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

A new cell-permeable calpain inhibitor

The ubiquitous calpains, μ- and m-calpain, are implicated in a variety of vital (patho)physiological processes and therefore cell-permeable specific inhibitors represent important tools for defining the role of calpains in cells and animal models. A synthetic N-acetylated 27-mer peptide derived from exon B of the human calpastatin inhibitory domain 1 is known to be the most potent and selective reversible inhibitor of calpains. To improve the membrane permeability of this peptidic inhibitor, it was N-terminally extended with or disulfide-linked to the C-terminal 7-mer fragment of penetratin, a well-established vector for cell membrane translocation of bioactive compounds. Despite the shorter penetratin sequence, both constructs showed increased cell permeability and retained their full calpain inhibitory potency. Copyright © 2006 European Peptide Society and John Wiley & Sons, Ltd

Biol. Chem.

Besides its physiological role in lysosomal protein breakdown, extralysosomal cathepsin B has recently been implicated in apoptotic cell death. Highly specific irreversible cathepsin B inhibitors that are readily cellpermeant should be useful tools to elucidate the effects of cathepsin B in the cytosol. We have covalently functionalised the poorly cellpermeant epoxysuccinyl based cathepsin B inhibitor [RGlyGlyLeu(2S, 3S)tEpsLeuProOH; R=OMe] with the Cterminal heptapeptide segment of penetratin (R=[epsilon]AhxArg ArgNleLysTrpLysLysNH(2)). The high inhibitory potency and selectivity for cathepsin B versus cathepsin L of the parent compound was not affected by the conjugation with the penetratin heptapeptide. The conjugate was shown to efficiently penetrate into MCF-7 cells as an active inhibitor, thereby circumventing an intracellular activation step that is required by other inhibitors, such as the prodruglike epoxysuccinyl peptides E64d and CA074Me

beta-cyclodextrin/epoxysuccinyl peptide conjugates: new drug targeting system for tumor cells

Schaschke N, Assfalg-Machleidt I, Machleidt W, Lassleben T, Sommerhoff CP, Moroder L. beta-cyclodextrin/epoxysuccinyl peptide conjugates: new drug targeting system for tumor cells. BIOORGANIC &amp; MEDICINAL CHEMISTRY LETTERS. 2000;10(7):677-680