Human tartrate-resistant acid phosphatase becomes an effective ATPase upon proteolytic activation

Proteolytic cleavage in an exposed loop of human tartrate-resistant acid phosphatase (TRAcP) with trypsin leads to a significant increase in activity. At each pH value between 3.25 and 8.0 the cleaved enzyme is more active. Substrate specificity is also influenced by proteolysis. Only the cleaved form is able to hydrolyze unactivated substrates efficiently, and at pH >6 cleaved TRAcP acquires a marked preference for ATP. The cleaved enzyme also has altered sensitivity to inhibitors. Interestingly, the magnitude and mode of inhibition by fluoride depends not only on the proteolytic state but also pH. The combined kinetic data imply a role of the loop residue D158 in catalysis in the cleaved enzyme. Notably, at low pH this residue may act as a proton donor for the leaving group. In this respect the mechanism of cleaved TRAcP resembles that of sweet potato purple acid phosphatase.     

Identification of latent procathepsins B and L in microsomal lumen: characterization of enzymatic activation and proteolytic processing in vitro

Procathepsins B and L in the hepatic endoplasmic lumen were identified as having a molecular weight of 39,000 by immunoblot analysis. The proenzymes were then purified to remove the mature enzymes by concanavalin A-Sepharose chromatography. The concanavalin A-adsorbed fractions containing the proenzymes showed no appreciable activities of cathepsins B and L. When those fractions were incubated at pH 3.0, the enzymatic activities markedly increased: the activities of cathepsins B and L after 36 h incubation were 60 and 210 times those of the controls, respectively. Immunoblot analysis showed that after 36 h incubation the proenzymes disappeared and the mature enzymes increased. Thus the proenzymes were processed to the mature enzymes under acidic conditions of pH 3.0. The marked increases of enzymatic activities and the conversion of the proenzymes to the mature forms were completely blocked with pepstatin, which is a potent inhibitor of aspartic proteases. The results strongly suggested that a processing protease for procathepsins B and L might be cathepsin D, a major lysosomal aspartic protease. Indeed, lysosomal cathepsin D could convert microsomal procathepsin B to the mature enzyme in vitro. Therefore, procathepsins B and L seem first to be synthesized as enzymatically inactive forms in endoplasmic reticulum and successively may be converted into active forms by cathepsin D in lysosomal compartments.     

Shiga toxin 1 acting on DNA in vitro is a heat-stable enzyme not requiring proteolytic activation

Shiga toxin 1 (Stx1) catalyses the removal of a specific adenine from 28S rRNA within ribosomes (RNA-N-glycosylase activity) and the removal of multiple adenines from DNA (DNA-glycosylase activity). For the in vitro activity the toxin requires activation by trypsin, urea and DTT which releases the enzymatically active A1 fragment. We show that activated Stx1 acts on DNA as a heat-stable enzyme. Moreover, heat-treatment of the pro-enzyme at acidic pH turns it into an enzymatically active species which efficiently depurinates DNA. Although the effect of this treatment is centred on the enzyme and not on DNA, we found no evidence for covalent modification of the holotoxin. We suggest that high temperatures and acidic buffer induce unfolding of the holotoxin allowing the substrate to gain access to the active site. Possible practical applications (rapid assay for Stx1 detection, use of the toxin for DNA sequencing) are discussed.     

Calpains: an elaborate proteolytic system

Calpain is an intracellular Ca(2+)-dependent cysteine protease (EC 3.4.22.17; Clan CA, family C02). Recent expansion of sequence data across the species definitively shows that calpain has been present throughout evolution; calpains are found in almost all eukaryotes and some bacteria, but not in archaebacteria. Fifteen genes within the human genome encode a calpain-like protease domain. Interestingly, some human calpains, particularly those with non-classical domain structures, are very similar to calpain homologs identified in evolutionarily distant organisms. Three-dimensional structural analyses have helped to identify calpain's unique mechanism of activation; the calpain protease domain comprises two core domains that fuse to form a functional protease only when bound to Ca(2+)via well-conserved amino acids. This finding highlights the mechanistic characteristics shared by the numerous calpain homologs, despite the fact that they have divergent domain structures. In other words, calpains function through the same mechanism but are regulated independently. This article reviews the recent progress in calpain research, focusing on those studies that have helped to elucidate its mechanism of action. This article is part of a Special Issue entitled: Proteolysis 50 years after the discovery of lysosome.     

The structural basis of proteolytic activation of bovine glutamate dehydrogenase

In this work, we re-examine the previously reported phenomenon of the creation of a superactive glutamate dehydrogenase by proteolytic modification by chymotrypsin and explore the various discrepancies that came to light during those studies. We find that superactivation is caused by cleavage at the N terminus of the protein and not the C-terminal allosteric site, as has previously been suggested. N-terminal sequencing reveals that TLCK-treated chymotrypsin cleaves bovine glutamate dehydrogenase at phenylalanine 10. We suggest that trypsin contamination in nontreated chymotrypsin may have led to the production of the larger 4–5 kDa digestion product, previously misinterpreted as having caused the activation. In line with some previous studies, we can confirm that GTP inhibition is attenuated to some extent by the proteolysis, while ADP activation is almost abolished. Utilizing the recently solved structures of bovine glutamate dehydrogenase, we illustrate the cleavage points.     

Structural analysis and proteolytic activation of Enterococcus faecalis cytolysin, a novel lantibiotic

Clinical isolates of Enterococcus faecalis more commonly produce a cytolysin than do commensal isolates. Epidemiologic evidence and animal-model studies have established a role for the cytolysin in the pathogenesis of enterococcal disease. The cytolysin consists of two structural subunits, CylL_L and CylL_s, that are activated by a third component, CylA. Genetic and biochemical characterization of CylA indicate that it is a serine protease, and that activation putatively results from cleavage of one or both cytolysin subunits. Genetic evidence also suggests that the cytolysin subunits are related to the rapidly growing class of bacteriocins termed lantibiotics. However, unlike lantibiotics, the cytolysin is lytic for eukaryotic as well as prokaryotic cells, and it consists of two structural subunits. This report describes the purification and characterization of the cytolysin subunits and detection of lanthionine-type post-translational modifications within their structures. Furthermore, the cleavage specificity of the CylA activator is reported and it is shown that proteolytic activation of both subunits is essential for activity.     

PhotoMEA: an opto-electronic biosensor for monitoring in vitro neuronal network activity

PhotoMEA is a biosensor useful for the analysis of an in vitro neuronal network, fully based on optical methods. Its function is based on the stimulation of neurons with caged glutamate and the recording of neuronal activity by Voltage-Sensitive fluorescent Dyes (VSD). The main advantage is that it will be possible to stimulate even at sub-single neuron level and to record with high resolution the activity of the entire network in the culture. A large-scale view of neuronal intercommunications offers a unique opportunity for testing the ability of drugs to affect neuronal properties as well as alterations in the behaviour of the entire network. The concept and a prototype for validation is described here in detail.     

Bioactive peptides encrypted in milk proteins: proteolytic activation and thropho-functional properties

The bioactivities of peptides encrypted in major milk proteins are latent until released and activated by enzymatic proteolysis, e.g. during gastrointestinal digestion or food processing. The proteolytic system of lactic acid bacteria can contribute to the liberation of bioactive peptides. In vitro, the purified cell wall proteinase of Lactococcus lactis was shown to liberate oligopeptides from - and -caseins which contain amino acid sequences present in casomorphins, casokinines, and immunopeptides. The further degradation of these peptides by endopeptidases and exopeptidases of lactic acid bacteria could lead to the liberation of bioactive peptides in fermented milk products. However, the sequences of practically all known biologically active peptides can also be cleaved by peptidases from lactic acid bacteria. Activated peptides are potential modulators of various regulatory processes in the body: Opioid peptides are opioid receptor ligands which can modulate ab sorption processes in the intestinal tract, angiotensin-I-converting enzyme (ACE)-inhibitory peptides are hemodynamic regulators and exert an antihypertensive effect, immunomodulating casein peptides stimulate the activities of cells of the immune system, antimicrobial peptides kill sensitive microorganisms, antithrombotic peptides inhibit aggregation of platelets and caseinophosphopeptides may function as carriers for different minerals, especially calcium. Bioactive peptides can interact with target sites at the luminal side of the intestinal tract. Furthermore, they can be absorbed and then reach peripheral organs. Food-derived bioactive peptides are claimed to be health enhancing components which can be used for functional food and pharmaceutical preparations.     

Translation of encephalomyocarditis virus RNA in vitro yields an active proteolytic processing enzyme.

In contrast to other cell-free translation systems, the mRNA-dependent reticulocyte lysate can translate encephalomyocarditis virus RNA efficiently and completely when supplemented with heterologous tRNA. Cleavage of the nascent polypeptide chain occurs, and one of the translation products appears to be a specific proteolytic enzyme which correctly processes the primary products. The identity of the proteins made in vitro was verified by comparison with infected cell proteins on dodecylsulphate/polyacrylamide gels, and by mapping their coding sequences on the viral genome.     

A three-step proteolytic cascade mediates the activation of the peptidoglycan-induced toll pathway in an insect

The recognition of lysine-type peptidoglycans (PG) by the PG recognition complex has been suggested to cause activation of the serine protease cascade leading to the processing of Sp?tzle and subsequent activation of the Toll signaling pathway. So far, two serine proteases involved in the lysine-type PG Toll signaling pathway have been identified. One is a modular serine protease functioning as an initial enzyme to be recruited into the lysine-type PG recognition complex. The other is the Drosophila Sp?tzle processing enzyme (SPE), a terminal enzyme that converts Sp?tzle pro-protein to its processed form capable of binding to the Toll receptor. However, it remains unclear how the initial PG recognition signal is transferred to Sp?tzle resulting in Toll pathway activation. Also, the biochemical characteristics and mechanism of action of a serine protease linking the modular serine protease and SPE have not been investigated. Here, we purified and cloned a novel upstream serine protease of SPE that we named SAE, SPE-activating enzyme, from the hemolymph of a large beetle, Tenebrio molitor larvae. This enzyme was activated by Tenebrio modular serine protease and in turn activated the Tenebrio SPE. The biochemical ordered functions of these three serine proteases were determined in vitro, suggesting that the activation of a three-step proteolytic cascade is necessary and sufficient for lysine-type PG recognition signaling. The processed Sp?tzle by this cascade induced antibacterial activity in vivo. These results demonstrate that the three-step proteolytic cascade linking the PG recognition complex and Sp?tzle processing is essential for the PG-dependent Toll signaling pathway.     

The relation of soluble lead to toxicity: an in vitro analysis.

Cells of human origin maintained in vitro in a phosphate-free medium (PFM) remain viable for over 24 h but lose viability rapidly in the presence of soluble lead. The threshold for this loss of viability is less than 1 ppm. In the presence of Ca-2+ in high concentrations, the cells maintain their viability. These data suggest that soluble lead is far more toxic than particulate lead, and that the protective action of the Ca-2+ ion in lead toxicity is not on a specific cell type.