Selective functional inhibition of a tumor-derived p53 mutant by cytosolic chaperones identified using split-YFP in budding yeast

Abstract

Life requires the oligomerization of individual proteins into higher-order assemblies. In order to form functional oligomers, monomers must adopt appropriate three-dimensional structures. Molecular chaperones transiently bind nascent or misfolded proteins to promote proper folding. Single missense mutations frequently cause disease by perturbing folding despite chaperone engagement. A misfolded mutant capable of oligomerizing with wild-type proteins can dominantly poison oligomer function. We previously found evidence that human-disease-linked mutations in Saccharomyces cerevisiae septin proteins slow folding and attract chaperones, resulting in a kinetic delay in oligomerization that prevents the mutant from interfering with wild-type function. Here we build upon our septin studies to develop a new approach for identifying chaperone interactions in living cells, and use it to expand our understanding of chaperone involvement, kinetic folding delays, and oligomerization in the recessive behavior of tumor-derived mutants of the tumor suppressor p53. We find evidence of increased binding of several cytosolic chaperones to a recessive, misfolding-prone mutant, p53(V272M). Similar to our septin results, chaperone overexpression inhibits the function of p53(V272M) with minimal effect on the wild type. Unlike mutant septins, p53(V272M) is not kinetically delayed under conditions in which it is functional. Instead, it interacts with wild-type p53 but this interaction is temperature sensitive. At high temperatures or upon chaperone overexpression, p53(V272M) is excluded from the nucleus and cannot function or perturb wild-type function. Hsp90 inhibition liberates mutant p53 to enter the nucleus. These findings provide new insights into the effects of missense mutations.

Selected humanization of yeast U1 snRNP leads to global suppression of pre-mRNA splicing and mitochondrial dysfunction in the budding yeast

“…Yeast cells were stained for viability using Evans Blue and mitochondrial ROS using dihydrorhodamine (Gomez et al 2014). All microscopy work was done using EVOSfl all-in-one Olympus microscope (Advanced Microscopy Group, Bothell, WA) as described (Denney et al 2021).…”

Simultaneous co‐overexpression of Saccharomyces cerevisiae septins Cdc3 and Cdc10 drives pervasive, phospholipid‐, and tag‐dependent plasma membrane localization

“…We saw no PM mislocalization or dominant-negative functional effects in previous studies with these same BiFC-tagged versions of Cdc3 and Cdc10 (Denney et al, 2021;Garcia et al, 2016;Weems & McMurray, 2017), but in those studies, the tagged septins were expressed at endogenous levels from the native promoters. We systematically manipulated individual features of the BiFC co- First, we made an improved version of the plasmid.…”

The APE2 Exonuclease Is a Client of the Hsp70–Hsp90 Axis in Yeast and Mammalian Cells

“…However, previous studies have shown that even a single divergent amino acid between Ssa1 and Ssa2 can produce differences in their ability to modulate prion propagation and protein degradation [ 36 ]. A recent study observed a parallel defect in septin levels in Ssa1-expressing yeast [ 37 ]. Future research, possibly involving a comparative interactome study of Ssa proteins, may shed light on this issue [ 34 ].…”