Aggregation-primed molten globule conformers of the p53 core domain provide potential tools for studying p53C aggregation in cancer.

The functionality of the tumor suppressor p53 is altered in more than 50% of human cancers, and many individuals with cancer exhibit amyloid-like buildups of aggregated p53. An understanding of what triggers the pathogenic amyloid conversion of p53 is required for the further development of cancer therapies. Here, perturbation of the p53 core domain (p53C) with subdenaturing concentrations of guanidine hydrochloride and high hydrostatic pressure revealed native-like molten globule (MG) states, a subset of which were highly prone to amyloidogenic aggregation. We found that MG conformers of p53C, probably representing population-weighted averages of multiple states, have different volumetric properties, as determined by pressure perturbation and size-exclusion chromatography. We also found that they bind the fluorescent dye 4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonic acid (bis-ANS) and have a native-like tertiary structure that occludes the single Trp residue in p53. Fluorescence experiments revealed conformational changes of the single Trp and Tyr residues before p53 unfolding and the presence of MG conformers, some of which were highly prone to aggregation. p53C exhibited marginal unfolding cooperativity, which could be modulated from unfolding to aggregation pathways with chemical or physical forces. We conclude that trapping amyloid precursor states in solution is a promising approach for understanding p53 aggregation in cancer. Our findings support the use of single-Trp fluorescence as a probe for evaluating p53 stability, effects of mutations, and the efficacy of therapeutics designed to stabilize p53.

Targeting the Prion-like Aggregation of Mutant p53 to Combat Cancer.

Prion-like behavior of several amyloidogenic proteins has been demonstrated in recent years. Despite having functional roles in some cases, irregular aggregation can have devastating consequences. The most commonly known amyloid diseases are Alzheimer's, Parkinson's, and Transmissible Spongiform Encephalopathies (TSEs). The pathophysiology of prion-like diseases involves the structural transformation of wild-type (wt) proteins to transmissible forms that can convert healthy proteins, generating aggregates. The mutant form of tumor suppressor protein, p53, has recently been shown to exhibit prion-like properties. Within the context of p53 aggregation and the search for ways to avert it, this review emphasizes discoveries, approaches, and research from our laboratory and others. Although its standard functions are strongly connected to tumor suppression, p53 mutants and aggregates are involved in cancer progression. p53 aggregates are heterogeneous assemblies composed of amorphous aggregates, oligomers, and amyloid-like fibrils. Evidence of these structures in tumor tissues, the in vitro capability for p53 mutants to coaggregate with wt protein, and the detection of cell-to-cell transmission indicate that cancer has the basic characteristics of prion and prion-like diseases. Various approaches aim to restore p53 functions in cancer. Methods include the use of small-molecule and peptide stabilizers of mutant p53, zinc administration, gene therapy, alkylating and DNA intercalators, and blockage of p53-MDM2 interaction. A primary challenge in developing small-molecule inhibitors of p53 aggregation is the large number of p53 mutations. Another issue is the inability to recover p53 function by dissociating mature fibrils. Consequently, efforts have emerged to target the intermediate species of the aggregation reaction. Φ-value analysis has been used to characterize the kinetics of the early phases of p53 aggregation. Our experiments using high hydrostatic pressure (HHP) and chemical denaturants have helped to clarify excited conformers of p53 that are prone to aggregation. Molecular dynamics (MD) and phasor analysis of single Trp fluorescence signals point toward the presence of preamyloidogenic conformations of p53, which are not observed for p63 or p73. Exploring the features of competent preamyloidogenic states of wt and different p53 mutants may provide a framework for designing personalized drugs for the restoration of p53 function. Protection of backbone hydrogen bonds (BHBs) has been shown to be an important factor for the stability of amyloidogenic proteins and was employed to identify and stabilize the structural defect resulting from the p53 Y220C mutation. Using MD simulations, we compared BHB protection factors between p53 family members to determine the donor-acceptor pairs in p53 that exhibit lower protection. The identification of structurally vulnerable sites in p53 should provide new insights into rational designs that can rapidly be screened using our experimental methodology. Through continued and combined efforts, the outlook is positive for the development of strategies for regulating p53 amyloid transformation.

Regulation of stress-inducible phosphoprotein 1 nuclear retention by protein inhibitor of activated STAT PIAS1.

Stress-inducible phosphoprotein 1 (STI1), a cochaperone for Hsp90, has been shown to regulate multiple pathways in astrocytes, but its contributions to cellular stress responses are not fully understood. We show that in response to irradiation-mediated DNA damage stress STI1 accumulates in the nucleus of astrocytes. Also, STI1 haploinsufficiency decreases astrocyte survival after irradiation. Using yeast two-hybrid screenings we identified several nuclear proteins as STI1 interactors. Overexpression of one of these interactors, PIAS1, seems to be specifically involved in STI1 nuclear retention and in directing STI1 and Hsp90 to specific sub-nuclear regions. PIAS1 and STI1 co-immunoprecipitate and PIAS1 can function as an E3 SUMO ligase for STI. Using mass spectrometry we identified five SUMOylation sites in STI1. A STI1 mutant lacking these five sites is not SUMOylated, but still accumulates in the nucleus in response to increased expression of PIAS1, suggesting the possibility that a direct interaction with PIAS1 could be responsible for STI1 nuclear retention. To test this possibility, we mapped the interaction sites between PIAS1 and STI1 using yeast-two hybrid assays and surface plasmon resonance and found that a large domain in the N-terminal region of STI1 interacts with high affinity with amino acids 450-480 of PIAS1. Knockdown of PIAS1 in astrocytes impairs the accumulation of nuclear STI1 in response to irradiation. Moreover, a PIAS1 mutant lacking the STI1 binding site is unable to increase STI1 nuclear retention. Interestingly, in human glioblastoma multiforme PIAS1 expression is increased and we found a significant correlation between increased PIAS1 expression and STI1 nuclear localization. These experiments provide evidence that direct interaction between STI1 and PIAS1 is involved in the accumulation of nuclear STI1. This retention mechanism could facilitate nuclear chaperone activity.

Stress-inducible phosphoprotein 1 has unique cochaperone activity during development and regulates cellular response to ischemia via the prion protein.

Stress-inducible phosphoprotein 1 (STI1) is part of the chaperone machinery, but it also functions as an extracellular ligand for the prion protein. However, the physiological relevance of these STI1 activities in vivo is unknown. Here, we show that in the absence of embryonic STI1, several Hsp90 client proteins are decreased by 50%, although Hsp90 levels are unaffected. Mutant STI1 mice showed increased caspase-3 activation and 50% impairment in cellular proliferation. Moreover, placental disruption and lack of cellular viability were linked to embryonic death by E10.5 in STI1-mutant mice. Rescue of embryonic lethality in these mutants, by transgenic expression of the STI1 gene, supported a unique role for STI1 during embryonic development. The response of STI1 haploinsufficient mice to cellular stress seemed compromised, and mutant mice showed increased vulnerability to ischemic insult. At the cellular level, ischemia increased the secretion of STI1 from wild-type astrocytes by 3-fold, whereas STI1 haploinsufficient mice secreted half as much STI1. Interesting, extracellular STI1 prevented ischemia-mediated neuronal death in a prion protein-dependent way. Our study reveals essential roles for intracellular and extracellular STI1 in cellular resilience.

The synthetic oleanane triterpenoid CDDO-Me binds and inhibits pyruvate kinase M2.

BACKGROUND: The M2 isoform of the glycolytic enzyme pyruvate kinase (PKM2) is one of the key components in the Warburg effect, and an important regulator of cancer cell metabolism. Elevated PKM2 expression is a hallmark of numerous tumor types, making it a promising target for cancer therapy. METHODS: Migration of H1299 lung tumor cells treated with synthetic oleanane triterpenoid derivatives CDDO-Me and CDDO-Im was monitored using scratch and transwell assays. Direct binding and inhibition of PKM2 activity by CDDO-Me was demonstrated by pull-down and activity assays. PKM2 localization in the absence and presence of CDDO-Me or CDDO-Im was determined by subcellular fractionation and immunofluorescence microscopy. Involvement of PKM2 in tumor cell migration was assessed using a stable PKM2 knockdown cell line. RESULTS: We demonstrate that migration of H1299 lung tumor cells is inhibited by CDDO-Me and CDDO-Im in scratch and transwell assays. CDDO-Me binds directly and specifically to recombinant PKM2, leading to a reduction of its catalytic activity. PKM2 knockdown cells exhibit significantly lower migration compared to control cells when subjected to glucose and oxygen deprivation, but not under regular conditions. CONCLUSIONS: The results suggest that PKM2 expression in a tumor-like environment contributes to cell migration, and that PKM2 activity can be down regulated by synthetic triterpenoid derivatives.

Backbone resonance assignments of the human p73 DNA binding domain.

p53, p63, p73 family of proteins are transcription factors with crucial roles in regulating cellular processes such apoptosis, proliferation, differentiation, and DNA damage response. The three family members have both overlapping and unique biological functions. Sequence and structural homology are greatest in the DNA binding domains (DBD), which is the site of the majority of p53 mutations. Structurally unstable p53 DBD mutants can associate with themselves or p63 and p73 DBDs, impeding tumor suppressor functions. Evidence suggests that these proteins associate to form amyloid-like oligomers and fibrils through an aggregation-prone sequence within the DBDs. Despite having high sequence and structure similarities, p63 and p73 DBDs appear to have considerably lower tendencies to be incorporated into p53 aggregates, relative to p53. The backbone resonance assignments of p73 DBD reported here complement those previously reported for p53 and p63, allowing comparisons and providing molecular insights into their biological functions and roles in aggregation and tumor development.

Aggregation tendencies in the p53 family are modulated by backbone hydrogen bonds.

The p53 family of proteins is comprised of p53, p63 and p73. Because the p53 DNA binding domain (DBD) is naturally unstable and possesses an amyloidogenic sequence, it is prone to form amyloid fibrils, causing loss of functions. To develop p53 therapies, it is necessary to understand the molecular basis of p53 instability and aggregation. Light scattering, thioflavin T (ThT) and high hydrostatic pressure (HHP) assays showed that p53 DBD aggregates faster and to a greater extent than p63 and p73 DBDs, and was more susceptible to denaturation. The aggregation tendencies of p53, p63, and p73 DBDs were strongly correlated with their thermal stabilities. Molecular Dynamics (MD) simulations indicated specific regions of structural heterogeneity unique to p53, which may be promoted by elevated incidence of exposed backbone hydrogen bonds (BHBs). The results indicate regions of structural vulnerability in the p53 DBD, suggesting new targetable sites for modulating p53 stability and aggregation, a potential approach to cancer therapy.

Aggregation and Prion-Like Properties of Misfolded Tumor Suppressors: Is Cancer a Prion Disease?

Prion diseases are disorders that share several characteristics that are typical of many neurodegenerative diseases. Recently, several studies have extended the prion concept to pathological aggregation in malignant tumors involving misfolded p53, a tumor-suppressor protein. The aggregation of p53 and its coaggregation with p53 family members, p63 and p73, have been shown. Certain p53 mutants exert a dominant-negative regulatory effect on wild-type (WT) p53. The basis for this dominant-negative effect is that amyloid-like mutant p53 converts WT p53 into an aggregated species, leading to a gain-of-function (GoF) phenotype and the loss of its tumor-suppressor function. Recently, it was shown that p53 aggregates can be internalized by cells and can coaggregate with endogenous p53, corroborating the prion-like properties of p53 aggregates. The prion-like behavior of oncogenic p53 mutants provides an explanation for its dominant-negative and GoF properties, including the high metastatic potential of cancer cells carrying p53 mutations. The inhibition of p53 aggregation appears to represent a promising target for therapeutic intervention in patients with malignant tumors.