Investigating the intrinsic aggregation potential of evolutionarily conserved segments in p53

Protein aggregation and amyloid formation are known to play a role both in diseases and in biological functions. Transcription factor p53 plays a major role in tumor suppression by maintaining genomic stability. Recent studies have suggested that amyloid formation of p53 could lead to its loss of physiological function as a tumor suppressor. Here, we investigated the intrinsic amyloidogenic nature of wild-type p53 using sequence analysis. We used bioinformatics and aggregation prediction algorithms to establish the evolutionarily conserved nature of aggregation-prone sequences in wild-type p53. Further, we analyzed the amyloid forming capacity of conserved and aggregation-prone p53-derived peptides PILTIITL and YFTLQI in vitro using various biophysical techniques, including all atom molecular dynamics simulation. Finally, we probed the seeding ability of the PILTIITL peptide on p53 aggregation in vitro and in cells. Our data demonstrate the intrinsic amyloid forming ability of a sequence stretch of the p53 DNA binding domain (DBD) and its aggregation templating behavior on full-length and p53 core domain. Therefore, p53 aggregation, instigated through an amyloidogenic segment in its DBD, could be a putative driving force for p53 aggregation in vivo

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

Investigating the Intrinsic Aggregation Potential of Evolutionarily Conserved Segments in p53

Protein aggregation and amyloid formation are known to play a role both in diseases and in biological functions. Transcription factor p53 plays a major role in tumor suppression by maintaining genomic stability. Recent studies have suggested that amyloid formation of p53 could lead to its loss of physiological function as a tumor suppressor. Here, we investigated the intrinsic amyloidogenic nature of wild-type p53 using sequence analysis. We used bioinformatics and aggregation prediction algorithms to establish the evolutionarily conserved nature of aggregation-prone sequences in wild-type p53. Further, we analyzed the amyloid forming capacity of conserved and aggregation-prone p53-derived peptides PILTIITL and YFTLQI <i>in vitro</i> using various biophysical techniques, including all atom molecular dynamics simulation. Finally, we probed the seeding ability of the PILTIITL peptide on p53 aggregation <i>in vitro</i> and in cells. Our data demonstrate the intrinsic amyloid forming ability of a sequence stretch of the p53 DNA binding domain (DBD) and its aggregation templating behavior on full-length and p53 core domain. Therefore, p53 aggregation, instigated through an amyloidogenic segment in its DBD, could be a putative driving force for p53 aggregation <i>in vivo</i>

Complexation of NAC-Derived Peptide Ligands with the C‑Terminus of α‑Synuclein Accelerates Its Aggregation

Aggregation of α-synuclein (α-Syn) into neurotoxic oligomers and amyloid fibrils is suggested to be the pathogenic mechanism for Parkinson’s disease (PD). Recent studies have indicated that oligomeric species of α-Syn are more cytotoxic than their mature fibrillar counterparts, which are responsible for dopaminergic neuronal cell death in PD. Therefore, the effective therapeutic strategies for tackling aggregation-associated diseases would be either to prevent aggregation or to modulate the aggregation process to minimize the formation of toxic oligomers during aggregation. In this work, we showed that arginine-substituted α-Syn ligands, based on the most aggregation-prone sequence of α-Syn, accelerate the protein aggregation in a concentration-dependent manner. To elucidate the mechanism by which Arg-substituted peptides could modulate α-Syn aggregation kinetics, we performed surface plasmon resonance (SPR) spectroscopy, nuclear magnetic resonance (NMR) studies, and all-atom molecular dynamics (MD) simulation. The SPR analysis showed a high binding potency of these peptides with α-Syn but one that was nonspecific in nature. The two-dimensional NMR studies suggest that a large stretch within the C-terminus of α-Syn displays a chemical shift perturbation upon interacting with Arg-substituted peptides, indicating C-terminal residues of α-Syn might be responsible for this class of peptide binding. This is further supported by MD simulation studies in which the Arg-substituted peptide showed the strongest interaction with the C-terminus of α-Syn. Overall, our results suggest that the binding of Arg-substituted ligands to the highly acidic C-terminus of α-Syn leads to reduced charge density and flexibility, resulting in accelerated aggregation kinetics. This may be a potentially useful strategy while designing peptides, which act as α-Syn aggregation modulators

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

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

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

Hydrophobic surface exposure of oligomers.

<p>Hydrophobic surface exposure in terms of NR binding by Mel and PP samples, incubated for two weeks in presence and absence of heparin. The data suggesting increased hydrophobic surface exposure during heparin-induced peptide oligomerization.</p