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Lara, Patricia
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Publikasjoner (10 av 14) Visa alla publikasjoner
Andersson, A., Kudva, R., Magoulopoulou, A., Lejarre, Q., Lara, P., Xu, P., . . . Tellgren-Roth, Å. (2020). Membrane integration and topology of RIFIN and STEVOR proteins of the Plasmodium falciparum parasite. The FEBS Journal, 287(13), 2744-2762
Åpne denne publikasjonen i ny fane eller vindu >>Membrane integration and topology of RIFIN and STEVOR proteins of the Plasmodium falciparum parasite
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2020 (engelsk)Inngår i: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 287, nr 13, s. 2744-2762Artikkel i tidsskrift (Fagfellevurdert) Published
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.

Emneord
membrane protein topology, N-linked glycosylation, Plasmodium, RIFIN protein, STEVOR protein
HSV kategori
Identifikatorer
urn:nbn:se:su:diva-177797 (URN)10.1111/febs.15171 (DOI)000504344200001 ()31821735 (PubMedID)
Tilgjengelig fra: 2020-01-21 Laget: 2020-01-21 Sist oppdatert: 2022-02-26bibliografisk kontrollert
Lara, P., Tellgren-Roth, Å., Behesti, H., Horn, Z., Schiller, N., Enquist, K., . . . Nilsson, I. (2019). Murine astrotactins 1 and 2 have a similar membrane topology and mature via endoproteolytic cleavage catalyzed by a signal peptidase. Journal of Biological Chemistry, 294(12), 4538-4545
Åpne denne publikasjonen i ny fane eller vindu >>Murine astrotactins 1 and 2 have a similar membrane topology and mature via endoproteolytic cleavage catalyzed by a signal peptidase
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2019 (engelsk)Inngår i: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 294, nr 12, s. 4538-4545Artikkel i tidsskrift (Fagfellevurdert) Published
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.

Emneord
signal peptidase, glycosylation, glycosylation inhibitor, cell migration, neuron, Astrotactin, central nervous system, neuronal migration, signal peptidase, topology
HSV kategori
Identifikatorer
urn:nbn:se:su:diva-168361 (URN)10.1074/jbc.RA118.007093 (DOI)000462969500022 ()30696770 (PubMedID)
Tilgjengelig fra: 2019-05-09 Laget: 2019-05-09 Sist oppdatert: 2022-03-23bibliografisk kontrollert
Renault, H., De Marothy, M., Jonasson, G., Lara, P., Nelson, D. R., Nilsson, I., . . . Werck-Reichhart, D. (2017). Gene Duplication Leads to Altered Membrane Topology of a Cytochrome P450 Enzyme in Seed Plants. Molecular biology and evolution, 34(8), 2041-2056
Åpne denne publikasjonen i ny fane eller vindu >>Gene Duplication Leads to Altered Membrane Topology of a Cytochrome P450 Enzyme in Seed Plants
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2017 (engelsk)Inngår i: Molecular biology and evolution, ISSN 0737-4038, E-ISSN 1537-1719, Vol. 34, nr 8, s. 2041-2056Artikkel i tidsskrift (Fagfellevurdert) Published
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.

Emneord
plant metabolism, membrane protein, metabolic complexity, cinnamic acid 4-hydroxylase, evolution of lignin metabolism
HSV kategori
Identifikatorer
urn:nbn:se:su:diva-147142 (URN)10.1093/molbev/msx160 (DOI)000406929700019 ()
Tilgjengelig fra: 2017-09-28 Laget: 2017-09-28 Sist oppdatert: 2022-03-23bibliografisk kontrollert
Lara, P., Öjemalm, K., Reithinger, J., Holgado, A., Maojun, Y., Hammed, A., . . . Nilsson, I. (2017). Refined topology model of the STT3/Stt3 protein subunit of the oligosaccharyltransferase complex. Journal of Biological Chemistry, 292(27), 11349-11360
Åpne denne publikasjonen i ny fane eller vindu >>Refined topology model of the STT3/Stt3 protein subunit of the oligosaccharyltransferase complex
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2017 (engelsk)Inngår i: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 292, nr 27, s. 11349-11360Artikkel i tidsskrift (Fagfellevurdert) Published
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.

HSV kategori
Identifikatorer
urn:nbn:se:su:diva-145793 (URN)10.1074/jbc.M117.779421 (DOI)000405119600019 ()28512128 (PubMedID)
Tilgjengelig fra: 2017-08-31 Laget: 2017-08-31 Sist oppdatert: 2022-03-23bibliografisk kontrollert
Öjemalm, K., Higuchi, T., Lara, P., Lindahl, E., Suga, H. & von Heijne, G. (2016). Energetics of side-chain snorkeling in transmembrane helices probed by nonproteinogenic amino acids. Proceedings of the National Academy of Sciences of the United States of America, 113(38), 10559-10564
Åpne denne publikasjonen i ny fane eller vindu >>Energetics of side-chain snorkeling in transmembrane helices probed by nonproteinogenic amino acids
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2016 (engelsk)Inngår i: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 113, nr 38, s. 10559-10564Artikkel i tidsskrift (Fagfellevurdert) Published
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.

Emneord
membrane protein, nonproteinogenic amino acids, translocon
HSV kategori
Identifikatorer
urn:nbn:se:su:diva-135178 (URN)10.1073/pnas.1606776113 (DOI)000383622600041 ()27601675 (PubMedID)
Tilgjengelig fra: 2016-11-24 Laget: 2016-11-01 Sist oppdatert: 2022-02-28bibliografisk kontrollert
Saenz, A., Presto, J., Lara, P., Akinyi-Oloo, L., Garcia-Fojeda, B., Nilsson, I., . . . Casals, C. (2015). Folding and Intramembraneous BRICHOS Binding of the Prosurfactant Protein C Transmembrane Segment. Journal of Biological Chemistry, 290(28), 17628-17641
Åpne denne publikasjonen i ny fane eller vindu >>Folding and Intramembraneous BRICHOS Binding of the Prosurfactant Protein C Transmembrane Segment
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2015 (engelsk)Inngår i: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 290, nr 28, s. 17628-17641Artikkel i tidsskrift (Fagfellevurdert) 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.

HSV kategori
Identifikatorer
urn:nbn:se:su:diva-119291 (URN)10.1074/jbc.M114.630343 (DOI)000357730900052 ()
Tilgjengelig fra: 2015-08-04 Laget: 2015-08-03 Sist oppdatert: 2022-03-23bibliografisk kontrollert
Lara Vasquez, P. (2015). Integration and topology of membrane proteins related to diseases. (Doctoral dissertation). Stockholm: Department of Biochemistry and Biophysics, Stockholm Univeristy
Åpne denne publikasjonen i ny fane eller vindu >>Integration and topology of membrane proteins related to diseases
2015 (engelsk)Doktoravhandling, med artikler (Annet vitenskapelig)
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.

sted, utgiver, år, opplag, sider
Stockholm: Department of Biochemistry and Biophysics, Stockholm Univeristy, 2015. s. 76
HSV kategori
Forskningsprogram
biokemi
Identifikatorer
urn:nbn:se:su:diva-113397 (URN)978-91-7649-094-5 (ISBN)
Disputas
2015-03-06, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B, Stockholm, 10:00 (engelsk)
Opponent
Veileder
Merknad

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

Tilgjengelig fra: 2015-02-12 Laget: 2015-01-29 Sist oppdatert: 2022-02-23bibliografisk kontrollert
Cuviello, F., Tellgren-Roth, Å., Lara, P., Ruud Selin, F., Monné, M., Bisaccia, F., . . . Ostuni, A. (2015). Membrane insertion and topology of the amino-terminal domain TMD0 of multidrug-resistance associated protein 6 (MRP6). FEBS Letters, 589(24), 3921-3928
Åpne denne publikasjonen i ny fane eller vindu >>Membrane insertion and topology of the amino-terminal domain TMD0 of multidrug-resistance associated protein 6 (MRP6)
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2015 (engelsk)Inngår i: FEBS Letters, ISSN 0014-5793, E-ISSN 1873-3468, Vol. 589, nr 24, s. 3921-3928Artikkel i tidsskrift (Fagfellevurdert) Published
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.

Emneord
ATP-binding cassette transporter, Multidrug-resistance associated protein 6, ABCC6, Transmembrane domain, Pseudoxanthoma elasticum, Membrane protein insertion
HSV kategori
Identifikatorer
urn:nbn:se:su:diva-124228 (URN)10.1016/j.febslet.2015.10.030 (DOI)000367232900019 ()26545497 (PubMedID)
Tilgjengelig fra: 2015-12-15 Laget: 2015-12-15 Sist oppdatert: 2022-02-23bibliografisk kontrollert
Goel, S., Palmkvist, M., Moll, K., Joannin, N., Lara, P., Akhouri, R. R., . . . Wahlgren, M. (2015). RIFINs are adhesins implicated in severe Plasmodium falciparum malaria. Nature Medicine, 21(4), 314-317
Åpne denne publikasjonen i ny fane eller vindu >>RIFINs are adhesins implicated in severe Plasmodium falciparum malaria
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2015 (engelsk)Inngår i: Nature Medicine, ISSN 1078-8956, E-ISSN 1546-170X, Vol. 21, nr 4, s. 314-317Artikkel i tidsskrift (Fagfellevurdert) Published
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.

HSV kategori
Identifikatorer
urn:nbn:se:su:diva-116951 (URN)10.1038/nm.3812 (DOI)000352493600012 ()25751816 (PubMedID)
Merknad

AuthorCount:21;

Tilgjengelig fra: 2015-08-17 Laget: 2015-05-04 Sist oppdatert: 2022-02-23bibliografisk kontrollert
Nilsson, I., Lara, P., Hessa, T., Johnson, A. E., von Heijne, G. & Karamyshev, A. L. (2015). The Code for Directing Proteins for Trans location across ER Membrane: SRP Cotranslationally Recognizes Specific Features of a Signal Sequence. Journal of Molecular Biology, 427(6), 1191-1201
Åpne denne publikasjonen i ny fane eller vindu >>The Code for Directing Proteins for Trans location across ER Membrane: SRP Cotranslationally Recognizes Specific Features of a Signal Sequence
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2015 (engelsk)Inngår i: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 427, nr 6, s. 1191-1201Artikkel i tidsskrift (Fagfellevurdert) Published
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.

HSV kategori
Identifikatorer
urn:nbn:se:su:diva-116784 (URN)10.1016/j.jmb.2014.06.014 (DOI)000351798500005 ()24979680 (PubMedID)
Merknad

AuthorCount:6;

Tilgjengelig fra: 2015-04-27 Laget: 2015-04-27 Sist oppdatert: 2022-02-23bibliografisk kontrollert
Organisasjoner