Prediction of Nucleating Sequences from Amyloidogenic Propensities of Tau-Related Peptides ² Federico A. Rojas Quijano, § Dana Morrow, § Barry M. Wise, ‡ Francesco L. Brancia, | and Warren J. Goux* ,§ Department of Chemistry, The UniVersity of Texas at Dallas, P.O. Box 830688 Richardson, Texas 75083-0688, Shimadzu Research Laboratory (Europe), Wharfside Trafford Wharf Road, Manchester, M17 1GP, U.K., and EigenVector Research, Inc., P.O. Box 561, Manson, WA 98831. ReceiVed NoVember 1, 2005; ReVised Manuscript ReceiVed February 8, 2006 ABSTRACT: Physical properties, including amyloid morphology, FTIR and CD spectra, enhancement of Congo red absorbance, polymerization rate, critical monomer concentration, free energy of stabilization, hydrophobicity, and the partition coefficient between soluble and amyloid states, were measured for the tau-related peptide Ac-VQIVYK amide (AcPHF6) and its single site mutants Ac-VQIVXK amide (X * Cys). Transmission electron microscopy showed that 15 out of the 19 peptides formed amyloid in buffer, with morphologies ranging from straight and twisted filaments to sheets and rolled sheets. Using principal component analysis (PCA), measured properties were treated in a comprehensive manner, and scores along the most significant principal components were used to define individual amino acid amyloidogenic propensities. Quantitative structure-activity modeling (QSAM) showed that residues with greater size and hydrophobicity made the largest contributions to the propensity of peptides to form amyloid. Using individual amino acid propensities, sequences within tau with high amyloid-forming potential were estimated and found to include 226 VAVVR 230 in the proline-rich region, 275 VQIINK 280 (PHF6*) and 306 VQIVYK 311 (PHF6) within the microtubule binding region, and 392 IVYK 395 in the C-tail region of the protein. The results suggest that regions outside the microtubule-binding region may play important roles in tau aggregation kinetics or paired helical filament structure. The deposition of insoluble protein deposits are charac- teristic of over 100 human diseases, including familial amyloidosis, type II diabetes, and numerous neurodegenera- tive diseases, including transmissible spongiform encepha- lopathies, Alzheimer’s disease (AD 1 ) and Parkinson’s disease (1-6). These deposits, named amyloid because they, like the polysaccharide amylose, bind iodine, display a yellow- green birefringence when stained with Congo red (CR) and a characteristic fluorescence when stained with thioflavin dyes (2, 7-10). In addition, amyloid is frequently character- ized by its cross- X-ray diffraction pattern and by its infrared (IR) or circular dichroism (CD) spectra, all of which suggest the presence of a high relative abundance of -structure (10-16). Although it was long believed that all amyloid existed as 3-15 nm wide straight unbranched fibers, more recent evidence, from our laboratory and others, suggests that amyloid may exist as twisted filaments, flat and rolled sheets, or spherical or annular particles (16-23). Hence, diverse morphodologies exist for amyloid, and a wide variety of methodologies have been used in the past to characterize it. Destabilization of the native conformation brought about by mutations or denaturing conditions of the media has been shown to induce globular proteins, some not normally associated with amyloid disease pathology, to form amyloid (24-29). This finding, and the lack of sequence homology among proteins associated with amyloid, has led some to suggest that the ability to form amyloid is a generic trait of all proteins, independent of their sequence (24, 30). However, it has been shown that certain defined sequences or “hot spots” nucleate amyloid formation in globular and nonglobu- lar proteins (3, 31-43). We refer to such sequences as “nucleating sequences” because they generate centers that can help seed amyloid and regulate its rate of formation (26, 27, 40, 43). Studies of short amyloidogenic peptides ho- mologous to these sequences suggest that amphiphilic motifs or π-stacking interactions of aromatic side chains play a crucial role in amyloid formation (3, 16, 32, 37-54). Tau is a microtubule-associated protein that regulates microtubule stability, neurite growth, and other microtubule- dependent functions. The human protein exists as six ² W.J.G. acknowledges The University of Texas at Dallas for their financial support. * To whom correspondence should be addressed. Tel: 972-883-2660. Fax: 972-883-2925. E-mail: wgoux@utdallas.edu. ‡ Eigenvector Research, Inc. § The University of Texas at Dallas. | Shimadzu Research Laboratory (Europe). 1 Abbreviations: PHFs, paired helical filaments; NFTs, neurofibril- lary tangles; FTDP-17, frontotemporal dementias with Parkinsonism linked to chromosome 17; AD, Alzheimer’s disease; MTBR, micro- tubule binding region of tau protein; R2, R3, or R4, the second, third, or fourth repeat motif of the tau microtubule binding region; 3R, 4R, tau protein or peptides containing three or four repeat regions; HFIP, hexafluoro2-propanol; MOPS, 3-[N-morpholino]propanesulfonic acid; ThS, thioflavin S; TFA, trifluoroacetic acid; Ac, acetyl group; CR, Congo red; RTs, retention times; TEM, transmission electron micros- copy; MALDI-TOF MS, matrix assisted laser desorption/ionization mass spectrometry; ESI, electrospray ionization; PCA, principal component analysis; PLS, partial least squares; QSAM, quantitative structure-activity modeling; LVs, latent variables; PHF6, 306 VQIVYK 311 ; PHF6*, 275 VQIINK 280 . 10.1021/bi052226q CCC: $33.50 © xxxx American Chemical Society PAGE EST: 14.4 Published on Web 03/16/2006