No abstract

Signal peptide peptidase (SPP) is an intramembrane aspartyl protease that cleaves remnant signal peptides after their release by signal peptidase. SPP contains active site motifs also found in presenilin, the catalytic component of the gamma-secretase complex of Alzheimer's disease. However, SPP has a membrane topology opposite that of presenilin, cleaves transmembrane substrates of opposite directionality, and does not require complexation with other proteins. Here we show that, upon isolation of membranes and solubilization with detergent, the biochemical characteristics of SPP are remarkably similar to gamma-secretase. The majority of the SPP-catalyzed cleavages occurred at a single site in a synthetic substrate based on the prolactin (Prl) signal sequence. However, as seen with cleavage of substrates by gamma-secretase, additional cuts at other minor sites are also observed. Like gamma-secretase, SPP is inhibited by helical peptidomimetics and apparently contains a substrate-binding site that is distinct from the active site. Surprisingly, certain nonsteroidal antiinflammatory drugs known to shift the site of proteolysis by gamma-secretase also alter the cleavage site of Prl by SPP. Together, these findings suggest that SPP and presenilin share certain biochemical properties, including a conserved drug-binding site for allosteric modulation of substrate proteolysis.

(17), and an E m ϭ -790 mV (18) was estimated for the 1ϩ͞0 couple, which is in line with E m estimates of all-ferrous Fe-S clusters by using discrete Fourier transform calculations (19). Indeed, this latter study raised the question of whether [Fe 4 S 4 ] 0 Av2 can even be made in vivo (1). A value of -790 mV is not consistent with turnover potential measured by Ti(III) and other reductants and with reported potentials for the Ti(IV)͞Ti(III) couple (7,20). If an E m ϭ -460 mV for the 1ϩ͞0 couple (16)

Presenilin (PS) is the presumptive catalytic component of the intramembrane aspartyl protease ␥-secretase complex. Recently a family of presenilin homologs was identified. One member of this family, signal peptide peptidase (SPP), has been shown to be a protease, which supports the hypothesis that PS and presenilin homologs are related intramembrane-cleaving aspartyl proteases. SPP has been reported as a glycoprotein of ϳ45 kDa. Our initial characterization of SPP isolated from human brain and cell lines demonstrated that SPP is primarily present as an SDS-stable ϳ95-kDa protein on Western blots. Upon heating or treatment of this ϳ95-kDa SPP band with acid, a ϳ45-kDa band could be resolved. Co-purification of two different epitopetagged forms of SPP from a stably transfected cell line expressing both tagged versions demonstrated that the ϳ95-kDa band is a homodimer of SPP. Pulse-chase metabolic labeling studies demonstrated that the SPP homodimer assembles rapidly and is metabolically stable. In a glycerol velocity gradient, SPP sedimented from ϳ100 -200 kDa. Significantly the SPP homodimer was specifically labeled by an active site-directed photoaffinity probe (III-63) for PS, indicating that the active sites of SPP and PS/␥-secretase are similar and providing strong evidence that the homodimer is functionally active. Collectively these data suggest that SPP exists in vivo as a functional dimer.

As a normal consequence of aging, men experience a significant decline in androgen levels. Although the neural consequences of age-related androgen depletion remain unclear, recent evidence suggests a link between low androgen levels and the development of Alzheimer's disease (AD). Here, we test the hypothesis that androgens act as endogenous modulators of b-amyloid protein (Ab) levels. To investigate this possibility, brain and plasma levels of Ab were measured in male rats with varying hormonal conditions. Depletion of endogenous sex steroid hormones via gonadectomy (GDX) resulted in increased brain levels of Ab in comparison to gonadally intact male rats. This GDX-induced increase in Ab levels was reversed by DHT supplementation, demonstrating a functional role for androgens in modulating brain levels of Ab. These findings suggest that age-related androgen depletion may result in accumulation of Ab in the male brain and thereby act as a risk factor for the development of AD. Keywords: aging, Alzheimer's disease, b-amyloid, androgen, dihydrotestosterone, estrogen. In contrast to abundant evidence that estrogen depletion is associated with increased risk of developing Alzheimer's disease (AD) (Hogervorst et al. 2000), little is known about the relationship between androgen depletion in men and AD. It is well established that aging men exhibit significant declines in bioavailable levels of testosterone and its active metabolite 5a-dihydrotestosterone (DHT) but maintain relatively normal levels of estrogen (Kaiser and Morley 1995). This normal, age-related reduction in androgen levels often results in functional impairments in androgen responsive tissues leading to a clinical syndrome termed 'androgen deficiency in aging men ' (Morley and Perry 2000). It is unknown as to how age-related androgen depletion affects the brain, an established target of androgen action. Interestingly, recent reports demonstrate lower circulating testosterone levels in AD vs. nondemented men (Hogervorst et al. 2001), suggesting that androgen depletion in males may promote AD pathogenesis.One mechanism by which sex steroid hormones may affect the development of AD is regulation of b-amyloid protein (Ab) levels. Estrogen has been shown to decrease Ab production in cultured cells (Jaffe et al. 1994;Xu et al. 1998) and in both wild type rodents and transgenic mouse models of AD (Petanceska et al. 2000; LevinAllerhand et al. 2002;Zheng et al. 2002). Unclear is whether androgens regulate Ab levels. This issue is complicated by the fact that the primary male sex steroid testosterone is converted to both DHT (via 5a-reductase) and estradiol (via aromatase) and thus activates androgen and estrogen signaling pathways. Although recent evidence shows that testosterone can increase non-amyloidogenic processing of amyloid precursor protein (APP) and thereby decrease Ab (Gouras et al. 2000), testosterone's modulation of APP processing is dependent upon its aromatization into estradiol (Goodenough et al. 2000).Here we directly investigate the ...

No abstract

The Alzheimer's disease-associated b-amyloid peptide is produced through cleavage of amyloid precursor protein by b-secretase and c-secretase. c-Secretase is a complex containing presenilin (PS) as the catalytic component and three essential cofactors: Nicastrin, anterior pharynx defective (APH-1) and presenilin enhancer-2 (PEN-2). PS and signal peptide peptidase (SPP) define a novel family of aspartyl proteases that cleave substrates within the transmembrane domain presumptively using two membrane-embedded aspartic acid residues for catalysis. Apart from the two aspartate-containing active site motifs, the only other region that is conserved between PS and SPP is a PAL sequence at the C-terminus. Although it has been well documented that this motif is essential for c-secretase activity, the mechanism underlying such a critical role is not understood. Here we show that mutations in this motif affect the conformation of the active site of c-secretase resulting in a complete loss of PS binding to a c-secretase transition state analog inhibitor, Merck C. Analogous mutations in SPP significantly inhibit its enzymatic activity. Furthermore, these mutations also abolish SPP binding to Merck C, indicating that SPP and c-secretase share a similar active site conformation, which is dependent on the PAL motif. Exploring the amino acid requirements within this motif reveals a very small side chain requirement, which is conserved during evolution. Together, these observations strongly support the hypothesis that the PAL motif contributes to the active site conformation of c-secretase and of SPP.

Signal peptide peptidase (SPP) is an intramembranecleaving protease identified by its cleavage of several type II membrane signal peptides after signal peptidase cleavage. Here we describe a novel, quantitative, cellbased SPP reporter assay. This assay utilizes a substrate consisting of the NH 2 terminus of the ATF6 transcription factor fused to a transmembrane domain susceptible to SPP cleavage in vitro. In cells, cleavage of the substrate releases ATF6 from the membrane. This cleavage can be monitored by detection of an epitope that is unmasked in the cleaved substrate or by luciferase activity induced by the cleaved ATF6 substrate binding to and activating an ATF6 luciferase reporter construct. Using this assay we show that (i) SPP is the first aspartyl intramembrane-cleaving protease whose activity increases proportionally to its overexpression and (ii) selectivity of various SPP and ␥-secretase inhibitors can be rapidly evaluated. Because this assay was designed based on data suggesting that SPP has an orientation distinct from presenilin and cleaves type II membrane proteins, we determined whether the segment of SPP located between the two presumptive catalytic aspartates was in the lumen or cytoplasm. Using site-directed mutagenesis to insert an N-linked glycosylation site we show that a portion of this region is present in the lumen. These data provide strong evidence that although the SPP and presenilin active sites have some similarities, their presumptive catalytic domains are inverted. This assay should prove useful for additional functional studies of SPP as well as evaluation of SPP and ␥-secretase inhibitors.