Cyclic dipeptide-based small molecules modulate zinc-mediated liquid–liquid phase separation of tau

Liquid–liquid phase separation (LLPS) is a complex physicochemical phenomenon mediated by multivalent transient weak interactions among macromolecules like polymers, proteins, and nucleic acids. It has implications in cellular physiology and disease conditions like cancer and neurodegenerative disorders. Many proteins associated with neurodegenerative disorders like RNA binding protein FUS (FUsed in Sarcoma), alpha-synuclein (α-Syn), TAR DNA binding protein 43 (TDP-43), and tau are shown to undergo LLPS. Recently, the tau protein responsible for Alzheimer's disease (AD) and other tauopathies is shown to phase separate into condensates in vitro and in vivo. The diverse noncovalent interactions among the biomolecules dictate the complex LLPS phenomenon. There are limited chemical tools to modulate protein LLPS which has therapeutic potential for neurodegenerative disorders. We have rationally designed cyclic dipeptide (CDP)-based small-molecule modulators (SMMs) by integrating multiple chemical groups that offer diverse chemical interactions to modulate tau LLPS. Among them, compound 1c effectively inhibits and dissolves Zn-mediated tau LLPS condensates. The SMM also inhibits tau condensate-to-fibril transition (tau aggregation through LLPS). This approach of designing SMMs of LLPS establishes a novel platform that has potential implication for the development of therapeutics for neurodegenerative disorders.     

Chemical probes for investigating protein liquid-liquid phase separation and aggregation

Protein liquid-liquid phase separation drives the dynamic assembly of membraneless organelles for fulfilling different physiological functions. Under diseased condition, protein may undergo liquid-to-solid condensation to form pathological amyloid aggregates closely associated with neurodegenerative diseases. Chemical probe serves as an important chemical tool not only for exploring the basic principle of the dynamic assembly of different protein condensates in vitro and in cell but also for clinical diagnosis and therapeutics of the related diseases. In this review, we first introduce chemical probes to image and regulate protein condensates. Then, we summarized three different categories of chemical probes including general amyloid dye, selective positron emission tomography tracer, and disaggregating binder, which feature distinct interaction pattern and activity upon binding to different pathological amyloid fibrillar aggregates. Next, we discuss the development of chemical probes for tracking protein amorphous aggregates in cells. Finally, we point out future direction in expanding the probes’ chemical space and applications.     

Liquid–liquid phase separation of Tau by self and complex coacervation

The liquid–liquid phase separation (LLPS) of Tau has been postulated to play a role in modulating the aggregation property of Tau, a process known to be critically associated with the pathology of a broad range of neurodegenerative diseases including Alzheimer''s Disease. Tau can undergo LLPS by homotypic interaction through self‐coacervation (SC) or by heterotypic association through complex‐coacervation (CC) between Tau and binding partners such as RNA. What is unclear is in what way the formation mechanisms for self and complex coacervation of Tau are similar or different, and the addition of a binding partner to Tau alters the properties of LLPS and Tau. A combination of in vitro experimental and computational study reveals that the primary driving force for both Tau CC and SC is electrostatic interactions between Tau‐RNA or Tau‐Tau macromolecules. The liquid condensates formed by the complex coacervation of Tau and RNA have distinctly higher micro‐viscosity and greater thermal stability than that formed by the SC of Tau. Our study shows that subtle changes in solution conditions, including molecular crowding and the presence of binding partners, can lead to the formation of different types of Tau condensates with distinct micro‐viscosity that can coexist as persistent and immiscible entities in solution. We speculate that the formation, rheological properties and stability of Tau droplets can be readily tuned by cellular factors, and that liquid condensation of Tau can alter the conformational equilibrium of Tau.     

Designer Condensates: A Toolkit for the Biomolecular Architect

Protein phase separation has emerged as a novel paradigm to explain the biogenesis of membraneless organelles and other so-called biomolecular condensates. While the implication of this physical phenomenon within cell biology is providing us with novel ways for understanding how cells compartmentalize biochemical reactions and encode function in such liquid-like assemblies, the newfound appreciation of this process also provides immense opportunities for designing and sculpting biological matter. Here, we propose that understanding the cell’s instruction manual of phase separation will enable bioengineers to begin creating novel functionalized biological materials and unprecedented tools for synthetic biology. We present FASE as the synthesis of the existing sticker-spacer framework, which explains the physical driving forces underlying phase separation, with quintessential principles of Scandinavian design. FASE serves both as a designer condensates catalogue and construction manual for the aspiring (membraneless) biomolecular architect. Our approach aims to inspire a new generation of bioengineers to rethink phase separation as an opportunity for creating reactive biomaterials with unconventional properties and to encode novel biological function in living systems. Although still in its infancy, several studies highlight how designer condensates have immediate and widespread potential applications in industry and medicine.     

Multiple Modes of Protein–Protein Interactions Promote RNP Granule Assembly

Eukaryotic cells are known to contain a wide variety of RNA–protein assemblies, collectively referred to as RNP granules. RNP granules form from a combination of RNA–RNA, protein–RNA, and protein–protein interactions. In addition, RNP granules are enriched in proteins with intrinsically disordered regions (IDRs), which are frequently appended to a well-folded domain of the same protein. This structural organization of RNP granule components allows for a diverse set of protein–protein interactions including traditional structured interactions between well-folded domains, interactions of short linear motifs in IDRs with the surface of well-folded domains, interactions of short motifs within IDRs that weakly interact with related motifs, and weak interactions involving at most transient ordering of IDRs and folded domains with other components. In addition, both well-folded domains and IDRs in granule components frequently interact with RNA and thereby can contribute to RNP granule assembly. We discuss the contribution of these interactions to liquid–liquid phase separation and the possible role of phase separation in the assembly of RNP granules. We expect that these principles also apply to other non-membrane bound organelles and large assemblies in the cell.     

Mechanics,Structure and Function of Biopolymer Condensates

The spontaneous nature of biopolymer phase separation in cells entails that the resulting condensates can be thermodynamic machines, which, in the process of condensing, can take on distinct forms themselves and deform neighboring cellular structures. We introduce here general notions of material and mechanical properties of protein condensates with an emphasis on how molecular arrangements and intermolecular interaction within condensates determine their ability to do work on their surroundings. We further propose functional implications of these concepts to cellular and subcellular morphology and biogenesis.     

Ubiquitin Modulates Liquid-Liquid Phase Separation of UBQLN2 via Disruption of Multivalent Interactions

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Who's In and Who's Out—Compositional Control of Biomolecular Condensates

Biomolecular condensates are two- and three-dimensional compartments in eukaryotic cells that concentrate specific collections of molecules without an encapsulating membrane. Many condensates behave as dynamic liquids and appear to form through liquid–liquid phase separation driven by weak, multivalent interactions between macromolecules. In this review, we discuss current models and data regarding the control of condensate composition, and we describe our current understanding of the composition of representative condensates including PML nuclear bodies, P-bodies, stress granules, the nucleolus, and two-dimensional membrane localized LAT and nephrin clusters. Specific interactions, such as interactions between modular binding domains, weaker interactions between intrinsically disorder regions and nucleic acid base pairing, and nonspecific interactions, such as electrostatic interactions and hydrophobic interactions, influence condensate composition. Understanding how specific condensate composition is determined is essential to understanding condensates as biochemical entities and ultimately discerning their cellular and organismic functions.     

The Role of RNA in Biological Phase Separations

Phase transitions that alter the physical state of ribonucleoprotein particles contribute to the spacial and temporal organization of the densely packed intracellular environment. This allows cells to organize biologically coupled processes as well as respond to environmental stimuli. RNA plays a key role in phase separation events that modulate various aspects of RNA metabolism. Here, we review the role that RNA plays in ribonucleoprotein phase separations.     

RGG/RG Motif Regions in RNA Binding and Phase Separation

RGG/RG motifs are RNA binding segments found in many proteins that can partition into membraneless organelles. They occur in the context of low-complexity disordered regions and often in multiple copies. Although short RGG/RG-containing regions can sometimes form high-affinity interactions with RNA structures, multiple RGG/RG repeats are generally required for high-affinity binding, suggestive of the dynamic, multivalent interactions that are thought to underlie phase separation in formation of cellular membraneless organelles. Arginine can interact with nucleotide bases via hydrogen bonding and π-stacking; thus, nucleotide conformers that provide access to the bases provide enhanced opportunities for RGG interactions. Methylation of RGG/RG regions, which is accomplished by protein arginine methyltransferase enzymes, occurs to different degrees in different cell types and may regulate the behavior of proteins containing these regions.