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Insoluble detergent-resistant aggregates form between pathological and nonpathological lengths of polyglutamine in mammalian cells.

ABSTRACT: Pathological degeneration of neurons in Huntington's disease and associated neurodegenerative disorders is directly correlated with the expansion of CAG repeats encoding polyglutamines of extended length. The physical properties of extended polyglutamines and the intracellular consequences of expression of polyglutamine expansion have been the object of intensive investigation. We have extended the range of lengths of polyglutamine produced by recombinant DNA methodology by constructing a library of CAG/CAA repeats coding for a range of 25-300 glutamine residues. We have investigated the subcellular localization, interaction with other polyglutamine-containing polypeptides, and the physical properties of aggregated forms of polyglutamine in the cell. Extended polyQ aggregated in the cytoplasm and was only transported to the nucleus when a strong nuclear localization signal was present. Polyglutamine below pathological lengths could be captured in aggregates and transported to ectopic cell locations. The CREB-binding protein (CBP), containing a homopolymeric stretch of 19 glutamines, was likewise found to coaggregate in a polyglutamine-dependent manner, suggesting that pathology in polyglutamine disease may result from cellular depletion of normal proteins containing polyglutamine. We have observed a striking detergent resistance in aggregates produced from polyglutamine of pathological length. This observation has led to the development of a fluorescence-based assay exploiting the detergent resistance of polyglutamine aggregates that should facilitate high-throughput screening for agents that suppress polyglutamine aggregation in cells.

SUBMITTER: Kazantsev A

PROVIDER: S-EPMC18046 | biostudies-literature | 1999 Sep

REPOSITORIES: biostudies-literature

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Insoluble detergent-resistant aggregates form between pathological and nonpathological lengths of polyglutamine in mammalian cells.

Kazantsev A A Preisinger E E Dranovsky A A Goldgaber D D Housman D D

Proceedings of the National Academy of Sciences of the United States of America 19990901 20

Pathological degeneration of neurons in Huntington's disease and associated neurodegenerative disorders is directly correlated with the expansion of CAG repeats encoding polyglutamines of extended length. The physical properties of extended polyglutamines and the intracellular consequences of expression of polyglutamine expansion have been the object of intensive investigation. We have extended the range of lengths of polyglutamine produced by recombinant DNA methodology by constructing a librar ...[more]

PMID: 10500189

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Project description:Proteins containing polyglutamine (polyQ) regions are found in almost all eukaryotes, albeit with various frequencies. In humans, proteins such as huntingtin (Htt) with abnormally expanded polyQ regions cause neurodegenerative diseases such as Huntington's disease (HD). To study how the presence of endogenous polyQ aggregation modulates polyQ aggregation and toxicity, we expressed polyQ expanded Htt fragments (polyQ Htt) in Schizosaccharomyces pombe In stark contrast to other unicellular fungi, such as Saccharomyces cerevisiae, S. pombe is uniquely devoid of proteins with more than 10 Q repeats. We found that polyQ Htt forms aggregates within S. pombe cells only with exceedingly long polyQ expansions. Surprisingly, despite the presence of polyQ Htt aggregates in both the cytoplasm and nucleus, no significant growth defect was observed in S. pombe cells. Further, PCR analysis showed that the repetitive polyQ-encoding DNA region remained constant following transformation and after multiple divisions in S. pombe, in contrast to the genetic instability of polyQ DNA sequences in other organisms. These results demonstrate that cells with a low content of polyQ or other aggregation-prone proteins can show a striking resilience with respect to polyQ toxicity and that genetic instability of repetitive DNA sequences may have played an important role in the evolutionary emergence and exclusion of polyQ expansion proteins in different organisms.ImportancePolyglutamine (polyQ) proteins encoded by repetitive CAG DNA sequences serve a variety of normal biological functions. Yet some proteins with abnormally expanded polyQ regions cause neurodegeneration through unknown mechanisms. To study how distinct cellular environments modulate polyQ aggregation and toxicity, we expressed CAG-expanded huntingtin fragments in Schizosaccharomyces pombe In stark contrast to many other eukaryotes, S. pombe is uniquely devoid of proteins containing long polyQ tracts. Our results show that S. pombe cells, despite their low content of endogenous polyQ proteins, exhibit striking and unexpected resilience with respect to polyQ toxicity and that genetic instability of repetitive DNA sequences may have played an important role in the emergence and expansion of polyQ domains in eukaryotic evolution.

Dataset Information

Insoluble detergent-resistant aggregates form between pathological and nonpathological lengths of polyglutamine in mammalian cells.

Publications

Insoluble detergent-resistant aggregates form between pathological and nonpathological lengths of polyglutamine in mammalian cells.

Similar Datasets

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