Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “PROTEASES”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 1,225 records · Page 68Linked to original sources
PubMed1989

Retrovirus protease characterized as a dimeric aspartic proteinase.

Retroviruses and retroviruslike elements have a protease for specific cleavage of their polyprotein precursors. On the basis of amino acid sequences conserved among species and the sensitivity to protease inhibitors, it was proposed that the retrovirus protease could be classified as an aspartic proteinase. Since the virus protease molecule is comparable to a single domain of aspartic proteinases having two symmetrical domains, we hypothesized and examined the dimer formation of the protease. The results of biochemical molecular mass determination and cross-linking experiments demonstrated that the virus protease molecules self-assemble into dimers. An inhibitory effect of fragmented protease molecules suggests the possibility that the intermolecular association is required for their activity. Other experiments of chemical inactivation suggest a close resemblance of the catalytic features of retrovirus and aspartic proteinases. Characterizations of these bovine and avian virus proteases would provide basic knowledge for the design of retrovirus protease-specific inhibitors, which is one of the possible strategies against human immunodeficiency virus infection.

Aspartic Acid Endopeptidases↗
PubMed1995

NS3-4A of hepatitis C virus is a chymotrypsin-like protease.

The polyprotein encoded by a single open reading frame of hepatitis C virus (HCV) is processed by host- and virus-encoded proteases. The viral protease NS3 is responsible for the cleavage of at least four sites (NS3/4A, NS4A/4B, NS4B/5A, and NS5A/5B junctions) in the nonstructural protein region. To characterize the protease function of NS3 and NS4 on various target sites, efficient cis- and trans-cleavage assay systems were developed by using in vitro transcription and translation. Deletion of the C-terminal two-thirds from NS3 in an NS3-NS4A-4B polypeptide (NS3 delta C-4A-4B) hampered cleavage of the NS3/4A junction but not that of the NS4A/4B junction. As a consequence, expression of NS3 delta C-4A-4B containing an internal deletion of NS3 results in an NS3 delta C-4A fusion protein. NS3 delta C-4A shows very efficient and specific trans-cleavage activity at NS4A/4B, NS4B/5A, and NS5A/5B junctions. In addition, the biochemical properties of HCV NS3 delta C-4A were further elucidated by adding known protease inhibitors in trans-cleavage reactions. The HCV protease NS3-4A is inhibited by chymotrypsin-specific inhibitors N-tosyl-L-phenylalanine chloromethyl ketone (TPCK), chymostatin, and Pefabloc SC but not by trypsin-like protease inhibitors antipain, leupeptin, and N-alpha-p-tosyl-L-lysine chloromethyl ketone (TLCK) or by the protease inhibitors E-64, bestatin, pepstatin, and phosphoramidon. This finding strongly suggests that HCV protease NS3-4A is a chymotrypsin-like serine protease.

Base Sequence↗
PubMed1995

Defining the level of human immunodeficiency virus type 1 (HIV-1) protease activity required for HIV-1 particle maturation and infectivity.

The human immunodeficiency virus type 1 (HIV-1) protease is the enzyme required for processing of the Gag and Gag-Pol polyproteins to yield mature, infectious virions. Although the complete absence of proteolytic activity prevents maturation, the level of activity sufficient for maturation and subsequent infectivity has not been determined. Amino acid substitutions that reduce catalytic activity without affecting substrate recognition have been engineered into the active site of the HIV-1 protease. The catalytic efficiency (kcat) of the HIV-1 protease is decreased 4-fold when threonine 26 is replaced by serine (T26S) and approximately 50-fold when alanine 28 is replaced by serine (A28S). Genes containing these mutations were cloned into a proviral vector for analysis of their effects on virion maturation and infectivity. The results show that virions containing the T26S protease variant, in which only 25% of the protease is active, are very similar to wild-type virions, although slight reductions in infectivity are observed. Virions containing the A28S protease variant are not infectious, even though a limited amount of polyprotein processing does occur. There appears to be a linear correlation between the level of protease activity and particle infectivity. Our observations suggest that a threshold of protease activity exists between a 4-fold and 50-fold reduction, below which processing is insufficient to yield infectious particles. Our data also suggest that a reduction of protease activity by 50-fold or greater is sufficient to prevent the formation of infectious particles.

Animals↗
PubMed1998

Mutagenesis of the NS3 protease of dengue virus type 2.

The flavivirus protease is composed of two viral proteins, NS2B and NS3. The amino-terminal portion of NS3 contains sequence and structural motifs characteristic of bacterial and cellular trypsin-like proteases. We have undertaken a mutational analysis of the region of NS3 which contains the catalytic serine, five putative substrate binding residues, and several residues that are highly conserved among flavivirus proteases and among all serine proteases. In all, 46 single-amino-acid substitutions were created in a cloned NS2B-NS3 cDNA fragment of dengue virus type 2, and the effect of each mutation on the extent of self-cleavage of the NS2B-NS3 precursor at the NS2B-NS3 junction was assayed in vivo. Twelve mutations almost completely or completely inhibited protease activity, 9 significantly reduced it, 14 decreased cleavage, and 11 yielded wild-type levels of activity. Substitution of alanine at ultraconserved residues abolished NS3 protease activity. Cleavage was also inhibited by substituting some residues that are conserved among flavivirus NS3 proteins. Two (Y150 and G153) of the five putative substrate binding residues could not be replaced by alanine, and only Y150 and N152 could be replaced by a conservative change. The two other putative substrate binding residues, D129 and F130, were more freely substitutable. By analogy with the trypsin model, it was proposed that D129 is located at the bottom of the substrate binding pocket so as to directly interact with the basic amino acid at the substrate cleavage site. Interestingly, we found that significant cleavage activity was displayed by mutants in which D129 was replaced by E, S, or A and that low but detectable protease activity was exhibited by mutants in which D129 was replaced by K, R, or L. Contrary to the proposed model, these results indicate that D129 is not a major determinant of substrate binding and that its interaction with the substrate, if it occurs at all, is not essential. This mutagenesis study provided us with an array of mutations that alter the cleavage efficiency of the dengue virus protease. Mutations that decrease protease activity without abolishing it are candidates for introduction into the dengue virus infectious full-length cDNA clone with the aim of creating potentially attenuated virus stocks.

Amino Acid Sequence↗
PubMed2005

Molecular models of NS3 protease variants of the Hepatitis C virus.

BACKGROUND: Hepatitis C virus (HCV) currently infects approximately three percent of the world population. In view of the lack of vaccines against HCV, there is an urgent need for an efficient treatment of the disease by an effective antiviral drug. Rational drug design has not been the primary way for discovering major therapeutics. Nevertheless, there are reports of success in the development of inhibitor using a structure-based approach. One of the possible targets for drug development against HCV is the NS3 protease variants. Based on the three-dimensional structure of these variants we expect to identify new NS3 protease inhibitors. In order to speed up the modeling process all NS3 protease variant models were generated in a Beowulf cluster. The potential of the structural bioinformatics for development of new antiviral drugs is discussed. RESULTS: The atomic coordinates of crystallographic structure 1CU1 and 1DY9 were used as starting model for modeling of the NS3 protease variant structures. The NS3 protease variant structures are composed of six subdomains, which occur in sequence along the polypeptide chain. The protease domain exhibits the dual beta-barrel fold that is common among members of the chymotrypsin serine protease family. The helicase domain contains two structurally related beta-alpha-beta subdomains and a third subdomain of seven helices and three short beta strands. The latter domain is usually referred to as the helicase alpha-helical subdomain. The rmsd value of bond lengths and bond angles, the average G-factor and Verify 3D values are presented for NS3 protease variant structures. CONCLUSIONS: This project increases the certainty that homology modeling is an useful tool in structural biology and that it can be very valuable in annotating genome sequence information and contributing to structural and functional genomics from virus. The structural models will be used to guide future efforts in the structure-based drug design of a new generation of NS3 protease variants inhibitors. All models in the database are publicly accessible via our interactive website, providing us with large amount of structural models for use in protein-ligand docking analysis.

Amino Acid Sequence↗
PubMed1998

[Protease inhibitory activities of nasal secretion on house dust mite allergen].

OBJECTIVE: To elucidate the interaction of protease inhibitors and house dust mite allergen in vitro. METHODS: Nasal secretion was collected by suction from patients with house dust mite nasal allergy, diluted immediately with PBS pH 7.0, mixed with vortex for 10 minutes, and then centrifuged at 2,000 g for 10 minutes. The supernatant was kept at -80 degrees C for use. Synthetic low molecular weight fluorogenic peptide substrates were applied to evaluate protease activities. Crude mite extract and other proteases were used, each of them was preincubated in the presence or absence of nasal fluid or other protease inhibitors at 37 degrees C for 30 minutes. The intensity of fluorescence of aminomethylcoumarin released from the cleaved substrate was measured. RESULTS: Nasal fluid exhibited mild trypsin-like and other protease activities. It inhibited the activities of trypsin and trypsin-like serine proteases and papain-like cysteine proteases in crude mite extract in a dose-dependent manner. CONCLUSIONS: Nasal fluid possibly plays a role in inhibiting penetration of allergen into the nasal mucosa by producing protease inhibitor-protease complex in the nasal mucus layer of patients with mite allergy.

Adolescent↗
PubMedDate not supplied

[Activity of ATP-dependent protease and steroid metabolism in the human placenta].

Placenta play the important role of nourishment of fetus and for the regulation of fetal and maternal steroid metabolism. Steroid metabolism is synthesized in mitochondria and levels of enzymes in steroid metabolism are regulated by the rate of synthesis and degradation of these enzymes. Therefore, we studied protease in human placental mitochondria to clarify regulation mechanism of steroid-synthesizing enzymes. 50 micrograms of human early and term placenta homogenate, proteins were analyzed by immunoblotting technique after SDS-polyacrylamid gel electrophoresis. ATP-dependent protease was detected using antiserum raised against purified bovine adrenocortical ATP-dependent protease and avidin-biotin complex method. ATP-dependent protease activity was assayed using 14C-Caseins a substrate. Five cell fractions of homogenate placenta were electrophorated and antibody-stained using ATP-dependent protease, cytochrome P-450 scc and adrenodoxin antibody after the blotting, and the quantity was analyzed by the densitometry and the method of De Douve. We had the following results: 1) ATP dependent protease is present in human placenta and localized in mitochondria. 2) ATP dependent protease decomposes adrenodoxin reductase in human placenta. 3) ATP dependent protease present in almost same consistent in early and term placenta and there is no significant difference. It is suggested as follows that the ATP dependent protease in human placenta participates in the regulation of steroid hormone through the metabolism of steroid synthetic enzyme.

ATP-Dependent Proteases↗
PubMed2005

Protease secreted by the infective larvae of angiostrongylus cantonensis and its role in the penetration of mouse intestine.

The infective third-stage larvae of Angiostrongylus cantonensis secrete a proteolytic enzyme that is thought to be essential for both larval penetration into the intestinal wall of the host and full development. Protease activity in these larvae during culture in vitro was determined by zymography, pH optimum, and substrate and inhibitor specificity. Excretory-secretory (ES) products of the third-stage larvae showed protease activity as three bands with molecular masses of 66, 30, and 23 kD by gelatin zymography. The optimal pH value for this protease activity was 10.0. The protease was found to have collagenolytic as well as elastinolytic activity, but these activities were inhibited by serine protease or metalloprotease inhibitors. The importance of this protease in larval penetration of the intestinal wall and entering the blood stream was observed in vitro by cocultured third-stage larvae of A. cantonensis with specific protease inhibitors in the intestines of BALB/c mice. The penetration rates of larvae significantly decreased when serine protease or metalloprotease inhibitors were added to the intestines. These results showed that serine protease and metalloprotease in ES products of A. cantonensis third-stage larvae are associated with larval penetration of the intestinal walls of mice.

Angiostrongylus cantonensis↗
PubMed1990

Recombinant HIV2 protease processes HIV1 Pr53gag and analogous junction peptides in vitro.

A synthetic DNA fragment encoding a protease precursor of the human immunodeficiency virus type 2 (HIV2) was cloned and expressed in bacteria and yeast. A recombinant plasmid encoding a hybrid polypeptide consisting of human superoxide dismutase and an HIV2 protease precursor of 113 amino acids was constructed for regulated intracellular expression in bacteria. Induction of this plasmid produced an autoprocessed form of the retroviral enzyme possessing the correct molecular weight. Overexpression and secretion of the protease from yeast was achieved with an expression vector encoding the yeast pheromone alpha-factor signal/leader sequence fused to a protease precursor of 115 amino acids. Amino-terminal sequence analysis confirmed that the viral enzyme exported from yeast was correctly processed from its precursor by cleavage of the predicted Ala-Pro peptide bond located at the NH2 terminus of the protease in the pol open reading frame. No additional amino acid residues were required at the COOH terminus of the protease for this autoproteolytic event. The HIV2 protease expressed in bacteria and yeast was active in an in vitro assay when tested on the HIV1 polyprotein precursor, myristylated Pr53gag. Two synthetic peptides representing junction sequences in the HIV1 gag-pol precursor were used to assay purified HIV2 protease. The enzyme exhibited a kcat/KM of 23.2 min-1 mM-1 on the HIV1 matrix-capsid junction peptide and a kcat/KM of 71.4 min-1 mM-1 on the protease-reverse transcriptase junction peptide. These rates show that the HIV2 enzyme is efficient at hydrolyzing the HIV1 peptide junctions, revealing the analogous nature of the substrate specificities of the two enzymes.

Aspartic Acid Endopeptidases↗
PubMed1986

Partial purification and characterization of serum protease from tumor-bearing rats which cleaves type IV collagen.

Activity of neutral protease was increased in sera of rats bearing ascites hepatoma AH109A compared to those of normal rats. The protease was isolated from serum protein and partially purified approximately 1,150 times in specific activity after sequential column chromatography of hemoglobin affinity, lysine-Sepharose, Ultrogel AcA34 and TSK-gel G2000SW in that order. The protease fraction still seemed to contain at least two kinds of proteases, serine and cysteine protease. It had a molecular weight of 18-21 kilodaltons with broad optimal pH range of 7.0-9.0, maximum at 8.0. Intradermal injection of the crude preparation of the neutral protease fraction induced extravascular emigration of circulating tumor cells in vivo. Moreover, partially purified protease degraded pepsin-treated chains of bovine glomerular type IV collagen in vitro, but such an in vitro action of the protease was inhibited by an addition of soybean trypsin inhibitor or mercuric chloride. It failed to cleave salt-extracted rat skin type I collagen under the same digestive conditions for bovine type IV collagen. The serum neutral proteases of tumor-bearing host may play some cooperative roles during extravascular emigration of tumor cells by destruction of vascular basement membrane.

Animals↗
PubMed1994

The encephalomyocarditis virus 3C protease is a substrate for the ubiquitin-mediated proteolytic system.

The encephalomyocarditis virus 3C protease has been shown to be rapidly degraded in infected cells and in vitro in rabbit reticulocyte lysate. The in vitro degradation, at least, is accomplished by a virus-independent, ATP-dependent proteolytic system. Here we identify this proteolytic system as the ubiquitin-mediated system. Incubation of the 3C protease in rabbit reticulocyte or cultured mouse cell lysate preparations, alone or in the presence of added ubiquitin or methylated ubiquitin, resulted in the generation of new higher molecular weight species. These new products were shown to be 3C protease-ubiquitin conjugates by their ability to bind antibodies against both the 3C protease and ubiquitin. Supplemental ubiquitin also stimulated the degradation of the 3C protease in these preparations. Large 3C protease-polyubiquitin conjugates were observed to accumulate in reticulocyte lysate in the presence of adenosine 5'-O-(3-thiotriphosphate), an inhibitor of the 26 S multicatalytic protease. This, combined with the fact that the proteolytic activity could be removed from the lysate by sedimentation, implicates the multicatalytic protease in the degradation of the 3C protease-ubiquitin conjugates. It was also found that the slow rate of degradation of a model polyprotein, which resembles the stable viral 3CD diprotein produced in vivo, is likely due to the fact that the polyprotein is a poor substrate for the ubiquitin-conjugating system.

3C Viral Proteases↗
PubMed1994

Prevention of carcinogenesis by protease inhibitors.

Protease inhibitors are very effective in their ability to suppress carcinogenesis in many different in vivo and in vitro assay systems. One particularly effective protease inhibitor, the soybean-derived Bowman-Birk inhibitor, has been extensively studied in our laboratory. Our results have indicated that Bowman-Birk inhibitor suppresses carcinogenesis 1) induced by several different types of carcinogens, 2) in three different species (mice, rats, and hamsters), 3) in several different tissues/organs [colon, liver, lung, esophagus, and cheek pouch (oral epithelium)], 4) when administered to animals by several different routes (including the diet), 5) involving several different types of tumors (squamous cell carcinomas, adenocarcinomas, angiosarcomas, etc.) and 6) in different cell types [epithelial cells (in the colon, liver, lung, esophagus, and cheek pouch) as well as connective tissue cells (fibroblasts, both in vitro and those in the liver which give rise to angiosarcomas)]. Thus, the remarkable ability of Bowman-Birk inhibitor to serve as an anticarcinogenic agent has been demonstrated in a variety of different carcinogenesis assay systems. Although the mechanism of action of protease inhibitors as anticarcinogenic agents is unknown, many hypotheses have been presented. Our results suggest that anticarcinogenic protease inhibitors are capable of reversing the initiating event in carcinogenesis, presumably by stopping an ongoing process begun by carcinogen exposure. We have observed several effects of protease inhibitors which are thought to be related to their anticarcinogenic activity; these include 1) the ability to affect the expression of certain oncogenes (e.g., c-myc and c-fos) and 2) the ability to affect the levels of certain types of proteolytic activities (e.g., N-t-butoxycarbonyl-Val-Pro-Arg-7-amino-4-methylcoumarin-hydrolyzing activity) which are elevated in carcinogen-exposed tissues. We have also observed other effects of anticarcinogenic protease inhibitors which may be related to carcinogenesis. For example, we have reported that the inhibitors can reduce the carcinogen-induced, elevated levels of gene amplification to nearly normal levels. While all of these effects of protease inhibitors may contribute to the prevention of carcinogenesis, the mechanism by which protease inhibitors prevent cancer cannot be determined with certainty until the mechanisms involved in cancer induction are known. While the mechanism remains unclear, it is clear that protease inhibitors can reverse a number of carcinogen-induced cellular changes which may play important roles in carcinogenesis.

Amino Acid Sequence↗
PubMed1993

Proteases--structures, mechanism and inhibitors.

Proteases have been divided into four mechanistic classes: serine proteases, metalloproteases, aspartic proteases, and cysteine proteases. Once a new enzyme is classified by the use of general inhibitors, it is possible to design reactive inhibitors by using mechanistic information learned through study of other members of the same protease family. The most useful types of inhibitors for serine proteases are transition-state inhibitors including alpha-ketoesters and phosphonates, and mechanism-based inhibitors such as heterocyclic isocoumarin inhibitors. Some of these inhibitors are quite specific toward individual target serine proteases. Many proteases are involved in various disease states, and potent inhibitors of these enzymes have the potential to be developed as new therapeutic agents. In the future, it is likely than numberous specific protease inhibitors will be tested clinically for the treatment of human disease.

Amino Acid Sequence↗
PubMed1998

Isolation and characterization of the phage T4 PinA protein, an inhibitor of the ATP-dependent lon protease of Escherichia coli.

The bacteriophage T4 PinA protein, expression of which leads to inhibition of protein degradation in Escherichia coli cells, has been purified from cells carrying multiple copies of the pinA gene. PinA is a heat-stable protein with a subunit Mr of 18,800 and an isoelectric point of 4.6. Under nondenaturing conditions on a gel filtration column, PinA migrated in two peaks corresponding to a dimer and a tetramer. Purified PinA inhibited ATP-dependent protein degradation by Lon protease in vitro; it did not inhibit the activity of other E. coli ATP-dependent proteases, ClpAP or ClpYQ. Furthermore, PinA did not inhibit ATP-independent proteolysis in E. coli cell extracts. PinA binds with high affinity to Lon protease (Kd approximately 10 nM for dimer binding), and a complex with approximately 1 dimer of PinA per tetramer of Lon protease could be isolated by gel filtration. Lon activity was partially restored upon dilution of the PinA-Lon complex to subnanomolar concentrations, indicating that inhibition was reversible and that PinA did not covalently modify Lon protease. PinA was not cleaved by Lon protease, and heating the Lon-PinA complex at 65 degrees C denatured Lon protease and released active PinA. The properties of PinA in vitro suggest that PinA inhibits protein degradation in vivo by forming a tight, reversible complex with Lon protease.

ATP-Dependent Proteases↗
PubMed2006

Inhibition of viral proteases by Zingiberaceae extracts and flavones isolated from Kaempferia parviflora.

In order to identify novel lead compounds with antiviral effect, methanol and aqueous extracts of eight medicinal plants in the Zingiberaceae family were screened for inhibition of proteases from human immunodeficiency virus type 1 (HIV-1), hepatitis C virus (HCV) and human cytomegalovirus (HCMV). In general, the methanol extracts inhibited the enzymes more effectively than the aqueous extracts. HIV-1 protease was strongly inhibited by the methanol extract of Alpinia galanga. This extract also inhibited HCV and HCMV proteases, but to a lower degree. HCV protease was most efficiently inhibited by the extracts from Zingiber officinale, with little difference between the aqueous and the methanol extracts. Many of the methanol extracts inhibited HCMV protease, but the aqueous extracts showed weak inhibition. In a first endeavor to identify the active constituents, eight flavones were isolated from the black rhizomes of Kaempferia parviflora. The most effective inhibitors, 5-hydroxy-7-methoxyflavone and 5,7-dimethoxyflavone, inhibited HIV-1 protease with IC50 values of 19 microM. Moreover, 5-hydroxy-3,7-dimethoxyflavone inhibited HCV protease and HCMV protease with IC50 values of 190 and 250 microM, respectively.

Cytomegalovirus↗
PubMed2004

Differential expression of neuropsin and protease M/neurosin in oligodendrocytes after injury to the spinal cord.

Neuropsin and protease M/neurosin are serine proteases expressed by neurons and glial cells, and serve a variety of functions in the central nervous system (CNS). The current study demonstrates changes in the expression of these proteases following hemisection of the mouse spinal cord. Within unlesioned spinal cord, neuropsin mRNA expression was occasionally observed in the gray but not white matter, while the level of protease M/neurosin mRNA was higher in the white matter. After injury to the spinal cord, neuropsin mRNA expression was induced in the white matter in the area immediately adjacent to the lesion, peaking at 4 days post-injury and disappearing by 14 days. Enhanced expression of protease M/neurosin mRNA was observed throughout the white and gray matter surrounding the lesion, peaking at 4 days and persisting for 14 days. Neuropsin mRNA was expressed predominantly by CNPase-positive oligodendrocytes. Furthermore, most of these cells were also associated with immunoreactivity for protease M/neurosin protein. Within unlesioned spinal cord, most protease M/neurosin mRNA-expressing cells were CNPase-positive oligodendrocytes, and a substantial fraction of these cells also showed immunoreactivity for NG2, a marker for oligodendrocyte progenitors. After injury, protease M/neurosin mRNA expression within NG2-positive cells was significantly decreased, while the constitutive expression in CNPase-positive oligodendrocytes appeared to be preserved. These findings suggest that each subpopulation of oligodendrocytes based on the expression of neuropsin and protease M/neurosin has different roles in the response of the spinal cord to injury as well as in normal homeostasis.

2',3'-Cyclic-Nucleotide Phosphodiesterases↗
PubMed1997

Processing/activation of CPP32-like proteases is involved in transforming growth factor beta1-induced apoptosis in rat hepatocytes.

Apoptosis induced in rat hepatocytes by transforming growth factor beta1 (TGF-beta1) was accompanied by the activation of interleukin-1beta converting enzyme (ICE)-like proteases. Cell lysates were isolated at various times after TGF-beta1 treatment and analyzed for ICE and CPP32-like activity, using N-acetyl-Tyr-Val-Ala-Asp-7-amino-4-methylcoumarin (Ac-YVAD.AMC) and benzyloxycarbonyl-Asp-Glu-Val-Asp-7-amino-4-trifluoromethylcoumarin (Z-DEVD.AFC), respectively. CPP32-like but not ICE protease activity increased in a time dependent manner and preceded the onset of apoptosis. Kinetic studies in cell lysates indicated that more than one CPP32-like protease was being activated. This was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)/Western blotting of TGF-beta1-treated cells, which showed limited processing of CPP32 as shown by the appearance of the catalytically active p17 subunit. Loss of pro-Mch3alpha was also observed but the catalytically active p19 subunit was not detected. Staurosporine, which induced a much greater level of hepatocyte apoptosis, produced a concomitant increase in CPP32/Mch3alpha processing as shown by the appearance of the p17/p19 subunits and the corresponding increase in CPP32-like protease activity. Apoptosis, CPP32/Mch3alpha processing and the increase in CPP32-like protease activity induced by TGF-beta1 and staurosporine were abolished in hepatocytes pretreated with Z-Asp-Glu-Val-Asp (OMe) fluoromethylketone (Z-DEVD.FMK) or Z-Val-Ala-Asp (OMe) fluoromethylketone (Z-VAD.FMK). These peptide analogues were potent inhibitors of CPP32-like protease activity in lysates. Pretreatment of hepatocytes with cycloheximide also blocked TGF-beta1-induced apoptosis and the increase in CPP32-like activity. Unlike Z-VAD.FMK and Z-DEVD.FMK, cycloheximide did not inhibit CPP32-like protease activity in cell lysates. Thus, cycloheximide may block apoptosis by inhibiting the synthesis of a protein, which is involved in the upstream events responsible for the activation of the CPP32-like protease activity. Our studies have identified two of the CPP32-like proteases, namely CPP32 and Mch3alpha, which are activated during the execution phase of hepatocyte apoptosis.

Animals↗
PubMed1996

Calpain inhibitors and serine protease inhibitors can produce apoptosis in HL-60 cells.

Recent investigations indicate that proteolysis is an important event in generation of the apoptosis phenotype. Although various proteases have been suggested to be candidates for this proteolysis, the results from different laboratories are inconsistent. In the present studies, HL-60 cells were treated with cycloheximide to investigate proteases involved in apoptosis. The calpain inhibitors benzyloxycarbonyl-Leu-Leu-Tyr diazomethylketone and acetyl-Leu-Leu-Nle aldehyde were not capable of preventing apoptosis induced by cycloheximide. In the absence of cycloheximide, these two inhibitors could initiate apoptosis in HL-60 cells. The thiol protease inhibitor benzyloxycarbonyl-Leu-Val-Gly diazomethylketone neither prevented nor produced apoptosis. The serine protease inhibitors 3,4-dichloroisocoumarin (DCI) and tosyl-Phe chloromethylketone (TPCK) also induced apoptosis in the absence of cycloheximide. On the other hand, the latter two inhibitors decreased cycloheximide-induced apoptosis, assessed either by cell morphologic changes or DNA ladder generation. Benzyloxycarbonyl-Val-Ala-Asp fluoromethyl ketone and iodoacetamide, inactivators of interleukin 1beta-converting enzyme (ICE)-like proteases, did not produce apoptosis and inhibited the induction of apoptosis by cycloheximide, calpain inhibitors, or serine protease inhibitors. These results are consistent with the ICE-like proteases having a central role in proteolysis during apoptosis, while calpain-like proteases and the serine proteases sensitive to DCI or TPCK are not required for generation of the apoptosis phenotype in HL-60 cells.

Apoptosis↗