An amyloidogenic hexapeptide from the cataract-associated γD-crystallin is a model for the full-length protein and is inhibited by naphthoquinone-tryptophan hybrids.

The eye lens is rich in proteins called crystallins, whose native conformation is crucial for preserving its transparency. With aging, crystallins may be exposed to environmental changes, which could lead to their aggregation and eventually to cataract development. Human γD-crystallin, among the most abundantly expressed γ-crystallins in the lens, was shown to form amyloid aggregates under denaturing conditions in vitro. However, the exact mechanism of aggregation remains to be clearly defined. Here, using prediction algorithms and biophysical methods, we identified a hexapeptide 41GCWMLY46 as a most aggregative fragment in human γD-crystallin. Two aromatic naphthoquinone-tryptophan hybrid molecules (NQTrp and Cl-NQTrp), inhibited the in vitro aggregation of this hexapeptide as well as full-length γD-crystallin in a dose-dependent manner, plausibly facilitated by hydrogen bonding and aromatic contacts with the hydrophobic residues. The two compounds had no toxic effect toward retinal cell culture and reduced the cytotoxicity induced by aggregates of the hexapeptide. In addition, NQTrp and Cl-NQTrp were able to disassemble pre-formed aggregates of the hexapeptide and the full-length γD-crystallin. Our results indicate that the amyloidogenic hexapeptide is a useful model for screening inhibitors of γD-crystallin and that the NQTrp hybrid scaffolds may serve as leads for developing new drugs for treating cataract.

Arginine refolds, stabilizes, and restores function of mutant pVHL proteins in animal model of the VHL cancer syndrome.

The von Hippel-Lindau (VHL) syndrome is a rare inherited cancer, caused by mutations in the VHL gene, many of which render the VHL protein (pVHL) unstable. pVHL is a tumor-suppressor protein implicated in a variety of cellular processes, most notably in response to changes in oxygen availability, due to its role as part of an E3-ligase complex which targets the hypoxia-inducible factor (HIF) for degradation. Previously we reported, using in silico and in vitro analyses, that common oncogenic VHL mutations render pVHL less stable than the wild-type protein, distort its core domain and as a result reduce the ability of the protein to bind its target HIF-1α. Among various chemical chaperones tested, arginine was the most effective in refolding mutant of pVHL. Here we examined the consequences of administering L- or D-arginine to a Drosophila VHL model and to human renal carcinoma cells, both expressing misfolded versions of human pVHL. Arginine treatment increased pVHL solubility in both models and increased the half-life of the mutant pVHL proteins in the cell culture. In both models, L- as well as D-arginine enhanced the ability of wild-type pVHL and certain misfolded mutant versions of pVHL to bind ODD, the HIF-derived target peptide, reflecting restoration of pVHL function. Moreover, continuous feeding of Drosophila expressing misfolded versions of pVHL either L- or D-arginine rich diet rescued their lethal phenotype. Collectively, these in vivo results suggest that arginine supplementation should be examined as a potential novel treatment for VHL cancer syndrome.

Rosmarinic Acid Restores Complete Transparency of Sonicated Human Cataract Ex Vivo and Delays Cataract Formation In Vivo.

Cataract, the leading cause of vision impairment worldwide, arises from abnormal aggregation of crystallin lens proteins. Presently, surgical removal is the only therapeutic approach. Recent findings have triggered renewed interest in development of non-surgical treatment alternatives. However, emerging treatments are yet to achieve full and consistent lens clearance. Here, the first ex vivo assay to screen for drug candidates that reduce human lenticular protein aggregation was developed. This assay allowed the identification of two leading compounds as facilitating the restoration of nearly-complete transparency of phacoemulsified cataractous preparation ex vivo. Mechanistic studies demonstrated that both compounds reduce cataract microparticle size and modify their amyloid-like features. In vivo studies confirmed that the lead compound, rosmarinic acid, delays cataract formation and reduces the severity of lens opacification in model rats. Thus, the ex vivo assay may provide an initial platform for broad screening of potential novel therapeutic agents towards pharmacological treatment of cataract.

Integrating in vitro and in silico approaches to evaluate the "dual functionality" of palmatine chloride in inhibiting and disassembling Tau-derived VQIVYK peptide fibrils.

BACKGROUND: Alzheimer's disease (AD) is the most common neurodegenerative disorder which is characterized by the deposits of intra-cellular tau protein and extra-cellular amyloid-ß (Aß) peptides in the human brain. Understanding the mechanism of protein aggregation and finding compounds that are capable of inhibiting its aggregation is considered to be highly important for disease therapy. METHODS: We used an in vitro High-Throughput Screening for the identification of potent inhibitors of tau aggregation using a proxy model; a highly aggregation-prone hexapeptide fragment 306VQIVYK311 derived from tau. Using ThS fluorescence assay we screened a library of 2401 FDA approved, bio-active and natural compounds in attempt to find molecules which can efficiently modulate tau aggregation. RESULTS: Among the screened compounds, palmatine chloride (PC) alkaloid was able to dramatically reduce the aggregation propensity of PHF6 at sub-molar concentrations. PC was also able to disassemble preformed aggregates of PHF6 and reduce the amyloid content in a dose-dependent manner. Insights obtained from MD simulation showed that PC interacted with the key residues of PHF6 responsible for ß-sheet formation, which could likely be the mechanism of inhibition and disassembly. Furthermore, PC could effectively inhibit the aggregation of full-length tau and disassemble preformed aggregates. CONCLUSIONS: We found that PC possesses "dual functionality" towards PHF6 and full-length tau, i.e. inhibit their aggregation and disassemble pre-formed fibrils. GENERAL SIGNIFICANCE: The "dual functionality" of PC is valuable as a disease modifying strategy for AD, and other tauopathies, by inhibiting their progress and reducing the effect of fibrils already present in the brain.