RESUMO
Although it has been known for more than 60 years that the cause of sickle cell disease is polymerization of a hemoglobin mutant, hydroxyurea is the only drug approved for treatment by the US Food and Drug Administration. This drug, however, is only partially successful, and the discovery of additional drugs that inhibit fiber formation has been hampered by the lack of a sensitive and quantitative cellular assay. Here, we describe such a method in a 96-well plate format that is based on laser-induced polymerization in sickle trait cells and robust, automated image analysis to detect the precise time at which fibers distort ("sickle") the cells. With this kinetic method, we show that small increases in cell volume to reduce the hemoglobin concentration can result in therapeutic increases in the delay time prior to fiber formation. We also show that, of the two drugs (AES103 and GBT440) in clinical trials that inhibit polymerization by increasing oxygen affinity, one of them (GBT440) also inhibits sickling in the absence of oxygen by two additional mechanisms.
Assuntos
Antidrepanocíticos/farmacologia , Tamanho Celular/efeitos dos fármacos , Eritrócitos/efeitos dos fármacos , Furaldeído/análogos & derivados , Anemia Falciforme/terapia , Eritrócitos/fisiologia , Furaldeído/farmacologia , Hemoglobina Falciforme/metabolismo , Humanos , Cinética , OxigênioRESUMO
AlphaB-Crystallin is a ubiquitous small heat-shock protein (sHsp) renowned for its chaperone ability to prevent target protein aggregation. It is stress-inducible and its up-regulation is associated with a number of disorders, including those linked to the deposition of misfolded proteins, such as Alzheimer's and Parkinson's diseases. We have characterised the formation of amyloid fibrils by human alphaB-crystallin in detail, and also that of alphaA-crystallin and the disease-related mutant R120G alphaB-crystallin. We find that the last 12 amino acid residues of the C-terminal region of alphaB-crystallin are predicted from their physico-chemical properties to have a very low propensity to aggregate. (1)H NMR spectroscopy reveals that this hydrophilic C-terminal region is flexible both in its solution state and in amyloid fibrils, where it protrudes from the fibrillar core. We demonstrate, in addition, that the equilibrium between different protofilament assemblies can be manipulated and controlled in vitro to select for particular alphaB-crystallin amyloid morphologies. Overall, this study suggests that there could be a fine balance in vivo between the native functional sHsp state and the formation of amyloid fibrils.
Assuntos
Amiloide/química , Amiloide/metabolismo , Cadeia A de alfa-Cristalina/química , Cadeia A de alfa-Cristalina/metabolismo , Cadeia B de alfa-Cristalina/química , Cadeia B de alfa-Cristalina/metabolismo , Amiloide/biossíntese , Amiloide/ultraestrutura , Humanos , Concentração de Íons de Hidrogênio , Cinética , Microscopia de Força Atômica , Ressonância Magnética Nuclear Biomolecular , Ligação Proteica , Cadeia A de alfa-Cristalina/ultraestrutura , Cadeia B de alfa-Cristalina/genética , Cadeia B de alfa-Cristalina/ultraestruturaRESUMO
We report the detailed mechanical characterization of individual amyloid fibrils by atomic force microscopy and spectroscopy. These self-assembling materials, formed here from the protein insulin, were shown to have a strength of 0.6 +/- 0.4 GPa, comparable to that of steel (0.6-1.8 GPa), and a mechanical stiffness, as measured by Young's modulus, of 3.3 +/- 0.4 GPa, comparable to that of silk (1-10 GPa). The values of these parameters reveal that the fibrils possess properties that make these structures highly attractive for future technological applications. In addition, analysis of the solution-state growth kinetics indicated a breakage rate constant of 1.7 +/- 1.3 x 10(-8) s(-1), which reveals that a fibril 10 mum in length breaks spontaneously on average every 47 min, suggesting that internal fracturing is likely to be of fundamental importance in the proliferation of amyloid fibrils and therefore for understanding the progression of their associated pathogenic disorders.
Assuntos
Amiloide/metabolismo , Amiloide/ultraestrutura , Nanoestruturas/química , Nanoestruturas/ultraestrutura , Amiloide/química , Animais , Bovinos , Insulina/química , Insulina/metabolismo , Cinética , Microscopia de Força Atômica , Ligação ProteicaRESUMO
Using atomic force microscopy height maps, we resolve and quantify torsional fluctuations in one-dimensional amyloid fibril aggregates self-assembled from three different representative polypeptide systems. Furthermore, we show that angular correlation in these nanoscale structures is maintained over several microns, corresponding to many thousands of molecules along the fibril axis. We model disorder in the fibril in respect of both thermal fluctuations and structural defects, and determine quantitative values for the defect density, as well as the energy scales involved in the fundamental interactions stabilizing these generic structures.