Effects of Membrane Lipids on the Activity and Processivity of Purified γ-Secretase

摘要

The 19-transmembrane multisubunit y-secretase complex gen-erates the amyloid β-peptide (Aβ) of Alzheimer's disease (AD) by intra-membrane proteolysis of theβ-amyloid precursor protein (APP). Despite substantial advances in elucidating how this protein complex functions, the effect of the local membrane lipid microenvironment on y-secretase cleavage of substrates is still poorly understood. Using detergent-free proteoliposomes to reconstitute purified human y-secretase, we examined the effects of fatty acyl (FA) chain length, saturation and double-bond isomerization, and membrane lipid polar headgroups on y-secretase function. We analyzed y-secretase activity and processivity [i.e., sequential cleavages in the APP transmembrane domain that convert longer Aβ species (e.g., Aβ_(46)) into shorter ones (e.g., Aβ_(40)] by quantifying the APP intracellular domain (AICD) and various Aβ peptides, including via a bicine/urea gel system that detects multiple Aβ lengths. These assays revealed several trends. (1) Switching from a cis to a trans isomer of a monounsaturated FA chain in phosphatidylcholine (PC) increased y-activity, did not affect Aβ_42:Aβ_(40) ratios, but decreased the ratio of long (≥42) versus short (≤41) Aβ peptides. (2) Increasing the FA carbon chain length (14, 16, 18, and 20) increased y-activity, reduced longer Aβ species, and reduced the Aβ_(42):Aβ_(4o) ratio. (3) Shifting the position of the double bond in 18:1(Δ9-cis) PC to the Δ6 position substantially reduced activity. (4) Gangliosides increased y-activity but decreased processivity, thus elevating the Aβ_(42):Aβ_(40) ratio. (5) Phosphatidylserine decreased y-activity but increased processivity. (6) Phosphatidylinositol strongly inhibited y-activity. Overall, our results show that subtle changes in membrane lipid composition can greatly influence y-secretase activity and processivity, suggesting that relatively small changes in lipid membrane composition may affect the risk of AD at least as much as presenilin or APP mutations do.