Characterization of Syntenin, a Syndecan-binding PDZ Protein, as a Component of Cell Adhesion Sites and Microfilaments

Syntenin is a PDZ protein that binds the cytoplasmic C-terminal FYA motif of the syndecans. Syntenin is widely expressed. In cell fractionation experiments, syntenin partitions between the cytosol and microsomes. Immunofluorescence microscopy localizes endogenous and epitope-tagged syntenin to cell adhesion sites, microfilaments, and the nucleus. Syntenin is composed of at least three domains. Both PDZ domains of syntenin are necessary to target reporter tags to the plasma membrane. The addition of a segment of 10 amino acids from the N-terminal domain of syntenin to these PDZ domains increases the localization of the tags to stress fibers and induces the formation of long, branching plasma membrane extensions. The addition of the complete N-terminal region, in contrast, reduces the localization of the tags to plasma membrane/adhesion sites and stress fibers, and reduces the morphotypical effects. Recombinant domains of syntenin with the highest plasma membrane localization display the lowest nuclear localization. Syndecan-1, E-cadherin, β-catenin, and α-catenin colocalize with syntenin at cell-cell contacts in epithelial cells, and coimmunoprecipitate with syntenin from extracts of these cells. These results suggest a role for syntenin in the composition of adherens junctions and the regulation of plasma membrane dynamics, and imply a potential role for syntenin in nuclear processes.

Vipp1 deletion mutant of Synechocystis: A connection between bacterial phage shock and thylakoid biogenesis?

Plant chloroplasts originated from an endosymbiotic event by which an ancestor of contemporary cyanobacteria was engulfed by an early eukaryotic cell and then transformed into an organelle. Oxygenic photosynthesis is the specific feature of cyanobacteria and chloroplasts, and the photosynthetic machinery resides in an internal membrane system, the thylakoids. The origin and genesis of thylakoid membranes, which are essential for oxygenic photosynthesis, are still an enigma. Vipp1 (vesicle-inducing protein in plastids 1) is a protein located in both the inner envelope and the thylakoids of Pisum sativum and Arabidopsis thaliana. In Arabidopsis disruption of the VIPP1 gene severely affects the plant's ability to form properly structured thylakoids and as a consequence to carry out photosynthesis. In contrast, Vipp1 in Synechocystis appears to be located exclusively in the plasma membrane. Yet, as in higher plants, disruption of the VIPP1 gene locus leads to the complete loss of thylakoid formation. So far VIPP1 genes are found only in organisms carrying out oxygenic photosynthesis. They share sequence homology with a subunit encoded by the bacterial phage shock operon (PspA) but differ from PspA by a C-terminal extension of about 30 amino acids. In two cyanobacteria, Synechocystis and Anabaena, both a VIPP1 and a pspA gene are present, and phylogenetic analysis indicates that VIPP1 originated from a gene duplication of the latter and thereafter acquired its new function. It also appears that the C-terminal extension that discriminates VIPP1 proteins from PspA is important for its function in thylakoid formation.

The arginine finger of RasGAP helps Gln-61 align the nucleophilic water in GAP-stimulated hydrolysis of GTP

The Ras family of GTPases is a collection of molecular switches that link receptors on the plasma membrane to signaling pathways that regulate cell proliferation and differentiation. The accessory GTPase-activating proteins (GAPs) negatively regulate the cell signaling by increasing the slow intrinsic GTP to GDP hydrolysis rate of Ras. Mutants of Ras are found in 25–30% of human tumors. The most dramatic property of these mutants is their insensitivity to the negative regulatory action of GAPs. All known oncogenic mutants of Ras map to a small subset of amino acids. Gln-61 is particularly important because virtually all mutations of this residue eliminate sensitivity to GAPs. Despite its obvious importance for carcinogenesis, the role of Gln-61 in the GAP-stimulated GTPase activity of Ras has remained a mystery. Our molecular dynamics simulations of the p21ras–p120GAP–GTP complex suggest that the local structure around the catalytic region can be different from that revealed by the x-ray crystal structure. We find that the carbonyl oxygen on the backbone of the arginine finger supplied in trans by p120GAP (Arg-789) interacts with a water molecule in the active site that is forming a bridge between the NH2 group of the Gln-61 and the γ-phosphate of GTP. Thus, Arg-789 may play a dual role in generating the nucleophile as well as stabilizing the transition state for P—O bond cleavage.

Interaction with mLin-7 Alters the Targeting of Endocytosed Transmembrane Proteins in Mammalian Epithelial Cells

To investigate the targeting mechanism for proteins bound to the mammalian Lin-7 (mLin-7) PDZ domain, we created receptor protein chimeras composed of the carboxyl-terminal amino acids of LET-23 fused to truncated nerve growth factor receptor/P75. mLin-7 bound to the chimera with a wild-type LET-23 carboxyl-terminal tail (P75t-Let23WT), but not a mutant tail (P75t-Let23MUT). In Madin-Darby canine kidney (MDCK) cells, P75t-Let23WT localized to the basolateral plasma membrane domain, whereas P75t-Let23MUT remained apical. Furthermore, mutant mLin-7 constructs acted as dominant interfering proteins and inhibited the basolateral localization of P75t-Let23WT. The mechanisms for this differential localization were examined further, and, initially, we found that P75t-Let23WT and P75t-Let23MUT were delivered equally to the apical and basolateral plasma membrane domains. Although basolateral retention of P75t-Let23WT, but not P75t-Let23MUT, was observed, the greatest difference in receptor localization was seen in the rapid trafficking of P75t-Let23WT to the basolateral plasma membrane domain after endocytosis, whereas P75t-Let23MUT was degraded in lysosomes, indicating that mLin-7 binding can alter the fate of endocytosed proteins. Altogether, these data support a model for basolateral protein targeting in mammalian epithelial cells dependent on protein–protein interactions with mLin-7, and also suggest a dynamic role for mLin-7 in endosomal sorting.

Manipulation of Glutathione and Amino Acid Biosynthesis in the Chloroplast1

Poplars (Populus tremula × Populus alba) were transformed to overexpress Escherichia coli γ-glutamylcysteine synthetase (γ-ECS) or glutathione synthetase in the chloroplast. Five independent lines of each transformant strongly expressed the introduced gene and possessed markedly enhanced activity of the gene product. Glutathione (GSH) contents were unaffected by high chloroplastic glutathione synthetase activity. Enhanced chloroplastic γ-ECS activity markedly increased γ-glutamylcysteine and GSH levels. These effects are similar to those previously observed in poplars overexpressing these enzymes in the cytosol. Similar to cytosolic γ-ECS overexpression, chloroplastic overexpression did not deplete foliar cysteine or methionine pools and did not lead to morphological changes. Light was required for maximal accumulation of GSH in poplars overexpressing γ-ECS in the chloroplast. High chloroplastic, but not cytosolic, γ-ECS activities were accompanied by increases in amino acids synthesized in the chloroplast. We conclude that (a) GSH synthesis can occur in the chloroplast and the cytosol and may be up-regulated in both compartments by increased γ-ECS activity, (b) interactions between GSH synthesis and the pathways supplying the necessary substrates are similar in both compartments, and (c) chloroplastic up-regulation of GSH synthesis is associated with an activating effect on the synthesis of specific amino acids formed in the chloroplast.

Direct evidence for modified solvent structure within the hydration shell of a hydrophobic amino acid.

Neutron scattering experiments are used to determine scattering profiles for aqueous solutions of hydrophobic and hydrophilic amino acid analogs. Solutions of hydrophobic solutes show a shift in the main diffraction peak to smaller angle as compared with pure water, whereas solutions of hydrophilic solutes do not. The same difference for solutions of hydrophobic and hydrophilic side chains is also predicted by molecular dynamics simulations. The neutron scattering curves of aqueous solutions of hydrophobic amino acids at room temperature are qualitatively similar to differences between the liquid molecular structure functions measured for ambient and supercooled water. The nonpolar solute-induced expansion of water structure reported here is also complementary to recent neutron experiments where compression of aqueous solvent structure has been observed at high salt concentration.

Tumor necrosis factor receptor associated factor 2 is a mediator of NF-kappa B activation by latent infection membrane protein 1, the Epstein-Barr virus transforming protein.

Latent infection membrane protein 1 (LMP1), the Epstein-Barr virus transforming protein, associates with tumor necrosis factor receptor (TNFR) associated factor 1 (TRAF1) and TRAF3. Since TRAF2 has been implicated in TNFR-mediated NF-kappa B activation, we have evaluated the role of TRAF2 in LMP1-mediated NF-kappa B activation. TRAF2 binds in vitro to the LMP1 carboxyl-terminal cytoplasmic domain (CT), coprecipitates with LMP1 in B lymphoblasts, and relocalizes to LMP1 plasma membrane patches. A dominant negative TRAF2 deletion mutant that lacks amino acids 6-86 (TRAF/ delta 6-86) inhibits NF-kappa B activation from the LMP1 CT and competes with TRAF2 for LMP1 binding. TRAF2 delta 6-86 inhibits NF-kappa B activation mediated by the first 45 amino acids of the LMP1 CT by more than 75% but inhibits NF-kappa B activation through the last 55 amino acids of the CT by less than 40%. A TRAF interacting protein, TANK, inhibits NF-kappa B activation by more than 70% from both LMP1 CT domains. These data implicate TRAF2 aggregation in NF-kappa B activation by the first 45 amino acids of the LMP1 CT and suggest that a different TRAF-related pathway may be involved in NF-kappa B activation by the last 55 amino acids of the LMP1 CT.

Mapping rat megalin: the second cluster of ligand binding repeats contains a 46-amino acid pathogenic epitope involved in the formation of immune deposits in Heymann nephritis.

Megalin (gp330), an epithelial endocytic receptor, is a major target antigen of Heymann nephritis (HN), an autoimmune disease in rats. To elucidate the mechanisms of HN, we have mapped a pathogenic epitope in megalin that binds anti-megalin antibodies. We focused our attention on four clusters of cysteine-rich, low density lipoprotein receptor (LDLR) ligand binding repeats in the extracellular domain of megalin because they represent putative ligand binding regions and therefore would be expected to be exposed in vivo and to be able to bind circulating antibodies. Rat megalin cDNA fragments I through IV encoding the first through fourth clusters of ligand-binding repeats, respectively, were expressed in a baculovirus system. All four expression products were detected by immunoblotting with two antisera capable of inducing passive HN (pHN). When antibodies eluted from glomeruli of rats with pHN were used for immunoblotting, only the expression product encoded by fragment II was detected. This indicates that the second cluster of LDLR ligand binding repeats is directly involved in binding anti-megalin antibodies and in the induction of pHN. To narrow the major epitope in this domain, fragment II was used to prepare proteins sequentially truncated from the C- and N-terminal ends by in vitro translation. Analysis of the truncated translation products by immunoprecipitation with anti-megalin IgG revealed that the fifth ligand-binding repeat (amino acids 1160-1205) contains the major epitope recognized. This suggests that a 46-amino acid sequence in the second cluster of LDLR ligand binding repeats contains a major pathogenic epitope that plays a key role in pHN. Identification of this epitope will facilitate studies on the pathogenesis of HN.

Crystal structure of D-amino acid oxidase: a case of active site mirror-image convergent evolution with flavocytochrome b2.

D-amino acid oxidase is the prototype of the FAD-dependent oxidases. It catalyses the oxidation of D-amino acids to the corresponding alpha-ketoacids. The reducing equivalents are transferred to molecular oxygen with production of hydrogen peroxide. We have solved the crystal structure of the complex of D-amino acid oxidase with benzoate, a competitive inhibitor of the substrate, by single isomorphous replacement and eightfold averaging. Each monomer is formed by two domains with an overall topology similar to that of p-hydroxybenzoate hydroxylase. The benzoate molecule lays parallel to the flavin ring and is held in position by a salt bridge with Arg-283. Analysis of the active site shows that no side chains are properly positioned to act as the postulated base required for the catalytic carboanion mechanism. On the contrary, the benzoate binding mode suggests a direct transfer of the substrate alpha-hydrogen to the flavin during the enzyme reductive half-reaction.The active site Of D-amino acid oxidase exhibits a striking similarity with that of flavocytochrome b2, a structurally unrelated FMN-dependent flavoenzyme. The active site groups (if these two enzymes are in fact superimposable once the mirror-image of the flavocytochrome b2 active site is generated with respect to the flavin plane. Therefore, the catalytic sites of D-amino acid oxidase and flavocytochrome b2 appear to have converged to a highly similar but enantiomeric architecture in order to catalvze similar reactions (oxidation of alpha-amino acids or alpha-hydroxy acids), although with opposite stereochemistry.

Brush border myosin-I truncated in the motor domain impairs the distribution and the function of endocytic compartments in an hepatoma cell line.

Myosins I, a ubiquitous monomeric class of myosins that exhibits actin-based motor properties, are associated with plasma and/or vesicular membranes and have been suggested as players for trafficking events between cell surface and intracellular membranous structures. To investigate the function of myosins 1, we have transfected a mouse hepatoma cell line (BWTG3) with cDNAs encoding the chicken brush border myosin-I (BBMI) and two variants truncated in the motor domain. One variant is deleted of the first 446 amino acids and thereby lacks the ATP binding site, whereas the other is deleted of the entire motor domain and lacks the ATP and actin binding sites. We have observed (i) that significant amounts of the truncated variants are recovered with membrane fractions after cell fractionation, (ii) that they codistribute with a compartment containing alpha2-macroglobulin internalized for 30 min as determined by fluorescent microscopy, (iii) that the production of BBMI-truncated variants impairs the distribution of the acidic compartment and ligands internalized for 30 min, and (iv) that the production of the truncated variant containing the actin binding site decreases the rate of alpha2-macroglobulin degradation whereas the production of the variant lacking the ATP binding site and the actin binding site increases the rate of a2-macroglobulin degradation. These observations indicate that the two truncated variants have a dominant negative effect on the distribution and the function of the endocytic compartments. We propose that an unidentified myosin-I might contribute to the distribution of endocytic compartments in a juxtanuclear position and/or to the regulation of the delivery of ligands to the degradative compartment in BWTG3 cells.

Matrix metalloproteinase 2 releases active soluble ectodomain of fibroblast growth factor receptor 1.

Recent studies have demonstrated the existence of a soluble fibroblast growth factor (FGF) receptor type 1 (FGFR1) extracellular domain in the circulation and in vascular basement membranes. However, the process of FGFR1 ectodomain release from the plasma membrane is not known. Here we report that the 72-kDa gelatinase A (matrix metalloproteinase type 2, MMP2) can hydrolyze the Val368-Met369 peptide bond of the FGFR1 ectodomain, eight amino acids upstream of the transmembrane domain, thus releasing the entire extracellular domain. Similar results were obtained regardless of whether FGF was first bound to the receptor or not. The action of MMP2 abolished binding of FGF to an immobilized recombinant FGFR1 ectodomain fusion protein and to Chinese hamster ovary cells overexpressing FGFR1 The released recombinant FGFR1 ectodomain was able to bind FGF after MMP2 cleavage, suggesting that the cleaved soluble receptor maintained its FGF binding capacity. The activity of MMP2 could not be reproduced by the 92-kDa gelatinase B (MMP9) and was inhibited by tissue inhibitor of metalloproteinase type 2. These studies demonstrate that FGFR1 may be a specific target for MMP2 on the cell surface, yielding a soluble FGF receptor that may modulate the mitogenic and angiogenic activities of FGF.

Posttranslational amino acid epimerization: enzyme-catalyzed isomerization of amino acid residues in peptide chains.

Since ribosomally mediated protein biosynthesis is confined to the L-amino acid pool, the presence of D-amino acids in peptides was considered for many years to be restricted to proteins of prokaryotic origin. Unicellular microorganisms have been responsible for the generation of a host of D-amino acid-containing peptide antibiotics (gramicidin, actinomycin, bacitracin, polymyxins). Recently, a series of mu and delta opioid receptor agonists [dermorphins and deltorphins] and neuroactive tetrapeptides containing a D-amino acid residue have been isolated from amphibian (frog) skin and mollusks. Amino acid sequences obtained from the cDNA libraries coincide with the observed dermorphin and deltorphin sequences, suggesting a stereospecific posttranslational amino acid isomerization of unknown mechanism. A cofactor-independent serine isomerase found in the venom of the Agelenopsis aperta spider provides the first major clue to explain how multicellular organisms are capable of incorporating single D-amino acid residues into these and other eukaryotic peptides. The enzyme is capable of isomerizing serine, cysteine, O-methylserine, and alanine residues in the middle of peptide chains, thereby providing a biochemical capability that, until now, had not been observed. Both D- and L-amino acid residues are susceptible to isomerization. The substrates share a common Leu-Xaa-Phe-Ala recognition site. Early in the reaction sequence, solvent-derived deuterium resides solely with the epimerized product (not substrate) in isomerizations carried out in 2H2O. Significant deuterium isotope effects are obtained in these reactions in addition to isomerizations of isotopically labeled substrates (2H at the epimerizeable serine alpha-carbon atom). The combined kinetic and structural data suggests a two-base mechanism in which abstraction of a proton from one face is concomitant with delivery from the opposite face by the conjugate acid of the second enzymic base.

Aromatic amino acid transamination and methionine recycling in trypanosomatids.

Although trypanosomatids are known to rapidly transaminate exogenous aromatic amino acids in vitro and in vivo, the physiological significance of this reaction is not understood. In postmitochondrial supernatants prepared from Trypanosoma brucei brucei and Crithidia fasciculata, we have found that aromatic amino acids were the preferred amino donors for the transamination of alpha-ketomethiobutyrate to methionine. Intact C. fasciculata grown in the presence of [15N]tyrosine were found to contain detectable [15N]methionine, demonstrating that this reaction occurs in situ in viable cells. This process is the final step in the recycling of methionine from methylthioadenosine, a product of decarboxylated S-adenosylmethionine from the polyamine synthetic pathway. Mammalian liver, in contrast, preferentially used glutamine for this reaction and utilized a narrower range of amino donors than seen with the trypanosomatids. Studies with methylthioadenosine showed that this compound was readily converted to methionine, demonstrating a fully functional methionine-recycling pathway in trypanosomatids.

Characterization of a second secreted IgE isoform and identification of an asymmetric pathway of IgE assembly.

A number of alternatively spliced epsilon transcripts have been detected in IgE-producing B cells, in addition to the mRNAs encoding the classical membrane and secreted IgE heavy (H) chains. In a recent study, we examined the protein products of three of these alternatively spliced isoforms and found that they are intracellularly retained and degraded because of their inability to assemble into complete IgE molecules. We have now similarly examined a more recently described epsilon mRNA species that is generated by splicing between a donor splice site immediately upstream of the stop codon in the H-chain constant region exon 4 (CH4) and an acceptor site located in the 3' part of the second membrane exon. We show that this isoform is efficiently secreted by both plasma cells and B lymphocytes and therefore represents a second secreted IgE isoform (epsilon S2). The epsilon S2 H chain is only six amino acids longer than the classical secreted Ig H chain (epsilon S1) and contains a C-terminal cysteine, which is a characteristic sequence feature of mu and alpha H chains. However, unlike IgM and IgA, the epsilon S2 C-terminal cysteine (Cys-554) does not induce polymerization of H2L2 molecules (where L is light chain), but rather creates a disulfide bond between the two H chains that increases the rate of association into covalently bound H2L2 monomers. This C-terminal cysteine also does not function as an intracellular retention element because the epsilon S2 isoform was secreted in amounts equal to that of the epsilon S1, both in B lymphocytes and in plasma cells. The epsilon S2 H chains secreted by B lymphocytes differed from the epsilon S1 H chains in the extent of glycosylation. Interestingly, a difference in glycosylation between B-lymphocytes and plasma cells was also noted for both isoforms. The presence of the Cys-554 also allowed the identification of a distinctive asymmetric pathway of IgE assembly, common to both types of epsilon H chains.

Mass spectrometric amino acid sequencing of a mixture of seed storage proteins (napin) from Brassica napus, products of a multigene family.

The amino acid sequences of a number of closely related proteins ("napin") isolated from Brassica napus were determined by mass spectrometry without prior separation into individual components. Some of these proteins correspond to those previously deduced (napA, BngNAP1, and gNa), chiefly from DNA sequences. Others were found to differ to a varying extent (BngNAP1', BngNAP1A, BngNAP1B, BngNAP1C, gNa', and gNaA). The short chains of gNa and gNa' and of BngNAP1 and BngNAP1' differ by the replacement of N-terminal proline by pyroglutamic acid; the long chains of gNaA and BngNAP1B contain a six amino acid stretch, MQGQQM, which is present in gNa (according to its DNA sequence) but absent from BngNAP1 and BngNAP1C. These alternations of sequences between napin isoforms are most likely due to homologous recombination of the genetic material, but some of the changes may also be due to RNA editing. The amino acids that follow the untruncated C termini of those napin chains for which the DNA sequences are known (napA, BngNAP1, and gNa) are aromatic amino acids. This suggests that the processing of the proprotein leading to the C termini of the two chains is due to the action of a protease that specifically cleaves a G/S-F/Y/W bond.