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Karyopherin-β2 Inhibits and Reverses Aggregation and Liquid-liquid Phase Separation of the ALS/FTD-Associated Protein FUS

核转运蛋白-β2抑制和ALS / FTD相关蛋白FUS的逆转聚集和液-液相分离

Emma Robinson Emma Robinson
James Shorter James Shorter
LG Lin Guo

发布: 2020年08月20日第10卷第16期 DOI: 10.21769/BioProtoc.3725 浏览次数: 4418

评审: Steven BoeynaemsIndranil MalikDonya Pakravan

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Abstract

The study of RNA-binding proteins (RBP) offers insight into the mechanisms of pathologic protein aggregation in neurodegenerative diseases. We developed a protocol for purifying an RBP FUS and a nuclear import receptor (NIR) Kapβ2 and testing the ability of Kapβ2 to mitigate FUS aggregation and liquid-liquid phase separation.

Keywords: FUS (FUS) search Liquid-liquid phase separation (液-液相分离) search Nuclear import receptor (核输入受体) search Kapβ2 (Kapβ2) search Protein aggregation (蛋白质聚集) search

Background

Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are neurodegenerative diseases characterized by the mislocalization and aggregation of several RNA-binding proteins (RBP) (Mackenzie et al., 2010; Da Cruz and Cleveland, 2011; King et al., 2012). One of these proteins is FUS (Fused in Sarcoma), a nuclear protein mislocalizes in the cytoplasm of neurons in ALS/FTD patients, where it undergoes liquid-liquid phase transition followed by aberrant phase transition to form insoluble aggregates (Altmeyer et al., 2015; Burke et al., 2015; Lin et al., 2015; Molliex et al., 2015; Murakami et al., 2015; Patel et al., 2015) (Figure 1). Karyopherin-β2 (Kapβ2), a nuclear import receptor (NIR), has been established as a chaperone and disaggregase of proteins with PY-nuclear localization sequences (NLS), such as FUS (Guo et al., 2018; Hofweber et al., 2018; Yoshizawa, et al., 2018). The following protocol was developed to test the ability of Kapβ2 to inhibit and reverse FUS aggregation and phase separation ex vivo (Guo et al., 2018).



Figure 1. Aberrant phase transition of FUS (10 μM) leads to the formation of fibrillar hydrogel. A. Domain architecture of FUS. B. DIC images show that FUS undergoes reversible liquid-liquid phase transition to form liquid droplets followed by aberrant phase transition to form hydrogels. Scale bar: 10 μm.

Materials and Reagents

  1. 1.5 ml tube holder
  2. 15 ml tube holder
  3. 50 ml tube holder
  4. Glass beads (MP Biomedicals, catalog number: MP07DP1070 )
  5. Nitrile gloves (FisherBrand, catalog number: 19-130-1597 )
  6. Ethanol spray bottle
  7. MilliQ water spray bottle
  8. Bleach spray bottle
  9. Ice bucket
  10. Foam liquid nitrogen container
  11. 1.5 ml Semi-Micro Plastic Cuvettes (Fisherbrand, catalog number: 14955127 )
  12. 96-well plate (Corning, catalog number: 08-757-230 )
  13. Lens paper (FisherBrand, catalog number: 11-995 )
  14. Glass microscope slides (Fisherbrand, catalog number: 12548B )
  15. 1.5 ml autoclaved Eppendorf tubes (Eppendorf, catalog number: 05-408-129 )
  16. 1.5 ml Protein LoBind Tubes (Eppendorf, catalog number: 22431081 )
  17. 15 ml conical tubes (Thermo Scientific, catalog number: 12-565-268 )
  18. 50 ml conical tubes (Thermo Scientific catalog number: 339652 )
  19. 50 ml plastic round-bottom centrifugal tubes (Thermo Scientific, catalog number: 05-529C )
  20. Pipette tips
    0.1-10 µl (USA Scientific, catalog number: 1121-3810 )
    2-20 µl (Fisherbrand, catalog number: 02-707-432 )
    20-200 µl (Fisherbrand, catalog number: 02-707-430 )
    200-1,000 µl (Fisherbrand, catalog number: 02-707-40 )
  21. Serological pipettor
  22. 5 ml, 10 ml, 25 ml, and 50 ml serological pipettes (Fisherbrand, model: 13-678-11 )
  23. BL21(DE3) Ril cells (Agilent, catalog number: 230245 )
  24. FUS expression construct in pDUET vector (Addgene Plasmid #29629) (Figure 2A)
  25. Kapβ2 expression construct in pGEX-Tev vector (Figure 2B)
  26. LB liquid media (Fisher Chemical, catalog number: BP9723-2 )
  27. Ampicillin (Gold Bio, catalog number: A-301-25 )
  28. LB + ampicillin agar plates
  29. Lysozyme (Gold Bio, catalog number: L-040-25 )
  30. Ice
  31. Liquid nitrogen
  32. Bradford Reagent (Thermo Scientific, catalog number: PI23236 )
  33. Microscope immersion oil (Carl Zeiss, catalog number: 12-070-397 )
  34. Bio-spin Chromatography Columns (Bio-Rad, catalog number: 7326008 )
  35. HiTrap-Q anion exchange chromatography column (GE Healthcare, catalog number: 17115301 )
  36. IPTG (GoldBio, catalog number: 367-93-1 )
  37. Phosphate-Buffered Saline tablets (Fisher Scientific, catalog number: BP2944100 )
  38. D-(+)-trehalose (Fisher Scientific, catalog number: BP2687100 )
  39. Glutathione Sepharose 4 Fast Flow beads (GE Healthcare, catalog number: 17-5132-01 )
  40. NaCl (Fisher Scientific, catalog number: S271-3 )
  41. EDTA (Fisher Scientific, catalog number: BP2482500 )
  42. Glycerol (Fisher Scientific, catalog number: BP2294 )
  43. DTT (Gold Bio, catalog number: DTT10 )
  44. Pepstatin (Gold Bio, catalog number: P-020-25
  45. EGTA (Millipore, catalog number: 324626-25GM )
  46. MgCl2 (Fisher BioReagents, catalog number: BP214-500 )
  47. Protease inhibitor tablets (Thermo Scientific, catalog number: A32955 )
  48. Imidazole (Fisher Scientific, catalog number: O3196-500 )
  49. Reduced glutathione (Fisher Scientific, catalog number: AC120000250 )
  50. FUS Wash Buffer (see Recipes)
  51. FUS Elution Buffer (see Recipes)
  52. Kapβ2 Resuspension Buffer (see Recipes)
  53. Kapβ2 ATP Buffer (see Recipes)
  54. Kapβ2 Buffer A (see Recipes)
  55. Kapβ2 Elution Buffer (see Recipes)
  56. TEV Buffer (see Recipes)


    Figure 2. Plasmid maps for the constructs used in the experiment. A. Plasmid map of FUS expression construct in pDUET vector. B. Plasmid map of Kapβ2 expression construct in pGEX-Tev vector.

Equipment

  1. 50 ml beakers (Pyrex, catalog number: CLS100050 )
  2. 100 ml beakers (Pyrex, catalog number: CLS100050 )
  3. 250 ml Erlenmeyer flask (Pyrex, catalog number: 4980250 )
  4. 2800 ml Erlenmeyer flasks (Pyrex, catalog number: CLS44202XL )
  5. Plastic nalgene 1 L centrifugal jars (Thermo Scientific, catalog number: 11-825B )
  6. 0.1-10 µl, 2-20 µl, 20-200 µl, and 200-1,000 µl pipettes
  7. Centrifuge 5424 R (Eppendorf, catalog number: 2231000655 )
  8. Centrifuge 5810 R (Eppendorf, catalog number: 0 22625004 )
  9. Sorvall Lynx 4000 Superspeed Centrifuge (Thermo Scientific, catalog number: 75-006-580 )
  10. Sorvall BP 8 Centrifuge (Thermo Scientific, catalog number: 75007681 )
  11. ThermomixerTM C with SmartBlock (Eppendorf, catalog number: 2231000667 )
  12. MaxQTM 4450 Benchtop Orbital Shaker (Thermo Scientific, catalog number: SHKE4450 )
  13. Innova® 44 Series Shaking Incubator (New Brunswick, catalog number: M1282 )
  14. HerathermTM General Protocol Microbiological Incubator (Thermo Scientific, catalog number: 51028063 )
  15. Tube Revolver/Rotator (Thermo Scientific, catalog number: 88881001 )
  16. Multi-Purpose Tube Rotator (Fisherbrand, catalog number: 88-861-049 )
  17. Digital Vortex Mixer (Fisherbrand, catalog number: 02-215-418 )
  18. Cimarec+TM Stirring Hotplate S (Thermo Scientific, catalog number: P88857100 )
  19. EntrisTM Precision Balance (SartoriusTM, catalog number: ENTRIS423i-1S )
  20. Accumet® AB150 pH Benchtop Meter (Fisherbrand, catalog number: 13-636-AB150 )
  21. Model 705 Sonic Dismembrator (Fisherbrand, catalog number: FB705110 ) 
  22. 160 UV-Vis Spectrophotometer (Biomate, catalog number: 840-301000 )
  23. TecanTM Spark® Multimode Microplate Reader
  24. HP Z230 Tower Workstation
  25. LEICATM DMi8 S Platform Inverted Microscope
  26. Dell Intel Core i7
  27. Cold room
  28. 4 °C Refrigerator
  29. -20 °C freezer
  30. -80 °C freezer
  31. AKTA pure FPLC

Software

  1. Excel (Microsoft Office)
  2. Tecan SparkControl 
  3. Leica LAS X Hardware
  4. AKTA Unicorn

Procedure

English
中文翻译

文章信息

版权信息

© 2020 The Authors; exclusive licensee Bio-protocol LLC.

如何引用

Robinson, E., Shorter, J. and Guo, L. (2020). Karyopherin-β2 Inhibits and Reverses Aggregation and Liquid-liquid Phase Separation of the ALS/FTD-Associated Protein FUS. Bio-protocol 10(16): e3725. DOI: 10.21769/BioProtoc.3725.

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分类

神经科学 > 神经系统疾病 > 神经退行性病变

生物化学 > 蛋白质 > 自组装

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