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Conformational targeting of fibrillar polyglutamine proteins in live cells escalates aggregation and cytotoxicity.

ABSTRACT: Misfolding- and aggregation-prone proteins underlying Parkinson's, Huntington's and Machado-Joseph diseases, namely alpha-synuclein, huntingtin, and ataxin-3 respectively, adopt numerous intracellular conformations during pathogenesis, including globular intermediates and insoluble amyloid-like fibrils. Such conformational diversity has complicated research into amyloid-associated intracellular dysfunction and neurodegeneration. To this end, recombinant single-chain Fv antibodies (scFvs) are compelling molecular tools that can be selected against specific protein conformations, and expressed inside cells as intrabodies, for investigative and therapeutic purposes.Using atomic force microscopy (AFM) and live-cell fluorescence microscopy, we report that a human scFv selected against the fibrillar form of alpha-synuclein targets isomorphic conformations of misfolded polyglutamine proteins. When expressed in the cytoplasm of striatal cells, this conformation-specific intrabody co-localizes with intracellular aggregates of misfolded ataxin-3 and a pathological fragment of huntingtin, and enhances the aggregation propensity of both disease-linked polyglutamine proteins. Using this intrabody as a tool for modulating the kinetics of amyloidogenesis, we show that escalating aggregate formation of a pathologic huntingtin fragment is not cytoprotective in striatal cells, but rather heightens oxidative stress and cell death as detected by flow cytometry. Instead, cellular protection is achieved by suppressing aggregation using a previously described intrabody that binds to the amyloidogenic N-terminus of huntingtin. Analogous cytotoxic results are observed following conformational targeting of normal or polyglutamine-expanded human ataxin-3, which partially aggregate through non-polyglutamine domains.These findings validate that the rate of aggregation modulates polyglutamine-mediated intracellular dysfunction, and caution that molecules designed to specifically hasten aggregation may be detrimental as therapies for polyglutamine disorders. Moreover, our findings introduce a novel antibody-based tool that, as a consequence of its general specificity for fibrillar conformations and its ability to function intracellularly, offers broad research potential for a variety of human amyloid diseases.

SUBMITTER: Kvam E

PROVIDER: S-EPMC2683928 | biostudies-literature | 2009 May

REPOSITORIES: biostudies-literature

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Conformational targeting of fibrillar polyglutamine proteins in live cells escalates aggregation and cytotoxicity.

Kvam Erik E Nannenga Brent L BL Wang Min S MS Jia Zongjian Z Sierks Michael R MR Messer Anne A

PloS one 20090528 5

<h4>Background</h4>Misfolding- and aggregation-prone proteins underlying Parkinson's, Huntington's and Machado-Joseph diseases, namely alpha-synuclein, huntingtin, and ataxin-3 respectively, adopt numerous intracellular conformations during pathogenesis, including globular intermediates and insoluble amyloid-like fibrils. Such conformational diversity has complicated research into amyloid-associated intracellular dysfunction and neurodegeneration. To this end, recombinant single-chain Fv antibod ...[more]

PMID: 19492089

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Project description:Neurodegenerative diseases are distinguished by characteristic protein aggregates initiated by disease-specific 'seed' proteins; however, roles of other co-aggregated proteins remain largely unexplored. Compact hippocampal aggregates were purified from Alzheimer's and control-subject pools using magnetic-bead immunoaffinity pulldowns. Their components were fractionated by electrophoretic mobility and analyzed by high-resolution proteomics. Although total detergent-insoluble aggregates from Alzheimer's and controls had similar protein content, within the fractions isolated by tau or A?1-42 pulldown, the protein constituents of Alzheimer-derived aggregates were more abundant, diverse, and post-translationally modified than those from controls. Tau- and A?-containing aggregates were distinguished by multiple components, and yet shared >90% of their protein constituents, implying similar accretion mechanisms. Alzheimer-specific protein enrichment in tau-containing aggregates was corroborated for individuals by three analyses. Five proteins inferred to co-aggregate with tau were confirmed by precise in situ methods, including proximity ligation amplification that requires co-localization within 40 nm. Nematode orthologs of 21 proteins, which showed Alzheimer-specific enrichment in tau-containing aggregates, were assessed for aggregation-promoting roles in C. elegans by RNA-interference 'knockdown'. Fifteen knockdowns (71%) rescued paralysis of worms expressing muscle A?, and 12 (57%) rescued chemotaxis disrupted by neuronal A? expression. Proteins identified in compact human aggregates, bound by antibody to total tau, were thus shown to play causal roles in aggregation based on nematode models triggered by A?1-42 . These observations imply shared mechanisms driving both types of aggregation, and/or aggregate-mediated cross-talk between tau and A?. Knowledge of protein components that promote protein accrual in diverse aggregate types implicates common mechanisms and identifies novel targets for drug intervention.

Project description:Polyglutamine expansion in the exon 1 domain of huntingtin leads to aggregation into beta-sheet-rich insoluble aggregates associated with Huntington's disease. We assessed eight polyglutamine peptides with different permutations of N-methylation of backbone and side chain amides as potential inhibitors of polyglutamine aggregation. Surprisingly, the most effective inhibitor, 5QMe(2) [Anth-K-Q-Q(Me(2))-Q-Q(Me(2))-Q-CONH(2), where Anth is N-methylanthranilic acid and Q(Me(2)) is side chain N-methyl Q], has only side chain methylations at alternate residues, highlighting the importance of side chain interactions in polyglutamine fibrillogenesis. Above a 1:1 stoichiometric ratio, 5QMe(2) can completely prevent fibrillation of a synthetic aggregating peptide, YAQ(12)A; it also shows significant inhibition at substoichiometric ratios. Surface plasmon resonance (SPR) measurements show a moderate K(d) with very fast k(on) and k(off) values. Sedimentation equilibrium analytical ultracentrifugation indicates that 5QMe(2) is predominantly or entirely monomeric at concentrations of <or=1 mM and that it forms a 1:1 stoichiometric complex with a fibril-forming target, YAQ(12)A. 5QMe(2) inhibits not only nucleation of YAQ(12)A but also fibril extension, as shown by the fact that it also inhibits seeded fibril growth where the nucleation steps are bypassed. 5QMe(2) acts on its targets only when they are in the PPII-like conformation, but not after they undergo a transition to beta-sheets. Thus, 5QMe(2) does not disassemble preformed YAQ(12)A; this contrasts with our previously described, backbone N-methylated inhibitors of beta-amyloid aggregation [Gordon, D. J., et al. (2001) Biochemistry 40, 8237-8245; Gordon, D. J., et al. (2002) J. Pept. Res. 60, 37-55]. The mode of action of 5QMe(2) is reminiscent of that of chaperones, because it binds and releases its targets very rapidly and maintains them in a nonaggregation-prone, monomeric state, in this case, the polyproline II (PPII)-like conformation, as shown by circular dichroism spectroscopy.

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Conformational targeting of fibrillar polyglutamine proteins in live cells escalates aggregation and cytotoxicity.

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Conformational targeting of fibrillar polyglutamine proteins in live cells escalates aggregation and cytotoxicity.

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