First-order rate-determining aggregation mechanism of p53 and its implications

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 ...

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“…S2D), indicating a reversible binding of ThT. We discuss further in the accompanying paper why different techniques give different kinetics (23).…”

Section: Resultsmentioning

confidence: 85%

“…In the accompanying paper, (23) we explore the consequences of the proposed mechanism for the aggregation of the core domain of Y220C and show the mechanism extends to the fulllength protein.…”

Section: Discussion Complex Kinetics Fits To a Simple Equation Other mentioning

confidence: 99%

Kinetic mechanism of p53 oncogenic mutant aggregation and its inhibition

Wilcken

Wang

Boeckler

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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112

137

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“…Increased crowding favors the formation of high-molecular-weight associates at the cost of correct folding, but the presence of chaperones tend to reduce aggregation even in such environment [7]. In vitro studies on protein aggregation indicated variability in the mechanisms of aggregation even for a given protein [8]. Kinetic analysis of in vitro protein aggregation [8][9][10] but also in vivo monitoring of IB formation [11] revealed that aggregate formation can be described as a pseudo-first-order process.…”

Section: Cellular Formation Of Ibsmentioning

confidence: 99%

“…In vitro studies on protein aggregation indicated variability in the mechanisms of aggregation even for a given protein [8]. Kinetic analysis of in vitro protein aggregation [8][9][10] but also in vivo monitoring of IB formation [11] revealed that aggregate formation can be described as a pseudo-first-order process. There are two possible mechanisms for IB formation, either IBs start growing from single or limited number of molecules acting as nucleation site or from smaller aggregates which assemble and form larger aggregates [12] .…”

Section: Cellular Formation Of Ibsmentioning

confidence: 99%

Bacterial Inclusion Bodies: Discovering Their Better Half

Rinas

García‐Fruitós

Corchero

et al. 2017

Trends in Biochemical Sciences

151

123

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Bacterial inclusion bodies are functional, non-toxic amyloids occurring in recombinant bacteria that show analogies with secretory granules of the mammalian endocrine system. The scientific interest in these mesoscale protein aggregates has been historically masked by their status as a hurdle in recombinant protein production.However, insights into their molecular organization and the progressive understanding on how the cell handles the quality of recombinant polypeptides have stimulated the interest on inclusion bodies and opened ways for their usability in diverse technological fields. The engineering and tailoring of inclusion bodies as functional protein particles for materials science and biomedicine is a good example of how formerly undesired bacterial by-products can be re-discovered as promising functional materials for a broad spectrum of applications.-2 - BACKGROUND

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“…15 in a mechanism that may be a basis for other proteins with multiple aggregation-prone sites. One molecule of p53 unfolds, partially or fully, to expose its aggregation-prone sequences (25,27,28,50). Another molecule is induced to unfold to give the elongation-competent state.…”

Section: Peptides and Peptide Combinations Selectively Kill Cancer Cementioning

confidence: 99%

Multisite aggregation of p53 and implications for drug rescue

Wang

Fersht

2017

Proc. Natl. Acad. Sci. U.S.A.

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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...

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First-order rate-determining aggregation mechanism of p53 and its implications

Cited by 61 publications

References 19 publications

Kinetic mechanism of p53 oncogenic mutant aggregation and its inhibition

Kinetic mechanism of p53 oncogenic mutant aggregation and its inhibition

Bacterial Inclusion Bodies: Discovering Their Better Half

Multisite aggregation of p53 and implications for drug rescue

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