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  • 1.
    Andersson, Annika
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Kudva, Renuka
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Magoulopoulou, Anastasia
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Lejarre, Quentin
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Lara, Patricia
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Xu, Peibo
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Goel, Suchi
    Pissi, Jennifer
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Ru, Xing
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Hessa, Tara
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Wahlgren, Mats
    von Heijne, Gunnar
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Karolinska Institutet, Sweden.
    Nilsson, IngMarie
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Tellgren-Roth, Åsa
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Membrane integration and topology of RIFIN and STEVOR proteins of the Plasmodium falciparum parasite2020In: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 287, no 13, p. 2744-2762Article in journal (Refereed)
    Abstract [en]

    The malarial parasite Plasmodium exports its own proteins to the cell surfaces of red blood cells (RBCs) during infection. Examples of exported proteins include members of the repetitive interspersed family (RIFIN) and subtelomeric variable open reading frame (STEVOR) family of proteins from Plasmodium falciparum. The presence of these parasite-derived proteins on surfaces of infected RBCs triggers the adhesion of infected cells to uninfected cells (rosetting) and to the vascular endothelium potentially obstructing blood flow. While there is a fair amount of information on the localization of these proteins on the cell surfaces of RBCs, less is known about how they can be exported to the membrane and the topologies they can adopt during the process. The first step of export is plausibly the cotranslational insertion of proteins into the endoplasmic reticulum (ER) of the parasite, and here, we investigate the insertion of three RIFIN and two STEVOR proteins into the ER membrane. We employ a well-established experimental system that uses N-linked glycosylation of sites within the protein as a measure to assess the extent of membrane insertion and the topology it assumes when inserted into the ER membrane. Our results indicate that for all the proteins tested, transmembranes (TMs) 1 and 3 integrate into the membrane, so that the protein assumes an overall topology of Ncyt-Ccyt. We also show that the segment predicted to be TM2 for each of the proteins likely does not reside in the membrane, but is translocated to the lumen.

  • 2. Cuviello, Flavia
    et al.
    Tellgren-Roth, Åsa
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Lara, Patricia
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Ruud Selin, Frida
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Monné, Magnus
    Bisaccia, Faustino
    Nilsson, IngMarie
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Ostuni, Angela
    Membrane insertion and topology of the amino-terminal domain TMD0 of multidrug-resistance associated protein 6 (MRP6)2015In: FEBS Letters, ISSN 0014-5793, E-ISSN 1873-3468, Vol. 589, no 24, p. 3921-3928Article in journal (Refereed)
    Abstract [en]

    The function of the ATP-binding cassette transporter MRP6 is unknown but mutations in its gene cause pseudoxanthoma elasticum. We have investigated the membrane topology of the N-terminal transmembrane domain TMD0 of MRP6 and the membrane integration and orientation propensities of its transmembrane segments (TMs) by glycosylation mapping. Results demonstrate that TMD0 has five TMs, an Nout-Cin topology and that the less hydrophobic TMs have strong preference for their orientation in the membrane that affects the neighboring TMs. Two disease-causing mutations changing the number of positive charges in the loops of TMD0 did not affect the membrane insertion efficiencies of the adjacent TMs.

  • 3. Gadalla, Salah-Eldin
    et al.
    Öjemalm, Karin
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Lara Vasquez, Patricia
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Nilsson, IngMarie
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Ericsson, Christer
    Zhao, Jian
    Nister, Monica
    EpCAM associates with endoplasmic reticulum aminopeptidase 2 (ERAP2) in breast cancer cells2013In: Biochemical and Biophysical Research Communications - BBRC, ISSN 0006-291X, E-ISSN 1090-2104, Vol. 439, no 2, p. 203-208Article in journal (Refereed)
    Abstract [en]

    Epithelial cell adhesion molecule (EpCAM) is an epithelial and cancer cell marker and there is a cumulative and growing evidence of its signaling role. Its importance has been recognized as part of the breast cancer stem cell phenotype, the tumorigenic breast cancer stem cell is EpCAM(+). In spite of its complex functions in normal cell development and cancer, relatively little is known about EpCAM-interacting proteins. We used breast cancer cell lines and performed EpCAM co-immunoprecipitation followed by mass spectrometry in search for novel potentially interacting proteins. The endoplasmic reticulum aminopeptidase 2 (ERAP2) was found to co-precipitate with EpCAM and to co-localize in the cytoplasm/ER and the plasma membrane. ERAP2 is a proteolytic enzyme set in the endoplasmic reticulum (ER) where it plays a central role in the trimming of peptides for presentation by MHC class I molecules. Expression of EpCAM and ERAP2 in vitro in the presence of dog pancreas rough microsomes (ER vesicles) confirmed N-linked glycosylation, processing in ER and the size of EpCAM. The association between ERAP2 and EpCAM is a unique and novel finding that provides new ideas on EpCAM processing and on how antigen presentation may be regulated in cancer.

  • 4. Goel, Suchi
    et al.
    Palmkvist, Mia
    Moll, Kirsten
    Joannin, Nicolas
    Lara, Patricia
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Akhouri, Reetesh R.
    Moradi, Nasim
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Öjemalm, Karin
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Westman, Mattias
    Angeletti, Davide
    Kjellin, Hanna
    Lehtio, Janne
    Blixt, Ola
    Ideström, Lars
    Gahmberg, Carl G.
    Storry, Jill R.
    Hult, Annika K.
    Olsson, Martin L.
    von Heijne, Gunnar
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Nilsson, IngMarie
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Wahlgren, Mats
    RIFINs are adhesins implicated in severe Plasmodium falciparum malaria2015In: Nature Medicine, ISSN 1078-8956, E-ISSN 1546-170X, Vol. 21, no 4, p. 314-317Article in journal (Refereed)
    Abstract [en]

    Rosetting is a virulent Plasmodium falciparum phenomenon associated with severe malaria. Here we demonstrate that P. falciparum-encoded repetitive interspersed families of polypeptides (RIFINs) are expressed on the surface of infected red blood cells (iRBCs), bind to RBCs-preferentially of blood group A-to form large rosettes and mediate microvascular binding of iRBCs. We suggest that RIFINs have a fundamental role in the development of severe malaria and thereby contribute to the varying global distribution of ABO blood groups in the human population.

  • 5.
    Lara, Patricia
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Tellgren-Roth, Åsa
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Behesti, Hourinaz
    Horn, Zachi
    Schiller, Nina
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Enquist, Karl
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Cammenberg, Malin
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Liljenström, Amanda
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Hatten, Mary E.
    von Heijne, Gunnar
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Nilsson, IngMarie
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Murine astrotactins 1 and 2 have a similar membrane topology and mature via endoproteolytic cleavage catalyzed by a signal peptidase2019In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 294, no 12, p. 4538-4545Article in journal (Refereed)
    Abstract [en]

    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.

  • 6.
    Lara, Patricia
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Öjemalm, Karin
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Reithinger, Johannes
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Holgado, Aurora
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Maojun, You
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Hammed, Abdessalem
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Mattle, Daniel
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Kim, Hyun
    Nilsson, IngMarie
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Refined topology model of the STT3/Stt3 protein subunit of the oligosaccharyltransferase complex2017In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 292, no 27, p. 11349-11360Article in journal (Refereed)
    Abstract [en]

    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.

  • 7.
    Lara Vasquez, Patricia
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Integration and topology of membrane proteins related to diseases2015Doctoral 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.

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  • 8. Lee, Hunsang
    et al.
    Lara, Patricia
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Ostuni, Angela
    Presto, Jenny
    Johansson, Janne
    Nilsson, IngMarie
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Kim, Hyun
    Live-cell topology assessment of URG7, MRP6(102) and SP-C using glycosylatable green fluorescent protein in mammalian cells2014In: Biochemical and Biophysical Research Communications - BBRC, ISSN 0006-291X, E-ISSN 1090-2104, Vol. 450, no 4, p. 1587-1592Article in journal (Refereed)
    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.

  • 9.
    Nilsson, IngMarie
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Lara, Patricia
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Hessa, Tara
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Johnson, Arthur E.
    von Heijne, Gunnar
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Karamyshev, Andrey L.
    The Code for Directing Proteins for Trans location across ER Membrane: SRP Cotranslationally Recognizes Specific Features of a Signal Sequence2015In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 427, no 6, p. 1191-1201Article in journal (Refereed)
    Abstract [en]

    The signal recognition particle (SRP) cotranslationally recognizes signal sequences of secretory proteins and targets ribosome-nascent chain complexes to the SRP receptor in the endoplasmic reticulum membrane, initiating translocation of the nascent chain through the Sec61 translocon. Although signal sequences do not have homology, they have similar structural regions: a positively charged N-terminus, a hydrophobic core and a more polar C-terminal region that contains the cleavage site for the signal peptidase. Here, we have used site-specific photocrosslinking to study SRP signal sequence interactions. A photoreactive probe was incorporated into the middle of wild-type or mutated signal sequences of the secretory protein preprolactin by in vitro translation of mRNAs containing an amber-stop codon in the signal peptide in the presence of the N-epsilon-(5-azido-2 nitrobenzoyl)-Lys-tRNA(amb) amber suppressor. A homogeneous population of SRP ribosome-nascent chain complexes was obtained by the use of truncated mRNAs in translations performed in the presence of purified canine SRP. Quantitative analysis of the photoadducts revealed that charged residues at the N-terminus of the signal sequence or in the early part of the mature protein have only a mild effect on the SRP signal sequence association. However, deletions of amino acid residues in the hydrophobic portion of the signal sequence severely affect SRP binding. The photocrosslinking data correlate with targeting efficiency and translocation across the membrane. Thus, the hydrophobic core of the signal sequence is primarily responsible for its recognition and binding by SRP, while positive charges fine-tune the SRP signal sequence affinity and targeting to the translocon.

  • 10. Ostuni, A.
    et al.
    Lara, Patricia
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Armentano, M. F.
    Miglionico, R.
    Salvia, A. M.
    Monnich, M.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Carmosino, M.
    Lasorsa, F. M.
    Monne, M.
    Nilsson, IngMarie
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Bisaccia, F.
    The hepatitis B x antigen anti-apoptotic effector URG7 is localized to the endoplasmic reticulum membrane2013In: FEBS Letters, ISSN 0014-5793, E-ISSN 1873-3468, Vol. 587, no 18, p. 3058-3062Article in journal (Refereed)
    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.

  • 11. Renault, Hugues
    et al.
    De Marothy, Minttu
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Jonasson, Gabriella
    Lara, Patricia
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Nelson, David R.
    Nilsson, IngMarie
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Andre, Francois
    von Heijne, Gunnar
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Werck-Reichhart, Danièle
    Gene Duplication Leads to Altered Membrane Topology of a Cytochrome P450 Enzyme in Seed Plants2017In: Molecular biology and evolution, ISSN 0737-4038, E-ISSN 1537-1719, Vol. 34, no 8, p. 2041-2056Article in journal (Refereed)
    Abstract [en]

    Evolution of the phenolic metabolism was critical for the transition of plants from water to land. A cytochrome P450, CYP73, with cinnamate 4-hydroxylase (C4H) activity, catalyzes the first plant-specific and rate-limiting step in this pathway. The CYP73 gene is absent from green algae, and first detected in bryophytes. A CYP73 duplication occurred in the ancestor of seed plants and was retained in Taxaceae and most angiosperms. In spite of a clear divergence in primary sequence, both paralogs can fulfill comparable cinnamate hydroxylase roles both in vitro and in vivo. One of them seems dedicated to the biosynthesis of lignin precursors. Its N-terminus forms a single membrane spanning helix and its properties and length are highly constrained. The second is characterized by an elongated and variable N-terminus, reminiscent of ancestral CYP73s. Using as proxies the Brachypodium distachyon proteins, we show that the elongation of the N-terminus does not result in an altered subcellular localization, but in a distinct membrane topology. Insertion in the membrane of endoplasmic reticulum via a double-spanning open hairpin structure allows reorientation to the lumen of the catalytic domain of the protein. In agreement with participation to a different functional unit and supramolecular organization, the protein displays modified heme proximal surface. These data suggest the evolution of divergent C4H enzymes feeding different branches of the phenolic network in seed plants. It shows that specialization required for retention of gene duplicates may result from altered protein topology rather than change in enzyme activity.

  • 12. Saenz, Alejandra
    et al.
    Presto, Jenny
    Lara, Patricia
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Akinyi-Oloo, Laura
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Garcia-Fojeda, Belen
    Nilsson, IngMarie
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Johansson, Jan
    Casals, Cristina
    Folding and Intramembraneous BRICHOS Binding of the Prosurfactant Protein C Transmembrane Segment2015In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 290, no 28, p. 17628-17641Article in journal (Refereed)
    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.

  • 13. Wanngren, Johanna
    et al.
    Lara Vasques, Patricia
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Öjemalm, Karin
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Maioli, Silvia
    Moradi, Nasim
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Chen, Lu
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Tjernberg, Lars O.
    Lundkvist, Johan
    Nilsson, IngMarie
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Karlström, Helena
    Changed membrane integration and catalytic site conformation are two mechanisms behind the increased Aβ42/Aβ40 ratio by presenilin 1 familial Alzheimer-linked mutations.2014In: FEBS Open Bio, E-ISSN 2211-5463, Vol. 4, p. 393-406Article in journal (Refereed)
    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.

  • 14.
    Öjemalm, Karin
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Higuchi, Takashi
    Lara, Patricia
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Lindahl, Erik
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Suga, Hiroaki
    von Heijne, Gunnar
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Energetics of side-chain snorkeling in transmembrane helices probed by nonproteinogenic amino acids2016In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 113, no 38, p. 10559-10564Article in journal (Refereed)
    Abstract [en]

    Cotranslational translocon-mediated insertion of membrane proteins into the endoplasmic reticulum is a key process in membrane protein biogenesis. Although the mechanism is understood in outline, quantitative data on the energetics of the process is scarce. Here, we have measured the effect on membrane integration efficiency of nonproteinogenic analogs of the positively charged amino acids arginine and lysine incorporated into model transmembrane segments. We provide estimates of the influence on the apparent free energy of membrane integration (Delta G(app)) of snorkeling of charged amino acids toward the lipid-water interface, and of charge neutralization. We further determine the effect of fluorine atoms and backbone hydrogen bonds (H-bonds) on Delta G(app). These results help establish a quantitative basis for our understanding of membrane protein assembly in eukaryotic cells.

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  • asciidoc
  • rtf