Mapping biophysical effects of the cellular environment on p53 and PTEN stability and aggregation - PROJECT SUMMARY/ABSTRACT Cancers are marked by misregulation of tumor suppressor proteins and changes to the cellular environment caused by grossly altered metabolic states. It is possible that the altered chemical environment of cancer cells induces destabilization of a protein’s folded state or protein misfolding and aggregation. Indeed, the crucial tumor suppressor proteins p53 and PTEN have been observed to form filamentous amyloid inclusions in both wild-type and cancer mutation-containing backgrounds, perhaps due to conformational landscapes changes caused by the altered cellular environment. Despite these long-established observations, the causes of p53 and PTEN amyloid formation remain elusive. Understanding the role that cellular environmental factors and protein posttranslational modifications (PTMs) play in destabilizing p53 and PTEN and driving amyloid aggregate formation would provide crucial mechanistic insight into cellular drivers of oncogenesis and uncover new routes for restoring p53 and PTEN function. Here, we propose to address this gap in knowledge by systematically testing an endogenous metabolite library and ubiquitination/phosphorylation PTMs for ability to alter p53 and PTEN thermodynamic stability and induce amyloid formation. Working with recombinantly purified p53 and PTEN, we will use differential scanning fluorimetry (DSF) to measure metabolite-induced changes in the apparent melting temperature (Tm), as reported by a fluorogenic dye that detects unfolded protein. Metabolites that decrease Tm will be considered candidates for destabilizing p53 and PTEN to loss of function, while metabolites that increase Tm may have a protective effect against sampling conformations that seed amyloid formation. We will also investigate the ability of metabolites in the library to induce amyloid formation for purified p53 and PTEN upon isothermal shaking, as measured by the fluorogenic dye Thioflavin T (ThT). Further, we will perform biophysical and conformational characterization of the resulting aggregates to map conformational heterogeneity and polymorphism in p53 and PTEN amyloid fibrils. For both soluble and amyloid protein, we will then screen the Aurora library of conformationally sensitive, fluorogenic dyes to identify potential chemical probes for molecular recognition of healthy vs. toxic p53 and PTEN proteoforms. In parallel, we will use a biochemical reconstitution of ubiquitination enzymes and genetically encodable site-specific phosphorylation system to create p53 and PTEN samples with these crucial regulatory PTMs. In addition to measuring the effects of ubiquitination and phosphorylation on thermodynamic stability and amyloid formation susceptibility, we will also investigate the effects of these modifications on protein function, measuring DNA binding ability/affinity for p53 and phosphatase activity for PTEN. Together, these studies will shed light on the connection between metabolic dysregulation in oncogenesis and tumor suppressor protein misfolding, dysfunction, and aggregation. This knowledge is important because a mechanistic understanding of this link will enable new strategies to block cancer development and create opportunities to tip the scales back towards tumor suppressor folding and function.
