Análise peptidômica dos venenos de vespas sociais neotropicais

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Stereoselective synthesis and structure determination of a bicyclo[3.3.2]decapeptide

By analogy to the structural diversity of covalent bond networks between atoms within organic molecules, one can design topologically diverse peptides from mathematical graphs by assigning amino acids to graph nodes and peptide bonds to graph edges. The key is to use diamino acids or amino diacids as equivalents of trivalent graph nodes, which enables a variety of graph topologies beyond the standard linear and monocyclic graphs in natural peptides. Here the bicyclic decapeptide A1FGk2VFPE1AG2 (1b) was prepared and crystallized to assign its bridge stereochemistry. The bridge configuration appears as planned by the chirality of the branching amino acids. Bicyclization furthermore depends on the presence of matched chiralities in the branching amino acids. The stereoselective formation of the second bridge opens the way for the synthesis of a large family of bicyclic peptides as promising new scaffolds for drug design.

Synthesis of unusual alpha-amino acids and study of the effect of their incorporation into antimicrobial peptides. Total synthesis of biactive marine natural products and analogues thereof

The principle theme of this thesis was the synthesis of bioactive compounds. To this end, this work was focus on two main projects. The first one, which was carried out in the Department of Chemistry of the University of Girona under the supervision of Dr Montserrat Heras, concerned the synthesis of new unnatural amino acids bearing a pyrimidine ring within their side chain for incorporation into the antimicrobial peptide BP100 following a rational design in order to improve its biological profile. On the other hand, the second chapter of this thesis was developed in collaboration with the Laboratoire de Chimie Organique (ESPCI-ParisTech, Paris, France) under the guidance of Pr Janine Cossy and Dr Arseniyadis. This chapter was centered on the total synthesis of three marine natural products with complex structures and interesting biological activities: acremolide B, (–) bitungolide F and lyngbouilloside.

Intracellular peptides as natural regulators of cell signaling

Protein degradation by the ubiquitin proteasome system releases large amounts of oligopeptides within cells. To investigate possible functions for these intracellularly generated oligopeptides, we fused them to a cationic transactivator peptide sequence using reversible disulfide bonds, introduced them into cells, and analyzed their effect on G protein-coupled receptor (GPCR) signal transduction. A mixture containing four of these peptides (20-80 mu M) significantly inhibited the increase in the extracellular acidification response triggered by angiotensin II (ang II) in CHO-S cells transfected with the ang II type 1 receptor (AT1R-CHO-S). Subsequently, either alone or in a mixture, these peptides increased luciferase gene transcription in AT1R-CHO-S cells stimulated with ang II and in HEK293 cells treated with isoproterenol. These peptides without transactivator failed to affect GPCR cellular responses. All four functional peptides were shown in vitro to competitively inhibit the degradation of a synthetic substrate by thimet oligopeptidase. Overexpression of thimet oligopeptidase in both CHO-S and HEK293 cells was sufficient to reduce luciferase activation triggered by a specific GPCR agonist. Moreover, using individual peptides as baits in affinity columns, several proteins involved in GPCR signaling were identified, including alpha-adaptin A and dynamin 1. These results suggest that before their complete degradation, intracellular peptides similar to those generated by proteasomes can actively affect cell signaling, probably representing additional bioactive molecules within cells.

Natural intracellular peptides can modulate the interactions of mouse brain proteins and thimet oligopeptidase with 14-3-3e and calmodulin

Protein interactions are crucial for most cellular process. Thus, rationally designed peptides that act as competitive assembly inhibitors of protein interactions by mimicking specific, determined structural elements have been extensively used in clinical and basic research. Recently, mammalian cells have been shown to contain a large number of intracellular peptides of unknown function. Here, we investigate the role of several of these natural intracellular peptides as putative modulators of protein interactions that are related to Ca2+-calmodulin (CaM) and 14-3-3 epsilon, which are proteins that are related to the spatial organization of signal transduction within cells. At concentrations of 1-50 mu M, most of the peptides that are investigated in this study modulate the interactions of CaM and 14-3-3 epsilon with proteins from the mouse brain cytoplasm or recombinant thimet oligopeptidase (EP24.15) in vitro, as measured by surface plasmon resonance. One of these peptides (VFDVELL; VFD-7) increases the cytosolic Ca2+ concentration in a dose-dependent manner but only if introduced into HEK293 cells, which suggests a wide biological function of this peptide. Therefore, it is exciting to suggest that natural intracellular peptides are novel modulators of protein interactions and have biological functions within cells.

Four p53 DNA-binding domain peptides bind natural p53-response elements and bend the DNA.

Recent structural studies of the minimal core DNA-binding domain of p53 (p53DBD) complexed to a single consensus pentamer sequence and of the isolated p53 tetramerization domain have provided valuable insights into their functions, but many questions about their interacting roles and synergism remain unanswered. To better understand these relationships, we have examined the binding of the p53DBD to two biologically important full-response elements (the WAF1 and ribosomal gene cluster sites) by using DNA circularization and analytical ultracentrifugation. We show that the p53DBD binds DNA strongly and cooperatively with p53DBD to DNA binding stoichiometries of 4:1. For the WAF1 element, the mean apparent Kd is (8.3 +/- 1.4) x 10(-8) M, and no intermediate species of lower stoichiometries can be detected. We show further that complex formation induces an axial bend of at least 60 degrees in both response elements. These results, taken collectively, demonstrate that p53DBD possesses the ability to direct the formation of a tight nucleoprotein complex having the same 4:1 DNA-binding stoichiometry as wild-type p53 which is accompanied by a substantial conformational change in the response-element DNA. This suggests that the p53DBD may play a role in the tetramerization function of p53. A possible role in this regard is proposed.

Cytomegalovirus evasion of natural killer cell responses

Natural killer (NK) cells are an important component of the innate cellular immune system. They are particularly important during the early immune responses following virus infection, prior to the induction of cytotoxic T cells (CTL). Unlike CTL, which recognize specific peptides displayed on the surface of cells by class I MHC, NK cells respond to aberrant expression of cell surface molecules, in particular class I MHC, in a non-specific manner. Thus, cells expressing low levels of surface class I MHC are susceptible to recognition by NK cells, with concomitant triggering of cytolytic and cytokine-mediated responses. Many viruses, including the cytomegaloviruses, downregulate cell surface MHC class I: this is likely to provide protection against CTL-mediated clearance of infected cells, but may also render infected cells sensitive to NK-cell attack. This review focuses upon cytomegalovirus-encoded proteins that are believed to promote evasion of NK-cell-mediated immunity. The class I MHC homologues, encoded by all cytomegaloviruses characterised to date, have been implicated as molecular 'decoys', which may mimic the ability of cellular MHC class I to inhibit NK-cell functions. Results from studies in vitro are not uniform, but in general they support the proposal that the class I homologues engage inhibitory receptors from NK cells and other cell types that normally interact with cellular class I. Consistent with this, in vivo studies of murine cytomegalovirus indicate that the class I homologue is required for efficient evasion of NK-cell-mediated clearance. Recently a second murine cytomegalovirus protein, a C-C chemokine homologue, has been implicated as promoting evasion of NK and T-cell-mediated clearance in vivo.

m144, a murine cytomegalovirus (MCMV)-encoded major histocompatibility complex class I homologue, confers tumor resistance to natural killer cell-mediated rejection

Until now, it has been unclear whether murine cytomegalovirus (MCMV)-encoded protein m144 directly regulates natural killer (NK) cell effector function and whether the effects of m144 are only strictly evident in the context of MCMV infection. We have generated clones of the transporter associated with antigen processing (TAP)-2-deficient RMA-S T lymphoma cell line and its parent cell line, RMA, that stably express significant and equivalent levels of m144. In vivo NK cell-mediated rejection of RMA-S-m144 lymphomas was reduced compared with rejection of parental or mock-transfected RMA-S clones, indicating the ability of m144 to regulate NK cell-mediated responses in vivo. Significantly, the accumulation of NK cells in the peritoneum was reduced in mice challenged with RMA-S-m144, as was the lytic activity of NK cells recovered from the peritoneum. Expression of m144 on RMA-S cells also conferred resistance to cytotoxicity mediated in vitro by interleukin 2-activated adherent spleen NK cells. In summary, the data demonstrate that m144 confers some protection from NK cell effector function mediated in the absence of target cell class I expression, but that in vivo the major effect of m144 is to regulate NK cell accumulation and activation at the site of immune challenge.

Computational binding assays of antigenic peptides

Computer models can be combined with laboratory experiments for the efficient determination of (i) peptides that bind MHC molecules and (ii) T-cell epitopes. For maximum benefit, the use of computer models must be treated as experiments analogous to standard laboratory procedures. This requires the definition of standards and experimental protocols for model application. We describe the requirements for validation and assessment of computer models. The utility of combining accurate predictions with a limited number of laboratory experiments is illustrated by practical examples. These include the identification of T-cell epitopes from IDDM-, melanoma- and malaria-related antigens by combining computational and conventional laboratory assays. The success rate in determining antigenic peptides, each in the context of a specific HLA molecule, ranged from 27 to 71%, while the natural prevalence of MHC-binding peptides is 0.1-5%.

Multiple conformational epitopes are recognized by natural and induced immunity to the E7 protein of human papilloma virus type 16 in man

The reactivity of sera from patients with cervical cancer with the E7 protein of human papilloma virus type 16 (HPV16) was estimated using a novel non-radioactive immunoprecipitation assay and four established protein-and peptide-based immunoassays. Six of 14 sera from patients with cervical cancer and 1 of 10 sera from healthy laboratory staff showed repeated reactivity with E7 in at least one assay. Four of the 7 reactive sera were consistently reactive in more than one assay, but only one was reactive in all four assays. Following immunization with E7, 2 of 5 patients with cervical cancer had increased E7-specific reactivity, measurable in one or more assays. No single assay was particularly sensitive for E7 reactivity, or predictive of cervical cancer. Mapping of E7 reactivity to specific E7 peptides was unsuccessful, suggesting that natural or induced E7 reactivity in human serum is commonly directed to conformational epitopes of E7, These results suggest that each assay employed with is study measures a different aspect of E7 reactivity, and that various reactivities to E7 may manifest following HPV infection or immunization. This finding is of significance for monitoring of E7 immunotherapy and for serological screening for cervical cancer. Copyright (C) 2000 S.Karger, AG. Basel.

Non-ribosomal peptides produced by Brazilian cyanobacterial isolates with antimicrobial activity

Cyanobacterial strains isolated from terrestrial and freshwater habitats in Brazil were evaluated for their antimicrobial and siderophore activities. Metabolites of fifty isolates were extracted from the supernatant culture media and cells using ethyl acetate and methanol, respectively. The extracts of 24 isolates showed antimicrobial activity against several pathogenic bacteria and one yeast. These active extracts were characterized by Q-TOF/MS. The cyanobacterial strains Cylindrospermopsis raciborskii 339-T3, Synechococcus elongatus PCC7942, Microcystis aeruginosa NPCD-1, M. panniformis SCP702 and Fischerella sp. CENA19 provided the most active extracts. The 50 cyanobacterial strains were also screened for the presence of non-ribosomal peptide synthetase (NRPS) and polyketide synthase (PKS) genes and microcystin production. Putative fragment genes coding for NRPS adenylation domains and PKS keto-synthase domains were successfully PCR amplified from 92% and 80% of cyanobacterial strains, respectively. The potential therapeutical compounds siderophores were detected in five cyanobacterial isolates. Microcystin production was detected by ELISA test in 26% of the isolates. Further a protease inhibitor substance was detected by LC-MS/MS in the M. aeruginosa NPLJ-4 extract and the presence of aeruginosin and cyanopeptolin was confirmed by PCR amplification using specific primers, and sequenced. This screening study showed that Brazilian cyanobacterial isolates are a rich source of natural products with potential for pharmacological and biotechnological applications. (C) 2010 Elsevier GmbH. All rights reserved.

Inhibition of natural killer cells by a cytomegalovirus MHC class I homologue in vivo

Herpesviruses, such as murine and human cytomegalovirus (MCMV and HCMV), can establish a persistent infection within the host and have diverse mechanisms as protection from host immune defences'. Several herpesvirus genes that are homologous to host immune modulators have been identified, and are implicated in viral evasion of the host immune response(2,3). The discovery of a viral major histocompatibility complex (MHC) class I homologue, encoded by HCMV(4), led to speculation that it might function as an immune modulator and disrupt presentation of peptides by MHC class I to cytotoxic T cells(5). However, there is no evidence concerning the biological significance of this gene during viral infection. Recent analysis of the MCMV genome has also demonstrated the presence of a MHC class I homologue(6). Here we show that a recombinant MCMV,in which. the gene encoding the class I homologue has been disrupted, has severely restricted replication during the acute stage of infection compared with wild-type MCMV, We demonstrate by in vivo depletion studies that natural killer (NK) cells are responsible for the attenuated phenotype of the mutant. Thus the viral MHC dass I homologue contributes to immune evasion through interference with NK cell-mediated clearance.

Congenital lactic acidosis: Evaluation of the properties of the A199T natural variant of human pyruvate dehydrogenase E1 alpha by in vitro mutation

One cause of congenital lactic acidosis is a mutation in the E1 alpha -subunit of the pyruvate dehydrogenase multienzyme complex. Little is known about the consequences of these mutations at the enzymatic level. Here we study the A199T mutation by expressing the protein in Escherichia coil. The specific activity is 25% of normal and the K-m for pyruvate is elevated by 10-fold. Inhibitors of lactate dehydrogenase might be a useful therapy for patients with such mutations. (C) 2001 Academic Press.

Geographic variation of natural products of tropical nudibranch Asteronotus cespitosus

Extracts of the dorid nudibranch Asteronotus cespitosus from two geographically separate regions of Australia and from the Philippines were compared using thin-layer, high-performance liquid and gas chromatography and H-1 NMR analysis. Halogenated metabolites were detected in all mollusk specimens. The major component detected in digestive tissue of specimens from the Great Barrier Reef in northeastern Australia was 4,6-dibromo2-(2',4'-dibromophenoxy)phenol (1), with minor amounts of 3,5-dibromo-2(3',5'-dibromo-20-methoxyphenoxy)phenol (2). In a specimen collected from northwestern Australia, only 3,5-dibromo-2-(3',5'-dibromo-2'-methoxyphenoxy)phenol was found. The specimen from the Philippines contained 2,3,4,5-tetrabromo-6-(2'-bromophenoxy) phenol (3) together with a novel chlorinated pyrrolidone (4). In addition, the sesquiterpenes dehydroherbadysidolide (5) and spirodysin (6) were detected in the digestive organs and mantle tissue of the nudibranchs from the Great Barrier Reef and from the Philippines, whereas these chemicals were not found in the specimen from northwestern Australia. All of the chemicals (1-3,5, and 6) have previously been isolated from the sponge Dysidea herbacea, as have chlorinated metabolites related to 4. This is the first time the characteristic halogenated metabolites that typify Dysidea herbacea have been reported from a carnivorous mollusk, which implies a dietary origin as opposed to de novo synthesis.

The cyclotides: Novel macrocyclic peptides as scaffolds in drug design

Cyclotides are a novel class of circular, disulfide-rich peptides (similar to 30 amino acids) that display a broad range of bioactivities and have exceptionally high stability. Their physical properties, which include resistance to thermal and enzymatic degradation, can be attributed to their unique cyclic backbone and knotted arrangement of disulfide bonds. The applicability of linear peptides as drugs is potentially limited by their susceptibility to proteolytic cleavage and poor bioavailability. Such limitations may be overcome by using the cyclotide framework as a scaffold onto which new activities may be engineered. The potential use of cyclotides for drug design is evaluated here, with reference to rapidly increasing knowledge of natural cyclotides and the emergence of new techniques in peptide engineering.