TITLE: Validation of Tankyrase Binding and PARylation in Full-Length Protein Context
AUTHORS: Katie Pollock, Michael Ranes, Ian Collins, Sebastian Guettler

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>This protocol is part of a collection: </span><a href="https://www.protocols.io/private/DA5F6F33163011EBAE360A58A9FEAC2A" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">Identifying and Validating Tankyrase Binders and Substrates: A Candidate Approach</span></a></div><br/><div class = "text-block"><span>The poly(ADP-ribose)polymerase (PARP) enzyme tankyrase (TNKS/ARTD5, TNKS2/ARTD6) uses its ankyrin repeat clusters (ARCs) to recognize degenerate peptide motifs in a wide range of proteins, thereby recruiting such proteins and their complexes for scaffolding and/or poly(ADP-ribosyl)ation. Here, we provide guidance for predicting putative tankyrase-binding motifs, based on the previously delineated peptide sequence rules and existing structural information. We present a general method for the expression and purification of tankyrase ARCs from</span><span style = "font-style:italic;">Escherichia coli</span><span>and outline a fluorescence polarization assay to quantitatively assess direct ARC–TBM peptide interactions. We provide a basic protocol for evaluating binding and poly(ADP-ribosyl)ation of full-length candidate interacting proteins by full-length tankyrase in mammalian cells.</span></div></div>

[STEPS]
?. [3.3 Validation of Tankyrase Binding and PARylation in Full-Length Protein Context]
This final section outlines a step-by-step approach to assess and validate the full-length candidate protein interaction with full-length tankyrase and the extent of its tankyrase-dependent PARylation. Mammalian expression constructs for MYC2-tagged TNKS2 (pLP-dMYC SD-TNKS2) and FLAG- or FLAG3-tagged candidate interacting proteins (pCMV-FLAG-MERIT40 and pLP-tripleFLAG SD-TRF1) are generated. Standard site-directed mutagenesis is performed to mutate the Gly at position 6 of the FP-validated TBMs to Arg. The FLAG-tagged candidate proteins and corresponding TBM mutant derivatives are next used as “baits” in co-immunoprecipitation experiments, to assess their binding to and their PARylation by MYC2-TNKS2, using catalytically inactive TNKS2 (G1032W) as control [31].
?. [3.3.1 Co-expression of TNKS2 and MERIT40 or TRF1 in HEK293T Cells]
Remove the media and gently rinse the cell culture plates with  to remove residual media, which could impede the efficient dissociation of the cells by trypsin.
[Versene]
?. [3.3.1 Co-expression of TNKS2 and MERIT40 or TRF1 in HEK293T Cells]
Add  to each plate and place the plates back in the incubator for . Gently tap the side of the plates to help the cells detach from the plate and dissociate from each other.
[trypsin-Versene]
?. [3.3.1 Co-expression of TNKS2 and MERIT40 or TRF1 in HEK293T Cells]
Add  to each plate; the FBS will inactivate the trypsin. Pipette the cell suspension up and down a few times to ensure proper dissociation of cell clumps into a homogenous cell suspension.
[DMEM media with 10% FBS]
?. [3.3.1 Co-expression of TNKS2 and MERIT40 or TRF1 in HEK293T Cells]
Measure the cell density, using either a hemocytometer or automated cell counter. Add 6 × 106 cells each to the required number of 10 cm cell culture plates for the co-transfection of different DNA constructs (Table 3). Add  to each plate and incubate cells .  ABCDEFGH1empty FLAG vectorMERIT40 WTMERIT40 G33RMERIT40 G53RMERIT40 GG33/53RRTRF1 WTTRF1 G18R2empty MYC2&nbsp;vector−+−−−+−3TNKS2 WT++++++++4TNKS2 G1032W−+−−−+−
[DMEM media with 10% FBS]
ABCDEFGH1empty FLAG vectorMERIT40 WTMERIT40 G33RMERIT40 G53RMERIT40 GG33/53RRTRF1 WTTRF1 G18R2empty MYC2&nbsp;vector−+−−−+−3TNKS2 WT++++++++4TNKS2 G1032W−+−−−+−
?. [3.3.1 Co-expression of TNKS2 and MERIT40 or TRF1 in HEK293T Cells]
The next day, co-transfect cells with 5 μg of each DNA construct (thus 10 μg total) per plate (Table 3):Pipette  in a 15 mL Falcon tube, add  followed by  and mix thoroughly. Subsequently, add , mix the solution thoroughly by pipetting up and down and incubate for – at .
[each DNA construct]
[2 M CaCl2]
[H2O]
[2× HBS]
0 Room temperature
It is important to add the different transfection reagents in the specified order to ensure proper calcium phosphate–DNA particle formation.
?. [3.3.1 Co-expression of TNKS2 and MERIT40 or TRF1 in HEK293T Cells]
Replace the growth media from the 10 cm plate with  supplemented with . Chloroquine inhibits degradation of endocytosed DNA and can increase transfection efficiency [21].
[fresh growth media]
[chloroquine]
?. [3.3.1 Co-expression of TNKS2 and MERIT40 or TRF1 in HEK293T Cells]
Gently add the transfection mix (4 mL each, dropwise) to each plate and incubate transfected cells for .
?. [3.3.1 Co-expression of TNKS2 and MERIT40 or TRF1 in HEK293T Cells]
The next day, remove the media and add . Harvest the cells by gently scraping them off the plate. Collect the scraped cell suspension in a 15 mL Falcon tube and pellet the cells by centrifugation at . Discard the supernatant; snap-freeze cell pellet in liquid nitrogen and store at  until further use.
[ice-cold PBS]
Avoid using micropipette tips to transfer the cell suspension after scraping. The small opening of the tips can cause cells to break due to shearing. You can cut off the tips to avoid this risk.
Centrifuge: 300 34, 5 min, 4 10
-80 °C
?. [3.3.2 Co-immunoprecipitation]
Add  (with freshly added DTT and protease inhibitors) to each cell pellet and resuspend cells . The freeze–thaw and the detergents will break open the cells. Transfer the lysates to 1.5 mL microcentrifuge tubes and incubate  for  for extraction.
[ice-cold RIPA buffer]
on ice
on ice
?. [3.3.2 Co-immunoprecipitation]
Sonicate lysates for , using a small tip sonicator at 25% amplitude output to shear chromatin.
on ice
?. [3.3.2 Co-immunoprecipitation]
Clear cell lysates by high-speed centrifugation at  and transfer supernatants to new 1.5 mL microcentrifuge tubes.
Centrifuge: 18000 34, 15 min, 4 10
?. [3.3.2 Co-immunoprecipitation]
Transfer  to a new microcentrifuge tube (input samples) and add . Boil samples at  for , collect by brief centrifugation , and store at  until analysis, if required.
[each cleared lysate ]
[4× SDS sample buffer]
95 °C
-20 °C
?. [3.3.2 Co-immunoprecipitation]
Pre-equilibrate a sufficient amount of anti-FLAG M2 Agarose resin ( per immunoprecipitation sample plus a minimum of 10% dead volume) with RIPA buffer and incubate resin with cleared lysates for  on a rotating wheel at .
[ packed-volume resin]
4 °C
?. [3.3.2 Co-immunoprecipitation]
Gently settle the resin by centrifugation at  and remove supernatant using a vacuum pump.
Centrifuge: 800 34, 5 min, 4 10
?. [3.3.2 Co-immunoprecipitation]
Wash the resin by adding  to each sample and incubating microcentrifuge tubes for  on a rotating wheel at .
[RIPA buffer]
4 °C
?. [3.3.2 Co-immunoprecipitation]
Settle the resin as before (step 16: Gently settle the resin by centrifugation at  and remove supernatant using a vacuum pump.) and repeat the wash step four more times:
Centrifuge: 800 34, 5 min, 4 10
?. [3.3.2 Co-immunoprecipitation]
After the last wash step, gently remove as much of buffer as possible and add . Boil samples at  for , collect briefly by centrifugation and store at  until analysis, if required (immunoprecipitate (IP) samples).
[2× SDS sample buffer]
95 °C
-20 °C
?. [3.3.2 Co-immunoprecipitation]
Gently settle the resin by centrifugation at  and remove supernatant using a vacuum pump. (Wash 1/4)
Centrifuge: 800 34, 5 min, 4 10
?. [3.3.2 Co-immunoprecipitation]
Gently settle the resin by centrifugation at  and remove supernatant using a vacuum pump. (Wash 2/4)
Centrifuge: 800 34, 5 min, 4 10
?. [3.3.2 Co-immunoprecipitation]
Gently settle the resin by centrifugation at  and remove supernatant using a vacuum pump. (Wash 3/4)
Centrifuge: 800 34, 5 min, 4 10
?. [3.3.2 Co-immunoprecipitation]
Gently settle the resin by centrifugation at  and remove supernatant using a vacuum pump. (Wash 4/4)
Centrifuge: 800 34, 5 min, 4 10
?. [3.3.3 SDS-PAGE and Immunoblotting]
Resolve  (step 14 above) and IP (step 19 above) samples on a pre-cast 4–15% Tris–glycine polyacrylamide gradient gel for SDS-PAGE analysis.
[boiled input]
?. [3.3.3 SDS-PAGE and Immunoblotting]
Transfer proteins from the gel onto a nitrocellulose membrane using a wet-transfer blotting system. Ponceau S can be used to assess the transfer quality.
?. [3.3.3 SDS-PAGE and Immunoblotting]
Incubate the nitrocellulose membrane for at least  at  on a horizontal shaking platform in 5% dry milk powder in PBS to block the membrane to reduce nonspecific binding of antibodies during the subsequent immunodetection steps.
0 Room temperature
?. [3.3.3 SDS-PAGE and Immunoblotting]
Incubate the membranes with the required antibody at the appropriate dilution in  at  on a horizontal shaking platform. (Antibody dilutions: anti-FLAG HRP-conjugated antibody, 1:1000; anti-MYC HRP -conjugated antibody, 1:1000; anti-PAR, 1:1000) .
[milk/PBS]
4 °C
?. [3.3.3 SDS-PAGE and Immunoblotting]
Wash the membrane three times for  with copious amounts PBS + 0.1% Tween 20 on a horizontal shaking platform:
?. [3.3.3 SDS-PAGE and Immunoblotting]
Develop Western blot by incubating membrane with ECL Western blotting substrate following the manufacturer’s instructions.
?. [3.3.3 SDS-PAGE and Immunoblotting]
Detect chemiluminescence signal by exposing membrane to X-ray film in a dark room and develop the film with an X-ray film developer. Fig. 4 shows the TBM-dependent interaction of FLAG3-TRF1 and FLAG-MERIT40 with MYC2-TNKS2, and the TNKS2-dependent PARylation of both TNKS2 interactors, in addition to TNKS2 auto-PARylation. Both MERIT40 TBMs contribute to tankyrase binding (Fig. 4b).
The Western blot protocol can be adapted for film-free chemiluminescence detection or fluorescence-based detection.
Attributing a PAR signal to a particular protein by molecular weight may be challenging, in part due to possible PAR-induced mobility shifts in SDS-PAGE. Since the tankyrase substrate candidates are immunoprecipitated from cell lysates, it is possible that the observed PAR signal corresponds to other PARylated proteins in a protein complex. Ultimate confirmation of substrates can be obtained from experiments with purified proteins or PAR site mapping by mass spectrometry, for example.
?. [3.3.3 SDS-PAGE and Immunoblotting]
Wash the membrane for  with copious amounts PBS + 0.1% Tween 20 on a horizontal shaking platform. (Wash 1/3)
?. [3.3.3 SDS-PAGE and Immunoblotting]
Wash the membrane for  with copious amounts PBS + 0.1% Tween 20 on a horizontal shaking platform. (Wash 2/3)
?. [3.3.3 SDS-PAGE and Immunoblotting]
Wash the membrane for  with copious amounts PBS + 0.1% Tween 20 on a horizontal shaking platform. (Wash 3/3)
?. [3.3.1 Co-expression of TNKS2 and MERIT40 or TRF1 in HEK293T Cells]
Grow a sufficient number (here two) 15 cm cell culture plates of HEK293T cells to a cell density of ≈90% confluence in standard DMEM media supplemented with 10% FBS.
?. [3.3.3 SDS-PAGE and Immunoblotting]
As required, incubate the membrane with the matching HRP-coupled secondary antibody, here diluted 1:5000 in  for  at  on a horizontal shaking platform (see Note 20).
[milk/PBS]
0 Room temperature
?. [3.3.3 SDS-PAGE and Immunoblotting]
Repeat wash: Wash the membrane three times for  with copious amounts PBS + 0.1% Tween 20 on a horizontal shaking platform:
?. [3.3.3 SDS-PAGE and Immunoblotting]
Wash the membrane for  with copious amounts PBS + 0.1% Tween 20 on a horizontal shaking platform. (Wash 1/3)
?. [3.3.3 SDS-PAGE and Immunoblotting]
Wash the membrane for  with copious amounts PBS + 0.1% Tween 20 on a horizontal shaking platform. (Wash 2/3)
?. [3.3.3 SDS-PAGE and Immunoblotting]
Wash the membrane for  with copious amounts PBS + 0.1% Tween 20 on a horizontal shaking platform. (Wash 3/3)