RESUMO
Increasing evidence suggests that oxidative injury is involved in the pathogenesis of many age-related neurodegenerative disorders, including Alzheimer's disease (AD). Identifying the protein targets of oxidative stress is critical to determine which proteins may be responsible for the neuronal impairments and subsequent cell death that occurs in AD. In this study, we have applied a high-throughput shotgun proteomic approach to identify the targets of protein carbonylation in both aged and PS1 + AbetaPP transgenic mice. However, because of the inherent difficulties associated with proteomic database searching algorithms, several newly developed bioinformatic tools were implemented to ascertain a probability-based discernment between correct protein assignments and false identifications to improve the accuracy of protein identification. Assigning a probability to each identified peptide/protein allows one to objectively monitor the expression and relative abundance of particular proteins from diverse samples, including tissue from transgenic mice of mixed genetic backgrounds. This robust bioinformatic approach also permits the comparison of proteomic data generated by different laboratories since it is instrument- and database-independent. Applying these statistical models to our initial studies, we detected a total of 117 oxidatively modified (carbonylated) proteins, 59 of which were specifically associated with PS1 + AbetaPP mice. Pathways and network component analyses suggest that there are three major protein networks that could be potentially altered in PS1 + AbetaPP mice as a result of oxidative modifications. These pathways are 1) iNOS-integrin signaling pathway, 2) CRE/CBP transcription regulation and 3) rab-lyst vesicular trafficking. We believe the results of these studies will help establish an initial AD database of oxidatively modified proteins and provide a foundation for the design of future hypothesis driven research in the areas of aging and neurodegeneration.
Assuntos
Doença de Alzheimer/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Modelos Animais de Doenças , Proteínas de Membrana/metabolismo , Estresse Oxidativo/fisiologia , Proteômica/métodos , Membranas Sinápticas/metabolismo , Fator 2 Ativador da Transcrição/metabolismo , Envelhecimento/fisiologia , Doença de Alzheimer/genética , Doença de Alzheimer/patologia , Animais , Morte Celular , Integrinas/metabolismo , Camundongos , Camundongos Transgênicos , Degeneração Neural/metabolismo , Degeneração Neural/patologia , Rede Nervosa/fisiologia , Óxido Nítrico Sintase/metabolismo , Fosfoproteínas/metabolismo , Presenilina-1 , Probabilidade , Carbonilação Proteica/fisiologia , Transdução de Sinais/fisiologia , Membranas Sinápticas/patologiaRESUMO
Abstract Oxidative stress imparted by reactive oxygen species (ROS) is implicated in the pathogenesis of Alzheimer's disease (AD). Given that amyloid beta (Abeta) itself generates ROS that can directly damage proteins, elucidating the functional consequences of protein oxidation can enhance our understanding of the process of Abeta-mediated neurodegeneration. In this study, we employed a biocytin hydrazide/streptavidin affinity purification methodology followed by two-dimensional liquid chromatography tandem mass spectrometry coupled with SEQUEST bioinformatics technology, to identify the targets of Abeta-induced oxidative stress in cultured primary cortical mouse neurons. The Golgi-resident enzyme glucuronyltransferase (GlcAT-P) was a carbonylated target that we investigated further owing to its involvement in the biosynthesis of HNK-1, a carbohydrate epitope expressed on cell adhesion molecules and implicated in modulating the effectiveness of synaptic transmission in the brain. We found that increasing amounts of Abeta, added exogenously to the culture media of primary cortical neurons, significantly decreased HNK-1 expression. Moreover, in vivo, HNK-1 immunoreactivity was decreased in brain tissue of a transgenic mouse model of AD. We conclude that a potential consequence of Abeta-mediated oxidation of GlcAT-P is impairment of its enzymatic function, thereby disrupting HNK-1 biosynthesis and possibly adversely affecting synaptic plasticity. Considering that AD is partly characterized by progressive memory impairment and disordered cognitive function, the data from our in vitro studies can be reconciled with results from in vivo studies that have demonstrated that HNK-1 modulates synaptic plasticity and is critically involved in memory consolidation.
Assuntos
Peptídeos beta-Amiloides/farmacologia , Antígenos CD57/metabolismo , Regulação para Baixo , Moléculas de Adesão de Célula Nervosa/biossíntese , Estresse Oxidativo/fisiologia , Fragmentos de Peptídeos/farmacologia , Proteômica , Transmissão Sináptica/fisiologia , Sequência de Aminoácidos , Peptídeos beta-Amiloides/genética , Animais , Antígenos CD57/biossíntese , Configuração de Carboidratos , Sequência de Carboidratos , Células Cultivadas , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/fisiologia , Glucuronosiltransferase/isolamento & purificação , Glucuronosiltransferase/metabolismo , Camundongos , Camundongos Transgênicos , Dados de Sequência Molecular , Moléculas de Adesão de Célula Nervosa/antagonistas & inibidores , Neurônios/efeitos dos fármacos , Neurônios/enzimologia , Estresse Oxidativo/efeitos dos fármacos , Fragmentos de Peptídeos/genética , Gravidez , Proteômica/métodos , Transmissão Sináptica/efeitos dos fármacosRESUMO
PURPOSE: Amyloid-beta (Abeta) is a self-aggregating protein found in senile plaques in Alzheimer's disease (AD) brain and is thought to play a major role in the disease process. Oxidative stress may be a predominant cause of the formation of these Abeta aggregates. This study aims at identifying possible sites of copper-catalyzed oxidation of Abeta1-40 using liquid chromatography tandem mass spectrometry (LC/MS/MS) and scoring algorithm for spectral analysis (SALSA). Traditionally, identification of post-translational modifications by tandem mass spectrometric analysis requires users to inspect manually thousands of MS/MS spectra, which can be a tedious and time-consuming process. With the use of SALSA, users can automatically search for post-translational modifications based on the spacing of the m/z values associated with the ion series of an amino acid sequence. METHODS: Abeta1-40 was subjected to copper-catalyzed oxidative stress. LC/MS/MS and SALSA analyses were used to determine the sites of post-translational modification within the tryptic fragments. RESULTS: Oxidation was found to occur preferentially at the histidine residues Hisl3 and Hisl4 and at the methionine residue (Met35) of Abeta1-40. CONCLUSIONS: The combination of LC/MS/MS and SALSA searches could dramatically improve the efficiency and accuracy of determining the specific sites of oxidation of in vitro, copper-oxidized Abeta1-40 as well as other oxidized proteins.
Assuntos
Algoritmos , Peptídeos beta-Amiloides/química , Fragmentos de Peptídeos/química , Peptídeos beta-Amiloides/síntese química , Catálise , Cromatografia Líquida/métodos , Cobre , Espectrometria de Massas/métodos , Oxirredução , Fragmentos de Peptídeos/síntese químicaRESUMO
PURPOSE: The major initiative of this study was to implement a novel proteomic approach in order to detect protein carbonylation in aged mouse brain. Several lines of evidence indicate that reactive oxygen species (ROS)-induced protein oxidation plays an essential role in the initiation of age-related neuropathologies. Therefore, the identification of free radical or peroxide substrates would provide further insight into key biochemical mechanisms that contribute to the progression of certain neurological disorders. METHODS: Historically, ROS targets have been identified by conventional immunological two-dimensional (2-D) gel electrophoresis and mass spectrometric analyses. However, specific classes of proteins, such as transmembrane-spanning proteins, high-molecular-weight proteins, and very acidic or basic proteins, are frequently excluded or underrepresented by these analyses. In order to fill this technologic gap, we have used a functional proteomics approach using a liquid chromatography tandem mass spectrometric (LC-MS/MS) analysis coupled with a hydrazide biotin-streptavidin methodology in order to identify protein carbonylation in aged mice. RESULTS: Our initial studies suggest an ability to identify at least 100 carbonylated proteins in a single LC-MS/MS experiment. In addition to high-abundance cytosolic proteins that have been previously identified by 2-D gel electrophoresis and mass spectrometric analyses, we are able to identify several low-abundance receptor proteins, mitochondrial proteins involved in glucose and energy metabolism, as well as a series of receptors and tyrosine phosphatases known to be associated with insulin and insulin-like growth factor metabolism and cell-signaling pathways. CONCLUSIONS: Here we describe a rapid and sensitive proteomic analysis for the identification of carbonylated proteins in mouse brain homogenates through the conjunction of liquid chromatography and tandem mass spectrometry methods. We believe the ability to detect these post-translationally modified proteins specifically associated with brain impairments during the course of aging should allow one to more closely and objectively monitor the efficacy of various clinical treatments. In addition, the discovery of these unique brain biomarkers could also provide a conceptual framework for the future design of alternative drugs in the treatment of a variety of age-related neurodegenerative disorders.