In search of g-secretase: Presenilin at the cutting edge

T effort to solve Alzheimer’s disease (AD) has led to a salutary intersection between the desire to prevent a major health problem and the fundamental study of membrane protein biology. It has become apparent in the last few years that a compelling strategy to treat and prevent AD may involve understanding how certain integral membrane proteins undergo unusual proteolytic cleavages within their transmembrane domains, and then inhibiting this process pharmacologically. The substrate of interest in AD is the bamyloid precursor protein (APP), but several other proteins apparently are cleaved by a highly similar or identical proteolytic activity within their respective transmembrane domains, including the Notch family of cell-surface receptors required for cell fate determination (1) and the Ire1 proteins that initiate signaling in the unfolded protein response pathway (2). The unknown protease that affects the intramembranous cleavage of APP (and perhaps Notch and Ire1) is referred to as g-secretase (reviewed in ref. 3). Its identity is being actively sought because it is the second of two proteases that sequentially cleave APP to release a small fragment called the amyloid b-protein (Ab). [The other protease, b-secretase, recently has been cloned (4–8).] Progressive accumulation of Ab in brain regions subserving memory and cognition is an early, invariant, and necessary step in the pathogenesis of AD. For these reasons, band g-secretases are considered important targets for the development of effective therapeutics to treat AD. In this issue of PNAS, Li and colleagues (9) report the solubilization and enrichment of g-secretase activity from cultured human cells and examine its relationship to the protein presenilin 1 (PS1). To understand the rationale for their experiments, we should review brief ly certain current tenets of AD pathobiology. The brains of patients dying with AD contain abundant deposits of fibrillar Ab (amyloid) surrounded by clusters of damaged axons and dendrites (the neuritic plaques), as well as many neuronal cell bodies containing abnormal filamentous assemblies of the microtubule-associated protein tau (the neurofibrillary tangles). Genetic research so far has confirmed three distinct genes, mutations that cause severe, autosomal dominant forms of AD (reviewed in ref. 10). Missense mutations in APP itself were the first genetic cause of AD to be identified, and these mutations are principally located at or near either the bor g-secretase cleavage sites. The mutations enhance these respective cleavages, resulting in overproduction of the highly amyloidogenic 42-residue form of Ab (Ab42). The other two causative genes encode homologous eight-transmembrane proteins, PS1 and PS2, and missense mutations again lead to excessive cellular production of Ab42 by somehow altering g-secretase activity. Deletion of PS1 in mice markedly reduces g-secretase activity, i.e., it lowers Ab production and increases levels of the C-terminal fragments of APP that are substrates for g-secretase (11). Thus, PS1 mediates most g-secretase activity, with the residual activity likely caused by the presence of PS2. Other findings increasingly suggested that g-secretase activity was intimately associated with presenilins. APP could be coimmunoprecipitated with PS1 and PS2 (12), and this interaction with PS recently was shown to occur for the APP C-terminal fragments that are the immediate substrates for g-secretase (13). Meanwhile, site-directed mutagenesis (14) and molecular modeling (15) supported a helical conformation of the g-secretase cleavage site within APP, typical of transmembrane regions and consistent with an intramembranous proteolysis. The development of transition state analogs that mimic the APP cleavage region and inhibit g-secretase suggested that this enzyme is an aspartyl protease (15). Together, these findings led to the observation of two conserved intramembranous aspartate residues in TM6 and TM7 of PS1, and their subsequent mutation to alanine or glutamate markedly decreased g-secretase cleavage of APP (16). As seen with the analogous g-secretase processing of APP, the putative intramembranous cleavage of Notch to release its cytoplasmic signaling domain to the nucleus was similarly inhibited by knockout of the PS1 gene (17), peptidomimetic g-secretase inhibitors (17), and PS Asp-to-Ala mutations (18, 19) (see Fig. 1). Moreover, mutation of the aspartates also abrogated the normal endoproteolysis of PS that creates its heterodimeric complexes, which are believed to be the biologically active form of the protein (16, 19, 20). These observations led to the hypothesis that the presenilins are either unique diaspartyl cofactors for g-secretase or are themselves the long-sought protease (16). Although g-secretase activity had been studied in intact cells and isolated microsomes, the solubilization of the enzyme and its biochemical enrichment had not been published. Now, Li et al. (9) have found that recovery of catalytically competent, soluble g-secretase activity from HeLa cells can be accomplished by using low concentrations of the detergents CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate) or CHAPSO (3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate) but not Triton X-100. Remarkably, the solubilized g-secretase activity faithfully reproduces the ratio of Ab42 to Ab40 peptides produced in intact cells (90:10), suggesting that Ab42 formation is a normal, intrinsic property of the protease and not primarily dependent on membrane composition or thickness. The solubilized activity is inhibited by pepstatin, a classic aspartyl protease inhibitor. Moreover, the protease activity is inhibited far more potently by a transition state analog closely related to known inhibitors of aspartyl proteases such as renin (21) and HIV protease (22). This pharmacological profile provides further strong evidence that g-secretase is an aspartyl protease. Li et al. further show that solubilized