Oncogenic Gain of Function in Glioblastoma Is Linked to Mutant p53 Amyloid Oligomers.

Tumor-associated p53 mutations endow cells with malignant phenotypes, including chemoresistance. Amyloid-like oligomers of mutant p53 transform this tumor suppressor into an oncogene. However, the composition and distribution of mutant p53 oligomers are unknown and the mechanism involved in the conversion is sparse. Here, we report accumulation of a p53 mutant within amyloid-like p53 oligomers in glioblastoma-derived cells presenting a chemoresistant gain-of-function phenotype. Statistical analysis from fluorescence fluctuation spectroscopy, pressure-induced measurements, and thioflavin T kinetics demonstrates the distribution of oligomers larger than the active tetrameric form of p53 in the nuclei of living cells and the destabilization of native-drifted p53 species that become amyloid. Collectively, these results provide insights into the role of amyloid-like mutant p53 oligomers in the chemoresistance phenotype of malignant and invasive brain tumors and shed light on therapeutic options to avert cancer.

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.

Second-Sphere Effects in Dinuclear FeIIIZnII Hydrolase Biomimetics: Tuning Binding and Reactivity Properties.

Herein, we report the synthesis and characterization of two dinuclear FeIIIZnII complexes [FeIIIZnIILP1] (1) and [FeIIIZnIILP2] (2), in which LP1 and LP2 are conjugated systems containing one and two pyrene groups, respectively, connected via the diamine -HN(CH2)4NH- spacer to the well-known N5O2-donor H2L ligand (H2L = 2-bis{[(2-pyridylmethyl)aminomethyl]-6-[(2-hydroxybenzyl)(2-pyridylmethyl)]aminomethyl}-4-methylphenol). The complex [FeIIIZnIIL1] (3), in which H2L was modified to H2L1, with a carbonyl group attached to the terminal phenol group, was included in this study for comparison purposes.1 Both complexes 1 and 2 were satisfactorily characterized in the solid state and in solution. Extended X-ray absorption fine structure data for 1 and 3 in an acetonitrile solution show that the multiply bridged structure seen in the solid state of 3 is retained in solution. Potentiometric and UV-vis titration of 1 and 2 show that electrostatic interaction between the protonated amino groups and coordinated water molecules significantly decreases the pKa of the iron(III)-bound water compared to those of 3. On the other hand, catalytic activity studies using 1 and 2 in the hydrolysis of the activated substrate bis(2,4-dinitrophenyl)phosphate (BDNPP) resulted in a significant increase in the association of the substrate (Kass ≅ 1/KM) compared to that of 3 because of electrostatic and hydrophobic interactions between BDNPP and the side-chain diaminopyrene of the ligands H2LP1 and H2LP2. In addition, the introduction of the pyrene motifs in 1 and 2 enhanced their activity toward DNA and as effective antitumor drugs, although the biochemical mechanism of the latter effect is currently under investigation. These complexes represent interesting examples of how to promote an increase in the activity of traditional artificial metal nucleases by introducing second-coordination-sphere effects.

Effect of internal cavities on folding rates and routes revealed by real-time pressure-jump NMR spectroscopy.

The time required to fold proteins usually increases significantly under conditions of high pressure. Taking advantage of this general property of proteins, we combined P-jump experiments with NMR spectroscopy to examine in detail the folding reaction of staphylococcal nuclease (SNase) and of some of its cavity-containing variants. The nearly 100 observables that could be measured simultaneously collectively describe the kinetics of folding as a function of pressure and denaturant concentration with exquisite site-specific resolution. SNase variants with cavities in the central core of the protein exhibit a highly heterogeneous transition-state ensemble (TSE) with a smaller solvent-excluded void volume than the TSE of the parent SNase. This heterogeneous TSE experiences Hammond behavior, becoming more native-like (higher molar volume) with increasing denaturant concentration. In contrast, the TSE of the L125A variant, which has a cavity at the secondary core, is only slightly different from that of the parent SNase. Because pressure acts mainly to eliminate solvent-excluded voids, which are heterogeneously distributed throughout structures, it perturbs the protein more selectively than chemical denaturants, thereby facilitating the characterization of intermediates and the consequences of packing on folding mechanisms. Besides demonstrating how internal cavities can affect the routes and rates of folding of a protein, this study illustrates how the combination of P-jump and NMR spectroscopy can yield detailed mechanistic insight into protein folding reactions with exquisite site-specific temporal information.

Entropy and volume change of dissociation in tobacco mosaic virus probed by high pressure.

Virus dissociation and inactivation by high pressure have been extensively studied in recent decades. Pressure-induced dissociation of viral particles involves a reduction in the Gibbs free energy of dissociation and a negative change in volume. In this work, we investigated the combined effect of high pressure and temperature on the dissociation of tobacco mosaic virus (TMV). We assumed the presence of two states of TMV with different tendencies to dissociate. Thus one form presents a low tendency (L) and the other a high tendency (H) to dissociate. Based on the model described here, the L-H transition was favored by an increase in pressure and a decrease in temperature. The volume change of dissociation was pressure- and temperature-dependent, with a highly negative value of -80 mL/mol being recorded at 0 °C and atmospheric pressure. The entropy and enthalpy of dissociation were very temperature- and pressure-dependent, with values of entropy of 450 to -1300 kJ/mol and values of enthalpy of 5.5 × 10(4) to 2.4 × 10(4) kJ/mol. The dissociation of TMV was enthalpy-driven at all temperatures and pressures investigated. Based on these findings, we conclude that the model presented allows accurate predictions of viral dissociation behavior in different experimental conditions.

Remodeling of the folding free energy landscape of staphylococcal nuclease by cavity-creating mutations.

The folding of staphylococcal nuclease (SNase) is known to proceed via a major intermediate in which the central OB subdomain is folded and the C-terminal helical subdomain is disordered. To identify the structural and energetic determinants of this folding free energy landscape, we have examined in detail, using high-pressure NMR, the consequences of cavity creating mutations in each of the two subdomains of an ultrastable SNase, Δ+PHS. The stabilizing mutations of Δ+PHS enhanced the population of the major folding intermediate. Cavity creation in two different regions of the Δ+PHS reference protein, despite equivalent effects on global stability, had very distinct consequences on the complexity of the folding free energy landscape. The L125A substitution in the C-terminal helix of Δ+PHS slightly suppressed the major intermediate and promoted an additional excited state involving disorder in the N-terminus, but otherwise decreased landscape heterogeneity with respect to the Δ+PHS background protein. The I92A substitution, located in the hydrophobic OB-fold core, had a much more profound effect, resulting in a significant increase in the number of intermediate states and implicating the entire protein structure. Denaturant (GuHCl) had very subtle and specific effects on the landscape, suppressing some states and favoring others, depending upon the mutational context. These results demonstrate that disrupting interactions in a region of the protein with highly cooperative, unfrustrated folding has very profound effects on the roughness of the folding landscape, whereas the effects are less pronounced for an energetically equivalent substitution in an already frustrated region.

Cavities determine the pressure unfolding of proteins.

It has been known for nearly 100 years that pressure unfolds proteins, yet the physical basis of this effect is not understood. Unfolding by pressure implies that the molar volume of the unfolded state of a protein is smaller than that of the folded state. This decrease in volume has been proposed to arise from differences between the density of bulk water and water associated with the protein, from pressure-dependent changes in the structure of bulk water, from the loss of internal cavities in the folded states of proteins, or from some combination of these three factors. Here, using 10 cavity-containing variants of staphylococcal nuclease, we demonstrate that pressure unfolds proteins primarily as a result of cavities that are present in the folded state and absent in the unfolded one. High-pressure NMR spectroscopy and simulations constrained by the NMR data were used to describe structural and energetic details of the folding landscape of staphylococcal nuclease that are usually inaccessible with existing experimental approaches using harsher denaturants. Besides solving a 100-year-old conundrum concerning the detailed structural origins of pressure unfolding of proteins, these studies illustrate the promise of pressure perturbation as a unique tool for examining the roles of packing, conformational fluctuations, and water penetration as determinants of solution properties of proteins, and for detecting folding intermediates and other structural details of protein-folding landscapes that are invisible to standard experimental approaches.