Inhibition of Polyglutamine Protein Aggregation and Cell Death by Novel Peptides Identified by Phage Display Screening

Abstract

Proteins with expanded polyglutamine domains cause eight inherited neurodegenerative diseases, including Huntington's, but the molecular mechanism(s) responsible for neuronal degeneration are not yet established. Expanded polyglutamine domain proteins possess properties that distinguish them from the same proteins with shorter glutamine repeats. Unlike proteins with short polyglutamine domains, proteins with expanded polyglutamine domains display unique protein interactions, form intracellular aggregates, and adopt a novel conformation that can be recognized by monoclonal antibodies. Any of these polyglutamine length-dependent properties could be responsible for the pathogenic effects of expanded polyglutamine proteins. To identify peptides that interfere with pathogenic polyglutamine interactions, we screened a combinatorial peptide library expressed on M13 phage pIII protein to identify peptides that preferentially bind pathologic-length polyglutamine domains. We identified six tryptophan-rich peptides that preferentially bind pathologic-length polyglutamine domain proteins. Polyglutamine-binding peptide 1 (QBP1) potently inhibits polyglutamine protein aggregation in an in vitro assay, while a scrambled sequence has no effect on aggregation. QBP1 and a tandem repeat of QBP1 also inhibit aggregation of polyglutamine-yellow fluorescent fusion protein in transfected COS-7 cells. Expression of QBP1 potently inhibits polyglutamine-induced cell death. Selective inhibition of pathologic interactions of expanded polyglutamine domains with themselves or other proteins may be a useful strategy for preventing disease onset or for slowing progression of the polyglutamine repeat diseases.Eight inherited neurodegenerative diseases, including Huntington's disease, dentatorubral pallidoluysian atrophy, spinobulbar muscular atrophy, and spinocerebellar ataxia types 1, 2, 3, 6 and 7, are caused by expanded CAG repeats in the coding region of the disease genes (1-3). The CAG codon is translated into glutamine, and the polyglutamine domain is the only region of homology among the eight disease proteins. The length of the repeat is the critical determinant of age-of-disease onset, with repeat length greater than 40 glutamines producing neurodegeneration in seven of the eight diseases (1-3).Proteins with pathologic-length polyglutamine domains display novel properties that are not present in these proteins when they contain a shorter polyglutamine domain. Length-dependent polyglutamine-protein interactions are reported for Huntington-associated protein 1, glyceraldehyde-3-phosphate dehydrogenase, leucine-rich acidic nuclear protein, vimentin, neurofilament, apopain, calmodulin, WW domain proteins, and Ras-related nuclear protein/ARA24 (4 -12). Proteins with expanded polyglutamine domains also aggregate, and aggregation is a pathologic hallmark of the polyglutamine repeat diseases (13,14). These polyglutamine length-dependent properties may arise from the ability of long polyglutamine domains to adopt unique three-dimensional confor...

The Two-stage Pathway of Ataxin-3 Fibrillogenesis Involves a Polyglutamine-independent Step

“…To confirm this, we have used the QBP1 peptide as a tool to explore the role of the poly(Q) tract in defining the at3(Q64) aggregation pathway. The QBP1 peptide is 11 amino acids in length and has been shown to inhibit poly(Q) aggregation both in vitro and in vivo (38,42). In Fig.…”

“…A number of reports have analyzed the aggregation properties of poly(Q) peptides (15,18), fragments of poly(Q) proteins (16,39,45), or fusion proteins containing poly(Q) tracts (38,52). However, little is known about the aggregation of intact naturally occurring poly(Q) proteins.…”

“…QBP1 Aggregation Assays-The QBP1 peptide was purchased from AusPep with the same sequence as described previously (38). The peptide was dissolved in Me 2 SO to a final concentration of 13.5 mM.…”

Detection of Polyglutamine Protein Oligomers in Cells by Fluorescence Correlation Spectroscopy

“…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 ϭ 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.…”

“…We here show a time-dependent increase in the diffusion time and particle size of expanded poly(Q)-green fluorescent protein (GFP) fusion proteins expressed in cultured cells by FCS, indicating oligomer formation. Furthermore, we show that the poly(Q)-binding peptide QBP1 (17) suppresses poly(Q)-GFP oligomer formation, whereas Congo red only inhibits the growth of oligomers, but not their initial formation, suggesting that FCS is capable of identifying poly(Q) oligomer inhibitors. We therefore conclude that FCS is a useful technique to monitor the oligomerization of disease-causing proteins in cells as well as inhibition of oligomerization in the conformational neurodegenerative diseases.…”