Extracellular proteinases of Candida species pathogenic yeasts.

The increased incidence of severe disseminated infections caused by the opportunistic yeast-like fungi Candida spp. highlights the urgent need for research into the major virulence factors of these pathogens-extracellular aspartic proteinases of the candidapepsin and yapsin families. Classically, these enzymes were considered to be generally destructive factors that damage host tissues and provide nutrients for pathogen propagation. However, in recent decades, novel and more specific functions have been suggested for extracellular candidal proteinases. These include contributions to cell wall maintenance and remodeling, the formation of polymicrobial biofilms, adhesion to external protective barriers of the host, the deregulation of host proteolytic cascades (such as the complement system, blood coagulation and the kallikrein-kinin system), a dysregulated host proteinase-inhibitor balance, the inactivation of host antimicrobial peptides, evasion of immune responses and the induction of inflammatory mediator release from host cells. Only a few of these activities recognized in Candida albicans candidapepsins have been also confirmed in other Candida species, and characterization of Candida glabrata yapsins remains limited.

Comparative cleavage sites within the reactive-site loop of native and oxidized alpha1-proteinase inhibitor by selected bacterial proteinases.

Human alpha1-proteinase inhibitor (alpha1-PI) is responsible for the tight control of neutrophil elastase activity which, if down regulated, may cause local excessive tissue degradation. Many bacterial proteinases can inactivate alpha1-PI by hydrolytic cleavage within its reactive site, resulting in the down regulation of elastase, and this mechanism is likely to contribute to the connective tissue damage often associated with bacterial infections. Another pathway of the inactivation of alpha1-PI is reversible and involves oxidation of a critical active-site methionine residue that may influence inhibitor susceptibility to proteolytic inactivation. Hence, the aim of this work was to determine whether this oxidation event might affectthe rate and pattern of the cleavage of the alpha1-PI reactive-site loop by selected bacterial proteinases, including thermolysin, aureolysin, serralysin, pseudolysin, Staphylococcus aureus serine proteinase, streptopain, and periodontain. A shift of cleavage specificity was observed after alpha1-PI oxidation, with a preference for the Glu354-Ala355 bond by most of the proteinases tested. Only aureolysin and serralysin cleave the oxidized form of alpha1-PI faster than the native inhibitor, suggesting that bacteria which secrete these metalloproteinases may specifically take advantage of the host defense oxidative mechanism to accelerate elimination of alpha1-PI and, consequently, tissue degradation by neutrophil elastase.

Ligand-protein interaction in plant seed thiamine-binding proteins. Binding of various thiamine analogues to the sepharose-immobilized buckwheat-seed protein.

Soluble thiamine-binding proteins occur in microorganisms, some animal tissues, and plant seeds. Their representative, the buckwheat-seed protein, was chosen as a model for chemical studies on the mechanism of ligand-protein interaction in these systems. In this work, in order to refine a concept of the chemical topography of the thiamine-binding center, the buckwheat seed protein was immobilized in Sepharose gel and probed with a new set of thiamine-related compounds. In terms of the standard change of Gibbs free energy on the complex formation, the following energetic contributions were specifically assigned to major structural features of the thiamine molecule: (i) 35-45% to the specific electronic structure of planar, unsaturated thiazolium ring with positive charge asymmetrically delocalized, one half of that contribution being attributable to the S(1) atom, (ii) 11-18% to nitrogen atoms and their electronic coupling within the pyrimidine ring, (iii) 15% to the 4'-amino group, and (iv) less than 10% to the hydroxyethyl chain.

Effect of oxidation of beta-amyloid precursor protein on its beta-secretase cleavage. A model study with synthetic peptides and candidate beta-secretases.

As the amyloidogenic processing of beta-amyloid precursor protein (betaAPP) proceeds under conditions of oxidative stress, the methionine-596 residue at the beta-secretase cleavage point is likely in an oxidized state. In the present work, possible consequences of the oxidation of Met-596 for the generation of the N-terminus of amyloid beta protein were modeled using synthetic peptide substrates, matching 587-606 sequence fragment of betaAPP and containing either intact methionine or methionine sulfoxide. Patterns and rates for the cleavage of these substrates by purified mast cell chymase, cathepsin G, cathepsin D, matrix metalloproteinase-3 and neutrophil elastase, were compared. Only the three first proteases, all previously suggested as candidate beta-secretases, preferentially cleaved the "intact" substrate after Met-596. For chymase and cathepsin G, the specificity of this cleavage increased upon a shift from optimal alkaline pH to acidic pH, which is also more compatible with the plausible intracellular localization of amyloidogenic betaAPP processing. The substitution of methionine sulfoxide for methionine in the substrate slowed down the cleavage rate for all the enzymes tested, by a factor of 6-15. This was associated with shifts of cleavage preferences to points of minor importance for the "intact" peptide, suggesting a specific resistance of the peptide bond after MetSO-596 against proteolysis.

A novel mechanism for bradykinin production at inflammatory sites. Diverse effects of a mixture of neutrophil elastase and mast cell tryptase versus tissue and plasma kallikreins on native and oxidized kininogens.

Coprocessing of kininogens by a mixture of human mast cell tryptase and neutrophil elastase was explored as a potential substitute for the kallikrein-dependent pathway for kinin generation during inflammation. Tryptase easily excised bradykinin from the synthetic heptadecapeptide, ISLMKRPPGFSPFRSSR, but was unable to produce significant amounts of kinin by proteolysis of kininogens. However, a mixture of tryptase and elastase released bradykinin from each protein with a yield comparable to that of human plasma kallikrein. Significantly, neither plasma nor tissue kallikrein was able to effectively process N-chlorosuccinimide-oxidized high molecular weight kininogen, an effect attributed to the oxidation of a methionine residue upstream from the N terminus of the kinin domain. In support of these results the model heptadecapetide, ISL(MO)KRPPGFSPFRSSR, was also resistant to hydrolysis by either kallikrein. In contrast, the release of bradykinin from oxidized peptide or protein substrates by the tryptase/elastase mixture was not altered. Because kininogen modification may occur at inflammatory sites, as a result of the oxidative burst of recruited neutrophils and macrophages, these results suggest an alternative pathway for kinin production and the necessity for the novel utilization of two specific proteinases known to be released from these cells during inflammatory episodes.

Mechanism of ligand-protein interaction in plant seed thiamine-binding proteins. Preliminary chemical identification of amino acid residues essential for thiamine binding to the buckwheat-seed protein.

Thiamine-binding protein, isolated from buckwheat seeds, was chemically modified in an attempt to identify amino acid residues involved in protein-thiamine interaction. No evidence was found in support of specific roles of arginine residues, sulfhydryl groups, amino groups and tyrosine residues. Under carefully controlled reaction conditions (Tris pH 5-6), the modification with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide caused a complete loss of thiamine-binding capacity. Thus, the carboxyl groups seemed to be essential for binding, possibly for ionic interaction with protein-bound thiamine cation. A selective modification of histidine residues using diethylpyrocarbonate correlated with a loss of thiamine-binding capacity; the modification and the loss of binding capacity could be reversed with hydroxylamine; some ligand-protection against modification was observed. From Tsou analysis of diethylpyrocarbonate modification and resulting loss of thiamine-binding it was suggested that 1-2 of 20 histidine residues of the protein were essential for thiamine binding. The essential histidine(s) might be present in the binding site and possibly were involved in hydrogen bonding(s) with protein-bound thiamine molecule.

Mechanism of ligand-protein interaction in plant seed thiamin-binding proteins. Probing the binding site of protein isolated from buckwheat seeds with a series of thiamin-related compounds.

Affinities of 14 thiamin derivatives or antagonists to a thiamin-binding protein isolated from buckwheat seeds were determined. A competitive displacement of radiolabeled thiamin by unlabeled ligand was analysed by a computerized model-fitting procedure. The dissociation constant of the thiamin-protein complex was 0.93 microM. Most modifications in ligand chemical structure weakened the ligand-protein interaction. A model of the thiamin-binding site is suggested. The hydroxyethyl-chain of thiamin while protein-bound appears to be excluded from the binding region. A positively charged quaternary nitrogen atom of the thiazolium ring probably interacts with some negative group(s) of protein. The rest of the thiazolium ring as well as the amino group of the pyrimidine fragment serve as additional anchors. The three structural features of the thiamin molecule accounting for binding contribute equally to overall binding energy by about 11-12 kJ/mol.