Characterization of Amyloid Formation by Glucagon-Like Peptides: Role of Basic Residues in Heparin-Mediated Aggregation

Glycosaminoglycans (GAGs) have been reported to play a significant role in amyloid formation of a wide range of proteins/peptides either associated with diseases or native biological functions. The exact mechanism by which GAGs influence amyloid formation is not clearly understood. Here, we studied two closely related peptides, glucagon-like peptide 1 (GLP1) and glucagon-like peptide 2 (GLP2), for their amyloid formation in the presence and absence of the representative GAG heparin using various biophysical and computational approaches. We show that the aggregation and amyloid formation by these peptides follow distinct mechanisms: GLP1 follows nucleation-dependent aggregation, whereas GLP2 forms amyloids without any significant lag time. Investigating the role of heparin, we also found that heparin interacts with GLP1, accelerates its aggregation, and gets incorporated within its amyloid fibrils. In contrast, heparin neither affects the aggregation kinetics of GLP2 nor gets embedded within its fibrils. Furthermore, we found that heparin preferentially influences the stability of the GLP1 fibrils over GLP2 fibrils. To understand the specific nature of the interaction of heparin with GLP1 and GLP2, we performed all-atom MD simulations. Our in silico results show that the basic-nonbasic-basic (B-X-B) motif of GLP1 (K28-G29-R30) facilitates the interaction between heparin and peptide monomers. However, the absence of such a motif in GLP2 could be the reason for a significantly lower strength of interaction between GLP2 and heparin. Our study not only helps to understand the role of heparin in inducing protein aggregation but also provides insight into the nature of heparin–protein interaction

Curcumin Modulates α‑Synuclein Aggregation and Toxicity

In human beings, Parkinson’s disease (PD) is associated with the oligomerization and amyloid formation of α-synuclein (α-Syn). The polyphenolic Asian food ingredient curcumin has proven to be effective against a wide range of human diseases including cancers and neurological disorders. While curcumin has been shown to significantly reduce cell toxicity of α-Syn aggregates, its mechanism of action remains unexplored. Here, using a series of biophysical techniques, we demonstrate that curcumin reduces toxicity by binding to preformed oligomers and fibrils and altering their hydrophobic surface exposure. Further, our fluorescence and two-dimensional nuclear magnetic resonance (2D-NMR) data indicate that curcumin does not bind to monomeric α-Syn but binds specifically to oligomeric intermediates. The degree of curcumin binding correlates with the extent of α-Syn oligomerization, suggesting that the ordered structure of protein is required for effective curcumin binding. The acceleration of aggregation by curcumin may decrease the population of toxic oligomeric intermediates of α-Syn. Collectively; our results suggest that curcumin and related polyphenolic compounds can be pursued as candidate drug targets for treatment of PD and other neurological diseases

Parkinson’s Disease Associated α‑Synuclein Familial Mutants Promote Dopaminergic Neuronal Death in <i>Drosophila melanogaster</i>

α-Synuclein (α-Syn) aggregation and amyloid formation are associated with loss of dopaminergic neurons in Parkinson’s disease (PD). In addition, familial mutations in α-Syn are shown to be one of the definite causes of PD. Here we have extensively studied familial PD associated α-Syn G51D, H50Q, and E46K mutations using <i>Drosophila</i> model system. Our data showed that flies expressing α-Syn familial mutants have a shorter lifespan and exhibit more climbing defects compared to wild-type (WT) flies in an age-dependent manner. The immunofluorescence studies of the brain from the old flies showed more dopaminergic neuronal cell death in all mutants compared to WT. This adverse effect of α-Syn familial mutations is highly correlated with the sustained population of oligomer production and retention in mutant flies. Furthermore, this was supported by our <i>in vitro</i> studies, where significantly higher amount of oligomer was observed in mutants compared to WT. The data suggest that the sustained population of oligomer formation and retention could be a major cause of cell death in α-Syn familial mutants

The Parkinson’s Disease-Associated H50Q Mutation Accelerates α‑Synuclein Aggregation <i>in Vitro</i>

α-Synuclein (α-Syn) aggregation is directly linked with Parkinson’s disease (PD) pathogenesis. Here, we analyzed the aggregation of newly discovered α-Syn missense mutant H50Q <i>in vitro</i> and found that this mutation significantly accelerates the aggregation and amyloid formation of α-Syn. This mutation, however, did not alter the overall secondary structure as suggested by two-dimensional nuclear magnetic resonance and circular dichroism spectroscopy. The initial oligomerization study by cross-linking and chromatographic techniques suggested that this mutant oligomerizes to an extent similar to that of the wild-type α-Syn protein. Understanding the aggregation mechanism of this H50Q mutant may help to establish the aggregation and phenotypic relationship of this novel mutant in PD

The Newly Discovered Parkinson’s Disease Associated Finnish Mutation (A53E) Attenuates α‑Synuclein Aggregation and Membrane Binding

α-Synuclein (α-Syn) oligomerization and amyloid formation are associated with Parkinson’s disease (PD) pathogenesis. Studying familial α-Syn mutants associated with early onset PD has therapeutic importance. Here we report the aggregation kinetics and other biophysical properties of a newly discovered PD associated Finnish mutation (A53E). Our <i>in vitro</i> study demonstrated that A53E attenuated α-Syn aggregation and amyloid formation without altering the major secondary structure and initial oligomerization tendency. Further, A53E showed reduced membrane binding affinity compared to A53T and WT. The present study would help to delineate the role of A53E mutation in early onset PD pathogenesis

Oligomerization prediction of Mel and PP.

<p>The intrinsic oligomerization ability of Mel and PP peptide was calculated (at pH 5.5) using Zyggregator software. The positive values (in red) represent aggregation propensity of corresponding amino acid.</p

Structural characterization of Mel and PP.

<p><b>(A)</b> Structural model of Mel (red, PDB ID: 2MLT) and PP (blue, bovine PDB ID: 1BBA). <b>(B)</b> CD spectra of Mel and PP at day 0 (d0) and after 15 days (d15) in presence and absence of heparin. After the addition of heparin and subsequent incubation for two weeks, the secondary structure of PP remained mostly unchanged (helical). <b>(C)</b> FTIR spectra of two weeks incubated PP and Mel (in the absence and presence of heparin). Y-axis represents the absorbance (AU) and X-axis represents the wavenumber (cm^-1). Wavenumbers corresponding to the maximum absorbance are represented with arrow marks. Consistent with CD data, FTIR study also showed that in the presence of heparin, unstructured Mel transformed into helical conformation, whereas PP remained mostly helical both in presence and absence of heparin after incubation.</p

Biophysical characterization of isolated Mel and PP oligomers.

<p><b>(A)</b> CD spectroscopy of isolated oligomers of Mel and PP in the presence of heparin. Both oligomers showed helical conformation in CD. <b>(B)</b> ThT fluorescence of the isolated Mel and PP oligomers showing moderate ThT binding. <b>(C)</b> CR binding of the isolated Mel and PP oligomers. <b>(D)</b> EM images showing large globular oligomeric morphology of the isolated Mel and PP oligomers formed in the presence of heparin. Scale bar is 500 nm.</p

Morphological characterization of Mel and PP oligomers.

<p>EM and AFM analysis were performed to visualize the morphology of two weeks incubated Mel and PP (in the presence of heparin). EM (left panel) and AFM (middle panel) images showing oligomer formation in the presence of heparin. The right panel shows 3D AFM height images of oligomer. Scale bars for EM images are 500 nm. Height scales for AFM images are also shown.</p