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.

Analysis of parotid glands of primary Sjögren's syndrome patients using proteomic technology reveals altered autoantigen composition and novel antigenic targets.

Sjögren's syndrome (SS) is an autoimmune disease characterized by lymphocytic infiltration, destruction of the salivary and lacrimal glands and production of autoantibodies against a variety of cellular proteins. The aberrant immune response against these autoantigens may begin or extend to other proteins that are not yet defined. Several studies have shown that autoantibody production is taking place in the affected salivary glands. In the present study, using proteomic approaches, we aimed to: (a) identify new autoantigens in the salivary glands of primary SS (pSS) patients and (b) evaluate the epigenetic changes of known autoantigens. Total parotid gland extracts of pSS patients were analysed using two-dimensional gel electrophoresis, sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblot with pSS patients' sera or purified autoantibodies and immunoprecipitation using homologous IgG. Identification of the unknown proteins was performed using mass spectrometry (MS). Immunoblot analysis on two-dimensional gels using purified anti-La/SSB antibodies revealed that pSS salivary glands contain high levels of post-translationally modified La/SSB autoantigen, while the native form of the protein is recognized faintly, in contrast to normal controls. Moreover, salivary glands of pSS patients contain post-translationally modified actin that becomes immunogenic in the microenviroment of the affected tissue. The alteration of the physicochemical properties of self-proteins could thus contribute to the break of immune tolerance against them.

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.

Calreticulin binds preferentially with B cell linear epitopes of Ro60 kD autoantigen, enhancing recognition by anti-Ro60 kD autoantibodies.

Calreticulin is a molecular chaperone to newly synthesized polypeptides. Previous studies suggested that calreticulin is probably a protein member of the Ro/La RNP complex. The aims of this study were (a) to investigate whether linear B cell epitopes of the Ro/La RNP complex are bound to calreticulin and (b) if the complex peptide-calreticulin is recognized specifically by anti-Ro autoantibodies. Calreticulin was isolated from either human or pig spleen using a multi-step purification method and found to interact preferentially with biotinylated peptides derived from the sequence of the Ro60 kD 175-184aa(10p) and 216-232aa(17p). The interaction of the peptide-calreticulin complex was favoured by the combination of heat treatment, divalent cations and ATP. La/SSB epitopes did not react with calreticulin. Peptides corresponding to La/SSB epitopes as well as the common epitope of Sm did not interact with calreticulin. Thirty-eight anti-Ro60 KD positive and 23 anti-Ro60 kD negative sera of patients with systemic lupus erythematosus (SLE) and primary Sjögren's syndrome (pSS) were tested. All anti-Ro60 kD positive sera bound the complex calreticulin-17p, while 95% of the same sera had activity against the complex calreticulin - 10p. Tested individually, calreticulin, pep10p and pep17p presented very low reactivity (8%, 11% and 29%, respectively) against anti-Ro60 kD positive sera. Anti-Ro60 KD negative sera did not exhibit significant reactivity either with calreticulin, 10rho and 17rho or with the complexes calreticulin - 10p and calreticulin-17p (<5%). These results suggest that calreticulin can induce conformation-dependent recognition of the Ro60 kD epitopes, leading eventually to their recognition by autoantibodies. This is the first time that such a relationship is shown between a chaperone protein and fragments of an intracellular autoantigen. This work also provides insights into the understanding of mechanisms for autoantibody production. Furthermore, this association can be proved useful for the development of new sensitive assays for autoantibody detection.

Fine specificity and subclasses of IgG anti-actin autoantibodies differ in health and disease.

Current opinions suggest that autoantibodies occurring in autoimmune diseases are generated by B-cells which primarily produce polyspecific natural autoantibodies, through either polyclonal activation or specific antigen selection of these B-cells. In this study, we compared the immunological properties (polyspecificity, fine specificity and IgG subclasses) between natural anti-actin antibodies (N-AAA) and disease-associated AAA (D-AAA). IgG AAA from sera of healthy donors, patients with autoimmune hepatitis type 1 (AIH-1) and patients with primary biliary cirrhosis (PBC) were affinity-purified on actin immunoadsorbent and tested initially for polyspecificity against various cytoskeleton proteins by enzyme-linked immunosorbent assay (ELISA). Fine specificity was studied by Western blotting using proteolytic peptides of actin and by ELISA using synthetic 12 mer peptides, spanning the 221-377 aa sequence of actin. Results showed that both N-AAA and D-AAA are polyspecific. Nevertheless, D-AAA from both diseases showed a specific reactivity pattern as compared to N-AAA, against the 16 kDa C-terminal (229-377 aa) proteolytic peptide of actin and more specifically against the P36 synthetic peptide (351-362 aa). Quantitation of AAA IgG subclasses revealed that IgG1 and IgG3 were specifically increased in D-AAA from AIH-1 and PBC, respectively, as compared to N-AAA. We conclude that D-AAA are differentiated from N-AAA in terms of fine specificity and IgG subclasses, probably through specific antigen selection of B-cells primarily producing N-AAA.

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.

Natural variants of human p85 alpha phosphoinositide 3-kinase in severe insulin resistance: a novel variant with impaired insulin-stimulated lipid kinase activity.

AIMS/HYPOTHESIS: Phosphoinositide 3-kinase (PI 3K) plays a central part in the mediation of insulin-stimulated glucose disposal. No genetic studies of this enzyme in human syndromes of severe insulin resistance have been previously reported. METHODS: Phosphoinositide 3-kinase p85 alpha regulatory subunit cDNA was examined in 20 subjects with syndromes of severe insulin resistance by single strand conformational polymorphism and restriction fragment length polymorphism analyses. Insulin-stimulated phosphoinositide 3-kinase activity and recruitment into phosphotyrosine complexes of variants of p85 alpha were studied in transiently transfected HEK293 cells. Phosphopeptide binding characteristics of wild-type and mutant p85 alpha-GST fusion proteins were examined by surface plasmon resonance. RESULTS: The common p85 alpha variant, Met326I1e, was identified in 9 of the 20 subjects. Functional studies of the Met326Ile variant showed it to have equivalent insulin-stimulated lipid kinase activity and phosphotyrosine recruitment as wild-type p85 alpha. A novel heterozygous mutation, Arg409Gln, was detected in one subject. Within the proband's family, carriers of the mutation had a higher median fasting plasma insulin (218 pmol/l) compared with wild-type relatives (72 mol/l) (n = 8 subjects, p = 0.06). The Arg409Gln p85 alpha subunit was associated with lower insulin-stimulated phosphoinositide 3-kinase activity compared with wild-type (mean reduction 15%, p < 0.05, n = 5). The recruitment of Arg409Gln p85 alpha into phosphotyrosine complexes was not significantly impaired. GST fusion proteins of wild-type and mutant p85 alpha showed identical binding to phosphopeptides in surface plasmon resonance studies. CONCLUSION/INTERPRETATION: Mutations in p85 alpha are uncommon in subjects with syndromes of severe insulin resistance. The Met326Ile p85 alpha variant appears to have no functional effect on the insulin-stimulated phosphoinositide 3-kinase activity. The impaired phosphoinositide 3-kinase activity of the Arg409Gln mutant suggests that it could contribute to the insulin resistance seen in this family.

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.

A read-ahead function in archaeal DNA polymerases detects promutagenic template-strand uracil.

Deamination of cytosine to uracil is the most common promutagenic change in DNA, and it is greatly increased at the elevated growth temperatures of hyperthermophilic archaea. If not repaired to cytosine prior to replication, uracil in a template strand directs incorporation of adenine, generating a G.C --> A.U transition mutation in half the progeny. Surprisingly, genomic analysis of archaea has so far failed to reveal any homologues of either of the known families of uracil-DNA glycosylases responsible for initiating the base-excision repair of uracil in DNA, which is otherwise universal. Here we show that DNA polymerases from several hyperthermophilic archaea (including Vent and Pfu) specifically recognize the presence of uracil in a template strand and stall DNA synthesis before mutagenic misincorporation of adenine. A specific template-checking function in a DNA polymerase has not been observed previously, and it may represent the first step in a pathway for the repair of cytosine deamination in archaea.

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.

Structural and biochemical evaluation of the interaction of the phosphatidylinositol 3-kinase p85alpha Src homology 2 domains with phosphoinositides and inositol polyphosphates.

Src homology 2 (SH2) domains exist in many intracellular proteins and have well characterized roles in signal transduction. SH2 domains bind to phosphotyrosine (Tyr(P))-containing proteins. Although tyrosine phosphorylation is essential for protein-SH2 domain interactions, the binding specificity also derives from sequences C-terminal to the Tyr(P) residue. The high affinity and specificity of this interaction is critical for precluding aberrant cross-talk between signaling pathways. The p85alpha subunit of phosphoinositide 3-kinase (PI 3-kinase) contains two SH2 domains, and it has been proposed that in competition with Tyr(P) binding they may also mediate membrane attachment via interactions with phosphoinositide products of PI 3-kinase. We used nuclear magnetic resonance spectroscopy and biosensor experiments to investigate interactions between the p85alpha SH2 domains and phosphoinositides or inositol polyphosphates. We reported previously a similar approach when demonstrating that some pleckstrin homology domains show binding specificity for distinct phosphoinositides (Salim, K., Bottomley, M. J., Querfurth, E., Zvelebil, M. J., Gout, I., Scaife, R., Margolis, R. L., Gigg, R., Smith, C. I., Driscoll, P. C., Waterfield, M. D., and Panayotou, G. (1996) EMBO J. 15, 6241-6250). However, neither SH2 domain exhibited binding specificity for phosphoinositides in phospholipid bilayers. We show that the p85alpha SH2 domain Tyr(P) binding pockets indiscriminately accommodate phosphoinositides and inositol polyphosphates. Binding of the SH2 domains to Tyr(P) peptides was only poorly competed for by phosphoinositides or inositol polyphosphates. We conclude that these ligands do not bind p85alpha SH2 domains with high affinity or specificity. Moreover, we observed that although wortmannin blocks PI 3-kinase activity in vivo, it does not affect the ability of tyrosine-phosphorylated proteins to bind to p85alpha. Consequently phosphoinositide products of PI 3-kinase are unlikely to regulate signaling through p85alpha SH2 domains.

Intermolecular interactions of the p85alpha regulatory subunit of phosphatidylinositol 3-kinase.

The regulatory subunit of phosphatidylinositol 3-kinase, p85, contains a number of well defined domains involved in protein-protein interactions, including an SH3 domain and two SH2 domains. In order to investigate in detail the nature of the interactions of these domains with each other and with other binding partners, a series of deletion and point mutants was constructed, and their binding characteristics and apparent molecular masses under native conditions were analyzed. The SH3 domain and the first proline-rich motif bound each other, and variants of p85 containing the SH3 and BH domains and the first proline-rich motif were dimeric. Analysis of the apparent molecular mass of the deletion mutants indicated that each of these domains contributed residues to the dimerization interface, and competition experiments revealed that there were intermolecular SH3 domain-proline-rich motif interactions and BH-BH domain interactions mediating dimerization of p85alpha both in vitro and in vivo. Binding of SH2 domain ligands did not affect the dimeric state of p85alpha. Recently, roles for the p85 subunit have been postulated that do not involve the catalytic subunit, and if p85 exists on its own we propose that it would be dimeric.

Binding of a diphosphotyrosine-containing peptide that mimics activated platelet-derived growth factor receptor beta induces oligomerization of phosphatidylinositol 3-kinase.

Phosphatidylinositol 3-kinase (PI3K) is a heterodimeric enzyme comprising a p110 catalytic subunit and a p85 regulatory subunit. We have recently shown that the isolated p85 subunit exists as a dimer; therefore, we examined whether the heterodimeric enzyme was capable of further self-association. Size-exclusion chromatography demonstrated that PI3K was a 1:1 complex of p85 and p110 under native conditions. However, binding of a diphosphotyrosine-containing peptide that mimics an activated platelet-derived growth factor receptor beta induced an increase in the apparent molecular mass of PI3K. This increase was due to dimerization of PI3K and was dependent on PI3K concentration but not diphosphopeptide concentration. Dimer formation was also observed directly using fluorescence resonance energy transfer. Diphosphopeptide-induced activation of PI3K (Carpenter, C. L., Auger, K. R., Chanudhuri, M., Yoakim, M., Schaffhausen, B., Shoelson, S., and Cantley, L. C. (1993) J. Biol. Chem. 268, 9478-9483; Rordorf-Nikolic, T., Van Horn, D. J., Chen, D., White, M. F., and Backer, J. M. (1995) J. Biol. Chem. 270, 3662-3666) was not a direct result of dimerization and occurred only when phosphatidylinositol, and not phosphatidylinositol-4,5-diphosphate, was the phosphorylation substrate. Binding of the tandem SH2 domains of the p85 regulatory subunit to activated receptor tyrosine kinases therefore induces dimerization of PI3K, which may be an early step in inositol lipid-mediated signal transduction.

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.

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.

Crystal structure of a G:T/U mismatch-specific DNA glycosylase: mismatch recognition by complementary-strand interactions.

G:U mismatches resulting from deamination of cytosine are the most common promutagenic lesions occurring in DNA. Uracil is removed in a base-excision repair pathway by uracil DNA-glycosylase (UDG), which excises uracil from both single- and double-stranded DNA. Recently, a biochemically distinct family of DNA repair enzymes has been identified, which excises both uracil and thymine, but only from mispairs with guanine. Crystal structures of the mismatch-specific uracil DNA-glycosylase (MUG) from E. coli, and of a DNA complex, reveal a remarkable structural and functional homology to UDGs despite low sequence identity. Details of the MUG structure explain its thymine DNA-glycosylase activity and the specificity for G:U/T mispairs, which derives from direct recognition of guanine on the complementary strand.