Hsp70 and hsp40 chaperones can inhibit self-assembly of polyglutamine proteins into amyloid-like fibrils

The deposition of protein aggregates in neurons is a hallmark of neurodegenerative diseases caused by polyglutamine (polyQ) proteins. We analyzed the effects of the heat shock protein (Hsp) 70 chaperone system on the aggregation of fragments of huntingtin (htt) with expanded polyQ tracts. In vitro, Hsp70 and its cochaperone Hsp40 suppressed the assembly of htt into detergent-insoluble amyloid-like fibrils in an ATP-dependent manner and caused the formation of amorphous, detergent-soluble aggregates. The chaperones were most active in preventing fibrillization when added during the lag phase of the polymerization reaction. Similarly, coexpression of Hsp70 or Hsp40 with htt in yeast inhibited the formation of large, detergent-insoluble polyQ aggregates, resulting in the accumulation of detergent-soluble inclusions. Thus, the recently established potency of Hsp70 and Hsp40 to repress polyQ-induced neurodegeneration may be based on the ability of these chaperones to shield toxic forms of polyQ proteins and to direct them into nontoxic aggregates

The cardiomyopathy and lens cataract mutation in αB-crystallin alters its protein structure, chaperone activity, and interaction withintermediate filaments in vitro

Desmin-related myopathy and cataract are both caused by the R120G mutation in αB-crystallin. Desmin-related myopathy is one of several diseases characterized by the coaggregation of intermediate filaments with αB-crystallin, and it identifies intermediate filaments as important physiological substrates for αB-crystallin. Using recombinant human αB-crystallin, the effects of the disease-causing mutation R120G upon the structure and the chaperone activities of αB-crystallin are reported. The secondary, tertiary, and quaternary structural features of αB-crystallin are all altered by the mutation as deduced by near- and far-UV circular dichroism spectroscopy, size exclusion chromatography, and chymotryptic digestion assays. The R120G αB-crystallin is also less stable than wild type αB-crystallin to heat-induced denaturation. These structural changes coincide with a significant reduction in thein vitro chaperone activity of the mutant αB-crystallin protein, as assessed by temperature-induced protein aggregation assays. The mutation also significantly altered the interaction of αB-crystallin with intermediate filaments. It abolished the ability of αB-crystallin to prevent those filament-filament interactions required to induce gel formation while increasing αB-crystallin binding to assembled intermediate filaments. These activities are closely correlated to the observed disease pathologies characterized by filament aggregation accompanied by αB-crystallin binding. These studies provide important insight into the mechanism of αB-crystallin-induced aggregation of intermediate filaments that causes disease

Green tea (-)-epigallocatechin-gallate modulates early events in huntingtin misfolding and reduces toxicity in Huntington's disease models

Huntington's disease (HD) is a progressive neurodegenerative disorder for which only symptomatic treatments of limited effectiveness are available. Preventing early misfolding steps and thereby aggregation of the polyglutamine (polyQ)-containing protein huntingtin (htt) in neurons of patients may represent an attractive therapeutic strategy to postpone the onset and progression of HD. Here, we demonstrate that the green tea polyphenol (-)-epigallocatechin-3-gallate (EGCG) potently inhibits the aggregation of mutant htt exon 1 protein in a dose-dependent manner. Dot-blot assays and atomic force microscopy studies revealed that EGCG modulates misfolding and oligomerization of mutant htt exon 1 protein in vitro, indicating that it interferes with very early events in the aggregation process. Also, EGCG significantly reduced polyQ-mediated htt protein aggregation and cytotoxicity in an yeast model of HD. When EGCG was fed to transgenic HD flies overexpressing a pathogenic htt exon 1 protein, photoreceptor degeneration and motor function improved. These results indicate that modulators of htt exon 1 misfolding and oligomerization like EGCG are likely to reduce polyQ-mediated toxicity in vivo. Our studies may provide the basis for the development of a novel pharmacotherapy for HD and related polyQ disorders

Binding of the molecular chaperone alpha B-crystallin to A beta amyloid fibrils Inhibits fibril elongation

The molecular chaperone aB-crystallin is a small heat-shock protein that is upregulated in response to a multitude of stress stimuli, and is found colocalized with Ab amyloid fibrils in the extracellular plaques that are characteristic of Alzheimer’s disease. We investigated whether this archetypical small heat-shock protein has the ability to interact with Ab fibrils in vitro. We find that aB-crystallin binds to wild-type Ab42 fibrils with micromolar affinity, and also binds to fibrils formed from the E22G Arctic mutation of Ab42. Immunoelectron microscopy confirms that binding occurs along the entire length and ends of the fibrils. Investigations into the effect of aB-crystallin on the seeded growth of Ab fibrils, both in solution and on the surface of a quartz crystal microbalance biosensor, reveal that the binding of aB-crystallin to seed fibrils strongly inhibits their elongation. Because the lag phase in sigmoidal fibril assembly kinetics is dominated by elongation and fragmentation rates, the chaperone mechanism identified here represents a highly effective means to inhibit fibril proliferation. Together with previous observations of aB-crystallin interaction with a-synuclein and insulin fibrils, the results suggest that this mechanism is a generic means of providing molecular chaperone protection against amyloid fibril formation.Sarah L. Shammas, Christopher A. Waudby, Shuyu Wang, Alexander K. Buell, Tuomas P. J. Knowles, Heath Ecroyd, Mark E. Welland, John A. Carver, Christopher M. Dobson, and Sarah Meehanhttp://www.cell.com/biophys