Cellular Vesicles: New Insights in Engineering Methods, Interaction with Cells and Potential for Brain Targeting.

Cellular vesicles (CVs) have been proposed as alternatives to exosomes for targeted drug delivery. CVs, prepared from human embryonic kidney 293 cells (HEK-293), C57BL/6 mouse B16F10 skin melanoma cells (B16F10), and immortalized human cerebral microvascular endothelial cells (hCMEC/D3) by liposome technology methods, were characterized for morphology, cytotoxicity, and cell uptake properties. CV brain-targeting potential was evaluated in vitro on the hCMEC/D3 blood-brain barrier (BBB) model, and in vivo/ex vivo. CV sizes were between 135 and 285 nm, and the ζ-potential was negative. The dehydration-rehydration method conferred highest calcein loading and latency to CVs compared with other methods. The increased calcein leakage from CVs when compared with liposomes indicated their poor integrity, which was increased by pegylation. The in vivo results confirmed lower liver uptake by PEG-CVs (compared with nonpegylated) proving that the calcein integrity test is useful for prediction of CV biodistribution, as used for liposomes. The cell uptake of homologous origin CVs was not always higher compared with that of non-homologous. Nevertheless, CVs from hCMEC/D3 demonstrated the highest BBB permeability (in vitro) compared with OX-26 targeted liposomes, and brain localization (in vivo). CVs from hCMEC/D3 cells grown in different media demonstrated decreased interaction with brain cells and brain localization. Significant differences in proteome of the two latter CV types were identified by proteomics, suggesting a potential methodology for identification of organotropism-determining CV components.

Identification of novel PTEN-binding partners: PTEN interaction with fatty acid binding protein FABP4.

PTEN is a tumor suppressor with dual protein and lipid-phosphatase activity, which is frequently deleted or mutated in many human advanced cancers. Recent studies have also demonstrated that PTEN is a promising target in type II diabetes and obesity treatment. Using C-terminal PTEN sequence in pEG202-NLS as bait, yeast two-hybrid screening on Mouse Embryo, Colon Cancer, and HeLa cDNA libraries was carried out. Isolated positive clones were validated by mating assay and identified through automated DNA sequencing and BLAST database searches. Sequence analysis revealed a number of PTEN-binding proteins linking this phosphatase to a number of different signaling cascades, suggesting that PTEN may perform other functions besides tumor-suppressing activity in different cell types. In particular, the interplay between PTEN function and adipocyte-specific fatty-acid-binding protein FABP4 is of notable interest. The demonstrable tautology of PTEN to FABP4 suggested a role for this phosphatase in the regulation of lipid metabolism and adipocyte differentiation. This interaction was further studied using coimmunoprecipitation and gel-filtration assays. Finally, based on Biacore assay, we have calculated the K(D) of PTEN-FABP4 complex, which is around 2.8 microM.

Phosphatidyl-inositol 3-kinase: a key enzyme in diverse signalling processes.

Phosphatidylinositol (PI) 3-kinase associates in signal-transducing complexes with activated growth factor receptors and other protein tyrosine kinases. The enzyme may also act downstream of receptors that are not tyrosine kinases in terminally differentiated cells. The recent cloning of the catalytic subunit of PI 3-kinase has revealed a structural similarity to a yeast protein important in vacuolar protein sorting. This finding provides some interesting clues to the function of PI3-kinase in diverse cellular responses.

Phosphoinositide 3-kinases: a conserved family of signal transducers.

Phosphoinositide 3-kinases (PI3Ks) generate lipids that are implicated in receptor-stimulated signalling and in the regulation of membrane traffic. Several distinct classes of PI3Ks have now been identified that have been conserved throughout eukaryotic evolution. Potential signalling pathways downstream of PI3Ks have been elucidated and PI3K function is now being characterised in several model organisms.

A novel recognition motif for phosphatidylinositol 3-kinase binding mediates its association with the hepatocyte growth factor/scatter factor receptor.

The pleiotropic effects (mitogenesis, motogenesis, and morphogenesis) elicited by hepatocyte growth factor/scatter factor (HGF/SF) are mediated by the activation of the tyrosine kinase receptor encoded by the MET proto-oncogene. Following autophosphorylation, the receptor associates with the p85/110 phosphatidylinositol (PI) 3-kinase complex in vivo and in vitro. By a combination of two complementary approaches, competition with synthetic phosphopeptides and association with Tyr-Phe receptor mutants, we have identified Y-1349 and Y-1356 in the HGF/SF receptor as the binding sites for PI 3-kinase. Y-1349VHV and Y-1356VNV do not conform to the canonical consensus sequence YXXM for PI 3-kinase binding and thus define YVXV as a novel recognition motif. Y-1349 and Y-1356 are located within the C-terminal portion of the HGF/SF receptor and are phosphorylation sites. The affinity of the N- and C-terminal src homology region 2 (SH2) domains of p85 for the phosphopeptides including Y-1349 and Y-1356 is 2 orders of magnitude lower than that measured for Y-751 in the platelet-derived growth factor receptor binding site. However, the closely spaced duplication of the novel recognition motif in the native HGF/SF receptor may allow binding with both SH2 domains of p85, thus generating an efficient docking site for PI 3-kinase. In agreement with this model, we have observed that a phosphopeptide including both Y-1349 and Y-1356 activates PI 3-kinase in vitro.

Wortmannin inactivates phosphoinositide 3-kinase by covalent modification of Lys-802, a residue involved in the phosphate transfer reaction.

Wortmannin at nanomolar concentrations is a potent and specific inhibitor of phosphoinositide (PI) 3-kinase and has been used extensively to demonstrate the role of this enzyme in diverse signal transduction processes. At higher concentrations, wortmannin inhibits the ataxia telangiectasia gene (ATM)-related DNA-dependent protein kinase (DNA-PKcs). We report here the identification of the site of interaction of wortmannin on the catalytic subunit of PI 3-kinase, p110alpha. At physiological pH (6.5 to 8) wortmannin reacted specifically with p110alpha. Phosphatidylinositol-4,5-diphosphate, ATP, and ATP analogs [adenine and 5'-(4-fluorosulfonylbenzoyl)adenine] competed effectively with wortmannin, while substances containing nucleophilic amino acid side chain functions had no effect at the same concentrations. This suggests that the wortmannin target site is localized in proximity to the substrate-binding site and that residues involved in wortmannin binding have an increased nucleophilicity because of their protein environment. Proteolytic fragments of wortmannin-treated, recombinant p110alpha were mapped with anti-wortmannin and anti-p110alpha peptide antibodies, thus limiting the target site within a 10-kDa fragment, colocalizing with the ATP-binding site. Site-directed mutagenesis of all candidate residues within this region showed that only the conservative Lys-802-to-Arg mutation abolished wortmannin binding. Inhibition of PI 3-kinase occurs, therefore, by the formation of an enamine following the attack of Lys-802 on the furan ring (at C-20) of wortmannin. The Lys-802-to-Arg mutant was also unable to bind FSBA and was catalytically inactive in lipid and protein kinase assays, indicating a crucial role for Lys-802 in the phosphotransfer reaction. In contrast, an Arg-916-to-Pro mutation abolished the catalytic activity whereas covalent wortmannin binding remained intact. Our results provide the basis for the design of novel and specific inhibitors of an enzyme family, including PI kinases and ATM-related genes, that play a central role in many physiological processes.

Interactions between SH2 domains and tyrosine-phosphorylated platelet-derived growth factor beta-receptor sequences: analysis of kinetic parameters by a novel biosensor-based approach.

The interaction between SH2 domains and phosphotyrosine-containing sequences was examined by real-time measurements of kinetic parameters. The SH2 domains of the p85 subunit of the phosphatidylinositol 3-kinase as well as of other signaling molecules were expressed in bacteria as glutathione S-transferase fusion proteins. Phosphotyrosine-containing peptides, corresponding to two autophosphorylation sites on the human platelet-derived growth factor beta-receptor that are responsible for phosphatidylinositol 3-kinase binding, were synthesized and used as capturing molecules, immobilized on a biosensor surface. The association and dissociation rate constants for binding to both sites were determined for intact p85 and the recombinant SH2 domains. High association rates were found to be coupled to very fast dissociation rates for all interactions studied. A binding specificity was observed for the two SH2 domains of p85, with the N-terminal SH2 binding with high affinity to the Tyr-751 site but not to the Tyr-740 site, and the C-terminal SH2 interacting strongly with both sites. This approach should be generally applicable to the study of the specificity inherent in the assembly of signaling complexes by activated protein-tyrosine kinase receptors.

SH2 domains of the p85 alpha subunit of phosphatidylinositol 3-kinase regulate binding to growth factor receptors.

The binding of cytoplasmic signaling proteins such as phospholipase C-gamma 1 and Ras GTPase-activating protein to autophosphorylated growth factor receptors is directed by their noncatalytic Src homology region 2 (SH2) domains. The p85 alpha regulatory subunit of phosphatidylinositol (PI) 3-kinase, which associates with several receptor protein-tyrosine kinases, also contains two SH2 domains. Both p85 alpha SH2 domains, when expressed individually as fusion proteins in bacteria, bound stably to the activated beta receptor for platelet-derived growth factor (PDGF). Complex formation required PDGF stimulation and was dependent on receptor tyrosine kinase activity. The bacterial p85 alpha SH2 domains recognized activated beta PDGF receptor which had been immobilized on a filter, indicating that SH2 domains contact autophosphorylated receptors directly. Several receptor tyrosine kinases within the PDGF receptor subfamily, including the colony-stimulating factor 1 receptor and the Steel factor receptor (Kit), also associate with PI 3-kinase in vivo. Bacterially expressed SH2 domains derived from the p85 alpha subunit of PI 3-kinase bound in vitro to the activated colony-stimulating factor 1 receptor and to Kit. We infer that the SH2 domains of p85 alpha bind to high-affinity sites on these receptors, whose creation is dependent on receptor autophosphorylation. The SH2 domains of p85 are therefore primarily responsible for the binding of PI 3-kinase to activated growth factor receptors.

Autoantibodies against aggrecan in systemic rheumatic diseases.

OBJECTIVE: This study was undertaken to investigate the presence of autoantibodies against the main cartilage proteoglycan, aggrecan, in systemic rheumatic disease sera, and to identify substructure(s) responsible for the autoimmune response. METHODS: Sera were obtained from 86 patients with various systemic rheumatic diseases, 14 with osteoarthritis (OA), 18 with cancer and 40 healthy individuals. The presence of autoantibodies against aggrecan was examined by a solid phase assay and by Western blotting, using proteoglycan aggregates treated with proteolytic enzymes. The positive bands were subjected to nanohigh performance liquid chromatography (nanoHPLC)-MS, in order to identify the aggrecan substructures involved in the autoimmune response. RESULTS: Autoantibodies against aggrecan were identified in all systemic rheumatic disease sera at a high titre, almost three times that observed in healthy controls. OA and cancer sera produced a reaction equal to that of the healthy. Western blotting analysis of aggrecan proteolytic fragments revealed the presence of a triple band, reacting with the patients' sera, of about 37 kDa, which also reacted with a polyclonal antibody against hyaluronan-binding region. NanoHPLC-MS analysis suggested that this band belonged to the G2 domain of aggrecan. CONCLUSION: At least a part of the autoimmune reaction to aggrecan, displayed by the systemic disease sera, involves the G2 domain. The significant difference observed between these sera and those from other diseases, especially cancer, may suggest a possible discriminatory role of anti-aggrecan antibodies. This may help in the differential diagnosis in complicated clinical cases. However, for this to be confirmed, studies in larger cohorts of patients should be performed.

EGF promotes in vivo tumorigenic growth of primary chicken embryo fibroblasts expressing v-myc and enhances in vitro transformation by the v-erbA oncogene.

We report that the activation of the endogenous chicken EGF receptor leads to the tumorigenic growth in vivo of early passage chicken embryo fibroblasts (CEFs) that express a nonsarcomagenic oncogene, v-myc. To provide a continuous paracrine source of this growth factor in vivo, we employed irradiated Rat-1 cells which had been stably transfected with a synthetic cDNA to human EGF. Expression of another non-sarcomagenic nuclear oncogene, v-erbA, prones the CEFs to in vitro transformation by EGF, but does not cause EGF dependent tumorigenicity in vivo. The short period of incubation in the in vivo assay employed by our study (10 days), together with the genetic stability of primary chicken embryo fibroblasts, make it very likely that the reported alterations in cellular behaviour are a direct and primary effect of the expression of the relevant oncogenes and their cooperation with the EGF induced response. Dose response and ligand binding assays suggest that the EGF response is transmitted via the chicken c-erbB molecule, which by virtue of its preference for TGF-alfa is distinct from the mammalian EGF receptors studied so far. The level of expression of the endogenous chicken EGF receptor is within the same range as that reported for primary human fibroblasts (5-7 x 10(3) per cell). The cooperative effect of v-myc with chicken c-erbB probably takes place at a post receptor level, as its expression did not affect the steady state level or affinity for ligand of the chicken EGF receptor.

A juxtamembrane autophosphorylation site in the Eph family receptor tyrosine kinase, Sek, mediates high affinity interaction with p59fyn.

The large subfamily of receptor tyrosine kinases (RTKs) for which EPH is the prototype have likely roles in intercellular communication during normal mammalian development, but the biochemical signalling pathways utilised by this family are poorly characterised. We have now identified two in vitro autophosphorylation sites within the juxtamembrane domain of the Eph family member Sek, and a candidate binding protein for the activated Sek kinase. Specific antibodies defined Sek as a 130 kDa glycoprotein with protein kinase activity expressed in keratinocytes, whilst a bacterially expressed gst-Sek kinase domain fusion protein autophosphorylated exclusively on tyrosine residues, confirming that Sek encodes an authentic protein tyrosine kinase. Two dimensional phosphopeptide mapping and site-directed mutagenesis defined juxtamembrane residue Y602 as a major site of in vitro autophosphorylation in Sek, whilst Y596 was phosphorylated to a lower stoichiometry. Complimentary approaches of in vitro binding assays and BIAcore analysis revealed a high affinity association between the Y602 Sek autophosphorylation site and the cytoplasmic tyrosine kinase p59fyn, an interaction mediated through the SH2 domain of this intracellular signalling molecule. Moreover, these data identify the novel phosphotyrosyl motif pYEDP as mediating high affinity association with fyn-SH2, extending the previously defined consensus motif for this interaction. The extensive conservation of this fyn-binding motif within the juxtamembrane domain of Eph family RTKs suggests that signalling through fyn, or fyn-related, tyrosine kinases may be utilised by many members of this large subclass of transmembrane receptors.

The motogenic and mitogenic responses to HGF are amplified by the Shc adaptor protein.

The receptor of Hepatocyte Growth Factor-Scatter Factor (HGF) is a tyrosine kinase which regulates cell motility and growth. After ligand-induced tyrosine phosphorylation, the HGF receptor associates with the Shc adaptor, via the SH2 domain. Site-directed mutagenesis of the HGF receptor indicates that phosphotyrosines Y1349VHV and Y1356VNV can work as docking sites for Shc. The Kd of this interaction, measured in real time using synthetic phosphopeptides and recombinant Shc on a BIAcore biosensor, is 150 nm for both sites. After stimulation of the HGF receptor, Shc is phosphorylated on Y317VNV, generating an high affinity binding site for Grb2 (Kd = 15 nM). This duplicates the high affinity binding site for Grb2 present on the HGF receptor (Y1356VNV). Thus HGF stimulation can trigger the Ras pathway by recruiting Grb2 both directly through the receptor, and indirectly, through Shc. Overexpression of wild-type Shc, but not of the Y317-->F mutant, enhances cell migration and growth in response to HGF. These data show that Shc is a relevant substrate of the HGF receptor, and works as an 'amplifier' of the motogenic as well as of the mitogenic response.

Phospholipid-binding protein domains.

Research into cellular mechanisms for signal transduction is currently one of the most exciting and rapidly advancing fields of biological study. It has been known for some time that numerous intracellular signals are transmitted by specific protein-protein interactions, as exemplified by those involving the Src homology domains. However, after some controversy, it has recently been widely accepted that specific protein-phospholipid interactions also play key roles in many signal transduction pathways. In this review, landmark discoveries and recent advances describing protein domains known to associate with phospholipids are discussed. Particular emphasis is placed on the interactions of proteins with phospholipids acting as second messengers in signalling pathways. For this purpose, the pleckstrin homology (PH) domain is highlighted, since studies of this domain provided some of the earliest, detailed data about protein-phospholipid interactions occurring downstream of growth factor-mediated receptor stimulation. Moreover, studies of PH domains have given insight into the mechanisms of certain diseases, revealed a number of intriguing functional variations on a common structural theme and recently culminated in providing the missing links in erstwhile mysteries of phosphoinositide-dependent signal transduction pathways. Finally, a short discussion is devoted to the developing field of protein-phospholipid interactions that influence cytoskeletal organisation.

Solution structure of the C-terminal SH2 domain of the p85 alpha regulatory subunit of phosphoinositide 3-kinase.

Heterodimeric class IA phosphoinositide 3-kinase (PI 3-kinase) plays a crucial role in a variety of cellular signalling events downstream of a number of cell-surface receptor tyrosine kinases. Activation of the enzyme is effected in part by the binding of two Src homology-2 domains (SH2) of the 85 kDa regulatory subunit to specific phosphotyrosine-containing peptide motifs within activated cytoplasmic receptor domains. The solution structure of the uncomplexed C-terminal SH2 (C-SH2) domain of the p85 alpha subunit of PI 3-kinase has been determined by means of multinuclear, double and triple-resonance NMR experiments and restrained molecular-dynamics simulated-annealing calculations. The solution structure clearly indicates that the uncomplexed C-SH2 domain conforms to the consensus polypeptide fold exhibited by other SH2 domains, with an additional short helical element at the N terminus. In particular, the C-SH2 structure is very similar to both the p85 alpha N-terminal SH2 domain (N-SH2) and the Src SH2 domain with a root mean square difference (rmsd) for 44 C alpha atoms of 1.09 and 0.89 A, respectively. The canonical BC, EF and BG loops are less well-defined by the experimental restraints and show greater variability in the ensemble of C-SH2 conformers. The lower level of definition in these regions may reflect the presence of conformational disorder, an interpretation supported by the absence or broadening of backbone and side-chain NMR resonances for some of these residues. NMR experiments were performed, where C-SH2 was titrated with phosphotyrosine-containing peptides corresponding to p85 alpha recognition sites in the cytoplasmic domain of the platelet-derived growth-factor receptor. The ligand-induced chemical-shift perturbations indicate the amino-acid residues in C-SH2 involved in peptide recognition follow the pattern predicted from homologous complexes. A series of C-SH2 mutants was generated and tested for phosphotyrosine peptide binding by surface plasmon resonance. Mutation of the invariant Arg36 (beta B5) to Met completely abolishes phosphopeptide binding. Mutation of each of Ser38, Ser39 or Lys40 in the BC loop to Ala reduces the affinity of C-SH2 for a cognate phosphopeptide, as does mutation of His93 (BG5) to Asn. These effects are consistent with the involvement of the BC loop and BG loops regions in ligation of phosphopeptide ligands. Mutation of Cys57 (beta D5) in C-SH2 to Ile, the corresponding residue type in the p85 alpha N-SH2 domain, results in a change in peptide binding selectivity of C-SH2 towards that demonstrated by p85 alpha N-SH2. This pattern of p85 alpha phosphopeptide binding specificity is interpreted in terms of a model of the p85 alpha/PDGF-receptor interaction.

Phosphopeptide binding to the N-terminal SH2 domain of the p85 alpha subunit of PI 3'-kinase: a heteronuclear NMR study.

The N-terminal src-homology 2 domain of the p85 alpha subunit of phosphatidylinositol 3' kinase (SH2-N) binds specifically to phosphotyrosine-containing sequences. Notably, it recognizes phosphorylated Tyr 751 within the kinase insert of the cytoplasmic domain of the activated beta PDGF receptor. A titration of a synthetic 12-residue phosphopeptide (ESVDY*VPMLDMK) into a solution of the SH2-N domain was monitored using heteronuclear 2D and 3D NMR spectroscopy. 2D-(15N-1H) heteronuclear single-quantum correlation (HSQC) experiments were performed at each point of the titration to follow changes in both 15N and 1H chemical shifts in NH groups. When mapped onto the solution structure of the SH2-N domain, these changes indicate a peptide-binding surface on the protein. Line shape analysis of 1D profiles of individual (15N-1H)-HSQC peaks at each point of the titration suggests a kinetic exchange model involving at least 2 steps. To characterize changes in the internal dynamics of the domain, the magnitude of the (15N-1H) heteronuclear NOE for the backbone amide of each residue was determined for the SH2-N domain with and without bound peptide. These data indicate that, on a nanosecond timescale, there is no significant change in the mobility of either loops or regions of secondary structure. A mode of peptide binding that involves little conformational change except in the residues directly involved in the 2 binding pockets of the p85 alpha SH2-N domain is suggested by this study.

Dual specificity antibodies using a double-stranded oligonucleotide bridge.

The covalent conjugation of oligonucleotides to antibody Fab' fragments was optimized by using oligonucleotides modified with a hexaethylene linker arm bearing three amino groups. One oligonucleotide was coupled to antibody of one specificity and a complementary oligonucleotide to antibody of a second specificity. The antibodies were then allowed to hybridize by base pairing of the complementary nucleotide sequences and the generation of bispecific antibody was analyzed on SDS-PAGE and confirmed using BIAcore analysis. The strategy of complementary oligonucleotide-linked bispecific molecules is not limited to antibodies but is applicable to linking any two molecules of different characteristics.

Ageing research in Greece.

Ageing research in Greece is well established. Research groups located in universities, research institutes or public hospitals are studying various and complementary aspects of ageing. These research activities include (a) functional analysis of Clusterin/Apolipoprotein J, studies in healthy centenarians and work on protein degradation and the role of proteasome during senescence at the National Hellenic Research Foundation; (b) regulation of cell proliferation and tissue formation, a nationwide study of determinants and markers of successful ageing in Greek centenarians and studies of histone gene expression and acetylation at the National Center for Scientific Research, Demokritos; (c) work on amyloid precursor protein and Presenilin 1 at the University of Athens; (d) oxidative stress-induced DNA damage and the role of oncogenes in senescence at the University of Ioannina; (e) studies in the connective tissue at the University of Patras; (f) proteomic studies at the Biomedical Sciences Research Center Alexander Fleming; (g) work on Caenorhabditis elegans at the Foundation for Research and Technology; (h) the role of ultraviolet radiation in skin ageing at Andreas Sygros Hospital; (i) follow-up studies in healthy elderly at the Athens Home for the Aged; and (j) socio-cultural aspects of ageing at the National School of Public Health. These research activities are well recognized by the international scientific community as it is evident by the group's very good publication records as well as by their direct funding from both European Union and USA. This article summarizes these research activities and discuss future directions and efforts towards the further development of the ageing field in Greece.

Antibody recognition of epitopes on wild-type and mutant beta-(1-->4)-galactosyltransferase-1.

The epitopes present on beta-(1-->4)-galactosyltransferase-1 (beta 4Gal-T1) have been explored using a panel of monoclonal antibodies (mAbs) raised against the soluble form of the human enzyme. Reactivity of the antibodies with site-specific and truncated mutants of human beta 4Gal-T1 suggests the presence of a major immunogenic epitope cluster consisting of four epitopes within the stem region and mapping between amino acids 42 and 115. The catalytic activity of the enzyme is increased in the presence of stem region-specific antibody. Two of the epitopes were further localized to a region between amino acids 42 and 77, sequences which are not shared with the recently cloned beta 4Gal-T2 and beta 4Gal-T3 enzymes. An epitope located close to or within the catalytic domain is also identified, and the mAb to this region binds synergistically with antibodies to the stem region.

The mechanism of DNA repair by uracil-DNA glycosylase: studies using nucleotide analogues.

2',4'-Dideoxy-4'-methyleneuridine incorporated into oligodeoxynucleotides forms regular B-DNA duplexes as shown by Tm and CD measurements. Such oligomers are not cleaved by the DNA repair enzyme, UDG, which cleaves the glycosylic bond in dU but not in dT nor in dC nucleosides in single stranded and double stranded DNA. Differential binding of oligomers containing carbadU, 4'-thiodU, and dU residues to wild type and mutant UDG proteins identify an essential role for the furanose 4'-oxygen in recognition and cleavage of dU residues in DNA.