␥-Secretase cleavage of ␤-amyloid precursor protein (APP) is crucial in the pathogenesis of Alzheimer disease, because it is the decisive step in the formation of the C terminus of ␤-amyloid protein (A␤). To better understand the molecular events involved in ␥-secretase cleavage of APP, in this study we report the identification of a new intracellular long A␤ species containing residues 1-46 (A␤ 46 ), which led to the identification of a novel -cleavage site between the known ␥-and ⑀-cleavage sites within the transmembrane domain of APP. Our data clearly demonstrate that the new -cleavage is a presenilin-dependent event. It is also noted that the new -cleavage site at A␤46 is the APP717 mutation site. Furthermore, we show that the new -cleavage is inhibited by ␥-secretase inhibitors known as transition state analogs but less affected by inhibitors known as non-transition state ␥-secretase inhibitors. Thus, the identification of A␤ 46 establishes a system to determine the specificity or the preference of the known ␥-secretase inhibitors by examining their effects on the formation or turnover of A␤ 46 .The amyloid deposits in the brain of Alzheimer disease (AD) 1 patients are principally composed of the 39 -43-amino acid residue amyloid ␤-peptide (A␤), which is derived from a large ␤-amyloid precursor protein (APP). In the amyloidogenic pathway, APP is first cleaved at the N terminus of A␤ sequence by ␤-secretase, to produce a soluble ectodomain, sAPP␤, and a membrane-anchored C-terminal fragment, CTF␤. CTF␤ is then subsequently cleaved within the transmembrane domain by ␥-secretase to produce the full-length A␤ and the intracellular domain (AICD) (1). ␤-Secretase has been identified as a type I membrane aspartyl protease (2, 3). The findings that knockout of presenilin 1 (PS1) and PS2 results in the abolishment of the ␥-secretase cleavage of APP and that two aspartate residues in two transmembrane domains of presenilin have been identified as critical for the ␥-secretase activity suggest that presenilin may be the ␥-secretase (4 -7). Recently, several other molecules, namely nicastrin, Aph-1, and Pen-2, have been identified as essential components of the ␥-secretase complex of which presenilin may function as the catalytic subunit (8).Most of the A␤ species contain 40 or 42 amino acids. Recently, sequence analysis revealed that the N terminus of AICD starts at residue 50 of the A␤ sequence, which is 7-9 amino acids away from the C termini of A␤ 40 and A␤ 42 . This led to the finding of the ⑀-cleavage site between A␤49 and A␤50 (9 -12). Now the cleavage at A␤40/42 has been specifically referred to as ␥-cleavage site (12). However, neither the intermediate A␤ peptide, which ends at the ⑀-cleavage site, nor the C-terminal fragment, which starts with an N terminus generated by ␥-cleavage, has ever been detected. One possibility is that ␥-and ⑀-cleavages occur simultaneously. The other possibility is that there may be additional cleavages(s) between ␥-and ⑀-cleavages. Here we report that, in our effort to determine th...

␤-Amyloid precursor protein apparently undergoes at least three major cleavages, ␥-, ⑀-, and the newly identified -cleavage, within its transmembrane domain to produce secreted ␤-amyloid protein (A␤). However, the roles of ⑀-and -cleavages in the formation of secreted A␤ and the relationship among these three cleavages, namely ⑀-, -, and ␥-cleavages, remain elusive. We investigated these issues by attempting to determine the formation and turnover of the intermediate products generated by these cleavages, in the presence or absence of known ␥-secretase inhibitors. By using a differential inhibition strategy, our data demonstrate that A␤ 46 is an intermediate precursor of secreted A␤. Our co-immunoprecipitation data also reveal that, as an intermediate, A␤ 46 is tightly associated with presenilin in intact cells. Furthermore, we identified a long A␤ species that is most likely the long sought after intermediate product, A␤ 49 , generated by ⑀-cleavage, and this A␤ 49 is further processed by -and ␥-cleavages to generate A␤ 46 and ultimately the secreted A␤ 40/42 . More interestingly, our data demonstrate that ␥-cleavage not only occurs last but also depends on -cleavage occurring prior to it, indicating that -cleavage is crucial for the formation of secreted A␤. Thus, we conclude that the C terminus of secreted A␤ is most likely generated by a series of sequential cleavages, namely first ⑀-cleavage which is then followed by -and ␥-cleavages, and that A␤ 46 produced by -cleavage is the precursor of secreted A␤ 40/42 .The mechanism of the formation of the ␤-amyloid protein (A␤) 2 is the central issue in Alzheimer disease research, not only because A␤ is the major constituent of senile plaques, one of the neuropathological hallmarks of Alzheimer disease, but also because A␤ formation may be a causative event in the disease (1). A␤ is proteolytically derived from a large single transmembrane protein, the ␤-amyloid precursor protein (APP), as a result of sequential cleavages by ␤-and ␥-secretases (1). ␤-Secretase has been identified as a type I membrane aspartyl protease (2, 3). Although the exact nature of ␥-secretase is still a matter of debate, accumulating evidence supports the idea that ␥-secretase is a multiple molecular complex composed of, at least, presenilins, nicastrin, Aph-1, and Pen-2 and that presenilin may function as the catalytic subunit (4).In understanding the mechanism by which the C termini of secreted A␤ are generated during the processing of APP, three major intramembranous cleavages have been established. The first one is the cleavage now specifically referred to as ␥-cleavage (5), which produces the C termini of most of the secreted A␤ species that end at amino acids 40 (A␤40) or 42 (A␤42) of the A␤ sequence. The second one is the ⑀-cleavage occurring between A␤ residues 49 and 50, which produces the N terminus of most of the APP intracellular domain (AICD) (5-8). The identification of this ⑀-cleavage site raises a question as to whether this ⑀-cleavage is obligatory for the generation of the...

We previously reported the phosphoinositide 3-kinase-dependent activation of the 5-AMP-activated kinase (AMPK) by peroxynitrite (ONOO ؊ ) and hypoxia-reoxygenation in cultured endothelial The AMP-activated protein kinase (AMPK) 2 is a serine/threonine kinase and a member of the Snf1/AMPK protein kinase family (1-3). Its activity is stimulated by an increase in intracellular AMP-to-ATP ratio in response to stresses such as exercise (4 -6), hypoxia (7,8), oxidant stress (9, 10), and glucose deprivation (11). AMPK activation switches on catabolic pathways that produce ATP and switches off anabolic pathways that consume ATP. The activation of AMPK leads to phosphorylation of a number of proteins that result in increased glucose uptake and metabolism as well as fatty acid oxidation and simultaneously in inhibition of hepatic lipogenesis, cholesterol synthesis, and glucose production (reviewed in Refs. 12-14). AMPK is also responsible for increased fatty acid oxidation in response to the adipocyte-derived hormones leptin (15) and adiponectin (16). Because AMPK activation could have beneficial metabolic consequences for diabetic patients, AMPK has emerged as a potential target for the treatment of obesity and type II diabetes (reviewed in Refs. 3 and 17). It has been demonstrated that two classes of anti-diabetic drugs, metformin (18, 19) and thiazolidinediones (20), can act at least in part through activation of AMPK in liver and muscle.AMPK is an obligatory heterotrimer containing catalytic ␣ subunit and regulatory ␤ and ␥ subunits, each of which occur in at least two isoforms. Activation of AMPK absolutely requires its phosphorylation at Thr 172 in the activation loop of ␣1 and ␣2 subunits by one or more upstream kinases (AMPKKs) (21, 22).The major breakthrough in identifying the first AMPKK came from research on the regulation of the AMPK ortholog Snf-1 in Saccharomyces cerevisiae (23,24). The T-loop residue of Snf-1 was phosphorylated by a group of three related protein kinases bearing homology to mammalian LKB1, which was subsequently identified by several laboratories as being the major upstream kinase for AMPK (25)(26)(27). LKB1 was found to co-purify with liver AMPK and to phosphorylate recombinant AMPK complexes. In addition, AMPK could not be activated in mammalian cells that lacked LKB1 expression or in cells that were treated with Hsp90 inhibitors, which decrease LKB1 expression (28, 29). Finally, LKB1 turned out to phosphorylate the T-loop of all the 12 human kinases that are phylogenetically related to AMPK (AMPK subfamily) (30). However, paradoxically, neither the activity of LKB1 itself nor that of AMPK-related kinases was regulated directly by the stimuli known to activate AMPK, like e.g. AMP, AICAR, or muscle contraction (31, 32). Thus, the question remained regarding how AMPK stimuli can lead to LKB1-dependent AMPK activation.We had previously reported that peroxynitrite (ONOO Ϫ ), a potent

Tissue factor (TF) gene expression is rapidly induced in epithelial cells by phorbol 12-myristate 13-acetate and serum. We have shown that this induction is mediated by a novel serum response region (SRR) (؊111 to ؉14 bp) within the human TF promoter. In this study, we characterized cis-acting genetic elements within the SRR that regulated basal and inducible expression of the TF gene in HeLa cells. Gel mobility shift assays using oligonucleotides spanning the entire SRR identified three 12-base pair (bp) motifs within subregions 1, 2, and 3 that bound constitutively expressed Sp1 and inducibly expressed EGR-1. Analysis of protein binding to these 12-bp motifs by competition with Sp1 and EGR-1 sites, mutation, and antibody supershift experiments indicated that they each contained distinct EGR-1 and Sp1 sites that overlapped by 6 bp. Functional studies using HeLa cells transfected with plasmids containing the wild-type TF promoter (؊111 to ؉14 bp) or derivatives containing mutations in the three Sp1 and/or EGR-1 sites examined basal and inducible expression. The Sp1 sites mediated basal promoter activity, and both Sp1 and EGR-1 sites were required for maximal induction of the TF promoter by phorbol 12-myristate 13-acetate or serum. These data indicated that TF gene expression in HeLa cells was regulated by both Sp1 and EGR-1.