Amidase domains from bacterial and phage autolysins define a family of gamma-D,L-glutamate-specific amidohydrolases

Several phage-encoded peptidoglycan hydrolases have been found to share a conserved amidase domain with a variety of bacterial autolysins (N-acetylmuramoyl-L-alanine amidases), bacterial and eukaryotic glutathionylspermidine amidases, gamma-D-glutamyl-L-diamino acid endopeptidase and NLP/P60 family proteins. All these proteins contain conserved cysteine and histidine residues and hydrolyze gamma-glutamyl-containing substrates. These cysteine residues have been shown to be essential for activity of several of these amidases and their thiol groups apparently function as the nucleophiles in the catalytic mechanisms of all enzymes containing this domain. The CHAP (cysteine, histidine-dependent amidohydrolases/peptidases) superfamily includes a variety of previously uncharacterized proteins, including the tail assembly protein K of phage lambda. Some members of this superfamily are important surface antigens in pathogenic bacteria and might represent drug and/or vaccine targets.     

Evolutionary lines of cysteine peptidases

The proteolytic enzymes that depend upon a cysteine residue for activity have come from at least seven different evolutionary origins, each of which has produced a group of cysteine peptidases with distinctive structures and properties. We show here that the characteristic molecular topologies of the peptidases in each evolutionary line can be seen not only in their three-dimensional structures, but commonly also in the two-dimensional structures. Clan CA contains the families of papain (C1), calpain (C2), streptopain (C10) and the ubiquitin-specific peptidases (C12, C19), as well as many families of viral cysteine endopeptidases. Clan CD contains the families of clostripain (C11), gingipain R (C25), legumain (C13), caspase-1 (C14) and separin (C50). These enzymes have specificities dominated by the interactions of the S1 subsite. Clan CE contains the families of adenain (C5) from adenoviruses, the eukaryotic Ulp1 protease (C48) and the bacterial YopJ proteases (C55). Clan CF contains only pyroglutamyl peptidase I (C15). The picornains (C3) in clan PA have probably evolved from serine peptidases, which still form the majority of enzymes in the clan. The cysteine peptidase activities in clans PB and CH are autolytic only. In conclusion, we suggest that although almost all the cysteine peptidases depend for activity on catalytic dyads of cysteine and histidine, it is worth noting some important differences that they have inherited from their distant ancestral peptidases.     

The unusual catalytic triad of poliovirus protease 3C

Picornaviruses are small pathogen RNA viruses, like poliovirus, hepatitis A virus, rhinovirus, and others. They produce a large polyprotein, which is cleaved by virally encoded cysteine peptidases, picornains 2A and 3C. Picornain 3C represents an intermediate between the serine peptidase chymotrypsin and the cysteine peptidase papain. Its steric structure resembles chymotrypsin, but its nucleophile is a thiol instead of the hydroxyl group. The histidine is a general base catalyst in chymotrypsin but forms a thiolate-imidazolium ion pair in papain. The third member of the catalytic triad is an acid (Glu71) as in chymotrypsin rather than an amide found in papain. Transformation of poliovirus 3C peptidase into a serine peptidase results in lower activity by a factor of 430, but the activity extends toward higher pH with the more basic hydroxyl group. The decrease in activity is caused by the less ordered active site, as supported by the unfavorable entropy of activation. At 25 degrees C the specificity rate constant for the thiol enzyme approaches k(1), the rate constant for the formation of the enzyme-substrate complex, but k(2), the acylation constant, becomes predominant with the increase in temperature. In contrast, for the serine peptidase the specificity constant is less than k(1) over the entire temperature range, and the transition state is controlled by both k(1) and k(2). The acidic component of the catalytic triad is essential for activity, but its negative charge does not influence the ionization of the thiol group.     

Cysteine digestive peptidases function as post-glutamine cleaving enzymes in tenebrionid stored-product pests

The major storage proteins in cereals, prolamins, have an abundance of the amino acids glutamine and proline. Storage pests need specific digestive enzymes to efficiently hydrolyze these storage proteins. Therefore, post-glutamine cleaving peptidases (PGP) were isolated from the midgut of the stored-product pest, Tenebrio molitor (yellow mealworm). Three distinct PGP activities were found in the anterior and posterior midgut using the highly-specific chromogenic peptide substrate N-benzyloxycarbonyl-L-Ala-L-Ala-L-Gln p-nitroanilide. PGP peptidases were characterized according to gel elution times, activity profiles in buffers of different pH, electrophoretic mobility under native conditions, and inhibitor sensitivity. The results indicate that PGP activity is due to cysteine and not serine chymotrypsin-like peptidases from the T. molitor larvae midgut. We propose that the evolutionary conservation of cysteine peptidases in the complement of digestive peptidases of tenebrionid stored-product beetles is due not only to the adaptation of insects to plants rich in serine peptidase inhibitors, but also to accommodate the need to efficiently cleave major dietary proteins rich in glutamine.     

Control of active B and L cathepsins in tissues of colorectal cancer using cystatins isolated from chicken egg proteins: in vitro studies

The activity of cysteine peptidases (cathepsins B and L) was estimated in homogenates of tissues sampled during surgery from 60 patients operated due to colorectal tumors. The results were compared to those obtained using tissues in which histopathology disclosed no tumorous cells, obtained from 20 patients of the same group, treated as a control. Activity of the enzymes was inhibited using cysteine peptidase inhibitors isolated from chicken egg proteins. Application of the inhibitors was found to inhibit activity of the enzymes which play a key role in tumor development. It is suggested that in future the inhibitors may provide a component of new generation drugs in the so-called inhibitor therapy.     

Clan CD cysteine peptidases of parasitic protozoa

Parasitic protozoa contain an abundance of cysteine peptidases that are crucial for a range of important biological processes. The most studied cysteine peptidases of parasitic protozoa belong to the group of papain-like enzymes known as clan CA. However, several more recently identified cysteine peptidases differ fundamentally from the clan CA enzymes and have been included together in clan CD. Enzymes of this clan have now been identified in parasitic protozoa. Many have important roles and also differ significantly from known mammalian counterparts. The main characteristics of clan CD enzymes are outlined here, in particular glycosylphosphatidylinositol (GPI):protein transamidase, metacaspase and separase, and their differences from the clan CA enzymes are described.     

Cysteine peptidases, secreted by Trichomonas gallinae, are involved in the cytopathogenic effects on a permanent chicken liver cell culture

Trichomonas gallinae, the aetiological agent of avian trichomonosis, was shown to secrete soluble factors involved in cytopathogenic effect on a permanent chicken liver (LMH) cell culture. The present study focused on the characterization of these molecules. The addition of specific peptidase inhibitors to the cell-free filtrate partially inhibited the monolayer destruction, which implied the presence of peptidases in the filtrate and their involvement in the cytopathogenic effect. One-dimensional substrate (gelatin) SDS-PAGE confirmed the proteolytic character of the filtrate by demonstrating the proteolytic activity within the molecular weight range from 38 to 110 kDa. In addition, the proteolytic activity was specifically inhibited by addition of TLCK and E-64 cysteine peptidase inhibitors implying their cysteine peptidase nature. Furthermore, variations in the intensity and the number of proteolytic bands were observed between cell-free filtrates of low and high passages of the same T. gallinae clonal culture. Two-dimensional substrate gel electrophoresis of concentrated T. gallinae cell-free filtrate identified at least six proteolytic spots. The mass spectrometric analysis of spots from 2-D gels identified the presence of at least two different Clan CA, family C1, cathepsin L-like cysteine peptidases in the cell-free filtrate of T. gallinae. In parallel, a PCR approach using degenerated primers based on the conserved amino acid sequence region of cysteine peptidases from Trichomonas vaginalis identified the coding sequences for four different Clan CA, family C1, cathepsin L-like cysteine peptidases. Finally, this is the first report analyzing molecules secreted by T. gallinae and demonstrating the ubiquity of peptidases secreted by this protozoon.     

Thiolate-imidazolium ion pair is not an obligatory catalytic entity of cysteine peptidases: the active site of picornain 3C

Cysteine peptidases are thought to attack the substrate by a thiolate-imidazolium ion-pair, as demonstrated with the most extensively studied papain. Picornavirus proteinases (picornains), a different family of cysteine peptidases, are structurally related to the trypsin family of serine peptidases, whose catalytically competent histidine operates as a general base catalyst. Measuring the absorbance change upon alkylation of picornains at 250 nm, where the nondissociated thiol group has a negligible absorbance relative to the ionized form, one can test the ionization state of the catalytic cysteine. For such studies, we have prepared and used a mutated variant of the poliovirus proteinase 3C, which contains a single thiol group. The pH dependence of the molar extinction coefficient has undoubtedly shown that picornain 3C contains an ordinary thiol group rather than the usual ion-pair. Therefore, the imidazole assistance, demonstrated in alkylation reactions, is presumably general base catalysis, as found with serine peptidases. Kinetic studies on k(cat)/K(m) gave large inverse deuterium isotope effects, which may overcompensate the reverse values characteristic of the potential general base catalysis. The inverse effects is associated with the stabilization of the protein structure in heavy water.     

Covalent modification of subtilisin Bacillus lentus cysteine mutants with enantiomerically pure chiral auxiliaries causes remarkable changes in activity

Methanethiosulfonate reagents may be used to introduce virtually unlimited structural modifications in enzymes via reaction with the thiol group of cysteine. The covalent coupling of enantiomerically pure (R) and (S) chiral auxiliary methanethiosulfonate ligands to cysteine mutants of subtilisin Bacillus lentus induces spectacular changes in catalytic activity between diastereomeric enzymes. Amidase and esterase kinetic assays using a low substrate approximation were used to establish kcat/KM values for the chemically modified mutants, and up to 3-fold differences in activity were found between diastereomeric enzymes. Changing the length of the carbon chain linking the phenyl or benzyl oxazolidinone ligand to the mutant N62C by a methylene unit reverses which diastereomeric enzyme is more active. Similarly, changing from a phenyl to benzyl oxazolidinone ligand at S166C reverses which diastereomeric enzyme is more active. Chiral modifications at S166C and L217C give CMMs having both high esterase kcat/KM's and high esterase to amidase ratios with large differences between diastereomeric enzymes.     

Identification of Peptidases in Highly Pathogenic vs. Weakly Pathogenic Naegleria fowleri Amebae

Naegleria fowleri, a free‐living ameba, is the causative agent of Primary Amebic Meningoencephalitis. Highly pathogenic mouse‐passaged amebae (Mp) and weakly pathogenic axenically grown (Ax) N. fowleri were examined for peptidase activity. Zymography and azocasein peptidase activity assays demonstrated that Mp and Ax N. fowleri exhibited a similar peptidase pattern. Prominent for whole cell lysates, membranes and conditioned medium (CM) from Mp and Ax amebae was the presence of an activity band of approximately 58 kDa that was sensitive to E64, a cysteine peptidase inhibitor. However, axenically grown N. fowleri demonstrated a high level of this peptidase activity in membrane preparations. The inhibitor E64 also reduced peptidase activity in ameba‐CM consistent with the presence of secreted cysteine peptidases. Exposure of Mp amebae to E64 reduced their migration through matrigel that was used as an extracellular matrix, suggesting a role for cysteine peptidases in invasion of the central nervous system (CNS). The collective results suggest that the profile of peptidases is not a discriminative marker for distinguishing Mp from Ax N. fowleri. However, the presence of a prominent level of activity for cysteine peptidases in N. fowleri membranes and CM, suggests that these enzymes may serve to facilitate passage of the amebae into the CNS.     

Characterization of novel cysteine proteases from germinating cotyledons of soybean [Glycine max (L.) Merrill].

The enzymatic properties of novel cysteine proteases D3-alpha and beta which were purified from germinating soybean cotyledons were investigated. The enzyme activities were exhibited in the presence of a thiol reagent, such as 2-mercaptoethanol, and apparently inhibited by E-64, a cysteine protease inhibitor. Hydrolytic activities toward carbobenzoxy-Phe-Arg-MCA were detected at a pH above 4.0. The optimum temperature for activities was about 40 degrees C. The isoelectric point of D3-alpha and beta was 4.4 and 4. 7, respectively. The molecular mass of D3-alpha and beta, measured by MALDI/TOF mass spectrometry, was 26,178 and 26,429 Da, respectively. The substrate specificities of the enzymes were examined using peptide-MCAs and peptides, and cathepsin L-like broad specificity was observed at pH 4.0. These results demonstrated that these enzymes are cysteine endopeptidases [EC 3.4.22.-] like papain [EC 3.4.22.2].     

A cysteine residue near the propionate side chain of heme is the radical site in ascorbate peroxidase

The radical scavenger 2,2,6,6-tetramethylpiperidinyl-1-oxy (TEMPO(*)) and the spin trap 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) were used in conjunction with mass spectrometry to identify the protein-based radical sites of the H(2)O(2)-tolerant ascorbate peroxidase (APX) of the red alga Galdieria partita and the H(2)O(2)-sensitive stromal APX of tobacco. A cysteine residue in the vicinity of the propionate side chain of heme in both enzymes was labeled with TEMPO(*) and DMPO in an H(2)O(2)-dependent manner, indicating that these cysteine residues form thiyl radicals through interaction of APX with H(2)O(2). TEMPO(*) bound to the cysteine thiyl radicals, and sulfinylated and sulfonylated them. Other oxidized cysteine residues were found in both APXs. Experiments with a cysteine-to-serine point mutation showed that formation of TEMPO adducts and subsequent oxidation of the cysteine residue located near the propionate group of heme leads to loss of enzyme activity, in particular in the Galdieria APX. When treated with glutathione and H(2)O(2), both cysteine residues in both enzymes were glutathionylated. These results suggest that, under oxidative stress in vivo, cysteine oxidation is involved in the inactivation of APXs in addition to the proposed H(2)O(2)-mediated crosslinking of heme to the distal tryptophan residue [Kitajima S, Shimaoka T, Kurioka M & Yokota A (2007) FEBS J274, 3013-3020], and that glutathione protects APX from irreversible oxidation of the cysteine thiol and loss of enzyme activity by binding to the cysteine thiol group.     

A potential role for ICP, a Leishmanial inhibitor of cysteine peptidases, in the interaction between host and parasite

The biological role of a natural inhibitor of cysteine peptidases (designated ICP) of Leishmania has been investigated by genetic manipulation of the parasite. Null mutants grew normally in vitro, were as infective to macrophages in vitro as wild-type parasites, but had reduced infectivity to mice. Mutants re-expressing ICP from a single gene gave partial restoration of virulence in vivo, whereas mutants overexpressing ICP secreted the inhibitor and showed markedly reduced virulence in mice. Promastigotes of the null mutants had similar cysteine peptidase activities as the wild-type parasites, suggesting that ICP is not required for the expression or processing of the enzymes. The only proteins found to bind to ICP in promastigote cell lysates were fully processed forms of CPA and CPB, showing that ICP does not bind in abundance either to zymogens of the cysteine peptidases or other leishmanial proteins. However, only a small proportion of ICP colocalized with CPA and CPB in the promastigote (in the endoplasmic reticulum and Golgi) and the majority of ICP resided in vesicles that are apparently distinct from endosomes and the multivesicular tubule (MVT)-lysosome. These data suggest that ICP has a role other than modulation of the activity of the parasite's own cysteine peptidases and their normal trafficking to the MVT-lysosome via the flagellar pocket. The finding that ICP partially colocalized with an endocytosed cysteine peptidase leads us to postulate that ICP has a role in protection of the parasite against the hydrolytic environment of the sandfly gut and/or the parasitophorous vacuole of host macrophages.     

Digestive proteolysis in the Colorado potato beetle,Leptinotarsa decemlineata: Activity-based profiling and imaging of a multipeptidase network

The Colorado potato beetle (CPB), Leptinotarsa decemlineata, is a major pest of potato plants, and its digestive system is a promising target for development of pest control strategies. This work focuses on functional proteomic analysis of the digestive proteolytic enzymes expressed in the CPB gut. We identified a set of peptidases using imaging with specific activity-based probes and activity profiling with selective substrates and inhibitors. The secreted luminal peptidases were classified as: (i) endopeptidases of cathepsin D, cathepsin L, and trypsin types and (ii) exopeptidases with aminopeptidase (cathepsin H), carboxypeptidase (serine carboxypeptidase, prolyl carboxypeptidase), and carboxydipeptidase (cathepsin B) activities. The proteolytic arsenal also includes non-luminal peptidases with prolyl oligopeptidase and metalloaminopeptidase activities. Our results indicate that the CPB gut employs a multienzyme network of peptidases with complementary specificities to efficiently degrade ingested proteins. This proteolytic system functions in both CPB larvae and adults and is controlled mainly by cysteine and aspartic peptidases and supported by serine and metallopeptidases. The component enzymes identified here are potential targets for inhibitors with tailored specificities that could be engineered into potato plants to confer resistance to CPB.     

Identification of peptidases in Nicotiana tabacum leaf intercellular fluid

Peptidases in the extracellular space might affect the integrity of recombinant proteins expressed in, and secreted from, plant cells. To identify extracellular peptidases, we recovered the leaf intercellular fluid from Nicotiana tabacum plants by an infiltration-centrifugation method. The activity of various peptidases was detected by an in vitro assay in the presence of specific inhibitors, using BSA and human serum gamma-globulin as substrates. Peptidases were detected by 1- and 2-D zymography in a polyacrylamide gel containing gelatin as substrate. Proteolytic activity was observed over a wide range of molecular masses equal to, or higher than, 45 kDa. To identify the peptidases, the extracellular proteins were digested with trypsin and analyzed by LC and MS. Seventeen peptides showing identity or similarity to predicted plant aspartic, cysteine, and serine peptidases have been identified. The extracellular localization of a cysteine peptidase aleurain homolog was also shown.     

Characterization of papain-like isoenzymes from latex of Asclepias curassavica by molecular biology validated by proteomic approach

Latices from Asclepias spp are used in wound healing and the treatment of some digestive disorders. These pharmacological actions have been attributed to the presence of cysteine proteases in these milky latices. Asclepias curassavica (Asclepiadaceae), “scarlet milkweed” is a perennial subshrub native to South America. In the current paper we report a new approach directed at the selective biochemical and molecular characterization of asclepain cI (acI) and asclepain cII (acII), the enzymes responsible for the proteolytic activity of the scarlet milkweed latex. SDS-PAGE spots of both purified peptidases were digested with trypsin and Peptide Mass Fingerprints (PMFs) obtained showed no equivalent peptides. No identification was possible by MASCOT search due to the paucity of information concerning Asclepiadaceae latex cysteine proteinases available in databases. From total RNA extracted from latex samples, cDNA of both peptidases was obtained by RT-PCR using degenerate primers encoding Asclepiadaceae cysteine peptidase conserved domains. Theoretical PMFs of partial polypeptide sequences obtained by cloning (186 and 185 amino acids) were compared with empirical PMFs, confirming that the sequences of 186 and 185 amino acids correspond to acI and acII, respectively. N-terminal sequences of acI and acII, characterized by Edman sequencing, were overlapped with those coming from the cDNA to obtain the full-length sequence of both mature peptidases (212 and 211 residues respectively). Alignment and phylogenetic analysis confirmed that acI and acII belong to the subfamily C1A forming a new group of papain-like cysteine peptidases together with asclepain f from Asclepias fruticosa. We conclude that PMF could be adopted as an excellent tool to differentiate, in a fast and unequivocal way, peptidases with very similar physicochemical and functional properties, with advantages over other conventional methods (for instance enzyme kinetics) that are time consuming and afford less reliable results.     

Complementary Proteomic and Biochemical Analysis of Peptidases in Lobster Gastric Juice Uncovers the Functional Role of Individual Enzymes in Food Digestion

Crustaceans are a diverse group, distributed in widely variable environmental conditions for which they show an equally extensive range of biochemical adaptations. Some digestive enzymes have been studied by purification/characterization approaches. However, global analysis is crucial to understand how digestive enzymes interplay. Here, we present the first proteomic analysis of the digestive fluid from a crustacean (Homarus americanus) and identify glycosidases and peptidases as the most abundant classes of hydrolytic enzymes. The digestion pathway of complex carbohydrates was predicted by comparing the lobster enzymes to similar enzymes from other crustaceans. A novel and unbiased substrate profiling approach was used to uncover the global proteolytic specificity of gastric juice and determine the contribution of cysteine and aspartic acid peptidases. These enzymes were separated by gel electrophoresis and their individual substrate specificities uncovered from the resulting gel bands. This new technique is called zymoMSP. Each cysteine peptidase cleaves a set of unique peptide bonds and the S2 pocket determines their substrate specificity. Finally, affinity chromatography was used to enrich for a digestive cathepsin D1 to compare its substrate specificity and cold-adapted enzymatic properties to mammalian enzymes. We conclude that the H. americanus digestive peptidases may have useful therapeutic applications, due to their cold-adaptation properties and ability to hydrolyze collagen.     

Mass spectrometry in studies of protein thiol chemistry and signaling: Opportunities and caveats

Mass spectrometry (MS) has become a powerful and widely utilized tool in the investigation of protein thiol chemistry, biochemistry, and biology. Very early biochemical studies of metabolic enzymes have brought to light the broad spectrum of reactivity profiles that distinguish cysteine thiols with functions in catalysis and protein stability from other cysteine residues in proteins. The development of MS methods for the analysis of proteins using electrospray ionization (ESI) or matrix-assisted laser desorption/ionization (MALDI) coupled with the emergence of high-resolution mass analyzers has been instrumental in advancing studies of thiol modifications, both in single proteins and within the cellular context. This article reviews MS instrumentation and methods of analysis employed in investigations of thiols and their reactivity toward a range of small biomolecules. A selected number of studies are detailed to highlight the advantages brought about by the MS technologies along with the caveats associated with these analyses.