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Review
. 2021 Aug 21;11(8):1248.
doi: 10.3390/biom11081248.

The Role of Methionine Residues in the Regulation of Liquid-Liquid Phase Separation

Juan Carlos Aledo  1
Affiliations
Review

The Role of Methionine Residues in the Regulation of Liquid-Liquid Phase Separation

Juan Carlos Aledo. Biomolecules. .

Abstract

Membraneless organelles are non-stoichiometric supramolecular structures in the micron scale. These structures can be quickly assembled/disassembled in a regulated fashion in response to specific stimuli. Membraneless organelles contribute to the spatiotemporal compartmentalization of the cell, and they are involved in diverse cellular processes often, but not exclusively, related to RNA metabolism. Liquid-liquid phase separation, a reversible event involving demixing into two distinct liquid phases, provides a physical framework to gain insights concerning the molecular forces underlying the process and how they can be tuned according to the cellular needs. Proteins able to undergo phase separation usually present a modular architecture, which favors a multivalency-driven demixing. We discuss the role of low complexity regions in establishing networks of intra- and intermolecular interactions that collectively control the phase regime. Post-translational modifications of the residues present in these domains provide a convenient strategy to reshape the residue-residue interaction networks that determine the dynamics of phase separation. Focus will be placed on those proteins with low complexity domains exhibiting a biased composition towards the amino acid methionine and the prominent role that reversible methionine sulfoxidation plays in the assembly/disassembly of biomolecular condensates.

Keywords: Pab1; TDP43; ataxin-2; biomolecular condensate; methionine sulfoxide; stress granule.

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Conflict of interest statement

The author declares no conflict of interest.

Figures

Figure 1
Figure 1
Cellular compartmentalization in eukaryotic cells. The main membranous organelles such as the nucleus (1), endoplasmic reticulum (2), lysosomes (3), endosomes (4), Golgi apparatus (5), vesicles (6), and mitochondria (7) are schematized using warm colors. On the other hand, nuclear MLOs such as nuclear pore (1), nuclear germs (2), PML bodies (3), paraspeckles (4), transcription puffs (5), Cajal bodies (6), nucleolus (7), heterochromatin (8), as well as extranuclear MLOs such as P bodies (9), SGs (10), membrane clusters (11), and cytoskeleton (12) are depicted in cold colors. The list of cellular compartments depicted here is not exhaustive. Some compartments occur only in specific cell types (i.e., synaptic densities and RNA transport granules in neurons; germ granules in germ cells; dicing bodies and photobodies in plant cells; carboxysomes in autotrophic bacteria, or pyrenoids in algae) and they are not shown in the figure.
Figure 2
Figure 2
Schematic phase diagrams; (A) The black curve separating the plane into two regions (one-phase in cyan and two-phase in green) informs about the conditions (macromolecular concentration; temperature, T; ionic strength, I; pH; etc.) at which the two regimes coexist in equilibrium. At a given value of the environmental variable, an increase in the concentration (black arrow) of the scaffold macromolecule leads from the one-phase to the two-phase state (squares 1 and 2, respectively, where the scaffold macromolecules have been represented in red). (B) Schematic representation of the results reported in [55]. In a population of cells expressing the fluorescent tagged proteins EGFP-DYRK3 (a kinase with dissolvase activity) and mCherry-SSRM1 (a nuclear substrate of the DYRK3 kinase), nuclear intensities of both signals can be recorded and plotted in a Cartesian plane using arbitrary units (AU). The black line splitting the plane in two regions is given by the kinase/substrate ratio above which the nuclear condensates containing SSRM1 melt. Thus, at a fixed value of kinase concentration, moving from lower to higher concentrations of the kinase substrate (black arrow) leads to the assembly of condensates (square 1 and 2, respectively). (C) Phase diagram of RNA-protein mixtures. The black arrow represents, at a given protein concentration, the direction in increasing RNA concentrations. An initial increase in RNA concentration drives phase transition from one phase (square 1) at low RNA levels to two phases (square 2) at intermediate levels of RNA. Further increasing the RNA concentration drives the second phase transition, now from two phases (square 2) to one phase (square 3) after dissolution of the condensates. This process, known as RNA-mediated reentrant phase transition, is thought to be driven by electrostatic forces.
Figure 3
Figure 3
Modular architecture of three scaffold proteins containing methionine-rich LC domains. Methionine positions in the primary structure are indicated by vertical black lines. (A) TDP-43 presents a folded N-terminal domain (green box) with six -strands [151], which has been shown to play an important role in the aggregation of TDP-43 monomer. This protein also has two RRMs (blue boxes) and a PrL domain (salmon box) that host a redox sensor formed by methionine residues (yellow box). (B) Pbp1, the yeast ataxin-2 orthologous, exhibits two RNA binding domain, Lsm and LsmAD, (blue boxes) and one methionine-rich LC regions (salmon box) containing a redox sensor (yellow box) that controls the aggregation ability of the protein. (C) Pab1 has 4 RRMs (blue boxes) one PrL domain (salmon box) enriched in methionine residues (yellow box) and a poly(A)-binding protein (PABP) domain towards the C-terminal of the polypeptide chain (green box). The red curves represent the score provided by the software PLAAC for each protein sequence. PLAAC uses Hidden Markov Models to compute the probability of a region from the analyzed protein being an LC region belonging to the PrLD category [156].
Figure 4
Figure 4
Schematic representation of H2O2-based footprinting of proteins. Three identical polypeptide chains, interacting by mean of a cross-β structure, are represented. The N- and C-terminal ends of each chain are supposed to be disordered and contain accessible methionines (Met-1 and Met-3). The central β-sheets also contain methionine (Met-2) but this residue is not accessible as it is protected by the laminated β-sheets that form the cross-β structure. The protein is initially exposed to limiting amounts of 16O-labeled H2O2, which will oxidize those methionines being accessible (MetO-1 and MetO-3). Afterwards, the protein is denatured and oxidized to completion with 18O-labeled H2O2, allowing the oxidation of Met-2 to MetO-2. Finally, samples are analyzed by mass spectrometry to determine the 18O/16O ratio of each methionine residue. This figure is a modification of the scheme found in the supplementary materials accompanying the paper [74].
Figure 5
Figure 5
Metabolic status and autophagy level. The figure outlines the mechanism that coordinates the metabolic state (fermentation versus respiration) with the levels of autophagy (low and high, respectively). Key to this mechanism is the redox state of specific methionine residues of the Pbp1 protein. When these residues are in their reduced form, as methionines, the protein is competent to form biomolecular condensates that retain TORC1 in a non-soluble compartment. Since TORC1 has an inhibitory effect on autophagy, its sequestration within the condensates leads to the upregulation of autophagy. On the contrary, when the target methionines are oxidized to MetO, Pbp1 is unable to aggregate, and the condensates melt releasing TORC1 in the cytoplasm where it inhibits autophagy. It should be noted that within cells, methionine oxidation and MetO reduction take place through two different reactions (angled lines). That is, one reaction is not the reverse of the other, and both reactions have unrelated equilibrium constants. Labels in blue represent the aspects of the proposed mechanism that require further investigation. For instance, if ROS are the oxidants of the methionine residues, is there a particular species whose production is favored during glycolysis? Is the ROS formation compartment-dependent? Is Ppb1 oxidized in an enzyme-catalyzed manner? How relevant is, in vivo, MsrB in the reduction of oxidized Pbp1?
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