| 41. |
- Tikhonova, Elena B., et al.
(författare)
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Silencing of Aberrant Secretory Protein Expression by Disease-Associated Mutations
- 2019
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Ingår i: Journal of Molecular Biology. - : Elsevier BV. - 0022-2836 .- 1089-8638. ; 431:14, s. 2567-2580
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Tidskriftsartikel (refereegranskat)abstract
- Signal recognition particle (SRP) recognizes signal sequences of secretory proteins and targets them to the endoplasmic reticulum membrane for translocation. Many human diseases are connected with defects in signal sequences. The current dogma states that the molecular basis of the disease-associated mutations in the secretory proteins is connected with defects in their transport. Here, we demonstrate for several secretory proteins with disease-associated mutations that the molecular mechanism is different from the dogma. Positively charged or helix-breaking mutations in the signal sequence hydrophobic core prevent synthesis of the aberrant proteins and lead to degradation of their mRNAs. The degree of mRNA depletion depends on the location and severity of the mutation in the signal sequence and correlates with inhibition of SRP interaction. Thus, SRP protects secretory protein mRNAs from degradation. The data demonstrate that if disease-associated mutations obstruct SRP interaction, they lead to silencing of the mutated protein expression.
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| 42. |
- Yoon, Jungmin, et al.
(författare)
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Integrative Structural Investigation on the Architecture of Human Importin4_Histone H3/1-14_Asf1a Complex and Its Histone H3 Tail Binding
- 2018
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Ingår i: Journal of Molecular Biology. - : Academic Press. - 0022-2836 .- 1089-8638. ; 430:6, s. 822-841
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Tidskriftsartikel (refereegranskat)abstract
- Importin4 transports histone H3/H4 in complex with Asf1a to the nucleus for chromatin assembly. Importin4 recognizes the nuclear localization sequence located at the N-terminal tail of histones. Here, we analyzed the structures and interactions of human Importin4, histones and Asf1a by cross-linking mass spectrometry, X-ray crystallography, negative-stain electron microscopy, small-angle X-ray scattering and integrative modeling. The cross-linking mass spectrometry data showed that the C-terminal region of Importin4 was extensively cross-linked with the histone H3 tail. We determined the crystal structure of the C-terminal region of Importin4 bound to the histone H3 peptide, thus revealing that the acidic patch in Importin4 accommodates the histone H3 tail, and that histone H3 Lys14 contributes to the interaction with Importin4. In addition, we show that Asf1a modulates the binding of histone H3/H4 to Importin4. Furthermore, the molecular architecture of the Importin4_histone H3/H4_Asf1a complex was produced through an integrative modeling approach. Overall, this work provides structural insights into how Importin4 recognizes histones and their chaperone complex.
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| 43. |
- Zawada, Katarzyna E., et al.
(författare)
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Influenza Hemifusion Phenotype Depends on Membrane Context : Differences in Cell-Cell and Virus-Cell Fusion
- 2018
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Ingår i: Journal of Molecular Biology. - : ACADEMIC PRESS LTD- ELSEVIER SCIENCE LTD. - 0022-2836 .- 1089-8638. ; 430:5, s. 594-601
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Tidskriftsartikel (refereegranskat)abstract
- Influenza viral entry into the host cell cytoplasm is accomplished by a process of membrane fusion mediated by the viral hemagglutinin protein. Hem agglutinin acts in a pH-triggered fashion, inserting a short fusion peptide into the host membrane followed by refolding of a coiled-coil structure to draw the viral envelope and host membranes together. Mutations to this fusion peptide provide an important window into viral fusion mechanisms and protein-membrane interactions. Here, we show that a well-described fusion peptide mutant, G1S, has a phenotype that depends strongly on the viral membrane context. The G1S mutant is well known to cause a "hemifusion" phenotype based on experiments in transfected cells, where cells expressing G1S hemagglutinin can undergo lipid mixing in a pH triggered fashion similar to virus but will not support fusion pores. We compare fusion by the G1S hemagglutinin mutant expressed either in cells or in influenza virions and show that this hemifusion phenotype occurs in transfected cells but that native virions are able to support full fusion, albeit at a slower rate and 10-100x reduced infectious titer. We explain this with a quantitative model where the G1S mutant, instead of causing an absolute block of fusion, alters the protein stoichiometry required for fusion. This change slightly slows fusion at high hemagglutinin density, as on the viral surface, but at lower hemagglutinin density produces a hemifusion phenotype. The quantitative model thus reproduces the observed virus-cell and cell-cell fusion phenotypes, yielding a unified explanation where membrane context can control the observed viral fusion phenotype. (C) 2018 Elsevier Ltd. All rights reserved.
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| 44. |
- Armistead, B., et al.
(författare)
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The Double Life of Group B Streptococcus: Asymptomatic Colonizer and Potent Pathogen
- 2019
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Ingår i: Journal of Molecular Biology. - : Elsevier BV. - 0022-2836. ; 431:16, s. 2914-2931
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Forskningsöversikt (refereegranskat)abstract
- Group B streptococcus (GBS) is a β-hemolytic gram-positive bacterium that colonizes the lower genital tract of approximately 18% of women globally as an asymptomatic member of the gastrointestinal and/or vaginal flora. If established in other host niches, however, GBS is highly pathogenic. During pregnancy, ascending GBS infection from the vagina to the intrauterine space is associated with preterm birth, stillbirth, and fetal injury. In addition, vertical transmission of GBS during or after birth results in life-threatening neonatal infections, including pneumonia, sepsis, and meningitis. Although the mechanisms by which GBS traffics from the lower genital tract to vulnerable host niches are not well understood, recent advances have revealed that many of the same bacterial factors that promote asymptomatic vaginal carriage also facilitate dissemination and virulence. Furthermore, highly pathogenic GBS strains have acquired unique factors that enhance survival in invasive niches. Several host factors also exist that either subdue GBS upon vaginal colonization or alternatively permit invasive infection. This review summarizes the GBS and host factors involved in GBS's state as both an asymptomatic colonizer and an invasive pathogen. Gaining a better understanding of these mechanisms is key to overcoming the challenges associated with vaccine development and identification of novel strategies to mitigate GBS virulence. © 2019 Elsevier Ltd
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| 45. |
- Cragnell, Carolina, et al.
(författare)
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Utilizing Coarse-Grained Modeling and Monte Carlo Simulations to Evaluate the Conformational Ensemble of Intrinsically Disordered Proteins and Regions
- 2018
-
Ingår i: Journal of Molecular Biology. - : Elsevier BV. - 0022-2836. ; 430:16, s. 2478-2492
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Tidskriftsartikel (refereegranskat)abstract
- In this study, we have used the coarse-grained model developed for the intrinsically disordered saliva protein (IDP) Histatin 5, on an experimental selection of monomeric IDPs, and we show that the model is generally applicable when electrostatic interactions dominate the intra-molecular interactions. Experimental and theoretically calculated small-angle X-ray scattering data are presented in the form of Kratky plots, and discussions are made with respect to polymer theory and the self-avoiding walk model. Furthermore, the impact of electrostatic interactions is shown and related to estimations of the conformational ensembles obtained from computer simulations and “Flexible-meccano.” Special attention is given to the form factor and how it is affected by the salt concentration, as well as the approximation of using the form factor obtained under physiological conditions to obtain the structure factor.
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| 46. |
- Henriques, João, et al.
(författare)
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On the Calculation of SAXS Profiles of Folded and Intrinsically Disordered Proteins from Computer Simulations
- 2018
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Ingår i: Journal of Molecular Biology. - : Elsevier BV. - 0022-2836. ; 430:16, s. 2521-2539
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Tidskriftsartikel (refereegranskat)abstract
- Solution techniques such as small-angle X-ray scattering (SAXS) play a central role in structural studies of intrinsically disordered proteins (IDPs); yet, due to low resolution, it is generally necessary to combine SAXS with additional experimental sources of data and to use molecular simulations. Computational methods for the calculation of theoretical SAXS intensity profiles can be separated into two groups, depending on whether the solvent is modeled implicitly as continuous electron density or considered explicitly. The former offers reduced computational cost but requires the definition of a number of free parameters to account for, for example, the excess density of the solvation layer. Overfitting can thus be an issue, particularly when the structural ensemble is unknown. Here, we investigate and show how small variations of the contrast of the hydration shell, δρ, severely affect the outcome, analysis and interpretation of computed SAXS profiles for folded and disordered proteins. For both the folded and disordered proteins studied here, using a default δρ may, in some cases, result in the calculation of non-representative SAXS profiles, leading to an overestimation of their size and a misinterpretation of their structural nature. The solvation layer of the different IDP simulations also impacts their size estimates differently, depending on the protein force field used. The same is not true for the folded protein simulations, suggesting differences in the solvation of the two classes of proteins, and indicating that different force fields optimized for IDPs may cause expansion of the polypeptide chain through different physical mechanisms.
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| 47. |
- Karademir Andersson, Ahu, et al.
(författare)
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Multiple DNA Interactions Contribute to the Initiation of Telomerase Elongation
- 2017
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Ingår i: Journal of Molecular Biology. - : Elsevier BV. - 0022-2836. ; 429:14, s. 2109-2123
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Tidskriftsartikel (refereegranskat)abstract
- Telomerase maintains telomere length and chromosome integrity by adding short tandem repeats of single-stranded DNA to the 3' ends, via reverse transcription of a defined template region of its RNA subunit. To further understand the telomerase elongation mechanism, we studied the primer utilization and extension activity of the telomerase from the budding yeast Naumovozyma castellii (Saccharomyces castellii), which displays a processive nucleotide and repeat addition polymerization. For the efficient initiation of canonical elongation, telomerase required 4-nt primer 3' end complementarity to the template RNA. This DNA-RNA hybrid formation was highly important for the stabilization of an initiation-competent telomerase-DNA complex. Anchor site interactions with the DNA provided additional stabilization to the complex. Our studies indicate three additional separate interactions along the length of the DNA primer, each providing different and distinct contributions to the initiation event. A sequence-independent anchor site interaction acts immediately adjacent to the base-pairing 3' end, indicating a protein anchor site positioned very close to the catalytic site. Two additional anchor regions further 5' on the DNA provide sequence-specific contributions to the initiation of elongation. Remarkably, a non-telomeric sequence in the distal 25- to 32-nt region negatively influences the initiation of telomerase elongation, suggesting an anchor site with a regulatory role in the telomerase elongation decision.
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| 48. |
- Nadeem, Aftab, et al.
(författare)
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Beta-sheet-specific interactions with heat shock proteins define a mechanism of delayed tumor cell death in response to HAMLET
- 2019
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Ingår i: Journal of Molecular Biology. - : Elsevier BV. - 0022-2836. ; 431:14, s. 2612-2627
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Tidskriftsartikel (refereegranskat)abstract
- As chaperones, heat shock proteins (HSPs)protect host cells against misfolded proteins that constitute a by-product of protein synthesis. Certain HSPs are also expressed on the surface of tumor cells, possibly to scavenge extracellular unfolded protein ligands and prevent them from becoming cytotoxic. HAMLET—a complex of partially unfolded alpha-lactalbumin and oleic acid—is relying on its N-terminal alpha-helical domain to perturb tumor cell membranes, and the cells die as a consequence of this interaction. Here we show that in parallel, cell surface HSPs bind the beta-sheet domain of alpha-lactalbumin and activate a temporarily protective loop, involving vesicular uptake and lysosomal accumulation. Later, HAMLET destroys lysosomal membrane integrity, and HAMLET release kills the remaining tumor cells. HSPs were identified as HAMLET targets in a proteomic screen and Hsp70-specific antibodies or shRNAs inhibited HAMLET uptake by tumor cells, which showed increased Hsp70 surface expression compared to differentiated cells. The results suggest that HAMLET engages tumor cells by two parallel recognition mechanisms, defined by alpha-helical- or beta-sheet domains of alpha-lactalbumin and resulting in an immediate death response, or a delay due to transient accumulation of the complex in the lysosomes. This dual response pattern was conserved among tumor cells but not seen in normal, differentiated cells. By two different mechanisms, HAMLET thus achieves a remarkably efficient elimination of tumor cells.
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| 49. |
- Rieloff, Ellen, et al.
(författare)
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Assessing the Intricate Balance of Intermolecular Interactions upon Self-Association of Intrinsically Disordered Proteins
- 2019
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Ingår i: Journal of Molecular Biology. - : Elsevier BV. - 0022-2836. ; 431:3, s. 511-523
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Tidskriftsartikel (refereegranskat)abstract
- Attractive interactions between intrinsically disordered proteins can be crucial for the functionality or, on the contrary, lead to the formation of harmful aggregates. For obtaining a molecular understanding of intrinsically disordered proteins and their interactions, computer simulations have proven to be a valuable complement to experiments. In this study, we present a coarse-grained model and its applications to a system dominated by attractive interactions, namely, the self-association of the saliva protein Statherin. SAXS experiments show that Statherin self-associates with increased protein concentration, and that both an increased temperature and a lower ionic strength decrease the size of the formed complexes. The model captures the observed trends and provides insight into the size distribution. Hydrophobic interaction is considered to be the major driving force of the self-association, while electrostatic repulsion represses the growth. In addition, the model suggests that the decrease of association number with increased temperature is of entropic origin.
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