Part 4 of our 11-part Eibsee conference series. See also Parts 1, 2, 3, 5, 6, 7, 8, 9, 10, 11, and complete PDF.
Mechanistic studies of γ-secretase, and the search for good modulators, would make a leap forward if the scientists could simply look at the protease and see its structure. With at least 18 transmembrane domains, the complex is too fiendishly complicated to have yielded to x-ray crystallography to date, and in the meantime, some groups are making do with electron microscopy. At the Eibsee, Dennis Selkoe described one of the first such attempts, performed with collaborator Huilin Li at Brookhaven National Laboratory in Upton, New Jersey, to use the synchrotron there to increase conventional EM resolution. The work just appeared in PNAS online (Lazarov et al., 2006; see comment by Boris Schmidt).
Joint first authors Vlado Lazarov of Brookhaven and Patrick Fraering of Selkoe’s group describe how they purified a cell-derived human γ-secretase complex, assured its proper composition and activity, labeled the nicastrin ectodomain with a lectin to orient the particles, and then developed averaged EM images as well as 3D surface reconstructions. The images show a slightly elongated particle about 12 nm across, with irregular bulges and indentations. At first glance, it resembles an ion channel because it features a hollow central chamber. This could shield the active site needed for hydrolysis from the hydrophobic environment of the membrane. The particle also has 2 nm-wide openings, one at the top and one at the bottom. Selkoe showed a movie of the particle (download at PNAS website), as well as a silhouette model of its surface contours into which the scientists had slotted its component proteins in a first-pass attempt of depicting how they might fit together. (Efforts to improve on these first images with cryoEM are ongoing, Selkoe noted.)
At the conference, a lively discussion ensued. One question concerned how the substrate would access the active site in the central chamber. It cannot flow in from the top or bottom, as is the case with ion channels, but must enter laterally from the lipid bilayer through an opening in the side wall, or some sort of “gating” mechanism. This initial EM study shows the γ-secretase complex in a closed form and cannot answer that question. Another question is where water would enter and the reaction products leave. Selkoe suggested that the two small holes might serve as conduits toward the extra- and intracellular compartments, where, in the case of APP, Aβ could exit through one hole and AICD through another. A third question was what additional proteins, or allosteric interactions, enabled the complex to plug up its central chamber and prevent water flooding in, which would kill the cell. Tom Rapoport’s seminal paper showing the structure of the sec61 protein channel featured a plug that seals the pore in the default state so the ER won’t collapse. It also proposes a mechanism for opening a lateral gate, (Van den Berg et al., 2004). For γ-secretase, the nicastrin ectodomain is one obvious candidate, Selkoe noted. Additional, regulatory, complex members are just beginning to be discovered (see St George-Hyslop talk below).
Haass’ group is approaching these questions with ongoing structural work using presenilin homologs from the SPP family, which have cropped up in database searches. Functioning as dimers and trimers, they are easier to study. Regina Fluhrer (who did double duty as conference organizer and presenter) presented data showing that, enzymatically, the active site of these SPP-like proteins (SPPLs) works just like that of γ-secretase. It uses the same cleavage mechanism even though its members do not form large complexes with nicastrin, Aph-1, and Pen-2. This suggests that such a complex may not be necessary for the formation—and visualization—of the central pore. A recent paper characterized the intracellular location and mutant phenotype of some of those SPPLs (Krawitz et al., 2005). At the Eibsee, Fluhrer showed new data suggesting that SPPLs tend to process type 2 transmembrane proteins, such as TNFα, whereas presenilins cleave type 1 transmembrane proteins (Fluhrer et al., 2006, in press). Type 2 transmembrane proteins are similar to the type 1 class in that they span the membrane once, but they run in the opposite orientation; that is, the amino-terminal side of their transmembrane domain reaches into the cytoplasmic side of the membrane and their COOH-terminal portion is exposed to the exterior. (Type 3 proteins span the membrane multiple times, like presenilin itself.) In other words, SPPLs act like “inside-out” presenilins, using the same enzymatic mechanism from an inverse orientation inside the membrane (see image by Haass et al. below).
When the Haass, Selkoe, and Takeshi Iwatsubo labs each reconstituted a functioning γ-secretase complex within a few months of each other, they learned that presenilin, nicastrin, Aph-1, and Pen-2 were the bare-bones requirement for activity in vitro. Yet in vivo, the complexes are larger, varied, and bound to be tweaked by a number of additional proteins. At the meeting, Peter St George-Hyslop introduced a new player in this area when he reported that his group had identified a fifth—and regulatory—complex component called TMP21. Itself a type 1 transmembrane protein (though not a γ-secretase substrate), TMP21 was previously identified as a member of a cargo protein family involved in proofreading material that is being shipped through the endoplasmic reticulum (ER) and trans-Golgi network (TGN) to the cell surface. Intriguingly, the TMP21 gene maps to chromosome 14 in a cluster including PS1. This cluster is conserved down to fugu (a poisonous Japanese pufferfish that is becoming a staple of comparative genomics), suggesting that transcription of these genes and their function might be conjoined.
St George-Hyslop’s team showed that besides being part of the p24 cargo complex, TMP21 also occurs in several different presenilin complexes, in which it functions quite differently. It selectively restrains γ cleavage (which generates Aβ from APP C100, for example, and Nβ from NEXT) but has no effect on ε cleavage (which generates the AICD product from C100, and NICD from NEXT). Removing TMP21 from the complex by siRNA increased Aβ/Nβ production but left AICD/NICD production unchanged. TMP21 binding to γ-secretase is highly specific, and its modulation of the γ cleavage is not due to a trafficking or assembly glitch but likely represents a direct interaction, St George-Hyslop said.
By identifying the first of what is likely to be a growing number of γ-secretase modulators, this work offers a fresh glimpse at the properties of the massive, 650 kDa γ-secretase, St. George Hyslop said. For example, the ligand-dependent ε cleavage, which is so crucial for signal transduction to the nucleus in embryonic and adult life, might well be under quite separate control from the γ cleavage, which is increasingly being associated with membrane “garbage” removal (Kopan and Ilagan, 2004). This is new because until recently, researchers have tended to view the different cleavages on nearby spots of a given substrate not as distinct activities but as more likely reflecting a single, loosely defined property. The work also offers a new approach for ongoing efforts to find therapeutic γ-secretase modulators. Rather than pointing to a way to distinguish between APP and Notch substrates per se, small-molecule TMP21 mimics might inhibit Aβ (and Nβ) production while sparing NICD and its signaling functions. This paper appeared last week in Nature (Chen et al., 2006).—Gabrielle Strobel.