bluuebunny/crossref_metadata_2025 huggingface dataset:
Identification and characterization of DdPDE3, a cGMP-selective phosphodiesterase from Dictyostelium
Authors: KUWAYAMA Hidekazu , SNIPPE Helena , DERKS Mari , ROELOFS Jeroen , HAASTERT Peter J. M. VAN
Published: August 2015

In Dictyostelium cAMP and cGMP have important functions as first and second messengers in chemotaxis and development. Two cyclic-nucleotide phosphodiesterases (DdPDE 1 and 2) have been identified previously, an extracellular dual-specificity enzyme and an intracellular cAMP-specific enzyme (encoded by the psdA and regA genes respectively). Biochemical data suggest the presence of at least one cGMP-specific phosphodiesterase (PDE) that is activated by cGMP. Using bioinformatics we identified a partial sequence in the Dictyostelium expressed sequence tag database that shows a high degree of amino acid sequence identity with mammalian PDE catalytic domains (DdPDE3). The deduced amino acid sequence of a full-length DdPDE3 cDNA isolated in this study predicts a 60kDa protein with a 300-residue C-terminal PDE catalytic domain, which is preceded by approx. 200 residues rich in asparagine and glutamine residues. Expression of the DdPDE3 catalytic domain in Escherichia coli shows that the enzyme has Michaelis–Menten kinetics and a higher affinity for cGMP (Km = 0.22µM) than for cAMP (Km = 145µM); cGMP does not stimulate enzyme activity. The enzyme requires bivalent cations for activity; Mn2+ is preferred to Mg2+, whereas Ca2+ yields no activity. DdPDE3 is inhibited by 3-isobutyl-1-methylxanthine with an IC50 of approx. 60µM. Overexpression of the DdPDE3 catalytic domain in Dictyostelium confirms these kinetic properties without indications of its activation by cGMP. The properties of DdPDE3 resemble those of mammalian PDE9, which also shows the highest sequence similarity within the catalytic domains. DdPDE3 is the first cGMP-selective PDE identified in lower eukaryotes.
Multiple polyamine transport systems on the vacuolar membrane in yeast
Authors: TOMITORI Hideyuki , KASHIWAGI Keiko , ASAKAWA Tomoko , KAKINUMA Yoshimi , MICHAEL Anthony J. , IGARASHI Kazuei
Published: August 2015

We recently identified a gene (TPO1, YLL028w) that encodes a polyamine transport protein on the vacuolar membrane in yeast [Tomitori, Kashiwagi, Sakata, Kakinuma and Igarashi (1999) J. Biol. Chem. 274, 3265–3267]. Because the existence of one or more other genes for a polyamine transport protein on the vacuolar membrane was expected, we searched sequence databases for homologues of the protein encoded by TPO1. Membrane proteins encoded by the open reading frames YGR138c (TPO2), YPR156c (TPO3) and YOR273c (TPO4) were postulated to be polyamine transporters and, indeed, were subsequently shown to be polyamine transport proteins on the vacuolar membrane. Cells overexpressing these genes were resistant to polyamine toxicity and showed an increase in polyamine uptake activity and polyamine content in vacuoles. Furthermore, cells in which these genes were disrupted showed an increased sensitivity to polyamine toxicity and a decrease in polyamine uptake activity and polyamine content in vacuoles. Resistance to polyamine toxicity in cells overexpressing the genes was overcome by bafilomycin A1, an inhibitor of the vacuolar H+-ATPase. Among the four polyamine transporters, those encoded by TPO2 and TPO3 were specific for spermine, whereas those encoded by TPO1 and TPO4 recognized spermidine and spermine. These results suggest that polyamine content in the cytoplasm of yeast is elaborately regulated by several polyamine transport systems in vacuoles. Furthermore, it was shown that Glu-207, Glu-324 (or Glu-323) and Glu-574 of TPO1 protein were important for the transport activity.
Anginex, a designed peptide that inhibits angiogenesis
Authors: GRIFFIOEN Arjan W. , VAN DER SCHAFT Daisy W. J. , BARENDSZ-JANSON Annemarie F. , COX Andrew , BOUDIER Harry A. J. STRUIJKER , HILLEN Harry F. P. , MAYO Kevin H.
Published: August 2015

Novel β-sheet-forming peptide 33-mers, βpep peptides, have been designed by using a combination approach employing basic folding principles and incorporating short sequences from the β-sheet domains of anti-angiogenic proteins. One of these designed peptides (βpep-25), named anginex, was observed to be potently anti-angiogenic. Anginex specifically inhibits vascular endothelial cell proliferation and induces apoptosis in these cells, as shown by flow-cytometric detection of sub-diploid cells, TUNEL (terminal deoxyribonucleotidyl transferase-mediated dUTP-nick-end labelling) analysis and cell morphology. Anginex also inhibits endothelial cell adhesion to and migration on different extracellular matrix components. Inhibition of angiogenesis in vitro is demonstrated in the sprout-formation assay and in vivo in the chick embryo chorio-allantoic membrane angiogenesis assay. Comparison of active and inactive βpep sequences allows structure–function relationships to be deduced. Five hydrophobic residues and two lysines appear to be crucial to activity. This is the first report of a designed peptide having a well-defined biological function as a novel cytokine, which may be an effective anti-angiogenic agent for therapeutic use against various pathological disorders, such as neoplasia, rheumatoid arthritis, diabetic retinopathy and restenosis.
Inositol phosphates from barley low-phytate grain mutants analysed by metal-dye detection HPLC and NMR
Authors: HATZACK Frank , HÜBEL Frank , ZHANG Wei , HANSEN Poul E. , RASMUSSEN Søren K.
Published: August 2015

Inositol phosphates from barley low-phytate grain mutants and their parent variety were analysed by metal-dye detection HPLC and NMR. Compound assignment was carried out by comparison of retention times using a chemical hydrolysate of phytate [Ins(1,2,3,4,5,6)P6] as a reference. Co-inciding retention times indicated the presence of phytate, D/L-Ins(1,2,3,4,5)P5, Ins(1,2,3,4,6)P5, D/L-(1,2,4,5,6)P5, D/L-(1,2,3,4)P4, D/L-Ins(1,2,5,6)P4 and D/L-Ins(1,4,5,6)P4 in PLP1B mutants as well as the parent variety. In grain extracts from mutant lines PLP1A, PLP2A and PLP3A unusual accumulations of D/L-Ins(1,3,4,5)P4 were observed whereas phytate and the above-mentioned inositol phosphates were present in relatively small amounts. Assignment of D/L-Ins(1,3,4,5)P4 was corroborated by precise co-chromatography with a commercial Ins(1,3,4,5)P4 standard and by NMR spectroscopy. Analysis of inositol phosphates during grain development revealed accumulation of phytate and D/L-Ins(1,3,4,5)P4, which suggested the tetrakisphosphate compound to be an intermediate of phytate synthesis. This assumption was strengthened further by phytate degradation assays showing that D/L-Ins(1,3,4,5)P4 did not belong to the spectrum of degradation products generated by endogenous phytase activity. Metabolic scenarios leading to accumulation of D/L-Ins(1,3,4,5)P4 in barley low-phytate mutants are discussed.
Delivery of oligonucleotides into mammalian cells by anionic peptides: comparison between monomeric and dimeric peptides
Authors: FREULON Isabelle , ROCHE Annie-Claude , MONSIGNY Michel , MAYER Roger
Published: August 2015

The use of antisense oligonucleotides as putative therapeutic agents is limited by their poor delivery into the cytosol and/or the nucleus because they are not able to efficiently cross lipid bilayers. To circumvent this pitfall, anionic amphipathic peptides derived from the influenza virus fusogenic peptide have been used to destabilize membranes in an acidic environment. In this paper, we compare the ability of a monomeric and a dimeric peptide to introduce oligonucleotides into the cytosol and nuclei of several types of cultured cells. Cells incubated at pH6.2 or at a slightly lower pH in the presence of the monomeric peptide but not the dimeric peptide were efficiently permeabilized. The location of fluorescent derivatives of peptides and of oligonucleotides was assessed by confocal microscopy. Both the peptides and oligonucleotides remained entrapped in vesicular compartments at neutral pH; at acidic pH, oligonucleotides in the presence of the monomeric peptide were mainly in the nucleus, while in the presence of the dimeric peptide they co-localized with the peptide into vesicles. The data are interpreted on the basis of the spectroscopic behaviour of monomeric and dimeric peptides in relation to the environmental pH.
Exonic Sp1 sites are required for neural-specific expression of the glycine receptor β subunit gene
Authors: TINTRUP Hartmut , FISCHER Maria , BETZ Heinrich , KUHSE Jochen
Published: August 2015

The gene encoding the β subunit of the inhibitory glycine receptor (GlyR) is widely expressed throughout the mammalian central nervous system. To unravel the elements regulating its transcription, we isolated its 5′ non-coding and upstream flanking regions from mouse. Sequence analysis revealed significant differences between the 5′ region of the β subunit gene and the corresponding regions of the homologous GlyR α subunit genes; it also identified a novel exon (exon 0) that encodes most of the 5′-untranslated portion of the GlyR β mRNA. Primer extension experiments disclosed multiple transcriptional start sites. Transfection experiments with luciferase reporter gene constructs showed that sequences encompassing 1.58kb of upstream flanking region and 180bp of exon 0 displayed high promoter activity in two neuroblastoma cell lines but not in non-neural cells. Analysis of various deletion constructs showed that the 5′ flanking region preceding the transcriptional start sites silences expression in non-neural cells but is not essential for general promoter activity. In contrast, the deletion of sequences within exon 0 drastically decreased or abolished transcription; the removal of sequences harbouring Sp1 consensus sequences within exon 0 decreased expression specifically in a neuroblastoma cell line. Band-shift assays confirmed the binding of Sp1 to sites within the deleted sequence. Our results indicate that neural-specific expression of the GlyR β subunit gene might depend on a direct interaction of Sp1 transcription factors with cis elements located downstream from transcription initiation sites.
Mechanism of malarial haem detoxification inhibition by chloroquine
Authors: PANDEY Amit V. , BISHT Himani , BABBARWAL Vinod K. , SRIVASTAVA Jaya , PANDEY Kailash C. , CHAUHAN Virander S.
Published: August 2015

The haem detoxification pathway of the malaria parasite Plasmodiumfalciparum is a potential biochemical target for drug development. Free haem, released after haemoglobin degradation, is polymerized by the parasite to form haemozoin pigment. Plasmodiumfalciparum histidine-rich protein-2 (Pfhrp-2) has been implicated as the catalytic scaffold for detoxification of haem in the malaria parasite. Previously we have shown that a hexapeptide repeat sequence (Ala-His-His-Ala-Ala-Asp), which appears 33 times in Pfhrp-2, may be the major haem binding site in this protein. The haem binding studies carried out by ourselves indicate that up to 18 equivalents of haem could be bound by this protein with an observed Kd of 0.94µM. Absorbance spectroscopy provides evidence that chloroquine is capable of extracting haem bound to Pfhrp-2. This was supported by the Kd value, of 37nM, observed for the haem-chloroquine complex. The native PAGE studies reveal that the formation of the haem-Pfhrp-2 complex is disrupted by chloroquine. These results indicate that chloroquine may be acting by inhibiting haem detoxification/binding to Pfhrp-2. Moreover, the higher affinity of chloroquine for haem than Pfhrp-2 suggests a possible mechanism of action for chloroquine; it may remove the haem bound to Pfhrp-2 and form a complex that is toxic to the parasite.
Expression of P-170 glycoprotein sensitizes lymphoblastoid CEM cells to mitochondria-mediated apoptosis
Authors: MATARRESE Paola , TESTA Ugo , CAUDA Roberto , VELLA Stefano , GAMBARDELLA Lucrezia , MALORNI Walter
Published: August 2015

Multidrug resistance caused by P-glycoprotein (P-170) is a phenomenon by which cells exposed to a single drug acquire resistance to other structurally and functionally unrelated drugs. This is a widespread phenomenon described in vivo in the management of infectious as well as non-infectious diseases. Several in vitro models have been developed in order to evaluate physiopathological properties of P-170. Among these are P-170-expressing variants of the human T-lymphoblastoid CEM cell line called VBL100. As a general rule, drug resistance normally results in resistance to apoptosis induction. By contrast, a paradoxical activity is exerted in this cell model by the cytokine tumour necrosis factor-α (TNF-α), which is capable of inducing apoptosis in P-170-expressing variants better than in wild-type (wt) cells. In the present study we partially address the mechanisms underlying this activity. In fact, the susceptibility of VBL100 cells to TNF-α appears to be specifically due to the depolarization of their mitochondrial membrane, a key factor for apoptotic induction. The same was observed with staurosporine, a specific mitochondrion-mediated proapoptotic chemical probe. Conversely, other proapoptotic stimuli, such as Fas/CD95 or the anti-cancer drug etoposide, did induce significant cell death in wild type cells only. Thus, schematically, mitochondrially dependent stimuli appeared to be more effective in VBL100-cell killing, while ‘physiological’ stimuli showed the opposite behaviour. Importantly, under steady-state conditions, VBL100 cells displayed per se a mitochondrial membrane hyperpolarization that appeared strictly related to their high susceptibility to specific apoptotic stimuli. In conclusion, the study of a well-established cell model such as that represented by the wt/VBL CEM lymphoid cell line seems to suggest that the multidrug resistance phenotype can specifically sensitize cells towards ‘unphysiological’, mitochondria-associated cell death cascade or, in the same fashion, it could shift cells from type I (mainly plasma membrane-associated) towards type II (mainly mitochondrial membrane-associated) phenotype.
The kinase DYRK1A phosphorylates the transcription factor FKHR at Ser329 in vitro, a novel in vivo phosphorylation site
Authors: WOODS Yvonne L. , RENA Graham , MORRICE Nick , BARTHEL Andreas , BECKER Walter , GUO Shaodong , UNTERMAN Terry G. , COHEN Philip
Published: August 2015

Forkhead in rhabdomyosarcoma (FKHR) is a transcription factor that has been implicated in the control of gene expression by insulin, as well as the regulation of apoptosis by survival factors. These signals trigger the protein kinase B (PKB)-catalysed phosphorylation of FKHR at three residues (Thr24, Ser256 and Ser319) by a phosphoinositide 3-kinase-dependent pathway that results in the nuclear exit and inactivation of this transcription factor. Here, we have identified a conserved residue (Ser329) as a novel in vivo phosphorylation site on FKHR. Ser329 phosphorylation also decreases the ability of FKHR to stimulate gene transactivation and reduces the proportion of FKHR present in the nucleus. However, unlike the residues targetted by PKB, Ser329 is phosphorylated in unstimulated HEK-293cells, and phosphorylation is not increased by stimulation with insulin-like growth factor-1 or by transfection with 3-phosphoinositide-dependent protein kinase-1. We have also purified a protein kinase to near homogeneity from rabbit skeletal muscle that phosphorylates FKHR at Ser329 specifically and identified it as DYRK1A (dual-specificity tyrosine-phosphorylated and regulated kinase 1A). We find that FKHR and DYRK1A co-localize in discrete regions of the nucleus and can be co-immunoprecipitated from cell extracts. These experiments suggest that DYRK1A may phosphorylate FKHR at Ser329in vivo.
Involvement of Hic-5 in platelet activation: integrin αIIbβ3-dependent tyrosine phosphorylation and association with proline-rich tyrosine kinase 2
Authors: OSADA Makoto , OHMORI Tsukasa , YATOMI Yutaka , SATOH Kaneo , HOSOGAYA Shigemi , OZAKI Yukio
Published: August 2015

Hic-5 and paxillin, members of the LIM protein family, have been shown to be localized in focal adhesion and to have a role in integrin-mediated signalling. In the present study we examined the involvement of Hic-5 in human platelet activation: platelets express Hic-5 but not paxillin, whereas human umbilical-vein vascular endothelial cells and MEG-01 cells express mainly paxillin. When platelets were stimulated with thrombin, collagen or the stable thromboxane A2 analogue U46619, Hic-5 was markedly tyrosine-phosphorylated, in a manner dependent on integrin αIIbβ3-mediated aggregation. In addition, direct activation of protein kinase C with PMA resulted in tyrosine phosphorylation of Hic-5 only when platelets were fully aggregated with the exogenous addition of fibrinogen. Furthermore, PMA-induced Hic-5 tyrosine phosphorylation was also observed when platelets adhered to immobilized fibrinogen. In studies on immunoprecipitation and immunodepletion, Hic-5 seemed to associate with proline-rich tyrosine kinase 2 (Pyk2) but only marginally with focal adhesion kinase. When platelets were stimulated with thrombin, both Hic-5 and Pyk2 translocated to the cytoskeleton from the cytosol and membrane fractions in a manner dependent on αIIbβ3-mediated aggregation. Finally, on stimulation with PMA, Hic-5, as well as Pyk2, translocated to the cell periphery, where a meshwork of actin filaments assembled after adhesion to immobilized fibrinogen. Our results suggest that Hic-5 might be important in platelet aggregation and adhesion, in a manner dependent on αIIbβ3-mediated outside-in signalling, through association with Pyk2.
Sound attenuation of polymerizing actin reflects supramolecular structures: viscoelastic properties of actin gels modified by cytochalasin D, profilin and α-actinin
Authors: WAGNER Oliver , SCHÜLER Herwig , HOFMANN Peter , LANGER David , DANCKER Peter , BEREITER-HAHN Juergen
Published: August 2015

Polymerization and depolymerization of cytoskeletal elements maintaining cytoplasmic stiffness are key factors in the control of cell crawling. Rheometry is a significant tool in determining the mechanical properties of the single elements in vitro. Viscoelasticity of gels formed by these polymers strongly depends on both the length and the associations of the filaments (e.g. entanglements, annealings and side-by-side associations). Ultrasound attenuation is related to viscosity, sound velocity and supramolecular structures in the sample. In combination with a small glass fibre (2mm×50µm), serving as a viscosity sensor, an acoustic microscope was used to measure the elasticity and acoustic attenuation of actin solutions. Changes in acoustic attenuation of polymerizing actin by far exceed the values expected from calculations based on changes in viscosity and sound velocity. During the lag-phase of actin polymerization, attenuation slightly decreases, depending on actin concentration. After the half-maximum viscosity is accomplished and elasticity turns into steady state, attenuation distinctly rises. Changes in ultrasound attenuation depend on actin concentration, and they are modulated by the addition of α-actinin, cytochalasin D and profilin. Thus absorption and scattering of sound on the polymerization of actin is related to the packing density of the actin net, entanglements and the length of the actin filaments. Shortening of actin filaments by cytochalasin D was also confirmed by electron micrographs and falling-ball viscosimetry. In addition to viscosity and elasticity, the attenuation of sound proved to be a valuable parameter in characterizing actin polymerization and the supramolecular associations of F-actin.
Suramin and the suramin analogue NF307 discriminate among calmodulin-binding sites
Authors: KLINGER Markus , BOFILL-CARDONA Elisa , MAYER Bernd , NANOFF Christian , FREISSMUTH Michael , HOHENEGGER Martin
Published: August 2015

Calmodulin-binding sites on target proteins show considerable variation in primary sequence; hence compounds that block the access of calmodulin to these binding sites may be more selective than compounds that inactivate calmodulin. Suramin and its analogue NF307 inhibit the interaction of calmodulin with the ryanodine receptor. We have investigated whether inhibition of calmodulin binding to target proteins is a general property of these compounds. Suramin inhibited binding of [125I]calmodulin to porcine brain membranes and to sarcoplasmic reticulum from skeletal muscle (IC50 = 4.9±1.2µM and 19.9±1.8µM, respectively) and blocked the cross-linking of [125I]calmodulin to some, but not all, target proteins in brain membranes by [125I]calmodulin. Four calmodulin-binding proteins were purified [ryanodine receptor-1 (RyR1) from rabbit skeletal muscle, neuronal NO synthase (nNOS) from Sf9 cells, G-protein βγ dimers (Gβγ) from porcine brain and a glutathione S-transferase-fusion protein comprising the C-terminal calmodulin-binding domain of the metabotropic glutamate receptor 7A (GST-CmGluR7A) from bacterial lysates]. Three of the proteins employed (Gβγ, GST-CmGluR7A and RyR1) display a comparable affinity for calmodulin (in the range of 50–70nM). Nevertheless, suramin and NF307 only blocked the binding of Gβγ and RyR1 to calmodulin–Sepharose. In contrast, the association of GST-CmGluR7A and nNOS was not impaired, whereas excess calmodulin uniformly displaced all proteins from the matrix. Thus suramin and NF307 are prototypes of a new class of calmodulin antagonists that do not interact directly with calmodulin but with calmodulin-recognition sites. In addition, these compounds discriminate among calmodulin-binding motifs.
Transcriptional regulation of the KEL gene and Kell protein expression in erythroid and non-erythroid cells
Authors: CAMARA-CLAYETTE Valérie , RAHUEL Cécile , LOPEZ Claude , HATTAB Claude , VERKARRE Virginie , BERTRAND Olivier , CARTRON Jean-Pierre
Published: August 2015

The Kell blood-group antigen was originally reported to be a protein expressed in erythroid tissue only. Transcriptional analysis of the KEL promoter activity in human erythroleukaemia K562 and epithelial HeLa cells by electrophoretic mobility-shift and supershift assays, chloramphenicol acetyltransferase assays, co-transfection studies and site-directed mutagenesis provided the following results: (i) the KEL promoter exhibits a strong transcriptional activity in K562 cells and, unexpectedly, a basal non-erythroid activity in HeLa cells, (ii) up-regulation of the 5′ distal promoter activity occurs only in the erythroid context, and (iii) two motifs localized in the exon 1 region, which bind the Sp1/Sp3 and the human GATA-1/Ku70/80 factors, were required for down-regulation of the promoter activity, but inhibition of the promoter activity by the repressing factors in HeLa cells was incomplete. KEL expression in HeLa cells was performed further by primer-extension analysis, which revealed the presence of a low amount of Kell transcript correlating with basal expression of the Kell protein in these cells, as shown by immunopurification and Western-blot analysis. DNA sequencing of the transcript revealed a sequence identical to that obtained from erythroid tissue. In human tissues, KEL expression was investigated by dot-blot analysis and revealed high levels of Kell mRNAs, particularly in brain tissues, testis and lymphoid tissues. Moreover, most tissues analysed exhibited low levels of Kell transcripts. The Kell protein was also detected by immunohistochemistry in the Sertoli cells of the testis and in lymphoid tissues like spleen and tonsil, specifically localized in the follicular dendritic cells. Altogether, the results indicated that KEL expression is not restricted to erythroid tissue.
Barley arabinoxylan arabinofuranohydrolases: purification, characterization and determination of primary structures from cDNA clones
Authors: LEE Robert C. , BURTON Rachel A. , HRMOVA Maria , FINCHER Geoffrey B.
Published: August 2015

A family 51 arabinoxylan arabinofuranohydrolase, designated AXAH-I, has been purified from extracts of 7-day-old barley (Hordeum vulgare L.) seedlings by fractional precipitation with (NH4)2SO4 and ion-exchange chromatography. The enzyme has an apparent molecular mass of 65kDa and releases l-arabinose from cereal cell wall arabinoxylans with a pH optimum of 4.3, a catalytic rate constant (kcat) of 6.9s−1 and a catalytic efficiency factor (kcat/Km) of 0.76 (ml·s−1·mg−1). Whereas the hydrolysis of α-l-arabinofuranosyl residues linked to C(O)3 of backbone (1 → 4)-β-xylosyl residues proceeds at the fastest rate, α-l-arabinofuranosyl residues on doubly substituted xylosyl residues are also hydrolysed, at lower rates. A near full-length cDNA encoding barley AXAH-I indicates that the mature enzyme consists of 626 amino acid residues and has a calculated pI of 4.8. A second cDNA, which is 81% identical with that encoding AXAH-I, encodes another barley AXAH, which has been designated AXAH-II. The barley AXAHs are likely to have key roles in wall metabolism in cereals and other members of the Poaceae. Thus the enzymes could participate in the modification of the fine structure of arabinoxylan during wall deposition, maturation or expansion, or in wall turnover and the hydrolysis of arabinoxylans in germinated grain.
Cloning and identification of MYPT3: a prenylatable myosin targetting subunit of protein phosphatase 1
Authors: SKINNER Jeffrey A. , SALTIEL Alan R.
Published: August 2015

To identify novel protein phosphatase 1 (PP1)-interacting proteins, a yeast two-hybrid 3T3-L1 adipocyte cDNA library was screened with the catalytic subunit of PP1 as bait. In the present work, the isolation, identification and initial biochemical characterization of a novel PP1-interacting protein, MYPT3, which is homologous with the myosin phosphatase targetting subunit (MYPT) family, is described. MYPT3 aligns > 99% with a region of mouse genomic DNA clone RP23-156P23 and localizes to chromosome 15, between markers at 44.1–46.5cM, as demonstrated by radiation hybrid mapping. The gene consists of ten exons that encode for a 524-amino acid sequence with a predicted molecular mass of 57529Da. The N-terminal region of MYPT3 consists of a consensus PP1-binding site and multiple ankyrin repeats. MYPT3 is distinguished from related ∼ 110–130kDa MYPT subunits by its molecular mass of 58kDa, and a unique C-terminal region that contains several potential signalling motifs and a CaaX prenylation site. We have shown that affinity-purified glutathione S-transferase (GST)–MYPT3 is prenylated by purified recombinant farnesyltransferase in vitro. Endogenous PP1 from 3T3-L1 lysates specifically interacts with MYPT3. Additionally, purified PP1 activity was inhibited by GST–MYPT3 toward phosphorylase a, myosin light chain and myosin substrate in vitro. Overall, our findings identify a novel prenylatable subunit of PP1 that defines a new subfamily of MYPT.
Functional coupling between the caffeine/ryanodine-sensitive Ca2+ store and mitochondria in rat aortic smooth muscle cells
Authors: VALLOT Olivier , COMBETTES Laurent , LOMPRÉ Anne-Marie
Published: August 2015

We investigated the role of mitochondria in the agonist-induced and/or caffeine-induced Ca2+ transients in rat aortic smooth muscle cells. We explored the possibility that proliferation modulates the coupling between mitochondria and endoplasmic reticulum. Ca2+ transients induced by either ATP or caffeine were measured in presence or absence of drugs interfering with mitochondrial activity in freshly dissociated cells (day 1) and in subconfluent primary culture (day 12). We found that the mitochondrial inhibitors, rotenone or carbonyl cyanide m-chlorophenylhydrazone, as well as the permeability transition pore inhibitor, cyclosporin A, had no effect on the ATP-induced Ca2+ transient at either day 1 or day 12, but prevented caffeine-induced cytosolic Ca2+ increase at day 12 but not at day 1. Close connections between ryanodine receptors and mitochondria were observed at both day 1 and 12. Thapsigargin (TG) prevented ATP- and caffeine-induced Ca2+ transients at day 1. At day 12, where only 50% of the cells were sensitive to caffeine, TG did not prevent the caffeine-induced Ca2+ transient, and prevented ATP-induced Ca2+ transient in only half of the cells. Together, these data demonstrate that rat aortic smooth muscle cells at day 1have an ATP- and caffeine-sensitive pool, which is functionally independent but physically closely linked to mitochondria and totally inhibited by TG. At day 12, we propose the existence of two cell populations: half contains IP3 receptors and TG-sensitive Ca2+ pumps only; the other half contains, in addition to the IP3-sensitive pool independent from mitochondria, a caffeine-sensitive pool. This latter pool is linked to mitochondria through the permeability transition pore and is refilled by both TG-sensitive and insensitive mechanisms.
Phenotype–genotype relationships in peroxisome biogenesis disorders of PEX1-defective complementation group 1 are defined by Pex1p–Pex6p interaction
Authors: TAMURA Shigehiko , MATSUMOTO Naomi , IMAMURA Atsushi , SHIMOZAWA Nobuyuki , SUZUKI Yasuyuki , KONDO Naomi , FUJIKI Yukio
Published: August 2015

The peroxisome biogenesis disorders (PBDs), including Zellweger syndrome (ZS), neonatal adrenoleucodystrophy (NALD) and infantile Refsum disease (IRD), are fatal autosomal recessive diseases caused by impaired peroxisome biogenesis, of which 12 genotypes have been reported. ZS patients manifest the severest clinical and biochemical abnormalities, whereas those with NALD and IRD show less severity and the mildest features respectively. We have reported previously that temperature-sensitive peroxisome assembly is responsible for the mildness of the clinical features of IRD. PEX1 is the causative gene for PBDs of complementation group E (CG-E, CG1 in the U.S.A. and Europe), the PBDs of highest incidence, encoding the peroxin Pex1p of the AAA ATPase family. It has been also reported that Pex1p and Pex6p interact with each other. In the present study we investigated phenotype–genotype relationships of CG1 PBDs. Pex1p from IRD such as Pex1p with the most frequently identified mutation at G843D was largely degraded in vivo at 37°C, whereas a normal level of Pex1p was detectable at the permissive temperature. In contrast, PEX1 proteins derived from ZS patients, including proteins with a mutation at L664P or the deletion of residues 634–690, were stably present at both temperatures. Pex1p-G843D interacted with Pex6p at approx. 50% of the level of normal Pex1p, whereas Pex1p from ZS patients mostly showing non-temperature-sensitive peroxisome biogenesis hardly bound to Pex6p. Taking these results together, we consider it most likely that the stability of Pex1p reflects temperature-sensitive peroxisome assembly in IRD fibroblasts. Failure in Pex1p–Pex6p interaction gives rise to more severe abnormalities, such as those manifested by patients with ZS.
Polyamines regulate gap junction communication in connexin 43-expressing cells
Authors: SHORE Leonard , McLEAN Pauline , GILMOUR Susan K. , HODGINS Malcolm B. , FINBOW Malcolm E.
Published: August 2015

The control of cell–cell communication through gap junctions is thought to be crucial in normal tissue function and during various stages of tumorigenesis. However, few natural regulators of gap junctions have been found. We show here that increasing the activity of ornithine decarboxylase, or adding polyamines to the outside of cells, increases the level of gap junction communication between various epithelial cells. Conversely, reduction of ornithine decarboxylase activity decreases the level of gap junction communication. This regulation is dependent upon the expression of connexin 43 (Cx43 or Cxα1), which is a major connexin expressed in many different cell types, and involves an increase in Cx43 and its cellular re-distribution.
Involvement of the chicken liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase sequence His444-Arg-Glu-Arg in modulation of the bisphosphatase activity by its kinase domain
Authors: ZHU Zheng , LING Song , YANG Qi-Heng , LI Lin
Published: August 2015

The bisphosphatase activity of the hepatic bifunctional enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase is repressed by its kinase domain, and regulated by cAMP-dependent protein kinase (PKA)-catalysed phosphorylation. In the present study, the mechanism by which the bisphosphatase activity is repressed by the kinase domain and regulated by phosphorylation was investigated. We found that truncation of the C-terminus of the enzyme by 25, but not 20, amino acids dramatically enhanced the catalytic rate of the bisphosphatase, abrogated the inhibition by the kinase domain, and eliminated the effect of PKA-mediated phosphorylation on activity. In addition, mutation of His444-Arg-Glu-Arg to Ala-Ala-Glu-Ala had similar effects as the deletion. Moreover, the mutations also significantly affected the phosphorylation-mediated regulation of the kinase activity of the enzyme. Furthermore, the mutations altered the pH-dependence of the bisphosphatase, and the mutant bisphosphatases were more sensitive to modification by diethyl pyrocarbonate and guanidine-induced inactivation than the wild-type enzyme. Taken together, these results demonstrate that the sequence His444-Arg-Glu-Arg plays a critical role in repression of the bisphosphatase activity by both the N-terminal kinase domain and the C-terminal tail itself. These results also explain the activation of the bisphosphatase activity by PKA-catalysed phosphorylation, by suggesting that phosphorylation may relieve the inhibitory effect of the kinase domain that is mediated by the three basic residues in this sequence.
Green fluorescent protein (GFP) tagged to the cytoplasmic tail of αIIb or β3 allows the expression of a fully functional integrin αIIbβ3: effect of β3GFP on αIIbβ3 ligand binding
Authors: PLANÇON Sébastien , MOREL-KOPP Marie-Christine , SCHAFFNER-RECKINGER Elisabeth , CHEN Ping , KIEFFER Nelly
Published: August 2015

Using green fluorescent protein (GFP) as an autofluorescent tag, we report the first successful visualization of a β3 integrin in a living cell. GFP fused in frame to the cytoplasmic tail of either αIIb or β3 allowed normal expression, heterodimerization, processing and surface exposure of αIIbGFPβ3 and αIIbβ3GFP receptors in Chinese hamster ovary (CHO) cells. Direct microscopic observation of the autofluorescent cells in suspension following antibody-induced αIIbβ3 capping revealed an intense autofluorescent cap corresponding to unlabelled immunoclustered GFP-tagged αIIbβ3. GFP-tagged αIIbβ3 receptors mediated fibrinogen-dependent cell adhesion, were readily detectable in focal adhesions of unstained living cells and triggered p125FAK tyrosine phosphorylation similar to wild-type αIIbβ3 (where FAK corresponds to focal adhesion kinase). However, GFP tagged to β3, but not to αIIb, induced spontaneous CHO cell aggregation in the presence of soluble fibrinogen, as well as binding of the fibrinogen mimetic monoclonal antibody PAC1 in the absence of αIIbβ3 receptor activation. Time-lapse imaging of living transfectants revealed a characteristic redistribution of GFP-tagged αIIbβ3 during the early stages of cell attachment and spreading, starting with αIIbβ3 clustering at the rim of the cell contact area, that gradually overlapped with the boundary of the attached cell, and, with the onset of cell spreading, to a reorganization of αIIbβ3 in focal adhesions. Taken together, our results demonstrate that (1) fusion of GFP to the cytoplasmic tail of either αIIb or β3 integrin subunits allows normal cell surface expression of a functional receptor, and (2) structural modification of the β3 integrin cytoplasmic tail, rather than the αIIb subunit, plays a major role in αIIbβ3 affinity modulation. With the successful direct visualization of functional αIIbβ3 receptors in living cells, the generation of autofluorescent integrins in transgenic animals will become possible, allowing new approaches to study the dynamics of integrin functions.
Extracellular processing of amphoterin generates a peptide active on erythroleukaemia cell differentiation
Authors: SPARATORE Bianca , PATRONE Mauro , PASSALACQUA Mario , PEDRAZZI Marco , GAGGERO Deborah , PONTREMOLI Sandro , MELLONI Edon
Published: August 2015

The release of amphoterin by murine erythroleukaemia cells exposed to the chemical inducer hexamethylenebisacetamide represents an essential step for the process of their terminal differentiation. Once exported in the culture medium, amphoterin undergoes limited proteolysis, catalysed by a serine proteinase also secreted by stimulated cells. The isolated proteinase is responsible for degradation of amphoterin, with the production of a 10-amino-acid-residue fragment, specifically retaining the cell-differentiation-stimulating activity of the native protein molecule. This peptide does not express other properties of amphoterin, such as protein kinase C-stimulating activity or systemic toxicity. These findings define a selective mechanism accounting for extracellular amphoterin functional maturation.
Multidrug-resistance P-glycoprotein (MDR1) secretes platelet-activating factor
Authors: RAGGERS René J. , VOGELS Ilse , van MEER Gerrit
Published: August 2015

The human multidrug-resistance (MDR1) P-glycoprotein (Pgp) is an ATP-binding-cassette transporter (ABCB1) that is ubiquitously expressed. Often its concentration is high in the plasma membrane of cancer cells, where it causes multidrug resistance by pumping lipophilic drugs out of the cell. In addition, MDR1 Pgp can transport analogues of membrane lipids with shortened acyl chains across the plasma membrane. We studied a role for MDR1 Pgp in transport to the cell surface of the signal-transduction molecule platelet-activating factor (PAF). PAF is the natural short-chain phospholipid 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine. [14C]PAF synthesized intracellularly from exogenous alkylacetylglycerol and [14C]choline became accessible to albumin in the extracellular medium of pig kidney epithelial LLC-PK1 cells in the absence of vesicular transport. Its translocation across the apical membrane was greatly stimulated by the expression of MDR1 Pgp, and inhibited by the MDR1 inhibitors PSC833 and cyclosporin A. Basolateral translocation was not stimulated by expression of the basolateral drug transporter MRP1 (ABCC1). It was insensitive to the MRP1 inhibitor indomethacin and to depletion of GSH which is required for MRP1 activity. While efficient transport of PAF across the apical plasma membrane may be physiologically relevant in MDR1-expressing epithelia, PAF secretion in multidrug-resistant tumours may stimulate angiogenesis and thereby tumour growth.
A fluorescence temperature-jump study of conformational transitions in myosin subfragment 1
Authors: URBANKE Claus , WRAY John
Published: August 2015

The tryptophan fluorescence of unmodified myosin subfragment 1 (S1) from rabbit and chicken skeletal muscle with various nucleotides and phosphate analogues bound was measured after rapid temperature jumps. The fluorescence decreased during the temperature rise. Under some conditions, this decrease was followed by an increase, reflecting structural transitions within the protein. With adenosine 5′-[β,γ-imido]triphosphate (p[NH]ppA) or with ADP and BeFx bound, this rise was very rapid (reciprocal time constant approx. 2000s−1) and varied only slightly with starting temperature, suggesting that, with these ligands, two different protein conformations were present in rapid equilibrium over a large temperature range. In the presence of ATP, the transient included several relaxation processes. Overall, the results suggest that complexes of S1 with ATP or with a number of other ligands exist as a mixture of two forms in temperature-dependent equilibrium. The results throw light on the finding of different forms of S1 in recent crystallographic studies and indicate a surprising lack of strong coupling between myosin's structural state and the nature of the nucleotide bound.
Subnuclear localization and mitotic phosphorylation of HIRA, the human homologue of Saccharomyces cerevisiae transcriptional regulators Hir1p/Hir2p
Authors: De LUCIA Filomena , LORAIN Stéphanie , SCAMPS Christine , GALISSON Frédéric , MACHOLD Jan , LIPINSKI Marc
Published: August 2015

The HIRA gene encodes a nuclear protein with histone-binding properties that have been conserved from yeast to humans. Hir1p and Hir2p, the two HIRA homologues in Saccharomyces cerevisiae, are transcriptional co-repressors whose action resides at the chromatin level and occurs in a cell-cycle-regulated fashion. In mammals, HIRA is an essential gene early during development, possibly through the control of specific gene-transcription programmes, but its exact function remains to be deciphered. Here we report on the subnuclear distribution and cell-cycle behaviour of the HIRA protein. Using both biochemical and immunofluorescence techniques, a minor fraction of HIRA was found tightly associated with the nuclear matrix, the material that remains after nuclease treatment and high-salt extraction. However, most HIRA molecules proved extractable. In non-synchronized cell populations, extraction from chromatin necessitated 300mM NaCl whereas 150mM was sufficient in mitotic cells. Immunofluorescence staining and microscopic examination of mitotic cells revealed HIRA as excluded from condensed chromosomes, confirming a lack of association with chromatin during mitosis. Western-blot analysis indicated that HIRA molecules were hyper-phosphorylated at this point in the cell cycle. Metabolic labelling and pulse-chase experiments characterized HIRA as a stable protein with a half-life of approx. 12h. The mitotic phosphorylation of HIRA could provide the dividing cell with a way to retarget HIRA-containing multi-protein complexes to different chromatin regions in daughter compared with parental cells.
Regulation of SulA cleavage by Lon protease by the C-terminal amino acid of SulA, histidine
Authors: ISHII Yoshiyuki , AMANO Fumio
Published: August 2015

SulA protein, a cell division inhibitor in Escherichia coli, is degraded by Lon protease. The C-terminal eight residues of SulA have been shown to be recognized by Lon; however, it remains to be elucidated which amino acid in the C-terminus of SulA is critical for the recognition of SulA by Lon. To clarify this point, we constructed mutants of SulA with changes in the C-terminal residues, and examined the accumulation and stability of the resulting mutant SulA proteins in vivo. Substitution of the extreme C-terminal histidine residue with another amino acid led to marked accumulation and high stability of SulA in lon+ cells. A SulA mutant in which the C-terminal eight residues were deleted (SulAC161) showed high accumulation and stability, but the addition of histidine to the C-terminus of SulAC161 (SulAC161+H) made it labile. Similarly, SulAC161+H fused to maltose-binding protein (MBP–SulAC161+H) formed a tight complex with and was degraded rapidly by Lon in vitro. Histidine competitively inhibited the degradation of MBP–SulA by Lon, while other amino acids did not. These results suggest that the histidine residue at the extreme C-terminus of SulA is recognized specifically by Lon, leading to a high-affinity interaction between SulA and Lon.
c-Jun N-terminal kinase-interacting protein 1 inhibits gene expression in response to hypertrophic agonists in neonatal rat ventricular myocytes
Authors: FINN Stephen G. , DICKENS Martin , FULLER Stephen J.
Published: August 2015

Gq-coupled receptor agonists, such as endothelin-1 (ET-1) and phenylephrine (PE), initiate a hypertrophic response in cardiac myocytes that is characterized by increased expression of atrial natriuretic factor (ANF), β-myosin heavy chain (β-MHC), skeletal muscle α-actin (SkαA) and ventricular myosin light chain-2 (vMLC2). ET-1 and PE activate both the extracellular signal-regulated kinases and c-Jun N-terminal kinases (JNKs) in cardiac myocytes, but the extent to which each contributes to the hypertrophic response is uncertain. Here we have used the JNK-binding domain of JNK-interacting protein 1 (JIP-1), a cytosolic scaffold protein that binds to JNK and inhibits its signalling when overexpressed, to assess the contribution of JNK activation to the hypertrophic response. Expression of JIP-1 inhibited the increase in ANF, β-MHC, SkαA and vMLC2 reporter gene expression in response to ET-1 (by 45–86%) and PE (by 56–60%). However, activation of these reporter genes by PMA, which does not activate JNK significantly in myocytes, was much less affected by overexpression of JIP-1. JIP-1 also failed to inhibit reporter gene activation in response to constitutively active Ras or Raf, but attenuated reporter gene activation induced by a constitutively active mutant of mitogen-activated protein kinase kinase kinase 1 (MEKK1), an upstream kinase that preferentially activates JNKs, by 50%. Overexpression of JIP-1 also significantly reduced the increase in cell area in response to PE from 63% to 56%, but had no effect on the increase in cell size in response to ET-1 (38%). These results suggest that activation of the JNK pathway contributes to the transcriptional and morphological responses to Gq receptor-coupled hypertrophic agonists.
Four Trypanosoma brucei fatty acyl-CoA synthetases: fatty acid specificity of the recombinant proteins
Authors: JIANG David W. , ENGLUND Paul T.
Published: August 2015

As part of our investigation of fatty acid metabolism in Trypanosoma brucei, we have expressed four acyl-CoA synthetase (TbACS) genes in Esherichia coli. The recombinant proteins, with His-tags on their C-termini, were purified to near homogeneity using nickel-chelate affinity chromatography. Although these enzymes are highly homologous, they have distinct specificities for fatty acid chain length. TbACS1 prefers saturated fatty acids in the range C11:0 to C14:0 and TbACS2 prefers shorter fatty acids, mainly C10:0. TbACS3 and 4, which have 95% sequence identity, have similar specificities, favouring fatty acids between C14:0 and C17:0. In addition, TbACS1, 3 and 4 function well with a variety of unsaturated fatty acids.
Lipolytic activity of ricin from Ricinus sanguineus and Ricinus communis on neutral lipids
Authors: LOMBARD Sophie , HELMY Mohamed E. , PIÉRONI Gérard
Published: August 2015

The present study was carried out with a view of determining ricin lipolytic activity on neutral lipids in emulsion and in a membrane-like model. Using 2,3-dimercapto-1-propanol tributyrate (BAL-TC4) as substrate, the lipolytic activity of ricin was found to be proportional to ricin and substrate concentrations, with an apparent Km (Km,app) of 2.4mM, a kcat of 200min−1 and a specific activity of 1.0unit/mg of protein. This work was extended to p-nitrophenyl (pNP) fatty acid esters containing two to twelve carbon atoms. Maximum lipolytic activity was registered on pNP decanoate (pNPC10), with a Km,app of 3.5mM, a kcat of 173min−1 and a specific activity of 3.5units/mg of protein. Ricin lipolytic activity is pH and galactose dependent, with a maximum at pH7.0 in the presence of 0.2M galactose. Using the monolayer technique with dicaprin as substrate, ricin showed a lipolytic activity proportional to the ricin concentration at 20mN/m, which is dependent on the surface pressure of the lipid monolayer and is detectable up to 30mN/m, a surface pressure that is of the same order of magnitude as that of natural cell membranes. The methods based on pNPC10 and BAL-TC4 hydrolysis are simple and reproducible; thus they can be used for routine studies of ricin lipolytic activity. Ricin from Ricinus communis and R. sanguineus were treated with diethyl p-nitrophenylphosphate, an irreversible serine esterase inhibitor, and their lipolytic activities on BAL-TC4 and pNPC10, and cytotoxic activity, were concurrently recorded. A reduction in lipolytic activity was accompanied by a decrease in cytotoxicity on Caco2 cells. These data support the idea that the lipolytic activity associated with ricin is relevant to a lipase whose activity is pH and galactose dependent, sensitive to diethyl p-nitrophenylphosphate, and that a lipolytic step may be involved in the process of cell poisoning by ricin. Both colorimetric tests used in this study are sensitive enough to be helpful in the detection of possible lipolytic activities associated with other cytotoxins or lectins.