Deciphering the Molecular Dance: Exploring the Dynamic Interplay Between Mouse Insulin B9-23 Peptides and their Variants.

Type 1 diabetes results from the autoimmune destruction of pancreatic insulin-producing ß-cells, primarily targeted by autoreactive T cells that recognize insulin B9-23 peptides as antigens. Using drift tube ion mobility spectrometry-mass spectrometry, transmission electron microscopy, and two-dimensional infrared spectroscopy, we characterized mouse insulin 1 B9-23 (Ins1 B9-23), insulin 2 B9-23 (Ins2 B9-23), along with two of their mutants, Ins2 B9-23 Y16A and Ins2 B9-23 C19S. Our findings indicate that Ins1 B9-23 and the Ins2 Y16A mutant exhibit rapid fibril formation, whereas Ins2 B9-23 and the Ins2 C19S mutant show slower fibrillization and a structural rearrangement from globular protofibrils to fibrillar aggregates. These differences in aggregation behaviors also manifest in interactions with (-)epigallocatechin gallate (EGCG), a canonical amyloid inhibitor. EGCG effectively disrupts the fibrils formed by Ins1 B9-23 and the Y16A mutant. However, it proves ineffective in preventing fibril formation of Ins2 B9-23 and the C19S mutant. These results establish a strong correlation between the aggregation behaviors of these peptides and their divergent effects on anti-islet autoimmunity.

Investigating a Novel Neurodegenerative Disease Toxic Mechanism Involving Lipid Binding Specificity of Amyloid Oligomers.

Exploring the mechanisms underlying the toxicity of amyloid oligomers (AOs) presents a significant opportunity for discovering cures and developing treatments for neurodegenerative diseases. Recently, using a combination of ion mobility spectrometry-mass spectrometry (IMS-MS) and X-ray crystallography (XRC), we showed that the peptide KVKVLWDVIEV, which is the G95W mutant of αB-Crystallin (90-100) and abbreviated as G6W, self-assembles up to a dodecamer that structurally resembles lipid transport proteins. The glycine to tryptophan mutation promotes not only larger oligomers and enhanced cytotoxicity in brain slices than the wild type but also a narrow hydrophobic cavity suitable for fatty acid or phospholipid binding. Here, we determine the plausibility of a novel cytotoxic mechanism where the G6W's structural motif could perturb lipid homeostasis by determining its lipid binding selectivity and specificity. We show that the G6W oligomers have a strong affinity toward unsaturated phospholipids with a preference toward phospholipids containing 16-C alkyl chains. Molecular dynamics simulations demonstrate how an unsaturated, 16-C phospholipid fits tightly inside and outside G6W's hydrophobic cavity. This binding is exclusive to the G6W peptide, as other amyloid oligomers with different atomic structures, including its wildtype αB-Crystallin (90-100) and several superoxide dismutase 1 (SOD1) peptides that are known to self-assemble into amyloid oligomers (SOD1P28K and SOD1WG-GW), do not experience the same strong binding affinity. While the existing chaperone-lipid hypothesis on amyloid toxicity suggests amyloid-lipid complexes perforate cell membranes, our work provides a new outlook, indicating that soluble amyloid oligomers disrupt lipid homeostasis via selective protein-ligand interactions. The toxic mechanisms may arise from the formation of unique amyloid oligomer structures assisted by lipid ligands or impaired lipid transports.

Cytotoxicity of α-Helical, Staphylococcus aureus PSMα3 Investigated by Post-Ion-Mobility Dissociation Mass Spectrometry.

Our knowledge of amyloid formation and cytotoxicity originating from self-assembly of α-helical peptides is incomplete. PSMα3 is the only system where high-resolution X-ray crystallography and toxicity data are available. Oligomers of multiple α-helical monomers are less stable than those of ß-strands, partially due to the lack of a consistent hydrogen-bonding network. It is challenging to preserve such oligomers in the gas phase where mass-selected structural studies using ion-mobility spectrometry mass spectrometry (IMS-MS) could be performed. As the oligomers fall apart after exiting the drift cell of the mass spectrometer, novel features that have shorter (a loss of charged species) or longer (a loss of neutral species) arrival times than expected are present together with those from the intact species. By obtaining a complete data set of PSMα3 peptides in solution and with n-dodecyl-ß-d-maltoside, a micelle-forming detergent, we are able to discern the dissociated from the intact oligomers and detergent-bound complexes and correlate the reported cytotoxicity to the peptide oligomeric structures and their interactions with membrane mimetics. The study sheds new insights into the interpretation of IMS-MS data from biomolecular self-assembly studies-an important and timely topic.

Hetero-oligomeric Amyloid Assembly and Mechanism: Prion Fragment PrP(106-126) Catalyzes the Islet Amyloid Polypeptide ß-Hairpin.

Protein aggregation is typically attributed to the association of homologous amino acid sequences between monomers of the same protein. Coaggregation of heterogeneous peptide species can occur, however, and is implicated in the proliferation of seemingly unrelated protein diseases in the body. The prion protein fragment (PrP106-126) and human islet amyloid polypeptide (hIAPP) serve as an interesting model of nonhomologous protein assembly as they coaggregate, despite a lack of sequence homology. We have applied ion-mobility mass spectrometry, atomic force microscopy, circular dichroism, and high-level molecular modeling to elucidate this important assembly process. We found that the prion fragment not only forms pervasive hetero-oligomeric aggregates with hIAPP but also promotes the transition of hIAPP into its amyloidogenic ß-hairpin conformation. Further, when PrP106-126 was combined with non-amyloidogenic rIAPP, the two formed nearly identical hetero-oligomers to those seen with hIAPP, despite rIAPP containing ß-sheet breaking proline substitutions. Additionally, while rIAPP does not natively form the amyloidogenic ß-hairpin structure, it did so in the presence of PrP106-126 and underwent a conformational transition to ß-sheet in solution. We also find that PrP106-126 forms hetero-oligomers with the IAPP8-20 fragment but not with the "aggregation hot spot" IAPP20-29 fragment. PrP106-126 apparently induces IAPP into a ß-hairpin structure within the PrP:IAPP heterodimer complex and then, through ligand exchange, catalytically creates the amyloidogenic ß-hairpin dimer of IAPP in significantly greater abundance than IAPP does on its own. This is a new mechanistic model that provides a critical foundation for the detailed study of hetero-oligomerization and prion-like proliferation in amyloid systems.