Biochemical characterization of prostasin, a channel activating protease

Human prostasin was recently identified as a potential regulator of epithelial sodium channel (ENaC) function. Through the use of positional scanning combinatorial substrate libraries, prostasin was shown to have a preference for poly-basic substrates: in position P4 preference was for arginine or lysine; in P3 preference was for histidine, lysine or arginine; in P2 preference was for basic or large hydrophobic amino acids; and in P1 preference was for arginine and lysine. P1', P2', and P3' displayed broad selectivity with the exception of a lack of activity for isoleucine, and P4' had a preference for small, unbranched, amino acids such as alanine and serine. A prostasin-preferred poly-basic cleavage site was found in the extracellular domains of the ENaC alpha- and beta-subunits, and may present a mechanism for prostasin activation. The absence of activity seen with substrates containing isoleucine in position P1' explains the inability of prostasin to autoactivate and suggests that prostasin proteolytic activity is regulated by an upstream protease. Prostasin activity was highly influenced by mono- and divalent metal ions which were potent inhibitors and substrate specific modulators of enzymatic activity. In the presence of sub-inhibitory concentrations of zinc, the activity of prostasin increased several-fold and its substrate specificity was significantly altered in favor of a strong preference for histidine in positions P3 or P4 of the substrate.     

Structural insights into the substrate specificity of human granzyme H: the functional roles of a novel RKR motif

Human granzyme H (GzmH) is constitutively expressed in human NK cells that have important roles in innate immune responses against tumors and viruses. GzmH is a chymotrypsin-like serine protease. Its substrate preference and its mechanism of substrate recognition are poorly understood. To provide structural insights into the substrate recognition mechanisms for GzmH, we solved the crystal structures of a D102N-GzmH mutant alone and in complex with a decapeptide substrate and an inhibitor to 2.2 ?, 2.4 ?, and 2.7 ?, respectively. The Thr(189), Gly(216), and Gly(226) specificity triad in the S1 pocket of GzmH defines its preference for bulky, aromatic residues (Tyr and Phe) at the P1 position. Notably, we discovered that an unusual RKR motif (Arg(39)-Lys(40)-Arg(41)), conserved only in GzmH, helps define the S3' and S4' binding regions, indicating the preference for acidic residues at the P3' and P4' sites. Disruption of the RKR motif or the acidic P3' and P4' residues in the substrate abolished the proteolytic activity of GzmH. We designed a tetrapeptide chloromethylketone inhibitor, Ac-PTSY-chloromethylketone, which can selectively and efficiently block the enzymatic and cytotoxic activity of GzmH, providing a useful tool for further studies on the function of GzmH.     

Optimization of peptide-based inhibitors of prostate-specific antigen (PSA) as targeted imaging agents for prostate cancer

Prostate-specific antigen (PSA) is a serine protease biomarker that may play a role in prostate cancer development and progression. The inhibition of PSA’s enzymatic activity with small molecule inhibitors is an attractive and, as of yet, unexploited target. Previously, we reported a series of peptidyl aldehyde and boronic acid based inhibitors of PSA. In this study, the structural requirements in the P2 and P3 positions of peptide-based PSA inhibitors are explored through the substitution of a series of natural and unnatural amino acids in these positions. This analysis demonstrated a preference for hydrophobic residues in the P2 position and amino acids with the potential to hydrogen bond in the P3 position. Using this information, a peptide boronic acid inhibitor with the sequence Cbz-Ser-Ser-Gln-Nle-(boro)-Leu was identified with a K_i for PSA of 25 nM. The attachment of a bulky metal chelating group to the amino terminal of this peptide did not adversely affect PSA inhibition. This result suggests that a platform of PSA inhibitor chelates could be developed as SPECT or PET-based imaging agents for prostate cancer.     

Specificity profiling of seven human tissue kallikreins reveals individual subsite preferences

Human tissue kallikreins (hKs) form a family of 15 closely related (chymo)trypsin-like serine proteinases. These tissue kallikreins are expressed in a wide range of tissues including the central nervous system, the salivary gland, and endocrine-regulated tissues, such as prostate, breast, or testis, and may have diverse physiological functions. For several tissue kallikreins, a clear correlation has been established between expression and different types of cancer. For example, the prostate-specific antigen (PSA or hK3) serves as tumor marker and is used to monitor therapy response. Using a novel strategy, we have cloned, expressed in Escherichia coli or in insect cells, refolded, activated, and purified the seven human tissue kallikreins hK3/PSA, hK4, hK5, hK6, hK7, hK10, and hK11. Moreover, we have determined their extended substrate specificity for the nonprime side using a positional scanning combinatorial library of tetrapeptide substrates. hK3/PSA and hK7 exhibited a chymotrypsin-like specificity preferring large hydrophobic or polar residues at the P1 position. In contrast, hK4, hK5, and less stringent hK6 displayed a trypsin-like specificity with strong preference for P1-Arg, whereas hK10 and hK11 showed an ambivalent specificity, accepting both basic and large aliphatic P1 residues. The extended substrate specificity profiles are in good agreement with known substrate cleavage sites but also in accord with experimentally solved (hK4, hK6, and hK7) or modeled structures. The specificity profiles may lead to a better understanding of human tissue kallikrein functions and assist in identifying their physiological protein substrates as well as in designing more selective inhibitors.     

Substrate profiling of tobacco etch virus protease using a novel fluorescence-assisted whole-cell assay

Site-specific proteolysis of proteins plays an important role in many cellular functions and is often key to the virulence of infectious organisms. Efficient methods for characterization of proteases and their substrates will therefore help us understand these fundamental processes and thereby hopefully point towards new therapeutic strategies. Here, a novel whole-cell in vivo method was used to investigate the substrate preference of the sequence specific tobacco etch virus protease (TEVp). The assay, which utilizes protease-mediated intracellular rescue of genetically encoded short-lived fluorescent substrate reporters to enhance the fluorescence of the entire cell, allowed subtle differences in the processing efficiency of closely related substrate peptides to be detected. Quantitative screening of large combinatorial substrate libraries, through flow cytometry analysis and cell sorting, enabled identification of optimal substrates for TEVp. The peptide, ENLYFQG, identical to the protease's natural substrate peptide, emerged as a strong consensus cleavage sequence, and position P3 (tyrosine, Y) and P1 (glutamine, Q) within the substrate peptide were confirmed as being the most important specificity determinants. In position P1', glycine (G), serine (S), cysteine (C), alanine (A) and arginine (R) were among the most prevalent residues observed, all known to generate functional TEVp substrates and largely in line with other published studies stating that there is a strong preference for short aliphatic residues in this position. Interestingly, given the complex hydrogen-bonding network that the P6 glutamate (E) is engaged in within the substrate-enzyme complex, an unexpectedly relaxed residue preference was revealed for this position, which has not been reported earlier. Thus, in the light of our results, we believe that our assay, besides enabling protease substrate profiling, also may serve as a highly competitive platform for directed evolution of proteases and their substrates.     

Substrate specificity of alkaline serine proteinase isolated from photosynthetic bacterium,Rubrivivax gelatinosus KDDS1

A novel type of fluorescence resonance energy transfer (FRET) combinatorial libraries were used for the characterization of alkaline serine proteinase produced from Rubrivivax gelatinosus KDDS1. This enzyme was the first serine proteinase characterized from photosynthetic bacteria. The proteinase was found to prefer Met and Phe at the P1 position, Ile and Lys at the P2 position, and Arg and Phe at the P3 position. To date, no serine proteinase has exhibited a preference for Met at the P1 position. Thus, the alkaline serine proteinase from R. gelatinosus KDDS1 is very unique in terms of substrate specificity. A highly sensitive substrate, Boc-Arg-Ile-Met-MCA, was synthesized for kinetic study based on the results reported here. The optimum pH of the enzyme for this substrate was pH 10.7, and the values of kcat, Km, and kcat/Km were 23.7 s(-1), 15.4 microM, and 1.54 microM(-1) s(-1), respectively.     

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.     

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.     

Substrate specificity of human kallikrein 2 (hK2) as determined by phage display technology.

Human glandular kallikrein 2 (hK2) is a trypsin-like serine protease expressed predominantly in the prostate epithelium. Recently, hK2 has proven to be a useful marker that can be used in combination with prostate specific antigen for screening and diagnosis of prostate cancer. The cleavage by hK2 of certain substrates in the proteolytic cascade suggest that the kallikrein may be involved in prostate cancer development; however, there has been very little other progress toward its biochemical characterization or elucidation of its true physiological role. In the present work, we adapt phage substrate technology to study the substrate specificity of hK2. A phage-displayed random pentapeptide library with exhaustive diversity was generated and then screened with purified hK2. Phages displaying peptides susceptible to hK2 cleavage were amplified in eight rounds of selection and genes encoding substrates were transferred from the phage to a fluorescent system using cyan fluorescent protein (derived from green fluorescent protein) that enables rapid determination of specificity constants. This study shows that hK2 has a strict preference for Arg in the P1 position, which is further enhanced by a Ser in P'1 position. The scissile bonds identified by phage display substrate selection correspond to those of the natural biological substrates of hK2, which include protein C inhibitor, semenogelins, and fibronectin. Moreover, three new putative hK2 protein substrates, shown elsewhere to be involved in the biology of the cancer, have been identified thus reinforcing the importance of hK2 in prostate cancer development.     

Studies on the subsite specificity of rat nardilysin (N-arginine dibasic convertase)

The subsite specificity of rat nardilysin was investigated using fluorogenic substrates of the type 2-aminobenzoyl-GGX(1)X(2)RKX(3)GQ-ethylenediamine-2,4- dinitrophenyl, where P(2), P(2)', and P(3) residues were varied. (The nomenclature of Schechter and Berger (Schechter, I., and Berger, A. (1967) Biochem. Biophys. Res. Commun. 27, 157-162) is used where cleavage of a peptide occurs between the P(1) and P(1)' residues, and adjacent residues are designated P(2), P(3), P(2)', P(3)', etc.) There was little effect on K(m) among different residues at any of these positions. In contrast, residues at each position affected k(cat), with P(2) residues having the greatest effect. The S(3), S(2), and S(2)' subsites differed in their amino acid preference. Tryptophan and serine, which produced poor substrates at the P(2) position, were among the best P(2)' residues. The specificity at P(3) was generally opposite that of P(2). Residues at P(2), and to a lesser extent at P(3), influenced the cleavage site. At the P(2) position, His, Phe, Tyr, Asn, or Trp produced cleavage at the amino side of the first basic residue. In contrast, a P(2) Ile or Val produced cleavage between the dibasic pair. Other residues produced intermediate effects. The pH dependence for substrate binding showed that the enzyme prefers to bind a protonated histidine. A comparison of the effect of arginine or lysine at the P(1)' or P(1) position showed that there is a tendency to cleave on the amino side of arginine and that this cleavage produces the highest k(cat) values.     

Crystal structure of a prostate kallikrein isolated from stallion seminal plasma: a homologue of human PSA

Prostate-specific kallikrein, a member of the gene family of serine proteases, was initially discovered in semen and is the most useful serum marker for prostate cancer diagnosis and prognosis. We report the crystal structure at 1.42A resolution of horse prostate kallikrein (HPK). This is the first structure of a serine protease purified from seminal plasma. HPK shares extensive sequence homology with human prostate-specific antigen (PSA), including a predicted chymotrypsin-like specificity, as suggested by the presence of a serine residue at position S1 of the specificity pocket. In contrast to other kallikreins, HPK shows a structurally distinct specificity pocket. Its entrance is blocked by the kallikrein loop, suggesting a possible protective or substrate-selective role for this loop. The HPK structure seems to be in an inactivated state and further processing might be required to allow the binding of substrate molecules. Crystal soaking experiments revealed a binding site for Zn(2+) and Hg(2+), two known PSA inhibitors.     

A human serine endopeptidase, purified with respect to activity against a peptide with phosphoserine in the P1' position, is apparently identical with prolyl endopeptidase

The present work describes the detection, purification, and characterization of a serine endopeptidase with preference for a phosphoserine in the P1' position of the substrate. During probing for the enzyme in crude extracts, as well as during its 64,000-fold purification, 32P-labeled guanidovaleryl-Arg-Ala-Ser(P)-isobutyl amide (I) was used to measure the cleavage of the Ala-Ser(P) bond. With this substrate, kcat was 1.7 s-1 and Km was 30 microM at the pH optimum, 7.5. The enzyme was classified as a serine peptidase from its reaction with a set of inhibitors, among which diisopropyl fluorophosphate was effective at low (20 microM) concentration. The endopeptidase showed an Mr of 74,000 under native as well as denaturing and reducing conditions, indicating that the native enzyme consists of only one major polypeptide chain. The molecular size and inhibition profile suggested identity of this enzyme with prolyl endopeptidase (EC 3.4.21.26). This was supported by its activity against specific substrates, such as succinyl-Gly-Pro-Leu-Pro-7-amido-4-methylcoumarin (kcat = 7.2 s-1 and Km = 290 microM), and by the inhibition of the latter activity by I. Compared with the cleavage of 100 microM I, Gly-Val-Leu-Arg-Arg-Ala-Ser-Val-Ala-Gln-Leu, after phosphorylation by cAMP-dependent protein kinase, was cleaved at the Ala-Ser(P) bond at a relative rate of 0.43, while cleavage of the Ala-Ser bond of the unphosphorylated undecapeptide was undetectable, i.e. less than 0.03. The pentapeptide Arg-Arg-Pro-Ser-Val was rapidly cleaved at the Pro-Ser bond (relative rate, 2.2). Still, the cleavage of the Pro-Ser(P) bond of the corresponding phosphorylated pentapeptide was even higher (relative rate, 4.0). These data suggest that phosphorylation of a serine residue in the P1' position of at least a few substrates of prolyl endopeptidase will increase the rate of their cleavage.     

Substrate specificity of human kallikreins 1 and 6 determined by phage display

The human tissue kallikrein (KLK) family contains 15 secreted serine proteases that are expressed in a wide range of tissues and have been implicated in different physiological functions and disease states. Of these, KLK1 has been shown to be involved in the regulation of multiple physiological processes such as blood pressure, smooth muscle contraction, and vascular cell growth. KLK6 is overexpressed in breast and ovarian cancer tissues and has been shown to cleave peptide derived from human myelin protein and Abeta amyloid peptide in vitro. Here we analyzed the substrate specificity of KLK1 and KLK6, by substrate phage display using a random octapeptide library. Consistent with earlier biochemical data, KLK1 was shown to exhibit both trypsin- and chymotrypsin-like selectivities with Tyr/Arg preferred at site P1, Ser/Arg strongly preferred at P1', and Phe/Leu at P2. KLK6 displayed trypsin-like activity, with the P1 position occupied only by Arg and a strong preference for Ser in P1'. Docking simulations of consensus peptide provide information on the identity of the enzyme residues that are responsible for substrate binding. Bioinformatic analysis suggested several putative KLK6 protein substrates, such as ionotropic glutamate receptor (GluR) and synphilin.     

Rapid determination of substrate specificity of Clostridium histolyticum beta-collagenase using an immobilized peptide library.

The molecular basis of the substrate specificity of Clostridium histolyticum beta-collagenase was investigated using a combinatorial method. An immobilized positional peptide library, which contains 24,000 sequences, was constructed with a 7-hydroxycoumarin-4-propanoyl (Cop) fluorescent group attached at the N terminus of each sequence. This immobilized peptide library was incubated with C. histolyticum beta-collagenase, releasing fluorogenic fragments in the solution phase. The relative substrate specificity (k(cat)/K(m)) for each member of the library was determined by measuring fluorescence intensity in the solution phase. Edman sequencing was used to assign structure to subsites of active substrate mixtures. Collectively, the substrate preference for subsites (P(3)-P(4)') of C. histolyticum beta-collagenase was determined. The last position on the C-terminal side in which the identity of the amino acids affects the activity of the enzyme is P(4)', and an aromatic side chain is preferred in this position. The optimal P(1)'-P(3)' extended substrate sequence is P(1)'-Gly/Ala, P(2)'-Pro/Xaa, and P(3)'-Lys/Arg/Pro/Thr/Ser. The Cop group in either the P(2) or P(3) position is required for a high substrate activity with C. histolyticum beta-collagenase. S(2) and S(3) sites of the protease play a dominant role in fixing the substrate specificity. The immobilized peptide library proved to be a powerful approach for assessing the substrate specificity of C. histolyticum beta-collagenase, so it may be applied to the study of other proteases of interest.     

The effect of changing the hydrophobic S1' subsite of thermolysin-like proteases on substrate specificity.

The hydrophobic S1' subsite is one of the major determinants of the substrate specificity of thermolysin and related M4 family proteases. In the thermolysin-like protease (TLP) produced by Bacillus stearothermophilus (TLP-ste), the hydrophobic S1' subsite is mainly formed by Phe130, Phe133, Val139 and Leu202. In the present study, we have examined the effects of replacing Leu202 by smaller (Gly, Ala, Val) and larger (Phe, Tyr) hydrophobic residues. The mutational effects showed that the wild-type S1' pocket is optimal for binding leucine side chains. Reduction of the size of residue 202 resulted in a higher efficiency towards substrates with Phe in the P1' position. Rather unexpectedly, the Leu202-->Phe and Leu202-->Tyr mutations, which were expected to decrease the size of the S1' subsite, resulted in a large increase in activity towards dipeptide substrates with Phe in the P1' position. This is probably due to the fact that 202Phe and 202Tyr adopt a second possible rotamer that opens up the subsite compared to Leu202, and also favours interactions with the substrate. To validate these results, we constructed variants of thermolysin with changes in the S1' subsite. Thermolysin and TLP-ste variants with identical S1' subsites were highly similar in terms of their preference for Phe vs. Leu in the P1' position.     

Molecular insights into substrate specificity of prostate specific antigen through structural modeling

Prostate Specific Antigen's (PSA) role as a biomarker for prostate cancer is well established but the physiological role of its serine protease activity in the pathobiology of normal prostate and prostate carcinogenesis remains largely unknown. In light of recent studies that implicate PSA's enzymatic activity in the initiation and/or progression of prostate cancer, we performed a molecular modeling study of substrate binding at the catalytic site of PSA wherein a PSA‐selective substrate (HSSKLQ) was docked in an acyl‐enzyme conformation to a three‐dimensional homology model of PSA. Additionally, virtual positional scanning studies were conducted to gain mechanistic insights into substrate recognition of PSA. Subsequently, 13 novel peptide substrates of 6‐aa length and four peptide substrates with varying length were synthesized and assayed for PSA hydrolysis to evaluate the experimental validity of docking insights. Additionally, six novel aldehyde‐containing transition state analog inhibitors were synthesized and tested for their inhibitory potencies. The experimental data on the hydrolysis rates of the newly synthesized substrates and inhibitory potencies of the aldehyde peptides agreed with the docking predictions, providing validation of the docking methodology and demonstrating its utility towards the design of substrate‐mimetic inhibitors that can be used to explore PSA's role in the pathobiology of prostate cancer. Proteins 2009. © 2009 Wiley‐Liss, Inc.     

Specificity of human cathepsin S determined by processing of peptide substrates and MHC class II-associated invariant chain

Cathepsin S (CatS) is a lysosomal cysteine protease of the papain family, the members of which possess relatively broad substrate specificities. It has distinct roles in major histocompatibility complex (MHC) class II-associated peptide loading and in antigen processing in both the MHC class I and class II pathways. It may therefore represent a target for interference with antigen presentation, which could be of value in the therapy of (auto)immune diseases. To obtain more detailed information on the specificity of CatS, we mapped its cleavage site preferences at subsites S3-S1' by in vitro processing of a peptide library. Only five amino acid residues at the substrate's P2 position allowed for cleavage by CatS under time-limited conditions. Preferences for groups of amino acid residues were also observed at positions P3, P1 and P1'. Based on these results, we developed highly CatS-sensitive peptides. After processing of MHC class II-associated invariant chain (Ii), a natural protein substrate of CatS, we identified CatS cleavage sites in Ii of which a majority matched the amino acid residue preference data obtained with peptides. These observed cleavage sites in Ii might be of relevance for its in vivo processing by CatS.     

Probing the substrate specificity of the dengue virus type 2 NS3 serine protease by using internally quenched fluorescent peptides

The NS3 (dengue virus non-structural protein 3) serine protease of dengue virus is an essential component for virus maturation, thus representing an attractive target for the development of antiviral drugs directed at the inhibition of polyprotein processing. In the present study, we have investigated determinants of substrate specificity of the dengue virus NS3 protease by using internally quenched fluorogenic peptides containing Abz (o-aminobenzoic acid; synonymous to anthranilic acid) and 3-nitrotyrosine (nY) representing both native and chimaeric polyprotein cleavage site sequences. By using this combinatorial approach, we were able to describe the substrate preferences and determinants of specificity for the dengue virus NS2B(H)-NS3pro protease. Kinetic parameters (kcat/K(m)) for the hydrolysis of peptide substrates with systematic truncations at the prime and non-prime side revealed a length preference for peptides spanning the P4-P3' residues, and the peptide Abz-RRRRSAGnY-amide based on the dengue virus capsid protein processing site was discovered as a novel and efficient substrate of the NS3 protease (kcat/K(m)=11087 M(-1) x s(-1)). Thus, while having confirmed the exclusive preference of the NS3 protease for basic residues at the P1 and P2 positions, we have also shown that the presence of basic amino acids at the P3 and P4 positions is a major specificity-determining feature of the dengue virus NS3 protease. Investigation of the substrate peptide Abz-KKQRAGVLnY-amide based on the NS2B/NS3 polyprotein cleavage site demonstrated an unexpected high degree of cleavage efficiency. Chimaeric peptides with combinations of prime and non-prime sequences spanning the P4-P4' positions of all five native polyprotein cleavage sites revealed a preponderant effect of non-prime side residues on the K(m) values, whereas variations at the prime side sequences had higher impact on kcat.     

Mechanism of inhibition of the retroviral protease by a Rous sarcoma virus peptide substrate representing the cleavage site between the gag p2 and p10 proteins.

The activity of the avian myeloblastosis virus (AMV) or the human immunodeficiency virus type 1 (HIV-1) protease on peptide substrates which represent cleavage sites found in the gag and gag-pol polyproteins of Rous sarcoma virus (RSV) and HIV-1 has been analyzed. Each protease efficiently processed cleavage site substrates found in their cognate polyprotein precursors. Additionally, in some instances heterologous activity was detected. The catalytic efficiency of the RSV protease on cognate substrates varied by as much as 30-fold. The least efficiently processed substrate, p2-p10, represents the cleavage site between the RSV p2 and p10 proteins. This peptide was inhibitory to the AMV as well as the HIV-1 and HIV-2 protease cleavage of other substrate peptides with Ki values in the 5-20 microM range. Molecular modeling of the RSV protease with the p2-p10 peptide docked in the substrate binding pocket and analysis of a series of single-amino acid-substituted p2-p10 peptide analogues suggested that this peptide is inhibitory because of the potential of a serine residue in the P1' position to interact with one of the catalytic aspartic acid residues. To open the binding pocket and allow rotational freedom for the serine in P1', there is a further requirement for either a glycine or a polar residue in P2' and/or a large amino acid residue in P3'. The amino acid residues in P1-P4 provide interactions for tight binding of the peptide in the substrate binding pocket.     

Engineering of human coagulation factor X variants activated by prostate-specific antigen

In this study, we present a novel approach for the induction of tumor vessel thrombosis using genetically modified coagulation factor X. Human factor X was engineered in its activation peptide in a way that it can be specifically activated by prostate-specific antigen (PSA), a tumor-specific proteinase secreted into the bloodstream by prostate cancer cells. For this purpose we inserted different sequences of known PSA cleavage sites from the natural substrate of PSA, semenogelin I, into the activation peptide of factor X. One FX variant (FX-V4) was further optimized by site-directed mutagenesis of the P2 position and the P5 position (FX-V4-P2YP5R). After preincubation with PSA, FX-V4-P2YP5R was able to efficiently induce coagulation in vitro. These FX variants should be useful for site-specific induction of blood coagulation in the tumor vasculature.