Aggregation of destabilized mutants of the tumor suppressor p53 is a major route for its loss of activity. In order to assay drugs that inhibit aggregation of p53, we established the basic kinetics of aggregation of its core domain, using the mutant Y220C that has a mutation-induced, druggable cavity. Aggregation monitored by light scattering followed lag kinetics. Electron microscopy revealed the formation of small aggregates that subsequently grew to larger amorphous aggregates. The kinetics of aggregation produced surprising results: progress curves followed either by the binding of Thioflavin T or the fluorescence of the protein at 340 nm fitted well to simple two-step sequential first-order lag kinetics with rate constants k 1 and k 2 that were independent of protein concentration, and not to classical nucleation-growth. We suggest a mechanism of first-order formation of an aggregation competent state as being rate determining followed by rapid polymerization with the higher order kinetics. By measuring the inhibition kinetics of k 1 and k 2 , we resolved that the process with the higher rate constant followed that of the lower. Further, there was only partial inhibition of k 1 and k 2 , which showed two parallel pathways of aggregation, one via a state that requires unfolding of the protein and the other of partial unfolding with the ligand still bound. Inhibition kinetics of ligands provides a useful tool for probing an aggregation mechanism.amyloid | misfolding | folding | cancer T he most frequently mutated gene in cancer is that of the tumor suppressor p53. Some 70% of types of human cancers have their p53 directly inactivated by mutation, and so its reactivation is an important therapeutic goal (1). The most common oncogenic mutations are of residues in the DNA binding domain (DBD, residues 94-312) that make direct contact with DNA (2). But about 30%-40% of oncogenic mutants are inactivated because the mutations lower the stability of the DBD so that it denatures at body temperature, although it can be fully active at lower temperatures (3-6). We are attempting to rescue those temperature-sensitive mutants of p53 by designing small molecules that bind specifically to the native state of the protein and stabilize it (7). The rationale is that small molecules that bind to the native state of a temperature-sensitive mutant and not the denatured states will raise the melting temperature by a mass action effect. Such a molecule will slow down the rate of unfolding if it binds weakly to the transition state for unfolding. The obvious binding sites to target are those already present that bind natural ligands. In theory, they can be targeted in a chaperone strategy in which a rescue drug will compete for a binding site (7). We have chosen, instead, as a particularly useful paradigm for these studies, the stability of the p53 mutant Y220C, because the mutation induces a druggable cavity that is remote from functional areas of the protein (8, 9). We have designed small molecules that raise the apparent T m of ...

Protein aggregation is involved in many diseases. Often, a unique aggregation-prone sequence polymerizes to form regular fibrils. Many oncogenic mutants of the tumor suppressor p53 rapidly aggregate but form amorphous fibrils. A peptide surrounding Ile254 is proposed to be the aggregation-driving sequence in cells. We identified several different aggregating sites from limited proteolysis of harvested aggregates and effects of mutations on kinetics and products of aggregation. We present a model whereby the amorphous nature of the aggregates results from multisite branching of polymerization after slow unfolding of the protein, which may be a common feature of aggregation of large proteins. Greatly lowering the aggregation propensity of any one single site, including the site of Ile254, by mutation did not inhibit aggregation in vitro because aggregation could still occur via the other sites. Inhibition of an individual site is, accordingly, potentially unable to prevent aggregation in vivo. However, cancer cells are specifically killed by peptides designed to inhibit the Ile254 sequence and further aggregation-driving sequences that we have found. Consistent with our proposed mechanism of aggregation, we found that such peptides did not inhibit aggregation of mutant p53 in vitro. The cytotoxicity was not eliminated by knockdown of p53 in 2D cancer cell cultures. The peptides caused rapid cell death, much faster than usually expected for p53-mediated transcription-dependent apoptosis. There may also be non-p53 targets for those peptides in cancer cells, such as p63, or the peptides may alter other interactions of partly denatured p53 with receptors.amyloid | mechanism | misfolding | disease T he tumor suppressor p53 is inactivated by mutation in a substantial number of tumors (1-3). Some 30-40% of those oncogenic mutants are simply destabilized by mutations in its core domain. Those mutants are temperature-sensitive, having a WT structure at lower temperatures, but melt at close to body temperature or below and rapidly aggregate (4-6). Protein aggregation occurs in many diseases (7-9). The best mechanistically characterized examples involve the polymerization of aggregationprone peptides (10, 11) or small proteins to give well-defined fibrils based on a regular repeat structure (12-18). The fibrillar aggregates have a characteristic cross-β X-ray diffraction pattern and bind such dyes as Congo Red and Thioflavine T (ThT) (16). The kinetics of aggregation usually follow a nucleation-growth mechanism, with very slow nucleation (19-21). WT p53 itself aggregates at body temperature (4-6, 22-24), and the oncogenic destabilized mutants aggregate even faster to give amorphous structures that display the characteristic diffraction pattern and bind those diagnostic dyes (23, 25), although under certain conditions, such as very high pressure, they will generate regular fibrils (23,26). The mechanism of initiation of aggregation of p53 differs from the usually studied examples. Two molecules of the core domain of p53 exten...

Aggregation of p53 is initiated by first-order processes that generate an aggregation-prone state with parallel pathways of major or partial unfolding. Here, we elaborate the mechanism and explore its consequences, beginning with the core domain and extending to the full-length p53 mutant Y220C. Production of large light-scattering particles was slower than formation of the Thioflavin T-binding state and simultaneous depletion of monomer. EDTA removes Zn 2þ to generate apo-p53, which aggregated faster than holo-p53. Apo-Y220C also aggregated by both partial and major unfolding. Apo-p53 was not an obligatory intermediate in the aggregation of holo-p53, but affords a parallel pathway that may be relevant to oncogenic mutants with impaired Zn 2þ binding. Full-length tetrameric Y220C formed the Thioflavin T-binding state with similar rate constants to those of core domain, consistent with a unimolecular initiation that is unaffected by neighboring subunits, but very slowly formed small light-scattering particles. Apo-Y220C and aggregated holo-Y220C had little, if any, seeding effect on the initial polymerization of holo-Y220C (measured by Thioflavin T binding), consistent with initiation being a unimolecular process. But apo-Y220C and aggregated holo-Y220C accelerated somewhat the subsequent formation of light-scattering particles from holo-protein, implying coaggregation. The implications for cancer cells containing wild-type and unstable mutant alleles are that aggregation of wild-type p53 (or homologs) might not be seeded by aggregated mutant, but it could coaggregate with p53 or other cellular proteins that have undergone the first steps of aggregation and speed up the formation of microscopically observable aggregates.kinetics | amyloid | folding | misfolding T he kinetics of aggregation of the core domain of the oncogenic p53 mutant Y220C is most unusual, as it does not appear to follow the conventional nucleation-growth mechanism. (1) The aggregation of p53 differs from that most frequently studied for other proteins (2-4): the kinetics is deceptively simple, fitting a simple sequential scheme of A → B → C with two first-order rate steps; the aggregate is amorphous, and not well-formed amyloid fibrils; and aggregation is fast and easily studied by continuous spectroscopic measurements over minutes, rather than hours or days. (1) We suggest that there is rate-determining first-order formation of an aggregation prone intermediate followed by fast polymerization events prior to very slow formation of light scattering large aggregates. That is, there is not rate-determining formation of an oligomeric nucleus, but initiation is unimolecular.Here, we explore the consequences of the suggested kinetic mechanism of aggregation of the core domain of Y220C, which might be a paradigm for amorphous aggregation, and extend the studies to full-length protein p53 (Flp53Y220C) with the Y220C mutation. Full-length p53 is a multidomain tetrameric protein with two folded domains: the core domain extending from residues 94-312 an...

Destabilized mutant p53s coaggregate with WT p53, p63, and p73 in cancer cell lines. We found that stoichiometric amounts of aggregation-prone mutants induced only small amounts of WT p53 to coaggregate, and preformed aggregates did not significantly seed the aggregation of bulk protein. Similarly, p53 mutants trapped only small amounts of p63 and p73 into their p53 aggregates. Tetrameric full-length protein aggregated at similar rates and kinetics to isolated core domains, but there was some induced aggregation of WT by mutants in hetero-tetramers. p53 aggregation thus differs from the usual formation of amyloid fibril or prion aggregates where tiny amounts of preformed aggregate rapidly seed further aggregation. The proposed aggregation mechanism of p53 of rate-determining sequential unfolding and combination of two molecules accounts for the difference. A molecule of fast-unfolding mutant preferentially reacts with another molecule of mutant and only occasionally traps a slower unfolding WT molecule. The mutant population rapidly selfaggregates before much WT protein is depleted. Subsequently, WT protein self-aggregates at its normal rate. However, the continual production of mutant p53 in a cancer cell would gradually trap more and more WT and other proteins, accounting for the observations of coaggregates in vivo. The mechanism corresponds more to trapping by cross-reaction and coaggregation rather than classical seeding and growth.protein | amyloid | folding | misfolding | cancer T he mechanism of aggregation of p53 (1) is different from that of the classical nucleation-growth of formation of amyloid fibrils (2-5). The classical mechanisms involve relatively slow nucleation events followed by rapid growth. The fibrils or their fragments act as seeds to accelerate greatly the polymerization of molecules from solution. However, the initiation of aggregation of p53 is relatively rapid, there appears to be little seeding from already polymerized molecules (6, 7), and stirring does not significantly speed up aggregation (1). An extensive Φ-value analysis of the aggregation of core domains of p53 suggests that the initial events involve the sequential unfolding of two molecules, involving a combination of first-order and second-order kinetics (1). Full-length p53, however, has a tetramerization domain, which gives a mixture of dimers and tetramers at normally encountered concentrations of protein. At 37°C, tetrameric p53 dissociates into dimers with a dissociation constant of 50 nM, and the dimers to monomers with a dissociation constant of 0.55 nM, with half lives of 20 and 50 min, respectively, for dissociation (8). Tetramerization of p53 may complicate the aggregation kinetics. The possibilities of intramolecular core-domain interactions could, for example, change the kinetic mechanism and accelerate the initial aggregation events by proximity effects of core domains in tetramers. Further, WT protein may form mixed hybrids with a destabilized, oncogenic mutant and facilitate seeding of the aggregation of WT d...