RESUMO
Hydrogels from self-assembling ionic complementary peptides have been receiving much interest from the scientific community as mimetics of the extracellular matrix that can offer three-dimensional support for cell growth or become vehicles for the delivery of stem cells or drugs. These scaffolds have also been proposed as bone substitutes for small defects as they promote beneficial effects on human osteoblasts. In order to develop a novel bioactive titanium implant, we propose the introduction of a layer of ionic-complementary self-assembling peptides (EAbuK) on Ti whose surface has been previously sandblasted and acid etched. The peptide layer is anchored to the metal by covalent functionalization of titania with self-assembling sequences. The peptide layer has also been enriched by the insulin-like growth factor-1 incorporated to the layer and/or a conjugate obtained by chemoselective ligation between EAbuK and a sequence of 25 residues containing four GRGDSP motifs per chain. X-ray photoelectron spectroscopy studies confirmed a change in the surface composition in agreement with the proposed decorations. An evaluation of the contact angle showed a substantial change in wettability induced by the peptide layer. The human osteoblast adhesion and proliferation assays showed an increase in adhesion for the surfaces enriched with conjugate at a concentration of 3.8 × 10(-7)m and an enhanced proliferation for samples enriched with insulin-like growth factor-1 at the highest concentration tested (2.1 × 10(-5)m).
Assuntos
Hidrogéis/química , Fator de Crescimento Insulin-Like I/fisiologia , Oligopeptídeos/química , Osteoblastos/fisiologia , Titânio/química , Sequência de Aminoácidos , Materiais Biocompatíveis/química , Adesão Celular , Técnicas de Cultura de Células , Proliferação de Células , Células Cultivadas , Meios de Cultura , Humanos , Masculino , Pessoa de Meia-Idade , Propriedades de Superfície , MolhabilidadeRESUMO
The most characterized stromal cell-derived factor-1 (SDF-1) variants are the isoform α, which is the predominant one but undergoes rapid proteolysis, and the ß isoform, which is more resistant. Through the interaction with a specific chemokine receptor called CXCR4, SDF-1 is able to regulate different physiological processes. The aim of this study was to verify the expression and potential functional role of SDF-1 and CXCR4 in the porcine ovary. Firstly, the expression of SDF-1 and its receptor in different ovarian districts was verified for the first time. Thereafter, the effect of SDF-1 ß isoform (51-72) fragment on functional parameters, such as proliferation, metabolic activity, redox status, nitric oxide production, and steroidogenic activity, was assessed on granulosa cells collected from follicles. In addition, the potential effect of this protein in vascular events was verified through investigations on porcine aortic (AOC) endothelial cells, such as the production of nitric oxide and viability tests. The proliferation and metabolic activity were not affected by treatment with the cytokine. As regard to steroidogenesis, the peptide stimulated both estrogen (P = 0.049) and progesterone production (P = 0.039). Redox status was affected by the examined substance since superoxide anion was inhibited (P = 0.001), while antioxidant power (P = 0.034), as well as nitric oxide generation, were stimulated (P = 0.034). Tests performed on AOCs showed significant stimulation of nitric oxide production (P = 0.004) by the examined peptide, while cell viability was unaffected. Therefore, the potential role of cytokine in the mechanisms involved in the regulation of follicular function can be hypothesized.
Assuntos
Quimiocina CXCL12/metabolismo , Folículo Ovariano/metabolismo , Receptores CXCR4/metabolismo , Células Estromais/metabolismo , Suínos , Animais , Quimiocina CXCL12/genética , Células Endoteliais/metabolismo , Feminino , Regulação da Expressão Gênica/fisiologia , Óxido Nítrico/metabolismo , Receptores CXCR4/genéticaRESUMO
Osteoblast cell adhesion to the extracellular matrix is established through two main pathways: one is mediated by the binding between integrin and a minimal adhesion sequence (RGD) on the extracellular protein, the other is based on the interactions between transmembrane proteoglycans and heparin-binding sequences found in many matrix proteins. The aim of this study is the evaluation in an in vivo endosseous implant model of the early osteogenic response of the peri-implant bone to a biomimetic titanium surface functionalized with the retro-inverso 2DHVP peptide, an analogue of Vitronectin heparin binding site. The experimental plan is based on a bilateral study design of Control and 2DHVP implants inserted respectively in the right and left femur distal metaphysis of adult male Wistar rats (n=16) weighing about 300grams and evaluated after 15days. Fluorochromic bone vital markers were given in a specific time frame, in order to monitor the dynamic of new bone deposition. The effect inducted by the peptidomimetic coating on the surrounding bone were qualitatively and quantitatively evaluated by means of static and dynamic histomorphometric analyses performed within three concentric and subsequent circular Regions of Interest (ROI) of equivalent thickness (220µm), ROI1 adjacent to the interface, ROI2, the middle, and ROI3 the farthest. The data indicated that these functionalized implants stimulated a higher bone apposition rate (p<0,01) and larger and rapid osteoblast activation in terms of mineralizing surface within ROI1 compared to the control (p<0,01). These higher osteoblast recruitment and activation leads to a greater bone-to-implant contact reached for DHVP samples (p<0,5). This represents an initial stimulus of the osteogenic activity that might results in a faster and better osteointegration process.
Assuntos
Osteogênese/efeitos dos fármacos , Peptidomiméticos , Próteses e Implantes , Titânio/química , Titânio/farmacologia , Sequência de Aminoácidos , Animais , Biomimética , Fêmur/anatomia & histologia , Fêmur/crescimento & desenvolvimento , Masculino , Osseointegração , Peptídeos/química , Ratos , Ratos Wistar , Receptores de Vitronectina/efeitos dos fármacos , Propriedades de SuperfícieRESUMO
Self-assembling peptides (SAPs) were investigated by means of XPS and Angular Dependent NEXAFS spectroscopies, with the aim to probe the influence of pH and Ionic Strength conditions on the chemical structure and molecular organization of SAPs anchored on titania surfaces. XPS at the C1s, N1s, O1s core levels allowed to study surfaces and biomolecule/substrate interfaces. NEXAFS data allowed ascertaining that SAPs molecular structure is preserved upon grafting to the titania surface. Angular Dependent NEXAFS was used to investigate the influence of environmental conditions on the molecular organization behaviour. The objective of our study was to establish a set of methodologies for obtaining arrangements of well-organized biomolecules on scaffolds surfaces as a basic technology to develop and optimize cells adhesion and proliferation for tissue engineering applications.
Assuntos
Materiais Biocompatíveis/química , Oligopeptídeos/química , Titânio/química , Concentração de Íons de Hidrogênio , Concentração Osmolar , Engenharia TecidualRESUMO
New experimental results are reported on the self-assembling behavior of EAK16-II, the first discovered ionic self-complementary peptide, incubated at ultralow concentration (10-6â¯M) at neutral pH onto differently charged surfaces. It is found that strongly negatively charged surfaces promote the self-assembly of flat, micrometer-long mono-molecular fibers of side-on assembled sequences, lying onto a continuous monolayer of flat-on EAK16-II molecules. These results suggest that the monomolecular EAK16-II self-assembly is driven by the peculiar matching condition between peptide and surface electrostatic properties. Molecular Mechanics simulations of the basic bimolecular interactions confirmed the experimental inferences, showing that the flat-on state is the most stable arrangement for two interacting EAK16-II sequences onto strongly negatively charged surfaces, where indeed EAK16-II ß-sheet conformation is stabilized, while the weak electrostatic interactions with mildly charged substrates promote an "entangled" EAK16-II geometry. Molecular Dynamics simulations further showed that the mobility and diffusional freedom of the peptides from the surfaces are ruled by the relative strength of peptide-surface electrostatic interactions, so that desorption probability for the peptide sequences is negligible from strongly-charged surfaces and high from mildly-charged surfaces. Furthermore, it has been found that an oligopeptide sequence lying onto two flat-on EAK16-II molecules, gains a remarkable lateral mobility, while remaining weakly bound to the surface, thus allowing the further molecular self-alignment responsible for the micrometer-long fiber formation. The reported results pave the way to the understanding and control of the subtle peptide-surface structural motifs matching enabling the formation of micrometer-long, but nanometer-wide monomolecular fibers.
Assuntos
Simulação de Dinâmica Molecular , Nanoestruturas/química , Oligopeptídeos/química , Estrutura Secundária de Proteína , Algoritmos , Sequência de Aminoácidos , Concentração de Íons de Hidrogênio , Microscopia de Força Atômica , Modelos Teóricos , Eletricidade Estática , Propriedades de SuperfícieRESUMO
Infectious HIV-1 requires gp160 cleavage by furin at the REKR511 downward arrow motif (site1) into the gp120/gp41 complex, whereas the KAKR503 (site2) sequence remains uncleaved. We synthesized 41mer and 51mer peptides, comprising site1 and site2, to study their conformation and in vitro furin processing. We found that, while the previously reported 19mer and 13mer analogues represent excellent in vitro furin substrates, the present extended sequences require heparin for optimal processing. Our data support the hypothesis of a direct binding of heparin with site1 and site2, allowing selective exposure/accessibility of the REKR sequence, which is only then optimally cleaved by furin.
Assuntos
Furina/metabolismo , Proteína gp160 do Envelope de HIV/metabolismo , Heparina/farmacologia , Peptídeos/metabolismo , Sequência de Aminoácidos , Cromatografia Líquida de Alta Pressão , Dicroísmo Circular , Proteína gp160 do Envelope de HIV/química , Humanos , Dados de Sequência Molecular , Peso Molecular , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Peptídeos/química , Especificidade por Substrato/efeitos dos fármacosRESUMO
HLA class II molecules present antigenic peptides to the T cell receptor of CD4+ T lymphocytes and interact with CD4 during the antigen recognition process. A major CD4 binding site encompassing amino acids (aa) 134-148 in the beta 2 domain of HLA-DR has been previously identified and residues located within the alpha 2 subunit of murine MHC class II I-Ad molecules have been shown to contribute to CD4-class II interaction. To characterize the alpha 2 region of HLA-DR molecules involved in the binding of CD4, we have synthesized overlapping linear and cyclic peptides derived from a region encompassing aa 121-143. We demonstrate that two linear peptides (aa 124-138 and 130-143) and a cyclic one (aa 121-138) specifically bind to CD4-sepharose affinity columns. Although cyclic analogues exhibit more ordered populations as detected by circular dichroism measurements, cyclization did not improve the activity of some peptides. Peptide sequence positioning in HLA-DR1 dimer model indicates that alpha 2 residues 124 to 136 form a solvent-exposed loop which faces the beta 2 loop delimited by residues 134-148. These data suggest that one CD4 molecule contacts both alpha 2 and beta 2 loops of the HLA-DR homodimer.
Assuntos
Antígenos CD4/metabolismo , Antígenos HLA-DR/química , Antígenos HLA-DR/metabolismo , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Sequência de Aminoácidos , Cromatografia de Afinidade , Dicroísmo Circular , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Fragmentos de Peptídeos/síntese química , Ligação Proteica/imunologia , SefaroseRESUMO
Several members of the ribonuclease superfamily possess a variety of interesting biological properties, including ribonucleolytic, angiogenic, antiproliferative, cytotoxic, embryotoxic, aspermatogenic and antitumoral activity. In this study, we report the purification from bovine milk of a protein with structural and enzymatic properties very similar to those of ribonuclease-4 (RNase-4), which is normally present in the liver and lungs, and examined its functional properties, biological activity and cytotoxic effects. RNase-4, at physiological concentrations, had a positive effect on the vitality and proliferation of human umbilical vein endothelial cells. Moreover, it induced an increase in cellular migration and the formation of in vitro capillary-like structures. We also evaluated the effect of RNase-4 in vitro on human breast, colorectal and cervical carcinoma cell lines. The protein was revealed to have a cytotoxic effect similar to that of RNase-A. We suggest that the positive effects of RNase-4 on normal cells were due to its particularly close interaction with RNase inhibitor, while good conformational stability and resistance to proteolytic degradation potentially favour ribonuclease cytotoxicity.
RESUMO
This paper describes a new method for the evaluation of conductimetric data collected during the in-line monitoring of the coupling step in solid-phase peptide synthesis. The control scheme relies on a feed-forward artificial neural network algorithm which can predict the final yield of the reaction within its initial 5 min by analyzing the conductivity signal profile. The yield values predicted by the artificial neural network algorithm result in good accordance with the data obtained by the commonly used ninhydrin test.
Assuntos
Algoritmos , Peptídeos/síntese química , Condutividade Elétrica , Redes Neurais de Computação , Ninidrina , Peptídeos/análise , Fotometria , Processamento de Sinais Assistido por ComputadorRESUMO
In previous studies we demonstrated that a synthetic peptide corresponding to the sequence in the (307-330) region of the gp120 principal neutralizing domain of the HIV-1 MN strain is able to bind sCD4 in an affinity chromatography assay and to enhance CD4 expression, CD4 affinity for gp120, and HIV-1 infection. This paper describes a photo affinity labeling experiment, designed to confirm the gp120 peptide-CD4 interaction and to locate the binding site of the synthetic peptide on the CD4 molecule. To this end two specifically marked analogues of the peptide patterned on the (307-330) region of HIV-MN-gp120, in which the TyrI residue is replaced with Phe(p-N3) or Phe(p-NO2), have been synthesized. Irradiation of CD4 solutions in the presence of both analogues produced a new component, the mass value of which confirms the formation of a covalent bond between the peptide and the protein.
Assuntos
Antígenos CD4/metabolismo , Proteína gp120 do Envelope de HIV/metabolismo , HIV-1/metabolismo , Fragmentos de Peptídeos/metabolismo , Sítios de Ligação , HIV-1/classificação , Marcadores de Fotoafinidade , Ligação Proteica , SolubilidadeRESUMO
The interaction between the viral envelope protein gp120 and the cellular surface antigen CD4 is a key event in HIV-1 infection. Reciprocal high affinity binding sites have been located in the first domain of CD4 and in the carboxy-terminal region of gp120, respectively. Upon infection, the membranes of the target cells fuse; sites of CD4 and gp120, distinct from their high affinity binding sites, play a role in the post-binding events leading to syncytia formation. We have studied the interactions of CD4 with gp120 and gp120-derived peptides using an in vitro assay based on immobilized recombinant soluble CD4 (sCD4). In this system CD4 binds to recombinant soluble gp120 and to anti-receptor peptides derived from the high affinity CD4-binding site of gp120, as well as to peptides corresponding to the principal neutralizing domain (PND) of the envelope protein, i.e., to the domain required for HIV-1-mediated syncytium formation. Competition experiments performed using epitope-specific mAbs and a variety of peptides indicated that PND-derived peptides are specifically recognized by a CD4 site adjacent to, but distinct from, the high affinity gp120-binding site of CD4. Synthetic peptides patterned on the PND of different viral isolates were retained onto sCD4-based affinity columns at different extent; some of the structural requirements for binding were analyzed. Studies performed on CD4+ T-cells showed that PND-derived peptides also interact with CD4 in its native membrane-bound conformation. These results indicate that a direct contact takes place between CD4 and the gp120 domain participating in HIV-induced syncytia formation.
Assuntos
Antígenos CD4/metabolismo , Proteína gp120 do Envelope de HIV/metabolismo , HIV-1/metabolismo , Fragmentos de Peptídeos/metabolismo , Sequência de Aminoácidos , Sítios de Ligação/fisiologia , Fusão Celular/fisiologia , Linhagem Celular , Cromatografia de Afinidade , Epitopos/metabolismo , Humanos , Dados de Sequência Molecular , Ensaio RadioliganteRESUMO
The H-Ala-Arg-(Ala)6-Lys-OH sequence is a biologically interesting 'difficult sequence' presenting N alpha-Fmoc deprotection and coupling problems. Different chemical conditions and synthetic strategies have been tested in order to overcome the problems due to sequence-dependent interactions. In particular, it was confirmed that different solvents in the deprotection step did not provide any significant improvement, but the use of a more efficient base in the deprotection mixture avoided insufficient unblocking of N alpha-protecting group; problems due to partial coupling in the last steps of the synthesis were solved by double coupling techniques. Moreover, the synthesis of the model peptide was carried out using both "continuous flow' and "batch' techniques. The present results demonstrate that on-line monitoring of the deprotection step by absorbance measurements represents a very effective tool to detect the onset of internal aggregations during the synthesis.
Assuntos
Oligopeptídeos/síntese química , Cromatografia Líquida de Alta Pressão , Métodos , Oligopeptídeos/químicaRESUMO
We previously demonstrated that a 23-amino-acid peptide derived from the V3 loop of the surface glycoprotein of human immunodeficiency virus (HIV-1) strain MN was able to bind soluble CD4 and to enhance HIV-1 infection. Further studies suggested that the peptide/CD4 interaction induces an increase in both CD4 expression and CD4/gp120 binding affinity. To facilitate identification of the complementary binding site for the peptide on cellular CD4, we designed an analogue carrying a single fluorescein moiety. The synthesis of this modified analogue presented several problems because of the presence of several amino acids in the sequence carrying potentially reactive groups in their side-chains, and the necessity of introducing only one marker per molecule in a position that would not affect biological activity. The side-chain of Lys19 was selected because separate studies demonstrated that its substitution with an uncharged amino acid does not reduce the peptide's biological activity. We compared the merits of various synthetic protocols used to condense the fluorescent marker with the peptide. Biological assays indicated that the presence of the fluorescein moiety did not compromise peptide binding to CD4; furthermore, binding of the labeled analogue was not abolished by trypsin treatment, suggesting that the peptide may interact with both CD4 and additional trypsin-resistant binding sites on the cell surface. Finally, we verified the preservation of HIV infection enhancing ability in the labeled peptide.
Assuntos
Antígenos CD4/metabolismo , Desenho de Fármacos , Fluoresceínas/síntese química , Corantes Fluorescentes , Proteína gp120 do Envelope de HIV/química , HIV-1/crescimento & desenvolvimento , Peptídeos/síntese química , Sequência de Aminoácidos , Sítios de Ligação , Linfócitos T CD4-Positivos/virologia , Linhagem Celular , Fluoresceína , Fluoresceínas/metabolismo , Fluoresceínas/farmacologia , Proteína gp120 do Envelope de HIV/metabolismo , HIV-1/química , HIV-1/efeitos dos fármacos , Humanos , Dados de Sequência Molecular , Peptídeos/metabolismo , Peptídeos/farmacologiaRESUMO
A key event in the biosynthesis of the human immunodeficiency virus is the maturation of the gp160 precursor generating gp120 and gp41, two proteins that are fundamental for the infective process. In vivo, gp160 is specifically cleaved at the 515-519 site (REKR decreases A), in spite of the presence in its sequence of another consensus sequence KAKR decreases R (residues 507-511). Comparative kinetic studies on synthetic peptides reproducing different sequences of gp160 by the enzymes PC1 and furin are reported in this paper. The data demonstrate the higher efficiency of furin in the cleavage of peptidic substrates with respect to PC1 and its preference for REKR decreases A vs. KAKR decreases R. Furthermore, furin and PC1 are unable to process peptides patterned on the sequence 307-330 of specific viral strains of the gp120 V3 loop.
Assuntos
Ácido Aspártico Endopeptidases/metabolismo , Produtos do Gene env/metabolismo , Precursores de Proteínas/metabolismo , Subtilisinas/metabolismo , Sequência de Aminoácidos , Sequência Consenso , Furina , Produtos do Gene env/química , HIV/metabolismo , Proteína gp160 do Envelope de HIV , Humanos , Cinética , Dados de Sequência Molecular , Peptídeos/síntese química , Peptídeos/química , Peptídeos/metabolismo , Pró-Proteína Convertases , Precursores de Proteínas/química , Especificidade por SubstratoRESUMO
We have previously demonstrated that a 23-amino acid peptide derived from the V3 loop of the surface glycoprotein of the HIV-1 strain MN is able to bind CD4 and to enhance HIV-1 infection. Further studies have suggested that the peptide/CD4 interaction induces an increase in both CD4 expression and CD4/gp120 binding affinity. This paper describes the biological and physico-chemical characterization of three analogues of reduced sequence that have been designed in order to identify the minimum active sequence of this peptide corresponding to the MN-HIV- 1 principal neutralizing domain. Biological studies indicate that the entire sequence is required for biological activity and that the sequence 1-18 presents an inhibitory activity. CD and FT-IR absorption data are discussed here in order to identify possible structure-function correlations.
Assuntos
Proteína gp120 do Envelope de HIV/química , HIV-1/patogenicidade , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/farmacologia , Sequência de Aminoácidos , Linhagem Celular/virologia , Dicroísmo Circular , Sequência Conservada , Relação Dose-Resposta a Droga , Humanos , Dados de Sequência Molecular , Conformação Proteica , Espectroscopia de Infravermelho com Transformada de Fourier , Relação Estrutura-AtividadeRESUMO
The HIV-1 envelope glycoprotein gp120 interacts consecutively with CD4 and CCR5 to mediate the entry of R5-HIV-1 strains into target cells. The N-terminus of CCR5, which contains several sulfated tyrosines, plays a critical role in gp120-CCR5 binding and, consequently, in viral entry. Here, we demonstrate that a tyrosine sulfated peptide, reproducing the entire N-terminal extracellular region of CCR5, its unsulfated analogue, and a point-mutated peptide are unable to inhibit R5-HIV-1 mediated infection, competing with the entire CCR5 in the formation of gp120-CD4-CCR5 complex. Surprisingly, these peptides show the capability of enhancing HIV-1 infection caused by X4 strains through the up-regulation of both CD4 and CXCR4 receptors.
Assuntos
HIV-1/patogenicidade , Receptores CCR5/química , Receptores CXCR4/metabolismo , Regulação para Cima , Sequência de Aminoácidos , Animais , Antígenos CD4/metabolismo , Linhagem Celular , Relação Dose-Resposta a Droga , Infecções por HIV/imunologia , Dados de Sequência Molecular , Peptídeos/química , Peptídeos/farmacologiaRESUMO
The principal neutralizing domain (PND) of Human Immunodeficiency Virus type 1 (HIV-1) is mapped to a 24-amino acid sequence located in the hypervariable V3 region of the viral envelope protein. The PND of HIV-1 isolates from infected individuals corresponds mostly to that of the HIV-1 MN strain. We found that a peptide designed from the PND of HIV-1 MN virus greatly enhanced viral infection, while a peptide-derived PND of HTLV-IIIB virus showed at least 10-fold less efficient activity; no such effect was exhibited by the other peptides tested, including one designed from the PND of HIV-1 RF strain. The observed enhancing effect occurred in the early steps of viral infection and was not strain-restricted as both MN- and IIIB-derived peptides increased heterologous virus expression, including that of the RF strain. The MN- and, to a lesser extent, IIIB-derived peptides also increased CD4 expression on the cell membrane and differentially inhibited CD4 down-regulation induced by the phorbol ester TPA and/or by the monosialoganglioside GM1; the peptides showing no viral infection enhancement had no such effects. These findings demonstrate that the viral enhancement observed took place through a CD4-dependent mechanism and suggest that the PND is involved in HIV-1 infection and spread.
Assuntos
Antígenos CD4/fisiologia , Transformação Celular Viral , HIV-1/fisiologia , Peptídeos/farmacologia , Proteínas do Envelope Viral/metabolismo , Sequência de Aminoácidos , Linhagem Celular , Cloranfenicol O-Acetiltransferase/genética , Cloranfenicol O-Acetiltransferase/metabolismo , Gangliosídeo G(M1)/farmacologia , HIV-1/efeitos dos fármacos , HIV-1/imunologia , Humanos , Dados de Sequência Molecular , Testes de Neutralização , Peptídeos/síntese química , Acetato de Tetradecanoilforbol/farmacologia , Proteínas do Envelope Viral/genética , Replicação Viral/efeitos dos fármacosRESUMO
Previous studies have indicated that proteolytic activation of pro-hormones and pro-proteins occurs most frequently at the level of basic amino acids arranged in doublets and that the dibasic sites are situated in or next to beta-turns. Investigations utilizing synthetic peptides reproducing the N-terminal processing domain of pro-oxytocin-neurophysin have suggested a close relationship between the secondary structure of the cleavage locus and enzyme recognition, the minimal recognized sequence being the -Pro-Leu-Gly-Gly-Lys-Arg-Ala-Val-Leu- segment of the native precursor. NMR investigations and energy minimization studies have demonstrated that this sequence is organized in two type-II beta-turns involving the -Pro-Leu-Gly-Gly- and -Lys-Arg-Ala-Val- sequences. To further strengthen the above reported hypothesis and to study the role of turn subtypes, a new proline containing cyclic substrate of the processing enzyme, in which the N-terminal side that comes before the Lys-Arg pair is constrained to adopt a type-lI beta-turn, has been synthesized. The presence of a type-II beta-turn structure in this cyclic peptide model has been demonstrated by a combined NMR, CD and FT-IR absorption investigation. A preliminary study shows that PC1 is able to recognize and process our constrained substrate.
Assuntos
Modelos Químicos , Oligopeptídeos/síntese química , Ocitocina/análogos & derivados , Ocitocina/química , Peptídeos Cíclicos/síntese química , Ácido Aspártico Endopeptidases/química , Dicroísmo Circular , Hidrólise , Espectroscopia de Ressonância Magnética/métodos , Conformação Molecular , Oligopeptídeos/química , Peptídeos Cíclicos/química , Prolina/química , Pró-Proteína Convertases , Espectroscopia de Infravermelho com Transformada de Fourier/métodosRESUMO
A series of peptides patterned on the principal neutralizing domain of the HIV-1 envelope glycoprotein gp 120 have been synthesized by solid-phase techniques. Interestingly, in vitro experiments have shown that some of these peptides specifically interact with CD4 and, in particular, that the peptide corresponding to the sequence 307-330 of the HIV-1 MN isolate was able to enhance infection in a dose-specific and not a strain-restricted way. To bypass problems observed in preliminary runs, peptides were synthesized by both Fmoc and Boc chemistry. Comparison of the two strategies has allowed the set up of convenient protocols for the preparation of the target peptides in good yield, and with the high-purity grade needed for biological and physiochemical studies. Since the biological effects were present in the carboxyl-free C-terminal linear peptide but not in the amidated C-terminal analogue, preliminary conformational studies by circular dichroism and nuclear magnetic resonance techniques were also performed in an attempt to correlate these effects with possible contributions of structured conformations as predicted by theoretical calculations. The possibility of a beta-turn structure for the crucial Gly-Pro-Gly-Arg sequence has been confirmed by 2D NMR experiments. Ongoing studies suggest the exploitation of the activating properties of the MN-derived peptides to design a more sensitive and innovative serological test based on the virus itself and not on anti-HIV antibodies, as is the case for the large majority of tests currently in use.
Assuntos
Proteína gp120 do Envelope de HIV/química , HIV-1/química , Fragmentos de Peptídeos/química , Conformação Proteica , Sequência de Aminoácidos , Dicroísmo Circular , Proteína gp120 do Envelope de HIV/isolamento & purificação , HIV-1/patogenicidade , Espectroscopia de Ressonância Magnética , Dados de Sequência Molecular , Fragmentos de Peptídeos/isolamento & purificaçãoRESUMO
In previous studies we have demonstrated that synthetic peptides, corresponding to sequences in the (307-330) region of the gp120 principal neutralizing domain of different HIV-1 isolates are specifically recognized by a site distinct from the high affinity gp120-binding site of CD4. Interestingly, a peptide designed from the HIV-1 MN strain is able to enhance viral infection, while a HTLV-IIIB derived analogue is at least ten-fold less efficient and no effect is shown by other tested peptides. This enhancing effect occurs in the early step of infection and it is not strain restricted. A correlation between structure and biological functions evidenced by CD, FT-IR, and preliminary mono and bidimensional NMR is presented in this paper. The experimental data are compared to the predictions obtained by theoretical calculations.