Cathepsin K induces platelet dysfunction and affects cell signaling in breast cancer - molecularly distinct behavior of cathepsin K in breast cancer.

BACKGROUND: Breast cancer comprises clinically and molecularly distinct tumor subgroups that differ in cell histology and biology and show divergent clinical phenotypes that impede phase III trials, such as those utilizing cathepsin K inhibitors. Here we correlate the epithelial-mesenchymal-like transition breast cancer cells and cathepsin K secretion with activation and aggregation of platelets. Cathepsin K is up-regulated in cancer cells that proteolyze extracellular matrix and contributes to invasiveness. Although proteolytically activated receptors (PARs) are activated by proteases, the direct interaction of cysteine cathepsins with PARs is poorly understood. In human platelets, PAR-1 and -4 are highly expressed, but PAR-3 shows low expression and unclear functions. METHODS: Platelet aggregation was monitored by measuring changes in turbidity. Platelets were immunoblotted with anti-phospho and total p38, Src-Tyr-416, FAK-Tyr-397, and TGFß monoclonal antibody. Activation was measured in a flow cytometer and calcium mobilization in a confocal microscope. Mammary epithelial cells were prepared from the primary breast cancer samples of 15 women with Luminal-B subtype to produce primary cells. RESULTS: We demonstrate that platelets are aggregated by cathepsin K in a dose-dependent manner, but not by other cysteine cathepsins. PARs-3 and -4 were confirmed as the cathepsin K target by immunodetection and specific antagonists using a fibroblast cell line derived from PARs deficient mice. Moreover, through co-culture experiments, we show that platelets activated by cathepsin K mediated the up-regulation of SHH, PTHrP, OPN, and TGFß in epithelial-mesenchymal-like cells from patients with Luminal B breast cancer. CONCLUSIONS: Cathepsin K induces platelet dysfunction and affects signaling in breast cancer cells.

Studies on the peptidase activity of transthyretin (TTR).

Transthyretin (TTR) is a plasma protein transporter of thyroxine (T(4)) and retinol and also has peptidase activity. In order to characterize TTR peptidase activity we used fluorescence resonance energy transfer (FRET) peptides derived from Abz-KLRSSK-Q-EDDnp and from two portion-mixing libraries as substrates. Most of the susceptible FRET peptides were cleaved at more than one peptide bond, without particular substrate specificity. The more relevant observation was that the peptides containing E or D were cleaved at only one peptide bond and TTR was competitively inhibited by glutathione analog peptide γ-E-A-G-OH that contains two free carboxyl groups. The dependence on ionic interactions of TTR hydrolytic activity was confirmed by the large inhibitory effects of salt and ionic surfactants. TTR was not inhibited by any usual peptidase inhibitors, except by ortho-phenanthroline and EDTA. The mechanism of TTR catalysis was explored by the pH-profile of TTR hydrolytic activity in different temperatures and by proton inventory. The obtained pK and heat of ionization values suggest that a carboxylate and an ammonium group, possibly from a lysine side chain are involved. These results support the recently proposed inducible metalloprotease mechanism for TTR based on its 3D structure in presence of Zn(2+) and a series of point mutations [Liz et al., Biochem. J 443 (2012) 769-778].

Foot and mouth disease leader protease (Lbpro): Investigation of prime side specificity allows the synthesis of a potent inhibitor.

Foot and mouth disease virus expresses its genetic information as a single polyprotein that is translated from the single-stranded RNA genome. Proteinases contained within the polyprotein then generate the mature viral proteins. The leader protease (Lb(pro)) performs the initial cleavage by freeing itself from the growing polypeptide chain; subsequently, Lb(pro) cleaves the two homologues of the host cell protein eukaryotic initiation factor 4G (eIF4G). We showed that Lb(pro) possesses specific binding sites at the non prime side from S(1) down to S(7) [Santos et al. (2009) Biochemistry, 48, 7948-7958]. Here, we demonstrate that Lb(pro) has high prime side specificity at least down to the S'(5) site. Lb(pro) is thus not only one of the smallest papain-like cysteine peptidases but also one of the most specific. It can still however cleave between both K↓G and G↓R pairs. We further determined the two-step irreversible inhibition (E + I ↔ EI→ E - I) kinetic parameters of two known irreversible epoxide-based inhibitors of cysteine proteinases, E64 and CA074 on Lb(pro) that show for the reversible step (E + I ↔ EI) K(i) = 3.4 µM and 11.6 µM, and for the irreversible step (EI→E-I) k(4) = 0.16 and 0.06 min(-1), respectively. Knowledge of the Lb(pro) specificity led us to extend E64 by addition of the dipeptide R-P. This compound, termed E64-R-P-NH(2), irreversibly inhibited Lb(pro) with a K(i) = 30 nM and k(4) = 0.01 min(-1) and can serve as the basis for design of specific inhibitors of FMDV replication.