Binding with Nucleic Acids or Glycosaminoglycans Converts Soluble Protein Oligomers to Amyloid

Abstract

Ample evidence suggests that almost all polypeptides can either adopt a native structure (folded or intrinsically disordered) or form misfolded amyloid fibrils. Soluble protein oligomers exist as an intermediate between these two states, and their cytotoxicity has been implicated in the pathology of multiple human diseases. However, the mechanism by which soluble protein oligomers develop into insoluble amyloid fibrils is not clear, and investigation of this important issue is hindered by the unavailability of stable protein oligomers. Here, we have obtained stabilized protein oligomers generated from common native proteins. These oligomers exert strong cytotoxicity and display a common conformational structure shared with known protein oligomers. They are soluble and remain stable in solution. Intriguingly, the stabilized protein oligomers interact preferentially with both nucleic acids and glycosaminoglycans (GAG), which facilitates their rapid conversion into insoluble amyloid. Concomitantly, binding with nucleic acids or GAG strongly diminished the cytotoxicity of the protein oligomers. EGCG, a small molecule that was previously shown to directly bind to protein oligomers, effectively inhibits the conversion to amyloid. These results indicate that stabilized oligomers of common proteins display characteristics similar to those of disease-associated protein oligomers and represent immediate precursors of less toxic amyloid fibrils. Amyloid conversion is potently expedited by certain physiological factors, such as nucleic acids and GAGs. These findings concur with reports of cofactor involvement with disease-associated amyloid and shed light on potential means to interfere with the pathogenic properties of misfolded proteins.

Rapid Generation of Amyloid from Native Proteins <em>In vitro</em>

“…In a control reaction, native HSA (1 mg) was mixed with DNA. The result demonstrates a general phenomenon of stabilized protein oligomer which gains ability to interact with nucleic acids, in contrast with its native form 16 . Click here to view larger image.…”

“…To ensure the quality of stabilized soluble protein oligomers, one can perform several simple tests. As characterized previously 16 , stabilized soluble protein oligomers can readily bind to nucleic acids, including both DNA and RNA, in a sequence-independent manner. Therefore, a gel shift assay can be performed to visualize the direct interaction between the stabilized soluble protein oligomers and DNA, which would result in the retarded migration of DNA on an agarose gel (Figure 2).…”

“…We have found that HSA oligomers can readily form amyloid the presence of sufficient amount of nucleic acids or heparin at a cofactor: protein ratio > 1:3 16 . At the same concentration, heparin is slightly more potent than DNA to inhibit the cytotoxicity of prion peptide and HSA oligomers, suggesting soluble protein oligomers may differentially interact with various cofactors.…”

“…The insoluble aggregates obtained display several characteristic properties of amyloid: they are stained positive with amyloid-specific dyes Thioflavin T, Thioflavin S, and Congo Red 16,27 . In addition, fibrous structures are visible when observed under electron microscopy.…”

“…However, as described earlier, almost any protein in principle can adopt amyloid conformation under the appropriate conditions. We recently established a method to prepare stabilized soluble oligomers of native proteins, which readily form amyloid in the presence of various cofactors 16 . The resulting amyloid fibrils reflect the complex nature of the terminal protein misfolding aggregates 17,18 .…”