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The relationship between aggregation and toxicity of polyglutamine-containing ataxin-3 in the intracellular environment of Escherichia coli.

ABSTRACT: Several neurodegenerative diseases are triggered by proteins containing a polyglutamine (polyQ) stretch expanded beyond a critical threshold. Among these, ataxin-3 (AT3) is the causative agent of spinocerebellar ataxia type-3. We expressed three authentic AT3 variants in Escherichia coli: one normal (AT3-Q24), one expanded (AT3-Q55) and one truncated immediately upstream of the polyQ (AT3-291?). Then, based on growth rate reduction, we quantified protein toxicity. We show that AT3-Q55 and -291? strongly reduced the growth rate in the early stages (2-4 h), unlike AT3-Q24. This correlated well with the appearance of soluble cytosolic oligomers, but not with the amount of insoluble protein in inclusion bodies (IBs). The impact of AT3-291? on cell growth suggests an intrinsic toxicity of the AT3 fragment. Besides the typical Fourier Transform Infrared Spectroscopy (FTIR) signal for intermolecular ?-sheets, the expanded form displayed an additional infrared signature, which was assigned to glutamine side-chain hydrogen bonding and associated with SDS-insoluble fibrils. The elongation of the latter was monitored by Atomic Force Microscopy (AFM). This mirrors the well-known in vitro two-step aggregation pattern of expanded AT3. We also demonstrated that final aggregates of strains expressing expanded or truncated AT3 play a protective role against toxicity. Furthermore, our findings suggest that the mechanisms of toxicity are evolutionarily conserved.

SUBMITTER: Invernizzi G

PROVIDER: S-EPMC3522584 | biostudies-literature | 2012

REPOSITORIES: biostudies-literature

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The relationship between aggregation and toxicity of polyglutamine-containing ataxin-3 in the intracellular environment of Escherichia coli.

Invernizzi Gaetano G Aprile Francesco A FA Natalello Antonino A Ghisleni Andrea A Penco Amanda A Relini Annalisa A Doglia Silvia M SM Tortora Paolo P Regonesi Maria E ME

PloS one 20121214 12

Several neurodegenerative diseases are triggered by proteins containing a polyglutamine (polyQ) stretch expanded beyond a critical threshold. Among these, ataxin-3 (AT3) is the causative agent of spinocerebellar ataxia type-3. We expressed three authentic AT3 variants in Escherichia coli: one normal (AT3-Q24), one expanded (AT3-Q55) and one truncated immediately upstream of the polyQ (AT3-291Δ). Then, based on growth rate reduction, we quantified protein toxicity. We show that AT3-Q55 and -291Δ ...[more]

PMID: 23251648

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Project description:Expansion of polyglutamine stretches leads to the formation of polyglutamine-containing neuronal aggregates and neuronal death in nine diseases for which there currently are no treatments or cures. This is largely due to a lack in understanding of the mechanisms by which expanded polyglutamine regions contribute to aggregation and disease. To complicate matters further, several of the polyglutamine-disease related proteins, including ataxin-3, have a multistage aggregation mechanism in which flanking domain self-assembly precedes polyglutamine aggregation yet is influenced by polyglutamine expansion. How polyglutamine expansion influences flanking domain aggregation is poorly understood. Here, we use a combination of mass spectrometry and biophysical approaches to investigate this issue for ataxin-3. We show that the conformational dynamics of the flanking Josephin domain in ataxin-3 with an expanded polyglutamine tract are altered in comparison to those exhibited by its nonexpanded counterpart, specifically within the aggregation-prone region of the Josephin domain (amino acid residues 73-96). Expansion thus exposes this region more frequently in ataxin-3 containing an expanded polyglutamine tract, providing a molecular explanation of why aggregation is accelerated upon polyglutamine expansion. Here, harnessing the power of ion mobility spectrometry-mass spectrometry, oligomeric species formed during aggregation are characterized and a model for oligomer growth proposed. The results suggest that a conformational change occurs at the dimer level that initiates self-assembly. New insights into ataxin-3 fibril architecture are also described, revealing the region of the Josephin domain involved in protofibril formation and demonstrating that polyglutamine aggregation proceeds as a distinct second step after protofibril formation without requiring structural rearrangement of the protofibril core. Overall, the results enable the effect of polyglutamine expansion on every stage of ataxin-3 self-assembly, from monomer through to fibril, to be described and a rationale for expedited aggregation upon polyglutamine expansion to be provided.

Dataset Information

The relationship between aggregation and toxicity of polyglutamine-containing ataxin-3 in the intracellular environment of Escherichia coli.

Publications

The relationship between aggregation and toxicity of polyglutamine-containing ataxin-3 in the intracellular environment of Escherichia coli.

Similar Datasets

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