TTLL12 has a potential oncogenic activity, suppression of ligation of nitrotyrosine to the C-terminus of detyrosinated α-tubulin, that can be overcome by molecules identified by screening a compound library

Tubulin tyrosine ligase 12 (TTLL12) is a promising target for therapeutic intervention since it has been implicated in tumour progression, the innate immune response to viral infection, ciliogenesis and abnormal cell division. It is the most mysterious of a fourteen-member TTL/TTLL family, since, although it is the topmost conserved in evolution, it does not have predicted enzymatic activities. TTLL12 seems to act as a pseudo-enzyme that modulates various processes indirectly. Given the need to target its functions, we initially set out to identify a property of TTLL12 that could be used to develop a reliable high-throughput screening assay. We discovered that TTLL12 suppresses the cell toxicity of nitrotyrosine (3-nitrotyrosine) and its ligation to the C-terminus of detyrosinated α-tubulin (abbreviated to ligated-nitrotyrosine). Nitrotyrosine is produced by oxidative stress and is associated with cancer progression. Ligation of nitrotyrosine has been postulated to be a check-point induced by excessive cell stress. We found that the cytotoxicities of nitrotyrosine and tubulin poisons are independent of one another, suggesting that drugs that increase nitrotyrosination could be complementary to current tubulin-directed therapeutics. TTLL12 suppression of nitrotyrosination of α-tubulin was used to develop a robust cell-based ELISA assay that detects increased nitrotyrosination in cells that overexpress TTLL12 We adapted it to a high throughput format and used it to screen a 10,000 molecule World Biological Diversity SETTM collection of low-molecular weight molecules. Two molecules were identified that robustly activate nitrotyrosine ligation at 1 μM concentration. This is the pioneer screen for molecules that modulate nitrotyrosination of α-tubulin. The molecules from the screen will be useful for the study of TTLL12, as well as leads for the development of drugs to treat cancer and other pathologies that involve nitrotyrosination.

We originally selected TTLL12 as a potential target for drug development from our multiomic dissection of human tumours and bioinformatic predictions of enzymatic activity.We found that TTLL12 is expressed in the proliferating layer of benign prostate and its expression increases during cancer progression [11].This overexpression in cancer could generate serum autoantibodies that recognise TTLL12 specifically in prostate cancer patients [13].TTLL12 expression and alterations have been linked to prognosis of various cancers, including prostate [14], stomach [15], lung adenocarcinoma [16], ulcerative colitis [17], ovarian cancer [18], and keloids in Chinese patients [19].A novel transcript isoform has been identified in human cancers [20].TTLL12 may contribute to tumour progression through effects on chromosomal ploidy [11], mitotic duration [10] and microtubule dynamics [12].Interestingly, TTLL12 is very highly conserved in evolution [10,21] and the only related protein in rice affects the dynamics and orientation of microtubules [22].
TTLL12 is also implicated in the innate immune response to viral infection.It acts as a negative regulator of RNA-virus induced type-I IFN expression through protein-protein interactions, rather than protein modifications [23].Repression of the innate immune response could explain different observations.TTLL12 is differentially expressed between HIV positive patients that have or have not been treated with highly active antiretroviral therapy, as well as healthy controls [24].TTLL12 activation improves lentiviral vector yields in HEK293 cells, which was found using genome-wide activator-and inhibitor-CRISP library screens to identify perturbations that improve viral yield [25]. Downregulation of TTLL12 gene transcription by the vitamin-D receptor / vitamin D has been proposed to alleviate the severity of SARS-CoV-2 infection [26].TTLL12 genetic variants are linked to the heritable gut microbiome induced by Salmonella Pullorum in chicken [27].However, the immune response is not obviously linked to other observations.TTLL12 is: a target for serum autoantibodies after acute angle closure glaucoma attack [28], a candidate gene for triglycerides in mice [29], and a gene that is differently expressed during lactogenic differentiation of buffalo mammary epithelial cells [30].Interestingly, TTLL12 is required for the formation of primary ciliary axonemes, which are critical sensory organelles in polarized epithelial cells [12].Amongst TTLL12's functions that can be targeted for drug development, we chose inhibition of tubulin retyrosination for various reasons.Such anti-TTLL12 drugs could be complementary to current therapeutic agents that target microtubules (review: [31]).They could target pathologies that involve dysregulation of α-tubulin retyrosination, such as cancer, neurodegenerative diseases, cardiovascular diseases and inflammatory disorder (reviews: [2,32,33]).They may also target proteins other than α-tubulin that are tyrosinated by TTL [13], as well as stress pathways that are proposed to involve ligation of nitrotyrosine (3-nitrotyrosine) to detyrosinated α-tubulin [34].Nitrotyrosine ligation may lead to microtubule dysfunction, as reported by some [34,35] but not all [36] studies.Nitrotyrosine is formed by a reaction between tyrosine in proteins and nitric oxide-derived reactive nitrogen species, such as peroxynitrite.Nitrotyrosine formation is a marker of oxidative and nitrosative stress, and has been implicated in various pathological conditions, including inflammation, neurodegenerative disease, cardiovascular diseases, and cancer (reviews: [37,38]).Nitrotyrosine levels increase with progression of various cancers, including prostate [39,40] and HNSCC [41].Nitrotyrosine, derived from degradation of nitrotyrosine containing proteins and ligation to α-tubulin, could be a "last" checkpoint leading to apoptosis, and escape from the checkpoint could lead to cancer [42,43].
We report that TTLL12 inhibits both nitrotyrosine incorporation into α-tubulin and nitrotyrosine induced cell toxicity.Using a validated high throughput screen (HTS), we identified hit compounds that relieve nitrotyrosination suppression by TTLL12.This is an important step towards developing molecules that could be used to probe the functions of TTLL12 and ultimately treat cancer and other pathologies by targeting novel pathways.

Transient transfections
siRNAs were transfected with Lipofectamine (Invitrogen Cat No. P/N50470) according to the manufacturer's protocol.In brief, 100,000 cells for HEp-2 and 50,000 cells for DU145 were seeded per well in a 12 well plate.After 18 h, cells were incubated for 3 h in OPTIMEM prior to transfection.Unless otherwise indicated, 10 nM siRNA was transfected per well (5 μl Lipofectamine/12 pM siRNA in OPTIMEM).Six hours after transfection, medium was changed to normal growth medium and incubated for the desired time period.Plasmids were transfected using Lipofectamine in 12 well plates as for siRNA (5 μl lipofectamine /0.5 μg plasmid DNA).

Nitrotyrosine treatment
Nitrotyrosine (3-Nitro-L-tyrosine; Sigma Cat No. 851914) was dissolved in 10 mM HCl to prepare a 10 mM stock.Cells were treated with nitrotyrosine by replacing the medium with nitrotyrosine containing medium or otherwise stated.Equivalent quantities of HCl were used in the medium as control.Medium with or without nitrotyrosine was refreshed every 48 h.

Cell proliferation
Cells were plated at a density of 2 X 10 5 cells for HEp-2 and TTLL12 stable clones, 1 X 10 5 per well for A549, PC3, DU145, PWR-1E, and RWPE1 cells, in 2 ml of medium per well in 6 well multidishes.Cells were allowed to attach and grow for 12 h, and then the medium was replaced with medium containing nitrotyrosine in the treated groups and medium containing an equivalent amount of solvent in the untreated groups, and incubated further as per the experimental requirements.Medium was replaced with fresh medium containing nitrotyrosine or solvent every 48 h.At selected time points, supernatant media in the wells were collected and cells were trypsinized with trypsin-EDTA.Supernatant medium combined with trypsinized cells were counted for viable cells using the tryphan blue exclusion method [44].Cell counts of untreated groups were considered as 100 percent growth.The percent growth of the treated cells over untreated cells was calculated.

High throughput assay to detect α-tubulin nitrotyrosination in cells
Microplate preparation.Exponentially growing TTLL12 and TTL clones were used to seed 96-well polystyrene tissue culture plates (BD MICROTEST TM 96 Cat.No.353072).The cells were seeded at 10,000 cells in 80 μl of medium per well.TTL clones served as the positive control and TTLL12 clones served as the screening line.Cells were allowed to grow for 12 hours before treatment.Nitrotyrosine was added to each well to get a final concentration of 400 μM.Control wells were treated with the equivalent amount of solvent and DMSO 0.1% final concentration.Background control wells did not receive nitrotyrosine and signal produced by these well were considered as noise and subtracted from the nitrotyrosine signals.

Test compound addition
Each well was supplemented with 10 μl MEM containing 100 μM test compounds to get a final test concentration of 10μM in the well, immediately after the addition of nitrotyrosine.Bioinactive controls were obtained with treatment corresponding to DMSO alone.The final concentration of DMSO in the well was 0.1%.After drug dispensing, cells were incubated for 18h at 37˚C, 5% CO 2 in a humidified incubator.

Results analysis
Raw data were exported from the software of the micro plate reader to the Excel file and calculated according to the following procedure.Signals corresponding to nitrotyrosine were obtained by subtracting the noise from cells not treated with nitrotyrosine.Fold increase in nitrotyrosine incorporation by the test substance was calculated by dividing the values of the signals from treated group, by the control group.

Assay performance
To evaluate the quality of the assay, the Z' factor was calculated using the following equation according to Zhang et al., 1999 [45]: where c+ is the positive control (TTL clones), c-is the screening line (TTLL12 clones), μ is the mean of signal and σ is the SD.In our experimental conditions, the Z' factor was above 0.5, indicating that the assay is good for high-throughput screening.In the high-throughput screen, "pseudo" Z' factors were calculated for each individual screening plate, with 85% of cases yielding "pseudo" Z' factors above 0.5.Positive hits were selected on a per-plate basis, only if the compound screening value was above a specific threshold (the average of the TTLL12+nitrotyrosine value plus 2 times SD).The same TTL +/-nitrotyrosine and TTLL12 +/-nitrotyrosine controls were present in quadruplicate on each plate.All identified positive hits underwent re-screening at three doses, leading to the selection of the two promising lead compounds.

Test compound library
For the screening, a library of 10,000 compounds was selected.This World Diversity Set is a selection that makes full use of Specs' global acquisition program.Compounds from 370 different sources were included, representing 36 countries.Chemical properties for drug-likeness were considered and applied loosely in order to maintain a high chemical diversity.The set was delivered in 96-well plates as a 10 mM DMSO solution by Specs (Zoetermeer, NL).

TTLL12 inhibits nitrotyrosine incorporation into α-tubulin
We previously showed that hTTLL12 affects tyrosination of α-tubulin in cells [10], suggesting that it may also affect nitrotyrosination of α-tubulin (

Nitrotyrosine inhibits growth of transformed cells
There are contradictory reports of the effects of nitrotyrosine on cell growth [34,36].Using conditions in which TTLL12 alters nitrotyrosination, we found that nitrotyrosine inhibits the growth of all of the cell lines tested, although there are differences in sensitivity.With 400 μM nitrotyrosine, growth of A549 cells decreased starting from 96 h (Fig 3A), whereas HEp-2 cells decreased earlier, starting from 72 h, and was lower after 96 h (Fig 3B).The effect of nitrotyrosine on cell proliferation was dose-dependent, since decreasing the amount of nitrotyrosine resulted in smaller effects on cell growth (Fig 3B ) as well as lower incorporation into α-tubulin (Fig 3C ), showing that there is a correlation between decreased cell growth and nitrotyrosination.To investigate whether transformation may affect sensitivity, we compared two immortalized lines derived from prostate epithelial cells (PWR-1E and RWPE-1) with two lines derived from prostate cancer metastases (DU145 and PC3).The cells were grown in a common medium, to exclude effects of medium composition, such as tyrosine levels.The two metastasis derived lines (DU145 and PC3) were more sensitive to nitrotyrosine, suggesting that transformation and cancer progression could increase sensitivity to nitrotyrosine toxicity (Fig 3D).

TTLL12 decreases the cell toxicity of nitrotyrosine
Our observation that TTLL12 inhibits nitrotyrosination of α-tubulin suggests that it could decrease the toxicity of nitotyrosine.To test this possibility, we incubated TTLL12

Tubulin poisons under non-saturating conditions do not affect nitrotyrosination of α-tubulin
Cytotoxicity induced by microtubule polymerizing and depolymerizing agents could affect nitrotyrosine cytotoxicity by altering incorporation into α-tubulin.We investigated this possibility with the polymerizing agent, paclitaxel, and the depolymerizer, nocodazole, under non- .These results show that the cytotoxic effects of nitrotyrosine and tubulin poisons can be cumulative, and suggest that the mechanisms could be different under limiting conditions.Encouragingly, for therapeutic applications, this suggests that manipulating nitrotyrosination could be used in addition to some tubulin poisons, since it would have an additional effect.

Validation of a cell-based assay to measure increases in nitrotyrosination above TTLL12-suppressed levels
The results described above indicate that TTLL12's suppression of nitrotyrosination is a promising target for the isolation of therapeutic drugs.Consequently, we searched for compounds that could increase nitrotyrosination of α-tubulin in cells that overexpress TTLL12.We used a cell-based ELISA (C-ELISA-HRP), in which cells can be treated with test compounds in the presence of nitrotyrosine and incorporation can be measured directly.Since there are no known inhibitors of TTLL12 and no compound that can increase tubulin nitrotyrosination, we used cell lines that incorporate high or low levels of nitrotyrosine, due to the stable expression of TTL or TTLL12, respectively, to provide a suitable detection window.The assay was adapted from the protocol published by Fonrose et al. [47] and uses a HRP conjugated secondary antibody and a TMB substrate for colorimetric detection.We optimized the assay for reproducibility and linearity, in order to achieve a Z' factor (> 0.5) that is suitable for a high throughput screen.The assay effectively detected increases in nitrotyrosinated α-tubulin with increasing time of incubation with nitrotyrosine (S1A Fig) .The difference between the high and low nitrotyrosination clones was maintained at all times, with a Z' factor > 0. We adapted C-ELISA-HRP to a high throughput platform.In particular, the platform uses a time-resolved fluorescence (TRF) detector and a Europium conjugated antibody (C-ELI-SA-Eu), which provides a higher dynamic range of detection as compared to the colorimetric readout.We optimized the conditions, including antibody dilutions, automated washing steps, time of incubation and buffer conditions

Identification of hits that relieve nitrotyrosination suppression by TTLL12
A library of 10,000 compounds (SPECS TM , World Biological Diversity Set) was screened using C-ELISA-Eu on the HTS platform.In the primary screen, nitrotyrosination was increased by 74 compounds (>1.33-fold) and decreased by 16 (<0.38-fold;Fig 7A; only six of the down regulators are show).These hits were rescreened at three different concentrations (10 μM, 1 μM and 0.1 μM).Of the eight selected compounds, six were considered to be of secondary appeal because they were inactive in the secondary screens at 10 μM, or at 1 μM and 0.1 μM (Fig 7B ), whereas, interestingly, 2 compounds retained activity at 1 μM (Fig 7C).These two most promising compounds also consistently increased nitrotyrosination of α-tubulin by immunoblotting (Fig 7D ), showing that the observed increases are reproducible and specific.These results show that C-ELISA-Eu is a reliable approach for the isolation of specific activators of nitrotyrosination of α-tubulin.The reason for the apparent discrepancy of fold inductions between the

Discussion
We report a proof of principle that molecules that relieve a potential tumour promoting activity of TTLL12 can be identified, as a step towards developing TTLL12 targeted therapeutic agents (Fig 1).We show that TTLL12 has a potential tumour promoting activity, namely inhibition of the toxicity of nitrotyrosine for transformed cells.Overcoming this activity could be used for therapy, since decreasing TTLL12 levels leads to increased nitrotyrosine toxicity.Manipulating nitrotyrosination of tubulin through TTLL12 appears to be a different microtubule directed therapeutic approach compared to classical tubulin poisons, since they act independently.These findings led us to test whether it was possible to identify molecules that overcome TTLL12 inhibition of nitrotyrosination.We developed a cell-based screen that successfully identified several promising hits from a 10,000 molecule World Biological Diversity Set library.These are original results first reported in a thesis [48].
The ability of TTLL12 to inhibit nitrotyrosine ligation to detyrosinated α-tubulin is consistent with other findings.We previously showed that decreasing TTLL12 levels increased C-terminally detyrosinated α-tubulin levels, similar to reducing TTL levels.However, we were not able to detect the converse changes in C-terminally tyrosinated α-tubulin levels, possibly due to the high endogenous levels of α-tyrosinated tubulin [10].Similar observations have been made with rice TTLL12 [22].Using the tyrosine analogue, nitrotyrosine, we show that there is the expected complementary effect, namely inhibition of nitrotyrosination.Nitrotyrosine is a known substrate of TTL [36,46], suggesting that nitrotyrosination and tyrosination enter into the same pathways.The mechanism by which TTLL12 inhibits nitrotyrosination remain to be discovered, and may involve protein-protein interactions rather than a direct enzymatic activity.Despite diverse attempts, TTLL12 has not been found to catalyse the various activities predicted from its resemblance to other proteins, [8][9][10]49] suggesting that it is a pseudo-enzyme [10].TTLL12 has been reported to bind α-tubulin [10,49], indicating that it may physically hinder tyrosine/nitrotyrosine ligation or promote removal.
An intriguing possibility is that TTLL12 could act as a tumour suppressor through suppression of nitrotyrosine ligation to the C-terminus of detyrosinated α-tubulin and the consequent cell toxicity.Idriss [42,43] proposed that cells have a checkpoint that is triggered by the incorporation of nitrotyrosinated α-tubulin into microtubules, constituting a "last check point" (LCP) that triggers apoptosis.Escape from the LCP leads to cancer.We hypothesized that suppression of nitrotyrosination of α-tubulin by TTLL12 could be one of the mechanisms by which this check point is attenuated.This concords with our and other observations.Nitrotyrosine (reviews: [37][38][39][40][41][50][51][52][53]) and TTLL12 [11,[14][15][16][17][18][19] levels increase with tumour progression, suggesting that they are linked.However, the comparison is indirect, since this nitrotyrosine is mainly found in proteins as a result of chemical modification during oxidative and nitrosative stress, rather than as free nitrotyrosine that is required for ligation to the C-terminus of detyrosinated α-tubulin.The "last check-point" appears to be activatable in transformed cells, since we found that increased nitrotyrosination resulting from decreased expression of TTLL12 was toxic for several transformed cell lines.
Drugs that affect TTLL12 repression of tubulin nitrotyrosination could be redundant with classical tubulin poisons used for therapy since they both impact the tyrosination-detyrosination cycle.Tubulin poisons affect the normal dynamics and stability of microtubules and are thought to thereby affect the extent of tyrosination, since detyrosination occurs on polymerized tubulin, whilst tyrosine is added to free α-β-tubulin dimers by TTL (reviews: [31,32,54].The effects of tubulin poisons on nitrotyrosine ligation are not well known.We found that the cytotoxicities of tubulin poisons and nitrotyrosine are independent, suggesting that they act through separate mechanism and could be targeted separately for therapy. Encouraged by these results, we set out to establish if we could develop a robust assay for a high throughput screen to identify activators of TTLL12 suppressed nitrotyrosine incorporation.One of the strengths of our approach is that it is based on nitrotyrosine detection, which is a very good target for the development of assays (see for example: [15,[55][56][57]) to follow oxidative stress (review: [58]).The advantages of our optimized cell-based ELISA are its: compatibility with high throughput screening, sensitivity to detect differences in incorporation with time, capability of detecting increases in tubulin nitrotyrosination induced by inhibition of TTLL12 and usefulness to follow other modifications of tubulin.The assay has a good Z' factor, which validates it as a high-throughput screening tool.In cell-based ELISAs test compounds can be tested directly on cells in 96-well plates, whereas sandwich ELISAs require additional steps, cell lysate preparation and protein quantification of the lysates.The current assay could be influenced by indirect effects of the tested molecules on cell division and survival, as well as direct substrate competition with nitrotyrosine.
In order to adapt the assay for a 10,000-molecule screen, a more automated system was developed and optimized.In particular, it uses a time-resolved fluorescence plate reader as the detection system, which has a higher dynamic detection range as compared to the colorimetric readers.The initial assay was developed using HRP conjugated rabbit IgG which provides a colorimetric readout, which was replaced with a Europium conjugated IgG to make the assay amenable to the platform requirements and increasing the dynamic range of detection.Seven compounds were identified that reproducible increase nitrotyrosination.Two of them active when diluted and increased nitrotyrosination in an independent assay (immunoblotting).These results show that C-ELISA-Eu can be used for additional screens and the identified compounds can be further investigated.
To the best of our knowledge, this is the first screen for molecules that modulate nitrotyrosination of tubulin.The molecules from the screen could be used to study the pathways involved in the novel signalling pathway that impinge on modification of the C-terminal tyrosine of α-tubulin, and also nitrosination of tyrosines in proteins.Nitrotyrosination is a novel target for cancer therapy.The molecules from the screen may have their uses in cancer therapy, and also in other pathologies that involve this modification (inflammation, neurodegenerative, and cardiovascular disorders).described in the Materials and Methods.The Z' factor was > 0.5.(D) C-ELISA-HRP detects increased levels of glu-tubulin induced by TTLL12 knockdown.HEp-2 cells were transfected with siTTLL12 or si-luciferase and seeded in 96-well plates.C-ELISA-HRP using glu-tubulin as the readout was performed as described in the Materials and Methods.Z' factor = 0.
Fig 1).To investigate this possibility, we studied the effects of over-or under-expression of TTLL12 in HEp-2 cells on nitrotyrosination of α-tubulin.The cells were treated with 400 μM of nitrotyrosine for 48 hours, lysates were separated on SDS PAGE and nitrotyrosine was immunodetected on blots.HEp-2 cell clones that overexpress TTLL12 incorporated less nitrotyrosine, as compared to the control clones (Fig 2A & Fig 2B; two representative clones are shown).Nitrotyrosine is incorporated into αtubulin, as shown by the size of the band, the co-migration with α-tubulin detected with specific antibodies (see below and data not shown), and as expected from previous studies [34, 36, 46].Cells in which TTLL12 was downregulated with siRNAs incorporated more nitrotyrosine into α-tubulin as compared to the control siRNAs, for 5 of the 6 siTLL12s tested (Fig 2C & Fig 2D).These results show that TTLL12 inhibits nitrotyrosination of α-tubulin.

Fig 1 .
Fig 1.Schematic summary.The diagram illustrates the detyrosination-tyrosination cycle of α-tubulin, in which TCP and TTL catalyse the detyrosination and religation of tyrosine (Y) to α-tubulin.Under oxidative (Oxy.)stress nitrotyrosine (NO2-Y) is ligated to detyrosinated α-tubulin (see box) to give nitrotyrosinated α-tubulin, which has been hypothesised to lead to cell death and suppression of this pathway could lead to oncogenesis.TTTLL12 inhibits ligation of nitrotyrosine to detyrosinated α-tubulin, which is a potential oncogenic activity.Inhibition of this process by small molecule inhibitors could lead to novel therapeutic drugs.https://doi.org/10.1371/journal.pone.0296960.g001

Fig 2 .
Fig 2. TTLL12 inhibits tubulin nitrotyrosination in HEp-2 cells.(A) Nitrotyrosinated α-tubulin levels in TTLL12 over-expressing clones.The TTLL12 over-expressing clones TTLL12_B and TTLL12_D, and the control clones control_B and control clone_D, were incubated with nitrotyrosine (400 μM) for 48 h.Total lysates were analyzed by immunoblotting with anti-TTLL12, anti-nitrotyrosine and anti-TBP.(B) Densitometric quantification of western blots.Signals corresponding to nitrotyrosine (NO 2 -tub) were normalized to anti-TBP.The average values of nitrotyrosinated tubulin levels of control clones_B and D are presented as 100%.Error bars represent ±SEM of three independent experiments.The symbol * indicates p values < 0.05 calculated with the Student's t-test.(C) Nitrotyrosinated α-tubulin levels in TTLL12 depleted cells.HEp-2 cells were transfected with 10 nM siRNAs targeting TTLL12 (_1 to _6) or controls (siLuciferase, siControl, siGFP or siSilencer) and treated with nitrotyrosine for 48 h post-transfection.Total cell lysates were analyzed by immunoblotting with anti-TBP, anti-TTLL12 and antinitrotyrosine antibodies.Two blots are shown side by side; the control siRNAs are the same on both blots, whereas the siRNAs that target TTLL12 are different.(D) Densitometric quantification of western blots.Signals corresponding to nitrotyrosine were normalized to TBP.Nitrotyrosinated α-tubulin levels of siTTLL12 samples are presented as % of the average of control siRNAs.Error bars represent ± SEM of two independent experiments.The symbol * indicates p < 0.05 calculated with the Student's t-test, comparing siTTLL12 to control siRNAs.https://doi.org/10.1371/journal.pone.0296960.g002

Fig 3 .
Fig 3. Nitrotyrosine inhibits cell growth.(A & B) Cytotoxicity of nitrotyrosine.A549 (A) or HEp-2 (B) cells were cultured in normal medium (control) or in medium supplemented with nitrotyrosine (A, 400 μM; B, 25-400 μM; control cells received the HCl solvent for nitrotyrosine) for 144 h.Media were replaced with corresponding fresh media after 72 h.Viable cells were counted using the tryphan blue exclusion assay and plotted against time.Values represent cells per well and the error bars represent the ± SEM of two independent experiments.p values were calculated with the Student's t-test, comparing treated with control samples at any particular time point.The symbol * signifies a p value < 0.05.(C) Nitrotyrosinated α-tubulin levels.HEp-2 cells were cultured in medium supplemented with nitrotyrosine (25-400 μM; control cells received HCl used as solvent for nitrotyrosine) for 120 h.Cells were replenished with respective fresh mediums after 72 h.Total cell lysates were analyzed by immunoblotting with antinitrotyrosine and anti-TBP antibodies.A representative blot is shown.(D) Cytotoxicity of nitrotyrosine for prostate cell lines.DU145, PC3, PWR1E and RWPE1 cells were cultured for 168 h in a 1:1 combination of media for PC3 and RWPE1 supplemented with either 400 μM of nitrotyrosine or solvent alone.Media were changed every 48 h.Viable cells were counted using the tryphan blue exclusion assay.Values represent % growth of treated cells normalized to untreated controls.The error bars represent the ± SEM of two independent experiments.https://doi.org/10.1371/journal.pone.0296960.g003

Fig 4 .Fig 5 .
Fig 4. Stable over-expression of TTLL12 in HEp-2 cells increases cell survival in the presence of nitrotyrosine.(A) Cell survival in the presence of nitrotyrosine.HEp-2 cell clones that over-express TTLL12 (TTLL12_B and TTLL12_D) and control clones (Vector control_B and Vector control_D) were cultured for 96 h in medium supplemented with nitrotyrosine (50 μM) or the equivalent amount of solvent.Viable cells were counted using the tryphan blue exclusion assay.Values represent cells per well in the nitrotyrosine treated and untreated groups (i) or % growth of treated cells normalized to untreated controls (ii).The error bars represent the ± SEM of two independent experiments done in duplicates.p values were calculated with the Student's t-test.The symbol * indicates p values < 0.05.(B) Nitrotyrosinated α-tubulin levels.Total lysates of clones that overexpress TTLL12 and control clones treated with 50 μM nitrotyrosine for 96 h were analyzed by immunoblotting with anti-TTLL12, anti-nitrotyrosine, anti-TTL, anti-TBP, anti-α-tubulin antibodies.A representative immunoblot is shown.https://doi.org/10.1371/journal.pone.0296960.g004 5. The C-ELISA-HRP signal precisely reflected specific bands intensities on immunoblots (S1B Fig).The C-ELISA-HRP efficiently detected the increases in nitrotyrosination that are expected from downregulation of TTLL12 with siRNA (S1C Fig), as well as glu tub (S1D Fig), which could eventually be used in secondary screens.These results validate C-ELISA-HRP for use in screens to select for inhibitors of TTLL12 which increase α-tubulin nitrotyrosination.
(S2 Fig), to maintain Z' factors >0.5.Antibodies dilutions of 1:1,000 and 1:2500 were found to be optimal (S2A Fig).Automatic washing effectively replaced manual washing (S2B and S2C Fig).Times of incubation from 3 to 18 h were suitable for the detection of differences in nitrotyrosination between the high and low nitrotyrosination clones (S2D Fig).The different washing buffers (PBS or Delphia) gave similar values (S2E represent the ± SEM of two independent experiments done in duplicates, p values were calculated with the Student's ttest (* indicates p < 0.05).(D) Effect of TTLL12 depletion in DU145 on nitrotyrosinated α-tubulin levels.Using the same culture and treatment conditions as (C), total cell lysates were prepared and analyzed by immunoblotting with anti-TTLL12, anti-nitrotyrosine and anti-TBP antibodies.A representative blot is shown.https://doi.org/10.1371/journal.pone.0296960.g005and S2F Fig).These results show that the high throughput screening platform (C-ELISA-Eu) gives similar results to the manual assay (C-ELISA-HRP).

Fig 6 .Fig 7 .
Fig 6.Effect of paclitaxel and nocodazole on cell viability and α-tubulin nitrotyrosination.(A) Growth curves with paclitaxel.HEp-2 cells were treated with nitrotyrosine (100 μM), paclitaxel (100 nM), both or neither (untreated).Viable cells were counted using the tryphan blue exclusion assay and plotted as cells per well against time.The error bars represent the ± SEM of two independent experiments with duplicates in each experiment.(B) Nitrotyrosinated α-tubulin levels with paclitaxel.HEp-2 cells were treated as in A. Total cell lysates were made at the stated times and analyzed by immunoblotting with anti-nitrotyrosine, anti-α-tubulin and anti-TBP antibodies.A representative immunoblot is shown.(C) Growth curves with nocodazole.HEp-2 cells were treated with nitrotyrosine (100 μM), nocodazole (1 or 10 nM), both or neither (untreated, with the equivalent amount of solvents).Viable cells were counted using the tryphan blue exclusion assay and cells per well plotted against time.The error bars represent the ± SEM of two independent experiments with duplicates in each experiment.(D) Nitrotyrosinated α-tubulin levels with nocodazole.HEp-2 cells were treated as in A. Total cell lysates were made at stated time intervals and analyzed by immunoblotting with anti-nitrotyrosine, anti-TBP or anti α-tubulin antibodies.A typical immunoblot is shown.https://doi.org/10.1371/journal.pone.0296960.g006 of C-ELISA-HRP to the HTS platform.(A) Europium conjugated secondary antibodies can be used for the cell-based assay.C-ELISA was performed with Europium conjugated rabbit IgG at different dilutions.The error bars represent the ± SEM of three wells in one experiment (*Z' factor > 0.5).(B, C) The washing steps were done either manually (B) or with an automated wash system (C).The error bars represent the ± SEM of three wells in one experiment (* Z' factor > 0.5).(D) C-ELISA-Eu detects the increase in α-tubulin nitrotyrosination with time.The error bars represent the ± SEM of three wells in one experiment (*Z' factor > 0.5).(E, F) Washing steps with PBS can be replaced with the Delphia TM buffers used for the C_ELISA-Eu.(E) Washing steps before the addition of rabbit IgG were done with PBS, and DELPHIA TM buffer was used thereafter.(F) All the washing steps were performed with DELPHIA TM buffer.The error bars represent the ± SEM of three wells in one experiment.The y-axis represents the time-resolved fluorescence values at emission/excitation wave lengths of 340/615 nm.The symbol * indicates Z' factors > 0.5.(TIF) S3 Fig. Structures of the two most promising compounds.(TIF)