Today's article is the first of a series, and is based on content reported in the ground-breaking peer-reviewed article "Melatonin: Regulation of Biomolecular Condensates in Neurodegenerative Disorders”By Doris Loh and Russel J. Reiter published at Antioxidants on September 17, 2021 .
[Source: Loh, Doris, and Russel J. Reiter 2021. “Melatonin: Regulation of Biomolecular Condensates in Neurodegenerative Disorders” Antioxidants 10, no. 9: 1483. https://doi.org/10.3390/antiox10091483]
There has been extensive research on the pleiotropic properties of melatonin in many different, important areas. A search at the NIH / PubMed archives revealed a total of 28,851 reports (as of October 1, 2021) published on melatonin since 1958, with 1,825 published in 2020 alone. Melatonin may be best described as an antioxidant; but when compared to another major endogenous antioxidant glutathione (GSH), melatonin articles comprise only ~ 17% of what has been published on GSH.
Even though it is possible that many of the mechanisms of action by glutathione are better understood while those for melatonin are less clearly elucidated, it is quite clear that the scope of influence by melatonin is as extensive, if not more, than glutathione and other endogenous antioxidants.
The fact that melatonin has been referred to in literature as a 'smart' molecule, with the ability to perform opposite functions in the same pathway such as autophagy where it has been reported to be protective in normal cells but cytotoxic in cancerous cells  truly captures the imagination as to how melatonin can exert such different properties in the same pathway under different contexts.
The in-depth, novel review "Melatonin: Regulation of Biomolecular Condensates in Neurodegenerative Disorders"May finally shed light on why and how melatonin can be a 'smart' molecule under different contexts.
The novel review by Loh and Reiter revealed for the first time in literature that melatonin is capable of influencing and regulating phase separation of biomolecular condensates that could result in different outcomes in health and disease. The extensive discussions on biomolecular condensate regulation by melatonin provides a deeper understanding of the fundamental relationships between melatonin, ATP, and RNA shaping the regulation of biomolecular condensates with the implication that this unique relationship between the three elements could have existed since the beginning of life on earth billions of years ago.
ATP & RNA in the RNA World
Most of you are familiar with adenosine triphosphate, commonly referred to as ATP or the energy currency produced by mitochondria in eukaryotes. However, did you know ATP existed before there was any mitochondrion?
In a time long before the existence of deoxyribonucleic acid (DNA), the earth was probably dominated by ribonucleic acid (RNA). This “RNA World” is believed to have existed more than 4 billion years ago. It is highly likely that DNA- and protein-based life forms were preceded by life forms based primarily on RNA. In the era known as the “RNA World”, genetic information was stored in RNA molecules instead of DNA sequences [3-6]. However, the synthesis of RNA requires ATP.
Adenosine triphosphate (ATP) is one of the four nucleotide monomers used during RNA synthesis 7. During the "RNA World", ATP existed as an important cofactor of a metabolic system composed of nucleic acid enzymes prior to the evolution of ribosomal protein synthesis. Coenzymes such as ATP, coenzyme A (CoA), s-adenosylmethionine (SAM), and nicotinamide adenine dinucleotide (NADH) supported the catalytic functions of RNA in addition to information processing 8,9. That is probably the reason why RNA has been observed to bind to ATP with high affinity and specificity 10.
ATP, RNA, and Biomolecular Condensates
The ability to form different compartments or complexes as rapid, organized responses to changing environmental stimuli was probably one of the most important drivers behind the first successful evolutionary steps of life on earth, long before the appearance of the first single-celled organism encapsulated within membranes [6-11]. To this date, these dynamic, self-assembling, liquid-like membraneless organelles (MLOs) or biomolecular condensates that contain protein, RNA, and other nucleic acids remain ubiquitous and essential in all cells of all life forms on earth [12-14] .
Liquid-Liquid Phase Separation (LLPS) Drives Biomolecular Condensates
LLPS is the physicochemical process  that is now believed to be the fundamental principle that is the basis of cellular processes such as gene regulation , metabolism , and stress response  in all living organisms.
Brangwynne et al. first discovered condensate droplets known as P-bodies that contained RNA and proteins in 2009 . Since then, science has discovered different features and functions of condensates formed by LLPS. One of these important areas of studies is the difference between membraneless organelles (MLOs) or biomolecular condensates which form as a result of external / endogenous stimuli into reversible, physiological droplets, and those that progress into irreversible pathological aggregates such as amyloids or prionoid-like fibrils associated with many diseases including neurodegeneration  and cancer .
Reversible, Physiological MLOs versus Irreversible, Pathological MLOs
[Source: Babinchak, WM; Surewicz, WK Liquid-Liquid Phase Separation and Its Mechanistic Role in Pathological Protein Aggregation. J. Mol. Biol. 2020, 432 (7), 1910–1925. https://doi.org/10.1016/j.jmb.2020.03.004]
What is Phase Separation?
Imagine a well-mixed salad dressing containing oil and vinegar to mirror the soluble state of intracellular biomolecules with different chemical characteristics. Phase separation allows the distinct separation of oil from vinegar, or a single phase of mutually soluble components, to 'demix' into two or more distinct phases to form dynamic, coherent droplet-like structures without any membranes. These droplets may act as effective biological compartments that can be regarded as bioreaction centers and filters in cells .
The ability to organize biomolecules such as RNA during early life may have conferred essential survival advantages to rapidly changing environments. If one extends this concept even further, then it becomes much easier to appreciate how cells use phase separation to sense and regulate intracellular and extracellular changes during the last ~ 3.5 billion years of evolution.
When external conditions become stressful and / or dangerous, organisms must first be able to sense and then rapidly adapt in order to survive and thrive during dramatic fluctuations in some of the potential primordial environmental stressors such as changes in temperature, oxygen concentration, and nutrient supply [23,24].
This ability to sense and respond rapidly to changes in environmental stressors is universally conserved, and the subsequent formation of MLOs in cells may also be universally conserved not only in eukaryotes [25,26], but also in prokaryotes as well .
Phase separation offers unique advantages to abrupt environmental / cellular changes as fast, on-off switching type responses to small changes that may be both temporally and energetically favorable because these rearrangements of existing cellular molecules can be achieved without the necessity for energetically costly creation or destruction processes . However, these rearrangements would become necessary on a longer time-scale as responses to continued stress inputs, resulting in changes in transcription, translation and protein turnover .
Phase Separation of a Single-phase Solution into Dense (droplet) and Dilute (surrounding medium) Phases
[Source: Yoo, H .; Triandafillou, C .; Drummond, DA Cellular Sensing by Phase Separation: Using the Process, Not Just the Products. J. Biol. Chem. 2019, 294 (18), 7151–7159. https://doi.org/10.1074/jbc.TM118.001191 ]
In yeast, the increase of a few degrees centigrade in heat can elevate heat-shock gene expressions by a 1000-fold . Heat-shock proteins (HSPs) have recently been associated with stress granules which are reversible MLOs that form under various cellular stress conditions . Stress granules are the “stress-coping” mechanisms used by cells to reprogram protein translation and modulate signaling pathways in order to ensure survival . Whether an organism can form and dissolve MLOs in a timely manner is probably key to solving unanswered questions in health and disease .
Formation and Dissolution of MLOs are ATP- and RNA-Dependent
Phase separation at its core is a thermodynamic process driven by the reduction or a negative change in global free energy [12,13]. The chemical process of LLPS is entropically unfavorable; therefore energetically supportive reactions are required to offset the energetic cost involved in successful LLPS .
ATP is the most favored molecule used by living organisms to capture and transfer free energy in order to drive LLPS. During hydrolysis, ATP is transformed into adenosine diphosphate (ADP) and inorganic phosphate (Pi) by ATPases. Cells use the associated release of −7.3 kcal / mol free energy during hydrolysis to fuel energetically favorable reactions that can control the nucleation, composition, and growth of MLOs [34,35].
It is believed that most proteins in the human proteome can undergo LLPS, and this constant exchange of substrates and information in the formation and dissolution of dynamic, reversible droplets are mediated not only by ATP-dependent processes, but also by ribonucleic acid (RNA) .
RNA are considered critical architectural components in MLOs that can fine-tune biophysical properties and regulate the formation, distribution, and disassembly of MLOs  such as stress granules. Stress granules (SGs) can be induced under stress conditions or different diseases such as viral infections, cancer and neurodegeneration. RNA itself can phase separate and nucleated into RNA granules in the absence of proteins. In the presence of proteins, RNA can act as efficient scaffolds to help assemble MLOs like SGs. That is why RNA is often the major component of SGs .
The presence and the amount of RNA usually can dictate whether MLOs are formed or dissolved. For example, Henninger and colleagues (2021)  showed the world how a low amount of RNA under constant protein concentration can induce the formation of MLOs, while increasing RNA levels actually dissolves those MLOs. Why?
[Source: Henninger, JE; Oksuz, O .; Shrinivas, K .; Sagi, I .; LeRoy, G .; Zheng, MM; Andrews, JO; Zamudio, AV; Lazaris, C .; Hannett, NM; Lee, TI; Sharp, PA; Cissé, II; Chakraborty, AK; Young, RA RNA-Mediated Feedback Control of Transcriptional Condensates. Cell 2021, 184 (1), 207-225.e24. https://doi.org/10.1016/j.cell.2020.11.030 ]
It's All About Charge!
Cells depend upon RNA to regulate MLOs because RNA molecules contain powerful electrostatic forces of the high negative charge densities buried in RNA phosphate backbones [39,40]. Therefore, when there is a low level of RNA with negative charge present, those RNA will interact with positively charged proteins to induce phase separation and form biomolecular condensates. On the other hand, if there is a lot of negatively charged RNA present, they would end up repelling proteins with positive charge and thus dissolve condensates . It is really just a simple story of opposites attract, but that attraction can result in the assembly of MLOs, which usually, is beneficial, except when the assembled complex is not subsequently dissolved and proceeds into the next stage of becoming irreversible aggregates often associated with diseases such as neurodegeneration and cancer.
How does melatonin affect phase separation and the formation and dissolution of biomolecular condensates in health and disease? The next article will take us deeper into the magical realm of melatonin and biomolecular condensates. Stay tuned….
If you can't wait for the next article, you can get a jumpstart and find out all about how melatonin can regulate biomolecular condensates at this link: https://www.mdpi.com/2076-3921/10/9/1483/htm.
In the meantime, have you had your MEL and AA today?
(1) Loh, D .; Reiter, RJ Melatonin: Regulation of Biomolecular Condensates in Neurodegenerative Disorders. Antioxidants (Basel) 2021, 10 (9), 1483. https://doi.org/10.3390/antiox10091483.
(2) Sagrillo-Fagundes, L .; Bienvenue-Pariseault, J .; Vaillancourt, C. Melatonin: The Smart Molecule That Differentially Modulates Autophagy in Tumor and Normal Placental Cells. PLoS One 2019, 14 (1), e0202458. https://doi.org/10.1371/journal.pone.0202458.
(3) Robertson, MP; Joyce, GF The Origins of the RNA World. Cold Spring Harb. Perspect. Biol. 2012, 4 (5). https://doi.org/10.1101/cshperspect.a003608.
(4) Joyce, GF The Antiquity of RNA-Based Evolution. Nature 2002, 418 (6894), 214–221. https://doi.org/10.1038/418214a.
(5) Joyce, GF RNA Evolution and the Origins of Life. Nature 1989, 338 (6212), 217–224. https://doi.org/10.1038/338217a0.
(6) Oparin, AI The Origin of Life on the Earth. The origin of life on the earth. 1957, No. 3rd Ed.
(7) Conaway, RC; Conaway, JW ATP Activates Transcription Initiation from Promoters by RNA Polymerase II in a Reversible Step prior to RNA Synthesis. J. Biol. Chem. 1988, 263 (6) 2962-2968.
(8) White, HB, 3rd. Coenzymes as Fossils of an Earlier Metabolic State. J. Mol. Evol. 1976, 7 (2), 101–104. https://doi.org/10.1007/BF01732468.
(9) Goldman, AD; Kacar, B. Cofactors Are Remnants of Life's Origin and Early Evolution. J. Mol. Evol. 2021, 89 (3), 127–133. https://doi.org/10.1007/s00239-020-09988-4.
(11) Deamer, D .; Dworkin, JP; Sandford, SA; Bernstein, MP; Allamandola, LJ The First Cell Membranes. Astrobiology 2002, 2 (4), 371–381. https://doi.org/10.1089/153110702762470482.
(12) Feng, Z .; Chen, X .; Wu, X .; Zhang, M. Formation of Biological Condensates via Phase Separation: Characteristics, Analytical Methods, and Physiological Implications. J. Biol. Chem. 2019, 294 (40), 14823–14835. https://doi.org/10.1074/jbc.REV119.007895.
(14) Weber, SC; Brangwynne, CP Getting RNA and Protein in Phase. Cell 2012, 149 (6), 1188–1191. https://doi.org/10.1016/j.cell.2012.05.022.
(15) Kamimura, YR; Kanai, M. Chemical Insights into Liquid-Liquid Phase Separation in Molecular Biology. BCSJ 2021, 94 (3), 1045–1058. https://doi.org/10.1246/bcsj.20200397.
(16) Guo, YE; Manteiga, JC; Henninger, JE; Sabari, BR; Dall'Agnese, A .; Hannett, NM; Spille, J.-H .; Afeyan, LK; Zamudio, AV; Shrinivas, K .; Abraham, BJ; Boija, A .; Decker, T.-M .; Rimel, JK; Fant, CB; Lee, TI; Cisse, II; Sharp, PA; Taatjes, DJ; Young, RA Pol II Phosphorylation Regulates a Switch between Transcriptional and Splicing Condensates. Nature 2019, 572 (7770), 543–548. https://doi.org/10.1038/s41586-019-1464-0.
(18) Riback, JA; Katanski, CD; Kear-Scott, JL; Pilipenko, EV; Rojek, AE; Sosnick, TR; Drummond, DA Stress-Triggered Phase Separation Is an Adaptive, Evolutionarily Tuned Response. Cell 2017, 168 (6), 1028–1040.e19. https://doi.org/10.1016/j.cell.2017.02.027.
(19) Brangwynne, CP; Eckmann, CR; Courson, DS; Rybarska, A .; Hoege, C .; Gharakhani, J .; Jülicher, F .; Hyman, AA Germline P Granules Are Liquid Droplets That Localize by Controlled Dissolution / condensation. Science 2009, 324 (5935), 1729–1732. https://doi.org/10.1126/science.1172046.
(20) Babinchak, WM; Surewicz, WK Liquid-Liquid Phase Separation and Its Mechanistic Role in Pathological Protein Aggregation. J.Mol. Biol. 2020, 432 (7), 1910–1925. https://doi.org/10.1016/j.jmb.2020.03.004.
(21) Matafora, V .; Farris, F .; Restuccia, U .; Tamburri, S .; Martano, G .; Bernardelli, C .; Sofia, A .; Pisati, F .; Casagrande, F .; Lazzari, L .; Marsoni, S .; Bonoldi, E .; Bachi, A. Amyloid Aggregates Accumulate in Melanoma Metastasis Modulating YAP Activity. EMBO Rep. 2020, 21 (9), and 50446. https://doi.org/10.15252/embr.202050446.
(22) Yoo, H .; Triandafillou, C .; Drummond, DA Cellular Sensing by Phase Separation: Using the Process, Not Just the Products. J. Biol. Chem. 2019, 294 (18), 7151–7159. https://doi.org/10.1074/jbc.TM118.001191.
(23) Lindquist, S. The Heat-Shock Response. Nodded. Rev. Biochem. 1986, 55, 1151–1191. https://doi.org/10.1146/annurev.bi.55.070186.005443.
(25) Mitrea, DM; Kriwacki, RW Phase Separation in Biology; Functional Organization of a Higher Order. Cell Commun. Signal. 2016, 14, 1. https://doi.org/10.1186/s12964-015-0125-7.
(26) Ning, W .; Guo, Y .; Lin, S .; Mei, B .; Wu, Y .; Jiang, P .; Tan, X .; Zhang, W .; Chen, G .; Peng, D .; Chu, L .; Xue, Y. DrLLPS: A Data Resource of Liquid – liquid Phase Separation in Eukaryotes. Nucleic Acid Res. 2019, 48 (D1), D288 – D295. https://doi.org/10.1093/nar/gkz1027.
(27) Ladouceur, AM; Parmar, BS Clusters of Bacterial RNA Polymerase Are Biomolecular Condensates That Assemble through Liquid – liquid Phase Separation. Proc. Natl. Academic Sci. USA 2020, 117 (31), 18540–18549. https://doi.org/10.1073/pnas.2005019117.
(28) Shamir, M .; Bar-On, Y .; Phillips, R .; Milo, R. SnapShot: Timescales in Cell Biology. Cell 2016, 164 (6), 1302–1302.e1. https://doi.org/10.1016/j.cell.2016.02.058.
(29) Morano, KA; Grant, CM; Moye-Rowley, WS The Response to Heat Shock and Oxidative Stress in Saccharomyces Cerevisiae. G 2012, 190 (4), 1157–1195. https://doi.org/10.1534/genetics.111.128033.
(30) van Leeuwen, W .; Rabouille, C. Cellular Stress Leads to the Formation of Membraneless Stress Assemblies in Eukaryotic Cells. Traffic 2019, 20 (9), 623–638. https://doi.org/10.1111/tra.12669.
(31) Verma, A .; Sumi, S .; Seervi, M. Heat Shock Proteins-Driven Stress Granule Dynamics: Yet Another Avenue for Cell Survival. Apoptosis 2021, 26 (7-8), 371–384. https://doi.org/10.1007/s10495-021-01678-w.
(32) Shin, Y .; Brangwynne, CP Liquid Phase Condensation in Cell Physiology and Disease. Science 2017, 357 (6357). https://doi.org/10.1126/science.aaf4382.
(33) Ahlers, J .; Adams, EM; Bader, V .; Pezzotti, S .; Winklhofer, KF; Tatzelt, J .; Havenith, M. The Key Role of Solvent in Condensation: Mapping Water in Liquid-Liquid Phase-Separated FUS. Biophys. J. 2021, 120 (7), 1266–1275. https://doi.org/10.1016 / j.bpj.2021.01.019.
(34) Lodish, H .; Berk, A .; Lawrence Zipursky, S .; Matsudaira, P .; Baltimore, D .; Darnell, J. Biochemical Energetics; WH Freeman, 2000.
(35) Manchester, KL Free Energy ATP Hydrolysis and Phosphorylation Potential. Biochem. Educ. 1980, 8 (3), 70–72. https://doi.org/10.1016/0307-4412(80)90043-6.
(36) Garcia-Jove Navarro, M .; Kashida, S .; Chouaib, R .; Souquere, S .; Pierron, G .; Weil, D .; Gueroui, Z. RNA Is a Critical Element for the Sizing and the Composition of Phase-Separated RNA-Protein Condensates. Nat. Common. 2019, 10 (1), 3230. https://doi.org/10.1038/s41467-019-11241-6.
(37) Campos-Melo, D .; Hawley, ZCE; Droppelmann, CA; Strong, MJ The Integral Role of RNA in Stress Granule Formation and Function. Front Cell Dev Biol 2021, 9, 621779. https://doi.org/10.3389/fcell.2021.621779.
(38) Henninger, JE; Oksuz, O .; Shrinivas, K .; Sagi, I .; LeRoy, G .; Zheng, MM; Andrews, JO; Zamudio, AV; Lazaris, C .; Hannett, NM; Lee, TI; Sharp, PA; Cissé, II; Chakraborty, AK; Young, RA RNA-Mediated Feedback Control of Transcriptional Condensates. Cell 2021, 184 (1), 207–225.e24. https://doi.org/10.1016/j.cell.2020.11.030.
(39) Drobot, B .; Iglesias-Artola, JM; Le Vay, K .; Mayr, V .; Kar, M .; Kreysing, M .; Mutschler, H .; Tang, T.-YD Compartmentalized RNA Catalysis in Membrane-Free Coacervate Protocells. Nat. Common. 2018, 9 (1), 3643. https://doi.org/10.1038/s41467-018-06072-w.
(40) Conn, GL; Gittis, AG; Lattman, EE; Misra, VK; Draper, DE A Compact RNA Tertiary Structure Contains a Buried Backbone-K + Complex. J.Mol. Biol. 2002, 318 (4), 963–973. https://doi.org/10.1016/S0022-2836(02)00147-X.