MINIREVIEW
The Role of Presenilins in gamma -Secretase Activity*

Michael S. WolfeDagger § and Christian Haass||

From the Dagger  Center for Neurologic Diseases, Harvard Medical School and Brigham and Women's Hospital, Boston, Massachusetts 02115 and  Ludwig-Maximilians-University Munich, Adolf-Butenandt-Institute, Department of Biochemistry, Schillerstrasse 44, D-80336 Muenchen, Germany



    INTRODUCTION
TOP
INTRODUCTION
Familial Alzheimer's Disease...
Presenilins and gamma -Secretases
Presenilins and Notch...
Biochemical Evidence That...
Perspective
REFERENCES

It is an aphorism that basic biological research lays the groundwork for solving important biomedical problems. However, the reverse is true as well: focusing on problems of human disease can lead to important new insights into the intricacies of nature. Research on Alzheimer's disease (AD)1 provides a case study for the latter, where the race to identify molecular players involved in pathogenesis has unveiled some unexpected and fascinating aspects of protease biochemistry and proteolysis-dependent signal transduction. The 40-42-residue amyloid-beta peptide (Abeta ) is implicated in the early pathogenic events that lead to AD, and this peptide is carved out of the amyloid-beta precursor protein (APP) by beta - and gamma -secretases (Fig. 1A) (1). These two proteases are considered such important drug targets that the search for inhibitors of Abeta production within pharmaceutical companies has gone on for years even though both beta - and gamma -secretases remained unknown until very recently. Last year, beta -secretase was identified as a new membrane-tethered member of the aspartyl protease family (2-6), a finding that should greatly facilitate drug discovery. alpha -Secretases, which prevent Abeta formation by cleaving in the middle of the amyloid domain (7), have now been shown to be members of the ADAM family (a disintegrin and metalloprotease) (8, 9). At the same time, the mysterious gamma -secretase is giving up its secrets as well.



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Fig. 1.   A, proteolytic processing of APP and Notch. Ectodomain shedding by alpha - or beta -secretase generates 83- or 99-residue membrane-associated C-terminal fragments (C83 or C99, respectively). Metalloproteases such as TACE and ADAM-10 appear to be alpha -secretases, and beta -secretase is a membrane-tethered aspartyl protease. C83 and C99 are further processed by gamma -secretase, which hydrolyzes these substrates in the middle of their transmembrane regions to form p3 from C83 and Abeta from C99. Notch is processed by a Furin-like protease, producing a heterodimeric species that is trafficked to the cell surface. After ligand binding, the Notch ectodomain is shed by TACE, and the remaining membrane-associated C terminus, NEXT, is cut within the transmembrane region by a gamma -secretase-like protease. B, topology and proteolytic processing of presenilins. Presenilins possess eight transmembrane regions and are snipped in the middle of the hydrophobic domain in the large cytosolic loop between TM 6 and TM 7, producing stable heterodimers by an unidentified "presenilinase" (PSase). Two conserved aspartates, one in TM 6 and one in TM 7, are each required for heterodimer formation and for gamma -secretase cleavage of APP and Notch, leading to the hypothesis that presenilins are proteases activated by autoproteolysis. NTF, N-terminal fragment; CTF, C-terminal fragment.



    Familial Alzheimer's Disease and Generation of Highly Amyloidogenic Abeta 42
TOP
INTRODUCTION
Familial Alzheimer's Disease...
Presenilins and gamma -Secretases
Presenilins and Notch...
Biochemical Evidence That...
Perspective
REFERENCES

Genetic studies of families susceptible to autosomal dominant, early onset (<60 years) AD underscore the importance of Abeta formation to the disease process (10, 11). Several different missense mutations in the APP gene on chromosome 21 cause familial AD (FAD), and these mutations are found immediately near the alpha -, beta -, or gamma -secretase processing sites. A Lys right-arrow Asn/Met right-arrow Leu double mutation near the beta -secretase cleavage site found in a Swedish pedigree leads to increased Abeta production (both Abeta 40 and Abeta 42) (12), and indeed purified beta -secretase cuts peptide substrates with the "Swedish mutation" better than peptides based on the wild-type sequence (2, 4, 6). The several FAD-causing mutations in APP near the gamma -secretase processing site also affect Abeta production, but in these cases the mutations alter the specificity of the protease, leading to increases in the 42-residue Abeta variant (13, 14). These findings bolstered the "amyloid hypothesis" of AD pathogenesis, because Abeta 42 is particularly prone to fibril formation (15) and is the protein initially deposited in the neuritic plaques that characterize the disease (16). Genes encoding the polytopic presenilin-1 and presenilin-2 (PS1 and PS2), found on chromosomes 14 and 1, respectively, were later identified as major loci for FAD (17, 18), and mutations in the presenilins were likewise found to increase Abeta 42 production in transfected cell lines (19-21), transgenic animals (19, 20, 22), and human plasma (23). Thus, presenilins somehow modulate gamma -secretase activity. In fact, deletion of PS1 in mice dramatically reduced gamma -secretase activity (24), and knockout of both PS1 and PS2 resulted in complete abolition of this APP processing event (25, 26), demonstrating that presenilins are absolutely required for proteolysis by gamma -secretase.

gamma -Secretase carries out an unusual proteolysis within the middle of the predicted transmembrane domain of its substrates, C99, generated by beta -secretase, and C83, generated through alternative processing by alpha -secretase (Fig. 1A). Indeed, a helical conformation of the gamma -secretase cleavage site in APP can explain why nearby FAD-causing mutations specifically increase Abeta 42 production; these mutations are immediately adjacent to the amide bond processed to give Abeta 42 (27). A phenylalanine-scanning study near the gamma -secretase cleavage site and the resulting effects on Abeta 40 and Abeta 42 production provided important experimental support for the idea that the substrate is in a helical conformation upon initial interaction with the protease (28). Other mutagenesis studies likewise indicated that gamma -secretase has loose sequence specificity (29-31).

Despite the loose sequence specificity, gamma -secretase nevertheless appears to process its substrate APP at specific sites. This apparent paradox can be resolved by considering distance within the membrane as a key determinant of the site of proteolysis, a concept that together with the helical model suggests that gamma -secretase might catalyze an intramembranous proteolysis. The development of substrate-based inhibitors led to further indirect characterization of gamma -secretase; inhibition by peptidomimetic transition-state analogues suggested an aspartyl protease mechanism (27). Moreover, gamma -secretase appears to have loose sequence specificity for its peptidomimetic inhibitors as it displays for its substrates (27). Distinctions are observed for the ability of a given compound to block Abeta 40 vis-à-vis Abeta 42, leading to the suggestion that these two Abeta species are generated by different gamma -secretases (32, 33).


    Presenilins and gamma -Secretases
TOP
INTRODUCTION
Familial Alzheimer's Disease...
Presenilins and gamma -Secretases
Presenilins and Notch...
Biochemical Evidence That...
Perspective
REFERENCES

The findings that PS mutations affect Abeta 42 generation and that PS knockouts prevent Abeta production raised the question if presenilins are identical with the gamma -secretases. Presenilins appear to bear no obvious sequence homology with known proteases, and their homology to Spe-4, a Caenorhabditis elegans protein apparently involved in vesicle transport in spermatozoa, initially suggested a less direct role in gamma -secretase processing of APP (17). Furthermore, overexpression of presenilins does not lead to increased gamma -secretase activity (20). However, presenilins themselves undergo proteolytic processing within the hydrophobic region of the large cytosolic loop between transmembrane domain (TM) 6 and TM 7 (Fig. 1B) to form stable heterodimeric complexes (39, 40). (Most studies support an eight-TM topology for the presenilins (34-36), although six- and seven-TM topologies have also been suggested (37, 38)). These heterodimers are only produced to limited levels even upon overexpression of the holoprotein (39, 41-43) and may be found at the cell surface (44). Expression of exogenous presenilins leads to replacement of endogenous presenilin heterodimers with the corresponding exogenous heterodimers, indicating competition for limiting cellular factors needed for stabilization and endoproteolysis (45).

FAD-causing presenilin mutants are likewise processed to stable heterodimers with one exception, a missense mutation in PS1 that leads to the aberrant splicing out of exon 9, a region that encodes the endoproteolytic cleavage site (39, 41). This PS1 Delta E9 variant is an active presenilin, like other FAD-causing presenilin mutants, causes increased Abeta 42 production (19, 46). Upon overexpression, most PS1 Delta E9 is rapidly degraded similar to unprocessed wild-type presenilins; however, a small portion of this PS1 variant is stabilized in cells (41, 47) and forms a high molecular weight complex like the N- and C-terminal fragments (40, 48), suggesting that it can interact with the same limiting cellular factors as wild-type presenilins. These observations are consistent with the idea that the bioactive form of presenilin is the heterodimer and that the hydrophobic region is an inhibitory domain. However, several artificial PS variants have been generated which are biologically active/inactive independent of their ability to undergo endoproteolysis (49, 50). Therefore, further evidence is required to finally demonstrate the role of PS endoproteolysis for their biological and pathological function.

Given the characterization of gamma -secretase as an aspartyl protease that might catalyze an unusual intramembranous proteolysis, the sequences of presenilins were inspected and found to contain two conserved aspartates predicted to lie within TM 6 and TM 7, flanking the large cytosolic loop (51). These aspartates appear to align with each other within the membrane and with the gamma -secretase cleavage sites in APP (Fig. 1B). Mutation of either aspartate to alanine completely prevented PS1 endoproteolysis, and these Asp right-arrow Ala PS1 mutants acted in a dominant-negative manner with respect to gamma -secretase processing of APP. Similar effects on APP processing were observed even when conservative mutations to glutamate (51) or asparagine (49, 52) were made, indicating the crucial identity of these two residues as aspartates and suggesting that the effects are not likely because of misfolding. These effects have been corroborated by several laboratories and have been seen for both PS1 and PS2 (48, 49, 51-54), although a recent study demonstrated that the TM 7 aspartate may be less tolerant of mutagenesis compared with the TM 6 aspartate (55).

The aspartates are critical for gamma -secretase independent of their role in presenilin endoproteolysis; aspartate mutation in the PS1 Delta E9 variant still blocked gamma -secretase activity, even though endoproteolysis is not required of this presenilin variant (51). Together these results suggested that presenilins might be the catalytic component of gamma -secretase; upon interaction with as yet unidentified limiting cellular factors, presenilin undergoes autoproteolysis via the two aspartates, and the two presenilin subunits remain together, each contributing one aspartate to the active site of gamma -secretase. The issue of autoproteolysis, however, needs further investigation, because heterologous expression of a worm PS homologue in human cells leads to a functional PS variant that is not cleaved (56), indicating that a separate "presenilinase" may at least be required to cleave worm PS.


    Presenilins and Notch Processing
TOP
INTRODUCTION
Familial Alzheimer's Disease...
Presenilins and gamma -Secretases
Presenilins and Notch...
Biochemical Evidence That...
Perspective
REFERENCES

The presenilins are also critical for processing of the Notch receptor, a signaling molecule crucial for cell-fate determination during embryogenesis (57). After translation in the ER, Notch is processed by a Furin-like protease, resulting in a heterodimeric receptor that is shuttled to the cell surface (Fig. 1A). Upon interaction with a cognate ligand, the ectodomain of Notch is shed by a metalloprotease apparently identical to tumor necrosis factor-alpha converting enzyme (TACE) (58, 59). Interestingly, metalloproteases such as TACE and ADAM-10 are among the identified alpha -secretases that shed the APP ectodomain (8, 9). The membrane-associated C terminus is then cut within the postulated transmembrane domain to release the Notch intracellular domain (NICD), which then translocates to the nucleus where it interacts with and activates the CSL (CBF1, SU(H), Lag-1) family of transcription factors (60). NICD formation is absolutely required for signaling from the Notch receptor; knock-in of a single point mutation near the transmembrane cleavage site in Notch1 results in an embryonic lethal phenotype in mice virtually identical to that observed upon knockout of the entire Notch1 protein (61).

The parallels between APP and Notch processing are striking. Not only are both cleaved by TACE, but also the transmembrane regions of both proteins are processed by a gamma -secretase-like protease that requires presenilins. Deletion of PS1 in mice is embryonic lethal, with a phenotype similar to that observed upon knockout of Notch1 (62, 63), and the PS1/PS2 double knockout phenotype is even more similar (64, 65). Deficiency in PS1 dramatically reduces NICD formation (66), and the complete absence of presenilins results in total abolition of NICD production (25, 26). Treatment of cells with gamma -secretase inhibitors designed from the transmembrane cleavage site within APP likewise blocks NICD production (66) and nuclear translocation (67) and reduces Notch signaling from a reporter gene (67). Moreover, the two conserved TM aspartates in presenilins are required for cleavage of the Notch TM domain; as seen with gamma -secretase inhibitors, expression of Asp mutant PS1 or PS2 results in reduction of NICD formation, translocation, and signaling (44, 54, 55, 67). Thus, if presenilins are the catalytic components of the gamma -secretases that process APP, they are also likely the catalytic components of the related proteases that clip the transmembrane region of Notch.

Subcellular localization presents a potential problem for the idea that presenilins could be gamma -secretases; presenilins are primarily found in the ER and early Golgi (68), whereas gamma -secretase activity apparently takes place on the cell surface and in the ER and Golgi (69-74). Small amounts of presenilin heterodimers, however, have been found at the cell surface in complexation with the membrane-associated C terminus of Notch (44), and only small amounts of this presumably bioactive form should be needed for catalysis. Nevertheless, this "spatial paradox" still needs clear resolution. Another perplexing phenomenon is that the holoproteins of APP and Notch form stable complexes with presenilins in the ER (75-77). APP and Notch themselves are not substrates for gamma -secretase, so why do they interact with presenilin if presenilin is the protease? However, as mentioned above, the membrane-associated C terminus of Notch likewise forms stable complexes (44), and APP-derived C83 and C99 can also complex with presenilins in the presence of a gamma -secretase inhibitor or when either critical presenilin aspartate is mutated (78). Perhaps presenilins mature together with APP or Notch. Other unresolved issues are the site and specificity of Notch cleavage vis à vis that of APP. Although Notch appears to be cleaved within its transmembrane region, the site of cleavage is close to the cytosolic edge, whereas the APP gamma -secretase site is apparently in the middle of the membrane. For either cleavage event, though, only one of the proteolytic products has been identified; characterizing the other fragments may clarify this issue. As for sequence specificity, a single Val to Leu change prevents proteolysis of the Notch transmembrane region (60), whereas a variety of mutations in the APP transmembrane region is tolerated by gamma -secretase. Examination of these proteolytic events in purified enzyme assays may offer insights to resolve this problem, especially since recent data suggest that not only APP but also Notch endoproteolysis occurs largely independent of sequence (79).


    Biochemical Evidence That Presenilins Are Proteases
TOP
INTRODUCTION
Familial Alzheimer's Disease...
Presenilins and gamma -Secretases
Presenilins and Notch...
Biochemical Evidence That...
Perspective
REFERENCES

Advancing the understanding of gamma -secretase and the role of presenilins in this activity had been hampered by the lack of an isolated enzyme assay. Recently, Li et al. (80) reported a solubilized gamma -secretase assay that faithfully reproduces the properties of the protease activity observed in whole cells. Isolated microsomes were solubilized with detergent, and gamma -secretase activity was determined by measuring Abeta production from a Flag-tagged version of C99. Abeta 40 and Abeta 42 were produced in the same ratio as seen in living cells (~9:1), and peptidomimetics that blocked Abeta 40 and Abeta 42 formation in cells likewise inhibited production of these Abeta species in the solubilized protease assay. After separation of the detergent-solubilized material by size-exclusion chromatography, gamma -secretase activity coeluted with the two subunits of PS1. Remarkably, immunoprecipitated PS1 heterodimers also produced Abeta from the Flag-tagged substrate, strongly suggesting that presenilins are part of a large gamma -secretase complex.

More direct evidence that presenilins are the catalytic components of gamma -secretases has recently come from affinity labeling studies using transition-state analogue inhibitors targeted to the active site of the protease. Shearman et al. (80, 81) identified a peptidomimetic that blocks gamma -secretase activity with an IC50 of 0.3 nM in the solubilized protease assay and contains a hydroxyethyl isostere, a transition-state mimicking moiety found in many aspartyl protease inhibitors. While the transition-state mimicking alcohol directs the compound to aspartyl proteases, flanking substructures determine specificity. Photoactivatable versions of this compound bound covalently to presenilin subunits exclusively (82). Interestingly, installation of the photoreactive group on one end of the inhibitor led to labeling of the N-terminal presenilin subunit, whereas installation on the other end resulted in the tagging of the C-terminal subunit. Moreover, whereas these agents did not label wild-type PS1 holoprotein, they did tag PS1 Delta E9, which (as described above) is not processed to heterodimers but nevertheless is active. Similarly, Esler et al. (83) identified peptidomimetic inhibitors containing a difluoro alcohol group, another type of transition-state mimicking moiety, and these compounds were developed starting from a substrate-based inhibitor designed from the gamma -secretase cleavage site in APP. Conversion of one such analogue to a reactive bromoacetamide provided an affinity reagent that likewise bound covalently and specifically to PS1 subunits in cell lysates, isolated microsomes, and whole cells. Either PS1 subunit so labeled could be brought down with antibodies to the other subunit under coimmunoprecipitation conditions, demonstrating that the inhibitor bound to heterodimeric PS1. Seiffert and colleagues (84) likewise identified presenilin subunits as the molecular target of novel peptidomimetic gamma -secretase inhibitors. These inhibitors, however, do not resemble known transition-state mimics, so it is not clear whether they would be expected to bind to the active site of the protease.

Taken together, these results strongly suggest that heterodimeric presenilins contain the catalytic component of gamma -secretase; inhibitors in two of the three studies are transition-state analogues targeted to the active site. The active site is likely at the PS heterodimeric interface; both subunits are labeled by gamma -secretase affinity reagents, and each contributes one critical aspartate. The affinity labeling studies also identify the protease responsible for the transmembrane cleavage of Notch, because substrate-based gamma -secretase inhibitors like those used by Esler et al. (83) also block this Notch proteolysis (see above). These findings reinforce the amyloid hypothesis of AD pathogenesis; all FAD-causing mutations identified to date (accounting for ~60% of all FAD cases) are either in the precursor protein leading to Abeta (APP) or appear to be located in the proteases that catalyze the final step in Abeta generation (presenilins/gamma -secretases). At the same time, an important target for drug development has been identified, although it is not yet clear whether toxic effects of blocking cleavage of other gamma -secretase substrates (e.g. Notch) will negate therapeutic effects. Although presenilins are essential for proper embryonic development, the role of these proteins in aging adults is not known.


    Perspective
TOP
INTRODUCTION
Familial Alzheimer's Disease...
Presenilins and gamma -Secretases
Presenilins and Notch...
Biochemical Evidence That...
Perspective
REFERENCES

The discovery that presenilins are likely gamma -secretases raises a number of fascinating questions with respect to protease biochemistry as well as protease evolution. To carry out hydrolysis in a lipid environment, the transmembrane regions likely form a pore- or channel-like topography, with polar residues facing inward to allow entry of and interaction with the catalytic water. With such a sequestered active site and the two-dimensional fluidity of the lipid bilayer, the substrate probably first interacts with a binding site on the outer surface of the protease, with subsequent conformational changes allowing entry of the substrate to the active site within the protease interior. Further, given that the conformation of the substrate upon initial interaction with the protease is apparently an alpha -helix, the enzyme must have some mechanism for unwinding or bending the substrate near the site of cleavage so that the catalytic water and protease residues can access the scissile amide bond.

As for the origin of presenilins and other intramembrane-cleaving proteases (I-CLiPs) (85), it seems likely that certain polytopic integral membrane proteins acquired a few catalytic residues essential for catalysis rather than that soluble proteases somehow acquired multiple transmembrane domains. This would explain why polytopic membrane proteases bear little or no sequence homology with soluble proteases. For instance, in the S2P family of proteases that process membrane-bound transcription factors in mammals and bacteria, each contains an essential HEXXH motif that is a consensus sequence for a number of metalloproteases (86). Otherwise, they bear little resemblance to other metalloproteases. Similarly, presenilins require two conserved aspartates for proteolytic function but otherwise do not look like typical aspartyl proteases. Apparently there are only a very limited number of efficient biochemical solutions to the problem of proteolysis, as mechanisms conferred to polytopic proteases are strikingly similar to those of soluble and membrane-tethered proteases. Interestingly, the presenilins contain a motif near one of the critical aspartates that is also found in the bacterial type 4 prepilin protease family (50, 87), polytopic aspartyl proteases that also have eight transmembrane domains and require two completely conserved aspartates (88). This finding further supports the idea that presenilins may indeed be polytopic aspartyl proteases.

Considerable work lies ahead to understand presenilins/gamma -secretases. Clearly, the top priority is to identify the other critical components of the gamma -secretase complex, allowing reconstitution of activity and development of a purified enzyme assay. Assembly of the complex into active gamma -secretase should allow more focused structural-functional studies than the current approach of expressing mutant proteins in whole cells or organisms. Also, understanding how the more than 70 different FAD mutations in presenilins cause specific increases in Abeta 42 production might shed some light on how this deleterious function can be skewed in favor of other less noxious Abeta isoforms. Such understanding should not only suggest novel therapeutic approaches to AD but should also undergird our appreciation of the workings of an emerging class of polytopic membrane proteases.


    FOOTNOTES

* This minireview will be reprinted in the 2001 Minireview Compendium, which will be available in December, 2001.

§ To whom correspondence may be addressed. E-mail: wolfe@cnd.bwh.harvard.edu.

|| To whom correspondence may be addressed. E-mail: chaass@pbm.med. uni-muenchen.de.

Published, JBC Papers in Press, December 29, 2000, DOI 10.1074/jbc.R000026200


    ABBREVIATIONS

The abbreviations used are: AD, Alzheimer's disease; Abeta , amyloid-beta peptide; APP, amyloid-beta precursor protein; FAD, familial AD; PS, presenilin; TM, transmembrane domain; TACE, tumor necrosis factor-alpha converting enzyme; NICD, Notch intracellular domain; ER, endoplasmic reticulum.


    REFERENCES
TOP
INTRODUCTION
Familial Alzheimer's Disease...
Presenilins and gamma -Secretases
Presenilins and Notch...
Biochemical Evidence That...
Perspective
REFERENCES


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