Watanabe N, Image Image II, Takagi S, Tominaga A, Image Image I, Tomita T, Iwatsubo T. Functional analysis of the transmembrane domains of presenilin 1: participation of transmembrane domains 2 and 6 in the formation of initial substrate-binding site of gamma-secretase. J Biol Chem. 2010 Jun 25;285(26):19738-46. PubMed.
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University of Kansas
This report provides a very thorough and systematic analysis of the role of the various transmembrane domains of PS1 in γ-secretase assembly, stability and activation. The evidence largely supports the role of specific TM domains in the various steps in the maturation of the protease complex as laid out schematically in Figure 9. This includes the role of TM2 and the luminal region of TM6 in the formation of the initial substrate binding site: swapping these regions with those of another unrelated membrane protein leads to the inability of a photoaffinity helical peptide probe targeted to this initial substrate binding site from labeling PS1. These two TM regions are apparently critical to the formation of this binding site, but whether they are both direct contributors to the binding site is not clear. It could be, for instance, that TM2 is not part of the initial substrate binding site but can nevertheless affect the conformation of this binding site. Some chemical crosslinking evidence is provided that TM2 is near TM9, the latter previously being implicated in binding and lateral gating of substrate. However, the evidence for TM2 proximity to TM9 is rather weak, and such proximity would not mean that TM2 is necessarily part of the substrate binding site.
These authors take one of the helices of presenilin at a time, replace it
with a helix of an unrelated protein and then measure its function. If the
helix they exchange is important for a certain function, then the modified
presenilin should no longer be able to show this function. It is like
exchanging the individual parts of a car with, say, parts of household
appliances. If you exchange the motor of the car with a vacuum cleaner and
then try to start the car and it won't, then you have found out that the
motor is necessary to drive the car. However if you push this car it will
still move, so the motor is obviously important for driving but not in
general for the ability of the car to be moved.
In essence, Watanabe et al do the same thing with presenilin and its
helices. The n-terminal domain has 6 helices (the C-terminal domain, of
which we have determined the structure has 3). By exchanging every domain
individually, the authors found that helix2 and helix6 are involved in
binding of helical peptides such as βamyloid precursor protein. They
also found that helix9, which is part of our structure, is involved in
substrate binding. Understanding where the substrate binds is very
important. As a field, we need lots of information about which sites bind
specific substrates and which are involved in processing of APP. Only with
such very detailed information will we able to develop drugs that inhibit
formation of the Aβ peptide but not other functions presenilin is
involved in.
In this sense both this and our paper provide complementary information
and provide another small but important piece of the puzzle.
The University of Tokyo
The main finding of this study is that using NMR studies in SDS micelles, the authors determined the structure of PS1 CTF. The structure fits well with previous studies using cysteine accessibility methods by us and by Bart de Strooper’s group. This is important because it is a first structure of a part of PS1 resolved at the atomic level. The study’s potential impact could come from an intriguing feature it shows around catalytic aspartate residue 385, that is, a half helix and extended structure kinked by a glycine residue located near the aspartate. This supports the notion that intramembrane cleavage is occurring within the water-accessible pore structure in the membrane.
At the same time, further study (e.g., using holoprotein) is required, as the PS1 N-terminal fragment (NTF) is also required for the proteolytic activity. Moreover, we have identified that TMD1 of PS1 also faces the catalytic pore (Takagi et al., unpublished result), suggesting that several TMDs in NTFs are involved in the formation of the catalytic pore.
Without an atomic structure, it is impossible to “rationally design” inhibitors or modulators. The Doetsch paper is a first step toward understanding the process by γ-secretase at an atomic level. The field has encountered difficulties for structural studies of this unusual membrane-bound protease; however, we also know that tackling this issue by whatever means provides hope for the future.
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