CAG Expansions Are Genetically Stable and Form Nontoxic Aggregates in Cells Lacking Endogenous Polyglutamine Proteins.

Ashley A Zurawel,James B Moseley,Kartikeya M Menon,Lin Deng,Martin L Duennwald,Surachai Supattapone,Ruth Kabeche,Sonja E Digregorio,Hannah Opalko,Tamara L Doering

doi:10.1128/mbio.01367-16

Abstract

ABSTRACTProteins 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.

Highlights

Proteins containing polyglutamine regions are found in almost all eukaryotes, albeit with various frequencies
We generated a series of constructs consisting of human huntingtin exon 1 with various lengths of CAG repeats, an amino-terminal FLAG epitope, and a C-terminal green fluorescent protein (GFP) tag
A very similar construct using cyan fluorescent protein (CFP) in place of GFP was expressed in S. cerevisiae cells under the control of the galactose-inducible GAL1 promoter and a very similar construct using cyan fluorescent protein in place of GFP was expressed in S. cerevisiae. (Note that the longest construct expressed in S. pombe was Htt-103Q, whereas the longest construct expressed in S. cerevisiae was Htt-97Q, due to the genetic instability of the polyQ-encoding region in S. cerevisiae upon integration.) Upon induction in these systems, Htt protein was expressed in S. cerevisiae and S. pombe at the expected molecular mass as judged by Western blotting

Summary

Introduction

Proteins containing polyglutamine (polyQ) regions are found in almost all eukaryotes, albeit with various frequencies. 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. The taxonomical distribution of polyQ proteins provides unique insights into the possible role of normal (i.e., non-diseaseassociated) polyQ proteins This distribution provides intriguing opportunities to study polyQ misfolding, aggregation, and toxicity in living cells with distinct proteomic environments. Recent work characterizing the effects of expanded polyQ Htt expression within D. discoideum [26] revealed that these cells effectively prevented pathogenic Htt aggregation and toxicity, except when stressed These results suggest that the D. discoideum protein quality control system may have adapted to the presence of a large number of polyQ proteins, possibly by coevolving a highly active network of chaperone proteins. The S. pombe protein quality control system presumably evolved under minimal selective pressure to regulate polyQ aggregation, thereby generating a unique model to examine the relationship between protein quality control systems and polyQ protein aggregation

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Results

Conclusion