ABSTRACT
Huntington's disease (HD) is caused by a polyglutamine expansion of the huntingtin protein, resulting in the formation of polyglutamine aggregates. The mechanisms of toxicity that result in the complex HD pathology remain only partially understood. Here, we show that nuclear polyglutamine aggregates induce nuclear envelope (NE) blebbing and ruptures that are often repaired incompletely. These ruptures coincide with disruptions of the nuclear lamina and lead to lamina scar formation. Expansion microscopy enabled resolving the ultrastructure of nuclear aggregates and revealed polyglutamine fibrils sticking into the cytosol at rupture sites, suggesting a mechanism for incomplete repair. Furthermore, we found that NE repair factors often accumulated near nuclear aggregates, consistent with stalled repair. These findings implicate nuclear polyQ aggregate-induced loss of NE integrity as a potential contributing factor to Huntington's disease and other polyglutamine diseases.
Subject(s)
Huntington Disease , Nuclear Envelope , Peptides , Nuclear Envelope/metabolism , Nuclear Envelope/ultrastructure , Humans , Peptides/metabolism , Peptides/genetics , Huntington Disease/metabolism , Huntington Disease/pathology , Huntington Disease/genetics , Animals , Huntingtin Protein/metabolism , Huntingtin Protein/genetics , Protein Aggregates , Nuclear Lamina/metabolism , Nuclear Lamina/ultrastructure , Cell Nucleus/metabolismABSTRACT
DNAJB6b is a molecular chaperone of the heat shock protein network, shown to play a crucial role in preventing aggregation of several disease-related intrinsically disordered proteins. Using homology modeling and microsecond-long all-atom molecular dynamics (MD) simulations, we show that monomeric DNAJB6b is a transiently interconverting protein cycling between three states: a closed state, an open state (both abundant), and a less abundant extended state. Interestingly, the reported regulatory autoinhibitory anchor between helix V in the G/F1 region and helices II/III of the J-domain, which obstructs the access of Hsp70 to the J-domain remains present in all three states. This possibly suggests a mechanistically intriguing regulation in which DNAJB6b only becomes exposed when loaded with substrates that require Hsp70 processing. Our MD results of DNAJB6b carrying mutations in the G/F1 region that are linked to limb-girdle muscular dystrophy type D1 (LGMDD1) show that this G/F1 region becomes highly dynamic, pointing towards a spontaneous release of the autoinhibitory helix V from helices II/III. This would increase the probability of non-functional Hsp70 interactions to DNAJB6b without substrates. Our cellular data indeed confirm that non-substrate loaded LGMDD1 mutants have aberrant interactions with Hsp70.
Subject(s)
Molecular Chaperones , Muscular Dystrophies, Limb-Girdle , Humans , Molecular Chaperones/metabolism , HSP70 Heat-Shock Proteins/metabolism , Heat-Shock Proteins/metabolism , Molecular Dynamics Simulation , Molecular Conformation , HSP40 Heat-Shock Proteins/metabolismABSTRACT
J-domain protein (JDP) molecular chaperones have emerged as central players that maintain a healthy proteome. The diverse members of the JDP family function as monomers/dimers and a small subset assemble into micron-sized oligomers. The oligomeric JDP members have eluded structural characterization due to their low-complexity, intrinsically disordered middle domains. This in turn, obscures the biological significance of these larger oligomers in protein folding processes. Here, we identified a short, aromatic motif within DNAJB8 that drives self-assembly through π-π stacking and determined its X-ray structure. We show that mutations in the motif disrupt DNAJB8 oligomerization in vitro and in cells. DNAJB8 variants that are unable to assemble bind to misfolded tau seeds more specifically and retain capacity to reduce protein aggregation in vitro and in cells. We propose a new model for DNAJB8 function in which the sequences in the low-complexity domains play distinct roles in assembly and substrate activity.
Subject(s)
HSP40 Heat-Shock Proteins , Protein Multimerization , Humans , HSP40 Heat-Shock Proteins/metabolism , HSP40 Heat-Shock Proteins/chemistry , HSP40 Heat-Shock Proteins/genetics , Models, Molecular , Amino Acid Motifs , Crystallography, X-Ray , Protein Binding , tau Proteins/metabolism , tau Proteins/chemistry , tau Proteins/genetics , Molecular Chaperones/metabolism , Molecular Chaperones/chemistry , Molecular Chaperones/genetics , Mutation , Protein FoldingABSTRACT
J-domain proteins (JDPs) are the largest family of chaperones in most organisms, but much of how they function within the network of other chaperones and protein quality control machineries is still an enigma. Here, we report on the latest findings related to JDP functions presented at a dedicated JDP workshop in Gdansk, Poland. The report does not include all (details) of what was shared and discussed at the meeting, because some of these original data have not yet been accepted for publication elsewhere or represented still preliminary observations at the time.