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- Gallego-Villarejo, Lucía, et al.
(author)
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Big dynorphin is a neuroprotector scaffold against amyloid β-peptide aggregation and cell toxicity
- 2022
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In: Computational and Structural Biotechnology Journal. - : Elsevier BV. - 2001-0370. ; 20, s. 5672-5679
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Journal article (peer-reviewed)abstract
- Amyloid β-peptide (Aβ) misfolding into β-sheet structures triggers neurotoxicity inducing Alzheimer’s disease (AD). Molecules able to reduce or to impair Aβ aggregation are highly relevant as possible AD treatments since they should protect against Aβ neurotoxicity. We have studied the effects of the interaction of dynorphins, a family of opioid neuropeptides, with Aβ40 the most abundant species of Aβ. Biophysical measurements indicate that Aβ40 interacts with Big Dynorphin (BigDyn), lowering the amount of hydrophobic aggregates, and slowing down the aggregation kinetics. As expected, we found that BigDyn protects against Aβ40 aggregates when studied in human neuroblastoma cells by cell survival assays. The cross-interaction between BigDyn and Aβ40 provides insight into the mechanism of amyloid pathophysiology and may open up new therapy possibilities.
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- Qureshi, Abdul Aziz, et al.
(author)
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The molecular basis for sugar import in malaria parasites
- 2020
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In: Nature. - : Springer Science and Business Media LLC. - 0028-0836 .- 1476-4687. ; 578:7794, s. 321-325
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Journal article (peer-reviewed)abstract
- Elucidating the mechanism of sugar import requires a molecular understanding of how transporters couple sugar binding and gating events. Whereas mammalian glucose transporters (GLUTs) are specialists1, the hexose transporter from the malaria parasite Plasmodium falciparum PfHT12,3 has acquired the ability to transport both glucose and fructose sugars as efficiently as the dedicated glucose (GLUT3) and fructose (GLUT5) transporters. Here, to establish the molecular basis of sugar promiscuity in malaria parasites, we determined the crystal structure of PfHT1 in complex with d-glucose at a resolution of 3.6 Å. We found that the sugar-binding site in PfHT1 is very similar to those of the distantly related GLUT3 and GLUT5 structures4,5. Nevertheless, engineered PfHT1 mutations made to match GLUT sugar-binding sites did not shift sugar preferences. The extracellular substrate-gating helix TM7b in PfHT1 was positioned in a fully occluded conformation, providing a unique glimpse into how sugar binding and gating are coupled. We determined that polar contacts between TM7b and TM1 (located about 15 Å from d-glucose) are just as critical for transport as the residues that directly coordinate d-glucose, which demonstrates a strong allosteric coupling between sugar binding and gating. We conclude that PfHT1 has achieved substrate promiscuity not by modifying its sugar-binding site, but instead by evolving substrate-gating dynamics.
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- Suades, Albert, et al.
(author)
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Establishing mammalian GLUT kinetics and lipid composition influences in a reconstituted-liposome system
- 2023
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In: Nature Communications. - : Springer Nature. - 2041-1723. ; 14:1
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Journal article (peer-reviewed)abstract
- Transport assays using purified glucose transporters (GLUTs) have proven to be difficult to implement, hampering deeper mechanistic insights. Here the authors have optimized a transport assay in liposomes that will provide insight to study other membrane transport proteins. Glucose transporters (GLUTs) are essential for organism-wide glucose homeostasis in mammals, and their dysfunction is associated with numerous diseases, such as diabetes and cancer. Despite structural advances, transport assays using purified GLUTs have proven to be difficult to implement, hampering deeper mechanistic insights. Here, we have optimized a transport assay in liposomes for the fructose-specific isoform GLUT5. By combining lipidomic analysis with native MS and thermal-shift assays, we replicate the GLUT5 transport activities seen in crude lipids using a small number of synthetic lipids. We conclude that GLUT5 is only active under a specific range of membrane fluidity, and that human GLUT1-4 prefers a similar lipid composition to GLUT5. Although GLUT3 is designated as the high-affinity glucose transporter, in vitro D-glucose kinetics demonstrates that GLUT1 and GLUT3 actually have a similar K-M,K- but GLUT3 has a higher turnover. Interestingly, GLUT4 has a high K-M for D-glucose and yet a very slow turnover, which may have evolved to ensure uptake regulation by insulin-dependent trafficking. Overall, we outline a much-needed transport assay for measuring GLUT kinetics and our analysis implies that high-levels of free fatty acid in membranes, as found in those suffering from metabolic disorders, could directly impair glucose uptake.
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- Yen, Hsin-Yung, et al.
(author)
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Electrospray ionization of native membrane proteins proceeds via a charge equilibration step
- 2022
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In: RSC Advances. - : Royal Society of Chemistry (RSC). - 2046-2069. ; 12:16, s. 9671-9680
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Journal article (peer-reviewed)abstract
- Electrospray ionization mass spectrometry is increasingly applied to study the structures and interactions of membrane protein complexes. However, the charging mechanism is complicated by the presence of detergent micelles during ionization. Here, we show that the final charge of membrane proteins can be predicted by their molecular weight when released from the non-charge reducing saccharide detergents. Our data indicate that PEG detergents lower the charge depending on the number of detergent molecules in the surrounding micelle, whereas fos-choline detergents may additionally participate in ion–ion reactions after desolvation. The supercharging reagent sulfolane, on the other hand, has no discernible effect on the charge of detergent-free membrane proteins. Taking our observations into the context of protein-detergent interactions in the gas phase, we propose a charge equilibration model for the generation of native-like membrane protein ions. During ionization of the protein-detergent complex, the ESI charges are distributed between detergent and protein according to proton affinity of the detergent, number of detergent molecules, and surface area of the protein. Charge equilibration influenced by detergents determines the final charge state of membrane proteins. This process likely contributes to maintaining a native-like fold after detergent release and can be harnessed to stabilize particularly labile membrane protein complexes in the gas phase.
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