RESUMEN
The pre-integration steps of the HIV-1 viral cycle are some of the most valuable targets of recent therapeutic innovations. HIV-1 integrase (IN) displays multiple functions, thanks to its considerable conformational flexibility. Recently, such flexible proteins have been characterized by their ability to form biomolecular condensates as a result of Liquid-Liquid-Phase-Separation (LLPS), allowing them to evolve in a restricted microenvironment within cells called membrane-less organelles (MLO). The LLPS context constitutes a more physiological approach to study the integration of molecular mechanisms performed by intasomes (complexes containing viral DNA, IN, and its cellular cofactor LEDGF/p75). We investigated here if such complexes can form LLPS in vitro and if IN enzymatic activities were affected by this LLPS environment. We observed that the LLPS formed by IN-LEDGF/p75 functional complexes modulate the in vitro IN activities. While the 3'-processing of viral DNA ends was drastically reduced inside LLPS, viral DNA strand transfer was strongly enhanced. These two catalytic IN activities appear thus tightly regulated by the environment encountered by intasomes.
Asunto(s)
Integrasa de VIH , VIH-1 , Integración Viral , Integrasa de VIH/metabolismo , Integrasa de VIH/química , Integrasa de VIH/genética , VIH-1/metabolismo , VIH-1/fisiología , Humanos , ADN Viral/metabolismo , ADN Viral/genética , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Péptidos y Proteínas de Señalización Intercelular/químicaRESUMEN
Integrons are adaptive bacterial devices that rearrange promoter-less gene cassettes into variable ordered arrays under stress conditions, thereby sampling combinatorial phenotypic diversity. Chromosomal integrons often carry hundreds of silent gene cassettes, with integrase-mediated recombination leading to rampant DNA excision and integration, posing a potential threat to genome integrity. How this activity is regulated and controlled, particularly through selective pressures, to maintain such large cassette arrays is unknown. Here, we show a key role of promoter-containing toxin-antitoxin (TA) cassettes as systems that kill the cell when the overall cassette excision rate is too high. These results highlight the importance of TA cassettes regulating the cassette recombination dynamics and provide insight into the evolution and success of integrons in bacterial genomes.
Asunto(s)
Integrones , Sistemas Toxina-Antitoxina , Integrones/genética , Sistemas Toxina-Antitoxina/genética , Bacterias/genética , Genoma Bacteriano , Recombinación GenéticaRESUMEN
Integrons are genetic elements involved in bacterial adaptation which capture, shuffle and express genes encoding adaptive functions embedded in cassettes. These events are governed by the integron integrase through site-specific recombination between attC and attI integron sites. Using computational and molecular genetic approaches, here we demonstrate that the integrase also catalyses cassette integration into bacterial genomes outside of its known att sites. Once integrated, these cassettes can be expressed if located near bacterial promoters and can be excised at the integration point or outside, inducing chromosomal modifications in the latter case. Analysis of more than 5 × 105 independent integration events revealed a very large genomic integration landscape. We identified consensus recombination sequences, named attG sites, which differ greatly in sequence and structure from classical att sites. These results unveil an alternative route for dissemination of adaptive functions in bacteria and expand the role of integrons in bacterial evolution.
Asunto(s)
Genoma Bacteriano , Integrones , Integrones/genética , Bacterias/genética , Bacterias/metabolismo , Integrasas/genética , Integrasas/metabolismo , GenómicaRESUMEN
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the infectious agent that has caused the current coronavirus disease (COVID) pandemic. Viral infection relies on the viral S (spike) protein/cellular receptor ACE2 interaction. Disrupting this interaction would lead to early blockage of viral replication. To identify chemical tools to further study these functional interfaces, 139,146 compounds from different chemical libraries were screened through an S/ACE2 in silico virtual molecular model. The best compounds were selected for further characterization using both cellular and biochemical approaches, reiterating SARS-CoV-2 entry and the S/ACE2 interaction. We report here two selected hits, bis-indolyl pyridine AB-00011778 and triphenylamine AB-00047476. Both of these compounds can block the infectivity of lentiviral vectors pseudotyped with the SARS-CoV-2 S protein as well as wild-type and circulating variant SARS-CoV-2 strains in various human cell lines, including pulmonary cells naturally susceptible to infection. AlphaLISA and biolayer interferometry confirmed a direct inhibitory effect of these drugs on the S/ACE2 association. A specific study of the AB-00011778 inhibitory properties showed that this drug inhibits viral replication with a 50% effective concentration (EC50) between 0.1 and 0.5 µM depending on the cell lines. Molecular docking calculations of the interaction parameters of the molecules within the S/ACE2 complex from both wild-type and circulating variants of the virus showed that the molecules may target multiple sites within the S/ACE2 interface. Our work indicates that AB-00011778 constitutes a good tool for modulating this interface and a strong lead compound for further therapeutic purposes.
Asunto(s)
Tratamiento Farmacológico de COVID-19 , SARS-CoV-2 , Enzima Convertidora de Angiotensina 2 , Humanos , Simulación del Acoplamiento Molecular , Peptidil-Dipeptidasa A/química , Peptidil-Dipeptidasa A/metabolismo , Peptidil-Dipeptidasa A/farmacología , Unión Proteica , Piridinas/farmacología , Glicoproteína de la Espiga del Coronavirus/metabolismo , Internalización del VirusRESUMEN
The stable insertion of the retroviral genome into the host chromosomes requires the association between integration complexes and cellular chromatin via the interaction between retroviral integrase and the nucleosomal target DNA. This final association may involve the chromatin-binding properties of both the retroviral integrase and its cellular cofactor LEDGF/p75. To investigate this and better understand the LEDGF/p75-mediated chromatin tethering of HIV-1 integrase, we used a combination of biochemical and chromosome-binding assays. Our study revealed that retroviral integrase has an intrinsic ability to bind and recognize specific chromatin regions in metaphase even in the absence of its cofactor. Furthermore, this integrase chromatin-binding property was modulated by the interaction with its cofactor LEDGF/p75, which redirected the enzyme to alternative chromosome regions. We also better determined the chromatin features recognized by each partner alone or within the functional intasome, as well as the chronology of efficient LEDGF/p75-mediated targeting of HIV-1 integrase to chromatin. Our data support a new chromatin-binding function of integrase acting in concert with LEDGF/p75 for the optimal association with the nucleosomal substrate. This work also provides additional information about the behavior of retroviral integration complexes in metaphase chromatin and the mechanism of action of LEDGF/p75 in this specific context.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/genética , Cromatina/metabolismo , Integrasa de VIH/genética , Histonas/genética , Interacciones Huésped-Patógeno/genética , Factores de Transcripción/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Cromatina/química , Clonación Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Regulación de la Expresión Génica , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Integrasa de VIH/metabolismo , Histonas/metabolismo , Humanos , Células K562 , Cultivo Primario de Células , Unión Proteica , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transducción de Señal , Linfocitos T/metabolismo , Linfocitos T/virología , Factores de Transcripción/metabolismoRESUMEN
Integrons confer a rapid adaptation capability to bacteria. Integron integrases are able to capture and shuffle novel functions embedded in cassettes. Here, we investigated cassette recruitment in the Vibrio cholerae chromosomal integron during horizontal transfer. We demonstrated that the endogenous integrase expression is sufficiently triggered, after SOS response induction mediated by the entry of cassettes during conjugation and natural transformation, to mediate significant cassette insertions. These insertions preferentially occur at the attIA site, despite the presence of about 180 attC sites in the integron array. Thanks to the presence of a promoter in the attIA site vicinity, all these newly inserted cassettes are expressed and prone to selection. We also showed that the RecA protein is critical for cassette recruitment in the V. cholerae chromosomal integron but not in mobile integrons. Moreover, unlike the mobile integron integrases, that of V. cholerae is not active in other bacteria. Mobile integrons might have evolved from the chromosomal ones by overcoming host factors, explaining their large dissemination in bacteria and their role in antibioresistance expansion.
Asunto(s)
Cromosomas/metabolismo , Transferencia de Gen Horizontal/genética , Integrasas/metabolismo , Integrones/genética , Vibrio cholerae/metabolismo , Cromosomas/genética , Escherichia coli/metabolismo , Regulación Bacteriana de la Expresión Génica/genética , Integrasas/genética , Rec A Recombinasas/genética , Rec A Recombinasas/metabolismo , Recombinación Genética/genética , Vibrio cholerae/genéticaRESUMEN
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the etiologic agent responsible for the recent coronavirus disease 2019 (COVID-19) pandemic. Productive SARS-CoV-2 infection relies on viral entry into cells expressing angiotensin-converting enzyme 2 (ACE2). Indeed, viral entry into cells is mostly mediated by the early interaction between the viral spike protein S and its ACE2 receptor. The S/ACE2 complex is, thus, the first contact point between the incoming virus and its cellular target; consequently, it has been considered an attractive therapeutic target. To further characterize this interaction and the cellular processes engaged in the entry step of the virus, we set up various in silico, in vitro and in cellulo approaches that allowed us to specifically monitor the S/ACE2 association. We report here a computational model of the SARS-CoV-2 S/ACE2 complex, as well as its biochemical and biophysical monitoring using pulldown, AlphaLISA and biolayer interferometry (BLI) binding assays. This led us to determine the kinetic parameters of the S/ACE2 association and dissociation steps. In parallel to these in vitro approaches, we developed in cellulo transduction assays using SARS-CoV-2 pseudotyped lentiviral vectors and HEK293T-ACE2 cell lines generated in-house. This allowed us to recapitulate the early replication stage of the infection mediated by the S/ACE2 interaction and to detect cell fusion induced by the interaction. Finally, a cell imaging system was set up to directly monitor the S/ACE2 interaction in a cellular context and a flow cytometry assay was developed to quantify this association at the cell surface. Together, these different approaches are available for both basic and clinical research, aiming to characterize the entry step of the original SARS-CoV-2 strain and its variants as well as to investigate the possible chemical modulation of this interaction. All these models will help in identifying new antiviral agents and new chemical tools for dissecting the virus entry step.
Asunto(s)
Enzima Convertidora de Angiotensina 2/metabolismo , COVID-19/virología , SARS-CoV-2/fisiología , Glicoproteína de la Espiga del Coronavirus/metabolismo , Internalización del Virus , Enzima Convertidora de Angiotensina 2/química , COVID-19/metabolismo , Simulación por Computador , Células HEK293 , Humanos , Técnicas In Vitro , Cinética , Modelos Moleculares , Simulación de Dinámica Molecular , Unión Proteica , Dominios Proteicos , SARS-CoV-2/metabolismo , Glicoproteína de la Espiga del Coronavirus/químicaRESUMEN
Integration of the HIV-1 DNA into the host genome is essential for viral replication and is catalyzed by the retroviral integrase. To date, the only substrate described to be involved in this critical reaction is the linear viral DNA produced in reverse transcription. However, during HIV-1 infection, two-long terminal repeat DNA circles (2-LTRcs) are also generated through the ligation of the viral DNA ends by the host cell's nonhomologous DNA end-joining pathway. These DNAs contain all the genetic information required for viral replication, but their role in HIV-1's life cycle remains unknown. We previously showed that both linear and circular DNA fragments containing the 2-LTR palindrome junction can be efficiently cleaved in vitro by recombinant integrases, leading to the formation of linear 3'-processed-like DNA. In this report, using in vitro experiments with purified proteins and DNAs along with DNA endonuclease and in vivo integration assays, we show that this circularized genome can also be efficiently used as a substrate in HIV-1 integrase-mediated integration both in vitro and in eukaryotic cells. Notably, we demonstrate that the palindrome cleavage occurs via a two-step mechanism leading to a blunt-ended DNA product, followed by a classical 3'-processing reaction; this cleavage leads to integrase-dependent integration, highlighted by a 5-bp duplication of the host genome. Our results suggest that 2-LTRc may constitute a reserve supply of HIV-1 genomes for proviral integration.
Asunto(s)
ADN Circular/química , ADN Viral/química , Integrasa de VIH/química , Duplicado del Terminal Largo de VIH , VIH-1/química , Integración Viral , ADN Circular/genética , ADN Viral/genética , ADN Viral/metabolismo , Integrasa de VIH/genética , Integrasa de VIH/metabolismo , VIH-1/genética , VIH-1/metabolismo , HumanosRESUMEN
The integration of the retroviral genome into the chromatin of the infected cell is catalysed by the integrase (IN)â¢viral DNA complex (intasome). This process requires functional association between the integration complex and the nucleosomes. Direct intasome/histone contacts have been reported to modulate the interaction between the integration complex and the target DNA (tDNA). Both prototype foamy virus (PFV) and HIV-1 integrases can directly bind histone amino-terminal tails. We have further investigated this final association by studying the effect of isolated histone tails on HIV-1 integration. We show here that the binding of HIV-1 IN to a peptide derived from the H4 tail strongly stimulates integration catalysis in vitro. This stimulation was not observed with peptide tails from other variants or with alpha-retroviral (RAV) and spuma-retroviral PFV integrases. Biochemical analyses show that the peptide tail induces both an increase in the IN oligomerization state and affinity for the target DNA, which are associated with substantial structural rearrangements in the IN carboxy-terminal domain (CTD) observed by NMR. Our data indicate that the H4 peptide tail promotes the formation of active strand transfer complexes (STCs) and support an activation step of the incoming intasome at the contact of the histone tail.
Asunto(s)
Integrasa de VIH/genética , VIH-1/genética , Histonas/genética , Integración Viral/genética , Catálisis , Cromatina/genética , Cromatina/virología , Genoma Viral/genética , VIH-1/patogenicidad , Interacciones Huésped-Patógeno/genética , Humanos , Nucleosomas/genética , Nucleosomas/virología , Spumavirus/genéticaRESUMEN
BACKGROUND: Stable insertion of the retroviral DNA genome into host chromatin requires the functional association between the intasome (integrase·viral DNA complex) and the nucleosome. The data from the literature suggest that direct protein-protein contacts between integrase and histones may be involved in anchoring the intasome to the nucleosome. Since histone tails are candidates for interactions with the incoming intasomes we have investigated whether they could participate in modulating the nucleosomal integration process. RESULTS: We show here that histone tails are required for an optimal association between HIV-1 integrase (IN) and the nucleosome for efficient integration. We also demonstrate direct interactions between IN and the amino-terminal tail of human histone H4 in vitro. Structure/function studies enabled us to identify amino acids in the carboxy-terminal domain of IN that are important for this interaction. Analysis of the nucleosome-binding properties of catalytically active mutated INs confirmed that their ability to engage the nucleosome for integration in vitro was affected. Pseudovirus particles bearing mutations that affect the IN/H4 association also showed impaired replication capacity due to altered integration and re-targeting of their insertion sites toward dynamic regions of the chromatin with lower nucleosome occupancy. CONCLUSIONS: Collectively, our data support a functional association between HIV-1 IN and histone tails that promotes anchoring of the intasome to nucleosomes and optimal integration into chromatin.
Asunto(s)
Integrasa de VIH/metabolismo , VIH-1/metabolismo , Histonas/metabolismo , Nucleosomas/metabolismo , Integración Viral , Línea Celular Transformada , Cromatina/virología , ADN Viral/metabolismo , Células HEK293 , VIH-1/genética , Histonas/química , Interacciones Huésped-Parásitos/fisiología , Humanos , Unión ProteicaRESUMEN
BACKGROUND: Insertion of retroviral genome DNA occurs in the chromatin of the host cell. This step is modulated by chromatin structure as nucleosomes compaction was shown to prevent HIV-1 integration and chromatin remodeling has been reported to affect integration efficiency. LEDGF/p75-mediated targeting of the integration complex toward RNA polymerase II (polII) transcribed regions ensures optimal access to dynamic regions that are suitable for integration. Consequently, we have investigated the involvement of polII-associated factors in the regulation of HIV-1 integration. RESULTS: Using a pull down approach coupled with mass spectrometry, we have selected the FACT (FAcilitates Chromatin Transcription) complex as a new potential cofactor of HIV-1 integration. FACT is a histone chaperone complex associated with the polII transcription machinery and recently shown to bind LEDGF/p75. We report here that a tripartite complex can be formed between HIV-1 integrase, LEDGF/p75 and FACT in vitro and in cells. Biochemical analyzes show that FACT-dependent nucleosome disassembly promotes HIV-1 integration into chromatinized templates, and generates highly favored nucleosomal structures in vitro. This effect was found to be amplified by LEDGF/p75. Promotion of this FACT-mediated chromatin remodeling in cells both increases chromatin accessibility and stimulates HIV-1 infectivity and integration. CONCLUSIONS: Altogether, our data indicate that FACT regulates HIV-1 integration by inducing local nucleosomes dissociation that modulates the functional association between the incoming intasome and the targeted nucleosome.
Asunto(s)
Cromatina/metabolismo , Integrasa de VIH/metabolismo , VIH-1/fisiología , Chaperonas de Histonas/metabolismo , Interacciones Huésped-Patógeno , Integración Viral/fisiología , Células Cultivadas , Ensamble y Desensamble de Cromatina/fisiología , Infecciones por VIH/genética , Infecciones por VIH/virología , VIH-1/genética , Humanos , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Nucleosomas/metabolismo , Unión ProteicaRESUMEN
Binding of ligand FasL to its receptor Fas triggers apoptosis via the caspase cascade. FasL itself is homotrimeric, and a productive apoptotic signal requires that FasL be oligomerized beyond the homotrimeric state. We generated a series of FasL chimeras by fusing FasL to domains of the Leukemia Inhibitory Factor receptor gp190 which confer homotypic oligomerization, and analyzed the capacity of these soluble chimeras to trigger cell death. We observed that the most efficient FasL chimera, called pFasL, was also the most polymeric, as it reached the size of a dodecamer. Using a cellular model, we investigated the structure-function relationships of the FasL/Fas interactions for our chimeras, and we demonstrated that the Fas-mediated apoptotic signal did not solely rely on ligand-mediated receptor aggregation, but also required a conformational adaptation of the Fas receptor. When injected into mice, pFasL did not trigger liver injury at a dose which displayed anti-tumor activity in a model of human tumor transplanted to immunodeficient animals, suggesting a potential therapeutic use. Therefore, the optimization of the FasL conformation has to be considered for the development of efficient FasL-derived anti-cancer drugs targeting Fas.
Asunto(s)
Apoptosis/genética , Proteína Ligando Fas/genética , Proteína Ligando Fas/metabolismo , Neoplasias/genética , Receptor fas/genética , Animales , Vectores Genéticos , Humanos , Células Jurkat , Ligandos , Ratones , Neoplasias/metabolismo , Neoplasias/patología , Proteínas de Fusión Oncogénica/genética , Proteínas de Fusión Oncogénica/metabolismo , Receptores OSM-LIF/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , TransfecciónRESUMEN
Esca is one of the major diseases affecting vineyards with direct impact on product yield; nevertheless, scientific studies concerning its impact on grape quality are scarce. As an attempt to better understand the mechanisms behind "Esca proper" development in grapes, this work focused on the identification of proteins whose expression is altered by the disease. 2-DEs were performed on protein extracts from grape skins at different stages of maturity for two consecutive vintages. Grapes were collected in 2009 and in 2010 from plants that did not present signs of infection by Esca proper since the 2004 vintage and from plants that presented cast leaf symptoms at least once since 2004. For the first time, 13 proteins were shown to be influenced by Esca proper during the ripening process. Extensive bioinformatics analysis allowed the grouping of proteins involved in (i) stress tolerance and defense response, (ii) oxidative phosphorylation, (iii) oxidation-reduction processes in mitochondria, and (iv) oxidation-reduction processes in chloroplasts. Of these 13 proteins, cysteine synthase is the only one implicated in a metabolic pathway of oenological interest. This study shows how foliar symptoms of Esca proper may impact stress-related pathways in grapes, which are characterized by modifications in the chain of oxidative phosphorylation and redox scavenging.
Asunto(s)
Hojas de la Planta , Proteínas/metabolismo , Vitis , Redes y Vías Metabólicas , Mitocondrias/metabolismo , Oxidación-Reducción , Estrés Oxidativo , Enfermedades de las Plantas , Hojas de la Planta/crecimiento & desarrollo , Hojas de la Planta/metabolismo , Vitis/crecimiento & desarrollo , Vitis/metabolismoRESUMEN
The secreted proteins (secretome) of fungi play a key role in interactions of pathogenic and symbiotic fungi with plants. Using the plant pathogenic fungus Leptosphaeria maculans and symbiont Laccaria bicolor grown in culture, we have established a proteomic protocol for extraction, concentration and resolution of the fungal secretome. As no proteomic data were available on mycelium tissues from both L. maculans and L. bicolor, mycelial proteins were studied; they also helped verifying the purity of secretome samples. The quality of protein extracts was initially assessed by both 1-DE and 2-DE using first a broad pH range for IEF, and then narrower acidic and basic pH ranges, prior to 2-DE. Compared with the previously published protocols for which only dozens of 2-D spots were recovered from fungal secretome samples, up to approximately 2000 2-D spots were resolved by our method. MS identification of proteins along several pH gradients confirmed this high resolution, as well as the presence of major secretome markers such as endopolygalacturonases, beta-glucanosyltransferases, pectate lyases and endoglucanases. Shotgun proteomic experiments evidenced the enrichment of secreted protein within the liquid medium. This is the first description of the proteome of L. maculans and L. bicolor, and the first application of liquid-phase IEF to any fungal extracts.
Asunto(s)
Electroforesis en Gel Bidimensional/métodos , Proteínas Fúngicas/análisis , Focalización Isoeléctrica/métodos , Proteómica/métodos , Ascomicetos/química , Diálisis , Liofilización , Proteínas Fúngicas/aislamiento & purificación , Proteínas Fúngicas/metabolismo , Laccaria/química , Micelio/química , Fragmentos de Péptidos/análisis , Mapeo Peptídico , Reproducibilidad de los ResultadosRESUMEN
Flavobacterium psychrophilum is an important infectious Gram-negative bacterium causing cold-water disease (CWD) and rainbow trout fry syndrome. Outer-membrane proteins (OMPs) are key molecules with regard to the interface between the cell and its environment. Therefore, we sought to define the outer-membrane (OM) subproteome of F. psychrophilum in order to gain insight into the biology and pathogenesis of this bacterium and to identify the dominant antigens targeted by the rainbow trout (Oncorhynchus mykiss) immune system during infection. First, OMs were prepared from a cell-envelope suspension by differential Sarkosyl (sodium lauryl sarcosinate) solubility. We then isolated the OMPs and identified 36 proteins from 34 spots resolved by two-dimensional electrophoresis and LC-MS/MS. An immunoproteomic approach using antibodies from CWD-convalescent rainbow trout was then used to identify 25 immunoreactive F. psychrophilum antigens that may be relevant in pathogenesis and diagnosis. These included the previously characterized surface-exposed OMPs OmpA, OmpH/P18 and FspA, as well as newly described antigenic proteins. This study provides a number of novel candidate proteins for developing vaccine(s) against flavobacteriosis infection in aquaculture.
Asunto(s)
Antígenos Bacterianos/inmunología , Antígenos Bacterianos/aislamiento & purificación , Proteínas de la Membrana Bacteriana Externa/metabolismo , Vacunas Bacterianas/inmunología , Flavobacterium/inmunología , Flavobacterium/metabolismo , Proteoma/metabolismo , Animales , Antígenos Bacterianos/genética , Proteínas de la Membrana Bacteriana Externa/química , Proteínas de la Membrana Bacteriana Externa/inmunología , Proteínas de la Membrana Bacteriana Externa/aislamiento & purificación , Electroforesis en Gel Bidimensional , Enfermedades de los Peces/microbiología , Infecciones por Flavobacteriaceae/microbiología , Infecciones por Flavobacteriaceae/veterinaria , Flavobacterium/genética , Oncorhynchus mykiss/inmunología , Oncorhynchus mykiss/microbiología , Proteoma/inmunologíaRESUMEN
Understanding the molecular basis of resistance to imatinib, a tyrosine kinase inhibitor used as front-line therapy in chronic myeloid leukemia, remains a challenge for successful treatment. In an attempt to identify new mechanisms of resistance, we performed a comparative proteomic analysis of an imatinib-resistant cell line generated from the erythroblastic cell line K562 (K562-r) for which no known mechanism of resistance has been detected. Bidimensional gel electrophoresis was carried out to compare the protein expression pattern of imatinib-sensitive and of imatinib-resistant K562 cells. Among the 400 matched spots on five pairs of gels, only 14 spots had a significantly increased or decreased expression leading to the identification of 24 proteins identified as scaffold proteins, metabolic enzymes, DNA translation and maturation, and chaperon proteins. Among the chaperon family, only Hsp70 and Hsc70 are overexpressed in K562-r, results confirmed by Western blotting. We recently reported the participation of Hsp70 overexpression in imatinib resistance whereas a role for Hsc70 has yet to be determined. Hsc70 is not involved in imatinib resistance as the inhibition of its expression by siRNA does not restore sensitivity to imatinib. In contrast, the induced decreased expression of Hsc70 was accompanied by a greater overexpression of Hsp70. This proteomic study therefore suggests opposing roles of Hsp70 and Hsc70 in imatinib resistance.
Asunto(s)
Antineoplásicos/uso terapéutico , Resistencia a Antineoplásicos/genética , Proteínas HSP70 de Choque Térmico/metabolismo , Piperazinas/uso terapéutico , Proteómica/métodos , Pirimidinas/uso terapéutico , Benzamidas , Línea Celular Tumoral , Humanos , Mesilato de Imatinib , Células K562 , Leucemia Mielógena Crónica BCR-ABL Positiva/tratamiento farmacológico , Leucemia Mielógena Crónica BCR-ABL Positiva/genética , Leucemia Mielógena Crónica BCR-ABL Positiva/patologíaRESUMEN
The characterization of proteins isolated from skin tissue is apparently an essential parameter for understanding grape ripening as this tissue contains the key compounds for wine quality. It has been particularly difficult to extract proteins from skins for analysis by two-dimensional electrophoresis gels and, therefore, a protocol for this purpose has been adapted. The focus was on the evolution of the proteome profile of grape skin during maturation. Proteome maps obtained at three stages of ripening were compared to assess the extent to which protein distribution differs in grape skin during ripening. The comparative analysis shows that numerous soluble skin proteins evolve during ripening and reveal specific distributions at different stages. Proteins involved in photosynthesis, carbohydrate metabolisms, and stress response are identified as being over-expressed at the beginning of colour-change. The end of colour-change is characterized by the over-expression of proteins involved in anthocyanin synthesis and, at harvest, the dominant proteins are involved in defence mechanisms. In particular, increases in the abundance of different chitinase and beta-1,3-glucanase isoforms were found as the berry ripens. This observation can be correlated with the increase of the activities of both of these enzymes during skin ripening. The differences observed in proteome maps clearly show that significant metabolic changes occur in grape skin during this crucial phase of ripening. This comparative analysis provides more detailed characterization of the fruit ripening process.
Asunto(s)
Frutas/metabolismo , Proteínas de Plantas/metabolismo , Proteoma , Vitis/metabolismo , Electroforesis en Gel Bidimensional , Frutas/crecimiento & desarrollo , Perfilación de la Expresión Génica , Proteínas de Plantas/clasificación , Vitis/crecimiento & desarrolloRESUMEN
Study of the complexome - all the protein complexes of the cell - is essential for a better understanding and more global vision of cell function. Using two-dimensional blue native/SDS-PAGE (2-D BN/SDS-PAGE) technology, the cytosolic and membrane protein complexes of Escherichia coli were separated. Then, the different partners of each protein complex were identified by LC-MS/MS. In this report, 306 protein complexes were separated and identified. Among these protein complexes, 50 heteromultimeric and 256 homomultimeric protein complexes were found. Among the 50 heteromultimeric protein complexes, 18 previously described protein complexes validate the technology. In this study, 109 new protein complexes were found, providing insight into the function of previously uncharacterized bacterial proteins.