Materials and Methods

Chemicals

The E1 ubiquitin-activating enzyme inhibitor PYR-41 and the DUB inhibitor ubiquitin aldehyde were obtained from Calbiochem, San Diego, CA.The oxidative indicator 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was obtained from Sigma Aldrich, St. Louis, MO.

Cloning of ubiquitin-containing targeted toxins

For cloning, ubiquitin and LFn were amplified by fusion PCR and cloned into pET19b via the NdeI and BamHI restriction sites.PEIII was inserted into this plasmid 3’ of ubiquitin.Finally, ubiquitin was substituted by other variants of ubiquitin (ubiquitin with an uncleavable sequence at the C-terminus of ubiquitin, ubiquitin with all lysine residues except Lys48 or Lys63 changed to arginine residues, and ubiquitin with all lysine residues changed to arginine residues) after amplification by PCR.The ubiquitin variants containing mutations were amplified from plasmids purchased from Addgene, Cambridge, MA ( constructs encoded an N-terminal 10 histidine tag, followed by the enterokinase cleavage site DDDDK for histidine tag removal. After histidine tag removal, all constructs obtained an N-terminal HMAGG sequence. All ubiquitin constructs contained in addition a GGGS linker at the C-terminus of LFn and a GS or AS linker preceding PEIII (Fig. 1).

Expression and purification of the ubiquitin-containing targeted toxins

All targeted toxins were expressed in 3l LB mediumsupplemented with 100 µg/ml ampicillin for 5 h at 37 °C in E. coli strain BL21 (DE3) Gold.Expression was induced by 1 mM isopropyl--D-thiogalactopyranoside after reaching an optical density (600 nm) of 0.8.Cells (6 g wet weight) were harvested by centrifugation, culture pellets resuspended in 120 ml sonication buffer (50 mM Tris, pH 8.0, 0.3 M KCl, 5 mM EDTA, 1 tablet protease inhibitor (Roche, Indianapolis, IN)/ 50 ml) supplemented with 1 mg/ml lysozyme, sonicated for 2 min and incubated 20 min on ice.After a subsequent centrifugation, the pellet was resuspended (in 35 ml 1.5% N-lauroylsarcosine, 1 % Triton-X 100, 50 mM Tris, pH 8.0, 0.3 M KCl), sonicated for 2 min, recentrifuged and 4 ml Ni-NTA-agarose (Qiagen, Valencia, CA) was added.The proteins were eluted by 0.5 M imidazole (in 50 mM Tris, pH 8.0, 10 % glycerol, 0.3 M KCl) after washing with 1 M and 0.3 M KCl (in 50 mM Tris, pH 8.0, 10 % glycerol, 20 mM imidazole).Fractions containing the TTs were pooled, dialyzed against 5 mM HEPES, pH 7.5, 0.5 mM EDTA overnight and loaded on a Q-Sepharose Fast Flow column for purification with an Äkta chromatography system (GE Healthcare, Waukesha, WI) and eluted using a linear gradient of 0-0.5 M NaCl in 20 mM Tris, pH 8.0, 0.5 M EDTA.Fractions containing the TTs were again dialyzed as described above, concentrated (Amicon Ultrafiltration devices, 30 kDa molecular weight cutoff, Millipore, Billerica, MA), filter sterilized, and stored in aliquots at -80 °C.All proteins were analyzed by electrospray ionization mass spectrometry to confirm that the masses matched those calculated from their sequences.

For cytotoxicity studies, enterokinase cleavage was performed to remove the N-terminal 10  histidine tag, exposing an N-terminal HMAGG sequence.The TTs (at 0.3 mg/ml) were incubated 24 h with 0.5 U/ml enterokinase (Stratagene, La Jolla, CA) in 50 mM Tris, pH 8.0, 50 mM NaCl, 2 mM CaCl2, 0.1% Triton X-100).The cleavage of the samples was confirmed by SDS-PAGE and the TTs were used in cytotoxicity assays without further purification.

Cell culture

HN6 and RAW264.7 cells were maintained in Dulbecco’s modified Eagle’s medium with Glutamax-1 (Gibco, Life Technologies, Grand Island, NY), CHO TEM8 T4 cells were maintained in modified Eagle’s medium alpha with Glutamax-1 (Gibco, Life Technologies, Grand Island, NY).All media were supplemented with 10% fetal bovine serum (Gibco, Life Technologies, Grand Island, NY) and 50 µg/ml gentamicin (Quality Biological, Gaithersburg, MD).CHO TEM 8 T4 cells were additionally grown in the presence of 500 µg/ml hygromycin (Invitrogen, Life Technologies, Grand Island, NY).

Cytotoxicity of TTs

Dose-response curves for the TTs were obtained by incubation on HN6 cells for 2 or 48 h in the presence of 0.25 µg/ml PA.The cells (10,000 cells per well in 100 µl medium) were seeded in 96-well plates and incubated at 37 °C for 6 h before addition of PA and the TTs.TTs were added in 100 µl to achieve final concentrations ranging from 0.3 pM-1 nM and the cells were incubated a further 48 h.For the experiments with 2 h TT exposure, the TT-containing medium was removed after 2 h and the cells were incubated a further 46 h in fresh medium.Cell survival was determined in an MTT assay by adding 30 µl MTT solution (5 mg/ml in medium without fetal bovine serum) to each well and incubation for 1 h at 37 °C.All medium was removed and the cells were treated with 50 µl solubilizer (90 % isopropanol, 1 % sodium dodecyl sulfate, 80 mM HCl).Plates were shaken for 1 min and absorbance was measured at 570 nm and 630 nm (for background subtraction for each sample) in a microplate reader (Spectra Max Gemini, Molecular Devices, Sunnyvale, CA).The relative cell survival, designated as survival index (SI), was calculated after blank subtraction (wells without cells) as the percentage of living cells in treated wells in relation to untreated cells (cells without toxin).For experiments using the inhibitor PYR-41, RAW264.7 cells (15,000 cells per well in 100 µl medium) were seeded in 96-well plates and incubated at 37 °C for 12 h.Cells were treated with 50 µl medium containing the inhibitor diluted so as to achieve the desired final 15.8 µM PYR-41 in 200 µl and incubated for 1 h at 37 °C.Subsequently, 50 µl medium supplemented with the TTs was added and incubated for a further 18 h at 37 °C before measurement of cell survival as described above.

Data analysis

The statistical significance of different 50 % survival indices (SI50, 50 % cell survival in comparison to untreated controls) values for cytotoxicity analyses was determined by a paired t-test using GraphPad Prism 5.02 and data obtained from a nonlinear regression curve fit.The method used for the nonlinear regression curve fit was “log(inhibitor) versus normalized response” (by using a least square fit).A two-tailed significance of p ≤ 0.05 was interpreted as being statistically significant.

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