Distinct Dibasic Cleavage Specificities of Neuropeptide-Producing Cathepsin L and Cathepsin V Cysteine Proteases Compared to PC1/3 and PC2 Serine Proteases

Abstract Protein C inhibitor (PCI) is a plasma serine protease inhibitor (serpin) that regulates several serine proteases in coagulation and fibrinolysis including thrombin and activated protein C (APC). The physiological role of PCI, however, remains under investigation since PCI both inhibits and promotes thrombin generation. The cysteine protease, cathepsin L, has been shown to play a role in many physiological processes including cardiovascular disease, atherosclerosis, blood vessel remodeling, and tumor cell invasion. Recently, several serpins have been described to inhibit both serine and cysteine proteases and they are termed “cross-class” inhibitors. The goal of this project was to determine if PCI inhibits cathepsin L activity and if so, does this inhibition process mimic the mechanism of serine proteases. Previous studies have shown that the prototypical serpin, antithrombin (AT), inhibits the cysteine proteases papain and cathepsin L. We found that PCI is a more efficient inhibitor of cathepsin L than AT with an inhibition rate (k2) of 1.5 × 106 M−1min−1 compared to 5.2 × 104 M−1min−1 for AT. Also, PCI is a more efficient inhibitor of cathepsin L than either thrombin or APC whose inhibition rates are 5.7 × 105 M−1min−1 and 3.4 × 104 M−1min−1, respectively. In contrast to AT, PCI does not inhibit papain. Thrombin inhibition by AT and PCI is accelerated in the presence of glycosaminoglycans such as heparin and heparan sulfate. The inhibition of cathepsin L by PCI is not accelerated in the presence of heparin suggesting either that cathepsin L does not bind heparin or that heparin is not required for accelerated inhibition of cysteine proteases. Interestingly, a reactive site P1 mutant (R354A) of PCI does not inhibit thrombin but does inhibit cathepsin L at rates comparable to wild-type PCI. This implies that the P1 residue of PCI does not determine specificity for inhibition of cathepsin L unlike for thrombin and APC. We believe that the specificity is primarily determined by the hydrophobic Phe residue located at the P2 position since other serpins that inhibit cathepsin L contain either a Phe or Val at the P2 position. Mutating the P14 residue (T341R; mutation in the hinge region) of PCI results in the conversion of PCI from an inhibitor to a substrate. As expected, the PCI-P14 mutant does not inhibit either thrombin or cathepsin L. Another characteristic of the serpin inhibition mechanism is formation of a bi-molecular SDS stable complex. We found that wild-type PCI and PCI-P1 mutant both form a stable complex with cathepsin L under non-reducing conditions. Lastly, the wild-type PCI-cathepsin L interaction has a stoichiometry of inhibition (SI) value of 1.6. This indicates that PCI is an effective and possibly a physiologically relevant inhibitor of cathepsin L. Regulating cathepsin L by serpins like PCI may be a novel and new pathway of regulation of hemostasis-thrombosis, cardiovascular and metastatic diseases.

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Reversed Proteolysis—Proteases as Peptide Ligases

Catalysts ◽

10.3390/catal11010033 ◽

2020 ◽

Vol 11 (1) ◽

pp. 33

Author(s):

Peter Goettig

Keyword(s):

Serine Proteases ◽

Academic Research ◽

Building Blocks ◽

Peptide Bond ◽

Cysteine Proteases ◽

Small Peptides ◽

Natural Peptide ◽

Peptide Esters ◽

Natural Amino Acids

Historically, ligase activity by proteases was theoretically derived due to their catalyst nature, and it was experimentally observed as early as around 1900. Initially, the digestive proteases, such as pepsin, chymotrypsin, and trypsin were employed to perform in vitro syntheses of small peptides. Protease-catalyzed ligation is more efficient than peptide bond hydrolysis in organic solvents, representing control of the thermodynamic equilibrium. Peptide esters readily form acyl intermediates with serine and cysteine proteases, followed by peptide bond synthesis at the N-terminus of another residue. This type of reaction is under kinetic control, favoring aminolysis over hydrolysis. Although only a few natural peptide ligases are known, such as ubiquitin ligases, sortases, and legumains, the principle of proteases as general catalysts could be adapted to engineer some proteases accordingly. In particular, the serine proteases subtilisin and trypsin were converted to efficient ligases, which are known as subtiligase and trypsiligase. Together with sortases and legumains, they turned out to be very useful in linking peptides and proteins with a great variety of molecules, including biomarkers, sugars or building blocks with non-natural amino acids. Thus, these engineered enzymes are a promising branch for academic research and for pharmaceutical progress.

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Inhibition of Staphylococcus aureus cysteine proteases by human serpin potentially limits staphylococcal virulence

Biological Chemistry ◽

10.1515/bc.2011.044 ◽

2011 ◽

Vol 392 (5) ◽

Cited By ~ 16

Author(s):

Tomasz Kantyka ◽

Karolina Plaza ◽

Joanna Koziel ◽

Danuta Florczyk ◽

Hennig R. Stennicke ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Serine Proteases ◽

Peptide Bond ◽

Cysteine Proteases ◽

Reactive Site ◽

Association Rate ◽

High Association ◽

Endogenous Protease ◽

Covalent Complex

AbstractBacterial proteases are considered virulence factors and it is presumed that by abrogating their activity, host endogenous protease inhibitors play a role in host defense against invading pathogens. Here we present data showing thatStaphylococcus aureuscysteine proteases (staphopains) are efficiently inhibited by Squamous Cell Carcinoma Antigen 1 (SCCA1), an epithelial-derived serpin. The high association rate constant (kass) for inhibitory complex formation (1.9×104m/s and 5.8×104 m/s for staphopain A and staphopain B interaction with SCCA1, respectively), strongly suggests that SCCA1 can regulate staphopain activityin vivoat epithelial surfaces infected/colonized byS. aureus. The mechanism of staphopain inhibition by SCCA1 is apparently the same for serpin interaction with target serine proteases whereby the formation of a covalent complex result in cleavage of the inhibitory reactive site peptide bond and associated release of the C-terminal serpin fragment. Interestingly, the SCCA1 reactive site closely resembles a motif in the reactive site loop of nativeS. aureus-derived inhibitors of the staphopains (staphostatins). Given thatS. aureusis a major pathogen of epithelial surfaces, we suggest that SCCA1 functions to temper the virulence of this bacterium by inhibiting the staphopains.

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High molecular weight kininogen: localization in the unstimulated and activated platelet and activation by a platelet calpain(s)

Blood ◽

10.1182/blood.v67.1.119.bloodjournal671119 ◽

1986 ◽

Vol 67 (1) ◽

pp. 119-130 ◽

Cited By ~ 1

Author(s):

AH Schmaier ◽

PM Smith ◽

AD Purdon ◽

JG White ◽

RW Colman

Keyword(s):

Serine Proteases ◽

Cysteine Proteases ◽

Subcellular Fractionation ◽

Enzyme Linked Immunosorbent Assay ◽

Contact Phase ◽

Normal Platelet ◽

Activated Platelets ◽

Coagulant Activity ◽

Platelet Enzymes ◽

Gray Platelet Syndrome

High mol wt kininogen (HMWK), the major cofactor-substrate of the contact phase of coagulation, is contained within and secreted by platelets. Studies have been performed to localize platelet HMWK in both the unstimulated and activated platelet and to ascertain the effect of platelet enzymes on HMWK itself. On platelet subcellular fractionation, platelet HMWK was localized to alpha-granules, and platelets from a patient with a deficiency of these granules (gray platelet syndrome) had 28% normal platelet HMWK. Platelet HMWK, in addition to being secreted from the platelet, was also localized to the surface of the platelet when activated. Using a competitive enzyme- linked immunosorbent assay for HMWK as an indirect antibody consumption assay, the external membrane of thrombin-activated platelets as well as the releasate from these stimulated platelets had 17 ng HMWK antigen/10(8) platelets available, whereas unstimulated platelets and their supernatant had only 4.9 and 4.2 ng HMWK/10(8) platelets present, respectively. The anti-HMWK antibody consumption by activated normal platelets was specific for membrane-expressed platelet HMWK, since activated platelets from a patient with total kininogen deficiency did not adsorb the anti-HMWK antibody. Enzymes in the cytosolic fraction of platelets cleaved 125I-HMWK (mol wt 120,000) into a mol wt 100,000 polypeptide as well as smaller products at mol wt 74,000, mol wt 62,000, mol wt 47,000, and a few components below mol wt 45,000. No cleavage products were observed when DFP and leupeptin were present. The cleavage of HMWK was specifically prevented by inhibitors of calcium-activated cysteine proteases (leupeptin, N-ethylmaleimide, iodoacetamide, and EDTA) but not by inhibitors of serine proteases (DFP, benzamidine, soybean trypsin inhibitor, or aprotinin). Platelet cytosol increased the coagulant activity of exogenous purified HMWK with maximum HMWK coagulant activity (35-fold) occurring within ten minutes of exposure to platelet cytosol. Treatment of platelet cytosol with leupeptin prevented the increase in the coagulant activity of exogenous HMWK. These studies indicate that activated platelets express platelet HMWK on their external membrane and platelet enzymes can cleave and increase the coagulant activity of exogenous HMWK.

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Molecular Characterization of a Novel Cathepsin L in Macrobrachium nipponense and Its Function in Ovary Maturation

Frontiers in Endocrinology ◽

10.3389/fendo.2021.816813 ◽

2022 ◽

Vol 12 ◽

Author(s):

Sufei Jiang ◽

Yiwei Xiong ◽

Wenyi Zhang ◽

Junpeng Zhu ◽

Dan Cheng ◽

...

Keyword(s):

Cathepsin L ◽

Cysteine Proteases ◽

Macrobrachium Nipponense ◽

Reading Frame ◽

Physiological Processes ◽

Gonad Somatic Index ◽

Oriental River Prawn ◽

Ovary Maturation

Cathepsin L genes, which belonged to cysteine proteases, were a series of multifunctional protease and played important roles in a lot of pathological and physiological processes. In this study, we analyzed the characteristics a cathepsin L (named Mn-CL2) in the female oriental river prawn, Macrobrachium nipponense which was involved in ovary maturation. The Mn-CL2 was1,582 bp in length, including a 978 bp open reading frame that encoded 326 amino acids. The Mn-CL2 was classified into the cathepsin L group by phylogenetic analysis. Real-time PCR (qPCR) analysis indicated that Mn-CL2 was highly expressed in the hepatopancreas and ovaries of female prawns. During the different ovarian stages, Mn-CL2 expression in the hepatopancreas and ovaries peaked before ovarian maturation. In situ hybridization studies revealed that Mn-CL2 was localized in the oocyte of the ovary. Injection of Mn-CL2 dsRNA significantly reduced the expression of vitellogenin. Changes in the gonad somatic index also confirmed the inhibitory effects of Mn-CL2 dsRNA on ovary maturation. These results suggest that Mn-CL2 has a key role in promoting ovary maturation.

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A possible alternative mechanism of kinin generation in vivo by cathepsin L

Biological Chemistry ◽

10.1515/bc.2005.081 ◽

2005 ◽

Vol 386 (7) ◽

pp. 699-704 ◽

Cited By ~ 11

Author(s):

Luciano Puzer ◽

Juliana Vercesi ◽

Marcio F.M. Alves ◽

Nilana M.T. Barros ◽

Mariana S. Araujo ◽

...

Keyword(s):

Blood Pressure ◽

Enzyme Inhibitor ◽

Alternative Pathway ◽

Cathepsin L ◽

Specific Inhibitor ◽

Cysteine Proteases ◽

Hypotensive Effect ◽

Alternative Mechanism

Abstract We investigated the ability of cathepsin L to induce a hypotensive effect after intravenous injection in rats and correlated this decrease in blood pressure with kinin generation. Simultaneously with blood pressure decrease, we detected plasma kininogen depletion in the treated rats. The effect observed in vivo was abolished by pre-incubation of cathepsin L with the cysteine peptidase-specific inhibitor E-64 (1 μM) or by previous administration of the bradykinin B2 receptor antagonist JE049 (4 mg/kg). A potentiation of the hypotensive effect caused by cathepsin L was observed by previous administration of the angiotensin I-converting enzyme inhibitor captopril (5 mg/kg). In vitro studies indicated that cathepsin L excised bradykinin from the synthetic fluorogenic peptide Abz-MTSVIRRPPGFSPFRAPRV-NH2, based on the Met375–Val393 sequence of rat kininogen (Abz=o-aminobenzoic acid). In conclusion, our data indicate that in vivo cathepsin L releases a kinin-related peptide, and in vitro experiments suggest that the kinin generated is bradykinin. Although it is well known that cysteine proteases are strongly inhibited by kininogen, cathepsin L could represent an alternative pathway for kinin production in pathological processes.

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High molecular weight kininogen: localization in the unstimulated and activated platelet and activation by a platelet calpain(s)

Blood ◽

10.1182/blood.v67.1.119.119 ◽

1986 ◽

Vol 67 (1) ◽

pp. 119-130 ◽

Cited By ~ 47

Author(s):

AH Schmaier ◽

PM Smith ◽

AD Purdon ◽

JG White ◽

RW Colman

Keyword(s):

Serine Proteases ◽

Cysteine Proteases ◽

Subcellular Fractionation ◽

Enzyme Linked Immunosorbent Assay ◽

Contact Phase ◽

Normal Platelet ◽

Activated Platelets ◽

Coagulant Activity ◽

Platelet Enzymes ◽

Gray Platelet Syndrome

Abstract High mol wt kininogen (HMWK), the major cofactor-substrate of the contact phase of coagulation, is contained within and secreted by platelets. Studies have been performed to localize platelet HMWK in both the unstimulated and activated platelet and to ascertain the effect of platelet enzymes on HMWK itself. On platelet subcellular fractionation, platelet HMWK was localized to alpha-granules, and platelets from a patient with a deficiency of these granules (gray platelet syndrome) had 28% normal platelet HMWK. Platelet HMWK, in addition to being secreted from the platelet, was also localized to the surface of the platelet when activated. Using a competitive enzyme- linked immunosorbent assay for HMWK as an indirect antibody consumption assay, the external membrane of thrombin-activated platelets as well as the releasate from these stimulated platelets had 17 ng HMWK antigen/10(8) platelets available, whereas unstimulated platelets and their supernatant had only 4.9 and 4.2 ng HMWK/10(8) platelets present, respectively. The anti-HMWK antibody consumption by activated normal platelets was specific for membrane-expressed platelet HMWK, since activated platelets from a patient with total kininogen deficiency did not adsorb the anti-HMWK antibody. Enzymes in the cytosolic fraction of platelets cleaved 125I-HMWK (mol wt 120,000) into a mol wt 100,000 polypeptide as well as smaller products at mol wt 74,000, mol wt 62,000, mol wt 47,000, and a few components below mol wt 45,000. No cleavage products were observed when DFP and leupeptin were present. The cleavage of HMWK was specifically prevented by inhibitors of calcium-activated cysteine proteases (leupeptin, N-ethylmaleimide, iodoacetamide, and EDTA) but not by inhibitors of serine proteases (DFP, benzamidine, soybean trypsin inhibitor, or aprotinin). Platelet cytosol increased the coagulant activity of exogenous purified HMWK with maximum HMWK coagulant activity (35-fold) occurring within ten minutes of exposure to platelet cytosol. Treatment of platelet cytosol with leupeptin prevented the increase in the coagulant activity of exogenous HMWK. These studies indicate that activated platelets express platelet HMWK on their external membrane and platelet enzymes can cleave and increase the coagulant activity of exogenous HMWK.

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Identification and biochemical characterisation of Acanthamoeba castellanii cysteine protease 3

Parasites & Vectors ◽

10.1186/s13071-020-04474-8 ◽

2020 ◽

Vol 13 (1) ◽

Author(s):

Zhixin Wang ◽

Duo Wu ◽

Hiroshi Tachibana ◽

Meng Feng ◽

Xun-jia Cheng

Keyword(s):

Cathepsin L ◽

Mrna Level ◽

Acanthamoeba Castellanii ◽

Cysteine Proteases ◽

Hydrolytic Activity ◽

Negative Control ◽

Host Cells ◽

Signalling Pathways ◽

Corneal Epithelial ◽

Biochemical Characterisation

Abstract Background Acanthamoeba spp. are free-living amoeba that are ubiquitously distributed in the environment. This study examines pathogenic Acanthamoeba cysteine proteases (AcCPs) belonging to the cathepsin L-family and explores the mechanism of AcCP3 interaction with host cells. Methods Six AcCP genes were amplified by polymerase chain reaction (PCR). Quantitative real-time PCR was used to analyse the relative mRNA expression of AcCPs during the encystation process and between pre- and post-reactivated trophozoites. To further verify the role of AcCP3 in these processes, AcCP3 recombinant proteins were expressed in Escherichia coli, and the hydrolytic activity of AcCP3 was determined. The influence of the AcCP3 on the hydrolytic activity of trophozoites and the toxicity of trophozoites to human corneal epithelial cells (HCECs) was examined by inhibiting AcCP3 expression using siRNA. Furthermore, the levels of p-Raf and p-Erk were examined in HCECs following coculture with AcCP3 gene knockdown trophozoites by Western blotting. Results During encystation, five out of six AcCPs exhibited decreased expression, and only AcCP6 was substantially up-regulated at the mRNA level, indicating that most AcCPs were not directly correlated to encystation. Furthermore, six AcCPs exhibited increased expression level following trophozoite reactivation with HEp-2 cells, particularly AcCP3, indicating that these AcCPs might be virulent factors. After refolding of recombinant AcCP3 protein, the 27 kDa mature protein from the 34 kDa pro-protein hydrolysed host haemoglobin, collagen and albumin and showed high activity in an acidic environment. After AcCP3 knockdown, the hydrolytic activity of trophozoite crude protein against gelatin was decreased, suggesting that these trophozoites had decreased toxicity. Compared with untreated trophozoites or negative control siRNA-treated trophozoites, AcCP3-knockdown trophozoites were less able to penetrate and damage monolayers of HCECs. Western blot analysis showed that the activation levels of the Ras/Raf/Erk/p53 signalling pathways in HCECs decreased after inhibiting the expression of trophozoite AcCP3. Conclusions AcCP6 was correlated to encystation. Furthermore, AcCP3 was a virulent factor in trophozoites and participated in the activation of the Ras/Raf/Erk/p53 signalling pathways of host cells.

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