| 1. |
- Cerrato, Carmine P. P., et al.
(författare)
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Monitoring Disassembly and Cargo Release of Phase-Separated Peptide Coacervates with Native Mass Spectrometry
- 2023
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Ingår i: Analytical Chemistry. - : American Chemical Society (ACS). - 0003-2700 .- 1520-6882. ; 95:29, s. 10869-10872
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Tidskriftsartikel (refereegranskat)abstract
- Engineering liquid-liquid phase separation (LLPS)of proteinsand peptides holds great promise for the development of therapeuticcarriers with intracellular delivery capability but requires accuratedetermination of their assembly properties in vitro, usually with fluorescently labeled cargo. Here, we use mass spectrometry(MS) to investigate redox-sensitive coacervate microdroplets (thedense phase formed during LLPS) assembled from a short His- and Tyr-richpeptide. We can monitor the enrichment of a reduced peptide in dilutephase as the microdroplets dissolve triggered by their redox-sensitiveside chain, thus providing a quantitative readout for disassembly.Furthermore, MS can detect the release of a short peptide from coacervatesunder reducing conditions. In summary, with MS, we can monitor thedisassembly and cargo release of engineered coacervates used as therapeuticcarriers without the need for additional labels.
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| 2. |
- Leppert, Axel, et al.
(författare)
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Liquid-Liquid Phase Separation Primes Spider Silk Proteins for Fiber Formation via a Conditional Sticker Domain
- 2023
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Ingår i: Nano Letters. - : American Chemical Society (ACS). - 1530-6984 .- 1530-6992. ; 23:12, s. 5836-5841
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Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)abstract
- Many protein condensates can convert to fibrillar aggregates, but the underlying mechanisms are unclear. Liquid-liquid phase separation (LLPS) of spider silk proteins, spidroins, suggests a regulatory switch between both states. Here, we combine microscopy and native mass spectrometry to investigate the influence of protein sequence, ions, and regulatory domains on spidroin LLPS. We find that salting out-effects drive LLPS via low-affinity stickers in the repeat domains. Interestingly, conditions that enable LLPS simultaneously cause dissociation of the dimeric C-terminal domain (CTD), priming it for aggregation. Since the CTD enhances LLPS of spidroins but is also required for their conversion into amyloid-like fibers, we expand the stickers and spacers-model of phase separation with the concept of folded domains as conditional stickers that represent regulatory units.
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| 3. |
- Saluri, Mihkel, et al.
(författare)
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A “grappling hook” interaction connects self-assembly and chaperone activity of Nucleophosmin 1
- 2023
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Ingår i: pnas nexus. - : Oxford University Press (OUP). - 2752-6542. ; 2:2
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Tidskriftsartikel (refereegranskat)abstract
- How the self-assembly of partially disordered proteins generates functional compartments in the cytoplasm and particularly in the nucleus is poorly understood. Nucleophosmin 1 (NPM1) is an abundant nucleolar protein that forms large oligomers and undergoes liquid-liquid phase separation by binding RNA or ribosomal proteins. It provides the scaffold for ribosome assembly but also prevents protein aggregation as part of the cellular stress response. Here, we use aggregation assays and native mass spectrometry (MS) to examine the relationship between the self-assembly and chaperone activity of NPM1. We find that oligomerization of full-length NPM1 modulates its ability to retard amyloid formation in vitro. Machine learning-based structure prediction and cryo-electron microscopy reveal fuzzy interactions between the acidic disordered region and the C-terminal nucleotide-binding domain, which cross-link NPM1 pentamers into partially disordered oligomers. The addition of basic peptides results in a tighter association within the oligomers, reducing their capacity to prevent amyloid formation. Together, our findings show that NPM1 uses a grappling hook mechanism to form a network-like structure that traps aggregation-prone proteins. Nucleolar proteins and RNAs simultaneously modulate the association strength and chaperone activity, suggesting a mechanism by which nucleolar composition regulates the chaperone activity of NPM1.
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