RESUMO
BACKGROUND: In healthy lungs, epithelial sodium channel (ENaC) is regulated by short, palate, lung, and nasal clone 1 (SPLUNC1). In cystic fibrosis (CF), ENaC is hyperactivated in part due to a loss of SPLUNC1 function. We have developed SPX-101 to replace the lost function of SPLUNC1 in the CF lung. METHODS: Expression of SPLUNC1 was determined in sputum from healthy and CF donors. Stability of SPLUNC1, S18 (the ENaC regulatory domain of SPLUNC1), and SPX-101 was determined in sputum from CF donors and towards neutrophil elastase. Activity of SPX-101 after exposure to CF sputum was determined in airway epithelial cells from CF donors and in the ßENaC transgenic mouse model. RESULTS: SPLUNC1 protein expression is significantly reduced in CF as compared to healthy sputum. SPLUNC1 is rapidly degraded in CF sputum as well as by a number of individual proteases known to be found in the sputum. SPX-101, but not S18, is stable in CF sputum. Finally, SPX-101 retains its ability to internalize ENaC, regulate airway surface liquid height, and increase survival of ßENaC mice after exposure to CF sputum. CONCLUSIONS: Our results demonstrate that SPX-101, but not SPLUNC1 or S18, is stable in CF sputum. These results support the therapeutic development of SPX-101 for the treatment of cystic fibrosis.
Assuntos
Fibrose Cística/metabolismo , Bloqueadores do Canal de Sódio Epitelial/farmacologia , Canais Epiteliais de Sódio/metabolismo , Glicoproteínas/metabolismo , Fosfoproteínas/metabolismo , Mucosa Respiratória , Animais , Células Cultivadas , Descoberta de Drogas , Humanos , Transporte de Íons/efeitos dos fármacos , Transporte de Íons/fisiologia , Elastase de Leucócito/metabolismo , Camundongos , Camundongos Transgênicos , Depuração Mucociliar/efeitos dos fármacos , Peptídeos/metabolismo , Mucosa Respiratória/efeitos dos fármacos , Mucosa Respiratória/enzimologia , Mucosa Respiratória/metabolismo , Escarro/metabolismoRESUMO
In cystic fibrosis (CF) lungs, epithelial Na+ channel (ENaC) hyperactivity causes a reduction in airway surface liquid volume, leading to decreased mucocilliary clearance, chronic bacterial infection, and lung damage. Inhibition of ENaC is an attractive therapeutic option. However, ENaC antagonists have failed clinically because of off-target effects in the kidney. The S18 peptide is a naturally occurring short palate lung and nasal epithelial clone 1 (SPLUNC1)-derived ENaC antagonist that restores airway surface liquid height for up to 24 h in CF human bronchial epithelial cultures. However, its efficacy and safety in vivo are unknown. To interrogate the potential clinical efficacy of S18, we assessed its safety and efficacy using human airway cultures and animal models. S18-mucus interactions were tested using superresolution microscopy, quartz crystal microbalance with dissipation, and confocal microscopy. Human and murine airway cultures were used to measure airway surface liquid height. Off-target effects were assessed in conscious mice and anesthetized rats. Morbidity and mortality were assessed in the ß-ENaC-transgenic (Tg) mouse model. Restoration of normal mucus clearance was measured in cystic fibrosis transmembrane conductance regulator inhibitor 172 [CFTR(inh)-172]-challenged sheep. We found that S18 does not interact with mucus and rapidly penetrated dehydrated CF mucus. Compared with amiloride, an early generation ENaC antagonist, S18 displayed a superior ability to slow airway surface liquid absorption, reverse CFTR(inh)-172-induced reduction of mucus transport, and reduce morbidity and mortality in the ß-ENaC-Tg mouse, all without inducing any detectable signs of renal toxicity. These data suggest that S18 is the first naturally occurring ENaC antagonist to show improved preclinical efficacy in animal models of CF with no signs of renal toxicity.
Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Glicoproteínas/metabolismo , Pneumopatias/tratamento farmacológico , Peptídeos/farmacologia , Fosfoproteínas/metabolismo , Mucosa Respiratória/efeitos dos fármacos , Animais , Células Cultivadas , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Humanos , Transporte de Íons , Pneumopatias/metabolismo , Pneumopatias/patologia , Masculino , Camundongos , Camundongos Transgênicos , Ratos , Ratos Sprague-Dawley , Mucosa Respiratória/metabolismoRESUMO
In addition to their role in glycosylation reactions, UDP-sugars are released from cells and activate widely distributed cell surface P2Y14 receptors (P2Y14R). However, the physiological/pathophysiological consequences of UDP-sugar release are incompletely defined. Here, we report that UDP-glucose levels are abnormally elevated in lung secretions from patients with cystic fibrosis (CF) as well as in a mouse model of CF-like disease, the ßENaC transgenic (Tg) mouse. Instillation of UDP-glucose into wild-type mouse tracheas resulted in enhanced neutrophil lung recruitment, and this effect was nearly abolished when UDP-glucose was co-instilled with the P2Y14R antagonist PPTN [4-(piperidin-4-yl)-phenyl)-7-(4-(trifluoromethyl)-phenyl-2-naphthoic acid]. Importantly, administration of PPTN to ßENaC-Tg mice reduced neutrophil lung inflammation. These results suggest that UDP-glucose released into the airways acts as a local mediator of neutrophil inflammation.
Assuntos
Fibrose Cística/metabolismo , Pulmão/efeitos dos fármacos , Infiltração de Neutrófilos/efeitos dos fármacos , Neutrófilos/efeitos dos fármacos , Uridina Difosfato Glucose/farmacologia , Trifosfato de Adenosina/metabolismo , Adulto , Animais , Fibrose Cística/imunologia , Citocinas/metabolismo , Modelos Animais de Doenças , Feminino , Humanos , Pulmão/imunologia , Pulmão/metabolismo , Masculino , Camundongos , Neutrófilos/imunologia , Neutrófilos/metabolismo , Escarro/imunologia , Escarro/metabolismo , Traqueia/efeitos dos fármacos , Traqueia/imunologia , Uridina Difosfato Glucose/metabolismo , Adulto JovemRESUMO
ATP in airway surface liquid (ASL) controls mucociliary clearance functions via the activation of airway epithelial purinergic receptors. However, abnormally elevated ATP levels have been reported in inflamed airways, suggesting that excessive ATP in ASL contributes to airway inflammation. Despite these observations, little is known about the mechanisms of ATP accumulation in the ASL covering inflamed airways. In this study, links between cystic fibrosis (CF)-associated airway inflammation and airway epithelial ATP release were investigated. Primary human bronchial epithelial (HBE) cells isolated from CF lungs exhibited enhanced IL-8 secretion after 6 to 11 days, but not 28 to 35 days, in culture, compared with normal HBE cells. Hypotonic cell swelling-promoted ATP release was increased in 6- to 11-day-old CF HBE cells compared with non-CF HBE cells, but returned to normal values after 28 to 35 days in culture. The exposure of non-CF HBE cells to airway secretions isolated from CF lungs, namely, sterile supernatants of mucopurulent material (SMM), also caused enhanced IL-8 secretion and increased ATP release. The SMM-induced increase in ATP release was sensitive to Ca(2+) chelation and vesicle trafficking/exocytosis inhibitors, but not to pannexin inhibition. Transcript levels of the vesicular nucleotide transporter, but not pannexin 1, were up-regulated after SMM exposure. SMM-treated cultures displayed increased basal mucin secretion, but mucin secretion was not enhanced in response to hypotonic challenge after the exposure of cells to either vehicle or SMM. We propose that CF airway inflammation up-regulates the capacity of airway epithelia to release ATP via Ca(2+)-dependent vesicular mechanisms not associated with mucin granule secretion.
Assuntos
Trifosfato de Adenosina/metabolismo , Sinalização do Cálcio , Fibrose Cística/metabolismo , Células Epiteliais/metabolismo , Pneumonia/metabolismo , Mucosa Respiratória/metabolismo , Vesículas Secretórias/metabolismo , Sinalização do Cálcio/efeitos dos fármacos , Tamanho Celular , Células Cultivadas , Quelantes/farmacologia , Conexinas/metabolismo , Fibrose Cística/imunologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/imunologia , Humanos , Mediadores da Inflamação/metabolismo , Interleucina-8/metabolismo , Mucinas/metabolismo , Depuração Mucociliar , Proteínas do Tecido Nervoso/metabolismo , Proteínas de Transporte de Nucleotídeos/metabolismo , Pressão Osmótica , Pneumonia/imunologia , Cultura Primária de Células , Mucosa Respiratória/efeitos dos fármacos , Mucosa Respiratória/imunologia , Vesículas Secretórias/efeitos dos fármacos , Vesículas Secretórias/imunologia , Fatores de TempoRESUMO
The nucleotide-sugar-activated P2Y14 receptor (P2Y14-R) is highly expressed in hematopoietic cells. Although the physiologic functions of this receptor remain undefined, it has been strongly implicated recently in immune and inflammatory responses. Lack of availability of receptor-selective high-affinity antagonists has impeded progress in studies of this and most of the eight nucleotide-activated P2Y receptors. A series of molecules recently were identified by Gauthier et al. (Gauthier et al., 2011) that exhibited antagonist activity at the P2Y14-R. We synthesized one of these molecules, a 4,7-disubstituted 2-naphthoic acid derivative (PPTN), and studied its pharmacological properties in detail. The concentration-effect curve of UDP-glucose for promoting inhibition of adenylyl cyclase in C6 glioma cells stably expressing the P2Y14-R was shifted to the right in a concentration-dependent manner by PPTN. Schild analyses revealed that PPTN-mediated inhibition followed competitive kinetics, with a KB of 434 pM observed. In contrast, 1 µM PPTN exhibited no agonist or antagonist effect at the P2Y1, P2Y2, P2Y4, P2Y6, P2Y11, P2Y12, or P2Y13 receptors. UDP-glucose-promoted chemotaxis of differentiated HL-60 human promyelocytic leukemia cells was blocked by PPTN with a concentration dependence consistent with the KB determined with recombinant P2Y14-R. In contrast, the chemotactic response evoked by the chemoattractant peptide fMetLeuPhe was unaffected by PPTN. UDP-glucose-promoted chemotaxis of freshly isolated human neutrophils also was blocked by PPTN. In summary, this work establishes PPTN as a highly selective high-affinity antagonist of the P2Y14-R that is useful for interrogating the action of this receptor in physiologic systems.
Assuntos
Quimiotaxia/efeitos dos fármacos , Neutrófilos/efeitos dos fármacos , Antagonistas do Receptor Purinérgico P2/farmacologia , Receptores Purinérgicos P2/metabolismo , Uridina Difosfato Glucose/metabolismo , Inibidores de Adenilil Ciclases , Adenilil Ciclases/metabolismo , Animais , Células CHO , Linhagem Celular Tumoral , Cricetinae , Glioma/metabolismo , Células HL-60 , Humanos , Leucemia Promielocítica Aguda/metabolismo , Neutrófilos/metabolismo , Agonistas do Receptor Purinérgico P2/farmacologia , Antagonistas do Receptor Purinérgico P2/síntese química , RatosRESUMO
Nucleotides within the airway surface liquid promote fluid secretion via activation of airway epithelial purinergic receptors. ATP is stored within and released from mucin granules as co-cargo with mucins, but the mechanism by which ATP, and potentially other nucleotides, enter the lumen of mucin granules is not known. We assessed the contribution of the recently identified SLC17A9 vesicle nucleotide transporter (VNUT) to the nucleotide availability within isolated mucin granules and further examined the involvement of VNUT in mucin granule secretion-associated nucleotide release. RT-PCR and Western blot analyses indicated that VNUT is abundantly expressed in airway epithelial goblet-like Calu-3 cells, migrating as a duplex with apparent mobility of 55 and 60 kDa. Subcellular fractionation studies indicated that VNUT55 was associated with high-density mucin granules, whereas VNUT60 was associated with low-density organelles. Immunofluorescence studies showed that recombinant VNUT localized to mucin granules and other organelles. Mucin granules isolated from VNUT short hairpin RNA-expressing cells exhibited a marked reduction of ATP, ADP, AMP, and UTP levels within granules. Ca(2+)-regulated vesicular ATP release was markedly reduced in these cells, but mucin secretion was not affected. These results suggest that VNUT is the relevant nucleotide transporter responsible for the uptake of cytosolic nucleotides into mucin granules. By controlling the entry of nucleotides into mucin granules, VNUT contributes to the release of purinergic signaling molecules necessary for the proper hydration of co-released mucins.
Assuntos
Células Caliciformes/metabolismo , Proteínas de Transporte de Nucleotídeos/metabolismo , Nucleotídeos/metabolismo , Sistema Respiratório/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Difosfato de Adenosina/biossíntese , Monofosfato de Adenosina/biossíntese , Trifosfato de Adenosina/biossíntese , Trifosfato de Adenosina/metabolismo , Transporte Biológico , Linhagem Celular , Grânulos Citoplasmáticos/metabolismo , Humanos , Mucinas/genética , Proteínas de Transporte de Nucleotídeos/biossíntese , RNA Interferente Pequeno , Vesículas Secretórias/metabolismo , Uridina Trifosfato/biossínteseRESUMO
The G(i)-coupled P2Y(14) receptor (P2Y(14)-R) is potently activated by UDP-sugars and UDP. Although P2Y(14)-R mRNA is prominently expressed in circulating neutrophils, the signaling pathways and functional responses associated with this receptor are undefined. In this study, we illustrate that incubation of isolated human neutrophils with UDP-glucose resulted in cytoskeleton rearrangement, change of cell shape, and enhanced cell migration. We also demonstrate that UDP-glucose promotes rapid, robust, and concentration-dependent activation of RhoA in these cells. Ecto-nucleotidases expressed on neutrophils rapidly hydrolyzed extracellular ATP, but incubation with UDP-glucose for up to 1 h resulted in negligible metabolism of the nucleotide-sugar. HL60 human promyelocytic leukemia cells do not express the P2Y(14)-R, but neutrophil differentiation of HL60 cells with DMSO resulted in markedly enhanced P2Y(14)-R expression. Accordingly, UDP-glucose, UDP-galactose, and UDP-N-acetylglucosamine promoted Rho activation in differentiated but not in undifferentiated HL60 cells. Stable expression of recombinant human P2Y(14)-R conferred UDP-sugar-promoted responses to undifferentiated HL60 cells. UDP-glucose-promoted RhoA activation also was accompanied by enhanced cell migration in differentiated HL60 cells, and these responses were blocked by Rho kinase inhibitors. These results support the notion that UDP-glucose is a stable and potent proinflammatory mediator that promotes P2Y(14)-R-mediated neutrophil motility via Rho/Rho kinase activation.
Assuntos
Quimiotaxia/fisiologia , Neutrófilos/metabolismo , Receptores Purinérgicos P2/metabolismo , Transdução de Sinais/fisiologia , Uridina Difosfato Glucose/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismo , Actinas/metabolismo , Forma Celular , Citoesqueleto , Regulação da Expressão Gênica/fisiologia , Células HEK293 , Humanos , Neutrófilos/citologia , Neutrófilos/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores Purinérgicos P2/genética , Proteína rhoA de Ligação ao GTP/genéticaRESUMO
ATP released from airway epithelial cells promotes purinergic receptor-regulated mucociliary clearance activities necessary for innate lung defense. Cell swelling-induced membrane stretch/strain is a common stimulus that promotes airway epithelial ATP release, but the mechanisms transducing cell swelling into ATP release are incompletely understood. Using knockdown and knockout approaches, we tested the hypothesis that pannexin 1 mediates ATP release from hypotonically swollen airway epithelia and investigated mechanisms regulating this activity. Well differentiated primary cultures of human bronchial epithelial cells subjected to hypotonic challenge exhibited enhanced ATP release, which was paralleled by the uptake of the pannexin probe propidium iodide. Both responses were reduced by pannexin 1 inhibitors and by knocking down pannexin 1. Importantly, hypotonicity-evoked ATP release from freshly excised tracheas and dye uptake in primary tracheal epithelial cells were impaired in pannexin 1 knockout mice. Hypotonicity-promoted ATP release and dye uptake in primary well differentiated human bronchial epithelial cells was accompanied by RhoA activation and myosin light chain phosphorylation and was reduced by the RhoA dominant negative mutant RhoA(T19N) and Rho and myosin light chain kinase inhibitors. ATP release and Rho activation were reduced by highly selective inhibitors of transient receptor potential vanilloid 4 (TRPV4). Lastly, knocking down TRPV4 impaired hypotonicity-evoked airway epithelial ATP release. Our data suggest that TRPV4 and Rho transduce cell membrane stretch/strain into pannexin 1-mediated ATP release in airway epithelia.
Assuntos
Trifosfato de Adenosina/metabolismo , Conexinas/metabolismo , Pulmão/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Mucosa Respiratória/metabolismo , Proteínas rho de Ligação ao GTP/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismo , Trifosfato de Adenosina/imunologia , Animais , Células Cultivadas , Conexinas/genética , Conexinas/imunologia , Células Epiteliais/citologia , Células Epiteliais/imunologia , Células Epiteliais/metabolismo , Técnicas de Silenciamento de Genes , Humanos , Imunidade Inata/fisiologia , Pulmão/citologia , Pulmão/imunologia , Camundongos , Camundongos Knockout , Mutação de Sentido Incorreto , Cadeias Leves de Miosina/genética , Cadeias Leves de Miosina/imunologia , Cadeias Leves de Miosina/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/imunologia , Fosforilação/fisiologia , Mucosa Respiratória/citologia , Mucosa Respiratória/imunologia , Canais de Cátion TRPV/genética , Canais de Cátion TRPV/imunologia , Canais de Cátion TRPV/metabolismo , Proteínas rho de Ligação ao GTP/genética , Proteínas rho de Ligação ao GTP/imunologia , Proteína rhoA de Ligação ao GTP/genética , Proteína rhoA de Ligação ao GTP/imunologiaRESUMO
The purinergic events regulating the airways' innate defenses are initiated by the release of purines from the epithelium, which occurs constitutively and is enhanced by chemical or mechanical stimulation. While the external triggers have been reviewed exhaustively, this chapter focuses on current knowledge of the receptors and signaling cascades mediating nucleotide release. The list of secreted purines now includes ATP, ADP, AMP and nucleotide sugars, and involves at least three distinct mechanisms reflecting the complexity of airway epithelia. First, the constitutive mechanism involves ATP translocation to the ER/Golgi complex as energy source for protein folding, and fusion of Golgi-derived vesicles with the plasma membrane. Second, goblet cells package ATP with mucins into granules, which are discharged in response to P2Y(2)R activation and Ca(2+)-dependent signaling pathways. Finally, non-mucous cells support a regulated mechanism of ATP release involving protease activated receptor (PAR)-elicited G(12/13) activation, leading to the RhoGEF-mediated exchange of GDP for GTP on RhoA, and cytoskeleton rearrangement. Together, these pathways provide fine tuning of epithelial responses regulated by purinergic signaling events.
Assuntos
Nucleotídeos de Adenina/metabolismo , Células Epiteliais/metabolismo , Mucosa Respiratória/metabolismo , Animais , Humanos , Receptores Purinérgicos/metabolismo , Transdução de SinaisRESUMO
Extracellular UDP-glucose is a natural purinergic receptor agonist, but its mechanisms of cellular release remain unclear. We studied these mechanisms in Saccharomyces cerevisiae, a simple model organism that releases ATP, another purinergic agonist. Similar to ATP, UDP-glucose was released by S. cerevisiae at a rate that was linear over time. However, unlike ATP release, UDP-glucose release was not dependent on glucose stimulation. This discrepancy was resolved by demonstrating the apparent glucose stimulation of ATP release reflected glucose-dependent changes in the intracellular pattern of adenine nucleotides, with AMP release dominating in the absence of glucose. Indeed, total adenine nucleotide release, like UDP-glucose release, did not vary with glucose concentration over the short term. The genetic basis of UDP-glucose release was explored through analysis of deletion mutants, aided by development of a novel bioassay for UDP-glucose based on signaling through heterologously expressed human P2Y 14 receptors. Using this assay, an elevated rate of UDP-glucose release was demonstrated in mutants lacking the putative Golgi nucleotide sugar transporter YMD8. An increased rate of UDP-glucose release in ymd8Delta was reduced by deletion of the YEA4 UDP- N-acetylglucosamine or the HUT1 UDP-galactose transporters, and overexpression of YEA4 or HUT1 increased the rate of UDP-glucose release. These findings suggest an exocytotic release mechanism similar to that of ATP, a conclusion supported by decreased rates of ATP, AMP, and UDP-glucose release in response to the secretory inhibitor Brefeldin A. These studies demonstrate the involvement of the secretory pathway in nucleotide and nucleotide sugar efflux in yeast and offer a powerful model system for further investigation.
Assuntos
Nucleotídeos de Adenina/metabolismo , Exocitose , Saccharomyces cerevisiae/metabolismo , Uridina Difosfato Glucose/metabolismo , Monofosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Brefeldina A/farmacologia , Exocitose/efeitos dos fármacos , Proteínas de Transporte de Nucleotídeos/metabolismoRESUMO
ADP-glucose pyrophosphorylase (ADP-Glc PPase) catalyzes the regulatory step in the pathway for synthesis of bacterial glycogen and starch in plants. ADP-Glc PPases from cyanobacteria (homotetramer) and from potato (Solanum tuberosum) tuber (heterotetramer) are activated by 3-phosphoglycerate and inhibited by inorganic orthophosphate. To study the function of two putative domains, chimeric enzymes were constructed. PSSANA contained the N-terminus (292 amino acids) of the potato tuber ADP-Glc PPase small subunit (PSS) and the C-terminus (159 residues) of the Anabaena PCC 7120 enzyme. ANAPSS was the inverse chimera. These constructs were expressed separately or together with the large subunit of the potato tuber ADP-Glc PPase (PLS), to obtain homo- and heterotetrameric chimeric proteins. Characterization of these forms showed that the N-terminus determines stability and regulatory redox-dependent properties. The chimeric forms exhibited intermediate 3-phosphoglycerate activation properties with respect to the wild-type homotetrameric enzymes, indicating that the interaction between the putative N- and C-domains determines the affinity for the activator. Characterization of the chimeric heterotetramers showed the functionality of the large subunit, mainly in modulating regulation of the enzyme by the coordinate action of 3-phosphoglycerate and inorganic orthophosphate.
Assuntos
Anabaena/enzimologia , Glucose-1-Fosfato Adenililtransferase/química , Glucose-1-Fosfato Adenililtransferase/metabolismo , Solanum tuberosum/enzimologia , Sequência de Aminoácidos , Ativação Enzimática/efeitos dos fármacos , Estabilidade Enzimática , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Glucose-1-Fosfato Adenililtransferase/genética , Ácidos Glicéricos/farmacologia , Dados de Sequência Molecular , Oxirredução , Fosfatos/farmacologia , Tubérculos/enzimologia , Plasmídeos , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismoRESUMO
ADPglucose pyrophosphorylase catalyzes the regulatory step in the pathway for bacterial glycogen synthesis. The enzymes from different organisms exhibit distinctive regulatory properties related to the main carbon metabolic pathway. Escherichia coli ADPglucose pyrophosphorylase is mainly activated by fructose 1,6-bisphosphate (FBP), whereas the Agrobacterium tumefaciens enzyme is activated by fructose 6-phosphate (F6P) and pyruvate. Little is known about the regions determining the specificity for the allosteric regulator. To study the function of different domains, two chimeric enzymes were constructed. "AE" contains the N-terminus (271 amino acids) of the A. tumefaciens ADPglucose pyrophosphorylase and the C-terminus (153 residues) of the E. coli enzyme, and "EA", the inverse construction. Expression of the recombinant wild-type and chimeric enzymes was performed using derivatives of the pET24a plasmid. Characterization of the purified chimeric enzymes showed that the C-terminus of the E. coli enzyme is relevant for the selectivity by FBP. However, this region seems to be less important for the specificity by F6P in the A. tumefaciens enzyme. The chimeric enzyme AE is activated by both FBP and F6P, neither of which affect EA. Pyruvate activates EA with higher apparent affinity than AE, suggesting that the C-terminus of the A. tumefaciens enzyme plays a role in the binding of this effector. The allosteric inhibitor site is apparently disrupted, as a marked desensitization toward AMP was observed in the chimeric enzymes.