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Cell surface proteins (CSPs) are valuable targets for therapeutic agents, but achieving highly selective CSP enrichment in cellular physiology remains a technical challenge. To address this challenge, we propose a newly developed sulfo-pyridinium ester (SPE) cross-linking probe, followed by two-step imaging and enrichment. The SPE probe showed higher efficiency in labeling proteins than similar NHS esters at the level of cell lysates and demonstrated specificity for Lys in competitive experiments. More importantly, this probe could selectively label the cell membranes in cell imaging with only negligible labeling of the intracellular compartment. Moreover, we successfully performed this strategy on MCF-7 live cells to label 425 unique CSPs from 1162 labeled proteins. Finally, we employed our probe to label the CSPs of insulin-cultured MCF-7, revealing several cell surface targets of key functional biomarkers and insulin-associated pathogenesis. The above results demonstrate that the SPE method provides a promising tool for the selective labeling of cell surface proteins and monitoring transient cell surface events.

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Inhibition of amyloid-ß peptide (Aß) aggregation is a promising therapeutic strategy for Alzheimer's disease (AD), as Aß aggregation is generally believed to trigger AD pathology. Pre-fibril Aß-oligomers induce membrane disruption and are crucial to neurotoxicity. We have previously designed a short peptide called cyclic helical amyloid surface inhibitor (cHASI) that can selectively bind to the Aß fibril surface. Here, we use cHASI to efficiently inhibit the surface-catalysed secondary nucleation process of Aß in a lipid membrane environment. By incubating Aß monomers with lipid vesicles, we show that during the assembly of Aß into amyloid fibrils, oligomers are formed that markedly disrupt the lipid bilayer. Remarkably, when Aß monomers are incubated with cHASI, although Aß forms amyloid fibrils via primary nucleation and elongation, this pathway to fibrils does not damage the lipid bilayer. This indicates that only oligomers produced during secondary surface nucleation disrupt membrane integrity. The protective effect of cHASI is confirmed by cytotoxicity assays. Our study highlights the therapeutic potential for inhibiting the secondary nucleation process in Aß aggregation, rather than inhibiting all pathways to fibril formation.

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Peptide self-assembly inspired by natural superhelical coiled coils has been actively pursued but remains challenging due to limited helicity of short peptides. Side chain stapling can strengthen short helices but is unexplored in design of self-assembled helical nanofibers as it is unknown how staples could be adapted to coiled coil architecture. Here, we demonstrate the feasibility of this design for pentapeptides using a computational method capable of predicting helicity and fiber-forming tendency of stapled peptides containing noncoded amino acids. Experiments showed that the best candidates, which carried an aromatically substituted staple and phenylalanine analogs, displayed exceptional helicity and assembled into nanofibers via specific head-to-tail hydrogen bonding and packing between staple and noncoded side chains. The fibers exhibited sheet-of-helix structures resembling the recently found collapsed coiled coils whose formation was sensitive to side chain flexibility. This study expands the chemical space of coiled coil assemblies and provides guidance for their design.

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Amyloid-ß (Aß) oligomers are well-known toxic molecular species associated with Alzheimer's disease. Recent discoveries of the ability of amyloid fibril surfaces to convert soluble proteins into toxic oligomers suggested that these surfaces could serve as therapeutic targets for intervention. We have shown previously that a short helical peptide could be a key structural motif that can specifically recognize the K16-E22 region of the Aß40 fibril surface with an affinity at the level of several micromolar. Here, we demonstrate that in-tether chiral center-induced helical stabilized peptides could also recognize the fibril surfaces, effectively inhibiting the surface-mediated oligomerization of Aß40. Moreover, through extensive computational sampling, we observed two distinct ways in which the peptide inhibitors recognize the fibril surface. Apart from a binding mode that, in accord with the original design, involves hydrophobic side chains at the binding interface, we observed much more frequently another binding mode in which the hydrophobic staple interacts directly with the fibril surface. The affinity of the peptides for the fibril surface could be adjusted by tuning the hydrophobicity of the staple. The best candidate investigated here exhibits a submicromolar affinity (∼0.75 µM). Collectively, this work opens an avenue for the rational design of candidate drugs with stapled peptides for amyloid-related disease.

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Amyloid fibril surfaces can convert soluble proteins into toxic oligomers and are attractive targets for intervention of protein aggregation diseases. Thus far, molecules identified with inhibitory activity are either large proteins or flat cyclic compounds lacking in specificity. The main design difficulty is flatness of amyloid surfaces and the lack of knowledge on binding interfaces. Here, we demonstrate, for the first time, a rational design of alpha-helical peptide inhibitors targeting the amyloid-beta 40 (Aß40) fibril surfaces, based on our in silico finding that a helical fragment of Aß40 interacts in a unique way with side-chain arrays on the fibril surface. We strengthen the fragment's binding capability through mutations and helicity enhancement with our Terminal Aspartic acid strategy. The resulting inhibitor shows micromolar affinity for the fibril surface, effectively impedes the surface-mediated oligomerization of Aß40, and mitigates its cytotoxicity. This work opens up an avenue to designing aggregation modulators for amyloid diseases.

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The molecular design of peptide-assembled nanostructures relies on extensive knowledge pertaining to the relationship between conformational features of peptide constituents and their behavior regarding self-assembly, and characterizing the conformational details of peptides during their self-assembly is experimentally challenging. Here, we demonstrate that a hybrid-resolution modeling method can be employed to investigate the role that conformation plays during the assembly of terminally capped diphenylalanines (FF) through microsecond simulations of hundreds or thousands of peptides. Our simulations discovered tubular or vesicular nanostructures that were consistent with experimental observation while reproducing critical self-assembly concentration and secondary structure contents in the assemblies that were measured in our experiments. The atomic details provided by our method allowed us to uncover diverse FF conformations and conformation dependence of assembled nanostructures. We found that the assembled morphologies and the molecular packing of FFs in the observed assemblies are linked closely with side-chain angle and peptide bond orientation, respectively. Of various conformations accessible to soluble FFs, only a select few are compatible with the assembled morphologies in water. A conformation resembling a FF crystal, in particular, became predominant due to its ability to permit highly ordered and energetically favorable FF packing in aqueous assemblies. Strikingly, several conformations incompatible with the assemblies arose transiently as intermediates, facilitating key steps of the assembly process. The molecular rationale behind the role of these intermediate conformations were further explained. Collectively, the structural details reported here advance the understanding of the FF self-assembly mechanism, and our method shows promise for studying peptide-assembled nanostructures and their rational design.

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