Cathepsin A knockdown decreases the proliferation and invasion of A549 lung adenocarcinoma cells.

Cathepsin A (CTSA) is a lysosomal protease that is abnormally expressed in various types of cancer; however, the function of CTSA in lung adenocarcinoma (LUAD) is unknown. The aim of the present study was to investigate the role of CTSA during LUAD development in vitro. The Cancer Genome Atlas (TCGA) database was used to analyze the expression of CTSA mRNA in LUAD tissues. CTSA was significantly upregulated in LUAD tissues compared with normal lung tissues. To explore the effect of CTSA on LUAD in vitro, LUAD A549 cells were transfected with CTSA small interfering RNA and the hallmarks of tumorigenesis were investigated using cell proliferation, cell cycle, wound healing, invasion and western blot assays. Following CTSA knockdown, proliferation of LUAD cells was decreased and an increased proportion of LUAD cells were arrested at the G0/G1 phase, with altered expression of critical cell cycle and proliferative marker proteins, including p53, p21 and proliferating cell nuclear antigen. Moreover, CTSA knockdown decreased the migration and invasion of A549 cells, as determined by wound healing, invasion, and western blotting assays. The expression levels of key proteins involved in epithelial­mesenchymal transition were analyzed by western blotting. CTSA knockdown enhanced the expression of E­cadherin, but decreased the expression of N­cadherin and ß­catenin in A549 cells. To the best of our knowledge, the present study suggested for the first time it has been identified that CTSA may serve as a tumor promoter in LUAD, enhancing the malignant progression of LUAD cells by promoting cell proliferation, migration and invasion. The results suggested that CTSA may serve as a novel therapeutic target for LUAD.

Structural and kinetic evidence of aging after organophosphate inhibition of human Cathepsin A.

Human Cathepsin A (CatA) is a lysosomal serine carboxypeptidase of the renin-angiotensin system (RAS) and is structurally similar to acetylcholinesterase (AChE). CatA can remove the C-terminal amino acids of endothelin I, angiotensin I, Substance P, oxytocin, and bradykinin, and can deamidate neurokinin A. Proteomic studies identified CatA and its homologue, SCPEP1, as potential targets of organophosphates (OP). CatA could be stably inhibited by low µM to high nM concentrations of racemic sarin (GB), soman (GD), cyclosarin (GF), VX, and VR within minutes to hours at pH 7. Cyclosarin was the most potent with a kinetically measured dissociation constant (KI) of 2 µM followed by VR (KI = 2.8 µM). Bimolecular rate constants for inhibition by cyclosarin and VR were 1.3 × 103 M-1sec-1 and 1.2 × 103 M-1sec-1, respectively, and were approximately 3-orders of magnitude lower than those of human AChE indicating slower reactivity. Notably, both AChE and CatA bound diisopropylfluorophosphate (DFP) comparably and had KIDFP = 13 µM and 11 µM, respectively. At low pH, greater than 85% of the enzyme spontaneously reactivated after OP inhibition, conditions under which OP-adducts of cholinesterases irreversibly age. At pH 6.5 CatA remained stably inhibited by GB and GF and <10% of the enzyme spontaneously reactivated after 200 h. A crystal structure of DFP-inhibited CatA was determined and contained an aged adduct. Similar to AChE, CatA appears to have a "backdoor" for product release. CatA has not been shown previously to age. These results may have implications for: OP-associated inflammation; cardiovascular effects; and the dysregulation of RAS enzymes by OP.

Cathepsin A inhibition attenuates myocardial infarction-induced heart failure on the functional and proteomic levels.

BACKGROUND: Myocardial infarction (MI) is a major cause of heart failure. The carboxypeptidase cathepsin A is a novel target in the treatment of cardiac failure. We aim to show that recently developed inhibitors of the protease cathepsin A attenuate post-MI heart failure. METHODS: Mice were subjected to permanent left anterior descending artery (LAD) ligation or sham operation. 24 h post-surgery, LAD-ligated animals were treated with daily doses of the cathepsin A inhibitor SAR1 or placebo. After 4 weeks, the three groups (sham, MI-placebo, MI-SAR1) were evaluated. RESULTS: Compared to sham-operated animals, placebo-treated mice showed significantly impaired cardiac function and increased plasma BNP levels. Cathepsin A inhibition prevented the increase of plasma BNP levels and displayed a trend towards improved cardiac functionality. Proteomic profiling was performed for the three groups (sham, MI-placebo, MI-SAR1). More than 100 proteins were significantly altered in placebo-treated LAD ligation compared to the sham operation, including known markers of cardiac failure as well as extracellular/matricellular proteins. This ensemble constitutes a proteome fingerprint of myocardial infarction induced by LAD ligation in mice. Cathepsin A inhibitor treatment normalized the marked increase of the muscle stress marker CA3 as well as of Igγ 2b and fatty acid synthase. For numerous further proteins, cathepsin A inhibition partially dampened the LAD ligation-induced proteome alterations. CONCLUSIONS: Our proteomic and functional data suggest that cathepsin A inhibition has cardioprotective properties and support a beneficial effect of cathepsin A inhibition in the treatment of heart failure after myocardial infarction.

Cathepsin A mediates susceptibility to atrial tachyarrhythmia and impairment of atrial emptying function in Zucker diabetic fatty rats.

AIMS: Type 2 diabetes (T2D) is an independent risk factor for atrial fibrillation (AF) and stroke. The serine protease cathepsin A (CatA) is up-regulated in diabetes and plays an important role in the degradation of extracellular peptides. This study sought to delineate the role of CatA for the development of atrial remodelling under diabetic conditions. METHODS AND RESULTS: Zucker Diabetic Fatty rats (ZDF) were treated with vehicle (n = 20) or CatA-inhibitor (SAR; 50 mg/kg; n = 20), and compared with age-matched non-diabetic littermates (Ctr, n = 20). Left-atrial (LA) emptying function [magnetic resonance imaging (MRI)] and atrial electrophysiological parameters were measured before sacrifice for histological and biochemical analysis. The impact of enhanced cardiac CatA expression on atrial remodelling was determined using CatA-transgenic mice. At the age of 9.5 months, atrial tissues of ZDF rats showed increased CatA gene expression and CatA-activity, along with increased AF-susceptibility and impaired LA-emptying function. CatA-inhibition reduced CatA-activity in ZDF comparable to Ctr values and decreased LA-fibrosis formation and connexin 43 lateralization. This was associated with shorter median duration of LA-tachyarrhythmia (12.0 ± 1.7 vs. 1.2 ± 0.47 s, P < 0.01) induced by burst pacing and diminished regions of slow conduction. Cardiac MRI revealed better LA-emptying function parameters (active per cent emptying: 29 ± 1 vs. 23 ± 2%, P < 0.01) after CatA-inhibition. CatA-inhibition reduced LA bradykinin-degrading activity in ZDF. Transgenic mice overexpressing CatA demonstrated enhanced atrial fibrosis formation and increased AF-susceptibility. CONCLUSION: T2D leads to arrhythmogenic atrial remodelling in ZDF rats. CatA-inhibition reduces LA bradykinin-degrading activity in ZDF and suppresses the development of atrial structural changes and AF-promotion, implicating CatA as an important mediator for AF-substrate in T2D.

Tolerability, safety, and pharmacokinetics of the novel cathepsin A inhibitor SAR164653 in healthy subjects.

Cathepsin A (CathA) is a lysosomal protein where it forms a stable complex with neuraminidase and ß-galactosidase. CathA also has enzymatic activity and is involved in the degradation of many peptides. CathA was recently discovered as a target for heart failure, fostering the development of CathA inhibitors with SAR164653 as a frontrunner. The first-in-man study investigated single oral doses from 20 to 800 mg of SAR164653 followed by repeat dose studies at doses up to 800 mg in healthy young and elderly subjects. SAR164653 was safe and well tolerated at doses up to 800 mg in healthy subjects, and a maximum tolerated dose could not be determined from the study. Activity of ß-galactosidase measured in leukocytes did not show any abnormalities. The tmax was 1.0 to 2.5 hours, and the t1/2 was ∼5-11 after single dosing; exposure increased less than dose proportional. Following multiple dosing, accumulation was not observed, Cmax and AUC0-24 increased in a dose-proportional manner, and t1/2 was around 14-20 hours. The novel CathA inhibitor SAR164653 was found to have a favorable safety profile in these early phase 1 studies, but further studies are required to confirm if SAR164653 is equally safe in patients undergoing long-term treatment.

Intracellular Activation of Tenofovir Alafenamide and the Effect of Viral and Host Protease Inhibitors.

Tenofovir alafenamide fumarate (TAF) is an oral phosphonoamidate prodrug of the HIV reverse transcriptase nucleotide inhibitor tenofovir (TFV). Previous studies suggested a principal role for the lysosomal serine protease cathepsin A (CatA) in the intracellular activation of TAF. Here we further investigated the role of CatA and other human hydrolases in the metabolism of TAF. Overexpression of CatA or liver carboxylesterase 1 (Ces1) in HEK293T cells increased intracellular TAF hydrolysis 2- and 5-fold, respectively. Knockdown of CatA expression with RNA interference (RNAi) in HeLa cells reduced intracellular TAF metabolism 5-fold. Additionally, the anti-HIV activity and the rate of CatA hydrolysis showed good correlation within a large set of TFV phosphonoamidate prodrugs. The covalent hepatitis C virus (HCV) protease inhibitors (PIs) telaprevir and boceprevir potently inhibited CatA-mediated TAF activation (50% inhibitory concentration [IC50] = 0.27 and 0.16 µM, respectively) in vitro and also reduced its anti-HIV activity in primary human CD4(+) T lymphocytes (21- and 3-fold, respectively) at pharmacologically relevant concentrations. In contrast, there was no inhibition of CatA or any significant effect on anti-HIV activity of TAF observed with cobicistat, noncovalent HIV and HCV PIs, or various prescribed inhibitors of host serine proteases. Collectively, these studies confirm that CatA plays a pivotal role in the intracellular metabolism of TAF, whereas the liver esterase Ces1 likely contributes to the hepatic activation of TAF. Moreover, this work demonstrates that a wide range of viral and host PIs, with the exception of telaprevir and boceprevir, do not interfere with the antiretroviral activity of TAF.

Crystal structure of cathepsin A, a novel target for the treatment of cardiovascular diseases.

The lysosomal serine carboxypeptidase cathepsin A is involved in the breakdown of peptide hormones like endothelin and bradykinin. Recent pharmacological studies with cathepsin A inhibitors in rodents showed a remarkable reduction in cardiac hypertrophy and atrial fibrillation, making cathepsin A a promising target for the treatment of heart failure. Here we describe the crystal structures of activated cathepsin A without inhibitor and with two compounds that mimic the tetrahedral intermediate and the reaction product, respectively. The structure of activated cathepsin A turned out to be very similar to the structure of the inactive precursor. The only difference was the removal of a 40 residue activation domain, partially due to proteolytic removal of the activation peptide, and partially by an order-disorder transition of the peptides flanking the removed activation peptide. The termini of the catalytic core are held together by the Cys253-Cys303 disulfide bond, just before and after the activation domain. One of the compounds we soaked in our crystals reacted covalently with the catalytic Ser150 and formed a tetrahedral intermediate. The other compound got cleaved by the enzyme and a fragment, resembling one of the natural reaction products, was found in the active site. These studies establish cathepsin A as a classical serine proteinase with a well-defined oxyanion hole. The carboxylate group of the cleavage product is bound by a hydrogen-bonding network involving one aspartate and two glutamate side chains. This network can only form if at least half of the carboxylate groups involved are protonated, which explains the acidic pH optimum of the enzyme.

Inhibition of CatA: an emerging strategy for the treatment of heart failure.

The lysosomal serine carboxypeptidase CatA has a very important and well-known structural function as well as a, so far, less explored catalytic function. A complete loss of the CatA protein results in the lysosomal storage disease galactosialidosis caused by intralysosomal degradation of ß-galactosidase and neuraminidase 1. However, mice with a catalytically inactive CatA enzyme show no signs of this disease. This observation establishes a clear distinction between structural and catalytic functions of the CatA enzyme. Recently, several classes of orally bioavailable synthetic inhibitors of CatA have been identified. Pharmacological studies in rodents indicate a remarkable influence of CatA inhibition on cardiovascular disease progression and identify CatA as a promising novel target for the treatment of heart failure.

Luminal Ca2+ depletion during the unfolded protein response in Xenopus oocytes: cause and consequence.

The endoplasmic reticulum (ER) is a Ca(2+) storing organelle that plays a critical role in the synthesis, folding and post-translational modifications of many proteins. The ER enters into a condition of stress when the load of newly synthesized proteins exceeds its folding and processing capacity. This activates a signal transduction pathway called the unfolded protein response (UPR) that attempts to restore homeostasis. The precise role of ER Ca(2+) in the initiation of the UPR has not been defined. Specifically, it has not been established whether ER Ca(2+) dysregulation is a cause or consequence of ER stress. Here, we report that partial depletion of ER Ca(2+) stores induces a significant induction of the UPR, and leads to the retention of a normally secreted protein Carboxypeptidase Y. Moreover, inhibition of protein glycosylation by tunicamycin rapidly induced an ER Ca(2+) leak into the cytosol. However, blockade of the translocon with emetine inhibited the tunicamycin-induced Ca(2+) release. Furthermore, emetine treatment blocked elF2α phosphorylation and reduced expression of the chaperone BiP. These findings suggest that Ca(2+) may be both a cause and a consequence of ER protein misfolding. Thus, it appears that ER Ca(2+) leak is a significant co-factor for the initiation of the UPR.

Novel ß-amino acid derivatives as inhibitors of cathepsin A.

Cathepsin A (CatA) is a serine carboxypeptidase distributed between lysosomes, cell membrane, and extracellular space. Several peptide hormones including bradykinin and angiotensin I have been described as substrates. Therefore, the inhibition of CatA has the potential for beneficial effects in cardiovascular diseases. Pharmacological inhibition of CatA by the natural product ebelactone B increased renal bradykinin levels and prevented the development of salt-induced hypertension. However, so far no small molecule inhibitors of CatA with oral bioavailability have been described to allow further pharmacological profiling. In our work we identified novel ß-amino acid derivatives as inhibitors of CatA after a HTS analysis based on a project adapted fragment approach. The new inhibitors showed beneficial ADME and pharmacokinetic profiles, and their binding modes were established by X-ray crystallography. Further investigations led to the identification of a hitherto unknown pathophysiological role of CatA in cardiac hypertrophy. One of our inhibitors is currently undergoing phase I clinical trials.

Identification of interaction site of propeptide toward mature carboxypeptidase Y (mCPY) based on the similarity between propeptide and CPY inhibitor (IC).

Both the propeptide in the precursor carboxypeptidase Y (proCPY) and the mature CPY (mCPY)-specific endogenous inhibitor (I(C)) inhibit CPY activity. The N-terminal inhibitory reactive site of I(C) (the N-terminal seven amino acids of I(C)) binds to the substrate-binding site of mCPY and is essential for mCPY inhibition, but the mechanism of mCPY inhibition by the propeptide is poorly understood. In this study, sequence alignment between I(C) and proCPY indicated that a sequence similar to the N-terminal region of I(C) was present in proCPY. In particular, a region including the C-terminus of the propeptide was similar to the N-terminal seven amino acids of I(C). In the presence of peptides identical to the N-terminus of I(C) and the C-terminus of the propeptide, CPY activity was competitively inhibited. The C-terminal region of the propeptide might bind to the substrate-binding site of mCPY.

Pepstatin A alters host cell autophagic machinery and leads to a decrease in influenza A virus production.

Autophagy is a survival mechanism that can take place in cells under metabolic stress and through which cells can recycle waste material. Disturbances in autophagic processes appear to be associated with a number of human pathologies, including viral infections. It has been hypothesized that viruses can subvert autophagy in order to penetrate the host cell and replicate. Because it has been suggested that autophagy is involved in influenza A virus replication, we analyzed the effects of two inhibitors of lysosomal proteases on the cellular control of influenza A virus replication. In particular, we used biochemical and morphological analyses to evaluate the modulation of influenza A/Puerto Rico/8/34 H1N1 virus production in the presence of CA074 and Pepstatin A, inhibitors of cathepsin proteases B and D, respectively. We found that Pepstatin A, but not CA074, significantly hindered influenza virus replication, probably by modulating host cell autophagic/apoptotic responses. These results are of potential interest to provide useful insights into the molecular pathways exploited by the influenza in order to replicate and to identify further cellular factors as targets for the development of innovative antiviral strategies.

Phosphoramidates of 2'-beta-D-arabinouridine (AraU) as phosphate prodrugs; design, synthesis, in vitro activity and metabolism.

2'-Beta-D-arabinouridine (AraU), the uridine analogue of the anticancer agent AraC, was synthesized and evaluated for antiviral activity and cytotoxicity. In addition, a series of AraU monophosphate prodrugs in the form of triester phosphoramidates (ProTides) were also synthesized and tested against a range of viruses, leukaemia and solid tumour cell lines. Unfortunately, neither the parent compound (AraU) nor any of its ProTides showed antiviral activity, nor potent inhibitory activity against any of the cancer cell lines. Therefore, the metabolism of AraU phosphoramidates to release AraU monophosphate was investigated. The results showed carboxypeptidase Y, hog liver esterase and crude CEM tumor cell extracts to hydrolyse the ester motif of phosphoramidates with subsequent loss of the aryl group, while molecular modelling studies suggested that the AraU l-alanine aminoacyl phosphate derivative might not be a good substrate for the phosphoramidase enzyme Hint-1. These findings are in agreement with the observed disappearance of intact prodrug and concomitant appearance of the corresponding phosphoramidate intermediate derivative in CEM cell extracts without measurable formation of araU monophosphate. These findings may explain the poor antiviral/cytostatic potential of the prodrugs.

Prediction of the mechanism of action of omuralide (clasto-lactacystin beta-lactone) on human cathepsin A based on a structural model of the yeast proteasome beta5/PRE2-subunit/omuralide complex.

Cathepsin A (CathA) is a lysosomal serine carboxypeptidase that exhibits homology and structural similarity to the yeast and wheat serine carboxypeptidases (CPY and CPW) belonging to the alpha/beta-hydrolase fold family. Human CathA (hCathA) and CPW have been demonstrated to be inhibited by a proteasome (threonine protease) inhibitor, lactacystin, and its active derivative, omuralide (clasto-lactacystin beta-lactone), as well as chymostatin. A hCathA/omuralide complex model constructed on the basis of the X-ray crystal structures of the CPW/chymostatin complex and the yeast proteasome beta-subunit (beta5/PRE2)/omuralide one predicted that the conformation of omuralide in the active-site cleft of proteasome beta5/PRE2 should be very similar to that of chymostatin at the S1 catalytic subsites in the hCathA- and CPW-complexes. The relative positions of the glycine residues, i.e., Gly57 in hCathA, Gly53 in CPW, and Gly47 in beta5/PRE2, present in the oxyanion hole of each enzyme were also highly conserved. These results suggest that omuralide might inhibit hCathA and CPW at the S1 subsite in their active-site clefts through direct binding to the active serine residue.

Ebelactone B, an inhibitor of extracellular cathepsin A-type enzyme, suppresses platelet aggregation ex vivo in renovascular hypertensive rats.

The present study was undertaken to investigate whether ebelactone B, an inhibitor of bradykinin and angiotensin I hydrolysis by serine carboxypeptidase Y-like enzymes, could influence platelet aggregation ex vivo in renovascular hypertensive rats (2-kidney, 1-clip Goldblatt model, 2K1C). We found that ebelactone B (5 mg/kg) administrated subcutaneously once a day for 5 days, 5 weeks after the development of hypertension, or a single dose of ebelactone B (0.5 mg/kg) injected intravenously into 2K1C hypertensive rats before the induction of arterial thrombosis, both markedly suppressed collagen-induced platelet aggregation in whole blood. In contrast, inhibition of collagen-induced platelet aggregation was not evident in vitro after pretreatment of the blood with ebelactone B, indicating that ex vivo the antiaggregatory action of this compound can proceed through an indirect mechanism. The injection of ebelactone B did not affect the mean blood pressure of 2K1C hypertensive rats but lowered an elevated extracellular serine carboxypeptidase cathepsin A-like activity. Thus, the data indicate that ebelactone B may be a promising antiaggregatory agent in renovascular hypertension and suggest that 1 of the possible mechanisms through which it exerts this effect may be related to the suppression of cathepsin A-like activity released locally during the development of renovascular hypertension.

Structure of the carboxypeptidase Y inhibitor IC in complex with the cognate proteinase reveals a novel mode of the proteinase-protein inhibitor interaction.

Carboxypeptidase Y (CPY) inhibitor, IC, shows no homology to any other known proteinase inhibitors and rather belongs to the phosphatidylethanolamine-binding protein (PEBP) family. We report here on the crystal structure of the IC-CPY complex at 2.7 A resolution. The structure of IC in the complex with CPY consists of one major beta-type domain and a N-terminal helical segment. The structure of the complex contains two binding sites of IC toward CPY, the N-terminal inhibitory reactive site (the primary CPY-binding site) and the secondary CPY-binding site, which interact with the S1 substrate-binding site of CPY and the hydrophobic surface flanked by the active site of the enzyme, respectively. It was also revealed that IC had the ligand-binding site, which is conserved among PEBPs and the putative binding site of the polar head group of phospholipid. The complex structure and analyses of IC mutants for inhibitory activity and the binding to CPY demonstrate that the N-terminal inhibitory reactive site is essential both for inhibitory function and the complex formation with CPY and that the binding of IC to CPY constitutes a novel mode of the proteinase-protein inhibitor interaction. The unique binding mode of IC toward the cognate proteinase provides insights into the inhibitory mechanism of PEBPs toward serine proteinases and into the specific biological functions of IC belonging to the PEBP family as well.

Microbial serine carboxypeptidase inhibitors--comparative analysis of actions on homologous enzymes derived from man, yeast and wheat.

The actions of peptidase inhibitors derived from Streptomycete on human cathepsin A (hCath A), yeast carboxypeptidase Y (CPY), and wheat carboxypeptidase II (CPW) were analyzed comparatively. Lactacystin and omuralide (clasto-lactacystin beta-lactone), well-known cytoplasmic proteasome inhibitors, both had a potent and non-competitive inhibitory effect on these homologous serine carboxypeptidases, although they inhibited CPW and hCath A more effectively than CPY in vitro. Ebelactone B exhibited a mixed non-competitive inhibitory effect and selectivity for CPY. Piperastatin A showed competitive inhibition of CPY and hCath A but had little effect on CPW. In contrast, chymostatin inhibited CPW efficiently, while it had less effect on hCath A and CPY. In cell culture system, lactacystin was the most potent as to inactivation of the intralysosomal recombinant hCath A activity expressed in a genetically engineered fibroblastic cell line with galactosialidosis (hCath A deficiency). These results suggest that the specific inhibitory effects of lactacystin and its derivatives on hCath A might be applicable to elucidate the pathophysiological roles in the human deficinecy.

The stress-induced Tfs1p requires NatB-mediated acetylation to inhibit carboxypeptidase Y and to regulate the protein kinase A pathway.

The Saccharomyces cerevisiae N-terminal acetyltransferase NatB consists of the subunits Nat3p and Mdm20p. We found by two-dimensional PAGE analysis that nat3Delta exhibited protein expression during growth in basal medium resembling protein expression in salt-adapted wild-type cells. The stress-induced carboxypeptidase Y (CPY) inhibitor and phosphatidylethanolamine-binding protein family member Tfs1p was identified as a novel NatB substrate. The N-terminal acetylation status of Tfs1p, Act1p, and Rnr4p in both wild type and nat3Delta was confirmed by tandem mass spectrometry. Furthermore it was found that unacetylated Tfs1p expressed in nat3Delta showed an approximately 100-fold decrease in CPY inhibition compared with the acetylated form, indicating that the N-terminal acetyl group is essential for CPY inhibition by Tfs1p. Phosphatidylethanolamine-binding proteins in other organisms have been reported to be involved in the regulation of cell signaling. Here we report that a number of proteins, whose expression has been shown previously to be dependent on the activity in the protein kinase A (PKA) signaling pathway, was found to be regulated in line with low PKA activity in the nat3Delta strain. The involvement of Nat3p and Tfs1p in PKA signaling was supported by caffeine growth inhibition studies. First, growth inhibition by caffeine addition (resulting in enhanced cAMP levels) was suppressed in tfs1Delta. Second, this suppression by tfs1Delta was abolished in the nat3Delta background, indicating that Tfs1p was not functional in the nat3Delta strain possibly because of a lack of N-terminal acetylation. We conclude that the NatB-dependent acetylation of Tfs1p appears to be essential for its inhibitory activity on CPY as well its role in regulating the PKA pathway.

Overexpression and functional characterization of a serine carboxypeptidase inhibitor (I(C)) from Saccharomyces cerevisiae.

Carboxypeptidase Y (CPY) inhibitor, I(C), a cytoplasmic inhibitor of vacuolar proteinases in yeast, Saccharomyces cerevisiae, was purified by means of a high-level expression system using a proteinase-deficient strain, BJ2168, and an expression vector with the promoter GAL1. The purified I(C) exists as a monomeric beta-protein in solution with a mole-cular weight of 24,398.4 as determined by gel filtration chromatography, MALDI-TOF mass spectrometry, and far-UV CD spectroscopy. The acetylated N-terminal methionine residue is the sole posttranslational modification. I(C) specifically inhibits both the peptidase and anilidase activities of CPY with inhibitor constants (K(i)) of approximately 1.0 x 10(-9) M. The chemical modification of I(C) with sulfhydryl reagents indicated that it lacks disulfide bonds and has two free SH groups, which are responsible, not for the inhibitory function, but, apparently, for the folding of the overall structure. The formation of a complex of I(C) with CPY was highly specific, as evidenced by no detectable interaction with pro-CPY. Chemical modification studies of the CPY-I(C) complex with specific reagents demonstrated that the catalytic Ser146 and S1 substrate-binding site of CPY are covered in the complex.

Separation of cathepsin A-like enzyme and the proteasome: evidence that lactacystin/beta-lactone is not a specific inhibitor of the proteasome.

Previous studies have described a human platelet cathepsin A-like enzyme with a number of similarities to the "acidic" and "neutral" chymotrypsin-like activities of the proteasome. This includes its strong inhibition by the highly specific proteasome inhibitor Lactacystin/beta-lactone, suggesting that either the Cbz-Phe-Ala-hydrolyzing activity attributed to cathepsin A was due to the chymotrypsin-like activity of the proteasome or that lactacystin was not a specific inhibitor of the proteasome. In the present study we discard the first possibility on the basis of the following findings: (a) human platelet cathepsin A, unlike proteasome, binds to concanavalin A, and does not bind to Heparin-Sepharose at pH 7.4; (b) neither the chymotrypsin-like activity of the proteasome, nor proteasome antigens are detected in the cathepsin A preparation; (c) purified proteasome does not exhibit Cbz-Phe-Ala-hydrolyzing activity; (d) Z-lle-Glu-(Ot-Bu)Ala-leucinal (PSI), a compound that selectively inhibits the chymotrypsin-like activity of the proteasome at a concentration of 10 microM has no inhibitory effect on the carboxypeptidase activity of cathepsin A; (e) cathepsin A, free of the proteasome, is completely inhibited by micromolar concentrations of lactacystin/beta-lactone. It is therefore concluded that lactacystin/beta-lactone is not a specific inhibitor of the proteasome.