18 research outputs found
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
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>
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
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
Amyloid Fibrils with Positive Charge Enhance Retroviral Transduction in Mammalian Cells
Amyloid fibrils are cross-β-sheet-rich
protein/peptide fibrils
that are typically associated with neurodegenerative diseases such
as Parkinson’s and Alzheimer’s disease. Recently, functional
amyloids have been discovered where amyloids are implicated in performing
normal physiological functions of the host organism rather than creating
diseases. The ability of amyloids to interact with the cell membrane
and other small biomolecules exhibits its great potential to be used
as a biomaterial for cell adhesion and gene delivery system. Given
the established ability of semen-derived amyloids to concentrate HIV
in semen and that of charged polymers as an enhancer of retroviral
gene transfer, we hypothesized that charged amyloid fibrils can augment
virus-mediated delivery system. We show that amyloids of α-synuclein
formed in the presence and absence of cationic polymers chitosan and
amyloid of poly-l-lysine can interact with lentiviral particles
and enhance transduction efficiency in cells. The amyloid nanofibrils
increase transduction efficiency up to ∼4 fold similar to widely
used cationic polymer Polybrene. This study shows that amyloid nanofibril
scaffolds may be used as targeted gene delivery systems
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
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
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
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