The misfolding and aberrant assembly of peptides and proteins into fibrillar aggregates is the hallmark of many pathologies. Fibril formation is accompanied by oligomeric species thought to be the primary pathogenic agents in many of these diseases. With the aim of identifying the structural determinants responsible for the toxicity of misfolded oligomers, we created 12 oligomeric variants from the N-terminal domain of the E. coli HypF protein (HypF-N) by replacing one or more charged amino acid residues with neutral apolar residues and allowing the mutated proteins to aggregate under two sets of conditions. The resulting oligomeric species have different degrees of cytotoxicity when added to the extracellular medium of the cells, as assessed by the extent of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction, apoptosis, and influx of Ca2+ into the cells. The structural properties of the oligomeric variants were characterized by evaluating their surface hydrophobicity with 8-anilinonaphthalene-1-sulfonate (ANS) binding and by measuring their size by means of turbidimetry as well as light scattering. We find that increases in the surface hydrophobicity of the oligomers following mutation can promote the formation of larger assemblies and that the overall toxicity correlates with a combination of both surface hydrophobicity and size, with the most toxic oligomers having high hydrophobicity and small size. These results have allowed the relationships between these three parameters to be studied simultaneously and quantitatively, and have enabled the generation of an equation that is able to rationalize and even predict toxicity of the oligomers resulting from their surface hydrophobicity and size.
SummaryIncreasing evidence suggests that the interaction of misfolded protein oligomers with cell membranes is a primary event resulting in the cytotoxicity associated with many protein-misfolding diseases, including neurodegenerative disorders. We describe here the results of a study on the relative contributions to toxicity of the physicochemical properties of protein oligomers and the cell membrane with which they interact. We altered the amount of cholesterol and the ganglioside GM1 in membranes of SH-SY5Y cells. We then exposed the cells to two types of oligomers of the prokaryotic protein HypF-N with different ultrastructural and cytotoxicity properties, and to oligomers formed by the amyloid-b peptide associated with Alzheimer's disease. We identified that the degree of toxicity of the oligomeric species is the result of a complex interplay between the structural and physicochemical features of both the oligomers and the cell membrane.
BackgroundVernal Keratoconjunctivitis (VKC) is an allergic conjunctivitis, often not easily diagnosed and properly treated. The disease is very debilitating for patients, may be complicated by corneal lesions and can evolve to keratoconus.PurposeThe exponential increase in VKC patients led us to start a close collaboration between pharmacists and allergists, ophthalmologists, and chemists. The goal was to address and solve problems caused by the lack of adequate knowledge of VKC, in order to find a diagnostic-therapeutic course, improve patient compliance and provide high-quality products as an alternative to conventional treatments.Material and methodsAfter discussions with allergists and ophthalmologists, pharmacists formulated 3 different kinds of eye drops as treatment: ciclosporin 1% and tacrolimus 0.1%, both in methylcellulose 0.15%, and ciclosporin 2% in sunflower oil. The stability of such formulations was demonstrated by using liquid chromatography coupled to a triple quadrupole mass spectrometer. The pharmacist now prepares a weekly supply of eye drops, after allergists pass on the number of children who will undergo eye examinations. Then, the pharmacist proceeds, after allergist confirmation, to arrange for eye drops to be sent directly to patients’ homes in the whole country.ResultsThe LC/MS/MS and sterility analysis results allowed the pharmacist to declare that formulations in methylcellulose 0.15% and in sunflower oil were safe for up to 45 days. Such formulations were chosen considering also patient compliance. Indeed, one of the results of the team collaboration has been the development of the formulation in sunflower oil, which can be stored at room temperature; thus leading to huge advantages in terms of patient compliance.ConclusionThe preparation of a galenical formulation of such quality has contributed to the efficacy of the treatment. Moreover, the sharing of information between medical doctors, pharmacists and nurses has led to personalised assistance that is highly responsive to health needs.ReferenceLeonardi A. Prog Retin Eye Res 2002;21(3):319–39No conflict of interest.
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