Abstract
gamma-Secretase is an unusual and ubiquitous aspartyl protease with an intramembrane catalytic site that cleaves many type-I integral membrane proteins, most notably APP and Notch. Several reports suggest that cleavage of APP to produce the Abeta peptide is regulated in part by lipids. As gamma-secretase is a multipass protein complex with 19 transmembrane domains, it is likely that the local lipid composition of the membrane can regulate gamma-activity. To determine the direct contribution of the lipid microenvironment to gamma-secretase activity, we purified the human protease from overexpressing mammalian cells, reconstituted it in vesicles of varying lipid composition, and examined the effects of individual phospholipids, sphingolipids, cholesterol, and complex lipid mixtures on substrate cleavage. A conventional gamma-activity assay was modified to include a detergent-removal step to facilitate proteoliposome formation, and this increased baseline activity over 2-fold. Proteoliposomes containing sphingolipids significantly increased gamma-secretase activity over a phosphatidylcholine-only baseline, whereas the addition of phosphatidylinositol significantly decreased activity. Addition of soluble cholesterol in the presence of phospholipids and sphingolipids robustly increased the cleavage of APP- and Notch-like substrates in a dose-dependent manner. Reconstitution of gamma-secretase in complex lipid mixtures revealed that a lipid raft-like composition supported the highest level of activity compared with other membrane compositions. Taken together, these results demonstrate that membrane lipid composition is a direct and potent modulator of gamma-secretase and that cholesterol, in particular, plays a major regulatory role.
Highlights
Scientific attention has focused on identifying and characterizing proteins that affect ␥-secretase activity or trafficking, there has been far less study of the lipid requirements and lipid modulation of the activity of ␥-secretase
In an effort to better understand the role of the lipid microenvironment on ␥-secretase activity in a system that supports lipid structure, we have assessed the effect of detergent removal on the activity of purified mammalian ␥-secretase and used a reductionist approach to systematically examine the effects of individual lipids and lipid mixtures directly on proteolytic activity
We found that vesicles containing 90% PC plus 10% of PE, PS, or phosphatidic acid (PA) had the greatest increase in amyloid -protein (A)40 production relative to the PC-only control, with a 1.3-fold increase in activity for each of the lipid mixtures (Fig. 3A)
Summary
Reagents—Lipids were purchased from Sigma (PC, PE, and sphingomyelin (SM) from bovine brain) or Avanti Polar Lipids (phosphatidylserine (PS), gangliosides (GS), and cerebrosides (CS) from porcine brain; phosphatidylinositol (PI) from bovine liver, phosphatidic acid (PA) from egg, and complex lipid mixtures from porcine brain, bovine liver, bovine heart, Escherichia coli, and soybean). Western Blotting and Antibodies—For Western blot analysis of the purified ␥-secretase complex from S-1 cells comprising PS1-NTF, PS1-CTF, Aph1␣2-HA, FLAG-Pen-2, and NCTGST, the samples were run on a 4 –20% Tris glycine polyacrylamide gel, transferred to a polyvinylidene difluoride membrane, and probed with Ab14 Samples from the ␥-secretase activity assays were run on 10 –20% Tris-Tricine gels and transferred to polyvinylidene difluoride membranes to detect AICDFLAG with M2 anti-FLAG antibody. Samples from ␥-secretase activity assays with the N100FLAG substrate were run on 4 –20% Tris glycine gels and transferred to polyvinylidene difluoride membranes to detect NICD-FLAG with the Notch Ab1744 antibody (1:1000, Cell Signaling Technology), which is selective for the free N terminus of the NICD. In experiments in which three or more groups were compared against each other, data were statistically analyzed by one-way analysis of variance with TukeyKramer post-testing
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