Effects of PH Alterations On Stress - and Aging-Ind

Cellular and Molecular Life Sciences (2022) 79:380
https://doi.org/10.1007/s00018-022-04393-0 Cellular and Molecular Life Sciences
REVIEW
Effects of pH alterations on stress‑ and aging‑induced protein phase

separation
Xuejiao Jin1 · Min Zhou1 · Shuxin Chen1 · Danqi Li1 · Xiuling Cao1 · Beidong Liu1,2,3
Received: 21 February 2022 / Revised: 26 April 2022 / Accepted: 21 May 2022 / Published online: 24 June 2022
© The Author(s) 2022
Abstract
Upon stress challenges, proteins/RNAs undergo liquid–liquid phase separation (LLPS) to fine-tune cell physiology and
metabolism to help cells adapt to adverse environments. The formation of LLPS has been recently linked with intracellular
pH, and maintaining proper intracellular pH homeostasis is known to be essential for the survival of organisms. However,
organisms are constantly exposed to diverse stresses, which are accompanied by alterations in the intracellular pH. Aging
processes and human diseases are also intimately linked with intracellular pH alterations. In this review, we summarize
stress-, aging-, and cancer-associated pH changes together with the mechanisms by which cells regulate cytosolic pH homeo-
stasis. How critical cell components undergo LLPS in response to pH alterations is also discussed, along with the functional
roles of intracellular pH fluctuation in the regulation of LLPS. Further studies investigating the interplay of pH with other
stressors in LLPS regulation and identifying protein responses to different pH levels will provide an in-depth understanding
of the mechanisms underlying pH-driven LLPS in cell adaptation. Moreover, deciphering aging and disease-associated pH
changes that influence LLPS condensate formation could lead to a deeper understanding of the functional roles of biomo-
lecular condensates in aging and aging-related diseases.
Keywords Acidification · Membrane-less compartment · Neurodegenerative disease · Tumorigenesis · Protein aggregation
Introduction separated from the solution to form a dense phase, while

these supersaturated components in the dilute phase are
Liquid–liquid phase separation (LLPS) refers to a demix- depleted. The macromolecular dense phase has liquid-like
ing transition of an initially homogeneous solution that properties and can exchange molecules rapidly with the
rearranges and separates into two phases that can coexist dilute phase [1]. Studies in recent years have shown that
stably in solution: a dense and a dilute phase. During this LLPS is the driving force for the assembly of nonmembrane
physiochemical process, supersaturated macromolecules are organelles and other functional biomolecular condensates,
which can achieve spatiotemporal control of their internal
Xuejiao Jin and Min Zhou contributed equally. complex biochemical reactions without physical barriers [2,
3].
* Xiuling Cao Currently, the formation mechanisms of the resulting
cxiuling@cau.edu.cn condensates formed by LLPS are preliminarily understood
* Beidong Liu and are thought to rely on a network of weak and multi-
beidong.liu@cmb.gu.se valent protein–protein interactions. Many proteins exhibit
1
State Key Laboratory of Subtropical Silviculture, School LLPS behaviors, and a common feature of such proteins is
of Forestry and Biotechnology, Zhejiang A&F University, the presence of multivalent binding domains. Among these,
Lin’an, Hangzhou 311300, China intrinsically disordered regions (IDRs) are the main driv-
2
Department of Chemistry and Molecular Biology, University ers that provide multivalent interactions [4]. Studies have
of Gothenburg, Medicinaregatan 9C, 413 90 Goteborg, shown that IDRs are rich in hydrophilic amino acids such as
Sweden asparagine, glycine, proline, serine, arginine, and aspartate,
3
Center for Large‑Scale Cell‑Based Screening, Faculty whereas they are lacking in hydrophobic amino acids such
of Science, University of Gothenburg, Medicinaregatan 9C, as valine, threonine, leucine, cysteine, isoleucine, histidine,
413 90 Goteborg, Sweden
13
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Page 2 of 23 X. Jin et al.
and tryptophan [4, 5]. The enrichment of only a few amino condensate formation, eventually providing new opportuni-
acids in these domains results in low complexity that could ties for the prevention and treatment of human diseases.
mediate weak interactions. Other proteins that contain oli- Phase separation of proteins is a multifactor dynamic
gomerization domains and multiple-folded modular domains process, and it occurs not only spontaneously under nor-
are also contribute to the multivalent interactions [5]. Fur- mal conditions, but also upon stimulation from an array of
thermore, emerging roles of RNA molecules in the assem- environmental factors, including changes in temperature,
bly of biomolecular condensates have been revealed. RNAs pH, ATP/energy, macromolecule concentration, and ionic
help establish the promiscuous interaction network through strength [14]. These physiological parameters constitute a
interactions with the RNA-binding domains of proteins, and continuous phase boundary, and crossing this boundary by
their intermolecular interactions and self-assembly define changing one or more parameters, such as by raising the
the compositions of higher-order condensates [6, 7]. Accu- temperature, depriving nutrients, lowering the pH, or chang-
mulating evidence shows that protein post-translational ing other factors, can cause phase separation and the forma-
modification (PTM) is another important mechanism that tion of condensates, which is an adaptive tuned response of
achieves cellular control of protein phase separation and cells [15]. Homeostasis of pH is a prerequisite for the normal
condensate formation through processes such as phospho- survival of organisms. Many proteins are very sensitive to
rylation, acetylation, SUMOylation, ubiquitination, meth- pH alterations, and a very small change in pH can induce
ylation, and ADP-ribosylation [8, 9]. These modifications phase transition of proteins. Phase separation in most pro-
can alter the weak multivalent interactions by changing the teins is triggered at low pH; while in others, it is induced by
charge, structure, hydrophobicity, and other properties of alkaline pH [16]. In vivo, the mechanism by which pH regu-
proteins, thus affecting phase separation behavior [8, 9]. In lates protein phase separation is not completely clear. Here,
addition, not only protein PTM but also RNA PTM affects we review the literature on stress-associated pH fluctuation
condensate dynamics. For instance, N6-methyladenosine in cells, how cells maintain and regulate cytosolic pH, and
(m6A) of RNA can modulate condensate formation and the effects of pH changes on protein phase separation. The
composition by regulating mRNA distribution into distinct mechanisms by which pH can mediate phase separation are
condensates or changing the phase separation behaviors of also discussed. Further research on these topics will not
its binding partners [10, 11]. only advance our understanding of compartment forma-
There is mounting evidence that LLPS and condensate tion affected by pH changes but will also provide important
formation are widely present in the cells and play important insight into the relationship between pH and a diverse set of
roles in a wide range of physiological processes (Fig. 1), human diseases.
including chromatin organization, cytoskeletal assembly,
signal transduction, transcriptional regulation, protein deg-
radation, cell division and differentiation, and environmental Diverse stresses induce intracellular pH
response and adaptation [2]. The condensates can transition fluctuation
into different material states such as gel- or solid-like states
[1, 12]. Therefore, maintenance of normal condensate mate- Cytosolic pH is a tightly controlled physiological param-
rial properties can ensure the assembly and disassembly of eter in all cellular systems, as almost all cellular processes
condensates in a tightly controlled manner to fulfill origi- depend on a constant pH for normal functions [17–21].
nal functions, while aberrant phase transition is causatively For instance, in yeast, pH is involved in replicative senes-
associated with the onset and development of age-related cence of mother cells and rejuvenation of nascent daugh-
neurodegenerative diseases and cancers (Fig. 1) [13]. In ter cells [22], and cytoplasmic acidification is critical for
recent research, the development of methods to study LLPS yeast cells to enter dormancy under stress conditions [23].
has become an important objective. A series of tools have In plants, intracellular pH changes are components of a
been developed to predict and analyze the phase separa- number of phytohormone signaling pathways, modulat-
tion capabilities of proteins [1]. Fluorescence microscopy ing gene expression and defence [21, 24]. In mammals,
observation techniques, including fluorescence recovery the maintenance of pH homeostasis is of key importance
after photobleaching (FRAP) and superresolution imaging, for the proper execution and regulation of neurotrans-
can also provide more detailed information on the material mission [25]. Small changes in cytosolic pH can lead to
properties, composition, and dynamics of biomolecular con- major changes in metabolism, signal transduction, protein
densates. In addition, in vitro reconstitution using purified folding, and protein–lipid interactions [19, 20]. However,
proteins is an accessory method used for studying LLPS [1]. organisms are often exposed to diverse adverse conditions
These methods can help researchers to further elucidate the throughout their life cycles, and stress-induced cytosolic
compositions of biological molecules and related biologi- pH fluctuations are broadly present in the cells; these fluc-
cal reactions and explore the factors that drive or influence tuations are induced, for example, by osmotic stress, heat
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Effects of pH alterations on stress‑ and aging‑induced protein phase separation Page 3 of 23 380
Fig. 1 Liquid–liquid phase separation in mammalian cells and its LLPS. a Mutations in the substrate recognition domain of the tumor
involvement in aging-related neurodegenerative diseases and can- suppressor SPOP prevent its binding to oncogenic substrates and sub-
cers. Under physiological conditions, scaffold biomacromolecules sequent condensate formation with ubiquitin ligase complex, causing
undergoing liquid–liquid phase separation (LLPS) can interact with a failure of oncogenic substrate ubiquitination and proteasomal deg-
and recruit other client molecules to form reversible liquid-like con- radation. b Mutation of p53 can accelerate its solid-phase transition
densates, which participate in a wide range of physiological pro- into amyloid aggregates, which is found in more than 50% of human
cesses. During aging, multiple factors, including protein mutation and cancers. c Chromosomal translocations lead to aberrant condensate
repeated expansions, cellular environmental and metabolic changes, formation of transcriptional regulators (TRs) at enhancers and pro-
damage to protein quality-control systems, and abnormal protein moters of oncogenes, driving abnormal oncogenic transcriptional pro-
localization and post-translational modification, can affect the LLPS grams. d Mutation or overexpression of signaling receptors alter the
process and promote aberrant gel-like condensate or pathological pro- formation of signaling clusters and activates aberrant signaling cas-
tein aggregate formation, ultimately leading to the onset and progres- cades, contributing to cancer development
sion of neurodegenerative diseases. Tumorigenesis is also related to
shock, and nutrient restriction [26–30]. Aging processes Temperature perturbation

and human diseases, including neurodegenerative diseases
and cancers, are also strongly linked with intracellular pH Proper environmental temperature is a critical factor for cell
alterations [22, 31, 32]. Table 1 summarizes the stresses survival. When the temperature becomes harsh, organisms
that can induce pH fluctuation in the cells of mammals, must respond rapidly to adapt and thrive. The best-known
plants, and microorganisms. Here, we describe the rela- stress response, the heat shock response (HSR), is a con-
tionships between pH changes and certain stresses, such served transcriptional program mediated by heat shock fac-
as temperature perturbation, starvation, and osmotic chal- tor 1, which is activated upon heat stress. It upregulates the
lenges, as well as aging and aging-related diseases, includ- transcription of a set of molecular chaperones to help the
ing neurodegenerative diseases and cancers. cell to manage the accumulation of heat-induced aberrantly
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Table 1 Effects of stresses on intracellular pH (pHi)
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Stress Species pHi Effects of stress The reason of pH change References
Page 4 of 23
Heat shock Yeast ↓ Membrane permeability increase Protons in the environment influx [19, 30, 33–35]
Drosophila Slight cell swelling and altered metabolic activ- Intracellular protons increase [28, 36, 37]
ity
Mammals Inhibit cell growth and change plasma mem- Inhibition of N
a+–H+ exchange and metabolic [38–42]
brane fluidity, permeability to small molecules, pathways
and membrane-bound enzyme activity
Starvation Yeast ↓ Decrease cytoplasmic mobility and volume, and Energy shortage to pump protons out [23, 43–47]
cell enters dormancy
Plasmodium Block mitosis Lack of energy to maintain the pH gradients [48]
inside and outside the cell
Osmotic stress Bacteria ↓ Change cell volume and metabolic processes Cell loses water and the concentration of protons [49, 50]
increases, and activate distinct OmpR-related
pathways
↑ Proton efflux and osmolarity-stimulated K
+ [51–53]
uptake
Protists ↓ Cells shrink, largely rearrange cellular proteins Cell loses water and secretes protons [54–56]
between compartments and decrease activity
Weak acid Yeast Bacteria ↓ Decrease cell growth rate and cell growth stasis Intracellular protons increase and inhibit the [45, 46, 57–59]
ability of cells to maintain normal pH
Hypoxia and anoxia Mammals Plants ↓ Cytoplasmic acidosis or cell death Metabolites produced by anaerobic fermenta- [26, 29, 60–62]
tion/ respiration, such as lactic acid
Alcohols Yeast ↓ Interfere with membrane transport by changing Proton permeability increase [63, 64]
the lipid composition of the plasma membrane
Pathogen Plants ↓ Cause pathological damage and activate defense Oxidation burst [21, 65]
responses
Light intensity Plants ↑ (light enhance), ↓ (light reduce) Affect photosynthesis Proton entering/leaving the thylakoids [21, 66]
Aging Yeast ↑(vacuole) Cells lose their physical integrity, resulting in Pma1 accumulates on the plasma membrane [22, 67]
Mammals (Rat ↓ impaired function (such as mitochondria/lyso- Na+–H+ exchange may be impaired [32]
hippocampus) some dysfunction) and increased risk of death
or diseases
Oxidative stress Plasmodium ↓, Lose pH control and decrease intracellular ATP Inhibition of V-ATPase activity [68]
↑ (vacuole) level

Mammals ↑ (bovine brain synaptic vesicle) Reduce neurotransmitter storage and release [69]
Cancer – ↑ – Change the expression and/or activity of plasma [31]
membrane ion pumps and transporters that
promote H+ efflux
a
Changes in pHi: increase (↑), decrease (↓)
X. Jin et al.
folded proteins and aggregates [70]. On the one hand, upreg- to changes in membrane permeability [90]. In turn, heat-
ulated chaperones can efficiently refold nonnative proteins induced proton influx and pH decreases can activate plasma
or promote the degradation of protein aggregates through membrane ATPase, whose activity is necessary for cell
autophagy or the ubiquitin–proteasome system (UPS). On survival under heat shock [33, 91]. The plasma membrane
the other hand, they can regulate the deposition of certain ATPase pumps intracellular protons out of the cell, partially
misfolded proteins into specialized cellular locations to offsetting the internal acidification resulting from the heat-
shield them from degradation and to refold them after stress induced increase in membrane permeability [33]. In mam-
[71]. In addition to HSR, another adaptive mechanism called malian cells, heat shock leads to a dramatic loss of plasma
the unfolded protein response induced by endoplasmic retic- membrane Na+–K+ ATPase activity, which then results in
ulum stress is also activated upon heat exposure and helps to loss of the inwardly directed electrochemical Na+ gradient
mitigate the damage caused by heat [72, 73]. Moreover, in across the membrane [39]. Therefore, it is speculated that
different research models, ubiquitination-dependent [74–77] Na+ gradient-dependent H+ export from the cytoplasm to
and autophagy-dependent degradation [78–82] have been the outside by Na+–K+ ATPase is affected and that the cyto-
observed to be induced after heat shock, and the activation plasm is acidified during heat stress.
of these degradation systems is essential for cell survival
and recovery from thermally induced protein aggregation Starvation
[74, 82].
In addition to the activation of evolutionarily conserved A decrease in cytosolic pH can also be caused by starva-
systems that contribute to thermotolerance, temperature tion. In yeast cells, numerous studies have shown that
change is often coupled with fluctuations in cytoplasmic pH the cytoplasmic pH decreases from approximately 7.4 to
[28, 83]. Some studies have shown that heat shock acidifies approximately 6.0 under starvation conditions [43, 45, 47].
the cytoplasm. For instance, in yeast, an intracellular pH Pma1, the plasma membrane-localized P-type H+-ATPase
drop can be induced by heat shock [35]. The same heat-asso- in yeast, is involved in pumping protons out of the cells and
ciated pH changes have also been observed in Drosophila is a primary contributor to the maintenance of cytosolic pH
melanogaster [28] and rat hepatoma cells [41]. Stress-asso- stability near neutrality [92, 93]. Importantly, its activation
ciated acidification is thought to be toxic to cells in some requires glucose-regulated phosphorylation [94]. In addition,
cases [29, 84]; whereas in other cases, it might be a cyto- other pumps, such as V-type H+-ATPases (V-ATPases), are
protective strategy that promotes cellular fitness under stress also responsible for cytosolic pH regulation [95]. They work
[23, 33, 85]. For example, cytosolic acidification is required by pumping excess protons into the vacuole to regulate cyto-
for HSR induction in translationally inhibited cells under solic pH homeostasis; they also maintain effective localiza-
heat shock, which allows the cells to adapt to high tempera- tion of Pma1 at the plasma membrane [95, 96]. Glucose is
ture by increasing the transcription of quality-control com- also required for V-ATPase activation because it mediates
ponents [35, 86]. Furthermore, some stress granule (SG) reversible associations between the V1 and V0 domains of
resident proteins, such as DEAD-box RNA helicase Ded1 V-ATPase [43, 97]. Under favorable conditions (with glu-
[87], poly(A)-binding protein 1 (Pab1) [34], and poly(U)- cose), V-ATPase cooperates with Pma1 to pump protons
binding protein 1 (Pub1) [88] in yeast, and Ras-GTPase- out of the cytoplasm and help cells stabilize cytoplasmic
activating protein SH3-domain-binding protein (G3BP1) pH in an ATP-dependent manner. However, upon glucose
in mammalian cells [89], have been reported to respond to depletion, a drop in cytoplasmic pH is observed, as starved
elevated temperatures to undergo LLPS. Likewise, they can yeast cells lack efficient H+-ATPase assembly and activation
also respond to low pH that mimics the pH conditions during to support the proton gradient across the membrane [46].
heat stress. Therefore, heat-induced acidification may play a Intracellular protons cannot be discharged outside of the
key role in protein LLPS following heat exposure and then cell. Instead, they accumulate inside the cell; thus, cytosolic
regulate SG dynamics and cell survival under or after stress. pH decreases. The increased concentrations of intracellular
The mechanism by which heat shock acidifies the cyto- protons cause the phase transition of the cytoplasm from a
plasm is not fully understood. However, evidence has shown fluid-like to a solid-like state, and such a dormant or qui-
that the compositions and structures of the cell membrane escent state is a protective strategy for cell survival under
are very sensitive to changes in temperature. In yeast, heat conditions of starvation [23]. Likewise, evidence suggests
shock increases membrane permeability, resulting in pro- that nutrient supply is also closely related to cytoplasmic
ton influx and a rapid decrease in intracellular pH [90]. pH in Physarum plasmodium. The cycle of intracellular
Studies have shown that intracellular pH disturbance is the pH corresponds to the period of the cell cycle of P. plas-
triggering mechanism of thermotolerance in yeast [33], modium. When P. plasmodium is growing in non-nutrient
and changes in plasma membrane compositions contribute medium, the intracellular pH remains stable and then begins
to the thermotolerance of cells, which may also be related to decline gradually, which serves to block normal mitosis.
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However, upon refeeding of starved P. plasmodium with the human body is often disturbed [115]. Thus, normal osmotic
nutrient medium, intracellular pH can recover to normal val- regulation is impaired, and this impairment is followed by
ues and the cell cycle resumes [48]. increases in the incidence and severity of diseases, such as
hypoosmolality and hyperosmolality [116].
Osmotic stress Interestingly, a growing body of evidence implicates
hyperosmotic stress as a factor leading to internal pH alter-
In addition to heat shock and starvation, osmotic stress is ation. In Listeria monocytogenes, a ubiquitous gram-pos-
another important environmental factor affecting cell sur- itive food-borne pathogen, the initial response to osmotic
vival and growth. Organisms including microbes, plants, stress caused by sorbitol or NaCl is a decrease in intracel-
and mammals, are commonly confronted with hyperos- lular pH [50]. Hyperosmotic stress also leads to cytosolic
motic conditions, which trigger a series of actions resulting acidification in Dictyostelium discoideum, which works as
in downregulation of cellular activity and progression of a novel signal mediator responsible for hyperosmotic stress
disease [98–100]. When osmolarity changes, cells adjust responses [54]. Moreover, another study has indicated that
their volumes accordingly in response to the changing envi- hyperosmotic shock elicits a transient increase in Escheri-
ronment. Cells mainly regulate volume changes by control- chia coli cytoplasmic pH, but the pH returns to normal val-
ling substance influx and efflux, which is usually manifests ues after osmotic adaptation [52]. However, whether and
as cell contraction or expansion, so that cells can return to how osmotic dysregulation in mammalian cells alters pH
a normal resting state [101–103]. A variety of membrane and whether it is relevant to human diseases remain unclear;
transporters are involved in this complex regulation process. thus, these aspects require further investigation to advance
For example, in a hypotonic environment, mammalian cells our understanding of pH-related condensate formation and
initially expand via water uptake and subsequently undergo diseases.
compensatory shrinkage to partially regulate volume reduc- Taken together, the evidence indicates that diverse envi-
tion, usually through efflux of KCl and organic osmolytes ronmental alterations contribute to intracellular pH fluctua-
[104, 105]. In contrast, in hypertonic environments, cells tion. Manipulating intracellular pH not only serves to main-
undergo transient dehydrating contraction by absorbing tain the morphology and function of cells to ensure normal
Na+, K+ and CI− and then pumping out N a+ to regulate the growth and metabolic activities, but also is associated with
increase in cell volume [104]. the preservation of cellular equilibrium in response to sev-
Intracellular osmotic homeostasis is necessary to maintain eral environmental factors, which could promote cellular
normal cell function and survival, and osmotic dysregulation fitness.
is the basis of many diseases and their complications, includ-
ing cataracts [106], epilepsy [107], inflammation [100, 108], Aging and neurodegenerative diseases
and hypernatremia [109]. For instance, in hyperglycemia
or hypergalactosemia, activated aldose reductase converts Aging is usually an irreversible biological process and is
glucose and lactose to galactose and sorbitol, respectively, considered to be a predominant risk factor for many neuro-
which accumulate in the lens and cause osmotic swelling, degenerative diseases [117]. Nine hallmarks of aging have
leading to diabetic cataracts [106]. In addition, cancer and been tentatively identified in different organisms, including
aging processes are also closely related to intracellular genomic instability, telomere attrition, epigenetic altera-
osmotic regulation. Many studies have shown that ion chan- tions, loss of proteostasis, deregulated nutrient sensing,
nels and ion pumps are beneficial to the development and mitochondrial dysfunction, cellular senescence, stem cell
progression of cancer [110]. Given the importance of ion exhaustion, and altered intercellular communication. These
channels for osmotic homeostasis and the abnormal expres- hallmarks can be classified into three layers: primary hall-
sion of transporters in many cancers [111], it is likely that marks, antagonistic hallmarks, and integrative hallmarks,
the original homeostasis in cells will be disrupted, creating which co-occur during aging and are usually interconnected
a more favorable internal environment for cancer develop- with each other; defining the exact relationships and causal
ment. Interestingly, Yes-associated protein (YAP), is a tran- network of these hallmarks may contribute to future studies
scriptional coactivator that is widely activated in cancer cells on aging and aging-related diseases [118].
[112], can sense the tumor microenvironment and modify In addition to the hallmarks of aging discussed above,
the physicochemical properties of the surrounding environ- growing evidence shows that intracellular pH alterations are
ment by activating transcription, thereby promoting tumor also intimately linked to aging processes and aging-related
development [113]. Moreover, YAP-activated transcription neurodegenerative diseases. In mammals, the intracellular
is mediated by the LLPS process, which also occurs under pH of central neurons is tightly regulated, and its fluctua-
hypertonic conditions [114]. Aging does not directly cause tions are important for signaling and synaptic plasticity [119,
disease, but in this process, the homeostasis of water in the 120]. Specifically, in cortical neurons, a mild intracellular
13

pH decrease occurs following an excitability increase, and (Fig. 2), which utilizes the inwardly directed electrochemical
this decrease acts as feedback to reduce local bioelectric Na+ gradient generated by Na+–K+ ATPase to export H+
activity and excitability. However, when the intracellular pH [32]. Moreover, limited ATP synthesis during aging might
is outside a certain range and reaches its limits, there may be also affect ATP-driven ion pumping, including N a+ gradi-
+ +
an increased risk of cell death [25, 119, 121, 122]. Impor- ent generation by N a –K ATPase [131]. The impacts of
tantly, a decrease in neural pH levels has been observed in a aging-related alterations on pH regulation are controversial.
number of neurodegenerative disorders [123, 124] and even A slight decrease in intracellular pH may provide neuropro-
in the normal aging process [32, 125, 126]. Moreover, acute tection [132], while successively greater acidification may
neuroinflammation has been observed to provoke intracellu- increase the vulnerability of brain tissue to stressful condi-
lar acidification in the mouse hippocampus [127]. For exam- tions [125, 133–135].
ple, in mammalian cortical neurons, intracellular pH is nega- In addition to cytosolic pH dysregulation, lysosomal/
tively correlated with aging, as evidenced by significantly vacuolar pH dysregulation has also been implicated in aging
lower pH in hippocampal slices from aged rats than in slices and aging-related neurodegenerative diseases. Evidence is
from young rats [32, 128]. Likewise, in human neurons, the now emerging that defective lysosomal function is a major
intracellular pH has also been observed to decrease with factor in the pathogeneses of different types of neurodegen-
aging [126, 129]. The mechanisms involved in decreased erative diseases, specifically, a failure of the maintenance
intracellular pH may be the disruption and overwhelmed of a highly acidic lysosomal/vacuolar pH [136]. There is
of pH regulatory systems through processes including also increasing evidence for aging-related compromise of
aging-related decreases in buffering capacity and disrup- lysosomal function [22]. In yeast, vacuolar pH is a critical
tion of diverse transmembrane acid/base-transporters [32, regulator of mitochondrial function and replicative lifespan.
128–130]. For instance, considering that Na+–H+ exchange Vacuolar acidity declines with aging, and reduced vacuolar
is the dominant regulatory mechanism for proton extru- acidity disrupts pH-dependent amino acid homeostasis in
sion in cultured hippocampal neurons, altered H+ homeo- the vacuolar lumen, resulting in age-related dysfunction
stasis might be attributable to impaired Na+–H+ exchange of mitochondria and a shortened lifespan [67]. In addition,
Fig. 2 Aging affects intracellular pH. When cells are young, P-type pH decreases. Moreover, cell buffering capacity is also impaired dur-
H+-ATPases distributed on the plasma membrane act in concert ing aging. V-type H+-ATPase is a target of oxidative stress in aging.
with V-type H+-ATPases localized on the lysosomal/vacuolar mem- Increased oxidative modification of V-type H+-ATPase might inhibit
brane to regulate intracellular pH. However, during aging, for exam- V-type H+-ATPase-mediated vacuolar acidification. Alternatively,
ple, in yeast, P-type H+-ATPase Pma1 accumulates on the plasma aging might alter lysosomal/vacuolar acidification by downregulat-
membrane, and excessive H + is pumped out of the cell, resulting in ing V-type H+-ATPase subunit expression, lowering the availability
reduced cytosolic H availability for V-type H+-ATPase. This leads
+
of V-type H +-ATPase. The solid lines represent normal ion transport.
to a decrease in vacuolar acidity. In other cases, such as in the aged The dashed lines represent impaired ion transport. In the young cell
rat hippocampus, the Na+–K+ pump and N a+–H+ exchange may be cytoplasm, yellow represents cytoplasm with a normal pH. In the
impaired; as a result, H+ accumulates in the cytoplasm, and cytosolic aged cell cytoplasm, red represents cytoplasm with a decreased pH
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380
lifespan extension via calorie restriction and methionine Cancers

restriction requires vacuolar acidification [67, 137, 138]. The
decrease in vacuolar acidification in yeast is due to excess In recent years, increasing evidence has linked LLPS and
accumulation of the major regulator of cytosolic pH, Pma1, condensates to tumorigenesis. A growing number of cancer-
in mother cells (Fig. 2). Vacuole acidity is thus antagonized associated proteins have been reported to have the ability to
by reduced cytosolic proton availability [22]. Importantly, undergo LLPS and form biomolecular condensates, such as
V-ATPase is implicated in lysosomal acidification. Muta- speckle-type POZ protein, which is involved in oncogenic
tions in V-ATPase or proteins that regulate V-ATPase func- substrate degradation [150]; p53-binding protein 1 (53BP1)
tion are observed in aging-related neurodegeneration [136]. and FET proteins, which are involved in the DNA damage
It is conceivable that during aging, oxidative stress might response and genomic stability [151, 152]; and EWS-FLI1,
impair V-ATPase activity through increased oxidative modi- β-catenin, YAP, and PDZ-binding motif (TAZ), which are
fication of V-ATPase (Fig. 2), which is inspired by the obser- involved in transcriptional regulation [114, 153–155]. In all
vation that hydrogen peroxide inhibits bovine brain synaptic of the above cases, disrupting functional condensate assem-
vesicle V-ATPase activity [69]. In fact, increased oxidative bly of tumor suppressors or promoting aberrant condensate
modification of V-ATPase subunits has been observed in assembly of oncoproteins contributes to the oncogenic pro-
aged rat brain tissue [139], and oxidative modification is cess (Fig. 1a–d). In addition, aberrant assembly of other
known to impair the activity of certain enzymes [140, 141]. membrane-less compartments formed by LLPS, including
Alternatively, aging might alter lysosomal/vacuolar acidifi- SGs [156, 157], PML bodies [158], paraspeckles [159],
cation via dynamic transcriptional regulation of V-ATPase and amyloid bodies [160], is also associated with cancer.
subunits (Fig. 2), a mechanism that is supported by the The tumor suppressor p53 has been the “star molecule” of
observation of reduced V-ATPase subunit mRNA levels in molecular biology and oncology since its discovery. It acts
hippocampal neurons in sporadic Alzheimer’s disease (AD) as a transcription factor, activating or inhibiting the tran-
[142]. In conclusion, intracellular pH alterations, including scription of various downstream target genes involved in
cytosolic pH changes and lysosomal/vacuolar pH dysregula- cell cycle regulation, senescence, and apoptosis [161, 162].
tion, are also striking features that occur during the aging p53 prevents tumor development through cell cycle arrest,
process and aging-related diseases onset. DNA repair, and antioxidant protein production to main-
The processes of aging and aging-related neurodegenera- tain genome integrity and limit cell proliferation under
tive diseases onset are typically accompanied by the forma- adverse conditions such as DNA damage, hypoxia, oncogene
tion of widespread intracellular protein aggregates [143]. expression, nutrient deprivation, and ribosomal dysfunction
Many RNA-binding proteins, such as fused in sarcoma [162–164]. Moreover, its mutation, which tends to result in
(FUS), tau, alpha synuclein (α-Syn), and TAR DNA-binding protein aggregation, is found in more than 50% of human
protein 43 (TDP-43), are the main components of protein cancers [165, 166]. Recent evidence has revealed that the
inclusions or aggregates in diverse neurodegenerative dis- p53 core domain can undergo LLPS and then undergo a
eases, including amyotrophic lateral sclerosis (ALS) [144], phase transition to the solid-like state. Mutation of p53 can
frontotemporal dementia (FTD) [144, 145], Parkinson’s dis- accelerate its solid-phase transition into amyloid aggregates
ease (PD)[146], and AD [147]. Furthermore, these disease- (Fig. 1b) [167]. Therefore, it is a reasonable assumption that
associated proteins are well known to undergo LLPS, and the differences in the tumor microenvironment compared to the
failure to maintain their liquid-phase homeostasis may serve microenvironment of normal differentiated cells may trig-
as a trigger of solid protein aggregate formation (Fig. 1) ger certain proteins to undergo LLPS and phase transition
[148]. Diverse layers of regulation may affect their transi- to solid aggregates, leading to further cancer progression.
tion from a liquid-like state with physiological function to As cancer cells grow at an uncontrolled high rate, they
solid pathological aggregates. Therefore, it is reasonable to are usually challenged with an adverse macroenvironment
speculate that alterations in the intracellular microenviron- characterized by hypoxia and nutrient starvation [168].
ment, such as pH changes during aging, provide these phase- Apart from this, considerable evidence links cancer directly
separated neurological disorder-related proteins with the to pH alterations since a higher intracellular pH and a lower
opportunity to change their phase separation behaviors and extracellular pH than those of normal differentiated cells
increase the risk of aggregation. In fact, evidence has already are observed in most cancers, regardless of tissue origin
shown that LLPS of α-Syn and its subsequent maturation and cell type [169, 170]. These differences may be attribut-
into protein aggregation are pH-mediated [149]. Therefore, able to changes in the expression and/or activity of plasma
further investigations on aging-induced pH dysregulation membrane ion pumps and transporters, as well as changes
will not only advance our understanding of aberrant LLPS in metabolic activities [169, 170]. In turn, the increased
and compartment formation but will also provide important intracellular pH and the decreased extracellular pH also
insight into the onset of aging-associated pathologies. synergistically enhance cancer progression. On the one
13

hand, the increased intracellular pH can increase cell prolif- of distinct transport pathways. For example, P-type proton
eration, facilitate apoptosis evasion, and promote cytoskel- pumps are widely distributed on eukaryotic cell membranes,
etal remodeling for cell migration. On the other hand, the and they are the main determinants of proton efflux and cyto-
acidified extracellular environment can increase the activi- plasmic pH control in plants and yeast [19, 173]. As mentioned
ties of acid-activated proteases and promote extracellular above, Pma1 is the most abundant protein in the plasma mem-
matrix degradation, thereby accelerating tumor cell invasion brane of yeast and actively coordinates with V-ATPases to
and dissemination [31]. However, during these processes, regulate cytosolic pH [19, 173]. V-ATPases can also acidify
whether and how pH alterations of cancer cells are related compartments in an ATP-dependent manner and are distrib-
to the aberrant phase behavior of cancer-related proteins or uted in acidic organelles such as the Golgi apparatus, vacuole/
aberrant formation of membrane-less compartments such lysosomes, and endosomes of all eukaryotic cells [180]. In
as SGs, PML bodies, paraspeckles, and amyloid bodies, yeast cells, V-ATPase activity is indispensable for vacuolar
remains unclear and requires further in-depth investiga- acidification during glucose metabolism and homeostasis of
tion. Such research will provide more knowledge about the cytoplasmic pH in the short term. In the long term, V-ATPase
molecular basis of cancer and facilitate the development of is very important for the stability of Pma1 localization [95].
new therapies. F-type proton pumps are mainly distributed in the bacterial
plasma membrane, the mitochondrial membrane, and the plant
endomembrane. In enterococci, when the cytoplasm is acidi-
Cytosolic pH control by metabolism‑based fied, the level and activity of F-type H+-ATPase increase syn-
and transporter‑based regulation chronously, leading to cytoplasmic alkalization [181]. When
the pH value is restored to the initial value, the decrease in the
Since pH control is a critical requirement for growth in all amount and activity of the enzymes terminates proton extru-
organisms, different organisms have adopted a number of sion. Thus, changes in the amount and activity of enzymes
common strategies to address the challenges of pH main- seem to be necessary for pH regulation [182].
tenance in the face of rapid metabolism and extracellular Moreover, these proton pumps act in concert with a large
environment changes [171–173]. Cells separate metabolites, array of other transporters. Increasing evidence indicates
proteins, and biochemical processes in a manner dependent that a number of ion/H+ exchangers are also important for
on compartmentalized membrane-bound organelles, each intracellular pH regulation in different organisms, including
of which has distinct pH requirements and pH regulation yeast, plants, and mammals [19, 172, 173]. These exchang-
mechanisms [172]. More importantly, cellular compartments ers couple the transfer of H + across biological membranes to
have inherent pH buffering capacities. This buffering is counter-transport of other cations, such as N a+ or K
+, to pro-
achieved by the presence of various intracellular weak acids tect against excess acidification. Furthermore, Na+-coupled
and bases, as well as the ionizable groups of macromol- HCO3− transporters, which are involved in the uptake of extra-
ecules such as side chains of amino acids [174]. Moreover, cellular HCO3−, have also been reported to play key roles in
cytosolic pH regulation also relies on metabolites produced the regulation of cytosolic pH. They contribute to the main-
by pH-dependent biological reactions [175]. Organic acids tenance of C O2–HCO3− equilibrium, the most important pH
such as malate can produce or consume H + via carboxylation buffering system [183, 184]. Although the importance of pro-
and decarboxylation reactions. Therefore, correct synthe- ton extrusion in pH control has been revealed, acid-importing
sis, degradation, and transport of organic acids through the transporters such as C l−–HCO3− exchangers, which allow
−
cytoplasm to other organelles are thought to be important HCO3 efflux, can efficiently prevent overalkalization of the
strategies to regulate intracellular pH [176, 177]. In addition, cells by working counter to C O2–HCO3− transporters to enable
the alternative pathways to glycolysis, the cyanide-resistant the fine control of cytosolic pH [185].
alternative respiration pathway and malate-derived lactic and In summary, cells exhibit a complicated pH regulation net-
alcoholic fermentation, which are unique to plants, jointly work dependent on the interplay among multiple transporters
regulate pH homeostasis in plants [175]. In mammalian cells that import or export proton equivalents and metabolism-based
and fermenting yeast, CO2 produced during metabolism can regulatory mechanisms, and this network can accurately regu-
diffuse freely through biological membranes. It can react late and maintain cytosolic pH. More details can be found in
with water to form H CO3−, which is an effective proton recent reviews [19, 171–173].
buffer and consumes protons to produce carbonic acid when
the cells are confronted with an acute drop in intracellular
pH [178, 179].
However, when cells are under long-term stress, the major
regulatory mechanism to maintain cytosolic pH homeostasis
is the membrane transport of H+, which involves a large array
13

380
pH‑dependent phase separation condensate switch in SG assembly. Its phase separation also occurs in
formation induced by stress an RNA-dependent manner under low pH and heat shock
[89, 203]. Moreover, members of the Asp–Glu–Ala–Asp
As discussed above, many types of stress cause a decrease (DEAD)-box ATPase (DDX)3 family are widely present
in cytoplasmic pH, and these stress conditions are known in both eukaryotes and prokaryotes [192, 194], and stud-
to induce phase separation of proteins to form conden- ies have suggested that many proteins in the DDX family
sates. Here, we summarize the proteins that are known to undergo LLPS in vivo or in vitro, including Ded1, Dbp1,
form phase separation condensates in response to pH stress and Dbp2 in yeast; DDX3X, DDX4, and DDX6 in humans;
together with other stresses in which phase separations are and DeaD, SrmB, and RhlE in E. coli [192, 193]. Ded1p,
mainly affected by pH alterations, such as heat shock and an ATP-dependent DEAD-box RNA helicase in yeast, is
starvation (Table 2). an indispensable translation initiation factor and a compo-
Many biomolecules undergo LLPS to form liquid-like nent of SGs [188]. It can parse the secondary structure of
condensates that mediate diverse cellular functions [222, mRNA 5′ untranslated regions for ribosomal scanning and
223]. For example, autophagosome formation is a pro- recognition of the initiation codon [186, 187]. Studies have
cess that is precisely regulated by protein phase separa- shown that Ded1p undergoes phase separation and forms
tion. Atg1 complex formation is a prerequisite for preau- condensates at elevated temperatures, or at lower tempera-
tophagosomal structure (PAS) assembly and autophagy tures when the pH is adjusted to that of the heat-stressed
initiation [202]. Recent research suggests that the PAS is cytosol (heat-shocked cells experience a decrease in cyto-
a liquid-like condensate formed by phase separation of solic pH). When in condensate form, Ded1p is translation-
the Atg1 complex, which is critical for further dynamic ally inactivated, which leads to a switch in translation from
recruitment of other proteins or factors during autophago- housekeeping transcripts to stress-responsive transcripts
some formation. Notably, this process occurs under low [87]. Therefore, heat shock-induced and temperature-asso-
pH and starvation conditions [200–202]. TORC1 is a ciated pH change-induced Ded1p condensation in SGs is
modulator of PAS organization that targets Atg1 complex an adaptive response to survive heat shock. It promotes an
assembly by regulating the phosphorylation/dephospho- evolutionarily conserved heat shock response that selec-
rylation of Atg13, a component of the Atg1 complex [202, tively translates housekeeping or heat shock transcripts
224]. Its activity is also modulated by phase-separated [87]. Similarly, another DDX family member in yeast,
compartments such as SGs. Under stressful conditions, Dhh1, is responsible for the assembly and disassembly
including heat, starvation, and osmotic stress, TORC1 is of RNA-containing membrane-less organelles. Dhh1 also
recruited into SGs; as a result, TORC1 signaling is inhib- exhibits enhanced phase separation at low pH, which mim-
ited [225–227]. For example, in yeast, TORC1 is parti- ics the pH conditions during glucose starvation [192].
tioned into heat shock-induced SGs, which then prevents Moreover, evidence indicates that in changed growth
an increase in the frequency of heat-induced DNA muta- conditions, enzyme activities can be acutely regulated
tions [225]. Under osmotic stress, TORC1 in mammalian through the formation of phase separation-induced enzyme
cells is similarly sequestered into SGs, thereby blocking its condensates, which restrict or promote specific biochemi-
signal transduction to downstream effectors [227]. cal reactions in membrane-less organelles, suggesting the
SGs are also dynamic membrane-less organelles, the importance of phase separation in regulating the metabo-
formation of which is driven by LLPS [228, 229]. It has lism of cells [47, 195]. For instance, glutamine synthetase
been reported that many proteins in SGs exhibit phase (Gln1) is an indispensable metabolic enzyme that catalyzes
separation behavior under stress-associated pH changes. the synthesis of glutamate and ammonium into glutamine,
Pab1, is an RNA-binding protein consisting of a short a process that requires ATP. Gln1 forms filaments during a
N-terminal sequence, four RNA recognition motifs state of advanced cellular starvation, and filament formation
(RRMs), a proline-rich low-complexity region (LCR) and leads to enzymatic inactivation [197]. Further evidence dem-
a C-terminal peptide-binding domain. It plays a key role onstrates that starvation-induced cytosolic acidification is
in controlling the polyadenylation, stability, and transla- the trigger for Gln1 condensate formation, and many meta-
tion of mRNA in yeast cells [34, 190]. Pub1 is similar bolic enzymes follow this principle to help cells endure and
to Pab1 in that it is an RNA-binding protein with three recover from severe starvation conditions [47].
RRMs and one LCR [191]. Both Pub1 and Pab1 are core In addition to the above-mentioned findings, there are
components of SGs and are prone to phase separation other proteins for which LLPS is directly or indirectly
when temperature increases, pH decreases, or nutrients affected by pH changes, increasing cell fitness or inducing
are lacking to help cells survive during stress [34, 88, diseases. For instance, Sup35 is a translation termination
189]. In addition, G3BP1 is a central node and molecular factor in budding yeast [198]. It can form condensates upon
energy depletion or at a low pH. This pH-dependent phase
13

Table 2 Proteins undergoing LLPS and response to stress-induced pH changes
Protein Function Domain function in RNAa Species Stress Effects of pH References
LLPS
Ded1p Initiate translation IDR + Yeast Acidic pH, heat shock − [87, 186–188]
Pab1 Control mRNA polyade- LCR, RRMs − Yeast Acidic pH, heat shock Act as signal messenger [34, 189, 190]
nylation, stability, and and affect electrostatic
translation interaction
Pub1 Regulate translation LCR, RRMs − Yeast Acidic pH, heat shock, Affect protein solubil- [88, 191]
glucose starvation ity and electrostatic
interaction
DDXs Coordinate mRNA LCDs + , − (if excess) Bacteria, Yeast, Mam- Acidic pH, glucose − [192–194]
de-capping and decay, mals starvation
regulate general trans-
lational repression
Gln1 Promote the conver- − − Bacteria, Yeast Acidic pH, glucose Act as signal messenger [47, 195–197]
sion of glutamate into starvation
glutamine
Sup35 Terminate translation The N-terminal prion − Yeast Acidic pH, glucose Act as signal messenger [44, 198]
domain and the elec- starvation
trically neutral domain
Atg1 complex Participate in PAS IDRs − Yeast Acidic pH, glucose Possible act as signal [199–202]
assembly starvation messenger
Effects of pH alterations on stress‑ and aging‑induced protein phase separation
G3BP1 Promote SG assembly IDRs, nuclear transport + Mammals Acidic pH, heat shock, Affect protein solubil- [89, 203]
and inhibit RNA factor like domain, osmotic stress ity and electrostatic
aggregation RBD interaction
SARS-CoV-2 N protein Participate in viral RNA IDR1 + Virus Acidic pH Affect electrostatic [204–206]
replication and virion interaction
packaging
α-Syn Act as a SNARE- The N-terminal region − Mammals Acidic pH Affect electrostatic [149, 207–209]
complex chaperone (most family disease interaction
and contribute to mutations occur) and
Parkinson’s disease the “non-amyloid-β
pathogenesis component” region
4R-Tau Induce tubulin assembly The microtubule-bind- − Mammals Lower-critical solution Affect electrostatic [16, 210–212]
and stabilize micro- ing repeats transition interaction
tubules
FUS Participate in DNA LCDs + , − (high ratios) Mammals Acidic pH, DNA dam- − [213, 214]

repair, transcription, age, heat shock
and RNA biogenesis
53BP1 Regulate the DNA dam- The oligomerization − Mammals Acidic pH, DNA dam- − [151, 215, 216]
age response and p53 domain and BRCT age, light
signaling domain
13
Page 11 of 23 380
380
separation of Sup35 can serve as a means for Sup35 to res-

References cue itself from stress-induced damage and promote recovery
[217–221]
of the yeast cell from stress [44]. The nucleocapsid protein
(N) of the severe acute respiratory syndrome coronavirus
(SARS-CoV-2) is a multivalent RNA-binding protein that
is essential for viral RNA replication and virion packag-
solubility and electro-
Possible affect protein
ing [206]. The N protein can partition into SGs and inter-
static interaction
act with G3BP1/2 to block the assembly of SGs through

RNA-dependent liquid phase separation and thus disrupt
Effects of pH
the immune response of host cells [204]. Notably, phase

separation of the N protein occurs under physiological con-
ditions and is enhanced at low pH [205]. α-Syn is an IDP
for which aggregation into amyloid-like fibrils is associated
Lower-critical solution
with PD pathology [207, 208]. One study found that α-Syn

initially undergoes phase separation and becomes rigid over
time and eventually transforms into solid-like aggregates.
transition
Low pH can promote α-Syn LLPS and further increase the

maturation and nucleation of α-Syn aggregates, which is
Stress
relevant to PD pathogenesis [149]. Additionally, pathologi-

cal inclusions of the microtubule-associated protein Tau
have been reported to accumulate in patients with several
Artificial synthesized
neurodegenerative diseases [210–212]. Evidence indicates

that the microtubule-binding repeats of the Tau protein have
a strong propensity for liquid demixing, which occurs over
Species
a wide range of pH values. The phase separation of these

four repeats at different pH values wound concentrate the
most aggregation-prone Tau residues and further promote
amyloid formation [16]. Interestingly, in addition to natu-
ral proteins, artificially constructed polypeptides can also
undergo phase separation. Elastin-like polypeptides (ELPs)
RNAa
are recombinant protein polymers composed of pentapeptide

(Val–Pro–Gly–Xaa–Gly)L repeat units, which are recurring
−
Effects of RNA on phase separation: promoting/requiring ( +), inhibiting (−)
motifs in tropoelastin in a wide range of species. ELPs are

often used as new biomaterials for drug delivery and tis-
Domain function in
sue engineering [218–221]. One study found that ELPs can

exhibit reversible phase separation triggered by a wide range
of pH values, and this pH responsiveness is controlled by
the type and number of ionizable residues and the molecular
LLPS
weight of the ELPs. This property of specific pH-controlled

−
ELP phase separation can be applied in drug delivery sys-

drug delivery and tis-
tems for local cancer therapy, as various tumors types usu-

New biomaterials for
ally have different pH values than healthy tissues [217].

sue engineering
Finally, as we discussed above, many cancer-associated

proteins have been reported to undergo LLPS and to be
Function
involved in biomolecular condensate formation. 53BP1 is a

binding partner of p53 [230] that can directly regulate p53
and affect p53 target gene expression [231]. It is also one of
the main regulators of the DNA damage response, loss of
Table 2 (continued)
which has been associated with apoptosis and cancer cell

proliferation [232]. Studies have found that 53BP1 under-
goes LLPS at DNA damage sites, forming DNA repair con-
Protein
densates that recruit and stabilize p53 [151]. If the expres-

ELPs
sion of 53BP1 is changed or LLPS behavior is affected, the

a
13

disruption of condensate formation leads to destabilization in response to these stresses [34, 88]. It is known that LLPS
of p53 and reduced induction of its target genes as well as is driven by multivalent weak macromolecular interactions
cell cycle arrest [151]. Interestingly, it has been reported (protein–protein, protein–RNA, and RNA–RNA interac-
that 53BP1 can respond to low pH to form 53BP1 droplets tions), disruption or alteration of which would affect pro-
[151]; thus, further studies on the relationships of pH regu- tein phase separation behaviors [5, 233]. Therefore, pH
lation and 53BP1 LLPS will help enhance our understand- changes can influence intramolecular or intermolecular pro-
ing of tumorigenesis. However, besides 53BP1, research on tein–protein/RNA interactions by changing the net charges
the relationships between cancer-associated proteins and of components, thereby driving LLPS (Fig. 3). For example,
pH dysregulation are limited. Considering that the physi- G3BP1 is a multidomain protein composed of two folded
ochemical properties and microenvironment of cancer cells domains and two IDRs. Under nonstress conditions, its
are different from those of normal cells [168], two impor- central negatively charged, glutamate-rich IDR can interact
tant research topics in the future are whether these proteins with the C-terminal positively charged RG-rich region to
undergo pH-regulated LLPS and how pH-regulated LLPS allow G3BP1 to fold into a compact state. This compact
is relevant to tumorigenesis. In addition, research on how state is an autoinhibitory conformation that disrupts G3BP1
microenvironmental changes in cancer cells affect the phase separation. However, at a low pH, protonation of the
dynamics of intracellular membrane-less organelles such as clustered glutamates changes the net charge of the acidic
SGs, PML bodies, paraspeckles, and amyloid bodies, whose IDR and disrupts its stable electrostatic interactions with
aberrant assembly is associated with cancer, is also needed. the RG-rich region, allowing G3BP1 to expand from its
Such research will provide further evidence regarding the original tightly self-inhibited state and release the RG-rich
links among pH, LLPS, and cancer. region. G3BP1 can then further facilitate intermolecular
protein–RNA/protein interactions to drive LLPS, which is
consistent with the observation that heterotypic interactions
Mechanisms underlying pH‑mediated phase among G3BP1 and RNA molecules drive SG assembly [89,
separation 203]. In addition, a low pH can directly trigger Pub1 assem-
bly, and this pH-dependent assembly formation is sensitive
pH changes mediate protein–protein/RNA to salt concentrations, suggesting that electrostatic interac-
interactions tions promote Pub1 assembly. Self-interactions among the
RRM domains are the main drivers for Pub1 phase sepa-
Some proteins or their specific domains possess the ability ration, and acidic pH may change the charge distribution
to sense stresses directly and thus undergo phase separation in the RRM domains, thereby mediating the electrostatic
Fig. 3 Roles of pH in biomolecular condensate formation. Under multiple functional roles in triggering liquid–liquid phase separation
nonstress conditions, proteins and RNAs are dispersed in the cyto- (LLPS)-driven condensate formation; for example, it affects protein–
plasm. When cells are exposed to stresses such as starvation, heat protein/RNA interactions, alters protein solubility, or acts as a mes-
shock, or acid stress, the intracellular pH changes, and this change senger to transmit stress signals. pH changes can also enhance phase
is accompanied by the formation of protein- and RNA-containing separation, which may gradually mature and result in transformation
biomolecular liquid-like condensates. During this process, pH plays into an irreversible gel-/solid-like state
13

380
interactions [88]. Moreover, the pH range that can induce changes in pH can affect the solubility of Pub1 and lead to
artificially recombinant ELP phase separation is related to the formation of stress-responsive Pub1 condensates [88].
the pKa. This suggests to a certain extent that pH can affect Changes in pH appear to be able to decrease the solubili-
protein–solution or protein–protein interactions by changing ties of many proteins. In yeast, a decrease in pH results in a
the number of ionizable residues of proteins, thus trigger- phase transition of cytoplasm from a fluid-like to a solid-like
ing phase separation [217]. Likewise, phase separation of state, which might be caused by decreased solubilities of a
Pab1 at low pH is also an electrostatically mediated process series of proteins and subsequent formation of intracellu-
[34]; thus, the principle of pH-dependent protein condensate lar solid-like assemblies, such as SGs [23, 235]. Moreover,
formation mediated by electrostatic interactions may be gen- the relationship between pH and protein isoelectric point is
eralizable to many proteins. closely related to solubility. The closer the pH value is to the
Notably, pH changes not only initiate protein LLPS by isoelectric point of the protein, the lower its solubility, and
facilitating intramolecular or intermolecular interactions the more likely it is that phase separation occurs [236, 237].
but also enhance phase separation and its further maturation This could explain to a certain degree why the microtubule-
into a gel state or a pathological solid state (Fig. 3). Phase binding repeats of Tau are most prone to phase separation
separation of α-Syn is mediated by an interplay of electro- when the pH is close to the isoelectric point but less prone
static interactions in the unstructured N-terminal domain to demixing when the protein solubility is increased in
and hydrophobic interactions in the central NAC domain, response to pH that is substantially higher or lower than the
while the charge distribution in these domains is strongly isoelectric point [16]. Therefore, it is believed that one of the
dependent on the pH value. A lower pH serves to change mechanisms by which pH triggers protein phase separation
the net charges and hydrophobicity of different domains as is the alteration of protein solubility (Fig. 3).
well as the interactions between these domains, leading to
significant structural reorganization. Thus, pH-mediated
diverse changes in α-Syn accelerate the maturation of phase pH changes act as messengers to transmit stress
separation and subsequent protein aggregation [149, 209]. signals
Similarly, a reduction in pH can enhance the intramolecular
interactions of the phase-separated SARS-CoV-2 N protein In the face of adverse conditions, intracellular pH might act
and lead to irregularly shaped assemblies with less liquid- as a messenger to signal changes in the environment, trig-
ity, in vitro [205]. Indeed, phase separation proteins that gering phase separation of proteins to promote cell fitness.
contain flexible LCDs are highly prone to forming patho- Upon heat shock, cells can integrate signals of different
genic aggregates. This could explain, to some extent, why temperatures and temperature-induced pH changes into a
hundreds of proteins are highly prone to forming aggregates unified response to provide a trigger for phase separation.
during aging. During aging or chronic pH stress, these phase For example, Pab1 undergoes LLPS autonomously through
separation proteins can transition into irreversible aggre- temperature-dependent structural changes under conditions
gates, which could then lead to persistent condensate for- of stressful temperatures [34].
mation, such as persistent SG formation, even after the stress However, how does a cell sense other stresses, such as
subsides. Persistent condensates typically exhibit solid-like starvation, to trigger LLPS to help cells survive diverse
properties; and as a consequence, other pathological changes adverse conditions? Previous studies have indicated that pro-
and neurodegenerative disorders occur [233]. teins and protein-associated condensates that undergo LLPS
under starvation conditions, such as Pub1, Gln1, Dhh1, and
pH changes affect protein solubility PAS, can also respond to low pH [47, 88, 192, 200, 201].
Considering that cytosolic pH is rapidly and reversibly reg-
In addition to engaging in the promiscuous interactions that ulated by glucose metabolism, the stress information per-
function in phase separation, macromolecules must reach ceived by these proteins is most likely transmitted through
a critical concentration threshold to start LLPS. Evidence pH. Evidence has shown that cytosolic pH is a second mes-
indicates that not all LCRs and IDRs function as autono- senger for glucose to mediate activation of the PKA pathway
mous modules that drive phase separation; instead, they through V-ATPase [43]. Therefore, a change in pH might
function as modifier sequences, regulating the solubility be an extremely sensitive readout of other changes in the
of phase-separating proteins and the material properties of environment, especially starvation, to induce protein LLPS
condensates [234]. Long-term evolutionary pressure has and cellular adaptive responses (Fig. 3).
tuned the solubility of Pub1 to be very close to the criti- In this way, pH is capable of playing diverse functional
cal threshold for phase separation. This not only endows roles in the regulation of LLPS, including by affecting pro-
Pub1 with a solubility that is conducive to growth but also tein–protein/RNA interactions, altering protein solubility,
enables Pub1 to quickly sense and respond to stress. In fact, and acting as a messenger to transmit stress signals.
13

Conclusion and perspective considerably by different stresses, and a given protein

might display different phase separation behaviors under
From viruses to prokaryotes and eukaryotes, the forma- different pH values. Therefore, the identification of pro-
tion of macromolecular condensates by phase separation teins that respond to different pH values or have behavior
is emerging as a principle means for cells to regulate cel- changes that accompany pH changes may provide vital
lular functions and adapt to environmental changes. Cells clues for investigation of the machineries involved in the
encounter a variety of stresses, some of which can cause influences of pH on cellular functions.
cytoplasmic pH fluctuations. In this review, we have sum- Finally, intracellular pH changes and phase separation
marized the relationships between pH changes and certain condensate formation are linked to aging, aging-related
stresses, such as heat shock, nutrient stress, and osmotic neurodegenerative diseases, and cancers. It would be
stress, and described which proteins or physiological interesting to further investigate how aging-induced pH
processes can respond to stress-associated pH changes changes affect protein phase separation. Importantly, inno-
through phase separation. We have also highlighted the vative drug delivery strategies could be developed for spe-
diverse ways by which pH fluctuation can influence protein cific local cancer therapy by exploiting the altered intracel-
phase separation. For example, pH can act as a signal to lular and extracellular pH in tumors. Attempts to modulate
transmit stress information, mediate protein–protein/RNA pH and SG formation could also spur the development of
interactions, and affect protein solubility, thereby regulat- innovative approaches for cancer therapy.
ing protein/RNA phase separation.
Despite the research progress concerning the relation-
ships between stress-associated pH changes and phase Funding Open access funding provided by University of Gothenburg.
This work was supported by grants from the National Natural Science
separation discussed in this review, further in-depth inves- Foundation of China (32000387) to XC, Scientific Research Founda-
tigations are still needed. It is worth noting that pH might tion of Zhejiang A&F University (2021LFR053) to XJ, and the Swed-
not be the sole determinant of stress-induced phase separa- ish Cancer Fund (Cancerfonden) [CAN 2017/643 and 19 0069] and the
tion and condensate formation. For stresses such as heat Swedish Natural Research Council (Vetenskapsrådet) [VR 2015-04984
and VR 2019-03604] to BL.
shock, changes in both intracellular temperature and pH
are involved, which can lead them to differences in protein
Declarations
phase separation behavior and condensate material proper-
ties [88]. The interplay of pH, temperature, ion strength, Conflict of interest The authors have no relevant financial or non-fi-
RNA concentration, protein concentration and other fac- nancial interests to disclose.
tors forms a sophisticated network that dynamically affects
the phase behavior of proteins. However, some questions Author contributions XJ and MZ wrote the manuscript. XJ, MZ, SC,
and DL compiled the tables and created the figures. Both XC and BL
remain. How does pH interact with other factors in this designed and edited the manuscript.
process? What are the differences and similarities in the
roles of pH among the different stress-induced phase sepa- Data availability Data sharing not applicable to this article as no data-
ration processes? Preliminary evidence suggests that the sets were generated or analyzed during the current study.
properties of different condensate materials formed by dif-
ferent groups of proteins can be used by cells to build a Open Access This article is licensed under a Creative Commons Attri-
hierarchical stress-adaptive system that is fine-tuned to dif- bution 4.0 International License, which permits use, sharing, adapta-
tion, distribution and reproduction in any medium or format, as long
ferent conditions [88]. In other words, when encountering as you give appropriate credit to the original author(s) and the source,
different types of stresses or the same stress with different provide a link to the Creative Commons licence, and indicate if changes
intensity or duration, a cell can regulate the activities of were made. The images or other third party material in this article are
multiple proteins to achieve specific biological functions included in the article’s Creative Commons licence, unless indicated
otherwise in a credit line to the material. If material is not included in
by concentrating specific cellular components in the con- the article’s Creative Commons licence and your intended use is not
densates (or excluding them from the condensates) for a permitted by statutory regulation or exceeds the permitted use, you will
favorable period of time. The cells can then determine need to obtain permission directly from the copyright holder. To view a
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sates can be used by the cells as a method to promote
adaptation to stress. Therefore, revealing the differences
and similarities among the various roles of pH in address-
ing different types of stress will provide insights into the References
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Cellular and Molecular Life Sciences (2022) 79:380

https://doi.org/10.1007/s00018-022-04393-0 Cellular and Molecular Life Sciences

Effects of pH alterations on stress‑ and aging‑induced protein phase

Keywords Acidification · Membrane-less compartment · Neurodegenerative disease · Tumorigenesis · Protein aggregation

Introduction separated from the solution to form a dense phase, while

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shock, and nutrient restriction [26–30]. Aging processes Temperature perturbation

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Table 1 Effects of stresses on intracellular pH (pHi)

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lifespan extension via calorie restriction and methionine Cancers

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separation of Sup35 can serve as a means for Sup35 to res-

act with G3BP1/2 to block the assembly of SGs through

the immune response of host cells [204]. Notably, phase

with PD pathology [207, 208]. One study found that α-Syn

Low pH can promote α-Syn LLPS and further increase the

relevant to PD pathogenesis [149]. Additionally, pathologi-

neurodegenerative diseases [210–212]. Evidence indicates

a wide range of pH values. The phase separation of these

are recombinant protein polymers composed of pentapeptide

Effects of RNA on phase separation: promoting/requiring ( +), inhibiting (−)

motifs in tropoelastin in a wide range of species. ELPs are

sue engineering [218–221]. One study found that ELPs can

weight of the ELPs. This property of specific pH-controlled

ELP phase separation can be applied in drug delivery sys-

tems for local cancer therapy, as various tumors types usu-

ally have different pH values than healthy tissues [217].

Finally, as we discussed above, many cancer-associated

involved in biomolecular condensate formation. 53BP1 is a

which has been associated with apoptosis and cancer cell

densates that recruit and stabilize p53 [151]. If the expres-

sion of 53BP1 is changed or LLPS behavior is affected, the

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Conclusion and perspective considerably by different stresses, and a given protein

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­ +-ATPase null mutants. J Biol Chem 275(51):40088–40095.

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Table 1 Effects of stresses on intracellular pH (pHi)

+-ATPase null mutants. J Biol Chem 275(51):40088–40095.