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How Intrinsically Disordered Proteins Modulate Biomolecular Condensates

Yanyan Wang , Limin Chen , Siyang Li , Luhua Lai

Progress in Chemistry ›› 2022, Vol. 34 ›› Issue (7) : 1610 -1618.

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Progress in Chemistry ›› 2022, Vol. 34 ›› Issue (7) : 1610-1618 DOI: 10.7536/PC220324
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How Intrinsically Disordered Proteins Modulate Biomolecular Condensates
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Abstract

Biomolecular condensates form various cellular membraneless organelles and play diverse biological functions as a result of their specific physicochemical properties. For example, biomolecular condensates are able to perceive changes in the external environment, regulate the cellular concentration of proteins, modulate different signaling pathways and selectively partition hub proteins as well as nucleic acids. Abnormal formation and changes of biomolecular condensates are closely related to human diseases, especially neurodegenerative diseases, cancers and viral diseases such as COVID-19. Intrinsically disordered proteins (IDPs) play key roles in the formation and regulation of biomolecular condensates. In this review, we analyze the roles that IDPs and small molecules play in biomolecular condensates formation and regulation, propose the possibility of rationally regulating biomolecular condensates through ligand design targeting IDPs, and discuss the challenges of understanding biomolecular condensate formation and regulation mechanisms and for discovering novel chemical compounds to modulate biomolecular condensates.

Contents

1 Introduction

1.1 Discovery of biomolecular condensates and their important functions

1.2 Brief introduction of intrinsically disordered proteins

2 Function of intrinsically disordered proteins in the formation of biomolecular condensates

3 Intrinsically disordered proteins participate in the regulation of biomolecular condensates

4 Regulatory roles of chemical compounds on biomolecular condensates

5 Drug design targeting intrinsically disordered proteins

6 Conclusion and perspective

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Keywords

intrinsically disordered proteins / biomolecular condensates / driving force / chemical compound regulation

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Yanyan Wang, Limin Chen, Siyang Li, Luhua Lai. How Intrinsically Disordered Proteins Modulate Biomolecular Condensates. Progress in Chemistry, 2022, 34(7): 1610-1618 DOI:10.7536/PC220324

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1 Introduction

1.1 Discovery of condensed phase in biological system and its important function

In order to survive, organisms must coordinate with each other in time and space, organize various complex biochemical reactions orderly in dense cell space, and ensure that the reactions are carried out efficiently and do not interfere with each other. The important means to maintain the order of complex biochemical reactions is to compartmentalize cells. However, many studies have shown that there are many kinds of non-membrane organelles in cells, such as nucleoli, Cajal bodies, PML nuclear bodies in the nucleus and Stress granules, P bodies in the cytoplasm[1]. Because these membraneless organelles lack a physical barrier separating their internal components from the surrounding medium, it has been impossible for many years to understand how they concentrate molecules, maintain and regulate molecular structure, control their components, and regulate internal biochemical activities. Recent studies have shown that these membraneless organelles are supramolecular assemblies of proteins and nucleic acids, which are isolated from the outside world by forming Biomolecular condensates[2,3].
The biomolecular condensed phase is a membrane-free structure that can participate in cell compartmentalization, concentrating cellular reactants. Unlike classical membranous organelles, such as the nucleus, mitochondria, and Golgi apparatus, these structures are not constrained by a lipid bilayer and do not have the stable characteristics that make up a cell. Instead, they tend to form reversibly and dynamically through phase separation. In 2009, Brangwynne and Hyman et al studied P particles of nematodes and found that P particles are not solid, but like droplets, they collide and fuse with each other, disperse into very small droplets after violent shaking, and then quickly fuse into large droplets[4]. Since then, scientists have found that many membraneless organelles in cells, such as nucleoli, Cajal bodies and stress granules, are generated by the phase transition of specific proteins and RNAs[5][6][7][8]. In 2012, Rosen et al. Reconstructed phase separation in vitro by multivalent proteins[9][9,10]. When the concentration of biomolecules exceeds a threshold concentration, the biological system produces a condensed phase through liquid-liquid phase separation or liquid-solid phase transition, and the condensed phase of biomolecules can have the properties of liquid, gel, or solid.Liquid condensed phases can age to have the properties of a gel or a solid, resulting in biomolecular condensed phases with different shapes, sizes, and behaviors[11].
The unique physical and chemical properties of biomolecular condensed phases enable them to perform a variety of functions, including sensing changes in the external environment and responding quickly to environmental changes[12⇓⇓~15]; Regulate the intracellular concentration of related proteins, such as the formation of local high-concentration proteins, thereby activating some biochemical reactions and activating related signal transduction pathways[16]; At the same time, it can isolate some proteins from their substrates and inhibit some biochemical reactions in cells. In addition, the biomolecular condensed phase can mediate the localization of some proteins to some organelles without membrane encapsulation, and selectively allow some proteins and RNAs to enter the specific biomolecular condensed phase[17]. Biomolecular condensations can also participate in transcriptional regulation, genome organization, immune response, and neuronal synaptic signaling, while playing an important role in human diseases, including neurodegenerative diseases, cancer, and viral diseases such as COVID-19[18].
Liquid-liquid phase separation of biomacromolecules is an emerging hot topic in the field of international life science in recent years. Abnormal phase separation is associated with a variety of human diseases such as neurodegenerative diseases and cancer. There have been many reviews on the study of biomolecular condensed phases, and more detailed information can be found in recent representative reviews. In July 2020, Zhang Hong, Zhang Mingjie, Zhu Xueliang and Li Pilong published a review, summarizing the recent major progress in the role of biomacromolecule phase separation in various biological pathways and diseases[19]. In addition to animals, plants can also produce phase separation. Xu Xiumei et al published a review focusing on the plant-specific biological processes involved in phase separation, including photosynthetic CO2 concentration mechanism, light signal perception and signal transduction, salicylic acid, auxin and other hormone signal transduction[20]. Li Pilong et al. analyzed the relationship between phase separation and biomolecular aggregates in vivo through in vitro reconstruction phase separation, and introduced the formation and regulation mechanism, composition and function of biomolecular phase separation[21]. Researcher Zhang Hong and others systematically summarized the existing research on the interaction between membrane-containing organelles and membrane-free organelles, and discussed how the two organelles interact and coordinate with each other to achieve various important life functions in cells[22]. By listing and summarizing the important functional systems of biomolecular phase separation, Zhang Changsheng et al discussed the relationship between the biological function of biomolecular phase separation and the physical and chemical mechanism from the basic properties, phase diagrams and microstructures of phase separation aggregates[23].
In addition, with the increasing understanding of biomolecular phase separation, many databases and prediction methods of biomolecular phase separation have been developed[24⇓⇓~27][28]. Li et al. Published a review on six biomolecular phase separation databases (LLPSDB, PhaSePro, PhaSepDB, DrLLPS, RNAgranuleDB, HUMAN CELL MAP) that have been developed so far, compared the data contents of different databases, analyzed the differences between them, and discussed the application scope of different databases[29]. Recently, based on the LLPSDB database, Chu et al. Developed a sequence-based phase separation protein prediction tool, PSPredictor, using machine learning methods to predict potential phase separation proteins[28].

1.2 Introduction to Disordered Proteins

Intrinsically disordered proteins (IDPs) are proteins that have no fixed secondary or tertiary structure under physiological conditions. Full-length proteins without fixed structure, as well as proteins with Intrinsically disordered regions (IDRs), are collectively referred to as natively disordered proteins[30]. Natural disordered proteins have been found to be abundant and to have important biological functions. The current understanding is that ordered domains and native disordered regions are two basic modules of protein functionality: native disordered proteins and ordered proteins can be functionally complementary, and native disordered regions and ordered domains can function synergistically when present on a protein at the same time[31]. Naturally disordered proteins have attracted much attention because of their key roles in cancer, neurodegenerative diseases, diabetes, and cardiovascular diseases[32].

2 The Role of Disordered Proteins in the Formation of Biological Condensed Phases

Disordered proteins play an important role in the formation of biological condensed phases. Natural disordered proteins that produce phase separation are rich in short linear interaction motifs and degenerate repetitive sequences, which are the main regions involved in protein-protein interactions[33]. For biological macromolecules, the multivalency of linear domains and motifs is a defining feature that drives protein or RNA phase transitions. One of the most extensive multivalent interactions comes from naturally disordered regions of proteins, sometimes called Low-complexity domains (LCDs), which can mediate not only intermolecular interactions, but also phase separation by mediating intramolecular interactions[34]. Briefly, the formation of condensed phases of biomolecules is attributed to specific weak multivalent interactions between molecules, including Electrostatic, π-π, Cation-π, and Hydrophobic interactions (Fig. 1)[35].
Electrostatic interactions play a key role in the formation of condensed phases in a variety of biological systems. For example, our work found that the transcriptional repressor VRN1 of Arabidopsis thaliana interacts with DNA through electrostatic interaction to produce phase separation, and the distribution of charged residues in the disordered region of VRN1 regulates the interaction strength between VRN1 and DNA, and the change of charge pattern leads to the interconversion between VRN1 and DNA in different phase separation forms (precipitate, droplet, and no phase separation)[36,37]. Under non-stress conditions, G3BP1 is stabilized in a compact self-inhibited state without phase separation by intramolecular electrostatic interactions between its disordered negatively charged region and its positively charged arginine-rich region. However, when the negatively charged mRNA interacts with the arginine-rich region of G3BP1, the self-inhibitory interaction of G3BP1 is destroyed, which leads to the conformational transition of G3 BP1 and promotes the aggregation of G3 BP1 through protein-RNA interaction, resulting in RNA/protein aggregates[38,39]. Another example is the tumor suppressor p53, which regulates the formation of phase separation through the multivalent electrostatic interaction between the N-terminal transcriptional activation region and the C-terminal tetramerization domain[40].
π-π and cation-π interactions are involved in the regulation of condensation in biological systems, especially the cation-π interaction between arginine and tyrosine, which is a key factor in the formation of disorder-driven phase separation. In 2018, Wang et al. carried out a large number of mutation experiments using FUS family proteins as a model system, compared in detail the driving ability of different forces and amino acids on phase separation, and found that the phase separation ability of FUS was lost whether the Y of FUS was mutated into S or R into G, indicating that the phase separation mediated by FUS proteins was probably driven by the cation-π interaction between R/Y[41]. At the same time, the phase separation ability of FUS and hnRNPA1α was significantly enhanced by mutating some polar residues to R. In addition, the researchers found that the π-π interaction between Y/Y can also drive the phase separation.
Hydrophobic interactions can also act as a driving force to promote the generation of biomolecular condensed phases. Many proteins in cells, including FUS, TDP-43, BRD4, SOX2 and Annexin A11, can form biomolecular condensed phase at low salt concentration, and the phase separation of these proteins can be destroyed by increasing salt concentration. But interestingly, by further increasing the salt concentration, these proteins can again produce phase separation[42]. By combining experiments and simulations, it is found that unlike the low-salt system, in which the condensed phase is stabilized by electrostatic interactions, the reentrant phase transition in the high-salt system is driven by hydrophobic and nonionic interactions.

3 Disordered Proteins Involved in the Regulation of Aggregation in Biological Systems

The aggregation properties and conformational changes of disordered proteins can regulate the formation of biological condensed phases. Natively disordered proteins can produce phase separation by themselves through the interaction between IDRs. For example, IDRs in FET family proteins, including hnRNPA1, hnRNPA2B1, RNA polymerase Ddx4, and Laf1, are sufficient by themselves to produce phase separation[43][44][45][46]. At the same time, many proteins in the condensed phase of biomolecules can also form the condensed phase by recruiting interacting proteins through IDRs. Different biomolecular condensed phase proteins have specific sequence characteristics of IDRs, for example, nucleoli contain arginine-rich IDRs, nuclear speckles contain serine/arginine-rich IDRs, and stress granule proteins contain a large number of polar residues and a small number of aromatic residues and charged residues, commonly known as prion-like domains[47,48].
Disordered protein IDRs can not only promote the formation of phase separation by their own condensation, but also regulate phase separation by regulating protein conformation. Native disordered proteins usually consist of disordered alternating regions of net negative and positive charge, which can produce long-range electrostatic interactions to regulate phase separation[49,50]. For example, we show that the Arabidopsis transcription factor VRN1 affects protein conformational regulatory phase separation through the protein intermediate disordered strand[37]. Transcription factor VR N1 is composed of two DNA binding regions and a disordered strand connected in the middle, which can interact with DNA to produce phase separation. Bioinformatics analysis of transcription factor VRN1 found that in the process of evolution, the protein with similar structure to VRN1 contains two DNA binding regions and a disordered strand in the middle, and the length of the disordered strand in the middle of these proteins is constantly changing, but there is a pair of relatively conserved positive and negative charge regions in the disordered strand. By constructing protein mutants with different disordered chain lengths and positive and negative charge regions, it was found that the disordered chain length could change the phase separation behavior (aggregation, liquid-liquid phase separation, no phase separation), but the presence of the positive and negative charge regions of the disordered chain could help VRN1 to produce stable liquid-liquid phase separation (Fig. 2). Further molecular dynamics simulations and fluorescence resonance energy transfer experiments show that the disordered chain regulates the phase separation behavior by changing the protein conformation through regulating the distance between the two DNA binding regions. In addition, IDRs can cooperate with folding domains to regulate liquid-liquid phase separation. Lin et al. Produced phase separation by fusing the IDRs of FUS protein to polySH3 domain protein (polySH3) and mixing with polyproline domain (polyPRM) ligand, and found that the IDRs of wild type FUS protein promoted the phase separation of polySH3-polyPRM system, which reduced the threshold concentration of phase separation by 8 times[51].
In addition, IDRs usually contain many sites capable of post-translational modification, which further participate in the regulation of biomolecular condensation phase. More and more studies have found that serine and tyrosine phosphorylation, arginine methylation, ubiquitination and m6A modification can control phase separation[52⇓~54][55,56][57][58,59]. Interestingly, proteins prone to phase separation all appear to be enriched in residues that can be post-translationally modified[60]. In fact, post-translational modification of proteins can significantly change the charge or other properties of IDRs/LCDs, thereby regulating phase separation. FUS protein, a key pathogenic protein in ALS, has reversible phase separation, which is formed by cation-π interaction between tyrosine in the low complex domain of FUS and arginine in the C-terminal domain, and this interaction is regulated by arginine methylation modification.Hypomethylated arginine forms a strong cation-π interaction, which promotes FUS protein phase separation and gelation, induces FUS to condense into stable, β-sheet-rich hydrogel particles, and destroys the function of Ribonucleoprotein granules (RNPs). However, when arginine is hypermethylated, the tyrosine cation-π interaction between arginine and low complex domain is weakened, which reduces the gelation of FUS protein[56]. In addition, there is phosphorylation modification in the low complex domain of FUS protein, which can destroy the binding and phase separation of FUS hydrogel and reduce the fibrillation of FUS protein. When serine and threonine residues are phosphorylated, the negative charge introduced by phosphorylation will produce repulsive force, which hinders the interaction of cross-β, reduces the stability of the polymer, and destroys the formation of hydrogel and phase separation of FUS[61]. In addition, Tau protein promotes liquid-liquid phase separation through phosphorylation modification and accelerates the formation of amyloid. Tau itself is a disordered, lysine-rich, highly soluble protein that binds to tubulin. When Tau is multisite phosphorylated, the positive charges bound to microtubules are shielded, and Tau forms droplets and condenses with negatively charged molecules, promoting amyloid formation[62].

4 Regulation of condensed phase of biomolecules by small chemical molecules

At present, it has been found that some small chemical molecules can regulate the condensed phase of biomolecules, but in general, there is still a lack of effective specific molecules, which need more research. Small chemical molecules can bind to natively disordered proteins, kinetically stabilizing protein monomers and reducing their propensity to aggregate[63]. At equilibrium, the population of the various States accessible to a protein depends on their relative free energies. For a simple two-state system, such as a globular protein with a corresponding extended state, the use of chemical chaperones to lock the globular state can stabilize the protein folded state structure by reducing the number of unfolded proteins, which can prevent protein degradation and aggregation. For natively disordered proteins capable of phase separation into biomolecular condensed phases, stabilization of the monomeric state with small molecules can inhibit the aggregation process[64].
In May 2019, Fang et al., through high-throughput drug screening in HEK293T and human iPSC-induced motor neuron cell lines, found that Mitoxantrone and other small molecules with planar structure can change the size, number and composition of stress granules, and inhibit the recruitment of RNA binding proteins related to amyotrophic lateral sclerosis (ALS) into stress granules[65]. In August of the same year, Wheeler et al screened more than 1600 small molecule drugs approved by the Food and Drug Administration (FDA) of the United States, and found that Lipoamide and its analogue Lipoic acid could regulate the phase separation of FUS and reduce the formation of intracellular stress granules, and verified that the drug had the effect of treating ALS in nematode and fruit fly models[66]. In 2020, Girdhar et al. Found that AIM4, which was reported to inhibit the in vitro aggregation of TDP-43 in ALS as early as 2016, also has the ability to disrupt its phase separation[67]. The research on the phase separation of the N protein of SARS-CoV-2 has made rapid progress, and the molecules that affect the phase separation behavior of the N protein have been reported. For example, Jack et al. Identified three classes of drug molecules that regulate N protein phase separation by screening 1200 FDA-approved drugs in vitro[68]. Zhao et al. Found that two small molecules, CVL218 and PJ34, could increase the size of N-protein phase-separated condensates[69].
Biomolecular condensed phases are not only regulated by small chemical molecules, but also can effectively enrich small drug molecules due to their unique physical and chemical properties, thus regulating their efficacy. In 2020, Young and his collaborators studied several cancer drugs and found that some anticancer drug molecules were selectively enriched by intracellular condensates, which provided a new possibility for understanding the efficacy of drugs[70]. They also found that phase separation may also be associated with drug resistance mechanisms in cancer. For example, the anticancer drug tamoxifen can expel the estrogen receptor ER from the condensate of MED1, thereby inhibiting the transcription of downstream oncogenes, which may be one of the mechanisms of tamoxifen's anticancer function. In tamoxifen-resistant cancer cells, the ER mutant was not driven out of the MED1 condensate by tamoxifen. In addition, for drug-resistant cancer cells with no ER mutation but MED1 overexpression, MED1 forms larger aggregates due to its high concentration, which dilutes the effective drug concentration in the aggregates and may have a negative effect on drug efficacy. However, because the anticancer drugs used in this study were labeled with fluorescent genes, this large hydrophobic label may induce the enrichment of compounds in the condensed phase, so the distribution of original drugs in cells under physiological conditions needs further study.
In the studies of small molecules regulating liquid-liquid phase separation reported at present, although some articles give the binding data of small molecules with proteins, most of the small molecules are obtained by experimental screening, and do not provide the experimental data of direct in vitro binding[67,69]. In 2019, Wheeler et al. Screened that Lipoamide could regulate the phase separation of FUS protein, and used NMR to study its interaction with protein, but no significant chemical shift changes were observed[66]. In a subsequent review, they proposed that the regulation of phase separation by small molecules is different from the traditional mode of protein ligand binding, and does not necessarily require strong interaction between small molecules and targets[71].
Small chemical molecules can not only regulate phase separation, but also effectively inhibit the formation of protein solid-state aggregates. Previous studies have focused on compounds that inhibit the formation of protein solid-state aggregates, which may be formed by liquid-liquid phase separation or directly from homogeneous solution. Small molecules regulate phase separation and the formation of solid-state aggregates, essentially affecting homotypic or heterotypic interactions between proteins. Weak multivalent interaction aims to emphasize that there are many sites for interaction, and the interaction at each site is weak, while in terms of chemical nature, the type of interaction is the same, that is, hydrophobic, electrostatic, π-π, cation-π, etc. Small molecules that inhibit the production of solid aggregates can also be used to regulate phase separation, such as AIM4, which inhibits the aggregation of TDP-43 protein[67]. However, compared with the regulation of phase separation, because the solid aggregate is more stable, the regulation of solid aggregate is relatively more difficult, and once the solid aggregate is formed, it is difficult to destroy it. Therefore, studies have focused on the discovery of small chemical molecules that can maintain proteins in a liquid-liquid phase separation state to reduce the formation of solid-state aggregates, so as to achieve the treatment of related diseases[72].

5 Drug design targeting disordered proteins

As mentioned above, IDPs are involved in many important cellular processes, especially in the formation and regulation of biomolecular condensed phases. The protein level of IDPs is strictly regulated by normal cells, and the abnormal expression of IDPs often leads to the occurrence of many diseases, such as ALS and neurodegenerative diseases, which are related to poor biomolecular aggregation. Therefore, targeting disordered proteins for drug design is expected to achieve the regulation of biomolecular condensed phases, and IDPs are very potential targets for drug design. It is worth mentioning that disordered proteins account for about 40% of the entire human proteome, and the existing clinical druggable targets are basically structured proteins, so the development of drug design strategies for disordered proteins can greatly increase the number of druggable targets[73].
At present, most of the existing drugs that can control the physiological processes involved in IDPs do not target IDPs directly, but indirectly by targeting enzymes that modify IDPs, ordered domains in IDPs, or ordered proteins that bind to IDPs. Due to their conformational diversity and rapid conformational transition ability, disordered proteins lack specific drug-binding pockets, and traditional structure-based drug design strategies are often not applicable. In recent years, some small molecules that directly target disordered proteins have been discovered, providing ideas for ligand design and mechanism of action of these "non-druggable" targets.
Ruan Hao et al. of our laboratory summarized and analyzed the published disordered protein small molecule ligands in 2019. It can be seen that most of the disordered protein ligands were found through high-throughput experimental screening, mainly based on typical protein-protein interactions and phenotype-based screening[74]. For example, the earliest c-Myc inhibitor was discovered by Berg et al. In 2002. They constructed the Fret system of Myc-CFP and Max-YFP in vitro and obtained compounds such as IIA6B17[75]. Yin et al. Found compounds such as 10058-F4 and 10074-A4 through yeast two-hybrid experiments, among which 10074-A4 has a clear NMR result to characterize its binding site with c-Myc protein[76]; Toretsky et al. Used SPR to screen small molecule compounds NSC635437 of EWS-FLI1 in vitro[77]. Subsequently, screening methods based on dynamic changes of disordered proteins have gradually emerged, such as Invest et al., which discovered the ligand ZZW-115 of NUPR1 based on thermal denaturation experiments[78]. Based on 1H WaterLOGSY and STD NMR, Luiji et al. Screened SJ403, an inhibitor of p27, and found its binding site with p27 by two-dimensional 1H-15N HSQC NMR[79]. In addition, the discovery of the c-Myc inhibitor EN4 has promoted the screening of covalent inhibitors of disordered proteins, and some new technologies, such as PROTACs targeting disordered proteins, are also being tried and developed[80].
On the other hand, people have gradually begun to develop rational design strategies based on integrated structural biology (Fig. 3), and force fields that can be used in the study of disordered proteins have received attention and attention in recent years.The development of new force fields has improved the accuracy of disordered protein conformation sampling, and the introduction and use of various enhanced sampling methods have increased the richness of disordered protein sampling[81⇓⇓~84]. Based on the NMR data of c-Myc polypeptide monomer and 10074-A4 and c-Myc polypeptide complex, Yu Chen et al. In our laboratory carried out molecular dynamics simulation, drug conformation and pocket analysis and inhibitor virtual screening, and found PKUMDL-YC-1205. On this basis, they proposed the strategy of "multi-conformation affinity"[85][85]. Its in vitro and intracellular targeting was characterized by in vitro CD, SPR experiments, cell proliferation inhibition, downstream gene transcription and other tests, and its action site was further characterized by nuclear magnetic resonance technology. By studying the dynamic binding process of 10074-A4 and c-Myc polypeptide, Jin Fan et al. proposed the concept of "ligand cloud surrounding protein cloud", pointing out that the disordered protein may still be disordered after binding with small molecule ligands, from which it can be seen that the optimization of disordered protein ligands is very challenging[86]. Ruan Hao et al. Successfully discovered a small molecule activator PKUMDL-HR-1047 targeting p53 TAD1 through a rational design strategy combining computation and experiment[87]. The discovery of these compounds shows the feasibility of targeting disordered proteins, and to some extent verifies the "druggability" of disordered proteins.
Generally speaking, compared with the more mature drug discovery targeting ordered proteins, the drug discovery targeting disordered proteins still needs continuous exploration and attempt. How the discovered small molecules targeting disordered proteins affect the conformational ensemble of disordered proteins in vitro and in vivo, and further affect the composition and properties of biomolecular condensed phases, need more research, such as whether the existing p53 activators can reasonably regulate the phase separation involved by p53 in cells. The development of more efficient sampling methods, the establishment and improvement of bioassay systems, the verification of intracellular targeting, the optimization of ligand structure and the improvement of specificity, and the characterization of binding sites are also the directions that need to be tried and challenged in the field of disordered proteins in the future.

6 Conclusion and prospect

Disordered proteins play an important role in the condensed phase of biomolecules, which mediate intramolecular and intermolecular interactions through short linear interaction motifs or degenerate repetitive sequences, and participate in the regulation of phase separation. Disordered proteins can participate in the regulation of biomolecular condensed phase through self-aggregation, continuous dynamic conformational changes and post-translational modifications. Natively disordered proteins that form condensed phases of biomolecules by liquid-liquid phase separation are thought to remain disordered and can diffuse across liquid-liquid interfaces[88,89]. Although it is known that the conformational change of disordered proteins is involved in the regulation of biomolecular condensed phase, it is often difficult to determine the conformational change of disordered proteins because they have no fixed structure and are in constant conformational change. How to realize the dynamic structure determination of disordered proteins in biomolecular condensed phase and deeply understand the regulation of disordered conformational changes on biomolecular condensed phase is an urgent problem to be solved.
Different from the physicochemical property analysis of simple monomolecular protein, the relationship between sequence and function can be accurately predicted by long-term simulation of monomolecular protein[90]. Biomolecular condensed phase is a large system containing thousands of complex molecules, which often needs to be simplified by coarse-grained models, and only a subset of interactions are modeled, which greatly reduces the accuracy of computational simulation analysis. Therefore, how to accurately analyze and simulate the physical and chemical properties of complex bio-condensed phase systems is also a problem to be solved.
Chemical small molecules can interact with disordered proteins to stabilize the conformation of disordered protein monomers or aggregates and regulate the formation of biomolecular condensed phases. At the same time, due to the unique physical and chemical properties of the biomolecular condensed phase, the distribution of small molecules in the biomolecular condensed phase can be changed, and the effective enrichment or expulsion of small molecules can be realized. Therefore, it is expected that the formation of biomolecular condensed phase can be regulated by changing the conformation of disordered proteins through the binding of small molecules to disordered proteins. However, in addition to the interaction between disordered proteins and small molecules that can affect phase separation, other proteins or nucleic acid components can also dynamically regulate the threshold concentration of phase separation. Disordered proteins can regulate phase separation not only through their own interactions, but also through heterotypic interactions with other proteins, resulting in highly variable saturation concentrations of biomolecular condensed phases such as nucleoli[91]. Therefore, whether to target individual protein binding or to regulate the protein components of the whole organism in the condensed phase needs further study. At present, there is a lack of understanding of the condensed phase properties of biomolecules (such as their composition, main driving forces, mechanical properties, etc.) And the tendency of chemical small molecules that can be enriched, which may require the development of more label-free experimental techniques and theoretical simulation methods for further study[92][93].

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Funding

National Natural Science Foundation of China(21633001)

Chinese Academy of Medical Sciences(2021-I2M-5-014)

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