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Cystatin C Properties Crucial for Uptake and Inhibition of Intracellular Target Enzymes

Wallin, Hanna ; Abrahamson, Magnus ; Ekström, Ulf

The Journal of biological chemistry, 2013-06, Vol.288 (23), p.17019-17029 [Periódico revisado por pares]

United States: Elsevier Inc

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Título:
Cystatin C Properties Crucial for Uptake and Inhibition of Intracellular Target Enzymes
Autor: Wallin, Hanna ; Abrahamson, Magnus ; Ekström, Ulf
Assuntos: Amino Acid Substitution ; Basic Medicine ; Binding Sites ; Breast Cancer ; Cathepsin B ; Cathepsin D - genetics ; Cathepsin D - metabolism ; Cell Line, Tumor ; Cystatin C - genetics ; Cystatin C - metabolism ; Cysteine Endopeptidases - genetics ; Cysteine Endopeptidases - metabolism ; Cysteine Protease ; Endocytosis ; Enzymology ; Farmakologi och toxikologi ; Humans ; Legumain ; Lysosomes - genetics ; Lysosomes - metabolism ; Läkemedelskemi ; Medical and Health Sciences ; Medicin och hälsovetenskap ; Medicinal Chemistry ; Medicinska och farmaceutiska grundvetenskaper ; Mutation, Missense ; Neoplasm Invasiveness ; Neoplasm Metastasis ; Neoplasm Proteins - genetics ; Neoplasm Proteins - metabolism ; Neoplasms - genetics ; Neoplasms - metabolism ; Neoplasms - pathology ; Pharmacology and Toxicology ; Protease Inhibitor ; Protein Chemistry
É parte de: The Journal of biological chemistry, 2013-06, Vol.288 (23), p.17019-17029
Descrição: 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. Background: The secreted protease inhibitor cystatin C is internalized into cancer cells. Results: Cystatin variants with altered uptake characteristics were identified, and shown to differently inhibit intracellular cathepsin B and legumain activities. Conclusion: The internalization process, and hence intracellular enzyme activity, can be modulated by selected cystatin variants. Significance: The cystatin C uptake system may be targeted to control cancer-promoting activities of tumor cells.
Editor: United States: Elsevier Inc
Idioma: Inglês

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Cystatin C Properties Crucial for Uptake and Inhibition of Intracellular Target Enzymes

Wallin, Hanna ; Abrahamson, Magnus ; Ekström, Ulf

United States: Elsevier Inc

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