RESUMO
The diarrheal disease cholera is caused by the versatile and responsive bacterium Vibrio cholerae, which is capable of adapting to environmental changes. Among others, the alternative sigma factor RpoS activates response pathways, including regulation of motility- and chemotaxis-related genes under nutrient-poor conditions in V. cholerae. Although RpoS has been well characterised, links between RpoS and other regulatory networks remain unclear. In this study, we identified the ArcAB two-component system to control rpoS transcription and RpoS protein stability in V. cholerae. In a manner similar to that seen in Escherichia coli, the ArcB kinase not only activates the response regulator ArcA but also RssB, the anti-sigma factor of RpoS. Our results demonstrated that, in V. cholerae, RssB is phosphorylated by ArcB, which subsequently activates RpoS proteolysis. Furthermore, ArcA acts as a repressor of rpoS transcription. Additionally, we determined that the cysteine residue at position 180 of ArcB is crucial for signal recognition and activity. Thus, our findings provide evidence linking RpoS response to the anoxic redox control system ArcAB in V. cholerae.
Assuntos
Proteínas de Bactérias , Regulação Bacteriana da Expressão Gênica , Fator sigma , Vibrio cholerae , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Quimiotaxia/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/genética , Redes Reguladoras de Genes , Fosforilação , Proteínas Repressoras/metabolismo , Proteínas Repressoras/genética , Fator sigma/metabolismo , Fator sigma/genética , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Vibrio cholerae/genética , Vibrio cholerae/metabolismoRESUMO
BACKGROUND: Biallelic pathogenic variants in the ANO10 gene cause autosomal recessive progressive ataxia (ATX-ANO10). METHODS: Following the MDSGene protocol, we systematically investigated genotype-phenotype relationships in ATX-ANO10 based on the clinical and genetic data from 82 published and 12 newly identified patients. RESULTS: Most patients (>80%) had loss-of-function (LOF) variants. The most common variant was c.1150_1151del, found in all 29 patients of Romani ancestry, who had a 14-year earlier mean age at onset than patients homozygous for other LOF variants. We identified previously undescribed clinical features of ATX-ANO10 (e.g., facial muscle involvement and strabismus) suggesting the involvement of brainstem pathology, and we propose a diagnostic algorithm that may aid clinical ATX-ANO10 diagnosis. CONCLUSIONS: The early disease onset in patients with c.1150_1151del may indicate the existence of genetic/environmental disease-modifying factors in the Romani population. Our findings will inform patient counseling and may improve our understanding of the disease mechanism. © 2024 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.
Assuntos
Anoctaminas , Ataxias Espinocerebelares , Adolescente , Adulto , Criança , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Adulto Jovem , Idade de Início , Anoctaminas/genética , Estudos de Associação Genética , Ataxias Espinocerebelares/genética , IdosoRESUMO
The pyruvate dehydrogenase complex regulator (PdhR) was originally identified as a repressor of the pdhR-aceEF-lpd operon, which encodes the pyruvate dehydrogenase complex (PDHc) and PdhR itself. According to previous reports, PdhR plays a regulatory role in the physiological and metabolic pathways of bacteria. At present, the function of PdhR in Plesiomonas shigelloides is still poorly understood. In this study, RNA sequencing (RNA-Seq) of the wild-type strain and the ΔpdhR mutant strains was performed for comparison to identify the PdhR-controlled pathways, revealing that PdhR regulates ~7.38% of the P. shigelloides transcriptome. We found that the deletion of pdhR resulted in the downregulation of practically all polar and lateral flagella genes in P. shigelloides; meanwhile, motility assay and transmission electron microscopy (TEM) confirmed that the ΔpdhR mutant was non-motile and lacked flagella. Moreover, the results of RNA-seq and quantitative Real-Time Polymerase Chain Reaction (qRT-PCR) showed that PdhR positively regulated the expression of the T3SS cluster, and the ΔpdhR mutant significantly reduced the ability of P. shigelloides to infect Caco-2 cells compared with the WT. Consistent with previous research, pyruvate-sensing PdhR directly binds to its promoter and inhibits pdhR-aceEF-lpd operon expression. In addition, we identified two additional downstream genes, metR and nuoA, that are directly negatively regulated by PdhR. Furthermore, we also demonstrated that ArcA was identified as being located upstream of pdhR and lpdA and directly negatively regulating their expression. Overall, we revealed the function and regulatory pathway of PdhR, which will allow for a more in-depth investigation into P. shigelloides pathogenicity as well as the complex regulatory network.
Assuntos
Proteínas de Escherichia coli , Plesiomonas , Humanos , Complexo Piruvato Desidrogenase/metabolismo , Proteínas de Escherichia coli/metabolismo , Plesiomonas/genética , Escherichia coli/metabolismo , Proteínas Repressoras/genética , Células CACO-2 , Perfilação da Expressão GênicaRESUMO
Objective: Minimally invasive direct coronary artery bypass grafting (MIDCAB) using the left internal thoracic artery to the left descending artery is a clinical routine in the treatment of coronary artery disease. Far less is known on right-sided MIDCAB (r-MIDCAB) using the right internal thoracic artery (RITA) to the right coronary artery (RCA). We aimed to present our experience in patients with complex coronary artery disease who underwent r-MIDCAB. Materials and Methods: Between October 2019 and January 2023, 11 patients received r-MIDCAB using RITA to RCA bypass in a minimally invasive technique via right anterior minithoracotomy without using a cardiopulmonary bypass. Underlying coronary disease was complex right coronary artery stenosis (n = 7) and anomalous right coronary artery (ARCA; n = 4). All procedure-related and outcome data were evaluated prospectively. Results: Successful minimally invasive revascularization was achieved in all patients (n = 11). There were no conversions to sternotomy and no re-explorations for bleeding. Furthermore, no myocardial infarction, no strokes, and, most importantly, no deaths were observed. During the follow-up period (median 24 months), all patients were alive and 90% were completely angina free. Two patients received a repeated revascularization after surgery but independently from the RITA-RCA bypass, which was fully competent in both patients. Conclusion: Right-sided MIDCAB can be performed safely and effectively in patients with expected technically challenging percutaneous coronary intervention of the RCA and in patients with ARCA. Mid-term results showed high freedom from angina in nearly all patients. Further studies with larger patient cohorts and more evidence are needed to provide the best revascularization strategy for patients suffering from isolated complex RCA stenosis and ARCA.
Assuntos
Doença da Artéria Coronariana , Humanos , Doença da Artéria Coronariana/cirurgia , Resultado do Tratamento , Procedimentos Cirúrgicos Minimamente Invasivos/métodos , Ponte de Artéria Coronária/métodosRESUMO
BACKGROUND: Vibrio cholerae, a Gram-negative bacterium, is highly motile owing to the presence of a single polar flagellum. The global anaerobiosis response regulator, ArcA regulates the expression of virulence factors and enhance biofilm formation in V. cholerae. However, the function of ArcA for the motility of V. cholerae is yet to be elucidated. CytR, which represses nucleoside uptake and catabolism, is known to play a chief role in V. cholerae pathogenesis and flagellar synthesis but the mechanism that CytR influences motility is unclear. RESULTS: In this study, we found that the ΔarcA mutant strain exhibited higher motility than the WT strain due to ArcA directly repressed flrA expression. We further discovered that CytR directly enhanced fliK expression, which explained why the ΔcytR mutant strain was retarded in motility. On the other hand, cytR was a direct ArcA target and cytR expression was directly repressed by ArcA. As expected, cytR expression was down-regulated. CONCLUSIONS: Overall, ArcA plays a critical role in V. cholerae motility by regulating flrA expression directly and fliK indirectly in the manner of cytR.
Assuntos
Proteínas de Bactérias/genética , Flagelos/fisiologia , Regulação Bacteriana da Expressão Gênica , Proteínas Repressoras/genética , Vibrio cholerae/genética , Flagelos/genética , Movimento , Proteínas Repressoras/classificação , Vibrio cholerae/metabolismo , Virulência , Fatores de VirulênciaRESUMO
The recent FDA approval of the mRNA vaccine for severe acute respiratory syndrome coronavirus (SARS-CoV-2) emphasizes the importance of mRNA as a powerful tool for therapeutic applications. The chemically modified mRNA cap analogs contain a unique cap structure, m7 G[5']ppp[5']N (where N=G, A, C or U), present at the 5'-end of many eukaryotic cellular and viral RNAs and several non-coding RNAs. The chemical modifications on cap analog influence orientation's nature, translational efficiency, nuclear stability, and binding affinity. The recent invention of a trinucleotide cap analog provides groundbreaking research in the area of mRNA analogs. Notably, trinucleotide cap analogs outweigh dinucleotide cap analogs in terms of capping efficiency and translational properties. This review focuses on the recent development in the synthesis of various dinucleotide cap analogs such as dinucleotide containing a triazole moiety, phosphorothiolate cap, biotinylated cap, cap analog containing N1 modification, cap analog containing N2 modification, dinucleotide containing fluorescence probe and TAT, bacterial caps, and trinucleotide cap analogs. In addition, the biological applications of these novel cap analogs are delineated.
Assuntos
COVID-19 , Vacinas , COVID-19/prevenção & controle , Humanos , Análogos de Capuz de RNA/química , Análogos de Capuz de RNA/metabolismo , RNA Mensageiro/química , SARS-CoV-2 , Vacinas Sintéticas , Vacinas de mRNARESUMO
Komagataeibacter xylinus is an aerobic strain that produces bacterial cellulose (BC). Oxygen levels play a critical role in regulating BC synthesis in K. xylinus, and an increase in oxygen tension generally means a decrease in BC production. Fumarate nitrate reduction protein (FNR) and aerobic respiration control protein A (ArcA) are hypoxia-inducible factors, which can signal whether oxygen is present in the environment. In this study, FNR and ArcA were used to enhance the efficiency of oxygen signaling in K. xylinus, and globally regulate the transcription of the genome to cope with hypoxic conditions, with the goal of improving growth and BC production. FNR and ArcA were individually overexpressed in K. xylinus, and the engineered strains were cultivated under different oxygen tensions to explore how their overexpression affects cellular metabolism and regulation. Although FNR overexpression did not improve BC production, ArcA overexpression increased BC production by 24.0% and 37.5% as compared to the control under oxygen tensions of 15% and 40%, respectively. Transcriptome analysis showed that FNR and ArcA overexpression changed the way K. xylinus coped with oxygen tension changes, and that both FNR and ArcA overexpression enhanced the BC synthesis pathway. The results of this study provide a new perspective on the effect of oxygen signaling on growth and BC production in K. xylinus and suggest a promising strategy for enhancing BC production through metabolic engineering. KEY POINTS: ⢠K. xylinus BC production increased after overexpression of ArcA ⢠The young's modulus is enhanced by the ArcA overexpression ⢠ArcA and FNR overexpression changed how cells coped with changes in oxygen tension.
Assuntos
Celulose , Gluconacetobacter xylinus , Humanos , Celulose/metabolismo , Nitratos/metabolismo , Gluconacetobacter xylinus/genética , Gluconacetobacter xylinus/metabolismo , Oxigênio/metabolismo , Fumaratos/metabolismo , HipóxiaRESUMO
Colorectal carcinoma (CRC) is one of the major causes of cancer-related incidence and deaths. Here, we identified a novel antitumor peptide, P6, with a molecular weight of 2794.8 Da from a marine Chinese medicine, Arca inflata Reeve. The full amino acid sequence and secondary structure of P6 were determined by tandem mass de novo sequencing and circular dichroism spectroscopy, respectively. P6 markedly inhibited cell proliferation and colony formation, and induced apoptosis in CRC cells. Mechanistically, transcriptomics analysis and a serial functional evaluation showed that P6 induced colon cancer cell apoptosis through the activation of the p38-MAPK signaling pathway. Moreover, it was demonstrated that P6 exhibited antitumor effects in a tumor xenograft model, and induced cell cycle arrest in CRC cells in a concentration-dependent mode. These findings provide the first line of indication that P6 could be a potential therapeutic agent for CRC treatment.
Assuntos
Antineoplásicos/farmacologia , Arcidae/química , Neoplasias Colorretais/tratamento farmacológico , Peptídeos/farmacologia , Animais , Antineoplásicos/química , Antineoplásicos/isolamento & purificação , Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Dicroísmo Circular , Células HT29 , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Peptídeos/química , Peptídeos/isolamento & purificação , Ensaios Antitumorais Modelo de Xenoenxerto , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
BACKGROUND: The anoxic redox control binary system plays an important role in the response to oxygen as a signal in the environment. In particular, phosphorylated ArcA, as a global transcription factor, binds to the promoter regions of its target genes to regulate the expression of aerobic and anaerobic metabolism genes. However, the function of ArcA in Plesiomonas shigelloides is unknown. RESULTS: In the present study, P. shigelloides was used as the research object. The differences in growth, motility, biofilm formation, and virulence between the WT strain and the ΔarcA isogenic deletion mutant strain were compared. The data showed that the absence of arcA not only caused growth retardation of P. shigelloides in the log phase, but also greatly reduced the glucose utilization in M9 medium before the stationary phase. The motility of the ΔarcA mutant strain was either greatly reduced when grown in swim agar, or basically lost when grown in swarm agar. The electrophoretic mobility shift assay results showed that ArcA bound to the promoter regions of the flaK, rpoN, and cheV genes, indicating that ArcA directly regulates the expression of these three motility-related genes in P. shigelloides. Meanwhile, the ability of the ΔarcA strain to infect Caco-2 cells was reduced by 40%; on the contrary, its biofilm formation was enhanced. Furthermore, the complementation of the WT arcA gene from pBAD33-arcA+ was constructed and all of the above features of the pBAD33-arcA+ complemented strain were restored to the WT level. CONCLUSIONS: We showed the effect of ArcA on the growth, motility, biofilm formation, and virulence of Plesiomonas shigelloides, and demonstrated that ArcA functions as a positive regulator controls the motility of P. shigelloides by directly regulating the expression of flaK, rpoN and cheV genes.
Assuntos
Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Biofilmes , Plesiomonas/genética , Plesiomonas/patogenicidade , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Virulência/genéticaRESUMO
Autosomal recessive cerebellar ataxia type 1 (ARCA-1) or spinocerebellar ataxia autosomal recessive type 8 (SCAR8) is a slowly progressive neurodegenerative disorder that occurs due to mutations in the spectrin repeat containing nuclear envelope protein 1 (SYNE1) gene. Previously considered a rare cause of ARCA, related to French-Canadian patients from Beauce, Quebec, Canada, SYNE1 ataxia is now known to be of worldwide distribution. We present the case report of a 54-year-old male patient with the genetic diagnosis of SYNE1 ataxia, presenting with a SYNE1 gene mutation never described in Chilean population before.
Assuntos
Ataxia Cerebelar , Canadá , Ataxia Cerebelar/diagnóstico por imagem , Ataxia Cerebelar/genética , Proteínas do Citoesqueleto/genética , Humanos , Masculino , Pessoa de Meia-Idade , Mutação , Proteínas do Tecido Nervoso/genética , Proteínas Nucleares/genéticaRESUMO
Mutations in the synaptic nuclear envelope protein 1 (SYNE1) gene have been reported to cause autosomal recessive cerebellar ataxia (ARCA) type 1 with highly variable clinical phenotypes. The aim of this study was to describe the phenotypic-genetic spectrum of SYNE1-related ARCA1 patients in the Chinese population. We screened 158 unrelated patients with autosomal recessive or sporadic ataxia for variants in SYNE1 using next-generation sequencing. Pathogenicity assessment of SYNE1 variants was interpreted according to the American College of Medical Genetics standards and guidelines. We identified eight truncating variants and two missense variants spreading throughout the SYNE1 gene from six unrelated families, including nine novel variants and one reported variant. Of the six index patients, two patients showed the classical pure cerebellar ataxia, while four patients exhibited non-cerebellar phenotypes, including motor neuron symptoms, cognitive impairment, or mental retardation. The variants associated with motor neuron or cognition involvement tend to be located in the C-terminal region of SYNE1 protein, compared with the variants related to pure cerebellar ataxia. Our data indicating SYNE1 mutation is one of the more common causes of recessive ataxia in the Chinese population. The use of next-generation sequencing has enabled the rapid analysis of recessive ataxia and further expanded our understanding of genotype-phenotype correlation.
Assuntos
Ataxia Cerebelar/genética , Proteínas do Citoesqueleto/genética , Proteínas do Tecido Nervoso/genética , Adolescente , Adulto , Idade de Início , Povo Asiático/genética , Ataxia Cerebelar/patologia , Criança , China , Disfunção Cognitiva/etiologia , Disfunção Cognitiva/genética , Disfunção Cognitiva/patologia , Feminino , Genes Recessivos , Variação Genética , Genótipo , Humanos , Deficiência Intelectual/etiologia , Deficiência Intelectual/genética , Deficiência Intelectual/patologia , Imageamento por Ressonância Magnética , Masculino , Doença dos Neurônios Motores/etiologia , Doença dos Neurônios Motores/genética , Doença dos Neurônios Motores/patologia , Mutação de Sentido Incorreto , Linhagem , Fenótipo , Sequenciamento do Exoma , Adulto JovemRESUMO
The F plasmid tra operon encodes most of the proteins required for bacterial conjugation. TraJ and ArcA are known activators of the tra operon promoter PY, which is subject to H-NS-mediated silencing. Donor ability and promoter activity assays indicated that PY is inactivated by silencers and requires both TraJ and ArcA for activation to support efficient F conjugation. The observed low-level, ArcA-independent F conjugation is caused by tra expression from upstream alternative promoters. Electrophoretic mobility shift assays showed that TraJ alone weakly binds to PY regulatory DNA; however, TraJ binding is significantly enhanced by ArcA binding to the same DNA, indicating cooperativity of the two proteins. Analysis of binding affinities between ArcA and various DNA fragments in the PY regulatory region defined a 22-bp tandem repeat sequence (from -76 to -55 of PY) sufficient for optimal ArcA binding, which is immediately upstream of the predicted TraJ-binding site (from -54 to -34). Deletion analysis of the PY promoter in strains deficient in TraJ, ArcA, and/or H-NS determined that sequences upstream of -103 are required by silencers including H-NS for PY silencing, whereas sequences downstream of -77 are targeted by TraJ and ArcA for activation. TraJ and ArcA appear not only to counteract PY silencers but also to directly activate PY in a cooperative manner. Our data reveal the cooperativity of TraJ and ArcA during PY activation and provide insights into the regulatory circuit controlling F-family plasmid-mediated bacterial conjugation.IMPORTANCE Conjugation is a major mechanism for dissemination of antibiotic resistance and virulence among bacterial populations. The tra operon in the F family of conjugative plasmids encodes most of the proteins involved in bacterial conjugation. This work reveals that activation of tra operon transcription requires two proteins, TraJ and ArcA, to bind cooperatively to adjacent sites immediately upstream of the major tra promoter PY The interaction of TraJ and ArcA with the tra operon not only relieves PY from silencers but also directly activates it. These findings provide insights into the regulatory circuit of the F-family plasmid-mediated bacterial conjugation.
Assuntos
Proteínas da Membrana Bacteriana Externa/metabolismo , Conjugação Genética , Proteínas de Escherichia coli/metabolismo , Escherichia coli/genética , Fator F , Regulação Bacteriana da Expressão Gênica , Óperon , Proteínas Repressoras/metabolismo , Proteínas da Membrana Bacteriana Externa/genética , DNA Bacteriano/metabolismo , Escherichia coli/enzimologia , Proteínas de Escherichia coli/genética , Deleção de Genes , Regiões Promotoras Genéticas , Ligação Proteica , Mapeamento de Interação de Proteínas , Proteínas Repressoras/genéticaRESUMO
l-Threonine is an important amino acid supplemented in food, medicine, or feed. Starting from glucose, l-threonine production in Escherichia coli involves the glycolysis, TCA cycle, and the l-threonine biosynthetic pathway. In this study, how the l-threonine production in an l-threonine producing E. coli TWF001 is controlled by the three regulators ArcA, Cra, and IclR, which control the expression of genes involved in the glycolysis and TCA cycle, has been investigated. Ten mutant strains were constructed from TWF001 by different combinations of deletion or overexpression of arcA, cra, iclR, and tdcC. l-Threonine production was increased in the mutants TWF015 (ΔarcAΔcra), TWF016 (ΔarcAPcra::Ptrc), TWF017 (ΔarcAΔiclR), TWF018 (ΔarcAΔiclRΔtdcC), and TWF019 (ΔarcAΔcraΔiclRΔtdcC). Among these mutant strains, the highest l-threonine production (26.0 g/L) was obtained in TWF018, which was a 109.7% increase compared with the control TWF001. In addition, TWF018 could consume glucose more efficiently than TWF001 and produce less acetate. The results suggest that deletion of arcA, iclR, and tdcC could efficiently increase l-threonine production in E. coli.
Assuntos
Sistemas de Transporte de Aminoácidos Neutros/metabolismo , Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Proteínas Repressoras/metabolismo , Treonina/biossíntese , Sistemas de Transporte de Aminoácidos Neutros/genética , Proteínas da Membrana Bacteriana Externa/genética , Proteínas de Escherichia coli/genética , Deleção de Genes , Mutação , Proteínas Repressoras/genéticaRESUMO
Diverse bioactive substances derived from marine organisms have been attracting growing attention. Besides small molecules and polypeptides, numerous studies have shown that marine proteins also exhibit antitumor activities. Small anticancer proteins can be expressed in vivo by viral vectors to exert local and long-term anticancer effects. Herein, we purified and characterized a novel protein (ASP-3) with unique antitumor activity from Arca subcrenata Lischke. The ASP-3 contains 179 amino acids with a molecular weight of 20.6 kDa. The spectral characterization of ASP-3 was elucidated using Fourier Transform infrared spectroscopy (FTIR) and Circular Dichroism (CD) spectroscopy. Being identified as a sarcoplasmic calcium-binding protein, ASP-3 exhibited strong inhibitory effects on the proliferation of Human hepatocellular carcinoma (HepG2) cells with an IC50 value of 171.18 ± 18.59 µg/mL, measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The RNA-seq analysis showed that ASP-3 regulated the vascular endothelial growth factor receptor (VEGFR) signaling pathway in HepG2 cells. Immunofluorescence results indicated that ASP-3 effectively reduced VEGFR2 phosphorylation in HepG2 cells and affected the downstream components of VEGF signaling pathways. The surface plasmon resonance (SPR) analysis further demonstrated that ASP-3 direct interacted with VEGFR2. More importantly, the therapeutic potential of ASP-3 as an anti-angiogenesis agent was further confirmed by an in vitro model using VEGF-induced tube formation assay of human umbilical vein endothelial cells (HUVECs), as well as an in vivo model using transgenic zebrafish model. Taken together, the ASP-3 provides a good framework for the development of even more potent anticancer proteins and provides important weapon for cancer treatment using novel approaches such as gene therapy.
Assuntos
Antineoplásicos/farmacologia , Arcidae/química , Produtos Biológicos/farmacologia , Proteínas/farmacologia , Inibidores da Angiogênese/farmacologia , Animais , Animais Geneticamente Modificados , Carcinoma Hepatocelular/tratamento farmacológico , Carcinoma Hepatocelular/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Feminino , Células Hep G2 , Células Endoteliais da Veia Umbilical Humana , Humanos , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/metabolismo , Masculino , Neovascularização Patológica/tratamento farmacológico , Neovascularização Patológica/metabolismo , Transdução de Sinais/efeitos dos fármacos , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto/métodos , Peixe-ZebraRESUMO
Biallelic nonsense mutations of SYNE1 underlie a variable array of cerebellar and non-cerebellar pathologies of unknown molecular etiology. SYNE1 encodes multiple isoforms of Nesprin1 that associate with the nuclear envelope, with large cerebellar synapses and with ciliary rootlets of photoreceptors. Using two novel mouse models, we determined the expression pattern of Nesprin1 isoforms in the cerebellum whose integrity and functions are invariably affected by SYNE1 mutations. We further show that a giant isoform of Nesprin1 associates with the ciliary rootlets of ependymal cells that line brain ventricles and establish that this giant ciliary isoform of Nesprin1 harbors a KASH domain. Whereas cerebellar phenotypes are not recapitulated in Nes1gSTOP/STOP mice, these mice display a significant increase of ventricular volume. Together, these data fuel novel hypotheses about the molecular pathogenesis of SYNE1 mutations and support that KASH proteins may localize beyond the nuclear envelope in vivo.
Assuntos
Cerebelo/metabolismo , Cílios/metabolismo , Epêndima/metabolismo , Proteínas do Tecido Nervoso/biossíntese , Proteínas Nucleares/biossíntese , Sequência de Aminoácidos , Animais , Proteínas de Ciclo Celular/biossíntese , Proteínas de Ciclo Celular/genética , Cerebelo/citologia , Proteínas do Citoesqueleto , Epêndima/citologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas do Tecido Nervoso/genética , Proteínas Nucleares/genética , Isoformas de Proteínas/biossíntese , Isoformas de Proteínas/genéticaRESUMO
Manipulation of global regulators is one of the strategies used for the construction of bacterial strains suitable for the synthesis of bioproducts. However, the pleiotropic effects of these regulators can vary under different conditions and are often strain dependent. This study analyzed the effects of ArcA, CreC, Cra, and Rob using single deletion mutants of the well-characterized and completely sequenced Escherichia coli strain BW25113. Comparison of the effects of each regulator on the synthesis of major extracellular metabolites, tolerance to several compounds, and synthesis of native and nonnative bioproducts under different growth conditions allowed the discrimination of the particular phenotypes that can be attributed to the individual mutants and singled out Cra and ArcA as the regulators with the most important effects on bacterial metabolism. These data were used to identify the most suitable backgrounds for the synthesis of the reduced bioproducts succinate and 1,3-propanediol (1,3-PDO). The Δcra mutant was further modified to enhance succinate synthesis by the addition of enzymes that increase NADH and CO2 availability, achieving an 80% increase compared to the parental strain. Production of 1,3-PDO in the ΔarcA mutant was optimized by overexpression of PhaP, which increased more than twice the amount of the diol compared to the wild type in a semidefined medium using glycerol, resulting in 24 g · liter-1 of 1,3-PDO after 48 h, with a volumetric productivity of 0.5 g · liter-1 h-1IMPORTANCE Although the effects of many global regulators, especially ArcA and Cra, have been studied in Escherichia coli, the metabolic changes caused by the absence of global regulators have been observed to differ between strains. This scenario complicates the identification of the individual effects of the regulators, which is essential for the design of metabolic engineering strategies. The genome of Escherichia coli BW25113 has been completely sequenced and does not contain additional mutations that could mask or interfere with the effects of the global regulator mutations. The uniform genetic background of the Keio collection mutants enabled the characterization of the physiological consequences of altered carbon and redox fluxes caused by each global regulator deletion, eliminating possible strain-dependent results. As a proof of concept, Δcra and ΔarcA mutants were subjected to further manipulations to obtain large amounts of succinate and 1,3-PDO, demonstrating that the metabolic backgrounds of the mutants were suitable for the synthesis of bioproducts.
Assuntos
Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Deleção de Genes , Regulação Bacteriana da Expressão Gênica , Proteínas Repressoras/metabolismo , Proteínas da Membrana Bacteriana Externa/genética , Proteínas de Bactérias/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Escherichia coli/genética , Glicerol/metabolismo , Engenharia Metabólica , Propilenoglicóis/metabolismo , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Proteínas Repressoras/genética , Ácido Succínico/metabolismoRESUMO
BACKGROUND: Acetyl-CoA-derived chemicals are suitable for multiple applications in many industries. The bio-production of these chemicals has become imperative owing to the economic and environmental problems. However, acetate overflow is the major drawback for acetyl-CoA-derived chemicals production. Approaches for overcoming acetate overflow may be beneficial for the production of acetyl-CoA-derived chemicals. RESULTS: In this study, a transcriptional regulator iclR was knocked out in E.coli BL21(DE3) to overcome acetate overflow and improve the chemicals production. Two important acetyl-CoA-derived chemicals, phloroglucinol (PG) and 3-hydroxypropionate (3HP) were used to evaluate it. It is revealed that knockout of iclR significantly increased expressions of aceBAK operon. The cell yields and glucose utilization efficiencies were higher than those of control strains. The acetate concentrations were decreased by more than 50% and the productions of PG and 3HP were increased more than twice in iclR mutants. The effects of iclR knockout on cell physiology, cell metabolism and production of acetyl-CoA-derived chemicals were similar to those of arcA knockout in our previous study. However, the arcA-iclR double mutants couldn't gain higher productions of PG and 3HP. The mechanisms are unclear and needed to be resolved in future. CONCLUSIONS: Knockout of iclR significantly increased gene expression of aceBAK operon and concomitantly activated glyoxylate pathway. This genetic modification may be a good way to overcome acetate overflow, and improve the production of a wide range of acetyl-CoA-derived chemicals.
Assuntos
Acetilcoenzima A/metabolismo , Proteínas da Membrana Bacteriana Externa/genética , Proteínas de Escherichia coli/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica , Proteínas Repressoras/genética , Deleção de Sequência/genética , Acetatos/metabolismo , Acetilcoenzima A/biossíntese , Escherichia coli/crescimento & desenvolvimento , Escherichia coli/fisiologia , Técnicas de Inativação de Genes , Glucose/metabolismo , Glioxilatos/metabolismo , Ácido Láctico/análogos & derivados , Ácido Láctico/metabolismo , Redes e Vias Metabólicas/genética , Óperon , Floroglucinol/metabolismoRESUMO
The continued emergence of antibiotic resistant bacteria in recent years is of great concern. The search for new classes of antibacterial agents has expanded to non-traditional sources such as shellfish. An antibacterial subunit of hemoglobin (Hb-I) was purified from the mantle of Arcainflata by phosphate extraction and ion exchange chromatography. A novel antibacterial peptide, AI-hemocidin 2, derived from Hb-I, was discovered using bioinformatics analysis. It displayed antibacterial activity across a broad spectrum of microorganisms, including several Gram-positive and Gram-negative bacteria, with minimal inhibitory concentration (MIC) values ranging from 37.5 to 300 µg/mL, and it exhibited minimal hemolytic or cytotoxic activities. The antibacterial activity of AI-hemocidin 2 was thermostable (25-100 °C) and pH resistant (pH 3-10). The cellular integrity was determined by flow cytometry. AI-hemocidin 2 was capable of permeating the cellular membrane. Changes in the cell morphology were observed with a scanning electron microscope. Circular dichroism spectra suggested that AI-hemocidin 2 formed an α-helix structure in the membrane mimetic environment. The results indicated that the anti-bacterial mechanism for AI-hemocidin 2 occurred through disrupting the cell membrane. AI-hemocidin 2 might be a potential candidate for tackling antibiotic resistant bacteria.
Assuntos
Antibacterianos/farmacologia , Arcidae/química , Hemoglobinas/farmacologia , Peptídeos/farmacologia , Animais , Antibacterianos/química , Antibacterianos/isolamento & purificação , Membrana Celular/efeitos dos fármacos , Cromatografia por Troca Iônica , Dicroísmo Circular , Farmacorresistência Bacteriana , Estabilidade de Medicamentos , Bactérias Gram-Negativas/efeitos dos fármacos , Bactérias Gram-Positivas/efeitos dos fármacos , Hemoglobinas/química , Hemoglobinas/isolamento & purificação , Hemólise/efeitos dos fármacos , Temperatura Alta , Concentração de Íons de Hidrogênio , Testes de Sensibilidade Microbiana , Peptídeos/química , Peptídeos/isolamento & purificaçãoRESUMO
Lipopolysaccharide (LPS) consists of three covalently linked domains: the lipid A, the core region and the O antigen (OAg), consisting of repeats of an oligosaccharide. Salmonella enterica serovar Enteritidis (S. Enteritidis) produces a LPS with two OAg preferred chain lengths: a long (L)-OAg controlled by WzzSE and a very long (VL)-OAg controlled by WzzfepE. In this work, we show that OAg produced by S. Enteritidis grown in E minimal medium also presented two preferred chain-lengths. However, a simultaneous and opposing change in the production of L-OAg and VL-OAg was observed in response to oxygen availability. Biochemical and genetics analyses indicate that this process is regulated by transcriptional factors Fnr and ArcA by means of controlling the transcription of genes encoding WzzSE and WzzfepE in response to oxygen availability. Thus, our results revealed a sophisticated regulatory mechanism involved in the adaptation of S. Enteritidis to one of the main environmental cues faced by this pathogen during infection.
Assuntos
Antígenos O/metabolismo , Oxigênio/metabolismo , Salmonella enterica/metabolismo , Eletroforese em Gel de Poliacrilamida , Genes Bacterianos , Antígenos O/química , Polimerização , Salmonella enterica/genéticaRESUMO
OBJECTIVE: Acetyl-CoA is used to produce many valuable metabolites in Escherichia coli. However, acetate overflow is a major shortcoming. Knockout of the global regulator gene, arcA, may solve this problem. RESULTS: The arcA gene of E. coli BL21(DE3) was knocked out, and the production of phloroglucinol (PG) and 3-hydroxypropionate (3HP), both derived from acetyl-CoA, were used to evaluate its effect. The arcA mutants had higher cell yields and higher glucose utilization efficiencies than the corresponding control strains, and the productions of PG and 3HP were 0.92 g/l and 0.27 g/l, respectively; more than twice that of the control strains. Furthermore, arcA knockout also showed significant repression on formation of acetate, the major byproduct in fermentation. Acetate concentrations were decreased 69.4 % and 87 % by arcA knockout during the production of PG and 3HP, respectively. CONCLUSIONS: The arcA gene knockout is a solution to acetate overflow and may improve production of a wide range of acetyl-CoA-derived metabolites.