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Affinity-based entrapment of the HER2 receptor in the endoplasmic reticulum using an affibody molecule
KTH, School of Biotechnology (BIO).
KTH, School of Biotechnology (BIO).
KTH, School of Biotechnology (BIO).ORCID iD: 0000-0001-7034-0850
KTH, School of Biotechnology (BIO).ORCID iD: 0000-0003-4214-6991
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2008 (English)In: Journal of immunological methods, ISSN 0022-1759, Vol. 338, 1-6 p.Article in journal (Refereed) Published
Abstract [en]

Interference with the export of cell surface receptors can be performed through co-expression of specific affinity molecules designed for entrapment in the endoplasmic reticulum during the export process. We describe the investigation of a small (6 kDa) non-immunoglobulin-based HER2 receptor binding affibody molecule (ZHER2:00477), for use in affinity mediated entrapment of the HER2 receptor in the ER. Constructs encoding ZHER2:00477 or a control affibody protein, with or without ER-retention peptide extensions (KDEL), were expressed in the HER2 over-expressing cell line SKOV-3. Intracellular expression of the full-length affibody constructs could be confirmed by probing cell extracts by Western blotting. Confocal immunofluorescence microscopy experiments showed extensive co-localization of the HER2 receptor and ZHER2:00477-KDEL in the ER, whereas the use of a KDEL-extended control affibody molecule resulted in distinct and separate signals from cell surface-localized HER2 receptor and ER-localized affibody protein. This indicated a capability of the ZHER2:00477-KDEL fusion protein to functionally interfere with the export process of HER2 receptor in a specific manner. Using flow cytometry and cell proliferation analyses, it could be shown that expression of the ZHER2:00477-KDEL fusion construct in the SKOV-3 cell line resulted both in a marked reduction in cell surface level of HER2 receptors and that the cell population doubling time was significantly increased. Expression of the ZHER2:00477-KDEL fusion protein in additional cell lines of different origin and with different expression levels of endogenous HER2 receptor compared to SKOV-3, also resulted in depletion of the cell surface levels of HER2 receptor. This indicated upon a general ability of the ZHER2:00477-KDEL fusion protein to functionally interfere with the export process of HER2.

Place, publisher, year, edition, pages
2008. Vol. 338, 1-6 p.
Keyword [en]
ErbB2; HER2; affibody molecule; endoplasmic reticulum; flow cytometry
National Category
Industrial Biotechnology
Identifiers
URN: urn:nbn:se:kth:diva-10075DOI: 10.1016/j.jim.2008.06.005ISI: 000259829600001PubMedID: 18671978Scopus ID: 2-s2.0-50949094371OAI: oai:DiVA.org:kth-10075DiVA: diva2:207399
Note
QC 20100813Available from: 2009-03-11 Created: 2009-03-11 Last updated: 2010-12-07Bibliographically approved
In thesis
1. Affinity protein based inhibition of cancer related signaling pathways
Open this publication in new window or tab >>Affinity protein based inhibition of cancer related signaling pathways
2009 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Dysregulation of protein activity, caused by alterations in protein sequence, expression, or localization, is associated with numerous diseases. In order to control the activity of harmful protein entities, affinity ligands such as proteins, oligonucleotides or small molecules can be engineered to specifically interact with them to modulate their function. In this thesis, non-immunoglobulin based affinity proteins known as affibody molecules are used to functionally inhibit proteins important for signaling through pathways that are overactive in different cancers.

 

In Paper I and Paper II, affibody molecules with high affinity for the receptor tyrosine kinases HER2 or EGFR are expressed in the secretory compartments of model cancer cell lines SKOV3 or A431 using a retrovirus-based gene delivery system. Equipping the affinity proteins with an ER retention tag, the affibody molecules together with their target protein are retained in the secretory compartments as shown by confocal fluorescence imaging. Flow cytometric analysis showed a 60 % or 80 % downregulation of surface located HER2 or EGFR in these cell lines, respectively. A significant decreased in proliferation rate of the cells was also observed, which for EGFR retention could be correlated with inhibition of phosphorylation in the kinase domain. In Paper III, novel affibody molecules interacting with the hormone binding site of the insulin growth factor-1 receptor were generated. One variant had high (1.2 nM) affinity for the receptor and could be used for immunofluorescence analysis and for receptor pull-out from cell lysates. Addition of this affibody molecule to MCF-7 cells had a dose dependent growth inhibitory effect on the cells. In Paper IV, novel affibody molecules against the intracellular oncoproteins H-Ras and Raf-1 were selected and characterized, and they proved to be specific for their target proteins. Mapping experiments showed that the affibody molecules selected against H-Ras interacted at over-lapping epitopes not affecting the interaction between Ras and Raf. In contrast, the predominant variant isolated during selection against Raf-1 could completely inhibit the Ras/Raf interaction in a real-time biospecific interaction analysis.

 

Taken together, the affibody molecules presented here and the strategies by which they are used to interfere with cancer related proteins and pathways may be valuable tools for further investigation of these systems and may possibly also be used to generate molecules suitable for cancer therapy.

Place, publisher, year, edition, pages
Stockholm: KTH, 2009. xii, 67 p.
Series
Trita-BIO-Report, ISSN 1654-2312 ; 2009:01
Keyword
Affibody, cancer, endoplasmic reticulum retention, EGFR, HER2, IGF-1R, Raf, Ras
National Category
Industrial Biotechnology
Identifiers
urn:nbn:se:kth:diva-10041 (URN)978-91-7415-232-6 (ISBN)
Public defence
2009-03-20, K1, KTH, Teknikringen 56, Stockholm, 10:00 (English)
Opponent
Supervisors
Note
QC 20100818Available from: 2009-03-11 Created: 2009-03-06 Last updated: 2010-08-18Bibliographically approved

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Publisher's full textPubMedScopushttp://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6T2Y-4T3CMF0-1&_user=4478132&_rdoc=1&_fmt=&_orig=search&_sort=d&view=c&_acct=C000034958&_version=1&_urlVersion=0&_userid=4478132&md5=3cd4484a10f861e7194f0391daf43137

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Lundberg, EmmaNygren, Per-ÅkeGräslund, Torbjörn

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