Cleavage Specificity Analysis of Six Type II Transmembrane Serine Proteases (TTSPs) Using PICS with Proteome-Derived Peptide Libraries

Background

Type II transmembrane serine proteases (TTSPs) are a family of cell membrane tethered serine proteases with unclear roles as their cleavage site specificities and substrate degradomes have not been fully elucidated. Indeed just 52 cleavage sites are annotated in MEROPS, the database of proteases, their substrates and inhibitors.

Methodology/Principal Finding

To profile the active site specificities of the TTSPs, we applied Proteomic Identification of protease Cleavage Sites (PICS). Human proteome-derived database searchable peptide libraries were assayed with six human TTSPs (matriptase, matriptase-2, matriptase-3, HAT, DESC and hepsin) to simultaneously determine sequence preferences on the N-terminal non-prime (P) and C-terminal prime (P’) sides of the scissile bond. Prime-side cleavage products were isolated following biotinylation and identified by tandem mass spectrometry. The corresponding non-prime side sequences were derived from human proteome databases using bioinformatics. Sequencing of 2,405 individual cleaved peptides allowed for the development of the family consensus protease cleavage site specificity revealing a strong specificity for arginine in the P1 position and surprisingly a lysine in P1′ position. TTSP cleavage between R↓K was confirmed using synthetic peptides. By parsing through known substrates and known structures of TTSP catalytic domains, and by modeling the remainder, structural explanations for this strong specificity were derived.

Conclusions

Degradomics analysis of 2,405 cleavage sites revealed a similar and characteristic TTSP family specificity at the P1 and P1′ positions for arginine and lysine in unfolded peptides. The prime side is important for cleavage specificity, thus making these proteases unusual within the tryptic-enzyme class that generally has overriding non-prime side specificity.     

A Tobacco Etch Virus Protease with Increased Substrate Tolerance at the P1' position

Site-specific proteases are important tools for in vitro and in vivo cleavage of proteins. They are widely used for diverse applications, like protein purification, assessment of protein–protein interactions or regulation of protein localization, abundance or activity. Here, we report the development of a procedure to select protease variants with altered specificity based on the well-established Saccharomyces cerevisiae adenine auxotrophy-dependent red/white colony assay. We applied this method on the tobacco etch virus (TEV) protease to obtain a protease variant with altered substrate specificity at the P1’ Position. In vivo experiments with tester substrates showed that the mutated TEV protease still efficiently recognizes the sequence ENLYFQ, but has almost lost all bias for the amino acid at the P1’ Position. Thus, we generated a site-specific protease for synthetic approaches requiring in vivo generation of proteins or peptides with a specific N-terminal amino acid.     

Enzymatic activity of the Staphylococcus aureus SplB serine protease is induced by substrates containing the sequence Trp-Glu-Leu-Gln

Proteases are of significant importance for the virulence of Staphylococcus aureus. Nevertheless, their subset, the serine protease-like proteins, remains poorly characterized. Here presented is an investigation of SplB protease catalytic activity revealing that the enzyme possesses exquisite specificity and only cleaves efficiently after the sequence Trp-Glu-Leu-Gln. To understand the molecular basis for such selectivity, we solved the three-dimensional structure of SplB to 1.8 Å. Modeling of substrate binding to the protease demonstrated that selectivity relies in part on a canonical specificity pockets-based mechanism. Significantly, the conformation of residues that ordinarily form the oxyanion hole, an essential structural element of the catalytic machinery of serine proteases, is not canonical in the SplB structure. We postulate that within SplB, the oxyanion hole is only formed upon docking of a substrate containing the consensus sequence motif. It is suggested that this unusual activation mechanism is used in parallel with classical determinants to further limit enzyme specificity. Finally, to guide future development, we attempt to point at likely physiological substrates and thus the role of SplB in staphylococcal physiology.     

A strategy to profile prime and non-prime proteolytic substrate specificity

A strategy was developed to determine the prime and non-prime substrate specificity of serine, threonine and cysteine proteases. ACC positional scanning technology was employed to determine the P4-P1 non-prime site substrate specificity. The data was used to synthesize biased donor-quencher positional scanning libraries to profile the P1'-P4' prime site substrate specificity. Directed sorting using the Irori Nanokan system allowed for the archiving of multiple P1'-P4' positional scanning libraries. From these libraries focused donor-quencher libraries incorporating P4-P1 data for each protease under study could be rapidly prepared. The profiling of thrombin and caspase-3 P4-P4' substrate specificity, comparison of the library specificity data to single substrates, and the analysis of physiological cleavage sites are described.     

Miropin,a Novel Bacterial Serpin from the Periodontopathogen Tannerella forsythia,Inhibits a Broad Range of Proteases by Using Different Peptide Bonds within the Reactive Center Loop

All prokaryotic genes encoding putative serpins identified to date are found in environmental and commensal microorganisms, and only very few prokaryotic serpins have been investigated from a mechanistic standpoint. Herein, we characterized a novel serpin (miropin) from the human pathogen Tannerella forsythia, a bacterium implicated in initiation and progression of human periodontitis. In contrast to other serpins, miropin efficiently inhibited a broad range of proteases (neutrophil and pancreatic elastases, cathepsin G, subtilisin, and trypsin) with a stoichiometry of inhibition of around 3 and second-order association rate constants that ranged from 2.7 × 10⁴ (cathepsin G) to 7.1 × 10⁵ m⁻¹s⁻¹ (subtilisin). Inhibition was associated with the formation of complexes that were stable during SDS-PAGE. The unusually broad specificity of miropin for target proteases is achieved through different active sites within the reactive center loop upstream of the P1-P1′ site, which was predicted from an alignment of the primary structure of miropin with those of well studied human and prokaryotic serpins. Thus, miropin is unique among inhibitory serpins, and it has apparently evolved the ability to inhibit a multitude of proteases at the expense of a high stoichiometry of inhibition and a low association rate constant. These characteristics suggest that miropin arose as an adaptation to the highly proteolytic environment of subgingival plaque, which is exposed continually to an array of host proteases in the inflammatory exudate. In such an environment, miropin may function as an important virulence factor by protecting bacterium from the destructive activity of neutrophil serine proteases. Alternatively, it may act as a housekeeping protein that regulates the activity of endogenous T. forsythia serine proteases.     

Rat mast cell protease 4 is a beta-chymase with unusually stringent substrate recognition profile

Activated mast cells release a variety of potent inflammatory mediators including histamine, cytokines, proteoglycans, and serine proteases. The serine proteases belong to either the chymase (chymotrypsin-like substrate specificity) or tryptase (trypsin-like specificity) family. In this report we have investigated the substrate specificity of a recently identified mast cell protease, rat mast cell protease-4 (rMCP-4). Based on structural homology, rMCP-4 is predicted to belong to the chymase family, although rMCP-4 has previously not been characterized at the protein level. rMCP-4 was expressed with an N-terminal His tag followed by an enterokinase site substituting for the native activation peptide. The enterokinase-cleaved fusion protein was labeled by diisopropyl fluorophosphate, demonstrating that it is an active serine protease. Moreover, rMCP-4 hydrolyzed MeO-Suc-Arg-Ala-Tyr-pNA, thus verifying that this protease belongs to the chymase family. rMCP-4 bound to heparin, and the enzymatic activity toward MeO-Suc-Arg-Ala-Tyr-pNA was strongly enhanced in the presence of heparin. Detailed analysis of the substrate specificity was performed using peptide phage display technique. After six rounds of amplification a consensus sequence, Leu-Val-Trp-Phe-Arg-Gly, was obtained. The corresponding peptide was synthesized, and rMCP-4 was shown to cleave only the Phe-Arg bond in this peptide. This demonstrates that rMCP-4 displays a striking preference for bulky/aromatic amino acid residues in both the P1 and P2 positions.     

Amino acid preferences of retroviral proteases for amino-terminal positions in a type 1 cleavage site

The specificities of the proteases of 11 retroviruses were studied using a series of oligopeptides with amino acid substitutions in the P1, P3, and P4 positions of a naturally occurring type 1 cleavage site (Val-Ser-Gln-Asn-Tyr↓Pro-Ile-Val-Gln) in human immunodeficiency virus type 1 (HIV-1). Previously, the substrate specificity of the P2 site was studied for the same representative set of retroviral proteases, which included at least one member from each of the seven genera of the family Retroviridae (P. Bagossi, T. Sperka, A. Fehér, J. Kádas, G. Zahuczky, G. Miklóssy, P. Boross, and J. Tözsér, J. Virol. 79:4213-4218, 2005). Our enzyme set comprised the proteases of HIV-1, HIV-2, equine infectious anemia virus, avian myeloblastosis virus (AMV), Mason-Pfizer monkey virus, mouse mammary tumor virus (MMTV), Moloney murine leukemia virus, human T-lymphotropic virus type 1, bovine leukemia virus, walleye dermal sarcoma virus, and human foamy virus. Molecular models were used to interpret the similarities and differences in specificity between these retroviral proteases. The results showed that the retroviral proteases had similar preferences (Phe and Tyr) for the P1 position in this sequence context, but differences were found for the P3 and P4 positions. Importantly, the sizes of the P3 and P4 residues appear to be a major contributor for specificity. The substrate specificities correlated well with the phylogenetic tree of the retroviruses. Furthermore, while the specificities of some enzymes belonging to different genera appeared to be very similar (e.g., those of AMV and MMTV), the specificities of the primate lentiviral proteases substantially differed from that observed for a nonprimate lentiviral protease.     

Purification and Functional Characterisation of Rhinocerase,a Novel Serine Protease from the Venom of Bitis gabonica rhinoceros

Background

Serine proteases are a major component of viper venoms and are thought to disrupt several distinct elements of the blood coagulation system of envenomed victims. A detailed understanding of the functions of these enzymes is important both for acquiring a fuller understanding of the pathology of envenoming and because these venom proteins have shown potential in treating blood coagulation disorders.

Methodology/Principal Findings

In this study a novel, highly abundant serine protease, which we have named rhinocerase, has been isolated and characterised from the venom of Bitis gabonica rhinoceros using liquid phase isoelectric focusing and gel filtration. Like many viper venom serine proteases, this enzyme is glycosylated; the estimated molecular mass of the native enzyme is approximately 36kDa, which reduces to 31kDa after deglycosylation. The partial amino acid sequence shows similarity to other viper venom serine proteases, but is clearly distinct from the sequence of the only other sequenced serine protease from Bitis gabonica. Other viper venom serine proteases have been shown to exert distinct biological effects, and our preliminary functional characterization of rhinocerase suggest it to be multifunctional. It is capable of degrading α and β chains of fibrinogen, dissolving plasma clots and of hydrolysing a kallikrein substrate.

Conclusions/Significance

A novel multifunctional viper venom serine protease has been isolated and characterised. The activities of the enzyme are consistent with the known in vivo effects of Bitis gabonica envenoming, including bleeding disorders, clotting disorders and hypotension. This study will form the basis for future research to understand the mechanisms of serine protease action, and examine the potential for rhinocerase to be used clinically to reduce the risk of human haemostatic disorders such as heart attacks and strokes.     

Substrate specifity profiling of the Aspergillus fumigatus proteolytic secretome reveals consensus motifs with predominance of Ile/Leu and Phe/Tyr

Background

The filamentous fungus Aspergillus fumigatus (AF) can cause devastating infections in immunocompromised individuals. Early diagnosis improves patient outcomes but remains challenging because of the limitations of current methods. To augment the clinician''s toolkit for rapid diagnosis of AF infections, we are investigating AF secreted proteases as novel diagnostic targets. The AF genome encodes up to 100 secreted proteases, but fewer than 15 of these enzymes have been characterized thus far. Given the large number of proteases in the genome, studies focused on individual enzymes may overlook potential diagnostic biomarkers.

Methodology and Principal Findings

As an alternative, we employed a combinatorial library of internally quenched fluorogenic probes (IQFPs) to profile the global proteolytic secretome of an AF clinical isolate in vitro. Comparative protease activity profiling revealed 212 substrate sequences that were cleaved by AF secreted proteases but not by normal human serum. A central finding was that isoleucine, leucine, phenylalanine, and tyrosine predominated at each of the three variable positions of the library (44.1%, 59.1%, and 57.0%, respectively) among substrate sequences cleaved by AF secreted proteases. In contrast, fewer than 10% of the residues at each position of cleaved sequences were cationic or anionic. Consensus substrate motifs were cleaved by thermostable serine proteases that retained activity up to 50°C. Precise proteolytic cleavage sites were reliably determined by a simple, rapid mass spectrometry-based method, revealing predominantly non-prime side specificity. A comparison of the secreted protease activities of three AF clinical isolates revealed consistent protease substrate specificity fingerprints. However, secreted proteases of A. flavus, A. nidulans, and A. terreus strains exhibited striking differences in their proteolytic signatures.

Conclusions

This report provides proof-of-principle for the use of protease substrate specificity profiling to define the proteolytic secretome of Aspergillus fumigatus. Expansion of this technique to protease secretion during infection could lead to development of novel approaches to fungal diagnosis.     

Expression of serine proteases in egg-parasitic nematophagous fungi during barley root colonization

Nematophagous fungi Pochonia chlamydosporia and P. rubescens colonize endophytically barley roots. During nematode infection, serine proteases are secreted. We have investigated whether such proteases are also produced during root colonization. Polyclonal antibodies against serine protease P32 of P. rubescens cross-reacted with a related protease (VCP1) of P. chlamydosporia, but not with barley proteases. These antibodies also detected an unknown ca. 65-kDa protein, labeled hyphae and appressoria of P. chlamydosporia and strongly reduced proteolytic activity of extracts from fungus-colonized roots. Mass spectrometry (MS) of 32-kDa protein bands detected peptides homologous to VCP1 only in Pochonia-colonized roots. Peptides homologous to barley serine carboxypeptidases were found in 65 kDa bands of all roots. RT-PCR detected expression of VCP1 and a new P. chlamydosporia serine carboxypeptidase (SCP1) genes only in fungus-colonized roots. SCP1 shared limited sequence homology with VCP1 and P32. Expression in roots of proteases from nematophagous fungi could be greatly relevant for nematode biocontrol.     

A New Class of Rhomboid Protease Inhibitors Discovered by Activity-Based Fluorescence Polarization

Rhomboids are intramembrane serine proteases that play diverse biological roles, including some that are of potential therapeutical relevance. Up to date, rhomboid inhibitor assays are based on protein substrate cleavage. Although rhomboids have an overlapping substrate specificity, substrates cannot be used universally. To overcome the need for substrates, we developed a screening assay using fluorescence polarization activity-based protein profiling (FluoPol ABPP) that is compatible with membrane proteases. With FluoPol ABPP, we identified new inhibitors for the E. coli rhomboid GlpG. Among these was a structural class that has not yet been reported as rhomboid inhibitors: β-lactones. They form covalent and irreversible complexes with the active site serine of GlpG. The presence of alkyne handles on the β-lactones also allowed activity-based labeling. Overall, these molecules represent a new scaffold for future inhibitor and activity-based probe development, whereas the assay will allow inhibitor screening of ill-characterized membrane proteases.     

The gene encoding dipeptidyl aminopeptidase BI from Pseudomonas sp. WO24: cloning,sequencing and expression in Escherichia coli

We have isolated the dipeptidyl aminopeptidase BI (DAP BI) gene from the plasmid library of Pseudomonas sp. WO24 chromosomal DNA by the enzymatic plate asaay using a chromogenic substrate. The DAP BI gene, designated dap b1, was further subcloned and sequenced. Sequence analysis of an approx. 3-kb fragment revealed an open reading frame of 2169 nucleotides, which was assigned to the dap b1 gene by N-terminal and internal amino acid sequences. The predicted amino acid sequence of DAP BI containing a serine protease Gly–X–Ser–X–Gly consensus motif displays extensive homologies to the several proteases belonging to the prolyl oligopeptidase family, a novel serine protease family possessing the catalytic triad with a specific array of Ser, Asp and His in this order, which is the hallmark of the member of this family including DAP IV. The dap b1 gene was expressed in Escherichia coli and the expressed enzyme was purified about 230-fold with 2.6% recovery from the cell-free extracts. The enzymatic properties such as molecular mass, substrate specificity and effect of inhibitor were similar to the native enzyme from Pseudomonas sp. WO24.     

Identification and Characterization of Fusolisin,the Fusobacterium nucleatum Autotransporter Serine Protease

Fusobacterium nucleatum is an oral anaerobe associated with periodontal disease, adverse pregnancy outcomes and colorectal carcinoma. A serine endopeptidase of 61–65 kDa capable of damaging host tissue and of inactivating immune effectors was detected previously in F. nucleatum. Here we describe the identification of this serine protease, named fusolisin, in three oral F. nucleatum sub-species. Gel zymogram revealed fusobacterial proteolytic activity with molecular masses ranging from 55–101 kDa. All of the detected proteases were inhibited by the serine protease inhibitor PMSF. analysis revealed that all of the detected proteases are encoded by genes encoding an open reading frame (ORF) with a calculated mass of approximately 115 kDa. Bioinformatics analysis of the identified ORFs demonstrated that they consist of three domains characteristic of autotransporters of the type Va secretion system. Our results suggest that the F. nucleatum fusolisins are derived from a precursor of approximately 115 kDa. After crossing the cytoplasmic membrane and cleavage of the leader sequence, the C-terminal autotransporter domain of the remaining 96–113 kDa protein is embedded in the outer membrane and delivers the N-terminal S8 serine protease passenger domain to the outer cell surface. In most strains the N-terminal catalytic 55–65 kDa domain self cleaves and liberates itself from the autotransporter domain after its transfer across the outer cell membrane. In F. nucleatum ATCC 25586 this autocatalytic activity is less efficient resulting in a full length membrane-anchored serine protease. The mature serine protease was found to cleave after Thr, Gly, Ala and Leu residues at the P1 position. Growth of F. nucleatum in complex medium was inhibited when serine protease inhibitors were used. Additional experiments are needed to determine whether fusolisin might be used as a target for controlling fusobacterial infections.     

Selective inhibition of the collagenolytic activity of human cathepsin K by altering its S2 subsite specificity

The primary specificity of papain-like cysteine proteases (family C1, clan CA) is determined by S2-P2 interactions. Despite the high amino acid sequence identities and structural similarities between cathepsins K and L, only cathepsin K is capable of cleaving interstitial collagens in their triple helical domains. To investigate this specificity, we have engineered the S2 pocket of human cathepsin K into a cathepsin L-like subsite. Using combinatorial fluorogenic substrate libraries, the P1-P4 substrate specificity of the cathepsin K variant, Tyr67Leu/Leu205Ala, was determined and compared with those of cathepsins K and L. The introduction of the double mutation into the S2 subsite of cathepsin K rendered the unique S2 binding preference of the protease for proline and leucine residues into a cathepsin L-like preference for bulky aromatic residues. Homology modeling and docking calculations supported the experimental findings. The cathepsin L-like S2 specificity of the mutant protein and the integrity of its catalytic site were confirmed by kinetic analysis of synthetic di- and tripeptide substrates as well as pH stability and pH activity profile studies. The loss of the ability to accept proline in the S2 binding pocket by the mutant protease completely abolished the collagenolytic activity of cathepsin K whereas its overall gelatinolytic activity remained unaffected. These results indicate that Tyr67 and Leu205 play a key role in the binding of proline residues in the S2 pocket of cathepsin K and are required for its unique collagenase activity.     

Purification and characterization of a neutral serine protease from non-sulfur purple photosynthetic bacterium

A neutral serine protease was purified as a homogeneous protein from the culture broth of photosynthetic bacterium T-20 by sequential chromatographies on columns of DEAE-cellulose, Toyopearl HW 55F, hydroxyapatite, and CM-cellulose. The molecular weight was estimated to be approximately 44,000 by SDS-PAGE, while the value of approximately 80,000 was obtained when the Hedrick-Smith method was used; this suggested that the enzyme consists of two identical subunits. The isoelectric point was determined to be 6.3 by isoelectric focusing. The enzyme had a pH optimum at 7.8. Maximal enzyme activity was detected at 50°C, and the activity was stable up to 50°C for 5 min at pH 7.0–7.2. The substrate specificity of the protease was investigated with a series of synthetic peptidyl-p-nitroanilide. The best substrate examined was Suc-Ala-Ala-Pro-Phe-pNA. The protease activity was inhibited by various inhibitors of serine protease such as chymostatin, PMSF, and DFP. EDTA, which is an inhibitor of metal protease, also inhibited the protease activity, whereas inhibitors of thiol and aspartic proteases had no significant effect.     

Purification and Characterization of a Keratinolytic Serine Proteinase from Streptomyces albidoflavus

Streptomyces strain K_1-02, which was identified as a strain of Streptomyces albidoflavus, secreted at least six extracellular proteases when it was cultured on feather meal-based medium. The major keratinolytic serine proteinase was purified to homogeneity by a two-step procedure. This enzyme had a molecular weight of 18,000 and was optimally active at pH values ranging from 6 to 9.5 and at temperatures ranging from 40 to 70°C. Its sensitivity to protease inhibitors, its specificity on synthetic substrates, and its remarkably high level of NH₂-terminal sequence homology with Streptomyces griseus protease B (SGPB) showed that the new enzyme, designated SAKase, was homologous to SGPB. We tested the activity of SAKase with soluble and fibrous substrates (elastin, keratin, and type I collagen) and found that it was very specific for keratinous substrates compared to SGPB and proteinase K.     

Study of Secondary Specificity of Enteropeptidase in Comparison with Trypsin

A comparative study of secondary specificities of enteropeptidase and trypsin was performed using peptide substrates with general formula A-(Asp/Glu) _n -Lys(Arg)--B, where n = 1-4. This was the first study to demonstrate that, similar to other serine proteases, enteropeptidase has an extended secondary binding site interacting with 6-7 amino acid residues surrounding the peptide bond to be hydrolyzed. However, in the case of typical enteropeptidase substrates containing four negatively charged Asp/Glu residues at positions P2-P5, electrostatic interaction between these residues and the secondary site Lys99 of the enteropeptidase light chain is the main factor that determines hydrolysis efficiency. The secondary specificity of enteropeptidase differs from the secondary specificity of trypsin. The chromophoric synthetic enteropeptidase substrate G₅DK-F(NO₂)G (k _cat/K _m = 2380 mM^–1·min^–1) is more efficient than the fusion protein PrAD₄K-P26 (k _cat/K _m = 1260 mM^–1·min^–1).     

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.     

A lead discovery strategy driven by a comprehensive analysis of proteases in the peptide substrate space

We present here a comprehensive analysis of proteases in the peptide substrate space and demonstrate its applicability for lead discovery. Aligned octapeptide substrates of 498 proteases taken from the MEROPS peptidase database were used for the in silico analysis. A multiple‐category naïve Bayes model, trained on the two‐dimensional chemical features of the substrates, was able to classify the substrates of 365 (73%) proteases and elucidate statistically significant chemical features for each of their specific substrate positions. The positional awareness of the method allows us to identify the most similar substrate positions between proteases. Our analysis reveals that proteases from different families, based on the traditional classification (aspartic, cysteine, serine, and metallo), could have substrates that differ at the cleavage site (P1–P1′) but are similar away from it. Caspase‐3 (cysteine protease) and granzyme B (serine protease) are previously known examples of cross‐family neighbors identified by this method. To assess whether peptide substrate similarity between unrelated proteases could reliably translate into the discovery of low molecular weight synthetic inhibitors, a lead discovery strategy was tested on two other cross‐family neighbors—namely cathepsin L2 and matrix metallo proteinase 9, and calpain 1 and pepsin A. For both these pairs, a naïve Bayes classifier model trained on inhibitors of one protease could successfully enrich those of its neighbor from a different family and vice versa, indicating that this approach could be prospectively applied to lead discovery for a novel protease target with no known synthetic inhibitors.     

Allosteric Control of βII-Tryptase by a Redox Active Disulfide Bond

The S1A serine proteases function in many key biological processes such as development, immunity, and blood coagulation. S1A proteases contain a highly conserved disulfide bond (Cys¹⁹¹–Cys²²⁰ in chymotrypsin numbering) that links two β-loop structures that define the rim of the active site pocket. Mast cell βII-tryptase is a S1A protease that is associated with pathological inflammation. In this study, we have found that the conserved disulfide bond (Cys²²⁰–Cys²⁴⁸ in βII-tryptase) exists in oxidized and reduced states in the enzyme stored and secreted by mast cells. The disulfide bond has a standard redox potential of −301 mV and is stoichiometrically reduced by the inflammatory mediator, thioredoxin, with a rate constant of 350 m⁻¹ s⁻¹. The oxidized and reduced enzymes have different substrate specificity and catalytic efficiency for hydrolysis of both small and macromolecular substrates. These observations indicate that βII-tryptase activity is post-translationally regulated by an allosteric disulfide bond. It is likely that other S1A serine proteases are similarly regulated.