Search

Bowman-Birk inhibitors (BBIs) are plant-derived serine proteinase inhibitors. Endogenously, they function as defense molecules against pathogens and insects, but they also have been explored for applications in cancer treatment and inflammatory disorders. Here, we isolated 15 novel BBIs from the bulb of Hyacinthus orientalis (termed HOSPIs). These isoinhibitors consisted of two or three chains, respectively, that are linked by disulfides bonds based on proposed cleavage sites in the canonical BBI reactive site loop. They strongly inhibited trypsin (Ki = 0.22-167 nM) and α-chymotrypsin (Ki = 19-1200 nM). Notably, HOSPI-B4 contains a six-residue reactive loop, which appears to be the smallest such motif discovered in BBIs to date. HOSPI-A6 and -A7 contain an unusual reactive site, i.e. Leu-Met at the P1-P1' position and have strong inhibitory activity against trypsin, α-chymotrypsin, and elastase. Analysis of the cDNA encoding HOSPIs revealed that the precursors have HOSPI-like domains repeated at least twice with a defined linker sequence connecting individual domains. Lastly, mutational analysis of HOSPIs suggested that the linker sequence does not affect the inhibitory activity, and a Thr residue at the P2 site and a Pro at the P3' site are crucial for elastase inhibition. Using mammalian proteases as representative model system, we gain novel insight into the sequence diversity and proteolytic activity of plant BBI. These results may aid the rational design of BBI peptides with potent and distinct inhibitory activity against human, pathogen, or insect serine proteinases., (© 2021 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

Aim: The aim of this study was to assess if the ovine articular cartilage serine proteinase inhibitors (SPIs) were related to the Kunitz inter-α-trypsin inhibitor (ITI) family., Methods: Ovine articular cartilage was finely diced and extracted in 6 M urea and SPIs isolated by sequential anion exchange, HA affinity and Sephadex G100 gel permeation chromatography. Selected samples were also subjected to chymotrypsin and concanavalin-A affinity chromatography. Eluant fractions from these isolation steps were monitored for protein and trypsin inhibitory activity. Inhibitory fractions were assessed by affinity blotting using biotinylated trypsin to detect SPIs and by Western blotting using antibodies to α1-microglobulin, bikunin, TSG-6 and 2-B-6 (+) CS epitope generated by chondroitinase-ABC digestion., Results: 2-B-6 (+) positive 250, 220,120, 58 and 36 kDa SPIs were detected. The 58 kDa SPI contained α1-microglobulin, bikunin and chondroitin-4-sulfate stub epitope consistent with an identity of α1-microglobulin-bikunin (AMBP) precursor and was also isolated by concanavalin-A lectin affinity chromatography indicating it had N -glycosylation. Kunitz protease inhibitor (KPI) species of 36, 26, 12 and 6 kDa were autolytically generated by prolonged storage of the 120 and 58 kDa SPIs; chymotrypsin affinity chromatography generated the 6 kDa SPI. KPI domain 1 and 2 SPIs were separated by concanavalin lectin affinity chromatography, domain 1 displayed affinity for this lectin indicating it had N -glycosylation. KPI 1 and 2 displayed potent inhibitory activity against trypsin, chymotrypsin, kallikrein, leucocyte elastase and cathepsin G. Localisation of versican, lubricin and hyaluronan (HA) in the surface regions of articular cartilage represented probable binding sites for the ITI serine proteinase inhibitors (SPIs) which may preserve articulatory properties and joint function., Discussion/conclusions: The Kunitz SPI proteins synthesised by articular chondrocytes are members of the ITI superfamily. By analogy with other tissues in which these proteins occur we deduce that the cartilage Kunitz SPIs may be multifunctional proteins. Binding of the cartilage Kunitz SPIs to HA may protect this polymer from depolymerisation by free radical damage and may also protect other components in the cartilage surface from proteolytic degradation preserving joint function.

Serine proteinase inhibitor (Serpin, SPI) is a vital superfamily of endogenous inhibitors that monitor proteolytic events active in a number of biological functions. In this study, we isolated a full length gene encoding a novel serine protease inhibitor of Schistosoma japonicum (SjSPI) and characterized its molecular properties. Our result showed that SjSPI contained an open reading frame of 1,218 bp, which encoded 405 amino acid residues. Chromosomal structure analysis showed that SjSPI gene was comprised of six exons separated by five introns. It had essential structural motifs which were well conserved among the Serpin superfamily and showed 17-33% sequence identities with Serpins from other helminthic parasites. Trematode Serpin diverged separately into two different subclades and that the SjSPI clustered Subclade I. Exon-intron structures of trematode Serpins were highly conserved, closely with cestode Serpins. No signal peptide but a strongly transmembrane domain was predicted to exist in SjSPI, suggesting that the protein might be a soluble membrane-associated protein. Homology modeling predicted in silico confirmed that the SjSPI structure also belonged to the Serpin superfamily, containing nine α-helices and a reactive central loop. The bacterially expressed recombinant GST-SjSPI protein effectively inhibited the activities of chymotrypsin, trypsin and thrombin. Expression of SjSPI was detected throughout various developmental stages of the parasite in host and reached its maximal levels at the adult and egg stages, which suggests that SjSPI may be possibly involved in maintaining the physiology of eggs by regulating endogenous serine proteases., (Copyright © 2018 Elsevier B.V. All rights reserved.)

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.)

A 5-domain Kazal type serine proteinase inhibitor SPIPm2 from Penaeus monodon is involved in innate immune defense against white spot syndrome virus (WSSV). To test which domains were involved, the 5 domains of SPIPm2 were over-expressed and tested against WSSV infection. By using hemocyte primary cell culture treated with each recombinant SPIPm2 domain along with WSSV, the expression of WSSV early genes ie1, WSV477 and late gene VP28 were substantially reduced as compared to other domains when the recombinant domain 2, rSPIPm2D2, was used. Injecting the WSSV along with rSPIPm2D2 but not with other domains caused delay in mortality rate of the infected shrimp. The results indicate that the SPIPm2D2 possesses strong antiviral activity and, hence, contributes predominantly to the antiviral activity of SPIPm2., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Purpose: To investigate the effects and possible mechanisms of serine protease inhibitor A3K (SERPINA3K) on the formation of ocular surface squamous metaplasia in a mouse dry eye model induced by topical benzalkonium chloride (BAC)., Methods: The eye drops containing SERPINA3K were topically administered during the induction of BAC-induced dry eye. The clinical indications of dry eye were evaluated on day (D)16, including tear break-up time (BUT), tear volume, corneal fluorescein staining, and inflammatory index. Global specimens were collected on D16 and the following examinations were performed: histologic investigation, immunostaining of cytokeratin 10 (K10), p63 and Ki67 in the cornea, and Western blot analysis of tumor necrosis factor-α (TNF-α)., Results: Serine protease inhibitor A3K suppressed the formation of BAC-induced dry eye, presenting with longer BUTs, lower corneal fluorescein staining scores, and inflammatory index, while no significant changes in tear volume. It also reduced the severity of abnormal differentiation and proliferation on ocular surface with lower expressions of K10, p63, and Ki67, and retained the number of goblet cells in the conjunctival fornix. Serine protease inhibitor A3K significantly decreased the levels of TNF-α in the cornea., Conclusions: Topical application of SERPINA3K ameliorated the severity of ocular surface squamous metaplasia and suppressed the formation of BAC-induced dry eye., (Copyright 2014 The Association for Research in Vision and Ophthalmology, Inc.)

Serine proteinase inhibitors represent an expanding superfamily of endogenous inhibitors that are regulate proteolytic events and involved in a variety of physiological and immunological processes. A five-domain Kazal-type serine proteinase inhibitor (poKSPI) was identified and characterized from pearl oyster Pinctada fucata based on expressed sequence tag (EST) analysis. The full-length cDNA was 737 bp with an open reading frame (ORF) 660 bp encoding a 219 amino acid protein a theoretical molecular weight (Mw) of 23.3 kDa and an isoelectric point (pI) of 8.40. A putative signal peptide of 19 amino acid residues and five tandem Kazal domains were identified. Four of the Kazal domains had the highly conserved motif sequences with six cysteine residues responsible for the formation of disulfide bridges. The deduced amino acid sequence of the poKSPI shared high homology with KSPIs from Hirudo medicinalis. The poKSPI mRNA could be detected in all examined tissues, the expression level of the poKSPI mRNA was the highest in mantle and gonad, while the lowest in haemocyte and intestine. After LPS challenge, the expression level of the poKSPI mRNA in digestive gland was significantly up-regulated at 4 h post-challenge and reached the peak at 12 h post-challenge, which was 4.23-fold higher than control group; the expression level of the poKSPI mRNA in gill was also significantly up-regulated at 8 and 12 h post-challenge, which were 4.48 and 2.26-fold higher than control group. After Vibrio alginolyticus challenge, the expression levels of the poKSPI mRNA in digestive gland were significantly up-regulated at 8, 12, 48 and 72 h post-challenge, which were 1.70, 1.79, 3.89 and 5.69-fold higher than control group, respectively; the expression level of the poKSPI mRNA in gill was significantly up-regulated at 24 h post-challenge, which was 5.30-fold higher than control group. The recombinant poKSPI protein could inhibit chymotrypsin and trypsin activities in dose-dependent manner, when the ratios of rpoKSPI to chymotrypsin and trypsin were 36:1 and 72:1, respectively, the proteinase activities of chymotrypsin and trypsin could be almost completely inhibited, but the rpoKSPI could not inhibit subtilisin., (Copyright © 2013. Published by Elsevier Ltd.)

Background: Plant protease inhibitors (PIs) constitute a diverse group of proteins capable of inhibiting proteases. Among PIs, serine PIs (SPIs) display stability and conformational restrictions of the reactive site loop by virtue of their compact size, and by the presence of disulfide bonds, hydrogen bonds, and other weak interactions., Scope of Review: The significance of various intramolecular interactions contributing to protein folding mechanism and their role in overall stability and activity of SPIs is discussed here. Furthermore, we have reviewed the effect of variation or manipulation of these interactions on the activity/stability of SPIs., Major Conclusions: The selective gain or loss of disulfide bond(s) in SPIs can be associated with their functional differentiation, which is likely to be compensated by non-covalent interactions (hydrogen bonding or electrostatic interactions). Thus, these intramolecular interactions are collectively responsible for the functional activity of SPIs, through the maintenance of scaffold framework, conformational rigidity and shape complementarities of reactive site loop., General Significance: Structural insight of these interactions will provide an in-depth understanding of kinetic and thermodynamic parameters involved in the folding and stability mechanisms of SPIs. These features can be explored for engineering canonical SPIs for optimizing their overall stability and functionality for various applications., (© 2013.)

Serpin or serine proteinase inhibitor is a family of protease inhibitors that are involved in controlling the proteolytic cascade in various biological processes. In shrimp, several serpins have been identified but only a few have been characterized. Herein, the PmSERPIN3 gene identified from Penaeus monodon EST database was studied. By using the 5'- and 3'-Rapid Amplification of cDNA Ends (RACE) techniques, the full-length of PmSERPIN3 cDNA was obtained. The cDNA contained an open reading frame of 1233 bp encoding for 410 amino acid residue protein. Genome sequence analysis revealed that the PmSERPIN3 was an intronless gene. RT-PCR analysis revealed that it was constitutively expressed in all developmental stages, all shrimp tissues tested, and upon pathogen infections. The recombinant mature PmSERPIN3 protein (rPmSERPIN3) produced in Escherichia coli exhibited inhibitory activity against subtilisin. The rPmSERPIN3 also inhibited the shrimp prophenoloxidase system activation in vitro. Injecting the rPmSERPIN3 along with Vibrio harveyi into the shrimp decreased the clearance rate of bacteria in the hemolymph. Potentially, the PmSERPIN3 functions as a regulator of the proPO activating system., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Serine proteinase inhibitors play important and diverse roles in biological processes such as coagulation, defense mechanisms, and immune responses. Here, we identified and characterized a Kunitz-type proteinase inhibitor, designated FcKuSPI, of the BPTI/Kunitz family of serine proteinase inhibitors from the hemocyte cDNA library of the shrimp Fenneropenaeus chinensis. The deduced amino acid sequence of FcKuSPI comprises 80 residues with a putative signal peptide of 15 amino acids. The predicted molecular weight of the mature peptide is 7.66 kDa and its predicted isoelectric point is 8.84. FcKuSPI includes a Kunitz domain containing six conserved cysteine residues that are predicted to form three disulfide bonds. FcKuSPI shares 44-53% homology with BPTI/Kunitz family members from other species. FcKuSPI mRNA was expressed highly in the hemocytes and moderately in muscle in healthy shrimp. Recombinant FcKuSPI protein demonstrated anti-protease activity against trypsin and anticoagulant activity against citrated human plasma in a dose-dependent manner in in vitro assays., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

In the intricate realm of animal biology, a multitude of vital processes heavily rely on precisely orchestrated proteinase cascades, but the potential for havoc makes proteinase inhibitors indispensable, with serine proteinase inhibitors (serpins) at the forefront, serving as custodians of homeostasis and participating in various critical biological processes. Importantly, there are still many unexplored facets of serpin functionality. In this study, we focused on the serpin family proteins from Marsupenaeus japonicus , utilizing a fine-tuned pretrained protein language model. This approach led to the identification and evolutionary validation of 28 serpins, one of which, referred to as Mj serpin -1, was both computationally and experimentally demonstrated to show potential as an antiviral and apoptosis inhibitor. Our research unveils exciting prospects for the fusion of state-of-the-art artificial intelligence and rich bioinformatics, holding the promise of significant discoveries that could pave the way for future therapeutic advancements. [ABSTRACT FROM AUTHOR]

Patients who undergo renal replacement therapy often exhibit a high plasma linezolid concentration. Linezolid is metabolized via oxidation. Nafamostat mesilate has antioxidant effects and is frequently used as an anticoagulant during renal replacement therapy. We aimed to investigate the effect of nafamostat mesilate on plasma linezolid concentration. We examined whether the co‐administration of linezolid and nafamostat had any effect on plasma linezolid concentration. Mice were randomly allocated to two groups (n = 18/group): linezolid (100 mg kg−1, subcutaneous injection) + nafamostat (30 mg kg−1, intraperitoneal injection) and linezolid + saline. At 5 hours, the linezolid concentration was significantly higher in the linezolid + nafamostat co‐administration group than that in the linezolid + saline group (20.6 ± 9.8 vs 3.6 ± 1.2 μg/mL, respectively P <.001). The antioxidant effects of nafamostat may inhibit linezolid metabolism, resulting in the adverse event of high linezolid concentration if both are administered concurrently during renal replacement therapy. [ABSTRACT FROM AUTHOR]

Low salinity is one of important environmental factors which often led to mass mortality of the noble scallop Chlamys nobilis cultivated in the South coast of China. It is well known that enzymic system and non‐enzymic system both play crucial roles in all living organisms against severe environments. To investigate how change about enzymic system and non‐enzymic system in the stenohaline marine bivalve under low salinity stress, an acute challenge lasting 48 hr was conducted using golden and brown noble scallops in the present study. The serine proteinase inhibitor from the noble scallop (CnSPI) was first cloned and expressed in different tissues. After low salinity stress, the gene expression levels were determined in haemocytes and compared between golden and brown scallops. Meanwhile, total carotenoids content (TCC) in adductor, superoxide dismutase (SOD) enzymatic activity and methylenedioxyamphetamine (MDA) content in gill and haemocytes were also determined and compared between the two colours scallops. Results showed that the CnSPI gene expression levels were significantly decreased after low salinity stress, and the golden scallops had higher gene expression levels than brown scallops (p < .05) at most times. Moreover, after low salinity stress, TCC, SOD enzymatic activity and MDA content also fluctuated, and the golden scallops contained higher TCC and SOD, but lower MDA than the brown ones. The present results indicated that enzymic system and non‐enzymic system were both changed under low salinity stress in the noble scallop and significantly different responses to the stress existed between golden and brown individuals. The SPI gene and carotenoids (CAR) both play a resistant role to low salinity stress in the noble scallop. [ABSTRACT FROM AUTHOR]

• Antiviral treatment did not show efficacy for hypoxemia and DIC in a COVID-19 patient. • Heparin and nafamostat combination therapy showed dramatic efficacy on the illness. • Mechanisms by which nafamostat is effective for COVID-19 have been fully explained. • This would be an instructional lecture on nafamostat therapy for COVID-19 patients. Nafamostat, a serine proteinase inhibitor with various actions including antithrombin, antiplasmin, and antitrypsin effects, has been used in clinical practice to treat disseminated intravascular coagulation (DIC) and pancreatitis. This case report describes the clinical course of a patient with COVID-19 pneumonia whose severe hypoxemia, probably caused by DIC and pulmonary embolism, showed remarkable improvement with combination heparin and nafamostat therapy. In addition, beneficial mechanisms of nafamostat against COVID-19 and the necessity of attention to hyperkalemia as an adverse effect are discussed. [ABSTRACT FROM AUTHOR]

Serine proteinase inhibitor, clade E member 2 (SERPINE2), also known as protease nexin-1 (PN-1), is a member of the serpin family. Despite several reported roles of SERPINE2 in tumor development the histological distribution of SERPINE2 and its expression levels in a large variety of tumors remains unclear. Through expressed sequence tag database analysis, immunohistochemical staining of tissue microarrays and a literature review, it was revealed that SERPINE2 expression varied according to growth stages and tissue types. SERPINE2 is differentially expressed in a number of tumors and their normal tissue counterparts. SERPINE2 is identified most abundantly in adenocarcinomas. SERPINE2 serves diverse roles in a variety of tumors and therefore may serve as a promising biomarker for tumor diagnosis and prognosis. [ABSTRACT FROM AUTHOR]

Abstract: A cDNA clone, namely PtKPI, coding for a one-kazal domain serine proteinase inhibitor (SPI) was identified from haemocyte cDNA library of the swimming crab Portunus trituberculatus. It is the first kazal-type SPI reported from crabs, also the first one-kazal domain SPI in crustaceans. The full-length cDNA sequence was 683bp and contained an open reading frame of 444bp, 36bp of 5′-untranslated region (UTR), and 203bp of 3′-UTR with a poly(A) tail. It encoded a polypeptide of 147 amino acids with an estimated molecular mass of 15.85kDa and a theoretical pI of 8.99. A signal peptide was defined at N-terminus with a putative cleavage site located after position 16 (GNA-YN). The domain of PtKPI with C-X3-C-X7-C-X6-Y-X3-C-X7-C-X12-C was similar to the consensus pattern of the kazal motif. One α-helix surrounded by an adjacent three-stranded β-sheet constructed the classical three-dimensional structure of kazal domain. PtKPI transcript was detected at a high level in the eyestalk and haemocytes that are involved in immune function. Upon challenge with three different microorganisms Vibrio alginolyticus, Micrococcus luteus and Pichia pastoris, the temporal expression of PtKPI in haemocytes showed different activation times against bacteria and fungi within an experimental period of 72h. These results indicate that PtKPI is potentially involved in acute response against invading bacteria and fungi in P. trituberculatus. [Copyright &y& Elsevier]

Abstract: Serine proteinase inhibitors (SERPINs or serpins) have been found in a diverse range of organisms. Herein, eight serpin genes, namely PmSERPIN1 – 8, were identified from the Penaeus monodon EST database (http://pmonodon.biotec.or.th/home.jsp). Among those, PmSERPIN6 was selected for further characterization. Tissue distribution analysis revealed that PmSERPIN6 transcripts were expressed in the lymphoid organ, hemocyte, heart and gill, but not in the hepatopancreas. Semi-quantitative RT-PCR analysis at 0–48 h after pathogen challenge demonstrated that the PmSERPIN6 gene transcript expression levels in hemocytes was slightly decreased after systemic white spot syndrome virus (WSSV) injection but remained unchanged upon Vibrio harveyi injection. Interestingly, immunocytochemistry using anti-PmSERPIN6 polyclonal antiserum showed an increase in the number of PmSERPIN6 producing hemocytes at 72 h after both WSSV and V. harveyi injections indicating that the expression of PmSERPIN6 responded to pathogen in the late phase of infection. Our results suggest a likely important function of PmSERPIN6 in the shrimp’s defense against invading pathogens. [Copyright &y& Elsevier]

Abstract: Proteinases and proteinase inhibitors are involved in several biological and physiological processes in all multicellular organisms. The proteinase inhibitors function as modulators for controlling the extent of deleterious proteinase activity. The Kazal-type proteinase inhibitors (KPIs) in family I1 are among the well-known families of proteinase inhibitors, widely found in mammals, avian and a variety of invertebrates. Like those classical KPIs, the invertebrate KPIs can be single or multiple domain proteins containing one or more Kazal inhibitory domains linked together by peptide spacers of variable length. All invertebrate Kazal domains of about 40–60 amino acids in length share a common structure which is dictated by six conserved cysteine residues forming three intra-domain disulfide cross-links despite the variability of amino acid sequences between the half-cystines. Invertebrate KPIs are strong inhibitors as shown by their extremely high association constant of 107–1013 M−1. The inhibitory specificity of a Kazal domain varies widely with a different reactive P1 amino acid. Different invertebrate KPI domains may arise from gene duplication but several KPI proteins can also be derived from alternative splicing. The invertebrate KPIs function as anticoagulants in blood-sucking animals such as leech, mosquitoes and ticks. Several KPIs are likely involved in protecting host from microbial proteinases while some from the parasitic protozoa help protecting the parasites from the host digestive proteinase enzymes. Silk moths produce KPIs to protect their cocoon from predators and microbial destruction. [Copyright &y& Elsevier]

Abstract: Crustacean hemocytes were found to produce a large number of transcripts coding for Kazal-type proteinase inhibitors (KPIs). A detailed study performed with the crayfish Pacifastacus leniusculus and the shrimp Penaeus monodon revealed the presence of at least 26 and 20 different Kazal domains from the hemocyte KPIs, respectively. Comparisons with KPIs from other taxa indicate that the sequences of these domains evolve rapidly. A few conserved positions, e.g. six invariant cysteines were present in all domain sequences whereas the position of P1 amino acid, a determinant for substrate specificity, varied highly. A study with a single crayfish animal suggested that even at the individual level considerable sequence variability among hemocyte KPIs produced exist. Expression analysis of four crayfish KPI transcripts in hematopoietic tissue cells and different hemocyte types suggest that some of these KPIs are likely to be involved in hematopoiesis or hemocyte release as they were produced in particular hemocyte types or maturation stages only. [Copyright &y& Elsevier]

Abstract: The mustard trypsin inhibitor 2, MTI2, was expressed in Escherichia coli. A specific procedure for its production and purification is described. The recombinant protein was recovered by protein extraction from the insoluble fraction, then renatured and purified by ion exchange and gel filtration chromatography. Finally, the inhibitory activity against trypsin was also determined. [Copyright &y& Elsevier]

Abstract: A potent serine proteinase inhibitor was isolated and characterized from the seeds of the tropical legume liana, Derris trifoliata (DtTCI) by ammonium sulfate precipitation, ion exchange chromatography and gel filtration chromatography. SDS–PAGE as well as MALDI-TOF analysis showed that DtTCI is a single polypeptide chain with a molecular mass of ∼20kDa. DtTCI has three isoinhibitors (pI: 4.55, 5.34 and 5.72) and, inhibited both trypsin and chymotrypsin in a 1:1 molar ratio. Both Dixon plots and Lineweaver–Burk double reciprocal plots revealed a competitive inhibition of trypsin and chymotrypsin activity, with inhibition constants (Ki ) of 1.7×10−10 and 1.25×10−10 M, respectively. N-terminal sequence of DtTCI showed over 50% similarity with numerous Kunitz-type inhibitors of the Papilionoideae subfamily. High pH amplitude and broad temperature optima were noted for DtTCI, and time course experiments indicated a gradual loss in inhibitory potency on treatment with dithiothreitol (DTT). Circular Dichroism (CD) spectrum of native DtTCI revealed an unordered structure whereas exposure to thermal-pH extremes, DTT and guanidine hydrochloride (Gdn HCl) suggested that an abundance of β-sheets along with intramolecular disulfide bonds provide conformational stability to the active site of DtTCI, and that severity of denaturants cause structural modifications promoting inhibitory inactivity. Antimalarial studies of DtTCI indicate it to be a potent antiparasitic agent. [Copyright &y& Elsevier]

Abstract: Squamous cell carcinoma antigen 1 (SCCA1), which belongs to serine proteinase inhibitor (serpin) superfamily, inhibits papain-like cysteine proteinase. Recently, it has been reported that SCCA1 acts not only as a proteinase inhibitor but also as an inhibitor of UV-induced apoptosis via suppression of the activity of c-Jun NH2-terminal kinase (JNK1). The present study determined the crystal structure of SCCA1, suggesting that the reactive center loop (RCL) of SCCA1, a recognition site of proteinase, is very flexible and located away form the main-body of SCCA1. We show that the inhibitory effect of SCCA1 on the kinase activity of JNK1 is lost when the RCL was truncated. Furthermore, we found that a mutant protein created by replacing one amino acid in RCL maintain the suppressive activity to JNK1, whereas the inhibitory effect to proteinase is obviously decreased. [Copyright &y& Elsevier]

Abstract: Tissue factor pathway inhibitor-2 (TFPI-2) is a serine proteinase inhibitor that induces caspase-mediated apoptosis when offered to a variety of tumor cells. In order to investigate the mechanism of TFPI-2-induced apoptosis, we initially studied the uptake and trafficking of TFPI-2 by HT-1080 cells. Exogenously offered TFPI-2 was rapidly internalized and distributed in both the cytosolic and nuclear fractions. Nuclear localization of TFPI-2 was also detected in a variety of endothelial cells constitutively expressing TFPI-2. Nuclear localization of TFPI-2 required a NLS sequence located in its Lys/Arg-rich C-terminal tail comprising residues 191–211, as a TFPI-2 construct lacking the C-terminal tail failed to localize to the nucleus. Complexes of TFPI-2 and importin-α were co-immunoprecipitated from cell lysates of HT-1080 cells either offered or overexpressing this protein, providing evidence that TFPI-2 was shuttled to the nucleus by the importin system. Our results provide the initial description of TFPI-2 internalization and translocation to the nucleus in a number of cells. [Copyright &y& Elsevier]

Human tissue factor pathway inhibitor-2 (TFPI-2) is a matrix-associated Kunitz-type serine proteinase inhibitor that is secreted by all cells of the vasculature, and presumably plays a role in the regulation of plasmin-mediated matrix remodeling. In this report, we describe the cloning and expression of a full-length cDNA for bovine TFPI-2 that exhibits 72% sequence identity with that of human TFPI-2. Following a 22 residue signal peptide, the mature protein contains 212 amino acids with 18 cysteines, three putative N-glycosylation sites, and one putative O-glycosylation site. The deduced sequence of mature bovine TFPI-2 revealed a short acidic amino-terminal region, three tandem Kunitz-type domains, and a carboxy-terminal tail highly enriched in basic amino acids. Recombinant bovine TFPI-2 was expressed in HEK 293 cells and resolved into two isoforms, designated as α-TFPI-2 (Mr 33 kDa) and β-TFPI-2 (Mr 31 kDa), which presumably represent differentially glycosylated forms of the inhibitor. Similar to human TFPI-2, both bovine TFPI-2 isoforms exhibited strong inhibitory activity towards trypsin and plasmin, and weak inhibitory activity towards the factor VIIa-tissue factor complex. [Copyright &y& Elsevier]

Aims/hypothesis. Kallikrein-binding protein (KBP) is a serine proteinase inhibitor (serpin). It specifically binds to tissue kallikrein and inhibits kallikrein activity. Our study was designed to test its effects on retinal neovascularization and vascular permeability. Methods. Endothelial cell proliferation was determined by [3H] thymidine incorporation assay and apoptosis quantified by Annexin V staining and flow cytometry. Effect on retinal neovascularization was determined by fluorescein angiography and count of pre-retinal vascular cells in an oxygen-induced retinopathy (OIR) model. Vascular permeability was assayed by the Evans blue method. Vascular endothelial growth factor (VEGF) was measured by Western blot analysis and ELISA. Results. Kallikrein-binding protein specifically inhibited proliferation and induced apoptosis in retinal capillary endothelial cells. Intravitreal injection of KBP inhibited retinal neovascularization in an OIR model. Moreover, KBP decreased vascular leakage in the retina, iris and choroid in rats with OIR. Blockade of kinin receptors by specific antagonists showed significantly weaker inhibition of endothelial cells, when compared to that of KBP, suggesting that the anti-angiogenic activity of KBP is not through inhibiting kallikrein activity or kinin production. KBP competed with 125I-VEGF for binding to endothelial cells and down-regulated VEGF production in endothelial cells and in the retina of the OIR rat model. Conclusion/interpretation. Kallikrein-binding protein is a multi-functional serpin, and its vascular activities are independent of its interactions with the kallikrein-kinin system. Inhibition of VEGF binding to its receptors and down-regulation of VEGF expression could represent a mechanism for the vascular activities of KBP. [ABSTRACT FROM AUTHOR]

Headpin (SERPINB13) is a novel member of the serine proteinase inhibitor (Serpin) gene family that was originally cloned from a keratinocyte cDNA library. Western blot analysis using a headpin-specific antiserum recognized a protein with the predicted Mr of 44 kDa in lysates derived from a transformed keratinocyte cell line known to express headpin mRNA. Similarity of the reactive-site loop (RSL) domain of headpin, notably at the P1–P1′ residues, with other serpins that inhibit cysteine and serine proteinases suggests that headpin may inhibit similar proteinases. This study demonstrates that recombinant headpin indeed inhibits cathepsins K and L, but not chymotrypsin, elastase, trypsin, subtilisin A, urokinase-type plasminogen activator, plasmin, or thrombin. The second-order rate constants (ka) for the inhibitory reactions of rHeadpin with cathepsins K and L were 5.1±0.6×104 and 4.1±0.8×104 M−1 s−1, respectively. Headpin formed SDS-stable complexes with cathepsins K and L, a characteristic property of inhibitory serpins. Interactions of the RSL domain of headpin with cathepsins K and L were indicated by cleavage of headpin near the predicted P1–P1′ residues by these proteinases. These results demonstrate that the serpin headpin possesses specificity for inhibiting lysosomal cysteine proteinases. [Copyright &y& Elsevier]

Heterozygosity for a G → A mutation converting Val384(GTG) to Met(ATG) associated with plasmin inhibitor (alpha2-antiplasmin) deficiency was identified in three family members with bleeding tendency. The proband had traumatic breast haematoma and per-/postoperative bleeds. An affected daughter required a blood transfusion after a normal delivery and a son had prolonged bleeding after tooth extraction. The plasma plasmin inhibitor activities of the affected family members were reduced to 49–66% of normal. The antigenic concentrations determined by electroimmunoassay were reduced to 57–68% of normal. Crossed immunoelectrophoresis of plasma from the proband showed a normal pattern. The amino acid Val384 is located eight residues C-terminal (P8′) of the P1 residue (Arg376) in the reactive site. The P8′ residues of bovine and mouse plasmin inhibitor are both Val. Among other serpins the P8′ residue is unconserved. The mutation was not present in the non-affected family member or 30 blood donors. In addition to the Val384Met mutation two new polymorphisms Ala-26(GCG)/Val(GTG) and Arg407(AGG)/Lys(AAG) and one previously reported polymorphism Arg6(CGG)/Trp(TGG) were identified in the plasmin inhibitor gene of the family. The allelic frequencies among 30 blood donors with normal values of plasma plasmin inhibitor (functional) were 0.84/0.16 for C/T in codon -26, 0.81/0.19 for C/T in codon 6 and 0.83/0.17 for G/A in codon 407. [ABSTRACT FROM AUTHOR]