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Integration and topology of membrane proteins related to diseases
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
2015 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

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.

Place, publisher, year, edition, pages
Stockholm: Department of Biochemistry and Biophysics, Stockholm Univeristy , 2015. , 76 p.
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
URN: urn:nbn:se:su:diva-113397ISBN: 978-91-7649-094-5 (print)OAI: oai:DiVA.org:su-113397DiVA: diva2:784680
Public defence
2015-03-06, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B, Stockholm, 10:00 (English)
Opponent
Supervisors
Note

At the time of the doctoral defense paper 3 was unpublished and had a status as manuscript.

Available from: 2015-02-12 Created: 2015-01-29 Last updated: 2016-02-23Bibliographically approved
List of papers
1. Changed membrane integration and catalytic site conformation are two mechanisms behind the increased Aβ42/Aβ40 ratio by presenilin 1 familial Alzheimer-linked mutations.
Open this publication in new window or tab >>Changed membrane integration and catalytic site conformation are two mechanisms behind the increased Aβ42/Aβ40 ratio by presenilin 1 familial Alzheimer-linked mutations.
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2014 (English)In: FEBS Open Bio, E-ISSN 2211-5463, Vol. 4, 393-406 p.Article in journal (Refereed) Published
Abstract [en]

The enzyme complex γ-secretase generates amyloid β-peptide (Aβ), a 37-43-residue peptide associated with Alzheimer disease (AD). Mutations in presenilin 1 (PS1), the catalytical subunit of γ-secretase, result in familial AD (FAD). A unifying theme among FAD mutations is an alteration in the ratio Aβ species produced (the Aβ42/Aβ40 ratio), but the molecular mechanisms responsible remain elusive. In this report we have studied the impact of several different PS1 FAD mutations on the integration of selected PS1 transmembrane domains and on PS1 active site conformation, and whether any effects translate to a particular amyloid precursor protein (APP) processing phenotype. Most mutations studied caused an increase in the Aβ42/Aβ40 ratio, but via different mechanisms. The mutations that caused a particular large increase in the Aβ42/Aβ40 ratio did also display an impaired APP intracellular domain (AICD) formation and a lower total Aβ production. Interestingly, seven mutations close to the catalytic site caused a severely impaired integration of proximal transmembrane/hydrophobic sequences into the membrane. This structural defect did not correlate to a particular APP processing phenotype. Six selected FAD mutations, all of which exhibited different APP processing profiles and impact on PS1 transmembrane domain integration, were found to display an altered active site conformation. Combined, our data suggest that FAD mutations affect the PS1 structure and active site differently, resulting in several complex APP processing phenotypes, where the most aggressive mutations in terms of increased Aβ42/Aβ40 ratio are associated with a decrease in total γ-secretase activity.

Keyword
Alzheimer disease, γ-Secretase, Membrane integration, Amyloid b-peptide, Protein structure
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:su:diva-109355 (URN)10.1016/j.fob.2014.04.006 (DOI)000346278200050 ()24918054 (PubMedID)
Available from: 2014-11-19 Created: 2014-11-19 Last updated: 2017-12-05Bibliographically approved
2. The hepatitis B x antigen anti-apoptotic effector URG7 is localized to the endoplasmic reticulum membrane
Open this publication in new window or tab >>The hepatitis B x antigen anti-apoptotic effector URG7 is localized to the endoplasmic reticulum membrane
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2013 (English)In: FEBS Letters, ISSN 0014-5793, E-ISSN 1873-3468, Vol. 587, no 18, 3058-3062 p.Article in journal (Refereed) Published
Abstract [en]

Hepatitis B x antigen up-regulates the liver expression of URG7 that contributes to sustain chronic virus infection and to increase the risk for hepatocellular carcinoma by its anti-apoptotic activity. We have investigated the subcellular localization of URG7 expressed in HepG2 cells and determined its membrane topology by glycosylation mapping in vitro. The results demonstrate that URG7 is N-glycosylated and located to the endoplasmic reticulum membrane with an N-lumen-C-cytosol orientation. The results imply that the anti-apoptotic effect of URG7 could arise from the C-terminal cytosolic tail binding a pro-apoptotic signaling factor and retaining it to the endoplasmic reticulum membrane.

Keyword
URG7, Hepatitis B x antigen, Apoptosis, Cellular localization, Topology, Endoplasmic reticulum
National Category
Biochemistry and Molecular Biology Biophysics Cell Biology
Identifiers
urn:nbn:se:su:diva-94175 (URN)10.1016/j.febslet.2013.07.042 (DOI)000324033700026 ()
Note

AuthorCount:11;

Funding Agencies:

Swedish Cancer Society;  Swedish Foundation for Strategic Research 

Available from: 2013-10-01 Created: 2013-09-30 Last updated: 2017-12-06Bibliographically approved
3. Folding and Intramembraneous BRICHOS Binding of the Prosurfactant Protein C Transmembrane Segment
Open this publication in new window or tab >>Folding and Intramembraneous BRICHOS Binding of the Prosurfactant Protein C Transmembrane Segment
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2015 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 290, no 28, 17628-17641 p.Article in journal (Refereed) Published
Abstract [en]

Surfactant protein C (SP-C) is a novel amyloid protein found in the lung tissue of patients suffering from interstitial lung disease (ILD) due to mutations in the gene of the precursor protein pro-SP-C. SP-C is a small alpha-helical hydrophobic protein with an unusually high content of valine residues. SP-C is prone to convert into beta-sheet aggregates, forming amyloid fibrils. Nature's way of solving this folding problem is to include a BRICHOS domain in pro-SP-C, which functions as a chaperone for SP-C during biosynthesis. Mutations in the pro-SP-C BRICHOS domain or linker region lead to amyloid formation of the SP-C protein and ILD. In this study, we used an in vitro transcription/translation system to study translocon-mediated folding of the WT pro-SP-C poly-Val and a designed poly-Leu transmembrane (TM) segment in the endoplasmic reticulum (ER) membrane. Furthermore, to understand how the pro-SP-C BRICHOS domain present in the ER lumen can interact with the TM segment of pro-SP-C, we studied the membrane insertion properties of the recombinant form of the pro-SP-C BRICHOS domain and two ILD-associated mutants. The results show that the co-translational folding of the WT pro-SP-C TM segment is inefficient, that the BRICHOS domain inserts into superficial parts of fluid membranes, and that BRICHOS membrane insertion is promoted by poly-Val peptides present in the membrane. In contrast, one BRICHOS and one non-BRICHOS ILD-associated mutant could not insert into membranes. These findings support a chaperone function of the BRICHOS domain, possibly together with the linker region, during pro-SP-C biosynthesis in the ER.

National Category
Biological Sciences
Identifiers
urn:nbn:se:su:diva-119291 (URN)10.1074/jbc.M114.630343 (DOI)000357730900052 ()
Available from: 2015-08-04 Created: 2015-08-03 Last updated: 2017-12-04Bibliographically approved
4. Live-cell topology assessment of URG7, MRP6(102) and SP-C using glycosylatable green fluorescent protein in mammalian cells
Open this publication in new window or tab >>Live-cell topology assessment of URG7, MRP6(102) and SP-C using glycosylatable green fluorescent protein in mammalian cells
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2014 (English)In: Biochemical and Biophysical Research Communications - BBRC, ISSN 0006-291X, E-ISSN 1090-2104, Vol. 450, no 4, 1587-1592 p.Article in journal (Refereed) Published
Abstract [en]

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.

Keyword
Endoplasmic reticulum, Membrane protein topology, Protein orientation, GFP, N-linked glycosylation
National Category
Biological Sciences
Identifiers
urn:nbn:se:su:diva-107994 (URN)10.1016/j.bbrc.2014.07.046 (DOI)000341338100058 ()
Note

AuthorCount:7;

Available from: 2014-10-06 Created: 2014-10-06 Last updated: 2017-12-05Bibliographically approved

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