Restoring wild-type p53 has long been viewed as a direct route to cancer therapy, yet clinical efforts have delivered only modest benefit. Here, we show that a critical but underappreciated aspect of p53 biology is its ability to form biomolecular condensates that can be manipulated to selectively enhance specific properties. The modest benefit of current p53-dependent therapeutics stems, in part, from the dual nature of p53; alongside canonical tumor-suppressor genes, wild-type p53 also boosts neuron-associated genes that can foster malignancy. Using live-cell super-resolution imaging we find that endogenous p53 nucleates novel ∼50-nm condensates. Coactivators reinforce droplets, whereas antagonists dissolve them, revealing a previously unappreciated regulatory layer. Guided by these mechanisms, we engineered liquid-liquid phase separation (LLPS)-enhanced p53 constructs with greater condensation propensity (LLPSEp53s). These condensates act as a mesoscale hub that biases promoter occupancy and partner selection. Intriguingly, LLPSEp53s tilt the transcriptome, preferentially strengthening tumor-suppressive transcription while dampening neuro-oncogenic outputs. LLPSEp53s suppress proliferation across multiple cancer cell lines, including those refractories to wild-type p53, and curtails melanoma growth in immunocompetent mice without added toxicity. Our findings establish phase separation as an adjustable layer of p53 target selectivity and highlight condensate engineering as a tractable strategy to unleash the full antitumor potential of p53.

Phase transitions of cellular proteins and lipids play a key role in governing the organisation and coordination of intracellular biology. Recent work has raised the intriguing prospect that phase transitions in proteins and lipids can be co-regulated. Here we investigate this possibility in the ribonucleoprotein (RNP) granule-ANXA11-lysosome ensemble, where ANXA11 tethers RNP granules to lysosomal membranes to enable their co-trafficking. We show that changes to the protein phase state within this system, driven by the low complexity ANXA11 N-terminus, induces a coupled phase state change in the lipids of the underlying membrane. We identify the ANXA11 interacting proteins ALG2 and CALC as potent regulators of ANXA11-based phase coupling and demonstrate their influence on the nanomechanical properties of the ANXA11-lysosome ensemble and its capacity to engage RNP granules. The phenomenon of protein-lipid phase coupling we observe within this system serves as a potential regulatory mechanism in RNA trafficking and offers an important template to understand other examples across the cell whereby biomolecular condensates closely juxtapose organellar membranes.

Long-distance RNA transport enables local protein synthesis at metabolically-active sites distant from the nucleus. This process ensures an appropriate spatial organization of proteins, vital to polarized cells such as neurons. Here, we present a mechanism for RNA transport in which RNA granules "hitchhike" on moving lysosomes. In vitro biophysical modeling, live-cell microscopy, and unbiased proximity labeling proteomics reveal that annexin A11 (ANXA11), an RNA granule-associated phosphoinositide-binding protein, acts as a molecular tether between RNA granules and lysosomes. ANXA11 possesses an N-terminal low complexity domain, facilitating its phase separation into membraneless RNA granules, and a C-terminal membrane binding domain, enabling interactions with lysosomes. RNA granule transport requires ANXA11, and amyotrophic lateral sclerosis (ALS)-associated mutations in ANXA11 impair RNA granule transport by disrupting their interactions with lysosomes. Thus, ANXA11 mediates neuronal RNA transport by tethering RNA granules to actively-transported lysosomes, performing a critical cellular function that is disrupted in ALS.

Recent work on the biophysics of proteins with low complexity, intrinsically disordered domains that have the capacity to form biological condensates has profoundly altered the concepts about the pathogenesis of inherited and sporadic neurodegenerative disorders associated with pathological accumulation of these proteins. In the present review, we use the FUS, TDP-43 and A11 proteins as examples to illustrate how missense mutations and aberrant post-translational modifications of these proteins cause amyotrophic lateral sclerosis (ALS) and fronto-temporal lobar degeneration (FTLD).

Many RNA binding proteins, including FUS, contain moderately repetitive, low complexity, intrinsically disordered domains. These sequence motifs have recently been found to underpin reversible liquid: liquid phase separation and gelation of these proteins, permitting them to reversibly transition from a monodispersed state to liquid droplet- or hydrogel-like states. This function allows the proteins to serve as scaffolds for the formation of reversible membraneless intracellular organelles such as nucleoli, stress granules and neuronal transport granules. Using FUS as an example, this review examines the biophysics of this physiological process, and reports on how mutations and changes in post-translational state alter phase behaviour, and lead to neurodegenerative diseases such as amyotrophic lateral sclerosis and frontotemporal lobar degeneration.

Reversible phase separation underpins the role of FUS in ribonucleoprotein granules and other membrane-free organelles and is, in part, driven by the intrinsically disordered low-complexity (LC) domain of FUS. Here, we report that cooperative cation-π interactions between tyrosines in the LC domain and arginines in structured C-terminal domains also contribute to phase separation. These interactions are modulated by post-translational arginine methylation, wherein arginine hypomethylation strongly promotes phase separation and gelation. Indeed, significant hypomethylation, which occurs in FUS-associated frontotemporal lobar degeneration (FTLD), induces FUS condensation into stable intermolecular β-sheet-rich hydrogels that disrupt RNP granule function and impair new protein synthesis in neuron terminals. We show that transportin acts as a physiological molecular chaperone of FUS in neuron terminals, reducing phase separation and gelation of methylated and hypomethylated FUS and rescuing protein synthesis. These results demonstrate how FUS condensation is physiologically regulated and how perturbations in these mechanisms can lead to disease.

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 thatwe 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.

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 self-aggregates 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.

Significance The tumor suppressor p53 is inactivated by aggregation in a substantial number of tumors, and those oncogenic mutants coaggregate with WT protein and other tumor suppressors. Inhibition of aggregation by small molecules is a possible drug therapy. p53 aggregation appears to have much simpler kinetics than commonly encountered in fibrillation, with two rate-determining sequential, apparently first-order, steps. We showed by combining mutagenesis and kinetics that the rate determining steps involve two molecules of p53 extensively unfolding and reacting in a bimolecular process that can appear first order. The mechanism provides a basis for understanding the progress of aggregation and coaggregation and points to the most effective drug targeting sites.

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 k1 and k2 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 k1 and k2, we resolved that the process with the higher rate constant followed that of the lower. Further, there was only partial inhibition of k1 and k2, 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.

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 Zn2+ 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 Zn2+ 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.

Oxidative refolding is a crucial step in the bioseparation process of bioactive RNase A from inclusion bodies (IBs) overexpressed in Escherichia coli, and it has been well recognized that RNase A refolding suffers from the slow formation of correct disulfide bonds with traditional redox agents such as reduced glutathione (GSH) and oxidized glutathione (GSSG). Hence, there is demand of developing new redox systems that can accelerate the oxidative refolding of this protein. In this work, a new redox system composed of 4-mercaptobenzeneacetate (ArSH) and hexanoyl cystamine (HCA) was proposed. It was found that the oxidative refolding of RNase A reached a refolding yield of 94% in 2h with ArSH/HCA at optimized concentrations, which was two times faster than that with GSH/GSSG. Moreover, the refolding yield of RNase A from IBs could also reach 89% in 3h with ArSH/HCA, much better than the performance with GSH/GSSG, which was only 69% in 8h refolding. Therefore, the use of the new redox system led to a significant increase of refolding yield and over 60% reduction in the refolding time in the inclusion body protein refolding. The results indicated that the new redox system was much more efficient in the oxidative refolding of RNase A than the traditional redox system.

Based on the structural characteristic of Protein disulfide isomerases and DsbA that have hydrophobic regions around the active sites, hydrophobic alkyl tails are linked to cystamine to create new small molecular foldase mimics, acyl cystamine. Both the oxidizing power and oxidation specificity of cystamine are enhanced by n ‐octanoyl or n ‐hexanoyl tail. N ‐octanoyl and n ‐hexanoyl cystamine are very effective to facilitate oxidative protein refolding at strong reducing environments. In the presence of 0.42 mM DTT, the activity recovery of lysozyme is over 90% by 90‐min refolding with 0.1 mM n ‐octanoyl cystamine and 0.1 mM cystamine as oxidant, while almost no activity is recovered with 0.2 mM GSSG by 160‐min refolding. For the refolding of 0.2 mg/mL lysozyme, with 0.6 mM n ‐hexanoyl cystamine and 1.12 mM residual DTT as redox agents, the activity recovery reaches as high as 93% after refolding for only 20 min. For ribonuclease A (RNase A) refolding, with 0.4 mM n ‐hexanoyl cystamine and 1.30 mM DTT, the recovery of activity reaches as high as 90% within 3 h. Thus, with n ‐octanoyl or n ‐hexanoyl cystamine as the oxidants, the necessity to remove excess DTT in the reduced and denatured protein solutions can be greatly alleviated. With a moderate hydrophobicity, n ‐hexanoyl cystamine is promising for application in oxidative protein refolding at an extensive concentration range. It is observed that in the oxidative refolding of 0.2 mg/mL lysozyme and RNase A, only about half of n ‐hexanoyl cystamine is needed when compared to cystamine to achieve the same kinetic effect. © 2011 American Institute of Chemical Engineers Biotechnol. Prog., 2011

Protein refolding is a crucial step for the production of therapeutic proteins expressed in bacteria as inclusion bodies. In vitro protein refolding is severely impeded by the aggregation of folding intermediates during the folding process, so inhibition of the aggregation is the most effective approach to high-efficiency protein refolding. We have herein found that electrostatic repulsion between like-charged protein and ion exchange gel beads can greatly suppress the aggregation of folding intermediates, leading to the significant increase of native protein recovery. This finding is extensively demonstrated with three different proteins and four kinds of ion-exchange resins when the protein and ion-exchange gel are either positively or negatively charged at the refolding conditions. It is remarkable that the enhancing effect is significant at very high protein concentrations, such as 4 mg/mL lysozyme (positively charged) and 2 mg/mL bovine serum albumin (negatively charged). Moreover, the folding kinetics is not compromised by the presence of the resins, so fast protein refolding is realized at high protein concentrations. It was not realistic by any other approaches. The working mechanism of the like-charged resin is considered due to the charge repulsion that could induce oriented alignment of protein molecules near the charged surface, leading to the inhibition of protein aggregation. The molecular crowding effect induced by the charge repulsion may also contribute to accelerating protein folding. The refolding method with like-charged ion exchangers is simple to perform, and the key material is easy to separate for recycling. Moreover, because ion exchangers can work as adsorbents of oppositely charged impurities, an operation of simultaneous protein refolding and purification is possible. All the characters are desirable for preparative refolding of therapeutic proteins expressed in bacteria as inclusion bodies.

Oxidative protein refolding is a big challenge for the recovery of disulfide bonds containing recombinant proteins expressed as inclusion bodies. In this study, the disulfide form of a small peptide mimic of protein disulfide isomerase, CGC, was explored to facilitate oxidative protein refolding with residual dithiothreitol (DTT) as the reductant. Working with DTT, CGC gave higher yield and folding rate than oxidized glutathione. Then, a new pentapeptide, RKCGC disulfide, was designed. It was found that RKCGC was more effective than CGC in increasing the folding rate and final yield, as demonstrated in the oxidative refolding of lysozyme and ribonuclease A. It was because the pentapeptide has lower pKa and higher reduction potential than CGC. Moreover, it efficiently facilitated protein refolding not only at neutral pH but also in a weak alkaline buffer. With CGC or RKCGC as the oxidant, the DTT removal step and the addition of other thiol reductants were not needed. This would benefit in decreasing the processing time and cost in protein refolding processes. This design has thus provided more efficient oxidant, and it may pave the way for the design of more effective thiol-disulfide redox agents.

Protein refolding from inclusion bodies is a crucial step in the downstream processing of therapeutic proteins. Because in vitro protein refolding operation is a kinetically competitive process between folding and aggregation, accelerating on-pathway folding and/or suppressing aggregation are effective approaches to high-performance protein refolding. Here, three examples of protein refolding methods are described, including (1) on-column refolding by artificial chaperone-assisted immobilized metal affinity chromatography that gives rise to simultaneous protein refolding and purification, (2) suppression of aggregation with like-charged ion exchange resin by electrostatic repulsion that leads to the significant increase of refolding yield, and (3) design of small-molecular foldase mimics that improves protein refolding yield by accelerating disulfide formation and reshuffling.

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Oxidative folding is of vital importance for producing therapeutic proteins in bacteria via recombinant DNA technology since disulfide bonds exist in most pharmaceutical proteins. Although oxidative protein folding has been extensively investigated in vitro, little is explored concerning the role of disulfide formation to protein conformational folding rate. The effects of oxidized (GSSG)/reduced (GSH) glutathione and pH on the conformational folding kinetics of denatured/reduced lysozyme have been studied herein by fluorescence and circular dichroism. It is found that 83% tryptophan residue burial requires disulfide formation, and increasing GSSG concentration greatly accelerates the tertiary structure formation. The fast phase folding rate constant (k1) is linearly related to GSSG concentration, indicating the rate-limiting role of mixed-disulfide formation. Moreover, k1=0.006(±0.001)s−1 is likely to be a critical value for judging the determinant of the slow phase folding rate (k2), namely, k2 is controlled by disulfide formation rate only at k1<0.006s−1. These findings have elucidated the determinants of different folding stages and thus may be beneficial for more efficient control of the oxidative folding of proteins.