RESUMO
In Archaea, an hexameric ATPase complex termed PAN promotes proteins unfolding and translocation into the 20 S proteasome. PAN is highly homologous to the six ATPases of the eukaryotic 19 S proteasome regulatory complex. Thus, insight into the mechanism of PAN function may reveal a general mode of action mutual to the eukaryotic 19 S proteasome regulatory complex. In this study we generated a three-dimensional model of PAN from tomographic reconstruction of negatively stained particles. Surprisingly, this reconstruction indicated that the hexameric complex assumes a two-ring structure enclosing a large cavity. Assessment of distinct three-dimensional functional states of PAN in the presence of adenosine 5'-O-(thiotriphosphate) and ADP and in the absence of nucleotides outlined a possible mechanism linking nucleotide binding and hydrolysis to substrate recognition, unfolding, and translocation. A novel feature of the ATPase complex revealed in this study is a gate controlling the "exit port" of the regulatory complex and, presumably, translocation into the 20 S proteasome. Based on our structural and biochemical findings, we propose a possible model in which substrate binding and unfolding are linked to structural transitions driven by nucleotide binding and hydrolysis, whereas translocation into the proteasome only depends upon the presence of an unfolded substrate and binding but not hydrolysis of nucleotide.
Assuntos
Adenosina Trifosfatases/química , Adenosina Trifosfatases/metabolismo , Proteínas Arqueais/química , Proteínas Arqueais/metabolismo , Modelos Moleculares , Complexo de Endopeptidases do Proteassoma/metabolismo , Hidrólise , Interações Hidrofóbicas e Hidrofílicas , Ligação ProteicaRESUMO
A major objective in identifying the mechanisms underlying neurobehavioral teratogenicity is the possibility of designing therapies that reverse or offset drug- or toxicant-induced neural damage. In our previous studies, we identified deficits in hippocampal muscarinic cholinergic receptor-induced membrane translocation of protein kinase C (PKC)gamma as the likely mechanism responsible for adverse behavioral effects of prenatal phenobarbital exposure. We therefore explored whether behavioral and synaptic defects could be reversed in adulthood by nicotine administration. Pregnant mice were given milled food containing phenobarbital to achieve a daily dose of 0.5-0.6 g/kg from gestational days 9-18. In adulthood, offspring showed deficits in the Morris maze, a behavior dependent on the integrity of septohippocampal cholinergic synaptic function, along with the loss of the PKCgamma response. Phenobarbital-exposed and control mice then received nicotine (10 mg/kg/day) for 14 days via osmotic minipumps. Nicotine reversed the behavioral deficits and restored the normal response of hippocampal PKCgamma to cholinergic receptor stimulation. The effects were regionally specific, as PKCgamma in the cerebellum was unaffected by either phenobarbital or nicotine; furthermore, in the hippocampus, PKC isoforms unrelated to the behavioral deficits showed no changes. Nicotine administration thus offers a potential therapy for reversing neurobehavioral deficits originating in septohippocampal cholinergic defects elicited by prenatal drug or toxicant exposures.
Assuntos
Comportamento Animal/efeitos dos fármacos , Hipnóticos e Sedativos/antagonistas & inibidores , Hipnóticos e Sedativos/toxicidade , Nicotina/uso terapêutico , Agonistas Nicotínicos/uso terapêutico , Fenobarbital/antagonistas & inibidores , Fenobarbital/toxicidade , Efeitos Tardios da Exposição Pré-Natal , Sinapses/efeitos dos fármacos , Animais , Biomarcadores , Tronco Encefálico/efeitos dos fármacos , Tronco Encefálico/metabolismo , Carbacol/farmacologia , Córtex Cerebral/efeitos dos fármacos , Córtex Cerebral/metabolismo , Citosol/efeitos dos fármacos , Citosol/metabolismo , Feminino , Hemicolínio 3/farmacologia , Isoenzimas/metabolismo , Aprendizagem em Labirinto/efeitos dos fármacos , Camundongos , Agonistas Muscarínicos/farmacologia , Neurônios/efeitos dos fármacos , Neurônios/patologia , Inibidores da Captação de Neurotransmissores/farmacologia , Sistema Nervoso Parassimpático/efeitos dos fármacos , Gravidez , Proteína Quinase C/metabolismo , Receptores Nicotínicos/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Natação/fisiologiaRESUMO
Although the actions of heroin on central nervous system (CNS) development are mediated through opioid receptors, the net effects converge on dysfunction of cholinergic systems. We explored the mechanisms underlying neurobehavioral deficits in mouse and avian (chick, Cayuga duck) models. In mice, prenatal heroin exposure (10 mg/kg on gestation days 9-18) elicited deficits in behaviors related to hippocampal cholinergic innervation, characterized by concomitant pre- and postsynaptic hyperactivity, but ending in a reduction of basal levels of protein kinase C (PKC) isoforms betaII and gamma and their desensitization to cholinergic receptor-induced activation. PKCalpha, which is not involved in the behaviors studied, was unaffected. Because mammalian models possess inherent confounding factors from maternal effects, we conducted parallel studies using avian embryos, evaluating hyperstriatal nucleus (intermedial part of the hyperstriatum ventrale, IMHV)-related, filial imprinting behavior. Heroin injection to the eggs (20 mg/kg) on incubation days 0 and 5 diminished the post-hatch imprinting ability and reduced PKCg and bII content in the IMHV membrane fraction. Two otherwise unrelated agents that converge on cholinergic systems, chlorpyrifos and nicotine, elicited the same spectrum of effects on PKC isoforms and imprinting but had more robust actions. Pharmacological characterization also excluded direct effects of opioid receptors on the expression of imprinting; instead, it indicated participation of serotonergic innervation. The avian models can provide rapid screening of neuroteratogens, exploration of common mechanisms of behavioral disruption, and the potential design of therapies to reverse neurobehavioral deficits.
Assuntos
Fixação Psicológica Instintiva/efeitos dos fármacos , Efeitos Tardios da Exposição Pré-Natal , Transdução de Sinais/efeitos dos fármacos , Teratogênicos/farmacologia , Animais , Fibras Colinérgicas/efeitos dos fármacos , Fibras Colinérgicas/fisiologia , Patos , Feminino , Heroína/farmacologia , Fixação Psicológica Instintiva/fisiologia , Camundongos , Naltrexona/farmacologia , Gravidez , Transdução de Sinais/fisiologiaRESUMO
The regulated degradation of damaged or misfolded proteins, as well as down-regulation of key signaling proteins, within eukaryotic and bacterial cells is catalyzed primarily by large, ATP-dependent multimeric proteolytic complexes, termed proteasomes. Inhibition of proteasomal activity affects a wide variety of physiological and pathological processes, and was found to be particularly effective for cancer therapy. We report here on the development of a novel high throughput assay for proteasome inhibition using a unique, highly sensitive live-cell screening, based on the cytoplasm-to-nucleus translocation of a fluorescent proteasome inhibition reporter (PIR) protein, consisting of nuclear localization signal-deficient p53 derivative. We further show here that mdm2, a key negative regulator of p53 plays a key role in the accumulation of PIR in the nucleus upon proteasome inhibition. Using this assay, we have screened the NCI Diversity Set library, containing 1,992 low molecular weight synthetic compounds, and identified four proteasome inhibitors. The special features of the current screen, compared to those of other approaches are discussed.