Presenilins are integral membrane protein involved in the production of amyloid ␤-protein. Mutations of the presenilin-1 and -2 gene are associated with familial Alzheimer's disease and are thought to alter ␥-secretase cleavage of the ␤-amyloid precursor protein, leading to increased production of longer and more amyloidogenic forms of A␤, the 4-kDa ␤-peptide. Here, we show that radiolabeled ␥-secretase inhibitors bind to mammalian cell membranes, and a benzophenone analog specifically photocross-links three major membrane polypeptides. A positive correlation is observed among these compounds for inhibition of cellular A␤ formation, inhibition of membrane binding and cross-linking. Immunological techniques establish N-and C-terminal fragments of presenilin-1 as specifically cross-linked polypeptides. Furthermore, binding of ␥-secretase inhibitors to embryonic membranes derived from presenilin-1 knockout embryos is reduced in a gene dose-dependent manner. In addition, C-terminal fragments of presenilin-2 are specifically cross-linked. Taken together, these results indicate that potent and selective ␥-secretase inhibitors block A␤ formation by binding to presenilin-1 and -2.␤-Amyloid precursor protein (␤APP) 1 is a transmembrane protein that undergoes processing to A␤ by proteolytic activities known as ␤-and ␥-secretases (for review, see Refs. 1-3). The ␤-secretase cleavage occurs in the extracellular domain by a recently identified aspartyl protease variously termed BACE, memapsin, and Asp2 (4 -9), whereas the heterogeneous ␥-secretase cleavage occurs in the transmembrane domain (2, 10). Dominant mutations in either of the two human presenilin (PS-1 and PS-2) genes lead to familial Alzheimer's disease (AD). PS-1 and -2 are polytopic membrane proteins (for review, see Refs. 11-13). Presenilins are proteolytic processed. In vivo, only small amounts of the holoprotein can be detected, primarily in the nuclear envelope, whereas 30-kDa N-terminal and 20-kDa C-terminal fragments of presenilin are observed in all mammalian tissues and cell lines analyzed so far. Coimmunoprecipitation experiments revealed that presenilin fragments are assembled into a high molecular weight complex together with other proteins (for review see 11-13). The proposed mechanism through which the presenilin mutations cause AD is an alteration in the predominant ␥-secretase cleavage site which increases the amount of the longer, more amyloidogenic A␤ 1-42(43) fragments produced (11-13). A null mutation of the mouse PS-1 selectively reduces ␥-secretase activity (14), and site-directed mutagenesis of PS-1 and PS-2 at two conserved aspartyl residues, which resemble the catalytic center of aspartyl proteases, also reduces ␥-secretase activity (15, 16). These observations indicate that PS-1 and PS-2 either stimulate the activity of ␥-secretase by trafficking to appropriate cellular compartments, serve as cofactors of the ␥-secretase, or are ␥-secretase themselves.Here, we report that a series of potent and selective ␥-secretase inhibitors bind to mam...

Presenilin (PS) proteins control the proteolytic cleavage that precedes nuclear access of the Notch intracellular domain. Here we observe that a partial activation of the HES1 promoter can be detected in PS1/PS2 (PS1/2) double null cells using Notch1⌬E constructs or following Delta1 stimulation, despite an apparent abolition of the production and nuclear accumulation of the Notch intracellular domain. PS1/2-independent Notch activation is sensitive to Numblike, a physiological inhibitor of Notch. PS1/2-independent Notch signaling is also inhibited by an active ␥-secretase inhibitor in the low micromolar range and is not inhibited by an inactive analogue, similar to PS-dependent Notch signaling. However, experiments using a Notch1-Gal4-VP16 fusion protein indicate that the PS1/2-independent activity does not release Gal4-VP16 and is therefore unlikely to proceed via an intramembranous cleavage. These data reveal that a novel PS1/2-independent mechanism plays a partial role in Notch signal transduction.

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Factor XIa is a serine protease which participates in both the extrinsic and intrinsic pathways of blood coagulation. In this work we used active site directed inhibitors to study the mechanism of factor IX activation by factor XIa. To this end, we developed a new sensitive method for the detection of factor IXa based on its affinity to antithrombin III. Using this assay, we found that the peptidic inhibitors, leupeptin and aprotinin, exhibited similar potencies in inhibiting factor IX activation and the cleavage of a tripeptidic chromogenic substrate by factor XIa. As expected, leupeptin and aprotinin were competitive with respect to the tripeptidic chromogenic substrate. However, the inhibition of factor IX activation was best described by mixed-type inhibition with the affinity of leupeptin and aprotinin to the factor XIa-factor IX complex only approximately 10-fold lower than their affinity toward factor XIa. These results, consistent with previous factor XI domain analyses, suggest that the active site of factor XIa does not contribute significantly to the affinity of factor XIa toward factor IX. The competitive component of the inhibition of factor IX activation suggests that binding of factor IX to factor XIa heavy chain affects the interactions of leupeptin and aprotinin with the active site.

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Obesity and related sequelae such as insulin resistance and diabetes present a growing health care burden throughout the world. One of the hallmarks of the disease process in these patients is the presence of a prothrombotic state that manifests itself as heightened levels of circulating thrombotic/platelet biomarkers such as thrombin-antithrombin complex (T-AT), soluble CD40 ligand (sCD40L) and inflammatory cytokines such as interleukin 6 (IL6). We have found that levels of these biomarkers are also elevated in genetic models of obesity and diabetes (db/db mice). Apixaban is a potent, highly selective, direct Factor Xa inhibitor that shows great promise as a safer and more efficacious therapy then the existing standard of care (ARISTOTLE trial). For this reason, we sought to study the differences in the ability of apixaban and current clinical therapies to lower plasma markers of thrombosis (T-AT and sCD40L) in diabetic mice. We administered either apixaban, warfarin or aspirin and clopidogrel to 12 week old, male db/db mice p.o. for 48 h prior to collection of blood. T-AT and sCD40L were measured in plasma by ELISA. db/db mice had 3-fold greater levels of T-AT (P<0.05) and 2-fold (P<0.05) greater levels of sCD40L in plasma as compared to C57Bl6 mice. Warfarin (0.3 mg/kg) dose-dependently decreased T-AT (P<0.05) to levels similar to normoglycemic C57/Bl6 controls. Apixaban (5 and 50 mg/kg) significantly reduced (P<0.05) elevated levels of these biomarkers when administered b.i.d. When administered q.d., apixaban (10 and 100 mg/kg) lowered T-AT levels but did not robustly affect sCD40L. The platelet therapies, aspirin (30 mg/kg) and clopidogrel (3 mg/kg) robustly decreased sCD40L (P<0.05) but did not affect T-AT levels in obese, diabetic mice. Overall, these data suggest that administration of apixaban is equally efficacious as warfarin in lowering elevated thrombotic biomarkers in diabetic mice. Anti-platelet therapies were efficacious on platelet biomarkers but did not affect markers associated with elevated thrombin activation. This study was funded by Bristol-Myers Squibb and Pfizer.