RESUMO
As the number of antibody drugs being approved and marketed increases, our knowledge of what makes potential drug candidates a successful product has increased tremendously. One of the critical parameters that have become clear in the field is the importance of mAb "developability." Efforts are being increasingly focused on simultaneously selecting molecules that exhibit both desirable biological potencies and manufacturability attributes. In the current study mutations to improve the developability profile of a problematic antibody that inconsistently precipitates in a batch scale-dependent fashion using a standard platform purification process are described. Initial bioinformatic analysis showed the molecule has no obvious sequence or structural liabilities that might lead it to precipitate. Subsequent analysis of the molecule revealed the presence of two unusual positively charged mutations on the light chain at the interface of VH and VL domains, which were hypothesized to be the primary contributor to molecule precipitation during process development. To investigate this hypothesis, straightforward reversion to the germline of these residues was carried out. The resulting mutants have improved expression titers and recovered stability within a forced precipitation assay, without any change to biological activity. Given the time pressures of drug development in industry, process optimization of the lead molecule was carried out in parallel to the "retrospective" mutagenesis approach. Bespoke process optimization for large-scale manufacturing was successful. However, we propose that such context-dependent sequence liabilities should be included in the arsenal of in silico developability screening early in development; particularly since this specific issue can be efficiently mitigated without the requirement for extensive screening of lead molecule variants.
Assuntos
Anticorpos Monoclonais , Engenharia de Proteínas , Anticorpos Monoclonais/biossíntese , Anticorpos Monoclonais/genética , Linhagem Celular , Humanos , SolubilidadeRESUMO
Protein post-translational modification (PTM) plays an important role in many biological processes; of which glycosylation is arguably one of the most complex and diverse modifications and is crucial for the safety and efficacy of biotherapeutic proteins. Mass spectrometric characterization of protein glycosylation is well established with clear advantages and disadvantages; on one hand it is precise and information-rich, as well as being relative inexpensive in terms of the reagents and consumables despite the instrumentation cost and, depending on the method, can give site specific information; on the other hand it generally suffers from low throughput, restriction to largely purified samples and is less quantitative, especially for sialylated glycan species. Here, we describe a high throughput, site-specific, targeted mass spectrometric peptide mapping approach to quickly screen/rank candidate production cell lines and culture conditions that give favourable glycosylation profiles directly from conditioned culture media for an Fc-fusion protein. The methodology is fully compatible with automation and combines the speed of 'top-down' mass spectrometry with the site-specific information of 'bottom-up' mass spectrometry. In addition, this strategy can be used for multi-attribute product quality screening/monitoring as an integral part of cell line selection and process development.
RESUMO
Human pharmaceuticals have been detected in wastewater treatment plants, rivers, and estuaries throughout Europe and the United States. It is widely acknowledged that there is insufficient information available to determine whether prolonged exposure to low levels of these substances is having an impact on the microbial ecology in such environments. In this study we attempt to measure the effects of exposing cultures of Pseudomonas putida KT2440 (UWC1) to six pharmaceuticals by looking at differences in metabolite levels. Initially, we used Fourier transform infrared (FT-IR) spectroscopy coupled with multivariate analysis to discriminate between cell cultures exposed to different pharmaceuticals. This suggested that on exposure to propranolol there were significant changes in the lipid complement of P. putida. Metabolic profiling with gas chromatography-mass spectrometry (GC-MS), coupled with univariate statistical analyses, was used to identify endogenous metabolites contributing to discrimination between cells exposed to the six drugs. This approach suggested that the energy reserves of exposed cells were being expended and was particularly evident on exposure to propranolol. Adenosine triphosphate (ATP) concentrations were raised in P. putida exposed to propranolol. Increased energy requirements may be due to energy dependent efflux pumps being used to remove propranolol from the cell.
Assuntos
Metaboloma , Metabolômica , Preparações Farmacêuticas , Pseudomonas putida/efeitos dos fármacos , Pseudomonas putida/metabolismo , Trifosfato de Adenosina/metabolismo , Análise de Variância , Cromatografia Gasosa-Espectrometria de Massas , Metabolômica/métodos , Propranolol/farmacologia , Espectroscopia de Infravermelho com Transformada de FourierRESUMO
Whilst development of medium and feeds has provided major advances in recombinant protein production in CHO cells, the fundamental understanding is limited. We have applied metabolite profiling with established robust (GC-MS) analytics to define the molecular loci by which two yield-enhancing feeds improve recombinant antibody yields from a model GS-CHO cell line. With data across core metabolic pathways, that report on metabolism within several cellular compartments, these data identify key metabolites and events associated with increased cell survival and specific productivity of cells. Of particular importance, increased process efficiency was linked to the functional activity of the mitochondria, with the amount and time course of use/production of intermediates of the citric acid cycle, for uses such as lipid biosynthesis, precursor generation and energy production, providing direct indicators of cellular status with respect to productivity. The data provide clear association between specific cellular metabolic indicators and cell process efficiency, extending from prior indications of the relevance of lactate metabolic balance to other redox sinks (glycerol, sorbitol and threitol). The information, and its interpretation, identifies targets for engineering cell culture efficiency, either from genetic or environmental perspectives, and greater understanding of the significance of specific medium components towards overall CHO cell bioprocessing.
Assuntos
Biotecnologia/métodos , Meios de Cultura/metabolismo , Metabolômica/métodos , Proteínas Recombinantes/metabolismo , Animais , Células CHO , Cricetinae , Cricetulus , Cromatografia Gasosa-Espectrometria de Massas , Espaço Intracelular/metabolismoRESUMO
UV resonance Raman (UVRR) spectroscopy combined with chemometric techniques was investigated as a physiochemical tool for monitoring secreted recombinant antibody production in cultures of Chinese hamster ovary (CHO) cells. Due to the enhanced selectivity of the UVRR, spectral variations arising from protein, small molecule substrates, and nucleic acid medium components could be measured simultaneously and we have successfully determined antibody titre. Medium samples were taken during culture of three CHO cell lines: two antibody-producing cell lines and a non-producing cell line, and analysed by UVRR spectroscopy using an excitation laser of 244 nm. Principal component analysis (PCA) was applied to the spectral sets and showed a linear trend over time for the antibody-producing cell lines that was not observed in the non-producing cell line. Partial least squares regression (PLSR) was used to predict antibody titres, glucose utilization and lactate accumulation, and compared very favourably with gold standard data acquired with the much slower techniques of ELISA and liquid chromatography. Further analysis of the UVRR spectral sets using two-dimensional correlation moving windows also revealed that spectral variations due to protein and nucleic acid concentrations in the medium during cell culture varied between each of the three cell lines investigated.
Assuntos
Formação de Anticorpos , Proteínas Recombinantes/análise , Análise Espectral Raman , Animais , Células CHO , Linhagem Celular , Cromatografia Líquida de Alta Pressão , Cricetinae , Cricetulus , Ensaio de Imunoadsorção Enzimática , Análise de Componente PrincipalRESUMO
Metabolite profiling of industrially important suspension-cultured mammalian cells is being increasingly used for rational improvement of bioprocesses. This requires the generation of global metabolite profiles that cover a broad range of metabolites and that are representative of the cells at the time of sampling. The protocol described here is a validated method for recovery of physiologically relevant amounts of key metabolites from suspension-cultured mammalian cells. The method is a two-step process consisting of initial quenching of the cells (to stop cellular metabolism and allow isolation of the cells) followed by extraction of the metabolites. The cells are quenched in 60% methanol supplemented with 0.85% (wt/vol) ammonium bicarbonate at -40 °C. Metabolites are then extracted from the quenched cells using two 100% methanol extractions followed by a single water extraction. Metabolite samples generated using this protocol are amenable to analysis by mass spectrometry-based techniques (e.g., gas chromatography-mass spectrometry, liquid chromatography-mass spectrometry), NMR spectroscopy and enzymatic assays.
Assuntos
Técnicas de Cultura de Células , Cricetulus/metabolismo , Metaboloma , Metabolômica/métodos , Animais , Células CHO , Cricetinae , Cromatografia Gasosa-Espectrometria de MassasRESUMO
Chinese hamster ovary (CHO) cells are the primary platform for commercial expression of recombinant therapeutic proteins. Obtaining maximum production from the expression platform requires optimal cell culture medium (and associated nutrient feeds). We have used metabolite profiling to define the balance of intracellular and extracellular metabolites during the production process of a CHO cell line expressing a recombinant IgG4 antibody. Using this metabolite profiling approach, it was possible to identify nutrient limitations, which acted as bottlenecks for antibody production, and subsequently develop a simple feeding regime to relieve these metabolic bottlenecks. This metabolite profiling-based strategy was used to design a targeted, low cost nutrient feed that increased cell biomass by 35% and doubled the antibody titer. This approach, with the potential for utilization in non-specialized laboratories, can be applied universally to the optimization of production of commercially important biopharmaceuticals.
Assuntos
Células CHO/química , Meios de Cultura/química , Imunoglobulina G/biossíntese , Metaboloma , Animais , Células CHO/metabolismo , Técnicas de Cultura de Células/métodos , Cricetinae , Cricetulus , Imunoglobulina G/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Tecnologia Farmacêutica/métodosRESUMO
Recombinant monoclonal antibodies (MAbs) are increasingly being used for therapeutic use and correct glycosylation of these MAbs is essential for their correct function. Glycosylation profiles are host cell- and antibody class-dependent and can change over culture time and environmental conditions. Therefore, rapid monitoring of glycan addition/status is of great importance for process validity. We describe two workflows of generally applicability for glycan profiling of purified and gel-purified MAbs produced in NS0 and CHO cells, in which small-scale antibody purification and buffer exchange is combined with PNGase F glycan cleavage and graphite HyperCarb desalting. MALDI-ToF mass spectrometry is used for sensitive detection of glycan forms, with the ability to confirm glycan structures by selective ion fragmentation. Both workflows are rapid, technically simple and amenable to automation, and use in multi-well formats.
Assuntos
Anticorpos Monoclonais/química , Anticorpos Monoclonais/isolamento & purificação , Polissacarídeos/análise , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Animais , Anticorpos Monoclonais/metabolismo , Linhagem Celular , Eletroforese em Gel de PoliacrilamidaRESUMO
Fourier transform infrared (FT-IR) spectroscopy combined with multivariate statistical analyses was investigated as a physicochemical tool for monitoring secreted recombinant antibody production in cultures of Chinese hamster ovary (CHO) and murine myeloma non-secreting 0 (NS0) cell lines. Medium samples were taken during culture of CHO and NS0 cells lines, which included both antibody-producing and non-producing cell lines, and analyzed by FT-IR spectroscopy. Principal components analysis (PCA) alone, and combined with discriminant function analysis (PC-DFA), were applied to normalized FT-IR spectroscopy datasets and showed a linear trend with respect to recombinant protein production. Loadings plots of the most significant spectral components showed a decrease in the C-O stretch from polysaccharides and an increase in the amide I band during culture, respectively, indicating a decrease in sugar concentration and an increase in protein concentration in the medium. Partial least squares regression (PLSR) analysis was used to predict antibody titers, and these regression models were able to predict antibody titers accurately with low error when compared to ELISA data. PLSR was also able to predict glucose and lactate amounts in the medium samples accurately. This work demonstrates that FT-IR spectroscopy has great potential as a tool for monitoring cell cultures for recombinant protein production and offers a starting point for the application of spectroscopic techniques for the on-line measurement of antibody production in industrial scale bioreactors.
Assuntos
Anticorpos/metabolismo , Biotecnologia/métodos , Meios de Cultura/química , Espectroscopia de Infravermelho com Transformada de Fourier , Animais , Linhagem Celular , Cricetinae , Cricetulus , Ensaio de Imunoadsorção Enzimática , Camundongos , Proteínas Recombinantes/metabolismoRESUMO
The galactokinase from Saccharomyces cerevisiae (ScGal1p) is a bifunctional protein. It is an enzyme responsible for the conversion of alpha-D-galactose into galactose 1-phosphate at the expense of ATP but can also function as a transcriptional inducer of the yeast GAL genes. For both of these activities, the protein requires two ligands; a sugar (galactose) and a nucleotide (ATP). Here we investigate the effect of these ligands on the stability and conformation of ScGal1p to determine how the ligands alter protein function. We show that nucleotide binding increases the thermal stability of ScGal1p, whereas binding of galactose alone had no effect on the stability of the protein. This nucleotide stabilization effect is also observed for the related proteins S. cerevisiae Gal3p and Kluyveromyces lactis Gal1p and suggests that nucleotide binding results in the formation of, or the unmasking of, the galactose-binding site. We also show that the increase in stability of ScGal1p does not result from a large conformational change but is instead the result of a smaller more energetically favorable stabilization event. Finally, we have used mutant versions of ScGal1p to show that the galactokinase and transcriptional induction functions of the protein are distinct and separable. Mutations resulting in constitutive induction do not function by mimicking the more stable active conformation but have highlighted a possible site of interaction between ScGal1p and ScGal80p. These data give significant insights into the mechanism of action of both a galactokinase and a transcriptional inducer.
Assuntos
Galactoquinase/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Fatores de Transcrição/metabolismo , Transcrição Gênica/fisiologia , Trifosfato de Adenosina/genética , Trifosfato de Adenosina/metabolismo , Sítios de Ligação/fisiologia , Estabilidade Enzimática/fisiologia , Galactoquinase/genética , Galactosefosfatos/genética , Galactosefosfatos/metabolismo , Kluyveromyces/enzimologia , Kluyveromyces/genética , Ligantes , Mutação , Ligação Proteica/fisiologia , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Fatores de Transcrição/genéticaRESUMO
Global metabolite analysis approaches, coupled with sophisticated data analysis and modeling procedures (metabolomics), permit a dynamic read-out of how cellular proteins interact with cellular and environmental conditions to determine cell status. This type of approach has profound potential for understanding, and subsequently manipulating, the regulation of cell function. As part of our study to define the regulatory events that may be used to maximize production of commercially valuable recombinant proteins from cultured mammalian cells, we have optimized the quenching process to allow retention of physiologically relevant intracellular metabolite profiles in samples from recombinant Chinese hamster ovary (CHO) cells. In a comparison of a series of candidate quenching procedures, we have shown that quenching in 60% methanol supplemented with 0.85% ammonium bicarbonate (AMBIC) at -40 degrees C generates a profile of metabolites that is representative of a physiological status based upon examination of key labile cellular metabolites. This represents a key feature for any metabolomic study with suspension cultured mammalian cells and provides confidence in the validity of subsequent data analysis and modeling procedures.
Assuntos
Metabolômica/métodos , Nucleotídeos de Adenina/análise , Nucleotídeos de Adenina/metabolismo , Animais , Bicarbonatos/química , Células CHO , Células Cultivadas , Cricetinae , Cricetulus , Cromatografia Gasosa-Espectrometria de Massas , Glucose-6-Fosfato/análise , Glucose-6-Fosfato/metabolismo , Metanol/química , NAD/análise , NAD/metabolismo , RatosRESUMO
The enzymes of the Leloir pathway catalyze the conversion of galactose to a more metabolically useful version, glucose-6-phosphate. This pathway is required as galactose itself cannot be used for glycolysis directly. In most organisms, including the yeast Saccharomyces cerevisiae, five enzymes are required to catalyze this conversion: a galactose mutarotase, a galactokinase, a galactose-1-phosphate uridyltransferase, a UDP-galactose-4-epimerase, and a phosphoglucomutase. In yeast, the genes encoding these enzymes are tightly controlled at the level of transcription and are only transcribed under specific sets of conditions. In the presence of glucose, the genes encoding the Leloir pathway enzymes (often called the GAL genes) are repressed through the action of a transcriptional repressor Mig1p. In the presence of galactose, but in the absence of glucose, the concerted actions of three other proteins Gal4p, Gal80p, and Gal3p, and two small molecules (galactose and ATP) enable the rapid and high-level activation of the GAL genes. The precise molecular mechanism of the GAL genetic switch is controversial. Recent work on solving the three-dimensional structures of the various GAL enzymes proteins and the GAL transcriptional switch proteins affords a unique opportunity to delve into the precise, and potentially unambiguous, molecular mechanism of a highly exploited transcriptional circuit. Understanding the details of the transcriptional and metabolic events that occur in this pathway can be used as a paradigm for understanding the integration of metabolism and transcriptional control more generally, and will assist our understanding of fundamental biochemical processes and how these might be exploited.
Assuntos
Proteínas Fúngicas/biossíntese , Galactose/metabolismo , Regulação Enzimológica da Expressão Gênica/fisiologia , Regulação Fúngica da Expressão Gênica/fisiologia , Saccharomycetales/fisiologia , Transcrição Gênica/fisiologiaRESUMO
In yeast, the GAL genes encode the enzymes required for normal galactose metabolism. Regulation of these genes in response to the organism being challenged with galactose has served as a paradigm for eukaryotic transcriptional control over the last 50 years. Three proteins, the activator Gal4p, the repressor Gal80p, and the ligand sensor Gal3p, control the switch between inert and active gene expression. Gal80p, the focus of this investigation, plays a pivotal role both in terms of repressing the activity of Gal4p and allowing the GAL switch to respond to galactose. Here we present the three-dimensional structure of Gal80p from Kluyveromyces lactis and show that it is structurally homologous to glucose-fructose oxidoreductase, an enzyme in the sorbitol-gluconate pathway. Our results clearly define the overall tertiary and quaternary structure of Gal80p and suggest that Gal4p and Gal3p bind to Gal80p at distinct but overlapping sites. In addition to providing a molecular basis for previous biochemical and genetic studies, our structure demonstrates that much of the enzymatic scaffold of the oxidoreductase has been maintained in Gal80p, but it is utilized in a very different manner to facilitate transcriptional regulation.
Assuntos
Regulação Fúngica da Expressão Gênica , Proteínas Repressoras/química , Proteínas Repressoras/fisiologia , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/fisiologia , Transcrição Gênica , Cristalografia por Raios X , Proteínas de Ligação a DNA , Dimerização , Kluyveromyces/metabolismo , Modelos Moleculares , Oxirredutases/química , Ligação Proteica , Conformação Proteica , Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/químicaRESUMO
The crystal structure of the yeast galactokinase, Gal1p, in the presence of its substrates has been solved recently. We systematically mutated each of the amino acid side chains that, from the structure, are implicated to be involved in direct contact with the hydroxyl groups of the galactose ring. One of these mutations, D62A, abolished all detectable galactokinase activity but retained the ability to use d-glucose as a substrate. Mutation of Asp-62 to either leucine, phenylalanine, or histidine resulted in the formation of protein with similar characteristics to D62A. Yeast galactokinase is highly similar to Gal3p, the ligand sensor and transcriptional inducer of the GAL genes. Equivalent mutations in Gal3p also abolished its ability to respond to galactose and uncovered its ability to respond to d-glucose. It therefore appears that Gal1p and Gal3p respond to their substrates in a similar, perhaps identical, fashion. This work also validates the approach of screening for mutants in an easily assayable system prior to mutant analysis in a more experimentally difficult transcriptional regulator.
Assuntos
Galactoquinase/química , Proteínas de Saccharomyces cerevisiae/química , Fatores de Transcrição/química , Cristalografia por Raios X , Galactoquinase/genética , Galactoquinase/metabolismo , Galactose/química , Cinética , Ligantes , Modelos Moleculares , Conformação Molecular , Mutação , Ligação Proteica , Conformação Proteica , Estrutura Terciária de Proteína , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Especificidade por Substrato , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
The recognition of changes in environmental conditions, and the ability to adapt to these changes, is essential for the viability of cells. There are numerous well characterized systems by which the presence or absence of an individual metabolite may be recognized by a cell. However, the recognition of a metabolite is just one step in a process that often results in changes in the expression of whole sets of genes required to respond to that metabolite. In higher eukaryotes, the signalling pathway between metabolite recognition and transcriptional control can be complex. Recent evidence from the relatively simple eukaryote yeast suggests that complex signalling pathways may be circumvented through the direct interaction between individual metabolites and regulators of RNA polymerase II-mediated transcription. Biochemical and structural analyses are beginning to unravel these elegant genetic control elements.
Assuntos
Ativação Transcricional , Sítios de Ligação/genética , Proteínas de Ligação a DNA , Células Eucarióticas , Genes Fúngicos , Modelos Genéticos , Prolina/metabolismo , RNA Polimerase II/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Transdução de Sinais , Transativadores/química , Transativadores/genética , Transativadores/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Gal1p of Saccharomyces cerevisiae is capable of performing two independent cellular functions. First, it is a key enzyme in the Leloir pathway for galactose metabolism where it catalyzes the conversion of alpha-d-galactose to galactose 1-phosphate. Second, it has the capacity to induce the transcription of the yeast GAL genes in response to the organism being challenged with galactose as the sole source of carbon. This latter function is normally performed by a highly related protein, Gal3p, but in its absence Gal1p can induce transcription, albeit inefficiently, both in vivo and in vitro. Here we report the x-ray structure of Gal1p in complex with alpha-d-galactose and Mg-adenosine 5'-(beta,gamma-imido)triphosphate (AMPPNP) determined to 2.4 Angstrom resolution. Overall, the enzyme displays a marked bilobal appearance with the active site being wedged between distinct N- and C-terminal domains. Despite being considerably larger than other galactokinases, Gal1p shares a similar molecular architecture with these enzymes as well as with other members of the GHMP superfamily. The extraordinary levels of similarity between Gal1p and Gal3p ( approximately 70% amino acid identity and approximately 90% similarity) have allowed a model for Gal3p to be constructed. By identifying the locations of mutations of Gal3p that result in altered transcriptional properties, we suggest potential models for Gal3p function and mechanisms for its interaction with the transcriptional inhibitor Gal80p. The GAL genetic switch has long been regarded as a paradigm for the control of gene expression in eukaryotes. Understanding the manner in which two of the proteins that function in transcriptional regulation interact with one another is an important step in determining the overall molecular mechanism of this switch.
Assuntos
Galactoquinase/metabolismo , Galactose/metabolismo , Regulação Fúngica da Expressão Gênica , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Transativadores/química , Transativadores/metabolismo , Adenilil Imidodifosfato/metabolismo , Sítios de Ligação , Cristalização , Cristalografia por Raios X , Humanos , Complexo Mediador , Modelos Químicos , Conformação Proteica , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Saccharomyces cerevisiae/genética , Transativadores/genética , Fatores de Transcrição/química , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transcrição GênicaRESUMO
The recognition of changes in environmental conditions, and the ability to adapt to these changes, is essential for the viability of cells. There are numerous well-characterized systems by which the presence or absence of an individual metabolite can be recognized by a cell. The recognition of a metabolite is, however, just one step of a process that often results in changes in the expression of sets of genes required to respond to that metabolite. The signalling pathway between metabolite recognition and transcriptional control is often complex. However, recent evidence from yeast suggests that complex signalling pathways might be circumvented via the direct interaction between individual metabolites and regulators of RNA polymerase II transcription.
Assuntos
Células Eucarióticas/metabolismo , Fenômenos Fisiológicos da Nutrição , Fatores de Transcrição , Modelos Biológicos , Estrutura Molecular , Prolina/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Transativadores/química , Transativadores/genética , Transativadores/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Saccharomyces cerevisiae are able to convert proline to glutamate so that it may be used as a source of nitrogen. Here, we show that the activator of the proline utilization genes, Put3p, is transcriptionally inert in the absence of proline but transcriptionally active in its presence. The activation of Put3p requires no additional yeast proteins and can occur in the presence of certain proline analogues: an unmodified pyrrolidine ring is able to activate Put3p as efficiently as proline itself. In addition, we show that a direct interaction occurs between Put3p and proline. These data, which represent direct control of transcriptional activator function by a metabolite, are discussed in terms of the regulation of proline-specific genes in yeast and as a general mechanism of the control of transcription.