Proteolytic processing of the amyloid precursor protein (APP) generates amyloid beta (Abeta) peptide, which is thought to be causal for the pathology and subsequent cognitive decline in Alzheimer's disease. Cleavage by beta-secretase at the amino terminus of the Abeta peptide sequence, between residues 671 and 672 of APP, leads to the generation and extracellular release of beta-cleaved soluble APP, and a corresponding cell-associated carboxy-terminal fragment. Cleavage of the C-terminal fragment by gamma-secretase(s) leads to the formation of Abeta. The pathogenic mutation K670M671-->N670L671 at the beta-secretase cleavage site in APP, which was discovered in a Swedish family with familial Alzheimer's disease, leads to increased beta-secretase cleavage of the mutant substrate. Here we describe a membrane-bound enzyme activity that cleaves full-length APP at the beta-secretase cleavage site, and find it to be the predominant beta-cleavage activity in human brain. We have purified this enzyme activity to homogeneity from human brain using a new substrate analogue inhibitor of the enzyme activity, and show that the purified enzyme has all the properties predicted for beta-secretase. Cloning and expression of the enzyme reveals that human brain beta-secretase is a new membrane-bound aspartic proteinase.

Several neurological diseases, includingThe importance of ␣-synuclein to the pathogenesis of Parkinson disease (PD) 4 and the related disorder, dementia with Lewy bodies (DLB), is suggested by its association with Lewy bodies and Lewy neurites, the inclusions that characterize these diseases (1)(2)(3), and demonstrated by the existence of mutations that cause syndromes mimicking sporadic PD and DLB (4 -6). Furthermore, three separate mutations cause early onset forms of PD and DLB. It is particularly telling that duplications or triplications of the gene (7-9), which increase levels of ␣-synuclein with no alteration in sequence, also cause PD or DLB.␣-Synuclein has been reported to be phosphorylated on serine residues, at Ser-87 and Ser-129 (10), although to date only the Ser-129 phosphorylation has been identified in the central nervous system (11,12). Phosphorylation at tyrosine residues has been observed by some investigators (13,14) but not by others (10 -12). Phosphorylation at Ser-129 (p-Ser-129) is of particular interest because the majority of synuclein in Lewy bodies contains this modification (15). In addition, p-Ser-129 was found to be the most extensive and consistent modification in a survey of synuclein in Lewy bodies (11). Results have been mixed from studies investigating the function of phosphorylation using S129A and S129D mutations to respectively block and mimic the modification. Although the phosphorylation mimic was associated with pathology in studies in Drosophila (16) and in transgenic mouse models (17, 18), studies using adeno-associated virus vectors to overexpress ␣-synuclein in rat substantia nigra found an exacerbation of pathology with the S129A mutation, whereas the S129D mutation was benign, if not protective (19). Interpretation of these studies is complicated by a recent study showing that the S129D and S129A mutations themselves have effects on the aggregation properties of ␣-synuclein independent of their effects on phosphorylation, with the S129A mutation stimulating fibril formation (20). Clearly, determination of the role of p-Ser-129 phosphorylation would be helped by identification of the responsible kinase. In addition, identification will provide a pathologically relevant way to increase phosphorylation in a cell or animal model.Several kinases have been proposed to phosphorylate ␣-synuclein, including casein kinases 1 and 2 (10, 12, 21) and members of the G-protein-coupled receptor kinase family (22). In this report, we offer evidence that a member of the polo-like kinase (PLK) family, PLK2 (or serum-inducible kinase, SNK), functions as an ␣-synuclein kinase. The ability of PLK2 to directly phosphorylate ␣-synuclein at Ser-129 is established by overexpression in cell culture and by in vitro reaction with the purified kinase. We show that PLK2 phosphorylates ␣-synuclein in cells, including primary neuronal cultures, using a series of kinase inhibitors as well as inhibition of expression with RNA interference. In addition, inhibitor and knock-out studies in mouse brai...

Recent genetic and biochemical studies have implicated cysteine-dependent aspartate-directed proteases (caspases) in the active phase of apoptosis. In the present study, three complementary techniques were utilized to follow caspase activation during the course of etoposide-induced apoptosis in HL-60 human leukemia cells. Immunoblotting revealed that levels of procaspase-2 did not change during etoposide-induced apoptosis, whereas levels of procaspase-3 diminished markedly 2-3 h after etoposide addition. At the same time, cytosolic peptidase activities that cleaved DEVDaminotrifluoromethylcoumarin and VEID-aminomethylcoumarin increased 100-and 20-fold, respectively; but there was only a 1.5-fold increase in YVAD-aminotrifluoromethylcoumarin cleavage activity. Affinity labeling with N-(N ␣ -benzyloxycarbonylglutamyl-N ⑀ -biotinyllysyl)-aspartic acid [(2,6-dimethylbenzoyl)oxy]methyl ketone indicated that multiple active caspase species sequentially appeared in the cytosol during the first 6 h after the addition of etoposide. Analysis on one-and twodimensional gels revealed that two species comigrated with caspase-6 and three comigrated with active caspase-3 species, suggesting that several splice or modification variants of these enzymes are active during apoptosis. Polypeptides that comigrate with the cytosolic caspases were also labeled in nuclei of apoptotic HL-60 cells. These results not only indicate that etoposide-induced apoptosis in HL-60 cells is accompanied by the selective activation of multiple caspases in cytosol and nuclei, but also suggest that other caspase precursors such as procaspase-2 are present but not activated during apoptosis.Recent studies (reviewed in Refs. 1-5) indicate that the cytotoxicity of virtually all chemotherapeutic agents is accompanied by apoptosis in susceptible cell lines. Likewise, experiments in animals (6 -9) and studies of circulating blasts from leukemia patients (10) have provided evidence that chemotherapy is accompanied by apoptosis in vivo. Moreover, it has been suggested that resistance to the cytotoxic effects of chemotherapeutic agents can result from resistance to chemotherapyinduced apoptosis (8,11,12). These observations highlight the potential importance of understanding the factors that control apoptosis.

The deposition of extracellular ␤-amyloid peptide (A␤) in the brain is a pathologic feature of Alzheimer's disease. The ␤-site amyloid precursor protein cleaving enzyme 1 (BACE1), an integral membrane aspartyl protease responsible for cleavage of amyloid precursor protein (APP) at the ␤-site, promotes A␤ production. A second integral membrane aspartyl protease related to BACE1, referred to as ␤-site amyloid precursor protein cleaving enzyme 2 (BACE2) has also been demonstrated to cleave APP at the ␤-cleavage site in transfected cells. The role of endogenous BACE2 in A␤ production remains unresolved. We investigated the role of endogenous BACE2 in A␤ production in cells by selective inactivation of its transcripts using RNA interference. We are able to reduce steady state levels for mRNA for each enzyme by >85%, and protein amounts by 88 -94% in cells. The production and deposition of insoluble A␤ 1 peptide in the brain results in the hallmark pathological feature of Alzheimer's disease (1). The cellular enzymes responsible for production of A␤ peptide are molecular targets for therapeutic intervention in Alzheimer's disease (2, 3). BACE1 (␤-site APP cleaving enzyme, ASP2, Memapsin 2, ␤-secretase), the enzyme responsible for cleavage of the amyloid precursor protein (APP) resulting in the amino terminus of A␤ peptide, is a novel integral membrane aspartyl protease (4 -8). Cellular antisense (6, 7) and knock-out mouse models (9 -11) have unambiguously confirmed the role of BACE1 in promoting A␤ production. A second integral membrane aspartyl protease related to BACE1, referred to as BACE2 (ASP1, Memapsin 1) has also been demonstrated to cleave APP at the ␤-cleavage site (7,8,(12)(13)(14)(15)(16). Both enzymes also cleave APP at a second site within the A␤ region. This second cleavage site for BACE1 is between A␤ residues 10 and 11 and between A␤ residues 19 and 20 for BACE2. Cleavage of APP by BACE1 at either site is amyloidogenic, whereas the second cleavage site for BACE2 on APP precludes formation of A␤.Overexpression of BACE2 in transfected cells produces intracellular carboxyl-terminal fragments (CTFs), as well as release of ␤-cleaved secreted APP (sAPP␤) from cells, consistent with cleavage of APP at the ␤-site by this enzyme (14, 16 -19). We have also observed decreased A␤ production from cells transfected with BACE2 (not shown), consistent with published observations (14, 16 -19). Hence, the reduction in secreted A␤ occurs in spite of the concomitant increased secretion of sAPP␤ from cells overexpressing BACE2. The decrease in A␤ production from BACE2 transfected cells has been attributed to the second cleavage site for BACE2 on APP (14, 16 -19). The observations on the cleavage specificity of BACE2 on APP substrate are derived from transfected cells that overexpress the enzyme. The role of BACE2 in A␤ production in cells expressing endogenous levels of the enzyme remains an unanswered question. Many tissues (including brain) and cell types co-express BACE1 and BACE2 mRNA (8,13,14,16). The therapeutic rele...

The capacity of recombinant human granulocyte–macrophage colony‐stimulating factor (GM‐CSF), glucocorticoids or all‐trans‐retinoic acid to modulate production of activin A by human monocytes was studied. It was shown that GM‐CSF stimulated monocytes to accumulate activin A RNA after as few as 4 hr of incubation, reaching a peak of stimulation at approximately 16 hr of incubation. The activin A transcripts accumulated in the monocytes after stimulation with only 5 U/ml of GM‐CSF and reached a maximum plateau level of expression between 25 and 50 U/ml of GM‐CSF. Biologically active activin A molecules were detected in the conditioned media by a bioassay, performed both in the absence and presence of a neutralizing antiserum for activin A. Accumulation of bioactive activin A in conditioned medium of monocyte cultures was detected after 24 hr of incubation with GM‐CSF and high levels of activin A were maintained for 72 hr. The production of the dimeric βAβA in these monocytes was further confirmed by a sandwich enzyme‐linked immunosorbent assay (ELISA) specific for activin A. In contrast to the stimulatory effect of GM‐CSF, hydrocortisone, dexamethasone or all‐trans‐retinoic acid at 1 × 10−7 to 1 × 10−5 m inhibited the constitutive expression of activin A and greatly suppressed the GM‐CSF‐stimulated production. Thus, the expression of activin A is modulated in monocytes by different agents. These observations may imply new roles for activin A at sites of inflammation where monocytes accumulate.