The true identity of γ-secretase, the enzyme that frees amyloid-β from its membrane-bound precursor, is one of the holy grails in the search for the cause of Alzheimer's disease. A battery of circumstantial evidence has recently focused attention on presenilin as the prime suspect. In today's Nature, Steven J. Gardell and his associates at Merck Research Laboratories add evidence that has some commentators saying the suspect has been apprehended.
Using an inhibitor targeted to the active site of aspartyl proteases, they first confirmed an earlier study indicating that γ-secretase is, in fact, an aspartyl protease. Antibodies against presenilin- 1 and presenilin-2, then appeared to identify that aspartyl protease as presenilin, although the authors acknowledge that their probes could be labeling a γ-secretase that is in a complex with presenilin.
Reactions to these latest data range from conversion to cautionary. Bart De Strooper, writing a News and Views letter accompanying the article, is now convinced. He accepts that there are a number of details to be worked out (e.g., how can hydrolysis of peptide bonds take place inside the hydrophobic cell membrane?), but he concludes that "presenilin ... is the culprit."
However, for Sam Sisodia of the University of Chicago, these data can be interpreted differently. "It's equally conceivable that presenilin is intimately associated with the γ-secretase and that in fact, the transition-state-analogue inhibitory site is actually blocking-inhibiting-the real enzyme, and the active site groups that are involved in the photoactivation are hitting presenilin which is close by. Thus, presenilin is actually an accessory factor. I think the definitive-proof experiment that presenilin is γ-secretase has yet to be published, and that is to reconstitute the reaction in vitro."—Hakon Heimer
Interview with Sangram Sisodia
ARF: Doesn't this paper confirm that presenilin is the gamma-secretase?
Sisodia: No. It's equally conceivable that presenilin is intimately associated with the γ-secretase and that in fact, the transition-state-analogue inhibitory site is actually blocking—inhibiting—the real enzyme, and the active site groups that are involved in the photoactivation are hitting presenilin which is close by. Thus, presenilin is actually an accessory factor. I think the definitive proof experiment that presenilin is γ-secretase has yet to be published (perhaps it's been tried), and that is to reconstitute the reaction in vitro. That is, to take the purified enzyme in a bilayer or artificial membrane of some sort, offer it the natural substrate and ask whether this activity can proceed.
ARF: Are there particular technical challenges to such an experiment?
Sisodia: There are always technical challenges and it's not trivial for presenilin because it spans the membrane several times. It's very fatty so biochemically it's hard to work with, to reinsert into artificial bilayers. Yes, it's technically challenging, but the burden of proof really rests on those who claim that presenilin is the γ-secretase.
There may be another technical challenge above and beyond that, in that presenilin may be part of a complex. One might need to reconstitute the complex to get activity; one would then have to go after the individual components of the complex and put those together. And that could take some time.
I think that presenilin does a myriad of things in mammalian cells. It affects cellular trafficking of several proteins. It affects the processing of several different proteins and it's hard for me to imagine—and I could be completely wrong—that it is a protease involved in γ-secretase processing. It would be unprecedented in biology, to begin with. But of course, that's the way biology is: there's always something new lurking around the corner. How you would get catalysis in the bilayer, where the water would come from if it really is an aspartyl protease, these are all the kinds of things that one would have to think about from a biochemical point of view. It could very well be true—I'm not going to discount it all outright—but I don't think the proof is there yet.
ARF: I'll quote you as being cautionary.
Sisodia: Yes. I've taken that view from the very beginning. We published a paper in Neuron about a year and half ago which suggests that protein trafficking of several membrane proteins is affected by loss of presenilin-1 function. So how could this molecule, which normally resides in the very early subcellular compartments—the endoplasmic reticulum and the early Golgi, perhaps—affect the maturation or proteolysis of APP in very late compartments? One has to try to understand that.
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- Li YM, Xu M, Lai MT, Huang Q, Castro JL, DiMuzio-Mower J, Harrison T, Lellis C, Nadin A, Neduvelil JG, Register RB, Sardana MK, Shearman MS, Smith AL, Shi XP, Yin KC, Shafer JA, Gardell SJ. Photoactivated gamma-secretase inhibitors directed to the active site covalently label presenilin 1. Nature. 2000 Jun 8;405(6787):689-94. PubMed.