Regulation of legumain in human cells

Abstract

The studies presented in this thesis aimed to investigate the regulation of the cysteine protease legumain in various human cells (paper I-IV), effects of statins (paper II and III) and a legumain-cleavable colchicine prodrug (paper IV). Legumain is the only known asparaginyl endoprotease and shows high structural similarities to the caspases. Legumain is reported to participate in extracellular matrix degradation by processing of pro-matrix metalloprotase-2, fibronectin, cathepsin B and L. The subcellular localization of legumain is thought to be mainly lysosomal, and dysregulation of legumain is suggested to be of consequence for proteolytic degradation and tissue remodeling. High legumain expression is correlated with cancer progression and malignancy as well as atherosclerotic plaque rupture, and pharmacological intervention targeting legumain may be useful. In the present work, legumain and/or cystatin E/M over-expressing HEK293 cells were established, and used to study regulation, subcellular localization and interplay of legumain and cystatin E/M (paper I). Legumain over-expressing HEK293 cells secreted prolegumain that was able to be internalized and subsequently activated intracellularly (paper I). Cystatin E/M (the most potent endogenous inhibitor of legumain) was also secreted and internalized, and thus able to regulate legumain activity both intra- and extracellularly (paper I). Macrophages are key regulators in cancer and atherosclerosis. In paper II, legumain expression, activity and secretion were highly up-regulated during monocyte-to-macrophage differentiation (paper II). Interestingly, 90 % of the legumain produced in M-CSF-stimulated macrophages was secreted as prolegumain, and surprisingly legumain was detected in human sera (paper II). Secreted legumain points to extracellular functions, and quantification may have diagnostic or prognostic value. Statins (HMG-CoA reductase inhibitors) have so-called pleiotropic effects beyond lowering the serum cholesterol level. These drugs are generally well tolerated, although skeletal muscle side effects are known, especially by simvastatin. In monocytes from patients treated with atorvastatin, legumain mRNA is shown to be down-regulated. Herein, statins were shown to inhibited legumain activity, expression, processing and secretion in various human cells types, such as macrophages (paper II), myotubes (paper III), and in legumain over-expressing HEK293 cells (Fig. 8), probably by interfering with the intracellular vesicular trafficking or by increasing the pH of the lysosomes (paper III). Legumain inhibition may contribute to the pleiotropic effects of statins. A novel pharmacological strategy is to construct a prodrug of a cytotoxic drug conjugated with a legumain-cleavable peptide (Ala-Ala-Asn), which upon cleavage by legumain becomes cytotoxic to legumain over-expressing cancer cells and tissues. In paper IV, a novel legumain-cleavable colchicine prodrug (Suc-Ala-Ala-Asn-Valcolchicine) were synthesized and showed cell-specific toxicity towards cells expressing active legumain (legumain over-expressing HEK293 cells and HCT116 colorectal cancer cell). In conclusion, the studies presented in this thesis have contributed to new knowledge about regulation of the cysteine protease legumain. It has demonstrated that regulation of legumain occur both extra- and intracellular by cystatin E/M. Pharmacological inhibition of legumain by statins or by utilizing legumain to release cytotoxic colchicine from a legumaincleavable prodrug may be of therapeutic value in preventing morbidity and mortality in cancer and atherosclerosis.