Published by De Gruyter

Volume 386 Issue 9

Issue of Biological Chemistry

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Contents
Journal Overview

September 9, 2005

Paper of the Year 2004: Award to Yin Wang and Iris Lorenzi

F. Ulrich Hartl

Page range: 815-815

Publicly Available September 9, 2005

Improving the levels of essential amino acids and sulfur metabolites in plants

Gad Galili, Rachel Amir, Rainer Hoefgen, Holger Hesse

Page range: 817-831

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Abstract

Plants represent the major source of food for humans, either directly or indirectly through their use as livestock feeds. Plant foods are not nutritionally balanced because they contain low proportions of a number of essential metabolites, such as vitamins and amino acids, which humans and a significant proportion of their livestock cannot produce on their own. Among the essential amino acids needed in human diets, Lys, Met, Thr and Trp are considered as the most important because they are present in only low levels in plant foods. In the present review, we discuss approaches to improve the levels of the essential amino acids Lys and Met, as well as of sulfur metabolites, in plants using metabolic engineering approaches. We also focus on specific examples for which a deeper understanding of the regulation of metabolic networks in plants is needed for tailor-made improvements of amino acid metabolism with minimal interference in plant growth and productivity.

Publicly Available September 9, 2005

Symmetry at the active site of the ribosome: structural and functional implications

Ilana Agmon, Anat Bashan, Raz Zarivach, Ada Yonath

Page range: 833-844

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Abstract

The sizable symmetrical region, comprising 180 ribosomal RNA nucleotides, which has been identified in and around the peptidyl transferase center (PTC) in crystal structures of eubacterial and archaeal large ribosomal subunits, indicates its universality, confirms that the ribosome is a ribozyme and evokes the suggestion that the PTC evolved by gene fusion. The symmetrical region can act as a center that coordinates amino acid polymerization by transferring intra-ribosomal signals between remote functional locations, as it connects, directly or through its extensions, the PTC, the three tRNA sites, the tunnel entrance, and the regions hosting elongation factors. Significant deviations from the overall symmetry stabilize the entire region and can be correlated with the shaping and guiding of the motion of the tRNA 3′-end from the A- into the P-site. The linkage between the elaborate PTC architecture and the spatial arrangements of the tRNA 3′-ends revealed the rotatory mechanism that integrates peptide bond formation, translocation within the PTC and nascent protein entrance into the exit tunnel. The positional catalysis exerted by the ribosome places the reactants in stereochemistry close to the intermediate state and facilitates the catalytic contribution of the P-site tRNA 2′-hydroxyl.

Publicly Available September 9, 2005

GPCR-induced migration of breast carcinoma cells depends on both EGFR signal transactivation and EGFR-independent pathways

Stefan Hart, Oliver M. Fischer, Norbert Prenzel, Esther Zwick-Wallasch, Matthias Schneider, Lothar Hennighausen, Axel Ullrich

Page range: 845-855

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Abstract

The epidermal growth factor receptor (EGFR) plays a key role in the regulation of important cellular processes under normal and pathophysiological conditions such as cancer. In human mammary carcinomas the EGFR is involved in regulating cell growth, survival, migration and metastasis and its activation correlates with the lack of response in hormone therapy. Here, we demonstrate in oestrogen receptor-positive and -negative human breast cancer cells and primary mammary epithelial cells a cross-communication between G protein-coupled receptors (GPCRs) and the EGFR. We present evidence that specific inhibition of ADAM15 or TACE blocks GPCR-induced and proHB-EGF-mediated EGFR tyrosine phosphorylation, downstream mitogenic signalling and cell migration. Notably, activation of the PI3K downstream mediator PKB/Akt by GPCR ligands involves the activity of sphingosine kinase (SPHK) and is independent of EGFR signal transactivation. We conclude that GPCR-induced chemotaxis of breast cancer cells is mediated by EGFR-dependent and -independent signalling pathways, with both parallel pathways having to act in concert to achieve a complete migratory response.

Publicly Available September 9, 2005

Developing an effective RNA interference strategy against a plus-strand RNA virus: silencing of coxsackievirus B3 and its cognate coxsackievirus-adenovirus receptor

Denise Werk, Steffen Schubert, Vanessa Lindig, Hans-Peter Grunert, Heinz Zeichhardt, Volker A. Erdmann, Jens Kurreck

Page range: 857-863

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Abstract

Coxsackievirus B3 (CVB-3) is a plus-strand RNA virus that is believed to be the most common causal agent of viral myocarditis. Since no specific treatment for CVB-3 infections is available to date, we and others have recently started to develop RNA interference (RNAi) approaches to prevent virus propagation. Here we describe our strategy for the development of efficient small interfering RNAs (siRNAs) against viral genomes. Initially, fusion constructs of a reporter (green fluorescent protein) and viral subgenomic fragments were employed to select active siRNAs against the virus. Moreover, in an attempt to achieve sustained virus silencing and reduce the risk of generating escape mutants, only highly efficient siRNAs directed against regions of the viral genome that are unlikely to tolerate mutations were considered for virus inhibition. Two siRNAs directed against the 3D RNA-dependent RNA polymerase were found to inhibit virus propagation by 80–90%. The protective effect of the efficient siRNAs lasted for several days. Furthermore, we have first evidence that inhibition of the cellular coxsackievirus-adenovirus receptor (CAR) by RNAi also reduces the virus titre. Our strategy is likely to be applicable to other (RNA) viruses as well.

Publicly Available September 9, 2005

NF-κB contributes to transcription of placenta growth factor and interacts with metal responsive transcription factor-1 in hypoxic human cells

Mirjam Cramer, Ivana Nagy, Brian J. Murphy, Max Gassmann, Michael O. Hottiger, Oleg Georgiev, Walter Schaffner

Page range: 865-872

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Abstract

Placenta growth factor (PlGF) is a member of the vascular endothelial growth factor family of cytokines that control vascular and lymphatic endothelium development. It has been implicated in promoting angiogenesis in pathological conditions via signaling to vascular endothelial growth factor receptor-1. PlGF expression is induced by hypoxia and proinflammatory stimuli. Metal responsive transcription factor 1 (MTF-1) was shown to take part in the hypoxic induction of PlGF in Ras-transformed mouse embryonic fibroblasts. Here we report that PlGF expression is also controlled by NF-κB. We identified several putative binding sites for NF-κB in the PlGF promoter/enhancer region by sequence analyses, and show binding and transcriptional activity of NF-κB p65 at these sites. Expression of NF-κB p65 from a plasmid vector in HEK293 cells caused a substantial increase of PlGF transcript levels. Furthermore, we found that hypoxic conditions induce nuclear translocation and interaction of MTF-1 and NF-κB p65 proteins, suggesting a role for this complex in hypoxia-induced transcription of PlGF.

September 9, 2005

Regulation of asparagine synthetase gene transcription by the basic region leucine zipper transcription factors ATF5 and CHOP

Jude Al Sarraj, Charles Vinson, Gerald Thiel

Page range: 873-879

Abstract

Asparagine synthetase catalyses the glutamine- and ATP-dependent conversion of aspartic acid to asparagine. In human hepatoma cells cultured in mediumcontaining amino acids, the mRNA of asparagine synthetase is not detectable by RNase protection mapping. However, maintaining the cells in amino acid-free Krebs-Ringer bicarbonate buffer strongly upregulated asparagine synthetase biosynthesis. The effect of amino acid deprivation on asparagine synthetase gene transcription is mediated by a genetic element termed the nutrient-sensing response unit. Previous studies revealed that the basic region leucine zipper (bZIP) transcription factor CREB2/ATF4 is involved in the nutrient deprivation-induced upregulation of asparagine synthetase gene transcription. Here we show that overexpression of the bZIP protein ATF5, a transcriptional activator, stimulates asparagine synthetase promoter/reporter gene transcription via the nutrient-sensing response unit. In contrast, ATF5 does not transactivate cAMP response element (CRE)-containing reporter genes. Overexpression of the C/EBP homologous transcription factor CHOP impaired transcriptional activation of the asparagine synthetase promoter following amino acid deprivation or over-expression of ATF5 or CREB2/ATF4. These data indicate that CHOP functions as a shut-off-device for nutrient deprivation-induced gene transcription.

September 9, 2005

Role of the N-terminal region and of β-sheet residue Thr29 on the activity of the ω₂ global regulator from the broad-host range Streptococcus pyogenes plasmid pSM19035

Karin Welfle, Florencia Pratto, Rolf Misselwitz, Joachim Behlke, Juan C. Alonso, Heinz Welfle

Page range: 881-894

Abstract

The dimeric regulatory protein wild-type ω (wt ω 2 ) binds to arrays of 7-bp sequences (heptads) present in the operator DNA region of copy control and partition functions of plasmid pSM19035. Each ω 2 protein probably binds with an antiparallel β-sheet structure in the major groove of the 7-bp subsite of the operator DNA. Exchange of threonine at position 29 to alanine (T29A) drastically affects the activity of variant protein ω 2 T29A both in vivo and in vitro , and reduces the thermodynamic stability Δ G u 0 , but does not change the conformation. Likewise, the binding affinity to DNA is reduced and the association of the two monomeric subunits of the ω 2 T29A dimer is weakened, as manifested by an increase in the dissociation constant from 3.2 μM for wt ω 2 to 6.3 μM for ω 2 T29A. Denatured dimers are formed upon thermal unfolding of wt ω 2 and ω 2 T29A at ca. 45 μM ( D n ↔ D u ). Removal of 8 (ω 2 ΔN8), or even 18 (ω 2 ΔN18) N-terminal amino acids has no obvious effect either on the core structure or on the activity in comparison to wt ω 2 . The stability of variants ω 2 ΔN8 and ω 2 ΔN18 is similar to that of wt ω 2 , and their binding to operator DNA is not impaired.

September 9, 2005

Lysine 3 acetylation regulates the phosphorylation of yeast 6-phosphofructo-2-kinase under hypo-osmotic stress

Hassan Dihazi, Renate Kessler, Gerhard A. Müller, Klaus Eschrich

Page range: 895-900

Abstract

N-terminal acetylation in the yeast Saccharomyces cerevisiae is catalysed by any of three N-terminal acetyltransferases (NAT), NatA, NatB, and NatC, which contain the catalytic subunits Ard1p, Nat3p and Mak3p, respectively. Yeast 6-phosphofructo-2-kinase (PFK2) was found to be acetylated at the amino acid lysine 3. The Lys3-Arg mutant was not acetylated and the mutation causes a slight decrease in enzyme activity. PFK2 from yeast cells exposed to hypo-osmotic stress is known to be phos-phorylated at Ser8 and Ser652 (Dihazi et al., 2001a). We have taken a mass spectrometric approach to investigate the influence of PFK2 acetylation on its phosphorylation. Wild-type PFK2 and the Lys3-Arg mutant were purified from hypo-osmotically stressed cells and analysed with MALDI-TOF MS for phosphorylation. Wild-type PFK2 without any tag sequence was found to be acetylated and two times phosphorylated at the N-terminal peptide T 1–40 carrying the acetylation. The same results were observed with C-terminally His-tagged PFK2. When the His-tag was added to the N-terminus of the protein PFK2, acetylation was found to be incomplete and only one phosphate was incorporated in the peptide T 1–41 . The Lys3-Arg mutant of PFK2 was not at all post-translationally modified at the N-terminal peptide. Our data indicate that Lys3 acetylation affects the N-terminal phosphorylation of PFK2 under hypo-osmotic stress.

Publicly Available September 9, 2005

Determination of the specificity of monoclonal antibodies against Schistosoma mansoni CAA glycoprotein antigen using neoglycoconjugate variants

Adriana Carvalho de Souza, Alexandra van Remoortere, Cornelis H. Hokke, André M. Deelder, Johannes F.G. Vliegenthart, Johannis P. Kamerling

Page range: 901-908

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Abstract

The immunogenic O -glycan of circulating anodic antigen (CAA) is a high-molecular-mass polysaccharide with the unique →6)-[β-D-Glc p A-(1→3)]-β-D-Gal p NAc-(1→ repeating unit. To obtain information at the molecular level about the specificity of monoclonal antibodies against CAA, the immunoreactivity of two series of bovine serum albumin-coupled synthetic oligosaccharides related to the CAA O -glycan was monitored using ELISA and surface plasmon resonance spectroscopy. The importance of the axial hydroxyl group of β-D-Gal p NAc for antibody binding was investigated using the following series of analogues: β-D-Glc p A-(1→3)-β-D-Glc p NAc-(1→O); β-D-Glc p NAc-(1→6)-[β-D-Glc p A-(1→3)]-β-D-Glc p NAc-(1→O); and β-D-Glc p A-(1→3)-β-D-Glc p NAc-(1→6)-[β-D-Glc p A-(1→3)]-β-D-Glc p NAc-(1→O). In the second series of analogues, β-D-Glc p 6 S -(1→3)-β-D-Gal p NAc-(1→O), β-D-Gal p NAc-(1→6)-[β-D-Glc p 6 S -(1→3)]-β-D-Gal p NAc-(1→O), and β-D-Glc p 6 S -(1→3)-β-D-Gal- p NAc-(1→6)-[β-D-Glc p 6 S -(1→3)]-β-D-Gal p NAc-(1→O), the native β-D-Glc p A moiety was replaced by β-D-Glc p 6 S to evaluate the influence of the nature of the charge on antibody recognition. Comparison of the immunoreactivity of these series with that measured for conjugates containing corresponding synthetic CAA fragments showed that the antibody binding levels can be correlated to the antibody specificity to CAA fragments. For the most reactive antibodies, the structural changes chosen (β-D-Gal p NAc replaced by β-D-Glc p NAc, and β-D-Glc p A replaced by β-D-Glc p 6 S ) completely eradicated the binding.

September 9, 2005

Cholesterol is the major component of native lipoproteins activating the p38 mitogen-activated protein kinases

Iveta Dobreva, Olaf Zschörnig, Gérard Waeber, Richard W. James, Christian Widmann

Page range: 909-918

Abstract

Elevated low-density lipoprotein (LDL) levels induce activation of the p38 mitogen-activated protein kinase (MAPK), a stress-activated protein kinase potentially participating in the development of atherosclerosis. The nature of the lipoprotein components inducing p38 MAPK activation has remained unclear however. We show here that both LDLs and high-density lipoproteins (HDLs) have the ability to stimulate the p38 MAPKs with potencies that correlate with their cholesterol content. Cholesterol solubilized in methyl-β-cyclodextrin was sufficient to activate the p38 MAPK pathway. Liposomes made of phosphatidylcholine (PC) or sphingomyelin, the two main phospholipids found in lipoproteins, were unable to stimulate the p38 MAPKs. In contrast, PC liposomes loaded with cholesterol potently activated this pathway. Reducing the cholesterol content of LDL particles lowered their ability to activate the p38 MAPKs. Cell lines representative of the three main cell types found in blood vessels (endothelial cells, smooth muscle cells and fibroblasts) all activated their p38 MAPK pathway in response to LDLs or cholesterol-loaded PC liposomes. These results indicate that elevated cholesterol content in lipoproteins, as seen in hypercholesterolemia, favors the activation of the stress-activated p38 MAPK pathway in cells from the vessel wall, an event that might contribute to the development of atherosclerosis.

September 9, 2005

Focal adhesion kinase is redistributed to focal complexes and mediates cell spreading in macrophages in response to M-CSF

Elisabetta Rovida, Benedetta Lugli, Valentina Barbetti, Serena Giuntoli, Massimo Olivotto, Persio Dello Sbarba

Page range: 919-929

Abstract

The macrophage colony-stimulating factor (M-CSF, CSF-1) regulates survival, proliferation and differentiation of mononuclear phagocytes, as well as macrophage motility and morphology. The latter features are usually regulated by ECM-mediated activation of integrins and subsequent tyrosine phosphorylation of cellular proteins, including focal adhesion kinase (FAK). FAK is phosphorylated by downstream receptor tyrosine kinases as well. We addressed the question whether M-CSF regulates FAK tyrosine phosphorylation in macrophages, and found that M-CSF induces FAK phosphorylation at all known tyrosine residues. This phosphorylation was dependent on Src. Extracellularly-regulated kinase (ERK), Jun N-terminal kinase (JNK) and phosphatidylinositol-3-kinase (PI3K) were found to be negatively involved in M-CSF-induced FAK phosphorylation, as their inhibition resulted in FAK hyper-phosphorylation. Following M-CSF treatment, FAK and the active forms of M-CSFR and Src were redistributed to the cytoskeleton, where active ERK, JNK and PI3K were detectable. Immunofluorescence showed the presence of FAK and its active form in focal complexes following M-CSF treatment. Moreover, cell spreading and adhesion were impaired when FAK tyrosine phosphorylation was abrogated by either transfection with FRNK, a dominant negative form of FAK, or treatment with a number of inhibitors of upstream FAK-activating signals. These results point to a relevant role for FAK in the regulation of cell spreading and adhesion in macrophages.

September 9, 2005

Inhibition of cathepsin B reduces β-amyloid production in regulated secretory vesicles of neuronal chromaffin cells: evidence for cathepsin B as a candidate β-secretase of Alzheimer's disease

Vivian Hook, Thomas Toneff, Matthew Bogyo, Doron Greenbaum, Katalin F. Medzihradszky, John Neveu, William Lane, Gregory Hook, Terry Reisine

Page range: 931-940

Abstract

The regulated secretory pathway of neurons is the major source of extracellular Aβ that accumulates in Alzheimer's disease (AD). Extracellular Aβ secreted from that pathway is generated by β-secretase processing of amyloid precursor protein (APP). Previously, cysteine protease activity was demonstrated as the major β-secretase activity in regulated secretory vesicles of neuronal chromaffin cells. In this study, the representative cysteine protease activity in these secretory vesicles was purified and identified as cathepsin B by peptide sequencing. Immunoelectron microscopy demonstrated colocalization of cathepsin B with Aβ in these vesicles. The selective cathepsin B inhibitor, CA074, blocked the conversion of endogenous APP to Aβ in isolated regulated secretory vesicles. In chromaffin cells, CA074Me (a cell permeable form of CA074) reduced by about 50% the extracellular Aβ released by the regulated secretory pathway, but CA074Me had no effect on Aβ released by the constitutive pathway. Furthermore, CA074Me inhibited processing of APP into the COOH-terminal β-secretase-like cleavage product. These results provide evidence for cathepsin B as a candidate β-secretase in regulated secretory vesicles of neuronal chromaffin cells. These findings implicate cathepsin B as β-secretase in the regulated secretory pathway of brain neurons, suggesting that inhibitors of cathepsin B may be considered as therapeutic agents to reduce Aβ in AD.

September 9, 2005

Activation processing of cathepsin H impairs recognition by its propeptide

Martin Horn, Lucie Dolečková-Marešová, Lubomír Rulíšek, Martin Máša, Olga Vasiljeva, Boris Turk, Tudeviin Gan-Erdene, Miroslav Baudyš, Michael Mareš

Page range: 941-947

Abstract

Free propeptides are known to function as inhibitors of the parental mature cysteine cathepsins. This general rule, however, does not apply to the aminopeptidase cathepsin H. Screening of propeptide fragments for their inhibitory potency revealed no significant effect on the native mature cathepsin H. On the other hand, inhibitory interaction was established with recombinant cathepsin H that displays endopeptidase activity due to a lack of the mini-chain. This finding suggests that the propeptide-binding region is structurally rearranged during maturation processing and mini-chain formation, which impairs the effective recognition of mature cathepsin H by its own propeptide.

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Objective
Biological Chemistry keeps you up-to-date with all new developments in the molecular life sciences. In addition to original research reports, authoritative reviews written by leading researchers in the field keep you informed about the latest advances in the molecular life sciences. Rapid, yet rigorous reviewing ensures fast access to recent research results of exceptional significance in the biological sciences.

Topics

general biochemistry
pathobiochemistry
structural biology
molecular and cellular biology
genetics and epigenetics
molecular medicine
cancer research
virology
immunology
plant molecular biology and biochemistry
novel experimental methodologies

Article formats
Research Articles, Short Communications, Reviews and Minireviews
Reviews are published by invitation only, but suggestions to the Editor-in-Chief are welcome.

The journal is associated with the German Society for Biochemistry and Molecular Biology (GBM).

Volume 386 Issue 9

Issue of Biological Chemistry

Paper of the Year 2004: Award to Yin Wang and Iris Lorenzi

Improving the levels of essential amino acids and sulfur metabolites in plants

Abstract

Symmetry at the active site of the ribosome: structural and functional implications

Abstract

GPCR-induced migration of breast carcinoma cells depends on both EGFR signal transactivation and EGFR-independent pathways

Abstract

Developing an effective RNA interference strategy against a plus-strand RNA virus: silencing of coxsackievirus B3 and its cognate coxsackievirus-adenovirus receptor

Abstract

NF-κB contributes to transcription of placenta growth factor and interacts with metal responsive transcription factor-1 in hypoxic human cells

Abstract

Regulation of asparagine synthetase gene transcription by the basic region leucine zipper transcription factors ATF5 and CHOP

Abstract

Role of the N-terminal region and of β-sheet residue Thr29 on the activity of the ω2 global regulator from the broad-host range Streptococcus pyogenes plasmid pSM19035

Abstract

Lysine 3 acetylation regulates the phosphorylation of yeast 6-phosphofructo-2-kinase under hypo-osmotic stress

Abstract

Determination of the specificity of monoclonal antibodies against Schistosoma mansoni CAA glycoprotein antigen using neoglycoconjugate variants

Abstract

Cholesterol is the major component of native lipoproteins activating the p38 mitogen-activated protein kinases

Abstract

Focal adhesion kinase is redistributed to focal complexes and mediates cell spreading in macrophages in response to M-CSF

Abstract

Inhibition of cathepsin B reduces β-amyloid production in regulated secretory vesicles of neuronal chromaffin cells: evidence for cathepsin B as a candidate β-secretase of Alzheimer's disease

Abstract

Activation processing of cathepsin H impairs recognition by its propeptide

Abstract

Role of the N-terminal region and of β-sheet residue Thr29 on the activity of the ω₂ global regulator from the broad-host range Streptococcus pyogenes plasmid pSM19035