Human Ras converting enzyme endoproteolytic specificity at the P2′ and P3′ positions of K-Ras-derived peptides

The membrane associated endoprotease, hRCE1, is responsible for one step in Ras membrane localization. The “CAAX” sequence at the C-terminal of farnesylated Ras proteins is cleaved by hRCE1 to yield an AAX tri-peptide. We found that an 8-aa K-Ras-derived “CAA” peptide, KSKTKC(farnesyl)VI, was a better substrate for hRCE1 than a KSKTKC(f)VIM “CAAX” peptide. When we examined hRCE1 activity on the same K-Ras core peptide with H-Ras (VLS) or N-Ras (VVM) C-terminal AAX sequences, we also found that in each case, the CAA peptides were better hRCE1 substrates. For each peptide set we examined, the P2′ (A) and P3′ (X) positions appeared independent in influencing hRCE1 activity on peptide substrates. We found that at the P3′ position, methionine was better than serine; while at the P2′ position, isoleucine and valine were better than leucine. Additionally, we found that a similar noncleaved peptide (modified at P′2 with a nitrophenyl group) could act as a competitive inhibitor of the reaction. Thus, hRCE1 has important functional interaction with the P2′ and P3′ substrate positions in addition to the farnesylated cysteine at the scissile bond site. This data could be useful in design of peptidomimetic inhibitors of hRCE1. Such inhibitors may be useful in treatment of cancer and inflammatory disease.     

Human Ras converting enzyme endoproteolytic specificity at the P2' and P3' positions of K-Ras-derived peptides

The membrane associated endoprotease, hRCE1, is responsible for one step in Ras membrane localization. The "CAAX" sequence at the C-terminal of farnesylated Ras proteins is cleaved by hRCE1 to yield an AAX tri-peptide. We found that an 8-aa K-Ras-derived "CAA" peptide, KSKTKC(farnesyl)VI, was a better substrate for hRCE1 than a KSKTKC(f)VIM "CAAX" peptide. When we examined hRCE1 activity on the same K-Ras core peptide with H-Ras (VLS) or N-Ras (VVM) C-terminal AAX sequences, we also found that in each case, the CAA peptides were better hRCE1 substrates. For each peptide set we examined, the P2' (A) and P3' (X) positions appeared independent in influencing hRCE1 activity on peptide substrates. We found that at the P3' position, methionine was better than serine; while at the P2' position, isoleucine and valine were better than leucine. Additionally, we found that a similar noncleaved peptide (modified at P'2 with a nitrophenyl group) could act as a competitive inhibitor of the reaction. Thus, hRCE1 has important functional interaction with the P2' and P3' substrate positions in addition to the farnesylated cysteine at the scissile bond site. This data could be useful in design of peptidomimetic inhibitors of hRCE1. Such inhibitors may be useful in treatment of cancer and inflammatory disease.     

Cloning and characterization of a mammalian prenyl protein-specific protease

Proteins containing C-terminal "CAAX" sequence motifs undergo three sequential post-translational processing steps: modification of the cysteine with either a 15-carbon farnesyl or 20-carbon geranylgeranyl isoprenyl lipid, proteolysis of the C-terminal -AAX tripeptide, and methylation of the carboxyl group of the now C-terminal prenylcysteine. A putative prenyl protein protease in yeast, designated Rce1p, was recently identified. In this study, a portion of a putative human homologue of RCE1 (hRCE1) was identified in a human expressed sequence tag data base, and the corresponding cDNA was cloned. Expression of hRCE1 was detected in all tissues examined. Both yeast and human RCE1 proteins were produced in Sf9 insect cells by infection with a recombinant baculovirus; membrane preparations derived from the infected Sf9 cells exhibited a high level of prenyl protease activity. Recombinant hRCE1 so produced recognized both farnesylated and geranylgeranylated proteins as substrates, including farnesyl-Ki-Ras, farnesyl-N-Ras, farnesyl-Ha-Ras, and the farnesylated heterotrimeric G protein Ggamma1 subunit, as well as geranylgeranyl-Ki-Ras and geranylgeranyl-Rap1b. The protease activity of hRCE1 activity was specific for prenylated proteins, because unprenylated peptides did not compete for enzyme activity. hRCE1 activity was also exquisitely sensitive to a prenyl peptide analogue that had been previously described as a potent inhibitor of the prenyl protease activity in mammalian tissues. These data indicate that both the yeast and the human RCE1 gene products are bona fide prenyl protein proteases and suggest that they play a major role in the processing of CAAX-type prenylated proteins.     

Identification of candidate substrates for ectodomain shedding by the metalloprotease-disintegrin ADAM8

ADAM proteases are type I transmembrane proteins with extracellular metalloprotease domains. As for most ADAM family members, ADAM8 (CD156a, MS2) is involved in ectodomain shedding of membrane proteins and is linked to inflammation and neurodegeneration. To identify potential substrates released under these pathologic conditions, we screened 10-mer peptides representing amino acid sequences from extracellular domains of various membrane proteins using the ProteaseSpot system. A soluble ADAM8 protease containing a pro- and metalloprotease domain was expressed in E. coli and purified as active protease owing to autocatalytic prodomain removal. From 34 peptides tested in the peptide cleavage assay, significant cleavage by soluble ADAM8 was observed for 14 peptides representing membrane proteins with functions in inflammation and neurodegeneration, among them the beta-amyloid precursor protein (APP). The in vivo relevance of the ProteaseSpot method was confirmed by cleavage of full-length APP with ADAM8 in human embryonic kidney 293 cells expressing tagged APP. ADAM8 cleaved APP with similar efficiency as ADAM10, whereas the inactive ADAM8 mutant did not. Exchanging amino acids at defined positions in the cleavage sequence of myelin basic protein (MBP) revealed sequence criteria for ADAM8 cleavage. Taken together, the results allowed us to identify novel candidate substrates that could be cleaved by ADAM8 in vivo under pathologic conditions.     

A high throughput assay for inhibitors of HIV-1 protease. Screening of microbial metabolites

A novel method for discovery of HIV-1 protease inhibitors in complex biological samples has been developed. The assay is based on two specific reagents: a recombinant protein constituted by a portion of the HIV-1 Gag polyprotein comprising the p17-p24 cleavage site, fused to E. coli beta-galactosidase, and a monoclonal antibody which binds the fusion protein in the Gag region. Binding occurs only if the fusion protein has not been cleaved by the HIV-1 protease. The assay has been adapted for the screening of large numbers of samples in standard 96-well microtiter plates. Using this method about 12000 microbial fermentation broths have been tested and several HIV-1 protease inhibitory activities have been detected. One of these has been studied in detail.     

Cleavage preference distinguishes the two-component NS2B-NS3 serine proteinases of Dengue and West Nile viruses

Regulated proteolysis of the polyprotein precursor by the NS2B-NS3 protease is required for the propagation of infectious virions. Unless the structural and functional parameters of NS2B-NS3 are precisely determined, an understanding of its functional role and the design of flaviviral inhibitors will be exceedingly difficult. Our objectives were to define the substrate recognition pattern of the NS2B-NS3 protease of West Nile and Dengue virises (WNV and DV respectively). To accomplish our goals, we used an efficient, 96-well plate format, method for the synthesis of 9-mer peptide substrates with the general P4-P3-P2-P1-P1'-P2'-P3'-P4'-Gly structure. The N-terminus and the constant C-terminal Gly of the peptides were tagged with a fluorescent tag and with a biotin tag respectively. The synthesis was followed by the proteolytic cleavage of the synthesized, tagged peptides. Because of the strict requirement for the presence of basic amino acid residues at the P1 and the P2 substrate positions, the analysis of approx. 300 peptide sequences was sufficient for an adequate representation of the cleavage preferences of the WNV and DV proteinases. Our results disclosed the strict substrate specificity of the WNV protease for which the (K/R)(K/R)R/GG amino acid motifs was optimal. The DV protease was less selective and it tolerated well the presence of a number of amino acid residue types at either the P1' or the P2' site, as long as the other position was occupied by a glycine residue. We believe that our data represent a valuable biochemical resource and a solid foundation to support the design of selective substrates and synthetic inhibitors of flaviviral proteinases.     

Fluorescent oligopeptide substrates for kinetic characterization of the specificity of Astacus protease

The design of fluorescent N-dansylated oligopeptides based on the tubulin cleavage pattern by Astacus protease yields substrates that are turned over up to 10(5) times faster than those presently available. On the basis of this study, an optimal substrate for Astacus protease contains seven or more amino acids and minimally requires at least five amino acids. Direct examination of the formation and breakdown of the ES complex shows its formation occurs within milliseconds at 25 degrees C. The best heptapeptide substrate, Dns-Pro-Lys-Arg-Ala-Pro-Trp-Val, is cleaved only between the Arg-Ala (P1-P1') bond with kinetic parameters kcat = 380 s-1 and Km = 3.7 x 10(-4) M. The presence of Lys or Arg in the P1 and P2 positions yields high-turnover substrates. In the P3 position, the enzyme prefers Pro greater than Val greater than Leu greater than Ala greater than Gly, following the same order of preference seen in the tubulin cleavage pattern. Substitution of Leu for Ala in P1' and of Ser for Pro in P2' decreases activity by 10(5)- and 10(2)-fold, respectively. In position P3', substitution of Trp for Leu leaves the activity unaltered. However, introduction of the Trp fluorophore greatly enhances the sensitivity of the assay due to a 10-fold increase in indole fluorescence for cleavage of any peptide bond between the tryptophan and the dansyl group. Such an energy-transfer-based assay should have widespread use for detection of neutral proteases.(ABSTRACT TRUNCATED AT 250 WORDS)     

Degradation of a signal peptide by protease IV and oligopeptidase A.

The degradation of the prolipoprotein signal peptide in vitro by membranes, cytoplasmic fraction, and two purified major signal peptide peptidases from Escherichia coli was followed by reverse-phase liquid chromatography (RPLC). The cytoplasmic fraction hydrolyzed the signal peptide completely into amino acids. In contrast, many peptide fragments accumulated as final products during the cleavage by a membrane fraction. Most of the peptides were similar to the peptides formed during the cleavage of the signal peptide by the purified membrane-bound signal peptide peptidase, protease IV. Peptide fragments generated during the cleavage of the signal peptide by protease IV and a cytoplasmic enzyme, oligopeptidase A, were identified from their amino acid compositions, their retention times during RPLC, and knowledge of the amino acid sequence of the signal peptide. Both enzymes were endopeptidases, as neither dipeptides nor free amino acids were formed during the cleavage reactions. Protease IV cleaved the signal peptide predominantly in the hydrophobic segment (residues 7 to 14). Protease IV required substrates with hydrophobic amino acids at the primary and the adjacent substrate-binding sites, with a minimum of three amino acids on either side of the scissile bond. Oligopeptidase A cleaved peptides (minimally five residues) that had either alanine or glycine at the P'1 (primary binding site) or at the P1 (preceding P'1) site of the substrate. These results support the hypothesis that protease IV is the major signal peptide peptidase in membranes that initiates the degradation of the signal peptide by making endoproteolytic cuts; oligopeptidase A and other cytoplasmic enzymes further degrade the partially degraded portions of the signal peptide that may be diffused or transported back into the cytoplasm from the membranes.     

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.     

Cloning and biochemical characterization of blisterase,a subtilisin-like convertase from the filarial parasite,Onchocerca volvulus

Blisterase is a subtilisin-like proprotein convertase of nematodes. The enzyme is named after the blistered cuticle found in Caenorhabditis elegans with the bli-4 e937 mutation. The critical role of the enzyme in cuticle production makes it a potential drug target for parasitic nematodes. We have cloned and expressed blisterase from the parasitic nematode Onchocerca volvulus, a major cause of blindness in Africa. The catalytic domain of the protease exhibits 84% identity with the corresponding domain of its closest homologue, C. elegans blisterase. O. volvulus blisterase expressed in insect cells has maximal activity in 1 mm calcium at neutral pH. The protease is inhibited by EDTA, the suicide substrate decanoyl-RVKR-chloromethylketone, alpha1-antitrypsin Portland and by its own propeptide. Substrate assays with fluorescent peptides show that O. volvulus blisterase requires a P4 arginine and a basic amino acid at P1 for cleavage. The kcat of blisterase on the peptide substrate, t-butyloxycarbonyl-RVRR-4-methylcoumaryl-7-amide was determined to be 0.018 s-1. In vitro cleavage studies with the nematode polyprotein antigen demonstrated that blisterase cleaved at tetrabasic (RRKR) but not at dibasic (KR) sites. This report describes the first biochemical characterization of the nematode specific protease, blisterase.     

Signal peptides having standard and nonstandard cleavage sites can be processed by Imp1p of the mitochondrial inner membrane protease

We have performed a site-directed mutagenesis study showing that residues comprising the type I signal peptidase signature in the two catalytic subunits of the yeast inner membrane protease, Imp1p and Imp2p, are functionally important, consistent with the idea that these subunits contain a serine/lysine catalytic dyad. Previous studies have shown that Imp1p cleaves signal peptides having asparagine at the -1 position, which deviates from the typical signal peptide possessing a small uncharged amino acid at this position. To determine whether asparagine is responsible for the nonoverlapping substrate specificities exhibited by the inner membrane protease subunits, we have substituted asparagine with 19 amino acids in the Imp1p substrate i-cytochrome (cyt) b(2). The resulting signal peptides containing alanine, serine, cysteine, leucine, and methionine can be cleaved efficiently by Imp1p. The remaining mutant signal peptides are cleaved inefficiently or not at all. Surprisingly, none of the amino acid changes results in the recognition of i-cyt b(2) by Imp2p, whose natural substrate, i-cyt c(1), has alanine at the -1 position. The data demonstrate that (i) although the -1 residue is important in substrates recognized by Imp1p, signal peptides having standard and nonstandard cleavage sites can be processed by Imp1p, and (ii) a -1 asparagine does not govern the substrate specificity of the inner membrane protease subunits.     

In vivo detection of aflatoxin-induced lipid free radicals in rat bile

Human kallikrein hK3 (prostate-specific antigen) is a chymotrypsin-like serine protease which is widely used in the diagnosis of prostate cancer. Assays of the enzymatic activity of hK3 in extracellular fluids have been limited by a lack of sensitive synthetic substrates. This report describes the design of a series of internally quenched fluorescent peptides containing an amino acid sequence based on preferential hK3 cleavage sites in semenogelins. Those were identified by 2-D gel electrophoresis analysis and N-terminal sequencing of semenogelin fragments generated by ex vivo proteolysis in freshly ejaculated semen. These peptides were cleaved by hK3 at the C-terminal of certain tyrosyl or glutaminyl residues with k(cat)/K(m) values of 15000-60000 M(-1) s(-1). The substrate Abz-SSIYSQTEEQ-EDDnp was cleaved at the Tyr-Ser bond with a specificity constant k(cat)/K(m) of 60000 M(-1) s(-1), making it the best substrate for hK3 described to date.     

Rapid assay to detect possible natural substrates of proteases in living cells

Proteolysis is a regulatory step in many physiological processes, but which proteases in what cellular sites are involved in activation or degradation of which peptides is not well known. We developed a rapid assay consisting of living cells and fluorogenic protease substrates to determine which bioactive peptides are possible natural substrates of a specific protease with the multifunctional or moonlighting protein CD26/dipeptidyl peptidase IV (DPPIV) as a model. CD26/DPPIV catalyzes cleavage of peptides from the amino terminus of peptides with proline at the penultimate position. Many biologically active peptides, such as beta-casomorphin1-5, contain proline in the penultimate position. We incubated living Jurkat cells, which are T cells that lack CD26/DPPIV, and CD26/DPPIV-transfected Jurkat cells in the presence of the fluorogenic substrate [Ala-Pro]2-cresyl violet (Magic Red) and beta-casomorphin1-5. Fluorescent cresyl violet was generated by CD26/DPPIV-transfected Jurkat cells but not by wild-type Jurkat cells with a Km of 3.7 microM. beta-Casomorphin1-5 appeared to be a possible natural substrate of CD26/DPPIV, because it inhibited production of fluorescence competitively (Ki = 60 microM). The assay using living cells and a fluorogenic protease substrate is an efficient system to determine whether specific peptides are possible natural substrates of a particular protease.     

A simple and convenient microtiter plate assay for the detection of bactericidal antibodies to Vibrio cholerae O1 and Vibrio cholerae O139

It is believed that the correlate of protection for cholera can be determined by the serum vibriocidal assay. The currently available vibriocidal assays, based on the conventional agar plating technique, are labor intensive. We developed a simple and convenient microtiter plate assay for the detection of vibriocidal antibodies that is equally as efficient for Vibrio cholerae O1 and for V. cholerae O139. The addition of succinate and neotetrazolium made it possible to measure the growth of surviving bacterial target cells by monitoring a color change. We evaluated assay parameters (target strains, growth of target cells, complement source and concentration) that may affect the reproducibility of the method for V. cholerae O139. The results obtained with the microtiter plate assay were uniformly similar to those obtained with the conventional agar plating assay, when testing both the Inaba and Ogawa serotypes of V. cholerae O1. The microtiter plate assay was also convenient for measuring the activity of animal sera and mouse monoclonal antibodies.     

Substrate specificity of human cathepsin D using internally quenched fluorescent peptides derived from reactive site loop of kallistatin

Kallistatin, a serpin that specifically inhibits human tissue kallikrein, was demonstrated to be cleaved at the Phe-Phe bond in its reactive site loop (RSL) by cathepsin D. Internally quenched fluorescent peptides containing the amino acid sequence of kallistatin RSL were highly susceptible to hydrolysis by cathepsin D. Surprisingly, these peptides were efficiently hydrolyzed at Phe-Phe bond, despite having Lys and Ser at P2 and P2' positions, respectively, which was reported to be very unfavorable for substrates for cathepsin D. Due to the importance of cathepsin D in several physiological and pathological processes, we took the peptide containing kallistatin RSL sequence, Abz-Ala-Ile-Lys-Phe-Phe-Ser-Arg-Gln-EDDnp, as a reference substrate for a systematic specificity study of S3 to S3' protease subsites (EDDnp=N-[2,4-dinitrophenyl]-ethylenediamine and Abz=ortho-amino benzoic acid). We present in this paper some internally quenched fluorescent peptides that were efficient substrates for cathepsin D. They essentially differ from other previously described substrates by their higher kcat/Km values due, mainly, to low Km values, such as the substrate Abz-Ala-Ile-Ala-Phe-Phe-Ser-Arg-Gln-EDDnp (Km=0.27 microM, kcat=16.25 s(-1), kcat/Km=60185 microM(-1) x s(-1)).     

Site-specific antibodies define a cleavage site conserved among arenavirus GP-C glycoproteins.

Arenaviruses share a common strategy for glycoprotein synthesis and processing in which a mannose-rich precursor glycoprotein, termed GP-C in lymphocytic choriomeningitis virus (LCMV), is posttranslationally processed by oligosaccharide trimming and proteolytic cleavage to yield two structural glycoproteins, GP-1 and GP-2. Mapping the orientation and proteolytic cleavage site(s) in such polyproteins has traditionally required direct protein sequencing of one or more of the cleaved products. This technique requires rigorous purification of the products for sequencing and may be complicated by amino-terminal modifications which interfere with sequence analysis. We used an alternative approach in which synthetic peptides corresponding to sequences bracketing a potential protease cleavage site were used to raise antisera which define the boundaries of the cleaved products. We found that cleavage of LCMV GP-C to yield GP-1 and GP-2 occurs within a 9-amino-acid stretch of GP-C which contains a paired basic amino acid group -Arg-Arg-, corresponding to amino acids 262 to 263 in the LCMV GP-C sequence. By comparison with the predicted amino acid sequences of a second LCMV strain, LCMV-WE, as well as with the deduced amino acid sequences of the New World arenavirus Pichinde and the Old World virus Lassa, we observed similar conservation of paired basic and flanking amino acid sequences among these viruses.     

Design of peptide substrates for nanosecond time-resolved fluorescence assays of proteases: 2,3-diazabicyclo[2.2.2]oct-2-ene as a noninvasive fluorophore

Fluorescence protease assays were investigated with peptide substrates containing a 2,3-diazabicyclo[2.2.2]oct-2-ene-labeled asparagine (Dbo) as a fluorescent amino acid. The special characteristic of the fluorophore Dbo is its exceedingly long fluorescence lifetime (ca. 300 ns in water under air), which allows the use of nanosecond time-resolved fluorescence (Nano-TRF) detection to efficiently suppress shorter-lived background emission. In addition, the natural amino acids tryptophan and tyrosine can be employed as intramolecular fluorescence quenchers, which facilitates substrate design. Fourteen synthetic peptide substrates (composed of 2-19 amino acids) and five enzymes (trypsin, pepsin, carboxypeptidase A, leucine aminopeptidase, and chymotrypsin) were investigated and, in all 28 examined combinations, enzymatic activity was detected by monitoring the increase in steady state fluorescence with time and determining the reaction rates as kcat/Km values, which ranged from 0.2 to 80x10(6) M-1 min-1. The results suggest an excellent compatibility of the very small and hydrophilic fluorescent probe Dbo with solid-phase peptide synthesis and the investigated proteases. For all 14 peptides the fluorescence lifetimes before and after enzymatic cleavage were measured and Nano-TRF measurements were performed in 384-well microplates. The fluorescence lifetimes of the different peptides provide the basis for the rational design of Dbo-based fluorescent substrates for protease assays. Measurements in Nano-TRF mode revealed, in addition to efficient suppression of background fluorescence, an increased differentiation between cleaved and uncleaved substrate. The Dbo-based assays can be adapted for high-throughput screening.     

Molecular determinants of substrate specificity for Semliki Forest virus nonstructural protease

The C-terminal cysteine protease domain of Semliki Forest virus nonstructural protein 2 (nsP2) regulates the virus life cycle by sequentially cleaving at three specific sites within the virus-encoded replicase polyprotein P1234. The site between nsP3 and nsP4 (the 3/4 site) is cleaved most efficiently. Analysis of Semliki Forest virus-specific cleavage sites with shuffled N-terminal and C-terminal half-sites showed that the main determinants of cleavage efficiency are located in the region preceding the cleavage site. Random mutagenesis analysis revealed that amino acid residues in positions P4, P3, P2, and P1 of the 3/4 cleavage site cannot tolerate much variation, whereas in the P5 position most residues were permitted. When mutations affecting cleavage efficiency were introduced into the 2/3 and 3/4 cleavage sites, the resulting viruses remained viable but had similar defects in P1234 processing as observed in the in vitro assay. Complete blockage of the 3/4 cleavage was found to be lethal. The amino acid in position P1' had a significant effect on cleavage efficiency, and in this regard the protease markedly preferred a glycine residue over the tyrosine natively present in the 3/4 site. Therefore, the cleavage sites represent a compromise between protease recognition and other requirements of the virus life cycle. The protease recognizes at least residues P4 to P1', and the P4 arginine residue plays an important role in the fast cleavage of the 3/4 site.