| 1. |
- Chen, Gefei, et al.
(författare)
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Molecular basis for different substrate-binding sites and chaperone functions of the BRICHOS domain
- 2024
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Ingår i: Protein Science. - : Wiley. - 0961-8368 .- 1469-896X. ; 33:7
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Tidskriftsartikel (refereegranskat)abstract
- Proteins can misfold into fibrillar or amorphous aggregates and molecular chaperones act as crucial guardians against these undesirable processes. The BRICHOS chaperone domain, found in several otherwise unrelated proproteins that contain amyloidogenic regions, effectively inhibits amyloid formation and toxicity but can in some cases also prevent non-fibrillar, amorphous protein aggregation. Here, we elucidate the molecular basis behind the multifaceted chaperone activities of the BRICHOS domain from the Bri2 proprotein. High-confidence AlphaFold2 and RoseTTAFold predictions suggest that the intramolecular amyloidogenic region (Bri23) is part of the hydrophobic core of the proprotein, where it occupies the proposed amyloid binding site, explaining the markedly reduced ability of the proprotein to prevent an exogenous amyloidogenic peptide from aggregating. However, the BRICHOS-Bri23 complex maintains its ability to form large polydisperse oligomers that prevent amorphous protein aggregation. A cryo-EM-derived model of the Bri2 BRICHOS oligomer is compatible with surface-exposed hydrophobic motifs that get exposed and come together during oligomerization, explaining its effects against amorphous aggregation. These findings provide a molecular basis for the BRICHOS chaperone domain function, where distinct surfaces are employed against different forms of protein aggregation.
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| 2. |
- Chen, Gefei, et al.
(författare)
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Short hydrophobic loop motifs in BRICHOS domains determine chaperone activity against amorphous protein aggregation but not against amyloid formation
- 2023
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Ingår i: Communications Biology. - : Springer Nature. - 2399-3642. ; 6:1
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Tidskriftsartikel (refereegranskat)abstract
- BRICHOS domain oligomerization exposes three short hydrophobic motifs that are necessary for efficient chaperone activity against amorphous protein aggregation. ATP-independent molecular chaperones are important for maintaining cellular fitness but the molecular determinants for preventing aggregation of partly unfolded protein substrates remain unclear, particularly regarding assembly state and basis for substrate recognition. The BRICHOS domain can perform small heat shock (sHSP)-like chaperone functions to widely different degrees depending on its assembly state and sequence. Here, we observed three hydrophobic sequence motifs in chaperone-active domains, and found that they get surface-exposed when the BRICHOS domain assembles into larger oligomers. Studies of loop-swap variants and site-specific mutants further revealed that the biological hydrophobicities of the three short motifs linearly correlate with the efficiency to prevent amorphous protein aggregation. At the same time, they do not at all correlate with the ability to prevent ordered amyloid fibril formation. The linear correlations also accurately predict activities of chimeras containing short hydrophobic sequence motifs from a sHSP that is unrelated to BRICHOS. Our data indicate that short, exposed hydrophobic motifs brought together by oligomerisation are sufficient and necessary for efficient chaperone activity against amorphous protein aggregation.
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| 3. |
- Abelein, Axel, et al.
(författare)
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Molecular Structure of Cu(II)-Bound Amyloid-β Monomer Implicated in Inhibition of Peptide Self-Assembly in Alzheimer’s Disease
- 2022
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Ingår i: JACS Au. - : American Chemical Society (ACS). - 2691-3704. ; 2:11, s. 2571-2584
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Tidskriftsartikel (refereegranskat)abstract
- Metal ions, such as copper and zinc ions, have been shown to strongly modulate the self-assembly of the amyloid-β (Aβ) peptide into insoluble fibrils, and elevated concentrations of metal ions have been found in amyloid plaques of Alzheimer’s patients. Among the physiological transition metal ions, Cu(II) ions play an outstanding role since they can trigger production of neurotoxic reactive oxygen species. In contrast, structural insights into Cu(II) coordination of Aβ have been challenging due to the paramagnetic nature of Cu(II). Here, we employed specifically tailored paramagnetic NMR experiments to determine NMR structures of Cu(II) bound to monomeric Aβ. We found that monomeric Aβ binds Cu(II) in the N-terminus and combined with molecular dynamics simulations, we could identify two prevalent coordination modes of Cu(II). For these, we report here the NMR structures of the Cu(II)–bound Aβ complex, exhibiting heavy backbone RMSD values of 1.9 and 2.1 Å, respectively. Further, applying aggregation kinetics assays, we identified the specific effect of Cu(II) binding on the Aβ nucleation process. Our results show that Cu(II) efficiently retards Aβ fibrillization by predominately reducing the rate of fibril-end elongation at substoichiometric ratios. A detailed kinetic analysis suggests that this specific effect results in enhanced Aβ oligomer generation promoted by Cu(II). These results can quantitatively be understood by Cu(II) interaction with the Aβ monomer, forming an aggregation inert complex. In fact, this mechanism is strikingly similar to other transition metal ions, suggesting a common mechanism of action of retarding Aβ self-assembly, where the metal ion binding to monomeric Aβ is a key determinant.
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| 4. |
- Arndt, Tina, et al.
(författare)
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Spidroin N-terminal domain forms amyloid-like fibril based hydrogels and provides a protein immobilization platform
- 2022
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Ingår i: Nature Communications. - : Springer Science and Business Media LLC. - 2041-1723. ; 13
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Tidskriftsartikel (refereegranskat)abstract
- Recombinant spider silk proteins (spidroins) have multiple potential applications in development of novel biomaterials, but their multimodal and aggregation-prone nature have complicated production and straightforward applications. Here, we report that recombinant miniature spidroins, and importantly also the N-terminal domain (NT) on its own, rapidly form self-supporting and transparent hydrogels at 37 °C. The gelation is caused by NT α-helix to β-sheet conversion and formation of amyloid-like fibrils, and fusion proteins composed of NT and green fluorescent protein or purine nucleoside phosphorylase form hydrogels with intact functions of the fusion moieties. Our findings demonstrate that recombinant NT and fusion proteins give high expression yields and bestow attractive properties to hydrogels, e.g., transparency, cross-linker free gelation and straightforward immobilization of active proteins at high density.
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| 5. |
- Kaldmäe, Margit, et al.
(författare)
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A “spindle and thread” mechanism unblocks p53 translation by modulating N-terminal disorder
- 2022
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Ingår i: Structure. - : Elsevier BV. - 0969-2126 .- 1878-4186. ; 30:5, s. 733-742, e1-e7
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Tidskriftsartikel (refereegranskat)abstract
- Disordered proteins pose a major challenge to structural biology. A prominent example is the tumor suppressor p53, whose low expression levels and poor conformational stability hamper the development of cancer therapeutics. All these characteristics make it a prime example of “life on the edge of solubility.” Here, we investigate whether these features can be modulated by fusing the protein to a highly soluble spider silk domain (NT∗). The chimeric protein displays highly efficient translation and is fully active in human cancer cells. Biophysical characterization reveals a compact conformation, with the disordered transactivation domain of p53 wrapped around the NT∗ domain. We conclude that interactions with NT∗ help to unblock translation of the proline-rich disordered region of p53. Expression of partially disordered cancer targets is similarly enhanced by NT∗. In summary, we demonstrate that inducing co-translational folding via a molecular “spindle and thread” mechanism unblocks protein translation in vitro.
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| 6. |
- Wallin, Cecilia, et al.
(författare)
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Metal ion coordination delays amyloid-β peptide self-assembly by forming an aggregation-inert complex
- 2020
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Ingår i: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 295:21, s. 7224-7234
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Tidskriftsartikel (refereegranskat)abstract
- A detailed understanding of the molecular pathways for amyloid-β (Aβ) peptide aggregation from monomers into amyloid fibrils, a hallmark of Alzheimer’s disease, is crucial for the development of diagnostic and therapeutic strategies. We investigate the molecular details of peptide fibrillization in vitro by perturbing this process through addition of differently charged metal ions. Here, we used a monovalent probe, the silver ion, that, similarly to divalent metal ions, binds to monomeric Aβ peptide and efficiently modulates Aβ fibrillization. On the basis of our findings, combined with our previous results on divalent zinc ions, we propose a model that links the microscopic metal ion binding to Aβ monomers to its macroscopic impact on the peptide self-assembly observed in bulk experiments. We found that sub-stoichiometric concentrations of the investigated metal ions bind specifically to the N-terminal region of Aβ, forming a dynamic, partially compact complex. The metal ion bound state appears to be incapable of aggregation, effectively reducing the available monomeric Aβ pool for incorporation into fibrils. This is especially reflected in a decreased fibril-end elongation rate. However, since the bound state is significantly less stable than the amyloid state, Aβ peptides are only transiently redirected from fibril formation and eventually almost all Aβ monomers are integrated into fibrils. Taken together, these findings unravel the mechanistic consequences of delaying Aβ aggregation via weak metal ion binding, quantitatively linking the contributions of specific interactions of metal ions with monomeric Aβ to their effects on bulk aggregation.
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