RESUMO
Huntington disease, a neurodegenerative disorder characterized by functional deficits and loss of striatal neurons, is linked to an expanded and unstable CAG trinucleotide repeat in the huntingtin gene (HTT). This DNA sequence translates to a polyglutamine repeat in the protein product, leading to mutant huntingtin (mHTT) protein aggregation. The aggregation of mHTT is inhibited in vitro and in vivo by the TCP-1 ring complex (TRiC) chaperonin. Recently, a novel complex comprised of a single type of TRiC subunit has been reported to inhibit mHTT aggregation. Specifically, the purified CCT5 homo-oligomer complex, when compared with TRiC, has a similar structure, ATP use, and substrate refolding activity, and, importantly, it also inhibits mHTT aggregation. Using an aggregation suppression assay and cryoelectron tomography coupled with a novel computational classification method, we uncover the interactions between the synthetic CCT5 complex (â¼ 1 MDa) and aggregates of mutant huntingtin exon 1 containing 46 glutamines (mHTTQ46-Ex1). We find that, in a similar fashion to TRiC, synthetic CCT5 complex caps mHTT fibrils at their tips and encapsulates mHTT oligomers, providing a structural description of the inhibition of mHTTQ46-Ex1 by CCT5 complex and a shared mechanism of mHTT inhibition between TRiC chaperonin and the CCT5 complex: cap and contain.
Assuntos
Chaperonina com TCP-1/química , Proteínas Mutantes/química , Proteínas do Tecido Nervoso/química , Chaperonina com TCP-1/genética , Chaperonina com TCP-1/ultraestrutura , Microscopia Crioeletrônica , Tomografia com Microscopia Eletrônica , Humanos , Proteína Huntingtina , Doença de Huntington/genética , Doença de Huntington/metabolismo , Modelos Moleculares , Proteínas Mutantes/genética , Proteínas Mutantes/ultraestrutura , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/ultraestrutura , Agregados Proteicos , Agregação Patológica de Proteínas/genética , Agregação Patológica de Proteínas/metabolismo , Domínios e Motivos de Interação entre Proteínas , Estrutura Quaternária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/ultraestruturaRESUMO
Protein misfolding and aggregation are thought to underlie the pathogenesis of many amyloid diseases, such as Alzheimer and Parkinson diseases, whereby a stepwise protein misfolding process begins with the conversion of soluble protein monomers to prefibrillar oligomers and progresses to the formation of insoluble amyloid fibrils. Drusen are extracellular deposits found in aging eyes and in eyes afflicted with age-related macular degeneration (AMD). Recent characterizations of drusen have revealed protein components that are shared with amyloid deposits. However, characteristic amyloid fibrils have thus far not been identified in drusen. In this study, we tested the hypothesis that nonfibrillar oligomers may be a common link in amyloid diseases. Oligomers consisting of distinct amyloidogenic proteins and peptides can be detected by a recently developed antibody that is thought to recognize a common structure. Notably, oligomers exhibit cellular toxicity, which suggests that they play a role in the pathogenesis of neurodegenerative diseases. Through use of the anti-oligomer antibody, we came to observe the presence of nonfibrillar, toxic oligomers in drusen. Conversely, no reactivity was observed in age-matched control eyes without drusen. These results suggest that amyloid oligomers may be involved in drusen biogenesis and that similar protein misfolding processes may occur in AMD and amyloid diseases.
Assuntos
Envelhecimento/fisiologia , Amiloide/química , Degeneração Macular/patologia , Conformação Proteica , Dobramento de Proteína , Drusas Retinianas/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Amiloide/toxicidade , Células Cultivadas , Feminino , Feto/anatomia & histologia , Humanos , Masculino , Pessoa de Meia-Idade , Epitélio Pigmentado Ocular/citologia , Epitélio Pigmentado Ocular/metabolismo , Epitélio Pigmentado Ocular/patologia , Drusas Retinianas/patologiaRESUMO
Oxidatively- or enzymatically-modified low-density lipoprotein (LDL) is intimately involved in the initiation and progression of atherosclerosis. The in vivo modified LDL is electro-negative (LDL(-)) and consists of peroxidized lipid and unfolded apoB-100 protein. This study was aimed at establishing specific protein modifications and conformational changes in LDL(-) assessed by liquid chromatography/tandem mass spectrometry (LC/MS/MS) and circular dichroism analyses, respectively. The functional significance of these chemical modifications and structural changes were validated with binding and uptake experiments to- and by bovine aortic endothelial cells (BAEC). The plasma LDL(-) fraction showed increased nitrotyrosine and lipid peroxide content as well as a greater cysteine oxidation as compared with native- and total-LDL. LC/MS/MS analyses of LDL(-) revealed specific modifications in the apoB-100 moiety, largely involving nitration of tyrosines in the alpha-helical structures and beta(2) sheet as well as cysteine oxidation to cysteic acid in beta(1) sheet. Circular dichroism analyses showed that the alpha-helical content of LDL(-) was substantially lower ( approximately 25%) than that of native LDL ( approximately 90%); conversely, LDL(-) showed greater content of beta-sheet and random coil structure, in agreement with unfolding of the protein. These results were mimicked by treatment of LDL subfractions with peroxynitrite (ONOO(-)) or SIN-1: similar amino acid modifications as well as conformational changes (loss of alpha-helical structure and gain in beta-sheet structure) were observed. Both LDL(-) and ONOO(-)-treated LDL showed a statistically significant increase in binding and uptake to- and by BAEC compared to native LDL. We further found that most binding and uptake in control-LDL was through LDL-R with minimal oxLDL-R-dependent uptake. ONOO(-)-treated LDL was significantly bound and endocytosed by LOX-1, CD36, and SR-A with minimal contribution from LDL-R. It is suggested that lipid peroxidation and protein nitration may account for the mechanisms leading to apoB-100 protein unfolding and consequential increase in modified LDL binding and uptake to and by endothelial cells that is dependent on oxLDL scavenger receptors.
Assuntos
Células Endoteliais/metabolismo , Lipoproteínas LDL/metabolismo , Animais , Aorta/citologia , Apolipoproteína B-100/química , Apolipoproteína B-100/metabolismo , Bovinos , Células Cultivadas , Cisteína/metabolismo , Relação Dose-Resposta a Droga , Endotélio Vascular/citologia , Endotélio Vascular/metabolismo , Humanos , Peroxidação de Lipídeos/efeitos dos fármacos , Peróxidos Lipídicos/análise , Lipoproteínas LDL/química , Lipoproteínas LDL/isolamento & purificação , Modelos Químicos , Molsidomina/análogos & derivados , Molsidomina/farmacologia , Oxirredução , Ácido Peroxinitroso/farmacologia , Conformação Proteica/efeitos dos fármacos , Desnaturação Proteica , Processamento de Proteína Pós-Traducional , Estrutura Secundária de Proteína , Proteínas/química , Proteínas/metabolismo , Reprodutibilidade dos Testes , Tirosina/análogos & derivados , Tirosina/metabolismoRESUMO
Biological membrane functions are coupled to membrane curvature, the regulation of which often involves membrane-associated proteins. The membrane-binding N-terminal amphipathic helix-containing BIN/Amphiphysin/Rvs (N-BAR) domain of amphiphysin is implicated in curvature generation and maintenance. Improving the mechanistic understanding of membrane curvature regulation by N-BAR domains requires quantitative experimental characterization. We have measured tube pulling force modulation by the N-BAR domain of Drosophila amphiphysin (DA-N-BAR) bound to tubular membranes pulled from micropipette-aspirated giant vesicles. We observed that fluorescently-labeled DA-N-BAR showed significantly higher protein density on tubules compared to the connected low-curvature vesicle membrane. Furthermore, we found the equilibrium tube pulling force to be systematically dependent on the aqueous solution concentration of DA-N-BAR, thereby providing the first quantitative assessment of spontaneous curvature generation. At sufficiently high protein concentrations, pulled tubes required no external force to maintain mechanical equilibrium, in agreement with the qualitative spontaneous tubulation previously reported for amphiphysin.