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• W1936775975 abstract "Cystatin C is a cysteine protease inhibitor, aimed for secretion, as it is produced with a signal peptide. Its target enzymes are thought to be the lysosomal cysteine cathepsins and legumain. Cystatin C has been considered to excert its enzyme inhibiting functions extracellularly, as a defense against enzymes from leaking lysosomes or invading pathogens. It was demonstrated by various techniques, including flow cytometry, confocal microscopy, ELISA and Western blotting, that cystatin C was internalised in cells of different cell lines after incubation with a physiological concentration of cystatin C. The internalised cystatin C was found in acidic endolysosomal vesicles and co-located with some potential target enzymes, in contrast to the endogenously produced inhibitor, which was mainly found in the endoplasmic reticulum. Cystatin C was non-degraded and still functional as an inhibitor of cysteine cathepsins after uptake, as the total enzyme inhibiting capacity of the cell lysates was increased, suggesting that intracellular cysteine protease activity can be regulated by the uptake. Invasion and migration of MCF-7 breast cancer cells were inhibited when cells were incubated in medium containing cystatin C. To pin-point the structural requirements for cellular uptake, twelve variants of cystatin C, including wild-type, were produced by site-directed mutagenesis and cleaving of the N-terminal. Positively charged amino acid residues on the surface of the molecule, and the amino acid at position 106 were shown to be important for internalisation. In most cases the uptake was decreased after molecular engineering, but for the variant W106F-cystatin C it was increased. The substitution of W106 affects the cathepsin-inhibiting properties of cystatin C, but it is still an efficient inhibitor of legumain. The increased uptake of this variant also induced an increased inhibition of legumain in lysates of cells after uptake." @default.
• W1936775975 created "2016-06-24" @default.
• W1936775975 creator A5042935371 @default.
• W1936775975 date "2013-01-01" @default.
• W1936775975 modified "2023-09-25" @default.
• W1936775975 title "Cellular Uptake of Cystatin C. Subcellular localisation and intracellular effects of a secreted cysteine protease inhibitor" @default.
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