Effect of pepstatin A on structure and polymerization of intermediate filament subunit proteins in vitro

Pepstatin A, a pentapeptide aspartyl protease inhibitor, can interact with intermediate filament (IF) subunit proteins and induce their polymerization in the absence of salt into long filaments with a rough surface and a diameter of 15-17 nm. This polymerization appears to be driven primarily by non-ionic interactions between pepstatin A and polymerization-competent forms of IF proteins, resulting in a composite filament. Proteolytic fragments of vimentin, lacking portions of only the head domain or of both the head and tail domains, failed to copolymerize with pepstatin A into long filaments under these conditions. Rather, these peptides, as well as control proteins like bovine serum albumin, were found to decorate pepstatin A polymers (filaments, ribbons, and sheets) by sticking to their surfaces. In addition to the electron microscopy experiments, UV difference spectra, ultracentrifugation, and SDS-PAGE analysis of in vitro cleavage products of vimentin obtained with HIV-1 protease all provided independent evidence for a direct association of pepstatin A with IF subunit proteins, with subsequent alterations in the IF subunit protein conformation. These data show that non-ionic interactions can substitute for the effect of salt and effectively drive the higher-order polymerization of IF subunit proteins.     

Linear non-competitive inhibition of solubilized human gamma-secretase by pepstatin A methylester,L685458, sulfonamides,and benzodiazepines

Cerebral deposition of amyloid beta-protein (A beta) is believed to play a key role in the pathogenesis of Alzheimer's disease. Because A beta is produced from the processing of amyloid beta-protein precursor (APP) by beta- and gamma-secretases, these enzymes are considered important therapeutic targets for identification of drugs to treat Alzheimer's disease. Unlike beta-secretase, which is a monomeric aspartyl protease, gamma-secretase activity resides as part of a membrane-bound, high molecular weight, macromolecular complex. Pepstatin and L685458 are among several structural classes of gamma-secretase inhibitors identified so far. These compounds possess a hydroxyethylene dipeptide isostere of aspartyl protease transition state analogs, suggesting gamma-secretase may be an aspartyl protease. However, the mechanism of inhibition of gamma-secretase by pepstatin and L685458 has not been elucidated. In this study, we report that pepstatin A methylester and L685458 unexpectedly displayed linear non-competitive inhibition of gamma-secretase. Sulfonamides and benzodiazepines, which do not resemble transition state analogs of aspartyl proteases, also displayed potent, non-competitive inhibition of gamma-secretase. Models to rationalize how transition state analogs inhibit their targets by non-competitive inhibition are discussed.     

Effects of pepstatin A on neutrophils; cross-deactivation with FMLP

The ability of pepstatin A, a protease inhibitor produced by Streptomyces testaceus, to elicit a number of responses by the human PMN has been studied. In lysozyme and beta-glucuronidase release, pepstatin A 10(-5)M is equivalent to the synthetic oligopeptide N-formyl-methionyl-leucyl-phenylalanine (FMLP) 10(-7)M. In superoxide release, pepstatin A 10(-5)M produces 80% of that originated by FMLP 10(-7). After two minutes of incubation the superoxide release is important, there being no further increase after 10 minutes. Preincubation of the cells with cytochalasin B before stimulation with pepstatin A elicits a noticeable increase in O2- release. In chemotaxis, pepstatin A 10(-6) originates the same cell motility as FMLP 10(-9). Pepstatin A produces a cross deactivation with FMLP which adds further evidence to the hypothesis that both stimuli compete for the same receptor in the PMN.     

Chemical modification studies of Rhizomucor miehei protease: evidence for the role of basic amino acids in enzyme catalysis.

The effect of chemical modification on milk clotting and proteolytic activities of aspartyl protease obtained from Rhizomucor miehei NRRL 3500 was examined in the absence and the presence of its specific inhibitor pepstatin A. The effect on the ratio of milk clotting activity (MC) to proteolytic activity (PA), an index of the quality of milk clotting proteases was also determined. Modification of the enzyme with trinitrobenzenesulfonic acid, diethylpyrocarbonate and phenylglyoxal produced an increase in the ratio of MC/PA, while modification with 2- hydroxy-5-nitrobenzyl bromide did not affect the ratio. Modification with N-acetylimidazole resulted in a marginal increase in MC/PA ratio. Protection using pepstatin A during modification with phenylglyoxal, N-acetylimidazole and 2-hydroxy-5-nitrobenzyl bromide, protected both MC and PA. In the case of modification by diethylpyrocarbonate, pepstatin A protected only MC. Pepstatin A did not protect both the activities on the modification of the enzyme by trinitrobenzene sulfonic acid. These observations indicate the presence of arginine, tyrosine and tryptophan at the catalytic site of the enzyme, for eliciting MC and PA of the enzyme. In general, modification of the positively charged residues increases the MC/PA ratio of the enzyme. In addition the modified lysine residues responsible for the inactivation of the enzyme were not involved in the active site of the enzyme. Thus the lysine residues might have a secondary role in enzyme catalysis. Further, histidine at the catalytic site was found to be exclusively involved in milk clotting activity. The enzyme with modified histidine residues were more susceptible to autocatalysis, indicating that histidine residues protect the enzyme against autolysis.     

Characterization of a serine protease activity in Sarcocystis neurona merozoites

Sarcocystis neurona merozoites were examined for their ability to invade and divide in bovine turbinate (BT) cell cultures after treatment with cysteine (iodoacetamide), aspartic (pepstatin A), metallo-(1,10-phenanthroline and ethylene glycol-bis(aminoethylether)-tetraacetic acid [EGTA]), or serine (4-[2-aminoethyl]-benzenesulfonyl fluoride hydrochloride [AEBSF], phenylmethane sulphonyl fluoride [PMSF], and tosyl lysyl chloramethyl ketone [TLCK]) protease inhibitors. Significant (P < 0.01) inhibition of serine protease activity by PMSF and TLCK led to a reduction of 86 and 78% in merozoites produced in BT cell cultures, respectively, whereas AEBSF (1 mM) led to a 68% reduction in merozoites produced in BT cell cultures and a reduction of 84 and 92% at higher AEBSF concentrations (2 and 3 mM, respectively). Pepstatin A and iodoacetamide failed to cause any inhibition in merozoite production, whereas 1,10-phenanthroline and EGTA caused slight, but not significant, inhibition at 6 and 17%, respectively. In zymograms, 2 bands of protease activity between 65- and 70-kDa molecular weight were seen. The protease activity was inhibited by AEBSF but not by E-64 (cysteine protease inhibitor), EGTA, iodoacetamide, or pepstatin A. In native zymograms, the protease activity was highest between a pH range of 8 and 10. These data suggest that merozoites of S. neurona have serine protease activity with a relative molecular weight range between 65 and 70 kDa and optimal pH range between 8 and 10, which is essential for host cell entry at least in vitro. The protease activity described here could be a potential target for chemotherapy development.     

Plasmodium falciparum: differential sensitivity in vitro to E-64 (cysteine protease inhibitor) and Pepstatin A (aspartyl protease inhibitor).

We investigated the effect of a cysteine proteinase inhibitor (E-64) and an aspartyl proteinase inhibitor (Pepstatin A) on asexual erythrocytic stages of Plasmodium falciparum in culture. These two protease inhibitors showed different patterns of activity. E-64 acted preferentially against trophozoite and schizont stages. After 48 h incubation at high concentrations of E-64 (28, 140, 280 microM), growth was totally abolished and the parasites presented characteristic enlarged food vacuoles. Morphological alterations were also seen after shorter incubation periods (6 h at 28 microM) or 12 h at the inhibitory concentration 50% (12 microM), but an additional culture period (24 h) in inhibitor-free medium allowed normal parasite development, demonstrating a parasitostatic effect. E-64 acts on parasite multiplication; the normal merozoite maturation was altered and the normal reinvasion process partially impaired. Pepstatin A used at the inhibitory concentration 50% (4 microM) killed the parasites before trophozoite development and had a major effect on schizonts maturation. No altered parasite development occurred during an additional culture period without Pepstatin A, demonstrating a parasiticidal effect. E-64 and Pepstatin A used in combination inhibit the parasite growth with a strong synergistic effect.     

Differential expression of Candida dubliniensis-secreted aspartyl proteinase genes (CdSAP1–4) under different physiological conditions and during infection of a keratinocyte culture

The in vitro and keratinocyte (HaCAT cells) culture expression of four putative genes coding for secreted aspartyl proteases of Candida dubliniensis – CdSAP1, CdSAP2, CdSAP3 , and CdSAP4 ( CdSAP1–4 ) – is reported for the first time. In addition, CdSAP7, 8, 9 , and 10 , orthologous genes of Candida albicans , were recognized in C. dubliniensis genome. There are no orthologs of C. albicans SAP5 and 6 in C. dubliniensis . The expression of CdSAP1 and 2 was independent of the morphological stage of C. dubliniensis ; they are expressed at both pH 4 and pH 7, and were induced with albumin as nitrogen source. CdSAP3 expression was regulated by the pH, and was related to the infection process of keratinocytes. Expression of CdSAP4 predominated during the mycelial phase and the initial stage of keratinocyte infection. During infection of the HaCaT cell line, only genes CdSAP3 – 4 were expressed, and keratinocytes were affected in their number and shape by the infection with C. dubliniensis ; however, this effect decreased in the presence of pepstatin A (aspartyl protease inhibitor). Pepstatin A was not able to inhibit keratinocyte damage. Based on the aforementioned, we suggest that the Saps from C. dubliniensis could be considered a virulence factor just as those from C. albicans , and participants in the nitrogen metabolism of the yeast for nutrient acquisition.     

Thiol and aspartyl proteolytic activities in secretory vesicles of bovine pituitary.

Thiol and aspartyl proteolytic activities in isolated secretory vesicles of neural (NL) and intermediate (IL) lobes of bovine pituitary were characterized with heterologous enkephalin and tachykinin precursor substrates, 35S-(Met)-preproenkephalin and 35S-(Met)-beta-preprotachykinin. IL and NL secretory vesicles contained thiol-dependent proteolytic activity that cleaved the enkephalin precursor with a pH optimum of 4.5; this activity resembled a novel "prohormone thiol protease' previously purified and characterized from adrenal medulla chromaffin granules. IL and NL vesicles also demonstrated aspartyl proteolytic activity with acidic pH optimum, as shown by pepstatin A inhibition of tachykinin and enkephalin precursor cleaving activity. This activity may be related to a previously characterized chromaffin granule aspartyl protease (CGAP) related to cathepsin D (2), as indicated by the presence of immunoreactive CGAP in NL secretory vesicles by anti-CGAP immunoblots. These results show that pituitary secretory vesicles, like chromaffin granules, may contain similar thiol-dependent and aspartyl proteolytic activities.     

Plasmodium falciparum: Differential Sensitivity In Vitro to E-64 (Cysteine Protease Inhibitor) and Pepstatin A (Aspartyl Protease Inhibitor)

ABSTRACT We investigated the effect of a cysteine proteinase inhibitor (E-64) and an aspartyl proteinase inhibitor (Pepstatin A) on asexual erythrocytic stages of Plasmodium falciparum in culture. These two protease inhibitors showed different patterns of activity. E-64 acted preferentially against trophozoite and schizont stages. After 48 h incubation at high concentrations of E-64 (28, 140, 280 μM), growth was totally abolished and the parasites presented characteristic enlarged food vacuoles. Morphological alterations were also seen after shorter incubation periods (6 h at 28 μM) or 12 h at the inhibitory concentration 50% (12 μM), but an additional culture period (24 h) in inhibitor-free medium allowed normal parasite development, demonstrating a parasitostatic effect. E-64 acts on parasite multiplication; the normal merozoite maturation was altered and the normal reinvasion process partially impaired. Pepstatin A used at the inhibitory concentration 50% (4 μM) killed the parasites before trophozoite development and had a major effect on schizonts maturation. No altered parasite development occurred during an additional culture period without Pepstatin A, demonstrating a parasiticidal effect. E-64 and Pepstatin A used in combination inhibit the parasite growth with a strong synergistic effect.     

The effect of pepstatin A, an inhibitor of the pro-opiomelanocortin (POMC)-converting enzyme, on POMC processing in mouse intermediate pituitary

In our previous studies, we have purified a unique, paired basic residue-specific, prohormone-converting enzyme from pituitary intermediate lobe secretory vesicles. This enzyme, an aspartyl protease, was shown to cleave the intermediate lobe prohormone, pro-opiomelanocortin (POMC), to adrenocorticotropin, beta-endorphin and a 16 kDa NH2-terminal glycopeptide, in vitro [(1985) J. Biol. Chem. 260, 7194-7205]. To provide some evidence that this enzyme plays a role in prohormone conversion in the intact cell, the ability of pepstatin A, an aspartyl protease inhibitor, to block POMC processing in the mouse intermediate pituitary was investigated. By the use of a radioactive pulse-chase paradigm, [3H]POMC processing was found to be inhibited by 36.4% in pepstatin A-treated intermediate lobes. This result is consistent with the inactivation of pro-opiomelanocortin-converting enzyme by pepstatin A in the intact pituitary and further supports a role of this enzyme in POMC processing in vivo.     

Identification and monitoring of protease activity in recombinant Saccharomyces cerevisiae

An assay for the detection of yeast (Saccharomyces cerevisiae) protease activity, using partially purified yeast-derived recombinant hepatitis B surface antigen (rHBsAg) as substrate, was developed to monitor proteolysis of rHBsAg that may occur through fermentation and isolation. The method consists of incubating small amounts of yeast lysate (protease source) with the substrate at 35 degrees C for about 16 h. Substrate proteolysis is assessed by subjecting the incubation mixtures to SDS-PAGE followed by silver-staining. The type of protease responsible for particular cleavages can be identified by treating the yeast lysates with specific protease inhibitors prior to incubation with substrate. The treatment of lysates with PMSF indicated that while many lysates possessed only serine protease activity (Protease B), some possessed proteolytic activity that could not be quenched with high levels of PMSF or other serine protease inhibitors. The use of the aspartyl protease inhibitor Pepstatin A in conjunction with PMSF virtually eliminated all proteolytic activity in these lysates, indicating that an aspartyl protease (Protease A) is expressed under some fermentation conditions. The relative amount of each protease in a lysate can be determined semiquantitatively by scanning the SDS gels densitometrically and plotting the ratio of degradates to intact antigen in the presence and absence of protease inhibitors. This method was used successfully to monitor the time-dependent expression of these proteases throughout production-scale fermentations. The impact of fermentation and purification changes on those proteases specifically responsible for the rHBsAg degradation can be easily evaluated.     

Synthetic HIV-2 protease cleaves the GAG precursor of HIV-1 with the same specificity as HIV-1 protease

A 99-amino acid protein having the deduced sequence of the protease from human immunodeficiency virus type 2 (HIV-2) was synthesized by the solid phase method and tested for specificity. The folded peptide catalyzes specific processing of a recombinant 43-kDa GAG precursor protein (F-16) of HIV-1. Although the protease of HIV-2 shares only 48% amino acid identity with that of HIV-1, the HIV-2 enzyme exhibits the same specificity toward the HIV-1 GAG precursor. Fragments of 34, 32, 24, 10, and 9 kDa were generated from F-16 GAG incubated with the protease. N-terminal amino acid sequence analysis of proteolytic fragments indicate that cleavage sites recognized by HIV-2 protease are identical to those of HIV-1 protease. The verified cleavage sites in F-16 GAG appear to be processed independently, as indicated by the formation of the intermediate fragments P32 and P34 in nearly equal ratios. The site nearest the amino terminus is quite conserved between the two viral GAG proteins (...VSQNY-PIVQN...in HIV-1,...KGGNY-PVQHV...in HIV-2). In contrast, the putative second site (...IPFAA-AQQKG...) of HIV-2 GAG shares minimal sequence identity with site 2 of HIV-1 GAG (...SATIM-MQRGN...). These sequence variations in the substrates suggest higher order structural features that may influence recognition by the proteases. Pepstatin A inhibits HIV-2 protease, whereas 1,10-phenanthroline and phenylmethylsulfonylfluoride do not; these results are in agreement with the finding that proteases of HIV and other retroviruses are aspartyl proteases.     

Crystal structures of Aspergillus oryzae aspartic proteinase and its complex with an inhibitor pepstatin at 1.9A resolution

The X-ray structures of Aspergillus oryzae aspartic proteinase (AOAP) and its complex with inhibitor pepstatin have been determined at 1.9A resolution. AOAP was crystallized in an orthorhombic system with the space group P2(1)2(1)2(1) and cell dimensions of a=49.4A, b=79.4A, and c=93.6A. By the soaking of pepstatin, crystals are transformed into a monoclinic system with the space group C2 and cell dimensions of a=106.8A, b=38.6A, c=78.7A, and beta=120.3 degrees. The structures of AOAP and AOAP/pepstatin complex were refined to an R-factor of 0.177 (R(free)=0.213) and of 0.185 (0.221), respectively. AOAP has a crescent-shaped structure with two lobes (N-lobe and C-lobe) and the deep active site cleft is constructed between them. At the center of the active site cleft, two Asp residues (Asp33 and Asp214) form the active dyad with a hydrogen bonding solvent molecule between them. Pepstatin binds to the active site cleft via hydrogen bonds and hydrophobic interactions with the enzyme. The structures of AOAP and AOAP/pepstatin complex including interactions between the enzyme and pepstatin are very similar to those of other structure-solved aspartic proteinases and their complexes with pepstatin. Generally, aspartic proteinases cleave a peptide bond between hydrophobic amino acid residues, but AOAP can also recognize the Lys/Arg residue as well as hydrophobic amino acid residues, leading to the activation of trypsinogen and chymotrypsinogen. The X-ray structure of AOAP/pepstatin complex and preliminary modeling show two possible sites of recognition for the positively charged groups of Lys/Arg residues around the active site of AOAP.     

Pepstatin A, an aspartic proteinase inhibitor, suppresses RANKL-induced osteoclast differentiation

Pepstatin A is well known to be an inhibitor of aspartic proteinases such as pepsin, cathepsins D and E. Except for its role as a proteinase inhibitor, however, the pharmacological action of pepstatin A upon cells remain unclear. In this study, we found that pepstatin A suppressed receptor activator of NF-kappaB ligand (RANKL)-induced osteoclast differentiation. Pepstatin A suppressed the formation of multinuclear osteoclasts dose-dependently. This inhibition of the formation only affected osteoclast cells, i.e., not osteoblast-like cells. Furthermore, pepstatin A also suppressed differentiation from pre-osteoclast cells to mononuclear osteoclast cells dose-dependently. This inhibition seems to be independent of the activities of proteinases such as cathepsin D, because the formation of osteoclasts was not suppressed with the concentration that inhibited the activity of cathepsin D. Cell signaling analysis indicated that the phosphorylation of ERK was inhibited in pepstatin A-treated cells, while the phosphorylation of IkappaB and Akt showed almost no change. Furthermore, pepstatin A decreased the expression of nuclear factor of activated T cells c1 (NFATc1). These results suggest that pepstatin A suppresses the differentiation of osteoclasts through the blockade of ERK signaling and the inhibition of NFATc1 expression.     

Receptor-mediated introduction of pepstatin-asialofetuin conjugate into lysosomes of rat hepatocytes

Pepstatin was linked through a carboxyl group to asialofetuin (PS-ASF). An analysis by separation of hepatocytes from nonparenchymal cells showed that PS-ASF was taken up by hepatocytes, following intravenous injection into rats. After the injection of PS-ASF, pepstatin concentration in the liver reached a maximum at 2 h and then decreased. In an analysis by differential centrifugation of the liver homogenate from rats injected with PS-ASF, pepstatin showed a lysosomal type subcellular distribution pattern. Isolation studies of tritosomes clearly demonstrated the exclusive accumulation of pepstatin within the lysosomes of livers from rats given PS-ASF (at 2 h after administration). Pepstatin contained in tritosomes was in a free form, as determined by column chromatography of Sephadex G-15. The activity of cathepsin D in the livers was markedly inhibited in rats given PS-ASF. However, the treatment of rats with PS-ASF had no effect on the hepatic lysosomal degradation of endocytosed FITC-labeled asialofetuin (FITC-ASF). Introduction of PS-ASF into the hepatocytes was followed by the immediate and time-dependent excretion of free pepstatin into the bile. Quantification of pepstatin excreted into the bile revealed that the biliary excretion route can account for the disappearance of pepstatin from the liver.     

Regulation of cellular alpha-MSH and beta-endorphin during stimulated secretion from intermediate pituitary cells: involvement of aspartyl and cysteine proteases in the control of cellular levels of alpha-MSH and beta-endorphin

The regulation of cellular levels of alpha-melanocyte stimulating factor (alpha-MSH) and beta-endorphin in response to stimulated secretion from intermediate pituitary cells in primary culture was investigated in this study. Regulation of the cell content of alpha-MSH and beta-endorphin occurred in two phases consisting of (a) initial depletion of cellular levels of these peptide hormones during short-term secretion (3 h) induced by isoproterenol, forskolin, or phorbol myristate acetate (PMA) which was followed by (b) long-term (24 h) increases in cellular levels of alpha-MSH and beta-endorphin in response to stimulated secretion induced by isoproterenol and PMA. In short-term experiments (3 h), cellular levels of alpha-MSH and beta-endorphin were reduced by 30-50% during stimulated secretion of these peptide hormones by isoproterenol (agonist for the beta-adrenergic receptor), forskolin that activates protein kinase A (PKA), and PMA that activates protein kinase C (PKC). Moreover, dopamine inhibited isoproterenol-induced depletion of cellular alpha-MSH and beta-endorphin. During long-term incubation of cells (24 h) with isoproterenol, cellular alpha-MSH and beta-endorphin were increased to twice that of controls (unstimulated cells). Treatment with PMA for 24 h also increased cellular levels of alpha-MSH and beta-endorphin. Moreover, cellular levels of alpha-MSH and beta-endorphin were decreased during long-term treatment of cells with an aspartyl protease inhibitor, pepstatin A, and with the cysteine protease inhibitor E64c. These results implicate aspartyl and cysteine proteases in the cellular production of alpha-MSH and beta-endorphin that requires proteolytic processing of their common precursor proopiomelanocortin (POMC). These findings demonstrate the parallel regulation of cellular levels of alpha-MSH and beta-endorphin during their cosecretion, which may involve aspartyl and cysteine proteases in the metabolism of these peptide hormones.     

Three structures associated with the nucleolus in male rat germinal cells: round body, coiled body, and "nubecula" and general presence of round body at male meiosis.

In addition to chromosomes and nucleoli, three structures, i.e., round body, coiled body, and nubecula, are encountered in the nucleus during the meiotic prophase in male rats. These structures have been examined by electron microscopy in random and serial sections. The round body is a finely fibrillar, proteinaceous structure closely associated with the granular component of a nucleolus in rat spermatocytes and young spermatids. A similar structure has been observed in man, the monkey Macaca mulatta, the gastropod Achatina fulica, and the insect Locusta migratoria. Together with evidence from the literature, these results support the view that the round body is of general occurrence in the male meiocytes of eukaryotes and may, therefore, play a role in meiosis. The coiled body is a group of electron-dense elements called "coils", which average 35 nm in width, except after mid-pachytene when their size almost doubles. The coils are composed of 2-nm-wide filaments and 8 to 10-nm-wide granules, both of which are ribonucleoprotein. The coiled bodies are interpreted to be groups of "spliceosomes", that is, structures containing heterogeneous RNA and small nuclear RNA. A remarkable feature of the coiled body is its temporary disappearance at early pachytene and its reappearance at late pachytene, possibly due to drastic changes in the turnover rate of its component RNAs. The nubecula is a newly identified nuclear inclusion, composed of weakly staining threads loosely organized into a 560 nm-wide spheroid. It has been observed only in early pachytene nuclei.     

Hypoglycosylation in the alg12delta yeast mutant destabilizes protease A and causes proteolytic loss of external invertase

The Saccharomyces cerevisiae alg12delta mutant accumulates oligosaccharide lipid with a Man(7)GlcNAc(2) oligosaccharide. To determine the N-glycan structures present on S. cerevisiae glycoproteins in the alg12delta strain, we made attempts to purify external invertase, a highly glycosylated secreted protein. These efforts revealed that, in the alg12delta background, external invertase was mildly hypoglycosylated and rapidly destroyed proteolytically. Although secreted alg9delta invertase was more severely hypoglycosylated than the alg12delta form, it was paradoxically stable during purification. The loss of periplasmic invertase was prevented by addition of pepstatin A to the cell cultures, suggesting that aspartyl proteases were active. We found that during overexpression of invertase in alg12delta yeast, sufficient protease A was mistargeted to the periplasmic space, where it hydrolyzed the invertase. Even though alg9delta invertase is underglycosylated in comparison to the alg12delta form, it is more stable because in this genetic background much less protease A is secreted compared to alg12delta cells. These observations may be relevant to studies using other extracellular proteins (e.g., mating factors, alpha-glucosidase) as probes when characterizing glycosylation defects in yeast.     

Presenilin endoproteolysis mediated by an aspartyl protease activity pharmacologically distinct from gamma-secretase

Presenilin (PS)-dependent gamma-secretase cleavage is the final proteolytic step in generating amyloid beta protein (A beta), a key peptide involved in the pathogenesis of Alzheimer's disease. PS undergoes endoproteolysis by an unidentified 'presenilinase' to generate the functional N-terminal and C-terminal fragment heterodimers (NTF/CTF) that may harbor the gamma-secretase active site. To better understand the relationship between presenilinase and gamma-secretase, we characterized the biochemical properties of presenilinase and compared them with those of gamma-secretase. Similar to gamma-secretase, presenilinase was most active at acidic pH 6.3. Aspartyl protease inhibitor pepstatin A blocked presenilinase activity with an IC50 of approximately 1 microM. Difluoroketone aspartyl protease transition state analogue MW167 was relatively selective for presenilinase (IC50 < 1 microM) over gamma-secretase (IC50-16 microM). Importantly, removing the transition state mimicking moiety simultaneously abolished both presenilinase and gamma-secretase inhibition, suggesting that presenilinase, like gamma-secretase, is an aspartyl protease. Interestingly, several of the most potent gamma-secretase inhibitors (IC50 = 0.3 or 20 nM) failed to block presenilinase activity. Although de novo generation of PS1 fragments coincided with production of A beta in vitro, blocking presenilinase activity without reducing pre-existing fragment levels permitted normal de novo generation of A beta and amyloid intracellular domain. Therefore, presenilinase has characteristics of an aspartyl protease, but this activity is distinct from gamma-secretase.     

Limited proteolysis of prolactin in secretory granules from the pituitary gland of estrogenized rats

The LTH-converting proteolytic activity in LTH granules isolated from estrogenized rat hypophysis was studied. Suspensions of granules were incubated at different values of pH for 4 hours at 37 degrees C. The reaction was controlled by SDS electrophoresis. Intensive proteolysis of LTH was observed at pH 6.0 and 3.9, which was accompanied by the formation of fragments with Mr 10, 12 and 17 kD and probably of smaller peptides. An inhibitory analysis revealed that the formation of the 17 kD fragment at pH 3.9 was partly and selectively inhibited by chloroquine, phenanthroline and phenylmethylsulfonyl fluoride. Pepstatin A fully inhibited the proteolysis, whereas leupeptin had no inhibiting influence. The data obtained testify to the presence in the granular fraction of the endopeptidase LTH-converting activity which is sensitive to pepstatin A, an aspartyl proteinase inhibitor as well as to chelators and a serine proteinase inhibitor.