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Sample records for e6 proteins modulate

  1. Papillomavirus E6 proteins

    PubMed Central

    Howie, Heather L; Katzenellenbogen, Rachel A; Galloway, Denise A

    2009-01-01

    The papillomaviruses are small DNA viruses that encode approximately eight genes, and require the host cell DNA replication machinery for their viral DNA replication. Thus papillomaviruses have evolved strategies to induce host cell DNA synthesis balanced with strategies to protect the cell from unscheduled replication. While the papillomavirus E1 and E2 genes are directly involved in viral replication by binding to and unwinding the origin of replication, the E6 and E7 proteins have auxillary functions that promote proliferation. As a consequence of disrupting the normal checkpoints that regulate cell cycle entry and progression, the E6 and E7 proteins play a key role in the oncogenic properties of human papillomaviruses with a high risk of causing anogenital cancers (HR HPVs). As a consequence, E6 and E7 of HR HPVs are invariably expressed in cervical cancers. This article will focus on the E6 protein and its numerous activities including inactivating p53, blocking apoptosis, activating telomerase, disrupting cell adhesion, polarity and epithelial differentiation, altering transcription and reducing immune recognition. PMID:19081593

  2. Papillomavirus E6 proteins

    SciTech Connect

    Howie, Heather L.; Katzenellenbogen, Rachel A.; Galloway, Denise A.

    2009-02-20

    The papillomaviruses are small DNA viruses that encode approximately eight genes, and require the host cell DNA replication machinery for their viral DNA replication. Thus papillomaviruses have evolved strategies to induce host cell DNA synthesis balanced with strategies to protect the cell from unscheduled replication. While the papillomavirus E1 and E2 genes are directly involved in viral replication by binding to and unwinding the origin of replication, the E6 and E7 proteins have auxillary functions that promote proliferation. As a consequence of disrupting the normal checkpoints that regulate cell cycle entry and progression, the E6 and E7 proteins play a key role in the oncogenic properties of human papillomaviruses with a high risk of causing anogenital cancers (HR HPVs). As a consequence, E6 and E7 of HR HPVs are invariably expressed in cervical cancers. This article will focus on the E6 protein and its numerous activities including inactivating p53, blocking apoptosis, activating telomerase, disrupting cell adhesion, polarity and epithelial differentiation, altering transcription and reducing immune recognition.

  3. E6 variants of human papillomavirus 18 differentially modulate the protein kinase B/phosphatidylinositol 3-kinase (akt/PI3K) signaling pathway

    SciTech Connect

    Contreras-Paredes, Adriana

    2009-01-05

    Intra-type genome variations of high risk Human papillomavirus (HPV) have been associated with a differential threat for cervical cancer development. In this work, the effect of HPV18 E6 isolates in Akt/PKB and Mitogen-associated protein kinase (MAPKs) signaling pathways and its implication in cell proliferation were analyzed. E6 from HPV types 16 and 18 are able to bind and promote degradation of Human disc large (hDlg). Our results show that E6 variants differentially modulate hDlg degradation, rebounding in levels of activated PTEN and PKB. HPV18 E6 variants are also able to upregulate phospho-PI3K protein, strongly correlating with activated MAPKs and cell proliferation. Data was supported by the effect of E6 silencing in HPV18-containing HeLa cells, as well as hDlg silencing in the tested cells. Results suggest that HPV18 intra-type variations may derive in differential abilities to activate cell-signaling pathways such as Akt/PKB and MAPKs, directly involved in cell survival and proliferation.

  4. The human papillomavirus type 11 and 16 E6 proteins modulate the cell-cycle regulator and transcription cofactor TRIP-Br1.

    PubMed

    Gupta, Sanjay; Takhar, Param Parkash S; Degenkolbe, Roland; Koh, Choon Heng; Zimmermann, Holger; Yang, Christopher Maolin; Guan Sim, Khe; Hsu, Stephen I-Hong; Bernard, Hans-Ulrich

    2003-12-05

    The genital human papillomaviruses (HPVs) are a taxonomic group including HPV types that preferentially cause genital and laryngeal warts ("low-risk types"), such as HPV-6 and HPV-11, or cancer of the cervix and its precursor lesions ("high-risk types"), such as HPV-16. The transforming processes induced by these viruses depend on the proteins E5, E6, and E7. Among these oncoproteins, the E6 protein stands out because it supports a particularly large number of functions and interactions with cellular proteins, some of which are specific for the carcinogenic HPVs, while others are shared among low- and high-risk HPVs. Here we report yeast two-hybrid screens with HPV-6 and -11 E6 proteins that identified TRIP-Br1 as a novel cellular target. TRIP-Br1 was recently detected by two research groups, which described two separate functions, namely that of a transcriptional integrator of the E2F1/DP1/RB cell-cycle regulatory pathway (and then named TRIP-Br1), and that of an antagonist of the cyclin-dependent kinase suppression of p16INK4a (and then named p34SEI-1). We observed that TRIP-Br1 interacts with low- and high-risk HPV E6 proteins in yeast, in vitro and in mammalian cell cultures. Transcription activation of a complex consisting of E2F1, DP1, and TRIP-Br1 was efficiently stimulated by both E6 proteins. TRIP-Br1 has an LLG E6 interaction motif, which contributed to the binding of E6 proteins. Apparently, E6 does not promote degradation of TRIP-Br1. Our observations imply that the cell-cycle promoting transcription factor E2F1/DP1 is dually targeted by HPV oncoproteins, namely (i) by interference of the E7 protein with repression by RB, and (ii) by the transcriptional cofactor function of the E6 protein. Our data reveal the natural context of the transcription activator function of E6, which has been predicted without knowledge of the E2F1/DP1/TRIP-Br/E6 complex by studying chimeric constructs, and add a function to the limited number of transforming properties shared

  5. The active form of E6-associated protein (E6AP)/UBE3A ubiquitin ligase is an oligomer.

    PubMed

    Ronchi, Virginia P; Klein, Jennifer M; Edwards, Daniel J; Haas, Arthur L

    2014-01-10

    Employing 125I-polyubiquitin chain formation as a functional readout of ligase activity, biochemical and biophysical evidence demonstrates that catalytically active E6-associated protein (E6AP)/UBE3A is an oligomer. Based on an extant structure previously discounted as an artifact of crystal packing forces, we propose that the fully active form of E6AP is a trimer, analysis of which reveals a buried surface of 7508Å2 and radially symmetric interacting residues that are conserved within the Hect (homologous to E6AP C terminus) ligase superfamily. An absolutely conserved interaction between Phe(727) and a hydrophobic pocket present on the adjacent subunit is critical for trimer stabilization because mutation disrupts the oligomer and decreases kcat 62-fold but fails to affect E2 ubiquitin binding or subsequent formation of the Hect domain Cys(820) ubiquitin thioester catalytic intermediate. Exogenous N-acetylphenylalanylamide reversibly antagonizes Phe(727)-dependent trimer formation and catalytic activity (Ki12 mM), as does a conserved-helical peptide corresponding to residues 474–490 of E6A Pisoform 1 (Ki22M) reported to bind the hydrophobic pocket of other Hect ligases, presumably blocking Phe(727) intercalation and trimer formation. Conversely, oncogenic human papillomavirus-16/18 E6 protein significantly enhances E6AP catalytic activity by promoting trimer formation (Kactivation 1.5 nM) through the ability of E6 to form homodimers. Recombinant E6 protein additionally rescues the kcat defect of the Phe(727) mutation and that of a specific loss-of-function Angelman syndrome mutation that promotes trimer destabilization. The present findings codify otherwise disparate observations regarding the mechanism of E6AP and related Hect ligases in addition to suggesting therapeutic approaches for modulating ligase activity.

  6. The HPV E6 oncoprotein targets histone methyltransferases for modulating specific gene transcription

    PubMed Central

    Hsu, C-H; Peng, K-L; Jhang, H-C; Lin, C-H; Wu, S-Y; Chiang, C-M; Lee, S-C; Yu, W C Y; Juan, L-J

    2012-01-01

    Expression of viral proteins causes important epigenetic changes leading to abnormal cell growth. Whether viral proteins directly target histone methyltransferases (HMTs), a key family enzyme for epigenetic regulation, and modulate their enzymatic activities remains elusive. Here we show that the E6 proteins of both low-risk and high-risk human papillomavirus (HPV) interact with three coactivator HMTs, CARM1, PRMT1 and SET7, and downregulate their enzymatic activities in vitro and in HPV-transformed HeLa cells. Furthermore, these three HMTs are required for E6 to attenuate p53 transactivation function. Mechanistically, E6 hampers CARM1- and PRMT1-catalyzed histone methylation at p53-responsive promoters, and suppresses the binding of p53 to chromatinized DNA independently of E6-mediated p53 degradation. p53 pre-methylated at lysine-372 (p53K372 mono-methylation) by SET7 protects p53 from E6-induced degradation. Consistently, E6 downregulates p53K372 mono-methylation and thus reduces p53 protein stability. As a result of the E6-mediated inhibition of HMT activity, expression of p53 downstream genes is suppressed. Together, our results not only reveal a clever approach for the virus to interfere with p53 function, but also demonstrate the modulation of HMT activity as a novel mechanism of epigenetic regulation by a viral oncoprotein. PMID:21963854

  7. The HPV E6 oncoprotein targets histone methyltransferases for modulating specific gene transcription.

    PubMed

    Hsu, C-H; Peng, K-L; Jhang, H-C; Lin, C-H; Wu, S-Y; Chiang, C-M; Lee, S-C; Yu, W C Y; Juan, L-J

    2012-05-03

    Expression of viral proteins causes important epigenetic changes leading to abnormal cell growth. Whether viral proteins directly target histone methyltransferases (HMTs), a key family enzyme for epigenetic regulation, and modulate their enzymatic activities remains elusive. Here we show that the E6 proteins of both low-risk and high-risk human papillomavirus (HPV) interact with three coactivator HMTs, CARM1, PRMT1 and SET7, and downregulate their enzymatic activities in vitro and in HPV-transformed HeLa cells. Furthermore, these three HMTs are required for E6 to attenuate p53 transactivation function. Mechanistically, E6 hampers CARM1- and PRMT1-catalyzed histone methylation at p53-responsive promoters, and suppresses the binding of p53 to chromatinized DNA independently of E6-mediated p53 degradation. p53 pre-methylated at lysine-372 (p53K372 mono-methylation) by SET7 protects p53 from E6-induced degradation. Consistently, E6 downregulates p53K372 mono-methylation and thus reduces p53 protein stability. As a result of the E6-mediated inhibition of HMT activity, expression of p53 downstream genes is suppressed. Together, our results not only reveal a clever approach for the virus to interfere with p53 function, but also demonstrate the modulation of HMT activity as a novel mechanism of epigenetic regulation by a viral oncoprotein.

  8. HPV E6 Proteins Target Ubc9, the SUMO Conjugating Enzyme

    PubMed Central

    Heaton, Phillip R.; Deyrieux, Adeline F.; Bian, Xue-Lin; Wilson, Van G.

    2011-01-01

    The human papillomavirus oncogenic protein, E6, interacts with a number of cellular proteins, and for some targets, E6 directs their degradation through the ubiquitin-proteasome pathway. Post-translational modification with ubiquitin-like modifiers, such as SUMO, also influences protein activities, protein-protein interactions, and protein stability. We report that the high risk HPVE6 proteins reduce the intracellular quantity of the sole SUMO conjugation enzyme, Ubc9, concomitant with decreased host sumoylation. E6 did not significantly influence transcription of Ubc9, indicating that the effects were likely at the protein level. Consistent with typical E6-mediated proteasomal degradation, E6 bound to Ubc9 in vitro, and required E6AP for reduction of Ubc9 levels. Under stable E6 expression conditions in differentiating keratinocytes there was a decrease in Ubc9 and a loss of numerous sumoylated targets indicating a significant perturbation of the normal sumoylation profile. While E6 is known to inhibit PIASy, a SUMO ligase, our results suggest that HPV E6 also targets the Ubc9 protein to modulate host cell sumoylation, suggesting that the sumoylation system may be an important target during viral reproduction and possibly the subsequent development of cervical cancer. PMID:21510985

  9. Papillomavirus E6 oncoproteins

    PubMed Central

    Vande Pol, Scott B.; Klingelhutz, Aloysius J.

    2013-01-01

    Papillomaviruses induce benign and malignant epithelial tumors, and the viral E6 oncoprotein is essential for full transformation. E6 contributes to transformation by associating with cellular proteins, docking on specific acidic LXXLL peptide motifs found on the associated cellular proteins. This review examines insights from recent studies of human and animal E6 proteins that determine the three-dimensional structure of E6 when bound to acidic LXXLL peptides. The structure of E6 is related to recent advances in the purification and identification of E6 associated protein complexes. These E6 protein-complexes, together with other proteins that bind to E6, alter a broad array of biological outcomes including modulation of cell survival, cellular transcription, host cell differentiation, growth factor dependence, DNA damage responses, and cell cycle progression. PMID:23711382

  10. The vaccinia virus E6 protein influences virion protein localization during virus assembly

    SciTech Connect

    Condit, Richard C. Moussatche, Nissin

    2015-08-15

    Vaccinia virus mutants in which expression of the virion core protein gene E6R is repressed are defective in virion morphogenesis. E6 deficient infections fail to properly package viroplasm into viral membranes, resulting in an accumulation of empty immature virions and large aggregates of viroplasm. We have used immunogold electron microscopy and immunofluorescence confocal microscopy to assess the intracellular localization of several virion structural proteins and enzymes during E6R mutant infections. We find that during E6R mutant infections virion membrane proteins and virion transcription enzymes maintain a normal localization within viral factories while several major core and lateral body proteins accumulate in aggregated virosomes. The results support a model in which vaccinia virions are assembled from at least three substructures, the membrane, the viroplasm and a “pre-nucleocapsid”, and that the E6 protein is essential for maintaining proper localization of the seven-protein complex and the viroplasm during assembly. - Highlights: • Mutation of E6 disrupts association of viral membranes with viral core proteins • Mutation of E6 does not perturb viral membrane biosynthesis • Mutation of E6 does not perturb localization of viral transcription enzymes • Mutation of E6 causes mis-localization and aggregation of viral core proteins • Vaccinia assembly uses three subassemblies: membranes, viroplasm, prenucleocapsid.

  11. The vaccinia virus E6 protein influences virion protein localization during virus assembly

    PubMed Central

    Condit, Richard C.; Moussatche, Nissin

    2015-01-01

    Vaccinia virus mutants in which expression of the virion core protein gene E6R is repressed are defective in virion morphogenesis. E6 deficient infections fail to properly package viroplasm into viral membranes, resulting in an accumulation of empty immature virions and large aggregates of viroplasm. We have used immunogold electron microscopy and immunofluorescence confocal microscopy to assess the intracellular localization of several virion structural proteins and enzymes during E6R mutant infections. We find that during E6R mutant infections virion membrane proteins and virion transcription enzymes maintain a normal localization within viral factories while several major core and lateral body proteins accumulate in aggregated virosomes. The results support a model in which vaccinia virions are assembled from at least three substructures, the membrane, the viroplasm and a “pre-nucleocapsid”, and that the E6 protein is essential for maintaining proper localization of the seven-protein complex and the viroplasm during assembly. PMID:25863879

  12. Localization of the E6-AP regions that direct human papillomavirus E6 binding, association with p53, and ubiquitination of associated proteins.

    PubMed Central

    Huibregtse, J M; Scheffner, M; Howley, P M

    1993-01-01

    E6-AP is a 100-kDa cellular protein that mediates the interaction of the human papillomavirus type 16 and 18 E6 proteins with p53. The association of p53 with E6 and E6-AP promotes the specific ubiquitination and subsequent proteolytic degradation of p53 in vitro. We recently isolated a cDNA encoding E6-AP and have now mapped functional domains of E6-AP involved in binding E6, association with p53, and ubiquitination of p53. The E6 binding domain consists of an 18-amino-acid region within the central portion of the molecule. Deletion of these 18 amino acids from E6-AP results in loss of both E6 and p53 binding activities. The region that directs p53 binding spans the E6 binding domain and consists of approximately 500 amino acids. E6-AP sequences in addition to those required for formation of a stable ternary complex with E6 and p53 are necessary to stimulate the ubiquitination of p53. These sequences lie within the C-terminal 84 amino acids of E6-AP. The entire region required for E6-dependent ubiquitination of p53 is also required for the ubiquitination of an artificial E6 fusion protein. Images PMID:8393140

  13. Interaction of the Human Papillomavirus E6 Oncoprotein with Sorting Nexin 27 Modulates Endocytic Cargo Transport Pathways

    PubMed Central

    Ganti, Ketaki; Massimi, Paola; Manzo-Merino, Joaquin; Tomaić, Vjekoslav; Pim, David; Playford, Martin P.; Lizano, Marcela; Roberts, Sally; Kranjec, Christian; Doorbar, John; Banks, Lawrence

    2016-01-01

    A subset of high-risk Human Papillomaviruses (HPVs) are the causative agents of a large number of human cancers, of which cervical is the most common. Two viral oncoproteins, E6 and E7, contribute directly towards the development and maintenance of malignancy. A characteristic feature of the E6 oncoproteins from cancer-causing HPV types is the presence of a PDZ binding motif (PBM) at its C-terminus, which confers interaction with cellular proteins harbouring PDZ domains. Here we show that this motif allows E6 interaction with Sorting Nexin 27 (SNX27), an essential component of endosomal recycling pathways. This interaction is highly conserved across E6 proteins from multiple high-risk HPV types and is mediated by a classical PBM-PDZ interaction but unlike many E6 targets, SNX27 is not targeted for degradation by E6. Rather, in HPV-18 positive cell lines the association of SNX27 with components of the retromer complex and the endocytic transport machinery is altered in an E6 PBM-dependent manner. Analysis of a SNX27 cargo, the glucose transporter GLUT1, reveals an E6-dependent maintenance of GLUT1 expression and alteration in its association with components of the endocytic transport machinery. Furthermore, knockdown of E6 in HPV-18 positive cervical cancer cells phenocopies the loss of SNX27, both in terms of GLUT1 expression levels and its vesicular localization, with a concomitant marked reduction in glucose uptake, whilst loss of SNX27 results in slower cell proliferation in low nutrient conditions. These results demonstrate that E6 interaction with SNX27 can alter the recycling of cargo molecules, one consequence of which is modulation of nutrient availability in HPV transformed tumour cells. PMID:27649450

  14. HPV16 E6 regulates annexin 1 (ANXA1) protein expression in cervical carcinoma cell lines

    SciTech Connect

    Calmon, Marilia Freitas; Sichero, Laura; Boccardo, Enrique; Villa, Luisa Lina; Rahal, Paula

    2016-09-15

    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-16 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.

  15. E6^E7, a Novel Splice Isoform Protein of Human Papillomavirus 16, Stabilizes Viral E6 and E7 Oncoproteins via HSP90 and GRP78

    PubMed Central

    Ajiro, Masahiko

    2015-01-01

    ABSTRACT Transcripts of human papillomavirus 16 (HPV16) E6 and E7 oncogenes undergo alternative RNA splicing to produce multiple splice isoforms. However, the importance of these splice isoforms is poorly understood. Here we report a critical role of E6^E7, a novel isoform containing the 41 N-terminal amino acid (aa) residues of E6 and the 38 C-terminal aa residues of E7, in the regulation of E6 and E7 stability. Through mass spectrometric analysis, we identified that HSP90 and GRP78, which are frequently upregulated in cervical cancer tissues, are two E6^E7-interacting proteins responsible for the stability and function of E6^E7, E6, and E7. Although GRP78 and HSP90 do not bind each other, GRP78, but not HSP90, interacts with E6 and E7. E6^E7 protein, in addition to self-binding, interacts with E6 and E7 in the presence of GRP78 and HSP90, leading to the stabilization of E6 and E7 by prolonging the half-life of each protein. Knocking down E6^E7 expression in HPV16-positive CaSki cells by a splice junction-specific small interfering RNA (siRNA) destabilizes E6 and E7 and prevents cell growth. The same is true for the cells with a GRP78 knockdown or in the presence of an HSP90 inhibitor. Moreover, mapping and alignment analyses for splicing elements in 36 alpha-HPVs (α-HPVs) suggest the possible expression of E6^E7 mostly by other oncogenic or possibly oncogenic α-HPVs (HPV18, -30, -31, -39, -42, -45, -56, -59, -70, and -73). HPV18 E6^E7 is detectable in HPV18-positive HeLa cells and HPV18-infected raft tissues. All together, our data indicate that viral E6^E7 and cellular GRP78 or HSP90 might be novel targets for cervical cancer therapy. PMID:25691589

  16. Human papillomavirus E6 proteins mediate resistance to interferon-induced growth arrest through inhibition of p53 acetylation.

    PubMed

    Hebner, Christy; Beglin, Melanie; Laimins, Laimonis A

    2007-12-01

    The high-risk human papillomavirus (HPV) E6 and E7 proteins act cooperatively to mediate multiple activities in viral pathogenesis. For instance, E7 acts to increase p53 levels while E6 accelerates its rate of turnover through the binding of the cellular ubiquitin ligase E6AP. Interferons are important antiviral agents that modulate both the initial and persistent phases of viral infection. The expression of HPV type 16 E7 was found to sensitize keratinocytes to the growth-inhibitory effects of interferon, while coexpression of E6 abrogates this inhibition. Treatment of E7-expressing cells with interferon ultimately resulted in cellular senescence through a process that is dependent upon acetylation of p53 by p300/CBP at lysine 382. Cells expressing mutant forms of E6 that are unable to bind p300/CBP or bind p53 failed to block acetylation of p53 at lysine 382 and were sensitive to growth arrest by interferon. In contrast, mutant forms of E6 that are unable to bind E6AP remain resistant to the effects of interferon, demonstrating that the absolute levels of p53 are not the major determinants of this activity. Finally, p53 acetylation at lysine 382 was found not to be an essential determinant of other types of senescence such as that induced by overexpression of Ras in human fibroblasts. This study identifies an important physiological role for E6 binding to p300/CBP in blocking growth arrest of human keratinocytes in the presence of interferon and so contributes to the persistence of HPV-infected cells.

  17. The E6 protein from vaccinia virus is required for the formation of immature virions

    SciTech Connect

    Boyd, Olga; Turner, Peter C.; Moyer, Richard W.; Condit, Richard C.; Moussatche, Nissin

    2010-04-10

    An IPTG-inducible mutant in the E6R gene of vaccinia virus was used to study the role of the E6 virion core protein in viral replication. In the absence of the inducer, the mutant exhibited a normal pattern DNA replication, concatemer resolution and late gene expression, but it showed an inhibition of virion structural protein processing it failed to produce infectious particles. Electron microscopic analysis showed that in the absence of IPTG viral morphogenesis was arrested before IV formation: crescents, aberrant or empty IV-like structures, and large aggregated virosomes were observed throughout the cytoplasm. The addition of IPTG to release a 12-h block showed that virus infectious particles could be formed in the absence of de novo DNA synthesis. Our observations show that in the absence of E6 the association of viroplasm with viral membrane crescents is impaired.

  18. Involvement of Novel Multifunction Steroid Hormone Receptor Coactivator, E6-Associated Protein, in Prostate Gland Tumorigenesis

    DTIC Science & Technology

    2009-01-01

    8. 25. Nawaz Z, Lonard DM, Smith CL, Lev-Lehman E, Tsai SY, Tsai MJ, O’Malley BW 1999 The Angelman syndrome-associated protein, E6-AP, is a...CM, Noebels JL, Eichele G, Sweatt JD, Beaudet AL 1998 Mutation of the Angelman ubiquitin ligase in mice causes increased 12 cytoplasmic p53 and

  19. Involvement of Novel Multifunctional Steroid Hormone Receptor Coactivator, E6-Associated Protein, in Prostate Gland Tumorigenesis

    DTIC Science & Technology

    2008-01-01

    Angelman syndrome-associated protein, E6-AP, is a coactivator for the nuclear hormone receptor superfamily. Mol Cell Biol 19:1182-9. 26. Hsiao PW, Chang C...Albrecht U, Atkins CM, Noebels JL, Eichele G, Sweatt JD, Beaudet AL 1998 Mutation of the Angelman ubiquitin ligase in mice causes increased cytoplasmic

  20. Involvement of Novel Multifunction Steroid Hormone Receptor Coactivator, E6-Associated Protein, in Prostate Gland Tumorigenesis

    DTIC Science & Technology

    2010-01-01

    synthesized RhoA G14V protein in NETN buffer (100 mM NaCl, 1 mM EDTA, 20 mM Tris (pH 8.0), and 0.5% Nonidet P - 40 ) for 2–3 hours at room...to test the effects of loss E6- AP on the normal development of the prostate gland ( 40 , 41). We have also generated E6-AP transgenic mouse line that...expression of p -Akt in all the lobes compared to the wild- type, there was no significant differences between the extents of p -Akt over expression between

  1. Expression of the highly conserved vaccinia virus E6 protein is required for virion morphogenesis

    SciTech Connect

    Resch, Wolfgang; Weisberg, Andrea S.; Moss, Bernard

    2009-04-10

    The vaccinia virus E6R gene (VACVWR062) is conserved in all members of the poxvirus family and encodes a protein associated with the mature virion. We confirmed this association and provided evidence for an internal location. An inducible mutant that conditionally expresses E6 was constructed. In the absence of inducer, plaque formation and virus production were severely inhibited in several cell lines, whereas some replication occurred in others. This difference could be due to variation in the stringency of repression, since we could not isolate a stable deletion mutant even in the more 'permissive' cells. Under non-permissive conditions, viral late proteins were synthesized but processing of core proteins was inefficient, indicative of an assembly block. Transmission electron microscopy of sections of cells infected with the mutant in the absence of inducer revealed morphogenetic defects with crescents and empty immature virions adjacent to dense inclusions of viroplasm. Mature virions were infrequent and cores appeared to have lucent centers.

  2. E6-AP/UBE3A protein acts as a ubiquitin ligase toward SOX9 protein.

    PubMed

    Hattori, Takako; Kishino, Tetsuya; Stephen, Shelley; Eberspaecher, Heidi; Maki, Sayumi; Takigawa, Masaharu; de Crombrugghe, Benoit; Yasuda, Hideyo

    2013-12-06

    SOX9 is a transcription factor that acts as a key regulator at various stages of cartilage differentiation. There is ample evidence that intracellular SOX9 protein levels are tightly regulated both by sumoylation and by degradation through the ubiquitin-proteasome pathway. Using a proteomics approach, here we report the identification of a SOX9-binding protein, E6-AP/UBE3A, that may act as a ubiquitin ligase toward Sox9. E6-AP bound SOX9 through the region consisting mostly of its high mobility group domain in vitro. In nuclear lysates, FLAG-tagged E6-AP coprecipitated with Sox9 and its high mobility group domain. This finding was estimated using nuclear lysates from a chondrocytic cell line that endogenously expresses E6-AP and SOX9. Accordingly, ectopically expressed E6-AP and SOX9 colocalized in the nucleus. We show that E6-AP ubiquitinates SOX9 in vitro and in vivo and that SOX9 levels are enhanced after addition of the proteasome inhibitor bortezomib. Similar, siRNA knockdown of E6-AP and the E2 ligase Ubc9 increased cellular SOX9 amounts, supporting the notion that SOX9 may be ubiquitinated in hypertrophic chondrocytes by E6-AP and degraded by proteasomes. This is in accordance with the distribution of SOX9 levels, which are high in proliferating and prehypertrophic chondrocytes but low in hypertrophic chondrocytes, whereas E6-AP levels are high in hypertrophic chondrocytes and low in prehypertrophic chondrocytes. Furthermore, E6-AP-deficient mice showed SOX9 accumulation in chondrocytes and the brain. These findings support the concept that E6-AP regulates SOX9 levels in developing cartilage by acting as a ubiquitin ligase.

  3. HPV 5 and 8 E6 Expression Reduces ATM Protein Levels and Attenuates LINE-1 Retrotransposition

    PubMed Central

    Wallace, Nicholas A.; Gasior, Stephen L.; Faber, Zachary J.; Howie, Heather L.; Deininger, Prescott L.; Galloway, Denise A.

    2013-01-01

    The expression of the E6 protein from certain members of the HPV genus β (β HPV 5 and 8 E6) can disrupt p53 signaling by diminishing the steady state levels of two p53 modifying enzymes, ATR and p300. Here, we show that β-HPV 5 and 8 E6 are also capable of reducing the steady state levels of another p53 modifying enzyme, ATM, and as a result restrict LINE-1 retrotransposition. Furthermore, we show that the reduction of both ATM and LINE-1 retrotransposition is dependent upon the ability of β-HPV 8 E6 to bind and degrade p300. We use inhibitors and dominant negative mutants to confirm that ATM is needed for efficient LINE-1 retrotransposition. Furthermore, neither sensitivity to LINE-1 expression nor LINE-1 induced DSB formation is altered in an ATM deficient background. Together, these data illustrate the broad impact some β-HPVs have on DNA damage signaling by promoting p300 degradation. PMID:23706308

  4. E6-Associated Protein Dependent Estrogen Receptor Regulation of Protein Kinase A Regulatory Subunit R2A Expression in Neuroblastoma.

    PubMed

    Obeid, Jean-Pierre; Zeidan, Youssef H; Zafar, Nawal; El Hokayem, Jimmy

    2017-02-18

    E6ap is a known transcriptional coregulator for estrogen receptor alpha (Er, Erα) in the presence of estrogen. Protein kinase A (PKA) contains two regulatory subunits derived from four genes. Recent evidence demonstrates that PKA regulates E6ap activity. Data generated in our lab indicated estrogen dependent regulation of Pkar2a levels. Our project sets to investigate a possible feedback mechanism constituting of Erα and E6ap transcriptional regulation of Pkar2a expression. Western blot evaluated protein regulation correlations with E2 in mouse neuroblastoma lines. Bioinformatics detected estrogen response element (ERE) sequences. quantitative polymerase chain reaction (qPCR) validated the western blot results. ERE oligonucleotides were synthesized. Reporter gene transcriptional activity was evaluated via Luciferase assay output. Electromobility shift assay (EMSA) assessed direct binding between Erα relevant sequences. Chromatin immunoprecipitation (ChIP) and Re-ChIP were conducted in quantifying protein complex recruitment levels. Pkar2a protein expression directly correlated with E2, and four putative ERE sequences were identified. Pkar2a mRNA expression reverted to baseline with either E2 or E6ap absent. In the presence of E2, ERE-1 and ERE-4 possessed Luciferase reporter gene transcriptional capabilities. ERE-1 portrayed band shifts, representing direct binding to Erα with E2 supplementation. With E2, ERE-1 significantly enhanced Erα and E6ap recruitment levels to the Pkar2a promoter. Pkar2a is directly regulated by Erα and E6ap in the presence of estrogen stimulus. This work indicates a feedback mechanism in the interplay between PKA and E6ap, which may prove crucial for the role of both proteins in cancers and neurogenetic diseases like Angelman syndrome.

  5. p53 Degradation Activity, Expression, and Subcellular Localization of E6 Proteins from 29 Human Papillomavirus Genotypes

    PubMed Central

    Mesplède, Thibault; Gagnon, David; Bergeron-Labrecque, Fanny; Azar, Ibrahim; Sénéchal, Hélène; Coutlée, François

    2012-01-01

    Human papillomaviruses (HPVs) are the etiological agents of cervical cancer and other human malignancies. HPVs are classified into high- and low-risk genotypes according to their association with cancer. Host cell transformation by high-risk HPVs relies in part on the ability of the viral E6 protein to induce the degradation of p53. We report the development of a cellular assay that accurately quantifies the p53 degradation activity of E6 in vivo, based on the fusion of p53 to Renilla luciferase (RLuc-p53). This assay was used to measure the p53 degradation activities of E6 proteins from 29 prevalent HPV types and variants of HPV type 16 (HPV16) and HPV33 by determining the amount of E6 expression vector required to reduce by half the levels of RLuc-p53 (50% effective concentration [EC50]). These studies revealed an unexpected variability in the p53 degradation activities of different E6 proteins, even among active types whose EC50s span more than 2 log units. Differences in activity were greater between types than between variants and did not correlate with differences in the intracellular localization of E6, with most being predominantly nuclear. Protein and mRNA expression of the 29 E6 proteins was also examined. For 16 high-risk types, spliced transcripts that encode shorter E6*I proteins of variable sizes and abundances were detected. Mutation of the splice donor site in five different E6 proteins increased their p53 degradation activity, suggesting that mRNA splicing can limit the activity of some high-risk E6 types. The quantification of p53 degradation in vivo represents a novel tool to systematically compare the oncogenic potentials of E6 proteins from different HPV types and variants. PMID:22013048

  6. Roles of the PDZ-binding motif of HPV 16 E6 protein in oncogenic transformation of human cervical keratinocytes.

    PubMed

    Yoshimatsu, Yuki; Nakahara, Tomomi; Tanaka, Katsuyuki; Inagawa, Yuki; Narisawa-Saito, Mako; Yugawa, Takashi; Ohno, Shin-Ichi; Fujita, Masatoshi; Nakagama, Hitoshi; Kiyono, Tohru

    2017-07-01

    The high-risk human papillomavirus E6 proteins have been shown to interact with and lead to degradation of PDZ-domain-containing proteins through its carboxy-terminal motif. This PDZ-binding motif plays important roles in transformation of cultured cells and carcinogenesis of E6-transgenic mice. However, its biological effects on the natural host cells have not been elucidated. We have examined its roles in an in vitro carcinogenesis model for cervical cancer, in which E6 and E7 together with activated HRAS (HRAS(G)(12V) ) can induce tumorigenic transformation of normal human cervical keratinocytes. In this model, E6Δ151 mutant, which is defective in binding to PDZ domains, almost lost tumorigenic ability, whereas E6SAT mutant, which is defective in p53 degradation showed activity close to wild-type E6. Interestingly, we found decreased expression of PAR3 in E6-expressing cells independently of E6AP, which has not been previously recognized. Therefore, we knocked down several PDZ-domain containing proteins including PAR3 in human cervical keratinocytes expressing E7, HRAS(G)(12V) and E6Δ151 to examine whether depletion of these proteins can restore the tumorigenic ability. Single knockdown of SCRIB, MAGI1 or PAR3 significantly but partially restored the tumorigenic ability. The combinatorial knockdown of SCRIB and MAGI1 cooperatively restored the tumorigenic ability, and additional depletion of PAR3 further enhanced the tumorigenic ability surpassing that induced by wild-type E6. These data highlight the importance of the carboxy-terminal motif of the E6 protein and downregulation of PAR3 in tumorigenic transformation of human cervical keratinocytes. © 2017 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.

  7. Impact of E6-associated protein on the proliferation and invasion of prostate cancer cells in bone metastasis

    PubMed Central

    Zhang, Liyan; Hu, Xiaoguang; Chen, Jiying; Fu, Guilian

    2015-01-01

    Purpose: To understand E6 associated protein (E6-AP)’s influence on prostate cancer cell proliferation and infiltration, thus providing the theoretical basis for developing therapeutic drugs for prostate cancer metastasis to the bone. Methods: Electroporation was performed to introduce linear regulatory plasmid PrevTet-off-in and conjugative plasmid PrevTRE2-flag-E6AP into prostate cancer cell line to establish wild-type E6-AP over-expressing transgenic LNCaP cell line; Western blot assay was adopted to examine expression levels of E6-AP, mammalian target of rapamycin (mTOR), protein kinase B (Akt), and phosphoinositide 3-kinase (PI3K); PI3K inhibitor LY294002 was applied to all the cells and MTT assay was used to measure cell proliferation; Matrigel invasion chamber assay was adopted to detect cancer cell migration and invasion. Results: Stably transfected LNCaP cells that over expressed E6-AP had higher expression levels of PI3K, Akt, and mTOR than control LNCaP cells; MTT assay showed that E6-AP-LNCaP cells were more responsive to the inhibitory effect of LY294002; Matrigel invasion chamber assay revealed increased cell crawling and adhesiveness of E6-AP-LNCaP cells. Conclusion: Stable over-expression of E6-AP increases the proliferation and invasion of LNCaP cells. PMID:26261538

  8. E6 protein of human papillomavirus 16 (HPV16) expressed in Escherichia coli sans a stretch of hydrophobic amino acids, enables purification of GST-ΔE6 in the soluble form and retains the binding ability to p53.

    PubMed

    Verma, Ravi Ranjan; Sriraman, Rajan; Rana, Samir Kumar; Ponnanna, N M; Rajendar, Burki; Ghantasala, Priyanka; Rajendra, Lingala; Matur, Ramesh V; Srinivasan, Villupanoor Alwar

    2013-11-01

    Recombinant E6 expressed in Escherichia coli is known to form recalcitrant inclusion bodies even when fused to the soluble GST protein. This study describes the modification of the HPV genotype-16 oncogenic protein E6 in order to obtain it in the soluble form. The modified proteinE6) was expressed in E. coli BL21 as an N-terminal fusion with GST (GST-ΔE6). ΔE6 was constructed by deleting the nucleotide sequences coding for IHDIIL (31-36 a.a), one of the highly hydrophobic peptide stretches, using splicing by overextension polymerase chain reaction (SOE-PCR). The removal of IHDIIL residues rendered the GST-ΔE6 soluble and amenable for purification involving a two step process a preliminary glutathione-GST affinity chromatography followed by gel-filtration chromatography. Evaluation of purified protein fractions by HPLC suggests that GST-ΔE6 exists as a monomer. Further, the ΔE6 in GST-ΔE6 seemed to retain the binding ability to p53 as determined by the glutathione-GST capture ELISA. Purified GST-ΔE6 we reckon, might find use as an essential reagent in immunological assays, in sero-epidemiological studies, and also in studies to delineate the structure and function of HPV16 E6. Copyright © 2013 Elsevier Inc. All rights reserved.

  9. Mutagenic Potential ofBos taurus Papillomavirus Type 1 E6 Recombinant Protein: First Description

    PubMed Central

    Araldi, Rodrigo Pinheiro; Mazzuchelli-de-Souza, Jacqueline; Modolo, Diego Grando; de Souza, Edislane Barreiros; de Melo, Thatiana Corrêa; Spadacci-Morena, Diva Denelle; Magnelli, Roberta Fiusa; de Carvalho, Márcio Augusto Caldas Rocha; de Sá Júnior, Paulo Luis; de Carvalho, Rodrigo Franco; Beçak, Willy; Stocco, Rita de Cassia

    2015-01-01

    Bovine papillomavirus (BPV) is considered a useful model to study HPV oncogenic process. BPV interacts with the host chromatin, resulting in DNA damage, which is attributed to E5, E6, and E7 viral oncoproteins activity. However, the oncogenic mechanisms of BPV E6 oncoprotein per se remain unknown. This study aimed to evaluate the mutagenic potential of Bos taurus papillomavirus type 1 (BPV-1) E6 recombinant oncoprotein by the cytokinesis-block micronucleus assay (CBMNA) and comet assay (CA). Peripheral blood samples of five calves were collected. Samples were subjected to molecular diagnosis, which did not reveal presence of BPV sequences. Samples were treated with 1 μg/mL of BPV-1 E6 oncoprotein and 50 μg/mL of cyclophosphamide (positive control). Negative controls were not submitted to any treatment. The samples were submitted to the CBMNA and CA. The results showed that BPV E6 oncoprotein induces clastogenesis per se, which is indicative of genomic instability. These results allowed better understanding the mechanism of cancer promotion associated with the BPV E6 oncoprotein and revealed that this oncoprotein can induce carcinogenesis per se. E6 recombinant oncoprotein has been suggested as a possible vaccine candidate. Results pointed out that BPV E6 recombinant oncoprotein modifications are required to use it as vaccine. PMID:26783529

  10. Genus Beta Human Papillomavirus E6 Proteins Vary in Their Effects on the Transactivation of p53 Target Genes

    PubMed Central

    White, Elizabeth A.; Walther, Johanna; Javanbakht, Hassan

    2014-01-01

    ABSTRACT The genus beta human papillomaviruses (beta HPVs) cause cutaneous lesions and are thought to be involved in the initiation of some nonmelanoma skin cancers (NMSCs), particularly in patients with the genetic disorder epidermodysplasia verruciformis (EV). We have previously reported that at least two of the genus beta HPV E6 proteins bind to and/or increase the steady-state levels of p53 in squamous epithelial cells. This is in contrast to a well-characterized ability of the E6 proteins of cancer-associated HPVs of genus alpha HPV, which inactivate p53 by targeting its ubiquitin-mediated proteolysis. In this study, we have investigated the ability of genus beta E6 proteins from eight different HPV types to block the transactivation of p53 target genes following DNA damage. We find that the E6 proteins from diverse beta HPV species and types vary in their capacity to block the induction of MDM2, p21, and proapoptotic genes after genotoxic stress. We conclude that some genus beta HPV E6 proteins inhibit at least some p53 target genes, although perhaps not by the same mechanism or to the same degree as the high-risk genus alpha HPV E6 proteins. IMPORTANCE This study addresses the ability of various human papillomavirus E6 proteins to block the activation of p53-responsive cellular genes following DNA damage in human keratinocytes, the normal host cell for HPVs. The E6 proteins encoded by the high-risk, cancer-associated HPV types of genus alpha HPV have a well-established activity to target p53 degradation and thereby inhibit the response to DNA damage. In this study, we have investigated the ability of genus beta HPV E6 proteins from eight different HPV types to block the ability of p53 to transactivate downstream genes following DNA damage. We find that some, but not all, genus beta HPV E6 proteins can block the transactivation of some p53 target genes. This differential response to DNA damage furthers the understanding of cutaneous HPV biology and may help

  11. Genus beta human papillomavirus E6 proteins vary in their effects on the transactivation of p53 target genes.

    PubMed

    White, Elizabeth A; Walther, Johanna; Javanbakht, Hassan; Howley, Peter M

    2014-08-01

    The genus beta human papillomaviruses (beta HPVs) cause cutaneous lesions and are thought to be involved in the initiation of some nonmelanoma skin cancers (NMSCs), particularly in patients with the genetic disorder epidermodysplasia verruciformis (EV). We have previously reported that at least two of the genus beta HPV E6 proteins bind to and/or increase the steady-state levels of p53 in squamous epithelial cells. This is in contrast to a well-characterized ability of the E6 proteins of cancer-associated HPVs of genus alpha HPV, which inactivate p53 by targeting its ubiquitin-mediated proteolysis. In this study, we have investigated the ability of genus beta E6 proteins from eight different HPV types to block the transactivation of p53 target genes following DNA damage. We find that the E6 proteins from diverse beta HPV species and types vary in their capacity to block the induction of MDM2, p21, and proapoptotic genes after genotoxic stress. We conclude that some genus beta HPV E6 proteins inhibit at least some p53 target genes, although perhaps not by the same mechanism or to the same degree as the high-risk genus alpha HPV E6 proteins. This study addresses the ability of various human papillomavirus E6 proteins to block the activation of p53-responsive cellular genes following DNA damage in human keratinocytes, the normal host cell for HPVs. The E6 proteins encoded by the high-risk, cancer-associated HPV types of genus alpha HPV have a well-established activity to target p53 degradation and thereby inhibit the response to DNA damage. In this study, we have investigated the ability of genus beta HPV E6 proteins from eight different HPV types to block the ability of p53 to transactivate downstream genes following DNA damage. We find that some, but not all, genus beta HPV E6 proteins can block the transactivation of some p53 target genes. This differential response to DNA damage furthers the understanding of cutaneous HPV biology and may help to explain the

  12. Structural and Functional Characterization of the R-modules in Alginate C-5 Epimerases AlgE4 and AlgE6 from Azotobacter vinelandii

    PubMed Central

    Buchinger, Edith; Knudsen, Daniel H.; Behrens, Manja A.; Pedersen, Jan Skov; Aarstad, Olav A.; Tøndervik, Anne; Valla, Svein; Skjåk-Bræk, Gudmund; Wimmer, Reinhard; Aachmann, Finn L.

    2014-01-01

    The bacterium Azotobacter vinelandii produces a family of seven secreted and calcium-dependent mannuronan C-5 epimerases (AlgE1–7). These epimerases are responsible for the epimerization of β-d-mannuronic acid (M) to α-l-guluronic acid (G) in alginate polymers. The epimerases display a modular structure composed of one or two catalytic A-modules and from one to seven R-modules having an activating effect on the A-module. In this study, we have determined the NMR structure of the three individual R-modules from AlgE6 (AR1R2R3) and the overall structure of both AlgE4 (AR) and AlgE6 using small angle x-ray scattering. Furthermore, the alginate binding ability of the R-modules of AlgE4 and AlgE6 has been studied with NMR and isothermal titration calorimetry. The AlgE6 R-modules fold into an elongated parallel β-roll with a shallow, positively charged groove across the module. Small angle x-ray scattering analyses of AlgE4 and AlgE6 show an overall elongated shape with some degree of flexibility between the modules for both enzymes. Titration of the R-modules with defined alginate oligomers shows strong interaction between AlgE4R and both oligo-M and MG, whereas no interaction was detected between these oligomers and the individual R-modules from AlgE6. A combination of all three R-modules from AlgE6 shows weak interaction with long M-oligomers. Exchanging the R-modules between AlgE4 and AlgE6 resulted in a novel epimerase called AlgE64 with increased G-block forming ability compared with AlgE6. PMID:25266718

  13. E6 viral protein ratio correlates with outcomes in human papillomavirus related oropharyngeal cancer

    PubMed Central

    Hong, Angela; Zhang, Xiaoying; Jones, Deanna; Zhang, Mei; Lee, C. Soon; Lyons, J. Guy; Veillard, Anne-Sophie; Rose, Barbara

    2016-01-01

    ABSTRACT Background: The study aimed to identify prognostic markers to improve the management of patients with HPV positive OSCC Methods: We determined the ratio of HPV E6*I and E6*II splice variants by quantitative RT-PCR in 177 HPV positive OSCC and correlated the findings with other clinicopathological data Results: There was no significant difference in locoregional recurrence (HR 1.72 p = 0.24) and death (HR 1.65, p = 0.13) among patients whose tumors had an E6*I/*II ratio ≥1 compared with an E6*I/*II ratio of <1. Univariate analysis showed that patients with E6*I/*II ≥1 OSCC were more likely to have an event. In the multivariable analysis, there was a trend for more events in patients with E6*I/*II ratio ≥1 (HR 1.70, 95% CI 0.95-3.03, p = 0.07) Conclusion: Our data suggest that the use of HPV 16 spliced transcripts may help to predict for poorer outcomes in patients with HPV positive OSCC. PMID:26575468

  14. High levels of p105 (NFKB1) and p100 (NFKB2) proteins in HPV16-transformed keratinocytes: role of E6 and E7 oncoproteins

    SciTech Connect

    Havard, L.; Rahmouni, S.; Boniver, J.; Delvenne, P. . E-mail: P.Delvenne@ulg.ac.be

    2005-01-20

    We have previously shown that functional components of the NF-{kappa}B signaling pathway are up-regulated and sequestered in the cytoplasm of human papillomavirus 16 (HPV16)-transformed cell lines leading to a reduced activity of NF-{kappa}B. In this study, we examined the expression of the NF-{kappa}B precursors p100 and p105 in keratinocytes transformed or not by HPV16. Western immunoblotting experiments demonstrated high levels of p100 and p105 proteins not only in HPV16{sup +} cervical carcinoma-derived keratinocytes but also in keratinocytes stably transfected by HPV16 E6 or E7 oncogenes. Moreover, p100 and p105 proteins were predominantly cytoplasmic and nuclear in keratinocytes expressing E7 and E6, respectively. A predominantly cytoplasmic localization of E7 protein was also detected in all keratinocytes expressing E7. Our results suggest that HPV16 E6 and E7 proteins modulate the expression and the subcellular localization of p100 and p105 NF-{kappa}B precursors.

  15. Expression and In Silico Analysis of the Recombinant Bovine Papillomavirus E6 Protein as a Model for Viral Oncoproteins Studies

    PubMed Central

    Mazzuchelli-de-Souza, J.; Carvalho, R. F.; Ruiz, R. M.; Melo, T. C.; Araldi, R. P.; Carvalho, E.; Thompson, C. E.; Sircili, M. P.; Beçak, W.; Stocco, R. C.

    2013-01-01

    Bovine papillomaviruses (BPVs) are recognized as the causal agents of economical relevant diseases in cattle, associated with the development of tumors in skin and mucosa. The oncogenesis process is mainly associated with different viral oncoprotein expressions, which are involved in cell transformation. The expression and characterization of recombinant viral oncoproteins represent an attractive strategy to obtain biotechnological products as antibodies and potential vaccines, Thus, the aim of this work was to clone and express the BPV-1 and BPV-2 E6 recombinant proteins and perform in silico analysis in order to develop a strategy for the systematic study of other papillomaviruses oncoproteins. The results demonstrated that BPV-1 and BPV-2 E6 recombinant proteins were expressed and purified from bacterial system as well as its in silico analysis was performed in order to explore and predict biological characteristics of these proteins. PMID:23878806

  16. Expression and in Silico analysis of the recombinant bovine papillomavirus E6 protein as a model for viral oncoproteins studies.

    PubMed

    Mazzuchelli-de-Souza, J; Carvalho, R F; Ruiz, R M; Melo, T C; Araldi, R P; Carvalho, E; Thompson, C E; Sircili, M P; Beçak, W; Stocco, R C

    2013-01-01

    Bovine papillomaviruses (BPVs) are recognized as the causal agents of economical relevant diseases in cattle, associated with the development of tumors in skin and mucosa. The oncogenesis process is mainly associated with different viral oncoprotein expressions, which are involved in cell transformation. The expression and characterization of recombinant viral oncoproteins represent an attractive strategy to obtain biotechnological products as antibodies and potential vaccines, Thus, the aim of this work was to clone and express the BPV-1 and BPV-2 E6 recombinant proteins and perform in silico analysis in order to develop a strategy for the systematic study of other papillomaviruses oncoproteins. The results demonstrated that BPV-1 and BPV-2 E6 recombinant proteins were expressed and purified from bacterial system as well as its in silico analysis was performed in order to explore and predict biological characteristics of these proteins.

  17. A conserved C-terminal sequence of high-risk cutaneous beta-human papillomavirus E6 proteins alters localization and signalling of β1-integrin to promote cell migration.

    PubMed

    Holloway, Amy; Storey, Alan

    2014-01-01

    Beta-human papillomaviruses (β-HPV) infect cutaneous epithelia, and accumulating evidence suggests that the virus may act as a co-factor with UV-induced DNA damage in the development and progression of non-melanoma skin cancer, although the molecular mechanisms involved are poorly understood. The E6 protein of cutaneous β-HPV types encodes functions consistent with a role in tumorigenesis, and E6 expression can result in papilloma formation in transgenic animals. The E6 proteins of high-risk α-HPV types, which are associated with the development of anogenital cancers, have a conserved 4 aa motif at their extreme C terminus that binds to specific PDZ domain-containing proteins to promote cell invasion. Likewise, the high-risk β-HPVs HPV5 and HPV8 E6 proteins also share a conserved C-terminal motif, but this is markedly different from that of α-HPV types, implying functional differences. Using binding and functional studies, we have shown that β-HPV E6 proteins target β1-integrin using this C-terminal motif. E6 expression reduced membrane localization of β1-integrin, but increased overall levels of β1-integrin protein and its downstream effector focal adhesion kinase in human keratinocytes. Altered β1-integrin localization due to E6 expression was associated with actin cytoskeleton rearrangement and increased cell migration that was abolished by point mutations in the C-terminal motif of E6. We concluded that modulation of β1-integrin signalling by E6 proteins may contribute towards the pathogenicity of these β-HPV types.

  18. The E7 protein of the cottontail rabbit papillomavirus immortalizes normal rabbit keratinocytes and reduces pRb levels, while E6 cooperates in immortalization but neither degrades p53 nor binds E6AP

    SciTech Connect

    Ganzenmueller, Tina; Matthaei, Markus; Muench, Peter; Scheible, Michael; Iftner, Angelika; Hiller, Thomas; Leiprecht, Natalie; Probst, Sonja; Stubenrauch, Frank; Iftner, Thomas

    2008-03-15

    Human papillomaviruses (HPVs) cause cervical cancer and are associated with the development of non-melanoma skin cancer. A suitable animal model for papillomavirus-associated skin carcinogenesis is the infection of domestic rabbits with the cottontail rabbit papillomavirus (CRPV). As the immortalizing activity of CRPV genes in the natural target cells remains unknown, we investigated the properties of CRPV E6 and E7 in rabbit keratinocytes (RK) and their influence on the cell cycle. Interestingly, CRPV E7 immortalized RK after a cellular crisis but showed no such activity in human keratinocytes. Co-expressed CRPV E6 prevented cellular crisis. The HPV16 or CRPV E7 protein reduced rabbit pRb levels thereby causing rabbit p19{sup ARF} induction and accumulation of p53 without affecting cellular proliferation. Both CRPV E6 proteins failed to degrade rabbit p53 in vitro or to bind E6AP; however, p53 was still inducible by mitomycin C. In summary, CRPV E7 immortalizes rabbit keratinocytes in a species-specific manner and E6 contributes to immortalization without directly affecting p53.

  19. Hepatitis C virus core protein inhibits E6AP expression via DNA methylation to escape from ubiquitin-dependent proteasomal degradation.

    PubMed

    Kwak, Juri; Shim, Joo Hee; Tiwari, Indira; Jang, Kyung Lib

    2016-09-28

    The E6-associated protein (E6AP) is a ubiquitin ligase that mediates ubiquitination and proteasomal degradation of hepatitis C virus (HCV) core protein. Given the role of HCV core protein as a major component of the viral nucleocapsid, as well as a multifunctional protein involved in viral pathogenesis and hepatocarcinogenesis, HCV has likely evolved a strategy to counteract the host anti-viral defense mechanism of E6AP and maximize its potential to produce infectious virus particles. In the present study, we found that HCV core protein derived from either ectopic expression or HCV infection inhibits E6AP expression via promoter hypermethylation in human hepatocytes. As a result, the potential of E6AP to ubiquitinate and degrade HCV core protein through the ubiquitin-proteasome system was severely impaired, which in turn led to stimulation of virus propagation. The effects of HCV core protein were almost completely abolished when the E6AP level was restored by ectopic expression of E6AP, treatment with a universal DNA methyltransferase (DNMT) inhibitor, 5-Aza-2'dC, or knock-down of DNMT1. In conclusion, HCV core protein inhibits E6AP expression via DNA methylation to protect itself from ubiquitin-dependent proteasomal degradation and stimulate virus propagation, providing a potential target for the development of anti-viral drugs against HCV. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  20. Human papillomavirus type 16 E2 and E6 are RNA-binding proteins and inhibit in vitro splicing of pre-mRNAs with suboptimal splice sites

    SciTech Connect

    Bodaghi, Sohrab; Jia Rong; Zheng Zhiming

    2009-03-30

    Human papillomavirus type 16 (HPV16) genome expresses six regulatory proteins (E1, E2, E4, E5, E6, and E7) which regulate viral DNA replication, gene expression, and cell function. We expressed HPV16 E2, E4, E6, and E7 from bacteria as GST fusion proteins and examined their possible functions in RNA splicing. Both HPV16 E2, a viral transactivator protein, and E6, a viral oncoprotein, inhibited splicing of pre-mRNAs containing an intron with suboptimal splice sites, whereas HPV5 E2 did not. The N-terminal half and the hinge region of HPV16 E2 as well as the N-terminal and central portions of HPV16 E6 are responsible for the suppression. HPV16 E2 interacts with pre-mRNAs through its C-terminal DNA-binding domain. HPV16 E6 binds pre-mRNAs via nuclear localization signal (NLS3) in its C-terminal half. Low-risk HPV6 E6, a cytoplasmic protein, does not bind RNA. Notably, both HPV16 E2 and E6 selectively bind to the intron region of pre-mRNAs and interact with a subset of cellular SR proteins. Together, these findings suggest that HPV16 E2 and E6 are RNA binding proteins and might play roles in posttranscriptional regulation during virus infection.

  1. Human papilloma virus early proteins E6 (HPV16/18-E6) and the cell cycle marker P16 (INK4a) are useful prognostic markers in uterine cervical carcinomas in Qassim Region--Saudi Arabia.

    PubMed

    Omran, O M; AlSheeha, M

    2015-01-01

    Cervical cancer is a common and an important public health problem for adult women in developing countries. In contrast, cervical cancer incidence is low in Saudi Arabia. High-risk types of human papilloma viruses (HPV16 and HPV18) are the most significant risk factors for cervical cancer. HPV16/18-E6 oncoprotein is associated with HPV etiology, viral persistence and epithelial transformation. Cell cycle protein p16 INK4a (p16) plays an important role in the pathophysiology of cervical carcinomas. The aims of this study were to investigate the expression of HPV16/18-E6 and p16 in uterine cervical carcinomas in Qassim Region--Saudi Arabia, and to relate the results to the established clinicopathological prognostic parameters (age of the patient, educational level, birth control methods, number of pregnancy, smoking status, degree of histological differentiation, clinical stage, and lymph node metastasis) The study included 40 specimens of uterine cervical squamous cell carcinomas diagnosed and confirmed by biopsy. Histopathological classification of cervical tumors cases was performed according to the International Federation of Gynecology and Obstetrics (FIGO). Immunohistochemical analysis for HPV16/18-E6 and p16 were carried out on formalin-fixed paraffin-embedded sections of cervical tissues using avidin-biotin peroxidase method. There was a significant statistical correlation between HPV16/18-E6 expression in cervical carcinoma and nationality, smoking status and size of the tumor. HPV16/18-E6 oncoprotein expression in normal lymphocytes and endothelial cells in the tumor tissues and the adjacent normal cervical tissues suggest the possibility that HPV infection might spread to other organs through blood circulation. P16 expression has been correlated with high grade, stage of cervical SCC and HPV16/18-E6 expression. The current study supports the critical function of p16 and HPV16/18-E6 as specific markers for cervical carcinoma. However the potential for usage

  2. The role of protein kinase A regulation of the E6 PDZ-binding domain during the differentiation-dependent life cycle of human papillomavirus type 18.

    PubMed

    Delury, Craig P; Marsh, Elizabeth K; James, Claire D; Boon, Siaw Shi; Banks, Lawrence; Knight, Gillian L; Roberts, Sally

    2013-09-01

    Human papillomavirus (HPV) E6 proteins of high-risk alpha types target a select group of PSD95/DLG1/ZO1 (PDZ) domain-containing proteins by using a C-terminal PDZ-binding motif (PBM), an interaction that can be negatively regulated by phosphorylation of the E6 PBM by protein kinase A (PKA). Here, we have mutated the canonical PKA recognition motif that partially overlaps with the E6 PBM in the HPV18 genome (E6153PKA) and compared the effect of this mutation on the HPVl8 life cycle in primary keratinocytes with the wild-type genome and with a second mutant genome that lacks the E6 PBM (E6ΔPDZ). Loss of PKA recognition of E6 was associated with increased growth of the genome-containing cells relative to cells carrying the wild-type genome, and upon stratification, a more hyperplastic phenotype, with an increase in the number of S-phase competent cells in the upper suprabasal layers, while the opposite was seen with the E6ΔPDZ genome. Moreover, the growth of wild-type genome-containing cells was sensitive to changes in PKA activity, and these changes were associated with increased phosphorylation of the E6 PBM. In marked contrast to E6ΔPDZ genomes, the E6153PKA mutation exhibited no deleterious effects on viral genome amplification or expression of late proteins. Our data suggest that the E6 PBM function is differentially regulated by phosphorylation in the HPV18 life cycle. We speculate that perturbation of protein kinase signaling pathways could lead to changes in E6 PBM function, which in turn could have a bearing on tumor promotion and progression.

  3. The Role of Protein Kinase A Regulation of the E6 PDZ-Binding Domain during the Differentiation-Dependent Life Cycle of Human Papillomavirus Type 18

    PubMed Central

    Delury, Craig P.; Marsh, Elizabeth K.; James, Claire D.; Boon, Siaw Shi; Banks, Lawrence; Knight, Gillian L.

    2013-01-01

    Human papillomavirus (HPV) E6 proteins of high-risk alpha types target a select group of PSD95/DLG1/ZO1 (PDZ) domain-containing proteins by using a C-terminal PDZ-binding motif (PBM), an interaction that can be negatively regulated by phosphorylation of the E6 PBM by protein kinase A (PKA). Here, we have mutated the canonical PKA recognition motif that partially overlaps with the E6 PBM in the HPV18 genome (E6153PKA) and compared the effect of this mutation on the HPVl8 life cycle in primary keratinocytes with the wild-type genome and with a second mutant genome that lacks the E6 PBM (E6ΔPDZ). Loss of PKA recognition of E6 was associated with increased growth of the genome-containing cells relative to cells carrying the wild-type genome, and upon stratification, a more hyperplastic phenotype, with an increase in the number of S-phase competent cells in the upper suprabasal layers, while the opposite was seen with the E6ΔPDZ genome. Moreover, the growth of wild-type genome-containing cells was sensitive to changes in PKA activity, and these changes were associated with increased phosphorylation of the E6 PBM. In marked contrast to E6ΔPDZ genomes, the E6153PKA mutation exhibited no deleterious effects on viral genome amplification or expression of late proteins. Our data suggest that the E6 PBM function is differentially regulated by phosphorylation in the HPV18 life cycle. We speculate that perturbation of protein kinase signaling pathways could lead to changes in E6 PBM function, which in turn could have a bearing on tumor promotion and progression. PMID:23804647

  4. Complexes of Human Papillomavirus Type 16 E6 Proteins Form Pseudo-Death-Inducing Signaling Complex Structures during Tumor Necrosis Factor-Mediated Apoptosis▿

    PubMed Central

    Filippova, Maria; Filippov, Valery A.; Kagoda, Mercy; Garnett, Theodore; Fodor, Nadya; Duerksen-Hughes, Penelope J.

    2009-01-01

    High-risk strains of human papillomavirus (HPV) such as HPV type 16 (HPV16) and HPV18 are causative agents of most human cervical carcinomas. E6, one of the oncogenes encoded by HPV16, possesses a number of biological and transforming functions. We have previously shown that the binding of E6 to host apoptotic proteins such as tumor necrosis factor (TNF) R1, the adaptor protein FADD, and procaspase 8 results in a significant modification of the normal flow of apoptotic events. For example, E6 can bind to and accelerate the degradation of FADD. In addition, full-length E6 binds to the TNF R1 death domain and can also bind to and accelerate the degradation of procaspase 8. In contrast, the binding of small splice isoforms known as E6* results in the stabilization of procaspase 8. In this report, we propose a model for the ability of HPV16 E6 to both sensitize and protect cells from TNF as well as to protect cells from Fas. We demonstrate that both the level of E6 expression and the ratio between full-length E6 and E6* are important factors in the modification of the host extrinsic apoptotic pathways and show that at high levels of E6 expression, the further sensitization of U2OS, NOK, and Ca Ski cells to TNF-mediated apoptosis is most likely due to the formation of a pseudo-death-inducing signaling complex structure that includes complexes of E6 proteins. PMID:18842714

  5. Pub1 acts as an E6-AP-like protein ubiquitiin ligase in the degradation of cdc25.

    PubMed Central

    Nefsky, B; Beach, D

    1996-01-01

    The level of the mitotic activating tyrosine phosphatase cdc25 is regulated by both transcriptional and post-transcriptional mechanisms in the fission yeast Schizosaccharomyces pombe. We have found that cdc25 is ubiquitinated and have cloned pub1, a gene which regulates this event. Pub1 contains a region highly homologous to the putative catalytic domain of the human protein ubiquitin ligase E6-AP. Disruption of pub1 elevates the level of cdc25 protein in vivo rendering cells relatively resistant to the cdc25-opposing tyrosine kinases wee1 and mik1. In addition, loss of wee1 activity in a pub1-disruption background results in a lethal premature entry into mitosis which can be rescued by loss of cdc25 function. A ubiquitin-thioester adduct of pub1 was isolated from fission yeast and disruption of pub1 dramatically reduced ubiquitination of cdc25 in vivo. These results suggest that pub1 directly ubiquitinates cdc25 in vivo. Images PMID:8635463

  6. High risk HPV E6 oncoproteins impair the subcellular distribution of the four and a half LIM-only protein 2 (FHL2).

    PubMed

    Manzo-Merino, Joaquin; Massimi, Paola; Banks, Lawrence; Lizano, Marcela

    2015-02-01

    HPVs are the causative agents of approximately 5% of all human cancers, with cervical cancer being the most predominant. To understand the mechanism of action of the viral E6 oncoprotein, we analysed the effects of E6 upon potential cellular target proteins. One candidate is FHL-2, involved in the regulation of signal transduction pathways from the multimeric complexes assembled at focal adhesions. We show that both HPV E6 and E6(⁎) can interact with FHL-2 in vitro, but unlike most E6 targets, FHL-2 does not appear to be an E6 degradation target. Analysis of the patterns of FHL-2 distribution within HPV-positive tumour-derived cells shows a significant alteration in the pattern of FHL-2 localisation when compared to non-HPV containing cells. This perturbation of FHL-2 distribution is proteasome-dependent and inhibition of E6 expression restores the normal distribution of FHL-2. These results confirm FHL-2 as a new interacting partner of the HPV-E6 oncoproteins. Copyright © 2014 Elsevier Inc. All rights reserved.

  7. Genetic stability of a recombinant adenovirus vaccine vector seed library expressing human papillomavirus type 16 E6 and E7 proteins.

    PubMed

    Wu, Jie; Chen, Ke-DA; Gao, Meng; Chen, Gang; Jin, Su-Feng; Zhuang, Fang-Cheng; Wu, Xiao-Hong; Jiang, Yun-Shui; Li, Jian-Bo

    2015-04-01

    The aim of the present study was to understand the genetic stability of a master seed bank (MSB) and a working seed bank (WSB) of an adenovirus vector vaccine expressing the human papillomavirus (HPV) type 16 E6 and E7 fusion proteins (Ad-HPV16E6E7). Microscopic examination and viral infectious efficacy were used to measure the infectious titers of the Ad-HPV16E6E7 MSB and WSB. Polymerase chain reaction was used to analyze the stability of the Ad-HPV16E6E7 target gene insertion, while western blot analysis and immunofluorescence were used to assess the expression levels of the Ad-HPV16E6E7 target protein. A C57BL/6 mouse TC-1 tumor cell growth inhibition model was used to evaluate the biological effect of Ad-HPV16E6E7 administration. The infectious titers of the Ad-HPV16E6E7 MSB and WSB were 6.31×10(9) IU/ml and 3.0×10(9) IU/ml, respectively. In addition, the expression levels of the inserted target genes and target proteins were found to be stable. In the mouse TC-1 tumor inhibition analysis, when the virus titers of the Ad-HPV16E6E7 MSB and WSB were 10(9) IU/ml, the tumor inhibition rate was 100%, which was significantly different when compared with the control group (χ(2)MSB=20.00 and χ(2)WSB=20.00; P<0.01). Therefore, the Ad-HPV16E6E7 vaccine seed bank is genetically stable and meets the requirements for vaccine development.

  8. Molecular screening of compounds to the predicted Protein-Protein Interaction site of Rb1-E7 with p53- E6 in HPV.

    PubMed

    Shaikh, Faraz; Sanehi, Parvish; Rawal, Rakesh

    2012-01-01

    Cervical cancer is malignant neoplasm of the cervix uteri or cervical area. Human Papillomaviruses (HPVs) which are heterogeneous groups of small double stranded DNA viruses are considered as the primary cause of cervical cancer, involved in 90% of all Cervical Cancers. Two early HPV genes, E6 and E7, are known to play crucial role in tumor formation. E6 binds with p53 and prevents its translocation and thereby inhibit the ability of p53 to activate or repress target genes. E7 binds to hypophosphorylated Rb and thereby induces cells to enter into premature S-phase by disrupting Rb-E2F complexes. The strategy of the research work was to target the site of interaction of Rb1 -E7 & p53-E6. A total of 88 compounds were selected for molecular screening, based on comprehensive literature survey for natural compounds with anti-cancer activity. Molecular docking analysis was carried out with Molegro Virtual Docker, to screen the 88 chosen compounds and rank them according to their binding affinity towards the site of interaction of the viral oncoproteins and human tumor suppressor proteins. The docking result revealed that Nicandrenone a member of Withanolides family of chemical compounds as the most likely molecule that can be used as a candidate drug against HPV induced cervical cancer. HPV - Human Papiloma Virus, HTSP - Human Tumor Suppressor Proteins, VOP - Viral oncoproteins.

  9. Molecular screening of compounds to the predicted Protein-Protein Interaction site of Rb1-E7 with p53- E6 in HPV

    PubMed Central

    Shaikh, Faraz; Sanehi, Parvish; Rawal, Rakesh

    2012-01-01

    Cervical cancer is malignant neoplasm of the cervix uteri or cervical area. Human Papillomaviruses (HPVs) which are heterogeneous groups of small double stranded DNA viruses are considered as the primary cause of cervical cancer, involved in 90% of all Cervical Cancers. Two early HPV genes, E6 and E7, are known to play crucial role in tumor formation. E6 binds with p53 and prevents its translocation and thereby inhibit the ability of p53 to activate or repress target genes. E7 binds to hypophosphorylated Rb and thereby induces cells to enter into premature S-phase by disrupting Rb-E2F complexes. The strategy of the research work was to target the site of interaction of Rb1 -E7 & p53-E6. A total of 88 compounds were selected for molecular screening, based on comprehensive literature survey for natural compounds with anti-cancer activity. Molecular docking analysis was carried out with Molegro Virtual Docker, to screen the 88 chosen compounds and rank them according to their binding affinity towards the site of interaction of the viral oncoproteins and human tumor suppressor proteins. The docking result revealed that Nicandrenone a member of Withanolides family of chemical compounds as the most likely molecule that can be used as a candidate drug against HPV induced cervical cancer. Abbreviations HPV - Human Papiloma Virus, HTSP - Human Tumor Suppressor Proteins, VOP - Viral oncoproteins. PMID:22829740

  10. The human papillomavirus 16 E6 protein binds to tumor necrosis factor (TNF) R1 and protects cells from TNF-induced apoptosis.

    PubMed

    Filippova, Maria; Song, Helen; Connolly, Jodi L; Dermody, Terence S; Duerksen-Hughes, Penelope J

    2002-06-14

    High risk strains of human papillomavirus (HPV), such as HPV 16, cause human cervical carcinoma. The E6 protein of HPV 16 mediates the rapid degradation of p53, although this is not the only function of E6 and cannot completely explain its transforming potential. Previous work in our laboratory has demonstrated that transfection of HPV 16 E6 into the tumor necrosis factor (TNF)-sensitive LM cell line protects expressing cells from TNF-induced apoptosis in a p53-independent manner, and the purpose of this study was to determine the molecular mechanism underlying this protection. Caspase 3 and caspase 8 activation were significantly reduced in E6-expressing cells, indicating that E6 acts early in the TNF apoptotic pathway. In fact, E6 binds directly to TNF R1, as shown both by co-immunoprecipitation and mammalian two-hybrid approaches. E6 requires the same C-terminal portion of TNF R1 for binding as does TNF R1-associated death domain, and TNF R1/TNF R1-associated death domain interactions are decreased in the presence of E6. HA-E6 also blocked cell death triggered by transfection of the death domain of TNF R1. Together, these results provide strong support for a model in which HPV E6 binding to TNF R1 interferes with formation of the death-inducing signaling complex and thus with transduction of proapoptotic signals. They also demonstrate that HPV, like several other viruses, has developed a method for evading the TNF-mediated host immune response.

  11. Expression of p53, MDM2, p21, heat shock protein 70, and HPV 16/18 E6 proteins in oral verrucous carcinoma and oral verrucous hyperplasia.

    PubMed

    Lin, Hung-Pin; Wang, Yi-Ping; Chiang, Chun-Pin

    2011-03-01

    Oral verrucous hyperplasia is a precancerous lesion of oral verrucous carcinoma. This study used immunohistochemistry to examine the expression of p53, murine double minute 2 (MDM2), p21, heat shock protein 70 (HSP 70), and human papillomavirus (HPV) 16/18 E6 proteins in 48 oral verrucous carcinoma and 30 oral verrucous hyperplasia samples. The mean labeling indices of p53, MDM2, p21, HSP 70, and HPV 16/18 E6 proteins in oral verrucous carcinoma samples were 21%, 31%, 7%, 17%, and 0.5%, respectively, and those in oral verrucous hyperplasia samples were 19%, 35%, 11%, 14%, and 0.3%, respectively. Immunohistochemistry with the above-cited 5 biomarkers could not help differentiate oral verrucous hyperplasia from oral verrucous carcinoma. The low expression of p21 may partially explain abnormal epithelial overgrowth in both verrucous lesions. The pathogenesis of both verrucous lesions may be at least partially attributed to the overexpression of MDM2 protein and moderate expression of HSP 70 protein in both lesions. Copyright © 2010 Wiley Periodicals, Inc.

  12. Human papillomavirus oncogenic E6 protein regulates human β-defensin 3 (hBD3) expression via the tumor suppressor protein p53

    PubMed Central

    Yue, Hong; Wang, Liming; Jin, Jessica; Ghosh, Santosh K.; Kawsar, Hameem I.; Zender, Chad; Androphy, Elliot J.; Weinberg, Aaron; McCormick, Thomas S.; Jin, Ge

    2016-01-01

    Human β-defensin-3 (hBD3) is an epithelial cell-derived innate immune regulatory molecule overexpressed in oral dysplastic lesions and fosters a tumor-promoting microenvironment. Expression of hBD3 is induced by the epidermal growth factor receptor signaling pathway. Here we describe a novel pathway through which the high-risk human papillomavirus type-16 (HPV-16) oncoprotein E6 induces hBD3 expression in mucosal keratinocytes. Ablation of E6 by siRNA induces the tumor suppressor p53 and diminishes hBD3 in HPV-16 positive CaSki cervical cancer cells and UM-SCC-104 head and neck cancer cells. Malignant cells in HPV-16-associated oropharyngeal cancer overexpress hBD3. HPV-16 E6 induces hBD3 mRNA expression, peptide production and gene promoter activity in mucosal keratinocytes. Reduction of cellular levels of p53 stimulates hBD3 expression, while activation of p53 by doxorubicin inhibits its expression in primary oral keratinocytes and CaSki cells, suggesting that p53 represses hBD3 expression. A p53 binding site in the hBD3 gene promoter has been identified by using electrophoretic mobility shift assays and chromatin immunoprecipitation (ChIP). In addition, the p63 protein isoform ΔNp63α, but not TAp63, stimulated transactivation of the hBD3 gene and was co-expressed with hBD3 in head and neck cancer specimens. Therefore, high-risk HPV E6 oncoproteins may stimulate hBD3 expression in tumor cells to facilitate tumorigenesis of HPV-associated head and neck cancer. PMID:27034006

  13. Expression of E6, p53 and p21 proteins and physical state of HPV16 in cervical cytologies with and without low grade lesions

    PubMed Central

    Tagle, Diana K Jiménez; Sotelo, Daniel Hernández; Illades-Aguiar, Berenice; Leyva-Vazquez, Marco A; Alfaro, Eugenia Flores; Coronel, Yaneth Castro; Hernández, Oscar del Moral; Romero, Luz del Carmen Alarcón

    2014-01-01

    The aim of this study was to determine the correlation between expression of HPV16 E6, p53 and p21 proteins and the physical state of HPV16 in cervical cytologies without squamous intraepithelial lesions (Non-SIL) and with low grade squamous intraepithelial lesions (LSIL), both with HPV16 infection. 101 liquid-based cytological samples were analyzed. 50 samples were without squamous intraepithelial lesions (Non-IL) and 51 samples of low grade squamous intraepithelial lesions (LSIL), both with HPV16 infection. HPV16 infection was determined by PCR-RFLP, and the physical state of HPV16 by in situ hybridization with tyramide-amplification. The expression of E6, p53 and p21 proteins was evaluated by immunocytochemistry. The expression of HPV16 E6 protein was significantly higher in LSIL that in Non-SIL samples (p=0.006). We found a significant correlation between E6 expression and the physical state of HPV16 in Non-SIL (p=0.049). Our results suggest that high expression of E6 in LSIL is an early event of cervical carcinogenesis and perhaps can be used as an early marker. PMID:24482706

  14. Expression of E6, p53 and p21 proteins and physical state of HPV16 in cervical cytologies with and without low grade lesions.

    PubMed

    Tagle, Diana K Jiménez; Sotelo, Daniel Hernández; Illades-Aguiar, Berenice; Leyva-Vazquez, Marco A; Alfaro, Eugenia Flores; Coronel, Yaneth Castro; Hernández, Oscar Del Moral; Romero, Luz Del Carmen Alarcón

    2014-01-01

    The aim of this study was to determine the correlation between expression of HPV16 E6, p53 and p21 proteins and the physical state of HPV16 in cervical cytologies without squamous intraepithelial lesions (Non-SIL) and with low grade squamous intraepithelial lesions (LSIL), both with HPV16 infection. 101 liquid-based cytological samples were analyzed. 50 samples were without squamous intraepithelial lesions (Non-IL) and 51 samples of low grade squamous intraepithelial lesions (LSIL), both with HPV16 infection. HPV16 infection was determined by PCR-RFLP, and the physical state of HPV16 by in situ hybridization with tyramide-amplification. The expression of E6, p53 and p21 proteins was evaluated by immunocytochemistry. The expression of HPV16 E6 protein was significantly higher in LSIL that in Non-SIL samples (p=0.006). We found a significant correlation between E6 expression and the physical state of HPV16 in Non-SIL (p=0.049). Our results suggest that high expression of E6 in LSIL is an early event of cervical carcinogenesis and perhaps can be used as an early marker.

  15. Analysis of protein expression changes of the Vero E6 cells infected with classic PEDV strain CV777 by using quantitative proteomic technique.

    PubMed

    Sun, Dongbo; Shi, Hongyan; Guo, Donghua; Chen, Jianfei; Shi, Da; Zhu, Qinghe; Zhang, Xin; Feng, Li

    2015-06-15

    Recent outbreaks of porcine epidemic diarrhea virus (PEDV) have caused widespread concern. The identification of proteins associated with PEDV infection might provide insight into PEDV pathogenesis and facilitate the development of novel antiviral strategies. We analyzed the differential protein profile of PEDV-infected Vero E6 cells using mass spectrometry and an isobaric tag for relative and absolute quantification. A total of 126 proteins were identified that were differentially expressed between the PEDV-infected and mock-infected groups (P<0.05, quantitative ratio ≥1.2), among which the expression of 58 proteins was up-regulated and that of 68 proteins was down-regulated in the PEDV-infected Vero E6 cells, involving in integrin β2/β3, cystatin-C. The Gene Ontology analysis indicated that the molecular function of the differentially expressed proteins (DEPs) was primarily related to binding and catalytic activity, and that the biological functions in which the DEPs are involved included metabolism, organismal systems, cellular processes, genetic information processing, environmental information processing, and diseases. Among the disease-related functions, certain anti-viral pathways and proteins, such as the RIG-I-like receptor, Rap1, autophagy, mitogen-activated protein kinase, PI3K-Akt and Jak-STAT signaling pathways, and integrin β2/β3 and cystatin-C proteins, represented potential factors in PEDV infection. Our findings provide valuable insight into PEDV-Vero E6 cell interactions.

  16. The Asian-American E6 variant protein of human papillomavirus 16 alone is sufficient to promote immortalization, transformation, and migration of primary human foreskin keratinocytes.

    PubMed

    Niccoli, Sarah; Abraham, Suraj; Richard, Christina; Zehbe, Ingeborg

    2012-11-01

    We examined how well the human papillomavirus (HPV) E6 oncogene can function in the absence of the E7 oncogene during the carcinogenic process in human keratinocytes using a common HPV variant strongly associated with cervical cancer: the Asian-American E6 variant (AAE6). This E6 variant is 20 times more frequently detected in cervical cancer than the prototype European E6 variant, as evidenced by independent epidemiological data. Using cell culture and cell-based functional assays, we assessed how this variant can perform crucial carcinogenesis steps compared to the prototype E6 variant. The ability to immortalize and transform primary human foreskin keratinocytes (PHFKs) to acquire resilient phenotypes and the ability to promote cell migration were evaluated. The immortalization capability was assayed based on population doublings, number of passages, surpassing mortality stages 1 and 2, human telomerase reverse transcriptase (hTERT) expression, and the ability to overcome G(1) arrest via p53 degradation. Transformation and migration efficiency were analyzed using a combination of functional cell-based assays. We observed that either AAE6 or prototype E6 proteins alone were sufficient to immortalize PHFKs, although AAE6 was more potent in doing so. The AAE6 variant protein alone pushed PHFKs through transformation and significantly increased their migration ability over that of the E6 prototype. Our findings are in line with epidemiological data that the AA variant of HPV16 confers an increased risk over the European prototype for cervical cancer, as evidenced by a superior immortalization, transformation, and metastatic potential.

  17. HPV-E6 protein enriches the CD55(+) population in cervical cancer cells promoting radio-resistance 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

    2017-09-25

    Accumulating evidence indicates that the human papilloma virus (HPV) E6 protein plays a crucial role in the development of cervical cancer. Sub-populations of cells that reside within tumors are responsible for tumor 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 exhibited self-renewal ability, from primary cervical tumors. Gene expression profiling revealed that CD55 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, while only few CD55(+) cells were found in HPV-negative cervical cancer and normal cervical epithelial cell lines. The isolated CD55(+) sub-population from the C33A cell line exhibited significant sphere-forming ability and enhanced tumorigenicity, cell migration and radio-resistance. In contrast, the suppression of CD55 in HPV-positive CaSki cells inhibited tumorigenicity both in vitro and in vivo and sensitized cells to irradiation treatment. In addition, ectopic expression of HPV-E6 in HPV-negative cervical cancer cells dramatically enriched the CD55(+) sub-population. CRISPR/Cas9 knockout of the CD55 gene in an HPV-E6-overexpressing stable clone abolished the tumorigenic properties exerted by HPV-E6. Taken together, our data suggest that HPV-E6 protein expression enriches the CD55(+) population, which contributes to tumorigenicity and radio-resistance 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. This article is protected by copyright. All rights reserved.

  18. Cutaneous HPV8 and MmuPV1 E6 Proteins Target the NOTCH and TGF-β Tumor Suppressors to Inhibit Differentiation and Sustain Keratinocyte Proliferation

    PubMed Central

    Meyers, Jordan M.; Grace, Miranda; Munger, Karl

    2017-01-01

    Cutaneous beta-papillomaviruses are associated with non-melanoma skin cancers that arise in patients who suffer from a rare genetic disorder, Epidermodysplasia verruciformis (EV) or after immunosuppression following organ transplantation. Recent studies have shown that the E6 proteins of the cancer associated beta human papillomavirus (HPV) 5 and HPV8 inhibit NOTCH and TGF-β signaling. However, it is unclear whether disruption of these pathways may contribute to cutaneous HPV pathogenesis and carcinogenesis. A recently identified papillomavirus, MmuPV1, infects laboratory mouse strains and causes cutaneous skin warts that can progress to squamous cell carcinoma. To determine whether MmuPV1 may be an appropriate model to mechanistically dissect the molecular contributions of cutaneous HPV infections to skin carcinogenesis, we investigated whether MmuPV1 E6 shares biological and biochemical activities with HPV8 E6. We report that the HPV8 and MmuPV1 E6 proteins share the ability to bind to the MAML1 and SMAD2/SMAD3 transcriptional cofactors of NOTCH and TGF-beta signaling, respectively. Moreover, we demonstrate that these cutaneous papillomavirus E6 proteins inhibit these two tumor suppressor pathways and that this ability is linked to delayed differentiation and sustained proliferation of differentiating keratinocytes. Furthermore, we demonstrate that the ability of MmuPV1 E6 to bind MAML1 is necessary for papilloma formation in experimentally infected mice. Our results, therefore, suggest that experimental MmuPV1 infection in mice will be a robust and useful experimental system to model key aspects of cutaneous HPV infection, pathogenesis and carcinogenesis. PMID:28107544

  19. Vaccination of full-length HPV16 E6 or E7 protein inhibits the growth of HPV16 associated tumors.

    PubMed

    Li, Yan-Li; Qiu, Xu-Hua; Shen, Chen; Liu, Jian-Ning; Zhang, Jing

    2010-11-01

    Cervical cancer is the second most common cancer in women worldwide. Human papillomavirus (HPV) is the primary etiologic agent of cervical cancer. Two HPV16 proteins, E6 and E7, are consistently expressed in tumor cells. Most therapeutic vaccines target one or both of these proteins. Taking the advantages of safety and no human leukocyte antigen restriction, protein vaccine has become the most popular form of HPV therapeutic vaccines. Here we demonstrate that immunization with full-length HPV16 E6 or E7 protein elicited specific immunological effect and inhibition of TC-1 cell growth using TC-1 mouse model. HPV16 E6 and E7 genes were cloned into pET-28a(+) and introduced into E. coli Rosetta. Expression of the genes was induced by IPTG. Proteins were purified by Ni-NTA agarose and they were detected by SDS-PAGE and Western blotting. C57BL/6 mice were vaccinated with 1.5 nmol HPV16 E6 or E7 protein. Then they were implanted with 1x10(5) TC-1 cells. No tumor was detected in any mouse vaccinated with E7 protein. Forty days later, the tumor-free mice and control mice were challenged with 2x10(5) TC-1 cells. All control mice developed tumors 6 days later, but E7 immunized mice were tumor free until 90 days. Tumor growth was slow in the E6 immunized mice, but 83% of the mice developed tumors and the survival percentage was not significantly different from the control. An adoptive immune model was used to demonstrate the therapeutic effect. Results showed that the development of TC-1 cells was obviously reduced by transfusion of T-cells but not serum from mice immunized with E7 protein. T-cells from E7 immunized mice also induced the lysis of TC-1 cells in the cytotoxic T lymphocyte assay. These findings show that immunization with HPV16 E6 or E7 protein was able to elicit specific protective immunity against TC-1 tumor growth.

  20. The E6/E7 promoter of human papillomavirus type 16 is activated in the absence of E2 proteins by a sequence-aberrant Sp1 distal element.

    PubMed Central

    Gloss, B; Bernard, H U

    1990-01-01

    The E6/E7 promoter of all genital human papillomaviruses is responsible for expression of the viral transforming genes. Centered 60 bp upstream of the transcription start, it contains a 20-bp segment with partially overlapping binding sites for the viral E2 proteins and for a cellular factor that was identified by footprint experiments. Bandshifts, bandshift competitions, and footprints revealed that protein complexes between nuclear extracts and these sequences have binding properties indistinguishable from those of the Sp1 factor that binds the simian virus 40 early promoter GC motif. Reactions of these complexes with anti-Sp1 antiserum were analyzed by superbandshifts and precipitation with protein A, and the results confirmed the identity of this transcription factor as Sp1. Sp1 binds in simian virus 40 and different human papillomavirus promoters the consensus sequence 5'-NGGNGN-3'. RNase protection analysis of in vitro or in vivo transcriptions with wild-type and mutant test vectors shows that the E6/E7 promoter of human papillomavirus type 16 is functionally dependent on the Sp1 distal promoter element. In all genital papillomaviruses, the Sp1 hexamer is invariably spaced by a single nucleotide from the distal E2 element, suggesting some precise interaction between Sp1 and E2 proteins. Published experimental evidence documents negative regulation of the E6/E7 promoter by E2 proteins through the proximal E2 element, whereas only minor quantitative differences in E6/E7 promoter function after cotransfection with E2 expression vectors were observed in this study. A detailed study of the interactions of Sp1 and E2 proteins with one another and with the corresponding three binding sites may reveal a complex modulation of this promoter. Images PMID:2170687

  1. Identification of host transcriptional networks showing concentration-dependent regulation by HPV16 E6 and E7 proteins in basal cervical squamous epithelial cells

    PubMed Central

    Smith, Stephen P.; Scarpini, Cinzia G.; Groves, Ian J.; Odle, Richard I.; Coleman, Nicholas

    2016-01-01

    Development of cervical squamous cell carcinoma requires increased expression of the major high-risk human-papillomavirus (HPV) oncogenes E6 and E7 in basal cervical epithelial cells. We used a systems biology approach to identify host transcriptional networks in such cells and study the concentration-dependent changes produced by HPV16-E6 and -E7 oncoproteins. We investigated sample sets derived from the W12 model of cervical neoplastic progression, for which high quality phenotype/genotype data were available. We defined a gene co-expression matrix containing a small number of highly-connected hub nodes that controlled large numbers of downstream genes (regulons), indicating the scale-free nature of host gene co-expression in W12. We identified a small number of ‘master regulators’ for which downstream effector genes were significantly associated with protein levels of HPV16 E6 (n = 7) or HPV16 E7 (n = 5). We validated our data by depleting E6/E7 in relevant cells and by functional analysis of selected genes in vitro. We conclude that the network of transcriptional interactions in HPV16-infected basal-type cervical epithelium is regulated in a concentration-dependent manner by E6/E7, via a limited number of central master-regulators. These effects are likely to be significant in cervical carcinogenesis, where there is competitive selection of cells with elevated expression of virus oncoproteins. PMID:27457222

  2. Identification of host transcriptional networks showing concentration-dependent regulation by HPV16 E6 and E7 proteins in basal cervical squamous epithelial cells.

    PubMed

    Smith, Stephen P; Scarpini, Cinzia G; Groves, Ian J; Odle, Richard I; Coleman, Nicholas

    2016-07-26

    Development of cervical squamous cell carcinoma requires increased expression of the major high-risk human-papillomavirus (HPV) oncogenes E6 and E7 in basal cervical epithelial cells. We used a systems biology approach to identify host transcriptional networks in such cells and study the concentration-dependent changes produced by HPV16-E6 and -E7 oncoproteins. We investigated sample sets derived from the W12 model of cervical neoplastic progression, for which high quality phenotype/genotype data were available. We defined a gene co-expression matrix containing a small number of highly-connected hub nodes that controlled large numbers of downstream genes (regulons), indicating the scale-free nature of host gene co-expression in W12. We identified a small number of 'master regulators' for which downstream effector genes were significantly associated with protein levels of HPV16 E6 (n = 7) or HPV16 E7 (n = 5). We validated our data by depleting E6/E7 in relevant cells and by functional analysis of selected genes in vitro. We conclude that the network of transcriptional interactions in HPV16-infected basal-type cervical epithelium is regulated in a concentration-dependent manner by E6/E7, via a limited number of central master-regulators. These effects are likely to be significant in cervical carcinogenesis, where there is competitive selection of cells with elevated expression of virus oncoproteins.

  3. Humoral immune response against proteins E6 and E7 in cervical carcinoma patients positive for human papilloma virus type 16 during treatment and follow-up.

    PubMed

    Baay, M F; Duk, J M; Burger, M P; de Bruijn, H W; Stolz, E; Herbrink, P

    1999-02-01

    To investigate the humoral immune response to transforming proteins E6 and E7 of human papillomavirus type 16 before and after treatment and during follow-up, consecutive serum samples from 36 cervical cancer patients whose tumours were found to contain human papillomavirus type 16 DNA by use of the polymerase chain reaction were tested using in vitro translated proteins E6 and E7 in a radioimmunoprecipitation assay and in an E7 synthetic peptide enzyme immunoassay. Antibody levels against E6 and E7 as measured by radioimmunoprecipitation assay showed a nearly identical pattern. Seronegative patients remained seronegative throughout treatment and follow-up. Seropositive patients showed either a decrease in antibody level or stable antibody levels during treatment. In contrast to patients without evidence of disease at the end of the study, the majority of patients with recurrent disease showed increasing antibody levels during the follow-up period. These results indicate that, in patients who are seropositive before treatment antibody levels against E6 and E7 of human papillomavirus type 16 after treatment are closely linked to treatment response. The use of the more sensitive radioimmunoprecipitation assay did not lead to a better correlation of antibody levels with clinical disease status of the patients than the use of the enzyme immunoassay.

  4. ERK Signaling Pathway Is Involved in HPV-16 E6 but not E7 Oncoprotein-Induced HIF-1α Protein Accumulation in NSCLC Cells.

    PubMed

    Liu, Fei; Lin, Bihua; Liu, Xin; Zhang, Wenzhang; Zhang, Erying; Hu, Liang; Ma, Yuefan; Li, Xiangyong; Tang, Xudong

    2016-01-01

    Extracellular signal-regulated kinase (ERK)1/2 signaling pathway plays a critical role in regulating tumor angiogenesis. Our previous studies have demonstrated that HPV-16 oncoproteins enhanced hypoxia-inducible factor-1α (HIF-1α) protein accumulation and vascular endothelial growth factor (VEGF) and interleukin-8 (IL-8) expression in non-small cell lung cancer (NSCLC) cells, thus contributing to angiogenesis. In this study, we further investigated the role of ERK1/2 signaling pathway in HPV-16 oncoprotein-induced HIF-1α, VEGF, and IL-8 expression and in vitro angiogenesis in NSCLC cells. Our results showed that HPV-16 E6 and HPV-16 E7 oncoproteins promoted the activation of ERK1/2 signaling pathway in A549 and NCI-H460 cells. Moreover, PD98059, a specific inhibitor of ERK1/2, blocked in vitro angiogenesis stimulated by HPV-16 E6 but not E7 oncoprotein. Additionally, HIF-1α protein accumulation and VEGF and IL-8 expression in NSCLC cells induced by HPV-16 E6 but not E7 oncoprotein were significantly inhibited by PD98059. Taken together, our results suggest that ERK1/2 signaling pathway is involved in HPV-16 E6 but not E7 oncoprotein-induced HIF-1α, VEGF, and IL-8 expression in NSCLC cells, leading to the enhanced angiogenesis in vitro.

  5. Sensitivity and specificity of antibodies against HPV16 E6 and other early proteins for the detection of HPV16-driven oropharyngeal squamous cell carcinoma.

    PubMed

    Holzinger, Dana; Wichmann, Gunnar; Baboci, Lorena; Michel, Angelika; Höfler, Daniela; Wiesenfarth, Manuel; Schroeder, Lea; Boscolo-Rizzo, Paolo; Herold-Mende, Christel; Dyckhoff, Gerhard; Boehm, Andreas; Del Mistro, Annarosa; Bosch, Franz X; Dietz, Andreas; Pawlita, Michael; Waterboer, Tim

    2017-06-15

    To determine the sensitivity and specificity of HPV16 serology as diagnostic marker for HPV16-driven oropharyngeal squamous cell carcinoma (OPSCC), 214 HNSCC patients from Germany and Italy with fresh-frozen tumor tissues and sera collected before treatment were included in this study. Hundred and twenty cancer cases were from the oropharynx and 94 were from head and neck cancer regions outside the oropharynx (45 oral cavity, 12 hypopharynx and 35 larynx). Serum antibodies to early (E1, E2, E6 and E7) and late (L1) HPV16 proteins were analyzed by multiplex serology and were compared to tumor HPV RNA status as the gold standard. A tumor was defined as HPV-driven in the presence of HPV16 DNA and HPV16 transformation-specific RNA transcript patterns (E6*I, E1(∧) E4 and E1C). Of 120 OPSCC, 66 (55%) were HPV16-driven. HPV16 E6 seropositivity was the best predictor of HPV16-driven OPSCC (diagnostic accuracy 97% [95%CI 92-99%], Cohen's kappa 0.93 [95%CI 0.8-1.0]). Of the 66 HPV-driven OPSCC, 63 were HPV16 E6 seropositive, compared to only one (1.8%) among the 54 non-HPV-driven OPSCC, resulting in a sensitivity of 96% (95%CI 88-98) and a specificity of 98% (95%CI 90-100). Of 94 HNSCC outside the oropharynx, six (6%) were HPV16-driven. In these patients, HPV16 E6 seropositivity had lower sensitivity (50%, 95%CI 19-81), but was highly specific (100%, 95%CI 96-100). In conclusion, HPV16 E6 seropositivity appears to be a highly reliable diagnostic marker for HPV16-driven OPSCC with very high sensitivity and specificity, but might be less sensitive for HPV16-driven HNSCC outside the oropharynx. © 2017 UICC.

  6. 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.

  7. 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 Central

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

    2010-01-01

    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. PMID:20053746

  8. 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

  9. Targeting the Human Papillomavirus E6 and E7 Oncogenes through Expression of the Bovine Papillomavirus Type 1 E2 Protein Stimulates Cellular Motility▿†

    PubMed Central

    Morrison, Monique A.; Morreale, Richard J.; Akunuru, Shailaja; Kofron, Matthew; Zheng, Yi; Wells, Susanne I.

    2011-01-01

    Expression of the high-risk human papillomavirus (HPV) E6 and E7 oncogenes is essential for the initiation and maintenance of cervical cancer. The repression of both was previously shown to result in activation of their respective tumor suppressor targets, p53 and pRb, and subsequent senescence induction in cervical cancer cells. Consequently, viral oncogene suppression is a promising approach for the treatment of HPV-positive tumors. One well-established method of E6/E7 repression involves the reexpression of the viral E2 protein which is usually deleted in HPV-positive cancer cells. Here, we show that, surprisingly, bovine papillomavirus type 1 (BPV1) E2 but not RNA interference-mediated E6/E7 repression in HPV-positive cervical cancer cells stimulates cellular motility and invasion. Migration correlated with the dynamic formation of cellular protrusions and was dependent upon cell-to-cell contact. While E2-expressing migratory cells were senescent, migration was not a general feature of cellular senescence or cell cycle arrest and was specifically observed in HPV-positive cervical cancer cells. Interestingly, E2-expressing cells not only were themselves motile but also conferred increased motility to admixed HeLa cervical cancer cells. Together, our data suggest that repression of the viral oncogenes by E2 stimulates the motility of E6/E7-targeted cells as well as adjacent nontargeted cancer cells, thus raising the possibility that E2 expression may unfavorably increase the local invasiveness of HPV-positive tumors. PMID:21835799

  10. Berberine alters epigenetic modifications, disrupts microtubule network, and modulates HPV-18 E6-E7 oncoproteins by targeting p53 in cervical cancer cell HeLa: a mechanistic study including molecular docking.

    PubMed

    Saha, Santu Kumar; Khuda-Bukhsh, Anisur Rahman

    2014-12-05

    Increased evidence of chemo-resistance, toxicity and carcinogenicity necessitates search for alternative approaches for determining next generation cancer therapeutics and targets. We therefore tested the efficacy of plant alkaloid berberine on human papilloma virus (HPV) -18 positive cervical cancer cell HeLa systematically-involving certain cellular, viral and epigenetic factors. We observed disruptions of microtubule network and changes in membrane topology due to berberine influx through confocal and atomic force microscopies (AFM). We examined nuclear uptake, internucleosomal DNA damages, mitochondrial membrane potential (MMP) alterations and cell migration assays to validate possible mode of cell death events. Analytical data on interactions of berberine with pBR322 through fourier transform infrared (FTIR) and gel migration assay strengthen berberine׳s biologically significant DNA binding abilities. We measured cellular uptake, DNA ploidy and DNA strand-breaks through fluorescence activated cell sorting (FACS). To elucidate epigenetic modifications, in support of DNA binding associated processes, if any, we conducted methylation-specific restriction enzyme (RE) assay, methylation specific-PCR (MSP) and expression studies of histone proteins. We also analyzed differential interactions and localization of cellular tumor suppressor p53 and viral oncoproteins HPV-18 E6-E7 through siRNA approach. We further made in-silico approaches to determine possible binding sites of berberine on histone proteins. Overall results indicated cellular uptake of berberine through cell membrane depolarization causing disruption of microtubule networks and its biological DNA binding abilities that probably contributed to epigenetic modifications. Results of modulation in p53 and viral oncoproteins HPV-18 E6-E7 by berberine further proved its potential as a promising chemotherapeutic agent in cervical cancer.

  11. Designed Spiroketal Protein Modulation

    PubMed Central

    Scheepstra, Marcel; Andrei, Sebastian A.; Unver, M. Yagiz; Hirsch, Anna K. H.; Leysen, Seppe; Ottmann, Christian

    2017-01-01

    Abstract Spiroketals are structural motifs found in many biologically active natural products, which has stimulated considerable efforts toward their synthesis and interest in their use as drug lead compounds. Despite this, the use of spiroketals, and especially bisbenzanulated spiroketals, in a structure‐based drug discovery setting has not been convincingly demonstrated. Herein, we report the rational design of a bisbenzannulated spiroketal that potently binds to the retinoid X receptor (RXR) thereby inducing partial co‐activator recruitment. We solved the crystal structure of the spiroketal–hRXRα–TIF2 ternary complex, and identified a canonical allosteric mechanism as a possible explanation for the partial agonist behavior of our spiroketal. Our co‐crystal structure, the first of a designed spiroketal–protein complex, suggests that spiroketals can be designed to selectively target other nuclear receptor subtypes. PMID:28407400

  12. Designed Spiroketal Protein Modulation.

    PubMed

    Scheepstra, Marcel; Andrei, Sebastian A; Unver, M Yagiz; Hirsch, Anna K H; Leysen, Seppe; Ottmann, Christian; Brunsveld, Luc; Milroy, Lech-Gustav

    2017-05-08

    Spiroketals are structural motifs found in many biologically active natural products, which has stimulated considerable efforts toward their synthesis and interest in their use as drug lead compounds. Despite this, the use of spiroketals, and especially bisbenzanulated spiroketals, in a structure-based drug discovery setting has not been convincingly demonstrated. Herein, we report the rational design of a bisbenzannulated spiroketal that potently binds to the retinoid X receptor (RXR) thereby inducing partial co-activator recruitment. We solved the crystal structure of the spiroketal-hRXRα-TIF2 ternary complex, and identified a canonical allosteric mechanism as a possible explanation for the partial agonist behavior of our spiroketal. Our co-crystal structure, the first of a designed spiroketal-protein complex, suggests that spiroketals can be designed to selectively target other nuclear receptor subtypes. © 2017 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.

  13. Protein modules conserved since LUCA.

    PubMed

    Sobolevsky, Yehoshua; Trifonov, Edward N

    2006-11-01

    Universal scale of the sequence conservation has been recently introduced based on omnipresence of the protein sequence motifs across species. A large spectrum of short sequences, up to eight residues has been found to reside in all or almost all prokaryotic organisms. By this discovery a principally novel quantitative approach is introduced to the problem of reconstruction of the last universal common ancestor (LUCA). The most conserved elements (protein modules) with defined structures and sequences harboring the omnipresent motifs are outlined in this work, by combining the sequence and protein crystal structure data. The structurally conserved modules involve 25-30 amino acid residues and have appearance of closed loops, loop-n-lock structures. This confirms earlier conclusions on the loop-fold structure of globular proteins. Many of the topmost conserved modules represent the primary closed loop prototypes, that have been derived by whole genome sequence searches. The data presented, thus, make a basis for further developments toward the earliest stages of protein evolution.

  14. E6 Gamma Decay

    SciTech Connect

    Brown, B. Alex; Rae, W. D. M.

    2011-05-06

    Rare electric hexacontatetrapole (E6) transitions are studied in the full (f{sub 7/2},f{sub 5/2},p{sub 3/2},p{sub 1/2}) shell-model basis. Comparison of theory to the results from the gamma decay in {sup 53}Fe and from inelastic electron scattering on {sup 52}Cr provides unique and interesting tests of the valence wavefunctions, the models used for energy density functionals and into the origin of effective charge.

  15. Proliferation of cells and expression of RARs, RXRs and HPV viral E6 and E7 proteins in cervical cancer cell lines after treatment with ATRA.

    PubMed

    Myga-Nowak, Magdalena; Pacholska-Bogalska, Joanna; Kwaśniewski, Wojciech; Kwaśniewska, Anna; Goździcka-Józefiak, Anna

    2011-01-01

    All-trans retinoic acid (ATRA) is considered to be a potential chemotherapeutic drug due to its capability to regulate cell growth and differentiation. The effects of ATRA on the proliferation of cells and gene regulation are mediated by retinoid receptors (RAR and RXR), which belong to the nuclear receptor superfamily of ligand- inducible transcription factors. ATRA can act either as a growth inhibitor or growth promoter, according to the functional state of retinoic receptors. Thus, we have established the effect of ATRA on the proliferation of cervical cancer cells line HeLa and CaSki and expression of retinoids receptors as well as the viral HPV oncogenic proteins E6 and E7. ATRA had no effect on proliferation CaSki cells, but it stimulated the growth of HeLa cells, which depended on the incubation time and the concentration of ATRA in cell culture. The overexpression of RAR alpha in HeLa cells after the administration of 10(-7) mM ATRA was also observed 72 hours, and the decrease of CaSki by 60-90%. In the study of cervical cancer cell lines, the very low levels of other endogenous RAR and RXR receptors were observed. ATRA does not repress the expression of two viral oncoproteins E6 and E7 HPV16/18, which play a key role in carcinogenesis of the cervix. Our results support the suggestions that the cell response to vitamin A, and other retinoids in the diet, may depend on cell type, and that the cancer cells are differentially resistant to retinoids. Thus, despite the important biological functions of retinoids, the effects of retinoids in a supplementation in supra-physiological doses as well as their physiological action are difficult to define.

  16. Human Papillomavirus Type 18 E6 Protein Binds the Cellular PDZ Protein TIP-2/GIPC, Which Is Involved in Transforming Growth Factor β Signaling and Triggers Its Degradation by the Proteasome

    PubMed Central

    Favre-Bonvin, Arnaud; Reynaud, Caroline; Kretz-Remy, Carole; Jalinot, Pierre

    2005-01-01

    Several viral proteins expressed by DNA or RNA transforming viruses have the particular property of binding via their C-terminal end to various cellular proteins with PDZ domains. This study is focused on the PDZ protein TIP-2/GIPC, which was originally identified in two-hybrid screens performed with two different baits: the human T-cell leukemia virus type 1 Tax oncoprotein and the regulator of G signaling RGS-GAIP. Further studies have shown that TIP-2/GIPC is also able to associate with the cytoplasmic domains of various transmembrane proteins. In this report we show that TIP-2/GIPC interacts with the E6 protein of human papillomavirus type 18 (HPV-18). This event triggers polyubiquitination and proteasome-mediated degradation of the cellular protein. In agreement with this observation, silencing of E6 by RNA interference in HeLa cells causes an increase in the intracellular TIP-2/GIPC level. This PDZ protein has been previously found to be involved in transforming growth factor β (TGF-β) signaling by favoring expression of the TGF-β type III receptor at the cell membrane. In line with this activity of TIP-2/GIPC, we observed that depletion of this protein in HeLa cells hampers induction of the Id3 gene by TGF-β treatment and also diminishes the antiproliferative effect of this cytokine. Conversely, silencing of E6 increases the expression of Id3 and blocks proliferation of HeLa cells. These results support the notion that HPV-18 E6 renders cells less sensitive to the cytostatic effect of TGF-β by lowering the intracellular amount of TIP-2/GIPC. PMID:15767424

  17. Human papillomavirus type 18 E6 protein binds the cellular PDZ protein TIP-2/GIPC, which is involved in transforming growth factor beta signaling and triggers its degradation by the proteasome.

    PubMed

    Favre-Bonvin, Arnaud; Reynaud, Caroline; Kretz-Remy, Carole; Jalinot, Pierre

    2005-04-01

    Several viral proteins expressed by DNA or RNA transforming viruses have the particular property of binding via their C-terminal end to various cellular proteins with PDZ domains. This study is focused on the PDZ protein TIP-2/GIPC, which was originally identified in two-hybrid screens performed with two different baits: the human T-cell leukemia virus type 1 Tax oncoprotein and the regulator of G signaling RGS-GAIP. Further studies have shown that TIP-2/GIPC is also able to associate with the cytoplasmic domains of various transmembrane proteins. In this report we show that TIP-2/GIPC interacts with the E6 protein of human papillomavirus type 18 (HPV-18). This event triggers polyubiquitination and proteasome-mediated degradation of the cellular protein. In agreement with this observation, silencing of E6 by RNA interference in HeLa cells causes an increase in the intracellular TIP-2/GIPC level. This PDZ protein has been previously found to be involved in transforming growth factor beta (TGF-beta) signaling by favoring expression of the TGF-beta type III receptor at the cell membrane. In line with this activity of TIP-2/GIPC, we observed that depletion of this protein in HeLa cells hampers induction of the Id3 gene by TGF-beta treatment and also diminishes the antiproliferative effect of this cytokine. Conversely, silencing of E6 increases the expression of Id3 and blocks proliferation of HeLa cells. These results support the notion that HPV-18 E6 renders cells less sensitive to the cytostatic effect of TGF-beta by lowering the intracellular amount of TIP-2/GIPC.

  18. The E6-Ap ubiquitin-protein ligase (UBE3A) gene is localized within a narrowed Angelman syndrome critical region.

    PubMed

    Sutcliffe, J S; Jiang, Y H; Galijaard, R J; Matsuura, T; Fang, P; Kubota, T; Christian, S L; Bressler, J; Cattanach, B; Ledbetter, D H; Beaudet, A L

    1997-04-01

    Angelman syndrome (AS) and Prader-Willi syndrome (PWS) are distinct clinical phenotypes resulting from maternal and paternal deficiencies, respectively, in human chromosome 15qll-q13. Although several imprinted, paternally expressed transcripts have been identified within the PWS candidate region, no maternally expressed gene has yet been identified within the AS candidate region. We have developed an integrated physical map spanning the PWS and AS candidate regions and localized two breakpoints, including a cryptic t(14;15) translocation associated with AS and a non-AS 15q deletion, which substantially narrow the AS candidate region to approximately 250 kb. Mapping data indicate that the entire transcriptional unit of the E6-AP ubiquitin-protein ligase (UBE3A) gene lies within the AS region. The UBE3A locus expresses a transcript of approximately 5 kb at low to moderate levels in all tissues tested. The mouse homolog of UBE3A was cloned and sequenced revealing a high degree of conservation at nucleotide and protein levels. Northern and RT-PCR analysis of Ube3a expression in mouse tissues from animals with segmental, paternal uniparental disomy failed to detect substantially reduced or absent expression compared to control animals, failing to provide any evidence for maternal-specific expression from this locus. Recent identification of de novo truncating mutations in UBE3A taken with these observations indicates that mutations in UBE3A can lead to AS and suggests that this locus may encode both imprinted and biallelically expressed products.

  19. Designed Proteins To Modulate Cellular Networks

    PubMed Central

    Cortajarena, Aitziber L.; Liu, Tina Y.; Hochstrasser, Mark; Regan, Lynne

    2012-01-01

    A major challenge of protein design is to create useful new proteins that interact specifically with biological targets in living cells. Such binding modules have many potential applications, including the targeted perturbation of protein networks. As a general approach to create such modules, we designed a library with approximately 109 different binding specificities based on a small 3-tetratricopeptide repeat (TPR) motif framework. We employed a novel strategy, based on split GFP reassembly, to screen the library for modules with the desired binding specificity. Using this approach, we identified modules that bind tightly and specifically to Dss1, a small human protein that interacts with the tumor suppressor protein BRCA2. We showed that these modules also bind the yeast homologue of Dss1, Sem1. Furthermore, we demonstrated that these modules inhibit Sem1 activity in yeast. This strategy will be generally applicable to make novel genetically encoded tools for systems/synthetic biology applications. PMID:20020775

  20. E6Tensors: A Mathematica package for E6 Tensors

    NASA Astrophysics Data System (ADS)

    Deppisch, Thomas

    2017-04-01

    We present the Mathematica package E6Tensors, a tool for explicit tensor calculations in E6 gauge theories. In addition to matrix expressions for the group generators of E6, it provides structure constants, various higher rank tensors and expressions for the representations 27, 78, 351 and 351‧. This paper comes along with a short manual including physically relevant examples. I further give a complete list of gauge invariant, renormalisable terms for superpotentials and Lagrangians.

  1. Requirement of E6AP and the features of human papillomavirus E6 necessary to support degradation of p53.

    PubMed

    Cooper, Brooke; Schneider, Steven; Bohl, Joanna; Jiang, Yong hui; Beaudet, Arthur; Vande Pol, Scott

    2003-02-01

    E6 oncoproteins from human papillomavirus type 16 (16E6) and Bovine Papillomavirus type 1 (BE6) bind to leucine rich peptides (called charged leucine, LXXLL, or signature peptides) found on target cellular proteins. BE6 and 16E6 both bind the product of the UBE3A gene called E6AP on a charged leucine peptide, LQELL. E6AP is an E3 ubiquitin ligase that together with 16E6 interacts with p53 to target p53 degradation. Although both BE6 and 16E6 bind the LQELL peptide of E6AP, only 16E6 acts as an adapter to then bring p53 to E6AP. In order to determine how E6 proteins function as adapters, 16E6, p53, and E6AP were expressed in yeast, and were shown to form a tri-molecular complex. 16E6 mutants were selected that retained interactions with E6AP yet were defective for interaction with p53. Such 16E6 mutations were typically within the amino-terminus of 16E6. Through the use of E6AP null cells, transfected E6AP was shown to be necessary and sufficient for the degradation of p53 in the presence of 16E6. However, the interaction of 16E6 with E6AP was complex. While BE6 interacts only with the LQELL motif of E6AP, an intact LQELL motif is not necessary either for interaction of 16E6 with E6AP or for p53 degradation. In addition, 16E6 mutants that fail to bind the LQELL motif of E6AP can support p53 degradation. These results indicate that 16E6 may have multiple modes of interaction with E6AP and that assembly of p53 containing complexes for targeted degradation by E6AP may occur in more than one way. These results have implications for potential targeting of the interaction of 16E6 and E6AP in the therapy of HPV-induced cancer.

  2. Module organization and variance in protein-protein interaction networks

    NASA Astrophysics Data System (ADS)

    Lin, Chun-Yu; Lee, Tsai-Ling; Chiu, Yi-Yuan; Lin, Yi-Wei; Lo, Yu-Shu; Lin, Chih-Ta; Yang, Jinn-Moon

    2015-03-01

    A module is a group of closely related proteins that act in concert to perform specific biological functions through protein-protein interactions (PPIs) that occur in time and space. However, the underlying module organization and variance remain unclear. In this study, we collected module templates to infer respective module families, including 58,041 homologous modules in 1,678 species, and PPI families using searches of complete genomic database. We then derived PPI evolution scores and interface evolution scores to describe the module elements, including core and ring components. Functions of core components were highly correlated with those of essential genes. In comparison with ring components, core proteins/PPIs were conserved across multiple species. Subsequently, protein/module variance of PPI networks confirmed that core components form dynamic network hubs and play key roles in various biological functions. Based on the analyses of gene essentiality, module variance, and gene co-expression, we summarize the observations of module organization and variance as follows: 1) a module consists of core and ring components; 2) core components perform major biological functions and collaborate with ring components to execute certain functions in some cases; 3) core components are more conserved and essential during organizational changes in different biological states or conditions.

  3. Characterization of a cluster of oncogenic mutations in E6 of a human papillomavirus 83 variant isolated from a high-grade squamous intraepithelial lesion.

    PubMed

    Cannavo, Isabelle; Benchetrit, Maxime; Loubatier, Céline; Michel, Gregory; Lemichez, Emmanuel; Giordanengo, Valérie

    2011-10-01

    We previously isolated human papillomavirus 83 (HPV83m) from a cervical smear. Sequence analysis of E6 and E7 proteins highlighted five mutations located in the second putative zinc-finger region of E6 (E6m), an important domain for protein-protein or protein-DNA interactions. Here, we show that E6m of HPV83m can trigger human primary cell proliferation and anchorage-independent growth properties, similarly to E6 of HPV16, a high-risk HPV (HR-HPV). Interestingly, we demonstrate that, in contrast to E6 of HPV16, E6m corrupts neither p53 stability nor telomerase activity, but acts as a specific modulator of the transcriptional machinery. By studying E6m reversion mutants, we confirmed the importance of the second zinc-finger domain in triggering the observed upregulation of cell growth and of the transcriptional machinery. Reversion of these mutations in E6m (to yield strain E6r) fully abolished the oncogenic potential of E6m, transforming the phenotype of E6 from a high-risk to a low-risk phenotype. Importantly, our data define the importance of a cluster of mutations in the second zinc finger of E6m in increasing the oncogenic potential of HPV83.

  4. Cellular Immunity Induced by a Novel HPV-18 DNA Vaccine Encoding an E6/E7 Fusion Consensus Protein in Mice and Rhesus Macaques

    PubMed Central

    Yan, Jian; Harris, Kristina; Khan, Amir S.; Draghia-Akli, Ruxandra; Sewell, Duane; Weiner, David B.

    2015-01-01

    Human papilloma-virus (HPV) infection is the major cause of cervical cancer. HPV 18 is the most prevalence high-risk HPV after type 16 that accounts for the largest number of cervical cancer cases worldwide. Currently, although prophylactic vaccines have been developed, there is still an urgent need to develop therapeutic HPV vaccines for targeting tumors post infection. In this study, we utilize a novel multi-phase strategy for HPV 18 antigen development with the goal of increasing anti-HPV18 cellular immunity. Our data show that this construct can induce strong cellular immune responses against HPV 18 E6 and E7 antigens in a murine model. Moreover, when applied to Rhesus monkeys, this construct is also able to elicit cellular immunity. These data suggest such DNA immunogens are candidates for further study in the eventual context of immunotherapy for HPV-associated cancers. PMID:18455277

  5. MiR-375 Is Epigenetically Downregulated by HPV-16 E6 Mediated DNMT1 Upregulation and Modulates EMT of Cervical Cancer Cells by Suppressing lncRNA MALAT1

    PubMed Central

    Liu, Shikai; Song, Lili; Yao, Hairong; Zhang, Liang; Xu, Dongkui; Gao, Fangyuan; Li, Qian

    2016-01-01

    Epigenetic modulation is an important mechanism of miRNA dysregulation in cervical cancer. In this study, we firstly studied how this mechanism contributes to miR-375 downregulation in cervical cancer cells. Then, we further studied the association between miR-375 and MALAT1 (metastasis associated lung adenocarcinoma transcript 1) in epithelial mesenchymal transition (EMT) of the cancer cells. HPV-16 positive SiHa and CaSki cells were used as in vitro model. Our data showed that HPV-16 E6 positively modulated DNMT1 expression in both SiHa and CaSki cells. Knockdown of DNMT1 partly restored miR-375 levels in the cells. The following methylation-specific PCR (MSP) assay and qRT-PCR analysis showed that methylation was common in the promoter region of miR-375 in both SiHa and CaSki cells and demethylation partly restored miR-375 levels in the cells. Therefore, we infer that miR-375 is downregulated partly due to promoter hypermethylation mediated by DNMT1 in HPV-16 positive cervical cancer cells. Our bioinformatics analysis showed that MALAT1 has three putative binding sites with miR-375 and the following dual luciferase assay confirmed two of them. QRT-PCR analysis showed that miR-375 overexpression significantly reduced MALAT1 expression, while MALAT1 overexpression reversely suppressed miR-375 levels. Therefore, we infer that there is a reciprocal regulation between miR-375 and MALAT1 in the cells. In SiHa cells, miR-375 overexpression or MALAT1 siRNA partly restored E-cadherin expression, significantly reduced N-cadherin and also reduced invasion capacity of SiHa cells. Therefore, these results suggest that miR-375 and MALAT1 form a functional axis modulating EMT in cervical cancer. PMID:27658300

  6. Combinations of ancestral modules in proteins.

    PubMed

    Sobolevsky, Yehoshua; Frenkel, Zakharia M; Trifonov, Edward N

    2007-12-01

    Twenty-seven protein sequence elements, six to nine amino acids long, were extracted from 15 phylogenetically diverse complete prokaryotic proteomes. The elements are present in all of these proteomes, with at least one copy each (omnipresent elements), and have presumably been conserved since the last universal common ancestor (LUCA). All these omnipresent elements are identified in crystallized protein structures as parts of highly conserved closed loops, 25-30 residues long, thus representing the closed-loop modules discovered in 2000 by Berezovsky et al. The omnipresent peptides make up seven distinct groups, of which the largest groups, Aleph and Beth, contain 18 and four elements, respectively, which are related but different, while five other groups are represented by only one element each. The LUCA modules appear with one or several copies per protein molecule in a variety of combinations depending on the functional identity of the corresponding protein. The functional involvement of individual LUCA modules is outlined on the basis of known protein annotations. Analyses of all the related sequences in a large, formatted protein sequence space suggest that many, if not all, of the 27 omnipresent elements have a common sequence origin. This sequence space network analysis may lead to elucidation of the earliest stages of protein evolution.

  7. Protein modules and signalling networks

    NASA Astrophysics Data System (ADS)

    Pawson, Tony

    1995-02-01

    Communication between cells assumes particular importance in multicellular organisms. The growth, migration and differentiation of cells in the embryo, and their organization into specific tissues, depend on signals transmitted from one cell to another. In the adult, cell signalling orchestrates normal cellular behaviour and responses to wounding and infection. The consequences of breakdowns in this signalling underlie cancer, diabetes and disorders of the immune and cardiovascular systems. Conserved protein domains that act as key regulatory participants in many of these different signalling pathways are highlighted.

  8. E6D25E, HPV16 Asian variant shows specific proteomic pattern correlating in cells transformation and suppressive innate immune response

    SciTech Connect

    Chopjitt, Peechanika; Pientong, Chamsai; Sunthamala, Nuchsupha; Kongyingyoes, Bunkerd; Haonon, Ornuma; Boonmars, Thidarut; Kikawa, Satomi; Nakahara, Tomomi; Kiyono, Tohru; Ekalaksananan, Tipaya

    2016-09-09

    HPV16 Asian variant (HPV16As) containing E6D25E oncogene, is commonly associated with cervical cancers of Asian populations. To explore a mechanism of E6D25E oncoprotein in carcinogenesis, we compared protein profiles in human keratinocytes expressing E6D25E with E6 of HPV16 prototype (E6Pro). A human cervical keratinocyte cell line, HCK1T, was transduced with retroviruses containing E6D25E or E6Pro genes. Biological properties of E6D25E or E6Pro transduced HCK1T cells were characterized. Protein profiles of the transduced HCK1T cells were analyzed using 2D-PAGE and characterized by mass spectrometry and western blotting. Reactomes of modulated proteins were analyzed by using the Reactome Knowledgebase. The E6D25E and E6Pro oncoproteins were comparable for their abilities to degrade p53 and suppress the induction of p21, and induce cell proliferation. Interestingly, the protein profiles of the HCK1T cells transduced with E6D25E showed specific proteomic patterns different from those with E6Pro. Among altered proteins, more than 1.5-fold up- or down- regulation was observed in E6D25E-expressing cells for gp96 and keratin7 which involved in activation of TLR signaling and transformation of squamocolumnar junction cells, respectively. This report describes new cellular proteins specifically targeted by E6D25E oncoprotein that may contribute to impair immune response against viral infection and cell transformation associated with oncogenic property of HPV16As variant. - Highlights: • E6D25E HPV16 specifically modulates protein profile of human keratinocytes. • E6D25E HPV16 modulates protein profile which involves in TLR signalling and transformation of squamocolumnar junction cells. • E6D25E oncoprotein may correlate to impair of immune response against viral infection and cells transformation.

  9. Positive modulator of bone morphogenic protein-2

    DOEpatents

    Zamora, Paul O.; Pena, Louis A.; Lin, Xinhua; Takahashi, Kazuyuki

    2009-01-27

    Compounds of the present invention of formula I and formula II are disclosed in the specification and wherein the compounds are modulators of Bone Morphogenic Protein activity. Compounds are synthetic peptides having a non-growth factor heparin binding region, a linker, and sequences that bind specifically to a receptor for Bone Morphogenic Protein. Uses of compounds of the present invention in the treatment of bone lesions, degenerative joint disease and to enhance bone formation are disclosed.

  10. Positive modulator of bone morphogenic protein-2

    DOEpatents

    Zamora, Paul O.; Pena, Louis A.; Lin, Xinhua; Kazuyuki, Takahashi

    2017-06-06

    Compounds of the present invention of formula I and formula II are disclosed in the specification and wherein the compounds are modulators of Bone Morphogenic Protein activity. Compounds are synthetic peptides having a non-growth factor heparin binding region, a linker, and sequences that bind specifically to a receptor for Bone Morphogenic Protein. Uses of compounds of the present invention in the treatment of bone lesions, degenerative joint disease and to enhance bone formation are disclosed.

  11. Modulation of protein synthesis by polyamines.

    PubMed

    Igarashi, Kazuei; Kashiwagi, Keiko

    2015-03-01

    Polyamines are ubiquitous small basic molecules that play important roles in cell growth and viability. Since polyamines mainly exist as a polyamine-RNA complex, we looked for proteins whose synthesis is preferentially stimulated by polyamines at the level of translation, and thus far identified 17 proteins in Escherichia coli and 6 proteins in eukaryotes. The mechanisms of polyamine stimulation of synthesis of these proteins were investigated. In addition, the role of eIF5A, containing hypusine formed from spermidine, on protein synthesis is described. These results clearly indicate that polyamines and eIF5A contribute to cell growth and viability through modulation of protein synthesis. © 2015 International Union of Biochemistry and Molecular Biology.

  12. 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

    SciTech Connect

    Vandermark, Erik R.; Deluca, Krysta A.; Gardner, Courtney R.; Marker, Daniel F.; Schreiner, Cynthia N.; Strickland, David A.; Wilton, Katelynn M.; Mondal, Sumona; Woodworth, Craig D.

    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 expression 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.

  13. 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.

    PubMed

    Vandermark, Erik R; Deluca, Krysta A; Gardner, Courtney R; Marker, Daniel F; Schreiner, Cynthia N; Strickland, David A; Wilton, Katelynn M; Mondal, Sumona; Woodworth, Craig D

    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 expression 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.

  14. HPV16 E6 and E7 proteins induce a chronic oxidative stress response via NOX2 that causes genomic instability and increased susceptibility to DNA damage in head and neck cancer cells.

    PubMed

    Marullo, Rossella; Werner, Erica; Zhang, Hongzheng; Chen, Georgia Z; Shin, Dong M; Doetsch, Paul W

    2015-11-01

    Human papillomavirus (HPV) is the causative agent of a subgroup of head and neck cancer characterized by an intrinsic radiosensitivity. HPV initiates cellular transformation through the activity of E6 and E7 proteins. E6 and E7 expression is necessary but not sufficient to transform the host cell, as genomic instability is required to acquire the malignant phenotype in HPV-initiated cells. This study reveals a key role played by oxidative stress in promoting genomic instability and radiosensitivity in HPV-positive head and neck cancer. By employing an isogenic human cell model, we observed that expression of E6 and E7 is sufficient to induce reactive oxygen species (ROS) generation in head and neck cancer cells. E6/E7-induced oxidative stress is mediated by nicotinamide adenine dinucleotide phosphate oxidases (NOXs) and causes DNA damage and chromosomal aberrations. This mechanism for genomic instability distinguishes HPV-positive from HPV-negative tumors, as we observed NOX-induced oxidative stress in HPV-positive but not HPV-negative head and neck cancer cells. We identified NOX2 as the source of HPV-induced oxidative stress as NOX2 silencing significantly reduced ROS generation, DNA damage and chromosomal aberrations in HPV-positive cells. Due to their state of chronic oxidative stress, HPV-positive cells are more susceptible to DNA damage induced by ROS and ionizing radiation (IR). Furthermore, exposure to IR results in the formation of complex lesions in HPV-positive cells as indicated by the higher amount of chromosomal breakage observed in this group of cells. These results reveal a novel mechanism for sustaining genomic instability in HPV-positive head and neck tumors and elucidate its contribution to their intrinsic radiosensitivity.

  15. How lipids modulate mitochondrial protein import.

    PubMed

    Böttinger, Lena; Ellenrieder, Lars; Becker, Thomas

    2016-04-01

    Mitochondria have to import the vast majority of their proteins, which are synthesized as precursors on cytosolic ribosomes. The translocase of the outer membrane (TOM complex) forms the general entry gate for the precursor proteins, which are subsequently sorted by protein machineries into the mitochondrial subcompartments: the outer and inner membrane, the intermembrane space and the mitochondrial matrix. The transport across and into the inner membrane is driven by the membrane potential, which is generated by the respiratory chain. Recent studies revealed that the lipid composition of mitochondrial membranes is important for the biogenesis of mitochondrial proteins. Cardiolipin and phosphatidylethanolamine exhibit unexpectedly specific functions for the activity of distinct protein translocases. Both phospholipids are required for full activity of respiratory chain complexes and thus to maintain the membrane potential for protein import. In addition, cardiolipin is required to maintain structural integrity of mitochondrial protein translocases. Finally, the low sterol content in the mitochondrial outer membrane may contribute to the targeting of some outer membrane proteins with a single α-helical membrane anchor. Altogether, mitochondrial lipids modulate protein import on various levels involving precursor targeting, membrane potential generation, stability and activity of protein translocases.

  16. Modulators of 14-3-3 Protein-Protein Interactions.

    PubMed

    Stevers, Loes M; Sijbesma, Eline; Botta, Maurizio; MacKintosh, Carol; Obsil, Tomas; Landrieu, Isabelle; Cau, Ylenia; Wilson, Andrew J; Karawajczyk, Anna; Eickhoff, Jan; Davis, Jeremy; Hann, Michael M; O'Mahony, Gavin; Doveston, Richard G; Brunsveld, Luc; Ottmann, Christian

    2017-10-02

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

  17. The irre cell recognition module (IRM) proteins.

    PubMed

    Fischbach, Karl-Friedrich; Linneweber, Gerit Arne; Andlauer, Till Felix Malte; Hertenstein, Alexander; Bonengel, Bernhard; Chaudhary, Kokil

    2009-01-01

    One of the most challenging problems in developmental neurosciences is to understand the establishment and maintenance of specific membrane contacts between axonal, dendritic, and glial processes in the neuropils, which eventually secure neuronal connectivity. However, underlying cell recognition events are pivotal in other tissues as well. This brief review focuses on the pleiotropic functions of a small, evolutionarily conserved group of proteins of the immunoglobulin superfamily involved in cell recognition. In Drosophila, this protein family comprises Irregular chiasm C/Roughest (IrreC/Rst), Kin of irre (Kirre), and their interacting protein partners, Sticks and stones (SNS) and Hibris (Hbs). For simplicity, we propose to name this ensemble of proteins the irre cell recognition module (IRM) after the first identified member of this family. Here, we summarize evidence that the IRM proteins function together in various cellular interactions, including myoblast fusion, cell sorting, axonal pathfinding, and target recognition in the optic neuropils of Drosophila. Understanding IRM protein function will help to unravel the epigenetic rules by which the intricate neurite networks in sensory neuropils are formed.

  18. Pathogen mimicry of host protein-protein interfaces modulates immunity.

    PubMed

    Guven-Maiorov, Emine; Tsai, Chung-Jung; Nussinov, Ruth

    2016-10-01

    Signaling pathways shape and transmit the cell's reaction to its changing environment; however, pathogens can circumvent this response by manipulating host signaling. To subvert host defense, they beat it at its own game: they hijack host pathways by mimicking the binding surfaces of host-encoded proteins. For this, it is not necessary to achieve global protein homology; imitating merely the interaction surface is sufficient. Different protein folds often interact via similar protein-protein interface architectures. This similarity in binding surfaces permits the pathogenic protein to compete with a host target protein. Thus, rather than binding a host-encoded partner, the host protein hub binds the pathogenic surrogate. The outcome can be dire: rewiring or repurposing the host pathways, shifting the cell signaling landscape and consequently the immune response. They can also cause persistent infections as well as cancer by modulating key signaling pathways, such as those involving Ras. Mapping the rewired host-pathogen 'superorganism' interaction network - along with its structural details - is critical for in-depth understanding of pathogenic mechanisms and developing efficient therapeutics. Here, we overview the role of molecular mimicry in pathogen host evasion as well as types of molecular mimicry mechanisms that emerged during evolution.

  19. The human papillomavirus (HPV) E6 oncoproteins promotes nuclear localization of active caspase 8

    SciTech Connect

    Manzo-Merino, Joaquin; Lizano, Marcela

    2014-02-15

    The HPV-16 E6 and E6{sup ⁎} proteins have been shown previously to be capable of regulating caspase 8 activity. We now show that the capacity of E6 to interact with caspase 8 is common to diverse HPV types, being also seen with HPV-11 E6, HPV-18 E6 and HPV-18 E6{sup ⁎}. Unlike most E6-interacting partners, caspase 8 does not appear to be a major proteasomal target of E6, but instead E6 appears able to stimulate caspase 8 activation, without affecting the overall apoptotic activity. This would appear to be mediated in part by the ability of the HPV E6 oncoproteins to recruit active caspase 8 to the nucleus. - Highlights: • Multiple HPV E6 oncoproteins interact with the caspase 8 DED domain. • HPV E6 stimulates activation of caspase 8. • HPV E6 promotes nuclear accumulation of caspase 8.

  20. Serum antibody response to Human papillomavirus (HPV) infections detected by a novel ELISA technique based on denatured recombinant HPV16 L1, L2, E4, E6 and E7 proteins

    PubMed Central

    Di Bonito, Paola; Grasso, Felicia; Mochi, Stefania; Accardi, Luisa; Donà, Maria Gabriella; Branca, Margherita; Costa, Silvano; Mariani, Luciano; Agarossi, Alberto; Ciotti, Marco; Syrjänen, Kari; Giorgi, Colomba

    2006-01-01

    Background Human papillomaviruses (HPVs) are the primary etiological agents of cervical cancer and are also involved in the development of other tumours (skin, head and neck). Serological survey of the HPV infections is important to better elucidate their natural history and to disclose antigen determinants useful for vaccine development. At present, the analysis of the HPV-specific antibodies has not diagnostic value for the viral infections, and new approaches are needed to correlate the antibody response to the disease outcome. The aim of this study is to develop a novel ELISA, based on five denatured recombinant HPV16 proteins, to be used for detection HPV-specific antibodies. Methods The HPV16 L1, L2, E4, E6 and E7 genes were cloned in a prokaryotic expression vector and expressed as histidine-tagged proteins. These proteins, in a denatured form, were used in ELISA as coating antigens. Human sera were collected from women with abnormal PAP smear enrolled during an ongoing multicenter HPV-PathogenISS study in Italy, assessing the HPV-related pathogenetic mechanisms of progression of cervical cancer precursor lesions. Negative human sera were collected from patients affected by other infectious agents. All the HPV-positive sera were also subjected to an avidity test to assess the binding strength in the antigen-antibody complexes. Results Most of the sera showed a positive reactivity to the denatured HPV16 proteins: 82% of the sera from HPV16 infected women and 89% of the sera from women infected by other HPV genotypes recognised at least one of the HPV16 proteins. The percentages of samples showing reactivity to L1, L2 and E7 were similar, but only a few serum samples reacted to E6 and E4. Most sera bound the antigens with medium and high avidity index, suggesting specific antigen-antibody reactions. Conclusion This novel ELISA, based on multiple denatured HPV16 antigens, is able to detect antibodies in women infected by HPV16 and it is not genotype

  1. Conserved intron positions in ancient protein modules

    PubMed Central

    de Roos, Albert DG

    2007-01-01

    Background The timing of the origin of introns is of crucial importance for an understanding of early genome architecture. The Exon theory of genes proposed a role for introns in the formation of multi-exon proteins by exon shuffling and predicts the presence of conserved splice sites in ancient genes. In this study, large-scale analysis of potential conserved splice sites was performed using an intron-exon database (ExInt) derived from GenBank. Results A set of conserved intron positions was found by matching identical splice sites sequences from distantly-related eukaryotic kingdoms. Most amino acid sequences with conserved introns were homologous to consensus sequences of functional domains from conserved proteins including kinases, phosphatases, small GTPases, transporters and matrix proteins. These included ancient proteins that originated before the eukaryote-prokaryote split, for instance the catalytic domain of protein phosphatase 2A where a total of eleven conserved introns were found. Using an experimental setup in which the relation between a splice site and the ancientness of its surrounding sequence could be studied, it was found that the presence of an intron was positively correlated to the ancientness of its surrounding sequence. Intron phase conservation was linked to the conservation of the gene sequence and not to the splice site sequence itself. However, no apparent differences in phase distribution were found between introns in conserved versus non-conserved sequences. Conclusion The data confirm an origin of introns deep in the eukaryotic branch and is in concordance with the presence of introns in the first functional protein modules in an 'Exon theory of genes' scenario. A model is proposed in which shuffling of primordial short exonic sequences led to the formation of the first functional protein modules, in line with hypotheses that see the formation of introns integral to the origins of genome evolution. Reviewers This article was

  2. Type IV Pilin Proteins: Versatile Molecular Modules

    PubMed Central

    Giltner, Carmen L.; Nguyen, Ylan

    2012-01-01

    Summary: Type IV pili (T4P) are multifunctional protein fibers produced on the surfaces of a wide variety of bacteria and archaea. The major subunit of T4P is the type IV pilin, and structurally related proteins are found as components of the type II secretion (T2S) system, where they are called pseudopilins; of DNA uptake/competence systems in both Gram-negative and Gram-positive species; and of flagella, pili, and sugar-binding systems in the archaea. This broad distribution of a single protein family implies both a common evolutionary origin and a highly adaptable functional plan. The type IV pilin is a remarkably versatile architectural module that has been adopted widely for a variety of functions, including motility, attachment to chemically diverse surfaces, electrical conductance, acquisition of DNA, and secretion of a broad range of structurally distinct protein substrates. In this review, we consider recent advances in this research area, from structural revelations to insights into diversity, posttranslational modifications, regulation, and function. PMID:23204365

  3. Analysis of the roles of E6 binding to E6TP1 and nuclear localization in the human papillomavirus type 31 life cycle.

    PubMed

    Lee, Choongho; Wooldridge, Tonia R; Laimins, Laimonis A

    2007-02-05

    The E6 oncoproteins of high-risk human papillomaviruses provide important functions not only for malignant transformation but also in the productive viral life cycle. E6 proteins have been shown to bind to a number of cellular factors, but only a limited number of analyses have investigated the effects of these interactions on the viral life cycle. In this study, we investigated the consequences of HPV 31 E6 binding to E6TP1, a putative Rap1 GAP protein. HPV 16 E6 has been shown to bind as well as induce the rapid turnover of E6TP1, and similar effects were observed with HPV 31 E6. Mutation of amino acid 128 in HPV 31 E6 was found to abrogate the ability to bind and degrade E6TP1 but did not alter binding to another alpha-helical domain protein, E6AP. When HPV 31 genomes containing mutations at amino acid 128 were transfected into human keratinocytes, the viral DNAs were not stably maintained as episomes indicating the importance of this residue for pathogenesis. Many E6 binding partners including E6TP1 are cytoplasmic proteins, but E6 has been also reported to be localized to the nucleus. We therefore investigated the importance of E6 localization to the nucleus in the viral life cycle. Using a fusion of E6 to Green Fluorescent Protein, we mapped one component of the nuclear localization sequences to residues 121 to 124 of HPV 31 E6. Mutation of these residues in the context of the HPV 31 genome abrogated the ability for episomes to be stably maintained and impaired the ability to extend the life span of cells. These studies identify two activities of HPV 31 E6 that are important for its function in the viral life cycle and for extension of cell life span.

  4. Analysis of the roles of E6 binding to E6TP1 and nuclear localization in the human papillomavirus type 31 life cycle

    SciTech Connect

    Lee, Choongho; Wooldridge, Tonia R.; Laimins, Laimonis A. . E-mail: l-laimins@northwestern.edu

    2007-02-05

    The E6 oncoproteins of high-risk human papillomaviruses provide important functions not only for malignant transformation but also in the productive viral life cycle. E6 proteins have been shown to bind to a number of cellular factors, but only a limited number of analyses have investigated the effects of these interactions on the viral life cycle. In this study, we investigated the consequences of HPV 31 E6 binding to E6TP1, a putative Rap1 GAP protein. HPV 16 E6 has been shown to bind as well as induce the rapid turnover of E6TP1, and similar effects were observed with HPV 31 E6. Mutation of amino acid 128 in HPV 31 E6 was found to abrogate the ability to bind and degrade E6TP1 but did not alter binding to another {alpha}-helical domain protein, E6AP. When HPV 31 genomes containing mutations at amino acid 128 were transfected into human keratinocytes, the viral DNAs were not stably maintained as episomes indicating the importance of this residue for pathogenesis. Many E6 binding partners including E6TP1 are cytoplasmic proteins, but E6 has been also reported to be localized to the nucleus. We therefore investigated the importance of E6 localization to the nucleus in the viral life cycle. Using a fusion of E6 to Green Fluorescent Protein, we mapped one component of the nuclear localization sequences to residues 121 to 124 of HPV 31 E6. Mutation of these residues in the context of the HPV 31 genome abrogated the ability for episomes to be stably maintained and impaired the ability to extend the life span of cells. These studies identify two activities of HPV 31 E6 that are important for its function in the viral life cycle and for extension of cell life span.

  5. HPV E6 antisense induces apoptosis in CaSki cells via suppression of E6 splicing.

    PubMed

    Cho, Cheong Weon; Poo, Haryoung; Cho, Young Sik; Cho, Min Chul; Lee, Kyung Ae; Lee, Shin Je; Park, Sue Nie; Kim, In Ki; Jung, Yong Keun; Choe, Yong Kyung; Yeom, Young I I; Choe, In Seong; Yoon, Do Young

    2002-05-31

    Cervical cancer is known to be highly associated with viral oncogene E6 and E7 of human papilloma virus. Down-regulation of oncogene expression by antisense-based gene therapy has been extensively studied. To investigate the effect of HPV 16 E6 antisense nucleic acid (AS) on cervical cancer cells, human cervical cancer cell lines, CaSki and SiHa cells harboring HPV 16 genome were transfected with plasmid containing E6(AS). The decreased viability and the apoptotic morphology were observed in E6(AS)-transfected cervical cancer cell lines. By 6 h after transfection, inhibition of E6 splicing, rapid upregulations of p53 and a p53-responsive protein, GADD45, were displayed in E6(AS)-transfected CaSki cells. Furthermore, E6(AS) induced loss of mitochondrial transmembrane potential, release of mitochondrial cytochrome c into the cytoplasm, and subsequent activation of caspase-9 and caspase-3. These results indicate that HPV 16 E6(AS) induces apoptosis in CaSki cells via upregulation of p53 and release of cytochrome c into cytoplasm, consequently activating procaspase-9 and procaspase-3.

  6. HPV16 E6 and E6AP differentially cooperate to stimulate or augment Wnt signaling

    SciTech Connect

    Sominsky, Sophia; Kuslansky, Yael; Shapiro, Beny; Jackman, Anna; Haupt, Ygal; Rosin-Arbesfeld, Rina; Sherman, Levana

    2014-11-15

    The present study investigated the roles of E6 and E6AP in the Wnt pathway. We showed that E6 levels are markedly reduced in cells in which Wnt signaling is activated. Coexpression of wild-type or mutant E6AP (C820A) in Wnt-activated cells stabilized E6 and enhanced Wnt/β-catenin/TCF transcription. Expression of E6AP alone in nonstimulated cells elevated β-catenin level, promoted its nuclear accumulation, and activated β-catenin/TCF transcription. A knockdown of E6AP lowered β-catenin levels. Coexpression with E6 intensified the activities of E6AP. Further experiments proved that E6AP/E6 stabilize β-catenin by protecting it from proteasomal degradation. This function was dependent on the catalytic activity of E6AP, the kinase activity of GSK3β and the susceptibility of β-catenin to GSK3β phosphorylation. Thus, this study identified E6AP as a novel regulator of the Wnt signaling pathway, capable of cooperating with E6 in stimulating or augmenting Wnt/β-catenin signaling, thereby possibly contributing to HPV carcinogenesis. - Highlights: • The roles of E6 and E6AP in the Wnt pathway were investigated. • E6AP stabilizes E6 and enhances E6 activity in augmentation of Wnt signaling. • E6AP cooperates with E6 to stabilize β-catenin and stimulate Wnt/β-catenin signaling. • E6AP and E6 act through different mechanisms to augment or stimulate Wnt signaling.

  7. Identification of Topological Network Modules in Perturbed Protein Interaction Networks.

    PubMed

    Sardiu, Mihaela E; Gilmore, Joshua M; Groppe, Brad; Florens, Laurence; Washburn, Michael P

    2017-03-08

    Biological networks consist of functional modules, however detecting and characterizing such modules in networks remains challenging. Perturbing networks is one strategy for identifying modules. Here we used an advanced mathematical approach named topological data analysis (TDA) to interrogate two perturbed networks. In one, we disrupted the S. cerevisiae INO80 protein interaction network by isolating complexes after protein complex components were deleted from the genome. In the second, we reanalyzed previously published data demonstrating the disruption of the human Sin3 network with a histone deacetylase inhibitor. Here we show that disrupted networks contained topological network modules (TNMs) with shared properties that mapped onto distinct locations in networks. We define TMNs as proteins that occupy close network positions depending on their coordinates in a topological space. TNMs provide new insight into networks by capturing proteins from different categories including proteins within a complex, proteins with shared biological functions, and proteins disrupted across networks.

  8. Identification of Topological Network Modules in Perturbed Protein Interaction Networks

    PubMed Central

    Sardiu, Mihaela E.; Gilmore, Joshua M.; Groppe, Brad; Florens, Laurence; Washburn, Michael P.

    2017-01-01

    Biological networks consist of functional modules, however detecting and characterizing such modules in networks remains challenging. Perturbing networks is one strategy for identifying modules. Here we used an advanced mathematical approach named topological data analysis (TDA) to interrogate two perturbed networks. In one, we disrupted the S. cerevisiae INO80 protein interaction network by isolating complexes after protein complex components were deleted from the genome. In the second, we reanalyzed previously published data demonstrating the disruption of the human Sin3 network with a histone deacetylase inhibitor. Here we show that disrupted networks contained topological network modules (TNMs) with shared properties that mapped onto distinct locations in networks. We define TMNs as proteins that occupy close network positions depending on their coordinates in a topological space. TNMs provide new insight into networks by capturing proteins from different categories including proteins within a complex, proteins with shared biological functions, and proteins disrupted across networks. PMID:28272416

  9. Hydrogen exchange, core modules, and new designed proteins.

    PubMed

    Carulla, Natàlia; Barany, George; Woodward, Clare

    2002-12-10

    A strategy for design of new proteins that mimic folding properties of native proteins is based on peptides modeled on the slow exchange cores of natural proteins. We have synthesized peptides, called core modules, that correspond to the elements of secondary structure that carry the very slowest exchanging amides in a protein. The expectation is that, if soluble in water, core modules will form conformational ensembles that favor native-like structure. Core modules modeled on natural bovine pancreatic trypsin inhibitor have been shown by NMR studies to meet this expectation. The next step toward production of a native state mimic is to further shift the conformational bias of a core module toward more ordered structure by promoting module-module interactions that are mutually stabilizing. For this, two core modules were incorporated into a single molecule by means of a long cross-link. From a panel of several two-module peptides, one very promising lead emerged; it is called BetaCore. BetaCore is monomeric in water and forms a new fold composed of a four-stranded, antiparallel beta-sheet. The single, dominant conformation of BetaCore is characterized by various NMR experiments. Here we compare the individual core module to the two-module BetaCore and discuss the progressive stabilization of intramodule structure and the formation of new intermodule interactions.

  10. O6-alkylguanine-DNA transferase (SNAP) as capture module for site-specific covalent bioconjugation of targeting protein on nanoparticles

    NASA Astrophysics Data System (ADS)

    Mazzucchelli, Serena; Colombo, Miriam; Galbiati, Elisabetta; Corsi, Fabio; Montenegro, Josè M.; Parak, Wolfgang J.; Prosperi, Davide

    2013-02-01

    A bimodular genetic fusion comprising a delivery module (scFv) and a capture module (SNAP) is proposed as a novel strategy for the biologically mediated site-specific covalent conjugation of targeting proteins to nanoparticles. ScFv800E6, an scFv mutant selective for HER2 antigen overexpressed in breast cancer cells was chosen as targeting ligand. The fusion protein SNAP-scFv was irreversibly immobilized on magnetofluorescent nanoparticles through the recognition between SNAP module and pegylated O6-alkylguanine derivative. The targeting efficiency of the resulting nanoparticle against HER2-positive breast cancer cells was assessed by flow cytometry and immunofluorescence.

  11. Processing incommensurately modulated protein diffraction data with Eval15

    SciTech Connect

    Porta, Jason; Lovelace, Jeffrey J.; Schreurs, Antoine M. M.; Kroon-Batenburg, Loes M. J.; Borgstahl, Gloria E. O.

    2011-07-01

    Data processing of an incommensurately modulated profilin–actin crystal is described. Recent challenges in biological X-ray crystallography include the processing of modulated diffraction data. A modulated crystal has lost its three-dimensional translational symmetry but retains long-range order that can be restored by refining a periodic modulation function. The presence of a crystal modulation is indicated by an X-ray diffraction pattern with periodic main reflections flanked by off-lattice satellite reflections. While the periodic main reflections can easily be indexed using three reciprocal-lattice vectors a*, b*, c*, the satellite reflections have a non-integral relationship to the main lattice and require a q vector for indexing. While methods for the processing of diffraction intensities from modulated small-molecule crystals are well developed, they have not been applied in protein crystallography. A recipe is presented here for processing incommensurately modulated data from a macromolecular crystal using the Eval program suite. The diffraction data are from an incommensurately modulated crystal of profilin–actin with single-order satellites parallel to b*. The steps taken in this report can be used as a guide for protein crystallographers when encountering crystal modulations. To our knowledge, this is the first report of the processing of data from an incommensurately modulated macromolecular crystal.

  12. Processing incommensurately modulated protein diffraction data with Eval15.

    PubMed

    Porta, Jason; Lovelace, Jeffrey J; Schreurs, Antoine M M; Kroon-Batenburg, Loes M J; Borgstahl, Gloria E O

    2011-07-01

    Recent challenges in biological X-ray crystallography include the processing of modulated diffraction data. A modulated crystal has lost its three-dimensional translational symmetry but retains long-range order that can be restored by refining a periodic modulation function. The presence of a crystal modulation is indicated by an X-ray diffraction pattern with periodic main reflections flanked by off-lattice satellite reflections. While the periodic main reflections can easily be indexed using three reciprocal-lattice vectors a*, b*, c*, the satellite reflections have a non-integral relationship to the main lattice and require a q vector for indexing. While methods for the processing of diffraction intensities from modulated small-molecule crystals are well developed, they have not been applied in protein crystallography. A recipe is presented here for processing incommensurately modulated data from a macromolecular crystal using the Eval program suite. The diffraction data are from an incommensurately modulated crystal of profilin-actin with single-order satellites parallel to b*. The steps taken in this report can be used as a guide for protein crystallographers when encountering crystal modulations. To our knowledge, this is the first report of the processing of data from an incommensurately modulated macromolecular crystal.

  13. Membrane shape modulates transmembrane protein distribution

    PubMed Central

    Aimon, Sophie; Callan-Jones, Andrew; Berthaud, Alice; Pinot, Mathieu; Toombes, Gilman E. S.; Bassereau, Patricia

    2014-01-01

    Summary Although membrane shape varies greatly throughout the cell, the contribution of membrane curvature to transmembrane protein targeting is unknown due to the numerous sorting mechanisms taking place concurrently in cells. To isolate the effect of membrane shape, cellsized Giant Unilamellar Vesicles (GUVs) containing either the potassium channel, KvAP, or water channel, AQP0, were used to form membrane nanotubes with controlled radii. While the AQP0 concentrations in flat and curved membranes were indistinguishable, KvAP was enriched in the tubes, with greater enrichment in more highly curved membranes. FRAP measurements showed that both proteins could freely diffuse through the neck between the tube and GUV, and the effect of each protein on membrane shape and stiffness was characterized using a thermodynamic sorting model. This study establishes the importance of membrane shape for targeting transmembrane proteins, and provides a method for determining the effective shape and flexibility of membrane proteins. PMID:24480645

  14. Star-PAP controls HPV E6 regulation of p53 and sensitizes cells to VP-16.

    PubMed

    Li, W; Anderson, R A

    2014-02-13

    Cervical cancer is the most common genital malignancy and the high-risk human papillomaviruses (HPV type 16, 18 and 31, and so on) are major agents for its cause. A key switch for the onset of cervical cancers by HPVs is the cellular degradation of the tumor-suppressor p53 that is mediated by the HPV-generated E6 protein. E6 forms a complex with the E3 ubiquitin-ligase E6-associated protein (E6AP) leading to p53 degradation. The components that control E6 expression and the mechanisms for regulation of the expression in host cells remain undefined. Here we show that the nuclear noncanonical poly(A) polymerase (PAP) speckle targeted PIPKIα regulated PAP (Star-PAP) controls E6 mRNA polyadenylation and expression and modulates wild-type p53 levels as well as cell cycle profile in high-risk HPV-positive cells. In the absence of Star-PAP, treatment of cells with the chemotherapeutic drug VP-16 dramatically reduced E6 and increased p53 levels. This diminished both cell proliferation and anchorage-independent growth required for cancer progression, indicating a synergism between VP-16 treatment and the loss of Star-PAP. This identifies Star-PAP as a potential drug target for the treatment of HPV-positive cancer cells. These data provide a mechanistic basis for increasing the sensitivity and efficiency of chemotherapy in the treatment of cancers that have low levels of wild-type p53.

  15. S100 Proteins Modulate Protein Phosphatase 5 Function

    PubMed Central

    Yamaguchi, Fuminori; Umeda, Yoshinori; Shimamoto, Seiko; Tsuchiya, Mitsumasa; Tokumitsu, Hiroshi; Tokuda, Masaaki; Kobayashi, Ryoji

    2012-01-01

    PP5 is a unique member of serine/threonine phosphatases comprising a regulatory tetratricopeptide repeat (TPR) domain and functions in signaling pathways that control many cellular responses. We reported previously that Ca2+/S100 proteins directly associate with several TPR-containing proteins and lead to dissociate the interactions of TPR proteins with their client proteins. Here, we identified protein phosphatase 5 (PP5) as a novel target of S100 proteins. In vitro binding studies demonstrated that S100A1, S100A2, S100A6, and S100B proteins specifically interact with PP5-TPR and inhibited the PP5-Hsp90 interaction. In addition, the S100 proteins activate PP5 by using a synthetic phosphopeptide and a physiological protein substrate, Tau. Overexpression of S100A1 in COS-7 cells induced dephosphorylation of Tau. However, S100A1 and permanently active S100P inhibited the apoptosis signal-regulating kinase 1 (ASK1) and PP5 interaction, resulting the inhibition of dephosphorylation of phospho-ASK1 by PP5. The association of the S100 proteins with PP5 provides a Ca2+-dependent regulatory mechanism for the phosphorylation status of intracellular proteins through the regulation of PP5 enzymatic activity or PP5-client protein interaction. PMID:22399290

  16. Development of small molecules designed to modulate protein-protein interactions.

    PubMed

    Che, Ye; Brooks, Bernard R; Marshall, Garland R

    2006-02-01

    Protein-protein interactions are ubiquitous, essential to almost all known biological processes, and offer attractive opportunities for therapeutic intervention. Developing small molecules that modulate protein-protein interactions is challenging, owing to the large size of protein-complex interface, the lack of well-defined binding pockets, etc. We describe a general approach based on the "privileged-structure hypothesis" [Che, Ph.D. Thesis, Washington University, 2003] - that any organic templates capable of mimicking surfaces of protein-recognition motifs are potential privileged scaffolds as protein-complex antagonists--to address the challenges inherent in the discovery of small-molecule inhibitors of protein-protein interactions.

  17. The Ribosome Modulates Nascent Protein Folding

    PubMed Central

    Kaiser, Christian M.; Goldman, Daniel H.; Chodera, John D.; Tinoco, Ignacio; Bustamante, Carlos

    2014-01-01

    Proteins are synthesized by the ribosome and generally must fold to become functionally active. Although it is commonly assumed that the ribosome affects the folding process, this idea has been extremely difficult to demonstrate. We have developed an experimental system to investigate the folding of single ribosome-bound stalled nascent polypeptides with optical tweezers. In T4 lysozyme, synthesized in a reconstituted in vitro translation system, the ribosome slows the formation of stable tertiary interactions and the attainment of the native state relative to the free protein. Incomplete T4 lysozyme polypeptides misfold and aggregate when free in solution, but they remain folding-competent near the ribosomal surface. Altogether, our results suggest that the ribosome not only decodes the genetic information and synthesizes polypeptides, but also promotes efficient de novo attainment of the native state. PMID:22194581

  18. Xanthophylls as modulators of membrane protein function.

    PubMed

    Ruban, Alexander V; Johnson, Matthew P

    2010-12-01

    This review discusses the structural aspect of the role of photosynthetic antenna xanthophylls. It argues that xanthophyll hydrophobicity/polarity could explain the reason for xanthophyll variety and help to understand their recently emerging function--control of membrane organization and the work of membrane proteins. The structure of a xanthophyll molecule is discussed in relation to other amphiphilic compounds like lipids, detergents, etc. Xanthophyll composition of membrane proteins, the role of their variety in protein function are discussed using as an example for the major light harvesting antenna complex of photosystem II, LHCII, from higher plants. A new empirical parameter, hydrophobicity parameter (H-parameter), has been introduced as an effective measure of the hydrophobicity of the xanthophyll complement of LHCII from different xanthophyll biosynthesis mutants of Arabidopsis. Photosystem II quantum efficiency was found to correlate well with the H-parameter of LHCII xanthophylls. PSII down-regulation by non-photochemical chlorophyll fluorescence quenching, NPQ, had optimum corresponding to the wild-type xanthophyll composition, where lutein occupies intrinsic sites, L1 and L2. Xanthophyll polarity/hydrophobicity alteration by the activity of the xanthophyll cycle explains the allosteric character of NPQ regulation, memory of illumination history and the hysteretic nature of the relationship between the triggering factor, ΔpH, and the energy dissipation process. Copyright © 2010 Elsevier Inc. All rights reserved.

  19. Modulation of signaling pathways by RNA virus capsid proteins.

    PubMed

    Urbanowski, Matthew D; Ilkow, Carolina S; Hobman, Tom C

    2008-07-01

    Capsid proteins are structural components of virus particles. They are nucleic acid-binding proteins whose main recognized function is to package viral genomes into protective structures called nucleocapsids. Research over the last 10 years indicates that in addition to their role as genome guardians, viral capsid proteins modulate host cell signaling networks. Disruption or alteration of intracellular signaling pathways by viral capsids may benefit replication of the virus by affecting innate immunity and in some cases, may underlie disease progression. In this review, we describe how the capsid proteins from medically relevant RNA viruses interact with host cell signaling pathways.

  20. Protein Interaction Networks—More Than Mere Modules

    PubMed Central

    Pinkert, Stefan; Schultz, Jörg; Reichardt, Jörg

    2010-01-01

    It is widely believed that the modular organization of cellular function is reflected in a modular structure of molecular networks. A common view is that a “module” in a network is a cohesively linked group of nodes, densely connected internally and sparsely interacting with the rest of the network. Many algorithms try to identify functional modules in protein-interaction networks (PIN) by searching for such cohesive groups of proteins. Here, we present an alternative approach independent of any prior definition of what actually constitutes a “module”. In a self-consistent manner, proteins are grouped into “functional roles” if they interact in similar ways with other proteins according to their functional roles. Such grouping may well result in cohesive modules again, but only if the network structure actually supports this. We applied our method to the PIN from the Human Protein Reference Database (HPRD) and found that a representation of the network in terms of cohesive modules, at least on a global scale, does not optimally represent the network's structure because it focuses on finding independent groups of proteins. In contrast, a decomposition into functional roles is able to depict the structure much better as it also takes into account the interdependencies between roles and even allows groupings based on the absence of interactions between proteins in the same functional role. This, for example, is the case for transmembrane proteins, which could never be recognized as a cohesive group of nodes in a PIN. When mapping experimental methods onto the groups, we identified profound differences in the coverage suggesting that our method is able to capture experimental bias in the data, too. For example yeast-two-hybrid data were highly overrepresented in one particular group. Thus, there is more structure in protein-interaction networks than cohesive modules alone and we believe this finding can significantly improve automated function prediction

  1. Deciphering peculiar protein-protein interacting modules in Deinococcus radiodurans

    PubMed Central

    Mezhoud, Karim; Sghaier, Haïtham; Barkallah, Insaf

    2009-01-01

    Interactomes of proteins under positive selection from ionizing-radiation-resistant bacteria (IRRB) might be a part of the answer to the question as to how IRRB, particularly Deinococcus radiodurans R1 (Deira), resist ionizing radiation. Here, using the Database of Interacting Proteins (DIP) and the Protein Structural Interactome (PSI)-base server for PSI map, we have predicted novel interactions of orthologs of the 58 proteins under positive selection in Deira and other IRRB, but which are absent in IRSB. Among these, 18 domains and their interactomes have been identified in DNA checkpoint and repair; kinases pathways; energy and nucleotide metabolisms were the important biological processes that were found to be involved. This finding provides new clues to the cellular pathways that can to be important for ionizing-radiation resistance in Deira. PMID:19356244

  2. Slob, a Slowpoke channel–binding protein, modulates synaptic transmission

    PubMed Central

    Zhang, Jiaming

    2011-01-01

    Modulation of ion channels by regulatory proteins within the same macromolecular complex is a well-accepted concept, but the physiological consequences of such modulation are not fully understood. Slowpoke (Slo), a potassium channel critical for action potential repolarization and transmitter release, is regulated by Slo channel–binding protein (Slob), a Drosophila melanogaster Slo (dSlo) binding partner. Slob modulates the voltage dependence of dSlo channel activation in vitro and exerts similar effects on the dSlo channel in Drosophila central nervous system neurons in vivo. In addition, Slob modulates action potential duration in these neurons. Here, we investigate further the functional consequences of the modulation of the dSlo channel by Slob in vivo, by examining larval neuromuscular synaptic transmission in flies in which Slob levels have been altered. In Slob-null flies generated through P-element mutagenesis, as well as in Slob knockdown flies generated by RNA interference (RNAi), we find an enhancement of synaptic transmission but no change in the properties of the postsynaptic muscle cell. Using targeted transgenic rescue and targeted expression of Slob-RNAi, we find that Slob expression in neurons (but not in the postsynaptic muscle cell) is critical for its effects on synaptic transmission. Furthermore, inhibition of dSlo channel activity abolishes these effects of Slob. These results suggest that presynaptic Slob, by regulating dSlo channel function, participates in the modulation of synaptic transmission. PMID:21282401

  3. Changes in global gene expression profiles induced by HPV 16 E6 oncoprotein variants in cervical carcinoma C33-A cells

    SciTech Connect

    Zacapala-Gómez, Ana Elvira; Del Moral-Hernández, Oscar; Villegas-Sepúlveda, Nicolás; Hidalgo-Miranda, Alfredo; Romero-Córdoba, Sandra Lorena; and others

    2016-01-15

    We analyzed the effects of the expression of HPV 16 E6 oncoprotein variants (AA-a, AA-c, E-A176/G350, E-C188/G350, E-G350), and the E-Prototype in global gene expression profiles in an in vitro model. E6 gene was cloned into an expression vector fused to GFP and was transfected in C33-A cells. Affymetrix GeneChip Human Transcriptome Array 2.0 platform was used to analyze the expression of over 245,000 coding transcripts. We found that HPV16 E6 variants altered the expression of 387 different genes in comparison with E-Prototype. The altered genes are involved in cellular processes related to the development of cervical carcinoma, such as adhesion, angiogenesis, apoptosis, differentiation, cell cycle, proliferation, transcription and protein translation. Our results show that polymorphic changes in HPV16 E6 natural variants are sufficient to alter the overall gene expression profile in C33-A cells, explaining in part the observed differences in oncogenic potential of HPV16 variants. - Highlights: • Amino acid changes in HPV16 E6 variants modulate the transciption of specific genes. • This is the first comparison of global gene expression profile of HPV 16 E6 variants. • Each HPV 16 E6 variant appears to have its own molecular signature.

  4. Searching for signatures of E6

    NASA Astrophysics Data System (ADS)

    Joglekar, Aniket; Rosner, Jonathan L.

    2017-07-01

    The grand unified group E6 is a predictive scheme for physics beyond the standard model (SM). It offers the possibility of extra Z bosons, new vectorlike fermions, sterile neutrinos, and neutral scalars in addition to the SM Higgs boson. Some previous discussions of these features are updated and extended. Their relevance to present searches at the CERN Large Hadron Collider and in patterns of neutrino masses is noted. Addition of a small set of scalar bosons at the TeV scale permits gauge unification near a scale of 1 016 GeV and leads to bounds on masses of particles beyond those in the standard model.

  5. Specific modulation of protein activity by using a bioorthogonal reaction.

    PubMed

    Warner, John B; Muthusamy, Anand K; Petersson, E James

    2014-11-24

    Unnatural amino acids with bioorthogonal reactive groups have the potential to provide a rapid and specific mechanism for covalently inhibiting a protein of interest. Here, we use mutagenesis to insert an unnatural amino acid containing an azide group (Z) into the target protein at positions such that a "click" reaction with an alkyne modulator (X) will alter the function of the protein. This bioorthogonally reactive pair can engender specificity of X for the Z-containing protein, even if the target is otherwise identical to another protein, allowing for rapid target validation in living cells. We demonstrate our method using inhibition of the Escherichia coli enzyme aminoacyl transferase by both active-site occlusion and allosteric mechanisms. We have termed this a "clickable magic bullet" strategy, and it should be generally applicable to studying the effects of protein inhibition, within the limits of unnatural amino acid mutagenesis.

  6. Chemical modulators working at pharmacological interface of target proteins.

    PubMed

    Jeon, Young Ho; Lee, Jin Young; Kim, Sunghoon

    2012-03-15

    For last few decades, the active site cleft and substrate-binding site of enzymes as well as ligand-binding site of the receptors have served as the main pharmacological space for drug discovery. However, rapid accumulation of proteome and protein network analysis data has opened a new therapeutic space that is the interface between the interacting proteins. Due to the complexity of the interaction modes and the numbers of the participating components, it is still challenging to identify the chemicals that can accurately control the protein-protein interactions at desire. Nonetheless, the number of chemical drugs and candidates working at the interface of the interacting proteins are rapidly increasing. This review addresses the current case studies and state-of-the-arts in the development of small chemical modulators controlling the interactions of the proteins that have pathological implications in various human diseases such as cancer, immune disorders, neurodegenerative and infectious diseases.

  7. Human protein status modulates brain reward responses to food cues.

    PubMed

    Griffioen-Roose, Sanne; Smeets, Paul Am; van den Heuvel, Emmy; Boesveldt, Sanne; Finlayson, Graham; de Graaf, Cees

    2014-07-01

    Protein is indispensable in the human diet, and its intake appears tightly regulated. The role of sensory attributes of foods in protein intake regulation is far from clear. We investigated the effect of human protein status on neural responses to different food cues with the use of functional magnetic resonance imaging (fMRI). The food cues varied by taste category (sweet compared with savory) and protein content (low compared with high). In addition, food preferences and intakes were measured. We used a randomized crossover design whereby 23 healthy women [mean ± SD age: 22 ± 2 y; mean ± SD body mass index (in kg/m(2)): 22.5 ± 1.8] followed two 16-d fully controlled dietary interventions involving consumption of either a low-protein diet (0.6 g protein · kg body weight(-1) · d(-1), ~7% of energy derived from protein, approximately half the normal protein intake) or a high-protein diet (2.2 g protein · kg body weight(-1) · d(-1), ~25% of energy, approximately twice the normal intake). On the last day of the interventions, blood oxygen level-dependent (BOLD) responses to odor and visual food cues were measured by using fMRI. The 2 interventions were followed by a 1-d ad libitum phase, during which a large array of food items was available and preference and intake were measured. When exposed to food cues (relative to the control condition), the BOLD response was higher in reward-related areas (orbitofrontal cortex, striatum) in a low-protein state than in a high-protein state. Specifically, BOLD was higher in the inferior orbitofrontal cortex in response to savory food cues. In contrast, the protein content of the food cues did not modulate the BOLD response. A low protein state also increased preferences for savory food cues and increased protein intake in the ad libitum phase as compared with a high-protein state. Protein status modulates brain responses in reward regions to savory food cues. These novel findings suggest that dietary protein status

  8. Protein-protein interactions and protein modules in the control of neurotransmitter release.

    PubMed Central

    Benfenati, F; Onofri, F; Giovedí, S

    1999-01-01

    Information transfer among neurons is operated by neurotransmitters stored in synaptic vesicles and released to the extracellular space by an efficient process of regulated exocytosis. Synaptic vesicles are organized into two distinct functional pools, a large reserve pool in which vesicles are restrained by the actin-based cytoskeleton, and a quantitatively smaller releasable pool in which vesicles approach the presynaptic membrane and eventually fuse with it on stimulation. Both synaptic vesicle trafficking and neurotransmitter release depend on a precise sequence of events that include release from the reserve pool, targeting to the active zone, docking, priming, fusion and endocytotic retrieval of synaptic vesicles. These steps are mediated by a series of specific interactions among cytoskeletal, synaptic vesicle, presynaptic membrane and cytosolic proteins that, by acting in concert, promote the spatial and temporal regulation of the exocytotic machinery. The majority of these interactions are mediated by specific protein modules and domains that are found in many proteins and are involved in numerous intracellular processes. In this paper, the possible physiological role of these multiple protein-protein interactions is analysed, with ensuing updating and clarification of the present molecular model of the process of neurotransmitter release. PMID:10212473

  9. Modulation of apoptosis by V protein mumps virus

    PubMed Central

    2011-01-01

    Background The Urabe AM9 vaccine strain of mumps virus contains two variants of V protein: VWT (of HN-A1081 viral population) and VGly (of HN-G1081). The V protein is a promoting factor of viral replication by blocking the IFN antiviral pathway. Findings We studied the relationship between V protein variants and IFN-α2b-induced apoptosis. V proteins decrease activation of the extrinsic IFN-α2b-induced apoptotic pathway monitored by the caspase 8 activity, being the effect greater with the VWT protein. Both V proteins decrease the activity of caspase 9 of the intrinsic apoptotic pathway. In a system without IFN, the VWT and VGly proteins expression promotes activation of caspases 3 and 7. However, when the cellular system was stimulated with IFN-α, this activity decreased partially. TUNEL assay shows that for treatment with IFN-α and ibuprofen of cervical adenocarcinoma cells there is nuclear DNA fragmentation but the V protein expression reduces this process. Conclusions The reduction in the levels of caspases and DNA fragmentation, suggesting that V protein, particularly VWT protein of Urabe AM9 vaccine strain, modulates apoptosis. In addition, the VWT protein shows a protective role for cell proliferation in the presence of antiproliferative signals. PMID:21569530

  10. Protein complexes and functional modules in molecular networks

    NASA Astrophysics Data System (ADS)

    Spirin, Victor; Mirny, Leonid A.

    2003-10-01

    Proteins, nucleic acids, and small molecules form a dense network of molecular interactions in a cell. Molecules are nodes of this network, and the interactions between them are edges. The architecture of molecular networks can reveal important principles of cellular organization and function, similarly to the way that protein structure tells us about the function and organization of a protein. Computational analysis of molecular networks has been primarily concerned with node degree [Wagner, A. & Fell, D. A. (2001) Proc. R. Soc. London Ser. B 268, 1803-1810; Jeong, H., Tombor, B., Albert, R., Oltvai, Z. N. & Barabasi, A. L. (2000) Nature 407, 651-654] or degree correlation [Maslov, S. & Sneppen, K. (2002) Science 296, 910-913], and hence focused on single/two-body properties of these networks. Here, by analyzing the multibody structure of the network of protein-protein interactions, we discovered molecular modules that are densely connected within themselves but sparsely connected with the rest of the network. Comparison with experimental data and functional annotation of genes showed two types of modules: (i) protein complexes (splicing machinery, transcription factors, etc.) and (ii) dynamic functional units (signaling cascades, cell-cycle regulation, etc.). Discovered modules are highly statistically significant, as is evident from comparison with random graphs, and are robust to noise in the data. Our results provide strong support for the network modularity principle introduced by Hartwell et al. [Hartwell, L. H., Hopfield, J. J., Leibler, S. & Murray, A. W. (1999) Nature 402, C47-C52], suggesting that found modules constitute the "building blocks" of molecular networks.

  11. Protein complexes and functional modules in molecular networks.

    PubMed

    Spirin, Victor; Mirny, Leonid A

    2003-10-14

    Proteins, nucleic acids, and small molecules form a dense network of molecular interactions in a cell. Molecules are nodes of this network, and the interactions between them are edges. The architecture of molecular networks can reveal important principles of cellular organization and function, similarly to the way that protein structure tells us about the function and organization of a protein. Computational analysis of molecular networks has been primarily concerned with node degree [Wagner, A. & Fell, D. A. (2001) Proc. R. Soc. London Ser. B 268, 1803-1810; Jeong, H., Tombor, B., Albert, R., Oltvai, Z. N. & Barabasi, A. L. (2000) Nature 407, 651-654] or degree correlation [Maslov, S. & Sneppen, K. (2002) Science 296, 910-913], and hence focused on single/two-body properties of these networks. Here, by analyzing the multibody structure of the network of protein-protein interactions, we discovered molecular modules that are densely connected within themselves but sparsely connected with the rest of the network. Comparison with experimental data and functional annotation of genes showed two types of modules: (i) protein complexes (splicing machinery, transcription factors, etc.) and (ii) dynamic functional units (signaling cascades, cell-cycle regulation, etc.). Discovered modules are highly statistically significant, as is evident from comparison with random graphs, and are robust to noise in the data. Our results provide strong support for the network modularity principle introduced by Hartwell et al. [Hartwell, L. H., Hopfield, J. J., Leibler, S. & Murray, A. W. (1999) Nature 402, C47-C52], suggesting that found modules constitute the "building blocks" of molecular networks.

  12. Altering protein surface charge with chemical modification modulates protein-gold nanoparticle aggregation

    NASA Astrophysics Data System (ADS)

    Jamison, Jennifer A.; Bryant, Erika L.; Kadali, Shyam B.; Wong, Michael S.; Colvin, Vicki L.; Matthews, Kathleen S.; Calabretta, Michelle K.

    2011-02-01

    Gold nanoparticles (AuNP) can interact with a wide range of molecules including proteins. Whereas significant attention has focused on modifying the nanoparticle surface to regulate protein-AuNP assembly or influence the formation of the protein "corona," modification of the protein surface as a mechanism to modulate protein-AuNP interaction has been less explored. Here, we examine this possibility utilizing three small globular proteins—lysozyme with high isoelectric point (pI) and established interactions with AuNP; α-lactalbumin with similar tertiary fold to lysozyme but low pI; and myoglobin with a different globular fold and an intermediate pI. We first chemically modified these proteins to alter their charged surface functionalities, and thereby shift protein pI, and then applied multiple methods to assess protein-AuNP assembly. At pH values lower than the anticipated pI of the modified protein, AuNP exposure elicits changes in the optical absorbance of the protein-NP solutions and other properties due to aggregate formation. Above the expected pI, however, protein-AuNP interaction is minimal, and both components remain isolated, presumably because both species are negatively charged. These data demonstrate that protein modification provides a powerful tool for modulating whether nanoparticle-protein interactions result in material aggregation. The results also underscore that naturally occurring protein modifications found in vivo may be critical in defining nanoparticle-protein corona compositions.

  13. Growing functional modules from a seed protein via integration of protein interaction and gene expression data.

    PubMed

    Maraziotis, Ioannis A; Dimitrakopoulou, Konstantina; Bezerianos, Anastasios

    2007-10-23

    Nowadays modern biology aims at unravelling the strands of complex biological structures such as the protein-protein interaction (PPI) networks. A key concept in the organization of PPI networks is the existence of dense subnetworks (functional modules) in them. In recent approaches clustering algorithms were applied at these networks and the resulting subnetworks were evaluated by estimating the coverage of well-established protein complexes they contained. However, most of these algorithms elaborate on an unweighted graph structure which in turn fails to elevate those interactions that would contribute to the construction of biologically more valid and coherent functional modules. In the current study, we present a method that corroborates the integration of protein interaction and microarray data via the discovery of biologically valid functional modules. Initially the gene expression information is overlaid as weights onto the PPI network and the enriched PPI graph allows us to exploit its topological aspects, while simultaneously highlights enhanced functional association in specific pairs of proteins. Then we present an algorithm that unveils the functional modules of the weighted graph by expanding a kernel protein set, which originates from a given 'seed' protein used as starting-point. The integrated data and the concept of our approach provide reliable functional modules. We give proofs based on yeast data that our method manages to give accurate results in terms both of structural coherency, as well as functional consistency.

  14. Timing Correlations in Proteins Predict Functional Modules and Dynamic Allostery.

    PubMed

    Lin, Milo M

    2016-04-20

    How protein structure encodes functionality is not fully understood. For example, long-range intraprotein communication can occur without measurable conformational change and is often not captured by existing structural correlation functions. It is shown here that important functional information is encoded in the timing of protein motions, rather than motion itself. I introduce the conditional activity function to quantify such timing correlations among the degrees of freedom within proteins. For three proteins, the conditional activities between side-chain dihedral angles were computed using the output of microseconds-long atomistic simulations. The new approach demonstrates that a sparse fraction of side-chain pairs are dynamically correlated over long distances (spanning protein lengths up to 7 nm), in sharp contrast to structural correlations, which are short-ranged (<1 nm). Regions of high self- and inter-side-chain dynamical correlations are found, corresponding to experimentally determined functional modules and allosteric connections, respectively.

  15. G protein modulation of recombinant P/Q-type calcium channels by regulators of G protein signalling proteins.

    PubMed

    Mark, M D; Wittemann, S; Herlitze, S

    2000-10-01

    1. Fast synaptic transmission is triggered by the activation of presynaptic Ca2+ channels which can be inhibited by Gbetagamma subunits via G protein-coupled receptors (GPCR). Regulators of G protein signalling (RGS) proteins are GTPase-accelerating proteins (GAPs), which are responsible for >100-fold increases in the GTPase activity of G proteins and might be involved in the regulation of presynaptic Ca2+ channels. In this study we investigated the effects of RGS2 on G protein modulation of recombinant P/Q-type channels expressed in a human embryonic kidney (HEK293) cell line using whole-cell recordings. 2. RGS2 markedly accelerates transmitter-mediated inhibition and recovery from inhibition of Ba2+ currents (IBa) through P/Q-type channels heterologously expressed with the muscarinic acetylcholine receptor M2 (mAChR M2). 3. Both RGS2 and RGS4 modulate the prepulse facilitation properties of P/Q-type Ca2+ channels. G protein reinhibition is accelerated, while release from inhibition is slowed. These kinetics depend on the availability of G protein alpha and betagamma subunits which is altered by RGS proteins. 4. RGS proteins unmask the Ca2+ channel beta subunit modulation of Ca2+ channel G protein inhibition. In the presence of RGS2, P/Q-type channels containing the beta2a and beta3 subunits reveal significantly altered kinetics of G protein modulation and increased facilitation compared to Ca2+ channels coexpressed with the beta1b or beta4 subunit.

  16. Novel drug form of chlorin e6

    NASA Astrophysics Data System (ADS)

    Abakumova, O. Y.; Baum, Rudolf P.; Ermakova, Natalia Y.; Gradyushko, A. T.; Guseva-Donskaya, T. N.; Karmenyan, Artashes V.; Koraboyev, U. M.; Laptev, V. P.; Mechkov, V. M.; Mikhailova, L. M.; Panferova, N. G.; Rebeko, Aleksei G.; Reshetnickov, Andrei V.; Ryabov, M. V.; Stranadko, Eugeny P.; Tsvetkova, Tatyana A.; Zhukova, O. S.

    1999-12-01

    A novel stable water-soluble form of well known photosensitizer chlorin e6 named `Photodithazine' has been obtained from Spirulina Platensis cyanobacteria as a noncovalent complex with N-methyl-D-glucosamine, and its biological characteristics evaluate, which proved to be as follows: in vitro photocytotoxicity was 1 (mu) M (EC50) as determined by the extent of DNA synthesis inhibition in CaOv cells after irradiation with 650 - 900 nm light, and 5 (mu) M (EC65) as determined using MTT test on PC12 cells after irradiation with 670 nm laser light at the doses of 15 and 20 J/cm2, respectively, with Al-sulfophthalocyanine `Photosense' (Russia; oligomerized hematoporphyrin-IX mixture `Photogen', Russia) being used as permitted reference drugs.

  17. TIMBAL v2: update of a database holding small molecules modulating protein-protein interactions.

    PubMed

    Higueruelo, Alicia P; Jubb, Harry; Blundell, Tom L

    2013-01-01

    TIMBAL is a database holding molecules of molecular weight <1200 Daltons that modulate protein-protein interactions. Since its first release, the database has been extended to cover 50 known protein-protein interactions drug targets, including protein complexes that can be stabilized by small molecules with therapeutic effect. The resource contains 14 890 data points for 6896 distinct small molecules. UniProt codes and Protein Data Bank entries are also included. Database URL: http://www-cryst.bioc.cam.ac.uk/timbal

  18. The Effect of Protein Mass Modulation on Human Dihydrofolate Reductase

    PubMed Central

    Francis, Kevin; Sapienza, Paul J.; Lee, Andrew L.; Kohen, Amnon

    2016-01-01

    Dihydrofolate reductase (DHFR) from Escherichia coli has long served as a model enzyme with which to elucidate possible links between protein dynamics and the catalyzed reaction. Such physical properties of its human counterpart have not been rigorously studied so far, but recent computer-based simulations suggest that these two DHFRs differ significantly in how closely coupled the protein dynamics and the catalyzed C-H→C hydride transfer step are. To test this prediction, two contemporary probes for studying the effect of protein dynamics on catalysis were combined here: temperature dependence of intrinsic kinetic isotope effects (KIEs) that are sensitive to the physical nature of the chemical step, and protein mass-modulation that slows down fast dynamics (femto- to picosecond timescale) throughout the protein. The intrinsic H/T KIEs of human DHFR, like those of E. coli DHFR, are shown to be temperature-independent in the range from 5–45 °C, indicating fast sampling of donor and acceptor distances (DADs) at the reaction’s transition state (or tunneling ready state – TRS). Mass modulation of these enzymes through isotopic labeling with 13C, 15N, and 2H at nonexchangeable hydrogens yield an 11% heavier enzyme. The additional mass has no effect on the intrinsic KIEs of the human enzyme. This finding indicates that the mass-modulation of the human DHFR affects neither DAD distribution nor the DAD’s conformational sampling dynamics. Furthermore, reduction in the enzymatic turnover number and the dissociation rate constant for the product indicate that the isotopic substitution affects kinetic steps that are not the catalyzed C-H→C hydride transfer. The findings are discussed in terms of fast dynamics and their role in catalysis, the comparison of calculations and experiments, and the interpretation of isotopically-modulated heavy enzymes in general. PMID:26813442

  19. Decreased Migration of Langerhans Precursor-Like Cells in Response to Human Keratinocytes Expressing HPV-16 E6/E7 is Related to Reduced Macrophage Inflammatory Protein-3Alpha Production

    DTIC Science & Technology

    2005-01-01

    infection. Introduction. Human papillomaviruses (HPVs) are small DNA viruses which exhibit tropism for cutaneous or mucosal epithelium. Commonly...retinoblastoma protein correlates with the transforming capacity of the E7 oncoproteins of the human papillomaviruses . Proc Natl Acad Sci U S A 89:4442-6

  20. ModuleRole: a tool for modulization, role determination and visualization in protein-protein interaction networks.

    PubMed

    Li, Guipeng; Li, Ming; Zhang, Yiwei; Wang, Dong; Li, Rong; Guimerà, Roger; Gao, Juntao Tony; Zhang, Michael Q

    2014-01-01

    Rapidly increasing amounts of (physical and genetic) protein-protein interaction (PPI) data are produced by various high-throughput techniques, and interpretation of these data remains a major challenge. In order to gain insight into the organization and structure of the resultant large complex networks formed by interacting molecules, using simulated annealing, a method based on the node connectivity, we developed ModuleRole, a user-friendly web server tool which finds modules in PPI network and defines the roles for every node, and produces files for visualization in Cytoscape and Pajek. For given proteins, it analyzes the PPI network from BioGRID database, finds and visualizes the modules these proteins form, and then defines the role every node plays in this network, based on two topological parameters Participation Coefficient and Z-score. This is the first program which provides interactive and very friendly interface for biologists to find and visualize modules and roles of proteins in PPI network. It can be tested online at the website http://www.bioinfo.org/modulerole/index.php, which is free and open to all users and there is no login requirement, with demo data provided by "User Guide" in the menu Help. Non-server application of this program is considered for high-throughput data with more than 200 nodes or user's own interaction datasets. Users are able to bookmark the web link to the result page and access at a later time. As an interactive and highly customizable application, ModuleRole requires no expert knowledge in graph theory on the user side and can be used in both Linux and Windows system, thus a very useful tool for biologist to analyze and visualize PPI networks from databases such as BioGRID. ModuleRole is implemented in Java and C, and is freely available at http://www.bioinfo.org/modulerole/index.php. Supplementary information (user guide, demo data) is also available at this website. API for ModuleRole used for this program can be

  1. Isoform-Specific Degradation of PR-B by E6-AP Is Critical for Normal Mammary Gland Development

    PubMed Central

    Ramamoorthy, Sivapriya; Dhananjayan, Sarath C.; Demayo, Francesco J.; Nawaz, Zafar

    2010-01-01

    E6-associated protein (E6-AP), which was originally identified as an ubiquitin-protein ligase, also functions as a coactivator of estrogen (ER-α) and progesterone (PR) receptors. To investigate the in vivo role of E6-AP in mammary gland development, we generated transgenic mouse lines that either overexpress wild-type (WT) human E6-AP (E6-APWT) or ubiquitin-protein ligase-defective E6-AP (E6-APC833S) in the mammary gland. Here we show that overexpression of E6-APWT results in impaired mammary gland development. In contrast, overexpression of E6-APC833S or loss of E6-AP (E6-APKO) increases lateral branching and alveolus-like protuberances in the mammary gland. We also show that the mammary phenotypes observed in the E6-AP transgenic and knockout mice are due, in large part, to the alteration of PR-B protein levels. We also observed alteration in ER-α protein level, which might contribute to the observed mammary phenotype by regulating PR expression. Furthermore, E6-AP regulates PR-B protein levels via the ubiquitin-proteasome pathway. Additionally, we also show that E6-AP impairs progesterone-induced Wnt-4 expression by decreasing the steady state level of PR-B in both mice and in human breast cancer cells. In conclusion, we present the novel observation that E6-AP controls mammary gland development by regulating PR-B protein turnover via the ubiquitin proteasome pathway. For the first time, we show that the E3-ligase activity rather than the coactivation function of E6-AP plays an important role in the mammary gland development, and the ubiquitin-dependent PR-B degradation is not required for its transactivation functions. This mechanism appears to regulate normal mammogenesis, and dysregulation of this process may be an important contributor to mammary cancer development and progression. PMID:20829392

  2. Plant LysM proteins: modules mediating symbiosis and immunity.

    PubMed

    Gust, Andrea A; Willmann, Roland; Desaki, Yoshitake; Grabherr, Heini M; Nürnberger, Thorsten

    2012-08-01

    Microbial glycans, such as bacterial peptidoglycans, fungal chitin or rhizobacterial Nod factors (NFs), are important signatures for plant immune activation or for the establishment of beneficial symbioses. Plant lysin motif (LysM) domain proteins serve as modules mediating recognition of these different N-acetylglucosamine (GlcNAc)-containing ligands, suggesting that this class of proteins evolved from an ancient sensor for GlcNAc. During early plant evolution, these glycans probably served as immunogenic patterns activating LysM protein receptor-mediated plant immunity and stopping microbial infection. The biochemical potential of plant LysM proteins for sensing microbial GlcNAc-containing glycans has probably since favored the evolution of receptors facilitating microbial infection and symbiosis. Copyright © 2012 Elsevier Ltd. All rights reserved.

  3. [Modulators of the regulatory protein activity acting at microdoses].

    PubMed

    Iamskova, V P; Krasnov, M S; Skripnikova, V S; Moliavka, A A; Il'ina, A P; Margasiuk, D V; Borisenko, A V; Berezin, B B; Iamskov, I A

    2009-01-01

    New, previously not studied bioregulators active in the ultra low doses corresponding of 10(-8) - 10(-17) mg/ml have been isolated from vitreoretinal tissue of eye. It has been shown that these bioregulators comprise some regulatory peptides-modulators represented by proteins with molecular weights 15-70 KDa one of which is bovine serum albumin. Correlation between the nanosize of bioregulators and their ability to show activity in ultra low doses is established.

  4. Modulating nanoparticle superlattice structure using proteins with tunable bond distributions

    DOE PAGES

    McMillan, Janet R.; Brodin, Jeffrey D.; Millan, Jaime A.; ...

    2017-01-25

    Here, we investigate the use of proteins with tunable DNA modification distributions to modulate nanoparticle superlattice structure. Using Beta-galactosidase (βgal) as a model system, we have employed the orthogonal chemical reactivities of surface amines and thiols to synthesize protein-DNA conjugates with 36 evenly distributed or 8 specifically positioned oligonucleotides. When assembled into crystalline superlattices with AuNPs, we find that the distribution of DNA modifications modulates the favored structure: βgal with uniformly distributed DNA bonding elements results in body-centered cubic crystals, whereas DNA functionalization of cysteines results in AB2 packing. We probe the role of protein oligonucleotide number and conjugate sizemore » on this observation, which revealed the importance of oligonucleotide distribution and number in this observed assembly behavior. These results indicate that proteins with defined DNA-modification patterns are powerful tools to control the nanoparticle superlattices architecture, and establish the importance of oligonucleotide distribution in the assembly behavior of protein-DNA conjugates.« less

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

    PubMed

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

    2013-03-01

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

  6. Protein dynamics modulated electron transfer kinetics in early stage photosynthesis.

    PubMed

    Kundu, Prasanta; Dua, Arti

    2013-01-28

    A recent experiment has probed the electron transfer kinetics in the early stage of photosynthesis in Rhodobacter sphaeroides for the reaction center of wild type and different mutants [Science 316, 747 (2007)]. By monitoring the changes in the transient absorption of the donor-acceptor pair at 280 and 930 nm, both of which show non-exponential temporal decay, the experiment has provided a strong evidence that the initial electron transfer kinetics is modulated by the dynamics of protein backbone. In this work, we present a model where the electron transfer kinetics of the donor-acceptor pair is described along the reaction coordinate associated with the distance fluctuations in a protein backbone. The stochastic evolution of the reaction coordinate is described in terms of a non-Markovian generalized Langevin equation with a memory kernel and Gaussian colored noise, both of which are completely described in terms of the microscopics of the protein normal modes. This model provides excellent fits to the transient absorption signals at 280 and 930 nm associated with protein distance fluctuations and protein dynamics modulated electron transfer reaction, respectively. In contrast to previous models, the present work explains the microscopic origins of the non-exponential decay of the transient absorption curve at 280 nm in terms of multiple time scales of relaxation of the protein normal modes. Dynamic disorder in the reaction pathway due to protein conformational fluctuations which occur on time scales slower than or comparable to the electron transfer kinetics explains the microscopic origin of the non-exponential nature of the transient absorption decay at 930 nm. The theoretical estimates for the relative driving force for five different mutants are in close agreement with the experimental estimates obtained using electrochemical measurements.

  7. Protein dynamics modulated electron transfer kinetics in early stage photosynthesis

    NASA Astrophysics Data System (ADS)

    Kundu, Prasanta; Dua, Arti

    2013-01-01

    A recent experiment has probed the electron transfer kinetics in the early stage of photosynthesis in Rhodobacter sphaeroides for the reaction center of wild type and different mutants [Science 316, 747 (2007)]. By monitoring the changes in the transient absorption of the donor-acceptor pair at 280 and 930 nm, both of which show non-exponential temporal decay, the experiment has provided a strong evidence that the initial electron transfer kinetics is modulated by the dynamics of protein backbone. In this work, we present a model where the electron transfer kinetics of the donor-acceptor pair is described along the reaction coordinate associated with the distance fluctuations in a protein backbone. The stochastic evolution of the reaction coordinate is described in terms of a non-Markovian generalized Langevin equation with a memory kernel and Gaussian colored noise, both of which are completely described in terms of the microscopics of the protein normal modes. This model provides excellent fits to the transient absorption signals at 280 and 930 nm associated with protein distance fluctuations and protein dynamics modulated electron transfer reaction, respectively. In contrast to previous models, the present work explains the microscopic origins of the non-exponential decay of the transient absorption curve at 280 nm in terms of multiple time scales of relaxation of the protein normal modes. Dynamic disorder in the reaction pathway due to protein conformational fluctuations which occur on time scales slower than or comparable to the electron transfer kinetics explains the microscopic origin of the non-exponential nature of the transient absorption decay at 930 nm. The theoretical estimates for the relative driving force for five different mutants are in close agreement with the experimental estimates obtained using electrochemical measurements.

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

    NASA Astrophysics Data System (ADS)

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

    2013-03-01

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

  9. Radiosensitization of Oropharyngeal Squamous Cell Carcinoma Cells by Human Papillomavirus 16 Oncoprotein E6*I

    SciTech Connect

    Pang, Ervinna; Delic, Naomi C.; Hong, Angela; Zhang Mei; Rose, Barbara R.; Lyons, J. Guy

    2011-03-01

    Purpose: Patients with oropharyngeal squamous cell carcinoma (OSCC) whose disease is associated with high-risk human papillomavirus (HPV) infection have a significantly better outcome than those with HPV-negative disease, but the reasons for the better outcome are not known. We postulated that they might relate to an ability of HPV proteins to confer a better response to radiotherapy, a commonly used treatment for OSCC. Methods and Materials: We stably expressed the specific splicing-derived isoforms, E6*I and E6*II, or the entire E6 open reading frame (E6total), which gives rise to both full length and E6*I isoforms, in OSCC cell lines. Radiation resistance was measured in clonogenicity assays, p53 activity was measured using transfected reporter genes, and flow cytometry was used to analyze cell cycle and apoptosis. Results: E6*I and E6total sensitized the OSCC cells to irradiation, E6*I giving the greatest degree of radiosensitization (approximately eightfold lower surviving cell fraction at 10 Gy), whereas E6*II had no effect. In contrast to radiosensitivity, E6*I was a weaker inhibitor than E6total of tumor suppressor p53 transactivator activity in the same cells. Flow cytometric analyses showed that irradiated E6*I expressing cells had a much higher G2M:G1 ratio than control cells, indicating that, after G2, cells were diverted from the cell cycle to programmed cell death. Conclusion: This study supports a role for E6*I in the enhanced responsiveness of HPV-positive oropharyngeal carcinomas to p53-independent radiation-induced death.

  10. The HPV16 E6 oncoprotein and UVB irradiation inhibit the tumor suppressor TGFβ pathway in the epidermis of the K14E6 transgenic mouse.

    PubMed

    Popoca-Cuaya, Marco; Diaz-Chavez, Jose; Hernandez-Monge, Jesus; Alvarez-Rios, Elizabeth; Lambert, Paul F; Gariglio, Patricio

    2015-06-01

    High-risk human papillomaviruses (HR-HPVs) are the causative agents of cervical cancer, and they are also associated with a subset of head and neck squamous cell carcinomas. In addition, HPVs have also been postulated in the development of non-melanoma skin cancers (NMSC). In these cancers, the oncogene E6 is best known for its ability to inactivate the tumor suppressor p53 protein. Interestingly, in transgenic mice for HPV16 E6 (K14E6), it was reported that E6 alone induced epithelial hyperplasia and delay in differentiation in skin epidermis independently of p53 inactivation. Transforming growth factor β (TGFβ) is an important regulator of cell growth/differentiation and apoptosis, and this pathway is often lost during tumorigenesis. Ultraviolet radiation B (UVB) exposure activates diverse cellular responses, including DNA damage and apoptosis. In this study, we investigated whether the E6 oncogene alone or in combination with UVB dysregulate some components of the TGFβ pathway in the epidermis of K14E6 mice. We used 8-day-old K14E6 and non-transgenic mice irradiated and unirradiated with a single dose of UVB. We found that the E6 oncogene and UVB irradiation impair the TGFβ pathway in epidermis of K14E6 mice by downregulation of the TGFβ type II receptor (TβRII). This loss of TβRII prevents downstream activation of Smad2 and target genes as p15, an important regulator of cell cycle progression. In summary, the TGFβ signalling in cells of the epidermis is downregulated in our mouse model by both the E6 oncoprotein and the UVB irradiation. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  11. Exosome engineering for efficient intracellular delivery of soluble proteins using optically reversible protein-protein interaction module.

    PubMed

    Yim, Nambin; Ryu, Seung-Wook; Choi, Kyungsun; Lee, Kwang Ryeol; Lee, Seunghee; Choi, Hojun; Kim, Jeongjin; Shaker, Mohammed R; Sun, Woong; Park, Ji-Ho; Kim, Daesoo; Heo, Won Do; Choi, Chulhee

    2016-07-22

    Nanoparticle-mediated delivery of functional macromolecules is a promising method for treating a variety of human diseases. Among nanoparticles, cell-derived exosomes have recently been highlighted as a new therapeutic strategy for the in vivo delivery of nucleotides and chemical drugs. Here we describe a new tool for intracellular delivery of target proteins, named 'exosomes for protein loading via optically reversible protein-protein interactions' (EXPLORs). By integrating a reversible protein-protein interaction module controlled by blue light with the endogenous process of exosome biogenesis, we are able to successfully load cargo proteins into newly generated exosomes. Treatment with protein-loaded EXPLORs is shown to significantly increase intracellular levels of cargo proteins and their function in recipient cells in vitro and in vivo. These results clearly indicate the potential of EXPLORs as a mechanism for the efficient intracellular transfer of protein-based therapeutics into recipient cells and tissues.

  12. Cell-specific modulation of surfactant proteins by ambroxol treatment

    SciTech Connect

    Seifart, Carola . E-mail: zwiebel@mailer.uni-marburg.de; Clostermann, Ursula; Seifart, Ulf

    2005-02-15

    Ambroxol [trans-4-(2-amino-3,5-dibromobenzylamino)-cyclohexanole hydrochloride], a mucolytic agent, was postulated to provide surfactant stimulatory properties and was previously used to prevent surfactant deficiency. Currently, the underlying mechanisms are not exactly clear. Because surfactant homeostasis is regulated by surfactant-specific proteins (SP), we analyzed protein amount and mRNA expression in whole lung tissue, isolated type II pneumocytes and bronchoalveolar lavage of Sprague-Dawley rats treated with ambroxol i.p. (75 mg/kg body weight, twice a day [every 12 h]). The methods used included competitive polymerase chain reaction (RT-PCR), Northern blotting, Western immunoblotting, and immunohistochemistry. In isolated type II pneumocytes of ambroxol-treated animals, SP-C protein and mRNA content were increased, whereas SP-A, -B and -D protein, mRNA, and immunoreactivity remained unaffected. However, ambroxol treatment resulted in a significant increase of SP-B and in a decrease of SP-D in whole lung tissue with enhanced immunostaining for SP-B in Clara Cells. SP-A and SP-D were significantly decreased in BAL fluid of ambroxol-treated animals. The data suggest that surfactant protein expression is modulated in a cell-specific manner by ambroxol, as type II pneumocytes exhibited an increase in SP-C, whereas Clara cells exhibited an increase in the immunoreactivity for SP-B accounting for the increased SP-B content of whole lung tissue. The results indicate that ambroxol may exert its positive effects, observed in the treatment of diseases related to surfactant deficiency, via modulation of surfactant protein expression.

  13. Cell-specific modulation of surfactant proteins by ambroxol treatment.

    PubMed

    Seifart, Carola; Clostermann, Ursula; Seifart, Ulf; Müller, Bernd; Vogelmeier, Claus; von Wichert, Peter; Fehrenbach, Heinz

    2005-02-15

    Ambroxol [trans-4-(2-amino-3,5-dibromobenzylamino)-cyclohexanole hydrochloride], a mucolytic agent, was postulated to provide surfactant stimulatory properties and was previously used to prevent surfactant deficiency. Currently, the underlying mechanisms are not exactly clear. Because surfactant homeostasis is regulated by surfactant-specific proteins (SP), we analyzed protein amount and mRNA expression in whole lung tissue, isolated type II pneumocytes and bronchoalveolar lavage of Sprague-Dawley rats treated with ambroxol i.p. (75 mg/kg body weight, twice a day [every 12 h]). The methods used included competitive polymerase chain reaction (RT-PCR), Northern blotting, Western immunoblotting, and immunohistochemistry. In isolated type II pneumocytes of ambroxol-treated animals, SP-C protein and mRNA content were increased, whereas SP-A, -B and -D protein, mRNA, and immunoreactivity remained unaffected. However, ambroxol treatment resulted in a significant increase of SP-B and in a decrease of SP-D in whole lung tissue with enhanced immunostaining for SP-B in Clara Cells. SP-A and SP-D were significantly decreased in BAL fluid of ambroxol-treated animals. The data suggest that surfactant protein expression is modulated in a cell-specific manner by ambroxol, as type II pneumocytes exhibited an increase in SP-C, whereas Clara cells exhibited an increase in the immunoreactivity for SP-B accounting for the increased SP-B content of whole lung tissue. The results indicate that ambroxol may exert its positive effects, observed in the treatment of diseases related to surfactant deficiency, via modulation of surfactant protein expression.

  14. ACC-FMD: ant colony clustering for functional module detection in protein-protein interaction networks.

    PubMed

    Ji, Junzhong; Liu, Hongxin; Zhang, Aidong; Liu, Zhijun; Liu, Chunnian

    2015-01-01

    Mining functional modules in Protein-Protein Interaction (PPI) networks is a very important research for revealing the structure-functionality relationships in biological processes. More recently, some swarm intelligence algorithms have been successfully applied in the field. This paper presents a new nature-inspired approach, ACC-FMD, which is based on ant colony clustering to detect functional modules. First, some proteins with the higher clustering coefficients are, respectively, selected as ant seed nodes. And then, the picking and dropping operations based on ant probabilistic models are developed and employed to assign proteins into the corresponding clusters represented by seeds. Finally, the best clustering result in each generation is used to perform the information transmission by updating the similarly function. Experimental results on some benchmarked datasets show that ACC-FMD outperforms the CFinder and MCODE algorithms and has comparative performance with the MINE, COACH, DPClus and Core algorithms in terms of the general evaluation metrics.

  15. Periodic and stochastic thermal modulation of protein folding kinetics

    SciTech Connect

    Platkov, Max; Gruebele, Martin

    2014-07-21

    Chemical reactions are usually observed either by relaxation of a bulk sample after applying a sudden external perturbation, or by intrinsic fluctuations of a few molecules. Here we show that the two ideas can be combined to measure protein folding kinetics, either by periodic thermal modulation, or by creating artificial thermal noise that greatly exceeds natural thermal fluctuations. We study the folding reaction of the enzyme phosphoglycerate kinase driven by periodic temperature waveforms. As the temperature waveform unfolds and refolds the protein, its fluorescence color changes due to FRET (Förster resonant Energy Transfer) of two donor/acceptor fluorophores labeling the protein. We adapt a simple model of periodically driven kinetics that nicely fits the data at all temperatures and driving frequencies: The phase shifts of the periodic donor and acceptor fluorescence signals as a function of driving frequency reveal reaction rates. We also drive the reaction with stochastic temperature waveforms that produce thermal fluctuations much greater than natural fluctuations in the bulk. Such artificial thermal noise allows the recovery of weak underlying signals due to protein folding kinetics. This opens up the possibility for future detection of a stochastic resonance for protein folding subject to noise with controllable amplitude.

  16. Periodic and stochastic thermal modulation of protein folding kinetics.

    PubMed

    Platkov, Max; Gruebele, Martin

    2014-07-21

    Chemical reactions are usually observed either by relaxation of a bulk sample after applying a sudden external perturbation, or by intrinsic fluctuations of a few molecules. Here we show that the two ideas can be combined to measure protein folding kinetics, either by periodic thermal modulation, or by creating artificial thermal noise that greatly exceeds natural thermal fluctuations. We study the folding reaction of the enzyme phosphoglycerate kinase driven by periodic temperature waveforms. As the temperature waveform unfolds and refolds the protein, its fluorescence color changes due to FRET (Förster resonant Energy Transfer) of two donor/acceptor fluorophores labeling the protein. We adapt a simple model of periodically driven kinetics that nicely fits the data at all temperatures and driving frequencies: The phase shifts of the periodic donor and acceptor fluorescence signals as a function of driving frequency reveal reaction rates. We also drive the reaction with stochastic temperature waveforms that produce thermal fluctuations much greater than natural fluctuations in the bulk. Such artificial thermal noise allows the recovery of weak underlying signals due to protein folding kinetics. This opens up the possibility for future detection of a stochastic resonance for protein folding subject to noise with controllable amplitude.

  17. Modulation of Sertoli cell secretory function by rat round spermatid protein(s).

    PubMed

    Onoda, M; Djakiew, D

    1990-10-01

    The influence of rat round spermatid protein(s) (RSP) on protein synthesis and secretory function of Sertoli cells was used in the bicameral chamber system. Round spermatids (RS) were purified from 90-day-old rats by centrifugal elutriation. RS were incubated in a supplement-enriched culture medium that lacked exogenous proteins. The RS-conditioned media were dialysed and lyophilized to obtain RSP. Most de novo protein synthesized under basal conditions by Sertoli cells (18-day-old) was secreted into the apical chamber (apical/basal ratio: 3.42). Follicle-stimulating hormone (FSH, 100 ng/ml) stimulated total protein secretion from Sertoli cells by a factor of 1.54. The RSP (100 micrograms/ml) stimulated total protein secretion from Sertoli cells by a factor of 2.33. The enhancement of total Sertoli cell protein secretion by FSH and RSP additively increased by a factor of 2.82. The combined effect of FSH and RSP on total protein secretion from Sertoli cells was dose dependent and saturated at approximately 200 micrograms/ml of RSP. Polarity of total protein secretion from Sertoli cells (apical/basal ratio: 3.42) was stimulated by RSP predominantly in the apical direction (apical/basal ratio: 8.48). The modulation of radiolabeled Sertoli cell secretory proteins (ceruloplasmin, CP; sulfated glycoprotein-2, SGP-2; testins and transferrin, Tf) by cold (non-labeled) RSP was investigated by immunoprecipitation followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The secretion of CP, SGP-2 and Tf was stimulated in a dose-dependent manner by the addition of RSP up to a saturating concentration of between 200 and 300 micrograms/ml, whereas the secretion of Sertoli cell testins did not reach saturation at 300 micrograms/ml RSP. These results indicate that FSH and RSP independently modulate Sertoli cell protein secretion, and that Sertoli cell secretory proteins may differentially respond to RSP stimulation.

  18. Functional modulation of AMP-activated protein kinase by cereblon.

    PubMed

    Lee, Kwang Min; Jo, Sooyeon; Kim, Hyunyoung; Lee, Jongwon; Park, Chul-Seung

    2011-03-01

    Mutations in cereblon (CRBN), a substrate binding component of the E3 ubiquitin ligase complex, cause a form of mental retardation in humans. However, the cellular proteins that interact with CRBN remain largely unknown. Here, we report that CRBN directly interacts with the α1 subunit of AMP-activated protein kinase (AMPK α1) and inhibits the activation of AMPK activation. The ectopic expression of CRBN reduces phosphorylation of AMPK α1 and, thus, inhibits the enzyme in a nutrient-independent manner. Moreover, AMPK α1 can be potently activated by suppressing endogenous CRBN using CRBN-specific small hairpin RNAs. Thus, CRBN may act as a negative modulator of the AMPK signaling pathway in vivo.

  19. Small-molecule modulators of 14-3-3 protein-protein interactions.

    PubMed

    Ottmann, Christian

    2013-07-15

    14-3-3 Proteins are eukaryotic adapter proteins that regulate a plethora of physiological processes by binding to several hundred partner proteins. They play a role in biological activities as diverse as signal transduction, cell cycle regulation, apoptosis, host-pathogen interactions and metabolic control. As such, 14-3-3s are implicated in disease areas like cancer, neurodegeneration, diabetes, pulmonary disease, and obesity. Targeted modulation of 14-3-3 protein-protein interactions (PPIs) by small molecules is therefore an attractive concept for disease intervention. In recent years a number of examples of inhibitors and stabilizers of 14-3-3 PPIs have been reported promising a vivid future in chemical biology and drug development for this remarkable class of proteins. Copyright © 2012 Elsevier Ltd. All rights reserved.

  20. Protein-solvent preferential interactions, protein hydration, and the modulation of biochemical reactions by solvent components

    PubMed Central

    Timasheff, Serge N.

    2002-01-01

    Solvent additives (cosolvents, osmolytes) modulate biochemical reactions if, during the course of the reaction, there is a change in preferential interactions of solvent components with the reacting system. Preferential interactions can be expressed in terms of preferential binding of the cosolvent or its preferential exclusion (preferential hydration). The driving force is the perturbation by the protein of the chemical potential of the cosolvent. It is shown that the measured change of the amount of water in contact with protein during the course of the reaction modulated by an osmolyte is a change in preferential hydration that is strictly a measure of the cosolvent chemical potential perturbation by the protein in the ternary water–protein–cosolvent system. It is not equal to the change in water of hydration, because water of hydration is a reflection strictly of protein–water forces in a binary system. There is no direct relation between water of preferential hydration and water of hydration. PMID:12097640

  1. Discovery, Synthesis and Biological Evaluation of Novel SMN Protein Modulators

    PubMed Central

    Xiao, Jingbo; Marugan, Juan J.; Zheng, Wei; Titus, Steve; Southall, Noel; Cherry, Jonathan J.; Evans, Matthew; Androphy, Elliot J.; Austin, Christopher P.

    2011-01-01

    Spinal Muscular Atrophy (SMA) is an autosomal recessive disorder affecting the expression or function of survival motor neuron protein (SMN) due to the homozygous deletion or rare point mutations in the survival motor neuron gene 1 (SMN1). The human genome includes a second nearly identical gene called SMN2 that is retained in SMA. SMN2 transcripts undergo alternative splicing with reduced levels of SMN. Up-regulation of SMN2 expression, modification of its splicing, or inhibition of proteolysis of the truncated protein derived from SMN2 have been discussed as potential therapeutic strategies for SMA. In this manuscript, we detail the discovery of a series of arylpiperidines as novel modulators of SMN protein. Systematic hit-to-lead efforts significantly improved potency and efficacy of the series in the primary and orthogonal assays. Structure property relationships including microsomal stability, cell permeability and in vivo pharmacokinetics (PK) studies were also investigated. We anticipate that a lead candidate chosen from this series may serve as a useful probe for exploring the therapeutic benefits of SMN protein up-regulation in SMA animal models, and a starting point for clinical development. PMID:21819082

  2. Capacitance-modulated transistor detects odorant binding protein chiral interactions

    NASA Astrophysics Data System (ADS)

    Mulla, Mohammad Yusuf; Tuccori, Elena; Magliulo, Maria; Lattanzi, Gianluca; Palazzo, Gerardo; Persaud, Krishna; Torsi, Luisa

    2015-01-01

    Peripheral events in olfaction involve odorant binding proteins (OBPs) whose role in the recognition of different volatile chemicals is yet unclear. Here we report on the sensitive and quantitative measurement of the weak interactions associated with neutral enantiomers differentially binding to OBPs immobilized through a self-assembled monolayer to the gate of an organic bio-electronic transistor. The transduction is remarkably sensitive as the transistor output current is governed by the small capacitance of the protein layer undergoing minute changes as the ligand-protein complex is formed. Accurate determination of the free-energy balances and of the capacitance changes associated with the binding process allows derivation of the free-energy components as well as of the occurrence of conformational events associated with OBP ligand binding. Capacitance-modulated transistors open a new pathway for the study of ultra-weak molecular interactions in surface-bound protein-ligand complexes through an approach that combines bio-chemical and electronic thermodynamic parameters.

  3. 29 CFR 2584.8477(e)-6 - Definitions.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 29 Labor 9 2010-07-01 2010-07-01 false Definitions. 2584.8477(e)-6 Section 2584.8477(e)-6 Labor... FOR THE ALLOCATION OF FIDUCIARY RESPONSIBILITY § 2584.8477(e)-6 Definitions. As used in this part: (a... appointed pursuant to 5 U.S.C. 8474; (e) Fiduciary duty and fiduciary responsibility mean any duty or...

  4. Carbohydrate coingestion delays dietary protein digestion and absorption but does not modulate postprandial muscle protein accretion.

    PubMed

    Gorissen, Stefan H M; Burd, Nicholas A; Hamer, Henrike M; Gijsen, Annemie P; Groen, Bart B; van Loon, Luc J C

    2014-06-01

    Dietary protein digestion and absorption is an important factor modulating muscle protein accretion. However, there are few data available on the effects of coingesting other macronutrients with protein on digestion and absorption kinetics and the subsequent muscle protein synthetic response. The objective of the study was to determine the impact of carbohydrate coingestion with protein on dietary protein digestion and absorption and muscle protein accretion in healthy young and older men. Twenty-four healthy young (aged 21± 1 y, body mass index 21.8 ±0.5 kg/m(2)) and 25 older (aged 75 ± 1 y, body mass index 25.4 ± 0.6 kg/m(2)) men received a primed continuous L-[ring-(2)H5]-phenylalanine and L-[ring-3,5-(2)H2]-tyrosine infusion and ingested 20 g intrinsically L-[1-(13)C]-phenylalanine-labeled protein with (Pro+CHO) or without (Pro) 60 g carbohydrate. Plasma samples and muscle biopsies were collected in a postabsorptive and postprandial state. Carbohydrate coingestion delayed the appearance of exogenous phenylalanine in the circulation (P = .001). Dietary protein-derived phenylalanine availability over the 5-hour postprandial period was lower in the older (62 ± 2%) when compared with the young subjects (74 ± 2%; P = .007), with no differences between conditions (P = .20). Carbohydrate coingestion did not modulate postprandial muscle protein synthesis rates (0.035 ± 0.003 vs 0.043 ± 0.004 and 0.033 ± 0.002 vs 0.035 ± 0.003%/h after Pro vs Pro+CHO in the young and older group, respectively). In accordance, no differences in muscle protein-bound L-[1-(13)C]-phenylalanine enrichments were observed between conditions (0.020 ± 0.002 vs 0.020 ± 0.002 and 0.019 ± 0.003 vs 0.022 ± 0.004 mole percent excess after Pro vs Pro+CHO in the young and older subjects, respectively). Carbohydrate coingestion with protein delays dietary protein digestion and absorption but does not modulate postprandial muscle protein accretion in healthy young or older men.

  5. Human Papillomavirus 16 E6 Antibodies in Individuals without Diagnosed Cancer: A Pooled Analysis.

    PubMed

    Lang Kuhs, Krystle A; Anantharaman, Devasena; Waterboer, Tim; Johansson, Mattias; Brennan, Paul; Michel, Angelika; Willhauck-Fleckenstein, Martina; Purdue, Mark P; Holcátová, Ivana; Ahrens, Wolfgang; Lagiou, Pagona; Polesel, Jerry; Simonato, Lorenzo; Merletti, Franco; Healy, Claire M; Kjaerheim, Kristina; Conway, David I; Macfarlane, Tatiana V; Thomson, Peter; Castellsagué, Xavier; Znaor, Ariana; Black, Amanda; Huang, Wen-Yi; Krogh, Vittorio; Trichopoulou, Antonia; Bueno-de-Mesquita, H B As; Clavel-Chapelon, Françoise; Weiderpass, Elisabete; Ekström, Johanna; Riboli, Elio; Tjønneland, Anne; Sánchez, María-José; Travis, Ruth C; Hildesheim, Allan; Pawlita, Michael; Kreimer, Aimée R

    2015-04-01

    The increasing incidence of oropharyngeal cancer in many developed countries has been attributed to human papillomavirus type 16 (HPV16) infections. Recently, HPV16 E6 serology has been identified as a promising early marker for oropharyngeal cancer. Therefore, characterization of HPV16 E6 seropositivity among individuals without cancer is warranted. A total of 4,666 controls were pooled from several studies of cancer and HPV seropositivity, all tested within the same laboratory. HPV16 E6 seropositive controls were classified as having (i) moderate [mean fluorescent intensity (MFI) ≥ 484 and <1,000] or (ii) high seroreactivity (MFI ≥ 1,000). Associations of moderate and high HPV16 E6 seroreactivity with (i) demographic risk factors; and seropositivity for (ii) other HPV16 proteins (E1, E2, E4, E7, and L1), and (iii) E6 proteins from non-HPV16 types (HPV6, 11, 18, 31, 33, 45, and 52) were evaluated. Thirty-two (0.7%) HPV16 E6 seropositive controls were identified; 17 (0.4%) with moderate and 15 (0.3%) with high seroreactivity. High HPV16 E6 seroreactivity was associated with former smoking [odds ratio (OR), 5.5; 95% confidence interval (CI), 1.2-51.8], and seropositivity against HPV16 L1 (OR, 4.8; 95% CI, 1.3-15.4); E2 (OR, 7.7; 95% CI, 1.4-29.1); multiple HPV16 proteins (OR, 25.3; 95% CI, 2.6-119.6 for three HPV16 proteins beside E6) and HPV33 E6 (OR, 17.7; 95% CI, 1.9-81.8). No associations were observed with moderate HPV16 E6 seroreactivity. High HPV16 E6 seroreactivity is rare among individuals without diagnosed cancer and was not explained by demographic factors. Some HPV16 E6 seropositive individuals without diagnosed HPV-driven cancer, especially those with seropositivity against other HPV16 proteins, may harbor a biologically relevant HPV16 infection. ©2015 American Association for Cancer Research.

  6. E6/E7-P53-POU2F1-CTHRC1 axis promotes cervical cancer metastasis and activates Wnt/PCP pathway

    PubMed Central

    Zhang, Rong; Lu, Huan; Lyu, Yuan-yuan; Yang, Xiao-mei; Zhu, Lin-yan; Yang, Guang-dong; Jiang, Peng-cheng; Re, Yuan; Song, Wei-wei; Wang, Jin-hao; Zhang, Can-can; Gu, Fei; Luo, Tian-jiao; Wu, Zhi-yong; Xu, Cong-jian

    2017-01-01

    Cervical cancer is an infectious cancer and the most common gynecologic cancer worldwide. E6/E7, the early genes of the high-risk mucosal human papillomavirus type, play key roles in the carcinogenic process of cervical cancer. However, little was known about its roles in modulating tumor microenvironment, particular extracellular matrix (ECM). In this study, we found that E6/E7 could regulate multiple ECM proteins, especially collagen triple helix repeat containing 1 (CTHRC1). CTHRC1 is highly expressed in cervical cancer tissue and serum and closely correlated with clinicopathological parameters. CTHRC1 promotes cervical cancer cell migration and invasion in vitro and metastasis in vivo. E6/E7 regulates the expression of CTHRC1 in cervical cancer by E6/E7-p53-POU2F1 (POU class 2 homeobox 1) axis. Futhermore, CTHRC1 activates Wnt/PCP signaling pathway. Take together, E6/E7-p53-POU2F1-CTHRC1 axis promotes cervical cancer cell invasion and metastasis and may act as a potential therapeutic target for interventions against cervical cancer invasion and metastasis. PMID:28303973

  7. Capacitance-modulated transistor detects odorant binding protein chiral interactions

    PubMed Central

    Mulla, Mohammad Yusuf; Tuccori, Elena; Magliulo, Maria; Lattanzi, Gianluca; Palazzo, Gerardo; Persaud, Krishna; Torsi, Luisa

    2015-01-01

    Peripheral events in olfaction involve odorant binding proteins (OBPs) whose role in the recognition of different volatile chemicals is yet unclear. Here we report on the sensitive and quantitative measurement of the weak interactions associated with neutral enantiomers differentially binding to OBPs immobilized through a self-assembled monolayer to the gate of an organic bio-electronic transistor. The transduction is remarkably sensitive as the transistor output current is governed by the small capacitance of the protein layer undergoing minute changes as the ligand–protein complex is formed. Accurate determination of the free-energy balances and of the capacitance changes associated with the binding process allows derivation of the free-energy components as well as of the occurrence of conformational events associated with OBP ligand binding. Capacitance-modulated transistors open a new pathway for the study of ultra-weak molecular interactions in surface-bound protein–ligand complexes through an approach that combines bio-chemical and electronic thermodynamic parameters. PMID:25591754

  8. HPV-16 E6 promotes cell growth of esophageal cancer via downregulation of miR-125b and activation of Wnt/β-catenin signaling pathway.

    PubMed

    Zang, Bao; Huang, Guojin; Wang, Xiaowei; Zheng, Shiying

    2015-01-01

    High-risk human papillomavirus (HPV) is a possible cause of esophageal cancer. However, the molecular pathogenesis of HPV-infected esophageal cancer remains unclear. The expression levels of some microRNAs including miR-125b have been negatively correlated with HPV infection, and miR-125b downregulation is associated with tumorigenesis. In addition, Wnt/β-catenin signaling pathway has been suggested to play an important role in esophageal cancer (EC). We examined miR-125b and Wnt/β-catenin signaling pathway in HPV-16 E6 promoted tumor progression in EC. HPV-16 E6 transfection decreased markedly the expression levels of miR-125b and promoted the colony formation in the Eca 109 and Kyse 150 cell lines, and restoration of miR-125b expression level antagonized the increased colony formation in HPV-16 E6 transfected cell lines. We also demonstrated that overexpression of E6 upregulated the Wnt/β-catenin signaling activity via modulating the multiple regulators including TLE1, GSK3β, and sFRP4. Overexpression of miR-125b restored the expression levels of these proteins. Expression of miR-125b was lower in HPV-16 E6 positive esophageal cancer tissues, and was negatively correlated with E6 mRNA levels. Our results indicate that HPV-16 E6 promotes tumorigenesis in EC via down-regulation of miR-125b, and this underlying mechanism may be involved in the activation of the Wnt/β-catenin signaling pathway.

  9. HPV-16 E6/E7 promotes cell migration and invasion in cervical cancer via regulating cadherin switch in vitro and in vivo.

    PubMed

    Hu, Dongxiao; Zhou, Jiansong; Wang, Fenfen; Shi, Haiyan; Li, Yang; Li, Baohua

    2015-12-01

    Cadherin switch, as a key hallmark of epithelial-mesenchymal transition (EMT), is characterized by reduced E-cadherin expression and increased N-cadherin or P-cadherin expression, and has been implicated in many aggressive tumors, but the importance and regulatory mechanism of cadherin switch in cervical cancer have not been investigated. Our study aimed to explore the role of cadherin switch by regulation of HPV-16 E6/E7 in progression and metastasis of cervical cancer. The expressions of E-cadherin and P-cadherin were examined by immunohistochemical staining in 40 cases of high-grade cervical lesions with HPV-16 infection only in which HPV-16 E6 and E7 expression had been detected using qRT-PCR method. Through modulating E6 and E7 expression using HPV-16 E6/E7 promoter-targeting siRNAs or expressed vector in vitro, cell growth, migration, and invasion were separately tested by MTT, wound-healing and transwell invasion assays, as well as the expressions of these cadherins by western blot analyses. Finally, the expressions of these cadherins in cancerous tissues of BALB/c-nu mouse model inoculated with the stable HPV-16 E6/E7 gene silencing Siha and Caski cells were also measured by immunohistochemical staining. Pearson correlation coefficient analyses showed the strongly inverse correlation of E-cadherin expression and strongly positive correlation of P-cadherin expression with E6/E7 level in 40 cases of high-grade cervical lesions. Furthermore, the modulation of HPV-16 E6/E7 expression remarkably influenced cell proliferation, migration, and invasion, as well as the protein levels of E-cadherin and P-cadherin in cervical cell lines. Finally, the reduction of HPV-16 E6/E7 expression led to up-regulated expression of E-cadherin and down-regulated expression of P-cadherin in BALB/c-nu mouse model in vivo assay. Our results unraveled the possibility that HPV-16 E6/E7 could promote cell invasive potential via regulating cadherin switching, and consequently contribute

  10. Comparison of module detection algorithms in protein networks and investigation of the biological meaning of predicted modules.

    PubMed

    Tripathi, Shailesh; Moutari, Salissou; Dehmer, Matthias; Emmert-Streib, Frank

    2016-03-18

    It is generally acknowledged that a functional understanding of a biological system can only be obtained by an understanding of the collective of molecular interactions in form of biological networks. Protein networks are one particular network type of special importance, because proteins form the functional base units of every biological cell. On a mesoscopic level of protein networks, modules are of significant importance because these building blocks may be the next elementary functional level above individual proteins allowing to gain insight into fundamental organizational principles of biological cells. In this paper, we provide a comparative analysis of five popular and four novel module detection algorithms. We study these module prediction methods for simulated benchmark networks as well as 10 biological protein interaction networks (PINs). A particular focus of our analysis is placed on the biological meaning of the predicted modules by utilizing the Gene Ontology (GO) database as gold standard for the definition of biological processes. Furthermore, we investigate the robustness of the results by perturbing the PINs simulating in this way our incomplete knowledge of protein networks. Overall, our study reveals that there is a large heterogeneity among the different module prediction algorithms if one zooms-in the biological level of biological processes in the form of GO terms and all methods are severely affected by a slight perturbation of the networks. However, we also find pathways that are enriched in multiple modules, which could provide important information about the hierarchical organization of the system.

  11. Natural Modulators of Amyloid-Beta Precursor Protein Processing

    PubMed Central

    Zhang, Can; Tanzi, Rudolph E.

    2013-01-01

    Alzheimer’s disease (AD) is a devastating neurodegenerative disease and the primary cause of dementia, with no cure currently available. The pathogenesis of AD is believed to be primarily driven by Aβ, the principal component of senile plaques. Aβ is an ~4 kDa peptide generated from the amyloid-β precursor protein (APP) through proteolytic secretases. Natural products, particularly those utilized in traditional Chinese medicine (TCM), have a long history alleviating common clinical disorders, including dementia. However, the cell/molecular pathways mediated by these natural products are largely unknown until recently when the underlying molecular mechanisms of the disorders begin to be elucidated. Here, the mechanisms with which natural products modulate the pathogenesis of AD are discussed, in particular, by focusing on their roles in the processing of APP. PMID:22998566

  12. Modulation of corneal and stromal matrix metalloproteinase by the mannose-induced Acanthamoeba cytolytic protein

    PubMed Central

    Alizadeh, Hassan; Li, Haochuan; Neelam, Sudha; Niederkorn, Jerry Y.

    2008-01-01

    The involvement of the mannose-induced Acanthamoeba cytopathic protein (MIP-133) in tissue injury and activation of metalloproteinase of corneal and stromal cells was examined in vitro. Activation of MMP-1, MMP-2, MMP-3, and MMP-9 induced by MIP-133 on human corneal epithelial and stromal cell cultures was examined by reverse transcriptase polymerase chain reaction (RT-PCR), and ELISA. MMP-1, MMP-2, MMP-3, and MMP-9 mRNA were expressed in both cultured human corneal epithelial and stromal cells. When the epithelial cells were exposed to MIP-133 protein, the mRNA expression for MMP-1 and MMP-9 was unchanged. However, the transcript for MMP-2 and MMP-3 was decreased by two fold. By contrast, the expression of MMP-2 and MMP-3 was significantly up-regulated (2–4 fold) in the corneal stromal cells 1, 4, and 8 hours after MIP-133 stimulation. At the protein level, there was no significant difference in the level of MMPs between the corneal epithelial cells before and after stimulation with MIP-133. By contrast, the levels of MMP-2 and MMP-3 were significantly higher in the corneal stromal cells stimulated with MIP-133. The supernatants from corneal stromal cells stimulated with MIP-133 were incubated with PMSF and MIP-133 antibody and the level of MMP-2 was measured by ELISA. Activation of MMP-2 by MIP-133 was inhibited in the supernatants pretreated with the serine protease inhibitor, PMSF, and anti-MIP-133. Supernatants pretreated with the cysteine protease inhibitor E6 or control antibody produced the same amount of MMP-2 as the untreated supernatants. To verify the possible of homology between MMPs and A. castellanii proteases, the mRNA from A. castellanii was prepared and analyzed for the expression of MMP genes by PT-PCR. The results showed that A. castellanii did not express mRNA for MMP-1, MMP-2, MMP-3, or MMP-9. Thus, A. castellanii mRNA does not cross react with human MMPs. Furthermore, ELISA was used to determine the cross reactivity of MMP antibodies with

  13. Protein Stability and Dynamics Modulation: The Case of Human Frataxin

    PubMed Central

    Gallo, Mariana; Salvay, Andres G.; Ferreiro, Diego U.; Santos, Javier

    2012-01-01

    Frataxin (FXN) is an α/β protein that plays an essential role in iron homeostasis. Apparently, the function of human FXN (hFXN) depends on the cooperative formation of crucial interactions between helix α1, helix α2, and the C-terminal region (CTR) of the protein. In this work we quantitatively explore these relationships using a purified recombinant fragment hFXN90–195. This variant shows the hydrodynamic behavior expected for a monomeric globular domain. Circular dichroism, fluorescence, and NMR spectroscopies show that hFXN90–195 presents native-like secondary and tertiary structure. However, chemical and temperature induced denaturation show that CTR truncation significantly destabilizes the overall hFXN fold. Accordingly, limited proteolysis experiments suggest that the native-state dynamics of hFXN90–195 and hFXN90–210 are indeed different, being the former form much more sensitive to the protease at specific sites. The overall folding dynamics of hFXN fold was further explored with structure-based protein folding simulations. These suggest that the native ensemble of hFXN can be decomposed in at least two substates, one with consolidation of the CTR and the other without consolidation of the CTR. Explicit-solvent all atom simulations identify some of the proteolytic target sites as flexible regions of the protein. We propose that the local unfolding of CTR may be a critical step for the global unfolding of hFXN, and that modulation of the CTR interactions may strongly affect hFXN physiological function. PMID:23049850

  14. Modulation of the Chromatin Phosphoproteome by the Haspin Protein Kinase*

    PubMed Central

    Maiolica, Alessio; de Medina-Redondo, Maria; Schoof, Erwin M.; Chaikuad, Apirat; Villa, Fabrizio; Gatti, Marco; Jeganathan, Siva; Lou, Hua Jane; Novy, Karel; Hauri, Simon; Toprak, Umut H.; Herzog, Franz; Meraldi, Patrick; Penengo, Lorenza; Turk, Benjamin E.; Knapp, Stefan; Linding, Rune; Aebersold, Ruedi

    2014-01-01

    Recent discoveries have highlighted the importance of Haspin kinase activity for the correct positioning of the kinase Aurora B at the centromere. Haspin phosphorylates Thr3 of the histone H3 (H3), which provides a signal for Aurora B to localize to the centromere of mitotic chromosomes. To date, histone H3 is the only confirmed Haspin substrate. We used a combination of biochemical, pharmacological, and mass spectrometric approaches to study the consequences of Haspin inhibition in mitotic cells. We quantified 3964 phosphorylation sites on chromatin-associated proteins and identified a Haspin protein-protein interaction network. We determined the Haspin consensus motif and the co-crystal structure of the kinase with the histone H3 tail. The structure revealed a unique bent substrate binding mode positioning the histone H3 residues Arg2 and Lys4 adjacent to the Haspin phosphorylated threonine into acidic binding pockets. This unique conformation of the kinase-substrate complex explains the reported modulation of Haspin activity by methylation of Lys4 of the histone H3. In addition, the identification of the structural basis of substrate recognition and the amino acid sequence preferences of Haspin aided the identification of novel candidate Haspin substrates. In particular, we validated the phosphorylation of Ser137 of the histone variant macroH2A as a target of Haspin kinase activity. MacroH2A Ser137 resides in a basic stretch of about 40 amino acids that is required to stabilize extranucleosomal DNA, suggesting that phosphorylation of Ser137 might regulate the interactions of macroH2A and DNA. Overall, our data suggest that Haspin activity affects the phosphorylation state of proteins involved in gene expression regulation and splicing. PMID:24732914

  15. Characterization and Modulation of Proteins Involved in SM Vesication

    DTIC Science & Technology

    2005-05-01

    lines such as HaCaT involve a number of undefined changes that occur over time in culture. We also found that NcoI cells , KC immortalized with HPV16 E6/7... lines such as HaCaT cells , we immortalized cells with a defined and physiologically relevant agent 21, HPV16 E6/7 (above), and utilized at passage 30...the retroviral vector, LXSN (Fig. 2A; Clontech). Following transient transfection of an amphotropic retrovirus packaging cell line (φNX; Gary Nolan

  16. Ankyrin-repeat proteins from sponge symbionts modulate amoebal phagocytosis.

    PubMed

    Nguyen, Mary T H D; Liu, Michael; Thomas, Torsten

    2014-03-01

    Bacteria-eukaryote symbiosis occurs in all stages of evolution, from simple amoebae to mammals, and from facultative to obligate associations. Sponges are ancient metazoans that form intimate symbiotic interactions with complex communities of bacteria. The basic nutritional requirements of the sponge are in part satisfied by the phagocytosis of bacterial food particles from the surrounding water. How bacterial symbionts, which are permanently associated with the sponge, survive in the presence of phagocytic cells is largely unknown. Here, we present the discovery of a genomic fragment from an uncultured gamma-proteobacterial sponge symbiont that encodes for four proteins, whose closest known relatives are found in a sponge genome. Through recombinant approaches, we show that these four eukaryotic-like, ankyrin-repeat proteins (ARP) when expressed in Eschericha coli can modulate phagocytosis of amoebal cells and lead to accumulation of bacteria in the phagosome. Mechanistically, two ARPs appear to interfere with phagosome development in a similar way to reduced vacuole acidification, by blocking the fusion of the early phagosome with the lysosome and its digestive enzymes. Our results show that ARP from sponge symbionts can function to interfere with phagocytosis, and we postulate that this might be one mechanism by which symbionts can escape digestion in a sponge host.

  17. C-reactive protein modulates human lung fibroblast migration.

    PubMed

    Kikuchi, Kazuhiko; Kohyama, Tadashi; Yamauchi, Yasuhiro; Kato, Jun; Takami, Kazutaka; Okazaki, Hitoshi; Desaki, Masashi; Nagase, Takahide; Rennard, Stephen I; Takizawa, Hajime

    2009-02-01

    C-reactive protein (CRP) has been classically used as a marker of inflammation. The aim of this study was to investigate the effect of CRP on migration of human fetal lung fibroblasts (HFL-1) to human plasma fibronectin (HFn). Using the blindwell chamber technique, CRP inhibited HFL-1 migration in a dose-dependent fashion (at 1 microg/mL, inhibition: 32.5% +/- 7.1%; P < .05). Western blot analysis showed that CRP inhibited the p38 mitogen-activated protein kinase (MAPK) activity in the presence of HFn. Moreover, the MAPK inhibitors SB202190 (25 microM) and SB203580 (25 microM) inhibited HFn-induced cell migration, suggesting an important role of p38 MAPK in HFn-induced migration. Taken together, these results suggest that the inhibitory effect of CRP is mediated by blocking MAPK. In summary, this study demonstrates that CRP directly modulates human lung fibroblasts migration. Thus, CRP may contribute to regulation of wound healing and may be endogenous antifibrotic factor acting on lung fibrosis.

  18. Cholinergic modulation of anaphylactic shock: plasma proteins influence.

    PubMed

    Nezhinskaya, Galina I; Vladykin, Aleksandr L; Sapronov, Nikolay S

    2007-05-30

    Cholinergic drugs can modulate anaphylactic shock and change lymphocyte functions. Plasma proteins modulate effects of muscarinic antagonists during anaphylactic shock. The present investigation was carried out to study the antianaphylactic activity of methacine (antagonist at muscarinic receptors) in combination with neostigmine (anticholinesterase drug). However, it is not known whether plasma proteins-albumin, C-reactive protein (CRP) and immunoglobulin G (IgG) - modify the effects of cholinergic drugs like methacine, serotonin (5-HT) level in the lymphoid organs and quantity of antibody-forming cells (AFC) in the spleen of guinea pigs during experimental anaphylactic shock. It was shown that administration of methacine with neostigmine (40 min and 15 min prior to shock induction, accordingly) at the pathochemical stage revokes shock development. By blocking cholinesterase endogenous acetylcholine is increased and methacine blocks muscarinic receptors and therewith unwanted side effects in the airways (bronchoconstriction) and heart (bradycardia). Administration of the combination of methacine with neostigmine at the immunological stage (guinea pig sensitization) does not affect the course of anaphylactic shock. Administration of methacine with IgG at the pathochemical stage of shock significantly decreases shock intensity, while administration of methacine with CRP or albumin has no influence on the shock. Administration of IgG or CRP (not albumin) at the immunological stage of shock and albumin or IgG (not CRP) at the pathochemical stage leads to reduction of the anaphylactic reaction. Application of methacine with neostigmine or IgG (effective combinations of drugs) results in normalization of antibody response in the spleen and 5-HT level in the lymphoid organs. Administration of methacine with CRP or albumin (ineffective combinations of drugs) leads to increase of antibody response in the spleen and 5-HT level in the lymphoid organs. Administration of

  19. α-Hemoglobin stabilizing protein: a modulating factor in thalassemias?

    PubMed

    Wajcman, Henri; Vasseur, Corinne; Pissard, Serge; Baudin-Creuza, Veronique

    2011-01-01

    α-Hemoglobin stabilizing protein (AHSP) is a small protein of 102 residues induced by GATA-1, Oct-1- and EKLF. It is synthesized at a high level in the red blood cell precursors and acts as a chaperone protecting the α-hemoglobin (α-Hb) chains against precipitation. α-Hemoglobin stabilizing protein forms a heterodimer complex with α-Hb, then displaying modified oxygen binding kinetics. In the absence of AHSP, α-Hb oxidizes and precipitates within the erythrocyte precursors of bone marrow leading to apoptosis and defective erythropoiesis. Several α-Hb variants with a structural abnormality, frequently located in the contact area between α-Hb and AHSP, exhibit instability and a thalassemia-like syndrome when they are associated with another α-thalassemia (α-thal) determinant. We suggest that this disorder could result from a disturbed interaction between the abnormal α-Hb chains and AHSP. Hb Groene Hart (Pro119>Ser) was one of the first examples in which we observed this abnormality. We later verified this mechanism in a list of several variants, now considered as being nondeletional α-thalassemias. Conversely, it was hypothesized from studies on knock-out mice, that a defect affecting AHSP could cause a thalassemia-like syndrome. This was supported in man by studies showing that a decreased expression of AHSP linked to specific genetic clades could act as a modulating factor in some thalassemia phenotypes. It was also supported by our observation of a family from Southeast Asia, in which a child homozygous for an AHSP mutant (Val56>Gly) displayed, in his first year of life, a moderate thalassemia syndrome. This mutant AHSP was expressed in vitro and demonstrated by biochemical and biophysical studies to display a clear defective interaction with α-Hb, which could support the hypothesis that the reb blood cell (RBC) disorders of the child resulted from this abnormality. It therefore appears that AHSP is a factor with a key role in the formation of Hb

  20. Detection of innate immune response modulating impurities in therapeutic proteins.

    PubMed

    Haile, Lydia Asrat; Puig, Montserrat; Kelley-Baker, Logan; Verthelyi, Daniela

    2015-01-01

    Therapeutic proteins can contain multiple impurities, some of which are variants of the product, while others are derived from the cell substrate and the manufacturing process. Such impurities, even when present at trace levels, have the potential to activate innate immune cells in peripheral blood or embedded in tissues causing expression of cytokines and chemokines, increasing antigen uptake, facilitating processing and presentation by antigen presenting cells, and fostering product immunogenicity. Currently, while products are tested for host cell protein content, assays to control innate immune response modulating impurities (IIRMIs) in products are focused mainly on endotoxin and nucleic acids, however, depending on the cell substrate and the manufacturing process, numerous other IIRMI could be present. In these studies we assess two approaches that allow for the detection of a broader subset of IIRMIs. In the first, we use commercial cell lines transfected with Toll like receptors (TLR) to detect receptor-specific agonists. This method is sensitive to trace levels of IIRMI and provides information of the type of IIRMIs present but is limited by the availability of stably transfected cell lines and requires pre-existing knowledge of the IIRMIs likely to be present in the product. Alternatively, the use of a combination of macrophage cell lines of human and mouse origin allows for the detection of a broader spectrum of impurities, but does not identify the source of the activation. Importantly, for either system the lower limit of detection (LLOD) of impurities was similar to that of PBMC and it was not modified by the therapeutic protein tested, even in settings where the product had inherent immune modulatory properties. Together these data indicate that a cell-based assay approach could be used to screen products for the presence of IIRMIs and inform immunogenicity risk assessments, particularly in the context of comparability exercises.

  1. Protein Modulator of Multidrug Efflux Gene Expression in Pseudomonas aeruginosa▿

    PubMed Central

    Daigle, Denis M.; Cao, Lily; Fraud, Sebastien; Wilke, Mark S.; Pacey, Angela; Klinoski, Rachael; Strynadka, Natalie C.; Dean, Charles R.; Poole, Keith

    2007-01-01

    nalC multidrug-resistant mutants of Pseudomonas aeruginosa show enhanced expression of the mexAB-oprM multidrug efflux system as a direct result of the production of a ca. 6,100-Da protein, PA3719, in these mutants. Using a bacterial two-hybrid system, PA3719 was shown to interact in vivo with MexR, a repressor of mexAB-oprM expression. Isothermal titration calorimetry (ITC) studies confirmed a high-affinity interaction (equilibrium dissociation constant [KD], 158.0 ± 18.1 nM) of PA3719 with MexR in vitro. PA3719 binding to and formation of a complex with MexR obviated repressor binding to its operator, which overlaps the efflux operon promoter, suggesting that mexAB-oprM hyperexpression in nalC mutants results from PA3719 modulation of MexR repressor activity. Consistent with this, MexR repression of mexA transcription in an in vitro transcription assay was alleviated by PA3719. Mutations in MexR compromising its interaction with PA3719 in vivo were isolated and shown to be located internally and distributed throughout the protein, suggesting that they impacted PA3719 binding by altering MexR structure or conformation rather than by having residues interacting specifically with PA3719. Four of six mutant MexR proteins studied retained repressor activity even in a nalC strain producing PA3719. Again, this is consistent with a PA3719 interaction with MexR being necessary to obviate MexR repressor activity. The gene encoding PA3719 has thus been renamed armR (antirepressor for MexR). A representative “noninteracting” mutant MexR protein, MexRI104F, was purified, and ITC confirmed that it bound PA3719 with reduced affinity (5.4-fold reduced; KD, 853.2 ± 151.1 nM). Consistent with this, MexRI104F repressor activity, as assessed using the in vitro transcription assay, was only weakly compromised by PA3719. Finally, two mutations (L36P and W45A) in ArmR compromising its interaction with MexR have been isolated and mapped to a putative C-terminal α-helix of the

  2. Features, processing states and heterologous protein interactions in the modulation of the retroviral nucleocapsid protein function

    PubMed Central

    Mirambeau, Gilles; Lyonnais, Sébastien

    2010-01-01

    Nucleocapsid (NC) is central to retroviral replication. Nucleic acid chaperoning is a key function for NC through the action of its conserved basic amino acids and zinc-finger structures. NC manipulates genomic RNA from its packaging in the producer cell to reverse transcription into the infected host cell. This chaperone function, in conjunction with NCs aggregating properties, is up-modulated by successive NC processing events, from the Gag precursor to the fully mature protein, resulting in the condensation of the nucleocapsid within the capsid shell. Reverse transcription also depends on NC processing, whereas this process provokes NC dissociation from double-stranded DNA, leading to a preintegration complex (PIC), competent for host chromosomal integration. In addition NC interacts with cellular proteins, some of which are involved in viral budding, and also with several viral proteins. All of these properties are reviewed here, focusing on HIV-1 as a paradigmatic reference and highlighting the plasticity of the nucleocapsid architecture. PMID:21045549

  3. Isoform-specific degradation of PR-B by E6-AP is critical for normal mammary gland development.

    PubMed

    Ramamoorthy, Sivapriya; Dhananjayan, Sarath C; Demayo, Francesco J; Nawaz, Zafar

    2010-11-01

    E6-associated protein (E6-AP), which was originally identified as an ubiquitin-protein ligase, also functions as a coactivator of estrogen (ER-α) and progesterone (PR) receptors. To investigate the in vivo role of E6-AP in mammary gland development, we generated transgenic mouse lines that either overexpress wild-type (WT) human E6-AP (E6-AP(WT)) or ubiquitin-protein ligase-defective E6-AP (E6-AP(C833S)) in the mammary gland. Here we show that overexpression of E6-AP(WT) results in impaired mammary gland development. In contrast, overexpression of E6-AP(C833S) or loss of E6-AP (E6-AP(KO)) increases lateral branching and alveolus-like protuberances in the mammary gland. We also show that the mammary phenotypes observed in the E6-AP transgenic and knockout mice are due, in large part, to the alteration of PR-B protein levels. We also observed alteration in ER-α protein level, which might contribute to the observed mammary phenotype by regulating PR expression. Furthermore, E6-AP regulates PR-B protein levels via the ubiquitin-proteasome pathway. Additionally, we also show that E6-AP impairs progesterone-induced Wnt-4 expression by decreasing the steady state level of PR-B in both mice and in human breast cancer cells. In conclusion, we present the novel observation that E6-AP controls mammary gland development by regulating PR-B protein turnover via the ubiquitin proteasome pathway. For the first time, we show that the E3-ligase activity rather than the coactivation function of E6-AP plays an important role in the mammary gland development, and the ubiquitin-dependent PR-B degradation is not required for its transactivation functions. This mechanism appears to regulate normal mammogenesis, and dysregulation of this process may be an important contributor to mammary cancer development and progression.

  4. A Drosophila Model of HPV E6-Induced Malignancy Reveals Essential Roles for Magi and the Insulin Receptor

    PubMed Central

    Padash Barmchi, Mojgan; Gilbert, Mary; Thomas, Miranda; Banks, Lawrence; Zhang, Bing; Auld, Vanessa J.

    2016-01-01

    Cervical cancer is one of the leading causes of cancer death in women worldwide. The causative agents of cervical cancers, high-risk human papillomaviruses (HPVs), cause cancer through the action of two oncoproteins, E6 and E7. The E6 oncoprotein cooperates with an E3 ubiquitin ligase (UBE3A) to target the p53 tumour suppressor and important polarity and junctional PDZ proteins for proteasomal degradation, activities that are believed to contribute towards malignancy. However, the causative link between degradation of PDZ proteins and E6-mediated malignancy is largely unknown. We have developed an in vivo model of HPV E6-mediated cellular transformation using the genetic model organism, Drosophila melanogaster. Co-expression of E6 and human UBE3A in wing and eye epithelia results in severe morphological abnormalities. Furthermore, E6, via its PDZ-binding motif and in cooperation with UBE3A, targets a suite of PDZ proteins that are conserved in human and Drosophila, including Magi, Dlg and Scribble. Similar to human epithelia, Drosophila Magi is a major degradation target. Magi overexpression rescues the cellular abnormalities caused by E6+UBE3A coexpression and this activity of Magi is PDZ domain-dependent. Drosophila p53 was not targeted by E6+UBE3A, and E6+UBE3A activity alone is not sufficient to induce tumorigenesis, which only occurs when E6+UBE3A are expressed in conjunction with activated/oncogenic forms of Ras or Notch. Finally, through a genetic screen we have identified the insulin receptor signaling pathway as being required for E6+UBE3A induced hyperplasia. Our results suggest a highly conserved mechanism of HPV E6 mediated cellular transformation, and establish a powerful genetic model to identify and understand the cellular mechanisms that underlie HPV E6-induced malignancy. PMID:27537218

  5. Prion protein self-peptides modulate prion interactions and conversion

    PubMed Central

    2009-01-01

    Background Molecular mechanisms underlying prion agent replication, converting host-encoded cellular prion protein (PrPC) into the scrapie associated isoform (PrPSc), are poorly understood. Selective self-interaction between PrP molecules forms a basis underlying the observed differences of the PrPC into PrPSc conversion process (agent replication). The importance of previously peptide-scanning mapped ovine PrP self-interaction domains on this conversion was investigated by studying the ability of six of these ovine PrP based peptides to modulate two processes; PrP self-interaction and conversion. Results Three peptides (octarepeat, binding domain 2 -and C-terminal) were capable of inhibiting self-interaction of PrP in a solid-phase PrP peptide array. Three peptides (N-terminal, binding domain 2, and amyloidogenic motif) modulated prion conversion when added before or after initiation of the prion protein misfolding cyclic amplification (PMCA) reaction using brain homogenates. The C-terminal peptides (core region and C-terminal) only affected conversion (increased PrPres formation) when added before mixing PrPC and PrPSc, whereas the octarepeat peptide only affected conversion when added after this mixing. Conclusion This study identified the putative PrP core binding domain that facilitates the PrPC-PrPSc interaction (not conversion), corroborating evidence that the region of PrP containing this domain is important in the species-barrier and/or scrapie susceptibility. The octarepeats can be involved in PrPC-PrPSc stabilization, whereas the N-terminal glycosaminoglycan binding motif and the amyloidogenic motif indirectly affected conversion. Binding domain 2 and the C-terminal domain are directly implicated in PrPC self-interaction during the conversion process and may prove to be prime targets in new therapeutic strategy development, potentially retaining PrPC function. These results emphasize the importance of probable PrPC-PrPC and required Pr

  6. Gonadal ecdysone titers are modulated by protein availability but do not impact protein appetite.

    PubMed

    Carvalho-Santos, Zita; Ribeiro, Carlos

    2017-08-24

    How animals survey internal nutrient availability to modulate specific appetites is currently largely unknown. Dietary proteins have a profound impact on the reproductive capacity and the selection of food sources in insects. When deprived of dietary proteins, insects stop producing eggs and develop strong protein appetites. In many adult insects, the ovaries are the site of synthesis of the ecdysone hormone. Therefore, an attractive hypothesis is that protein availability changes the gonadal production of ecdysone, which instructs the brain to increase its preference for yeast. We combine quantitative feeding assays, dietary manipulations, hormonal measurements, and genetic germline manipulations to test this hypothesis in Drosophila melanogaster. Our results show that upon yeast deprivation mated adult female Drosophila develop a strong yeast appetite and strongly reduce their egg production. This dietary manipulation also leads to a drastic reduction in ecdysone titers. However, the drop in ecdysone is not linked to the increase in yeast appetite as mutants with impaired oogenesis are able to adapt yeast intake to their nutrient state while displaying a constitutive low ecdysone titer. Interestingly, a low ecdysone titer is correlated with a lower level of overall food intake. Our data therefore show that in mated females the level of ecdysone reflects the level of protein in the diet and the physiological state of the ovaries. While the ovaries and ecdysone are unlikely to instruct the brain to develop a yeast appetite upon protein deprivation, they seem to be able to control overall food intake. Copyright © 2017. Published by Elsevier Ltd.

  7. The Golgi Protein ACBD3, an Interactor for Poliovirus Protein 3A, Modulates Poliovirus Replication

    PubMed Central

    Téoulé, François; Brisac, Cynthia; Pelletier, Isabelle; Vidalain, Pierre-Olivier; Jégouic, Sophie; Mirabelli, Carmen; Bessaud, Maël; Combelas, Nicolas; Autret, Arnaud; Tangy, Frédéric; Delpeyroux, Francis

    2013-01-01

    We have shown that the circulating vaccine-derived polioviruses responsible for poliomyelitis outbreaks in Madagascar have recombinant genomes composed of sequences encoding capsid proteins derived from poliovaccine Sabin, mostly type 2 (PVS2), and sequences encoding nonstructural proteins derived from other human enteroviruses. Interestingly, almost all of these recombinant genomes encode a nonstructural 3A protein related to that of field coxsackievirus A17 (CV-A17) strains. Here, we investigated the repercussions of this exchange, by assessing the role of the 3A proteins of PVS2 and CV-A17 and their putative cellular partners in viral replication. We found that the Golgi protein acyl-coenzyme A binding domain-containing 3 (ACBD3), recently identified as an interactor for the 3A proteins of several picornaviruses, interacts with the 3A proteins of PVS2 and CV-A17 at viral RNA replication sites, in human neuroblastoma cells infected with either PVS2 or a PVS2 recombinant encoding a 3A protein from CV-A17 [PVS2-3A(CV-A17)]. The small interfering RNA-mediated downregulation of ACBD3 significantly increased the growth of both viruses, suggesting that ACBD3 slowed viral replication. This was confirmed with replicons. Furthermore, PVS2-3A(CV-A17) was more resistant to the replication-inhibiting effect of ACBD3 than the PVS2 strain, and the amino acid in position 12 of 3A was involved in modulating the sensitivity of viral replication to ACBD3. Overall, our results indicate that exchanges of nonstructural proteins can modify the relationships between enterovirus recombinants and cellular interactors and may thus be one of the factors favoring their emergence. PMID:23926333

  8. 42 CFR 52e.6 - How will NIH evaluate applications?

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 52e.6 Public Health PUBLIC HEALTH SERVICE, DEPARTMENT OF HEALTH AND HUMAN SERVICES GRANTS NATIONAL HEART, LUNG, AND BLOOD INSTITUTE GRANTS FOR PREVENTION AND CONTROL PROJECTS § 52e.6 How will NIH... the prevention, diagnosis, or treatment of heart, blood vessel, lung, or blood diseases of...

  9. 42 CFR 52e.6 - How will NIH evaluate applications?

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 52e.6 Public Health PUBLIC HEALTH SERVICE, DEPARTMENT OF HEALTH AND HUMAN SERVICES GRANTS NATIONAL HEART, LUNG, AND BLOOD INSTITUTE GRANTS FOR PREVENTION AND CONTROL PROJECTS § 52e.6 How will NIH... the prevention, diagnosis, or treatment of heart, blood vessel, lung, or blood diseases of...

  10. 42 CFR 52e.6 - How will NIH evaluate applications?

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 52e.6 Public Health PUBLIC HEALTH SERVICE, DEPARTMENT OF HEALTH AND HUMAN SERVICES GRANTS NATIONAL HEART, LUNG, AND BLOOD INSTITUTE GRANTS FOR PREVENTION AND CONTROL PROJECTS § 52e.6 How will NIH... the prevention, diagnosis, or treatment of heart, blood vessel, lung, or blood diseases of...

  11. 42 CFR 52e.6 - How will NIH evaluate applications?

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 52e.6 Public Health PUBLIC HEALTH SERVICE, DEPARTMENT OF HEALTH AND HUMAN SERVICES GRANTS NATIONAL HEART, LUNG, AND BLOOD INSTITUTE GRANTS FOR PREVENTION AND CONTROL PROJECTS § 52e.6 How will NIH... the prevention, diagnosis, or treatment of heart, blood vessel, lung, or blood diseases of...

  12. 42 CFR 52e.6 - How will NIH evaluate applications?

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 52e.6 Public Health PUBLIC HEALTH SERVICE, DEPARTMENT OF HEALTH AND HUMAN SERVICES GRANTS NATIONAL HEART, LUNG, AND BLOOD INSTITUTE GRANTS FOR PREVENTION AND CONTROL PROJECTS § 52e.6 How will NIH... the prevention, diagnosis, or treatment of heart, blood vessel, lung, or blood diseases of...

  13. Acidic bile salts modulate the squamous epithelial barrier function by modulating tight junction proteins.

    PubMed

    Chen, Xin; Oshima, Tadayuki; Tomita, Toshihiko; Fukui, Hirokazu; Watari, Jiro; Matsumoto, Takayuki; Miwa, Hiroto

    2011-08-01

    Experimental models for esophageal epithelium in vitro either suffer from poor differentiation or complicated culture systems. An air-liquid interface system with normal human bronchial epithelial cells can serve as a model of esophageal-like squamous epithelial cell layers. Here, we explore the influence of bile acids on barrier function and tight junction (TJ) proteins. The cells were treated with taurocholic acid (TCA), glycocholic acid (GCA), or deoxycholic acid (DCA) at different pH values, or with pepsin. Barrier function was measured by transepithelial electrical resistance (TEER) and the diffusion of paracellular tracers (permeability). The expression of TJ proteins, including claudin-1 and claudin-4, was examined by Western blotting of 1% Nonidet P-40-soluble and -insoluble fractions. TCA and GCA dose-dependently decreased TEER and increased paracellular permeability at pH 3 after 1 h. TCA (4 mM) or GCA (4 mM) did not change TEER and permeability at pH 7.4 or pH 4. The combination of TCA and GCA at pH 3 significantly decreased TEER and increased permeability at lower concentrations (2 mM). Pepsin (4 mg/ml, pH 3) did not have any effect on barrier function. DCA significantly decreased the TEER and increased permeability at pH 6, a weakly acidic condition. TCA (4 mM) and GCA (4 mM) significantly decreased the insoluble fractions of claudin-1 and claudin-4 at pH 3. In conclusion, acidic bile salts disrupted the squamous epithelial barrier function partly by modulating the amounts of claudin-1 and claudin-4. These results provide new insights for understanding the role of TJ proteins in esophagitis.

  14. Modulation of nociceptive ion channels and receptors via protein-protein interactions: implications for pain relief

    PubMed Central

    Rouwette, Tom; Avenali, Luca; Sondermann, Julia; Narayanan, Pratibha; Gomez-Varela, David; Schmidt, Manuela

    2015-01-01

    In the last 2 decades biomedical research has provided great insights into the molecular signatures underlying painful conditions. However, chronic pain still imposes substantial challenges to researchers, clinicians and patients alike. Under pathological conditions, pain therapeutics often lack efficacy and exhibit only minimal safety profiles, which can be largely attributed to the targeting of molecules with key physiological functions throughout the body. In light of these difficulties, the identification of molecules and associated protein complexes specifically involved in chronic pain states is of paramount importance for designing selective interventions. Ion channels and receptors represent primary targets, as they critically shape nociceptive signaling from the periphery to the brain. Moreover, their function requires tight control, which is usually implemented by protein-protein interactions (PPIs). Indeed, manipulation of such PPIs entails the modulation of ion channel activity with widespread implications for influencing nociceptive signaling in a more specific way. In this review, we highlight recent advances in modulating ion channels and receptors via their PPI networks in the pursuit of relieving chronic pain. Moreover, we critically discuss the potential of targeting PPIs for developing novel pain therapies exhibiting higher efficacy and improved safety profiles. PMID:26039491

  15. Amyloid precursor protein at node of Ranvier modulates nodal formation

    PubMed Central

    Xu, De-En; Zhang, Wen-Min; Yang, Zara Zhuyun; Zhu, Hong-Mei; Yan, Ke; Li, Shao; Bagnard, Dominique; Dawe, Gavin S; Ma, Quan-Hong; Xiao, Zhi-Cheng

    2014-01-01

    Amyloid precursor protein (APP), commonly associated with Alzheimer disease, is upregulated and distributes evenly along the injured axons, and therefore, also known as a marker of demyelinating axonal injury and axonal degeneration. However, the physiological distribution and function of APP along myelinated axons was unknown. We report that APP aggregates at nodes of Ranvier (NOR) in the myelinated central nervous system (CNS) axons but not in the peripheral nervous system (PNS). At CNS NORs, APP expression co-localizes with tenascin-R and is flanked by juxtaparanodal potassium channel expression demonstrating that APP localized to NOR. In APP-knockout (KO) mice, nodal length is significantly increased, while sodium channels are still clustered at NORs. Moreover, APP KO and APP-overexpressing transgenic (APP TG) mice exhibited a decreased and an increased thickness of myelin in spinal cords, respectively, although the changes are limited in comparison to their littermate WT mice. The thickness of myelin in APP KO sciatic nerve also increased in comparison to that in WT mice. Our observations indicate that APP acts as a novel component at CNS NORs, modulating nodal formation and has minor effects in promoting myelination. PMID:25482638

  16. Modulation of PML protein expression regulates JCV infection

    SciTech Connect

    Gasparovic, Megan L.; Maginnis, Melissa S.; O'Hara, Bethany A.; Dugan, Aisling S.; Atwood, Walter J.

    2009-08-01

    JC virus (JCV) is a human polyomavirus that infects the majority of the human population worldwide. It is responsible for the fatal demyelinating disease Progressive Multifocal Leukoencephalopathy. JCV binds to cells using the serotonin receptor 5-HT{sub 2A}R and alpha(2-6)- or alpha(2-3)-linked sialic acid. It enters cells using clathrin-dependent endocytosis and traffics to the early endosome and possibly to the endoplasmic reticulum. Viral DNA is then delivered to the nucleus where transcription, replication, and assembly of progeny occur. We found that the early regulatory protein large T antigen accumulates in microdomains in the nucleus adjacent to ND-10 or PML domains. This observation prompted us to explore the role of these domains in JCV infection. We found that a reduction of nuclear PML enhanced virus infection and that an increase in nuclear PML reduced infection. Infection with JCV did not directly modulate nuclear levels of PML but our data indicate that a host response involving interferon beta is likely to restrict virus infection by increasing nuclear PML.

  17. Mechanisms of Protein-Ligand Association and Its Modulation by Protein Mutations

    PubMed Central

    Held, Martin; Metzner, Philipp; Prinz, Jan-Hendrik; Noé, Frank

    2011-01-01

    Protein-ligand interactions are essential for nearly all biological processes, and yet the biophysical mechanism that enables potential binding partners to associate before specific binding occurs remains poorly understood. Fundamental questions include which factors influence the formation of protein-ligand encounter complexes, and whether designated association pathways exist. To address these questions, we developed a computational approach to systematically analyze the complete ensemble of association pathways. Here, we use this approach to study the binding of a phosphate ion to the Escherichia coli phosphate-binding protein. Various mutants of the protein are considered, and their effects on binding free-energy profiles, association rates, and association pathway distributions are quantified. The results reveal the existence of two anion attractors, i.e., regions that initially attract negatively charged particles and allow them to be efficiently screened for phosphate, which is subsequently specifically bound. Point mutations that affect the charge on these attractors modulate their attraction strength and speed up association to a factor of 10 of the diffusion limit, and thus change the association pathways of the phosphate ligand. It is demonstrated that a phosphate that prebinds to such an attractor neutralizes its attraction effect to the environment, making the simultaneous association of a second phosphate ion unlikely. This study suggests ways in which structural properties can be used to tune molecular association kinetics so as to optimize the efficiency of binding, and highlights the importance of kinetic properties. PMID:21281585

  18. Protein kinase A phosphorylation modulates transport of the polypyrimidine tract-binding protein

    PubMed Central

    Xie, Jiuyong; Lee, Ji-Ann; Kress, Tracy L.; Mowry, Kimberly L.; Black, Douglas L.

    2003-01-01

    The heterogeneous nuclear ribonucleoprotein particle (hnRNP) proteins play important roles in mRNA processing in eukaryotes, but little is known about how they are regulated by cellular signaling pathways. The polypyrimidine-tract binding protein (PTB, or hnRNP I) is an important regulator of alternative pre-mRNA splicing, of viral RNA translation, and of mRNA localization. Here we show that the nucleo-cytoplasmic transport of PTB is regulated by the 3′,5′-cAMP-dependent protein kinase (PKA). PKA directly phosphorylates PTB on conserved Ser-16, and PKA activation in PC12 cells induces Ser-16 phosphorylation. PTB carrying a Ser-16 to alanine mutation accumulates normally in the nucleus. However, export of this mutant protein from the nucleus is greatly reduced in heterokaryon shuttling assays. Conversely, hyperphosphorylation of PTB by coexpression with the catalytic subunit of PKA results in the accumulation of PTB in the cytoplasm. This accumulation is again specifically blocked by the S16A mutation. Similarly, in Xenopus oocytes, the phospho-Ser-16-PTB is restricted to the cytoplasm, whereas the non-Ser-16-phosphorylated PTB is nuclear. Thus, direct PKA phosphorylation of PTB at Ser-16 modulates the nucleo-cytoplasmic distribution of PTB. This phosphorylation likely plays a role in the cytoplasmic function of PTB. PMID:12851456

  19. Therapeutic design of peptide modulators of protein-protein interactions in membranes.

    PubMed

    Stone, Tracy A; Deber, Charles M

    2017-04-01

    Membrane proteins play the central roles in a variety of cellular processes, ranging from nutrient uptake and signalling, to cell-cell communication. Their biological functions are directly related to how they fold and assemble; defects often lead to disease. Protein-protein interactions (PPIs) within the membrane are therefore of great interest as therapeutic targets. Here we review the progress in the application of membrane-insertable peptides for the disruption or stabilization of membrane-based PPIs. We describe the design and preparation of transmembrane peptide mimics; and of several categories of peptidomimetics used for study, including d-enantiomers, non-natural amino acids, peptoids, and β-peptides. Further aspects of the review describe modifications to membrane-insertable peptides, including lipidation and cyclization via hydrocarbon stapling. These approaches provide a pathway toward the development of metabolically stable, non-toxic, and efficacious peptide modulators of membrane-based PPIs. This article is part of a Special Issue entitled: Lipid order/lipid defects and lipid-control of protein activity edited by Dirk Schneider.

  20. Network analysis reveals common host protein/s modulating pathogenesis of neurotropic viruses

    PubMed Central

    Ghosh, Sourish; Mukherjee, Sriparna; Sengupta, Nabonita; Roy, Arunava; Dey, Dhritiman; Chakraborty, Surajit; Chattopadhyay, Dhrubajyoti; Banerjee, Arpan; Basu, Anirban

    2016-01-01

    Network analysis through graph theory provides a quantitative approach to characterize specific proteins and their constituent assemblies that underlie host-pathogen interactions. In the present study, graph theory was used to analyze the interactome designed out of 50 differentially expressing proteins from proteomic analysis of Chandipura Virus (CHPV, Family: Rhabdoviridae) infected mouse brain tissue to identify the primary candidates for intervention. Using the measure of degree centrality, that quantifies the connectedness of a single protein within a milieu of several other interacting proteins, DJ-1 was selected for further molecular validation. To elucidate the generality of DJ-1’s role in propagating infection its role was also monitored in another RNA virus, Japanese Encephalitis Virus (JEV, Family: Flaviviridae) infection. Concurrently, DJ-1 got over-expressed in response to reactive oxygen species (ROS) generation following viral infection which in the early phase of infection migrated to mitochondria to remove dysfunctional mitochondria through the process of mitophagy. DJ-1 was also observed to modulate the viral replication and interferon responses along with low-density lipoprotein (LDL) receptor expression in neurons. Collectively these evidences reveal a comprehensive role for DJ-1 in neurotropic virus infection in the brain. PMID:27581498

  1. Amyloid precursor protein modulates β-catenin degradation

    PubMed Central

    Chen, Yuzhi; Bodles, Angela M

    2007-01-01

    Background The amyloid precursor protein (APP) is genetically associated with Alzheimer's disease (AD). Elucidating the function of APP should help understand AD pathogenesis and provide insights into therapeutic designs against this devastating neurodegenerative disease. Results We demonstrate that APP expression in primary neurons induces β-catenin phosphorylation at Ser33, Ser37, and Thr41 (S33/37/T41) residues, which is a prerequisite for β-catenin ubiquitinylation and proteasomal degradation. APP-induced phosphorylation of β-catenin resulted in the reduction of total β-catenin levels, suggesting that APP expression promotes β-catenin degradation. In contrast, treatment of neurons with APP siRNAs increased total β-catenin levels and decreased β-catenin phosphorylation at residues S33/37/T41. Further, β-catenin was dramatically increased in hippocampal CA1 pyramidal cells from APP knockout animals. Acute expression of wild type APP or of familial AD APP mutants in primary neurons downregulated β-catenin in membrane and cytosolic fractions, and did not appear to affect nuclear β-catenin or β-catenin-dependent transcription. Conversely, in APP knockout CA1 pyramidal cells, accumulation of β-catenin was associated with the upregulation of cyclin D1, a downstream target of β-catenin signaling. Together, these data establish that APP downregulates β-catenin and suggest a role for APP in sustaining neuronal function by preventing cell cycle reactivation and maintaining synaptic integrity. Conclusion We have provided strong evidence that APP modulates β-catenin degradation in vitro and in vivo. Future studies may investigate whether APP processing is necessary for β-catenin downregulation, and determine if excessive APP expression contributes to AD pathogenesis through abnormal β-catenin downregulation. PMID:18070361

  2. Protein kinase C modulates ventilatory patterning in the developing rat.

    PubMed

    Bandla, H P; Simakajornboon, N; Graff, G R; Gozal, D

    1999-03-01

    Protein kinase C (PKC) mediates important components of signal transduction pathways underlying neuronal excitability and modulates respiratory timing mechanisms in adult rats. To determine ventilatory effects of systemic PKC inhibition during development, whole-body plethysmographic recordings were conducted in 2-3-d (n = 11), 5-6-d (n = 19), 10-12-d (n = 14), and 20-21-d-old (n = 14) rat pups after treatment with vehicle and Ro 32-0432 (100 mg/kg, intraperitoneally). Ro 32-0432 decreased minute ventilation (V E) by 51.0 +/- 5.5% (mean +/- SEM) in youngest pups (p < 0.01) but only 19.1 +/- 6.8% in 20-21-d-old pups (p < 0.01). V E decreases were always due to frequency reductions with tidal volume (VT) remaining unaffected. Respiratory rate decreases primarily resulted from marked expiratory time (TE) prolongations being more pronounced in 2-3-d-old (115.5 +/- 28.9%) compared with 20-21-d old (36.6 +/- 10.9%; p < 0.002 analysis of variance [ANOVA] ). Expression of the PKC isoforms alpha, beta, gamma, delta, iota, and mu was further examined in brainstem and cortex by immunoblotting and revealed different patterns with postnatal age and location. We conclude that endogenous PKC inhibition elicits age-dependent ventilatory reductions which primarily affect timing mechanisms rather than changes in volume drive. This effect on ventilation abates with increasing postnatal age suggesting that the neural substrate mediating overall respiratory output may be more critically dependent on PKC activity in the immature animal.

  3. Human papillomavirus 16E6 and NFX1-123 potentiate notch signaling and differentiation without activating cellular arrest

    SciTech Connect

    Vliet-Gregg, Portia A.; Hamilton, Jennifer R.; Katzenellenbogen, Rachel A.

    2015-04-15

    High-risk human papillomavirus (HR HPV) oncoproteins bind host cell proteins to dysregulate and uncouple apoptosis, senescence, differentiation, and growth. These pathways are important for both the viral life cycle and cancer development. HR HPV16 E6 (16E6) interacts with the cellular protein NFX1-123, and they collaboratively increase the growth and differentiation master regulator, Notch1. In 16E6 expressing keratinocytes (16E6 HFKs), the Notch canonical pathway genes Hes1 and Hes5 were increased with overexpression of NFX1-123, and their expression was directly linked to the activation or blockade of the Notch1 receptor. Keratinocyte differentiation genes Keratin 1 and Keratin 10 were also increased, but in contrast their upregulation was only indirectly associated with Notch1 receptor stimulation and was fully unlinked to growth arrest, increased p21{sup Waf1/CIP1}, or decreased proliferative factor Ki67. This leads to a model of 16E6, NFX1-123, and Notch1 differently regulating canonical and differentiation pathways and entirely uncoupling cellular arrest from increased differentiation. - Highlights: • 16E6 and NFX1-123 increased the Notch canonical pathway through Notch1. • 16E6 and NFX1-123 increased the differentiation pathway indirectly through Notch1. • 16E6 and NFX1-123 increased differentiation gene expression without growth arrest. • Increased NFX1-123 with 16E6 may create an ideal cellular phenotype for HPV.

  4. A generalised module for the selective extracellular accumulation of recombinant proteins

    PubMed Central

    2012-01-01

    Background It is widely believed that laboratory strains of Escherichia coli, including those used for industrial production of proteins, do not secrete proteins to the extracellular milieu. Results Here, we report the development of a generalised module, based on an E. coli autotransporter secretion system, for the production of extracellular recombinant proteins. We demonstrate that a wide variety of structurally diverse proteins can be secreted as soluble proteins when linked to the autotransporter module. Yields were comparable to those achieved with other bacterial secretion systems. Conclusions The advantage of this module is that it relies on a relatively simple and easily manipulated secretion system, exhibits no apparent limitation to the size of the secreted protein and can deliver proteins to the extracellular environment at levels of purity and yields sufficient for many biotechnological applications. PMID:22640772

  5. Abiotic regulation: a common way for proteins to modulate their functions.

    PubMed

    Zou, Zhi; Fu, Xinmiao

    2015-01-01

    Modulation of protein intrinsic activity in cells is generally carried out via a combination of four common ways, i.e., allosteric regulation, covalent modification, proteolytic cleavage and association of other regulatory proteins. Accumulated evidence indicate that changes of certain abiotic factors (e.g., temperature, pH, light and mechanical force) within or outside the cells directly influence protein structure and thus profoundly modulate the functions of a wide range of proteins, termed as abiotic regulatory proteins (e.g., heat shock factor, small heat shock protein, hemoglobin, zymogen, integrin, rhodopsin). Such abiotic regulation apparently differs from the four classic ways in perceiving and response to the signals. Importantly, it enables cells to directly and also immediately response to extracellular stimuli, thus facilitating the ability of organisms to resist against and adapt to the abiotic stress and thereby playing crucial roles in life evolution. Altogether, abiotic regulation may be considered as a common way for proteins to modulate their functions.

  6. Toxicity, pharmacokinetics, and photodynamic properties of chlorin e6

    NASA Astrophysics Data System (ADS)

    Kostenich, Gennady; Zhuravkin, Ivan N.; Gurinovich, G. P.; Zhavrid, Edvard A.

    1993-03-01

    Toxicity, pharmacokinetics, and the tumor damage effect of chlorin e6 after light irradiation were studied. The results show that chlorin e6 LD50 value in C57Bl mice was 189 +/- 10 mg/kg, in non-inbred white rats it was 99 +/- 14 mg/kg 14 days after the agent iv injection. The concentration of chlorin e6 in blood, liver, kidney, spleen, and tumors (sarcoma M-1 and sarcoma 45) of the rats was determined by the fluorescence method 3, 6, 12, 18, 24, 48, and 72 hours after the agent iv injection at the dose of 10 mg/kg. For this purpose chlorin e6 was extracted from tissues by detergent triton X-100. The depth of necrosis spreading in tumor tissue was evaluated after chlorin e6 injection at the doses of 1 - 10 mg/kg and subsequent irradiation by a krypton laser with light energy density of 90 J/cm2, using the method of vital staining with Evans blue. It was found that depending on the agent dose and time interval between chlorin e6 injection and photoradiation, the depth of tumor necrosis varied from 4.0 to 16.6 mm in sarcoma M-1 and from 5.0 to 15.0 in sarcoma 45.

  7. E6 inspired supersymmetric models with exact custodial symmetry

    NASA Astrophysics Data System (ADS)

    Nevzorov, Roman

    2013-01-01

    The breakdown of E6 gauge symmetry at high energies may lead to supersymmetric models based on the standard model gauge group together with extra U(1)ψ and U(1)χ gauge symmetries. To ensure anomaly cancellation the particle content of these E6 inspired models involves extra exotic states that generically give rise to nondiagonal flavor transitions and rapid proton decay. We argue that a single discrete Z˜2H symmetry can be used to forbid tree-level flavor changing transitions, as well as the most dangerous baryon and lepton number violating operators. We present 5D and 6D orbifold grand unified theory constructions that lead to the E6 inspired supersymmetric models of this type. The breakdown of U(1)ψ and U(1)χ gauge symmetries that preserves E6 matter parity assignment guarantees that ordinary quarks and leptons and their superpartners, as well as the exotic states which originate from 27 representations of E6, survive to low energies. These E6 inspired models contain two dark matter candidates and must also include additional TeV scale vectorlike lepton or vectorlike down-type quark states to render the lightest exotic quark unstable. We examine gauge coupling unification in these models and discuss their implications for collider phenomenology and cosmology.

  8. Carboranyl-Chlorin e6 as a Potent Antimicrobial Photosensitizer

    PubMed Central

    Omarova, Elena O.; Nazarov, Pavel A.; Firsov, Alexander M.; Strakhovskaya, Marina G.; Arkhipova, Anastasia Yu.; Moisenovich, Mikhail M.; Agapov, Igor I.; Ol’shevskaya, Valentina A.; Zaitsev, Andrey V.; Kalinin, Valery N.; Kotova, Elena A.; Antonenko, Yuri N.

    2015-01-01

    Antimicrobial photodynamic inactivation is currently being widely considered as alternative to antibiotic chemotherapy of infective diseases, attracting much attention to design of novel effective photosensitizers. Carboranyl-chlorin-e6 (the conjugate of chlorin e6 with carborane), applied here for the first time for antimicrobial photodynamic inactivation, appeared to be much stronger than chlorin e6 against Gram-positive bacteria, such as Bacillus subtilis, Staphyllococcus aureus and Mycobacterium sp. Confocal fluorescence spectroscopy and membrane leakage experiments indicated that bacteria cell death upon photodynamic treatment with carboranyl-chlorin-e6 is caused by loss of cell membrane integrity. The enhanced photobactericidal activity was attributed to the increased accumulation of the conjugate by bacterial cells, as evaluated both by centrifugation and fluorescence correlation spectroscopy. Gram-negative bacteria were rather resistant to antimicrobial photodynamic inactivation mediated by carboranyl-chlorin-e6. Unlike chlorin e6, the conjugate showed higher (compared to the wild-type strain) dark toxicity with Escherichia coli ΔtolC mutant, deficient in TolC-requiring multidrug efflux transporters. PMID:26535905

  9. Screening of Protein-Protein Interaction Modulators via Sulfo-Click Kinetic Target-Guided Synthesis

    PubMed Central

    Kulkarni, Sameer S.; Hu, Xiangdong; Doi, Kenichiro; Wang, Hong-Gang

    2011-01-01

    Kinetic Target-Guided Synthesis (TGS) and in situ click chemistry are among unconventional discovery strategies having the potential to streamline the development of protein-protein interaction modulators (PPIMs). In kinetic TGS and in situ click chemistry, the target is directly involved in the assembly of its own potent, bidentate ligand from a pool of reactive fragments. Herein, we report the use and validation of kinetic TGS based on the sulfo-click reaction between thio acids and sulfonyl azides as a screening and synthesis platform for the identification of high-quality PPIMs. Starting from a randomly designed library consisting of 9 thio acids and 9 sulfonyl azides leading to 81 potential acylsulfonamides, the target protein, Bcl-XL selectively assembled four PPIMs, acylsulfonamides SZ4TA2, SZ7TA2, SZ9TA1, and SZ9TA5, which have been shown to modulate Bcl-XL/BH3 interactions. To further investigate the Bcl-XL templation effect, control experiments were carried out using two mutants of Bcl-XL. In one mutant, phenylalanine Phe131 and aspartic acid Asp133, which are critical for the BH3 domain binding, have been substituted by alanines, while arginine Arg139, a residue identified to play a crucial role in the binding of ABT-737, a BH3 mimetic, has been replaced by an alanine in the other mutant. Incubation of these mutants with the reactive fragments and subsequent LC/MS-SIM analysis confirmed that these building block combinations yield the corresponding acylsulfonamides at the BH3 binding site, the actual “hot spot” of Bcl-XL. These results validate kinetic TGS using the sulfo-click reaction as a valuable tool for the straightforward identification of high-quality PPIMs. PMID:21506574

  10. Human Papillomavirus 16 E6 Upregulates APOBEC3B via the TEAD Transcription Factor

    PubMed Central

    Takeuchi, Takamasa; Ishii, Yoshiyuki; Yugawa, Takashi; Kiyono, Tohru; Nishina, Hiroshi; Kukimoto, Iwao

    2017-01-01

    ABSTRACT The cytidine deaminase APOBEC3B (A3B) underlies the genetic heterogeneity of several human cancers, including cervical cancer, which is caused by human papillomavirus (HPV) infection. We previously identified a region within the A3B promoter that is activated by the viral protein HPV16 E6 in human keratinocytes. Here, we discovered three sites recognized by the TEAD family of transcription factors within this region of the A3B promoter. Reporter assays in HEK293 cells showed that exogenously expressed TEAD4 induced A3B promoter activation through binding to these sites. Normal immortalized human keratinocytes expressing E6 (NIKS-E6) displayed increased levels of TEAD1/4 protein compared to parental NIKS. A series of E6 mutants revealed that E6-mediated degradation of p53 was important for increasing TEAD4 levels. Knockdown of TEADs in NIKS-E6 significantly reduced A3B mRNA levels, whereas ectopic expression of TEAD4 in NIKS increased A3B mRNA levels. Finally, chromatin immunoprecipitation assays demonstrated increased levels of TEAD4 binding to the A3B promoter in NIKS-E6 compared to NIKS. Collectively, these results indicate that E6 induces upregulation of A3B through increased levels of TEADs, highlighting the importance of the TEAD-A3B axis in carcinogenesis. IMPORTANCE The expression of APOBEC3B (A3B), a cellular DNA cytidine deaminase, is upregulated in various human cancers and leaves characteristic, signature mutations in cancer genomes, suggesting that it plays a prominent role in carcinogenesis. Viral oncoproteins encoded by human papillomavirus (HPV) and polyomavirus have been reported to induce A3B expression, implying the involvement of A3B upregulation in virus-associated carcinogenesis. However, the molecular mechanisms causing A3B upregulation remain unclear. Here, we demonstrate that exogenous expression of the cellular transcription factor TEAD activates the A3B promoter. Further, the HPV oncoprotein E6 increases the levels of endogenous

  11. Human Papillomavirus 16 E6 Upregulates APOBEC3B via the TEAD Transcription Factor.

    PubMed

    Mori, Seiichiro; Takeuchi, Takamasa; Ishii, Yoshiyuki; Yugawa, Takashi; Kiyono, Tohru; Nishina, Hiroshi; Kukimoto, Iwao

    2017-03-15

    The cytidine deaminase APOBEC3B (A3B) underlies the genetic heterogeneity of several human cancers, including cervical cancer, which is caused by human papillomavirus (HPV) infection. We previously identified a region within the A3B promoter that is activated by the viral protein HPV16 E6 in human keratinocytes. Here, we discovered three sites recognized by the TEAD family of transcription factors within this region of the A3B promoter. Reporter assays in HEK293 cells showed that exogenously expressed TEAD4 induced A3B promoter activation through binding to these sites. Normal immortalized human keratinocytes expressing E6 (NIKS-E6) displayed increased levels of TEAD1/4 protein compared to parental NIKS. A series of E6 mutants revealed that E6-mediated degradation of p53 was important for increasing TEAD4 levels. Knockdown of TEADs in NIKS-E6 significantly reduced A3B mRNA levels, whereas ectopic expression of TEAD4 in NIKS increased A3B mRNA levels. Finally, chromatin immunoprecipitation assays demonstrated increased levels of TEAD4 binding to the A3B promoter in NIKS-E6 compared to NIKS. Collectively, these results indicate that E6 induces upregulation of A3B through increased levels of TEADs, highlighting the importance of the TEAD-A3B axis in carcinogenesis.IMPORTANCE The expression of APOBEC3B (A3B), a cellular DNA cytidine deaminase, is upregulated in various human cancers and leaves characteristic, signature mutations in cancer genomes, suggesting that it plays a prominent role in carcinogenesis. Viral oncoproteins encoded by human papillomavirus (HPV) and polyomavirus have been reported to induce A3B expression, implying the involvement of A3B upregulation in virus-associated carcinogenesis. However, the molecular mechanisms causing A3B upregulation remain unclear. Here, we demonstrate that exogenous expression of the cellular transcription factor TEAD activates the A3B promoter. Further, the HPV oncoprotein E6 increases the levels of endogenous TEAD1

  12. Involvement of Nuclear Export in Human Papillomavirus Type 18 E6-Mediated Ubiquitination and Degradation of p53

    PubMed Central

    Stewart, Deborah; Ghosh, Anirban; Matlashewski, Greg

    2005-01-01

    The E6 protein from high-risk human papillomaviruses (HPVs) targets the p53 tumor suppressor for degradation by the proteasome pathway. This ability contributes to the oncogenic potential of these viruses. However, several aspects concerning the mechanism of E6-mediated p53 degradation at the cellular level remain to be clarified. This study therefore examined the role of cell localization and ubiquitination in the E6-mediated degradation of p53. As demonstrated within, following coexpression both p53 and high-risk HPV type 18 (HPV-18) E6 (18E6) shuttle from the nucleus to the cytoplasm. Mutation of the C-terminal nuclear export signal (NES) of p53 or treatment with leptomycin B inhibited the 18E6-mediated nuclear export of p53. Impairment of nuclear export resulted in only a partial reduction in 18E6-mediated degradation, suggesting that both nuclear and cytoplasmic proteasomes can target p53 for degradation. This was also consistent with the observation that 18E6 mediated the accumulation of polyubiquitinated p53 in the nucleus. In comparison, a p53 isoform that localizes predominantly to the cytoplasm was not targeted for degradation by 18E6 in vivo but could be degraded in vitro, arguing that nuclear p53 is the target for E6-mediated degradation. This study supports a model in which (i) E6 mediates the accumulation of polyubiquitinated p53 in the nucleus, (ii) E6 is coexported with p53 from the nucleus to the cytoplasm via a CRM1 nuclear export mechanism involving the C-terminal NES of p53, and (iii) E6-mediated p53 degradation can be mediated by both nuclear and cytoplasmic proteasomes. PMID:15994771

  13. All repeats are not equal: a module-based approach to guide repeat protein design.

    PubMed

    Sawyer, Nicholas; Chen, Jieming; Regan, Lynne

    2013-05-27

    Repeat proteins composed of tandem arrays of a short structural motif often mediate protein-protein interactions. Past efforts to design repeat protein-based molecular recognition tools have focused on the creation of templates from the consensus of individual repeats, regardless of their natural context. Such an approach assumes that all repeats are essentially equivalent. In this study, we present the results of a "module-based" approach in which modules composed of tandem repeats are aligned to identify repeat-specific features. Using this approach to analyze tetratricopeptide repeat modules that contain three tandem repeats (3TPRs), we identify two classes of 3TPR modules with distinct structural signatures that are correlated with different sets of functional residues. Our analyses also reveal a high degree of correlation between positions across the entire ligand-binding surface, indicative of a coordinated, coevolving binding surface. Extension of our analyses to different repeat protein modules reveals more examples of repeat-specific features, especially in armadillo repeat modules. In summary, the module-based analyses that we present effectively capture key repeat-specific features that will be important to include in future repeat protein design templates.

  14. Heat shock protein coinducers with no effect on protein denaturation specifically modulate the membrane lipid phase

    PubMed Central

    Török, Zsolt; Tsvetkova, Nelly M.; Balogh, Gábor; Horváth, Ibolya; Nagy, Enikő; Pénzes, Zoltán; Hargitai, Judit; Bensaude, Olivier; Csermely, Péter; Crowe, John H.; Maresca, Bruno; Vígh, László

    2003-01-01

    The hydroxylamine derivative bimoclomol (BM) has been shown to activate natural cytoprotective homeostatic responses by enhancing the capability of cells to cope with various pathophysiological conditions. It exerts its effect in synergy with low levels of stress to induce the synthesis of members of major stress protein families. We show here that the presence of BM does not influence protein denaturation in the cells. BM and its derivatives selectively interact with acidic lipids and modulate their thermal and dynamic properties. BM acts as a membrane fluidizer at normal temperature, but it is a highly efficient membrane stabilizer, inhibiting the bilayer–nonbilayer phase transitions during severe heat shock. We suggest that BM and the related compounds modify those domains of membrane lipids where the thermally or chemically induced perturbation of lipid phase is sensed and transduced into a cellular signal, leading to enhanced activation of heat shock genes. BM may be a prototype for clinically safe membrane-interacting drug candidates that rebalance the level and composition of heat shock proteins. PMID:12615993

  15. Regulating the ethylene response of a plant by modulation of F-box proteins

    DOEpatents

    Guo, Hongwei [Beijing, CN; Ecker, Joseph R [Carlsbad, CA

    2014-01-07

    The relationship between F-box proteins and proteins invovled in the ethylene response in plants is described. In particular, F-box proteins may bind to proteins involved in the ethylene response and target them for degradation by the ubiquitin/proteasome pathway. The transcription factor EIN3 is a key transcription factor mediating ethylne-regulated gene expression and morphological responses. EIN3 is degraded through a ubiquitin/proteasome pathway mediated by F-box proteins EBF1 and EBF2. The link between F-box proteins and the ethylene response is a key step in modulating or regulating the response of a plant to ethylene. Described herein are transgenic plants having an altered sensitivity to ethylene, and methods for making transgenic plant haing an althered sensitivity to ethylene by modulating the level of activity of F-box proteins. Methods of altering the ethylene response in a plant by modulating the activity or expression of an F-box protein are described. Also described are methods of identifying compounds that modulate the ethylene response in plants by modulating the level of F-box protein expression or activity.

  16. Combining sequence and Gene Ontology for protein module detection in the Weighted Network.

    PubMed

    Yu, Yang; Liu, Jie; Feng, Nuan; Song, Bo; Zheng, Zeyu

    2017-01-07

    Studies of protein modules in a Protein-Protein Interaction (PPI) network contribute greatly to the understanding of biological mechanisms. With the development of computing science, computational approaches have played an important role in locating protein modules. In this paper, a new approach combining Gene Ontology and amino acid background frequency is introduced to detect the protein modules in the weighted PPI networks. The proposed approach mainly consists of three parts: the feature extraction, the weighted graph construction and the protein complex detection. Firstly, the topology-sequence information is utilized to present the feature of protein complex. Secondly, six types of the weighed graph are constructed by combining PPI network and Gene Ontology information. Lastly, protein complex algorithm is applied to the weighted graph, which locates the clusters based on three conditions, including density, network diameter and the included angle cosine. Experiments have been conducted on two protein complex benchmark sets for yeast and the results show that the approach is more effective compared to five typical algorithms with the performance of f-measure and precision. The combination of protein interaction network with sequence and gene ontology data is helpful to improve the performance and provide a optional method for protein module detection. Copyright © 2016 Elsevier Ltd. All rights reserved.

  17. Combining Sequence and Gene Ontology for Protein Module Detection in the Weighted Network.

    PubMed

    Yu, Yang; Liu, Jie; Feng, Nuan; Song, Bo; Zheng, Zeyu

    2016-10-29

    Studies of protein modules in a Protein-Protein Interaction (PPI) network contribute greatly to the understanding of biological mechanisms. With the development of computing science, computational approaches have played an important role in locating protein modules. In this paper, a new approach combining Gene Ontology and amino acid background frequency is introduced to detect the protein modules in the weighted PPI networks. The proposed approach mainly consists of three parts: the feature extraction, the weighted graph construction and the protein complex detection. Firstly, the topology-sequence information is utilized to present the feature of protein complex. Secondly, six types of the weighed graph are constructed by combining PPI network and Gene Ontology information. Lastly, protein complex algorithm is applied to the weighted graph, which locates the clusters based on three conditions, including density, network diameter and the included angle cosine. Experiments have been conducted on two protein complex benchmark sets for yeast and the results show that the approach is more effective compared to five typical algorithms with the performance of f-measure and precision. The combination of protein interaction network with sequence and gene ontology data is helpful to improve the performance and provide a optional method for protein module detection.

  18. HPV16 E6 upregulates Aurora A expression

    PubMed Central

    Guo, Yi; Ma, Jiaming; Zheng, Yahong; Li, Lu; Gui, Xiaowei; Wang, Qian; Meng, Xiangkai; Shang, Hong

    2016-01-01

    Overexpression of Aurora A kinase occurs in certain types of cancer, and therefore results in chromosome instability and phosphorylation-mediated ubiquitylation and degradation of p53 for tumorigenesis. The high-risk subtype human papillomavirus (HPV)16 early oncoprotein E6 is a major contributor inducing host cell immortalization and transformation through interaction with a number of cellular factors. In the present study, co-immunoprecipitation, glutathione S-transferase pull-down and immunostaining were used to show that HPV16 E6 and Aurora A bind to each other in vivo and in vitro. Western blotting and reverse transcription-polymerase chain reaction were used to reveal that HPV16 E6 inhibited cell apoptosis by stabilizing Aurora A expression. The present study may report a new mechanism for the involvement of HPV16 E6 in carcinogenesis, as HPV16 E6 elevates Aurora A expression and the latter may be a common target for oncogenic viruses that result in cell carcinogenesis. PMID:27446442

  19. Multiple display of catalytic modules on a protein scaffold: nano-fabrication of enzyme particles.

    PubMed

    Heyman, Arnon; Barak, Yoav; Caspi, Jonathan; Wilson, David B; Altman, Arie; Bayer, Edward A; Shoseyov, Oded

    2007-09-30

    Self assembly is a prerequisite for fabricating nanoscale structures. Here we present a new fusion protein based on the stress-responsive homo-oligomeric protein, SP1. This ring-shaped protein is a highly stable homododecamer, which can be potentially utilized to self-assemble different modules and enzymes in a predicted and oriented manner. For that purpose, a cohesin module (a component of the bacterial cellulosome) was selected, its gene fused in-frame to SP1, and the fusion protein was expressed in Escherichia coli. The cohesin module, specialized to incorporate different enzymes through specific recognition of a dockerin modular counterpart, is used to display new moieties on the SP1 scaffold. The SP1 scaffold displayed 12 active cohesin modules and specific binding to a dockerin-fused cellulase enzyme from Thermobifida fusca. Moreover, we found a significant increase in specific activity of the scaffold-displayed enzymes.

  20. Effect of surfaces in modulating protein folding mechanisms

    NASA Astrophysics Data System (ADS)

    Shea, Joan

    2014-03-01

    Protein-surface interactions are ubiquitous in the crowded cytosol, where proteins encounter a variety of surfaces, ranging from membranes surfaces, to the surfaces presented by chaperone molecules. Protein-surface interactions are also at the heart of a number of emerging technologies, including protein micro-arrays, biosensors and biomaterials. The effect of surfaces on protein structure and stability can vary substantially depending on the chemical composition of the surface. In this talk, I will present detailed atomistic simulations of the folding of a small beta-sheet protein in the presence of graphite and titanium oxide surfaces. The role of water-mediated and direct protein-surface interactions in governing protein conformations will be discussed.

  1. Optical protein modulation via quantum dot coupling and use of a hybrid sensor protein.

    PubMed

    Griep, Mark; Winder, Eric; Lueking, Donald; Friedrich, Craig; Mallick, Govind; Karna, Shashi

    2010-09-01

    Harnessing the energy transfer interactions between the optical protein bacteriorhodopsin (bR) and CdSe/ZnS quantum dots (QDs) could provide a novel bio-nano electronics substrate with a variety of applications. In the present study, a polydimethyldiallyammonium chloride based I-SAM technique has been utilized to produce bilayers, trilayers and multilayers of alternating monolayers of bR, PDAC and QD's on a conductive ITO substrate. The construction of multilayer systems was directly monitored by measuring the unique A570 nm absorbance of bR, as well as QD fluorescence emission. Both of these parameters displayed a linear relationship to the number of monolayers present on the ITO substrate. The photovoltaic response of bilayers of bR/PDAC was observed over a range of 3 to 12 bilayers and the ability to efficiently create an electrically active multilayered substrate composed of bR and QDs has been demonstrated for the first time. Evaluation of QD fluorescence emission in the multilayer system strongly suggests that FRET coupling is occurring and, since the I-SAM technique provide a means to control the bR/QD separation distance on the nanometer scale, this technique may prove highly valuable for optimizing the distance dependent energy transfer effects for maximum sensitivity to target molecule binding by a biosensor. Finally, preliminary studies on the production of a sensor protein/bR hybrid gene construct are presented. It is proposed that the energy associated with target molecule binding to a hybrid sensor protein would provide a means to directly modulate the electrical output from a sensor protein/bR biosensor platform.

  2. Human papillomavirus E6 and E7 oncoproteins affect the expression of cancer-related microRNAs: additional evidence in HPV-induced tumorigenesis.

    PubMed

    Chiantore, Maria Vincenza; Mangino, Giorgio; Iuliano, Marco; Zangrillo, Maria Simona; De Lillis, Ilaria; Vaccari, Gabriele; Accardi, Rosita; Tommasino, Massimo; Columba Cabezas, Sandra; Federico, Maurizio; Fiorucci, Gianna; Romeo, Giovanna

    2016-08-01

    Human papillomaviruses (HPVs) are the causative agents of cervical cancer and are also associated with other types of cancers. HPVs can modulate microRNAs (miRNAs) expressed by infected cells. The production of extracellular vesicles is deregulated in cancer, and their cargo delivered to the microenvironment can promote tumorigenesis. The involvement of HPV oncoproteins on miRNA expression in cells and exosomes was analyzed in keratinocytes transduced with E6 and E7 from mucosal HPV-16 or cutaneous HPV-38 (K16 and K38). MiRNAs were investigated through the TaqMan Array Human MicroRNA Cards, followed by real-time RT-PCR assay for specific miRNAs. Selected miRNA targets were analyzed by Western blot and correlated to the HPV oncoproteins by specifically silencing E6 and E7 expression. Exosomes, isolated from K16 and K38 supernatants by differential centrifugations, were quantified through the vesicle-associated acetylcholinesterase activity. MiRNAs deregulated in K16 and K38 cells were identified. HPV-16 and/or HPV-38 E6 and E7 single proteins can modify the expression of selected miRNAs involved in the tumorigenesis, in particular miR-18a, -19a, -34a and -590-5p. The analysis of the content of exosomes isolated from HPV-positive cells revealed the presence of E6 and E7 mRNAs and few miRNAs. MiR-222, a key miRNA deregulated in many cancers, was identified in exosomes from K16 cells. HPV E6 and/or E7 oncoprotein expression can induce the deregulation of some miRNAs. Through the production and function of exosomes, HPV oncogenes as well as HPV-deregulated miRNAs can potentiate the virus oncogenic effects in the tumor cell microenvironment.

  3. Modulation of Protein Fragmentation Through Carbamylation of Primary Amines

    NASA Astrophysics Data System (ADS)

    Greer, Sylvester M.; Holden, Dustin D.; Fellers, Ryan; Kelleher, Neil L.; Brodbelt, Jennifer S.

    2017-08-01

    We evaluate the impact of carbamylation of the primary amines of the side-chains of Lys and the N-termini on the fragmentation of intact protein ions and the chromatographic properties of a mixture of E. coli ribosomal proteins. The fragmentation patterns of the six unmodified and carbamylated proteins obtained by higher energy collision dissociation (HCD) and ultraviolet photodissociation (UVPD) were compared. Carbamylation significantly reduced the total number of protons retained by the protein owing to the conversion of basic primary amines to non-basic carbamates. Carbamylation caused a significant negative impact on fragmentation of the protein by HCD (i.e., reduced sequence coverage and fewer diagnostic fragment ions) consistent with the mobile proton model, which correlates peptide fragmentation with charge distribution and the opportunity for charge-directed pathways. In addition, fragmentation was enhanced near the N- and C-termini upon HCD of carbamylated proteins. For LCMS/MS analysis of E. coli ribosomal proteins, the retention times increased by 16 min on average upon carbamylation, an outcome attributed to the increased hydrophobicity of the proteins after carbamylation. As noted for both the six model proteins and the ribosomal proteins, carbamylation had relatively little impact on the distribution or types of fragment ions product by UVPD, supporting the proposition that the mechanism of UVPD for intact proteins does not reflect the mobile proton model.

  4. Intracellular scFvs against the viral E6 oncoprotein provoke apoptosis in human papillomavirus-positive cancer cells

    SciTech Connect

    Lagrange, Magali; Boulade-Ladame, Charlotte; Mailly, Laurent; Weiss, Etienne; Orfanoudakis, Georges; Deryckere, Francois . E-mail: francois.deryckere@esbs.u-strasbg.fr

    2007-09-21

    The E6 protein of human papillomavirus type 16 (16E6) is involved in the tumorigenesis of human cervical cells by targeting numerous cellular proteins. We have designed a strategy for neutralizing 16E6 based on the intracellular expression of single-chain Fv antibodies (scFvs) specific to 16E6. Recombinant adenovirus vectors were constructed to allow expression of two 16E6-binding scFvs and one 16E6-non-binding scFv in HPV16-positive and -negative cells. Expression of the scFvs provoked two types of effects: (i) inhibition of proliferation of all cell lines tested, this aspecific toxicity being likely due to the aggregation of unfolded scFvs; and (ii) apoptosis observed only in HPV16-positive cervical cancer cell lines after expression of 16E6-binding scFvs, this specific effect being proportional to the intracellular solubility of the scFvs. These data demonstrate the feasibility of intracellular immunization with anti-16E6 scFvs and highlight the importance of the solubility of the intracellular antibodies.

  5. Myofibrillar Z-discs Are a Protein Phosphorylation Hot Spot with Protein Kinase C (PKCα) Modulating Protein Dynamics.

    PubMed

    Reimann, Lena; Wiese, Heike; Leber, Yvonne; Schwäble, Anja N; Fricke, Anna L; Rohland, Anne; Knapp, Bettina; Peikert, Christian D; Drepper, Friedel; van der Ven, Peter F M; Radziwill, Gerald; Fürst, Dieter O; Warscheid, Bettina

    2017-03-01

    The Z-disc is a protein-rich structure critically important for the development and integrity of myofibrils, which are the contractile organelles of cross-striated muscle cells. We here used mouse C2C12 myoblast, which were differentiated into myotubes, followed by electrical pulse stimulation (EPS) to generate contracting myotubes comprising mature Z-discs. Using a quantitative proteomics approach, we found significant changes in the relative abundance of 387 proteins in myoblasts versus differentiated myotubes, reflecting the drastic phenotypic conversion of these cells during myogenesis. Interestingly, EPS of differentiated myotubes to induce Z-disc assembly and maturation resulted in increased levels of proteins involved in ATP synthesis, presumably to fulfill the higher energy demand of contracting myotubes. Because an important role of the Z-disc for signal integration and transduction was recently suggested, its precise phosphorylation landscape further warranted in-depth analysis. We therefore established, by global phosphoproteomics of EPS-treated contracting myotubes, a comprehensive site-resolved protein phosphorylation map of the Z-disc and found that it is a phosphorylation hotspot in skeletal myocytes, underscoring its functions in signaling and disease-related processes. In an illustrative fashion, we analyzed the actin-binding multiadaptor protein filamin C (FLNc), which is essential for Z-disc assembly and maintenance, and found that PKCα phosphorylation at distinct serine residues in its hinge 2 region prevents its cleavage at an adjacent tyrosine residue by calpain 1. Fluorescence recovery after photobleaching experiments indicated that this phosphorylation modulates FLNc dynamics. Moreover, FLNc lacking the cleaved Ig-like domain 24 exhibited remarkably fast kinetics and exceedingly high mobility. Our data set provides research community resource for further identification of kinase-mediated changes in myofibrillar protein interactions

  6. A broken E6 solution to the solar neutrino problem

    NASA Astrophysics Data System (ADS)

    Ross, G. G.; Segrè, G. C.

    1987-10-01

    Broken E6 models, as suggested by superstrings, may have stable massive neutrinos in matter multiplets. These can be candidates for the dark matter of the universe. If we choose an additional Z' in the E6 gauge multiplet to couple to these neutrinos, but not ordinary leptons, we may also solve the solar neutrino problem, without violating known experimental bounds. The Z' must have a mass comparable to the ordinary Z mass. On sabbatical leave from Department of Physics, University of Pennsylvania, Philadelphia, PA 19104, USA.

  7. HMGA proteins as modulators of chromatin structure during transcriptional activation

    PubMed Central

    Ozturk, Nihan; Singh, Indrabahadur; Mehta, Aditi; Braun, Thomas; Barreto, Guillermo

    2013-01-01

    High mobility group (HMG) proteins are the most abundant non-histone chromatin associated proteins. HMG proteins bind to DNA and nucleosome and alter the structure of chromatin locally and globally. Accessibility to DNA within chromatin is a central factor that affects DNA-dependent nuclear processes, such as transcription, replication, recombination, and repair. HMG proteins associate with different multi-protein complexes to regulate these processes by mediating accessibility to DNA. HMG proteins can be subdivided into three families: HMGA, HMGB, and HMGN. In this review, we will focus on recent advances in understanding the function of HMGA family members, specifically their role in gene transcription regulation during development and cancer. PMID:25364713

  8. TRAF Family Proteins Regulate Autophagy Dynamics by Modulating AUTOPHAGY PROTEIN6 Stability in Arabidopsis[OPEN

    PubMed Central

    Qi, Hua; Xia, Fan-Nv; Xie, Li-Juan; Yu, Lu-Jun; Chen, Qin-Fang; Jiang, Liwen

    2017-01-01

    Eukaryotic cells use autophagy to recycle cellular components. During autophagy, autophagosomes deliver cytoplasmic contents to the vacuole or lysosome for breakdown. Mammalian cells regulate the dynamics of autophagy via ubiquitin-mediated proteolysis of autophagy proteins. Here, we show that the Arabidopsis thaliana Tumor necrosis factor Receptor-Associated Factor (TRAF) family proteins TRAF1a and TRAF1b (previously named MUSE14 and MUSE13, respectively) help regulate autophagy via ubiquitination. Upon starvation, cytoplasmic TRAF1a and TRAF1b translocated to autophagosomes. Knockout traf1a/b lines showed reduced tolerance to nutrient deficiency, increased salicylic acid and reactive oxygen species levels, and constitutive cell death in rosettes, resembling the phenotypes of autophagy-defective mutants. Starvation-activated autophagosome accumulation decreased in traf1a/b root cells, indicating that TRAF1a and TRAF1b function redundantly in regulating autophagosome formation. TRAF1a and TRAF1b interacted in planta with ATG6 and the RING finger E3 ligases SINAT1, SINAT2, and SINAT6 (with a truncated RING-finger domain). SINAT1 and SINAT2 require the presence of TRAF1a and TRAF1b to ubiquitinate and destabilize AUTOPHAGY PROTEIN6 (ATG6) in vivo. Conversely, starvation-induced SINAT6 reduced SINAT1- and SINAT2-mediated ubiquitination and degradation of ATG6. Consistently, SINAT1/SINAT2 and SINAT6 knockout mutants exhibited increased tolerance and sensitivity, respectively, to nutrient starvation. Therefore, TRAF1a and TRAF1b function as molecular adaptors that help regulate autophagy by modulating ATG6 stability in Arabidopsis. PMID:28351989

  9. Force modulated conductance of artificial coiled-coil protein monolayers.

    PubMed

    Atanassov, Alexander; Hendler, Ziv; Berkovich, Inbal; Ashkenasy, Gonen; Ashkenasy, Nurit

    2013-01-01

    Studies of charge transport through proteins bridged between two electrodes have been the subject of intense research in recent years. However, the complex structure of proteins makes it difficult to elucidate transport mechanisms, and the use of simple peptide oligomers may be an over simplified model of the proteins. To bridge this structural gap, we present here studies of charge transport through artificial parallel coiled-coil proteins conducted in dry environment. Protein monolayers uniaxially oriented at an angle of ∼ 30° with respect to the surface normal were prepared. Current voltage measurements, obtained using conductive-probe atomic force microscopy, revealed the mechano-electronic behavior of the protein films. It was found that the low voltage conductance of the protein monolayer increases linearly with applied force, mainly due to increase in the tip contact area. Negligible compression of the films for loads below 26 nN allowed estimating a tunneling attenuation factor, β(0) , of 0.5-0.6 Å(-1) , which is akin to charge transfer by tunneling mechanism, despite the comparably large charge transport distance. These studies show that mechano-electronic behavior of proteins can shed light on their complex charge transport mechanisms, and on how these mechanisms depend on the detailed structure of the proteins. Such studies may provide insightful information on charge transfer in biological systems.

  10. Immunohistochemical Evaluation of E6/E7 HPV Oncoproteins Staining in Cervical Cancer.

    PubMed

    Stiasny, Annika; Kuhn, Christina; Mayr, Doris; Alexiou, Christoph; Janko, Christina; Wiest, Irmi; Jeschke, Udo; Kost, Bernd

    2016-06-01

    High-risk human papillomavirus (HPV) subtypes (i.e. 16 and 18) lead to uterine cervical cancer as well as HPV-positive oropharyngeal cancer (OSCC), a form of head and neck cancer. The induction of HPV-induced cancer is driven by virus-specific oncoproteins E6 and E7. E6 protein of HPV types 16 and 18 interacts with the E3 ubiquitin protein ligase, resulting in ubiquitination and proteolysis of tumor protein p53. E7 inactivates retinoblastoma protein (Rb) by phosphorylation followed by an increase of free eukaryotic transcription factor E2F (E2F) in the cell. This leads to an increase of cyclin-dependent kinase inhibitor p16, that is used as an immunohistochemical marker of HPV-associated OSCC. Unfortunately, p16 is not exclusively increased by E7 oncoprotein in carcinogenesis. Therefore, the aim of this study was to develop an immunohistochemical approach for the direct detection of E6/E7 oncoproteins in uterine cervical cancer as well as in OSCC. Paraffin sections of uterine cervical cancer and 130 were analyzed. Immunohistochemical staining protocols were evaluated with tissue slides from patients with cervical dysplasia (CIN III) and squamous epithelial carcinoma tissue with HPV infection. Liver and placental tissues were used as negative controls. E6-Specific antibody (Biorbyt) was used as primary antibody. The polymer staining method and diaminobenzidine were applied for further development. Panels of E7-specific antibodies were tested. Again, the polymer staining method and diaminobenzidine were applied for further development. E6-Specific antibody revealed specific and intense staining after pre-incubation of tissue slides with citrate buffer solution. Only the E7 antibody obtained from Chemicon showed intense and specific staining in patients with CIN III and squamous epithelial carcinoma tissue. Pre-incubation with proteinase K diminished non-specific reaction. Our results revealed a useful staining protocol for the immunohistochemical evaluation of E6/E7

  11. Exosome engineering for efficient intracellular delivery of soluble proteins using optically reversible protein–protein interaction module

    PubMed Central

    Yim, Nambin; Ryu, Seung-Wook; Choi, Kyungsun; Lee, Kwang Ryeol; Lee, Seunghee; Choi, Hojun; Kim, Jeongjin; Shaker, Mohammed R.; Sun, Woong; Park, Ji-Ho; Kim, Daesoo; Do Heo, Won; Choi, Chulhee

    2016-01-01

    Nanoparticle-mediated delivery of functional macromolecules is a promising method for treating a variety of human diseases. Among nanoparticles, cell-derived exosomes have recently been highlighted as a new therapeutic strategy for the in vivo delivery of nucleotides and chemical drugs. Here we describe a new tool for intracellular delivery of target proteins, named ‘exosomes for protein loading via optically reversible protein–protein interactions' (EXPLORs). By integrating a reversible protein–protein interaction module controlled by blue light with the endogenous process of exosome biogenesis, we are able to successfully load cargo proteins into newly generated exosomes. Treatment with protein-loaded EXPLORs is shown to significantly increase intracellular levels of cargo proteins and their function in recipient cells in vitro and in vivo. These results clearly indicate the potential of EXPLORs as a mechanism for the efficient intracellular transfer of protein-based therapeutics into recipient cells and tissues. PMID:27447450

  12. Identification of critical functional determinants of kainate receptor modulation by auxiliary protein Neto2.

    PubMed

    Griffith, Theanne N; Swanson, Geoffrey T

    2015-11-15

    Kainate receptors (KARs) are ionotropic glutamate receptors (iGluRs) that modulate synaptic transmission and intrinsic neuronal excitability. KARs associate with the auxiliary proteins neuropilin- and tolloid-like 1 and 2 (Neto1 and Neto2), which act as allosteric modulators of receptor function impacting all biophysical properties of these receptors studied to date. M3-S2 linkers play a critical role in KAR gating; we found that individual residues in these linkers bidirectionally influence Neto2 modulation of KAR desensitization in an agonist specific manner. We also identify the D1 dimer interface as a novel site of Neto2 modulation and functionally correlate the actions of Neto2 modulation of desensitization with modulation of cation sensitivity. We identify these domains as determinants of Neto2 modulation. Thus, our work contributes to the understanding of auxiliary subunit modulation of KAR function and could aid the development of KAR-specific modulators to alter receptor function. Kainate receptors (KARs) are important modulators of synaptic transmission and intrinsic neuronal excitability in the CNS. Their activity is shaped by the auxiliary proteins Neto1 and Neto2, which impact KAR gating in a receptor subunit- and Neto isoform-specific manner. The structural basis for Neto modulation of KAR gating is unknown. Here we identify the M3-S2 gating linker as a critical determinant contributing to Neto2 modulation of KARs. M3-S2 linkers control both the valence and magnitude of Neto2 modulation of homomeric GluK2 receptors. Furthermore, a single mutation in this domain abolishes Neto2 modulation of heteromeric receptor desensitization. Additionally, we found that cation sensitivity of KAR gating is altered by Neto2 association, suggesting that stability of the D1 dimer interface in the ligand-binding domain (LBD) is an important determinant of Neto2 actions. Moreover, modulation of cation sensitivity was eliminated by mutations in the M3-S2 linkers, thereby

  13. Disorder transitions and conformational diversity cooperatively modulate biological function in proteins

    PubMed Central

    Zea, Diego Javier; Monzon, Alexander Miguel; Gonzalez, Claudia; Fornasari, María Silvina; Tosatto, Silvio C. E.

    2016-01-01

    Abstract Structural differences between conformers sustain protein biological function. Here, we studied in a large dataset of 745 intrinsically disordered proteins, how ordered‐disordered transitions modulate structural differences between conformers as derived from crystallographic data. We found that almost 50% of the proteins studied show no transitions and have low conformational diversity while the rest show transitions and a higher conformational diversity. In this last subset, 60% of the proteins become more ordered after ligand binding, while 40% more disordered. As protein conformational diversity is inherently connected with protein function our analysis suggests differences in structure‐function relationships related to order‐disorder transitions. PMID:27038125

  14. Metal ion modulated electron transfer in photosynthetic proteins.

    SciTech Connect

    Utschig, L. M.; Thurnauer, M. C.; Chemistry

    2004-07-01

    Photosynthetic purple bacterial reaction center (RC) proteins are ideal native systems for addressing basic questions regarding the nature of biological electron transfer because both the protein structure and the electron-transfer reactions are well-characterized. Metal ion binding to the RC can affect primary photochemistry and provides a probe for understanding the involvement of local protein environments in electron transfer. The RC has two distinct transition metal ion binding sites, the well-known non-heme Fe{sup 2+} site buried in the protein interior and a recently discovered Zn{sup 2+} site located on the surface of the protein. Fe{sup 2+} removal and Zn{sup 2+} binding systematically affect different electron-transfer steps in the RC. Factors involved in the metal ion alteration of RC electron transfer may provide a paradigm for other biological systems involved in electron transfer.

  15. Utilization of alkyne bioconjugations to modulate protein function.

    PubMed

    Maza, Johnathan C; Howard, Christina A; Vipani, Megha A; Travis, Christopher R; Young, Douglas D

    2017-01-01

    The ability to introduce or modify protein function has widespread application to multiple scientific disciplines. The introduction of unique unnatural amino acids represents an excellent mechanism to incorporate new functionality; however, this approach is limited by ability of the translational machinery to recognize and incorporate the chemical moiety. To overcome this potential limitation, we aimed to exploit the functionality of existing unnatural amino acids to perform bioorthogonal reactions to introduce the desired protein modification, altering its function. Specifically, via the introduction of a terminal alkyne containing unnatural amino acid, we demonstrated chemically programmable protein modification through the Glaser-Hay coupling to other terminal alkynes, altering the function of a protein. In a proof-of-concept experiment, this approach has been utilized to modify the fluorescence spectrum of green fluorescent protein.

  16. Allosteric modulation of protein oligomerization: an emerging approach to drug design

    PubMed Central

    Gabizon, Ronen; Friedler, Assaf

    2014-01-01

    Many disease-related proteins are in equilibrium between different oligomeric forms. The regulation of this equilibrium plays a central role in maintaining the activity of these proteins in vitro and in vivo. Modulation of the oligomerization equilibrium of proteins by molecules that bind preferentially to a specific oligomeric state is emerging as a potential therapeutic strategy that can be applied to many biological systems such as cancer and viral infections. The target proteins for such compounds are diverse in structure and sequence, and may require different approaches for shifting their oligomerization equilibrium. The discovery of such oligomerization-modulating compounds is thus achieved based on existing structural knowledge about the specific target proteins, as well as on their interactions with partner proteins or with ligands. In silico design and combinatorial tools such as peptide arrays and phage display are also used for discovering compounds that modulate protein oligomerization. The current review highlights some of the recent developments in the design of compounds aimed at modulating the oligomerization equilibrium of proteins, including the “shiftides” approach developed in our lab. PMID:24790978

  17. Engineered carbohydrate-binding module (CBM) protein-suspended single-walled carbon nanotubes in water.

    PubMed

    Xu, Qi; Song, Qing; Ai, Xin; McDonald, Timothy J; Long, Hai; Ding, Shi-You; Himmel, Michael E; Rumbles, Garry

    2009-01-21

    Engineered protein, CtCBM4, the first carbohydrate-binding module (CBM) protein is successfully used to debundle and suspend single-walled carbon nanotubes (SWNTs) effectively in aqueous solution, which opens up a new avenue in further functionalizing and potential selectively fractionating SWNTs for diverse biology- and/or energy-related applications.

  18. Allosteric Modulation of protein oligomerization: an emerging approach to drug design

    NASA Astrophysics Data System (ADS)

    Gabizon, Ronen; Friedler, Assaf

    2014-03-01

    Many disease-related proteins are in equilibrium between different oligomeric forms. The regulation of this equilibrium plays a central role in maintaining the activity of these proteins in vitro and in vivo. Modulation of the oligomerization equilibrium of proteins by molecules that bind preferentially to a specific oligomeric state is emerging as a potential therapeutic strategy that can be applied to many biological systems such as cancer and viral infections. The target proteins for such compounds are diverse in structure and sequence, and may require different approaches for shifting their oligomerization equilibrium. The discovery of such oligomerization-modulating compounds is thus achieved based on existing structural knowledge about the specific target proteins, as well as on their interactions with partner proteins or with ligands. In silico design and combinatorial tools such as peptide arrays and phage display are also used for discovering compounds that modulate protein oligomerization. The current review highlights some of the recent developments in the design of compounds aimed at modulating the oligomerization equilibrium of proteins, including the "shiftides" approach developed in our lab.

  19. Dynamic functional modules in co-expressed protein interaction networks of dilated cardiomyopathy

    PubMed Central

    2010-01-01

    Background Molecular networks represent the backbone of molecular activity within cells and provide opportunities for understanding the mechanism of diseases. While protein-protein interaction data constitute static network maps, integration of condition-specific co-expression information provides clues to the dynamic features of these networks. Dilated cardiomyopathy is a leading cause of heart failure. Although previous studies have identified putative biomarkers or therapeutic targets for heart failure, the underlying molecular mechanism of dilated cardiomyopathy remains unclear. Results We developed a network-based comparative analysis approach that integrates protein-protein interactions with gene expression profiles and biological function annotations to reveal dynamic functional modules under different biological states. We found that hub proteins in condition-specific co-expressed protein interaction networks tended to be differentially expressed between biological states. Applying this method to a cohort of heart failure patients, we identified two functional modules that significantly emerged from the interaction networks. The dynamics of these modules between normal and disease states further suggest a potential molecular model of dilated cardiomyopathy. Conclusions We propose a novel framework to analyze the interaction networks in different biological states. It successfully reveals network modules closely related to heart failure; more importantly, these network dynamics provide new insights into the cause of dilated cardiomyopathy. The revealed molecular modules might be used as potential drug targets and provide new directions for heart failure therapy. PMID:20950417

  20. Inactivation of p53 Rescues the Maintenance of High Risk HPV DNA Genomes Deficient in Expression of E6

    PubMed Central

    Lorenz, Laurel D.; Rivera Cardona, Jessenia; Lambert, Paul F.

    2013-01-01

    The human papillomavirus DNA genome undergoes three distinct stages of replication: establishment, maintenance and amplification. We show that the HPV16 E6 protein is required for the maintenance of the HPV16 DNA genome as an extrachromosomal, nuclear plasmid in its natural host cell, the human keratinocyte. Based upon mutational analyses, inactivation of p53 by E6, but not necessarily E6-mediated degradation of p53, was found to correlate with the ability of E6 to support maintenance of the HPV16 genome as a nuclear plasmid. Inactivation of p53 with dominant negative p53 rescued the ability of HPV16 E6STOP and E6SAT mutant genomes to replicate as extrachromosomal genomes, though not to the same degree as observed for the HPV16 E6 wild-type (WT) genome. Inactivation of p53 also rescued the ability of HPV18 and HPV31 E6-deficient genomes to be maintained at copy numbers comparable to that of HPV18 and HPV31 E6WT genomes at early passages, though upon further passaging copy numbers for the HPV18 and 31 E6-deficient genomes lessened compared to that of the WT genomes. We conclude that inactivation of p53 is necessary for maintenance of HPV16 and for HPV18 and 31 to replicate at WT copy number, but that additional functions of E6 independent of inactivating p53 must also contribute to the maintenance of these genomes. Together these results suggest that re-activation of p53 may be a possible means for eradicating extrachromosomal HPV16, 18 or 31 genomes in the context of persistent infections. PMID:24204267

  1. Rubella virus capsid protein modulation of viral genomic and subgenomic RNA synthesis

    SciTech Connect

    Tzeng, W.-P.; Frey, Teryl K. . E-mail: tfrey@gsu.edu

    2005-07-05

    The ratio of the subgenomic (SG) to genome RNA synthesized by rubella virus (RUB) replicons expressing the green fluorescent protein reporter gene (RUBrep/GFP) is substantially higher than the ratio of these species synthesized by RUB (4.3 for RUBrep/GFP vs. 1.3-1.4 for RUB). It was hypothesized that this modulation of the viral RNA synthesis was by one of the virus structural protein genes and it was found that introduction of the capsid (C) protein gene into the replicons as an in-frame fusion with GFP resulted in an increase of genomic RNA production (reducing the SG/genome RNA ratio), confirming the hypothesis and showing that the C gene was the moiety responsible for the modulation effect. The N-terminal one-third of the C gene was required for the effect of be exhibited. A similar phenomenon was not observed with the replicons of Sindbis virus, a related Alphavirus. Interestingly, modulation was not observed when RUBrep/GFP was co-transfected with either other RUBrep or plasmid constructs expressing the C gene, demonstrating that modulation could occur only when the C gene was provided in cis. Mutations that prevented translation of the C protein failed to modulate RNA synthesis, indicating that the C protein was the moiety responsible for modulation; consistent with this conclusion, modulation of RNA synthesis was maintained when synonymous codon mutations were introduced at the 5' end of the C gene that changed the C gene sequence without altering the amino acid sequence of the C protein. These results indicate that C protein translated in proximity of viral replication complexes, possibly from newly synthesized SG RNA, participate in regulating the replication of viral RNA.

  2. A predictor for toxin-like proteins exposes cell modulator candidates within viral genomes

    PubMed Central

    Naamati, Guy; Askenazi, Manor; Linial, Michal

    2010-01-01

    Motivation: Animal toxins operate by binding to receptors and ion channels. These proteins are short and vary in sequence, structure and function. Sporadic discoveries have also revealed endogenous toxin-like proteins in non-venomous organisms. Viral proteins are the largest group of quickly evolving proteomes. We tested the hypothesis that toxin-like proteins exist in viruses and that they act to modulate functions of their hosts. Results: We updated and improved a classifier for compact proteins resembling short animal toxins that is based on a machine-learning method. We applied it in a large-scale setting to identify toxin-like proteins among short viral proteins. Among the ∼26 000 representatives of such short proteins, 510 sequences were positively identified. We focused on the 19 highest scoring proteins. Among them, we identified conotoxin-like proteins, growth factors receptor-like proteins and anti-bacterial peptides. Our predictor was shown to enhance annotation inference for many ‘uncharacterized’ proteins. We conclude that our protocol can expose toxin-like proteins in unexplored niches including metagenomics data and enhance the systematic discovery of novel cell modulators for drug development. Availability: ClanTox is available at http://www.clantox.cs.huji.ac.il Contact: michall@cc.huji.ac.il PMID:20823311

  3. Saturated fatty acids modulate autophagy's proteins in the hypothalamus.

    PubMed

    Portovedo, Mariana; Ignacio-Souza, Letícia M; Bombassaro, Bruna; Coope, Andressa; Reginato, Andressa; Razolli, Daniela S; Torsoni, Márcio A; Torsoni, Adriana S; Leal, Raquel F; Velloso, Licio A; Milanski, Marciane

    2015-01-01

    Autophagy is an important process that regulates cellular homeostasis by degrading dysfunctional proteins, organelles and lipids. In this study, the hypothesis that obesity could lead to impairment in hypothalamic autophagy in mice was evaluated by examining the hypothalamic distribution and content of autophagic proteins in animal with obesity induced by 8 or 16 weeks high fat diet to induce obesity and in response to intracerebroventricular injections of palmitic acid. The results showed that chronic exposure to a high fat diet leads to an increased expression of inflammatory markers and downregulation of autophagic proteins. In obese mice, autophagic induction leads to the downregulation of proteins, such as JNK and Bax, which are involved in the stress pathways. In neuron cell-line, palmitate has a direct effect on autophagy even without inflammatory activity. Understanding the cellular and molecular bases of overnutrition is essential for identifying new diagnostic and therapeutic targets for obesity.

  4. Alcohol modulation of G-protein-gated inwardly rectifying potassium channels: from binding to therapeutics

    PubMed Central

    Bodhinathan, Karthik; Slesinger, Paul A.

    2014-01-01

    Alcohol (ethanol)-induced behaviors may arise from direct interaction of alcohol with discrete protein cavities within brain proteins. Recent structural and biochemical studies have provided new insights into the mechanism of alcohol-dependent activation of G protein-gated inwardly rectifying potassium (GIRK) channels, which regulate neuronal responses in the brain reward circuit. GIRK channels contain an alcohol binding pocket formed at the interface of two adjacent channel subunits. Here, we discuss the physiochemical properties of the alcohol pocket and the roles of G protein βγ subunits and membrane phospholipid PIP2 in regulating the alcohol response of GIRK channels. Some of the features of alcohol modulation of GIRK channels may be common to other alcohol-sensitive brain proteins. We discuss the possibility of alcohol-selective therapeutics that block alcohol access to the pocket. Understanding alcohol recognition and modulation of brain proteins is essential for development of therapeutics for alcohol abuse and addiction. PMID:24611054

  5. Modulation of Ionic Channel Function by Protein Phosphorylation

    DTIC Science & Technology

    1992-11-12

    and chemical synthesis of oligomeric channel proteins. In: Membrane Electrochemistry. I. Vodyanoy and M. Blank, ed(s). American Chemical Society, ACS...A250 (1992). Grove, A., J.M. Tomich, T. Iwamoto, G.L. Reddy, S. Marrer, M.S. Montal and M. Montal. Design and synthesis of four-helix bundle channel... dihydropyridine - sensitive calcium channel protein. In: Miles Symposium on Nimodipine. On calcium channel antagonists in the central nervous system

  6. Modulation of collagen metabolism by the nucleolar protein fibrillarin.

    PubMed

    Lefèvre, F; Garnotel, R; Georges, N; Gillery, P

    2001-11-15

    Metabolic functions of fibroblasts are tightly regulated by the extracellular environment. When cultivated in tridimensional collagen lattices, fibroblasts exhibit a lowered activity of protein synthesis, especially concerning extracellular matrix proteins. We have previously shown that extracellular collagen impaired the processing of ribosomal RNA (rRNA) in nucleoli by generating changes in the expression of nucleolar proteins and a premature degradation of neosynthesized rRNA. In this study, we have investigated whether inhibiting the synthesis of fibrillarin, a major nucleolar protein with decreased expression in collagen lattices, could mimic the effects of extracellular matrix. Monolayer-cultured fibroblasts were transfected with anti-fibrillarin antisense oligodeoxynucleotides, which significantly decreased fibrillarin content. Downregulation of fibrillarin expression inhibited procollagen secretion into the extracellular medium, without altering total collagen production. No changes of pro1(I)collagen mRNA expression or proline hydroxylation were found. A concomitant intracellular retention of collagen and its chaperone protein HSP47 was found, but no effect on the production of other extracellular matrix macromolecules or remodelling enzymes was observed. These data show that collagen processing depends on unknown mechanisms, involving proteins primarily located in the nucleolar compartment with other demonstrated functions, and suggest specific links between nucleolar machinery and extracellular matrix.

  7. Ingestion of Casein in a Milk Matrix Modulates Dietary Protein Digestion and Absorption Kinetics but Does Not Modulate Postprandial Muscle Protein Synthesis in Older Men.

    PubMed

    Churchward-Venne, Tyler A; Snijders, Tim; Linkens, Armand M A; Hamer, Henrike M; van Kranenburg, Janneau; van Loon, Luc J C

    2015-07-01

    The slow digestion and amino acid absorption kinetics of isolated micellar casein have been held responsible for its relatively lower postprandial muscle protein synthetic response compared with rapidly digested proteins such as isolated whey. However, casein is normally consumed within a milk matrix. We hypothesized that protein digestion and absorption kinetics and the subsequent muscle protein synthetic response after micellar casein ingestion are modulated by the milk matrix. The aim of this study was to determine the impact of a milk matrix on casein protein digestion and absorption kinetics and postprandial muscle protein synthesis in older men. In a parallel-group design, 32 healthy older men (aged 71 ± 1 y) received a primed continuous infusion of L-[ring-(2)H5]-phenylalanine, L-[ring-3,5-(2)H2]-tyrosine, and L-[1-(13)C]-leucine, and ingested 25 g intrinsically L-[1-(13)C]-phenylalanine and L-[1-(13)C]-leucine labeled casein dissolved in bovine milk serum (Cas+Serum) or water (Cas). Plasma samples and muscle biopsies were collected in the postabsorptive state and for 300 min in the postprandial period to examine whole-body and skeletal muscle protein metabolism. Casein ingestion increased plasma leucine and phenylalanine concentrations and L-[1-(13)C]-phenylalanine enrichments, with a more rapid rise after Cas vs. Cas+Serum. Nonetheless, dietary protein-derived phenylalanine availability did not differ between Cas+Serum (47 ± 2%, mean ± SEM) and Cas (46 ± 3%) when assessed over the 300-min postprandial period (P = 0.80). The milk matrix did not modulate postprandial myofibrillar protein synthesis rates from 0 to 120 min (0.038 ± 0.005 vs. 0.031 ± 0.007%/h) or from 120 to 300 min (0.052 ± 0.004 vs. 0.067 ± 0.005%/h) after Cas+Serum vs. Cas. Similarly, no treatment differences in muscle protein-bound L-[1-(13)C]-phenylalanine enrichments were observed at 120 min (0.003 ± 0.001 vs. 0.002 ± 0.001) or 300 min (0.015 ± 0.002 vs. 0.016 ± 0.002 mole

  8. Calcium-binding modulator protein from the unfertilized egg of the sea urchin Arbacia punctulata

    PubMed Central

    1979-01-01

    We have purified and partly characterized a calcium-binding protein from the unfertilized egg of the sea urchin Arbacia punctulata. This protein closely resembles the calcium-binding modulator protein of bovine brain in its molecular weight, electrophoretic mobility, amino acid analysis, and peptide map. It activates bovine brain phosphodiesterase in the presence of calcium but has no effect on the phosphodiesterase of the Arbacia egg. Densitometric scanning of acrylamide gels of arbacia egg homogenates shows the modulator protein to represent 0.1% of the total protein of the egg. At 10(-4) M free calcium, the protein binds four calcium ions per 17,000-dalton molecule. We have used a column of rabbit skeletal muscle troponin-I covalently coupled to Sepharose 4B as an affinity column to selectively purify the Arbacia egg calcium-binding protein. This column has also been used to purify bovine brain modulator protein and may prove of general use in isolating similar proteins from other sources. The technique may be particularly helpful when only small quantities of starting material are available. PMID:217882

  9. Modulation of protein quality control systems by food phytochemicals.

    PubMed

    Murakami, Akira

    2013-05-01

    There is compelling evidence showing that dietary phytochemicals have exhibited pronounced bioactivities in a number of experimental models. In addition, a variety of epidemiological surveys have demonstrated that frequent ingestion of vegetables and fruits, which contain abundant phytochemicals, lowers the risk of onset of some diseases. However, the action mechanisms by which dietary phytochemicals show bioactivity remain to be fully elucidated and a fundamental question is why this class of chemicals has great potential for regulating health. Meanwhile, maintenance and repair of biological proteins by molecular chaperones, such as heat shock proteins, and clearance of abnormal proteins by the ubiquitin-proteasome system and autophagy play central roles in health, some disease prevention, and longevity. Interestingly, several recent studies have revealed that phytochemicals, including curcumin (yellow pigment in turmeric), resveratrol (phytoalexin in grapes), quercetin (general flavonol in onions and others), and isothiocyanates (preferentially present in cruciferous vegetables, such as broccoli and cabbage), are remarkable regulators of protein quality control systems, suggesting that their physiological and biological functions are exerted, at least in part, through activation of such unique mechanisms. This review article highlights recent findings regarding the effects of representative phytochemicals on protein quality control systems and their possible molecular mechanisms.

  10. Calcium phosphate bioceramics induce mineralization modulated by proteins.

    PubMed

    Wang, Kefeng; Leng, Yang; Lu, Xiong; Ren, Fuzeng

    2013-08-01

    Proteins play an important role in the process of biomineralization, which is considered the critical process of new bone formation. The calcium phosphate (Ca-P) mineralization happened on hydroxyapatite (HA), β-tricalcium phosphate (β-TCP) and biphasic calcium phosphate (BCP) when proteins presented were investigated systematically. The results reveal that the presence of protein in the revised simulated body fluid (RSBF) did not alter the shape and crystal structure of the precipitated micro-crystals in the Ca-P layer formed on the three types of bioceramics. However, the morphology of the Ca-P precipitates was regulated but the structure of Ca-P crystal was unchanged in vivo. The presence of proteins always inhibits Ca-P mineralization in RSBF and the degree of inhibitory effect is concentration dependent. Furthermore, Protein presence can increase the possibility of HA precipitation in vitro and in vivo. The results obtained in this study can be helpful for better understanding the mechanism of biomineralization induced by the Ca-P bioceramics.

  11. Intrinsic disorder modulates protein self-assembly and aggregation.

    PubMed

    De Simone, Alfonso; Kitchen, Craig; Kwan, Ann H; Sunde, Margaret; Dobson, Christopher M; Frenkel, Daan

    2012-05-01

    Protein molecules have evolved to adopt distinctive and well-defined functional and soluble states under physiological conditions. In some circumstances, however, proteins can self-assemble into fibrillar aggregates designated as amyloid fibrils. In vivo these processes are normally associated with severe pathological conditions but can sometimes have functional relevance. One such example is the hydrophobins, whose aggregation at air-water interfaces serves to create robust protein coats that help fungal spores to resist wetting and thus facilitate their dispersal in the air. We have performed multiscale simulations to address the molecular determinants governing the formation of functional amyloids by the class I fungal hydrophobin EAS. Extensive samplings of full-atom replica-exchange molecular dynamics and coarse-grained simulations have allowed us to identify factors that distinguish aggregation-prone from highly soluble states of EAS. As a result of unfavourable entropic terms, highly dynamical regions are shown to exert a crucial influence on the propensity of the protein to aggregate under different conditions. More generally, our findings suggest a key role that specific flexible structural elements can play to ensure the existence of soluble and functional states of proteins under physiological conditions.

  12. Dark matter in a constrained E 6 inspired SUSY model

    NASA Astrophysics Data System (ADS)

    Athron, P.; Harries, D.; Nevzorov, R.; Williams, A. G.

    2016-12-01

    We investigate dark matter in a constrained E 6 inspired supersymmetric model with an exact custodial symmetry and compare with the CMSSM. The breakdown of E 6 leads to an additional U(1) N symmetry and a discrete matter parity. The custodial and matter symmetries imply there are two stable dark matter candidates, though one may be extremely light and contribute negligibly to the relic density. We demonstrate that a predominantly Higgsino, or mixed bino-Higgsino, neutralino can account for all of the relic abundance of dark matter, while fitting a 125 GeV SM-like Higgs and evading LHC limits on new states. However we show that the recent LUX 2016 limit on direct detection places severe constraints on the mixed bino-Higgsino scenarios that explain all of the dark matter. Nonetheless we still reveal interesting scenarios where the gluino, neutralino and chargino are light and discoverable at the LHC, but the full relic abundance is not accounted for. At the same time we also show that there is a huge volume of parameter space, with a predominantly Higgsino dark matter candidate that explains all the relic abundance, that will be discoverable with XENON1T. Finally we demonstrate that for the E 6 inspired model the exotic leptoquarks could still be light and within range of future LHC searches.

  13. Chemosensitization of Prostate Cancer by Modulating Bcl-2 Family Proteins

    PubMed Central

    Karnak, David; Xu, Liang

    2010-01-01

    A major challenge in oncology is the development of chemoresistance. This often occurs as cancer progresses and malignant cells acquire mechanisms to resist insults that would normally induce apoptosis. The onset of androgen independence in advanced prostate cancer is a prime example of this phenomenon. Overexpression of the pro-survival/anti-apoptotic proteins Bcl-2, Bcl-xL, and Mcl-1 are hallmarks of this transition. Here we outline the evolution of therapeutics designed to either limit the source or disrupt the interactions of these pro-survival proteins. By either lessening the stoichiometric abundance of Bcl-2/xL/Mcl-1 in reference to their pro-apoptotic foils or freeing these pro-apoptotic proteins from their grip, these treatments aim to sensitize cells to chemotherapy by priming cells for death. DNA anti-sense and RNA interference have been effectively employed to decrease Bcl-2 family mRNA and protein levels in cell culture models of advanced prostate cancer. However, clinical studies are lagging due to in vivo delivery challenges. The burgeoning field of nanoparticle delivery holds great promise in helping to overcome the challenge of administering highly labile nucleic acid based therapeutics. On another front, small molecule inhibitors that block the hetero-dimerization of pro-survival with pro-apoptotic proteins have significant clinical advantages and have advanced farther in clinical trials with promising early results. Most recently, a peptide has been discovered that can convert Bcl-2 from a pro-survival to a pro-apoptotic protein. The future may lie in targeting multiple steps of the apoptotic pathway, including Bcl-2/xL/Mcl-1, to debilitate the survival capacity of cancer cells and make chemotherapy induced death their only option. PMID:20298153

  14. Amyloid Precursor Protein Expression Modulates Intestine Immune Phenotype

    PubMed Central

    Puig, Kendra L.; Swigost, Adam J.; Zhou, Xudong; Sens, MaryAnn; Combs, Colin K.

    2014-01-01

    Amyloid precursor protein (APP) is widely expressed across many tissue and cell types. Proteolytic processing of the protein gives rise to a plethora of protein fragments with varied biological activities. Although a large amount of data has been generated describing the metabolism of the protein in neurons, its role in regulating the phenotype of other cells remains unclear. Based upon prior work demonstrating that APP regulates the activation phenotype of monocytic lineage cells, we hypothesized that APP can regulate macrophage activation phenotype in tissues other than brain. Ileums of the small intestines from C57BL6/J wild type and APP−/− mice were compared as a representative tissue normally associated with abundant macrophage infiltration. APP−/− intestines demonstrated diminished CD68 immunoreactivity compared to wild type mice. This correlated with significantly less cycloxygenase-2 (cox-2), CD68, CD40, CD11c, and βIII-tubulin protein levels. Peritoneal macrophage from APP−/− mice demonstrated decreased in vitro migratory ability compared to wild type cells and diminished basal KC cytokine secretion. Whereas, APP−/− intestinal macrophage had an increase in basal KC cytokine secretion compared to wild type cells. Conversely, there was a significant decrease in multiple cytokine levels in APP−/− compared to wild type ileums. Finally, APP−/− mice demonstrated impaired absorption and increased motility compared to wild type mice. These data demonstrate the APP expression regulates immune cell secretions and phenotype and intestinal function. This data set describes a novel function for this protein or its metabolites that may be relevant not only for Alzheimer’s disease but a range of immune-related disorders. PMID:22124967

  15. Estrogen Modulates Expression of Tight Junction Proteins in Rat Vagina.

    PubMed

    Oh, Kyung-Jin; Lee, Hyun-Suk; Ahn, Kyuyoun; Park, Kwangsung

    2016-01-01

    Background. The objectives of this study were to investigate the localization of tight junctions and the modulation of zonula occludens- (ZO-) 1, occludin and claudin-1 expression by estrogen in castrated female rat vagina. Female Sprague-Dawley rats (230-240 g, n = 45) were divided into three groups and subjected to a sham operation (control group, n = 15), bilateral ovariectomy (Ovx group, n = 15), or bilateral ovariectomy followed by daily subcutaneous injection of 17β-estradiol (50 μg/kg/day, Ovx + Est group, n = 15). The cellular localization and expression of ZO-1, occludin, and claudin-1 were determined in each group by immunohistochemistry and western blot. Results. Expression of ZO-1 was diffuse in all groups, with the highest intensity in the superficial epithelium in the control group. Occludin was localized in the intermediate and basal epithelium. Claudin-1 was most intense in the superficial layer of the vaginal epithelium in the control group. Expression of ZO-1, occludin, and claudin-1 was significantly decreased after ovariectomy and was restored to the level of the control after estrogen replacement. Conclusions. Tight junctions are distinctly localized in rat vagina, and estrogen modulates the expression of tight junctions. Further researches are needed to clarify the functional role of tight junctions in vaginal lubrication.

  16. Estrogen Modulates Expression of Tight Junction Proteins in Rat Vagina

    PubMed Central

    Oh, Kyung-Jin; Ahn, Kyuyoun

    2016-01-01

    Background. The objectives of this study were to investigate the localization of tight junctions and the modulation of zonula occludens- (ZO-) 1, occludin and claudin-1 expression by estrogen in castrated female rat vagina. Female Sprague-Dawley rats (230–240 g, n = 45) were divided into three groups and subjected to a sham operation (control group, n = 15), bilateral ovariectomy (Ovx group, n = 15), or bilateral ovariectomy followed by daily subcutaneous injection of 17β-estradiol (50 μg/kg/day, Ovx + Est group, n = 15). The cellular localization and expression of ZO-1, occludin, and claudin-1 were determined in each group by immunohistochemistry and western blot. Results. Expression of ZO-1 was diffuse in all groups, with the highest intensity in the superficial epithelium in the control group. Occludin was localized in the intermediate and basal epithelium. Claudin-1 was most intense in the superficial layer of the vaginal epithelium in the control group. Expression of ZO-1, occludin, and claudin-1 was significantly decreased after ovariectomy and was restored to the level of the control after estrogen replacement. Conclusions. Tight junctions are distinctly localized in rat vagina, and estrogen modulates the expression of tight junctions. Further researches are needed to clarify the functional role of tight junctions in vaginal lubrication. PMID:27127786

  17. Tumor prevention in HPV8 transgenic mice by HPV8-E6 DNA vaccination.

    PubMed

    Marcuzzi, Gian Paolo; Awerkiew, Sabine; Hufbauer, Martin; Schädlich, Lysann; Gissmann, Lutz; Eming, Sabine; Pfister, Herbert

    2014-06-01

    The genus beta human papillomavirus 8 (HPV8) is involved in the development of cutaneous squamous cell carcinomas (SCCs) in individuals with epidermodysplasia verruciformis. Immunosuppressed transplant recipients are prone to harbor particularly high betapapillomavirus DNA loads, which may contribute to their highly increased risk of SCC. Tumor induction in HPV8 transgenic mice correlates with increased expression of viral oncogenes E6 and E2. In an attempt to prevent skin tumor development, we evaluated an HPV8-E6-DNA vaccine, which was able to stimulate a detectable HPV8-E6-specific cell-mediated immune response in 8/15 immunized mice. When skin of HPV8 transgenic mice was grafted onto non-transgenic littermates, the grafted HPV8 transgenic tissue was not rejected and papillomas started to grow within 14 days all over the transplant of 9/9 non-vaccinated and 7/15 not successfully vaccinated mice. In contrast, no papillomas developed in 6/8 successfully vaccinated mice. In the other two of these eight mice, a large ulcerative lesion developed within the initial papilloma growth or papilloma development was highly delayed. As the vaccine completely or partially prevented papilloma development without rejecting the transplanted HPV8 positive skin, the immune system appears to attack only keratinocytes with increased levels of E6 protein, which would give rise to papillomas.

  18. Structural basis for modulation of a G-protein-coupled receptor by allosteric drugs.

    PubMed

    Dror, Ron O; Green, Hillary F; Valant, Celine; Borhani, David W; Valcourt, James R; Pan, Albert C; Arlow, Daniel H; Canals, Meritxell; Lane, J Robert; Rahmani, Raphaël; Baell, Jonathan B; Sexton, Patrick M; Christopoulos, Arthur; Shaw, David E

    2013-11-14

    The design of G-protein-coupled receptor (GPCR) allosteric modulators, an active area of modern pharmaceutical research, has proved challenging because neither the binding modes nor the molecular mechanisms of such drugs are known. Here we determine binding sites, bound conformations and specific drug-receptor interactions for several allosteric modulators of the M2 muscarinic acetylcholine receptor (M2 receptor), a prototypical family A GPCR, using atomic-level simulations in which the modulators spontaneously associate with the receptor. Despite substantial structural diversity, all modulators form cation-π interactions with clusters of aromatic residues in the receptor extracellular vestibule, approximately 15 Å from the classical, 'orthosteric' ligand-binding site. We validate the observed modulator binding modes through radioligand binding experiments on receptor mutants designed, on the basis of our simulations, either to increase or to decrease modulator affinity. Simulations also revealed mechanisms that contribute to positive and negative allosteric modulation of classical ligand binding, including coupled conformational changes of the two binding sites and electrostatic interactions between ligands in these sites. These observations enabled the design of chemical modifications that substantially alter a modulator's allosteric effects. Our findings thus provide a structural basis for the rational design of allosteric modulators targeting muscarinic and possibly other GPCRs.

  19. Structural basis for modulation of a G-protein-coupled receptor by allosteric drugs

    NASA Astrophysics Data System (ADS)

    Dror, Ron O.; Green, Hillary F.; Valant, Celine; Borhani, David W.; Valcourt, James R.; Pan, Albert C.; Arlow, Daniel H.; Canals, Meritxell; Lane, J. Robert; Rahmani, Raphaël; Baell, Jonathan B.; Sexton, Patrick M.; Christopoulos, Arthur; Shaw, David E.

    2013-11-01

    The design of G-protein-coupled receptor (GPCR) allosteric modulators, an active area of modern pharmaceutical research, has proved challenging because neither the binding modes nor the molecular mechanisms of such drugs are known. Here we determine binding sites, bound conformations and specific drug-receptor interactions for several allosteric modulators of the M2 muscarinic acetylcholine receptor (M2 receptor), a prototypical family A GPCR, using atomic-level simulations in which the modulators spontaneously associate with the receptor. Despite substantial structural diversity, all modulators form cation-π interactions with clusters of aromatic residues in the receptor extracellular vestibule, approximately 15Å from the classical, `orthosteric' ligand-binding site. We validate the observed modulator binding modes through radioligand binding experiments on receptor mutants designed, on the basis of our simulations, either to increase or to decrease modulator affinity. Simulations also revealed mechanisms that contribute to positive and negative allosteric modulation of classical ligand binding, including coupled conformational changes of the two binding sites and electrostatic interactions between ligands in these sites. These observations enabled the design of chemical modifications that substantially alter a modulator's allosteric effects. Our findings thus provide a structural basis for the rational design of allosteric modulators targeting muscarinic and possibly other GPCRs.

  20. The RecX protein interacts with the RecA protein and modulates its activity in Herbaspirillum seropedicae

    PubMed Central

    Galvão, C.W.; Souza, E.M.; Etto, R.M.; Pedrosa, F.O.; Chubatsu, L.S.; Yates, M.G.; Schumacher, J.; Buck, M.; Steffens, M.B.R.

    2012-01-01

    DNA repair is crucial to the survival of all organisms. The bacterial RecA protein is a central component in the SOS response and in recombinational and SOS DNA repairs. The RecX protein has been characterized as a negative modulator of RecA activity in many bacteria. The recA and recX genes of Herbaspirillum seropedicae constitute a single operon, and evidence suggests that RecX participates in SOS repair. In the present study, we show that the H. seropedicae RecX protein (RecXHs) can interact with the H. seropedicae RecA protein (RecAHs) and that RecAHs possesses ATP binding, ATP hydrolyzing and DNA strand exchange activities. RecXHs inhibited 90% of the RecAHs DNA strand exchange activity even when present in a 50-fold lower molar concentration than RecAHs. RecAHs ATP binding was not affected by the addition of RecX, but the ATPase activity was reduced. When RecXHs was present before the formation of RecA filaments (RecA-ssDNA), inhibition of ATPase activity was substantially reduced and excess ssDNA also partially suppressed this inhibition. The results suggest that the RecXHs protein negatively modulates the RecAHs activities by protein-protein interactions and also by DNA-protein interactions. PMID:23044625

  1. Modulation of lipoprotein receptor functions by intracellular adaptor proteins.

    PubMed

    Stolt, Peggy C; Bock, Hans H

    2006-10-01

    Members of the low density lipoprotein (LDL) receptor gene family are critically involved in a wide range of physiological processes including lipid and vitamin homeostasis, cellular migration, neurodevelopment, and synaptic plasticity, to name a few. Lipoprotein receptors exert these diverse biological functions by acting as cellular uptake receptors or by inducing intracellular signaling cascades. It was discovered that a short sequence in the intracellular region of all lipoprotein receptors, Asn-Pro-X-Tyr (NPXY) is important for mediating either endocytosis or signal transduction events, and that this motif serves as a binding site for phosphotyrosine-binding (PTB) domain containing scaffold proteins. These molecular adaptors connect the transmembrane receptors with the endocytosis machinery and regulate cellular trafficking, or function as assembly sites for dynamic multi-protein signaling complexes. Whereas the LDL receptor represents the archetype of an endocytic lipoprotein receptor, the structurally closely related apolipoprotein E receptor 2 (apoER2) and very low density lipoprotein (VLDL) receptor activate a kinase-dependent intracellular signaling cascade after binding to the neuronal signaling molecule Reelin. This review focuses on two related PTB domain containing adaptor proteins that mediate these divergent lipoprotein receptor responses, ARH (autosomal recessive hypercholesterolemia protein) and Dab1 (disabled-1), and discusses the structural and molecular basis of this different behaviour.

  2. TNFα modulates protein degradation pathways in rheumatoid arthritis synovial fibroblasts.

    PubMed

    Connor, Alison M; Mahomed, Nizar; Gandhi, Rajiv; Keystone, Edward C; Berger, Stuart A

    2012-03-14

    Rheumatoid arthritis (RA) is a chronic inflammatory and destructive disease of the joint. The synovial lining consists of two main types of cells: synovial fibroblasts and macrophages. The macrophage-derived cytokine TNFα stimulates RA synovial fibroblasts to proliferate and produce growth factors, chemokines, proteinases and adhesion molecules, making them key players in the RA disease process. If proteins are not correctly folded, cellular stress occurs that can be relieved in part by increased degradation of the aberrant proteins by the proteasome or autophagy. We hypothesized that the activity of the protein degradation pathways would be increased in response to TNFα stimulation in RA synovial fibroblasts compared with control fibroblasts. Endoplasmic reticulum (ER) stress markers were examined in synovial fibroblasts by immunoblotting and PCR. Use of the autophagy and proteasome protein degradation pathways in response to TNFα stimulation was determined using a combination of experiments involving chemical inhibition of the autophagy or proteasome pathways followed by immunoblotting for the autophagy marker LC3, measurement of proteasome activity and long-lived protein degradation, and determination of cellular viability. RA synovial fibroblasts are under acute ER stress, and the stress is increased in the presence of TNFα. Autophagy is the main pathway used to relieve the ER stress in unstimulated fibroblasts, and both autophagy and the proteasome are more active in RA synovial fibroblasts compared with control fibroblasts. In response to TNFα, the autophagy pathway but not the proteasome is consistently stimulated, yet there is an increased dependence on the proteasome for cell viability. If autophagy is blocked in the presence of TNFα, an increase in proteasome activity occurs in RA synovial fibroblasts but not in control cells. TNFα stimulation of synovial fibroblasts results in increased expression of ER stress markers. Survival of synovial

  3. Use of hydrostatic pressure for modulation of protein chemical modification and enzymatic selectivity.

    PubMed

    Makarov, Alexey A; Helmy, Roy; Joyce, Leo; Reibarkh, Mikhail; Maust, Mathew; Ren, Sumei; Mergelsberg, Ingrid; Welch, Christopher J

    2016-05-11

    Using hydrostatic pressure to induce protein conformational changes can be a powerful tool for altering the availability of protein reactive sites and for changing the selectivity of enzymatic reactions. Using a pressure apparatus, it has been demonstrated that hydrostatic pressure can be used to modulate the reactivity of lysine residues of the protein ubiquitin with a water-soluble amine-specific homobifunctional coupling agent. Fewer reactive lysine residues were observed when the reaction was carried out under elevated pressure of 3 kbar, consistent with a pressure-induced conformational change of ubiquitin that results in fewer exposed lysine residues. Additionally, modulation of the stereoselectivity of an enzymatic transamination reaction was observed at elevated hydrostatic pressure. In one case, the minor diasteromeric product formed at atmospheric pressure became the major product at elevated pressure. Such pressure-induced alterations of protein reactivity may provide an important new tool for enzymatic reactions and the chemical modification of proteins.

  4. Effector proteins that modulate plant--insect interactions.

    PubMed

    Hogenhout, Saskia A; Bos, Jorunn I B

    2011-08-01

    Insect herbivores have highly diverse life cycles and feeding behaviors. They establish close interactions with their plant hosts and suppress plant defenses. Chewing herbivores evoke characteristic defense responses distinguishable from general mechanical damage. In addition, piercing-sucking hemipteran insects display typical feeding behavior that suggests active suppression of plant defense responses. Effectors that modulate plant defenses have been identified in the saliva of these insects. Tools for high-throughput effector identification and functional characterization have been developed. In addition, in some insect species it is possible to silence gene expression by RNAi. Together, this technological progress has enabled the identification of insect herbivore effectors and their targets that will lead to the development of novel strategies for pest resistances in plants.

  5. Design, Immune Responses and Anti-Tumor Potential of an HPV16 E6E7 Multi-Epitope Vaccine.

    PubMed

    de Oliveira, Liliane Maria Fernandes; Morale, Mirian Galliote; Chaves, Agatha A Muniz; Cavalher, Aline Marques; Lopes, Aline Soriano; Diniz, Mariana de Oliveira; Schanoski, Alessandra Soares; de Melo, Robson Lopes; Ferreira, Luís Carlos de Souza; de Oliveira, Maria Leonor S; Demasi, Marilene; Ho, Paulo Lee

    2015-01-01

    Cervical cancer is a common type of cancer among women worldwide and infection with high-risk human papillomavirus (HPVs) types represents the major risk factor for the etiopathogenesis of the disease. HPV-16 is the most frequently identified HPV type in cervical lesions and expression of E6 and E7 oncoproteins is required for the uncontrolled cellular proliferation. In the present study we report the design and experimental testing of a recombinant multi-epitope protein containing immunogenic epitopes of HPV-16 E6 and E7. Tumor preventive assays, based on the engraftment of TC-1 cells in mice, showed that the E6E7 multi-epitope protein induced a full preventive anti-tumor protection in wild-type mice, as well as in mice deficient in expression of CD4+ T cells and TLR4 receptor. Nonetheless, no anti-tumor protection was observed in mice deficient in CD8+ T cells. Also, the vaccine promoted high activation of E6/E7-specific T cells and in a therapeutic-approach, E6E7 protein conferred full anti-tumor protection in mice. These results show a potential use of this E6E7 multi-epitope antigen as a new and promising antigen for the development of a therapeutic vaccine against tumors induced by HPV.

  6. Repeat-modulated population genetic effects in fungal proteins.

    PubMed

    Braun, F N; Liberles, D A

    2004-07-01

    A number of fungal lineages, notably N. crassa, have evolved a novel mechanism of processing genomic duplication events known as repeat-induced point (RIP) mutation. This mechanism appears, on the one hand, to act as a conservative genomic safeguard, by introducing stop codons into duplicated nucleotide sequences, thereby preempting consequences such as dosage effects. However, it also typically performs further nonsynonymous (i.e., amino acid-changing) nucleotide substitutions, the significance of which is unclear. We explore here the possibility that RIP-mutated genes which evade silencing may have some microevolutionary impact on functional sequences. Our approach focuses on structurally important hydrophobic/polar (HP) amino-acid substitutions effected by RIP. We exploit a simple generic protein folding model to predict the associated emergence of increased protein-structural stability and variance within a large population.

  7. Modulation of Auxin-Binding Proteins in Cell Suspensions 1

    PubMed Central

    LoSchiavo, Fiorella; Filippini, Francesco; Cozzani, Fabrizio; Vallone, Daniela; Terzi, Mario

    1991-01-01

    This paper shows that the level of 2,4-dichlorophenoxyacetic acid (2,4-D) in the medium determines the level of auxin-binding proteins in the membranes of carrot, Daucus carota, cells grown in suspension. This induction takes slightly more than 2 hours to complete and can be elicited by natural as well as synthetic auxins. The auxin binding sites thus generated, which are pronase-sensitive, bind 2,4-D, indoleacetic acid, and naphthalene-acetic acid (NAA) equally well. However both α- and β-NAA bind, whereas only α-NAA is effective in the inductive process. Cells committed to embryogeny (proembryogenic masses) do not respond to auxin, i.e. their level of auxin-binding proteins remains very low, and they do not seem to synthesize the hormone, as indicated by inhibitor studies. Sensitivity to, and production of, auxin, begins when the embryo becomes polarized, i.e. at postglobular stage. PMID:16668416

  8. Effects of estrogen receptor modulators on cytoskeletal proteins in the central nervous system.

    PubMed

    Segura-Uribe, Julia J; Pinto-Almazán, Rodolfo; Coyoy-Salgado, Angélica; Fuentes-Venado, Claudia E; Guerra-Araiza, Christian

    2017-08-01

    Estrogen receptor modulators are compounds of interest because of their estrogenic agonistic/antagonistic effects and tissue specificity. These compounds have many clinical applications, particularly for breast cancer treatment and osteoporosis in postmenopausal women, as well as for the treatment of climacteric symptoms. Similar to estrogens, neuroprotective effects of estrogen receptor modulators have been described in different models. However, the mechanisms of action of these compounds in the central nervous system have not been fully described. We conducted a systematic search to investigate the effects of estrogen receptor modulators in the central nervous system, focusing on the modulation of cytoskeletal proteins. We found that raloxifene, tamoxifen, and tibolone modulate some cytoskeletal proteins such as tau, microtuble-associated protein 1 (MAP1), MAP2, neurofilament 38 (NF38) by different mechanisms of action and at different levels: neuronal microfilaments, intermediate filaments, and microtubule-associated proteins. Finally, we emphasize the importance of the study of these compounds in the treatment of neurodegenerative diseases since they present the benefits of estrogens without their side effects.

  9. Ion-specific modulation of protein interactions: Anion-induced, reversible oligomerization of a fusion protein

    PubMed Central

    Gokarn, Yatin R; Fesinmeyer, R Matthew; Saluja, Atul; Cao, Shawn; Dankberg, Jane; Goetze, Andrew; Remmele, Richard L; Narhi, Linda O; Brems, David N

    2009-01-01

    Ions can significantly modulate the solution interactions of proteins. We aim to demonstrate that the salt-dependent reversible heptamerization of a fusion protein called peptibody A or PbA is governed by anion-specific interactions with key arginyl and lysyl residues on its peptide arms. Peptibody A, an E. coli expressed, basic (pI = 8.8), homodimer (65.2 kDa), consisted of an IgG1-Fc with two, C-terminal peptide arms linked via penta-glycine linkers. Each peptide arm was composed of two, tandem, active sequences (SEYQGLPPQGWK) separated by a spacer (GSGSATGGSGGGASSGSGSATG). PbA was monomeric in 10 mM acetate, pH 5.0 but exhibited reversible self-association upon salt addition. The sedimentation coefficient (sw) and hydrodynamic diameter (DH) versus PbA concentration isotherms in the presence of 140 mM NaCl (A5N) displayed sharp increases in sw and DH, reaching plateau values of 9 s and 16 nm by 10 mg/mL PbA. The DH and sedimentation equilibrium data in the plateau region (>12 mg/mL) indicated the oligomeric ensemble to be monodisperse (PdI = 0.05) with a z-average molecular weight (Mz) of 433 kDa (stoichiometry = 7). There was no evidence of reversible self-association for an IgG1-Fc molecule in A5N by itself or in a mixture containing fluorescently labeled IgG1-Fc and PbA, indicative of PbA self-assembly being mediated through its peptide arms. Self-association increased with pH, NaCl concentration, and anion size (I− > Br− > Cl− > F−) but could be inhibited using soluble Trp-, Phe-, and Leu-amide salts (Trp > Phe > Leu). We propose that in the presence of salt (i) anion binding renders PbA self-association competent by neutralizing the peptidyl arginyl and lysyl amines, (ii) self-association occurs via aromatic and hydrophobic interactions between the..xx..xxx..xx.. motifs, and (iii) at >10 mg/mL, PbA predominantly exists as heptameric clusters. PMID:19177361

  10. Homeodomain Interacting Protein Kinases Modulate Hypoxic Adaptation and Chemoresistance

    DTIC Science & Technology

    2015-08-01

    established cell lines. Our objectives were to (1) ectopically express or silence HIPK2 and HIPK3 in PCa cell lines (LnCAP & LnCAP-abl) , (2) define the...small RNAs for silencing . Technical difficulties prevented our group from evaluating the impact on HIPKs in PCa as ectopic expression produced a...protein of incorrect size and silencing proved ineffective. We will resolve these issue and continue to examine HIPKs in PCa in future studies. 15

  11. Modulation of membrane protein lateral mobility by polyphosphates and polyamines.

    PubMed

    Schindler, M; Koppel, D E; Sheetz, M P

    1980-03-01

    The lateral mobility of fluorescein-labeled membrane glycoproteins was measured in whole unlysed erythrocytes and erythrocyte ghosts by the technique of "fluorescence redistribution after fusion." Measurements were made on polyethylene glycol-fused cell pairs in which only one member of the couplet was initially fluorescently labeled. Diffusion coefficients were estimated from the rate of fluorescence redistribution determined from successive scans with a focused laser beam across individual fused pairs. This technique allows for the analysis of diffusion within cell membranes without the possible damaging photochemical events caused by photobleaching. It was found that lateral mobility of erythrocyte proteins can be increased by the addition of polyphosphates (i.e., ATP and 2,3-diphosphoglycerate) and decreased by the addition of organic polyamines (i.e., neomycin and spermine). This control is exerted by these molecules only when they contact the cytoplasmic side of the membrane and is not dependent upon high-energy phosphates. Microviscosity experiments employing diphenylhexatriene demonstrated no changes in membrane lipid state as a function of these reagents. Our results, in conjunction with data on the physical interactions of cytoskeletal proteins, suggest that the diffusion effector molecules alter the lateral mobility of erythrocyte membrane proteins through modifications of interactions in the shell, which is composed of spectrin, actin, and component 4.1.

  12. Whey protein concentrate (WPC) and glutathione modulation in cancer treatment.

    PubMed

    Bounous, G

    2000-01-01

    The glutathione (GSH) antioxidant system is foremost among the cellular protective mechanisms. Depletion of this small molecule is a common consequence of increased formation of reactive oxygen species during increased cellular activities. This phenomenon can occur in the lymphocytes during the development of the immune response and in the muscular cells during strenuous exercise. It is not surprising that so much research has been done, and is still being done on this small tripeptide molecule. Whey protein concentrate has been shown to represent an effective and safe cysteine donor for GSH replenishment during GSH depletion in immune deficiency states. Cysteine is the crucial limiting amino acid for intracellular GSH synthesis. Animal experiments showed that the concentrates of whey proteins also exhibit anti-carcinogenesis and anticancer activity. They do this via their effect on increasing GSH concentration in relevant tissues, and may have anti-tumor effect on low volume of tumor via stimulation of immunity through the GSH pathway. It is considered that oxygen radical generation is frequently a critical step in carcinogenesis, hence the effect of GSH on free radicals as well as carcinogen detoxification, could be important in inhibiting carcinogenesis induced by a number of different mechanisms. Case reports are presented which strongly suggest an anti-tumor effect of a whey protein dietary supplement in some urogenital cancers. This non toxic dietary intervention, which is not based on the principles of current cancer chemotherapy, will hopefully attract the attention of laboratory and clinical oncologists.

  13. Modulation of Membrane Protein Lateral Mobility by Polyphosphates and Polyamines

    NASA Astrophysics Data System (ADS)

    Schindler, Melvin; Koppel, Dennis E.; Sheetz, Michael P.

    1980-03-01

    The lateral mobility of fluorescein-labeled membrane glycoproteins was measured in whole unlysed erythrocytes and erythrocyte ghosts by the technique of ``fluorescence redistribution after fusion.'' Measurements were made on polyethylene glycol-fused cell pairs in which only one member of the couplet was initially fluorescently labeled. Diffusion coefficients were estimated from the rate of fluorescence redistribution determined from successive scans with a focused laser beam across individual fused pairs. This technique allows for the analysis of diffusion within cell membranes without the possible damaging photochemical events caused by photobleaching. It was found that lateral mobility of erythrocyte proteins can be increased by the addition of polyphosphates (i.e., ATP and 2,3-diphosphoglycerate) and decreased by the addition of organic polyamines (i.e., neomycin and spermine). This control is exerted by these molecules only when they contact the cytoplasmic side of the membrane and is not dependent upon high-energy phosphates. Microviscosity experiments employing diphenylhexatriene demonstrated no changes in membrane lipid state as a function of these reagents. Our results, in conjunction with data on the physical interactions of cytoskeletal proteins, suggest that the diffusion effector molecules alter the lateral mobility of erythrocyte membrane proteins through modifications of interactions in the shell, which is composed of spectrin, actin, and component 4.1.

  14. NMR solution structure of Ole e 6, a major allergen from olive tree pollen.

    PubMed

    Treviño, Miguel Angel; García-Mayoral, María Flor; Barral, Patricia; Villalba, Mayte; Santoro, Jorge; Rico, Manuel; Rodríguez, Rosalía; Bruix, Marta

    2004-09-10

    Ole e 6 is a pollen protein from the olive tree (Olea europaea) that exhibits allergenic activity with a high prevalence among olive-allergic individuals. The three-dimensional structure of Ole e 6 has been determined in solution by NMR methods. This is the first experimentally determined structure of an olive tree pollen allergen. The structure of this 50-residue protein is based on 486 upper limit distance constraints derived from nuclear Overhauser effects and 24 torsion angle restraints. The global fold of Ole e 6 consists of two nearly antiparallel alpha-helices, spanning residues 3-19 and 23-33, that are connected by a short loop and followed by a long, unstructured C-terminal tail. Viewed edge-on, the structured N terminus has a dumbbell-like shape with the two helices on the outside and with the hydrophobic core, mainly composed of 3 aromatic and 6 cysteine residues, on the inside. All the aromatic rings lie on top of and pack against the three disulfide bonds. The lack of thermal unfolding, even at 85 degrees C, indicates a high conformational stability. Based on the analysis of the molecular surface, we propose five plausible epitopes for IgE recognition. The results presented here provide the structural foundation for future experiments to verify the antigenicity of the proposed epitopes, as well as to design novel hypoallergenic forms of the protein suitable for diagnosis and treatment of type-I allergies. In addition, three-dimensional structure features of Ole e 6 are discussed to provide a basis for future functional studies.

  15. Inferring modules of functionally interacting proteins using the Bond Energy Algorithm

    PubMed Central

    Watanabe, Ryosuke LA; Morett, Enrique; Vallejo, Edgar E

    2008-01-01

    Background Non-homology based methods such as phylogenetic profiles are effective for predicting functional relationships between proteins with no considerable sequence or structure similarity. Those methods rely heavily on traditional similarity metrics defined on pairs of phylogenetic patterns. Proteins do not exclusively interact in pairs as the final biological function of a protein in the cellular context is often hold by a group of proteins. In order to accurately infer modules of functionally interacting proteins, the consideration of not only direct but also indirect relationships is required. In this paper, we used the Bond Energy Algorithm (BEA) to predict functionally related groups of proteins. With BEA we create clusters of phylogenetic profiles based on the associations of the surrounding elements of the analyzed data using a metric that considers linked relationships among elements in the data set. Results Using phylogenetic profiles obtained from the Cluster of Orthologous Groups of Proteins (COG) database, we conducted a series of clustering experiments using BEA to predict (upper level) relationships between profiles. We evaluated our results by comparing with COG's functional categories, And even more, with the experimentally determined functional relationships between proteins provided by the DIP and ECOCYC databases. Our results demonstrate that BEA is capable of predicting meaningful modules of functionally related proteins. BEA outperforms traditionally used clustering methods, such as k-means and hierarchical clustering by predicting functional relationships between proteins with higher accuracy. Conclusion This study shows that the linked relationships of phylogenetic profiles obtained by BEA is useful for detecting functional associations between profiles and extending functional modules not found by traditional methods. BEA is capable of detecting relationship among phylogenetic patterns by linking them through a common element shared in

  16. Modulation of protein stability and aggregation properties by surface charge engineering.

    PubMed

    Raghunathan, Govindan; Sokalingam, Sriram; Soundrarajan, Nagasundarapandian; Madan, Bharat; Munussami, Ganapathiraman; Lee, Sun-Gu

    2013-09-01

    An attempt to alter protein surface charges through traditional protein engineering approaches often affects the native protein structure significantly and induces misfolding. This limitation is a major hindrance in modulating protein properties through surface charge variations. In this study, as a strategy to overcome such a limitation, we attempted to co-introduce stabilizing mutations that can neutralize the destabilizing effect of protein surface charge variation. Two sets of rational mutations were designed; one to increase the number of surface charged amino acids and the other to decrease the number of surface charged amino acids by mutating surface polar uncharged amino acids and charged amino acids, respectively. These two sets of mutations were introduced into Green Fluorescent Protein (GFP) together with or without stabilizing mutations. The co-introduction of stabilizing mutations along with mutations for surface charge modification allowed us to obtain functionally active protein variants (s-GFP(+15-17) and s-GFP(+5-6)). When the protein properties such as fluorescent activity, folding rate and kinetic stability were assessed, we found the possibility that the protein stability can be modulated independently of activity and folding by engineering protein surface charges. The aggregation properties of GFP could also be altered through the surface charge engineering.

  17. Structural and functional discussion of the tetra-trico-peptide repeat, a protein interaction module.

    PubMed

    Zeytuni, Natalie; Zarivach, Raz

    2012-03-07

    Tetra-trico-peptide repeat (TPR) domains are found in numerous proteins, where they serve as interaction modules and multiprotein complex mediators. TPRs can be found in all kingdoms of life and regulate diverse biological processes, such as organelle targeting and protein import, vesicle fusion, and biomineralization. This review considers the structural features of TPR domains that permit the great ligand-binding diversity of this motif, given that TPR-interacting partners display variations in both sequence and secondary structure. In addition, tools for predicting TPR-interacting partners are discussed, as are the abilities of TPR domains to serve as protein-protein interaction scaffolds in biotechnology and therapeutics.

  18. Positive Lysosomal Modulation As a Unique Strategy to Treat Age-Related Protein Accumulation Diseases

    PubMed Central

    Wisniewski, Meagan L.; Butler, David

    2012-01-01

    Abstract Lysosomes are involved in degrading and recycling cellular ingredients, and their disruption with age may contribute to amyloidogenesis, paired helical filaments (PHFs), and α-synuclein and mutant huntingtin aggregation. Lysosomal cathepsins are upregulated by accumulating proteins and more so by the modulator Z-Phe-Ala-diazomethylketone (PADK). Such positive modulators of the lysosomal system have been studied in the well-characterized hippocampal slice model of protein accumulation that exhibits the pathogenic cascade of tau aggregation, tubulin breakdown, microtubule destabilization, transport failure, and synaptic decline. Active cathepsins were upregulated by PADK; Rab proteins were modified as well, indicating enhanced trafficking, whereas lysosome-associated membrane protein and proteasome markers were unchanged. Lysosomal modulation reduced the pre-existing PHF deposits, restored tubulin structure and transport, and recovered synaptic components. Further proof-of-principle studies used Alzheimer disease mouse models. It was recently reported that systemic PADK administration caused dramatic increases in cathepsin B protein and activity levels, whereas neprilysin, insulin-degrading enzyme, α-secretase, and β-secretase were unaffected by PADK. In the transgenic models, PADK treatment resulted in clearance of intracellular amyloid beta (Aβ) peptide and concomitant reduction of extracellular deposits. Production of the less pathogenic Aβ1–38 peptide corresponded with decreased levels of Aβ1–42, supporting the lysosome's antiamyloidogenic role through intracellular truncation. Amelioration of synaptic and behavioral deficits also indicates a neuroprotective function of the lysosomal system, identifying lysosomal modulation as an avenue for disease-modifying therapies. From the in vitro and in vivo findings, unique lysosomal modulators represent a minimally invasive, pharmacologically controlled strategy against protein accumulation disorders

  19. A DEK domain-containing protein modulates chromatin structure and function in Arabidopsis.

    PubMed

    Waidmann, Sascha; Kusenda, Branislav; Mayerhofer, Juliane; Mechtler, Karl; Jonak, Claudia

    2014-11-01

    Chromatin is a major determinant in the regulation of virtually all DNA-dependent processes. Chromatin architectural proteins interact with nucleosomes to modulate chromatin accessibility and higher-order chromatin structure. The evolutionarily conserved DEK domain-containing protein is implicated in important chromatin-related processes in animals, but little is known about its DNA targets and protein interaction partners. In plants, the role of DEK has remained elusive. In this work, we identified DEK3 as a chromatin-associated protein in Arabidopsis thaliana. DEK3 specifically binds histones H3 and H4. Purification of other proteins associated with nuclear DEK3 also established DNA topoisomerase 1α and proteins of the cohesion complex as in vivo interaction partners. Genome-wide mapping of DEK3 binding sites by chromatin immunoprecipitation followed by deep sequencing revealed enrichment of DEK3 at protein-coding genes throughout the genome. Using DEK3 knockout and overexpressor lines, we show that DEK3 affects nucleosome occupancy and chromatin accessibility and modulates the expression of DEK3 target genes. Furthermore, functional levels of DEK3 are crucial for stress tolerance. Overall, data indicate that DEK3 contributes to modulation of Arabidopsis chromatin structure and function. © 2014 American Society of Plant Biologists. All rights reserved.

  20. Analysis of Multiple HPV E6 PDZ Interactions Defines Type-Specific PDZ Fingerprints That Predict Oncogenic Potential

    PubMed Central

    Thomas, Miranda; Myers, Michael P.; Guarnaccia, Corrado; Banks, Lawrence

    2016-01-01

    The high-risk Human Papillomavirus (HPV) E6 oncoproteins are characterised by the presence of a class I PDZ-binding motif (PBM) on their extreme carboxy termini. The PBM is present on the E6 proteins derived from all cancer-causing HPV types, but can also be found on some related non-cancer-causing E6 proteins. We have therefore been interested in investigating the potential functional differences between these different E6 PBMs. Using an unbiased proteomic approach in keratinocytes, we have directly compared the interaction profiles of these different PBMs. This has allowed us to identify the potential PDZ target fingerprints of the E6 PBMs from 7 different cancer-causing HPV types, from 3 HPV types with weak cancer association, and from one benign HPV type that possesses an ancestral PBM. We demonstrate a striking increase in the number of potential PDZ targets bound by each E6 PBM as cancer-causing potential increases, and show that the HPV-16 and HPV-18 PBMs have the most flexibility in their PDZ target selection. Furthermore, the specific interaction with hScrib correlates directly with increased oncogenic potential. In contrast, hDlg is bound equally well by all the HPV E6 PBMs analysed, indicating that this is an evolutionarily conserved interaction, and was most likely one of the original E6 PBM target proteins that was important for the occupation of a potential new niche. Finally, we present evidence that the cell junction components ZO-2 and β-2 syntrophin are novel PDZ domain–containing targets of a subset of high-risk HPV types. PMID:27483446

  1. Antitumor efficacy of Venezuelan equine encephalitis virus replicon particles encoding mutated HPV16 E6 and E7 genes.

    PubMed

    Cassetti, M Cristina; McElhiney, Sue P; Shahabi, Vafa; Pullen, Jeffrey K; Le Poole, I Caroline; Eiben, Gretchen L; Smith, Larry R; Kast, W Martin

    2004-01-02

    An effective vaccine for treating human papillomavirus (HPV)-associated malignancies such as cervical cancer should elicit strong T cell-mediated immunity (CMI) against the E6 and/or E7 proteins necessary for the malignant state. We have developed Venezuelan equine encephalitis (VEE) virus replicon particle (VRP) vaccines encoding the HPV16 E6 and E7 genes and tested their immunogenicity and antitumor efficacy. The E6 and E7 genes were fused to create one open reading frame and mutated at four or at five amino acid positions to inactivate their oncogenic potential. VRP encoding mutant or wild type E6 and E7 proteins elicited comparable cytotoxic T lymphocyte (CTL) responses to an immunodominant E7(49-57) epitope and generated comparable antitumor responses in several HPV16 E6(+)E7(+) tumor challenge models: protection from either C3 or TC-1 tumor challenge was observed in 100% of VRP-vaccinated mice. Eradication of C3 tumors was observed in approximately 90% of mice following therapeutic VRP vaccination. Eradication of HLF16 tumors lacking the E7(49-57) epitope was observed in 90% of human leukocyte antigen (HLA)-A(*)0201 transgenic mice following therapeutic VRP vaccination. Finally, the predicted inactivation of E6 and E7 oncogenic potential was confirmed by demonstrating normal levels of both p53 and retinoblastoma proteins in human mammary epithelial cells (MEC) infected with VRP expressing mutant E6 and E7 genes. These promising results support the continued development of mutant E6 and E7 VRP as safe and effective candidates for clinical evaluation against HPV-associated disease.

  2. E5/E6 Target Generation Study (TARGEN)

    DTIC Science & Technology

    1988-07-01

    submit DD Form 1498 (Research and Technology Work Unit Summary) and final study documents to Defense Technical Information Center DTIC). 8 B-8 CAA-SR...OAA-SR-88-19 cv) 0 E5/E6 TARGET GENERATION STUDY ev (TARGEN) DTIC JULY 1988 LJAN2198 9’S. PREPARED BY FORCE SYSTEMS DIRECTORATE US ARMY CONCEPTS...MBF 6c. ADDRESS (City, State, and ZIP Code) 10. SOURCE OF FUNDING NUMBERS Headquarters, Department of the Army PROGRAM PROJECT TASK WORK UNIT

  3. Cellular prion protein and NMDA receptor modulation: protecting against excitotoxicity

    PubMed Central

    Black, Stefanie A. G.; Stys, Peter K.; Zamponi, Gerald W.; Tsutsui, Shigeki

    2014-01-01

    Although it is well established that misfolding of the cellular prion protein (PrPC) into the β-sheet-rich, aggregated scrapie conformation (PrPSc) causes a variety of transmissible spongiform encephalopathies (TSEs), the physiological roles of PrPC are still incompletely understood. There is accumulating evidence describing the roles of PrPC in neurodegeneration and neuroinflammation. Recently, we identified a functional regulation of NMDA receptors by PrPC that involves formation of a physical protein complex between these proteins. Excessive NMDA receptor activity during conditions such as ischemia mediates enhanced Ca2+ entry into cells and contributes to excitotoxic neuronal death. In addition, NMDA receptors and/or PrPC play critical roles in neuroinflammation and glial cell toxicity. Inhibition of NMDA receptor activity protects against PrPSc-induced neuronal death. Moreover, in mice lacking PrPC, infarct size is increased after focal cerebral ischemia, and absence of PrPC increases susceptibility of neurons to NMDA receptor-dependent death. Recently, PrPC was found to be a receptor for oligomeric beta-amyloid (Aβ) peptides, suggesting a role for PrPC in Alzheimer's disease (AD). Our recent findings suggest that Aβ peptides enhance NMDA receptor current by perturbing the normal copper- and PrPC-dependent regulation of these receptors. Here, we review evidence highlighting a role for PrPC in preventing NMDA receptor-mediated excitotoxicity and inflammation. There is a need for more detailed molecular characterization of PrPC-mediated regulation of NMDA receptors, such as determining which NMDA receptor subunits mediate pathogenic effects upon loss of PrPC-mediated regulation and identifying PrPC binding site(s) on the receptor. This knowledge will allow development of novel therapeutic interventions for not only TSEs, but also for AD and other neurodegenerative disorders involving dysfunction of PrPC. PMID:25364752

  4. Wheat ROP proteins modulate defense response through lignin metabolism.

    PubMed

    Ma, Qing-Hu; Zhu, Hai-Hao; Han, Jia-Qi

    2017-09-01

    ROP is a subfamily of small GTP-binding proteins that uniquely exist in plants. It acts as versatile molecular switches that regulate various developmental processes. Some ROP proteins are also reported to affect defense responses, although their exact mechanism is not fully understood. Herein, ROP members in wheat were mined; the functions of three wheat ROP proteins were studied. RT-PCR results showed that the expression of TaRac1 was rapidly and strongly induced after leaf rust infection. TaRac1 interacted with TaCCR in yeast-hybridization assay. The overexpression of TaRac1 in tobacco promoted CCR and CAD gene expression, increased the total lignin content and sinapyl lignin proportion, and then enhanced resistance to tobacco black shank and bacterial wilt diseases. In contrast, TaRac3 and TaRac4 did not show to interact with TaCCR. Furthermore, the overexpression of TaRac3 and TaRac4 did not increase lignin gene expression and lignin accumulation either. Unlike TaRac1, the overexpression of TaRac3 increased susceptibility to both black shank and bacterial wilt pathogens, while overexpression of TaRac4 showed no effect on disease resistance but promoted the root growth in tobacco seedling. These data collectively suggest that TaRac1 in Group II is mainly involved in regulating lignin metabolism which, in turn, responsible for the observed roles in pathogen resistance. TaRac3 and TaRac4 have the minor roles in defense response but may act on regulation in plant developmental processes. These results shed light on the complexity and diverse function of ROP in plant defense pathway. Copyright © 2017 Elsevier B.V. All rights reserved.

  5. Cellular factors modulating the mechanism of tau protein aggregation

    PubMed Central

    Fontaine, Sarah N.; Sabbagh, Jonathan J.; Baker, Jeremy; Martinez-Licha, Carlos R.; Darling, April

    2015-01-01

    Pathological accumulation of the microtubule-associated protein tau, in the form of neurofibrillary tangles, is a major hallmark of Alzheimer’s disease, the most prevalent neurodegenerative condition worldwide. In addition to Alzheimer’s disease, a number of neurodegenerative diseases, called tauopathies, are characterized by the accumulation of aggregated tau in a variety of brain regions. While tau normally plays an important role in stabilizing the microtubule network of the cytoskeleton, its dissociation from microtubules and eventual aggregation into pathological deposits is an area of intense focus for therapeutic development. Here we discuss the known cellular factors that affect tau aggregation, from post-translational modifications to molecular chaperones. PMID:25666877

  6. Tripping the light fantastic: blue-light photoreceptors as examples of environmentally modulated protein-protein interactions.

    PubMed

    Zoltowski, Brian D; Gardner, Kevin H

    2011-01-11

    Blue-light photoreceptors play a pivotal role in detecting the quality and quantity of light in the environment, controlling a wide range of biological responses. Several families of blue-light photoreceptors have been characterized in detail using biophysics and biochemistry, beginning with photon absorption, through intervening signal transduction, to regulation of biological activities. Here we review the light oxygen voltage, cryptochrome, and sensors of blue light using FAD families, three different groups of proteins that offer distinctly different modes of photochemical activation and signal transduction yet play similar roles in a vast array of biological responses. We cover mechanisms of light activation and propagation of conformational responses that modulate protein-protein interactions involved in biological signaling. Discovery and characterization of these processes in natural proteins are now allowing the design of photoregulatable engineered proteins, facilitating the generation of novel reagents for biochemical and cell biological research.

  7. Nuclear actin-binding proteins as modulators of gene transcription.

    PubMed

    Gettemans, Jan; Van Impe, Katrien; Delanote, Veerle; Hubert, Thomas; Vandekerckhove, Joël; De Corte, Veerle

    2005-10-01

    Dynamic transformations in the organization of the cellular microfilament system are the driving force behind fundamental biological processes such as cellular motility, cytokinesis, wound healing and secretion. Eukaryotic cells express a plethora of actin-binding proteins (ABPs) allowing cells to control the organization of the actin cytoskeleton in a flexible manner. These structural proteins were, not surprisingly, originally described as (major) constituents of the cytoplasm. However, in recent years, there has been a steady flow of reports detailing not only translocation of ABPs into and out of the nucleus but also describing their role in the nuclear compartment. This review focuses on recent developments pertaining to nucleocytoplasmic transport of ABPs, including their mode of translocation and nuclear function. In particular, evidence that structurally and functionally unrelated cytoplasmic ABPs regulate transcription activation by various nuclear (steroid hormone) receptors is steadily accruing. Furthermore, the recent finding that actin is a necessary component of the RNA polymerase II-containing preinitiation complex opens up new opportunities for nuclear ABPs in gene transcription regulation.

  8. Keratins modulate colonocyte electrolyte transport via protein mistargeting

    PubMed Central

    Toivola, Diana M.; Krishnan, Selvi; Binder, Henry J.; Singh, Satish K.; Omary, M. Bishr

    2004-01-01

    The function of intestinal keratins is unknown, although keratin 8 (K8)–null mice develop colitis, hyperplasia, diarrhea, and mistarget jejunal apical markers. We quantified the diarrhea in K8-null stool and examined its physiologic basis. Isolated crypt-units from K8-null and wild-type mice have similar viability. K8-null distal colon has normal tight junction permeability and paracellular transport but shows decreased short circuit current and net Na absorption associated with net Cl secretion, blunted intracellular Cl/HCO3-dependent pH regulation, hyperproliferation and enlarged goblet cells, partial loss of the membrane-proximal markers H,K-ATPase-β and F-actin, increased and redistributed basolateral anion exchanger AE1/2 protein, and redistributed Na-transporter ENaC-γ. Diarrhea and protein mistargeting are observed 1–2 d after birth while hyperproliferation/inflammation occurs later. The AE1/2 changes and altered intracellular pH regulation likely account, at least in part, for the ion transport defects and hyperproliferation. Therefore, colonic keratins have a novel function in regulating electrolyte transport, likely by targeting ion transporters to their cellular compartments. PMID:15007064

  9. Tula hantavirus triggers pro-apoptotic signals of ER stress in Vero E6 cells.

    PubMed

    Li, Xiao-Dong; Lankinen, Hilkka; Putkuri, Niina; Vapalahti, Olli; Vaheri, Antti

    2005-03-01

    Tula virus is a member of the Hantavirus genus of the family Bunyaviridae. Viruses of this family have an unusual pattern of intracellular maturation at the ER-Golgi compartment. We recently found that Tula virus, similar to several other hantaviruses, is able to induce apoptosis in cultured cells [Li, X.D., Kukkonen, S., Vapalahti, O., Plyusnin, A., Lankinen, H., Vaheri, A., 2004. Tula hantavirus infection of Vero E6 cells induces apoptosis involving caspase 8 activation. J. Gen. Virol. 85, 3261-3268.]. However, the cellular mechanisms remain to be clarified. In this study, we demonstrate that the progressive replication of Tula virus in Vero E6 cells initiates several death programs that are intimately associated with ER stress: (1) early activation of ER-resident caspase-12; (2) phosphorylation of Jun NH2-terminal kinase (JNK) and its downstream target transcriptional factor, c-jun; (3) induction of the pro-apoptotic transcriptional factor, growth arrest- and DNA damage-inducible gene 153, or C/EBP homologous protein (Gadd153/chop); and (4) changes in the ER-membrane protein BAP31 implying cross-talk with the mitochondrial apoptosis pathway. Furthermore, we confirmed that a sustained ER stress was induced marked by an increased expression of an ER chaperone Grp78/BiP. Taken together, we have identified involvement of ER stress-mediated death program in Tula virus-infected Vero E6 cells which provides a new approach to understand the mechanisms in hantavirus-induced apoptosis.

  10. Cooperative DNA Recognition Modulated by an Interplay between Protein-Protein Interactions and DNA-Mediated Allostery.

    PubMed

    Merino, Felipe; Bouvier, Benjamin; Cojocaru, Vlad

    2015-06-01

    Highly specific transcriptional regulation depends on the cooperative association of transcription factors into enhanceosomes. Usually, their DNA-binding cooperativity originates from either direct interactions or DNA-mediated allostery. Here, we performed unbiased molecular simulations followed by simulations of protein-DNA unbinding and free energy profiling to study the cooperative DNA recognition by OCT4 and SOX2, key components of enhanceosomes in pluripotent cells. We found that SOX2 influences the orientation and dynamics of the DNA-bound configuration of OCT4. In addition SOX2 modifies the unbinding free energy profiles of both DNA-binding domains of OCT4, the POU specific and POU homeodomain, despite interacting directly only with the first. Thus, we demonstrate that the OCT4-SOX2 cooperativity is modulated by an interplay between protein-protein interactions and DNA-mediated allostery. Further, we estimated the change in OCT4-DNA binding free energy due to the cooperativity with SOX2, observed a good agreement with experimental measurements, and found that SOX2 affects the relative DNA-binding strength of the two OCT4 domains. Based on these findings, we propose that available interaction partners in different biological contexts modulate the DNA exploration routes of multi-domain transcription factors such as OCT4. We consider the OCT4-SOX2 cooperativity as a paradigm of how specificity of transcriptional regulation is achieved through concerted modulation of protein-DNA recognition by different types of interactions.

  11. Cooperative DNA Recognition Modulated by an Interplay between Protein-Protein Interactions and DNA-Mediated Allostery

    PubMed Central

    Merino, Felipe; Bouvier, Benjamin; Cojocaru, Vlad

    2015-01-01

    Highly specific transcriptional regulation depends on the cooperative association of transcription factors into enhanceosomes. Usually, their DNA-binding cooperativity originates from either direct interactions or DNA-mediated allostery. Here, we performed unbiased molecular simulations followed by simulations of protein-DNA unbinding and free energy profiling to study the cooperative DNA recognition by OCT4 and SOX2, key components of enhanceosomes in pluripotent cells. We found that SOX2 influences the orientation and dynamics of the DNA-bound configuration of OCT4. In addition SOX2 modifies the unbinding free energy profiles of both DNA-binding domains of OCT4, the POU specific and POU homeodomain, despite interacting directly only with the first. Thus, we demonstrate that the OCT4-SOX2 cooperativity is modulated by an interplay between protein-protein interactions and DNA-mediated allostery. Further, we estimated the change in OCT4-DNA binding free energy due to the cooperativity with SOX2, observed a good agreement with experimental measurements, and found that SOX2 affects the relative DNA-binding strength of the two OCT4 domains. Based on these findings, we propose that available interaction partners in different biological contexts modulate the DNA exploration routes of multi-domain transcription factors such as OCT4. We consider the OCT4-SOX2 cooperativity as a paradigm of how specificity of transcriptional regulation is achieved through concerted modulation of protein-DNA recognition by different types of interactions. PMID:26067358

  12. Safety, efficacy, and immunogenicity of VGX-3100, a therapeutic synthetic DNA vaccine targeting human papillomavirus 16 and 18 E6 and E7 proteins for cervical intraepithelial neoplasia 2/3: a randomised, double-blind, placebo-controlled phase 2b trial

    PubMed Central

    Trimble, Cornelia L; Morrow, Matthew P; Kraynyak, Kimberly A; Shen, Xuefei; Dallas, Michael; Yan, Jian; Edwards, Lance; Parker, R Lamar; Denny, Lynette; Giffear, Mary; Brown, Ami Shah; Marcozzi-Pierce, Kathleen; Shah, Divya; Slager, Anna M; Sylvester, Albert J; Khan, Amir; Broderick, Kate E; Juba, Robert J; Herring, Timothy A; Boyer, Jean; Lee, Jessica; Sardesai, Niranjan Y; Weiner, David B; Bagarazzi, Mark L

    2016-01-01

    Summary Background Despite preventive vaccines for oncogenic human papillomaviruses (HPVs), cervical intraepithelial neoplasia (CIN) is common, and current treatments are ablative and can lead to long-term reproductive morbidity. We assessed whether VGX-3100, synthetic plasmids targeting HPV-16 and HPV-18 E6 and E7 proteins, delivered by electroporation, would cause histopathological regression in women with CIN2/3. Methods Efficacy, safety, and immunogenicity of VGX-3100 were assessed in CIN2/3 associated with HPV-16 and HPV-18, in a randomised, double-blind, placebo-controlled phase 2b study. Patients from 36 academic and private gynaecology practices in seven countries were randomised (3:1) to receive 6 mg VGX-3100 or placebo (1 mL), given intramuscularly at 0, 4, and 12 weeks. Randomisation was stratified by age (<25 vs ≥25 years) and CIN2 versus CIN3 by computer-generated allocation sequence (block size 4). Funder and site personnel, participants, and pathologists were masked to treatment. The primary efficacy endpoint was regression to CIN1 or normal pathology 36 weeks after the first dose. Per-protocol and modified intention-to-treat analyses were based on patients receiving three doses without protocol violations, and on patients receiving at least one dose, respectively. The safety population included all patients who received at least one dose. The trial is registered at ClinicalTrials.gov (number NCT01304524) and EudraCT (number 2012-001334-33). Findings Between Oct 19, 2011, and July 30, 2013, 167 patients received either VGX-3100 (n=125) or placebo (n=42). In the per-protocol analysis 53 (49.5%) of 107 VGX-3100 recipients and 11 (30.6%) of 36 placebo recipients had histopathological regression (percentage point difference 19.0 [95% CI 1.4–36.6]; p=0.034). In the modified intention-to-treat analysis 55 (48.2%) of 114 VGX-3100 recipients and 12 (30.0%) of 40 placebo recipients had histopathological regression (percentage point difference 18.2 [95% CI

  13. HPV 5 and 8 E6 Abrogate ATR Activity Resulting in Increased Persistence of UVB Induced DNA Damage

    PubMed Central

    Wallace, Nicholas A.; Robinson, Kristin; Howie, Heather L.; Galloway, Denise A.

    2012-01-01

    The role of the E6 oncoprotein from high-risk members of the α human papillomavirus genus in anogenital cancer has been well established. However, far less is known about the E6 protein from the β human papillomavirus genus (β-HPVs). Some β-HPVs potentially play a role in non-melanoma skin cancer development, although they are not required for tumor maintenance. Instead, they may act as a co-factor that enhances the carcinogenic potential of UV damage. Indeed, the E6 protein from certain β-HPVs (HPV 5 and 8) promotes the degradation of p300, a histone acetyl transferase involved in UV damage repair. Here, we show that the expression of HPV 5 and 8 E6 increases thymine dimer persistence as well as the likelihood of a UVB induced double strand break (DSB). Importantly, we provide a mechanism for the increased DNA damage by showing that both extended thymine dimer persistence as well as elevated DSB levels are dependent on the ability of HPV 8 E6 to promote p300 degradation. We further demonstrate that HPV 5 and 8 E6 expression reduces the mRNA and protein levels of ATR, a PI3 kinase family member that plays a key role in UV damage signaling, but that these levels remain unperturbed in cells expressing a mutated HPV 8 E6 incapable of promoting p300 degradation. We confirm that the degradation of p300 leads to a reduction in ATR protein levels, by showing that ATR levels rebound when a p300 mutant resistant to HPV 8 mediated degradation and HPV 8 E6 are co-transfected. Conversely, we show that ATR protein levels are reduced when p300 is targeted for degradation by siRNA. Moreover, we show the reduced ATR levels in HPV 5 and 8 E6 expressing cells results in delayed ATR activation and an attenuated ability of cells to phosphorylate, and as a result accumulate, p53 in response to UVB exposure, leading to significantly reduced cell cycle arrest. In conclusion, these data demonstrate that β-HPV E6 expression can enhance the carcinogenic potential of UVB exposure by

  14. Intra- and intermembrane distribution of chlorin e6 derivatives

    NASA Astrophysics Data System (ADS)

    Zorin, Vladimir P.; Zorina, Tatyana E.; Mikhalovsky, Iosif S.; Khludeyev, Ivan I.

    1995-01-01

    The parameters of chlorin e6 and trimethylester of chlorin e6 incorporation and distribution in suspensions of unilamellar liposomes of DMPC, DPPC, and DSPC, as well as efficiency of the pigment redistribution from liposomes to cellular membranes have been studied. Determination of the fraction of pigments' fluorescence which is accessible to quenching by a watersoluble quencher indicates that for both chlorins the outer monolayer of the liposomal membrane is more populated than the inner one. Gel-liquid crystalline phase transition induces a shift of a part of the pigments' molecules toward the inner monolayer. By means of ultrafiltration technique it is shown that chlorins binding to liposomal membrane occurs as partitioning between water and lipid phases. The partition coefficient is affected strongly by the type of pigment, the phase state of the lipid bilayer. Similar results were obtained when the influence of the physical state of the lipid bilayer on the rate of chlorins redistribution from liposomes to cellular membrane was studied. These findings show that diffusive mobility of the sensitizer in suspensions of cellular and model membranes is a complex process which is dependent on structural features of both the pigment and its biological carriers.

  15. Transitive closure and metric inequality of weighted graphs:detecting protein interaction modules using cliques

    SciTech Connect

    Ding, Chris; He, Xiaofeng; Xiong, Hui; Peng, Hanchuan; Holbrook,Stephen R.

    2006-06-02

    We study transitivity properties of edge weights in complex networks. We show that enforcing transitivity leads to a transitivity inequality which is equivalent to ultra-metric inequality. This can be used to define transitive closure on weighted undirected graphs, which can be computed using a modified Floyd-Warshall algorithm. We outline several applications and present results of detecting protein functional modules in a protein interaction network.

  16. Modulation of the yeast protein interactome in response to DNA damage.

    PubMed

    Rochette, Samuel; Gagnon-Arsenault, Isabelle; Diss, Guillaume; Landry, Christian R

    2014-04-04

    Cells deploy diverse mechanisms to physiologically adapt to potentially detrimental perturbations. These mechanisms include changes in the organization of protein-protein interaction networks (PINs). Most PINs characterized to date are portrayed in a single environmental condition and are thus likely to miss important connections among biological processes. In this report, we show that the yeast DHFR-PCA on high-density arrays allows to detects modulations of protein-protein interactions (PPIs) in different conditions by testing more than 1000 PPIs in standard and in a drug-inducing DNA damage conditions. We identify 156 PPIs that show significant modulation in response to DNA damage. We provide evidence that modulated PPIs involve essential genes (NOP7, EXO84 and LAS17) playing critical roles in response to DNA damage. Additionally, we show that a significant proportion of PPI changes are likely explained by changes in protein localization and, to a lesser extent, protein abundance. The protein interaction modules affected by changing PPIs support the role of mRNA stability and translation, protein degradation and ubiquitylation and the regulation of the actin cytoskeleton in response to DNA damage. Overall, we provide a valuable tool and dataset for the study of the rewiring of PINs in response to environmental perturbations. We show that the DHFR-PCA is a high-throughput method that allows the detection of changes in PPIs associated with different environmental conditions using DNA damage response as a testbed. We provide a valuable resource for the study of DNA damage in eukaryotic cells. This article is part of a Special Issue: Can Proteomics Fill the Gap Between Genomics and Phenotypes? Copyright © 2013 Elsevier B.V. All rights reserved.

  17. O-linked GlcNAcylation elevated by HPV E6 mediates viral oncogenesis

    PubMed Central

    Zeng, Qinghua; Zhao, Rui-Xun; Chen, Jianfeng; Li, Yining; Li, Xiang-Dong; Liu, Xiao-Long; Zhang, Wei-Ming; Quan, Cheng-Shi; Wang, Yi-Shu; Zhai, Ying-Xian; Wang, Jian-Wei; Youssef, Mariam; Cui, Rutao; Liang, Jiyong; Genovese, Nicholas; Chow, Louise T.; Li, Yu-Lin; Xu, Zhi-Xiang

    2016-01-01

    High-risk human papillomaviruses (HPVs) are causative agents of anogenital cancers and a fraction of head and neck cancers. The mechanisms involved in the progression of HPV neoplasias to cancers remain largely unknown. Here, we report that O-linked GlcNAcylation (O-GlcNAc) and O-GlcNAc transferase (OGT) were markedly increased in HPV-caused cervical neoplasms relative to normal cervix, whereas O-GlcNAcase (OGA) levels were not altered. Transduction of HPV16 oncogene E6 or E6/E7 into mouse embryonic fibroblasts (MEFs) up-regulated OGT mRNA and protein, elevated the level of O-GlcNAc, and promoted cell proliferation while reducing cellular senescence. Conversely, in HPV-18–transformed HeLa cervical carcinoma cells, inhibition of O-GlcNAc with a low concentration of a chemical inhibitor impaired the transformed phenotypes in vitro. We showed that E6 elevated c-MYC via increased protein stability attributable to O-GlcNAcylation on Thr58. Reduction of HPV-mediated cell viability by a high concentration of O-GlcNAc inhibitor was partially rescued by elevated c-MYC. Finally, knockdown of OGT or O-GlcNAc inhibition in HeLa cells or in TC-1 cells, a mouse cell line transformed by HPV16 E6/E7 and activated K-RAS, reduced c-MYC and suppressed tumorigenesis and metastasis. Thus, we have uncovered a mechanism for HPV oncoprotein-mediated transformation. These findings may eventually aid in the development of effective therapeutics for HPV-associated malignancies by targeting aberrant O-GlcNAc. PMID:27482104

  18. O-linked GlcNAcylation elevated by HPV E6 mediates viral oncogenesis.

    PubMed

    Zeng, Qinghua; Zhao, Rui-Xun; Chen, Jianfeng; Li, Yining; Li, Xiang-Dong; Liu, Xiao-Long; Zhang, Wei-Ming; Quan, Cheng-Shi; Wang, Yi-Shu; Zhai, Ying-Xian; Wang, Jian-Wei; Youssef, Mariam; Cui, Rutao; Liang, Jiyong; Genovese, Nicholas; Chow, Louise T; Li, Yu-Lin; Xu, Zhi-Xiang

    2016-08-16

    High-risk human papillomaviruses (HPVs) are causative agents of anogenital cancers and a fraction of head and neck cancers. The mechanisms involved in the progression of HPV neoplasias to cancers remain largely unknown. Here, we report that O-linked GlcNAcylation (O-GlcNAc) and O-GlcNAc transferase (OGT) were markedly increased in HPV-caused cervical neoplasms relative to normal cervix, whereas O-GlcNAcase (OGA) levels were not altered. Transduction of HPV16 oncogene E6 or E6/E7 into mouse embryonic fibroblasts (MEFs) up-regulated OGT mRNA and protein, elevated the level of O-GlcNAc, and promoted cell proliferation while reducing cellular senescence. Conversely, in HPV-18-transformed HeLa cervical carcinoma cells, inhibition of O-GlcNAc with a low concentration of a chemical inhibitor impaired the transformed phenotypes in vitro. We showed that E6 elevated c-MYC via increased protein stability attributable to O-GlcNAcylation on Thr58. Reduction of HPV-mediated cell viability by a high concentration of O-GlcNAc inhibitor was partially rescued by elevated c-MYC. Finally, knockdown of OGT or O-GlcNAc inhibition in HeLa cells or in TC-1 cells, a mouse cell line transformed by HPV16 E6/E7 and activated K-RAS, reduced c-MYC and suppressed tumorigenesis and metastasis. Thus, we have uncovered a mechanism for HPV oncoprotein-mediated transformation. These findings may eventually aid in the development of effective therapeutics for HPV-associated malignancies by targeting aberrant O-GlcNAc.

  19. TNF Superfamily Protein–Protein Interactions: Feasibility of Small-Molecule Modulation

    PubMed Central

    Song, Yun; Buchwald, Peter

    2015-01-01

    The tumor necrosis factor (TNF) superfamily (TNFSF) contains about thirty structurally related receptors (TNFSFRs) and about twenty protein ligands that bind to one or more of these receptors. Almost all of these cell surface protein-protein interactions (PPIs) represent high-value therapeutic targets for inflammatory or immune modulation in autoimmune diseases, transplant recipients, or cancers, and there are several biologics including antibodies and fusion proteins targeting them that are in various phases of clinical development. Small-molecule inhibitors or activators could represent possible alternatives if the difficulties related to the targeting of protein-protein interactions by small molecules can be addressed. Compounds proving the feasibility of such approaches have been identified through different drug discovery approaches for a number of these TNFSFR-TNFSF type PPIs including CD40-CD40L, BAFFR-BAFF, TRAIL-DR5, and OX40-OX40L. Corresponding structural, signaling, and medicinal chemistry aspects are briefly reviewed here. While none of these small-molecule modulators identified so far seems promising enough to be pursued for clinical development, they provide proof-of-principle evidence that these interactions are susceptible to small-molecule modulation and can serve as starting points toward the identification of more potent and selective candidates. PMID:25706111

  20. Dissection of human papillomavirus E6 and E7 function in transgenic mouse models of cervical carcinogenesis.

    PubMed

    Riley, Rebeccah R; Duensing, Stefan; Brake, Tiffany; Münger, Karl; Lambert, Paul F; Arbeit, Jeffrey M

    2003-08-15

    Human cervix cancer is caused by high-risk human papillomaviruses encoding E6 and E7 oncoproteins, each of which alter function of distinct targets regulating the cell cycle, apoptosis, and differentiation. Here we determined the molecular contribution of E6 or E7 to neoplastic progression and malignant growth in a transgenic mouse model of cervical carcinogenesis. E7 increased proliferation and centrosome copy number, and produced progression to multifocal microinvasive cervical cancers. E6 elevated centrosome copy number and eliminated detectable p53 protein, but did not produce neoplasia or cancer. E6 plus E7 additionally elevated centrosome copy number and created large, extensively invasive cancers. Centrosome copy number increases and p53 loss likely contributed to malignant growth; however, dysregulated proliferation and differentiation were required for carcinogenic progression.

  1. Bio::Homology::InterologWalk--a Perl module to build putative protein-protein interaction networks through interolog mapping.

    PubMed

    Gallone, Giuseppe; Simpson, T Ian; Armstrong, J Douglas; Jarman, Andrew P

    2011-07-18

    Protein-protein interaction (PPI) data are widely used to generate network models that aim to describe the relationships between proteins in biological systems. The fidelity and completeness of such networks is primarily limited by the paucity of protein interaction information and by the restriction of most of these data to just a few widely studied experimental organisms. In order to extend the utility of existing PPIs, computational methods can be used that exploit functional conservation between orthologous proteins across taxa to predict putative PPIs or 'interologs'. To date most interolog prediction efforts have been restricted to specific biological domains with fixed underlying data sources and there are no software tools available that provide a generalised framework for 'on-the-fly' interolog prediction. We introduce Bio::Homology::InterologWalk, a Perl module to retrieve, prioritise and visualise putative protein-protein interactions through an orthology-walk method. The module uses orthology and experimental interaction data to generate putative PPIs and optionally collates meta-data into an Interaction Prioritisation Index that can be used to help prioritise interologs for further analysis. We show the application of our interolog prediction method to the genomic interactome of the fruit fly, Drosophila melanogaster. We analyse the resulting interaction networks and show that the method proposes new interactome members and interactions that are candidates for future experimental investigation. Our interolog prediction tool employs the Ensembl Perl API and PSICQUIC enabled protein interaction data sources to generate up to date interologs 'on-the-fly'. This represents a significant advance on previous methods for interolog prediction as it allows the use of the latest orthology and protein interaction data for all of the genomes in Ensembl. The module outputs simple text files, making it easy to customise the results by post-processing, allowing the

  2. Development of anti-E6 pegylated lipoplexes for mucosal application in the context of cervical preneoplastic lesions.

    PubMed

    Lechanteur, Anna; Furst, Tania; Evrard, Brigitte; Delvenne, Philippe; Hubert, Pascale; Piel, Géraldine

    2015-04-10

    Cervical cancer induced by human papillomavirus (HPV) is the fourth highest mortality causing cancer in women despite the use of prophylactic vaccines. E6 targeting represents an attractive strategy to treat this cancer. Indeed, oncoprotein E6 is produced by keratinocytes infected by HPV and is partially responsible for carcinogenesis. E6 interferes with the apoptosis process in stressed cells by degradation of p53 tumor suppressor gene. Our strategy consists in using E6 siRNA complexed with pegylated lipoplexes. The addition of hydrophilic polymer around the nanoparticles is crucial to use them by vaginal application on account of cervicovaginal mucus. Physicochemical characteristics were evaluated and in vitro assays were performed to evaluate transfection potential, E6 mRNA extinction and p53 re-expression. Cationic liposomes DOTAP/Cholesterol/DOPE 1/0.75/0.5 (N/P 2.5) with or without 50% DSPE-PEG2000 and associated with siE6 have demonstrated good physicochemical characteristics in terms of complexation, size, surface charge and stability. Both lipoplexes have been tested on CaSki cell line (HPV 16+) with 50 nM and 100 nM of siE6. Lipoplexes formulations induce 30-40% of E6 mRNA extinction and induce the re-expression of p53. In conclusion, pegylated anti-E6 lipoplexes have demonstrated their efficiency to cross the cellular membrane and to release siRNA into the cytoplasm confirmed by final p53 protein production. Copyright © 2015 Elsevier B.V. All rights reserved.

  3. Histone Code Modulation by Oncogenic PWWP-Domain Protein in Breast Cancers

    DTIC Science & Technology

    2014-08-01

    Wysocka J. Methylation of lysine 4 on his- e H3: intricacy of writing and reading a single epigenetic mark. l Cell 2007;25:15–30. erna SD, Li H...important for telomeric si- lencing and Sir protein association. Genes Dev 2002; 16: 1518-1527. [10] van Leeuwen F. Dot1p modulates silencing in

  4. MODULATION OF EASTERN OYSTER HEMOCYTE ACTIVITIES BY PERKINSUS MARINUS EXTRACELLULAR PROTEINS

    EPA Science Inventory

    The oyster pathogen Perkinsus marinusproduces many extracellular proteins (ECP) in vitro. Analysis of this ECP revealed a battery of hydrolytic enzymes. Some of these enzymes are known to modulate the activity of host defense cells. Although information on the effects of P. marin...

  5. MODULATION OF EASTERN OYSTER HEMOCYTE ACTIVITIES BY PERKINSUS MARINUS EXTRACELLULAR PROTEINS

    EPA Science Inventory

    The oyster pathogen Perkinsus marinusproduces many extracellular proteins (ECP) in vitro. Analysis of this ECP revealed a battery of hydrolytic enzymes. Some of these enzymes are known to modulate the activity of host defense cells. Although information on the effects of P. marin...

  6. Mapping the interactome of HPV E6 and E7 oncoproteins with the ubiquitin-proteasome system.

    PubMed

    Poirson, Juline; Biquand, Elise; Straub, Marie-Laure; Cassonnet, Patricia; Nominé, Yves; Jones, Louis; van der Werf, Sylvie; Travé, Gilles; Zanier, Katia; Jacob, Yves; Demeret, Caroline; Masson, Murielle

    2017-08-08

    Protein ubiquitination and its reverse reaction, deubiquitination, regulate protein stability, protein binding activity, and their subcellular localization. These reactions are catalyzed by the enzymes E1, E2, and E3 ubiquitin (Ub) ligases and deubiquitinases (DUBs). The Ub-proteasome system (UPS) is targeted by viruses for the sake of their replication and to escape host immune response. To identify novel partners of human papillomavirus 16 (HPV16) E6 and E7 proteins, we assembled and screened a library of 590 cDNAs related to the UPS by using the Gaussia princeps luciferase protein complementation assay. HPV16 E6 was found to bind to the homology to E6AP C terminus-type Ub ligase (E6AP), three really interesting new gene (RING)-type Ub ligases (MGRN1, LNX3, LNX4), and the DUB Ub-specific protease 15 (USP15). Except for E6AP, the binding of UPS factors did not require the LxxLL-binding pocket of HPV16 E6. LNX3 bound preferentially to all high-risk mucosal HPV E6 tested, whereas LNX4 bound specifically to HPV16 E6. HPV16 E7 was found to bind to several broad-complex tramtrack and bric-a-brac domain-containing proteins (such as TNFAIP1/KCTD13) that are potential substrate adaptors of Cullin 3-RING Ub ligases, to RING-type Ub ligases implicated in innate immunity (RNF135, TRIM32, TRAF2, TRAF5), to the substrate adaptor DCAF15 of Cullin 4-RING Ub ligase and to some DUBs (USP29, USP33). The binding to UPS factors did not require the LxCxE motif but rather the C-terminal region of HPV16 E7 protein. The identified UPS factors interacted with most of E7 proteins across different HPV types. This study establishes a strategy for the rapid identification of interactions between host or pathogen proteins and the human ubiquitination system. © 2017 Federation of European Biochemical Societies.

  7. Positive modulation of RNA polymerase III transcription by ribosomal proteins

    SciTech Connect

    Dieci, Giorgio; Carpentieri, Andrea; Amoresano, Angela; Ottonello, Simone

    2009-02-06

    A yeast nuclear fraction of unknown composition, named TFIIIE, was reported previously to enhance transcription of tRNA and 5S rRNA genes in vitro. We show that TFIIIE activity co-purifies with a specific subset of ribosomal proteins (RPs) which, as revealed by chromatin immunoprecipitation analysis, generally interact with tRNA and 5S rRNA genes, but not with a Pol II-specific promoter. Only Rpl6Ap and Rpl6Bp, among the tested RPs, were found associated to a TATA-containing tRNA{sup Ile}(TAT) gene. The RPL6A gene also emerged as a strong multicopy suppressor of a conditional mutation in the basal transcription factor TFIIIC, while RPL26A and RPL14A behaved as weak suppressors. The data delineate a novel extra-ribosomal role for one or a few RPs which, by influencing 5S rRNA and tRNA synthesis, could play a key role in the coordinate regulation of the different sub-pathways required for ribosome biogenesis and functionality.

  8. Zinc ions modulate protein tyrosine phosphatase 1B activity.

    PubMed

    Bellomo, Elisa; Massarotti, Alberto; Hogstrand, Christer; Maret, Wolfgang

    2014-07-01

    Protein tyrosine phosphatases (PTPs) are key enzymes in cellular regulation. The 107 human PTPs are regulated by redox signalling, phosphorylation, dimerisation, and proteolysis. Recent findings of very strong inhibition of some PTPs by zinc ions at concentrations relevant in a cellular environment suggest yet another mechanism of regulation. One of the most extensively investigated PTPs is PTP1B (PTPN1). It regulates the insulin and leptin signalling pathway and is implicated in cancer and obesity/diabetes. The development of novel assay conditions to investigate zinc inhibition of PTP1B provides estimates of about 5.6 nM affinity for inhibitory zinc(II) ions. Analysis of three PTP1B 3D structures (PDB id: 2CM2, 3I80 and 1A5Y) identified putative zinc binding sites and supports the kinetic studies in suggesting an inhibitory zinc only in the closed and cysteinyl-phosphate intermediate forms of the enzyme. These observations gain significance with regard to recent findings of regulatory roles of zinc ions released from the endoplasmic reticulum.

  9. Rassf Proteins as Modulators of Mst1 Kinase Activity

    PubMed Central

    Bitra, Aruna; Sistla, Srinivas; Mariam, Jessy; Malvi, Harshada; Anand, Ruchi

    2017-01-01

    Rassf1A/5 tumor suppressors serve as adaptor proteins possessing a modular architecture with the C-terminal consisting of a coiled-coil SARAH (Salvador-Rassf-Hippo) domain and the central portion being composed of Ras associated (RA) domain. Here, we investigate the effect of Rassf effectors on Mst1 function by mapping the interaction of various domains of Rassf1A/5 and Mst1 kinase using surface plasmon resonance (SPR). The results revealed that apart from the C-terminal SARAH domain of Mst1 which interacts to form heterodimers with Rassf1A/5, the N-terminal kinase domain of Mst1 plays a crucial role in the stabilization of this complex. In addition, SPR experiments show that the RA domains play an important role in fine-tuning the Mst1-Rassf interaction, with Rassf5 being a preferred partner over a similar Rassf1A construct. It was also demonstrated that the activity profile of Mst1 in presence of Rassf adaptors completely switches. A Rassf-Mst1 complexed version of the kinase becomes apoptotic by positively regulating Mst1-H2B mediated serine 14 histone H2B phosphorylation, a hallmark of chromatin condensation. In contrast, the heterodimerization of Mst1 with Rassf1A/5 suppresses the phosphorylation of FoxO, thereby inhibiting the downstream Mst1-FoxO signalling pathway. PMID:28327630

  10. Rassf Proteins as Modulators of Mst1 Kinase Activity.

    PubMed

    Bitra, Aruna; Sistla, Srinivas; Mariam, Jessy; Malvi, Harshada; Anand, Ruchi

    2017-03-22

    Rassf1A/5 tumor suppressors serve as adaptor proteins possessing a modular architecture with the C-terminal consisting of a coiled-coil SARAH (Salvador-Rassf-Hippo) domain and the central portion being composed of Ras associated (RA) domain. Here, we investigate the effect of Rassf effectors on Mst1 function by mapping the interaction of various domains of Rassf1A/5 and Mst1 kinase using surface plasmon resonance (SPR). The results revealed that apart from the C-terminal SARAH domain of Mst1 which interacts to form heterodimers with Rassf1A/5, the N-terminal kinase domain of Mst1 plays a crucial role in the stabilization of this complex. In addition, SPR experiments show that the RA domains play an important role in fine-tuning the Mst1-Rassf interaction, with Rassf5 being a preferred partner over a similar Rassf1A construct. It was also demonstrated that the activity profile of Mst1 in presence of Rassf adaptors completely switches. A Rassf-Mst1 complexed version of the kinase becomes apoptotic by positively regulating Mst1-H2B mediated serine 14 histone H2B phosphorylation, a hallmark of chromatin condensation. In contrast, the heterodimerization of Mst1 with Rassf1A/5 suppresses the phosphorylation of FoxO, thereby inhibiting the downstream Mst1-FoxO signalling pathway.

  11. MAE-FMD: multi-agent evolutionary method for functional module detection in protein-protein interaction networks.

    PubMed

    Ji, Jun Zhong; Jiao, Lang; Yang, Cui Cui; Lv, Jia Wei; Zhang, Ai Dong

    2014-09-30

    Studies of functional modules in a Protein-Protein Interaction (PPI) network contribute greatly to the understanding of biological mechanisms. With the development of computing science, computational approaches have played an important role in detecting functional modules. We present a new approach using multi-agent evolution for detection of functional modules in PPI networks. The proposed approach consists of two stages: the solution construction for agents in a population and the evolutionary process of computational agents in a lattice environment, where each agent corresponds to a candidate solution to the detection problem of functional modules in a PPI network. First, the approach utilizes a connection-based encoding scheme to model an agent, and employs a random-walk behavior merged topological characteristics with functional information to construct a solution. Next, it applies several evolutionary operators, i.e., competition, crossover, and mutation, to realize information exchange among agents as well as solution evolution. Systematic experiments have been conducted on three benchmark testing sets of yeast networks. Experimental results show that the approach is more effective compared to several other existing algorithms. The algorithm has the characteristics of outstanding recall, F-measure, sensitivity and accuracy while keeping other competitive performances, so it can be applied to the biological study which requires high accuracy.

  12. Modulation of apoptosis and immune signaling pathways by the Hantaan virus nucleocapsid protein

    SciTech Connect

    Ontiveros, Steven J.; Li Qianjun; Jonsson, Colleen B.

    2010-06-05

    Herein, we show a direct relationship between the Hantaan virus (HTNV) nucleocapsid (N) protein and the modulation of apoptosis. We observed an increase in caspase-7 and -8, but not -9 in cells expressing HTNV N protein mutants lacking amino acids 270-330. Similar results were observed for the New World hantavirus, Andes virus. Nuclear factor kappa B (NF-kappaB) was sequestered in the cytoplasm after tumor necrosis factor receptor (TNFR) stimulation in cells expressing HTNV N protein. Further, TNFR stimulated cells expressing HTNV N protein inhibited caspase activation. In contrast, cells expressing N protein truncations lacking the region from amino acids 270-330 were unable to inhibit nuclear import of NF-kappaB and the mutants also triggered caspase activity. These results suggest that the HTNV circumvents host antiviral signaling and apoptotic response mediated by the TNFR pathway through host interactions with the N protein.

  13. Affinity Modulation of Small-Molecule Ligands by Borrowing Endogenous Protein Surfaces

    NASA Astrophysics Data System (ADS)

    Briesewitz, Roger; Ray, Gregory T.; Wandless, Thomas J.; Crabtree, Gerald R.

    1999-03-01

    A general strategy is described for improving the binding properties of small-molecule ligands to protein targets. A bifunctional molecule is created by chemically linking a ligand of interest to another small molecule that binds tightly to a second protein. When the ligand of interest is presented to the target protein by the second protein, additional protein-protein interactions outside of the ligand-binding sites serve either to increase or decrease the affinity of the binding event. We have applied this approach to an intractable target, the SH2 domain, and demonstrate a 3-fold enhancement over the natural peptide. This approach provides a way to modulate the potency and specificity of biologically active compounds.

  14. Amyloid precursor protein modulates macrophage phenotype and diet-dependent weight gain

    PubMed Central

    Puig, Kendra L.; Brose, Stephen A.; Zhou, Xudong; Sens, Mary A.; Combs, Gerald F.; Jensen, Michael D.; Golovko, Mikhail Y.; Combs, Colin K.

    2017-01-01

    It is well known that mutations in the gene coding for amyloid precursor protein are responsible for autosomal dominant forms of Alzheimer’s disease. Proteolytic processing of the protein leads to a number of metabolites including the amyloid beta peptide. Although brain amyloid precursor protein expression and amyloid beta production are associated with the pathophysiology of Alzheimer’s disease, it is clear that amyloid precursor protein is expressed in numerous cell types and tissues. Here we demonstrate that amyloid precursor protein is involved in regulating the phenotype of both adipocytes and peripheral macrophages and is required for high fat diet-dependent weight gain in mice. These data suggest that functions of this protein include modulation of the peripheral immune system and lipid metabolism. This biology may have relevance not only to the pathophysiology of Alzheimer’s disease but also diet-associated obesity. PMID:28262782

  15. Application of a micromembrane chromatography module to the examination of protein adsorption equilibrium.

    PubMed

    Káňavová, Natália; Kosior, Anna; Antošová, Monika; Faber, René; Polakovič, Milan

    2012-11-01

    A micromembrane chromatography module based on a 96-well plate design and enabling fast and simple separation of small amounts of proteins was used for the determination of binding capacities of lysozyme, bovine serum albumin, ovalbumin, bovine γ-globulin, and human immunoglobulin G on a hydrophobic membrane Sartobind® Phenyl. Dependence of the binding capacity of the proteins on the ammonium sulfate concentration was examined in the salt concentration range of 0.5-2.0 mol L(-1). An exponential increase of the binding capacity was observed for all proteins. Simple Langmuir one-component isotherm was found suitable for the characterization of the effect of protein concentration in all cases. A combined effect of protein and salt concentrations was expressed via the Langmuir exponential isotherm and fitted the adsorption data for three of the investigated proteins well.

  16. Roles of the PDZ domain-binding motif of the human papillomavirus type 16 E6 on the immortalization and differentiation of primary human foreskin keratinocytes.

    PubMed

    Choi, Moonju; Lee, Sungjin; Choi, Taekyu; Lee, Choongho

    2014-04-01

    A number of PDZ domain-containing proteins have been identified as binding partners for the oncoprotein E6 of the high-risk type human papillomaviruses (HPVs). These include hDlg, hScrib, MAGI1, MAGI2, and MAGI3, MUPP1, 14-3-3zeta, Na/H exchange regulatory factor 1, PTPN13, TIP-2/GIPC, Tip-1, and PATJ. The PDZ domain-binding motif (-X-T-X-V) at the carboxy terminus of E6 is essential for targeting PDZ proteins for proteasomal degradation. However, contribution of degradation of PDZ proteins by E6 to HPV-induced oncogenesis is still controversial. In order to clarify potential roles of molecular interactions between high-risk HPV E6 and one of best characterized PDZ proteins, hDlg in HPV-induced transformation, we used a retroviral infection system to overexpress HPV16 E7 gene alone or together with either HPV16 E6 wild type or E6 mutant gene lacking the PDZ domain-binding motif and investigated the effect of mutating the PDZ domain-binding motif of E6 on the immortalization and differentiation of human foreskin keratinocytes (HFKs) by the high-risk type HPV E6 and E7. Although the PDZ domain-binding motif of E6 was found to be required for the efficient growth of HFKs, it was not necessary for the E6 and E7-induced immortalization of HFKs. Furthermore, the overexpression of E6 and E7 neither induced degradation nor altered cellular localization of hDlg in undifferentiated or differentiated HFKs. These data indicate that the PDZ domain-binding motif of E6 contributes to the efficient cellular growth through mechanisms other than degradation and changes in the subcellular localizations of hDlg.

  17. Tough Coating Proteins: Subtle Sequence Variation Modulates Cohesion

    PubMed Central

    Das, Saurabh; Miller, Dusty R.; Kaufman, Yair; Martinez Rodriguez, Nadine R.; Pallaoro, Alessia; Harrington, Matthew J.; Gylys, Maryte; Israelachvili, Jacob N.; Waite, J. Herbert

    2015-01-01

    Mussel foot protein-1 (mfp-1) is an essential constituent of the protective cuticle covering all exposed portions of the byssus (plaque and the thread) that marine mussels use to attach to intertidal rocks. The reversible complexation of Fe3+ by the 3,4-dihydroxyphenylalanine (Dopa) side chains in mfp-1 in Mytilus californianus cuticle is responsible for its high extensibility (120%) as well as its stiffness (2 GPa) due to the formation of sacrificial bonds that help to dissipate energy and avoid accumulation of stresses in the material. We have investigated the interactions between Fe3+ and mfp-1 from two mussel species, M. californianus (Mc) and M. edulis (Me), using both surface sensitive and solution phase techniques. Our results show that although mfp-1 homologues from both species bind Fe3+, mfp-1 (Mc) contains Dopa with two distinct Fe3+-binding tendencies and prefers to form intramolecular complexes with Fe3+. In contrast, mfp-1 (Me) is better adapted to intermolecular Fe3+ binding by Dopa. Addition of Fe3+ did not significantly increase the cohesion energy between the mfp-1 (Mc) films at pH 5.5. However, iron appears to stabilize the cohesive bridging of mfp-1 (Mc) films at the physiologically relevant pH of 7.5, where most other mfps lose their ability to adhere reversibly. Understanding the molecular mechanisms underpinning the capacity of M. californianus cuticle to withstand twice the strain of M. edulis cuticle is important for engineering of tunable strain tolerant composite coatings for biomedical applications. PMID:25692318

  18. Tough coating proteins: subtle sequence variation modulates cohesion.

    PubMed

    Das, Saurabh; Miller, Dusty R; Kaufman, Yair; Martinez Rodriguez, Nadine R; Pallaoro, Alessia; Harrington, Matthew J; Gylys, Maryte; Israelachvili, Jacob N; Waite, J Herbert

    2015-03-09

    Mussel foot protein-1 (mfp-1) is an essential constituent of the protective cuticle covering all exposed portions of the byssus (plaque and the thread) that marine mussels use to attach to intertidal rocks. The reversible complexation of Fe(3+) by the 3,4-dihydroxyphenylalanine (Dopa) side chains in mfp-1 in Mytilus californianus cuticle is responsible for its high extensibility (120%) as well as its stiffness (2 GPa) due to the formation of sacrificial bonds that help to dissipate energy and avoid accumulation of stresses in the material. We have investigated the interactions between Fe(3+) and mfp-1 from two mussel species, M. californianus (Mc) and M. edulis (Me), using both surface sensitive and solution phase techniques. Our results show that although mfp-1 homologues from both species bind Fe(3+), mfp-1 (Mc) contains Dopa with two distinct Fe(3+)-binding tendencies and prefers to form intramolecular complexes with Fe(3+). In contrast, mfp-1 (Me) is better adapted to intermolecular Fe(3+) binding by Dopa. Addition of Fe(3+) did not significantly increase the cohesion energy between the mfp-1 (Mc) films at pH 5.5. However, iron appears to stabilize the cohesive bridging of mfp-1 (Mc) films at the physiologically relevant pH of 7.5, where most other mfps lose their ability to adhere reversibly. Understanding the molecular mechanisms underpinning the capacity of M. californianus cuticle to withstand twice the strain of M. edulis cuticle is important for engineering of tunable strain tolerant composite coatings for biomedical applications.

  19. Spontaneous CP Violation in E6 GUT with horizontal symmetry

    NASA Astrophysics Data System (ADS)

    Maekawa, Nobuhiro

    2010-02-01

    We consider spontaneous CP violation in E6 grand unified theory (GUT) with horizontal symmetry and anomalous U(1)A gauge symmetry in order to solve the SUSY CP problem. To realize the sufficiently small phases of SUSY Higgs mass μ and mixing parameter B, an additional discrete symmetry is introduced. The discrete symmetry plays multiple roles in explaining various things. By the symmetry, the up-type Yukawa couplings become real, which is important in satisfying the Chromo-EDM constraints to the imaginary part of the off-diagonal elements of squark mass matrices, and the down-type Yukawa couplings become complex, which is important in obtaining the Kobayashi-Maskawa phase. Moreover, this symmetry improves the smallness of up quark mass, and reduces the number of O(1) coefficients. One of the interesting predictions is Vub˜γ4, which is quite good agreement with the measured value. This talk is based on the works in Ref. [1].

  20. Modulation of AraC family member activity by protein ligands.

    PubMed

    Plano, Gregory V

    2004-10-01

    A number of AraC family transcriptional activators bind low-molecular-weight ligands that modulate the activity of these proteins. Recently, it has become clear that the activity of several virulence-related AraC family members is regulated through the direct interaction of protein ligands. These interactions, in general, function to activate or repress the transcription of virulence genes in response to specific extracellular stimuli. The identification and characterization of several protein ligands that modify the activity of AraC family members in Pseudomonas aeruginosa and Salmonella enterica are discussed herein.

  1. Regulating the ethylene response of a plant by modulation of F-box proteins

    DOEpatents

    Guo, Hongwei [Beijing, CN; Ecker, Joseph R [Carlsbad, CA

    2011-03-08

    The invention relates to transgenic plants having reduced sensitivity to ethylene as a result of having a recombinant nucleic acid encoding an F-box protein that interacts with a EIN3 involved in an ethylene response of plants, and a method of producing a transgenic plant with reduced ethylene sensitivity by transforming the plant with a nucleic acid sequence encoding an F-box protein. The inventions also relates to methods of altering the ethylene response in a plant by modulating the activity or expression of an F-box protein.

  2. A Novel Assay Principle for Modulators of Protein-Protein Interactions and its Application to non-ATP-Competitive Ligands Targeting Protein Kinase A

    PubMed Central

    Saldanha, S. Adrian; Kaler, Gregory; Cottam, Howard B.; Abagyan, Ruben

    2012-01-01

    Targeting sites that modulate protein-protein interactions represents an ongoing challenge for drug discovery. We have devised an assay principle, named Ligand-Regulated Competition (LiReC), in an effort to find non-ATP competitive small molecule regulators for type Iα cAMP-dependent Protein Kinase A (PKA-Iα), a protein complex that is implicated in disease. Our assay based on the LiReC principle utilizes a competitive fluorescent peptide probe to assess the integrity of the PKA-Iα complex upon introduction of an allosteric ligand. The developed fluorescence polarization method screens for small molecules that specifically protect (antagonists) or conversely activate (agonists) this protein complex. In high throughput format, various cyclic nucleotide-derived agonists and antagonists are successfully detected with high precision. Furthermore, assay performance (Z’-factors above 0.7) far exceeds the minimum requirement for small molecule screening. To identify compounds that operate through novel modes of action, our method shields the ATP binding site and purposely excludes ATP-competitive ligands. These proof-of-principle experiments highlight the potential of the LiReC technique and suggest its application to other protein complexes, thereby providing a novel approach to identify and characterize modulators (small molecules, proteins, peptides, or nucleic acids) of protein-protein systems. PMID:17165815

  3. C-terminal tyrosine residues modulate the fusion activity of the Hendra virus fusion protein

    PubMed Central

    Popa, Andreea; Pager, Cara Teresia; Dutch, Rebecca Ellis

    2011-01-01

    The paramyxovirus family includes important human pathogens such as measles, mumps, respiratory syncytial virus and the recently emerged, highly pathogenic Hendra and Nipah viruses. The viral fusion (F) protein plays critical roles in infection, promoting both the viral-cell membrane fusion events needed for viral entry as well as cell-cell fusion events leading to syncytia formation. We describe the surprising finding that addition of the short epitope HA tag to the cytoplasmic tail (CT) of the Hendra virus F protein leads to a significant increase in cell-cell membrane fusion. This increase was not due to alterations in surface expression, cleavage state, or association with lipid microdomains. Addition of a Myc tag of similar length did not alter Hendra F fusion activity, indicating that the observed stimulation was not solely a result of lengthening the CT. Three tyrosine residues within the HA tag were critical for the increase in fusion, suggesting C-terminal tyrosines may modulate Hendra fusion activity. The effects of HA tag addition varied with other fusion proteins, as parainfluenza virus 5 F-HA showed decreased surface expression and no stimulation in fusion. These results indicate that additions to the C-terminal end of the F protein CT can modulate protein function in a sequence specific manner, reinforcing the need for careful analysis of epitope tagged glycoproteins. In addition, our results implicate C-terminal tyrosine residues in modulation of the membrane fusion reaction promoted by these viral glycoproteins. PMID:21175223

  4. Enumeration of condition-dependent dense modules in protein interaction networks

    PubMed Central

    Georgii, Elisabeth; Dietmann, Sabine; Uno, Takeaki; Pagel, Philipp; Tsuda, Koji

    2009-01-01

    Motivation: Modern systems biology aims at understanding how the different molecular components of a biological cell interact. Often, cellular functions are performed by complexes consisting of many different proteins. The composition of these complexes may change according to the cellular environment, and one protein may be involved in several different processes. The automatic discovery of functional complexes from protein interaction data is challenging. While previous approaches use approximations to extract dense modules, our approach exactly solves the problem of dense module enumeration. Furthermore, constraints from additional information sources such as gene expression and phenotype data can be integrated, so we can systematically mine for dense modules with interesting profiles. Results: Given a weighted protein interaction network, our method discovers all protein sets that satisfy a user-defined minimum density threshold. We employ a reverse search strategy, which allows us to exploit the density criterion in an efficient way. Our experiments show that the novel approach is feasible and produces biologically meaningful results. In comparative validation studies using yeast data, the method achieved the best overall prediction performance with respect to confirmed complexes. Moreover, by enhancing the yeast network with phenotypic and phylogenetic profiles and the human network with tissue-specific expression data, we identified condition-dependent complex variants. Availability: A C++ implementation of the algorithm is available at http://www.kyb.tuebingen.mpg.de/~georgii/dme.html. Contact: koji.tsuda@tuebingen.mpg.de Supplementary information: Supplementary data are available at Bioinformatics online. PMID:19213739

  5. Modulation of neurotransmitter receptors and synaptic differentiation by proteins containing complement-related domains.

    PubMed

    Nakayama, Minoru; Hama, Chihiro

    2011-02-01

    Neurotransmitter receptors play central roles in basic neurotransmission and synaptic plasticity. Recent studies have revealed that some transmembrane and extracellular proteins bind to neurotransmitter receptors, forming protein complexes that are required for proper synaptic localization or gating of core receptor molecules. Consequently, the components of these complexes contribute to long-term potentiation, a process that is critical for learning and memory. Here, we review factors that regulate neurotransmitter receptors, with a focus on proteins containing CUB (complement C1r/C1s, Uegf, Bmp1) or CCP (complement control protein) domains, which are frequently found in complement system proteins. Proteins that contain these domains are structurally distinct from TARPs (transmembrane AMPA receptor regulatory proteins), and may constitute new protein families that modulate either the localization or function of neurotransmitter receptors. In addition, other CCP domain-containing proteins participate in dendritic patterning and/or synaptic differentiation, although current evidence has not identified any direct activities on neurotransmitter receptors. Some of these proteins are involved in pathologic conditions such as epileptic seizure and mental retardation. Together, these lines of information have shown that CUB and CCP domain-containing proteins contribute to a wide variety of neuronal events that ultimately establish neural circuits.

  6. Atomic basis for the species-specific inhibition of αV integrins by monoclonal antibody 17E6 is revealed by the crystal structure of αVβ3 ectodomain-17E6 Fab complex.

    PubMed

    Mahalingam, Bhuvaneshwari; Van Agthoven, Johannes F; Xiong, Jian-Ping; Alonso, José Luis; Adair, Brian D; Rui, Xianliang; Anand, Saurabh; Mehrbod, Mehrdad; Mofrad, Mohammad R K; Burger, Christa; Goodman, Simon L; Arnaout, M Amin

    2014-05-16

    The function-blocking, non-RGD-containing, and primate-specific mouse monoclonal antibody 17E6 binds the αV subfamily of integrins. 17E6 is currently in phase II clinical trials for treating cancer. To elucidate the structural basis of recognition and the molecular mechanism of inhibition, we crystallized αVβ3 ectodomain in complex with the Fab fragment of 17E6. Protein crystals grew in presence of the activating cation Mn(2+). The integrin in the complex and in solution assumed the genuflected conformation. 17E6 Fab bound exclusively to the Propeller domain of the αV subunit. At the core of αV-Fab interface were interactions involving Propeller residues Lys-203 and Gln-145, with the latter accounting for primate specificity. The Propeller residue Asp-150, which normally coordinates Arg of the ligand Arg-Gly-Asp motif, formed contacts with Arg-54 of the Fab that were expected to reduce soluble FN10 binding to cellular αVβ3 complexed with 17E6. This was confirmed in direct binding studies, suggesting that 17E6 is an allosteric inhibitor of αV integrins.

  7. Atomic Basis for the Species-specific Inhibition of αV Integrins by Monoclonal Antibody 17E6 Is Revealed by the Crystal Structure of αVβ3 Ectodomain-17E6 Fab Complex*

    PubMed Central

    Mahalingam, Bhuvaneshwari; Van Agthoven, Johannes F.; Xiong, Jian-Ping; Alonso, José Luis; Adair, Brian D.; Rui, Xianliang; Anand, Saurabh; Mehrbod, Mehrdad; Mofrad, Mohammad R. K.; Burger, Christa; Goodman, Simon L.; Arnaout, M. Amin

    2014-01-01

    The function-blocking, non-RGD-containing, and primate-specific mouse monoclonal antibody 17E6 binds the αV subfamily of integrins. 17E6 is currently in phase II clinical trials for treating cancer. To elucidate the structural basis of recognition and the molecular mechanism of inhibition, we crystallized αVβ3 ectodomain in complex with the Fab fragment of 17E6. Protein crystals grew in presence of the activating cation Mn2+. The integrin in the complex and in solution assumed the genuflected conformation. 17E6 Fab bound exclusively to the Propeller domain of the αV subunit. At the core of αV-Fab interface were interactions involving Propeller residues Lys-203 and Gln-145, with the latter accounting for primate specificity. The Propeller residue Asp-150, which normally coordinates Arg of the ligand Arg-Gly-Asp motif, formed contacts with Arg-54 of the Fab that were expected to reduce soluble FN10 binding to cellular αVβ3 complexed with 17E6. This was confirmed in direct binding studies, suggesting that 17E6 is an allosteric inhibitor of αV integrins. PMID:24692540

  8. Community Structure Detection for Overlapping Modules through Mathematical Programming in Protein Interaction Networks

    PubMed Central

    Bennett, Laura; Kittas, Aristotelis; Liu, Songsong; Papageorgiou, Lazaros G.; Tsoka, Sophia

    2014-01-01

    Community structure detection has proven to be important in revealing the underlying properties of complex networks. The standard problem, where a partition of disjoint communities is sought, has been continually adapted to offer more realistic models of interactions in these systems. Here, a two-step procedure is outlined for exploring the concept of overlapping communities. First, a hard partition is detected by employing existing methodologies. We then propose a novel mixed integer non linear programming (MINLP) model, known as OverMod, which transforms disjoint communities to overlapping. The procedure is evaluated through its application to protein-protein interaction (PPI) networks of the rat, E. coli, yeast and human organisms. Connector nodes of hard partitions exhibit topological and functional properties indicative of their suitability as candidates for multiple module membership. OverMod identifies two types of connector nodes, inter and intra-connector, each with their own particular characteristics pertaining to their topological and functional role in the organisation of the network. Inter-connector proteins are shown to be highly conserved proteins participating in pathways that control essential cellular processes, such as proliferation, differentiation and apoptosis and their differences with intra-connectors is highlighted. Many of these proteins are shown to possess multiple roles of distinct nature through their participation in different network modules, setting them apart from proteins that are simply ‘hubs’, i.e. proteins with many interaction partners but with a more specific biochemical role. PMID:25412367

  9. A Two-Stage Model for Lipid Modulation of the Activity of Integral Membrane Proteins

    PubMed Central

    Dodes Traian, Martín M.; Cattoni, Diego I.; Levi, Valeria; González Flecha, F. Luis

    2012-01-01

    Lipid-protein interactions play an essential role in the regulation of biological function of integral membrane proteins; however, the underlying molecular mechanisms are not fully understood. Here we explore the modulation by phospholipids of the enzymatic activity of the plasma membrane calcium pump reconstituted in detergent-phospholipid mixed micelles of variable composition. The presence of increasing quantities of phospholipids in the micelles produced a cooperative increase in the ATPase activity of the enzyme. This activation effect was reversible and depended on the phospholipid/detergent ratio and not on the total lipid concentration. Enzyme activation was accompanied by a small structural change at the transmembrane domain reported by 1-aniline-8-naphtalenesulfonate fluorescence. In addition, the composition of the amphipilic environment sensed by the protein was evaluated by measuring the relative affinity of the assayed phospholipid for the transmembrane surface of the protein. The obtained results allow us to postulate a two-stage mechanistic model explaining the modulation of protein activity based on the exchange among non-structural amphiphiles at the hydrophobic transmembrane surface, and a lipid-induced conformational change. The model allowed to obtain a cooperativity coefficient reporting on the efficiency of the transduction step between lipid adsorption and catalytic site activation. This model can be easily applied to other phospholipid/detergent mixtures as well to other membrane proteins. The systematic quantitative evaluation of these systems could contribute to gain insight into the structure-activity relationships between proteins and lipids in biological membranes. PMID:22723977

  10. Community structure detection for overlapping modules through mathematical programming in protein interaction networks.

    PubMed

    Bennett, Laura; Kittas, Aristotelis; Liu, Songsong; Papageorgiou, Lazaros G; Tsoka, Sophia

    2014-01-01

    Community structure detection has proven to be important in revealing the underlying properties of complex networks. The standard problem, where a partition of disjoint communities is sought, has been continually adapted to offer more realistic models of interactions in these systems. Here, a two-step procedure is outlined for exploring the concept of overlapping communities. First, a hard partition is detected by employing existing methodologies. We then propose a novel mixed integer non linear programming (MINLP) model, known as OverMod, which transforms disjoint communities to overlapping. The procedure is evaluated through its application to protein-protein interaction (PPI) networks of the rat, E. coli, yeast and human organisms. Connector nodes of hard partitions exhibit topological and functional properties indicative of their suitability as candidates for multiple module membership. OverMod identifies two types of connector nodes, inter and intra-connector, each with their own particular characteristics pertaining to their topological and functional role in the organisation of the network. Inter-connector proteins are shown to be highly conserved proteins participating in pathways that control essential cellular processes, such as proliferation, differentiation and apoptosis and their differences with intra-connectors is highlighted. Many of these proteins are shown to possess multiple roles of distinct nature through their participation in different network modules, setting them apart from proteins that are simply 'hubs', i.e. proteins with many interaction partners but with a more specific biochemical role.

  11. Modification of proteins by cyclopentenone prostaglandins is differentially modulated by GSH in vitro.

    PubMed

    Gayarre, Javier; Avellano, M Isabel; Sánchez-Gómez, Francisco J; Carrasco, M Jesús; Cañada, F Javier; Pérez-Sala, Dolores

    2007-01-01

    Prostanoids with cyclopentenone structure (cyP) display a potent anti-inflammatory and antiproliferative activity. CyP are reactive compounds, which may modulate cellular functions by multiple mechanisms, including the direct covalent modification of cysteine residues by Michael addition. This interaction displays selectivity since only a subset of cellular proteins is modified by cyP. Several factors have been proposed to influence the selectivity and/or extent of cyP addition to proteins, including determinants related to protein and cyP structure, and levels of cellular thiols, such as glutathione (GSH). Here we have explored the ability of biotinylated cyP analogs to modify several recombinant proteins in vitro, and the influence of GSH in these effects. We have observed that protein modification by cyP is protein- and cyP-selective. Under our conditions, biotinylated 15-deoxy-Delta(12,14)-PGJ(2) (15d-PGJ(2)-B) was more efficient than biotinylated PGA(1) (PGA(1)-B) at forming adducts with components of the transcription factors NF-kappaB and activator protein-1 (AP-1). However, both biotinylated cyP were nearly equipotent at modifying human GSTP1-1. Interestingly, the presence of GSH differentially modulated the formation of protein-cyP adducts. Under our conditions, GSH reduced the incorporation of cyP into GST, but improved their binding to p50, more intensely in the case of PGA(1)-B. These results evidence the importance of GSH-cyP and/or GSH-protein interactions for the selectivity of protein modification by cyP and suggest a complex role for GSH that may be related to its ability to prevent protein oxidation or induce conformational alterations. This may shed light on the factors involved in the pleiotropic effects of electrophiles with therapeutic potential.

  12. Seropositivity of HPV 16 E6 and E7 and the risk of oral cancer.

    PubMed

    Wong, G R; Ha, K O; Himratul-Aznita, W H; Yang, Y H; Wan Mustafa, W M; Yuen, K M; Abraham, M T; Tay, K K; Karen-Ng, L P; Cheong, S C; Zain, R B

    2014-11-01

    The objective of the study was to determine the prevalence of HPV seropositivity among patients with oral squamous cell carcinoma (OSCC) and healthy individuals and to correlate the association between HPV 16 seropositivity and risk of OSCC. HPV 16 E6 and E7 plasmids were constructed for the production of recombinant protein, which was used as the antigen in ELISA. HPV ELISA was performed on serum samples from 50 healthy individuals and 50 patients with OSCC. Using the HPV ELISA, 30% (OR = 2.25, 95% CI = 0.85-5.93) and 18% (OR = 1.61, 95% CI = 0.53-4.92) of patients with oral cancer were found to be HPV 16 E6 and E7 seropositive, respectively. Significant association was found between HPV 16 seropositivity and increased risk of OSCC in men, but not in male subjects. A similar trend was observed in non-betel quid chewers. Potential associations between HPV 16 E6/E7 seropositivity and oral cancer were revealed in men and non-betel quid chewer subjects, suggesting a possible etiological role of HPV 16 in subgroup of patients with OSCC in Malaysia. © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  13. Towards the identification of protein complexes and functional modules by integrating PPI network and gene expression data

    PubMed Central

    2012-01-01

    Background Identification of protein complexes and functional modules from protein-protein interaction (PPI) networks is crucial to understanding the principles of cellular organization and predicting protein functions. In the past few years, many computational methods have been proposed. However, most of them considered the PPI networks as static graphs and overlooked the dynamics inherent within these networks. Moreover, few of them can distinguish between protein complexes and functional modules. Results In this paper, a new framework is proposed to distinguish between protein complexes and functional modules by integrating gene expression data into protein-protein interaction (PPI) data. A series of time-sequenced subnetworks (TSNs) is constructed according to the time that the interactions were activated. The algorithm TSN-PCD was then developed to identify protein complexes from these TSNs. As protein complexes are significantly related to functional modules, a new algorithm DFM-CIN is proposed to discover functional modules based on the identified complexes. The experimental results show that the combination of temporal gene expression data with PPI data contributes to identifying protein complexes more precisely. A quantitative comparison based on f-measure reveals that our algorithm TSN-PCD outperforms the other previous protein complex discovery algorithms. Furthermore, we evaluate the identified functional modules by using “Biological Process” annotated in GO (Gene Ontology). The validation shows that the identified functional modules are statistically significant in terms of “Biological Process”. More importantly, the relationship between protein complexes and functional modules are studied. Conclusions The proposed framework based on the integration of PPI data and gene expression data makes it possible to identify protein complexes and functional modules more effectively. Moveover, the proposed new framework and algorithms can distinguish

  14. Functional Module Search in Protein Networks based on Semantic Similarity Improves the Analysis of Proteomics Data*

    PubMed Central

    Boyanova, Desislava; Nilla, Santosh; Klau, Gunnar W.; Dandekar, Thomas; Müller, Tobias; Dittrich, Marcus

    2014-01-01

    The continuously evolving field of proteomics produces increasing amounts of data while improving the quality of protein identifications. Albeit quantitative measurements are becoming more popular, many proteomic studies are still based on non-quantitative methods for protein identification. These studies result in potentially large sets of identified proteins, where the biological interpretation of proteins can be challenging. Systems biology develops innovative network-based methods, which allow an integrated analysis of these data. Here we present a novel approach, which combines prior knowledge of protein-protein interactions (PPI) with proteomics data using functional similarity measurements of interacting proteins. This integrated network analysis exactly identifies network modules with a maximal consistent functional similarity reflecting biological processes of the investigated cells. We validated our approach on small (H9N2 virus-infected gastric cells) and large (blood constituents) proteomic data sets. Using this novel algorithm, we identified characteristic functional modules in virus-infected cells, comprising key signaling proteins (e.g. the stress-related kinase RAF1) and demonstrate that this method allows a module-based functional characterization of cell types. Analysis of a large proteome data set of blood constituents resulted in clear separation of blood cells according to their developmental origin. A detailed investigation of the T-cell proteome further illustrates how the algorithm partitions large networks into functional subnetworks each representing specific cellular functions. These results demonstrate that the integrated network approach not only allows a detailed analysis of proteome networks but also yields a functional decomposition of complex proteomic data sets and thereby provides deeper insights into the underlying cellular processes of the investigated system. PMID:24807868

  15. Functional module search in protein networks based on semantic similarity improves the analysis of proteomics data.

    PubMed

    Boyanova, Desislava; Nilla, Santosh; Klau, Gunnar W; Dandekar, Thomas; Müller, Tobias; Dittrich, Marcus

    2014-07-01

    The continuously evolving field of proteomics produces increasing amounts of data while improving the quality of protein identifications. Albeit quantitative measurements are becoming more popular, many proteomic studies are still based on non-quantitative methods for protein identification. These studies result in potentially large sets of identified proteins, where the biological interpretation of proteins can be challenging. Systems biology develops innovative network-based methods, which allow an integrated analysis of these data. Here we present a novel approach, which combines prior knowledge of protein-protein interactions (PPI) with proteomics data using functional similarity measurements of interacting proteins. This integrated network analysis exactly identifies network modules with a maximal consistent functional similarity reflecting biological processes of the investigated cells. We validated our approach on small (H9N2 virus-infected gastric cells) and large (blood constituents) proteomic data sets. Using this novel algorithm, we identified characteristic functional modules in virus-infected cells, comprising key signaling proteins (e.g. the stress-related kinase RAF1) and demonstrate that this method allows a module-based functional characterization of cell types. Analysis of a large proteome data set of blood constituents resulted in clear separation of blood cells according to their developmental origin. A detailed investigation of the T-cell proteome further illustrates how the algorithm partitions large networks into functional subnetworks each representing specific cellular functions. These results demonstrate that the integrated network approach not only allows a detailed analysis of proteome networks but also yields a functional decomposition of complex proteomic data sets and thereby provides deeper insights into the underlying cellular processes of the investigated system.

  16. The Drosophila Homologue of the Amyloid Precursor Protein Is a Conserved Modulator of Wnt PCP Signaling

    PubMed Central

    Soldano, Alessia; Okray, Zeynep; Janovska, Pavlina; Tmejová, Kateřina; Reynaud, Elodie; Claeys, Annelies; Yan, Jiekun; Atak, Zeynep Kalender; De Strooper, Bart; Dura, Jean-Maurice; Bryja, Vítězslav; Hassan, Bassem A.

    2013-01-01

    Wnt Planar Cell Polarity (PCP) signaling is a universal regulator of polarity in epithelial cells, but it regulates axon outgrowth in neurons, suggesting the existence of axonal modulators of Wnt-PCP activity. The Amyloid precursor proteins (APPs) are intensely investigated because of their link to Alzheimer's disease (AD). APP's in vivo function in the brain and the mechanisms underlying it remain unclear and controversial. Drosophila possesses a single APP homologue called APP Like, or APPL. APPL is expressed in all neurons throughout development, but has no established function in neuronal development. We therefore investigated the role of Drosophila APPL during brain development. We find that APPL is involved in the development of the Mushroom Body αβ neurons and, in particular, is required cell-autonomously for the β-axons and non-cell autonomously for the α-axons growth. Moreover, we find that APPL is a modulator of the Wnt-PCP pathway required for axonal outgrowth, but not cell polarity. Molecularly, both human APP and fly APPL form complexes with PCP receptors, thus suggesting that APPs are part of the membrane protein complex upstream of PCP signaling. Moreover, we show that APPL regulates PCP pathway activation by modulating the phosphorylation of the Wnt adaptor protein Dishevelled (Dsh) by Abelson kinase (Abl). Taken together our data suggest that APPL is the first example of a modulator of the Wnt-PCP pathway specifically required for axon outgrowth. PMID:23690751

  17. The Drosophila homologue of the amyloid precursor protein is a conserved modulator of Wnt PCP signaling.

    PubMed

    Soldano, Alessia; Okray, Zeynep; Janovska, Pavlina; Tmejová, Kateřina; Reynaud, Elodie; Claeys, Annelies; Yan, Jiekun; Atak, Zeynep Kalender; De Strooper, Bart; Dura, Jean-Maurice; Bryja, Vítězslav; Hassan, Bassem A

    2013-01-01

    Wnt Planar Cell Polarity (PCP) signaling is a universal regulator of polarity in epithelial cells, but it regulates axon outgrowth in neurons, suggesting the existence of axonal modulators of Wnt-PCP activity. The Amyloid precursor proteins (APPs) are intensely investigated because of their link to Alzheimer's disease (AD). APP's in vivo function in the brain and the mechanisms underlying it remain unclear and controversial. Drosophila possesses a single APP homologue called APP Like, or APPL. APPL is expressed in all neurons throughout development, but has no established function in neuronal development. We therefore investigated the role of Drosophila APPL during brain development. We find that APPL is involved in the development of the Mushroom Body αβ neurons and, in particular, is required cell-autonomously for the β-axons and non-cell autonomously for the α-axons growth. Moreover, we find that APPL is a modulator of the Wnt-PCP pathway required for axonal outgrowth, but not cell polarity. Molecularly, both human APP and fly APPL form complexes with PCP receptors, thus suggesting that APPs are part of the membrane protein complex upstream of PCP signaling. Moreover, we show that APPL regulates PCP pathway activation by modulating the phosphorylation of the Wnt adaptor protein Dishevelled (Dsh) by Abelson kinase (Abl). Taken together our data suggest that APPL is the first example of a modulator of the Wnt-PCP pathway specifically required for axon outgrowth.

  18. New integrative modules for multicolor-protein labeling and live-cell imaging in Saccharomyces cerevisiae.

    PubMed

    Malcova, Ivana; Farkasovsky, Marian; Senohrabkova, Lenka; Vasicova, Pavla; Hasek, Jiri

    2016-05-01

    Live-imaging analysis is performed in many laboratories all over the world. Various tools have been developed to enable protein labeling either in plasmid or genomic context in live yeast cells. Here, we introduce a set of nine integrative modules for the C-terminal gene tagging that combines three fluorescent proteins (FPs)-ymTagBFP, mCherry and yTagRFP-T with three dominant selection markers: geneticin, nourseothricin and hygromycin. In addition, the construction of two episomal modules for Saccharomyces cerevisiae with photostable yTagRFP-T is also referred to. Our cassettes with orange, red and blue FPs can be combined with other fluorescent probes like green fluorescent protein to prepare double- or triple-labeled strains for multicolor live-cell imaging. Primers for PCR amplification of the cassettes were designed in such a way as to be fully compatible with the existing PCR toolbox representing over 50 various integrative modules and also with deletion cassettes either for single or repeated usage to enable a cost-effective and an easy exchange of tags. New modules can also be used for biochemical analysis since antibodies are available for all three fluorescent probes. © FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  19. Protein Profiles Associated With Context Fear Conditioning and Their Modulation by Memantine*

    PubMed Central

    Ahmed, Md. Mahiuddin; Dhanasekaran, A. Ranjitha; Block, Aaron; Tong, Suhong; Costa, Alberto C. S.; Gardiner, Katheleen J.

    2014-01-01

    Analysis of the molecular basis of learning and memory has revealed details of the roles played by many genes and the proteins they encode. Because most individual studies focus on a small number of proteins, many complexities of the relationships among proteins and their dynamic responses to stimulation are not known. We have used the technique of reverse phase protein arrays (RPPA) to assess the levels of more than 80 proteins/protein modifications in subcellular fractions from hippocampus and cortex of mice trained in Context Fear Conditioning (CFC). Proteins include components of signaling pathways, several encoded by immediate early genes or involved in apoptosis and inflammation, and subunits of glutamate receptors. At one hour after training, levels of more than half the proteins had changed in one or more fractions, among them multiple components of the Mitogen-activated protein kinase, MAPK, and Mechanistic Target of Rapamycin, MTOR, pathways, subunits of glutamate receptors, and the NOTCH pathway modulator, NUMB homolog (Drosophila). Levels of 37 proteins changed in the nuclear fraction of hippocampus alone. Abnormalities in levels of thirteen proteins analyzed have been reported in brains of patients with Alzheimer's Disease. We therefore further investigated the protein profiles of mice treated with memantine, a drug approved for treatment of AD. In hippocampus, memantine alone induced many changes similar to those seen after CFC and altered the levels of seven proteins associated with Alzheimer's Disease abnormalities. Lastly, to further explore the relevance of these datasets, we superimposed responses to CFC and memantine onto components of the long term potentiation pathway, a process subserving learning and memory formation. Fourteen components of the long term potentiation pathway and 26 proteins interacting with components responded to CFC and/or memantine. Together, these datasets provide a novel view of the diversity and complexity in protein

  20. A Chemically Inducible Helper Module for Detecting Protein-Protein Interactions with Tunable Sensitivity Based on KIPPIS.

    PubMed

    Kashima, Daiki; Kawade, Raiji; Nagamune, Teruyuki; Kawahara, Masahiro

    2017-04-13

    As protein-protein interactions (PPIs) play essential roles in regulating their functional consequences in cells, methods to detect PPIs in living cells are desired for correct understanding of intracellular PPIs and pharmaceutical development therefrom. Here we demonstrate a c-kit-based PPI screening (KIPPIS) system in combination with a chemically inducible helper module for detecting PPIs in living mammalian cells. In this system, a mutant of FK506-binding protein 12 (FKBPF36 V) is fused with a protein of interest and the intracellular domain of a receptor tyrosine kinase c-kit. Constitutive expression of two fusion proteins with interacting proteins of interest in interleukin-3 (IL-3)-dependent cells results in dimerization and subsequent activation of the c-kit intracellular domains, which allows cell proliferation in a culture medium devoid of IL-3. A helper ligand, a small synthetic chemical that homodimerizes FKBPF36 V, assists the formation of stable complexes of the fusion proteins and serves as a tuner for sensitivity of the system. Using this system, two model PPIs were successfully detected on the basis of cell proliferation, which was featured by the helper-ligand- and PPI-dependent phosphorylation of the Src family kinases, a hallmark of the c-kit signaling. Notably, the inclusion of the helper module enabled PPI detection with tunable sensitivity. The helper-assisted KIPPIS allows us to configure various affinity thresholds by changing the concentration of the helper ligand, which may be applied to select affinity-matured variants using the advantage of cell proliferation.

  1. FunMod: a Cytoscape plugin for identifying functional modules in undirected protein-protein networks.

    PubMed

    Natale, Massimo; Benso, Alfredo; Di Carlo, Stefano; Ficarra, Elisa

    2014-08-01

    The characterization of the interacting behaviors of complex biological systems is a primary objective in protein-protein network analysis and computational biology. In this paper we present FunMod, an innovative Cytoscape version 2.8 plugin that is able to mine undirected protein-protein networks and to infer sub-networks of interacting proteins intimately correlated with relevant biological pathways. This plugin may enable the discovery of new pathways involved in diseases. In order to describe the role of each protein within the relevant biological pathways, FunMod computes and scores three topological features of the identified sub-networks. By integrating the results from biological pathway clustering and topological network analysis, FunMod proved to be useful for the data interpretation and the generation of new hypotheses in two case studies.

  2. In Vitro Calcite Crystal Morphology Is Modulated by Otoconial Proteins Otolin-1 and Otoconin-90

    PubMed Central

    Moreland, K. Trent; Hong, Mina; Lu, Wenfu; Rowley, Christopher W.; Ornitz, David M.; De Yoreo, James J.; Thalmann, Ruediger

    2014-01-01

    Otoconia are formed embryonically and are instrumental in detecting linear acceleration and gravity. Degeneration and fragmentation of otoconia in elderly patients leads to imbalance resulting in higher frequency of falls that are positively correlated with the incidence of bone fractures and death. In this work we investigate the roles otoconial proteins Otolin-1 and Otoconin 90 (OC90) perform in the formation of otoconia. We demonstrate by rotary shadowing and atomic force microscopy (AFM) experiments that Otolin-1 forms homomeric protein complexes and self-assembled networks supporting the hypothesis that Otolin-1 serves as a scaffold protein of otoconia. Our calcium carbonate crystal growth data demonstrate that Otolin-1 and OC90 modulate in vitro calcite crystal morphology but neither protein is sufficient to produce the shape of otoconia. Coadministration of these proteins produces synergistic effects on crystal morphology that contribute to morphology resembling otoconia. PMID:24748133

  3. Recent insights into Pasteurella multocida toxin and other G-protein-modulating bacterial toxins.

    PubMed

    Wilson, Brenda A; Ho, Mengfei

    2010-08-01

    Over the past few decades, our understanding of the bacterial protein toxins that modulate G proteins has advanced tremendously through extensive biochemical and structural analyses. This article provides an updated survey of the various toxins that target G proteins, ending with a focus on recent mechanistic insights in our understanding of the deamidating toxin family. The dermonecrotic toxin from Pasteurella multocida (PMT) was recently added to the list of toxins that disrupt G-protein signal transduction through selective deamidation of their targets. The C3 deamidase domain of PMT has no sequence similarity to the deamidase domains of the dermonecrotic toxins from Escherichia coli (cytotoxic necrotizing factor [CNF]1-3), Yersinia (CNFY) and Bordetella (dermonecrotic toxin). The structure of PMT-C3 belongs to a family of transglutaminase-like proteins, with active site Cys-His-Asp catalytic triads distinct from E. coli CNF1.

  4. Review article: insights into colonic protein fermentation, its modulation and potential health implications.

    PubMed

    Yao, C K; Muir, J G; Gibson, P R

    2016-01-01

    Beneficial effects of carbohydrate fermentation on gastrointestinal health are well established. Conversely, protein fermentation generates harmful metabolites but their relevance to gastrointestinal health is poorly understood. To review the effects of increased protein fermentation on biomarkers of colonic health, factors influencing fermentative activity and potential for dietary modulation to minimise detrimental effects. A literature search was performed in PubMed, Medline, EMBASE and Google scholar for clinical and pre-clinical studies using search terms - 'dietary protein', 'fermentation', 'putrefaction', 'phenols', 'sulphide', 'branched-chain fatty acid', 'carbohydrate fermentation', 'gastrointestinal'. High protein, reduced carbohydrate diets alter the colonic microbiome, favouring a potentially pathogenic and pro-inflammatory microbiota profile, decreased short-chain fatty acid production and increased ammonia, phenols and hydrogen sulphide concentrations. These metabolites largely compromise the colonic epithelium structure, causing mucosal inflammation but may also directly modulate the enteric nervous system and intestinal motility. Increased protein fermentation as a result of a high-protein intake can be attenuated by addition of oligosaccharides, resistant starch and nonstarch polysaccharides and a reduction in total protein or specifically, aromatic and sulphur-containing amino acids. These factors may have clinical importance as novel therapeutic approaches to problems, in which protein fermentation may be implicated, such as malodorous flatus, irritable bowel syndrome, ulcerative colitis and prevention of colorectal cancer. The direct clinical relevance of excessive protein fermentation in the pathogenesis of irritable bowel syndrome, malodorous flatus and ulcerative colitis are underexplored. Manipulating dietary carbohydrate and protein intake have potential therapeutic applications in such settings and warrant further clinical studies. © 2015

  5. An Artificial Reaction Promoter Modulates Mitochondrial Functions via Chemically Promoting Protein Acetylation

    PubMed Central

    Shindo, Yutaka; Komatsu, Hirokazu; Hotta, Kohji; Ariga, Katsuhiko; Oka, Kotaro

    2016-01-01

    Acetylation, which modulates protein function, is an important process in intracellular signalling. In mitochondria, protein acetylation regulates a number of enzymatic activities and, therefore, modulates mitochondrial functions. Our previous report showed that tributylphosphine (PBu3), an artificial reaction promoter that promotes acetylransfer reactions in vitro, also promotes the reaction between acetyl-CoA and an exogenously introduced fluorescent probe in mitochondria. In this study, we demonstrate that PBu3 induces the acetylation of mitochondrial proteins and a decrease in acetyl-CoA concentration in PBu3-treated HeLa cells. This indicates that PBu3 can promote the acetyltransfer reaction between acetyl-CoA and mitochondrial proteins in living cells. PBu3-induced acetylation gradually reduced mitochondrial ATP concentrations in HeLa cells without changing the cytoplasmic ATP concentration, suggesting that PBu3 mainly affects mitochondrial functions. In addition, pyruvate, which is converted into acetyl-CoA in mitochondria and transiently increases ATP concentrations in the absence of PBu3, elicited a further decrease in mitochondrial ATP concentrations in the presence of PBu3. Moreover, the application and removal of PBu3 reversibly alternated mitochondrial fragmentation and elongation. These results indicate that PBu3 enhances acetyltransfer reactions in mitochondria and modulates mitochondrial functions in living cells. PMID:27374857

  6. A conserved patch of hydrophobic amino acids modulates Myb activity by mediating protein-protein interactions.

    PubMed

    Dukare, Sandeep; Klempnauer, Karl-Heinz

    2016-07-01

    The transcription factor c-Myb plays a key role in the control of proliferation and differentiation in hematopoietic progenitor cells and has been implicated in the development of leukemia and certain non-hematopoietic tumors. c-Myb activity is highly dependent on the interaction with the coactivator p300 which is mediated by the transactivation domain of c-Myb and the KIX domain of p300. We have previously observed that conservative valine-to-isoleucine amino acid substitutions in a conserved stretch of hydrophobic amino acids have a profound effect on Myb activity. Here, we have explored the function of the hydrophobic region as a mediator of protein-protein interactions. We show that the hydrophobic region facilitates Myb self-interaction and binding of the histone acetyl transferase Tip60, a previously identified Myb interacting protein. We show that these interactions are affected by the valine-to-isoleucine amino acid substitutions and suppress Myb activity by interfering with the interaction of Myb and the KIX domain of p300. Taken together, our work identifies the hydrophobic region in the Myb transactivation domain as a binding site for homo- and heteromeric protein interactions and leads to a picture of the c-Myb transactivation domain as a composite protein binding region that facilitates interdependent protein-protein interactions of Myb with regulatory proteins. Copyright © 2016 Elsevier B.V. All rights reserved.

  7. Control of B cell production by the adaptor protein lnk. Definition Of a conserved family of signal-modulating proteins.

    PubMed

    Takaki, S; Sauer, K; Iritani, B M; Chien, S; Ebihara, Y; Tsuji, K; Takatsu, K; Perlmutter, R M

    2000-11-01

    Lnk is an SH2 domain-containing adaptor protein expressed preferentially in lymphocytes. To illuminate the importance of Lnk, we generated lnk(-/-) mice. Whereas T cell development was unaffected, pre-B and immature B cells accumulated in the spleens. In the bone marrow, B-lineage cells were proportionately increased, reflecting enhanced production of pro-B cells that resulted in part from hypersensitivity of precursors to SCF, the ligand for c-kit. Hence, Lnk ordinarily acts to regulate B cell production. Further characterization of lnk(-/-) mice also revealed that full-length Lnk is a 68 kDa protein containing a conserved proline-rich region and a PH domain. Lnk is a representative of a multigene adaptor protein family whose members act, by analogy with Lnk, to modulate intracellular signaling.

  8. Temperature-induced protein secretion by Leishmania mexicana modulates macrophage signalling and function.

    PubMed

    Hassani, Kasra; Antoniak, Elisabeth; Jardim, Armando; Olivier, Martin

    2011-05-03

    Protozoan parasites of genus Leishmania are the causative agents of leishmaniasis. These digenetic microorganisms undergo a marked environmental temperature shift (TS) during transmission from the sandfly vector (ambient temperature, 25-26°C) to the mammalian host (37°C). We have observed that this TS induces a rapid and dramatic increase in protein release from Leishmania mexicana (cutaneous leishmaniasis) within 4 h. Proteomic identification of the TS-induced secreted proteins revealed 72 proteins, the majority of which lack a signal peptide and are thus thought to be secreted via nonconventional mechanisms. Interestingly, this protein release is accompanied by alterations in parasite morphology including an augmentation in the budding of exovesicles from its surface. Here we show that the exoproteome of L. mexicana upon TS induces cleavage and activation of the host protein tyrosine phosphatases, specifically SHP-1 and PTP1-B, in a murine bone-marrow-derived macrophage cell line. Furthermore, translocation of prominent inflammatory transcription factors, namely NF-κB and AP-1 is altered. The exoproteome also caused inhibition of nitric oxide production, a crucial leishmanicidal function of the macrophage. Overall, our results provide strong evidence that within early moments of interaction with the mammalian host, L. mexicana rapidly releases proteins and exovesicles that modulate signalling and function of the macrophage. These modulations can result in attenuation of the inflammatory response and deactivation of the macrophage aiding the parasite in the establishment of infection.

  9. Ubx4 modulates cdc48 activity and influences degradation of misfolded proteins of the endoplasmic reticulum.

    PubMed

    Alberts, Sven M; Sonntag, Caroline; Schäfer, Antje; Wolf, Dieter H

    2009-06-12

    Misfolded proteins of the secretory pathway are recognized in the endoplasmic reticulum (ER), retrotranslocated into the cytoplasm, and degraded by the ubiquitin-proteasome system. Right after retrotranslocation and polyubiquitination, they are extracted from the cytosolic side of the ER membrane through a complex consisting of the AAA ATPase Cdc48 (p97 in mammals), Ufd1, and Npl4. This complex delivers misfolded proteins to the proteasome for final degradation. Extraction, delivery, and processing of ERAD (ER-associated degradation) substrates to the proteasome requires additional cofactors of Cdc48. Here we characterize the UBX domain containing protein Ubx4 (Cui1) as a crucial factor for the degradation of polyubiquitinated proteins via ERAD. Ubx4 modulates the Cdc48-Ufd1-Npl4 complex to guarantee its correct function. Mutant variants of Ubx4 lead to defective degradation of misfolded proteins and accumulation of polyubiquitinated proteins bound to Cdc48. We show the requirement of the UBX domain of Ubx4 for its function in ERAD. The observation that Ubx2 and Ubx4 are not found together in one complex with Cdc48 suggests several distinct steps in modulating the activity and localization of Cdc48 in ERAD.

  10. Adhesion-modulating/matricellular ECM protein families: a structural, functional and evolutionary appraisal.

    PubMed

    Mosher, Deane F; Adams, Josephine C

    2012-04-01

    The thrombospondins are a family of secreted, oligomeric glycoproteins that interact with cell surfaces, multiple components of the extracellular matrix, growth factors and proteases. These interactions underlie complex roles in cell interactions and tissue homeostasis in animals. Thrombospondins have been grouped functionally with SPARCs, tenascins and CCN proteins as adhesion-modulating or matricellular components of the extracellular milieu. Although all these multi-domain proteins share various commonalities of domains, the grouping is not based on structural homologies. Instead, the terms emphasise the general observations that these proteins do not form large-scale ECM structures, yet act at cell surfaces and function in coordination with the structural ECM and associated extracellular proteins. The designation of adhesion-modulation thus depends on observed tissue and cell culture ECM distributions and on experimentally identified functional properties. To date, the evolutionary relationships of these proteins have not been critically compared: yet, knowledge of their evolutionary histories is clearly relevant to any consideration of functional similarities. In this article, we survey briefly the structural and functional knowledge of these protein families, consider the evolution of each family, and outline a perspective on their functional roles.

  11. E6 and E7 Gene Polymorphisms in Human Papillomavirus Types-58 and 33 Identified in Southwest China

    PubMed Central

    Wen, Qiang; Wang, Tao; Mu, Xuemei; Chenzhang, Yuwei; Cao, Man

    2017-01-01

    Cancer of the cervix is associated with infection by certain types of human papillomavirus (HPV). The gene variants differ in immune responses and oncogenic potential. The E6 and E7 proteins encoded by high-risk HPV play a key role in cellular transformation. HPV-33 and HPV-58 types are highly prevalent among Chinese women. To study the gene intratypic variations, polymorphisms and positive selections of HPV-33 and HPV-58 E6/E7 in southwest China, HPV-33 (E6, E7: n = 216) and HPV-58 (E6, E7: n = 405) E6 and E7 genes were sequenced and compared to others submitted to GenBank. Phylogenetic trees were constructed by Maximum-likelihood and the Kimura 2-parameters methods by MEGA 6 (Molecular Evolutionary Genetics Analysis version 6.0). The diversity of secondary structure was analyzed by PSIPred software. The selection pressures acting on the E6/E7 genes were estimated by PAML 4.8 (Phylogenetic Analyses by Maximun Likelihood version4.8) software. The positive sites of HPV-33 and HPV-58 E6/E7 were contrasted by ClustalX 2.1. Among 216 HPV-33 E6 sequences, 8 single nucleotide mutations were observed with 6/8 non-synonymous and 2/8 synonymous mutations. The 216 HPV-33 E7 sequences showed 3 single nucleotide mutations that were non-synonymous. The 405 HPV-58 E6 sequences revealed 8 single nucleotide mutations with 4/8 non-synonymous and 4/8 synonymous mutations. Among 405 HPV-58 E7 sequences, 13 single nucleotide mutations were observed with 10/13 non-synonymous mutations and 3/13 synonymous mutations. The selective pressure analysis showed that all HPV-33 and 4/6 HPV-58 E6/E7 major non-synonymous mutations were sites of positive selection. All variations were observed in sites belonging to major histocompatibility complex and/or B-cell predicted epitopes. K93N and R145 (I/N) were observed in both HPV-33 and HPV-58 E6. PMID:28141822

  12. Biophysical Insights into How Surfaces, Including Lipid Membranes, Modulate Protein Aggregation Related to Neurodegeneration

    PubMed Central

    Burke, Kathleen A.; Yates, Elizabeth A.; Legleiter, Justin

    2013-01-01

    There are a vast number of neurodegenerative diseases, including Alzheimer’s disease (AD), Parkinson’s disease (PD), and Huntington’s disease (HD), associated with the rearrangement of specific proteins to non-native conformations that promotes aggregation and deposition within tissues and/or cellular compartments. These diseases are commonly classified as protein-misfolding or amyloid diseases. The interaction of these proteins with liquid/surface interfaces is a fundamental phenomenon with potential implications for protein-misfolding diseases. Kinetic and thermodynamic studies indicate that significant conformational changes can be induced in proteins encountering surfaces, which can play a critical role in nucleating aggregate formation or stabilizing specific aggregation states. Surfaces of particular interest in neurodegenerative diseases are cellular and subcellular membranes that are predominately comprised of lipid components. The two-dimensional liquid environments provided by lipid bilayers can profoundly alter protein structure and dynamics by both specific and non-specific interactions. Importantly for misfolding diseases, these bilayer properties can not only modulate protein conformation, but also exert influence on aggregation state. A detailed understanding of the influence of (sub)cellular surfaces in driving protein aggregation and/or stabilizing specific aggregate forms could provide new insights into toxic mechanisms associated with these diseases. Here, we review the influence of surfaces in driving and stabilizing protein aggregation with a specific emphasis on lipid membranes. PMID:23459674

  13. Expression of Yes-associated protein modulates Survivin expression in primary liver malignancies.

    PubMed

    Bai, Haibo; Gayyed, Mariana F; Lam-Himlin, Dora M; Klein, Alison P; Nayar, Suresh K; Xu, Yang; Khan, Mehtab; Argani, Pedram; Pan, Duojia; Anders, Robert A

    2012-09-01

    Hepatocellular carcinoma and intrahepatic cholangiocarcinoma account for 95% of primary liver cancer. For each of these malignancies, the outcome is dismal; incidence is rapidly increasing, and mechanistic understanding is limited. We observed abnormal proliferation of both biliary epithelium and hepatocytes in mice after genetic manipulation of Yes-associated protein, a transcription coactivator. Here, we comprehensively documented Yes-associated protein expression in the human liver and primary liver cancers. We showed that nuclear Yes-associated protein expression is significantly increased in human intrahepatic cholangiocarcinoma and hepatocellular carcinoma. We found that increased Yes-associated protein levels in hepatocellular carcinoma are due to multiple mechanisms including gene amplification and transcriptional and posttranscriptional regulation. Survivin, a member of the inhibitors-of-apoptosis protein family, has been reported as an independent prognostic factor for poor survival in both hepatocellular carcinoma and intrahepatic cholangiocarcinoma. We found that nuclear Yes-associated protein expression correlates significantly with nuclear Survivin expression for both intrahepatic cholangiocarcinoma and hepatocellular carcinoma. Furthermore, using mice engineered to conditionally overexpress Yes-associated protein in the liver, we found that Survivin messenger RNA expression depends upon Yes-associated protein levels. Our findings suggested that Yes-associated protein contributes to primary liver tumorigenesis and likely mediates its oncogenic effects through modulating Survivin expression. Copyright © 2012 Elsevier Inc. All rights reserved.

  14. Role of inhibitory proteins as modulators of oscillations in NFB signalling.

    PubMed

    Nikolov, S; Vera, J; Rath, O; Kolch, W; Wolkenhauer, O

    2009-03-01

    The authors discuss the role of the Raf kinase inhibitory protein (RKIP) as a modulator of oscillations in NFB signalling. A mathematical model of the NFB signalling pathway was derived and the Lyapunov-Andronov theory was used to analyse dynamical properties of the system. The analytical results were complemented by predictive numerical simulations. Our results suggest that the nature of oscillations, emerging under sustained stimulation of the system, depends on the interplay between the IB kinase (IKK) stimulation and the inhibitory action of RKIP. The authors found a mathematical relation that defines isoclines in IKK and RKIP levels for which the properties of oscillations are conserved and changes in the stimulation can be compensated by modulating RKIP inhibition. On the other hand, the shifting from the current isocline provokes modulation in either the amplitude (for stronger stimulation) or the frequency (for weaker stimulation).

  15. Regulator of G protein signaling proteins differentially modulate signaling of μ and δ opioid receptors

    PubMed Central

    Xie, Zhihua; Li, Zhisong; Guo, Lei; Ye, Caiying; Li, Juan; Yu, Xiaoli; Yang, Huifen; Wang, Yulin; Chen, Chongguang; Zhang, Dechang; Liu-Chen, Lee-Yuan

    2009-01-01

    Effects of regulator of G protein signaling (RGS) proteins on μ and δ opioid receptors were investigated in HEK293 cells. Co-expression of RGS1, RGS2, RGS4, RGS9, RGS10 or RGS19 (Gα-interacting protein (GAIP)) significantly reduced [Tyr-D-Ala-Gly-N-methyl-Phe-Gly-ol]-Enkephalin (DAMGO)-induced inhibition of adenylyl cyclase (AC) mediated by μ opioid receptor, but only RGS9 decreased the effects of [Tyr-D-Pen-Gly-p-Chloro-Phe-D-Pen]-Enkephalin (DPDPE) mediated by δ opioid receptor. When C-tails of the receptors were exchanged (μ/δC and δ/μC chimeras), RGS proteins decreased δ/μC-mediated AC inhibition, but none had significant effects on that via μ/δC receptor. Thus, the C-terminal domains of the receptors are critical for the differential effects of RGS proteins, which may be due to differences in receptor - G protein - RGS protein interactions in signaling complexes. PMID:17433292

  16. Reishi immuno-modulation protein induces interleukin-2 expression via protein kinase-dependent signaling pathways within human T cells.

    PubMed

    Hsu, Hsien-Yeh; Hua, Kuo-Feng; Wu, Wei-Chi; Hsu, Jason; Weng, Shih-Ting; Lin, Tsai-Leng; Liu, Chun-Yi; Hseu, Ruey-Shyang; Huang, Ching-Tsan

    2008-04-01

    Ganoderma lucidum, a medicinal fungus is thought to possess and enhance a variety of human immune functions. An immuno-modulatory protein, Ling Zhi-8 (LZ-8) isolated from G. lucidum exhibited potent mitogenic effects upon human peripheral blood lymphocytes (PBL). However, LZ-8-mediated signal transduction in the regulation of interleukin-2 (IL-2) gene expression within human T cells is largely unknown. Here we cloned the LZ-8 gene of G. lucidum, and expressed the recombinant LZ-8 protein (rLZ-8) by means of a yeast Pichia pastoris protein expression system. We found that rLZ-8 induces IL-2 gene expression via the Src-family protein tyrosine kinase (PTK), via reactive oxygen species (ROS), and differential protein kinase-dependent pathways within human primary T cells and cultured Jurkat T cells. In essence, we have established the nature of the rLZ-8-mediated signal-transduction pathways, such as PTK/protein kinase C (PKC)/ROS, PTK/PLC/PKCalpha/ERK1/2, and PTK/PLC/PKCalpha/p38 pathways in the regulation of IL-2 gene expression within human T cells. Our current results of analyzing rLZ-8-mediated signal transduction in T cells might provide a potential application for rLZ-8 as a pharmacological immune-modulating agent.

  17. Modulating protein behaviors on responsive surface by external electric fields: A molecular dynamics study

    NASA Astrophysics Data System (ADS)

    Xie, Yun; Pan, Yufang; Zhang, Rong; Liang, Ying; Li, Zhanchao

    2015-01-01

    Molecular dynamics simulations were employed to investigate the modulation of protein behaviors on the electrically responsive zwitterionic phosphorylcholine self-assembled monolayers (PC-SAMs). Results show that PC-SAMs could sensitively respond to the applied electric fields and exhibit three states with different charge distributions, namely both the negatively charged phosphate groups and the positively charged choline groups are exposed to the solution in the absence of electric fields (state 1), phosphate groups exposed in the presence of positive electric fields (state 2), and choline groups exposed in the presence of negative electric fields (state 3). Under state 1, the adsorption of Cyt c on the PC-SAM is reversible and the orientations of Cyt c are randomly distributed. Under state 2, the adsorption of Cyt c is enhanced due to the electrostatic attractions between the exposed phosphate groups and the positively charged protein; when adsorbed on the PC-SAMs, Cyt c tends to adopt the orientation with the heme plane perpendicular to the surface plane, and the percentage of this orientation increases as the field strength rises up. Under state 3, the adsorption of Cyt c is retarded because of the electrostatic repulsions between the exposed choline groups and the protein; however, if the gaps between PC chains are large enough, Cyt c could insert into the PC-SAM and access the phosphate groups after overcoming a slight energy barrier. Under three states, the basic backbone structures of Cyt c are well kept within the simulation time since the conformation of Cyt c is mainly affected by the surface-generated electric fields, whose strengths are modulated by the external electric fields and are not strong enough to deform protein. The results indicate the possibility of regulating protein behaviors, including promoting or retarding protein adsorption and regulating protein orientations, on responsive surfaces by applying electric fields on the surfaces without

  18. Molecular and cellular factors control signal transduction via switchable allosteric modulator proteins (SAMPs).

    PubMed

    Babel, Heiko; Bischofs, Ilka B

    2016-04-27

    Rap proteins from Bacilli directly target response regulators of bacterial two-component systems and modulate their activity. Their effects are controlled by binding of signaling peptides to an allosteric site. Hence Raps exemplify a class of monomeric signaling receptors, which we call switchable allosteric modulator proteins (SAMPs). These proteins have potential applications in diverse biomedical and biotechnical settings, but a quantitative understanding of the impact of molecular and cellular factors on signal transduction is lacking. Here we introduce mathematical models that elucidate how signals are propagated though the network upon receptor stimulation and control the level of active response regulator. Based on a systematic parameter analysis of the models, we show that key features of the dose-response behavior at steady state are controlled either by the molecular properties of the modulator or the signaling context. In particular, we find that the biochemical activity (i.e. non-enzymatic vs. enzymatic) and allosteric properties of the modulator control the response amplitude. The Hill coefficient and the EC50 are controlled in addition by the relative ligand affinities. By tuning receptor properties, either graded or more switch-like (memory-less) response functions can be fashioned. Furthermore, we show that other contextual factors (e.g. relative concentrations of network components and kinase activity) have a substantial impact on the response, and we predict that there exists a modulator concentration which is optimal for response amplitude. We discuss data on Rap-Phr systems in B. subtilis to show how our models can contribute to an integrated view of SAMP signaling by combining biochemical, structural and physiological insights. Our results also suggest that SAMPs could be evolved or engineered to implement diverse response behaviors. However-without additional regulatory controls-they can generate rather variable cellular outputs.

  19. Peptide interactions stabilize and restructure human papillomavirus type 16 E6 to interact with p53.

    PubMed

    Ansari, Tina; Brimer, Nicole; Vande Pol, Scott B

    2012-10-01

    Human papillomavirus type 16 (HPV-16) E6 (16E6) binds the E3 ubiquitin ligase E6AP and p53, thereby targeting degradation of p53 (M. Scheffner, B. A. Werness, J. M. Huibregtse, A. J. Levine, and P. M. Howley, Cell 63:1129-1136, 1990). Here we show that minimal 16E6-binding LXXLL peptides reshape 16E6 to confer p53 interaction and stabilize 16E6 in vivo but that degradation of p53 by 16E6 requires E6AP expression. These experiments establish a general mechanism for how papillomavirus E6 binding to LXXLL peptides reshapes E6 to then act as an adapter molecule.

  20. Peptide Interactions Stabilize and Restructure Human Papillomavirus Type 16 E6 To Interact with p53

    PubMed Central

    Ansari, Tina; Brimer, Nicole

    2012-01-01

    Human papillomavirus type 16 (HPV-16) E6 (16E6) binds the E3 ubiquitin ligase E6AP and p53, thereby targeting degradation of p53 (M. Scheffner, B. A. Werness, J. M. Huibregtse, A. J. Levine, and P. M. Howley, Cell 63:1129–1136, 1990). Here we show that minimal 16E6-binding LXXLL peptides reshape 16E6 to confer p53 interaction and stabilize 16E6 in vivo but that degradation of p53 by 16E6 requires E6AP expression. These experiments establish a general mechanism for how papillomavirus E6 binding to LXXLL peptides reshapes E6 to then act as an adapter molecule. PMID:22896608

  1. Single-step affinity purification of recombinant proteins using a self-excising module from Neisseria meningitidis FrpC

    PubMed Central

    Sadilkova, Lenka; Osicka, Radim; Sulc, Miroslav; Linhartova, Irena; Novak, Petr; Sebo, Peter

    2008-01-01

    Purification of recombinant proteins is often a challenging process involving several chromatographic steps that must be optimized for each target protein. Here, we developed a self-excising module allowing single-step affinity chromatography purification of untagged recombinant proteins. It consists of a 250-residue-long self-processing module of the Neisseria meningitidis FrpC protein with a C-terminal affinity tag. The N terminus of the module is fused to the C terminus of a target protein of interest. Upon binding of the fusion protein to an affinity matrix from cell lysate and washing out contaminating proteins, site-specific cleavage of the Asp–Pro bond linking the target protein to the self-excising module is induced by calcium ions. This results in the release of the target protein with only a single aspartic acid residue added at the C terminus, while the self-excising affinity module remains trapped on the affinity matrix. The system was successfully tested with several target proteins, including glutathione-S-transferase, maltose-binding protein, β-galactosidase, chloramphenicol acetyltransferase, and adenylate cyclase, and two different affinity tags, chitin-binding domain or poly-His. Moreover, it was demonstrated that it can be applied as an alternative to two currently existing systems, based on the self-splicing intein of Saccharomyces cerevisiae and sortase A of Staphylococcus aureus. PMID:18662906

  2. Tat is a multifunctional viral protein that modulates cellular gene expression and functions.

    PubMed

    Clark, Evan; Nava, Brenda; Caputi, Massimo

    2017-02-07

    The human immunodeficiency virus type I (HIV-1) has developed several strategies to condition the host environment to promote viral replication and spread. Viral proteins have evolved to perform multiple functions, aiding in the replication of the viral genome and modulating the cellular response to the infection. Tat is a small, versatile, viral protein that controls transcription of the HIV genome, regulates cellular gene expression and generates a permissive environment for viral replication by altering the immune response and facilitating viral spread to multiple tissues. Studies carried out utilizing biochemical, cellular, and genomic approaches show that the expression and activity of hundreds of genes and multiple molecular networks are modulated by Tat via multiple mechanisms.

  3. Protein Mass-Modulated Effects in the Catalytic Mechanism of Dihydrofolate Reductase: Beyond Promoting Vibrations

    PubMed Central

    2015-01-01

    The role of fast protein dynamics in enzyme catalysis has been of great interest in the past decade. Recent “heavy enzyme” studies demonstrate that protein mass-modulated vibrations are linked to the energy barrier for the chemical step of catalyzed reactions. However, the role of fast dynamics in the overall catalytic mechanism of an enzyme has not been addressed. Protein mass-modulated effects in the catalytic mechanism of Escherichia coli dihydrofolate reductase (ecDHFR) are explored by isotopic substitution (13C, 15N, and non-exchangeable 2H) of the wild-type ecDHFR (l-DHFR) to generate a vibrationally perturbed “heavy ecDHFR” (h-DHFR). Steady-state, pre-steady-state, and ligand binding kinetics, intrinsic kinetic isotope effects (KIEint) on the chemical step, and thermal unfolding experiments of both l- and h-DHFR show that the altered protein mass affects the conformational ensembles and protein–ligand interactions, but does not affect the hydride transfer at physiological temperatures (25–45 °C). Below 25 °C, h-DHFR shows altered transition state (TS) structure and increased barrier-crossing probability of the chemical step compared with l-DHFR, indicating temperature-dependent protein vibrational coupling to the chemical step. Protein mass-modulated vibrations in ecDHFR are involved in TS interactions at cold temperatures and are linked to dynamic motions involved in ligand binding at physiological temperatures. Thus, mass effects can affect enzymatic catalysis beyond alterations in promoting vibrations linked to chemistry. PMID:24820793

  4. Docosahexaenoic acid induces the degradation of HPV E6/E7 oncoproteins by activating the ubiquitin–proteasome system

    PubMed Central

    Jing, K; Shin, S; Jeong, S; Kim, S; Song, K-S; Park, J-H; Heo, J-Y; Seo, K-S; Park, S-K; Kweon, G-R; Wu, T; Park, J-I; Lim, K

    2014-01-01

    The oncogenic human papillomavirus (HPV) E6/E7 proteins are essential for the onset and maintenance of HPV-associated malignancies. Here, we report that activation of the cellular ubiquitin–proteasome system (UPS) by the omega-3 fatty acid, docosahexaenoic acid (DHA), leads to proteasome-mediated degradation of E6/E7 viral proteins and the induction of apoptosis in HPV-infected cancer cells. The increases in UPS activity and degradation of E6/E7 oncoproteins were associated with DHA-induced overproduction of mitochondrial reactive oxygen species (ROS). Exogenous oxidative stress and pharmacological induction of mitochondrial ROS showed effects similar to those of DHA, and inhibition of ROS production abolished UPS activation, E6/E7 viral protein destabilization, and apoptosis. These findings identify a novel role for DHA in the regulation of UPS and viral proteins, and provide evidence for the use of DHA as a mechanistically unique anticancer agent for the chemoprevention and treatment of HPV-associated tumors. PMID:25393480

  5. 78 FR 42530 - Prospective Grant of an Exclusive License: Human Papillomavirus 16 E2 and E6 Peptides for...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-07-16

    ... generated from amino acid positions 69-77 (ALQAIELQL) and 138-147 (YICEEASVTV) bind to HLA.A2 and elicit CD8... peptides of 0-11 amino acids in length comprising contiguous HPV 18 E6 amino acid sequences) protein that...

  6. FAT10 protein binds to polyglutamine proteins and modulates their solubility.

    PubMed

    Nagashima, Yu; Kowa, Hisatomo; Tsuji, Shoji; Iwata, Atsushi

    2011-08-26

    Expansion of polyglutamine (pQ) chain by expanded CAG repeat causes dominantly inherited neurodegeneration such as Huntington disease, dentatorubral-pallidoluysian atrophy (DRPLA), and numbers of other spinocerebellar ataxias. Expanded pQ disrupts the stability of the pQ-harboring protein and increases its susceptibility to aggregation. Aggregated pQ protein is recognized by the ubiquitin proteasome system, and the enzyme ubiquitin ligase covalently attaches ubiquitin, which serves as a degradation signal by the proteasome. However, accumulation of the aggregated proteins in the diseased brain suggests insufficient degradation machinery. Ubiquitin has several functionally related proteins that are similarly attached to target proteins through its C terminus glycine residue. They are called ubiquitin-like molecules, and some of them are similarly related to the protein degradation pathway. One of the ubiquitin-like molecules, FAT10, is known to accelerate protein degradation through a ubiquitin-independent manner, but its role in pQ aggregate degradation is completely unknown. Thus we investigated its role in a Huntington disease cellular model and found that FAT10 molecules were covalently attached to huntingtin through their C terminus glycine. FAT10 binds preferably to huntingtin with a short pQ chain and completely aggregated huntingtin was FAT10-negative. In addition, ataxin-1,3 and DRPLA proteins were both positive for FAT10, and aggregation enhancement was observed upon FAT10 knockdown. These findings were similar to those for huntingtin. Our new finding will provide a new role for FAT10 in the pathogenesis of polyglutamine diseases.

  7. Surface Density of the Hendra G Protein Modulates Hendra F Protein-Promoted Membrane Fusion: Role for Hendra G Protein Trafficking and Degradation

    PubMed Central

    Whitman, Shannon D.; Dutch, Rebecca Ellis

    2007-01-01

    Hendra virus, like most paramyxoviruses, requires both a fusion (F) and attachment (G) protein for promotion of cell-cell fusion. Recent studies determined that Hendra F is proteolytically processed by the cellular protease cathepsin L after endocytosis. This unique cathepsin L processing results in a small percentage of Hendra F on the cell surface. To determine how the surface densities of the two Hendra glycoproteins affect fusion promotion, we performed experiments that varied the levels of glycoproteins expressed in transfected cells. Using two different fusion assays, we found a marked increase in fusion when expression of the Hendra G protein was increased, with a 1:1 molar ratio of Hendra F:G on the cell surface resulting in optimal membrane fusion. Our results also showed that Hendra G protein levels are modulated by both more rapid protein turnover and slower protein trafficking than is seen for Hendra F. PMID:17328935

  8. Surface density of the Hendra G protein modulates Hendra F protein-promoted membrane fusion: Role for Hendra G protein trafficking and degradation

    SciTech Connect

    Whitman, Shannon D.; Dutch, Rebecca Ellis . E-mail: rdutc2@uky.edu

    2007-07-05

    Hendra virus, like most paramyxoviruses, requires both a fusion (F) and attachment (G) protein for promotion of cell-cell fusion. Recent studies determined that Hendra F is proteolytically processed by the cellular protease cathepsin L after endocytosis. This unique cathepsin L processing results in a small percentage of Hendra F on the cell surface. To determine how the surface densities of the two Hendra glycoproteins affect fusion promotion, we performed experiments that varied the levels of glycoproteins expressed in transfected cells. Using two different fusion assays, we found a marked increase in fusion when expression of the Hendra G protein was increased, with a 1:1 molar ratio of Hendra F:G on the cell surface resulting in optimal membrane fusion. Our results also showed that Hendra G protein levels are modulated by both more rapid protein turnover and slower protein trafficking than is seen for Hendra F.

  9. Transcriptional profiling of Vero E6 cells over-expressing SARS-CoV S2 subunit: Insights on viral regulation of apoptosis and proliferation

    SciTech Connect

    Yeung, Y.-S. Yip, C.-W. Hon, C.-C. Chow, Ken Y.C. Ma, Iris C.M. Zeng Fanya Leung, Frederick C.C.

    2008-02-05

    We have previously demonstrated that over-expression of spike protein (S) of severe acute respiratory syndrome coronavirus (SARS-CoV) or its C-terminal subunit (S2) is sufficient to induce apoptosis in vitro. To further investigate the possible roles of S2 in SARS-CoV-induced apoptosis and pathogenesis of SARS, we characterized the host expression profiles induced upon S2 over-expression in Vero E6 cells by oligonucleotide microarray analysis. Possible activation of mitochondrial apoptotic pathway in S2 expressing cells was suggested, as evidenced by the up-regulation of cytochrome c and down-regulation of the Bcl-2 family anti-apoptotic members. Inhibition of Bcl-2-related anti-apoptotic pathway was further supported by the diminution of S2-induced apoptosis in Vero E6 cells over-expressing Bcl-xL. In addition, modulation of CCN E2 and CDKN 1A implied the possible control of cell cycle arrest at G1/S phase. This study is expected to extend our understanding on the pathogenesis of SARS at a molecular level.

  10. Design of Protein-Peptide Interaction Modules for Assembling Supramolecular Structures in Vivo and in Vitro.

    PubMed

    Speltz, Elizabeth B; Nathan, Aparna; Regan, Lynne

    2015-09-18

    Synthetic biology and protein origami both require protein building blocks that behave in a reliable, predictable fashion. In particular, we require protein interaction modules with known specificity and affinity. Here, we describe three designed TRAP (Tetratricopeptide Repeat Affinity Protein)-peptide interaction pairs that are functional in vivo. We show that each TRAP binds to its cognate peptide and exhibits low cross-reactivity with the peptides bound by the other TRAPs. In addition, we demonstrate that the TRAP-peptide interactions are functional in many cellular contexts. In extensions of these designs, we show that the binding affinity of a TRAP-peptide pair can be systematically varied. The TRAP-peptide pairs we present thus represent a powerful set of new building blocks that are suitable for a variety of applications.

  11. Modulation of heat-shock protein 27 (Hsp27) anti-apoptotic activity by methylglyoxal modification.

    PubMed

    Sakamoto, Hiroshi; Mashima, Tetsuo; Yamamoto, Kazuo; Tsuruo, Takashi

    2002-11-29

    Methylglyoxal (MG) is one of the side-products in glycolysis, and it reacts with proteins under physiological conditions. Here, we identified heat-shock protein 27 (Hsp27) as a major MG-modified protein in cells. MG modification of Hsp27 selectively occurs at Arg-188 to form argpyrimidine, and mutation in the residue represses the formation of a large oligomer. This modification process is essential to its repressing activity for cytochrome c-mediated caspase activation. Inhibition of MG modification of Hsp27 causes sensitization of the cells to anti-tumor drug-induced apoptosis. Thus, MG is a novel modulator of cell survival by directly incorporating with the specific protein residue.

  12. Modulation of Rho GTPases and the actin cytoskeleton by Yersinia outer proteins (Yops).

    PubMed

    Aepfelbacher, M; Heesemann, J

    2001-09-01

    Pathogenic species of the genus Yersinia employ a type III secretion apparatus to inject up to six effector proteins (Yersinia outer proteins; Yops) into host cells. Thereby yersiniae disarm the immune cell system of the host to proliferate extracellularly. At least four of the Yop effectors (YopE, YpkA/YopO, YopT and YopH) are involved in the rearrangement of the actin cytoskeleton: YopE, YopT and YpkA/YopO modulate the activity of actin-regulating Rho GTP-binding proteins, whereas YopH dephosphorylates phospho-tyrosine residues in focal adhesion proteins. In this review we will focus on recent evidence implicating Rho GTPases and the actin cytoskeleton as major targets of Yersinia Yops.

  13. Protein Interactions of the MLL PHD Fingers Modulate MLL Target Gene Regulation in Human Cells

    PubMed Central

    Fair, Keri; Anderson, Melanie; Bulanova, Elena; Mi, Huaifeng; Tropschug, Maximilian; Diaz, Manuel O.

    2001-01-01

    The PHD fingers of the human MLL and Drosophila trx proteins have strong amino acid sequence conservation but their function is unknown. We have determined that these fingers mediate homodimerization and binding of MLL to Cyp33, a nuclear cyclophilin. These two proteins interact in vitro and in vivo in mammalian cells and colocalize at specific nuclear subdomains. Overexpression of the Cyp33 protein in leukemia cells results in altered expression of HOX genes that are targets for regulation by MLL. These alterations are suppressed by cyclosporine and are not observed in cell lines that express a mutant MLL protein without PHD fingers. These results suggest that binding of Cyp33 to MLL modulates its effects on the expression of target genes. PMID:11313484

  14. Protein interactions of the MLL PHD fingers modulate MLL target gene regulation in human cells.

    PubMed

    Fair, K; Anderson, M; Bulanova, E; Mi, H; Tropschug, M; Diaz, M O

    2001-05-01

    The PHD fingers of the human MLL and Drosophila trx proteins have strong amino acid sequence conservation but their function is unknown. We have determined that these fingers mediate homodimerization and binding of MLL to Cyp33, a nuclear cyclophilin. These two proteins interact in vitro and in vivo in mammalian cells and colocalize at specific nuclear subdomains. Overexpression of the Cyp33 protein in leukemia cells results in altered expression of HOX genes that are targets for regulation by MLL. These alterations are suppressed by cyclosporine and are not observed in cell lines that express a mutant MLL protein without PHD fingers. These results suggest that binding of Cyp33 to MLL modulates its effects on the expression of target genes.

  15. SENP1-modulated sumoylation regulates retinoblastoma protein (RB) and Lamin A/C interaction and stabilization.

    PubMed

    Sharma, P; Kuehn, M R

    2016-12-15

    The retinoblastoma tumor suppressor protein (RB) plays a critical role in cell proliferation and differentiation and its inactivation is a frequent underlying factor in tumorigenesis. While the regulation of RB function by phosphorylation is well studied, proteasome-mediated RB protein degradation is emerging as an important regulatory mechanism. Although our understanding of RB turnover is currently limited, there is evidence that the nuclear lamina filament protein Lamin A/C protects RB from proteasomal degradation. Here we show that SUMO1 conjugation of RB and Lamin A/C is modulated by the SUMO protease SENP1 and that sumoylation of both proteins is required for their interaction. Importantly, this SUMO1-dependent complex protects both RB and Lamin A/C from proteasomal turnover.

  16. An allosteric modulator to control endogenous G protein-coupled receptors with light.

    PubMed

    Pittolo, Silvia; Gómez-Santacana, Xavier; Eckelt, Kay; Rovira, Xavier; Dalton, James; Goudet, Cyril; Pin, Jean-Philippe; Llobet, Artur; Giraldo, Jesús; Llebaria, Amadeu; Gorostiza, Pau

    2014-10-01

    Controlling drug activity with light offers the possibility of enhancing pharmacological selectivity with spatial and temporal regulation, thus enabling highly localized therapeutic effects and precise dosing patterns. Here we report on the development and characterization of what is to our knowledge the first photoswitchable allosteric modulator of a G protein-coupled receptor. Alloswitch-1 is selective for the metabotropic glutamate receptor mGlu5 and enables the optical control of endogenous mGlu5 receptors.

  17. Trypanosoma cruzi modulates gene expression of plasma membrane repair-related proteins.

    PubMed

    Brígido, Rebecca Tavares E Silva; Tavares, Paula Cristina Brígido; Santos, Marlus Alves Dos; Santos, Júlia de Gouveia; Souza, Maria Aparecida de; Goulart, Isabela Maria Bernardes; Silva, Claudio Vieira da

    2017-10-01

    Plasma membrane injury and repair is particularly prevalent in muscle cells. Here, we aimed to verify dysferlin, acid sphingomyelinase and transcriptional factor EB gene expression during Trypanosoma cruzi infection in vitro and in vivo. Our results showed that the parasite modulates gene expression of these proteins in a way dependent on the number of plasma membrane interacting parasites and in a rapamycin-sensitive manner. Copyright © 2016 Elsevier B.V. All rights reserved.

  18. Encapsulated Cellular Implants for Recombinant Protein Delivery and Therapeutic Modulation of the Immune System

    PubMed Central

    Lathuilière, Aurélien; Mach, Nicolas; Schneider, Bernard L.

    2015-01-01

    Ex vivo gene therapy using retrievable encapsulated cellular implants is an effective strategy for the local and/or chronic delivery of therapeutic proteins. In particular, it is considered an innovative approach to modulate the activity of the immune system. Two recently proposed therapeutic schemes using genetically engineered encapsulated cells are discussed here: the chronic administration of monoclonal antibodies for passive immunization against neurodegenerative diseases and the local delivery of a cytokine as an adjuvant for anti-cancer vaccines. PMID:26006227

  19. Pili-like proteins of Akkermansia muciniphila modulate host immune responses and gut barrier function

    PubMed Central

    Reunanen, Justus; Meijerink, Marjolein; Pietilä, Taija E.; Kainulainen, Veera; Klievink, Judith; Huuskonen, Laura; Aalvink, Steven; Skurnik, Mikael; Boeren, Sjef; Satokari, Reetta; Mercenier, Annick; Palva, Airi; Smidt, Hauke; de Vos, Willem M.; Belzer, Clara

    2017-01-01

    Gut barrier function is key in maintaining a balanced response between the host and its microbiome. The microbiota can modulate changes in gut barrier as well as metabolic and inflammatory responses. This highly complex system involves numerous microbiota-derived factors. The gut symbiont Akkermansia muciniphila is positively correlated with a lean phenotype, reduced body weight gain, amelioration of metabolic responses and restoration of gut barrier function by modulation of mucus layer thickness. However, the molecular mechanisms behind its metabolic and immunological regulatory properties are unexplored. Herein, we identify a highly abundant outer membrane pili-like protein of A. muciniphila MucT that is directly involved in immune regulation and enhancement of trans-epithelial resistance. The purified Amuc_1100 protein and enrichments containing all its associated proteins induced production of specific cytokines through activation of Toll-like receptor (TLR) 2 and TLR4. This mainly leads to high levels of IL-10 similar to those induced by the other beneficial immune suppressive microorganisms such as Faecalibacterium prausnitzii A2-165 and Lactobacillus plantarum WCFS1. Together these results indicate that outer membrane protein composition and particularly the newly identified highly abundant pili-like protein Amuc_1100 of A. muciniphila are involved in host immunological homeostasis at the gut mucosa, and improvement of gut barrier function. PMID:28249045

  20. Systematic identification of transcriptional regulatory modules from protein–protein interaction networks

    PubMed Central

    Diez, Diego; Hutchins, Andrew Paul; Miranda-Saavedra, Diego

    2014-01-01

    Transcription factors (TFs) combine with co-factors to form transcriptional regulatory modules (TRMs) that regulate gene expression programs with spatiotemporal specificity. Here we present a novel and generic method (rTRM) for the reconstruction of TRMs that integrates genomic information from TF binding, cell type-specific gene expression and protein–protein interactions. rTRM was applied to reconstruct the TRMs specific for embryonic stem cells (ESC) and hematopoietic stem cells (HSC), neural progenitor cells, trophoblast stem cells and distinct types of terminally differentiated CD4+ T cells. The ESC and HSC TRM predictions were highly precise, yielding 77 and 96 proteins, of which ∼75% have been independently shown to be involved in the regulation of these cell types. Furthermore, rTRM successfully identified a large number of bridging proteins with known roles in ESCs and HSCs, which could not have been identified using genomic approaches alone, as they lack the ability to bind specific DNA sequences. This highlights the advantage of rTRM over other methods that ignore PPI information, as proteins need to interact with other proteins to form complexes and perform specific functions. The prediction and experimental validation of the co-factors that endow master regulatory TFs with the capacity to select specific genomic sites, modulate the local epigenetic profile and integrate multiple signals will provide important mechanistic insights not only into how such TFs operate, but also into abnormal transcriptional states leading to disease. PMID:24137002

  1. Stk38 Modulates Rbm24 Protein Stability to Regulate Sarcomere Assembly in Cardiomyocytes

    PubMed Central

    Liu, Jing; Kong, Xu; Yew Mun, Lee; Meng Kai, Zhang; Li Yan, Guo; Lin, Yu; Lim, Teck Kwang; Lin, Qingsong; Xu, Xiu Qin

    2017-01-01

    RNA-binding protein Rbm24 is a key regulator of heart development and required for sarcomere assembly and heart contractility. Yet, its underlying mechanism remains unclear. Here, we link serine/threonine kinase 38 (Stk38) signaling to the regulation of Rbm24 by showing that Rbm24 phosphorylation and its function could be modulated by Stk38. Using co-immunoprecipitation coupled with mass spectrometry technique, we identified Stk38 as an endogenous binding partner of Rbm24. Stk38 knockdown resulted in decreased Rbm24 protein level in cardiomyocytes. Further studies using Stk38 kinase inhibitor or activator showed that Rbm24 protein stability was regulated in a kinase activity-dependent manner. Deficiency of Stk38 caused reduction of sarcomere proteins and disarrangement of sarcomere, suggesting that Stk38 is essential for Rbm24 to regulate sarcomere assembly. Our results revealed that Stk38 kinase catalyzes the phosphorylation of Rbm24 during sarcomerogensis and this orchestrates accurate sarcomere alignment. This furthers our understanding of the regulatory mechanism of cardiac sarcomere assembly in both physiologic and pathologic contexts, and uncovers a potential novel pathway to cardiomyopathy through modulating the Stk38/Rbm24 protein activity. PMID:28322254

  2. Protein conformational modulation by photons: a mechanism for laser treatment effects.

    PubMed

    Liebert, Ann D; Bicknell, Brian T; Adams, Roger D

    2014-03-01

    Responsiveness to low-level laser treatment (LLTT) at a wavelength of 450-910 nm has established it as an effective treatment of medical, veterinary and dental chronic pain, chronic inflammation conditions (arthritis and macular degeneration), wound repair, and lymphoedema, yet the mechanisms underlying the effectiveness of LLLT remain unclear. However, there is now sufficient evidence from recent research to propose an integrated model of LLLT action. The hypothesis presented in this paper is that external applications of photons (through laser at an appropriate dose) modulates the nervous system through an integrated mechanism. This stimulated mechanism involves protein-to-protein interaction, where two or more proteins bind together to facilitate molecular processes, including modification of proteins by members of SUMO (small ubiquitin-related modifier proteins) and also protein phosphorylation and tyrosination. SUMO has been shown to have a role in multiple nuclear and perinuclear targets, including ion channels, and in the maintenance of telomeres and the post-translational modification of genes. The consequence of laser application in treatment, therefore, can be seen as influencing the transmission of neural information via an integrated and rapid modulation of ion channels, achieved through both direct action on photo-acceptors (such as cytochrome c-oxidase) and through indirect modulation via enzymes, including tyrosine hydroxylase (TH), tyrosine kinases and tyrosine kinase receptors. This exogenous action then facilitates an existing photonic biomodulation mechanism within the body, and initiates ion channel modulation both in the periphery and the central nervous system (CNS). Evidence indicates that the ion channel modulation functions predominately through the potassium channels, including two pore leak channels (K2P), which act as signal integrators from the periphery to the cortex. Photonic action also transforms SUMOylation processes at the cell

  3. NTTMUNSW BioC modules for recognizing and normalizing species and gene/protein mentions.

    PubMed

    Dai, Hong-Jie; Singh, Onkar; Jonnagaddala, Jitendra; Su, Emily Chia-Yu

    2016-01-01

    In recent years, the number of published biomedical articles has increased as researchers have focused on biological domains to investigate the functions of biological objects, such as genes and proteins. However, the ambiguous nature of genes and their products have rendered the literature more complex for readers and curators of molecular interaction databases. To address this challenge, a normalization technique that can link variants of biological objects to a single, standardized form was applied. In this work, we developed a species normalization module, which recognizes species names and normalizes them to NCBI Taxonomy IDs. Unlike most previous work, which ignored the prefix of a gene name that represents an abbreviation of the species name to which the gene belongs, the recognition results of our module include the prefixed species. The developed species normalization module achieved an overall F-score of 0.954 on an instance-level species normalization corpus. For gene normalization, two separate modules were respectively employed to recognize gene mentions and normalize those mentions to their Entrez Gene IDs by utilizing a multistage normalization algorithm developed for processing full-text articles. All of the developed modules are BioC-compatible .NET framework libraries and are publicly available from the NuGet gallery.Database URL: https://sites.google.com/site/hjdairesearch/Projects/isn-corpus.

  4. E6 and E7 fusion immunoglobulin from human papilloma virus 16 induces dendritic cell maturation and antigen specific activation of T helper 1 response.

    PubMed

    Kim, Sang-Hoon; Hur, Yu Jin; Lee, Suk Jun; Kim, Sang Joon; Park, Chung-Gyu; Oh, Yu-Koung; Jung, Woon-Won; Seo, Jong Bok; Nam, Myung Hee; Choi, Inho; Chun, Taehoon

    2011-04-01

    Human papilloma virus (HPV) 16 causes cervical cancer. Induction of oncogenesis by HPV 16 is primarily dependent on the function of E6 and E7 proteins, which inactivate the function of p53 and pRB, respectively. Thus, blocking the activity of the E6 and E7 proteins from HPV 16 is critical to inhibiting oncogenesis during infection. We have expressed and purified soluble HPV 16 E6 and E7 fusion immunoglobulin (Ig), which were combined with the constant region of an Ig heavy chain, in a mammalian system. To assess whether soluble E6 and E7 fusion Igs induce effective cellular immune responses, immature dendritic cells (DCs) were treated with these fusion proteins. Soluble E6 and E7 fusion Igs effectively induced maturation of DCs. Furthermore, immunization with soluble E6 and E7 fusion Igs in mice resulted in antigen-specific activation of T helper 1 (Th1) cells. This is the first comprehensive study to show the molecular basis of how soluble HPV 16 E6 or E7 fusion Igs induces Th1 responses through the maturation of DCs. In addition, we show that DC therapy using soluble HPV E6 and E7 fusion Igs may be a valuable tool for controlling the progress of cervical cancer.

  5. Membrane Curvature Sensing by Amphipathic Helices Is Modulated by the Surrounding Protein Backbone

    PubMed Central

    Doucet, Christine M.; Esmery, Nina; de Saint-Jean, Maud; Antonny, Bruno

    2015-01-01

    Membrane curvature is involved in numerous biological pathways like vesicle trafficking, endocytosis or nuclear pore complex assembly. In addition to its topological role, membrane curvature is sensed by specific proteins, enabling the coordination of biological processes in space and time. Amongst membrane curvature sensors are the ALPS (Amphipathic Lipid Packing Sensors). ALPS motifs are short peptides with peculiar amphipathic properties. They are found in proteins targeted to distinct curved membranes, mostly in the early secretory pathway. For instance, the ALPS motif of the golgin GMAP210 binds trafficking vesicles, while the ALPS motif of Nup133 targets nuclear pores. It is not clear if, besides curvature sensitivity, ALPS motifs also provide target specificity, or if other domains in the surrounding protein backbone are involved. To elucidate this aspect, we studied the subcellular localization of ALPS motifs outside their natural protein context. The ALPS motifs of GMAP210 or Nup133 were grafted on artificial fluorescent probes. Importantly, ALPS motifs are held in different positions and these contrasting architectures were mimicked by the fluorescent probes. The resulting chimeras recapitulated the original proteins localization, indicating that ALPS motifs are sufficient to specifically localize proteins. Modulating the electrostatic or hydrophobic content of Nup133 ALPS motif modified its avidity for cellular membranes but did not change its organelle targeting properties. In contrast, the structure of the backbone surrounding the helix strongly influenced targeting. In particular, introducing an artificial coiled-coil between ALPS and the fluorescent protein increased membrane curvature sensitivity. This coiled-coil domain also provided membrane curvature sensitivity to the amphipathic helix of Sar1. The degree of curvature sensitivity within the coiled-coil context remains correlated to the natural curvature sensitivity of the helices. This suggests

  6. Membrane Curvature Sensing by Amphipathic Helices Is Modulated by the Surrounding Protein Backbone.

    PubMed

    Doucet, Christine M; Esmery, Nina; de Saint-Jean, Maud; Antonny, Bruno

    2015-01-01

    Membrane curvature is involved in numerous biological pathways like vesicle trafficking, endocytosis or nuclear pore complex assembly. In addition to its topological role, membrane curvature is sensed by specific proteins, enabling the coordination of biological processes in space and time. Amongst membrane curvature sensors are the ALPS (Amphipathic Lipid Packing Sensors). ALPS motifs are short peptides with peculiar amphipathic properties. They are found in proteins targeted to distinct curved membranes, mostly in the early secretory pathway. For instance, the ALPS motif of the golgin GMAP210 binds trafficking vesicles, while the ALPS motif of Nup133 targets nuclear pores. It is not clear if, besides curvature sensitivity, ALPS motifs also provide target specificity, or if other domains in the surrounding protein backbone are involved. To elucidate this aspect, we studied the subcellular localization of ALPS motifs outside their natural protein context. The ALPS motifs of GMAP210 or Nup133 were grafted on artificial fluorescent probes. Importantly, ALPS motifs are held in different positions and these contrasting architectures were mimicked by the fluorescent probes. The resulting chimeras recapitulated the original proteins localization, indicating that ALPS motifs are sufficient to specifically localize proteins. Modulating the electrostatic or hydrophobic content of Nup133 ALPS motif modified its avidity for cellular membranes but did not change its organelle targeting properties. In contrast, the structure of the backbone surrounding the helix strongly influenced targeting. In particular, introducing an artificial coiled-coil between ALPS and the fluorescent protein increased membrane curvature sensitivity. This coiled-coil domain also provided membrane curvature sensitivity to the amphipathic helix of Sar1. The degree of curvature sensitivity within the coiled-coil context remains correlated to the natural curvature sensitivity of the helices. This suggests

  7. Amino-terminal precursor sequence modulates canine distemper virus fusion protein function.

    PubMed

    von Messling, Veronika; Cattaneo, Roberto

    2002-05-01

    The fusion (F) proteins of most paramyxoviruses are classical type I glycoproteins with a short hydrophobic leader sequence closely following the translation initiation codon. The predicted reading frame of the canine distemper virus (CDV) F protein is more complex, with a short hydrophobic sequence beginning 115 codons downstream of the first AUG. To verify if the sequence between the first AUG and the hydrophobic region is translated, we produced a specific antiserum that indeed detected a short-lived F protein precursor that we named PreF(0). A peptide resulting from PreF(0) cleavage was identified and named Pre, and its half-life was measured to be about 30 min. PreF(0) cleavage was completed before proteolytic activation of F(0) into its F(1) and F(2) subunits by furin. To test the hypothesis that the Pre peptide may influence protein activity, we compared the function of F proteins synthesized with that peptide to that of F proteins synthesized with a shorter amino-terminal signal sequence. F proteins synthesized with the Pre peptide were more stable and less active. Thus, the Pre peptide modulates the function of the CDV F protein. Interestingly, a distinct two-hit activation process has been recently described for human respiratory syncytial virus, another paramyxovirus.

  8. Exploring protein structure and dynamics through a project-oriented biochemistry laboratory module.

    PubMed

    Lipchock, James M; Ginther, Patrick S; Douglas, Bonnie B; Bird, Kelly E; Patrick Loria, J

    2017-09-01

    Here, we present a 10-week project-oriented laboratory module designed to provide a course-based undergraduate research experience in biochemistry that emphasizes the importance of biomolecular structure and dynamics in enzyme function. This module explores the impact of mutagenesis on an important active site loop for a biomedically-relevant human enzyme, protein tyrosine phosphatase 1B (PTP1B). Over the course of the semester students guide their own mutant of PTP1B from conception to characterization in a cost-effective manner and gain exposure to fundamental techniques in biochemistry, including site-directed DNA mutagenesis, bacterial recombinant protein expression, affinity column purification, protein quantitation, SDS-PAGE, and enzyme kinetics. This project-based approach allows an instructor to simulate a research setting and prepare students for productive research beyond the classroom. Potential modifications to expand or contract this module are also provided. © 2017 by The International Union of Biochemistry and Molecular Biology, 45(5):403-410, 2017. © 2017 The International Union of Biochemistry and Molecular Biology.

  9. Modulator of Apoptosis 1 (MOAP-1) Is a Tumor Suppressor Protein Linked to the RASSF1A Protein*

    PubMed Central

    Law, Jennifer; Salla, Mohamed; Zare, Alaa; Wong, Yoke; Luong, Le; Volodko, Natalia; Svystun, Orysya; Flood, Kayla; Lim, Jonathan; Sung, Miranda; Dyck, Jason R. B.; Tan, Chong Teik; Su, Yu-Chin; Yu, Victor C.; Mackey, John; Baksh, Shairaz

    2015-01-01

    Modulator of apoptosis 1 (MOAP-1) is a BH3-like protein that plays key roles in cell death or apoptosis. It is an integral partner to the tumor suppressor protein, Ras association domain family 1A (RASSF1A), and functions to activate the Bcl-2 family pro-apoptotic protein Bax. Although RASSF1A is now considered a bona fide tumor suppressor protein, the role of MOAP-1 as a tumor suppressor protein has yet to be determined. In this study, we present several lines of evidence from cancer databases, immunoblotting of cancer cells, proliferation, and xenograft assays as well as DNA microarray analysis to demonstrate the role of MOAP-1 as a tumor suppressor protein. Frequent loss of MOAP-1 expression, in at least some cancers, appears to be attributed to mRNA down-regulation and the rapid proteasomal degradation of MOAP-1 that could be reversed utilizing the proteasome inhibitor MG132. Overexpression of MOAP-1 in several cancer cell lines resulted in reduced tumorigenesis and up-regulation of genes involved in cancer regulatory pathways that include apoptosis (p53, Fas, and MST1), DNA damage control (poly(ADP)-ribose polymerase and ataxia telangiectasia mutated), those within the cell metabolism (IR-α, IR-β, and AMP-activated protein kinase), and a stabilizing effect on microtubules. The loss of RASSF1A (an upstream regulator of MOAP-1) is one of the earliest detectable epigenetically silenced tumor suppressor proteins in cancer, and we speculate that the additional loss of function of MOAP-1 may be a second hit to functionally compromise the RASSF1A/MOAP-1 death receptor-dependent pathway and drive tumorigenesis. PMID:26269600

  10. Markov state models provide insights into dynamic modulation of protein function.

    PubMed

    Shukla, Diwakar; Hernández, Carlos X; Weber, Jeffrey K; Pande, Vijay S

    2015-02-17

    CONSPECTUS: Protein function is inextricably linked to protein dynamics. As we move from a static structural picture to a dynamic ensemble view of protein structure and function, novel computational paradigms are required for observing and understanding conformational dynamics of proteins and its functional implications. In principle, molecular dynamics simulations can provide the time evolution of atomistic models of proteins, but the long time scales associated with functional dynamics make it difficult to observe rare dynamical transitions. The issue of extracting essential functional components of protein dynamics from noisy simulation data presents another set of challenges in obtaining an unbiased understanding of protein motions. Therefore, a methodology that provides a statistical framework for efficient sampling and a human-readable view of the key aspects of functional dynamics from data analysis is required. The Markov state model (MSM), which has recently become popular worldwide for studying protein dynamics, is an example of such a framework. In this Account, we review the use of Markov state models for efficient sampling of the hierarchy of time scales associated with protein dynamics, automatic identification of key conformational states, and the degrees of freedom associated with slow dynamical processes. Applications of MSMs for studying long time scale phenomena such as activation mechanisms of cellular signaling proteins has yielded novel insights into protein function. In particular, from MSMs built using large-scale simulations of GPCRs and kinases, we have shown that complex conformational changes in proteins can be described in terms of structural changes in key structural motifs or "molecular switches" within the protein, the transitions between functionally active and inactive states of proteins proceed via multiple pathways, and ligand or substrate binding modulates the flux through these pathways. Finally, MSMs also provide a theoretical

  11. Markov State Models Provide Insights into Dynamic Modulation of Protein Function

    PubMed Central

    2015-01-01

    Conspectus Protein function is inextricably linked to protein dynamics. As we move from a static structural picture to a dynamic ensemble view of protein structure and function, novel computational paradigms are required for observing and understanding conformational dynamics of proteins and its functional implications. In principle, molecular dynamics simulations can provide the time evolution of atomistic models of proteins, but the long time scales associated with functional dynamics make it difficult to observe rare dynamical transitions. The issue of extracting essential functional components of protein dynamics from noisy simulation data presents another set of challenges in obtaining an unbiased understanding of protein motions. Therefore, a methodology that provides a statistical framework for efficient sampling and a human-readable view of the key aspects of functional dynamics from data analysis is required. The Markov state model (MSM), which has recently become popular worldwide for studying protein dynamics, is an example of such a framework. In this Account, we review the use of Markov state models for efficient sampling of the hierarchy of time scales associated with protein dynamics, automatic identification of key conformational states, and the degrees of freedom associated with slow dynamical processes. Applications of MSMs for studying long time scale phenomena such as activation mechanisms of cellular signaling proteins has yielded novel insights into protein function. In particular, from MSMs built using large-scale simulations of GPCRs and kinases, we have shown that complex conformational changes in proteins can be described in terms of structural changes in key structural motifs or “molecular switches” within the protein, the transitions between functionally active and inactive states of proteins proceed via multiple pathways, and ligand or substrate binding modulates the flux through these pathways. Finally, MSMs also provide a

  12. Systematic expression profiling analysis mines dys-regulated modules in active tuberculosis based on re-weighted protein-protein interaction network and attract algorithm.

    PubMed

    Sun, Ying; Weng, Yan; Zhang, Ying; Yan, Xiang; Guo, Lei; Wang, Jia; Song, Xin; Yuan, Ying; Chang, Fu-Ye; Wang, Chun-Ling

    2017-03-18

    About 90% of tuberculosis (TB) patients latently infected with Mycobacterium tuberculosis (Mtb) show no symptoms, yet have a 10% chance in lifetime to progress active TB. Nevertheless, current diagnosis approaches need improvement in efficiency and sensitivity. The objective of this work was to detect potential signatures for active TB to further improve the understanding of the biological roles of functional modules involved in this disease. First, targeted networks of active TB and control groups were established via re-weighting protein-protein interaction (PPI) networks using Pearson's correlation coefficient (PCC). Candidate modules were detected from the targeted networks, and the modules with Jaccard score >0.7 were defined as attractors. After that, identification of dys-regulated modules was conducted from the attractors using attract method, Subsequently, gene oncology (GO) enrichment analyses were implemented for genes in the dys-regulated modules. We obtained 33 and 65 candidate modules from the targeted networks of control and active TB groups, respectively. Overall, 13 attractors were identified. Using the cut-off criteria of false discovery rate <0.05, there were 4 dys-regulated modules (Module 1, 2, 3, and 4). Based on the GO annotation results, genes in Modules 1, 2 and 4 were only involved in translation. Most genes in Module 1, 2 and 4 were associated with ribosomes. Accordingly, these dys-regulated modules might serve as potential biomarkers of active TB, facilitating the development for a more efficient, and sensitive diagnostic assay for active TB.

  13. Context-based retrieval of functional modules in protein-protein interaction networks.

    PubMed

    Dobay, Maria Pamela; Stertz, Silke; Delorenzi, Mauro

    2017-03-27

    Various techniques have been developed for identifying the most probable interactants of a protein under a given biological context. In this article, we dissect the effects of the choice of the protein-protein interaction network (PPI) and the manipulation of PPI settings on the network neighborhood of the influenza A virus (IAV) network, as well as hits in genome-wide small interfering RNA screen results for IAV host factors. We investigate the potential of context filtering, which uses text mining evidence linked to PPI edges, as a complement to the edge confidence scores typically provided in PPIs for filtering, for obtaining more biologically relevant network neighborhoods. Here, we estimate the maximum performance of context filtering to isolate a Kyoto Encyclopedia of Genes and Genomes (KEGG) network Ki from a union of KEGG networks and its network neighborhood. The work gives insights on the use of human PPIs in network neighborhood approaches for functional inference.

  14. Modulation of protein-protein interactions as a therapeutic strategy for the treatment of neurodegenerative tauopathies.

    PubMed

    Ballatore, C; Brunden, K R; Trojanowski, J Q; Lee, V M-Y; Smith, A B; Huryn, D M

    2011-01-01

    The recognition that malfunction of the microtubule (MT) associated protein tau is likely to play a defining role in the onset and/or progression of a number of neurodegenerative diseases, including Alzheimer's disease, has resulted in the initiation of drug discovery programs that target this protein. Tau is an endogenous MT-stabilizing agent that is highly expressed in the axons of neurons. The MT-stabilizing function of tau is essential for the axonal transport of proteins, neurotransmitters and other cellular constituents. Under pathological conditions, tau misfolding and aggregation results in axonal transport deficits that appear to have deleterious consequences for the affected neurons, leading to synapse dysfunction and, ultimately, neuronal loss. This review focuses on both progress and unresolved issues surrounding the development of novel therapeutics for the treatment of neurodegenerative tauopathies, which are based on (A) MT-stabilizing agents to compensate for the loss of normal tau function, and (B) small molecule inhibitors of tau aggregation.

  15. DENSE: efficient and prior knowledge-driven discovery of phenotype-associated protein functional modules

    PubMed Central

    2011-01-01

    Background Identifying cellular subsystems that are involved in the expression of a target phenotype has been a very active research area for the past several years. In this paper, cellular subsystem refers to a group of genes (or proteins) that interact and carry out a common function in the cell. Most studies identify genes associated with a phenotype on the basis of some statistical bias, others have extended these statistical methods to analyze functional modules and biological pathways for phenotype-relatedness. However, a biologist might often have a specific question in mind while performing such analysis and most of the resulting subsystems obtained by the existing methods might be largely irrelevant to the question in hand. Arguably, it would be valuable to incorporate biologist's knowledge about the phenotype into the algorithm. This way, it is anticipated that the resulting subsytems would not only be related to the target phenotype but also contain information that the biologist is likely to be interested in. Results In this paper we introduce a fast and theoretically guranteed method called DENSE (Dense and ENriched Subgraph Enumeration) that can take in as input a biologist's prior knowledge as a set of query proteins and identify all the dense functional modules in a biological network that contain some part of the query vertices. The density (in terms of the number of network egdes) and the enrichment (the number of query proteins in the resulting functional module) can be manipulated via two parameters γ and μ, respectively. Conclusion This algorithm has been applied to the protein functional association network of Clostridium acetobutylicum ATCC 824, a hydrogen producing, acid-tolerant organism. The algorithm was able to verify relationships known to exist in literature and also some previously unknown relationships including those with regulatory and signaling functions. Additionally, we were also able to hypothesize that some uncharacterized

  16. Modulation of Kaposi's sarcoma-associated herpesvirus interleukin-6 function by hypoxia-upregulated protein 1.

    PubMed

    Giffin, Louise; Yan, Feng; Ben Major, M; Damania, Blossom

    2014-08-01

    Kaposi's sarcoma-associated herpesvirus (KSHV, also called human herpesvirus 8) is linked to the development of Kaposi's sarcoma (KS), primary effusion lymphoma (PEL), and multicentric Castleman's disease (MCD). KSHV expresses several proteins that modulate host cell signaling pathways. One of these proteins is viral interleukin-6 (vIL-6), which is a homolog of human IL-6 (hIL-6). vIL-6 is able to prevent apoptosis and promote proinflammatory signaling, angiogenesis, and cell proliferation. Although it can be secreted, vIL-6 is mainly an intracellular protein that is retained in the endoplasmic reticulum (ER). We performed affinity purification and mass spectrometry to identify novel vIL-6 binding partners and found that a cellular ER chaperone, hypoxia-upregulated protein 1 (HYOU1), interacts with vIL-6. Immunohistochemical staining reveals that both PEL and KS tumor tissues express significant amounts of HYOU1. We also show that HYOU1 increases endogenous vIL-6 protein levels and that HYOU1 facilitates vIL-6-induced JAK/STAT signaling, migration, and survival in endothelial cells. Furthermore, our data suggest that HYOU1 also modulates vIL-6's ability to induce CCL2, a chemokine involved in cell migration. Finally, we investigated the impact of HYOU1 on cellular hIL-6 signaling. Collectively, our data indicate that HYOU1 is important for vIL-6 function and may play a role in the pathogenesis of KSHV-associated cancers. KSHV vIL-6 is detectable in all KSHV-associated malignancies and promotes tumorigenesis and inflammation. We identified a cellular protein, called hypoxia-upregulated protein 1 (HYOU1), that interacts with KSHV vIL-6 and is present in KSHV-infected tumors. Our data suggest that HYOU1 facilitates the vIL-6-induced signaling, migration, and survival of endothelial cells. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

  17. Brucella Modulates Secretory Trafficking via Multiple Type IV Secretion Effector Proteins

    PubMed Central

    Myeni, Sebenzile; Child, Robert; Ng, Tony W.; Kupko, John J.; Wehrly, Tara D.; Porcella, Stephen F.; Knodler, Leigh A.; Celli, Jean

    2013-01-01

    The intracellular pathogenic bacterium Brucella generates a replicative vacuole (rBCV) derived from the endoplasmic reticulum via subversion of the host cell secretory pathway. rBCV biogenesis requires the expression of the Type IV secretion system (T4SS) VirB, which is thought to translocate effector proteins that modulate membrane trafficking along the endocytic and secretory pathways. To date, only a few T4SS substrates have been identified, whose molecular functions remain unknown. Here, we used an in silico screen to identify putative T4SS effector candidate proteins using criteria such as limited homology in other bacterial genera, the presence of features similar to known VirB T4SS effectors, GC content and presence of eukaryotic-like motifs. Using β-lactamase and CyaA adenylate cyclase reporter assays, we identified eleven proteins translocated into host cells by Brucella, five in a VirB T4SS-dependent manner, namely BAB1_0678 (BspA), BAB1_0712 (BspB), BAB1_0847 (BspC), BAB1_1671 (BspE) and BAB1_1948 (BspF). A subset of the translocated proteins targeted secretory pathway compartments when ectopically expressed in HeLa cells, and the VirB effectors BspA, BspB and BspF inhibited protein secretion. Brucella infection also impaired host protein secretion in a process requiring BspA, BspB and BspF. Single or combined deletions of bspA, bspB and bspF affected Brucella ability to replicate in macrophages and persist in the liver of infected mice. Taken together, these findings demonstrate that Brucella modulates secretory trafficking via multiple T4SS effector proteins that likely act coordinately to promote Brucella pathogenesis. PMID:23950720

  18. Polypeptide Modulators of Caspase Recruitment Domain (CARD)-CARD-mediated Protein-Protein Interactions*

    PubMed Central

    Palacios-Rodríguez, Yadira; García-Laínez, Guillermo; Sancho, Mónica; Gortat, Anna; Orzáez, Mar; Pérez-Payá, Enrique

    2011-01-01

    The caspase recruitment domain (CARD) is present in a large number of proteins. Initially, the CARD was recognized as part of the caspase activation machinery. CARD-CARD interactions play a role in apoptosis and are responsible for the Apaf-1-mediated activation of procaspase-9 in the apoptosome. CARD-containing proteins mediate the inflammasome-dependent activation of proinflammatory caspase-1. More recently, new roles for CARD-containing proteins have been reported in signaling pathways associated with immune responses. The functional role of CARD-containing proteins and CARDs in coordinating apoptosis and inflammatory and immune responses is not completely understood. We have explored the putative cross-talk between apoptosis and inflammation by analyzing the modulatory activity on both the Apaf-1/procaspase-9 interaction and the inflammasome-mediated procaspase-1 activation of CARD-derived polypeptides. To this end, we analyzed the activity of individual recombinant CARDs, rationally designed CARD-derived peptides, and peptides derived from phage display. PMID:22065589

  19. Activation of human papillomavirus type 18 E6-E7 oncogene expression by transcription factor Sp1.

    PubMed Central

    Hoppe-Seyler, F; Butz, K

    1992-01-01

    The human papillomavirus 18 (HPV18) E6 and E7 proteins are considered to be primarily responsive for the transforming activity of the virus. In order to analyse the molecular mechanisms resulting in viral oncoprotein expression, it is necessary to identify the factors involved in the transcriptional regulation of the E6/E7 genes. Here we define by gel retardation experiments a sequence aberrant Sp1 binding site present in the promoter proximal part of the viral transcriptional control region (Upstream Regulatory Region, URR). Functional analyses employing transient reporter assays reveal that this Sp1 element is required for an efficient stimulation of the HPV18 E6/E7-promoter. Mutation of the Sp1 element in the natural context of the HPV18 URR leads to a strong decrease in the activity of the E6/E7-promoter in several cell lines. The magnitude of reduction varies between different cell types and is higher in cell lines of epithelial origin when compared with nonepithelial cells. Cotransfection assays using Sp1 expression vector systems further define the promoter proximal HPV18 Sp1 binding motif as a functional Sp1 element in vivo and show that its integrity is essential for the stimulation of the E6/E7-promoter by augmented levels of Sp1. These results indicate, that the cellular transcription factor Sp1 plays an important role for the stimulation of the E6/E7-promoter by the viral URR and represents a major determinant for the expression of HPV18 transforming genes E6 and E7. Images PMID:1336181

  20. Use of folding modulators to improve heterologous protein production in Escherichia coli

    PubMed Central

    Kolaj, Olga; Spada, Stefania; Robin, Sylvain; Wall, J Gerard

    2009-01-01

    Despite the fundamental importance of E. coli in the manufacture of a wide range of biotechnological and biomedical products, extensive process and/or target optimisation is routinely required in order to achieve functional yields in excess of low mg/l levels. Molecular chaperones and folding catalysts appear to present a panacea for problems of heterologous protein folding in the organism, due largely to their broad substrate range compared with, e.g., protein-specific mutagenesis approaches. Painstaking investigation of chaperone overproduction has, however, met with mixed – and largely unpredictable – results to date. The past 5 years have nevertheless seen an explosion in interest in exploiting the native folding modulators of E. coli, and particularly cocktails thereof, driven largely by the availability of plasmid systems that facilitate simultaneous, non-rational screening of multiple chaperones during recombinant protein expression. As interest in using E. coli to produce recombinant membrane proteins and even glycoproteins grows, approaches to reduce aggregation, delay host cell lysis and optimise expression of difficult-to-express recombinant proteins will become even more critical over the coming years. In this review, we critically evaluate the performance of molecular chaperones and folding catalysts native to E. coli in improving functional production of heterologous proteins in the bacterium and we discuss how they might best be exploited to provide increased amounts of correctly-folded, active protein for biochemical and biophysical studies. PMID:19173718

  1. Protein thermal denaturation is modulated by central residues in the protein structure network.

    PubMed

    Souza, Valquiria P; Ikegami, Cecília M; Arantes, Guilherme M; Marana, Sandro R

    2016-03-01

    Network structural analysis, known as residue interaction networks or graphs (RIN or RIG, respectively) or protein structural networks or graphs (PSN or PSG, respectively), comprises a useful tool for detecting important residues for protein function, stability, folding and allostery. In RIN, the tertiary structure is represented by a network in which residues (nodes) are connected by interactions (edges). Such structural networks have consistently presented a few central residues that are important for shortening the pathways linking any two residues in a protein structure. To experimentally demonstrate that central residues effectively participate in protein properties, mutations were directed to seven central residues of the β-glucosidase Sfβgly (β-D-glucoside glucohydrolase; EC 3.2.1.21). These mutations reduced the thermal stability of the enzyme, as evaluated by changes in transition temperature (Tm ) and the denaturation rate at 45 °C. Moreover, mutations directed to the vicinity of a central residue also caused significant decreases in the Tm of Sfβgly and clearly increased the unfolding rate constant at 45 °C. However, mutations at noncentral residues or at surrounding residues did not affect the thermal stability of Sfβgly. Therefore, the data reported in the present study suggest that the perturbation of the central residues reduced the stability of the native structure of Sfβgly. These results are in agreement with previous findings showing that networks are robust, whereas attacks on central nodes cause network failure. Finally, the present study demonstrates that central residues underlie the functional properties of proteins. © 2016 Federation of European Biochemical Societies.

  2. IQGAP1 Binds to Yes-associated Protein (YAP) and Modulates Its Transcriptional Activity *

    PubMed Central

    Sayedyahossein, Samar; Li, Zhigang; Hedman, Andrew C.; Morgan, Chase J.

    2016-01-01

    During development, the Hippo signaling pathway regulates key physiological processes, such as control of organ size, regeneration, and stem cell biology. Yes-associated protein (YAP) is a major transcriptional co-activator of the Hippo pathway. The scaffold protein IQGAP1 interacts with more than 100 binding partners to integrate diverse signaling pathways. In this study, we report that IQGAP1 binds to YAP and modulates its activity. IQGAP1 and YAP co-immunoprecipitated from cells. In vitro analysis with pure proteins demonstrated a direct interaction between IQGAP1 and YAP. Analysis with multiple fragments of each protein showed that the interaction occurs via the IQ domain of IQGAP1 and the TEAD-binding domain of YAP. The interaction between IQGAP1 and YAP has functional effects. Knock-out of endogenous IQGAP1 significantly increased the formation of nuclear YAP-TEAD complexes. Transcription assays were performed with IQGAP1-null mouse embryonic fibroblasts and HEK293 cells with IQGAP1 knockdown by CRISPR/Cas9. Quantification demonstrated that YAP-TEAD-mediated transcription in cells lacking IQGAP1 was significantly greater than in control cells. These data reveal that IQGAP1 binds to YAP and modulates its co-transcriptional function, suggesting that IQGAP1 participates in Hippo signaling. PMID:27440047

  3. IQGAP1 Binds to Yes-associated Protein (YAP) and Modulates Its Transcriptional Activity.

    PubMed

    Sayedyahossein, Samar; Li, Zhigang; Hedman, Andrew C; Morgan, Chase J; Sacks, David B

    2016-09-09

    During development, the Hippo signaling pathway regulates key physiological processes, such as control of organ size, regeneration, and stem cell biology. Yes-associated protein (YAP) is a major transcriptional co-activator of the Hippo pathway. The scaffold protein IQGAP1 interacts with more than 100 binding partners to integrate diverse signaling pathways. In this study, we report that IQGAP1 binds to YAP and modulates its activity. IQGAP1 and YAP co-immunoprecipitated from cells. In vitro analysis with pure proteins demonstrated a direct interaction between IQGAP1 and YAP. Analysis with multiple fragments of each protein showed that the interaction occurs via the IQ domain of IQGAP1 and the TEAD-binding domain of YAP. The interaction between IQGAP1 and YAP has functional effects. Knock-out of endogenous IQGAP1 significantly increased the formation of nuclear YAP-TEAD complexes. Transcription assays were performed with IQGAP1-null mouse embryonic fibroblasts and HEK293 cells with IQGAP1 knockdown by CRISPR/Cas9. Quantification demonstrated that YAP-TEAD-mediated transcription in cells lacking IQGAP1 was significantly greater than in control cells. These data reveal that IQGAP1 binds to YAP and modulates its co-transcriptional function, suggesting that IQGAP1 participates in Hippo signaling.

  4. Application of TZERO calibrated modulated temperature differential scanning calorimetry to characterize model protein formulations.

    PubMed

    Badkar, Aniket; Yohannes, Paulos; Banga, Ajay

    2006-02-17

    The objective of this study was to evaluate the feasibility of using T(ZERO) modulated temperature differential scanning calorimetry (MDSC) as a novel technique to characterize protein solutions using lysozyme as a model protein and IgG as a model monoclonal antibody. MDSC involves the application of modulated heating program, along with the standard heating program that enables the separation of overlapping thermal transitions. Although characterization of unfolding transitions for protein solutions requires the application of high sensitive DSC, separation of overlapping transitions like aggregation and other exothermic events may be possible only by use of MDSC. A newer T(ZERO) calibrated MDSC model from TA instruments that has improved sensitivity than previous models was used. MDSC analysis showed total, reversing and non-reversing heat flow signals. Total heat flow signals showed a combination of melting endotherms and overlapping exothermic events. Under the operating conditions used, the melting endotherms were seen in reversing heat flow signal while the exothermic events were seen in non-reversing heat flow signal. This enabled the separation of overlapping thermal transitions, improved data analysis and decreased baseline noise. MDSC was used here for characterization of lysozyme solutions, but its feasibility for characterizing therapeutic protein solutions needs further assessment.

  5. Almond protein hydrolysate fraction modulates the expression of proinflammatory cytokines and enzymes in activated macrophages.

    PubMed

    Udenigwe, Chibuike C; Je, Jae-Young; Cho, Young-Sook; Yada, Rickey Y

    2013-04-30

    Simulated gastrointestinal treatment of almond proteins with pepsin and pancreatic proteases resulting in 16.6% degree of hydrolysis or 1.33 milliequivalent leucine per g protein yielded a hydrolysate that modulated excessive nitric oxide production in lipopolysaccharide-activated RAW264.7 macrophages. After fractionation, a resulting fraction of molecular size > 5 kDa retained the nitric oxide modulatory effect observed initially in the crude hydrolysate. The high molecular size fraction was found to modulate levels of proinflammatory cytokines, interleukin (IL)-6, IL-1β, and tumour necrosis factor (TNF)-α in the activated cells. Immunoblotting analysis indicated that the hydrolysate fraction decreased the expression levels of inflammatory enzyme indicators, inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX)-2 in the activated cells. RT-PCR analysis showed that treatment of the activated cells with the hydrolysate fraction resulted in the inhibition of relative gene expressions of proinflammatory IL-6, IL-1β, TNF-α, iNOS and COX-2. These results indicate a potential application of almond protein hydrolysates against inflammatory conditions, and will contribute to delineating the possible contributions of proteins to health benefits attributed to almond consumption.

  6. Grb7 Protein Stability Modulated by Pin1 in Association with Cell Cycle Progression

    PubMed Central

    Tai, Yu-Ling; Tung, Li-Hsuan; Lin, Yu-Chi; Lu, Pei-Jung; Chu, Pei-Yu; Wang, Ming-Yang; Huang, Wei-Pang; Chen, Ko-Chien; Lee, Hsinyu; Shen, Tang-Long

    2016-01-01

    Growth factor receptor bound protein-7 (Grb7) is a multi-domain adaptor protein that is co-opted by numerous tyrosine kinases involved in various cellular signaling and functions. The molecular mechanisms underlying the regulation of Grb7 remain unclear. Here, we revealed a novel negative post-translational regulation of Grb7 by the peptidyl-prolyl cis/trans isomerase, Pin1. Our data show that phosphorylation of Grb7 protein on the Ser194-Pro motif by c-Jun N-terminal kinase facilitates its binding with the WW domain of Pin1. Subsequently, Grb7 is degraded by the ubiquitin- and proteasome-dependent proteolytic pathway. Indeed, we found that Pin1 exerts its peptidyl-prolyl cis/trans isomerase activity in the modulation of Grb7 protein stability in regulation of cell cycle progression at the G2-M phase. This study illustrates a novel regulatory mechanism in modulating Grb7-mediated signaling, which may take part in pathophysiological consequences. PMID:27658202

  7. Reversible protein phosphorylation modulates nucleotide excision repair of damaged DNA by human cell extracts.

    PubMed

    Ariza, R R; Keyse, S M; Moggs, J G; Wood, R D

    1996-02-01

    Nucleotide excision repair of DNA in mammalian cells uses more than 20 polypeptides to remove DNA lesions caused by UV light and other mutagens. To investigate whether reversible protein phosphorylation can significantly modulate this repair mechanism we studied the effect of specific inhibitors of Ser/Thr protein phosphatases. The ability of HeLa cell extracts to carry out nucleotide excision repair in vitro was highly sensitive to three toxins (okadaic acid, microcystin-LR and tautomycin), which block PP1- and PP2A-type phosphatases. Repair was more sensitive to okadaic acid than to tautomycin, suggesting the involvement of a PP2A-type enzyme, and was insensitive to inhibitor-2, which exclusively inhibits PP1-type enzymes. In a repair synthesis assay the toxins gave 70% inhibition of activity. Full activity could be restored to toxin-inhibited extracts by addition of purified PP2A, but not PP1. The p34 subunit of replication protein A was hyperphosphorylated in cell extracts in the presence of phosphatase inhibitors, but we found no evidence that this affected repair. In a coupled incision/synthesis repair assay okadaic acid decreased the production of incision intermediates in the repair reaction. The formation of 25-30mer oligonucleotides by dual incision during repair was also inhibited by okadaic acid and inhibition could be reversed with PP2A. Thus Ser/Thr- specific protein phosphorylation plays an important role in the modulation of nucleotide excision repair in vitro.

  8. Modulation of function in a minimalist heme-binding membrane protein.

    PubMed

    Shinde, Sandip; Cordova, Jeanine M; Woodrum, Brian W; Ghirlanda, Giovanna

    2012-04-01

    De novo designed heme-binding proteins have been used successfully to recapitulate features of natural hemoproteins. This approach has now been extended to membrane-soluble model proteins. Our group designed a functional hemoprotein, ME1, by engineering a bishistidine binding site into a natural membrane protein, glycophorin A (Cordova et al. in J Am Chem Soc 129:512-518, 2007). ME1 binds iron(III) protoporphyrin IX with submicromolar affinity, has a redox potential of -128 mV, and displays peroxidase activity. Here, we show the effect of aromatic residues in modulating the redox potential in the context of a membrane-soluble model system. We designed aromatic interactions with the heme through a single-point mutant, G25F, in which a phenylalanine is designed to dock against the porphyrin ring. This mutation results in roughly tenfold tighter binding to iron(III) protoporphyrin IX (K(d,app) = 6.5 × 10(-8) M), and lowers the redox potential of the cofactor to -172 mV. This work demonstrates that specific design features aimed at controlling the properties of bound cofactors can be introduced in a minimalist membrane hemoprotein model. The ability to modulate the redox potential of cofactors embedded in artificial membrane proteins is crucial for the design of electron transfer chains across membranes in functional photosynthetic devices.

  9. Modulating the midpoint potential of the [4Fe-4S] cluster of the nitrogenase Fe protein.

    PubMed

    Jang, S B; Seefeldt, L C; Peters, J W

    2000-02-01

    Protein-bound [FeS] clusters function widely in biological electron-transfer reactions, where their midpoint potentials control both the kinetics and thermodynamics of these reactions. The polarity of the protein environment around [FeS] clusters appears to contribute largely to modulating their midpoint potentials, with local protein dipoles and water dipoles largely defining the polarity. The function of the [4Fe-4S] cluster containing Fe protein in nitrogenase catalysis is, at least in part, to serve as the nucleotide-dependent electron donor to the MoFe protein which contains the sites for substrate binding and reduction. The ability of the Fe protein to function in this manner is dependent on its ability to adopt the appropriate conformation for productive interaction with the MoFe protein and on its ability to change redox potentials to provide the driving force required for electron transfer. Phenylalanine at position 135 is located near the [4Fe-4S] cluster of nitrogenase Fe protein and has been suggested by amino acid substitution studies to participate in defining both the midpoint potential and the nucleotide-induced changes in the [4Fe-4S] cluster. In the present study, the crystal structure of the Azotobacter vinelandii nitrogenase Fe protein variant having phenylalanine at position 135 substituted by tryptophan has been determined by X-ray diffraction methods and refined to 2.4 A resolution. A comparison of available Fe protein structures not only provides a structural basis for the more positive midpoint potential observed in the tryptophan substituted variant but also suggests a possible general mechanism by which the midpoint potential could be controlled by nucleotide interactions and nitrogenase complex formation.

  10. Modulation of neurosteroid potentiation by protein kinases at synaptic- and extrasynaptic-type GABAA receptors

    PubMed Central

    Adams, Joanna M.; Thomas, Philip; Smart, Trevor G.

    2015-01-01

    GABAA receptors are important for inhibition in the CNS where neurosteroids and protein kinases are potent endogenous modulators. Acting individually, these can either enhance or depress receptor function, dependent upon the type of neurosteroid or kinase and the receptor subunit combination. However, in vivo, these modulators probably act in concert to fine-tune GABAA receptor activity and thus inhibition, although how this is achieved remains unclear. Therefore, we investigated the relationship between these modulators at synaptic-type α1β3γ2L and extrasynaptic-type α4β3δ GABAA receptors using electrophysiology. For α1β3γ2L, potentiation of GABA responses by tetrahydro-deoxycorticosterone was reduced after inhibiting protein kinase C, and enhanced following its activation, suggesting this kinase regulates neurosteroid modulation. In comparison, neurosteroid potentiation was reduced at α1β3S408A,S409Aγ2L receptors, and unaltered by PKC inhibitors or activators, indicating that phosphorylation of β3 subunits is important for regulating neurosteroid activity. To determine whether extrasynaptic-type GABAA receptors were similarly modulated, α4β3δ and α4β3S408A,S409Aδ receptors were investigated. Neurosteroid potentiation was reduced at both receptors by the kinase inhibitor staurosporine. By contrast, neurosteroid-mediated potentiation at α4S443Aβ3S408A,S409Aδ receptors was unaffected by protein kinase inhibition, strongly suggesting that phosphorylation of α4 and β3 subunits is required for regulating neurosteroid activity at extrasynaptic receptors. Western blot analyses revealed that neurosteroids increased phosphorylation of β3S408,S409 implying that a reciprocal pathway exists for neurosteroids to modulate phosphorylation of GABAA receptors. Overall, these findings provide important insight into the regulation of GABAA receptors in vivo, and into the mechanisms by which GABAergic inhibitory transmission may be simultaneously tuned by

  11. Diversity-oriented synthetic strategy for developing a chemical modulator of protein-protein interaction

    NASA Astrophysics Data System (ADS)

    Kim, Jonghoon; Jung, Jinjoo; Koo, Jaeyoung; Cho, Wansang; Lee, Won Seok; Kim, Chanwoo; Park, Wonwoo; Park, Seung Bum

    2016-10-01

    Diversity-oriented synthesis (DOS) can provide a collection of diverse and complex drug-like small molecules, which is critical in the development of new chemical probes for biological research of undruggable targets. However, the design and synthesis of small-molecule libraries with improved biological relevance as well as maximized molecular diversity represent a key challenge. Herein, we employ functional group-pairing strategy for the DOS of a chemical library containing privileged substructures, pyrimidodiazepine or pyrimidine moieties, as chemical navigators towards unexplored bioactive chemical space. To validate the utility of this DOS library, we identify a new small-molecule inhibitor of leucyl-tRNA synthetase-RagD protein-protein interaction, which regulates the amino acid-dependent activation of mechanistic target of rapamycin complex 1 signalling pathway. This work highlights that privileged substructure-based DOS strategy can be a powerful research tool for the construction of drug-like compounds to address challenging biological targets.

  12. Modulation of neurite branching by protein phosphorylation in cultured rat hippocampal neurons.

    PubMed

    Audesirk, G; Cabell, L; Kern, M

    1997-09-20

    The control of branching of axons and dendrites is poorly understood. It has been hypothesized that branching may be produced by changes in the cytoskeleton [F.J. Diez-Guerra, J. Avila, MAP2 phosphorylation parallels dendrite arborization in hippocampal neurones in culture, NeuroReport 4 (1993) 412-419; P. Friedrich, A. Aszodi, MAP2: a sensitive cross-linker and adjustable spacer in dendritic architecture, FEBS Lett. 295 (1991) 5-9]. The assembly and stability of microtubules, which are prominent cytoskeletal elements in both axons and dendrites, are regulated by microtubule-associated proteins, including tau (predominantly found in axons) and MAP2 (predominantly found in dendrites). The phosphorylation state of tau and MAP2 modulates their interactions with microtubules. In their low-phosphorylation states, tau and MAP2 bind to microtubules and increase microtubule assembly and/or stability. Increased phosphorylation decreases these effects. Diez-Guerra and Avila [F.J. Diez-Guerra, J. Avila, MAP2 phosphorylation parallels dendrite arborization in hippocampal neurones in culture, NeuroReport 4 (1993) 412-419] found that protein phosphorylation correlates with neurite branching in cultured rat hippocampal neurons, and hypothesized that increased protein phosphorylation stimulates neurite branching. To test this hypothesis, we cultured rat hippocampal neurons in the presence of specific modulators of serine-threonine protein kinases and phosphatases. Inhibitors of several protein kinases, which would be expected to decrease protein phosphorylation, reduced branching. KT5720, an inhibitor of cyclic AMP-dependent protein kinase, and KN62, an inhibitor of Ca(2+)-calmodulin-dependent protein kinases, inhibited branching of both axons and dendrites. Calphostin C and chelerythrine, inhibitors of protein kinase C, inhibited branching of axons but not dendrites. Treatments that would be expected to increase protein phosphorylation, including inhibitors of protein

  13. Integrating Protein Engineering and Bioorthogonal Click Conjugation for Extracellular Vesicle Modulation and Intracellular Delivery

    PubMed Central

    Wang, Ming; Altinoglu, Sarah; Takeda, Yuji S.; Xu, Qiaobing

    2015-01-01

    Exosomes are small, cell-secreted vesicles that transfer proteins and genetic information between cells. This intercellular transmission regulates many physiological and pathological processes. Therefore, exosomes have emerged as novel biomarkers for disease diagnosis and as nanocarriers for drug delivery. Here, we report an easy-to-adapt and highly versatile methodology to modulate exosome composition and conjugate exosomes for intracellular delivery. Our strategy combines the metabolic labeling of newly synthesized proteins or glycan/glycoproteins of exosome-secreting cells with active azides and bioorthogonal click conjugation to modify and functionalize the exosomes. The azide-integrated can be conjugated to a variety of small molecules and proteins and can efficiently deliver conjugates into cells. The metabolic engineering of exosomes diversifies the chemistry of exosomes and expands the functions that can be introduced into exosomes, providing novel, powerful tools to study the roles of exosomes in biology and expand the biomedical potential of exosomes. PMID:26529317

  14. PIPE: a protein–protein interaction passage extraction module for BioCreative challenge

    PubMed Central

    Chu, Chun-Han; Su, Yu-Chen; Chen, Chien Chin; Hsu, Wen-Lian

    2016-01-01

    Identifying the interactions between proteins mentioned in biomedical literatures is one of the frequently discussed topics of text mining in the life science field. In this article, we propose PIPE, an interaction pattern generation module used in the Collaborative Biocurator Assistant Task at BioCreative V (http://www.biocreative.org/) to capture frequent protein-protein interaction (PPI) patterns within text. We also present an interaction pattern tree (IPT) kernel method that integrates the PPI patterns with convolution tree kernel (CTK) to extract PPIs. Methods were evaluated on LLL, IEPA, HPRD50, AIMed and BioInfer corpora using cross-validation, cross-learning and cross-corpus evaluation. Empirical evaluations demonstrate that our method is effective and outperforms several well-known PPI extraction methods. Database URL: PMID:27524807

  15. Interferons modulate mitogen-induced protein synthesis in airway smooth muscle

    PubMed Central

    Goncharova, Elena A.; Lim, Poay N.; Chisolm, Amelia; Fogle, Homer W.; Taylor, Jerome H.; Goncharov, Dmitry A.; Eszterhas, Andrew; Panettieri, Reynold A.

    2010-01-01

    Severe asthma is characterized by increased airway smooth muscle (ASM) mass due, in part, to ASM cell growth and contractile protein expression associated with increased protein synthesis. Little is known regarding the combined effects of mitogens and interferons on ASM cytosolic protein synthesis. We demonstrate that human ASM mitogens including PDGF, EGF, and thrombin stimulate protein synthesis. Surprisingly, pleiotropic cytokines IFN-β and IFN-γ, which inhibit ASM proliferation, also increased cytosolic protein content in ASM cells. Thus IFN-β alone significantly increased protein synthesis by 1.62 ± 0.09-fold that was further enhanced by EGF to 2.52 ± 0.17-fold. IFN-γ alone also stimulated protein synthesis by 1.91 ± 0.15-fold; treatment of cells with PDGF, EGF, and thrombin in the presence of IFN-γ stimulated protein synthesis by 2.24 ± 0.3-, 1.25 ± 0.17-, and 2.67 ± 0.34-fold, respectively, compared with growth factors alone. The mammalian target of rapamycin (mTOR)/S6 kinase 1 (S6K1) inhibition with rapamycin inhibited IFN- and EGF-induced protein synthesis, suggesting that IFN-induced protein synthesis is modulated by mTOR/S6K1 activation. Furthermore, overexpression of tumor suppressor protein tuberous sclerosis complex 2 (TSC2), which is an upstream negative regulator of mTOR/S6K1 signaling, also inhibited mitogen-induced protein synthesis in ASM cells. IFN-β and IFN-γ stimulated miR143/145 microRNA expression and increased SM α-actin accumulation but had little effect on ASM cell size. In contrast, EGF increased ASM cell size but had little effect on miR143/145 expression. Our data demonstrate that both IFNs and mitogens stimulate protein synthesis but have differential effects on cell size and contractile protein expression and suggest that combined effects of IFNs and mitogens may contribute to ASM cell growth, contractile protein expression, and ASM remodeling in asthma. PMID:20382746

  16. Phylogenetic and functional analysis of sequence variation of human papillomavirus type 31 E6 and E7 oncoproteins.

    PubMed

    Ferenczi, Annamária; Gyöngyösi, Eszter; Szalmás, Anita; László, Brigitta; Kónya, József; Veress, György

    2016-09-01

    High-risk human papillomaviruses (HPV) are the causative agents of cervical and other anogenital cancers as well as a subset of head and neck cancers. The E6 and E7 oncoproteins of HPV contribute to oncogenesis by associating with the tumour suppressor protein p53 and pRb, respectively. For HPV types 16 and 18, intratypic sequence variation was shown to have biological and clinical significance. The functional significance of sequence variation among HPV 31 variants was studied less intensively. HPV 31 variants belonging to different variant lineages were found to have differences in persistence and in the ability to cause high grade cervical intraepithelial neoplasia. In the present study, we started to explore the functional effects of natural sequence variation of HPV 31 E6 and E7 oncoproteins. The E6 variants were tested for their effects on p53 protein stability and transcriptional activity, while the E7 variants were tested for their effects on pRb protein level and also on the transcriptional activity of E2F transcription factors. HPV 31 E7 variants displayed uniform effects on pRb stability and also on the activity of E2F transcription factors. HPV 31 E6 variants had remarkable differences in the ability to inhibit the trans-activation function of p53 but not in the ability to induce the in vivo degradation of p53. Our results indicate that natural sequence variation of the HPV 31 E6 protein may be involved in the observed differences in the oncogenic potential between HPV 31 variants.

  17. Acute exercise modulates BDNF and pro-BDNF protein content in immune cells.

    PubMed

    Brunelli, Andrea; Dimauro, Ivan; Sgrò, Paolo; Emerenziani, Gian Pietro; Magi, Fiorenza; Baldari, Carlo; Guidetti, Laura; Di Luigi, Luigi; Parisi, Paolo; Caporossi, Daniela

    2012-10-01

    Although several studies have shown that immune cells stimulated by in vitro stress are capable to produce neurotrophins, there is still no evidence whether physiological stress, such as exercise, can modulate the in vivo levels of brain-derived neurotrophic factor (BDNF) in peripheral blood mononuclear cells (PBMCs). This work investigated whether acute exercise modulates the expression of BDNF, pro-BDNF, and p75(NTR) in the PBMCs of 10 healthy young men who performed a cycling incremental test to exhaustion (MAX) or exercised at individual anaerobic threshold (IAT). The PBMC expression of stress response proteins and the level of circulating BDNF, vascular endothelial growth growth factor, platelet-derived growth factor subunit B, basic fibroblast growth factor pro-inflammatory, and anti-inflammatory cytokines were analyzed as well. A major finding is that both sessions of acute exercise regulated the content of BDNF isoforms within PBMCs in a manner related to the physiological stress exerted. Although the pro-BDNF increased after both MAX and IAT protocols, BDNF showed a kinetics dependent on exercise type: MAX induced a 54% protein increase immediately after exercise, followed by a significant drop 60 min after its conclusion (38% lower than the baseline). Differently, in the IAT, BDNF increased significantly up to 75% from the baseline throughout the recovery phase. All physiological parameters, as well as the p75(NTR) receptor and the stress-inducible proteins, were also differently regulated by the two exercise conditions. These data supported the hypothesis that PBMCs might produce and secrete BDNF isoforms, as well as modulate the proteins p75(NTR) , Bcl-xL, hsp90, hsp27, and αB-crystallin, as part of the physiological stress response induced by acute exercise, offering a novel example of bidirectional interaction between nervous and immune systems.

  18. G-Protein-Coupled Receptor Kinase 2 as a Potential Modulator of the Hallmarks of Cancer.

    PubMed

    Nogués, Laura; Reglero, Clara; Rivas, Verónica; Neves, María; Penela, Petronila; Mayor, Federico

    2017-03-01

    Malignant features-such as sustained proliferation, refractoriness to growth suppressors, resistance to cell death or aberrant motility, and metastasis-can be triggered by a variety of mutations and signaling adaptations. Signaling nodes can act as cancer-associated factors by cooperating with oncogene-governed pathways or participating in compensatory transduction networks to strengthen tumor properties. G-protein-coupled receptor kinase 2 (GRK2) is arising as one of such nodes. Via its complex network of connections with other cellular proteins, GRK2 contributes to the modulation of basic cellular functions-such as cell proliferation, survival, or motility-and is involved in metabolic homeostasis, inflammation, or angiogenic processes. Moreover, altered GRK2 levels are starting to be reported in different tumoral contexts and shown to promote breast tumorigenesis or to trigger the tumoral angiogenic switch. The ability to modulate several of the hallmarks of cancer puts forward GRK2 as an oncomodifier, able to modulate carcinogenesis in a cell-type specific way.

  19. Modulation of the bilayer thickness of exocytic pathway membranes by membrane proteins rather than cholesterol

    NASA Astrophysics Data System (ADS)

    Mitra, Kakoli; Ubarretxena-Belandia, Iban; Taguchi, Tomohiko; Warren, Graham; Engelman, Donald M.

    2004-03-01

    A biological membrane is conceptualized as a system in which membrane proteins are naturally matched to the equilibrium thickness of the lipid bilayer. Cholesterol, in addition to lipid composition, has been suggested to be a major regulator of bilayer thickness in vivo because measurements in vitro have shown that cholesterol can increase the thickness of simple phospholipid/cholesterol bilayers. Using solution x-ray scattering, we have directly measured the average bilayer thickness of exocytic pathway membranes, which contain increasing amounts of cholesterol. The bilayer thickness of membranes of the endoplasmic reticulum, the Golgi, and the basolateral and apical plasma membranes, purified from rat hepatocytes, were determined to be 37.5 ± 0.4 Å, 39.5 ± 0.4 Å, 35.6 ± 0.6 Å, and 42.5 ± 0.3 Å, respectively. After cholesterol depletion using cyclodextrins, Golgi and apical plasma membranes retained their respective bilayer thicknesses whereas the bilayer thickness of the endoplasmic reticulum and the basolateral plasma membrane decreased by 1.0 Å. Because cholesterol was shown to have a marginal effect on the thickness of these membranes, we measured whether membrane proteins could modulate thickness. Protein-depleted membranes demonstrated changes in thickness of up to 5 Å, suggesting that (i) membrane proteins rather than cholesterol modulate the average bilayer thickness of eukaryotic cell membranes, and (ii) proteins and lipids are not naturally hydrophobically matched in some biological membranes. A marked effect of membrane proteins on the thickness of Escherichia coli cytoplasmic membranes, which do not contain cholesterol, was also observed, emphasizing the generality of our findings.

  20. Calcium-regulatory proteins as modulators of chemotherapy in human neuroblastoma.

    PubMed

    Florea, Ana-Maria; Varghese, Elizabeth; McCallum, Jennifer E; Mahgoub, Safa; Helmy, Irfan; Varghese, Sharon; Gopinath, Neha; Sass, Steffen; Theis, Fabian J; Reifenberger, Guido; Büsselberg, Dietrich

    2017-02-11

    Neuroblastoma (NB) is a pediatric cancer treated with poly-chemotherapy including platinum complexes (e.g. cisplatin (CDDP), carboplatin), DNA alkylating agents, and topoisomerase I inhibitors (e.g. topotecan (TOPO)). Despite aggressive treatment, NB may become resistant to chemotherapy. We investigated whether CDDP and TOPO treatment of NB cells interacts with the expression and function of proteins involved in regulating calcium signaling. Human neuroblastoma cell lines SH-SY5Y, IMR-32 and NLF were used to investigate the effects of CDDP and TOPO on cell viability, apoptosis, calcium homeostasis, and expression of selected proteins regulating intracellular calcium concentration ([Ca2+]i). In addition, the impact of pharmacological inhibition of [Ca2+]i-regulating proteins on neuroblastoma cell survival was studied. Treatment of neuroblastoma cells with increasing concentrations of CDDP (0.1-10 μM) or TOPO (0.1 nM-1 μM) induced cytotoxicity and increased apoptosis in a concentration- and time-dependent manner. Both drugs increased [Ca2+]i over time. Treatment with CDDP or TOPO also modified mRNA expression of selected genes encoding [Ca2+]i-regulating proteins. Differentially regulated genes included S100A6, ITPR1, ITPR3, RYR1 and RYR3. With FACS and confocal laser scanning microscopy experiments we validated their differential expression at the protein level. Importantly, treatment of neuroblastoma cells with pharmacological modulators of [Ca2+]i-regulating proteins in combination with CDDP or TOPO increased cytotoxicity. Thus, our results confirm an important role of calcium signaling in the response of neuroblastoma cells to chemotherapy and suggest [Ca2+]i modulation as a promising strategy for adjunctive treatment.

  1. Diversity of bone matrix adhesion proteins modulates osteoblast attachment and organization of actin cytoskeleton.

    PubMed

    Demais, V; Audrain, C; Mabilleau, G; Chappard, D; Baslé, M F

    2014-06-01

    Interaction of cells with extracellular matrix is an essential event for differentiation, proliferation and activity of osteoblasts. In bone, binding of osteoblasts to bone matrix is required to determine specific activities of the cells and to synthesize matrix bone proteins. Integrins are the major cell receptors involved in the cell linkage to matrix proteins such as fibronectin, type I collagen and vitronectin, via the RGD-sequences. In this study, cultures of osteoblast-like cells (Saos-2) were done on coated glass coverslips in various culture conditions: DMEM alone or DMEM supplemented with poly-L-lysine (PL), fetal calf serum (FCS), fibronectin (FN), vitronectin (VN) and type I collagen (Col-I). The aim of the study was to determine the specific effect of these bone matrix proteins on cell adherence and morphology and on the cytoskeleton status. Morphological characteristics of cultured cells were studied using scanning electron microscopy and image analysis. The heterogeneity of cytoskeleton was studied using fractal analysis (skyscrapers and blanket algorithms) after specific preparation of cells to expose the cytoskeleton. FAK and MAPK signaling pathways were studied by western blotting in these various culture conditions. Results demonstrated that cell adhesion was reduced with PL and VN after 240 min. After 60 min of adhesion, cytoskeleton organization was enhanced with FN, VN and Col-I. No difference in FAK phosphorylation was observed but MAPK phosphorylation was modulated by specific adhesion on extracellular proteins. These results indicate that culture conditions modulate cell adhesion, cytoskeleton organization and intracellular protein pathways according to extracellular proteins present for adhesion.

  2. Modulation of the bilayer thickness of exocytic pathway membranes by membrane proteins rather than cholesterol

    PubMed Central

    Mitra, Kakoli; Ubarretxena-Belandia, Iban; Taguchi, Tomohiko; Warren, Graham; Engelman, Donald M.

    2004-01-01

    A biological membrane is conceptualized as a system in which membrane proteins are naturally matched to the equilibrium thickness of the lipid bilayer. Cholesterol, in addition to lipid composition, has been suggested to be a major regulator of bilayer thickness in vivo because measurements in vitro have shown that cholesterol can increase the thickness of simple phospholipid/cholesterol bilayers. Using solution x-ray scattering, we have directly measured the average bilayer thickness of exocytic pathway membranes, which contain increasing amounts of cholesterol. The bilayer thickness of membranes of the endoplasmic reticulum, the Golgi, and the basolateral and apical plasma membranes, purified from rat hepatocytes, were determined to be 37.5 ± 0.4 Å, 39.5 ± 0.4 Å, 35.6 ± 0.6 Å, and 42.5 ± 0.3 Å, respectively. After cholesterol depletion using cyclodextrins, Golgi and apical plasma membranes retained their respective bilayer thicknesses whereas the bilayer thickness of the endoplasmic reticulum and the basolateral plasma membrane decreased by 1.0 Å. Because cholesterol was shown to have a marginal effect on the thickness of these membranes, we measured whether membrane proteins could modulate thickness. Protein-depleted membranes demonstrated changes in thickness of up to 5 Å, suggesting that (i) membrane proteins rather than cholesterol modulate the average bilayer thickness of eukaryotic cell membranes, and (ii) proteins and lipids are not naturally hydrophobically matched in some biological membranes. A marked effect of membrane proteins on the thickness of Escherichia coli cytoplasmic membranes, which do not contain cholesterol, was also observed, emphasizing the generality of our findings. PMID:15016920

  3. Isozyme-Specific Effects of Protein Kinase C in Pain Modulation

    PubMed Central

    Zhao, Chengshui; Leitges, Michael; Gereau, Robert W.

    2011-01-01

    Background Protein kinase C (PKC) is a family of serine/threonine kinases that contains more than 10 isozymes. Evidence suggests that PKC may play important roles in pain modulation, but the isozyme-specific effects of PKC on different aspects of pain modulation are not fully understood. We hypothesize that different PKC isozymes play different roles in different aspects of pain modulation. Methods The nociceptive behaviors of mice with deletion of PKC α, β, γ, or δ in multiple pain models were compared with their respective wild type littermates. Also, the morphine analgesia and the development of morphine tolerance in mice with deletion of PKC γ were compared with their respective wild type littermates. Results Thermal hyperalgesia induced by complete Freund’s adjuvant injection was significantly attenuated by the deletion of PKC β, γ or δ, but not PKC α. Deletion of PKC γ significantly attenuated neuropathic mechanical allodynia induced by spared nerve injury, whereas deletion of PKC α enhanced this allodynia. Baseline thermal and mechanical sensitivity, nociceptive behaviors induced by formalin, mechanical allodynia induced by complete Freund’s adjuvant injection, were not altered by deletion of PKC α, β, γ or δ. Finally, morphine analgesia and the development of morphine tolerance were not altered in PKC γ-deficient mice. Conclusions PKC plays isozyme-specific effects in pain modulation. PMID:22042410

  4. Different non-synonymous polymorphisms modulate the interaction of the WRN protein to its protein partners and its enzymatic activities

    PubMed Central

    Gagné, Jean-Philippe; Lachapelle, Sophie; Garand, Chantal; Tsofack, Serges P.; Coulombe, Yan; Caron, Marie-Christine; Poirier, Guy G.; Masson, Jean-Yves; Lebel, Michel

    2016-01-01

    Werner syndrome (WS) is characterized by the premature onset of several age-associated pathologies including cancer. The protein defective in WS patients (WRN) is a helicase/exonuclease involved in DNA replication and repair. Here, we present the results of a large-scale proteome analysis that has been undertaken to determine protein partners of different polymorphic WRN proteins found with relatively high prevalence in the human population. We expressed different fluorescently tagged-WRN (eYFP-WRN) variants in human 293 embryonic kidney cells (HEK293) and used a combination of affinity-purification and mass spectrometry to identify different compositions of WRN-associated protein complexes. We found that a WRN variant containing a phenylalanine residue at position 1074 and an arginine at position 1367 (eYFP-WRN(F-R)) possesses more affinity for DNA-PKc, KU86, KU70, and PARP1 than a variant containing a leucine at position 1074 and a cysteine at position 1367 (eYFP-WRN(L-C)). Such results were confirmed in a WRN-deficient background using WS fibroblasts. Interestingly, the exonuclase activity of WRN recovered from immunoprecipitated eYFP-WRN(L-C) variant was lower than the eYFP-WRN(F-R) in WS cells. Finally, HEK293 cells and WS fibroblasts overexpressing the eYFP-WRN(F-R) variant were more resistant to the benzene metabolite hydroquinone than cells expressing the eYFP-WRN(L-C) variant. These results indicate that the protein-protein interaction landscape of WRN is subject to modulation by polymorphic amino acids, a characteristic associated with distinctive cell survival outcome. PMID:27863399

  5. A new role for laminins as modulators of protein toxicity in Caenorhabditis elegans.

    PubMed

    Jensen, Louise T; Møller, Tine H; Larsen, Simon A; Jakobsen, Helle; Olsen, Anders

    2012-02-01

    Protein misfolding is a common theme in aging and several age-related diseases such as Alzheimer's and Parkinson's disease. The processes involved in the development of these diseases are many and complex. Here, we show that components of the basement membrane (BM), particularly laminin, affect protein integrity of the muscle cells they support. We knocked down gene expression of epi-1, a laminin α-chain, and found that this resulted in increased proteotoxicity in different Caenorhabditis elegans transgenic models, expressing aggregating proteins in the body wall muscle. The effect could partially be rescued by decreased insulin-like signaling, known to slow the aging process and the onset of various age-related diseases. Our data points to an underlying molecular mechanism involving proteasomal degradation and HSP-16 chaperone activity. Furthermore, epi-1-depleted animals had altered synaptic function and displayed hypersensitivity to both levamisole and aldicarb, an acetylcholine receptor agonist and an acetylcholinesterase inhibitor, respectively. Our results implicate the BM as an extracellular modulator of protein homeostasis in the adjacent muscle cells. This is in agreement with previous research showing that imbalance in neuromuscular signaling disturbs protein homeostasis in the postsynaptic cell. In our study, proteotoxicity may indeed be mediated by the neuromuscular junction which is part of the BM, where laminins are present in high concentration, ensuring the proper microenvironment for neuromuscular signaling. Laminins are evolutionarily conserved, and thus the BM may play a much more causal role in protein misfolding diseases than currently recognized.

  6. Large-scale modulation of thermodynamic protein folding barriers linked to electrostatics

    PubMed Central

    Halskau, Øyvind; Perez-Jimenez, Raul; Ibarra-Molero, Beatriz; Underhaug, Jarl; Muñoz, Victor; Martinez, Aurora; Sanchez-Ruiz, Jose M.

    2008-01-01

    Protein folding barriers, which range from zero to the tens of RT that result in classical two-state kinetics, are primarily determined by protein size and structural topology [Plaxco KW, Simons KT, Baker D (1998) J Mol Biol 277:985–994]. Here, we investigate the thermodynamic folding barriers of two relatively large proteins of the same size and topology: bovine α-lactalbumin (BLA) and hen-egg-white lysozyme (HEWL). From the analysis of differential scanning calorimetry experiments with the variable-barrier model [Muñoz V, Sanchez-Ruiz JM (2004) Proc Natl Acad Sci USA 101:17646–17651] we obtain a high barrier for HEWL and a marginal folding barrier for BLA. These results demonstrate a remarkable tuning range of at least 30 kJ/mol (i.e., five to six orders of magnitude in population) within a unique protein scaffold. Experimental and theoretical analyses on these proteins indicate that the surprisingly small thermodynamic folding barrier of BLA arises from the stabilization of partially unfolded conformations by electrostatic interactions. Interestingly, there is clear reciprocity between the barrier height and the biological function of the two proteins, suggesting that the marginal barrier of BLA is a product of natural selection. Electrostatic surface interactions thus emerge as a mechanism for the modulation of folding barriers in response to special functional requirements within a given structural fold. PMID:18550823

  7. Macromolecular Crowding Modulates Folding Mechanism of α/β Protein Apoflavodoxin

    PubMed Central

    Homouz, Dirar; Stagg, Loren; Wittung-Stafshede, Pernilla; Cheung, Margaret S.

    2009-01-01

    Abstract Protein dynamics in cells may be different from those in dilute solutions in vitro, because the environment in cells is highly concentrated with other macromolecules. This volume exclusion because of macromolecular crowding is predicted to affect both equilibrium and kinetic processes involving protein conformational changes. To quantify macromolecular crowding effects on protein folding mechanisms, we investigated the folding energy landscape of an α/β protein, apoflavodoxin, in the presence of inert macromolecular crowding agents, using in silico and in vitro approaches. By means of coarse-grained molecular simulations and topology-based potential interactions, we probed the effects of increased volume fractions of crowding agents (ϕc) as well as of crowding agent geometry (sphere or spherocylinder) at high ϕc. Parallel kinetic folding experiments with purified Desulfovibro desulfuricans apoflavodoxin in vitro were performed in the presence of Ficoll (sphere) and Dextran (spherocylinder) synthetic crowding agents. In conclusion, we identified the in silico crowding conditions that best enhance protein stability, and discovered that upon manipulation of the crowding conditions, folding routes experiencing topological frustrations can be either