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Pin-II protease inhibitors (PIs) are the focus of research interest because of their large structural-functional diversity and relevance in plant defense. Two representative Capsicum annuum PI genes (CanPI-15 and -7) comprising one and four inhibitory repeat domains, respectively, were expressed and recombinant proteins were characterized. β-Sheet and unordered structure was found predominant in CanPI-15 while -7 also displayed the signatures of polyproline fold, as revealed by circular dichroism studies. Inhibition kinetics against bovine trypsin indicated three times higher potency of CanPI-7 (K(i)~57 μM) than -15 (~184 μM). Activity and structural stability of these CanPIs were revealed under various conditions of pH, temperature and denaturing agent. Structure prediction, docking studies with proteases and mass spectroscopy revealed the organization of multiple reactive site loops of multi domain PIs in space as well as the steric hindrances imposed while binding to proteases due to their close proximity., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Here we describe the isolation of a novel C-terminally amidated octadecapeptide--SVIGCWTKSIPPRPCFVK-amide--that contains a disulphide loop between Cys(5) and Cys(15) that is consistent with a Bowman-Birk type protease inhibitor, from the skin secretion of the Chinese Bamboo odorous frog, Huia versabilis. Named HV-BBI, the peptide is encoded by a single precursor of 62 amino acid residues whose primary structure was deduced from cloned skin cDNA. The precursor exhibits the typical organization of that encoding an amphibian skin peptide with a highly-conserved signal peptide, an intervening acidic amino acid residue-rich domain and a single HV-BBI-encoding domain located towards the C-terminus. A synthetic replicate of HV-BBI, with the wild-type K (Lys-8) residue in the presumed P1 position, was found to be a potent inhibitor of trypsin with a K(i) just slightly less than 19 nM. Substitution at this site with R (Arg) resulted in a significant reduction in potency (K(i) 57 nM), whereas replacement of K with F (Phe) resulted in the complete abolition of trypsin inhibitory activity. Thus, HV-BBI is a potent inhibitor of trypsin and the lysyl (K) residue that occupies the P1 position appears to be optimal for potency of action against this protease.

MTJ1/ERdj1 and its human homologue HTJ1 are membrane proteins that interact with the molecular chaperone BiP through their J-domain. HTJ1 also contains a C-terminal cytosolic region of unknown function that consists of two SANT domains separated by a spacer region. We recently showed that the second SANT domain of HTJ1 (SANT2) binds to alpha1-antichymotrypsin and alters its serpin activity [B. Kroczynska, C.M. Evangelista, S.S. Samant, E.C. Elguindi, S.Y. Blond, The SANT2 domain of the murine tumor cell DnaJ-like protein 1 human homologue interacts with alpha1-antichymotrypsin and kinetically interferes with its serpin inhibitory activity, J. Biol. Chem. 279 (2004) 11432-11443]. Here, we identified a new variant of human inter-alpha-trypsin inhibitor heavy chain 4 (ITIH4) that also interacts with the SANT2 domain of HTJ1. Biochemical, mutagenesis, and fluorescence studies demonstrate that SANT2 binds to a carboxyl-terminal fragment that corresponds to the last third of the new ITIH4 isoform sequence (residues 588-930). ITIH4 and MTJ1 co-immunoprecipitate from total liver protein extracts and SANT2 protects the ITIH4(588-930) recombinant fragment from being processed by kallikrein in vitro. This work reveals that the SANT2 domain of HTJ1 is a genuine protein-protein interaction module.

Serine proteinase inhibitors are widely distributed in nature and inhibit the activity of enzymes like trypsin and chymotrypsin. These proteins interfere with the physiological processes such as germination, maturation and form the first line of defense against the attack of seed predator. The most thoroughly examined plant serine proteinase inhibitors are found in the species of the families Leguminosae, Graminae, and Solanaceae. Leucaena leucocephala belongs to the family Leguminosae. It is widely used both as an ornamental tree as well as cattle food. We have constructed a three-dimensional model of a serine proteinase inhibitor from L. leucocephala seeds (LTI) complexed with trypsin. The model was built based on its comparative homology with soybean trypsin inhibitor (STI) using the program, MODELLER6. The quality of the model was assessed stereochemically by PROCHECK. LTI shows structural features characteristic of the Kunitz type trypsin inhibitor and shows 39% residue identity with STI. LTI consists of 172 amino acid residues and is characterized by two disulfide bridges. The protein is a dimer with the two chains being linked by a disulfide bridge. Despite the high similarity in the overall tertiary structure, significant differences exist at the active site between STI and LTI. The present study aims at analyzing these interactions based on the available amino acid sequences and structural data. We have also studied some functional sites such as phosphorylation, myristoylation, which can influence the inhibitory activity or complexation with other molecules. Some of the differences observed at the active site and functional sites can explain the unique features of LTI.

Sonic hedgehog (Shh) is a secreted signaling protein that plays important roles in a variety of developmental processes and also in pathogenesis of some human cancers and congenital diseases. Molecules that function downstream of Shh, however, still remain elusive. Here we searched for Shh-responsive genes by using an in-house cDNA microarray. Two genes were newly identified to be Shh responsive in neuroepithelial cell line MNS-70: the metal-binding protein Ceruloplasmin (Cp) and the serine protease inhibitor inter-alpha-trypsine inhibitor heavy chain H3 (ITIH3). In MNS-70 cells, expression of ITIH3 was regulated by Gli zinc-finger transcription factors downstream of Shh, whereas Cp appeared to be regulated by Gli-independent pathways. Cp mRNA was detected in the developing mouse brain, where its expression domain was closely adjacent to that of Shh. These results demonstrate that microarray technology provides a useful tool for studying expression of developmentally regulated genes.

An isolation procedure comprising affinity chromatography on Affi-gel blue gel, ion exchange chromatography on SP-Toyopearl, and fast protein liquid chromatography on Mono S was used to purify a peptide from broad beans which manifested antifungal activity toward Mycosphaerella arachidicola, Fusarium oxysporum, and Botrytis cinerea. The peptide demonstrated a molecular mass of 7.5 kDa. N-terminal sequence analysis disclosed the identity of the antifungal peptide to be a trypsin-chymotrypsin inhibitor. The trypsin-chymotrypsin inhibitor also exerted an inhibitory action on chymotrypsin activity and HIV-1 reverse transcriptase activity. Proliferation of murine splenocytes was stimulated in the presence of the trypsin-chymotrypsin inhibitor. This report constitutes the first observation of antifungal activity of a leguminous peptidic protease inhibitor., (Copyright 2001 Academic Press.)

The mustard trypsin inhibitor MTI-2 is a potential tool in the study of interactions between pest insects and plants. It can be applied to study the adaptations of digestive proteases in pest insects. Phage display allows a rapid and exhaustive system for the selection of heterologous protein variants with novel specificities. Here we describe a bacteriophage expression system which permits functional expression of MTI-2 variants. Active and inactive mutants of MTI-2 are constructed and displayed on phage. These are used to demonstrate that an active variant can be selected from a background of 10,000 inactive mutants in four rounds of selection and amplification., (Copyright 2001 Academic Press.)

The design of chimeric proteins is a major field of interest in structural biology and biotechnology. The successful design of the chimeric protein composed by the minimized reactive site domain of the low-molecular-mass trypsin inhibitor from Brassica napus (var. oleifera) seed (Ser3-Lys35; mini-RTI-III) and murine dihydrofolate reductase (DHFR) is reported here. The DHFR-mini-RTI-III chimeric protein was expressed in Escherichia coli, purified by metal-chelate affinity chromatography and oxidatively refolded. The affinity of the purified and refolded DHFR-mini-RTI-III for bovine trypsin (K = 5.0 x 10(-10) M) was closely similar to that determined for native RTI-III (K = 2.9 x 10(-10) M), at pH 8.2 and 22.0 degrees C. DHFR-mini-RTI-III may be regarded as a tool in structure-function studies and for developing multifunctional and multidomain proteinase inhibitors., (Copyright 2000 Academic Press.)

Metallothionein (MT) can modulate transcriptional activity in vitro. We examined whether the absence of MT affects gene expression in vivo. We compared the hepatic RNA profiles of wild-type and MT-null neonatal mice using improved differential display. The hepatic MT level was maximal during neonatal development. We identified five cDNA fragments that were expressed in MT-null mice at different levels from those in wild-type mice. Two were fragments of MT-I and mutant MT-I cDNA. The sequences of the other cDNA fragments were identical to those of contrapsin, transketolase, and vanin-3. The latter two were up-regulated, whereas contrapsin was down-regulated in neonatal MT-null mice. These mRNA levels were remarkably different between the two strains of neonatal mice. Further characterization of the regulated mRNA identified here will determine whether or not they are primary or secondary effects of an MT deficiency., (Copyright 2000 Academic Press.)

The newly developed techniques of peptide libraries have become a conventional and efficient method in screening ligands of proteins of interest. We present here the successful results of selection of trypsin inhibitors in a phage hexapeptide library. After affinity selection and activity assay, peptide sequences, deduced from DNA sequencing of the phage peptides with the most striking trypsin activity, share some common features with trypsin inhibitors reported. All of the phage peptides selected out and those native and synthetic trypsin inhibitors reported are composed of three parts: (a) positively charged part (Arg, Lys or their analogs); (b) polar part that may form hydrogen bonds with Ser in the active site of trypsin; (c) hydrophobic part that interacts with the nonpolar region of trypsin active site.

Cucurbita maxima trypsin inhibitor-V (CMTI-V) is also a specific inhibitor of human blood coagulation factor beta-factor XIIa. A recombinant version of CMTI-V has allowed probing of roles of individual amino acid residues including the reactive site residue, lysine (P1), by site-directed mutagenesis. The K44R showed at least a 5-fold increase in inhibitory activity toward human beta-factor XIIa, while there was no change toward bovine trypsin. This result demonstrates that beta-factor-XIIa prefers an arginine residue over lysine residue, while trypsin is non-specific to lysine or arginine in its binding pocket. On the other hand, the specificity of CMTI-V could be changed from trypsin to chymotrypsin inhibition by mutation of the P1 residue to either leucine or methionine (K44L or K44M).

Three highly homologous serine protease inhibitors, SPI-1, SPI-2 and SPI-3 (contrapsins), are synthesized in rat liver. Their expression is regulated differently in healthy and inflamed animals. We found that interleukin 6 (IL-6), a major acute phase cytokine, and to a lesser extent leukemia inhibitory factor (LIF), both together with glucocorticoids, are responsible for the regulation of expression of the contrapsins in rat hepatocytes in primary culture. The effect of IL-6 is time- and dose-dependent. IL-1, TGF beta 1, HGF, PMA and IL-8 did not have any effect on contrapsin mRNA levels. We postulate that SPI-1, SPI-2 and SPI-3 belong to the class II acute phase proteins. Additionally, we show induction of SPI-3 mRNA in rat liver by in situ hybridization using a specific oligonucleotide probe.

Synthetic oligonucleotides corresponding to all possible sequences of N-terminal and C-terminal region of Acacia confusa trypsin inhibitor were used to generate ACTI-related sequences using the polymerase chain reaction on the cDNAs encoding ACTI of the seeds of legume, A. confusa. The deduced amino acid sequence agreed with that determined by the peptide analysis except an extra amino acid residue, serine, was found at the junction of A and B chain, which was removed by post-translation processing with specific protease(s). The substrate specificity of the protease(s) was found to cleave at the C-terminal sites of asparagine and serine, which was also shown to be the same case for another plant protein, abrin, isolated from legume, Abrus precatorius.

The putative inhibitor domain of Alzheimer's disease amyloid protein precursor was purified from E. coli containing a synthetic gene encoding the Kunitz domain. The purified protein (A4 inhibitor) inhibited the activity of trypsin, forming a 1:1 molar complex with the enzyme. It also strongly inhibited plasmin (Ki = 7.5 x 10(-11) M) from human serum and tryptase (Ki = 2.2 x 10(-10) M) from rat mast cells (tryptase M). In addition, it inhibited rat pancreatic trypsin, alpha-chymotrypsin and kallikrein and human serum kallikrein, but did not inhibit rat chymase, pancreatic elastase, alpha-thrombin, urokinase, papain or cathepsin B.

We have isolated and sequenced a cDNA clone coding for the human pancreatic secretory trypsin inhibitor (PSTI) from a human pancreatic cDNA library. The predicted product consists of 79 amino acids, and contains no apparent functional polypeptide other than PSTI. Southern blot analysis suggests that there is one copy of PSTI gene per haploid genome. This gene seems to be expressed not only in pancreas, but also in gastric mucosa, since a Northern blot analysis demonstrated the presence of a poly(A) RNA of the same size as in the pancreas. A comparison of sequences between human PSTI mRNA and mouse epidermal growth factor (EGF) mRNA revealed a high homology, suggesting that they share a common ancestral DNA sequence.

Seven microproteins analogous to Ecballium elaterium Trypsin Inhibitor II. were synthesized. The study of their inhibiting power showed a change in selectivity from trypsin to elastase for 5 of the compounds and to alpha-chymotrypsin for another one. A striking characteristic that appeared during this synthetic approach was the ability of the 28 amino acid peptides to refold and close correctly the 3 disulfide bridges, giving in each case an active compound.

Two types of cDNA, which code for the two types of rat pancreatic secretory trypsin inhibitors (PSTIs), were cloned and sequenced. Both predicted amino acid sequences consisting of 79 amino acids, with the secretion signal peptide consisting of 18 and 23 amino acids for PSTI-I and PSTI-II, respectively. The nucleotide sequences were 91% homologous between the two cDNAs, but 68% and 65% homologous, respectively, when compared with human PSTI cDNA. Northern blot analyses showed that PSTI-I is expressed in the pancreas, whereas PSTI-II is expressed in the pancreas and the liver using the same promoter. Southern blot analyses suggested that both PSTI-I and PSTI-II genes are single copy genes per haploid genome. Duplication of rat PSTI gene seems to have occurred recently, after the divergence of humans and rats.

The human pancreatic secretory trypsin inhibitor (PSTI) gene was isolated from a human gene library. Restriction endonuclease mapping and DNA sequencing analysis revealed that this gene is approximately 7.5 kb long and is separated into four exons by three introns. The gene has multiple transcription start points and examination with a single-laser cell-sorter showed that it is located on chromosome 5.