Chaperone-like N-Methyl Peptide Inhibitors of Polyglutamine Aggregation

Abstract

Polyglutamine expansion in the exon 1 domain of huntingtin leads to aggregation into β-sheet-rich insoluble aggregates associated with Huntington's Disease. We assessed eight polyglutamine peptides with different permutations of N-methylation of backbone and side chain amides as potential inhibitors of polyglutamine aggregation. Surprisingly, the most effective inhibitor, 5QMe 2 (Anth-K-Q-Q(Me2)-Q-Q(Me 2 )-Q-CONH 2 , Anth = N-methyl anthranilic acid, Q(Me 2 ) = side chain N-methyl Q) has only side chain methylations at alternate residues, highlighting the importance of side chain interactions in polyglutamine fibrillogenesis. Above a 1:1 stoichiometric ratio, 5QMe 2 can completely prevent fibrillation of a synthetic aggregating peptide YAQ 12 A; it also shows significant inhibition at substoichiometric ratios. Surface plasmon resonance (SPR) measurements show a moderate K d with very fast k on and k off . Sedimentation equilibrium analytical ultracentrifugation indicates that 5QMe 2 is predominantly or entirely monomeric at concentrations up to 1 mM, and that it forms a 1:1 stoichiometric complex with a fibril-forming target, YAQ 12 A. 5QMe 2 inhibits not only nucleation of YAQ 12 A, but also fibril extension, as shown by the fact that it also inhibits seeded fibril growth where the nucleation steps are bypassed. 5QMe 2 acts on its targets only when they are in the PPII-like conformation, but not after they undergo a transition to β-sheets. Thus 5QMe 2 does not disassemble pre-formed YAQ 12 A; this contrasts with our previously described, backbone N-methylated inhibitors of β-amyloid aggregation (16,17). The mode of action of 5QMe 2 is reminiscent of chaperones, since it binds and releases its targets very rapidly, and maintains them in a non-aggregation-prone, monomeric state, in this case, the polyproline II (PPII)-like conformation, as shown by CD spectroscopy.Expanded polyglutamine (polyQ) tracts are responsible for at least nine neurodegenerative diseases, including Huntington's Disease (HD). HD occurs when the polyQ domain of exon † We acknowledge NIH Medical Scientist Training Program Grant (T32 GM07281 JDL), NIH Cardiovascular Pathophysiology Training Grant (HL07237 JDL) and NIH (NS042852 SCM) and the Alzheimer's Association (IIRG-06-27794). * To whom correspondence should be addressed: Department of Pathology, 5841 S. Maryland Ave., Chicago, IL 60637. Tel: 773-702-1267. Fax: 773-834-5251. scmeredi@uchicago.edu . Supporting Information Available The following items are available as supporting information: A derivation of the equations and other details needed for analysis of equilibrium sedimentation analytical ultracentrifugation of two dissimilar interacting species; determination of for YAQ 12 A and 5QMe 2 ; reverse phase HPLC isocratic analysis of YAQ 12 A remaining in solution, in the presence or absence of 5QMe 2 ; experiments showing the effect of added fibril seeds on fibrillation of YAQ 12 A; electron micrograph of fibril seed slurry used in preceding experiments; example of analytical ...

Strategies for Inhibiting Protein Aggregation: Therapeutic Approaches to Protein-Aggregation Diseases

“…This peptide was, in fact, their most effective inhibitor, and thus demonstrates that high affi nity is not the best criterion for predicting the effi cacy of an aggregation inhibitor. As discussed below, similar results were also obtained by another group (Lanning et al 2010) for an inhibitor of polyGln peptide aggregation. These peptides appear to act not by binding to and "capping" growth sites.…”

“…In all of these cases, self-association occurs mainly through the hydrophobic effect, i.e., the shielding of hydrophobic groups from the aqueous medium. In contrast, PolyGln peptides interact through hydrogen bonds, not only between peptide backbone groups, as in all amyloids, but also between side-chain amides (Starikov et al 1999;Esposito et al 2008 ;Lanning et al 2010;Masino 2004 ) . In solution, short PolyGln peptides adopt a polyPro-II-helixlike structure, with formation of oligomers that also have polyPro-II-helix-like structure (Darnell et al 2007(Darnell et al , 2009 , and thus, the conversion to fi brils may require a transition from this structure to b -sheet.…”

“…One attempt at rational design of PolyGln inhibitor examined eight permutations of N -methylation of short PolyGln peptides as potential PolyGln aggregation inhibitors (Lanning et al 2010). Since PolyGln peptides contain both backbone and sidechain amides, it is not clear, a priori , which amide should be methylated to inhibit aggregation, and which should be retained to allow binding to the target PolyGln peptide.…”

Switching the Immunogenicity of Peptide Assemblies Using Surface Properties

“…23 In addition, two different peptides with N, N-dimethylated glutamine residues in the Q11 domain were also studied, where the dimethylated Gln residues were expected to disrupt β-sheet formation by disrupting side chain hydrogen bonding. 40 …”

Backbone Engineering within a Latent β-Hairpin Structure to Design Inhibitors of Polyglutamine Amyloid Formation

“…Inhibitory activity was modest, however, requiring an approximate 4:1 ratio of inhibitor to K 2 Q 50 K 2 for good inhibition. Lanning et al prepared a Q 5 - d -Pro-Gly-Q 5 derivative in which both Q 5 arms contained multiple backbone N-Me-Gln modifications; this peptide partially inhibited aggregation of a Q 12 peptide when present in a 40:1 excess over Q 12 61 . The vastly improved inhibitory activities of the latent β-hairpin derivatives βHP- NMe Q 20 or βHP-P 20 described here (Fig.…”