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1.
FASEB J ; 28(7): 2840-51, 2014 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-24671707

RESUMO

Autoantibodies to nuclear antigens arise in human autoimmune diseases, but a unifying pathogenetic mechanism remains elusive. Recently we reported that exposure of neutrophils to inflammatory conditions induces the citrullination of core histones by peptidylarginine deiminase 4 (PAD4) and that patients with autoimmune disorders produce autoantibodies that recognize such citrullinated histones. Here we identify histone H1 as an additional substrate of PAD4, localize H1 within neutrophil extracellular traps, and detect autoantibodies to citrullinated H1 in 6% of sera from patients with systemic lupus erythematosus and Sjögren's syndrome. No preference for deiminated H1 was observed in healthy control sera and sera from patients with scleroderma or rheumatoid arthritis. We map binding to the winged helix of H1 and determine that citrulline 53 represents a key determinant of the autoantibody epitope. In addition, we quantitate RNA for H1 histone subtypes in mature human neutrophils and identify citrulline residues by liquid chromatography and tandem mass spectrometry. Our results indicate that deimination of linker histones generates new autoantibody epitopes with enhanced potential for stimulating autoreactive human B cells.-Dwivedi, N., Neeli, I., Schall, N., Wan, H., Desiderio, D. M., Csernok, E., Thompson, P. R., Dali, H., Briand, J.-P., Muller, S., Radic, M. Deimination of linker histones links neutrophil extracellular trap release with autoantibodies in systemic autoimmunity.


Assuntos
Autoanticorpos/imunologia , Autoimunidade/imunologia , Histonas/imunologia , Neutrófilos/imunologia , Sequência de Aminoácidos , Autoantígenos/imunologia , Doenças Autoimunes/imunologia , Epitopos/imunologia , Humanos , Imunoglobulina G/imunologia , Dados de Sequência Molecular , Alinhamento de Sequência
2.
J Biol Chem ; 287(52): 43685-93, 2012 Dec 21.
Artigo em Inglês | MEDLINE | ID: mdl-23109338

RESUMO

Blockage of the metastasis process remains a significant clinical challenge, requiring innovative therapeutic approaches. For this purpose, molecules that inhibit matrix metalloproteinases activity or induce the expression of their natural inhibitor, the tissue inhibitor of metalloproteinases (TIMPs), are potentially interesting. In a previous study, we have shown that synthetic ligands binding to cell surface nucleolin/nucleophosmin and known as HB 19 for the lead compound and NucAnt 6L (N6L) for the most potent analog, inhibit both tumor growth and angiogenesis. Furthermore, they prevent metastasis in a RET transgenic mice model which develops melanoma. Here, we investigated the effect of N6L on the invasion capacity of MDA-MB-435 melanoma cells. Our results show that the multivalent pseudopeptide N6L inhibited Matrigel invasion of MDA-MB-435 cells in a modified Boyden chamber model. This was associated with an increase in TIMP-3 in the cell culture medium without a change in TIMP-3 mRNA expression suggesting its release from cell surface and/or extracellular matrix. This may be explained by our demonstrated N6L interaction with sulfated glycosaminoglycans and consequently the controlled bioavailability of glycosaminoglycan-bound TIMP-3. The implication of TIMP-3 in N6L-induced inhibition of cell invasion was evidenced by siRNA silencing experiments showing that the loss of TIMP-3 expression abrogated the effect of N6L. The inhibition of tumor cell invasion by N6L demonstrated in this study, in addition to its previously established inhibitory effect on tumor growth and angiogenesis, suggests that N6L represents a promising anticancer drug candidate warranting further investigation.


Assuntos
Antineoplásicos/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Chumbo/farmacologia , Proteínas de Neoplasias/biossíntese , Neoplasias/metabolismo , Peptídeos/farmacologia , Inibidor Tecidual de Metaloproteinase-3/biossíntese , Animais , Antineoplásicos/química , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Chumbo/química , Camundongos , Camundongos Transgênicos , Invasividade Neoplásica , Proteínas de Neoplasias/genética , Neoplasias/tratamento farmacológico , Neoplasias/genética , Neoplasias/patologia , Peptídeos/química , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , RNA Neoplásico/biossíntese , Inibidor Tecidual de Metaloproteinase-3/genética
3.
BMC Cell Biol ; 13: 32, 2012 Nov 13.
Artigo em Inglês | MEDLINE | ID: mdl-23146273

RESUMO

BACKGROUND: Nucleolin is a protein over-expressed on the surface of activated cells. Recent studies have underlined the involvement of cell surface nucleolin in angiogenesis processes. This cell surface molecule serves as a receptor for various ligands implicated in pathophysiological processes such as growth factors, cell adhesion molecules like integrins, selectins or laminin-1, lipoproteins and viruses. N6L is a synthetic multimeric pseudopeptide that binds cell surface expressed nucleolin and inhibits cell proliferation. RESULTS: In the present work, we further investigated the mechanisms of action of pseudopeptide N6L on angiogenesis using HUVECs. We provide evidence that N6L inhibits the in vitro adhesion, proliferation and migration of HUVECs without inducing their apoptosis. In addition, we found that N6L downregulates MMP-2 in HUVECs. The above biological actions are regulated by SRC, ERK1/2, AKT and FAK kinases as we found that N6L inhibits their activation in HUVECs. Finally, down regulation of nucleolin using siRNA demonstrated the implication of nucleolin in the biological actions of these peptides. CONCLUSIONS: Taken together, these results indicate that N6L could constitute an interesting therapeutic tool for treating diseases associated with excessive angiogenesis.


Assuntos
Inibidores da Angiogênese/farmacologia , Peptídeos/farmacologia , Fosfoproteínas/metabolismo , Proteínas de Ligação a RNA/metabolismo , Inibidores da Angiogênese/síntese química , Inibidores da Angiogênese/química , Adesão Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Regulação para Baixo/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana , Humanos , Metaloproteinase 2 da Matriz/metabolismo , Peptídeos/síntese química , Peptídeos/química , Fosfoproteínas/antagonistas & inibidores , Fosfoproteínas/genética , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Proteínas de Ligação a RNA/antagonistas & inibidores , Proteínas de Ligação a RNA/genética , Nucleolina
4.
J Autoimmun ; 39(3): 143-53, 2012 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-22727561

RESUMO

After a long period where the potential of therapeutic peptides was let into oblivion and even dismissed, there is a revival of interest in peptides as potential drug candidates. Novel strategies for limiting metabolism and improve their bioavailability, and alternative routes of administration have emerged. This resulted in a large number of peptide-based drugs that are now being marketed in different indications. Regarding autoimmunity, successful data have been reported in numerous mouse models of autoimmune inflammation, yet relatively few clinical trials based on synthetic peptides are currently underway. This review reports on peptides that show much promises in appropriate mouse models of autoimmunity and describes in more detail clinical trials based on peptides for treating autoimmune patients. A particular emphasis is given to the 21-mer peptide P140/Lupuzor that has completed successfully phase I, phase IIa and phase IIb clinical trials for systemic lupus erythematosus.


Assuntos
Doenças Autoimunes/tratamento farmacológico , Fatores Imunológicos/farmacocinética , Lúpus Eritematoso Sistêmico/tratamento farmacológico , Fragmentos de Peptídeos/farmacocinética , Sequência de Aminoácidos , Animais , Doenças Autoimunes/imunologia , Autoimunidade/efeitos dos fármacos , Disponibilidade Biológica , Ensaios Clínicos como Assunto , Vias de Administração de Medicamentos , Esquema de Medicação , Humanos , Fatores Imunológicos/administração & dosagem , Lúpus Eritematoso Sistêmico/imunologia , Lúpus Eritematoso Sistêmico/mortalidade , Camundongos , Dados de Sequência Molecular , Fragmentos de Peptídeos/administração & dosagem , Taxa de Sobrevida
5.
J Immunol ; 184(7): 3937-45, 2010 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-20181885

RESUMO

An important hallmark of systemic lupus erythematosus is the production of autoantibodies specific for nuclear Ags, among which nucleosomes and their constituents, DNA and histones. It is widely admitted that some of these autoantibodies contribute largely in lupus pathogenesis because of their nephritogenic potential. However, the underlying mechanisms are still debated. In this study, we analyzed the autoimmune response against histone H2B during the course of the disease in lupus-prone (NZBxNZW)F1 mice, both in lymphoid organs and kidneys, and we assessed its potential involvement in lupus pathogenicity. We found that the N-terminal region of histone H2B represents a preferential target for circulating autoantibodies, which kinetics of appearance positively correlates with disease development. Furthermore, immunization of preautoimmune (NZBxNZW)F1 mice with H2B peptide 1-25 accelerates the disease. Kidney eluates from diseased (NZBxNZW)F1 mice do contain IgG Abs reacting with this peptide, and this H2B sequence was found to be accessible to specific Ab probes in Ag-containing deposits detected in nephritic kidneys. Finally, compared with control normal mice and to young preautoimmune (NZBxNZW)F1 animals, the frequency of cells secreting autoantibodies reacting with peptide 1-25 was significantly raised in the spleen and bone marrow and most importantly on a pathophysiological point of view, locally, in nephritic kidneys of diseased (NZBxNZW)F1 mice. Altogether our results demonstrate the existence in (NZBxNZW)F1 mice of both a systemic and local B cell response targeting the N-terminal region of histone H2B, and highlight the potential implication of this nuclear domain in lupus pathology.


Assuntos
Autoanticorpos/imunologia , Linfócitos B/imunologia , Histonas/imunologia , Nefrite Lúpica/imunologia , Animais , Autoanticorpos/sangue , Autoantígenos/imunologia , Ensaio de Imunoadsorção Enzimática , Feminino , Immunoblotting , Imuno-Histoquímica , Nefrite Lúpica/sangue , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos NZB
6.
Ann Rheum Dis ; 70(5): 837-43, 2011 May.
Artigo em Inglês | MEDLINE | ID: mdl-21173017

RESUMO

BACKGROUND: The P140 phosphopeptide issued from the spliceosomal U1-70K small nuclear ribonucleoprotein protein displays protective properties in MRL/lpr lupus-prone mice. It binds both major histocompatibility class II (MHCII) and HSC70/Hsp73 molecules. P140 peptide increases MRL/lpr peripheral blood lymphocyte apoptosis and decreases autoepitope recognition by T cells. OBJECTIVE: To explore further the mode of action of P140 peptide on HSC70+ antigen-presenting cells. METHODS: P140 biodistribution was monitored in real time using an imaging system and by fluorescence and electron microscopy. Fluorescence activated cell sorting and Western blotting experiments were used to evaluate the P140 effects on autophagic flux markers. RESULTS: P140 fluorescence accumulated especially in the lungs and spleen. P140 peptide reduced the number of peripheral and splenic T and B cells without affecting these cells in normal mice. Remaining MRL/lpr B cells responded normally to mitogens. P140 peptide decreased the expression levels of HSC70/Hsp73 chaperone and stable MHCII dimers, which are both increased in MRL/lpr splenic B cells. It impaired refolding properties of chaperone HSC70. In MRL/lpr B cells, it increased the accumulation of the autophagy markers p62/SQSTM1 and LC3-II, consistent with a downregulated lysosomal degradation during autophagic flux. CONCLUSION: The study results suggest that after P140 peptide binding to HSC70, the endogenous (auto)antigen processing might be greatly affected in MRL/lpr antigen-presenting B cells, leading to the observed decrease of autoreactive T-cell priming and signalling via a mechanism involving a lysosomal degradation pathway. This unexpected mechanism might explain the beneficial effect of P140 peptide in treated MRL/lpr mice.


Assuntos
Autofagia/efeitos dos fármacos , Proteínas de Choque Térmico HSC70/antagonistas & inibidores , Antígenos de Histocompatibilidade Classe II/efeitos dos fármacos , Lúpus Eritematoso Sistêmico/patologia , Fragmentos de Peptídeos/farmacologia , Animais , Apresentação de Antígeno/efeitos dos fármacos , Linfócitos B/patologia , Proteínas de Choque Térmico HSC70/metabolismo , Antígenos de Histocompatibilidade Classe II/metabolismo , Lúpus Eritematoso Sistêmico/tratamento farmacológico , Lúpus Eritematoso Sistêmico/imunologia , Camundongos , Camundongos Endogâmicos MRL lpr , Fragmentos de Peptídeos/farmacocinética , Fragmentos de Peptídeos/uso terapêutico , Transdução de Sinais/efeitos dos fármacos , Distribuição Tecidual
7.
BMC Cancer ; 10: 325, 2010 Jun 24.
Artigo em Inglês | MEDLINE | ID: mdl-20573279

RESUMO

BACKGROUND: The importance of cell-surface nucleolin in cancer biology was recently highlighted by studies showing that ligands of nucleolin play critical role in tumorigenesis and angiogenesis. By using a specific antagonist that binds the C-terminal tail of nucleolin, the HB-19 pseudopeptide, we recently reported that HB-19 treatment markedly suppressed the progression of established human breast tumor cell xenografts in the athymic nude mice without apparent toxicity. METHODS: The in vivo antitumoral action of HB-19 treatment was assessed on the spontaneous development of melanoma in the RET transgenic mouse model. Ten days old RET mice were treated with HB-19 in a prophylactic setting that extended 300 days. In parallel, the molecular basis for the action of HB-19 was investigated on a melanoma cell line (called TIII) derived from a cutaneous nodule of a RET mouse. RESULTS: HB-19 treatment of RET mice caused a significant delay in the onset of cutaneous tumors, several-months delay in the incidence of large tumors, a lower frequency of cutaneous nodules, and a reduction of visceral metastatic nodules while displaying no toxicity to normal tissue. Moreover, microvessel density was significantly reduced in tumors recovered from HB-19 treated mice compared to corresponding controls. Studies on the melanoma-derived tumor cells demonstrated that HB-19 treatment of TIII cells could restore contact inhibition, impair anchorage-independent growth, and reduce their tumorigenic potential in mice. Moreover, HB-19 treatment caused selective down regulation of transcripts coding matrix metalloproteinase 2 and 9, and tumor necrosis factor-alpha in the TIII cells and in melanoma tumors of RET mice. CONCLUSIONS: Although HB-19 treatment failed to prevent the development of spontaneous melanoma in the RET mice, it delayed for several months the onset and frequency of cutaneous tumors, and exerted a significant inhibitory effect on visceral metastasis. Consequently, HB-19 could provide a novel therapeutic agent by itself or as an adjuvant therapy in association with current therapeutic interventions on a virulent cancer like melanoma.


Assuntos
Membrana Celular/metabolismo , Neoplasias Pulmonares/prevenção & controle , Melanoma/prevenção & controle , Fragmentos de Peptídeos/farmacologia , Fosfoproteínas/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-ret/fisiologia , Proteínas de Ligação a RNA/antagonistas & inibidores , Neoplasias Cutâneas/prevenção & controle , Animais , Western Blotting , Proliferação de Células , Ensaio de Unidades Formadoras de Colônias , Citometria de Fluxo , Imunofluorescência , Humanos , Técnicas Imunoenzimáticas , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/secundário , Melanoma/metabolismo , Melanoma/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Fosfoproteínas/metabolismo , RNA Mensageiro/genética , Proteínas de Ligação a RNA/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Neoplasias Cutâneas/metabolismo , Neoplasias Cutâneas/patologia , Taxa de Sobrevida , Nucleolina
8.
Methods ; 49(4): 328-33, 2009 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-19303048

RESUMO

Immunostimulatory ODN CpGs have extensively been tested as adjuvants and immunotherapeutics and hold a lot of promise for human use. In our studies we took advantage of their negative charge to study their biological activities after being complexed with carbon nanotubes, a novel vector for vaccine delivery and Tat protein of HIV, a target protein for therapeutic or prophylactic intervention. In the case of carbon nanotubes, ODN CpGs were able to form stable complexes based on charge interaction and exert increased immunostimulatory activity in vitro. With regard to the Tat protein, ODN CpGs were shown to bind effectively through the basic domain of the protein representing residues 44-61. Moreover, using surface Plasmon Resonance Technology and an in vitro cellular system, ODN CpGs were shown to inhibit the interaction of Tat protein with the transactivation responsive element, a bulged RNA hairpin structure. However, when ODN CpGs were complexed with Tat they readily increased the apoptotic properties of this protein as studied in CD3-stimulated Jurkat cells. Overall, our findings together with published data support the view that for harnessing the beneficial effects of ODN CpGs a careful consideration has to be given depending on the target intervention.


Assuntos
Ilhas de CpG/imunologia , Fatores Imunológicos/imunologia , Nanotubos de Carbono , Oligodesoxirribonucleotídeos/imunologia , Poliaminas/imunologia , Animais , Humanos , Fatores Imunológicos/administração & dosagem , Oligodesoxirribonucleotídeos/administração & dosagem , Poliaminas/administração & dosagem , Polieletrólitos , Ativação Transcricional/efeitos dos fármacos , Ativação Transcricional/imunologia
9.
Exp Cell Res ; 315(2): 357-69, 2009 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-19026635

RESUMO

Nucleolin is an ubiquitous nucleolar phosphoprotein involved in fundamental aspects of transcription regulation, cell proliferation and growth. It has also been described as a shuttling molecule between nucleus, cytosol and the cell surface. Several studies have demonstrated that surface nucleolin serves as a receptor for various extracellular ligands implicated in cell proliferation, differentiation, adhesion, mitogenesis and angiogenesis. Previously, we reported that nucleolin in the extranuclear cell compartment is a glycoprotein containing N- and O-glycans. In the present study, we show that glycosylation is an essential requirement for surface nucleolin expression, since it is prevented when cells are cultured in the presence of tunicamycin, an inhibitor of N-glycosylation. Accordingly, surface but not nuclear nucleolin is radioactively labeled upon metabolic labeling of cells with [(3)H]glucosamine. Besides its well-demonstrated role in the internalization of specific ligands, here we show that ligand binding to surface nucleolin could also induce Ca(2+) entry into cells. Indeed, by flow cytometry, microscopy and patch-clamp experiments, we show that the HB-19 pseudopeptide, which binds specifically surface nucleolin, triggers rapid and intense membrane Ca(2+) fluxes in various types of cells. The use of several drugs then indicated that Store-Operated Ca(2+) Entry (SOCE)-like channels are involved in the generation of these fluxes. Taken together, our findings suggest that binding of an extracellular ligand to surface nucleolin could be involved in the activation of signaling pathways by promoting Ca(2+) entry into cells.


Assuntos
Cálcio/metabolismo , Glicoproteínas/fisiologia , Fosfoproteínas/fisiologia , Proteínas de Ligação a RNA/fisiologia , Anticorpos/imunologia , Anticorpos/farmacologia , Transporte Biológico/efeitos dos fármacos , Transporte Biológico/fisiologia , Complexo CD3/imunologia , Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cálcio/fisiologia , Sinalização do Cálcio/efeitos dos fármacos , Sinalização do Cálcio/fisiologia , Linhagem Celular Tumoral , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Núcleo Celular/metabolismo , Proliferação de Células/efeitos dos fármacos , Citoplasma/metabolismo , Ácido Egtázico/farmacologia , Glucosamina/metabolismo , Glicoproteínas/antagonistas & inibidores , Glicoproteínas/biossíntese , Glicosilação/efeitos dos fármacos , Humanos , Células Jurkat , Técnicas de Patch-Clamp , Peptídeos/farmacologia , Fosfoproteínas/antagonistas & inibidores , Fosfoproteínas/biossíntese , Proteínas de Ligação a RNA/antagonistas & inibidores , Proteínas de Ligação a RNA/biossíntese , Tunicamicina/farmacologia , Nucleolina
10.
Apoptosis ; 14(10): 1190-203, 2009 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-19693674

RESUMO

Dengue viruses belong to the Flavivirus family and are responsible for hemorrhagic fever in Human. Dengue virus infection triggers apoptosis especially through the expression of the small membrane (M) protein. Using isolated mitochondria, we found that synthetic peptides containing the C-terminus part of the M ectodomain caused apoptosis-related mitochondrial membrane permeabilization (MMP) events. These events include matrix swelling and the dissipation of the mitochondrial transmembrane potential (DeltaPsi(m)). Protein M Flavivirus sequence alignments and helical wheel projections reveal a conserved distribution of charged residues. Moreover, when combined to the cell penetrating HIV-1 Tat peptide transduction domain (Tat-PTD), this sequence triggers a caspase-dependent cell death associated with DeltaPsi(m) loss and cytochrome c release. Mutational approaches coupled to functional screening on isolated mitochondria resulted in the selection of a protein M derived sequence containing nine residues with potent MMP-inducing properties on isolated mitochondria. A chimeric peptide composed of a Tat-PTD linked to the 9-mer entity triggers MMP and cell death. Finally, local administration of this chimeric peptide induces growth inhibition of xenograft prostate PC3 tumors in immuno-compromised mice, and significantly enhances animal survival. Together, these findings support the notion of using viral genomes as valuable sources to discover mitochondria-targeted sequences that may lead to the development of new anticancer compounds.


Assuntos
Flavivirus/química , Membranas Mitocondriais/efeitos dos fármacos , Membranas Mitocondriais/metabolismo , Peptídeos/farmacologia , Proteínas Virais/química , Ensaios Antitumorais Modelo de Xenoenxerto , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Humanos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Camundongos , Camundongos Nus , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Dilatação Mitocondrial/efeitos dos fármacos , Dados de Sequência Molecular , Peptídeos/química , Permeabilidade/efeitos dos fármacos , Estrutura Terciária de Proteína , Análise de Sobrevida , Produtos do Gene tat do Vírus da Imunodeficiência Humana/farmacologia
11.
Mol Immunol ; 45(7): 1963-75, 2008 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-18054388

RESUMO

The potential caveolin-1 binding domain (CBD), referred to as CBD1 and CBD2, is highly conserved in the transmembrane envelope glycoprotein of various HIV-1 and HIV-2 isolates, respectively. However, HIV-1 neutralizing antibodies raised against the synthetic CBD1 peptide (SLEQIWNNMTWMQWDK) do not cross-react with the CBD2 peptide (SLTPDWNNMTWQEWER) and have no effect on HIV-2 infection. Here we show that the CBD2 peptide is not immunogenic under similar immunization conditions as the CBD1 peptide. Moreover, the CBD1 but not the CBD2 peptide has the capacity to bind caveolin-1 in crude cell extracts thus suggesting the existence of structural and/or conformational differences between CBD1 and CBD2. Accordingly, circular dichroism spectroscopy and fluorimetry analysis indicated that CBD1 but not CBD2 could adopt a defined secondary structure and form a complex with a peptide corresponding to the caveolin-1 scaffolding domain, which is the site of interaction of caveolin-1 with various proteins. In line with these observations, CBD1 but not CBD2 binds cells and forms large aggregates at the plasma membrane by colocalizing with cytofacial caveolin-1. This latter is dependent on the lipid raft integrity of the plasma membrane. Supporting that the ability to penetrate into plasma membranes is sustained by folding at the interface, CBD1 but not CBD2 has the capacity to insert into lipid monolayers, penetrate into artificial membranes and adopt a beta-sheet conformation in presence of lipid vesicles. These structural determinants and membrane partitioning properties could account for the immunogenicity of the CBD1 peptide in various animals.


Assuntos
Caveolina 1/imunologia , Membrana Celular/metabolismo , Proteína gp41 do Envelope de HIV/química , Proteína gp41 do Envelope de HIV/imunologia , Peptídeos/imunologia , Sequência de Aminoácidos , Animais , Cobaias , Células HeLa , Humanos , Microdomínios da Membrana , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Peptídeos/química , Ligação Proteica , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Transporte Proteico , Relação Estrutura-Atividade
12.
Angew Chem Int Ed Engl ; 48(9): 1625-8, 2009.
Artigo em Inglês | MEDLINE | ID: mdl-19170154

RESUMO

The perfect blend: A new class of self-assembling cyclooligomers with mixed urea/amide backbone is described (see figure). A high level of hierarchical and directional control is achieved: depending on the level of backbone preorganization, columnar or tubular arrangements with either parallel or antiparallel growing modes can be selected.


Assuntos
Amidas/química , Ureia/química , Cristalografia por Raios X , Dimerização , Compostos Macrocíclicos/química , Nanotubos/química
13.
BMC Microbiol ; 8: 145, 2008 Sep 12.
Artigo em Inglês | MEDLINE | ID: mdl-18789156

RESUMO

BACKGROUND: Francisella tularensis, the causative agent of tularemia, is one of the most infectious human bacterial pathogens. It is phagocytosed by immune cells, such as monocytes and macrophages. The precise mechanisms that initiate bacterial uptake have not yet been elucidated. Participation of C3, CR3, class A scavenger receptors and mannose receptor in bacterial uptake have been already reported. However, contribution of an additional, as-yet-unidentified receptor for F. tularensis internalization has been suggested. RESULTS: We show here that cell-surface expressed nucleolin is a receptor for Francisella tularensis Live Vaccine Strain (LVS) and promotes LVS binding and infection of human monocyte-like THP-1 cells. The HB-19 pseudopeptide that binds specifically carboxy-terminal RGG domain of nucleolin inhibits LVS binding and infection of monocyte-like THP-1 cells. In a pull-down assay, elongation factor Tu (EF-Tu), a GTP-binding protein involved in protein translation, usually found in cytoplasm, was recovered among LVS bacterial membrane proteins bound on RGG domain of nucleolin. A specific polyclonal murine antibody was raised against recombinant LVS EF-Tu. By fluorescence and electron microscopy experiments, we found that a fraction of EF-Tu could be detected at the bacterial surface. Anti-EF-Tu antibodies reduced LVS binding to monocyte-like THP-1 cells and impaired infection, even in absence of complement and complement receptors. Interaction between EF-Tu and nucleolin was illustrated by two different pull-down assays using recombinant EF-Tu proteins and either RGG domain of nucleolin or cell solubilized nucleolin. DISCUSSION: Altogether, our results demonstrate that the interaction between surface nucleolin and its bacterial ligand EF-Tu plays an important role in Francisella tularensis adhesion and entry process and may therefore facilitate invasion of host tissues. Since phagosomal escape and intra-cytosolic multiplication of LVS in infected monocytes are very similar to those of human pathogenic F. tularensis ssp tularensis, the mechanism of entry into monocyte-like THP-1 cells, involving interaction between EF-Tu and nucleolin, might be similar in the two subspecies. Thus, the use of either nucleolin-specific pseudopeptide HB-19 or recombinant EF-Tu could provide attractive therapeutic approaches for modulating F. tularensis infection.


Assuntos
Francisella tularensis/fisiologia , Proteínas de Membrana/metabolismo , Monócitos/metabolismo , Monócitos/microbiologia , Fator Tu de Elongação de Peptídeos/metabolismo , Fosfoproteínas/metabolismo , Proteínas de Ligação a RNA/metabolismo , Animais , Anticorpos Antibacterianos/metabolismo , Sítios de Ligação , Linhagem Celular , Regulação para Baixo , Francisella tularensis/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Ligantes , Antígeno de Macrófago 1/genética , Camundongos , Peptídeos/farmacologia , Proteínas Recombinantes/metabolismo , Tularemia/microbiologia , Nucleolina
14.
Mol Immunol ; 44(11): 3024-36, 2007 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-17292472

RESUMO

Recent studies have revealed that peptide analogues containing modified peptide bonds might replace poorly stable natural peptides in therapeutic strategies. Using the model peptide 88-99 of histone H4, which contains a supradominant epitope recognized by Th cells induced to nucleosomes, we have generated twelve analogues containing aza-beta(3)-amino acid residue substitutions. The ability of this new class of peptidomimetics corresponding to the Psi[CONHNRCH(2)] modification to be recognized by T cells primed with the parent peptide was examined in BALB/c mice. An Ala-scan study revealed that residues 88 to 92 were essential for keeping antigenic activity of the nominal peptide. In good agreement, the six aza-beta(3)-analogues encompassing substitutions in the region 89-92 were antigenically inactive. Analogues PsiG94 and PsiG99 were both antigenic and immunogenic, though at levels that were slightly lower to that of the parent peptide. However, the remaining analogues PsiR95, PsiL97, PsiY98 and PsiL97-Y98 were strongly recognized by T cells generated to the homologous peptides. The PsiL97-Y98 analogue, in particular, strongly activated CD4(+) T cells as visualized in CFSE dilution assay. T cells primed to these four analogues and recalled with the nominal peptide secreted high levels of either IL-2 (PsiR95, PsiY98) or IFN-gamma (PsiL97, PsiL97-Y98). This result, supported by molecular modeling, suggests that TCRs of T cells primed to these four analogues recognized the parent peptide associated with the MHC I-A(d)/I-E(d) molecules. Since these T cells produce a distinct cytokine pattern when they are recalled with the parent sequence, this new class of analogues may have valuable applications in the context of self-tolerance and autoimmunity.


Assuntos
Linfócitos T CD4-Positivos/imunologia , Epitopos de Linfócito T , Modelos Moleculares , Mimetismo Molecular , Peptídeos/imunologia , Animais , Apresentação de Antígeno , Autoimunidade , Epitopos de Linfócito T/química , Epitopos de Linfócito T/imunologia , Feminino , Camundongos , Camundongos Endogâmicos BALB C , Modelos Imunológicos , Nucleossomos/imunologia , Peptídeos/química
15.
Sci Rep ; 8(1): 16820, 2018 11 14.
Artigo em Inglês | MEDLINE | ID: mdl-30429537

RESUMO

The nuclear translocation of endogenous heat shock cognate protein HSPA8 is a requisite for cell survival during oxidative and heat shock stress. Upon these events, cytoplasmic HSPA8 is thought to concentrate within the nucleus and nucleolus. When the situation returns to normal, HSPA8 is released from its nuclear/nucleolar anchors and redistributes into the cytoplasm. By using different stress conditions and a 21-mer phosphopeptide tool called P140, which binds HSPA8 and hampers its chaperone properties, we deciphered the cellular and molecular effects arising during this vital cytoplasmic-nuclear-cytoplasmic shuttling process. Using the non-metastatic fibroblastoid cell line MRL/N-1 derived from a MRL/MpTn-gld/gld lupus-prone mouse, we discovered that P140 treatment neutralized the egress of HSPA8 from nucleus to cytoplasm in the cell recovery phase. This lack of relocation of HSPA8 into the cytoplasm of heat-shocked MRL/N-1 cells altered the ability of these cells to survive when a second mild oxidative stress mimicking inflammatory conditions was applied. Crosslinking experiments followed by proteomics studies showed that P140 binds regions close to nuclear import and export signal sequences encompassed within the HSPA8 structure. These data are consistent with HSPA8 having a crucial cell protective role against reactive oxygen species (ROS) production by mitochondria during inflammatory conditions.


Assuntos
Transporte Ativo do Núcleo Celular , Sobrevivência Celular , Proteínas de Choque Térmico HSC70/metabolismo , Resposta ao Choque Térmico , Transporte Ativo do Núcleo Celular/efeitos dos fármacos , Animais , Sítios de Ligação , Linhagem Celular , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Camundongos , Estresse Oxidativo , Ligação Proteica , Sinais Direcionadores de Proteínas
16.
Biochim Biophys Acta ; 1757(9-10): 1312-23, 2006.
Artigo em Inglês | MEDLINE | ID: mdl-16928356

RESUMO

For many years, medical drug discovery has extensively exploited peptides as lead compounds. Currently, novel structures of therapeutic peptides are derived from active pre-existing peptides or from high-throughput screening, and optimized following a rational drug design approach. Molecules of interest may prove their ability to influence the disease outcome in animal models and must respond to a set of criteria based on toxicity studies, ease of administration, the cost of their synthesis, and logistic for clinical use to validate it as a good candidate in a therapeutic perspective. This applies to the potential use of peptides to target one central intracellular organelle, the mitochondrion, to modulate (i.e. activate or prevent) apoptosis. Putative mitochondrial protein targets and the strategies already elaborated to correct the defects linked to these proteins (overexpression, inactivation, mutation..., etc.) are described, and recent advances that led or may lead to the conception of therapeutic peptides via a specific action on these mitochondrial targets in the future are discussed.


Assuntos
Apoptose/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Peptídeos/farmacologia , Sequência de Aminoácidos , Animais , Humanos , Dilatação Mitocondrial/efeitos dos fármacos , Dados de Sequência Molecular , Peptídeos/química , Permeabilidade/efeitos dos fármacos
17.
J Clin Invest ; 111(6): 851-7, 2003 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-12639991

RESUMO

Accumulating evidence favors a role for proinsulin as a key autoantigen in diabetes. In the mouse, two proinsulin isoforms coexist. Most studies point to proinsulin 2 as the major isoform recognized by T cells in the NOD mouse. We studied mice in which a null proinsulin 2 mutation was transferred from proinsulin 2-deficient 129 mice onto the NOD background along with 16 genetic markers (including I-A(g7) MHC molecule) associated with diabetes. Intercross mice from the fourth backcross generation showed that proinsulin 2(-/-) mice develop accelerated insulitis and diabetes. The high prevalence of anti-insulin autoantibodies in proinsulin 2(-/-) mice indicates that diabetes acceleration relates to altered recognition of proinsulin. The prevalence of anti-glutamic acid decarboxylase autoantibodies and of sialitis is not increased in proinsulin 2(-/-) mice. We give evidence that proinsulin 2 expression leads to silencing of T cells specific for an epitope shared by proinsulin 1 and proinsulin 2. In the human, alleles located in the VNTR region flanking the insulin gene control beta cell response to glucose and proinsulin expression in the thymus and are key determinants of diabetes susceptibility. Proinsulin 2(-/-) NOD mice provide a model to study the role of thymic expression of insulin in susceptibility to diabetes.


Assuntos
Diabetes Mellitus Tipo 1/etiologia , Proinsulina/fisiologia , Transferência Adotiva , Sequência de Aminoácidos , Animais , Autoimunidade , Diabetes Mellitus Tipo 1/imunologia , Glutamato Descarboxilase/imunologia , Anticorpos Anti-Insulina/sangue , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Dados de Sequência Molecular , Proinsulina/deficiência
18.
Chem Biol ; 13(5): 531-8, 2006 May.
Artigo em Inglês | MEDLINE | ID: mdl-16720274

RESUMO

Unnatural oligomeric scaffolds designed to adopt defined secondary structures (e.g., helices), while retaining the chemical diversity of amino acid side chains, are of practical value to elaborate functional mimetics of bioactive alpha-polypeptides. Enantiopure N,N'-linked oligoureas as short as seven residues long have been previously shown to fold into a stable helical structure, stabilized by 12- and 14-membered H-bonded rings. We now report that eight-residue oligoureas designed to mimic globally amphiphilic alpha-helical host-defense peptides are effective against both gram-negative and gram-positive bacteria (including methicillin-resistant Staphylococcus aureus [MRSA]) and exhibit selectivity for bacterial versus mammalian cells. Circular dichroism (CD) spectroscopy studies suggest enhanced helical propensity of oligoureas in the presence of phospholipid vesicles. The utility of this new class of nonpeptidic foldamers for biological applications is highlighted by high resistance to proteolytic degradation.


Assuntos
Antibacterianos/química , Biopolímeros , Mimetismo Molecular , Peptídeos/química , Sequência de Aminoácidos , Cromatografia Líquida de Alta Pressão , Dicroísmo Circular , Resistência a Meticilina , Estrutura Secundária de Proteína , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Staphylococcus aureus/efeitos dos fármacos
19.
Chem Commun (Camb) ; (11): 1182-4, 2006 Mar 21.
Artigo em Inglês | MEDLINE | ID: mdl-16518484

RESUMO

Multi-walled carbon nanotubes have been covalently functionalized via 1,3-dipolar cycloaddition of azomethine ylides with orthogonally protected amino functions that can be selectively deprotected and subsequently modified with drugs and fluorescent probes.


Assuntos
Compostos Azo/química , Portadores de Fármacos/química , Nanotubos de Carbono/química , Preparações Farmacêuticas/administração & dosagem , Ciclização , Microscopia de Tunelamento , Solubilidade
20.
Chem Commun (Camb) ; (39): 4069-71, 2006 Oct 21.
Artigo em Inglês | MEDLINE | ID: mdl-17024251

RESUMO

Enantiopure dipeptide-derived 1,3,5-triazepan-2,6-diones and form H-bonded 3(1) helical molecular tapes with P chirality in the solid state; in the case of , these columnar tapes self-assemble through aromatic-aromatic interactions to give hollow tubular structures.


Assuntos
Azepinas , Dipeptídeos/química , Azepinas/síntese química , Azepinas/química , Cristalografia por Raios X , Ligação de Hidrogênio , Microscopia Eletrônica de Transmissão , Modelos Moleculares , Estrutura Molecular , Porosidade , Solventes/química , Estereoisomerismo
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