Grafting of features of cystatins C or B into the N-terminal region or second binding loop of cystatin A (stefin A) substantially enhances inhibition of cysteine proteinases

Replacement of the three N-terminal residues preceding the conserved Gly of cystatin A by the corresponding 10-residue long segment of cystatin C increased the affinity of the inhibitor for the major lysosomal cysteine proteinase, cathepsin B, by approximately 15-fold. This tighter binding was predominantly due to a higher overall association rate constant. Characterization of the interaction with an inactive Cys29 to Ala variant of cathepsin B indicated that the higher rate constant was a result of an increased ability of the N-terminal region of the chimeric inhibitor to promote displacement of the cathepsin B occluding loop in the second binding step. The low dissociation rate constant for the binding of cystatin A to cathepsin B was retained by the chimeric inhibitor, which therefore had a higher affinity for this enzyme than any natural cystatin identified so far. In contrast, the N-terminal substitution negligibly affected the ability of cystatin A to inhibit papain. However, substitutions of Gly75 in the second binding loop of cystatin A by Trp or His, making the loop similar to those of cystatins C or B, respectively, increased the affinity for papain by approximately 10-fold. This enhanced affinity was due to both a higher association rate constant and a lower dissociation rate constant. Modeling of complexes between the two variants and papain indicated the possibility of favorable interactions being established between the substituting residues and the enzyme. The second-loop substitutions negligibly affected or moderately reduced the affinity for cathepsin B. Together, these results show that the inhibitory ability of cystatins can be substantially improved by protein engineering.     

The contribution of N-terminal region residues of cystatin A (stefin A) to the affinity and kinetics of inhibition of papain, cathepsin B, and cathepsin L.

The affinity and kinetics of binding of three N-terminally truncated variants of the cysteine proteinase inhibitor cystatin A to cysteine proteinases were characterized. Deletion of Met-1 only minimally altered the inhibitory properties of the protein. However, deletion also of Ile-2 resulted in reduced affinities of 900-, >/=3-, and 200-fold for papain and cathepsins L and B, respectively. Further truncation of Pro-3 substantially increased the inhibition constants to approximately 0.5 microM for papain and cathepsin L and to 60 microM for cathepsin B, reflecting additionally 2 x 10(3)-, 2 x 10(4)-, and 400-fold decreased affinities, respectively. The reductions in affinity shown by the latter mutant indicate that the N-terminal region contributes about 40% of the total free energy of binding of cystatin A to cysteine proteinases. Moreover, Pro-3 and to a lesser extent Ile-2 are the residues responsible for this binding energy. The reduced affinities for papain and cathepsin L were due only to higher dissociation rate constants, whereas both lower association and higher dissociation rate constants contributed to the decreased affinity for cathepsin B. These differential effects indicate that the N-terminal portion of cystatin A primarily functions by stabilizing the complexes with enzymes having easily accessible active-site clefts, e.g., papain and cathepsin L. In contrast, the N-terminal region is required also for an initial binding of cystatin A to cathepsin B, presumably by promoting the displacement of the occluding loop and allowing facile interaction of the rest of the inhibiting wedge with the active-site cleft of the enzyme.     

Importance of the second binding loop and the C-terminal end of cystatin B (stefin B) for inhibition of cysteine proteinases.

The importance of residues in the second hairpin loop and the C-terminal end of mammalian cystatin B for binding of proteinases was elucidated by mutagenesis of the bovine inhibitor. Bovine cystatin B was modeled onto the crystal structure of the human inhibitor in complex with papain with minimal structural changes. Substitution of the two deduced contact residues in the second hairpin loop, Leu-73 and His-75, with Gly resulted in appreciably reduced affinities for papain and cathepsins H and B. These losses indicated that the two residues together contribute 20-30% of the free energy of binding of cystatin B to these enzymes and that Leu-73 is responsible for most of this contribution. In contrast, the small decrease in the affinity for cathepsin L suggested that the second hairpin loop is less important for inhibition of this proteinase. Replacement of the contact residue in the C-terminal end, Tyr-97, with Ala resulted in losses in affinity for papain and cathepsins L and H that were consistent with Tyr-97 contributing 6-12% of the energy of binding of cystatin B to these enzymes. However, this substitution minimally affected the affinity for cathepsin B, indicating that the C-terminal end is of limited importance for binding of this proteinase. All affinity decreases were due predominantly to increased dissociation rate constants. These results show that both the second hairpin loop and the C-terminal end of cystatin B contribute to anchoring the inhibitor to target proteinases, each of the two regions interacting with a different domain of the enzyme. However, the relative contributions of these two interactions vary with the proteinase.     

Contributions of individual residues in the N-terminal region of cystatin B (stefin B) to inhibition of cysteine proteinases

The importance of individual residues in the N-terminal region of cystatin B for proteinase inhibition was elucidated by measurements of the affinity and kinetics of binding of N-terminally truncated, recombinant variants of the bovine inhibitor to cysteine proteinases. Removal of Met-1 caused an 8- to 10-fold lower affinity for papain and cathepsin B, decreased the affinity also for cathepsin L but only minimally affected cathepsin H affinity. Additional truncation of Met-2 further weakened the binding to papain and cathepsin B by 40-70-fold, whereas the affinity for cathepsins L and H was essentially unaffected. Removal of Cys-3 had the most drastic effects on the interactions, resulting in a further affinity decrease of approximately 1500-fold for papain, approximately 700-fold for cathepsin L and approximately 15-fold for cathepsin H; the binding to cathepsin B could not be assessed. The binding kinetics could only be evaluated for papain and cathepsin H and showed that the reduced affinities for these enzymes were predominantly due to increased dissociation rate constants. These results demonstrate that the N-terminal region of cystatin B contributes appreciably to proteinase inhibition, in contrast to previous proposals. It is responsible for 12-40% of the total binding energy of the inhibitor to the proteinases investigated, being of least importance for cathepsin H binding. Cys-3 is the most important residue of the N-terminal region for inhibition of papain, cathepsin L and cathepsin H, the role of the other residues of this region varying with the target proteinase.     

Role of the single cysteine residue, Cys 3, of human and bovine cystatin B (stefin B) in the inhibition of cysteine proteinases

Cystatin B is unique among cysteine proteinase inhibitors of the cystatin superfamily in having a free Cys in the N-terminal segment of the proteinase binding region. The importance of this residue for inhibition of target proteinases was assessed by studies of the affinity and kinetics of interaction of human and bovine wild-type cystatin B and the Cys 3-to-Ser mutants of the inhibitors with papain and cathepsins L, H, and B. The wild-type forms from the two species had about the same affinity for each proteinase, binding tightly to papain and cathepsin L and more weakly to cathepsins H and B. In general, these affinities were appreciably higher than those reported earlier, perhaps because of irreversible oxidation of Cys 3 in previous work. The Cys-to-Ser mutation resulted in weaker binding of cystatin B to all four proteinases examined, the effect varying with both the proteinase and the species variant of the inhibitor. The affinities of the human inhibitor for papain and cathepsin H were decreased by threefold to fourfold and that for cathepsin B by approximately 20-fold, whereas the reductions in the affinities of the bovine inhibitor for papain and cathepsins H and B were approximately 14-fold, approximately 10-fold and approximately 300-fold, respectively. The decreases in affinity for cathepsin L could not be properly quantified but were greater than threefold. Increased dissociation rate constants were responsible for the weaker binding of both mutants to papain. By contrast, the reduced affinities for cathepsins H and B were due to decreased association rate constants. Cys 3 of both human and bovine cystatin B is thus of appreciable importance for inhibition of cysteine proteinases, in particular cathepsin B.     

Differential effect toward inhibition of papain and cathepsin C by recombinant human salivary cystatin SN and its variants produced by a baculovirus system

Human salivary cystatin SN (CsnSN) is a member of the cystatin superfamily of cysteine proteinase inhibitors. In this study we used a baculovirus expression system to produce a full-length unaltered CsnSN and its variants. The variants were constructed with the changes in the three predicted proteinase-binding regions: the N-terminus (variant N(12-13), G12A-G13A), beta-hairpin loop I (variant L(56-58), Q56G-T57G-V58G) and beta-hairpin loop II (variant L(106-107), P106G-W107G). The secreted CsnSNs were purified using sequential spiral cartridge ultrafiltration and DE-52 radial flow chromatography. The purified proteins were examined for papain- and cathepsin C-inhibition. The wild-type CsnSN, and variants N(12-13) and L(106-107) bound tightly to papain (K(i) < 10 pM), whereas mutation in the loop I reduced binding affinity 5700-fold (K(i) = 57 nM). On the other hand, the wild-type CsnSN bound to cathepsin C less tightly (K(i) = 100 nM). The mutation in the N-terminus or loop I reduced binding affinity by 16 (K(i) = 1.6 microM)- and 19-fold (K(i) = 1.9 microM), respectively, while mutation in loop II resulted in an ineffective cathepsin C inhibitor (K(i) = 14 microM). Collectively, these results suggest that the N-terminal G12-G13 residues of CsnSN are not essential for papain inhibition but play a role in cathepsin C inhibition; residues Q56-T57-V58 in the loop I are essential for both papain and cathepsin C inhibitions, and residues P106-W107 in the loop II are not important for papain inhibition but essential for cathepsin C inhibition. These results demonstrated that CsnSN variants have different effects toward different cysteine proteinases.     

Inhibition of mammalian legumain by some cystatins is due to a novel second reactive site.

We have investigated the inhibition of the recently identified family C13 cysteine peptidase, pig legumain, by human cystatin C. The cystatin was seen to inhibit enzyme activity by stoichiometric 1:1 binding in competition with substrate. The Ki value for the interaction was 0.20 nM, i.e. cystatin C had an affinity for legumain similar to that for the papain-like family C1 cysteine peptidase, cathepsin B. However, cystatin C variants with alterations in the N-terminal region and the "second hairpin loop" that rendered the cystatin inactive against cathepsin B, still inhibited legumain with Ki values 0.2-0.3 nM. Complexes between cystatin C and papain inhibited legumain activity against benzoyl-Asn-NHPhNO2 as efficiently as did cystatin C alone. Conversely, cystatin C inhibited papain activity against benzoyl-Arg-NHPhNO2 whether or not the cystatin had been incubated with legumain, strongly indicating that the cystatin inhibited the two enzymes with non-overlapping sites. A ternary complex between legumain, cystatin C, and papain was demonstrated by gel filtration supported by immunoblotting. Screening of a panel of cystatin superfamily members showed that type 1 inhibitors (cystatins A and B) and low Mr kininogen (type 3) did not inhibit pig legumain. Of human type 2 cystatins, cystatin D was non-inhibitory, whereas cystatin E/M and cystatin F displayed strong (Ki 0.0016 nM) and relatively weak (Ki 10 nM) affinity for legumain, respectively. Sequence alignments and molecular modeling led to the suggestion that a loop located on the opposite side to the papain-binding surface, between the alpha-helix and the first strand of the main beta-pleated sheet of the cystatin structure, could be involved in legumain binding. This was corroborated by analysis of a cystatin C variant with substitution of the Asn39 residue in this loop (N39K-cystatin C); this variant showed a slight reduction in affinity for cathepsin B (Ki 1.5 nM) but >5,000-fold lower affinity for legumain (Ki >1,000 nM) than wild-type cystatin C.     

A novel type of bifunctional inhibitor directed against proteolytic activity and receptor/ligand interaction. Cystatin with a urokinase receptor binding site

Cancer invasion and metastasis is a process requiring a coordinated series of (anti-)adhesive, migratory, and pericellular proteolytic events involving various proteases such as urokinase-type plasminogen activator (uPA)/plasmin, cathepsins B and L, and matrix metalloproteases. Novel types of double-headed inhibitors directed to different tumor-associated proteolytic systems were generated by substitution of a loop in chicken cystatin, which is nonessential for cysteine protease inhibition, with uPA-derived peptides covering the human uPA receptor binding sequence uPA-(19-31). The inhibition constants of these hybrids toward cysteine proteases are similar to those of wild-type cystatin (K(i), papain (pm), 1.9-2.4; K(i), cathepsin B (nm), 1.0-1.7; K(i), cathepsin L (pm), 0.12-0.61). FACS analyses revealed that the hybrids compete for binding of uPA to the cell surface-associated uPA receptor (uPAR) expressed on human U937 cells. The simultaneous interaction of the hybrid molecules with papain and uPAR was analyzed by surface plasmon resonance. The measured K(D) value of a papain-bound cystatin variant harboring the uPAR binding sequence of uPA (chCys-uPA-(19-31)) and soluble uPAR was 17 nm (K(D) value for uPA/uPAR interaction, 5 nm). These results indicate that cystatins with a uPAR binding site are efficient inhibitors of cysteine proteases and uPA/uPAR interaction at the same time. Therefore, these compact and small bifunctional inhibitors may represent promising agents for the therapy of solid tumors.     

Recombinant chicken egg white cystatin variants of the QLVSG region.

Using recombinant DNA methods, seven cystatin variants were produced by cassette mutagenesis of a chicken egg white cystatin variant which already contains the mutations Ala3, Glu2, Phe1, Ser1-->Met, Met29-->and Met 89-->Leu. When characterized by structural and functional studies, they were all found to harbour mutations in the first hairpin loop, the so-called 'QXVXG' region, which is highly conserved within the cystatin superfamily and thought to be important for its inhibitory activity towards cysteine proteinases. They were purified to more than 90% homogeneity and analysed by SDS/PAGE, HPLC, tryptic peptide mapping, N-terminal amino acid sequencing and ELISA. Structural model building of the variants and their complexes with papain was performed using computer graphics based on the crystallographic coordinates of chicken egg white cystatin and the papain-stefin complex. Only minor conformational changes were required for modelling the mutants or complexes. Equilibrium dissociation constants and rate constants of complex formation of the variants with papain, actinidin as well as cathepsin B and L were determined by kinetic measurements using fluorogenic substrates. The single exchanges Gln53-->Glu, Gln53-->Asn, Val44-->Asp, Gly57-->Ala and the double exchanges Arg52-->Leu, Gln53-->Glu, Gln53-->Asn, Ser56-->Ala, Leu54-->Met, Gly57-->Ala reduced the inhibition of papain, actinidin and cathespin B significantly by 10-1000-fold. With the exception of the Val55-->Asp variant, the differences in the Ki values are mainly due to larger k off values, whereas the kon values seem to be more or less unaffected by the selected mutations. The effect on the inhibition of papain is generally smaller than the effects on actinidin and cathepsin B inhibition. Cathepsin L inhibition is strikingly insensitive to all mutations. These distinct effects of the inhibitor variants indicate differences in proteinase-inhibitor-protein interactions between closely related cysteine proteinases. In addition, the results verify the prediction, made earlier from sequence alignment studies and from a docking model of the chicken cystatin-papain complex, that the first hairpin loop of cystatins is essential for effective inhibition.     

Synthetic domains of cystatins linked to enkephalins are novel inhibitors of brain cathepsins L/B

Cystatin domains or homologous sequences were synthesized and tested as inhibitors of papain, and rat brain cathepsins B and L. These domains included: I, an enzyme substrate binding site containing a -GG- cleavage site (YGGFL); II, known cystatin consensus sequences (-QVVAG- or -QLVSG-); and III, the proposed ancillary site for binding of chicken cystatin to papain (-IPWLN-). A Domain II analog QVVAG(K-NH2) inhibited cathepsin L and papain with Ki 1-4 X 10(-4) M but was inactive towards cathepsin B. A peptide containing Domains I and II, YGGFL-QVVAG(K-NH2), inhibited papain and cathepsin B with Ki 10(-4)-10(-5) M, and cathepsin L with Ki 10(-6) M. The presence of Domain III in the analog YGGFL-QVVAG-IPWLN(K-NH2) resulted in a 10-fold increase in potency towards papain. These data demonstrated that putative cystatin domains are: 1) probably proximal in the intact cystatins; 2) can be linked directly to each other to yield smaller peptides active as inhibitors; 3) showed some specificity towards the three cysteine proteinases.     

A novel glycosyltransferase with a polyglutamine repeat; a new candidate for GD1alpha synthase (ST6GalNAc V)(1)

Cystatin C with the 11 N-terminal amino acids truncated shows a much lower affinity for cysteine proteinases than the intact inhibitor. Such truncation of cystatin C is recorded after action of glycyl endopeptidase and cathepsin L. Incubation of cystatin C with papain, cathepsin B or cathepsin H led to no changes in the cystatin C molecule. Isoelectric focusing of the cathepsin L and cystatin C mixture showed the formation of two new bands. One of them appeared whether E-64 or PMSF was added or not, evidently representing a cystatin C/cathepsin L complex. The other band is the truncated cystatin C molecule. N-terminal sequencing after separation by HPLC showed that cystatin C is cleaved by cathepsin L at the Gly11-Gly12 bond. The action of cathepsin L on cystatin C may be explained by the cleavage of the scissile bond in an inappropriate complex.     

Endogenous thiol protease inhibitor from rat liver

A thiol protease inhibitor was purified from rat liver by a rapid procedure involving heat treatment of the post-lysosomal fraction, affinity chromatography on papain-Sepharose 4B and Sephadex G-75. The purified inhibitor appeared homogeneous on sodium dodecyl sulfate electrophoresis. The inhibitor had a molecular weight of about 11,500 and consisted of three forms (pI 4.9, 5.2 and 5.6). The preparation inhibited thiol proteases, such as papain, cathepsin H, cathepsin B and cathepsin L, but not serine proteases (trypsin, chymotrypsin, mast cell protease and cathepsin A) or cathepsin D.     

Down-regulation of human extracellular cysteine protease inhibitors by the secreted staphylococcal cysteine proteases, staphopain A and B

Of seven human cystatins investigated, none inhibited the cysteine proteases staphopain A and B secreted by the human pathogen Staphylococcus aureus. Rather, the extracellular cystatins C, D and E/M were hydrolyzed by both staphopains. Based on MALDI-TOF time-course experiments, staphopain A cleavage of cystatin C and D should be physiologically relevant and occur upon S. aureus infection. Staphopain A hydrolyzed the Gly11 bond of cystatin C and the Ala10 bond of cystatin D with similar Km values of approximately 33 and 32 microM, respectively. Such N-terminal truncation of cystatin C caused >300-fold lower inhibition of papain, cathepsin B, L and K, whereas the cathepsin H activity was compromised by a factor of ca. 10. Similarly, truncation of cystatin D caused alleviated inhibition of all endogenous target enzymes investigated. The normal activity of the cystatins is thus down-regulated, indicating that the bacterial enzymes can cause disturbance of the host protease-inhibitor balance. To illustrate the in vivo consequences, a mixed cystatin C assay showed release of cathepsin B activity in the presence of staphopain A. Results presented for the specificity of staphopains when interacting with cystatins as natural protein substrates could aid in the development of therapeutic agents directed toward these proteolytic virulence factors.     

Human cathepsin X: A cysteine protease with unique carboxypeptidase activity.

Cathepsin X is a novel cysteine protease which was identified recently from the EST (expressed sequence tags) database. In a homology model of the mature cathepsin X, a unique three residue insertion between the Gln22 of the oxyanion hole and the active site Cys31 was found to be located in the primed region of the binding cleft as part of a surface loop corresponding to residues His23 to Tyr27, which we have termed the "mini-loop". From the model, it became apparent that this distinctive structural feature might confer exopeptidase activity to the enzyme. To verify this hypothesis, human procathepsin X was expressed in Pichia pastoris and converted to mature cathepsin X using small amounts of human cathepsin L. Cathepsin X was found to display excellent carboxypeptidase activity against the substrate Abz-FRF(4NO(2)), with a k(cat)/K(M) value of 1.23 x 10(5) M(-)(1) s(-)(1) at the optimal pH of 5.0. However, the activity of cathepsin X against the substrates Cbz-FR-MCA and Abz-AFRSAAQ-EDDnp was found to be extremely low, with k(cat)/K(M) values lower than 70 M(-)(1) s(-)(1). Therefore, cathepsin X displays a stricter exopeptidase activity than cathepsin B. No inhibition of cathepsin X by cystatin C could be detected up to a concentration of 4 microM of inhibitor. From a model of the protease complexed with Cbz-FRF, the bound carboxypeptidase substrate is predicted to establish a number of favorable contacts within the cathepsin X binding site, in particular with residues His23 and Tyr27 from the mini-loop. The presence of the mini-loop restricts the accessibility of cystatin C as well as of the endopeptidase and MCA substrates in the primed subsites of the protease. The marked structural and functional differences of cathepsin X relative to other members of the papain family of cysteine proteases will be of great value in designing specific inhibitors useful as research tools to investigate the physiological and potential pathological roles of this novel enzyme.     

Potency and selectivity of inhibition of cathepsin K, L and S by their respective propeptides.

The prodomains of several cysteine proteases of the papain family have been shown to be potent inhibitors of their parent enzymes. An increased interest in cysteine proteases inhibitors has been generated with potential therapeutic targets such as cathepsin K for osteoporosis and cathepsin S for immune modulation. The propeptides of cathepsin S, L and K were expressed as glutathione S-transferase-fusion proteins in Escherichia coli. The proteins were purified on glutathione affinity columns and the glutathione S-transferase was removed by thrombin cleavage. All three propeptides were tested for inhibitor potency and found to be selective within the cathepsin L subfamily (cathepsins K, L and S) compared with cathepsin B or papain. Inhibition of cathepsin K by either procathepsin K, L or S was time-dependent and occurred by an apparent one-step mechanism. The cathepsin K propeptide had a Ki of 3.6-6.3 nM for each of the three cathepsins K, L and S. The cathepsin L propeptide was at least a 240-fold selective inhibitor of cathepsin K (Ki = 0.27 nM) and cathepsin L (Ki = 0.12 nM) compared with cathepsin S (Ki = 65 nM). Interestingly, the cathepsin S propeptide was more selective for inhibition of cathepsin L (Ki = 0.46 nM) than cathepsin S (Ki = 7.6 nM) itself or cathepsin K (Ki = 7.0 nM). This is in sharp contrast to previously published data demonstrating that the cathepsin S propeptide is equipotent for inhibition of human cathepsin S and rat and paramecium cathepsin L [Maubach, G., Schilling, K., Rommerskirch, W., Wenz, I., Schultz, J. E., Weber, E. & Wiederanders, B. (1997), Eur J. Biochem. 250, 745-750]. These results demonstrate that limited selectivity of inhibition can be measured for the procathepsins K, L and S vs. the parent enzymes, but selective inhibition vs. cathepsin B and papain was obtained.     

Internalization of cystatin C in human cell lines

Altered protease activity is considered important for tumour invasion and metastasis, processes in which the cysteine proteases cathepsin B and L are involved. Their natural inhibitor cystatin C is a secreted protein, suggesting that it functions to control extracellular protease activity. Because cystatins added to cell cultures can inhibit polio, herpes simplex and coronavirus replication, which are intracellular processes, the internalization and intracellular regulation of cysteine proteases by cystatin C should be considered. The extension, mechanism and biological importance of this hypothetical process are unknown. We investigated whether internalization of cystatin C occurs in a set of human cell lines. Demonstrated by flow cytometry and confocal microscopy, A-431, MCF-7, MDA-MB-453, MDA-MB-468 and Capan-1 cells internalized fluorophore-conjugated cystatin C when exposed to physiological concentrations (1 microm). During cystatin C incubation, intracellular cystatin C increased after 5 min and accumulated for at least 6 h, reaching four to six times the baseline level. Western blotting showed that the internalized inhibitor was not degraded. It was functionally intact and extracts of cells exposed to cystatin C showed a higher capacity to inhibit papain and cathepsin B than control cells (decrease in enzyme activity of 34% and 37%, respectively). The uptake of labelled cystatin C was inhibited by unlabelled inhibitor, suggesting a specific pathway for the internalization. We conclude that the cysteine protease inhibitor cystatin C is internalized in significant quantities in various cancer cell lines. This is a potentially important physiological phenomenon not previously described for this group of inhibitors.     

DNA accelerates the inhibition of human cathepsin V by serpins

A balance between proteolytic activity and protease inhibition is crucial to the appropriate function of many biological processes. There is mounting evidence for the presence of both papain-like cysteine proteases and serpins with a corresponding inhibitory activity in the nucleus. Well characterized examples of cofactors fine tuning serpin activity in the extracellular milieu are known, but such modulation has not been studied for protease-serpin interactions within the cell. Accordingly, we present an investigation into the effect of a DNA-rich environment on the interaction between model serpins (MENT and SCCA-1), cysteine proteases (human cathepsin V and human cathepsin L), and cystatin A. DNA was indeed found to accelerate the rate at which MENT inhibited cathepsin V, a human orthologue of mammalian cathepsin L, up to 50-fold, but unexpectedly this effect was primarily effected via the protease and secondarily by the recruitment of the DNA as a "template" onto which cathepsin V and MENT are bound. Notably, the protease-mediated effect was found to correspond both with an altered substrate turnover and a conformational change within the protease. Consistent with this, cystatin inhibition, which relies on occlusion of the active site rather than the substrate-like behavior of serpins, was unaltered by DNA. This represents the first example of modulation of serpin inhibition of cysteine proteases by a co-factor and reveals a mechanism for differential regulation of cathepsin proteolytic activity in a DNA-rich environment.     

High throughput substrate specificity profiling of serine and cysteine proteases using solution-phase fluorogenic peptide microarrays

Proteases regulate numerous biological processes with a degree of specificity often dictated by the amino acid sequence of the substrate cleavage site. To map protease/substrate interactions, a 722-member library of fluorogenic protease substrates of the general format Ac-Ala-X-X-(Arg/Lys)-coumarin was synthesized (X=all natural amino acids except cysteine) and microarrayed with fluorescent calibration standards in glycerol nanodroplets on glass slides. Specificities of 13 serine proteases (activated protein C, plasma kallikrein, factor VIIa, factor IXabeta, factor XIa and factor alpha XIIa, activated complement C1s, C1r, and D, tryptase, trypsin, subtilisin Carlsberg, and cathepsin G) and 11 papain-like cysteine proteases (cathepsin B, H, K, L, S, and V, rhodesain, papain, chymopapain, ficin, and stem bromelain) were obtained from 103,968 separate microarray fluorogenic reactions (722 substrates x 24 different proteases x 6 replicates). This is the first comprehensive study to report the substrate specificity of rhodesain, a papain-like cysteine protease expressed by Trypanasoma brucei rhodesiense, a parasitic protozoa responsible for causing sleeping sickness. Rhodesain displayed a strong P2 preference for Leu, Val, Phe, and Tyr in both the P1=Lys and Arg libraries. Solution-phase microarrays facilitate protease/substrate specificity profiling in a rapid manner with minimal peptide library or enzyme usage.