RNA-binding protein (RBP) TLS/FUS plays a major role in induction of phase separation/phase transition and aggregation in relation to familial amyotrophic lateral sclerosis (ALS). Recently, organelles without lipid-bilayer membrane including stress granule, Cajal body, and nucleolus are found to be formed by the phase separation. The phase separation is an event that solutions with two solvents separate into two distinctive phases. The phase separation is prone to have solid phase and forms harmful precipitation or aggregation against living cells, indicating that the phase separation has both benefit and risk on cellular programs. Thus, it is essential for utilization of the phase separation in divergent cellular programs to control or inhibit the undesirable precipitation in living cells. Here, we analyze an inhibitory mechanism of the phase separation and precipitation. Inhibition of the phase separation is one of a critical regulatory step to prevent dysregulation of the phase separation and resulting deleterious precipitations. An inhibitory agent against the phase separation, amphiphilic alcohol, 1,6-hexanediol (1,6-HD) has been reported to examine function of phase separations. Thus, affinity chromatography of biotinylated 1,6-HD is employed to identify an initial event induced by 1,6-HD. Upon successful synthesis of biotinylated compounds of 1,6-HD (bio-1,6-HD), the affinity chromatography with bio-1,6-HD has been established at this study. The bio-1,6-HD captured protein bands on SDS-PAGE gel from HeLa cell nuclear extracts. The bands were analyzed with mass spectrometric analyses, showing that the proteins should be cytoskeleton related proteins. Further analysis using specific antibodies revealed one of the bands as human beta actin. It has been shown that beta actin is involved in divergent cellular activities including the phase separation and also neuronal functions like long-term potentiation. Therefore, beta actin might initiate the 1,6-HD-induced inhibition of the precipitation, although more experiments should be required to test whether it actually works in living cells.
| Published in | Biomedical Sciences (Volume 6, Issue 4) |
| DOI | 10.11648/j.bs.20200604.13 |
| Page(s) | 89-97 |
| Creative Commons |
This is an Open Access article, distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution and reproduction in any medium or format, provided the original work is properly cited. |
| Copyright |
Copyright © The Author(s), 2024. Published by Science Publishing Group |
TLS, FUS, Phase Separation, Phase Transition, Aggregation, Precipitation, 1,6-hexanediol
| [1] | Vance C, Rogelj B, Hortobagyi T, De Vos KJ, Nishimura AL, Sreedharan J, Hu X, Smith B, Ruddy D, Wright P, et al. (2009) Mutations in FUS, an RNA processing protein, cause familial amyotrophic lateral sclerosis type 6. Science 323, 1208-1211, doi: 323/5918/1208 [pii] 10.1126/science.1165942. |
| [2] | Kwiatkowski TJ, Jr., Bosco DA, Leclerc AL, Tamrazian E, Vanderburg CR, Russ C, Davis A, Gilchrist J, Kasarskis EJ, Munsat T, et al. (2009) Mutations in the FUS/TLS gene on chromosome 16 cause familial amyotrophic lateral sclerosis. Science 323, 1205-1208, doi: 323/5918/1205 [pii] 10.1126/science.1166066. |
| [3] | Mackenzie IR & Neumann M (2016) Molecular neuropathology of frontotemporal dementia: insights into disease mechanisms from postmortem studies. Journal of neurochemistry 138 Suppl 1, 54-70, doi: 10.1111/jnc.13588. |
| [4] | Ludolph AC, Brettschneider J & Weishaupt JH (2012) Amyotrophic lateral sclerosis. Current opinion in neurology 25, 530-535, doi: 10.1097/WCO.0b013e328356d328. |
| [5] | Arbab M, Baars S & Geijsen N (2014) Modeling motor neuron disease: the matter of time. Trends in neurosciences 37, 642-652, doi: 10.1016/j.tins.2014.07.008. |
| [6] | Rosen DR, Siddique T, Patterson D, Figlewicz DA, Sapp P, Hentati A, Donaldson D, Goto J, O'Regan JP, Deng HX, et al. (1993) Mutations in Cu/Zn superoxide dismutase gene are associated with familial amyotrophic lateral sclerosis. Nature 362, 59-62, doi: 10.1038/362059a0. |
| [7] | Sreedharan J, Blair IP, Tripathi VB, Hu X, Vance C, Rogelj B, Ackerley S, Durnall JC, Williams KL, Buratti E, et al. (2008) TDP-43 mutations in familial and sporadic amyotrophic lateral sclerosis. Science 319, 1668-1672, doi: 10.1126/science.1154584. |
| [8] | Lagier-Tourenne C & Cleveland DW (2009) Rethinking ALS: the FUS about TDP-43. Cell 136, 1001-1004, doi: S0092-8674(09)00263-3 [pii]10.1016/j.cell.2009.03.006. |
| [9] | Taylor JP, Brown Jr RH & Cleveland DW (2016) Decoding ALS: from genes to mechanism. Nature 539, 197-206, doi: 10.1038/nature20413. |
| [10] | Sadek H, Hannack B, Choe E, Wang J, Latif S, Garry MG, Garry DJ, Longgood J, Frantz DE, Olson EN, et al. (2008) Cardiogenic small molecules that enhance myocardial repair by stem cells. Proceedings of the National Academy of Sciences 105, 6063-6068, doi: 10.1073/pnas.0711507105. |
| [11] | Kato M, Han TW, Xie S, Shi K, Du X, Wu LC, Mirzaei H, Goldsmith EJ, Longgood J, Pei J, et al. (2012) Cell-free formation of RNA granules: low complexity sequence domains form dynamic fibers within hydrogels. Cell 149, 753-767, doi: 10.1016/j.cell.2012.04.017. |
| [12] | Kurokawa R & Bando T (2016) Three-Dimensional Structure of RNA-Binding Protein TLS Co-Crystallized with Biotinylated Isoxazole. Biomedical Sciences 2, 1-10, doi: 10.11648/j.rnat.20160201.11. |
| [13] | Ueda N, Kashiwazaki G, Bando T & Kurokawa R (2017) Biotin-Lys-His Blocks Aggregation of RNA-binding Protein TLS, a Cause of Amyotrophic Lateral Sclerosis. Biomedical Sciences 3, 67-77, doi: doi: 10.11648/j.bs.20170304.11. |
| [14] | Han TW, Kato M, Xie S, Wu LC, Mirzaei H, Pei J, Chen M, Xie Y, Allen J, Xiao G, et al. (2012) Cell-free formation of RNA granules: bound RNAs identify features and components of cellular assemblies. Cell 149, 768-779, doi: 10.1016/j.cell.2012.04.016. |
| [15] | Lorković ZJ & Barta A (2002) Genome analysis: RNA recognition motif (RRM) and K homology (KH) domain RNA-binding proteins from the flowering plant Arabidopsis thaliana. Nucleic Acids Research 30, 623-635, doi: 10.1093/nar/30.3.623. |
| [16] | Farina KL, Hüttelmaier S, Musunuru K, Darnell R & Singer RH (2003) Two ZBP1 KH domains facilitate β-actin mRNA localization, granule formation, and cytoskeletal attachment. J Cell Biol 160, 77-87, doi: 10.1083/jcb.200206003. |
| [17] | Gerstberger S, Hafner M & Tuschl T (2014) A census of human RNA-binding proteins. Nature Reviews Genetics 15, 829, doi: 10.1038/nrg3813https://www.nature.com/articles/nrg3813#supplementary-information. |
| [18] | Patel A, Lee Hyun O, Jawerth L, Maharana S, Jahnel M, Hein Marco Y, Stoynov S, Mahamid J, Saha S, Franzmann Titus M, et al. (2015) A Liquid-to-Solid Phase Transition of the ALS Protein FUS Accelerated by Disease Mutation. Cell 162, 1066-1077, doi: 10.1016/j.cell.2015.07.047. |
| [19] | Niaki AG, Sarkar J, Cai X, Rhine K, Vidaurre V, Guy B, Hurst M, Lee JC, Koh HR, Guo L, et al. (2020) Loss of Dynamic RNA Interaction and Aberrant Phase Separation Induced by Two Distinct Types of ALS/FTD-Linked FUS Mutations. Molecular Cell 77, 82-94. e84, doi: https://doi.org/10.1016/j.molcel.2019.09.022. |
| [20] | Yoshizawa T, Nozawa R-S, Jia TZ, Saio T & Mori E (2020) Biological phase separation: cell biology meets biophysics. Biophysical Reviews 12, 519-539, doi: 10.1007/s12551-020-00680-x. |
| [21] | Yoshizawa T, Ali R, Jiou J, Fung HYJ, Burke KA, Kim SJ, Lin Y, Peeples WB, Saltzberg D, Soniat M, et al. (2018) Nuclear Import Receptor Inhibits Phase Separation of FUS through Binding to Multiple Sites. Cell 173, 693-705.e622, doi: https://doi.org/10.1016/j.cell.2018.03.003. |
| [22] | Qamar S, Wang G, Randle SJ, Ruggeri FS, Varela JA, Lin JQ, Phillips EC, Miyashita A, Williams D, Ströhl F, et al. (2018) FUS Phase Separation Is Modulated by a Molecular Chaperone and Methylation of Arginine Cation-π Interactions. Cell 173, 720-734.e715, doi: 10.1016/j.cell.2018.03.056. |
| [23] | Sun S, Ling S-C, Qiu J, Albuquerque CP, Zhou Y, Tokunaga S, Li H, Qiu H, Bui A, Yeo GW, et al. (2015) ALS-causative mutations in FUS/TLS confer gain and loss of function by altered association with SMN and U1-snRNP. Nature communications 6, doi: 10.1038/ncomms7171. |
| [24] | Turner MR, Hardiman O, Benatar M, Brooks BR, Chio A, de Carvalho M, Ince PG, Lin C, Miller RG, Mitsumoto H, et al. (2013) Controversies and priorities in amyotrophic lateral sclerosis. The Lancet Neurology 12, 310-322, doi: 10.1016/S1474-4422(13)70036-X. |
| [25] | Murakami T, Qamar S, Lin JQ, Schierle GS, Rees E, Miyashita A, Costa AR, Dodd RB, Chan FT, Michel CH, et al. (2015) ALS/FTD Mutation-Induced Phase Transition of FUS Liquid Droplets and Reversible Hydrogels into Irreversible Hydrogels Impairs RNP Granule Function. Neuron 88, 678-690, doi: 10.1016/j.neuron.2015.10.030. |
| [26] | Lagier-Tourenne C, Polymenidou M & Cleveland DW (2010) TDP-43 and FUS/TLS: emerging roles in RNA processing and neurodegeneration. Hum Mol Genet 19, R46-64, doi: ddq137 [pii] 10.1093/hmg/ddq137. |
| [27] | DeJesus-Hernandez M, Mackenzie IR, Boeve BF, Boxer AL, Baker M, Rutherford NJ, Nicholson AM, Finch NA, Flynn H, Adamson J, et al. (2011) Expanded GGGGCC hexanucleotide repeat in noncoding region of C9ORF72 causes chromosome 9p-linked FTD and ALS. Neuron 72, 245-256, doi: 10.1016/j.neuron.2011.09.011. |
| [28] | Renton AE, Majounie E, Waite A, Simon-Sanchez J, Rollinson S, Gibbs JR, Schymick JC, Laaksovirta H, van Swieten JC, Myllykangas L, et al. (2011) A hexanucleotide repeat expansion in C9ORF72 is the cause of chromosome 9p21-linked ALS-FTD. Neuron 72, 257-268, doi: 10.1016/j.neuron.2011.09.010. |
| [29] | Jucker M & Walker LC (2013) Self-propagation of pathogenic protein aggregates in neurodegenerative diseases. Nature 501, 45-51, doi: 10.1038/nature12481. |
| [30] | Jain A & Vale RD (2017) RNA phase transitions in repeat expansion disorders. Nature 546, 243-247, doi: 10.1038/nature22386. |
| [31] | Patel SS, Belmont BJ, Sante JM & Rexach MF (2007) Natively unfolded nucleoporins gate protein diffusion across the nuclear pore complex. Cell 129, 83-96, doi: 10.1016/j.cell.2007.01.044. |
| [32] | Updike DL, Hachey SJ, Kreher J & Strome S (2011) P granules extend the nuclear pore complex environment in the C. elegans germ line. J Cell Biol 192, 939-948, doi: 10.1083/jcb.201010104. |
| [33] | Kroschwald S, Maharana S, Mateju D, Malinovska L, Nuske E, Poser I, Richter D & Alberti S (2015) Promiscuous interactions and protein disaggregases determine the material state of stress-inducible RNP granules. Elife 4, e06807, doi: 10.7554/eLife.06807. |
| [34] | Lin Y, Mori E, Kato M, Xiang S, Wu L, Kwon I & McKnight SL (2016) Toxic PR Poly-Dipeptides Encoded by the C9orf72 Repeat Expansion Target LC Domain Polymers. Cell 167, 789-802.e712, doi: 10.1016/j.cell.2016.10.003. |
| [35] | Wang X, Arai S, Song X, Reichart D, Du K, Pascual G, Tempst P, Rosenfeld MG, Glass CK & Kurokawa R (2008) Induced ncRNAs allosterically modify RNA-binding proteins in cis to inhibit transcription. Nature 454, 126-130, doi: nature06992 [pii]10.1038/nature06992. |
| [36] | Cui W, Yoneda R, Ueda N & Kurokawa R (2018) Arginine methylation of translocated in liposarcoma (TLS) inhibits its binding to long noncoding RNA, abrogating TLS-mediated repression of CBP/p300 activity. J Biol Chem 293, 10937-10948, doi: 10.1074/jbc.RA117.000598. |
| [37] | Yoneda R, Suzuki S, Mashima T, Kondo K, Nagata T, Katahira M & Kurokawa R (2016) The binding specificity of Translocated in LipoSarcoma/FUsed in Sarcoma with lncRNA transcribed from the promoter region of cyclin D1. Cell & bioscience 6, 4, doi: 10.1186/s13578-016-0068-8. |
| [38] | Yoneda R, Ueda N, Uranishi K, Hirasaki M & Kurokawa R (2020) Long noncoding RNA pncRNA-D reduces cyclin D1 gene expression and arrests cell cycle through RNA m (6) A modification. J Biol Chem 295, 5626-5639, doi: 10.1074/jbc.RA119.011556. |
| [39] | Du K, Arai S, Kawamura T, Matsushita A & Kurokawa R (2011) TLS and PRMT1 synergistically coactivate transcription at the survivin promoter through TLS arginine methylation. Biochem Biophys Res Commun 404, 991-996, doi: S0006-291X(10)02335-1[pii] 10.1016/j.bbrc.2010.12.097. |
| [40] | Shin Y, Chang Y-C, Lee DSW, Berry J, Sanders DW, Ronceray P, Wingreen NS, Haataja M & Brangwynne CP (2018) Liquid Nuclear Condensates Mechanically Sense and Restructure the Genome. Cell 175, 1481-1491.e1413, doi: 10.1016/j.cell.2018.10.057. |
| [41] | Hyman AA, Weber CA & Jülicher F (2014) Liquid-Liquid Phase Separation in Biology. Annual Review of Cell and Developmental Biology 30, 39-58, doi: 10.1146/annurev-cellbio-100913-013325. |
| [42] | Baumgart T, Hammond AT, Sengupta P, Hess ST, Holowka DA, Baird BA & Webb WW (2007) Large-scale fluid/fluid phase separation of proteins and lipids in giant plasma membrane vesicles. Proceedings of the National Academy of Sciences 104, 3165-3170, doi: 10.1073/pnas.0611357104. |
| [43] | Strom AR, Emelyanov AV, Mir M, Fyodorov DV, Darzacq X & Karpen GH (2017) Phase separation drives heterochromatin domain formation. Nature 547, 241-245, doi: 10.1038/nature22989. |
| [44] | Alberti S, Gladfelter A & Mittag T (2019) Considerations and Challenges in Studying Liquid-Liquid Phase Separation and Biomolecular Condensates. Cell 176, 419-434, doi: https://doi.org/10.1016/j.cell.2018.12.035. |
| [45] | Boeynaems S, Alberti S, Fawzi NL, Mittag T, Polymenidou M, Rousseau F, Schymkowitz J, Shorter J, Wolozin B, Van Den Bosch L, et al. (2018) Protein Phase Separation: A New Phase in Cell Biology. Trends in Cell Biology 28, 420-435, doi: https://doi.org/10.1016/j.tcb.2018.02.004. |
| [46] | Brangwynne CP, Eckmann CR, Courson DS, Rybarska A, Hoege C, Gharakhani J, Jülicher F & Hyman AA (2009) Germline P Granules Are Liquid Droplets That Localize by Controlled Dissolution/Condensation. Science 324, 1729-1732, doi: 10.1126/science.1172046. |
| [47] | Brangwynne CP, Mitchison TJ & Hyman AA (2011) Active liquid-like behavior of nucleoli determines their size and shape in Xenopus laevis oocytes. Proceedings of the National Academy of Sciences 108, 4334-4339, doi: 10.1073/pnas.1017150108. |
| [48] | Wippich F, Bodenmiller B, Trajkovska MG, Wanka S, Aebersold R & Pelkmans L (2013) Dual specificity kinase DYRK3 couples stress granule condensation/dissolution to mTORC1 signaling. Cell 152, 791-805, doi: 10.1016/j.cell.2013.01.033. |
| [49] | Strzelecka M, Trowitzsch S, Weber G, Lührmann R, Oates AC & Neugebauer KM (2010) Coilin-dependent snRNP assembly is essential for zebrafish embryogenesis. Nature Structural & Molecular Biology 17, 403-409, doi: 10.1038/nsmb.1783. |
| [50] | Weirich KL, Banerjee S, Dasbiswas K, Witten TA, Vaikuntanathan S & Gardel ML (2017) Liquid behavior of cross-linked actin bundles. Proceedings of the National Academy of Sciences 114, 2131-2136, doi: 10.1073/pnas.1616133114. |
| [51] | Case LB, Zhang X, Ditlev JA & Rosen MK (2019) Stoichiometry controls activity of phase-separated clusters of actin signaling proteins. Science 363, 1093-1097, doi: 10.1126/science.aau6313. |
| [52] | Boija A, Klein IA, Sabari BR, Dall’Agnese A, Coffey EL, Zamudio AV, Li CH, Shrinivas K, Manteiga JC, Hannett NM, et al. (2018) Transcription Factors Activate Genes through the Phase-Separation Capacity of Their Activation Domains. Cell 175, 1842-1855.e1816, doi: 10.1016/j.cell.2018.10.042. |
| [53] | Wei MT, Chang YC, Shimobayashi SF, Shin Y, Strom AR & Brangwynne CP (2020) Nucleated transcriptional condensates amplify gene expression. Nat Cell Biol, doi: 10.1038/s41556-020-00578-6. |
| [54] | Nair SJ, Yang L, Meluzzi D, Oh S, Yang F, Friedman MJ, Wang S, Suter T, Alshareedah I, Gamliel A, et al. (2019) Phase separation of ligand-activated enhancers licenses cooperative chromosomal enhancer assembly. Nature Structural & Molecular Biology 26, 193-203, doi: 10.1038/s41594-019-0190-5. |
| [55] | Kanaan NM, Hamel C, Grabinski T & Combs B (2020) Liquid-liquid phase separation induces pathogenic tau conformations in vitro. Nature Communications 11, 2809, doi: 10.1038/s41467-020-16580-3. |
| [56] | Ambadipudi S, Biernat J, Riedel D, Mandelkow E & Zweckstetter M (2017) Liquid–liquid phase separation of the microtubule-binding repeats of the Alzheimer-related protein Tau. Nature Communications 8, 275, doi: 10.1038/s41467-017-00480-0. |
| [57] | Wegmann S, Eftekharzadeh B, Tepper K, Zoltowska KM, Bennett RE, Dujardin S, Laskowski PR, MacKenzie D, Kamath T, Commins C, et al. (2018) Tau protein liquid–liquid phase separation can initiate tau aggregation. Embo J 37, e98049, doi: 10.15252/embj.201798049. |
| [58] | Mackenzie IR, Nicholson AM, Sarkar M, Messing J, Purice MD, Pottier C, Annu K, Baker M, Perkerson RB, Kurti A, et al. (2017) TIA1 Mutations in Amyotrophic Lateral Sclerosis and Frontotemporal Dementia Promote Phase Separation and Alter Stress Granule Dynamics. Neuron 95, 808-816.e809, doi: https://doi.org/10.1016/j.neuron.2017.07.025. |
| [59] | Hofweber M, Hutten S, Bourgeois B, Spreitzer E, Niedner-Boblenz A, Schifferer M, Ruepp M-D, Simons M, Niessing D, Madl T, et al. (2018) Phase Separation of FUS Is Suppressed by Its Nuclear Import Receptor and Arginine Methylation. Cell 173, 706-719. e713, doi: https://doi.org/10.1016/j.cell.2018.03.004. |
| [60] | Monahan Z, Ryan VH, Janke AM, Burke KA, Rhoads SN, Zerze GH, O'Meally R, Dignon GL, Conicella AE, Zheng W, et al. (2017) Phosphorylation of the FUS low-complexity domain disrupts phase separation, aggregation, and toxicity. Embo J 36, 2951-2967, doi: 10.15252/embj.201696394. |
| [61] | Molliex A, Temirov J, Lee J, Coughlin M, Kanagaraj AP, Kim HJ, Mittag T & Taylor JP (2015) Phase separation by low complexity domains promotes stress granule assembly and drives pathological fibrillization. Cell 163, 123-133, doi: 10.1016/j.cell.2015.09.015. |
| [62] | Finkelstein A (1976) Water and nonelectrolyte permeability of lipid bilayer membranes. J Gen Physiol 68, 127-135, doi: 10.1085/jgp.68.2.127. |
| [63] | Kwon SC, Yi H, Eichelbaum K, Fohr S, Fischer B, You KT, Castello A, Krijgsveld J, Hentze MW & Kim VN (2013) The RNA-binding protein repertoire of embryonic stem cells. Nature structural & molecular biology 20, 1122-1130, doi: 10.1038/nsmb.2638. |
| [64] | Ribbeck K & Gorlich D (2002) The permeability barrier of nuclear pore complexes appears to operate via hydrophobic exclusion. Embo J 21, 2664-2671, doi: 10.1093/emboj/21.11.2664. |
| [65] | Wheeler JR, Matheny T, Jain S, Abrisch R & Parker R (2016) Distinct stages in stress granule assembly and disassembly. Elife 5, doi: 10.7554/eLife.18413. |
| [66] | Schatzle P, Esteves da Silva M, Tas RP, Katrukha EA, Hu HY, Wierenga CJ, Kapitein LC & Hoogenraad CC (2018) Activity-Dependent Actin Remodeling at the Base of Dendritic Spines Promotes Microtubule Entry. Curr Biol 28, 2081-2093 e2086, doi: 10.1016/j.cub.2018.05.004. |
APA Style
Naomi Ueda, Yuki Hirose, Ryoma Yoneda, Toshikazu Bando, Riki Kurokawa. (2020). Potential Inhibitor Against Phase Separation, 1,6-hexanediol Specifically Binds to Beta Actin in Nuclear Extract of Human Cell Line. Biomedical Sciences, 6(4), 89-97. https://doi.org/10.11648/j.bs.20200604.13
ACS Style
Naomi Ueda; Yuki Hirose; Ryoma Yoneda; Toshikazu Bando; Riki Kurokawa. Potential Inhibitor Against Phase Separation, 1,6-hexanediol Specifically Binds to Beta Actin in Nuclear Extract of Human Cell Line. Biomed. Sci. 2020, 6(4), 89-97. doi: 10.11648/j.bs.20200604.13
AMA Style
Naomi Ueda, Yuki Hirose, Ryoma Yoneda, Toshikazu Bando, Riki Kurokawa. Potential Inhibitor Against Phase Separation, 1,6-hexanediol Specifically Binds to Beta Actin in Nuclear Extract of Human Cell Line. Biomed Sci. 2020;6(4):89-97. doi: 10.11648/j.bs.20200604.13
Share This Article
Twitter
Linked In
Facebook
Copy
Download@article{10.11648/j.bs.20200604.13,
author = {Naomi Ueda and Yuki Hirose and Ryoma Yoneda and Toshikazu Bando and Riki Kurokawa},
title = {Potential Inhibitor Against Phase Separation, 1,6-hexanediol Specifically Binds to Beta Actin in Nuclear Extract of Human Cell Line},
journal = {Biomedical Sciences},
volume = {6},
number = {4},
pages = {89-97},
doi = {10.11648/j.bs.20200604.13},
url = {https://doi.org/10.11648/j.bs.20200604.13},
eprint = {https://article.sciencepublishinggroup.com/pdf/10.11648.j.bs.20200604.13},
abstract = {RNA-binding protein (RBP) TLS/FUS plays a major role in induction of phase separation/phase transition and aggregation in relation to familial amyotrophic lateral sclerosis (ALS). Recently, organelles without lipid-bilayer membrane including stress granule, Cajal body, and nucleolus are found to be formed by the phase separation. The phase separation is an event that solutions with two solvents separate into two distinctive phases. The phase separation is prone to have solid phase and forms harmful precipitation or aggregation against living cells, indicating that the phase separation has both benefit and risk on cellular programs. Thus, it is essential for utilization of the phase separation in divergent cellular programs to control or inhibit the undesirable precipitation in living cells. Here, we analyze an inhibitory mechanism of the phase separation and precipitation. Inhibition of the phase separation is one of a critical regulatory step to prevent dysregulation of the phase separation and resulting deleterious precipitations. An inhibitory agent against the phase separation, amphiphilic alcohol, 1,6-hexanediol (1,6-HD) has been reported to examine function of phase separations. Thus, affinity chromatography of biotinylated 1,6-HD is employed to identify an initial event induced by 1,6-HD. Upon successful synthesis of biotinylated compounds of 1,6-HD (bio-1,6-HD), the affinity chromatography with bio-1,6-HD has been established at this study. The bio-1,6-HD captured protein bands on SDS-PAGE gel from HeLa cell nuclear extracts. The bands were analyzed with mass spectrometric analyses, showing that the proteins should be cytoskeleton related proteins. Further analysis using specific antibodies revealed one of the bands as human beta actin. It has been shown that beta actin is involved in divergent cellular activities including the phase separation and also neuronal functions like long-term potentiation. Therefore, beta actin might initiate the 1,6-HD-induced inhibition of the precipitation, although more experiments should be required to test whether it actually works in living cells.},
year = {2020}
}
TY - JOUR T1 - Potential Inhibitor Against Phase Separation, 1,6-hexanediol Specifically Binds to Beta Actin in Nuclear Extract of Human Cell Line AU - Naomi Ueda AU - Yuki Hirose AU - Ryoma Yoneda AU - Toshikazu Bando AU - Riki Kurokawa Y1 - 2020/11/04 PY - 2020 N1 - https://doi.org/10.11648/j.bs.20200604.13 DO - 10.11648/j.bs.20200604.13 T2 - Biomedical Sciences JF - Biomedical Sciences JO - Biomedical Sciences SP - 89 EP - 97 PB - Science Publishing Group SN - 2575-3932 UR - https://doi.org/10.11648/j.bs.20200604.13 AB - RNA-binding protein (RBP) TLS/FUS plays a major role in induction of phase separation/phase transition and aggregation in relation to familial amyotrophic lateral sclerosis (ALS). Recently, organelles without lipid-bilayer membrane including stress granule, Cajal body, and nucleolus are found to be formed by the phase separation. The phase separation is an event that solutions with two solvents separate into two distinctive phases. The phase separation is prone to have solid phase and forms harmful precipitation or aggregation against living cells, indicating that the phase separation has both benefit and risk on cellular programs. Thus, it is essential for utilization of the phase separation in divergent cellular programs to control or inhibit the undesirable precipitation in living cells. Here, we analyze an inhibitory mechanism of the phase separation and precipitation. Inhibition of the phase separation is one of a critical regulatory step to prevent dysregulation of the phase separation and resulting deleterious precipitations. An inhibitory agent against the phase separation, amphiphilic alcohol, 1,6-hexanediol (1,6-HD) has been reported to examine function of phase separations. Thus, affinity chromatography of biotinylated 1,6-HD is employed to identify an initial event induced by 1,6-HD. Upon successful synthesis of biotinylated compounds of 1,6-HD (bio-1,6-HD), the affinity chromatography with bio-1,6-HD has been established at this study. The bio-1,6-HD captured protein bands on SDS-PAGE gel from HeLa cell nuclear extracts. The bands were analyzed with mass spectrometric analyses, showing that the proteins should be cytoskeleton related proteins. Further analysis using specific antibodies revealed one of the bands as human beta actin. It has been shown that beta actin is involved in divergent cellular activities including the phase separation and also neuronal functions like long-term potentiation. Therefore, beta actin might initiate the 1,6-HD-induced inhibition of the precipitation, although more experiments should be required to test whether it actually works in living cells. VL - 6 IS - 4 ER -
Information
Email: