Analysis and comparison of amorphous & amyloid aggregates of the tumor suppressor p53.

Abstract

Retention of a function of the tumor suppressor protein p53 is important in inhibiting tumorigenesis. In fact, p53 is mutated in around half of all cancers. The hotspot mutations are highly localized in the DNA-binding domain of p53 (p53-DBD), one of the domains in p53. The wild-type p53-DBD has been reported to be aggregation-prone and hotspot mutations increase the aggregation. Furthermore, it has been proposed that when p53 forms aggregates, the active wild-type p53 is incorporated into the aggregates, causing it to lose its function. p53 forms at least two types of aggregates - amorphous aggregates and amyloid aggregates - simultaneously. In this study, we hypothesized that there are factors that affect the formation of two types of p53 aggregates in the cell since intracellular p53 actually functions. We investigated the effects of intracellular osmolytes on p53 aggregation and function. The DNA binding and the degree of aggregation of p53-DBD were evaluated by gel shift assay and by monitoring the fluorescence of thioflavin T and 8,1-ANS, respectively. The shape of the aggregates was observed by fluorescence microscopy and the structural transition was monitored by tryptophan fluorescence detection. We found that intracellular osmolytes inhibit amorphous aggregation and denaturation of p53. Furthermore, these osmolytes suppressed the loss of DNA-binding ability associated with aggregation. We have revealed not only the effects of intracellular osmolytes but also the differences in the properties of the two types of aggregates. Interestingly, amyloid aggregates of p53-DBD were hardly formed under conditions of loss of function and were promoted under conditions where the function is retained. Our results overturn the established hypothesis that amyloid is the main cause of loss of function of p53.