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 ProteínasRESUMO
Persistent exposure of the immune system to death cell debris leads to autoantibodies against chromatin in patients with systemic lupus erythematosus (SLE). Deposition of anti-chromatin/chromatin complexes can instigate inflammation in multiple organs including the kidney. Previously we identified specific cell death-associated histone modifications as targets of autoantibodies in SLE. In this study we addressed, in a large cohort of SLE patients and controls, the question whether plasma reactivities with specific histone peptides associated with serology and clinical features. Plasma from SLE patients with and without lupus nephritis, disease controls, and healthy controls, were tested in ELISA with histone H4 peptide acetylated at lysines 8, 12 and 16 (H4pac), H2B peptide acetylated at lysine 12 (H2Bpac), H3 peptide trimethylated at lysine 27 (H3pme), and their unmodified equivalents. SLE patients displayed a higher reactivity with the modified equivalent of each peptide. Reactivity with H4pac showed both a high sensitivity (89%) and specificity (91%) for SLE, while H2Bpac exhibited a high specificity (96%) but lower sensitivity (69%). Reactivity with H3pme appeared not specific for SLE. Anti-H4pac and anti-H2Bpac reactivity demonstrated a high correlation with disease activity. Moreover, patients reacting with multiple modified histone peptides exhibited higher SLEDAI and lower C3 levels. SLE patients with renal involvement showed higher reactivity with H2B/H2Bpac and a more pronounced reactivity with the modified equivalent of H3pme and H2Bpac. In conclusion, reactivity with H4pac and H2Bpac is specific for SLE patients and correlates with disease activity, whereas reactivity with H2Bpac is in particular associated with lupus nephritis.
Assuntos
Autoanticorpos/sangue , Histonas/imunologia , Lúpus Eritematoso Sistêmico/patologia , Nefrite Lúpica/patologia , Acetilação , Adulto , Sequência de Aminoácidos , Área Sob a Curva , Autoanticorpos/imunologia , Estudos de Casos e Controles , Estudos Transversais , Progressão da Doença , Ensaio de Imunoadsorção Enzimática , Feminino , Histonas/química , Humanos , Lúpus Eritematoso Sistêmico/complicações , Lúpus Eritematoso Sistêmico/metabolismo , Nefrite Lúpica/complicações , Nefrite Lúpica/metabolismo , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Peptídeos/química , Peptídeos/imunologia , Curva ROCRESUMO
The P140 peptide, a 21-mer linear peptide (sequence 131-151) generated from the spliceosomal SNRNP70/U1-70K protein, contains a phosphoserine residue at position 140. It significantly ameliorates clinical manifestations in autoimmune patients with systemic lupus erythematosus and enhances survival in MRL/lpr lupus-prone mice. Previous studies showed that after P140 treatment, there is an accumulation of autophagy markers sequestosome 1/p62 and MAP1LC3-II in MRL/lpr B cells, consistent with a downregulation of autophagic flux. We now identify chaperone-mediated autophagy (CMA) as a target of P140 and demonstrate that its inhibitory effect on CMA is likely tied to its ability to alter the composition of HSPA8/HSC70 heterocomplexes. As in the case of HSPA8, expression of the limiting CMA component LAMP2A, which is increased in MRL/lpr B cells, is downregulated after P140 treatment. We also show that P140, but not the unphosphorylated peptide, uses the clathrin-dependent endo-lysosomal pathway to enter into MRL/lpr B lymphocytes and accumulates in the lysosomal lumen where it may directly hamper lysosomal HSPA8 chaperoning functions, and also destabilize LAMP2A in lysosomes as a result of its effect on HSP90AA1. This dual effect may interfere with the endogenous autoantigen processing and loading to major histocompatibility complex class II molecules and as a consequence, lead to lower activation of autoreactive T cells. These results shed light on mechanisms by which P140 can modulate lupus disease and exert its tolerogenic activity in patients. The unique selective inhibitory effect of the P140 peptide on CMA may be harnessed in other pathological conditions in which reduction of CMA activity would be desired.
Assuntos
Autofagia , Chaperonas Moleculares/química , Fragmentos de Peptídeos/química , Fosfopeptídeos/química , Ribonucleoproteína Nuclear Pequena U1/química , Animais , Autoimunidade , Linfócitos B/imunologia , Endocitose , Endossomos/metabolismo , Proteínas de Choque Térmico HSC70/química , Antígenos de Histocompatibilidade Classe II/metabolismo , Humanos , Lúpus Eritematoso Sistêmico/imunologia , Lisossomos/química , Lisossomos/metabolismo , Camundongos , Camundongos Endogâmicos MRL lpr , Células NIH 3T3 , Fosforilação , Serina/química , Linfócitos T/imunologiaRESUMO
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 SequênciaRESUMO
Synthetic peptides are attracting increasing attention as therapeutics. Despite their potential, however, only a few selected peptides have been able to enter in clinical trials for chronic autoimmune diseases and systemic lupus erythematosus (SLE) in particular. Here, we describe and discuss a series of assays, which may help in characterizing valuable candidate peptides that were applied in our laboratory to develop the lupus P140 peptide program. The different steps of selection include the choice of the initial autoantigen, the design, synthesis and purification of peptides, their preliminary screen by measuring cytokines produced ex vivo by T cells and their binding to major histocompatibility complex class II (MHCII) molecules, their capacity to lower peripheral cell hyperproliferation in lupus-prone MRL/lpr mice, and, as a final step, their ability to slow down the development of lupus disease in model animals.
Assuntos
Lúpus Eritematoso Sistêmico/tratamento farmacológico , Lúpus Eritematoso Sistêmico/imunologia , Peptídeos/uso terapêutico , Animais , Autoantígenos/química , Autoantígenos/imunologia , Citocinas/biossíntese , Modelos Animais de Doenças , Ensaio de Imunoadsorção Enzimática , Epitopos de Linfócito T/química , Epitopos de Linfócito T/imunologia , Epitopos de Linfócito T/metabolismo , Feminino , Citometria de Fluxo , Antígenos de Histocompatibilidade Classe II/imunologia , Antígenos de Histocompatibilidade Classe II/metabolismo , Camundongos , Camundongos Endogâmicos MRL lpr , Peptídeos/imunologia , Peptídeos/metabolismo , Ligação Proteica/imunologiaRESUMO
BACKGROUND: Nucleolin is a protein over-expressed on the surface of tumor and endothelial cells. Recent studies have underlined the involvement of cell surface nucleolin in tumor growth and angiogenesis. 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 (HIV and coxsackie B). HB-19 is a synthetic multimeric pseudopeptide that binds cell surface expressed nucleolin and inhibits both tumor growth and angiogenesis. METHODOLOGY/PRINCIPAL FINDINGS: In the present work, we further investigated the biological actions of pseudopeptide HB-19 on HUVECs. In a previous work, we have shown that HB-19 inhibits the in vivo angiogenesis on the chicken embryo CAM assay. We now provide evidence that HB-19 inhibits the in vitro adhesion, migration and proliferation of HUVECs without inducing their apoptosis. The above biological actions seem to be regulated by SRC, ERK1/2, AKT and FAK kinases as we found that HB-19 inhibits their activation in HUVECs. Matrix metalloproteinases (MMPs) play crucial roles in tumor growth and angiogenesis, so we investigated the effect of HB-19 on the expression of MMP-2 and we found that HB-19 downregulates MMP-2 in HUVECs. Finally, down regulation of nucleolin using siRNA confirmed the implication of nucleolin in the biological actions of these peptides. CONCLUSIONS/SIGNIFICANCE: Taken together, these results indicate that HB-19 could constitute an interesting tool for tumor therapy strategy, targeting cell surface nucleolin.
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 , NucleolinaRESUMO
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/genéticaRESUMO
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 SobrevidaRESUMO
OBJECTIVE: Both the early steps and the high recurrence of autoimmunity once the disease is established are unexplained in human type 1 diabetes. Because CD8(+) T cells are central and insulin is a key autoantigen in the disease process, our objective was to characterize HLA class I-restricted autoreactive CD8(+) T cells specific for preproinsulin (PPI) in recent-onset and long-standing type 1 diabetic patients and healthy control subjects. RESEARCH DESIGN AND METHODS: We used HLA-A*02:01 tetramers complexed to PPI peptides to enumerate circulating PPI-specific CD8(+) T cells in patients and characterize them using membrane markers and single-cell PCR. RESULTS: Most autoreactive CD8(+) T cells detected in recent-onset type 1 diabetic patients are specific for leader sequence peptides, notably PPI(6-14), whereas CD8(+) T cells in long-standing patients recognize the B-chain peptide PPI(33-42) (B(9-18)). Both CD8(+) T-cell specificities are predominantly naïve, central, and effector memory cells, and their gene expression profile differs from cytomegalovirus-specific CD8(+) T cells. PPI(6-14)-specific CD8(+) T cells detected in one healthy control displayed Il-10 mRNA expression, which was not observed in diabetic patients. CONCLUSIONS: PPI-specific CD8(+) T cells in type 1 diabetic patients include central memory and target different epitopes in new-onset versus long-standing disease. Our data support the hypothesis that insulin therapy may contribute to the expansion of autoreactive CD8(+) T cells in the long term.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Diabetes Mellitus Tipo 1/imunologia , Diabetes Mellitus Tipo 1/metabolismo , Insulina/metabolismo , Adulto , Células Cultivadas , Feminino , Perfilação da Expressão Gênica , Humanos , Insulina/genética , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
The multimeric DNA sliding clamps confer high processivity to replicative DNA polymerases and are also binding platforms for various enzymes involved in DNA metabolism. These enzymes interact with the clamp through a small peptide that binds into a hydrophobic pocket which is a potential target for the development of new antibacterial compounds. Starting from a generic heptapeptide, we used a structure-based strategy to improve the design of new peptide ligands. Chemical modifications at specific residues result in a dramatic increase of the interaction as measured by SPR and ITC. The affinity of our best hits was improved by 2 orders of magnitude as compared to the natural ligand, reaching 10(-8) M range. The molecular basis of the interactions was analyzed by solving the co-crystal structures of the most relevant peptides bound to the clamp and reveals how chemical modifications establish new contacts and contributes to an increased affinity of the ligand.
Assuntos
DNA Polimerase III/química , DNA Polimerase beta/química , Proteínas de Escherichia coli/química , Escherichia coli/química , Oligopeptídeos/síntese química , Cristalografia por Raios X , Desenho de Fármacos , Ligantes , Modelos Moleculares , Oligopeptídeos/química , Ligação Proteica , Relação Estrutura-Atividade , TermodinâmicaRESUMO
Recent studies have implicated the involvement of cell surface forms of nucleolin in tumor growth. In this study, we investigated whether a synthetic ligand of cell-surface nucleolin known as N6L could exert antitumor activity. We found that N6L inhibits the anchorage-dependent and independent growth of tumor cell lines and that it also hampers angiogenesis. Additionally, we found that N6L is a proapoptotic molecule that increases Annexin V staining and caspase-3/7 activity in vitro and DNA fragmentation in vivo. Through affinity isolation experiments and mass-spectrometry analysis, we also identified nucleophosmin as a new N6L target. Notably, in mouse xenograft models, N6L administration inhibited human tumor growth. Biodistribution studies carried out in tumor-bearing mice indicated that following administration N6L rapidly localizes to tumor tissue, consistent with its observed antitumor effects. Our findings define N6L as a novel anticancer drug candidate warranting further investigation.
Assuntos
Neoplasias/tratamento farmacológico , Peptídeos/farmacologia , Animais , Apoptose/efeitos dos fármacos , Processos de Crescimento Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Nucléolo Celular/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Células Endoteliais/efeitos dos fármacos , Humanos , Ligantes , Linfoma/tratamento farmacológico , Linfoma/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Terapia de Alvo Molecular/métodos , Neoplasias/metabolismo , Peptídeos/farmacocinética , Fosfoproteínas/metabolismo , Proteínas de Ligação a RNA/metabolismo , Distribuição Tecidual , Ensaios Antitumorais Modelo de Xenoenxerto , NucleolinaRESUMO
The P140 phosphopeptide encompassing residues 131-151 of the spliceosomal U1-70K snRNP protein displays protective properties in lupus patients and MRL/lpr mice. It increases peripheral blood lymphocyte apoptosis via a mechanism involving γδ T cells. After intravenous administration, P140 accumulates in the lungs and spleen. It binds both the HSC70/Hsp73 chaperone and MHC class II (MHCII) molecules, which colocalize in splenic MRL/lpr B cells. Expression of HSC70 and MHCII, which is increased in MRL/lpr splenic B cells, is diminished after P140 administration. P140 impairs refolding properties of HSC70 and alters expression of stable MHCII molecules in B lymphocytes. In MRL/lpr B cells, P140 increases the accumulation of the autophagy markers p62/SQSTM1 and LC3-II, consistent with a downregulation of autophagic flux. Our study reveals a very unique property of P140 peptide that alters the autophagy pathway leading to a defect of endogenous (auto)antigen processing in MRL/lpr antigen-presenting B cells and a decrease of T cell priming and signaling.
Assuntos
Autofagia/efeitos dos fármacos , Linfócitos B/metabolismo , Antígenos de Histocompatibilidade Classe II/metabolismo , Lúpus Eritematoso Sistêmico/imunologia , Peptídeos/farmacologia , Animais , Autofagia/imunologia , Linfócitos B/efeitos dos fármacos , Linfócitos B/imunologia , Proteínas de Choque Térmico HSC70/metabolismo , Antígenos de Histocompatibilidade Classe II/química , Antígenos de Histocompatibilidade Classe II/imunologia , Humanos , Lúpus Eritematoso Sistêmico/metabolismo , Lúpus Eritematoso Sistêmico/patologia , Lúpus Eritematoso Sistêmico/terapia , Camundongos , Camundongos Endogâmicos MRL lpr , Peptídeos/imunologia , Peptídeos/uso terapêutico , Estabilidade Proteica/efeitos dos fármacosRESUMO
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 TecidualRESUMO
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 , NucleolinaRESUMO
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 NZBRESUMO
Ongoing clinical trials are exploring anticancer approaches based on signaling by TRAIL, a ligand for the cell death receptors DR4 and DR5. In this study, we report on the selective apoptotic effects of multivalent DR5 binding peptides (TRAIL(mim/DR5)) on cancer cells in vitro and in vivo. Surface plasmon resonance revealed up to several thousand-fold increased affinities of TRAIL(mim/DR5)-receptor complexes on generation of divalent and trivalent molecules, the latter of which was achieved with a conformationally restricted adamantane core. Notably, only multivalent molecules triggered a substantial DR5-dependent apoptotic response in vitro. In tumor models derived from human embryonic kidney cells or primary foreskin fibroblasts, TRAIL(mim/DR5) peptides exerted a cancer cell-selective action that could synergize with resveratrol in a manner independent of p53. In a xenograft model of human colon cancer, a divalent TRAIL(mim/DR5) peptide inhibited tumor growth. Our results offer a proof-of-principle for the development of synthetic small molecules to trigger the TRAIL apoptosis pathway for cancer therapy.
Assuntos
Apoptose/efeitos dos fármacos , Oligopeptídeos/farmacologia , Transdução de Sinais/efeitos dos fármacos , Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Sequência de Aminoácidos , Animais , Antineoplásicos Fitogênicos/farmacologia , Western Blotting , Linhagem Celular , Células Cultivadas , Neoplasias do Colo/tratamento farmacológico , Neoplasias do Colo/metabolismo , Neoplasias do Colo/patologia , Sinergismo Farmacológico , Feminino , Células HCT116 , Humanos , Camundongos , Camundongos Nus , Dados de Sequência Molecular , Oligopeptídeos/química , Oligopeptídeos/metabolismo , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/química , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Resveratrol , Estilbenos/farmacologia , Ressonância de Plasmônio de Superfície , Ligante Indutor de Apoptose Relacionado a TNF/química , Ligante Indutor de Apoptose Relacionado a TNF/farmacologia , Carga Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Current pharmacologic treatments for inflammatory diseases are largely palliative rather than curative. Most of them result in nonspecific immunosuppression. This can be associated with disruption of natural and induced immunity with significant, sometimes dramatic, adverse effects. Among the novel strategies that are under development, tools that target specific molecular pathways and cells, and more precisely modulate the immune system to restore normal tolerance mechanisms are central. In these approaches, peptide therapeutics constitute a valuable class of therapeutic agents. They possess a number of intrinsic properties that are favorable for long-term treatments. They are also versatile components that can be modified to improve their capacities without affecting their bioactivity. Peptide-mediated immunotherapy has been evaluated in several appropriate experimental animal models. A few peptides are currently evaluated in clinical trials for the treatment of human chronic inflammatory diseases. In this review we describe a number of these emerging peptide therapeutics. We also discuss future challenges that, in addition to include selection of appropriate peptide drugs, also involve the optimization of peptide dosage and route of administration as well as the improvement of peptide stability for adequate bioavailability and specific targeting.
Assuntos
Doenças Autoimunes/tratamento farmacológico , Sistemas de Liberação de Medicamentos/métodos , Drogas em Investigação/uso terapêutico , Imunossupressores/uso terapêutico , Inflamação/tratamento farmacológico , Peptídeos/administração & dosagem , Animais , Doenças Autoimunes/imunologia , Descoberta de Drogas/métodos , Humanos , Inflamação/imunologia , Peptídeos/farmacologiaAssuntos
Anti-Infecciosos/química , Anti-Infecciosos/farmacologia , Peptídeos/química , Peptídeos/farmacologia , Amidas/síntese química , Amidas/química , Candida/efeitos dos fármacos , Membrana Celular/química , Membrana Celular/efeitos dos fármacos , Desenho de Fármacos , Escherichia coli/efeitos dos fármacos , Testes de Sensibilidade Microbiana , Microscopia Imunoeletrônica , Conformação Molecular , Mimetismo Molecular , Dobramento de Proteína , Estrutura Secundária de Proteína , Pseudomonas aeruginosa/efeitos dos fármacos , Staphylococcus aureus/efeitos dos fármacos , Relação Estrutura-Atividade , Ureia/síntese química , Ureia/químicaRESUMO
OBJECTIVE: Nucleosomes have been identified as a key autoantigen in systemic lupus erythematosus (SLE). Nucleosomes are present in the circulation due to a disturbed apoptosis and/or an insufficient clearance in SLE. During apoptosis, histones can be modified, thereby making them more immunogenic. Recently, we showed the importance of apoptosis-induced acetylation of histone H4 in the pathogenesis of SLE. The lupus-derived antibody LG11-2 was previously shown to react with the N-terminal tail of histone H2B, which contains amino acid residues that can be modified including phosphorylation of serine 14, known to occur during apoptosis. Here, we evaluate whether apoptosis-induced histone modifications on H2B exist that are targeted by LG11-2 or lupus-derived plasmas. METHODS: Immunofluorescence staining and western immunoblot analysis of control, apoptotic and trichostatin A-treated cells/chromatin were performed with monoclonal antibody LG11-2. Reactivity of LG11-2 and plasmas from lupus mice and SLE patients with acetylated and/or phosphorylated H2B peptides was determined in competition ELISA. RESULTS: LG11-2 showed enhanced reactivity with apoptotic and hyperacetylated H2B compared to normal H2B. This enhanced reactivity was due to the acetylation of lysine 12 in H2B. This modification was also recognized by autoantibodies from pre-diseased lupus mice, but to a lesser extent by plasmas of diseased lupus mice and lupus patients. CONCLUSIONS: The apoptosis-induced acetylation on H2BK12 is a target for autoantibodies in SLE. Since the anti-H2BK12ac reactivity was mainly found in pre-diseased lupus mice, this epitope seems important in the early phase of the anti-chromatin autoimmune response with subsequent epitope spreading to unmodified H2B.