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PubMedDate not supplied

Kynostatin and 17beta-estradiol prevent the apoptotic death of human neuroblastoma cells exposed to HIV-1 protease.

A significant number of adult male patients with acquired immunodeficiency syndrome develop cerebral atrophy and progressive brain disorders such as dementia complex and neuropsychiatric problems. Upon entering the brain via activated macrophages or microglias, the human immunodeficiency type 1 virus (HIV-1) may produce cytotoxic factors such as HIV-1 envelope protein (gp120) and protease. Owing to significant proteolysis of nonviral proteins, the protease derived from HIV-1 may be detrimental to brain cells and neurons. Our results revealed that HIV-1 protease, at nanomolar concentrations, was as potent as gp120 in causing neurotoxicity in human neuroblastoma neurotypic SH-SY5Y cells. As shown by the Oncor ApopTag staining procedure, HIV-1 protease significantly increased the number of apoptotic cells over the serum-free controls. Moreover, HIV-1 protease-induced neurotoxicity was blocked by a selective protease inhibitor, kynostatin (KNI-272). Antioxidants such as 17beta-estradiol, melatonin, and S-nitrosoglutathione also prevented protease-induced neurotoxicity. These findings indicate that oxidative proteolysis may mediate HIV-1 protease-induced apoptosis and the degeneration of neurons and other brain cells. Centrally active protease inhibitors and antioxidants may play an important role in preventing cerebral atrophy and associated dementia complex caused by HIV-1.

Antioxidants↗
PubMed1998

A rapid and sensitive bacterial assay to determine the inhibitory effect of 'interface' peptides on HIV-1 protease co-expressed in Escherichia coli.

The HIV-1 protease is essential for maturation of virus particles and is, therefore, an attractive target for antiviral drugs. The function of this protease depends on the dimerization of two identical subunits. Commonly used protease inhibitors are directed mainly against the active site of the enzyme which often leads to viral resistance. To determine the inhibitory effect of peptides interfering with the dimerization site of the HIV-1 protease, a recombinant bacterial screening assay was established. Escherichia coli was co-transformed with two different plasmids, expressing the 'interface' peptide and an active HIV-1 protease toxic for the bacteria. Co-expression of inhibitory peptides overcomes the incomplete membrane transmission of supplemented inhibitors and leads to a direct interaction of the inhibitory peptide and the HIV-1 protease. The inhibitory effect of co-expressed peptides was measured by an increased growth of co-transformed bacteria, compared with a slowly growing E. coli control culture only expressing the HIV-1 protease. Using this assay several penta- and hexa-peptides were screened for their ability to inhibit HIV-1 protease activity. One of these peptides showed a significant inhibitory effect on co-expressed recombinant HIV-1 protease.

Amino Acid Sequence↗
PubMed2000

Probing the S1/S1' substrate binding pocket geometry of HIV-1 protease with modified aspartic acid analogues.

Aspartates 25 and 125, the active site residues of HIV-1 protease, participate functionally in proteolysis by what is believed to be a general acid-general base mechanism. However, the structural role that these residues may play in the formation and maintenance of the neighboring S1/S1' substrate binding pockets remains largely unstudied. Because the active site aspartic acids are essential for catalysis, alteration of these residues to any other naturally occurring amino acid by conventional site-directed mutagenesis renders the protease inactive, and hence impossible to characterize functionally. To investigate whether Asp-25 and Asp-125 may also play a structural role that influences substrate processing, a series of active site protease mutants has been produced in a cell-free protein synthesizing system via readthrough of mRNA nonsense (UAG) codons by chemically misacylated suppressor tRNAs. The suppressor tRNAs were activated with the unnatural aspartic acid analogues erythro-beta-methylaspartic acid, threo-beta-methylaspartic acid, or beta,beta-dimethylaspartic acid. On the basis of the specific activity measurements of the mutants that were produced, the introduction of the beta-methyl moiety was found to alter protease function to varying extents depending upon its orientation. While a beta-methyl group in the erythro orientation was the least deleterious to the specific activity of the protease, a beta-methyl group in the threo orientation, present in the modified proteins containing threo-beta-methylaspartate and beta,beta-dimethylaspartate, resulted in specific activities between 0 and 45% of that of the wild type depending upon the substrate and the substituted active site position. Titration studies of pH versus specific activity and inactivation studies, using an aspartyl protease specific suicide inhibitor, demonstrated that the mutant proteases maintained bell-shaped pH profiles, as well as suicide-inhibitor susceptibilities that are characteristic of aspartyl proteases. A molecular dynamics simulation of the beta-substituted aspartates in position 25 of HIV-1 protease indicated that the threo-beta-methyl moiety may partially obstruct the adjacent S1' binding pocket, and also cause reorganization within the pocket, especially with regard to residues Val-82 and Ile-84. This finding, in conjunction with the biochemical studies, suggests that the active site aspartate residues are in proximity to the S1/S1' binding pocket and may be spatially influenced by the residues presented in these pockets upon substrate binding. It thus seems possible that the catalytic residues cooperatively interact with the residues that constitute the S1/S1' binding pockets and can be repositioned during substrate binding to orient the active site carboxylates with respect to the scissile amide bond, a process that likely affects the facility of proteolysis.

Aspartic Acid↗
PubMed1993

A symmetric inhibitor binds HIV-1 protease asymmetrically.

Potential advantages of C2-symmetric inhibitors designed for the symmetric HIV-1 protease include high selectivity, potency, stability, and bioavailability. Pseudo-C2-symmetric monools and C2-symmetric diols, containing central hydroxymethylene and (R,R)-dihydroxyethylene moieties flanked by a variety of hydrophobic P1/P1' side chains, were studied as HIV-1 protease inhibitors. The monools and diols were synthesized in 8-10 steps from D-(+)-arabitol and D-(+)-mannitol, respectively. Monools with ethyl or isobutyl P1/P1' side chains were weak inhibitors of recombinant HIV-1 protease (Ki > 10 microM), while benzyl P1/P1' side chains afforded a moderately potent inhibitor (apparent Ki = 230 nM). Diols were 100-10,000x more potent than analogous monools, and a wider range of P1/P1' side chains led to potent inhibition. Both classes of compounds exhibited lower apparent Ki values under high-salt conditions. Surprisingly, monool and diol HIV-1 protease inhibitors were potent inhibitors of porcine pepsin, a prototypical asymmetric monomeric aspartic protease. These results were evaluated in the context of the pseudosymmetric structure of monomeric aspartic proteases and their evolutionary kinship with the retroviral proteases. The X-ray crystal structure of HIV-1 protease complexed with a symmetric diol was determined at 2.6 A. Contrary to expectations, the diol binds the protease asymmetrically and exhibits 2-fold disorder in the electron density map. Molecular dynamics simulations were conducted beginning with asymmetric and symmetric HIV-1 protease/inhibitor model complexes. A more stable trajectory resulted from the asymmetric complex, in agreement with the observed asymmetric binding mode. A simple four-point model was used to argue more generally that van der Waals and electrostatic force fields can commonly lead to an asymmetric association between symmetric molecules.

Alcohols↗
PubMed1994

The HIV-1 protease as enzyme and substrate: mutagenesis of autolysis sites and generation of a stable mutant with retained kinetic properties.

Site-directed mutagenesis of autolysis sites in the human immunodeficiency virus type 1 (HIV-1) protease was applied in an analysis of enzyme specificity; the protease served, therefore, as both enzyme and substrate in this study. Inspection of natural substrates of all retroviral proteases revealed the absence of beta-branched amino acids at the P1 site and of Lys anywhere from P2 through P2'. Accordingly, several mutants of the HIV-1 protease were engineered in which these excluded amino acids were substituted at their respective P positions at the three major sites of autolysis in the wild-type protease (Leu5-Trp6, Leu33-Glu34, and Leu63-Ile64), and the mutant enzymes were evaluated in terms of their resistance to autodegradation. All of the mutant HIV-1 proteases, expressed as inclusion bodies in Escherichia coli, were enzymatically active after refolding, and all showed greatly diminished rates of cleavage at the altered autolysis sites. Some, however, were not viable enzymatically because of poor physical characteristics. This was the case for mutants having Lys replacements of Glu residues at P2' and for another in which all three P1 leucines were replaced by Ile. However, one of the mutant proteases, Q7K/L33I/L63I, was highly resistant to autolysis, while retaining the physical properties, specificity, and susceptibility to inhibition of the wild-type enzyme. Q7K/L33I/L63I should find useful application as a stable surrogate of the HIV-1 protease. Overall, our results can be interpreted relative to a model in which the active HIV-1 protease dimer is in equilibrium with monomeric, disordered species which serve as the substrates for autolysis.

Amino Acid Sequence↗
PubMed2002

Amplification of the effects of drug resistance mutations by background polymorphisms in HIV-1 protease from African subtypes.

The vast majority of HIV-1 infections worldwide are caused by the C and A viral subtypes rather than the B subtype prevalent in the United States and Western Europe. Genomic differences between subtypes give rise to sequence variations in the encoded proteins, including those identified as targets for antiretroviral therapies. In the case of the HIV-1 protease, we reported earlier [Velazquez-Campoy et al. (2001) Proc. Natl. Acad. Sci. U.S.A. 98, 6062-6067] that proteases from the C and A subtypes exhibit a higher biochemical fitness in the presence of widely prescribed protease inhibitors. In this paper we present a complete thermodynamic dissection of the differences between proteases from different subtypes and the effects of the V82F/I84V drug-resistant mutation within the framework of the B, C, and A subtypes. These studies involved four inhibitors in clinical use (indinavir, saquinavir, ritonavir, and nelfinavir) and a second-generation protease inhibitor (KNI-764). Naturally occurring amino acid polymorphisms found in proteases from the C and A subtypes lower the binding affinities of existing clinical inhibitors by factors ranging between 2 and 7.5 which by themselves are not enough to cause drug resistance. The preexisting lower affinity in the C and A subtypes, however, significantly amplifies the effects of the drug-resistant mutation. Relative to the wild-type B subtype protease, the V82F/I84V drug-resistant mutation within the C and A subtypes lowers the binding affinity of inhibitors by factors ranging between 40 and 3000. When the enzyme kinetic properties (k(cat) and K(m)) are included in the analysis, the biochemical fitness of the C and A subtype drug-resistant mutants can be up to 1000-fold higher than that of the wild-type B subtype protease in the presence of the studied inhibitors. From a thermodynamic standpoint, the combined effects of the drug-resistant mutations and the natural amino acid polymorphisms on the Gibbs energy are additive and involve significant alterations in the enthalpy and entropy changes associated with inhibitor binding. At the biochemical level, the combined effects of naturally existing polymorphisms and drug-resistant mutations might have important consequences on the long-term viability of current HIV-1 protease inhibitors.

Africa↗
PubMed1997

Kinetic properties of saquinavir-resistant mutants of human immunodeficiency virus type 1 protease and their implications in drug resistance in vivo.

In order to study the basis of resistance of human immunodeficiency virus, type 1 (HIV-1), to HIV-1 protease inhibitor saquinavir, the catalytic and inhibition properties of the wild-type HIV-1 protease and three saquinavir resistant mutants, G48V, L90M, and G48V/L90M, were compared. The kinetic parameter kcat/Km was determined for these proteases using eight peptide substrates whose sequences were derived from the natural processing site sequences of HIV-1. The kcat/Km values were determined using conventional steady-state kinetics as well as initial velocities of mixed substrate cleavages under the condition where the substrate concentrations [S]o << Km. The independently determined kcat and Km values for some of the substrates confirmed the accuracy of the mixed-substrate method and also permitted the calculation in all cases of true rather than relative kcat/Km values. The Ki values were also determined. Using a previously described kinetic model [Tang, J., & Hartsuck, J. A. (1995) FEBS Lett. 367, 112-116], the relative processing activities of HIV-1 protease variants were estimated in the saquinavir concentration range of 0-10(-7) M. Although the protease activity of G48V, L90M, and G48V/L90M are only about 10, 7, and 3% of that of the wild-type HIV-1 protease in the absence of inhibitor, the resistance tendencies of the three mutants are clearly manifest by relatively less activity loss as inhibitor concentration becomes higher. Also, the ratios of the activities of the four protease species at certain saquinavir concentrations appear to correlate with the population ratios of the four protease species at different time points of clinical trials. This correlation suggests that the population ratio of the protease species is driven by in vivo saquinavir concentration, which appears to be in the range 10(-10)-10(-9) M during the clinical trials.

Dose-Response Relationship, Drug↗
PubMed1998

Evidence for a novel ATP-dependent protease from the rat liver mitochondrial intermembrane space: purification and characterisation.

An ATP-dependent protease in the intermembrane space of rat liver mitochondria, MISP I (mitochondrial intermembrane space protease), was partially purified and characterised. The protease complex has a molecular mass of 200 kDa and appears to be an oligomeric enzyme complex. The proteolytic activity of the enzyme can be stimulated up to 3-fold by Mg2+ATP. The Km for ATP is 200 microM. Nucleoside triphosphates, but not ADP, AMP, or nonhydrolysable ATP analogues, can substitute for ATP. The protease exhibits multicatalytic properties with chymotrypsin-like, peptidyl-glutamyl-hydrolysing, and trypsin-like activities. Of the latter the trypsin-like activity is not enhanced by ATP. In addition to the hydrolysis of fluorogenic peptide substrates the protease is able to degrade radiolabeled model proteins. The ATP-dependent mitochondrial protease was characterised as a cysteine protease sensitive to hemine. The cross reactivity of an anti-human-S4 antibody raised against an ATPase subunit of the PA700 complex with a component of MISP I indicated a structural relationship. Furthermore, ATP-agarose-binding assays revealed the connection of the peptide hydrolysing activity with an ATP binding domain. The data presented here and a comparison with known ATP-dependent mitochondrial proteases demonstrated that MISP I represents a novel ATP-dependent protease in the mitochondrial intermembrane space of rat liver.

ATP-Dependent Proteases↗
PubMed1995

Cross-resistance analysis of human immunodeficiency virus type 1 variants individually selected for resistance to five different protease inhibitors.

Human immunodeficiency virus type 1 (HIV-1) protease inhibitor-resistant variants, isolated on passage of HIV-1HXB2 in MT-4 cells with five different protease inhibitors, have been examined for cross-resistance to five inhibitors. The protease inhibitors studied were Ro 31-8959, A-77003, XM323, L-735,524, and VX-478. Resistant variants with two to four mutations within their protease sequence and 9- to 40-fold-decreased susceptibility were selected for all five inhibitors within six to eight passes in cell culture. Passage of a zidovudine-resistant mutant in Ro 31-8959 generated a dual reverse transcriptase- and protease-resistant virus. Variants were cloned directly into a modified pHXB2-D infectious clone for cross-resistance analysis. Although the resistant variants selected possessed different combinations of protease mutations for each inhibitor, many showed cross-resistance to the other inhibitors, and one showed cross-resistance to all five inhibitors. Interestingly, some mutants showed increased susceptibility to some inhibitors. Further HIV passage studies in the combined presence of two protease inhibitors demonstrated that in vitro it was possible to delay significantly selection of mutations producing resistance to one or both inhibitors. These studies indicate that there may be some rationale for combining different protease inhibitors as well as protease and reverse transcriptase inhibitors in HIV combination therapy.

Amino Acid Sequence↗
PubMed2005

Molecular basis for increased susceptibility of isolates with atazanavir resistance-conferring substitution I50L to other protease inhibitors.

Protease inhibitors (PIs) are highly effective drugs against the human immunodeficiency virus (HIV), yet long-term therapeutic use is limited by emergence of HIV type 1 (HIV-1) protease substitutions that confer cross-resistance to multiple protease inhibitor drugs. Atazanavir is a highly potent HIV protease inhibitor with a distinct resistance profile that includes effectiveness against most HIV-1 isolates resistant to one or two PIs. The signature resistance substitution for atazanavir is I50L, and it is frequently (53%) accompanied by a compensatory A71V substitution that helps restore viability and increases atazanavir resistance levels. We measured the binding affinities of wild-type (WT) and I50L/A71V HIV-1 proteases to atazanavir and other currently approved PIs (ritonavir, lopinavir, saquinavir, nelfinavir, indinavir, and amprenavir) by isothermal titration calorimetry. Remarkably, we find that all of the PIs have 2- to 10-fold increased affinities for I50L/A71V protease, except for atazanavir. The results are also manifested by thermal stability measures of affinity for WT and I50L/A71V proteases. Additional biophysical and enzyme kinetics experiments show I50L/A71V protease is a stable enzyme with catalytic activity that is slightly reduced (34%) relative to the WT. Computational modeling reveals that the unique resistance phenotype of I50L/A71V protease likely originates from bulky tert-butyl groups at P2 and P2' (specific to atazanavir) that sterically clash with methyl groups on residue L50. The results of this study provide a molecular understanding of the novel hypersusceptibility of atazanavir-resistant I50L/A71V-containing clinical isolates to other currently approved PIs.

Amino Acid Substitution↗
PubMed2000

Sensitive genetic screen for protease activity based on a cyclic AMP signaling cascade in Escherichia coli.

We describe a genetic system that allows in vivo screening or selection of site-specific proteases and of their cognate-specific inhibitors in Escherichia coli. This genetic test is based on the specific proteolysis of a signaling enzyme, the adenylate cyclase (AC) of Bordetella pertussis. As a model system we used the human immunodeficiency virus (HIV) protease. When an HIV protease processing site, p5, was inserted in frame into the AC polypeptide, the resulting ACp5 protein retained enzymatic activity and, when expressed in an E. coli cya strain, restored the Cya(+) phenotype. The HIV protease coexpressed in the same cells resulted in cleavage and inactivation of ACp5; the cells became Cya(-). When the entire HIV protease, including its adjacent processing sites, was inserted into the AC polypeptide, the resulting AC-HIV-Pr fusion protein, expressed in E. coli cya, was autoproteolysed and inactivated: the cells displayed Cya(-) phenotype. In the presence of the protease inhibitor indinavir or saquinavir, AC-HIV-Pr autoproteolysis was inhibited and the AC activity of the fusion protein was preserved; the cells were Cya(+). Protease variants resistant to particular inhibitors could be easily distinguished from the wild type, as the cells displayed a Cya(-) phenotype in the presence of these inhibitors. This genetic test could represent a powerful approach to screen for new proteolytic activities and for novel protease inhibitors. It could also be used to detect in patients undergoing highly active antiretroviral therapy the emergence of HIV variants harboring antiprotease-resistant proteases.

Adenylyl Cyclases↗
PubMed2006

Role of invariant Thr80 in human immunodeficiency virus type 1 protease structure, function, and viral infectivity.

Sequence variability associated with human immunodeficiency virus type 1 (HIV-1) is useful for inferring structural and/or functional constraints at specific residues within the viral protease. Positions that are invariant even in the presence of drug selection define critically important residues for protease function. While the importance of conserved active-site residues is easily understood, the role of other invariant residues is not. This work focuses on invariant Thr80 at the apex of the P1 loop of HIV-1, HIV-2, and simian immunodeficiency virus protease. In a previous study, we postulated, on the basis of a molecular dynamics simulation of the unliganded protease, that Thr80 may play a role in the mobility of the flaps of protease. In the present study, both experimental and computational methods were used to study the role of Thr80 in HIV protease. Three protease variants (T80V, T80N, and T80S) were examined for changes in structure, dynamics, enzymatic activity, affinity for protease inhibitors, and viral infectivity. While all three variants were structurally similar to the wild type, only T80S was functionally similar. Both T80V and T80N had decreased the affinity for saquinavir. T80V significantly decreased the ability of the enzyme to cleave a peptide substrate but maintained infectivity, while T80N abolished both activity and viral infectivity. Additionally, T80N decreased the conformational flexibility of the flap region, as observed by simulations of molecular dynamics. Taken together, these data indicate that HIV-1 protease functions best when residue 80 is a small polar residue and that mutations to other amino acids significantly impair enzyme function, possibly by affecting the flexibility of the flap domain.

Amino Acid Substitution↗
PubMed2006

HIV-2 Protease resistance defined in yeast cells.

BACKGROUND: Inhibitors of the HIV-1 Protease currently used in therapeutic protocols, have been found to inhibit, although at higher concentrations, the HIV-2 encoded enzyme homologue. Similar to observations in HIV-1 infected individuals, therapeutic failure has also been observed for some patients infected with HIV-2 as a consequence of the emergence of viral strains resistant to the anti-retroviral molecules. In order to be able to define the specific mutations in the Protease that confer loss of susceptibility to Protease Inhibitors, we set up an experimental model system based in the expression of the viral protein in yeast. RESULTS: Our results show that the HIV-2 Protease activity kills the yeast cell, and this process can be abolished by inhibiting the viral enzyme activity. Since this inhibition is dose dependent, IC50 values can be assessed for each anti-retroviral molecule tested. We then defined the susceptibility of HIV-2 Proteases to Protease Inhibitors by comparing the IC50 values of Proteases from 7 infected individuals to those of a sensitive wild type laboratory adapted strain. CONCLUSION: This functional assay allowed us to show for the first time that the L90M substitution, present in a primary HIV-2 isolate, modifies the HIV-2 Protease susceptibility to Saquinavir but not Lopinavir. Developing a strategy based on the proposed yeast expressing system will contribute to define amino acid substitutions conferring HIV-2 Protease resistance.

Amino Acid Sequence↗
PubMed1987

Degradation in vitro of bacteriophage lambda N protein by Lon protease from Escherichia coli.

Lon protease from Escherichia coli degraded lambda N protein in a reaction mixture consisting of the two homogeneous proteins, ATP, and MgCl2 in 50 mM Tris, Ph 8.0. Genetic and biochemical data had previously indicated that N protein is a substrate for Lon protease in vivo (Gottesman, S., Gottesman, M., Shaw, J. E., and Pearson, M. L. (1981) Cell 24, 225-233). Under conditions used for N protein degradation, several lambda and E. coli proteins, including native proteins, oxidatively modified proteins, and cloned fragments of native proteins, were not degraded by Lon protease. Degradation of N protein occurred with catalytic amounts of Lon protease and required the presence of ATP or an analog of ATP. This is the first demonstration of the selective degradation of a physiological substrate by Lon protease in vitro. The turnover number for N protein degradation was approximately 60 +/- 10 min-1 at pH 8.0 in 50 mM Tris/HCl, 25 mM MgCl2 and 4 mM ATP. By comparison the turnover number for oxidized insulin B chain was 20 min-1 under these conditions. Kinetic studies suggest that N protein (S0.5 = 13 +/- 5 microM) is intermediate between oxidized insulin B chain (S0.5 = 160 +/- 10 microM) and methylated casein (S0.5 = 2.5 +/- 1 microM) in affinity for Lon protease. N protein was extensively degraded by Lon protease with an average of approximately six bonds cleaved per molecule. In N protein, as well as in oxidized insulin B chain and glucagon, Lon protease preferentially cut at bonds at which the carboxy group was contributed by an amino acid with an aliphatic side chain (leucine or alanine). However, not all such bonds of the substrates were cleaved, indicating that sequence or conformational determinants beyond the cleavage site affect the ability of Lon protease to degrade a protein.

ATP-Dependent Proteases↗
PubMed1985

Effects of protease inhibitors on adenylate cyclase activation and aldosterone production in rat adrenal zona glomerulosa cells.

It has been shown that serine proteases are involved in aldosterone and 18-hydroxycorticosterone production by the rat adrenal zona glomerulosa in response to a variety of stimulants. From evidence presented for various tissues, including the rat adrenal cortex, the observation that adenylate cyclase can be activated by proteolytic enzymes and inhibited by protease inhibitors has led to the suggestion that serine proteases may also be involved in the hormonal stimulation of adenylate cyclase. In studies designed to test this hypothesis using protease inhibitors, only high concentrations (greater than 10(-4) M) of TAME (p-tosyl-L-arginine methyl ester) inhibited ACTH stimulated steroid and cAMP production in rat adrenal glomerulosa cells. TPCK (tosyl-L-phenylalanine chloromethylketone) and TLCK (tosyl-L-lysine chloromethylketone) were found to have a similar effect at very high concentrations (10(-2) M) but had no effect at the serine protease inhibitory concentration of 5 X 10(-6) M. Other protease inhibitors tested had no effect on ACTH-stimulated cAMP but the inhibitory effect of high concentrations of protease inhibitors on ACTH-stimulated adenylate cyclase was duplicated by the polyanion dextran sulphate. The results suggest that the inhibitors act through non-specific membrane effects and that proteases are not involved in the activation of zona glomerulosa adenylate cyclase by ACTH. In view of these findings it is concluded that a more rigorous approach should be applied to the use of protease inhibitors in whole cell systems, and that the concept of hormonal activation of adenylate cyclase via proteolytic events, which is based on studies with such inhibitors, should be reconsidered.

Adenylyl Cyclases↗
PubMed2005

Reverse zymography using fluorogenic substrates for protease inhibitor detection.

A novel, sensitive method for detecting protease inhibitors by using fluorescent protease substrates in gels is described. The protease inhibitors were separated on sodium dodecyl sulfate (SDS)-polyacrylamide gels containing a copolymerized peptide substrate, namely 4-methyl-coumaryl-7-amide (MCA). As the incorporated substrates in the gel, Boc-Phe Ser-Arg-MCA was used for trypsin, Suc-Ala-Ala-Pro-Phe-MCA for alpha-chymotrypsin, and Z-Phe-Arg-MCA for papain. After electrophoresis, washing and incubating the gel with the target protease solutions allowed the substrate to be cleaved by the protease, and the release of the fluorescent 7 amino-4 methyl-coumarin (AMC), which was detected under a UV transilluminator. The uncleaved peptide-MCA substrate remained where the inhibitors were present, and was visualized as dark blue bands on the light-green fluorescent background gel. This new method offers several advantages over other previous methods including: (i) greatly increased sensitivity can be achieved in a shorter period of time, which may be useful for discovering new protease inhibitors in small amounts of crude material; (ii) the procedure is quite simple and quick since the incubation period is very short and no time is needed for staining and destaining steps; (iii) since these probes using substrate specificity/target proteases, they are excellent tools for detection and discrimination of unknown protease inhibitors for various target proteases.

Animals↗
PubMedDate not supplied

Sulfur mustard-stimulated protease: a target for antivesicant drugs.

One of the mechanisms of the skin blistering effect (vesication) of sulfur mustard (bis-(2-chloroethyl)sulfide, HD) is believed to be via the stimulation of specific protease(s) at the dermal-epidermal junction. Cultured normal human epidermal keratinocytes (NHEK) were used as a model to study and characterize protease stimulated by the mustards 2-chloroethyl ethyl sulfide (CEES), 2-chloro-N-(2-chloroethyl)-N-methylethanamine hydrochloride (nitrogen mustard, HN(2)) and HD. The results obtained using a chromozym (TRY) peptide substrate protease assay revealed the optimum mustard concentrations and time for protease stimulation to be about 200 microM (CEES), 100 microM (HN(2)) and 100 microM (HD) and 16 h. The mustard-stimulated protease was membrane bound and was inhibited by adding a Ca(2+) chelator (either 2 mM EGTA (ethylene glycol-bis(amino ethyl ether) N,N,N',N' tetraacetic acid) or 50 microM BAPTA AM (1,2-bis(z-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, tetraacetoxy methyl ester) alone or in combination), a serine protease inhibitor diisopropyl fluoro-phosphate (DFP, 1 mM), or a protein synthesis inhibitor cycloheximide (35 microM) in the extracellular medium. These results suggest that mustard toxicity may involve the stimulation of trypsin/chymotrypsin-like serine protease, dependent on Ca(2+) and new protein synthesis. Protein purification by gel exclusion and hydrophobic chromatography produced a 70-80 kDa protease, which had an amino acid sequence homologous with a mammalian-type bacterial serine endopeptidase. Based on this information, research is in progress to identify the protease stimulated by HD in NHEK and to determine whether its inhibitors are useful as prospective antivesicant drugs.

Amino Acid Sequence↗
PubMed1989

Identification of HTLV-I gag protease and its sequential processing of the gag gene product.

The full-length provirus of human T-cell leukemia virus type I (HTLV-I) was isolated from MT-2, a lymphoid cell line producing HTLV-I. In transfected cells, structural proteins of HTLV-I, the gag and env products, were formed and processed in the same manner as observed in MT-2 cells. The nucleotide sequence was determined for a region between the gag and pol genes of the proviral DNA clone containing an open-reading frame. The deduced amino acid sequences show that this open-reading frame encodes a putative HTLV-I protease. The protease gene (pro) of HTLV-I was investigated using a vaccinia virus expression vector. Processing of 53k gag precursor polyprotein into mature p19, p24, and p15 gag structural proteins was detectable with a recombinant plasmid harboring the entire gag- and protease-coding sequence. We demonstrated that the protease processed the gag precursor polyprotein in a trans-action. A change in the sequence Asp(64)-Thr-Gly, the catalytic core sequence among aspartyl proteases, to Gly-Thr-Gly was shown to abolish correct processing, suggesting that HTLV-I protease may belong to the aspartyl protease group. The 76k gag-pro precursor polyprotein was identified, implying that a cis-acting function of HTLV-I protease may be necessary to trigger the initial cleavage event for its own release from a precursor protein, followed by the release of p53 gag precursor protein. The p53 gag precursor protein is then processed by the trans-action of the released protease to form p19, p24, and p15.

Amino Acid Sequence↗