RESUMEN
The mechanisms by which gene expression is regulated by oxygen are of considerable interest from basic science and therapeutic perspectives. Using mass spectrometric analyses of Saccharomyces cerevisiae ribosomes, we found that the amino acid residue in closest proximity to the decoding center, Pro-64 of the 40S subunit ribosomal protein Rps23p (RPS23 Pro-62 in humans) undergoes posttranslational hydroxylation. We identify RPS23 hydroxylases as a highly conserved eukaryotic subfamily of Fe(II) and 2-oxoglutarate dependent oxygenases; their catalytic domain is closely related to transcription factor prolyl trans-4-hydroxylases that act as oxygen sensors in the hypoxic response in animals. The RPS23 hydroxylases in S. cerevisiae (Tpa1p), Schizosaccharomyces pombe and green algae catalyze an unprecedented dihydroxylation modification. This observation contrasts with higher eukaryotes, where RPS23 is monohydroxylated; the human Tpa1p homolog OGFOD1 catalyzes prolyl trans-3-hydroxylation. TPA1 deletion modulates termination efficiency up to â¼10-fold, including of pathophysiologically relevant sequences; we reveal Rps23p hydroxylation as its molecular basis. In contrast to most previously characterized accuracy modulators, including antibiotics and the prion state of the S. cerevisiae translation termination factor eRF3, Rps23p hydroxylation can either increase or decrease translational accuracy in a stop codon context-dependent manner. We identify conditions where Rps23p hydroxylation status determines viability as a consequence of nonsense codon suppression. The results reveal a direct link between oxygenase catalysis and the regulation of gene expression at the translational level. They will also aid in the development of small molecules altering translational accuracy for the treatment of genetic diseases linked to nonsense mutations.
Asunto(s)
Biosíntesis de Proteínas/fisiología , Procesamiento Proteico-Postraduccional/fisiología , Proteínas Ribosómicas/metabolismo , Ribosomas/fisiología , Chlorophyta , Codón de Terminación/genética , Humanos , Hidroxilación , Espectrometría de Masas , Oxigenasas/genética , Oxigenasas/metabolismo , Ribosomas/metabolismo , Saccharomyces cerevisiae , Schizosaccharomyces , Especificidad de la EspecieRESUMEN
Genome sequences predict the presence of many 2-oxoglutarate (2OG)-dependent oxygenases of unknown biochemical and biological functions in Drosophila. Ribosomal protein hydroxylation is emerging as an important 2OG oxygenase catalyzed pathway, but its biological functions are unclear. We report investigations on the function of Sudestada1 (Sud1), a Drosophila ribosomal oxygenase. As with its human and yeast homologs, OGFOD1 and Tpa1p, respectively, we identified Sud1 to catalyze prolyl-hydroxylation of the small ribosomal subunit protein RPS23. Like OGFOD1, Sud1 catalyzes a single prolyl-hydroxylation of RPS23 in contrast to yeast Tpa1p, where Pro-64 dihydroxylation is observed. RNAi-mediated Sud1 knockdown hinders normal growth in different Drosophila tissues. Growth impairment originates from both reduction of cell size and diminution of the number of cells and correlates with impaired translation efficiency and activation of the unfolded protein response in the endoplasmic reticulum. This is accompanied by phosphorylation of eIF2α and concomitant formation of stress granules, as well as promotion of autophagy and apoptosis. These observations, together with those on enzyme homologs described in the companion articles, reveal conserved biochemical and biological roles for a widely distributed ribosomal oxygenase.
Asunto(s)
Proteínas de Drosophila/metabolismo , Drosophila/enzimología , Homeostasis/fisiología , Prolil Hidroxilasas/metabolismo , Biosíntesis de Proteínas/fisiología , Proteínas Ribosómicas/metabolismo , Animales , Animales Modificados Genéticamente , Apoptosis/genética , Autofagia/genética , Western Blotting , Pesos y Medidas Corporales , Cromatografía Liquida , Cartilla de ADN/genética , Proteínas de Drosophila/genética , Cuerpo Adiposo/citología , Femenino , Técnicas de Silenciamiento del Gen , Hidroxilación , Prolil Hidroxilasas/genética , Procesamiento Proteico-Postraduccional/fisiología , Interferencia de ARN , Reacción en Cadena en Tiempo Real de la Polimerasa , Proteínas Ribosómicas/genética , Espectrometría de Masas en Tándem , Respuesta de Proteína Desplegada/genéticaRESUMEN
2-Oxoglutarate (2OG) and Fe(II)-dependent oxygenase domain-containing protein 1 (OGFOD1) is predicted to be a conserved 2OG oxygenase, the catalytic domain of which is related to hypoxia-inducible factor prolyl hydroxylases. OGFOD1 homologs in yeast are implicated in diverse cellular functions ranging from oxygen-dependent regulation of sterol response genes (Ofd1, Schizosaccharomyces pombe) to translation termination/mRNA polyadenylation (Tpa1p, Saccharomyces cerevisiae). However, neither the biochemical activity of OGFOD1 nor the identity of its substrate has been defined. Here we show that OGFOD1 is a prolyl hydroxylase that catalyzes the posttranslational hydroxylation of a highly conserved residue (Pro-62) in the small ribosomal protein S23 (RPS23). Unusually OGFOD1 retained a high affinity for, and forms a stable complex with, the hydroxylated RPS23 substrate. Knockdown or inactivation of OGFOD1 caused a cell type-dependent induction of stress granules, translational arrest, and growth impairment in a manner complemented by wild-type but not inactive OGFOD1. The work identifies a human prolyl hydroxylase with a role in translational regulation.
Asunto(s)
Proteínas Portadoras/metabolismo , Proteínas Nucleares/metabolismo , Prolil Hidroxilasas/metabolismo , Biosíntesis de Proteínas/fisiología , Procesamiento Proteico-Postraduccional/fisiología , Proteínas Ribosómicas/metabolismo , Análisis de Varianza , Proteínas Portadoras/genética , Biología Computacional , Técnica del Anticuerpo Fluorescente , Técnicas de Silenciamiento del Gen , Humanos , Hidroxilación , Immunoblotting , Inmunoprecipitación , Ácidos Cetoglutáricos/metabolismo , Luciferasas , Proteínas Nucleares/genética , Prolina/metabolismo , Biosíntesis de Proteínas/genética , LevadurasRESUMEN
The finding that oxygenase-catalyzed protein hydroxylation regulates animal transcription raises questions as to whether the translation machinery and prokaryotic proteins are analogously modified. Escherichia coli ycfD is a growth-regulating 2-oxoglutarate oxygenase catalyzing arginyl hydroxylation of the ribosomal protein Rpl16. Human ycfD homologs, Myc-induced nuclear antigen (MINA53) and NO66, are also linked to growth and catalyze histidyl hydroxylation of Rpl27a and Rpl8, respectively. This work reveals new therapeutic possibilities via oxygenase inhibition and by targeting modified over unmodified ribosomes.
Asunto(s)
Proteínas de Escherichia coli/metabolismo , Oxigenasas de Función Mixta/metabolismo , Oxigenasas/metabolismo , Células Procariotas/metabolismo , Ribosomas/metabolismo , Animales , Arginina/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Dioxigenasas , Inhibidores Enzimáticos/farmacología , Escherichia coli/metabolismo , Proteínas de Escherichia coli/antagonistas & inhibidores , Histidina/metabolismo , Histona Demetilasas , Humanos , Hidroxilación , Espectroscopía de Resonancia Magnética , Oxigenasas de Función Mixta/antagonistas & inhibidores , Proteínas Nucleares/metabolismo , Oxigenasas/antagonistas & inhibidores , Proteínas Ribosómicas/metabolismoRESUMEN
Inhibition of the hypoxia-inducible factor (HIF) prolyl hydroxylases (PHD or EGLN enzymes) is of interest for the treatment of anemia and ischemia-related diseases. Most PHD inhibitors work by binding to the single ferrous ion and competing with 2-oxoglutarate (2OG) co-substrate for binding at the PHD active site. Non-specific iron chelators also inhibit the PHDs, both in vitro and in cells. We report the identification of dual action PHD inhibitors, which bind to the active site iron and also induce the binding of a second iron ion at the active site. Following analysis of small-molecule iron complexes and application of non-denaturing protein mass spectrometry to assess PHD2·iron·inhibitor stoichiometry, selected diacylhydrazines were identified as PHD2 inhibitors that induce the binding of a second iron ion. Some compounds were shown to inhibit the HIF hydroxylases in human hepatoma and renal carcinoma cell lines.
Asunto(s)
Hidrazinas/química , Hidrazinas/farmacología , Hierro/metabolismo , Procolágeno-Prolina Dioxigenasa/antagonistas & inhibidores , Procolágeno-Prolina Dioxigenasa/metabolismo , Dominio Catalítico , Línea Celular Tumoral , Humanos , Prolina Dioxigenasas del Factor Inducible por Hipoxia , Simulación del Acoplamiento Molecular , Procolágeno-Prolina Dioxigenasa/química , Unión Proteica/efectos de los fármacos , Espectrometría de Masa por Ionización de ElectrosprayRESUMEN
Jumonji-C domain-containing protein 5 (JMJD5) is a 2-oxoglutarate (2OG)-dependent oxygenase that plays important roles in development, circadian rhythm, and cancer through unclear mechanisms. JMJD5 has been reported to have activity as a histone protease, as an Nε-methyl lysine demethylase, and as an arginine residue hydroxylase. Small-molecule JMJD5-selective inhibitors will be useful for investigating its (patho)physiological roles. Following the observation that the broad-spectrum 2OG oxygenase inhibitor pyridine-2,4-dicarboxylic acid (2,4-PDCA) is a 2OG-competing JMJD5 inhibitor, we report that 5-aminoalkyl-substituted 2,4-PDCA derivatives are potent JMJD5 inhibitors manifesting selectivity for JMJD5 over other human 2OG oxygenases. Crystallographic analyses with five inhibitors imply induced fit binding and reveal that the 2,4-PDCA C5 substituent orients into the JMJD5 substrate-binding pocket. Cellular studies indicate that the lead compounds display similar phenotypes as reported for clinically observed JMJD5 variants, which have a reduced catalytic activity compared to wild-type JMJD5.
Asunto(s)
Histonas , Neoplasias , Humanos , Ritmo Circadiano , Piridinas/farmacología , Oxigenasas/metabolismo , Histona Demetilasas con Dominio de Jumonji/metabolismoRESUMEN
Fluoride assays for oxygenases: The 2-oxoglutarate-dependent oxygenase BBOX catalyses the final step in carnitine biosynthesis and is a medicinal chemistry target. We report that BBOX can hydroxylate fluorinated substrates analogues with subsequent release of a fluoride ion, thereby enabling an efficient fluorescence-based assay.
Asunto(s)
Fluoruros/análisis , gamma-Butirobetaína Dioxigenasa/análisis , gamma-Butirobetaína Dioxigenasa/metabolismo , Biocatálisis , Fluorescencia , Humanos , Hidrocarburos Fluorados/química , Hidrocarburos Fluorados/metabolismo , Espectroscopía de Resonancia Magnética , Estructura Molecular , ProtonesRESUMEN
5-Hydroxymethylcytosine has recently been characterized as the 'sixth base' in human DNA. To enable research on this DNA modification, we report an improved method for the synthesis of 5-hydroxymethyl-2'-deoxycytidine ((5-HOMe)dC) phosphoramidite for site-specific incorporation into oligonucleotides. To minimize manipulations we employed a temporary protecting group-free 2'-deoxyuridine to 2'-deoxycytidine conversion procedure that utilizes phase transfer catalysis. The desired (5-HOMe)dC phosphoramidite is obtained in six steps and 24% overall yield from 2'-deoxyuridine.
Asunto(s)
Desoxicitidina/análogos & derivados , Desoxicitidina/química , Desoxiuridina/química , Oligonucleótidos/síntesis química , Compuestos Organofosforados/química , Electroforesis Capilar , Humanos , Oligonucleótidos/química , Compuestos Organofosforados/síntesis química , Espectrometría de Masa por Ionización de ElectrosprayRESUMEN
Based on structural analysis of the human 2-oxoglutarate (2OG) dependent JMJD2 histone N(ε)-methyl lysyl demethylase family, 3-substituted pyridine 2,4-dicarboxylic acids were identified as potential inhibitors with possible selectivity over other human 2OG oxygenases. Microwave-assisted palladium-catalysed cross coupling methodology was developed to install a diverse set of substituents on the sterically demanding C-3 position of a pyridine 2,4-dicarboxylate scaffold. The subsequently prepared di-acids were tested for in vitro inhibition of the histone demethylase JMJD2E and another human 2OG oxygenase, prolyl-hydroxylase domain isoform 2 (PHD2, EGLN1). A subset of substitution patterns yielded inhibitors with selectivity for JMJD2E over PHD2, demonstrating that structure-based inhibitor design can enable selective inhibition of histone demethylases over related human 2OG oxygenases.
Asunto(s)
Ácidos Carboxílicos/química , Histona Demetilasas con Dominio de Jumonji/antagonistas & inhibidores , Piridinas/química , Ácidos Carboxílicos/farmacología , Catálisis , Humanos , Histona Demetilasas con Dominio de Jumonji/química , Modelos Moleculares , Estructura Molecular , Relación Estructura-ActividadRESUMEN
The human 2-oxoglutarate (2OG)-dependent oxygenase aspartate/asparagine-ß-hydroxylase (AspH) is a potential medicinal chemistry target for anticancer therapy. AspH is present on the cell surface of invasive cancer cells and accepts epidermal growth factor-like domain (EGFD) substrates with a noncanonical (i. e., Cys 1-2, 3-4, 5-6) disulfide pattern. We report a concise synthesis of C-3-substituted derivatives of pyridine-2,4-dicarboxylic acid (2,4-PDCA) as 2OG competitors for use in SAR studies on AspH inhibition. AspH inhibition was assayed by using a mass spectrometry-based assay with a stable thioether analogue of a natural EGFD AspH substrate. Certain C-3-substituted 2,4-PDCA derivatives were potent AspH inhibitors, manifesting selectivity over some, but not all, other tested human 2OG oxygenases. The results raise questions about the use of pyridine-carboxylate-related 2OG analogues as selective functional probes for specific 2OG oxygenases, and should aid in the development of AspH inhibitors suitable for inâ vivo use.
Asunto(s)
Ácidos Carboxílicos/farmacología , Inhibidores Enzimáticos/farmacología , Oxigenasas de Función Mixta/antagonistas & inhibidores , Piridinas/farmacología , Bibliotecas de Moléculas Pequeñas/farmacología , Ácidos Carboxílicos/síntesis química , Ácidos Carboxílicos/química , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/química , Humanos , Oxigenasas de Función Mixta/metabolismo , Estructura Molecular , Piridinas/síntesis química , Piridinas/química , Bibliotecas de Moléculas Pequeñas/síntesis química , Bibliotecas de Moléculas Pequeñas/química , Relación Estructura-ActividadRESUMEN
Post-translational ribosomal protein hydroxylation is catalyzed by 2-oxoglutarate (2OG) and ferrous iron dependent oxygenases, and occurs in prokaryotes and eukaryotes. OGFOD1 catalyzes trans-3 prolyl hydroxylation at Pro62 of the small ribosomal subunit protein uS12 (RPS23) and is conserved from yeasts to humans. We describe crystal structures of the human uS12 prolyl 3-hydroxylase (OGFOD1) and its homolog from Saccharomyces cerevisiae (Tpa1p): OGFOD1 in complex with the broad-spectrum 2OG oxygenase inhibitors; N-oxalylglycine (NOG) and pyridine-2,4-dicarboxylate (2,4-PDCA) to 2.1 and 2.6 Å resolution, respectively; and Tpa1p in complex with NOG, 2,4-PDCA, and 1-chloro-4-hydroxyisoquinoline-3-carbonylglycine (a more selective prolyl hydroxylase inhibitor) to 2.8, 1.9, and 1.9 Å resolution, respectively. Comparison of uS12 hydroxylase structures with those of other prolyl hydroxylases, including the human hypoxia-inducible factor (HIF) prolyl hydroxylases (PHDs), reveals differences between the prolyl 3- and prolyl 4-hydroxylase active sites, which can be exploited for developing selective inhibitors of the different subfamilies.
Asunto(s)
Proteínas Portadoras/química , Proteínas Nucleares/química , Inhibidores de Prolil-Hidroxilasa/farmacología , Proteínas de Saccharomyces cerevisiae/química , Secuencia de Aminoácidos , Sitios de Unión , Proteínas Portadoras/antagonistas & inhibidores , Proteínas Portadoras/metabolismo , Humanos , Simulación del Acoplamiento Molecular , Datos de Secuencia Molecular , Proteínas Nucleares/antagonistas & inhibidores , Proteínas Nucleares/metabolismo , Unión Proteica , Saccharomyces cerevisiae/enzimología , Proteínas de Saccharomyces cerevisiae/antagonistas & inhibidores , Proteínas de Saccharomyces cerevisiae/metabolismo , Especificidad por SustratoRESUMEN
A convenient method for the synthesis of fluoromethylated carnitine biosynthesis intermediates, i.e. fluorinated derivatives of γ-butyrobetaine and trimethyllysine, is described. The fluoromethylated probes were useful in both in vitro and cell based assays employing (19)F NMR and LC-MS analyses.
Asunto(s)
Carnitina/química , Flúor/química , Línea Celular , Escherichia , Colorantes Fluorescentes/síntesis química , Colorantes Fluorescentes/química , Humanos , Metilación , Estructura MolecularRESUMEN
Transcription factor binding and high resolution crystallographic studies (1.3 Å) of Dickerson-Drew duplexes with cytosine, methylcytosine and hydroxymethylcytosine bases provide evidence that C-5 cytosine modifications could regulate transcription by context dependent effects on DNA transcription factor interactions.
Asunto(s)
Citosina/análogos & derivados , Metilación de ADN , ADN/química , ADN/metabolismo , Factores de Transcripción/metabolismo , 5-Metilcitosina/análogos & derivados , Citosina/metabolismo , Modelos Moleculares , Conformación de Ácido Nucleico , Oxidación-Reducción , Unión ProteicaRESUMEN
2-Oxoglutarate (2OG) and ferrous iron dependent oxygenases catalyze two-electron oxidations of a range of small and large molecule substrates, including proteins/peptides/amino acids, nucleic acids/bases, and lipids, as well as natural products including antibiotics and signaling molecules. 2OG oxygenases employ variations of a core double-stranded ß-helix (DSBH; a.k.a. jelly-roll, cupin or jumonji C (JmjC)) fold to enable binding of Fe(II) and 2OG in a subfamily conserved manner. The topology of the DSBH limits regions directly involved in substrate binding: commonly the first, second and eighth strands, loops between the second/third and fourth/fifth DSBH strands, and the N-terminal and C-terminal regions are involved in primary substrate, co-substrate and cofactor binding. Insights into substrate recognition by 2OG oxygenases will help to enable selective inhibition and bioengineering studies.
Asunto(s)
Oxigenasas/química , Oxigenasas/metabolismo , Animales , Inhibidores Enzimáticos/farmacología , Humanos , Oxigenasas/antagonistas & inhibidores , Oxigenasas/genética , Unión Proteica , Ingeniería de Proteínas , Estructura Secundaria de ProteínaRESUMEN
The reaction of enol(ate)s with electrophiles is used extensively in organic synthesis for stereoselective C-C bond formation. Protein-based catalysts have had comparatively limited application for the stereoselective formation of C-C bonds of choice via enolate chemistry. We describe protein engineering studies on 5-carboxymethylproline synthases, members of the crotonase superfamily, aimed at enabling stereoselective C-C bond formation leading to N-heterocycles via control of trisubstituted enolate intermediates. Active site substitutions, including at the oxyanion binding site, enable the production of substituted N-heterocycles in high diastereomeric excesses via stereocontrolled enolate formation and reaction. The results reveal the potential of the ubiquitous crotonase superfamily as adaptable catalysts for the control of enolate chemistry.
Asunto(s)
Liasas de Carbono-Carbono/metabolismo , Carbono/metabolismo , Ingeniería de Proteínas , Secuencia de Aminoácidos , Liasas de Carbono-Carbono/química , Catálisis , Dominio Catalítico , Modelos Moleculares , Datos de Secuencia Molecular , Resonancia Magnética Nuclear Biomolecular , Homología de Secuencia de AminoácidoRESUMEN
Cytosine-5-methylation stabilises DNA duplexes and is associated with transcriptional repression; 5-methylcytosine undergoes hydroxylation to 5-hydroxymethylcytosine, a modification of unknown biological function. Spectroscopic and calorimetric analyses show that 5-hydroxymethylcytosine introduction reverses the stabilising effect of 5-methylcytosine, suggesting that in some contexts, 5-methylcytosine hydroxylation may, along with other factors, contribute to the alleviation of transcriptional repression.
Asunto(s)
5-Metilcitosina/química , Nucleótidos de Citosina/química , Citosina/análogos & derivados , ADN/química , Fosfatos de Dinucleósidos/química , Secuencia de Bases , Citosina/química , ADN/metabolismo , Metilación de ADN , Humanos , Hidroxilación , Metilación , Datos de Secuencia Molecular , Estructura Molecular , Reacción en Cadena de la Polimerasa/métodosRESUMEN
Human 2-oxoglutarate oxygenases catalyse a range of biological oxidations including the demethylation of histone and nucleic acid substrates and the hydroxylation of proteins and small molecules. Some of these processes are centrally involved in regulation of cellular responses to hypoxia. The ALKBH proteins are a sub-family of 2OG oxygenases that are defined by homology to the Escherichia coli DNA-methylation repair enzyme AlkB. Here we report evidence that ALKBH5 is probably unique amongst the ALKBH genes in being a direct transcriptional target of hypoxia inducible factor-1 (HIF-1) and is induced by hypoxia in a range of cell types. We show that purified recombinant ALKBH5 is a bona fide 2OG oxygenase that catalyses the decarboxylation of 2OG but appears to have different prime substrate requirements from those so far defined for other ALKBH family members. Our findings define a new class of HIF-transcriptional target gene and suggest that ALKBH5 may have a role in the regulation of cellular responses to hypoxia.
Asunto(s)
Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Ácidos Cetoglutáricos/metabolismo , Oxigenasas/metabolismo , Desmetilasa de ARN, Homólogo 5 de AlkB , Línea Celular , Dioxigenasas , Humanos , Hipoxia , Proteínas de la Membrana , Proteínas Nucleares/genética , Proteínas Nucleares/fisiología , Oxigenasas/genética , Oxigenasas/fisiología , Activación TranscripcionalRESUMEN
Photoactivated cross-linking of peptides to proteins is a useful strategy for identifying enzyme-substrate and protein-protein interactions in cell lysates as demonstrated by studies on the human hypoxia inducible factor system.
Asunto(s)
Reactivos de Enlaces Cruzados/farmacología , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Fragmentos de Péptidos/efectos de la radiación , Fotoquímica , Proteína Supresora de Tumores del Síndrome de Von Hippel-Lindau/metabolismo , Células Cultivadas , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/química , Riñón/citología , Riñón/metabolismo , Sondas Moleculares , Fragmentos de Péptidos/química , Mapeo de Interacción de Proteínas , Espectrometría de Masa por Ionización de Electrospray , Proteína Supresora de Tumores del Síndrome de Von Hippel-Lindau/químicaRESUMEN
JMJD2A, a 2-oxoglutarate dependent N(epsilon)-methyl lysine histone demethylase, is inhibited by disruption of its Zn-binding site by Zn-ejecting compounds including disulfiram and ebselen; this observation may enable the development of inhibitors selective for this subfamily of 2OG dependent oxygenases that do not rely on binding to the highly-conserved Fe(ii)-containing active site.
Asunto(s)
Histona Demetilasas con Dominio de Jumonji/metabolismo , Zinc/química , Azoles/química , Sitios de Unión , Dominio Catalítico , Cristalografía por Rayos X , Disulfiram/química , Isoindoles , Histona Demetilasas con Dominio de Jumonji/antagonistas & inhibidores , Compuestos de Organoselenio/química , Selenio/química , Espectrometría de Masa por Ionización de ElectrosprayRESUMEN
Triacylglycerol metabolism in Saccharomyces cerevisiae was analyzed quantitatively using a systems biological approach. Cellular growth, glucose uptake and ethanol secretion were measured as a function of time and used as input for a dynamic flux-balance model. By combining dynamic mass balances for key metabolites with a detailed steady-state analysis, we trained a model network and simulated the time-dependent degradation of cellular triacylglycerol and its interaction with fatty acid and membrane lipid synthesis. This approach described precisely, both qualitatively and quantitatively, the time evolution of various key metabolites in a consistent and self-contained manner, and the predictions were found to be in excellent agreement with experimental data. We showed that, during pre-logarithmic growth, lipolysis of triacylglycerol allows for the rapid synthesis of membrane lipids, whereas de novo fatty acid synthesis plays only a minor role during this growth phase. Progress in triacylglycerol hydrolysis directly correlates with an increase in cell size, demonstrating the importance of lipolysis for supporting efficient growth initiation.