This paper revisits the issue of presenilin topology, providing further evidence of a 7-TM model with an orientation opposite those suggested from many other studies. With new antibodies in hand, Dewji and colleagues detected the PS1 N-terminus and a large internal loop as being outside the cell by immunofluorescent techniques. In contrast, the C-terminus was not detected, suggesting that this region is intracellular and that PS has an odd number of TM domains. The study included endogenous PS1, avoiding possible artifacts as a consequence of tagging or overexpression. However, there are caveats.

The failure to detect the C-terminus outside the cell using antibodies means only that this domain was not detected outside the cell using antibodies. The C-terminus is well-known from mutagenesis studies to be essential for PS function. Moreover, while immunoprecipitation with antibodies to the PS1 N-terminus brings down nicastrin, Aph-1, and Pen-1 as well as γ-secretase activity, antibodies to the C-terminus do not, suggesting that the C-terminus is unavailable for interaction...  Read more

This paper revisits the issue of presenilin topology, providing further evidence of a 7-TM model with an orientation opposite those suggested from many other studies. With new antibodies in hand, Dewji and colleagues detected the PS1 N-terminus and a large internal loop as being outside the cell by immunofluorescent techniques. In contrast, the C-terminus was not detected, suggesting that this region is intracellular and that PS has an odd number of TM domains. The study included endogenous PS1, avoiding possible artifacts as a consequence of tagging or overexpression. However, there are caveats.

The failure to detect the C-terminus outside the cell using antibodies means only that this domain was not detected outside the cell using antibodies. The C-terminus is well-known from mutagenesis studies to be essential for PS function. Moreover, while immunoprecipitation with antibodies to the PS1 N-terminus brings down nicastrin, Aph-1, and Pen-1 as well as γ-secretase activity, antibodies to the C-terminus do not, suggesting that the C-terminus is unavailable for interaction with antibodies.

As mentioned by Kim and Schekman in their accompanying commentary in PNAS, it is possible that PS is present in the membrane in more than one topology, reconciling the otherwise contradictory results from different labs, and it is interesting to consider the possibility that an “inside-out” presenilin may have functions that are distinct from a flipped counterpart. Kim and Schekman are also careful to point out the limitations of the molecular techniques so far employed to elucidate the topology of PS, and that the techniques of structural biology are needed to unequivocally address this issue.

Dewji and Singer suggest in their new paper that their topological model does not support the hypothesis that presenilin contains the active site of γ-secretase, and that this hypothesis is still controversial The authors ignore a large body of evidence in support of the “presenilin-as-protease” hypothesis, and the fact that a number of other multi-pass membrane proteins that otherwise do not resemble classical proteases can cleave the transmembrane regions of other proteins.

The model of Dewji and Singer remains unproven. Very little new evidence in support of this model has been reported in nearly six years, and as pointed out by Kim and Schekman, the inside-out 7-TM model is not consistent with other studies on PS (e.g., its ability to be cleaved by caspases, its direct association with β-catenin). Moreover, the suggestion that PS is a 7-TM G-protein-coupled receptor is an old idea that remains speculative. In contrast, the 8-TM model that has generally been favored has explained a variety of otherwise disparate data and continues to advance our understanding of presenilin biology. Given the choice between two apparently conflicting models, it is not unreasonable to favor the one that explains a variety of phenomena and leads to coherent and testable hypotheses that are supported by experiments.

View all comments by Michael Wolfe

That PS1 keeps a most amazing profile is no longer a surprise, but the results reported now by J. Singer and colleagues will certainly invigorate the debate about the "firmness" of some concepts.

First, let's recall the data of this same group demonstrating PS1 at the cell-surface back in 1997. It seemed unbelievable and was not believed—and played into the concept of the "spatial paradox." This imaginary or virtual concept was never based on very hard data, so not surprisingly, it turned out to be untenable. That PS1 is (or can be) located in "all" cellular compartments, directed there by its molecular partners (nicastrin, APH2, Pen1...) and even by some of its (many) substrates, is now accepted and underscored by experimental data.

We have developed the idea that PS1 and γ-secretase (to us, both names equate to the same active entity) is actually "a-channel-turned-proteinase" (Dewachter et al., 2001). The topology, however interesting it is, has not been our major concern, as we focus on the...  Read more

That PS1 keeps a most amazing profile is no longer a surprise, but the results reported now by J. Singer and colleagues will certainly invigorate the debate about the "firmness" of some concepts.

First, let's recall the data of this same group demonstrating PS1 at the cell-surface back in 1997. It seemed unbelievable and was not believed—and played into the concept of the "spatial paradox." This imaginary or virtual concept was never based on very hard data, so not surprisingly, it turned out to be untenable. That PS1 is (or can be) located in "all" cellular compartments, directed there by its molecular partners (nicastrin, APH2, Pen1...) and even by some of its (many) substrates, is now accepted and underscored by experimental data.

We have developed the idea that PS1 and γ-secretase (to us, both names equate to the same active entity) is actually "a-channel-turned-proteinase" (Dewachter et al., 2001). The topology, however interesting it is, has not been our major concern, as we focus on the functional implications that are widespread and diverse. However, that both are linked goes without question, and the new data prove that both sides of the coin should continue to be studied with vigor and tenacity.

Based on biochemical, cellular, and electrophysiological data, we recently proposed that PS1 is also at the synapse, involved in regulation of capacitive calcium entry (CCE) and long-term potentiation (LTP) (Ris et al., 2003), based on our experimental data and those of others.

We would not be surprised to learn that the γ-secretase complex is a structurally "loose" complex in which the interplay of composition, both in terms of lipids and proteins (partners and substrates) actually defines or changes "shape and function." The analysis of the structure-function relationship of γ-secretase will be center-stage for some time to come, including its modulation by such diverse actors as the GSK-3's, cholesterol, cytoskeletal elements, NSAIDs, and more.

This will not only help to understand some fundamental processes acting in our body—from conception to death and from "head to tail"—including brain physiology and pathology as in Alzheimer's disease, but also in CADASIL and other diseases. This closes the circle and brings us back to APP, the somewhat forgotten player in the flurry surrounding PS1.

The structure-function aspects of APP are functionally directly related to those of PS1, and are by no means less interesting. They might be even more complex, stretching from extracellular interaction with ECM and proteinase inhibition (serine and metalloproteinases involved in synaptic remodeling, i.e., tPA/plasmin and MMP9, respectively) to nuclear activities in gene regulation (via APPs and AICD).

No wonder that tampering with the biochemistry of APP and PS1 (by mutations or old age) results in devastation of the most essential brain functions and in dementia.

View all comments by Fred Van Leuven

By this date, three comments followed the on-line publication of our paper in the Proc. Natl. Acad. Sci. USA (1). The one by Erene Mina was a brief review of our paper; the second consisted of some interesting comments on PS and ß-APP by Fred Van Leuven; and the third by Michael Wolfe. We consider here only the last one, since it is the only one critical of our conclusion that the PS molecule has a 7-transmembrane (7-TM) topology in the surface membranes of cells, and not the 8-TM that is currently widely accepted. Dr. Wolfe argues that there is a great deal of evidence that has been adduced to support the 8-TM model, and therefore that the 7-TM model, which is apparently inconsistent with this data, cannot be correct. We do not think that the number of publications supporting one or the other model is a criterion by which to judge which one is correct. Rather, it is the inability to find an experimental or logical fault with a particular piece or set of evidence supporting a conclusion. In this respect, Dr. Wolfe does not refer to any significant fault with our data or...  Read more

By this date, three comments followed the on-line publication of our paper in the Proc. Natl. Acad. Sci. USA (1). The one by Erene Mina was a brief review of our paper; the second consisted of some interesting comments on PS and ß-APP by Fred Van Leuven; and the third by Michael Wolfe. We consider here only the last one, since it is the only one critical of our conclusion that the PS molecule has a 7-transmembrane (7-TM) topology in the surface membranes of cells, and not the 8-TM that is currently widely accepted. Dr. Wolfe argues that there is a great deal of evidence that has been adduced to support the 8-TM model, and therefore that the 7-TM model, which is apparently inconsistent with this data, cannot be correct. We do not think that the number of publications supporting one or the other model is a criterion by which to judge which one is correct. Rather, it is the inability to find an experimental or logical fault with a particular piece or set of evidence supporting a conclusion. In this respect, Dr. Wolfe does not refer to any significant fault with our data or their interpretation favoring the 7-TM model, but instead raises the multiple lines of evidence in the literature favoring the 8-TM model. We are of the opinion, however, that our recent immunofluorescence data are irrefutable. If our critics think otherwise, we would appreciate learning the basis of their contrary opinion. In reference to whether PS is itself the g-secretase enzyme, we state in our paper that in the 7 TM model, Asp 385 is outside the membrane in the large loop region following helix VI. The model of an intramembranous two-aspartyl transition complex is therefore not supported by the 7-TM topography. However, it is not altogether ruled out, in that the moderately hydrophobic domain labeled VII’ in Fig. 1 of our paper may be partially inserted into the extracellular half of the membrane, placing Asp 385 closer to Asp 257 than is schematically represented. Even more to the point is the recent X-ray structure of the 7-TM bovine rhodopsin molecule (2), for example, which reveals a remarkably more complex organization than the usual schematic 2-dimensional representation of the PS molecular structure. The TM helical domains of rhodopsin are arranged in an overall roughly ovoid (not linear) arrangement in the plane of the membrane, but with the helices positioned at quite different angles to one another, and in relative proximities that are altogether unrelated to their successive sequences in the molecule. Different helices also exhibit considerably different degrees of internal kinking at their proline and other residues. Furthermore, the extracellular and cytoplasmic interhelical loop domains each show a complex set of domain interactions and a unique coordinated conformation on their respective sides of the membrane. The structure of rhodopsin, by extension, reveals the obvious fact that the usual 2-dimensional representation of PS membrane protein topography depicted for the 8-TM model, with the two Asp residues closely adjacent to each other in the plane of the membrane, is pure fiction. Only X-ray structure determination will settle this problem. (An additional aside by S. J. Singer: I am the senior author of these studies with Dr. Nazneen Dewji only in age. Dr. Dewji is the senior author and PI of the NIH grants that support this work.) Nazneen N. Dewji. ndewji@ucsd.edu S.J. Singer. ssinger@ucsd.edu

References:
1. Dewji NN, Valdez D, Singer SJ. The presenilins turned inside out: Implications for their structures and functions. PNAS 101(4): 1057-1062 (2004). 2. Palczewski K, Kumasaka T, Hori T, Behnke CA, Motoshima H. et al., Science 289: 739-45 (2000).

View all comments by Nazneen Dewji