RESUMO
Expression of many disease-related aggregation-prone proteins results in cytotoxicity and the formation of large intracellular inclusion bodies. To gain insight into the role of inclusions in pathology and the in situ structure of protein aggregates inside cells, we employ advanced cryo-electron tomography methods to analyze the structure of inclusions formed by polyglutamine (polyQ)-expanded huntingtin exon 1 within their intact cellular context. In primary mouse neurons and immortalized human cells, polyQ inclusions consist of amyloid-like fibrils that interact with cellular endomembranes, particularly of the endoplasmic reticulum (ER). Interactions with these fibrils lead to membrane deformation, the local impairment of ER organization, and profound alterations in ER membrane dynamics at the inclusion periphery. These results suggest that aberrant interactions between fibrils and endomembranes contribute to the deleterious cellular effects of protein aggregation. VIDEO ABSTRACT.
Assuntos
Doença de Huntington/patologia , Corpos de Inclusão/patologia , Neurônios/patologia , Neurônios/ultraestrutura , Peptídeos/metabolismo , Amiloide/química , Animais , Microscopia Crioeletrônica , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/patologia , Feminino , Células HeLa , Humanos , Proteína Huntingtina/genética , Proteína Huntingtina/metabolismo , Corpos de Inclusão/química , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Eletrônica de Transmissão , Mutação , Agregação Patológica de Proteínas , Tomografia/métodosRESUMO
Aggregation of proteins containing expanded polyglutamine (polyQ) repeats is the cytopathologic hallmark of a group of dominantly inherited neurodegenerative diseases, including Huntington's disease (HD). Huntingtin (Htt), the disease protein of HD, forms amyloid-like fibrils by liquid-to-solid phase transition. Macroautophagy has been proposed to clear polyQ aggregates, but the efficiency of aggrephagy is limited. Here, we used cryo-electron tomography to visualize the interactions of autophagosomes with polyQ aggregates in cultured cells in situ. We found that an amorphous aggregate phase exists next to the radially organized polyQ fibrils. Autophagosomes preferentially engulfed this amorphous material, mediated by interactions between the autophagy receptor p62/SQSTM1 and the non-fibrillar aggregate surface. In contrast, amyloid fibrils excluded p62 and evaded clearance, resulting in trapping of autophagic structures. These results suggest that the limited efficiency of autophagy in clearing polyQ aggregates is due to the inability of autophagosomes to interact productively with the non-deformable, fibrillar disease aggregates.
Assuntos
Amiloide , Autofagossomos , Autofagia , Proteína Huntingtina , Doença de Huntington , Peptídeos , Agregados Proteicos , Proteína Sequestossoma-1 , Peptídeos/metabolismo , Peptídeos/química , Peptídeos/genética , Humanos , Proteína Huntingtina/metabolismo , Proteína Huntingtina/genética , Proteína Huntingtina/química , Autofagossomos/metabolismo , Autofagossomos/ultraestrutura , Proteína Sequestossoma-1/metabolismo , Proteína Sequestossoma-1/genética , Amiloide/metabolismo , Amiloide/química , Amiloide/genética , Doença de Huntington/metabolismo , Doença de Huntington/genética , Doença de Huntington/patologia , Microscopia Crioeletrônica , Animais , Agregação Patológica de Proteínas/metabolismo , Agregação Patológica de Proteínas/genéticaRESUMO
Osmolytes stabilize protein structure and suppress protein aggregation. The mechanism of how osmolytes impact polyglutamine (polyQ) aggregation implicated in Huntington's disease was studied. By using a reverse-phase chromatography assay, we show that methylamines-trimethylamine N-oxide and betaine are generic in enhancing polyQ aggregation, while a disaccharide trehalose and an amino acid citrulline moderately retard polyQ aggregation in a sequence specific manner. Despite the altered kinetics, the fundamental nucleation mechanism of polyQ aggregation and the nature of end stage aggregates remains unaffected. These results highlight the importance of using osmolytes as modulatory agents of polyQ aggregation.
Assuntos
Peptídeos/química , Agregados Proteicos , Humanos , Cinética , Concentração Osmolar , Estabilidade ProteicaRESUMO
Amyloid-like aggregates of the microtubule-associated protein Tau are associated with several neurodegenerative disorders including Alzheimer's disease. The existence of cellular machinery for the removal of such aggregates has remained unclear, as specialized disaggregase chaperones are thought to be absent in mammalian cells. Here we show in cell culture and in neurons that the hexameric ATPase valosin-containing protein (VCP) is recruited to ubiquitylated Tau fibrils, resulting in their efficient disaggregation. Aggregate clearance depends on the functional cooperation of VCP with heat shock 70 kDa protein (Hsp70) and the ubiquitin-proteasome machinery. While inhibition of VCP activity stabilizes large Tau aggregates, disaggregation by VCP generates seeding-active Tau species as byproduct. These findings identify VCP as a core component of the machinery for the removal of neurodegenerative disease aggregates and suggest that its activity can be associated with enhanced aggregate spreading in tauopathies.