RESUMO
Deposition of insoluble protein aggregates is a hallmark of neurodegenerative diseases. The universal presence of ß-amyloid and tau in Alzheimer's disease (AD) has facilitated advancement of the amyloid cascade and tau hypotheses that have dominated AD pathogenesis research and therapeutic development. However, the underlying etiology of the disease remains to be fully elucidated. Here we report a comprehensive study of the human brain-insoluble proteome in AD by mass spectrometry. We identify 4,216 proteins, among which 36 proteins accumulate in the disease, including U1-70K and other U1 small nuclear ribonucleoprotein (U1 snRNP) spliceosome components. Similar accumulations in mild cognitive impairment cases indicate that spliceosome changes occur in early stages of AD. Multiple U1 snRNP subunits form cytoplasmic tangle-like structures in AD but not in other examined neurodegenerative disorders, including Parkinson disease and frontotemporal lobar degeneration. Comparison of RNA from AD and control brains reveals dysregulated RNA processing with accumulation of unspliced RNA species in AD, including myc box-dependent-interacting protein 1, clusterin, and presenilin-1. U1-70K knockdown or antisense oligonucleotide inhibition of U1 snRNP increases the protein level of amyloid precursor protein. Thus, our results demonstrate unique U1 snRNP pathology and implicate abnormal RNA splicing in AD pathogenesis.
Assuntos
Processamento Alternativo/fisiologia , Doença de Alzheimer/fisiopatologia , Encéfalo/metabolismo , Proteoma/metabolismo , Ribonucleoproteína Nuclear Pequena U1/metabolismo , Spliceossomos/metabolismo , Processamento Alternativo/genética , Western Blotting , Cromatografia Líquida , Imunofluorescência , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Imuno-Histoquímica , Proteoma/genética , Proteômica , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Espectrometria de Massas em TandemRESUMO
Presynaptic synthesis of acetylcholine (ACh) requires a steady supply of choline, acquired by a plasma membrane, hemicholinium-3-sensitive (HC-3) choline transporter (CHT). A significant fraction of synaptic choline is recovered from ACh hydrolyzed by acetylcholinesterase (AChE) after vesicular release. Although antecedent neuronal activity is known to dictate presynaptic CHT activity, the mechanisms supporting this regulation are unknown. We observe an exclusive localization of CHT to cholinergic neurons and demonstrate that the majority of CHTs reside on small vesicles within cholinergic presynaptic terminals in the rat and mouse brain. Furthermore, immunoisolation of presynaptic vesicles with multiple antibodies reveals that CHT-positive vesicles carry the vesicular acetylcholine transporter (VAChT) and synaptic vesicle markers such as synaptophysin and Rab3A and also contain acetylcholine. Depolarization of synaptosomes evokes a Ca2+-dependent botulinum neurotoxin C-sensitive increase in the Vmax for HC-3-sensitive choline uptake that is accompanied by an increase in the density of CHTs in the synaptic plasma membrane. Our study leads to the novel hypothesis that CHTs reside on a subpopulation of synaptic vesicles in cholinergic terminals that can transit to the plasma membrane in response to neuronal activity to couple levels of choline re-uptake to the rate of ACh release.
Assuntos
Proteínas de Membrana Transportadoras/metabolismo , Terminações Pré-Sinápticas/metabolismo , Vesículas Sinápticas/metabolismo , Proteínas de Transporte Vesicular , Animais , Especificidade de Anticorpos , Biomarcadores/análise , Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Fibras Colinérgicas/química , Fibras Colinérgicas/metabolismo , Técnicas de Imunoadsorção , Proteínas de Membrana Transportadoras/genética , Camundongos , Camundongos Endogâmicos C57BL , Neurônios/química , Neurônios/metabolismo , Células PC12 , Terminações Pré-Sinápticas/química , Transporte Proteico , Ratos , Frações Subcelulares/química , Vesículas Sinápticas/química , Vesículas Sinápticas/classificação , Proteínas Vesiculares de Transporte de AcetilcolinaRESUMO
BACKGROUND: Genetic, epidemiologic, and biochemical evidence suggests that apolipoprotein E, low-density lipoprotein receptors, and lipid metabolism play important roles in sporadic Alzheimer disease (AD). OBJECTIVE: To identify novel candidate genes associated with sporadic AD. DESIGN: We performed an unbiased microarray screen for genes differentially expressed in lymphoblasts of patients with sporadic AD and prioritized 1 gene product for further characterization in AD brain. SETTING: Emory University, Atlanta, Ga. SUBJECTS: Cell lines were used from 14 patients with AD and 9 normal human control subjects. RESULTS: Six genes were differentially expressed in lymphoblasts of 2 independent groups of patients with probable AD and autopsy-proven AD. We hypothesized that 1 of the genes, termed low-density lipoprotein receptor relative with 11 binding repeats (LR11) (reduced 1.8- and 2.5-fold in AD lymphoblasts vs controls), might be associated with sporadic AD on the basis of its function as neuronal apolipoprotein E receptor. We found dramatic and consistent loss of immunocytochemical staining for LR11 in histologically normal-appearing neurons in AD brains. This reduction of LR11 protein was confirmed by quantitative Western blotting (P =.01). CONCLUSIONS: There is loss of the microarray-derived candidate, LR11, in neurons of AD brains. This study shows that microarray analysis of widely available lymphoblasts derived from patients with AD holds promise as a primary screen for candidate genes associated with AD.
Assuntos
Doença de Alzheimer/metabolismo , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Receptores de LDL/metabolismo , Proteínas Adaptadoras de Transporte Vesicular , Idoso , Idoso de 80 Anos ou mais , Doença de Alzheimer/genética , Encéfalo/metabolismo , Linhagem Celular Transformada , Feminino , Humanos , Proteínas Relacionadas a Receptor de LDL , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade/biossíntese , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade/genética , Linfócitos/metabolismo , Masculino , Glicoproteínas de Membrana/biossíntese , Proteínas de Membrana Transportadoras/biossíntese , Proteínas de Membrana Transportadoras/genética , Pessoa de Meia-Idade , Proteínas do Tecido Nervoso/biossíntese , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Receptores de LDL/biossíntese , Receptores de LDL/genéticaRESUMO
Activation of innate and adaptive immune responses is tightly regulated, as insufficient activation could result in defective clearance of pathogens, while excessive activation might lead to lethal systemic inflammation or autoimmunity. A20 functions as a negative regulator of innate and adaptive immunity by inhibiting NF-κB activation. A20 mediates its inhibitory function in a complex with other proteins including RNF11 and Itch, both E3 ubiquitin ligases and TAX1BP1, an adaptor protein. Since NF-κB has been strongly implicated in various neuronal functions, we predict that its inhibitor, the A20 complex, is also present in the nervous system. In efforts to better understand the role of A20 complex and NF-κB signaling pathway, we determined regional distribution of A20 mRNA as well as protein expression levels and distribution of RNF11, TAX1BP1 and Itch, in different brain regions. The distribution of TRAF6 was also investigated since TRAF6, also an E3 ligase, has an important role in NF-κB signaling pathway. Our investigations, for the first time, describe and demonstrate that the essential components of the A20 ubiquitin-editing complex are present and mainly expressed in neurons. The A20 complex components are also differentially expressed throughout the human brain. This study provides useful information about region specific expression of the A20 complex components that will be invaluable while determining the role of NF-κB signaling pathway in neuronal development and degeneration.
Assuntos
Encéfalo/metabolismo , Proteínas de Ligação a DNA/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Neurônios/metabolismo , Proteínas Nucleares/metabolismo , Ubiquitina/metabolismo , Proteínas de Transporte/metabolismo , Proteínas de Ligação a DNA/genética , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas Nucleares/genética , RNA Mensageiro/metabolismo , Proteínas Repressoras/metabolismo , Fator 6 Associado a Receptor de TNF/metabolismo , Proteína 3 Induzida por Fator de Necrose Tumoral alfa , Ubiquitina-Proteína Ligases/metabolismoRESUMO
BACKGROUND: Detergent-insoluble protein accumulation and aggregation in the brain is one of the pathological hallmarks of neurodegenerative diseases. Here, we describe the identification of septin 11 (SEPT11), an enriched component of detergent-resistant fractions in frontotemporal lobar degeneration with ubiquitin-immunoreactive inclusions (FTLD-U), using large-scale unbiased proteomics approaches. RESULTS: We developed and applied orthogonal quantitative proteomic strategies for the unbiased identification of disease-associated proteins in FTLD-U. Using these approaches, we proteomically profiled detergent-insoluble protein extracts prepared from frontal cortex of FTLD-U cases, unaffected controls, or neurologic controls (i.e. Alzheimer's disease; AD). Among the proteins altered specifically in FTLD-U, we identified TAR DNA binding protein-43 (TDP-43), a known component of ubiquitinated inclusions. Moreover, we identified additional proteins enriched in detergent-resistant fractions in FTLD-U, and characterized one of them, SEPT11, in detail. Using independent highly sensitive targeted proteomics approaches, we confirmed the enrichment of SEPT11 in FTLD-U extracts. We further showed that SEPT11 is proteolytically cleaved into N-terminal fragments and, in addition to its prominent glial localization in normal brain, accumulates in thread-like pathology in affected cortex of FTLD-U patients. CONCLUSIONS: The proteomic discovery of insoluble SEPT11 accumulation in FTLD-U, along with novel pathological associations, highlights a role for this cytoskeleton-associated protein in the pathogenesis of this complex disorder.
Assuntos
Degeneração Lobar Frontotemporal/metabolismo , Septinas/química , Septinas/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Detergentes/química , Feminino , Degeneração Lobar Frontotemporal/genética , Degeneração Lobar Frontotemporal/patologia , Células HEK293 , Humanos , Masculino , Espectrometria de Massas/métodos , Pessoa de Meia-Idade , Proteômica/métodos , Septinas/genéticaRESUMO
Muscarinic acetylcholine receptors (mAChRs) are drug targets for multiple neurodegenerative and neuropsychiatric disorders, but the full therapeutic potential of mAChR-targeted drugs has not been realized, mainly because of a lack of subtype-selective agonists. Recent advances have allowed the development of highly selective agonists that bind to an allosteric site on the M(1) mAChR that is spatially distinct from the orthosteric acetylcholine binding site, but less is known about the profile of intracellular signals activated by orthosteric versus allosteric M(1) mAChR agonists. We investigated the activation and regulatory mechanisms of two structurally distinct allosteric M(1) mAChR agonists, AC260584 and TBPB. We show that allosteric agonists potently activate multiple signal transduction pathways linked to the M(1) mAChR receptor but, compared to orthosteric agonists, much less efficiently recruit arrestin 3, a protein involved in regulation of G-protein coupled receptor signaling. Consistent with decreased arrestin recruitment, both allosteric agonists showed blunted responses in measurements of receptor desensitization, internalization, and downregulation. These results advance the understanding of mAChR biology and may shed light on unanticipated differences in the pharmacology of orthosteric vs. allosteric agonists that might be capitalized upon for drug development for the treatment of CNS diseases.
RESUMO
Yeast and animal homotypic fusion and vacuole protein sorting (HOPS) complexes contain conserved subunits, but HOPS-mediated traffic in animals might require additional proteins. Here, we demonstrate that SPE-39 homologues, which are found only in animals, are present in RAB5-, RAB7-, and RAB11-positive endosomes where they play a conserved role in lysosomal delivery and probably function via their interaction with the core HOPS complex. Although Caenorhabditis elegans spe-39 mutants were initially identified as having abnormal vesicular biogenesis during spermatogenesis, we show that these mutants also have disrupted processing of endocytosed proteins in oocytes and coelomocytes. C. elegans SPE-39 interacts in vitro with both VPS33A and VPS33B, whereas RNA interference of VPS33B causes spe-39-like spermatogenesis defects. The human SPE-39 orthologue C14orf133 also interacts with VPS33 homologues and both coimmunoprecipitates and cosediments with other HOPS subunits. SPE-39 knockdown in cultured human cells altered the morphology of syntaxin 7-, syntaxin 8-, and syntaxin 13-positive endosomes. These effects occurred concomitantly with delayed mannose 6-phosphate receptor-mediated cathepsin D delivery and degradation of internalized epidermal growth factor receptors. Our findings establish that SPE-39 proteins are a previously unrecognized regulator of lysosomal delivery and that C. elegans spermatogenesis is an experimental system useful for identifying conserved regulators of metazoan lysosomal biogenesis.
Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Proteínas de Transporte/metabolismo , Lisossomos/metabolismo , Complexos Multiproteicos/metabolismo , Animais , Caenorhabditis elegans/ultraestrutura , Endocitose , Endossomos/metabolismo , Células HeLa , Humanos , Masculino , Camundongos , Ligação Proteica , Processamento de Proteína Pós-Traducional , Transporte Proteico , RNA Interferente Pequeno/metabolismo , Receptor IGF Tipo 2/metabolismo , Espermatócitos/ultraestrutura , Espermatogênese , Proteínas de Transporte Vesicular/metabolismoRESUMO
This unit details the use of bacterially produced fusion proteins for the production of antisera, allowing for the large-scale generation of affinity-purified antibodies to specific, targeted epitopes. The use of pET vectors containing a polyhistidine (His) or glutathione-S-transferase (GST) tag to construct bacterial expression plasmids are provided as prototypical examples of fusion protein methodology. The basic protocols provided in this unit describe: (1) transformation of E. coli for high-yield production of soluble fusion protein, (2) purification of soluble fusion proteins for use in immunization using chelated nickel or glutathione affinity chromatography (for His- and GST-tagged fusion proteins, respectively), (3) immunization of rabbits with purified fusion protein and collection of antisera, and (4) characterization of antisera for antibody specificity using immunoblotting techniques. Support protocols describe the purification of His-tagged insoluble fusion proteins for animal immunization and the construction and use of affinity columns for purifying antibodies using soluble fusion proteins.