Cystatin C properties crucial for uptake and inhibition of intracellular target enzymes

Hanna Wallin, Magnus Abrahamson, Ulf Ekström
Journal of Biological Chemistry 2013 June 7, 288 (23): 17019-29
To elucidate the molecular requirements for cancer cell internalization of the extracellular cysteine protease inhibitor cystatin C, 12 variants of the protein were produced and used for uptake experiments in MCF-7 cells. Variants with alterations in the cysteine cathepsin binding region ((Δ1-10)-, K5A-, R8G-, (R8G,L9G,V10G)-, (R8G,L9G,V10G,W106G)-, and W106G-cystatin C) were internalized to a very low extent compared with the wild-type inhibitor. Substitutions of N39 in the legumain binding region (N39K- and N39A-cystatin C) decreased the internalization and (R24A,R25A)-cystatin C, with substitutions of charged residues not involved in enzyme inhibition, was not taken up at all. Two variants, W106F- and K75A-cystatin C, showed that the internalization can be positively affected by engineering of the cystatin molecule. Microscopy revealed vesicular co-localization of internalized cystatin C with the lysosomal marker proteins cathepsin D and legumain. Activities of both cysteine cathepsins and legumain, possible target enzymes associated with cancer cell invasion and metastasis, were down-regulated in cell homogenates following cystatin C uptake. A positive effect on regulation of intracellular enzyme activity by a cystatin variant selected from uptake properties was illustrated by incubating cells with W106F-cystatin C. This resulted in more efficient down-regulation of intracellular legumain activity than when cells were incubated with wild-type cystatin C. Uptake experiments in prostate cancer cells corroborated that the cystatin C internalization is generally relevant and confirmed an increased uptake of W106F-cystatin C, in PC3 cells. Thus, intracellular cysteine proteases involved in cancer-promoting processes might be controled by cystatin uptake.

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