Engineered NLS-chimera downregulates expression of aggregation-prone endogenous FUS.
Journal
Nature communications
ISSN: 2041-1723
Titre abrégé: Nat Commun
Pays: England
ID NLM: 101528555
Informations de publication
Date de publication:
10 Sep 2024
10 Sep 2024
Historique:
received:
08
12
2023
accepted:
27
08
2024
medline:
10
9
2024
pubmed:
10
9
2024
entrez:
9
9
2024
Statut:
epublish
Résumé
Importin β-superfamily nuclear import receptors (NIRs) mitigate mislocalization and aggregation of RNA-binding proteins (RBPs), like FUS and TDP-43, which are implicated in neurodegenerative diseases. NIRs potently disaggregate RBPs by recognizing their nuclear localization signal (NLS). However, disease-causing mutations in NLS compromise NIR binding and activity. Here, we define features that characterize the anti-aggregation activity of NIR and NLS. We find that high binding affinity between NIR and NLS, and optimal NLS location relative to the aggregating domain plays a role in determining NIR disaggregation activity. A designed FUS chimera (FUS
Identifiants
pubmed: 39251571
doi: 10.1038/s41467-024-52151-6
pii: 10.1038/s41467-024-52151-6
doi:
Substances chimiques
RNA-Binding Protein FUS
0
Nuclear Localization Signals
0
beta Karyopherins
0
FUS protein, human
0
Protein Aggregates
0
Recombinant Fusion Proteins
0
Types de publication
Journal Article
Langues
eng
Sous-ensembles de citation
IM
Pagination
7887Subventions
Organisme : U.S. Department of Health & Human Services | NIH | National Institute of Neurological Disorders and Stroke (NINDS)
ID : RF1NS121143
Organisme : U.S. Department of Health & Human Services | NIH | National Institute of Neurological Disorders and Stroke (NINDS)
ID : R21NS128396
Organisme : U.S. Department of Health & Human Services | NIH | National Institute of General Medical Sciences (NIGMS)
ID : R35GM138109
Organisme : NIGMS NIH HHS
ID : T32 GM144302
Pays : United States
Organisme : U.S. Department of Health & Human Services | NIH | National Institute of General Medical Sciences (NIGMS)
ID : R35GM140733
Informations de copyright
© 2024. The Author(s).
Références
Portz, B., Lee, B. L. & Shorter, J. FUS and TDP-43 phases in health and disease. Trends Biochem. Sci. 46, 550–563 (2021).
pubmed: 33446423
pmcid: 8195841
doi: 10.1016/j.tibs.2020.12.005
Carey, J. L. & Guo, L. Liquid-liquid phase separation of TDP-43 and FUS in physiology and pathology of neurodegenerative diseases. Front. Mol. Biosci. 9, 826719 (2022).
pubmed: 35187086
pmcid: 8847598
doi: 10.3389/fmolb.2022.826719
Harrison, A. F. & Shorter, J. RNA-binding proteins with prion-like domains in health and disease. Biochem. J. 474, 1417–1438 (2017).
pubmed: 28389532
doi: 10.1042/BCJ20160499
Purice, M. D. & Taylor, J. P. Linking hnRNP function to ALS and FTD pathology. Front. Neurosci. 12, 326 (2018).
pubmed: 29867335
pmcid: 5962818
doi: 10.3389/fnins.2018.00326
Hock, E. M. et al. Hypertonic stress causes cytoplasmic translocation of neuronal, but not astrocytic, FUS due to impaired transportin function. Cell Rep. 24, 987–1000.e1007 (2018).
pubmed: 30044993
doi: 10.1016/j.celrep.2018.06.094
Wood, A., Gurfinkel, Y., Polain, N., Lamont, W. & Rea, L. S. Molecular mechanisms underlying TDP-43 pathology in cellular and animal models of ALS and FTLD. Int. J. Mol. Sci. 22, 4705 (2021).
pubmed: 33946763
pmcid: 8125728
doi: 10.3390/ijms22094705
Dormann, D. et al. ALS-associated fused in sarcoma (FUS) mutations disrupt Transportin-mediated nuclear import. EMBO J. 29, 2841–2857 (2010).
pubmed: 20606625
pmcid: 2924641
doi: 10.1038/emboj.2010.143
Bosco, D. A. et al. Mutant FUS proteins that cause amyotrophic lateral sclerosis incorporate into stress granules. Hum. Mol. Genet. 19, 4160–4175 (2010).
pubmed: 20699327
pmcid: 2981014
doi: 10.1093/hmg/ddq335
Gitler, A. D. & Shorter, J. RNA-binding proteins with prion-like domains in ALS and FTLD-U. Prion 5, 179–187 (2011).
pubmed: 21847013
pmcid: 3226045
doi: 10.4161/pri.5.3.17230
Sun, Z. et al. Molecular determinants and genetic modifiers of aggregation and toxicity for the ALS disease protein FUS/TLS. PLoS Biol. 9, e1000614 (2011).
pubmed: 21541367
pmcid: 3082519
doi: 10.1371/journal.pbio.1000614
Johnson, B. S. et al. TDP-43 is intrinsically aggregation-prone, and amyotrophic lateral sclerosis-linked mutations accelerate aggregation and increase toxicity. J. Biol. Chem. 284, 20329–20339 (2009).
pubmed: 19465477
pmcid: 2740458
doi: 10.1074/jbc.M109.010264
Fang, Y. S. et al. Full-length TDP-43 forms toxic amyloid oligomers that are present in frontotemporal lobar dementia-TDP patients. Nat. Commun. 5, 4824 (2014).
pubmed: 25215604
doi: 10.1038/ncomms5824
Guo, L. et al. Nuclear-import receptors reverse aberrant phase transitions of RNA-binding proteins with prion-like domains. Cell 173, 677–692.e620 (2018).
pubmed: 29677512
pmcid: 5911940
doi: 10.1016/j.cell.2018.03.002
Hofweber, M. et al. Phase separation of FUS is suppressed by its nuclear import receptor and arginine methylation. Cell 173, 706–719.e713 (2018).
pubmed: 29677514
doi: 10.1016/j.cell.2018.03.004
Qamar, S. et al. FUS phase separation is modulated by a molecular chaperone and methylation of arginine cation-π interactions. Cell 173, 720–734.e715 (2018).
pubmed: 29677515
pmcid: 5927716
doi: 10.1016/j.cell.2018.03.056
Yoshizawa, T. et al. Nuclear import receptor inhibits phase separation of FUS through binding to multiple sites. Cell 173, 693–705.e622 (2018).
pubmed: 29677513
pmcid: 6234985
doi: 10.1016/j.cell.2018.03.003
Girdhar, A. & Guo, L. Regulating phase transition in neurodegenerative diseases by nuclear import receptors. Biology 11, 1009 (2022).
pubmed: 36101390
pmcid: 9311884
doi: 10.3390/biology11071009
Guo, L., Fare, C. M. & Shorter, J. Therapeutic dissolution of aberrant phases by nuclear-import receptors. Trends Cell Biol. 29, 308–322 (2019).
pubmed: 30660504
pmcid: 6750949
doi: 10.1016/j.tcb.2018.12.004
Chook, Y. M. & Süel, K. E. Nuclear import by karyopherin-βs: recognition and inhibition. Biochim. Biophys. Acta 1813, 1593–1606 (2011).
pubmed: 21029754
doi: 10.1016/j.bbamcr.2010.10.014
Soniat, M. & Chook, Y. M. Nuclear localization signals for four distinct karyopherin-β nuclear import systems. Biochem. J. 468, 353–362 (2015).
pubmed: 26173234
doi: 10.1042/BJ20150368
Lee, B. J. et al. Rules for nuclear localization sequence recognition by karyopherinβ2. Cell 126, 543–558 (2006).
pubmed: 16901787
pmcid: 3442361
doi: 10.1016/j.cell.2006.05.049
Pumroy, R. A. & Cingolani, G. Diversification of importin-α isoforms in cellular trafficking and disease states. Biochem. J. 466, 13–28 (2015).
pubmed: 25656054
doi: 10.1042/BJ20141186
Winton, M. J. et al. Disturbance of nuclear and cytoplasmic TAR DNA-binding protein (TDP-43) induces disease-like redistribution, sequestration, and aggregate formation. J. Biol. Chem. 283, 13302–13309 (2008).
pubmed: 18305110
pmcid: 2442318
doi: 10.1074/jbc.M800342200
Nishimura, A. L. et al. Nuclear import impairment causes cytoplasmic trans-activation response DNA-binding protein accumulation and is associated with frontotemporal lobar degeneration. Brain 133, 1763–1771 (2010).
pubmed: 20472655
doi: 10.1093/brain/awq111
Chook, Y. M. & Blobel, G. Karyopherins and nuclear import. Curr. Opin. Struct. Biol. 11, 703–715 (2001).
pubmed: 11751052
doi: 10.1016/S0959-440X(01)00264-0
Doll, S. G. et al. Recognition of the TDP-43 nuclear localization signal by importin α1/β. Cell Rep. 39, 111007 (2022).
pubmed: 35767952
pmcid: 9290431
doi: 10.1016/j.celrep.2022.111007
Fare, C. M., Rhine, K., Lam, A., Myong, S. & Shorter, J. A minimal construct of nuclear-import receptor Karyopherin-β2 defines the regions critical for chaperone and disaggregation activity. J. Biol. Chem. 299, 102806 (2023).
Dormann, D. et al. Arginine methylation next to the PY-NLS modulates Transportin binding and nuclear import of FUS. EMBO J. 31, 4258–4275 (2012).
pubmed: 22968170
pmcid: 3501225
doi: 10.1038/emboj.2012.261
Gonzalez, A. et al. Mechanism of karyopherin-β2 binding and nuclear import of ALS variants FUS(P525L) and FUS(R495X). Sci. Rep. 11, 3754 (2021).
pubmed: 33580145
pmcid: 7881136
doi: 10.1038/s41598-021-83196-y
Zhang, Z. C. & Chook, Y. M. Structural and energetic basis of ALS-causing mutations in the atypical proline-tyrosine nuclear localization signal of the Fused in Sarcoma protein (FUS). Proc. Natl Acad. Sci. USA 109, 12017–12021 (2012).
pubmed: 22778397
pmcid: 3409756
doi: 10.1073/pnas.1207247109
Brelstaff, J. et al. Transportin1: a marker of FTLD-FUS. Acta Neuropathol. 122, 591–600 (2011).
pubmed: 21847626
doi: 10.1007/s00401-011-0863-6
Troakes, C. et al. Transportin 1 colocalization with Fused in Sarcoma (FUS) inclusions is not characteristic for amyotrophic lateral sclerosis-FUS confirming disrupted nuclear import of mutant FUS and distinguishing it from frontotemporal lobar degeneration with FUS inclusions. Neuropathol. Appl. Neurobiol. 39, 553–561 (2013).
pubmed: 22934812
doi: 10.1111/j.1365-2990.2012.01300.x
Neumann, M. et al. Transportin 1 accumulates specifically with FET proteins but no other transportin cargos in FTLD-FUS and is absent in FUS inclusions in ALS with FUS mutations. Acta Neuropathol. 124, 705–716 (2012).
pubmed: 22842875
doi: 10.1007/s00401-012-1020-6
Snowden, J. S. et al. The most common type of FTLD-FUS (aFTLD-U) is associated with a distinct clinical form of frontotemporal dementia but is not related to mutations in the FUS gene. Acta Neuropathol. 122, 99–110 (2011).
pubmed: 21424531
doi: 10.1007/s00401-011-0816-0
Mackenzie, I. R., Rademakers, R. & Neumann, M. TDP-43 and FUS in amyotrophic lateral sclerosis and frontotemporal dementia. Lancet Neurol. 9, 995–1007 (2010).
pubmed: 20864052
doi: 10.1016/S1474-4422(10)70195-2
Mikhaleva, S. & Lemke, E. A. Beyond the transport function of import receptors: what’s all the FUS about? Cell 173, 549–553 (2018).
pubmed: 29677508
pmcid: 7611746
doi: 10.1016/j.cell.2018.04.002
Zhou, Y., Liu, S., Liu, G., Öztürk, A. & Hicks, G. G. ALS-associated FUS mutations result in compromised FUS alternative splicing and autoregulation. PLoS Genet. 9, e1003895 (2013).
pubmed: 24204307
pmcid: 3814325
doi: 10.1371/journal.pgen.1003895
Imasaki, T. et al. Structural basis for substrate recognition and dissociation by human transportin 1. Mol. Cell 28, 57–67 (2007).
pubmed: 17936704
doi: 10.1016/j.molcel.2007.08.006
Cansizoglu, A. E., Lee, B. J., Zhang, Z. C., Fontoura, B. M. & Chook, Y. M. Structure-based design of a pathway-specific nuclear import inhibitor. Nat. Struct. Mol. Biol. 14, 452–454 (2007).
pubmed: 17435768
pmcid: 3437620
doi: 10.1038/nsmb1229
Wang, J. et al. A molecular grammar governing the driving forces for phase separation of prion-like RNA binding proteins. Cell 174, 688–699.e616 (2018).
pubmed: 29961577
pmcid: 6063760
doi: 10.1016/j.cell.2018.06.006
Choi, J. M., Holehouse, A. S. & Pappu, R. V. Physical principles underlying the complex biology of intracellular phase transitions. Annu Rev. Biophys. 49, 107–133 (2020).
pubmed: 32004090
pmcid: 10715172
doi: 10.1146/annurev-biophys-121219-081629
Lin, Y., Currie, S. L. & Rosen, M. K. Intrinsically disordered sequences enable modulation of protein phase separation through distributed tyrosine motifs. J. Biol. Chem. 292, 19110–19120 (2017).
pubmed: 28924037
pmcid: 5704491
doi: 10.1074/jbc.M117.800466
Bourgeois, B. et al. Nonclassical nuclear localization signals mediate nuclear import of CIRBP. Proc. Natl Acad. Sci. USA 117, 8503–8514 (2020).
pubmed: 32234784
pmcid: 7165476
doi: 10.1073/pnas.1918944117
Schmidt, H. B., Barreau, A. & Rohatgi, R. Phase separation-deficient TDP43 remains functional in splicing. Nat. Commun. 10, 4890 (2019).
pubmed: 31653829
pmcid: 6814767
doi: 10.1038/s41467-019-12740-2
Conicella, A. E., Zerze, G. H., Mittal, J. & Fawzi, N. L. ALS mutations disrupt phase separation mediated by α-helical structure in the TDP-43 low-complexity C-terminal domain. Structure 24, 1537–1549 (2016).
pubmed: 27545621
pmcid: 5014597
doi: 10.1016/j.str.2016.07.007
Conicella, A. E. et al. TDP-43 α-helical structure tunes liquid-liquid phase separation and function. Proc. Natl Acad. Sci. USA 117, 5883–5894 (2020).
pubmed: 32132204
pmcid: 7084079
doi: 10.1073/pnas.1912055117
Zhang, Y. J. et al. The dual functions of the extreme N-terminus of TDP-43 in regulating its biological activity and inclusion formation. Hum. Mol. Genet. 22, 3112–3122 (2013).
pubmed: 23575225
pmcid: 3699067
doi: 10.1093/hmg/ddt166
Baade, I. et al. The RNA-binding protein FUS is chaperoned and imported into the nucleus by a network of import receptors. J. Biol. Chem. 296, 100659 (2021).
pubmed: 33857479
pmcid: 8131929
doi: 10.1016/j.jbc.2021.100659
Waldmann, I., Wälde, S. & Kehlenbach, R. H. Nuclear import of c-Jun is mediated by multiple transport receptors. J. Biol. Chem. 282, 27685–27692 (2007).
pubmed: 17652081
doi: 10.1074/jbc.M703301200
Arnold, M., Nath, A., Hauber, J. & Kehlenbach, R. H. Multiple importins function as nuclear transport receptors for the rev protein of human immunodeficiency virus type 1. J. Biol. Chem. 281, 20883–20890 (2006).
pubmed: 16704975
doi: 10.1074/jbc.M602189200
Cingolani, G., Bednenko, J., Gillespie, M. T. & Gerace, L. Molecular basis for the recognition of a nonclassical nuclear localization signal by importin beta. Mol. Cell 10, 1345–1353 (2002).
pubmed: 12504010
doi: 10.1016/S1097-2765(02)00727-X
Kosugi, S. et al. Design of peptide inhibitors for the importin alpha/beta nuclear import pathway by activity-based profiling. Chem. Biol. 15, 940–949 (2008).
pubmed: 18804031
doi: 10.1016/j.chembiol.2008.07.019
Lott, K. & Cingolani, G. The importin β binding domain as a master regulator of nucleocytoplasmic transport. Biochim. Biophys. Acta 1813, 1578–1592 (2011).
pubmed: 21029753
doi: 10.1016/j.bbamcr.2010.10.012
Andersson, M. K. et al. The multifunctional FUS, EWS and TAF15 proto-oncoproteins show cell type-specific expression patterns and involvement in cell spreading and stress response. BMC Cell Biol. 9, 37 (2008).
pubmed: 18620564
pmcid: 2478660
doi: 10.1186/1471-2121-9-37
Ribbeck, K., Lipowsky, G., Kent, H. M., Stewart, M. & Gorlich, D. NTF2 mediates nuclear import of Ran. EMBO J. 17, 6587–6598 (1998).
pubmed: 9822603
pmcid: 1171005
doi: 10.1093/emboj/17.22.6587
Lowe, A. R. et al. Importin-beta modulates the permeability of the nuclear pore complex in a Ran-dependent manner. Elife https://doi.org/10.7554/eLife.04052 (2015).
Humphrey, J. et al. FUS ALS-causative mutations impair FUS autoregulation and splicing factor networks through intron retention. Nucleic Acids Res. 48, 6889–6905 (2020).
pubmed: 32479602
pmcid: 7337901
doi: 10.1093/nar/gkaa410
Dini Modigliani, S., Morlando, M., Errichelli, L., Sabatelli, M. & Bozzoni, I. An ALS-associated mutation in the FUS 3’-UTR disrupts a microRNA-FUS regulatory circuitry. Nat. Commun. 5, 4335 (2014).
pubmed: 25004804
doi: 10.1038/ncomms5335
Ling, S.-C. et al. Overriding FUS autoregulation in mice triggers gain-of-toxic dysfunctions in RNA metabolism and autophagy-lysosome axis. eLife 8, e40811 (2019).
pubmed: 30747709
pmcid: 6389288
doi: 10.7554/eLife.40811
Qiu, H. et al. ALS-associated mutation FUS-R521C causes DNA damage and RNA splicing defects. J. Clin. Invest. 124, 981–999 (2014).
pubmed: 24509083
pmcid: 3938263
doi: 10.1172/JCI72723
Miyamoto, Y., Yamada, K. & Yoneda, Y. Importin α: a key molecule in nuclear transport and non-transport functions. J. Biochem. 160, 69–75 (2016).
pubmed: 27289017
doi: 10.1093/jb/mvw036
Doll, S. G. & Cingolani, G. Importin α/β and the tug of war to keep TDP-43 in solution: quo vadis? Bioessays 44, e2200181 (2022).
pubmed: 36253101
pmcid: 9969346
doi: 10.1002/bies.202200181
Khalil, B. et al. Nuclear import receptors are recruited by FG-nucleoporins to rescue hallmarks of TDP-43 proteinopathy. Mol. Neurodegener. 17, 80 (2022).
pubmed: 36482422
pmcid: 9733332
doi: 10.1186/s13024-022-00585-1
Li, Y. R., King, O. D., Shorter, J. & Gitler, A. D. Stress granules as crucibles of ALS pathogenesis. J. Cell Biol. 201, 361–372 (2013).
pubmed: 23629963
pmcid: 3639398
doi: 10.1083/jcb.201302044
Sanjuan-Ruiz, I. et al. Wild-type FUS corrects ALS-like disease induced by cytoplasmic mutant FUS through autoregulation. Mol. Neurodegener. 16, 61 (2021).
pubmed: 34488813
pmcid: 8419956
doi: 10.1186/s13024-021-00477-w
Görlich, D., Panté, N., Kutay, U., Aebi, U. & Bischoff, F. R. Identification of different roles for RanGDP and RanGTP in nuclear protein import. EMBO J. 15, 5584–5594 (1996).
pubmed: 8896452
pmcid: 452303
doi: 10.1002/j.1460-2075.1996.tb00943.x
Avendaño-Vázquez, S. E. et al. Autoregulation of TDP-43 mRNA levels involves interplay between transcription, splicing, and alternative polyA site selection. Genes Dev. 26, 1679–1684 (2012).
pubmed: 22855830
pmcid: 3418585
doi: 10.1101/gad.194829.112
Ayala, Y. M. et al. TDP-43 regulates its mRNA levels through a negative feedback loop. EMBO J. 30, 277–288 (2011).
pubmed: 21131904
doi: 10.1038/emboj.2010.310
Suzuki, H. & Matsuoka, M. hnRNPA1 autoregulates its own mRNA expression to remain non-cytotoxic. Mol. Cell. Biochem. 427, 123–131 (2017).
pubmed: 28000042
doi: 10.1007/s11010-016-2904-x
McGlincy, N. J. et al. Expression proteomics of UPF1 knockdown in HeLa cells reveals autoregulation of hnRNP A2/B1 mediated by alternative splicing resulting in nonsense-mediated mRNA decay. BMC Genomics 11, 565 (2010).
pubmed: 20946641
pmcid: 3091714
doi: 10.1186/1471-2164-11-565
Wang, A. et al. A single N-terminal phosphomimic disrupts TDP-43 polymerization, phase separation, and RNA splicing. EMBO J 37, e97452 (2018).
Britton, S. et al. DNA damage triggers SAF-A and RNA biogenesis factors exclusion from chromatin coupled to R-loops removal. Nucleic Acids Res. 42, 9047–9062 (2014).
pubmed: 25030905
pmcid: 4132723
doi: 10.1093/nar/gku601
Gruijs da Silva, L. A. et al. Disease-linked TDP-43 hyperphosphorylation suppresses TDP-43 condensation and aggregation. EMBO J. 41, e108443 (2022).
pubmed: 35112738
pmcid: 9016352
doi: 10.15252/embj.2021108443
Hallegger, M. et al. TDP-43 condensation properties specify its RNA-binding and regulatory repertoire. Cell 184, 4680–4696.e4622 (2021).
pubmed: 34380047
pmcid: 8445024
doi: 10.1016/j.cell.2021.07.018
Schneider, C. A., Rasband, W. S. & Eliceiri, K. W. NIH Image to ImageJ: 25 years of image analysis. Nat. Methods 9, 671–675 (2012).
pubmed: 22930834
pmcid: 5554542
doi: 10.1038/nmeth.2089
Schindelin, J. et al. Fiji: an open-source platform for biological-image analysis. Nat. Methods 9, 676–682 (2012).
pubmed: 22743772
doi: 10.1038/nmeth.2019
Stirling, D. R. et al. CellProfiler 4: improvements in speed, utility and usability. BMC Bioinform. 22, 433 (2021).
doi: 10.1186/s12859-021-04344-9
Kuchipudi, S. V. et al. 18S rRNA is a reliable normalisation gene for real time PCR based on influenza virus infected cells. Virol. J. 9, 230 (2012).
pubmed: 23043930
pmcid: 3499178
doi: 10.1186/1743-422X-9-230