| 21. |
- 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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| 22. |
- 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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| 23. |
- Kronqvist, Nina, et al.
(författare)
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Combining phage and staphylococcal surface display for generation of ErbB3-specific Affibody molecules
- 2011
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Ingår i: Protein Engineering Design & Selection. - : Oxford University Press (OUP). - 1741-0126 .- 1741-0134. ; 24:4, s. 385-396
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Tidskriftsartikel (refereegranskat)abstract
- Emerging evidence suggests that the catalytically inactive ErbB3 (HER3) protein plays a fundamental role in normal tyrosine kinase receptor signaling as well as in aberrant functioning of these signaling pathways, resulting in several forms of human cancers. ErbB3 has recently also been implicated in resistance to ErbB2-targeting therapies. Here we report the generation of high-affinity ErbB3-specific Affibody molecules intended for future molecular imaging and biotherapeutic applications. Using a high-complexity phage-displayed Affibody library, a number of ErbB3 binders were isolated and specific cell-binding activity was demonstrated in immunofluorescence microscopic studies. Subsequently, a second-generation library was constructed based on sequences of the candidates from the phage display selection. By exploiting the sensitive affinity discrimination capacity of a novel bacterial surface display technology, the affinity of candidate Affibody molecules was further increased down to subnanomolar affinity. In summary, the demonstrated specific targeting of native ErbB3 receptor on human cancer cell lines as well as competition with the heregulin/ErbB3 interaction indicates that these novel biological agents may become useful tools for diagnostic and therapeutic targeting of ErbB3-expressing cancers. Our studies also highlight the powerful approach of combining the advantages of different display technologies for generation of functional high-affinity protein-based binders. Potential future applications, such as radionuclide-based diagnosis and treatment of human cancers are discussed.
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| 24. |
- Lara, Patricia, et al.
(författare)
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Murine astrotactins 1 and 2 have a similar membrane topology and mature via endoproteolytic cleavage catalyzed by a signal peptidase
- 2019
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Ingår i: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 294:12, s. 4538-4545
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Tidskriftsartikel (refereegranskat)abstract
- Astrotactin 1 (Astn1) and Astn2 are membrane proteins that function in glial-guided migration, receptor trafficking, and synaptic plasticity in the brain as well as in planar polarity pathways in the skin. Here we used glycosylation mapping and protease protection approaches to map the topologies of mouse Astn1 and Astn2 in rough microsomal membranes and found that Astn2 has a cleaved N-terminal signal peptide, an N-terminal domain located in the lumen of the rough microsomal membranes (topologically equivalent to the extracellular surface in cells), two transmembrane helices, and a large C-terminal lumenal domain. We also found that Astn1 has the same topology as Astn2, but we did not observe any evidence of signal peptide cleavage in Astn1. Both Astn1 and Astn2 mature through endoproteolytic cleavage in the second transmembrane helix; importantly, we identified the endoprotease responsible for the maturation of Astn1 and Astn2 as the endoplasmic reticulum signal peptidase. Differences in the degree of Astn1 and Astn2 maturation possibly contribute to the higher levels of the C-terminal domain of Astn1 detected on neuronal membranes of the central nervous system. These differences may also explain the distinct cellular functions of Astn1 and Astn2, such as in membrane adhesion, receptor trafficking, and planar polarity signaling.
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| 25. |
- Lara, Patricia, et al.
(författare)
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Refined topology model of the STT3/Stt3 protein subunit of the oligosaccharyltransferase complex
- 2017
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Ingår i: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 292:27, s. 11349-11360
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Tidskriftsartikel (refereegranskat)abstract
- The oligosaccharyltransferase complex, localized in the endoplasmic reticulum (ER) of eukaryotic cells, is responsible for the N-linked glycosylation of numerous protein substrates. The membrane protein STT3 is a highly conserved part of the oligosaccharyltransferase and likely contains the active site of the complex. However, understanding the catalytic determinants of this system has been challenging, in part because of a discrepancy in the structural topology of the bacterial versus eukaryotic proteins and incomplete information about the mechanism of membrane integration. Here, we use a glycosylation mapping approach to investigate these questions. We measured the membrane integration efficiency of the mouse STT3-A and yeast Stt3p transmembrane domains (TMDs) and report a refined topology of the N-terminal half of the mouse STT3-A. Our results show that most of the STT3 TMDs are well inserted into the ER membrane on their own or in the presence of the natural flanking residues. However, for the mouse STT3-A hydrophobic domains 4 and 6 and yeast Stt3p domains 2, 3a, 3c, and 6 we measured reduced insertion efficiency into the ER membrane. Furthermore, we mapped the first half of the STT3-A protein, finding two extra hydrophobic domains between the third and the fourthTMD. This result indicates that the eukaryotic STT3 has 13 transmembrane domains, consistent with the structure of the bacterial homolog of STT3 and setting the stage for future combined efforts to interrogate this fascinating system.
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| 26. |
- Lara Vasquez, Patricia, 1982-
(författare)
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Integration and topology of membrane proteins related to diseases
- 2015
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Membranes are boundaries that separate the cell from the external environment. Membrane proteins can function as e.g. receptors and channels, allowing cells to communicate with the exterior and molecules to pass through the membrane. The biogenesis of membrane proteins involves a protein-conducting channel that aids the hydrophobic segments to partition into the membrane and translocate the hydrophilic loops. Membrane proteins need to fold to its native conformation including post-translational modifications and assembly with other proteins and/or cofactors. If this regulated pathway goes wrong the degradation machinery degrades the protein. If the system is failing can result in serious disorders. The main focus in this thesis is membrane proteins associated to diseases.We have studied mutations in the gene of presenilin 1, which is involved in Alzheimer’s disease. We found that some mutations affect the structure and other the function of the PS1. URG7 is an unknown protein associated with liver cancer. We suggest it is localized and targeted to the ER membrane, having an NoutCin topology. SP-C is important for our lungs to function. Mutations can cause the protein to aggregate. We have studied the highly Val-rich transmembrane segment (poly-Val) and its analogue (poly-Leu) and show that poly-Leu folds into a more compact conformation than poly-Val. We show that the C-terminal chaperon-like BRICHOS domain interacts with the ER membrane, suggesting an involvement in poly-Val folding. We have also confirmed the topology of URG7, MRP6 and SP-C poly-Val/Leu using gGFP that is fused to the C-terminal of the protein.
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| 27. |
- Lee, Hunsang, et al.
(författare)
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Live-cell topology assessment of URG7, MRP6(102) and SP-C using glycosylatable green fluorescent protein in mammalian cells
- 2014
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Ingår i: Biochemical and Biophysical Research Communications - BBRC. - : Elsevier BV. - 0006-291X .- 1090-2104. ; 450:4, s. 1587-1592
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Tidskriftsartikel (refereegranskat)abstract
- Experimental tools to determine membrane topology of a protein are rather limited in higher eukaryotic organisms. Here, we report the use of glycosylatable GFP (gGFP) as a sensitive and versatile membrane topology reporter in mammalian cells. gGFP selectively loses its fluorescence upon N-linked glycosylation in the ER lumen. Thus, positive fluorescence signal assigns location of gGFP to the cytosol whereas no fluorescence signal and a glycosylated status of gGFP map the location of gGFP to the ER lumen. By using mammalian gGFP, the membrane topology of disease-associated membrane proteins, URG7, MRP6(102), SP-C(Val) and SP-C(Leu) was confirmed. URG7 is partially targeted to the ER, and inserted in C-in, form. MRP6(102) and SP-C(Leu/Val) are inserted into the membrane in C-out form. A minor population of untargeted SP-C is removed by proteasome dependent quality control system.
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| 28. |
- Lerch-Bader, Mirjam, et al.
(författare)
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Contribution of positively charged flanking residues to the insertion of transmembrane helices into the endoplasmic reticulum
- 2008
-
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. ; 105:11, s. 4127-4132
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Tidskriftsartikel (refereegranskat)abstract
- Positively charged residues located near the cytoplasmic end of hydrophobic segments in membrane proteins promote membrane insertion and formation of transmembrane alpha-helices. A quantitative understanding of this effect has been lacking, however. Here, using an in vitro transcription-translation system to study the insertion of model hydrophobic segments into dog pancreatic rough microsomes, we show that a single Lys or Arg residue typically contributes approximately -0.5 kcal/mol to the apparent free energy of membrane insertion (DeltaG(app)) when placed near the cytoplasmic end of a hydrophobic segment and that stretches of 3-6 Lys residues can contribute significantly to DeltaG(app) from a distance of up to approximately 13 residues away.
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| 29. |
- Lundin, Carolina, et al.
(författare)
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Membrane topology of the Drosophila OR83b odorant receptor
- 2007
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Ingår i: FEBS Letters. - : Wiley. - 0014-5793 .- 1873-3468. ; 581:29, s. 5601-5604
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Tidskriftsartikel (refereegranskat)abstract
- By analogy to mammals, odorant receptors (ORs) in insects, such as Drosophila melanogaster, have long been thought to belong to the G-protein coupled receptor (GPCR) superfamily. However, recent work has cast doubt on this assumption and has tentatively suggested an inverted topology compared to the canonical N(out) - C(in) 7 transmembrane (TM) GPCR topology, at least for some Drosophila ORs. Here, we report a detailed topology mapping of the Drosophila OR83b receptor using engineered glycosylation sites as topology markers. Our results are inconsistent with a classical GPCR topology and show that OR83b has an intracellular N-terminus, an extracellular C-terminus, and 7TM helices.
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| 30. |
- Lundin, Carolina, et al.
(författare)
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Membrane topology of the human seipin protein
- 2006
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Ingår i: FEBS Letters. - : Wiley. - 0014-5793 .- 1873-3468. ; 580:9, s. 2281-2284
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Tidskriftsartikel (refereegranskat)abstract
- The Berardinelli-Seip congenital lipodystrophy type 2 (BSCL2) gene encodes an integral membrane protein, called seipin, of unknown function localized to the endoplasmic reticulum of eukaryotic cells. Seipin is associated with the heterogeneous genetic disease BSCL2, and mutations in an N-glycosylation motif links the protein to two other disorders, autosomal-dominant distal hereditary motor neuropathy type V and Silver syndrome. Here, we report a topological study of seipin using an in vitro topology mapping assay. Our results suggest that the predominant form of seipin is 462 residues long and has an N-cyt-C-cyt orientation with a long luminal loop between the two transmembrane helices.
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