bluuebunny/crossref_metadata_2025 huggingface dataset:
The generation and characterisation of antagonist RNA aptamers to MCP‐1
Authors: Rhodes Andrew , Smithers Nick , Chapman Trevor , Parsons Sarah , Rees Stephen
Published: October 2002

Monocyte chemoattractant protein‐1 (MCP‐1) has been implicated as a powerful pro‐inflammatory mediator and may represent a potentially important, therapeutic opportunity for treatment of inflammatory disease and atherosclerosis. To further investigate the role of MCP‐1 in inflammatory disorders we have isolated a series of RNA aptamers that bind specifically to mouse MCP‐1. The highest affinity aptamers, designated ADR7 and ADR22, have been functionally characterised in vitro and in cell based assays. ADR7 and ADR22 have an affinity of 180 pM and 370 pM respectively for mouse MCP‐1, they can antagonise MCP‐1 binding to heparin and specifically antagonise MCP‐1 induced chemotaxis in a cell based assay. An interesting feature of ADR22 but not ADR7 is that it is capable of antagonising the function of human MCP‐1, demonstrating the high level of specificity of these aptamers and that the aptamers recognise MCP‐1 in different ways. The aptamers may be used as a tool to further investigate the role of MCP‐1 in inflammatory disorders and may also have a role as a therapeutic agent.
Inhibition of myogenesis enables adipogenic trans‐differentiation in the C2C12 myogenic cell line
Authors: Yeow Karen , Phillips Blaine , Dani Christian , Cabane Candice , Amri Ez Zoubir , Dérijard Benoît
Published: October 2002

C2C12 cells are a well‐established model system for studying myogenesis. We examined whether inhibiting the process of myogenesis via expression of dominant negative (DN) mitogen‐activated protein kinase kinase‐3 (MKK3) facilitated the trans‐differentiation of these cells into adipocytes. Cells expressing DN MKK3 respond to rosiglitazone, resulting in adipocyte formation. The effects of rosiglitazone appear to be potentiated through peroxisome proliferator activating receptor‐γ. This trans‐differentiation is inhibited by the use of the phosphoinositide‐3 (PI3) kinase inhibitor, LY294002. These results indicate that preventing myogenesis through expression of DN MKK3 facilitates adipocytic trans‐differentiation, and involves PI3 kinase signalling.
Arabidopsis thaliana ferrochelatase‐I and ‐II are not imported into Arabidopsis mitochondria
Authors: Lister Ryan , Chew Orinda , Rudhe Charlotta , Lee May-Nee , Whelan James
Published: October 2002

Using in vitro import assays into purified mitochondria and chloroplasts we found that Arabidopsis ferrochelatase‐I and ferrochelatase‐II were not imported into mitochondria purified from Arabidopsis (or several other plants) but were imported into pea leaf chloroplasts. Other dual targeted proteins could be imported into purified mitochondria from Arabidopsis. As only two ferrochelatase genes are present in the completed Arabidopsis genome, the presence of ferrochelatase activity in plant mitochondria needs to be re‐evaluated. Previous reports of Arabidopsis ferrochelatase‐I import into pea mitochondria are due to the fact that pea leaf (and root) mitochondria appear to import a variety, but not all chloroplast proteins. Thus pea mitochondria are not a suitable system to either study dual targeting, or to distinguish between isozymes present in mitochondria and chloroplasts.
Heterologous HIV‐nef mRNA trans‐splicing: a new principle how mammalian cells generate hybrid mRNA and protein molecules
Authors: Caudevilla C. , Da Silva-Azevedo L. , Berg B. , Guhl E. , Graessmann M. , Graessmann A.
Published: October 2002

Heterologous trans‐splicing is a messenger RNA (mRNA) processing mechanism, that joins RNA segments from separate transcripts to generate functional mRNA molecules. We present here for the first time experimental evidence that the proximal segment of the HIV‐nef RNA segment can be trans‐spliced to both viral (e.g. SV40 T‐antigen) and cellular transcripts. Following either microinjection of in vitro synthesized HIV‐nef and SV40 T‐antigen pre‐mRNA or transfection of the HIV‐nef DNA into T‐antigen positive cells (CV1‐B3; Cos7), it was found that recipient cells synthesized HIV‐nef/T‐antigen hybrid mRNA and protein molecules. To generate the hybrid mRNA, the cells utilized the 5′ cryptic splice sites of the HIV‐nef (5′cry 66 and 5′cry 74) and the SV40 T/t‐antigen 3′ splice site. To demonstrate that heterologous trans‐splicing also occurs between the HIV‐nef RNA and cellular transcripts, a cDNA library was established from HIV‐nef positive CV1‐B3 cells (CV1‐B3/13 cells) and screened for hybrid mRNA molecules. Reverse transcription‐PCR and Northern blot analysis revealed that a significant portion of the HIV‐nef transcript is involved in heterologous trans‐splicing. To date, eight independent HIV‐nef/cellular hybrid mRNA molecules have been identified. Five of these isolates contain segments from known cellular genes (KIAA1454, PTPκ, Alu and transposon gene families), while three hybrid segments contain sequences of not yet known cellular genes (genes 1–3).
The twoXenopus Gbx2genes exhibit similar, but not identical expression patterns and can affect head formation
Authors: Tour Ella , Pillemer Graciela , Gruenbaum Yosef , Fainsod Abraham
Published: October 2002

Gbx2homeobox genes are important for formation and function of the midbrain/hindbrain boundary, namely the isthmic organizer. TwoGbx2genes were identified inXenopus laevis, differing in 13 amino acids, including a change in the homeodomain.Xgbx2ais activated earlier during gastrulation and reaches higher levels of expression whileXgbx2bis expressed later, at lower levels and has an additional domain in the ventral blood islands. Their overexpression results in microcephalic embryos with shortened axes and defects in brain and notochord formation. Both genes encode functionally homologous proteins, which differ primarily in their temporal and spatial expression patterns.
Activation of the extracellular signal‐regulated protein kinase (ERK) cascade by membrane‐type‐1 matrix metalloproteinase (MT1‐MMP)
Authors: Gingras Denis , Bousquet-Gagnon Nathalie , Langlois Stéphanie , Lachambre Marie-Paule , Annabi Borhane , Béliveau Richard
Published: October 2002

The mechanisms underlying membrane‐type‐1 matrix metalloproteinase (MT1‐MMP)‐dependent induction of cell migration were investigated. Overexpression of MT1‐MMP induced a marked increase in cell migration, this increase being dependent on the presence of the cytoplasmic domain of the protein. MT1‐MMP‐dependent migration was inhibited by a mitogen‐activated protein kinase kinase 1 inhibitor, suggesting the involvement of the extracellular signal‐regulated protein kinase (ERK) cascade in the induction of migration. Accordingly, MT1‐MMP overexpression induced the activation of ERK, this process being also dependent on the presence of its cytoplasmic domain. MT1‐MMP‐induced activation of both migration and ERK required the catalytic activity of the enzyme as well as attachment of the cells to matrix proteins. The MT1‐MMP‐dependent activation of ERK was correlated with the activation of transcription through the serum response element, whereas other promoters were unaffected. Taken together, these results indicate that MT1‐MMP trigger important changes in cellular signal transduction events, leading to cell migration and to gene transcription, and that these signals possibly originate from the cytoplasmic domain of the protein.
Antisense inhibition of a BRI1 receptor reveals additional protein kinase signaling components downstream to the perception of brassinosteroids in rice
Authors: Sharma Arun , Matsuoka Makoto , Tanaka Hiroshi , Komatsu Setsuko
Published: October 2002

Plants express a variety of proteins at the cell surface responsible for the transduction of regulatory information into the cell via receptors. In the present study, an attempt has been made to identify the components of the brassinosteroids (BRs) signaling transduction cascades in transgenic rice (Oryza sativa) expressing the antisense strand of OsBRI1 transcript. A 60 kDa protein, immunologically characterized as mitogen‐activated protein kinase (MAPK), showed reduced phosphorylation activity in the membrane fractions of the OsBRI1 antisense rice over control, demonstrating the inhibition in the perception of BRs by the BRI1 receptor, when compared with the exogenously applied brassinolide. The phosphorylation activity of the 50 kDa Ca2+‐dependent protein kinase was however increased in the cytosolic fractions of OsBRI1 antisense over control. The data obtained suggest that MAPK and Ca2+‐dependent protein kinase in rice are discrete but parallel signaling cascades and might involve receptors other than BRI1 in response to BR stimulus.
Tetrathiomolybdate inhibition of the Enterococcus hirae CopB copper ATPase
Authors: Bissig Karl-Dimiter , Voegelin Thomas Christophe , Solioz Marc
Published: October 2002

Tetrathiomolybdate (TTM) avidly interacts with copper and has recently been employed to reduce excess copper in patients with Wilson disease. We found that TTM inhibits the purified Enterococcus hirae CopB copper ATPase with an IC50 of 34 nM. Dithiomolybdate and trithiomolybdate, which commonly contaminate TTM, inhibited the copper ATPases with similar potency. Inhibition could be reversed by copper or silver, suggesting inhibition by substrate binding. These findings for the first time allowed an estimate of the high affinity of CopB for copper and silver. TTM is a new tool for the study of copper ATPases.
Involvement of phospholipase D in oxidative stress‐induced necrosis of vascular smooth muscle cells
Authors: Shin Eun-Young , Min Do Sik , Shin Ji-Cheol , Shin Kyung-Sun , Hyun Min-Soo , Ha Kwon-Soo , Kim Hun-Sik , Ahn Hee-Yul , Kim Eung-Gook
Published: October 2002

Phospholipase D (PLD) has been associated with necrosis. However, it is not clear whether PLD plays a causative role in this cellular process. We investigated the role of PLD in oxidative stress‐induced necrosis of vascular smooth muscle cells (VSMCs). Pervanadate (hydrogen peroxide plus orthovanadate) but not hydrogen peroxide alone activated PLD in a dose‐ and time‐dependent manner. Exposure of VSMCs to pervanadate resulted in necrosis. Pretreatment with butan‐1‐ol, a PLD inhibitor, attenuated both pervanadate‐induced necrosis and increase of intracellular Ca2+. Removal of extracellular Ca2+ inhibited pervanadate‐induced necrosis by 50%. These results suggest that PLD activation mediates pervanadate‐induced necrosis of VSMCs, which is at least partly due to Ca2+ toxicity.
Myosin VI, a new force in clathrin mediated endocytosis
Authors: Buss Folma , Luzio J.Paul , Kendrick-Jones John
Published: October 2002

The integrity of the actin cytoskeleton and associated motor proteins are essential for the efficient functioning of clathrin mediated endocytosis at least in polarised cells. Myosin VI, the only motor protein so far identified that moves towards the minus end of actin filaments, is the first motor protein to be shown to associate with clathrin coated pits/vesicles at the plasma membrane and to modulate clathrin mediated endocytosis. Recent kinetic studies suggest that myosin VI may move processively along actin filaments providing clues about its functions in the cell. The possible role(s) of myosin VI in the sequential steps involved in receptor mediated endocytosis are discussed.
Identification of a structural motif of 23S rRNA interacting with 5S rRNA
Authors: Ko Jaehyeong , Lee Younghoon , Park Inwon , Cho Bongrae
Published: October 2002

To identify RNA motifs interacting with 5S rRNA, a systematic evolution of ligands by exponential enrichment experiment was applied. Some of the resulting RNA aptamers contained a consensus sequence similar to the sequence in the loop region of helix 89 of 23S rRNA. We show that the synthetic helix 89 RNA motif indeed interacted with 5S rRNA and that the region around loop B of 5S rRNA was involved in this interaction. These results suggest the presence of a novel RNA–RNA interaction between 23S rRNA and 5S rRNA which may play an important role in the ribosome function.
Hydrogen peroxide is a regulator of ABI1, a protein phosphatase 2C from Arabidopsis
Authors: Meinhard Michael , Grill Erwin
Published: October 2002

Protein phosphatases 2C (PP2Cs) exhibit diverse regulatory functions in signalling pathways of animals, yeast and plants. ABI1 is a PP2C of Arabidopsis that exerts negative control on signalling of the phytohormone abscisic acid (ABA). Characterisation of the redox sensitivity of ABI1 revealed a strong enzymatic inactivation by hydrogen peroxide (H2O2) which has recently been implicated as a secondary messenger of ABA signalling. H2O2 reversibly inhibited ABI1 activity in vitro with an IC50 of approximately 140 μM in the presence of physiological concentrations of glutathione. In addition, ABI1 was highly susceptible to inactivation by phenylarsine oxide (IC50=3–4 μM) indicative for the facile oxidation of vicinal cysteine residues. Thus, H2O2 generated during ABA signalling seems to inactivate the negative regulator of the ABA response.
Two parameters improve efficiency of mitochondrial uptake of adenylate kinase: decreased folding velocity and increased propensity of N‐terminal α‐helix formation
Authors: Angermayr Michaela , Strobel Gertrud , Zollner Alfred , Korber Dorothee , Bandlow Wolfhard
Published: October 2002

The long isoform of eukaryotic adenylate kinase has a dual subcellular location in the cytoplasm and in the mitochondrial intermembrane space. Protein sequences and modifications are identical in both locations. In yeast, the bulk of the major form of adenylate kinase (Aky2p) is in the cytoplasm and, in the steady state, only 5–8% is sorted to the mitochondrial intermembrane space. Since the reasons for exclusion from mitochondrial import are unclear, we have constructed aky2 mutants with elevated mitochondrial uptake efficiency of Aky2p in vivo and in vitro. We have analyzed the effect of the mutations on secondary structure prediction in silico and have tested folding velocity and folding stability. One type of mutants displayed decreased proteolytic stability and retarded renaturation kinetics after chaotropic denaturation implying that deterioration of folding leads to prolonged presentation of target information to mitochondrial import receptors, thereby effecting improved uptake. In a second type of mutants, increased import efficiency was correlated with an increased probability of formation of an α‐helix with increased amphipathic moment at the N‐terminus suggesting that targeting interactions with mitochondrial import receptors had been improved at the level of binding affinity.
Clusterin/apolipoprotein J is a novel biomarker of cellular senescence that does not affect the proliferative capacity of human diploid fibroblasts
Authors: Petropoulou Chariklia , Trougakos Ioannis P. , Kolettas Evangelos , Toussaint Olivier , Gonos Efstathios S.
Published: October 2002

Normal human fibroblasts have a limited replicative potential in culture and eventually reach a state of irreversible growth arrest, termed senescence. In a previous study aiming to identify genes that are differentially regulated during cellular senescence we have cloned clusterin/apolipoprotein J (Apo J), a 80 kDa secreted glycoprotein. In the current report we pursue our studies and show that senescence of human diploid fibroblasts is accompanied by up‐regulation of both Apo J mRNA and protein levels, but with no altered biogenesis, binding partner profile or intracellular distribution of the two Apo J forms detected. To analyze the causal relationship between senescence and Apo J protein accumulation, we stably overexpressed the Apo J gene in primary as well as in SV40 T antigen‐immortalized human fibroblasts and we showed no alteration of the proliferative capacity of the transduced cells. Despite previous reports on tumor‐derived cell lines, overexpression of Apo J in human fibroblasts did not provide protection against apoptosis or growth arrest induced by hydrogen peroxide. Overall, our results suggest that Apo J overexpression does not induce senescence but it is rather a secondary consequence of the senescence phenotype. To our knowledge this is the first report that provides a functional analysis of human Apo J during replicative senescence.
A point mutant of human sphingosine kinase 1 with increased catalytic activity
Authors: M. Pitson Stuart , Moretti Paul A.B. , Zebol Julia R. , Vadas Mathew A. , D'Andrea Richard J. , Wattenberg Binks W.
Published: October 2002

Sphingosine kinase (SK) catalyses the formation of sphingosine 1‐phosphate, a lipid second messenger that has been implicated in mediating such fundamental biological processes as cell growth and survival. Very little is currently known regarding the structure or mechanisms of catalysis and activation of SK. Here we have tested the functional importance of Gly113, a highly conserved residue of human sphingosine kinase 1 (hSK), by site‐directed mutagenesis. Surprisingly, a Gly113→Ala substitution generated a mutant that had 1.7‐fold greater catalytic activity than wild‐type hSK (hSKWT). Our data suggests that the Gly113→Ala mutation increases catalytic efficiency of hSK, probably by inducing a conformational change that increases the efficiency of phosphoryl transfer. Interestingly, hSKG113A activity could be stimulated in HEK293T cells by cell agonists to a comparable extent to hSKWT.
Phosphatidylglycerophosphate synthases from Arabidopsis thaliana
Authors: Müller Frank , Frentzen Margrit
Published: October 2002

Two Arabidopsis thaliana genes were shown to encode phosphatidylglycerophosphate synthases (PGPS) of 25.4 and 32.2 kDa, respectively. Apart from their N‐terminal regions, the two proteins exhibit high sequence similarity. Functional expression studies in yeast provided evidence that the 25.4 kDa protein is a microsomal PGPS while the 32.2 kDa protein represents a preprotein which can be imported into yeast mitochondria and processed to a mature PGPS. The two isozymes were solubilized and purified as fusion proteins carrying a His tag at their C‐terminus. Enzyme assays with both membrane fractions and purified enzyme fractions revealed that the two A. thaliana isozymes have similar properties but differ in their CDP‐diacylglycerol species specificity.
A new family of CoA‐transferases
Authors: Heider Johann
Published: October 2002

CoA‐transferases are found in organisms from all lines of descent. Most of these enzymes belong to two well‐known enzyme families, but recent work on unusual biochemical pathways of anaerobic bacteria has revealed the existence of a third family of CoA‐transferases. The members of this enzyme family differ in sequence and reaction mechanism from CoA‐transferases of the other families. Currently known enzymes of the new family are a formyl‐CoA: oxalate CoA‐transferase, a succinyl‐CoA: (R)‐benzylsuccinate CoA‐transferase, an (E)‐cinnamoyl‐CoA: (R)‐phenyllactate CoA‐transferase, and a butyrobetainyl‐CoA: (R)‐carnitine CoA‐transferase. In addition, a large number of proteins of unknown or differently annotated function from Bacteria, Archaea and Eukarya apparently belong to this enzyme family. Properties and reaction mechanisms of the CoA‐transferases of family III are described and compared to those of the previously known CoA‐transferases.
Differential localization of non‐muscle myosin II isoforms and phosphorylated regulatory light chains in human MRC‐5 fibroblasts
Authors: Saitoh Takayuki , Takemura Shuhei , Ueda Kozue , Hosoya Hiroshi , Nagayama Masafumi , Haga Hisashi , Kawabata Kazushige , Yamagishi Akihiko , Takahashi Masayuki
Published: October 2002

We investigated the localization of non‐muscle myosin II isoforms and mono‐ (at serine 19) and diphosphorylated (at serine 19 and threonine 18) regulatory light chains (RLCs) in motile and non‐motile MRC‐5 fibroblasts. In migrating cells, myosin IIA localized to the lamella and throughout the posterior region. Myosin IIB colocalized with myosin IIA to the posterior region except at the very end. Diphosphorylated RLCs were detected in the restricted region where myosin IIA was enriched. In non‐motile cells, myosin IIA was enriched in peripheral stress fibers with diphosphorylated RLCs, but myosin IIB was not. Our results suggest that myosin IIA may be highly activated by diphosphorylation of RLCs and primarily involved in cell migration.
F‐actin assembly in Dictyostelium cell locomotion and shape oscillations propagates as a self‐organized reaction–diffusion wave
Authors: Vicker Michael G
Published: October 2002

The crawling locomotion and shape of eukaryotic cells have been associated with the stochastic molecular dynamics of actin and its protein regulators, chiefly Arp2/3 and Rho family GTPases, in making a cytoskeleton meshwork within cell extensions. However, the cell's actin‐dependent oscillatory shape and extension dynamics may also yield insights into locomotory mechanisms. Confocal observations of live Dictyostelium cells, expressing a green fluorescent protein–actin fusion protein, demonstrate oscillating supramolecular patterns of filamentous actin throughout the cell, which generate pseudopodia at the cell edge. The distinctively dissipative spatio‐temporal behavior of these structures provides strong evidence that reversible actin filament assembly propagates as a self‐organized, chemical reaction–diffusion wave.
Differing substrate specificities of members of the DYRK family of arginine‐directed protein kinases
Authors: Campbell Linda E , Proud Christopher G
Published: October 2002

The mammalian DYRK (dual specificity tyrosine phosphorylated and regulated kinase) family of protein kinases comprises a number of related, but poorly understood enzymes. DYRK1A is nuclear while DYRKs 2 and 3 are cytoplasmic. We recently showed that DYRK2 phosphorylates the translation initiation factor eIF2B at Ser539 in its ϵ‐subunit and thereby ‘primes’ its phosphorylation by glycogen synthase kinase‐3. Here we have used peptides based on the sequence around Ser539 to help define the specificity of DYRK2/3 in comparison with DYRK1A. These kinases require an arginine N‐terminal to the target residue for efficient substrate phosphorylation. This cannot be replaced even by lysine. A peptide with arginine at −2 is phosphorylated much less well by all three kinases than one with arginine at −3. Replacement of the +1 proline by alanine almost completely eliminates substrate phosphorylation, but valine here does allow phosphorylation especially by DYRK2. This study reveals both similarities and differences in the specificities of these arginine‐dependent protein kinases.
Expression and functional role of bTRPC1 channels in native endothelial cells
Authors: Antoniotti Susanna , Lovisolo Davide , Fiorio Pla Alessandra , Munaron Luca
Published: October 2002

We have analyzed the expression and localization of bovine transient receptor potential‐C1 (bTRPC1) in bovine aortic endothelial cells, and its possible involvement in the store‐independent calcium influx induced by basic fibroblast growth factor (bFGF). RT‐PCR experiments confirmed the existence of two btrpc1 mRNA isoforms; conversely, the btrpc3 gene was not transcribed. Anti‐TRPC1 antibody revealed the presence of the protein in the membrane‐rich compartment only. Application of anti‐TRPC1 during the response to bFGF caused a partial but significant reduction of calcium entry. This is the first evidence of TRP channel involvement in a non‐capacitative calcium influx induced by a biologically relevant agonist such as the angiogenic factor bFGF in native endothelial cells.
A strategy for the generation of non‐aggregating mutants of Anthozoa fluorescent proteins
Authors: Yanushevich Yurii G. , Staroverov Dmitry B. , Savitsky Alexander P. , Fradkov Arkady F. , Gurskaya Nadya G. , Bulina Maria E. , Lukyanov Konstantin A. , Lukyanov Sergey A.
Published: October 2002

Recently, we cloned several fluorescent proteins of different colors homologous to Aequorea victoria green fluorescent protein, which have great biotechnological potential as in vivo markers of gene expression. However, later investigations revealed severe drawbacks in the use of novel fluorescent proteins (FPs), in particular, the formation of tetramers (tetramerization) and high molecular weight aggregates (aggregation). In this report, we employ a mutagenic approach to resolve the problem of aggregation. The elimination of basic residues located near the N‐termini of FPs results in the generation of non‐aggregating versions of several FPs, specifically, drFP583 (DsRed), DsRed‐Timer, ds/drFP616, zFP506, zFP538, amFP486, and asFP595.
Insulin receptor activation inhibits insulin secretion from human islets of Langerhans
Authors: Persaud Shanta J , Asare-Anane Henry , Jones Peter M
Published: October 2002

There is no consensus on the role of insulin secreted from pancreatic β‐cells in regulating its own secretion, either in rodent islets or in human islets. We have now investigated whether there is an autocrine signalling role for insulin in human islets by determining insulin receptor expression and assessing the effects of insulin receptor activation using a non‐peptidyl insulin mimetic termed L‐783,281. Human insulin receptor mRNA was detected by PCR amplification of human islet cDNA, and translation of the message in human islets was confirmed by Western blotting. Perifusion experiments revealed that both glucose‐stimulated and basal insulin secretion were significantly inhibited following human islet insulin receptor activation with L‐783,281, and that signalling through phosphatidylinositol 3‐kinase (PI 3‐kinase) was responsible, at least in part, for this inhibitory effect. These studies indicate that human islets express insulin receptors and that they are functionally coupled to a PI 3‐kinase‐dependent inhibition of insulin secretion.
Protein domain analysis in the era of complete genomes
Authors: Copley Richard R. , Doerks Tobias , Letunic Ivica , Bork Peer
Published: October 2002

Domains present one of the most useful levels at which to understand protein function, and domain family‐based analysis has had a profound impact on the study of individual proteins. Protein domain discovery has been progressing steadily over the past 30 years. What are the realistically achievable goals of sequence‐based domain analysis, and how far off are they for the sequences encoded in eukaryotic genomes? Here we address some of the issues involved in better coverage of sequence‐based domain annotation, and the integration of these results within the wider context of genomes, structures and function.
Normalization of nomenclature for peptide motifs as ligands of modular protein domains
Authors: Aasland Rein , Abrams Charles , Ampe Christophe , Ball Linda J. , Bedford Mark T. , Cesareni Gianni , Gimona Mario , Hurley James H. , Jarchau Thomas , Lehto Veli-Pekka , Lemmon Mark A. , Linding Rune , Mayer Bruce J. , Nagai Makoto , Sudol Marius , Walter Ulrich , Winder Steve J.
Published: October 2002

We propose a normalization of symbols and terms used to describe, accurately and succinctly, the detailed interactions between amino acid residues of pairs of interacting proteins at protein:protein (or protein:peptide) interfaces. Our aim is to unify several diverse descriptions currently in use in order to facilitate communication in the rapidly progressing field of signaling by protein domains. In order for the nomenclature to be convenient and widely used, we also suggest a parallel set of symbols restricted to the ASCII format allowing accurate parsing of the nomenclature to a computer‐readable form. This proposal will be reviewed in the future and will therefore be open for the inclusion of new rules, modifications and changes.
Signaling to the Rho GTPases: networking with the DH domain
Authors: Hoffman Gregory R , Cerione Richard A
Published: October 2002

The Dbl homology (DH) domain was first identified in the Dbl oncogene product as the limit region required for mediating guanine nucleotide exchange on the Rho family GTPase Cdc42. Since the initial biochemical characterization of the DH domain, this conserved motif has been identified in a large family of proteins. In each case, a pleckstrin homology (PH) domain immediately follows the DH domain and this tandem DH–PH module is the signature motif of the Dbl family of guanine nucleotide exchange factors (GEFs). Recent structural studies have provided significant insight into the molecular basis of guanine nucleotide exchange by Dbl family GEFs, opening the door for understanding the specificity of the DH/GTPase interaction as well as providing a starting point for understanding how the exchange activity of these proteins is modulated to achieve specific biological outcomes in the cell.
Point mutations confer loss of ATP‐induced human P2X7 receptor function
Authors: Worthington R.A. , Smart M.L. , Gu B.J. , Williams D.A. , Petrou S. , Wiley J.S. , Barden J.A.
Published: October 2002

Residues considered essential for ATP binding to the human P2X7 receptor (hP2X7R) were investigated. HEK293 cells or Xenopus oocytes were transfected with wild‐type or site‐directed mutants of hP2X7R constructs and channel/pore activity measured in the presence of ATP or 2′,3′‐O‐(4‐benzoylbenzoyl)‐ATP (BzATP). Barium uptake and ethidium influx into HEK293 cells were abolished in cells expressing K193A and K311A mutants, and were partially reduced in cells expressing mutant P210A. K193A and K311A mutations also completely abolished responses to ATP and BzATP in Xenopus oocytes as measured by electrophysiology. These results indicate that K193 and K311 are essential residues in ATP binding in the hP2X7R.
Conversion of L‐tryptophan to serotonin and melatonin in human melanoma cells
Authors: Slominski Andrzej , Semak Igor , Pisarchik Alexander , Sweatman Trevor , Szczesniewski Andre , Wortsman Jacobo
Published: October 2002

We showed in human melanoma cells tryptophan hydroxylase (TPH) and hydroxyindole methyltransferase genes expression with the sequential enzymatic activities of TPH, serotonin (Ser) N‐acetyltransferase and hydroxyindole methyltransferase. The presence of the products Ser, 5OH‐tryptophan, N‐acetylserotonin, melatonin (Mel), 5‐methoxytryptamine and 5‐methoxytryptophol was documented by liquid chromatography–mass spectrometry. Thus, human melanoma cells can synthesize and metabolize Ser and Mel.
Carbonyl formation on a copper‐bound prion protein fragment, PrP23–98, associated with its dopamine oxidase activity
Authors: Shiraishi Noriyuki , Nishikimi Morimitsu
Published: October 2002

The amino‐terminal part of prion protein (PrP), containing a series of octapeptide repeats with the consensus sequence PHGGGWGQ, has been implicated in the binding of copper ion. This region possesses amino acid residues susceptible to oxidation, such as histidine, lysine, arginine and proline. In this study, we have investigated copper‐catalyzed oxidation of an N‐terminal part of human PrP, PrP23–98, that was prepared by the recombinant DNA technique. Carbonyl formations on copper‐bound PrP23–98 induced by dopamine and L‐ascorbate were analyzed kinetically, and it was found that the redox cycling of PrP23–98‐bound copper, especially induced by dopamine, was coupled to the formation of carbonyls on the protein.
Frataxin knockin mouse
Authors: Miranda Carlos J , Santos Manuela M , Ohshima Keiichi , Smith Julie , Li Liangtao , Bunting Michaeline , Cossée Mireille , Koenig Michael , Sequeiros Jorge , Kaplan Jerry , Pandolfo Massimo
Published: October 2002

Friedreich ataxia is the consequence of frataxin deficiency, most often caused by a GAA repeat expansion in intron 1 of the corresponding gene. Frataxin is a mitochondrial protein involved in iron homeostasis. As an attempt to generate a mouse model of the disease, we introduced a (GAA)230 repeat within the mouse frataxin gene by homologous recombination. GAA repeat knockin mice were crossed with frataxin knockout mice to obtain double heterozygous mice expressing 25–36% of wild‐type frataxin levels. These mice were viable and did not develop anomalies of motor coordination, iron metabolism or response to iron loading. Repeats were meiotically and mitotically stable.
Single synonymous codon substitution eliminates pausing during chloramphenicol acetyl transferase synthesis on Escherichia coli ribosomes in vitro
Authors: Ramachandiran Vasanthi , Kramer Gisela , Horowitz Paul M , Hardesty Boyd
Published: October 2002

The coding sequence for chloramphenicol acetyl transferase (CAT) contains several rare codons; three of them are ATA encoding isoleucine in positions 13, 84 and 119 of the amino acid sequence. Expression of CAT on Escherichia coli ribosomes in vitro results in mostly full‐length product but also distinct smaller polypeptides from less than 3 kDa to over 20 kDa. As reported earlier, the smaller polypeptides are the predominant products, if translation is initiated with fluorophore‐Met‐tRNAf. All this translational pausing is eliminated when the first ATA codon is mutated to ATC, a frequently used codon for isoleucine in E. coli. Addition of large amounts of E. coli tRNA to the coupled transcription/translation reaction does not reduce the number of pause‐site peptides seen in the expression of wild‐type CAT. Thus we hypothesize that the mRNA structure may be an important determinant for translational pausing.
SNARE expression and distribution during 3T3‐L1 adipocyte differentiation
Authors: Torrejón-Escribano Benjamı́n , Gómez de Aranda Inmaculada , Blasi Juan
Published: October 2002

Differentiation of 3T3‐L1 cells into adipocytes presupposes the expression of the glucose transporter isoform GLUT4 and the acquisition of insulin‐dependent GLUT4 translocation from intracellular storage vesicles to plasma membrane. This ability to translocate GLUT4 depends on the presence of a set of proteins of the SNARE category that are essential in the fusion step. The expression and levels of some of these SNARE proteins are altered during 3T3‐L1 differentiation. Levels of the v‐SNARE protein cellubrevin and of the t‐SNARE protein syntaxin 4 were increased in this process in parallel to GLUT4. However, the levels of SNAP‐23, another t‐SNARE, were maintained during differentiation. Immunofluorescence images of SNAP‐23 showed the initial distribution of this protein in a perinuclear region before differentiation and its redistribution towards plasma membrane in the adipocyte form. These results suggest a capital role in the expression levels and cellular distribution, during 3T3‐L1 differentiation, of SNARE proteins involved in the late steps of GLUT4 translocation.
Pin1 modulates the dephosphorylation of the RNA polymerase II C‐terminal domain by yeast Fcp1
Authors: Kops Oliver , Zhou Xiao Zhen , Lu Kun Ping
Published: October 2002

The reversible phosphorylation of serine and threonine residues N‐terminal to proline (pSer/Thr–Pro) is an important signaling mechanism in the cell. The pSer/Thr–Pro moiety exists in the two distinct cis and trans conformations, whose conversion is catalyzed by the peptidyl–prolyl isomerase (PPIase) Pin1. Among others, Pin1 binds to the phosphorylated C‐terminal domain (CTD) of the largest subunit of the RNA polymerase II, but the biochemical and functional relevance of this interaction is unknown. Here we confirm that the CTD phosphatase Fcp1 can suppress a Pin1 mutation in yeast. Furthermore, this genetic interaction requires the phosphatase domain as well as the BRCT domain of Fcp1, suggesting a critical role of the Fcp1 localization. Based on these observations, we developed a new in vitro assay to analyze the CTD dephosphorylation by Fcp1 that uses only recombinant proteins and mimics the in vivo situation. This assay allows us to present strong evidence that Pin1 is able to stimulate CTD dephosphorylation by Fcp1 in vitro, and that this stimulation depends on Pin1's PPIase activity. Finally, Pin1 significantly increased the dephosphorylation of the CTD on the Ser5–Pro motif, but not on Ser2–Pro in yeast, which can be explained with Pin1's substrate specificity. Together, our results indicate a new role for Pin1 in the regulation of CTD phosphorylation and present a further example for prolyl isomerization‐dependent protein dephosphorylation.
Transit of tRNA through the Escherichia coli ribosome: cross‐linking of the 3′ end of tRNA to ribosomal proteins at the P and E sites
Authors: Kirillov Stanislav V , Wower Jacek , Hixson Stephen S , Zimmermann Robert A
Published: October 2002

Photoreactive derivatives of yeast tRNAPhe containing 2‐azidoadenosine at their 3′ termini were used to trace the movement of tRNA across the 50S subunit during its transit from the P site to the E site of the 70S ribosome. When bound to the P site of poly(U)‐programmed ribosomes, deacylated tRNAPhe, Phe‐tRNAPhe and N‐acetyl‐Phe‐tRNAPhe probes labeled protein L27 and two main sites within domain V of the 23S RNA. In contrast, deacylated tRNAPhe bound to the E site in the presence of poly(U) labeled protein L33 and a single site within domain V of the 23S rRNA. In the absence of poly(U), the deacylated tRNAPhe probe also labeled protein L1. Cross‐linking experiments with vacant 70S ribosomes revealed that deacylated tRNA enters the P site through the E site, progressively labeling proteins L1, L33 and, finally, L27. In the course of this process, tRNA passes through the intermediate P/E binding state. These findings suggest that the transit of tRNA from the P site to the E site involves the same interactions, but in reverse order. Moreover, our results indicate that the final release of deacylated tRNA from the ribosome is mediated by the F site, for which protein L1 serves as a marker. The results also show that the precise placement of the acceptor end of tRNA on the 50S subunit at the P and E sites is influenced in subtle ways both by the presence of aminoacyl or peptidyl moieties and, more surprisingly, by the environment of the anticodon on the 30S subunit.
Cadherin‐like receptor binding facilitates proteolytic cleavage of helix α‐1 in domain I and oligomer pre‐pore formation of Bacillus thuringiensis Cry1Ab toxin
Authors: Gómez Isabel , Sánchez Jorge , Miranda Raúl , Bravo Alejandra , Soberón Mario
Published: October 2002

Cry toxins form lytic pores in the insect midgut cells. The role of receptor interaction in the process of protoxin activation was analyzed. Incubation of Cry1Ab protoxin with a single chain antibody that mimics the cadherin‐like receptor and treatment with Manduca sexta midgut juice or trypsin, resulted in toxin preparations with high pore‐forming activity in vitro. This activity correlates with the formation of a 250 kDa oligomer that lacks the helix α‐1 of domain I. The oligomer, in contrast with the 60 kDa monomer, was capable of membrane insertion as judged by 8‐anilino‐1‐naphthalenesulfonate binding. Cry1Ab protoxin was also activated to a 250 kDa oligomer by incubation with brush border membrane vesicles, presumably by the action of a membrane‐associated protease. Finally, a model where receptor binding allows the efficient cleavage of α‐1 and formation of a pre‐pore oligomeric structure that is efficient in pore formation, is presented.
Cysteinyl‐tRNA formation and prolyl‐tRNA synthetase
Authors: Jacquin-Becker Clarisse , Ahel Ivan , Ambrogelly Alexandre , Ruan Benfang , Söll Dieter , Stathopoulos Constantinos
Published: October 2002

Aminoacyl‐tRNA (AA‐tRNA) formation is a key step in protein biosynthesis. This reaction is catalyzed with remarkable accuracy by the AA‐tRNA synthetases, a family of 20 evolutionarily conserved enzymes. The lack of cysteinyl‐tRNA (Cys‐tRNA) synthetase in some archaea gave rise to the discovery of the archaeal prolyl‐tRNA (Pro‐tRNA) synthetase, an enzyme capable of synthesizing Pro‐tRNA and Cys‐tRNA. Here we review our current knowledge of this fascinating process.
The putative effector‐binding site of Leishmania mexicana pyruvate kinase studied by site‐directed mutagenesis
Authors: Hannaert Véronique , Yernaux Cédric , Rigden Daniel J , Fothergill-Gilmore Linda A , Opperdoes Fred R , Michels Paul A.M
Published: October 2002

The activity of pyruvate kinase of Leishmania mexicana is allosterically regulated by fructose 2,6‐bisphosphate (F‐2,6‐P2), contrary to the pyruvate kinases from other eukaryotes that are usually stimulated by fructose 1,6‐bisphosphate (F‐1,6‐P2). Based on the comparison of the three‐dimensional structure of Saccharomyces cerevisiae pyruvate kinase crystallized with F‐1,6‐P2 present at the effector site (R‐state) and the L. mexicana enzyme crystallized in the T‐state, two residues (Lys453 and His480) were proposed to bind the 2‐phospho group of the effector. This hypothesis was tested by site‐directed mutagenesis. The allosteric activation by F‐2,6‐P2 appeared to be entirely abrogated in the mutated enzymes confirming our predictions.
Accelerated α‐synuclein fibrillation in crowded milieu
Authors: Uversky Vladimir N , M. Cooper Elisa , Bower Kiowa S , Li Jie , Fink Anthony L
Published: October 2002

Parkinson's disease is the second most common age‐related neurodegenerative disease, resulting from loss of dopaminergic neurons in the substantia nigra. The aggregation and fibrillation of α‐synuclein has been implicated as a causative factor in the disease, and the process of fibril formation has been intensively studied in vitro with dilute protein solutions. However, the intracellular environment of proteins is crowded with other macromolecules, whose concentration can reach 400 g/l. To address this discrepancy, the effect of molecular crowding on α‐synuclein fibrillation has being studied. The addition of high concentrations of different polymers (proteins, polysaccharides and polyethylene glycols) dramatically accelerated α‐synuclein fibrillation in vitro. The magnitude of the accelerating effect depended on the nature of the polymer, its length and concentration. Our results suggest that the major factor responsible for the accelerated fibrillation under crowded conditions is the excluded volume.
Extended longevity of Caenorhabditis elegans by knocking in extra copies of hsp70F, a homolog of mot‐2 (mortalin)/mthsp70/Grp75
Authors: Yokoyama Ken , Fukumoto Keiji , Murakami Tatsuya , Harada Shin-ichi , Hosono Ryuji , Wadhwa Renu , Mitsui Youji , Ohkuma Shoji
Published: October 2002

The Caenorhabditis elegans homolog of mortalin/mthsp70/Grp75 (called mot‐2 hereafter) was isolated by screening of a nematode cDNA library with mouse mot‐2 cDNA. The isolated clone matched to hsp70F of C. elegans. Analysis with two of the antibodies raised against hsp70F revealed that unlike mammalian mot‐2, it is heat inducible. Transient induction of hsp70F by heat shock led to a slight (<13%) extension in the C. elegans life span. The transgenic worms that constitutively over‐expressed hsp70F predominantly in muscle showed life span extension (∼43% for mean and ∼45% for maximum life span) as compared to the wild‐type and green fluorescent protein‐transgenic worms. Life span extension of human cells was obtained by over‐expression of mot‐2 [Kaul et al. (2000) FEBS Lett. 474, 159–164]. Our results show, for the first time, that this member of the hsp70 family governs the longevity of worms and thus there are common pathways that determine mammalian and worm longevity.
Synthesis of novel internal standards for the quantitative determination of plasma ceramide trihexoside in Fabry disease by tandem mass spectrometry
Authors: Mills Kevin , Johnson Andrew , Winchester Bryan
Published: October 2002

The concentration of globotriaosylceramide (ceramide trihexoside (CTH)) in the plasma of patients with Fabry disease has been determined quantitatively by tandem mass spectrometry (MS) using novel internal standards, [D4]C‐16 CTH and C‐17 CTH, which were synthesised enzymically from lyso‐CTH using the reverse reaction of sphingolipid ceramide N‐deacylase. C‐17 CTH was also synthesised chemically from lyso‐CTH. This strategy has also been used to prepare standards for the quantitative determination by MS of other glycosphingolipids.
Lamellipodial localization of Dictyostelium myosin heavy chain kinase A is mediated via F‐actin binding by the coiled‐coil domain
Authors: Steimle Paul A , Licate Lucila , Côté Graham P , Egelhoff Thomas T
Published: October 2002

Myosin heavy chain kinase A (MHCK A) modulates myosin II filament assembly in the amoeba Dictyostelium discoideum. MHCK A localization in vivo is dynamically regulated during chemotaxis, phagocytosis, and other polarized cell motility events, with preferential recruitment into anterior filamentous actin (F‐actin)‐rich structures. The current work reveals that an amino‐terminal segment of MHCK A, previously identified as forming a coiled‐coil, mediates anterior localization. MHCK A co‐sediments with F‐actin, and deletion of the amino‐terminal domain eliminated actin binding. These results indicate that the anterior localization of MHCK A is mediated via direct binding to F‐actin, and reveal the presence of an actin‐binding function not previously detected by primary sequence evaluation of the coiled‐coil domain.
Human fibroblasts in vitro senesce with a donor‐specific telomere length
Authors: Serra Violeta , von Zglinicki Thomas
Published: October 2002

In human fibroblasts, replicative senescence, telomere length and donor age are closely interrelated. We analyzed these relationships for fibroblast strains from 14 healthy human donors in the age range of 28–90 years. In vitro replicative capacity was correlated more closely with donor age than with telomere length ex vivo, especially for healthy donors. Telomere length at senescence was as variable as at cell explantation and increased with donor age. The data suggest a donor‐specific, age‐dependent regulation of the telomere length threshold that triggers senescence in human fibroblasts.
Presence of a novel subset of NKT cells bearing an invariant Vα19.1–Jα26 TCR α chain
Authors: Shimamura Michio , Huang Yi-Ying
Published: October 2002

CD1d‐deficient (CD1d−/−) mouse lymphocytes were analyzed to classify the natural killer T (NKT) cells without reactivity to CD1d. The cells bearing a Vα19.1–Jα26 (AV19–AJ33) invariant TCR α chain, originally found in the peripheral blood lymphocytes, were demonstrated to be abundant in the NK1.1+ but not NK1.1− T cell population isolated from CD1d−/− mice. Moreover, more than half (11/21) of the hybrid cell lines established from CD1d−/− NKT cells expressed the Vα19.1–Jα26 invariant TCR α chain. The expression of the invariant Vα19.1–Jα26 mRNA was absent in β2‐microglobulin‐deficient mice. Collectively, the present findings suggest the presence of a second NKT cell repertoire characterized by an invariant TCR α chain (Vα19.1–Jα26) that is selected by an MHC class I‐like molecule other than CD1d.
Sucrose metabolism: Anabaena sucrose‐phosphate synthase and sucrose‐phosphate phosphatase define minimal functional domains shuffled during evolution
Authors: Cumino Andrea , Curatti Leonardo , Giarrocco Laura , Salerno Graciela L.
Published: October 2002

Based on the functional characterization of sucrose biosynthesis related proteins [SBP: sucrose‐phosphate synthase (SPS), sucrose‐phosphate phosphatase (SPP), and sucrose synthase (SuS)] in Anabaena sp. PCC7120 and sequence analysis, we have shown that SBP are restricted to cyanobacterium species and plants, and that they are multidomain proteins with modular architecture. Anabaena SPS, a minimal catalytic SPS unit, defines a glucosyltransferase domain present in all SPSs and SuSs. Similarly, Anabaena SPP defines a phosphohydrolase domain characteristic of all SPPs and some SPSs. Phylogenetic analysis points towards the evolution of modern cyanobacterial and plant SBP from a bidomainal common ancestral SPS‐like gene.
The nascent polypeptide‐associated complex (NAC) promotes interaction of ribosomes with the mitochondrial surface in vivo
Authors: George Rebecca , Walsh Peter , Beddoe Travis , Lithgow Trevor
Published: October 2002

The nascent polypeptide‐associated complex (NAC) is a peripheral component of cytoplasmic ribosomes, and interacts with nascent chains as they leave the ribosome. Yeast mutants lacking NAC translate polypeptides normally, but have fewer ribosomes associated with the mitochondrial surface. The mutants lacking NAC suffer mitochondrial defects and have decreased levels of proteins like fumarase, normally targeted to mitochondria co‐translationally. NAC might contribute to a ribosomal environment in which amino‐terminal, mitochondrial targeting sequences can effectively adopt their appropriate conformation.