Molecular Mechanism of Mutant p53 Stabilization: The Role of HSP70 and MDM2

Abstract

Numerous p53 missense mutations possess gain-of-function activities. Studies in mouse models have demonstrated that the stabilization of p53 R172H (R175H in human) mutant protein, by currently unknown factors, is a prerequisite for its oncogenic gain-of-function phenotype such as tumour progression and metastasis. Here we show that MDM2-dependent ubiquitination and degradation of p53 R175H mutant protein in mouse embryonic fibroblasts is partially inhibited by increasing concentration of heat shock protein 70 (HSP70/HSPA1-A). These phenomena correlate well with the appearance of HSP70-dependent folding intermediates in the form of dynamic cytoplasmic spots containing aggregate-prone p53 R175H and several molecular chaperones. We propose that a transient but recurrent interaction with HSP70 may lead to an increase in mutant p53 protein half-life. In the presence of MDM2 these pseudoaggregates can form stable amyloid-like structures, which occasionally merge into an aggresome. Interestingly, formation of folding intermediates is not observed in the presence of HSC70/HSPA8, the dominant-negative K71S variant of HSP70 or HSP70 inhibitor. In cancer cells, where endogenous HSP70 levels are already elevated, mutant p53 protein forms nuclear aggregates without the addition of exogenous HSP70. Aggregates containing p53 are also visible under conditions where p53 is partially unfolded: 37°C for temperature-sensitive variant p53 V143A and 42°C for wild-type p53. Refolding kinetics of p53 indicate that HSP70 causes transient exposure of p53 aggregate-prone domain(s). We propose that formation of HSP70- and MDM2-dependent protein coaggregates in tumours with high levels of these two proteins could be one of the mechanisms by which mutant p53 is stabilized. Moreover, sequestration of p73 tumour suppressor protein by these nuclear aggregates may lead to gain-of-function phenotypes.

Chaperone-mediated autophagy degrades mutant p53

“…It has recently been shown that mutant p53 proteins may form amyloid-like aggregates in cancer cells (Ano Bom et al 2012), and the ubiquitination and proteasomal degradation of mutant p53 is inhibited by chaperone proteins that promote their aggregation (Wiech et al 2012). Since different alleles of mutant p53 showed a different propensity to form amyloid-like structures (Ano Bom et al 2012), we hypothesize that the aggregation of mutant p53 may show allele specificity, which may affect its ubiquitination.…”

“…In addition, mutant p53 has been reported to misfold and form amyloid oligomers and fibrils (Ano Bom et al 2012), which might be intrinsically resistant to proteasomal degradation. Indeed, interaction of mutant p53 with chaperone proteins such as Hsp70 has been shown to inhibit its ubiquitination, mediated by MDM2 and proteasomal degradation, and promote its aggregation (Wiech et al 2012). Thus, how to effectively promote the degradation of mutant p53 in cancer cells is an important question to be investigated.…”

The Molecular Chaperone HSP70 Binds to and Stabilizes NOD2, an Important Protein Involved in Crohn Disease

“…There are many examples in the literature supporting this idea. For example, p53 and mutants of this protein are protected from degradation in the presence of excess HSP70 (50). In addition, human ether-a-go-go-related gene, a subunit of the potassium current I protein, is stabilized when HSP70 is overexpressed (51).…”

The aggregation of mutant p53 produces prion-like properties in cancer

“…(2) MDM2 and HSP70 might stabilize mutant p53 by forming structures named "pseudo-aggregates," which can be involved in p53 intracellular aggregation. 71 (3) The co-aggregation of p53 with its partalogs, p63 and p73, has been described by Xu et al 9 (4) p53 displays prionoid characteristics, since mutp53 aggregates are able to induce wTp53 conversion to the misfolded, aggregation-prone conformation. 8 These p53 aggregates might be captured by other cells through macropinocitosis.…”

“…69,70 Certain interactions have been proposed to stabilize p53 mutants in cells. The transient but recurrent interaction between p53 and the cellular chaperone HSP70 (Heat-Shock Protein 70) may promote an increase in the half-life of mutant p53 protein; in addition, in the presence of MDM2, these "pseudoaggregates" can form stable amyloid-like structures 71 that appear to be associated with an altered structural conformation of p53.…”