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Biomedical subjects

P Auvinen

Publications and source records attributed to P Auvinen.

At least 19 recordsLinked to original sources
PubMed2005

Expression of CD 44 s, CD 44 v 3 and CD 44 v 6 in benign and malignant breast lesions: correlation and colocalization with hyaluronan.

AIMS: To examine the expression of CD 44 s, CD 44 v 3 and CD 44 v 6 in breast lesions, and to correlate it with the expression of hyaluronan (HA). METHODS AND RESULTS: CD 44 expression was studied in 75 breast tissue samples, consisting of benign, premalignant and malignant breast lesions, using immunohistochemistry. CD 44 s, but not CD 44 v 3 or CD 44 v 6, was found in the stromal cells, and it was similar in benign and malignant tumours. In benign lesions CD 4 v 6 was detected in 20-30% of the ductal epithelial cells, while C 44 v 3 and CD 44 s were not expressed. CD 44 s, CD 44 v 3 and CD 44 v 6 were all up-regulated in the in situ carcinomas and invasive carcinomas. The level of CD 44 expression in carcinoma cells did not correlate with the type or differentiation of the tumours. CD 44 and HA expression levels were not closely linked in the benign or malignant breast lesions, because HA was overexpressed later in breast cancer progression than CD 44. However, in breast carcinomas CD 44 and HA positivity was often found in the same areas of the sections, and the dual staining confirmed actual colocalization of CD 44 s and HA in the same cells. CONCLUSIONS: CD 44 s, CD 44 v 3 and CD 44 v 6 are up-regulated earlier than HA in breast carcinoma progression, and in later stages they often colocalize with cell surface HA.

Breast Neoplasms↗
PubMed2004

Human papillomavirus type 16 E5 protein colocalizes with the antiapoptotic Bcl-2 protein.

Human papillomavirus type 16 E5 protein contributes to cellular transformation by increasing the mitogenic stimulus from growth factor receptors to the nucleus. In order to study the biological mechanisms of the E5 protein we performed site-directed mutagenesis of the E5 gene. Wild-type as well as mutant E5 proteins were transiently expressed in human cervical epithelial cells, and cell morphology, expression of proteins involved in cell adhesion, and localization of the different proteins were studied. Little differences in cell morphology or expression kinetics were observed between the different E5 proteins, except for relocalization of a mutant E5 protein where a hydrophobic leucine membrane anchor was mutated to positively charged amino acids. This mutant E5 protein localized to lamellipodia, which are motility-associated structures at the leading edge of motile cells. In our experimental conditions, 100% of E5-expressing epithelial cells died by four days of expression, possibly due to toxicity or disturbance of the membrane compartment by the E5 protein. Most interestingly, a remarkable colocalization of the E5 protein with the Bcl-2 antiapoptotic protein on intracellular membranes was established.

Amino Acid Substitution↗
PubMed2001

Virus-specific mRNA capping enzyme encoded by hepatitis E virus.

Hepatitis E virus (HEV), a positive-strand RNA virus, is an important causative agent of waterborne hepatitis. Expression of cDNA (encoding amino acids 1 to 979 of HEV nonstructural open reading frame 1) in insect cells resulted in synthesis of a 110-kDa protein (P110), a fraction of which was proteolytically processed to an 80-kDa protein. P110 was tightly bound to cytoplasmic membranes, from which it could be released by detergents. Immunopurified P110 catalyzed transfer of a methyl group from S-adenosylmethionine (AdoMet) to GTP and GDP to yield m(7)GTP or m(7)GDP. GMP, GpppG, and GpppA were poor substrates for the P110 methyltransferase. There was no evidence for further methylation of m(7)GTP when it was used as a substrate for the methyltransferase. P110 was also a guanylyltransferase, which formed a covalent complex, P110-m(7)GMP, in the presence of AdoMet and GTP, because radioactivity from both [alpha-(32)P]GTP and [(3)H-methyl]AdoMet was found in the covalent guanylate complex. Since both methyltransferase and guanylyltransferase reactions are strictly virus specific, they should offer optimal targets for development of antiviral drugs. Cap analogs such as m(7)GTP, m(7)GDP, et(2)m(7)GMP, and m(2)et(7)GMP inhibited the methyltransferase reaction. HEV P110 capping enzyme has similar properties to the methyltransferase and guanylyltransferase of alphavirus nsP1, tobacco mosaic virus P126, brome mosaic virus replicase protein 1a, and bamboo mosaic virus (a potexvirus) nonstructural protein, indicating there is a common evolutionary origin of these distantly related plant and animal virus families.

Cell Line↗
PubMed2001

Biogenesis of the Semliki Forest virus RNA replication complex.

The nonstructural (ns) proteins nsP1 to -4, the components of Semliki Forest virus (SFV) RNA polymerase, were localized in infected cells by confocal microscopy using double labeling with specific antisera against the individual ns proteins. All ns proteins were associated with large cytoplasmic vacuoles (CPV), the inner surfaces of which were covered by small invaginations, or spherules, typical of alphavirus infection. All ns proteins were localized by immuno-electron microscopy (EM) to the limiting membranes of CPV and to the spherules, together with newly labeled viral RNA. Along with earlier observations by EM-autoradiography (P. M. Grimley, I. K. Berezesky, and R. M. Friedman, J. Virol. 2:326-338, 1968), these results suggest that individual spherules represent template-associated RNA polymerase complexes. Immunoprecipitation of radiolabeled ns proteins showed that each antiserum precipitated the other three ns proteins, implying that they functioned as a complex. Double labeling with organelle-specific and anti-ns-protein antisera showed that CPV were derivatives of late endosomes and lysosomes. Indeed, CPV frequently contained endocytosed bovine serum albumin-coated gold particles, introduced into the medium at different times after infection. With time, increasing numbers of spherules were also observed on the cell surfaces; they were occasionally released into the medium, probably by secretory lysosomes. We suggest that the spherules arise by primary assembly of the RNA replication complexes at the plasma membrane, guided there by nsP1, which has affinity to lipids specific for the cytoplasmic leaflet of the plasma membrane. Endosomal recycling and fusion of CPV with the plasma membrane can circulate spherules between the plasma membrane and the endosomal-lysosomal compartment.

Animals↗
PubMed2000

Membrane binding mechanism of an RNA virus-capping enzyme.

The RNA replication complex of Semliki Forest virus is bound to cytoplasmic membranes via the mRNA-capping enzyme Nsp1. Here we have studied the structure and liposome interactions of a synthetic peptide (245)GSTLYTESRKLLRSWHLPSV(264) corresponding to the membrane binding domain of Nsp1. The peptide interacted with liposomes only if negatively charged lipids were present that induced a structural change in the peptide from a random coil to a partially alpha-helical conformation. NMR structure shows that the alpha-helix is amphipathic, the hydrophobic surface consisting of several leucines, a valine, and a tryptophan moiety (Trp-259). Fluorescence studies revealed that this tryptophan intercalates in the bilayer to the depth of the ninth and tenth carbons of lipid acyl chains. Mutation W259A altered the mode of bilayer association of the peptide and abolished its ability to compete for membrane association of intact Nsp1, demonstrating its crucial role in the membrane association and function of Nsp1.

Amino Acid Sequence↗
PubMed2000

Identification of a novel function of the alphavirus capping apparatus. RNA 5'-triphosphatase activity of Nsp2.

Both genomic and subgenomic RNAs of the Alphavirus have m(7)G(5')ppp(5')N (cap0 structure) at their 5' end. Previously it has been shown that Alphavirus-specific nonstructural protein Nsp1 has guanine-7N-methyltransferase and guanylyltransferase activities needed in the synthesis of the cap structure. During normal cap synthesis the 5' gamma-phosphate of the nascent viral RNA chain is removed by a specific RNA 5'-triphosphatase before condensation with GMP, delivered by the guanylyltransferase. Using a novel RNA triphosphatase assay, we show here that nonstructural protein Nsp2 (799 amino acids) of Semliki Forest virus specifically cleaves the gamma,beta-triphosphate bond at the 5' end of RNA. The same activity was demonstrated for Nsp2 of Sindbis virus, as well as for the amino-terminal fragment of Semliki Forest virus Nsp2-N (residues 1-470). The carboxyl-terminal part of Semliki Forest virus Nsp2-C (residues 471-799) had no RNA triphosphatase activity. Replacement of Lys-192 by Asn in the nucleotide-binding site completely abolished RNA triphosphatase and nucleoside triphosphatase activities of Semliki Forest virus Nsp2 and Nsp2-N. Here we provide biochemical characterization of the newly found function of Nsp2 and discuss the unique properties of the entire Alphavirus-capping apparatus.

Acid Anhydride Hydrolases↗
PubMed2000

Hyaluronan in peritumoral stroma and malignant cells associates with breast cancer spreading and predicts survival.

Hyaluronan (HA) is an extracellular matrix polysaccharide that promotes cell migration through its cell surface receptors and by effecting changes in the physical environment. HA expression is frequently increased in malignant tumors, whereas its association with the invasive potential and patient outcome in breast cancer has not been reported. The localization and signal intensity of HA was analyzed in 143 paraffin-embedded tumor samples of human breast carcinoma using a biotinylated HA-specific probe. In the immediate peritumoral stroma, HA signal was moderately or strongly increased in 39% and 56% of the cases, respectively. Normal ductal epithelium showed no HA, whereas in 57% of the tumors at least some of the carcinoma cells were HA positive. The intensity of the stromal HA signal and the presence of cell-associated HA were both significantly related to poor differentiation of the tumors, axillary lymph node positivity, and short overall survival of the patients. In Cox's multivariate analysis, both the intensity of stromal HA signal alone and that combined with the HA positivity in tumor cells were independent prognostic factors for overall survival. These results suggest that HA is directly involved in the spreading of breast cancer and may offer a potential target for new therapies.

Axilla↗
PubMed2000

Effects of palmitoylation of replicase protein nsP1 on alphavirus infection.

The membrane-associated alphavirus RNA replication complex contains four virus-encoded subunits, the nonstructural proteins nsP1 to nsP4. Semliki Forest virus (SFV) nsP1 is hydrophobically modified by palmitoylation of cysteines 418 to 420. Here we show that Sindbis virus nsP1 is also palmitoylated on the same site (cysteine 420). When mutations preventing nsP1 palmitoylation were introduced into the genomes of these two alphaviruses, the mutant viruses remained viable and replicated to high titers, although their growth was slightly delayed. The subcellular distribution of palmitoylation-defective nsP1 was altered in the mutant: it no longer localized to filopodial extensions, and a fraction of it was soluble. The ultrastructure of the alphavirus replication sites appeared normal, and the localization of the other nonstructural proteins was unaltered in the mutants. In both wild-type- and mutant-virus-infected cells, SFV nsP3 and nsP4 could be extracted from membranes only by alkaline solutions whereas the nsP2-membrane association was looser. Thus, the membrane binding properties of the alphavirus RNA replication complex were not determined by the palmitoylation of nsP1. The nsP1 palmitoylation-defective alphaviruses produced normal plaques in several cell types, but failed to give rise to plaques in HeLa cells, although they induced normal apoptosis of these cells. The SFV mutant was apathogenic in mice: it caused blood viremia, but no infectious virus was detected in the brain.

Alphavirus Infections↗
PubMed2000

Mouse A6/twinfilin is an actin monomer-binding protein that localizes to the regions of rapid actin dynamics.

In our database searches, we have identified mammalian homologues of yeast actin-binding protein, twinfilin. Previous studies suggested that these mammalian proteins were tyrosine kinases, and therefore they were named A6 protein tyrosine kinase. In contrast to these earlier studies, we did not find any tyrosine kinase activity in our recombinant protein. However, biochemical analysis showed that mouse A6/twinfilin forms a complex with actin monomer and prevents actin filament assembly in vitro. A6/twinfilin mRNA is expressed in most adult tissues but not in skeletal muscle and spleen. In mouse cells, A6/twinfilin protein is concentrated to the areas at the cell cortex which overlap with G-actin-rich actin structures. A6/twinfilin also colocalizes with the activated forms of small GTPases Rac1 and Cdc42 to membrane ruffles and to cell-cell contacts, respectively. Furthermore, expression of the activated Rac1(V12) in NIH 3T3 cells leads to an increased A6/twinfilin localization to nucleus and cell cortex, whereas a dominant negative form of Rac1(V12,N17) induces A6/twinfilin localization to cytoplasm. Taken together, these studies show that mouse A6/twinfilin is an actin monomer-binding protein whose localization to cortical G-actin-rich structures may be regulated by the small GTPase Rac1.

Actins↗
PubMed1999

Virus-specific capping of tobacco mosaic virus RNA: methylation of GTP prior to formation of covalent complex p126-m7GMP.

In capping cellular mRNAs, a covalent GMP-enzyme intermediate leads to formation of G(5')ppp(5')N at the 5' end of the RNA, which is modified by methylation catalyzed by guanine-7-methyltransferase. Here we show that isolated membranes from tobacco mosaic virus (TMV)-infected plant or insect cells expressing TMV replicase protein p126, synthesized m7GTP using S-adenosylmethionine (AdoMet) as the methyl donor, and catalyzed the formation of a covalent guanylate-p126 complex in the presence of AdoMet. The methyl group from AdoMet was incorporated into p126, suggesting that the complex consisted of m7GMP-p126. Thus, TMV and alphaviruses, despite their evolutionary distance, share the same virus-specific capping mechanism.

Amino Acid Sequence↗
PubMed1999

Semliki Forest virus mRNA capping enzyme requires association with anionic membrane phospholipids for activity.

The replication complexes of all positive strand RNA viruses of eukaryotes are associated with membranes. In the case of Semliki Forest virus (SFV), the main determinant of membrane attachment seems to be the virus-encoded non-structural protein NSP1, the capping enzyme of the viral mRNAs, which has guanine-7-methyltransferase and guanylyltransferase activities. We show here that both enzymatic activities of SFV NSP1 are inactivated by detergents and reactivated by anionic phospholipids, especially phosphatidylserine. The region of NSP1 responsible for binding to membranes as well as to liposomes was mapped to a short segment, which is conserved in the large alphavirus-like superfamily of viruses. A synthetic peptide of 20 amino acids from the putative binding site competed with in vitro synthesized NSP1 for binding to liposomes containing phosphatidylserine. These findings suggest a molecular mechanism by which RNA virus replicases attach to intracellular membranes and why they depend on the membranous environment.

Amino Acid Sequence↗
PubMed1999

Intracellular distribution of rubella virus nonstructural protein P150.

Antiserum prepared against an amino-terminal fragment of rubella virus (RUB) nonstructural polyprotein was used to study RUB-infected Vero cells. Replicase protein P150 was associated with vesicles and vacuoles of endolysosomal origin and later with large, convoluted, tubular membrane structures. Newly incorporated bromouridine was associated with the same structures and specifically with small membrane invaginations, spherules, indicating that these structures may be the sites of viral RNA synthesis.

Animals↗
PubMed1998

Alphavirus replicase protein NSP1 induces filopodia and rearrangement of actin filaments.

Expression of the NSP1 protein of Semliki Forest virus and Sindbis virus in cultured cells induced filopodia-like extensions containing NSP1 but not F actin. The actin stress fibers disappeared, whereas vimentin, keratin, and tubulin networks remained intact. The effects of NSP1 were dependent on its palmitoylation but not on its enzymatic activities and were also observed in virus-infected cells.

Actins↗
PubMed1996

Endosome dynamics regulated by a Rho protein.

Vesicular transport is a dynamic process that requires coordinated interactions between membrane and cytoskeleton. The mechanisms and molecules integrating these interactions are unclear. A Rho protein, RhoD, might provide a molecular link between membrane traffic and the cytoskeleton. Activated RhoD causes rearrangements of the actin cytoskeleton and cell surface, and governs early endosome motility and distribution.

Actins↗
PubMed1996

Functional characterization of mouse syndecan-1 promoter.

The members of the syndecan family are temporally and spatially expressed heparan sulfate proteoglycans of various tissues, where they mediate extracellular influences on cell morphology and behavior. Functional characterization of the mouse syndecan-1 promoter was carried out in order to elucidate the mechanisms involved in the maintenance of the high transcription levels of syndecan-1 gene in various epithelia. For that 9.5 kilobase pairs of the upstream region of mouse syndecan-1 gene were cloned, sequenced, and used to prepare chimaeric constructs with a reporter gene followed by transient or stable transfections into NMuMG cells, cultured either in the presence or absence of serum, the 2.5-kilobase pair promoter region resulted in the constitutive transcription activity, whereas in 3T3 cells the serum depletion decreased the promoter activity significantly. Deletion of the upstream sequences to -437 base pairs relative to the translation initiation site had little effect on this promoter activity. Further deletion to -365 base pairs removed three GT boxes and slightly increased the promoter activity, whereas the deletion of the next two GC boxes (to -326 base pair) reduced the promoter activity dramatically. All of the GC or GT box sequences bound the same set of Sp1-like nuclear protein in gel shift assays. Nuclear protein binding was also demonstrated around both of the most intense transcription initiation sites. Mutation of these regions separately resulted in total loss of transcription initiation from the deleted site and decreased the promoter activity in relation to the intensity of the abolished start site. This indicates that the transcription initiation of the syndecan-1 gene is directed through initiator-like elements directly overlapping the start sites, as shown for several TATA-less housekeeping and growth regulated genes. We assume that the constitutive high level gene expression in epithelial cells is achieved by the proximal promoter, which is controlled by members of Sp1 transcription factor family.

3T3 Cells↗
PubMed1996

Rab8 promotes polarized membrane transport through reorganization of actin and microtubules in fibroblasts.

Rab8 is a small Ras-like GTPase that regulates polarized membrane transport to the basolateral membrane in epithelial cells and to the dendrites in neurons. It has recently been demonstrated that fibroblasts sort newly synthesized proteins into two different pathways for delivery to the cell surface that are equivalent to the apical and the basolateral post-Golgi routes in epithelial cells (Yoshimori, T., P. Keller, M.G. Roth, and K. Simons. 1996. J. Cell Biol. 133:247-256). To determine the role of Rab8 in fibroblasts, we used both transient expression systems and stable cell lines expressing mutant or wild-type (wt) Rab8. A dramatic change in cell morphology occurred in BHK cells expressing both the wt Rab8 and the activated form of the GTPase, the Rab8Q67L mutant. These cells formed processes as a result of a reorganization of both their actin filaments and microtubules. Newly synthesized vesicular stomatitis virus G glycoprotein, a basolateral marker protein in MDCK cells, was preferentially delivered into these cell outgrowths. Based on these findings, we propose that Rab8 provides a link between the machinery responsible for the formation of cell protrusions and polarized biosynthetic membrane traffic.

Actin Cytoskeleton↗
PubMed1995

CDC42 and Rac1 control different actin-dependent processes in the Drosophila wing disc epithelium.

Cdc42 and Rac1 are members of the rho family of small guanosinetriphosphatases and are required for a diverse set of cytoskeleton-membrane interactions in different cell types. Here we show that these two proteins contribute differently to the organization of epithelial cells in the Drosophila wing imaginal disc. Drac1 is required to assemble actin at adherens junctions. Failure of adherens junction actin assembly in Drac1 dominant-negative mutants is associated with increased cell death. Dcdc42, on the other hand, is required for processes that involve polarized cell shape changes during both pupal and larval development. In the third larval instar, Dcdc42 is required for apico-basal epithelial elongation. Whereas normal wing disc epithelial cells increase in height more than twofold during the third instar, cells that express a dominant-negative version of Dcdc42 remain short and are abnormally shaped. Dcdc42 localizes to both apical and basal regions of the cell during these events, and mediates elongation, at least in part, by effecting a reorganization of the basal actin cytoskeleton. These observations suggest that a common cdc42-based mechanism may govern polarized cell shape changes in a wide variety of cell types.

Actins↗