Comparison of Synthetic Neuronal Model Membrane Mimics in Amyloid Aggregation at Atomic Resolution.

Alzheimer's disease (AD) is a severe neurodegenerative disorder caused by abnormal accumulation of toxic amyloid plaques of the amyloid-beta (Aß) or the tau proteins in the brain. The plaque deposition leading to the collapse of the cellular integrity is responsible for a myriad of surface phenomena acting at the neuronal lipid interface. Recent years have witnessed dysfunction of the blood-brain barriers (BBB) associated with AD. Several studies support the idea that BBB acts as a platform for the formation of misfolded Aß peptide, promoting oligomerization and fibrillation, compromising the overall integrity of the central nervous system. While the amyloid plaque deposition has been known to be responsible for the collapse of the BBB membrane integrity, the causal effect relationship between BBB and Aß amyloidogenesis remains unclear. In this study, we have used physiologically relevant synthetic model membrane systems to gain atomic insight into the functional aspects of the lipid interface. Here, we have used a minimalist BBB mimic, POPC/POPG/cholesterol/GM1, to compare with the native BBB (total lipid brain extract (TLBE)), to understand the molecular events occurring in the membrane-induced Aß40 amyloid aggregation. Our study showed that the two membrane models accelerated the Aß40 aggregation kinetics with differential secondary structural transitions of the peptide. The observed structural transitions are defined by the lipid compositions, which in turn undermines the differences in lipid surface phenomena, leading to peptide induced cellular toxicity in the neuronal membrane.

Lipopolysaccharide from Gut Microbiota Modulates α-Synuclein Aggregation and Alters Its Biological Function.

Altered intestinal permeability has been correlated with Parkinson's pathophysiology in the enteric nervous system, before manifestations in the central nervous system (CNS). The inflammatory endotoxin or lipopolysaccharide (LPS) released by gut bacteria is known to modulate α-synuclein amyloidogenesis through the formation of intermediate nucleating species. Here, biophysical techniques in conjunction with microscopic images revealed the molecular interaction between lipopolysaccharide and α-synuclein that induce rapid nucleation events. This heteromolecular interaction stabilizes the α-helical intermediates in the α-synuclein aggregation pathway. Multitude NMR studies probed the residues involved in the LPS-binding structural motif that modulates the nucleating forms, affecting the cellular internalization and associated cytotoxicity. Collectively, our data characterizes this heteromolecular interaction associated with an alternative pathway in Parkinson's disease progression.

Bipartite Role of Heat Shock Protein 90 (Hsp90) Keeps CRAF Kinase Poised for Activation.

CRAF kinase maintains cell viability, growth, and proliferation by participating in the MAPK pathway. Unlike BRAF, CRAF requires continuous chaperoning by Hsp90 to retain MAPK signaling. However, the reason behind the continuous association of Hsp90 with CRAF is still elusive. In this study, we have identified the bipartite role of Hsp90 in chaperoning CRAF kinase. Hsp90 facilitates Ser-621 phosphorylation of CRAF and prevents the kinase from degradation. Co-chaperone Cdc37 assists in this phosphorylation event. However, after folding, the stability of the kinase becomes insensitive to Hsp90 inhibition, although the physical association between Hsp90 and CRAF remains intact. We observed that overexpression of Hsp90 stimulates MAPK signaling by activating CRAF. The interaction between Hsp90 and CRAF is substantially increased under an elevated level of cellular Hsp90 and in the presence of either active Ras (RasV12) or EGF. Surprisingly, enhanced binding of Hsp90 to CRAF occurs prior to the Ras-CRAF association and facilitates actin recruitment to CRAF for efficient Ras-CRAF interaction, which is independent of the ATPase activity of Hsp90. However, monomeric CRAF (CRAFR401H) shows abrogated interaction with both Hsp90 and actin, thereby affecting Hsp90-dependent CRAF activation. This finding suggests that stringent assemblage of Hsp90 keeps CRAF kinase equipped for participating in the MAPK pathway. Thus, the role of Hsp90 in CRAF maturation and activation acts as a limiting factor to maintain the function of a strong client like CRAF kinase.

Inhibition of Insulin Amyloid Fibrillation by a Novel Amphipathic Heptapeptide: MECHANISTIC DETAILS STUDIED BY SPECTROSCOPY IN COMBINATION WITH MICROSCOPY.

The aggregation of insulin into amyloid fibers has been a limiting factor in the development of fast acting insulin analogues, creating a demand for excipients that limit aggregation. Despite the potential demand, inhibitors specifically targeting insulin have been few in number. Here we report a non-toxic and serum stable-designed heptapeptide, KR7 (KPWWPRR-NH2), that differs significantly from the primarily hydrophobic sequences that have been previously used to interfere with insulin amyloid fibrillation. Thioflavin T fluorescence assays, circular dichroism spectroscopy, and one-dimensional proton NMR experiments suggest KR7 primarily targets the fiber elongation step with little effect on the early oligomerization steps in the lag time period. From confocal fluorescence and atomic force microscopy experiments, the net result appears to be the arrest of aggregation in an early, non-fibrillar aggregation stage. This mechanism is noticeably different from previous peptide-based inhibitors, which have primarily shifted the lag time with little effect on later stages of aggregation. As insulin is an important model system for understanding protein aggregation, the new peptide may be an important tool for understanding peptide-based inhibition of amyloid formation.

Biophysical Characterization of Essential Phosphorylation at the Flexible C-Terminal Region of C-Raf with 14-3-3ζ Protein.

Phosphorylation at the C-terminal flexible region of the C-Raf protein plays an important role in regulating its biological activity. Auto-phosphorylation at serine 621 (S621) in this region maintains C-Raf stability and activity. This phosphorylation mediates the interaction between C-Raf and scaffold protein 14-3-3ζ to activate the downstream MEK kinase pathway. In this study, we have defined the interaction of C-terminal peptide sequence of C-Raf with 14-3-3ζ protein and determined the possible structural adaptation of this region. Biophysical elucidation of the interaction was carried out using phosphopeptide (residue number 615-630) in the presence of 14-3-3ζ protein. Using isothermal titration calorimetry (ITC), a high binding affinity with micro-molar range was found to exist between the peptide and 14-3-3ζ protein, whereas the non-phosphorylated peptide did not show any appreciable binding affinity. Further interaction details were investigated using several biophysical techniques such as circular dichroism (CD), fluorescence, and nuclear magnetic resonance (NMR) spectroscopy, in addition to molecular modeling. This study provides the molecular basis for C-Raf C-terminal-derived phosphopeptide interaction with 14-3-3ζ protein as well as structural insights responsible for phosphorylated S621-mediated 14-3-3ζ binding at an atomic resolution.