| 1. |
- Orlova, Anna, et al.
(författare)
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Tumor imaging using a picomolar affinity HER2 binding affibody molecule
- 2006
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Ingår i: Cancer Research. - 0008-5472 .- 1538-7445. ; 66:8, s. 4339-48
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Tidskriftsartikel (refereegranskat)abstract
- The detection of cell-bound proteins that are produced due to aberrant gene expression in malignant tumors can provide important diagnostic information influencing patient management. The use of small radiolabeled targeting proteins would enable high-contrast radionuclide imaging of cancers expressing such antigens if adequate binding affinity and specificity could be provided. Here, we describe a HER2-specific 6 kDa Affibody molecule (hereinafter denoted Affibody molecule) with 22 pmol/L affinity that can be used for the visualization of HER2 expression in tumors in vivo using gamma camera. A library for affinity maturation was constructed by re-randomization of relevant positions identified after the alignment of first-generation variants of nanomolar affinity (50 nmol/L). One selected Affibody molecule, Z(HER2:342) showed a >2,200-fold increase in affinity achieved through a single-library affinity maturation step. When radioiodinated, the affinity-matured Affibody molecule showed clear, high-contrast visualization of HER2-expressing xenografts in mice as early as 6 hours post-injection. The tumor uptake at 4 hours post-injection was improved 4-fold (due to increased affinity) with 9% of the injected dose per gram of tissue in the tumor. Affibody molecules represent a new class of affinity molecules that can provide small sized, high affinity cancer-specific ligands, which may be well suited for tumor imaging.
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| 2. |
- Boekel, Carolina, 1977-
(författare)
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Integration and topology of membrane proteins
- 2009
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Membrane proteins comprise around 20-30% of most proteomes. They play important roles in most biochemical pathways. All receptors and ion channels are membrane proteins, which make them attractive targets for drug design. Membrane proteins insert and fold co-translationally into the endoplasmic reticular membrane of eukaryotic cells. The protein-conducting channel that inserts the protein into the membrane is called Sec61 translocon, which is a hetero-oligomeric channel that allows transmembrane segments to insert laterally into the lipid bilayer. The focus of this thesis is how the translocon recognizes the transmembrane helices and integrates them into the membrane.We have investigated the sequence requirements for the translocon-mediated integration of a transmembrane α-helix into the ER by challenging the Sec61 translocon with designed polypeptide segments in an in vitro expression system that allows a quantitative assessment of membrane insertion efficiency. Our studies suggest that helices might interact with each other already during the membrane-insertion step, possibly forming helical hairpins that partition into the membrane as a single unit. Further, the insertion efficiency for Nin-Cout vs. Nout-Cin transmembrane helices and the integration efficiency of Alzheimer’s Aβ-peptide fragments has been investigated.Finally, detailed topology mapping was performed on two biologically interesting proteins with unknown topology, the human seipin protein and Drosophila melanogaster odorant receptor OR83b.
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| 3. |
- Enquist, Karl, et al.
(författare)
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Membrane-integration characteristics of two ABC transporters, CFTR and P-glycoprotein
- 2009
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Ingår i: Journal of Molecular Biology. - : Elsevier BV. - 0022-2836 .- 1089-8638. ; 387:5, s. 1153-1164
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Tidskriftsartikel (refereegranskat)abstract
- To what extent do corresponding transmembrane helices in related integral membrane proteins have different membrane-insertion characteristics? Here, we compare, side-by-side, the membrane insertion characteristics of the 12 transmembrane helices in the adenosine triphosphate-binding cassette (ABC) transporters, P-glycoprotein (P-gp) and the cystic fibrosis transmembrane conductance regulator (CFTR). Our results show that 10 of the 12 CFTR transmembrane segments can insert independently into the ER membrane. In contrast, only three of the P-gp transmembrane segments are independently stable in the membrane, while the majority depend on the presence of neighboring loops and/or transmembrane segments for efficient insertion. Membrane-insertion characteristics can thus vary widely between related proteins.
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| 4. |
- Friedman, Mikaela, et al.
(författare)
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Directed evolution to low nanomolar affinity of a tumor-targeting epidermal growth factor receptor-binding affibody molecule
- 2008
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Ingår i: Journal of Molecular Biology. - : Elsevier BV. - 0022-2836 .- 1089-8638. ; 376:5, s. 1388-1402
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Tidskriftsartikel (refereegranskat)abstract
- The epidermal growth factor receptor 1 (EGFR) is overexpressed in various malignancies and is associated with a poor patient prognosis. A small, receptor-specific, high-affinity imaging agent would be a useful tool in diagnosing malignant tumors and in deciding upon treatment and assessing the response to treatment. We describe here the affinity maturation procedure for the generation of Affibody molecules binding with high affinity and specificity to EGFR. A library for affinity maturation was constructed by rerandomization of selected positions after the alignment of first-generation binding variants. New binders were selected with phage display technology, using a single oligonucleotide in a single-library effort, and the best second-generation binders had an approximately 30-fold improvement in affinity (K(d)=5-10 nM) for the soluble extracellular domain of EGFR in biospecific interaction analysis using Biacore. The dissociation equilibrium constant, K(d), was also determined for the Affibody with highest affinity using EGFR-expressing A431 cells in flow cytometric analysis (K(d)=2.8 nM). A retained high specificity for EGFR was verified by a dot blot assay showing staining only of EGFR proteins among a panel of serum proteins and other EGFR family member proteins (HER2, HER3, and HER4). The EGFR-binding Affibody molecules were radiolabeled with indium-111, showing specific binding to EGFR-expressing A431 cells and successful targeting of the A431 tumor xenografts with 4-6% injected activity per gram accumulated in the tumor 4 h postinjection.
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| 5. |
- Friedman, Mikaela, et al.
(författare)
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Phage display selection of Affibody molecules with specific binding to the extracellular domain of the epidermal growth factor receptor
- 2007
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Ingår i: Protein Engineering Design & Selection. - : Oxford University Press (OUP). - 1741-0126 .- 1741-0134. ; 20:4, s. 189-199
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Tidskriftsartikel (refereegranskat)abstract
- Affibody molecules specific for the epidermal growth factor receptor (EGFR) have been selected by phage display technology from a combinatorial protein library based on the 58-residue, protein A-derived Z domain. EGFR is overexpressed in various malignancies and is frequently associated with poor patient prognosis, and the information provided by targeting this receptor could facilitate both patient diagnostics and treatment. Three selected Affibody variants were shown to selectively bind to the extracellular domain of EGFR (EGFR-ECD). Kinetic biosensor analysis revealed that the three monomeric Affibody molecules bound with similar affinity, ranging from 130 to 185 nM. Head-to-tail dimers of the Affibody molecules were compared for their binding to recombinant EGFR-ECD in biosensor analysis and in human epithelial cancer A431 cells. Although the dimeric Affibody variants were found to bind in a range of 25-50 nM affinities in biosensor analysis, they were found to be low nanomolar binders in the cellular assays. Competition assays using radiolabeled Affibody dimers confirmed specific EGFR-binding and demonstrated that the three Affibody molecules competed for the same epitope. Immunofluorescence microscopy demonstrated that the selected Affibody dimers were initially binding to EGFR at the cell surface of A431, and confocal microscopy analysis showed that the Affibody dimers could thereafter be internalized. The potential use of the described Affibody molecules as targeting agents for radionuclide based imaging applications in various carcinomas is discussed.
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| 6. |
- Hessa, Tara, et al.
(författare)
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Molecular code for transmembrane-helix recognition by the Sec61 translocon
- 2007
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Ingår i: Nature. - : Springer Science and Business Media LLC. - 0028-0836 .- 1476-4687. ; 450:7172, s. 1026-1030
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Tidskriftsartikel (refereegranskat)abstract
- Transmembrane alpha-helices in integral membrane proteins are recognized co-translationally and inserted into the membrane of the endoplasmic reticulum by the Sec61 translocon. A full quantitative description of this phenomenon, linking amino acid sequence to membrane insertion efficiency, is still lacking. Here, using in vitro translation of a model protein in the presence of dog pancreas rough microsomes to analyse a large number of systematically designed hydrophobic segments, we present a quantitative analysis of the position- dependent contribution of all 20 amino acids to membrane insertion efficiency, as well as of the effects of transmembrane segment length and flanking amino acids. The emerging picture of translocon- mediated transmembrane helix assembly is simple, with the critical sequence characteristics mirroring the physical properties of the lipid bilayer.
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| 7. |
- Hessa, Tara, et al.
(författare)
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Recognition of transmembrane helices by the endoplasmic reticulum translocon
- 2005
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Ingår i: Nature. - : Springer Science and Business Media LLC. - 0028-0836 .- 1476-4687. ; 433:7024, s. 377-381
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Tidskriftsartikel (refereegranskat)abstract
- Membrane proteins depend on complex translocation machineries for insertion into target membranes. Although it has long been known that an abundance of nonpolar residues in transmembrane helices is the principal criterion for membrane insertion, the specific sequence-coding for transmembrane helices has not been identified. By challenging the endoplasmic reticulum Sec61 translocon with an extensive set of designed polypeptide segments, we have determined the basic features of this code, including a 'biological' hydrophobicity scale. We find that membrane insertion depends strongly on the position of polar residues within transmembrane segments, adding a new dimension to the problem of predicting transmembrane helices from amino acid sequences. Our results indicate that direct protein - lipid interactions are critical during translocon-mediated membrane insertion.
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| 8. |
- Jaud, Simon, et al.
(författare)
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Insertion of short transmembrane helices by the Sec61 translocon
- 2009
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Ingår i: Proceedings of the National Academy of Sciences of the United States of America. - : Proceedings of the National Academy of Sciences. - 0027-8424 .- 1091-6490. ; 106:28, s. 11588-11593
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Tidskriftsartikel (refereegranskat)abstract
- The insertion efficiency of transmembrane (TM) helices by the Sec61 translocon depends on helix amino acid composition, the positions of the amino acids within the helix, and helix length. We have used an in vitro expression system to examine systematically the insertion efficiency of short polyleucine segments (L(n), n = 4 ... 12) flanked at either end by 4-residue sequences of the form XXPX-L(n)-XPXX with X = G, N, D, or K. Except for X = K, insertion efficiency (p) is <10% for n < 8, but rises steeply to 100% for n = 12. For X = K, p is already close to 100% for n = 10. A similar pattern is observed for synthetic peptides incorporated into oriented phospholipid bilayer arrays, consistent with the idea that recognition of TM segments by the translocon critically involves physical partitioning of nascent peptide chains into the lipid bilayer. Molecular dynamics simulations suggest that insertion efficiency is determined primarily by the energetic cost of distorting the bilayer in the vicinity of the TM helix. Very short lysine-flanked leucine segments can reduce the energetic cost by extensive hydrogen bonding with water and lipid phosphate groups (snorkeling) and by partial unfolding.
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| 9. |
- Karamyshev, Andrey L., et al.
(författare)
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Mapping the interaction of the STT3 subunit of the oligosaccharyl transferase complex with nascent polypeptide chains
- 2005
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Ingår i: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 280:49, s. 40489-40493
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Tidskriftsartikel (refereegranskat)abstract
- Many secretory and membrane proteins are N-glycosylated by the oligosaccharyl transferase complex during their translocation across the endoplasmic reticulum membrane. Several experimental observations suggest that the highly conserved STT3 subunit contains the active site of the oligosaccharyl transferase. Here, we report a detailed study of the interaction between the active site of the STT3 protein and nascent polypeptide chains using an in vitro photocrosslinking technique. Our results show that the addition of a glycan moiety in a stretch of similar to 15 residues surrounding a QK*T cross-linking site impairs the interaction between the nascent chain and STT3.
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| 10. |
- Kim, Hyun, et al.
(författare)
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Membrane topology of the STT3 subunit of the oligosaccharyl transferase complex
- 2005
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Ingår i: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 280:21, s. 20261-20267
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Tidskriftsartikel (refereegranskat)abstract
- The highly conserved membrane protein STT3 is part of the oligosaccharyl transferase complex in the endoplasmic reticulum of eukaryotic cells. Various experimental observations strongly suggest that STT3 contains the active site of the complex. Here, we report a detailed topology study of STT3 from two different organisms, Saccharomyces cerevisiae and mouse, using in vivo and in vitro topology mapping assays. Our results suggest that STT3 has 11 transmembrane helices and an overall N-cyt-C-lum orientation.
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