Cellular Uptake of Cystatin C. Subcellular localisation and intracellular effects of a secreted cysteine protease inhibitor

Hanna Wallin

Cellular Uptake of Cystatin C. Subcellular localisation and intracellular effects of a secreted cysteine protease inhibitor

Hanna Wallin

Avdelningen för klinisk kemi och farmakologi

Forskningsoutput: Avhandling › Doktorsavhandling (sammanläggning)

384 Nedladdningar (Pure)

Sammanfattning

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.

Originalspråk	engelska
Kvalifikation	Doktor
Tilldelande institution	Avdelningen för klinisk kemi och farmakologi
Handledare	Abrahamson, Magnus, handledare Ekström, Ulf, handledare
Tilldelningsdatum	2013 sep. 13
Förlag	Division of Clinical Chemistry and Pharmacology, Faculty of Medicine, Lund University
ISBN (tryckt)	978-91-87449-57-4
Status	Published - 2013

Bibliografisk information

Defence details

Date: 2013-09-13
Time: 13:15
Place: Segerfalksalen, BMC, Sölvegatan 17, Lund

External reviewer(s)

Name: Theodorsson, Elvar
Title: [unknown]
Affiliation: Department of Clinical and Experimental Medicine, Division of Clinical Chemistry, Linköping University

---

Ämnesklassifikation (UKÄ)

Farmakologi och toxikologi
Läkemedelskemi

FN:s Globala mål

Denna forskningsoutput relaterar till följande Globala mål

Tillgång till dokument

Avhandling_130807_hw

3 Artikel i vetenskaplig tidskrift

Cystatin C properties crucial for uptake and inhibition of intracellular target enzymes.
Wallin, H., Abrahamson, M. & Ekström, U., 2013, I: Journal of Biological Chemistry. 288, 23, s. 17019-17029
Forskningsoutput: Tidskriftsbidrag › Artikel i vetenskaplig tidskrift › Peer review

Fil
196 Nedladdningar (Pure)
Cystatins - Extra- and Intracellular Cysteine Protease Inhibitors: High-level Secretion and Uptake of Cystatin C in Human Neuroblastoma Cells.
Wallin, H., Bjarnadottir, M., Vogel, L. K., Wasselius, J., Ekström, U. & Abrahamson, M., 2010, I: Biochimie. 92, s. 1625-1634
Forskningsoutput: Tidskriftsbidrag › Artikel i vetenskaplig tidskrift › Peer review
Internalization of cystatin C in human cell lines.
Ekström, U., Wallin, H., Lorenzo, J., Holmqvist, B., Abrahamson, M. & Avilés, F. X., 2008, I: The FEBS Journal. Aug 9, s. 4571-4582
Forskningsoutput: Tidskriftsbidrag › Artikel i vetenskaplig tidskrift › Peer review

Citera det här

@phdthesis{9baf9542ca69496f8a4bf2f1e49c8ea5,

title = "Cellular Uptake of Cystatin C. Subcellular localisation and intracellular effects of a secreted cysteine protease inhibitor",

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.",

keywords = "cathepsin, cell line, co-localisation, cystatin C, internalisation, legumain",

author = "Hanna Wallin",

note = "Defence details Date: 2013-09-13 Time: 13:15 Place: Segerfalksalen, BMC, S{\"o}lvegatan 17, Lund External reviewer(s) Name: Theodorsson, Elvar Title: [unknown] Affiliation: Department of Clinical and Experimental Medicine, Division of Clinical Chemistry, Link{\"o}ping University ---",

year = "2013",

language = "English",

isbn = "978-91-87449-57-4",

series = "Lund University Faculty of Medicine Doctoral Dissertation Series ",

publisher = "Division of Clinical Chemistry and Pharmacology, Faculty of Medicine, Lund University",

type = "Doctoral Thesis (compilation)",

school = "Division of Clinical Chemistry and Pharmacology",

}

TY - THES

T1 - Cellular Uptake of Cystatin C. Subcellular localisation and intracellular effects of a secreted cysteine protease inhibitor

AU - Wallin, Hanna

N1 - Defence details Date: 2013-09-13 Time: 13:15 Place: Segerfalksalen, BMC, Sölvegatan 17, Lund External reviewer(s) Name: Theodorsson, Elvar Title: [unknown] Affiliation: Department of Clinical and Experimental Medicine, Division of Clinical Chemistry, Linköping University ---

PY - 2013

Y1 - 2013

N2 - 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.

AB - 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.

KW - cathepsin

KW - cell line

KW - co-localisation

KW - cystatin C

KW - internalisation

KW - legumain

M3 - Doctoral Thesis (compilation)

SN - 978-91-87449-57-4

T3 - Lund University Faculty of Medicine Doctoral Dissertation Series

PB - Division of Clinical Chemistry and Pharmacology, Faculty of Medicine, Lund University

ER -

Cellular Uptake of Cystatin C. Subcellular localisation and intracellular effects of a secreted cysteine protease inhibitor

Sammanfattning

Bibliografisk information

Ämnesklassifikation (UKÄ)

FN:s Globala mål

Tillgång till dokument

Fingeravtryck

Forskningsoutput

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

Cystatins - Extra- and Intracellular Cysteine Protease Inhibitors: High-level Secretion and Uptake of Cystatin C in Human Neuroblastoma Cells.

Internalization of cystatin C in human cell lines.

Citera det här