Protein misfolding followed by aggregation is the major cause of neurodegenerative diseases such as Alzheimer’s, Parkinson’s, familial amyloid poly neuropathy (FAP), Huntington’s, type-II diabetes, etc. Common aspect of all protein aggregation diseases is the altered protein conformation known as partially unfolded amyloidogenic intermediate that is capable of assembly into amyloid structures. Recently discovered mesoscopic protein-rich clusters may act as crucial precursors for the nucleation of ordered protein solids, such as crystals, sickle hemoglobin polymers, and amyloid fibrils. These clusters challenge settled paradigms of protein condensation as the constituent protein molecules present features characteristic of both partially misfolded and native proteins. Some of their unusual features include the kinetically determined size, thermodynamically controlled number, and their distinct nature from aggregation triggered by reduction of the intramolecular S−S bonds and amyloid aggregates. We investigated the role of protein structural flexibility on its ability to induce formation of mesoscopic clusters for multiple proteins including the p53, known as guardian of genome, which contains multi dis-ordered and b-sheet rich domains; hemoglobin A, which is the major component of red blood cells and contains a compact structure rich in a-helices; antimicrobial enzyme lysozyme which is a robust model in study of protein aggregation. Whereas lysozyme and hemoglobin A demonstrate mesoscopic clusters at high protein concentrations, p53, whose aggregation is tied to cancer development, exhibits clustering at physiological temperatures for low concentrations of the protein. These findings suggest that the clusters are a product of limited protein structural flexibility. Furthermore, we discovered that the crowding environment of the inside cell significantly promotes clustering of intrinsic disordered proteins (IDPs) such as p53. About half of human cancers are associated with mutations of the tumor suppressor p53. Mutated p53 emerges as a powerful oncogene, which blocks the activity of wild-type p53 and several distinct anticancer pathways. The gained functions of the mutant have been related to the aggregation behaviors of wild-type and mutant p53. Our data reveals that in presence of crowders, the p53 clusters can capture some of the crowder molecules, which causes steric hindrance effects and raises the nucleation barrier of the aggregation. Thus these clusters can potentially act as storage of proteins and protect them from formation of toxic amyloid aggregates by providing sufficient time for the proteomic and chaperonin machinery to clear out or refold the misfolded aggregated species in the cell. The nucleation of p53 fibrils deviates from the accepted mechanism of sequential association of single solute molecule. We find the mesoscopic clusters serve as a pre-assembled precursor of high p53 concentration that facilitate fibril assembly. Fibril nucleation hosted by precursors represents a novel biological pathway, which awards unexplored avenues to suppression of protein fibrillation in aggregation diseases.
We established the mechanisms of action in blocking hematin crystallization of several classes of antimalarial drugs and related compounds. The results of my colleagues on the mechanism of hematin crystallization, concurrent with studies of parasite physiology, suggest that hemozoin crystals form in organic sub-phases comprised of a blend of several lipids, which may be suspended in the digestive vacuole or lining its walls. They have demonstrated that the formation of hematin crystals follows a classical mechanism whereby new crystal layers are nucleated on top of existing ones and grow by the association of solute molecules that leads to spreading of the layers until they cover the entire face. They have shown that quinoline-class antimalarials possess an extremely efficient pathway of inhibition of hematin crystallization by binding to specific crystal surface sites and impeding the incorporation of soluble hematin.