Altered intestinal permeability has been correlated with Parkinson’s pathophysiology in the enteric nervous system, before manifestations in the central nervous system (CNS). The inflammatory endotoxin or lipopolysaccharide (LPS) released by gut bacteria is known to modulate α-synuclein amyloidogenesis through the formation of intermediate nucleating species. Here, biophysical techniques in conjunction with microscopic images revealed the molecular interaction between lipopolysaccharide and α-synuclein that induce rapid nucleation events. This heteromolecular interaction stabilizes the α-helical intermediates in the α-synuclein aggregation pathway. Multitude NMR studies probed the residues involved in the LPS-binding structural motif that modulates the nucleating forms, affecting the cellular internalization and associated cytotoxicity. Collectively, our data characterizes this heteromolecular interaction associated with an alternative pathway in Parkinson’s disease progression.

The aggregation of insulin into amyloid fibers has been a limiting factor in the development of fast acting insulin analogues, creating a demand for excipients that limit aggregation. Despite the potential demand, inhibitors specifically targeting insulin have been few in number. Here we report a non-toxic and serum stable-designed heptapeptide, KR7 (KPWWPRR-NH), that differs significantly from the primarily hydrophobic sequences that have been previously used to interfere with insulin amyloid fibrillation. Thioflavin T fluorescence assays, circular dichroism spectroscopy, and one-dimensional proton NMR experiments suggest KR7 primarily targets the fiber elongation step with little effect on the early oligomerization steps in the lag time period. From confocal fluorescence and atomic force microscopy experiments, the net result appears to be the arrest of aggregation in an early, non-fibrillar aggregation stage. This mechanism is noticeably different from previous peptide-based inhibitors, which have primarily shifted the lag time with little effect on later stages of aggregation. As insulin is an important model system for understanding protein aggregation, the new peptide may be an important tool for understanding peptide-based inhibition of amyloid formation.

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Insulin and lysozyme share the common features of being prone to aggregate and having biomedical importance. Encapsulating lysozyme and insulin in micellar nanoparticles probably would prevent aggregation and facilitate oral drug delivery. Despite the vivid structural knowledge of lysozyme and insulin, the environment-dependent oligomerization (dimer, trimer, and multimer) and associated structural dynamics remain elusive. The knowledge of the intra- and intermolecular interaction profiles has cardinal importance for the design of encapsulation protocols. We have employed various biophysical methods such as NMR spectroscopy, X-ray crystallography, Thioflavin T fluorescence, and atomic force microscopy in conjugation with molecular modeling to improve the understanding of interaction dynamics during homo-oligomerization of lysozyme (human and hen egg) and insulin (porcine, human, and glargine). The results obtained depict the atomistic intra- and intermolecular interaction details of the homo-oligomerization and confirm the propensity to form fibrils. Taken together, the data accumulated and knowledge gained will further facilitate nanoparticle design and production with insulin or lysozyme-related protein encapsulation.