Natural Protease Research Articles

SLPI and trappin-2 as therapeutic agents to target airway serine proteases in inflammatory lung diseases: current and future directions

It is now clear that NSPs (neutrophil serine proteases), including elastase, Pr3 (proteinase 3) and CatG (cathepsin G) are major pathogenic determinants in chronic inflammatory disorders of the lungs. Two unglycosylated natural protease inhibitors, SLPI (secretory leucocyte protease inhibitor) and elafin, and its precursor trappin-2 that are found in the lungs, have therapeutic potential for reducing the protease-induced inflammatory response. This review examines the multifaceted roles of SLPI and elafin/trappin-2 in the context of their possible use as inhaled drugs for treating chronic lung diseases such as CF (cystic fibrosis) and COPD (chronic obstructive pulmonary disease).

Enzymes for the laundry industries: tapping the vast metagenomic pool of alkaline proteases

SummaryIn the wide field of laundry and cleaning applications, there is an unbroken need for novel detergent proteases excelling in high stability and activity and a suitable substrate range. We demonstrated the large amount of highly diverse subtilase sequences present in metagenomic DNA by recovering 57 non‐redundant subtilase sequence tags with degenerate primers. Furthermore, an activity‐ as well as a sequence homology‐based screening of metagenomic DNA libraries was carried out, using alkaline soil and habitat enrichments as a source of DNA. In this way, 18 diverse full‐length protease genes were recovered, sharing only 37–85% of their amino acid residues with already known protease genes. Active clones were biochemically characterized and subjected to a laundry application assay, leading to the identification of three promising detergent proteases. According to sequence similarity, two proteases (HP53 and HP70) can be classified as subtilases, while the third enzyme (HP23) belongs to chymotrypsin‐like S1 serine proteases, a class of enzymes that has not yet been described for the use in laundry and cleaning applications.

Fluorescence properties of native proteinase K and in the presence of SDS and DTAB

Fluorescence properties of native proteinase K and in the presence of SDS and DTAB

The Chemical Modification of Protease Isolated from Locale Bacteria Isolate Bacillus subtilis ITBCCB148 with Nitrophenolcarbonate-Polyethylene Glycol (NPC-PEG)

The research aims to study the effect of chemical modification on the stability of protease enzyme from a local bacteria isolate B. subtilis ITBCCB148 with NPC-PEG. The result showed that the native enzyme has optimum pH and temperature of 6.5 and 60°C, respectively. The stability test of the native protease at pH 6.5 and temperature 60°C for 360 minutes produce the following results: the residual activity of 5.75%, t½= 84.5 min., ki = 0.0082 min.-1, and ?Gi = 106.508 kJ mol-1. The modified enzyme with NPC-PEG (33%, 42%, and 75%), showed that the optimum pH did not changed, however, the optimum temperature shifted from 60°C to 65°C. The stability tests of the modified enzyme with NPC-PEG have increased of 2.06; 2.24; and 2.31 times, respectively, than that of the native one. The decrease of ki, the increase of t½ and ?Gi of the modified enzymes with NPC-PEG demonstrated that upon modification, the enzyme became more stable. This might due to rigidity increase of the modified enzyme, so the active structure of the enzyme is maintained and is protected from unfolding process.

Cleavage of IgG1 and IgG3 by gingipain K from Porphyromonas gingivalis may compromise host defense in progressive periodontitis.

Degradation of immunoglobulins is an effective strategy of bacteria to evade the immune system. We have tested whether human IgG is a substrate for gingipain K of Porphyromonas gingivalis and found that the enzyme can hydrolyze subclass 1 and 3 of human IgG. The heavy chain of IgG(1) was cleaved at a single site within the hinge region, generating Fab and Fc fragments. IgG(3) was also cleaved within the heavy chain, but at several sites around the CH2 region. Investigation of the enzyme kinetics of IgG proteolysis by gingipain K, using FPLC- and isothermal titration calorimetry-based assays followed by Hill plots, revealed non-Michaelis-Menten kinetics involving a mechanism of positive cooperativity. In ex vivo studies, it was shown that gingipain K retained its IgG hydrolyzing activity in human plasma despite the high content of natural protease inhibitors; that IgG(1) cleavage products were detected in gingival crevicular fluid samples from patients with severe periodontitis; and that gingipain K treatment of serum samples from patients with high antibody titers against P. gingivalis significantly hindered opsonin-dependent phagocytosis of clinical isolates of P. gingivalis by neutrophils. Altogether, these findings underline a biological function of gingipain K as an IgG protease of pathophysiological importance.

Visualization of Early Events in Acetic Acid Denaturation of HIV-1 Protease: A Molecular Dynamics Study

Protein denaturation plays a crucial role in cellular processes. In this study, denaturation of HIV-1 Protease (PR) was investigated by all-atom MD simulations in explicit solvent. The PR dimer and monomer were simulated separately in 9 M acetic acid (9 M AcOH) solution and water to study the denaturation process of PR in acetic acid environment. Direct visualization of the denaturation dynamics that is readily available from such simulations has been presented. Our simulations in 9 M AcOH reveal that the PR denaturation begins by separation of dimer into intact monomers and it is only after this separation that the monomer units start denaturing. The denaturation of the monomers is flagged off by the loss of crucial interactions between the α-helix at C-terminal and surrounding β-strands. This causes the structure to transit from the equilibrium dynamics to random non-equilibrating dynamics. Residence time calculations indicate that denaturation occurs via direct interaction of the acetic acid molecules with certain regions of the protein in 9 M AcOH. All these observations have helped to decipher a picture of the early events in acetic acid denaturation of PR and have illustrated that the α-helix and the β-sheet at the C-terminus of a native and functional PR dimer should maintain both the stability and the function of the enzyme and thus present newer targets for blocking PR function.

Coordinate expression of multiple proteins in plant cells by exploiting endogenous kex2p‐like protease activity

Simultaneous expression of multiple proteins in plants finds ample applications. Here, we examined the biotechnological application of native kex2p-like protease activity in plants for coordinate expression of multiple secretory proteins from a single transgene encoding a cleavable polyprotein precursor. We expressed a secretory red fluorescent protein (DsRed) or human cytokine (GMCSF), fused to a downstream green fluorescent protein (GFP) by a linker containing putative recognition sites of the kex2p-like protease in tobacco cells and referred to them as RKG and GKG cells, respectively. Our analyses showed that GFP is cleaved off the fusion proteins and secreted into the media by both RKG and GKG cells. The cleaved GFP product displayed the expected fluorescence characteristics. Using GFP immunoprecipitation and fluorescence analysis, the cleaved DsRed product in the RKG cells was found to be functional as well. However, DsRed was not detected in the RKG culture medium, possibly due to its tetramer formation. Cleaved and biologically active GMCSF could also be detected in GKG cell extracts, but secreted GMCSF was found to be only at a low level, likely because of instability of GMCSF protein in the medium. Processing of polyprotein precursors was observed to be similarly effective in tobacco leaf, stem and root tissues. Importantly, we also demonstrated that, via agroinfiltration, polyprotein precursors can be efficiently processed in plant species other than tobacco. Collectively, our results demonstrate the utility of native kex2p-like protease activity for the expression of multiple secretory proteins in plant cells using cleavable polyprotein precursors containing kex2p linker(s).

Characterization of plasmin, plasminogen and plasminogen activator in goat milk

Plasmin (PL), a serine-proteinase, appears to be the predominant native proteinase in milk and it is mainly associated to casein micelles which represent its substrate (Bastian and Brown, 1996). Plasmin occurs in milk together with its inactive zymogene, plasminogen (PG) (Bastian et al. 1991). The cascade of reactions leading to plasminogen activation is regulated by a complex network of molecular interactions between plasminogen activators (PA) (tissue-type and urokinase-type) and at least three types of specific PA inhibitors (Politis, 1996). Stage of lactation affects PL and PA activities: late lactation is associated with higher activity of PL and PA (Baldi et al., 1996). Plasmin in milk is responsible for the hydrolysis of α- and β-caseins (Aslam and Hurley, 1997; Trujillo et al., 1997)...

The in vitro secretome of Mycosphaerella fijiensis induces cell death in banana leaves

The hemibiotrophic filamentous fungus Mycosphaerella fijiensis causes the banana foliar disease known as black Sigatoka, responsible for major worldwide losses in the banana fruit industry. In this work the in vitro secretome of M. fijiensis was characterized. Native and denaturant polyacrylamide gel protease assays showed the M. fijiensis secretome contains protease activity capable of degrading gelatin. Necrotic lesions on leaves were produced by application of the in vitro secretome to the surface of one black Sigatoka-resistant banana wild species, one susceptible cultivar and the non-host plant Carica papaya. To distinguish if necrosis by the secretome is produced by phytotoxins or proteins, the latter ones were precipitated with ammonium sulfate and applied in native or denatured forms onto leaves of the same three plant species. Proteins applied in both preparations were able to produce necrotic lesions. Application of Pronase, a commercial bacterial protease suggested that the necrosis was, at least in part, caused by protease activity from the M. fijiensis secretome. The ability to cause necrotic lesions between M. fijiensis secreted- and ammonium sulfate-precipitated proteins, and purified lipophilic or hydrophilic phytotoxins, was compared. The results suggested that leaf necrosis arises from the combined action of non-host specific hydrolytic activities from the secreted proteins and the action of phytotoxins. This is the first characterization of the M. fijiensis protein secretome produced in vitro but, more importantly, it is also the first time the M. fijiensis secretome has been shown to contain virulence factors capable of causing necrosis to its natural host.

Protective cross talk between activated protein C and TNF signaling in vascular endothelial cells: implication of EPCR, noncanonical NF-κB, and ERK1/2 MAP kinases

Activated protein C (APC) is a natural anticoagulant protease that displays cytoprotective and antiinflammatory activities and has been demonstrated to reduce mortality of patients with severe sepsis. However, APC signaling is not fully understood. This study further investigated the antiinflammatory effects of APC in vascular endothelial cells (EC) and examined the cross talk between APC and TNF signaling. Analysis of the regulatory mechanisms mediated by APC on vascular human EC shows that APC impairs TNF signaling by triggering a preemptive activation of intracellular pathways. We found that APC signaling causes a moderate but significant induction of cell adhesion molecules (CAMs) including VCAM-1 at mRNA and protein levels. Activation of the noncanonical NF-κB and ERK1/2 are both pivotal to APC signaling leading to VCAM-1 expression. APC upregulates TNF receptor-associated factor 2 (TRAF2) and phosphorylates NF-κB p65 at Ser276 and Ser536 independently of IκB degradation. The ultimate protective antiinflammatory effect of APC in response to TNF is associated with a sustained activation of ERK1/2 and Akt while phosphorylation of NF-κB p65 is precluded. Inhibitors of ERK (PD98059 and U0126) abolish the antiinflammatory signal mediated by APC. Blocking antibodies and silencing assays also suggest that, in EC, protease-activated receptor 1 and endothelial protein C receptor (EPCR) both conduct ERK activation and VCAM-1 induction in response to APC. To conclude, APC protects EC by attenuating CAM expression during inflammation. APC engages a regulatory cross talk involving EPCR, ERK, and NF-κB that impairs TNF signaling.

Sugarcane Cystatin CaneCPI-4 inhibits Melanoma Growth by Angiogenesis Disruption

Study background: Cathepsins are lysosomal cysteine proteases that have increased expression in tumor cells, may translocate to the cell surface and be secreted. They play a role in tumor angiogenesis. Cystatins are natural cysteine protease inhibitors that can inhibit tumor development, growth and metastasis. In the present work we evaluated the potential therapeutic use of sugarcane cystatin CaneCPI-4 as an anticancer drug. Methods: Viability, migration, invasion and anchorage-independent growth were investigated in B16F10-Nex2 melanoma and HUVEC cells in the presence of CaneCPI-4. The in vivo effect of CanceCPI-4 on tumor development was assessed using a murine model. Angiogenesis in vitro was evaluated using HUVEC cells plated on Matrigel. Immunohistochemical analysis of CD34 expression in primary melanoma was also carried out. Results: Sugarcane cystatin CaneCPI-4 was not cytotoxic to melanoma or HUVEC cells growing in vitro, but efficiently inhibited melanoma cell development in vivo. CaneCPI-4 inhibited melanoma and endothelial cell migration and tumor invasion in vitro. Using a Matrigel angiogenesis assay, CaneCPI-4 at 1 mM was able to completely abolished endothelial cell sprouting in vitro. Angiogenesis inhibition was confirmed in vivo by immunohistochemistry. Conclusions: Sugarcane cystatin CaneCPI-4 inhibits melanoma development in vivo by angiogenesis disruption and inhibition of melanoma invasion, migration and anchorage-independent growth.

EMBM − A New Enzyme Mechanism-Based Method for Rational Design of Chemical Sites of Covalent Inhibitors

We introduce an enzyme mechanism-based method (EMBM) aimed at rational design of chemical sites (CS) of reaction coordinate analog inhibitors. The energy of valence reorganization of CS, caused by the formation of the enzyme-inhibitor covalent complex, is accounted for by new covalent descriptors W1 and W2. We considered CS fragments with a carbonyl reactivity center, like in native protease substrates. The W1 and W2 descriptors are calculated quantum mechanically on small molecular clusters simulating the reaction core of the formed covalent tetrahedral complex, anionic TC(O-) or neutral TC(OH). The modeling on a reaction core allows generation of various CS and corresponding TC(O-) and TC(OH) as universal building blocks of real inhibitors and their covalent complexes with serine or cysteine hydrolases. Moreover, the approach avoids the need for 3D structure of the target enzyme, so EMBM may be used for ligand-based design. We have built a chemical site of inhibitors (CSI) databank with pairs of W1 and W2 descriptors precalculated for both CH₃O(-) and CH₃S(-) nucleophiles for every collected CS fragment. We demonstrated that contribution of a CS fragment to the binding affinity of an inhibitor depends on both its covalent reorganization during the chemical transformation and its noncovalent interactions in the enzyme active site. Consequently, prediction of inhibitors binding trend can be done only by accounting for all of these factors, using W1 and W2 in combination with noncovalent QSAR descriptors.

Increased virulence using engineered protease-chitin binding domain hybrid expressed in the entomopathogenic fungus Beauveria bassiana

Insect cuticles consist mainly of interlinked networks of proteins and the highly insoluble polysaccharide, chitin. Entomopathogenic fungi, such as Beauveria bassiana, invade insects by direct penetration of host cuticles via the action of diverse hydrolases including proteases and chitinases coupled to mechanical pressure. In order to better target cuticle protein-chitin structures and accelerate penetration speed, a hybrid protease (CDEP-BmChBD) was constructed by fusion of a chitin binding domain BmChBD from Bombyx mori chitinase to the C-terminal of CDEP-1, a subtilisin-like protease from B. bassiana. Compared to the wild-type, the hybrid protease was able to bind chitin and released greater amounts of peptides/proteins from insect cuticles. The insecticidal activity of B. bassiana was enhanced by including proteases, CDEP-1 or CDEP:BmChBD produced in Pichia pastoris, as an additive, however, the augment effect of CDEP:BmChBD was significantly higher than that of CDEP-1. Expression of the hybrid protease in B. bassiana also significantly increased fungal virulence compared to wild-type and strains overexpressing the native protease. These results demonstrate that rational design virulence factor is a potential strategy for strain improvement by genetic engineering.

Substrate-induced Changes in Protease Active Site Conformation Impact on Subsequent Reactions with Substrates

Enzymatic catalysis of biochemical reactions is essential to all living systems. The "lock and key" and "induced fit" models were early contributions to our understanding of the mechanisms involved in the reaction between an enzyme and its substrate. However, whether a given substrate-induced conformation is rigid or remains flexible has not yet been determined. By measuring the enzyme activity and intrinsic fluorescence of a nonspecific Eisenia fetida protease-I with different chromogenic substrates, we show that in subsequent reactions of protease with substrates, both the "lock and key" and "induced fit" mechanisms are used depending on the degree of conformational change required. Chromozym-Th- or chromosym-Ch-induced protease conformations were unable to bind chromozym-U. The chromosym-U-induced protease conformation remained flexible and could be further induced by chromozym-Th and chromozym-Ch. When low concentrations of guanidine HCl were used to disturb the conformation of the enzyme, only small changes in intrinsic fluorescence of the chromozym-Th-induced protease were detected, in contrast to the native enzyme whose intrinsic fluorescence markedly increased. This indicates that the substrate-induced enzyme was relatively rigid compared with the native protease. Utilizing a lock and key mechanism for secondary substrate reactions may have adaptive value in that it facilitates high efficiency in enzymatic reactions.

A Protease‐Based Strategy for the Controlled Release of Therapeutic Peptides

A polypeptide containing an albumin-binding domain (ABD), an amino acid sequence linker, and glucagon-like peptide-1 (GLP-1), a potential antidiabetic agent, is designed to slowly release GLP-1 through sequential cleavage by native plasma proteases (see picture). The half-life of the polypeptide in the bloodstream is extended by its association with human serum albumin (HSA).

The effect of calciums on molecular motions of proteinase K

The native serine protease proteinase K binds two calcium cations. It has been reported that Ca(2+) removal decreased the enzyme's thermal stability and to some extent the substrate affinity, but has discrepant effects on catalytic activity of the enzyme. Molecular dynamics simulations were performed on the Ca(2+)-bound and Ca(2+)-free proteases to investigate the mechanism by which the calciums affect the structural stability, molecular motions, and catalytic activity of proteinase K. Very similar structural properties were observed between these two forms of proteinase K during simulations; and several long-lived hydrogen bonds and salt bridges common to both forms of proteinase K were found to be crucial in maintaining the local conformations around these two Ca(2+) sites. Although Ca(2+) removal enhanced the overall flexibility of proteinase K, the flexibility in a limited number of segments surrounding the substrate-binding pockets decreased. The largest differences in the equilibrium structures of the two simulations indicate that, upon the removal of Ca(2+), the large concerted motion originating from the Ca1 site can transmit to the substrate-binding regions but not to the catalytic triad residues. In conjunction with the large overlap of the essential subspaces between the two simulations, these results not only provide insight into the dynamics of the underlying molecular mechanism responsible for the unchanged enzymatic activity as well as the decreased thermal stability and substrate affinity of proteinase K upon Ca(2+) removal, but also complement the experimentally determined structural and biochemical data.

Substrate-Induced Eisenia Fetida Protease Reactions Involve Both “Induced Fit” and “Lock and Key” Mechanisms

The coupling between ligand binding and protein conformational change is the heart of biological network. “Lock and key” theory and “induced fit” theory were early contributions to our understanding for explaining how an enzyme binds to a substrate. It was accepted that the binding of a substrate to an enzyme is often accompanied by conformational changes of the enzyme. However, whether the substrate-induced complementary conformation is flexible or rigid after a catalytic reaction remains to be determined. By testing the enzyme activity and intrinsic fluorescence of a substrate non-specific Eisenia fetida protease-I with different substrates, we show that when this enzyme reacts with a first substrate, it utilises the “induced fit” mechanism. However, in its reaction with further substrates, either the “lock and key” or “induced fit” mechanisms will be used depending on the degree of conformational change required. In contrast to the high activity of the native protease, the chromozym-Th (or -Ch)-induced protease was unable to react with chromozym-U. Chromozym-U-induced enzyme, however, had high activity with chromozym-Th and chromozym-Ch. When low concentrations of GuHCl were used to disturb the conformation of the enzyme, only small changes in intrinsic fluorescence of the CTH-induced protease were detected, in contrast to the native enzyme whose intrinsic fluorescence markedly increased. This indicates a relatively high rigidity of the substrate-induced enzyme compared with the native protease. It also indicates that the “induced fit” theory is not an adequate explanation of the mechanisms involved in such reactions. Utilising a “lock and key” mechanism for such secondary reactions may have adaptive value in that it facilitates high efficiency in enzymatic reactions.

Effect of Flap Mutation I54L/M in Inhibition of Human Immunodeficiency Virus Type 1 Protease: Relationship to Drug Resistance

The HIV-1 protease enzyme is one of the prime and an utmost essentially important target towards the HIV therapy. However, one of the most complex mechanisms found in this enzyme is that, it produces resistance toward most of the drugs due to mutational changes, but still maintains activity with their natural substrates. This work focuses on mechanism of Darunavir resistance HIV-1 protease flap mutant I54M and I54L. To gain insight into why mutations confer such resistance, Docking analysis, binding energetics analysis and Molecular Dynamics simulations in explicit solvent were performed on drug resistant mutants and native HIV-1 protease. The flap mutation I54M and I54L lowers the binding affinity of Darunavir by altering the position of binding site residues in 3D space. It decreases the electrostatic and van der waals interaction energy and further reduction in total receptor-ligand interaction energy. In Darunavir resistance, the contribution of I54M mutant was more than I54L mutant. The results summarized in this paper emphasize the importance of shape complementarity and flexibility of binding residues in drug design.

Identification, expression and immunolocalization of cathepsin B3, a stage-specific antigen expressed by juvenile Fasciola gigantica

To identify antigens that could potentially be developed as vaccines against Fasciola gigantica, somatic antigens were analyzed by immunoprecipitation using pooled sera from rats infected with F. gigantica metacercariae. A prominent antigen of the newly excysted juveniles (NEJ), cathepsin B3 protease (FgCatB3), was identified by N-terminal sequencing and PCR screening of a cDNA library. Recombinant FgCatB3 (rFgCatB3) was expressed in Pichia pastoris, and shown to catalyse the digestion of gelatin, the fluorometric substrate Z-Phe-Arg-AMC and native fibronectin, suggesting that this enzyme may be involved in digesting host connective tissues during the fluke's penetration and migration in the host. Rabbit polyclonal sera against rFgCatB3 was produced and used to determine the distribution of the native cathepsin B3 protease in various developmental stages of F. gigantica. By Western blotting, cathepsin B3 was detected in the whole body (WB) extract of metacercariae and NEJ but not in 4-week-old juveniles or adult parasites which confirmed the stage-specific characteristics of cathepsin B3. Immunolocalization of cathepsin B3 protease in each parasite stage showed that high levels of FgCatB3 were present in the caecal epithelium of the metacercariae and NEJ. The differential distribution of FgCatB3 in the different life cycle stages suggests that this protease is functionally important for the juvenile stage of F. gigantica.

Reply to Klemba: Intracellular processing of the membrane-bound Pf A-M1 neutral aminopeptidase, a target for new antimalarials

In reply to Michael Klemba's letter (1), we investigated the cellular location of the PfA-M1 aminopeptidase by transfecting Plasmodium falciparum malaria with a GFP-PfA-M1 conjugate and by probing parasite-infected erythrocytes with antibodies prepared against the recombinant protein; these antibodies should bind to native full-length protease (2). Consistent with published studies (3), we observed reactivity in the cytosolic compartment only.