Cellular Uptake of Cystatin C. Subcellular localisation and intracellular effects of a secreted cysteine protease inhibitor
(2013) In Lund University Faculty of Medicine Doctoral Dissertation Series 2013:85.- 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... (More) - 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. (Less) - Abstract (Swedish)
- Popular Abstract in English
The secreted cysteine protease inhibitor cystatin C has been thought to exert its functions extracellularly, as a defense against enzymes from leaking lysosomes or invading pathogens. In this thesis we demonstrate that regulation of intracellular proteases by cystatin C is possible, as a result of cellular uptake, and that the uptake can be modulated by molecular engineering of wild-type cystatin C. The uptake was seen in different epithelial and neuroblastoma cancer cell lines and was shown by various techniques, including flow cytometry, confocal microscopy, ELISA and Western blotting. Intracellular cysteine proteases involved in cancer-promoting processes may thus be controlled by cystatin C... (More) - Popular Abstract in English
The secreted cysteine protease inhibitor cystatin C has been thought to exert its functions extracellularly, as a defense against enzymes from leaking lysosomes or invading pathogens. In this thesis we demonstrate that regulation of intracellular proteases by cystatin C is possible, as a result of cellular uptake, and that the uptake can be modulated by molecular engineering of wild-type cystatin C. The uptake was seen in different epithelial and neuroblastoma cancer cell lines and was shown by various techniques, including flow cytometry, confocal microscopy, ELISA and Western blotting. Intracellular cysteine proteases involved in cancer-promoting processes may thus be controlled by cystatin C uptake. (Less)
Please use this url to cite or link to this publication:
https://lup.lub.lu.se/record/3991196
- author
- Wallin, Hanna LU
- supervisor
-
- Magnus Abrahamson LU
- Ulf Ekström LU
- opponent
-
- Theodorsson, Elvar, Department of Clinical and Experimental Medicine, Division of Clinical Chemistry, Linköping University
- organization
- publishing date
- 2013
- type
- Thesis
- publication status
- published
- subject
- keywords
- cathepsin, cell line, co-localisation, cystatin C, internalisation, legumain
- in
- Lund University Faculty of Medicine Doctoral Dissertation Series
- volume
- 2013:85
- pages
- 58 pages
- publisher
- Division of Clinical Chemistry and Pharmacology, Faculty of Medicine, Lund University
- defense location
- Segerfalksalen, BMC, Sölvegatan 17, Lund
- defense date
- 2013-09-13 13:15:00
- ISSN
- 1652-8220
- ISBN
- 978-91-87449-57-4
- language
- English
- LU publication?
- yes
- id
- 9baf9542-ca69-496f-8a4b-f2f1e49c8ea5 (old id 3991196)
- date added to LUP
- 2016-04-01 13:27:40
- date last changed
- 2023-04-18 20:36:21
@phdthesis{9baf9542-ca69-496f-8a4b-f2f1e49c8ea5,
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. <br/><br>
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. <br/><br>
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.}},
author = {{Wallin, Hanna}},
isbn = {{978-91-87449-57-4}},
issn = {{1652-8220}},
keywords = {{cathepsin; cell line; co-localisation; cystatin C; internalisation; legumain}},
language = {{eng}},
publisher = {{Division of Clinical Chemistry and Pharmacology, Faculty of Medicine, Lund University}},
school = {{Lund University}},
series = {{Lund University Faculty of Medicine Doctoral Dissertation Series}},
title = {{Cellular Uptake of Cystatin C. Subcellular localisation and intracellular effects of a secreted cysteine protease inhibitor}},
url = {{https://lup.lub.lu.se/search/files/3387001/3992325.pdf}},
volume = {{2013:85}},
year = {{2013}},
}