S303p, CANXS583p, MCM2S41p, POGZS425p, SRRM2T983p, and PRPF4BS431p were significantly correlated with the overall survival of colon cancer patients. Further pull-down assays identified 23 proteins, such as THRAP3, BCLAF1, and STAU1, showing strong binding affinity to GSK3α. The interaction between THRAP3 and GSK3α was verified by biochemical experiments. Notably, among the 18 phosphosites of THRAP3, phosphorylation at S248, S253, and S682 is specifically mediated by GSK3α. Mutation of S248 to D (S248D), which mimics the effect of phosphorylation, obviously increased cancer cell migration and the binding affinity to proteins related to DNA damage repair. Collectively, this work not only discloses the specific function of GSK3α as a kinase but also suggests GSK3α as a promising therapeutic target for colon cancer.GSK3α and GSK3β are two GSK3 isoforms with 84% overall identity and 98% identity in their catalytic domains. GSK3β plays important roles in the pathogenesis of cancer, while GSK3α has long been considered a functionally redundant protein of GSK3β. Few studies have specifically investigated the functions of GSK3α. In this study, unexpectedly, we found that the expression of GSK3α, but not GSK3β, was significantly correlated with the overall survival of colon cancer patients in 4 independent cohorts. To decipher the roles of GSK3α in colon cancer, we profiled the phosphorylation substrates of GSK3α and uncovered 156 phosphosites from 130 proteins specifically regulated by GSK3α. A number of these GSK3α-mediated phosphosites have never been reported before or have been incorrectly identified as substrates of GSK3β. Among them, the levels of HSF1  •High levels of GSK3α predict poor prognosis of colon cancer patients.•Identification of 156 phosphosites specifically regulated by GSK3α, not GSK3β.•THRAP3 has been identified as a phospho-substrate of GSK3α.•GSK3α-mediated phosphorylation of THRAP3 at S248 promotes cancer cell migration. In vitro and in cell line phosphoproteomics identified 156 phosphosites specifically regulated by GSK3α. Notably, phosphorylation of THRAP3S248 remarkably enhanced colon cancer cell migration. These findings suggest that GSK3α may have unique and important roles in colon cancer development and progression.GSK3α and GSK3β are distinct isoforms, with GSK3β studied in cancer development while GSK3α was considered redundant. Yet, our research has revealed that GSK3α, rather than GSK3β, is significantly correlated with colon cancer patients' survival. Integrative  Colorectal cancer (CRC) is the third most common cancer and has the second highest mortality rate worldwide . DistantGSK3A and GSK3B, respectively. The two GSK3 isoforms have 84% overall identity and 98% identity in their catalytic domains  to mimic the effect of its phosphorylation obviously increased the migratory ability of cancer cells and the binding affinity to proteins related to DNA damage repair (DDR).In this study, we screened the key kinases that affect the survival of colon cancer patients and may be potential targets. Based on our proteome and immunostaining data and publicly available proteome data from 4 independent colon cancer cohorts , 26, we This work was conducted according to the Declaration of Helsinki ethical principles. Paired colon cancer samples from 78 treatment-naïve patients were obtained from West China Hospital. The tissues were quickly frozen in liquid nitrogen after surgery and stored in a −80 °C refrigerator until use. The pathological sections were processed by the Department of Pathology of West China Hospital, a laboratory accredited by the College of American Pathologists. Both colon cancer tissues used for omics and colon cancer tissues used for immunohistochemistry were obtained with informed consent and the approval of the Ethics Committee of Biology Research, West China Hospital at Sichuan University (Permission number: 2020(374)). Clinical information, including age, sex, tumor stage, and follow-up information, was collected.g and 4 °C for 30 min. The supernatants were collected and the protein concentrations were measured.Tissue and cell samples were lysed in radioimmunoprecipitation assay buffer . Tissue samples were homogenized with a gentleMACS Dissociator (Miltenyi Biotec GmbH) using the procedure “protein 01. 01”. The crude lysates were sonicated  and then centrifuged at 20,000A tandem mass tag (TMT)-based strategy was used for quantitative proteomics and phosphoproteomics analysis of tumors and distant normal tissues (DNTs). For proteomics, 78 paired tumors and DNTs were divided into 21 TMT-labeled batches with common reference samples. Fifty micrograms of protein lysates were reduced with 10 mM tris (2-carboxyethyl) phosphine (Sigma) at 56 °C for 1 h and alkylated with 20 mM iodoacetamide in the dark at room temperature for 30 min. Subsequently, the protein lysates were precipitated with methanol/chloroform/water and the precipitate was resuspended in 50 mM triethylammonium bicarbonate (Sigma) and digested with MS sequencing grade trypsin  at 37 °C overnight. The trypsin was then inactivated by heating at 95 °C for 2 min. The digested peptides and the common reference sample were labeled by TMT10plex reagents (Thermo Fisher Scientific). After quenching with hydroxylamine, the colon cancer samples and the reference sample in the same batch were combined. The combined peptides were desalted by reversed-phase C18 SPE columns  and separated into 120 fractions by reversed-phase HPLC (RP-HPLC) at a flow rate of 1 ml/min. These fractions were combined into 15 fractions before MS analysis.For phosphoproteomics of colon cancer tissues, 70 paired tumors and DNTs were divided into 18 TMT-labeled batches with common reference samples. Forty micrograms of digested peptides from each sample were labeled with TMT reagents. The combined TMT-labeled peptides were separated into 15 fractions by C18 SPE columns  and then combined into 5 fractions, which were further used for phosphopeptide enrichment.PureCube Fe-nitrilotriacetic acid (NTA)-agarose beads were used for phosphopeptide enrichment and purchased from Cube Biotech. The desalted peptides were dissolved in loading buffer (85% acetonitrile (ACN), 0.1% TFA) and then incubated with Fe-NTA agarose beads at 25 °C for 1 h. After incubation, the agarose beads were washed 4 times with washing buffer . Then, the phosphopeptides were eluted from the agarose beads by elution buffer . The eluted peptides were acidized with 10% TFA and dried. The samples were desalted and further analyzed by LC-MS/MS.For proteomics profiling of colon cancer tissues, desalted peptides of each fraction were analyzed using an EASY-nanoLC 1200 system (Thermo Fisher Scientific) coupled to an Orbitrap Exploris 480 mass spectrometer (Thermo Fisher Scientific). The peptides were separated in the analytical column with a 65 min gradient with 4% to 100% buffer B  at a flow rate of 300 nl/min. Under the data-dependent acquisition mode, MS spectra were acquired from 350 to 1800 m/z with a resolution of 60,000 at m/z = 200. The normalized automatic gain control (AGC) target (%) was set as 300%. For the MS2 analysis, a 0.7 m/z isolation window and normalized collision energy (NCE) of 36% were selected, with exclusion of precursor ions with charge states of z = 1 or 8 or unassigned charge states.For phosphoproteomics of colon cancer tissues, the enriched phosphopeptides were analyzed by an EASY-nanoLC 1200 system coupled to a Q Exactive HF-X mass spectrometer. The flow rate of the analytical column was 330 nl/min with a 65 min gradient from 6% to 100% buffer B . For the full MS scans, ions with m/z ranging from 350 to 1600 were acquired under data-dependent acquisition mode by orbitrap with a resolution of 60,000 at m/z = 200. For the MS2 scans, the top 20 most intense parent ions were selected with an isolation window of 0.6 m/z and fragmented with stepped NCE values of 25% and 31%.in vitro phosphoproteomics analysis, the enriched phosphopeptides were desalted with ZipTip (Merck Millipore) and analyzed by an EASY-nanoLC 1000 coupled to a Q Exactive Plus mass spectrometer (Thermo Fisher Scientific). The flow rate of the analytical column was 330 nl/min with a 65 min gradient from 10% to 90% buffer B . For the full MS, ions with m/z ranging from 350 to 1800 were acquired by an orbitrap resolution of 60,000 at m/z = 200. For the MS2 scans, the top 20 most intense parent ions were selected for MS2 analysis and fragmented with stepped NCE values of 25% and 27%. The maximum injection time for MS2 was 64 ms and the dynamic exclusion time was 50 s.For cellular phosphoproteomics analysis, the enriched phosphopeptides were desalted with ZipTip (Merck Millipore) and analyzed by an EASY-nLC 1200 system coupled to a Q Exactive HF-X mass spectrometer (Thermo Fisher Scientific). The flow rate of the analytical column was 330 nl/min with a 90 min gradient from 12% to 100% buffer B . For the full MS, ions with m/z ranging from 350 to 1800 m/z were acquired by orbitrap resolution of 60,000 at m/z = 200. For the MS2 scans, the top 20 most intense parent ions were selected with an isolation window of 1.6 m/z and fragmented with stepped NCE values of 25% and 27%. The maximum injection time for MS2 was 64 ms and the dynamic exclusion time was 50 s. For 6, with a maximum fill time of 20 ms. For the MS/MS scans, the top 20 most intense parent ions were selected with an isolation window of 1.6 m/z and fragmented with an NCE of 27%. The AGC was set at 1e6, with a maximum fill time of 64 ms and a resolution of 17,500.For analysis of coimmunoprecipitated proteins, the digested peptides were desalted with ZipTip (Merck Millipore) and analyzed by an EASY-nanoLC 1000 coupled to a Q Exactive Plus mass spectrometer (Thermo Fisher Scientific). Peptides were separated in the analytical column with a 65 min gradient from 10% to 90% buffer B  at a flow rate of 330 nl/min. For the full MS, the mass range was from 350 to 1800 m/z with a resolution of 60,000 at 200 m/z. The AGC value was set at 3ehttps://www.maxquant.org) and searched against the Swiss-Prot human protein sequence database . The proteolytic enzyme was trypsin/p and two missed cleavage sites were allowed. Oxidation of methionine and acetylation of the protein N terminus was set as variable modifications. Cysteine carbamidomethylation was set as a fixed modification. The maximum peptide mass was set to 12,000 Da and the minimum amino acid length was set to 6. The first search mass tolerance was set to 20 ppm and the main search peptide tolerance was set to 4.5 ppm. The mass tolerance for fragment ions was set at 0.5 Da. The phosphoproteomics data were searched with the same database and similar parameters. Phosphorylation at S, T, or Y residues was also set as a variable modification. For label-free phosphoproteomics, the “match between runs” was enabled.The proteomics data were analyzed with MaxQuant  or <0.1 (for samples in each tumor-node-metastasis [TNM] stage), and HR ≥ 2 or ≤0.5.For processing the output datasheets of proteome data of colon cancer tissues, the contaminant and reverse hits were first removed. The proteins with two or more unique peptides were kept. The total protein intensity of each sample as well as the reference sample was then normalized in each batch. The protein intensity of the sample was divided by that of the reference sample in the same batch and then the proteome data of 21 batches were combined. The proteins with missing values in less than 50% of the samples were used for subsequent data analysis. Kaplan–Meier survival curves (log-rank test) and the Cox proportional hazard model for overall survival (OS) analyses were implemented to identify the most important kinases. The above analyses were achieved through the “survival” and “survminer” packages in R. The filter criteria for kinase biomarkers were as follows: log-rank p value <0.05, Cox p value <0.05, and HR > 1 or <1. The functions of phosphoproteins were analyzed by Metascape. For processing the output datasheets of phosphoproteome data in vitro or in cell lines, the contaminant and reverse hits were first removed. The phosphopeptides with localization probabilities greater than 0.75 were assigned to reliable identification. The phosphosites with ratio (GSK3α/Control) >1.5 and p value <0.05 (Student’s t test) or only detected in the GSK3α-treated group and p value <0.05 (Student’s t test) were defined as phosphosites significantly upregulated by GSK3α in vitro. Similarly, phosphosites with ratio (GSK3A-KO/WT) <0.667 and p value <0.05 (Student’s t test) or only detected in the control group and p value <0.05 (Student’s t test) were defined as phosphosites significantly regulated by GSK3α in the cell lines. The functions of phosphoproteins were analyzed by Metascape.For processing the output datasheets of phosphoproteome data of colon cancer tissues, the contaminant and reverse hits were first removed. The phosphopeptides with localization probabilities greater than 0.75 were assigned to reliable identification. The total phosphopeptide intensity of each sample in the same batch was normalized to the same level. The phosphopeptide intensity of the sample was divided by that of the reference sample in the same batch and then the phosphoproteome data of 18 batches were combined. The phosphosites with missing values in less than 50% of the samples were used for subsequent data analysis. Kaplan–Meier survival curves (log-rank test) and Cox proportional hazard models for OS analyses were carried out to identify the most important phosphosites. The filter criteria for important phosphosites were as follows: log-rank t test, p < 0.05) or only identified in the GSK3α or GSK3β overexpression groups  were supposed to be the binding proteins of GSK3α or GSK3β. For the binding proteins of THRAP3 mutants, the proteins with foldchanges >1.5  or exclusively identified in that group were considered the binding proteins of the corresponding THRAP3 mutant. The functions of binding proteins were analyzed by Metascape.For processing the output table of coimmunoprecipitation proteome data, the contaminant and reverse hits were first removed. Proteins with ≥2 unique peptides were selected for data analysis. Based on the label-free quantification intensities, the quantified proteins with ratio (GSK3α/control) >1.5 or ratio (GSK3β/control) >1.5  , 100 U/ml penicillin and 100 μg/ml streptomycin .The protein samples were separated by 10% or 12% SDS-PAGE and transferred to polyvinylidene fluoride membranes . After blocking with 4% nonfat milk, the polyvinylidene fluoride membranes were incubated with primary antibody (1:2000) at 4 °C overnight and then incubated with HRP secondary antibody  at room temperature for 1 h. Antibodies against the following proteins were used: GAPDH , GSK3α , GSK3β , Flag , Myc , THRAP3 , E-cadherin , N-cadherin , and vimentin .GSK3A (1–483 aa) and GSK3B (1–420 aa) were cloned into the pGEX-6P1 vector and transduced into Escherichia coli BL21-Gold (DE3) cells. The recombinant proteins GST-GSK3α and GST-GSK3β were extracted by lysis buffer A , and lysis buffer B , respectively. The recombinant proteins were purified by GST affinity resin .Human 2, 5 mM DTT, 25 μM ATP, pH = 7.5) and incubated at 30 °C for 0, 1, 2, or 3 h. The reaction products were mixed with the Kinase-Lumi detection reagent and the fluorescence was detected by Multikinetic microplate reader (Biotech).The kinase activity was measured by a Kinase-Lumi assay Kit . Five micrograms of extracted HCT116 protein as the substrate was mixed with 0, 0.01, 0.02, 0.05, 0.1, 0.2, 0.5, and 1.0 μg of recombinant GST-GSK3α protein. The mixture was added to the kinase reaction buffer  and incubated at 30 °C for 2 h. A reaction without recombinant GST-GSK3α was used as a control. The experimental and control assays were repeated three times. The phosphopeptides of each sample were enriched by Fe-NTA agarose beads and analyzed by LC-MS/MS.Two hundred micrograms of extracted proteins from HCT116 cells were mixed with the recombinant GST-GSK3α protein and kinase reaction buffer  HCT116 or DLD-1 cell lines were generated using a CRISPR/Cas9 system. There were two guide RNAs targeting the human GSK3A gene and two gRNAs targeting the human THRAP3 gene. The HCT116 and DLD-1 cells after knockdown of the target genes were screened by puromycin . To obtain GSK3A-KO HCT116 cells, a limiting dilution strategy was used to obtain monoclonal cells. The gRNA sequence was as follows:GSK3A-gRNA1-sense: 5′-CACCGGGCGGAAAGGCATCTGTCG-3′GSK3A-gRNA1-antisense: 5′-AAACCGACAGATGCCTTTCCGCCC-3′GSK3A-gRNA2-sense: 5′-CACCGGCACTAGCTTCCCGCCGCC-3′GSK3A-gRNA2-antisense: 5′-AAACGGCGGCGGGAAGCTAGTGCC-3′THRAP3-gRNA1-sense: 5′-CACCGCGAGAGAGAGATCGGCTTC-3′THRAP3-gRNA1-antisense: 5′-AAACGAAGCCGATCTCTCTCTCGC-3′THRAP3-gRNA2-sense: 5′-CACCGCTCTCGTTCAAGGAAGCGC-3′THRAP3-gRNA2-antisense: 5′-AAACGCGCTTCCTTGAACGAGAGC-3′GSK3A and THRAP3 mutants in GSK3A and THRAP3-KD HCT116 cell lines, respectively, we inserted the complementary DNA into a vector (pCDH-CMV-MCS-EF1) and abrogated the binding of gRNAs by altering the protospacer adjacent motif (PAM) sequence with the following primers:To reconstruct the GSK3A-PAM1-sense: 5′-AGGCATCTGTCGGAGCCATGGGT-3′GSK3A-PAM1-antisense: 5′-ACCCATGGCTCCGACAGATGCCT-3′THRAP3-PAM2-sense: 5′-TTCAAGGAAGCGCAGACTGAGTTCTAGGT-3′THRAP3-PAM2-antisense: 5′-ACCTAGAACTCAGTCTGCGCTTCCTTGAA-3′THRAP3 mutants, 6 paired primers were used and designed as follows:To construct the THRAP3S248A-sense: 5′-AGTCCTCGGGAGCGAGCCCCAGCTCTCAAAA-3′THRAP3S248A-antisense: 5′-TTTTGAGAGCTGGGGCTCGCTCCCGAGGACT-3′THRAP3S248D-sense: 5′-AGTCCTCGGGAGCGAGACCCAGCTCTCAAAA-3′THRAP3S248D-antisense: 5′-TTTTGAGAGCTGGGTCTCGCTCCCGAGGACT-3′THRAP3S253A-sense: 5′-AGCCCAGCTCTCAAAGCACCCCTCCAGTCTGT-3′THRAP3S253A-antisense: 5′-CAGACTGGAGGGGTGCTTTGAGAGCTGGGCT-3′THRAP3S253D-sense: 5′-AGCCCAGCTCTCAAAGATCCCCTCCAGTCTGTG-3′THRAP3S253D-antisense: 5′-CACAGACTGGAGGGGATCTTTGAGAGCTGGGCT-3′THRAP3S248/253A-sense: 5′-AGCGAGCACCAGCTCTCAAAGCACCCCTCCA-3′THRAP3S248/253A-antisense: 5′-TGGAGGGGTGCTTTGAGAGCTGGTGCTCGCT-3′THRAP3S248/253D-sense: 5′-AGCGAGATCCAGCTCTCAAAGATCCCCTCCA-3′THRAP3S248/253D-antisense: 5′-TGGAGGGGATCTTTGAGAGCTGGATCTCGCT-3′GSK3A-KD (or KO) cells were seeded into 6-well plates and incubated for approximately 10 days. The cells were fixed with 4% paraformaldehyde at room temperature for 30 min and then stained with 1% (w/v) crystal violet. Colonies were imaged and counted by ImageJ (https://imagej.net).For the colony formation assay, empty vector-transduced cells, and 5) or DLD-1 cells (1 × 105) were plated in an 8.0 μm, 24-well cell culture insert  in 200 μl of medium containing 1% FBS serum, with medium containing 10% FBS at the bottom of the insert. The HCT116 and DLD-1 cells were incubated for 24 h and 20 h, respectively. Then, the cells were fixed with 4% paraformaldehyde at room temperature for 30 min and stained with 1% (w/v) crystal violet. The migrated cells were imaged and counted by ImageJ.HCT116 cells (2 × 10https://www.meyerinst.com/brand/mediacybernetics).A colon cancer tissue microarray was obtained from the West China Hospital Biobank of Sichuan University. Immunohistochemistry was performed using an anti-GSK3α antibody . The expression level of GSK3α in each spot in the microarray was evaluated by Image-Pro Plus 6.0 software , and THRAP3 proteins were pulled down with an anti-THRAP3 antibody . The interacting proteins were eluted by elution buffer  and heated at 95 °C for 5 min. The eluted products were analyzed by Western blots or LC-MS/MS.Cells were harvested 48 h after transfection in cold PBS and lysed with lysis buffer  on ice for 30 min and centrifuged at 20,000in vitro or in cell lines, each experiment had 3 replicates. The phosphosites that were identified in at least 4 of 6 samples or only identified in at least 2 of 3 samples in the control or experimental group were used for difference analysis by Student’s t test. For the identification of binding proteins of GSK3α-Flag or GSK3β-Flag, each experiment had 3 replicates. The proteins that were identified in at least 4 of 6 samples or only identified in at least 2 of 3 samples in the control or experimental group with ≥2 unique peptides were used for difference analysis by Student’s t test. For the identification of binding proteins of THRAP3 mutants , each experiment had 4 replicates. Proteins identified in more than 50% of samples or only identified in the control or experimental group were used for difference analysis by Student’s t test.For the proteomics of colon cancer tissues, 156 samples were divided into 21 batches with a common reference sample and 12 quality control (QC) samples were used to monitor the stability of the machine. For the phosphoproteomics of colon cancer tissues, 140 samples were divided into 18 batches with a common reference sample. To ensure strict analysis, proteins quantified in more than 50% of samples with ≥2 unique peptides or phosphosites quantified in more than 50% of samples with localization probability ≥0.75 were used for subsequent data analysis. The significance of differences between groups was calculated by the Wilcoxon test. The significance of survival analysis was calculated by the log-rank test and the best cutoff was applied. For phosphoproteomics of GSK3α A) (B). In addition, correlation analysis of the common reference samples in different batches also indicated high reproducibility of the data acquisition (C). Principal component analysis showed that these samples were well separated into DNT and tumor groups (D), without an obvious batch effect (E).Paired tumors and DNTs collected from 78 treatment-naive colon cancer patients were subjected to TMT-based proteomics A 27). T. TA) (27A) B. In adduisition C. Princir groups D, withouh effect E.A). However, the hazard ratios of MAP2K1 in stage II-IV patients were inconsistent , indicating that GSK3α is a key kinase in colon cancer. Notably, in the tumors of colon cancer patients grouped into high-GSK3α based on the best cutoff , the expression differences of GSK3α between tumors and DNTs were much more significant , although some stages were no longer significant after TNM staging, probably due to the limited number of samples. The survival correlation result was confirmed in the Clinical Proteomic Tumor Analysis Consortium (CPTAC) cohort  and the Cancer Genome Atlas (TCGA) cohort . Notably, no significant correlation was found between the levels of GSK3β in tumors and the prognosis of colon cancer patients . Collectively, these results indicate that GSK3α is an important kinase in colon cancer and that high expression of GSK3α predicts unfavorable OS in colon cancer patients.Next, we screened the kinases significantly correlated with the OS of colon cancer patients. Out of the 5322 proteins with 2 or more unique peptides that were identified in more than 50% of samples, 158 kinases were identified. Based on Kaplan–Meier survival analysis and Cox proportional hazards regression modeling in all 78 tumors, the levels of 63 kinases were found to be significantly correlated with the prognosis of colon cancer patients. The clinical significance of the 63 kinases was further analyzed separately in the tumors of each TNM stage. As a result, out of the 63 kinases, 0 kinases in stage I tumors, 25 kinases in stage II tumors, 32 kinases in stage III tumors, and 4 kinases in stage IV tumors were found to be significantly associated with the OS of colon cancer patients . OverlapFig. 1nsistent . In contA and B and supplemental Fig. S3C). To reveal the functions of GSK3α, silencing of GSK3A in DLD-1 and HCT116 colon cell lines was carried out by a CRISPR/Cas9 system. The colony formation abilities (A and B) and the migratory abilities (A and S4C) of HCT116 and DLD-1 cells were significantly reduced when GSK3A was silenced, while the phenotypes were restored by re-expressing GSK3A (B and S4D). Immunoblotting detection of epithelial-mesenchymal transition markers showed increased E-cadherin and reduced vimentin and N-cadherin expression after GSK3A silencing (C and S4E). The expression of these markers could be recovered by restoring GSK3α expression (D), consistent with the observed cell phenotypes. Collectively, these results suggest that the high expression of GSK3α in tumors is disastrous and may promote colon cancer malignancy.To further confirm the clinical significance of GSK3α, we next performed immunohistochemical straining of colon cancer tissue microarrays from another independent colon cancer cohort using a specific anti-GSK3α antibody . Then, the purified GST-GSK3α and ATP were incubated with the extracted whole cell lysate. After tryptic digestion, the phosphopeptides were enriched with Fe-NTA agarose beads and analyzed by MS (B). In total, we identified 1236 phosphosites increased by 1.5-fold from 843 proteins in response to GST-GSK3α treatment  (C and D and in vitro mainly occurred on serine residues (E). Motif analysis of GSK3α-mediated phosphosites showed that proline residues were preferred at the +1 and +2 positions (F). Approximately 13% of phosphosites might be primed by serine phosphorylation at the +4 position (G) (H).To clarify the downstream phospho-signals regulated by GSK3α, we next performed reported , the subreported . To idenST-GSK3α A. Then, ed by MS B. In totresidues E. Motif ositions F. Approxposition G 29). I. Iin vittion (G) , global tion (G) H.Fig. 3Pin vitro, we then carried out the kinase reaction with active recombinant GST-GSK3β in vitro in duplicate (A). Pearson’s correlation coefficient of the phosphoproteome between the two repeats was 0.683 (p < 0.0001) (B). A total of 571 phosphosites, which had ratio (GSK3β/Control) > 1.5 in duplicate or were only identified in both GSK3β-treated groups, were the most reliable phospho-substrates of GSK3β in vitro . Moreover, proline residues were preferred at the +1 positions of the phosphosites and some phosphorylation events were also primed by serine phosphorylation at the +4 position . Unexpectedly, only 49 phosphosites were common substrates of GSK3α and GSK3β in vitro, indicating the huge difference between GSK3α and GSK3β as kinases . Collectively, these in vitro enzymatic results suggest significant functional differences between GSK3β and GSK3α as kinases.To compare the phospho-substrates of GSK3α with those of GSK3β uplicate A. Pearso 0.0001) B. A totaIn vitro phosphorylation assays may not represent in vivo regulation. Therefore, by using WT and GSK3A-KO colon cancer cell lines, we screened phospho-substrates exclusively regulated by GSK3α in cell lines with three biological repeats. Principal component analysis showed that the WT and GSK3A-KO samples could be clearly separated based on the phosphoproteome data (A). A total of 9501 phosphosites  on 3541 proteins were identified, of which 8069 phosphosites on 3255 proteins were quantified (B). Compared with the GSK3A-KO group, a total of 988 phosphosites were increased by 1.5-fold in the WT group  . By analyzing the ΔPs of phosphoproteins, we identified 420 proteins with significantly downregulated total phosphorylation levels in the GSK3A-KO group . Overlapping analysis identified 161 GSK3α-mediated phosphosites in vitro and in cell lines, of which 156 were specifically regulated by GSK3α but not GSK3β . The 156 phosphosites belonged to 130 proteins. Further biological process and molecular function analyses of the 130 phosphoproteins showed that RNA binding, protein binding, cadherin binding, RNA splicing, mRNA processing, DNA repair, and transcriptional regulation were most enriched . Collectively, we reveal 156 phosphosites specifically regulated by GSK3α.ome data A. A totaantified B. ComparA). We found that 11 out of the 34 phosphosites, such as THRAP3S682p and THRAP3S248p, were increased in tumors   and were involved in DNA repair, DNA binding, RNA binding, ATP binding, apoptotic process, and positive regulation of transcription by RNA polymerase II . Survival analysis showed that high levels of 5 phosphosites, including HSF1S303p, CANXS583p, MCM2S41p, POGZS425p, and SRRM2T983p, were significantly correlated with poor prognosis, while high levels of PRPF4BS431p predicted good prognosis .To analyze the clinical significance of 156 phosphosites specifically regulated by GSK3α. We analyzed the phosphoproteome data of 70 colon cancer patients . OverlapFig. 5GSK3A-Flag and GSK3B-Flag vectors in the 293T cell line (A–C) and performed pull-down experiments with an anti-Flag antibody (D). As a result, 23 proteins specifically bound to GSK3α but not GSK3β . Notably, of the 156 phosphosites specifically regulated by GSK3α, 9 phosphosites were derived from THRAP3, BCLAF1, and STAU1. Among the 9 phosphosites, 4 sites, including THRAP3S248p, THRAP3S682p, BCLAF1S512p, and BCLAF1S531p, were also identified in the phosphoproteomics analysis of paired tissues from 70 colon cancer patients (A). However, none of them showed a significant correlation with OS in our cohort of 70 colon cancer patients. Interestingly, of the 4 phosphosites, the levels of THRAP3S248p and BCLAF1S531p were significantly correlated with OS in another colon cancer cohort termed the mCRC cohort (G and H), in which more metastatic colon cancer patients were included .We speculated that of the 156 phosphosites specifically regulated by GSK3αand their corresponding proteins might have different binding affinities to GSK3α. To identify the protein substrates most tightly bound to GSK3α, we overexpressed ell line , A–C andantibody . The binantibody D. As a rpatients A. HoweveC cohort  . Because THRAP3S248p but not BCLAF1S531p was significantly increased in tumors compared with DNTs (B), the function of THRAP3S248p was further investigated. Based on the evidence from the mCRC cohort . Retro-pull-down of GSK3α and GSK3β by THRAP3 was also performed. The results showed that GSK3α but not GSK3β could be pulled down by THRAP3 (L and M). To exclude the influence of the tag on the pull-down results, we used an anti-THRAP3 antibody to enrich endogenous GSK3. As expected, GSK3α, but not GSK3β, was specifically enriched by the anti-THRAP3 antibody (N).THRAP3 is a protein with multiple phosphorylation sites. Of the 18 identified phosphosites in this work, phosphorylation at S248, S253, and S682 was specifically mediated by GSK3α ll lines J. Becausith DNTs B, the fuC cohort , we assublotting K. Retro-y THRAP3 , L and Mantibody N.S253p in colon cancer was also investigated for comparison. To disclose the functions of THRAP3S248p and THRAP3S253p in colon cancer, we silenced the THRAP3 gene in the HCT116 cell line (A) and then overexpressed two pairs of THRAP3 mutants, Flag-THRAP3S248A and Flag-THRAP3S248D, as well as Flag-THRAP3S253A and Flag-THRAP3S253D, in the THRAP3-KD HCT116 cell line (B). Of these mutants, Flag-THRAP3S248A and Flag-THRAP3S253A mimicked the effect of nonphosphorylation, and Flag-THRAP3S248D and Flag-THRAP3S253D mimicked the effect of phosphorylation. As a result, we did not observe a significant difference in clonogenic growth capability between cancer cell lines overexpressing THRAP3S248A and THRAP3S248D mutants (A). However, compared with THRAP3S248A, THRAP3S248D mimicking phosphorylation was able to enhance the migration of HCT116 cells (C), consistent with our assumption that THRAP3S248p is closely linked to tumor metastasis. It should be mentioned that both THRAP3S253D and THRAP3S248/253D had no effect on cancer cell migration (B–D).Because the S248 and S253 residues of THRAP3 are very close in the sequence, the function of THRAP3ell line A and theell line B. Of the mutants A. Howeve16 cells C, consisigration , B–D.FigS248p-binding proteins and identified 37 proteins that tended to bind to THRAP3S248D and 0 proteins that tended to bind to THRAP3S248A  and cadherin binding (p = 7.85E-03) . In contrast, 17 and 25 proteins preferred to bind to THRAP3S253D and THRAP3S253A, respectively . Collectively, the above results show the potential roles of THRAP3S248p in regulating colon cancer cell migration and DDR.We next performed pull-down experiments to identify the THRAP3in vitro and in colon cancer cell lines. Furthermore, a number of phospho-substrates of GSK3α have been identified in tissues from colon cancer patients and some of them show a significant correlation with the OS of colon cancer patients. Third, we investigated the potential roles of THRAP3S248p, a phosphosite regulated by GSK3α, in colon cancer and showed that THRAP3S248p may function in the regulation of colon cancer cell migration and DDR. Collectively, this work not only discloses the specific function of GSK3α as a kinase but also suggests GSK3α as a promising therapeutic target for colon cancer.In summary, we investigated the kinase functions of GSK3α and its specific phospho-substrates in colon cancer. Several new achievements have been gained. First, we demonstrated that high expression of GSK3α, but not GSK3β, was significantly correlated with unfavorable outcomes in colon cancer patients from four independent cohorts. Second, we identified 156 phospho-substrates specifically regulated by GSK3α, but not GSK3β, E) and phospho-substrates (F) in colon cancer cells, although we cannot exclude the possibility that the phospho-substrates of GSK3α and GSK3β may differ in different cell types. GSK3α was more closely associated with colon cancer progression and unfavorable outcomes than GSK3β (A and B and 1F). Therefore, we assume that specific inhibition or targeted degradation of GSK3α by proteolysis-targeting chimeras may be an effective strategy for the treatment of colon cancer . The reason for these results may be that the investigators used GSK3 inhibitors but classified the affected phosphosites as substrates of GSK3β. In addition, we also found that some phosphosites previously clearly reported as substrates of GSK3β can also be regulated by GSK3α in vitro, such as SRFS224p  to the cell line phosphoproteome data (B), it was observed that many more phosphosites and phosphoproteins were identified in the cell lines, indicating that the phosphorylation events are more complex in cells than in vitro. Moreover, secondary phosphorylation reactions in the cells can further increase the complexity. For example, some identified phosphoproteins, which are substrates of GSK3α, are also kinases, such as CDK1, CDK11B, and MAPK1. GSK3α-mediated phosphorylation of these kinases may further phosphorylate downstream protein substrates, which are substrates of downstream kinases rather than direct substrates of GSK3α. Notably, the number of phosphosites regulated by GSK3α was greater in the in vitro enzymatic assays (F). Previous studies have suggested that kinases may be less specific in vitro than in vivo . In addition, we also revealed the roles of THRAP3S248p in regulating cancer cell migration. Although THRAP3S248p and THRAP3S253p are very close in sequence, no synergistic effect in regulating cancer cell migration was observed (D).THRAP3, also called TRAP150 and BCLAF2, is a member of the BRCA1-interacting RNA splicing complex involved in promoting pre-mRNA splicing and subsequent transcript stability in response to DNA damage , 49. THR process D. In addobserved D.S290p is also a known phosphosite involved in the regulation of the DNA damage response  via the iProX partner repository, with the dataset identifiers PXD038081 (the phosphoproteome data of the 70 paired colon cancer tissues) and PXD038830 (other omics data except the phosphoproteome data of the 70 paired colon cancer tissues).The annotated spectra has been deposited to the MS-Viewer with the unique search key for each batch of colon cancer proteomic data provided in the in vitro phosphoproteomic data of GSK3α, and GSK3β provided in the S248A and THRAP3S248D, THRAP3S253A and THRAP3S253D) provided in the All MS raw data and the output tables have been deposited to the ProteomeXchange Consortium (This article contains There is no competing interest.


Alphaherpesviruses cause genital lesions in both animals and humans. Ozone (O3) has a strong virucidal action on enveloped and naked viruses. The aim of this study was to test the in vitro virucidal and antiviral activity of an ozone/oxygen (O3/O2) gaseous mixture against caprine herpesvirus type 1 (CpHV-1). To test the virucidal activity, the virus was exposed to different concentrations (20 and 50 μg/mL) of the gaseous mixture at different time points, and a decrease in the viral titer by up to 2.0 log10 TCID50/50 μL was observed. To test the antiviral activity, the virus was exposed to different non-cytotoxic concentrations of the gaseous mixture. When MDBK cell monolayers were treated with the gas mixture after infection with CpHV-1 at a concentration of 50 μg/mL, significant antiviral activity was observed with a decrease in viral titer of 2.0 log10 TCID50/50 μL. These findings aid future studies aimed at assessing if topical treatment of genital herpes lesions in vivo with O3/O2 gaseous mixture could be a valid and safe therapeutic option in an animal model, with possible translational applications in the therapy of human herpes simplex virus type 2 (HSV-2), which shares several biological similarities with CpHV-1.Alphaherpesviruses cause genital lesions and reproductive failure in both humans and animals. Their control is mainly based on prevention using hygienic prophylactic measures due to the absence of vaccines and limitations of antiviral drug therapy. Ozone is an oxidating gas showing a strong microbicidal activity on bacteria, fungi, viruses, and protozoa. The present study assessed the in vitro virucidal and antiviral activity of ozone against caprine herpesvirus type 1 (CpHV-1). The virucidal activity of a gaseous mixture containing O3 at 20 and 50 μg/mL was assessed against the virus at different contact times . Antiviral activity of a gaseous mixture containing O3 at 20 and 50 μg/mL was evaluated against the virus after 30 s and 60 s. Ozone displayed significant virucidal activity when used at all the tested concentrations whilst significant antiviral activity was observed using ozone at 50 μg/mL. The gaseous mixture, tested in the present study, showed virucidal and antiviral activity against CpHV-1 in a dose- and time contact-dependent fashion. Ozone therapy could be evaluated in vivo for the treatment of CpHV-1-induced genital lesions in goats using topical applications. Alphaherpesviruses  cause genital lesions and abortus in both humans and animals [Alphaherpesviruses are large, enveloped DNA viruses characterized by rapid, lytic growth cycles [Viral infections of the reproductive system are endemic in mammals and have negative repercussions on sexual and reproductive performances. Among them, the  animals ,2. Alphah cycles . Some heh cycles .In humans, herpes simplex virus type 2 (HSV-2) is a major cause of genital infection, inducing painful genital ulcers, with 13% of the population aged 15–49 years being infected [The control of HSV-2 is mainly based on prevention (through information and education) and on the use of viral DNA polymerase inhibitors . These mAlphaherpesviruses also cause reproductive failure in farm animals and economic loss for the livestock industry [industry . Caprineindustry . Abortioindustry . CpHV-1 industry . Interesindustry .On goat farms, the control of CpHV-1 is based on prevention and eradication. Different types of vaccines have been investigated since the 2000s. However, vaccines for CpHV-1 have not been released as this pharmaceutical market is not economically profitable. Consequently, the control of this infection relies on hygienic prophylactic measures , and theCpHV-1 has a significant biological similarity to HSV-2 considering its ability to induce latent infection in the sacral ganglia and similar genital lesions . This haMelissa officinalis Lamiaceae effectively inhibited HSV-2 replication [The immunosuppressive drug, mizoribine, when combined with aciclovir has been evaluated in vitro, proving effective against CpHV-1 . The admlication . Ginger lication . Moreovelication . In addilication . However3) is an alternative therapy that uses O3 in a mixture with oxygen (O2) for medical purposes [3 is an allotropic form of oxygen, composed by three oxygen atoms, organized in a relatively unstable cyclic structure that makes it a powerful oxidant agent [3 causes structural damage by protein and lipid peroxidation of the envelope and capsid, respectively, and by the destruction of nucleic acids [3 in air treatment at low concentrations . Lipid peroxidation is the main procedure used to inactivate enveloped viruses. In a study conducted by Wells et al. [3 in a dose-dependent manner. Severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) viral titer on different materials  significantly decreased after 30 min/2 h of exposure to O3 in a plexiglass chamber  [3 in air for 80 min was able to trigger a 4-fold reduction of influenza A virus titer. Conversely, this mixture was ineffective against respiratory syncytial virus [Treatment with ozone  . A gaseoal virus .3 displayed in vitro virucidal activity on HSV-1 and bovid herpesvirus type 1 (BoHV-1), inducing viral inhibition by more than 90% after 3 h of exposure [3 against CpHV-1 and HSV-2 are not available.Oexposure . Neverth3 has been used for systemic treatment using auto-haemo [3 has been used to treat postpartum pathologies [3 therapy seemingly matched or outperformed antibiotics treatments, reducing the risks of antimicrobial resistance [3 therapy, and they are mainly focused on reproductive [In large animal veterinary medicine, Oto-haemo ,32 or toto-haemo  administhologies  and to ihologies ,35 and thologies . O3 thersistance ,37 and woductive  and milkoductive  performa3/O2 gaseous mixture against CpHV-1.The aim of this study was to evaluate the in vitro virucidal and antiviral effects of a medical O3 medical generator  was used to produce an ozone/oxygen (O3/O2) gas mixture. After connection to an electrical source and to an O2 cylinder, the generator produced electrical discharges that convert O2 (substrate) into O3. The generator can produce a gas mixture containing 20 and 50 µg of O3/mL.An O3 gas flow, as previously described [An in-house method was developed to expose the Petri dishes to the Oescribed .3 generator and the output tube to a drainpipe.Two silicon tubes were assembled on the cover of a polypropylene hermetic box. The tube for the incoming flow was connected to the OAfter placing the uncovered Petry dishes inside the box, the box was hermetically sealed, and the ozone generator was switched on. The generated ozonized gas mixture entered the box through tube 1. Subsequently, the gas mixture came into contact with the Petri dishes and exited through tube 2, allowing a continuous gas flow . The box2) atmosphere in Dulbecco minimum essential medium (D-MEM) supplemented with 10% foetal bovine serum, 100 IU/mL penicillin, 0.1 mg/mL streptomycin, and 2 mM l-glutamine. The same medium was used for the antiviral assays. The CpHV-1 strain Ba-1, previously isolated from vaginitis in goat, was cultured and titrated in MDBK cells. The virus stock with a titer of 7.25 log10 tissue culture infectious dose (TCID50)/50 μL was stored at −80 °C and used for the experiments. The CpHV-1 viral suspension used in the experiments underwent preliminary centrifugation at 4000× g for 15 min to separate cellular debris.Madin–Darby bovine kidney cells (MDBK) were kindly provided by the Cell Substrate Center of the Experimental Zooprofilactic Institute of Lombardy and Emilia–Romagna. The cells were cultured at 37 °C in a 5% carbon dioxide (CO3 (O3 concentration in the gas mixture and exposure time) for the antiviral activity tests. Confluent 24 h monolayers of MDBK cells grown in 35 mm Petri dishes and maintained in D-MEM were exposed to the O3/O2 gas mixture containing different concentrations of O3 (20 and 50 μg/mL) at room temperature for 30 s (T1), 60 s (T2), 90 s (T3), 120 s (T4), 180 s (T5), and 300 s (T6). Negative controls were prepared by adding cells inside the hermetic box at the same temperature and for the same time intervals without exposure to O3/O2 gas mixture. Cytotoxicity was assessed using both direct microscopic examination of cell morphology  [A cytotoxicity assay was carried out in order to determine the conditions of cell exposure to Oborders)  and indiborders)  by folloThe cytophatic effect of CpHV-1 was evaluated on MDBK cells using an inverted microscope with live-cell imaging and hematoxylin eosin staining.3/O2 gaseous mixture against CpHV-1 was assessed at 20 and 50 μg/mL O3 concentration. The virucidal activity of O3/O2 gas mixture in the modified hermetic box at room temperature. At different time intervals (T1 to T6), 100 µL of the treated viral suspension was collected for subsequent viral titration.One mL of CpHV-1 stock virus was poured in a 35 mm Petri dishes and directly exposed to the OA 1 mL aliquot of CpHV-1 stock virus was left untreated at room temperature and similarly sampled for viral titration, serving as virus control. The experiments were performed in triplicates.3/O2 gaseous mixture containing O3 at 20 and 50 μg/mL for different exposure times (T1 and T2). To identify the step of viral inhibition by O3 against CpHV-1, two different protocols (A and B) were carried out as detailed below. All experiments were performed in triplicate.On the basis of the cytotoxicity assay results, the antiviral activity against the CpHV-1 strain Ba-1 was evaluated using the O50 CpHV-1. After virus adsorption for 1 h at 37 °C, the viral inoculum was removed, and cell monolayers were washed once with D-MEM before adding 1 mL of maintenance medium (D-MEM). Then, cell monolayers were treated with the O3/O2 gaseous mixture. Untreated infected cells were used as virus control. After 72 h, aliquots of the supernatants were collected for subsequent viral titration. Confluent monolayers of MDBK cells of 24 h in 24-well plates were used. Cells were infected with 100 μL of viral suspension containing 100 TCID3/O2 gaseous mixture. Then, the monolayers were washed once with D-MEM and infected with 100 μL viral suspension containing 100 TCID50 CpHV-1. After virus adsorption for 1 h at 37 °C, the inoculum was removed and the monolayers were washed with D-MEM before adding 1 mL of maintenance medium (D-MEM). Untreated infected cells were used as virus control. After 72 h, aliquots of each supernatants were collected for subsequent viral titration.Confluent monolayers of MDBK cells of 24 h in 24-well plates were used. Cells were treated with the O−8) of each supernatant were titrated in quadruplicates in 96-well plates containing MDBK cells. The plates were incubated for 72 h at 37 °C in 5% CO2. Cytopathic effect of CpHV-1 on MDBK cells was evaluated using an inverted microscope with live-cell imaging or using haematoxylin eosin staining. Based on the cytopathic effect, TCID50/50 μL was calculated by following the Reed–Muench method [Ten-fold dilutions .All data were expressed as mean ± SD and analyzed using GraphPad Prism (v 9.5.0) program . To assess the normality of distribution, Shapiro–Wilk test was performed. Two-way factorial ANOVA, with concentration * time as factors, and Tukey test as post hoc test were applied to cytotoxicity results. Student’s 3/O2 gas mixture containing O3 at 20 and 50 μg/mL did not produce any changes in cell morphology at T1 and T2, whereas morphological signs of cytotoxicity were consistently observed in cells exposed to O3 at 20 and 50 μg/mL for longer time intervals .Direct exposure of MDBK cells to O3 at 20 and 50 μg/mL at different time intervals (T1 to T6) resulted in increasing cytotoxicity in a dose- and time contact-dependent fashion (3 at 20 μg/mL at T1 and T2 induced cytotoxicity of 0.53% (±0.15) and 3.64% (±0.8), respectively, which was below the cytotoxic threshold. Higher cytotoxicity of 31.03% (±1.1), 36.78% (±1.2), 40.10% (±1.3), and 81.52% (±2.3) was observed at T3, T4, T5, and T6, respectively  and 3.61 % (±0.95), respectively, which was below the cytotoxic threshold. Higher cytotoxicity of 59.77% (±1.3), 65.51% (±1.6), 82.57% (±1.8), and 85.39% (±2.6) was observed at T3, T4, T5, and T6, respectively  and 50  μg/mL between different time intervals (T1–T6). Using a two-by-two comparison of cytotoxicity induced by O3 at 20 and 50 μg/mL, a statistically significant decrease in cytotoxicity was consistently observed at different time intervals (T1–T6). Conversely, the comparison between O3 at 20 μg/mL at T4 and T5 and between O3 at 50 μg/mL at T5 and T6 lacked statistical significance (p > 0.05). The ANOVA model showed a statistically significant decrease in cytotoxicity in MDBK cells treated with O3 at 20 and 50 μg/mL at T1 and T2, which were below the cytotoxicity threshold.On the basis of these results, the antiviral activity assays were carried out using OCytopathic effect of CpHV-1 on MDBK cells is displayed in p > 0.05). Data from the virucidal activity assay showed that the O3/O2 gaseous mixture containing O3 at 20 μg/mL significantly reduced CpHV-1 titer by 1.25 log10 TCID50/50 μL (p < 0.05) at T1 and T2, 1.50 log10 TCID50/50 μL (p < 0.05) at T3 to T5, and 2.00 log10 TCID50/50 μL at T6 (p < 0.0001) when compared with the untreated control.Data obtained were analyzed using Shapiro–Wilk test, confirming the normality of distribution  at T1 and T2, 1.50 log10 TCID50/50 μL (p < 0.05) at T3 to T4, 1,75 log10 TCID50/50 μL (p < 0.05) at T5, and 2.00 log10 TCID50/50 μL at T6 (p < 0.0001) when compared with the untreated control  with the infected cells treated with the O3/O2 gas mixture containing O3 at 20 μg/mL at T1 and T2 (7.00 ± 0.25 log10 TCID50/50 μL), a slight decrease in viral titer (0.25 log10) was observed, however, with no statistical significance (p > 0.05). Comparing the viral titer of the untreated infected cells (7.25 ± 0.25 log10 TCID50/50 μL) and of the infected cells treated with O3 at 50 μg/mL at T1 and T2 (6.00 ±0.25 log10 TCID50/50 μL), a significant decrease in the viral titer (1.25 log10) was observed (p < 0.05)  . 10 TCID50/50 μL) with the viral titer of the infected cells pre-treated with the O3/O2 gas mixture containing O3 at 20 and 50 μg/mL at T1 and T2 (7.25 ± 0.25 log10 TCID50/50 μL), no decrease in viral titer was observed  to decipher a possible field application in veterinary and human medicine. Indeed, CpHV-1 and HSV-2 share important biological characteristics, and the infection by CpHV-1 in goats is considered a valid animal model for the study of infection of HSV-2 and its therapy in humans, .3 in treating genital infections of farm animals [3. Inoculation of O3 using foams into the vagina and uterus of cows affected by urovagina has been shown to decrease the “open days” and the number of artificial inseminations required to induce pregnancy in cows affected by urovagina. Moreover, the beneficial role of O3 in the repair process of the vaginal and cervical mucosa was observed [There are several in vivo and in vitro studies published in the literature, addressing/demonstrating the therapeutic potential of O animals ,35 and dobserved . The int3 has been reported for different viruses. This gas has a potent oxidant action on microorganisms [3 could inactivate viruses by destroying guanine residues of nucleic acids [The virucidal effect of Organisms ,27,28,29rganisms . In addiic acids  as demonic acids ,43. 3/O2 gas mixture containing O3 at 20 and 50 μg/mL was evaluated against CpHV-1 at different time points (T1 to T6) to assess its virucidal properties. Furthermore, the in vitro antiviral activity of the O3/O2 gas mixture containing O3 at 20 and 50 μg/mL against CpHV-1 was evaluated at two different time points, T1 and T2. The concentrations of 20 and 50 μg/mL were chosen based on the cytotoxic activity obtained using XTT test on MDBK cells for different time points (T1–T6). Both O3 concentrations were regarded as non-cytotoxic (below the cytotoxicity threshold of 20%) at T1 and T2. At later time points, starting from T3, an increase in cytotoxicity was observed primarily at the concentration of 50 μg/mL (over 60%).In this study, an O3 in O3/O2 gas mixture  were assessed in other cell lines, i.e., HeLa [3 at 100 μg/mL [In other studies, concentrations from 10 to 20 μg/mL of Oe., HeLa  and SH-Se., HeLa . These c00 μg/mL .3 because they are protected by the presence of albumin, which with its reducing group -SH is one of the most protective compounds [3 concentration is crucial as high concentrations could overwhelm this protective mechanism, leading to cell damage and death [Eukaryotic cells demonstrate a certain resistance in vitro to the prooxidant effect of Oompounds . Of cournd death .10 TCID50/50 μL at T6.In the virucidal activity assay, exposure of CpHV-1 to the gas mixture was able to reduce significantly the viral titer in a time-dependent manner, leading to a decrease in viral titer of up to 2.00 log3 at 20 and 50 μg/mL at T1 and T2, in order to identify the phase in which viral replication might be inhibited, cells were infected with CpHV-1 before (protocol A) and after (protocol B) the treatment with O3.To evaluate the antiviral activity at the maximum non-cytotoxic dose of O3 was used at a concentration of 20 μg/mL, we observed a very slight and statistically insignificant reduction in viral titer (0.25 log10 TCID50/50 μL), suggesting that O3 was not able to inhibit virus replication. O3 at a 50 μg/mL concentration induced a statistically significant reduction of viral titer (1.25 log10 TCID50/50 μL). In protocol A, when O3 at 20 and 50 μg/mL (protocol B) did not reduce the viral titer, hinting a lack of inhibition of O3 in virus uptake and replication.Pre-treatment of the cells with O3 in vivo could be implemented, primarily in the veterinary field. Future studies could address the use of O3 in CpHV-1-infected goats to gain more translational information for human herpesvirus genital infection. In a previous report, the inactivation of herpes viruses (HSV-1 and BoHV-1) with O3 was achieved by applying a long exposure time (1 to 3 h) [3/O2 gas mixture required to trigger significant effects against CpHV-1 was lower, and this could be an advantage for in vivo experiments. Long treatment times would not be ideal due to excessive stress induced to animals, mainly for ones in animal containment.Overall, as significant results were obtained with short exposure times, the use of O to 3 h) . Compare to 3 h) ,30,31,323/O2 gaseous mixture against CpHV-1. A short exposure of the virus to O3 at low concentration (20 μg /mL) was required to achieve partial virus inactivation. This study represents the first step to assess the clinical efficacy of O3 therapy for the treatment of genital herpes infection. Further essential steps will be the evaluation of the in vitro effects on vulvar and vaginal epithelial cells as well as of the efficacy of treatment of CpHV-1-associated genital lesions in infected goats in vivo. Furthermore, it might be interesting to test whether O3 is also effective on HSV-2 given the close biological similarity with CpHV-1.We reported the in vitro virucidal and antiviral activity of a medical O


Xenopus laevis oocyte swelling assays. We found that HvNIP2;1 permeates water, boric and germanic acids, sucrose, and lactose but not d-glucose or d-fructose. Other saccharides, such as neutral  and charged  aldoses, disaccharides , trisaccharide raffinose, and urea, glycerol, and acyclic polyols, were permeated to a much lower extent. We observed apparent permeation of hydrated KCl and MgSO4 ions, while CH3COONa and NaNO3 permeated at significantly lower rates. Our experiments with boric acid and sucrose revealed no apparent interaction between solutes when permeated together, and AgNO3 or H[AuCl4] blocked the permeation of all solutes. Docking of sucrose in HvNIP2;1 and spinach water-selective SoPIP2;1 aquaporins revealed the structural basis for sucrose permeation in HvNIP2;1 but not in SoPIP2;1, and defined key residues interacting with this permeant. In a biological context, sucrose transport could constitute a novel element of plant saccharide-transporting machinery. Phylogenomic analyses of 164 Viridiplantae and 2993 Archaean, bacterial, fungal, and Metazoan aquaporins rationalized solute poly-selectivity in NIP3 sub-clade entries and suggested that they diversified from other sub-clades to acquire a unique specificity of saccharide transporters. Solute specificity definition in NIP aquaporins could inspire developing plants for food production.Aquaporins can facilitate the passive movement of water, small polar molecules, and some ions. Here, we examined solute selectivity for the barley Nodulin 26-like Intrinsic Protein  embedded in liposomes and examined through stopped-flow light scattering spectrophotometry and  Physcomitrella and lycopods (Aquaporins (AQPs) are fundamental to water and other solute movements in nearly all living organisms. AQPs are membrane proteins classified in the family of major intrinsic proteins (MIPs) that transport water and neutral solutes , 2, althlycopods . AQPs frlycopods , 6, 7.2 (3 (2 (2O2 but not water (aqpN) gene was acquired by some plants via horizontal transfer shedding light on NIPs solute evolution and selectivity , an abundant protein in the peribacteroid membrane of nitrogen-fixing root nodules, are functional equivalents of aquaglyceroporins  and occu2 , NH3  in SoPIP2;1 (PDB-Protein Data Bank 2B5F) (6qim) (5i32) (7cjs and 7nl4)  , 39, AtP) (6qim) , AtTIP2;) (5i32)  and recend 7nl4) , 43 havend 7nl4) .To understand the function of HvNIP2;1 in the context of metalloid toxicity, it is fundamental to understand that this trait is a major problem in cereal crops around the world . AlthougPichia pastoris membranes through embedding in liposomes. The selectivity of HvNIP2;1 towards neutral solutes and ion pairs is examined, establishing that this AQP permeated water, BA, germanic acid, sucrose, and MgSO4 and KCl ion pairs at relatively high rates, but also permeated at low rates acyclic polyols, urea and glycerol, other ion pairs, and some mono- and disaccharides of various stereo chemistries. Molecular docking was performed on the monomeric HvNIP2;1 3D AlphaFold 3D model (accession AF-D8V828) to provide molecular-level descriptions of permeation, including identifying structural elements that underlie solute poly-specificity. These data are compared to those of water-selective SoPIP2;1 AQP in an open state conformation . HvNIP2;1 was solubilized in styrene-maleic anhydride co-polymer 3:1 (SMA) (A) was reconstituted in 1, 2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) liposomes that were floated on an iohexol (Accudenz) gradient (B) used to define permeation properties of HvNIP2;1.The native sequence of HvNIP2;1 was cloned into the A fusion , 47, 48 :1 (SMA)  and puri:1 (SMA) , 47. Nea:1 (SMA) A was recgradient , 51 to ogradient B used toA). The inspection of the tetrameric model of HvNIP2;1, built on the coordinates of OsNIP2;1 .Electrophoretic profiles of purified HvNIP2;1 in the form of diffused bands suggested that HvNIP2;1 was N-glycosylated at putative S26 although O-glycosylation sites could also be occupied. No degradation of purified HvNIP2;1 was observed after 3 weeks at 4 °C indicating that it was amenable for permeation studies. To confirm that HvNIP2;1 was incorporated in DMPC liposomes and not simply aggregated during HvNIP2;1 reconstitution, liposomes with embedded HvNIP2;1 were subjected to floatation by iohexol density gradient ultra-centrifugation. As indicated, fractions 2 to 4 containing proteo-liposomes with HvNIP2;1, detected with anti-His antibody, floated near the top of the gradient, and most of HvNIP2;1 incorporated in DMPC liposomes B.3, a potent blocker of AQPs (C). HvNIP2;1 permeated BA and germanic acid at higher rates, while glycerol, d-mannitol and d-sorbitol, and urea were permeated at low rates , and hydrated ion pairs KCl and MgSO4, while lactose, the CH3COONa, and NaNO3 ion pairs were transported at lower rates. Conversely, NaF, the neutral monosaccharides d-glucopyranose, d-fructopyranose, d-galactopyranose, d-mannopyranose, and d-mannopyranopheptaose, charged monosaccharides d-glucosamine, N-acetyl β-d-glucosamine and d-glucuronic acid, disaccharides trehalose, cellobiose and gentiobiose, and trisaccharide raffinose were permeated at even lower rates (P)   for wate2O >> BA > germanic acid.Water and metalloids: Hd-mannitol > d-sorbitol.Polyols and urea: urea > glycerol > l-arabinofuranose > lactose > d-glucose > d-fructose.Saccharides: sucrose > 4 > KCl > CH3COONa > NaNO3.Ion pairs: MgSO2O >> MgSO4 ≥ BA > KCl ≥ sucrose > urea > glycerol > CH3COONa > d-mannitol ≥ lactose > germanic acid > NaNO3 > d-sorbitol > d-glucose > d-fructose.All permeants: HXenopus laevis oocytes that were injected with cRNA of HvNIP2;1 or water as controls (A) confirming water permeation by HvNIP2;1, and isosmotic BA, sucrose, and d-glucose concentration gradients (B and C), while d-glucose permeation did not occur (D), as also shown using the stopped-flow light scattering analyses (C). In oocytes, incubated with 0.5 mM H[AuCl4] , and the plant structural homologs AtTIP2;1 (PDB 5i32), AtPIP2;4 (PDB 6qim), and OsNIP2;1 (PDB 7cjs and 7nl4). As shown in Figure 4A, the HvNIP2;1 model has nearly an identical secondary structure distribution to that of the crystal structures of Lsi1 (A). We also utilised the 3D AlphaFold HvNIP2;1 model . Thus, the respective 0.67 Å and 0.45 Å root-mean-square deviation values between OsNIP2;1 and Lsi1, at the sequence similarity/identity of 74%/68% and 80%/71% , while the residues of the two conserved NPA1 and NPA2 motifs  were located deeper in the pore, in accordance with the positions of these structural elements in both crystal structures. A single solute-conducting pore in HvNIP2;1, investigated through Caver .To investigate the structural features of HvNIP2;1 and work out the structural basis of sucrose permeation, we used the structural bioinformatics approach and aligned the sequences of HvNIP2;1 and SoPIP2;1 , which gorithm; ) were ungh Caver  in monomtructure , 39 , by means of HDOCK, which is based on a hybrid algorithm of template-based modeling and ab initio free docking ; these interactions were formed at separations between 2.2 Å and 3.2 Å.To investigate the permeation of sucrose, observed in HvNIP2;1 through stopped-flow light scattering spectrophotometry and oocyte swelling, this disaccharide molecule was docked in HvNIP2;1 B, by mea docking . The docB; right-top panel; SoPIP2;1 – yellow cartoon). With SoPIP2;1, the sucrose disaccharide could not localise inside the pore (approximate diameter range between 1.0 Å and 2.1 Å) and it was always contained within the surface or at the entry or exit to the pore of the SoPIP2;1 in all examined 100 poses. Here, the prominent GGGANSVALGNYK loop (residues 149–161), was localized at the entry of SoPIP2;1, which obstructed the pore and prevented the passage of the sucrose molecule through the pore. Underneath this loop, the second TGTGINPAR loop (residues 217–225) was identified, consisting of nine residues, which also carried the NPA motif and highly conserved R225. Residues from both loops, more specifically A152, N153 and S154 (first loop) and T217 and R225 (second loop) made close contacts with sucrose at separations between 2.2 Å and 3.5 Å, immobilised it and prevented its passage through the SoPIP2;1 pore . Additionally, it would also be impossible for sucrose to bypass the aromatic/R constriction region point of around 2.1 Å  phylogenetic analysis as a tool. This widely used approach embodies a fast-maximum likelihood tree search algorithm that reconstructs phylogenetic trees with exceptional likelihood scores. Here, we analyzed 164 Viridiplantae MIP sequences from  vulgare . These a vulgare  and addi vulgare  clustereA. thaliana and A. trichopoda. We also indicated that HvNIP2;1 split from P. patens that carried the FAAR signature, and that the three NIP1, NIP2, and NIP3 representative clades emerged prior to the evolution of tracheophytes (ferns and seed plants), where the NIP2 and NIP3 clades contained basal Physcomitrella sequences, and where a NIP1 representative was perhaps lost. The functional evolution of the NIP clades was evident, and this was indicated by the fact that the selectivity filter in Physcomitrella (FAAR) diversified into the GSGR signature, specifically in the NIP3 sub-clade, following the tracheophyte/Physcomitrella split. This finding contrasted with the PIP entries, where the FHTR signature was highly conserved in all examined PIP entries , purines (adenine), pyrimidines , and thiourea, but not cyclic saccharides  and charged  monosaccharides, disaccharides , and the trisaccharide raffinose were permeated at lower rates than sucrose, l-arabinofuranose and lactose  and AtPIP2;2, HvPIP2;8 and OsPIP1;3, when expressed in oocytes or Human Embryonic Kidney (HEK) 293 cells , as these small molecules could permeate passively through lipid bilayers (Pwater = 3.98 × 10−2 cm s−1) was 240-fold higher than that of empty liposomes (Escherichia coli water-permeable aquaporin-Z (Pwater = 2.83 × 10−2 cm s−1) (Pwater = 1.2 × 10−2 cm s−1)  involved in pollen cell wall formation   but high cm s−1) . The comormation , indicatormation , althougormation  and soybormation , where lX. laevis, where water, BA, and sucrose transport but not glucose transport occurred . To explain the inability of HvNIP2;1 to permeate smaller monomeric d-glucose or d-fructose molecules compared to the disaccharide sucrose (a larger molecule) that we observed, we suggest that the voluminous HvNIP2;1 pore is decorated with hydrophobic and hydrophilic residues. We assume that in the HvNIP2;1 pore, hydrophilic glucose could get trapped in pockets, while sucrose, due to its larger size avoids this trapping and slides through the pore. Conversely, a narrower and less capacious SoPIP2;1 would collectively offer an unfavorable milieu for saccharide molecules to pass through.Sucrose permeation by the 3D AlphaFold model of HvNIP2;1 was confirmed by docking, where HvNIP2;1 contributed with M107, H160, G216, S217, and N219 as pivotal residues , while nFig. 4In planta, HvNIP2;1 AQP is expressed in roots and localized to epidermal and cortical cells of seminal roots and hypodermal cells in lateral roots , 10. TheTo investigate the evolutionary origin of NIP AQPs, the phylogenetic reconstruction of 164 Viridiplantae MIPs was conducted to reveal that PIP, TIP, and SIP clades diversified before the chlorophyte  and embryophyte (land plants) split. This finding proposes that a single duplication event resulted in SIPs, or that multiple duplication events occurred before the chlorophyte and embryophyte split. These analyses used a relatively complex substitution model avoiding insufficient data for maximum likelihood but still kept accuracy during reconstruction, although there was little support for establishing deep relationships in MIPs as these sequences are relatively short. Hence, we restricted our maximum likelihood analyses to Viridiplantae only , and sepNotably, and relevantly to the experimental permeation data obtained in this work, the NIP3 sub-clade segregated clearly and carried the GSSR selectivity filter signature. This NIP3 clade was lost or reduced in eudicots, although the deep phylogenetic relationships of the AQP clades were less supported . NeverthIn conclusion, our phylogenomic analyses of sequences comprising 3157 AQPs  and S3 pPichia cells, urea/alkali treatment of microsomal membrane fractions, solubilization from the urea/alkali-treated microsomal membrane fractions of HvNIP2;1 and other analytical techniques are detailed in Materials and procedures used for fractionation and enzymatic digestion and disruption of HvNIP2;1-Myc-6xHis-pPICZ-B DNA fusion, destined to be transformed in P. pastoris, and Pichia clone selection were conducted as described  in the pPICZ (frame B) (Invitrogen) expression vector yielding the native escribed , 47, 48.P. pastoris cells (Invitrogen) transformed with the linearized HvNIP2;1-Myc-6xHis-pPICZ-B were streaked on the YPD plates  containing 100 μg/ml zeocin (InvivoGen) and incubated for 2 days at 28 °C , pellets resuspended in 10% (v/v) glycerol, and stored at −80 °C.Competent X-33 The SMA-solubilized preparation  was incu2 to disrupt the SMA polymer. The 50 mM MgCl2 concentration was maintained in all the buffers after this step. No protein precipitation was observed during the reconstitution of HvNIP2;1 in DMPC liposomes.DMPC lipids were dissolved at 10 mg/ml in chloroform, dried on a rotary evaporator under vacuum for 30 min, and rehydrated in the liposome buffer (LB) [20 mM Tris-HCl (pH 8.0) containing 100 mM KCl]. The lipid mixture was sonicated until clear and filtered through 100 nm pores (Avanti) of a uniform size using the LiposoFast Hand Extruder (Avestin). The SMA-solubilized HvNIP2;1 protein preparation and filtered DMPC liposomes were mixed at a ratio of 1:50 on a weight basis, mixed by gentle shaking at room temperatures for 15 min and the mixture was dialyzed in LB containing 50 mM MgClg, 4 h, four °C) in the L-80XP ultra-centrifuge using the SW55Ti swinging-bucket rotor (Beckman Coulter). After ultra-centrifugation, 60 μl fractions were sequentially collected from the top of the gradient to the bottom and examined by SDS-PAGE and IB. Selected fractions of liposomes with embedded HvNIP2;1 were pooled, centrifuged , resuspended in LB, dialyzed for 18 h in LB at 4 °C using 10-kD cut-off Slide-Alizarine dialysis cups (Thermo Fischer Scientific) and used in stopped-flow light scattering recordings. Fractionated proteo-liposomes stored on crushed ice remained stable for up to 5 days.Equal volumes 50 μl) of liposomes with reconstituted HvNIP2;1 and 80% (w/v) iohexol in the 25 mM HEPES-NaOH buffer (pH 7.5) containing 100 mM NaCl and 10% (w/v) glycerol were mixed  μl of li. The mix3 at 0.5 mM was used  × rate constant/(Vw × Co) (Psolutes = (V/A) × rate constant   and permconstant , where V2, and 1 mM MgCl2) supplemented with solutes at 160 mM concentration, which equaled to 200 mOsmol/kg osmolarity. 0.5 mM H[AuCl4]  were taken from the AlphaFold Protein Structure Database . In this AlphaFold HvNIP2;1 model, six residues were positioned in disallowed regions, which were all positioned at the N-terminal loop  and which was not used in docking or structural evaluations of permeation pores.The coordinates of a monomeric 3D HvNIP2;1 model from barley (Spinacia oleracea L.) SoPIP2;1 (PDB accession 2B5F) in an open state conformation (7cjs) (7nl4)   OsNIP2;1) (7nl4)  and wereThe root-mean-square deviation values of 0.67 Å and 0.45 Å, between the HvNIP2;1 AlphaFold model and OsNIP2;1 or Lsi1, indicated high similarity at structural levels of all three AQPs, which was underlined by high sequence identities amongst three AQPs  and used for analyses of sucrose dispositions.Docking of sucrose into HvNIP2;1 and SoPIP2;1 was performed using the HDOCK server , which iPermeation tunnels in HvNIP2;1 and SoPIP2;1 and their properties such as lengths and diameters, were evaluated by Caver v.3.0.3   (https://www.ebi.ac.uk/Tools/hmmer/search/hmmsearch)    . Archaeamsearch) , 83. Barmsearch) . Excessimsearch)  was usedmsearch)  was usedmsearch)  was usedAs MIP sequences were relatively short, data could not support the fit for complex, parameter-rich models to deep alignments. Thus, maximum-likelihood analyses were restricted to Viridiplantae, while the analyses of Virdiplantae, archaean, bacterial, fungal, and metazoan sequences were limited to distance methods. Substitution model selection for the Viridiplantae and all domain data was performed using ModelTest-NG  with LG+All data are included within the manuscript and its This article contains The authors declare that they have no conflicts of interest with the contents of this article.


Plasmodium falciparum requires purine nucleoside uptake from host cells. The indispensable nucleoside transporter ENT1 of P. falciparum facilitates nucleoside uptake in the asexual blood stage. Specific inhibitors of PfENT1 prevent the proliferation of P. falciparum at submicromolar concentrations. However, the substrate recognition and inhibitory mechanism of PfENT1 are still elusive. Here, we report cryo-EM structures of PfENT1 in apo, inosine-bound, and inhibitor-bound states. Together with in vitro binding and uptake assays, we identify that inosine is the primary substrate of PfENT1 and that the inosine-binding site is located in the central cavity of PfENT1. The endofacial inhibitor GSK4 occupies the orthosteric site of PfENT1 and explores the allosteric site to block the conformational change of PfENT1. Furthermore, we propose a general “rocker switch” alternating access cycle for ENT transporters. Understanding the substrate recognition and inhibitory mechanisms of PfENT1 will greatly facilitate future efforts in the rational design of antimalarial drugs.By lacking de novo purine biosynthesis enzymes,  PfENT1 is a promising antimalarial drug target. Here, authors report cryo-EM structures of PfENT1 that, together with biochemical work, suggests PfENT1 is an inosine transporter and describe the inhibitory mechanism of the endofacial inhibitor, GSK4. Plasmodium parasites, remains a severe infectious disease affecting human health worldwide. According to the World Health Organization, there were 241 million infection cases worldwide, and 627 000 people died from malaria in 20201. Over the past several decades, many antimalarial drugs, including chloroquine and artemisinin, have been used to eradicate malaria3. However, the emergence drug-resistant Plasmodium parasites essentially delay malaria elimination6. As a result, there is a pressing need to develop antimalarial drugs9.Malaria, caused by infection with 11. Consequently, the salvage pathway mediated by nucleoside/nucleobase transporters is essential to maintaining DNA/RNA synthesis and proliferation of Plasmodium parasites14. P. falciparum nucleoside transporter 1 , located predominantly in the parasite plasma membrane (PPM), is the primary nucleoside transporter mediating nucleoside uptake from erythrocytes18. Previous investigations have clarified that targeting the purine uptake pathway interferes with the survival of Plasmodium20. Crucially, PfENT1, a member of equilibrative nucleoside transporters  family, shares only 17% sequence identity with the human equilibrative nucleoside transporter ENT1 (hENT1) and is unsusceptible to hENT1 inhibitors16, which provides an opportunity to design inhibitors targeting PfENT1 specifically. As expected, the reported PfENT1 inhibitors, including GSK4 (5-methyl-N-[2-(2-oxo-1-azepanyl)ethyl]−2-phenyl-1,3-oxazole-4-carbox-amide), disrupt the transport activity of PfENT1 and prevent the proliferation of parasites22. Hence, PfENT1 is a promising target to be exploited for novel antimalarial drug development25.Malaria parasites, missing the de novo purine synthesis pathway, are purine auxotrophs26. Moreover, the structure of PfENT1 in complex with GSK4 provides a detailed inhibitory mechanism of the specific inhibitor. Our investigation will firmly deepen the understanding of the nucleoside transport mechanism and facilitate antimalarial drug design.Here, we report three cryo-EM structures of PfENT1 in the apo, inosine-bound, and inhibitor-bound states at a resolution of 3.3 Å, 3.1 Å, and 4 Å, respectively. With extensive biochemical analysis, we decipher the inosine-binding site of PfENT1. Our structures also suggest the “rocker switch” transport model for ENTs28, yet the preferred substrate is controversial. Therefore, we overexpressed wild-type PfENT1 in Sf9 insect cells, purified the monodisperse peak in solution  yeast strain  are the primary substrates of PfENT1, which is different from the human equilibrative nucleoside transporter (hENT1) with the specific recognition of the adenine base of nucleoside35.Considering inosine and hypoxanthine as the major purines in human bloodY190A mutant with a similar inosine-binding affinity presented better solution properties  and PfENT1Y190A-Nb48 (inosine-bound) at 3.3 Å and 3.1 Å, respectively  helixes with pseudosymmetry Fig. . TM1-6 aY190A-Nb48 complex in the presence of inosine. Comparing the density of PfENTY190A-Nb19, the omitted density indicative of one inosine molecule was clearly visible in the central cavity of the PfENT1Y190A-Nb48 structure  from the C domain of PfENT1 involved in ribose recognition directly. The lower availability of residues interacting with the ribose group is probably responsible for the nucleobase transport activity and substrate specificity of PfENT141. In addition, we found four conserved polar residues  from the C domain, which are all within 6 Å of the ribose group of inosine  and concentrative nucleoside transporters (CNTs) recognize the ribose moiety of nucleoside via extensive polar contacts37. The structural alignments between the individual amino or carboxyl domains of PfENT1 to hENT1 are similar to the r.m.s.d. value at approximately 3 Å  shares the similar conformation with hENT1  was synthesized by GENEWIZ. For PfENT1GFP, the cDNA of GFP was cloned into PfENT1 between K370 and K371. All constructs were subcloned into a modified pFastBac1 with an N-terminal 10 X His tag. The baculovirus was generated in DH10Bac for recombinant protein expression. After 72 h of infection, Sf9 insect cells were harvested. For structural determination of the PfENT1 protein, Sf9 cells were solubilized in lysis buffer , 1% (w/v) lauryl maltose neopentyl glycol , and protease inhibitor cocktail  at 4 °C for 2 h. After centrifugation at 20,000 × g for 60 min at 4 °C, the detergent-soluble supernatant was collected via centrifugation at 4 °C for 60 min and incubated with Ni-NTA resin (QIAGEN) at 4 °C for 30 min. The resin was further washed with 25 mM Tris-HCl pH 8.0, 150 mM NaCl, 10 mM imidazole, and 0.06% (w/v) glyc-diosgenin  and eluted with buffer containing 25 mM Tris-HCl pH 8.0, 150 mM NaCl, 250 mM imidazole, and 0.06% (w/v) GDN.The codon-optimized cDNA of E. coli BL21(DE3) cells. For recombinant expression, the cells were cultured at 37 °C and induced by 0.2 mM isopropyl β-D-thiogalactoside (IPTG) at 22 °C overnight once the cell density reached an OD600 of 0.8. The cells were harvested and homogenized in lysis buffer . The cells were lysed by a high-pressure homogenizer (ATS) and clarified via centrifugation. The supernatant was applied to Ni-NTA resin (QIAGEN) and eluted from the resin using buffer containing 25 mM Tris-HCl (pH 8.0), 30 mM NaCl, and 300 mM imidazole. The elution was further applied to ion-exchange chromatography  and gel filtration . Finally, nanobodies were eluted in buffer containing 25 mM Tris-HCl (pH 8.0) and 30 mM NaCl and concentrated to ~50 mg/ml for further purification.The nanobodies for PfENT1 were generated by Chengdu NB Biolab Co., Ltd. Subsequently, the cDNA of nanobodies was subcloned into pET21b and overexpressed in The purified PfENT1 proteins were concentrated and incubated with nanobody at a molar ratio of 1:1.5 at 4 °C for 1 h. The PfENT1-nanobody complex was subjected to size-exclusion chromatography  in buffer containing 25 mM MES pH 6.0, 150 mM NaCl, and 0.06% (w/v) GDN. The peak fractions were pooled and concentrated to 8-12 mg/mL for cryo-EM analysis.GFP protein, Sf9 cells  were solubilized in lysis buffer , 2% (w/v) n-dodecyl-β-D-maltopyranoside (DDM), and protease inhibitors  at 4 °C for 1.5 h. After centrifugation at 18,000 × g for 30 min at 4 °C, the detergent-soluble supernatant was collected and incubated with Ni-NTA resin (QIAGEN) at 4 °C°C for 30 min. The resin was further washed with 25 mM Tris-HCl pH 8.0, 150 mM NaCl, 30 mM imidazole, and 0.02% (w/v) n-dodecyl-β-D-maltopyranoside  and eluted with buffer containing 25 mM Tris-HCl pH 8.0, 150 mM NaCl, 300 mM imidazole, and 0.02% (w/v) n-dodecyl-β-D-maltopyranoside . The purified protein was concentrated and subjected to size-exclusion chromatography  in buffer containing 25 mM Tris-HCl pH 8.0, 150 mM NaCl, and 0.02% (w/v) n-dodecyl-β-D-maltopyranoside . The peak fractions were pooled and concentrated to 4-12 mg/mL for cryo-EM analysis.For structural determination of PfENT1For the in vitro binding assay, Sf9 cells were solubilized in lysis buffer containing 2% (w/v) n-dodecyl-β-D-maltopyranoside  and protease inhibitor cocktail  at 4 °C for 2 h. The soluble supernatant was collected via centrifugation and incubated with Ni-NTA resin (QIAGEN) at 4 °C for 1 h. The resin was washed and eluted using lysis buffer containing 0.05% (w/v) DDM and 30 mM/300 mM imidazole. The protein was further purified by size-exclusion chromatography  in buffer containing 25 mM MES (pH 6.0), 150 mM NaCl, and 0.05% (w/v) DDM. The peak fractions were pooled and concentrated to 10~50 μM for the nucleoside-binding assay.2Cl2 at room temperature, oxalyl chloride (2.0 equiv) and 2 drops of DMF were added, and the mixture was stirred at room temperature overnight. The resulting mixture was concentrated under reduced pressure to quantitatively afford acid chloride 1.The synthesis process is summarized in Supplementary Fig. 3 was stirred with BocNH(CH2)2NH22 (1.0 equiv) and K2CO3 (1.5 equiv) in CH2Cl2 at 0 °C for 30 min and 25 °C overnight. The sample was filtered to remove solids and washed with water, 1 M HCl, and brine. The residue was concentrated under reduced pressure to afford pale yellow crystals 4.6-Bromohexanoic acyl chloride 4 (1.0 equiv.) in dry THF was added dropwise at rt over 30 minutes. The mixture was then stirred for an additional 12 h. The mixture was then cooled (ice), acidified (4 M HCl), and extracted with DCM. The combined organic extracts were washed with brine, dried, concentrated in vacuo, and purified by column chromatography to yield compound 5.A suspension of sodium hydride in oil  was suspended in dry THF, and a solution of 5  in 5 mL DCM. The solution was stirred at room temperature for 2-3 h. The reaction was completely detected by TLC. The reaction mixture was quenched by the addition of 2 M NaOH and extracted with ethyl acetate. The combined organic layers were washed with brine, dried over Na2SO4, filtered, and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography in ethyl acetate/petroleum to give desired compound 6.TFA (1.5 mL) was added to a solution of compound 6 (1.05 equiv.) and triethylamine (2.0 equiv.) were combined in dichloromethane under nitrogen, giving a colorless suspension. Benzoyl chloride 1 (1.0 equiv.) in dichloromethane was added dropwise over 10 min, and the solution was stirred overnight. The reaction mixture was quenched by the addition of water, and the mixture was extracted with CH2Cl2. The combined organic layers were dried over Na2SO4, filtered, and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography in ethyl acetate/petroleum to give desired compound 7.Compound 10 mg GSK-4 compound dissolved in 0.5 mL CDCl3 and tested for 1H NMR spectra on an AMX 400 NMR spectrometer from Bruker, Germany. 1H NMR was reported as follows: chemical shift, multiplicity , coupling constant  in Hz and integration. Chemical shifts (δ) were reported with respect to the corresponding solvent residual peak at 7.26 ppm for CDCl3 for 1H NMR.The binding affinity between wild-type and mutant PfENT1 with nucleosides and GSK4 was measured using a Micro-Cal ITC200 . All proteins were prepared in 25 mM MES (pH 6.0), 150 mM NaCl, and 0.05% (w/v) DDM. Nucleosides (5~10 mM) and GSK4 (100-200 μM) dissolved in the same buffer were titrated against 20–50 μM PfENT1 proteins at 25 °C. PEAQ-ITC analysis software  was employed to analyze the data. All titrations were repeated three times.S. cerevisiae strain  BY4741 (MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0) was used as the starting yeast strain. Yeast strains were cultured in YPD medium . The antibiotic G418 was supplemented at a concentration of 250 μg/mL to select strains carrying the KanMX marker. E. coli DH5α was used for subcloning.The FUI1 gene were amplified from BY4741 genomic DNA. The gene cassette of the ‘TEF1p promoter-PfENT1-PGK1t terminator’ together with the two homologous arms was cloned into the PUC19 plasmid through Gibson assembly52, generating the final plasmid PUC19: PfENT1. PfENT1 mutations were generated using point mutation and verified by Sanger sequencing. The spacer targeting the FUI1 gene (5ʹ-ACTCGAAGTTCGTCAAATTG-3ʹ) was cloned into the pCas-BsaI plasmid to generate the pCas-FUI1 plasmid as described previously53.Codon-optimized PfENT1 (for expression in yeast) was synthesized by Sangon Biotech. The carboxyl terminus of PfENT1 was HA-tagged through PCR. The TEF1p promoter and PGK1t terminator were amplified from BY4741 genomic DNA. Overlap PCR was applied to generate the gene overexpression cassette of PfENT1 (TEF1p promoter-PfENT1-PGK1t terminator). The two 600-bp homologous arms corresponding to the upstream and downstream regions of the fui1Δ and PfENT1 (or its mutations) overexpression in the S. cerevisiae strain, 1 μg pCas-FUI1 plasmid and 15 μg PUC19:PfENT1 were transformed into yeast using the LiAc/ssDNA/PEG method54. Then, the strains were plated on a YPD plate supplemented with G418. Clones were screened using PCR and finally verified through Sanger sequencing.To obtain 8 cells in mid-log phase growth were harvested by centrifugation at 3000 × g for 8 min at 4 °C. Pellets were washed with 2 ml lysis buffer (25 mM Tris 8.0 150 mM NaCl) and resuspended in 20% Snailase (v/v) with reaction buffer overnight at 37 °C. The digested yeast was harvested with 100 μl lysis buffer and prepare samples. For western blots, the following parameters were used: wet transfer (25 mM Tris 190 mM glycine 20% [v/v] methanol in transfer buffer) for 90 min / 100 V / 4 °C, PVDF membrane (0.22 μm). Blocking was applied for 2 h at room temperature using 5% (w/v) non-fat dry milk in TBST . Primary antibodies  were applied overnight at 4 °C in 5% (w/v) non-fat dry milk in TBST. Secondary antibodies  were applied for 1.5 h at room temperature using 5% (w/v) non-fat dry milk in TBST.Approximately 2 × 10g for 5 min. Cells were washed three times in 2% glucose. The yeast pellets were resuspended in uptake assay buffer  to a final concentration of 2 × 108 cells mL–1. The uptake assay was initiated by combining 100 μL of prewarmed (30 °C) cells and 100 μL uptake assay buffer mixture: 25 mM Tris-HCl 8.0, 150 mM NaCl, 2% glucose, 200 nM [3H] Inosine . Cells were removed onto a membrane filter at the appropriate times to generate the time course. After washing by vacuum filtration with 2 ml ice-cold 2% glucose, the radioactivity retained on the filter was incubated with 1 mL of ULTIMA GOLD scintillation fluid . Counts were measured using a liquid scintillation analyzer .Cells were grown overnight in YPD medium to mid-log phase and harvested by centrifugation at 3000 × For competition assays, the indicated nucleosides and nucleobase were added into the external uptake assay buffer  to a final concentration of 5 mM, and the transport reaction was stopped at 30 min. The reading of the competition assays was normalized against the one without a competitor, which had the uptake of radiolabeled inosine set as 100%. All uptake assays were performed at 30 °C and repeated three times. Error bars represent the s.d. The final result was analyzed in the GraphPad Prism 8 software (version 7.0.0).Y190A-Nb48 complex and 8.0 mg/mL for the PfENT1Y190A-Nb19 complex) was applied to glow-discharged, holey carbon-coated grids . The grids were blotted for 3 s and flash-frozen in liquid ethane using a Vitrobot . Images were recorded on a 300 kV Titan Krios G3i electron microscope (Thermo Fisher Scientific) equipped with a Gatan K3 Summit direct detector and a GIF Quantum energy filter (slit width 20 eV). Movie stacks were collected using SerialEM55 in counting mode at a magnification of 105,000x with a corresponding pixel size of 0.83 Å. Each movie stack of PfENT1Y190A-Nb19 complex with 50 frames was exposed for 3 s at a dose rate of 13.2 e/px/s, resulting in a total dose of 57.5 per Å2 over 50 frames. The defocus range was set from −1.2 to −1.8 μm. Each movie stack of the PfENT1Y190A-Nb48-inosine complex was exposed for 2.5 s at a dose rate of approximately 15.5 e/px/s, resulting in a total dose of approximately 56.3 per Å2 over 50 frames. The defocus range was set from −1.0 to −1.8 μm. A total of 4631 movie stacks were collected for the PfENT1Y190A-Nb19 complex. A total of 6916 movie stacks were collected for the PfENT1Y190A-Nb48 complex.A total of 3.5 μL of the concentrated complex at approximately 8-12 mg/mL . The grids were blotted for 2.5 s and flash-frozen in liquid ethane using a Vitrobot . Images were recorded on a 300 kV Titan Krios G3i electron microscope (Thermo Fisher Scientific) equipped with a Gatan K2 Summit direct detector and a GIF Quantum energy filter (slit width 20 eV). Movie stacks were collected using EPU in counting mode at a magnification of 130,000x with a corresponding pixel size of 1.1 Å. Each movie stack of the PfENT1GFP-GSK4 complex with 30 frames was exposed for 6 s at a dose rate of 6.463 e/px/s, resulting in a total dose of 52.452 per Å2 over 30 frames. The defocus range was set from −1.1 μm to −1.8 μm. A total of 9044 movie stacks were collected for the PfENT1GFP-GSK4 complex.For the PfENT156 with 5 by 5 patches. Micrograph contrast transfer function (CTF) estimations were performed by CTFFind457 using micrographs without dose-weighting. The dose-weighted micrographs were used for particle picking and further processing. Particles were automatically picked by Gautomatch. For data of the PfENT1Y190A-Nb48-inosine complex, 4,651,677 particles were picked. Micrographs with estimated Ctf Max Resolution (> 5 Å) and Ctf Figure Of Merit (> 0.3) were selected for further processing. A total of 6778 micrographs were selected with 4,572,619 particles. Particles were extracted by Relion3.158 with a box size of 72 pixels and a pixel size of 3.32 Å. Iterative 2D classifications were performed using cryoSPARC v.2.14.259. A total of 1,129,242 particles were selected and then recentered and re-extracted by Relion with a box size of 144 pixels and a pixel size of 1.66 Å. Three parallel ab initio reconstructions were performed in cryoSPARC. A total of 854,399 particles from combined good classes were selected. These particles were subjected to one round of heterogeneous refinement, and 492,596 particles were selected. Next, particles were subjected to one round of nonuniform refinement, yielding a 3.8 Å map. Three rounds of heterogeneous refinement were performed, and 243,894 particles were selected. One round of nonuniform refinement was performed, yielding a 3.4 Å map. Particles were recentered and re-extracted by Relion with a box size of 288 pixels and a pixel size of 0.83 Å. Particles were imported into cryoSPARC, and one round of heterogeneous refinement was performed. A total of 183,797 particles were selected. Finally, one round of nonuniform refinement and local refinement was performed, yielding a 3.11 Å map. For data of the PfENT1Y190A-Nb19 complex, 2,488,266 particles were picked. A total of 4174 micrographs were selected with 2,292,368 particles. A similar processing procedure was applied. 89,264 particles were selected for final reconstruction. After particles re-extraction, particles were imported into cryoSPARC v.3.3.1. One round of ab inito reconstruction, nonuniform refinement, and local refinement was performed, yielding a 3.34 Å map. Conversions of particle star files between Relion and cryoSPARC were performed mainly using the pyem script60. The FSC between the map and model was calculated by Phenix.mtriage58.Movies frames were aligned using MotionCor2GFP data, data processing was performed using cryoSPARC. Movies frames were aligned using Patch motion. CTF estimation was performed using Patch CTF. Particles were first picked using a blob picker with partial micrographs. 2D templates were generated by 2D classification. Particle picking of all micrographs was performed by a template picker. A total of 6,684,066 particles from 4508 micrographs were extracted using a box size of 196 pixels and cropped into 98 pixels. After four rounds of 2D classification, 1,801,045 particles were selected. Ab initio reconstruction using partial particles and five rounds of heterogeneous refinement was performed and generated a dataset with 386,720 particles. One round of nonuniform and local refinement was performed. A total about 6,746,245 particles from 4536 micrographs were extracted using a box size of 196 pixels and cropped into 98 pixels. After four rounds of 2D classification, 1,255,179 particles were selected, followed by ab initio reconstruction. Two rounds of heterogeneous refinement were performed using combined selected particle sets, generated a new dataset with 757,172 particles. Two rounds of ab initio reconstruction were performed, generated a new dataset with 598,813 particles. One round of nonuniform and local refinement was performed, followed by particle re-extraction with a box size of 196 pixels, ab initio reconstruction, and new round of nonuniform and local refinement. A 4.37 Å map was obtained. Finally, after three rounds of local refinement with a mask excluded GFP, a 4.15 Å map was obtained. Three rounds of local refinement with a mask excluded GFP generated a 4.15 Å map. 204,716 particles were selected after one round of ab initio reconstruction. Finally, one round of nonuniform generated a 4.04 Å map.For PfENT1SWISS-MODEL using the structures of hENT1 (PDB code: 6OB6) and nanobody (PDB code: 3EZJ) as templates, respectively. The two models were then docked into the density map and manually adjusted and rebuilt by COOT61. The restraint files of inosine GSK4 were generated by the Phenix.elbow package62. The complete model was finally refined in Phenix using real-space refinement with secondary structure and geometry restraints63. The atomic model of the PfENT1Y190A-Nb19 complex and PfENT1GFP –GSK4 complex is built similarly to that of PfENT1Y190A-Nb48. Overfitting of model was diagnosed by refining model using one of the two independent maps from gold-standard refinement, and calculated FSC against both two half maps64. The final structure was validated using Molprobity65.The PfENT1 and Nb48 models were first generated by Further information on research design is available in the Reporting SummarySupplementary information