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.