Biomedical subjects

Enrico Weidauer

Publications and source records attributed to Enrico Weidauer.

3 recordsLinked to original sources

PubMed2004

Cathepsin K: a cysteine protease with unique kinin-degrading properties.

Taking into account a previous report of an unidentified enzyme from macrophages acting as a kininase, the ability of cysteine proteases to degrade kinins has been investigated. Wild-type fibroblast lysates from mice, by contrast with cathepsin K-deficient lysates, hydrolysed BK (bradykinin), and released two metabolites, BK-(1-4) and BK-(5-9). Cathepsin K, but not cathepsins B, H, L and S, cleaved kinins at the Gly4-Phe5 bond and the bradykinin-mimicking substrate Abz (o-aminobenzoic acid)-RPPGFSPFR-3-NO2-Tyr (3-nitrotyrosine) more efficiently (pH 6.0: kcat/K(m)=12500 mM(-1) x s(-1); pH 7.4: kcat/K(m)=6930 mM(-1) x s(-1)) than angiotensin-converting enzyme hydrolysed BK. Conversely Abz-RPPGFSPFR-3-NO2-Tyr was not cleaved by the Y67L (Tyr67-->Leu)/L205A (Leu205-->Ala) cathepsin K mutant, indicating that kinin degradation mostly depends on the S2 substrate specificity. Kininase activity was further evaluated on bronchial smooth muscles. BK, but not its metabolites BK(1-4) and BK(5-9), induced a dose-dependent contraction, which was abolished by Hoe140, a B2-type receptor antagonist. Cathepsin K impaired BK-dependent contraction of normal and chronic hypoxic rats, whereas cathepsins B and L did not. Taking together vasoactive properties of kinins and the potency of cathepsin K to modulate BK-dependent contraction of smooth muscles, the present data support the notion that cathepsin K may act as a kininase, a unique property among mammalian cysteine proteases.

Animals↗

PubMed2004

Response of rat alveolar type II cells and human lung tumor cells towards oxidative stress induced by hydrogen peroxide and paraquat.

The expression of MDR1b coding mRNA is increased in alveolar type II cells from juvenile rat lung in culture. Hydrogen peroxide and paraquat-induced further upregulation supporting that oxidative stress mediated mechanisms are involved in the regulation of MDR1b in rat lung. The expression rates of mRNA for catalase, Cu/Zn-superoxide dismutase (Cu/Zn-SOD) and Mn-superoxide dismutase (Mn-SOD) remains constant during culture and were not modulated by hydrogen peroxide or paraquat. Thus, antioxidative enzymes in primary A II cells from rat lung are not regulated by reactive oxygen species dependent mechanisms. Primary A II cells were substantially more sensitive towards paraquat-induced cytotoxicity and lipid peroxidation than the permanent human lung tumor cell lines H322 and H358. A 100 microM hydrogen peroxide for 2h induces substantial DNA damage which is not paralleled by an increased rate of lipid peroxidation. The expression rate of mRNA coding for catalase and Mn-SOD was not changed and almost the same is true for the activity of catalase and Cu/Zn-SOD. Only 50 microM paraquat induced a significant decrease in catalase activity and an increase in Cu/Zn-SOD activity.

ATP Binding Cassette Transporter, Subfamily B↗

PubMed2003

Probing cathepsin K activity with a selective substrate spanning its active site.

The limited availability of highly selective cathepsin substrates seriously impairs studies designed to monitor individual cathepsin activities in biological samples. Among mammalian cysteine proteases, cathepsin K has a unique preference for a proline residue at P2, the primary determinant of its substrate specificity. Interestingly, congopain from Trypanosoma congolense also accommodates a proline residue in its S2 subsite. Analysis of a congopain model showed that amino acids forming its S2 subsite are identical with those of cathepsin K, except Leu67 which is replaced by a tyrosine residue in cathepsin K. Furthermore, amino acid residues of the congopain S2' binding pocket, which accepts a proline residue, are strictly identical with those of cathepsin K. Abz-HPGGPQ-EDN2ph [where Abz represents o-aminobenzoic acid and EDN2ph (=EDDnp) represents N -(2,4-dinitrophenyl)-ethylenediamine], a substrate initially developed for trypanosomal enzymes, was efficiently cleaved at the Gly-Gly bond by cathepsin K (kcat/ K(m)=426000 M(-1) x s(-1)). On the other hand, Abz-HPGGPQ-EDN2ph was resistant to hydrolysis by cathepsins B, F, H, L, S and V (20 nM enzyme concentration) and the Y67L (Tyr67-->Leu)/L205A cathepsin K mutant (20 nM), but still acted as a competitive inhibitor. Taken together, the selectivity of Abz-HPGGPQ-EDN2ph to cathepsin K primarily depends on the S2 and S2' subsite specificities of cathepsin K and the ionization state of histidine at P3. Whereas Abz-HPGGPQ-EDN2ph was hydrolysed by wild-type mouse fibroblast lysates, its hydrolysis was completely abolished in the cathepsin K-deficient samples, indicating that Abz-HPGGPQ-EDN2ph can be used to monitor selectively cathepsin K activity in physiological fluids and cell lysates.

Animals↗