Journal of Biological Chemistry (J Biol Chem)

Publications in this journal

• Article: Differential Role of PTEN Phosphatase in Chemotactic Growth Cone Guidance

  Steven J. Henle, Lucas P. Carlstrom, Thomas R. Cheever, John R. Henley
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  ABSTRACT: Negatively targeting the tumor suppressor and phosphoinositide phosphatase PTEN promotes axon re-growth after injury. How PTEN functions in axon guidance has remained unknown. Here we report the differential role of PTEN in chemotactic guidance of axonal growth cones. Downregulating PTEN expression in Xenopus laevis spinal neurons selectively abolished growth cone chemorepulsion but permitted chemoattraction. These findings persisted during cAMP-dependent switching of turning behaviors. Live-cell imaging using a GFP biosensor revealed rapid PTEN-dependent depression of phosphatidylinositol 3,4,5-trisphosphate (PIP3) levels in the growth cone induced by the repellent myelin-associated glycoprotein (MAG). Moreover, downregulating PTEN expression blocked negative remodeling of β1-integrin adhesions triggered by MAG, yet permitted integrin clustering by a positive chemotropic treatment. Thus, PTEN negatively regulates growth cone PIP3 levels and mediates chemorepulsion, whereas chemoattraction is PTEN independent. Regenerative therapies targeting PTEN may therefore suppress growth cone repulsion to soluble cues while permitting attractive guidance, an essential feature for re-forming functional neural circuits.
  Journal of Biological Chemistry 06/2013;
• Article: Translation Initiation Requires Cell Division Cycle 123 (Cdc123) to Facilitate Biogenesis of the Eukaryotic Initiation Factor 2 (eIF2).

  Angelika F Perzlmaier, Frank Richter, Wolfgang Seufert
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  ABSTRACT: The eukaryotic translation initiation factor 2 (eIF2) is central to the onset of protein synthesis and its modulation in response to physiological demands. eIF2, a heterotrimeric G-protein, is activated by guanine nucleotide exchange to deliver the initiator methionyl-tRNA to the ribosome. Here we report that assembly of the eIF2 complex in vivo depends on Cdc123, a cell proliferation protein conserved among eukaryotes. Mutations of CDC123 in budding yeast reduced the association of eIF2 subunits, diminished polysome levels, and increased GCN4 expression indicating that Cdc123 is critical for eIF2 activity. Cdc123 bound the unassembled eIF2γ subunit, but not the eIF2 complex, and the C-terminal domain III region of eIF2γ was both necessary and sufficient for Cdc123 binding. Alterations of the binding site revealed a strict correlation between Cdc123 binding, the biological function of eIF2γ, and its ability to assemble with eIF2α and eIF2β. Interestingly, high levels of Cdc123 neutralized the assembly defect and restored the biological function of an eIF2γ mutant. Moreover, the combined overexpression of eIF2 subunits rescued an otherwise inviable cdc123 deletion mutant. Thus, Cdc123 is a specific eIF2 assembly factor indispensable for the onset of protein synthesis. Human Cdc123 is encoded by a disease risk locus and, therefore, eIF2 biogenesis control by Cdc123 may prove relevant for normal cell physiology and human health. This work identifies a novel step in the eukaryotic translation initiation pathway and assigns a biochemical function to a protein that is essential for growth and viability of eukaryotic cells.
  Journal of Biological Chemistry 06/2013;
• Article: A reaction centre-dependent photoprotection mechanism in a highly robust photosystem II from an extremophilic red alga Cyanidioschyzon merolae.

  Tomasz Krupnik, Eva Kotabov Aacute, Laura S van Bezouwen, Radoslaw Mazur, Maciej Garstka, Peter J Nixon, Jim Barber, Radek Kana, Egbert J Boekema, Joanna Kargul
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  ABSTRACT: Members of the rhodophytan order Cyanidiales are unique among phototrophs in their ability to live in extremely low pH levels and moderately high temperatures. The photosynthetic apparatus of the red alga Cyanidioschyzon merolae represents an intermediate type between cyanobacteria and higher plants, suggesting that this alga may provide the evolutionary link between prokaryotic and eukaryotic phototrophs. Although we now have a detailed structural model of photosystem II (PSII) from cyanobacteria at an atomic resolution, no corresponding structure of the eukaryotic PSII complex has been published to date. Here we report the isolation and characterization of a highly active and robust dimeric PSII complex from C. merolae. We show that this complex is highly stable across a range of extreme light, temperature and pH conditions. By measuring fluorescence quenching properties of the isolated C. merolae PSII complex, we provide the first direct evidence of pH-dependent non-photochemical quenching (NPQ) in the red algal PSII reaction centre. This type of NPQ together with high zeaxanthin content appear to underlie photoprotection mechanisms that are efficiently employed by this robust natural water-splitting complex under excess irradiance. In order to provide structural details of this eukaryotic form of PSII, we have employed electron microscopy (EM) and single particle analyses to obtain a 17 Å map of the C. merolae PSII dimer in which we locate the position of the protein mass corresponding to the additional extrinsic protein stabilizing the OEC, PsbQ'. We conclude that this lumenal subunit is present in the vicinity of the CP43 protein, close to the membrane plane.
  Journal of Biological Chemistry 06/2013;
• Article: SGEF is overexpressed in high grade gliomas and promotes TWEAK-Fn14-induced cell migration and invasion via TRAF2.

  Shannon P Fortin Ensign, Ian T Mathews, Jennifer M Eschbacher, Joseph C Loftus, Marc H Symons, Nhan L Tran
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  ABSTRACT: Glioblastoma (GB) is the highest grade of primary adult brain tumors, characterized by a poorly defined and highly invasive cell population. Importantly, these invading cells are attributed with having a decreased sensitivity to radiation and chemotherapy. TWEAK-Fn14 ligand-receptor signaling is one mechanism in GB that promotes cell invasiveness and survival, and is dependent upon the activity of multiple Rho GTPases including Rac1. Here we report that SGEF, a RhoG-specific guanine nucleotide exchange factor (GEF), is overexpressed in GB tumors and promotes TWEAK-Fn14-mediated glioma invasion. Importantly, levels of SGEF expression in GB tumors inversely correlate with patient survival. SGEF mRNA expression is increased in GB cells at the invasive rim relative to those in the tumor core, and knockdown of SGEF expression by shRNA decreases glioma cell migration in vitro and invasion ex vivo. Furthermore, we showed that upon TWEAK stimulation, SGEF is recruited to the Fn14 cytoplasmic tail via TRAF2. Mutation of the Fn14-TRAF domain site or depletion of TRAF2 expression by siRNA oligonucleotides blocked SGEF recruitment to Fn14, and inhibited SGEF activity and subsequent GB cell migration. We also showed that knockdown of either SGEF or RhoG diminished TWEAK activation of Rac1 and subsequent lamellipodia formation. Together, these results indicate that SGEF-RhoG is an important downstream regulator of TWEAK-Fn14-driven GB cell migration and invasion.
  Journal of Biological Chemistry 06/2013;
• Article: Foxm1 Expression in Prostate Epithelial Cells is Essential for Prostate Carcinogenesis.

  Yuqi Cai, David Balli, Vladimir Ustiyan, Logan A Fulford, Andrea Hiller, Vinko Misetic, Yufang Zhang, Andrew M Paluch, Susan E Waltz, Susan Kasper, Tanya V Kalin
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  ABSTRACT: The treatment of prostate cancer (PCa) remains a challenge. Identification of new molecular mechanisms regulating PCa initiation and progression would provide targets for the new cancer treatments. Foxm1 transcription factor is highly upregulated in tumor cells and cells of tumor microenvironment. However, its functions in different cell populations of PCa lesions are unknown. To determine the role of Foxm1 in tumor cells, we generated two novel mouse models, with Foxm1 gain-of-function and Foxm1 loss-of-function under the control of prostate epithelial-specific probasin promoter. In the transgenic adenocarcinoma mouse prostate (TRAMP) model of PCa, loss of Foxm1 decreased tumor growth and metastasis. Decreased tumorigenesis was associated with decreased tumor cell proliferation and down-regulation of genes critical for proliferation and metastasis, including Cdc25b, Cyclin B1, Plk-1, LOX and Versican. Tumor-associated angiogenesis was decreased, coinciding with reduced VEGF-A expression. The mRNA and protein levels of 11β-Hsd2, an enzyme playing an important role in tumor cell proliferation, were down-regulated in Foxm1-deficient PCa tumors in vivo and in Foxm1-depleted TRAMP C2 cells in vitro. Foxm1 bound to, and increased transcriptional activity of, the mouse 11β-Hsd2 promoter through the -706/-873 region, indicating that 11β-Hsd2 was a direct transcriptional target of Foxm1. Without TRAMP, over-expression of Foxm1 either alone or in combination with inhibition of p19ARF tumor suppressor caused a robust epithelial hyperplasia, but was insufficient to induce progression from hyperplasia to PCa. Foxm1 expression in prostate epithelial cells is critical for prostate carcinogenesis, suggesting that inhibition of Foxm1 is a promising therapeutic approach for prostate cancer chemotherapy.
  Journal of Biological Chemistry 06/2013;
• Article: Biochemical characterization of Hpa2 and Hpa3-two small closely related acetyltransferases from S. cerevisiae.

  Vinaya Sampath, Bingsheng Liu, Stefan Tafrov, Madhusudhan Srinivasan, Robert Rieger, Emily I Chen, Rolf Sternglanz
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  ABSTRACT: Based on their sequence Saccharomyces cerevisiae Hpa2 and Hpa3 proteins are annotated as two closely related members of the Gcn5 acetyltransferase family. Here we describe the biochemical characterization of Hpa2 and Hpa3 as bona fide acetyltransferases with different substrate specificities. Mutational and MALDI-TOF analyses show that Hpa3 translation initiates primarily from methionine (M) 19 rather than the annotated start M1, with a minor product starting at M27. When expressed in E.coli and assayed in vitro, Hpa2 and Hpa3 (from M19) acetylate histones and polyamines. While Hpa2 acetylates histones H3 and H4 (at H3 K14, H4 K5 and H4 K12), Hpa3 acetylates only histone H4 (at H4 K8). Additionally, Hpa2, but not Hpa3, acetylates certain small basic proteins. Hpa3, but not Hpa2, has been reported to acetylate D-amino acids and we present results consistent with that. Overexpression of Hpa2 or Hpa3 is toxic to yeast cells. However, their deletions do not show any standard phenotypic defects. These results suggest that Hpa2 and Hpa3 are similar but distinct acetyltransferases that might have overlapping roles with other known acetyltransferases in vivo to acetylate histones and other small proteins.
  Journal of Biological Chemistry 06/2013;
• Article: The multifaceted Proprotein Convertases: their unique, redundant, complementary and opposite functions.

  Nabil G Seidah, Mohamad Seyed Sadr, Michel Chretien, Majambu Mbikay
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  ABSTRACT: The secretory proprotein convertases (PCs) family comprises 9 members: PC1/3, PC2, furin, PC4, PC5/6, PACE4, PC7, SKI-1/S1P and PCSK9. The first 7 PCs cleave their substrates at single or paired basic residues, SKI-1/S1P cleaves its substrates at non-basic residues in the Golgi; PCSK9 cleaves itself once, and the secreted inactive protease escorts specific receptors for lysosomal degradation. It regulates the levels of circulating LDL-cholesterol and is considered a major therapeutic target in phase III clinical trials. In vivo, PCs exhibit unique and often essential functions during development and/or in adulthood, but certain convertases also exhibit complementary, redundant or opposite functions.
  Journal of Biological Chemistry 06/2013;
• Article: A novel function of Onecut1 as a negative regulator of MafA.

  Kaoru Yamamoto, Takaaki Matsuoka, Satoshi Kawashima, Satomi Takebe, Noriyo Kubo, Takeshi Miyatsuka, Hideaki Kaneto, Iichirou Shimomura
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  ABSTRACT: The transcription factor MafA is a key regulator of insulin gene expression and maturation of islet β cells. In spite of its importance, the regulatory mechanism of MafA gene expression is still unclear. To identify the transcriptional regulators of MafA, we examined various transcription factors, which are potentially involved in β cell differentiation. An adenovirus-mediated overexpression study clearly demonstrated that Onecut1 suppresses the promoter activity of MafA through the Foxa2 binding cis-element on the Mafa enhancer region (named areaA). However, ChIP analysis showed that Foxa2 but not Onecut1 could directly bind to areaA. Furthermore, overexpression of Onecut1 inhibited the binding of Foxa2 onto areaA upon ChIP analysis. Importantly, insertion of a mutation in the Foxa2 binding site of areaA significantly decreased the promoter activity of MafA. These findings suggest that Onecut1 suppresses MafA gene expression through the Foxa2 binding site. In the mouse pancreas, MafA expression was first detected at the latest stage of β cell differentiation and was scarcely observed in Onecut1 positive cells during pancreas development. In addition, Onecut1 expression was significantly increased in the islets of diabetic db/db mice, while MafA expression was markedly decreased. The improved glucose levels of db/db mice with insulin injections significantly reduced Onecut1 expression and rescued the reduction of MafA expression. These in vivo experiments also suggest that Onecut1 is a negative regulator of Mafa gene expression. The present study implicates the novel role of Onecut1 in the control of normal β cell differentiation, and its involvement in β cell dysfunction under diabetic conditions by suppressing MafA gene expression.
  Journal of Biological Chemistry 06/2013;
• Article: Differential Role of PTEN Phosphatase in Chemotactic Growth Cone Guidance.

  Steven J Henle, Lucas P Carlstrom, Thomas R Cheever, John R Henley
  [show abstract] [hide abstract]
  ABSTRACT: Negatively targeting the tumor suppressor and phosphoinositide phosphatase PTEN promotes axon re-growth after injury. How PTEN functions in axon guidance has remained unknown. Here we report the differential role of PTEN in chemotactic guidance of axonal growth cones. Downregulating PTEN expression in Xenopus laevis spinal neurons selectively abolished growth cone chemorepulsion but permitted chemoattraction. These findings persisted during cAMP-dependent switching of turning behaviors. Live-cell imaging using a GFP biosensor revealed rapid PTEN-dependent depression of phosphatidylinositol 3,4,5-trisphosphate (PIP3) levels in the growth cone induced by the repellent myelin-associated glycoprotein (MAG). Moreover, downregulating PTEN expression blocked negative remodeling of β1-integrin adhesions triggered by MAG, yet permitted integrin clustering by a positive chemotropic treatment. Thus, PTEN negatively regulates growth cone PIP3 levels and mediates chemorepulsion, whereas chemoattraction is PTEN independent. Regenerative therapies targeting PTEN may therefore suppress growth cone repulsion to soluble cues while permitting attractive guidance, an essential feature for re-forming functional neural circuits.
  Journal of Biological Chemistry 06/2013;
• Article: Formation of dynamic soluble surfactant-induced amyloid β peptide aggregation intermediates.

  Axel Abelein, Jørn Døvling Kaspersen, Søren Bang Nielsen, Grethe Vestergaard Jensen, Gunna Christiansen, Jan Skov Pedersen, Jens Danielsson, Daniel E Otzen, Astrid Gräslund
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  ABSTRACT: Intermediate amyloidogenic states along the amyloid β peptide (Aβ) aggregation pathway have been shown to be linked to neurotoxicity. To shed more light on the different structures that may arise during Aβ aggregation, we here investigate surfactant-induced Aβ aggregation. This process leads to co-aggregates featuring a β-structure motif that is characteristic for mature amyloid-like structures. Surfactants induce secondary structure in Aβ in a concentration-dependent manner, from predominantly random coil at low surfactant concentration, via β-structure to fully formed α-helical state at high surfactant concentration. The β-rich state is the most aggregation prone as monitored by Thioflavin T fluorescence. Small angle X-ray scattering reveals initial globular structures of surfactant-Aβ co-aggregated oligomers and formation of elongated fibrils during a slow aggregation process. Alongside this slow (min to h time scale) fibrillation process, much faster dynamic exchange (kex ~ 1100 s(-1)) takes place between free and co-aggregate-bound peptide. The two hydrophobic segments of the peptide are directly involved in the chemical exchange and interact with the hydrophobic part of the co-aggregates. Our findings suggest a model for surfactant-induced aggregation where free peptide and surfactant initially co-aggregate to dynamic globular oligomers and eventually form elongated fibrils. When interacting with β-structure promoting substances, such as surfactants, Aβ is kinetically driven towards an aggregation prone state.
  Journal of Biological Chemistry 06/2013;
• Article: A crayfish insulin-like-binding protein: Another piece in the androgenic gland insulin-like hormone puzzle is revealed.

  Ohad Rosen, Simy Weil, Rivka Manor, Ziv Roth, Isam Khalaila, Amir Sagi
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  ABSTRACT: Across the animal kingdom, the involvement of insulin-like peptide (ILP) signaling in sex-related differentiation processes is attracting increasing attention. Recently, a gender-specific ILP was identified as the androgenic sex hormone in Crustacea. However, moieties modulating the actions of this androgenic insulin-like growth factor were yet to be revealed. Through molecular screening of an androgenic gland (AG) cDNA library prepared from the crayfish Cherax quadricarinatus, we have identified a novel insulin-like growth factor-binding protein (IGFBP) termed Cq-IGFBP. Based on bioinformatic analyses, the deduced Cq-IGFBP was shown to share high sequence homology with IGFBP-family members from both invertebrates and vertebrates. The protein also includes a sequence determinant proven crucial for ligand binding, which, according to 3D modeling, is assigned to the exposed outer surface of the protein. Recombinant Cq-IGFBP (rCq-IGFBP) protein was produced and, using a 'pull-down' methodology, was shown to specifically interact with the insulin-like AG hormone of the crayfish (Cq-IAG). Particularly, using both mass spectral analysis and an immunological tool, rCq-IGFBP was shown to bind the Cq-IAG prohormone. Furthermore, a peptide corresponding to residues 23-38 of the Cq-IAG A-chain was found sufficient for in vitro recognition by rCq-IGFBP. Cq-IGFBP is the first IGFBP family member shown to specifically interact with a gender-specific ILP. Unlike their ILP ligands, IGFBPs are highly conserved across evolution, from ancient arthropods, like crustaceans, to humans. Such conservation places ILP signaling at the center of sex-related phenomena in early animal development.
  Journal of Biological Chemistry 06/2013;
• Article: Ca2+-dependent structural changes in the B-cell receptor CD23 increase its affinity for human Immunoglobulin E.

  Daopeng Yuan, Anthony H Keeble, Richard G Hibbert, Stella Fabiane, Hannah J Gould, James M McDonnell, Andrew J Beavil, Brian J Sutton, Balvinder Dhaliwal
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  ABSTRACT: Immunoglobulin E (IgE) antibodies play a fundamental role in allergic disease and are a target for therapeutic intervention. IgE functions principally through two receptors, FcεRI and CD23 (FcεRII). Minute amounts of allergen trigger mast cell or basophil degranulation by cross-linking IgE-bound FcεRI, leading to an inflammatory response. The interaction between IgE and CD23 on B-cells regulates IgE synthesis. CD23 is unique amongst Ig receptors in that it belongs to the C-type (calcium- dependent) lectin-like superfamily. While the interaction of CD23 with IgE is carbohydrate-independent, calcium has been reported to increase the affinity for IgE, but the structural basis for this activity has hitherto been unknown. We have determined the crystal structures of the human lectin-like head domain of CD23 in its Ca2+-free and Ca2+-bound forms, as well as the crystal structure of the Ca2+-bound head domain of CD23 in complex with a sub-fragment of IgE-Fc consisting of the dimer of Cε3 and Cε4 domains (Fcε3-4). Together with site-directed mutagenesis, the crystal structures of four Ca2+ ligand mutants, ITC, SPR and stopped-flow analysis, we demonstrate that Ca2+ binds at the principal, evolutionarily conserved binding site in CD23. Ca2+ binding drives Pro250, at the base of an IgE-binding loop (loop 4), from the trans to the cis configuration with a concomitant conformational change and ordering of residues in the loop. These Ca2+-induced structural changes in CD23 lead to additional interactions with IgE, a more entropically favorable interaction, and a thirty-fold increase in affinity of a single head domain of CD23 for IgE. Taken together, these results suggest that binding of Ca2+ brings an extra degree of modulation to CD23 function.
  Journal of Biological Chemistry 06/2013;
• Article: PP2Ac promotes DNA hypomethylation by suppressing the pMEK/pERK/DNMT1 pathway in T cells from controls and SLE patients.

  Katsue Sunahori, Kamalpreet Nagpal, Christian M Hedrich, Masayuki Mizui, Lisa M Fitzgerald, George C Tsokos
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  ABSTRACT: DNA hypomethylation is a characteristic feature of SLE immune cells. Numerous reports have implicated the involvement of the mitogen-activated protein kinase kinase 1 (MEK)/extracellular signal-regulated kinase (ERK) pathway in the reduction of DNA methyltransferase (DNMT) expression, hence inducing the transcription of methylation-sensitive genes in SLE patients. However, the molecular mechanisms involved remain unclear. Here, we investigated whether the catalytic subunit of protein phosphatase 2A (PP2Ac) which is overexpressed in SLE T cells contributes to reduced DNA methylation. We show that both chemical suppression and siRNA silencing of PP2Ac in T cells results in sustained phosphorylation of MEK and ERK following stimulation with PMA and ionomycin. Furthermore, PP2Ac suppression resulted in increased DNMT enzymatic activity, DNA hypermethylation and decreased expression of methylation-sensitive genes. Similarly, in SLE T cells, suppression of PP2Ac resulted in increased MEK/ERK phosphorylation, enhanced DNMT1 expression and suppressed expression of the methylation-sensitive CD70 gene. Our results demonstrate that PP2A regulates DNA methylation by influencing the phosphorylation of MEK/ERK. We propose that enhanced PP2Ac in SLE T cells may dephosphorylate and activate the signaling pathway upstream of DNMT1, thus disturbing the tight control of methylation-sensitive genes, which are involved in SLE pathogenesis.
  Journal of Biological Chemistry 06/2013;
• Article: Increased cAMP in monocytes augments Notch signaling mechanisms by elevating RBP-J and Transducin-like Enhancer of Split (TLE).

  Jason L Larabee, Salika M Shakir, Soumitra Barua, Jimmy D Ballard
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  ABSTRACT: In cells of the innate immune system, pathological increases in intracellular cAMP attenuate immune responses and contribute to infections by bacteria such as Bacillus anthracis. In this work, cAMP from B. anthracis edema toxin (ET) is found to activate the Notch signaling pathway in both mouse macrophages and human monocytes. ET as well as a cell permeable activator of PKA induce Notch target genes (HES1, HEY1, IL2RA, and IL7R) and are able to significantly enhance the induction of these Notch target genes by a Toll-like receptor (TLR) ligand. Elevated cAMP also resulted in increased levels of Groucho/Transducin-like Enhancer of Split (TLE) and led to increased amounts of a transcriptional repressor complex consisting of TLE and the Notch target gene, Hes1. To address the mechanism used by ET to activate Notch signaling, components of Notch signaling were examined and results revealed that ET increased levels of RBP-J, a DNA binding protein and principal transcriptional regulator of Notch signaling. Overexpression studies indicated that RBP-J was sufficient to activate Notch signaling and potentiate LPS induced Notch signaling. Further examination of the mechanism used by ET to activate Notch signaling revealed that C/EBP β, a transcription factor activated by cAMP, helped activate Notch signaling and upregulated RBP-J. These studies demonstrate that cAMP activates Notch signaling and increases the expression of TLE, which could be an important mechanism utilized by cAMP to suppress immune responses.
  Journal of Biological Chemistry 06/2013;
• Article: Ribosomal protein S14 negatively regulates c-Myc activity.

  Xiang Zhou, Qian Hao, Jun-Ming Liao, Peng Liao, Hua Lu
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  ABSTRACT: The ribosomal gene RPS14 is associated with the cancer-prone 5q-syndrome, which is caused by an interstitial deletion of the long arm of human chromosome 5. Previously, we found that ribosomal protein S14 (RPS14) binds to and inactivates MDM2, consequently leading to p53-dependent cell-cycle arrest and growth inhibition. However, it remains elusive whether RPS14 regulates cell proliferation in a p53-independent manner. Here, we show that RPS14 interacts with the Myc homology box II (MBII) and the C-terminal basic helix-loop-helix leucine zipper (bHLH-LZ) domains of the oncoprotein c-Myc. Further, RPS14 inhibited c-Myc transcriptional activity by preventing the recruitment of c-Myc and its cofactor, TRRAP, to the target gene promoters, as thus suppressing c-Myc-induced cell proliferation. Also, siRNA-mediated RPS14 depletion elevated c-Myc transcriptional activity determined by its target gene, Nucleolin, expression. Interestingly, RPS14 depletion also resulted in the induction of c-Myc mRNA and subsequent protein levels. Consistent with this, RPS14 promoted c-Myc mRNA turnover through an Argonaute 2 (Ago2)- and microRNA- mediated pathway. Taken together, our study demonstrates that RPS14 negates c-Myc functions by directly inhibiting its transcriptional activity and mediating its mRNA degradation via miRNA.
  Journal of Biological Chemistry 06/2013;
• Article: Generating S-nitrosothiols from Hemoglobin: Mechanisms, Conformational Dependence and Physiological Relevance.

  Camille J Roche, Maria Belen Cassera, David Dantsker, Rhoda Elison Hirsch, Joel M Friedman
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  ABSTRACT: In vitro, ferrous deoxy hemes in hemoglobin (Hb) react with nitrite to generate nitric oxide (NO) through a nitrite reductase (NR) reaction. In vivo studies indicate Hb with nitrite can be a source of NO bioactivity. The NR reaction does not appear to account fully for this activity since free NO is short lived especially within the red blood cell. Thus, the exporting of NO bioactivity both out of the RBC and over a large distance requires an additional mechanism. A nitrite anhydrase (NA) reaction in which N2O3, a potent S-nitrosating agent, is produced through the reaction of NO with ferric heme-bound nitrite has been proposed (Gladwin, Kim Shapiro and collaborators) as a possible mechanism. Legitimate concerns including physiological relevance and the nature of the mechanism have been raised concerning the NA reaction. The present study addresses these concerns demonstrating NO and nitrite with ferric hemes under near physiological conditions yields an intermediate having the properties of the purported NA heme bound N2O3 intermediate. The results indicate that ferric heme sites, traditionally viewed as a source of potential toxicity, can be functionally significant, especially for partially oxygenated/partially met R state Hb that arises from the NO dioxygenation reaction. In the presence of low levels of nitrite and either NO or a suitable reductant such as L-Cysteine, these ferric heme sites can function as a generator for the formation of S-nitrosothiols such as S-nitrosoglutathione (GSNO) and, as such, should be considered as a source of RBC derived and exportable bioactive NO.
  Journal of Biological Chemistry 06/2013;
• Article: CREB and NF-κB are activated during prolonged hypoxia and cooperatively regulate the induction of matrix metalloproteinase MMP1.

  Koh Nakayama
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  ABSTRACT: Responses to low levels of oxygen (hypoxia) are essential to maintain homeostasis. During the hypoxic response, gene expression is altered by various transcription factors. The transcription factor, hypoxia-inducible factor (HIF), plays a central role in the hypoxic response. The α subunit of HIF, which is actively degraded during normoxia, becomes stabilized during hypoxia, which leads to HIF activation. A microarray analysis of HeLa cells showed that expression of matrix metalloproteinase 1 (MMP1) was markedly induced during prolonged hypoxia. CREB and NF-κB-binding sites were identified in the MMP1 promoter region between 1945 and 1896 nucleotides upstream of the transcription start site. Assays with luciferase reporters demonstrated that HIF activity was induced during the early phase of hypoxia, while CREB and NF-κB were activated during the later (prolonged) phase. Depletion of CREB and/or NF-κB reduced MMP1 induction during prolonged hypoxia, both at the mRNA and protein levels. A chromatin immunoprecipitation assay demonstrated binding of CREB and NF-κB to the MMP1 promoter. Finally, cell migration and invasion on a collagen matrix, and pulmonary metastasis in nude mice were inhibited following depletion of CREB and NF-κB in MDA-MB-231 cells. Taken together, these results suggest that the cooperative action of CREB and NF-κB plays an important role to induce MMP1 expression during prolonged hypoxia and regulates cell migration and invasion in cancer cells.
  Journal of Biological Chemistry 06/2013;
• Article: Processive ATP-driven disassembly of SNARE complexes by the N-ethylmaleimide sensitive factor molecular machine.

  Daniel J Cipriano, Jaemyeong Jung, Sandro Vivona, Timothy D Fenn, Axel T Brunger, Zev Bryant
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  ABSTRACT: SNARE proteins promote membrane fusion by forming a 4-stranded parallel helical bundle that brings the membranes into close proximity. Post fusion, the complex is disassembled by an AAA+ ATPase called N-ethylmaleimide sensitive factor (NSF). We present evidence that NSF uses a processive unwinding mechanism to disassemble SNARE proteins. Using a real-time disassembly assay based on fluorescence dequenching, we correlate NSF-driven disassembly rates with the SNARE-activated ATPase activity of NSF. Neuronal SNAREs activate the ATPase rate of NSF by ~26-fold. One SNARE complex takes an average of ~5 seconds to disassemble in a process that consumes ~50 ATP. Investigations of substrate requirements show that NSF is capable of disassembling a truncated SNARE substrate consisting of only the core SNARE domain, but not an unrelated four-stranded coiled coil. NSF can also disassemble an engineered double-length SNARE complex, suggesting a processive unwinding mechanism. We further investigated processivity using single turnover experiments, which show that SNAREs can be unwound in a single encounter with NSF. We propose a processive helicase-like mechanism for NSF in which ~1 residue is unwound for every hydrolyzed ATP molecule.
  Journal of Biological Chemistry 06/2013;
• Article: Functional Selectivity of 6'-guanidinonaltrindole (6'-GNTI) at Kappa Opioid Receptors in Striatal Neurons.

  Cullen L Schmid, John M Streicher, Chad E Groer, Thomas A Munro, Lei Zhou, Laura M Bohn
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  ABSTRACT: There is considerable evidence to suggest that drug actions at the kappa opioid receptor (KOR) may represent a means to control pain perception and modulate reward thresholds. As a G protein-coupled receptor (GPCR), the activation of KOR promotes Gαi/o protein coupling and the recruitment of βarrestins. It has become increasingly evident that GPCRs can transduce signals that originate independently via G protein pathways and βarrestin pathways; the ligand-dependent bifurcation of such signaling is referred to as ″functional selectivity″ or ″signaling bias.″ Recently, a KOR agonist, 6'-guanidinonaltrindole (6'-GNTI), was shown to display bias towards the activation of G protein-mediated signaling over βarrestin2 recruitment. Therefore, we investigated whether such ligand bias was preserved in striatal neurons. While the reference KOR agonist, U69,593 induces the phosphorylation of ERK1/2 and Akt, 6'-GNTI only activates the Akt pathway in striatal neurons. Using pharmacological tools and βarrestin2 knockout mice, we show that KOR-mediated ERK1/2 phosphorylation in striatal neurons requires βarrestin2, while Akt activation depends upon G protein signaling. These findings reveal a point of KOR signal bifurcation that can be observed in an endogenous neuronal setting and may prove to be an important indicator when developing biased agonists at the KOR.
  Journal of Biological Chemistry 06/2013;
• Article: Nuclear factor-erythroid 2 (NF-E2) p45-related factor-2 (Nrf2) modulates dendritic cell immune function through regulation of p38MAPK-CREB/ATF1 signalling.

  Laith M A Al-Huseini, Han Xian Aw Yeang, Swaminathan Sethu, Naif Alhumeed, Junnat M Hamdam, Yulia Tingle, Laiche Djouhri, Neil Kitteringham, B Kevin Park, Christopher E Goldring, Jean G Sathish
  [show abstract] [hide abstract]
  ABSTRACT: Nrf2 is a redox responsive transcription factor that has been implicated in the regulation of DC immune function. Loss of Nrf2 results in increased co-stimulatory molecule expression, enhanced T cell stimulatory capacity and increased reactive oxygen species (ROS) levels in murine immature DCs (iDCs). It is unknown whether altered DC function in Nrf2 deficient DCs (Nrf2-/- iDCs) is due to elevated ROS levels. Furthermore, it is unclear what intracellular signalling pathways are involved in Nrf2-mediated regulation of DC function. Using antioxidant vitamins to reset ROS levels in Nrf2-/- iDCs, we show that elevated ROS is not responsible for the altered phenotype and function of these DCs. Pharmacological inhibitors were used to explore the role of key MAPKs in mediating the altered phenotype and function in Nrf2-/- iDCs. We demonstrate that the increased co-stimulatory molecule expression (MHC II and CD86) and antigen specific T cell activation capacity observed in Nrf2-/- iDCs was reversed by inhibition of p38MAPK but not JNK. Importantly, we provide evidence for increased phosphorylation of cAMP-responsive element binding protein (CREB) and activating transcription factor 1 (ATF1), transcription factors that are downstream of p38MAPK. The increased phosphorylation of CREB/ATF1 in Nrf2-/- iDCs was sensitive to p38MAPK inhibition. We also show data to implicate heme oxygenase-1 as a potential molecular link between Nrf2 and CREB/ATF1. These results indicate that dysregulation of p38MAPK-CREB/ATF1 signalling axis underlies the altered function and phenotype in Nrf2 deficient DCs. Our findings provide new insights into the mechanisms by which Nrf2 mediates regulation of DC function.
  Journal of Biological Chemistry 06/2013;