Inhibition of cathepsin L-like proteases by cathepsin V propeptide

The N-terminal propeptide domains of several cathepsin L-like cysteine proteases have been shown to possess potent inhibitory activity. Here we report the first kinetic characterisation of the inhibition properties of the cathepsin V propeptide (CatV PP). Using a facile recombinant approach we demonstrate expression, purification and evaluation of the CatV PP. This propeptide was found to behave as a tight-binding inhibitor against CatV (K (i) 10.2 nm). It also functions as an inhibitor against other members of the CatL-like subclass (CatL, 9.8 nm; CatS, 10.7 nm; and CatK, 149 nm) and had no discernible effects upon the more distantly related CatB.

Recombinant cathepsin S propeptide attenuates cell invasion by inhibition of cathepsin L-like proteases in tumor microenvironment

Human cathepsin L along with cathepsin S, K, and V are collectively known as cathepsin L-like proteases due to their high homology. The overexpression and aberrant activity of each of these proteases has been implicated in tumorigenesis. These proteases contain propeptide domains that can potently inhibit both their cognate protease and other proteases within the cathepsin L-like subfamily. In this investigation, we have produced the cathepsin S propeptide recombinantly and have shown that it is a potent inhibitor of the peptidolytic, elastinolytic, and gelatinolytic activities of the cathepsin L-like proteases. In addition, we show that this peptide is capable of significantly attenuating tumor cell invasion in a panel of human cancer cell lines. Furthermore, fusion of an IgG Fc-domain to the COOH terminus of the propeptide resulted in a chimeric protein with significantly enhanced ability to block tumor cell invasion. This Fc fusion protein exhibited enhanced stability in cell-based assays in comparison with the unmodified propeptide species. This approach for the combined inhibition of the cathepsin L-like proteases may prove useful for the further study in cancer and other conditions where their aberrant activity has been implicated. Furthermore, this strategy for simultaneous inhibition of multiple cysteine cathepsins may represent the basis for novel therapeutics to attenuate tumorigenesis.

Antibody-Mediated Inhibition of Cathepsin S Blocks Colorectal Tumor Invasion and Angiogenesis

Purpose: Cathepsin S is a cysteine protease that promotes the invasion of tumor and endothelial cells during cancer progression. Here we investigated the potential to target cathepsin S using an antagonistic antibody, Fsn0503, to block these tumorigenic effects.
Experimental Design: A panel of monoclonal antibodies was raised to human cathepsin S. The effects of a selected antibody were subsequently determined using invasion and proteolysis assays. Endothelial cell tube formation and aorta sprouting assays were done to examine antiangiogenic effects. In vivo effects were also evaluated using HCT116 xenograft studies.
Results: A selected cathepsin S antibody, Fsn0503, significantly blocked invasion of a range of tumor cell lines, most significantly HCT116 colorectal carcinoma cells, through inhibition of extracellular cathepsin S–mediated proteolysis. We subsequently found enhanced expression of cathepsin S in colorectal adenocarcinoma biopsies when compared with normal colon tissue. Moreover, Fsn0503 blocked endothelial cell capillary tube formation and aortic microvascular sprouting. We further showed that administration of Fsn0503 resulted in inhibition of tumor growth and neovascularization of HCT116 xenograft tumors.
Conclusions: These results show that blocking the invasive and proangiogenic effects of cathepsin S with antibody inhibitors may have therapeutic utility upon further preclinical and clinical evaluation.

USP17 Regulates Ras Activation and Cell Proliferation by Blocking RCE1 Activity

The proto-oncogene Ras undergoes a series of post-translational modifications at its carboxyl-terminal CAAX motif that are essential for its proper membrane localization and function. One step in this process is the cleavage of the CAAX motif by the enzyme Ras-converting enzyme 1 (RCE1). Here we show that the deubiquitinating enzyme USP17 negatively regulates the activity of RCE1. We demonstrate that USP17 expression blocks Ras membrane localization and activation, thereby inhibiting phosphorylation of the downstream kinases MEK and ERK. Furthermore, we show that this effect is caused by the loss of RCE1 catalytic activity as a result of its deubiquitination by USP17. We also show that USP17 and RCE1 co-localize at the endoplasmic reticulum and that USP17 cannot block proliferation or Ras membrane localization in RCE1 null cells. These studies demonstrate that USP17 modulates Ras processing and activation, at least in part, by regulating RCE1 activity.