Urša Pečar Fonović

Cathepsin X cleaves the C-terminal dipeptide of alpha- and gamma-enolase and impairs survival and neuritogenesis of neuronal cells

The cysteine carboxypeptidase cathepsin X has been recognized as an important player in degenerative processes during normal aging and in pathological conditions. In this study we identify isozymes alpha- and gamma-enolases as targets for cathepsin X. Cathepsin X sequentially cleaves C-terminal amino acids of both isozymes, abolishing their neurotrophic activity. Neuronal cell survival and neuritogenesis are, in this way, regulated, as shown on pheochromocytoma cell line PC12. Inhibition of cathepsin X activity increases generation of plasmin, essential for neuronal differentiation and changes the length distribution of neurites, especially in the early phase of neurite outgrowth. Moreover, cathepsin X inhibition increases neuronal survival and reduces serum deprivation induced apoptosis, particularly in the absence of nerve growth factor. On the other hand, the proliferation of cells is decreased, indicating induction of differentiation. Our study reveals enolase isozymes as crucial neurotrophic factors that are regulated by the proteolytic activity of cathepsin X.

Cathepsin H Mediates the Processing of Talin and Regulates Migration of Prostate Cancer Cells

Background: Cathepsin H (CtsH) is an aminopeptidase that is involved in tumor progression. Results: CtsH cleaves talin, and its inhibition reduces the migration of prostate cancer cells. Conclusion: CtsH affects cell migration by influencing the activity of integrins, a process that could be regulated by talin cleavage. Significance: Identification of novel CtsH proteolytic targets is important to understand and control tumor progression. The cytoskeletal protein talin, an actin- and β-integrin tail-binding protein, plays an important role in cell migration by promoting integrin activation and focal adhesion formation. Here, we show that talin is a substrate for cathepsin H (CtsH), a lysosomal cysteine protease with a strong aminopeptidase activity. Purified active CtsH sequentially cleaved a synthetic peptide representing the N terminus of the talin F0 head domain. The processing of talin by CtsH was determined also in the metastatic PC-3 prostate cancer cell line, which exhibits increased expression of CtsH. The attenuation of CtsH aminopeptidase activity by a specific inhibitor or siRNA-mediated silencing significantly reduced the migration of PC-3 cells on fibronectin and invasion through Matrigel. We found that in migrating PC-3 cells, CtsH was co-localized with talin in the focal adhesions. Furthermore, specific inhibition of CtsH increased the activation of αvβ3-integrin on PC-3 cells. We propose that CtsH-mediated processing of talin might promote cancer cell progression by affecting integrin activation and adhesion strength.

Three-dimensional invasion of macrophages is mediated by cysteine cathepsins in protrusive podosomes

Podosomes, specialized actin‐rich structures in macrophages (Mfs), degrade the extra‐cellular matrix (ECM) and are involved in cell migration. On two‐dimensional (2D) surfaces Mfs form spot‐like podosomes at the ventral cell surface that develop into protrusive structures in a three‐dimensional (3D) environment resembling the ECM. We have shown that the tips of these protrusive podosomes are characterized by increased accumulation of cysteine cathepsins (Cts) B, X, S, H, and L, both in human blood Mfs and in human monocytic cell line U‐937. Monocyte‐to‐Mf differentiation induces an increase in cysteine cathepsin expression and activity, promoting their translocation to the cell surface, where they interact with ECM. This group of proteases is crucial for the extracellular as well as intracellular degradation of ECM, as demonstrated by quantitative monitoring of collagen IV degradation. Furthermore, inhibiting CtsB, X, and S significantly impairs Mf invasion through the 3D matrix. Time‐lapse live‐cell imaging of CtsB activity revealed that the extracellular and the intracellular ECM degradation are associated with extensive endocytosis at the tip of protrusive podosomes. The targeting of cysteine cathepsins, as the major mediators of human Mf 3D invasion, could be an approach to the treatment of inflammatory and cancerous diseases.

Intracellular signaling by cathepsin X: molecular mechanisms and diagnostic and therapeutic opportunities in cancer.

Cathepsin X is a cysteine carboxypeptidase, localized predominantly in immune cells, regulating their proliferation, maturation, migration and adhesion. It has recently been confirmed as a significant promoter of malignant progression. Its role in signal transduction was first implied through the interaction with integrin receptors, either by binding with the RGD motif or by proteolytic cleavage of the C-terminal amino acids of the cytosolic part of the integrin beta chain. Several other molecules, involved in cellular signaling, have since been shown to be targets for cathepsin X, such as γ-enolase, chemokine CXCL-12, bradykinin, kallidin, huntingtin and profilin 1. In cancer, cathepsin X regulates adhesion of tumor and endothelial cells and their migration and invasion through the extracellular matrix. It also promotes tumor progression by bypassing cellular senescence and by inducing an epithelial-mesenchymal transition. The high RNA and protein levels of cathepsin X, found in tumor samples and bodily fluids of patients with various cancer types, further support its active role in tumor progression. Its prognostic value and relation to response to chemotherapy confirm cathepsin X as a new target for improving diagnosis and treating cancer patients.

Profilin 1 as a target for cathepsin X activity in tumor cells.

Cathepsin X has been reported to be a tumor promotion factor in various types of cancer; however, the molecular mechanisms linking its activity with malignant processes are not understood. Here we present profilin 1, a known tumor suppressor, as a target for cathepsin X carboxypeptidase activity in prostate cancer PC-3 cells. Profilin 1 co-localizes strongly with cathepsin X intracellularly in the perinuclear area as well as at the plasma membrane. Selective cleavage of C-terminal amino acids was demonstrated on a synthetic octapeptide representing the profilin C-terminal region, and on recombinant profilin 1. Further, intact profilin 1 binds its poly-L-proline ligand clathrin significantly better than it does the truncated one, as shown using cathepsin X specific inhibitor AMS-36 and immunoprecipitation of the profilin 1/clathrin complex. Moreover, the polymerization of actin, which depends also on the binding of poly-L-proline ligands to profilin 1, was promoted by AMS-36 treatment of cells and by siRNA cathepsin X silencing. Our results demonstrate that increased adhesion, migration and invasiveness of tumor cells depend on the inactivation of the tumor suppressive function of profilin 1 by cathepsin X. The latter is thus designated as a target for development of new antitumor strategies.

Cathepsin S generates soluble CX3CL1 (fractalkine) in vascular smooth muscle cells.

Abstract CX3CL1 chemokine (fractalkine) is highly expressed by vascular smooth muscle cells (VSMCs) in atherosclerotic lesions. Its membrane-bound form promotes cell-cell interactions, whereas the soluble form induces chemotaxis of CX3CR1- expressing leukocytes. We show that the cysteine protease cathepsin S, expressed by VSMCs, is able to cleave membrane-anchored CX3CL1, releasing a 55-kDa fragment to the medium, thus regulating the adhesion of VSMCs and the capture of monocytes to the sites of atherogenesis. Moreover, strong co-localization of cathepsin S and CX3CL1 with a recycling endosome marker Rab11a suggests a processing of CX3CL1 in recycling endosomes during its redistribution to the plasma membrane.

Cathepsin X Cleaves Profilin 1 C-Terminal Tyr139 and Influences Clathrin-Mediated Endocytosis

Cathepsin X, a cysteine carboxypeptidase, is upregulated in several types of cancer. Its molecular target in tumor cells is profilin 1, a known tumor suppressor and regulator of actin cytoskeleton dynamics. Cathepsin X cleaves off the C-terminal Tyr139 of profilin 1, affecting binding of poly-L-proline ligands and, consequently, tumor cell migration and invasion. Profilin 1 with mutations at the C-terminus, transiently expressed in prostate cancer cells PC-3, showed that Tyr139 is important for proper function of profilin 1 as a tumor suppressor. Cleaving off Tyr139 prevents the binding of clathrin, a poly-L-proline ligand involved in endocytosis. More profilin 1—clathrin complexes were present in PC-3 cells when cathepsin X was inhibited by its specific inhibitor AMS36 or silenced by siRNA. As a consequence, the endocytosis of FITC-labeled dextran and transferrin conjugate was significantly increased. These results constitute the first report of the regulation of clathrin-mediated endocytosis in tumor cells through proteolytic processing of profilin 1.

Cysteine cathepsins B and X promote epithelial-mesenchymal transition of tumor cells

Cathepsins B and X are lysosomal cysteine carboxypeptidases suggested as having a redundant role in cancer. They are involved in a number of processes leading to tumor progression but their role in the epithelial-mesenchymal transition (EMT) remains unknown. We have investigated the contribution of both cathepsins B and X in EMT using tumor cell lines differing in their expression of epithelial and mesenchymal markers and cell morphology. Higher levels of both cathepsins are shown to promote EMT and are associated with the mesenchymal-like cell phenotype. Moreover, simultaneous knockdown of the two peptidases triggers a reverse, mesenchymal to epithelial transition. Of the two cathepsins, cathepsin B appears to be the stronger promotor of EMT. Furthermore, we evaluated the involvement of cathepsin B and X in the transforming growth factor-β1 (TGF-β1) signaling pathway, one of the key signaling mechanisms triggering EMT in cancer. In MCF-7 cells the expression of cathepsin B was shown to depend on their activation with TGF-β1 while, for cathepsin X, a TGF-β1 independent mechanism of induction during EMT is indicated. EMT is thus shown to be another mechanism linking cathepsins B and X with tumor progression. With silencing of their expression or inhibition of enzymatic activity, the tumor cells could be reverted to less aggressive epithelial-like phenotype.

Identification and characterization of the novel reversible and selective cathepsin X inhibitors

Cathepsin X is a cysteine peptidase involved in the progression of cancer and neurodegenerative diseases. Targeting this enzyme with selective inhibitors opens a new possibility for intervention in several therapeutic areas. In this study triazole-based reversible and selective inhibitors of cathepsin X have been identified. Their selectivity and binding is enhanced when the 2,3-dihydrobenzo[b][1,4]dioxine moiety is present as the R1 substituent. Of a series of selected triazole-benzodioxine derivatives, compound 22 is the most potent inhibitor of cathepsin X carboxypeptidase activity (Ki = 2.45 ± 0.05 μM) with at least 100-fold greater selectivity in comparison to cathepsin B or other related cysteine peptidases. Compound 22 is not cytotoxic to prostate cancer cells PC-3 or pheochromocytoma PC-12 cells at concentrations up to 10 μM. It significantly inhibits the migration of tumor cells and increases the outgrowth of neurites, both processes being under the control of cathepsin X carboxypeptidase activity. Compound 22 and other characterized triazole-based inhibitors thus possess a great potential for further development resulting in several in vivo applications.

Increased Plasma Cathepsin S at the Time of Percutaneous Transluminal Angioplasty is Associated with 6-Months’ Restenosis of the Femoropopliteal Artery

Summary Background: We tested the hypothesis that increased levels of cathepsin S and decreased levels of cystatin C in plasma at the time of percutaneous transluminal angioplasty (PTA) are associated with the occurrence of 6-months’ restenosis of the femoropopliteal artery (FPA). Methods: 20 patients with restenosis and 24 matched patients with patent FPA after a 6-months follow-up were in - cluded in this study. They all exhibited disabling claudication or critical limb ischemia and had undergone technically successful PTA. They were all receiving statins and ACE in hi - bitors (or angiotensin II receptor antagonist) before the PTA and the therapy did not change throughout the observational period. Plasma concentrations of C-reactive protein were < 10 mg/L and of creatinine within the reference range at the time of the PTA. Plasma concentration and activity of cathepsin S, together with its potent inhibitor cystatin C, were measured the day before and the day after the PTA. Results: The increased plasma concentration and activity of cathepsin S at the time of PTA was associated with the occurrence of 6-months’ restenosis of FPA, independently of established risk factors (lesion complexity, infrapopliteal run-off vessels, type of PTA, age, gender, smoking, diabetes, lipids) and of cystatin C. Plasma cystatin C concentration was not associated with restenosis and did not correlate with cathepsin S activity and concentration in the plasma. Conclusion: Increased level of plasma cathepsin S at the time of PTA is associated with 6-months’ restenosis of PTA, independently of established risk factors.