Novel cysteine proteinase inhibitors homologous to the proregions of cysteine proteinases

Propeptides of papain-like cysteine proteinases such as papain, cathepsins B, L and S are potent inhibitors of their cognate cysteine proteinases with Ki values in the nanomolar range, and they exhibit highest inhibition selectivity for enzymes from which they originate. Recent studies have identified novel inhibitor proteins that are homologous to the proregions of papain-like cysteine proteinases. Mouse activated T-lymphocytes express cytotoxic T-lymphocyte antigen (CTLA-2), which is homologous to the proregion of mouse cathepsin L. CTLA-2 exhibits inhibitory activities to several cysteine proteinases. We have also identified a similar propeptide-like cysteine proteinase inhibitor, Bombyx cysteine proteinase inhibitor (BCPI), in the silkmoth Bombyx mori. BCPI is a slow and tight binding inhibitor of cathepsin L-like cysteine proteinases with Ki values in picomolar range, and the inhibition is highly selective towards these proteinases just like the propeptides. Recent genome analyses have shown the expression of similar propeptide-like proteins in Drosophila and rat, suggesting the presence of a novel class of cysteine proteinase inhibitors in a variety of organisms. Studies of the gene structures and phylogenetic analysis have shown that genes of the propeptide-like cysteine proteinase inhibitors have emerged from ancestor genes of their parental enzymes.     

Preparation and preliminary characterization of poly(ethylene glycol)-pepstatin conjugate

The carboxyl function of pepstatin has been coupled, through an amide bond, to methoxypoly(ethylene glycol) (5 kDa), to which an amino function had been previously grafted. The mPEG-pepstatin conjugate inhibits hog pepsin (aspartic proteinase) in vitro as pepstatin itself, however, with a 400 times higher apparent K_i. The conjugate apparently does not inhibit proteinases belonging to other proteinase families such as serine (trypsin, carboxypeptidase Y), cysteine (Papaya proteinase III), or metallo (collagenase) proteinases.     

Plant protein proteinase inhibitors: structure and mechanism of inhibition

This review outlines known examples of the three-dimensional structures of protein proteinase inhibitors from plants. Three families of enzymes, serine proteinases, carboxypeptidases and cysteine proteinases, are targeted by at least a dozen inhibitor families, with the majority of them adopting the standard mechanism of inhibition towards the serine proteinases. All of the inhibitors discussed maintain compact and stable inhibitory domains that bind to the active site of their target proteinases and prevent access to the substrate molecules. One interesting highlight is the knottin group. Three separate inhibitor families utilize the overall knottin fold in a different way. This fold can accommodate extensive sequence variation and for each of the squash, Mirabilis and Potato carboxypeptidase families, the proteinase-binding residues are found at a different location. Plants have also evolved additional strategies to regulate proteinase activity, such as linking inhibitory domains and targeting multiple enzymes at once. The structural aspects of these strategies are discussed in the review.     

Renin inhibition by substituted piperidines: a novel paradigm for the inhibition of monomeric aspartic proteinases?

BACKGROUND: The aspartic proteinase renin catalyses the first and rate-limiting step in the conversion of angiotensinogen to the hormone angiotensin II, and therefore plays an important physiological role in the regulation of blood pressure. Numerous potent peptidomimetic inhibitors of this important drug target have been developed, but none of these compounds have progressed past clinical phase II trials. Limited oral bioavailability or excessive production costs have prevented these inhibitors from becoming new antihypertensive drugs. We were interested in developing new nonpeptidomimetic renin inhibitors. RESULTS: High-throughput screening of the Roche compound library identified a simple 3, 4-disubstituted piperidine lead compound. We determined the crystal structures of recombinant human renin complexed with two representatives of this new class. Binding of these substituted piperidine derivatives is accompanied by major induced-fit adaptations around the enzyme's active site. CONCLUSIONS: The efficient optimisation of the piperidine inhibitors was facilitated by structural analysis of the renin active site in two renin-inhibitor complexes (some of the piperidine derivatives have picomolar affinities for renin). These structural changes provide the basis for a novel paradigm for inhibition of monomeric aspartic proteinases.     

Partial primary structure of a fibrinogenase from the venom of the snake Lachesis stenophrys.

The partial primary structure of an Mr 24,000 non-haemorrhagic metalloproteinase isolated from the venom of the snake Lachesis stenophrys has been determined. The native proteinase was resistant to Edman degradation exhibiting the N-terminal blockade. The pyridylethylated or native proteinase was chemically and enzymatically fragmented and the obtained peptides were separated by gel or reversed-phase chromatography, and sequenced. The metalloproteinase from Lachesis stenophrys contains a putative zinc-chelating sequence HELGHNLGMKH, characteristic for the reprolysin family of zinc-metalloproteinases. It contains six cysteine residues in the standard positions for this group of proteins suggesting the same disulfide bonding. Interestingly, it has almost identical sequence as the metalloproteinase from Lachesis muta muta, LHF-II, which is, however, haemorrhagic. The main structural differences between the two molecules were found in their N-terminal parts and in glycosylation. As the substrate-binding regions of both proteinases are practically identical, we suggest that the absence of haemorrhagicity in Lachesis stenophrys enzyme is due to its lower affinity for the matrix proteins and not due to different substrate specificity.     

An electroblotting, two-step procedure for the detection of proteinases and the study of proteinase/inhibitor complexes in gelatin-containing polyacrylamide gels.

A two-step gelatin/polyacrylamide gel electrophoresis (gelatin/PAGE) procedure was devised for the detection of proteinases and the study of proteinase/inhibitor interactions in complex biological extracts. The proteins are first resolved by sodium dodecyl sulfate (SDS)-PAGE under reducing or nonreducing conditions, and electrotransferred into a 0.75 mm-thick accompanying polyacrylamide slab gel containing 0.1% w/v porcine gelatin. The active proteinase bands are developed by a gelatin proteolysis step in the accompanying gel in the presence or absence of diagnostic proteinase inhibitors, allowing the assessment of proteinase classes and the visual discrimination of inhibitor-'sensitive' and -'insensitive' proteinases in complex extracts. Alternatively, protein extracts are preincubated with specific reversible inhibitors before electrophoresis, allowing a rapid discrimination of strong and weak interactions implicating proteinases and reversible inhibitors. In comparison with the standard gelatin/PAGE procedure, that involves copolymerization of gelatin with acrylamide in the resolving gel, this new procedure simplifies proteinase patterns, avoids overestimation of proteinase numbers in complex extracts, and allows in certain conditions the estimation of proteinase molecular weights. Stem bromelain (EC 3.4.22.32), bovine trypsin (EC 3.4.21.4), papain (EC 3.4.22.2), and the extracellular (digestive) cysteine proteinases of five herbivorous pests are used as model enzymes to illustrate the usefulness of this approach in detecting proteinases and in studying their interactions with specific proteinaceous inhibitors potentially useful in biotechnology.     

Structural and functional properties of kunitz proteinase inhibitors from leguminosae: a mini review

Seed proteins that inhibit proteinases are classified in families based on amino acid sequence similarity, nature of reactive site and mechanism of action, and are used as tools for investigating proteinases in physiological and pathological events. More recently, the plant Kunitz family of inhibitors with two disulphide bridges was enlarged with members containing variable number of cysteine residues, ranging from no cysteine at all to more than four residues. The characteristic of these proteins, as well the interactions with their target proteinases, are briefly discussed.     

Porphyromonas gingivalis gingipains: the molecular teeth of a microbial vampire

The gingipains are cell surface Arg- and Lys-specific proteinases of the bacterium Porphyromons gingivalis, which has been associated with periodontitis, a disease that results in the destruction of the teeth-s supporting tissues. The proteinases are encoded by three genes designated rgpA, rgpB and kgp. Arg-specific proteolytic activity is encoded by rgpA/B and the Lys-specific activity by kgp. RgpA and Kgp are polyproteins comprising proteinases with C-terminal adhesin domains that are proteolytically processed. After processing, the domains remain non-covalently associated as complexes on the cell surface. RgpB is also a cell surface proteinase but does not associate with adhesin domains. Using gene knockout P. gingivalis mutants, the proteolytic processing of the gingipain domains has been shown to involve the gingipains themselves as well as C-terminal processing by a carboxypeptidase. A motif in the C-terminal domain of each protein/polyprotein has been identified that is suggested to be involved in attachment to LPS on the cell surface. RgpB lacks a C-terminal adhesin binding motif found in the catalytic domains of RgpA and Kgp. This adhesin binding motif is proposed to be responsible for the non-covalent association of the RgpA and Kgp catalytic domains into the cell surface complexes with the processed adhesin domains. The RgpA-Kgp proteinase-adhesin complexes, through the adhesin domains A1 and A3, have been implicated in colonization of P. gingivalis by binding to other bacteria in subgingival plaque and also binding to crevicular epithelial cells. The RgpA-Kgp complexes also bind to fibrinogen, laminin, collagen type V, fibronectin and hemoglobin. Amino acid sequences likely to be involved in binding to these host proteins have been identified in adhesin domains A1 and A3. It is proposed that these adhesins target the proteolytic activity to host cell surface matrix proteins and receptors. The continual cycle of binding and degradation of the surface proteins/receptors on epithelial, fibroblast and endothelial cells by the RgpA-Kgp complexes in the gingival tissue leading to cell death would contribute to inflammation, tissue destruction and vascular disruption (bleeding). P. gingivalis has an obligate growth requirement for iron and protoporphyrin IX, which it preferentially utilizes in the form of hemoglobin. Kgp proteolytic activity is essential for rapid hydrolysis of hemoglobin and it is suggested therefore that a major role of the RgpA-Kgp complexes is in vascular disruption and the binding and rapid degradation of hemoglobin for heme assimilation by P. gingivalis. The RgpA-Kgp complexes also have a major role in the evasion and dysregulation of the host-s immune response. It is proposed that host pro-inflammatory cytokines and cellular receptors close to the infection site may be rapidly and efficiently degraded by the gingipains while the proteinases at lower concentrations distally could result in the promotion of an inflammatory response through activation of proteinase-activated receptors and cytokine release. The culmination of this dysregulation would be tissue destruction and bone resorption. In animal models of disease the RgpA-Kgp complex when used as a vaccine to produce a high titre antibody response protects against challenge with P. gingivalis. Using recombinant domains of RgpA and Kgp as vaccines, it has been demonstrated that the A1 and A3 domains confer protection.     

Inhibition of unwanted proteolysis during sample preparation: evaluation of its efficiency in challenge experiments

Measures to counteract proteolysis during sample preparation are widely used; among them, protein extraction at a basic pH (Tris pH 11.0), sample boiling in sodium dodecyl sulfate (SDS), extraction in denaturing lysis solutions and the use of proteinase inhibitors combined with some of these approaches. Here, we tested their efficiency under stringent conditions using a high proteinase (trypsin and a mixture of pancreatic proteinases) contamination and as substrate, streptokinase, a protein highly sensitive to proteolytic degradation. Total degradation was observed in Tris pH 11.0. There was an efficient inhibition for the pancreatic proteinases after boiling in 1% SDS, 1% dithiothreitol (DTT), while trypsin inhibition was dependent on the enzyme-to-substrate ratio. A panel of 21 lysis solutions with variable concentrations of urea, thiourea and detergents was essayed for the ability to counteract proteolysis. In all solutions containing 7-9 M urea, detergents and proteinase inhibitors but not containing thiourea, there was a strong proteolysis. However, in all samples containing 2 M thiourea, proteolysis was inhibited. Moreover, inhibition was dependent on the thiourea concentration. According to these results, we are prompted to consider that the well-known benefits of incorporating thiourea into the lysis solution are a result of two factors, its efficiency in solubilizing proteins and the inhibition of the proteolysis of sensitive substrates; both contributing to the detection of a higher number of species in two-dimensional electrophoresis (2-DE) gels.     

Identification of amyloidogenic peptide sequences using a coarse-grained physicochemical model

Cross-beta amyloid is implicated in over 20 human diseases. Experiments suggest that specific sequence elements within amyloidogenic proteins play a major role in seeding amyloid formation. Identifying these seeding sequences is important for rationalizing the molecular mechanisms of amyloid formation and for elaborating therapeutic strategies that target amyloid. Theoretical techniques play an important role in facilitating the identification and structural characterization of putative seeding sequences; most amyloid species are not amenable to high resolution experimental structure techniques. In this study we have combined a coarse-grained physicochemical protein model with a highly efficient Monte Carlo sampling technique to identify amyloidogenic sequences in four proteins for which respective experimental peptide fragmentation data exist. Peptide sequences were defined as amyloidogenic if the ensemble structure predicted for three interacting peptides described a stable and regular three-stranded beta-sheet. For such peptides, free energies were calculated to provide a measure of amyloid propensity. The overall agreement between the experimental and predicted data is good, and we correctly identify several self-recognition motifs proposed to define the cross-beta amyloid fibril architectures of two of the proteins. Our results compare very favorably with those obtained using atomistic molecular dynamics methods, though our simulations are 30-40 times faster.     

Suppression of virulence of Porphyromonas gingivalis by potent inhibitors specific for gingipains

Porphyromonas gingivalis is a Gram-negative anaerobic bacterium that is implicated as a major etiologic agent of adult periodontal disease. This bacterium is asaccharolytic and possesses strong potency for proteolysis. It produces a novel class of cysteine proteinases, termed gingipains, in the cell-associated and secretory forms. Gingipains consist of arginine-X-specific cysteine proteinases (Arg-gingipains, Rgps) and lysine-X-specific cysteine proteinase (Lys-gingipain, Kgp). Previous studies using various P. gingivalis mutants deficient in Rgp- and/or Kgp-encoding genes have revealed that both enzymes are important for the bacterium both to exhibit its virulence and to survive in periodontal pockets. Mammalian internal proteinase inhibitors such as cystatins, a1-antichymotrypsin, and tissue inhibitor of metalloproteinases (TIMPs) have little or no effects on the proteolytic activities of these enzymes, suggesting the evasion of the bacterium from host defense mechanisms. Recent epidemiological reports have shown a significant relation between periodontal diseases and systemic diseases such as cardiovascular diseases and diabetes. Thus, the development of potent inhibitors specific for gingipains provides new therapeutic approaches to treat periodontal diseases and the related systemic diseases. More recently, we have developed novel synthetic inhibitors specific for Rgp and Kgp, based on the specificity and efficacy of cleavage of histatins by each enzyme. We have also isolated a novel and potent inhibitor of Rgp from the culture supernatant of Streptomyces species strain FA-70, now designated as FA-70C1. Here we summarized the usefulness of these new inhibitors in providing a broader application in studies of this important class of enzymes.     

Nepenthesin, a unique member of a novel subfamily of aspartic proteinases: enzymatic and structural characteristics

Carnivorous plants are known to secrete acid proteinases to digest prey, mainly insects, for nitrogen uptake. In our recent study, we have purified, for the first time, to homogeneity two acid proteinases, nepenthesin I (Nep I) and nepenthesin II (Nep II) from the pitcher fluid of Nepenthes distillatoria and investigated their enzymatic and structural characteristics. Both enzymes were optimally active at pH approx. 2.6 toward acid-denatured hemoglobin; the specificity of Nep I toward oxidized insulin B chain appears to be similar, but slightly wider than those of other aspartic proteinases (APs). At or below 50 degrees C both enzymes were remarkably stable; especially Nep I was extremely stable over a wide range of pH from 3 to 10 for over 30 days. This suggests an evolutionary adaptation of the enzymes to their specific habitat. We have also cloned the cDNAs and deduced the complete amino acid sequences of the precursors of Nep I and Nep II from the pitcher tissue of Nepenthes gracilis. Although the corresponding mature enzymes are homologous with ordinary pepsin-type APs, both enzymes had a high content of cysteine residues (12 residues per molecule), which are assumed to form six unique disulfide bonds as suggested by computer modeling and are thought to contribute toward the remarkable stability of Neps. Moreover, the amino acid sequence identity of Neps with ordinary APs, including plant vacuolar APs, are remarkably low (approx. 20%), and phylogenetic comparison shows that Neps are distantly related to them to form a novel subfamily of APs with a high content of cysteine residues and a characteristic insertion, named 'the Nep-type AP (NAP)-specific insertion', including a large number of novel, orthologous plant APs emerging in the gene/protein databases.     

Creating functional artificial proteins

Much is now known about how protein folding occurs, through the sequence analysis of proteins of known folding geometry and the sequence/structural analysis of proteins and their mutants. This has allowed not only the modification of natural proteins but also the construction of de novo polypeptides with predictable folding patterns. Structure/function analysis of natural proteins is used to construct derived versions that retain a degree of biological activity. The constructed versions made of either natural or artificial sequences contain critical residues for activity such as receptor binding. In some cases, the functionality is introduced by incorporating binding sites for other elements, such as organic cofactors or transition metals, into the protein scaffold. While these modified proteins can mimic the function of natural proteins, they can also be constructed to have novel activities. Recently engineered photoactive proteins are good examples of such systems in which a light-induced electron transfer can be established in normally light-insensitive proteins. The present review covers some aspects of protein design that have been used to investigate protein receptor binding, cofactor binding and biological electron transfer.     

Molecular mechanics study of myelin basic protein peptide 87-118: Some local energy minima

Myelin basic protein (MBP) is the major extrinsic protein of the myelin sheath in the central nervous system. It is this protein that is destroyed in such demyelinating diseases as multiple sclerosis. We have examined the predicted structures of one segment of MBP using the molecular mechanics program ECEPP83 developed by Scheraga and co-workers as modified by Chuman, Momany, and Schafer. We have focused upon a segment, 87-118, containing the Pro-Pro-Pro sequence (residues 100–102), which has been predicted from standard algorithms to exist in a hairpin loop connecting anti-parallel beta-strands. Several local energy minima have been found and reported. Tripoline sequences are not rare in proteins, but their structure and function is still uncertain.     

Studies on the dephosphorylation of phosphotyrosine-containing peptides during post-source decay in matrix-assisted laser desorption/ionization

Phosphorylation of tyrosine residues in proteins is a common regulatory mechanism, although it accounts for less than 1% of the total O-phosphate content in proteins. Whereas aromatic phosphorylation sites can be identified by a number of different analytical techniques, sequence analysis of phosphotyrosine-containing proteins at the low picomole or even femtomole level is still a challenging task. This paper describes the post-source decay in matrix-assisted laser desorption/ionization mass spectrometry of phosphotyrosine-containing model peptides by comparing their fragmentation behavior with sequence-homologous unphosphorylated peptides. Whereas the parent ions showed significant losses of HPO3, all phosphorylated fragment ions of the b- and y-series displayed only minor dephosphorylated signals, which often were not detectable. Surprisingly, one of the studied phosphotyrosine-containing sequences displayed, in addition to the [M + H - 80]+ ion, a more abundant [M + H - 98]+ ion, which could be explained by elimination of phosphoric acid. This dephosphorylation pattern was very similar to the patterns obtained for phosphoserine- and phosphothreonine-containing peptides. Because the dephosphorylation pattern of the parent ion is often used to identify modified amino acids in peptides, we investigated possible dephosphorylation mechanisms in detail. Therefore, we substituted single trifunctional amino acid residues and incorporated deuterated phosphotyrosine residues. After excluding direct elimination of phosphoric acid from tyrosine, we could show that the obtained loss of H3PO4 depends on aspartic acid and arginine residues. Most likely the HPO3 group is transferred to aspartic acid followed by cleavage of phosphoric acid forming a succinimide. On the other hand, arginine appears to induce the H3PO4 loss by protonation of phosphotyrosine leaving a phenyl cation.     

MMM-QSAR recognition of ribonucleases without alignment: comparison with an HMM model and isolation from Schizosaccharomyces pombe, prediction, and experimental assay of a new sequence

The study of type III RNases constitutes an important area in molecular biology. It is known that the pac1+ gene encodes a particular RNase III that shares low amino acid similarity with other genes despite having a double-stranded ribonuclease activity. Bioinformatics methods based on sequence alignment may fail when there is a low amino acidic identity percentage between a query sequence and others with similar functions (remote homologues) or a similar sequence is not recorded in the database. Quantitative structure-activity relationships (QSAR) applied to protein sequences may allow an alignment-independent prediction of protein function. These sequences of QSAR-like methods often use 1D sequence numerical parameters as the input to seek sequence-function relationships. However, previous 2D representation of sequences may uncover useful higher-order information. In the work described here we calculated for the first time the spectral moments of a Markov matrix (MMM) associated with a 2D-HP-map of a protein sequence. We used MMMs values to characterize numerically 81 sequences of type III RNases and 133 proteins of a control group. We subsequently developed one MMM-QSAR and one classic hidden Markov model (HMM) based on the same data. The MMM-QSAR showed a discrimination power of RNAses from other proteins of 97.35% without using alignment, which is a result as good as for the known HMM techniques. We also report for the first time the isolation of a new Pac1 protein (DQ647826) from Schizosaccharomyces pombe strain 428-4-1. The MMM-QSAR model predicts the new RNase III with the same accuracy as other classical alignment methods. Experimental assay of this protein confirms the predicted activity. The present results suggest that MMM-QSAR models may be used for protein function annotation avoiding sequence alignment with the same accuracy of classic HMM models.     

Amino acid sequences of 60B8 antigens induced in HL-60 cells by 1,25-dihydroxyvitamin D3. The antigens are identical with macrophage-related protein-14 and -8

A differentiation antigen 60B8 appeared in human promyelocytic leukemia HL-60 cells which had been induced to differentiate into macrophage-like cells by treatment with 1,25-dihydroxyvitamin D3. The antigen was purified by immunoaffinity chromatography and separated into two proteins, 60B8-A and -B antigens, by reverse-phase high performance liquid chromatography (HPLC). Both proteins were digested with proteases, and the resulting peptides were subjected to amino acid sequence analysis after purification by reverse-phase HPLC. The amino acid sequences of 60B8-A and -B antigens were identical with those of the proteins MRP-14 and -8, respectively, which were recently predicted from the nucleotide sequences of their complementary deoxyribonucleic acid (cDNA) clones by Odink et al. (Nature (London), 330, 80 (1987)). Although they did not characterize the chemical properties of the two proteins, our results clearly indicate that macrophage-related protein (MRP)-14 and -8 are expressed without post-translational modification, except that the amino-terminus of MRP-14 is blocked, in differentiated HL-60 cells.     

Proteomic analysis of the Arabidopsis thaliana cell wall

With the completion of the Arabidopsis genome, many hypothetical proteins have been predicted without any information on their expression, subcellular localisation and function. We have performed proteomic analysis of proteins sequentially extracted from enriched Arabidopsis cell wall fractions and separated by two-dimensional gel electrophoresis (2-DE). The proteins were identified by peptide mass fingerprinting using matrix-assisted laser desorption/ionisation-time of flight (MALDI-TOF) mass spectrometry and genomic database searches. This is part of a targeted exercise to establish the entire Arabidopsis secretome database. We report evidence for new proteins of unknown function whose existence had been predicted from genomic sequences and, furthermore, localise them to the cell wall. In addition, we observed an unexpected presence in the cell wall preparations of proteins whose known biochemical activity has never been associated with this compartment hitherto. We discuss the implications of these findings and present results suggesting a possible involvement of cell wall kinases in plant responses to pathogen attack.     

Serine proteinase inhibitors in the skin: role in homeostasis and disease

Serine proteinases fulfill and facilitate a broad spectrum of biological processes. They are held in check by different specific inhibitors. This delicate balance can be disturbed by genetic defects or exogenous influences and has been shown as the underlying or promoting cause for a large number of different diseases. For instance, proteinases are under investigation as drug targets for cancer, infections, neurodegenerative diseases, osteoporosis, inflammatory disorders and many more. Dermatological research has contributed greatly to the appreciation of the complex regulatory network between serine proteinases and serine proteinase inhibitors. In addition, proteolytically trimmed proteinase-activated receptors (PARs) trigger keratinocyte proliferation and differentiation as well as leukocyte attraction and activation. New insights have been gained particularly concerning the progression of inflammatory disorders of the skin. This review summarizes the role of serine proteinase inhibitors in physiology and pathophysiology of the skin.     

Computational methods for protein-protein interaction and their application

Protein-protein interactions play a central role in numerous processes in cell and are one of the main research fields in current functional proteomics. The increase of finished genomic sequences has greatly stimulated the progress for detecting the functions of the genes and their encoded proteins. As complementary ways to the high through-put experimental methods, various methods of bioinformatics have been developed for the study of the protein-protein interaction. These methods range from the sequence homology-based to the genomic-context based. Recently, it tends to integrate the data from different methods to build the protein-protein interaction network, and to predict the protein function from the analysis of the network structure. Efforts are ongoing to improve these methods and to search for novel aspects in genomes that could be exploited for function prediction. This review highlights the recent advances of the bioinformatics methods in protein-protein interaction researches. In the end, the application of the protein-protein interaction has also been discussed.