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1.
Chem Biol Interact ; 175(1-3): 101-7, 2008 Sep 25.
Artigo em Inglês | MEDLINE | ID: mdl-18538755

RESUMO

The classical function of acetylcholinesterase (AChE) is to terminate synaptic transmission at cholinergic synapses by rapidly hydrolyzing the neurotransmitter acetylcholine (ACh). Non-classical functions of AChE involve accelerating the assembly of Abeta peptide into amyloid fibrils and participating in haematopoiesis and neurite growth. Although numerous antibodies have been raised against AChE, many researchers have questioned their reliability to identify the AChE in situ, especially with the regard to its non-classical roles. Researchers attended the Ninth International Meeting on Cholinesterase raised this question by showing different Western blot patterns of AChE detected by different Abs. Producing more effective and reliable Abs for measuring AChE in vivo or in situ has become an important issue in many scientific fields. In this paper, we introduce a monoclonal antibody raised against synaptic AChE that we identified by Western blot assays, immunofluorescent staining and immunoprecipitation of AChE, and mass spectrometry. Our results strongly demonstrate the specificity of our monoclonal antibody to recognize synaptic AChE; hence our antibody can be used as an effective tool to study the various functions of AChE. Since the apoptosis-related AChE was its synaptic form, our antibody can be used as a tool to detect apoptotic cells.


Assuntos
Acetilcolinesterase/imunologia , Anticorpos Monoclonais/imunologia , Apoptose , Sinapses/enzimologia , Animais , Linhagem Celular , Eletroforese em Gel de Poliacrilamida , Humanos , Masculino , Ratos , Ratos Sprague-Dawley
2.
J Mol Cell Biol ; 3(4): 250-9, 2011 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-21377978

RESUMO

Acetylcholinesterase (AChE) is emerging as an important contributor to apoptosis in various cell types. However, overexpression of AChE does not initiate apoptosis, and cells which express AChE at basal levels grow normally, suggesting that AChE may function differently between normal and apoptotic conditions. In this study, we determined that an AChE-derived protein (∼55 kDa) positively correlated with cellular apoptotic levels. The 55 kDa AChE protein was not a result of a novel splice variant of the AChE primary transcript. Instead, it was determined to be a cleaved fragment of the full-length 68 kDa AChE protein that could not be inhibited by cycloheximide (CHX) but could be suppressed by caspase inhibitors in apoptotic PC-12 cells. Furthermore, activation of the Akt cascade abolished the 55 kDa protein, and both AChE protein forms (68 and 55 kDa) accumulated in the nucleus during apoptosis. In a mouse model for ischemia/reperfusion (I/R)-induced acute renal failure, the 55 kDa AChE protein was detected in the impaired organs but not in the normal ones, and its levels correlated with the genotype of the mice. In summary, a 55 kDa AChE protein resulting from the cleavage of 68 kDa AChE is induced during apoptosis, and it is negatively regulated by the Akt pathway. This study suggests that an alternative form of AChE may play a role in apoptosis.


Assuntos
Acetilcolinesterase/metabolismo , Apoptose , Regulação Enzimológica da Expressão Gênica , Proteínas Proto-Oncogênicas c-akt/metabolismo , Acetilcolinesterase/química , Acetilcolinesterase/genética , Animais , Linhagem Celular , Cicloeximida/farmacologia , Modelos Animais de Doenças , Ativação Enzimática , Humanos , Camundongos , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Ratos , Proteínas Recombinantes de Fusão/antagonistas & inibidores , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Traumatismo por Reperfusão/metabolismo , Transdução de Sinais
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