A method to detect proteinase activity using unprocessed X-ray films.

Routine assays to detect proteinases in biological samples are generally tedious and time-consuming. To expedite the recognition of proteinases, we have developed an assay utilizing the gelatin on the surface of an unprocessed Kodak X-Omat AR film as the proteolytic substrate. A positive reaction is indicated by a clear zone on the film after it has been rinsed with running water. This proteinase assay has been found to be inexpensive, rapid, and simple. Besides its ease of use, this assay has been found to be quantitatively reproducible with a well-defined endpoint. More importantly, this assay method is applicable to a variety of proteolytic enzymes under diverse pH (5-8.5) and salt conditions (up to 5 M NaCl) and has a sensitivity similar to that of azocoll. Since the assay does not require sophisticated equipment, it is useful as a general laboratory procedure.     

鱼糜凝胶劣化现象相关蛋白水解酶

鱼糜凝胶劣化现象发生在鱼糜凝胶化过程中,导致鱼糜制品凝胶弹性下降,品质降低。研究表明,引起鱼糜凝胶劣化现象的原因是鱼糜内含的蛋白水解酶的作用。与该现象相关的蛋白水解酶类有肌浆型和肌原纤维结合型之分。大部分肌浆型蛋白水解酶为水溶性的,可以在鱼糜制备过程中漂洗去除。肌原纤维结合型蛋白酶不能通过漂洗有效去除,是引发鱼糜凝胶劣化现象的重要相关酶类。已证实的凝胶劣化相关肌原纤维结合型酶类包括丝氨酸型蛋白酶和溶酶体型组织蛋白酶。     

Molecular mechanisms of proteolytic processing of viral proteins and the problem of antiviral chemotherapy and vaccine design

Proteolytic cleavage of virus-specific proteins is a universal phenomenon, which is widely expanded among different viruses including bacterial, plant, animal, and human viruses. Proteolytic processing of viral proteins involves the cleavage in strictly specific sites (proteolytic sites) of polyprotein molecules. Specificity of this processing is a doubly dependent event controlled by the amino acids of proteolytic sites and the presence of adequate proteinases. Host-originated and/or virus-coded proteinases are known to perform the cleavage of viral polypeptides. Conformational and functional behaviour of many virus proteins is regulated by proteolytic modification; as a result, the reproduction of mature virions and the infection pathways are directly controlled. Molecular mechanisms of site-specific proteolytic processing of viral proteins are proposed as a target to be attacked for chemotherapeutic virus inhibition and to be modified for vaccine design. The approaches are analysed to realise this antiviral strategy, and prospects for its development are discussed.     

A new approach to the use of fluorogenic dinitrophenyl-containing substrates for determining the proteolytic activity of aspartyl proteinases

A method for the determination of proteolytic activity of aspartyl proteinases using known colored fluorogenic substrates was developed. The technique utilizes the chromophore properties of the dinitrophenyl (DNP) group. The approach proposed comprises separation of the initial peptide and subsequent measurement of absorption of the solution of the DNP-containing C-terminal fragment, produced by its enzymatic cleavage, at 360 nm. This method was used to determine the activity of calf chymosin, the pepsins from various sources, and the commercial preparations containing a mixture of enzymes without preliminary desalting. The method is simple and applicable under plant conditions.     

Proteolysis in eukaryotic cells. Identification of multiple proteolytic enzymes in yeast

A previous study led to the discovery of new proteinases in yeast (Achstetter, T., Ehmann, C., and Wolf, D. H. (1981) Arch. Biochem. Biophys. 207, 445-454). The search for proteolytic enzymes active in the neutral pH range has been extended. Studies were done on a mutant lacking four well-known proteinases involved in protein degradation, the two endoproteinases A and B and the two carboxypeptidases Y and S. Twenty-nine chromogenic peptides (amino terminally blocked peptidyl-4-nitroanilides) as well as [3H]methylcasein were used as substrates in this search. For the detection of endoproteolytic activity using chromogenic peptide substrates two versions of the assay were used. In one system the direct cleavage of the 4-nitroanilide bond was measured. In the second, the cleavage of the chromogenic peptide at some site other than the 4-nitroanilide bond was measured. Both variations led to the discovery of multiple proteinase activities. Regulation of these proteolytic activities under different growth conditions of cells was observed. Proteolytic activity on [3H]methylcasein was also found. Ion-exchange chromatography and gel filtration were used for the reproducible separation of the multiple proteolytic activities.     

Chromogenic and fluorogenic peptide substrates of proteolytic enzymes

The review describes approaches to designing chromogenic and fluorogenic substrates for proteolytic enzymes, mainly for assay of serine proteinases. Principles of substrate polypeptide chain construction and some methods for detection of chromogenic and fluorogenic products of their hydrolysis are considered. The use of these substrates for the study of blood clotting enzymes and for clinical diagnostics is briefly treated. Methodology of chemical synthesis of principal chromogenic and fluorogenic substrates is also discussed.     

Prediction of Protein Cleavage Site with Feature Selection by Random Forest

Proteinases play critical roles in both intra and extracellular processes by binding and cleaving their protein substrates. The cleavage can either be non-specific as part of degradation during protein catabolism or highly specific as part of proteolytic cascades and signal transduction events. Identification of these targets is extremely challenging. Current computational approaches for predicting cleavage sites are very limited since they mainly represent the amino acid sequences as patterns or frequency matrices. In this work, we developed a novel predictor based on Random Forest algorithm (RF) using maximum relevance minimum redundancy (mRMR) method followed by incremental feature selection (IFS). The features of physicochemical/biochemical properties, sequence conservation, residual disorder, amino acid occurrence frequency, secondary structure and solvent accessibility were utilized to represent the peptides concerned. Here, we compared existing prediction tools which are available for predicting possible cleavage sites in candidate substrates with ours. It is shown that our method makes much more reliable predictions in terms of the overall prediction accuracy. In addition, this predictor allows the use of a wide range of proteinases.     

A new approach to the use of fluorogenic dinitrophenyl-containing substrates for determining the proteolytic activity of aspartyl proteinases

A method for the determination of proteolytic activity of aspartyl proteinases using known colored fluorogenic substrates was developed. The technique utilizes the chromophore properties of the dinitrophenyl (DNP) group. The approach proposed comprises separation of the initial peptide and subsequent measurement of absorption of the solution of the DNP-containing C-terminal fragment, produced by its enzymatic cleavage, at 360 nm. This method was used to determine the activity of calf chymosin, the pepsins from various sources, and the commercial preparations containing a mixture of enzymes without preliminary desalting. The method is simple and applicable under plant conditions.     

An assay for 26S proteasome activity based on fluorescence anisotropy measurements of dye-labeled protein substrates

The 26S proteasome is the molecular machine at the center of the ubiquitin proteasome system and is responsible for adjusting the concentrations of many cellular proteins. It is a drug target in several human diseases, and assays for the characterization of modulators of its activity are valuable. The 26S proteasome consists of two components: a core particle, which contains the proteolytic sites, and regulatory caps, which contain substrate receptors and substrate processing enzymes, including six ATPases. Current high-throughput assays of proteasome activity use synthetic fluorogenic peptide substrates that report directly on the proteolytic activity of the proteasome, but not on the activities of the proteasome caps that are responsible for protein recognition and unfolding. Here, we describe a simple and robust assay for the activity of the entire 26S proteasome using fluorescence anisotropy to follow the degradation of fluorescently labeled protein substrates. We describe two implementations of the assay in a high-throughput format and show that it meets the expected requirement of ATP hydrolysis and the presence of a canonical degradation signal or degron in the target protein.     

A simple fluorescence labeling method for studies of protein oxidation, protein modification, and proteolysis

Proteins are sensitive to oxidation, and oxidized proteins are excellent substrates for degradation by proteolytic enzymes such as the proteasome and the mitochondrial Lon protease. Protein labeling is required for studies of protein turnover. Unfortunately, most labeling techniques involve (3)H or (14)C methylation, which is expensive, exposes researchers to radioactivity, generates large amounts of radioactive waste, and allows only single-point assays because samples require acid precipitation. Alternative labeling methods have largely proven unsuitable, either because the probe itself is modified by the oxidant(s) being studied or because the alternative labeling techniques are too complex or too costly for routine use. What is needed is a simple, quick, and cheap labeling technique that uses a non-radioactive marker, binds strongly to proteins, is resistant to oxidative modification, and emits a strong signal. We have devised a new reductive method for labeling free carboxyl groups of proteins with the small fluorophore 7-amino-4-methycoumarin (AMC). When bound to target proteins, AMC fluoresces very weakly but when AMC is released by proteinases, proteases, or peptidases, it fluoresces strongly. Thus, without acid precipitation, the proteolysis of any target protein can be studied continuously, in multiwell plates. In direct comparisons, (3)H-labeled proteins and AMC-labeled proteins exhibited essentially identical degradation patterns during incubation with trypsin, cell extracts, and purified proteasome. AMC-labeled proteins are well suited to studying increased proteolytic susceptibility after protein modification, because the AMC-protein bond is resistant to oxidizing agents such as hydrogen peroxide and peroxynitrite and is stable over time and to extremes of pH, temperature (even boiling), freeze-thaw, mercaptoethanol, and methanol.     

A mass spectrometry-based strategy for detecting and characterizing endogenous proteinase activities in complex biological samples

Endogenous proteinases in biological fluids such as human saliva produce a rich peptide repertoire that reflects a unique combination of enzymes, substrates, and inhibitors/activators. Accordingly, this subproteome is an interesting source of biomarkers for disease processes that either directly or indirectly involve proteolysis. However, the relevant proteinases, typically very low abundance molecules, are difficult to classify and identify. We hypothesized that a sensitive technique for monitoring accumulated peptide products in an unbiased, global manner would be very useful for detecting and profiling proteolytic activities in complex biological samples. Building on the longstanding use of 18O isotope-based approaches for the classification of proteolytic and other enzymatic processes we devised a new method for evaluating endogenous proteinases. Specifically, we showed that upon ex vivo incubation endogenous proteinases in human parotid saliva introduced 18O from isotopically enriched water into the C-terminal carboxylic groups of their peptide products. Subsequent peptide sequence determination and inhibitor profiling enabled the detection of discrete subsets of proteolytic products that were generated by different enzymes. As a proof-of-principle we used one of these fingerprints to identify the relevant activity as tissue kallikrein. We termed this technique PALeO. Our results suggest that PALeO is a rapid and highly sensitive method for globally assessing proteinase activities in complex biological samples.     

Grass group I pollen allergens (beta-expansins) lack proteinase activity and do not cause wall loosening via proteolysis.

Group I grass pollen allergens make up a subgroup of the beta-expansin family of cell wall loosening proteins in plants. A recent study reported that recombinant Phl p 1, the group I allergen from timothy grass pollen, was associated with papain-like proteinase activity and suggested that expansins loosen the plant cell wall via proteolysis. We tested this idea with three experimental approaches. First, we evaluated three purified native group I allergens from timothy grass, ryegrass and maize (Phl p 1, Lol p 1, Zea m 1) using five proteinase assays with a variety of substrates. The proteins had substantial wall loosening activity, but no detectable proteolytic activity. Thus we cannot confirm proteolytic activity in the pollen allergen class of beta-expansins. Second, we tested the ability of proteinases to induce cell wall extension in vitro. Tests included cysteine proteinases, serine proteinases, aspartic proteinases, metallo proteinases, and aggressive proteinase mixtures, none of which induced wall extension in vitro. Thus, wall proteins are unlikely to be important load-bearing components of the plant cell wall. Third, we tested the sensitivity of beta-expansin activity and native wall extension activity to proteinase inhibitors. The results show that a wide range of proteinase inhibitors (phenylmethanesulfonyl fluoride, N-ethylmaleimide, iodoacetic acid, Pefabloc SC, and others) inhibited neither activity. From these three sets of results we conclude proteolysis is not a likely mechanism of plant cell wall loosening and that the pollen allergen class of beta-expansins do not loosen cell walls via a proteolytic mechanism.     

Phagocytosis and intracellular digestion of collagen, its role in turnover and remodelling

Summary Collagens of most connective tissues are subject to continuous remodelling and turnover, a phenomenon which occurs under both physiological and pathological conditions. Degradation of these proteins involves participation of a variety of proteolytic enzymes including members of the following proteinase classes: matrix metalloproteinases (e.g. collagenase, gelatinase and stromelysin), cysteine proteinases (e.g. cathepsin B and L) and serine proteinases (e.g. plasmin and plasminogen activator). Convincing evidence is available indicating a pivotal role for matrix metalloproteinases, in particular collagenase, in the degradation of collagen under conditions of rapid remodelling, e.g. inflammation and involution of the uterus. Under steady state conditions, such as during turnover of soft connective tissues, involvement of collagenase has yet to be demonstrated. Under these circumstances collagen degradation is likely to take place particularly within the lysosomal apparatus after phagocytosis of the fibrils. We propose that this process involves the following steps: (i) recognition of the fibril by membranebound receptors (integrins?), (ii) segregation of the fibril, (iii) partial digestion of the fibril and/or its surrounding noncollagenous proteins by matrix metalloproteinases (possibly gelatinase), and finally (iv) lysosomal digestion by cysteine proteinases, such as cathepsin B and/or L. Modulation of this pathway is carried out under the influence of growth factors and cytokines, including transforming growth factor β and interleukin 1α.     

An o-phthalaldehyde spectrophotometric assay for proteinases

A rapid and convenient spectrophotometric assay has been devised to measure proteolysis. The assay is based on the reaction of o-phthalaldehyde (OPA) and 2-mercaptoethanol with amino groups released during proteolysis of a protein substrate. The reaction is specific for primary amines in amino acids, peptides, and proteins, approaches completion within 1 to 2 min at 25 degrees C (half-times of approx 10-15 s), and requires no preliminary heating or separation of the hydrolyzed products from the undegraded protein substrate prior to performing the assay. The OPA assay was relatively as successful as a 2,4,6-trinitrobenzenesulfonic acid (TNBS) procedure in predicting the extent of hydrolysis of a protein substrate. The utility of the OPA method was demonstrated by measuring the degree of proteolytic degradation caused by trypsin, subtilisin, Pronase, and chymotrypsin of various soluble protein substrates. Ethanethiol (instead of 2-mercaptoethanol) or 50% of dimethyl sulfoxide can be included in the assay solution to stabilize certain OPA-amine products. The present method approaches the sensitivity of ninhydrin and TNBS procedures, is more convenient and rapid, and could substitute for these reagents in most assay systems.     

On the substrate specificity of cathepsin L

A view is given on the maximal hydrolysis of proteins by cathepsin L (EC 3.4.22.15) in dependence on the pH. The overall degradation of several proteins at pH values lower than pH 6.0 implies a very broad specificity, whereas at pH 7.0 and 7.5 cathepsin L seems to act on proteins cleaving only restricted specific peptide bonds. Some kinetic constants are given for the three synthetic substrates of cathepsin L which are known so far: Bz-Arg-NH2, Z-Lys-OPhNO2 and Z-Phe-Arg-NMec. They cannot be used as completely specific substrates of cathepsin L, because all of them are hydrolysed by cathepsin B and also other proteinases.     

Measurement of casein digestion by a fluorometric method

A qualitative and quantitative method to assay proteolytic degradation of casein with a spectrofluorometer was developed. Proteolysis produced by different pure or mixed proteinases in a pH range 2 to 7.4 quenches the fluorescence emitted at a wavelength of 350 nm by casein excited at 300 nm in less than 5 min. This method is very sensitive, fast, and requires minimal sample preparation. Proteinases that do not generate peptides appropriate for fluorescence quenching cannot be detected with this assay and proteinases with intrinsic fluorescence may require special adjustments of the spectrofluorometer. This method monitors the disappearance of intact substrate proteins continuously, omitting the separation step necessary in other methods to measure product peptides.     

Assessment of proteasome activity in cell lysates and tissue homogenates using peptide substrates

The ubiquitin-proteasome pathway is a major route of degradation of cell proteins. It also plays an essential role in maintaining cell homeostasis by degrading many rate-limiting enzymes and critical regulatory proteins. Alterations in proteasome activity have been implicated in a number of pathologies including Parkinson's disease, Alzheimer's disease and diabetes. The eukaryotic proteasome is a multicatalytic protease characterized by three activities with distinct specificities against peptide substrates. Although substrates were identified which could selectively measure the individual activities in the purified proteasome little data is available on how specific those substrates are for proteasomal activity when used with biological samples which may contain many other active peptidases. Here we examine the three major peptidase activities in lysates of two cell types and in a liver cytosol fraction in the presence of specific proteasome inhibitors and after fractionation by gel permeation chromatography. We demonstrate that other proteinases present in these preparations can degrade the commonly used proteasome substrates under the standard assay conditions. We develop a simple method for separating the proteasome from the lower molecular weight proteases using a 500kDa molecular weight cut-off membrane. This allows proteasome activity to be accurately measured in crude biological samples and may have quite broad applicability. We also identify low molecular weight tryptic activity in both the cell and tissue preparations which could not be inhibited by the proteasome inhibitor epoxomycin but was inhibitable by two cysteine proteinase inhibitors and by lactacystin suggesting that lactacystin may not be completely proteasome specific.     

Degradation of extracellular matrix proteins by hemorrhagic metalloproteinases

The proteolytic activity of four hemorrhagic metalloproteinases (Ht-a, c, d, and e) isolated from the venom of the Western diamondback rattlesnake (Crotalus atrox) was investigated using isolated extracellular matrix (ECM) proteins. We determined that all of the proteinases are capable of cleaving fibronectin, laminin, type IV collagen, nidogen (entactin), and gelatins. However, none of the proteinases were proteolytic against the interstitial collagen types I and III or type V collagen. With all of the substrates listed above Ht-c and Ht-d produced identical digestion patterns, as would be expected for these isoenzymes. With fibronectin, Ht-a produces a different ratio of products from Ht-c and Ht-d, while Ht-e produces a unique pattern of digestion. Ht-e and Ht-a produced nonidentical patterns with the laminin/nidogen preparation although some similarity was shared between them as well as with the Ht-c/d digestion pattern. Similar results were also observed for these proteinases with nidogen 150 as the substrate. The type IV collagen digestion patterns by Ht-e and Ht-a were similar to the pattern observed with Ht-c/d but differed by two bands. The digestion patterns of the three gelatins produced by the proteinases show differences between Ht-c and Ht-d when compared to Ht-e and Ht-a. This investigation clearly shows that several of the ECM proteins are efficiently digested by these toxins. The proteinases have some digestion sites in common but show differing specificities. In addition, the range of ECM proteins digested by these hemorrhagic proteinases is nearly identical to that demonstrated by the ECM proteinase stromelysin (MMP-3). From these data, and the knowledge of the roles these ECM proteins have in maintaining basement membrane structural/functional integrity, one can envision that the degradation of these ECM proteins could readily lead to loss of capillary integrity resulting in hemorrhage occurring at those sites.     

A BODIPY fluorescent microplate assay for measuring activity of calpains and other proteases

The use of 4,4-difluoro-5,7-dimethyl-4-bora-3a, 4a-diaza-s-indacene-3-propionic acid (BODIPY-FL) labeled casein in autoquenching assays of proteolytic activity has been recently described, and we have adapted this assay to measurement of calpain activity. BODIPY-FL coupled to casein at a ratio of 8 mol of BODIPY-FL/mol of casein or higher produces a BODIPY-FL-casein substrate that can be used in an autoquenching assay of calpain proteolytic activity. This assay has a number of advantages for measuring calpain activity. (1) The procedure does not require precipitation and removal of undegraded protein, so it is much faster than other procedures that require a precipitation step, and it can be used directly in kinetic assays of proteolytic activity. (2) The BODIPY-FL-casein assay is easily adapted to a microtiter plate format, so it can be used to screen large numbers of samples. (3) Casein is an inexpensive and readily available protein substrate that more closely mimics the natural substrates of endoproteinases, such as the calpains, than synthetic peptide substrates do. Casein has K(m) values for micro- and m-calpain that are similar to those of other substrates such as fodrin or MAP2 that may be "natural" substrates for the calpains, and there is no reason to believe that calpain hydrolysis of casein is inherently different from hydrolysis of fodrin or MAP2, which are much less accessible as substrates for protease assays. (4) The BODIPY-FL-casein assay is capable of detecting 10 ng ( approximately 5 nM) of calpain and is nearly as sensitive as the most sensitive calpain assay reported thus far. (5) The BODIPY-FL-casein assay is as reproducible as the FITC-casein assay, whose reproducibility is comparable to or better than the reproducibility of other methods used to assay calpain activity. The BODIPY-FL-casein assay is a general assay for proteolytic activity and can be used with any protease that cleaves casein.     

ClpS is the recognition component for Escherichia coli substrates of the N-end rule degradation pathway

The N-end rule degradation pathway states that the half-life of a protein is determined by the nature of its N-terminal residue. In Escherichia coli the adaptor protein ClpS directly interacts with destabilizing N-terminal residues and transfers them to the ClpA/ClpP proteolytic complex for degradation. The crucial role of ClpS in N-end rule degradation is currently under debate, since ClpA/ClpP was shown to process selected N-terminal degrons harbouring destabilizing residues in the absence of ClpS. Here, we investigated the contribution of ClpS to N-end rule degradation by two approaches. First, we performed a systematic mutagenesis of selected N-degron model substrates, demonstrating that ClpS but not ClpA specifically senses the nature of N-terminal residues. Second, we identified two natural N-end rule substrates of E. coli : Dps and PATase (YgjG). The in vivo degradation of both proteins strictly relied on ClpS, thereby establishing the function of ClpS as the essential discriminator of the E. coli N-end rule pathway.