| 1. |
- Ch'ng, Jun-Hong, et al.
(författare)
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Epitopes of anti-RIFIN antibodies and characterization of rif-expressing Plasmodium falciparum parasites by RNA sequencing
- 2017
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Ingår i: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 7
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Tidskriftsartikel (refereegranskat)abstract
- Variable surface antigens of Plasmodium falciparum have been a major research focus since they facilitate parasite sequestration and give rise to deadly malaria complications. Coupled with its potential use as a vaccine candidate, the recent suggestion that the repetitive interspersed families of polypeptides (RIFINs) mediate blood group A rosetting and influence blood group distribution has raised the research profile of these adhesins. Nevertheless, detailed investigations into the functions of this highly diverse multigene family remain hampered by the limited number of validated reagents. In this study, we assess the specificities of three promising polyclonal anti-RIFIN antibodies that were IgG-purified from sera of immunized animals. Their epitope regions were mapped using a 175,000-peptide microarray holding overlapping peptides of the P. falciparum variable surface antigens. Through immunoblotting and immunofluorescence imaging, we show that different antibodies give varying results in different applications/assays. Finally, we authenticate the antibody-based detection of RIFINs in two previously uncharacterized non-rosetting parasite lines by identifying the dominant rif transcripts using RNA sequencing.
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| 2. |
- Andersson, Annika, et al.
(författare)
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Membrane integration and topology of RIFIN and STEVOR proteins of the Plasmodium falciparum parasite
- 2020
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Ingår i: The FEBS Journal. - : Wiley. - 1742-464X .- 1742-4658. ; 287:13, s. 2744-2762
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 3. |
- Bañó-Polo, Manuel, et al.
(författare)
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Transmembrane but not soluble helices fold inside the ribosome tunnel
- 2018
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Ingår i: Nature Communications. - : Springer Science and Business Media LLC. - 2041-1723. ; 9
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Tidskriftsartikel (refereegranskat)abstract
- Integral membrane proteins are assembled into the ER membrane via a continuous ribosome-translocon channel. The hydrophobicity and thickness of the core of the membrane bilayer leads to the expectation that transmembrane (TM) segments minimize the cost of harbouring polar polypeptide backbones by adopting a regular pattern of hydrogen bonds to form a-helices before integration. Co-translational folding of nascent chains into an a-helical conformation in the ribosomal tunnel has been demonstrated previously, but the features governing this folding are not well understood. In particular, little is known about what features influence the propensity to acquire a-helical structure in the ribosome. Using in vitro translation of truncated nascent chains trapped within the ribosome tunnel and molecular dynamics simulations, we show that folding in the ribosome is attained for TM helices but not for soluble helices, presumably facilitating SRP (signal recognition particle) recognition and/or a favourable conformation for membrane integration upon translocon entry.
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| 4. |
- 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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| 5. |
- Cuviello, Flavia, et al.
(författare)
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Membrane insertion and topology of the amino-terminal domain TMD0 of multidrug-resistance associated protein 6 (MRP6)
- 2015
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Ingår i: FEBS Letters. - : Wiley. - 0014-5793 .- 1873-3468. ; 589:24, s. 3921-3928
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 6. |
- Dowaidar, Moataz
(författare)
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In-silico design of peptide-based transfection systems, in-vitro validation, and up-take pathways investigation
- 2017
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Licentiatavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Cell-penetrating peptide-based transfection systems (PBTS) are a promising group of drug delivery vectors. Cell-penetrating peptides (CPPs) are short cationic peptides that are able of transporting cell non-permeant cargos into different cell types. Some CPPs can be used to form non-covalent complexes with oligonucleotides for gene delivery applications. For the potential use of CPPs as drug delivery tools, it is important to understand the mechanism of uptake. Here, a fragment quantitative structure–activity relationships (FQSAR) model is generated to predict novel peptides based on approved alpha helical conformers and assisted model construction with energy refinement molecular mechanics simulations of former peptides. The modeled peptides were examined for plasmid transfection efficiency and compared with their predicted biological activity. The best predicted peptides were efficient for plasmid transfection with significant enhancement compared to the former group of peptides. Our results confirm that FQSAR model refinement is an efficient method for optimizing PBTS for improved biological activity. Additionally, using RNA sequencing, we demonstrated the involvement of autophagy pathways in PBTS uptake.
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| 7. |
- 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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| 8. |
- Gadalla, Salah-Eldin, et al.
(författare)
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EpCAM associates with endoplasmic reticulum aminopeptidase 2 (ERAP2) in breast cancer cells
- 2013
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Ingår i: Biochemical and Biophysical Research Communications - BBRC. - : Elsevier BV. - 0006-291X .- 1090-2104. ; 439:2, s. 203-208
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 9. |
- Goel, Suchi, et al.
(författare)
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RIFINs are adhesins implicated in severe Plasmodium falciparum malaria
- 2015
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Ingår i: Nature Medicine. - : Springer Science and Business Media LLC. - 1078-8956 .- 1546-170X. ; 21:4, s. 314-317
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 10. |
- Hedin, Linnea E., et al.
(författare)
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Membrane Insertion of Marginally Hydrophobic Transmembrane Helices Depends on Sequence Context
- 2010
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Ingår i: Journal of Molecular Biology. - : Elsevier BV. - 0022-2836 .- 1089-8638. ; 396:1, s. 221-229
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Tidskriftsartikel (refereegranskat)abstract
- In mammalian cells, most integral membrane proteins are initially inserted into the endoplasmic reticulum membrane by the so-called Sec61 translocon. However, recent predictions suggest that many transmembrane helices (TMHs) in multispanning membrane proteins are not sufficiently hydrophobic to be recognized as such by the translocon. In this study, we have screened 16 marginally hydrophobic TMHs from membrane proteins of known three-dimensional structure. Indeed, most of these TMHs do not insert efficiently into the endoplasmic reticulum membrane by themselves. To test if loops or TMHs immediately upstream or downstream of a marginally hydrophobic helix might influence the insertion efficiency, insertion of marginally hydrophobic helices was also studied in the presence of their neighboring loops and helices. The results show that flanking loops and nearest-neighbor TMHs are sufficient to ensure the insertion of many marginally hydrophobic helices. However, for at least two of the marginally hydrophobic helices, the local interactions are not enough, indicating that post-insertional rearrangements are involved in the folding of these proteins.
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