Oligomeric proteins: Toxicity, aggregation, and polyvalent inhibition.

摘要

While oligomeric proteins are ubiquitous in nature, there are adverse effects associated with some oligomeric proteins. Of interest to us are two such oligomeric proteins: Serum Amyloid A (SAA) and the heptameric receptor-binding subunit of anthrax toxin [PA63]7. Although the exact role of SAA in disease is poorly understood, its misfolding and fibrillation has been linked to the disease Reactive Amyloidosis and several other diseases associated with chronic inflammation. On the other hand, [PA63]7 helps transport the enzyme lethal factor (LF) across the cell membrane, where it exerts its toxic effects and causes cell death in anthrax.;We studied the oligomerization, misfolding, and aggregation properties of different isoforms of SAA found in mice and humans. We found that a pathologically-relevant isoform of human SAA, hSAA1.1, forms alpha helix-rich native oligomers which misfold and form cross beta rich aggregates. Using techniques ranging from fluorescence spectroscopy to atomic force microscopy, solubility measurement, circular dichroism, and immunoblot studies we identified a possible fibrillation pathway for this protein. We also studied the interactions of SAA amyloid aggregates with model lipid membranes---both planar lipid bilayers and liposomes. We found that a single amino-acid change in the N-terminus of hSAA1.1 significantly modulates its fibrillation and interactions with lipid membranes. In a separate study, we used a variety of bioanalytical characterization techniques like size exclusion chromatography and dynamic light scattering to investigate the role of the carboxy terminus of SAA in the oligomerization and misfolding of this protein. We found that the proline-rich and presumably disordered carboxy-terminus of SAA can also influence native oligomer formation and the rate of fibril formation by inhibiting misfolding of SAA into other structures.;Finally, we designed polyvalent molecules that bind with high affinity to protein oligomers. Specifically, we used a structure-based design strategy to engineer monodisperse and well-defined polyvalent inhibitors of anthrax lethal toxin using polypeptide scaffolds. Specifically, we expressed and purified polypeptides incorporating multiple copies of an inhibitory [PA63] 7-binding peptide separated by peptide linkers. A characteristic feature of this design was the ability to control precisely and independently the valency and the spacing between ligands. We used computational studies to guide our choice of linker lengths and tested the validity of the design using cell viability studies. We found that the resulting polypeptides were five orders of magnitude more potent than monovalent ligands in inhibiting anthrax lethal toxin in vitro. We have therefore demonstrated a simple strategy to rationally design monodisperse polyvalent molecules that could be broadly applicable for the inhibition of toxins and pathogens.