RESUMEN
Fighting viral infections is hampered by the scarcity of viral targets and their variability, resulting in development of resistance. Viruses depend on cellular molecules-which are attractive alternative targets-for their life cycle, provided that they are dispensable for normal cell functions. Using the model organism Drosophila melanogaster, we identify the ribosomal protein RACK1 as a cellular factor required for infection by internal ribosome entry site (IRES)-containing viruses. We further show that RACK1 is an essential determinant for hepatitis C virus translation and infection, indicating that its function is conserved for distantly related human and fly viruses. Inhibition of RACK1 does not affect Drosophila or human cell viability and proliferation, and RACK1-silenced adult flies are viable, indicating that this protein is not essential for general translation. Our findings demonstrate a specific function for RACK1 in selective mRNA translation and uncover a target for the development of broad antiviral intervention.
Asunto(s)
Dicistroviridae/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/virología , Proteínas de Unión al GTP/metabolismo , Hepatocitos/virología , Virus de Insectos/metabolismo , Proteínas de Neoplasias/metabolismo , Receptores de Superficie Celular/metabolismo , Receptores Citoplasmáticos y Nucleares/metabolismo , Animales , Línea Celular Tumoral , Drosophila melanogaster/metabolismo , Hepacivirus/metabolismo , Hepatocitos/metabolismo , Humanos , Modelos Moleculares , Factores de Iniciación de Péptidos/metabolismo , Biosíntesis de Proteínas , Receptores de Cinasa C Activada , Secuencias Reguladoras de Ácido Ribonucleico , Replicación ViralRESUMEN
The metaproteomic approach is an attractive way to describe a microbiome at the functional level, allowing the identification and quantification of proteins across a broad dynamic range as well as the detection of post-translational modifications. However, it remains relatively underutilized, mainly due to technical challenges that should be addressed, including the complexity of extracting proteins from heterogeneous microbial communities. Here, we show that a ChipFilter microfluidic device coupled to a liquid chromatography tandem mass spectrometry (LC-MS/MS) setup can be successfully used for the identification of microbial proteins. Using cultures of Escherichia coli, Bacillus subtilis, and Saccharomyces cerevisiae, we have shown that it is possible to directly lyse the cells and digest the proteins in the ChipFilter to allow the identification of a higher number of proteins and peptides than that by standard protocols, even at low cell density. The peptides produced are overall longer after ChipFilter digestion but show no change in their degree of hydrophobicity. Analysis of a more complex mixture of 17 species from the gut microbiome showed that the ChipFilter preparation was able to identify and estimate the amounts of 16 of these species. These results show that ChipFilter can be used for the proteomic study of microbiomes, particularly in the case of a low volume or cell density. The mass spectrometry data have been deposited on the ProteomeXchange Consortium via the PRIDE partner repository with the data set identifier PXD039581.
Asunto(s)
Consorcios Microbianos , Microfluídica , Cromatografía Liquida , Proteómica , Espectrometría de Masas en Tándem , Escherichia coli/genética , Saccharomyces cerevisiae/genética , PéptidosRESUMEN
Endogenous viral elements derived from nonretroviral RNA viruses have been described in various animal genomes. Whether they have a biological function, such as host immune protection against related viruses, is a field of intense study. Here, we investigated the repertoire of endogenous flaviviral elements (EFVEs) in Aedes mosquitoes, the vectors of arboviruses such as dengue and chikungunya viruses. Previous studies identified three EFVEs from Aedes albopictus cell lines and one from Aedes aegypti cell lines. However, an in-depth characterization of EFVEs in wild-type mosquito populations and individual mosquitoes in vivo has not been performed. We detected the full-length DNA sequence of the previously described EFVEs and their respective transcripts in several A. albopictus and A. aegypti populations from geographically distinct areas. However, EFVE-derived proteins were not detected by mass spectrometry. Using deep sequencing, we detected the production of PIWI-interacting RNA-like small RNAs, in an antisense orientation, targeting the EFVEs and their flanking regions in vivo The EFVEs were integrated in repetitive regions of the mosquito genomes, and their flanking sequences varied among mosquito populations. We bioinformatically predicted several new EFVEs from a Vietnamese A. albopictus population and observed variation in the occurrence of those elements among mosquitoes. Phylogenetic analysis of an A. aegypti EFVE suggested that it integrated prior to the global expansion of the species and subsequently diverged among and within populations. The findings of this study together reveal the substantial structural and nucleotide diversity of flaviviral integrations in Aedes genomes. Unraveling this diversity will help to elucidate the potential biological function of these EFVEs.IMPORTANCE Endogenous viral elements (EVEs) are whole or partial viral sequences integrated in host genomes. Interestingly, some EVEs have important functions for host fitness and antiviral defense. Because mosquitoes also have EVEs in their genomes, characterizing these EVEs is a prerequisite for their potential use to manipulate the mosquito antiviral response. In the study described here, we focused on EVEs related to the Flavivirus genus, to which dengue and Zika viruses belong, in individual Aedes mosquitoes from geographically distinct areas. We show the existence in vivo of flaviviral EVEs previously identified in mosquito cell lines, and we detected new ones. We show that EVEs have evolved differently in each mosquito population. They produce transcripts and small RNAs but not proteins, suggesting a function at the RNA level. Our study uncovers the diverse repertoire of flaviviral EVEs in Aedes mosquito populations and contributes to an understanding of their role in the host antiviral system.
Asunto(s)
Aedes/genética , Aedes/virología , ADN Viral/análisis , Flavivirus/genética , Genoma de los Insectos , ARN Viral/análisis , Animales , Biología Computacional , ADN Viral/genética , Evolución Molecular , Secuenciación de Nucleótidos de Alto Rendimiento , Espectrometría de Masas , ARN Viral/genética , Recombinación Genética , Proteínas Virales/análisis , Integración ViralRESUMEN
The Drosophila defense against pathogens largely relies on the activation of two signaling pathways: immune deficiency (IMD) and Toll. The IMD pathway is triggered mainly by Gram-negative bacteria, whereas the Toll pathway responds predominantly to Gram-positive bacteria and fungi. The activation of these pathways leads to the rapid induction of numerous NF-κB-induced immune response genes, including antimicrobial peptide genes. The IMD pathway shows significant similarities with the TNF receptor pathway. Recent evidence indicates that the IMD pathway is also activated in response to various noninfectious stimuli (i.e., inflammatory-like reactions). To gain a better understanding of the molecular machinery underlying the pleiotropic functions of this pathway, we first performed a comprehensive proteomics analysis to identify the proteins interacting with the 11 canonical members of the pathway initially identified by genetic studies. We identified 369 interacting proteins (corresponding to 291 genes) in heat-killed Escherichia coli-stimulated Drosophila S2 cells, 92% of which have human orthologs. A comparative analysis of gene ontology from fly or human gene annotation databases points to four significant common categories: (i) the NuA4, nucleosome acetyltransferase of H4, histone acetyltransferase complex, (ii) the switching defective/sucrose nonfermenting-type chromatin remodeling complex, (iii) transcription coactivator activity, and (iv) translation factor activity. Here we demonstrate that sumoylation of the IκB kinase homolog immune response-deficient 5 plays an important role in the induction of antimicrobial peptide genes through a highly conserved sumoylation consensus site during bacterial challenge. Taken together, the proteomics data presented here provide a unique avenue for a comparative functional analysis of proteins involved in innate immune reactions in flies and mammals.
Asunto(s)
Proteínas de Drosophila/inmunología , Drosophila/inmunología , Drosophila/microbiología , Transducción de Señal/inmunología , Secuencia de Aminoácidos , Animales , Animales Modificados Genéticamente , Ensamble y Desensamble de Cromatina/genética , Ensamble y Desensamble de Cromatina/inmunología , Drosophila/genética , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Escherichia coli/inmunología , Genes de Insecto , Histona Acetiltransferasas/genética , Histona Acetiltransferasas/inmunología , Histona Acetiltransferasas/metabolismo , Interacciones Huésped-Patógeno/genética , Interacciones Huésped-Patógeno/inmunología , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Mapas de Interacción de Proteínas , Homología de Secuencia de AminoácidoRESUMEN
Coxsackievirus B (CVB) infection of pancreatic ß cells is associated with ß cell autoimmunity and type 1 diabetes. We investigated how CVB affects human ß cells and anti-CVB T cell responses. ß cells were efficiently infected by CVB in vitro, down-regulated human leukocyte antigen (HLA) class I, and presented few, selected HLA-bound viral peptides. Circulating CD8+ T cells from CVB-seropositive individuals recognized a fraction of these peptides; only another subfraction was targeted by effector/memory T cells that expressed exhaustion marker PD-1. T cells recognizing a CVB epitope cross-reacted with ß cell antigen GAD. Infected ß cells, which formed filopodia to propagate infection, were more efficiently killed by CVB than by CVB-reactive T cells. Our in vitro and ex vivo data highlight limited CD8+ T cell responses to CVB, supporting the rationale for CVB vaccination trials for type 1 diabetes prevention. CD8+ T cells recognizing structural and nonstructural CVB epitopes provide biomarkers to differentially follow response to infection and vaccination.
Asunto(s)
Infecciones por Coxsackievirus , Diabetes Mellitus Tipo 1 , Células Secretoras de Insulina , Humanos , Linfocitos T CD8-positivos , Anticuerpos , Epítopos , Péptidos , AntiviralesRESUMEN
Coxsackievirus B (CVB) infection of pancreatic ß cells is associated with ß-cell autoimmunity. We investigated how CVB impacts human ß cells and anti-CVB T-cell responses. ß cells were efficiently infected by CVB in vitro, downregulated HLA Class I and presented few, selected HLA-bound viral peptides. Circulating CD8+ T cells from CVB-seropositive individuals recognized only a fraction of these peptides, and only another sub-fraction was targeted by effector/memory T cells that expressed the exhaustion marker PD-1. T cells recognizing a CVB epitope cross-reacted with the ß-cell antigen GAD. Infected ß cells, which formed filopodia to propagate infection, were more efficiently killed by CVB than by CVB-reactive T cells. Thus, our in-vitro and ex-vivo data highlight limited T-cell responses to CVB, supporting the rationale for CVB vaccination trials for type 1 diabetes prevention. CD8+ T cells recognizing structural and non-structural CVB epitopes provide biomarkers to differentially follow response to infection and vaccination.
RESUMEN
In type 1 diabetes, autoimmune ß-cell destruction may be favored by neoantigens harboring posttranslational modifications (PTMs) such as citrullination. We studied the recognition of native and citrullinated glucose-regulated protein (GRP)78 peptides by CD8+ T cells. Citrullination modulated T-cell recognition and, to a lesser extent, HLA-A2 binding. GRP78-reactive CD8+ T cells circulated at similar frequencies in healthy donors and donors with type 1 diabetes and preferentially recognized either native or citrullinated versions, without cross-reactivity. Rather, the preference for native GRP78 epitopes was associated with CD8+ T cells cross-reactive with bacterial mimotopes. In the pancreas, a dominant GRP78 peptide was instead preferentially recognized when citrullinated. To further clarify these recognition patterns, we considered the possibility of citrullination in the thymus. Citrullinating peptidylarginine deiminase (Padi) enzymes were expressed in murine and human medullary epithelial cells (mTECs), with citrullinated proteins detected in murine mTECs. However, Padi2 and Padi4 expression was diminished in mature mTECs from NOD mice versus C57BL/6 mice. We conclude that, on one hand, the CD8+ T cell preference for native GRP78 peptides may be shaped by cross-reactivity with bacterial mimotopes. On the other hand, PTMs may not invariably favor loss of tolerance because thymic citrullination, although impaired in NOD mice, may drive deletion of citrulline-reactive T cells.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Citrulinación/fisiología , Diabetes Mellitus Tipo 1/inmunología , Chaperón BiP del Retículo Endoplásmico/inmunología , Epítopos de Linfocito T/metabolismo , Adolescente , Adulto , Animales , Niño , Citrulinación/inmunología , Diabetes Mellitus Tipo 1/metabolismo , Chaperón BiP del Retículo Endoplásmico/química , Chaperón BiP del Retículo Endoplásmico/metabolismo , Epítopos de Linfocito T/química , Femenino , Humanos , Activación de Linfocitos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos NOD , Persona de Mediana Edad , Procesamiento Proteico-Postraduccional/inmunología , Procesamiento Proteico-Postraduccional/fisiología , Adulto JovenRESUMEN
The antigenic peptides processed by ß-cells and presented through surface HLA class I molecules are poorly characterized. Each HLA variant (e.g., the most common being HLA-A2 and HLA-A3) carries some peptide-binding specificity. Hence, features that, despite these specificities, remain shared across variants may reveal factors favoring ß-cell immunogenicity. Building on our previous description of the HLA-A2/A3 peptidome of ß-cells, we analyzed the HLA-A3-restricted peptides targeted by circulating CD8+ T cells. Several peptides were recognized by CD8+ T cells within a narrow frequency (1-50/106), which was similar in donors with and without type 1 diabetes and harbored variable effector/memory fractions. These epitopes could be classified as conventional peptides or neoepitopes, generated either via peptide cis-splicing or mRNA splicing (e.g., secretogranin-5 [SCG5]-009). As reported for HLA-A2-restricted peptides, several epitopes originated from ß-cell granule proteins (e.g., SCG3, SCG5, and urocortin-3). Similarly, H-2Kd-restricted CD8+ T cells recognizing the murine orthologs of SCG5, urocortin-3, and proconvertase-2 infiltrated the islets of NOD mice and transferred diabetes into NOD/scid recipients. The finding of granule proteins targeted in both humans and NOD mice supports their disease relevance and identifies the insulin granule as a rich source of epitopes, possibly reflecting its impaired processing in type 1 diabetes.
Asunto(s)
Cromograninas/metabolismo , Diabetes Mellitus Tipo 1/metabolismo , Adulto , Empalme Alternativo , Animales , Linfocitos T CD8-positivos , Estudios de Casos y Controles , Cromograninas/genética , Simulación por Computador , Minería de Datos , Diabetes Mellitus Tipo 1/genética , Epítopos , Femenino , Regulación de la Expresión Génica , Antígeno HLA-A3 , Humanos , Insulina , Masculino , Ratones , Ratones Endogámicos NOD , Proteína 7B2 Secretora Neuroendocrina/genética , Proteína 7B2 Secretora Neuroendocrina/metabolismo , Unión Proteica , ARN Mensajero/genética , Urocortinas/genética , Urocortinas/metabolismo , Adulto JovenRESUMEN
Although CD8+ T-cell-mediated autoimmune ß cell destruction occurs in type 1 diabetes (T1D), the target epitopes processed and presented by ß cells are unknown. To identify them, we combined peptidomics and transcriptomics strategies. Inflammatory cytokines increased peptide presentation in vitro, paralleling upregulation of human leukocyte antigen (HLA) class I expression. Peptide sources featured several insulin granule proteins and all known ß cell antigens, barring islet-specific glucose-6-phosphatase catalytic subunit-related protein. Preproinsulin yielded HLA-A2-restricted epitopes previously described. Secretogranin V and its mRNA splice isoform SCG5-009, proconvertase-2, urocortin-3, the insulin gene enhancer protein ISL-1, and an islet amyloid polypeptide transpeptidation product emerged as antigens processed into HLA-A2-restricted epitopes, which, as those already described, were recognized by circulating naive CD8+ T cells in T1D and healthy donors and by pancreas-infiltrating cells in T1D donors. This peptidome opens new avenues to understand antigen processing by ß cells and for the development of T cell biomarkers and tolerogenic vaccination strategies.
Asunto(s)
Presentación de Antígeno , Linfocitos T CD8-positivos/inmunología , Diabetes Mellitus Tipo 1/inmunología , Epítopos de Linfocito T/inmunología , Transcriptoma/inmunología , Animales , Biomarcadores/metabolismo , Linfocitos T CD8-positivos/citología , Linfocitos T CD8-positivos/metabolismo , Estudios de Casos y Controles , Línea Celular , Hormona Liberadora de Corticotropina/metabolismo , Citocinas/metabolismo , Antígenos HLA/metabolismo , Humanos , Insulina/metabolismo , Polipéptido Amiloide de los Islotes Pancreáticos/metabolismo , Ratones , Proteína 7B2 Secretora Neuroendocrina/metabolismo , Proproteína Convertasa 2/metabolismo , Precursores de Proteínas/metabolismo , Proteómica/métodos , Urocortinas/metabolismoRESUMEN
Identification of fox (Vulpes vulpes) sperm antigens was carried out to assess their interest as a potential target for a contraceptive vaccine. We report here the cloning and sequencing of fSP8, a fox sperm protein of 14.7 kd. fSP8 was isoantigenic in foxes, as it was recognized by sera of both male and female foxes immunized with fox sperm proteins. No glycosylation was detected, on fSP8, as shown both by deglycosylation assay and lectin labeling. To determine the fSP8 sequence, the NH2-terminal sequence was first obtained, and a piece of cDNA was amplified from testicular RNA by Rapid Amplification of cDNA extremities polymerase chain reaction. This piece was used to screen a cDNA library from fox testis by Southern blot. A sequence of 879 base pairs was obtained, which includes a major open reading frame coding for 128 amino acids. Mass spectrometric analyses have confirmed the position of the open reading frame. Analysis of the predicted amino acids sequence revealed no apparent transmembrane regions. Comparison of the protein sequence with the Prosite database demonstrated a homology with the Zinc binding site of the subunit Vb of the cytochrome c oxidase. On the C-terminal extremity, fSP8 presents a high homology to the Vb polypeptide of the cytochrome c oxidase from bovine, mouse, and human; however the 34 amino acids on the NH2-extremity were specific to fSP8. Moreover, it was demonstrated that this sequence was testis-specific. This could contribute to the antigenicity of this protein. fSP8 is one of the first fox sperm antigens to be cloned and sequenced.
Asunto(s)
ADN Complementario/genética , Complejo IV de Transporte de Electrones/genética , Zorros/genética , Espermatozoides/química , Testículo/química , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , Complejo IV de Transporte de Electrones/inmunología , Femenino , Biblioteca de Genes , Masculino , Datos de Secuencia Molecular , Especificidad de Órganos , Subunidades de Proteína/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Espermatozoides/inmunología , Testículo/inmunología , Vacunas Anticonceptivas/inmunologíaRESUMEN
Nonenzymatic deamidation of asparaginyl residues can occur spontaneously under physiological conditions principally when a glycyl residue is at the carboxyl side of Asn and leads to formation of aspartyl and isoaspartyl residues. This modification can change the biological activity of proteins or peptides and trigger an auto-immune response. The α-lactalbumins of members of the Camelidae family are the only of described α-lactalbumins that carry two AsnGly sequences. In the present study, high-resolution mass spectrometry, which enables accurate mass measurement has shown that Asn(16) and Asn(45) underwent a nonenzymatic deamidation, the sequence Asn(45)-Gly(46) being deamidated spontaneously at near-neutral and basic pH and Asn(16)-Gly(17) rather at basic pH. The 16-17 sequence was probably stabilized at near-neutral pH by hydrogen bonds according to the molecular modelisation performed with the camel protein.
Asunto(s)
Lactalbúmina/análisis , Leche/química , Secuencia de Aminoácidos , Animales , Asparagina/química , Camelus , Electroforesis en Gel Bidimensional , Concentración de Iones de Hidrógeno , Espectrometría de Masas , Modelos Moleculares , Datos de Secuencia Molecular , Péptidos/química , Conformación ProteicaRESUMEN
Tau is a central player in Alzheimer's disease (AD) and related Tauopathies, where it is found as aggregates in degenerating neurons. Abnormal post-translational modifications, such as truncation, are likely involved in the pathological process. A major step forward in understanding the role of Tau truncation would be to identify the precise cleavage sites of the several truncated Tau fragments that are observed until now in AD brains, especially those truncated at the N-terminus, which are less characterized than those truncated at the C-terminus. Here, we optimized a proteomics approach and succeeded in identifying a number of new N-terminally truncated Tau species from the human brain. We initiated cell-based functional studies by analyzing the biochemical characteristics of two N-terminally truncated Tau species starting at residues Met11 and Gln124 respectively. Our results show, interestingly, that the Gln124-Tau fragment displays a stronger ability to bind and stabilize microtubules, suggesting that the Tau N-terminal domain could play a direct role in the regulation of microtubule stabilization. Future studies based on our new N-terminally truncated-Tau species should improve our knowledge of the role of truncation in Tau biology as well as in the AD pathological process.
Asunto(s)
Enfermedad de Alzheimer/patología , Microtúbulos/fisiología , Tubulina (Proteína)/metabolismo , Proteínas tau/genética , Acetilación , Enfermedad de Alzheimer/genética , Encéfalo/patología , Línea Celular , Humanos , Degeneración Nerviosa/metabolismo , Fosforilación , Unión Proteica/genética , Procesamiento Proteico-Postraduccional , Proteómica , Proteínas tau/metabolismoRESUMEN
The binding of amyloid beta peptides (Abeta) to plasma membranes appears to be a promising point of intervention in the events leading to the development of Alzheimer's disease (AD). This binding has been studied as regards the direct toxicity of Abeta on neurons, and the activation of a local inflammation phase involving microglia. By virtue of its structure, Abeta is able to bind to a variety of biomolecules, including lipids, proteoglycans and proteins. This review focuses on the membrane proteins that can mediate the interaction between Abeta and the plasma membranes in AD. On neurons, these are APP (amyloid precursor protein), the NMDA-R (N-methyl-D-aspartate receptor), integrins, the alpha7nicotinic acetylcholine receptor (alpha7nAChR), the P75 neurotrophin receptor (P75NTR) and the CLAC-P/collagen type XXV (collagen-like Alzheimer amyloid plaque component precursor/collagen XXV). On glial cells, FPRL1 (formyl peptide receptor-like 1), the scavenger receptors A, BI (SR-A, SR-BI) and CD36, a complex involving CD36, alpha(6)beta(1)-integrin and CD47, and heparan sulfate proteoglycans have been reported to bind Abeta. It should be noted that integrins, RAGE (receptor for advanced glycosylation end-products), the Serpin-enzyme complex receptor (SEC-R) and the insulin receptor can bind Abeta and are present on neurons and on glial cells. After a presentation of the structure and the function of each of these proteins, the method used to prove their binding to Abeta is described, and the implication of this binding in AD is discussed. Finally, it is underlined that multireceptor complexes containing integrins may be involved in this interaction.
Asunto(s)
Enfermedad de Alzheimer/etiología , Péptidos beta-Amiloides/metabolismo , Membrana Celular/metabolismo , Fragmentos de Péptidos/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Antígenos CD36 , Proteoglicanos de Heparán Sulfato/metabolismo , Humanos , Integrinas/metabolismo , Datos de Secuencia Molecular , Neuroglía/metabolismo , Neuronas/metabolismo , Receptor para Productos Finales de Glicación Avanzada , Receptor de Insulina/metabolismo , Receptores de Superficie Celular/metabolismo , Receptores de Formil Péptido/metabolismo , Receptores Inmunológicos/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Receptores Depuradores , Receptores Depuradores de Clase A , Regulación hacia ArribaRESUMEN
The aim of this work was to identify antigenic surface proteins on fox spermatozoa. Six foxes were inguitinally vasectomised, and the time course of antibody response in the sera was studied. Five out of the six foxes reacted to vasectomy with a production of antisperm antibodies. The number of bands recognised by Western blot was maximal 120-150 days after the vasectomy, at the end of the reproductive season. On the whole, 30 bands were recognised between 9 and 150 kDa. The pattern of recognised proteins varied from one fox to another. The humoral response was studied in one fox 2 years after the vasectomy, before, during and after the breeding season. The same proteins were recognised, but the intensity of staining was increased during the testis regression. Using FITC-labelling on sperm smears with fox sera, antigens were localised at or near the sperm surface, either on the acrosome or both on the acrosome and on the flagellum. Using two-dimensional gel electrophoresis and Western blotting, we identified eight areas containing major antigens, recognised by at least two sera. The molecular weights (kDa) and isoelectric points of the repeated antigens were, respectively [150, 6.6-6.0]; [105-98, 6.0-5.5]; [97, 4.6-4.3]; [95, 5.0]; [85-80, 5.4-5.1]; [42, 5.0-4.8]; [17-15, 6.5-5.9]; and [17-15, 5.5-4.8]. The results of this study can be used to characterise more precisely fox sperm auto-antigens by microsequencing the selected proteins.
Asunto(s)
Autoantígenos/análisis , Espermatozoides/inmunología , Vasectomía , Animales , Autoantígenos/fisiología , Western Blotting , Electroforesis en Gel Bidimensional , Zorros , MasculinoRESUMEN
The aim of this work was to identify antigenic proteins on fox spermatozoa. Fox spermatozoa proteins were injected into 3 female rabbits and into 3 male and 3 female foxes. In rabbits, a rapid humoral response was observed. Using rabbit sera for Western blotting, 23 fox sperm protein bands were recognized between 10 and 110 kd. In foxes, the time course of antibody response was studied in the same manner. The number of recognized bands was maximal on day 75 for 2 foxes, on day 90 for 3 foxes, and on day 120 for 1 fox. Western blot patterns varied from one fox to another. On the whole, 25 protein bands between 10 and 110 kd were recognized. Using fluorescein isothiocyanate (FITC) labeling on fox spermatozoa with rabbit and fox sera, we showed that several antigens recognized by the antisera were located at or near the surface of the spermatozoa. By two-dimensional electrophoresis and gel-purification, we have selected 6 highly antigenic proteins with molecular weights of 11.4, 14.7, 16.4, 16.4, 16.8, and 16.9 kd, and isoelectric points of 6.0, 6.0, 6.2, 5.5, 5.3, and 5.8, respectively, and one antigenic protein at 97 kd with an isoelectric point of 4.3 to 4.6. The results of this study can be used to characterize these 7 antigens selected more precisely by microsequencing or mass spectrometry.
Asunto(s)
Zorros , Proteínas de Plasma Seminal/análisis , Espermatozoides/química , Animales , Formación de Anticuerpos , Antígenos de Superficie/análisis , Antígenos de Superficie/inmunología , Electroforesis en Gel Bidimensional , Zorros/inmunología , Masculino , Espectrometría de Masas , Proteínas de Plasma Seminal/inmunologíaRESUMEN
A prerequisite to dephosphorylation at Ser-Pro or Thr-Pro motifs is the isomerization of the imidic peptide bond preceding the proline. The peptidyl-prolyl cis/trans isomerase named Pin1 catalyzes this mechanism. Through isomerization, Pin1 regulates the function of a growing number of targets including the microtubule-associated tau protein and is supposed to be deregulated Alzheimer's disease (AD). Using proteomics, we showed that Pin1 is posttranslationally modified on more than 5 residues, comprising phosphorylation, N-acetylation, and oxidation. Although Pin1 expression remained constant, Pin1 posttranslational two-dimensional pattern was modified by tau overexpression in a tau-inducible neuroblastoma cell line, in our THY-Tau22 mouse model of tauopathy as well as in AD. Interestingly, in all of these systems, Pin1 modifications were very similar. In AD brain tissue when compared with control, Pin1 is hyperphosphorylated at serine 16 and found in the most insoluble hyperphosphorylated tau fraction of AD brain tissue. Furthermore, in all tau pathology conditions, acetylation of Pin1 may also contribute to the differences observed. In conclusion, Pin1 displays several posttranslational modifications, which are specific in tauopathies and may be useful as biomarker.
Asunto(s)
Encéfalo/metabolismo , Isomerasa de Peptidilprolil/metabolismo , Procesamiento Proteico-Postraduccional/fisiología , Tauopatías/metabolismo , Proteínas tau/metabolismo , Acetilación , Adulto , Anciano , Anciano de 80 o más Años , Enfermedad de Alzheimer/metabolismo , Animales , Biomarcadores/metabolismo , Línea Celular Tumoral , Modelos Animales de Enfermedad , Femenino , Humanos , Masculino , Ratones , Ratones Transgénicos , Persona de Mediana Edad , Peptidilprolil Isomerasa de Interacción con NIMA , Oxidación-Reducción , Fosforilación/fisiología , Prolina/metabolismo , Proteoma , Serina/metabolismoRESUMEN
This study aims to characterize the immune response against bacteria in Drosophila melanogaster. Obtaining a description of the in vivo state of protein complexes requires their isolation as a snapshot of physiological conditions before their identification. Affinity purification with streptavidin-biotin system is widely used to address this issue. However, because of the extraordinary stability of the interaction between streptavidin and biotin, the release of biotin-labeled bait remains a challenge. We transfected Drosophila cells with a DNA construct encoding a biotin-tagged Dredd protein (ortholog of caspase 8). After affinity purification, different strategies were evaluated, and proteins analyzed by LC-MS/MS mass spectrometry. The on-bead digestion allowed the identification of more proteins associated to the Dredd complex than different protocols using competitive or acid elution. A functional assay showed that a large part of the proteins specifically identified in the Dredd sample are functionally involved in the activation of the Imd pathway. These proteins are immune response proteins (BG4, Q9VP57), stress response proteins (HSP7C, Q9VXQ5), structural proteins (TBB1, CP190), a protein biosynthesis protein (Q9W1B9) and an antioxidant system protein (SODC). Our results clearly show that on-bead digestion of proteins is an attractive procedure for the study of protein complexes by mass spectrometry. This article is part of a Special Issue entitled: Translational Proteomics.
Asunto(s)
Bacterias , Infecciones Bacterianas/metabolismo , Proteínas de Drosophila/metabolismo , Espectrometría de Masas/métodos , Proteolisis , Proteoma/metabolismo , Proteómica/métodos , Animales , Infecciones Bacterianas/inmunología , Línea Celular , Proteínas de Drosophila/inmunología , Drosophila melanogaster , Proteoma/inmunologíaRESUMEN
Synthetic peptides are widely used in immunological research as epitopes to stimulate their cognate T cells. These preparations are never completely pure, but trace contaminants are commonly revealed by mass spectrometry quality controls. In an effort to characterize novel major histocompatibility complex (MHC) Class I-restricted ß-cell epitopes in non-obese diabetic (NOD) mice, we identified islet-infiltrating CD8+ T cells recognizing a contaminating peptide. The amount of this contaminant was so small to be undetectable by direct mass spectrometry. Only after concentration by liquid chromatography, we observed a mass peak corresponding to an immunodominant islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP)(206-214) epitope described in the literature. Generation of CD8+ T-cell clones recognizing IGRP(206-214) using a novel method confirmed the identity of the contaminant, further underlining the immunodominance of IGRP(206-214). If left undetected, minute impurities in synthetic peptide preparations may thus give spurious results.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Espectrometría de Masas/métodos , Péptidos/inmunología , Secuencia de Aminoácidos , Animales , Células Clonales , Epítopos de Linfocito T/inmunología , Glucosa-6-Fosfatasa/inmunología , Antígenos de Histocompatibilidad Clase I/inmunología , Humanos , Interferón gamma/inmunología , Islotes Pancreáticos/inmunología , Ratones , Ratones Endogámicos NOD , Datos de Secuencia Molecular , Péptidos/química , Proteínas/inmunología , Reproducibilidad de los Resultados , Análisis de Secuencia de ProteínaRESUMEN
Mitochondrial dysfunction and brain metabolic alteration are early neurofunctional aspects in Alzheimer's disease (AD). Regional brain metabolism was analyzed by cytochrome c oxidase (COX) histochemistry in PS1-A246E mouse mutants, a model of autosomal dominant AD overexpressing beta-amyloid (Abeta) peptide without amyloidosis or cell degeneration. Immunohistochemical samples were analyzed on adjacent sections for regional Abeta1-42 levels, as well as DNA oxidative damage with 8-hydroxy-2-deoxyguanosine (8-OHdG). COX activity increased in the basal forebrain nuclear complex, specific parts of the amygdala and hippocampus, as well as in striatum and connected regions. On the contrary, a hypometabolism was observed in midline thalamic, interpeduncular, and pedonculopontine nuclei. The integration of these regions in circuitries subserving emotions, arousal, and cognitive functions may explain why neurochemical alterations in specific brain regions were linearly correlated with psychomotor slowing and disinhibition previously reported in the mutant. As the PS1-A246E model appears to mimick prodromal AD, the results support the existence of mitochondrial abnormalities prior to AD-related cognitive deficits. However, since affected PS1-A246E brain regions were not primarily those altered in AD-associated histopathological features and did not systematically display either Abeta overexpression or higher 8-OHdG immunolabelling, the hypermetabolism observed seems to comprise a compensatory reaction to early mitochondrial abnormalities; furthermore, neuronal synaptic function should be considered as particularly relevant in COX activity changes.
Asunto(s)
Enfermedad de Alzheimer/metabolismo , Conducta Animal/fisiología , Encéfalo/metabolismo , Complejo IV de Transporte de Electrones/metabolismo , Metabolismo Energético/genética , Estrés Oxidativo/genética , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/fisiopatología , Péptidos beta-Amiloides/genética , Péptidos beta-Amiloides/metabolismo , Animales , Metabolismo Basal/genética , Biomarcadores/análisis , Biomarcadores/metabolismo , Encéfalo/anatomía & histología , Encéfalo/fisiopatología , Química Encefálica/genética , Trastornos de los Cromosomas/genética , Trastornos de los Cromosomas/metabolismo , Trastornos de los Cromosomas/fisiopatología , Daño del ADN/genética , Modelos Animales de Enfermedad , Complejo IV de Transporte de Electrones/análisis , Femenino , Genes Dominantes/genética , Histocitoquímica/métodos , Humanos , Masculino , Ratones , Ratones Noqueados , Ratones Transgénicos , Mitocondrias/genética , Mitocondrias/metabolismo , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Presenilina-1/genética , Desempeño Psicomotor/fisiología , Regulación hacia Arriba/genéticaRESUMEN
Advances in the understanding of AD pathogenesis have recently provided strong support for a modified Abeta protein cascade hypothesis, stating that several different Abeta assemblies contribute to the triggering of a complex pathological cascade leading to neurodegeneration. Both in vitro and in vivo, Abeta rapidly forms fibrils (fAbeta), which are able to interact with various molecular partners, including proteins, lipids and proteoglycans. In a previous study aimed to identify some of these molecular partners of fAbeta, we demonstrated that the GAPDH was specifically coprecipitated with fAbeta. The aim of this study was to characterize this interaction. First, it was shown by TEM that synthetic GAPDH directly binds fAbeta 1-42. Then rat synaptosomal proteins were purified and incubated with different forms of Abeta in various conditions, and the presence of GAPDH among the proteins coprecipitated with Abeta was studied by western blotting. Results showed that the interaction between GAPDH and fAbeta 1-42 is nonionic, as is not impaired by increasing salt concentrations. GAPDH is coprecipitated not only by fAbeta, but also by nonfibrillar forms of Abeta 1-42. The 41-42 Abeta sequence seems to be important in the interaction of GAPDH and Abeta, as more GAPDH was coprecipitated with fAbeta 1-42 than with fAbeta 1-40. GAPDH extracted from various subcellular fractions including mitochondria, was shown to interact with fAbeta. Our data demonstrate a direct interaction between Abeta and GAPDH and support the possibility that this interaction has an important pathogenic role in AD.