RESUMO
Ceramides are a family of lipids constituted by a sphingoid base and a fatty acid. In the skin, they are mainly present in the stratum corneum where, with cholesterol and free fatty acids, they constitute the inter-corneocyte lipids. With the other lipid groups, they play a key role in the formation of dense lamellar structures between adjacent corneocytes, collectively ensuring the vital efficient barrier to water evaporation and protection from foreign agents´ penetration. Changes in ceramide level and relative composition, with potential impairment of lipid arrangement, have been evidenced in different skin conditions and skin diseases. Therefore, use of suitably formulated ceramides has been proposed for topical treatment to help re-structure damaged lipid arrangement and repair impaired skin barrier function. Nonetheless, the formulation of ceramides in products necessitates specific processes such as heating to high temperature before their introduction in the final formula. In this review on the structure, the role and the potential of ceramides for skincare, we point out the necessity of rigorous process when formulating ceramides into the final product. We demonstrate the counterproductive effects of undissolved ceramides on skin barrier repair capacity of the formulas, when assessed in different in vitro models of disrupted skin barrier.
Les céramides sont une famille de lipides constituée d'une base sphingoïde et d'un acide gras. Dans la peau, ils sont principalement présents dans la couche cornée où, avec le cholestérol et les acides gras libres, ils constituent les lipides intercornéocytes. Avec les autres groupes de lipides, ils jouent un rôle clé dans la formation de structures lamellaires denses entre les cornéocytes adjacents, assurant collectivement la barrière efficace vitale contre l'évaporation de l'eau et la protection contre la pénétration des agents étrangers. Des modifications du taux de céramides et de la composition relative, avec une altération potentielle de l'arrangement lipidique, ont été observées dans différentes affections cutanées et maladies cutanées. Par conséquent, l'utilisation de céramides formulés de manière appropriée a été proposée pour un traitement topique afin d'aider à restructurer la disposition des lipides endommagés et à réparer la fonction de barrière cutanée altérée. Néanmoins, la formulation des céramides dans les produits nécessite des processus spécifiques tels que le chauffage à température élevée avant leur introduction dans la formule finale. Dans cette revue sur la structure, le rôle et le potentiel des céramides pour les soins de la peau, nous soulignons la nécessité d'un processus rigoureux lors de la formulation des céramides dans le produit final. Nous démontrons les effets contreproductifs des céramides non dissous sur la capacité de réparation de la barrière cutanée des formules, lorsqu'ils sont évalués dans différents modèles in vitro de barrière cutanée perturbée.
Assuntos
Ceramidas , Higiene da Pele , Pele , Ceramidas/química , Humanos , Pele/metabolismo , Higiene da Pele/métodosRESUMO
Sphingolipids are well known to promote keratinocyte differentiation and to induce ceramide production. In addition, they show anti-inflammatory and antimicrobial activities. Thus, the aim of this study is to investigate the potential effect of sphinganine on prolonging the hair anagen rate and improving the overall hair quality and scalp health. The inhibitory potential of sphinganine toward 5-α-reductase was studied using an in vitro assay. The stimulation of the antimicrobial peptide HBD2 by sphinganine was measured by real-time polymerase chain reaction and immunostaining. Sphinganine bioavailability was studied ex vivo using a pig skin model. A placebo-controlled, double-blind study was designed to evaluate the efficacy of sphinganine on hair loss and hair/scalp quality in vivo. In vitro results showed that sphinganine is a potent inhibitor of 5-α-reductase type I that prevents the conversion of testosterone to dihydrotestosterone, a key factor of androgenetic male baldness. In vivo results demonstrated efficacy in reducing non-illness-related hair loss among males. In terms of expert rating, all hair quality and scalp parameters improved after application of sphinganine. Improved scalp health might be linked to the observed increase of the antimicrobial peptide HBD2. Thus, sphinganine is well suited as a topical alternative for the improvement of scalp health and hair quality and anti-hair loss application.
RESUMO
Ceramides are the major lipid of lamellar sheets present in intercellular spaces of the stratum corneum contributing to epidermal barrier properties. Therefore, ceramides and their analogues have been studied for barrier enhancing and water-holding properties for decades. In vitro studies have indicated cytotoxic potential for cell-permeable ceramides thereby raising the question whether topical ceramide application might contribute to UVB-induced apoptosis. Phytosphingosine, N-hexanoyl-phytosphingosine and N-stearoylphytosphingosine (ceramide III) in concentrations ≤5 µm have been used for co-stimulation with low (160 J/m(2) ) or high (600 J/m(2) ) UVB doses in subconfluent basal and confluent differentiating keratinocytes. Significantly, increased caspase-3 activity was observed in basal keratinocytes irradiated with 600 J/m(2) UVB and in differentiating keratinocytes with both UVB doses. Co-stimulation with the named ceramides did not further increase (i) caspase-3 activity and (ii) nucleosomal fragmentation in differentiating keratinocytes. Moreover, co-stimulation with 1-mm ceramides did not further affect viability/lactate dehydrogenase release in UVB-irradiated reconstructed human epidermis corroborating the safety of these ceramides.
Assuntos
Administração Tópica , Apoptose , Ceramidas/administração & dosagem , Epiderme/efeitos da radiação , Queratinócitos/efeitos da radiação , Caspase 3/metabolismo , Diferenciação Celular , Sobrevivência Celular , Ceramidas/química , Epiderme/efeitos dos fármacos , Humanos , Queratinócitos/citologia , Queratinócitos/efeitos dos fármacos , Pele , Esfingosina/análogos & derivados , Esfingosina/química , Raios Ultravioleta , Água/químicaRESUMO
Keratinocyte sphingolipids are structural elements of epidermal permeability barrier and potential regulators of epidermal functions. We tested the influence of sphingoid bases sphinganine, sphingosine and phytosphingosine on in vitro keratinocyte differentiation. Lipidomic and transcriptomic analysis after treatment emphasizes sphinganine and phytosphingosine as potent modulators of keratinocyte differentiation and lipid metabolism. Sphinganine treatment regulated differentiation and sphingolipid metabolism-related genes, and also increased all major ceramide species. Sphingosine treatment increased ceramide and phytoceramide pools without changes in dihydroceramides. Phytosphingosine treatment markedly increased phytoceramide pools without raising ceramide or dihydroceramide levels. Sphinganine treatment increased specifically very long chain ceramides essential for intact barrier function. In summary, sphingoid bases, especially sphinganine, promote differentiation and ceramide production in keratinocytes. Free sphinganine may serve as a dermatological and cosmetic agent by enhancing formation and maintenance of an intact epidermal lipid barrier, with beneficial effects for skin and hair care applications.
Assuntos
Diferenciação Celular/efeitos dos fármacos , Queratinócitos/citologia , Queratinócitos/efeitos dos fármacos , Pele/efeitos dos fármacos , Esfingolipídeos/química , Células Cultivadas , Ceramidas/química , Cosméticos , Ácidos Graxos/química , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Lipídeos/química , Permeabilidade , Pele/metabolismo , Esfingosina/análogos & derivados , Esfingosina/química , Fatores de TempoRESUMO
Wickerhamomyces ciferrii is a microorganism characterized by the production and secretion of large amounts of acetylated sphingoid bases, in particular tetraacetyl phytosphingosine. Here, we present the 15.90-Mbp draft genome sequence of W. ciferrii NRRL Y-1031 F-60-10 generated by pyrosequencing and de novo assembly. The draft genome sequence comprising 364 contigs in 150 scaffolds was annotated and covered 6,702 protein-coding sequences. This information will contribute to the metabolic engineering of this yeast to improve the yield and spectrum of acetylated sphingoid bases in biotechnological production.
Assuntos
Genoma Fúngico , Pichia/genética , Sequência de Bases , Mapeamento de Sequências Contíguas , Bases de Dados Genéticas , Anotação de Sequência Molecular , Dados de Sequência MolecularRESUMO
The study describes the identification of sphingolipid biosynthesis genes in the non-conventional yeast Pichia ciferrii, the development of tools for its genetic modification as well as their application for metabolic engineering of P. ciferrii with the goal to generate strains capable of producing the rare sphingoid bases sphinganine and sphingosine. Several canonical genes encoding ceramide synthase (encoded by PcLAG1 and PcLAF1), alkaline ceramidase (PcYXC1) and sphingolipid C-4-hydroxylase(PcSYR2), as well as structural genes for dihydroceramide Δ(4)-desaturase (PcDES1) and sphingolipid Δ(8)-desaturase (PcSLD1) were identified, indicating that P. ciferrii would be capable of synthesizing desaturated sphingoid bases, a property not ubiquitously found in yeasts. In order to convert the phytosphingosine-producing P. ciferrii wildtype into a strain capable of producing predominantly sphinganine, Syringomycin E-resistant mutants were isolated. A stable mutant almost exclusively producing high levels of acetylated sphinganine was obtained and used as the base strain for further metabolic engineering. A metabolic pathway required for the three-step conversion of sphinganine to sphingosine was implemented in the sphinganine producing P. ciferrii strain and subsequently enhanced by screening for the appropriate heterologous enzymes, improvement of gene expression and codon optimization. These combined efforts led to a strain capable of producing 240mgL(-1) triacetyl sphingosine in shake flask, with tri- and diacetyl sphinganine being the main by-products. Lab-scale fermentation of this strain resulted in production of up to 890mgkg(-1) triacetyl sphingosine. A third by-product was unequivocally identified as triacetyl sphingadienine. It could be shown that inactivation of the SLD1 gene in P. ciferrii efficiently suppresses triacetyl sphingadienine formation. Further improvement of the described P. ciferrii strains will enable a biotechnological route to produce sphinganine and sphingosine for cosmetic and pharmaceutical applications.
Assuntos
Engenharia Metabólica/métodos , Pichia/enzimologia , Esfingosina/análogos & derivados , Esfingosina/biossíntese , Ceramidase Alcalina/genética , Ceramidase Alcalina/metabolismo , Oxigenases de Função Mista/genética , Oxigenases de Função Mista/metabolismo , Oxirredutases/genética , Oxirredutases/metabolismo , Pichia/genética , Esfingosina/genéticaRESUMO
Uneven skin pigmentation is a significant cosmetic concern, and the identification of topically applicable molecules to address this issue is of general interest. We report that the tetrapeptide PKEK (Pro-Lys-Glu-Lys) can exert skin whitening effects based on one in vitro and four double-blinded vehicle-controlled in vivo studies. (i) Treatment of human keratinocytes with PKEK significantly reduced UVB-stimulated mRNA expression of interleukin (IL)-6, IL-8 and TNF-α and, most importantly, proopiomelanocorticotropin (POMC), i.e. a gene encoding the pigmentation-inducing soluble mediator α- (α-MSH). (ii) PKEK treatment significantly inhibited UVB-induced upregulation of genes encoding for IL-1α, IL-6, IL-8, TNF-α as well as POMC and tyrosinase in 10 healthy volunteers pretreated with PKEK for 4 weeks once daily. (iii) In a study enrolling 39 Caucasian women, facial pigment spots significantly faded after 6 weeks when PKEK was combined with the skin whitener sodium ascorbyl phosphate (SAP), whereas PKEK or SAP alone led to less pronounced fading of the pigment spots. (iv) Addition of PKEK enhanced the skin whitening potency of a SAP-containing preparation if applied for 8 weeks to the back of hands of 19 Caucasians. (v) 27 Japanese women were treated on their faces twice daily with an SAP only or a PKEK+SAP-containing formulation for 8 weeks. Application of PKEK+SAP significantly reduced skin pigmentation by 26% and by 18% according to SCINEXA score. We demonstrate that PKEK has the capacity to reduce UVB-induced skin pigmentation and may be suited to serve as a skin tone-modulating agent in cosmetic products.
Assuntos
Oligopeptídeos/farmacologia , Oligopeptídeos/uso terapêutico , Pigmentação da Pele/efeitos dos fármacos , Pele/efeitos dos fármacos , Adulto , Idoso , Ácido Ascórbico/análogos & derivados , Ácido Ascórbico/farmacologia , Ácido Ascórbico/uso terapêutico , Povo Asiático , Células Cultivadas , Colorimetria , Método Duplo-Cego , Feminino , Expressão Gênica/efeitos dos fármacos , Expressão Gênica/efeitos da radiação , Humanos , Interleucina-1beta/genética , Interleucina-6/genética , Interleucina-8/genética , Queratinócitos/efeitos dos fármacos , Queratinócitos/metabolismo , Queratinócitos/efeitos da radiação , Masculino , Pessoa de Meia-Idade , Monofenol Mono-Oxigenase/genética , Pró-Opiomelanocortina/metabolismo , Pele/anatomia & histologia , Pele/metabolismo , Pele/efeitos da radiação , Envelhecimento da Pele/efeitos dos fármacos , Pigmentação da Pele/efeitos da radiação , Resultado do Tratamento , Fator de Necrose Tumoral alfa/genética , Raios Ultravioleta , População BrancaRESUMO
BACKGROUND: Due to its strong water-binding potential, hyaluronic acid (HA) is a well-known active ingredient for cosmetic applications. Native HA is proposed to help the skin to retain and maintain elasticity, turgor and moisture. OBJECTIVE: To observe the efficacy of topical application of 0.1% hyaluronan formulations of different molecular weights (MW) (50, 130, 300, 800 and 2000 kDa, respectively) in the periocular area as anti-wrinkle treatment. MATERIAL AND METHODS: Seventy-six female subjects between 30 and 60 years of age with clinical signs of periocular wrinkles applied one of the formulations twice-daily to the area of interest in a randomized fashion for 60 days. Around the other eye, a vehicle control cream was applied. Measurements of skin hydration and skin elasticity were performed before treatment, 30 and 60 days thereafter. At similar time points negative replicas were taken and evaluated by semi-automated morphometry. RESULTS: All HA-based creams utilized in this study demonstrated a significant improvement in skin hydration and overall elasticity values (R2) when compared to placebo. Measurements of wrinkle depth using mean roughness (Ra) and maximum roughness (Rz) values revealed significant improvement in the 130 and the 50 kDa HA group after 60 days of treatment compared to placebo-treated area. CONCLUSION: Topical application of all 0.1% HA formulations used in this study led to significant improvement in skin hydration and elasticity. Application of low-molecular-weight (LMW) HA was associated with significant reduction of wrinkle depth, which may be due to better penetration abilities of LMW HA.
Assuntos
Técnicas Cosméticas , Ácido Hialurônico/administração & dosagem , Absorção Cutânea , Envelhecimento da Pele/efeitos dos fármacos , Administração Cutânea , Adulto , Elasticidade/efeitos dos fármacos , Feminino , Humanos , Ácido Hialurônico/química , Ácido Hialurônico/farmacocinética , Pessoa de Meia-Idade , Peso Molecular , Permeabilidade , Fatores de Tempo , Resultado do TratamentoRESUMO
BACKGROUND: Irregular skin pigmentation may be a substantial contributor to the signs of aging and to a person's lack of psychological well-being. Although a large number of skin-lightening agents are available, the opportunity exists to identify more efficacious agents, agents that target alternative biological mechanisms. AIMS: To provide clinical evidence of the skin-lightening effect of the tetrapeptide, Pro-Lys-Glu-Lys (PKEK), on subjects with skin types V-VI living in South Africa. METHODS: Pro-Lys-Glu-Lys was evaluated in a double-blind and vehicle-controlled clinical study using expert grading of digital images by comparing its effects in subjects with skin types V-VI suffering from facial melasma and postinflammatory hyperpigmentation. RESULTS: This study demonstrated the efficacy of PKEK on subjects with skin types V-VI. On comparing the two treatments, the skin-lightening peptide-containing formulation was significantly superior to the vehicle at 12 weeks on overall appearance (P < 0.05) and evenness of skin tone (P < 0.01). CONCLUSIONS: The tetrapeptide, PKEK, has proven skin-lightening benefits on skin discoloration from melasma and postinflammatory hyperpigmentation. These studies have been conducted on subjects with skin types V-VI living in South Africa, but we believe this technology to be suitable for all racial groups.
Assuntos
Fármacos Dermatológicos/uso terapêutico , Melanose/tratamento farmacológico , Oligopeptídeos/uso terapêutico , Adulto , Método Duplo-Cego , Face , Feminino , Ácido Glutâmico/administração & dosagem , Humanos , Lisina/administração & dosagem , Pessoa de Meia-Idade , Prolina/administração & dosagem , Pigmentação da Pele/efeitos dos fármacos , África do Sul , Resultado do TratamentoRESUMO
The 'matrikine' concept claims that processing of the precursors for collagen results in the formation of peptides such as KTTKS which in turn augments extracellular matrix (ECM) production. In the present study, we show the development of an anti-ageing active from an in silico approach by molecular design resulting in the tetrapeptide GEKG derived from ECM proteins. The efficacy of the peptide to significantly induce collagen production of the protein level and mRNA level has been demonstrated in vitro in human dermal fibroblasts and in vivo in a double-blind, randomized, placebo-controlled study enroling 10 volunteers with an average age of 48.2 years. The effect of GEKG on facial wrinkles was studied in 30 volunteers using state of the art fringe projection, which allows determination of surface roughness in three-dimensions. Here, only GEKG but not the placebo was able to significantly decrease skin roughness as a measure for wrinkles.
Assuntos
Matriz Extracelular/metabolismo , Oligopeptídeos/farmacologia , Envelhecimento da Pele/efeitos dos fármacos , Adulto , Células Cultivadas , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Cadeia alfa 1 do Colágeno Tipo I , Fármacos Dermatológicos/farmacologia , Fármacos Dermatológicos/uso terapêutico , Método Duplo-Cego , Elasticidade/efeitos dos fármacos , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Fibronectinas/genética , Fibronectinas/metabolismo , Expressão Gênica/genética , Glucuronosiltransferase/genética , Humanos , Hialuronan Sintases , Ácido Hialurônico/metabolismo , Oligopeptídeos/uso terapêutico , Pró-Colágeno/metabolismo , Pele/efeitos dos fármacos , Pele/metabolismo , Fenômenos Fisiológicos da Pele/efeitos dos fármacosRESUMO
Keratinocyte differentiation plays a pivotal role in the epidermal barrier. Single keratinocyte differentiation genes have already been studied, but many important constituents of this process may have been missed so far. Gene expression profiling by microarray was carried out in cultured normal human epidermal keratinocytes undergoing confluence-induced differentiation to find novel differentiation genes. Candidate gene lists were established and genes of potential dermatological interest were validated by quantitative reverse transcription polymerase chain reaction and immunohistochemical analysis. Some of these points lead to the identification of counter-regulation of heme oxygenase and biliverdin reductase as well as glutaredoxin and glutathione reductase indicative of potential novel redox signaling in differentiating human keratinocytes. Others indicate a strong concert down-regulation of interleukin-1 signaling at previously unidentified levels during keratinocyte differentiation. We believe that identified genes contribute to a more comprehensive understanding of the complicated epidermal differentiation process and lead to better understanding of dermatological diseases.
Assuntos
Diferenciação Celular/genética , Perfilação da Expressão Gênica , Queratinócitos/citologia , Queratinócitos/metabolismo , Redes Reguladoras de Genes , Genoma Humano , Humanos , Técnicas In Vitro , Análise de Sequência com Séries de OligonucleotídeosRESUMO
Sphingolipids are important components of the water permeability barrier of the skin. Moreover, ceramides were also shown to influence keratinocyte differentiation and regulate cellular signalling. A confluence-induced differentiation model of normal human keratinocytes was established to allow evaluation of pro- and anti-differentiation effects of exogenous compounds. The effects of phytosphingosine (PS), sphingosine (SO), sphinganine (SA) and their hexanoyl (-C6), stearoyl (-C18) and salicyl (-SLC) derivatives, C12-alkylamine-salicylate (C12-SLC), salicylate (SLC) along with vitamin D3 (VD3) and retinol as control substances were tested in this system. Cytotoxicity assays were carried out to optimize the incubation conditions of compounds and whole genome expression changes were monitored by DNA-microarray on days 0, 1 and 4. Geometric means of gene expression levels of a subset of known keratinocyte differentiation-related genes were calculated from the microarray data to compare effects of the sphingolipid derivatives. Compound treatment-induced transcriptional changes were analysed by the ExPlain software (BIOBASE GmbH). Five of the assayed substances (SA, SO-C6, PS-C6, SO-SLC, PS-SLC) were found to be potent promoters of keratinocyte differentiation compared with VD3, and C12-SLC revealed potential anti-differentiation properties. ExPlain analysis found a different regulatory profile in the computed transcriptional networks of the sphingoid bases versus their -C6 and especially -SLC derivatives suggesting that the change in their keratinocyte differentiation modifying potential is due to a unique effect of the covalent attachment of the salicylic acid. Taken together, these results demonstrate the gene regulatory potential of sphingolipid species that could be valuable for dermatological or cosmetic applications.
Assuntos
Diferenciação Celular/efeitos dos fármacos , Queratinócitos/efeitos dos fármacos , Esfingolipídeos/farmacologia , Adulto , Antígenos de Diferenciação/genética , Sequência de Bases , Sítios de Ligação , Diferenciação Celular/genética , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Células Cultivadas , Colecalciferol/farmacologia , Feminino , Proteínas Filagrinas , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/efeitos dos fármacos , Glicoproteínas/genética , Humanos , Peptídeos e Proteínas de Sinalização Intercelular , Proteínas de Filamentos Intermediários/genética , Queratina-10/genética , Queratinócitos/citologia , Queratinócitos/metabolismo , Pessoa de Meia-Idade , Modelos Genéticos , Dados de Sequência Molecular , Análise de Sequência com Séries de Oligonucleotídeos , Regiões Promotoras Genéticas/genética , Salicilatos/farmacologia , Transglutaminases/genética , Vitamina A/farmacologiaRESUMO
AmtR, the master regulator of nitrogen control in Corynebacterium glutamicum, represses transcription of a number of genes during nitrogen surplus. Repression is released by an interaction of AmtR with signal transduction protein GlnK. As shown by pull-down assays and gel retardation experiments, only adenylylated GlnK, which is present in the cells during nitrogen limitation, is able to bind to AmtR. The AmtR regulon was characterized in this study by a combination of bioinformatics, transcriptome and proteome analyses. At least 33 genes are directly controlled by the repressor protein including those encoding transporters and enzymes for ammonium assimilation (amtA, amtB, glnA, gltBD), urea and creatinine metabolism (urtABCDE, ureABCEFGD, crnT, codA), a number of biochemically uncharacterized enzymes and transport systems (NCgl1099, NCgl1100, NCgl 1915-1918) as well as signal transduction proteins (glnD, glnK). For the AmtR regulon, an AmtR box has been defined which comprises the sequence tttCTATN6AtAGat/aA. Furthermore, the transcriptional organization of AmtR-regulated genes and operons was characterized.
Assuntos
Corynebacterium glutamicum/metabolismo , Regulação Bacteriana da Expressão Gênica , Regulon , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sequência de Bases , Biologia Computacional/métodos , Corynebacterium glutamicum/genética , Perfilação da Expressão Gênica , Dados de Sequência Molecular , Família Multigênica , Nitrogênio/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Proteoma/análise , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Transdução de Sinais/fisiologia , Transcrição GênicaRESUMO
The amino acid producing Corynebacterium glutamicum grows aerobically on a variety of carbohydrates and organic acids as single or combined sources of carbon and energy. Among the substrates metabolized are glucose and acetate which both can also serve as substrates for amino acid production. Based on biochemical, genetic and regulatory studies and on quantitative determination of metabolic fluxes during utilization of acetate and/or glucose, this review summarizes the present knowledge on the different steps of the fundamental pathways of acetate utilization in C. glutamicum, namely, on acetate transport, acetate activation, tricarboxylic acid (TCA) cycle, glyoxylate cycle and gluconeogenesis. It becomes evident that, although the pathways of acetate utilization follow the same theme in many bacteria, important biochemical, genetic and regulatory peculiarities exist in C. glutamicum. Recent genome wide and comparative expression analyses in C. glutamicum cells grown on glucose and on acetate substantiated previously identified transcriptional regulation of acetate activating enzymes and of glyoxylate cycle enzymes. Additionally, a variety of genes obviously also under transcriptional control in response to the presence or absence of acetate in the growth medium were uncovered. These genes, thus also belonging to the acetate stimulon of C. glutamicum, include genes coding for TCA cycle enzymes (e.g. aconitase and succinate dehydrogenase), for gluconeogenesis (phosphoenolpyruvate carboxykinase), for glycolysis (pyruvate dehydrogenase E1) and genes coding for proteins with hitherto unknown function. Although the basic mechanism of transcriptional regulation of the enzymes involved in acetate metabolism is not yet understood, some recent findings led to a better understanding of the adaptation of C. glutamicum to acetate at the molecular level.
Assuntos
Acetatos/metabolismo , Proteínas de Bactérias/metabolismo , Ciclo do Ácido Cítrico/fisiologia , Corynebacterium/genética , Corynebacterium/metabolismo , Regulação Bacteriana da Expressão Gênica/fisiologia , Gluconeogênese/fisiologia , Complexos Multienzimáticos/metabolismo , Adaptação Fisiológica/fisiologia , Proteínas de Bactérias/genética , Corynebacterium/crescimento & desenvolvimento , Glioxilatos/metabolismo , Homeostase/fisiologiaRESUMO
In this study, the phosphoproteome of Corynebacterium glutamicum, an industrially important soil bacterium of the Corynebacterium/Mycobacterium/Nocardia (CMN) group of Gram-positive bacteria, was investigated by two different detection methods: first, by in vivo radio-labeling using [(33)P]-phosphoric acid with subsequent autoradiography and second, by immunostaining with phosphoamino acid-specific monoclonal antibodies. After two-dimensional gel electrophoresis (2-DE), around 60 [(33)P]-labeled protein spots were visualized and around 90 antibody-decorated protein spots detected; 31 of the protein spots were detected with both methods. By peptide mass fingerprinting, 41 different proteins were identified, namely 5-enolpyruvylshikimate 3-phosphate synthase, aconitase, acyl-CoA carboxylase, acyl-CoA synthetase, ATP (synthase alpha- and beta-chain), carbamoyl-phosphate synthase, citrate synthase, cysteine synthase, DnaK, the elongation factors G, P, Ts and Tu, enolase, fructose bisphosphate aldolase, fumarase, Gap dehydrogenase, glutamine synthetase I, glycine hydroxymethyltransferase, GroEL2, GTPase, heat-inducible transcriptional repressor DnaJ2, inorganic pyrophosphatase, isocitrate dehydrogenase, ketol-acid reductoisomerase, lactate dehydrogenase, leucine-tRNA ligase, lipoamide dehydrogenase, methionine synthase, O-acetylhomoserine sulfhydrylase, pyruvate carboxylase, pyruvate kinase, pyruvate oxidase, ribosomal protein S1, RNA polymerase (beta-subunit), succinyl-CoA:CoA transferase, transketolase and UDP-N-acetylmuramoyl-L-alanine ligase, besides a hypothetical 35k protein and a hypothetical glucose kinase. Both detection techniques were used to create a phosphoproteome map. Additionally, the influence of nitrogen deprivation on the phosphoproteome of C. glutamicum was investigated.