RESUMO
Organophosphates (OPs) are a class of neurotoxic acetylcholinesterase inhibitors including widely used pesticides as well as nerve agents such as VX and VR. Current treatment of these toxins relies on reactivating acetylcholinesterase, which remains ineffective. Enzymatic scavengers are of interest for their ability to degrade OPs systemically before they reach their target. Here we describe a library of computationally designed variants of phosphotriesterase (PTE), an enzyme that is known to break down OPs. The mutations G208D, F104A, K77A, A80V, H254G, and I274N broadly improve catalytic efficiency of VX and VR hydrolysis without impacting the structure of the enzyme. The mutation I106â A improves catalysis of VR and L271E abolishes activity, likely due to disruptions of PTE's structure. This study elucidates the importance of these residues and contributes to the design of enzymatic OP scavengers with improved efficiency.
Assuntos
Hidrolases de Triester Fosfórico , Hidrolases de Triester Fosfórico/metabolismo , Hidrolases de Triester Fosfórico/química , Inibidores da Colinesterase/química , Inibidores da Colinesterase/metabolismo , Compostos Organotiofosforados/química , Compostos Organotiofosforados/metabolismo , Mutação , Hidrólise , Modelos MolecularesRESUMO
Kinetic enhancement of organophosphate hydrolysis is a long-standing challenge in catalysis. For prophylactic treatment against organophosphate exposure, enzymatic hydrolysis needs to occur at high rates in the presence of low substrate concentrations and enzymatic activity should persist over days and weeks. Here, the conjugation of small DNA scaffolds was used to introduce substrate binding sites with micromolar affinity to VX, paraoxon, and methyl-parathion in close proximity to the enzyme phosphotriesterase (PTE). The result was a decrease in KM and increase in the rate at low substrate concentrations. An optimized system for paraoxon hydrolysis decreased KM by 11-fold, with a corresponding increase in second-order rate constant. The initial rates of VX and methyl-parathion hydrolysis were also increased by 3.1- and 6.7-fold, respectively. The designed scaffolds not only increased the local substrate concentration, but they also resulted in increased stability and PTE-DNA particle size tuning between 25 and ~150 nm. The scaffold engineering approach taken here is focused on altering the local chemical and physical microenvironment around the enzyme and is therefore compatible with active site engineering via combinatorial and computational approaches.
Assuntos
Substâncias para a Guerra Química/metabolismo , Agentes Neurotóxicos/metabolismo , Compostos Organotiofosforados/metabolismo , Animais , Sítios de Ligação , Linhagem Celular , Substâncias para a Guerra Química/química , DNA/química , DNA/metabolismo , Expressão Gênica , Humanos , Hidrólise , Nanoestruturas/química , Nanotecnologia , Hidrolases de Triester Fosfórico/química , Hidrolases de Triester Fosfórico/metabolismo , Especificidade por SubstratoRESUMO
Human butyrylcholinesterase (E.C. 3.1.1.8) purified from blood plasma has previously been shown to provide protection against up to five and a half times the median lethal dose of an organophosphorus nerve agent in several animal models. In this study the stoichiometric nature of the protection afforded by human butyrylcholinesterase against organophosphorus nerve agents was investigated in guinea pigs. Animals were administered human butyrylcholinesterase (26.15â¯mg/kgâ¯≡â¯308â¯nmol/kg) by the intravascular or intramuscular route. Animals were subsequently dosed with either soman or VX in accordance with a stage-wise adaptive dose design to estimate the modified median lethal dose in treated animals. Human butyrylcholinesterase (308â¯nmol/kg) increased the median lethal dose of soman from 154â¯nmol/kg to 770â¯nmol/kg. Comparing the molar ratio of agent molecules to enzyme active sites yielded a stoichiometric protective ratio of 2:1 for soman, likely related to the similar stereoselectivity the enzyme has compared to the toxic target, acetylcholinesterase. In contrast, human butyrylcholinesterase (308â¯nmol/kg) increased the median lethal dose of VX from 30â¯nmol/kg to 312â¯nmol/kg, resulting in a stoichiometric protective ratio of only 1:1, suggesting a lack of stereoselectivity for this agent.
Assuntos
Butirilcolinesterase/administração & dosagem , Substâncias para a Guerra Química/intoxicação , Agentes Neurotóxicos/intoxicação , Intoxicação/prevenção & controle , Animais , Área Sob a Curva , Butirilcolinesterase/sangue , Butirilcolinesterase/química , Substâncias para a Guerra Química/química , Cobaias , Humanos , Injeções Intramusculares , Injeções Intravenosas , Dose Letal Mediana , Masculino , Taxa de Depuração Metabólica , Fármacos Neuroprotetores/administração & dosagem , Fármacos Neuroprotetores/química , Fármacos Neuroprotetores/farmacocinética , Compostos Organotiofosforados/química , Compostos Organotiofosforados/intoxicação , Soman/química , Soman/intoxicação , EstereoisomerismoRESUMO
Nerve agents are a class of organophosphorus compounds (OPs) that blocks communication between nerves and organs. Because of their acute neurotoxicity, it is extremely difficult to rescue the victims after exposure. Numerous efforts have been devoted to search for an effective prophylactic nerve agent bioscavenger to prevent the deleterious effects of these compounds. However, low scavenging efficiency, unfavorable pharmacokinetics, and immunological problems have hampered the development of effective drugs. Here, we report the development and testing of a nanoparticle-based nerve agent bioscavenger (nanoscavenger) that showed long-term protection against OP intoxication in rodents. The nanoscavenger, which catalytically breaks down toxic OP compounds, showed a good pharmacokinetic profile and negligible immune response in a rat model of OP intoxication. In vivo administration of the nanoscavenger before or after OP exposure in animal models demonstrated protective and therapeutic efficacy. In a guinea pig model, a single prophylactic administration of the nanoscavenger effectively prevented lethality after multiple sarin exposures over a 1-week period. Our results suggest that the prophylactic administration of the nanoscavenger might be effective in preventing the toxic effects of OP exposure in humans.
Assuntos
Nanopartículas/química , Agentes Neurotóxicos/toxicidade , Substâncias Protetoras/farmacologia , Administração Intravenosa , Animais , Feminino , Cobaias , Masculino , Nanopartículas/administração & dosagem , Paraoxon/toxicidade , Substâncias Protetoras/administração & dosagem , Substâncias Protetoras/farmacocinética , Ratos Sprague-Dawley , Sarina/toxicidade , Análise de Sobrevida , Fatores de Tempo , Distribuição TecidualRESUMO
The atypical butyrylcholinesterase (aBuChE) from Oryzias latipes shares approximately 65% sequence similarity to both acetylcholinesterase and butyrylcholinesterase and was studied for its capacity to spontaneously reactivate following inhibition by organophosphorus nerve agents. Like other cholinesterases, aBuChE was inhibited by all G- and V-type nerve agents. Interestingly, aBuChE was able to undergo spontaneous reactivation after inhibition with VR (t1/2 = 5.5 ± 0.2 h). Mass spectrometry of aBuChE after VR inhibition confirmed the presence of a covalently bound adduct of the size expected for non-aged VR on the peptide containing the active site serine. To understand the effect of substrate volume on rates of reactivation, the capacity of aBuChE to bind and spontaneously reactivate after inhibition with five V-agent analogues was examined. No appreciable reactivation was detected for enzyme inhibited by V2 (VX with O-isopropyl on retained group), V4 (VX with N-diethyl leaving group termination), or V5 (VX with N-dimethyl leaving group termination). Minimal reactivation was detected with V1 (VX with O-propyl on retained group). Conversely, spontaneous reactivation was observed when aBuChE was inhibited by V3 (VX with O-isobutyl on retained group; t1/2 = 6.3 ± 0.4 h). The data suggest that the ability of aBuChE to spontaneously reactivate after inhibition by V-agent analogues is related to the structure of the retained group. These results provide structural information that may shed light on the design of improved small molecule reactivators of nerve agent-inhibited acetylcholinesterase or butyrylcholinesterase, and further suggest that re-engineering the active site of a cholinesterase could result in enzymes with clinically relevant rates of nerve agent hydrolysis.
Assuntos
Butirilcolinesterase/química , Compostos Organotiofosforados/química , Animais , Butirilcolinesterase/genética , Butirilcolinesterase/metabolismo , Domínio Catalítico , Meia-Vida , Cinética , Larva/metabolismo , Espectrometria de Massas , Mariposas/crescimento & desenvolvimento , Mariposas/metabolismo , Compostos Organotiofosforados/metabolismo , Oryzias/metabolismo , Ligação Proteica , Proteínas Recombinantes/sangue , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificaçãoRESUMO
Human paraoxonase-1 (HuPON1) has been proposed as a catalytic bioscavenger of organophosphorus (OP) pesticides and nerve agents. We assessed the potential of this enzyme to protect against OP poisoning using two different paradigms. First, recombinant HuPON1 purified from cabbage loopers (iPON1; Trichoplusia ni) was administered to guinea pigs, followed by exposure to at least 2 times the median lethal dose (LD(50)) of the OP nerve agents tabun (GA), sarin (GB), soman (GD), and cyclosarin (GF), or chlorpyrifos oxon, the toxic metabolite of the OP pesticide chlorpyrifos. In the second model, mice were infected with an adenovirus that induced expression of HuPON1 and then exposed to sequential doses of GD, VX, or (as reported previously) diazoxon, the toxic metabolite of the OP pesticide diazinon. In both animal models, the exogenously added HuPON1 protected animals against otherwise lethal doses of the OP pesticides but not against the nerve agents. Together, the results support prior modeling and in vitro activity data which suggest that wild-type HuPON1 does not have sufficient catalytic activity to provide in vivo protection against nerve agents.
Assuntos
Arildialquilfosfatase/administração & dosagem , Substâncias para a Guerra Química/toxicidade , Compostos Organofosforados/toxicidade , Praguicidas/toxicidade , Animais , Antídotos/administração & dosagem , Antídotos/farmacocinética , Arildialquilfosfatase/genética , Arildialquilfosfatase/isolamento & purificação , Arildialquilfosfatase/farmacocinética , Clorpirifos/análogos & derivados , Clorpirifos/toxicidade , Cobaias , Humanos , Masculino , Camundongos , Mariposas , Organofosfatos/toxicidade , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/farmacocinética , Sarina/toxicidade , Soman/toxicidadeRESUMO
In an effort to discover novel catalytic bioscavengers of organophosphorus (OP) nerve agents, cell lysates from a diverse set of bacterial strains were screened for their capacity to hydrolyze the OP nerve agents VX, VR, and soman (GD). The library of bacterial strains was identified using both random and rational approaches. Specifically, two representative strains from eight categories of extremophiles were chosen at random. For the rational approach, the protein sequence of organophosphorus hydrolase (OPH) from Brevundimonas diminuta was searched against a non-redundant protein database using the Basic Local Alignment Search Tool to find regions of local similarity between sequences. Over 15 protein sequences with significant sequence similarity to OPH were identified from a variety of bacterial strains. Some of these matches were based on predicted protein structures derived from bacterial genome sequences rather than from bona fide proteins isolated from bacteria. Of the 25 strains selected for nerve agent testing, three bacterial strains had measurable levels of OP hydrolase activity. These strains are Ammoniphilus oxalaticus, Haloarcula sp., and Micromonospora aurantiaca. Lysates from A. oxalaticus had detectable hydrolysis of VR; Haloarcula sp. had appreciable hydrolysis of VX and VR, whereas lysates from M. aurantiaca had detectable hydrolysis of VR and GD.
Assuntos
Arildialquilfosfatase/metabolismo , Proteínas de Bactérias/metabolismo , Substâncias para a Guerra Química/metabolismo , Compostos Organofosforados/metabolismo , Antídotos/isolamento & purificação , Antídotos/metabolismo , Antídotos/farmacologia , Arildialquilfosfatase/genética , Arildialquilfosfatase/isolamento & purificação , Bacillales/enzimologia , Bacillales/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Substâncias para a Guerra Química/toxicidade , Descoberta de Drogas , Avaliação Pré-Clínica de Medicamentos , Haloarcula/enzimologia , Haloarcula/genética , Hidrólise , Micromonospora/enzimologia , Micromonospora/genética , Compostos Organofosforados/toxicidade , Compostos Organotiofosforados/metabolismo , Compostos Organotiofosforados/toxicidade , Paraoxon/metabolismo , Paraoxon/toxicidade , Soman/metabolismo , Soman/toxicidadeRESUMO
Paraoxonase-1 (PON1) is a serum protein, the activity of which is related to susceptibility to cardiovascular disease and intoxication by organophosphorus (OP) compounds. It may also be involved in innate immunity, and it is a possible lead molecule in the development of a catalytic bioscavenger of OP pesticides and nerve agents. Human PON1 expressed in E. coli is mostly found in the insoluble fraction, which motivated the engineering of soluble variants, such as G2E6, with more than 50 mutations from huPON1. We examined the effect on the solubility, activity, and stability of three sets of mutations designed to solubilize huPON1 with fewer overall changes: deletion of the N-terminal leader, polar mutations in the putative HDL binding site, and selection of the subset of residues that became more polar in going from huPON1 to G2E6. All three sets of mutations increase the solubility of huPON1; the HDL-binding mutant has the largest effect on solubility, but it also decreases the activity and stability the most. Based on the G2E6 polar mutations, we "humanized" an engineered variant of PON1 with high activity against cyclosarin (GF) and found that it was still very active against GF with much greater similarity to the human sequence.
RESUMO
The use of whole insect larvae as a source of recombinant proteins offers a more cost-effective method of producing large quantities of human proteins than conventional cell-culture approaches. Human carboxylesterase 1 has been produced in and isolated from whole Trichoplusia ni larvae. The recombinant protein was crystallized and its structure was solved to 2.2 resolution. The results indicate that the larvae-produced enzyme is essentially identical to that isolated from cultured Sf21 cells, supporting the use of this expression system to produce recombinant enzymes for crystallization studies.
Assuntos
Carboxilesterase/química , Animais , Carboxilesterase/genética , Carboxilesterase/isolamento & purificação , Carboxilesterase/metabolismo , Linhagem Celular , Humanos , Hidrólise , Larva/genética , Larva/metabolismo , Modelos Moleculares , Mariposas/genética , Mariposas/metabolismo , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismoRESUMO
Human Serum paraoxonase 1 (HuPON1) is an enzyme that has been shown to hydrolyze a variety of chemicals including the nerve agent VX. While wildtype HuPON1 does not exhibit sufficient activity against VX to be used as an in vivo countermeasure, it has been suggested that increasing HuPON1's organophosphorous hydrolase activity by one or two orders of magnitude would make the enzyme suitable for this purpose. The binding interaction between HuPON1 and VX has recently been modeled, but the mechanism for VX hydrolysis is still unknown. In this study, we created a transition state model for VX hydrolysis (VX(ts)) in water using quantum mechanical/molecular mechanical simulations, and docked the transition state model to 22 experimentally characterized HuPON1 variants using AutoDock Vina. The HuPON1-VX(ts) complexes were grouped by reaction mechanism using a novel clustering procedure. The average Vina interaction energies for different clusters were compared to the experimentally determined activities of HuPON1 variants to determine which computational procedures best predict how well HuPON1 variants will hydrolyze VX. The analysis showed that only conformations which have the attacking hydroxyl group of VX(ts) coordinated by the sidechain oxygen of D269 have a significant correlation with experimental results. The results from this study can be used for further characterization of how HuPON1 hydrolyzes VX and design of HuPON1 variants with increased activity against VX.
Assuntos
Arildialquilfosfatase/metabolismo , Compostos Organotiofosforados/química , Compostos Organotiofosforados/metabolismo , Arildialquilfosfatase/genética , Humanos , Hidrólise , Modelos MolecularesRESUMO
Organophosphorus (OP) nerve agents are potent suicide inhibitors of the essential neurotransmitter-regulating enzyme acetylcholinesterase. Due to their acute toxicity, there is significant interest in developing effective countermeasures to OP poisoning. Here we impart nerve agent hydrolysis activity into the human drug metabolism enzyme carboxylesterase 1. Using crystal structures of the target enzyme in complex with nerve agent as a guide, a pair of histidine and glutamic acid residues were designed proximal to the enzyme's native catalytic triad. The resultant variant protein demonstrated significantly increased rates of reactivation following exposure to sarin, soman, and cyclosarin. Importantly, the addition of these residues did not alter the high affinity binding of nerve agents to this protein. Thus, using two amino acid substitutions, a novel enzyme was created that efficiently converted a group of hemisubstrates, compounds that can start but not complete a reaction cycle, into bona fide substrates. Such approaches may lead to novel countermeasures for nerve agent poisoning.
Assuntos
Hidrolases de Éster Carboxílico/metabolismo , Substâncias para a Guerra Química/farmacocinética , Compostos Organofosforados/farmacocinética , Hidrolases de Éster Carboxílico/antagonistas & inibidores , Hidrolases de Éster Carboxílico/química , Hidrolases de Éster Carboxílico/genética , Domínio Catalítico , Substâncias para a Guerra Química/metabolismo , Humanos , Hidrólise , Modelos Moleculares , Mutação , Compostos Organofosforados/metabolismo , Conformação ProteicaRESUMO
The concept of using cholinesterase bioscavengers for prophylaxis against organophosphorous nerve agents and pesticides has progressed from the bench to clinical trial. However, the supply of the native human proteins is either limited (e.g., plasma-derived butyrylcholinesterase and erythrocytic acetylcholinesterase) or nonexisting (synaptic acetylcholinesterase). Here we identify a unique form of recombinant human butyrylcholinesterase that mimics the native enzyme assembly into tetramers; this form provides extended effective pharmacokinetics that is significantly enhanced by polyethylene glycol conjugation. We further demonstrate that this enzyme (but not a G117H/E197Q organophosphorus acid anhydride hydrolase catalytic variant) can prevent morbidity and mortality associated with organophosphorous nerve agent and pesticide exposure of animal subjects of two model species.
Assuntos
Butirilcolinesterase/farmacologia , Substâncias para a Guerra Química/toxicidade , Fármacos Neuroprotetores/farmacologia , Nicotiana/metabolismo , Compostos Organofosforados/toxicidade , Praguicidas/toxicidade , Animais , Butirilcolinesterase/metabolismo , Butirilcolinesterase/farmacocinética , Substâncias para a Guerra Química/metabolismo , Cromatografia Líquida de Alta Pressão , Cobaias , Humanos , Immunoblotting , Cinética , Camundongos , Fármacos Neuroprotetores/metabolismo , Fármacos Neuroprotetores/farmacocinética , Compostos Organofosforados/metabolismo , Praguicidas/metabolismo , Polietilenoglicóis/metabolismo , Engenharia de ProteínasRESUMO
Expression and purification of recombinant human paraoxonase-1 (rHuPON1) from bacterial systems have proven elusive. Most systems for successful production of recombinant PON1 have relied on either eukaryotic expression in baculovirus or prokaryotic expression of synthetic, gene-shuffled rabbit-mouse-human PON1 hybrid molecules. We review here methods and protocols for the production of pure, native rHuPON1 using an E. coli expression system followed by conventional column chromatographic purification. The resulting rHuPON1 is stable, active, and capable of protecting PON1 knockout mice (PON1(-/-)) from exposure to high levels of the organophosphorus (OP) compound diazoxon. Bacterially-derived rHuPON1 can be produced in large quantities and lacks the glycosylation of eukaryotic systems that produces immunogenic complications when used as a therapeutic. The rHuPON1 should be useful for treating insecticide OP exposures and reducing risks of other diseases resulting from low PON1 status. The ease of mutagenesis in bacterial systems will also allow for the generation and screening of rHuPON1 variants with enhanced catalytic efficiencies against nerve agents and other OP compounds.
Assuntos
Arildialquilfosfatase/metabolismo , Escherichia coli/metabolismo , Engenharia Genética/métodos , Animais , Arildialquilfosfatase/genética , Catálise , Glicosilação , Humanos , Inseticidas/farmacologia , Cinética , Camundongos , Camundongos Knockout , Compostos Organofosforados/farmacologia , Proteínas Recombinantes/químicaRESUMO
Human serum paraoxonase-1 (HuPON1) is difficult to either purify from plasma or functionally express in high yield from recombinant sources. Here, we describe the characterization of functional HuPON1 expressed and purified from Trichoplusia ni (T. ni) larvae infected with an orally active form of baculovirus. SDS-PAGE and anti-HuPON1 Western blot analyses yielded only three bands of approximately 41, 42, and 44 kDa. MALDI-TOF confirmed the identity of each of these bands as HuPON1 with greater than 95% confidence. These isoforms result from differential glycosylation of the enzyme as indicated by peptide mapping, mass analysis, and PNGase F deglycosylation experiments. Recombinant insect-produced HuPON1 hydrolyzed phenyl acetate, paraoxon, and the nerve agents GF, VX, and VR. The enzyme had dramatic stereoselectivity for the P+ isomers of VX and VR. T. ni larvae expressing HuPON1 were remarkably resistant to the pesticide chlorpyrifos. Together, these results demonstrate that the caterpillar of the T. ni moth can be used as an expression system to produce large quantities of functional recombinant HuPON1. Insect production of HuPON1 may provide a source for both in vitro enzymatic and crystallographic studies and in vivo stability and anti-nerve agent efficacy testing.
Assuntos
Arildialquilfosfatase/biossíntese , Arildialquilfosfatase/metabolismo , Lepidópteros/genética , Animais , Arildialquilfosfatase/genética , Arildialquilfosfatase/isolamento & purificação , Baculoviridae/genética , Baculoviridae/fisiologia , Clorpirifos/metabolismo , Expressão Gênica , Humanos , Hidrólise , Cinética , Larva/genética , Larva/virologia , Lepidópteros/virologia , Compostos Organotiofosforados/química , Compostos Organotiofosforados/metabolismo , Praguicidas/metabolismo , Estereoisomerismo , Especificidade por SubstratoRESUMO
Organophosphorus (OP) nerve agents are potent toxins that inhibit cholinesterases and produce a rapid and lethal cholinergic crisis. Development of protein-based therapeutics is being pursued with the goal of preventing nerve agent toxicity and protecting against the long-term side effects of these agents. The drug-metabolizing enzyme human carboxylesterase 1 (hCE1) is a candidate protein-based therapeutic because of its similarity in structure and function to the cholinesterase targets of nerve agent poisoning. However, the ability of wild-type hCE1 to process the G-type nerve agents sarin and cyclosarin has not been determined. We report the crystal structure of hCE1 in complex with the nerve agent cyclosarin. We further use stereoselective nerve agent analogs to establish that hCE1 exhibits a 1700- and 2900-fold preference for the P(R) enantiomers of analogs of soman and cyclosarin, respectively, and a 5-fold preference for the P(S) isomer of a sarin analog. Finally, we show that for enzyme inhibited by racemic mixtures of bona fide nerve agents, hCE1 spontaneously reactivates in the presence of sarin but not soman or cyclosarin. The addition of the neutral oxime 2,3-butanedione monoxime increases the rate of reactivation of hCE1 from sarin inhibition by more than 60-fold but has no effect on reactivation with the other agents examined. Taken together, these data demonstrate that hCE1 is only reactivated after inhibition with the more toxic P(S) isomer of sarin. These results provide important insights toward the long-term goal of designing novel forms of hCE1 to act as protein-based therapeutics for nerve agent detoxification.
Assuntos
Hidrolases de Éster Carboxílico/química , Substâncias para a Guerra Química/química , Inibidores Enzimáticos/química , Compostos Organofosforados/química , Sarina/química , Hidrolases de Éster Carboxílico/antagonistas & inibidores , Cristalização , Humanos , Hidrólise , Modelos Moleculares , Compostos Organofosforados/farmacologia , Oximas/farmacologia , Sarina/farmacologia , EstereoisomerismoRESUMO
The enzyme human paraoxonase 1 (huPON1) has demonstrated significant potential for use as a bioscavenger for treatment of exposure to organophosphorus (OP) nerve agents. Herein we report the development of protein models for the human isoform derived from a crystal structure of a chimeric version of the protein (pdb ID: 1V04) and a homology model derived from the related enzyme diisopropylfluorophosphatase (pdb ID: 1XHR). From these structural models, binding modes for OP substrates are predicted, and these poses are found to orient substrates in proximity to residues known to modulate specificity of the enzyme. Predictions are made with regard to the role that residues play in altering substrate binding and turnover, in particular with regard to the stereoselectivity of the enzyme, and the known differences in activity related to a natural polymorphism in the enzyme. Potential mechanisms of action of the protein for catalytic hydrolysis of OP substrates are also evaluated in light of the proposed binding modes.
RESUMO
Human serum paraoxonase-1 (HuPON1) has the capacity to hydrolyze aryl esters, lactones, oxidized phospholipids, and organophosphorus (OP) compounds. HuPON1 and bacterially expressed chimeric recombinant PON1s (G2E6 and G3C9) differ by multiple amino acids, none of which are in the putative enzyme active site. To address the importance of these amino acid differences, the abilities of HuPON1, G2E6, G3C9, and several variants to hydrolyze phenyl acetate, paraoxon, and V-type OP nerve agents were examined. HuPON1 and G2E6 have a 10-fold greater catalytic efficiency toward phenyl acetate than G3C9. In contrast, bacterial PON1s are better able to promote hydrolysis of paraoxon, whereas HuPON1 is considerably better at catalyzing the hydrolysis of nerve agents VX and VR. These studies demonstrate that mutations distant from the active site of PON1 have large and unpredictable effects on the substrate specificities and possibly the hydrolytic mechanisms of HuPON1, G2E6, and G3C9. The replacement of residue H115 in the putative active site with tryptophan (H115W) has highly disparate effects on HuPON1 and G2E6. In HuPON1, variant H115W loses the ability to hydrolyze VR but has improved activity toward paraoxon and VX. The H115W variant of G2E6 has paraoxonase activity similar to that of wild-type G2E6, modest activity with phenyl acetate and VR, and enhanced VX hydrolysis. VR inhibits H115W HuPON1 competitively when paraoxon is the substrate and noncompetitively when VX is the substrate. We have identified the first variant of HuPON1, H115W, that displays significantly enhanced catalytic activity against an authentic V-type nerve agent.
Assuntos
Arildialquilfosfatase/metabolismo , Proteínas Recombinantes/metabolismo , Arildialquilfosfatase/química , Arildialquilfosfatase/genética , Bactérias/genética , Bactérias/metabolismo , Hidrolases de Éster Carboxílico/genética , Hidrolases de Éster Carboxílico/metabolismo , Linhagem Celular , Humanos , Modelos Biológicos , Mutação , Paraoxon/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Especificidade por Substrato/genéticaRESUMO
Microarray gene expression profiling was used to identify bone morphogenetic protein-4 (BMP-4) responsive factors involved in late stages of adipocyte commitment in C3H10T1/2 cells. The analysis revealed that the matrix metalloproteinase-3 (MMP-3) gene decreased 100-fold after BMP-4 treatment, and expression of MMP-13 decreased 19.5-fold. Uncommitted C3H10T1/2 cells exhibit dramatic up-regulation of MMP-3 and MMP-13 genes as cells become confluent. Real-time RT-PCR demonstrated that BMP-4 blocks expression of both transcripts. Likewise, a stable committed preadipocyte line derived from C3H10T1/2 cells did not express MMP-3 or MMP-13 at confluence, despite never receiving BMP-4. Active forms of both proteins were detected in media from confluent C3H10T1/2 cells but not in BMP-4 treated cells. Addition of BMP-4 to confluent C3H10T1/2 cells repressed the expression of both genes but did not induce adipocyte differentiation. The findings indicate that BMP-4-induced down-regulation of MMP-3 and MMP-13 is associated with commitment, but is insufficient to induce adipogenesis.
Assuntos
Proteínas Morfogenéticas Ósseas/farmacologia , Células-Tronco Embrionárias/efeitos dos fármacos , Perfilação da Expressão Gênica , Metaloproteinase 13 da Matriz/genética , Metaloproteinase 3 da Matriz/genética , Células 3T3-L1 , Adipócitos/citologia , Adipócitos/efeitos dos fármacos , Adipócitos/metabolismo , Animais , Proteína Morfogenética Óssea 4 , Diferenciação Celular/genética , Linhagem Celular , Eletroforese em Gel de Poliacrilamida , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Expressão Gênica/efeitos dos fármacos , Immunoblotting , Metaloproteinase 13 da Matriz/metabolismo , Metaloproteinase 3 da Matriz/metabolismo , Camundongos , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Tempo , Transcrição Gênica/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacosRESUMO
Previous studies showed that exposure of C3H10T1/2 stem cells to bone morphogenetic protein-4 (BMP-4) produced cells that convert into adipocytes at high frequency when treated with differentiation inducers. In the present investigation, an independent approach shows that BMP-4 is required for stable commitment of pluripotent stem cells to the adipocyte lineage. Exposure of proliferating 10T1/2 stem cells to 5-azacytidine, a potent DNA methylation inhibitor, gave rise to a subpopulation of cells that can be cloned and that have the capacity to undergo conversion into adipocytes upon treatment with terminal differentiation inducers. Detailed studies performed with a cloned committed subline, the A33 line, verified stable adipocyte lineage determination in the absence of exogenous BMP-4. Remarkably, this cell line expresses and secretes BMP-4 during proliferation in the same time window that exogenous BMP-4 must be added to naïve 10T1/2 cells to induce maximal adipocyte commitment. Furthermore, exposure of A33 cells to noggin, a naturally occurring BMP-4-binding antagonist, during this critical time window blocks subsequent differentiation. The role of BMP-4 in adipocyte lineage commitment is further strengthened by gene expression profiling of proliferating 10T1/2 stem cells and A33 preadipocytes. These findings revealed changes in the molecular circuitry, specifically coordinated changes in the expression of members of the BMP-4 signaling pathway, that distinguish A33 preadipocytes from uncommitted parental 10T1/2 stem cells. Together, these studies provide compelling evidence for the participation of BMP-4 in adipocyte lineage determination.
Assuntos
Adipócitos/citologia , Proteínas Morfogenéticas Ósseas/genética , Linhagem da Célula , Metilação de DNA , Células-Tronco/citologia , Adipócitos/efeitos dos fármacos , Animais , Azacitidina/farmacologia , Proteína Morfogenética Óssea 4 , Proteínas Morfogenéticas Ósseas/antagonistas & inibidores , Proteínas Morfogenéticas Ósseas/metabolismo , Proteínas de Transporte/metabolismo , Contagem de Células , Linhagem da Célula/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Metilação de DNA/efeitos dos fármacos , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Mesoderma/citologia , Camundongos , Fenótipo , Regiões Promotoras Genéticas/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transdução de Sinais/efeitos dos fármacos , Células-Tronco/efeitos dos fármacosRESUMO
Cell culture models have been developed to study commitment and subsequent differentiation of preadipocytes into adipocytes. Bone morphogenetic protein 4 commits mesenchymal stem cells to the adipose lineage. Other factors, including Wnt signaling, cell density, and cell shape, play a role in lineage commitment. Following commitment to the adipose lineage, growth-arrested preadipocytes can differentiate to adipocytes by treatment with insulin-like growth factor 1, glucocorticoid and an agent that increases cAMP level. This process is characterized by a rapid and transient increase in CCAAT/enhancer binding protein (C/EBP) beta and synchronous re-entry into the cell cycle. Acquisition of DNA-binding by C/EBPbeta occurs after the transcription factor becomes phosphorylated. The cells enter a growth-arrested state and begin terminal differentiation. C/EBPalpha, peroxisome proliferator-activated receptor gamma, and adipocyte determination, and differentiation-dependent factor 1 coordinate the expression of genes that create and maintain the adipocyte phenotype.