␥-Secretase is a membrane-associated protease that cleaves within the transmembrane region of amyloid precursor protein to generate the C termini of the two A␤ peptide isoforms, A␤40 and A␤42. Here we report the detergent solubilization and partial characterization of ␥-secretase. The activity of solubilized ␥-secretase was measured with a recombinant substrate, C100Flag, consisting largely of the C-terminal fragment of amyloid precursor protein downstream of the ␤-secretase cleavage site. Cleavage of C100Flag by ␥-secretase was detected by electrochemiluminescence using antibodies that specifically recognize the A␤40 or A␤42 termini. Incubation of C100Flag with HeLa cell membranes or detergent-solubilized HeLa cell membranes generates both the A␤40 and A␤42 termini. Recovery of catalytically competent, soluble ␥-secretase critically depends on the choice of detergent; CHAPSO (3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate) but not Triton X-100 is suitable. Solubilized ␥-secretase activity is inhibited by pepstatin and more potently by a novel aspartyl protease transition-state analog inhibitor that blocks formation of A␤40 and A␤42 in mammalian cells. Upon gel exclusion chromatography, solubilized ␥-secretase activity coelutes with presenilin 1 (PS1) at an apparent relative molecular weight of approximately 2.0 ؋ 10 6 . Anti-PS1 antibody immunoprecipitates ␥-secretase activity from the solubilized ␥-secretase preparation. These data suggest that ␥-secretase activity is catalyzed by a PS1-containing macromolecular complex.

␤-Secretase (BACE) is a membrane-bound aspartyl protease that cleaves the amyloid precursor protein to generate the N terminus of the amyloid ␤ peptide. BACE is expressed as a precursor protein containing Pre, Pro, protease, transmembrane, and cytosolic domains. A soluble BACE derivative (PreProBACE460) that is truncated between the protease and transmembrane domains was produced by baculovirus-mediated expression. ProBACE460 was purified from conditioned media of infected insect cells using immobilized concanavalin A and immobilized BACE inhibitor, P10-P4 Stat(Val). Furin cleaves ProBACE460 between the Pro and protease regions to generate mature BACE460. The k cat /K m of ProBACE460 when assayed with a polypeptide substrate is only 2.3-fold less than that of BACE460. This finding and the similar inhibitory potency of P10-P4 Stat(Val) for ProBACE460 and BACE460 suggest that the Pro domain has little effect on the BACE active site. Exposure of ProBACE460 to guanidine denaturation/ renaturation results in a 7-fold higher recovery of BACE activity than when BACE460 is similarly treated. The presence of free BACE Pro peptide during renaturation of BACE460 but not ProBACE460 increases recovery of activity. These findings show that the Pro domain in ProBACE460 does not suppress activity as in a strict zymogen but does appear to facilitate proper folding of an active protease domain.

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ABSTRACT:The mechanism underlying subcutaneous absorption of macromolecules and factors that can influence this process were studied in rats using PEGylated erythropoietins (EPOs) as model compounds. Using a thoracic lymph duct cannulation (LDC) model, we showed that PEGylated EPO was absorbed from the subcutaneous injection site mainly via the lymphatic system in rats, which is similar to previous reports in sheep. After subcutaneous administration, the serum exposure was reduced by ϳ70% in LDC animals compared with that in the control animals, and most of the systemically available dose was recovered in the lymph. In both LDC and intact rats, the total radioactivity recoveries in excreta after subcutaneous administration were high (70-80%), indicating that catabolism, not poor absorption, was the main cause for the observed low bioavailability (30-40%). Moreover, catabolism of PEGylated EPO was found with both rat subcutaneous tissue homogenate and lymph node cell suspensions, and a significant amount of dose-related breakdown fragments was found in the lymph of LDC rats. In addition, the bioavailability of PEGylated EPOs was shown to be 2-to 4-fold lower in "fat rats," indicating that physiologic features pertinent to lymphatic transport can have a profound impact on subcutaneous absorption. Limited studies in dogs also suggested similar subcutaneous absorption mechanisms. Collectively, our results suggest that the lymphatic absorption mechanism for macromolecules is probably conserved among commonly used preclinical species, e.g., rats and dogs, and that mechanistic understanding of the subcutaneous absorption mechanism and associated determinants should be helpful in biologic drug discovery and development.

N1-guanyl-1,7-diaminoheptane (GC7) is a potent inhibitor of deoxyhypusine synthase (DHS), the enzyme that catalyzes the first step in the hypusination of eukaryotic translation initiation factor 5A (eIF-5A). Since eIF-5A is the only known cellular substrate for DHS and GC7 has been reported to block the proliferation of CHO cells, it has been suggested that DHS may be a novel target for anti-cancer therapy. In the present study we investigated the antiproliferative effect of GC7 on several tumorigenic cell lines under various growth conditions. We found that this compound inhibits the proliferation of H9 cells in suspension culture and the growth of HeLa cells and v-src-transformed NIH3T3 cells under both anchorage-dependent and anchorage-independent conditions. Moreover, studies with NIH3T3 cells and v-src-transformed NIH3T3 cells show that GC7 inhibits the growth of both cell lines in monolayer culture with similar potency and could not reverse the transformed phenotype. In addition, the v-src-transformed cells grown under both anchorage-dependent and anchorage-independent conditions showed similar sensitivity toward GC7. These data indicate that GC7 acts as a general antiproliferative agent and does not appear to preferentially target tumorigenic cell types. Cell cycle analysis show that GC7 reduces the CHO-K1 cell population in the G1-phase of the cell cycle by 42% and increases the number of cells in the S-phase by 44%. This cell cycle distribution profile strikingly resembles the distribution of cells treated with puromycin. This result supports the hypothesis that the synthesis of a subset of proteins important for the S-phase progression of CHO-K1 cells might be dependent upon hypusinated eIF-5A. Thus the antiproliferative effect of GC7 appears to be related to its interference with the progression of cell cycle, which also provides a possible explanation for the lack of selectivity of GC7 between nontransformed and transformed cell types tested in this study.