An Extracellular Protein from Phytophthora parasitica var nicotianae Is Associated with Stress Metabolite Accumulation in Tobacco Callus

The most abundant extracellular protein produced by Phytophthora parasitica var nicotianae at early stages of rapid growth in culture has a molecular weight of 46 kilodaltons and has been designated Ppn 46e. Culture conditions for the production of this protein have been optimized and the protein has been purified by gel filtration and ion-exchange chromatography. Ppn 46e is a soluble, acidic protein (pI 4.67). The amino acids Asx (aspartic acid or asparagine), alanine, glycine, Glx (glutamic acid or glutamine), and serine are the most abundant at 13.4%, 12.3%, 12.1%, 9.3%, and 9.3% of the residues, respectively. The purified protein is, by weight, 1.8% glucose, 1.6% mannose, and 0.5% galactose. A bioassay for Ppn 46e based on tobacco callus has been developed. In this assay as little as 20 nanograms (4.3 × 10⁻¹³ mole) Ppn 46e causes the accumulation of the sesquiterpenoid phytoalexin, capsidiol, as estimated by gas chromatography. Levels of capsidiol of 25 micrograms per gram fresh weight were elicited by 80 nanograms Ppn 46e per callus piece. Pretreatment of the protein with either pronase or by boiling resulted in a loss of elicitor activity. Periodate treatment, which inactivates glucan elicitors, did not affect the ability of Ppn 46e to cause capsidiol accumulation. Monospecific antibodies to Ppn 46e were raised in mice. Western blotting experiments employing these antibodies showed that Ppn 46e was present in infected tobacco plants. Dot blotting experiments revealed the presence of the Ppn 46e epitope(s) in Phytophthora megasperma, P. cactorum, P. cinnamomi, and P. infestans but not in Fusarium.     

Purification and characterization of the yeast glycosylphosphatidylinositol-anchored, monobasic-specific aspartyl protease yapsin 2 (Mkc7p).

The Saccharomyces cerevisiae YPS2 (formerly MKC7) gene product is a glycosylphosphatidylinositol-linked aspartyl protease that functions as a yeast secretase. Here, the glycosylphosphatidylinositol-linked form of yapsin 2 (Mkc7p) was purified to homogeneity from the membrane fraction of an overexpressing yeast strain. Purified yapsin 2 migrated diffusely in SDS-polyacrylamide gel electrophoresis (molecular mass approximately 200 kDa), suggesting extensive, heterogeneous glycosylation. Studies using internally quenched fluorogenic peptide substrates revealed cleavage by the enzyme carboxyl to Lys or Arg. No cleavage was seen when both Lys and Arg were absent. No significant enhancement was seen with multiple basic residues. However, cleavage always occurred carboxyl to the most COOH-terminal basic residue. V(max)/K(m) was insensitive to P(2) and P(3) residues except that Pro at P(2) blocked cleavage entirely. These results suggest that yapsin 2 is a monobasic amino acid-specific protease that requires a basic residue at P(1) and excludes basic residues from P(1)'. The pH dependence of V(max)/K(m) for a substrate containing a pro-alpha factor cleavage site was bell-shaped, with a maximum near pH 4.0. However, V(max)/K(m) for a substrate mimicking the alpha-secretase site in human beta amyloid precursor protein was optimal near pH 6.0, consistent with cleavage of beta amyloid precursor protein by yapsin 2 when expressed in yeast.     

Human kallikrein 4: enzymatic activity, inhibition, and degradation of extracellular matrix proteins

Human kallikrein 4 (hK4) is a member of the expanded family of human kallikreins, a group of 15 secreted proteases. While this protein has been associated with ovarian and prostate cancer prognosis, only limited functional information exists. Therefore, we have undertaken an investigation of its enzymatic properties regarding substrate preference, degradation of extracellular matrix proteins, and its inhibition by various inhibitors. We successfully expressed and purified active recombinant hK4 from supernatants of the Pichia pastoris expression system. This enzyme seems to cleave more efficiently after Arg compared to Lys at the P1 position and exhibits modest specificity for amino acids at positions P2 and P3. hK4 forms complexes with alpha1-antitrypsin, alpha2-antiplasmin and alpha2-macroglobulin. The protease mediates limited degradation of extracellular matrix proteins such as collagen I and IV, and more efficient degradation of the alpha-chain of fibrinogen. The cleavage of extracellular matrix proteins by hK4 suggests that this enzyme may play a role in tissue remodeling and cancer metastasis.     

Competitive inhibition of the dengue virus NS3 serine protease by synthetic peptides representing polyprotein cleavage sites

The NS3 serine protease of dengue virus is required for the maturation of the viral polyprotein and consequently represents a promising target for the development of antiviral inhibitors. However, the substrate specificity of this enzyme has been characterized only to a limited extent. In this study, we have investigated product inhibition of the NS3 protease by synthetic peptides derived from the P6-P1 and the P1'-P5' regions of the natural polyprotein substrate. N-terminal cleavage site peptides corresponding to the P6-P1 region of the polyprotein were found to act as competitive inhibitors of the enzyme with K(i) values ranging from 67 to 12 microM. The lowest K(i) value was found for the peptide representing the NS2A/NS2B cleavage site, RTSKKR. Inhibition by this cleavage site sequence was analyzed by using shorter peptides, SKKR, KKR, KR, AGRR, and GKR. With the exception of the peptide AGRR which did not inhibit the protease at a concentration of 1mM, all other peptides displayed K(i) values in the range from 188 to 22 microM. Peptides corresponding to the P1'-P5' region of the polyprotein cleavage sites had no effect on enzymatic activity at a concentration of 1mM. Molecular docking data of peptide inhibitors to a homology-based model of the dengue virus type 2 NS2B(H)-NS3p co-complex indicate that binding of the non-prime site product inhibitors is similar to ground-state binding of the corresponding substrates.     

Peptide substrate profiling defines fibroblast activation protein as an endopeptidase of strict Gly(2)-Pro(1)-cleaving specificity

Fibroblast activation protein (FAP) is a serine protease of undefined endopeptidase specificity implicated in tumorigenesis. To characterize FAP's P(4)-P(2)(') specificity, we synthesized intramolecularly quenched fluorescent substrate sets based on the FAP cleavage site in alpha(2)-antiplasmin (TSGP-NQ). FAP required substrates with Pro at P(1) and Gly or d-amino acids at P(2) and preferred small, uncharged amino acids at P(3), but tolerated most amino acids at P(4), P(1)(') and P(2)('). These substrate preferences allowed design of peptidyl-chloromethyl ketones that inhibited FAP, but not the related protease, dipeptidyl peptidase-4. Thus, FAP is a narrow specificity endopeptidase and this can be exploited for inhibitor design.     

Proteolytic modification of calcium-dependent protease 1 in erythrocytes treated with ionomycin and calcium

In vitro, limited proteolytic cleavage of the subunits of the purified calcium-dependent proteases [also known as calpains (EC 3.4.22.17) or calcium-activated neutral proteinases (CANPs)] appears to be required for enzyme activity. It has not yet been demonstrated if similar processing of the protease subunits occurs in vivo. To directly assess proteolytic modification of these proteases in cells, we have measured the loss of the proenzyme form of the regulatory subunit (a 26-kDa protein) and/or the appearance of the modified regulatory subunit (a 17-kDa protein) by densitometric analysis of immunoblots. In rat erythrocytes, proteolytic modification of the endogenous calcium-dependent protease (calcium-dependent protease 1, mu CANP) occurs in vivo in response to ionomycin and calcium. The extent of enzyme modification was dependent on time, ionomycin concentration, and calcium concentration, suggesting that in this cellular model Ca2+ regulates proteolytic modification of the enzyme.     

In vitro characterization of a purified NS2/3 protease variant of hepatitis C virus

The cleavage of the hepatitis C virus polyprotein between the nonstructural proteins NS2 and NS3 is mediated by the NS2/3 protease, whereas the NS3 protease is responsible for the cleavage of the downstream proteins. Purification and in vitro characterization of the NS2/3 protease has been hampered by its hydrophobic nature. NS2/3 protease activity could only be detected in cells or in in vitro translation assays with the addition of microsomal membranes or detergent. To facilitate purification of this poorly characterized protease, we truncated the N-terminal hydrophobic domain, resulting in an active enzyme with improved biophysical properties. We define a minimal catalytic region of NS2/3 protease retaining autocleavage activity that spans residues 904-1206 and includes the C-terminal half of NS2 and the N-terminal NS3 protease domain. The NS2/3 (904-1206) variant was purified from Escherichia coli inclusion bodies and refolded by gel filtration chromatography. The purified inactive form of NS2/3 (904-1206) was activated by the addition of glycerol and detergent to induce autocleavage at the predicted site between Leu(1026) and Ala(1027). NS2/3 (904-1206) activity was dependent on zinc ions and could be inhibited by NS4A peptides, peptides that span the cleavage site, or an N-terminal peptidic cleavage product. This NS2/3 variant will facilitate the development of an assay suitable for identifying inhibitors of HCV replication.     

Holliday junction resolvase in Schizosaccharomyces pombe has identical endonuclease activity to the CCE1 homologue YDC2.

A novel Holliday junction resolving activity has been identified in fractionated cell extracts of the fission yeast Schizosaccharomyces pombe . The enzyme catalyses endonucleolytic cleavage of Holliday junction-containing chi DNA and synthetic four-way DNA junctions. The activity cuts with high specificity a synthetic four-way junction containing a 12 bp core of homologous sequences but has no activity on another four-way junction (with a fixed crossover point), a three-way junction, linear duplex DNA or duplex DNA containing six mismatched nucleotides in the centre. The major cleavage sites map as single nicks in the vicinity of the crossover point, 3' of a thymidine residue. These data indicate that the activity has a strong DNA structure selectivity as well as a limited sequence preference; features similar to the Holliday junction resolving enzymes RuvC of Escherichia coli and the mitochondrial CCE1 (cruciform-cuttingenzyme 1) of Saccharomyces cerevisiae. A putative homologue of CCE1 in S.pombe (YDC2_SCHPO) has been identified through a search of the sequence database. The open reading frame of this gene has been cloned and the encoded protein, YDC2, expressed in E.coli . The purified recombinant YDC2 exhibits Holliday junction resolvase activity and is, therefore, a functional S.pombe homologue of CCE1. The resolvase YDC2 shows the same substrate specificity and produces identical cleavage sites as the activity obtained from S. pombe cells. Both YDC2 and the cellular activity cleave Holliday junctions in both orientations to give nicks that can be ligated in vitro. The partially purified Holliday junction resolving enzyme in fission yeast is biochemically indistinguishable from recombinant YDC2 and appears to be the same protein.     

Substrate specificity of the Escherichia coli outer membrane protease OmpT

OmpT is a surface protease of gram-negative bacteria that has been shown to cleave antimicrobial peptides, activate human plasminogen, and degrade some recombinant heterologous proteins. We have analyzed the substrate specificity of OmpT by two complementary substrate filamentous phage display methods: (i) in situ cleavage of phage that display protease-susceptible peptides by Escherichia coli expressing OmpT and (ii) in vitro cleavage of phage-displayed peptides using purified enzyme. Consistent with previous reports, OmpT was found to exhibit a virtual requirement for Arg in the P1 position and a slightly less stringent preference for this residue in the P1' position (P1 and P1' are the residues immediately prior to and following the scissile bond). Lys, Gly, and Val were also found in the P1' position. The most common residues in the P2' position were Val or Ala, and the P3 and P4 positions exhibited a preference for Trp or Arg. Synthetic peptides based upon sequences selected by bacteriophage display were cleaved very efficiently, with kcat/Km values up to 7.3 x 10(6) M(-1) s(-1). In contrast, a peptide corresponding to the cleavage site of human plasminogen was hydrolyzed with a kcat/Km almost 10(6)-fold lower. Overall, the results presented in this work indicate that in addition to the P1 and P1' positions, additional amino acids within a six-residue window (between P4 and P2') contribute to the binding of substrate polypeptides to the OmpT binding site.     

Characterization of a soluble stable human cytomegalovirus protease and inhibition by M-site peptide mimics.

The human cytomegalovirus (HCMV) protease is a potential target for antiviral chemotherapeutics; however, autoprocessing at internal sites, particularly at positions 143 and 209, hinders the production of large quantities of stable enzyme for either screening or structural studies. Using peptides encompassing the sequence of the natural M-site substrate (P5-P5', GVVNA/SCRLA), we previously demonstrated that substitution of glycine for valine at the P3 position in the substrate abrogates processing by the recombinant protease in vitro. We now demonstrate that introduction of the V-to-G substitution in the P3 positions of the two major internal processing sites, positions 143 and 209, in the mature HCMV protease renders the enzyme stable to autoprocessing. When expressed in Escherichia coli, the doubly substituted protease was produced almost exclusively as the 30-kDa full-length protein. The full-length V141G, V207G (V-to-G changes at positions 141 and 207) protease was purified as a soluble protein by a simple two-step procedure, ammonium sulfate precipitation followed by DEAE ion-exchange chromatography, resulting in 10 to 15 mg of greater than 95% pure enzyme per liter. The stabilized enzyme was characterized kinetically and was indistinguishable from the wild-type recombinant protease, exhibiting Km and catalytic constant values of 0.578 mM and 13.18/min, respectively, for the maturation site (M-site) peptide substrate, GVVNASCRLARR (underlined residues indicate additions to or substitutions from peptides derived from the wild-type substrate). This enzyme was also used to perform inhibition studies with a series of truncated and/or substituted maturation site peptides. Short nonsubstrate M-site-derived peptides were demonstrated to be competitive inhibitors of cleavage in vitro, and these analyses defined amino acids VVNA, P4 through P1 in the substrate, as the minimal substrate binding and recognition sequence for the HCMV protease.     

Generation of Lys-gamma 3-melanotropin from pro-opiomelanocortin 1-77 by a bovine intermediate lobe secretory vesicle membrane-associated aspartic protease and purified pro-opiomelanocortin converting enzyme

The ability of bovine intermediate lobe secretory vesicle membrane-associated enzyme(s) and purified, soluble paired basic residue-specific, pro-opiomelanocortin converting enzyme (Loh, Y.P., Parish, D. C., and Tuteja, R. (1985) J. Biol. Chem. 260, 7194-7205) to cleave bovine NH2-terminal pro-opiomelanocortin1-77 (N-POMC 1-77) was investigated. Purified pro-opiomelanocortin converting enzyme and an enzyme activity associated with the secretory vesicle membrane were shown to cleave bovine N-POMC1-77 to two major products: N-POMC1-49 and Lys-gamma 3-melanotropin (MSH), and one minor product, gamma 3-MSH. These products were identified by their retention times on high performance liquid chromatography, immunological characteristics, and for Lys-gamma 3-MSH, amino acid composition. The products generated indicate cleavage preferentially between Arg 49-Lys 50 of bN-POMC1-77 (where b indicates bovine), which is identical to the processing pattern found in the bovine intermediate lobe in situ. The membrane converting activity was shown to be stimulated by 5 mM Ca2+ and has a pH optimum of 4-5 and an inhibitor profile characteristic of an aspartic protease. This suggests that the membrane-associated enzyme involved is very similar or identical to the purified, soluble pro-opiomelanocortin converting enzyme, which has previously been reported to be an acidic, aspartic protease responsible for the initial steps of POMC processing. The results of this study lead to the proposal that the lack of processing of the Arg49-Lys50 site in POMC in the anterior lobe versus the intermediate lobe of the pituitary in vivo may be due to other regulatory mechanisms rather than invoking the existence in the intermediate lobe of another enzyme specific for this site, different from pro-opiomelanocortin converting enzyme.     

Release of highly active Fet3 from membranes of the yeast Pichia pastoris by limited proteolysis

A soluble derivative of Fet3 has been obtained from the methylotrophic yeast Pichia pastoris by limited proteolysis of membrane suspensions with trypsin. The soluble protein and the membrane-bound parent Fet3 have been purified to apparent homogeneity. Soluble Fet3 had molecular mass 100 kDa, while the full-length protein had molecular mass 110 kDa, in line with the expected decrease for cleavage and loss of a single transmembrane helix and a small cytoplasmic domain. The optical and EPR spectra of Fet3 were typical of the multicopper oxidases, indicating the presence of one type 1 blue copper site and a type 2/type 3 copper trinuclear cluster. V(max) values for iron oxidation by P. pastoris Fet3 were obtained similar to human ceruloplasmin and much higher than those reported for Saccharomyces cerevisiae Fet3.     

Rapid purification of active gamma-secretase, an intramembrane protease implicated in Alzheimer's disease

γ-Secretase is an unconventional aspartyl protease that processes many type 1 membrane proteins within the lipid bilayer. Because its cleavage of amyloid-β precursor protein generates the amyloid-β protein (Aβ) of Alzheimer's disease, partially inhibiting γ-secretase is an attractive therapeutic strategy, but the structure of the protease remains poorly understood. We recently used electron microscopy and single particle image analysis on the purified enzyme to generate the first 3D reconstruction of γ-secretase, but at low resolution (15 Å). The limited amount of purified γ-secretase that can be produced using currently available cell lines and procedures has prevented the achievement of a high resolution crystal structure by X-ray crystallography or 2D crystallization. We report here the generation and characterization of a new mammalian cell line (S-20) that overexpresses strikingly high levels of all four γ-secretase components (presenilin, nicastrin, Aph-1 and Pen-2). We then used these cells to develop a rapid protocol for the high-grade purification of proteolytically active γ-secretase. The cells and purification methods detailed here provide a key step towards crystallographic studies of this ubiquitous enzyme.     

An uniquely purified HCV NS3 protease and NS4A(21-34) peptide form a highly active serine protease complex in peptide hydrolysis.

The N-terminal domain of the hepatitis C virus (HCV) polyprotein containing the NS3 protease (residues 1027 to 1206) was expressed in Escherichia coli as a soluble protein under the control of the T7 promoter. The enzyme has been purified to homogeneity with cation exchange (SP-Sepharose HR) and heparin affinity chromatography in the absence of any detergent. The purified enzyme preparation was soluble and remained stable in solution for several weeks at 4 degrees C. The proteolytic activity of the purified enzyme was examined, also in the absence of detergents, using a peptide mimicking the NS4A/4B cleavage site of the HCV polyprotein. Hydrolysis of this substrate at the expected Cys-Ala scissile bond was catalyzed by the recombinant protease with a pseudo second-order rate constant (k(cat)/K(M)) of 205 and 196,000 M(-1) s(-1), respectively, in the absence and presence of a central hydrophobic region (sequence represented by residues 21 to 34) of the NS4A protein. The rate constant in the presence of NS4A peptide cofactor was two orders of magnitude greater than reported previously for the NS3 protease domain. A significantly higher activity of the NS3 protease-NS4A cofactor complex was also observed with a substrate mimicking the NS4B/5A site (k(cat)/K(M) of 5180 +/- 670 M(-1) s(-1)). Finally, the optimal formation of a complex between the NS3 protease domain and the cofactor NS4A was critical for the high proteolytic activity observed.     

Human immunodeficiency virus protease. Bacterial expression and characterization of the purified aspartic protease

The protease of human immunodeficiency virus has been expressed in Escherichia coli and purified to apparent homogeneity. Immunoreactivity toward anti-protease peptide sera copurified with an activity that cleaved the structural polyprotein gag p55 and the peptide corresponding to the sequence gag 128-135. The enzyme expressed as a nonfusion protein exhibits proteolytic activity with a pH optimum of 5.5 and is inhibited by the aspartic protease inhibitor pepstatin with a Ki of 1.1 microM. Replacement of the conserved residue Asp-25 with an Asn residue eliminates proteolytic activity. Analysis of the minimal peptide substrate size indicates that 7 amino acids are required for efficient peptide cleavage. Size exclusion chromatography is consistent with a dimeric enzyme and circular dichroism spectra of the purified enzyme are consistent with a proposed structure of the protease (Pearl, L.H., and Taylor, W.R. (1987) Nature 329, 351-354). These data support the classification of the human immunodeficiency virus protease as an aspartic protease, likely to be structurally homologous with the well characterized family that includes pepsin and renin.     

Isolation and characterization of a serine protease from the sprouts of Pleioblastus hindsii Nakai

An endopeptidase has been purified from sprouts of bamboo (Pleioblastus hindsii Nakai) to electrophoretic homogeneity by four purification steps. Its Mr was estimated to be 82 kDa by SDS-PAGE. Enzyme activity was inhibited strongly by diisopropyl fluorophosphate, and weakly by p-chloromercuriphenylsulfonic acid, but not at all by EDTA or pepstatin, indicating that it was a serine protease. The preferential cleavage sites for this protease were found to be large hydrophobic and amide residues at the P1 position. The specificity of the bamboo serine protease differed from that of cucumisin [EC 3.4.21.25], which cleaved the charged amino acid residues at the P1 position.     

Recombinant chymotrypsin inhibitor 2: expression, kinetic analysis of inhibition with alpha-chymotrypsin and wild-type and mutant subtilisin BPN', and protein engineering to investigate inhibitory specificity and mechanism

The serine protease inhibitor chymotrypsin inhibitor 2 (CI2 or BSPI2) has been expressed in Escherichia coli with the pINIIIompA3 expression vector to produce 20-40 mg/L of culture. Recombinant CI2 purified from this system has been characterized and found to be identical with CI2 from barley. Slow-binding kinetics were observed for the interaction between CI2 and subtilisin BPN', with Ki = 2.9 x 10(-12) M. Analysis of slow-binding data indicates that binding of the inhibitor follows the simplest model of E + I = EI with no kinetically detectable intermediate steps or proteolytic cleavage of the reactive site bond in CI2 (Met-59-Glu-60). This, in agreement with crystallographic data, indicates that the enzyme-inhibitor adduct is the Michaelis complex, which is not chemically processed by the enzyme. Three mutant CI2 molecules with new P1 residues have also been examined with a range of serine proteases, including a mutant subtilisin. In agreement with earlier studies, we find the P1 amino acid an important determinant of specificity. CI2 Met----Lys-59 was found to be a temporary inhibitor of subtilisin BPN' but an effective inhibitor of subtilisin Carlsberg and subtilisin BPN'(Glu----Ser-156). The structural reasons for this are discussed in relation to mechanisms of inhibition of serine proteases.     

Purification and characterization of protease III from Escherichia coli.

An endoproteolytic enzyme of Escherichia coli, designated protease III, has been purified about 9,600-fold to homogeneity with a 6% yield. The purified enzyme consists of a single polypeptide chain of Mr 110,000 and is most active at pH 7.4. Protease III is very sensitive to metal-chelating agents and reducing agents. The EDTA-inactivated enzyme can be reactivated by Zn2+, Co2+ or Mn2+. Protease III is devoid of activity toward aminopeptidase, carboxypeptidase, or esterase substrates but rapidly degrades small proteins. When fragments of beta-galactosidase are used as substrates for protease III, the enzyme preferentially degrades proteins with molecular weights of less than 7,000. Protease III cleaves the oxidized insulin B chain at two sites with an initial rapid cleavage at Tyr-Leu (16-17) and a second slower cut at Phe-Tyr (25-26).     

Activity of recombinant dengue 2 virus NS3 protease in the presence of a truncated NS2B co-factor, small peptide substrates, and inhibitors

Recombinant forms of the dengue 2 virus NS3 protease linked to a 40-residue co-factor, corresponding to part of NS2B, have been expressed in Escherichia coli and shown to be active against para-nitroanilide substrates comprising the P6-P1 residues of four substrate cleavage sequences. The enzyme is inactive alone or after the addition of a putative 13-residue co-factor peptide but is active when fused to the 40-residue co-factor, by either a cleavable or a noncleavable glycine linker. The NS4B/NS5 cleavage site was processed most readily, with optimal processing conditions being pH 9, I = 10 mm, 1 mm CHAPS, 20% glycerol. A longer 10-residue peptide corresponding to the NS2B/NS3 cleavage site (P6-P4') was a poorer substrate than the hexapeptide (P6-P1) para-nitroanilide substrate under these conditions, suggesting that the prime side substrate residues did not contribute significantly to protease binding. We also report the first inhibitors of a co-factor-complexed, catalytically active flavivirus NS3 protease. Aprotinin was the only standard serine protease inhibitor to be active, whereas a number of peptide substrate analogues were found to be competitive inhibitors at micromolar concentrations.