A functional interaction of Ku with Werner exonuclease facilitates digestion of damaged DNA

Werner syndrome (WS) is a premature aging disorder where the affected individuals appear much older than their chronological age. The single gene that is defective in WS encodes a protein (WRN) that has ATPase, helicase and 3′→5′ exonuclease activities. Our laboratory has recently uncovered a physical and functional interaction between WRN and the Ku heterodimer complex that functions in double-strand break repair and V(D)J recombination. Importantly, Ku specifically stimulates the exonuclease activity of WRN. We now report that Ku enables the Werner exonuclease to digest through regions of DNA containing 8-oxoadenine and 8-oxoguanine modifications, lesions that have previously been shown to block the exonuclease activity of WRN alone. These results indicate that Ku significantly alters the exonuclease function of WRN and suggest that the two proteins function concomitantly in a DNA damage processing pathway. In support of this notion we also observed co-localization of WRN and Ku, particularly after DNA damaging treatments.

Protonation dynamics of the extracellular and cytoplasmic surface of bacteriorhodopsin in the purple membrane.

The dynamics of proton binding to the extracellular and the cytoplasmic surfaces of the purple membrane were measured by laser-induced proton pulses. Purple membranes, selectively labeled by fluorescein at Lys-129 of bacteriorhodopsin, were pulsed by protons released in the aqueous bulk from excited pyranine (8-hydroxy-1,3,6-pyrenetrisulfonate) and the reaction of protons with the indicators was measured. Kinetic analysis of the data imply that the two faces of the membrane differ in their buffer capacities and in their rates of interaction with bulk protons. The extracellular surface of the purple membrane contains one anionic proton binding site per protein molecule with pK = 5.1. This site is within a Coulomb cage radius (approximately 15 A) from Lys-129. The cytoplasmic surface of the purple membrane bears 4-5 protonable moieties (pK = 5.1) that, due to close proximity, function as a common proton binding site. The reaction of the proton with this cluster is at a very fast rate (3.10(10) M-1.s-1). The proximity between the elements is sufficiently high that even in 100 mM NaCl they still function as a cluster. Extraction of the chromophore retinal from the protein has a marked effect on the carboxylates of the cytoplasmic surface, and two to three of them assume positions that almost bar their reaction with bulk protons. The protonation dynamics determined at the surface of the purple membrane is of relevance both for the vectorial proton transport mechanism of bacteriorhodopsin and for energy coupling, not only in halobacteria, but also in complex chemiosmotic systems such as mitochondrial and thylakoid membranes.

Urea-inducible Egr-1 transcription in renal inner medullary collecting duct (mIMCD3) cells is mediated by extracellular signal-regulated kinase activation.

Urea (200-400 milliosmolar) activates transcription, translation of, and trans-activation by the immediate-early gene transcription factor Egr-1 in a renal epithelial cell-specific fashion. The effect at the transcriptional level has been attributed to multiple serum response elements and their adjacent Ets motifs located within the Egr-1 promoter. Elk-1, a principal ternary complex factor and Ets domain-containing protein, is a substrate of the extracellular signal-regulated kinase (ERK) mitogen-activated protein kinases. In the renal medullary mIMCD3 cell line, urea (200-400 milliosmolar) activated both ERK1 and ERK2 as determined by in-gel kinase assay and immune-complex kinase assay of epitope-tagged] ERK1 and ERK2. Importantly, urea did not affect abundance of either ERK. Urea-inducible Egr-1 transcription was a consequence of ERK activation because the ERK-specific inhibitor, PD98059, abrogated transcription from the murine Egr-1 promoter in a luciferase reported gene assay. In addition, activators of protein kinase A, including forskolin and 8-Br-cAMP, which are known to inhibit ERK-mediated events, also inhibited urea-inducible Egr-1 transcription. Furthermore, urea-inducible activation of the physiological ERK substrate and transcription factor, Elk-1, was demonstrated through transient cotransfection of a chimeric Elk-1/GAL4 expression plasmid and a GAL4-driven luciferase reporter plasmid. Taken together, these data indicate that, in mIMCD3 cells, urea activates ERKs and the ERK substrate, Elk-1, and that ERK inhibition abrogates urea-inducible Egr-1 transcription. These data are consistent with a model of urea-inducible renal medullary gene expression wherein sequential activation of ERKs and Elk-1 results in increased transcription of Egr-1 through serum response element/Ets motifs.

Mutations affecting conserved cysteine residues within the extracellular domain of Neu promote receptor dimerization and activation.

Overexpression of the Neu/ErbB-2 receptor tyrosine kinase has been implicated in the genesis of human breast cancer. Indeed, expression of either activated or wild-type neu in the mammary epithelium of transgenic mice results in the induction of mammary tumors. Previously, we have shown that many of the mammary tumors arising in transgenic mice expressing wild-type neu occur through somatic activating mutations within the neu transgene itself. Here we demonstrate that these mutations promote dimerization of the Neu receptor through the formation of disulfide bonds, resulting in its constitutive activation. To explore the role of conserved cysteine residues within the region deleted in these altered Neu proteins, we examined the transforming potential of a series of Neu receptors in which the individual cysteine residues were mutated. These analyses indicated that mutation of certain cysteine residues resulted in the oncogenic activation of Neu. The increased transforming activity displayed by the altered receptors correlated with constitutive dimerization that occurred in a disulfide bond-dependent manner. We further demonstrate that addition of 2-mercaptoethanol to the culture medium interfered with the specific transforming activity of the mutant Neu receptors. These observations suggest that oncogenic activation of Neu results from constitutive disulfide bond-dependent dimerization.

Human fetal enterocytes in vitro: modulation of the phenotype by extracellular matrix.

The differentiation of small intestinal epithelial cells may require stimulation by microenvironmental factors in vivo. In this study, the effects of mesenchymal and luminal elements in nonmalignant epithelia] cells isolated from the human fetus were studied in vitro. Enterocytes from the human fetus were cultured and microenvironmental factors were added in stages, each stage more closely approximating the microenvironment in vivo. Four stages were examined: epithelial cells derived on plastic from intestinal culture and grown as a cell clone, the same cells grown on connective tissue support, primary epithelial explants grown on fibroblasts with a laminin base, and primary epithelial explants grown on fibroblasts and laminin with n-butyrate added to the incubation medium. The epithelial cell clone dedifferentiated when grown on plastic; however, the cells expressed cytokeratins and villin as evidence of their epithelial cell origin. Human connective tissue matrix from Engelbreth-Holm-Swarm sarcoma cells (Matrigel) modulated their phenotype: alkaline phosphatase activity increased, microvilli developed on their apical surface, and the profile of insulin-like growth factor binding proteins resembled that secreted by differentiated enterocytes. Epithelial cells taken directly from the human fetus as primary cultures and grown as explants on fibroblasts and laminin expressed greater specific enzyme activities in brush border membrane fractions than the cell clone. These activities were enhanced by the luminal molecule sodium butyrate. Thus the sequential addition of connective tissue and luminal molecules to nonmalignant epithelia] cells in vitro induces a spectrum of changes in the epithelial cell phenotype toward full differentiation.

Linker histones affect patterns of digestion of supercoiled plasmids by single-strand-specific nucleases.

The effect of histone H1 binding on the cleavage of superhelical plasmids by single-strand-specific nucleases was investigated. Mapping of P1 cleavage sites in pBR322, achieved by EcoRI digestion after the original P1 attack, showed an intriguing phenomenon: preexisting susceptible sites became "protected," whereas some new sites appeared at high levels of H1. Similar results were obtained with another single-strand-specific nuclease, S1. Disappearance of cutting at preexisting sites and appearance of new sites was also observed in a derivative plasmid that contains a 36-bp stretch of alternating d(AT) sequence that is known to adopt an altered P1-sensitive conformation. On the other hand, H1 titration of a dimerized version of the d(AT)18-containing plasmid led to protection of all preexisting sites except the d(AT)18 inserts, which were still cut even at high H1 levels; in this plasmid no new sites appeared. The protection of preexisting sites is best explained by long-range effects of histone H1 binding on the superhelical torsion of the plasmid. The appearance of new sites, on the other hand, probably also involves a local effect of stabilization of specific sequences in Pl-sensitive conformation, due to direct H1 binding to such sequences. That such binding involves linker histone N- and/or C-terminal tails is indicated by the fact that titration with the globular domain of H5, while causing disappearance of preexisting sites, does not lead to the appearance of any new sites.

The glycine binding site of the N-methyl-D-aspartate receptor subunit NR1: identification of novel determinants of co-agonist potentiation in the extracellular M3-M4 loop region.

The N-methyl-D-aspartate (NMDA) subtype of ionotropic glutamate receptors is a heterooligomeric membrane protein composed of homologous subunits. Here, the contribution of the M3-M4 loop of the NR1 subunit to the binding of glutamate and the co-agonist glycine was investigated by site-directed mutagenesis. Substitution of the phenylalanine residues at positions 735 or 736 of the M3-M4 loop produced a 15- to 30-fold reduction in apparent glycine affinity without affecting the binding of glutamate and the competitive glycine antagonist 7-chlorokynurenic acid; mutation of both residues caused a >100-fold decrease in glycine affinity. These residues are found in a C-terminal region of the M3-M4 loop that shows significant sequence similarity to bacterial amino acid-binding proteins. Epitope tagging revealed both the N-terminus and the M3-M4 loop to be exposed extracellularly, whereas a C-terminal epitope was localized intracellularly. These results indicate that the M3-M4 loop is part of the ligand-binding pocket of the NR1 subunit and provide the basis for a refined model of the glycine-binding site of the NMDA receptor.

The rat extracellular superoxide dismutase dimer is converted to a tetramer by the exchange of a single amino acid.

Extracellular superoxide dismutase (EC-SOD) is a secreted Cu and Zn-containing glycoprotein. While EC-SOD from most mammals is tetrameric and has a high affinity for heparin and heparan sulfate, rat EC-SOD has a low affinity for heparin, does not bind to heparan sulfate in vivo, and is apparently dimeric. To examine the molecular basis of the deviant physical properties of rat EC-SOD, the cDNAs of the rat and mouse EC-SODs were isolated and the deduced amino acid sequences were compared with that of human EC-SOD. Comparison of the sequences offered no obvious explanation of the differences. Analysis of a series of chimeric and point mutated EC-SODs showed that the N-terminal region contributes to the oligomeric state of the EC-SODs, and that a single amino acid, a valine (human amino acid position 24), is essential for the tetramerization. This residue is replaced by an aspartate in the rat. Rat EC-SOD carrying an Asp --> Val mutation is tetrameric and has a high heparin affinity, while mouse EC-SOD with a Val --> Asp mutation is dimeric and has lost its high heparin affinity. Thus, the rat EC-SOD dimer is converted to a tetramer by the exchange of a single amino acid. Furthermore, the cooperative action of four heparin-binding domains is necessary for high heparin affinity. These results also suggest that tetrameric EC-SODs are not symmetrical tetrahedrons, but composed of two interacting dimers, further supporting an evolutionary relationship with the dimeric cytosolic Cu and Zn-containing SODs.

Ca2+ channel blockers modulate metabolism of collagens within the extracellular matrix.

The extracellular matrix (ECM) is an intricate network composed of an array of macromolecules capable of regulating the functional responsiveness of cells. Its composition greatly varies among different types of tissue, and dysregulation of its metabolism may contribute to vascular remodeling during the pathogenesis of various diseases, including atherosclerosis. In view of their antiatherosclerotic effects, the role of Ca2+ channel blockers in the metabolism of ECM was examined. Nanomolar concentrations of the five Ca2+ channel blockers amlodipine, felodipine, manidipine, verapamil, or diltiazem significantly decreased both the constitutive and platelet-derived growth factor BB-dependent collagen deposition in the ECM formed by human vascular smooth muscle cells and fibroblasts. The drugs inhibited the expression of fibrillar collagens type I and III and of basement membrane type IV collagen. Furthermore, Ca2+ channel blockers specifically increased the proteolytic activity of the 72-kDa type IV collagenase as shown by gelatin zymography and inhibited the transcription of tissue inhibitor of metalloproteinases-2.

Signal transduction by activated mNotch: importance of proteolytic processing and its regulation by the extracellular domain.

Previous studies imply that the intracellular domain of Notch1 must translocate to the nucleus for its activity. In this study, we demonstrate that a mNotch1 mutant protein that lacks its extracellular domain but retains its membrane-spanning region becomes proteolytically processed on its intracellular surface and, as a result, the activated intracellular domain (mNotchIC) is released and can move to the nucleus. Proteolytic cleavage at an intracellular site is blocked by protease inhibitors. Intracellular cleavage is not seen in cells transfected with an inactive variant, which includes the extracellular lin-Notch-glp repeats. Collectively, the studies presented here support the model that mNotch1 is proteolytically processed and the cleavage product is translocated to the nucleus for mNotch1 signal transduction.

Antibodies against specific extracellular epitopes of the glucagon receptor block glucagon binding.

Polyclonal antibodies were prepared against synthetic peptides corresponding to four different extramembrane segments of the rat glucagon receptor. The antibodies bound specifically to native glucagon receptor as judged by immunofluorescence microscopy of cultured cells expressing a synthetic gene for the receptor. Antibodies to peptides designated PR-15 and DK-12 were directed against amino acid residues 103-117 and 126-137, respectively, of the extracellular N-terminal tail. Antibody to peptide KD-14 was directed against residues 206-219 of the first extracellular loop, and antibody to peptide ST-18, against the intracellular C-terminal tail, residues 468-485. The DK-12 and KD-14 antibodies, but not the PR-15 and ST-18 antibodies, could effectively block binding of 125I-labeled glucagon to its receptor in liver membranes. Incubation of these antibodies with rat liver membranes resulted in both a decrease in the maximal hormonal binding capacity and an apparent decrease in glucagon affinity for its receptor. These effects were abolished in the presence of excess specific peptide antigen. In addition, DK-12 and KD-14 antibodies, but not PR-15 and ST-18 antibodies, interfered with glucagon-induced adenylyl cyclase activation in rat liver membranes and behaved as functional glucagon antagonists. These results demonstrate that DK-12 and KD-14 antibodies are pharmacologically active glucagon antagonists and strongly suggest that residues 126-137 of the N-terminal tail and residues 206-219 of the first extracellular loop contain determinants of ligand binding and may comprise the primary ligand-binding site on the glucagon receptor.

Estrogens increase the expression of fibulin-1, an extracellular matrix protein secreted by human ovarian cancer cells.

Ovarian cancers have a high ability to invade the peritoneal cavity and some are stimulated by estrogens. In an attempt to understand the mode of action of estrogens on these cancer cells and to develop new markers, we have characterized estrogen-regulated proteins. This study was aimed at identifying a protein secreted by ovarian cancer cells whose level was increased by estradiol [Galtier-Dereure, F., Capony, F., Maudelonde, T. & Rochefort, H. (1992) J. Clin. Endocrinol. Metab. 75, 1497-1502]. By using microprotein sequencing, the 110-kDa protein was identified as fibulin-1, a protein of the extracellular matrix that binds to fibronectin, laminin, and nidogen. The amount of immunoprecipitated fibulin-1 secreted into the medium and present in the cell extract was increased up to 10-fold by estradiol in three estrogen-responsive ovarian cancer cell lines. By immunohistochemistry fibulin-1 was located in the stroma of several ovarian cancers and cysts. The findings highlight a potential role for fibulin-1 in the spread of ovarian cancer in the peritoneal cavity and/or in distal metastases.

The peptide binding site of the substance P (NK-1) receptor localized by a photoreactive analogue of substance P: presence of a disulfide bond.

Substance P (SP) is a neuropeptide that mediates multiple physiological responses including transmission of painful stimuli and inflammation via an interaction with a receptor of known primary sequence. To identify the regions of the SP receptor, also termed the NK-1 receptor, involved in peptide recognition, we are using analogues of SP containing the photoreactive amino acid p-benzoyl-L-phenylalanine (Bpa). In the present study, we used radioiodinated Bpa8-SP to covalently label with high efficiency the rat SP receptor expressed in a transfected mammalian cell line. To identify the amino acid residue that serves as the site of covalent attachment, a membrane preparation of labeled receptor was subjected to partial enzymatic cleavage by trypsin. A major digestion product of 22 kDa was identified. Upon reduction with 2-mercaptoethanol the mass of this product decreased to 14 kDa. The 22-kDa tryptic fragment was purified in excellent yield by preparative SDS/PAGE under nonreducing conditions. Subcleavage with Staphylococcus aureus V8 protease and endoproteinase ArgC yielded fragments of 8.2 and 9.0 kDa, respectively. Upon reductive cleavage, the V8 protease fragment decreased to 3.0 kDa while the endoproteinase ArgC fragment decreased to 3.2 kDa. Taking into consideration enzyme specificity, molecular size, determination of the presence or absence of N-glycosylation sites, and recognition by antibodies to specific sequences of the SP receptor, the V8 protease fragment is Thr-173 to Glu-183, while the endoproteinase ArgC fragment is Val-178 to Arg-190. These two fragments share the common sequence Val-Val-Cys-Met-Ile-Glu (residues 178-183). The site of covalent attachment of radioiodinated Bpa8-SP is thus restricted to a residue within this overlap sequence. The data presented here also establish that the cysteine residue in this sequence Cys-180, which is positioned in the middle of the second extracellular loop, participates in a disulfide bond that links the first and second extracellular loops of the receptor.

Intravenous cocaine, morphine, and amphetamine preferentially increase extracellular dopamine in the "shell" as compared with the "core" of the rat nucleus accumbens.

The nucleus accumbens is considered a critical target of the action of drugs of abuse. In this nucleus a "shell" and a "core" have been distinguished on the basis of anatomical and histochemical criteria. The present study investigated the effect in freely moving rats of intravenous cocaine, amphetamine, and morphine on extracellular dopamine concentrations in the nucleus accumbens shell and core by means of microdialysis with vertically implanted concentric probes. Doses selected were in the range of those known to sustain drug self-administration in rats. Morphine, at 0.2 and 0.4 mg/kg, and cocaine, at 0.5 mg/kg, increased extracellular dopamine selectivity in the shell. Higher doses of cocaine (1.0 mg/kg) and the lowest dose of amphetamine tested (0.125 mg/kg) increased extracellular dopamine both in the shell and in the core, but the effect was significantly more pronounced in the shell compared with the core. Only the highest dose of amphetamine (0.250 mg/kg) increased extracellular dopamine in the shell and in the core to a similar extent. The present results provide in vivo neurochemical evidence for a functional compartmentation within the nucleus accumbens and for a preferential effect of psychostimulants and morphine in the shell of the nucleus accumbens at doses known to sustain intravenous drug self-administration.

Glucose stimulation of insulin release in the absence of extracellular Ca2+ and in the absence of any increase in intracellular Ca2+ in rat pancreatic islets.

Insulin secretion has been studied in isolated rat pancreatic islets under stringent Ca(2+)-depleted, Ca(2+)-free conditions. Under these conditions, the effect of 16.7 mM glucose to stimulate insulin release was abolished. Forskolin, which activates adenylyl cyclase, also failed to stimulate release in the presence of either low or high glucose concentrations. A phorbol ester (phorbol 12-myristate 13-acetate; PMA) increased the release rate slightly and this was further increased by 16.7 mM glucose. Remarkably, in the presence of both forskolin and PMA, 16.7 mM glucose strongly augmented insulin release. The augmentation was concentration dependent and monophasic and had a temporal profile similar to the "second phase" of glucose-stimulated insulin release, which is seen under normal conditions when Ca2+ is present. Metabolism is required for the effect because mannoheptulose abolished the glucose response. Other nutrient secretagogues, alpha-ketoisocaproate, and the combination of leucine and glutamine augmented release under the same conditions. Norepinephrine, a physiological inhibitor of insulin secretion, totally blocked the stimulation of release by forskolin and PMA and the augmentation of release by glucose. Thus, under the stringent Ca(2+)-free conditions imposed, the stimulation of insulin release by forskolin and PMA, as well as the augmentation of release by glucose, is under normal physiological control. As no increase in intracellular [Ca2+] was observed, the results demonstrate that glucose can increase the rate of exocytosis and insulin release by pancreatic islets in a Ca(2+)-independent manner. This interesting pathway of stimulus-secretion coupling for glucose appears to exert its effect at a site beyond the usual elevation of intracellular [Ca2+] and is not due to an activation by glucose of protein kinase A or C.