RESUMO
Inclusion body myositis and Alzheimer's disease are age-related disorders characterized in part by the appearance of intracellular lesions composed of filamentous aggregates of the microtubule-associated protein tau. Abnormal tau phosphorylation accompanies tau aggregation and may be an upstream pathological event in both diseases. Enzymes implicated in tau hyperphosphorylation in Alzheimer's disease include members of the casein kinase 1 family of phosphotransferases, a group of structurally related protein kinases that frequently function in tandem with the ubiquitin modification system. To determine whether casein kinase 1 isoforms associate with degenerating muscle fibers of inclusion body myositis, muscle biopsy sections isolated from sporadic disease cases were subjected to double-label fluorescence immunohistochemistry using selective anti-casein kinase 1 and anti-phospho-tau antibodies. Results showed that the alpha isoform of casein kinase 1, but not the delta or epsilon isoforms, stained degenerating muscle fibers in all eight inclusion body myositis cases examined. Staining was almost exclusively localized to phospho-tau-bearing inclusions. These findings, which extend the molecular similarities between inclusion body myositis muscle and Alzheimer's disease brain, implicate casein kinase 1 alpha as one of the phosphotransferases potentially involved in tau hyperphosphorylation.
Assuntos
Caseína Quinase I/metabolismo , Fibras Musculares Esqueléticas/metabolismo , Miosite de Corpos de Inclusão/metabolismo , Proteínas tau/metabolismo , Adulto , Idoso , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Miosite de Corpos de Inclusão/patologia , Enzimas Ativadoras de Ubiquitina/metabolismoRESUMO
Alzheimer's Disease (AD) is characterized by the appearance of neurofibrillary and granulovacuolar lesions in the brains of affected individuals. The former is composed of hyperphosphorylated aggregates of the microtubule-associated protein tau. The latter is poorly characterized but reacts strongly with anti-phosphoepitope antibodies indicating that it too accumulates phosphoproteins. Both lesions react strongly with antibodies directed against members of the casein kinase-1 family of phosphotransferases, a group of closely related protein kinases that frequently function in tandem with the ubiquitin modification system. To determine whether individual members of the casein kinase-1 family differentially associate with AD lesions, hippocampal sections isolated from late stage cases of AD were subjected to double-label fluorescence immunohistochemistry using a panel of selective anti-casein kinase 1 antibodies and small-molecule fluorochromes thioflavin S and thiazin red. The resultant colocalization patterns revealed that the alpha CK1 isoform strongly correlated with thioflavin S and thiazin red fluorescence, indicating that it preferentially associated with neurofibrillary lesions. In contrast, the delta isoform staining pattern was dominated by colocalization with granulovacuolar degeneration bodies. These findings suggest that granulovacuolar and neurofibrillary lesions occupy separate populations of neurons, and implicate CK1 isoforms in the generation of lesion-associated phosphoepitopes. They also suggest a nexus between the phosphorylation and ubiquitination modifications found in both lesions.
Assuntos
Doença de Alzheimer/enzimologia , Caseína Quinase I/metabolismo , Emaranhados Neurofibrilares/enzimologia , Vacúolos/enzimologia , Idoso de 80 Anos ou mais , Doença de Alzheimer/patologia , Especificidade de Anticorpos/imunologia , Caseína Quinase I/genética , Caseína Quinase I/imunologia , Feminino , Regulação Enzimológica da Expressão Gênica , Hipocampo/enzimologia , Hipocampo/patologia , Humanos , Imuno-Histoquímica/métodos , Isoenzimas/genética , Isoenzimas/imunologia , Isoenzimas/metabolismo , Masculino , Peptidilprolil Isomerase de Interação com NIMA , Degeneração Neural , Emaranhados Neurofibrilares/patologia , Peptidilprolil Isomerase/genética , Peptidilprolil Isomerase/metabolismo , Vacúolos/patologiaRESUMO
To understand the role of RNA-binding proteins (RBPs) in the regulation of gene expression, methods are needed for the in vivo identification of RNA-protein interactions. We have developed the peptide nucleic acid (PNA)-assisted identification of RBP technology to enable the identification of proteins that complex with a target RNA in vivo. Specific regions of the 3' and 5' UTRs of ankylosis mRNA were targeted by antisense PNAs transported into cortical neurons by the cell-penetrating peptide transportan 10. An array of proteins was isolated in complex with or near the targeted regions of the ankylosis mRNA through UV-induced crosslinking of the annealed PNA-RNA-RBP complex. The first evidence for pharmacological modulation of these specific protein-RNA associations was observed. These data show that the PNA-assisted identification of the RBP technique is a reliable method to rapidly identify proteins interacting in vivo with the target RNA.
Assuntos
RNA/química , Ribonucleoproteínas/química , Aminoácidos/química , Animais , Sequência de Bases , Células Cultivadas , Reagentes de Ligações Cruzadas/farmacologia , DNA/química , Primers do DNA/química , Corantes Fluorescentes/farmacologia , Hipocampo/metabolismo , Imuno-Histoquímica , Imunoprecipitação , Hibridização In Situ , Espectrometria de Massas , Microscopia de Fluorescência , Dados de Sequência Molecular , Neurônios/metabolismo , Ácidos Nucleicos Peptídicos/química , Peptídeos/química , Ligação Proteica , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/química , Ratos , Ribonucleoproteínas/metabolismo , Fatores de Tempo , Raios UltravioletaRESUMO
Immunoprecipitation of mRNA-protein complexes is a method that can be used to study RNA binding protein (RBP)-RNA interactions. In this protocol, an antibody targeting an RBP of interest is used to immunoprecipitate the RBP and any interacting molecules from a cell lysate. Reverse transcription followed by PCR is then used to identify individual mRNAs isolated with the RBP. This method focuses on examining an association between a specific RBP-mRNA complex, and it is best suited for a small scale screening of known or putative binding partners. It can also be used as a second, independent method to verify RBP-mRNA interactions discovered through more universal screening techniques. We describe the immunoprecipitation protocol in practical detail and discuss variations of the method as well as issues associated with it. The procedure takes three days to complete.
Assuntos
Imunoprecipitação/métodos , RNA Mensageiro/química , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/metabolismo , Animais , Células Cultivadas , Córtex Cerebral/citologia , Neurônios/citologia , Neurônios/metabolismo , RatosRESUMO
All aspects of RNA metabolism are regulated by RNA-binding proteins (RBPs) that directly associate with the RNA. Some aspects of RNA biology such as RNA abundance can be readily assessed using standard hybridization technologies. However, identification of RBPs that specifically associate with selected RNAs has been more difficult, particularly when attempting to assess this in live cells. The peptide nucleic acid (PNA)-assisted identification of RBPs (PAIR) technology has recently been developed to overcome this issue. The PAIR technology uses a cell membrane-penetrating peptide (CPP) to efficiently deliver into the cell its linked PNA oligomer that complements the target mRNA sequence. The PNA will then anneal to its target mRNA in the living cell, and then covalently couple to the mRNA-RBP complexes subsequent to an ultraviolet (UV) cross-linking step. The resulting PNA-RNA-RBP complex can be isolated using sense oligonucleotide magnetic beads, and the RBPs can then be identified by mass spectrometry (MS). This procedure can usually be completed within 3 d. The use of the PAIR procedure promises to provide insight into the dynamics of RNA processing, transport, degradation and translation in live cells.
Assuntos
Ácidos Nucleicos Peptídicos/metabolismo , Proteínas de Ligação a RNA/análise , Proteínas de Ligação a RNA/metabolismo , RNA/metabolismo , Animais , Sobrevivência Celular , Células Cultivadas , Reagentes de Ligações Cruzadas , Hibridização In Situ , RatosAssuntos
Encéfalo/fisiologia , Perfilação da Expressão Gênica/métodos , Expressão Gênica , RNA Mensageiro/genética , Envelhecimento/genética , Animais , Química Encefálica , Encefalopatias/genética , Encefalopatias/fisiopatologia , Sistema Nervoso Central/fisiologia , Sistema Nervoso Central/fisiopatologia , DNA/genética , Humanos , Doenças Neurodegenerativas/genética , Doenças Neurodegenerativas/fisiopatologia , RNA Mensageiro/análiseRESUMO
New methods for analyzing tau fibrillization have yielded insights into the biochemical transitions involved in the process. Here we review the parallels between the sequential progression of tau fibrillization observed macroscopically in Alzheimer's disease (AD) lesions and the pathway of tau aggregation observed in vitro with purified tau preparations. In addition, pharmacological agents for further dissection of fibrillization mechanism and lesion formation are discussed.