Discoidin, CUB, and LCCL Domain-containing (DCBLD) 2 is a neuropilin-like transmembrane scaffolding receptor with known and anticipated roles in vascular remodeling and neuronal positioning. DCBLD2 is also upregulated in several cancers and can drive glioblastomas downstream of activated Epidermal Growth Factor Receptor. While a few studies have shown either a positive or negative role for DCBLD2 in regulating growth factor receptor signaling, little is known about the conserved signaling features of DCBLD family members that drive their molecular activities. We previously identified DCBLD2 tyrosine phosphorylation sites in intracellular YxxP motifs that are required for the phosphorylation-dependent binding of the signaling adaptors CRK and CRKL (CT10 regulator of kinase and CRK-Like). These intracellular YxxP motifs are highly conserved across vertebrates and between DCBLD family members. Here, we demonstrate that, as for DCBLD2, DCBLD1 YxxP motifs are required for CRKL-SH2 binding. We report Src family kinases (SFKs) and Abl differentially promote the interaction between the CRKL-SH2 domain and DCBLD1 and DCBLD2, and while SFKs and Abl each promotes DCBLD1 and DCBLD2 binding to the CRKL-SH2 domain, the effect of Abl is more pronounced for DCBLD1. Using high performance liquid chromatography coupled with tandem mass spectrometry, we quantified phosphorylation at several YxxP sites in DCBLD1 and DCBLD2, mapping site-specific preferences for SFKs and Abl. Together these data provide a platform to decipher the signaling mechanisms by which these novel receptors drive their biological activities. Proper neurodevelopment requires precise temporal and spatial regulation of a complex array of signaling molecules, the regulation of which remains largely uncharacterized. CT10 regulator of kinase (CRK) and CRK-like (CRKL) are ubiquitously expressed intracellular signaling adaptors critical to neuronal positioning in the embryonic brain, as well as to many fundamental cellular processes such as proliferation, differentiation, apoptosis, and focal adhesion dynamics . CRK andDiscoidin, CUB, and LCCL domain-containing 2  as a novel phosphotyrosine-dependent CRKL-SH2 binding partner. DCBLD2 possesses a similar ectodomain structure to that of neuropilins, critical co-receptors for guidance cues in neuronal pathfinding. The seven YxxP motifs residing within the intracellular sequence of DCBLD2 are highly conserved among vertebrates, and are essential for the phosphorylation-dependent binding of DCBLD2 to the CRKL SH2 domain. Furthermore, it was determined that SFKs were sufficient for, but not the only tyrosine kinases capable of, inducing the interaction of DCBLD2 with CRKL. Our initial study also identified four specific sites on DCBLD2 of regulated tyrosine phosphorylation, three of which were in YxxP motifs [Previously, we reported a proteomics screen for novel Src Family Kinase (SFK) substrates that, when phosphorylated, would bind to the CRKL SH2 domain . This scP motifs .DCBLD2 has been reported to be involved in vasculature remodeling –10 and iIn the mechanisms described above, the roles of DCBLD2 tyrosine phosphorylation were not explored. However, in the case of oncogenic EGFR signaling, DCBLD2 was shown to be phosphorylated at Tyr750 thereby activating a TRAF6-Akt pathway , and bothomo sapiens , mus musculus , rattus norvegicus , danio rerio (Dr F1QGI1); DCBLD2 Hs (Q96PD2), Mm (Q91ZV3), Rn (Q91ZV2), Dr (B0S5V9).Multiple sequence alignments were constructed with the UniProt Align tool . Conservation of intracellular amino acids and YxxP motifs was examined across representative model vertebrates: DCBLD1 13C6, 15N4) and L-lysine  were obtained from Cambridge Isotope Laboratories Inc. , and unlabeled L-arginine  and L-lysine  were purchased from MP Biomedicals . The ProFection® Mammalian Transfection System kit for calcium phosphate transfections and the trypsin used in enzymatic digests prior to LC-MS/MS analysis were from Promega . Calf Intestinal Phosphatase (CIP) was obtained from New England Biolabs . The SFK inhibitor Src-1 was purchased from EMD-Calbiochem  and Imatinib (STI571), the Abl-specific inhibitor, was acquired from Selleckchem . The BSA standard for Bradford assays and the Bradford Reagent were obtained from Amresco Life Sciences, LLC. . For the development of Western blots, enhanced chemiluminescence (ECL) reagents were purchased from Pierce , and x-ray film was from Denville scientific . Synthetic stable isotope-labeled peptide standards (SL peptides) were from Cell Signaling Technologies . Packing material used for HPLC was 5 μm C18-coated silica beads, 200 Å pore size, purchased from Michrom Bioresources Inc. . Nitrocellulose membranes were from GVS Life Sciences . All additional reagents were purchased from Sigma  unless otherwise noted.Penicillin/Streptomycin 100X solution and Dulbecco’s Modified Eagle Medium (DMEM) were obtained from Mediatech . SILAC media and kanamycin sulfate were acquired from Thermo Scientific , and fetal bovine serum (FBS), dialyzed FBS for SILAC experiments, and cosmic calf serum (CCS) were purchased from Hyclone . Heavy labeled L-arginine  and DCBLD1 (Mm) in pCMV6-Entry, tagged with Flag and Myc sequences at C-termini, were obtained from Origene . Wild-type (WT) FYN . DCBLD2 The mouse α-Flag (M2) antibody (Ab) and Affinity Gel were from Sigma and the free Ab was used for Western blotting at a concentration of 0.5 μg/mL. Cell Signaling Technologies Inc.  was the source of the following antibodies, used at the indicated dilution or concentration: α-Flag , α-Fyn , α-Src , α-pTyr416-Src , α-pTyr412-Abl , α-alpha-tubulin (1:1000), and α-pan-actin (0.1 μg/mL). The rabbit α-Abl antibody  was from Santa Cruz Biotechnology . The α-phosphotyrosine  was from EMD Millipore  and the α-Myc  was obtained from American Type Tissue Collection . All primary antibodies were diluted in 1.5% BSA in Tris-Buffered Saline  with 0.05% Tween 20 (TBS-T) and containing 0.005% sodium azide. Horseradish peroxidase (HRP)-conjugated secondary antibodies were obtained from EMD Millipore and used at the following concentrations: goat α-mouse IgG-HRP , light-chain-specific goat α-mouse IgG-HRP  and goat α-rabbit IgG-HRP . All secondary antibodies were diluted in TBS-T.2. For experiments involving stable isotope labeling of amino acids in cell culture (SILAC), HEK 293 cells were cultured in labeled (heavy) or unlabeled (light) growth medium for at least one week prior to transfection to ensure full incorporation of stable isotopes into proteins. SILAC media, lacking L-lysine and L-arginine were supplemented with 10% dialyzed FBS and antibiotics as stated above, 60 mg/L unlabeled L-proline, 100 mg/L of L-lysine either unlabeled or labeled , and 100 mg/L of L-arginine either unlabeled or labeled .Adenovirus early region-transformed HEK 293 cells were cultured in DMEM supplemented with 5% each of FBS and CCS, 50 U/mL penicillin, and 50 μg/mL streptomycin at 37 °C in 5% atmospheric CO2O2 . Cells were placed on ice immediately following H2O2 stimulation and washed with PBS (4 °C) prior to lysis in Brain Complex Lysis Buffer , 10 μg/mL each leupeptin and pepstatin-A). Lysates were centrifuged and the supernatant was reserved and stored at −20 °C for further analysis.HEK 293 cells were grown to 60% of confluence prior to transfection via calcium phosphate precipitation. The following amount of plasmid was transfected per 10 cm dish: wild type and mutant DCBLD1 and DCBLD2 (6 μg), wild type FYN (1.5 μg), kinase-dead FYN (2.5 μg), and wild type c-ABL (2 μg). Six hours post-transfection, cells were washed with phosphate-buffered saline (PBS) and returned to full medium overnight before lysis. Cells treated with inhibitors received 2 μM Src-1 and/or 20 μM STI571 in dimethyl sulfoxide (DMSO) for 15 min at standard culture conditions, prior to stimulation with 8.8 mM H3 μg total protein in 750 μL) were incubated with α-Flag Affinity Gel (10 μL of a 50 % slurry in BCLB) overnight, rocking at 4 °C. The beads were washed four times with BCLB, after which bound proteins were eluted and denatured in 25 μL sample buffer  at 95 °C for 5 min. For Western blotting of whole cell lysates, samples were denatured in sample buffer and 15 μg total protein was loaded per lane. Immunoprecipitations, pull-downs, whole cell lysates, and phosphatase assays were separated on 10% acrylamide gels (30% w/v and 37.5:1 acrylamide:bis-acrylamide) with 4.2% acrylamide stacking gels. Current was maintained at 20 mA and 30 mA per gel through the stacking and separating layers, respectively. Following separation, proteins were either transferred to a nitrocellulose membrane for Western blotting or stained with Coomassie for mass spectrometric analysis. Transfers to nitrocellulose membranes were in a submersible transfer unit at 4 °C (400 mA for 2 hours) in 1.13% glycine, 0.25% Tris-base and 20% methanol. Membranes were stained with a reversible Ponceau stain (0.5% Ponceau and 1% acetic acid in H2O) to assess total protein levels and were then blocked with 5% non-fat dry milk in TBS-T. Primary antibody solutions were incubated overnight at 4 °C. Membranes were then washed three times with TBS-T and incubated with HRP-conjugated secondary antibody solution for three hours at 25 °C. After three final washes in TBS-T, membranes were briefly incubated in ECL reagents and exposed to x-ray filmProtein concentration was determined using an Eppendorf BioPhotometer Plus  with BSA standards. For immunoprecipitations, normalized lysates  with or without 2.5 units of CIP, after which all samples were incubated at 37 °C on a dry block for 3 hours. Proteins were eluted and denatured at 95 °C after the addition of 4x protein sample buffer. Denatured proteins were prior subjected to SDS-PAGE and Western blotting.Anti-Flag resin was added to cell extracts containing 1 mg of protein in 500 μL BCLB as described above. After washing three times with BCLB and once with PBS, the resin from each immunoprecipitation was divided equally into two separate tubes and drained. Resins were resuspended in 50 μL Calf Intestinal Phosphatase (CIP) Buffer  to 1 mM, followed by an additional 4 hours of incubation. Cultures were then centrifuged at 4 °C . The supernatants were decanted and the pellets were stored at −20 °C. Pellets were resuspended in 10 mL Bacterial Lysis Buffer . Cells were sonicated in six 30 sec intervals, intermitted with equal rest periods on ice, after which 1 mL of 10% Triton X-100 was added. Lysates were mixed and centrifuged at 4 °C  and pellets were discarded. Glutathione resin (300 μL of a 50% slurry in BLB) was added to the GST-CRKL-SH2-enriched supernatant and rocked overnight at 4 °C. Beads were washed three times in BLB, once in BCLB, and twice in PBS and then stored in PBS at 4 °C. For pull-down assays, HEK 293 cell lysates were rocked with 15 μL GST-CRKL-SH2-conjugated glutathione beads (30% slurry in PBS) at 4 °C. Beads were washed three times with BCLB and proteins were eluted and denatured in 25 μL of sample buffer at 95 °C for 5 min prior to analysis via SDS-PAGE and Western blotting. GST-ABL-SH2-conjugated beads were obtained using the protocol outlined above, after which the purified protein was eluted with 10 mM glutathione.To generate GST-CRKL-SH2-conjugated glutathione beads, 50 mL cultures of Luna Broth  containing 50 μg/mL ampicillin were inoculated with 2O and then de-stained in 50 mM ammonium bicarbonate (NH4HCO3) and 50% acetonitrile (MeCN) at 37 °C for 30 minutes. De-stain was then removed and gel pieces were dehydrated in 100% MeCN. On ice, dried gel pieces were rehydrated in 25 μL of 12 ng/μL sequencing grade modified trypsin in 50 mM NH4HCO3. After a 20-minute incubation at 4 °C, an additional 25–50 μL of 50 mM NH4HCO3 were added to submerge gel pieces, followed by overnight incubation at 37 °C. After centrifugation, supernatants were transferred to new microcentrifuge tubes. Remaining peptides were extracted from gel pieces with the addition of 50% MeCN, 2.5% formic acid (FA). Supernatants were combined with the initial tryptic digest supernatants, and gel pieces were dehydrated in 100% MeCN. The final extraction was combined with the previous two extractions and peptides were dried in a speed-vac.Bands containing immunoprecipitated proteins of interest were excised from Coomassie-stained acrylamide gels, diced to 1 mm cubes and transferred to separate microcentrifuge tubes. Gel pieces were washed with HPLC-grade H4, scan speed=1 Hz) mass spectrometer fitted with a Finnigan Surveyor Pump Plus and Micro AS autosampler  and controlled with Xcalibur™ 2.1 Software . Following a 15 min loading phase (flow rate=100 μL/min) onto the C18 column in Solvent A, peptides were eluted using a 0–50% gradient of Solvent B  over 38 min and electrosprayed (2.1 kV) into the mass spectrometer. This gradient was followed by 7 min at 100% Solvent B before a 10 min equilibration in 100% Solvent A.Peptides were resuspended in 2.5% MeCN/0.15% FA (Solvent A) and separated on a reverse-phase HPLC column (length=12 cm × 100 μm) packed in house with 5 μm C18 beads (pore size=200 Å) prior to analysis via a linear ion trap-orbitrap  was followed by ten low energy collision-induced dissociation (CID) tandem mass spectra. CID spectra were acquired for the top two most abundant ions in the precursor scan  followed by eight targeted scans for DCBLD1 or DCBLD2 peptides of interest , oxidation of methionine (+15.9949 Da), and acrylamidation of cysteine (+71.0371 Da). Mass additions in experiments employing isotopically labeled residues were as follows: heavy lysine (+8.0142 Da) and arginine (+10.0083 Da) in SILAC analyses, and heavy leucine (+7.0172 Da) and valine (+6.0138 Da) when using SL peptides.2O2-stimulation and paired with heavy cultures subjected to H2O2-stimulation alone. Heavy and light samples were immunoprecipitated separately and eluted proteins were combined as heavy and light pairs before SDS-PAGE.Peptides were manually quantified in Xcalibur using the precursor scan monoisotopic peak intensities averaged across full width at half maximum (FWHM) of the elution window. In the case of SILAC and SL peptides, the heavy-to-light ratio (H:L) or L:H of monoisotopic peak intensities, for SILAC or SL quantification respectively, were determined. Ratios were normalized to reference peptides of DCBLD1 or DCBLD2 that were not found modified . For SILa) mock, b) DCBLD(X), c) DCBLD(X) with H2O2, d) DCBLD(X) with WT FYN or Src-1/H2O2, e) DCBLD(X) with c-ABL or STI571/H2O2, f) DCBLD(X) with WT FYN/c-ABL or Src-1/STI571/H2O2. Approximately 300 fmol of each SL peptide (m/z tabulated in Each set of SL peptides or label-free (LF) experiments had six conditions with three replicates for each condition as follows:  peptide  were spi peptide . In labe peptide . Referen2O2, kinases, or inhibitors, normalized L:H (SL peptides) or LF intensities were divided by the condition in which we predicted to find to the maximum signal per peptide. It was predicted that the maximum number of unphosphorylated peptides would be found in the unstimulated condition, and that of phosphorylated peptides would be observed in the co-expression of Fyn and c-Abl with DCBLD(X) in kinase expression experiments, or in the H2O2-stimulated condition in the case of inhibitor treatments. Changes in the number of observed ions across conditions were reported as a percent relative to the condition that would yield the predicted maximum. The percent change relative to the treatment deemed the predicted max was determined as described above. Two and three trials were conducted for each condition with DCBLD1 and DCBLD2, respectively.To quantify changes in phosphorylation in the presence of HP < 0.05) was used to compare each experimental condition to the others. Methods of SL peptides and LF quantification for a given peptide and experimental condition were compared using a t-test (P < 0.05), with the exception of Tyr565. Four distinct methods of quantification were considered for the peptide harboring Tyr565 due to alternative cleavage patterns and the use of both SL and LF methods, necessitating the use of a Tukey-HSD (P < 0.05) analysis. The four methods used to quantify phosphorylation at this site were compared using a one-way ANOVA and Tukey-HSD (P < 0.05). Statistical analyses were performed in JMP Pro 12 Statistical Software .Quantified changes in phosphorylation state among experimental conditions were analyzed for significance with a one-way ANOVA test. The Tukey-HSD  of the extracts were incubated with or without calf intestinal phosphatase (CIP). Regulated tyrosine phosphorylation on DCBLD1 and DCBLD2 was demonstrated following immunoblotting for phosphotyrosine (2O2-stimulated tyrosine phosphorylation observed in the WCE was lost when CIP was incubated with the immune complexes. We next tested if H2O2-induced tyrosine phosphorylation on DCBLD1 would render it capable of binding to the CRKL-SH2 domain. A GST-CRKL-SH2 pull-down (PD) was performed with lysates from untreated and H2O2-stimulated cells expressing either DCBLD1 or DCBLD2. Both paralogs showed H2O2-induced binding to the CRKL-SH2 domain . WT DCBLD1, but not DCBLD1 Y8F exhibited regulated binding to the CRKL-SH2 domain in response to Hmulation . These rOur previous characterization of the DCBLD2-CRKL interaction demonstrated that co-expression of the SFK Fyn was sufficient to induce DCBLD2 to bind to the CRKL-SH2 domain . This le2O2 stimulation to investigate whether the presence of SFK or Abl inhibitors counteracted the effect of tyrosine phosphatase inhibition on the pull-down assay. Appropriate inhibitor concentrations were determined from a titration in which endogenous SFK and Abl activities were monitored after treatments of increasing concentrations of Src-1 and STI571, individually  demonstrate high specificity for tyrosine residues in YxxP motifs , 18. Thevidually . This wavidually . Sanguiny  and STI571 (20 μM) treatment on the binding of CRKL-SH2 to DCBLD1 and DCBLD2 . Src-1 seraction , the comeraction .2O2-induced interaction between the CRKL-SH2 domain and DCBLD proteins, Abl co-expression with DCBLD1 and DCBLD2 was investigated to determine sufficiency while using Fyn as a positive control as established in 2O2 stimulation or Abl in inducing DCBLD1 and DCBLD2 to bind to CRKL-SH2. Interestingly, co-expression of DCBLD proteins with Abl significantly reduced the mobility of both DCBLD family members in SDS-PAGE. This could be the result of dramatically higher levels of phosphorylation on DCBLD proteins generally, Abl-specific phosphorylation events, or additional Abl-specific modifications. Fyn and Abl both can drive DCBLD family members to bind to the CRKL-SH2 domain. However, these kinases demonstrate preferences with the binding of DCBLD1 more strongly affected by Abl, suggesting that SFKs and Abl differentially target DCBLD protein YxxP sites.Upon establishing that activities of endogenous SFKs and Abl are necessary for the HIn order to identify sites that may be differentially regulated by SFKs and Abl, we immunoprecipitated DCBLD1 or DCBLD2 from HEK 293 cell lysates under kinase active or inhibited conditions and monitored the phosphorylation of several of the eight DCBLD1 and seven DCBLD2 intracellular YxxP sites  using taSILAC was used as an initial survey of differential phosphorylation . HEK 2932O2-stimulation, (ii) Fyn co-expression, (iii) Abl co-expression, or (iv) Fyn and Abl co-expression) we detected phosphorylation of Tyr589, Tyr600, and Tyr621 in YxxP sites on DCBLD1 and all were dominantly phosphorylated by Abl. When Abl was co-expressed with DCBLD1 alone or in combination with Fyn, phosphorylation of all three tyrosines were induced between 16 and over 100 fold. None of these sites was observed phosphorylated by Fyn co-expression alone  Hon alone . H2O2-stf Tyr600 . Notablyf Tyr600 . For phof Tyr600 . Furtherf Tyr600 .2O2 stimulation  quantification of monoisotopic ion intensities for more rapid replication. This was accompanied by the use of synthetic stable isotope-labeled peptide standards (SL peptides) corresponding to select DCBLD2 tryptic peptides. The chosen peptides harbored phosphorylated or unphosphorylated Tyr565, Tyr715, and Tyr750. We selected these peptides given that, on the one hand tryptic peptides containing phosphorylated Tyr715 and Tyr750 were readily detected, and on the other hand the tryptic peptide containing phosphorylated Tyr565 was more challenging to detect, due in part to the RKKK sequence creating a “ragged” N-terminal side of the tryptic peptide. The SL peptides that were chosen harboring Tyr565 had a single missed cleavage, KTEGTYDLPWDR, as this peptide would likely be a fraction of the fully-tryptic peptide and we found it in a previous study [2O2 stimulation when inhibitors were used. Unphosphorylated peptides (blue) were normalized to the unstimulated condition regardless of treatment. Y-axes for unphosphorylated peptides were inverted to best visualize the shift in phosphorylation state of the given peptide. Clusters of bars represent different quantification methods for a given condition. Significant differences between conditions were determined by a one-way ANOVA and Tukey HSD, and are either indicated  or are tabulated in P < 0.05). Additional DCBLD2 phospho-tyrosine residues that were identified, but not quantified, are listed in 2O2 stimulation. Further, application of Src-1 and STI571 together reduced the phosphorylation state beyond that of STI571 alone, suggesting that both Fyn and Abl are involved in the phosphorylation of Tyr715. Tyr750 . In the simple identification or verification of site-specific PTMs that are present in low abundances, standard addition would present an advantage over SILAC or LF methods. However, in the quantification of multiple sites across a variety of conditions as described here, SL peptides can artificially inflate native-to-standard ratios in cases where no modified peptide is identified. For such quantitative comparisons, the LF method is advantageous, as it provides the more accurate representation as minimal-to-no signal above the level of noise. Overall, LF and SL quantification yielded similar results, demonstrating the merits of LF quantification. In low-complexity samples, such as peptides derived from an immunoprecipitated band as used here, the known challenges of ion suppression in LF quantification  are miniAdditional merits of LF quantification over SL peptides include the ability to quantify different enzymatic cleavage patterns. Using the quantification of DCBLD2 pTyr565 as an example, the summed raw intensities of KTEGTYDLPYWDR and TEGTYDLPYWDR were not statistically different than the most abundant fully cleaved peptide. This demonstrates that quantification of the most abundant cleavage pattern will generally yield an accurate measurement in cases where the PTM falls far from cleavage sites. However, PTMs close to the enzyme cleavage site can reduce the specificity of the enzyme for that site, as was observed with the tryptic cleavage of DCBLD1 Tyr621, and it may be necessary to sum the intensities of fully cleaved and miss-cleaved peptides. Using LF methods in such cases allows the investigator flexibility in quantification post-LC-MS/MS. These findings demonstrate the importance in choice of quantification method, and the need to consider the peptide harboring the PTM when determining which method is most appropriate.2O2 stimulation in DCBLD2-transfected cells, endogenous Abl activity was maintained above that in untransfected cells stimulated with H2O2 by a possible binding interaction between the Abl-SH2 domain and at least one of the DCBLD2 YxxP motifs. This activity was reduced to baseline levels observed in the mock when all seven YxxP sites were mutated to FxxP (Future work will characterize whether phosphorylation of any individual tyrosine residue reported here is dependent upon phosphorylation of other sites. As we observed with H to FxxP . Others Our working DCBLD signaling model is summarized in While the biological roles of DCBLD family tyrosine phosphorylation sites remain to be elucidated, their strong conservation across vertebrates implies they mediate important signaling events. Our characterization here of the overlapping roles of SFKs and Abl in DCBLD family phosphorylation and how this multisite phosphorylation induces CRKL binding provides a framework for further cellular biochemical studies of this new receptor family.


Arabidopsis thaliana, the lncRNA COOLAIR is transcribed antisense from FLOWERING LOCUS C (FLC) in response to cold. Despite relatively low sequence similarity, the COOLAIR expression pattern and in vitro RNA secondary structure are highly conserved across the family Brassicaceae, which originated some 50 mya. It is unclear, however, whether COOLAIR functions in distantly related species such as monocots, which diverged some 150 mya. Here, we identified antisense lncRNAs from FLC homologs in various monocot species that share no sequence similarity with A. thaliana COOLAIR. Yet similar to COOLAIR, we found that BdODDSOC1 antisense (BdCOOLAIR1) and BdODDSOC2 antisense (BdCOOLAIR2) are induced by cold in a Brachypodium distachyon winter accession. Across B. distachyon accessions, the sequences of BdCOOLAIR1 and BdCOOLAIR2 are less conserved than exons but more conserved than flanking regions, suggesting a function for the transcript itself. Knock down of the BdODDSOC2 non-overlapping BdCOOLAIR2 transcript did not show a morphological phenotype, but did result in significantly higher BdODDSOC2 expression during cold, indicating that BdCOOLAIR2 performs a role in cis in the rate of BdODDSOC2 silencing. This functional similarity between eudicot and monocot species reveals ancient conservation or convergent evolution of FLC antisense transcription. Either scenario supports its functional importance.Functional conservation of RNAs between different species is a key argument for their importance. While few long non-coding RNAs are conserved at the sequence level, many long non-coding RNAs have been identified that only share a position relative to other genes. It remains largely unknown whether and how these lncRNAs are conserved beyond their position. In  XIST (HOTAIR (TUNA (cyrano (Thousands of long non-coding RNAs (lncRNAs) have been identified and new lncRNAs are continuously being discovered. Yet, the functional importance of these transcripts remains debated. One reason is that lncRNAs evolve much faster than protein coding genes, which has been observed frequently by comparing genome and transcriptome sequences between various species . While sXIST , HOTAIR   in Arabidopsis thaliana , while repressed by FCA. In the vernalization pathway, FLC expression is reduced by prolonged cold and the locus is epigenetically silenced. COOLAIR is involved in both of these pathways. In the autonomous pathway, FCA, FY, CstF64, and CstF77 promote the use of the proximal splice site of COOLAIR to represses FLC through an FLD-dependent reduction in H3K4me2 . For qPCR, root trainers were putted in 8 h/16 h light/dark at 23°C for 3 weeks, then 8 h/16 h light/dark at 4°C for 2 weeks, and finally transferred to 8 h/16 h light/dark at 23°C for 1 week. For phenotyping, root trainers were placed in 8 h/16 h light/dark at 23°C for 3 weeks, then transferred to 8 h/16 h light/dark at 4°C for 2 weeks (Bd21-3) and 6 weeks (BdTR3C), respectively, then transferred to 20h/4h light/dark at 23–25°C until flowering.RNA extraction was performed following standard TRIsure protocol. DNA was removed using TURBO DNA free kit (Ambion-Applied), first single strand cDNA was synthesized by SensiFast cDNA Synthesis Kit of Bioline (GC Biotech BIO-65054). qPCR was performed with SensiFast SYBR Hi-Rox Kit (GC Biotech: BIO-92020), at least three biological replicates and two technical replicates were used for each time point.Brachypodium distachyon pan-genome sequences were downloaded from the COGE database. Alignment was performed by CLC sequence viewer 7.7, with gap open cost 10.0, gap extension cost 1.0. DNA polymorphisms were calculated by DNASP, sites with alignment gaps were excluded. The figure was drawn with a 50-sites sliding window and 25 sites step size.BdCOOLAIR2, around 200 bp of BdCOOLAIR2 was first cloned into a pENTR2B vector (Invitrogen), with BamHI (Promega) and EcoRV (Promega) restriction enzymes. Then, the BdCOOLAR2 sequence was inserted into a binary destination vector pIPK007 with the Zea mays UBIQUITIN promoter (TM LR ClonaseTM Enzyme mix (Invitrogen). The primers for BdCOOLAIR2 cloning are as follows: BdCOOLAIR2_RNAi_F: TGGGTC GGATCCGTCCGGAGGCACACAAAT and BdCOOLAIR2_RNAi_R: CAAACTGATATCGGGACCTGAAGAACACGAGA. B. distachyon transformation was performed according to the Agrobacterium-mediated transformation protocol  behind the stop codon of BdODDSOC1 and BdODDSOC2, based on the RNA-seq and EST data in the phytozome browser  has high confidence with a full-length of 2581 bp. For BdODDSOC2, there are 2 annotated antisense ESTs, both with high confidence. The length of those is 475 bp (Bradi2g59186.1) and 372 bp (Bradi2g59186.2), respectively. The coding potential for these three antisense transcripts is very low, –1.130 (Bradi2g59124.5), -1.034 (Bradi2g59186.1) and –1.023 (Bradi2g59186.2), further suggesting that they could function as non-coding transcripts.To investigate whether there are non-coding RNAs generated from  browser   and reaches a maximal level after 3 days of cold. To investigate whether BdCOOLAIR2 is specifically induced by cold, we compared its expression to non-cold treatment control conditions. Indeed, without cold, BdODDSOC2 antisense expression is not induced  after 1 day  of 0.008. The intronic and gene flanking region NDV is 0.0209 and 0.0217, respectively. The antisense value (0.009) is higher than the value for exons, but lower than introns and flanking regions. Thus, in BdODDSOC2, the antisense region evolved slightly faster than exons but much more slowly than regions under more neutral selection (introns and flanking regions). This suggests a function for the RNA transcript and not just for the act of transcription  evolved faster than exons (NDV is 0.0035) and introns (NDV is 0.0045), but more slowly than flanking regions (NDV is 0.015). Therefore, we wanted to test whether BdCOOLAIR2 performs a function at the transcript level rather than testing whether the action of transcription is important.cription . BdODDSOBdCOOLAIR2 does not overlap with BdODDSOC2  = 4.3258, p < 0.001, BdCOOLAIR2 reached its maximum expression level, supporting a role for BdCOOLAIR2 in regulating BdODDSOC2 during cold treatment. Although BdODDSOC2 is still repressed in these RNAi lines, the silencing rate is slower compared with control lines . This further confirms the antagonistic relationship between BdCOOLAIR2 and BdODDSOC2.To further test the antagonistic relationship between COOLAIR2 , we inveCOOLAIR2 . We obseways low . This suways low . We founCOOLAIR and its structure is conserved across closely related Brassicaceae  has evolved rapidly in Brassicaceae (COLDAIR has not been found beyond A. thaliana. We found some transcripts based on RNA-Seq, EST and our RT-PCR data. These transcripts show a similar expression pattern as sense coding transcripts, and they are downregulated during prolonged cold. We propose that these transcripts might be unspliced sense FLC transcripts. However, we cannot exclude the existence of non-coding transcripts in the introns. The reason might be that we did not use suitable accessions or conditions that would induce lncRNAs expression.We tried to identify sense intronic lncRNAs, but we did not reach a conclusion. The VRE region (promoter of sicaceae , with veA. thaliana, the sequence of COOLAIR overlaps with FLC, which makes the use of RNAi limited to understand its function. Therefore, FLC terminator/COOLAIR promotor exchange lines were created to truncate the expression of COOLAIR (B. distachyon, BdCOOLAIR2 does not overlap with BdODDSOC2 and we generated RNAi lines specifically targeting the antisense lncRNA. In no-cold and post-cold conditions, we did not see a clear effect on BdODDSOC2, however at 3 days of cold, BdODDSOC2 expression is higher in antisense knockdown lines compared with controls. Thus, we demonstrated that BdCOOLAIR2 functions in cis, and at the RNA level. BdCOOLAIR2 knock down does not affect the overall expression of BdODDSOC2 but slows down the repression process. This effect apparently was not strong enough to affect flowering time. However, considering the slow repression of FLC caused by splicing of the distally polyadenylated COOLAIR in the slowly vernalizing accession Var2-6 (COOLAIR works more importantly in natural conditions in which temperature and other factors fluctuate widely (B. distachyon.In  COOLAIR . In the n Var2-6 , it has e widely . It woulBdODDSOC1 and BdODDSOC2 introns. FJ and KG wrote the manuscript.KG and FJ designed the experiments. FJ, KP, and MM carried out the experiments and analyzed the data. ND designed the primers for The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Clozapine is an antipsychotic agent prescribed to psychotic patients exhibiting tolerance and/or resistance to the conventional antipsychotic medications that mainly drive monoamine antagonism. As the pharmacological fundamentals of its unique antipsychotic profile have been unrevealed, here, we attempted to obtain hints at this question. Here, we found that clozapine directly acts on ErbB kinases to downregulate epidermal growth factor (EGF)/neuregulin signaling. In cultured cell lines and cortical neurons, EGF-triggered ErbB1 phosphorylation was diminished by 30 μM clozapine, but not haloperidol, risperidone, or olanzapine. The neuregulin-1-triggered ErbB4 phosphorylation was attenuated by 10 μM clozapine and 30 μM haloperidol. We assumed that clozapine may directly interact with the ErbB tyrosine kinases and affect their enzyme activity. To test this assumption, we performed in vitro kinase assays using recombinant truncated ErbB kinases. Clozapine (3–30 μM) significantly decreased the enzyme activity of the truncated ErbB1, B2, and B4 kinases. Acute in vivo administration of clozapine (20 mg/kg) to adult rats significantly suppressed the basal phosphorylation levels of ErbB4 in the brain, although we failed to detect effects on basal ErbB1 phosphorylation. Altogether with the previous findings that quinazoline inhibitors for ErbB kinases harbor antipsychotic potential in animal models for schizophrenia, our present observations suggest the possibility that the micromolar concentrations of clozapine can attenuate the activity of ErbB receptor kinases, which might illustrate a part of its unique antipsychotic psychopharmacology. Clozapine exhibits a unique antipsychotic profile represented by its pharmacological efficacy on the negative symptoms and reduced extra pyramidal effects3. In contrast, typical antipsychotic agents such as haloperidol, fluphenazine, and phenothiazines derivatives were developed to block dopamine neurotransmission but produce extra pyramidal effects and weight gain5. The discovery of clozapine has led to the development of atypical antipsychotic drugs such as risperidone, olanzapine, and quetiapine7. Unlike other atypical antipsychotic agents, clozapine exhibits the unique antipsychotic pharmacology of suicide prevention and is applicable to the patients exhibiting tolerance to the conventional antipsychotic drugs8. In spite of these favorable effects of clozapine, the prescription of clozapine is highly regulated owing to severe side effects such as agranulocytosis and myocarditis12. The unconventional characteristics of clozapine are presumably attributed to neither dopamine or serotonin antagonism and thus, the molecular mechanisms underlying this unique pharmacology remain to be characterized15.Clozapine was synthesized in 1958 as a new generation antipsychotic drug18. For example, chlorpromazine and thioridazine produce exocytotoxic influences on various cancer cells, recruiting antiproliferative signaling or autophagy cascades20. Aripiprazole is reported to exert anti-cancer actions, inhibiting Src kinase and PI3 kinase21. However, the kinase-inhibiting activities of antipsychotic compounds appear to be controversial especially in in vivo conditions. Haloperidol and clozapine act on neurons to induce neurite extension, stimulating Ras-MAP kinase signaling24. Treatment with several antipsychotics results in the increases in Akt signaling in the brain28. These reports propose the possibility that several antipsychotic compounds such as clozapine carry unrevealed biological activities on various protein kinases. However, it is poorly understood whether these novel activities of antipsychotic agents can be ascribed to their monoaminergic function or novel chemical actions not involving neurotransmission29.Several antipsychotic compounds including chlorpromazine, aripiprazole, and clozapine possess biological activities that cannot be illustrated by their anti-monoaminergic actions31. According to this hypothesis, we previously tested the antipsychotic potential of various kinase inhibitors for epidermal growth factor receptor family (ErbB) and found that these kinase inhibitors can ameliorate the behavioral deficits of several animal models for schizophrenia34. The present experiments were designed on the basis of the hypothesis that ErbB kinases might be potential molecular targets of clozapine.The upregulation of epidermal growth factor (EGF) and/or neuregulin signaling has been implicated in the neuropathology of schizophrenia, as well as in its animal modeling31. In particular, the use of the non-neuronal cancer cell lines that lack neurotransmitter synthesis and release allowed us to minimize the anti-monoaminergic actions of antipsychotic drugs in our assays. Among many antipsychotic drugs, we selected olanzapine, haloperidol, and risperidone as control compounds, all of which have been well characterized with respect to their receptor pharmacology and neurobiology. Based on the obtained results in culture and in vitro, we attempted to illustrate the unique antipsychotic actions and the side effects of clozapine on schizophrenic patients.Employing cancer cells as well as primary cultured neurons, we compared the effects of various antipsychotic compounds on cell growth/survival and explored the molecular targets of clozapine, focusing on their actions on EGF/neuregulin receptor kinases The following four antipsychotic compounds were used: olanzapine and haloperidol , clozapine , and risperidone . All antipsychotic compounds except risperidone were dissolved in dimethyl sulfoxide (DMSO). Alternatively, antipsychotic compounds were dissolved in Triton X-100 in the in vitro kinase assay.36. Cells were grown in DMEM or RPMI-1640 containing 10% fetal bovine serum and kanamycin (50 μg/mL). Cancer cells were plated at a cell density of 103 onto the 96-well culture plates and grown for 24 h. Cell growth/survival for the following 48 h was measured using the Cell Counting Kit-8 . For phosphorylation assays, cells were starved in serum-free DMEM for 10–12 h and then treated with 0–100 µM of the antipsychotic compounds for 20–30 min followed by the stimulation of EGF or neuregulin-1  for 5 min. The used neuregulin-1 was a core EGF domain peptide whose amino acid sequence is shared by all splice variants of neuregulin-130Human astroglioma cells (U87MG) were obtained from the Health Protection Agency . Human epidermoid cells (A431) were obtained from the RIKEN Cell Bank . Human breast cancer cells (MDA-MB-453) was a kind gift from Dr. Higashiyama6 cells/dish, and grown in the serum-free condition as described previously37. Cortical cultures were maintained for 5 days and subjected to treatment with EGF or neuregulin-1 as described above.Whole cerebral neocortices of fetal rats  were enzymatically dissociated, plated on 35 mm-diameter dishes at a density of 1.5 × 10Clozapine was dissolved in sodium-citrate buffer (pH 5.5) and intraperitoneally administered to adult rats  at the dose of 20 mg/kg. One hour after administration, rats were euthanized and subjected to brain dissection. The brain regions such as hippocampus and frontal cortex were obtained and frozen in dry ice. The treatment of these animals was in accordance with the local and international guidelines on the ethical use of laboratory animals. All animal procedures adopted in this study were approved by and conducted under the control of the Niigata University Animal Care and Use Committee.Randomization and blinding of individual rats were not applied to this animal experiment.2VO4, and protease inhibitor cocktail , and 1 mM phenylmethylsulfonyl fluoride (PMSF). Cell lysates were heat-denatured as described previously37. Protein concentrations were determined using a Micro BCA kit . Typically, protein samples (20 µg/lane) were loaded and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Proteins were transferred to a polyvinylidene difluoride membrane (PVDF) by electrophoresis in transfer buffer37. The membranes were incubated overnight with the indicated primary antibodies at 4 °C. The dilution ratio of the primary antibodies was 1:1000 unless specified below. Membranes were rinsed with the wash buffer  and subsequently incubated with peroxidase-conjugated secondary antibodies . The immunoreactivity was visualized with a chemiluminescence reaction using Western Lightning Plus-ECL  or ImmunoStar LD (Wako) as a substrate. Chemiluminescence images were taken at various time points by a 16-bit CCD camera system, G:Box Chemi XRQ . The intensity of the immunoreactive band was normalized to the background signal and automatically quantitated with an image analysis software, GeneTools (Syngene).Cultured cells or frozen tissue were homogenized with the lysis buffer containing 1% lithium dodecyl sulfate, 50 mM Tris (pH 6.8), 500 µM EDTA, 1 mM NaF, 1 mM Na38. Additionally, the following primary antibodies were used; anti-phospho-Tyr1139-ErbB2 , anti-ErbB2 , anti-phospho-Tyr1328-ErbB3 , phospho-Tyr1173-ErbB1 , anti-ErbB3 , anti-phospho-Tyr1056-ErbB4 , anti-phospho-Tyr1242-ErbB4 , and anti-ErbB4 39. No randomization or blinding was used for sample loading.The primary antibodies against phospho-Ser473-Akt , Akt , phospho-Thr 202/Tyr 204-Erk1/2 , Erk1/2 , phospho-Tyr845-ErbB1 , phospho-Tyr1045-ErbB1 , ErbB1 , phospho-Tyr1196-ErbB2 , phospho-Tyr1248-ErbB2 , phospho-Tyr1197-ErbB3 , phospho-Tyr1289-ErbB3 , and phospho-Tyr1284-ErbB4  were obtained from Cell Signaling 3VO4, 10 mM MgCl2, and 5 mM MnCl2. The enzyme stabilizer dithiothreitol was not further supplemented in addition to the carryover of dithiothreitol present in the kinase stock solution. It was presumed that this strong reducing agent could chemically react with or inhibit the action of the antipsychotic compounds used40. The tyrosine kinase reaction was initiated with the addition of 4 mM ATP, maintained at 37 °C for 30 min and terminated with a stopping buffer containing 10 mM EDTA, 1% bovine serum albumin, and 0.5% gelatin. Tyrosine-phosphorylation of the substrate was detected by a peroxide-conjugated anti-phosphotyrosine antibody . The trapped immunoreactivity was measured with a peroxidase substrate mixture  at 37 °C for 15 min and terminated by adding 0.3 M phosphoric acid. The optical absorbance was measured at 450 nm and compared with that of the standard reactions obtained with fixed amounts of the recombinant ErbB kinase. No randomization or blinding was used.A 96-well plate was precoated with tyrosine-glutamate heteropolymer  followed by a non-protein membrane blocking agent, Can Gel Signal Block . After washing, the recombinant enzyme, namely, truncated ErbB1 kinase , truncated ErbB2 kinase , or truncated ErbB4 kinase  was added to the wells along with the kinase reaction buffer (100 µL) comprising 0.2% Triton X-100, 40 mM Tris HCl (pH 7.5), 5 mM β-glycerophosphate, 0.1 mM Nad > 0.8 and η2 > 0.14), which can be implicated biologically and pharmacologically. For this purpose, we set-up the minimum number of samples (n = 3–6), lowering the type I error level (α) but not minimizing the type II error level (β). With this respect, some experimental data sets, which carried the lower statistical power , are indicated in figure legends to warn about their type II error risk. Kolmogolov–Smirnov test was applied to individual data sets (n > 3) and failed to reject the null hypothesis that the data variation fits the normal distribution (data not shown). Accordingly, all data were subjected to parametric analyses and are expressed as means ± SEM. Brown–Forsythe test failed to reject the homogeniety of data variance in almost all the experiments as indicated in figure legends. When this test rejected the null hypothesis for the data variance, those data were directly analyzed by planned multiple comparisons of Welch t-test (two tails) with Bonferroni’s compensation. When univariate data with more than two groups harbored similar data distributions, those were subjected to analysis of variance (ANOVA) to assess the statistical differences among the experimental groups and among doses, followed by a Tukey’s post-hoc test for multiple comparisons. Alternatively, Student t-test or Welch t-test (both two tails) was applied to univariate data from two groups. Statistical analyses were performed using SPSS software . A P-value of less than the α level (0.05/N) was considered as statistically significant .As our preliminary studies found marked effects of clozapine on ErbB kinase activity, the present study focused on such large effects of clozapine  under investigation, namely, olanzapine, clozapine, haloperidol, or risperidone to U87MG and A431 cultures  = 24.86, P < 0.001 for U87MG; F = 28.42, P < 0.001 for A431]. The growth inhibitory effect of clozapine on those cells was dose-dependent  = 6.127, P = 0.009]. Similarly, in cultured cortical neurons, treatment with 30 µM clozapine significantly attenuated ErbB1 phosphorylation (Tyr1173)  = 7.480, P = 0.005]. The phosphorylation of ErbB4 (Tyr1284), which was induced by application of neuregulin-1, was attenuated by clozapine and haloperidol (30 µM each)  but not by olanzapine or risperidone , ErbB1 phosphorylation was triggered by EGF application to cultured U87MG cells, as well as to rat cortical neurons Fig. . Clozapi73) Fig.  . In contrast, the basal phosphorylation of ErbB4 (Tyr1284) was diminished by clozapine and haloperidol as seen in the above ligand-stimulated condition .We also investigated the effects of antipsychotic compounds alone on basal phosphorylation levels of ErbB1 and ErbB4, using cultured cortical neurons  = 12.10, P < 0.001 for main effects and F = 1.839, P = 0.201 for their interaction).Ligand interaction leads to the dimerization of the ErbB receptor tyrosine kinases, followed by trans-phosphorylation of various tyrosine residues of the partner ErbB molecules as substrates45. We examined the dose effects of clozapine on the EGF-triggered ErbB1 phosphorylation as well as neuregulin-induced ErbB4 phosphorylation in cultured U87MG cells and cortical neurons, respectively. In U87MG cells, a reduction in ErbB1 phosphorylation was dose-dependent   = 36.69, P < 0.001]  of clozapine was ~47 µM for Tyr1173-phosphorylation of ErbB1 and ~16 µM for Tyr1284-phosphorylation of ErbB4. The inhibitory action of clozapine on ErbB1 phosphorylation was more modest in A431 cells, however  = 12.49, P = 0.001 for Erk1; F = 7.508, P = 0.005 for Erk2; F = 52.81, P < 0.001 for Akt]  = 8.909, P = 0.002 for Erk1; F = 8.47, P = 0.003 for Erk2; F = 318.8, P < 0.001 for Akt] .These results suggest that clozapine exerts dose-dependent negative effects not only on ErbB1/4 kinases but also on their ligand-induced downstream signaling, at least, at cellular levels. We may argue that the employed concentrations of clozapine in these culture experiments were relatively high with respect to clinical pharmacology of drug dosingEscherichia coli-derived recombinant proteins of truncated ErbB kinases, which lack the extracellular domains for their interaction with EGF/neuregulins, are commercially available and often utilized for cancer drug screening. Using glutamate-tyrosine heteropolymers as a substrate, the enzyme activity of the truncated ErbB1, B2, and B4 kinases was assessed in vitro in the presence of various concentrations of clozapine  = 7.91, P = 0.001 for ErbB1 and F = 16.47, P < 0.001 for ErbB4]. The tyrosine kinase activity of ErbB2 was significantly reduced at 3 μM clozapine .Several potential mechanisms may illustrate the kinase-inhibitory activity of clozapine: (1) competitive interaction with ligands for ErbB receptors, (2) inhibition of ErbB dimerization, and (3) enzymatic inhibition of ErbB kinases. Currently, ine Fig. . The tyr48 and reported to give a rise to a clinically relevant concentration in rat blood50 . The administration of clozapine significantly reduced the basal phosphorylation levels of ErbB4 in the hippocampus and frontal cortex  to adult rats. The dose suitable for this animal experiment was selected from the doses that have been employed in the previous animal studiestex Fig. . The eff24. In the present study, we attempted to elucidate these unique actions of antipsychotic compounds that cannot be illustrated by established dopaminergic and/or serotonergic antagonism. To exclude the influences of the anti-monoaminergic actions of clozapine, we employed the cultures of cancer cells, where neurotransmission is not involved. First, we compared the antiproliferative effects of four antipsychotic compounds . Subsequently, we explored the molecular target(s) responsible for their antiproliferative function. In particular, we focused on the ErbB receptor tyrosine kinases since the inhibition of ErbB kinases is suggested to produce antipsychotic drug-like effects34. Among the antipsychotic compounds, clozapine gained immense attention, which possesses the unique psycopharmacological feature8. In agreement with our assumption, clozapine produced the most remarkable inhibition of ErbB kinase activities in this study, which might be implicated in its unique antipsychotic property and side effects.Several antipsychotic compounds are known to exhibit the non-psychiatric or non-monoaminergic actions on various types of tissues and cells, such as the anti-carcinogenic actionsIn the present experiments, we made the following observations. (1) The growth/survival rates of U87MG and A431 cells in culture were decreased by clozapine and haloperidol. (2) The acute application of clozapine inhibited the ligand-induced phosphorylation of ErbB1 and that of ErbB4 in dose-dependent manners. (3) Clozapine also attenuated the ligand-dependent activation of Akt and Erk1/2 within its micromolar concentration ranges in culture. (5) Clozapine-dependent inhibition of ErbB1 and ErbB4 kinases was reproducible in vitro, suggesting that clozapine can directly inhibit the kinase enzymes of ErbB1 and ErbB4. (6) In vivo application of clozapine similarly downregulated the basal phosphorylation of ErbB4, but not that of ErbB1 in rat brain. Although some controversy of the clozapine action on basal ErbB phosphorylation still remains, the present results suggest that clozapine targets ErbB kinases to interfere the ligand-dependent ErbB kinase actions.53 although the etiological contributions of the up- or downregulation of ErbB signaling to schizophrenia remain to be controversial54. The expression of EGF, neuregulins, or ErbB receptors is reported to increase in the brain of patients with schizophrenia as well as in the blood of the patients57. Conversely, EGF concentrations in blood are decreased in patients with schizophrenia52. Consistent with the hypotheses, the ligands for ErbB1 and ErbB4 are known to induce various behavioral deficits in the animal models relevant to schizophrenia. Such deficits include decreased prepulse inhibition, lowed social interaction, elevated sensitivity to psychostimulants, and reduced mismatch negativity for EGF/ErbB160 and for neuregulin-1/ErbB464.Several postmortem studies have suggested that the hyper-signaling of ErbBs is associated with the neuropathology of schizophrenia33. Similarly, 6-hydroxyantorathene (emodin), an attenuator of ErbB signaling, normalizes such behavioral deficits of the animal model for schizophrenia65. Recently, Tadmor et al., provided supportive evidence for this argument; chronic administration of an ErbB inhibitor, JNJ28871063, ameliorates the social withdrawal of phencyclidine-treated rats66.The inhibitors for ErbB1 kinases exert opposite functions to ameliorate the above behavioral deficits. The anti-cancer agents of ErbB inhibitors namely, ZD1839, PD153035 and OSI-774, all ameliorate the deficits of acoustic prepulse inhibition in the animal models established by neonatal EGF injection and hippocampal lesion without apparent adverse influence67. The anti-cancer drugs targeting ErbB kinases can produce agranulocytosis and/or neutropenia69. However, the present results do not rule out that these side effects of clozapine may involve other protein kinases or signal transducers.Of note, the kinase-inhibitory activity of clozapine might also be implicated in its characteristic side effects; myocarditis and agranulocytosis. These deficits are inducible by the inhibitors for ErbB kinases as well. The attenuation of ErbB kinase signals is known to impair the normal development of cardiac myocytes and result in myocarditis in adult animalsCollectively, all previous reports agree with the present assumption that the unique antipsychotic action and potentially the side effects of clozapine might be associated with the observed negative action of clozapine on ErbB kinases.70, the concentration 30 μM of clozapine, olanzapine, haloperidol, and risperidone, used in the present cell culture experiments, are equivalent to 20, 188, 565, and 615 mg/kg of chlorpromazine, respectively. This calculation conversely suggests that the molar concentration of clozapine in cultures was the lowest and that of haloperidol was the highest with respect to conventional antipsychotic pharmacology. For example, the conventional clinical dose range of haloperidol is 0.75–6 mg/day/person, which results in the concentrations of 1–4 nM in the bloodstream of patients72. Thus, the concentration of haloperidol in the present culture experiments was markedly higher than the clinical dose used for patients with schizophrenia. Thus, a regular clinical use of haloperidol is unlikely to influence ErbB signaling in patients.The present culture results revealed that the acute application of 10–30 μM clozapine and 30 μM haloperidol significantly suppressed the ligand-triggered phosphorylation of ErbB1 and ErbB4. The in vitro kinase study revealed that the minimum effective concentration of clozapine was as low as 3–30 μM. These concentrations of clozapine in the present study raise an important question of whether the corresponding concentrations can be attained clinically in human patients with schizophrenia. According to the reported minimum effective dose of antipsychotics73. Using adult rats, Baldessarini et al.50 reported that single intraperitoneal injection of clozapine  to rats resulted in 2.2 μM concentration in blood and 47 μM concentration in brain, which is higher than the effective dose  obtained in the present culture experiments. Moreover, chronic administration of clozapine is reported to further elevate its local concentrations in the brain owing to its lipophilic accumulation75. Thus, we speculate that the clinical use of clozapine, in particular, in case of high dosing and chronic administration, would allow it to reach the micromolar concentrations and affect ErbB signaling in human brain.In contrast, the accepted daily clinical dose of clozapine  is much higher than that of haloperidol. In contrast to haloperidol, the effective concentration of clozapine used in the present culture study, 10–30 μM, appears to be clinically acceptable and/or attainable. The conventional dose range of clozapine in patients’ blood is around 0.3–7.8 µMIn the present experiments, the ligand-dependency of the clozapine actions differed between its target molecules of ErbB1 and ErbB4 kinases. In culture as well as in vivo, clozapine inhibited both the neuregulin-triggered and untriggered  phosphorylation of ErbB4. In contrast, clozapine failed to affect the basal phosphorylation of ErbB1. Thus, clozapine appears to be ineffective on the ligand-independent basal phosphorylation of ErbB1.31. The above controversy in clozapine actions on basal phosphorylation of ErbB1 might be ascribed to clozapine-insensitive tyrosine kinases, other than ErbB kinases.Is this phenomenon discrepant against the result from the in vitro kinase assay of ErbB1 kinase? The ligand-induced phosphorylation of ErbB receptors mainly depend on the enzyme activity of their receptor kinases. However, it is not necessary that the basal phosphorylation of ErbB receptors should depend on their homodimerization and trans-phosphorylation. It is because several other tyrosine kinases such as Src, Met, or PDGFR are known to induce ErbB1 phosphorylation through their heteromeric dimerization or binding27. When ErbB1 was stimulated by its full ligand EGF, clozapine works as an inhibitor for the kinase in the present study, while clozapine itself is reported to stimulate ErbB1 kinase or Erk/Akt signaling in vivo49. Smith et al.27 proposed that the clozapine-triggered activation of Akt signaling involves insulin secretion in vivo while Pereira et al.22 suggest that the clozapine-triggered activation of Erk signaling in vivo is mediated by ErbB1 (EGFR) transactivation. In the brain in vivo, where robust strengths of neurotransmission occur, the direct and indirect effects on ErbB signaling and monoaminergic neurotransmission would lead to such complex and controversial phenomena. Even in the present experiments, the controversial results of the clozapine actions were obtained; in cultured A431 cells, 100 μM clozapine reduced EGF-trigged ErbB1 phosphorylation to 66.2 ± 8.3% (Supplementary Fig. We also attempted to illustrate the paradoxical positive and negative actions of clozapine on ErbB phosphorylation and signaling76. However, as the expression of ErbB kinases is relatively limited to the subpopulation of brain cells, it would be difficult for the agonistic action of clozapine on ErbB receptors to fully illustrate the reported global effects of clozapine on Akt signaling in the brain27. The molecular mechanism underlying these bi-directional actions of clozapine on ErbB kinases remains to be characterized.The agonistic action of clozapine on ErbB kinases is in agreement with the previous argument that the activation of Akt signaling in the brain is beneficial for antipsychotic medication77, future studies hopefully warrant molecular understanding of the interaction between clozapine and ErbB kinases.In summary, the present study proposes the possibility that clozapine has a novel biological reactivity with ErbB1 and ErbB4 kinases, which might be associated with the unique psychopharmacological profiles of this drug. With the reported unique chemical reactivity of clozapineSupplemental Figure S1- S6


Numerous reports have shown that rigid spinal fixation contributes to a series of unwanted complications in lumbar fusion procedure. This innovative micro-dynamic pedicle screw study was designed to investigate the biomechanical performance of lumbar implants using numerical simulation technique and biomechanical experiment.Instrumented finite element models of three configurations  using a functional L3-L4 lumbar unit were developed, to compare the range of motion of the lumbar spine and stress values on the endplate and implants. An in vitro experiment was simultaneously conducted using 18 intact porcine lumbar spines and segmental motion analyses were performed as well.Simulation results indicated that the dynamic fixation and the hybrid fixation models respectively increased the range of motion of the lumbar spine by 95 and 60% in flexion and by 83 and 55% in extension, compared with the rigid fixation model. The use of micro-dynamic pedicle screw led to higher stress on endplates and lower stress on pedicle screws. The outcome of the in vitro experiment demonstrated that the micro-dynamic pedicle screw could provide better range of motion at the instrumented segments than a rigid fixation.The micro-dynamic pedicle screw has the advantage of providing better range of motion than conventional pedicle screw in flexion-extension, without compromising stabilization, and has the potential of bringing the load transfer behavior of fusional segment closer to normal and also lowers the stress values of pedicle screws. Treatment with rigid fixation and spinal fusion has been regarded as the gold standard for spine surgery. Traditional transpedicular rigid fixation is widely accepted in clinical practice not only because it is easier and safer, but it also offers better biomechanic stability , 2. FromTo address the problems caused by rigid fixation, motion preservation techniques have gradually emerged, which may stabilize the bridged segments and preserve the segment motion to a certain extent. The basic mechanics of dynamic stabilization devices is to reduce stiffness of the instrumentation to allow for more physiologic load transmission at the instrumented levels , 6. At pThe micro-dynamic pedicle screw (MDPS) is a novel design where the uniaxial head coupling to the screw forms a screw-hinge joint. The screws are used to anchor the anterior vertebral bodies with the uniaxial head and then linked by titanium rods. This do not only have the ability of reducing pathologic motion or hypermobility of the instrumented spine, but also can preserve moderate motion at the instrumented levels. In this paper, both in vitro animal experiments and finite element (FE) analysis techniques were employed to gain insight into the lumbar biomechanics of MDPS, in comparison to clinically proven rigid systems. This research involving human data and animal specimens has been approved by Medical Ethics Committee, Shengjing Hospital of China Medical University, and the reference number is 2017PS059K.Since subtle ROM for lumbar spine fusion in flexion-extension may have unexpected positive effects, the overall design philosophy of MDPS is to achieve an approximate release of angle constraint in the sagittal plane. The first step was to create a functional interface between screw seat and screw ball that provides minimal motion of the pedicle screws in the sagittal plane, with respect to rods after locking of the screw. The opening shape of the screw seat can give a good angular-control ability of the pedicle screw. A limited angle was initially set at 30°, based on the experiences of predecessors . HoweverThis study was supported by Shengjing Hospital of China Medical University. A detailed, anatomically accurate three-dimensional non-linear finite element model(FEM) of L3-L4 spine was first developed. The vertebral geometry data for the intact lumbar spine were obtained from a 0.5 mm thick, transverse slices CT scans of a 25-year-old healthy male volunteer. Briefly, the model contained the functional spinal unit L3-L4 with tension-only, linear ligaments, incompressible nucleus pulposus and surface-to-surface non-linear frictional contact in the facets. The vertebral bodies were composed of a cancellous bone core, a 0.5-mm thick cortical shell surrounding and posterior bone regions. The intervertebral disc was modeled as a composite structure including annulus fibrosis and nucleus pulposus. All the six major ligaments, interspinous (ISL), supraspinous (SSL), intertransverse (ITL), posterior longitudinal (PLL), anterior longitudinal (ALL) and ligamentum flavum (LF) were simulated in the model. Besides, the upper and lower endplates with 1-mm thickness were constructed lying at the interlamination between vertebral body and intervertebral disc , 14. AllBefore utilizing this model to evaluate the biomechanical performance of MDPS, its validation had to be operated via comparing the predicted data obtained by the current model with the results of in vitro tests performed by Yamamoto as well as the results of FE tests performed by Rohlmann , 16. TheThe FEM of intact L3-L4 segment was employed to develop the instrumented models, as shown in Fig. In vitro studies are necessary to support the biomechanical findings obtained from finite element analysis (FEA) technique. The animal experiments were set up to investigate the feasibility and safety of MDPS as well as investigate the ROMs of the three configurations  instrumented models using a pure-moment flexibility testing protocol. The experiments were performed at the minimally invasive medical device engineering research center of Ministry of Education at Shanghai University of Science and Technology. The specimens were purchased from the local livestock farmers. During this matter,animal welfare and experimental procedures were carried out in accordance with the Guide for the Care and Use of Laboratory Animals , and were approved by the animal ethics committee of Shengjing Hospital of China Medical University.Eighteen fresh-frozen porcine lumbar spine specimens with L1 to L5 segment were used in this investigation. Each specimen was radiographically imaged, to exclude specimens demonstrating any anomalies or fractures. The specimens were thawed at room temperature, and the tissue surrounding the spine, except the ligaments and facet joints were carefully removed. Each spine was embedded in a self-solidifying dental base acrylic resin liquid and dental base acrylic resin powder cranially at L1 and caudally at L5 to get smooth end faces. Finally, optical markers were firmly attached to the anterior surface of each vertebral body to check for any posttest deformity, and then tested in the four loading directions. Figure P < 0.05. Statistical analyses was performed using SPSS Statistics, version 23.0 .First, we randomly selected six uninstrumented spines and tested in the four loading directions to determine the baseline ROM. Subsequently, eighteen specimens were equally divided into three groups to perform instrumentation according to the three configurations . Spines were regularly sprayed with normal saline to prevent moisture loss until all tests was completed. Multiple comparisons of the obtained ROMs of the implanted segment for each of the three configurations were made using paired t-tests for each loading scheme . Significance levels was set at The dynamic fixator MDPS is designed to increase the ROM appropriately without compromising stability. ROM of L3-L4 motion segments for the three configurations are summarized in Fig. The stress shielding effect during transition was evaluated in terms of stress on the endplate. Compared with L3PS + L4PS model, the endplate stress in the L3MDPS + L4MDPS model increased by 6.5, 4.5, 4.8, and 8.0%in flexion, extension, lateral bending, and axial rotation respectively, while that of the L3MDPS + L4PS model increased by 2.3, 0.4, 3.7, and 4.0%, respectively.Stresses on the implants were also quantified. Compared to the L3PS + L4PS model, the L3MDPS + L4MDPS model had decreased maximum Von Mises stress (MVMS) on screws in flexion, extension and lateral bending, which decreased by 15.9, 16.2 and 9.2% respectively, but increased on screws in axial rotation by 22.6%. Meanwhile, the L3MDPS + L4MDPS model had increased MVMS on rods in all loading conditions. The L3MDPS + L4PS model demonstrated intermediate values of MVMS on screws and rods, between the other two models.The mean ROM of each level for all grouped specimens in F, E, LB and RB are shown graphically in Fig. A limited number of studies have investigated dynamic devices based on dynamic pedicle screw. The MDPS was designed to preserve index level mobility and thereby avoiding the cascade of adjacent segment degeneration (ASD). Cakir et al. reported in their study that increase segmental ROM in flexion/extension was positively correlated with better clinical outcome . TherefoThe FE results of ROM control showed that all three patterns of fixation could provide adequate stabilization at the instrumented segments. The difference was that the introduction of MDPS increased the ROM in flexion and extension. In comparison with theL3PS + L4PS model, the L3MDPS + L4MDPS model increased the ROM by 95 and 83% in flexion and extension respectively. This finding suggests that, MDPS may offer better motion preservation at implanted segment. Although the increased ROM value was relatively small in both flexion and extension (0.74° and 0.63° respectively), which is due to the moderate design, this small change can induce significant alteration of the mechanical environment of the segmental fusion , 21.For years, rigid fixation has been proven to lead to adverse stress-shielding effects of the interbody space, on account of excessive stabilization . The MDPPedicle screw systems have to provide segmental stabilization and load transfer throughout its life. During this period, they would be subjected to constant cyclic dynamic loading after surgical operation. Implant failure is an important consideration to make when designing an implant. The stress level on the implant has a significant impact on its endurance limit as well. Von Mises equivalent stresses of the pedicle screws and rods for the L3PS + L4PS versus L3MDPS + L4MDPS models are shown graphically in Fig. Because of the limitation by test conditions, we only investigated the ROMs of specimens only in this study. There was some difference between our test results and previously reported data, may be owing to the different experimental conditions and experimental methods used. A study about quantitative biomechanical properties of the whole porcine was conducted by Wilke et al. . The ROMPosterior dynamic stabilization (PDS) is now a popular form of surgical intervention, which may relieve symptoms of spinal stenosis as well as discogenic pain by reducing pathologic motion and improving the distribution of loads while preserving the integrity of the native disk and facet joints. PDS devices are FDA-approved for use as adjuncts to spinal fusion. The MDPS does not only inherit the merit of design of ball-socket, but also combines the concept of hinge joint of cosmic system. However, the only downside to MDPS is that kinematic in the uniaxial sagittal plane may lead to malalignment between the rod and the fixed-angle screw head. To overcome the problem of uniaxial malalignment between the rod and fixed angle screw head of MDPS, hybrid use with a conventional pedicle screw is recommended. The biomechanical effects of the L3MDPS + L4PS model was intermediate in the comparison, but this pattern provided more freedom on the instrumentation to facilitate easier rod seating into the screw head saddle. Therefore, the hybrid fixation may be a promising alternative option to treat spinal stenosis or chronic low-back pain conservatively. It is of clinical significance that the choice of MDPS and rigid system might depend on the desired stability of the surgical segment. A surgical segment with more severe degeneration can be protected by using the MDPS due to its higher motion and load transfer abilities. In other situations, the rigid system is more appropriate in response to higher joint instability.We have to note some potential limitations of the current study. Firstly, the design of ±15° rotational degrees of freedom for MDPS, which plays a significant role in controlling the segmental stability of the spine, stemmed from a former pedicle hinged screw–rod system . An in-depth clinical investigation into the angles is lacking in the literature. Therefore, a large accumulation of clinical data to make the support of its role is required. Secondly, the FE study was limited by simplifications in several ways: the use of linear elastic and homogeneous materials for the entire vertebral body and ligaments, tie contact between screw and surrounding bone, absence of the tension of lower-back muscles, and so on. All the above assumption would have fundamental impact on the simulation result. However, the goal of this study was to simply compare the biomechanical stability of the devices, therefore the final results obtained may not have been influenced much by the above limitation. Finally, porcine lumbar spines were employed in the in-vitro biomechanical experiments due to lack of human cadaver specimens. The ROMs data could be limited by the anatomic differences between human and pig. The porcine lumbar spine had little ROM in axial rotation, that may have a notable impact on the biomechanical evaluation of the instrumentations.Biomechanical evaluation of a MDPS for spinal fusion has been demonstrated through finite element simulation and in vitro animal experiments. Our results indicate that MDPS has the advantage of providing larger ROM in flexion and extension without compromising stabilization of the bridged segments, as compared to conventional pedicle screw. The dynamic effect of MDPS has the potential to make the load transfer behavior of fusional segments closer to that of normal, and lowers the stress values of pedicle screws.


Due to rising costs and the difficulty to identify new targets, drug repurposing appears as a viable strategy for the development of new anti-cancer treatments. Although the interest of disulfiram (DSF), an anti-alcohol drug, to treat cancer was reported for many years, it is only very recently that one anticancer mechanism-of-action was highlighted. This would involve the inhibition of the p97 segregase adaptor NPL4, which is essential for the turnover of proteins involved in multiple regulatory and stress-response intracellular pathways. However, recently DSF was also reported as one of the first phosphoglycerate dehydrogenase (PHGDH) inhibitors, a tetrameric enzyme catalyzing the initial step of the serine synthetic pathway that is highly expressed in numerous cancer types. Here, we investigated the structure-activity relationships (SAR) of PHGDH inhibition by disulfiram analogues as well as the mechanism of action of DSF on PHGDH via enzymatic and cell-based evaluation, mass spectrometric and mutagenesis experiments. Its mechanism of action probably involves an increase of the body’s sensitivity to ethanol by inhibition of the enzyme acetaldehyde dehydrogenase (ALDH)2.Disulfiram (bis(diethylthiocarbamoyl) disulfide = DSF), commercially known as Antabuse, is used since 1948 (FDA-approved in 1951) as an alcohol-aversive agent for the treatment of alcohol dependence4 and its repurposing in the therapy of cancer is foreseen. This would provide a new effective drug, avoiding expensive development phases before its commercialization6, DSF having a well-controlled ADME profile7 and a fairly broad efficiency on various tumor lines in pre-clinical models8.Starting from the 2000s, numerous studies have reported anti-tumoral properties for DSF9. Other data evidenced that DSF was able to induce apoptotic cell death of breast cancer cell lines by inhibition of the proteasomal machinery3. However it is only very recently that a clear anticancer mechanism for DSF was detailed when Skrott et al. demonstrated that an in vivo metabolite of DSF could act as an inhibitor of NPL4, an adaptor of segregase p97 , essential for the recycling of proteins involved in multiple regulatory and stress-response intracellular pathways10. In fact, in the body, DSF is metabolized to ditiocarb  and other metabolites. It is also known that DSF chelates bivalent metals and forms complexes with copper (Cu), which enhances its anti-tumour activity. The group of Bartek actually demonstrated that a DTC–copper complex named bis(diethyldithiocarbamate)–copper (CuET) forms in vivo, thereby providing the anti-cancer metabolite of DSF10.Different mechanisms accounting for the anticancer activity of DSF were suggested. The group of Cassidy showed for instance in 2003 that DSF was able to inhibit nuclear factor-kappa B (NF-κB), a protein implicated in immune response, hence preventing the resistance of cancer cells to 5-fluorouracil (5-FU)11, the Cantley lab demonstrated that DSF was also a potent inhibitor of phosphoglycerate dehydrogenase (PHGDH), the first and limiting step of the so-called serine synthetic pathway (SSP)12, thus suggesting that DSF itself could display anticancer properties via an alternative mechanism-of-action. In fact, in 2011, the group of Possemato et al. demonstrated that PHGDH silencing leads to a significant decrease in tumor proliferation in several PHGDH-overexpressing cells13. However, tumorigenesis supported by PHGDH still need to be detailed.Finally, in 201614 and, similarly to the Cantley lab, DSF was identified with an IC50 of 0.59 µM. Given the importance of PHGDH in cancer metabolism and the growing interest in repurposing DSF for cancer therapy, we set out to examine structure–activity relationships (SAR) in a series of DSF analogs and to elucidate its mechanism-of-action.Recent results in our lab led to the identification of new PHGDH inhibitors following a drug screening campaign15 by our team was screened on purified PHGDH using an isolated enzymes inhibition assay14. Compared to the parent compound (DSF = 1), apart from compound 2, all the attempts to replace the diethylamino side chain in DSF (3–16) afforded similarly active compounds (Table 17), a thioacetamido carbamodithioate (18), or a methylene dicarbamidodithioate (19–20) led to compounds that inhibit PHGDH only weakly or inactive compounds. These relatively flat SAR for DSF analogues possessing the symmetrical bis(dithiocarbamate) central core  led us to suspect either (i) unspecific binding, (ii) binding at a site distant from the PHGDH active site  or possibly (iii) covalent inhibition. Puzzled by this question we set out to detail the mechanism-of-action of DSF on PHGDH.As a first step to detail our understanding of the binding of DSF on PHGDH, some preliminary SAR were investigated around the bis(dithiocarbamate) central core. To this end, a library of 20 DSF analogues developed earliervia unspecific binding, and measuring a similar IC50  is responsible for the anti-cancer activity through inhibiting the p97 segregase adaptor NPL4, we verified whether PHGDH inhibition could result from the formation of this copper complex. To this end, we synthesized the diethyldithiocarbamate (DTC)-copper complex CuEt and analyzed wtPHGDH inhibition. As a result, an IC50 in the 10 µM range, that is about 16-fold weaker compared to DSF itself, was obtained . At the end of the incubation, chloroacetamide was added and incubated 15′ in large excess in order to block the remaining free cysteines residues for the following analysis. The protein was separated from the medium  followed by trypsinization and analysis of the peptides by nanoUHPLC/MS.To unambiguously confirm this hypothesis and possibly identify the cysteine residues involved in this interaction, mass spectrometry experiments were undertaken. Briefly, PHGDH was incubated for 30 min with various concentrations of disulfiram , although at very different levels . Interestingly, this mutant is known to retain a catalytic activity comparable to the wild type enzymeWith a view to understand the interaction of DSF with PHGDH on Cys116 at the molecular level, we focused our interest on an already described X-ray crystal structure of a truncated form of PHGDH  intermediate as exemplified on Fig. 19, the results obtained here suggest that DSF would inhibit PHGDH by disruption of the active tetramer either into an inactive dimer resulting from the formation of a disulfide bridge between two Cys116 residue on two adjacent monomers, or to a lesser extent to an inactive diethyl(dithiocarbamate) intermediate monomer.To confirm this hypothesis, a cross-linking experiment, using bis-sulfosuccinimidyl suberate (BS3) as cross-linker, was finally undertaken with PHGDH alone or PHGDH after treatment with increasing concentrations of DSF. As clearly observed from Fig. Finally to detail the relationship between the ability of DSF to disrupt PHGDH tetramer and its anti-cancer activity, we set out additional experiments aiming to assess the effect of DSF on two cancerous cell lines: UM-UC-3 human transitional cell carcinoma that constitutively express PHGDH (UM-UC-3-PHGDH+) and a variant of these cells that do not express PHGDH (UM-UC-3-PHGDH-) Fig. .Figure 850) of 3,64 µM is obtained, whereas cells expressing PHGDH (UM-UC-3-PHGDH+) appear to be more susceptible to DSF treatment with an IC50 of 0,77 µM. Although the difference remains weak (~5-fold), probably because other anti-proliferative mechanisms are involved upon DSF treatment, tumor cell proliferation inhibition is higher for cells expressing PHGDH.When cells that do not express PHGDH (UM-UC-3-PHGDH-) are treated with DSF, a tumor cell proliferation inhibition  into notably the diethyldithiocarbamate (DTC) metabolite which undergoes copper complexation, and hence provides anticancer activity for DSF, our results suggest that the non-metabolized circulating DSF could also provide anticancer activity but through a completely different mechanism of action involving PHGDH inhibition. Also, the maximum plasma concentration of DSF (Cmax) being reported to be 1.28 µM, that is around 2-fold higher than the IC50 of DSF on PHGDH in the cell-based assay (0.77 µM), one can hypothesized that exposure to DSF in vivo could be sufficient to provide anticancer activity via PHGDH inhibition.Because it is known that DSF is metabolized 14. NADH fluorescence emission (Ex 340 nm/Em 460 nm) was followed over time. Assays were performed in PHGDH assay buffer . Substrates and enzyme concentrations were as follows: 3-PG, 240 μM; NAD+, 120 μM; glutamate, 30 mM; PHGDH, 12 ng/μL; PSAT1, 20 ng/μL. The final concentration of DMSO in the assay mixture was set to 5%.Enzymatic assay was adapted from a previously described procedure14. DSF (5 μM) or DMSO control was incubated with PHGDH for 45 min at 37 °C. Undiluted DSF (5 μM) was included as a positive control for inhibition.Dilution experiment was conducted following a reported procedureEscherichia coli. A single colony was grown to an OD600 0.6 in 1 L of Luria broth. Protein expression was induced with 1 mM isopropyl thiogalactopyranoside (IPTG). The culture was chilled on ice for 30 min, cultured for 18 h at room temperature and pelleted . Pellets were resuspended in 60 mL of lysis buffer  and sonicated, and cell debris were pelleted by centrifugation . The supernatant was collected and purified using Akta purifier on HisTrapTM FF column . After column equilibration with wash buffer , bound proteins were eluted with elution buffer  and collected (1-mL fractions). Fraction protein content was measured via a Bradford assay. The most concentrated fractions were pooled and dialyzed overnight into 4 L of dialysis buffer . Protein purity was assessed via SDS/PAGE and Coomassie staining.pET28a human PHGDH and pET28a human C116S PHGDH were transformed into BL21 21. A local protein database containing the human PHGDH sequence (accession Uniprot O43175) was used to process the obtained MS/MS data. Mass error was set to 15 ppm for precursor ions and 0.6 Da for fragment ions. Oxidation on Met; disulfiram, (+147.025 Da) and carbamidomethyl (+57.021 Da) were considered as variable modifications on Cys.Mass spectrometry experiments were carried out on an Orbitrap Lumos, following a previously reported procedure, with some modifications+ in 18 μL total volume for 30 min on ice. BS3  cross-linker dissolved in PBS was added to a final concentration of 5 mM and incubated for 30 min under shaking at room temperature. The reaction was then quenched for 15 min by adding 1 M Tris, pH 7.5, to a final concentration of 28 mM. Cross-linked proteins were mixed with sample buffer, boiled for 5 min, and run on SDS/PAGE. Gels were stained with Gelcode blue stain reagent  overnight and destained according to the manufacturer’s instructions.PHGDH (3 μg) was incubated with DSF  or vehicle control (DMSO) in 25 mM HEPES, pH 7.3, and 1 mM NADUM-UC-3 (PHGDH+ and PHGDH-) bladder cancer cells were purchased from the ATCC and cultured in DMEM medium supplemented with 10% heat-inactivated Fetal Bovine Serum (FBS) and 1% penicillin/streptomycin. Cells were seeded at 2500 cells/well in 96-well plates in serine depleted media (MEM). DSF or vehicle (DSMO) was added and cells were grown for 48 hours. Viability was assessed using Presto Blue reagent (Life Technologies) according to manufacturer’s instructions.22.All reagents were purchased from chemical suppliers and used without purification. Copper/DSF complex was obtained according a previously described procedure23.Western blot and immunoblot analysis were performed according a reported procedureSupplementary information


This novel approach fills the gap between conventional in vitro assays for inhibitors targeting C4 key enzymes and in vivo experiments on whole plants. The assay addresses inhibition of the target enzymes in a plant context thereby taking care of any reduced target inhibition due to metabolization or inadequate uptake of small molecule inhibitors across plant cell walls and membranes. Known small molecule inhibitors targeting C4 photosynthesis were used to validate the approach. To this end, we tested pyruvate phosphate dikinase inhibitor bisindolylmaleimide IV and phosphoenolpyruvate carboxylase inhibitor okanin. Both inhibitors show inhibition of plant photosynthesis at half-maximal inhibitory concentrations in the sub-mM range and confirm their potential to act as a new class of C4 selective inhibitors.The identification of novel herbicides is of crucial importance to modern agriculture. We developed an efficient  Each of them is specialized to accommodate different environmental conditions with C3 photosynthesis being the most common one. CAM and C4 plants adapted to hot and dry climate by establishing CO2 concentrating mechanisms that circumvent the oxygenase reactivity of ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) which is the key enzyme of photosynthetic carbon fixation within the Calvin-Benson-Bassham (CBB) cycle. In C4 plants, this CO2 concentrating mechanism is implemented by a spatial separation of CO2 uptake from the CBB cycle. Normally, this separation is established across two cell types: bundle sheath (BS) cells which are surrounded by a layer of leaf mesophyll (LM) cells forming the so-called Kranz anatomy. Primary CO2 fixation takes place in the LM cells, where atmospheric CO2 is fixed after conversion to HCO3− by phosphoenolpyruvate carboxylase (PEPC) yielding the four-carbon organic acid oxaloacetate that is subsequently converted species-specifically into malate or aspartate. These diffuse to the BS cells where CO2 is released by decarboxylation and hereby enriched within the BS cells. The local increase in CO2 concentration greatly disfavors the oxygenase activity of RuBisCO which then uses the released CO2 during the CBB cycle to form triose phosphates. Pyruvate formed in the process of decarboxylation diffuses back into the LM cells and is regenerated to phosphoenolpyruvate (PEP) by the pyruvate phosphate dikinase (PPDK). The regenerated PEP then may be reused to fix another molecule of CO2. With the discovery of plants capable of performing single-cell C4 photosynthesis (SCC4), it has been shown however that Kranz anatomy is not a mandatory requirement for C4 photosynthesis  as well as biological (free diffusion of compounds) limits confine their application. Here, we present a novel in vivo test system using intact plant cell suspensions that builds up on previous studies using leaf cuttings  as previously described (−2 s−1. Plants were watered twice a week. Liquid fertilizer (0.2 % (v/v), NPK 12-4-6, Wuxal Top N, Manna, DE), and 100 mM NaCl were provided once per week while watering the plants. Zea mays was grown in the greenhouse under natural light conditions.escribed  with dayB. sinuspersici plants. Leaves were gently squeezed with mortar and pestle in cold extraction buffer  for 5 to 10 min. A total volume of 5 ml buffer was used for the extraction of 50 leaves per preparation. The obtained cell suspension was centrifuged twice . The supernatant of each step was discarded to segregate intact cells from cell debris and free chloroplasts. The green-colored sediment was re-suspended in the same volume of degassed extraction buffer used for the initial cell extraction step  trypan blue staining solution  were added to 1 ml B. sinuspersici chlorenchyma cell suspension in extraction buffer to prevent substrate limitation. The sample was then incubated at 30°C for 5 min and added to the pre-heated (30°C) electrode chamber. Oxygen evolution in the dark was monitored until stability (dark rate). Two factors contribute to the dark rate: oxygen depletion by the electrode (electrode drift) and mitochondrial respiration. Subsequently the electrode chamber was illuminated at 1,000 µmol m−2 s−1 and light-dependent oxygen evolution (light rate) was recorded after signal stabilization. The POE which is indicative for the photosynthetic activity was calculated as the difference between light and dark rate.Measurements were performed using a Clark-type oxygen electrode  . PyruvatInhibition experiments were performed in the presence of known inhibitors of PEPC or PPDK. Here we used the chalcone okanin  — an allosteric inhibitor of PEPC activity — and bisindolylmaleimide IV (BIM4)  — an active-site inhibitor competing with ATP for the nucleotide binding site of PPDK.Inhibitors were added to the electrode chamber after the system reached equilibrium. The rate was recorded (inhibited light rate), once a stable POE was reached again after addition of the compound. The relative inhibition was then calculated according to equation 1.For okanin a substantial light-independent oxygen consumption was observed immediately after addition of the compound. However, this oxygen scavenging effect is well-known for other hydroxylated chalcones . To corr50) were calculated by measuring the activity in the presence of different concentrations of each compound. Nine inhibitor concentrations in the range of 0 to 350 µM (BIM4) or 0 to 3,000 µM (okanin) were measured in triplicates as described above. A log-logistic dose response curve with three parameters (LL3) was globally fitted to the individual replicates using the R software collection v3.5.0  inhibitory concentrations , 50 μM MnCl2, and 2.5 mM dithiothreitol (4-driven photosynthesis. POE rates were monitored as stated before Leaf slices were recovered from the chamber and transferred to 1 ml of 80 % acetone (v/v) for chlorophyll extraction. Samples in acetone were kept in the dark at 4°C for 48 h. Absorption was measured using a DU800 spectrophotometer  at 645 and 663 nm and the total chlorophyll amount was calculated as stated in equation 2 , Ft , So , Sb , Zm , Ah , Ec , and Si . Figures of multiple sequence alignment (MSA) were prepared using TeXshade  or the 4′-carbon atom of 2′-Br-dAppNHp (BsPPDK), respectively. Energy minimized docking poses were subsequently ranked according to their predicted binding affinities. Figures of molecular models were prepared using PyMOL  corresponds to arginine, whereas the Flaveria C4 model used in the in vitro studies .To explore whether the cell suspension assay is able to yield more accurate data compared to leaf cutting assays, measurements of POE were repeated on different days over the course of 6 h as described in as described in Material and Methods For better comparability, POE rates obtained from both methods were normalized to the respective mean of each measurement series Figure 3B. sinuspersici cell suspension assay presented in this study is that key enzymes and mechanisms in the SCC4 plant function in a similar manner as in conventional Kranz-type C4 plants. To identify possibly meaningful differences on sequence level, a MSA of the two key enzymes of C4 photosynthesis — PEPC and PPDK — was computed for a range of C4 species  for PEPC. This region is part of the PEPC allosteric aspartate/malate inhibitor site and is likewise involved in binding the inhibitor okanin . The docking suggests that BIM4 is bound via polar side chain contacts to R174, T187, and T405  under Germany´s Excellence Strategy — EXC 2048/1 — Project ID: 390686111. Work in the S.O. lab was funded by the “Cooperative Research Program for Agriculture Science & Technology Development (Project No. 0109532016)”. Rural Development Administration, Republic of Korea.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.