In vitro AMPylation/Adenylylation of Alpha-synuclein by HYPE/FICD

Materials and Reagents

1. Kimwipes (Kimberly-Clark Professional, KimTech Science Brand, catalog number: 34155 )
2. Set of micropipettes, with volumes ranging from 0.5 to 1,000 μl (generic)
3. Appropriately sized sterile micropipette tips (generic)
4. 2 ml pipette bulbs (VWR, catalog number: 82024-554 )
5. Pasteur pipette (VWR, catalog number: 14672-380 )
6. Glass thermometer, 20-100 °C range (generic)
7. 1.5 ml tubes (Eppendorf Safe-lock microcentrifuge tubes, catalog number: 0 22363204 )
8. Gel loading pipet tips (Fisher Scientific, Fisher Brand, catalog number: 02-707-181 )
9. MTC Stop-Pop locking tubes clips (MTC Bio, catalog number: C2086 )
10. Bacterially purified HYPE_102-456 (WT, E234G, and E234G/H363A) (Sanyal et al., 2019)
11. Bacterially purified full-length α-syn (Sanyal et al., 2019)
12. ddH₂O (ELGA LabWater, PURELAB Flex 2 Dispenser, model number: PF2XXXXM1 )
13. HEPES (Sigma-Aldrich, Sigma-Millipore, catalog number: H4034 )
14. Manganese (II) chloride tetrahydrate (Sigma-Aldrich, Sigma Millipore, catalog number: M3634 )
15. 12.1 N Hydrochloric acid (Fisher Scientific, Fisher Chemical, catalog number: A144-212 )
16. 50% (w/w) Sodium hydroxide solution (Fisher Scientific, Fisher Chemical, catalog number: SS254-4 )
17. Tris base (Fisher Scientific, Fisher Bioreagents, catalog number: L-15863 )
18. Sodium chloride (Fisher Scientific, Fisher Chemical, catalog number: L-11620 )
19. Glycerol (Fisher Scientific, Fisher Bioreagents, catalog number: L-13751 )
20. α-³²P-ATP 3000Ci/mmol 10mCi/ml, 250 µCi (Perkin Elmer, BLU003H250UC)
21. 4-20% Precast SDS-PAGE gel (Bio-Rad, Mini-PROTEAN TGX Gels, catalog number: 456-1091 )
22. Protein ladder (Bio-Rad, Precision Plus Protein Dual Color Standards, catalog number: 1610374TGX )
23. 2-Mercaptoethanol (Sigma-Aldrich, Sigma Life Sciences, catalog number: M3148 )
24. Sodium dodecyl sulfate (Sigma-Aldrich, Sigma Life Sciences, catalog number: L4390 )
25. Bromophenol Blue sodium salt, ACS (Alfa Aesar, catalog number:32639)
26. Methanol (Fisher Scientific, Fisher Chemical, catalog number: L-21063 )
27. Acetic acid, glacial (Fisher Scientific, Fisher Chemical, catalog number: A38C-212 )
28. Glycine (Fisher Scientific, Fisher Bioreagents, catalog number: L-15696 )
29. Coomassie Brilliant Blue R-250 (Amresco, catalog number: 0472-25G )
30. Phosphor screen (General Electric, Kodak Phosphor Storage Screen, catalog number: 00 146931 )
31. Protein Storage Buffer (see Recipes)
32. 10x AMPylation Buffer (see Recipes)
33. 4x SDS-PAGE Loading Buffer (see Recipes)
34. 1x SDS-PAGE Running Buffer (see Recipes)
35. Coomassie Stain (see Recipes)
36. Destain (see Recipes)
    

Equipment

1. Plexiglass or Lucite shield, 0.5 inches thick (Nalgene Acrylic Benchtop Beta Radiation Shield, Thermo Fisher, catalog number: 6700-1812 )
2. Geiger counter (Model 3 Survey Meter, Ludlum Measurements, model: 44-7)
3. ddH₂O dispenser (PURELAB Flex 2 Dispenser, ELGA LabWater, model: PF2XXXXM1 )
4. Heatblock (Analog Heatblock, VWR, catalog number: 12621-108 )
5. pH meter (Seven Easy, Mettler Toledo, catalog number: 1227396066 )
6. Centrifuge (Eppendorf, model: Centrifuge 5417C , catalog number: 00 23210 )
7. Power supply ( PowerPac 300 /DCode, Bio-Rad, model: PowerPac 300 )
8. PAGE system (Mini-PROTEAN Tetra Cell, 4-Gel System, Bio-Rad, catalog number: 1658004 )
9. Fluorescent Image Analyzer (Typhoon FLA 9500, General Electric, model: Typhoon FLA 9000 )
10. Phosphor screen eraser ( FLA Image Eraser , General Electric, model: FLA Image Eraser )
11. Rocker (VWR, Rocking Platform, model: 200 )
12. Image scanner (Epson, Epson Perfection V700 Photo)
    

Software

1. Typhoon FLA 9500 (General Electric)

Procedure

1. AMPylation reaction
   Note: This protocol requires use of radioactive α-³²P-ATP. As such, all radioactive materials or materials in contact with them must be handled, stored, and disposed of in accordance with your institution’s radioactive safety guidelines. Radioactivity must be handled behind a radiation shield at all times. A Geiger counter should be used to constantly monitor work environment for radioactive contamination. Appropriate personal protective equipment should be worn at all times.
   1. Pipette calculated volume of ddH₂O into designated 1.5 ml tubes.
   2. Pipette calculated volume of 10x AMPylation Buffer into designated 1.5 ml tubes.
      Note: Components of 10x AMPylation Buffer may be modified as needed to accommodate differing experimental conditions, such as enzymes, substrates, etc.
   3. Pipette 2 μg of purified, recombinant HYPE enzyme to designated 1.5 ml tubes.
      Notes:
      1. Reactions were performed at a final volume of 20 µl. The actual reaction amounts may be modified so long as a ≥ 10:1 molar substrate:enzyme ratio is maintained, and a detectable amount of each protein (i.e., ≥ 0.1 μg) is loaded onto the gel.
      2. All proteins are purified as previously described (Sanyal et al., 2019) and come dissolved in protein storage buffer.
      3. It is advisable to prepare twice the amount of required sample in case the gel needs to be rerun.
   4. Pipette 20 μg of purified, recombinant α-syn (or other substrate) into designated 1.5 ml tubes.
      Note: When performing kinetic experiments or comparing different substrates side-by-side, molar concentrations should be used in order to ensure that the relative numbers of molecules in the reaction are consistent.
   5. Pipette 2.5 μCi α-³²P-ATP into designated 1.5 ml tubes.
      Notes:
      1. Reaction start time begins with α-³²P-ATP addition. As α-³²P-ATP is the only radioactive component, the other sample ingredients may be added at a standard lab bench, and samples can then be transported to a designated radioactive area for α-³²P-ATP addition.
      2. Alternative concentrations of α-³²P-ATP may be used depending on the catalytic efficiencies of the test enzyme and/or the freshness of the nucleotide. These concentrations should be determined empirically. AMPylation reactions should ideally occur within the first half-life (14.3 days) of α-³²P-ATP.
   6. Incubate the samples in a heat block set to 30 °C for 30 min.
      Notes:
      1. Be sure to place the heat block behind a radiation shield.
      2. Different AMPylases may require different reaction temperatures or durations for optimal enzymatic activity.
      3. Samples may be snap-centrifuged for 5 s at 2,000 x g in order to consolidate any sample components not at the bottom of the tube.
   7. Quench reaction with an appropriate volume of 4x SDS Loading Buffer.
   8. Heat samples for 10 min at 95 °C on a heat block.
      Note: Ensure that tube lids are firmly closed to avoid lids popping open during heating. A weighted sample tray may be placed flatly over tube lids to prevent opening. Alternatively, MTC Stop-Pop locking tubes clips (MTC Bio, Inc.) could be used.
   9. Snap centrifuge samples for 5 s at 2,000 x g.
      
      
      
2. SDS-PAGE separation
   1. Carefully remove well comb and green seal tape from precast gel cassette.
   2. Assemble gel cassette and tank according to manufacturers’ instructions.
      Note: Fresh 1x Running Buffer should be used inside of the gel cassette assembly, although used 1x Running Buffer may be used to fill the tank.
   3. Using gel loading pipette tips, carefully load 2-4 μl of protein ladder into the first lane.
   4. Using gel loading pipette tips, carefully load 10-30 μl of each sample into lanes adjacent to the first lane.
   5. Securely close tank lid.
   6. Run gel at constant 200 V for 45 min. Alternatively, better band resolution can be obtained by first running the gel at 80 V for ~30 min (until the samples clear the stacking layer), then ramping up to 120 V for ~1 h (until the dye front reaches the reference line on the gel cassette).
      Note: Ensure proper current flow via production of bubbles in the running buffer and migration of the dye front towards the bottom of the gel.
      
      
      
3. Phosphor imaging
   1. Carefully remove gel using a cassette lever.
      Note: The gel is radioactive at this point and should be handled and disposed of in accordance with your institution’s regulations/guidelines for radioactive materials.
   2. Place gel in between a clean, plastic cover sheet. Smooth out any air bubbles in the gel.
   3. Place cover sheet with gel on top of the imaging side (white) of a clean Phosphor screen.
      Notes:
      1. Phosphor screen should be erased with the Phosphor screen eraser after each use to prevent signal contamination.
      2. Avoid touching the imaging side of the Phosphor screen, as this can damage the screen.
   4. Carefully place Phosphor screen and gel inside of the foam phosphor screen storage container.
   5. Expose the screen for 3 h at room temperature.
      Note: Duration of Phosphor screen exposure is dependent on the AMPylation reaction parameters and must therefore be determined empirically.
   6. Place the Phosphor screen imaging-face down in an imager.
      Note: Ensure imager screen is clear of debris before imaging, as these may mask sample signal. Kimwipes and ddH₂O should be used to clean imager screen as needed.
   7. Open the Typhoon FLA 9500 software.
   8. Under “Mode Settings”, select the “Phosphorimaging” tab.
   9. Under “Reader Settings,” go to the “Method” dropdown tab and select “[Phosphor]” option.
      Note: The “[Phosphor]” option will be colored red and correspond to a wavelength of 673 nm.
   10. Under “Reader Settings,” go to “PMT” (Photo-multiplier Tube) and select a value between 250-1,000 V.
       Note: A PMT of 500 V is usually a good starting point to gauge band intensity; the higher the PMT, the stronger the signal.
   11. Under “Reader Settings,” go to “Area” and select the grid area corresponding to where the gel is placed on the screen. Alternatively, the entire screen may be imaged, but this takes additional time.
   12. Under “Reader Settings” go to “Pixel Size” and Select “50 μm” pixel size.
       Note: Lower or higher resolutions may be selected based on specific image quality needs.
   13. Select “Scan” to scan image.
   14. Select “Save As” to save image as a Tiff file in the appropriate folder.
       
       
       
4. Coomassie staining
   1. Once exposure of the Phosphor screen to the gel is complete, incubate the gel in Coomassie Stain with a rocker set to low to medium speed for 10 min.
      Note: The amount of stain used will vary depending on the shape and dimensions of the container used for incubation but should be enough to fully immerse the gel.
   2. Pour off Coomassie Stain in its original container for reuse.
   3. Incubate gel in Destain on rocker for 30 min or until sufficient contrast between protein bands and gel background is established.
   4. Discard Destain in appropriate waste container.
   5. Scan gel on generic document scanner between cover sheet, or with phone camera or scanning application.
      Notes:
      1. Coomassie staining is done to control for protein loading, and should yield bands that validate experimental amounts of protein (see Figure 1, bottom).
      2. If reactions are set up using molar ratios instead of mass-based concentrations, the apparent band sizes may vary with the differing molecular weights of the proteins.
      
      
      Figure 1. HYPE AMPylates α-syn in vitro. AMPylation competent E234G-HYPE robustly AMPylates α-syn and itself (auto-AMPylation) with α-³²P-AMP (top). Intrinsically autoinhibited WT-HYPE and a catalytically dead E234G/H363A mutant show no auto-AMPylation or substrate AMPylation (top). 1 μg of enzyme and 10 μg of substrate from the AMPylation reaction were loaded on the gel (bottom). Phosphor screen was exposed for 3 h (top).

Recipes

Note: Dissolve the following solutions in ddH₂O. Always add solvent before addition of other components. No pH adjustments are required unless explicitly stated. Adjust pH to appropriate value with 12.1 N HCl or 50% NaOH with pipette and rubber suction bulb. Diluted acid or base may also be prepared to avoid overshooting target pH.

1. Protein Storage Buffer
   50 mM Tris base
   300 mM NaCl
   10% (v/v) glycerol
   Adjust to pH 7.5
2. 10x AMPylation Buffer
   50 mM HEPES
   10 mM MnCl₂
   Adjust to pH 7.5
3. 4x SDS-PAGE Loading Buffer
   2.86 M BME
   250 mM Tris, pH 6.8
   10% (w/v) SDS
   0.008% (v/v) bromophenol blue
   40% (v/v) glycerol
4. 1x SDS-PAGE Running Buffer
   0.3% Tris base
   1.4% glycine
   0.1% (w/v) SDS
5. Coomassie Stain
   50% (v/v) methanol
   10% (v/v) acetic acid
   0.1% Coomassie Brilliant Blue
6. Destain
   50% (v/v) methanol
   10% (v/v) acetic acid
   

Acknowledgments

This publication was made possible with partial support from the Indiana CTSI TL1 Predoctoral Fellowship (Grant # UL1TR002529 [A. Shekhar, PI], 5/18/2018-4/30/2023, and Grant # TL1TR002531 [T. Hurley, PI], 5/18/2018-4/30/2023, from the National Institutes of Health, National Center for Advancing Translational Sciences, Clinical and Translational Sciences Award) to A.C.; and Eli Lilly-Stark Neuroscience Research Institute-CTSI predoctoral fellowship to S.D.; a grant from the Branfman Family Foundation to J.C.R.; and the National Institute of General Medical Sciences of the National Institute of Health (R01GM10092), an Indiana Clinical and Translational Research Grant (CTSI-106564), and a Purdue Institute for Inflammation, Immunology, and Infectious Disease Core Start Grant (PI4D-209263) to S.M. The authors would also like to thank members of the Rochet and Mattoo laboratories for their helpful discussions.

Competing interests

The authors declare no conflicting or competing interests.

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