RESUMO
Glioblastoma is a devastating tumor of the central nervous system characterized by a poor survival and an extremely dark prognosis, making its diagnosis, treatment and monitoring highly challenging. Numerous studies have highlighted extracellular vesicles (EVs) as key players of tumor growth, invasiveness and resistance, as they carry and disseminate oncogenic material in the local tumor microenvironment and at distance. However, whether their quality and quantity reflect individual health status and changes in homeostasis is still not fully elucidated. Here, we separated EVs from plasma collected at different time points alongside with the clinical management of GBM patients. Our findings confirm that plasmatic EVs could be separated and characterized with standardized protocols, thereby ensuring the reliability of measuring vesiclemia, i.e. extracellular vesicle concentration in plasma. This unveils that vesiclemia is a dynamic parameter, which could be reflecting tumor burden and/or response to treatments. Further label-free liquid chromatography tandem mass spectrometry unmasks the von Willebrand Factor (VWF) as a selective protein hallmark for GBM-patient EVs. Our data thus support the notion that EVs from GBM patients showed differential protein cargos that can be further surveyed in circulating EVs, together with vesiclemia.
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Biomarcadores Tumorais/metabolismo , Neoplasias Encefálicas/patologia , Vesículas Extracelulares/metabolismo , Glioblastoma/patologia , Proteoma/metabolismo , Microambiente Tumoral/imunologia , Fator de von Willebrand/metabolismo , Idoso , Idoso de 80 Anos ou mais , Neoplasias Encefálicas/sangue , Neoplasias Encefálicas/imunologia , Neoplasias Encefálicas/metabolismo , Estudos de Casos e Controles , Feminino , Seguimentos , Glioblastoma/sangue , Glioblastoma/imunologia , Glioblastoma/metabolismo , Humanos , Masculino , Pessoa de Meia-Idade , Prognóstico , Proteoma/análiseRESUMO
Although rare, glioblastoma is a devastating tumor of the central nervous system characterized by a poor survival and an extremely dark prognosis, making its diagnosis, treatment, and monitoring highly challenging. Numerous studies have highlighted extracellular vesicles (EVs) as key players of tumor growth, invasiveness, and resistance, as they carry oncogenic material. Moreover, EVs have been shown to communicate locally in a paracrine way but also at remote throughout the organism. Indeed, recent reports demonstrated the presence of brain tumor-derived EVs into body fluids such as plasma and cerebrospinal fluid. Fluid-associated EVs have indeed been suspected to reflect quantitative and qualitative information about the status and fate of the tumor and can potentially act as a resource for noninvasive biomarkers that might assist in diagnosis, treatment, and follow-up of glioblastoma patients. Here, we coined the name vesiclemia to define the concentration of plasmatic EVs, an intuitive term to be directly transposed in the clinical jargon.
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Neoplasias Encefálicas/metabolismo , Vesículas Extracelulares/metabolismo , Glioblastoma/metabolismo , Animais , Transporte Biológico , Biomarcadores Tumorais , Neoplasias Encefálicas/sangue , Neoplasias Encefálicas/diagnóstico , Neoplasias Encefálicas/genética , Comunicação Celular , Fracionamento Celular/métodos , Gerenciamento Clínico , Progressão da Doença , Glioblastoma/sangue , Glioblastoma/diagnóstico , Glioblastoma/genética , Humanos , Técnicas de Diagnóstico Molecular , Microambiente Tumoral/genéticaRESUMO
Myocardial infarction is one of the leading causes of mortality and morbidity worldwide. Whereas transplantation of several cell types into the infarcted heart has produced promising preclinical results, clinical studies using analogous human cells have shown limited structural and functional benefits. In dogs and humans, we have described a type of muscle-derived stem cells termed MuStem cells that efficiently promoted repair of injured skeletal muscle. Enhanced survival rate, long-term engraftment, and participation in muscle fiber formation were reported, leading to persistent tissue remodeling and clinical benefits. With the consideration of these features that are restricted or absent in cells tested so far for myocardial infarction, we wanted to investigate the capacity of human MuStem cells to repair infarcted hearts. Their local administration in immunodeficient rats 1 week after induced infarction resulted in reduced fibrosis and increased angiogenesis 3 weeks post-transplantation. Importantly, foci of human fibers were detected in the infarct site. Treated rats also showed attenuated left-ventricle dilation and preservation of contractile function. Interestingly, no spontaneous arrhythmias were observed. Our findings support the potential of MuStem cells, which have already been proposed as therapeutic candidates for dystrophic patients, to treat myocardial infarction and position them as an attractive tool for muscle-regenerative medicine.
RESUMO
BACKGROUND: Canine MuStem cells have demonstrated regenerative efficacy in a dog model of muscular dystrophy, and the recent characterization of human counterparts (hMuStem) has highlighted the therapeutic potential of this muscle-derived stem cell population. To date, these cells have only been generated in research-grade conditions. However, evaluation of the clinical efficacy of any such therapy will require the production of hMuStem cells in compliance with good manufacturing practices (GMPs). Because the current use of fetal bovine serum (FBS) to isolate and expand hMuStem cells raises several ethical, safety, and supply concerns, we assessed the use of two alternative xeno-free blood derivatives: human serum (HS) and a human platelet lysate (hPL). METHODS: hMuStem cells were isolated and expanded in vitro in either HS-supplemented or hPL-supplemented media and the proliferation rate, clonogenicity, myogenic commitment potential, and oligopotency compared with that observed in FBS-supplemented medium. Flow cytometry and high-throughput 3'-digital gene expression RNA sequencing were used to characterize the phenotype and global gene expression pattern of hMuStem cells cultured with HS or hPL. RESULTS: HS-supplemented and hPL-supplemented media both supported the isolation and long-term proliferation of hMuStem cells. Compared with FBS-based medium, both supplements enhanced clonogenicity and allowed for a reduction in growth factor supplementation. Neither supplement altered the cell lineage pattern of hMuStem cells. In vitro differentiation assays revealed a decrease in myogenic commitment and in the fusion ability of hMuStem cells when cultured with hPL. In return, this reduction of myogenic potential in hPL-supplemented cultures was rapidly reversed by substitution of hPL with HS or fibrinogen-depleted hPL. Moreover, culture of hMuStem cells in hPL hydrogel and fibrinogen-depleted hPL demonstrated that myogenic differentiation potential is maintained in heparin-free hPL derivatives. CONCLUSIONS: Our findings indicate that HS and hPL are efficient and viable alternatives to FBS for the preparation of hMuStem cell batches in compliance with GMPs.
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Plaquetas/metabolismo , Terapia Baseada em Transplante de Células e Tecidos/métodos , Soro/química , Adolescente , Adulto , Animais , Diferenciação Celular , Proliferação de Células , Cães , Feminino , Humanos , Masculino , Adulto JovemRESUMO
After intra-arterial delivery in the dystrophic dog, allogeneic muscle-derived stem cells, termed MuStem cells, contribute to long-term stabilization of the clinical status and preservation of the muscle regenerative process. However, it remains unknown whether the human counterpart could be identified, considering recent demonstrations of divergent features between species for several somatic stem cells. Here, we report that MuStem cells reside in human skeletal muscle and display a long-term ability to proliferate, allowing generation of a clinically relevant amount of cells. Cultured human MuStem (hMuStem) cells do not express hematopoietic, endothelial, or myo-endothelial cell markers and reproducibly correspond to a population of early myogenic-committed progenitors with a perivascular/mesenchymal phenotypic signature, revealing a blood vessel wall origin. Importantly, they exhibit both myogenesis in vitro and skeletal muscle regeneration after intramuscular delivery into immunodeficient host mice. Together, our findings provide new insights supporting the notion that hMuStem cells could represent an interesting therapeutic candidate for dystrophic patients.
Assuntos
Músculo Esquelético/fisiologia , Mioblastos Esqueléticos/citologia , Mioblastos Esqueléticos/transplante , Regeneração , Transplante de Células-Tronco , Células-Tronco Adultas , Animais , Diferenciação Celular , Proliferação de Células , Células Cultivadas , Humanos , Camundongos , Desenvolvimento Muscular , Distrofia Muscular Animal/terapia , Distrofia Muscular de Duchenne/terapia , Medicina RegenerativaRESUMO
Proteomic profiling plays a decisive role in the elucidation of molecular signatures representative of a specific clinical context. MuStem cell based therapy represents a promising approach for clinical applications to cure Duchenne muscular dystrophy (DMD). To expand our previous studies collected in the clinically relevant DMD animal model, we decided to investigate the skeletal muscle proteome 4 months after systemic delivery of allogenic MuStem cells. Quantitative proteomics with isotope-coded protein labeling was used to compile quantitative changes in the protein expression profiles of muscle in transplanted Golden Retriever muscular dystrophy (GRMD) dogs as compared to Golden Retriever muscular dystrophy dogs. A total of 492 proteins were quantified, including 25 that were overrepresented and 46 that were underrepresented after MuStem cell transplantation. Interestingly, this study demonstrates that somatic stem cell therapy impacts on the structural integrity of the muscle fascicle by acting on fibers and its connections with the extracellular matrix. We also show that cell infusion promotes protective mechanisms against oxidative stress and favors the initial phase of muscle repair. This study allows us to identify putative candidates for tissue markers that might be of great value in objectively exploring the clinical benefits resulting from our cell-based therapy for DMD. All MS data have been deposited in the ProteomeXchange with identifier PXD001768 (http://proteomecentral.proteomexchange.org/dataset/PXD001768).
Assuntos
Terapia Baseada em Transplante de Células e Tecidos/métodos , Células Musculares/transplante , Distrofia Muscular Animal/terapia , Proteoma/genética , Transplante de Células-Tronco , Células-Tronco/metabolismo , Animais , Cães , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Ontologia Genética , Internet , Anotação de Sequência Molecular , Células Musculares/citologia , Células Musculares/metabolismo , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Distrofia Muscular Animal/genética , Distrofia Muscular Animal/metabolismo , Distrofia Muscular Animal/patologia , Estresse Oxidativo , Proteoma/metabolismo , Proteômica/métodos , Software , Células-Tronco/citologia , Resultado do TratamentoRESUMO
BACKGROUND: Duchenne muscular dystrophy (DMD) is an X-linked muscle disease that leads to fibre necrosis and progressive paralysis. At present, DMD remains a lethal disease without any effective treatment, requiring a better understanding of the pathophysiological processes and comprehensive assessment of the newly identified therapeutic strategies. MicroRNAs including members of the muscle-specific myomiR family have been identified as being deregulated in muscle of DMD patients and in mdx mice used as a model for DMD. In recent years, the Golden Retriever muscular dystrophy (GRMD) dog has appeared as the crucial animal model for objectively assessing the potential of new innovative approaches. Here, we first aim at establishing the muscle expression pattern of five selected miRNAs in this clinically relevant model to determine if they are similarly affected compared with other DMD contexts. Second, we attempt to show whether these miRNAs could be impacted by the systemic delivery of a promising stem cell candidate (referred to as MuStem cells) to implement our knowledge on its mode of action and/or identify markers associated with cell therapy efficacy. METHODS: A comparative study of miRNAs expression levels and cellular localization was performed on 9-month-old healthy dogs, as well as on three sub-sets of GRMD dog (without immunosuppression or cell transplantation, with continuous immunosuppressive regimen and with MuStem cell transplantation under immunosuppression), using RT-qPCR and in situ hybridization. RESULTS: We find that miR-222 expression is markedly up-regulated in GRMD dog muscle compared to healthy dog, while miR-486 tends to be down-expressed. Intriguingly, the expression of miR-1, miR-133a and miR-206 does not change. In situ hybridization exploration reveals, for the first time, that miR-486 and miR-206 are mainly localized in newly regenerated fibres in GRMD dog muscle. In addition, we show that cyclosporine-based immunosuppression, classically used in allogeneic cell transplantation, exclusively impacts the miR-206 expression. Finally, we demonstrate that intra-arterial administration of MuStem cells results in up-regulation of miR-133a and miR-222 concomitantly with a down-expression of two sarcomeric proteins corresponding to miR-222 targets. CONCLUSION: We point out a differential muscle expression of miR-222 and miR-486 associated with the pathophysiology of the clinically relevant GRMD dog model with a tissue localization focused on regenerated fibres. We also establish a modified expression of miR-133a and miR-222 subsequent to MuStem cell infusion.
Assuntos
MicroRNAs/metabolismo , Células Musculares/transplante , Distrofia Muscular Animal/genética , Distrofia Muscular Animal/terapia , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/terapia , Transplante de Células-Tronco/métodos , Animais , Biomarcadores/metabolismo , Ciclosporina/farmacologia , Ciclosporina/uso terapêutico , Modelos Animais de Doenças , Cães , Regulação para Baixo , Imunofluorescência , Humanos , Terapia de Imunossupressão/métodos , Imunossupressores/farmacologia , Imunossupressores/uso terapêutico , Hibridização In Situ , Injeções Intra-Arteriais , Camundongos , Camundongos Endogâmicos mdx , MicroRNAs/efeitos dos fármacos , Células Musculares/metabolismo , Músculo Esquelético/citologia , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Distrofia Muscular Animal/patologia , Distrofia Muscular de Duchenne/patologia , Cadeias Pesadas de Miosina/metabolismo , Células-Tronco/metabolismo , Regulação para CimaRESUMO
BACKGROUND: Several adult stem cell populations exhibit myogenic regenerative potential, thus representing attractive candidates for therapeutic approaches of neuromuscular diseases such as Duchenne Muscular Dystrophy (DMD). We have recently shown that systemic delivery of MuStem cells, skeletal muscle-resident stem cells isolated in healthy dog, generates the remodelling of muscle tissue and gives rise to striking clinical benefits in Golden Retriever Muscular Dystrophy (GRMD) dog. This global effect, which is observed in the clinically relevant DMD animal model, leads us to question here the molecular pathways that are impacted by MuStem cell transplantation. To address this issue, we compare the global gene expression profile between healthy, GRMD and MuStem cell treated GRMD dog muscle, four months after allogenic MuStem cell transplantation. RESULTS: In the dystrophic context of the GRMD dog, disease-related deregulation is observed in the case of 282 genes related to various processes such as inflammatory response, regeneration, calcium ion binding, extracellular matrix organization, metabolism and apoptosis regulation. Importantly, we reveal the impact of MuStem cell transplantation on several molecular and cellular pathways based on a selection of 31 genes displaying signals specifically modulated by the treatment. Concomitant with a diffuse dystrophin expression, a histological remodelling and a stabilization of GRMD dog clinical status, we show that cell delivery is associated with an up-regulation of genes reflecting a sustained enhancement of muscle regeneration. We also identify a decreased mRNA expression of a set of genes having metabolic functions associated with lipid homeostasis and energy. Interestingly, ubiquitin-mediated protein degradation is highly enhanced in GRMD dog muscle after systemic delivery of MuStem cells. CONCLUSIONS: Overall, our results provide the first high-throughput characterization of GRMD dog muscle and throw new light on the complex molecular/cellular effects associated with muscle repair and the clinical efficacy of MuStem cell-based therapy.
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Perfilação da Expressão Gênica , Músculo Esquelético/patologia , Distrofia Muscular Animal/genética , Distrofia Muscular Animal/terapia , Transplante de Células-Tronco , Animais , Modelos Animais de Doenças , Cães , Seguimentos , Humanos , Músculo Esquelético/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Controle de Qualidade , Reação em Cadeia da Polimerase em Tempo Real , Reprodutibilidade dos TestesRESUMO
Duchenne muscular dystrophy (DMD) is caused by null mutations in the dystrophin gene, leading to progressive and unrelenting muscle loss. Although the genetic basis of DMD is well resolved, the cellular mechanisms associated with the physiopathology remain largely unknown. Increasing evidence suggests that secondary mechanisms, as the alteration of key signaling pathways, may play an important role. In order to identify reliable biomarkers and potential therapeutic targets, and taking advantage of the clinically relevant Golden Retriever Muscular Dystrophy (GRMD) dog model, a proteomic study was performed. Isotope-coded affinity tag (ICAT) profiling was used to compile quantitative changes in protein expression profiles of the vastus lateralis muscles of 4-month old GRMD vs healthy dogs. Interestingly, the set of under-expressed proteins detected appeared primarily composed of metabolic proteins, many of which have been shown to be regulated by the transcriptional peroxisome proliferator-activated receptor-gamma co-activator 1 alpha (PGC-1α). Subsequently, we were able to showed that PGC1-α expression is dramatically reduced in GRMD compared to healthy muscle. Collectively, these results provide novel insights into the molecular pathology of the clinically relevant animal model of DMD, and indicate that defective energy metabolism is a central hallmark of the disease in the canine model.
Assuntos
Biomarcadores/metabolismo , Metabolismo Energético/fisiologia , Músculo Esquelético/metabolismo , Distrofia Muscular de Duchenne/metabolismo , Proteoma/metabolismo , Proteômica/métodos , Animais , Primers do DNA/genética , Cães , Immunoblotting , Imuno-Histoquímica , Espectrometria de Massas , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transativadores/metabolismoRESUMO
Duchenne muscular dystrophy is the most common and severe form of muscular dystrophy, and although the genetic basis of this disease is well defined, the overall mechanisms that define its pathogenesis remain obscure. Alterations in individual signaling pathways have been described, but little information is available regarding their putative implications in Duchenne muscular dystrophy pathogenesis. Here, we studied the status of various major signaling pathways in the Golden Retriever muscular dystrophy dog that specifically reproduces the full spectrum of human pathology. Using antibody arrays, we found that Akt1, glycogen synthase kinase-3beta (GSK3beta), 70-kDa ribosomal protein S6 kinase (p70S6K), extracellular signal-regulated kinases 1/2, and p38delta and p38gamma kinases all exhibited decreased phosphorylation in muscle from a 4-month-old animal with Golden Retriever muscular dystrophy, revealing a deep alteration of the phosphatidylinositol 3-kinase (PI3K)/Akt and mitogen-activated protein kinase pathways. Immunohistochemistry analysis revealed the presence of muscle fibers exhibiting a cytosolic accumulation of Akt1, GSK3beta, and phosphatidylinositol-3,4,5-trisphosphate 3-phosphatase (PTEN), an enzyme counteracting PI3K-mediated Akt activation. Enzymatic assays established that these alterations in phosphorylation and expression levels were associated with decreased Akt and increased GSK3beta and PTEN activities. PTEN/GSK3beta-positive fibers were also observed in muscle sections from 3- and 36-month-old animals, indicating long-term PI3K/Akt pathway alteration. Collectively, our data suggest that increased PTEN expression and activity play a central role in PI3K/Akt/GSK3beta and p70S6K pathway modulation, which could exacerbate the consequences of dystrophin deficiency.
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Distrofia Muscular Animal/fisiopatologia , PTEN Fosfo-Hidrolase/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Animais , Western Blotting , Cães , Distrofina/deficiência , Quinase 3 da Glicogênio Sintase/metabolismo , Glicogênio Sintase Quinase 3 beta , Humanos , Imuno-Histoquímica , Microscopia Confocal , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Distrofia Muscular Animal/metabolismo , Distrofia Muscular Animal/patologia , Distrofia Muscular de Duchenne/metabolismo , Distrofia Muscular de Duchenne/patologia , Distrofia Muscular de Duchenne/fisiopatologia , Fosforilação , Proteínas Quinases S6 Ribossômicas 70-kDa/metabolismo , Transdução de Sinais/fisiologiaRESUMO
Adult skeletal muscle possesses remarkable regenerative capacity that has conventionally been attributed to the satellite cells. These precursor cells were thought to contain distinct populations with varying myogenic potential. Recently, the identification of multipotent stem cells capable of new myofiber formation has expanded the general view on the muscle regenerative process. Here we examined the characteristics of turkey skeletal muscle-derived cell (MDC) populations that were separated according to their adhesion abilities. We sought to determine whether these abilities could be a potential tool for separating cells with different myogenic commitment. Using the preplate technique, we showed that MDCs display a wide range of adhesion ability, allowing us to isolate a marginal fraction with initial adhesion defect. Methodological investigations revealed that this defect represents an intrinsic and well-established biological feature for these cells. In vitro behavioral and morphological analyses showed that late adherent cells (LACs) share several primitive cell characteristics. Phenotypic assessment indicated that LACs contain early stage myogenic cells and immature progenitors of satellite cells, whereas early adherent cells consist mainly of fully committed precursors. Overall, our findings demonstrate for the first time in an avian model that differential MDC adhesion properties could be used to efficiently purify cells with varying myogenic commitment, including immature progenitor cells. This manuscript contains online supplemental material at http://www.jhc.org. Please visit this article online to view these materials.
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Diferenciação Celular , Mioblastos Esqueléticos/citologia , Animais , Caderinas/análise , Adesão Celular , Técnicas de Cultura de Células/métodos , Proliferação de Células , Desmina/análise , Fibroblastos/citologia , Imuno-Histoquímica , Microscopia Confocal , Mioblastos Esqueléticos/química , Mioblastos Esqueléticos/fisiologia , Cadeias Pesadas de Miosina/análise , Fator de Transcrição PAX7/análise , Fatores de Tempo , PerusRESUMO
We have previously reported that CD34(+) cells purified from mouse fetal muscles can differentiate into skeletal muscle in vitro and in vivo when injected into muscle tissue of dystrophic mdx mice. In this study, we investigate the ability of such donor cells to restore dystrophin expression, and to improve the functional muscle capacity of the extensor digitorum longus muscle (EDL) of mdx mice. For this purpose green fluorescent-positive fetal GFP(+)/CD34(+) cells or desmin(+)/(-)LacZ/CD34(+) cells were transplanted into irradiated or non-irradiated mdx EDL muscle. Donor fetal muscle-derived cells predominantly fused with existing fibers. Indeed more than 50% of the myofibers of the host EDL contained donor nuclei delivering dystrophin along 80-90% of the length of their sarcolemma. The presence of significant amounts of dystrophin (about 60-70% of that found in a control wild-type mouse muscle) was confirmed by Western blot analyses. Dystrophin expression also outcompeted that of utrophin, as revealed by a spatial shift in the distribution of utrophin. At 1 month post-transplant, the recipient muscle appeared to have greater resistance to fatigue than control mdx EDL muscle during repeated maximal contractions.
Assuntos
Antígenos CD34/metabolismo , Células Musculares/transplante , Distrofia Muscular Animal/terapia , Animais , Fusão Celular , Desmina/metabolismo , Distrofina/metabolismo , Fadiga/induzido quimicamente , Feminino , Proteínas de Fluorescência Verde/genética , Óperon Lac , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos mdx , Células Musculares/fisiologia , Músculos/embriologia , Músculos/efeitos da radiaçãoRESUMO
The alpha subunit (alphaTm) of Thermotoga maritima RNA polymerase has been characterized to investigate its role in transcriptional regulation in one of the few known anaerobic hyperthermophilic bacteria. The highly thermostable alphaTm shares 54% similarity with its Escherichia coli analogue (alphaEc). The T. maritima rpoA gene coding the alpha subunit does not complement the thermosensitive rpoA112 mutation of E. coli. However, alphaTm and alphaEc show similar folding patterns as determined by circular dichroism. Purified alphaTm binds to the T. maritima PargGo promoter region (probably to a UP-element) and Arg282 appears to be crucial for DNA binding. The thermostable protein is also able to interact with transcription regulatory proteins, like ArgR from T. neapolitana or CRP from E. coli. These data indicate that the RNA polymerase alpha subunit might play a crucial role in the modulation of gene expression in hyperthermophiles.
Assuntos
RNA Polimerases Dirigidas por DNA/genética , Escherichia coli/genética , Thermotoga maritima/genética , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Sequência de Bases , Proteínas de Ligação a DNA/genética , RNA Polimerases Dirigidas por DNA/fisiologia , Regulação Bacteriana da Expressão Gênica , Genes Bacterianos , Teste de Complementação Genética/métodos , Dados de Sequência Molecular , Regiões Promotoras Genéticas , Ligação Proteica , Homologia de Sequência de Aminoácidos , Fatores de Transcrição/genéticaRESUMO
The protein array methodology is used to study DNA-protein and protein-protein interactions governing gene expression from the Bacillus stearothermophilus PargCo promoter-operator region. Using probes labelled with near-infrared fluorescence dyes with exitation characteristics close to 700 or 800 nm, it is possible to detect signals from proteins (purified or non-purified in Escherichia coli cell extracts) immobilised on a nitrocellulose membrane with a high sensitivity (almost 12 amol of a spotted protein for protein-DNA interactions). Protein array data are confirmed by other methods indicating that molecular interactions of the order 10(-7) M can be monitored with the proposed protein array approach. We show that the PargCo region is a target for binding at least three types of regulatory proteins, ArgR repressors from thermophilic bacteria, the E. coli RNA polymerase alpha subunit and cyclic AMP binding protein CRP. We also demonstrate that the high strength of the PargC promoter is related to an upstream element that binds to the E. coli RNA polymerase alpha subunit.
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Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , Análise Serial de Proteínas/métodos , Sequência de Bases , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Genes Bacterianos , Geobacillus stearothermophilus/genética , Geobacillus stearothermophilus/metabolismo , Dados de Sequência Molecular , Regiões Operadoras Genéticas , Regiões Promotoras Genéticas , Análise Serial de Proteínas/estatística & dados numéricos , Ligação Proteica , Proteômica , Sensibilidade e Especificidade , Espectroscopia de Luz Próxima ao Infravermelho , Transcrição GênicaRESUMO
Bacillus stearothermophilus ArgR binds efficiently to the Escherichia coli carAB operator, whereas the E. coli repressor binds very poorly to the argCo operator of B. stearothermophilus. In order to elucidate this contradictory behavior between ArgRs, we constructed chimeric proteins by swapping N-terminal DNA-binding and C-terminal oligomerization domains or by exchanging the linker peptide. Chimeras carrying the E. coli DNA-binding domain and the B. stearothermophilus oligomerization domain showed sequence-nonspecific rather than sequence-specific interactions with arg operators. Chimeras carrying the B. stearothermophilus DNA-binding domain and E. coli oligomerization domain exhibited a high DNA-binding affinity for the B. stearothermophilus argCo and E. coli carAB operators and repressed the reporter-gene transcription from the B. stearothermophilus PargCo control region in vitro; arginine had no effect on, and indeed even decreased, their DNA-binding affinity. With the protein array method, we showed that the wild-type B. stearothermophilus ArgR and derivatives of it containing only the exchanged linker from E. coli ArgR or carrying the B. stearothermophilus DNA-binding domain along with the linker and the alpha4 regions were able to bind argCo containing the single Arg box. This binding was weaker than binding to the two-box operator but was no longer arginine dependent. Several lines of observations indicate that the alpha4 helix in the oligomerization domain and the linker peptide can contribute to the recognition of single or double Arg boxes and therefore to the operator DNA-binding specificity in similar but not identical ArgR repressors from two distant bacteria.