Metastability is associated with diseases
It costs to form metastable LLPS in cells. The LLPS of IDPs derives from a state of supersaturated compositional proteins wherein their cellular concentrations are high relative to their solubilities67,83,108, a phenomenon that can drive the aberrant protein aggregation in neurodegenerative diseases108,109. The liquid condensed phase is intrinsically metastable and vulnerable to uncontrolled aggregation leading to pathological consequences 9,10,14,37,110. The metastable liquid MLOs is susceptible to alteration simply over time9,12, which is commonly expedited under pathological conditions 12,19,111. The transition from metastable MLOs to stable aggregates can further lead to the gain of biological changes, including cancer, neurodegenerative diseases and aging4,11,118,119,35,75,112–117. A wide array of factors can engender the alteration of metastability, such as PTMs, mutations and overexpression of proteins (Figure 3B ), which are reviewed below.
Loss of metastability can be triggered by aberrant PTMs120. George-Hyslop et al. 11showed hypomethylation of arginine can drive the formation of hydrogel structure from FUS protein, which can disrupt ribonucleoprotein (RNP) granule function and damage protein synthesis in neuron terminals, thus suggesting a plausible mechanism for frontotemporal lobar degeneration (FTLD), as well as other neurodegenerative diseases. Conversely, metastability can be preserved by specific PTMs. Fawzi et al.85 revealed that arginine methylation can help to maintain the metastability of hnRNPA2 protein by disrupting interactions between arginine guanidyl group and aromatic residues, thereby pointing out a possible method for the regulation of solid-like assemblies and modify the toxicity. Fawzi et al. 39investigated the impact of phosphorylation using phosphomimetic mutations. All the twelve serine/threonine–glutamine (S/TQ) sites of FUS protein were mutated to glutamate–glutamine (EQ) sites. The disordered nature of LCDs was preserved, whilst allowing the ability to form LLPS in vitro . Distinctively, the aggregation-prone characteristics were reduced. With one-day orbital agitation to induce maturation of IDPs, most of the droplets formed from wild-type FUS protein evolved into irregular assemblies, while the droplets formed from phosphomimetic variant 12E remained spherical in vitro . They further demonstrated both the aggregation propensity and cytotoxicity in yeast Saccharomyces cerevisiae were reduced, suggesting that maintaining metastability of liquid-like MLOs can be an effective way to tackle neurodegeneration caused by aberrant protein aggregation. Ferreonet al. 121 found PTM can counteract LLPS-initiated protein aggregation. Tau protein underwent LLPS under physiology-relevant conditions in vitro , whilst the liquid-to-solid transition can be induced in the presence of heparin. The 24-hour maturation process was visualized by morphological change into solid-like irregular aggregates and quantified by prominent fluorescence increase of Thioflavin T (ThT) fluorescence assay. Tau protein was also hyperacetylated with p300 histone acetyltransferase. Distinctively, hyperacetylated Tau protein forms much less irregular solid after 24-hour maturation. The aggregation propensity of Tau is largely decreased by acetylation, suggesting a way to ameliorate neurodegenerative diseases associated with toxic Tau aggregation, including Alzheimer’s disease.
Disease-associated mutations often strengthen homotypic interactions of IDPs, thereby impairing the liquidity of MLOs, decreasing dynamics and promoting protein aggregation 112. Neurodegenerative diseases have been widely reported to link with aberrant transition of MLOs. Alberti et al. 12 showed the maturation of FUS protein liquid droplets can be drastically accelerated by mutations associated with amyotrophic lateral sclerosis (ALS). After 8-hour maturation in vitro , droplets formed by the LLPS of FUS protein was mostly preserved, only demonstrating minute solidification. By contrast, droplets from the G156E mutant of FUS protein maturated into stable fibrous aggregates (a state without metastability) almost completely. How is the loss of metastability linked with disease? Da Cruz et al. 122 further clarified that ALS/frontotemporal dementia (FTD)-linked mutant of FUS protein drives disease by a gain of toxicity, rather than a loss of function. Tayloret al. 19 investigated the hnRNPA1 mutant, D262V, which is associated with multisystem proteinopathy and ALS123. At the initial state, D262V mutant formed LLPS comparable with wild-type hnRNPA1, demonstrating similar propensity of droplet formation and critical melting temperature. Repeated heating-cooling cycling was applied to both D262V mutant and wild-type hnRNPA1. Notably, within minutes of maturation, the reversible droplet formation of D262V mutant was concomitant with the fibrilization and formation of irregular solid-like assemblies, which is stable, amyloid-like and ThT-positive. By contrast, no such liquid-to-solid conversion was observed for wild-type hnRNPA1, thus suggesting the metastability of MLOs was impaired by the D262V mutation, which is associated with neurodegeneration. Fawzi et al.85 studied the P298L and D290V mutants of hnRNPA2 protein with connections to Paget’s disease and multisystem proteinopathy (MSP), respectively. Freshly prepared wild-type hnRNPA2, P298L and D290V mutants all formed similar spherical droplets with liquidity and comparable dynamics, as was quantified by FRAP. Nonetheless, P298L and D290V mutants prominently transited into stable solid-like assemblies after 30-min maturation in vitro , in stark contrast to the preservation of liquid droplets of wild-type hnRNPA2 for at least 120 mins, thereby indicating disease-associated mutations impaired metastability of MLOs. Fawzi et al. 86reported A321V, an ALS variant of C-terminal domain (CTD) of TDP-43, formed liquid droplets similar to wild-type TDP-43, as was confirmed by turbidity, spherical shape and FRAP. After one hour incubation in vitro , A321V variant changed into irregular assemblies which is distinct from the spherical shape of wild type TDP-43. Similar phenomena have also been found for P362L mutants in the LCD of T cell-restricted intracellular antigen-1 (TIA1) 124, and A4V mutants in superoxide dismutase 1 (SOD1) 45, which are associated with ALS. Taylor et al. 118 studied the expansion of a hexanucleotide repeat GGGGCC in C9ORF72, which is the most common cause of ALS and FTD, presumably through the expression of toxic dipeptide repeat proteins. They identified that arginine-containing dipeptide repeat proteins, namely, polyGly-Arg and polyPro-Arg, can interact with LCDs of IDPs to alter the biophysical properties of MLOs. Both polyGly-Arg and polyPro-Arg can impair the liquidity, dynamics and metastability of MLOs in vitro and in living HeLa cells, as was substantiated by FRAP and change of spherical shape. Moreover, the polyGly-Arg and polyPro-Arg can promote the assembly of stress granules lacking metastability in HeLa cells, which can inhibit cellular translation and increase the risk of cell death events. Vale et al. 125 further showed RNAs harboring CAG and CUG repeats (length 31, 47 and 66 repeats) underwent LLPS event into droplets in vitro , albeit rapidly evolving into solid-like RNA hydrogels maintaining sphere-like shape. RNAs harboring 47 CAG repeats can form liquid-like nuclear foci in living U-2OS cells. By contrast, RNAs with 10 or 23 GGGGCC repeats forms irregular solid-like gels in vitro , and RNAs with 29 GGGGCC repeats forms irregular solid-like gels in living U-2OS cells. This report demonstrated that expansion of the GGGGCC in the C9ORF72 gene can trigger formation of solid-like RNA gels with impaired dynamics and metastability. As the accumulation of the repeat-containing transcripts into aberrant RNA foci in the nucleus is a common feature in neurodegeneration, this research suggests sequence-specific gelation of RNAs can be one cause of neurodegenerative diseases. Hsp27 is molecular chaperone that is vital in maintaining the metastability and dynamics of liquid droplets of stress granules from FUS protein, whilst mutations of chaperons can directly engender hereditary motor neuron diseases42. Liu et al. 42 mutated residues (I120, H124, 126–131, I134, F136, R140 and T143) of Hsp27 to alanine to synthesize Hsp27-A, which can largely change the binding surface between Hsp27 and FUS protein. Hsp27 bound to FUS LCD to preserve the liquid phase against amyloid fibril formation, whilst the mutant Hsp27-A had little inhibitory effect on the amyloid-like aggregation of FUS LCD, as has been quantified by the ThT fluorescence assay. This study suggests loss of metastability can lead to diseases through the mutations of chaperone. Besides neurodegenerative diseases, Mittag et al. 126 investigated the impact of cancer mutations on LLPS and functioning of MLOs. Tumor suppressor speckle-type POZ protein (SPOP) formed liquid-like MLOs with death-domain-associated protein (DAXX) in living HeLa cells, and CUL3 ubiquitin ligase activity is found in the MLOs formed. By contrast, cancer mutants (W131G and F133V) of SPOP failed to co-localize and form liquid-like MLOs with DAXX in living HeLa cells, resulting into reduced protein ubiquitination, thus suggesting cancer mutations are associated with loss of function of MLOs.
The overexpression of IDPs can also trigger the alteration of metastable MLOs. Carra et al. 127 revealed overexpression of small heat shock proteins (HSPBs) engendered aberrant MLOs in nucleus, which can mislocalize nuclear intermediate filament protein lamin-A/C (LMNA) and chromatin, resulting in the abnormal distribution of LMNA and chromatin and thereby damaging the integrity and function of nucleus.