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Oligopeptidases are enzymes involved in the degradation of short peptides (generally less than 30 amino acids in size) which help pathogens evade the host defence mechanisms. Leptospira is a zoonotic pathogen and causes leptospirosis in mammals. Proteome analysis of Leptospira revealed the presence of oligopeptidase A (OpdA) among other membrane proteins. To study the role of oligopeptidase in leptospirosis, the OpdA of L. interrogans was cloned and expressed in Escherichia coli with a histidine tag (His-tag). The protein showed maximum expression at 37 °C with 0.5 mM of IPTG after 2 h of induction. Recombinant OpdA protein was purified to homogeneity using Ni-affinity chromatography. The purified OpdA showed more than 80% inhibition with a serine protease inhibitor but the activity was reduced to 30% with the cysteine protease inhibitor. The peptidase activity was increased significantly in the presence of Zn 2+ at a neutral pH. Inhibitor assay indicate the presence of more than one active sites for peptidase activity as reported with the OpdA of E. coli and Salmonella. Over-expression of OpdA in E. coli BL21 (DE3) did not cause any negative effects on normal cell growth and viability. The role of OpdA as virulence factor in Leptospira and its potential as a therapeutic and diagnostic target in leptospirosis is yet to be identified.

Triatomines are hematophagous arthropod vectors of Trypanosoma cruzi, the causative agent of Chagas Disease. Panstrongylus lignarius, also known as Panstrongylus herreri, is considered one of the most versatile triatomines because it can parasitize different hosts, it is found in different habitats and countries, it has sylvatic, peridomestic and domestic behavior and it is a very important vector of Chagas disease, especially in Peru. Molecules produced and secreted by salivary glands and fat body are considered of important adaptational value for triatomines because, among other functions, they subvert the host haemostatic, inflammatory and immune systems and detoxify or protect them against environmental aggressors. In this context, the elucidation of the molecules produced by these tissues is highly valuable to understanding the ability of this species to adapt and transmit pathogens. Here, we use high-throughput sequencing techniques to assemble and describe the coding sequences resulting from the transcriptome of the fat body and salivary glands of P. lignarius. The final assembly of both transcriptomes together resulted in a total of 11,507 coding sequences (CDS), which were mapped from a total of 164,676,091 reads. The CDS were subdivided according to their 10 folds overexpression on salivary glands (513 CDS) or fat body (2073 CDS). Among the families of proteins found in the salivary glands, lipocalins were the most abundant. Other ubiquitous families of proteins present in other sialomes were also present in P. lignarius, including serine protease inhibitors, apyrase and antigen-5. The unique transcriptome of fat body showed proteins related to the metabolic function of this organ. Remarkably, nearly 20% of all reads mapped to transcripts coded by Triatoma virus. The data presented in this study improve the understanding on triatomines' salivary glands and fat body function and reveal important molecules used in the interplay between vectors and vertebrate hosts.

Protease inhibitors have important function during homeostasis, inflammation and tissue injury. In this study, we described the role of Schistosoma mansoni SmKI-1 serine protease inhibitor in parasite development and as a molecule capable of regulating different models of inflammatory diseases. First, we determine that recombinant (r) SmKI-1 and its Kunitz domain but not the C-terminal region possess inhibitory activity against trypsin and neutrophil elastase (NE). To better understand the molecular basis of NE inhibition by SmKI-1, molecular docking studies were also conducted. Docking results suggest a complete blockage of NE active site by SmKI-1 Kunitz domain. Additionally, rSmKI-1 markedly inhibited the capacity of NE to kill schistosomes. In order to further investigate the role of SmKI-1 in the parasite, we designed specific siRNA to knockdown SmKI-1 in S. mansoni. SmKI-1 gene suppression in larval stage of S. mansoni robustly impact in parasite development in vitro and in vivo. To determine the ability of SmKI-1 to interfere with neutrophil migration and function, we tested SmKI-1 anti-inflammatory potential in different murine models of inflammatory diseases. Treatment with SmKI-1 rescued acetaminophen (APAP)-mediated liver damage, with a significant reduction in both neutrophil recruitment and elastase activity. In the model of gout arthritis, this protein reduced neutrophil accumulation, IL-1β secretion, hypernociception, and overall pathological score. Finally, we demonstrated the ability of SmKI-1 to inhibit early events that trigger neutrophil recruitment in pleural cavities of mice in response to carrageenan. In conclusion, SmKI-1 is a key protein in S. mansoni survival and it has the ability to inhibit neutrophil function as a promising therapeutic molecule against inflammatory diseases.

Neutrophils contain high levels of chymotrypsin-like serine proteases (NSPs) within their azurophilic granules that have a multitude of functions within the immune system. In response, the pathogen Staphylococcus aureus has evolved three potent inhibitors (Eap, EapH1, and EapH2) that protect the bacterium as well as several of its secreted virulence factors from the degradative action of NSPs. We previously showed that these so-called EAP domain proteins represent a novel class of NSP inhibitors characterized by a non-covalent inhibitory mechanism and a distinct target specificity profile. Based upon high levels of structural homology amongst the EAP proteins and the NSPs, as well as supporting biochemical data, we predicted that the inhibited complex would be similar for all EAP/NSP pairs. However, we present here evidence that EapH1 and EapH2 bind the canonical NSP, Neutrophil Elastase (NE), in distinct orientations. We discovered that alteration of EapH1 residues at the EapH1/NE interface caused a dramatic loss of affinity and inhibition of NE, while mutation of equivalent positions in EapH2 had no effect on NE binding or inhibition. Surprisingly, mutation of residues in an altogether different region of EapH2 severely impacted both the NE binding and inhibitory properties of EapH2. Even though EapH1 and EapH2 bind and inhibit NE and a second NSP, Cathepsin G, equally well, neither of these proteins interacts with the structurally related, but non-proteolytic granule protein, azurocidin. These studies expand our understanding of EAP/NSP interactions and suggest that members of this immune evasion protein family are capable of diverse target recognition modes., (© 2017 The Protein Society.)

In this study, we reported a jellyfish-derived Kazal-type serine protease inhibitor, named CcKPI1, from Cyanea capillata. CcKPI1 has a calculated molecular mass of 19.02kDa and contains three typical Kazal domains. Soluble recombinant CcKPI1 (rCcKPI1) was successfully expressed and purified. rCcKPI1 exhibited significant inhibitory activities against elastase, subtilisin A and proteinase K, but not against trypsin or chymotrypsin. Kinetic studies showed that all of the inhibitory effects of rCcKPI1 were competitive, indicating that it may be a microbial serine protease inhibitor and can exhibit antimicrobial activity. As predicted, rCcKPI1 directly bound to various microorganisms, including the Gram-positive bacteria Staphylococcus aureus and Bacillus subtilis, Gram-negative bacteria Escherichia coli, marine pathogenic vibrios Vibrio vulnificus, Vibrio cholerae, Vibrio natriegens, Vibrio mimicus, Vibrio alginolyticus and Vibrio parahaemolyticus, and fungi Candida albicans, Candida parapsilokis and Candida glabrata. In addition, rCcKPI1 inhibited the growth of most of the tested microorganisms that it bound to. These findings indicate that CcKPI1 possesses marked antibacterial and antifungal activities and may play an important role in the immune defence of C. capillata, providing a novel view for the understanding of the immune system of jellyfish and also facilitating future research on antimicrobial agents from marine natural products., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Background: Plants and insects have coexisted for million years and evolved a set of interactions which affect both organisms at different levels. Plants have developed various morphological and biochemical adaptations to cope with herbivores attacks. However, Tuta absoluta (Meyrick) (Lepidoptera: Gelechiidae) has become the major pest threatening tomato crops worldwide and without the appropriated management it can cause production losses between 80 to 100%., Results: The aim of this study was to investigate the in vivo effect of a serine proteinase inhibitor (BTI-CMe) and a cysteine proteinase inhibitor (Hv-CPI2) from barley on this insect and to examine the effect their expression has on tomato defensive responses. We found that larvae fed on tomato transgenic plants co-expressing both proteinase inhibitors showed a notable reduction in weight. Moreover, only 56% of these larvae reached the adult stage. The emerged adults showed wings deformities and reduced fertility. We also investigated the effect of proteinase inhibitors ingestion on the insect digestive enzymes. Our results showed a decrease in larval trypsin activity. Transgenes expression had no harmful effect on Nesidiocoris tenuis (Reuter) (Heteroptera: Miridae), a predator of Tuta absoluta, despite transgenic tomato plants attracted the mirid. We also found that barley cystatin expression promoted plant defense by inducing the expression of the tomato endogenous wound inducible Proteinase inhibitor 2 (Pin2) gene, increasing the production of glandular trichomes and altering the emission of volatile organic compounds., Conclusion: Our results demonstrate the usefulness of the co-expression of different proteinase inhibitors for the enhancement of plant resistance to Tuta absoluta.

During feeding with blood meal, female Aedes aegypti can transmit infectious agents, such as dengue, yellow fever, chikungunya and Zika viruses. Dengue virus causes human mortality in tropical regions of the world, and there is no specific treatment or vaccine with maximum efficiency being used for these infections. In the vector-virus interaction, the production of several molecules is modulated by both mosquitoes and invading agents. However, little information is available about these molecules in the Ae. aegypti mosquito during dengue infection. Inhibitors of the pacifastin family have been described to participate in the immune response of insects and Pac2 is the only gene of this family present in Ae. aegypti being then chosen for investigation. Pac2 was expressed in E. coli, purified and analyzed by mass spectrometry and SDS-PAGE. The Pac2 transcript was detected by qPCR, and its protein levels were assessed by Western blotting. The inhibitory activity of Pac2 was measured using its K i , IC 50 and zymography. Mosquito infections with DENV were introduced with the Brazilian ACS-46 DENV-2 strain propagated in C6/36 cells. In the present work, we showed that it is possibly involved in the interaction of the mosquitoes with the dengue virus. The Pac2 transcript was detected in larvae and in both the salivary gland and midgut of Ae. aegypti females, while the native protein was identified in females 3 h post-blood meal. Pac2 is a strong inhibitor of trypsin-like and thrombin-like proteases, which are present in 4th instar larvae midgut and females 24 h after blood meal. During DENV infection, up regulation of Pac2 expression occurs in the salivary gland and midgut. Pac2 is the first Pacifastin inhibitor member described in mosquitoes. Our results suggest that Pac2 acts on mosquito serine proteases, mainly the trypsin-like type, and is under transcriptional control by virus infection signals to allow its survival in the vector or by the mosquito as a defense mechanism against virus infection., (Copyright © 2017 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.)

Background: Serpins are a superfamily of serine peptidase inhibitors that participate in the regulation of many physiological and cell peptidase-mediated processes in all organisms (e.g. in blood clotting, complement activation, fibrinolysis, inflammation, and programmed cell death). It was postulated that in the blood-feeding members of the monogenean family Diplozoidae, serpins could play an important role in the prevention of thrombus formation, activation of complement, inflammation in the host, and/or in the endogenous regulation of protein degradation., Results: In silico analysis showed that the DNA and primary protein structures of serpin from Eudiplozoon nipponicum (EnSerp1) are similar to other members of the serpin superfamily. The inhibitory potential of EnSerp1 on four physiologically-relevant serine peptidases (trypsin, factor Xa, kallikrein, and plasmin) was demonstrated and its presence in the worm's excretory-secretory products (ESPs) was confirmed., Conclusion: EnSerp1 influences the activity of peptidases that play a role in blood coagulation, fibrinolysis, and complement activation. This inhibitory potential, together with the serpin's presence in ESPs, suggests that it is likely involved in host-parasite interactions and could be one of the molecules involved in the control of feeding and prevention of inflammatory responses., (© P. Roudnický et al., published by EDP Sciences, 2018.)

Thermoactinomyces sp. strain YT06 was isolated from poultry compost and observed to degrade integral chicken feathers completely at 60°C, resulting in the formation of 3.24 mg/ml of free amino acids from 50 ml of culture containing 10 g/l chicken feathers. Strain YT06 could grow and secrete keratinase using feather as the only carbon and nitrogen sources without other supplement, but complementation of 10 g/l sucrose and 4 g/l NaNO₃ increased the production of the keratinolytic enzyme. The maximum protease activity obtained was 110 U/ml and for keratinase was 42 U/ml. The keratinase maintained active status over a broad pH (pH 8-11) and temperature (60-75°C). It was inhibited by serine protease inhibitors and most metal ions; however, it could be stimulated by Mn²⁺ and the surfactant Tween-20. A reductive agent (β-mercaptoethanol) was observed to cleave the disulfide bond of keratin and improve the access of the enzyme to the keratinaceous substrate. Zymogram analysis showed that strain YT06 primarily secreted keratinase with a molecular mass of approximately 35 kDa. The active band was assessed by MALDI-TOF mass spectrometry and was observed to be completely identical to an alkaline serine protease from Thermoactinomyces sp. Gus2-1. Thermoactinomyces sp. strain YT06 shows great potential as a novel candidate in enzymatic processing of hard-to-degrade proteins into high-value products, such as keratinous wastes.

Effects of serine protease inhibitor from squid ovary (SOSPI) and egg white powder (EWP) on gel properties of surimi from Indian mackerel (Rastrelliger kanagurta) were investigated. Breaking force of gel increased when SOSPI levels increased up to 2%. However, EWP showed higher efficiency in increasing breaking force of resulting surimi gel. Nevertheless, SOSPI (0.5-3%) had no effect on deformation. Trichloroacetic acid-soluble peptide content in surimi gel decreased when the levels of SOSPI and EWP increased (p < .05). This was coincidental with more retained myosin heavy chain. Water holding capacity was increased with the addition of these protein additives, but resulted in lower whiteness. Microstructure study revealed that surimi gel added with 2% EWP was denser and had higher connectivity as compared to that containing 2% SOSPI. The SOSPI had no negative effect on sensory attributes and could serve as the alternative protein additive to improve gel strength of surimi., Practical Applications: Surimi from dark-fleshed fish is prone to degradation of myofibrillar proteins caused by the endogenous proteases, thereby lowering textural property. To tackle the problem, protease inhibitors (egg white, porcine plasma protein, and bovine plasma protein) have been used by the surimi industries. Due to drawbacks of some particular inhibitors (religious constraints, off-white color, off odor, etc.), the novel additives are still in demand. Ovary from squid processing industry, commonly discarded as the waste, can be an alternative natural protease inhibitor, which can improve the gel properties of surimi, especially produced from dark-fleshed fish., (© 2017 Wiley Periodicals, Inc.)

Serine protease inhibitors (SPIs) regulate protease-mediated activities by inactivating their cognate proteinases, and are involved in multiple physiological processes. SPIs have been extensively studied in vertebrates and invertebrates; however, little SPI information is available in parasitoids. Herein, we identified 57 SPI genes in total through the genome of a parasitoid wasp, Pteromalus puparum. Gene structure analyses revealed that these SPIs contain 7 SPI domains. Depending on their mode of action, these SPIs can be categorized into serpins, canonical inhibitors and alpha-2-macroglobulins (A2Ms). For serpins and canonical inhibitors, we predicted their putative inhibitory activities to trypsin/chymotrypsin/elastase-like enzymes based on the amino acids in cleaved reactive sites. Sequence alignment and phylogenetic tree indicated that some serpins similar to known functional inhibitory serpins may participate in immune responses. Transcriptome analysis also showed some canonical SPI genes displayed distinct expression patterns in the venom gland and this was confirmed by quantitative real-time PCR (qPCR) analysis, suggesting their specific physiological functions as venom proteins in suppressing host immune responses. The study provides valuable information to clarify the functions of SPIs in digestion, development, reproduction and innate immunity.

The serine protease inhibitor neuroserpin regulates the activity of tissue-type plasminogen activator (tPA) in the nervous system. Neuroserpin expression is particularly prominent at late stages of neuronal development in most regions of the central nervous system (CNS), whereas it is restricted to regions related to learning and memory in the adult brain. The physiological expression pattern of neuroserpin, its high degree of colocalization with tPA within the CNS, together with its dysregulation in neuropsychiatric disorders, suggest a role in formation and refinement of synapses. In fact, studies in cell culture and mice point to a role for neuroserpin in dendritic branching, spine morphology, and modulation of behavior. In this study, we investigated the physiological role of neuroserpin in the regulation of synaptic density, synaptic plasticity, and behavior in neuroserpin-deficient mice. In the absence of neuroserpin, mice show a significant decrease in spine-synapse density in the CA1 region of the hippocampus, while expression of the key postsynaptic scaffold protein PSD-95 is increased in this region. Neuroserpin-deficient mice show decreased synaptic potentiation, as indicated by reduced long-term potentiation (LTP), whereas presynaptic paired-pulse facilitation (PPF) is unaffected. Consistent with altered synaptic plasticity, neuroserpin-deficient mice exhibit cognitive and sociability deficits in behavioral assays. However, although synaptic dysfunction is implicated in neuropsychiatric disorders, we do not detect alterations in expression of neuroserpin in fusiform gyrus of autism patients or in dorsolateral prefrontal cortex of schizophrenia patients. Our results identify neuroserpin as a modulator of synaptic plasticity, and point to a role for neuroserpin in learning and memory., (© 2017 Reumann et al.; Published by Cold Spring Harbor Laboratory Press.)

Dysregulation of pericellular proteolysis is often required for tumor invasion and cancer progression. It has been shown that down-regulation of hepatocyte growth factor activator inhibitor-2 (HAI-2) results in activation of matriptase (a membrane-anchored serine protease), human prostate cancer cell motility and tumor growth. In this study, we further characterized if HAI-2 was a cognate inhibitor for matriptase and identified which Kunitz domain of HAI-2 was required for inhibiting matriptase and human prostate cancer cell motility. Our results show that HAI-2 overexpression suppressed matriptase-induced prostate cancer cell motility. We demonstrate that HAI-2 interacts with matriptase on cell surface and inhibits matriptase proteolytic activity. Moreover, cellular HAI-2 harnesses its Kunitz domain 1 (KD1) to inhibit matriptase activation and prostate cancer cell motility although recombinant KD1 and KD2 of HAI-2 both show an inhibitory activity and interaction with matriptase protease domain. The results together indicate that HAI-2 is a cognate inhibitor of matriptase, and KD1 of HAI-2 plays a major role in the inhibition of cellular matritptase activation as well as human prostate cancer invasion.

Dengue infection is prevailing among the people not only from the developing countries but also from the developed countries due to its high morbidity rate around the globe. Hence, due to the unavailability of any suitable vaccine for rigorous dengue virus (DENV), the only mode of its treatment is prevention. The circumstances require an urgent development of efficient and practical treatment to deal with these serotypes. The severe effects and cost of synthetic vaccines simulated researchers to find anti-viral agents from medicinal plants. Flavonoids present in medicinal plants, holds anti-viral activity and can be used as vaccine against viruses. Therefore, present study was planned to find anti-viral potential of 2500 flavonoids inhibitors against the DENVNS2B/NS3 protease through computational screening which can hinder the viral replication within the host cell. By using molecular docking, it was revealed that flavonoids showed strong and stable bonding in the binding pocket of DENV NS2B/NS3 protease and had strong interactions with catalytic triad. Drug capability and anti-dengue potential of the flavonoids was also evaluated by using different bioinformatics tools. Some flavonoids effectively blocked the catalytic triad of DENV NS2B/NS3 protease and also passed through drug ability evaluation. It can be concluded from this study that these flavonoids could act as potential inhibitors to stop the replication of DENV and there is a need to study the action of these molecules in-vitro to confirm their action and other properties.

Proteolytic activity is fundamental to survival, so it is not surprising that all living organisms have proteases, especially seine protease. This enzyme in its numerous isoforms and homologues, constitutes the quintessential offence and defence factors, in the form of surface proteins, secreted molecules, gut digestive enzymes, venom in specialised glands or plant latex, among other manifestations. Occurring as trypsin, chymotrypsin, elastase, collagenase, thrombin, subtilisin etc., it mediates a diverse array of functions, including pathological roles as inflammatory, coagulatory to haemorrhagic. This review emphasizes that despite the superficial differences in mechanisms, most health issues, be they infectious, allergic, metabolic, or neural have a common conduit. This enzyme, in its various glycosylated forms leads to signal misinterpretations, wreaking havoc. However, organisms are endowed with serine protease inhibitors which might restrain this ubiquitous yet deleterious enzyme. Hence, serine proteases-driven pathogenesis and antagonising role of inhibitors is the focal point of this critical review., (Copyright © 2016 SEICAP. Published by Elsevier España, S.L.U. All rights reserved.)

Matriptase-2 plays a pivotal role in keeping iron concentrations within a narrow physiological range in humans. The opportunity to reduce matriptase-2 proteolytic activity may open a novel possibility to treat iron overload diseases, such as hereditary hemochromatosis and thalassemia. Here, we present 23 new analogues of trypsin inhibitor SFTI-1 designed to inhibit human matriptase-2. Influence of the modifications Gly1Lys, Ile10Arg, and Phe12His, as well as the introduction of Narg in P1 or P1 and P4 positions were examined. Selected peptides were further analyzed, together with previously reported peptides, for their inhibitory activity against related human proteases, that are, matriptase-1, plasmin, thrombin and trypsin. A highly potent inhibitor of matriptase-2, the bicycylic [Arg 5 , Arg 10 , His 12 ]SFTI-1, with a K i value of 15 nm was obtained., (© 2017 Wiley Periodicals, Inc.)

Bee venom contains a variety of peptide constituents, including low-molecular-weight protease inhibitors. While the putative low-molecular-weight serine protease inhibitor Api m 6 containing a trypsin inhibitor-like cysteine-rich domain was identified from honeybee (Apis mellifera) venom, no anti-fibrinolytic or anti-microbial roles for this inhibitor have been elucidated. In this study, we identified an Asiatic honeybee (A. cerana) venom serine protease inhibitor (AcVSPI) that was shown to act as a microbial serine protease inhibitor and plasmin inhibitor. AcVSPI was found to consist of a trypsin inhibitor-like domain that displays ten cysteine residues. Interestingly, the AcVSPI peptide sequence exhibited high similarity to the putative low-molecular-weight serine protease inhibitor Api m 6, which suggests that AcVSPI is an allergen Api m 6-like peptide. Recombinant AcVSPI was expressed in baculovirus-infected insect cells, and it demonstrated inhibitory activity against trypsin, but not chymotrypsin. Additionally, AcVSPI has inhibitory effects against plasmin and microbial serine proteases; however, it does not have any detectable inhibitory effects on thrombin or elastase. Consistent with these inhibitory effects, AcVSPI inhibited the plasmin-mediated degradation of fibrin to fibrin degradation products. AcVSPI also bound to bacterial and fungal surfaces and exhibited anti-microbial activity against fungi as well as gram-positive and gram-negative bacteria. These findings demonstrate the anti-fibrinolytic and anti-microbial roles of AcVSPI as a serine protease inhibitor., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Essentials Corn Trypsin Inhibitor (CTI) is a selective inhibitor of coagulation Factor XII (FXII). Molecular modelling of the CTI-FXIIa complex suggested a canonical inhibitor binding mode. Mutagenesis revealed the CTI inhibitory loop and helices α1 and α2 mediate the interaction. This confirms that CTI inhibits FXII in canonical fashion and validates the molecular model., Summary: Background Corn trypsin inhibitor (CTI) has selectivity for the serine proteases coagulation factor XII and trypsin. CTI is in widespread use as a reagent that specifically inhibits the intrinsic pathway of blood coagulation but not the extrinsic pathway. Objectives To investigate the molecular basis of FXII inhibition by CTI. Methods We performed molecular docking of CTI, using its known crystal structure, with a model of the activated FXII (FXIIa) protease domain. The interaction model was verified by use of a panel of recombinant CTI variants tested for their ability to inhibit FXIIa enzymatic activity in a substrate cleavage assay. Results The docking predicted that: (i) the CTI central inhibitory loop P1 Arg34 side chain forms a salt bridge with the FXIIa S1 pocket Asp189 side chain; (ii) Trp22 from CTI helix α1 interacts with the FXIIa S3 pocket; and (iii) Arg43 from CTI helix α2 forms a salt bridge with FXIIa H1 pocket Asp60A. CTI amino acid substitution R34A negated all inhibitory activity, whereas the G32W, L35A, W22A and R42A/R43A substitutions reduced activity by large degrees of 108-fold, 41-fold, 158-fold, and 100-fold, respectively; the R27A, W37A, W39A and R42A substitutions had no effect. Synthetic peptides spanning CTI residues 20-44 had inhibitory activity that was three-fold to 4000-fold less than that of full-length CTI. Conclusions The data confirm the validity of a canonical model of the FXIIa-CTI interaction, with helix α1 (Trp22), central inhibitory loop (Arg34) and helix α2 (Arg43) of CTI being required for effective binding by contacting the S1, S3 and H1 pockets of FXIIa, respectively., (© 2017 The Authors. Journal of Thrombosis and Haemostasis published by Wiley Periodicals, Inc. on behalf of International Society on Thrombosis and Haemostasis.)

Neuroserpin is a serine protease inhibitor that regulates the activity of plasmin and its activators in the neuronal tissues. This study provides novel evidence of regulatory effect of the neuroserpin on plasmin proteolytic activity in the retina in glaucoma. Human retinal and vitreous tissues from control and glaucoma subjects as well as retinas from experimental glaucoma rats were analysed to establish changes in plasmin and neuroserpin activity. Neuroserpin undergoes oxidative inactivation in glaucoma which leads to augmentation of plasmin activity. Neuroserpin contains several methionine residues in addition to a conserved reactive site methionine and our study revealed enhanced oxidation of Met residues in the serpin under glaucoma conditions. Met oxidation was associated with loss of neuroserpin inhibitory activity and similar findings were observed in the retinas of superoxide dismutase (SOD) mutant mice that have increased oxidative stress. Treatment of purified neuroserpin with H2O2 further established that Met oxidation inversely correlated with its plasmin inhibitory activity. Dysregulation of the plasmin proteolytic system associated with increased degradation of the extracellular matrix (ECM) proteins in the retina. Collectively, these findings delineate a novel molecular basis of plasmin activation in glaucoma and potentially for other neuronal disorders with implications in disease associated ECM remodelling.

Aphids are phloem feeders that cause large damage globally as pest insects. They induce a variety of responses in the host plant, but not much is known about which responses are promoting or inhibiting aphid performance. Here, we investigated whether one of the responses induced in barley by the cereal aphid, bird cherry-oat aphid ( Rhopalosiphum padi L.) affects aphid performance in the model plant Arabidopsis thaliana L. A barley cDNA encoding the protease inhibitor CI2c was expressed in A. thaliana and aphid performance was studied using the generalist green peach aphid ( Myzus persicae Sulzer). There were no consistent effects on aphid settling or preference or on parameters of life span and long-term fecundity. However, short-term tests with apterous adult aphids showed lower fecundity on three of the transgenic lines, as compared to on control plants. This effect was transient, observed on days 5 to 7, but not later. The results suggest that the protease inhibitor is taken up from the tissue during probing and weakly inhibits fecundity by an unknown mechanism. The study shows that a protease inhibitor induced in barley by an essentially monocot specialist aphid can inhibit a generalist aphid in transgenic Arabidopsis., Competing Interests: The authors declare that they have no conflict of interest.

Proteins are the most prominent bioactive component in deer antler velvet. The aim of the present study was to track the fate of protein of antler velvet by protein digestomics. The peptide profile identified by LC-MS/MS and the in vitro bioactivity of antler velvet aqueous extract (AAE) were investigated in simulated gastrointestinal digestion. A total of 23, 387 and 417 peptides in AAE, gastric and pancreatic digests were identified using LC-MS/MS, respectively. Collagens, the predominant proteins, released 34 peptides in gastric digests and 146 peptides in pancreatic digests. The gastric and pancreatic digests presented dipeptidyl peptidase IV (DPP-IV) and prolyl endopeptidase (PEP) inhibition activities. Four peptides from digests were proved to be DPP-IV and PEP inhibitory peptides. The results showed that the peptides released from antler velvet protein contributed to the bioactivity of antler velvet during digestion., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Background: In human skin, the serine proteases kallikrein-related peptidase (KLK)5 and KLK7 degrade corneodesmosome proteins, leading to desquamation. Serine protease activity of the skin is tightly regulated by the interplay between such proteases and serine protease inhibitors, including lymphoepithelial Kazal-type related inhibitor (LEKTI), encoded by SPINK5; secretory leucocyte peptidase inhibitor (SLPI); and elafin. Expression of KLK5 and KLK7 is controlled and upregulated by stimulants such as calcium, 1,25-dihydroxyvitamin D 3 [1,25(OH) 2 VD 3 ] and retinoic acid (RA)., Objectives: To understand the effect of calcium, 1,25(OH) 2 VD 3 and RA on the expression of serine protease inhibitors in epidermal keratinocytes., Methods: We stimulated normal human epidermal keratinocytes (NHEKs) with high calcium, 1,25(OH) 2 VD 3 or RA, and then analysed the expression of serine protease inhibitors using quantitative real-time polymerase chain reaction, enzyme-linked immunosorbent assay and immunocytofluorescence. We also analysed trypsin- and chymotrypsin-like serine protease activities in stimulated NHEKs., Results: High calcium, but not 1,25(OH) 2 VD 3 or RA, significantly induced the expression of LEKTI, SLPI and elafin at both transcript and protein levels in NHEKs. These inductions were time- and dose-dependent. The activities of trypsin- and chymotrypsin-like serine proteases were significantly up- and downregulated by high calcium, respectively, in NHEKs., Conclusions: High calcium, but not 1,25(OH) 2 VD 3 or RA, increases the expression of serine protease inhibitors in epidermal keratinocytes. Our findings contribute to the understanding of the mechanisms by which serine protease activities are regulated by serine proteases and related inhibitors in epidermal keratinocytes., (© 2016 British Association of Dermatologists.)

The propeptides of subtilisin-like serine proteinases (subtilases, SBTs) serve dual functions as intramolecular chaperones that are required for enzyme folding and as inhibitors of the mature proteases. SBT propeptides are homologous to the I9 family of protease inhibitors that have only been described in fungi. Here we report the identification and characterization of subtilisin propeptide-like inhibitor 1 (SPI-1) from Arabidopsis thaliana Sequence similarity and the shared β-α-β-β-α-β core structure identified SPI-1 as a member of the I9 inhibitor family and as the first independent I9 inhibitor in higher eukaryotes. SPI-1 was characterized as a high-affinity, tight-binding inhibitor of Arabidopsis subtilase SBT4.13 with K d and K i values in the picomolar range. SPI-1 acted as a stable inhibitor of SBT4.13 over the physiologically relevant range of pH, and its inhibitory profile included many other SBTs from plants but not bovine chymotrypsin or bacterial subtilisin A. Upon binding to SBT4.13, the C-terminal extension of SPI-1 was proteolytically cleaved. The last four amino acids at the newly formed C terminus of SPI-1 matched both the cleavage specificity of SBT4.13 and the consensus sequence of Arabidopsis SBTs at the junction of the propeptide with the catalytic domain. The data suggest that the C terminus of SPI-1 acts as a competitive inhibitor of target proteases as it remains bound to the active site in a product-like manner. SPI-1 thus resembles SBT propeptides with respect to its mode of protease inhibition. However, in contrast to SBT propeptides, SPI-1 could not substitute as a folding assistant for SBT4.13., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

Protease inhibitors and their cognate proteases regulate growth, development and defense. Serine protease inhibitors (serpins) constitute a large family of genes in most metazoans and plants. Drosophila NECROTIC (NEC) gene and its homologues in the mammalian system are well-characterized serpins, which play a role in regulating proteases that participate in cell death pathways. Although the Arabidopsis genome contains several serpin homologs, biological function is not known for most of them. Here we show that two Arabidopsis serpins, AtSRP4 and AtSRP5, are closest sequence homologue of Drosophila NEC protein, and are involved in stress-induced cell death and defense. Expression of both AtSRP4 and AtSRP5 genes induced upon ultra-violet (UV)-treatment and inoculation with avirulent pathogens. The knockout mutants and amiRNA lines of AtSRP4 and AtSRP5 exaggerated UV- and hypersensitive response (HR)-induced cell death. Over-expression of AtSRP4 reduced UV- and HR-induced cell death. Mutants of AtSRP4 and AtSRP5 suppressed whereas over-expression of AtSRP4 supported the growth of bacterial pathogen Pseudomonas syringae pv. tomato DC3000 carrying the AvrRpt2 effector, but not other avirulent or virulent pathogens. Results altogether identified AtSRP4 and AtSRP5 as negative regulators of stress-induced cell death and AvrRpt2-triggered immunity; however, the influence of AtSRP4 was more prominent than AtSRP5., (© 2016 Scandinavian Plant Physiology Society.)

Protease inhibitors are an extremely diverse group of proteins that control the proteolytic activities of proteases and play a crucial role in biological processes including host defenses. The I84 family of protease inhibitors in the MEROPS database currently consists of cvSI-1 and cvSI-2, two novel serine protease inhibitors purified and characterized from the eastern oyster Crassostrea virginica plasma and believed to play a role in host defense and disease resistance. In the present study, a third member of I84 family, named cvSI-3, was identified from C. virginica by cDNA cloning and sequencing. The full cvSI-3 cDNA was composed of 342 bp including a 255 bp open reading frame (ORF) that encodes an 84-amino acid peptide. The mature cvSI-3 molecule was predicted to have 68 amino acid residues after removal of a 16-amino acid signal peptide, with a calculated molecular mass of 7724.5 Da and a theoretical isoelectric point (pI) of 6.28. CvSI-3 amino acid sequence shared 41% identity with cvSI-2 and 37% identity with cvSI-1, which included 12 conserved cysteines. Quantitative real-time PCR determined that cvSI-3 gene expressed primarily in oyster digestive glands. Real-time PCR also detected that cvSI-1, cvSI-2 and cvSI-3 expression levels in digestive glands varied significantly, with cvSI-2 showing the highest expression level and cvSI-3 the lowest. Additionally, a significant correlation was detected between cvSI-2 and cvSI-3 mRNAs levels. Searches into sequence databases using cvSI-1, cvSI-2 and cvSI-3 as queries retrieved ESTs suggesting the possible existence of at least 9 more I84 family members in eastern oysters and of I84 family protease inhibitors in various bivalve and gastropod species. Moreover, orthologs of all C. virginica I84 family members or potential member genes were found to be present in the C. gigas genome, and their distributions among species provided important information about the evolution of the I84 family of protease inhibitors. It appears that the I84 family of protease inhibitors is widely distributed and actively evolving in the Phylum Mollusca., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

In order to predict the potencies of mechanism-based reversible covalent inhibitors, the relationships between calculated Gibbs free energy of hydrolytic water molecule in acyl-trypsin intermediates and experimentally measured catalytic rate constants (k cat ) were investigated. After obtaining representative solution structures by molecular dynamics (MD) simulations, hydration thermodynamics analyses using WaterMap™ were conducted. Consequently, we found for the first time that when Gibbs free energy of the hydrolytic water molecule was lower, logarithms of k cat were also lower. The hydrolytic water molecule with favorable Gibbs free energy may hydrolyze acylated serine slowly. Gibbs free energy of hydrolytic water molecule might be a useful descriptor for computer-aided discovery of mechanism-based reversible covalent inhibitors of hydrolytic enzymes.

The presence of the low-molecular-mass serine protease inhibitor Kazal-type (Spink) is a characteristic feature of vertebrate semen. Its main function is control of the serine protease in the acrosome, acrosin. Here we showed for the first time that Spink is present in the seminal plasma of carp, which have anacrosomal spermatozoa. Using a three-step isolation procedure that consisted in gel filtration and RP-HPLC and re-RP-HPLC, we isolated this inhibitor and identified it as serine protease inhibitor Kazal-type 2 (Spink2), a reproductive-derived member of the Spink family. The cDNA sequence of this inhibitor obtained from carp testis encoded 77 amino acids, including a 17 amino acids signal peptide; this sequence was distinct from fish Kazal-type inhibitors. The mRNA expression analysis showed that Spink2 is expressed predominantly in carp testis and spermatic duct. Immunohistochemical analysis demonstrated its localization in testis in Sertoli, Leydig and germ cells at all developmental stages (with the exception of spermatozoa) and in the epithelium of the spermatic duct. Aside from strong inhibition of trypsin, this inhibitor acts strongly against subtilisin and possesses bacteriostatic activities against Lactobacillus subtilis, Escherichia coli and Aeromonas hydrophila. The localization of Spink2 in carp reproductive tract suggests an important function in spermatogenesis and in maintenance of the microenvironment in which sperm maturation occurs and sperm are stored. Our results suggest that Spink2 from carp seminal plasma may play a role in antibacterial semen defense, protecting semen against unwanted proteolysis within the reproductive tract., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Some serine protease inhibitor (serpin) regulators of essential life pathways bind glycosaminoglycans (GAGs) to enhance inhibitory functions and achieve physiologically relevant rates. This study demonstrates that highly conserved Amblyomma americanum tick saliva serpin 19 (AAS19), a broad-spectrum inhibitor of hemostasis and inflammation system proteases and anticoagulant, can bind heparan sulfate/heparin (HS)GAGs and that this interaction alters its function. Substrate hydrolysis and unpaired t-test analyses revealed that HSGAG binding caused rAAS19 inhibitory activity to: (i) significantly increase against blood clotting factors (f) IIa (thrombin) and fIXa, (ii) significantly reduce against fXa and fXIIa and (iii) moderate to no effect against trypsin, kallikrein, papain, and plasmin. Stoichiometry of inhibition (SI) analyses show that HSGAG binding improved the rAAS19 inhibitory efficiency against thrombin 2.7-4.3 fold as revealed by SI change from 13.19 in absence of HSGAGs to 4.83-3.04 in their presence. Our data show that HSGAG binding dramatically enhanced rAAS19 anticoagulant function. In the recalcification time assay, rAAS19 pre-incubated with HSGAGs prior to the assay, delayed plasma clotting by an additional 176-457 s above HSGAGs or rAAS19 alone. Our data suggest that formation of the HSGAGs and rAAS19 complex is important for the observed enhanced anticoagulant effect. Delay of plasma clotting was higher when HSGAGs and rAAS19 were co-incubated to allow complex formation prior to blood clotting assay as opposed to no co-incubation. We have discussed our finding with reference to tick feeding physiology and utility of the rAAS19 in blood clotting disorder therapy., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Pupae inside cocoons rarely suffer from disease. It is apparent that some factors in the cocoon exert antimicrobial effects whereby the pupae inside can be protected from microbial infection. In the present study, we investigated the expression of cocoon protease inhibitors using immunoblotting and activity staining. Enzymatic hydrolysis of cocoon proteins in vitro was performed to characterize their roles in protecting the cocoon from microbial proteases. We found that some protease inhibitors, particularly trypsin inhibitor-like (TIL)-type protease inhibitors, can be secreted into the cocoon layer during the spinning process, thereby providing effective protection to the cocoon and pupa by inhibiting the extracellular proteases that can be secreted by pathogens., (© 2015 Institute of Zoology, Chinese Academy of Sciences.)

Serine protease inhibitors (SPIs) play a crucial role in regulation of both host and bacterial serine protease. They are classified into several protein families, where Kazal-type inhibitors are one of families with multi-domain. In the present study, the polymorphism of AiSPI from Bay scallop Argopecten irradians was found to be associated with disease resistance of bay scallop against Listonella anguillarum. Nine single nucleotide polymorphisms (SNPs) were identified in the exon region of AiSPI, where five SNPs were non-synonymous mutation. Three of these mutations were located in "kazal-like 3"domain, two SNP loci positioned at +536, +1312 were selected for further association studies. For the locus +536, the genotype frequency of A/G in the resistant stock (12.8%) was significantly lower (p < 0.05) than that in the susceptible stock (35.1%), while, the genotype A/A in the resistant stock (87.2%) was significantly higher in comparison with susceptible stock (64.9%) (p < 0.05). The G allele frequencies were 6.4% and 17.6% in resistant stock and susceptible stock, respectively, and χ 2 -test revealed a significant difference in the frequency distribution between the two stocks (p < 0.05). But there was no significant association between the mutation C-T at locus +1312 with either resistant or susceptible group (p > 0.05). The genotype frequencies of T/T, T/C, C/C at locus +1312 were 94.6%, 2.7% and 2.7% respectively in the susceptible stock, while 100%, 0% and 0% respectively in the resistant stock. The amino acid change for the mutation at locus +536 A-G was from asparagine to serine, and the predicted homology model of this amino acid variation could affect its function as well as the structural integrity of the domain. In vitro elastase inhibition assay of the protein variants at locus +536 was conducted to explicate the effect of SNP. The increasing concentration of protein (0 mmol/L- 2.93 mmol/L) was incubated with 80 nmol/L elastase where the residual enzyme activity values for rAiSPI (N) with A variant and rAiSPI (S) with G variant were started to reduce from 0.40 to 0.215 and 0.435 to 0.356, respectively. The elastase inhibition ability of rAiSPI (N) variant was significantly higher than that of rAiSPI (S) (p < 0.01). The results suggested that the mutation at locus +536A/A significantly associated with disease resistance of bay scallop would shed light for selective breeding program., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

A novel antistasin/WAP-like serine protease inhibitor, named as StmAW-SPI, was identified from sea cucumber (Stichopus monotuberculatus) and functionally characterized in this study. The full-length cDNA of StmAW-SPI is 1917 bp in length with a 72 bp 5'-untranslated region (UTR), a 294 bp 3'-UTR and a 1551 bp open reading frame (ORF) encoding a protein of 516 amino acids with a deduced molecular weight of 54.56 kDa. The StmAW-SPI protein has 5-fold internal repeats (IRs) of antistasin domain and 6-fold IRs of WAP domain. For the gene structure, StmAW-SPI contains 10 exons separated by 9 introns. The StmAW-SPI mRNA expression pattern was determined using quantitative real-time PCR. The highest level of StmAW-SPI was found in the intestine, followed by coelomocytes, gonad, body wall and respiratory tree. The StmAW-SPI expressions were significantly up-regulated after polyriboinosinic polyribocytidylic acid [Poly (I:C)] or lipopolysaccharides (LPS) challenge in in vitro experiments performed in primary coelomocytes. In addition, the serine protease inhibitory activity and bacterial protease inhibitory activity of StmAW-SPI were examined, and the antibacterial activity was also demonstrated in this study. Our study, as a whole, suggested that StmAW-SPI might play a critical role in the innate immune defense of sea cucumber against microbial infections, by not only inactivating the serine protease but also inhibiting the growth of pathogens., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Background: In eukaryotes, the serpins constitute a wide family of protease inhibitors regulating many physiological pathways. Many reports stressed the key role of serpins in several human physiopathologies including mainly the inflammatory bowel diseases. In this context, eukaryotic serpins were largely studied and their use to limit inflammation was reported. In comparison to that, bacterial serpins and mainly those from human gut microbiota remain poorly studied., Results: The two genes encoding for putative serpins from the human gut bacterium Eubacterium sireaum, display low sequence identities. These genes were overexpressed and the encoded proteins, named Siropins, were purified. Activity studies demonstrated that both purified proteins inhibited serine proteases but surprisingly they preferentially inhibited two human serine proteases (Human Neutrophil Elastase and Proteinase3). The biochemical characterization of these Siropins revealed that Siropin 1 was the most active and stable at low pH values while Siropin 2 was more thermoactive and thermostable. Kinetic analysis allowed the determination of the stoichiometry of inhibition (SI) which was around 1 and of the association rate constants of 7.7 × 10 4 for the Human Neutrophil Elastase and 2.6 × 10 5 for the Proteinase3. Moreover, both Siropins displayed the ability to inhibit proteases usually present in fecal waters. Altogether our data indicate the high efficiency of Siropins and their probable involvement in the control of the overall intestine protease activity., Conclusions: Here we report the purification and the biochemical characterization of two novel serpins originated from Eubacterium sireaum, a human gastro-intestinal tract commensal bacteria. These proteins that we called Siropins, efficiently inhibited two human proteases reported to be associated with inflammatory bowel diseases. The determination of the biochemical properties of these enzymes revealed different temperature and pH behaviours that may reflect adaptation of this human commensal bacterium to different ecological environments. To the best of our knowledge, it is the first bacterial serpins showing an attractive inhibition of fecal proteases recovered from a mice group with chemically induced inflammation. Altogether our data highlight the interesting potential of Siropins, and serpins from the human gut microbiota in general, to be used as new alternative to face inflammatory diseases.

Objective: To investigate the effect of temperature and protease inhibitor on adrenocorticotrophin (ACTH) stability., Methods: We collected blood specimens from 10 donors into K3 EDTA (ethylenediaminetetraacetic acid) and K3 EDTA + aprotinin tubes. Plasma specimens were held at room temperature and at 2 °C to 8[Formula: see text] for 0, 2, 4, 8, 24, and 72 hours. ACTH concentrations in 4 storage conditions were compared with repeated-measures analysis of variance (ANOVA) at each time point., Results: ACTH concentrations substantially decreased after a 72-hour time period. At the 72nd hour, standard EDTA tubes and EDTA + aprotinin tubes stored at 4[Formula: see text] showed the optimal stability. EDTA + aprotinin tubes stored at room temperature performed more strongly than standard EDTA tubes stored at room temperature., Conclusion: Our results show that refrigerated storage is more effective than protease inhibitor addition to maintain ACTH stability. We recommend using aprotinin tubes, especially for specimens standing at room temperature., (© American Society for Clinical Pathology, 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Although bortezomib is successfully used against multiple myeloma, the pharmacodynamics of proteasome inhibition and its association with efficacy or resistance is poorly understood. Using ultra performance liquid chromatography coupled to tandem mass spectrometry, site-specific luminogenic substrate assays and 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazoliumbromide (MTT) assays, effects of bortezomib on cellular drug concentrations, chymotrypsin- , caspase- , and trypsin-like activities, and cytotoxic efficacy were evaluated in eight myeloma cell lines directly after 1 h of exposure and additionally after a 23-h washout phase. Bortezomib accumulated in myeloma cells by up to 100-fold and concentration-dependently inhibited the proteasomal activities with the chymotrypsin-like activity being the most sensitive. Whereas intracellular concentrations correlated with the inhibition of the chymotrypsin- and the caspase-like activities of the proteasome, the cytotoxic efficacy of bortezomib did not correlate with either intracellular concentrations or proteasomal inhibition. However, the ratio of concentrations measured directly after the exposure and after the washout phase (indicating drug disposition) correlated with efficacy, suggesting that the cell's ability to dispose bortezomib at least in part influences bortezomib's cytotoxicity. In conclusion, this data argues against a direct association of intracellular concentration or proteasomal inhibition with cytotoxic efficacy but advocates for an important role of cellular drug disposition. Moreover, this study underlines the pleiotropic mode of action of bortezomib going beyond proteasome inhibition.

Snake venom Kunitz-type serine protease inhibitors (KSPIs) exhibit various biological functions including anticoagulant activity. This study elucidates the occurrence and subunit stoichiometry of a putative complex formed between two KSPIs (Rusvikunin and Rusvikunin-II) purified from the native Rusvikunin complex of Pakistan Russell's Viper (Daboia russelii russelii) venom (RVV). The protein components of the Rusvikunin complex were identified by LC-MS/MS analysis. The non-covalent interaction between two major components of the complex (Rusvikunin and Rusvikunin-II) at 1:2 stoichiometric ratio to form a stable complex was demonstrated by biophysical techniques such as spectrofluorometric, classical gel-filtration, equilibrium gel-filtration, circular dichroism (CD), dynamic light scattering (DLS), RP-HPLC and SDS-PAGE analyses. CD measurement showed that interaction between Rusvikunin and Rusvikunin-II did not change their overall secondary structure; however, the protein complex exhibited enhanced hydrodynamic diameter and anticoagulant activity as compared to the individual components of the complex. This study may lay the foundation for understanding the basis of protein complexes in snake venoms and their role in pathophysiology of snakebite., (Copyright © 2016 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.)

Sugarcane's (Saccharum spp.) response to Diatraea saccharalis (F.) (Lepidoptera: (Crambidae) herbivory was investigated using a macroarray spotted with 248 sugarcane Expressed Sequence Tags (ESTs) encoding serine peptidase inhibitors, serine peptidases. and Clp protease system subunits. Our results showed that after nine hours of herbivory, 13 sugarcane genes were upregulated and nine were downregulated. Among the upregulated genes, nine were similar to serine peptidase inhibitors and four were similar to Bowman-Birk Inhibitors (BBIs). Phylogenetic analysis revealed that these sequences belong to a phylogenetic group of sugarcane BBIs that are potentially involved in plant defense against insect predation. The remaining four upregulated genes included serine peptidases and one homolog to the Arabidopsis AAA+ chaperone subunit ClpD, which is a member of the Clp protease system. Among the downregulated genes, five were homologous to serine peptidases and four were homologous to Arabidopsis Clp subunits (three homologous to Clp AAA+ chaperones and one to a ClpP-related ClpR subunit). Although the roles of serine peptidase inhibitors in plant defenses against herbivory have been extensively investigated, the roles of plant serine peptidases and the Clp protease system represent a new and underexplored field of study. The up- and downregulated D. saccharalis genes presented in this study may be candidate genes for the further investigation of the sugarcane response to herbivory., Competing Interests: The authors declare no conflicts of interest.

Serine peptidase inhibitor (serpin) is the name given to the superfamily of proteins with wide range of biological functions, and that the main feature is the inhibition of serine proteases. Here we describe the inhibitory characterization of a serpin from Gloeobacter violaceus that we named vioserpin. The serpin presented a high specificity to inhibit trypsin-like enzymes with a rapid inhibition rate constant (2.1 × 10(6) M(-1) s(-1)). We also demonstrated that the inhibitory activity of the vioserpin is influenced by the concentration of heparin, and this finding may throw a new light on understanding the molecular evolution of serpins., (Copyright © 2016 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.)

The only oral direct thrombin inhibitors that have reached the market, ximelagatran and dabigatran etexilat, are double prodrugs with low bioavailability in humans. We have evaluated an alternative strategy: the preparation of a nonpeptidic, polar direct thrombin inhibitor as a single, macrocyclic esterase-cleavable (acyloxy)alkoxy prodrug. Two homologous prodrugs were synthesized and displayed high solubilities and Caco-2 cell permeabilities, suggesting high absorption from the intestine. In addition, they were rapidly and completely converted to the active zwitterionic thrombin inhibitor in human hepatocytes. Unexpectedly, the most promising prodrug displayed only moderately higher oral bioavailability in rat than the polar direct thrombin inhibitor, most likely due to rapid metabolism in the intestine or the intestinal wall. To the best of our knowledge, this is the first in vivo ADME study of macrocyclic (acyloxy)alkoxy prodrugs, and it remains to be established if the modest increase in bioavailability is a general feature of this category of prodrugs or not.

A peptide segment that binds the active site of a serine protease in a substrate-like manner may behave like an inhibitor or a substrate. However, there is sparse information on which factors determine the behavior a particular peptide segment will exhibit. Here, we describe the first x-ray crystal structure of a nanobody in complex with a serine protease. The nanobody displays a new type of interaction between an antibody and a serine protease as it inserts its complementary determining region-H3 loop into the active site of the protease in a substrate-like manner. The unique binding mechanism causes the nanobody to behave as a strong inhibitor as well as a poor substrate. Intriguingly, its substrate behavior is incomplete, as 30-40% of the nanobody remained intact and inhibitory after prolonged incubation with the protease. Biochemical analysis reveals that an intra-loop interaction network within the complementary determining region-H3 of the nanobody balances its inhibitor versus substrate behavior. Collectively, our results unveil molecular factors, which may be a general mechanism to determine the substrate versus inhibitor behavior of other protease inhibitors., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

The major protease inhibitor from the sea anemone Stichodactyla helianthus (ShPI-1) is a non-specific inhibitor that binds trypsin and other trypsin-like enzymes, as well as chymotrypsin, and human neutrophil elastase. We performed site-directed mutagenesis of ShPI-1 to produce two variants (rShPI-1/K13L and rShPI/Y15S) that were expressed in Pichia pastoris, purified, and characterized. After a single purification step, 65 mg and 15 mg of protein per liter of culture supernatant were obtained for rShPI-1/K13L and rShPI/Y15S, respectively. Functional studies demonstrated a 100-fold decreased trypsin inhibitory activity as result of the K13L substitution at the reactive (P1) site. This protein variant has a novel tight-binding inhibitor activity of pancreatic elastase and increased activity toward neutrophil elastase in comparison to rShPI-1A. In contrast, the substitution Y15S at P2' site did not affect the Ki value against trypsin, but did reduce activity 10-fold against chymotrypsin and neutrophil elastase. Our results provide two new ShPI-1 variants with modified inhibitory activities, one of them with increased biomedical potential. This study also offers new insight into the functional impact of the P1 and P2' sites on ShPI-1 specificity., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Matriptase-2, a type II transmembrane serine protease, plays a key role in human iron homeostasis. Inhibition of matriptase-2 is considered as an attractive strategy for the treatment of iron-overload diseases, such as hemochromatosis and β-thalassemia. In the present study, synthetic routes to nine dipeptidomimetic inactivators were developed. Five active compounds (41-45) were identified and characterized kinetically as irreversible inhibitors of matriptase-2. In addition to a phosphonate warhead, these dipeptides possess two benzguanidine moieties as arginine mimetics to provide affinity for matriptase-2 by binding to the S1 and S3/S4 subpockets, respectively. This binding mode was strongly supported by covalent docking analysis. Compounds 41-45 were obtained as mixtures of two diastereomers and were therefore separated into the single epimers. Compound 45 A, with S configuration at the N-terminal amino acid and R configuration at the phosphonate carbon atom, was the most potent matriptase-2 inactivator with a rate constant of inactivation of 2790 m(-1)  s(-1) and abolished the activity of membrane-bound matriptase-2 on the surface of intact cells. Based on the chemotyp of phosphono bisbenzguanidines, the design and synthesis of a fluorescent probe (51 A) by insertion of a coumarin label is described. The in-gel fluorescence detection of matriptase-2 was demonstrated by applying 51 A as the first activity-based probe for this enzyme., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Serine peptidase inhibitor, Kazal type 3 (SPINK3) is a trypsin inhibitor, and also a growth factor that has an identical structure to epidermal growth factor (EGF), which could combine with epidermal growth factor receptor (EGFR) to promote cell proliferation. To shed light on the role and regulation mechanism of SPINK3 in rat liver regeneration (LR), Rat Genome 230 2.0 assay was used to detect the expression profiles of LR genes after partial hepatectomy (PH). The results showed that Spink3 was significantly up-regulated at 2-24 h and 72-168 h after PH. In the present study, RT-PCR and immunoblotting were used to validate the assay results. Ingenuity Pathway Analysis 9.0 (IPA) software was used to build the SPINK3 signaling regulating LR and analyze the possible mechanism. And then the expression of cell proliferation-associated gene Ccna2 was examined by RT-PCR in normal rat liver cell line BRL-3A in which Spink3 was overexpressed. The results showed that Ccna2 was significantly up-regulated in BRL-3A in which Spink3 was over-expressed. SPINK3 combining with EGFR accelerated cell proliferation during rat liver regeneration via P38, PKC, JAK-STAT and AKT pathways. Thus, SPINK3 was likely to promote hepatocytes proliferation in LR through P38, PKC, JAK-STAT and AKT.

Many different types of serine proteinase inhibitors have been involved in several kinds of plant physiological processes, including defense mechanisms against phytopathogens. Kazal-type serine proteinase inhibitors, which are included in the serine proteinase inhibitor family, are present in several organisms. These proteins play a regulatory role in processes that involve serine proteinases like trypsin, chymotrypsin, thrombin, elastase and/or subtilisin. In the present work, we characterized two putative Kazal-type serine proteinase inhibitors from Arabidopsis thaliana, which have a single putative Kazal-type domain. The expression of these inhibitors is transiently induced in response to leaf infection by Botrytis cinerea, suggesting that they play some role in defense against pathogens. We also evaluated the inhibitory specificity of one of the Kazal-type serine proteinase inhibitors, which resulted to be induced during the local response to B. cinerea infection. The recombinant Kazal-type serine proteinase inhibitor displayed high specificity for elastase and subtilisin, but low specificity for trypsin, suggesting differences in its selectivity. In addition, this inhibitor exhibited a strong antifungal activity inhibiting the germination rate of B. cinerea conidia in vitro. Due to the important role of proteinase inhibitors in plant protection against pathogens and pests, the information about Kazal-type proteinase inhibitors described in the present work could contribute to improving current methods for plant protection against pathogens., (Copyright © 2016 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.)

Neutrophils store large quantities of neutrophil serine proteases (NSPs) that contribute, via multiple mechanisms, to antibacterial immune defences. Even though neutrophils are indispensable in fighting Staphylococcus aureus infections, the importance of NSPs in anti-staphylococcal defence is yet unknown. However, the fact that S. aureus produces three highly specific inhibitors for NSPs [the extracellular adherence proteins (EAPs) Eap, EapH1 and EapH2], suggests that these proteases are important for host defences against this bacterium. In this study we demonstrate that NSPs can inactivate secreted virulence factors of S. aureus and that EAP proteins function to prevent this degradation. Specifically, we find that a large group of S. aureus immune-evasion proteins is vulnerable to proteolytic inactivation by NSPs. In most cases, NSP cleavage leads to functional inactivation of virulence proteins. Interestingly, proteins with similar immune-escape functions appeared to have differential cleavage sensitivity towards NSPs. Using targeted mutagenesis and complementation analyses in S. aureus, we demonstrate that all EAP proteins can protect other virulence factors from NSP degradation in complex bacterial supernatants. These findings show that NSPs inactivate S. aureus virulence factors. Moreover, the protection by EAP proteins can explain why this antibacterial function of NSPs was masked in previous studies. Furthermore, our results indicate that therapeutic inactivation of EAP proteins can help to restore the natural host immune defences against S. aureus., (© 2015 John Wiley & Sons Ltd.)

Rhipicephalus microplus is an ectoparasite responsible for transmissions of babesiosis and anaplasmosis causing large losses to livestock production. To survive R. microplus tick produces several active molecules, such as protease inhibitors. This ectoparasite has been described as a rich source of serine protease inhibitors most of them are Kunitz-BPTI members named BmTIs which have no clear function yet. In the present work, we described the expression and functional characterization of rBmTI-A which showed to be similar to the native BmTI-A, a double-headed Kunitz-BPTI inhibitor, capable to inhibit trypsin, human neutrophil elastase (HNE), human plasma kalikrein (HuPK) and human plasmin. rBmTI-A was able to cause a decrease of HUVEC cell viability. Besides, the rBmTI-A showed to be a potent inhibitor of "in vitro" vessel formation. Our results suggested that BmTI-A may participate in the blood acquisition process interfering in the vessel formation during the tick parasite life stage, around 20 days. In conclusion, BmTI-A is a promising molecule to be used in the drug design and development of new method of R. microplus control., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Plant lectins and protease inhibitors constitute a class of proteins which plays a crucial role in plant defense. In our continuing investigations on lectins from plants, we have isolated, purified and characterized a protein of about 20 kDa, named PotHg, showing hemagglutination activity from tubers of Indian potato, Solanum tuberosum. De novo sequencing and MS/MS analysis confirmed that the purified protein was a Kunitz-type serine protease inhibitor having two chains (15 kDa and 5 kDa). SDS and native PAGE analysis showed that the protein was glycosylated and was a heterodimer of about 15 and 5 kDa subunits. PotHg agglutinated rabbit erythrocytes with specific activity of 640 H.U./mg which was inhibited by complex sugars like fetuin. PotHg retained hemagglutination activity over a pH range 4-9 and up to 80°C. Mannose and galactose interacted with the PotHg with a dissociation constant (Kd) of 1.5×10(-3) M and 2.8×10(-3) M, respectively as determined through fluorescence studies. Fluorescence studies suggested the involvement of a tryptophan in sugar binding which was further confirmed through modification of tryptophan residues using N-bromosuccinimide. Circular dichroism (CD) studies showed that PotHg contains mostly β sheets (∼45%) and loops which is in line with previously characterized protease inhibitors and modeling studies. There are previous reports of Kunitz-type protease inhibitors showing lectin like activity from Peltophorum dubium and Labramia bojeri. This is the first report of a Kunitz-type protease inhibitor showing lectin like activity from a major crop plant and this makes PotHg an interesting candidate for further investigation., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Since years, serine proteases and their inhibitors were an enigma to meat scientists. They were indeed considered to be extracellular and to play no role in postmortem muscle proteolysis. In the 1990's, we observed that protease inhibitors levels in muscles are a better predictor of meat tenderness than their target enzymes. From a practical point of view, we therefore choose to look for serine protease inhibitors rather than their target enzymes, i.e. serine proteases and the purpose of this report was to overview the findings obtained. Fractionation of a muscle crude extract by gel filtration revealed three major trypsin inhibitory fractions designed as F1 (Mr:50-70 kDa), F2 (Mr:40-60 kDa) and F3 (Mr:10-15kD) which were analyzed separately. Besides antithrombin III, an heparin dependent thrombin inhibitor, F1 and F2 comprised a large set of closely related trypsin inhibitors encoded by at least 8 genes bovSERPINA3-1 to A3-8 and able to inhibit also strongly initiator and effector caspases. They all belong to the serpin superfamily, known to form covalent complexes with their target enzymes, were located within muscle cells and found in all tissues and fluids examined irrespective of the animal species. Potential biological functions in living and postmortem muscle were proposed for all of them. In contrast to F1 and F2 which have been more extensively investigated only preliminary findings were provided for F3. Taken together, these results tend to ascertain the onset of apoptosis in postmortem muscle. However, the exact mechanisms driving the cell towards apoptosis and how apoptosis, an energy dependent process, can be completed postmortem remain still unclear.

Plants have a wide range of chemical defenses against predation, including substances that target digestive serine proteinases of herbivorous. Previous works demonstrated that lepidopteran insects have digestive serine proteinases resistant to plant proteinase inhibitors (PPIs) and ketone modifications, while coleopteran ones are sensitive to those plant defenses. This paper focuses on molecular aspects that lead lepidopteran serine proteinases to PPI and ketone modification resistance. Using biochemical experiments and computer 3D modeling we demonstrated that lepidopteran trypsins are more hydrophobic than coleopteran ones, a feature associated to trypsin oligomerization and decreased inhibition by PPI. Moreover, the determination of pKa values of chymotrypsin catalytic residues obtained by TPCK modification indicates that the environment around the active site of ketone-resistant and -sensitive chymotrypsins are different. Structural analysis using resistant and sensitive chymotrypsins data allowed us to point 2 hotspot regions around the active site that could explain the observed differences. Our set of results highlights features of serine proteinases important for understanding the resistance of insects to plant chemical defenses., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Serine protease inhibitors (SPIs) are an important group of protease inhibitors involved in a variety of biological processes. In the present study, a Kazal-type serine protease inhibitor homolog gene (designated as CsKPI) was identified from a Cyclina sinensis cDNA library. The open reading frame consists of 456 bp and encodes a protein of 151 amino acid residues with a theoretical molecular mass of 16.85 kDa and an isoelectric point of 5.74. Furthermore, using quantitative real-time PCR, we focused on the expression patterns of CsKPI found in tissues and on the stimulation of this gene's expression by bacteria. The results show that a higher-level mRNA expression of CsKPI was detected in hemocytes (P < 0.05) and was significantly upregulated at 3 h (P < 0.01) upon receiving bacterial challenges with Vibrio anguillarum. In addition, after the CsKPI gene was silenced by RNA interference, the expression of the CsTLR2 and CsMyD88 genes was extremely significantly decreased (P < 0.01) in C. sinensis. Finally, the recombinant CsKPI (rCsKPI) protein was purified and shown to exhibit less inhibitory activity than C-lyz against V. anguillarum in vitro. Hence, we propose that CsKPI plays an important role in the innate immunity and mediates TLR2 and MyD88-dependent pathway initiation in C. sinensis., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Background: Dengue, transmitted primarily by the bites of infected Aedes aegypti L., is transmitted to millions of individuals each year in tropical and subtropical areas. Dengue control strategies are primarily based on controlling the vector, using insecticides, but the appearance of resistance poses new challenges. Recently, highly selective protease inhibitors by phage display were obtained for digestive enzymes of the 4th instar larvae (L4) midgut. These mutants were not confirmed as a larvicide due to the low yield of the expression of these inhibitors. In the present study, chimera molecules were constructed based on the mutations at positions P1-P4' selected previously. The T6, T23 and T149 mutants were mixed with another Kunitz inhibitor, domain 1 of the inhibitor boophilin (D1)., Methods: The chimeras T6/D1, T149/D1 and T23/D1 were expressed at high levels in P. pastoris yeast, purified by ionic exchange chromatography and their homogeneity was analyzed by SDS-PAGE. The chimera inhibitors were assayed against larval trypsin, chymotrypsin and elastase using specific chromogenic substrates. The inhibitors were assayed for their larvicide potential against L4., Results: The chimeras exhibited strong inhibitory activities against the larval digestive enzymes in a dose-dependent manner. T6/D1, T149/D1 and T23/D1 exhibited strong larvicidal activity against L4 of Ae. aegypti with inhibitor concentrations in the μM range. A synergistic increase in mortality was observed when a mixture of the three chimeric inhibitors was tested., Conclusions: The strategy for constructing the chimeric inhibitors was successful. The chimeras showed strong larvicidal activity against Ae. aegypti. In the future, our findings can be used to design synthetic inhibitors for larvae digestive enzymes as an alternative method to control the dengue vector.