RESUMO
Human Adipose-Derived Mesenchymal Stem/Stromal Cells (hAD-MSCs) have great potential for tissue regeneration. Since transplanted hAD-MSCs are likely to be placed in a hypoxic environment, culturing the cells under hypoxic conditions might improve their post-transplantation survival and regenerative performance. The combination of hAD-MSCs and PCL-nHA nanofibers synergically improves the contribution of both components for osteoblast differentiation. In this work, we hypothesized that this biomaterial constitutes a hypoxic environment for hAD-MSCs. We studied the cellular re-arrangement and the subcellular ultrastructure by Transmission Electron Microscopy (TEM) of hAD-MSCs grown into PCL-nHA nanofibers, and we compared them with the same cells grown in two-dimensional cultures, over tissue culture-treated plastic, or glass coverslips. Among the most evident changes, PCL-nHA grown cells showed enlarged mitochondria, and accumulation of glycogen granules, consistent with a hypoxic environment. We observed a 3.5 upregulation (p = 0.0379) of Hypoxia Inducible Factor (HIF)-1A gene expression in PCL-nHA grown cells. This work evidences for the first time intra-cellular changes in three-dimensional compared to two-dimensional cultures, which are adaptive responses of the cells to an environment more closely resembling that of the in vivo niche after transplantation, thus PCL-nHA nanofibers are adequate for hAD-MSCs pre-conditioning.
Assuntos
Células-Tronco Mesenquimais , Nanofibras , Humanos , Alicerces Teciduais/química , Durapatita/química , Durapatita/metabolismo , Poliésteres/química , Materiais Biocompatíveis/química , Diferenciação Celular , Nanofibras/química , Engenharia Tecidual/métodosRESUMO
En el campo de la odontología, prevalecen actualmente alternativas terapéuticas con una filosofía conservadora. Sin embargo, con el advenimiento de los tratamientos con células madre (CM), se amplían las posibilidades terapéuticas, que buscan la combinación y el equilibrio entre la intervención tradicional y las posibilidades de reposición de estructuras anatómicas dañadas, a través de la regeneración de tejidos utilizando células madre o sus derivados (AU)
In the dentistry field, therapeutic alternatives with a conservative philosophy currently prevail. However, with the advent of stem cell (SC) treatments, therapeutic possibilities are expanding, seeking a combination and balance between traditional intervention and the pos- sibility of replacing damaged anatomical structures through tissue regeneration, using stem cells or their derivatives (AU)
Assuntos
Humanos , Células-Tronco , Engenharia Tecidual , Células-Tronco Mesenquimais/fisiologia , Ligamento Periodontal/fisiologia , Regeneração/fisiologia , Dente/citologia , Germe de Dente/fisiologia , Materiais Biocompatíveis/uso terapêutico , Regeneração Óssea/fisiologia , Polpa Dentária/fisiologia , Alicerces Teciduais , COVID-19/terapiaRESUMO
The 14-3-3 protein family binds and regulates hundreds of serine/threonine phosphorylated proteins as an essential component of many signaling networks. Specific biological functions are currently been discovered for each of its seven isoforms in mammals. These proteins have been traditionally considered unregulated; however, its acetylation in an essential lysine residue, causing its inactivation, was recently published. Here, we studied the acetylation state of this lysine 49/51 during the osteogenic differentiation of human adipose-derived stem cells. We found that during this process, the levels of 14-3-3ß (but not its isoform 14-3-3γ) acK49/51 increase, representing the first report linking this PTM to a specific isoform and a cellular process. Our results suggested that this posttranslational modification could be catalyzed by the HBO1 acetyltransferase, as overexpression of HBO1 increased specifically 14-3-3 acK49/51 acetylation. Acetylated 14-3-3 proteins are located primarily in the nucleus, where their active state has been described to bind H3 histones and many transcription factors. The inhibition of the expression of different isoforms showed that the specific silencing of the 14-3-3ß gene, but not γ, increased significantly the osteogenic potential of the cells. This result correlated to the increase in acetylation of 14-3- 3ß Lys 49/51 during osteogenesis. The possible role of this PTM in osteogenesis is discussed.
Assuntos
Proteínas 14-3-3/metabolismo , Diferenciação Celular , Osteogênese , Células-Tronco/metabolismo , Células 3T3-L1 , Acetilação , Animais , Humanos , Camundongos , Células NIH 3T3RESUMO
The SARS-CoV-2 N protein binds several cell host proteins including 14-3-3γ, a well-characterized regulatory protein. However, the biological function of this interaction is not completely understood. We analyzed the variability of â¼90 000 sequences of the SARS-CoV-2 N protein, particularly, its mutations in disordered regions containing binding motifs for 14-3-3 proteins. We studied how these mutations affect the binding energy to 14-3-3γ and found that changes positively affecting the predicted interaction with 14-3-3γ are the most successfully spread, with the highest prevalence in the phylogenetic tree. Although most residues are highly conserved within the 14-3-3 binding site, compensatory mutations to maintain the interaction energy of N-14-3-3γ were found, including half of the current variants of concern and interest. Our results suggest that binding of N to 14-3-3γ is beneficial for the virus, thus targeting this viral-host protein-protein interaction seems an attractive approach to explore antiviral strategies.
Assuntos
Proteínas 14-3-3/metabolismo , Proteínas do Nucleocapsídeo de Coronavírus/análise , Proteínas do Nucleocapsídeo de Coronavírus/metabolismo , Sítios de Ligação , Proteínas do Nucleocapsídeo de Coronavírus/genética , Humanos , Mutação/genética , Fosfoproteínas/análise , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Fosforilação , Filogenia , Ligação ProteicaRESUMO
3D printing has emerged as vanguard technique of biofabrication to assemble cells, biomaterials and biomolecules in a spatially controlled manner to reproduce native tissues. In this work, gelatin methacrylate (GelMA)/alginate hydrogel scaffolds were obtained by 3D printing and 14-3-3ε protein was encapsulated in the hydrogel to induce osteogenic differentiation of human adipose-derived mesenchymal stem cells (hASC). GelMA/alginate-based grid-like structures were printed and remained stable upon photo-crosslinking. The viscosity of alginate allowed to control the pore size and strand width. A higher viscosity of hydrogel ink enhanced the printing accuracy. Protein-loaded GelMA/alginate-based hydrogel showed a clear induction of the osteogenic differentiation of hASC cells. The results are relevant for future developments of GelMA/alginate for bone tissue engineering given the positive effect of 14-3-3ε protein on both cell adhesion and proliferation.
Assuntos
Proteínas 14-3-3/química , Hidrogéis/química , Osteogênese/fisiologia , Impressão Tridimensional , Tecido Adiposo/metabolismo , Alginatos/química , Adesão Celular , Diferenciação Celular , Proliferação de Células , Reagentes de Ligações Cruzadas , Gelatina , Humanos , Tinta , Células-Tronco Mesenquimais/metabolismo , Metacrilatos/química , Osteogênese/efeitos dos fármacos , Proteínas Recombinantes/química , ViscosidadeRESUMO
Currently, membrane curvature is understood as an active mechanism to control cells spatial organization and activity. Protein processes involved in sensing and generating curvature are therefore of major interest. In this work, we have studied α-synuclein interactions with a model lipid bilayer, inducing curvature in a controlled manner and describing protein responses at molecular level. We show that the intrinsically disordered region of α-synuclein binds to the bilayer as an acknowledgment to the induced curvature, a mechanism used by the interacting protein-membrane assembly to relieve free energy. We have calculated free energies for bending the bilayer with α-synuclein adsorbed on the surface and we have established the crucial role of the intrinsically disordered region, suggesting that a dynamic order/disorder interplay takes place as the bilayer reorganizes to bend.
Assuntos
Bicamadas Lipídicas/química , alfa-Sinucleína/química , Bicamadas Lipídicas/metabolismo , Modelos Teóricos , Ligação Proteica , Propriedades de Superfície , Termodinâmica , alfa-Sinucleína/metabolismoRESUMO
Are the dimerization of transmembrane (TM) domains and the reorganization of the lipid bilayer two independent events? Does one event induce or interfere with the other? In this work, we have performed well-tempered metadynamics simulations to calculate the free energy cost to bend a model ternary lipid bilayer in the presence of a TM peptide in its dimer form. We have compared this result with the free energy cost needed to bend a bilayer-only system. Additionally, we have calculated the free energy cost to form a model TM peptide dimer quantitatively describing how lipids reorganize themselves in response to the increase of the membrane curvature and to the lipid-peptide interactions. Our results indicate that the formation of the peptide dimer inside the bilayer increases the cost of the membrane bending due to the spontaneous clustering of cholesterol molecules.
Assuntos
Colesterol/química , Bicamadas Lipídicas/química , Modelos Biológicos , Domínios Proteicos/fisiologia , Dimerização , Metabolismo Energético , Simulação de Dinâmica MolecularRESUMO
Human Adipose-derived mesenchymal stem/stromal cells (hASCs) are of great interest because of their potential for therapeutic approaches. The method described here covers every single step necessary for hASCs isolation from subcutaneous abdominal adipose tissue, multicolor phenotyping by flow cytometry, and quantitative determination of adipogenic differentiation status by means of lipid droplets (LDs) accumulation, and Western blot analysis. Moreover, to simultaneously analyze both LDs accumulation and cellular proteins localization by fluorescence microscopy, we combined Oil Red O (ORO) staining with immunofluorescence detection. For LDs quantification we wrote a program for automatic ORO-stained digital image processing implemented in Octave, a freely available software package. Our method is based on the use of the traditional low cost neutral lipids dye ORO, which can be imaged both by bright-field and fluorescence microscopy. The utilization of ORO instead of other more expensive lipid-specific dyes, together with the fact that the whole method has been designed employing cost-effective culture reagents (standard culture medium and serum), makes it affordable for tight-budget research laboratories. These may be replaced, if necessary or desired, by defined xeno-free reagents for clinical research and applications.
RESUMO
Twenty years ago, a novel concept in protein structural biology was discovered: the intrinsically disordered regions (IDRs). These regions remain largely unstructured under native conditions and the more are studied, more properties are attributed to them. Possibly, one of the most important is their ability to conform a new type of protein-protein interaction. Besides the classical domain-to-domain interactions, IDRs follow a 'fly-casting' model including 'induced folding'. Unfortunately, it is only possible to experimentally explore initial and final states. However, the complete movie of conformational changes of protein regions and their characterization can be addressed by in silico experiments. Here, we simulate the binding of two proteins to describe how the phosphorylation of a single residue modulates the entire process. 14-3-3 protein family is considered a master regulator of phosphorylated proteins and from a modern point-of-view, protein phosphorylation is a three component system, with writers (kinases), erasers (phosphatases) and readers. This later biological role is attributed to the 14-3-3 protein family. Our molecular dynamics results show that phosphorylation of the key residue Thr31 in a partner of 14-3-3, the aralkylamine N-acetyltransferase, releases the fly-casting mechanism during binding. On the other hand, the non-phosphorylation of the same residue traps the proteins, systematically and repeatedly driving the simulations into wrong protein-protein conformations.
Assuntos
Proteínas 14-3-3/metabolismo , Arilalquilamina N-Acetiltransferase/metabolismo , Fluorescência , Simulação de Dinâmica Molecular , FosforilaçãoRESUMO
The 14-3-3 protein family interacts with more than 2000 different proteins in mammals, as a result of its specific phospho-serine/phospho-threonine binding activity. Seven paralogs are strictly conserved in mammalian species. Here, we show that during adipogenic differentiation of 3T3-L1 preadipocytes, the level of each 14-3-3 protein paralog is regulated independently. For instance 14-3-3ß, γ, and η protein levels are increased compared to untreated cells. In contrast, 14-3-3ε protein levels decreased after differentiation while others remained constant. In silico analysis of the promoter region of each gene showed differences that explain the results obtained at mRNA and protein levels.
RESUMO
The understanding of networks is a common goal of an unprecedented array of traditional disciplines. One of the protein network properties most influenced by the structural contents of its nodes is the inter-connectivity. Recent studies in which structural information was included into the topological analysis of protein networks revealed that the content of intrinsic disorder in the nodes could modulate the network topology, rewire networks, and change their inter-connectivity, which is defined by its clustering coefficient. Here, we review the role of intrinsic disorder present in the partners of the highly conserved 14-3-3 protein family on its interaction networks. The 14-3-3s are phospho-serine/threonine binding proteins that have strong influence in the regulation of metabolism and signal transduction networks. Intrinsic disorder increases the clustering coefficients, namely the inter-connectivity of the nodes within each 14-3-3 paralog networks. We also review two new ideas to measure intrinsic disorder independently of the primary sequence of proteins, a thermodynamic model and a method that uses protein structures and their solvent environment. This new methods could be useful to explain unsolved questions about versatility and fixation of intrinsic disorder through evolution. The relation between the intrinsic disorder and network topologies could be an interesting model to investigate new implicitness of the graph theory into biology.
RESUMO
The 14-3-3 protein family interacts with more than 700 different proteins in mammals, in part as a result of its specific phospho-serine/phospho-threonine binding activity. Upon binding to 14-3-3, the stability, subcellular localization and/or catalytic activity of the ligands are modified. Seven paralogs are strictly conserved in mammalian species. Although initially thought as redundant, the number of studies showing specialization is growing. We created a protein-protein interaction network for 14-3-3, kinases and their substrates signaling in human cells. We included information of phosphorylation, acetylation and other PTM sites, obtaining a complete representation of the 14-3-3 binding partners and their modifications. Using a computational system approach we found that networks of each 14-3-3 isoform are statistically different. It was remarkable to find that Tyr was the most phosphorylatable amino acid in domains of 14-3-3 epsilon partners. This, together with the over-representation of SH3 and Tyr_Kinase domains, suggest that epsilon could be involved in growth factors receptors signaling pathways particularly. We also found that within zeta's network, the number of acetylated partners (and the number of modify lysines) is significantly higher compared with each of the other isoforms. Our results imply previously unreported hidden differences of the 14-3-3 isoforms interaction networks. The phosphoproteome and lysine acetylome within each network revealed post-transcriptional regulation intertwining phosphorylation and lysine acetylation. A global understanding of these networks will contribute to predict what could occur when regulatory circuits become dysfunctional or are modified in response to external stimuli.
Assuntos
Proteínas 14-3-3/metabolismo , Lisina/metabolismo , Acetilação , Regulação da Expressão Gênica , Humanos , Fosforilação , Serina/metabolismo , Treonina/metabolismoRESUMO
Disordered regions are segments of a protein that do not fold completely and thus remain flexible. These regions have key physiological roles, particularly in phospho-proteins, which are enriched in disorder-promoting residues surrounding their phosphorylation sites. 14-3-3 proteins are ordered hubs that interact with multiple and diverse intrinsically disordered phosphorylated targets. This provides 14-3-3 with the ability to participate in and to regulate multiple signalling networks. Here, I review the effect of structural disorder on the mechanism involved in 14-3-3 protein-protein interactions and how 14-3-3 impacts cell biology through disordered ligands. How 14-3-3 proteins constitute an advantageous system to identify novel classes of biological tools is discussed with a special emphasis on a particular-and innovative-use of small molecules to stabilize 14-3-3 protein complexes, useful to study gene expression, cancer signalling and neurodegenerative diseases.
Assuntos
Proteínas 14-3-3/química , Proteínas 14-3-3/metabolismo , Dobramento de Proteína , Mapas de Interação de Proteínas , Animais , Humanos , Ligação Proteica , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , Fatores de Transcrição/metabolismoRESUMO
Transcriptome profiling of the pineal gland has revealed night/day differences in the expression of a major fraction of the genes active in this tissue, with two-thirds of these being nocturnal increases. A set of over 600 transcripts exhibit two-fold to >100-fold daily differences in abundance. These changes appear to be primarily attributable to adrenergic-cyclic-AMP-dependent mechanisms, which are controlled via a neural pathway that includes the suprachiasmatic nucleus, the master circadian oscillator. In addition to melatonin synthesis, night/day differences in gene expression impact genes associated with several specialized functions, including the immune/inflammation response, photo-transduction, and thyroid hormone/retinoic acid biology. The following nonspecialized cellular features are also affected: adhesion, cell cycle/cell death, cytoskeleton, DNA modification, endothelium, growth, RNA modification, small molecule biology, transcription factors, vesicle biology, signaling involving Ca(2+), cyclic nucleotides, phospholipids, mitogen-activated protein kinases, the Wnt signaling pathway, and protein phosphorylation.
Assuntos
Ritmo Circadiano , Perfilação da Expressão Gênica , Glândula Pineal/metabolismo , Animais , Regulação da Expressão Gênica , Humanos , Glândula Pineal/anatomia & histologiaRESUMO
14-3-3 proteins participate in many key cellular processes after binding to disordered phospho-partners. Usually, the phosphorylated state is an essential target for the binding. Here, we show for the first time residues other than those in the 14-3-3 binding motif that are essential for the binding between 14-3-3 and a phosphorylated partner. Results support that phosphorylation, although necessary, is not sufficient for 14-3-3's complex formation, as structurally constrained anchor residues play a critical function in stabilizing the protein-protein interaction.
Assuntos
Proteínas 14-3-3/química , Proteínas 14-3-3/metabolismo , Arilalquilamina N-Acetiltransferase/química , Arilalquilamina N-Acetiltransferase/metabolismo , Mapeamento de Interação de Proteínas , Animais , Sítios de Ligação , Cinética , Modelos Moleculares , Fosforilação , Ligação Proteica , OvinosRESUMO
Glyceraldehyde-3-phosphate dehydrogenases catalyze key steps in energy and reducing power partitioning in cells of higher plants. Because non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase (NP-Ga3PDHase) is involved in the production of reductive power (NADPH) in the cytosol, its behavior under oxidative stress conditions was analyzed. The specific activity of the enzyme was found to increase up to 2-fold after oxidative conditions imposed by methylviologen in wheat and maize seedlings. Under moderate oxidant concentration, lack of mRNA induction was observed. The increase in specific activity would thus be a consequence of a significant stability of NP-Ga3PDHase. Our results suggest that the enzyme could be modified by oxidation of cysteine residues, but formation of disulfide bridges is dependent on levels of divalent cations and 14-3-3 proteins. The latter differential effect could be critical to relatively maintain energy and reductant levels in the cytoplasm of plant cells under oxidative stress.
Assuntos
Gliceraldeído-3-Fosfato Desidrogenases/metabolismo , Estresse Oxidativo , Triticum/enzimologia , Zea mays/enzimologia , Estabilidade Enzimática/efeitos dos fármacos , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Gliceraldeído-3-Fosfato Desidrogenases/genética , Peróxido de Hidrogênio/farmacologia , Oxidantes/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Paraquat/farmacologia , Fosforilação/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Plântula/efeitos dos fármacos , Plântula/enzimologia , Triticum/efeitos dos fármacos , Triticum/genética , Zea mays/efeitos dos fármacos , Zea mays/genéticaRESUMO
Proteins named 14-3-3 can bind more than 200 different proteins, mostly (but not exclusively) when they are at a phosphorylated state. These partner proteins are involved in different cellular processes, such as cell signaling, transcription factors, cellular morphology, and metabolism; this suggests pleiotropic functionality for 14-3-3 proteins. Recent efforts to establish a rational classification of 14-3-3 binding partners showed neither structural nor functional relatedness in this group of proteins. Using three natural predictors of disorder in proteins, and the structural available information, we show that >90% of 14-3-3 protein partners contain disordered regions. This percentage is significantly high when compared with recent studies on cell signaling and cancer-related proteins or RNA chaperons. More important, almost all 14-3-3-binding sites are inside disordered regions, this reinforcing the importance of structural disorder in this class of proteins. We also propose that a disorder-to-order transition occurs in the binding of 14-3-3 proteins with their partners. We discuss the consequences of the latter for consensus binding sequences, specificity, affinity, and thermodynamic control.
Assuntos
Proteínas 14-3-3/química , Biologia Computacional/métodos , Proteômica/métodos , Animais , Sítios de Ligação , Caenorhabditis elegans , Drosophila melanogaster , Humanos , Modelos Moleculares , Modelos Teóricos , Conformação Molecular , Fosforilação , Polyomavirus/genética , Ligação Proteica , Conformação Proteica , Desnaturação Proteica , Dobramento de Proteína , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , RNA/química , Transdução de Sinais , Relação Estrutura-Atividade , TermodinâmicaRESUMO
Phosphorylated non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.9; GAPN) found in heterotrophic cells of wheat is activated by MgCl(2). The divalent cation disrupts the interaction between GAPN and a 14-3-3 regulatory protein. This effect is quite remarkable, since it has previously been shown that 14-3-3 binding to a target protein requires divalent cations as Mg(2+) or Ca(2+). Binding of the divalent cation to 14-3-3 causes an increase in surface hydrophobicity. Crystal structure of a 14-3-3-target protein complex has been only determined for serotinin N-acetyltransferase. We utilized a model of a subunit of plant GAPN and the crystallographic structure of human 14-3-3zeta to shape the complex between theses two proteins. Initial dockings were performed with the BiGGER program, which allows an exhaustive search of translational and rotational space. A filtering procedure was then applied to reduce the number of complexes to a manageable number. We predict the structural characteristics of GAPN-14-3-3zeta binding process, proposing that the main attractive force in this complex derives from electrostatic interactions. The predicted model was corroborated by analysis of kinetic behavior of GAPN and its relationship with pH and ionic strength conditions. This study provides a variant on the interaction of 14-3-3 with target proteins, thus affording a wider scenario to establish possible structural models for this remarkable family of regulatory proteins.
Assuntos
Proteínas 14-3-3/química , Gliceraldeído 3-Fosfato Desidrogenase (NADP+)/química , Modelos Moleculares , Proteínas de Plantas/química , Plantas/enzimologia , Sequência de Aminoácidos , Humanos , Concentração de Íons de Hidrogênio , Cinética , Dados de Sequência Molecular , Estrutura Terciária de Proteína , Eletricidade EstáticaRESUMO
Glyceraldehyde-3-phosphate dehydrogenases catalyze key steps in energy and reducing power partitioning in cells of higher plants. Phosphorylated non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase (GAPN) present in heterotrophic cells of wheat (Triticum aestivum) was activated up to 3-fold by MgCl2. The effect was not observed with the non-phosphorylated enzyme found in leaves. The divalent cation also affected the response of the enzyme from endosperm and shoots to adenine nucleotides and inorganic pyrophosphate. Gel filtration chromatography, co-immunoprecipitation followed by immunostaining, and the use of a phosphopeptide containing a canonical binding motif showed that MgCl2 actually disrupted the interaction between GAPN and a 14-3-3 regulatory protein. After interaction with 14-3-3, phosphorylated GAPN exhibits a 3-fold lower Vmax and higher sensitivity to inhibition by ATP and pyrophosphate. Results suggest that GAPN is a target for regulation by phosphorylation, levels of divalent cations, and 14-3-3 proteins. The regulatory mechanism could be critical to maintain levels of energy and reductants in the cytoplasm of heterotrophic plant cells.
Assuntos
Gliceraldeído-3-Fosfato Desidrogenases/metabolismo , Triticum/metabolismo , Tirosina 3-Mono-Oxigenase/metabolismo , Proteínas 14-3-3 , Difosfato de Adenosina/metabolismo , Monofosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Cátions Bivalentes/farmacologia , Células Cultivadas , Cromatografia em Gel , Difosfatos/metabolismo , Gliceraldeído-3-Fosfato Desidrogenases/isolamento & purificação , Cinética , Fosfoproteínas/metabolismo , Fosforilação , Proteínas de Plantas/metabolismoRESUMO
In wheat, non-phosphorylating, NADP-dependent glyceraldehyde-3-phosphate dehydrogenase (GAPN) was found to be encoded by one gene giving rise to a single protein. However, Western blots revealed two different subunits of about 58 and 60 kDa in endosperm and shoots. The latter was attributed to in vivo phosphorylation of shoot GAPN. No modification occurred in leaves, where the enzyme is composed by a single 58 kDa polypeptide. GAPN partially purified from shoots and endosperm was dephosphorylated in vitro with alkaline phosphatase. Phosphorylated GAPN exhibited similar affinity for substrates but a lower V(max) compared to the non-phosphorylated enzyme. Results suggest that reversible phosphorylation of GAPN could regulate NADPH production in the cytosol of heterotrophic plant cells.