15 research outputs found
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>
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
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
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
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
Hydrodynamic radius of oligomers.
<p>Dynamic light scattering (DLS) was performed to obtain the hydrodynamic radius (Rh) of peptide samples incubated for two weeks in presence and absence of heparin. The Rh values of peptides incubated in the presence of heparin increased considerably.</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<sup>-1</sup>). 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
Cytotoxic Helix-Rich Oligomer Formation by Melittin and Pancreatic Polypeptide
<div><p>Conversion of amyloid fibrils by many peptides/proteins involves cytotoxic helix-rich oligomers. However, their toxicity and biophysical studies remain largely unknown due to their highly dynamic nature. To address this, we chose two helical peptides (melittin, Mel and pancreatic polypeptide, PP) and studied their aggregation and toxicity. Mel converted its random coil structure to oligomeric helical structure upon binding to heparin; however, PP remained as helix after oligomerization. Interestingly, similar to Parkinson’s associated α-synuclein (AS) oligomers, Mel and PP also showed tinctorial properties, higher hydrophobic surface exposure, cellular toxicity and membrane pore formation after oligomerization in the presence of heparin. We suggest that helix-rich oligomers with exposed hydrophobic surface are highly cytotoxic to cells irrespective of their disease association. Moreover as Mel and PP (in the presence of heparin) instantly self-assemble into stable helix-rich amyloidogenic oligomers; they could be represented as models for understanding the biophysical and cytotoxic properties of helix-rich intermediates in detail.</p></div