclase-cAMP pathway activation. Family B1 GPCRs also interact with a few accessory proteins which play a role in cell signaling, receptor expression and/or pharmacological profiles of receptors. These accessory proteins may represent new targets for the design of new drugs. Here, we review the current knowledge regarding: i) the structure of family B1 GPCR binding domain for natural ligands and ii) the interaction of family B1 GPCRs with accessory proteins.
Disruption of Genes Encoding eIF4E Binding Proteins-1 And -2 Does Not Alter Basal or Sepsis-Induced Changes in Skeletal Muscle Protein Synthesis in Male or Female Mice

PubMed Central

Steiner, Jennifer L.; Pruznak, Anne M.; Deiter, Gina; Navaratnarajah, Maithili; Kutzler, Lydia; Kimball, Scot R.; Lang, Charles H.

2014-01-01

Sepsis decreases skeletal muscle protein synthesis in part by impairing mTOR activity and the subsequent phosphorylation of 4E-BP1 and S6K1 thereby controlling translation initiation; however, the relative importance of changes in these two downstream substrates is unknown. The role of 4E-BP1 (and -BP2) in regulating muscle protein synthesis was assessed in wild-type (WT) and 4E-BP1/BP2 double knockout (DKO) male mice under basal conditions and in response to sepsis. At 12 months of age, body weight, lean body mass and energy expenditure did not differ between WT and DKO mice. Moreover, in vivo rates of protein synthesis in gastrocnemius, heart and liver did not differ between DKO and WT mice. Sepsis decreased skeletal muscle protein synthesis and S6K1 phosphorylation in WT and DKO male mice to a similar extent. Sepsis only decreased 4E-BP1 phosphorylation in WT mice as no 4E-BP1/BP2 protein was detected in muscle from DKO mice. Sepsis decreased the binding of eIF4G to eIF4E in WT mice; however, eIF4E•eIF4G binding was not altered in DKO mice under either basal or septic conditions. A comparable sepsis-induced increase in eIF4B phosphorylation was seen in both WT and DKO mice. eEF2 phosphorylation was similarly increased in muscle from WT septic mice and both control and septic DKO mice, compared to WT control values. The sepsis-induced increase in muscle MuRF1 and atrogin-1 (markers of proteolysis) as well as TNFα and IL-6 (inflammatory cytokines) mRNA was greater in DKO than WT mice. The sepsis-induced decrease in myocardial and hepatic protein synthesis did not differ between WT and DKO mice. These data suggest overall basal protein balance and synthesis is maintained in muscle of mice lacking both 4E-BP1/BP2 and that sepsis-induced changes in mTOR signaling may be mediated by a down-stream mechanism independent of 4E-BP1 phosphorylation and eIF4E•eIF4G binding. PMID:24945486
Disruption of genes encoding eIF4E binding proteins-1 and -2 does not alter basal or sepsis-induced changes in skeletal muscle protein synthesis in male or female mice.

PubMed

Steiner, Jennifer L; Pruznak, Anne M; Deiter, Gina; Navaratnarajah, Maithili; Kutzler, Lydia; Kimball, Scot R; Lang, Charles H

2014-01-01

Sepsis decreases skeletal muscle protein synthesis in part by impairing mTOR activity and the subsequent phosphorylation of 4E-BP1 and S6K1 thereby controlling translation initiation; however, the relative importance of changes in these two downstream substrates is unknown. The role of 4E-BP1 (and -BP2) in regulating muscle protein synthesis was assessed in wild-type (WT) and 4E-BP1/BP2 double knockout (DKO) male mice under basal conditions and in response to sepsis. At 12 months of age, body weight, lean body mass and energy expenditure did not differ between WT and DKO mice. Moreover, in vivo rates of protein synthesis in gastrocnemius, heart and liver did not differ between DKO and WT mice. Sepsis decreased skeletal muscle protein synthesis and S6K1 phosphorylation in WT and DKO male mice to a similar extent. Sepsis only decreased 4E-BP1 phosphorylation in WT mice as no 4E-BP1/BP2 protein was detected in muscle from DKO mice. Sepsis decreased the binding of eIF4G to eIF4E in WT mice; however, eIF4E•eIF4G binding was not altered in DKO mice under either basal or septic conditions. A comparable sepsis-induced increase in eIF4B phosphorylation was seen in both WT and DKO mice. eEF2 phosphorylation was similarly increased in muscle from WT septic mice and both control and septic DKO mice, compared to WT control values. The sepsis-induced increase in muscle MuRF1 and atrogin-1 (markers of proteolysis) as well as TNFα and IL-6 (inflammatory cytokines) mRNA was greater in DKO than WT mice. The sepsis-induced decrease in myocardial and hepatic protein synthesis did not differ between WT and DKO mice. These data suggest overall basal protein balance and synthesis is maintained in muscle of mice lacking both 4E-BP1/BP2 and that sepsis-induced changes in mTOR signaling may be mediated by a down-stream mechanism independent of 4E-BP1 phosphorylation and eIF4E•eIF4G binding.
Active sites prediction and binding analysis E1-E2 protein human papillomavirus with biphenylsulfonacetic acid

NASA Astrophysics Data System (ADS)

Iryani, I.; Amelia, F.; Iswendi, I.

2018-04-01

Cervix cancer triggered by Human papillomavirus infection is the second cause to woman death in worldwide. The binding site of E1-E2 protein of HPV 16 is not known from a 3-D structure yet, so in this study we address this issue to study the structure of E1-E2 protein from Human papillomavirus type 16 and to find its potential binding sites using biphenylsulfonacetic acid as inhibitor. Swiss model was used for 3D structure prediction and PDB: 2V9P (E1 protein) and 2NNU (E2 protein) having 52.32% and 100% identity respectively was selected as a template. The 3D model structure developed of E1 and E2 in the core and allowed regions were 99.2% and 99.5%. The ligand binding sites were predicted using online server meta pocket 2.0 and MOE 2009.10 was used for docking. E1-and E2 protein of HPV-16 has three potential binding site that can interact with the inhibitors. The Docking biphenylsulfonacetic acid using these binding sites shows that ligand interact with the protein through hydrogen bonds on Lys 403, Arg 410, His 551 in the first pocket, on Tyr 32, Leu 99 in the second pocket, and Lys 558m Lys 517 in the third pocket.
Myostatin induces insulin resistance via Casitas B-lineage lymphoma b (Cblb)-mediated degradation of insulin receptor substrate 1 (IRS1) protein in response to high calorie diet intake.

PubMed

Bonala, Sabeera; Lokireddy, Sudarsanareddy; McFarlane, Craig; Patnam, Sreekanth; Sharma, Mridula; Kambadur, Ravi

2014-03-14

To date a plethora of evidence has clearly demonstrated that continued high calorie intake leads to insulin resistance and type-2 diabetes with or without obesity. However, the necessary signals that initiate insulin resistance during high calorie intake remain largely unknown. Our results here show that in response to a regimen of high fat or high glucose diets, Mstn levels were induced in muscle and liver of mice. High glucose- or fat-mediated induction of Mstn was controlled at the level of transcription, as highly conserved carbohydrate response and sterol-responsive (E-box) elements were present in the Mstn promoter and were revealed to be critical for ChREBP (carbohydrate-responsive element-binding protein) or SREBP1c (sterol regulatory element-binding protein 1c) regulation of Mstn expression. Further molecular analysis suggested that the increased Mstn levels (due to high glucose or fatty acid loading) resulted in increased expression of Cblb in a Smad3-dependent manner. Casitas B-lineage lymphoma b (Cblb) is an ubiquitin E3 ligase that has been shown to specifically degrade insulin receptor substrate 1 (IRS1) protein. Consistent with this, our results revealed that elevated Mstn levels specifically up-regulated Cblb, resulting in enhanced ubiquitin proteasome-mediated degradation of IRS1. In addition, over expression or knock down of Cblb had a major impact on IRS1 and pAkt levels in the presence or absence of insulin. Collectively, these observations strongly suggest that increased glucose levels and high fat diet, both, result in increased circulatory Mstn levels. The increased Mstn in turn is a potent inducer of insulin resistance by degrading IRS1 protein via the E3 ligase, Cblb, in a Smad3-dependent manner.
20 CFR 655.700 - What statutory provisions govern the employment of H-1B, H-1B1, and E-3 nonimmigrants and how do...

Code of Federal Regulations, 2012 CFR

2012-04-01

... employment of H-1B, H-1B1, and E-3 nonimmigrants and how do employers apply for H-1B, H-1B1, and E-3 visas... Requirements for Employers Seeking To Employ Nonimmigrants on H-1b Visas in Specialty Occupations and as Fashion Models, and Requirements for Employers Seeking To Employ Nonimmigrants on H-1b1 and E-3 Visas in...

20 CFR 655.700 - What statutory provisions govern the employment of H-1B, H-1B1, and E-3 nonimmigrants and how do...

Code of Federal Regulations, 2013 CFR

2013-04-01

... employment of H-1B, H-1B1, and E-3 nonimmigrants and how do employers apply for H-1B, H-1B1, and E-3 visas... Requirements for Employers Seeking To Employ Nonimmigrants on H-1b Visas in Specialty Occupations and as Fashion Models, and Requirements for Employers Seeking To Employ Nonimmigrants on H-1b1 and E-3 Visas in...
20 CFR 655.700 - What statutory provisions govern the employment of H-1B, H-1B1, and E-3 nonimmigrants and how do...

Code of Federal Regulations, 2014 CFR

2014-04-01

... employment of H-1B, H-1B1, and E-3 nonimmigrants and how do employers apply for H-1B, H-1B1, and E-3 visas... Requirements for Employers Seeking To Employ Nonimmigrants on H-1b Visas in Specialty Occupations and as Fashion Models, and Requirements for Employers Seeking To Employ Nonimmigrants on H-1b1 and E-3 Visas in...
20 CFR 655.700 - What statutory provisions govern the employment of H-1B, H-1B1, and E-3 nonimmigrants and how do...

Code of Federal Regulations, 2011 CFR

2011-04-01

... employment of H-1B, H-1B1, and E-3 nonimmigrants and how do employers apply for H-1B, H-1B1, and E-3 visas... Requirements for Employers Seeking To Employ Nonimmigrants on H-1b Visas in Specialty Occupations and as Fashion Models, and Requirements for Employers Seeking To Employ Nonimmigrants on H-1b1 and E-3 Visas in...
20 CFR 655.700 - What statutory provisions govern the employment of H-1B, H-1B1, and E-3 nonimmigrants and how do...

Code of Federal Regulations, 2010 CFR

2010-04-01

... employment of H-1B, H-1B1, and E-3 nonimmigrants and how do employers apply for H-1B, H-1B1, and E-3 visas... Requirements for Employers Seeking To Employ Nonimmigrants on H-1b Visas in Specialty Occupations and as Fashion Models, and Requirements for Employers Seeking To Employ Nonimmigrants on H-1b1 and E-3 Visas in...
Changes in Gene Expression during Tomato Fruit Ripening 1

PubMed Central

Biggs, M. Scott; Harriman, Robert W.; Handa, Avtar K.

1986-01-01

Total proteins from pericarp tissue of different chronological ages from normally ripening tomato (Lycopersicon esculentum Mill. cv Rutgers) fruits and from fruits of the isogenic ripening-impaired mutants rin, nor, and Nr were extracted and separated by sodium dodecylsulfate-polyacrylamide gel electrophoresis. Analysis of the stained bands revealed increases in 5 polypeptides (94, 44, 34, 20, and 12 kilodaltons), decreases in 12 polypeptides (106, 98, 88, 76, 64, 52, 48, 45, 36, 28, 25, and 15 kilodaltons), and fluctuations in 5 polypeptides (85, 60, 26, 21, and 16 kilodaltons) as normal ripening proceeded. Several polypeptides present in ripening normal pericarp exhibited very low or undetectable levels in developing mutant pericarp. Total RNAs extracted from various stages of Rutgers pericarp and from 60 to 65 days old rin, nor, and Nr pericarp were fractionated into poly(A)+ and poly(A)− RNAs. Peak levels of total RNA, poly(A)+ RNA, and poly(A)+ RNA as percent of total RNA occurred between the mature green to breaker stages of normal pericarp. In vitro translation of poly(A)+ RNAs from normal pericarp in rabbit reticulocyte lysates revealed increases in mRNAs for 9 polypeptides (116, 89, 70, 42, 38, 33, 31, 29, and 26 kilodaltons), decreases in mRNAs for 2 polypeptides (41 and 35 kilodaltons), and fluctuations in mRNAs for 5 polypeptides (156, 53, 39, 30, and 14 kilodaltons) during normal ripening. Analysis of two-dimensional separation of in vitro translated polypeptides from poly(A)+ RNAs isolated from different developmental stages revealed even more extensive changes in mRNA populations during ripening. In addition, a polygalacturonase precursor (54 kilodaltons) was immunoprecipitated from breaker, turning, red ripe, and 65 days old Nr in vitro translation products. Images Fig. 1 Fig. 3 Fig. 5 Fig. 6 Fig. 7 PMID:16664828
HTLV-1 Tax upregulates early growth response protein 1 through nuclear factor-κB signaling

PubMed Central

Han, Jingxian; Liu, Xihong; Lv, Zhuangwei; Li, Huanhuan; Yuan, Lixiang; Li, Xiangping; Sun, Shuming; Wang, Hui; Huang, Xinxiang

2017-01-01

Human T cell leukemia virus type 1 (HTLV-1) is a complex retrovirus that causes adult T cell leukemia (ATL) in susceptible individuals. The HTLV-1-encoded oncoprotein Tax induces persistent activation of the nuclear factor-κB (NF-κB) pathway. Early growth response protein 1 (EGR1) is overexpressed in HTLV-1-infected T cell lines and ATL cells. Here, we showed that both Tax expression and HTLV-1 infection promoted EGR1 overexpression. Loss of the NF-κB binding site in the EGR1 promotor or inhibition of NF-κB activation reduced Tax-induced EGR1 upregulation. Tax mutants unable to activate NF-κB induced only slight EGR1 upregulation as compared with wild-type Tax, confirming NF-κB pathway involvement in EGR1 regulation. Tax also directly interacted with the EGR1 protein and increased endogenous EGR1 stability. Elevated EGR1 in turn promoted p65 nuclear translocation and increased NF-κB activation. These results demonstrate a positive feedback loop between EGR1 expression and NF-κB activation in HTLV-1-infected and Tax-expressing cells. Both NF-κB activation and Tax-induced EGR1 stability upregulated EGR1, which in turn enhanced constitutive NF-κB activation and facilitated ATL progression in HTLV-1-infected cells. These findings suggest EGR1 may be an effective anti-ATL therapeutic target. PMID:28881635
HTLV-1 Tax upregulates early growth response protein 1 through nuclear factor-κB signaling.

PubMed

Huang, Qingsong; Niu, Zhiguo; Han, Jingxian; Liu, Xihong; Lv, Zhuangwei; Li, Huanhuan; Yuan, Lixiang; Li, Xiangping; Sun, Shuming; Wang, Hui; Huang, Xinxiang

2017-08-01

Human T cell leukemia virus type 1 (HTLV-1) is a complex retrovirus that causes adult T cell leukemia (ATL) in susceptible individuals. The HTLV-1-encoded oncoprotein Tax induces persistent activation of the nuclear factor-κB (NF-κB) pathway. Early growth response protein 1 (EGR1) is overexpressed in HTLV-1-infected T cell lines and ATL cells. Here, we showed that both Tax expression and HTLV-1 infection promoted EGR1 overexpression. Loss of the NF-κB binding site in the EGR1 promotor or inhibition of NF-κB activation reduced Tax-induced EGR1 upregulation. Tax mutants unable to activate NF-κB induced only slight EGR1 upregulation as compared with wild-type Tax, confirming NF-κB pathway involvement in EGR1 regulation. Tax also directly interacted with the EGR1 protein and increased endogenous EGR1 stability. Elevated EGR1 in turn promoted p65 nuclear translocation and increased NF-κB activation. These results demonstrate a positive feedback loop between EGR1 expression and NF-κB activation in HTLV-1-infected and Tax-expressing cells. Both NF-κB activation and Tax-induced EGR1 stability upregulated EGR1, which in turn enhanced constitutive NF-κB activation and facilitated ATL progression in HTLV-1-infected cells. These findings suggest EGR1 may be an effective anti-ATL therapeutic target.
UV-B induction of the E3 ligase ARIADNE12 depends on CONSTITUTIVELY PHOTOMORPHOGENIC 1

PubMed Central

Xie, Lisi; Lang-Mladek, Christina; Richter, Julia; Nigam, Neha; Hauser, Marie-Theres

2015-01-01

The UV-B inducible ARIADNE12 (ARI12) gene of Arabidopsis thaliana is a member of the RING-between-RING (RBR) family of E3 ubiquitin ligases for which a novel ubiquitination mechanism was identified in mammalian homologs. This RING-HECT hybrid mechanism needs a conserved cysteine which is replaced by serine in ARI12 and might affect the E3 ubiquitin ligase activity. We have shown that under photomorphogenic UV-B, ARI12 is a downstream target of the classical ultraviolet B (UV-B) UV RESISTANCE LOCUS 8 (UVR8) pathway. However, under high fluence rate of UV-B ARI12 was induced independently of UVR8 and the UV-A/blue light and red/far-red photoreceptors. A key component of several light signaling pathways is CONSTITUTIVELY PHOTOMORPHOGENIC 1 (COP1). Upon UV-B COP1 is trapped in the nucleus through interaction with UVR8 permitting the activation of genes that regulate the biosynthesis of UV-B protective metabolites and growth adaptations. To clarify the role of COP1 in the regulation of ARI12 mRNA expression and ARI12 protein stability, localization and interaction with COP1 was assessed with and without UV-B. We found that COP1 controls ARI12 in white light, low and high fluence rate of UV-B. Furthermore we show that ARI12 is indeed an E3 ubiquitin ligase which is mono-ubiquitinated, a prerequisite for the RING-HECT hybrid mechanism. Finally, genetic analyses with transgenes expressing a genomic pmARI12:ARI12-GFP construct confirm the epistatic interaction between COP1 and ARI12 in growth responses to high fluence rate UV-B. PMID:25817546
Hepatic protein phosphatase 1 regulatory subunit 3B (Ppp1r3b) promotes hepatic glycogen synthesis and thereby regulates fasting energy homeostasis.

PubMed

Mehta, Minal B; Shewale, Swapnil V; Sequeira, Raymond N; Millar, John S; Hand, Nicholas J; Rader, Daniel J

2017-06-23

Maintenance of whole-body glucose homeostasis is critical to glycemic function. Genetic variants mapping to chromosome 8p23.1 in genome-wide association studies have been linked to glycemic traits in humans. The gene of known function closest to the mapped region, PPP1R3B (protein phosphatase 1 regulatory subunit 3B), encodes a protein (G L ) that regulates glycogen metabolism in the liver. We therefore sought to test the hypothesis that hepatic PPP1R3B is associated with glycemic traits. We generated mice with either liver-specific deletion ( Ppp1r3b Δ hep ) or liver-specific overexpression of Ppp1r3b The Ppp1r3b deletion significantly reduced glycogen synthase protein abundance, and the remaining protein was predominantly phosphorylated and inactive. As a consequence, glucose incorporation into hepatic glycogen was significantly impaired, total hepatic glycogen content was substantially decreased, and mice lacking hepatic Ppp1r3b had lower fasting plasma glucose than controls. The concomitant loss of liver glycogen impaired whole-body glucose homeostasis and increased hepatic expression of glycolytic enzymes in Ppp1r3b Δ hep mice relative to controls in the postprandial state. Eight hours of fasting significantly increased the expression of two critical gluconeogenic enzymes, phosphoenolpyruvate carboxykinase and glucose-6-phosphatase, above the levels in control livers. Conversely, the liver-specific overexpression of Ppp1r3b enhanced hepatic glycogen storage above that of controls and, as a result, delayed the onset of fasting-induced hypoglycemia. Moreover, mice overexpressing hepatic Ppp1r3b upon long-term fasting (12-36 h) were protected from blood ketone-body accumulation, unlike control and Ppp1r3b Δ hep mice. These findings indicate a major role for Ppp1r3b in regulating hepatic glycogen stores and whole-body glucose/energy homeostasis. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.
A modified MacConkey agar for selective enumeration of necrotoxigenic E. coli O55 and probiotic E. coli Nissle 1917.

PubMed

Splichalova, Alla; Splichal, Igor; Sonnenborn, Ulrich; Rada, Vojtech

2014-09-01

An agar selective enumeration of necrotoxigenic Escherichia coli O55 (NTEC2) and probiotic E. coli Nissle 1917, using modified MacConkey agar, was developed to study bacterial interference between these E. coli strains in a gnotobiotic piglet model. Replacement of lactose with saccharose in the agar enables the direct visual enumeration of red colonies of E. coli O55 and yellow colonies of E. coli Nissle 1917 that are co-cultured in the same Petri dish. A total of 336 colonies (168 for each color) were subjected to strain-specific PCR identification with LNA probes. Sensitivity, specificity, and positive and negative predictive values were 96.43%, 95.83%, 95.86% and 96.41% respectively in E. coli O55, and 98.21%, 97.02%, 97.06% and 98.19% respectively in E. coli Nissle 1917. Color-based enumeration of both E. coli strains in colonic contents and mesenteric lymph nodes homogenates of gnotobiotic piglets demonstrated the applicability of this method for the gnotobiotic piglet model of enteric diseases. Copyright © 2014 Elsevier B.V. All rights reserved.
Human papillomavirus type 16 E6 and E 7 proteins alter NF-kB in cultured cervical epithelial cells and inhibition of NF-kB promotes cell growth and immortalization

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

Vandermark, Erik R.; Deluca, Krysta A.; Gardner, Courtney R.

2012-03-30

The NF-kB family of transcription factors regulates important biological functions including cell growth, survival and the immune response. We found that Human Papillomavirus type 16 (HPV-16) E7 and E6/E7 proteins inhibited basal and TNF-alpha-inducible NF-kB activity in human epithelial cells cultured from the cervical transformation zone, the anatomic region where most cervical cancers develop. In contrast, HPV-16 E6 regulated NF-kB in a cell type- and cell growth-dependent manner. NF-kB influenced immortalization of cervical cells by HPV16. Inhibition of NF-kB by an IkB alpha repressor mutant increased colony formation and immortalization by HPV-16. In contrast, activation of NF-kB by constitutive expressionmore » of p65 inhibited proliferation and immortalization. Our results suggest that inhibition of NF-kB by HPV-16 E6/E7 contributes to immortalization of cells from the cervical transformation zone.« less
A family of glycoproteins (GP55), which inhibit neurite outgrowth, are members of the Ig superfamily and are related to OBCAM, neurotrimin, LAMP and CEPU-1.

PubMed

Wilson, D J; Kim, D S; Clarke, G A; Marshall-Clarke, S; Moss, D J

1996-12-01

We have previously identified a glycosylphosphatidylinositol-linked glycoprotein of 55 kDa (GP55) which inhibits neurite outgrowth. We now provide evidence that GP55, isolated from adult chick brain, consists of at least two bands, both of which are active, i.e., block outgrowth of neurites from chick dorsal root ganglion neurons. An antiserum raised against the adult proteins reverses the inhibition and preliminary experiments suggest that GP55 is restricted to the nervous system, increases during development from very low levels at embryonic day 10 and is most abundant after hatching. Immunofluorescence reveals that GP55 is expressed on neurons cultured from an embryonic day 14 chick brain but is barely detectable on embryonic day 10 dorsal root ganglion neurons or embryonic day 8 forebrain neurons; the neurons which respond to substrate-bound GP55. Peptide sequencing revealed considerable homology with OBCAM, a protein previously identified on the basis of binding opiates. Nested polymerase chain reaction using primers to the OBCAM sequence and internal primers to GP55 peptides produced two different polymerase chain reaction fragments with homology to OBCAM. A full length clone (E19S) corresponding to one polymerase chain reaction product and a partial length clone (E14S) corresponding to the second have been isolated from an embryonic chick brain library. Both are members of the immunoglobulin superfamily and have (or are expected to have) three C2 domains. E19S has 90% homology with LAMP at the amino acid level. This sequence only partially matches the peptides from the adult protein and hence is probably not a major component of the adult proteins. E14S (GP55-A) has 83% homology to OBCAM at the amino acid level over the region sequenced. The sequence matches several of the peptides from the adult protein and is hence likely to correspond to a major component of the adult proteins. Thus members of the GP55 family are related to OBCAM, neurotrimin, LAMP and a
Phase Curve Analysis of Super-Earth 55 Cancri e

NASA Astrophysics Data System (ADS)

Angelo, Isabel; Hu, Renyu

2018-01-01

One of the primary questions when characterizing Earth-sized and super-Earth-sized exoplanets is whether they have a substantial atmosphere like Earth and Venus, or a bare-rock surface that may come with a tenuous atmosphere like Mercury. Phase curves of the planets in thermal emission provide clues to this question, because a substantial atmosphere would transport heat more efficiently than a bare-rock surface. Analyzing phase curve photometric data around secondary eclipse has previously been used to study energy transport in the atmospheres of hot Jupiters. Here we use phase curve, Spitzer time-series photometry to study the thermal emission properties of the super-Earth exoplanet 55 Cancri e. We utilize a previously developed semi-analytical framework to fit a physical model to infrared photometric data of host star 55 Cancri from the Spitzer telescope IRAC 2 band at 4.5 μm. The model uses various parameters of planetary properties including Bond albedo, heat redistribution efficiency (i.e., the ratio between the radiative timescale and advective timescale of the photosphere), and atmospheric greenhouse factor. The phase curve of 55 Cancri e is dominated by thermal emission with an eastward-shifted hot spot located on the planet surface. We determine the heat redistribution efficiency to be ≈1.47, which implies that the advective timescale is on the same order as the radiative timescale. This requirement from the phase curve cannot be met by the bare-rock planet scenario, because heat transport by currents of molten lava would be too slow. The phase curve thus favors the scenario with a substantial atmosphere. Our constraints on the heat redistribution efficiency translate to a photosphere pressure of ~1.4 bar. The Spitzer IRAC 2 band is thus a window into the deep atmosphere of the planet 55 Cancri e.
Interaction between the glutamate transporter GLT1b and the synaptic PDZ domain protein PICK1

PubMed Central

Bassan, Merav; Liu, Hongguang; Madsen, Kenneth L.; Armsen, Wencke; Zhou, Jiayi; DeSilva, Tara; Chen, Weizhi; Paradise, Allison; Brasch, Michael A.; Staudinger, Jeff; Gether, Ulrik; Irwin, Nina; Rosenberg, Paul A.

2015-01-01

Synaptic plasticity is implemented by the interaction of glutamate receptors with PDZ domain proteins. Glutamate transporters provide the only known mechanism of clearance of glutamate from excitatory synapses, and GLT1 is the major glutamate transporter. We show here that GLT1 interacts with the PDZ domain protein PICK1, which plays a critical role in regulating the expression of glutamate receptors at excitatory synapses. A yeast two-hybrid screen of a neuronal library using the carboxyl tail of GLT1b yielded clones expressing PICK1. The GLT1b C-terminal peptide bound to PICK1 with high affinity (Ki = 6.5 ± 0.4 μm) in an in vitro fluorescence polarization assay. We also tested peptides based on other variants of GLT1 and other glutamate transporters. GLT1b co-immunoprecipitated with PICK1 from rat brain lysates and COS7 cell lysates derived from cells transfected with plasmids expressing PICK1 and GLT1b. In addition, expression of GLT1b in COS7 cells changed the distribution of PICK1, bringing it to the surface. GLT1b and PICK1 co-localized with each other and with synaptic markers in hippocampal neurons in culture. Phorbol ester, an activator of protein kinase C (PKC), a known PICK1 interactor, had no effect on glutamate transport in rat forebrain neurons in culture. However, we found that exposure of neurons to a myristolated decoy peptide with sequence identical to the C-terminal sequence of GLT1b designed to block the PICK1–GLT1b interaction rendered glutamate transport into neurons responsive to phorbol ester. These results suggest that the PICK1–GLT1b interaction regulates the modulation of GLT1 function by PKC. PMID:18184314
E2F mediates induction of the Sp1-controlled promoter of the human DNA polymerase ɛ B-subunit gene POLE2

PubMed Central

Huang, Deqi; Jokela, Maarit; Tuusa, Jussi; Skog, Sven; Poikonen, Kari; Syväoja, Juhani E.

2001-01-01

The B-subunits of replicative DNA polymerases from Archaea to humans belong to the same protein family, suggesting that they share a common fundamental function. We report here the gene structure for the B-subunit of human DNA polymerase ɛ (POLE2), whose expression and transcriptional regulation is typical for replication proteins with some unique features. The 75 bp core promoter region, located within exon 1, contains an Sp1 element that is a critical determinant of promoter activity as shown by the luciferase reporter, electrophoretic mobility shift and DNase I footprinting assays. Two overlapping E2F elements adjacent to the Sp1 element are essential for full promoter activity and serum response. Binding sites for E2F1 and NF-1 reside immediately downstream from the core promoter region. Our results suggest that human POLE2 is regulated by two E2F–pocket protein complexes, one associated with Sp1 and the other with NF-1. So far, only one replicative DNA polymerase B-subunit gene promoter, POLA2 encoding the B-subunit of DNA polymerase α, has been characterized. Mitogenic activation of the POLE2 promoter by an E2F-mediated mechanism resembles that of POLA2, but the regulation of basal promoter activity is different between these two genes. PMID:11433027
Flexible docking-based molecular dynamics/steered molecular dynamics calculations of protein-protein contacts in a complex of cytochrome P450 1A2 with cytochrome b5.

PubMed

Jeřábek, Petr; Florián, Jan; Stiborová, Marie; Martínek, Václav

2014-10-28

Formation of transient complexes of cytochrome P450 (P450) with another protein of the endoplasmic reticulum membrane, cytochrome b5 (cyt b5), dictates the catalytic activities of several P450s. Therefore, we examined formation and binding modes of the complex of human P450 1A2 with cyt b5. Docking of soluble domains of these proteins was performed using an information-driven flexible docking approach implemented in HADDOCK. Stabilities of the five unique binding modes of the P450 1A2-cyt b5 complex yielded by HADDOCK were evaluated using explicit 10 ns molecular dynamics (MD) simulations in aqueous solution. Further, steered MD was used to compare the stability of the individual P450 1A2-cyt b5 binding modes. The best binding mode was characterized by a T-shaped mutual orientation of the porphyrin rings and a 10.7 Å distance between the two redox centers, thus satisfying the condition for a fast electron transfer. Mutagenesis studies and chemical cross-linking, which, in the absence of crystal structures, were previously used to deduce specific P450-cyt b5 interactions, indicated that the negatively charged convex surface of cyt b5 binds to the positively charged concave surface of P450. Our simulations further elaborate structural details of this interface, including nine ion pairs between R95, R100, R138, R362, K442, K455, and K465 side chains of P450 1A2 and E42, E43, E49, D65, D71, and heme propionates of cyt b5. The universal heme-centric system of internal coordinates was proposed to facilitate consistent classification of the orientation of the two porphyrins in any protein complex.
Characterization of the cryptic plasmid pOfk55 from Legionella pneumophila and construction of a pOfk55-derived shuttle vector.

PubMed

Nishida, Takashi; Watanabe, Kenta; Tachibana, Masato; Shimizu, Takashi; Watarai, Masahisa

2017-03-01

In this study, a cryptic plasmid pOfk55 from Legionella pneumophila was isolated and characterized. pOfk55 comprised 2584bp with a GC content of 37.3% and contained three putative open reading frames (ORFs). orf1 encoded a protein of 195 amino acids and the putative protein shared 39% sequence identity with a putative plasmid replication protein RepL. ORF1 was needed for replication in L. pneumophila but pOfk55 did not replicate in Escherichia coli. orf2 and orf3 encoded putative hypothetical proteins of 114 amino acids and 78 amino acids, respectively, but the functions of the putative proteins ORF2 and OFR3 are not clear. The transfer mechanism for pOfk55 was independent on the type IVB secretion system in the original host. A L. pneumophila-E. coli shuttle vector, pNT562 (5058bp, Km R ), was constructed by In-Fusion Cloning of pOfk55 with a kanamycin-resistance gene from pUTmini-Tn5Km and the origin of replication from pBluescript SK(+) (pNT561). Multiple cloning sites from pBluescript SK(+) as well as the tac promoter region and lacI gene from pAM239-GFP were inserted into pNT561 to construct pNT562. The transformation efficiency of pNT562 in L. pneumophila strains ranged from 1.6×10 1 to 1.0×10 5 CFU/ng. The relative number of pNT562 was estimated at 5.7±1.0 copies and 73.6% of cells maintained the plasmid after 1week in liquid culture without kanamycin. A green fluorescent protein (GFP) expression vector, pNT563, was constructed by ligating pNT562 with the gfpmut3 gene from pAM239-GFP. pNT563 was introduced into L. pneumophila Lp02 and E. coli DH5α, and both strains expressed GFP successfully. These results suggest that the shuttle vector is useful for genetic studies in L. pneumophila. Copyright © 2017 Elsevier Inc. All rights reserved.
Neuroprotective Effects of Protein Tyrosine Phosphatase 1B Inhibition against ER Stress-Induced Toxicity

PubMed Central

Jeon, Yu-Mi; Lee, Shinrye; Kim, Seyeon; Kwon, Younghwi; Kim, Kiyoung; Chung, Chang Geon; Lee, Seongsoo; Lee, Sung Bae; Kim, Hyung-Jun

2017-01-01

Several lines of evidence suggest that endoplasmic reticulum (ER) stress plays a critical role in the pathogenesis of many neurodegenerative diseases such as Alzheimer’s disease, Parkinson’s disease, and amyotrophic lateral sclerosis. Protein tyrosine phosphatase 1B (PTP1B) is known to regulate the ER stress signaling pathway, but its role in neuronal systems in terms of ER stress remains largely unknown. Here, we showed that rotenone-induced toxicity in human neuroblastoma cell lines and mouse primary cortical neurons was ameliorated by PTP1B inhibition. Moreover, the increase in the level of ER stress markers (eIF2α phosphorylation and PERK phosphorylation) induced by rotenone treatment was obviously suppressed by concomitant PTP1B inhibition. However, the rotenone-induced production of reactive oxygen species (ROS) was not affected by PTP1B inhibition, suggesting that the neuroprotective effect of the PTP1B inhibitor is not associated with ROS production. Moreover, we found that MG132-induced toxicity involving proteasome inhibition was also ameliorated by PTP1B inhibition in a human neuroblastoma cell line and mouse primary cortical neurons. Consistently, downregulation of the PTP1B homologue gene in Drosophila mitigated rotenone- and MG132-induced toxicity. Taken together, these findings indicate that PTP1B inhibition may represent a novel therapeutic approach for ER stress-mediated neurodegenerative diseases. PMID:28359145
Structure of a BMI-1-Ring1B Polycomb Group Ubiquitin Ligase Complex

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

Li,Z.; Cao, R.; Wang, M.

2006-01-01

Polycomb group (PcG) proteins Bmi-1 and Ring1B are core subunits of the PRC1 complex which plays important roles in the regulation of Hox gene expression, X-chromosome inactivation, tumorigenesis and stem cell self-renewal. The RING finger protein Ring1B is an E3 ligase that participates in the ubiquitination of lysine 119 of histone H2A, and the binding of Bmi-1 stimulates the E3 ligase activity. We have mapped the regions of Bmi-1 and Ring1B required for efficient ubiquitin transfer and determined a 2.5 Angstroms structure of the Bmi-1-Ring1B core domain complex. The structure reveals that Ring1B 'hugs' Bmi-1 through extensive RING domain contactsmore » and its N-terminal tail wraps around Bmi-1. The two regions of interaction have a synergistic effect on the E3 ligase activity. Our analyses suggest a model where the Bmi-1-Ring1B complex stabilizes the interaction between the E2 enzyme and the nucleosomal substrate to allow efficient ubiquitin transfer.« less
Protection against gamma-radiation injury by protein tyrosine phosphatase 1B.

PubMed

Mojena, Marina; Pimentel-Santillana, María; Povo-Retana, Adrián; Fernández-García, Victoria; González-Ramos, Silvia; Rada, Patricia; Tejedor, Alberto; Rico, Daniel; Martín-Sanz, Paloma; Valverde, Angela M; Boscá, Lisardo

2018-07-01

Protein tyrosine phosphatase 1B (PTP1B) is widely expressed in mammalian tissues, in particular in immune cells, and plays a pleiotropic role in dephosphorylating many substrates. Moreover, PTP1B expression is enhanced in response to pro-inflammatory stimuli and to different cell stressors. Taking advantage of the use of mice deficient in PTP1B we have investigated the effect of γ-radiation in these animals and found enhanced lethality and decreased respiratory exchange ratio vs. the corresponding wild type animals. Using bone-marrow derived macrophages and mouse embryonic fibroblasts (MEFs) from wild-type and PTP1B-deficient mice, we observed a differential response to various cell stressors. PTP1B-deficient macrophages exhibited an enhanced response to γ-radiation, UV-light, LPS and S-nitroso-glutathione. Macrophages exposed to γ-radiation show DNA damage and fragmentation, increased ROS production, a lack in GSH elevation and enhanced acidic β-galactosidase activity. Interestingly, these differences were not observed in MEFs. Differential gene expression analysis of WT and KO macrophages revealed that the main pathways affected after irradiation were an up-regulation of protein secretion, TGF-β signaling and angiogenesis among other, and downregulation of Myc targets and Hedgehog signaling. These results demonstrate a key role for PTP1B in the protection against the cytotoxicity of irradiation in intact animal and in macrophages, which might be therapeutically relevant. Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.

Optimised purification and characterisation of lipid transfer protein 1 (LTP1) and its lipid-bound isoform LTP1b from barley malt.

PubMed

Nieuwoudt, Melanie; Lombard, Nicolaas; Rautenbach, Marina

2014-08-15

In beer brewing, brewers worldwide strive to obtain product consistency in terms of flavour, colour and foam. Important proteins contributing to beer foam are lipid transfer proteins (LTPs), in particular LTP1 and its lipid-bound isoform LTP1b, which are known to transport lipids in vivo and prevent lipids from destabilising the beer foam. LTP1 and LTP1b were successfully purified using only five purification steps with a high purified protein yield (160 mg LTP1 and LTP1b from 200 g barley). Circular dichroism of LTP1 and LTP1b confirmed that both proteins are highly tolerant to high temperatures (>90 °C) and are pH stable, particularly at a neutral to a more basic pH. Only LTP1 exhibited antiyeast and thermo-stable lytic activity, while LTP1b was inactive, indicating that the fatty acid moiety compromised the antimicrobial activity of LTP1. This lack in antiyeast activity and the positive foam properties of LTP1b would benefit beer fermentation and quality. Copyright © 2014 Elsevier Ltd. All rights reserved.
Epitomics: IgG-epitome decoding of E6, E7 and L1 proteins from oncogenic human papillomavirus type 58

PubMed Central

Xu, Wan-Xiang; Wang, Jian; Tang, Hai-Ping; He, Ya-Ping; Zhu, Qian-Xi; Gupta, Satish K.; Gu, Shao-Hua; Huang, Qiang; Ji, Chao-Neng; Liu, Ling-Feng; Li, Gui-Ling; Xu, Cong-Jian; Xie, Yi

2016-01-01

To enable rational multi-epitope vaccine and diagnostic antigen design, it is imperative to delineate complete IgG-epitome of the protein. Here, we describe results of IgG-epitome decoding of three proteins from high-risk (HR-) oncogenic human papillomavirus type 58 (HPV58). To reveal their entire epitomes, employing peptide biosynthetic approach, 30 precise linear B-cell epitopes (BCEs) were mapped on E6, E7 and L1 proteins using rabbits antisera to the respective recombinant proteins. Using sequence alignment based on BCE minimal motif, the specificity and conservativeness of each mapped BCE were delineated mainly among known HR-HPVs, including finding 3 broadly antibody cross-reactive BCEs of L1 that each covers almost all HR-HPVs. Western blots revealed that 13 of the 18 BCEs within L1-epitome were recognized by murine antisera to HPV58 virus-like particles, suggesting that these are antibody accessible BCEs. Also, a highly conserved epitope (YGD/XTL) of E6 was found to exist only in known common HR-HPVs, which could be used as the first peptide reference marker for judging HR-HPVs. Altogether, this study provides systemic and exhaustive information on linear BCEs of HR-HPV58 that will facilitate development of novel multi-epitope diagnostic reagents/chips for testing viral antibodies and ‘universal’ preventive HPV peptide vaccine based on L1 conserved BCEs. PMID:27708433
Protein tyrosine phosphatase 1B inhibitory activity of lavandulyl flavonoids from roots of Sophora flavescens.

PubMed

Sasaki, Tatsunori; Li, Wei; Higai, Koji; Quang, Tran Hong; Kim, Young Ho; Koike, Kazuo

2014-05-01

Protein tyrosine phosphatase 1B is a non-transmembrane protein tyrosine phosphatase and major negative regulator in insulin signaling cascades, and much attention has been paid to protein tyrosine phosphatase 1B inhibitors as potential therapies for diabetes. The screening of a natural compound library led to the discovery of five lavandulyl flavonoids, which were isolated from the roots of Sophora flavescens, as novel PTP1B inhibitors: kuraridin (1), norkurarinone (2), kurarinone (3), 2'-methoxykurarinone (4), and kushenol T (5). The three most potent compounds, 1, 2, and 4 (IC50 < 30 µM), were demonstrated to be noncompetitive inhibitors of protein tyrosine phosphatase 1B based on a kinetic analysis, and they exhibited different inhibitory selectivities against four homologous protein tyrosine phosphatases (T cell protein tyrosine phosphatase, vaccinia H1-related phosphatase, and Src homology domain 2-containing protein tyrosine phosphatases 1 and 2). Compounds 1, 2, and 4 also exhibited cellular activity in the insulin signaling pathway by increasing the insulin-stimulated Akt phosphorylation level in human hepatocellular liver carcinoma HepG2 cells, suggesting their potential for new anti-insulin-resistant drug developments. Georg Thieme Verlag KG Stuttgart · New York.
Respiratory syncytial virus M2-1 protein induces the activation of nuclear factor kappa B

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

Reimers, Kerstin; Buchholz, Katja; Werchau, Hermann

2005-01-20

Respiratory syncytial virus (RSV) induces the production of a number of cytokines and chemokines by activation of nuclear factor kappa B (NF-{kappa}B). The activation of NF-{kappa}B has been shown to depend on viral replication in the infected cells. In this study, we demonstrate that expression of RSV M2-1 protein, a transcriptional processivity and anti-termination factor, is sufficient to activate NF-{kappa}B in A549 cells. Electromobility shift assays show increased NF-{kappa}B complexes in the nuclei of M2-1-expressing cells. M2-1 protein is found in nuclei of M2-1-expressing cells and in RSV-infected cells. Co-immunoprecipitations of nuclear extracts of M2-1-expressing cells and of RSV-infected cellsmore » revealed an association of M2-1 with Rel A protein. Furthermore, the activation of NF-{kappa}B depends on the C-terminus of the RSV M2-1 protein, as shown by NF-{kappa}B-induced gene expression of a reporter gene construct.« less
Caged xanthones displaying protein tyrosine phosphatase 1B (PTP1B) inhibition from Cratoxylum cochinchinense.

PubMed

Li, Zuo Peng; Lee, Hyeong-Hwan; Uddin, Zia; Song, Yeong Hun; Park, Ki Hun

2018-08-01

Four new caged xanthones (1-4) and two known compounds (5, 6) were isolated from the roots of Cratoxylum cochinchinense, a polyphenol rich plant, collected in China. The structures of the isolated compounds (1-6) were characterized by obtaining their detailed spectroscopic data. In particular, compounds 1 and 6 were fully identified by X-ray crystallographic data. The isolated compounds (1-6) were evaluated against protein tyrosine phosphatase 1B (PTP1B), which plays an important role in diabetes, obesity, and cancer. Among these compounds, 3, 4, and 6 displayed significant inhibition with IC 50 values of 76.3, 43.2, and 6.6 µM, respectively. A detailed kinetic study was conducted by determining K m , V max , and the ratio of K ik and K iv , which revealed that all the compounds behaved as competitive inhibitors. Copyright © 2018 Elsevier Inc. All rights reserved.
Differential expression and molecular characterisation of Lmo7, Myo1e, Sash1, and Mcoln2 genes in Btk-defective B-cells.

PubMed

Lindvall, Jessica M; Blomberg, K Emelie M; Wennborg, Anders; Smith, C I Edvard

2005-05-01

Bruton's tyrosine kinase is crucial for B-lymphocyte development. By the use of gene expression profiling, we have identified four expressed sequence tags among 38 potential Btk target genes, which have now been characterised. Bioinformatics tools including data mining of additional unpublished gene expression profiles, sequence verification of PCR products and qualitative RT-PCR were used. Stimulations targeting the B-cell receptor and the protein kinase C were used to activate whole B-cell splenocytes. Target genes were characterised as Lim domain only 7 (Lmo7); Myosin1e (Myo1e); SAM and SH3 domain containing 1 (Sash1); and Mucolipin2 (Mcoln2). Expression was found in cell lines of different origin and developmental stages as well as in whole B-cell splenocytes and Transitional type 1 (T1) splenic B-cells from wild type and Btk-defective mice, respectively. By the use of semi-quantitative RT-PCR we found Sash1 not to be expressed in the investigated haematopoietic cell lines, while transcripts were found in whole splenic B-cells from both wild type and Btk-defective mice, whereas Lmo7, Myo1e, and Mcoln2 were expressed in both B-cell lines and primary B-lymphocytes. Except for Lmo7, the transcript level was similarly affected by stimulation in control and Btk-defective cells.
Growth suppression of colorectal cancer by plant-derived multiple mAb CO17-1A × BR55 via inhibition of ERK1/2 phosphorylation.

PubMed

Kwak, Dong Hoon; Moussavou, Ghislain; Lee, Ju Hyoung; Heo, Sung Youn; Ko, Kisung; Hwang, Kyung-A; Jekal, Seung-Joo; Choo, Young-Kug

2014-11-14

We have generated the transgenic Tabaco plants expressing multiple monoclonal antibody (mAb) CO7-1A × BR55 by cross-pollinating with mAb CO17-1A and mAb BR55. We have demonstrated the anti-cancer effect of plant-derived multiple mAb CO17-1A × BR55. We find that co-treatment of colorectal mAbs (anti-epithelial cellular adhesion molecule (EpCAM), plant-derived monoclonal antibody (mAb(P)) CO17-1A and mAb(P) CO17-1A × BR55) with RAW264.7 cells significantly inhibited the cell growth in SW620 cancer cells. In particular, multi mAb(P) CO17-1A × BR55 significantly and efficiently suppressed the growth of SW620 cancer cells compared to another mAbs. Apoptotic death-positive cells were significantly increased in the mAb(P) CO17-1A × BR55-treated. The mAb(P) CO17-1A × BR55 treatment significantly decreased the expression of B-Cell lymphoma-2 (BCl-2), but the expression of Bcl-2-associated X protein (Bax), and cleaved caspase-3 were markedly increased. In vivo, the mAb(P) CO17-1A × BR55 significantly and efficiently inhibited the growth of colon tumors compared to another mAbs. The apoptotic cell death and inhibition of pro-apoptotic proteins expression were highest by treatment with mAb(P) CO17-1A × BR55. In addition, the mAb(P) CO17-1A × BR55 significantly inhibited the extracellular signal-regulated kinase 1 and 2 (ERK1/2) phosphorylation in cancer cells and tumors. Therefore, this study results suggest that multiple mAb(P) CO17-1A × BR55 has a significant effect on apoptosis-mediated anticancer by suppression of ERK1/2 phosphorylation in colon cancer compared to another mAbs. In light of these results, further clinical investigation should be conducted on mAb(P) CO17-1A × BR55 to determine its possible chemopreventive and/or therapeutic efficacy against human colon cancer.
Reduction of protein tyrosine phosphatase 1B increases insulin-dependent signaling in ob/ob mice.

PubMed

Gum, Rebecca J; Gaede, Lori L; Koterski, Sandra L; Heindel, Matthew; Clampit, Jill E; Zinker, Bradley A; Trevillyan, James M; Ulrich, Roger G; Jirousek, Michael R; Rondinone, Cristina M

2003-01-01

Protein tyrosine phosphatase 1B (PTP1B) is a negative regulator of insulin receptor (IR) signal transduction and a drug target for treatment of type 2 diabetes. Using PTP1B antisense oligonucleotides (ASOs), effects of decreased PTP1B levels on insulin signaling in diabetic ob/ob mice were examined. Insulin stimulation, prior to sacrifice, resulted in no significant activation of insulin signaling pathways in livers from ob/ob mice. However, in PTP1B ASO-treated mice, in which PTP1B protein was decreased by 60% in liver, similar stimulation with insulin resulted in increased tyrosine phosphorylation of the IR and IR substrate (IRS)-1 and -2 by threefold, fourfold, and threefold, respectively. IRS-2-associated phosphatidylinositol 3-kinase activity was also increased threefold. Protein kinase B (PKB) serine phosphorylation was increased sevenfold in liver of PTP1B ASO-treated mice upon insulin stimulation, while phosphorylation of PKB substrates, glycogen synthase kinase (GSK)-3alpha and -3beta, was increased more than twofold. Peripheral insulin signaling was increased by PTP1B ASO, as evidenced by increased phosphorylation of PKB in muscle of insulin-stimulated PTP1B ASO-treated animals despite the lack of measurable effects on muscle PTP1B protein. These results indicate that reduction of PTP1B is sufficient to increase insulin-dependent metabolic signaling and improve insulin sensitivity in a diabetic animal model.
Endoplasmic reticulum degradation-enhancing α-mannosidase-like protein 1 targets misfolded HLA-B27 dimers for endoplasmic reticulum-associated degradation.

PubMed

Guiliano, David B; Fussell, Helen; Lenart, Izabela; Tsao, Edward; Nesbeth, Darren; Fletcher, Adam J; Campbell, Elaine C; Yousaf, Nasim; Williams, Sarah; Santos, Susana; Cameron, Amy; Towers, Greg J; Kellam, Paul; Hebert, Daniel N; Gould, Keith G; Powis, Simon J; Antoniou, Antony N

2014-11-01

HLA-B27 forms misfolded heavy chain dimers, which may predispose individuals to inflammatory arthritis by inducing endoplasmic reticulum (ER) stress and the unfolded protein response (UPR). This study was undertaken to define the role of the UPR-induced ER-associated degradation (ERAD) pathway in the disposal of HLA-B27 dimeric conformers. HeLa cell lines expressing only 2 copies of a carboxy-terminally Sv5-tagged HLA-B27 were generated. The ER stress-induced protein ER degradation-enhancing α-mannosidase-like protein 1 (EDEM1) was overexpressed by transfection, and dimer levels were monitored by immunoblotting. EDEM1, the UPR-associated transcription factor X-box binding protein 1 (XBP-1), the E3 ubiquitin ligase hydroxymethylglutaryl-coenzyme A reductase degradation 1 (HRD1), and the degradation-associated proteins derlin 1 and derlin 2 were inhibited using either short hairpin RNA or dominant-negative mutants. The UPR-associated ERAD of HLA-B27 was confirmed using ER stress-inducing pharamacologic agents in kinetic and pulse chase assays. We demonstrated that UPR-induced machinery can target HLA-B27 dimers and that dimer formation can be controlled by alterations to expression levels of components of the UPR-induced ERAD pathway. HLA-B27 dimers and misfolded major histocompatibility complex class I monomeric molecules bound to EDEM1 were detected, and overexpression of EDEM1 led to inhibition of HLA-B27 dimer formation. EDEM1 inhibition resulted in up-regulation of HLA-B27 dimers, while UPR-induced ERAD of dimers was prevented in the absence of EDEM1. HLA-B27 dimer formation was also enhanced in the absence of XBP-1, HRD1, and derlins 1 and 2. The present findings indicate that the UPR ERAD pathway can dispose of HLA-B27 dimers, thus presenting a potential novel therapeutic target for modulation of HLA-B27-associated inflammatory disease. Copyright © 2014 by the American College of Rheumatology.
Aurora kinases and protein phosphatase 1 mediate chromosome congression through regulation of CENP-E

PubMed Central

Kim, Yumi; Holland, Andrew J.; Lan, Weijie; Cleveland, Don W.

2010-01-01

Summary Opposing roles of Aurora kinases and protein phosphatase 1 (PP1) during mitosis have long been suggested. Here we demonstrate that Aurora kinases A and B phosphorylate a single residue on the kinetochore motor CENP-E. PP1 binds CENP-E via a motif overlapping this phosphorylation site and binding is disrupted by Aurora phosphorylation. Phosphorylation of CENP-E by the Auroras is enriched at spindle poles, disrupting binding of PP1 and reducing CENP-E’s affinity for individual microtubules. This phosphorylation is required for CENP-E-mediated towing of initially polar chromosomes toward the cell center. Kinetochores on such chromosomes cannot make subsequent stable attachment to spindle microtubules when dephosphorylation of CENP-E or rebinding of PP1 to CENP-E is blocked. Thus, an Aurora/PP1 phosphorylation switch modulates CENP-E motor activity as an essential feature of chromosome congression from poles and localized PP1 delivery by CENP-E to the outer kinetochore is necessary for stable microtubule capture by those chromosomes. PMID:20691903
HTLV-I Tax protein binds to MEKK1 to stimulate IkappaB kinase activity and NF-kappaB activation.

PubMed

Yin, M J; Christerson, L B; Yamamoto, Y; Kwak, Y T; Xu, S; Mercurio, F; Barbosa, M; Cobb, M H; Gaynor, R B

1998-05-29

NF-kappaB, a key regulator of the cellular inflammatory and immune response, is activated by the HTLV-I transforming and transactivating protein Tax. We show that Tax binds to the amino terminus of the protein kinase MEKK1, a component of an IkappaB kinase complex, and stimulates MEKK1 kinase activity. Tax expression increases the activity of IkappaB kinase beta (IKKbeta) to enhance phosphorylation of serine residues in IkappaB alpha that lead to its degradation. Dominant negative mutants of both IKKbeta and MEKK1 prevent Tax activation of the NF-kappaB pathway. Furthermore, recombinant MEKK1 stimulates IKKbeta phosphorylation of IkappaB alpha. Thus, Tax-mediated increases in NF-kappaB nuclear translocation result from direct interactions of Tax and MEKK1 leading to enhanced IKKbeta phosphorylation of IkappaB alpha.
Spectrophotometric determination of total protein in serum using a novel near-infrared cyanine dye, 5,5'-dicarboxy-1,1'-disulfobutyl-3,3,3',3'-tetramethylindotricarbocyanine.

PubMed

Wang, Hong; Li, Wei-Rong; Guo, Xiao-Feng; Zhang, Hua-Shan

2007-04-01

The application of near-infrared (NIR) dyes (lambda (em) > 750 nm) to the analysis of biological samples shows much promise, because the long emission wavelengths of such dyes allow interferences from biomolecule matrices to be minimized. In this paper, a novel NIR dye, 5,5'-dicarboxy-1,1'-disulfobutyl-3,3,3',3'-tetramethylindotricarbocyanine (DCDSTCY) has been developed for the spectrophotometric determination of total protein in serum. Under acidic conditions, the binding of DCDSTCY to proteins caused a new peak at 878 nm, the height of which was proportional to the concentration of protein. The linear range of the method was found to be 0.04-0.5 microg mL(-1) for bovine serum albumin (BSA) and human serum albumin (HSA), and detection limits of 5 ng mL(-1) were obtained for these substances. The maximum binding number of BSA with DCDSTCY was measured to be 133. The method proposed here has been applied to the quantitation of total protein in serum, and recoveries of 96.6-104% were achieved. Figure Near-infrared probe for protein determination.
Role of the disaggregase ClpB in processing of proteins aggregated as inclusion bodies.

PubMed

Zblewska, Kamila; Krajewska, Joanna; Zolkiewski, Michal; Kędzierska-Mieszkowska, Sabina

2014-08-01

Overproduction of heterologous proteins in bacterial systems often results in the formation of insoluble inclusion bodies (IBs), which is a major impediment in biochemical research and biotechnology. In principle, the activity of molecular chaperones could be employed to gain control over the IB formation and to improve the recombinant protein yields, but the potential of each of the major bacterial chaperones (DnaK/J, GroEL/ES, and ClpB) to process IBs has not been fully established yet. We investigated the formation of inclusion bodies (IBs) of two aggregation-prone proteins, VP1LAC and VP1GFP, overproduced in Escherichiacoli in the presence and absence of the chaperone ClpB. We found that both ClpB isoforms, ClpB95 and ClpB80 accumulated in E. coli cells during the production of IBs. The amount of IB proteins increased in the absence of ClpB. ClpB supported the resolubilization and reactivation of the aggregated VP1LAC and VP1GFP in E. coli cells. The IB disaggregation was optimal in the presence of both ClpB95 and ClpB80. Our results indicate an essential role of ClpB in controlling protein aggregation and inclusion body formation in bacteria. Copyright © 2014 Elsevier Inc. All rights reserved.
Ultrafast transient photocarrier dynamics of the bulk-insulating topological insulator B i1.5S b0.5T e1.7S e1.3

NASA Astrophysics Data System (ADS)

Choi, Young Gwan; Zhung, Chan June; Park, Sun-Hee; Park, Joonbum; Kim, Jun Sung; Kim, Seongheun; Park, Jaehun; Lee, J. S.

2018-02-01

Using optical-pump terahertz-probe spectroscopy, we investigated an ultrafast photocarrier relaxation behavior in a B i1.5S b0.5T e1.7S e1.3 (BSTS) single crystal, which is one of the most bulk-insulating topological insulators. Compared to n -type bulk-metallic B i2S e3 , we found that BSTS endows distinct behaviors in its photocarrier dynamics; the relaxation time turns out to be an order of magnitude longer, and the transient conductance spectrum exhibits a nonlinear increase as a function of the pumping power. Also, we observed an abrupt reduction of the photocarrier scattering rate in several picoseconds after the initial photoexcitation. We discuss these intriguing experimental observations based on a bulk-to-surface carrier injection assisted by the built-in electric field near the surface and electron-phonon scattering.
Interaction of the amyloid precursor protein-like protein 1 (APLP1) E2 domain with heparan sulfate involves two distinct binding modes

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

Dahms, Sven O., E-mail: sdahms@fli-leibniz.de; Mayer, Magnus C.; Miltenyi Biotec GmbH, Robert-Koch-Strasse 1, 17166 Teterow

2015-03-01

Two X-ray structures of APLP1 E2 with and without a heparin dodecasaccharide are presented, revealing two distinct binding modes of the protein to heparan sulfate. The data provide a mechanistic explanation of how APP-like proteins bind to heparan sulfates and how they specifically recognize nonreducing structures of heparan sulfates. Beyond the pathology of Alzheimer’s disease, the members of the amyloid precursor protein (APP) family are essential for neuronal development and cell homeostasis in mammals. APP and its paralogues APP-like protein 1 (APLP1) and APP-like protein 2 (APLP2) contain the highly conserved heparan sulfate (HS) binding domain E2, which effects variousmore » (patho)physiological functions. Here, two crystal structures of the E2 domain of APLP1 are presented in the apo form and in complex with a heparin dodecasaccharide at 2.5 Å resolution. The apo structure of APLP1 E2 revealed an unfolded and hence flexible N-terminal helix αA. The (APLP1 E2){sub 2}–(heparin){sub 2} complex structure revealed two distinct binding modes, with APLP1 E2 explicitly recognizing the heparin terminus but also interacting with a continuous heparin chain. The latter only requires a certain register of the sugar moieties that fits to a positively charged surface patch and contributes to the general heparin-binding capability of APP-family proteins. Terminal binding of APLP1 E2 to heparin specifically involves a structure of the nonreducing end that is very similar to heparanase-processed HS chains. These data reveal a conserved mechanism for the binding of APP-family proteins to HS and imply a specific regulatory role of HS modifications in the biology of APP and APP-like proteins.« less
Identification and Application of Neutralizing Epitopes of Human Adenovirus Type 55 Hexon Protein

PubMed Central

Tian, Xingui; Ma, Qiang; Jiang, Zaixue; Huang, Junfeng; Liu, Qian; Lu, Xiaomei; Luo, Qingming; Zhou, Rong

2015-01-01

Human adenovirus type 55 (HAdV55) is a newly identified re-emergent acute respiratory disease (ARD) pathogen with a proposed recombination of hexon gene between HAdV11 and HAdV14 strains. The identification of the neutralizing epitopes is important for the surveillance and vaccine development against HAdV55 infection. In this study, four type-specific epitope peptides of HAdV55 hexon protein, A55R1 (residues 138 to 152), A55R2 (residues 179 to 187), A55R4 (residues 247 to 259) and A55R7 (residues 429 to 443), were predicted by multiple sequence alignment and homology modeling methods, and then confirmed with synthetic peptides by enzyme-linked immunosorbent assay (ELISA) and neutralization tests (NT). Finally, the A55R2 was incorporated into human adenoviruses 3 (HAdV3) and a chimeric adenovirus rAd3A55R2 was successfully obtained. The chimeric rAd3A55R2 could induce neutralizing antibodies against both HAdV3 and HAdV55. This current study will contribute to the development of novel adenovirus vaccine candidate and adenovirus structural analysis. PMID:26516903
Immunogold-agglutination assay for direct detection of HPV-16 E6 and L1 proteins from clinical specimens.

PubMed

Bhattarakosol, Parvapan; Plaignam, Kamolwan; Sereemaspun, Amornpun

2018-05-01

HPV-16 infection is the most common cause of cervical cancer. As HPV-16 transforms the cell, E6 oncoprotein is over-expressed. Therefore, molecular detection of HPV-16 E6 mRNA is now being used for diagnosis and prediction of cancer development. Besides detecting E6 mRNA, a rapid lateral flow detecting the E6 protein using enzyme immunoassay is also now on market with a sensitivity of 53.5% for cervical intraepithelial neoplasia (CIN)-3 or more severe (CIN-3+). Here, an immunogold-agglutination assay was developed to detect not only HPV-16 E6 protein but also L1, a major capsid protein found in the productive stage of the virus. Evaluation of this test using HPV-16 DNA positive cervical samples showed that the HPV-16 E6 immunogold-agglutination assay results correlated well with the progression of the cervical lesions, i.e., 10.34% of CIN-1, 68.75% of CIN-3 and 80% of cancer (CaCx) and none for healthy normal samples. Interestingly, the HPV-16 L1 protein was found in most of the cases with cancer indicating the possibility of virion production. Immunogold-agglutination assay for E6 protein is simpler, easier to be performed with a sensitivity of 73.1% for CIN-3+ suggesting a good method for laboratory diagnostic use. Copyright © 2018 Elsevier B.V. All rights reserved.
Covalent Allosteric Inactivation of Protein Tyrosine Phosphatase 1B (PTP1B) by an Inhibitor-Electrophile Conjugate.

PubMed

Punthasee, Puminan; Laciak, Adrian R; Cummings, Andrea H; Ruddraraju, Kasi Viswanatharaju; Lewis, Sarah M; Hillebrand, Roman; Singh, Harkewal; Tanner, John J; Gates, Kent S

2017-04-11

Protein tyrosine phosphatase 1B (PTP1B) is a validated drug target, but it has proven difficult to develop medicinally useful, reversible inhibitors of this enzyme. Here we explored covalent strategies for the inactivation of PTP1B using a conjugate composed of an active site-directed 5-aryl-1,2,5-thiadiazolidin-3-one 1,1-dioxide inhibitor connected via a short linker to an electrophilic α-bromoacetamide moiety. Inhibitor-electrophile conjugate 5a caused time-dependent loss of PTP1B activity consistent with a covalent inactivation mechanism. The inactivation occurred with a second-order rate constant of (1.7 ± 0.3) × 10 2 M -1 min -1 . Mass spectrometric analysis of the inactivated enzyme indicated that the primary site of modification was C121, a residue distant from the active site. Previous work provided evidence that covalent modification of the allosteric residue C121 can cause inactivation of PTP1B [Hansen, S. K., Cancilla, M. T., Shiau, T. P., Kung, J., Chen, T., and Erlanson, D. A. (2005) Biochemistry 44, 7704-7712]. Overall, our results are consistent with an unusual enzyme inactivation process in which noncovalent binding of the inhibitor-electrophile conjugate to the active site of PTP1B protects the nucleophilic catalytic C215 residue from covalent modification, thus allowing inactivation of the enzyme via selective modification of allosteric residue C121.
Adaptor protein 1 B mu subunit does not contribute to the recycling of kAE1 protein in polarized renal epithelial cells.

PubMed

Almomani, Ensaf Y; Touret, Nicolas; Cordat, Emmanuelle

2018-04-13

Mutations in the gene encoding the kidney anion exchanger 1 (kAE1) can lead to distal renal tubular acidosis (dRTA). dRTA mutations reported within the carboxyl (C)-terminal tail of kAE1 result in apical mis-targeting of the exchanger in polarized renal epithelial cells. As kAE1 physically interacts with the μ subunit of epithelial adaptor protein 1 B (AP-1B), we investigated the role of heterologously expressed μ1B subunit of the AP-1B complex for kAE1 retention to the basolateral membrane in polarized porcine LLC-PK1 renal epithelial cells that are devoid of endogenous AP-1B. We confirmed the interaction and close proximity between kAE1 and μ1B using immunoprecipitation and proximity ligation assay, respectively. Expressing the human μ1B subunit in these cells decreased significantly the amount of cell surface kAE1 at the steady state, but had no significant effect on kAE1 recycling and endocytosis. We show that (i) heterologous expression of μ1B displaces the physical interaction of endogenous GAPDH with kAE1 WT supporting that both AP-1B and GAPDH proteins bind to an overlapping site on kAE1 and (ii) phosphorylation of tyrosine 904 within the potential YDEV interaction motif does not alter the kAE1/AP-1B interaction. We conclude that μ1B subunit is not involved in recycling of kAE1.
Lysosomal-associated Transmembrane Protein 4B (LAPTM4B) Decreases Transforming Growth Factor β1 (TGF-β1) Production in Human Regulatory T Cells.

PubMed

Huygens, Caroline; Liénart, Stéphanie; Dedobbeleer, Olivier; Stockis, Julie; Gauthy, Emilie; Coulie, Pierre G; Lucas, Sophie

2015-08-14

Production of active TGF-β1 is one mechanism by which human regulatory T cells (Tregs) suppress immune responses. This production is regulated by glycoprotein A repetitions predominant (GARP), a transmembrane protein present on stimulated Tregs but not on other T lymphocytes (Th and CTLs). GARP forms disulfide bonds with proTGF-β1, favors its cleavage into latent inactive TGF-β1, induces the secretion and surface presentation of GARP·latent TGF-β1 complexes, and is required for activation of the cytokine in Tregs. We explored whether additional Treg-specific protein(s) associated with GARP·TGF-β1 complexes regulate TGF-β1 production in Tregs. We searched for such proteins by yeast two-hybrid assay, using GARP as a bait to screen a human Treg cDNA library. We identified lysosomal-associated transmembrane protein 4B (LAPTM4B), which interacts with GARP in mammalian cells and is expressed at higher levels in Tregs than in Th cells. LAPTM4B decreases cleavage of proTGF-β1, secretion of soluble latent TGF-β1, and surface presentation of GARP·TGF-β1 complexes by Tregs but does not contribute to TGF-β1 activation. Therefore, LAPTM4B binds to GARP and is a negative regulator of TGF-β1 production in human Tregs. It may play a role in the control of immune responses by decreasing Treg immunosuppression. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

GOLGI TRANSPORT 1B Regulates Protein Export from the Endoplasmic Reticulum in Rice Endosperm Cells[OPEN

PubMed Central

Liu, Feng; Wang, Yunlong; Liu, Xi; Wang, Di; Zhu, Xiaopin; Jing, Ruonan; Wu, Mingming; Hao, Yuanyuan; Jiang, Ling; Wang, Chunming

2016-01-01

Coat protein complex II (COPII) mediates the first step of anterograde transport of newly synthesized proteins from the endoplasmic reticulum (ER) to other endomembrane compartments in eukaryotes. A group of evolutionarily conserved proteins (Sar1, Sec23, Sec24, Sec13, and Sec31) constitutes the basic COPII coat machinery; however, the details of how the COPII coat assembly is regulated remain unclear. Here, we report a protein transport mutant of rice (Oryza sativa), named glutelin precursor accumulation4 (gpa4), which accumulates 57-kD glutelin precursors and forms two types of ER-derived abnormal structures. GPA4 encodes the evolutionarily conserved membrane protein GOT1B (also known as GLUP2), homologous to the Saccharomyces cerevisiae GOT1p. The rice GOT1B protein colocalizes with Arabidopsis thaliana Sar1b at Golgi-associated ER exit sites (ERESs) when they are coexpressed in Nicotiana benthamiana. Moreover, GOT1B physically interacts with rice Sec23, and both proteins are present in the same complex(es) with rice Sar1b. The distribution of rice Sar1 in the endomembrane system, its association with rice Sec23c, and the ERES organization pattern are significantly altered in the gpa4 mutant. Taken together, our results suggest that GOT1B plays an important role in mediating COPII vesicle formation at ERESs, thus facilitating anterograde transport of secretory proteins in plant cells. PMID:27803308
Conformational Rigidity and Protein Dynamics at Distinct Timescales Regulate PTP1B Activity and Allostery.

PubMed

Choy, Meng S; Li, Yang; Machado, Luciana E S F; Kunze, Micha B A; Connors, Christopher R; Wei, Xingyu; Lindorff-Larsen, Kresten; Page, Rebecca; Peti, Wolfgang

2017-02-16

Protein function originates from a cooperation of structural rigidity, dynamics at different timescales, and allostery. However, how these three pillars of protein function are integrated is still only poorly understood. Here we show how these pillars are connected in Protein Tyrosine Phosphatase 1B (PTP1B), a drug target for diabetes and cancer that catalyzes the dephosphorylation of numerous substrates in essential signaling pathways. By combining new experimental and computational data on WT-PTP1B and ≥10 PTP1B variants in multiple states, we discovered a fundamental and evolutionarily conserved CH/π switch that is critical for positioning the catalytically important WPD loop. Furthermore, our data show that PTP1B uses conformational and dynamic allostery to regulate its activity. This shows that both conformational rigidity and dynamics are essential for controlling protein activity. This connection between rigidity and dynamics at different timescales is likely a hallmark of all enzyme function. Copyright © 2017 Elsevier Inc. All rights reserved.
24 CFR 55.2 - Terminology.

Code of Federal Regulations, 2012 CFR

2012-04-01

... or inoperative during flood and storm events (e.g., data storage centers, generating plants... appropriate data sources.) (4) Floodway means that portion of the floodplain which is effective in carrying.... (See § 55.2(b)(1) for appropriate data sources.) (5) Functionally dependent use means a land use that...
Protein tyrosine phosphatase, PTP1B, expression and activity in rat corneal endothelial cells

PubMed Central

Harris, Deshea L.

2007-01-01

Purpose The current studies were conducted to determine whether the protein tyrosine phosphatase, PTP1B, plays a role in regulating epidermal growth factor receptor (EGFR) Tyr992 phosphorylation and cell cycle entry in rat corneal endothelial cells. Methods Corneas were obtained from male Sprague-Dawley rats. PTP1B mRNA and protein expression were compared in confluent and subconfluent cells by RT-PCR and western blots. Immunocytochemistry was used to determine the subcellular localization of both PTP1B and EGFR following epidermal growth factor (EGF) stimulation. Western blots were used to analyze the time-dependent effect of EGF on phosphorylation of EGFR Tyr992 plus or minus CinnGEL 2Me, an inhibitor of PTP1B activity. The effect of PTP1B inhibition on cell cycle entry was determined by calculating the percent of Ki67-positive cells following EGF treatment. Results PTP1B mRNA expression was similar in confluent and subconfluent cells, but PTP1B protein was expressed at 3 fold higher levels in subconfluent cells. Positive staining for PTP1B was localized in vesicular structures below the plasma membrane. EGFR staining was located at cell-cell borders in untreated endothelium, but was mainly cytoplasmic by 15 min after EGF treatment. In control cultures, phosphorylation of EGFR Tyr992 peaked by 5 min following EGF stimulation and rapidly decreased to basal levels by 30 min. In cultures pretreated with CinnGEL 2Me, Tyr992 phosphorylation peaked 2 min following EGF addition and was consistently sustained at a higher level than controls until 60 min after treatment. By 18 h following EGF treatment, cultures pretreated with CinnGEL 2Me exhibited a 1.7 fold increase in the number of Ki67-positive cells compared with control cultures. Conclusions Comparison of PTP1B mRNA and protein levels indicates that PTP1B expression is regulated mainly at the protein level and is higher in subconfluent cells. PTP1B was located in vesicles below the plasma membrane. The fact that
The calcium-binding protein ALG-2 regulates protein secretion and trafficking via interactions with MISSL and MAP1B proteins.

PubMed

Takahara, Terunao; Inoue, Kuniko; Arai, Yumika; Kuwata, Keiko; Shibata, Hideki; Maki, Masatoshi

2017-10-13

Mobilization of intracellular calcium is essential for a wide range of cellular processes, including signal transduction, apoptosis, and vesicular trafficking. Several lines of evidence have suggested that apoptosis-linked gene 2 (ALG-2, also known as PDCD6 ), a calcium-binding protein, acts as a calcium sensor linking calcium levels with efficient vesicular trafficking, especially at the endoplasmic reticulum (ER)-to-Golgi transport step. However, how ALG-2 regulates these processes remains largely unclear. Here, we report that M APK1- i nteracting and s pindle- s tabilizing (MISS)- l ike (MISSL), a previously uncharacterized protein, interacts with ALG-2 in a calcium-dependent manner. Live-cell imaging revealed that upon a rise in intracellular calcium levels, GFP-tagged MISSL (GFP-MISSL) dynamically relocalizes in a punctate pattern and colocalizes with ALG-2. MISSL knockdown caused disorganization of the components of the ER exit site, the ER-Golgi intermediate compartment, and Golgi. Importantly, knockdown of either MISSL or ALG-2 attenuated the secretion of se creted a lkaline p hosphatase (SEAP), a model secreted cargo protein, with similar reductions in secretion by single- and double-protein knockdowns, suggesting that MISSL and ALG-2 act in the same pathway to regulate the secretion process. Furthermore, ALG-2 or MISSL knockdown delayed ER-to-Golgi transport of procollagen type I. We also found that ALG-2 and MISSL interact with microtubule-associated protein 1B (MAP1B) and that MAP1B knockdown reverts the reduced secretion of SEAP caused by MISSL or ALG-2 depletion. These results suggest that a change in the intracellular calcium level plays a role in regulation of the secretory pathway via interaction of ALG-2 with MISSL and MAP1B. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.
Red light-regulated growth. I. Changes in the abundance of indoleacetic acid and a 22-kilodalton auxin-binding protein in the maize mesocotyl

NASA Technical Reports Server (NTRS)

Jones, A. M.; Cochran, D. S.; Lamerson, P. M.; Evans, M. L.; Cohen, J. D.

1991-01-01

We examined the changes in the levels of indoleacetic acid (IAA), IAA esters, and a 22-kilodalton subunit auxin-binding protein (ABP1) in apical mesocotyl tissue of maize (Zea mays L.) during continuous red light (R) irradiation. These changes were compared with the kinetics of R-induced growth inhibition in the same tissue. Upon the onset of continuous irradiation, growth decreased in a continuous manner following a brief lag period. The decrease in growth continued for 5 hours, then remained constant at 25% of the dark rate. The abundance of ABP1 and the level of free IAA both decreased in the mesocotyl. Only the kinetics of the decrease in IAA within the apical mesocotyl correlated with the initial change in growth, although growth continued to decrease even after IAA content reached its final level, 50% of the dark control. This decrease in IAA within the mesocotyl probably occurs primarily by a change in its transport within the shoot since auxin applied as a pulse move basipetally in R-irradiated tissue at the same rate but with half the area as dark control tissue. In situ localization of auxin in etiolated maize shoots revealed that R-irradiated shoots contained less auxin in the epidermis than the dark controls. Irradiated mesocotyl grew 50% less than the dark controls even when incubated in an optimal level of auxin. However, irradiated and dark tissue contained essentially the same amount of radioactivity after incubation in [14C]IAA indicating that the light treatment does not affect the uptake into the tissue through the cut end, although it is possible that a small subset of cells within the mesocotyl is affected. These observations support the hypothesis that R causes a decrease in the level of auxin in epidermal cells of the mesocotyl, consequently constraining the growth of the entire mesocotyl.
Gestational Protein Restriction Reduces Expression of Hsd17b2 in Rat Placental Labyrinth1

PubMed Central

Gao, Haijun; Yallampalli, Uma; Yallampalli, Chandra

2012-01-01

ABSTRACT Accumulating evidence strongly supports the premise that testosterone may be a key player in fetal programming on hypertension. Studies have shown that gestational protein restriction doubles the plasma testosterone levels in pregnant rats. In this study, we hypothesized that elevated testosterone levels in response to gestational protein restriction were caused by enhanced expression of steroidogenic enzymes or impaired expression of Hsd17b2, a known testosterone inactivator that converts testosterone to androstenedione in placenta. Pregnant Sprague-Dawley rats were fed normal (20% protein, control; n = 10) or a low-protein diet (6% protein, PR; n = 10) from Day 1 of pregnancy until killed at Days 14, 18, or 21. Junctional (JZ) and labyrinth (LZ) zones of placenta were collected for expression assay on steroidogenic genes (Cyp11a1, Hsd3b1, Cyp17a1, Hsd17b2, and Srd5a1) by real-time PCR. The main findings include the following: 1) expressions of Cyp11a1, Hsd3b1, and Cyp17a1 in JZ were not affected by diet but were affected by day of pregnancy; 2) expression of Hsd17b2 in both female and male JZs was remarkably increased by PR at Days 18 and 21 of pregnancy; 3) expressions of Hsd17b2 were reduced by PR in both female and male LZ at Day 18 of pregnancy and in female LZ at Day 21 of pregnancy; and 4) expression of Srd5a1in LZ was not affected by day of pregnancy, gender, or diet. These results indicate that in response to gestational protein restriction, Hsd17b2 may be a key regulator of testosterone levels and associated activities in placental zones, apparently in a paradoxical manner. PMID:22837477
Identification of drugs and drug metabolites as substrates of multidrug resistance protein 2 (MRP2) using triple-transfected MDCK-OATP1B1-UGT1A1-MRP2 cells

PubMed Central

Fahrmayr, C; König, J; Auge, D; Mieth, M; Fromm, MF

2012-01-01

BACKGROUND AND PURPOSE The coordinate activity of hepatic uptake transporters [e.g. organic anion transporting polypeptide 1B1 (OATP1B1)], drug-metabolizing enzymes [e.g. UDP-glucuronosyltransferase 1A1 (UGT1A1)] and efflux pumps (e.g. MRP2) is a crucial determinant of drug disposition. However, limited data are available on transport of drugs (e.g. ezetimibe, etoposide) and their glucuronidated metabolites by human MRP2 in intact cell systems. EXPERIMENTAL APPROACH Using monolayers of newly established triple-transfected MDCK-OATP1B1-UGT1A1-MRP2 cells as well as MDCK control cells, single- (OATP1B1) and double-transfected (OATP1B1-UGT1A1, OATP1B1-MRP2) MDCK cells, we therefore studied intracellular concentrations and transcellular transport after administration of ezetimibe or etoposide to the basal compartment. KEY RESULTS Intracellular accumulation of ezetimibe was significantly lower in MDCK-OATP1B1-UGT1A1-MRP2 triple-transfected cells compared with all other cell lines. Considerably higher amounts of ezetimibe glucuronide were found in the apical compartment of MDCK-OATP1B1-UGT1A1-MRP2 monolayers compared with all other cell lines. Using HEK cells, etoposide was identified as a substrate of OATP1B1. Intracellular concentrations of etoposide equivalents (i.e. parent compound plus metabolites) were affected only to a minor extent by the absence or presence of OATP1B1/UGT1A1/MRP2. In contrast, apical accumulation of etoposide equivalents was significantly higher in monolayers of both cell lines expressing MRP2 (MDCK-OATP1B1-MRP2, MDCK-OATP1B1-UGT1A1-MRP2) compared with the single-transfected (OATP1B1) and the control cell line. CONCLUSIONS AND IMPLICATIONS Ezetimibe glucuronide is a substrate of human MRP2. Moreover, etoposide and possibly also its glucuronide are substrates of MRP2. These data demonstrate the functional interplay between transporter-mediated uptake, phase II metabolism and export by hepatic proteins involved in drug disposition. PMID:21923755
Double mushroom 1.55-μm waveguide photodetectors for integrated E-band (60-90 GHz) wireless transmitter modules

NASA Astrophysics Data System (ADS)

Rymanov, Vitaly; Tekin, Tolga; Stöhr, Andreas

2012-03-01

High data rate photonic wireless systems operating at millimeter wave carrier frequencies are considered as a disruptive technology e.g. for reach extension in optical access networks and for mobile backhauling. Recently, we demonstrated 60 GHz photonic wireless systems with record data rates up to 27 Gbit/s. Because of the oxygen absorption at 60 GHz, it is beneficial for fixed wireless systems with spans exceeding 1 km to operate at even higher frequencies. Here, the recently regulated 10 GHz bandwidth within the E-band (60-90 GHz) is of particular interest, covering the 71-76 GHz and 81-86 GHz allocations for multi-gigabit wireless transmission. For this purpose, wideband waveguide photodetectors with high external quantum efficiency are required. Here, we report on double mushroom 1.55 μm waveguide photodetectors for integration in an E-band wireless transmitter module. The developed photodetector consists of a partially p-doped, partly non-intentionally doped absorbing layer centered in a mushroom-type optical waveguide, overcoming the compromise between the junction capacitance and the series resistance. For efficient fiber-chip coupling, a second mushroom-type passive optical waveguide is used. In contrast to the conventional shallow ridge waveguide approach, the mushroom-type passive waveguide allows to shift the center of the optical mode further away from the top surface, thus reducing waveguide losses due to the surface roughness. Experimentally, a very flat frequency response with a deviation up to +/-1 dB in the entire E-band has been found together with an output power level of -15.7 dBm at 10 mA photocurrent and at a frequency of 73 GHz.
Porcine Mx1 Protein Inhibits Classical Swine Fever Virus Replication by Targeting Nonstructural Protein NS5B.

PubMed

Zhou, Jing; Chen, Jing; Zhang, Xiao-Min; Gao, Zhi-Can; Liu, Chun-Chun; Zhang, Yun-Na; Hou, Jin-Xiu; Li, Zhao-Yao; Kan, Lin; Li, Wen-Liang; Zhou, Bin

2018-04-01

Mx proteins are interferon (IFN)-induced GTPases that have broad antiviral activity against a wide range of RNA and DNA viruses; they belong to the dynamin superfamily of large GTPases. In this study, we confirmed the anti-classical swine fever virus (CSFV) activity of porcine Mx1 in vitro and showed that porcine Mx2 (poMx2), human MxA (huMxA), and mouse Mx1 (mmMx1) also have anti-CSFV activity in vitro Small interfering RNA (siRNA) experiments revealed that depletion of endogenous poMx1 or poMx2 enhanced CSFV replication, suggesting that porcine Mx proteins are responsible for the antiviral activity of interferon alpha (IFN-α) against CSFV infection. Confocal microscopy, immunoprecipitation, glutathione S -transferase (GST) pulldown, and bimolecular fluorescence complementation (BiFC) demonstrated that poMx1 associated with NS5B, the RNA-dependent RNA polymerase (RdRp) of CSFV. We used mutations in the poMx1 protein to elucidate the mechanism of their anti-CSFV activity and found that mutants that disrupted the association with NS5B lost all anti-CSV activity. Moreover, an RdRp activity assay further revealed that poMx1 undermined the RdRp activities of NS5B. Together, these results indicate that porcine Mx proteins exert their antiviral activity against CSFV by interacting with NS5B. IMPORTANCE Our previous studies have shown that porcine Mx1 (poMx1) inhibits classical swine fever virus (CSFV) replication in vitro and in vivo , but the molecular mechanism of action remains largely unknown. In this study, we dissect the molecular mechanism of porcine Mx1 and Mx2 against CSFV in vitro Our results show that poMx1 associates with NS5B, the RNA-dependent RNA polymerase of CSFV, resulting in the reduction of CSFV replication. Moreover, the mutants of poMx1 further elucidate the mechanism of their anti-CSFV activities. Copyright © 2018 American Society for Microbiology.
ACBD3 functions as a scaffold to organize the Golgi stacking proteins and a Rab33b-GAP.

PubMed

Yue, Xihua; Bao, Mengjing; Christiano, Romain; Li, Siyang; Mei, Jia; Zhu, Lianhui; Mao, Feifei; Yue, Qiang; Zhang, Panpan; Jing, Shuaiyang; Rothman, James E; Qian, Yi; Lee, Intaek

2017-09-01

Golgin45 plays important roles in Golgi stack assembly and is known to bind both the Golgi stacking protein GRASP55 and Rab2 in the medial-Golgi cisternae. In this study, we sought to further characterize the cisternal adhesion complex using a proteomics approach. We report here that Acyl-CoA binding domain containing 3 (ACBD3) is likely to be a novel binding partner of Golgin45. ACBD3 interacts with Golgin45 via its GOLD domain, while its co-expression significantly increases Golgin45 targeting to the Golgi. Furthermore, ACBD3 recruits TBC1D22, a Rab33b GTPase activating protein (GAP), to a large multi-protein complex containing Golgin45 and GRASP55. These results suggest that ACBD3 may provide a scaffolding to organize the Golgi stacking proteins and a Rab33b-GAP at the medial-Golgi. © 2017 Federation of European Biochemical Societies.
Protein tyrosine phosphatase-1B (PTP1B) helps regulate EGF-induced stimulation of S-phase entry in human corneal endothelial cells

PubMed Central

Ishino, Yutaka; Zhu, Cheng; Harris, Deshea L.

2008-01-01

Purpose Human corneal endothelial cells (HCEC), particularly from older donors, only proliferate weakly in response to EGF. The protein tyrosine phosphatase, PTP1B, is known to negatively regulate EGF-induced signaling in several cell types by dephosphorylating the epidermal growth factor receptor (EGFR). The current studies were conducted to determine whether PTP1B plays a role in regulating cell cycle entry in HCEC in response to EGF stimulation. Methods Donor corneas were obtained from the National Disease Research Interchange and accepted for study based on established exclusion criteria. PTP1B was localized in the endothelium of ex vivo corneas and in cultured cells by immunocytochemistry. Western blot analysis verified PTP1B protein expression in HCEC and then compared the relative expression of EGFR and PTP1B in HCEC from young (<3 years old) and older donors (>60 years old). The effect of inhibiting the activity of PTP1B on S-phase entry was tested by comparing time-dependent BrdU incorporation in subconfluent HCEC incubated in the presence or absence of the PTP1B inhibitor, CinnGEL 2Me, before EGF stimulation. Results PTP1B was localized in a punctate pattern mainly within the cytoplasm of HCEC in ex vivo corneas and cultured cells. Western blots revealed the presence of three PTP1B-positive bands in HCEC and the control. Further western blot analysis showed no significant age-related difference in expression of EGFR (p=0.444>0.05); however, PTP1B expression was significantly higher in HCEC from older donors (p=0.024<0.05). Pre-incubation of HCEC with the PTP1B inhibitor significantly increased (p=0.019<0.05) the number of BrdU positive cells by 48 h after EGF stimulation. Conclusions Both immunolocalization and western blot studies confirmed that PTP1B is expressed in HCEC. Staining patterns strongly suggest that at least a subset of PTP1B is localized to the cytoplasm and most likely to the endoplasmic reticulum, the known site of EGFR/PTP1B interaction
β-Adrenergic Stimulation Induces Histone Deacetylase 5 (HDAC5) Nuclear Accumulation in Cardiomyocytes by B55α-PP2A-Mediated Dephosphorylation.

PubMed

Weeks, Kate L; Ranieri, Antonella; Karaś, Agnieszka; Bernardo, Bianca C; Ashcroft, Alexandra S; Molenaar, Chris; McMullen, Julie R; Avkiran, Metin

2017-03-25

Class IIa histone deacetylase (HDAC) isoforms such as HDAC5 are critical signal-responsive repressors of maladaptive cardiomyocyte hypertrophy, through nuclear interactions with transcription factors including myocyte enhancer factor-2. β-Adrenoceptor (β-AR) stimulation, a signal of fundamental importance in regulating cardiac function, has been proposed to induce both phosphorylation-independent nuclear export and phosphorylation-dependent nuclear accumulation of cardiomyocyte HDAC5. The relative importance of phosphorylation at Ser259/Ser498 versus Ser279 in HDAC5 regulation is also controversial. We aimed to determine the impact of β-AR stimulation on the phosphorylation, localization, and function of cardiomyocyte HDAC5 and delineate underlying molecular mechanisms. A novel 3-dimensional confocal microscopy method that objectively quantifies the whole-cell nuclear/cytoplasmic distribution of green fluorescent protein tagged HDAC5 revealed the β-AR agonist isoproterenol to induce β 1 -AR-mediated and protein kinase A-dependent HDAC5 nuclear accumulation in adult rat cardiomyocytes, which was accompanied by dephosphorylation at Ser259/279/498. Mutation of Ser259/Ser498 to Ala promoted HDAC5 nuclear accumulation and myocyte enhancer factor-2 inhibition, whereas Ser279 ablation had no such effect and did not block isoproterenol-induced nuclear accumulation. Inhibition of the Ser/Thr phosphatase PP2A blocked isoproterenol-induced HDAC5 dephosphorylation. Co-immunoprecipitation revealed a specific interaction of HDAC5 with the PP2A targeting subunit B55α, as well as catalytic and scaffolding subunits, which increased >3-fold with isoproterenol. Knockdown of B55α in neonatal cardiomyocytes attenuated isoproterenol-induced HDAC5 dephosphorylation. β-AR stimulation induces HDAC5 nuclear accumulation in cardiomyocytes by a mechanism that is protein kinase A-dependent but requires B55α-PP2A-mediated dephosphorylation of Ser259/Ser498 rather than protein kinase A
Identification and characterization of a gene product that regulates type 1 piliation in Escherichia coli.

PubMed Central

Orndorff, P E; Falkow, S

1984-01-01

The recombinant plasmid pSH2 confers type 1 piliation (Pil+) on a nonpiliated (Pil-) strain of Escherichia coli K-12. At least four plasmid-encoded gene products are involved in pilus biosynthesis and expression. We present evidence which indicates that one gene encodes an inhibitor of piliation. Hyperpiliated (Hyp) mutants were isolated after Tn5 insertion mutagenesis of pSH2 and introduction of the plasmid DNA into a Pil- strain of E. coli as unique small, compact colonies. Also, Hyp mutants clumped during growth in static broth and were piliated under several cultural conditions that normally suppressed piliation. Electron microscopic examination of Hyp mutants associated an observed 40-fold increase in pilin antigen with an increase in the number and length of pili per cell. All Hyp mutants examined failed to produce a 23-kilodalton protein that was encoded by a gene adjacent to the structural (pilin) gene for type 1 pili, and all Tn5 insertion mutations that produced the Hyp phenotype mapped in this region (hyp). Piliation in Hyp mutants could be reduced to near parental levels by introducing a second plasmid containing a parental hyp gene. Thus the 23-kilodalton (hyp) protein appears to act in trans to regulate the level of piliation. Images PMID:6148338
Myelin protein zero gene sequencing diagnoses Charcot-Marie-Tooth Type 1B disease

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

Su, Y.; Zhang, H.; Madrid, R.

1994-09-01

Charcot-Marie-Tooth disease (CMT), the most common genetic neuropathy, affects about 1 in 2600 people in Norway and is found worldwide. CMT Type 1 (CMT1) has slow nerve conduction with demyelinated Schwann cells. Autosomal dominant CMT Type 1B (CMT1B) results from mutations in the myelin protein zero gene which directs the synthesis of more than half of all Schwann cell protein. This gene was mapped to the chromosome 1q22-1q23.1 borderline by fluorescence in situ hybridization. The first 7 of 7 reported CMT1B mutations are unique. Thus the most effective means to identify CMT1B mutations in at-risk family members and fetuses ismore » to sequence the entire coding sequence in dominant or sporadic CMT patients without the CMT1A duplication. Of the 19 primers used in 16 pars to uniquely amplify the entire MPZ coding sequence, 6 primer pairs were used to amplify and sequence the 6 exons. The DyeDeoxy Terminator cycle sequencing method used with four different color fluorescent lables was superior to manual sequencing because it sequences more bases unambiguously from extracted genomic DNA samples within 24 hours. This protocol was used to test 28 CMT and Dejerine-Sottas patients without CMT1A gene duplication. Sequencing MPZ gene-specific amplified fragments identified 9 polymorphic sites within the 6 exons that encode the 248 amino acid MPZ protein. The large number of major CMT1B mutations identified by single strand sequencing are being verified by reverse strand sequencing and when possible, by restriction enzyme analysis. This protocol can be used to distringuish CMT1B patients from othre CMT phenotypes and to determine the CMT1B status of relatives both presymptomatically and prenatally.« less
Purification and Thermal Dependence of Glutathione Reductase from Two Forage Legume Species 1

PubMed Central

Kidambi, Saranga P.; Mahan, James R.; Matches, Arthur G.

1990-01-01

Alfalfa (Medicago sativa L.) and sainfoin (Onobrychis viciifolia Scop.) are forage legumes that differ in their responses to high and low temperature stresses. Thermal limitations on the function of glutathione reductase (EC 1.6.4.2) could adversely affect the ability of the plant to cope with adverse temperatures. Our objectives were to (a) purify glutathione reductase from `Cimarron' alfalfa and `PI 212241' sainfoin and (b) investigate the intraspecies variation in the thermal dependency of glutathione reductase from each of three cultivars of alfalfa and two cultivars and an introduction of sainfoin. Glutathione reductase was purified 1222-and 1948-fold to a specific activity of 281 and 273 units per milligram of protein, from one species each of alfalfa and sainfoin, respectively. The relative molecular mass of the protein was approximately 140 kilodaltons with subunits of 57 and 37 kilodaltons under denaturing conditions. The activation energies were approximately 50 kilojoules per mole for both species. Over a 5 to 45°C temperature gradient, large variation among species and genotypes within species was found for: (a) the minimum apparent Michaelis constant (0.6-2.1 micromoles of NADPH), (b) the temperature at which the minimum apparent Michaelis constant was observed (10-25°C), and (c) the thermal kinetic windows (6-19°C width). Future studies will focus on relating the thermal dependence of the Michaelis constant of the glutathione reductases and plant growth rates and forage quality of these species throughout the growing season. PMID:16667283
Altered CSNK1E, FABP4 and NEFH protein levels in the dorsolateral prefrontal cortex in schizophrenia.

PubMed

Pinacho, Raquel; Villalmanzo, Núria; Meana, J Javier; Ferrer, Isidre; Berengueras, Adriana; Haro, Josep M; Villén, Judit; Ramos, Belén

2016-11-01

Schizophrenia constitutes a complex disease. Negative and cognitive symptoms are enduring and debilitating components of the disorder, highly associated to disability and burden. Disrupted neurotransmission circuits in dorsolateral prefrontal cortex (DLPFC) have been related to these symptoms. To identify candidates altered in schizophrenia, we performed a pilot proteomic analysis on postmortem human DLPFC tissue from patients with schizophrenia (n=4) and control (n=4) subjects in a pool design using differential isotope peptide labelling followed by liquid chromatography tandem mass spectrometry (LC-MS/MS). We quantified 1315 proteins with two or more unique peptides, 116 of which showed altered changes. Of these altered proteins, we selected four with potential roles on cell signaling, neuronal development and synapse functioning for further validation: casein kinase I isoform epsilon (CSNK1E), fatty acid-binding protein 4 (FABP4), neurofilament triplet H protein (NEFH), and retinal dehydrogenase 1 (ALDH1A1). Immunoblot validation confirmed our proteomic findings of these proteins being decreased in abundance in the schizophrenia samples. Additionally, we conducted immunoblot validation of these candidates on an independent sample cohort comprising 23 patients with chronic schizophrenia and 23 matched controls. In this second cohort, CSNK1E, FABP4 and NEFH were reduced in the schizophrenia group while ALDH1A1 did not significantly change. This study provides evidence indicating these proteins are decreased in schizophrenia: CSNK1E, involved in circadian molecular clock signaling, FABP4 with possible implication in synapse functioning, and NEFH, important for cytoarchitecture organization. Hence, these findings suggest the possible implication of these proteins in the cognitive and/or negative symptoms in schizophrenia. Copyright © 2016 Elsevier B.V. All rights reserved.
Identification and characterization of genes determining receptor binding and pilus length of Escherichia coli type 1 pili.

PubMed Central

Maurer, L; Orndorff, P E

1987-01-01

We describe the identification and characterization of two genes and their gene products responsible for determining receptor binding and pilus length in type 1-piliated Escherichia coli. One gene, pilE, conferred the ability of piliated cells to agglutinate guinea pig erythrocytes. The other gene, pilF, determined pilus length, in that mutants having lesions in pilF had very long pili. The two genes were detected after Tn5 mutagenesis of a cloned segment of DNA that normally complemented a pilE lesion in the chromosome. Thus, lesions in pilE or pilF on the cloned segment resulted in mutants having the PilE- phenotype (piliated but unable to agglutinate erythrocytes). Introduction of the plasmid-encoded mutant alleles of pilE and pilF into the chromosome followed by electron microscopic examination of the mutants showed that only lesions in pilF conferred the striking increase in pilus length. Mutations in pilF could be complemented in trans by the original cloned segment to produce cells with parental-length pili. Minicell transcription and translation of the cloned pilE and pilF genes having representative Tn5 insertion mutations showed that the pilE gene product was a protein of ca. 31 kilodaltons and that the pilF gene product was a protein of ca. 18 kilodaltons. We believe that the pilF gene product may act as a competitive inhibitor of pilus polymerization. Thus, pilus length may be controlled by the ratio of pilin to pilF gene product present within the cell. Images PMID:2879830
Loss of endophilin-B1 exacerbates Alzheimer’s disease pathology

PubMed Central

Wang, David B.; Kinoshita, Yoshito; Kinoshita, Chizuru; Uo, Takuma; Sopher, Bryce L.; Cudaback, Eiron; Keene, C. Dirk; Bilousova, Tina; Gylys, Karen; Case, Amanda; Jayadev, Suman; Wang, Hong-Gang; Garden, Gwenn A.

2015-01-01

Endophilin-B1, also known as Bax-interacting factor 1 (Bif-1, and encoded by SH3GLB1), is a multifunctional protein involved in apoptosis, autophagy and mitochondrial function. We recently described a unique neuroprotective role for neuron-specific alternatively spliced isoforms of endophilin-B1. To examine whether endophilin-B1-mediated neuroprotection could be a novel therapeutic target for Alzheimer’s disease we used a double mutant amyloid precursor protein and presenilin 1 (APPswe/PSEN1dE9) mouse model of Alzheimer’s disease and observed that expression of neuron-specific endophilin-B1 isoforms declined with disease progression. To determine if this reduction in endophilin-B1 has a functional role in Alzheimer’s disease pathogenesis, we crossed endophilin-B1−/− mice with APPswe/PSEN1dE9 mice. Deletion of endophilin-B1 accelerated disease onset and progression in 6-month-old APPswe/PSEN1dE9/endophilin-B1−/− mice, which showed more plaques, astrogliosis, synaptic degeneration, cognitive impairment and mortality than APPswe/PSEN1dE9 mice. In mouse primary cortical neuron cultures, overexpression of neuron-specific endophilin-B1 isoforms protected against amyloid-β-induced apoptosis and mitochondrial dysfunction. Additionally, protein and mRNA levels of neuron-specific endophilin-B1 isoforms were also selectively decreased in the cerebral cortex and in the synaptic compartment of patients with Alzheimer’s disease. Flow sorting of synaptosomes from patients with Alzheimer’s disease demonstrated a negative correlation between amyloid-β and endophilin-B1 levels. The importance of endophilin-B1 in neuronal function was further underscored by the development of synaptic degeneration and cognitive and motor impairment in endophilin-B1−/− mice by 12 months. Our findings suggest that endophilin-B1 is a key mediator of a feed-forward mechanism of Alzheimer’s disease pathogenesis where amyloid-β reduces neuron-specific endophilin-B1, which in turn
Word Frequency Analysis. MOS: 55B. Skill Levels 1 & 2.

DTIC Science & Technology

1981-05-01

AO-AX17 176 ARMY TRAINING DEVELOPMENT$ INS? FORT MONROE VA F/G 5/9WORD FREQUENCY ANALYSIS. NMIS 55. SKILL LEVELS I 2.(U~) MAY SI A A LONSO...UNCLASSIFIED MOS-558 NLSill AD A1171 7 6 HEADQUARTERS DATA CONTROL NNE UNITED STATES ARMY TRAINING AND DOCTRINE COMMAND FORT MONROE, VIRGINIA 23651 . 1w 0 a~t L...O.OANIZATION HA.ME AND ADDRESS 0. PROGRAM ELEMENT. P’IUJECT. TASKAREA At WO K UNIT HM;lEHS Training Developments Institute R ATTN: ATTG-DOR Fort Monroe. VA 71651

Role of SKP1-CUL1-F-Box-Protein (SCF) E3 Ubiquitin Ligases in Skin Cancer

PubMed Central

Xie, Chuan-Ming; Wei, Wenyi; Sun, Yi

2013-01-01

Many biological processes such as cell proliferation, differentiation, and cell death depend precisely on the timely synthesis and degradation of key regulatory proteins. While protein synthesis can be regulated at multiple levels, protein degradation is mainly controlled by the ubiquitin—proteasome system (UPS), which consists of two distinct steps: (1) ubiquitylation of targeted protein by E1 ubiquitin-activating enzyme, E2 ubiquitin-conjugating enzyme and E3 ubiquitin ligase, and (2) subsequent degradation by the 26S proteasome. Among all E3 ubiquitin ligases, the SCF (SKP1-CUL1-F-box protein) E3 ligases are the largest family and are responsible for the turnover of many key regulatory proteins. Aberrant regulation of SCF E3 ligases is associated with various human diseases, such as cancers, including skin cancer. In this review, we provide a comprehensive overview of all currently published data to define a promoting role of SCF E3 ligases in the development of skin cancer. The future directions in this area of research are also discussed with an ultimate goal to develop small molecule inhibitors of SCF E3 ligases as a novel approach for the treatment of human skin cancer. Furthermore, altered components or substrates of SCF E3 ligases may also be developed as the biomarkers for early diagnosis or predicting prognosis. PMID:23522382
33 CFR 55.1 - Purpose.

Code of Federal Regulations, 2010 CFR

2010-07-01

... 33 Navigation and Navigable Waters 1 2010-07-01 2010-07-01 false Purpose. 55.1 Section 55.1 Navigation and Navigable Waters COAST GUARD, DEPARTMENT OF HOMELAND SECURITY PERSONNEL CHILD DEVELOPMENT... Child Development Services. ...
33 CFR 55.1 - Purpose.

Code of Federal Regulations, 2012 CFR

2012-07-01

... 33 Navigation and Navigable Waters 1 2012-07-01 2012-07-01 false Purpose. 55.1 Section 55.1 Navigation and Navigable Waters COAST GUARD, DEPARTMENT OF HOMELAND SECURITY PERSONNEL CHILD DEVELOPMENT... Child Development Services. ...
33 CFR 55.1 - Purpose.

Code of Federal Regulations, 2011 CFR

2011-07-01

... 33 Navigation and Navigable Waters 1 2011-07-01 2011-07-01 false Purpose. 55.1 Section 55.1 Navigation and Navigable Waters COAST GUARD, DEPARTMENT OF HOMELAND SECURITY PERSONNEL CHILD DEVELOPMENT... Child Development Services. ...
33 CFR 55.1 - Purpose.

Code of Federal Regulations, 2013 CFR

2013-07-01

... 33 Navigation and Navigable Waters 1 2013-07-01 2013-07-01 false Purpose. 55.1 Section 55.1 Navigation and Navigable Waters COAST GUARD, DEPARTMENT OF HOMELAND SECURITY PERSONNEL CHILD DEVELOPMENT... Child Development Services. ...
33 CFR 55.1 - Purpose.

Code of Federal Regulations, 2014 CFR

2014-07-01

... 33 Navigation and Navigable Waters 1 2014-07-01 2014-07-01 false Purpose. 55.1 Section 55.1 Navigation and Navigable Waters COAST GUARD, DEPARTMENT OF HOMELAND SECURITY PERSONNEL CHILD DEVELOPMENT... Child Development Services. ...
Interaction of Human Cytomegalovirus Tegument Proteins ppUL35 and ppUL35A with Sorting Nexin 5 Regulates Glycoprotein B (gpUL55) Localization.

PubMed

Maschkowitz, Gregor; Gärtner, Sabine; Hofmann-Winkler, Heike; Fickenscher, Helmut; Winkler, Michael

2018-05-01

Human cytomegalovirus (HCMV) is a widespread human pathogen that causes asymptomatic infection in healthy individuals but poses a serious threat to immunocompromised patients. During the late phase of HCMV infection, the viral capsid is transported to the cytoplasmic viral assembly center (cVAC), where it is enclosed by the tegument protein layer and the viral envelope. The cVAC consists of circularly arranged vesicles from the trans -Golgi and endosomal networks. The HCMV gene UL35 encodes ppUL35 and its shorter form, ppUL35A. We have previously shown that the UL35 gene is involved in HCMV assembly, but it is unknown how UL35 proteins regulate viral assembly. Here we show that sorting nexin 5 (SNX5), a component of the retromer and part of the retrograde transport pathway, interacts with UL35 proteins. Expression of wild-type proteins but not mutants defective in SNX5 binding resulted in the cellular redistribution of the cation-independent mannose-6-phosphate receptor (CI-M6PR), indicating that UL35 proteins bind and negatively regulate SNX5 to modulate cellular transport pathways. Furthermore, binding of UL35 proteins to SNX5 was required for efficient viral replication and for transport of the most abundant HCMV glycoprotein B (gB; gpUL55) to the cVAC. These results indicate that ppUL35 and ppUL35A control the localization of the essential gB through the regulation of a retrograde transport pathway. Thus, this work is the first to define a molecular interaction between a tegument protein and a vesicular transport factor to regulate glycoprotein localization. IMPORTANCE Human cytomegalovirus is ubiquitously present in the healthy population, but reactivation or reinfection can cause serious, life-threatening infections in immunocompromised patients. For completion of its lytic cycle, human cytomegalovirus induces formation of an assembly center where mature virus particles are formed from multiple viral proteins. Viral glycoproteins use separate vesicular
Structural model of the hUbA1-UbcH10 quaternary complex: in silico and experimental analysis of the protein-protein interactions between E1, E2 and ubiquitin.

PubMed

Correale, Stefania; de Paola, Ivan; Morgillo, Carmine Marco; Federico, Antonella; Zaccaro, Laura; Pallante, Pierlorenzo; Galeone, Aldo; Fusco, Alfredo; Pedone, Emilia; Luque, F Javier; Catalanotti, Bruno

2014-01-01

UbcH10 is a component of the Ubiquitin Conjugation Enzymes (Ubc; E2) involved in the ubiquitination cascade controlling the cell cycle progression, whereby ubiquitin, activated by E1, is transferred through E2 to the target protein with the involvement of E3 enzymes. In this work we propose the first three dimensional model of the tetrameric complex formed by the human UbA1 (E1), two ubiquitin molecules and UbcH10 (E2), leading to the transthiolation reaction. The 3D model was built up by using an experimentally guided incremental docking strategy that combined homology modeling, protein-protein docking and refinement by means of molecular dynamics simulations. The structural features of the in silico model allowed us to identify the regions that mediate the recognition between the interacting proteins, revealing the active role of the ubiquitin crosslinked to E1 in the complex formation. Finally, the role of these regions involved in the E1-E2 binding was validated by designing short peptides that specifically interfere with the binding of UbcH10, thus supporting the reliability of the proposed model and representing valuable scaffolds for the design of peptidomimetic compounds that can bind selectively to Ubcs and inhibit the ubiquitylation process in pathological disorders.
Early events of polyoma infection: adsorption, penetration and nuclear transport

NASA Technical Reports Server (NTRS)

Consigli, R. A.; Haynes, J. I. Jr; Chang, D.; Grenz, L.; Richter, D.; Spooner, B. S. (Principal Investigator)

1992-01-01

Polyoma virions have different attachment proteins which are responsible for hemagglutination of erythrocytes and attachment to cultured mouse kidney cells (MKC). Virion binding studies demonstrated that MKC possess specific (productive infection) and nonspecific (nonproductive) receptors. Empty polyoma capsids have hemagglutination activity and bind to non-specific MKC receptors, but they are not capable of competing for specific virion cell receptors or preventing productive infection. Isoelectric focusing of the virion major capsid protein, VP1, separated this protein into six species (A through F). These species had identical amino acid sequences, but differed in degree of modification (phosphorylation, acetylation, sulfation and hydroxylation). Evidence based upon precipitation with specific antisera supports the view that VP1 species E is required for specific adsorption and that D and F are required for hemagglutination. The virion attachment domain has been localized to an 18 kilodalton fragment of the C-terminal region of VP1. Monopinocytotic vesicles containing 125I-labeled polyoma virions were isolated from infected MKC. A crosslinker was used to bind the MKC cell receptor(s) covalently to VP1 attachment protein, and a new 120 kilodalton band was identified by SDS-PAGE. An anti-idiotype antibody prepared against a neutralizing polyoma monoclonal antiody was used to identify a putative 50 kilodalton receptor protein from a detergent extract of MKC, as well as from MKC membrane preparation.
Using mass spectrometry to study the photo-affinity labeling of protein tyrosine phosphatase 1B

NASA Astrophysics Data System (ADS)

Leriche, Tammy; Skorey, Kathryn; Roy, Patrick; McKay, Dan; Bateman, Kevin P.

2004-11-01

Protein tyrosine phosphatase 1B (PTP1B) is a potential target for the treatment of Type II diabetes and several companies are developing small molecule inhibitors of this enzyme. Part of the characterization of these compounds as PTP1B inhibitors is the understanding of how they bind in the enzyme active site. The use of photo-activated inhibitors that target the active site can provide such insight. This paper describes the characterization of a photoprobe directed at the active site of PTP1B. Mass spectrometry revealed the specific binding of the probe to the intact protein. Digestion of the labeled protein followed by LC-MS and LC-MS/MS was used to show that the photoprobe binds to a specific active site amino acid. This was confirmed by comparison with the X-ray structure of PTP1B with a PTP1B inhibitor. The probe labels a conserved acidic residue (Asp) that is required for catalytic activity. This photoprobe may prove to be a useful tool for the development of a PTP1B inhibitor or for the study of PTPs in general.
High Performance Liquid Chromatography Resolution of Ubiquitin Pathway Enzymes from Wheat Germ 1

PubMed Central

Sullivan, Michael L.; Callis, Judy; Vierstra, Richard D.

1990-01-01

The highly conserved protein ubiquitin is involved in several cellular processes in eukaryotes as a result of its covalent ligation to a variety of target proteins. Here, we describe the purification of several enzymatic activities involved in ubiquitin-protein conjugate formation and disassembly from wheat germ (Triticum vulgare) by a combination of ubiquitin affinity chromatography and anion-exchange high performance liquid chromatography. Using this procedure, ubiquitin activating enzyme (E1), several distinct ubiquitin carrier proteins (E2s) with molecular masses of 16, 20, 23, 23.5, and 25 kilodaltons, and a ubiquitin-protein hydrolase (isopeptidase) were isolated. Purified E1 formed a thiol ester linkage with 125I-ubiquitin in an ATP-dependent manner and transferred bound ubiquitin to the various purified E2s. The ubiquitin protein hydrolase fraction was sensitive to hemin, and in an ATP-independent reaction, was capable of removing the ubiquitin moiety from both ubiquitin 125I-lysozyme conjugates (ε-amino or isopeptide linkage) and the ubiquitin 52-amino acid extension protein fusion (α-amino or peptide linkage). Using this procedure, wheat germ represents an inexpensive source from which enzymes involved in the ubiquitin pathway may be isolated. Images Figure 1 Figure 2 Figure 3 Figure 4 PMID:16667769
Stimulation of dihydroxyacetone and glycerol kinase activity in Streptococcus faecalis by phosphoenolpyruvate-dependent phosphorylation catalyzed by enzyme I and HPr of the phosphotransferase systems

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

Deutscher, J.; Sauerwald, H.

1986-06-01

Recently a report was given of the phosphoenolpyruvate (PEP)-dependent phosphorylation of a 55-kilodalton protein of Streptococus faecalis catalyzed by enzyme I and histidine-containing protein (HPr) of the phosphotransferase system. The purified 55-kilodalton protein was found to exhibit dihydroxyacetone kinase activity. Glycerol was six times more slowly phosphorylated than dihydroxyacetone. The K/sub m/s were found to 0.7 mM for ATP, 0.45 mM for dihydroxyacetone, and 0.9 MM for glycerol. PEP-dependent phosphorylation of dihydroxyacetone kinase stimulated phosphorylation of both substrates about 10-fold. Fructose 1,6-diphosphate at concentrations higher than 2 mM inhibited the activity of phosphorylated and unphosphorylated dihydroxyacetone kinase in a noncompetitivemore » manner. The rate of PEP-dependent phosphorylation of dihydroxyacetone kinase was about 200-fold slower than the phosphorylation rate of III proteins (also called enzyme III or factor III), which so far have been considered the only phosphoryl acceptors of histidyl-phosphorylated HPr. P-Dihydroxyacetone kinase was found to be able to transfer its phosphoryl group in a backward reaction to HPr. Following (/sup 32/P)PEP-dependent phosphorylation and tryptic digestion of dihydroxyacetone kinase, the authors isolated a labeled peptide composed of 37 amino acids, as determined by amino acid analysis. The single histidyl residue of this peptide most likely carries the phosphoryl group in phosphorylated dihydroxyacetone kinase.« less
HPV16 E6 regulates annexin 1 (ANXA1) protein expression in cervical carcinoma cell lines

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

Calmon, Marilia Freitas; Sichero, Laura; Boccardo, Enrique

Annexin 1 (ANXA1) is a substrate for E6AP mediated ubiquitylation. It has been hypothesized that HPV 16 E6 protein redirects E6AP away from ANXA1, increasing its stability and possibly contributing to viral pathogenesis. We analyzed ANXA1 expression in HPV-positive and negative cervical carcinoma-derived cells, in cells expressing HPV-16 oncogenes and in cells transduced with shRNA targeting E6AP. We observed that ANXA1 protein expression increased in HPV-16-positive tumor cells, in keratinocytes expressing HPV-16 E6wt (wild-type) or E6/E7 and C33 cells expressing HPV-16 E6wt. ANXA1 protein expression decreased in cells transfected with E6 Dicer-substrate RNAs (DsiRNA) and C33 cells cotransduced with HPV-16more » E6wt and E6AP shRNA. Moreover, colony number and proliferation rate decreased in HPV16-positive cells transduced with ANXA1 shRNA. We observed that in cells infected with HPV16, the E6 binds to E6AP to degrade p53 and upregulate ANXA1. We suggest that ANXA1 may play a role in HPV-mediated carcinogenesis. - Highlights: • ANXA1 upregulation requires the presence of E6 and E6AP and is dependent on E6 integrity. • E6 binds to E6AP to degrade p53 and upregulate ANXA1 in cells infected with HPV16. • ANXA1 plays a role in cell proliferation in HPV-positive cervical cells.« less
Biochemical characterization of the prolyl 3-hydroxylase 1.cartilage-associated protein.cyclophilin B complex.

PubMed

Ishikawa, Yoshihiro; Wirz, Jackie; Vranka, Janice A; Nagata, Kazuhiro; Bächinger, Hans Peter

2009-06-26

The rough endoplasmic reticulum-resident protein complex consisting of prolyl 3-hydroxylase 1 (P3H1), cartilage-associated protein (CRTAP), and cyclophilin B (CypB) can be isolated from chick embryos on a gelatin-Sepharose column, indicating some involvement in the biosynthesis of procollagens. Prolyl 3-hydroxylase 1 modifies a single proline residue in the alpha chains of type I, II, and III collagens to (3S)-hydroxyproline. The peptidyl-prolyl cis-trans isomerase activity of cyclophilin B was shown previously to catalyze the rate of triple helix formation. Here we show that cyclophilin B in the complex shows peptidyl-prolyl cis-trans isomerase activity and that the P3H1.CRTAP.CypB complex has another important function: it acts as a chaperone molecule when tested with two classical chaperone assays. The P3H1.CRTAP.CypB complex inhibited the thermal aggregation of citrate synthase and was active in the denatured rhodanese refolding and aggregation assay. The chaperone activity of the complex was higher than that of protein-disulfide isomerase, a well characterized chaperone. The P3H1.CRTAP.CypB complex also delayed the in vitro fibril formation of type I collagen, indicating that this complex is also able to interact with triple helical collagen and acts as a collagen chaperone.
Ppm1E is an in cellulo AMP-activated protein kinase phosphatase.

PubMed

Voss, Martin; Paterson, James; Kelsall, Ian R; Martín-Granados, Cristina; Hastie, C James; Peggie, Mark W; Cohen, Patricia T W

2011-01-01

Activation of 5'-AMP-activated protein kinase (AMPK) is believed to be the mechanism by which the pharmaceuticals, metformin and phenformin, exert their beneficial effects for treatment of type 2 diabetes. These biguanide drugs elevate 5'-AMP, which allosterically activates AMPK and promotes phosphorylation on Thr172 of AMPK catalytic α subunits. Although kinases phosphorylating this site have been identified, phosphatases that dephosphorylate it are unknown. The aim of this study is to identify protein phosphatase(s) that dephosphorylate AMPKα-Thr172 within cells. Our initial data indicated that members of the protein phosphatase Mg/Mn(2+)-dependent [corrected] (PPM) family and not those of the PPP family of protein serine/threonine phosphatases may be directly or indirectly inhibited by phenformin. Using antibodies raised to individual Ppm phosphatases that facilitated the assessment of their activities, phenformin stimulation of cells was found to decrease the Mg(2+)/Mn(2+)-dependent [corrected] protein serine/threonine phosphatase activity of Ppm1E and Ppm1F, but not that attributable to other PPM family members, including Ppm1A/PP2Cα. Depletion of Ppm1E, but not Ppm1A, using lentiviral-mediated stable gene silencing, increased AMPKα-Thr172 phosphorylation approximately three fold in HEK293 cells. In addition, incubation of cells with low concentrations of phenformin and depletion of Ppm1E increased AMPK phosphorylation synergistically. Ppm1E and the closely related Ppm1F interact weakly with AMPK and assays with lysates of cells stably depleted of Ppm1F suggest [corrected] that this phosphatase contributes to dephosphorylation of AMPK. The data indicate that Ppm1E and probably PpM1F are in cellulo AMPK phosphatases and that Ppm1E is a potential anti-diabetic drug target. Copyright © 2010 Elsevier Inc. All rights reserved.
Bromodomain and Extraterminal (BET) Protein Inhibition Suppresses Human T Cell Leukemia Virus 1 (HTLV-1) Tax Protein-mediated Tumorigenesis by Inhibiting Nuclear Factor κB (NF-κB) Signaling*

PubMed Central

Wu, Xuewei; Qi, Jun; Bradner, James E.; Xiao, Gutian; Chen, Lin-Feng

2013-01-01

The etiology of human T cell leukemia virus 1 (HTLV-1)-mediated adult T cell leukemia is associated with the ability of viral oncoprotein Tax to induce sustained NF-κB activation and the expression of many NF-κB target genes. Acetylation of the RelA subunit of NF-κB and the subsequent recruitment of bromodomain-containing factor Brd4 are important for the expression of NF-κB target genes in response to various stimuli. However, their contributions to Tax-mediated NF-κB target gene expression and tumorigenesis remain unclear. Here we report that Tax induced the acetylation of lysine 310 of RelA and the binding of Brd4 to acetylated RelA to facilitate Tax-mediated transcriptional activation of NF-κB. Depletion of Brd4 down-regulated Tax-mediated NF-κB target gene expression and cell proliferation. Inhibiting the interaction of Brd4 and acetylated RelA with the bromodomain extraterminal protein inhibitor JQ1 suppressed the proliferation of Tax-expressing rat fibroblasts and Tax-positive HTLV-1-infected cells and Tax-mediated cell transformation and tumorigenesis. Moreover, JQ1 attenuated the Tax-mediated transcriptional activation of NF-κB, triggering the polyubiquitination and proteasome-mediated degradation of constitutively active nuclear RelA. Our results identify Brd4 as a key regulator for Tax-mediated NF-κB gene expression and suggest that targeting epigenetic regulators such as Brd4 with the bromodomain extraterminal protein inhibitor might be a potential therapeutic strategy for cancers and other diseases associated with HTLV-1 infection. PMID:24189064
The vacuolar ATPase from Entamoeba histolytica: molecular cloning of the gene encoding for the B subunit and subcellular localization of the protein.

PubMed

Meléndez-Hernández, Mayra Gisela; Barrios, María Luisa Labra; Orozco, Esther; Luna-Arias, Juan Pedro

2008-12-23

Entamoeba histolytica is a professional phagocytic cell where the vacuolar ATPase plays a key role. This enzyme is a multisubunit complex that regulates pH in many subcellular compartments, even in those that are not measurably acidic. It participates in a wide variety of cellular processes such as endocytosis, intracellular transport and membrane fusion. The presence of a vacuolar type H+-ATPase in E. histolytica trophozoites has been inferred previously from inhibition assays of its activity, the isolation of the Ehvma1 and Ehvma3 genes, and by proteomic analysis of purified phagosomes. We report the isolation and characterization of the Ehvma2 gene, which encodes for the subunit B of the vacuolar ATPase. This polypeptide is a 55.3 kDa highly conserved protein with 34 to 80% identity to orthologous proteins from other species. Particularly, in silico studies showed that EhV-ATPase subunit B displays 78% identity and 90% similarity to its Dictyostelium ortholog. A 462 bp DNA fragment of the Ehvma2 gene was expressed in bacteria and recombinant polypeptide was used to raise mouse polyclonal antibodies. EhV-ATPase subunit B antibodies detected a 55 kDa band in whole cell extracts and in an enriched fraction of DNA-containing organelles named EhkOs. The V-ATPase subunit B was located by immunofluorescence and confocal microscopy in many vesicles, in phagosomes, plasma membrane and in EhkOs. We also identified the genes encoding for the majority of the V-ATPase subunits in the E. histolytica genome, and proposed a putative model for this proton pump. We have isolated the Ehvma2 gene which encodes for the V-ATPase subunit B from the E. histolytica clone A. This gene has a 154 bp intron and encodes for a highly conserved polypeptide. Specific antibodies localized EhV-ATPase subunit B in many vesicles, phagosomes, plasma membrane and in EhkOs. Most of the orthologous genes encoding for the EhV-ATPase subunits were found in the E. histolytica genome, indicating the
Novel Mixed-Type Inhibitors of Protein Tyrosine Phosphatase 1B. Kinetic and Computational Studies.

PubMed

Sarabia-Sánchez, Marie Jazmín; Trejo-Soto, Pedro Josué; Velázquez-López, José Miguel; Carvente-García, Carlos; Castillo, Rafael; Hernández-Campos, Alicia; Avitia-Domínguez, Claudia; Enríquez-Mendiola, Daniel; Sierra-Campos, Erick; Valdez-Solana, Mónica; Salas-Pacheco, José Manuel; Téllez-Valencia, Alfredo

2017-12-20

The Atlas of Diabetes reports 415 million diabetics in the world, a number that has surpassed in half the expected time the twenty year projection. Type 2 diabetes is the most frequent form of the disease; it is characterized by a defect in the secretion of insulin and a resistance in its target organs. In the search for new antidiabetic drugs, one of the principal strategies consists in promoting the action of insulin. In this sense, attention has been centered in the protein tyrosine phosphatase 1B (PTP1B), a protein whose overexpression or increase of its activity has been related in many studies with insulin resistance. In the present work, a chemical library of 250 compounds was evaluated to determine their inhibition capability on the protein PTP1B. Ten molecules inhibited over the 50% of the activity of the PTP1B, the three most potent molecules were selected for its characterization, reporting Ki values of 5.2, 4.2 and 41.3 µM, for compounds 1 , 2 , and 3 , respectively. Docking and molecular dynamics studies revealed that the three inhibitors made interactions with residues at the secondary binding site to phosphate, exclusive for PTP1B. The data reported here support these compounds as hits for the design more potent and selective inhibitors against PTP1B in the search of new antidiabetic treatment.
1.55 Å resolution X-ray crystal structure of Rv3902c from Mycobacterium tuberculosis

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

Reddy, Bharat G.; Moates, Derek B.; Kim, Heung-Bok

The 1.55 Å resolution X-ray crystal structure of Rv3902c from M. tuberculosis reveals a novel fold. The crystallographic structure of the Mycobacterium tuberculosis (TB) protein Rv3902c (176 residues; molecular mass of 19.8 kDa) was determined at 1.55 Å resolution. The function of Rv3902c is unknown, although several TB genes involved in bacterial pathogenesis are expressed from the operon containing the Rv3902c gene. The unique structural fold of Rv3902c contains two domains, each consisting of antiparallel β-sheets and α-helices, creating a hand-like binding motif with a small binding pocket in the palm. Structural homology searches reveal that Rv3902c has an overallmore » structure similar to that of the Salmonella virulence-factor chaperone InvB, with an r.m.s.d. for main-chain atoms of 2.3 Å along an aligned domain.« less
Concomitant changes in high temperature tolerance and heat-shock proteins in desert succulents. [Agave deserti, Carnegiea gigantea, Ferocactus acanthodes

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

Kee, S.C.; Nobel, P.S.

1986-02-01

Raising the day/night air temperatures from 30/sup 0/C/20/sup 0/C to 50/sup 0/C/40/sup 0/C increases the high temperature tolerated by Agave deserti, Carnegiea gigantea, and Ferocactus acanthodes by 6/sup 0/C to 8/sup 0/C; the increase is about half completed in 3 days and fully completed in 10 days. A 25 to 27 kilodalton protein concomitantly accumulates for all three desert succulents upon transfer to 50/sup 0/C/40/sup 0/C, while accumulation of other heat heat-shock proteins is species specific. Some of the induced proteins are more abundant at 3 days, while others (including the 25-27 kilodalton protein) remain after completion of high temperaturemore » acclimation.« less

α1B-Adrenergic Receptors Differentially Associate with Rab Proteins during Homologous and Heterologous Desensitization

PubMed Central

Castillo-Badillo, Jean A.; Sánchez-Reyes, Omar B.; Alfonzo-Méndez, Marco A.; Romero-Ávila, M. Teresa; Reyes-Cruz, Guadalupe; García-Sáinz, J. Adolfo

2015-01-01

Internalization of G protein-coupled receptors can be triggered by agonists or by other stimuli. The process begins within seconds of cell activation and contributes to receptor desensitization. The Rab GTPase family controls endocytosis, vesicular trafficking, and endosomal fusion. Among their remarkable properties is the differential distribution of its members on the surface of various organelles. In the endocytic pathway, Rab 5 controls traffic from the plasma membrane to early endosomes, whereas Rab 4 and Rab 11 regulate rapid and slow recycling from early endosomes to the plasma membrane, respectively. Moreover, Rab 7 and Rab 9 regulate the traffic from late endosomes to lysosomes and recycling to the trans-Golgi. We explore the possibility that α1B-adrenergic receptor internalization induced by agonists (homologous) and by unrelated stimuli (heterologous) could involve different Rab proteins. This possibility was explored by Fluorescence Resonance Energy Transfer (FRET) using cells coexpressing α1B-adrenergic receptors tagged with the red fluorescent protein, DsRed, and different Rab proteins tagged with the green fluorescent protein. It was observed that when α1B-adrenergic receptors were stimulated with noradrenaline, the receptors interacted with proteins present in early endosomes, such as the early endosomes antigen 1, Rab 5, Rab 4, and Rab 11 but not with late endosome markers, such as Rab 9 and Rab 7. In contrast, sphingosine 1-phosphate stimulation induced rapid and transient α1B-adrenergic receptor interaction of relatively small magnitude with Rab 5 and a more pronounced and sustained one with Rab 9; interaction was also observed with Rab 7. Moreover, the GTPase activity of the Rab proteins appears to be required because no FRET was observed when dominant-negative Rab mutants were employed. These data indicate that α1B-adrenergic receptors are directed to different endocytic vesicles depending on the desensitization type (homologous vs
p55PIK regulates alpha-fetoprotein expression through the NF-κB signaling pathway.

PubMed

Ye, Guoguo; Sun, Ge; Cheng, Zhikui; Zhang, Lei; Hu, Kanghong; Xia, Xianmin; Zhou, Yin

2017-12-15

Alpha-fetoprotein (AFP) is regarded as a diagnostic and prognostic biomarker and a potential therapeutic target for hepatocellular carcinoma (HCC). However, the regulation of AFP expression in HCC remains poorly understood. This study aimed to investigate the mechanism by which AFP expression is regulated by p55PIK, an isoform of PI3K. Human HCC cell lines (HepG2 and Huh-7) were treated with p55PIK specific competitive inhibitor or shRNA, or p55PIK overexpression vector, in the absence or presence of NF-κB inhibitor PDTC. AFP expression was detected by quantitative real-time PCR and Western blotting. NF-κB responsive elements in AFP enhancer region were characterized by luciferase reporter assay. p55PIK significantly stimulated the expression of AFP by activating NF-κB signaling pathway in HCC cells. Furthermore, two NF-κB binding sites in AFP enhancer region were identified to be primarily responsible for p55PIK mediated upregulation of AFP expression. p55PIK/NF-κB signaling plays an important role in the upregulation of AFP expression in HCC. Copyright © 2017 Elsevier Inc. All rights reserved.
EBV latent membrane protein 1 activates Akt, NFkappaB, and Stat3 in B cell lymphomas.

PubMed

Shair, Kathy H Y; Bendt, Katherine M; Edwards, Rachel H; Bedford, Elisabeth C; Nielsen, Judith N; Raab-Traub, Nancy

2007-11-01

Latent membrane protein 1 (LMP1) is the major oncoprotein of Epstein-Barr virus (EBV). In transgenic mice, LMP1 promotes increased lymphoma development by 12 mo of age. This study reveals that lymphoma develops in B-1a lymphocytes, a population that is associated with transformation in older mice. The lymphoma cells have deregulated cell cycle markers, and inhibitors of Akt, NFkappaB, and Stat3 block the enhanced viability of LMP1 transgenic lymphocytes and lymphoma cells in vitro. Lymphoma cells are independent of IL4/Stat6 signaling for survival and proliferation, but have constitutively activated Stat3 signaling. These same targets are also deregulated in wild-type B-1a lymphomas that arise spontaneously through age predisposition. These results suggest that Akt, NFkappaB, and Stat3 pathways may serve as effective targets in the treatment of EBV-associated B cell lymphomas.
Influence of NaCl on Growth, Proline, and Phosphoenolpyruvate Carboxylase Levels in Mesembryanthemum crystallinum Suspension Cultures 1

PubMed Central

Thomas, John C.; De Armond, Richard L.; Bohnert, Hans J.

1992-01-01

The facultative halophyte Mesembryanthemum crystallinum responds to salt stress by increasing the levels of phosphoenolpyruvate carboxylase (PEPCase) and other enzymes associated with Crassulacean acid metabolism. A more common response to salt stress in sensitive and tolerant species, including M. crystallinum, is the accumulation of proline. We have established M. crystallinum suspension cultures to investigate whether both these salt-induced responses occur at the cellular level. Leaf-and root-derived cultures maintain 5% of the total soluble amino acids as proline. Cell culture growth slows upon addition of 400 millimolar NaCl, and proline levels increase to 40% of the total soluble amino acids. These results suggest a functional salt-stress and response program in Mesembryanthemum cells. Suspension cultures grown with or without 400 millimolar NaCl have PEPCase levels that compare with those from roots and unstressed leaves. The predominant protein cross-reacting with an anti-PEPCase antibody corresponds to 105 kilodaltons (apparent molecular mass), whereas a second species of approximately 110 kilodaltons is present at low levels. In salt-stressed leaves, the 110 kilodalton protein is more prevalent. Levels of mRNA for both ppc1 (salt stress induced in leaves) and ppc2 (constitutive) genes in salt-treated suspensions cultures are equal to unstressed leaves, and only twice the levels found in untreated suspension cultures. Whereas cells accumulate proline in response to NaCl, PEPCase protein amounts remain similar in salt-treated and untreated cultures. The induction upon salt stress of the 110 kilodalton PEPCase protein and other Crassulacean acid metabolism enzymes in organized tissues is not observed in cell culture and may depend on tissue-dependent or photoautotrophy-dependent programs. ImagesFigure 4Figure 5 PMID:16668687
Analysis of complete genomes of the rubella virus genotypes 1E and 2B which circulated in China, 2000–2013

PubMed Central

Zhu, Zhen; Chen, Min-hsin; Abernathy, Emily; Icenogle, Joseph; Zhou, Shujie; Wang, Changyin; Zhao, Chunfang; Wang, Yan; Chen, Haiyun; Si, Yuan; Xu, Wenbo

2016-01-01

Rubella viruses of genotypes 1E and 2B are currently the most frequently detected wild-type viruses in the world. Genotype 1E viruses from China have been genetically distinct from genotype 1E viruses found elsewhere, while genotype 2B viruses found in China are not distinguishable from genotype 2B viruses from other areas. Genetic clusters of viruses of both genotypes were defined previously using sequences of the 739-nt genotyping window. Here we report phylogenic analysis using whole genomic sequences from seven genotype 1E and three genotype 2B viruses which were isolated in China between 2000 and 2013 and confirm the subgrouping of current circulating genotypes 1E and 2B viruses. In addition, the whole genomic characterization of Chinese rubella viruses was clarified. The results indicated that the Chinese rubella viruses were highly conserved at the genomic level, and no predicted amino acid variations were found at positions where functional domains of the proteins were identified. Therefore, it gives us the idea that the rubella control and elimination goal should be achieved if vaccine immunization coverage continues maintaining at the high level. PMID:27959338
Synthesis, crystal structure, DFT studies, acid dissociation constant, and antimicrobial activity of methyl 2-(4-chlorophenyl)-7a-((4-chlorophenyl)carbamothioyl)-1-oxo-5,5-diphenyl-3-thioxo-hexahydro-1H-pyrrolo[1,2-e]imidazole-6-carboxylate

NASA Astrophysics Data System (ADS)

Nural, Yahya; Gemili, Muge; Seferoglu, Nurgul; Sahin, Ertan; Ulger, Mahmut; Sari, Hayati

2018-05-01

A novel bicyclic thiohydantoin fused to pyrrolidine compound, methyl 2-(4-chlorophenyl)-7a-((4-chlorophenyl)carbamothioyl)-1-oxo-5,5-diphenyl-3-thioxo-hexahydro-1H-pyrrolo[1,2-e]imidazole-6-carboxylate, was synthesized by the cyclization reaction of dimethyl 5,5-diphenylpyrrolidine-2,4-dicarboxylate and 4-chlorophenyl isothiocyanate in the presence of 4-(dimethylamino)pyridine to form methyl 2-(4-chlorophenyl)-1-oxo-5,5-diphenyl-3-thioxo-hexahydro-1H-pyrrolo[1,2-e]imidazole-6-carboxylate with concomitant addition reaction of the 4-chlorophenyl isothiocyanate in 79% yield. The structural characterization was performed by NMR, FT-IR, MS and HRMS techniques, and the stereochemistry of the compound was determined by single crystal X-ray diffraction study. In addition, the molecular structure and 1H and 13C NMR chemical shifts of the compound were obtained with the density functional theory and Hartree-Fock calculations. Acid dissociation constants of the compound were determined using potentiometric titration method in 25% (v/v) dimethyl sulfoxide-water hydroorganic solvent at 25 ± 0.1 °C, at an ionic background of 0.1 mol/L of NaCl using the HYPERQUAD computer program. Four acid dissociation constants were obtained for the compound, and we suggest that these acid dissociation constants are related to the NH, for two groups of enthiols and enol groups. Antimicrobial activity study was performed against S. aureus, B. subtilis, A. hydrophila, E. coli and A. baumannii as bacterial standard strains, and against M. tuberculosis H37Rv as mycobacterial strain. The compound exhibited antibacterial activity in the range of 31.25-62.5 μg/mL, and antimycobacterial activity with a MIC value of 40 μg/mL against the indicated strains.
Detection of an Atmosphere Around the Super-Earth 55 Cancri e

NASA Astrophysics Data System (ADS)

Tsiaras, A.; Rocchetto, M.; Waldmann, I. P.; Venot, O.; Varley, R.; Morello, G.; Damiano, M.; Tinetti, G.; Barton, E. J.; Yurchenko, S. N.; Tennyson, J.

2016-04-01

We report the analysis of two new spectroscopic observations in the near-infrared of the super-Earth 55 Cancri e, obtained with the WFC3 camera on board the Hubble Space Telescope. 55 Cancri e orbits so close to its parent star that temperatures much higher than 2000 K are expected on its surface. Given the brightness of 55 Cancri, the observations were obtained in scanning mode, adopting a very long scanning length and a very high scanning speed. We use our specialized pipeline to take into account systematics introduced by these observational parameters when coupled with the geometrical distortions of the instrument. We measure the transit depth per wavelength channel with an average relative uncertainty of 22 ppm per visit and find modulations that depart from a straight line model with a 6σ confidence level. These results suggest that 55 Cancri e is surrounded by an atmosphere, which is probably hydrogen-rich. Our fully Bayesian spectral retrieval code, { T }-REx, has identified HCN to be the most likely molecular candidate able to explain the features at 1.42 and 1.54 μm. While additional spectroscopic observations in a broader wavelength range in the infrared will be needed to confirm the HCN detection, we discuss here the implications of such a result. Our chemical model, developed with combustion specialists, indicates that relatively high mixing ratios of HCN may be caused by a high C/O ratio. This result suggests this super-Earth is a carbon-rich environment even more exotic than previously thought.
Erythrocyte concentrations of B1, B2, B6 but not plasma C and E are reliable indicators of nutrition status in the presence of systemic inflammation.

PubMed

Ghashut, Rawia A; McMillan, Donald C; Kinsella, John; Talwar, Dinesh

2017-02-01

There is increasing evidence that the plasma concentration of vitamin D, carotenoids, zinc and selenium are associated with the magnitude of the systemic inflammatory response. In order to examine whether other vitamins may be affected and whether red cell concentrations are less affected by systemic inflammation the aim of the present study was to examine the effect of the systemic inflammatory response on red cell measurements of vitamins B1, B2 and B6, and plasma concentration of vitamin C and E in a large cohort of patients referred for a nutritional screen. Patients referred for nutritional assessment of B1 (n = 551), B2 (n = 251), B6 (n = 313), ascorbic acid (n = 494) and α-tocopherol (n = 395) concentrations. These vitamins were measured using routine laboratory methods. The median concentrations of vitamin B1 grouped according to C-reactive protein concentrations ≤10, 11-80 and >80 mg/L were 543, 664 and 766 ng/g Hb respectively (p < 0.001, 41% higher). The median concentration of vitamin B1 grouped according to albumin concentrations ≥35, 25-34 and <25 g/l were 547, 664 and 701 ng/g Hb respectively (p < 0.001, 28% higher). The median concentrations of red cell vitamin B2 grouped according to CRP concentrations ≤10, 11-80 and >80 mg/L were 2.2, 2.3 and 2.4 nmol/g Hb respectively (p < 0.001, 9% higher). The median red cell concentrations of vitamin B2 grouped according to albumin concentrations ≥35, 25-34 and <25 g/l were 2.1, 2.4 and 2.3 nmol/g Hb respectively (p < 0.001, 14% higher). The median concentrations of red cell vitamin B6 grouped according to CRP concentrations ≤10, 11-80 and >80 mg/L were 534, 548 and 767 pmol/g Hb respectively (p < 0.001, 44% higher). The median red cell concentrations of vitamin B6 grouped according to albumin concentrations ≥35, 25-34 and <25 g/l were 462, 644 and 840 pmol/g Hb respectively (p < 0.001, 82% higher). In contrast, the median plasma concentrations of ascorbic acid
Four new neolignans isolated from Eleutherococcus senticosus and their protein tyrosine phosphatase 1B inhibitory activity (PTP1B).

PubMed

Zhang, Le; Li, Ban-Ban; Li, Hao-Ze; Meng, Xiao; Lin, Xin; Jiang, Yi-Yu; Ahn, Jong-Seog; Cui, Long

2017-09-01

Four new compounds, erythro-7'E-4-hydroxy-3,3'-dimethoxy-8,5'-oxyneoligna-7'-ene-7,9-diol-9'-al (1), (7S,8S)-4-hydroxy-3,1',3'-trimethoxy-4',7-epoxy-8,5'-neolign-9-ol (5), (7S,8S,7'E)-5-hydroxy-3,3'-dimethoxy-4',7-epoxy-8,5'-neolign-7'-ene-9,9'-diol (6) and (7S,8S,7'E)-5-hydroxy-3,3',9'-trimethoxy-4'-7-epoxy-8,5'-neolign-7'-ene-9-ol (7). Along with four known compounds (2-4, 8) were isolated from the EtOAc-soluble extract of Eleutherococcus senticosus. Their structures were elucidated on the basis of spectroscopic and physicochemical analyses. All the compounds were evaluated for in vitro inhibitory activity against PTP1B, VHR and PP1. Among them, compounds 1-4 and 6-8 were found to exhibit selective inhibitory activity on PTP1B with IC 50 values ranging from 17.2±1.6 to 32.7±1.2μM. Copyright © 2017 Elsevier B.V. All rights reserved.
Immunoglobulin subunits of murine B lymphocytes: structure and associations with other membrane proteins.

PubMed Central

Vogel, L; Haustein, D

1989-01-01

The Ig subunit structure of murine B lymphocytes was studied by employing different radiolabelling techniques in combination with chemical cross-linking. The main membrane structure of IgM was a half molecule that was disulphide-linked to proteins with MW 30,000, 45,000 and 55,000, respectively. Small amounts of mu 2L2, microL disulphide-linked to a protein with MW 50,000, and free microL were also detected. The main IgD structures were half molecules disulphide-linked to two proteins with MW 14,000 and two proteins with MW 16,000. Furthermore, IgD half molecules disulphide-linked to a protein with MW 16,000 and free half molecules could be demonstrated. Labelling with hydrophobic reagents showed that all Ig molecules and the protein with MW 50,000, linked to microL, penetrated the lipid bilayer, whereas the other IgM- and IgD-linked proteins probably did not. Additional proteins which were associated exclusively with IgM were detected by chemical cross-linking. These findings offer new possibilities for the investigation of the function(s) of antigen receptors on B cells. Images Figure 1 Figure 2 Figure 4 Figure 5 PMID:2787780
Generation of a Stable Transgenic Swine Model Expressing a Porcine Histone 2B-eGFP Fusion Protein for Cell Tracking and Chromosome Dynamics Studies

PubMed Central

Simpson, Sean; Collins, Bruce; Sommer, Jeff; Petters, Robert M.; Caballero, Ignacio; Platt, Jeff L.

2017-01-01

Transgenic pigs have become an attractive research model in the field of translational research, regenerative medicine, and stem cell therapy due to their anatomic, genetic and physiological similarities with humans. The development of fluorescent proteins as molecular tags has allowed investigators to track cell migration and engraftment levels after transplantation. Here we describe the development of two transgenic pig models via SCNT expressing a fusion protein composed of eGFP and porcine Histone 2B (pH2B). This fusion protein is targeted to the nucleosomes resulting a nuclear/chromatin eGFP signal. The first model (I) was generated via random insertion of pH2B-eGFP driven by the CAG promoter (chicken beta actin promoter and rabbit Globin poly A; pCAG-pH2B-eGFP) and protected by human interferon-β matrix attachment regions (MARs). Despite the consistent, high, and ubiquitous expression of the fusion protein pH2B-eGFP in all tissues analyzed, two independently generated Model I transgenic lines developed neurodegenerative symptoms including Wallerian degeneration between 3–5 months of age, requiring euthanasia. A second transgenic model (II) was developed via CRISPR-Cas9 mediated homology-directed repair (HDR) of IRES-pH2B-eGFP into the endogenous β-actin (ACTB) locus. Model II transgenic animals showed ubiquitous expression of pH2B-eGFP on all tissues analyzed. Unlike the pCAG-pH2B-eGFP/MAR line, all Model II animals were healthy and multiple pregnancies have been established with progeny showing the expected Mendelian ratio for the transmission of the pH2B-eGFP. Expression of pH2B-eGFP was used to examine the timing of the maternal to zygotic transition after IVF, and to examine chromosome segregation of SCNT embryos. To our knowledge this is the first viable transgenic pig model with chromatin-associated eGFP allowing both cell tracking and the study of chromatin dynamics in a large animal model. PMID:28081156
46 CFR 112.55-1 - General.

Code of Federal Regulations, 2010 CFR

2010-10-01

... 46 Shipping 4 2010-10-01 2010-10-01 false General. 112.55-1 Section 112.55-1 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) ELECTRICAL ENGINEERING EMERGENCY LIGHTING AND POWER SYSTEMS Storage Battery Installation § 112.55-1 General. Each storage battery installation must meet Subpart 111...
46 CFR 112.55-1 - General.

Code of Federal Regulations, 2014 CFR

2014-10-01

... 46 Shipping 4 2014-10-01 2014-10-01 false General. 112.55-1 Section 112.55-1 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) ELECTRICAL ENGINEERING EMERGENCY LIGHTING AND POWER SYSTEMS Storage Battery Installation § 112.55-1 General. Each storage battery installation must meet Subpart 111...
46 CFR 112.55-1 - General.

Code of Federal Regulations, 2013 CFR

2013-10-01

... 46 Shipping 4 2013-10-01 2013-10-01 false General. 112.55-1 Section 112.55-1 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) ELECTRICAL ENGINEERING EMERGENCY LIGHTING AND POWER SYSTEMS Storage Battery Installation § 112.55-1 General. Each storage battery installation must meet Subpart 111...
46 CFR 112.55-1 - General.

Code of Federal Regulations, 2012 CFR

2012-10-01

... 46 Shipping 4 2012-10-01 2012-10-01 false General. 112.55-1 Section 112.55-1 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) ELECTRICAL ENGINEERING EMERGENCY LIGHTING AND POWER SYSTEMS Storage Battery Installation § 112.55-1 General. Each storage battery installation must meet Subpart 111...
46 CFR 112.55-1 - General.

Code of Federal Regulations, 2011 CFR

2011-10-01

... 46 Shipping 4 2011-10-01 2011-10-01 false General. 112.55-1 Section 112.55-1 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) ELECTRICAL ENGINEERING EMERGENCY LIGHTING AND POWER SYSTEMS Storage Battery Installation § 112.55-1 General. Each storage battery installation must meet Subpart 111...
Discovery and study of novel protein tyrosine phosphatase 1B inhibitors

NASA Astrophysics Data System (ADS)

Zhang, Qian; Chen, Xi; Feng, Changgen

2017-10-01

Protein tyrosine phosphatase 1B (PTP1B) is considered to be a target for therapy of type II diabetes and obesity. So it is of great significance to take advantage of a computer aided drug design protocol involving the structured-based virtual screening with docking simulations for fast searching small molecule PTP1B inhibitors. Based on optimized complex structure of PTP1B bound with specific inhibitor of IX1, structured-based virtual screening against a library of natural products containing 35308 molecules, which was constructed based on Traditional Chinese Medicine database@ Taiwan (TCM database@ Taiwan), was conducted to determine the occurrence of PTP1B inhibitors using the Lubbock module and CDOCKER module from Discovery Studio 3.1 software package. The results were further filtered by predictive ADME simulation and predictive toxic simulation. As a result, 2 good drug-like molecules, namely para-benzoquinone compound 1 and Clavepictine analogue 2 were identified ultimately with the dock score of original inhibitor (IX1) and the receptor as a threshold. Binding model analyses revealed that these two candidate compounds have good interactions with PTP1B. The PTP1B inhibitory activity of compound 2 hasn't been reported before. The optimized compound 2 has higher scores and deserves further study.
Mapping the Complement Factor H-Related Protein 1 (CFHR1):C3b/C3d Interactions

PubMed Central

Laskowski, Jennifer; Thurman, Joshua M.; Hageman, Gregory S.; Holers, V. Michael

2016-01-01

Complement factor H-related protein 1 (CFHR1) is a complement regulator which has been reported to regulate complement by blocking C5 convertase activity and interfering with C5b surface association. CFHR1 also competes with complement factor H (CFH) for binding to C3b, and may act as an antagonist of CFH-directed regulation on cell surfaces. We have employed site-directed mutagenesis in conjunction with ELISA-based and functional assays to isolate the binding interaction that CFHR1 undertakes with complement components C3b and C3d to a single shared interface. The C3b/C3d:CFHR1 interface is identical to that which occurs between the two C-terminal domains (SCR19-20) of CFH and C3b. Moreover, we have been able to corroborate that dimerization of CFHR1 is necessary for this molecule to bind effectively to C3b and C3d, or compete with CFH. Finally, we have established that CFHR1 competes with complement factor H-like protein 1 (CFHL-1) for binding to C3b. CFHL-1 is a CFH gene splice variant, which is almost identical to the N-terminal 7 domains of CFH (SCR1-7). CFHR1, therefore, not only competes with the C-terminus of CFH for binding to C3b, but also sterically blocks the interaction that the N-terminus of CFH undertakes with C3b, and which is required for CFH-regulation. PMID:27814381
46 CFR 111.55-1 - General.

Code of Federal Regulations, 2010 CFR

2010-10-01

... 46 Shipping 4 2010-10-01 2010-10-01 false General. 111.55-1 Section 111.55-1 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) ELECTRICAL ENGINEERING ELECTRIC SYSTEMS-GENERAL REQUIREMENTS Switches § 111.55-1 General. (a) Each switch must meet Article 404 of NFPA NEC 2002 (incorporated by...
46 CFR 111.55-1 - General.

Code of Federal Regulations, 2014 CFR

2014-10-01

... 46 Shipping 4 2014-10-01 2014-10-01 false General. 111.55-1 Section 111.55-1 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) ELECTRICAL ENGINEERING ELECTRIC SYSTEMS-GENERAL REQUIREMENTS Switches § 111.55-1 General. (a) Each switch must meet Article 404 of NFPA NEC 2002 (incorporated by...

46 CFR 111.55-1 - General.

Code of Federal Regulations, 2012 CFR

2012-10-01

... 46 Shipping 4 2012-10-01 2012-10-01 false General. 111.55-1 Section 111.55-1 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) ELECTRICAL ENGINEERING ELECTRIC SYSTEMS-GENERAL REQUIREMENTS Switches § 111.55-1 General. (a) Each switch must meet Article 404 of NFPA NEC 2002 (incorporated by...
46 CFR 111.55-1 - General.

Code of Federal Regulations, 2011 CFR

2011-10-01

... 46 Shipping 4 2011-10-01 2011-10-01 false General. 111.55-1 Section 111.55-1 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) ELECTRICAL ENGINEERING ELECTRIC SYSTEMS-GENERAL REQUIREMENTS Switches § 111.55-1 General. (a) Each switch must meet Article 404 of NFPA NEC 2002 (incorporated by...
46 CFR 111.55-1 - General.

Code of Federal Regulations, 2013 CFR

2013-10-01

... 46 Shipping 4 2013-10-01 2013-10-01 false General. 111.55-1 Section 111.55-1 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) ELECTRICAL ENGINEERING ELECTRIC SYSTEMS-GENERAL REQUIREMENTS Switches § 111.55-1 General. (a) Each switch must meet Article 404 of NFPA NEC 2002 (incorporated by...
JAK1 kinase forms complexes with interleukin-4 receptor and 4PS/insulin receptor substrate-1-like protein and is activated by interleukin-4 and interleukin-9 in T lymphocytes.

PubMed

Yin, T; Tsang, M L; Yang, Y C

1994-10-28

Interleukin (IL)-4 and IL-9 regulate the proliferation of T lymphocytes through interactions with their receptors. Previous studies have shown that unknown tyrosine kinases are involved in the proliferative signaling triggered by IL-4 and IL-9. Here we show that IL-4 and IL-9 induce overlapping (170, 130, and 125 kilodalton (kDa)) and distinct (45 and 88/90 kDa, respectively) protein tyrosine phosphorylation in T lymphocytes. We further identify the 170-kDa tyrosine-phosphorylated protein as 4PS/insulin receptor substrate-1-like (IRS-1L) protein and 130-kDa protein as JAK1 kinase. Furthermore, we demonstrate for the first time that JAK1 forms complexes with the IL-4 receptor and 4PS/IRS-1L protein following ligand-receptor interaction. In addition, we demonstrate that IL-9, but not IL-4, induced tyrosine phosphorylation of Stat 91 transcriptional factor. The overlapping and distinct protein tyrosine phosphorylation and activation of the same JAK1 kinase in T lymphocytes strongly suggests that IL-4 and IL-9 share the common signal transduction pathways and that the specificity for each cytokine could be achieved through the unique tyrosine-phosphorylated proteins triggered by individual cytokines.
Identification of a conserved B-cell epitope on duck hepatitis A type 1 virus VP1 protein.

PubMed

Wu, Xiaoying; Li, Xiaojun; Zhang, Qingshan; Wulin, Shaozhou; Bai, Xiaofei; Zhang, Tingting; Wang, Yue; Liu, Ming; Zhang, Yun

2015-01-01

The VP1 protein of duck hepatitis A virus (DHAV) is a major structural protein that induces neutralizing antibodies in ducks; however, B-cell epitopes on the VP1 protein of duck hepatitis A genotype 1 virus (DHAV-1) have not been characterized. To characterize B-cell epitopes on VP1, we used the monoclonal antibody (mAb) 2D10 against Escherichia coli-expressed VP1 of DHAV-1. In vitro, mAb 2D10 neutralized DHAV-1 virus. By using an array of overlapping 12-mer peptides, we found that mAb 2D10 recognized phages displaying peptides with the consensus motif LPAPTS. Sequence alignment showed that the epitope 173LPAPTS178 is highly conserved among the DHAV-1 genotypes. Moreover, the six amino acid peptide LPAPTS was proven to be the minimal unit of the epitope with maximal binding activity to mAb 2D10. DHAV-1-positive duck serum reacted with the epitope in dot blotting assay, revealing the importance of the six amino acids of the epitope for antibody-epitope binding. Competitive inhibition assays of mAb 2D10 binding to synthetic LPAPTS peptides and truncated VP1 protein fragments, detected by Western blotting, also verify that LPAPTS was the VP1 epitope. We identified LPAPTS as a VP1-specific linear B-cell epitope recognized by the neutralizing mAb 2D10. Our findings have potential applications in the development of diagnostic techniques and epitope-based marker vaccines against DHAV-1.
Vaccination with Eimeria tenella elongation factor-1α recombinant protein induces protective immunity against E. tenella and E. maxima infections.

PubMed

Lin, Rui-Qing; Lillehoj, Hyun S; Lee, Seung Kyoo; Oh, Sungtaek; Panebra, Alfredo; Lillehoj, Erik P

2017-08-30

Avian coccidiosis is caused by multiple species of the apicomplexan protozoan, Eimeria, and is one of the most economically devastating enteric diseases for the poultry industry worldwide. Host immunity to Eimeria infection, however, is relatively species-specific. The ability to immunize chickens against different species of Eimeria using a single vaccine will have a major beneficial impact on commercial poultry production. In this paper, we describe the molecular cloning, purification, and vaccination efficacy of a novel Eimeria vaccine candidate, elongation factor-1α (EF-1α). One day-old broiler chickens were given two subcutaneous immunizations one week apart with E. coli-expressed E. tenella recombinant (r)EF-1α protein and evaluated for protection against challenge infection with E. tenella or E. maxima. rEF-1α-vaccinated chickens exhibited increased body weight gains, decreased fecal oocyst output, and greater serum anti-EF-1α antibody levels following challenge infection with either E. tenella or E. maxima compared with unimmunized controls. Vaccination with EF-1α may represent a new approach to inducing cross-protective immunity against avian coccidiosis in the field. Published by Elsevier B.V.
Fusicoccin-Binding Proteins in Arabidopsis thaliana (L.) Heynh. 1

PubMed Central

Meyer, Christiane; Feyerabend, Martin; Weiler, Elmar W.

1989-01-01

Using the novel radioligand, [3H]-9′-nor-fusicoccin-8′-alcohol, high affinity binding sites for fusicoccin were characterized in preparations from leaves of Arabidopsis thaliana (L.) Heynh. The binding site copartitioned with the plasmalemma marker, vanadate-sensitive K+, Mg2+-ATPase, when microsomal fractions were further purified by aqueous two-phase partitioning in polyethylene glycol-dextran phase systems and sedimented at an equilibrium density of 1.17 grams per cubic centimeter in continuous sucrose density gradients, as did the ATPase marker. The binding of [3H]-9′-nor-fusicoccin-8′-alcohol was saturable and Scatchard analysis revealed a biphasic plot with two apparent dissociation constants (KD), KD1 = 1.5 nanomolar and KD2 = 42 nanomolar, for the radioligand. Binding was optimal at pH 6, thermolabile, and was reduced by 70% when the membrane vesicles were pretreated with trypsin. The data are consistent with the presence of one or several binding proteins for fusicoccin at the plasma membrane of A. thaliana. Binding of the radioligand was unaffected by pretreatment of the sites with various alkylating and reducing agents, but was reduced by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, diethylpyrocarbonate, chloramine T, and periodate. A number of detergents were tested to find optimum conditions for solubilization. Nonanoyl-N-methylglucamide (50 millimolar) solubilized 70% of the radioligand-binding protein complex in undissociated form. Photoaffinity labeling of membrane preparations with a tritiated azido analog of fusicoccin resulted in the labeling of a 34 ± 1 kilodalton polypeptide. Labeling of this polypeptide, presumably the fusicoccin-binding protein, was severely reduced in the presence of unlabeled fusicoccin. PMID:16666603
Thionin-D4E1 chimeric protein protects plants against bacterial infections

DOEpatents

Stover, Eddie W; Gupta, Goutam; Hao, Guixia

2017-08-08

The generation of a chimeric protein containing a first domain encoding either a pro-thionon or thionin, a second domain encoding D4E1 or pro-D4E1, and a third domain encoding a peptide linker located between the first domain and second domain is described. Either the first domain or the second domain is located at the amino terminal of the chimeric protein and the other domain (second domain or first domain, respectively) is located at the carboxyl terminal. The chimeric protein has antibacterial activity. Genetically altered plants and their progeny expressing a polynucleotide encoding the chimeric protein resist diseases caused by bacteria.
The Burkholderia pseudomallei Proteins BapA and BapC Are Secreted TTSS3 Effectors and BapB Levels Modulate Expression of BopE

PubMed Central

Treerat, Puthayalai; Alwis, Priyangi; D’Cruze, Tanya; Cullinane, Meabh; Vadivelu, Jamunarani; Devenish, Rodney J.; Prescott, Mark; Adler, Ben; Boyce, John D.

2015-01-01

Many Gram-negative pathogens use a type III secretion system (TTSS) for the injection of bacterial effector proteins into host cells. The injected effector proteins play direct roles in modulation of host cell pathways for bacterial benefit. Burkholderia pseudomallei, the causative agent of melioidosis, expresses three different TTSSs. One of these systems, the TTSS3, is essential for escape from host endosomes and therefore intracellular survival and replication. Here we have characterized three putative TTSS3 proteins; namely BapA, BapB and BapC. By employing a tetracysteine (TC)-FlAsH™ labelling technique to monitor the secretion of TC-tagged fusion proteins, BapA and BapC were shown to be secreted during in vitro growth in a TTSS3-dependant manner, suggesting a role as TTSS3 effectors. Furthermore, we constructed B. pseudomallei bapA, bapB and bapC mutants and used the well-characterized TTSS3 effector BopE as a marker of secretion to show that BapA, BapB and BapC are not essential for the secretion process. However, BopE transcription and secretion were significantly increased in the bapB mutant, suggesting that BapB levels modulate BopE expression. In a BALB/c mouse model of acute melioidosis, the bapA, bapB and bapC mutants showed a minor reduction of in vivo fitness. Thus, this study defines BapA and BapC as novel TTSS3 effectors, BapB as a regulator of BopE production, and all three as necessary for full B. pseudomallei in vivo fitness. PMID:26624293
The Burkholderia pseudomallei Proteins BapA and BapC Are Secreted TTSS3 Effectors and BapB Levels Modulate Expression of BopE.

PubMed

Treerat, Puthayalai; Alwis, Priyangi; D'Cruze, Tanya; Cullinane, Meabh; Vadivelu, Jamunarani; Devenish, Rodney J; Prescott, Mark; Adler, Ben; Boyce, John D

2015-01-01

Many Gram-negative pathogens use a type III secretion system (TTSS) for the injection of bacterial effector proteins into host cells. The injected effector proteins play direct roles in modulation of host cell pathways for bacterial benefit. Burkholderia pseudomallei, the causative agent of melioidosis, expresses three different TTSSs. One of these systems, the TTSS3, is essential for escape from host endosomes and therefore intracellular survival and replication. Here we have characterized three putative TTSS3 proteins; namely BapA, BapB and BapC. By employing a tetracysteine (TC)-FlAsH™ labelling technique to monitor the secretion of TC-tagged fusion proteins, BapA and BapC were shown to be secreted during in vitro growth in a TTSS3-dependant manner, suggesting a role as TTSS3 effectors. Furthermore, we constructed B. pseudomallei bapA, bapB and bapC mutants and used the well-characterized TTSS3 effector BopE as a marker of secretion to show that BapA, BapB and BapC are not essential for the secretion process. However, BopE transcription and secretion were significantly increased in the bapB mutant, suggesting that BapB levels modulate BopE expression. In a BALB/c mouse model of acute melioidosis, the bapA, bapB and bapC mutants showed a minor reduction of in vivo fitness. Thus, this study defines BapA and BapC as novel TTSS3 effectors, BapB as a regulator of BopE production, and all three as necessary for full B. pseudomallei in vivo fitness.
Pharmacology, signaling and physiological relevance of the G protein-coupled receptor 55.

PubMed

Balenga, Nariman A B; Henstridge, Christopher M; Kargl, Julia; Waldhoer, Maria

2011-01-01

According to The European Monitoring Centre for Drugs and Drug Addiction (EMCDDA), ∼70 million European adults have consumed cannabis on at least one occasion. Cannabis consumption leads to a variety of psychoactive effects due to the presence of the constituent Δ(9)-tetrahydrocannabinol (Δ(9)-THC). Δ(9)-THC interacts with the endocannabinoid system (ECS), which consists of the seven transmembrane spanning (7TM)/G protein-coupled receptors (GPCRs) CB(1) and CB(2), their respective ligands (endocannabinoids), and enzymes involved in their biosynthesis and degradation. This system plays a critical role in many physiological processes such as learning and memory, appetite control, pain sensation, motor coordination, lipogenesis, modulation of immune response, and the regulation of bone mass. Therefore, a huge effort has been spent trying to fully elucidate the composition and function of the ECS. The G protein-coupled receptor 55 (GPR55) was recently proposed as a novel component of this system; however, its classification as a cannabinoid receptor has been significantly hampered by its complex pharmacology, signaling, and cellular function. GPR55 is phylogenetically distinct from the traditional cannabinoid receptors, but in some experimental paradigms, it is activated by endocannabinoids, phytocannabinoids, and synthetic cannabinoid ligands. However, the most potent compound appears to be a lysophospholipid known as lysophosphatidylinositol (LPI). Here, we provide a comprehensive evaluation of the current pharmacology and signaling of GPR55 and review the proposed role of this receptor in a number of physiological and pathophysiological processes. Copyright © 2011 Elsevier Inc. All rights reserved.
Protein Phosphotyrosine Phosphatase 1B (PTP1B) in Calpain-dependent Feedback Regulation of Vascular Endothelial Growth Factor Receptor (VEGFR2) in Endothelial Cells: IMPLICATIONS IN VEGF-DEPENDENT ANGIOGENESIS AND DIABETIC WOUND HEALING.

PubMed

Zhang, Yixuan; Li, Qiang; Youn, Ji Youn; Cai, Hua

2017-01-13

The VEGF/VEGFR2/Akt/eNOS/NO pathway is essential to VEGF-induced angiogenesis. We have previously discovered a novel role of calpain in mediating VEGF-induced PI3K/AMPK/Akt/eNOS activation through Ezrin. Here, we sought to identify possible feedback regulation of VEGFR2 by calpain via its substrate protein phosphotyrosine phosphatase 1B (PTP1B), and the relevance of this pathway to VEGF-induced angiogenesis, especially in diabetic wound healing. Overexpression of PTP1B inhibited VEGF-induced VEGFR2 and Akt phosphorylation in bovine aortic endothelial cells, while PTP1B siRNA increased both, implicating negative regulation of VEGFR2 by PTP1B. Calpain inhibitor ALLN induced VEGFR2 activation, which can be completely blocked by PTP1B overexpression. Calpain activation induced by overexpression or Ca/A23187 resulted in PTP1B cleavage, which can be blocked by ALLN. Moreover, calpain activation inhibited VEGF-induced VEGFR2 phosphorylation, which can be restored by PTP1B siRNA. These data implicate calpain/PTP1B negative feedback regulation of VEGFR2, in addition to the primary signaling pathway of VEGF/VEGFR2/calpain/PI3K/AMPK/Akt/eNOS. We next examined a potential role of PTP1B in VEGF-induced angiogenesis. Endothelial cells transfected with PTP1B siRNA showed faster wound closure in response to VEGF. Aortic discs isolated from PTP1B siRNA-transfected mice also had augmented endothelial outgrowth. Importantly, PTP1B inhibition and/or calpain overexpression significantly accelerated wound healing in STZ-induced diabetic mice. In conclusion, our data for the first time demonstrate a calpain/PTP1B/VEGFR2 negative feedback loop in the regulation of VEGF-induced angiogenesis. Modulation of local PTP1B and/or calpain activities may prove beneficial in the treatment of impaired wound healing in diabetes. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.
Crystal Structure of the Minimalist Max-E47 Protein Chimera

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

Ahmadpour, Faraz; Ghirlando, Rodolfo; De Jong, Antonia T.

Max-E47 is a protein chimera generated from the fusion of the DNA-binding basic region of Max and the dimerization region of E47, both members of the basic region/helix-loop-helix (bHLH) superfamily of transcription factors. Like native Max, Max-E47 binds with high affinity and specificity to the E-box site, 5'-CACGTG, both in vivo and in vitro. We have determined the crystal structure of Max-E47 at 1.7 Å resolution, and found that it associates to form a well-structured dimer even in the absence of its cognate DNA. Analytical ultracentrifugation confirms that Max-E47 is dimeric even at low micromolar concentrations, indicating that the Max-E47more » dimer is stable in the absence of DNA. Circular dichroism analysis demonstrates that both non-specific DNA and the E-box site induce similar levels of helical secondary structure in Max-E47. These results suggest that Max-E47 may bind to the E-box following the two-step mechanism proposed for other bHLH proteins. In this mechanism, a rapid step where protein binds to DNA without sequence specificity is followed by a slow step where specific protein:DNA interactions are fine-tuned, leading to sequence-specific recognition. Collectively, these results show that the designed Max-E47 protein chimera behaves both structurally and functionally like its native counterparts.« less
Antagonists for the orphan G-protein-coupled receptor GPR55 based on a coumarin scaffold.

PubMed

Rempel, Viktor; Volz, Nicole; Gläser, Franziska; Nieger, Martin; Bräse, Stefan; Müller, Christa E

2013-06-13

The orphan G-protein-coupled receptor GPR55, which is activated by 1-lysophosphatidylinositol and interacts with cannabinoid (CB) receptor ligands, has been proposed as a new potential drug target for the treatment of diabetes, Parkinson's disease, neuropathic pain, and cancer. We applied β-arrestin assays to identify 3-substituted coumarins as a novel class of antagonists and performed an extensive structure-activity relationship study for GPR55. Selectivity versus the related receptors CB1, CB2, and GPR18 was assessed. Among the 7-unsubstituted coumarins selective, competitive GPR55 antagonists were identified, such as 3-(2-hydroxybenzyl)-5-isopropyl-8-methyl-2H-chromen-2-one (12, PSB-SB-489, IC50 = 1.77 μM, pA2 = 0.547 μM). Derivatives with long alkyl chains in position 7 were potent, possibly allosteric GPR55 antagonists which showed ancillary CB receptor affinity. 7-(1,1-Dimethyloctyl)-5-hydroxy-3-(2-hydroxybenzyl)-2H-chromen-2-one (69, PSB-SB-487, IC50 = 0.113 μM, KB = 0.561 μM) and 7-(1,1-dimethylheptyl)-5-hydroxy-3-(2-hydroxybenzyl)-2H-chromen-2-one (67, PSB-SB-1203, IC50 = 0.261 μM) were the most potent GPR55 antagonists of the present series.
A novel immediate-early response gene of endothelium is induced by cytokines and encodes a secreted protein.

PubMed

Holzman, L B; Marks, R M; Dixit, V M

1990-11-01

We have previously described the cloning of a group of novel cellular immediate-early response genes whose expression in human umbilical vein endothelial cells is induced by tumor necrosis factor alpha in the presence of cycloheximide. These genes are likely to participate in mediating the response of the vascular endothelium to proinflammatory cytokines. In this study, we further characterized one of these novel gene products named B61. Sequence analysis of cDNA clones encoding B61 revealed that its protein product has no significant homology to previously described proteins. Southern analysis suggested that B61 is an evolutionarily conserved single-copy gene. B61 is primarily a hydrophilic molecule but contains both a hydrophobic N-terminal and a hydrophobic C-terminal region. The N-terminal region is typical of a signal peptide, which is consistent with the secreted nature of the protein. The mature form of the predicted protein consists of 187 amino acid residues and has a molecular weight of 22,000. Immunoprecipitation of metabolically labeled human umbilical vein endothelial cell preparations revealed that B61 is a 25-kilodalton secreted protein which is markedly induced by tumor necrosis factor.
A novel immediate-early response gene of endothelium is induced by cytokines and encodes a secreted protein.

PubMed Central

Holzman, L B; Marks, R M; Dixit, V M

1990-01-01

We have previously described the cloning of a group of novel cellular immediate-early response genes whose expression in human umbilical vein endothelial cells is induced by tumor necrosis factor alpha in the presence of cycloheximide. These genes are likely to participate in mediating the response of the vascular endothelium to proinflammatory cytokines. In this study, we further characterized one of these novel gene products named B61. Sequence analysis of cDNA clones encoding B61 revealed that its protein product has no significant homology to previously described proteins. Southern analysis suggested that B61 is an evolutionarily conserved single-copy gene. B61 is primarily a hydrophilic molecule but contains both a hydrophobic N-terminal and a hydrophobic C-terminal region. The N-terminal region is typical of a signal peptide, which is consistent with the secreted nature of the protein. The mature form of the predicted protein consists of 187 amino acid residues and has a molecular weight of 22,000. Immunoprecipitation of metabolically labeled human umbilical vein endothelial cell preparations revealed that B61 is a 25-kilodalton secreted protein which is markedly induced by tumor necrosis factor. Images PMID:2233719
Cdk1-cyclin B1-mediated phosphorylation of tumor-associated microtubule-associated protein/cytoskeleton-associated protein 2 in mitosis.

PubMed

Hong, Kyung Uk; Kim, Hyun-Jun; Kim, Hyo-Sil; Seong, Yeon-Sun; Hong, Kyeong-Man; Bae, Chang-Dae; Park, Joobae

2009-06-12

During mitosis, establishment of structurally and functionally sound bipolar spindles is necessary for maintaining the fidelity of chromosome segregation. Tumor-associated microtubule-associated protein (TMAP), also known as cytoskeleton-associated protein 2 (CKAP2), is a mitotic spindle-associated protein whose level is frequently up-regulated in various malignancies. Previous reports have suggested that TMAP is a potential regulator of mitotic spindle assembly and dynamics and that it is required for chromosome segregation to occur properly. So far, there have been no reports on how its mitosis-related functions are regulated. Here, we report that TMAP is hyper-phosphorylated at the C terminus specifically during mitosis. At least four different residues (Thr-578, Thr-596, Thr-622, and Ser-627) were responsible for the mitosis-specific phosphorylation of TMAP. Among these, Thr-622 was specifically phosphorylated by Cdk1-cyclin B1 both in vitro and in vivo. Interestingly, compared with the wild type, a phosphorylation-deficient mutant form of TMAP, in which Thr-622 had been replaced with an alanine (T622A), induced a significant increase in the frequency of metaphase cells with abnormal bipolar spindles, which often displayed disorganized, asymmetrical, or narrow and elongated morphologies. Formation of these abnormal bipolar spindles subsequently resulted in misalignment of metaphase chromosomes and ultimately caused a delay in the entry into anaphase. Moreover, such defects resulting from the T622A mutation were associated with a decrease in the rate of protein turnover at spindle microtubules. These findings suggest that Cdk1-cyclin B1-mediated phosphorylation of TMAP is important for and contributes to proper regulation of microtubule dynamics and establishment of functional bipolar spindles during mitosis.
Cdk1-Cyclin B1-mediated Phosphorylation of Tumor-associated Microtubule-associated Protein/Cytoskeleton-associated Protein 2 in Mitosis*

PubMed Central

Uk Hong, Kyung; Kim, Hyun-Jun; Kim, Hyo-Sil; Seong, Yeon-Sun; Hong, Kyeong-Man; Bae, Chang-Dae; Park, Joobae

2009-01-01

During mitosis, establishment of structurally and functionally sound bipolar spindles is necessary for maintaining the fidelity of chromosome segregation. Tumor-associated microtubule-associated protein (TMAP), also known as cytoskeleton-associated protein 2 (CKAP2), is a mitotic spindle-associated protein whose level is frequently up-regulated in various malignancies. Previous reports have suggested that TMAP is a potential regulator of mitotic spindle assembly and dynamics and that it is required for chromosome segregation to occur properly. So far, there have been no reports on how its mitosis-related functions are regulated. Here, we report that TMAP is hyper-phosphorylated at the C terminus specifically during mitosis. At least four different residues (Thr-578, Thr-596, Thr-622, and Ser-627) were responsible for the mitosis-specific phosphorylation of TMAP. Among these, Thr-622 was specifically phosphorylated by Cdk1-cyclin B1 both in vitro and in vivo. Interestingly, compared with the wild type, a phosphorylation-deficient mutant form of TMAP, in which Thr-622 had been replaced with an alanine (T622A), induced a significant increase in the frequency of metaphase cells with abnormal bipolar spindles, which often displayed disorganized, asymmetrical, or narrow and elongated morphologies. Formation of these abnormal bipolar spindles subsequently resulted in misalignment of metaphase chromosomes and ultimately caused a delay in the entry into anaphase. Moreover, such defects resulting from the T622A mutation were associated with a decrease in the rate of protein turnover at spindle microtubules. These findings suggest that Cdk1-cyclin B1-mediated phosphorylation of TMAP is important for and contributes to proper regulation of microtubule dynamics and establishment of functional bipolar spindles during mitosis. PMID:19369249
A Phosphoproteomic Comparison of B-RAFV600E and MKK1/2 Inhibitors in Melanoma Cells.

PubMed

Stuart, Scott A; Houel, Stephane; Lee, Thomas; Wang, Nan; Old, William M; Ahn, Natalie G

2015-06-01

Inhibitors of oncogenic B-RAF(V600E) and MKK1/2 have yielded remarkable responses in B-RAF(V600E)-positive melanoma patients. However, the efficacy of these inhibitors is limited by the inevitable onset of resistance. Despite the fact that these inhibitors target the same pathway, combination treatment with B-RAF(V600E) and MKK1/2 inhibitors has been shown to improve both response rates and progression-free survival in B-RAF(V600E) melanoma patients. To provide insight into the molecular nature of the combinatorial response, we used quantitative mass spectrometry to characterize the inhibitor-dependent phosphoproteome of human melanoma cells treated with the B-RAF(V600E) inhibitor PLX4032 (vemurafenib) or the MKK1/2 inhibitor AZD6244 (selumetinib). In three replicate experiments, we quantified changes at a total of 23,986 phosphosites on 4784 proteins. This included 1317 phosphosites that reproducibly decreased in response to at least one inhibitor. Phosphosites that responded to both inhibitors grouped into networks that included the nuclear pore complex, growth factor signaling, and transcriptional regulators. Although the majority of phosphosites were responsive to both inhibitors, we identified 16 sites that decreased only in response to PLX4032, suggesting rare instances where oncogenic B-RAF signaling occurs in an MKK1/2-independent manner. Only two phosphosites were identified that appeared to be uniquely responsive to AZD6244. When cells were treated with the combination of AZD6244 and PLX4032 at subsaturating concentrations (30 nm), responses at nearly all phosphosites were additive. We conclude that AZD6244 does not substantially widen the range of phosphosites inhibited by PLX4032 and that the benefit of the drug combination is best explained by their additive effects on suppressing ERK1/2 signaling. Comparison of our results to another recent ERK1/2 phosphoproteomics study revealed a surprising degree of variability in the sensitivity of phosphosites to
Requirement for the eIF4E binding proteins for the synergistic down-regulation of protein synthesis by hypertonic conditions and mTOR inhibition.

PubMed

Clemens, Michael J; Elia, Androulla; Morley, Simon J

2013-01-01

The protein kinase mammalian target of rapamycin (mTOR) regulates the phosphorylation and activity of several proteins that have the potential to control translation, including p70S6 kinase and the eIF4E binding proteins 4E-BP1 and 4E-BP2. In spite of this, in exponentially growing cells overall protein synthesis is often resistant to mTOR inhibitors. We report here that sensitivity of wild-type mouse embryonic fibroblasts (MEFs) to mTOR inhibitors can be greatly increased when the cells are subjected to the physiological stress imposed by hypertonic conditions. In contrast, protein synthesis in MEFs with a double knockout of 4E-BP1 and 4E-BP2 remains resistant to mTOR inhibitors under these conditions. Phosphorylation of p70S6 kinase and protein kinase B (Akt) is blocked by the mTOR inhibitor Ku0063794 equally well in both wild-type and 4E-BP knockout cells, under both normal and hypertonic conditions. The response of protein synthesis to hypertonic stress itself does not require the 4E-BPs. These data suggest that under certain stress conditions: (i) translation has a greater requirement for mTOR activity and (ii) there is an absolute requirement for the 4E-BPs for regulation by mTOR. Importantly, dephosphorylation of p70S6 kinase and Akt is not sufficient to affect protein synthesis acutely.

The Gab1 protein is a docking site for multiple proteins involved in signaling by the B cell antigen receptor.

PubMed

Ingham, R J; Holgado-Madruga, M; Siu, C; Wong, A J; Gold, M R

1998-11-13

Gab1 is a member of the docking/scaffolding protein family which includes IRS-1, IRS-2, c-Cbl, p130(cas), and p62(dok). These proteins contain a variety of protein-protein interaction motifs including multiple tyrosine residues that when phosphorylated can act as binding sites for Src homology 2 (SH2) domain-containing signaling proteins. We show in the RAMOS human B cell line that Gab1 is tyrosine-phosphorylated in response to B cell antigen receptor (BCR) engagement. Moreover, tyrosine phosphorylation of Gab1 correlated with the binding of several SH2-containing signaling proteins to Gab1 including Shc, Grb2, phosphatidylinositol 3-kinase, and the SHP-2 tyrosine phosphatase. Far Western analysis showed that the SH2 domains of Shc, SHP-2, and the p85 subunit of phosphatidylinositol 3-kinase could bind directly to tyrosine-phosphorylated Gab1 isolated from activated RAMOS cells. In contrast, the Grb2 SH2 domain did not bind directly to Gab1 but instead to the Shc and SHP-2 associated with Gab1. We also show that Gab1 is present in the membrane-enriched particulate fraction of RAMOS cells and that Gab1/signaling protein complexes are found in this fraction after BCR engagement. Thus, tyrosine-phosphorylated Gab1 may recruit cytosolic signaling proteins to cellular membranes where they can act on membrane-bound targets. This may be a critical step in the activation of multiple BCR signaling pathways.
Cep55 overexpression causes male-specific sterility in mice by suppressing Foxo1 nuclear retention through sustained activation of PI3K/Akt signaling.

PubMed

Sinha, Debottam; Kalimutho, Murugan; Bowles, Josephine; Chan, Ai-Leen; Merriner, D Jo; Bain, Amanda L; Simmons, Jacinta L; Freire, Raimundo; Lopez, J Alejandro; Hobbs, Robin M; O'Bryan, Moira K; Khanna, Kum Kum

2018-04-17

Spermatogenesis is a dynamic process involving self-renewal and differentiation of spermatogonial stem cells, meiosis, and ultimately, the differentiation of haploid spermatids into sperm. Centrosomal protein (CEP)-55 is necessary for somatic cell abscission during cytokinesis. It facilitates equal segregation of cytoplasmic contents between daughter cells by recruiting endosomal sorting complex required for transport machinery (ESCRT) at the midbody. In germ cells, CEP55, in partnership with testes expressed-14 protein (TEX14), has also been shown to be an integral component of intercellular bridge before meiosis. Various in vitro studies have demonstrated a role for CEP55 in multiple cancers and other diseases. However, its oncogenic potential in vivo remains elusive. To investigate, we generated ubiquitously overexpressing Cep55 transgenic mice ( Cep55 Tg/Tg ) aiming to characterize its oncogenic role in cancer. Unexpectedly, we found that Cep55 Tg/Tg male mice were sterile and had severe and progressive defects in spermatogenesis related to spermatogenic arrest and lack of spermatids in the testes. In this study, we characterized this male-specific phenotype and showed that excessively high levels of Cep55 results in hyperactivation of PI3K/protein kinase B (Akt) signaling in testis. In line with this finding, we observed increased phosphorylation of forkhead box protein O1 (FoxO1), and suppression of its nuclear retention, along with the relative enrichment of promyelocytic leukemia zinc finger (PLZF) -positive cells. Independently, we observed that Cep55 amplification favored upregulation of ret ( Ret) proto-oncogene and glial-derived neurotrophic factor family receptor α-1 ( Gfra1). Consistent with these data, we observed selective down-regulation of genes associated with germ cell differentiation in Cep55-overexpressing testes at postnatal day 10, including early growth response-4 ( Egr4) and spermatogenesis and oogenesis specific basic helix-loop-helix-1
Hepatitis B X-interacting protein promotes cisplatin resistance and regulates CD147 via Sp1 in ovarian cancer.

PubMed

Zou, Wei; Ma, Xiangdong; Yang, Hong; Hua, Wei; Chen, Biliang; Cai, Guoqing

2017-03-01

Ovarian cancer is the highest mortality rate of all female reproductive malignancies. Drug resistance is a major cause of treatment failure in malignant tumors. Hepatitis B X-interacting protein acts as an oncoprotein, regulates cell proliferation, and migration in breast cancer. We aimed to investigate the effects and mechanisms of hepatitis B X-interacting protein on resistance to cisplatin in human ovarian cancer cell lines. The mRNA and protein levels of hepatitis B X-interacting protein were detected using RT-PCR and Western blotting in cisplatin-resistant and cisplatin-sensitive tissues, cisplatin-resistant cell lines A2780/CP and SKOV3/CP, and cisplatin-sensitive cell lines A2780 and SKOV3. Cell viability and apoptosis were measured to evaluate cellular sensitivity to cisplatin in A2780/CP cells. Luciferase reporter gene assay was used to determine the relationship between hepatitis B X-interacting protein and CD147. The in vivo function of hepatitis B X-interacting protein on tumor burden was assessed in cisplatin-resistant xenograft models. The results showed that hepatitis B X-interacting protein was highly expressed in ovarian cancer of cisplatin-resistant tissues and cells. Notably, knockdown of hepatitis B X-interacting protein significantly reduced cell viability in A2780/CP compared with cisplatin treatment alone. Hepatitis B X-interacting protein and cisplatin cooperated to induce apoptosis and increase the expression of c-caspase 3 as well as the Bax/Bcl-2 ratio. We confirmed that hepatitis B X-interacting protein up-regulated CD147 at the protein expression and transcriptional levels. Moreover, we found that hepatitis B X-interacting protein was able to activate the CD147 promoter through Sp1. In vivo, depletion of hepatitis B X-interacting protein decreased the tumor volume and weight induced by cisplatin. Taken together, these results indicate that hepatitis B X-interacting protein promotes cisplatin resistance and regulated CD147 via Sp1 in
IgG1 memory B cells keep the memory of IgE responses.

PubMed

He, Jin-Shu; Subramaniam, Sharrada; Narang, Vipin; Srinivasan, Kandhadayar; Saunders, Sean P; Carbajo, Daniel; Wen-Shan, Tsao; Hidayah Hamadee, Nur; Lum, Josephine; Lee, Andrea; Chen, Jinmiao; Poidinger, Michael; Zolezzi, Francesca; Lafaille, Juan J; Curotto de Lafaille, Maria A

2017-09-21

The unique differentiation of IgE cells suggests unconventional mechanisms of IgE memory. IgE germinal centre cells are transient, most IgE cells are plasma cells, and high affinity IgE is produced by the switching of IgG1 cells to IgE. Here we investigate the function of subsets of IgG1 memory B cells in IgE production and find that two subsets of IgG1 memory B cells, CD80 + CD73 + and CD80 - CD73 - , contribute distinctively to the repertoires of high affinity pathogenic IgE and low affinity non-pathogenic IgE. Furthermore, repertoire analysis indicates that high affinity IgE and IgG1 plasma cells differentiate from rare CD80 + CD73 + high affinity memory clones without undergoing further mutagenesis. By identifying the cellular origin of high affinity IgE and the clonal selection of high affinity memory B cells into the plasma cell fate, our findings provide fundamental insights into the pathogenesis of allergies, and on the mechanisms of antibody production in memory B cell responses.IgE is an important mediator of protective immunity as well as allergic reaction, but how high affinity IgE antibodies are produced in memory responses is not clear. Here the authors show that IgE can be generated via class-switch recombination in IgG1 memory B cells without additional somatic hypermutation.
55. E.M.C., photographer July 3, 1936 HUMBOLDT COUNTY, SECTION J, ...

Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

55. E.M.C., photographer July 3, 1936 HUMBOLDT COUNTY, SECTION J, HIGHWAY 1. 1-HUM-I-J #80, BITUMULS PORTION OF EXPERIMENTAL SECTION, E.M.C, 7-3-36. BACK READS: C/PROCESSING STABILIZING BITUMULS WITH BLADE AFTER BEING PROCESSED WITH SPRING TOOTH HARROW. Stamped office copy. - Redwood National & State Parks Roads, California coast from Crescent City to Trinidad, Crescent City, Del Norte County, CA
Heat Stress Response in Pea Involves Interaction of Mitochondrial Nucleoside Diphosphate Kinase with a Novel 86-Kilodalton Protein1

PubMed Central

Escobar Galvis, Martha L.; Marttila, Salla; Håkansson, Gunilla; Forsberg, Jens; Knorpp, Carina

2001-01-01

In this work we have further characterized the first mitochondrial nucleoside diphosphate kinase (mtNDPK) isolated from plants. The mitochondrial isoform was found to be especially abundant in reproductive and young tissues. Expression of the pea (Pisum sativum L. cv Oregon sugarpod) mtNDPK was not affected by different stress conditions. However, the pea mtNDPK was found to interact with a novel 86-kD protein, which is de novo synthesized in pea leaves upon exposure to heat. Thus, we have evidence for the involvement of mtNDPK in mitochondrial heat response in pea in vivo. Studies on oligomerization revealed that mtNDPK was found in complexes of various sizes, corresponding to the sizes of e.g. hexamers, tetramers, and dimers, indicating flexibility in oligomerization. This flexibility, also found for other NDPK isoforms, has been correlated with the ability of this enzyme to interact with other proteins. We believe that the mtNDPK is involved in heat stress response in pea, possibly as a modulator of the 86-kD protein. PMID:11351071
Increased hypothalamic protein tyrosine phosphatase 1B contributes to leptin resistance with age.

PubMed

Morrison, Christopher D; White, Christy L; Wang, Zhong; Lee, Seung-Yub; Lawrence, David S; Cefalu, William T; Zhang, Zhong-Yin; Gettys, Thomas W

2007-01-01

Animals at advanced ages exhibit a reduction in central leptin sensitivity. However, changes in growth, metabolism, and obesity risk occur much earlier in life, particularly during the transition from youth to middle age. To determine when initial decreases in central leptin sensitivity occur, leptin-dependent suppression of food intake was tested in 8-, 12-, and 20-wk-old male, chow-fed Sprague Dawley rats. Intracerebroventricular leptin injection (3 microg) suppressed 24-h food intake in 8- and 12-wk-old rats (P < 0.05) but not 20-wk-old rats. To identify potential cellular mediators of this resistance, we focused on protein tyrosine phosphatase 1B (PTP1B), a recently described inhibitor of leptin signaling. PTP1B protein levels, as determined by Western blot, were significantly higher in mediobasal hypothalamic punches collected from 20-wk-old rats, compared with 8-wk-old rats (P < 0.05). When 20-wk-old rats were fasted for 24 h, levels of hypothalamic PTP1B decreased (P < 0.05), coincident with a restoration of leptin sensitivity. To directly test whether inhibition of PTP1B restores leptin sensitivity, 20-wk-old chow-fed rats were pretreated with a pharmacological PTP1B inhibitor 1 h before leptin, and 24-h food intake was recorded. As expected, leptin alone produced a small but nonsignificant reduction in food intake. However, pretreatment with the PTP1B inhibitor resulted in a marked improvement in leptin-dependent suppression of food intake (P < 0.05). These data are consistent with the hypothesis that increases in PTP1B contribute to hypothalamic leptin resistance as rats transition into middle age.
Cell cycle-dependent regulation of Greatwall kinase by protein phosphatase 1 and regulatory subunit 3B.

PubMed

Ren, Dapeng; Fisher, Laura A; Zhao, Jing; Wang, Ling; Williams, Byron C; Goldberg, Michael L; Peng, Aimin

2017-06-16

Greatwall (Gwl) kinase plays an essential role in the regulation of mitotic entry and progression. Mitotic activation of Gwl requires both cyclin-dependent kinase 1 (CDK1)-dependent phosphorylation and its autophosphorylation at an evolutionarily conserved serine residue near the carboxyl terminus (Ser-883 in Xenopus ). In this study we show that Gwl associates with protein phosphatase 1 (PP1), particularly PP1γ, which mediates the dephosphorylation of Gwl Ser-883. Consistent with the mitotic activation of Gwl, its association with PP1 is disrupted in mitotic cells and egg extracts. During mitotic exit, PP1-dependent dephosphorylation of Gwl Ser-883 occurs prior to dephosphorylation of other mitotic substrates; replacing endogenous Gwl with a phosphomimetic S883E mutant blocks mitotic exit. Moreover, we identified PP1 regulatory subunit 3B (PPP1R3B) as a targeting subunit that can direct PP1 activity toward Gwl. PPP1R3B bridges PP1 and Gwl association and promotes Gwl Ser-883 dephosphorylation. Consistent with the cell cycle-dependent association of Gwl and PP1, Gwl and PPP1R3B dissociate in M phase. Interestingly, up-regulation of PPP1R3B facilitates mitotic exit and blocks mitotic entry. Thus, our study suggests PPP1R3B as a new cell cycle regulator that functions by governing Gwl dephosphorylation. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.
Glucagon Like Peptide-1 (GLP-1) Modulates OVA-Induced Airway Inflammation and Mucus Secretion Involving a Protein Kinase A (PKA)-Dependent Nuclear Factor-κB (NF-κB) Signaling Pathway in Mice.

PubMed

Zhu, Tao; Wu, Xiao-Ling; Zhang, Wei; Xiao, Min

2015-08-26

Asthma is a common chronic pulmonary inflammatory disease, featured with mucus hyper-secretion in the airway. Recent studies found that glucagon like peptide-1 (GLP-1) analogs, including liraglutide and exenatide, possessed a potent anti-inflammatory property through a protein kinase A (PKA)-dependent signaling pathway. Therefore, the aim of current study was to investigate the value of GLP-1 analog therapy liraglutide in airway inflammation and mucus secretion in a murine model of ovalbumin (OVA)-induced asthma, and its underlying molecular mechanism. In our study, BALB/c mice were sensitized and challenged by OVA to induce chronic asthma. Pathological alterations, the number of cells and the content of inflammatory mediators in bronchoalveolar lavage fluid (BALF), and mucus secretion were observed and measured. In addition, the mRNA and protein expression of E-selectin and MUC5AC were analyzed by qPCR and Western blotting. Then, the phosphorylation of PKA and nuclear factor-κB (NF-κB) p65 were also measured by Western blotting. Further, NF-κB p65 DNA binding activity was detected by ELISA. OVA-induced airway inflammation, airway mucus hyper-secretion, the up-regulation of E-selectin and MUC5AC were remarkably inhibited by GLP-1 in mice (all p < 0.01). Then, we also found that OVA-reduced phosphorylation of PKA, and OVA-enhanced NF-κB p65 activation and NF-κB p65 DNA binding activity were markedly improved by GLP-1 (all p < 0.01). Furthermore, our data also figured out that these effects of GLP-1 were largely abrogated by the PKA inhibitor H-89 (all p < 0.01). Taken together, our results suggest that OVA-induced asthma were potently ameliorated by GLP-1 possibly through a PKA-dependent inactivation of NF-κB in mice, indicating that GLP-1 analogs may be considered an effective and safe drug for the potential treatment of asthma in the future.
10 CFR 55.1 - Purpose.

Code of Federal Regulations, 2014 CFR

2014-01-01

... 10 Energy 2 2014-01-01 2014-01-01 false Purpose. 55.1 Section 55.1 Energy NUCLEAR REGULATORY... of utilization facilities licensed under the Atomic Energy Act of 1954, as amended, or Section 202 of the Energy Reorganization Act of 1974, as amended, and part 50, part 52, or part 54 of this chapter...
10 CFR 55.1 - Purpose.

Code of Federal Regulations, 2010 CFR

2010-01-01

... 10 Energy 2 2010-01-01 2010-01-01 false Purpose. 55.1 Section 55.1 Energy NUCLEAR REGULATORY... of utilization facilities licensed under the Atomic Energy Act of 1954, as amended, or Section 202 of the Energy Reorganization Act of 1974, as amended, and part 50, part 52, or part 54 of this chapter...
Amyloid Precursor Protein (APP) May Act as a Substrate and a Recognition Unit for CRL4CRBN and Stub1 E3 Ligases Facilitating Ubiquitination of Proteins Involved in Presynaptic Functions and Neurodegeneration.

PubMed

Del Prete, Dolores; Rice, Richard C; Rajadhyaksha, Anjali M; D'Adamio, Luciano

2016-08-12

The amyloid precursor protein (APP), whose mutations cause Alzheimer disease, plays an important in vivo role and facilitates transmitter release. Because the APP cytosolic region (ACR) is essential for these functions, we have characterized its brain interactome. We found that the ACR interacts with proteins that regulate the ubiquitin-proteasome system, predominantly with the E3 ubiquitin-protein ligases Stub1, which binds the NH2 terminus of the ACR, and CRL4(CRBN), which is formed by Cul4a/b, Ddb1, and Crbn, and interacts with the COOH terminus of the ACR via Crbn. APP shares essential functions with APP-like protein-2 (APLP2) but not APP-like protein-1 (APLP1). Noteworthy, APLP2, but not APLP1, interacts with Stub1 and CRL4(CRBN), pointing to a functional pathway shared only by APP and APLP2. In vitro ubiquitination/ubiquitome analysis indicates that these E3 ligases are enzymatically active and ubiquitinate the ACR residues Lys(649/650/651/676/688) Deletion of Crbn reduces ubiquitination of Lys(676) suggesting that Lys(676) is physiologically ubiquitinated by CRL4(CRBN) The ACR facilitated in vitro ubiquitination of presynaptic proteins that regulate exocytosis, suggesting a mechanism by which APP tunes transmitter release. Other dementia-related proteins, namely Tau and apoE, interact with and are ubiquitinated via the ACR in vitro This, and the evidence that CRBN and CUL4B are linked to intellectual disability, prompts us to hypothesize a pathogenic mechanism, in which APP acts as a modulator of E3 ubiquitin-protein ligase(s), shared by distinct neuronal disorders. The well described accumulation of ubiquitinated protein inclusions in neurodegenerative diseases and the link between the ubiquitin-proteasome system and neurodegeneration make this concept plausible. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.
Purification of family B G protein-coupled receptors using nanodiscs: Application to human glucagon-like peptide-1 receptor.

PubMed

Cai, Yingying; Liu, Yuting; Culhane, Kelly J; DeVree, Brian T; Yang, Yang; Sunahara, Roger K; Yan, Elsa C Y

2017-01-01

Family B G protein-coupled receptors (GPCRs) play vital roles in hormone-regulated homeostasis. They are drug targets for metabolic diseases, including type 2 diabetes and osteoporosis. Despite their importance, the signaling mechanisms for family B GPCRs at the molecular level remain largely unexplored due to the challenges in purification of functional receptors in sufficient amount for biophysical characterization. Here, we purified the family B GPCR human glucagon-like peptide-1 (GLP-1) receptor (GLP1R), whose agonists, e.g. exendin-4, are used for the treatment of type 2 diabetes mellitus. The receptor was expressed in HEK293S GnTl- cells using our recently developed protocol. The protocol incorporates the receptor into the native-like lipid environment of reconstituted high density lipoprotein (rHDL) particles, also known as nanodiscs, immediately after the membrane solubilization step followed by chromatographic purification, minimizing detergent contact with the target receptor to reduce denaturation and prolonging stabilization of receptor in lipid bilayers without extra steps of reconstitution. This method yielded purified GLP1R in nanodiscs that could bind to GLP-1 and exendin-4 and activate Gs protein. This nanodisc purification method can potentially be a general strategy to routinely obtain purified family B GPCRs in the 10s of microgram amounts useful for spectroscopic analysis of receptor functions and activation mechanisms.
Purification of family B G protein-coupled receptors using nanodiscs: Application to human glucagon-like peptide-1 receptor

PubMed Central

Cai, Yingying; Liu, Yuting; Culhane, Kelly J.; DeVree, Brian T.; Yang, Yang; Sunahara, Roger K.; Yan, Elsa C. Y.

2017-01-01

Family B G protein-coupled receptors (GPCRs) play vital roles in hormone-regulated homeostasis. They are drug targets for metabolic diseases, including type 2 diabetes and osteoporosis. Despite their importance, the signaling mechanisms for family B GPCRs at the molecular level remain largely unexplored due to the challenges in purification of functional receptors in sufficient amount for biophysical characterization. Here, we purified the family B GPCR human glucagon-like peptide-1 (GLP-1) receptor (GLP1R), whose agonists, e.g. exendin-4, are used for the treatment of type 2 diabetes mellitus. The receptor was expressed in HEK293S GnTl- cells using our recently developed protocol. The protocol incorporates the receptor into the native-like lipid environment of reconstituted high density lipoprotein (rHDL) particles, also known as nanodiscs, immediately after the membrane solubilization step followed by chromatographic purification, minimizing detergent contact with the target receptor to reduce denaturation and prolonging stabilization of receptor in lipid bilayers without extra steps of reconstitution. This method yielded purified GLP1R in nanodiscs that could bind to GLP-1 and exendin-4 and activate Gs protein. This nanodisc purification method can potentially be a general strategy to routinely obtain purified family B GPCRs in the 10s of microgram amounts useful for spectroscopic analysis of receptor functions and activation mechanisms. PMID:28609478
C/EBPβ-LAP*/LAP Expression Is Mediated by RSK/eIF4B-Dependent Signalling and Boosted by Increased Protein Stability in Models of Monocytic Differentiation

PubMed Central

Christmann, Martin; Friesenhagen, Judith; Westphal, Andreas; Pietsch, Daniel; Brand, Korbinian

2015-01-01

The transcription factor C/EBPβ plays a key role in monocytic differentiation and inflammation. Its small isoform LIP is associated with proliferation at early premonocytic developmental stages and regulated via mTOR-dependent signalling. During later stages of (pre)monocytic differentiation there is a considerable increase in the large C/EBPβ isoforms LAP*/LAP which inhibit proliferation thus supporting terminal differentiation. Here, we showed in different models of monocytic differentiation that this dramatic increase in the LAP*/LAP protein and LAP/LIP ratio was accompanied by an only modest/retarded mRNA increase suggesting an important role for (post)translational mechanisms. We found that LAP*/LAP formation was induced via MEK/RSK-dependent cascades, whereas mTOR/S6K1 were not involved. Remarkably, LAP*/LAP expression was dependent on phosphorylated eIF4B, an acceleratory protein of RNA helicase eIF4A. PKR inhibition reduced the expression of eIF4B and C/EBPβ in an eIF2α-independent manner. Furthermore, under our conditions a marked stabilisation of LAP*/LAP protein occurred, accompanied by reduced chymotrypsin-like proteasome/calpain activities and increased calpastatin levels. Our study elucidates new signalling pathways inducing LAP*/LAP expression and indicates new alternative PKR functions in monocytes. The switch from mTOR- to RSK-mediated signalling to orchestrate eIF4B-dependent LAP*/LAP translation, accompanied by increased protein stability but only small mRNA changes, may be a prototypical example for the regulation of protein expression during selected processes of differentiation/proliferation. PMID:26646662
Structural rationale for the cross-resistance of tumor cells bearing the A399V variant of elongation factor eEF1A1 to the structurally unrelated didemnin B, ternatin, nannocystin A and ansatrienin B

NASA Astrophysics Data System (ADS)

Sánchez-Murcia, Pedro A.; Cortés-Cabrera, Álvaro; Gago, Federico

2017-10-01

At least four classes of structurally distinct natural products with potent antiproliferative activities target the translation elongation factor eEF1A1, which is best known as the G-protein that delivers amino acyl transfer RNAs (aa-tRNAs) to ribosomes during mRNA translation. We present molecular models in atomic detail that provide a common structural basis for the high-affinity binding of didemnin B, ternatin, ansatrienin B and nannocystin A to eEF1A1, as well as a rationale based on molecular dynamics results that accounts for the deleterious effect of replacing alanine 399 with valine. The proposed binding site, at the interface between domains I and III, is eminently hydrophobic and exists only in the GTP-bound conformation. Drug binding at this site is expected to disrupt neither loading of aa-tRNAs nor GTP hydrolysis but would give rise to stabilization of this particular conformational state, in consonance with reported experimental findings. The experimental solution of the three-dimensional structure of mammalian eEF1A1 has proved elusive so far and the highly homologous eEF1A2 from rabbit muscle has been crystallized and solved only as a homodimer in a GDP-bound conformation. Interestingly, in this dimeric structure the large interdomain cavity where the drugs studied are proposed to bind is occupied by a mostly hydrophobic α-helix from domain I of the same monomer. Since binding of this α-helix and any of these drugs to domain III of eEF1A(1/2) is, therefore, mutually exclusive and involves two distinct protein conformations, one intriguing possibility that emerges from our study is that the potent antiproliferative effect of these natural products may arise not only from inhibition of protein synthesis, which is the current dogma, but also from interference with some other non-canonical functions. From this standpoint, this type of drugs could be considered antagonists of eEF1A1/2 oligomerization, a hypothesis that opens up novel areas of research.
A POSSIBLE CARBON-RICH INTERIOR IN SUPER-EARTH 55 Cancri e

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

Madhusudhan, Nikku; Lee, Kanani K. M.; Mousis, Olivier, E-mail: Nikku.Madhusudhan@yale.edu

Terrestrial planets in the solar system, such as the Earth, are oxygen-rich, with silicates and iron being the most common minerals in their interiors. However, the true chemical diversity of rocky planets orbiting other stars is yet unknown. Mass and radius measurements are used to constrain the interior compositions of super-Earths (exoplanets with masses of 1-10 M{sub Circled-Plus }), and are typically interpreted with planetary interior models that assume Earth-centric oxygen-rich compositions. Using such models, the super-Earth 55 Cancri e (mass 8 M{sub Circled-Plus }, radius 2 R{sub Circled-Plus }) has been suggested to bear an interior composition consisting ofmore » Fe, silicates, and an envelope ({approx}> 10% by mass) of supercritical water. We report that the mass and radius of 55 Cancri e can also be explained by a carbon-rich solid interior made of Fe, C, SiC, and/or silicates and without a volatile envelope. While the data allow Fe mass fractions of up to 40%, a wide range of C, SiC, and/or silicate mass fractions are possible. A carbon-rich 55 Cancri e is also plausible if its protoplanetary disk bore the same composition as its host star, which has been reported to be carbon-rich. However, more precise estimates of the stellar elemental abundances and observations of the planetary atmosphere are required to further constrain its interior composition. The possibility of a C-rich interior in 55 Cancri e opens a new regime of geochemistry and geophysics in extraterrestrial rocky planets, compared to terrestrial planets in the solar system.« less
Structural basis for midbody targeting of spastin by the ESCRT-III protein CHMP1B

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

Yang, Dong; Rimanchi, Neggy; Renvoise, Benoit

2009-01-15

The endosomal sorting complex required for transport (ESCRT) machinery, including ESCRT-III, localizes to the midbody and participates in the membrane-abscission step of cytokinesis. The ESCRT-III protein charged multivesicular body protein 1B (CHMP1B) is required for recruitment of the MIT domain-containing protein spastin, a microtubule-severing enzyme, to the midbody. The 2.5-{angstrom} structure of the C-terminal tail of CHMP1B with the MIT domain of spastin reveals a specific, high-affinity complex involving a noncanonical binding site between the first and third helices of the MIT domain. The structural interface is twice as large as that of the MIT domain of the VPS4-CHMP complex,more » consistent with the high affinity of the interaction. A series of unique hydrogen-bonding interactions and close packing of small side chains discriminate against the other ten human ESCRT-III subunits. Point mutants in the CHMP1B binding site of spastin block recruitment of spastin to the midbody and impair cytokinesis.« less
The Zinc-finger protein ASCIZ regulates B cell development via DYNLL1 and Bim

PubMed Central

Jurado, Sabine; Gleeson, Kimberly; O’Donnell, Kristy; Izon, David J.; Walkley, Carl R.; Strasser, Andreas; Tarlinton, David M.

2012-01-01

Developing B lymphocytes expressing defective or autoreactive pre-B or B cell receptors (BCRs) are eliminated by programmed cell death, but how the balance between death and survival signals is regulated to prevent immunodeficiency and autoimmunity remains incompletely understood. In this study, we show that absence of the essential ATM (ataxia telangiectasia mutated) substrate Chk2-interacting Zn2+-finger protein (ASCIZ; also known as ATMIN/ZNF822), a protein with dual functions in the DNA damage response and as a transcription factor, leads to progressive cell loss from the pre-B stage onwards and severely diminished splenic B cell numbers in mice. This lymphopenia cannot be suppressed by deletion of p53 or complementation with a prearranged BCR, indicating that it is not caused by impaired DNA damage responses or defective V(D)J recombination. Instead, ASCIZ-deficient B cell precursors contain highly reduced levels of DYNLL1 (dynein light chain 1; LC8), a recently identified transcriptional target of ASCIZ, and normal B cell development can be restored by ectopic Dynll1 expression. Remarkably, the B cell lymphopenia in the absence of ASCIZ can also be fully suppressed by deletion of the proapoptotic DYNLL1 target Bim. Our findings demonstrate a key role for ASCIZ in regulating the survival of developing B cells by activating DYNLL1 expression, which may then modulate Bim-dependent apoptosis. PMID:22891272
An Arabidopsis Prenylated Rab Acceptor 1 Isoform, AtPRA1.B6, Displays Differential Inhibitory Effects on Anterograde Trafficking of Proteins at the Endoplasmic Reticulum1[W][OA

PubMed Central

Lee, Myoung Hui; Jung, Chanjin; Lee, Junho; Kim, Soo Youn; Lee, Yongjik; Hwang, Inhwan

2011-01-01

Prenylated Rab acceptors (PRAs), members of the Ypt-interacting protein family of small membrane proteins, are thought to aid the targeting of prenylated Rabs to their respective endomembrane compartments. In plants, the Arabidopsis (Arabidopsis thaliana) PRA1 family contains 19 members that display varying degrees of sequence homology to animal PRA1 and localize to the endoplasmic reticulum (ER) and/or endosomes. However, the exact role of these proteins remains to be fully characterized. In this study, the effect of AtPRA1.B6, a member of the AtPRA1 family, on the anterograde trafficking of proteins targeted to various endomembrane compartments was investigated. High levels of AtPRA1.B6 resulted in differential inhibition of coat protein complex II vesicle-mediated anterograde trafficking. The trafficking of the vacuolar proteins sporamin:GFP (for green fluorescent protein) and AALP:GFP, the secretory protein invertase:GFP, and the plasma membrane proteins PMP:GFP and H+-ATPase:GFP was inhibited in a dose-dependent manner, while the trafficking of the Golgi-localized proteins ST:GFP and KAM1(ΔC):mRFP was not affected. Conversely, in RNA interference plants displaying lower levels of AtPRA1.B6 transcripts, the trafficking efficiency of sporamin:GFP and AALP:GFP to the vacuole was increased. Localization and N-glycan pattern analyses of cargo proteins revealed that AtPRA1.B6-mediated inhibition of anterograde trafficking occurs at the ER. In addition, AtPRA1.B6 levels were controlled by cellular processes, including 26S proteasome-mediated proteolysis. Based on these results, we propose that AtPRA1.B6 is a negative regulator of coat protein complex II vesicle-mediated anterograde trafficking for a subset of proteins at the ER. PMID:21828250

Characterisation of Antigen B Protein Species Present in the Hydatid Cyst Fluid of Echinococcus canadensis G7 Genotype

PubMed Central

Folle, Ana Maite; Kitano, Eduardo S.; Lima, Analía; Gil, Magdalena; Cucher, Marcela; Mourglia-Ettlin, Gustavo; Iwai, Leo K.; Rosenzvit, Mara; Batthyány, Carlos

2017-01-01

The larva of cestodes belonging to the Echinococcus granulosus sensu lato (s.l.) complex causes cystic echinococcosis (CE). It is a globally distributed zoonosis with significant economic and public health impact. The most immunogenic and specific Echinococcus-genus antigen for human CE diagnosis is antigen B (AgB), an abundant lipoprotein of the hydatid cyst fluid (HF). The AgB protein moiety (apolipoprotein) is encoded by five genes (AgB1-AgB5), which generate mature 8 kDa proteins (AgB8/1-AgB8/5). These genes seem to be differentially expressed among Echinococcus species. Since AgB immunogenicity lies on its protein moiety, differences in AgB expression within E. granulosus s.l. complex might have diagnostic and epidemiological relevance for discriminating the contribution of distinct species to human CE. Interestingly, AgB2 was proposed as a pseudogene in E. canadensis, which is the second most common cause of human CE, but proteomic studies for verifying it have not been performed yet. Herein, we analysed the protein and lipid composition of AgB obtained from fertile HF of swine origin (E. canadensis G7 genotype). AgB apolipoproteins were identified and quantified using mass spectrometry tools. Results showed that AgB8/1 was the major protein component, representing 71% of total AgB apolipoproteins, followed by AgB8/4 (15.5%), AgB8/3 (13.2%) and AgB8/5 (0.3%). AgB8/2 was not detected. As a methodological control, a parallel analysis detected all AgB apolipoproteins in bovine fertile HF (G1/3/5 genotypes). Overall, E. canadensis AgB comprised mostly AgB8/1 together with a heterogeneous mixture of lipids, and AgB8/2 was not detected despite using high sensitivity proteomic techniques. This endorses genomic data supporting that AgB2 behaves as a pseudogene in G7 genotype. Since recombinant AgB8/2 has been found to be diagnostically valuable for human CE, our findings indicate that its use as antigen in immunoassays could contribute to false negative results in
Characterisation of Antigen B Protein Species Present in the Hydatid Cyst Fluid of Echinococcus canadensis G7 Genotype.

PubMed

Folle, Ana Maite; Kitano, Eduardo S; Lima, Analía; Gil, Magdalena; Cucher, Marcela; Mourglia-Ettlin, Gustavo; Iwai, Leo K; Rosenzvit, Mara; Batthyány, Carlos; Ferreira, Ana María

2017-01-01

The larva of cestodes belonging to the Echinococcus granulosus sensu lato (s.l.) complex causes cystic echinococcosis (CE). It is a globally distributed zoonosis with significant economic and public health impact. The most immunogenic and specific Echinococcus-genus antigen for human CE diagnosis is antigen B (AgB), an abundant lipoprotein of the hydatid cyst fluid (HF). The AgB protein moiety (apolipoprotein) is encoded by five genes (AgB1-AgB5), which generate mature 8 kDa proteins (AgB8/1-AgB8/5). These genes seem to be differentially expressed among Echinococcus species. Since AgB immunogenicity lies on its protein moiety, differences in AgB expression within E. granulosus s.l. complex might have diagnostic and epidemiological relevance for discriminating the contribution of distinct species to human CE. Interestingly, AgB2 was proposed as a pseudogene in E. canadensis, which is the second most common cause of human CE, but proteomic studies for verifying it have not been performed yet. Herein, we analysed the protein and lipid composition of AgB obtained from fertile HF of swine origin (E. canadensis G7 genotype). AgB apolipoproteins were identified and quantified using mass spectrometry tools. Results showed that AgB8/1 was the major protein component, representing 71% of total AgB apolipoproteins, followed by AgB8/4 (15.5%), AgB8/3 (13.2%) and AgB8/5 (0.3%). AgB8/2 was not detected. As a methodological control, a parallel analysis detected all AgB apolipoproteins in bovine fertile HF (G1/3/5 genotypes). Overall, E. canadensis AgB comprised mostly AgB8/1 together with a heterogeneous mixture of lipids, and AgB8/2 was not detected despite using high sensitivity proteomic techniques. This endorses genomic data supporting that AgB2 behaves as a pseudogene in G7 genotype. Since recombinant AgB8/2 has been found to be diagnostically valuable for human CE, our findings indicate that its use as antigen in immunoassays could contribute to false negative results in
Purification and characterization of Escherichia coli MreB protein.

PubMed

Nurse, Pearl; Marians, Kenneth J

2013-02-01

The actin homolog MreB is required in rod-shaped bacteria for maintenance of cell shape and is intimately connected to the holoenzyme that synthesizes the peptidoglycan layer. The protein has been reported variously to exist in helical loops under the cell surface, to rotate, and to move in patches in both directions around the cell surface. Studies of the Escherichia coli protein in vitro have been hampered by its tendency to aggregate. Here we report the purification and characterization of native E. coli MreB. The protein requires ATP hydrolysis for polymerization, forms bundles with a left-hand twist that can be as long as 4 μm, forms sheets in the presence of calcium, and has a critical concentration for polymerization of 1.5 μM.
Purification and Characterization of Escherichia coli MreB Protein*

PubMed Central

Nurse, Pearl; Marians, Kenneth J.

2013-01-01

The actin homolog MreB is required in rod-shaped bacteria for maintenance of cell shape and is intimately connected to the holoenzyme that synthesizes the peptidoglycan layer. The protein has been reported variously to exist in helical loops under the cell surface, to rotate, and to move in patches in both directions around the cell surface. Studies of the Escherichia coli protein in vitro have been hampered by its tendency to aggregate. Here we report the purification and characterization of native E. coli MreB. The protein requires ATP hydrolysis for polymerization, forms bundles with a left-hand twist that can be as long as 4 μm, forms sheets in the presence of calcium, and has a critical concentration for polymerization of 1.5 μm. PMID:23235161
Coevolution of the ATPase ClpV, the Sheath Proteins TssB and TssC, and the Accessory Protein TagJ/HsiE1 Distinguishes Type VI Secretion Classes*

PubMed Central

Förster, Andreas; Planamente, Sara; Manoli, Eleni; Lossi, Nadine S.; Freemont, Paul S.; Filloux, Alain

2014-01-01

The type VI secretion system (T6SS) is a bacterial nanomachine for the transport of effector molecules into prokaryotic and eukaryotic cells. It involves the assembly of a tubular structure composed of TssB and TssC that is similar to the tail sheath of bacteriophages. The sheath contracts to provide the energy needed for effector delivery. The AAA+ ATPase ClpV disassembles the contracted sheath, which resets the systems for reassembly of an extended sheath that is ready to fire again. This mechanism is crucial for T6SS function. In Vibrio cholerae, ClpV binds the N terminus of TssC within a hydrophobic groove. In this study, we resolved the crystal structure of the N-terminal domain of Pseudomonas aeruginosa ClpV1 and observed structural alterations in the hydrophobic groove. The modification in the ClpV1 groove is matched by a change in the N terminus of TssC, suggesting the existence of distinct T6SS classes. An accessory T6SS component, TagJ/HsiE, exists predominantly in one of the classes. Using bacterial two-hybrid approaches, we showed that the P. aeruginosa homolog HsiE1 interacts strongly with ClpV1. We then resolved the crystal structure of HsiE1 in complex with the N terminus of HsiB1, a TssB homolog and component of the contractile sheath. Phylogenetic analysis confirmed that these differences distinguish T6SS classes that resulted from a functional co-evolution between TssB, TssC, TagJ/HsiE, and ClpV. The interaction of TagJ/HsiE with the sheath as well as with ClpV suggests an alternative mode of disassembly in which HsiE recruits the ATPase to the sheath. PMID:25305017
Search for the decays B_{(s)};{0} --> e;{+} micro;{-} and B_{(s)};{0} --> e;{+} e;{-} in CDF run II.

PubMed

Aaltonen, T; Adelman, J; Akimoto, T; Alvarez González, B; Amerio, S; Amidei, D; Anastassov, A; Annovi, A; Antos, J; Apollinari, G; Apresyan, A; Arisawa, T; Artikov, A; Ashmanskas, W; Attal, A; Aurisano, A; Azfar, F; Azzurri, P; Badgett, W; Barbaro-Galtieri, A; Barnes, V E; Barnett, B A; Bartsch, V; Bauer, G; Beauchemin, P-H; Bedeschi, F; Beecher, D; Behari, S; Bellettini, G; Bellinger, J; Benjamin, D; Beretvas, A; Beringer, J; Bhatti, A; Binkley, M; Bisello, D; Bizjak, I; Blair, R E; Blocker, C; Blumenfeld, B; Bocci, A; Bodek, A; Boisvert, V; Bolla, G; Bortoletto, D; Boudreau, J; Boveia, A; Brau, B; Bridgeman, A; Brigliadori, L; Bromberg, C; Brubaker, E; Budagov, J; Budd, H S; Budd, S; Burke, S; Burkett, K; Busetto, G; Bussey, P; Buzatu, A; Byrum, K L; Cabrera, S; Calancha, C; Campanelli, M; Campbell, M; Canelli, F; Canepa, A; Carls, B; Carlsmith, D; Carosi, R; Carrillo, S; Carron, S; Casal, B; Casarsa, M; Castro, A; Catastini, P; Cauz, D; Cavaliere, V; Cavalli-Sforza, M; Cerri, A; Cerrito, L; Chang, S H; Chen, Y C; Chertok, M; Chiarelli, G; Chlachidze, G; Chlebana, F; Cho, K; Chokheli, D; Chou, J P; Choudalakis, G; Chuang, S H; Chung, K; Chung, W H; Chung, Y S; Chwalek, T; Ciobanu, C I; Ciocci, M A; Clark, A; Clark, D; Compostella, G; Convery, M E; Conway, J; Cordelli, M; Cortiana, G; Cox, C A; Cox, D J; Crescioli, F; Cuenca Almenar, C; Cuevas, J; Culbertson, R; Cully, J C; Dagenhart, D; Datta, M; Davies, T; de Barbaro, P; De Cecco, S; Deisher, A; De Lorenzo, G; Dell'orso, M; Deluca, C; Demortier, L; Deng, J; Deninno, M; Derwent, P F; di Giovanni, G P; Dionisi, C; Di Ruzza, B; Dittmann, J R; D'Onofrio, M; Donati, S; Dong, P; Donini, J; Dorigo, T; Dube, S; Efron, J; Elagin, A; Erbacher, R; Errede, D; Errede, S; Eusebi, R; Fang, H C; Farrington, S; Fedorko, W T; Feild, R G; Feindt, M; Fernandez, J P; Ferrazza, C; Field, R; Flanagan, G; Forrest, R; Frank, M J; Franklin, M; Freeman, J C; Furic, I; Gallinaro, M; Galyardt, J; Garberson, F; Garcia, J E; Garfinkel, A F; Genser, K; Gerberich, H; Gerdes, D; Gessler, A; Giagu, S; Giakoumopoulou, V; Giannetti, P; Gibson, K; Gimmell, J L; Ginsburg, C M; Giokaris, N; Giordani, M; Giromini, P; Giunta, M; Giurgiu, G; Glagolev, V; Glenzinski, D; Gold, M; Goldschmidt, N; Golossanov, A; Gomez, G; Gomez-Ceballos, G; Goncharov, M; González, O; Gorelov, I; Goshaw, A T; Goulianos, K; Gresele, A; Grinstein, S; Grosso-Pilcher, C; Grundler, U; Guimaraes da Costa, J; Gunay-Unalan, Z; Haber, C; Hahn, K; Hahn, S R; Halkiadakis, E; Han, B-Y; Han, J Y; Happacher, F; Hara, K; Hare, D; Hare, M; Harper, S; Harr, R F; Harris, R M; Hartz, M; Hatakeyama, K; Hays, C; Heck, M; Heijboer, A; Heinrich, J; Henderson, C; Herndon, M; Heuser, J; Hewamanage, S; Hidas, D; Hill, C S; Hirschbuehl, D; Hocker, A; Hou, S; Houlden, M; Hsu, S-C; Huffman, B T; Hughes, R E; Husemann, U; Hussein, M; Huston, J; Incandela, J; Introzzi, G; Iori, M; Ivanov, A; James, E; Jang, D; Jayatilaka, B; Jeon, E J; Jha, M K; Jindariani, S; Johnson, W; Jones, M; Joo, K K; Jun, S Y; Jung, J E; Junk, T R; Kamon, T; Kar, D; Karchin, P E; Kato, Y; Kephart, R; Keung, J; Khotilovich, V; Kilminster, B; Kim, D H; Kim, H S; Kim, H W; Kim, J E; Kim, M J; Kim, S B; Kim, S H; Kim, Y K; Kimura, N; Kirsch, L; Klimenko, S; Knuteson, B; Ko, B R; Kondo, K; Kong, D J; Konigsberg, J; Korytov, A; Kotwal, A V; Kreps, M; Kroll, J; Krop, D; Krumnack, N; Kruse, M; Krutelyov, V; Kubo, T; Kuhr, T; Kulkarni, N P; Kurata, M; Kwang, S; Laasanen, A T; Lami, S; Lammel, S; Lancaster, M; Lander, R L; Lannon, K; Lath, A; Latino, G; Lazzizzera, I; Lecompte, T; Lee, E; Lee, H S; Lee, S W; Leone, S; Lewis, J D; Lin, C-S; Linacre, J; Lindgren, M; Lipeles, E; Lister, A; Litvintsev, D O; Liu, C; Liu, T; Lockyer, N S; Loginov, A; Loreti, M; Lovas, L; Lucchesi, D; Luci, C; Lueck, J; Lujan, P; Lukens, P; Lungu, G; Lyons, L; Lys, J; Lysak, R; Macqueen, D; Madrak, R; Maeshima, K; Makhoul, K; Maki, T; Maksimovic, P; Malde, S; Malik, S; Manca, G; Manousakis-Katsikakis, A; Margaroli, F; Marino, C; Marino, C P; Martin, A; Martin, V; Martínez, M; Martínez-Ballarín, R; Maruyama, T; Mastrandrea, P; Masubuchi, T; Mathis, M; Mattson, M E; Mazzanti, P; McFarland, K S; McIntyre, P; McNulty, R; Mehta, A; Mehtala, P; Menzione, A; Merkel, P; Mesropian, C; Miao, T; Miladinovic, N; Miller, R; Mills, C; Milnik, M; Mitra, A; Mitselmakher, G; Miyake, H; Moggi, N; Moon, C S; Moore, R; Morello, M J; Morlock, J; Movilla Fernandez, P; Mülmenstädt, J; Mukherjee, A; Muller, Th; Mumford, R; Murat, P; Mussini, M; Nachtman, J; Nagai, Y; Nagano, A; Naganoma, J; Nakamura, K; Nakano, I; Napier, A; Necula, V; Nett, J; Neu, C; Neubauer, M S; Neubauer, S; Nielsen, J; Nodulman, L; Norman, M; Norniella, O; Nurse, E; Oakes, L; Oh, S H; Oh, Y D; Oksuzian, I; Okusawa, T; Orava, R; Osterberg, K; Griso, S Pagan; Palencia, E; Papadimitriou, V; Papaikonomou, A; Paramonov, A A; Parks, B; Pashapour, S; Patrick, J; Pauletta, G; Paulini, M; Paus, C; Peiffer, T; Pellett, D E; Penzo, A; Phillips, T J; Piacentino, G; Pianori, E; Pinera, L; Pitts, K; Plager, C; Pondrom, L; Poukhov, O; Pounder, N; Prakoshyn, F; Pronko, A; Proudfoot, J; Ptohos, F; Pueschel, E; Punzi, G; Pursley, J; Rademacker, J; Rahaman, A; Ramakrishnan, V; Ranjan, N; Redondo, I; Renton, P; Renz, M; Rescigno, M; Richter, S; Rimondi, F; Ristori, L; Robson, A; Rodrigo, T; Rodriguez, T; Rogers, E; Rolli, S; Roser, R; Rossi, M; Rossin, R; Roy, P; Ruiz, A; Russ, J; Rusu, V; Rutherford, B; Saarikko, H; Safonov, A; Sakumoto, W K; Saltó, O; Santi, L; Sarkar, S; Sartori, L; Sato, K; Savoy-Navarro, A; Schlabach, P; Schmidt, A; Schmidt, E E; Schmidt, M A; Schmidt, M P; Schmitt, M; Schwarz, T; Scodellaro, L; Scribano, A; Scuri, F; Sedov, A; Seidel, S; Seiya, Y; Semenov, A; Sexton-Kennedy, L; Sforza, F; Sfyrla, A; Shalhout, S Z; Shears, T; Shepard, P F; Shimojima, M; Shiraishi, S; Shochet, M; Shon, Y; Shreyber, I; Sidoti, A; Sinervo, P; Sisakyan, A; Slaughter, A J; Slaunwhite, J; Sliwa, K; Smith, J R; Snider, F D; Snihur, R; Soha, A; Somalwar, S; Sorin, V; Spalding, J; Spreitzer, T; Squillacioti, P; Stanitzki, M; St Denis, R; Stelzer, B; Stelzer-Chilton, O; Stentz, D; Strologas, J; Strycker, G L; Stuart, D; Suh, J S; Sukhanov, A; Suslov, I; Suzuki, T; Taffard, A; Takashima, R; Takeuchi, Y; Tanaka, R; Tecchio, M; Teng, P K; Terashi, K; Thom, J; Thompson, A S; Thompson, G A; Thomson, E; Tipton, P; Ttito-Guzmán, P; Tkaczyk, S; Toback, D; Tokar, S; Tollefson, K; Tomura, T; Tonelli, D; Torre, S; Torretta, D; Totaro, P; Tourneur, S; Trovato, M; Tsai, S-Y; Tu, Y; Turini, N; Ukegawa, F; Vallecorsa, S; van Remortel, N; Varganov, A; Vataga, E; Vázquez, F; Velev, G; Vellidis, C; Vidal, M; Vidal, R; Vila, I; Vilar, R; Vine, T; Vogel, M; Volobouev, I; Volpi, G; Wagner, P; Wagner, R G; Wagner, R L; Wagner, W; Wagner-Kuhr, J; Wakisaka, T; Wallny, R; Wang, S M; Warburton, A; Waters, D; Weinberger, M; Weinelt, J; Wenzel, H; Wester, W C; Whitehouse, B; Whiteson, D; Wicklund, A B; Wicklund, E; Wilbur, S; Williams, G; Williams, H H; Wilson, P; Winer, B L; Wittich, P; Wolbers, S; Wolfe, C; Wright, T; Wu, X; Würthwein, F; Xie, S; Yagil, A; Yamamoto, K; Yamaoka, J; Yang, U K; Yang, Y C; Yao, W M; Yeh, G P; Yoh, J; Yorita, K; Yoshida, T; Yu, G B; Yu, I; Yu, S S; Yun, J C; Zanello, L; Zanetti, A; Zhang, X; Zheng, Y; Zucchelli, S

2009-05-22

We report results from a search for the lepton flavor violating decays B_{s};{0} --> e;{+} micro;{-} and B;{0} --> e;{+} micro;{-}, and the flavor-changing neutral-current decays B_{s};{0} --> e;{+} e;{-} and B;{0} --> e;{+} e;{-}. The analysis uses data corresponding to 2 fb;{-1} of integrated luminosity of pp[over ] collisions at sqrt[s] = 1.96 TeV collected with the upgraded Collider Detector (CDF II) at the Fermilab Tevatron. The observed number of B0 and B_{s};{0} candidates is consistent with background expectations. The resulting Bayesian upper limits on the branching ratios at 90% credibility level are B(B_{s};{0} --> e;{+} micro;{-}) < 2.0 x 10;{-7}, B(B;{0} --> e;{+} micro;{-}) < 6.4 x 10;{-8}, B(B_{s};{0} --> e;{+} e;{-}) < 2.8 x 10;{-7}, and B(B;{0} --> e;{+} e;{-}) < 8.3 x 10;{-8}. From the limits on B(B_{(s)};{0} --> e;{+} micro;{-}), the following lower bounds on the Pati-Salam leptoquark masses are also derived: M_{LQ}(B_{s};{0} --> e;{+} micro;{-}) > 47.8 TeV/c;{2}, and M_{LQ}(B;{0} --> e;{+} micro;{-}) > 59.3 TeV / c;{2}, at 90% credibility level.
Identification of helper T cell epitopes of dengue virus E-protein.

PubMed

Leclerc, C; Dériaud, E; Megret, F; Briand, J P; Van Regenmortel, M H; Deubel, V

1993-05-01

The T cell proliferative response to dengue 2 (Jamaica) E-glycoprotein (495 amino acids) was analyzed in vitro using either killed virus or E-protein fragments or synthetic peptides. Inactivated dengue virus stimulated dengue-specific lymph node (LN) CD4+T cell proliferation in BALB/c (H-2d), C3H (H-2k) and DBA/1 (H-2q) but not in C57BL/6 (H-2b) mice. Moreover, LN cells from dengue-virus primed BALB/c mice proliferated in vitro in response to three purified non-overlapping E-protein fragments expressed in E. coli as polypeptides fused to trpE (f22-205, f267-354, f366-424). To further determine T cell epitopes in the E-protein, synthetic peptides were selected using prediction algorithms for T cell epitopes. Highest proliferative responses were obtained after in vitro exposure of virus-primed LN cells to peptides p135-157, p270-298, p295-307 and p337-359. Peptide p59-78 was able to induce specific B and T cell responses in peptide-primed mice of H-2d, H-2q and H-2k haplotypes. Two peptides p59-78 corresponding to two dengue (Jamaica and Sri Lanka) isolates and differing only at position 71 cross-reacted at the B but not at the T cell level in H-2b mice. This analysis of murine T helper cell response to dengue E-protein may be of use in dengue subunit vaccine design.
THE E1 PROTEINS

PubMed Central

Bergvall, Monika; Melendy, Thomas; Archambault, Jacques

2013-01-01

E1, an ATP-dependent DNA helicase, is the only enzyme encoded by papillomaviruses (PVs). It is essential for replication and amplification of the viral episome in the nucleus of infected cells. To do so, E1 assembles into a double-hexamer at the viral origin, unwinds DNA at the origin and ahead of the replication fork and interacts with cellular DNA replication factors. Biochemical and structural studies have revealed the assembly pathway of E1 at the origin and how the enzyme unwinds DNA using a spiral escalator mechanism. E1 is tightly regulated in vivo, in particular by post-translational modifications that restrict its accumulation in the nucleus. Here we review how different functional domains of E1 orchestrate viral DNA replication, with an emphasis on their interactions with substrate DNA, host DNA replication factors and modifying enzymes. These studies have made E1 one of the best characterized helicases and provided unique insights on how PVs usurp different host-cell machineries to replicate and amplify their genome in a tightly controlled manner. PMID:24029589
Functional characterisation of a TLR accessory protein, UNC93B1, in Atlantic salmon (Salmo salar).

PubMed

Lee, P T; Zou, J; Holland, J W; Martin, S A M; Scott, C J W; Kanellos, T; Secombes, C J

2015-05-01

Toll-like receptors (TLRs) are indispensable components of the innate immune system, which recognise conserved pathogen associated molecular patterns (PAMPs) and induce a series of defensive immune responses to protect the host. Biosynthesis, localisation and activation of TLRs are dependent on TLR accessory proteins. In this study, we identified the accessory protein, UNC93B1, from Atlantic salmon (Salmo salar) whole-genome shotgun (WGS) contigs aided by the conserved gene synteny of genes flanking UNC93B1 in fish, birds and mammals. Phylogenetic analysis showed that salmon UNC93B1 grouped with other vertebrate UNC93B1 molecules, and had highest amino acid identity and similarity to zebrafish UNC93B1. The salmon UNC93B1 gene organisation was also similar in structure to mammalian UNC93B1. Our gene expression studies revealed that salmon UNC93B1 was more highly expressed in spleen, liver and gill tissues but was expressed at a lower level in head kidney tissue in post-smolts relative to parr. Moreover, salmon UNC93B1 mRNA transcripts were up-regulated in vivo in spleen tissue from polyI:C treated salmon and in vitro in polyI:C or IFNγ stimulated Salmon Head Kidney-1 (SHK-1) cells. Initial studies into the functional role of salmon UNC93B1 in fish TLR signalling found that both wild type salmon UNC93B1 and a molecule with a site-directed mutation (H424R) co-immunoprecipitated with salmon TLR19, TLR20a and TLR20d. Overall, these data illustrate the potential importance of UNC93B1 as an accessory protein in fish TLR signalling. Copyright © 2014 Elsevier Ltd. All rights reserved.
Ubiquitination and sumoylation of the HTLV-2 Tax-2B protein regulate its NF-κB activity: a comparative study with the HTLV-1 Tax-1 protein

PubMed Central

2012-01-01

Background Retroviruses HTLV-1 and HTLV-2 have homologous genomic structures but differ significantly in pathogenicity. HTLV-1 is associated with Adult T cell Leukemia (ATL), whereas infection by HTLV-2 has no association with neoplasia. Transformation of T lymphocytes by HTLV-1 is linked to the capacity of its oncoprotein Tax-1 to alter cell survival and cell cycle control mechanisms. Among these functions, Tax-1-mediated activation of cellular gene expression via the NF-κB pathway depends on Tax-1 post-translational modifications by ubiquitination and sumoylation. The Tax-2 protein of HTLV-2B (Tax-2B) is also modified by ubiquitination and sumoylation and activates the NF-κB pathway to a level similar to that of Tax-1. The present study aims to understand whether ubiquitination and sumoylation modifications are involved in Tax-2B-mediated activation of the NF-κB pathway. Results The comparison of Tax-1 and Tax-2B lysine to arginine substitution mutants revealed conserved patterns and levels of ubiquitination with notable difference in the lysine usage for sumoylation. Neither Tax-1 nor Tax-2B ubiquitination and sumoylation deficient mutants could activate the NF-κB pathway and fusion of ubiquitin or SUMO-1 to the C-terminus of the ubiquitination and sumoylation deficient Tax-2B mutant strikingly restored transcriptional activity. In addition, ubiquitinated forms of Tax-2B colocalized with RelA and IKKγ in prominent cytoplasmic structures associated with the Golgi apparatus, whereas colocalization of Tax-2B with the RelA subunit of NF-κB and the transcriptional coactivator p300 in punctate nuclear structures was dependent on Tax-2B sumoylation, as previously observed for Tax-1. Conclusions Both Tax-1 and Tax-2 activate the NF-κB pathway via similar mechanisms involving ubiquitination and sumoylation. Therefore, the different transforming potential of HTLV-1 and HTLV-2 is unlikely to be related to different modes of activation of the canonical NF-κB pathway
The 5.5 protein of phage T7 inhibits H-NS through interactions with the central oligomerization domain.

PubMed

Ali, Sabrina S; Beckett, Emily; Bae, Sandy Jeehoon; Navarre, William Wiley

2011-09-01

The 5.5 protein (T7p32) of coliphage T7 (5.5(T7)) was shown to bind and inhibit gene silencing by the nucleoid-associated protein H-NS, but the mechanism by which it acts was not understood. The 5.5(T7) protein is insoluble when expressed in Escherichia coli, but we find that 5.5(T7) can be isolated in a soluble form when coexpressed with a truncated version of H-NS followed by subsequent disruption of the complex during anion-exchange chromatography. Association studies reveal that 5.5(T7) binds a region of H-NS (residues 60 to 80) recently found to contain a distinct domain necessary for higher-order H-NS oligomerization. Accordingly, we find that purified 5.5(T7) can disrupt higher-order H-NS-DNA complexes in vitro but does not abolish DNA binding by H-NS per se. Homologues of the 5.5(T7) protein are found exclusively among members of the Autographivirinae that infect enteric bacteria, and despite fairly low sequence conservation, the H-NS binding properties of these proteins are largely conserved. Unexpectedly, we find that the 5.5(T7) protein copurifies with heterogeneous low-molecular-weight RNA, likely tRNA, through several chromatography steps and that this interaction does not require the DNA binding domain of H-NS. The 5.5 proteins utilize a previously undescribed mechanism of H-NS antagonism that further highlights the critical importance that higher-order oligomerization plays in H-NS-mediated gene repression. Copyright © 2011, American Society for Microbiology. All Rights Reserved.
Iron regulatory protein 1 is not required for the modulation of ferritin and transferrin receptor expression by iron in a murine pro-B lymphocyte cell line

PubMed Central

Schalinske, Kevin L.; Blemings, Kenneth P.; Steffen, Daniel W.; Chen, Opal S.; Eisenstein, Richard S.

1997-01-01

Iron regulatory proteins (IRPs) are cytoplasmic RNA binding proteins that are central components of a sensory and regulatory network that modulates vertebrate iron homeostasis. IRPs regulate iron metabolism by binding to iron responsive element(s) (IREs) in the 5′ or 3′ untranslated region of ferritin or transferrin receptor (TfR) mRNAs. Two IRPs, IRP1 and IRP2, have been identified previously. IRP1 exhibits two mutually exclusive functions as an RNA binding protein or as the cytosolic isoform of aconitase. We demonstrate that the Ba/F3 family of murine pro-B lymphocytes represents the first example of a mammalian cell line that fails to express IRP1 protein or mRNA. First, all of the IRE binding activity in Ba/F3-gp55 cells is attributable to IRP2. Second, synthesis of IRP2, but not of IRP1, is detectable in Ba/F3-gp55 cells. Third, the Ba/F3 family of cells express IRP2 mRNA at a level similar to other murine cell lines, but IRP1 mRNA is not detectable. In the Ba/F3 family of cells, alterations in iron status modulated ferritin biosynthesis and TfR mRNA level over as much as a 20- and 14-fold range, respectively. We conclude that IRP1 is not essential for regulation of ferritin or TfR expression by iron and that IRP2 can act as the sole IRE-dependent mediator of cellular iron homeostasis. PMID:9380695
A Case for an Atmosphere on Super-Earth 55 Cancri e

NASA Astrophysics Data System (ADS)

Angelo, Isabel; Hu, Renyu

2017-12-01

One of the primary questions when characterizing Earth-sized and super-Earth-sized exoplanets is whether they have a substantial atmosphere like Earth and Venus or a bare-rock surface like Mercury. Phase curves of the planets in thermal emission provide clues to this question, because a substantial atmosphere would transport heat more efficiently than a bare-rock surface. Analyzing phase-curve photometric data around secondary eclipses has previously been used to study energy transport in the atmospheres of hot Jupiters. Here we use phase curve, Spitzer time-series photometry to study the thermal emission properties of the super-Earth exoplanet 55 Cancri e. We utilize a semianalytical framework to fit a physical model to the infrared photometric data at 4.5 μm. The model uses parameters of planetary properties including Bond albedo, heat redistribution efficiency (I.e., ratio between radiative timescale and advective timescale of the atmosphere), and the atmospheric greenhouse factor. The phase curve of 55 Cancri e is dominated by thermal emission with an eastward-shifted hotspot. We determine the heat redistribution efficiency to be {1.47}-0.25+0.30, which implies that the advective timescale is on the same order as the radiative timescale. This requirement cannot be met by the bare-rock planet scenario because heat transport by currents of molten lava would be too slow. The phase curve thus favors the scenario with a substantial atmosphere. Our constraints on the heat redistribution efficiency translate to an atmospheric pressure of ˜1.4 bar. The Spitzer 4.5 μm band is thus a window into the deep atmosphere of the planet 55 Cancri e.
Protein Tyrosine Phosphatase 1B Regulates Pyruvate Kinase M2 Tyrosine Phosphorylation*

PubMed Central

Bettaieb, Ahmed; Bakke, Jesse; Nagata, Naoto; Matsuo, Kosuke; Xi, Yannan; Liu, Siming; AbouBechara, Daniel; Melhem, Ramzi; Stanhope, Kimber; Cummings, Bethany; Graham, James; Bremer, Andrew; Zhang, Sheng; Lyssiotis, Costas A.; Zhang, Zhong-Yin; Cantley, Lewis C.; Havel, Peter J.; Haj, Fawaz G.

2013-01-01

Protein-tyrosine phosphatase 1B (PTP1B) is a physiological regulator of glucose homeostasis and adiposity and is a drug target for the treatment of obesity and diabetes. Here we identify pyruvate kinase M2 (PKM2) as a novel PTP1B substrate in adipocytes. PTP1B deficiency leads to increased PKM2 total tyrosine and Tyr105 phosphorylation in cultured adipocytes and in vivo. Substrate trapping and mutagenesis studies identify PKM2 Tyr-105 and Tyr-148 as key sites that mediate PTP1B-PKM2 interaction. In addition, in vitro analyses illustrate a direct effect of Tyr-105 phosphorylation on PKM2 activity in adipocytes. Importantly, PTP1B pharmacological inhibition increased PKM2 Tyr-105 phosphorylation and decreased PKM2 activity. Moreover, PKM2 Tyr-105 phosphorylation is regulated nutritionally, decreasing in adipose tissue depots after high-fat feeding. Further, decreased PKM2 Tyr-105 phosphorylation correlates with the development of glucose intolerance and insulin resistance in rodents, non-human primates, and humans. Together, these findings identify PKM2 as a novel substrate of PTP1B and provide new insights into the regulation of adipose PKM2 activity. PMID:23640882
The serum and glucocorticoid-regulated protein kinases (SGK) stimulate bovine herpesvirus 1 and herpes simplex virus 1 productive infection.

PubMed

Kook, Insun; Jones, Clinton

2016-08-15

Serum and glucocorticoid-regulated protein kinases (SGK) are serine/threonine protein kinases that contain a catalytic domain resembling other protein kinases: AKT/protein kinase B, protein kinase A, and protein kinase C-Zeta for example. Unlike these constitutively expressed protein kinases, SGK1 RNA and protein levels are increased by growth factors and corticosteroids. Stress can directly stimulate SGK1 levels as well as stimulate bovine herpesvirus 1 (BoHV-1) and herpes simplex virus 1 (HSV-1) productive infection and reactivation from latency suggesting SGK1 can stimulate productive infection. For the first time, we provide evidence that a specific SGK inhibitor (GSK650394) significantly reduced BoHV-1 and HSV-1 replication in cultured cells. Proteins encoded by the three BoHV-1 immediate early genes (bICP0, bICP4, and bICP22) and two late proteins (VP16 and gE) were consistently reduced by GSK650394 during early stages of productive infection. In summary, these studies suggest SGK may stimulate viral replication following stressful stimuli. Copyright © 2016 Elsevier B.V. All rights reserved.
Antigenicity analysis of human parvovirus B19-VP1u protein in the induction of anti-phospholipid syndrome.

PubMed

Lin, Chun-Yu; Chiu, Chun-Ching; Cheng, Ju; Lin, Chia-Yun; Shi, Ya-Fang; Tsai, Chun-Chou; Tzang, Bor-Show; Hsu, Tsai-Ching

2018-01-01

Mounting evidence suggests a connection between human parvovirus B19 (B19) and autoimmune diseases, and especially an association between the B19-VP1 unique region (VP1u) and anti-phospholipid syndrome (APS). However, little is known about the antigenicity of B19-VP1u in the induction of APS-like syndrome. To elucidate the antigenicity of B19-VP1u in the induction of APS, N-terminal truncated B19-VP1u (tVP1u) proteins were prepared to immunize Balb/c mice to generate antibodies against B19-tVP1u proteins. The secreted phospholipase A2 (sPLA2) activities and binding specificity of mice anti-B19-tVP1u antibodies with cardiolipin (CL) and beta-2-glycoprotein I (β2GPI) were evaluated by performing immunoblot, ELISA and absorption experiments. A mice model of passively induced APS was adopted. Although sPLA2 activities were identified in all B19-tVP1u proteins, only amino acid residues 61-227 B19-tVP1u exhibited a higher sPLA2 activity. Autoantibodies against CL and β2GPI exhibited binding activities with all B19-tVP1u proteins. IgG that was purified from mice that had been immunized with amino acid residues 21-227 to 121-227 B19-tVP1u proteins exhibited significantly higher binding activity with CL. IgG that was purified from mice that had been immunized with amino acid residues 21-227, 31-227, 82-227 and 91-227 B19-tVP1u proteins exhibited significantly higher binding activity with β2GPI. Accordingly, significantly higher binding inhibition of CL was detected in the presence of amino acid residues 61-227 and 101-227 B19-tVP1u. Significantly higher binding inhibition of β2GPI was detected in the presence of amino acid residues 21-227, 31-227, 82-227 and 91-227 B19-tVP1u. The mice that received amino acid residues 31-227 or 61-227 anti-tB19-VP1u IgG revealed significant thrombocytopenia and those that received amino acid residues 21-227, 31-227, 61-227, 71-227, 82-227, 91-227, 101-227 or 114-227 anti-tB19-VP1u IgG exhibited significantly prolonged aPTT. These
Poliovirus replication proteins: RNA sequence encoding P3-1b and the sites of proteolytic processing

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

Semler, B.L.; Anderson, C.W.; Kitamura, N.

1981-06-01

A partial amino-terminal amino acid sequence of each of the major proteins encoded by the replicase region of the poliovirus genome has been determined. A comparison of this sequence information with the amino acid sequence predicted from the RNA sequence that has been determined for the 3' region of the poliovirus genome has allowed us to locate precisely the proteolytic cleavage sites at which the initial polyprotein is processed to create the poliovirus products P3-1b (NCVP1b), P3-2 (NCVP2), P3-4b (NCVP4b), and P3-7c (NCVP7c). For each of these products, as well as for the small genome-linked protein VPg, proteolytic cleavage occursmore » between a glutamine and a glycine residue to create the amino terminus of each protein. This result suggests that a single proteinase may be responsible for all of these cleavages. The sequence data also allow the precise positioning of the genome-linked protein VPg within the precursor P3-1b just proximal to the amino terminus of polypeptide P3-2.« less
Mycobacterium tuberculosis WhiB1 is an essential DNA-binding protein with a nitric oxide sensitive iron-sulphur cluster

PubMed Central

Smith, Laura J.; Stapleton, Melanie R.; Fullstone, Gavin J. M.; Crack, Jason C.; Thomson, Andrew J.; Le Brun, Nick E.; Hunt, Debbie M.; Harvey, Evelyn; Adinolfi, Salvatore; Buxton, Roger S.; Green, Jeffrey

2010-01-01

Mycobacterium tuberculosis is a major pathogen that has the ability to establish, and emerge from, a persistent state. Wbl family proteins are associated with developmental processes in actinomycetes, and M. tuberculosis has seven such proteins. Here it is shown that the M. tuberculosis H37Rv whiB1 gene is essential. The WhiB1 protein possesses a [4Fe-4S]2+ cluster that is stable in air but reacts rapidly with eight equivalents of nitric oxide to yield two dinuclear dinitrosyl-iron thiol complexes. The [4Fe-4S] form of WhiB1 did not bind whiB1 promoter DNA, but the reduced and oxidized apo-WhiB1, and nitric oxide-treated holo-WhiB1 did bind to DNA. Mycobacterium smegmatis RNA polymerase induced transcription of whiB1 in vitro; however in the presence of apo-WhiB1 transcription was severely inhibited, irrespective of the presence or absence of the CRP protein Rv3676, which is known to activate whiB1 expression. Footprinting suggested that autorepression of whiB1 is achieved by apo-WhiB1 binding at a region that overlaps the core promoter elements. A model incorporating regulation of whiB1 expression in response to nitric oxide and cAMP is discussed with implications for sensing two important signals in establishing M. tuberculosis infections. PMID:20929442
Biophysical Analysis of Anopheles gambiae Leucine-Rich Repeat Proteins APL1A1, APL1B and APL1C and Their Interaction with LRIM1

DOE PAGES

Williams, Marni; Summers, Brady J.; Baxter, Richard H. G.; ...

2015-03-16

Natural infection of Anopheles gambiae by malaria-causing Plasmodium parasites is significantly influenced by the APL1 genetic locus. The locus contains three closely related leucine-rich repeat (LRR) genes, APL1A, APL1B and APL1C. Multiple studies have reported the participation of APL1A—C in the immune response of A. gambiae to invasion by both rodent and human Plasmodium isolates. APL1C forms a heterodimer with the related LRR protein LRIM1 via a C-terminal coiled-coil domain that is also present in APL1A and APL1B. The LRIM1/APL1C heterodimer protects A. gambiae from infection by binding the complement-like protein TEP1 to form a stable and active immune complex.more » We report solution x-ray scatting data for the LRIM1/APL1C heterodimer, the oligomeric state of LRIM1/APL1 LRR domains in solution and the crystal structure of the APL1B LRR domain. The LRIM1/APL1C heterodimeric complex has a flexible and extended structure in solution. In contrast to the APL1A, APL1C and LRIM1 LRR domains, the APL1B LRR domain is a homodimer. The crystal structure of APL1B-LRR shows that the homodimer is formed by an N-terminal helix that complements for the absence of an N-terminal capping motif in APL1B, which is a unique distinction within the LRIM1/APL1 protein family. Full-length APL1A 1 and APL1B form a stable complex with LRIM1. Our results support a model in which APL1A 1, APL1B and APL1C can all form an extended, flexible heterodimer with LRIM1, providing a repertoire of functional innate immune complexes to protect A. gambiae from a diverse array of pathogens.« less
Biophysical Analysis of Anopheles gambiae Leucine-Rich Repeat Proteins APL1A1, APL1B and APL1C and Their Interaction with LRIM1

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

Williams, Marni; Summers, Brady J.; Baxter, Richard H. G.

Natural infection of Anopheles gambiae by malaria-causing Plasmodium parasites is significantly influenced by the APL1 genetic locus. The locus contains three closely related leucine-rich repeat (LRR) genes, APL1A, APL1B and APL1C. Multiple studies have reported the participation of APL1A—C in the immune response of A. gambiae to invasion by both rodent and human Plasmodium isolates. APL1C forms a heterodimer with the related LRR protein LRIM1 via a C-terminal coiled-coil domain that is also present in APL1A and APL1B. The LRIM1/APL1C heterodimer protects A. gambiae from infection by binding the complement-like protein TEP1 to form a stable and active immune complex.more » We report solution x-ray scatting data for the LRIM1/APL1C heterodimer, the oligomeric state of LRIM1/APL1 LRR domains in solution and the crystal structure of the APL1B LRR domain. The LRIM1/APL1C heterodimeric complex has a flexible and extended structure in solution. In contrast to the APL1A, APL1C and LRIM1 LRR domains, the APL1B LRR domain is a homodimer. The crystal structure of APL1B-LRR shows that the homodimer is formed by an N-terminal helix that complements for the absence of an N-terminal capping motif in APL1B, which is a unique distinction within the LRIM1/APL1 protein family. Full-length APL1A 1 and APL1B form a stable complex with LRIM1. Our results support a model in which APL1A 1, APL1B and APL1C can all form an extended, flexible heterodimer with LRIM1, providing a repertoire of functional innate immune complexes to protect A. gambiae from a diverse array of pathogens.« less

Hepatitis B virus X promotes hepatocellular carcinoma development via nuclear protein 1 pathway.

PubMed

Bak, Yesol; Shin, Hye-jun; Bak, In seon; Yoon, Do-young; Yu, Dae-Yeul

2015-10-30

Hepatocellular carcinoma (HCC) is one of the most common malignancies and chronic hepatitis B virus (HBV) infection is a major risk factor for HCC. Hepatitis B virus X (HBx) protein relates to trigger oncogenesis. HBx has oncogenic properties with a hyperproliferative response to HCC. Nuclear protein 1 (NUPR1) is a stress-response protein, frequently upregulated in several cancers. Recent data revealed that NUPR1 is involved in tumor progression, but its function in HCC is not revealed yet. Here we report HBx can induce NUPR1 in patients, mice, and HCC cell lines. In an HBx transgenic mouse model, we found that HBx overexpression upregulates NUPR1 expression consistently with tumor progression. Further, in cultured HBV positive cells, HBx knockdown induces downregulation of NUPR1. Smad4 is a representative transcription factor, regulated by HBx, and we showed that HBx upregulates NUPR1 by Smad4 dependent way. We found that NUPR1 can inhibit cell death and induce vasculogenic mimicry in HCC cell lines. Moreover, NUPR1 silencing in HepG2-HBx showed reduced cell motility. These results suggest that HBx can modulate NUPR1 expression through the Smad4 pathway and NUPR1 has a role in hepatocellular carcinoma progression. Copyright © 2015 Elsevier Inc. All rights reserved.
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
40 CFR 205.55-1 - General requirements.

Code of Federal Regulations, 2010 CFR

2010-07-01

... 40 Protection of Environment 24 2010-07-01 2010-07-01 false General requirements. 205.55-1 Section 205.55-1 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) NOISE ABATEMENT PROGRAMS TRANSPORTATION EQUIPMENT NOISE EMISSION CONTROLS Medium and Heavy Trucks § 205.55-1 General...
DISC1, PDE4B, and NDE1 at the centrosome and synapse

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

Bradshaw, Nicholas J.; Ogawa, Fumiaki; Antolin-Fontes, Beatriz

Disrupted-In-Schizophrenia 1 (DISC1) is a risk factor for schizophrenia and other major mental illnesses. Its protein binding partners include the Nuclear Distribution Factor E Homologs (NDE1 and NDEL1), LIS1, and phosphodiesterases 4B and 4D (PDE4B and PDE4D). We demonstrate that NDE1, NDEL1 and LIS1, together with their binding partner dynein, associate with DISC1, PDE4B and PDE4D within the cell, and provide evidence that this complex is present at the centrosome. LIS1 and NDEL1 have been previously suggested to be synaptic, and we now demonstrate localisation of DISC1, NDE1, and PDE4B at synapses in cultured neurons. NDE1 is phosphorylated by cAMP-dependantmore » Protein Kinase A (PKA), whose activity is, in turn, regulated by the cAMP hydrolysis activity of phosphodiesterases, including PDE4. We propose that DISC1 acts as an assembly scaffold for all of these proteins and that the NDE1/NDEL1/LIS1/dynein complex is modulated by cAMP levels via PKA and PDE4.« less
Structure of the immature HIV-1 capsid in intact virus particles at 8.8 Å resolution

NASA Astrophysics Data System (ADS)

Schur, Florian K. M.; Hagen, Wim J. H.; Rumlová, Michaela; Ruml, Tomáš; Müller, Barbara; Kräusslich, Hans-Georg; Briggs, John A. G.

2015-01-01

Human immunodeficiency virus type 1 (HIV-1) assembly proceeds in two stages. First, the 55 kilodalton viral Gag polyprotein assembles into a hexameric protein lattice at the plasma membrane of the infected cell, inducing budding and release of an immature particle. Second, Gag is cleaved by the viral protease, leading to internal rearrangement of the virus into the mature, infectious form. Immature and mature HIV-1 particles are heterogeneous in size and morphology, preventing high-resolution analysis of their protein arrangement in situ by conventional structural biology methods. Here we apply cryo-electron tomography and sub-tomogram averaging methods to resolve the structure of the capsid lattice within intact immature HIV-1 particles at subnanometre resolution, allowing unambiguous positioning of all α-helices. The resulting model reveals tertiary and quaternary structural interactions that mediate HIV-1 assembly. Strikingly, these interactions differ from those predicted by the current model based on in vitro-assembled arrays of Gag-derived proteins from Mason-Pfizer monkey virus. To validate this difference, we solve the structure of the capsid lattice within intact immature Mason-Pfizer monkey virus particles. Comparison with the immature HIV-1 structure reveals that retroviral capsid proteins, while having conserved tertiary structures, adopt different quaternary arrangements during virus assembly. The approach demonstrated here should be applicable to determine structures of other proteins at subnanometre resolution within heterogeneous environments.
Site-Specific Protein Adducts of 4-Hydroxy-2(E)-Nonenal in Human THP-1 Monocytic Cells: Protein Carbonylation Is Diminished by Ascorbic Acid

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

Chavez, Juan; Chung, Woon-Gye; Miranda, Cristobal L.

2010-01-18

The protein targets and sites of modification by 4-hydroxy-2(E)-nonenal (HNE) in human monocytic THP-1 cells after exogenous exposure to HNE were examined using a multipronged proteomic approach involving electrophoretic, immunoblotting, and mass spectrometric methods. Immunoblot analysis using monoclonal anti-HNE antibodies showed several proteins as targets of HNE adduction. Pretreatment of THP-1 cells with ascorbic acid resulted in reduced levels of HNE-protein adducts. Biotinylation of Michael-type HNE adducts using an aldehyde-reactive hydroxylamine-functionalized probe (aldehyde-reactive probe, ARP) and subsequent enrichment facilitated the identification and site-specific assignment of the modifications by LC-MS/MS analysis. Sixteen proteins were unequivocally identified as targets of HNE adduction,more » and eighteen sites of HNE modification at Cys and His residues were assigned. HNE exposure of THP-1 cells resulted in the modification of proteins involved in cytoskeleton organization and regulation, proteins associated with stress responses, and enzymes of the glycolytic and other metabolic pathways. Finally, this study yielded the first evidence of site-specific adduction of HNE to Cys-295 in tubulin α-1B chain, Cys-351 and Cys-499 in α-actinin-4, Cys-328 in vimentin, Cys-369 in d-3-phosphoglycerate dehydrogenase, and His-246 in aldolase A.« less
Biochemical Characterization of the Prolyl 3-Hydroxylase 1·Cartilage-associated Protein·Cyclophilin B Complex*

PubMed Central

Ishikawa, Yoshihiro; Wirz, Jackie; Vranka, Janice A.; Nagata, Kazuhiro; Bächinger, Hans Peter

2009-01-01

The rough endoplasmic reticulum-resident protein complex consisting of prolyl 3-hydroxylase 1 (P3H1), cartilage-associated protein (CRTAP), and cyclophilin B (CypB) can be isolated from chick embryos on a gelatin-Sepharose column, indicating some involvement in the biosynthesis of procollagens. Prolyl 3-hydroxylase 1 modifies a single proline residue in the α chains of type I, II, and III collagens to (3S)-hydroxyproline. The peptidyl-prolyl cis-trans isomerase activity of cyclophilin B was shown previously to catalyze the rate of triple helix formation. Here we show that cyclophilin B in the complex shows peptidyl-prolyl cis-trans isomerase activity and that the P3H1·CRTAP·CypB complex has another important function: it acts as a chaperone molecule when tested with two classical chaperone assays. The P3H1·CRTAP·CypB complex inhibited the thermal aggregation of citrate synthase and was active in the denatured rhodanese refolding and aggregation assay. The chaperone activity of the complex was higher than that of protein-disulfide isomerase, a well characterized chaperone. The P3H1·CRTAP·CypB complex also delayed the in vitro fibril formation of type I collagen, indicating that this complex is also able to interact with triple helical collagen and acts as a collagen chaperone. PMID:19419969
Sequence conservation, HLA-E-Restricted peptide, and best-defined CTL/CD8+ epitopes in gag P24 (capsid) of HIV-1 subtype B

NASA Astrophysics Data System (ADS)

Prasetyo, Afiono Agung; Dharmawan, Ruben; Sari, Yulia; Sariyatun, Ratna

2017-02-01

Human immunodeficiency virus type 1 (HIV-1) remains a cause of global health problem. Continuous studies of HIV-1 genetic and immunological profiles are important to find strategies against the virus. This study aimed to conduct analysis of sequence conservation, HLA-E-restricted peptide, and best-defined CTL/CD8+ epitopes in p24 (capsid) of HIV-1 subtype B worldwide. The p24-coding sequences from 3,557 HIV subtype B isolates were aligned using MUSCLE and analysed. Some highly conserved regions (sequence conservation ≥95%) were observed. Two considerably long series of sequences with conservation of 100% was observed at base 349-356 and 550-557 of p24 (HXB2 numbering). The consensus from all aligned isolates was precisely the same as consensus B in the Los Alamos HIV Database. The HLA-E-restricted peptide in amino acid (aa) 14-22 of HIV-1 p24 (AISPRTLNA) was found in 55.9% (1,987/3,557) of HIV-1 subtype B worldwide. Forty-four best-defined CTL/CD8+ epitopes were observed, in which VKNWMTETL epitope (aa 181-189 of p24) restricted by B*4801 was the most frequent, as found in 94.9% of isolates. The results of this study would contribute information about HIV-1 subtype B and benefits for further works willing to develop diagnostic and therapeutic strategies against the virus.
77 FR 3380 - Airworthiness Directives; Eurocopter France (ECF) Model AS350B, B1, B2, B3, BA, and D; and AS355E...

Federal Register 2010, 2011, 2012, 2013, 2014

2012-01-24

... Airworthiness Directives; Eurocopter France (ECF) Model AS350B, B1, B2, B3, BA, and D; and AS355E, F, F1, F2... Register as of February 8, 2012. We must receive comments on this AD by March 26, 2012. ADDRESSES: You may..., 2009, specifying MOD 073330 for the Model SA350B, B1, B2, B3, BA, BB, D, and L1 helicopters. ECF has...
[Cloning and sequence analysis of 55 K protein of egg drop syndrome virus].

PubMed

Zhu, L; Jin, Q; Zeng, L

1999-06-30

For understanding the characteristics of genomic structure of egg drop syndrome virus(EDSV). Nucleic acid was extracted using routine method from weak virulent strain AA-2 of EDSV isolated from Chinese sick hens. Construction of the whole genomic library was by hydrolysis with Hind III, strand encoding 55 K gene locating in Hind III--A segment was sequenced and analyzed. The open reading frame has a length of 1,014 nt and codes a polypeptide of 337 amino acids with molecular weight of 38,200. Analysis of the amino acid sequence revealed a homology from 25.5%-32.4% to the 55 K protein of human adenovirus types 2, 12, 40, canine adenovirus and fowl adenoviruses of group 1, whereas to ovine adenovirus is 46.4%. The genomic structure of EDSV has some relationship with adenoviruses.
The Role of Protein Elongation Factor eEF1A2 in Breast Cancer

DTIC Science & Technology

2006-09-01

serve as regulators of multiple signaling pathways (15-18). PIs are composed of an inositol ring covalently bound to a lipid phosphatidic acid ...mouse model of aristolochic acid nephropathy, and human kidney-proximal tubule cells. Satisfyingly, one of these targets is Dishevelled 2 (DVL2...Rho signaling proteins together. The two human eEF1A isoforms (eEF1A2 and eEF1A2) are very similar proteins (92% amino acid identity). The two
Endosperm Protein Synthesis and l-[35S]Methionine Incorporation in Maize Kernels Cultured In Vitro1

PubMed Central

Cully, David E.; Gengenbach, Burle G.; Smith, Jane A.; Rubenstein, Irwin; Connelly, James A.; Park, William D.

1984-01-01

This study was conducted to examine protein synthesis and l-[35S] methionine incorporation into the endosperm of Zea mays L. kernels developing in vitro. Two-day-old kernels of the inbred line W64A were placed in culture on a defined medium containing 10 microCuries l-[35S] methionine per milliliter (13 milliCuries per millimole) and harvested at 10, 15, 20, 25, 30, 35, and 40 days after pollination. Cultured kernels attained a final endosperm mass of 120 milligrams compared to 175 milligrams for field-grown controls. Field and cultured kernels had similar concentrations (microgram per milligram endospern) for total protein, albumin plus globulin, zein, and glutelin fractions at most kernel ages. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and isoelectric focusing patterns for endosperm proteins were similar for field and cultured kernels throughout development. By 15 days, over 70% of the l-[35S]methionine taken up was present in endosperm proteins. Label incorporation visualized by fluorography generally followed the protein intensity of the stained gels. The high methionine content, low molecular weight zeins (i.e. 15 and 9 kilodaltons) were highly labeled. All of the radioactivity in hydrolyzed zein samples was recovered in the methionine peak indicating minimal conversion to l-[35S]cysteine. The procedure described here is suitable for long term culture and labeling experiments in which continued kernel development is required. Images Fig. 2 Fig. 3 Fig. 4 PMID:16663428
Characterization of a Complement-Binding Protein, DRS, from Strains of Streptococcus pyogenes Containing the emm12 and emm55 Genes

PubMed Central

Binks, Michael; Sriprakash, K. S.

2004-01-01

An extracellular protein of Streptococcus pyogenes, streptococcal inhibitor of complement (SIC), and its variant, called DRS (distantly related to SIC), are expressed by some S. pyogenes strains. SIC from type 1 (M1) isolates of S. pyogenes interferes with complement-mediated cell lysis, reportedly via its interaction with complement proteins. In this study we demonstrate that S. pyogenes strains carrying emm12 and emm55 (the genes for the M12 and M55 proteins, respectively) express and secrete DRS. This protein, like SIC, binds to the C6 and C7 complement proteins, and competition enzyme-linked immunosorbent assay experiments demonstrate that DRS competes with SIC for C6 and C7 binding. Similarly, SIC competes with DRS for binding to the complement proteins. Despite this, the recombinant DRS preparation showed no significant effect on complement function, as determined by lysis of sensitized sheep erythrocytes. Furthermore, the presence of DRS is not inhibitory to SIC activity. PMID:15213143
Characterization of a complement-binding protein, DRS, from strains of Streptococcus pyogenes containing the emm12 and emm55 genes.

PubMed

Binks, Michael; Sriprakash, K S

2004-07-01

An extracellular protein of Streptococcus pyogenes, streptococcal inhibitor of complement (SIC), and its variant, called DRS (distantly related to SIC), are expressed by some S. pyogenes strains. SIC from type 1 (M1) isolates of S. pyogenes interferes with complement-mediated cell lysis, reportedly via its interaction with complement proteins. In this study we demonstrate that S. pyogenes strains carrying emm12 and emm55 (the genes for the M12 and M55 proteins, respectively) express and secrete DRS. This protein, like SIC, binds to the C6 and C7 complement proteins, and competition enzyme-linked immunosorbent assay experiments demonstrate that DRS competes with SIC for C6 and C7 binding. Similarly, SIC competes with DRS for binding to the complement proteins. Despite this, the recombinant DRS preparation showed no significant effect on complement function, as determined by lysis of sensitized sheep erythrocytes. Furthermore, the presence of DRS is not inhibitory to SIC activity.
PA1b, an insecticidal protein extracted from pea seeds (Pisum sativum): 1H-2-D NMR study and molecular modeling.

PubMed

Jouvensal, Laurence; Quillien, Laurence; Ferrasson, Eric; Rahbé, Yvan; Guéguen, Jacques; Vovelle, Françoise

2003-10-21

PA1b (pea albumin 1, subunit b) is a 37-amino acid cysteine-rich plant defense protein isolated from pea seeds (Pisum sativum). It induces short-term mortality in several pests, among which the cereal weevils Sitophilus sp. (Sitophilus oryzae, Sitophilus granarius, and Sitophilus zeamais) that are a major nuisance for stored cereals, all over the world. As such, PA1b is the first genuine protein phytotoxin specifically toxic to insects, which makes it a promising tool for seed weevil damage control. We have determined the 3-D solution structure of PA1b, using 2-D homonuclear proton NMR methods and molecular modeling. The primary sequence of the protein does not share similarities with other known toxins. It includes six cysteines forming three disulfide bridges. However, because of PA1b resistance to protease cleavage, conventional methods failed to establish the connectivity pattern. Our first attempts to assign the disulfide network from NOE data alone remained unsuccessful due to the tight packing of the cysteine residues within the core of the molecule. Yet, the use of ambiguous disulfide restraints within ARIA allowed us to establish that PA1b belongs to the inhibitor cystine-knot family. It exhibits the structural features that are characteristic of the knottin fold, namely, a triple-stranded antiparallel beta-sheet with a long flexible loop connecting the first to the second strand and a series of turns. A comparison of the structural properties of PA1b with that of structurally related proteins adopting a knottin fold and exhibiting a diverse range of biological activities shows that the electrostatic and lipophilic potentials at the surface of PA1b are very close to those found for the spider toxin ACTX-Hi:OB4219, thereby suggesting activity on ion channels.
Walleye dermal sarcoma virus Orf B functions through receptor for activated C kinase (RACK1) and protein kinase C

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

Daniels, Candelaria C.; Rovnak, Joel; Quackenbush, Sandra L.

2008-06-05

Walleye dermal sarcoma virus is a complex retrovirus that is associated with walleye dermal sarcomas that are seasonal in nature. Fall developing tumors contain low levels of spliced accessory gene transcripts A and B, suggesting a role for the encoded proteins, Orf A and Orf B, in oncogenesis. In explanted tumor cells the 35 kDa Orf B accessory protein is localized to the cell periphery in structures similar to focal adhesions and along actin stress fibers. Similar localization was observed in mammalian cells. The cellular protein, receptor for activated C kinase 1 (RACK1), bound Orf B in yeast two-hybrid assaysmore » and in cell culture. Sequence analysis of walleye RACK1 demonstrated high conservation to other known RACK1 sequences. RACK1 binds to activated protein kinase C (PKC). Orf B associates with PKC{alpha}, which is constitutively activated and localized at the membrane. Activated PKC promoted cell survival, proliferation, and increased cell viability in Orf B-expressing cells.« less
The low density lipoprotein receptor-related protein 1B retains beta-amyloid precursor protein at the cell surface and reduces amyloid-beta peptide production.

PubMed

Cam, Judy A; Zerbinatti, Celina V; Knisely, Jane M; Hecimovic, Silva; Li, Yonghe; Bu, Guojun

2004-07-09

The low density lipoprotein (LDL) receptor-related protein 1B (LRP1B) is a newly identified member of the LDL receptor family that shares high homology with the LDL receptor-related protein (LRP). LRP1B was originally described as a putative tumor suppressor in lung cancer cells; however, its expression profile in several regions of adult human brain suggests it may have additional functions in the central nervous system. Since LRP1B has overlapping ligand binding properties with LRP, we investigated whether LRP1B, like LRP, could interact with the beta-amyloid precursor protein (APP) and modulate its processing to amyloid-beta peptides (Abetas). Using an LRP1B minireceptor (mLRP1B4) generated to study the trafficking of LRP1B, we found that mLRP1B4 and APP form an immunoprecipitable complex. Furthermore mLRP1B4 bound and facilitated the degradation of a soluble isoform of APP containing a Kunitz proteinase inhibitor domain but not soluble APP lacking a Kunitz proteinase inhibitor domain. A functional consequence of mLRP1B4 expression was a significant accumulation of APP at the cell surface, which is likely related to the slow endocytosis rate of LRP1B. More importantly, mLRP1B4-expressing cells that accumulated cell surface APP produced less Abeta and secreted more soluble APP. These findings reveal that LRP1B is a novel binding partner of APP that functions to decrease APP processing to Abeta. Consequently LRP1B expression could function to protect against the pathogenesis of Alzheimer's disease.
Zinc finger protein 521 antagonizes early B-cell factor 1 and modulates the B-lymphoid differentiation of primary hematopoietic progenitors.

PubMed

Mega, Tiziana; Lupia, Michela; Amodio, Nicola; Horton, Sarah J; Mesuraca, Maria; Pelaggi, Daniela; Agosti, Valter; Grieco, Michele; Chiarella, Emanuela; Spina, Raffaella; Moore, Malcolm A S; Schuringa, Jan Jacob; Bond, Heather M; Morrone, Giovanni

2011-07-01

Zinc finger protein 521 (EHZF/ZNF521) is a multi-functional transcription co-factor containing 30 zinc fingers and an amino-terminal motif that binds to the nucleosome remodelling and histone deacetylase (NuRD) complex. ZNF521 is believed to be a relevant player in the regulation of the homeostasis of the hematopoietic stem/progenitor cell compartment, however the underlying molecular mechanisms are still largely unknown. Here, we show that this protein plays an important role in the control of B-cell development by inhibiting the activity of early B-cell factor-1 (EBF1), a master factor in B-lineage specification. In particular, our data demonstrate that: (1) ZNF521 binds to EBF1 via its carboxyl-terminal portion and this interaction is required for EBF1 inhibition; (2) NuRD complex recruitment by ZNF521 is not essential for the inhibition of transactivation of EBF1-dependent promoters; (3) ZNF521 represses EBF1 target genes in a human B-lymphoid molecular context; and (4) RNAi-mediated silencing of ZNF521/Zfp521 in primary human and murine hematopoietic progenitors strongly enhances the generation of B-lymphocytes in vitro. Taken together, our data indicate that ZNF521 can antagonize B-cell development and lend support to the notion that it may contribute to conserve the multipotency of primitive lympho-myeloid progenitors by preventing or delaying their EBF1-driven commitment toward the B-cell lineage.
Antihypertensive effect of formononetin through regulating the expressions of eNOS, 5-HT2A/1B receptors and α1-adrenoceptors in spontaneously rat arteries.

PubMed

Sun, Tao; Wang, Jie; Huang, Lin-Hong; Cao, Yong-Xiao

2013-01-15

One of the main pathological changes of hypertension is the dysfunction of blood vessels. We have found in our previous study that formononetin, one kind of phytoestrogens, has an acute antihypertensive effect. Therefore, we hypothesized that formononetin might produce a chronic antihypertensive effect through regulating the expressions of contractile receptors and endothelial nitric oxide synthase (eNOS) in artery. The present study was conducted to verify this effect. Male spontaneously hypertensive rats (SHRs) were divided into two groups, orally administrated formononetin (50mg/kg per day) and Tween 80 vehicle, respectively, for 8 weeks. The blood pressure was measured by tail-cuff method. Isometric tension of arterial rings was recorded by a myograph system. The mRNA and protein expression in arteries was determined with quantitative real-time polymerase chain reaction and immunohistochemistry, respectively. Results showed that the systolic blood pressure of SHRs decreased significantly in formononetin group compared to Tween 80 group. The vasoconstriction induced by phenylephrine or 5-hydroxytryptamine (5-HT) in the mesenteric artery segments in formononetin group was decreased, and the relaxation induced by acetylcholine was increased compared with that in Tween 80 group. In the mesenteric arteries of the formononetin-treated SHRs, the expressions of α(1)-adrenoceptors and 5-HT(2A/1B) receptors at both mRNA and protein levels decreased, while the mRNA and protein expressions of eNOS increased. In conclusion, formononetin has a chronic antihypertensive effect in SHRs. The antihypertensive mechanism may be associated with the down-regulation of α(1)-adrenoceptors and 5-HT(2A/1B) receptors, and the up-regulation of eNOS expression in arteries. Copyright © 2012 Elsevier B.V. All rights reserved.
46 CFR 97.55-1 - Master's responsibility.

Code of Federal Regulations, 2013 CFR

2013-10-01

... 46 Shipping 4 2013-10-01 2013-10-01 false Master's responsibility. 97.55-1 Section 97.55-1... Carried § 97.55-1 Master's responsibility. Before loading bulk grain or any bulk solid cargo to which § 148.435 of this chapter applies, the master shall have the lighting circuits to cargo compartments in...

46 CFR 97.55-1 - Master's responsibility.

Code of Federal Regulations, 2012 CFR

2012-10-01

... 46 Shipping 4 2012-10-01 2012-10-01 false Master's responsibility. 97.55-1 Section 97.55-1... Carried § 97.55-1 Master's responsibility. Before loading bulk grain or any bulk solid cargo to which § 148.435 of this chapter applies, the master shall have the lighting circuits to cargo compartments in...
46 CFR 97.55-1 - Master's responsibility.

Code of Federal Regulations, 2014 CFR

2014-10-01

... 46 Shipping 4 2014-10-01 2014-10-01 false Master's responsibility. 97.55-1 Section 97.55-1... Carried § 97.55-1 Master's responsibility. Before loading bulk grain or any bulk solid cargo to which § 148.435 of this chapter applies, the master shall have the lighting circuits to cargo compartments in...
Alzheimer risk genes modulate the relationship between plasma apoE and cortical PiB binding

DOE PAGES

Lazaris, Andreas; Hwang, Kristy S.; Goukasian, Naira; ...

2015-10-15

Objective: We investigated the association between apoE protein plasma levels and brain amyloidosis and the effect of the top 10 Alzheimer disease (AD) risk genes on this association. Methods: Our dataset consisted of 18 AD, 52 mild cognitive impairment, and 3 cognitively normal Alzheimer's Disease Neuroimaging Initiative 1 (ADNI1) participants with available [ 11C]-Pittsburgh compound B (PiB) and peripheral blood protein data. We used cortical pattern matching to study associations between plasma apoE and cortical PiB binding and the effect of carrier status for the top 10 AD risk genes. Results: Low plasma apoE was significantly associated with high PiBmore » SUVR, except in the sensorimotor and entorhinal cortex. For BIN1 rs744373, the association was observed only in minor allele carriers. For CD2AP rs9349407 and CR1 rs3818361, the association was preserved only in minor allele noncarriers. We did not find evidence for modulation by CLU, PICALM, ABCA7, BIN1, and MS4A6A. Conclusions: Our data show that BIN1 rs744373, CD2AP rs9349407, and CR1 rs3818361 genotypes modulate the association between apoE protein plasma levels and brain amyloidosis, implying a potential epigenetic/downstream interaction.« less
26 CFR 1.55-1 - Alternative minimum taxable income.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 26 Internal Revenue 1 2010-04-01 2010-04-01 true Alternative minimum taxable income. 1.55-1... TAXES Tax Surcharge § 1.55-1 Alternative minimum taxable income. (a) General rule for computing alternative minimum taxable income. Except as otherwise provided by statute, regulations, or other published...
Purification and characterization of human mitochondrial transcription factor 1.

PubMed Central

Fisher, R P; Clayton, D A

1988-01-01

We purified to near homogeneity a transcription factor from human KB cell mitochondria. This factor, designated mitochondrial transcription factor 1 (mtTF1), is required for the in vitro recognition of both major promoters of human mitochondrial DNA by the homologous mitochondrial RNA polymerase. Furthermore, it has been shown to bind upstream regulatory elements of the two major promoters. After separation from RNA polymerase by phosphocellulose chromatography, mtTF1 was chromatographed on a MonoQ anion-exchange fast-performance liquid chromatography column. Analysis of mtTF1-containing fractions by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a single major polypeptide with an Mr of approximately 25,000. Centrifugation in analytical glycerol gradients indicated a sedimentation coefficient of approximately 2.5 S, consistent with a monomeric 25-kilodalton protein. Finally, when the 25-kilodalton polypeptide was excised from a stained sodium dodecyl sulfate-polyacrylamide gel and allowed to renature, it regained DNA-binding and transcriptional stimulatory activities at both promoters. Although mtTF1 is the only mitochondrial DNA-binding transcription factor to be purified and characterized, its properties, such as a high affinity for random DNA and a weak specificity for one of its target sequences, may typify this class of regulatory proteins. Images PMID:3211148
Structural Relationships Between Minor and Major Proteins of Hepatitis B Surface Antigen

PubMed Central

Stibbe, Werner; Gerlich, Wolfram H.

1983-01-01

The minor glycoproteins from hepatitis B surface antigen, GP33 and GP36, contain at their carboxy-terminal part the sequence of the major protein P24. They have 55 additional amino acids at the amino-terminal part which are coded by the pre-S region of the viral DNA. Images PMID:6842680
Depletion of the cellular levels of Bag-1 proteins attenuates phorbol ester-induced downregulation of I{kappa}B{alpha} and nuclear accumulation of NF-{kappa}B

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

Maier, Jana V., E-mail: Jana.maier@kit.edu; Volz, Yvonne; Berger, Caroline

2010-10-22

Research highlights: {yields}Bag-1 depletion only marginally affects the action of the glucocorticoid receptor but strongly regulates the activity of NF-{kappa}B. {yields}Bag-1 depletion attenuates phosphorylation and degradation of I{kappa}B{alpha} and nuclear accumulation of NF-{kappa}B p65 and p50. {yields}Bag-1 interacts with I{kappa}B{alpha} and partially restores I{kappa}B{alpha} and NF-{kappa}B activation in Bag-1 depleted cells. -- Abstract: Bag-1 consists in humans of four isoforms generated from the same RNA by alternative translation. Overexpression of single Bag-1 isoforms has identified Bag-1 as a negative regulator of action of many proteins including the glucocorticoid receptor (GR). Here we have analysed the ability of Bag-1 to regulatemore » the transrepression function of the GR. Silencing Bag-1 expression only marginally affects the transrepression action of the GR but decreased the action of the transcription factor NF-{kappa}B. Furthermore phosphorylation and degradation of the inhibitor protein I{kappa}B{alpha} and nuclear accumulation of p65 and p50 NF-{kappa}B proteins in response to phorbol ester was attenuated following Bag-1 depletion in HeLa cells. Reconstitution of Bag-1 in depleted cells partially restored I{kappa}B{alpha} and NF-{kappa}B activation. Knock-down of Bag-1 expression also did not significantly alter GR-mediated transactivation but affected the basal transcription of some of the target genes. Thus Bag-1 proteins function as regulators of the action of selective transcription factors.« less
Functional Evolution of Influenza Virus NS1 Protein in Currently Circulating Human 2009 Pandemic H1N1 Viruses.

PubMed

Clark, Amelia M; Nogales, Aitor; Martinez-Sobrido, Luis; Topham, David J; DeDiego, Marta L

2017-09-01

In 2009, a novel H1N1 influenza virus emerged in humans, causing a global pandemic. It was previously shown that the NS1 protein from this human 2009 pandemic H1N1 (pH1N1) virus was an effective interferon (IFN) antagonist but could not inhibit general host gene expression, unlike other NS1 proteins from seasonal human H1N1 and H3N2 viruses. Here we show that the NS1 protein from currently circulating pH1N1 viruses has evolved to encode 6 amino acid changes (E55K, L90I, I123V, E125D, K131E, and N205S) with respect to the original protein. Notably, these 6 residue changes restore the ability of pH1N1 NS1 to inhibit general host gene expression, mainly by their ability to restore binding to the cellular factor CPSF30. This is the first report describing the ability of the pH1N1 NS1 protein to naturally acquire mutations that restore this function. Importantly, a recombinant pH1N1 virus containing these 6 amino acid changes in the NS1 protein (pH1N1/NSs-6mut) inhibited host IFN and proinflammatory responses to a greater extent than that with the parental virus (pH1N1/NS1-wt), yet virus titers were not significantly increased in cell cultures or in mouse lungs, and the disease was partially attenuated. The pH1N1/NSs-6mut virus grew similarly to pH1N1/NSs-wt in mouse lungs, but infection with pH1N1/NSs-6mut induced lower levels of proinflammatory cytokines, likely due to a general inhibition of gene expression mediated by the mutated NS1 protein. This lower level of inflammation induced by the pH1N1/NSs-6mut virus likely accounts for the attenuated disease phenotype and may represent a host-virus adaptation affecting influenza virus pathogenesis. IMPORTANCE Seasonal influenza A viruses (IAVs) are among the most common causes of respiratory infections in humans. In addition, occasional pandemics are caused when IAVs circulating in other species emerge in the human population. In 2009, a swine-origin H1N1 IAV (pH1N1) was transmitted to humans, infecting people then and up
Functional Evolution of Influenza Virus NS1 Protein in Currently Circulating Human 2009 Pandemic H1N1 Viruses

PubMed Central

Clark, Amelia M.; Nogales, Aitor; Martinez-Sobrido, Luis

2017-01-01

ABSTRACT In 2009, a novel H1N1 influenza virus emerged in humans, causing a global pandemic. It was previously shown that the NS1 protein from this human 2009 pandemic H1N1 (pH1N1) virus was an effective interferon (IFN) antagonist but could not inhibit general host gene expression, unlike other NS1 proteins from seasonal human H1N1 and H3N2 viruses. Here we show that the NS1 protein from currently circulating pH1N1 viruses has evolved to encode 6 amino acid changes (E55K, L90I, I123V, E125D, K131E, and N205S) with respect to the original protein. Notably, these 6 residue changes restore the ability of pH1N1 NS1 to inhibit general host gene expression, mainly by their ability to restore binding to the cellular factor CPSF30. This is the first report describing the ability of the pH1N1 NS1 protein to naturally acquire mutations that restore this function. Importantly, a recombinant pH1N1 virus containing these 6 amino acid changes in the NS1 protein (pH1N1/NSs-6mut) inhibited host IFN and proinflammatory responses to a greater extent than that with the parental virus (pH1N1/NS1-wt), yet virus titers were not significantly increased in cell cultures or in mouse lungs, and the disease was partially attenuated. The pH1N1/NSs-6mut virus grew similarly to pH1N1/NSs-wt in mouse lungs, but infection with pH1N1/NSs-6mut induced lower levels of proinflammatory cytokines, likely due to a general inhibition of gene expression mediated by the mutated NS1 protein. This lower level of inflammation induced by the pH1N1/NSs-6mut virus likely accounts for the attenuated disease phenotype and may represent a host-virus adaptation affecting influenza virus pathogenesis. IMPORTANCE Seasonal influenza A viruses (IAVs) are among the most common causes of respiratory infections in humans. In addition, occasional pandemics are caused when IAVs circulating in other species emerge in the human population. In 2009, a swine-origin H1N1 IAV (pH1N1) was transmitted to humans, infecting people
Stimulation of the human immunodeficiency virus type 1 enhancer by the human T-cell leukemia virus type I tax gene product involves the action of inducible cellular proteins.

PubMed

Böhnlein, E; Siekevitz, M; Ballard, D W; Lowenthal, J W; Rimsky, L; Bogérd, H; Hoffman, J; Wano, Y; Franza, B R; Greene, W C

1989-04-01

The human immunodeficiency virus type 1 (HIV-1) preferentially infects CD4+ T lymphocytes and may exist as a latent provirus within these cells for extended periods. The transition to a productive retroviral infection results in T-cell death and clinically may lead to the acquired immune deficiency syndrome. Accelerated production of infectious HIV-1 virions appears to be closely linked to a heightened state of T-cell activation. The transactivator (Tax) protein of the type I human T-cell leukemia virus (HTLV-I) can produce such an activated T-cell phenotype and augments activity of the HIV-1 long terminal repeat. One Tax-responsive region within the HIV-1 long terminal repeat has been mapped to a locus composed of two 10-base-pair direct repeats sharing homology with the binding site for the eucaryotic transcription factor NF-kappaB (GGGACTTTCC). Tax-expressing Jurkat T cells contain one or more inducible cellular proteins that specifically associate with the HIV-1 enhancer at these binding sites. Microscale DNA affinity precipitation assays identified a Tax-inducible 86-kilodalton protein, HIVEN86A, as one of these HIV-1 enhancer-binding factors. The interaction of HIVEN86A, and presumably other cellular proteins, with the HIV-1 enhancer appears functionally important as oligonucleotides corresponding to this enhancer were sufficient to impart Tax inducibility to an unresponsive heterologous promoter. These findings suggest that the Tax-inducible cellular protein HIVEN86A plays an important role in the transcriptional activation of the HIV-1 enhancer. These specific protein-DNA interactions may also be important for the transition of HIV-1 from a latent to a productive mode of infection. Furthermore, these findings highlight an intriguing biological interplay between HTLV-1 and HIV-1 through a cellular transcriptional pathway that is normally involved in T-cell activation and growth.
Antisense protein tyrosine phosphatase 1B reverses activation of p38 mitogen-activated protein kinase in liver of ob/ob mice.

PubMed

Gum, Rebecca J; Gaede, Lori L; Heindel, Matthew A; Waring, Jeffrey F; Trevillyan, James M; Zinker, Bradley A; Stark, Margery E; Wilcox, Denise; Jirousek, Michael R; Rondinone, Cristina M; Ulrich, Roger G

2003-06-01

Phosphorylation of stress-activated kinase p38, a MAPK family member, was increased in liver of ob/ob diabetic mice relative to lean littermates. Treatment of ob/ob mice with protein tyrosine phosphatase 1B (PTP1B) antisense oligonucleotides (ASO) reduced phosphorylation of p38 in liver-to below lean littermate levels-and normalized plasma glucose while reducing plasma insulin. Phosphorylation of ERK, but not JNK, was also decreased in ASO-treated mice. PTP1B ASO decreased TNFalpha protein levels and phosphorylation of the transcription factor cAMP response element binding protein (CREB) in liver, both of which can occur through decreased phosphorylation of p38 and both of which have been implicated in insulin resistance or hyperglycemia. Decreased p38 phosphorylation was not directly due to decreased phosphorylation of the kinases that normally phosphorylate p38-MKK3 and MKK6. Additionally, p38 phosphorylation was not enhanced in liver upon insulin stimulation of ASO-treated ob/ob mice (despite increased activation of other signaling molecules) corroborating that p38 is not directly affected via the insulin receptor. Instead, decreased phosphorylation of p38 may be due to increased expression of MAPK phosphatases, particularly the p38/ERK phosphatase PAC1 (phosphatase of activated cells). This study demonstrates that reduction of PTP1B protein using ASO reduces activation of p38 and its substrates TNFalpha and CREB in liver of diabetic mice, which correlates with decreased hyperglycemia and hyperinsulinemia.
Development of candidate combination vaccine for hepatitis E and hepatitis B: a liposome encapsulation approach.

PubMed

Shrivastava, Shubham; Lole, Kavita S; Tripathy, Anuradha S; Shaligram, Umesh S; Arankalle, Vidya A

2009-11-05

To reduce extra injections, cost and ensure better coverage, use of combination vaccines is preferable. An attempt was made to evaluate the encapsulation of hepatitis E virus neutralizing epitope (NE) region and hepatitis B virus surface antigen (HBsAg) in liposomes as DNAs, proteins and DNA+protein. Mice groups were immunized with different liposome-encapsulated formulations and monitored for anti-HEV and anti-HBs titres, IgG subtypes, antigen-specific lymphocyte proliferation and cytokine levels. The protective levels of anti-HBs and in vitro virus-binding capacity of anti-HEV antibodies were assessed. Liposome-encapsulated DNA either singly or in combination did not elicit antibody response. Anti-HEV and anti-HBs IgG titres of individual component of protein alone (Lipo-E-P/Lipo-B-P) or DNA+protein formulations (Lipo-E-DP/Lipo-B-DP) were comparable to respective titres in combination vaccine of protein (Lipo-BE-P) and DNA+protein formulations (Lipo-BE-DP). IgG1 levels were significantly higher in Lipo-BE-P group whereas, equivalent levels of IgG1 and IgG2a were observed in Lipo-BE-DP group against both components of the vaccine. Combination vaccine group showed mixed Th1/Th2 cytokine profile. Liposome entrapped NE and HBsAg in protein and DNA+protein formats induce excellent immune response to both the components and need to be evaluated in higher animals.
The HIV-1 envelope protein gp120 is captured and displayed for B cell recognition by SIGN-R1+ lymph node macrophages

PubMed Central

Park, Chung; Arthos, James; Cicala, Claudia; Kehrl, John H

2015-01-01

The HIV-1 envelope protein gp120 is both the target of neutralizing antibodies and a major focus of vaccine efforts; however how it is delivered to B cells to elicit an antibody response is unknown. Here, we show that following local gp120 injection lymph node (LN) SIGN-R1+ sinus macrophages located in interfollicular pockets and underlying SIGN-R1+ macrophages form a cellular network that rapidly captures gp120 from the afferent lymph. In contrast, two other antigens, phycoerythrin and hen egg lysozyme, were not captured by these cells. Intravital imaging of mouse LNs revealed persistent, but transient interactions between gp120 bearing interfollicular network cells and both trafficking and LN follicle resident gp120 specific B cells. The gp120 specific, but not the control B cells repetitively extracted gp120 from the network cells. Our findings reveal a specialized LN antigen delivery system poised to deliver gp120 and likely other pathogen derived glycoproteins to B cells. DOI: http://dx.doi.org/10.7554/eLife.06467.001 PMID:26258881
Logarithmic phase Escherichia coli K1 efficiently avoids serum killing by promoting C4bp-mediated C3b and C4b degradation

PubMed Central

Wooster, David G; Maruvada, Ravi; Blom, Anna M; Prasadarao, Nemani V

2006-01-01

Meningitis caused by Escherichia coli K1 is a serious illness in neonates with neurological sequelae in up to 50% of survivors. A high degree of bacteremia is required for E. coli K1 to cross the blood–brain barrier, which suggests that the bacterium must evade the host defence mechanisms and survive in the bloodstream. We previously showed that outer membrane protein A (OmpA) of E. coli binds C4b-binding protein (C4bp), an inhibitor of complement activation via the classical pathway. Nevertheless, the exact mechanism by which E. coli K1 survives in serum remains elusive. Here, we demonstrate that log phase (LP) OmpA+E. coli K1 avoids serum bactericidal activity more effectively than postexponential phase bacteria. OmpA–E. coli cannot survive in serum grown to either phase. The increased serum resistance of LP OmpA+E. coli is the result of increased binding of C4bp, with a concomitant decrease in the deposition of C3b and the downstream complement proteins responsible for the formation of the membrane attack complex. C4bp bound to E. coli K1 acts as a cofactor to factor I in the cleavage of both C3b and C4b, which shuts down the ensuing complement cascade. Accordingly, a peptide corresponding to the complement control protein domain 3 of C4bp sequence, was able to compete with C4bp binding to OmpA and cause increased deposition of C3b. Thus, binding of C4bp appears to be responsible for survival of E. coli K1 in human serum. PMID:16556262
Localisation of the Putative Magnetoreceptive Protein Cryptochrome 1b in the Retinae of Migratory Birds and Homing Pigeons.

PubMed

Bolte, Petra; Bleibaum, Florian; Einwich, Angelika; Günther, Anja; Liedvogel, Miriam; Heyers, Dominik; Depping, Anne; Wöhlbrand, Lars; Rabus, Ralf; Janssen-Bienhold, Ulrike; Mouritsen, Henrik

2016-01-01

Cryptochromes are ubiquitously expressed in various animal tissues including the retina. Some cryptochromes are involved in regulating circadian activity. Cryptochrome proteins have also been suggested to mediate the primary mechanism in light-dependent magnetic compass orientation in birds. Cryptochrome 1b (Cry1b) exhibits a unique carboxy terminus exclusively found in birds so far, which might be indicative for a specialised function. Cryptochrome 1a (Cry1a) is so far the only cryptochrome protein that has been localised to specific cell types within the retina of migratory birds. Here we show that Cry1b, an alternative splice variant of Cry1a, is also expressed in the retina of migratory birds, but it is primarily located in other cell types than Cry1a. This could suggest different functions for the two splice products. Using diagnostic bird-specific antibodies (that allow for a precise discrimination between both proteins), we show that Cry1b protein is found in the retinae of migratory European robins (Erithacus rubecula), migratory Northern Wheatears (Oenanthe oenanthe) and pigeons (Columba livia). In all three species, retinal Cry1b is localised in cell types which have been discussed as potentially well suited locations for magnetoreception: Cry1b is observed in the cytosol of ganglion cells, displaced ganglion cells, and in photoreceptor inner segments. The cytosolic rather than nucleic location of Cry1b in the retina reported here speaks against a circadian clock regulatory function of Cry1b and it allows for the possible involvement of Cry1b in a radical-pair-based magnetoreception mechanism.
African Swine Fever Virus pB119L Protein Is a Flavin Adenine Dinucleotide-Linked Sulfhydryl Oxidase

PubMed Central

Rodríguez, Irene; Redrejo-Rodríguez, Modesto; Rodríguez, Javier M.; Alejo, Alí; Salas, José; Salas, María L.

2006-01-01

Protein pB119L of African swine fever virus belongs to the Erv1p/Alrp family of sulfhydryl oxidases and has been described as a late nonstructural protein required for correct virus assembly. To further our knowledge of the function of protein pB119L during the virus life cycle, we have investigated whether this protein possesses sulfhydryl oxidase activity, using a purified recombinant protein. We show that the purified protein contains bound flavin adenine dinucleotide and is capable of catalyzing the formation of disulfide bonds both in a protein substrate and in the small molecule dithiothreitol, the catalytic activity being comparable to that of the Erv1p protein. Furthermore, protein pB119L contains the cysteines of its active-site motif CXXC, predominantly in an oxidized state, and forms noncovalently bound dimers in infected cells. We also show in coimmunoprecipitation experiments that protein pB119L interacts with the viral protein pA151R, which contains a CXXC motif similar to that present in thioredoxins. Protein pA151R, in turn, was found to interact with the viral structural protein pE248R, which contains disulfide bridges and belongs to a class of myristoylated proteins related to vaccinia virus L1R, one of the substrates of the redox pathway encoded by this virus. These results suggest the existence in African swine fever virus of a system for the formation of disulfide bonds constituted at least by proteins pB119L and pA151R and identify protein pE248R as a possible final substrate of this pathway. PMID:16537584
African swine fever virus pB119L protein is a flavin adenine dinucleotide-linked sulfhydryl oxidase.

PubMed

Rodríguez, Irene; Redrejo-Rodríguez, Modesto; Rodríguez, Javier M; Alejo, Alí; Salas, José; Salas, María L

2006-04-01

Protein pB119L of African swine fever virus belongs to the Erv1p/Alrp family of sulfhydryl oxidases and has been described as a late nonstructural protein required for correct virus assembly. To further our knowledge of the function of protein pB119L during the virus life cycle, we have investigated whether this protein possesses sulfhydryl oxidase activity, using a purified recombinant protein. We show that the purified protein contains bound flavin adenine dinucleotide and is capable of catalyzing the formation of disulfide bonds both in a protein substrate and in the small molecule dithiothreitol, the catalytic activity being comparable to that of the Erv1p protein. Furthermore, protein pB119L contains the cysteines of its active-site motif CXXC, predominantly in an oxidized state, and forms noncovalently bound dimers in infected cells. We also show in coimmunoprecipitation experiments that protein pB119L interacts with the viral protein pA151R, which contains a CXXC motif similar to that present in thioredoxins. Protein pA151R, in turn, was found to interact with the viral structural protein pE248R, which contains disulfide bridges and belongs to a class of myristoylated proteins related to vaccinia virus L1R, one of the substrates of the redox pathway encoded by this virus. These results suggest the existence in African swine fever virus of a system for the formation of disulfide bonds constituted at least by proteins pB119L and pA151R and identify protein pE248R as a possible final substrate of this pathway.
Functional analysis of the glycogen binding subunit CG9238/Gbs-70E of protein phosphatase 1 in Drosophila melanogaster.

PubMed

Kerekes, Éva; Kókai, Endre; Páldy, Ferenc Sándor; Dombrádi, Viktor

2014-06-01

The product of the CG9238 gene that we termed glycogen binding subunit 70E (Gbs-70E) was characterized by biochemical and molecular genetics methods. The interaction between Gbs-70E and all catalytic subunits of protein phosphatase 1 (Pp1-87B, Pp1-9C, Pp1-96A and Pp1-13C) of Drosophila melanogaster was confirmed by pairwise yeast two-hybrid tests, co-immunoprecipitation and pull down experiments. The binding of Gbs-70E to glycogen was demonstrated by sedimentation analysis. With RT-PCR we found that the mRNAs coding for the longer Gbs-70E PB/PC protein were expressed in all developmental stages of the fruit flies while the mRNA for the shorter Gbs-70E PA was restricted to the eggs and the ovaries of the adult females. The development specific expression of the shorter splice variant was not conserved in different Drosophila species. The expression level of the gene was manipulated by P-element insertions and gene deletion to analyze the functions of the gene product. A small or moderate reduction in the gene expression resulted in no significant changes, however, a deletion mutant expressing very low level of the transcript lived shorter and exhibited reduced glycogen content in the imagos. In addition, the gene deletion decreased the fertility of the fruit flies. Our results prove that Gbs-70E functions as the glycogen binding subunit of protein phosphatase 1 that regulates glycogen content and plays a role in the development of eggs in D. melanogaster. Copyright © 2014 Elsevier Ltd. All rights reserved.
ATP1B3 Protein Modulates the Restriction of HIV-1 Production and Nuclear Factor κ Light Chain Enhancer of Activated B Cells (NF-κB) Activation by BST-2*

PubMed Central

Nishitsuji, Hironori; Sugiyama, Ryuichi; Abe, Makoto; Takaku, Hiroshi

2016-01-01

Here, we identify ATP1B3 and fibrillin-1 as novel BST-2-binding proteins. ATP1B3 depletion in HeLa cells (BST-2-positive cells), but not 293T cells (BST-2-negative cells), induced the restriction of HIV-1 production in a BST-2-dependent manner. In contrast, fibrillin-1 knockdown reduced HIV-1 production in 293T and HeLa cells in a BST-2-independent manner. Moreover, NF-κB activation was enhanced by siATP1B3 treatment in HIV-1- and HIV-1ΔVpu-infected HeLa cells. In addition, ATP1B3 silencing induced high level BST-2 expression on the surface of HeLa cells. These results indicate that ATP1B3 is a co-factor that accelerates BST-2 degradation and reduces BST-2-mediated restriction of HIV-1 production and NF-κB activation. PMID:26694617
[Effect of NF-κB on the expression of interleukin-6 induced by lipopolysaccharides of Porphyromonas endodontalis in MC3T3-E1 cells].

PubMed

Yu, Ya-qiong; Guo, Jia-jie; Qiu, Li-hong; Lv, You; Jia, Ge; Guo, Yan

2013-08-01

To investigate the effect of NF-κB signaling on the expression of interleukin-6(IL-6) induced by lipopolysaccharides(LPS) extracted from Porphyromonas endodontalis(P.e) in MC3T3-El cells. MC3T3-E1 cells were pretreated with BAY-117082 for 1 h, and then were treated with 10 mg/L P.e-LPS for different times. The translocation of NF-κB was observed by immunofluorescence. The expression of IL-6 was detected by reverse transcription polymerse chain reaction (RT-PCR) and enzyme-linked immuno sorbent assay (ELISA). Statistical analysis was performed using multi-way ANOVA and Dunnett's t test with SPSS 13.0 software package. The staining of NF-κB was mostly in cytoplasm in untreated cells. Rapid translocation of NF-κB into nucleus was observed in the cells stimulated for 30 min and mostly relocalization of NF-κB from nucleus to cytoplasm was observed after 60 min. Pretreatment with 10 μmol/L BAY-117082 for 1h significantly inhibited P.e-LPS-induced translocation of NF-κB .The mRNA and proteins of IL-6 decreased significantly after pretreatment with 10 μmol/L BAY-117082 and the expression of IL-6 proteins was reduced from (774.983±6.585) ng/L to (377.384±14.620) ng/L (P<0.01). The group of treatment with BAY-117082 alone had no significant difference from the blank control group. P.e-LPS can induce translocation of NF-κB in mouse osteoblast MC3T3-El, and P.e-LPS may induce the expression of IL-6 in mouse osteoblast through the signaling of NF-κB.

Mutations in STX1B, encoding a presynaptic protein, cause fever-associated epilepsy syndromes.

PubMed

Schubert, Julian; Siekierska, Aleksandra; Langlois, Mélanie; May, Patrick; Huneau, Clément; Becker, Felicitas; Muhle, Hiltrud; Suls, Arvid; Lemke, Johannes R; de Kovel, Carolien G F; Thiele, Holger; Konrad, Kathryn; Kawalia, Amit; Toliat, Mohammad R; Sander, Thomas; Rüschendorf, Franz; Caliebe, Almuth; Nagel, Inga; Kohl, Bernard; Kecskés, Angela; Jacmin, Maxime; Hardies, Katia; Weckhuysen, Sarah; Riesch, Erik; Dorn, Thomas; Brilstra, Eva H; Baulac, Stephanie; Møller, Rikke S; Hjalgrim, Helle; Koeleman, Bobby P C; Jurkat-Rott, Karin; Lehman-Horn, Frank; Roach, Jared C; Glusman, Gustavo; Hood, Leroy; Galas, David J; Martin, Benoit; de Witte, Peter A M; Biskup, Saskia; De Jonghe, Peter; Helbig, Ingo; Balling, Rudi; Nürnberg, Peter; Crawford, Alexander D; Esguerra, Camila V; Weber, Yvonne G; Lerche, Holger

2014-12-01

Febrile seizures affect 2-4% of all children and have a strong genetic component. Recurrent mutations in three main genes (SCN1A, SCN1B and GABRG2) have been identified that cause febrile seizures with or without epilepsy. Here we report the identification of mutations in STX1B, encoding syntaxin-1B, that are associated with both febrile seizures and epilepsy. Whole-exome sequencing in independent large pedigrees identified cosegregating STX1B mutations predicted to cause an early truncation or an in-frame insertion or deletion. Three additional nonsense or missense mutations and a de novo microdeletion encompassing STX1B were then identified in 449 familial or sporadic cases. Video and local field potential analyses of zebrafish larvae with antisense knockdown of stx1b showed seizure-like behavior and epileptiform discharges that were highly sensitive to increased temperature. Wild-type human syntaxin-1B but not a mutated protein rescued the effects of stx1b knockdown in zebrafish. Our results thus implicate STX1B and the presynaptic release machinery in fever-associated epilepsy syndromes.
On the anisotropy of E 0 ⩾ 5.5× 1019 eV cosmic rays according to data of the Pierre Auger Collaboration

NASA Astrophysics Data System (ADS)

Glushkov, A. V.

2012-02-01

The Pierre Auger Collaboration discovered, in a solid angle of radius about 18°, a local group of cosmic rays having energies in the region E 0 ≥ 5.5 × 1019 eV and coming from the region of the Gen A radio galaxy, whose galactic coordinates are l G = 309.5° and b G = 19.4°. Near it, there is the Centaur supercluster of galaxies, its galactic coordinates being l G = 302.4° and b G = 21.6°. It is noteworthy that the Great Attractor, which may have a direct bearing on the observed picture, is also there.
Coordinated Regulation of Insulin Signaling by the Protein Tyrosine Phosphatases PTP1B and TCPTP

PubMed Central

Galic, Sandra; Hauser, Christine; Kahn, Barbara B.; Haj, Fawaz G.; Neel, Benjamin G.; Tonks, Nicholas K.; Tiganis, Tony

2005-01-01

The protein tyrosine phosphatase PTP1B is a negative regulator of insulin signaling and a therapeutic target for type 2 diabetes. Our previous studies have shown that the closely related tyrosine phosphatase TCPTP might also contribute to the regulation of insulin receptor (IR) signaling in vivo (S. Galic, M. Klingler-Hoffmann, M. T. Fodero-Tavoletti, M. A. Puryer, T. C. Meng, N. K. Tonks, and T. Tiganis, Mol. Cell. Biol. 23:2096-2108, 2003). Here we show that PTP1B and TCPTP function in a coordinated and temporally distinct manner to achieve an overall regulation of IR phosphorylation and signaling. Whereas insulin-induced phosphatidylinositol 3-kinase/Akt signaling was prolonged in both TCPTP−/− and PTP1B−/− immortalized mouse embryo fibroblasts (MEFs), mitogen-activated protein kinase ERK1/2 signaling was elevated only in PTP1B-null MEFs. By using phosphorylation-specific antibodies, we demonstrate that both IR β-subunit Y1162/Y1163 and Y972 phosphorylation are elevated in PTP1B−/− MEFs, whereas Y972 phosphorylation was elevated and Y1162/Y1163 phosphorylation was sustained in TCPTP−/− MEFs, indicating that PTP1B and TCPTP differentially contribute to the regulation of IR phosphorylation and signaling. Consistent with this, suppression of TCPTP protein levels by RNA interference in PTP1B−/− MEFs resulted in no change in ERK1/2 signaling but caused prolonged Akt activation and Y1162/Y1163 phosphorylation. These results demonstrate that PTP1B and TCPTP are not redundant in insulin signaling and that they act to control both common as well as distinct insulin signaling pathways in the same cell. PMID:15632081
The 32-Kilodalton Subunit of Replication Protein A Interacts with Menin, the Product of the MEN1 Tumor Suppressor Gene

PubMed Central

Sukhodolets, Karen E.; Hickman, Alison B.; Agarwal, Sunita K.; Sukhodolets, Maxim V.; Obungu, Victor H.; Novotny, Elizabeth A.; Crabtree, Judy S.; Chandrasekharappa, Settara C.; Collins, Francis S.; Spiegel, Allen M.; Burns, A. Lee; Marx, Stephen J.

2003-01-01

Menin is a 70-kDa protein encoded by MEN1, the tumor suppressor gene disrupted in multiple endocrine neoplasia type 1. In a yeast two-hybrid system based on reconstitution of Ras signaling, menin was found to interact with the 32-kDa subunit (RPA2) of replication protein A (RPA), a heterotrimeric protein required for DNA replication, recombination, and repair. The menin-RPA2 interaction was confirmed in a conventional yeast two-hybrid system and by direct interaction between purified proteins. Menin-RPA2 binding was inhibited by a number of menin missense mutations found in individuals with multiple endocrine neoplasia type 1, and the interacting regions were mapped to the N-terminal portion of menin and amino acids 43 to 171 of RPA2. This region of RPA2 contains a weak single-stranded DNA-binding domain, but menin had no detectable effect on RPA-DNA binding in vitro. Menin bound preferentially in vitro to free RPA2 rather than the RPA heterotrimer or a subcomplex consisting of RPA2 bound to the 14-kDa subunit (RPA3). However, the 70-kDa subunit (RPA1) was coprecipitated from HeLa cell extracts along with RPA2 by menin-specific antibodies, suggesting that menin binds to the RPA heterotrimer or a novel RPA1-RPA2-containing complex in vivo. This finding was consistent with the extensive overlap in the nuclear localization patterns of endogenous menin, RPA2, and RPA1 observed by immunofluorescence. PMID:12509449
Competitive protein tyrosine phosphatase 1B (PTP1B) inhibitors, prenylated caged xanthones from Garcinia hanburyi and their inhibitory mechanism.

PubMed

Tan, Xue Fei; Uddin, Zia; Park, Chanin; Song, Yeong Hun; Son, Minky; Lee, Keun Woo; Park, Ki Hun

2017-04-15

Protein tyrosine phosphatase 1B (PTP1B) plays important role in diabetes, obesity and cancer. The methanol extract of the gum resin of Garcinia hanburyi (G. hanburyi) showed potent PTP1B inhibition at 10µg/ml. The active compounds were identified as prenylated caged xanthones (1-9) which inhibited PTP1B in dose-dependent manner. Carboxybutenyl group within caged motif (A ring) was found to play a critical role in enzyme inhibition such as 1-6 (IC 50 s=0.47-4.69µM), whereas compounds having hydroxymethylbutenyl 7 (IC 50 =70.25µM) and methylbutenyl 8 (IC 50 >200µM) showed less activity. The most potent inhibitor, gambogic acid 1 (IC 50 =0.47µM) showed 30-fold more potency than ursolic acid (IC 50 =15.5µM), a positive control. In kinetic study, all isolated xanthones behaved as competitive inhibitors which were fully demonstrated with K m , V max and K ik /K iv ratio. It was also proved that inhibitor 1 operated under the enzyme isomerization model having k 5 =0.0751µM - 1 S - 1 , k 6 =0.0249µM - 1 S - 1 and K i app =0.499µM. To develop a pharmacophore model, we explored the binding sites of compound 1 and 7 in PTP1B. These modeling results were in agreement with our findings, which revealed that the inhibitory activities are tightly related to caged motif and prenyl group in A ring. Copyright © 2017 Elsevier Ltd. All rights reserved.
Model of OSBP-Mediated Cholesterol Supply to Aichi Virus RNA Replication Sites Involving Protein-Protein Interactions among Viral Proteins, ACBD3, OSBP, VAP-A/B, and SAC1.

PubMed

Ishikawa-Sasaki, Kumiko; Nagashima, Shigeo; Taniguchi, Koki; Sasaki, Jun

2018-04-15

Positive-strand RNA viruses, including picornaviruses, utilize cellular machinery for genome replication. Previously, we reported that each of the 2B, 2BC, 2C, 3A, and 3AB proteins of Aichi virus (AiV), a picornavirus, forms a complex with the Golgi apparatus protein ACBD3 and phosphatidylinositol 4-kinase IIIβ (PI4KB) at viral RNA replication sites (replication organelles [ROs]), enhancing PI4KB-dependent phosphatidylinositol 4-phosphate (PI4P) production. Here, we demonstrate AiV hijacking of the cellular cholesterol transport system involving oxysterol-binding protein (OSBP), a PI4P-binding cholesterol transfer protein. AiV RNA replication was inhibited by silencing cellular proteins known to be components of this pathway, OSBP, the ER membrane proteins VAPA and VAPB (VAP-A/B), the PI4P-phosphatase SAC1, and PI-transfer protein β. OSBP, VAP-A/B, and SAC1 were present at RNA replication sites. We also found various previously unknown interactions among the AiV proteins (2B, 2BC, 2C, 3A, and 3AB), ACBD3, OSBP, VAP-A/B, and SAC1, and the interactions were suggested to be involved in recruiting the component proteins to AiV ROs. Importantly, the OSBP-2B interaction enabled PI4P-independent recruitment of OSBP to AiV ROs, indicating preferential recruitment of OSBP among PI4P-binding proteins. Protein-protein interaction-based OSBP recruitment has not been reported for other picornaviruses. Cholesterol was accumulated at AiV ROs, and inhibition of OSBP-mediated cholesterol transfer impaired cholesterol accumulation and AiV RNA replication. Electron microscopy showed that AiV-induced vesicle-like structures were close to ER membranes. Altogether, we conclude that AiV directly recruits the cholesterol transport machinery through protein-protein interactions, resulting in formation of membrane contact sites between the ER and AiV ROs and cholesterol supply to the ROs. IMPORTANCE Positive-strand RNA viruses utilize host pathways to modulate the lipid composition of
Flavonoids as potent allosteric inhibitors of protein tyrosine phosphatase 1B: molecular dynamics simulation and free energy calculation.

PubMed

Zargari, Farshid; Lotfi, Maryam; Shahraki, Omolbanin; Nikfarjam, Zahra; Shahraki, Jafar

2017-12-11

Protein tyrosine phosphatase 1B (PTP1B) is a member of the PTP superfamily which is considered to be a negative regulator of insulin receptor (IR) signaling pathway. PTP1B is a promising drug target for the treatment of type 2 diabetes, obesity, and cancer. The existence of allosteric site in PTP1B has turned the researcher's attention to an alternate strategy for inhibition of this enzyme. Herein, the molecular interactions between the allosteric site of PTP1B with three non-competitive flavonoids, (MOR), (MOK), and (DPO) have been investigated. Three ligands were docked into allosteric site of the enzyme. The resulting protein-ligand complexes were used for molecular dynamics studies. Principal component and free-energy landscape (FEL) as well as cluster analyses were used to investigate the conformational and dynamical properties of the protein and identify representative enzyme substrates bounded to the inhibitors. Per residue energy decomposition analysis attributed dissimilar affinities of three inhibitors to the several hydrogen bonds and non-bonded interactions. In conclusion, our results exhibited an inhibitory pattern of the ligands against PTP1B.
Curcumin modulates cellular AP-1, NF-kB, and HPV16 E6 proteins in oral cancer.

PubMed

Mishra, Alok; Kumar, Rakesh; Tyagi, Abhishek; Kohaar, Indu; Hedau, Suresh; Bharti, Alok C; Sarker, Subhodeep; Dey, Dipankar; Saluja, Daman; Das, Bhudev

2015-01-01

In this study, we investigated the effects of the natural antioxidant curcumin on the HPV16-positive oral carcinoma cell line 93VU147T and demonstrated that curcumin is not only a potent inhibitor for the activity of host nuclear transcription factors AP-1 and NF-kB but it also selectively suppresses transcription of the HPV16/E6 oncogene during the carcinogenic process in oral cancer cells. This study suggests a therapeutic potential of curcumin for high-risk human papilloma virus (HPV)-infected oral cancers.
Curcumin modulates cellular AP-1, NF-kB, and HPV16 E6 proteins in oral cancer

PubMed Central

Mishra, Alok; Kumar, Rakesh; Tyagi, Abhishek; Kohaar, Indu; Hedau, Suresh; Bharti, Alok C; Sarker, Subhodeep; Dey, Dipankar; Saluja, Daman; Das, Bhudev

2015-01-01

In this study, we investigated the effects of the natural antioxidant curcumin on the HPV16-positive oral carcinoma cell line 93VU147T and demonstrated that curcumin is not only a potent inhibitor for the activity of host nuclear transcription factors AP-1 and NF-kB but it also selectively suppresses transcription of the HPV16/E6 oncogene during the carcinogenic process in oral cancer cells. This study suggests a therapeutic potential of curcumin for high-risk human papilloma virus (HPV)-infected oral cancers. PMID:25932049
Roux-en-Y gastric bypass surgery suppresses hypothalamic PTP1B protein level and alleviates leptin resistance in obese rats

PubMed Central

Liu, Jia-Yu; Mu, Song; Zhang, Shu-Ping; Guo, Wei; Li, Qi-Fu; Xiao, Xiao-Qiu; Zhang, Jun; Wang, Zhi-Hong

2017-01-01

The present study aimed to explore the effect of Roux-en-Y gastric bypass (RYGB) surgery on protein tyrosine phosphatase 1B (PTP1B) expression levels and leptin activity in hypothalami of obese rats. Obese rats induced by a high-fat diet (HFD) that underwent RYGB (n=11) or sham operation (SO, n=9), as well as an obese control cohort (Obese, n=10) and an additional normal-diet group (ND, n=10) were used. Food efficiency was measured at 8 weeks post-operation. Plasma leptin levels were evaluated and hypothalamic protein tyrosine phosphatase 1B (PTP1B) levels and leptin signaling activity were examined at the genetic and protein levels. The results indicated that food efficiency was typically lower in RYGB rats compared with that in the Obese and SO rats. In the RYGB group, leptin receptor expression and proopiomelanocortin was significantly higher, while Neuropeptide Y levels were lower than those in the Obese and SO groups. Furthermore, the gene and protein expression levels of PTP1B in the RYGB group were lower, while levels of phosphorylated signal transducer and activator of transcription 3 protein were much higher compared with those in the Obese and SO groups. In conclusion, RYGB surgery significantly suppressed hypothalamic PTP1B protein expression. PTP1B regulation may partially alleviate leptin resistance. PMID:28947917
Roux-en-Y gastric bypass surgery suppresses hypothalamic PTP1B protein level and alleviates leptin resistance in obese rats.

PubMed

Liu, Jia-Yu; Mu, Song; Zhang, Shu-Ping; Guo, Wei; Li, Qi-Fu; Xiao, Xiao-Qiu; Zhang, Jun; Wang, Zhi-Hong

2017-09-01

The present study aimed to explore the effect of Roux-en-Y gastric bypass (RYGB) surgery on protein tyrosine phosphatase 1B (PTP1B) expression levels and leptin activity in hypothalami of obese rats. Obese rats induced by a high-fat diet (HFD) that underwent RYGB (n=11) or sham operation (SO, n=9), as well as an obese control cohort (Obese, n=10) and an additional normal-diet group (ND, n=10) were used. Food efficiency was measured at 8 weeks post-operation. Plasma leptin levels were evaluated and hypothalamic protein tyrosine phosphatase 1B (PTP1B) levels and leptin signaling activity were examined at the genetic and protein levels. The results indicated that food efficiency was typically lower in RYGB rats compared with that in the Obese and SO rats. In the RYGB group, leptin receptor expression and proopiomelanocortin was significantly higher, while Neuropeptide Y levels were lower than those in the Obese and SO groups. Furthermore, the gene and protein expression levels of PTP1B in the RYGB group were lower, while levels of phosphorylated signal transducer and activator of transcription 3 protein were much higher compared with those in the Obese and SO groups. In conclusion, RYGB surgery significantly suppressed hypothalamic PTP1B protein expression. PTP1B regulation may partially alleviate leptin resistance.
Genetically engineered mouse models for functional studies of SKP1-CUL1-F-box-protein (SCF) E3 ubiquitin ligases.

PubMed

Zhou, Weihua; Wei, Wenyi; Sun, Yi

2013-05-01

The SCF (SKP1 (S-phase-kinase-associated protein 1), Cullin-1, F-box protein) E3 ubiquitin ligases, the founding member of Cullin-RING ligases (CRLs), are the largest family of E3 ubiquitin ligases in mammals. Each individual SCF E3 ligase consists of one adaptor protein SKP1, one scaffold protein cullin-1 (the first family member of the eight cullins), one F-box protein out of 69 family members, and one out of two RING (Really Interesting New Gene) family proteins RBX1/ROC1 or RBX2/ROC2/SAG/RNF7. Various combinations of these four components construct a large number of SCF E3s that promote the degradation of many key regulatory proteins in cell-context, temporally, and spatially dependent manners, thus controlling precisely numerous important cellular processes, including cell cycle progression, apoptosis, gene transcription, signal transduction, DNA replication, maintenance of genome integrity, and tumorigenesis. To understand how the SCF E3 ligases regulate these cellular processes and embryonic development under in vivo physiological conditions, a number of mouse models with transgenic (Tg) expression or targeted deletion of components of SCF have been established and characterized. In this review, we will provide a brief introduction to the ubiquitin-proteasome system (UPS) and the SCF E3 ubiquitin ligases, followed by a comprehensive overview on the existing Tg and knockout (KO) mouse models of the SCF E3s, and discuss the role of each component in mouse embryogenesis, cell proliferation, apoptosis, carcinogenesis, as well as other pathogenic processes associated with human diseases. We will end with a brief discussion on the future directions of this research area and the potential applications of the knowledge gained to more effective therapeutic interventions of human diseases.
Immunoblot detection of class-specific humoral immune response to outer membrane proteins isolated from Salmonella typhi in humans with typhoid fever.

PubMed Central

Ortiz, V; Isibasi, A; García-Ortigoza, E; Kumate, J

1989-01-01

The studies reported here were undertaken to assess the ability of the outer membrane proteins (OMPs) of Salmonella typhi to induce a humoral immune response in humans with typhoid fever. OMPs were isolated with the nonionic detergent Triton X-100 and were found to be contaminated with approximately 4% lipopolysaccharide. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis patterns showed protein bands with molecular size ranges from 17 to 70 kilodaltons; the major groups of proteins were those that correspond to the porins and OmpA of gram-negative bacteria. Rabbit antiserum to OMPs or to S. typhi recognized OMPs after absorption with lipopolysaccharide. Sera from patients with typhoid fever contained immunoglobulin M antibodies which reacted with a protein of 28 kilodaltons and immunoglobulin G antibodies which reacted mainly with the porins, as determined by immunoblotting. These results indicate that the porins are the major immunogenic OMPs from S. typhi and that the immune response induced in the infection could be related to the protective status. Images PMID:2768450
X-1E launch from B-50 mothership

NASA Technical Reports Server (NTRS)

1950-01-01

it was to be launched on its second flight. It had to be jettisoned to the Muroc desert. Shop experiments soon determined that the deadly explosive culprit for the X-1D, the X-1 #3, and the X-1A was the ulmer leather gasket material used in contact with the liquid oxygen. The loss of the X-1 #3 and the X-1D led the NACA to rebuild the X-1 #2 into a new aircraft. By December 1955, the redesignated X-1E was ready. It featured a new, very thin 4-percent wing along with the existing 8-percent tail, with an efficient low-pressure turbo-pump for the engine. It also contained an ejection seat for the pilot, unlike the original X-1. On October 8, 1957, the aircraft with NACA pilot Joseph A. Walker achieved a speed of Mach 2.24 (1,478 mph). During its second flight career, the new X-1E allowed NACA to gather significant data on high Mach flight and stability questions and to demonstrate improved engine and production technology for incorporation into new USAF aircraft. The X-1E was also used to obtain in-flight data on the improvements achieved with the high-speed wing. These wings, made by Stanley Aircraft, wereonly 3 - 3/8-in. thick at the thickest point and had 343 gauges installed in them for measurement of structural loads and aerodynamic heating. Like the original X-1 it was air launched. This movie clip running about 10 seconds shows a drop from the B-50 mothership, accelerating away under rocket power and at speed making a high altitude contrail.
Pharmacology of GPR55 in yeast and identification of GSK494581A as a mixed-activity glycine transporter subtype 1 inhibitor and GPR55 agonist.

PubMed

Brown, Andrew J; Daniels, Dion A; Kassim, Mumta; Brown, Sue; Haslam, Carl P; Terrell, Victoria R; Brown, Jason; Nichols, Paula L; Staton, Penny C; Wise, Alan; Dowell, Simon J

2011-04-01

GPR55 is a G protein-coupled receptor activated by L-α-lysophosphatidylinositol and suggested to have roles in pain signaling, bone morphogenesis, and possibly in vascular endothelial cells. It has affinity for certain cannabinoids (molecules that interact with the cannabinoid CB(1) and CB(2) receptors), but investigation of its functional role in cell-based systems and in tissue has been limited by a lack of selective pharmacological tools. Here, we present our characterization of GPR55 in the yeast Saccharomyces cerevisiae and in human embryonic kidney (HEK293) cells. We describe GSK494581A (1-{2-fluoro-4-[1-(methyloxy)ethyl]phenyl}-4-{[4'-fluoro-4-(methylsulfonyl)-2-biphenylyl]carbonyl}piperazine), a selective small-molecule ligand of GPR55 identified through diversity screening. GSK494581A is one of a series of benzoylpiperazines originally identified and patented as inhibitors of the glycine transporter subtype 1 (GlyT1). The structure-activity relationship between GPR55 and GlyT1 is divergent across this series. The most GPR55-selective example is GSK575594A (3-fluoro-4-(4-{[4'-fluoro-4-(methylsulfonyl)-2-biphenylyl]carbonyl}-1-piperazinyl)aniline), which is approximately 60-fold selective for GPR55 (pEC(50) = 6.8) over GlyT1 (pIC(50) = 5.0). Several exemplars with activity at GPR55 and GlyT1 have been profiled at a broad range of other molecular targets and are inactive at cannabinoid receptors and all other targets tested. The benzoylpiperazine agonists activate human GPR55 but not rodent GPR55, suggesting that the relatively low level of sequence identity between these orthologs (75%) translates to important functional differences in the ligand-binding site.
Modulation of E2F activity in primary mouse B cells following stimulation via surface IgM and CD40 receptors.

PubMed

Lam, E W; Glassford, J; van der Sman, J; Banerji, L; Pizzey, A R; Shaun, N; Thomas, B; Klaus, G G

1999-10-01

Since signals via CD40 and the B cell receptor are known to synergize to induce B cell activation, we have analyzed the pocket protein/E2F complexes in mouse B lymphocytes following stimulation by anti-IgM, anti-CD40, alone or together. We find that E2F4 and DP1 form the predominant E2F heterodimers in the G0 and G1 phases of the cell cycle, complexed with hypophosphorylated p130. During late G1 and S phase this complex is replaced by at least three different E2F complexes, one of which is an E2F complex containing p107 or pRB as well as two "free" E2F complexes consisting of E2F4/DP1 and E2F1-3/DP1. These effects were mirrored by the levels and phosphorylation status of the three pocket proteins. We also observed an increase in electrophoretic mobility of DP1 and E2F4 as B cells progressed from G0 into early G1, resulting from their dephosphorylation. This is known to correlate with a decrease in DNA binding capacity of these proteins and could also be important for derepression of genes negatively regulated through E2F sites in their promoters. These results therefore indicate that the pRB/E2F pathway integrates proliferative signals emanating from the sIgM and CD40 receptors.
Insight from TonB Hybrid Proteins into the Mechanism of Iron Transport through the Outer Membrane▿

PubMed Central

Kaserer, Wallace A.; Jiang, Xiaoxu; Xiao, Qiaobin; Scott, Daniel C.; Bauler, Matthew; Copeland, Daniel; Newton, Salete M. C.; Klebba, Phillip E.

2008-01-01

We created hybrid proteins to study the functions of TonB. We first fused the portion of Escherichia coli tonB that encodes the C-terminal 69 amino acids (amino acids 170 to 239) of TonB downstream from E. coli malE (MalE-TonB69C). Production of MalE-TonB69C in tonB+ bacteria inhibited siderophore transport. After overexpression and purification of the fusion protein on an amylose column, we proteolytically released the TonB C terminus and characterized it. Fluorescence spectra positioned its sole tryptophan (W213) in a weakly polar site in the protein interior, shielded from quenchers. Affinity chromatography showed the binding of the TonB C-domain to other proteins: immobilized TonB-dependent (FepA and colicin B) and TonB-independent (FepAΔ3-17, OmpA, and lysozyme) proteins adsorbed MalE-TonB69C, revealing a general affinity of the C terminus for other proteins. Additional constructions fused full-length TonB upstream or downstream of green fluorescent protein (GFP). TonB-GFP constructs had partial functionality but no fluorescence; GFP-TonB fusion proteins were functional and fluorescent. The activity of the latter constructs, which localized GFP in the cytoplasm and TonB in the cell envelope, indicate that the TonB N terminus remains in the inner membrane during its biological function. Finally, sequence analyses revealed homology in the TonB C terminus to E. coli YcfS, a proline-rich protein that contains the lysin (LysM) peptidoglycan-binding motif. LysM structural mimicry occurs in two positions of the dimeric TonB C-domain, and experiments confirmed that it physically binds to the murein sacculus. Together, these findings infer that the TonB N terminus remains associated with the inner membrane, while the downstream region bridges the cell envelope from the affinity of the C terminus for peptidoglycan. This architecture suggests a membrane surveillance model of action, in which TonB finds occupied receptor proteins by surveying the underside of
Monoclonal Antibody Analysis and Insecticidal Spectrum of Three Types of Lepidopteran-Specific Insecticidal Crystal Proteins of Bacillus thuringiensis

PubMed Central

Höfte, Herman; Van Rie, Jeroen; Jansens, Stefan; Van Houtven, Annemie; Vanderbruggen, Hilde; Vaeck, Mark

1988-01-01

We have investigated the protein composition and the insecticidal spectrum of crystals of 29 Bacillus thuringiensis strains active against lepidopteran larvae. All crystals contained proteins of 130 to 140 kilodaltons (kDa) which could be grouped into three types by the molecular weight of the protoxin and the trypsin-activated core fragment. Proteins of the three types showed a characteristic insecticidal spectrum when tested against five lepidopteran species. Type A crystal proteins were protoxins of 130 or 133 kDa, which were processed into 60-kDa toxins by trypsin. Several genes encoding crystal proteins of this type have been cloned and sequenced earlier. They are highly conserved in the N-terminal half of the toxic fragment and were previously classified in three subtypes (the 4.5-, 5.3-, and 6.6-kilobase subtypes) based on the restriction map of their genes. The present study shows that different proteins of these three subtypes were equally toxic against Manduca sexta and Pieris brassicae and had no detectable activity against Spodoptera littoralis. However, the 4.5-, 5.3-, and 6.6-kilobase subtypes differed in their toxicity against Heliothis virescens and Mamestra brassicae. Type B crystal proteins consisted of 140-kDa protoxins with a 55-kDa tryptic core fragment. These were only active against one of the five insect species tested (P. brassicae). The protoxin and the trypsin-activated toxin of type C were 135- and 63-kDa proteins, respectively. Proteins of this type were associated with high toxicity against S. littoralis and M. brassicae. A panel of 35 monoclonal antibodies was used to compare the structural characteristics of crystal proteins of the three different types and subtypes. Each type of protein could be associated with a typical epitope structure, indicating an unambiguous correlation between antigenic structure and insect specificity. Images PMID:16347711
Bacterially produced human B7-1 protein encompassing its complete extracellular domain maintains its costimulatory activity in vitro.

PubMed

Shen, W; Wang, Y; Geng, Y; Si, L

2000-08-01

To investigate which of the two immunoglobulin (Ig)-like domains, immunoglobulin variable region homologous domain IgV (hB7-1 IgV), or immunoglobulin constant region homologous domain IgC (hB7-1 IgC) on human B7-1 molecule contain the receptor binding sites, and to evaluate if the B7-1 molecule expressed in bacteria has biological activity. PCR was used to amplify three fragments of hB7-1 IgV, hB7-1 IgC and complete extracellular region of human B7-1 containing both the IgV and IgC domains (hB7-1 IgV + IgC). Three recombinants, pQE9-hB7-1 IgV, pQE9-hB7-1 IgC and pQE9-Hb7-1 (IgV + IgC) were generated by cloning the PCR products into a prokaryote expression plasmid (pQE-9) and were introduced into the host stain M15. The relevant target hexahistidine-tagged proteins were identified by SDS-PAGE and Western blotting. With the presence of the first signal imitated by anti-CD3 antibody, T cell activation was observed by exposing purified T lymphocytes to each soluble form of the three bacterially-produced human B7-1 proteins and [3H]-TdR incorporation. Three recombinant proteins of human B7-1, hB7-1 IgV, hB7-1 IgC and hB7-1 (IgV + IgC) were produced and detected in both soluble and inclusive body forms from engineered bacterial cells. With the presence of anti-CD3 antibody, T lymphocytes proliferated when co-stimulated by bacterially produced hB7-1 (IgV + IgC), but not by either hB7-1 IgV or hB7-1 IgC. Functional glycoprotein human B7-1 could be produced in bacterial cells. Both extracellular immunoglobulin-like domains are necessary for B7-1 to react with its counter receptors.
The FupA/B protein uniquely facilitates transport of ferrous iron and siderophore-associated ferric iron across the outer membrane of Francisella tularensis live vaccine strain

PubMed Central

Sen, Bhaswati

2014-01-01

Francisella tularensis is a highly infectious Gram-negative pathogen that replicates intracellularly within the mammalian host. One of the factors associated with virulence of F. tularensis is the protein FupA that mediates high-affinity transport of ferrous iron across the outer membrane. Together with its paralogue FslE, a siderophore–ferric iron transporter, FupA supports survival of the pathogen in the host by providing access to the essential nutrient iron. The FupA orthologue in the attenuated live vaccine strain (LVS) is encoded by the hybrid gene fupA/B, the product of an intergenic recombination event that significantly contributes to attenuation of the strain. We used 55Fe transport assays with mutant strains complemented with the different paralogues to show that the FupA/B protein of LVS retains the capacity for high-affinity transport of ferrous iron, albeit less efficiently than FupA of virulent strain Schu S4. 55Fe transport assays using purified siderophore and siderophore-dependent growth assays on iron-limiting agar confirmed previous findings that FupA/B also contributes to siderophore-mediated ferric iron uptake. These assays further demonstrated that the LVS FslE protein is a weaker siderophore–ferric iron transporter than the orthologue from Schu S4, and may be a result of the sequence variation between the two proteins. Our results indicate that iron-uptake mechanisms in LVS differ from those in Schu S4 and that functional differences in the outer membrane iron transporters have distinct effects on growth under iron limitation. PMID:24307666

Changes in the Polypeptide Patterns of Barley Seedlings Exposed to Jasmonic Acid and Salinity 1

PubMed Central

Maslenkova, Liliana Todorova; Miteva, Tania Simeonova; Popova, Losanka P.

1992-01-01

Soluble and thylakoid membrane proteins of jasmonic acid (JA)-treated and salt-stressed barley (Hordeum vulgare L.) seedlings were investigated using 15% sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis. High JA concentrations induced marked quantitative and qualitative changes in polypeptide profiles concerning mainly the proteins with approximately equal mobility, as in NaCl-stressed plants. The most obvious increase in thylakoid polypeptide band intensity was at 55 to 57 kilodaltons (kD). The relative share of some polypeptides with apparent molecular masses above 66 kD and of polypeptides with lower molecular masses in the region of 20.5 to 15 kD was enhanced. At the same time, one new band at 31 to 31.5 kD was well expressed at 25 and 250 micromolar JA concentrations and became discernible in the 100 micromolar NaCl-treated plants. The intensity of some polypeptides of soluble proteins (molecular masses of 60, 47, 37, 30, and 23.4 kD) increased with increasing JA concentration, whereas the intensities of other polypeptide bands (55, 21.4, and 15 kD) decreased. Enhanced levels of 60-, 47-, 34-, and 30-kD polypeptides and reduced levels of 55- and 15-kD polypeptides were present in NaCl-treated plants. The appearance of one new polypeptide, of 25.1 kD, was observed only in NaCl-treated plants. At 100 millimolar NaCl, an eightfold increase in proline content was observed while at 250 micromolar JA, the proline content was threefold over the control. It is hypothesized that exogenously applied jasmonates act as stress agents. As such, they provoke alterations in the proline content and they can modulate typical stress responses by induction of stress proteins. ImagesFigure 1Figure 4Figure 5 PMID:16668698
N-acetyl cysteine inhibits lipopolysaccharide-mediated induction of interleukin-6 synthesis in MC3T3-E1 cells through the NF-kB signaling pathway.

PubMed

Guo, Ling; Zhang, Hui; Li, Wangyang; Zhan, Danting; Wang, Min

2018-06-06

Interleukin-6 (IL-6) is a potent stimulator of osteoclastic activity. Lipopolysaccharide (LPS) has been shown to regulate the expression of potent inflammatory factors, including TNF-α and IL-6. Currently, effective therapeutic treatments for bacteria-caused bone destruction are limited. N-acetyl cysteine (NAC) is an antioxidant small molecule that possibly modulates osteoblastic differentiation. However, whether NAC can affect the LPS-mediated reduction of IL-6 synthesis in MC3T3-E1 cells is still unknown. The aim of this study was to investigate the role of NAC in the LPS -mediated reduction of IL-6 synthesis by MC3T3-E1 cells and to explore the underlying molecular mechanisms. In addition, we aimed to determine the involvement of the NF-kB pathway in any changes in IL-6 expression observed in response to LPS and NAC. MC3T3-E1 cells (ATCC, CRL-2593) were cultured in α-minimum essential medium. Cells were stimulated using NAC or LPS at various concentrations. Cell proliferation was observed at multiple time points using a cell counting kit 8 (CCK-8). IL-6 mRNA expression and protein synthesis were determined using quantitative polymerase chain reaction (qPCR) and enzyme-linked immunosorbent assay analyses. NF-kB mRNA expression and protein synthesis was determined using qPCR and Western blots analyses. The results demonstrate that LPS induced IL-6 and NF-kB mRNA expression and protein synthesis in the cultured MC3T3-E1 cells. However, these effects were abolished following pre-treatment with NAC. Pretreatment with NAC (1 mmol/l) or BAY11-7082 (10 μmol/l) both significantly inhibited the NF-kB activity induced by LPS. NAC inhibits the LPS-mediated induction of IL-6 synthesis in MC3T3-E1 cells through the NF-kB pathway. Copyright © 2018 Elsevier Ltd. All rights reserved.
46 CFR 91.55-1 - General.

Code of Federal Regulations, 2012 CFR

2012-10-01

... 46 Shipping 4 2012-10-01 2012-10-01 false General. 91.55-1 Section 91.55-1 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) CARGO AND MISCELLANEOUS VESSELS INSPECTION AND CERTIFICATION Plan... 44th Street, New York, NY 10036, on the Internet at http://www.icgb.com. [CGFR 65-50, 30 FR 16974, Dec...
46 CFR 91.55-1 - General.

Code of Federal Regulations, 2011 CFR

2011-10-01

... 46 Shipping 4 2011-10-01 2011-10-01 false General. 91.55-1 Section 91.55-1 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) CARGO AND MISCELLANEOUS VESSELS INSPECTION AND CERTIFICATION Plan... 44th Street, New York, NY 10036, on the Internet at http://www.icgb.com. [CGFR 65-50, 30 FR 16974, Dec...
46 CFR 91.55-1 - General.

Code of Federal Regulations, 2013 CFR

2013-10-01

... 46 Shipping 4 2013-10-01 2013-10-01 false General. 91.55-1 Section 91.55-1 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) CARGO AND MISCELLANEOUS VESSELS INSPECTION AND CERTIFICATION Plan... 44th Street, New York, NY 10036, on the Internet at http://www.icgb.com. [CGFR 65-50, 30 FR 16974, Dec...
46 CFR 91.55-1 - General.

Code of Federal Regulations, 2014 CFR

2014-10-01

... 46 Shipping 4 2014-10-01 2014-10-01 false General. 91.55-1 Section 91.55-1 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) CARGO AND MISCELLANEOUS VESSELS INSPECTION AND CERTIFICATION Plan... 44th Street, New York, NY 10036, on the Internet at http://www.icgb.com. [CGFR 65-50, 30 FR 16974, Dec...
VIP1 is very important/interesting protein 1 regulating touch responses of Arabidopsis.

PubMed

Tsugama, Daisuke; Liu, Shenkui; Takano, Tetsuo

2016-06-02

VIP1 (VIRE2-INTERACTING PROTEIN 1) is a bZIP transcription factor in Arabidopsis thaliana. VIP1 and its close homologs (i.e., Arabidopsis group I bZIP proteins) are present in the cytoplasm under steady conditions, but are transiently localized to the nucleus when cells are exposed to hypo-osmotic conditions, which mimic mechanical stimuli such as touch. Recently we have reported that overexpression of a repression domain-fused form of VIP1 represses the expression of some touch-responsive genes, changes structures and/or local auxin responses of the root cap cells, and enhances the touch-induced root waving. This raises the possibility that VIP1 suppresses touch-induced responses. VIP1 should be useful to further characterize touch responses of plants. Here we discuss 2 seemingly interesting perspectives about VIP1: (1) What factors are involved in regulating the nuclear localization of VIP1?; (2) What can be done to further characterize the physiological functions of VIP1 and other Arabidopsis group I bZIP proteins?
Studies of the mechanism of selectivity of protein tyrosine phosphatase 1B (PTP1B) bidentate inhibitors using molecular dynamics simulations and free energy calculations.

PubMed

Fang, Lei; Zhang, Huai; Cui, Wei; Ji, Mingjun

2008-10-01

Bidentate inhibitors of protein tyrosine phosphatase 1B (PTP1B) are considered as a group of ideal inhibitors with high binding potential and high selectivity in treating type II diabetes. In this paper, the binding models of five bidentate inhibitors to PTP1B, TCPTP, and SHP-2 were investigated and compared by using molecular dynamics (MD) simulations and free energy calculations. The binding free energies were computed using the Molecular Mechanics/Poisson-Boltzmann Surface Area (MM/PBSA) methodology. The calculation results show that the predicted free energies of the complexes are well consistent with the experimental data. The Molecular Mechanics/Generalized Born Surface Area (MM/GBSA) free energy decomposition analysis indicates that the residues ARG24, ARG254, and GLN262 in the second binding site of PTP1B are essential for the high selectivity of inhibitors. Furthermore, the residue PHE182 close to the active site is also important for the selectivity and the binding affinity of the inhibitors. According to our analysis, it can be concluded that in most cases the polarity of the portion of the inhibitor that binds to the second binding site of the protein is positive to the affinity of the inhibitors while negative to the selectivity of the inhibitors. We expect that the information we obtained here can help to develop potential PTP1B inhibitors with more promising specificity.
The cannabinoid receptor CB1 modulates the signaling properties of the lysophosphatidylinositol receptor GPR55.

PubMed

Kargl, Julia; Balenga, Nariman; Parzmair, Gerald P; Brown, Andrew J; Heinemann, Akos; Waldhoer, Maria

2012-12-28

The G protein-coupled receptor (GPCR) 55 (GPR55) and the cannabinoid receptor 1 (CB1R) are co-expressed in many tissues, predominantly in the central nervous system. Seven transmembrane spanning (7TM) receptors/GPCRs can form homo- and heteromers and initiate distinct signaling pathways. Recently, several synthetic CB1 receptor inverse agonists/antagonists, such as SR141716A, AM251, and AM281, were reported to activate GPR55. Of these, SR141716A was marketed as a promising anti-obesity drug, but was withdrawn from the market because of severe side effects. Here, we tested whether GPR55 and CB1 receptors are capable of (i) forming heteromers and (ii) whether such heteromers could exhibit novel signaling patterns. We show that GPR55 and CB1 receptors alter each others signaling properties in human embryonic kidney (HEK293) cells. We demonstrate that the co-expression of FLAG-CB1 receptors in cells stably expressing HA-GPR55 specifically inhibits GPR55-mediated transcription factor activation, such as nuclear factor of activated T-cells and serum response element, as well as extracellular signal-regulated kinases (ERK1/2) activation. GPR55 and CB1 receptors can form heteromers, but the internalization of both receptors is not affected. In addition, we observe that the presence of GPR55 enhances CB1R-mediated ERK1/2 and nuclear factor of activated T-cell activation. Our data provide the first evidence that GPR55 can form heteromers with another 7TM/GPCR and that this interaction with the CB1 receptor has functional consequences in vitro. The GPR55-CB1R heteromer may play an important physiological and/or pathophysiological role in tissues endogenously co-expressing both receptors.
The Cannabinoid Receptor CB1 Modulates the Signaling Properties of the Lysophosphatidylinositol Receptor GPR55*

PubMed Central

Kargl, Julia; Balenga, Nariman; Parzmair, Gerald P.; Brown, Andrew J.; Heinemann, Akos; Waldhoer, Maria

2012-01-01

The G protein-coupled receptor (GPCR) 55 (GPR55) and the cannabinoid receptor 1 (CB1R) are co-expressed in many tissues, predominantly in the central nervous system. Seven transmembrane spanning (7TM) receptors/GPCRs can form homo- and heteromers and initiate distinct signaling pathways. Recently, several synthetic CB1 receptor inverse agonists/antagonists, such as SR141716A, AM251, and AM281, were reported to activate GPR55. Of these, SR141716A was marketed as a promising anti-obesity drug, but was withdrawn from the market because of severe side effects. Here, we tested whether GPR55 and CB1 receptors are capable of (i) forming heteromers and (ii) whether such heteromers could exhibit novel signaling patterns. We show that GPR55 and CB1 receptors alter each others signaling properties in human embryonic kidney (HEK293) cells. We demonstrate that the co-expression of FLAG-CB1 receptors in cells stably expressing HA-GPR55 specifically inhibits GPR55-mediated transcription factor activation, such as nuclear factor of activated T-cells and serum response element, as well as extracellular signal-regulated kinases (ERK1/2) activation. GPR55 and CB1 receptors can form heteromers, but the internalization of both receptors is not affected. In addition, we observe that the presence of GPR55 enhances CB1R-mediated ERK1/2 and nuclear factor of activated T-cell activation. Our data provide the first evidence that GPR55 can form heteromers with another 7TM/GPCR and that this interaction with the CB1 receptor has functional consequences in vitro. The GPR55-CB1R heteromer may play an important physiological and/or pathophysiological role in tissues endogenously co-expressing both receptors. PMID:23161546
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
Reduction of adenovirus E1A mRNA by RNAi results in enhanced recombinant protein expression in transiently transfected HEK293 cells.

PubMed

Hacker, David L; Bertschinger, Martin; Baldi, Lucia; Wurm, Florian M

2004-10-27

Human embryonic kidney 293 (HEK293) cells, a widely used host for large-scale transient expression of recombinant proteins, are transformed with the adenovirus E1A and E1B genes. Because the E1A proteins function as transcriptional activators or repressors, they may have a positive or negative effect on transient transgene expression in this cell line. Suspension cultures of HEK293 EBNA (HEK293E) cells were co-transfected with a reporter plasmid expressing the GFP gene and a plasmid expressing a short hairpin RNA (shRNA) targeting the E1A mRNAs for degradation by RNA interference (RNAi). The presence of the shRNA in HEK293E cells reduced the steady state level of E1A mRNA up to 75% and increased transient GFP expression from either the elongation factor-1alpha (EF-1alpha) promoter or the human cytomegalovirus (HCMV) immediate early promoter up to twofold. E1A mRNA depletion also resulted in a twofold increase in transient expression of a recombinant IgG in both small- and large-scale suspension cultures when the IgG light and heavy chain genes were controlled by the EF-1alpha promoter. Finally, transient IgG expression was enhanced 2.5-fold when the anti-E1A shRNA was expressed from the same vector as the IgG light chain gene. These results demonstrated that E1A has a negative effect on transient gene expression in HEK293E cells, and they established that RNAi can be used to enhance recombinant protein expression in mammalian cells.
Differential effects of human SP-A1 and SP-A2 variants on phospholipid monolayers containing surfactant protein B

PubMed Central

Wang, Guirong; Taneva, Svetla; Keough, Kevin M.W.; Floros, Joanna

2010-01-01

Summary Surfactant protein A (SP-A), the most abundant protein in the lung alveolar surface, has multiple activities, including surfactant-related functions. SP-A is required for the formation of tubular myelin and the lung surface film. The human SP-A locus consists of two functional SP-A genes, SP-A1 and SP-A2, with a number of alleles characterized for each gene. We have found that the human in vitro expressed variants, SP-A1 (6A2) and SP-A2 (1A0), and the coexpressed SP-A1/SP-A2 (6A2/1A0) protein have a differential influence on the organization of phospholipid monolayers containing surfactant protein B (SP-B). Lipid films containing SP-B and SP-A2 (1A0) showed surface features similar to those observed in lipid films with SP-B and native human SP-A. Fluorescence images revealed the presence of characteristic fluorescent probe-excluding clusters coexisting with the traditional lipid liquid-expanded and liquid-condensed phase. Images of the films containing SP-B and SP-A1 (6A2) showed different distribution of the proteins. The morphology of lipid films containing SP-B and the coexpressed SP-A1/SP-A2 (6A2/1A0) combined features of the individual films containing the SP-A1 or SP-A2 variant. The results indicate that human SP-A1 and SP-A2 variants exhibit differential effects on characteristics of phospholipid monolayers containing SP-B. This may differentially impact surface film activity. PMID:17678872
Resolution of the diadenosine 5',5"'-P1,P4-tetraphosphate binding subunit from a multiprotein form of HeLa cell DNA polymerase alpha.

PubMed Central

Baril, E; Bonin, P; Burstein, D; Mara, K; Zamecnik, P

1983-01-01

A diadenosine 5',5"'-P1,P4-tetraphosphate (Ap4A) binding subunit has been resolved from a high molecular weight (640,000) multiprotein form of DNA polymerase alpha [deoxynucleoside triphosphate:DNA nucleotidyltransferase (DNA-directed), EC 2.7.7.7] from HeLa cells [DNA polymerase alpha 2 of Lamothe, P., Baril, B., Chi, A., Lee, L. & Baril, E. (1981) Proc. Natl. Acad. Sci. USA 78, 4723-4727]. The Ap4A binding activity copurifies with the DNA polymerizing activity during the course of purification. Hydrophobic chromatography on butylagarose resolves the Ap4A binding activity from the DNA polymerase. The Ap4A binding activity is protein in nature since the binding of Ap4A is abolished by treatment of the isolated binding activity with proteinase K but is insensitive to treatment with DNase or RNase. The molecular weight of the Ap4A binding protein, as determined by polyacrylamide gel electrophoresis under nondenaturing conditions or by NaDodSO4/polyacrylamide gel electrophoresis after photoaffinity labeling of the protein with [32P]Ap4A is 92,000 or 47,000. The binding activity of this protein is highly specific for Ap4A. Images PMID:6576366
Sesquiterpenoids with PTP1B Inhibitory Activity and Cytotoxicity from the Edible Mushroom Pleurotus citrinopileatus.

PubMed

Tao, Qiao-Qiao; Ma, Ke; Bao, Li; Wang, Kai; Han, Jun-Jie; Wang, Wen-Zhao; Zhang, Jin-Xia; Huang, Chen-Yang; Liu, Hong-Wei

2016-05-01

One new perhydrobenzannulated 5,5-spiroketal sesquiterpene, pleurospiroketal F (1), as well as six new modified bisabolene sesquiterpenes pleurotins A-F (2-7) were isolated from solid-state fermentation of Pleurotus citrinopileatus. The structures of compounds 1-7 were determined by NMR and MS spectroscopic analysis. The absolute configuration of 1 was determined by X-ray diffraction analysis, while the absolute configurations of 3-7 were assigned using the in situ dimolybdenum circular dichroism method and circular dichroism data comparison. Protein tyrosine phosphatase 1B plays a crucial role as a negative regulator of the insulin-dependent signal cascades. Therefore, the protein tyrosine phosphatase 1B inhibitor can be used for treating type 2 diabetes mellitus and obesity. Compounds 2 and 6 showed moderate inhibitory effects on protein tyrosine phosphatase 1B with IC50 s of 32.1 µM and 30.5 µM, respectively. The kinetic study confirmed compound 2 to be a noncompetitive inhibitor. Compounds 1-7 did not show cytotoxic activity against cancer cell lines (IC50 > 50 µM). Georg Thieme Verlag KG Stuttgart · New York.
Glial S100B protein modulates mutant ataxin-1 aggregation and toxicity: TRTK12 peptide, a potential candidate for SCA1 therapy.

PubMed

Vig, Parminder J S; Hearst, Scoty; Shao, Qingmei; Lopez, Mariper E; Murphy, Henry A; Safaya, Eshan

2011-06-01

Non-cell autonomous involvement of glial cells in the pathogenesis of polyglutamine diseases is gaining recognition in the ataxia field. We previously demonstrated that Purkinje cells (PCs) in polyglutamine disease spinocerebellar ataxia-1 (SCA1) contain cytoplasmic vacuoles rich in Bergmann glial protein S100B. The vacuolar formation in SCA1 PCs is accompanied with an abnormal morphology of dendritic spines. In addition, S100B messenger RNA (mRNA) expression levels are significantly high in the cerebella of asymptomatic SCA1 transgenic (Tg) mice and increase further with age when compared with the age-matched wild-type animals. This higher S100B mRNA expression positively correlates with an increase in the number of vacuoles. To further characterize the function of S100B in SCA1 pathology, we explored the effects of S100B protein on GFP-ataxin-1 (ATXN1) with expanded polyglutamines [82Q] in HEK stable cell line. Externally added S100B protein to these cells induced S100B-positive vacuoles similar to those seen in SCA1 PCs in vivo. Further, we found that both externally added and internally expressed S100B significantly reduced GFP-ATXN1[82Q] inclusion body formation. In contrast, the addition of S100B inhibitory peptide TRTK12 reversed S100B-mediated effects. Interestingly, in SCA1 Tg mice, PCs containing S100B vacuoles also showed the lack of nuclear inclusions, whereas PCs without vacuoles contained nuclear inclusions. Additionally, TRTK12 treatment reduced abnormal dendritic growth and morphology of PCs in cerebellar slice cultures prepared from SCA1 Tg mice. Moreover, intranasal administration of TRTK12 to SCA1 Tg mice reduced cerebellar S100B levels in the particulate fractions, and these mice displayed a significant improvement in their performance deficit on the Rotarod test. Taken together, our results suggest that glial S100B may augment degenerative changes in SCA1 PCs by modulating mutant ataxin-1 toxicity/solubility through an unknown signaling pathway.
Glial S100B protein modulates mutant ataxin-1 aggregation and toxicity: TRTK12 peptide, a potential candidate for SCA1 therapy

PubMed Central

Vig, Parminder J.S.; Hearst, Scoty; Shao, Qingmei; Lopez, Maripar E; Murphy, Henry A; Safaya, Eshan

2011-01-01

Non-cell autonomous involvement of glial cells in the pathogenesis of polyglutamine diseases is gaining recognition in the ataxia field. We previously demonstrated that Purkinje cells (PCs) in polyglutamine disease spinocerebellar ataxia-1 (SCA1) contain cytoplasmic vacuoles rich in Bergmann glial (BG) protein S100B. The vacuolar formation in SCA1 PCs is accompanied with an abnormal morphology of dendritic spines. In addition, S100B mRNA expression levels are significantly high in the cerebella of asymptomatic SCA1 transgenic (Tg) mice and increase further with age when compared with the age-matched wildtype animals. This higher S100B mRNA expression positively correlates with an increase in the number of vacuoles. To further characterize the function of S100B in SCA1 pathology, we explored the effects of S100B protein on GFP-ataxin-1 (ATXN1) with expanded polyglutamines [82Q] in HEK stable cell line. Externally added S100B protein to these cells induced S100B positive vacuoles similar to those seen in SCA1 PCs in vivo. Further, we found that both externally added and internally expressed S100B significantly reduced GFP-ATXN1[82Q] inclusion body formation. In contrast, the addition of S100B inhibitory peptide TRTK12 reversed S100B mediated effects. Interestingly, in SCA1 Tg mice, PCs containing S100B vacuoles also showed the lack of nuclear inclusions, whereas, PCs without vacuoles contained nuclear inclusions. Additionally, TRTK12 treatment reduced abnormal dendritic growth and morphology of PCs in cerebellar slice cultures prepared from SCA1 Tg mice. Moreover, intranasal administration of TRTK12 to SCA1 Tg mice reduced cerebellar S100B levels in the particulate fractions and these mice displayed a significant improvement in their performance deficit on the Rotarod test. Taken together our results suggest that glial S100B may augment degenerative changes in SCA1 PCs by modulating mutant ataxin-1 toxicity/solubility through an unknown signaling pathway. PMID
MSE55, a Cdc42 effector protein, induces long cellular extensions in fibroblasts

PubMed Central

Burbelo, Peter D.; Snow, Dianne M.; Bahou, Wadie; Spiegel, Sarah

1999-01-01

Cdc42 is a member of the Rho GTPase family that regulates multiple cellular activities, including actin polymerization, kinase-signaling activation, and cell polarization. MSE55 is a nonkinase CRIB (Cdc42/Rac interactive-binding) domain-containing molecule of unknown function. Using glutathione S-transferase-capture experiments, we show that MSE55 binds to Cdc42 in a GTP-dependent manner. MSE55 binding to Cdc42 required an intact CRIB domain, because a MSE55 CRIB domain mutant no longer interacted with Cdc42. To study the function of MSE55 we transfected either wild-type MSE55 or a MSE55 CRIB mutant into mammalian cells. In Cos-7 cells, wild-type MSE55 localized at membrane ruffles and increased membrane actin polymerization, whereas expression of the MSE55 CRIB mutant showed fewer membrane ruffles. In contrast to these results, MSE55 induced the formation of long, actin-based protrusions in NIH 3T3 cells as detected by immunofluorescence and live-cell video microscopy. MSE55-induced protrusion formation was blocked by expression of dominant-negative N17Cdc42, but not by expression of dominant-negative N17Rac. These findings indicate that MSE55 is a Cdc42 effector protein that mediates actin cytoskeleton reorganization at the plasma membrane. PMID:10430899
Epstein-Barr Virus-Encoded Latent Membrane Protein 1 Upregulates Glucose Transporter 1 Transcription via the mTORC1/NF-κB Signaling Pathways

PubMed Central

Zhang, Jun; Jia, Lin; Lin, Weitao; Yip, Yim Ling; Lo, Kwok Wai; Lau, Victoria Ming Yi; Zhu, Dandan; Tsang, Chi Man; Zhou, Yuan; Deng, Wen; Lung, Hong Lok; Lung, Maria Li; Cheung, Lai Man

2017-01-01

ABSTRACT Accumulating evidence indicates that oncogenic viral protein plays a crucial role in activating aerobic glycolysis during tumorigenesis, but the underlying mechanisms are largely undefined. Epstein-Barr virus (EBV)-encoded latent membrane protein 1 (LMP1) is a transmembrane protein with potent cell signaling properties and has tumorigenic transformation property. Activation of NF-κB is a major signaling pathway mediating many downstream transformation properties of LMP1. Here we report that activation of mTORC1 by LMP1 is a key modulator for activation of NF-κB signaling to mediate aerobic glycolysis. NF-κB activation is involved in the LMP1-induced upregulation of glucose transporter 1 (Glut-1) transcription and growth of nasopharyngeal carcinoma (NPC) cells. Blocking the activity of mTORC1 signaling effectively suppressed LMP1-induced NF-κB activation and Glut-1 transcription. Interfering NF-κB signaling had no effect on mTORC1 activity but effectively altered Glut-1 transcription. Luciferase promoter assay of Glut-1 also confirmed that the Glut-1 gene is a direct target gene of NF-κB signaling. Furthermore, we demonstrated that C-terminal activating region 2 (CTAR2) of LMP1 is the key domain involved in mTORC1 activation, mainly through IKKβ-mediated phosphorylation of TSC2 at Ser939. Depletion of Glut-1 effectively led to suppression of aerobic glycolysis, inhibition of cell proliferation, colony formation, and attenuation of tumorigenic growth property of LMP1-expressing nasopharyngeal epithelial (NPE) cells. These findings suggest that targeting the signaling axis of mTORC1/NF-κB/Glut-1 represents a novel therapeutic target against NPC. IMPORTANCE Aerobic glycolysis is one of the hallmarks of cancer, including NPC. Recent studies suggest a role for LMP1 in mediating aerobic glycolysis. LMP1 expression is common in NPC. The delineation of essential signaling pathways induced by LMP1 in aerobic glycolysis contributes to the understanding of NPC
Translation elongation factor EF-Tu modulates filament formation of actin-like MreB protein in vitro.

PubMed

Defeu Soufo, Hervé Joël; Reimold, Christian; Breddermann, Hannes; Mannherz, Hans G; Graumann, Peter L

2015-04-24

EF-Tu has been shown to interact with actin-like protein MreB and to affect its localization in Escherichia coli and in Bacillus subtilis cells. We have purified YFP-MreB in an active form, which forms filaments on glass slides in vitro and was active in dynamic light-scattering assays, polymerizing in milliseconds after addition of magnesium. Purified EF-Tu enhanced the amount of MreB filaments, as seen by sedimentation assays, the speed of filament formation and the length of MreB filaments in vitro. EF-Tu had the strongest impact on MreB filaments in a 1:1 ratio, and EF-Tu co-sedimented with MreB filaments, revealing a stoichiometric interaction between both proteins. This was supported by cross-linking assays where 1:1 species were well detectable. When expressed in E. coli cells, B. subtilis MreB formed filaments and induced the formation of co-localizing B. subtilis EF-Tu structures, indicating that MreB can direct the positioning of EF-Tu structures in a heterologous cell system. Fluorescence recovery after photobleaching analysis showed that MreB filaments have a higher turnover in B. subtilis cells than in E. coli cells, indicating different filament kinetics in homologous or heterologous cell systems. The data show that MreB can direct the localization of EF-Tu in vivo, which in turn positively affects the formation and dynamics of MreB filaments. Thus, EF-Tu is a modulator of the activity of a bacterial actin-like protein. Copyright © 2015. Published by Elsevier Ltd.

Deficiency in Protein Tyrosine Phosphatase PTP1B Shortens Lifespan and Leads to Development of Acute Leukemia.

PubMed

Le Sommer, Samantha; Morrice, Nicola; Pesaresi, Martina; Thompson, Dawn; Vickers, Mark A; Murray, Graeme I; Mody, Nimesh; Neel, Benjamin G; Bence, Kendra K; Wilson, Heather M; Delibegović, Mirela

2018-01-01

Protein tyrosine phosphatase PTP1B is a critical regulator of signaling pathways controlling metabolic homeostasis, cell proliferation, and immunity. In this study, we report that global or myeloid-specific deficiency of PTP1B in mice decreases lifespan. We demonstrate that myeloid-specific deficiency of PTP1B is sufficient to promote the development of acute myeloid leukemia. LysM-PTP1B -/- mice lacking PTP1B in the innate myeloid cell lineage displayed a dysregulation of bone marrow cells with a rapid decline in population at midlife and a concomitant increase in peripheral blood blast cells. This phenotype manifested further with extramedullary tumors, hepatic macrophage infiltration, and metabolic reprogramming, suggesting increased hepatic lipid metabolism prior to overt tumor development. Mechanistic investigations revealed an increase in anti-inflammatory M2 macrophage responses in liver and spleen, as associated with increased expression of arginase I and the cytokines IL10 and IL4. We also documented STAT3 hypersphosphorylation and signaling along with JAK-dependent upregulation of antiapoptotic proteins Bcl2 and BclXL. Our results establish a tumor suppressor role for PTP1B in the myeloid lineage cells, with evidence that its genetic inactivation in mice is sufficient to drive acute myeloid leukemia. Significance: This study defines a tumor suppressor function for the protein tyrosine phosphatase PTP1B in myeloid lineage cells, with evidence that its genetic inactivation in mice is sufficient to drive acute myeloid leukemia. Cancer Res; 78(1); 75-87. ©2017 AACR . ©2017 American Association for Cancer Research.
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
VPAC2 receptor agonist BAY 55-9837 increases SMN protein levels and moderates disease phenotype in severe spinal muscular atrophy mouse models.

PubMed

Hadwen, Jeremiah; MacKenzie, Duncan; Shamim, Fahad; Mongeon, Kevin; Holcik, Martin; MacKenzie, Alex; Farooq, Faraz

2014-01-09

Spinal Muscular Atrophy (SMA) is one of the most common inherited causes of infant death and is caused by the loss of functional survival motor neuron (SMN) protein due to mutations or deletion in the SMN1 gene. One of the treatment strategies for SMA is to induce the expression of the protein from the homologous SMN2 gene, a rescuing paralog for SMA. Here we demonstrate the promise of pharmacological modulation of SMN2 gene by BAY 55-9837, an agonist of the vasoactive intestinal peptide receptor 2 (VPAC2), a member of G protein coupled receptor family. Treatment with BAY 55-9837 lead to induction of SMN protein levels via activation of MAPK14 or p38 pathway in vitro. Importantly, BAY 55-9837 also ameliorated disease phenotype in severe SMA mouse models. Our findings suggest the VPAC2 pathway is a potential SMA therapeutic target.
An Analysis of Transiting Hot Jupiters Observed with K2: WASP-55b and WASP-75b

NASA Astrophysics Data System (ADS)

Clark, B. J. M.; Anderson, D. R.; Hellier, C.; Turner, O. D.; Močnik, T.

2018-03-01

We present our analysis of the K2 short-cadence data of two previously known hot Jupiter exoplanets: WASP-55b and WASP-75b. The high precision of the K2 light curves enabled us to search for transit timing and duration variations, rotational modulation, starspots, phase-curve variations and additional transiting planets. We identified stellar variability in the WASP-75 light curve which may be an indication of rotational modulation, with an estimated period of 11.2 ± 1.5 days. We combined this with the spectroscopically measured v\\sin ({i}* ) to calculate a possible line of sight projected inclination angle of {i}* =41^\\circ +/- 16^\\circ . We also perform a global analysis of K2 and previously published data to refine the system parameters.
Protein Kinase C Controls Binding of Igo/ENSA Proteins to Protein Phosphatase 2A in Budding Yeast.

PubMed

Thai, Vu; Dephoure, Noah; Weiss, Amit; Ferguson, Jacqueline; Leitao, Ricardo; Gygi, Steven P; Kellogg, Douglas R

2017-03-24

Protein phosphatase 2A (PP2A) plays important roles in controlling mitosis in all eukaryotic cells. The form of PP2A that controls mitosis is associated with a conserved regulatory subunit that is called B55 in vertebrates and Cdc55 in budding yeast. The activity of this form of PP2A can be inhibited by binding of conserved Igo/ENSA proteins. Although the mechanisms that activate Igo/ENSA to bind and inhibit PP2A are well understood, little is known about how Igo/Ensa are inactivated. Here, we have analyzed regulation of Igo/ENSA in the context of a checkpoint pathway that links mitotic entry to membrane growth in budding yeast. Protein kinase C (Pkc1) relays signals in the pathway by activating PP2A Cdc55 We discovered that constitutively active Pkc1 can drive cells through a mitotic checkpoint arrest, which suggests that Pkc1-dependent activation of PP2A Cdc55 plays a critical role in checkpoint signaling. We therefore used mass spectrometry to determine how Pkc1 modifies the PP2A Cdc55 complex. This revealed that Pkc1 induces changes in the phosphorylation of multiple subunits of the complex, as well as dissociation of Igo/ENSA. Pkc1 directly phosphorylates Cdc55 and Igo/ENSA, and phosphorylation site mapping and mutagenesis indicate that phosphorylation of Cdc55 contributes to Igo/ENSA dissociation. Association of Igo2 with PP2A Cdc55 is regulated during the cell cycle, yet mutation of Pkc1-dependent phosphorylation sites on Cdc55 and Igo2 did not cause defects in mitotic progression. Together, the data suggest that Pkc1 controls PP2A Cdc55 by multiple overlapping mechanisms. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.
Protein-Tyrosine Phosphatase-1B Mediates Sleep Fragmentation-Induced Insulin Resistance and Visceral Adipose Tissue Inflammation in Mice.

PubMed

Gozal, David; Khalyfa, Abdelnaby; Qiao, Zhuanghong; Akbarpour, Mahzad; Maccari, Rosanna; Ottanà, Rosaria

2017-09-01

Sleep fragmentation (SF) is highly prevalent and has emerged as an important contributing factor to obesity and metabolic syndrome. We hypothesized that SF-induced increases in protein tyrosine phosphatase-1B (PTP-1B) expression and activity underlie increased food intake, inflammation, and leptin and insulin resistance. Wild-type (WT) and ObR-PTP-1b-/- mice (Tg) were exposed to SF and control sleep (SC), and food intake was monitored. WT mice received a PTP-1B inhibitor (RO-7d; Tx) or vehicle (Veh). Upon completion of exposures, systemic insulin and leptin sensitivity tests were performed as well as assessment of visceral white adipose tissue (vWAT) insulin receptor sensitivity and macrophages (ATM) polarity. SF increased food intake in either untreated or Veh-treated WT mice. Leptin-induced hypothalamic STAT3 phosphorylation was decreased, PTP-1B activity was increased, and reduced insulin sensitivity emerged both systemic and in vWAT, with the latter displaying proinflammatory ATM polarity changes. All of the SF-induced effects were abrogated following PTP-1B inhibitor treatment and in Tg mice. SF induces increased food intake, reduced leptin signaling in hypothalamus, systemic insulin resistance, and reduced vWAT insulin sensitivity and inflammation that are mediated by increased PTP-1B activity. Thus, PTP-1B may represent a viable therapeutic target in the context of SF-induced weight gain and metabolic dysfunction. © Sleep Research Society 2017. Published by Oxford University Press on behalf of the Sleep Research Society. All rights reserved. For permissions, please e-mail journals.permissions@oup.com.
Brachyspira hyodysenteriae and B. pilosicoli Proteins Recognized by Sera of Challenged Pigs.

PubMed

Casas, Vanessa; Rodríguez-Asiain, Arantza; Pinto-Llorente, Roberto; Vadillo, Santiago; Carrascal, Montserrat; Abian, Joaquin

2017-01-01

The spirochetes Brachyspira hyodysenteriae and B. pilosicoli are pig intestinal pathogens that are the causative agents of swine dysentery (SD) and porcine intestinal spirochaetosis (PIS), respectively. Although some inactivated bacterin and recombinant vaccines have been explored as prophylactic treatments against these species, no effective vaccine is yet available. Immunoproteomics approaches hold the potential for the identification of new, suitable candidates for subunit vaccines against SD and PIS. These strategies take into account the gene products actually expressed and present in the cells, and thus susceptible of being targets of immune recognition. In this context, we have analyzed the immunogenic pattern of two B. pilosicoli porcine isolates (the Spanish farm isolate OLA9 and the commercial P43/6/78 strain) and one B. hyodysenteriae isolate (the Spanish farm V1). The proteins from the Brachyspira lysates were fractionated by preparative isoelectric focusing, and the fractions were analyzed by Western blot with hyperimmune sera from challenged pigs. Of the 28 challenge-specific immunoreactive bands detected, 21 were identified as single proteins by MS, while the other 7 were shown to contain several major proteins. None of these proteins were detected in the control immunoreactive bands. The proteins identified included 11 from B. hyodysenteriae and 28 from the two B. pilosicoli strains. Eight proteins were common to the B. pilosicoli strains (i.e., elongation factor G, aspartyl-tRNA synthase, biotin lipoyl, TmpB outer membrane protein, flagellar protein FlaA, enolase, PEPCK, and VspD), and enolase and PEPCK were common to both species . Many of the identified proteins were flagellar proteins or predicted to be located on the cell surface and some of them had been previously described as antigenic or as bacterial virulence factors. Here we report on the identification and semiquantitative data of these immunoreactive proteins which constitute a unique
Hepatitis B virus X promotes hepatocellular carcinoma development via nuclear protein 1 pathway

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

Bak, Yesol; Shin, Hye-jun; Bak, In seon

Hepatocellular carcinoma (HCC) is one of the most common malignancies and chronic hepatitis B virus (HBV) infection is a major risk factor for HCC. Hepatitis B virus X (HBx) protein relates to trigger oncogenesis. HBx has oncogenic properties with a hyperproliferative response to HCC. Nuclear protein 1 (NUPR1) is a stress-response protein, frequently upregulated in several cancers. Recent data revealed that NUPR1 is involved in tumor progression, but its function in HCC is not revealed yet. Here we report HBx can induce NUPR1 in patients, mice, and HCC cell lines. In an HBx transgenic mouse model, we found that HBxmore » overexpression upregulates NUPR1 expression consistently with tumor progression. Further, in cultured HBV positive cells, HBx knockdown induces downregulation of NUPR1. Smad4 is a representative transcription factor, regulated by HBx, and we showed that HBx upregulates NUPR1 by Smad4 dependent way. We found that NUPR1 can inhibit cell death and induce vasculogenic mimicry in HCC cell lines. Moreover, NUPR1 silencing in HepG2-HBx showed reduced cell motility. These results suggest that HBx can modulate NUPR1 expression through the Smad4 pathway and NUPR1 has a role in hepatocellular carcinoma progression. - Highlights: • NUPR1 is overexpressed in HBx transgenic mouse and HCC patients. • NUPR1 inactivation hampers the HBx induced growth, VM formation, and migration of HepG2 cells in vitro. • NUPR1 has a role for survival of HCC and mechanistically NUPR1 is activated by HBx-Smad4 axis.« less
Murine T-Cell Response to Native and Recombinant Protein Antigens of Rickettsia Tsutsugamushi

DTIC Science & Technology

1993-02-01

Wright, and J. Sadoff. 1985. 18-kilodalton protein of Mycobacterium leprae recognized by Immunoenzymatic analysis by monoclonal antibodies of bacte- Vo...determinants and closely resembles T-cell antigenic determinants, Rothbard and Taylor, by the GroEL homolog (65 kDa) of Mycobacterium tuberculo- analysis of...not be completely present in protein that is recognized by 20% of the mycobacterium - peptide 91-110. If this were the core of the antigenic deter
Cannabinoid receptor agonist WIN55,212-2 and fatty acid amide hydrolase inhibitor URB597 ameliorate neuroinflammatory responses in chronic cerebral hypoperfusion model by blocking NF-κB pathways.

PubMed

Su, Shao-Hua; Wu, Yi-Fang; Lin, Qi; Hai, Jian

2017-12-01

The present study explored the protective effects of cannabinoid receptor agonist WIN55,212-2 (WIN) and fatty acid amide hydrolase inhibitor URB597 (URB) against neuroinflammation in rats with chronic cerebral hypoperfusion (CCH). Activated microglia, astrocytes, and nuclear factor kappa B (NF-κB) p65-positive cells were measured by immunofluorescence. Reactive oxygen species (ROS) was assessed by dihydroethidium staining. The protein levels of cluster of differentiation molecule 11b (OX-42), glial fibrillary acidic protein (GFAP), NF-κB p65, inhibitor of kappa B alpha (IκB-a), IκB kinase a/β (IKK a/β), phosphorylated IKK a/β (p-IKK a/β), cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS), tumor necrosis factor (TNF)-α, and interleukin-1β (IL-1β) were examined by western blotting or enzyme-linked immunosorbent assay. All the protein levels of OX-42, GFAP, TNF-a, IL-1β, COX-2, and iNOS are increased in CCH rats. WIN and URB downregulated the levels of OX-42, GFAP, TNF-α, IL-1β, COX-2 and iNOS and inhibited CCH-induced ROS accumulation in CCH rats, indicating that WIN and URB might exert their neuroprotective effects by inhibiting the neuroinflammatory response. In addition, the NF-κB signaling pathway was activated by CCH in frontal cortex and hippocampus, while the aforementioned changes were reversed by WIN and URB treatment. These findings suggest that WIN and URB treatment ameliorated CCH-induced neuroinflammation through inhibition of the classical pathway of NF-κB activation, resulting in mitigation of chronic ischemic injury.
The M Phase Kinase Greatwall (Gwl) Promotes Inactivation of PP2A/B55δ, a Phosphatase Directed Against CDK Phosphosites

PubMed Central

Castilho, Priscila V.; Williams, Byron C.; Mochida, Satoru; Zhao, Yong

2009-01-01

We have previously shown that Greatwall kinase (Gwl) is required for M phase entry and maintenance in Xenopus egg extracts. Here, we demonstrate that Gwl plays a crucial role in a novel biochemical pathway that inactivates, specifically during M phase, “antimitotic” phosphatases directed against phosphorylations catalyzed by cyclin-dependent kinases (CDKs). A major component of this phosphatase activity is heterotrimeric PP2A containing the B55δ regulatory subunit. Gwl is activated during M phase by Cdk1/cyclin B (MPF), but once activated, Gwl promotes PP2A/B55δ inhibition with no further requirement for MPF. In the absence of Gwl, PP2A/B55δ remains active even when MPF levels are high. The removal of PP2A/B55δ corrects the inability of Gwl-depleted extracts to enter M phase. These findings support the hypothesis that M phase requires not only high levels of MPF function, but also the suppression, through a Gwl-dependent mechanism, of phosphatase(s) that would otherwise remove MPF-driven phosphorylations. PMID:19793917
Sequence preservation of osteocalcin protein and mitochondrial DNA in bison bones older than 55 ka

NASA Astrophysics Data System (ADS)

Nielsen-Marsh, Christina M.; Ostrom, Peggy H.; Gandhi, Hasand; Shapiro, Beth; Cooper, Alan; Hauschka, Peter V.; Collins, Matthew J.

2002-12-01

We report the first complete sequences of the protein osteocalcin from small amounts (20 mg) of two bison bone (Bison priscus) dated to older than 55.6 ka and older than 58.9 ka. Osteocalcin was purified using new gravity columns (never exposed to protein) followed by microbore reversed-phase high-performance liquid chromatography. Sequencing of osteocalcin employed two methods of matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS): peptide mass mapping (PMM) and post-source decay (PSD). The PMM shows that ancient and modern bison osteocalcin have the same mass to charge (m/z) distribution, indicating an identical protein sequence and absence of diagenetic products. This was confirmed by PSD of the m/z 2066 tryptic peptide (residues 1 19); the mass spectra from ancient and modern peptides were identical. The 129 mass unit difference in the molecular ion between cow (Bos taurus) and bison is caused by a single amino-acid substitution between the taxa (Trp in cow is replaced by Gly in bison at residue 5). Bison mitochondrial control region DNA sequences were obtained from the older than 55.6 ka fossil. These results suggest that DNA and protein sequences can be used to directly investigate molecular phylogenies over a considerable time period, the absolute limit of which is yet to be determined.
Dissecting the Role of E2 Protein Domains in Alphavirus Pathogenicity.

PubMed

Weger-Lucarelli, James; Aliota, Matthew T; Wlodarchak, Nathan; Kamlangdee, Attapon; Swanson, Ryan; Osorio, Jorge E

2015-12-16

Alphaviruses represent a diverse set of arboviruses, many of which are important pathogens. Chikungunya virus (CHIKV), an arthritis-inducing alphavirus, is the cause of a massive ongoing outbreak in the Caribbean and South America. In contrast to CHIKV, other related alphaviruses, such as Venezuelan equine encephalitis virus (VEEV) and Semliki Forest virus (SFV), can cause encephalitic disease. E2, the receptor binding protein, has been implicated as a determinant in cell tropism, host range, pathogenicity, and immunogenicity. Previous reports also have demonstrated that E2 contains residues important for host range expansions and monoclonal antibody binding; however, little is known about what role each protein domain (e.g., A, B, and C) of E2 plays on these factors. Therefore, we constructed chimeric cDNA clones between CHIKV and VEEV or SFV to probe the effect of each domain on pathogenicity in vitro and in vivo. CHIKV chimeras containing each of the domains of the E2 (ΔDomA, ΔDomB, and ΔDomC) from SFV, but not VEEV, were successfully rescued. Interestingly, while all chimeric viruses were attenuated compared to CHIKV in mice, ΔDomB virus showed similar rates of infection and dissemination in Aedes aegypti mosquitoes, suggesting differing roles for the E2 protein in different hosts. In contrast to CHIKV; ΔDomB, and to a lesser extent ΔDomA, caused neuron degeneration and demyelination in mice infected intracranially, suggesting a shift toward a phenotype similar to SFV. Thus, chimeric CHIKV/SFV provide insights on the role the alphavirus E2 protein plays on pathogenesis. Chikungunya virus (CHIKV) has caused large outbreaks of acute and chronic arthritis throughout Africa and Southeast Asia and has now become a massive public health threat in the Americas, causing an estimated 1.2 million human cases in just over a year. No approved vaccines or antivirals exist for human use against CHIKV or any other alphavirus. Despite the threat, little is known about
A single amino acid substitution within the transmembrane domain of the human immunodeficiency virus type 1 Vpu protein renders simian-human immunodeficiency virus (SHIV{sub KU-1bMC33}) susceptible to rimantadine

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

Hout, David R.; Gomez, Lisa M.; Pacyniak, Erik

2006-05-10

Previous studies from our laboratory have shown that the transmembrane domain (TM) of the Vpu protein of human immunodeficiency virus type 1 (HIV-1) contributes to the pathogenesis of SHIV{sub KU-1bMC33} in macaques and that the TM domain of Vpu could be replaced with the M2 protein viroporin from influenza A virus. Recently, we showed that the replacement of the TM domain of Vpu with that of the M2 protein of influenza A virus resulted in a virus (SHIV{sub M2}) that was sensitive to rimantadine [Hout, D.R., Gomez, M.L., Pacyniak, E., Gomez, L.M., Inbody, S.H., Mulcahy, E.R., Culley, N., Pinson, D.M.,more » Powers, M.F., Wong, S.W., Stephens, E.B., 2006. Substitution of the transmembrane domain of Vpu in simian human immunodeficiency virus (SHIV{sub KU-1bMC33}) with that of M2 of influenza A results in a virus that is sensitive to inhibitors of the M2 ion channel and is pathogenic for pig-tailed macaques. Virology 344, 541-558]. Based on previous studies of the M2 protein which have shown that the His-X-X-X-Trp motif within the M2 is essential to the function of the M2 proton channel, we have constructed a novel SHIV in which the alanine at position 19 of the TM domain was replaced with a histidine residue resulting in the motif His-Ile-Leu-Val-Trp. The SHIV{sub VpuA19H} replicated with similar kinetics as the parental SHIV{sub KU-1bMC33} and pulse-chase analysis revealed that the processing of viral proteins was similar to SHIV{sub KU-1bMC33}. This SHIV{sub VpuA19H} virus was found to be more sensitive to the M2 ion channel blocker rimantadine than SHIV{sub M2}. Electron microscopic examination of SHIV{sub VpuA19H}-infected cells treated with rimantadine revealed an accumulation of viral particles at the cell surface and within intracellular vesicles, which was similar to that previously observed to SHIV{sub M2}-infected cells treated with rimantadine. These data indicate that the Vpu protein of HIV-1 can be converted into a rimantadine-sensitive ion channel
76 FR 70046 - Airworthiness Directives; Eurocopter France Model AS350B, B1, B2, B3, BA, C, D, and D1; and...

Federal Register 2010, 2011, 2012, 2013, 2014

2011-11-10

... Airworthiness Directives; Eurocopter France Model AS350B, B1, B2, B3, BA, C, D, and D1; and AS355E, F, F1, F2, N... France (Eurocopter) Model AS350B, B1, B2, B3, BA, C, D, and D1 helicopters; and Model AS355E, F, F1, F2... (AD 2003- 22-06), for Eurocopter Model AS350B, B1, B2, B3, BA, C, D, and D1; and Model AS355E, F, F1...
C-Terminal Helical Domains of Dengue Virus Type 4 E Protein Affect the Expression/Stability of prM Protein and Conformation of prM and E Proteins

PubMed Central

Tsai, Wen-Yang; Hsieh, Szu-Chia; Lai, Chih-Yun; Lin, Hong-En; Nerurkar, Vivek R.; Wang, Wei-Kung

2012-01-01

Background The envelope (E) protein of dengue virus (DENV) is the major immunogen for dengue vaccine development. At the C-terminus are two α-helices (EH1 and EH2) and two transmembrane domains (ET1 and ET2). After synthesis, E protein forms a heterodimer with the precursor membrane (prM) protein, which has been shown as a chaperone for E protein and could prevent premature fusion of E protein during maturation. Recent reports of enhancement of DENV infectivity by anti-prM monoclonal antibodies (mAbs) suggest the presence of prM protein in dengue vaccine is potentially harmful. A better understanding of prM-E interaction and its effect on recognition of E and prM proteins by different antibodies would provide important information for future design of safe and effective subunit dengue vaccines. Methodology/Principal Findings In this study, we examined a series of C-terminal truncation constructs of DENV4 prME, E and prM. In the absence of E protein, prM protein expressed poorly. In the presence of E protein, the expression of prM protein increased in a dose-dependent manner. Radioimmunoprecipitation, sucrose gradient sedimentation and pulse-chase experiments revealed ET1 and EH2 were involved in prM-E interaction and EH2 in maintaining the stability of prM protein. Dot blot assay revealed E protein affected the recognition of prM protein by an anti-prM mAb; truncation of EH2 or EH1 affected the recognition of E protein by several anti-E mAbs, which was further verified by capture ELISA. The E protein ectodomain alone can be recognized well by all anti-E mAbs tested. Conclusions/Significance A C-terminal domain (EH2) of DENV E protein can affect the expression and stability of its chaperone prM protein. These findings not only add to our understanding of the interaction between prM and E proteins, but also suggest the ectodomain of E protein alone could be a potential subunit immunogen without inducing anti-prM response. PMID:23300717
Indoxyl sulfate enhances IL-1β-induced E-selectin expression in endothelial cells in acute kidney injury by the ROS/MAPKs/NFκB/AP-1 pathway.

PubMed

Shen, Wen-Ching; Liang, Chan-Jung; Huang, Tao-Ming; Liu, Chen-Wei; Wang, Shu-Huei; Young, Guang-Huar; Tsai, Jaw-Shiun; Tseng, Ying-Chin; Peng, Yu-Sen; Wu, Vin-Cent; Chen, Yuh-Lien

2016-11-01

Uremic toxins are considered a risk factor for cardiovascular disorders in kidney diseases, but it is not known whether, under inflammatory conditions, they affect adhesion molecule expression on endothelial cells, which may play a critical role in acute kidney injury (AKI). In the present study, in cardiovascular surgery-related AKI patients, who are known to have high plasma levels of the uremic toxin indoxyl sulfate (IS), plasma levels of IL-1β were found to be positively correlated with plasma levels of the adhesion molecule E-selectin. In addition, high E-selectin and IL-1β expression were seen in the kidney of ischemia/reperfusion mice in vivo. We also examined the effects of IS on E-selectin expression by IL-1β-treated human umbilical vein endothelial cells (HUVECs) and the underlying mechanism. IS pretreatment of HUVECs significantly increased IL-1β-induced E-selectin expression, monocyte adhesion, and the phosphorylation of mitogen-activated protein kinases (ERK, p38, and JNK) and transcription factors (NF-κB and AP-1), and phosphorylation was decreased by pretreatment with inhibitors of ERK1/2 (PD98059), p38 MAPK (SB202190), and JNK (SP600125). Furthermore, IS increased IL-1β-induced reactive oxygen species (ROS) production and this effect was inhibited by pretreatment with N-acetylcysteine (a ROS scavenger) or apocynin (a NADPH oxidase inhibitor). Gel shift assays and ChIP-PCR demonstrated that IS enhanced E-selectin expression in IL-1-treated HUVECs by increasing NF-κB and AP-1 DNA-binding activities. Moreover, IS-enhanced E-selectin expression in IL-1β-treated HUVECs was inhibited by Bay11-7082, a NF-κB inhibitor. Thus, IS may play an important role in the development of cardiovascular disorders in kidney diseases during inflammation by increasing endothelial expression of E-selectin.
Inhibition of protein tyrosine phosphatase 1B (PTP1B) and α-glucosidase by xanthones from Cratoxylum cochinchinense, and their kinetic characterization.

PubMed

Li, Zuo Peng; Song, Yeong Hun; Uddin, Zia; Wang, Yan; Park, Ki Hun

2018-02-01

Cratoxylum cochinchinense displayed significant inhibition against protein tyrosine phosphatase 1B (PTP1B) and α-glucosidase, both of which are key target enzymes to attenuate diabetes and obesity. The compounds responsible for both enzymes inhibition were identified as twelve xanthones (1-12) among which compounds 1 and 2 were found to be new ones. All of them simultaneously inhibited PTP1B with IC 50 s of (2.4-52.5 µM), and α-glucosidase with IC 50 values of (1.7-72.7 µM), respectively. Cratoxanthone A (3) and γ-mangostin (7) were estimated to be most active inhibitors against both PTP1B (IC 50  = 2.4 µM for 3, 2.8 µM for 7) and α-glucosidase (IC 50  = 4.8 µM for 3, 1.7 µM for 7). In kinetic studies, all isolated xanthones emerged to be mixed inhibitors of α-glucosidase, whereas they behaved as competitive inhibitors of PTP1B. In time dependent experiments, compound 3 showed isomerization inhibitory behavior with following kinetic parameters: K i app  = 2.4 µM; k 5  = 0.05001 µM -1  S -1 and k 6  = 0.02076 µM -1  S -1 . Copyright © 2017 Elsevier Ltd. All rights reserved.
Control of SIV infection and subsequent induction of pandemic H1N1 immunity in rhesus macaques using an Ad5 [E1-, E2b-] vector platform.

PubMed

Gabitzsch, Elizabeth S; Balint-Junior, Joseph P; Xu, Younong; Balcaitis, Stephanie; Sanders-Beer, Brigitte; Karl, Julie; Weinhold, Kent J; Paessler, Slobodan; Jones, Frank R

2012-11-26

Anti-vector immunity mitigates immune responses induced by recombinant adenovirus vector vaccines, limiting their prime-boost capabilities. We have developed a novel gene delivery and expression platform (Ad5 [E1-, E2b-]) that induces immune responses despite pre-existing and/or developed concomitant Ad5 immunity. In the present study, we evaluated if this new Ad5 platform could overcome the adverse condition of pre-existing Ad5 immunity to induce effective immune responses in prime-boost immunization regimens against two different infectious diseases in the same animal. Ad5 immune rhesus macaques (RM) were immunized multiple times with the Ad5 [E1-, E2b-] platform expressing antigens from simian immunodeficiency virus (SIV). Immunized RM developed cell-mediated immunity against SIV antigens Gag, Pol, Nef and Env as well as antibody against Env. Vaccinated and vector control RMs were challenged intra-rectally with homologous SIVmac239. During a 7-week follow-up, there was perturbation of SIV load in some immunized RM. At 7 weeks post-challenge, eight immunized animals (53%) did not have detectable SIV, compared to two RM controls (13%) (P<0.02; log-rank Mantel-Cox test). There was no correlation of protective MHC contributing to infection control. The RM without detectable circulating SIV, now hyper immune to Ad5, were then vaccinated with the same Ad5 [E1-, E2b-] platform expressing H1N1 influenza hemagglutinin (HA). Thirty days post Ad5 [E1-, E2b-]-HA vaccination, significant levels of influenza neutralizing antibody were induced in all animals that increased after an Ad5 [E1-, E2b-]-HA homologous boost. These data demonstrate the versatility of this new vector platform to immunize against two separate disease targets in the same animal despite the presence of immunity against the delivery platform, permitting homologous repeat immunizations with an Ad5 gene delivery platform. Copyright © 2012 Elsevier Ltd. All rights reserved.
26 CFR 55.6091-1 - Place for filing Chapter 44 tax returns.

Code of Federal Regulations, 2010 CFR

2010-04-01

... (CONTINUED) MISCELLANEOUS EXCISE TAXES (CONTINUED) EXCISE TAX ON REAL ESTATE INVESTMENT TRUSTS AND REGULATED INVESTMENT COMPANIES Procedure and Administration § 55.6091-1 Place for filing Chapter 44 tax returns. Except... estate investment trust or regulated investment company. (b) Returns filed with service centers or by...

26 CFR 55.6091-1 - Place for filing Chapter 44 tax returns.

Code of Federal Regulations, 2011 CFR

2011-04-01

... (CONTINUED) MISCELLANEOUS EXCISE TAXES (CONTINUED) EXCISE TAX ON REAL ESTATE INVESTMENT TRUSTS AND REGULATED INVESTMENT COMPANIES Procedure and Administration § 55.6091-1 Place for filing Chapter 44 tax returns. Except... estate investment trust or regulated investment company. (b) Returns filed with service centers or by...
26 CFR 55.6091-1 - Place for filing Chapter 44 tax returns.

Code of Federal Regulations, 2013 CFR

2013-04-01

... (CONTINUED) MISCELLANEOUS EXCISE TAXES (CONTINUED) EXCISE TAX ON REAL ESTATE INVESTMENT TRUSTS AND REGULATED INVESTMENT COMPANIES Procedure and Administration § 55.6091-1 Place for filing Chapter 44 tax returns. Except... estate investment trust or regulated investment company. (b) Returns filed with service centers or by...
26 CFR 55.6091-1 - Place for filing Chapter 44 tax returns.

Code of Federal Regulations, 2014 CFR

2014-04-01

... (CONTINUED) MISCELLANEOUS EXCISE TAXES (CONTINUED) EXCISE TAX ON REAL ESTATE INVESTMENT TRUSTS AND REGULATED INVESTMENT COMPANIES Procedure and Administration § 55.6091-1 Place for filing Chapter 44 tax returns. Except... estate investment trust or regulated investment company. (b) Returns filed with service centers or by...
26 CFR 55.6091-1 - Place for filing Chapter 44 tax returns.

Code of Federal Regulations, 2012 CFR

2012-04-01

... (CONTINUED) MISCELLANEOUS EXCISE TAXES (CONTINUED) EXCISE TAX ON REAL ESTATE INVESTMENT TRUSTS AND REGULATED INVESTMENT COMPANIES Procedure and Administration § 55.6091-1 Place for filing Chapter 44 tax returns. Except... estate investment trust or regulated investment company. (b) Returns filed with service centers or by...
Characterization of Protein Tyrosine Phosphatase 1B Inhibition by Chlorogenic Acid and Cichoric Acid.

PubMed

Lipchock, James M; Hendrickson, Heidi P; Douglas, Bonnie B; Bird, Kelly E; Ginther, Patrick S; Rivalta, Ivan; Ten, Nicholas S; Batista, Victor S; Loria, J Patrick

2017-01-10

Protein tyrosine phosphatase 1B (PTP1B) is a known regulator of the insulin and leptin signaling pathways and is an active target for the design of inhibitors for the treatment of type II diabetes and obesity. Recently, cichoric acid (CHA) and chlorogenic acid (CGA) were predicted by docking methods to be allosteric inhibitors that bind distal to the active site. However, using a combination of steady-state inhibition kinetics, solution nuclear magnetic resonance experiments, and molecular dynamics simulations, we show that CHA is a competitive inhibitor that binds in the active site of PTP1B. CGA, while a noncompetitive inhibitor, binds in the second aryl phosphate binding site, rather than the predicted benzfuran binding pocket. The molecular dynamics simulations of the apo enzyme and cysteine-phosphoryl intermediate states with and without bound CGA suggest CGA binding inhibits PTP1B by altering hydrogen bonding patterns at the active site. This study provides a mechanistic understanding of the allosteric inhibition of PTP1B.
The identification of the heat-stable microsomal protein required for methoxyflurane metabolism as cytochrome b5.

PubMed

Canova-Davis, E; Waskell, L

1984-02-25

Methoxyflurane is an anesthetic whose metabolism by cytochrome P-450LM2 has been shown to be dependent upon a heat-stable microsomal protein (Canova-Davis, E., and Waskell, L. A. (1982) Biochem. Biophys. Res. Commun. 108, 1264-1270). Treatment of this protein with diethylpyrocarbonate, which modifies selected amino acids, caused a dose-dependent loss in its ability to effect the metabolism of methoxyflurane by purified cytochrome P-450LM2. This protein factor has been identified as cytochrome b5 by demonstrating that cytochrome b5 and the heat-stable factor coelute during cytochrome b5 purification. Neither ferriheme nor apocytochrome b5 was able to substitute for the activating factor, while cytochrome b5 reconstituted from apocytochrome b5 and heme exhibited an activity similar to that of native b5. Examination of the cytochrome b5 molecule by computer graphics suggested that diethylpyrocarbonate did not inactivate b5 by reacting with the anionic surface of the cytochrome b5 molecule. Maximal rates of methoxyflurane metabolism were obtained at a ratio of 1:1:1 of the three proteins, cytochrome P-450LM2:reductase:cytochrome b5. In summary, it has been demonstrated that the heat-stable protein, cytochrome b5, is obligatory for the metabolism of methoxyflurane by cytochrome P-450LM2. These data also suggest that cytochrome b5 may be acting as an electron donor to P-450LM2 in the O-demethylation of methoxyflurane.
UNC93B1 mediates differential trafficking of endosomal TLRs

PubMed Central

Lee, Bettina L; Moon, Joanne E; Shu, Jeffrey H; Yuan, Lin; Newman, Zachary R; Schekman, Randy; Barton, Gregory M

2013-01-01

UNC93B1, a multipass transmembrane protein required for TLR3, TLR7, TLR9, TLR11, TLR12, and TLR13 function, controls trafficking of TLRs from the endoplasmic reticulum (ER) to endolysosomes. The mechanisms by which UNC93B1 mediates these regulatory effects remain unclear. Here, we demonstrate that UNC93B1 enters the secretory pathway and directly controls the packaging of TLRs into COPII vesicles that bud from the ER. Unlike other COPII loading factors, UNC93B1 remains associated with the TLRs through post-Golgi sorting steps. Unexpectedly, these steps are different among endosomal TLRs. TLR9 requires UNC93B1-mediated recruitment of adaptor protein complex 2 (AP-2) for delivery to endolysosomes while TLR7, TLR11, TLR12, and TLR13 utilize alternative trafficking pathways. Thus, our study describes a mechanism for differential sorting of endosomal TLRs by UNC93B1, which may explain the distinct roles played by these receptors in certain autoimmune diseases. DOI: http://dx.doi.org/10.7554/eLife.00291.001 PMID:23426999
Role of cytochrome P450 2E1 in protein nitration and ubiquitin-mediated degradation during acetaminophen toxicity.

PubMed

Abdelmegeed, Mohamed A; Moon, Kwan-Hoon; Chen, Chi; Gonzalez, Frank J; Song, Byoung-Joon

2010-01-01

It is well established that following a toxic dose of acetaminophen (APAP), nitrotyrosine protein adducts (3-NT), a hallmark of peroxynitrite production, were colocalized with necrotic hepatic centrilobular regions where cytochrome P450 2E1 (CYP2E1) is highly expressed, suggesting that 3-NT formation may be essential in APAP-mediated toxicity. This study was aimed at investigating the relationship between CYP2E1 and nitration (3-NT formation) followed by ubiquitin-mediated degradation of proteins in wild-type and Cyp2e1-null mice exposed to APAP (200 and 400mg/kg) for 4 and 24h. Markedly increased centrilobular liver necrosis and 3-NT formation were only observed in APAP-exposed wild-type mice in a dose- and time-dependent manner, confirming an important role for CYP2E1 in APAP biotransformation and toxicity. However, the pattern of 3-NT protein adducts, not accompanied by concurrent activation of nitric oxide synthase (NOS), was similar to that of protein ubiquitination. Immunoblot analysis further revealed that immunoprecipitated nitrated proteins were ubiquitinated in APAP-exposed wild-type mice, confirming the fact that nitrated proteins are more susceptible than the native proteins for ubiquitin-dependent degradation, resulting in shorter half-lives. For instance, cytosolic superoxide dismutase (SOD1) levels were clearly decreased and immunoprecipitated SOD1 was nitrated and ubiquitinated, likely leading to its accelerated degradation in APAP-exposed wild-type mice. These data suggest that CYP2E1 appears to play a key role in 3-NT formation, protein degradation, and liver damage, which is independent of NOS, and that decreased levels of many proteins in the wild-type mice (compared with Cyp2e1-null mice) likely contribute to APAP-related toxicity.
Immunization with mutant HPV16 E7 protein inhibits the growth of TC-1 cells in tumor-bearing mice.

PubMed

Li, Yan-Li; Ma, Zhong-Liang; Zhao, Yue; Zhang, Jing

2015-04-01

Two human papillomavirus (HPV) 16 oncogenic proteins, E6 and E7, are co-expressed in the majority of HPV16-induced cervical cancer cells. Thus, the E6 and E7 proteins are good targets for developing therapeutic vaccines for cervical cancer. In the present study, immunization with the mutant non-transforming HPV16 E7 (mE7) protein was demonstrated to inhibit the growth of TC-1 cells in the TC-1 mouse model. The HPV16 mE7 gene was amplified by splicing overlap extension polymerase chain reaction using pET-28a(+)-E7 as a template, and the gene was cloned into pET-28a(+) to form pET-28a(+)-mE7. Compared with the E7 protein, mE7 lacks amino acid residues 94-98, and at residue 24, there is a Cys to Gly substitution. pET-28a(+)-mE7 was then introduced into Escherichia coli Rosetta. The expression of mE7 was induced by isopropyl β-D-1-thiogalactopyranoside. The mE7 protein was purified using Ni-NTA agarose and detected by SDS-PAGE and western blot analysis. In the tumor prevention model, no tumor was detected in the mice vaccinated with the mE7 protein. After 40 days, the tumor-free mice and control mice were challenged with 2×10 5 TC-1 cells. All control mice developed tumors six days later, but mE7 immunized mice were tumor free until 90 days. In the tumor therapy model, the TC-1 cells were initially injected subcutaneously, and the mice were subsequently vaccinated. Vaccination against the mE7 protein may significantly inhibit TC-1 cell growth compared to the control. These results demonstrated that immunization with the HPV16 mE7 protein elicited a long-term protective immunity against TC-1 tumor growth and generated a significant inhibition of TC-1 growth in a TC-1 mouse model.
Evaluation of synergistic antimicrobial effect of vitamins (A, B1, B2, B6, B12, C, D, E and K) with antibiotics against resistant bacterial strains.

PubMed

Shahzad, Shakeel; Ashraf, M Adnan; Sajid, M; Shahzad, Aqeel; Rafique, Azhar; Mahmood, M Shahid

2018-02-02

Multiple drug resistant super bugs of Acinetobacter baumannii and methicillin-resistant Staphylococcus aureus (MRSA) are becoming challenge for healthcare professionals. In this study, vitamins were evaluated for synergistic activity with the antibiotics. Synergistic effect between antibiotic and stock solutions of vitamins is evaluated by using Kirby-Bauer disc diffusion assay. Distilled water and propylene glycol were used as solvent for water soluble vitamins and fat-soluble vitamins respectively. The final concentration of 10mg/ml of each water-soluble vitamin B1 (Thiamine), B2 (Riboflavin), B6 (Pyridoxine) B12 (Methylcobalamin), C (Ascorbic acid) and 0.1mg/ml of each fat soluble vitamin A (retinol), D (cholecalciferol) E (αTocopherol) K (Menadione) were used with the antibiotics. The results depicted that vitamin K and E have better synergistic activity with piperacillin-tazobactam, imipenem and doripenem antibiotics against A. baumannii. While vitamin B1, B2 and B12 showed remarkable synergistic activity with linezolid against MRSA. Vitamin B1 was further tested to have better synergism with antibiotics oxacillin, tetracycline, rifampicin and linezolid against MRSA. The fat-soluble vitamins E and K were good in synergism against Gram negative A. baumannii while water soluble vitamins as B1, B2 and B12 were effective against MRSA but not against A. baumannii. This synergistic action of vitamins with the antibiotics can be used as a tool to treat multiple drug resistant super bugs with further evaluation at molecular level. Copyright © 2018 International Society for Chemotherapy of Infection and Cancer. Published by Elsevier Ltd. All rights reserved.
Chemical and biological activities of a 64-kilodalton outer sheath protein from Treponema denticola strains.

PubMed Central

Weinberg, A; Holt, S C

1991-01-01

This study examined the distribution of the major outer sheath proteins (MOSP) in several Treponema denticola strains and reports the isolation of a 64-kDa protein from the outer sheath of human clinical isolate T. denticola GM-1. The outer sheath was isolated by freeze-thaw procedures, and the distribution of outer sheath proteins was examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). T. denticola GM-1, MS25, SR-5, and three low-passage clinical isolates possessed an MOSP with a relative molecular mass of 60 to 64 kDa. This MOSP was absent in T. denticola ATCC 35404 (TD-4) and clinical isolate SR-4. The latter possessed an MOSP of 58 kDa. 125I labeling revealed both MOSP to be dissociated forms of higher-molecular-mass oligomeric units between 116 and 162 kDa. Two-dimensional SDS-PAGE confirmed the modifiability of these MOSP. Isoelectric focusing of the 64-kDa MOSP indicated a pI of 6.7. Immunoblots with antiserum to GM-1 whole cells revealed the 64-kDa protein to be immunogenic and not cross-reactive with the MOSP of TD-4 or SR-4, and monospecific antibody to the 64-kDa protein recognized common epitopes on the high-molecular-weight oligomeric protein. These antibodies did not react with any component of TD-4 whole cells in immunoblots or in immunogold electron microscopy. Fab fragments inhibited the adherence of T. denticola GM-1 to human gingival fibroblasts by 78% (1:1,600; 0.72 micrograms of protein per ml), while TD-4 adherence was not inhibited. Amino acid analysis revealed a slightly acidic protein, devoid of cysteine, with 36% hydrophobic residues. Cyanogen bromide fragmentation of the 64-kDa protein revealed that a 42-kDa fragment contained a T-L-D-L-A-L-D segment which was 100% homologous with an integrin alpha subunit of a human leukocyte adhesion glycoprotein p 150,95. Images FIG. 1 FIG. 2 FIG. 3 FIG. 4 FIG. 5 FIG. 6 FIG. 7 FIG. 8 FIG. 9 FIG. 10 FIG. 11 FIG. 12 FIG. 13 FIG. 14 PMID:1938897
Exploring the Lead Compounds for Zika Virus NS2B-NS3 Protein: an e-Pharmacophore-Based Approach.

PubMed

Rohini, K; Agarwal, Pratika; Preethi, B; Shanthi, V; Ramanathan, K

2018-06-18

The rapid spread of the Zika virus and its association with the abnormal brain development constitute a global health emergency. With a continuing spread of the mosquito vector, the exposure is expected to accelerate in the coming years. Despite number of efforts, there is still no proper vaccine or medicine to combat this virus. Of note, the NS2B-NS3 protein is proven to be the potential target for the Zika virus therapeutics. Hence, e-pharmacophore-based drug design strategy was employed to identify potent inhibitors of NS2B-NS3 protein from ASINEX database consisting of 467,802 molecules. A 3D e-pharmacophore model was generated using PHASE module of Schrödinger Suite. The generated model consists of one hydrogen bond acceptor (A), two hydrogen bond donors (D), and two aromatic rings (R), ADDRR. The model was further evaluated for its ability to screen actives using enrichment analysis. Subsequently, high-throughput virtual screening protocol was employed, and the resultant hit molecules were also examined for its binding free energies and ADME properties using Prime MM-GBSA and Qikprop module of Schrodinger packages, respectively. Finally, the screened hit molecule was subjected to molecular dynamics simulation to examine its stability. Overall, the results from our analysis suggest that compound BAS 19192837 could be a potent inhibitor for the NS2B-NS3 protein of the Zika virus. It is also noteworthy to mention that our results are in good agreement with literature evidences. We hope that this result is of immense importance in designing potential drug molecules to combat the spread of Zika virus in the near future.
Precision Measurements of the B(E1) Strengths in 11Be

NASA Astrophysics Data System (ADS)

Kwan, E.; Wu, C. Y.; Summers, N. C.; Hackman, G.; Drake, T. E.; Andreoiu, C.; Ashley, R.; Ball, G. C.; Bender, P. C.; Boston, A. J.; Boston, H. C.; Chester, A.; Close, A.; Cline, D.; Cross, D. S.; Dunlop, R.; Finley, A.; Garnsworthy, A.; Hayes, A. B.; Laffoley, A. T.; Nano, T.; Navrátil, P.; Pearson, C. J.; Pore, J.; Starosta, K.; Thompson, I. J.; Voss, P.; Williams, S. J.; Wang, Z. M.

2014-09-01

The electromagnetic transition strength between the two bound states were measured in the one-neutron halo nucleus 11Be from Coulomb excitation on 196Pt at projectile energies of 1.727 and 2.086 MeV/nucleon at TRIUMF. A B(E1) strength of 0.102(2) e2fm2, deduced from the forward-scattering data, is consistent with previous Coulomb excitation measurements at intermediate projectile energies with a model-dependent analysis.
Complementary phosphorylation sites in the adaptor protein SLP-76 promote synergistic activation of natural killer cells.

PubMed

Kim, Hun Sik; Long, Eric O

2012-07-10

The cytotoxic effects of natural killer (NK) cells and their ability to secrete cytokines require synergistic signals from specific pairs of co-activation receptors, such as CD314 (also known as NKG2D) and CD244 (2B4), which bind to distinct ligands present on target cells. These signals are required to overcome inhibition mediated by the E3 ubiquitin ligase c-Cbl of the guanine nucleotide exchange factor Vav1, which promotes activation of NK cells. Here, we showed that the adaptor protein SLP-76 (Src homology 2 domain-containing leukocyte phosphoprotein of 76 kilodaltons) was required for this synergy and that distinct tyrosine residues in SLP-76 were phosphorylated by each member of a pair of synergistic receptors. Selective phosphorylation of tyrosine 113 or tyrosine 128 in SLP-76 enabled binding of SLP-76 to Vav1. Selective phosphorylation of SLP-76 at these residues was restricted to receptors that stimulated ligand-dependent target cell killing; antibody-dependent stimulation of the Fc receptor CD16 promoted phosphorylation at both sites. Knockdown and reconstitution experiments with SLP-76 mutant proteins showed the distinct role of each tyrosine in the synergistic mobilization of Ca2+, revealing an unexpected degree of selectivity in the phosphorylation of SLP-76 by NK cell co-activation receptors. Together, these data suggest that combined phosphorylation of separate tyrosine residues in SLP-76 forms the basis of synergistic NK cell activation.
Ethanol metabolism by alcohol dehydrogenase or cytochrome P450 2E1 differentially impairs hepatic protein trafficking and growth hormone signaling.

PubMed

Doody, Erin E; Groebner, Jennifer L; Walker, Jetta R; Frizol, Brittnee M; Tuma, Dean J; Fernandez, David J; Tuma, Pamela L

2017-12-01

The liver metabolizes alcohol using alcohol dehydrogenase (ADH) and cytochrome P 450 2E1 (CYP2E1). Both enzymes metabolize ethanol into acetaldehyde, but CYP2E1 activity also results in the production of reactive oxygen species (ROS) that promote oxidative stress. We have previously shown that microtubules are hyperacetylated in ethanol-treated polarized, hepatic WIF-B cells and livers from ethanol-fed rats. We have also shown that enhanced protein acetylation correlates with impaired clathrin-mediated endocytosis, constitutive secretion, and nuclear translocation and that the defects are likely mediated by acetaldehyde. However, the roles of CYP2E1-generated metabolites and ROS in microtubule acetylation and these alcohol-induced impairments have not been examined. To determine if CYP2E1-mediated alcohol metabolism is required for enhanced acetylation and the trafficking defects, we coincubated cells with ethanol and diallyl sulfide (DAS; a CYP2E1 inhibitor) or N -acetyl cysteine (NAC; an antioxidant). Both agents failed to prevent microtubule hyperacetylation in ethanol-treated cells and also failed to prevent impaired secretion or clathrin-mediated endocytosis. Somewhat surprisingly, both DAS and NAC prevented impaired STAT5B nuclear translocation. Further examination of microtubule-independent steps of the pathway revealed that Jak2/STAT5B activation by growth hormone was prevented by DAS and NAC. These results were confirmed in ethanol-exposed HepG2 cells expressing only ADH or CYP2E1. Using quantitative RT-PCR, we further determined that ethanol exposure led to blunted growth hormone-mediated gene expression. In conclusion, we determined that alcohol-induced microtubule acetylation and associated defects in microtubule-dependent trafficking are mediated by ADH metabolism whereas impaired microtubule-independent Jak2/STAT5B activation is mediated by CYP2E1 activity. NEW & NOTEWORTHY Impaired growth hormone-mediated signaling is observed in ethanol
Aldo-keto reductase family 1 B10 protein detoxifies dietary and lipid-derived alpha, beta-unsaturated carbonyls at physiological levels.

PubMed

Zhong, Linlin; Liu, Ziwen; Yan, Ruilan; Johnson, Stephen; Zhao, Yupei; Fang, Xiubin; Cao, Deliang

2009-09-18

Alpha, beta-unsaturated carbonyls are highly reactive mutagens and carcinogens to which humans are exposed on a daily basis. This study demonstrates that aldo-keto reductase family 1 member B10 (AKR1B10) is a critical protein in detoxifying dietary and lipid-derived unsaturated carbonyls. Purified AKR1B10 recombinant protein efficiently catalyzed the reduction to less toxic alcohol forms of crotonaldehyde at 0.90 microM, 4-hydroxynonenal (HNE) at 0.10 microM, trans-2-hexanal at 0.10 microM, and trans-2,4-hexadienal at 0.05 microM, the concentrations at or lower than physiological exposures. Ectopically expressed AKR1B10 in 293T cells eliminated immediately HNE at 1 (subtoxic) or 5 microM (toxic) by converting to 1,4-dihydroxynonene, protecting the cells from HNE toxicity. AKR1B10 protein also showed strong enzymatic activity toward glutathione-conjugated carbonyls. Taken together, our study results suggest that AKR1B10 specifically expressed in the intestine is physiologically important in protecting the host cell against dietary and lipid-derived cytotoxic carbonyls.
Cell Transformation by PTP1B Truncated Mutants Found in Human Colon and Thyroid Tumors.

PubMed

Mei, Wenhan; Wang, Kemin; Huang, Jian; Zheng, Xinmin

2016-01-01

Expression of wild-type protein tyrosine phosphatase (PTP) 1B may act either as a tumor suppressor by dysregulation of protein tyrosine kinases or a tumor promoter through Src dephosphorylation at Y527 in human breast cancer cells. To explore whether mutated PTP1B is involved in human carcinogenesis, we have sequenced PTP1B cDNAs from human tumors and found splice mutations in ~20% of colon and thyroid tumors. The PTP1BΔE6 mutant expressed in these two tumor types and another PTP1BΔE5 mutant expressed in colon tumor were studied in more detail. Although PTP1BΔE6 revealed no phosphatase activity compared with wild-type PTP1B and the PTP1BΔE5 mutant, its expression induced oncogenic transformation of rat fibroblasts without Src activation, indicating that it involved signaling pathways independent of Src. The transformed cells were tumourigenic in nude mice, suggesting that the PTP1BΔE6 affected other molecule(s) in the human tumors. These observations may provide a novel therapeutic target for colon and thyroid cancer.
Conversion from CUL4-based COP1–SPA E3 apparatus to UVR8–COP1–SPA complexes underlies a distinct biochemical function of COP1 under UV-B

PubMed Central

Huang, Xi; Ouyang, Xinhao; Yang, Panyu; Lau, On Sun; Chen, Liangbi; Wei, Ning; Deng, Xing Wang

2013-01-01

The evolutionarily conserved CONSTITUTIVE PHOTOMORPHOGENESIS 1 (COP1) is a RING and WD40 protein that functions as a substrate receptor of CULLIN4–DAMAGED DNA BINDING PROTEIN 1 (CUL4–DDB1)–based E3 ubiquitin ligases in both plants and animals. In Arabidopsis, COP1 is a central repressor of photomorphogenesis in the form of COP1–SUPPRESSOR OF PHYA (SPA) complex(es). CUL4–DDB1–COP1–SPA suppresses the photomorphogenic program by targeting the transcription factor ELONGATED HYPOCOTYL 5 for degradation. Intriguingly, under photomorphogenic UV-B light, COP1 reverses its repressive role and promotes photomorphogenesis. However, the mechanism by which COP1 is functionally switched is still obscure. Here, we demonstrate that UV-B triggers the physical and functional disassociation of the COP1–SPA core complex(es) from CUL4–DDB1 and the formation of a unique complex(es) containing the UV-B receptor UV RESISTANCE LOCUS 8 (UVR8). The establishment of this UV-B–dependent COP1 complex(es) is associated with its positive modulation of ELONGATED HYPOCOTYL 5 stability and activity, which sheds light on the mechanism of COP1’s promotive action in UV-B–induced photomorphogenesis. PMID:24067658
Cloning and sequencing of a gene encoding a glutamate and aspartate carrier of Escherichia coli K-12.

PubMed Central

Wallace, B; Yang, Y J; Hong, J S; Lum, D

1990-01-01

A gene encoding a carrier protein for glutamate and aspartate was cloned into Escherichia coli K-12 strain BK9MDG by using the high-copy-number plasmid pBR322. The gene (designated gltP) is probably identical to a gene recently cloned from E. coli B (Y. Deguchi, I. Yamato, and Y. Anraku, J. Bacteriol. 171:1314-1319). A 1.6-kilobase DNA fragment containing gltP was subcloned into the expression plasmids pT7-5 and pT7-6, and its product was identified by a phage T7 RNA polymerase-T7 promoter coupled system (S. Tabor and C. C. Richardson, Proc. Natl. Acad. Sci. USA 82:1074-1078) as a polypeptide with an apparent mass of 38 kilodaltons. A portion of the gltP polypeptide was associated with the cytoplasmic membrane. The nucleotide sequence of the 1.6-kilobase fragment was determined. It contained an open reading frame capable of encoding a highly hydrophobic polypeptide of 395 amino acids, containing four possible transmembrane segments. Uptake of glutamate and aspartate was increased 5.5- and 4.5-fold, respectively, in strains containing gltP plasmids. Glutamate uptake was insensitive to the concentration of Na+ and was inhibited by L-cysteate and beta-hydroxyaspartate. These results suggest that gltP is a structural gene for a carrier protein of the Na(+)-independent, binding-protein-independent glutamate-aspartate transport system. Images PMID:1971622
Salvianolic Acid B (Sal B) Protects Retinal Pigment Epithelial Cells from Oxidative Stress-Induced Cell Death by Activating Glutaredoxin 1 (Grx1)

PubMed Central

Liu, Xiaobin; Xavier, Christy; Jann, Jamieson; Wu, Hongli

2016-01-01

Protein glutathionylation, defined as the formation of protein mixed disulfides (PSSG) between cysteine residues and glutathione (GSH), can lead to cell death. Glutaredoxin 1 (Grx1) is a thiol repair enzyme which catalyzes the reduction of PSSG. Therefore, Grx1 exerts strong anti-apoptotic effects by improving the redox state, especially in times of oxidative stress. However, there is currently no compound that is identified as a Grx1 activator. In this study, we identified and characterized Salvianolic acid B (Sal B), a natural compound, as a Grx1 inducer, which potently protected retinal pigment epithelial (RPE) cells from oxidative injury. Our results showed that treatment with Sal B protected primary human RPE cells from H2O2-induced cell damage. Interestingly, we found Sal B pretreatment upregulated Grx1 expression in RPE cells in a time- and dose-dependent manner. Furthermore, NF-E2-related factor 2 (Nrf2), the key transcription factor that regulates the expression of Grx1, was activated in Sal B treated RPE cells. Further investigation showed that knockdown of Grx1 by small interfering RNA (siRNA) significantly reduced the protective effects of Sal B. We conclude that Sal B protects RPE cells against H2O2-induced cell injury through Grx1 induction by activating Nrf2 pathway, thus preventing lethal accumulation of PSSG and reversing oxidative damage. PMID:27827892

The Scaffold Protein TANK/I-TRAF Inhibits NF-κB Activation by Recruiting Polo-like Kinase 1

PubMed Central

Zhang, Wanqiao; Zhang, Ying; Yuan, Yanzhi; Guan, Wei; Jin, Chaozhi; Chen, Hui; Wang, Xiaohui

2010-01-01

TANK/I-TRAF is a TRAF-binding protein that negatively regulates NF-κB activation. The underlying mechanism of this activity remains unclear. Here we show that TANK directly interacts with PLK1, a conserved cell cycle–regulated kinase. PLK1 inhibits NF-κB transcriptional activation induced by TNF-α, IL-1β, or several activators, but not by nuclear transcription factor p65. PLK1 expression reduces the DNA-binding activity of NF-κB induced by TNF-α. Moreover, endogenous activation of PLK1 reduces the TNF-induced phosphorylation of endogenous IκBα. PLK1 is bound to NEMO (IKKγ) through TANK to form a ternary complex in vivo. We describe a new regulatory mechanism for PLK1: PLK1 negatively regulates TNF-induced IKK activation by inhibiting the ubiquitination of NEMO. These findings reveal that the scaffold protein TANK recruits PLK1 to negatively regulate NF-κB activation and provide direct evidence that PLK1 is required for the repression function of TANK. PMID:20484576
BAG3 regulates total MAP1LC3B protein levels through a translational but not transcriptional mechanism.

PubMed

Rodríguez, Andrea E; López-Crisosto, Camila; Peña-Oyarzún, Daniel; Salas, Daniela; Parra, Valentina; Quiroga, Clara; Morawe, Tobias; Chiong, Mario; Behl, Christian; Lavandero, Sergio

2016-01-01

Autophagy is mainly regulated by post-translational and lipid modifications of ATG proteins. In some scenarios, the induction of autophagy is accompanied by increased levels of certain ATG mRNAs such as MAP1LC3B/LC3B, ATG5 or ATG12. However, little is known about the regulation of ATG protein synthesis at the translational level. The cochaperone of the HSP70 system BAG3 (BCL2-associated athanogene 3) has been associated to LC3B lipidation through an unknown mechanism. In the present work, we studied how BAG3 controls autophagy in HeLa and HEK293 cells. Our results showed that BAG3 regulates the basal amount of total cellular LC3B protein by controlling its mRNA translation. This effect was apparently specific to LC3B because other ATG protein levels were not affected. BAG3 knockdown did not affect LC3B lipidation induced by nutrient deprivation or proteasome inhibition. We concluded that BAG3 maintains the basal amount of LC3B protein by controlling the translation of its mRNA in HeLa and HEK293 cells.
BAG3 regulates total MAP1LC3B protein levels through a translational but not transcriptional mechanism

PubMed Central

Rodríguez, Andrea E.; López-Crisosto, Camila; Peña-Oyarzún, Daniel; Salas, Daniela; Parra, Valentina; Quiroga, Clara; Morawe, Tobias; Chiong, Mario; Behl, Christian; Lavandero, Sergio

2016-01-01

ABSTRACT Autophagy is mainly regulated by post-translational and lipid modifications of ATG proteins. In some scenarios, the induction of autophagy is accompanied by increased levels of certain ATG mRNAs such as MAP1LC3B/LC3B, ATG5 or ATG12. However, little is known about the regulation of ATG protein synthesis at the translational level. The cochaperone of the HSP70 system BAG3 (BCL2-associated athanogene 3) has been associated to LC3B lipidation through an unknown mechanism. In the present work, we studied how BAG3 controls autophagy in HeLa and HEK293 cells. Our results showed that BAG3 regulates the basal amount of total cellular LC3B protein by controlling its mRNA translation. This effect was apparently specific to LC3B because other ATG protein levels were not affected. BAG3 knockdown did not affect LC3B lipidation induced by nutrient deprivation or proteasome inhibition. We concluded that BAG3 maintains the basal amount of LC3B protein by controlling the translation of its mRNA in HeLa and HEK293 cells. PMID:26654586
Apple RING E3 ligase MdMIEL1 inhibits anthocyanin accumulation by ubiquitinating and degrading MdMYB1 protein.

PubMed

An, Jian-Ping; Liu, Xin; Li, Hao-Hao; You, Chun-Xiang; Wang, Xiao-Fei; Hao, Yu-Jin

2017-11-01

MdMYB1 is an important regulator for anthocyanin accumulation in apple (Malus × domestica). Here, an apple RING E3 ligase, MdMIEL1, was screened out as a partner of MdMYB1 with a yeast two-hybrid approach. Pull-down, bimolecular fluorescence complementation and coimmunoprecipitation assays further verified the interaction between MdMIEL1 and MdMYB1 proteins. Subsequently, in vitro and in vivo experiments indicated that MdMIEL1 functioned as a ubiquitin E3 ligase to ubiquitinate MdMYB1 protein, followed by degradation through a 26S proteasome pathway. Furthermore, transgenic studies in apple calli and Arabidopsis demonstrated that MdMIEL1 negatively regulated anthocyanin accumulation by modulating the degradation of MdMYB1 protein. Taken together, our findings provide a new insight into the molecular mechanism by which MdMIEL1 negatively regulates anthocyanin biosynthesis by ubiquitinating and degrading MdMYB1 protein. © The Author 2017. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.
Effect of fluxing treatment on the properties of Fe66Co15Mo1P7.5C5.5B2Si3 bulk metallic glass by water quenching

NASA Astrophysics Data System (ADS)

Li, Jin-Feng; Wang, Xin; Liu, Xue; Zhao, Shao-Fan; Yao, Ke-Fu

2018-01-01

The effect of fluxing treatment on the properties of Fe66Co15Mo1P7.5C5.5B2Si3bulk amorphous alloy (BAA) has been investigated. Prepared by a combination method of flux treatment and water quenching, the Fe66Co15Mo1P7.5C5.5B2Si3 BAA exhibits better glass-forming ability, thermal stability, soft magnetic properties and ductility than those of the one prepared by direct water quenching. This indicates that fluxing treatment can play a potential role in improving the properties of Fe-based BAA due to the effective elimination of the impurities within the alloy.
A Study on the Interaction of Rhodamine B with Methylthioadenosine Phosphorylase Protein Sourced from an Antarctic Soil Metagenomic Library

PubMed Central

Bujacz, Anna; Wierzbicka-Woś, Anna; Kur, Józef

2013-01-01

The presented study examines the phenomenon of the fluorescence under UV light excitation (312 nm) of E. coli cells expressing a novel metagenomic-derived putative methylthioadenosine phosphorylase gene, called rsfp, grown on LB agar supplemented with a fluorescent dye rhodamine B. For this purpose, an rsfp gene was cloned and expressed in an LMG194 E. coli strain using an arabinose promoter. The resulting RSFP protein was purified and its UV-VIS absorbance spectrum and emission spectrum were assayed. Simultaneously, the same spectroscopic studies were carried out for rhodamine B in the absence or presence of RSFP protein or native E. coli proteins, respectively. The results of the spectroscopic studies suggested that the fluorescence of E. coli cells expressing rsfp gene under UV illumination is due to the interaction of rhodamine B molecules with the RSFP protein. Finally, this interaction was proved by a crystallographic study and then by site-directed mutagenesis of rsfp gene sequence. The crystal structures of RSFP apo form (1.98 Å) and complex RSFP/RB (1.90 Å) show a trimer of RSFP molecules located on the crystallographic six fold screw axis. The RSFP complex with rhodamine B revealed the binding site for RB, in the pocket located on the interface between symmetry related monomers. PMID:23383268
Cytoskeleton changes following differentiation of N1E-115 neuroblastoma cell line.

PubMed

Oh, J-E; Karlmark Raja, K; Shin, J-H; Pollak, A; Hengstschläger, M; Lubec, G

2006-10-01

No systematic approach to detect expression of differentiation-related elements was published so far. The undifferentiated N1E-115 neuroblastoma cell line was switched into a neuronal phenotype by DMSO treatment and used for proteomic experiments. We used two-dimensional gel electrophoresis followed by unambiguous mass spectrometrical identification of proteins to generate a map of cytoskeleton proteins (CPs), i.e., to search for differentiation-related structures. Alpha-actin, actin-like protein 6A, gamma-tubulin complex component 2, tubulin alpha 3/alpha 7, CLIP associating protein 2, B4 integrin interactor homolog were detectable in the undifferentiated cell line exclusively and neuron-specific CPs drebrin and presynaptic density protein 95, actin-related protein 2/3, alpha and beta-centractin, PDZ-domain actin binding protein, actinin alpha 1, profilin II, ezrin, coactosin-like protein, transgelin 2, myosin light polypeptide 6, tubulin alpha 2, 6 and 7, beta tubulin (94% similar with tubulin beta-2), tubulin beta 3, tubulin tyrosine ligase-like protein 1, lamin B1 and keratin 20 were observed in the differentiated cell line only. We herein identified differentiation-related expressional patterns thus providing new evidence for the role of CPs in the process of neuronal differentiation.
Rice phytochrome-interacting factor protein OsPIF14 represses OsDREB1B gene expression through an extended N-box and interacts preferentially with the active form of Phytochrome B

PubMed Central

Cordeiro, André M.; Figueiredo, Duarte D.; Tepperman, James; Borba, Ana Rita; Lourenço, Tiago; Abreu, Isabel A.; Ouwerkerk, Pieter B.F.; Quail, Peter H.; Oliveira, M. Margarida; Saibo, Nelson J. M.

2016-01-01

DREB1/CBF genes, known as major regulators of plant stress responses, are rapidly and transiently induced by low temperatures. Using a Yeast one Hybrid screening, we identified a putative Phytochrome-Interacting bHLH Factor (OsPIF14), as binding to the OsDREB1B promoter. bHLH proteins are able to bind to hexameric E-box (CANNTG) or N-box (CACG(A/C)G) motifs, depending on transcriptional activity. We have shown that OsPIF14 binds to the OsDREB1B promoter through two N-boxes and that the flanking regions of the hexameric core are essential for protein-DNA interaction and stability. We also showed that OsPIF14 down-regulates OsDREB1B gene expression in rice protoplasts, corroborating the OsPIF14 repressor activity observed in the transactivation assays using Arabidopsis protoplasts. In addition, we showed that OsPIF14 is indeed a Phytochrome Interacting Factor, which preferentially binds to the active form (Pfr) of rice phytochrome B. This raises the possibility that OsPIF14 activity might be modulated by light. However, we did not observe any regulation of the OsDREB1B gene expression by light under control conditions. Moreover, OsPIF14 gene expression was shown to be modulated by different treatments, such as drought, salt, cold and ABA. Interestingly, OsPIF14 showed also a specific cold-induced alternative splicing. All together, these results suggest the possibility that OsPIF14 is involved in cross-talk between light and stress signaling through interaction with the OsDREB1B promoter. Although in the absence of stress, OsDREB1B gene expression was not regulated by light, given previous reports, it remains possible that OsPIF14 has a role in light modulation of stress responses. PMID:26732823
Phosphatase control of 4E-BP1 phosphorylation state is central for glycolytic regulation of retinal protein synthesis.

PubMed

Gardner, Thomas W; Abcouwer, Steven F; Losiewicz, Mandy K; Fort, Patrice E

2015-09-15

Control of protein synthesis in insulin-responsive tissues has been well characterized, but relatively little is known about how this process is regulated in nervous tissues. The retina exhibits a relatively high protein synthesis rate, coinciding with high basal Akt and metabolic activities, with the majority of retinal ATP being derived from aerobic glycolysis. We examined the dependency of retinal protein synthesis on the Akt-mTOR signaling and glycolysis using ex vivo rat retinas. Akt inhibitors significantly reduced retinal protein synthesis but did not affect glycolytic lactate production. Surprisingly, the glycolytic inhibitor 2-deoxyglucose (2-DG) markedly inhibited Akt1 and Akt3 activities, as well as protein synthesis. The effects of 2-DG, and 2-fluorodeoxyglucose (2-FDG) on retinal protein synthesis correlated with inhibition of lactate production and diminished ATP content, with all these effects reversed by provision of d-mannose. 2-DG treatment was not associated with increased AMPK, eEF2, or eIF2α phosphorylation; instead, it caused rapid dephosphorylation of 4E-BP1. 2-DG reduced total mTOR activity by 25%, but surprisingly, it did not reduce mTORC1 activity, as indicated by unaltered raptor-associated mTOR autophosphorylation and ribosomal protein S6 phosphorylation. Dephosphorylation of 4E-BP1 was largely prevented by inhibition of PP1/PP2A phosphatases with okadaic acid and calyculin A, and inhibition of PPM1 phosphatases with cadmium. Thus, inhibition of retinal glycolysis diminished Akt and protein synthesis coinciding with accelerated dephosphorylation of 4E-BP1 independently of mTORC1. These results demonstrate a novel mechanism regulating protein synthesis in the retina involving an mTORC1-independent and phosphatase-dependent regulation of 4E-BP1. Copyright © 2015 the American Physiological Society.
Characterization of the Eimeria maxima sporozoite surface protein IMP1.

PubMed

Jenkins, M C; Fetterer, R; Miska, K; Tuo, W; Kwok, O; Dubey, J P

2015-07-30

The purpose of this study was to characterize Eimeria maxima immune-mapped protein 1 (IMP1) that is hypothesized to play a role in eliciting protective immunity against E. maxima infection in chickens. RT-PCR analysis of RNA from unsporulated and sporulating E. maxima oocysts revealed highest transcription levels at 6-12h of sporulation with a considerable downregulation thereafter. Alignment of IMP1 coding sequence from Houghton, Weybridge, and APU-1 strains of E. maxima revealed single nucleotide polymorphisms that in some instances led to amino acid changes in the encoded protein sequence. The E. maxima (APU-1) IMP1 cDNA sequence was cloned and expressed in 2 different polyHis Escherichia coli expression vectors. Regardless of expression vector, recombinant E. maxima IMP1 (rEmaxIMP1) was fairly unstable in non-denaturing buffer, which is consistent with stability analysis of the primary amino acid sequence. Antisera specific for rEmaxIMP1 identified a single 72 kDa protein or a 61 kDa protein by non-reducing or reducing SDS-PAGE/immunoblotting. Immunofluorescence staining with anti-rEmaxIMP1, revealed intense surface staining of E. maxima sporozoites, with negligible staining of merozoite stages. Immuno-histochemical staining of E. maxima-infected chicken intestinal tissue revealed staining of E. maxima developmental stages in the lamnia propia and crypts at both 24 and 48 h post-infection, and negligible staining thereafter. The expression of IMP1 during early stages of in vivo development and its location on the sporozoite surface may explain in part the immunoprotective effect of this protein against E. maxima infection. Published by Elsevier B.V.
XAFS and Protein Crystallography Beamline BL38B1 at SPring-8

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

Tanida, Hajime; Miura, Keiko; Takeshita, Kunikazu

2004-05-12

The SPring-8 bending magnet beamline BL38B1 is designed for R and D of optics, detectors, experiments for XAFS and protein X-ray crystallography (PX). This beamline has a multi-purpose hutch for two experimental stations of XAFS and PX, and removable optical benches used for R and D of detectors and instruments. The design and the performance of the beamline are presented.
The adenovirus E4-ORF3 protein functions as a SUMO E3 ligase for TIF-1γ sumoylation and poly-SUMO chain elongation.

PubMed

Sohn, Sook-Young; Hearing, Patrick

2016-06-14

The adenovirus (Ad) early region 4 (E4)-ORF3 protein regulates diverse cellular processes to optimize the host environment for the establishment of Ad replication. E4-ORF3 self-assembles into multimers to form a nuclear scaffold in infected cells and creates distinct binding interfaces for different cellular target proteins. Previous studies have shown that the Ad5 E4-ORF3 protein induces sumoylation of multiple cellular proteins and subsequent proteasomal degradation of some of them, but the detailed mechanism of E4-ORF3 function remained unknown. Here, we investigate the role of E4-ORF3 in the sumoylation process by using transcription intermediary factor (TIF)-1γ as a substrate. Remarkably, we discovered that purified E4-ORF3 protein stimulates TIF-1γ sumoylation in vitro, demonstrating that E4-ORF3 acts as a small ubiquitin-like modifier (SUMO) E3 ligase. Furthermore, E4-ORF3 significantly increases poly-SUMO3 chain formation in vitro in the absence of substrate, showing that E4-ORF3 has SUMO E4 elongase activity. An E4-ORF3 mutant, which is defective in protein multimerization, exhibited severely decreased activity, demonstrating that E4-ORF3 self-assembly is required for these activities. Using a SUMO3 mutant, K11R, we found that E4-ORF3 facilitates the initial acceptor SUMO3 conjugation to TIF-1γ as well as poly-SUMO chain elongation. The E4-ORF3 protein displays no SUMO-targeted ubiquitin ligase activity in our assay system. These studies reveal the mechanism by which E4-ORF3 targets specific cellular proteins for sumoylation and proteasomal degradation and provide significant insight into how a small viral protein can play a role as a SUMO E3 ligase and E4-like SUMO elongase to impact a variety of cellular responses.
Disruption of protein-tyrosine phosphatase 1B expression in the pancreas affects β-cell function.

PubMed

Liu, Siming; Xi, Yannan; Bettaieb, Ahmed; Matsuo, Kosuke; Matsuo, Izumi; Kulkarni, Rohit N; Haj, Fawaz G

2014-09-01

Protein-tyrosine phosphatase 1B (PTP1B) is a physiological regulator of glucose homeostasis and energy balance. However, the role of PTP1B in pancreatic endocrine function remains largely unknown. To investigate the metabolic role of pancreatic PTP1B, we generated mice with pancreas PTP1B deletion (panc-PTP1B KO). Mice were fed regular chow or a high-fat diet, and metabolic parameters, insulin secretion and glucose tolerance were determined. On regular chow, panc-PTP1B KO and control mice exhibited comparable glucose tolerance whereas aged panc-PTP1B KO exhibited mild glucose intolerance. Furthermore, high-fat feeding promoted earlier impairment of glucose tolerance and attenuated glucose-stimulated insulin secretion in panc-PTP1B KO mice. The secretory defect in glucose-stimulated insulin secretion was recapitulated in primary islets ex vivo, suggesting that the effects were likely cell-autonomous. At the molecular level, PTP1B deficiency in vivo enhanced basal and glucose-stimulated tyrosyl phosphorylation of EphA5 in islets. Consistently, PTP1B overexpression in the glucose-responsive MIN6 β-cell line attenuated EphA5 tyrosyl phosphorylation, and substrate trapping identified EphA5 as a PTP1B substrate. In summary, these studies identify a novel role for PTP1B in pancreatic endocrine function.
2,4-Dihydroxychalcone derivatives as novel potent cell division cycle 25B phosphatase inhibitors and protein tyrosine phosphatase 1B inhibitors.

PubMed

Xie, Chao; Sun, Yuan; Pan, Cheng-Yan; Tang, Li-Ming; Guan, Li-Ping

2014-04-01

Eleven 2,4-dihydroxychalcone compounds were synthesized and identified as reversible and competitive cell division cycle 25 (CDC25) B and protein tyrosine phosphatase (PTP) 1B inhibitors with inhibition values in the micromolar range. The results showed that nine compounds significantly inhibited CDC25B phosphatase, whereas seven compounds inhibited the activity against PTP1B in vitro. Compound 8 had the greatest inhibition activity against CDC25B and PTP1B in vitro, with percentage inhibition values of 97.5% and 96.3% at a dose of 20 microg/mL, respectively. Cytotoxic activity assays revealed that compound 8 was the most potent against HCT116, HeLa, and A549 cells. Furthermore, compound 8 exhibited potent antitumor activity in a colo205 xenograft model.
Atypical Responsiveness of the Orphan Receptor GPR55 to Cannabinoid Ligands*

PubMed Central

Kapur, Ankur; Zhao, Pingwei; Sharir, Haleli; Bai, Yushi; Caron, Marc G.; Barak, Larry S.; Abood, Mary E.

2009-01-01

The cannabinoid receptor 1 (CB1) and CB2 cannabinoid receptors, associated with drugs of abuse, may provide a means to treat pain, mood, and addiction disorders affecting widespread segments of society. Whether the orphan G-protein coupled receptor GPR55 is also a cannabinoid receptor remains unclear as a result of conflicting pharmacological studies. GPR55 has been reported to be activated by exogenous and endogenous cannabinoid compounds but surprisingly also by the endogenous non-cannabinoid mediator lysophosphatidylinositol (LPI). We examined the effects of a representative panel of cannabinoid ligands and LPI on GPR55 using a β-arrestin-green fluorescent protein biosensor as a direct readout of agonist-mediated receptor activation. Our data demonstrate that AM251 and SR141716A (rimonabant), which are cannabinoid antagonists, and the lipid LPI, which is not a cannabinoid receptor ligand, are GPR55 agonists. They possess comparable efficacy in inducing β-arrestin trafficking and, moreover, activate the G-protein-dependent signaling of protein kinase CβII. Conversely, the potent synthetic cannabinoid agonist CP55,940 acts as a GPR55 antagonist/partial agonist. CP55,940 blocks GPR55 internalization, the formation of β-arrestin GPR55 complexes, and the phosphorylation of ERK1/2; CP55,940 produces only a slight amount of protein kinase CβII membrane recruitment but does not stimulate membrane remodeling like LPI, AM251, or rimonabant. Our studies provide a paradigm for measuring the responsiveness of GPR55 to a variety of ligand scaffolds comprising cannabinoid and novel compounds and suggest that at best GPR55 is an atypical cannabinoid responder. The activation of GPR55 by rimonabant may be responsible for some of the off-target effects that led to its removal as a potential obesity therapy. PMID:19723626
Jumonji/Arid1b (Jarid1b) protein modulates human esophageal cancer cell growth

PubMed Central

KANO, YOSHIHIRO; KONNO, MASAMITSU; OHTA, KATSUYA; HARAGUCHI, NAOTSUGU; NISHIKAWA, SHIMPEI; KAGAWA, YOSHINORI; HAMABE, ATSUSHI; HASEGAWA, SHINICHIRO; OGAWA, HISATAKA; FUKUSUMI, TAKAHITO; NOGUCHI, YUKO; OZAKI, MIYUKI; KUDO, TOSHIHIRO; SAKAI, DAISUKE; SATOH, TAROH; ISHII, MASARU; MIZOHATA, EIICHI; INOUE, TAKESHI; MORI, MASAKI; DOKI, YUICHIRO; ISHII, HIDESHI

2013-01-01

Although esophageal cancer is highly heterogeneous and the involvement of epigenetic regulation of cancer stem cells is highly suspected, the biological significance of epigenetically modified molecules that regulate different subpopulations remains to be firmly established. Using esophageal cancer cells, we investigated the functional roles of the H3K4 demethylase Jumonji/Arid1b (Jarid1b) (Kdm5b/Plu-1/Rbp2-h1), an epigenetic factor that is required for continuous cell growth in melanoma. JARID1B knockdown resulted in the suppression of esophageal cancer cell growth, sphere formation and invasion ability and was associated with loss of epithelial marker expression. However, these inhibitory effects observed on tumor formation were reverted subsequent to subcutaneous inoculation of these cells into immune-deficient mice. These results indicated that JARID1B plays a role in maintaining cancer stem cells in the esophagus and justifies the rationale for studying the effects of continuous inhibition of this epigenetic factor in esophageal cancer. PMID:24649241
Characterization and comparison of Fumonisin B(1)-protein conjugates by six methods.

PubMed

Wang, Ying; He, Cheng-Hua; Zheng, Hao; Zhang, Hai-Bin

2012-01-01

In order to generate an antibody against a small hapten molecule, the hapten is cross-linked with carrier protein to make it immunogenic. In this study, the hapten (Fumonisin B(1), FB(1)) was coupled to ovalbumin (OVA) and bovine serum albumin (BSA), respectively by a short cross-linker reagent (glutaraldehyde, GA). To develop a technique for detecting the conjugation, the hapten-protein conjugates (FB(1)-OVA and FB(1)-BSA) were characterized thoroughly by ultraviolet (UV) spectroscopy, Fourier transform infrared (FT-IR) spectroscopy, gel electrophoresis and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS), respectively. The molecular weights of FB(1)-BSA and FB(1)-OVA were 74,355.301 Da and 48,009.212 Da, respectively determined by the method of MALDI-TOF-MS. The molecular coupling ratios were 11 and 5 in FB(1)-BSA and FB(1)-OVA, respectively. In this experiment, MALDI-TOF-MS was selected as the most efficient method to evaluate the cross-linking effect and calculate the molecular coupling ratio.
Various plus unique: Viral protein U as a plurifunctional protein for HIV-1 replication.

PubMed

Soper, Andrew; Juarez-Fernandez, Guillermo; Aso, Hirofumi; Moriwaki, Miyu; Yamada, Eri; Nakano, Yusuke; Koyanagi, Yoshio; Sato, Kei

2017-04-01

Human immunodeficiency virus type 1 (HIV-1), the causative agent of acquired immunodeficiency syndrome, encodes four accessory genes, one of which is viral protein U (Vpu). Recently, the study of Vpu has been of great interest. For instance, various cellular proteins are degraded (e.g. CD4) and down-modulated (e.g. tetherin) by Vpu. Vpu also antagonizes the function of tetherin and inhibits NF-κB. Moreover, Vpu is a viroporin forming ion channels and may represent a promising target for anti-HIV-1 drugs. In this review, we summarize the domains/residues that are responsible for Vpu's functions, describe the current understanding of the role of Vpu in HIV-1-infected cells, and review the effect of Vpu on HIV-1 in replication and pathogenesis. Future investigations that simultaneously assess a combination of Vpu functions are required to clearly delineate the most important functions for viral replication. Impact statement Viral protein U (Vpu) is a unique protein encoded by human immunodeficiency virus type 1 (HIV-1) and related lentiviruses, playing multiple roles in viral replication and pathogenesis. In this review, we briefly summarize the most up-to-date knowledge of HIV-1 Vpu.
Phosphorylated 4E binding protein 1: a hallmark of cell signaling that correlates with survival in ovarian cancer.

PubMed

Castellvi, Josep; Garcia, Angel; Rojo, Federico; Ruiz-Marcellan, Carmen; Gil, Antonio; Baselga, Jose; Ramon y Cajal, Santiago

2006-10-15

Growth factor receptors and cell signaling factors play a crucial role in human carcinomas and have been studied in ovarian tumors with varying results. Cell signaling involves multiple pathways and a myriad of factors that can be mutated or amplified. Cell signaling is driven through the mammalian target of rapamycin (mTOR) and extracellular regulated kinase (ERK) pathways and by some downstream molecules, such as 4E binding protein 1 (4EBP1), eukaryotic initiation factor 4E, and p70 ribosomal protein S6 kinase (p70S6K). The objectives of this study were to analyze the real role that these pathways play in ovarian cancer, to correlate them with clinicopathologic characteristics, and to identify the factors that transmit individual proliferation signals and are associated with pathologic grade and prognosis, regardless specific oncogenic alterations upstream. One hundred twenty-nine ovarian epithelial tumors were studied, including 20 serous cystadenomas, 7 mucinous cystadenomas, 11 serous borderline tumors, 16 mucinous borderline tumors, 29 serous carcinomas, 16 endometrioid carcinomas, 15 clear cell carcinomas, and 15 mucinous carcinomas. Tissue microarrays were constructed, and immunohistochemistry for the receptors epidermal growth factor receptor (EGFR) and c-erb-B2 was performed and with phosphorylated antibodies for protein kinase B (AKT), 4EBP1, p70S6K, S6, and ERK. Among 129 ovarian neoplasms, 17.8% were positive for c-erb-B2, 9.3% were positive for EGFR, 47.3% were positive for phosphorylated AKT (p-AKT), 58.9% were positive for p-ERK, 41.1% were positive for p-4EBP1, 26.4% were positive for p70S6K, and 15.5% were positive for p-S6. Although EGFR, p-AKT, and p-ERK expression did not differ between benign, borderline, or malignant tumors, c-erb-B2, p-4EBP1, p-p70S6K, and p-S6 were expressed significantly more often in malignant tumors. Only p-4EBP1 expression demonstrated prognostic significance (P = .005), and only surgical stage and p-4EBP1 expression
N-terminus conservation in the terminal pigment of phycobilisomes from a prokaryotic and eukaryotic alga. [Porphyridium cruentum; Nostoc

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

Gantt, E.; Cunningham, F.X. Jr.; Lipschultz, C.A.

1988-04-01

High molecular weight polypeptides from phycobilisomes, believed to be involved in facilitating the energy flow from phycobilisomes to thylakoids, are conserved in the prokaryote Nostoc sp. and the eukaryote Porphyridium cruentum. Partial N-terminal sequence analysis of the phycobilisome-polypeptides of Nostoc (94 kilodalton) and Porphyridium (92 kilodalton) revealed 55% identity in the first 20 residues, but no significant homology with sequences of other phycobiliproteins or phycobilisome-linkers. Polypeptides (94 and 92 kilodalton) from Nostoc thylakoids free of phycobilisomes, previously presumed to be involved in the phycobilisome-thylakoid linkage exhibit the same immunocrossreactivity but are different from the 94 kilodalton-phycobilisome polypeptide by having blockedmore » N-termini and a different amino acid composition.« less

Cloning and expression of Bartonella henselae sucB gene encoding an immunogenic dihydrolipoamide succinyltransferase homologous protein.

PubMed

Kabeya, Hidenori; Maruyama, Soichi; Hirano, Kouji; Mikami, Takeshi

2003-01-01

Immunoscreening of a ZAP genomic library of Bartonella henselae strain Houston-1 expressed in Escherichia coli resulted in the isolation of a clone containing 3.5 kb BamHI genomic DNA fragment. This 3.5 kb DNA fragment was found to contain a sequence of a gene encoding a protein with significant homology to the dihydrolipoamide succinyltransferase of Brucella melitensis (sucB). Subsequent cloning and DNA sequence analysis revealed that the deduced amino acid sequence from the cloned gene showed 66.5% identity to SucB protein of B. melitensis, and 43.4 and 47.2% identities to those of Coxiella burnetii and E. coli, respectively. The gene was expressed as a His-Nus A-tagged fusion protein. The recombinant SucB protein (rSucB) was shown to be an immunoreactive protein of about 115 kDa by Western blot analysis with sera from B. henselae-immunized mice. Therefore the rSucB may be a candidate antigen for a specific serological diagnosis of B. henselae infection.
The effects of clobazam treatment in rats on the expression of genes and proteins encoding glucronosyltransferase 1A/2B (UGT1A/2B) and multidrug resistance‐associated protein-2 (MRP2), and development of thyroid follicular cell hypertrophy

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

Miyawaki, Izuru, E-mail: izuru-miyawaki@ds-pharma.co.jp; Tamura, Akitoshi; Matsumoto, Izumi

Clobazam (CLB) is known to increase hepatobiliary thyroxine (T4) clearance in Sprague–Dawley (SD) rats, which results in hypothyroidism followed by thyroid follicular cell hypertrophy. However, the mechanism of the acceleration of T4-clearance has not been fully investigated. In the present study, we tried to clarify the roles of hepatic UDP-glucronosyltransferase (UGT) isoenzymes (UGT1A and UGT2B) and efflux transporter (multidrug resistance–associated protein-2; MRP2) in the CLB-induced acceleration of T4-clearance using two mutant rat strains, UGT1A-deficient mutant (Gunn) and MRP2-deficient mutant (EHBR) rats, especially focusing on thyroid morphology, levels of circulating hormones (T4 and triiodothyronine (T3)) and thyroid-stimulating hormone (TSH), and mRNAmore » or protein expressions of UGTs (Ugt1a1, Ugt1a6, and Ugt2b1/2) and MRP2 (Mrp). CLB induced thyroid morphological changes with increases in TSH in SD and Gunn rats, but not in EHBR rats. T4 was slightly decreased in SD and Gunn rats, and T3 was decreased in Gunn rats, whereas these hormones were maintained in EHBR rats. Hepatic Ugt1a1, Ugt1a6, Ugt2b1/2, and Mrp2 mRNAs were upregulated in SD rats. In Gunn rats, UGT1A mRNAs (Ugt1a1/6) and protein levels were quite low, but UGT2B mRNAs (Ugt2b1/2) and protein were prominently upregulated. In SD and Gunn rats, MRP2 mRNA and protein were upregulated to the same degree. These results suggest that MRP2 is an important contributor in development of the thyroid cellular hypertrophy in CLB-treated rats, and that UGT1A and UGT2B work in concert with MRP2 in the presence of MRP2 function to enable the effective elimination of thyroid hormones. -- Highlights: ► Role of UGT and MRP2 in thyroid pathology was investigated in clobazam-treated rats. ► Clobazam induced thyroid cellular hypertrophy in SD and Gunn rats, but not EHBR rats. ► Hepatic Mrp2 gene and protein were upregulated in SD and Gunn rats, but not EHBR rats. ► Neither serum thyroid hormones
52. G.E.H. photographer August 1930 1HUM1B #59, OILING OPERATIONS, GEH ...

Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

52. G.E.H. photographer August 1930 1-HUM-1-B #59, OILING OPERATIONS, GEH 8-30. Stamped office copy. - Redwood National & State Parks Roads, California coast from Crescent City to Trinidad, Crescent City, Del Norte County, CA
Over-activation of AKT signaling leading to 5-Fluorouracil resistance in SNU-C5/5-FU cells

PubMed Central

Kim, Eun-Ji; Kang, Gyeoung-Jin; Kang, Jung-Il; Boo, Hye-Jin; Hyun, Jin Won; Koh, Young Sang; Chang, Weon-Young; Kim, Young Ree; Kwon, Jung-Mi; Maeng, Young Hee; Yoo, Eun-Sook; Lee, Chang Hoon; Kang, Hee-Kyoung

2018-01-01

Here, we investigated whether over-activation of AKT pathway is important in the resistance to 5-fluorouracil (5-FU) in SNU-C5/5-FU cells, 5-FU-resistant human colon cancer cells. When compared to wild type SNU-C5 cells (WT), SNU-C5/5-FU cells showed over-activation of PI3K/AKT pathway, like increased phosphorylation of AKT, mTOR, and GSK-3β, nuclear localization of β-catenin, and decreased E-cadherin. Moreover, E-cadherin level was down-regulated in recurrent colon cancer tissues compared to primary colon cancer tissues. Gene silencing of AKT1 or treatment of LY294002 (PI3 kinase inhibitor) increased E-cadherin, whereas decreased phospho-GSK-3β. LY294002 also reduced protein level of β-catenin with no influence on mRNA level. PTEN level was higher in SNU-C5/WT than SNU-C5/5-FU cells, whereas the loss of PETN in SNU-C5/WT cells induced characteristics of SNU-C5/5-FU cells. In SNU-C5/5-FU cells, NF-κB signaling was activated, along with the overexpression of COX-2 and stabilization of survivin. However, increased COX-2 contributed to the stabilization of survivin, which directly interacts with cytoplasmic procaspase-3, while the inhibition of AKT reduced this cascade. We finally confirmed that combination treatment with 5-FU and LY294002 or Vioxx could induce apoptosis in SNU-C5/5-FU cells. These data suggest that inhibition of AKT activation may overcome 5-FU-resistance in SNU-C5/5-FU cells. These findings provide evidence that over-activation of AKT is crucial for the acquisition of resistance to anticancer drugs and AKT pathway could be a therapeutic target for cancer treatment. PMID:29731993
Identification of a conserved neutralizing linear B-cell epitope in the VP1 proteins of duck hepatitis A virus type 1 and 3.

PubMed

Zhang, Ruihua; Zhou, Guomei; Xin, Yinghao; Chen, Junhao; Lin, Shaoli; Tian, Ye; Xie, Zhijing; Jiang, Shijin

2015-11-18

Duck virus hepatitis (DVH), mainly caused by duck hepatitis A virus (DHAV), is a severe disease threaten to duck industry and has worldwide distribution. As the major structural protein, the VP1 protein of DHAV is able to induce neutralizing antibody in ducks. In this study, a monoclonal antibody (mAb) 4F8 against the intact DHAV-1 particles was used to identify the possible epitope in the three serotypes of DHAV. The mAb 4F8 had weak neutralizing activities to both DHAV-1 and DHAV-3, and reacted with the conserved linear B-cell epitopes of (75)GEIILT(80) in DHAV-1 VP1 and (75)GEVILT(80) in DHAV-3 VP1 protein, respectively, while not with DHAV-2 VP1. This was the first report about identification of the common conserved neutralizing linear B-cell epitope of DHAV-1 and DHAV-3, which will facilitate understanding of the antigenic structure of VP1 and the serologic diagnosis of DHAV infection. Copyright © 2015 Elsevier B.V. All rights reserved.
Iron acquisition in Pasteurella haemolytica: expression and identification of a bovine-specific transferrin receptor.

PubMed Central

Ogunnariwo, J A; Schryvers, A B

1990-01-01

Seven type 1 field isolates of Pasteurella haemolytica were screened for their ability to use different transferrins as a source of iron for growth. All seven strains were capable of using bovine but not human, porcine, avian, or equine transferrin. A screening assay failed to detect siderophore production in any of the strains tested. Iron-deficient cells from these strains expressed a binding activity, specific for bovine transferrin, that was regulated by the level of iron in the medium. Inhibition of expression by translation and transcription inhibitors suggested that iron regulation was occurring at the gene level. Affinity isolation of receptor proteins from all seven strains with biotinylated bovine transferrin identified a 100-kilodalton iron-regulated outer membrane protein as the bovine transferrin receptor. Iron-regulated outer membrane proteins of 71 and 77 kilodaltons were isolated along with the 100-kilodalton protein when less stringent washing procedures were employed in the affinity isolation procedure. Images PMID:2365453
Analytical Ultracentrifugation Sedimentation Velocity for the Characterization of Detergent-Solubilized Membrane Proteins Ca++-ATPase and ExbB

PubMed Central

Salvay, Andrés G.; Santamaria, Monica; le Maire, Marc

2008-01-01

We have investigated the potential of new methods of analysis of sedimentation velocity (SV) analytical ultracentrifugation (AUC) for the characterization of detergent-solubilized membrane proteins. We analyze the membrane proteins Ca++-ATPase and ExbB solubilized with DDM (dodecyl-β-d-maltoside). SV is extremely well suited for characterizing sample heterogeneity. DDM micelles (s20w = 3.1 S) and complexes (Ca++-ATPase: s20w = 7.3 S; ExbB: s20w = 4 S) are easily distinguished. Using different detergent and protein concentrations, SV does not detect any evidence of self-association for the two proteins. An estimate of bound detergent of 0.9 g/g for Ca++-ATPase and 1.5 g/g for ExbB is obtained from the combined analysis of SV profiles obtained using absorbance and interference optics. Combining s20w with values of the hydrodynamic radius, Rs = 5.5 nm for Ca++-ATPase or Rs = 3.4 nm for ExbB, allows the determination of buoyant molar masses, Mb. In view of their Mb and composition, Ca++-ATPase and ExbB are monomers in our experimental conditions. We conclude that one of the main advantages of SV versus other techniques is the possibility to ascertain the homogeneity of the samples and to focus on a given complex even in the presence of other impurities or aggregates. The relative rapidity of SV measurements also allows experiments on unstable samples. PMID:19669527
Heterochromatin protein 1 gamma and IκB kinase alpha interdependence during tumour necrosis factor gene transcription elongation in activated macrophages.

PubMed

Thorne, James L; Ouboussad, Lylia; Lefevre, Pascal F

2012-09-01

IκB kinase α (IKKα) is part of the cytoplasmic IKK complex regulating nuclear factor-κB (NF-κB) release and translocation into the nucleus in response to pro-inflammatory signals. IKKα can also be recruited directly to the promoter of NF-κB-dependent genes by NF-κB where it phosphorylates histone H3 at serine 10, triggering recruitment of the bromodomain-containing protein 4 and the positive transcription elongation factor b. Herein, we report that IKKα travels with the elongating form of ribonucleic acid polymerase II together with heterochromatin protein 1 gamma (HP1γ) at NF-κB-dependent genes in activated macrophages. IKKα binds to and phosphorylates HP1γ, which in turn controls IKKα binding to chromatin and phosphorylation of the histone variant H3.3 at serine 31 within transcribing regions. Downstream of transcription end sites, IKKα accumulates with its inhibitor the CUE-domain containing protein 2, suggesting a link between IKKα inactivation and transcription termination.
Polycyclic phloroglucinols as PTP1B inhibitors from Hypericum longistylum: Structures, PTP1B inhibitory activities, and interactions with PTP1B.

PubMed

Cao, Xiangrong; Yang, Xueyuan; Wang, Peixia; Liang, Yue; Liu, Feng; Tuerhong, Muhetaer; Jin, Da-Qing; Xu, Jing; Lee, Dongho; Ohizumi, Yasushi; Guo, Yuanqiang

2017-12-01

Protein tyrosine phosphatase 1B (PTP1B) has been regarded asa target for the research and development of new drugs to treat type II diabetes and PTP1B inhibitors are potential lead compounds for this type of new drugs. A phytochemical investigation to obtain new PTP1B inhibitors resulted in the isolation of four new phloroglucinols, longistyliones A-D (1-4) from the aerial parts of Hypericum longistylum. The structures of 1-4 were elucidated on the basis of extensive 1D and 2D NMR spectroscopic data analysis, and the absolute configurations of these compounds were established by comparing their experimental electronic circular dichroism (ECD) spectra with those calculated by the time-dependent density functional theory method. Compounds 1-4 possess a rare polycyclic phloroglucinol skeleton. The following biological evaluation revealed that all of the compounds showed PTP1B inhibitory effects. The further molecular docking studies indicated the strong interactions between these bioactive compounds with the PTP1B protein, which revealed the possible mechanism of PTP1B inhibition of bioactive compounds. All of the results implied that these compounds are potentially useful for the treatment of type II diabetes. Copyright © 2017 Elsevier Inc. All rights reserved.
SalB inactivation modulates culture supernatant exoproteins and affects autolysis and viability in Enterococcus faecalis OG1RF.

PubMed

Shankar, Jayendra; Walker, Rachel G; Wilkinson, Mark C; Ward, Deborah; Horsburgh, Malcolm J

2012-07-01

The culture supernatant fraction of an Enterococcus faecalis gelE mutant of strain OG1RF contained elevated levels of the secreted antigen SalB. Using differential fluorescence gel electrophoresis (DIGE) the salB mutant was shown to possess a unique complement of exoproteins. Differentially abundant exoproteins were identified using matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry. Stress-related proteins including DnaK, Dps family protein, SOD, and NADH peroxidase were present in greater quantity in the OG1RF salB mutant culture supernatant. Moreover, several proteins involved in cell wall synthesis and cell division, including d-Ala-d-Lac ligase and EzrA, were present in reduced quantity in OG1RF salB relative to the parent strain. The salB mutant displayed reduced viability and anomalous cell division, and these phenotypes were exacerbated in a gelE salB double mutant. An epistatic relationship between gelE and salB was not identified with respect to increased autolysis and cell morphological changes observed in the salB mutant. SalB was purified as a six-histidine-tagged protein to investigate peptidoglycan hydrolytic activity; however, activity was not evident. High-pressure liquid chromatography (HPLC) analysis of reduced muropeptides from peptidoglycan digested with mutanolysin revealed that the salB mutant and OG1RF were indistinguishable.
[Incidence of exercise-induced bronchospasm (E.I.B.) and its correlation with clinical history in children with allergic asthma].

PubMed

Novembre, E; Dini, L; Veneruso, G; Lombardi, E; Bernardini, R; Vierucci, A

1993-01-01

In this study, 65 allergic asthmatic children were evaluated for the presence of exercise induced bronchospasm (E.I.B.). Pulmonary function (volume at 1 second of forced expiration--FEV1; maximal mid-expiratory flow--FEF 25-75; peak expiratory flow--PEF) was assessed before and serially for 20' following treadmill exercise. Only 18 children (27.6%) presented a fall in FEV1 > or = 20%. There was a relationship between severity of asthma and incidence of E.I.B., as children with episodic, frequent and chronic asthma presented respectively 13/55 (23.4%), 2/6 (33.3%) and 3/4 (75%) of positive responses. The presence of E.I.B. cannot be predicted by clinical history, as 15/36 (41.7%) of subjects with positive clinical history had E.I.B., against 3/29 (10.3%) of subjects with negative clinical history (p = n.s.). E.I.B. in asthmatic children is less frequent as reported, correlates with severity of asthma and cannot be accurately predicted from the history.
Isolation and Characterization of Protein Tyrosine Phosphatase 1B (PTP1B) Inhibitory Polyphenolic Compounds From Dodonaea viscosa and Their Kinetic Analysis.

PubMed

Uddin, Zia; Song, Yeong Hun; Ullah, Mahboob; Li, Zuopeng; Kim, Jeong Yoon; Park, Ki Hun

2018-01-01

Diabetes mellitus is one of a major worldwide concerns, regulated by either defects in secretion or action of insulin, or both. Insulin signaling down-regulation has been related with over activity of protein tyrosine phosphatase 1B (PTP1B) enzyme, which has been a promising target for the treatment of diabetes mellitus. Herein, activity guided separation of methanol extract (95%) of Dodonaea viscosa aerial parts afforded nine ( 1 - 9 ) polyphenolic compounds, all of them were identified through spectroscopic data including 2D NMR and HREIMS. Subsequently, their PTP1B inhibitory potentials were evaluated, in which all of the isolates exhibited significant dose-dependent inhibition with IC 50 13.5-57.9 μM. Among them, viscosol ( 4 ) was found to be the most potent compound having IC 50 13.5 μM. In order to unveil the mechanistic behavior, detailed kinetic study was carried out, in which compound 4 was observed as a reversible, and mixed type I inhibitor of PTP1B with inhibitory constant ( K i ) value of 4.6 μM. Furthermore, we annotated the major metabolites through HPLC-DAD-ESI/MS analysis, in which compounds 3 , 6 , 7 , and 9 were found to be the most abundant metabolites in D. viscosa extract.
Isolation and characterization of protein tyrosine phosphatase 1B (PTP1B) inhibitory polyphenolic compounds from Dodonaea viscosa and their kinetic analysis

NASA Astrophysics Data System (ADS)

Uddin, Zia; Song, Yeong Hun; Ullah, Mahboob; Li, Zuopeng; Kim, Jeong Yoon; Park, Ki Hun

2018-03-01

Diabetes mellitus is one of a major worldwide concerns, regulated by either defects in secretion or action of insulin, or both. Insulin signaling down-regulation has been related with over activity of protein tyrosine phosphatase 1B (PTP1B) enzyme, which has been a promising target for the treatment of diabetes mellitus. Herein, activity guided separation of methanol extract (95%) of Dodonaea viscosa aerial parts afforded nine (1-9) polyphenolic compounds, all of them were identified through spectroscopic data including 2D NMR and HREIMS. Subsequently, their PTP1B inhibitory potentials were evaluated, in which all of the isolates exhibited significant dose-dependent inhibition with IC50 13.5-57.9 μM. Among them, viscosol (4) was found to be the most potent compound having IC50 13.5 μM. In order to unveil the mechanistic behavior, detailed kinetic study was carried out, in which compound 4 was observed as a reversible, and mixed type I inhibitor of PTP1B with inhibitory constant (Ki) value of 4.6 μM. Furthermore, we annotated the major metabolites through HPLC-DAD-ESI/MS analysis, in which compounds 3, 6, 7 and 9 were found to be the most abundant metabolites in D.viscosa extract.
Orpinomyces cellulase CelE protein and coding sequences

DOEpatents

Li, Xin-Liang; Ljungdahl, Lars G.; Chen, Huizhong

2000-08-29

A CDNA designated celE cloned from Orpinomyces PC-2 encodes a polypeptide (CelE) of 477 amino acids. CelE is highly homologous to CelB of Orpinomyces (72.3% identity) and Neocallimastix (67.9% identity), and like them, it has a non-catalytic repeated peptide domain (NCRPD) at the C-terminal end. The catalytic domain of CelE is homologous to glycosyl hydrolases of Family 5, found in several anaerobic bacteria. The gene of celE is devoid of introns. The recombinant proteins CelE and CelB of Orpinomyces PC-2 randomly hydrolyze carboxymethylcellulose and cello-oligosaccharides in the pattern of endoglucanases.
Regulation of HTLV-1 Gag budding by Vps4A, Vps4B, and AIP1/Alix

PubMed Central

Urata, Shuzo; Yokosawa, Hideyoshi; Yasuda, Jiro

2007-01-01

Background HTLV-1 Gag protein is a matrix protein that contains the PTAP and PPPY sequences as L-domain motifs and which can be released from mammalian cells in the form of virus-like particles (VLPs). The cellular factors Tsg101 and Nedd4.1 interact with PTAP and PPPY, respectively, within the HTLV-1 Gag polyprotein. Tsg101 forms a complex with Vps28 and Vps37 (ESCRT-I complex) and plays an important role in the class E Vps pathway, which mediates protein sorting and invagination of vesicles into multivesicular bodies. Nedd4.1 is an E3 ubiquitin ligase that binds to the PPPY motif through its WW motif, but its function is still unknown. In the present study, to investigate the mechanism of HTLV-1 budding in detail, we analyzed HTLV-1 budding using dominant negative (DN) forms of the class E proteins. Results Here, we report that DN forms of Vps4A, Vps4B, and AIP1 inhibit HTLV-1 budding. Conclusion These findings suggest that HTLV-1 budding utilizes the MVB pathway and that these class E proteins may be targets for prevention of mother-to-infant vertical transmission of the virus. PMID:17601348
A parapoxviral virion protein targets the retinoblastoma protein to inhibit NF-κB signaling

PubMed Central

Nagendraprabhu, Ponnuraj; Khatiwada, Sushil; Chaulagain, Sabal

2017-01-01

Poxviruses have evolved multiple strategies to subvert signaling by Nuclear Factor κB (NF-κB), a crucial regulator of host innate immune responses. Here, we describe an orf virus (ORFV) virion-associated protein, ORFV119, which inhibits NF-κB signaling very early in infection (≤ 30 min post infection). ORFV119 NF-κB inhibitory activity was found unimpaired upon translation inhibition, suggesting that virion ORFV119 alone is responsible for early interference in signaling. A C-terminal LxCxE motif in ORFV119 enabled the protein to interact with the retinoblastoma protein (pRb) a multifunctional protein best known for its tumor suppressor activity. Notably, experiments using a recombinant virus containing an ORFV119 mutation which abrogates its interaction with pRb together with experiments performed in cells lacking or with reduced pRb levels indicate that ORFV119 mediated inhibition of NF-κB signaling is largely pRb dependent. ORFV119 was shown to inhibit IKK complex activation early in infection. Consistent with IKK inhibition, ORFV119 also interacted with TNF receptor associated factor 2 (TRAF2), an adaptor protein recruited to signaling complexes upstream of IKK in infected cells. ORFV119-TRAF2 interaction was enhanced in the presence of pRb, suggesting that ORFV119-pRb complex is required for efficient interaction with TRAF2. Additionally, transient expression of ORFV119 in uninfected cells was sufficient to inhibit TNFα-induced IKK activation and NF-κB signaling, indicating that no other viral proteins are required for the effect. Infection of sheep with ORFV lacking the ORFV119 gene led to attenuated disease phenotype, indicating that ORFV119 contributes to virulence in the natural host. ORFV119 represents the first poxviral protein to interfere with NF-κB signaling through interaction with pRb. PMID:29244863
Aldo-keto reductase family 1 B10 protein detoxifies dietary and lipid-derived alpha, beta-unsaturated carbonyls at physiological levels

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

Zhong, Linlin; Department of Neurobiology and Anatomy, China Medical University, Shenyang 110001; Liu, Ziwen

2009-09-18

Alpha, beta-unsaturated carbonyls are highly reactive mutagens and carcinogens to which humans are exposed on a daily basis. This study demonstrates that aldo-keto reductase family 1 member B10 (AKR1B10) is a critical protein in detoxifying dietary and lipid-derived unsaturated carbonyls. Purified AKR1B10 recombinant protein efficiently catalyzed the reduction to less toxic alcohol forms of crotonaldehyde at 0.90 {mu}M, 4-hydroxynonenal (HNE) at 0.10 {mu}M, trans-2-hexanal at 0.10 {mu}M, and trans-2,4-hexadienal at 0.05 {mu}M, the concentrations at or lower than physiological exposures. Ectopically expressed AKR1B10 in 293T cells eliminated immediately HNE at 1 (subtoxic) or 5 {mu}M (toxic) by converting to 1,4-dihydroxynonene,more » protecting the cells from HNE toxicity. AKR1B10 protein also showed strong enzymatic activity toward glutathione-conjugated carbonyls. Taken together, our study results suggest that AKR1B10 specifically expressed in the intestine is physiologically important in protecting the host cell against dietary and lipid-derived cytotoxic carbonyls.« less
24 CFR 55.2 - Terminology.

Code of Federal Regulations, 2014 CFR

2014-04-01

... or inoperative during flood and storm events (e.g., data storage centers, generating plants...” (§ 55.2(b)(5)). When FEMA provides interim flood hazard data, such as Advisory Base Flood Elevations... data may be used as “best available information” in accordance with Executive Order 11988. However, a...
The Potyviral P3 Protein Targets Eukaryotic Elongation Factor 1A to Promote the Unfolded Protein Response and Viral Pathogenesis1[OPEN

PubMed Central

Shine, M.B.; Cui, Xiaoyan; Chen, Xin; Ma, Na; Kachroo, Pradeep; Zhi, Haijan; Kachroo, Aardra

2016-01-01

The biochemical function of the potyviral P3 protein is not known, although it is known to regulate virus replication, movement, and pathogenesis. We show that P3, the putative virulence determinant of soybean mosaic virus (SMV), targets a component of the translation elongation complex in soybean. Eukaryotic elongation factor 1A (eEF1A), a well-known host factor in viral pathogenesis, is essential for SMV virulence and the associated unfolded protein response (UPR). Silencing GmEF1A inhibits accumulation of SMV and another ER-associated virus in soybean. Conversely, endoplasmic reticulum (ER) stress-inducing chemicals promote SMV accumulation in wild-type, but not GmEF1A-knockdown, plants. Knockdown of genes encoding the eEF1B isoform, which is important for eEF1A function in translation elongation, has similar effects on UPR and SMV resistance, suggesting a link to translation elongation. P3 and GmEF1A promote each other’s nuclear localization, similar to the nuclear-cytoplasmic transport of eEF1A by the Human immunodeficiency virus 1 Nef protein. Our results suggest that P3 targets host elongation factors resulting in UPR, which in turn facilitates SMV replication and place eEF1A upstream of BiP in the ER stress response during pathogen infection. PMID:27356973
55 Cancri e with Atmosphere (Artist's Concept)

NASA Image and Video Library

2017-11-16

The super-Earth exoplanet 55 Cancri e, depicted with its star in this artist's concept, likely has an atmosphere thicker than Earth's, with ingredients that could be similar to those of Earth's atmosphere, according to a 2017 study using data from NASA's Spitzer Space Telescope. Scientists say the planet may be entirely covered in lava. The planet is so close to its star that one face of the planet consistently faces the star, resulting in a dayside and a nightside. https://photojournal.jpl.nasa.gov/catalog/PIA22069

The Role of the Rab Coupling Protein in ErbB2-Driven Mammary Tumorigenesis and Metastasis

DTIC Science & Technology

2014-10-01

Coupling Protein/Rab11FIP1/RCP, Epithelial Mesenchymal Transition , Cell junctions , Cell Proliferation, Senescence. 16. SECURITY CLASSIFICATION OF: 17...Tyrosine Kinase, Her/ErbB2 signaling, Rab Coupling Protein/Rab11FIP1/RCP, Epithelial Mesenchymal Transition , Cell junctions , Cell Proliferation...lines included RCP condition to internalization and detection of E-cadherin, a well-known adherent junction and epithelial mesenchymal transition
Therapeutic Strategies against Cyclin E1-Amplified Ovarian Cancers

DTIC Science & Technology

2016-10-01

AWARD NUMBER: W81XWH-15-1-0564 TITLE : Therapeutic Strategies against Cyclin E1-Amplified Ovarian Cancers PRINCIPAL INVESTIGATOR: Panagiotis A...4. TITLE AND SUBTITLE 5a. CONTRACT NUMBER Therapeutic Strategies against Cyclin E1-Amplified Ovarian Cancers 5b. GRANT NUMBER W81XWH-15-1-0564 5c... box protein M1, Retinoblastoma 16. SECURITY CLASSIFICATION OF: 17. LIMITATION OF ABSTRACT 18. NUMBER OF PAGES 19a. NAME OF RESPONSIBLE PERSON
PrkC-mediated phosphorylation of overexpressed YvcK protein regulates PBP1 protein localization in Bacillus subtilis mreB mutant cells.

PubMed

Foulquier, Elodie; Pompeo, Frédérique; Freton, Céline; Cordier, Baptiste; Grangeasse, Christophe; Galinier, Anne

2014-08-22

The YvcK protein has been shown to be necessary for growth under gluconeogenic conditions in Bacillus subtilis. Amazingly, its overproduction rescues growth and morphology defects of the actin-like protein MreB deletion mutant by restoration of PBP1 localization. In this work, we observed that YvcK was phosphorylated at Thr-304 by the protein kinase PrkC and that phosphorylated YvcK was dephosphorylated by the cognate phosphatase PrpC. We show that neither substitution of this threonine with a constitutively phosphorylated mimicking glutamic acid residue or a phosphorylation-dead mimicking alanine residue nor deletion of prkC or prpC altered the ability of B. subtilis to grow under gluconeogenic conditions. However, we observed that a prpC mutant and a yvcK mutant were more sensitive to bacitracin compared with the WT strain. In addition, the bacitracin sensitivity of strains in which YvcK Thr-304 was replaced with either an alanine or a glutamic acid residue was also affected. We also analyzed rescue of the mreB mutant strain by overproduction of YvcK in which the phosphorylation site was substituted. We show that YvcK T304A overproduction did not rescue the mreB mutant aberrant morphology due to PBP1 mislocalization. The same observation was made in an mreB prkC double mutant overproducing YvcK. Altogether, these data show that YvcK may have two distinct functions: 1) in carbon source utilization independent of its phosphorylation level and 2) in cell wall biosynthesis and morphogenesis through its phosphorylation state. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.
27 CFR 1.55 - Recalling permits for correction.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 27 Alcohol, Tobacco Products and Firearms 1 2010-04-01 2010-04-01 false Recalling permits for correction. 1.55 Section 1.55 Alcohol, Tobacco Products and Firearms ALCOHOL AND TOBACCO TAX AND TRADE BUREAU... upon demand of the appropriate TTB officer. ...
Escherichia coli K1 inhibits proinflammatory cytokine induction in monocytes by preventing NF-kappaB activation.

PubMed

Selvaraj, Suresh K; Prasadarao, Nemani V

2005-08-01

Phagocytes are well-known effectors of the innate immune system to produce proinflammatory cytokines and chemokines such as tumor necrosis factor alpha (TNF-alpha), interleukin (IL)-1beta, and IL-8 during infections. Here, we show that infection of monocytes with wild-type Escherichia coli K1, which causes meningitis in neonates, suppresses the production of cytokines and chemokines (TNF-alpha, regulated on activation, normal T expressed and secreted, macrophage-inflammatory protein-1beta, IL-1beta, and IL-8). In contrast, infection of monocytes with a mutant E. coli, which lacks outer membrane protein A (OmpA- E. coli) resulted in robust production of cytokines and chemokines. Wild-type E. coli K1 (OmpA+ E. coli) prevented the phosphorylation and its degradation of inhibitor of kappaB, thereby blocking the translocation of nuclear factor (NF)-kappaB to the nucleus. OmpA+ E. coli-infected cells, subsequently subjected to lipopolysaccharide challenge, were crippled severely in their ability to activate NF-kappaB to induce cytokine/chemokine production. Selective inhibitors of the extracellular signal-regulated kinase (ERK) 1/2 pathway and p38 mitogen-activated protein kinase (MAPK), but not Jun N-terminal kinase, significantly reduced the activation of NF-kappaB and the production of cytokines and chemokines induced by OmpA- E. coli, indicating a role for these kinases in the NF-kappaB/cytokine pathway. It is interesting that the phosphorylation of ERK 1/2 and p38 MAPK was notably reduced in monocytes infected with OmpA+ E. coli when compared with monocytes infected with OmpA- E. coli, suggesting that the modulation of upstream events common for NF-kappaB and MAPKs by the bacterium is possible. The ability of OmpA+ E. coli K1 to inhibit the macrophage response temporarily may enable bacterial survival and growth within the host for the onset of meningitis by E. coli K1.
GLYOXALASE I A111E, PARAOXONASE 1 Q192R AND L55M POLYMORPHISMS IN ITALIAN PATIENTS WITH SPORADIC CEREBRAL CAVERNOUS MALFORMATIONS: A PILOT STUDY.

PubMed

Rinaldi, C; Bramanti, P; Famà, A; Scimone, C; Donato, L; Antognelli, C; Alafaci, C; Tomasello, F; D'Angelo, R; Sidoti, A

2015-01-01

It is already known that the conditions of increased oxidative stress are associated to a greater susceptibility to vascular malformations including cerebral cavernous malformations (CCMs). These are vascular lesions of the CNS characterized by abnormally enlarged capillary cavities that can occur sporadically or as a familial autosomal dominant condition with incomplete penetrance and variable clinical expression attributable to mutations in three different genes: CCM1(Krit1), CCM2 (MGC4607) and CCM3 (PDCD10). Polymorphisms in the genes encoding for enzymes involved in the antioxidant systems such as glyoxalase I (GLO I) and paraoxonase I (PON I) could influence individual susceptibility to the vascular malformations. A single nucleotide polymorphism was identified in the exon 4 of GLO 1 gene that causes an amino acid substitution of Ala for Glu (Ala111Glu). Two common polymorphisms have been described in the coding region of PON1, which lead to glutamine → arginine substitution at 192 (Q192R) and a leucine → methionine substitution at 55 (L55M). The polymorphisms were characterized in 59 patients without mutations in the CCM genes versus 213 healthy controls by PCR/RFLP methods using DNA from lymphocytes. We found that the frequency of patients carrying the GLO1 A/E genotype among the case group (56%) was four-fold higher than among the controls (14.1%). In the cohort of CCM patients, an increase in the frequency of PON192 Q/R genotype was observed (39% in the CCM group versus 3.7% in the healthy controls). Similarly, an increase was observed in the proportion of individuals with the genotype R/R in the disease group (5%) in respect to the normal healthy cohort (0.5%). Finally, the frequency of the PON55 heterozygotes L/M genotype was 29% in patients with CCMs and 4% in the healthy controls. The same trend was observed in PON55 homozygous M/M genotype frequency (CCMs 20% vs controls 10%). The present study aimed to investigate the possible association of GLO1
Levels of the E2 interacting protein TopBP1 modulate papillomavirus maintenance stage replication

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

Kanginakudru, Sriramana, E-mail: skangina@iu.edu; DeSmet, Marsha, E-mail: mdesmet@iupui.edu; Thomas, Yanique, E-mail: ysthomas@umail.iu.edu

2015-04-15

The evolutionarily conserved DNA topoisomerase II beta-binding protein 1 (TopBP1) functions in DNA replication, DNA damage response, and cell survival. We analyzed the role of TopBP1 in human and bovine papillomavirus genome replication. Consistent with prior reports, TopBP1 co-localized in discrete nuclear foci and was in complex with papillomavirus E2 protein. Similar to E2, TopBP1 is recruited to the region of the viral origin of replication during G1/S and early S phase. TopBP1 knockdown increased, while over-expression decreased transient virus replication, without affecting cell cycle. Similarly, using cell lines harboring HPV-16 or HPV-31 genome, TopBP1 knockdown increased while over-expression reducedmore » viral copy number relative to genomic DNA. We propose a model in which TopBP1 serves dual roles in viral replication: it is essential for initiation of replication yet it restricts viral copy number. - Highlights: • Protein interaction study confirmed In-situ interaction between TopBP1 and E2. • TopBP1 present at papillomavirus ori in G1/S and early S phase of cell cycle. • TopBP1 knockdown increased, over-expression reduced virus replication. • TopBP1 protein level change did not influence cell survival or cell cycle. • TopBP1 displaced from papillomavirus ori after initiation of replication.« less
Perk Ablation Ameliorates Myelination in S63del-Charcot–Marie–Tooth 1B Neuropathy

PubMed Central

Musner, Nicolò; Sidoli, Mariapaola; Zambroni, Desireè; Del Carro, Ubaldo; Ungaro, Daniela; D’Antonio, Maurizio; Feltri, Maria L.

2016-01-01

In peripheral nerves, P0 glycoprotein accounts for more than 20% of myelin protein content. P0 is synthesized by Schwann cells, processed in the endoplasmic reticulum (ER) and enters the secretory pathway. However, the mutant P0 with S63 deleted (P0S63del) accumulates in the ER lumen and induces a demyelinating neuropathy in Charcot–Marie–Tooth disease type 1B (CMT1B)–S63del mice. Accumulation of P0S63del in the ER triggers a persistent unfolded protein response. Protein kinase RNA-like endoplasmic reticulum kinase (PERK) is an ER stress sensor that phosphorylates eukaryotic initiation factor 2 alpha (eIF2alpha) in order to attenuate protein synthesis. We have shown that increasing phosphophorylated-eIF2alpha (P-eIF2alpha) is a potent therapeutic strategy, improving myelination and motor function in S63del mice. Here, we explore the converse experiment: Perk haploinsufficiency reduces P-eIF2alpha in S63del nerves as expected, but surprisingly, ameliorates, rather than worsens S63del neuropathy. Motor performance and myelin abnormalities improved in S63del//Perk+/− compared with S63del mice. These data suggest that mechanisms other than protein translation might be involved in CMT1B/S63del neuropathy. In addition, Perk deficiency in other cells may contribute to demyelination in a non–Schwann-cell autonomous manner. PMID:27095827
Kinesin 1 regulates cilia length through an interaction with the Bardet-Biedl syndrome related protein CCDC28B.

PubMed

Novas, Rossina; Cardenas-Rodriguez, Magdalena; Lepanto, Paola; Fabregat, Matías; Rodao, Magela; Fariello, María Inés; Ramos, Mauricio; Davison, Camila; Casanova, Gabriela; Alfaya, Lucía; Lecumberry, Federico; González-Sapienza, Gualberto; Irigoín, Florencia; Badano, Jose L

2018-02-14

Bardet-Biedl syndrome (BBS) is a ciliopathy characterized by retinal degeneration, obesity, polydactyly, renal disease and mental retardation. CCDC28B is a BBS-associated protein that we have previously shown plays a role in cilia length regulation whereby its depletion results in shortened cilia both in cells and Danio rerio (zebrafish). At least part of that role is achieved by its interaction with the mTORC2 component SIN1, but the mechanistic details of this interaction and/or additional functions that CCDC28B might play in the context of cilia remain poorly understood. Here we uncover a novel interaction between CCDC28B and the kinesin 1 molecular motor that is relevant to cilia. CCDC28B interacts with kinesin light chain 1 (KLC1) and the heavy chain KIF5B. Notably, depletion of these kinesin 1 components results in abnormally elongated cilia. Furthermore, through genetic interaction studies we demonstrate that kinesin 1 regulates ciliogenesis through CCDC28B. We show that kinesin 1 regulates the subcellular distribution of CCDC28B, unexpectedly, inhibiting its nuclear accumulation, and a ccdc28b mutant missing a nuclear localization motif fails to rescue the phenotype in zebrafish morphant embryos. Therefore, we uncover a previously unknown role of kinesin 1 in cilia length regulation that relies on the BBS related protein CCDC28B.
La-related protein 1 (LARP1) binds the mRNA cap, blocking eIF4F assembly on TOP mRNAs

PubMed Central

Lahr, Roni M; Fonseca, Bruno D; Ciotti, Gabrielle E; Al-Ashtal, Hiba A; Jia, Jian-Jun; Niklaus, Marius R; Blagden, Sarah P; Alain, Tommy; Berman, Andrea J

2017-01-01

The 5’terminal oligopyrimidine (5’TOP) motif is a cis-regulatory RNA element located immediately downstream of the 7-methylguanosine [m7G] cap of TOP mRNAs, which encode ribosomal proteins and translation factors. In eukaryotes, this motif coordinates the synchronous and stoichiometric expression of the protein components of the translation machinery. La-related protein 1 (LARP1) binds TOP mRNAs, regulating their stability and translation. We present crystal structures of the human LARP1 DM15 region in complex with a 5’TOP motif, a cap analog (m7GTP), and a capped cytidine (m7GpppC), resolved to 2.6, 1.8 and 1.7 Å, respectively. Our binding, competition, and immunoprecipitation data corroborate and elaborate on the mechanism of 5’TOP motif binding by LARP1. We show that LARP1 directly binds the cap and adjacent 5’TOP motif of TOP mRNAs, effectively impeding access of eIF4E to the cap and preventing eIF4F assembly. Thus, LARP1 is a specialized TOP mRNA cap-binding protein that controls ribosome biogenesis. DOI: http://dx.doi.org/10.7554/eLife.24146.001 PMID:28379136
3-Phenylpropanoic acid-based phosphotyrosine (pTyr) mimetics: hit evolution to a novel orally active protein tyrosine phosphatase 1B (PTP1B) inhibitor.

PubMed

Tang, Yan-Bo; Liu, Jun-Zheng; Zhang, Shu-En; Du, Xin; Nie, Feilin; Tian, Jin-Ying; Ye, Fei; Huang, Kai; Hu, Jin-Ping; Li, Yan; Xiao, Zhiyan

2014-05-01

Protein tyrosine phosphatase 1B (PTP1B) is a promising therapeutic target for type 2 diabetes. Herein, we report the evolution of a previously identified 3-phenylpropanoic acid-based PTP1B inhibitor to an orally active lead compound. A series of 3-phenylpropanoic acid-based PTP1B inhibitors were synthesized, and three of them, 3-(4-(9H-carbazol-9-yl)phenyl)-5-(3,5-di-tert-butyl-4-methoxyphenyl)-5-oxopentanoic acid (9), 3-(4-(9H-carbazol-9-yl)phenyl)-5-(4'-bromo-[1,1'-biphenyl]-4-yl)-5-oxopentanoic acid (10) and 3-(4-(9H-carbazol-9-yl)-2-fluorophenyl)-5-(4-cyclohexylphenyl)-5-oxopentanoic acid (16), showed IC50 values at sub-micromolar level. Further in vivo evaluation indicated the sodium salt of 9 not only exhibited significant insulin-sensitizing and hypoglycemia effects, but also decreased the serum levels of triglyceride and total cholesterol in high-fat-diet-induced insulin resistance model mice. Preliminary in vivo pharmacokinetic studies on the sodium salt of 9 revealed its pharmacokinetic profile after oral administration in rats. These results provide proof-of-concept for the dual effects of PTP1B inhibitors on both glucose and lipid metabolisms. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
Insulin sparing action of adenovirus 36 and its E4orf1 protein.

PubMed

Dhurandhar, Nikhil V

2013-01-01

Additional drugs are required to effectively manage diabetes and its complications. Recent studies have revealed protective effects of Ad36, a human adenovirus, and its E4orf1 protein on glucose disposal, which may be creatively harnessed to develop novel anti-diabetic agents. Experimental Ad36 infection improves hyperglycemia in animal models and natural Ad36 infection in humans is associated with better glycemic control. Available data indicate distinctive advantages for a drug that may mimic the action of Ad36/E4orf1. The key features of such a potential drug include the ability to increase glucose uptake by adipose tissue and skeletal muscle, to reduce hepatic glucose output independent of proximal insulin signaling, and to up-regulate adiponectin and its hepatic action. The effect of Ad36/E4orf1 on hepatocyte metabolism suggests a role for treating hepatic steatosis. Despite these potential advantages, considerable research is required before such a drug is developed. The in vivo efficacy and safety of E4orf1 in improving hyperglycemia remain unknown, and an appropriate drug delivery system is required. Nonetheless, Ad36 E4orf1 offers a research opportunity to develop a new anti-diabetic agent with multiple potential advantages and conceptually advances the use of a rather unconventional source, microbial proteins, for anti-diabetic drug development. Copyright © 2013 Elsevier Inc. All rights reserved.
HIV-1 Tat protein induces DNA damage in human peripheral blood B-lymphocytes via mitochondrial ROS production.

PubMed

El-Amine, Rawan; Germini, Diego; Zakharova, Vlada V; Tsfasman, Tatyana; Sheval, Eugene V; Louzada, Ruy A N; Dupuy, Corinne; Bilhou-Nabera, Chrystèle; Hamade, Aline; Najjar, Fadia; Oksenhendler, Eric; Lipinski, Marс; Chernyak, Boris V; Vassetzky, Yegor S

2018-05-01

Human immunodeficiency virus (HIV) infection is associated with B-cell malignancies in patients though HIV-1 is not able to infect B-cells. The rate of B-cell lymphomas in HIV-infected individuals remains high even under the combined antiretroviral therapy (cART) that reconstitutes the immune function. Thus, the contribution of HIV-1 to B-cell oncogenesis remains enigmatic. HIV-1 induces oxidative stress and DNA damage in infected cells via multiple mechanisms, including viral Tat protein. We have detected elevated levels of reactive oxygen species (ROS) and DNA damage in B-cells of HIV-infected individuals. As Tat is present in blood of infected individuals and is able to transduce cells, we hypothesized that it could induce oxidative DNA damage in B-cells promoting genetic instability and malignant transformation. Indeed, incubation of B-cells isolated from healthy donors with purified Tat protein led to oxidative stress, a decrease in the glutathione (GSH) levels, DNA damage and appearance of chromosomal aberrations. The effects of Tat relied on its transcriptional activity and were mediated by NF-κB activation. Tat stimulated oxidative stress in B-cells mostly via mitochondrial ROS production which depended on the reverse electron flow in Complex I of respiratory chain. We propose that Tat-induced oxidative stress, DNA damage and chromosomal aberrations are novel oncogenic factors favoring B-cell lymphomas in HIV-1 infected individuals. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.
Flowering-Related RING Protein 1 (FRRP1) Regulates Flowering Time and Yield Potential by Affecting Histone H2B Monoubiquitination in Rice (Oryza Sativa).

PubMed

Du, Yiwei; He, Wei; Deng, Changwang; Chen, Xi; Gou, Lanming; Zhu, Fugui; Guo, Wei; Zhang, Jianfu; Wang, Tao

2016-01-01

Flowering time is a critical trait for crops cultivated under various temperature/photoperiod conditions around the world. To understand better the flowering time of rice, we used the vector pTCK303 to produce several lines of RNAi knockdown transgenic rice and investigated their flowering times and other agronomic traits. Among them, the heading date of FRRP1-RNAi knockdown transgenic rice was 23-26 days earlier than that of wild-type plants. FRRP1 is a novel rice gene that encodes a C3HC4-type Really Interesting Novel Gene (RING) finger domain protein. In addition to the early flowering time, FRRP1-RNAi knockdown transgenic rice caused changes on an array of agronomic traits, including plant height, panicle length and grain length. We analyzed the expression of some key genes associated with the flowering time and other agronomic traits in the FRRP1-RNAi knockdown lines and compared with that in wild-type lines. The expression of Hd3a increased significantly, which was the key factor in the early flowering time. Further experiments showed that the level of histone H2B monoubiquitination (H2Bub1) was noticeably reduced in the FRRP1-RNAi knockdown transgenic rice lines compared with wild-type plants and MBP-FRRP1-F1 was capable of self-ubiquitination. The results indicate that Flowering Related RING Protein 1 (FRRP1) is involved in histone H2B monoubiquitination and suggest that FRRP1 functions as an E3 ligase in vivo and in vitro. In conclusion, FRRP1 probably regulates flowering time and yield potential in rice by affecting histone H2B monoubiquitination, which leads to changes in gene expression in multiple processes.
Flowering-Related RING Protein 1 (FRRP1) Regulates Flowering Time and Yield Potential by Affecting Histone H2B Monoubiquitination in Rice (Oryza Sativa)

PubMed Central

Deng, Changwang; Chen, Xi; Gou, Lanming; Zhu, Fugui; Guo, Wei; Zhang, Jianfu; Wang, Tao

2016-01-01

Flowering time is a critical trait for crops cultivated under various temperature/photoperiod conditions around the world. To understand better the flowering time of rice, we used the vector pTCK303 to produce several lines of RNAi knockdown transgenic rice and investigated their flowering times and other agronomic traits. Among them, the heading date of FRRP1-RNAi knockdown transgenic rice was 23–26 days earlier than that of wild-type plants. FRRP1 is a novel rice gene that encodes a C3HC4-type Really Interesting Novel Gene (RING) finger domain protein. In addition to the early flowering time, FRRP1-RNAi knockdown transgenic rice caused changes on an array of agronomic traits, including plant height, panicle length and grain length. We analyzed the expression of some key genes associated with the flowering time and other agronomic traits in the FRRP1-RNAi knockdown lines and compared with that in wild-type lines. The expression of Hd3a increased significantly, which was the key factor in the early flowering time. Further experiments showed that the level of histone H2B monoubiquitination (H2Bub1) was noticeably reduced in the FRRP1-RNAi knockdown transgenic rice lines compared with wild-type plants and MBP-FRRP1-F1 was capable of self-ubiquitination. The results indicate that Flowering Related RING Protein 1 (FRRP1) is involved in histone H2B monoubiquitination and suggest that FRRP1 functions as an E3 ligase in vivo and in vitro. In conclusion, FRRP1 probably regulates flowering time and yield potential in rice by affecting histone H2B monoubiquitination, which leads to changes in gene expression in multiple processes. PMID:26934377
Effects of ZEB1 on regulating osteosarcoma cells via NF-κB/iNOS.

PubMed

Xu, X-M; Liu, W; Cao, Z-H; Liu, M-X

2017-03-01

Osteosarcoma is one common malignant bone tumors, as it frequently has invasion, metastasis and recurrence, causing unfavorable prognosis of patients. Osteosarcoma has complicated pathogenesis, which has not been elucidated fully. Therefore, the identification of effective molecular target of osteosarcoma onset can help to improve treatment efficacy and prognosis of osteosarcoma. Zinc finger E-box binding homeobox 1 (ZEB1) protein is one member of zinc finger E-box binding protein family, and participates in embryonic genesis and development. A recent study found the participation of ZEB1 in mediating multiple tumor onset and its up-regulation of osteosarcoma. The regulatory mechanism of ZEB1 in osteosarcoma has not been illustrated yet. In vitro cultured osteosarcoma MG-63 cells were transfected with ZEB1 siRNA. Real-time PCR and Western blot were tested for ZEB1 mRNA/protein expression. MTT was used to test MG-63 cell proliferation, whilst cell invasion was used to describe the effect of ZEB1 on MG-63 cells. Caspase-3 activity assay was employed to test MG-63 cell apoptosis. Western blot was employed to detect nuclear factor kappa B (NF-kB) and inducible nitric oxide synthase (iNOS) protein expression. After transfecting with ZEB1 siRNA, MG-63 cell proliferation or invasion was inhibited accompanied with lower ZEB1 mRNA/protein expression. Caspase3 activity was also increased after transfection (p < 0.05), along with down-regulation of NF-kB and iNOS proteins in MG-63 cells (p < 0.05). Inhibition of ZEB1 can facilitate osteosarcoma cell apoptosis and inhibit cell proliferation or invasion via down-regulating NF-kB/iNOS signal pathway.
Expression of the Bacillus anthracis protective antigen gene by baculovirus and vaccinia virus recombinants.

PubMed Central

Iacono-Connors, L C; Schmaljohn, C S; Dalrymple, J M

1990-01-01

The gene encoding Bacillus anthracis protective antigen (PA) was modified by site-directed mutagenesis, subcloned into baculovirus and vaccinia virus plasmid transfer vectors (pAcYM1 and pSC-11, respectively), and inserted via homologous recombinations into baculovirus Autographa californica nuclear polyhedrosis virus or vaccinia virus (strains WR and Connaught). Expression of PA was detected in both systems by immunofluorescence assays with antisera from rabbits immunized with B. anthracis PA. Western blot (immunoblot) analysis showed that the expressed product of both systems was slightly larger (86 kilodaltons) than B. anthracis-produced PA (83.5 kilodaltons). Analysis of trypsin digests of virus-expressed and authentic PA suggested that the size difference was due to the presence of a signal sequence remaining with the virus-expressed protein. Immunization of mice with either recombinant baculovirus-infected Spodoptera frugiperda cells or with vaccinia virus recombinants elicited a high-titer, anti-PA antibody response. Images PMID:2105271
B-2 Extremely High Frequency SATCOM and Computer Increment 1 (B-2 EHF Inc 1)

DTIC Science & Technology

2015-12-01

Confidence Level Confidence Level of cost estimate for current APB: 55% This APB reflects cost and funding data based on the B-2 EHF Increment I SCP...This cost estimate was quantified at the Mean (~55%) confidence level . Total Quantity Quantity SAR Baseline Production Estimate Current APB...Production Estimate Econ Qty Sch Eng Est Oth Spt Total 33.624 -0.350 1.381 0.375 0.000 -6.075 0.000 -0.620 -5.289 28.335 Current SAR Baseline to Current
Discovery and structure-activity relationship of oxalylarylaminobenzoic acids as inhibitors of protein tyrosine phosphatase 1B.

PubMed

Liu, Gang; Szczepankiewicz, Bruce G; Pei, Zhonghua; Janowick, David A; Xin, Zhili; Hajduk, Philip J; Abad-Zapatero, Cele; Liang, Heng; Hutchins, Charles W; Fesik, Stephen W; Ballaron, Steve J; Stashko, Mike A; Lubben, Tom; Mika, Amanda K; Zinker, Bradley A; Trevillyan, James M; Jirousek, Michael R

2003-05-22

Protein Tyrosine phosphatase 1B (PTP1B) has been implicated as a key negative regulator of both insulin and leptin signaling pathways. Using an NMR-based screening approach with 15N- and 13C-labeled PTP1B, we have identified 2,3-dimethylphenyloxalylaminobenzoic acid (1) as a general, reversible, and competitive PTPase inhibitor. Structure-based approach guided by X-ray crystallography facilitated the development of 1 into a novel series of potent and selective PTP1B inhibitors occupying both the catalytic site and a portion of the noncatalytic, second phosphotyrosine binding site. Interestingly, oral biovailability has been observed in rats for some compounds. Furthermore, we demonstrated in vivo plasma glucose lowering effects with compound 12d in ob/ob mice.
Identification of caffeoylquinic acid derivatives as natural protein tyrosine phosphatase 1B inhibitors from Artemisia princeps.

PubMed

Zhang, Jie; Sasaki, Tatsunori; Li, Wei; Nagata, Kazuya; Higai, Koji; Feng, Feng; Wang, Jian; Cheng, Maosheng; Koike, Kazuo

2018-04-15

Considerable attention has been paid to protein tyrosine phosphatase 1B (PTP1B) inhibitors as a potential therapy for diabetes, obesity, and cancer. Ten caffeoylquinic acid derivatives (1-10) from leaves of Artemisia princeps Pamp. (Asteraceae) were identified as natural PTP1B inhibitors. Among them, chlorogenic acid (3) showed the most potent inhibitory activity (IC 50 11.1 μM). Compound 3 was demonstrated to be a noncompetitive inhibitor by a kinetic analysis. Molecular docking simulation suggested that compound 3 bound to the allosteric site of PTP1B. Furthermore, compound 3 showed remarkable selectivity against four homologous PTPs. According to these findings, compound 3 might be potentially valuable for further drug development. Copyright © 2018 Elsevier Ltd. All rights reserved.

Annexin A1, Annexin A2, and Dyrk 1B are upregulated during GAS1-induced cell cycle arrest.

PubMed

Pérez-Sánchez, Gilberto; Jiménez, Adriana; Quezada-Ramírez, Marco A; Estudillo, Enrique; Ayala-Sarmiento, Alberto E; Mendoza-Hernández, Guillermo; Hernández-Soto, Justino; Hernández-Hernández, Fidel C; Cázares-Raga, Febe E; Segovia, Jose

2018-05-01

GAS1 is a pleiotropic protein that has been investigated because of its ability to induce cell proliferation, cell arrest, and apoptosis, depending on the cellular or the physiological context in which it is expressed. At this point, we have information about the molecular mechanisms by which GAS1 induces proliferation and apoptosis; but very few studies have been focused on elucidating the mechanisms by which GAS1 induces cell arrest. With the aim of expanding our knowledge on this subject, we first focused our research on finding proteins that were preferentially expressed in cells arrested by serum deprivation. By using a proteomics approach and mass spectrometry analysis, we identified 17 proteins in the 2-DE protein profile of serum deprived NIH3T3 cells. Among them, Annexin A1 (Anxa1), Annexin A2 (Anxa2), dual specificity tyrosine-phosphorylation-regulated kinase 1B (Dyrk1B), and Eukaryotic translation initiation factor 3, F (eIf3f) were upregulated at transcriptional the level in proliferative NIH3T3 cells. Moreover, we demonstrated that Anxa1, Anxa2, and Dyrk1b are upregulated at both the transcriptional and translational levels by the overexpression of GAS1. Thus, our results suggest that the upregulation of Anxa1, Anxa2, and Dyrk1b could be related to the ability of GAS1 to induce cell arrest and maintain cell viability. Finally, we provided further evidence showing that GAS1 through Dyrk 1B leads not only to the arrest of NIH3T3 cells but also maintains cell viability. © 2017 Wiley Periodicals, Inc.
Induction of mesenchymal cell phenotypes in lung epithelial cells by adenovirus E1A.

PubMed

Behzad, A R; Morimoto, K; Gosselink, J; Green, J; Hogg, J C; Hayashi, S

2006-12-01

Epithelial-mesenchymal transformation is now recognised as an important feature of tissue remodelling. The present report concerns the role of adenovirus infection in inducing this transformation in an animal model of chronic obstructive pulmonary disease. Guinea pig primary peripheral lung epithelial cells (PLECs) transfected with adenovirus E1A (E1A-PLECs) were compared to guinea pig normal lung fibroblasts (NLFs) transfected with E1A (E1A-NLFs). These cells were characterised by PCR, immunocytochemistry, electron microscopy, and Western and Northern blot analyses. Electrophoretic mobility shift assays were performed in order to examine nuclear factor (NF)-kappaB and activator protein (AP)-1 binding activities. E1A-PLECs and E1A-NLFs positive for E1A DNA, mRNA and protein expressed cytokeratin and vimentin but not smooth muscle alpha-actin. Both exhibited cuboidal morphology and junctional complexes, but did not contain lamellar bodies or express surfactant protein A, B or C mRNAs. These two cell types differed, however, in their NF-kappaB and AP-1 binding after lipopolysaccharide stimulation, possibly due to differences in the expression of the subunits that comprise these transcriptional complexes. E1A transfection results in the transformation of peripheral lung epithelial cells and normal lung fibroblasts to a phenotype intermediate between that of the two primary cells. It is postulated that this intermediate phenotype may play a major role in the remodelling of the airways in chronic obstructive pulmonary disease associated with persistence of adenovirus E1A DNA.
Analysis of in vitro interactions of protein tyrosine phosphatase 1B with insulin receptors.

PubMed

Wang, X Y; Bergdahl, K; Heijbel, A; Liljebris, C; Bleasdale, J E

2001-02-28

One strategy to treat the insulin resistance that is central to type II diabetes mellitus may be to maintain insulin receptors (IR) in the active (tyrosine phosphorylated) form. Because protein tyrosine phosphatase 1B (PTP1B) binds and subsequently dephosphorylates IR, inhibitors of PTP1B-IR binding are potential insulin 'sensitizers.' A Scintillation Proximity Assay (SPA) was developed to characterize and quantitate PTP1B-IR binding. Human IR were solubilized and captured on wheat germ agglutinin (WGA)-coated SPA beads. Subsequent binding of human, catalytically inactive [35S] PTP1B Cys(215)/Ser (PTP1B(C215S)) to the lectin-anchored IR results in scintillation from the SPA beads that can be quantitated. Binding of PTP1B to IR was pH- and divalent cation-sensitive. Ca(2+) and Mn(2+), but not Mg(2+), dramatically attenuated the loss of PTP1B-IR binding observed when pH was raised from 6.2 to 7.8. PTP1B binding to IR from insulin-stimulated cells was much greater than to IR from unstimulated cells and was inhibited by either an antiphosphotyrosine antibody or treatment of IR with alkaline phosphatase, suggesting that tyrosine phosphorylation of IR is required for PTP1B binding. Phosphopeptides modeled after various IR phosphotyrosine domains each only partially inhibited PTP1B-IR binding, indicating that multiple domains of IR are likely involved in binding PTP1B. However, competitive displacement of [35S]PTP1B(C215S) by PTP1B(C215S) fitted best to a single binding site with a K(d) in the range 100-1000 nM, depending upon pH and divalent cations. PNU-200898, a potent and selective inhibitor of PTP1B whose orientation in the active site of PTP1B has been solved, competitively inhibited catalysis and PTP1B-IR binding with equal potency. The results of this novel assay for PTP1B-IR binding suggest that PTP1B binds preferentially to tyrosine phosphorylated IR through its active site and that binding may be susceptible to therapeutic disruption by small molecules.
Sulfone-stabilized carbanions for the reversible covalent capture of a posttranslationally-generated cysteine oxoform found in protein tyrosine phosphatase 1B (PTP1B).

PubMed

Parsons, Zachary D; Ruddraraju, Kasi Viswanatharaju; Santo, Nicholas; Gates, Kent S

2016-06-15

Redox regulation of protein tyrosine phosphatase 1B (PTP1B) involves oxidative conversion of the active site cysteine thiolate into an electrophilic sulfenyl amide residue. Reduction of the sulfenyl amide by biological thiols regenerates the native cysteine residue. Here we explored fundamental chemical reactions that may enable covalent capture of the sulfenyl amide residue in oxidized PTP1B. Various sulfone-containing carbon acids were found to react readily with a model peptide sulfenyl amide via attack of the sulfonyl carbanion on the electrophilic sulfur center in the sulfenyl amide. Both the products and the rates of these reactions were characterized. The results suggest that capture of a peptide sulfenyl amide residue by sulfone-stabilized carbanions can slow, but not completely prevent, thiol-mediated generation of the corresponding cysteine-containing peptide. Sulfone-containing carbon acids may be useful components in the construction of agents that knock down PTP1B activity in cells via transient covalent capture of the sulfenyl amide oxoform generated during insulin signaling processes. Copyright © 2016 Elsevier Ltd. All rights reserved.
Protein tyrosine phosphatase 1B inhibitory activity of alkaloids from Rhizoma Coptidis and their molecular docking studies.

PubMed

Choi, Jae Sue; Ali, Md Yousof; Jung, Hyun Ah; Oh, Sang Ho; Choi, Ran Joo; Kim, Eon Ji

2015-08-02

Rhizoma Coptidis (the rhizome of Coptis chinensis Franch) has commonly been used for treatment of diabetes mellitus in traditional Chinese medicine due to its blood sugar-lowering properties and therapeutic benefits which highly related to the alkaloids therein. However, a limited number of studies focused on the Coptis alkaloids other than berberine. In the present study, we investigated the anti-diabetic potential of Coptis alkaloids, including berberine (1), epiberberine (2), magnoflorine (3), and coptisine (4), by evaluating the ability of these compounds to inhibit protein tyrosine phosphatase 1B (PTP1B), and ONOO(-)-mediated protein tyrosine nitration. We scrutinized the potentials of Coptis alkaloids as PTP1B inhibitors via enzyme kinetics and molecular docking simulation. The Coptis alkaloids 1-4 exhibited remarkable inhibitory activities against PTP1B with the IC50 values of 16.43, 24.19, 28.14, and 51.04 μM, respectively, when compared to the positive control ursolic acid. These alkaloids also suppressed ONOO(-)-mediated tyrosine nitration effectively in a dose dependent manner. In addition, our kinetic study using the Lineweaver-Burk and Dixon plots revealed that 1 and 2 showed a mixed-type inhibition against PTP1B, while 3 and 4 noncompetitively inhibited PTP1B. Moreover, molecular docking simulation of these compounds demonstrated negative binding energies (Autodock 4.0=-6.7 to -7.8 kcal/mol; Fred 2.0=-59.4 to -68.2 kcal/mol) and a high proximity to PTP1B residues, including Phe182 and Asp181 in the WPD loop, Cys215 in the active sites and Tyr46, Arg47, Asp48, Val49, Ser216, Ala217, Gly218, Ile219, Gly220, Arg221 and Gln262 in the pocket site, indicating a higher affinity and tighter binding capacity of these alkaloids for the active site of the enzyme. Our results clearly indicate the promising anti-diabetic potential of Coptis alkaloids as inhibitors on PTP1B as well as suppressors of ONOO(-)-mediated protein tyrosine nitration, and thus hold
The Homeodomain of PDX-1 Mediates Multiple Protein-Protein Interactions in the Formation of a Transcriptional Activation Complex on the Insulin Promoter

PubMed Central

Ohneda, Kinuko; Mirmira, Raghavendra G.; Wang, Juehu; Johnson, Jeffrey D.; German, Michael S.

2000-01-01

Activation of insulin gene transcription specifically in the pancreatic β cells depends on multiple nuclear proteins that interact with each other and with sequences on the insulin gene promoter to build a transcriptional activation complex. The homeodomain protein PDX-1 exemplifies such interactions by binding to the A3/4 region of the rat insulin I promoter and activating insulin gene transcription by cooperating with the basic-helix-loop-helix (bHLH) protein E47/Pan1, which binds to the adjacent E2 site. The present study provides evidence that the homeodomain of PDX-1 acts as a protein-protein interaction domain to recruit multiple proteins, including E47/Pan1, BETA2/NeuroD1, and high-mobility group protein I(Y), to an activation complex on the E2A3/4 minienhancer. The transcriptional activity of this complex results from the clustering of multiple activation domains capable of interacting with coactivators and the basal transcriptional machinery. These interactions are not common to all homeodomain proteins: the LIM homeodomain protein Lmx1.1 can also activate the E2A3/4 minienhancer in cooperation with E47/Pan1 but does so through different interactions. Cooperation between Lmx1.1 and E47/Pan1 results not only in the aggregation of multiple activation domains but also in the unmasking of a potent activation domain on E47/Pan1 that is normally silent in non-β cells. While more than one activation complex may be capable of activating insulin gene transcription through the E2A3/4 minienhancer, each is dependent on multiple specific interactions among a unique set of nuclear proteins. PMID:10629047
Interaction of the PA2G4 (EBP1) protein with ErbB-3 and regulation of this binding by heregulin

PubMed Central

Yoo, J-Y; Wang, X W; Rishi, A K; Lessor, T; Xia, X-M; Gustafson, T A; Hamburger, A W

2000-01-01

The processes by which ErbB-3, an inactive tyrosine kinase, exerts its biological effects are poorly understood. Using the yeast two-hybrid system, we have isolated an ErbB-3 binding protein (Ebp1) that interacts with the juxtamembrane domain of ErbB-3. This protein is identical to that predicted to be encoded for by the human PA2G4 gene. Ebp1 is the human homologue of a previously identified cell cycle-regulated mouse protein p38-2G4. Two transcripts of ebp1 mRNA (1.7 and 2.2 kb) were detected in several normal human organs. The interaction of Ebp1 with ErbB-3 was examined in vitro and in vivo. The first 15 amino acids of the juxtamembrane domain of ErbB-3 were essential for Ebp1 binding in vitro. Treatment of AU565 cells with the ErbB-3 ligand heregulin resulted in dissociation of Ebp1 from ErbB-3. Ebp1 translocated from the cytoplasm into the nucleus following heregulin stimulation. These findings suggest that Ebp1 may be a downstream member of an ErbB-3-regulated signal transduction pathway. © 2000 Cancer Research Campaign PMID:10682683
Alcohol binding in the C1 (C1A + C1B) domain of protein kinase C epsilon

PubMed Central

Pany, Satyabrata; Das, Joydip

2015-01-01

Background Alcohol regulates the expression and function of protein kinase C epsilon (PKCε). In a previous study we identified an alcohol binding site in the C1B, one of the twin C1 subdomains of PKCε. Methods In this study, we investigated alcohol binding in the entire C1 domain (combined C1A and C1B) of PKCε. Fluorescent phorbol ester, SAPD and fluorescent diacylglycerol (DAG) analog, dansyl-DAG were used to study the effect of ethanol, butanol, and octanol on the ligand binding using fluorescence resonance energy transfer (FRET). To identify alcohol binding site(s), PKCεC1 was photolabeled with 3-azibutanol and 3-azioctanol, and analyzed by mass spectrometry. The effects of alcohols and the azialcohols on PKCε were studied in NG108-15 cells. Results In the presence of alcohol, SAPD and dansyl-DAG showed different extent of FRET, indicating differential effects of alcohol on the C1A and C1B subdomains. Effects of alcohols and azialcohols on PKCε in NG108-15 cells were comparable. Azialcohols labeled Tyr-176 of C1A and Tyr-250 of C1B. Inspection of the model structure of PKCεC1 reveals that these residues are 40 Å apart from each other indicating that these residues form two different alcohol binding sites. Conclusions The present results provide evidence for the presence of multiple alcohol-binding sites on PKCε and underscore the importance of targeting this PKC isoform in developing alcohol antagonists. PMID:26210390
HTLV-1 Tax Stimulates Ubiquitin E3 Ligase, Ring Finger Protein 8, to Assemble Lysine 63-Linked Polyubiquitin Chains for TAK1 and IKK Activation.

PubMed

Ho, Yik-Khuan; Zhi, Huijun; Bowlin, Tara; Dorjbal, Batsukh; Philip, Subha; Zahoor, Muhammad Atif; Shih, Hsiu-Ming; Semmes, Oliver John; Schaefer, Brian; Glover, J N Mark; Giam, Chou-Zen

2015-08-01

Human T lymphotropic virus type 1 (HTLV-1) trans-activator/oncoprotein, Tax, impacts a multitude of cellular processes, including I-κB kinase (IKK)/NF-κB signaling, DNA damage repair, and mitosis. These activities of Tax have been implicated in the development of adult T-cell leukemia (ATL) in HTLV-1-infected individuals, but the underlying mechanisms remain obscure. IKK and its upstream kinase, TGFβ-activated kinase 1 (TAK1), contain ubiquitin-binding subunits, NEMO and TAB2/3 respectively, which interact with K63-linked polyubiquitin (K63-pUb) chains. Recruitment to K63-pUb allows cross auto-phosphorylation and activation of TAK1 to occur, followed by TAK1-catalyzed IKK phosphorylation and activation. Using cytosolic extracts of HeLa and Jurkat T cells supplemented with purified proteins we have identified ubiquitin E3 ligase, ring finger protein 8 (RNF8), and E2 conjugating enzymes, Ubc13:Uev1A and Ubc13:Uev2, to be the cellular factors utilized by Tax for TAK1 and IKK activation. In vitro, the combination of Tax and RNF8 greatly stimulated TAK1, IKK, IκBα and JNK phosphorylation. In vivo, RNF8 over-expression augmented while RNF8 ablation drastically reduced canonical NF-κB activation by Tax. Activation of the non-canonical NF-κB pathway by Tax, however, is unaffected by the loss of RNF8. Using purified components, we further demonstrated biochemically that Tax greatly stimulated RNF8 and Ubc13:Uev1A/Uev2 to assemble long K63-pUb chains. Finally, co-transfection of Tax with increasing amounts of RNF8 greatly induced K63-pUb assembly in a dose-dependent manner. Thus, Tax targets RNF8 and Ubc13:Uev1A/Uev2 to promote the assembly of K63-pUb chains, which signal the activation of TAK1 and multiple downstream kinases including IKK and JNK. Because of the roles RNF8 and K63-pUb chains play in DNA damage repair and cytokinesis, this mechanism may also explain the genomic instability of HTLV-1-transformed T cells and ATL cells.
HTLV-1 Tax Stimulates Ubiquitin E3 Ligase, Ring Finger Protein 8, to Assemble Lysine 63-Linked Polyubiquitin Chains for TAK1 and IKK Activation

PubMed Central

Ho, Yik-Khuan; Zhi, Huijun; Bowlin, Tara; Dorjbal, Batsukh; Philip, Subha; Zahoor, Muhammad Atif; Shih, Hsiu-Ming; Semmes, Oliver John; Schaefer, Brian; Glover, J. N. Mark; Giam, Chou-Zen

2015-01-01

Human T lymphotropic virus type 1 (HTLV-1) trans-activator/oncoprotein, Tax, impacts a multitude of cellular processes, including I-κB kinase (IKK)/NF-κB signaling, DNA damage repair, and mitosis. These activities of Tax have been implicated in the development of adult T-cell leukemia (ATL) in HTLV-1-infected individuals, but the underlying mechanisms remain obscure. IKK and its upstream kinase, TGFβ-activated kinase 1 (TAK1), contain ubiquitin-binding subunits, NEMO and TAB2/3 respectively, which interact with K63-linked polyubiquitin (K63-pUb) chains. Recruitment to K63-pUb allows cross auto-phosphorylation and activation of TAK1 to occur, followed by TAK1-catalyzed IKK phosphorylation and activation. Using cytosolic extracts of HeLa and Jurkat T cells supplemented with purified proteins we have identified ubiquitin E3 ligase, ring finger protein 8 (RNF8), and E2 conjugating enzymes, Ubc13:Uev1A and Ubc13:Uev2, to be the cellular factors utilized by Tax for TAK1 and IKK activation. In vitro, the combination of Tax and RNF8 greatly stimulated TAK1, IKK, IκBα and JNK phosphorylation. In vivo, RNF8 over-expression augmented while RNF8 ablation drastically reduced canonical NF-κB activation by Tax. Activation of the non-canonical NF-κB pathway by Tax, however, is unaffected by the loss of RNF8. Using purified components, we further demonstrated biochemically that Tax greatly stimulated RNF8 and Ubc13:Uev1A/Uev2 to assemble long K63-pUb chains. Finally, co-transfection of Tax with increasing amounts of RNF8 greatly induced K63-pUb assembly in a dose-dependent manner. Thus, Tax targets RNF8 and Ubc13:Uev1A/Uev2 to promote the assembly of K63-pUb chains, which signal the activation of TAK1 and multiple downstream kinases including IKK and JNK. Because of the roles RNF8 and K63-pUb chains play in DNA damage repair and cytokinesis, this mechanism may also explain the genomic instability of HTLV-1-transformed T cells and ATL cells. PMID:26285145
Kinetics of B cell responses to Plasmodium falciparum erythrocyte membrane protein 1 in Ghanaian women naturally exposed to malaria parasites.

PubMed

Ampomah, Paulina; Stevenson, Liz; Ofori, Michael F; Barfod, Lea; Hviid, Lars

2014-06-01

Naturally acquired protective immunity to Plasmodium falciparum malaria takes years to develop. It relies mainly on Abs, particularly IgG specific for Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) proteins on the infected erythrocyte surface. It is only partially understood why acquisition of clinical protection takes years to develop, but it probably involves a range of immune-evasive parasite features, not least of which are PfEMP1 polymorphism and clonal variation. Parasite-induced subversion of immunological memory and expansion of "atypical" memory B cells may also contribute. In this first, to our knowledge, longitudinal study of its kind, we measured B cell subset composition, as well as PfEMP1-specific Ab levels and memory B cell frequencies, in Ghanaian women followed from early pregnancy up to 1 y after delivery. Cell phenotypes and Ag-specific B cell function were assessed three times during and after pregnancy. Levels of IgG specific for pregnancy-restricted, VAR2CSA-type PfEMP1 increased markedly during pregnancy and declined after delivery, whereas IgG levels specific for two PfEMP1 proteins not restricted to pregnancy did not. Changes in VAR2CSA-specific memory B cell frequencies showed typical primary memory induction among primigravidae and recall expansion among multigravidae, followed by contraction postpartum in all. No systematic changes in the frequencies of memory B cells specific for the two other PfEMP1 proteins were identified. The B cell subset analysis confirmed earlier reports of high atypical memory B cell frequencies among residents of P. falciparum-endemic areas, and indicated an additional effect of pregnancy. Our study provides new knowledge regarding immunity to P. falciparum malaria and underpins efforts to develop PfEMP1-based vaccines against this disease. Copyright © 2014 by The American Association of Immunologists, Inc.
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
Antibody response to Streptococcus pneumoniae proteins PhtD, LytB, PcpA, PhtE and Ply after nasopharyngeal colonization and acute otitis media in children.

PubMed

Pichichero, Michael E; Kaur, Ravinder; Casey, Janet R; Xu, Qingfu; Almudevar, Anthony; Ochs, Martina

2012-06-01

We prospectively compared serum antibody levels of 5 Streptococcus pneumoniae (Spn) proteins: PcpA PhtD, PhtE Ply and LytB associated with nasopharyngeal (NP) colonization and acute otitis media (AOM) infection in a cohort of 6-30 mo old children. Antigen-specific antibody titers were determined by ELISA. A total of 731 visits among 168 children were studied. There were 301 Spn NP colonization episodes documented in 109 (65%) children and 42 Spn AOM episodes in 34 (20%) children. IgG antibody titers to the 5 proteins were significantly different among children over time (p < 0.001), with a rank order as follows: PcpA > PhtE = PhtD > Ply > LytB Characterization of IgG and IgM acute and convalescent serum antibody levels of Spn AOM infection showed the kinetics of the response differed among children, with the same rank order of antibody levels over time. Individual data showed that some children responded to AOM with an antibody increase to one or more of these Spn proteins but some children failed to respond. We conclude that antibody levels to Spn proteins PcpA PhtD, PhtE, Ply and LytB, all rise over time in children age 6 to 30 mo following natural exposure to Spn after NP colonization and AOM; however, there were significant differences in quantity of antibody elicited among these potential vaccine antigens.
Functional and evolutionary insight from the crystal structure of rubella virus protein E1.

PubMed

DuBois, Rebecca M; Vaney, Marie-Christine; Tortorici, M Alejandra; Kurdi, Rana Al; Barba-Spaeth, Giovanna; Krey, Thomas; Rey, Félix A

2013-01-24

Little is known about the three-dimensional organization of rubella virus, which causes a relatively mild measles-like disease in children but leads to serious congenital health problems when contracted in utero. Although rubella virus belongs to the same family as the mosquito-borne alphaviruses, in many respects it is more similar to other aerosol-transmitted human viruses such as the agents of measles and mumps. Although the use of the triple MMR (measles, mumps and rubella) live vaccine has limited its incidence in western countries, congenital rubella syndrome remains an important health problem in the developing world. Here we report the 1.8 Å resolution crystal structure of envelope glycoprotein E1, the main antigen and sole target of neutralizing antibodies against rubella virus. E1 is the main player during entry into target cells owing to its receptor-binding and membrane-fusion functions. The structure reveals the epitope and the neutralization mechanism of an important category of protecting antibodies against rubella infection. It also shows that rubella virus E1 is a class II fusion protein, which had hitherto only been structurally characterized for the arthropod-borne alphaviruses and flaviviruses. In addition, rubella virus E1 has an extensive membrane-fusion surface that includes a metal site, reminiscent of the T-cell immunoglobulin and mucin family of cellular proteins that bind phosphatidylserine lipids at the plasma membrane of cells undergoing apoptosis. Such features have not been seen in any fusion protein crystallized so far. Structural comparisons show that the class II fusion proteins from alphaviruses and flaviviruses, despite belonging to different virus families, are closer to each other than they are to rubella virus E1. This suggests that the constraints on arboviruses imposed by alternating cycles between vertebrates and arthropods resulted in more conservative evolution. By contrast, in the absence of this constraint, the
Staphylococcus aureus FepA and FepB proteins drive heme iron utilization in Escherichia coli.

PubMed

Turlin, Evelyne; Débarbouillé, Michel; Augustyniak, Katarzyna; Gilles, Anne-Marie; Wandersman, Cécile

2013-01-01

EfeUOB-like tripartite systems are widespread in bacteria and in many cases they are encoded by genes organized into iron-regulated operons. They consist of: EfeU, a protein similar to the yeast iron permease Ftrp1; EfeO, an extracytoplasmic protein of unknown function and EfeB, also an extracytoplasmic protein with heme peroxidase activity, belonging to the DyP family. Many bacterial EfeUOB systems have been implicated in iron uptake, but a prefential iron source remains undetermined. Nevertheless, in the case of Escherichia coli, the EfeUOB system has been shown to recognize heme and to allow extracytoplasmic heme iron extraction via a deferrochelation reaction. Given the high level of sequence conservations between EfeUOB orthologs, we hypothesized that heme might be the physiological iron substrate for the other orthologous systems. To test this hypothesis, we undertook characterization of the Staphylococcus aureus FepABC system. Results presented here indicate: i) that the S. aureus FepB protein binds both heme and PPIX with high affinity, like EfeB, the E. coli ortholog; ii) that it has low peroxidase activity, comparable to that of EfeB; iii) that both FepA and FepB drive heme iron utilization, and both are required for this activity and iv) that the E. coli FepA ortholog (EfeO) cannot replace FepA in FepB-driven iron release from heme indicating protein specificity in these activities. Our results show that the function in heme iron extraction is conserved in the two orthologous systems.
Inhibition of protein tyrosine phosphatase 1B by flavonoids: A structure - activity relationship study.

PubMed

Proença, Carina; Freitas, Marisa; Ribeiro, Daniela; Sousa, Joana L C; Carvalho, Félix; Silva, Artur M S; Fernandes, Pedro A; Fernandes, Eduarda

2018-01-01

The classical non-transmembrane protein tyrosine phosphatase 1B (PTP1B) has emerged as a key negative regulator of insulin signaling pathways that leads to insulin resistance, turning this enzyme a promising therapeutic target in the management of type 2 diabetes mellitus (T2DM). In the present work, the in vitro inhibitory activity of a panel of structurally related flavonoids, for recombinant human PTP1B was studied and the type of inhibition of the most active compounds further evaluated. The majority of the studied flavonoids was tested in this work for the first time, including flavonoid C13, which was the most potent inhibitor. It was observed that the ability to inhibit PTP1B depends on the nature, position and number of substituents in the flavonoid structure, as the presence of both 7- and 8-OBn groups in the A ring, together with the presence of both 3' and 4'-OMe groups in the B ring and the 3-OH group in the C ring; these substituents increase the flavonoids' ability to inhibit PTP1B. In conclusion, some of the tested flavonoids seem to be promising PTP1B inhibitors and potential effective agents in the management of T2DM, by increasing insulin sensitivity. Copyright © 2017 Elsevier Ltd. All rights reserved.
Purification and characterization of a 14-kilodalton protein that is bound to the surface of polyhydroxyalkanoic acid granules in Rhodococcus ruber.

PubMed Central

Pieper-Fürst, U.; Madkour, M. H.; Mayer, F.; Steinbüchel, A.

1994-01-01

The N-terminal amino acid sequence of the polyhydroxyalkanoic acid (PHA) granule-associated M(r)-15,500 protein of Rhodococcus ruber (the GA14 protein) was analyzed. The sequence revealed that the corresponding structural gene is represented by open reading frame 3, encoding a protein with a calculated M(r) of 14,175 which was recently localized downstream of the PHA synthase gene (U. Pieper and A. Steinbüchel, FEMS Microbiol. Lett. 96:73-80, 1992). A recombinant strain of Escherichia coli XL1-Blue carrying the hybrid plasmid (pSKXA10*) with open reading frame 3 overexpressed the GA14 protein. The GA14 protein was subsequently purified in a three-step procedure including chromatography on DEAE-Sephacel, phenyl-Sepharose CL-4B, and Superose 12. Determination of the molecular weight by gel filtration as well as electron microscopic studies indicates that a tetrameric structure of the recombinant, native GA14 protein is most likely. Immunoelectron microscopy demonstrated a localization of the GA14 protein at the periphery of PHA granules as well as close to the cell membrane in R. ruber. Investigations of PHA-leaky and PHA-negative mutants of R. ruber indicated that expression of the GA14 protein depended strongly on PHA synthesis. Images PMID:8021220
Protective effects of murine monoclonal antibodies in experimental septicemia: E. coli antibodies protect against different serotypes of E. coli.

PubMed

Salles, M F; Mandine, E; Zalisz, R; Guenounou, M; Smets, P

1989-04-01

Murine monoclonal antibodies that bind outer membrane antigens of the J5 mutant of Escherichia coli O111:B4 were derived from spleen cells of BALB/c mice immunized with killed whole cells and boosted with lipopolysaccharide (LPS) and LPS-associated proteins. Seven hybridomas were selected for their reactivity against the J5 LPS; they cross-reacted with O111, O55, O127, and O128 E. coli LPS. One (B7B3) also reacted with the Serratia marcescens LPS and Klebsiella pneumoniae lipid A. A protective effect was obtained with D6B4 antibody in a lethal endotoxemia model induced by LPS from O111, O127, and O128 E. coli serotypes in D-galactosamine-sensitized mice. D6B4 and D6B3 antibodies protected mice infected with E. coli O111:B4, when administered before infection. The D6B4 antibody was also protective when administered after infection. The antibodies D6B3 and D4B5 were protective in heterologous infection induced by E. coli O2:K1.
La-related protein 1 (LARP1) binds the mRNA cap, blocking eIF4F assembly on TOP mRNAs.

PubMed

Lahr, Roni M; Fonseca, Bruno D; Ciotti, Gabrielle E; Al-Ashtal, Hiba A; Jia, Jian-Jun; Niklaus, Marius R; Blagden, Sarah P; Alain, Tommy; Berman, Andrea J

2017-04-07

The 5'terminal oligopyrimidine (5'TOP) motif is a cis -regulatory RNA element located immediately downstream of the 7-methylguanosine [m 7 G] cap of TOP mRNAs, which encode ribosomal proteins and translation factors. In eukaryotes, this motif coordinates the synchronous and stoichiometric expression of the protein components of the translation machinery. La-related protein 1 (LARP1) binds TOP mRNAs, regulating their stability and translation. We present crystal structures of the human LARP1 DM15 region in complex with a 5'TOP motif, a cap analog (m 7 GTP), and a capped cytidine (m 7 GpppC), resolved to 2.6, 1.8 and 1.7 Å, respectively. Our binding, competition, and immunoprecipitation data corroborate and elaborate on the mechanism of 5'TOP motif binding by LARP1. We show that LARP1 directly binds the cap and adjacent 5'TOP motif of TOP mRNAs, effectively impeding access of eIF4E to the cap and preventing eIF4F assembly. Thus, LARP1 is a specialized TOP mRNA cap-binding protein that controls ribosome biogenesis.

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