ABSTRACT
Many of the discoveries of functional amyloid to date have occurred via serendipity in scientific research, often in laboratories that had other, initially unrelated projects on pathogenic amyloid, or staff with prior experience in the pathogenic amyloid field. It is thus highly plausible that amyloid occurs far more commonly in nature than is currently apparent, but has remained unidentified. This may primarily be due to continuing problems with definitive identification of amyloid and the difficulties associated with analysing the composition of highly insoluble amyloid fibrils. Currently, there are a broad range of techniques that are used to suggest the presence of amyloid including Thioflavin T fluorescence, Congo red binding, luminescent conjugated polyelectrolyte probes, circular dichroism spectroscopy, Raman spectroscopy, and Fourier transform infrared spectroscopy, fibril observation in electron microscopy or atomic force microscopy images, X-ray fibre diffraction and the use of amyloid specific antibodies. However, no single technique is accepted in isolation as providing a definitive identification of amyloid due to the large number of false positives that can occur and the often highly specific requirements of the sample preparation not to incur a false negative. As a consequence the onus is on the researcher to obtain as many positive indications of amyloid as is possible for a particular sample using a number of the available techniques. This
can often be problematic as it is unlikely that access to numerous techniques will be available at a single location, and thus convincing identification of amyloid remains a major hurdle in the development of the field, especially in the case of serendipitous discoveries. The isolation of fibrils from natural materials is tremendously challenging, unlike the use of fibrils formed in vitro from commercially available peptides.11 In addition, the extreme structural stability of functional amyloid makes it difficult to apply conventional protein science approaches to their purification and characterization. Special techniques such as preparative gel electrophoresis and specific solubilization by organic acids are often required. A disproportionate amount of time can be spent optimizing these steps, severely delaying the subsequent characterization of amyloid. However, this characterization is often a crucial step in proving that the amyloid serves a biological function for the organism, rather than merely the coincidental presence of amyloid in a sample of natural material. Due to the breadth of functions identified to date, it is likely that there are significant structural differences at the molecular level between fibrils that have been harvested from different living organisms. However, the challenges of purification and characterization will need to be overcome before the full extent of these differences can be explored and understood.
