The function of proteins relies on their compact three-dimensional (3D) structures. Nascent polypeptides achieve their correct folding structure as dictated by their peptide sequence. The folding of proteins into their discrete 3D structures is the most fundamental example of biological self-assembly. Amyloid fibrils are thread like highly ordered protein aggregates with a core region formed by repetitive arrays of β-sheets oriented perpendicularly to the fibril axis. Aggregation of misfolded proteins into amyloid conformations that escape cellular quality-control mechanisms is a feature of wide range of debilitating diseases. The polypeptides adopt a common fold, cross-β-sheet which displays a signature “cross,” as seen in X-ray diffraction patterns. The conversion of peptides and proteins into aggregates is associated with several pathological conditions in humans, including Alzheimer’s disease (AD), Parkinson’s disease, transmissible sporadic encephalopathies cataract, and diabetes type II (Invernizzi et al., 2012
). Alzheimer's disease is a fatal progressive neurodegenerative disorder and the underlying pathogenesis is the amyloid aggregation specifically β-amyloid peptide (Aβ) which accumulate in the brain as extracellular amyloid deposits. The aggregates exert cytotoxic effects by triggering a cascade of reactions resulting in neuronal dysfunction and dementia leading to death. Such disorders are predicted to cause a crisis in public health in the near future. Thus, there is an increasing need to understand the mechanism of protein misfolding and protein aggregation and this area has emerged as a high priority in biological and medical research which would lead to developing of effective therapies.
Methods of Amyloid detection
Many approaches exist to study amyloids in vitro
, but the techniques available for the study of amyloid aggregation in cells are limited and non-specific. A gold standard in vitro
method to screen for protein aggregation inhibitors employs a purified recombinant protein and Thioflavin-T (ThT) fluorometric assay to monitor aggregation. One of the limitations of in vitro
evaluation of compounds against isolated protein targets is the failure to reproduce when assayed in vivo
. However, fluorescence from ThT dyes have a spectral overlap of intrinsic fluorescence with cellular constituents such as Flavin and NADPH. An alternative is to use animal models to screen chemical libraries at early stages of drug discovery, but they are cost prohibitive and the assays are complex. Prokaryotic cells have recently emerged as suitable model systems to study mechanisms of amyloid formation and its inhibition.
Advantages of bacterial model system with PROTEOSTAT® dyes technology
Navarro and colleagues
from University of Barcelona have demonstrated the successful application of bacterial systems as an effective and alternative system to screen for amyloid inhibitors on fluorescence spectroscopy and flow cytometry utilizing the advantages of novel red fluorescent Enzo’s PROTEOSTAT® dye. They expressed Aβ 40 and Aβ 42 in E.coli
systems and established a screening protocol by benchmarking known amyloid inhibitors. Though bacterial systems do not recapitulate human multicellular systems, they have been shown to be of value in expression of aggregation-prone proteins. Relying on newly synthesized polypeptide mimics is physiologically closer than using a purified protein/peptide in in vitro
aggregation assay. PROTEOSTAT® dye has a larger dynamic range and its fluorescence is not influenced by nucleic acids, its ability to discriminate between intracellular-amyloid like deposits and non-ordered aggregates. The combined use of PROTEOSTAT® with bacterial model systems has the potential to supplement the existing methods to screen small molecules to develop lead candidates for the treatment of amyloidogenic disease. Enzo Life Sciences offers comprehensive tools for advancing your research and detecting protein aggregates.