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Eglin C, a small protein from the medicinal leech, has been long considered a general high‐affinity inhibitor of chymotrypsins and elastases. Here, we demonstrate that eglin C inhibits human chymotrypsin‐like protease (CTRL) weaker by several orders of magnitude than other chymotrypsins. In order to identify the underlying structural aspects of this unique deviation, we performed comparative molecular dynamics simulations on experimental and AlphaFold model structures of bovine CTRA and human CTRL. Our results indicate that in CTRL, the primary determinants of the observed weak inhibition are amino‐acid positions 192 and 218 (using conventional chymotrypsin numbering), which participate in shaping the S1 substrate‐binding pocket and thereby affect the stability of the protease‐inhibitor complexes. [ABSTRACT FROM AUTHOR]

Chymotrypsin-like protease (CTRL) is one of the four chymotrypsin isoforms expressed in the human exocrine pancreas. Human genetic and experimental evidence indicate that chymotrypsins B1, B2, and C (CTRB1, CTRB2 and CTRC) are important not only for protein digestion but also for protecting the pancreas against pancreatitis by degrading potentially harmful trypsinogen. CTRL has not been reported to play a similar role, possibly due to its low abundance and/or different substrate specificity. To address this problem, we investigated the specificity of the substrate-binding groove of CTRL by evolving the substrate-like canonical loop of the Schistocerca gregaria proteinase inhibitor 2 (SGPI-2), a small-protein reversible chymotrypsin inhibitor to bind CTRL. We found that phage-associated SGPI-2 variants with strong affinity to CTRL were similar to those evolved previously against CTRB1, CTRB2 or bovine chymotrypsin A (bCTRA), indicating comparable substrate specificity. When tested as recombinant proteins, SGPI-2 variants inhibited CTRL with similar or slightly weaker affinity than bCTRA, confirming that CTRL is a typical chymotrypsin. Interestingly, an SGPI-2 variant selected with a Thr29His mutation in its reactive loop was found to inhibit CTRL strongly, but it was digested rapidly by bCTRA. Finally, CTRL was shown to degrade human anionic trypsinogen, however, at a much slower rate than CTRB2, suggesting that CTRL may not have a significant role in the pancreatic defense mechanisms against inappropriate trypsinogen activation and pancreatitis. [ABSTRACT FROM AUTHOR]

Comparing the genomes of mammals which evolved to have poor vision identifies an important gene for eyesight.

Despite decades of research, knowledge about the genes that are important for development and function of the mammalian eye and are involved in human eye disorders remains incomplete. During mammalian evolution, mammals that naturally exhibit poor vision or regressive eye phenotypes have independently lost many eye-related genes. This provides an opportunity to predict novel eye-related genes based on specific evolutionary gene loss signatures. Building on these observations, we performed a genome-wide screen across 49 mammals for functionally uncharacterized genes that are preferentially lost in species exhibiting lower visual acuity values. The screen uncovered several genes, including SERPINE3, a putative serine proteinase inhibitor. A detailed investigation of 381 additional mammals revealed that SERPINE3 is independently lost in 18 lineages that typically do not primarily rely on vision, predicting a vision-related function for this gene. To test this, we show that SERPINE3 has the highest expression in eyes of zebrafish and mouse. In the zebrafish retina, serpine3 is expressed in Müller glia cells, a cell type essential for survival and maintenance of the retina. A CRISPR-mediated knockout of serpine3 in zebrafish resulted in alterations in eye shape and defects in retinal layering. Furthermore, two human polymorphisms that are in linkage with SERPINE3 are associated with eye-related traits. Together, these results suggest that SERPINE3 has a role in vertebrate eyes. More generally, by integrating comparative genomics with experiments in model organisms, we show that screens for specific phenotype-associated gene signatures can predict functions of uncharacterized genes.

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.

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]

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]

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]

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. [ABSTRACT FROM AUTHOR]

Plants and insects have been coexisting for more than 350 million years. During this time, both have evolved many strategies to successfully exploit or respond to reciprocal adaptation and defense reactions. Plants tend to minimize the damage caused by pest feeding, while pests tend to manipulate plant response by suppressing plant defense mechanisms or developing strategies to overcome plant defense systems. Plants recognize insect pests by the wounding that they cause and elicitors present in pest oral secretions (saliva and/or regurgitant). These elicitors or insect-associated microorganisms can modulate plant response to the benefit of their insect hosts. In this article, we have undertaken an analysis of gene expression in serine and cysteine proteinase inhibitors (SerPI and CysPI, respectively) in wheat (Triticum aestivum) plants exposed to cereal leaf beetle (CLB, Oulema melanopus, Coleoptera, Chrysomelidae) larvae feeding, and the impact of microbes associated with CLB on the expression levels of these genes. Using three wheat varieties and antibiotic-treated and untreated CLB larvae, we found that SerPI plays a more important role than CysPIs in plant defense against CLB larvae. Additionally, higher levels of SerPI gene expression were observed in systemic leaves in comparison to the wounded leaves (local response). Each of the tested wheat varieties reacted in a specific way to the particular treatment. Moreover, the presence of microbial components associated with insects influenced plant response to CLB larvae feeding. [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]

A chymotrypsin inhibitor was isolated and purified from the seeds of Enterolobium saman (Leguminaceae family) by extraction with 100 mM phosphate buffer, heat treatment, ammonium sulphate precipitation, ion-exchange chromatography on DEAEcellulose and filtration through Sephadex G-75. The final preparation appeared to be homogeneous by both chromatographic and electrophoretic analyses. ESCI had a molecular weight of about 17,890 and an isoelectric point of 5.8. ESCI inhibited bovine chymotrypsin at an inhibitor-enzyme molar ratio of 1:2. The inhibition mode of chymotrypsin inhibitor was competitive on bovine chymotrypsin. Investigation has been carried out on the complex formed between chymotrypsin and chymotrypsin inhibitor by physico-chemical methods. An apparent dissociation constant (Ki) of 9.05 X 10-8 M has been calculated for the complex. This enzyme- inhibitor complex was isolated by gel filtration on Sephadex G-75 and a molecular weight of 43.000 was estimated for the complex. The inhibitor did not have any effect on other proteinases, such as papain, bromelin, elastase, α -amylase, trypsin and pepsin. The chemical modification of lysine residues indicated that –NH2 groups are not essential for the activity of ESCI toward chymotrypsin. The inhibitor was an acidic protein and was stable over a wide pH range of 2-12 and temperature range of 10o C-97o C.

Chymotrypsin-like protease (CTRL) is one of the four chymotrypsin isoforms expressed in the human exocrine pancreas. Human genetic and experimental evidence indicate that chymotrypsins B1, B2, and C (CTRB1, CTRB2 and CTRC) are important not only for protein digestion but also for protecting the pancreas against pancreatitis by degrading potentially harmful trypsinogen. CTRL has not been reported to play a similar role, possibly due to its low abundance and/or different substrate specificity. To address this problem, we investigated the specificity of the substrate-binding groove of CTRL by evolving the substrate-like canonical loop of the Schistocerca gregaria proteinase inhibitor 2 (SGPI-2), a small-protein reversible chymotrypsin inhibitor to bind CTRL. We found that phage-associated SGPI-2 variants with strong affinity to CTRL were similar to those evolved previously against CTRB1, CTRB2 or bovine chymotrypsin A (bCTRA), indicating comparable substrate specificity. When tested as recombinant proteins, SGPI-2 variants inhibited CTRL with similar or slightly weaker affinity than bCTRA, confirming that CTRL is a typical chymotrypsin. Interestingly, an SGPI-2 variant selected with a Thr29His mutation in its reactive loop was found to inhibit CTRL strongly, but it was digested rapidly by bCTRA. Finally, CTRL was shown to degrade human anionic trypsinogen, however, at a much slower rate than CTRB2, suggesting that CTRL may not have a significant role in the pancreatic defense mechanisms against inappropriate trypsinogen activation and pancreatitis., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

Serine protease inhibitor protease nexin-1 (PN-1) is encoded by the SERPINE2 gene. It belongs to a member of serine protease inhibitor superfamily, and is a kind of glycoprotein which is secreted by a variety of cells. PN-1 is a serpin that is barely detectable in the plasma but is discovered in many organs and cell types, which plays a key role in the physiological and pathological processes, such as the blood congealing, immunoreactions, plasmin fusion, inflammation and tumor suppressor. In recent years, the research of PN-1 has been paid more attention to. This article will summarize the latest research progress of PN-1 in the circulatory system, cancer, nervous system and lung-related diseases. [ABSTRACT FROM AUTHOR]

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Expression of recombinant therapeutic proteins in transgenic plants has a tremendous impact on safe and economical production of biomolecules for biopharmaceutical industry. The major limitation in their production is downstream processing of recombinant protein to obtain higher yield and purity of the final product. In this study, a simple and rapid process has been developed for purification of therapeutic recombinant α-proteinase inhibitor (rα-PI) from transgenic tomato plants, which is an abundant serine protease inhibitor in human serum and chiefly inhibits the activity of neutrophil elastase in lungs. We have expressed rα-PI with modified synthetic gene in transgenic tomato plants at a very high level (≃3.2 % of total soluble protein). The heterologous protein was extracted with (NH)SO precipitation, followed by chromatographic separation on different matrices. However, only immunoaffinity chromatography resulted into homogenous preparation of rα-PI with 54 % recovery. The plant-purified rα-PI showed molecular mass and structural conformation comparable to native serum α-PI, as shown by mass spectrometry and optical spectroscopy. The results of elastase inhibition assay revealed biological activity of the purified rα-PI protein. This work demonstrates a simple and efficient one-step purification of rα-PI from transgenic plants, which is an essential prerequisite for further therapeutic development. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Pacifastin is a recently classified family of serine proteinase inhibitors that play essential roles in various biological processes, including in the regulation of the melanization cascade. Here, a novel pacifastin-related gene, termed Pm Pacifastin-like, was identified from a reverse suppression subtractive hybridization (SSH) cDNA library created from hemocytes of the prophenoloxidase Pm proPO1/2 co-silenced black tiger shrimp Penaeus monodon . The full-length sequences of Pm Pacifastin-like and its homologue Lv Pacifastin-like from the Pacific white shrimp Litopenaeus vannamei were determined. Sequence analysis revealed that both sequences contained thirteen conserved pacifastin light chain domains (PLDs), followed by two putative kunitz domains. Expression analysis demonstrated that the Pm Pacifastin-like transcript was expressed in all tested shrimp tissues and larval developmental stages, and its expression responded to Vibrio harveyi challenge. To gain insight into the functional roles of Pm Pacifastin-like protein, the in vivo RNA interference experiment was employed; the results showed that Pm Pacifastin-like depletion strongly increased PO activity. Interestingly, suppression of Pm Pacifastin-like also down-regulated the expression of the proPO-activating enzyme Pm PPAE2 transcript; the Pm Pacifastin-like transcript was down-regulated after the Pm proPO1/2 transcripts were silenced. Taken together, these results suggest that Pm Pacifastin-like is important in the shrimp proPO system and may play an essential role in shrimp immune defense against bacterial infection. These results also expand the knowledge of how pacifastin-related protein participates in the negative regulation of the proPO system in shrimp. [ABSTRACT FROM AUTHOR]

Jatropha curcas seed cake is a low-value by-product resulting from biodiesel production. The seed cake is highly toxic, but it has great potential for biotechnology applications as it is a repository of biomolecules that could be important in agriculture, medicine and industry. To explore this potential, a novel trypsin inhibitor called JcTI-I was purified by fractionation of the crude extract with trichloroacetic acid (2.5%, v/v) followed by affinity chromatography (Trypsin-Sepharose 4B) and molecular exclusion (Sephacryl S-200). Non-reducing SDS-PAGE and gel filtration showed that JcTI-I has approximately 20.0 kDa. Mass spectrometry analysis revealed that the intact molecular mass of JcTI-I is 10.252 kDa. Moreover, JcTI-I is a glycoprotein with 6.4% (m/m) carbohydrates, pI of 6.6, N-terminal sequence similarity around 60% to plant albumins and high stability to heat, pH and salinity. JcTI-I presented antibacterial activity against the human pathogenic bacteria Salmonella enterica subspecies enterica serovar choleraesuis and Staphylococcus aureus, with minimum inhibitory concentration (MIC) less than 5 µg/mL. Furthermore, JcTI-I did have inhibitory activity against the serine proteases from the tested bacteria. Otherwise, no hemolytic activity of human erythrocytes and signs of acute toxicity to mice were observed for JcTI-I. The results demonstrate the benefits of J. curcas seed cake as a source of trypsin inhibitor with potential for biotechnological application as a new antimicrobial agent against human pathogenic bacteria.

A 5-domain Kazal type serine proteinase inhibitor SPI Pm 2 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 SPI Pm 2 were over-expressed and tested against WSSV infection. By using hemocyte primary cell culture treated with each recombinant SPI Pm 2 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, rSPI Pm 2D2, was used. Injecting the WSSV along with rSPI Pm 2D2 but not with other domains caused delay in mortality rate of the infected shrimp. The results indicate that the SPI Pm 2D2 possesses strong antiviral activity and, hence, contributes predominantly to the antiviral activity of SPI Pm 2. [ABSTRACT FROM AUTHOR]

Abstract: 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 &y& Elsevier]

Abstract: 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. [Copyright &y& Elsevier]

Highlights: [•] A full-length cDNA of PmSERPIN3 gene were identified. [•] The PmSERPIN3 gene was constitutively expressed in all tissues tested and upon pathogen infection. [•] The rPmSERPIN3 protein inhibited subtilisin and prophenoloxidase activation. [•] The rPmSERPIN3 decreased the clearance efficacy of bacteria in the shrimp hemolymph. [•] The PmSERPIN3 was produced in all three types of shrimp hemocytes. [ABSTRACT FROM AUTHOR]

Abstract: 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 &y& Elsevier]

Expression pattern of a sugar beet serine proteinase inhibitor gene, BvSTI, was characterized in response to mechanical and fall armyworm (FAW; Spodoptera frugiperda J.E. Smith) induced wounding. BvSTI expression was analyzed in three breeding lines moderately resistant to sugar beet root maggot (Tetanops myopaeformis Roder) and in a susceptible line, F1010. Increased mechanical wound induced levels of BvSTI expression were observed in all resistant lines as compared to F1010 during the first 24 h. The most intensive response to wounding was observed in one of the resistant lines, F1016, with a maximum 5- and 2.5-fold increase of BvSTI transcript levels over non-wounded roots and leaves, respectively. In contrast, slight increase of BvSTI transcript levels in leaves and even an initial decrease in roots were observed in F1010. BvSTI transcript accumulation in F1016 and F1010 tissues wounded by FAW showed a similar gene expression pattern, but it was delayed and less intense than the response incited by abiotic wounding. On the protein level, BvSTI specific antibody confirmed increased accumulation of the 30 kDa BvSTI protein in wounded leaves but not in roots of F1016 and F1010. Using trypsin inhibition assays, the activity of BvSTI was confirmed in F1016 roots and leaves and F1010 leaves. In F1010 roots BvSTI activity was completely lacking. We conclude that BvSTI gene expression was wound induced in the insect resistant germplasm suggesting that BvSTI can be used in biotechnological approaches or in breeding programs for improving insect resistance. [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]

Kallikrein-related peptidases (KLKs) are secreted serine proteinases with trypsin or chymotrypsin-like activity. Several family members, such as KLKs 6 and 10, are potential ovarian cancer biomarkers. Recently, using a newly developed assay for active KLK6, we found that only a very small proportion of immunoreactive KLK6 in tumor-derived clinical samples (malignant ascites fluid), in cerebrospinal fluid, and in cancer cell line supernatants is enzymatically active. We therefore hypothesized that a proportion of other immunoreactive KLKs in such samples could be present, but might be partly complexed to endogenous serine proteinase inhibitors. Using a combination of immunological isolation of the enzymes, activity-based probe analysis and proteomics, we identified active KLK10 in ovarian cancer ascites and we provide preliminary data that the activity of other KLKs present in these samples can be decreased by known proteinase inhibitors (e.g., α2-macroglobulin, α1-antitrypsin). Our data suggest that the enzymatic activity of ovarian cancer-released KLKs that are detected by regular immunoassays is low in vivo and very likely regulated by proteinase inhibitors. [ABSTRACT FROM AUTHOR]

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]

Proteinase inhibitors play a significant role in plant defense against insect pests and phytopathogens by inhibiting their proteases. A thermotolerant monomeric trypsin inhibitor with molecular weight ∼18kD was purified from Cocculus hirsutus (ChTI) using trypsin sepharose affinity column. Western blot analysis using ChTI IgY revealed its presence in vegetative parts and seeds. The second and third instar larvae of H. armigera fed with ChTI (5000TIU/ml) resulted in 84.59 and 58.71% reduction in mean larval weight respectively. An increase in the larval growth period was observed in ChTI fed larvae at all instars and inhibitor fed larvae could not complete their life cycle. ChTI caused 74 and 59.53% inhibition of bovine trypsin and Helicoverpa gut proteases respectively. ChTI exhibited strain specificity and inhibited growth and development of plant fungal pathogens. Bioassay studies on yeast strains indicated that ΔYNK and MNN1 are more sensitive to ChTI. The results suggest that phosphodiester linkage in cell wall components is likely to be the key determinants for binding of ChTI. Taken together, these studies indicate that ChTI is a potential candidate for development of transgenic plants against foliar diseases and insect pests. [ABSTRACT FROM AUTHOR]

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: Serine proteinase inhibitors (SPIs) in multi-cellular organisms are important modulators of proteinase activities in various biological processes. A five-domain Kazal-type SPI SPIPm2 from the black tiger shrimp Penaeus monodon is presumably involved in innate immune response. The SPIPm2 with the domain P1 residues T, A, E, K and E was isolated from the hemocyte cDNA libraries and found to strongly inhibit subtilisin and elastase, and weakly inhibit trypsin. To unravel further the inhibitory activity of each domain, we subcloned, over-expressed and purified each individual SPI domain. Their inhibitory specificities against trypsin, subtilisin and elastase were determined. Domain 1 was found to be inactive. Domains 2, 3 and 5 inhibited subtilisin. Domain 2 inhibited also elastase. Domain 4 weakly inhibited subtilisin and trypsin. The intact SPIPm2 inhibitor was found to possess bacteriostatic activity against the Bacillus subtilis but not the Bacillus megaterium, Staphylococcus aureus, Vibrio harveyi 639 and Escherichia coli JM109. Domains 2, 4 and 5 contributed to this bacteriostatic activity. [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]

Abstract: Esophageal Cancer-Related Gene 2 (ECRG2) is a novel member of the KAZAL-type serine proteinase inhibitor family and plays an important role in the inhibition of human esophageal cancer cell proliferation. The previous studies have shown that ECRG2 can bind the urokinase-type plasminogen activator (uPA)/plasmin system and inhibit its activity. In this study, the strategy of cloning, overexpression, and purification of ECRG2 for obtaining a properly folded ECRG2 with accurately formed disulfide bonds was established. The heteronuclear NMR experiments were performed with isotope labeled ECRG2 to investigate the binding interface of the protein with uPA. The sequence regions of ECRG2 for uPA binding were determined. Analysis indicates that the uPA-binding loops of ECRG2 are in correspondence with the reactive site loops for binding of serine proteinase in turkey ovomucoid third domain (OMTKY3). The structural similarity of ECRG2 to OMTKY3 was identified and a model for ECRG2 was proposed. [Copyright &y& Elsevier]

Abstract: Snake venom Kunitz/BPTI members are good tools for understanding of structure–functional relationship between serine proteases and their inhibitors. A novel dual Kunitz/BPTI serine proteinase inhibitor named OH-TCI (trypsin- and chymotrypsin-dual inhibitor from Ophiophagus hannah) was isolated from king cobra venom by three chromatographic steps of gel filtration, trypsin affinity and reverse phase HPLC. OH-TCI is composed of 58 amino acid residues with a molecular mass of 6339Da. Successful expression of OH-TCI was performed as the maltose-binding fusion protein in E. coli DH5α. Much different from Oh11-1, the purified native and recombinant OH-TCI both had strong inhibitory activities against trypsin and chymotrypsin although the sequence identity (74.1%) between them is very high. The inhibitor constants (K i) of recombinant OH-TCI were 3.91×10−7 and 8.46×10−8 M for trypsin and chymotrypsin, respectively. To our knowledge, it was the first report of Kunitz/BPTI serine proteinase inhibitor from snake venom that had equivalent trypsin and chymotrypsin inhibitory activities. [Copyright &y& Elsevier]

Abstract: By Sephadex G-50 gel filtration, Resource Q anionic exchange and C4 reversed phase liquid high performance liquid chromatography, a proteinase inhibitor protein (Ranaserpin) was identified and purified from the eggs of the odour frog, Rana grahami. The protein displayed a single band adjacent to the molecular weight marker of 14.4 kDa analyzed by SDS-PAGE. The inhibitor protein homogeneity and its molecular weight were confirmed again by MALDI-TOF mass spectrometry analysis. The MALDI-TOF mass spectrum analysis gave this inhibitor protein an m/z of 14422.26 that was matched well with the result from SDS-PAGE. This protein is a serine proteinase inhibitor targeting multiple proteinases including trypsin, elastase, and subtilisin. Ranaserpin inhibited the proteolytic activities of trypsin, elastase, and subtilisin. It has an inhibitory constant (K i) of 6.2×10− 8 M, 2.7×10− 7 M and 2.2×10− 8 M for trypsin, elastase, and subtilisin, respectively. This serine proteinase inhibitor exhibited bacteriostatic effect on Gram-positive bacteria Bacillus subtilis (ATCC 6633). It was suggested that ranaserpin might act as a defensive role in resistance to invasion of pests or pathogens. This is the first report of serine proteinase inhibitor and its direct defensive role from amphibian eggs. [Copyright &y& Elsevier]

Abstract: A partial clone coding for a two-WAP domain protein was isolated from a Litopenaeus vannamei haemocytes cDNA library. The complete sequence was obtained by RACE, and the full-length cDNA sequence is 0.8Kb long and encodes for a 116-amino acid protein. The domain composition is similar to the mammalian WFDC5 (WAP four disulfide core) and secretory leukocyte proteinase inhibitor (SLPI). Modifications in expression were determined by real-time PCR, after injection of Vibrio alginolyticus, suggesting its participation in the shrimp immune response. Structural and phylogenetic analyses showed close similarity between shrimp and mammalian SLPI, indicating a probable common ancestor. This is the first report of a mammalian SLPI-like protein in an invertebrate. [Copyright &y& Elsevier]

The complete amino acid sequence obtained by electrospray ionization tandem mass spectrometry of the proteinase inhibitor CmPI-II isolated from Cenchritis muricatus is described. CmPI-II is a 5480-Da protein with three disulfide bridges that inhibits human neutrophil elastase (HNE) ( Ki 2.6±0.2 nM), trypsin ( Ki 1.1±0.9 nM), and other serine proteinases such as subtilisin A ( Ki 30.8±1.2 nM) and pancreatic elastase ( Ki 145.0±4.4 nM); chymotrypsin, pancreatic and plasma kallikreins, thrombin and papain are not inhibited. CmPI-II shares homology with the Kazal-type domain and may define a new group of ‘non-classical’ Kazal inhibitors according to its CysI-CysV disulfide bridge position. The 3D model of CmPI-II exhibits similar secondary structure characteristics to Kazal-type inhibitors and concurs with circular dichroism experiments. A 3D model of the CmPI-II/HNE complex provides a structural framework for the interpretation of its experimentally determined Ki value. The model shows both similar and different contacts at the primary binding sites in comparison with the structure of turkey ovomucoid third domain (OMTKY3)/HNE used as template. Additional contacts calculated at the protease-inhibitor interface could also contribute to the association energy of the complex. This inhibitor represents an exception in terms of specificity owing to its ability to strongly inhibit elastases and trypsin. [ABSTRACT FROM AUTHOR]

Two new serine proteinase inhibitors (RmIn I and RmIn II) from the tropical sea anemone Radianthus macrodactylus have been isolated and characterized. The purification procedure includes polychrome-1 hydrophobic chromatography, Superdex™ Peptide 10/30 FPLC, and Nucleosil C18 reverse-phase HPLC. The molecular masses of RmIn I, RmIn II, and the complexes RmIn II/trypsin and RmIn I,II/α-chymotrypsin have been determined. The K i values of RmIn I and RmIn II for trypsin and α-chymotrypsin have been determined. The polypeptides RmIn I and RmIn II are shown to be nontoxic and to exhibit antihistamine activity. The N-terminal amino acid sequences of RmIn I (GICSEPIVVGPCKAG-) and RmIn II (GSTCLEPKVVGPCKA-) have been determined. A high homology of the amino acid sequences is demonstrated for the proteinase inhibitors produced by such evolutionarily distant species as coelenterates, reptiles, and mammals. [ABSTRACT FROM AUTHOR]

Abstract: A novel five-domain Kazal-type serine proteinase inhibitor, SPIPm2, identified from the hemocyte cDNA library of black tiger shrimp Penaeus monodon was successfully expressed in the Escherichia coli expression system. The expressed recombinant SPIPm2 (rSPIPm2) as inclusion bodies was solubilized with a sodium carbonate buffer, pH10, and purified by gel filtration chromatography. The molecular mass of rSPIPm2 was determined using MALDI-TOF mass spectrometry to be 29.065kDa. The inhibitory activities of rSPIPm2 were tested against trypsin, α-chymotrypsin, subtilisin and elastase. The inhibitor exhibited potent inhibitory activities against subtilisin and elastase, weak inhibitory activity against trypsin, and did not inhibit chymotrypsin. Tight-binding inhibition assay suggested that the molar ratios of SPIPm2 to subtilisin and elastase were 1:2 and 1:1, respectively. The inhibition against subtilisin and elastase was a competitive type with inhibition constants (K i) of 0.52 and 3.27nM, respectively. The inhibitory activity of SPIPm2 against subtilisin implies that, in shrimp, it may function as a defense component against proteinases from pathogenic bacteria but the elastase inhibitory function is not known. [Copyright &y& Elsevier]