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Proceedings of the 31 st European Peptide SymposiumMichal Lebl, Morten Meldal, Knud J. Jensen, Thomas Hoeg-Jensen (Editors)European Peptide Society, 2010Designed Hairpins Modulate the Amyloidogenesis of AlphaSynuclein: Inhibition and Diversion to Non-Amyloid AggregatesNiels H. Andersen 1 , Kelly N.L. Huggins 1 , Marco Bisaglio 2 , andLuigi Bubacco 21 Department of Chemistry, University of Washington, Seattle, WA, 98195, U.S.A.;2 Department of Biology, University of Padova, Padova, 35121, ItalyIntroductionAt the previous EPS symposium we reported that designed β hairpins bearing cross-strandpairs of Trp and Tyr residues inhibit the aggregation of human pancreatic amylin (hAM);the inhibition was evidenced by 2 to 8-fold increases in the lag time to amyloid fibrilformation as measured by thioflavin-T (ThT) fluorescence and the timescale for theevolution of a β-structure CD spectrum [1]. Those conclusions have since been confirmedby additional studies including TEM imaging of the fibrils. The effects of the hairpins usedin that study, and additional peptides, upon the aggregation of α synuclein (α-syn, theamyloid-producing species associated with Parkinson’s disease) has now been examined.Hairpins also modulate the aggregation α-syn, but by a different mechanism and withdifferent structure activity relationships.Results and DiscussionTo date, the effects of 18 7-18 residue peptides on amyloid fibril formation by α-syn havebeen examined. In this preliminary account, we will present the data for two hairpins(WW2 and YY2) as well as a species with a Trp-flanked turn (μPro1, with the sameYY2 KKLTVY-IpGK-YITVSAWW2 KKLTVW-IpGK-WITVSAcontrol KKLTVW-IμPro1 C 2 H 5 CO-W-IpGK-WTGScheme 1. Peptide names and sequenceslowercase p = D-Pro.WIpGKW core sequence as WW2),comparing this to the aggregation assayresults for α-syn in the absence of addedpeptides as well as in the presence ofselected peptide controls. The peptidesequences are collected in Scheme 1. In thecase of hAM, WW2 was the most potentinhibitor of hAM aggregation, showingeffects at equimolar concentrations while ratamylin [2] required a 6-fold concentration to effect any retardation of aggregation onset.Hairpin WW2 effected at 70% reduction in hAM fibril yield and increased the lag time by afactor of 6 when present in a 2-fold excess. In contrast, μPro1 addition increased the fibrilyield (120% of control by the ThT assay) and caused a decrease in the lag time.Our α-syn aggregation assay conditions (100 μM α-syn in 1.5 vol-% HFIP) yieldreproducible amyloid formation with the onset of β structuring (by CD and ThTfluorescence) occurring in 6 h and complete after 16 h (Figure 1). While many hairpinsdrastically altered the course and results in this assay, μPro1 (which enhanced fibrilformation for hAM) was entirely without effects on α-syn aggregation. This (or a smallincrease in the lag time coupled with modest levels of inhibition as measured in the ThTfluorescence assay) was also observed for other hairpins bearing a W-loop-W unit but withFig. 1. The course of amyloid formation and the fibril TEM for α-synuclein controls.22