Biochemical and Biophysical Research Communications
Disruption of the toxic conformation of the expanded polyglutamine stretch leads to suppression of aggregate formation and cytotoxicity☆
Section snippets
Materials and methods
Introduction of proline coding CCGs into the CAG repeat. Proline coding CCGs were introduced into the CAG repeat by a strategy using polymerase chain reaction (PCR) mutagenesis and digestion with a type IIS restriction enzyme, as shown in Supplementary Fig. 1[16]. The pure polyQ stretches and polyQ stretches with proline interruptions (QPQ) used in this study are listed in Table 1.
Construction of plasmid vectors. Plasmid vectors for expression of polyQ stretches fused with a thioredoxin
Interruption of the expanded polyQ stretch by proline insertion disrupts its conformation
The 1C2 antibody, which specifically recognizes the unique conformation of expanded polyQ stretches, was used to determine the effect of proline insertion on the conformation of the expanded polyQ stretch (Fig. 1A). The immunoreactivities of 1C2 against thio-QPQ decreased in correlation with the number of proline interruptions compared with thio-Q57, while an anti-thioredoxin antibody showed similar immunoreactivity to all the proteins, which is consistent with a previous study showing that an
Acknowledgements
We thank Dr. Kazuhisa Nakayama for helpful discussions and Mr. Timothy Tucker for his technical assistance. This work was supported in part by a Grant-in-Aid for Scientific Research on Priority Areas (C)—Advanced Brain Science Project (to Y.N.), a Grant-in-Aid for Scientific Research on Priority Areas (A)—Life of Proteins (to Y.N.), and the 21st Century COE program (to T.T.), from the Ministry of Education, Culture, Sports, Science and Technology, Japan, and a Grant-in-Aid for the Research
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2012, Journal of Molecular BiologyAggregation kinetics of interrupted polyglutamine peptides
2011, Journal of Molecular BiologyCitation Excerpt :Interruptions of the polyQ sequence have interesting effects on the aggregation behavior. Proline33–35 and histidine36 interruptions have been found to slow aggregation, although the impact of proline residues can be lessened if they are positioned such that they are easily incorporated into a β-turn.33 Conversely, peptides containing d-proline glycine, a β-turn template, display more rapid aggregation kinetics.33
Structural characterization of polyglutamine fibrils by solid-state NMR spectroscopy
2011, Journal of Molecular BiologyCitation Excerpt :Fibril formation in these constructs was less efficient, and through-space backbone–side chain correlations indicative of a well-ordered side-chain arrangement were absent, indicating that these mutations indeed interfered with formation of the β-sheet fibrillar core (data not shown). However, another mutation study found reduced aggregation levels in proline mutants of polyglutamine peptides even if the proline residues were inserted at positions that should be within loop regions according to our model.65 Further mutational studies will be necessary to resolve this issue and delineate the exact influence of residue position and type on fibril formation.
Location trumps length: Polyglutamine-mediated changes in folding and aggregation of a host protein
2011, Biophysical JournalCitation Excerpt :We therefore chose to conduct our studies using a host protein that is not associated with disease. Precedence for this approach was established by previous studies that used glutathione S-transferase (20–22), thioredoxin (23,24), CRABP1 (8,17), Staphylococcal protein A (14), and Sup35 (25). This approach is also justified biologically by experiments wherein symptoms of classic polyQ neurodegenerative disease were induced by incorporation of expanded polyQ domains into an otherwise innocuous protein (26).
Detection of polyglutamine protein oligomers in cells by fluorescence correlation spectroscopy
2007, Journal of Biological ChemistryCitation Excerpt :Cell Culture, DNA Transfection, and Microscopy—COS-7 cells were grown and maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum in a 5% CO2 humidified atmosphere at 37 °C. Cells were transfected with expression vectors for various length pure poly(Q) stretches or poly(Q) stretches with proline insertions fused with enhanced green/yellow/cyan fluorescent protein ((Q)n-GFP/YFP/CFP; n = 19, 45 or 81) (17, 18), or GFP alone as a control, using FuGENE6 transfection reagent (Roche Applied Science) according to the manufacturer’s instructions. To evaluate the inhibitory effects of the poly(Q)-binding peptide QBP1 (SNWKWWPGIFD) (17) on poly(Q) protein oligomerization/aggregation, expression vectors for a tandem repeat of QBP1 fused with CFP (QBP1) or a scrambled sequence of QBP1 fused with CFP (SCR), used as a control, were co-transfected with poly(Q)-GFP or GFP.
Chemical and Physical Properties of Polyglutamine Repeat Sequences
2006, Genetic Instabilities and Neurological Diseases
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Supplementary data associated with this article can be found, in the online version, at doi: 10.1016/j.bbrc.2004.03.161.
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These authors contributed equally to this work.