Large Proteins Have a Great Tendency to Aggregate but a Low Propensity to Form Amyloid Fibrils

The assembly of soluble proteins into ordered fibrillar aggregates with cross-β structure is an essential event of many human diseases. The polypeptides undergoing aggregation are generally small in size. To explore if the small size is a primary determinant for the formation of amyloids under pathological conditions we have created two databases of proteins, forming amyloid-related and non-amyloid deposits in human diseases, respectively. The size distributions of the two protein populations are well separated, with the systems forming non-amyloid deposits appearing significantly larger. We have then investigated the propensity of the 486-residue hexokinase-B from Saccharomyces cerevisiae (YHKB) to form amyloid-like fibrils in vitro. This size is intermediate between the size distributions of amyloid and non-amyloid forming proteins. Aggregation was induced under conditions known to be most effective for amyloid formation by normally globular proteins: (i) low pH with salts, (ii) pH 5.5 with trifluoroethanol. In both situations YHKB aggregated very rapidly into species with significant β-sheet structure, as detected using circular dichroism and X-ray diffraction, but a weak Thioflavin T and Congo red binding. Moreover, atomic force microscopy indicated a morphology distinct from typical amyloid fibrils. Both types of aggregates were cytotoxic to human neuroblastoma cells, as indicated by the MTT assay. This analysis indicates that large proteins have a high tendency to form toxic aggregates, but low propensity to form regular amyloid in vivo and that such a behavior is intrinsically determined by the size of the protein, as suggested by the in vitro analysis of our sample protein

Vestnik statistiki : ezemesjacnyj teoreticeskij naucno-prakticeskij zurnal Gosudarstvennogo Komiteta SSSR po Statistike

Chaperones are the primary regulators of the proteostasis network and are known to facilitate protein folding, inhibit protein aggregation, and promote disaggregation and clearance of misfolded aggregates inside cells. We have tested the effects of five chaperones on the toxicity of misfolded oligomers preformed from three different proteins added extracellularly to cultured cells. All the chaperones were found to decrease oligomer toxicity significantly, even at very low chaperone/protein molar ratios, provided that they were added extracellularly rather than being overexpressed in the cytosol. Infrared spectroscopy and site-directed labeling experiments using pyrene ruled out structural reorganizations within the discrete oligomers. Rather, confocal microscopy, SDS-PAGE, and intrinsic fluorescence measurements indicated tight binding between oligomers and chaperones. Moreover, atomic force microscopy imaging indicated that larger assemblies of oligomers are formed in the presence of the chaperones. This suggests that the chaperones bind to the oligomers and promote their assembly into larger species, with consequent shielding of the reactive surfaces and a decrease in their diffusional mobility. Overall, the data indicate a generic ability of chaperones to neutralize extracellular misfolded oligomers efficiently and reveal that further assembly of protein oligomers into larger species can be an effective strategy to neutralize such extracellular species

A causative link between the structure of aberrant protein oligomers and their toxicity.

The aberrant assembly of peptides and proteins into fibrillar aggregates proceeds through oligomeric intermediates that are thought to be the primary pathogenic species in many protein deposition diseases. We describe two types of oligomers formed by the HypF-N protein that are morphologically and tinctorially similar, as detected with atomic force microscopy and thioflavin T assays, though one is benign when added to cell cultures whereas the other is toxic. Structural investigation at a residue-specific level using site-directed labeling with pyrene indicated differences in the packing of the hydrophobic interactions between adjacent protein molecules in the oligomers. The lower degree of hydrophobic packing was found to correlate with a higher ability to penetrate the cell membrane and cause an influx of Ca2+ ions. Our findings suggest that structural flexibility and hydrophobic exposure are primary determinants of the ability of oligomeric assemblies to cause cellular dysfunction and its consequences, such as neurodegeneration