Promotion of Amyloid β Protein Misfolding and Fibrillogenesis by a Lipid Oxidation Product Liu Liu, Hiroaki Komatsu, Ian V. J. Murray and Paul H. Axelsen⁎ Department of Pharmacology, University of Pennsylvania, Philadelphia, PA 19104, USA Department of Biochemistry and Biophysics, University of Pennsylvania, Philadelphia, PA 19104, USA Department of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA Received 18 December 2007; received in revised form 13 January 2008; accepted 21 January 2008 Available online 30 January 2008 Oxidatively damaged lipid membranes are known to promote the aggregation of amyloid β proteins and fibril formation. Oxidative damage typically produces 4-hydroxy-2-nonenal when lipid membranes contain ω-6 polyunsaturated fatty acyl chains, and this compound is known to modify the three His residues in Aβ proteins by Michael addition. In this report, the ability of 4-hydroxy-2-nonenal to reproduce the previously observed amyloidogenic effects of oxidative lipid damage on amyloid β proteins is demonstrated and the mechanism by which it exerts these effects is examined. Results indicate that 4-hydroxy-2-nonenal modifies the three His residues in amyloid beta proteins, which increases their membrane affinity and causes them to adopt a conformation on membranes that is similar to their conformation in a mature amyloid fibril. As a consequence, fibril formation is accelerated at relatively low protein concentrations, and the ability to seed the formation of fibrils by unmodified amyloid beta proteins is enhanced. These in vitro findings linking oxidative stress to amyloid fibril formation may be significant to the in vivo mechanism by which oxidative stress is linked to the formation of amyloid plaques in Alzheimer's disease. © 2008 Elsevier Ltd. All rights reserved. Edited by J. Bowie Keywords: Alzheimer's disease; internal reflection infrared spectroscopy; mass spectrometry; surface plasmon resonance; hydroxynonenal Introduction Amyloid β (Aβ) proteins form fibrils and accu- mulate as dense “senile plaques” in the cortical brain tissues of patients with Alzheimer's disease (AD). The 40-residue form of Aβ (Aβ40) along with its various amino-terminal derivatives appears to pre- dominate in fully developed plaques. 1 The 42- residue form (Aβ42), on the other hand, appears to predominate in early-stage diffuse plaques. 2 There- fore, it has been proposed that Aβ42 serves to nucleate amyloid plaque formation. 3–6 Neverthe- less, the fundamental reason amyloid plaques form in patients with sporadic AD remains elusive. Oxidative stress has been frequently implicated in the pathogenesis of AD, 7–16 although not directly in the formation of amyloid plaques. Polyunsaturated fatty acyl chains are abundant in lipid membranes of the brain 17 and they are highly vulnerable to oxidative stress. When considering which com- pounds are likely to arise during lipid oxidation, interact with Aβ proteins, explain the effects of oxidatively damaged membranes, and have a role in the pathogenesis of AD, 4-hydroxy-2-nonenal (HNE) is an obvious candidate. HNE is derived from ω-6 fatty acyl chains via nonenzymatic degradation pathways from lipid hydroperoxide intermediates. 18 It is a highly reactive compound that tends to form Michael adducts with the His side chains in a protein. 19–26 HNE concentrations ranging from 8 to 20 μM have been reported in normal human plasma 27 and cerebral ventricular fluid, 28 along with evidence of increased levels of HNE in AD. 28–30 Spontaneous *Corresponding author. E-mail address: axe@pharm.med.upenn.edu. Present address: I. V. J. Murray, Department of Neuroscience and Experimental Medicine, Texas A&M Health Science Center, College Station, TX 77840, USA. Abbreviations used: Aβ, amyloid β proteins; AD, Alzheimer's disease; Aβ40, the 40-residue form of Aβ; Aβ42, the 42-residue form of Aβ; amide I′, the prime indicates a spectrum collected in D 2 O; HNE, 4-hydroxy-2- nonenal; HHE, 4-hydroxy-2-hexenal; IR, infrared spectroscopy; PATIR-FTIR, polarized attenuated total internal reflection IR spectroscopy; DMPC, 1,2- dimyristoyl-sn-glycero-3-phosphocholine; CR, Congo Red; HFIP, hexafluoro-2-propanol; PICUP, photoinduced cross-linking of unmodified proteins. doi:10.1016/j.jmb.2008.01.057 J. Mol. Biol. (2008) 377, 1236–1250 Available online at www.sciencedirect.com 0022-2836/$ - see front matter © 2008 Elsevier Ltd. All rights reserved.