Human Cysteine Cathepsins Are Not Reliable Markers of Infection by Pseudomonas aeruginosa in Cystic Fibrosis

Cysteine cathepsins have emerged as new players in inflammatory lung disorders. Their activities are dramatically increased in the sputum of cystic fibrosis (CF) patients, suggesting that they are involved in the pathophysiology of CF. We have characterized the cathepsins in CF expectorations and evaluated their use as markers of colonization by Pseudomonas aeruginosa. The concentrations of active cathepsins B, H, K, L and S were the same in P. aeruginosa-positive (19 Ps+) and P. aeruginosa-negative (6 Ps−) samples, unlike those of human neutrophil elastase. Also the cathepsin inhibitory potential and the cathepsins/cathepsin inhibitors imbalance remained unchanged and similar (∼2-fold) in the Ps+ and Ps− groups (p<0.001), which correlated with the breakdown of their circulating cystatin-like inhibitors (kininogens). Procathepsins, which may be activated autocatalytically, are a potential proteolytic reservoir. Immunoblotting and active-site labeling identified the double-chain cathepsin B, the major cathepsin in CF sputum, as the main molecular form in both Ps+ and Ps− samples, despite the possible release of the ∼31 kDa single-chain form from procathepsin B by sputum elastase. Thus, the hydrolytic activity of cysteine cathepsins was not correlated with bacterial colonization, indicating that cathepsins, unlike human neutrophil elastase, are not suitable markers of P. aeruginosa infection

Mycobacterial lipolytic enzymes: A gold mine for tuberculosis research

International audienceTuberculosis (TB) is one of the deadliest infectious diseases worldwide with a strong impact in developing countries. Mycobacterium tuberculosis, the etiological agent of TB, has a high capacity to evade the host immune system and establish a chronic, asymptomatic and latent infection. In a latent TB infection, persistent bacilli are present in a non-replicating dormant state within host granulomas. During reactivation, bacilli start replicating again leading to an active TB infection that can be highly contagious. Mycobacterial lipids and lipolytic enzymes are thought to play important physiological roles during dormancy and reactivation. The role of lipolytic enzymes in the physiology of M. tuberculosis and physiopathology of the disease will be discussed in this review, with an emphasis on the secreted or cell wall-associated, surface exposed lipolytic enzymes characterized to date. Studies on the localization, enzymatic activity and immunological properties of these enzymes highlighted their possible usefulness as new diagnostic markers in the fight against TB

NEW SUBSTRATES of PAPAIN, BASED ON the CONSERVED SEQUENCE of NATURAL INHIBITORS of the CYSTATIN FAMILY

A series of peptide substrates with different fluorogenic leaving groups has been synthesized. the peptide moiety in these substrates mimics a highly conserved sequence (QVVAG) in the natural reversible inhibitors of cysteine proteinases, the cystatins, that participates to the tight binding of target proteinases. This sequence is invariably cleaved at the A-G bond when synthetic peptides containing it were incubated with papain. AEC and AMC fluorophores were therefore attached to the Ala residue to construct new substrates for cysteine proteinases. the solubility of the resulting substrates was improved by attaching a N-terminal gluconoyl group, or by introducing an arginyl residue at P5 (nomenclature of Schechter I, Berger A (1967) Biochem Biophys Res Commun 27, 157-162). Neither induced significant changes in the k(cat)/K-m values with papain. Those values were all in the 10(5) M(-1) s(-1) range. the k(cat)/K-m was increased 10-50-fold by using substrates with intramolecularly quenched fluorescence. With these, the enzyme specificity on both sides of the scissile bond can be investigated. the substrate Abz-QVVAGA-EDDnp is among the most sensitive papain substrates ever reported, with a k(cat)/K-m, value of 29 10(6) M(-1) s(-1). the positioning and conformation of the bound QVVA moiety within the active site of papain were predicted by molecular modelling using the X-ray coordinates of a peptide inhibitor-papain complex.UNIV TOURS, ENZYMOL & CHIM PROT LAB, CNRS, URA 1334, F-37032 TOURS, FRANCEESCOLA PAULISTA MED, INFAR, DEPT BIOPHYS, BR-04044 São Paulo, BRAZILCTR BIOPHYS MOLEC, BIOCHIM GLYCOCONJUGUES & LECTINES ENDOGENES LAB, CNRS, F-45071 ORLEANS, FRANCEESCOLA PAULISTA MED, INFAR, DEPT BIOPHYS, BR-04044 São Paulo, BRAZILWeb of Scienc

Functional expression of the catalytic domains of two cysteine proteinases from Trypanosoma congolense

The catalytic domains of two closely related cysteine proteinases (CP l and CP2) from Trypanosoma congolense, referred to as Cl and C2, were expressed as proforms in Escherichia coli (C 1) and in the baculovirus system (C I and C2). While the bacterial expression system did not allow recovery of active C 1, the baculovirus system led to secretion of inactive zymogens which could be processed at acidic pH into mature enzymes. Active Cl and C2 were purified from serum-free culture supernatants by anion-exchange chromatography and characterised. Their kinetic parameters and pH activity profiles confirmed the relatedness between C2 and native CP2 (congopain). These properties also underline major functional differences between Cl and C2, that appear to relate to discrete but essential sequence differences. It is likely that these two enzymes perform distinct roles in vivo, in the parasite and/or in the host-parasite relationships