Mechanistic insight into functionally different human islet polypeptide (hIAPP) amyloid: the intrinsic role of the C-terminal structural motifs.

Targeting amyloidosis requires high-resolution insight into the underlying mechanisms of amyloid aggregation. The sequence-specific intrinsic properties of a peptide or protein largely govern the amyloidogenic propensity. Thus, it is essential to delineate the structural motifs that define the subsequent downstream amyloidogenic cascade of events. Additionally, it is important to understand the role played by extrinsic factors, such as temperature or sample agitation, in modulating the overall energy barrier that prompts divergent nucleation events. Consequently, these changes can affect the fibrillation kinetics, resulting in structurally and functionally distinct amyloidogenic conformers associated with disease pathogenesis. Here, we have focused on human Islet Polypeptide (hIAPP) amyloidogenesis for the full-length peptide along with its N- and C-terminal fragments, under different temperatures and sample agitation conditions. This helped us to gain a comprehensive understanding of the intrinsic role of specific functional epitopes in the primary structure of the peptide that regulates amyloidogenesis and subsequent cytotoxicity. Intriguingly, our study involving an array of biophysical experiments and ex vivo data suggests a direct influence of external changes on the C-terminal fibrillating sequence. Furthermore, the observations indicate a possible collaborative role of this segment in nucleating hIAPP amyloidogenesis in a physiological scenario, thus making it a potential target for future therapeutic interventions.

Targeted inhibition of amyloidogenesis using a non-toxic, serum stable strategically designed cyclic peptide with therapeutic implications.

Amyloidogenic disorders are currently rising as a global health issue, prompting more and more studies dedicated to the development of effective targeted therapeutics. The innate affinity of these amyloidogenic proteins towards the biomembranes adds further complexities to the systems. Our previous studies have shown that biologically active peptides can effectively target amyloidogenesis serving as an efficient therapeutic alternative in several amyloidogenic disorders. The structural uniqueness of the PWWP motif in the de novo designed heptapeptide, KR7 (KPWWPRR-NH2) was demonstrated to target insulin fiber elongation specifically. By working on insulin, an important model system in amyloidogenic studies, we gained several mechanistic insights into the inhibitory actions at the protein-peptide interface. Here, we report a second-generation non-toxic and serum stable cyclic peptide, based primarily on the PWWP motif that resulted in complete inhibition of insulin fibrillation both in the presence and absence of the model membranes. Using both low- and high-resolution spectroscopic techniques, we could delineate the specific mechanism of inhibition, at atomistic resolution. Our studies put forward an effective therapeutic intervention that redirects the default aggregation kinetics towards off-pathway fibrillation. Based on the promising results, this novel cyclic peptide can be considered an excellent lead to design pharmaceutical molecules against amyloidogenesis.

Design, Synthesis, Antibacterial Potential, and Structural Characterization of N-Acylated Derivatives of the Human Autophagy 16 Polypeptide.

A synthetic antimicrobial peptide library based on the human autophagy 16 polypeptide has been developed. Designed acetylated peptides bearing lipids of different chain lengths resulted in peptides with enhanced potency compared to the parent Atg16. A 21-residue fragment of Atg16 conjugated to 4-methylhexanoic acid (K30) emerged as the most potent antibacterial, with negligible hemolysis. Several studies, including microscopy, dye leakage, and ITC, were conducted to gain insight into the antibacterial mechanism of action of the peptide. Visual inspection using both SEM and TEM revealed the membranolytic effect of the peptide on bacterial cells. The selectivity of the peptide against bacterial cell membranes was also proven using dye leakage assays. ITC analysis revealed the exothermic nature of the binding interaction of the peptide to D8PG micelles. The three-dimensional solution NMR structure of K30 in complex with dioctanoylphosphatidylglycerol (D8PG) micelles revealed that the peptide adopts a helix-loop-helix structure in the presence of anionic membrane lipids mimicking bacterial membranes. Intermolecular NOEs between the peptide and lipid deciphered the location of the peptide in the bound state, which was subsequently supported by the paramagnetic relaxation enhancement (PRE) NMR experiment. Collectively, these results describe the structure-function relationship of the peptide in the bacterial membrane.