Site-specific Effects of Peptide Lipidation on β-Amyloid Aggregation and Cytotoxicity

Abstract

␤-Amyloid (A␤) aggregates at low concentrations in vivo, and this may involve covalently modified forms of these peptides. Modification of A␤ by 4-hydroxynonenal (4-HNE) initially increases the hydrophobicity of these peptides and subsequently leads to additional reactions, such as peptide cross-linking. To model these initial events, without confounding effects of subsequent reactions, we modified A␤ at each of its amino groups using a chemically simpler, close analogue of 4-HNE, the octanoyl group: K16-octanoic acid (OA)-A␤, K28-OA-A␤, and N␣-OA-A␤. Octanoylation of these sites on A␤-(1-40) had strikingly different effects on fibril formation. K16-OA-A␤ and K28-OA-A␤, but not N␣-OA-A␤, had increased propensity to aggregate. The type of aggregate (electron microscopic appearance) differed with the site of modification. The ability of octanoyl-A␤ peptides to cross-seed solutions of A␤ was the inverse of their ability to form fibrils on their own (i.e. A␤ ≈ N␣-OA-A␤ Ͼ Ͼ K16-OA-A␤ Ͼ Ͼ K28-OA-A␤). By CD spectroscopy, K16-OA-A␤ and K28-OA-A␤ had increased ␤-sheet propensity compared with A␤-(1-40) or N␣-OA-A␤. K16-OA-A␤ and K28-OA-A␤ were more amphiphilic than A␤-(1-40) or N␣-OA-A␤, as shown by lower "critical micelle concentrations" and higher monolayer collapse pressures. Finally, K16-OA-A␤ and K28-OA-A␤ are much more cytotoxic to N2A cells than A␤-(1-40) or N␣-OA-A␤. The greater cytotoxicity of K16-OA-A␤ and K28-OA-A␤ may reflect their greater amphiphilicity. We conclude that lipidation can make A␤ more prone to aggregation and more cytotoxic, but these effects are highly site-specific.Alzheimer disease, the most prevalent neurodegenerative disorder, leads to progressive memory loss, disability and eventually death (1). It is characterized by the accumulation of extracellular plaques of amyloid ␤ (A␤) 2 (2). Although aggregation of A␤ appears to be important in the pathogenesis of Alzheimer disease, one of the unanswered questions is how A␤ peptides aggregate at the low concentrations at which they exist in the central nervous system. The concentration of A␤ in the cerebrospinal fluid has been estimated as in the nanomolar range or even lower (3-12). This is below the "critical micelle concentration" for A␤ (13) and at the margin of the critical concentration (C r ), at which A␤ solutions can extend fibrillar seeds (14, 15). One recently proposed answer to this question is that an aggregation-prone, covalently modified A␤ could catalyze aggregation of the unmodified peptide. In addition to catalyzing peptide aggregation, aldehyde-modified A␤ could also be cytotoxic to neurons by itself. Oxidative stress, a possible pathogenic factor in Alzheimer disease, can lead to the formation of reactive lipid aldehydes, such as 4-HNE (16 -21) and cholesterol oxides (22), among others. Indeed, 4-HNE immunoreactivity is detected in plaques and cerebrospinal fluid of patients with Alzheimer disease (19,20) and is believed to play a role in many other neurodegenerative diseases, including Parkinson disease (23-26), Lewy body dis...

Seeded growth of β-amyloid fibrils from Alzheimer's brain-derived fibrils produces a distinct fibril structure

“…Seeding with brain amyloid produced significantly greater ThT fluorescence and non-monotonic kinetics, possibly because of fibril-fibril association and blockage of ThT binding sites at long times. Similar nonmonotonic kinetics have been reported for a modified A␤ , i.e., K28-octanoyl-A␤ (15). Figure 2B shows that material extracted from brain tissue of two patients without AD, which contained nonfibrillar particles but no amyloid fibrils detectable by TEM, did not seed or accelerate A␤ 1-40 fibril growth.…”

“…Additional 2D fpRFDR spectra were obtained from brain 1/generation 3 and brain 2/generation 3 samples with uniform 15 N, 13 Clabeling of D23, K28, G29, I32, and V36 (Fig. S4).…”

“…The ability of preformed fibrils to propagate their structures through seeded growth in vitro has enabled detailed structural characterization of two distinct A␤ fibril structures, based largely on solid-state NMR measurements on a series of 15 N-and 13 C-labeled samples (4,5). Self-propagating, molecular-level structural polymorphism of amyloid fibrils underlies the phenomenon of strains in yeast prions and possibly mammalian prions (6)(7)(8).…”

Oxidative Stress and Cell Membranes in the Pathogenesis of Alzheimer's Disease

“…The effects of HNE on A␤ fibril formation depend on the size or hydrophobicity of the modification because the corresponding analog from -3 PUFA chains, 4-hydroxy-2(E)-hexenal, does not have this effect (187). The addition of an octanoyl group to specific Lys residues of A␤ proteins also appears to have a pro-amyloidogenic effect (258).…”

Lipidation Effect on Surface Adsorption and Associated Fibrillation of the Model Protein Insulin

“…In bulk assays, lipidation also affected the fibrillation tendency in a site-specific manner of proteins prone to form amyloid fibrils 23,24 . In the present work, we study the adsorption and subsequent fibrillation behavior of the lipidated human insulin variant Lys B29 -tetradecanoyl des-(B30) insulin, (insulin_lip), given the 14-carbon fatty acid chain covalently bound to the lysine residue (Figure 1), and we compare it to human insulin.…”