Cystatin C properties crucial for uptake and inhibition of intracellular target enzymes.
Författare
Summary, in English
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 to 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.
Avdelning/ar
- Avdelningen för klinisk kemi och farmakologi
- Proteasinhibitorforskning
- BioCARE: Biomarkers in Cancer Medicine improving Health Care, Education and Innovation
Publiceringsår
2013
Språk
Engelska
Sidor
17019-17029
Publikation/Tidskrift/Serie
Journal of Biological Chemistry
Volym
288
Issue
23
Fulltext
- Available as PDF - 20 MB
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Länkar
Dokumenttyp
Artikel i tidskrift
Förlag
American Society for Biochemistry and Molecular Biology
Ämne
- Medicinal Chemistry
- Pharmacology and Toxicology
Status
Published
Forskningsgrupp
- Protease Inhibitor Research
ISBN/ISSN/Övrigt
- ISSN: 1083-351X