Precision medicine requests accurate serological inspections to precisely stratify patients for targeted treatment. Intact transition epitope mapping analysis proved surrogate seroconversion of a model organism’s serum when spiked with a monoclonal murine anti-Ovalbumin antibody (mAb) with epitope resolution. Isolation of the IgG fraction from blood serum applied two consecutive protein precipitation steps followed by ultrafiltration and resulted in an ESI-MS analysis-ready IgG preparation. For epitope mapping by epitope extraction, the Ovalbumin antigen was digested with trypsin. After desalting, the peptide mixture was added to the ESI-MS-ready IgG preparation from mAb-spiked serum and the solution was incubated to form an immune complex between the Ovalbumin-derived epitope peptide and the anti-Ovalbumin mAb. Then, the entire mixture of proteins and peptides was directly electrosprayed. Sorting of ions in the mass spectrometer’s gas phase, dissociation of the immune complex ions by collision-induced dissociation, and recording of the epitope peptide ion that had been released from the immune complex proved the presence of the anti-Ovalbumin mAb in serum. Mass determination of the complex-released epitope peptide ion with isotope resolution is highly accurate, guaranteeing high specificity of this novel analysis approach, which is termed Intact Transition Epitope Mapping—Serological Inspections by Epitope EXtraction (ITEM—SIX). Chlamydia trachomatis infection, thereby assessing the associated risk of infertility , a commercially available monoclonal antibody (mAb) that recognizes chicken Ovalbumin (P01012), which itself is a readily available food allergen. Mass spectrometric characterization of the anti-Ovalbumin mAb dissolved in ammonium acetate buffer (solution 1a) by nanoESI-MS afforded a mass spectrum with the typical charge distribution of antibody ion signals with the most abundant 23+ charged ion signal . From thFor surrogate seroconversion, the anti-Ovalbumin antibody had been spiked into rabbit serum. Then, the mAb-spiked serum was subjected to IgG preparation using the newly developed fractionated precipitation procedure and the respective IgG mixture (solution 1b) was analyzed by nanoESI-MS . The masNext, the Ovalbumin antigen solution (solution 2) was inspected for its molecular integrity, and SDS-PAGE analysis under non-reducing conditions provided a single strongly stained protein band at an apparent molecular mass of ca. 45 kDa at the expected location in the gel . Upon trm/z 1400. However, despite the fact that peptide ion signals above m/z 1400 were present in only low intensities, they were unambiguously assigned to the respective partial sequences of chicken Ovalbumin. The known post-translational modifications, such as acetylation of the N-terminal glycine residue (G1) and phosphorylations at amino acid residues S68 and S344, were confirmed. Despite the fact that (i) there remained unassigned peptide ion signals An epitope mapping experiment typically starts with testing immune complex formation efficiency with a standard immune analysis assay, such as Western blotting. The after SDS-PAGE separation blotted Ovalbumin (solution 2) was readily decorated by the anti-Ovalbumin mAb (solution 1a) and detected upon addition of the fluorescence-labeled secondary anti-murine IgG antibody using a fluorescence imaging system .m/z values (cut-off setting: x = m/z 2000) was suppressed and only ions with high m/z values ) were able to reach the detector with more or less unmuted intensities. The ITEM—ONE mass spectrum showed multiply charged ion signals (21+ to 25+) of the antibody, which were accompanied by multiply charged satellite ion signals (21+ to 25+) on the high mass sides of the ion signals  with the Ovalbumin-derived peptide mixture (solution 2′) upon electrospraying the entire mixture. The mass spectrum was recorded under ITEM—ONE conditions, which means that transmission of ions with low  signals . From th signals , indicatTherefore, ITEM—ONE analysis was applied. Immune complex dissociation in the collision cell of the mass spectrometer and subsequent mass determination of the complex-released peptide ion provided the required information with the requested accuracy. After increasing the collision cell voltage difference (ΔCV), the complex-released epitope peptide was analyzed by the mass spectrometer’s second mass analyzer with isotope resolution .m/z 1687.74 was the only ion signal that appeared with strongly increased intensity in the low mass range of the mass spectrum upon increasing ΔCV, this ion signal was determined to represent the epitope peptide that had been released from the immune complex by CID. The mass of the singly protonated ion signal with m/z 1687.74 matched precisely with the calculated mass of the chicken Ovalbumin partial amino acid sequence GGLEPINFQTAADQAR (sequence range 127–142). This peptide had been produced by tryptic cleavage at amino acid residues R126 and R142  was mixed with the IgG preparation that had been prepared from blood serum (solution 0). Under ITEM—ONE conditions, i.e., with blocking transmission of low detected . Obviousdetected .m/z 1687.71 to the chicken Ovalbumin partial amino acid sequence GGLEPINFQTAADQAR, a mass spectrometric fragmentation experiment of this ion was performed. The MS/MS spectrum showed intense Y-type ion signals that were unambiguously assigned by comparing the experimentally determined masses with the fragments’ theoretical masses  was mixed with the IgG preparation obtained from mAb-spiked blood serum (solution 1b) and the entire mixture (solution 3b) was analyzed by nanoESI-MS using ITEM conditions .m/z range (m/z 500–2500) just a few ion signals above m/z 1400, all with very little intensities, indicating that blocking of ions with m/z values below m/z 2000 was incomplete for ions with m/z values higher than 1400. By contrast, transmission of ions with m/z values below 1400 was completely suppressed. Nevertheless, upon increasing ΔCV, there appeared an additional singly protonated ion signal at m/z 1688.06, which is the only ion signal that increased in intensity in the mass spectrum and thus is assigned as a complex-released peptide ion signal. No other low abundant ion signal changed intensity with increasing ΔCV.The mass spectrum that was recorded at low ΔCV showed in the low m/z values above m/z 5000  with the IgG preparation which had been prepared from blood serum (solution 0). Again, an increase of ΔCV in the collision cell caused no additional ion signals to appear in the low mass range. The pattern of incompletely suppressed low abundant ion signals maintained the same. Multiply charged antibody ion signals with high expected . Ovalbumin from chicken is a major allergen against which susceptible humans may generate antibodies (IgG and/or IgE), leading to food intolerance and/or allergic reactions towards the chickens’ eggs’ ingredients ,34. The Because of targeting an antigen’s epitope, ITEM—SIX has the potential to move current antigen-based serology to the next level of future epitope-based serological investigations. This move towards fine-tuning of biomedical assays, which detect an antibody via its defined recognition motif in a crude mixture, such as blood serum, enables to dissect patient cohorts not only according to the presence or absence of antigen-specific antibodies but instead based on the presence or absence of antibodies that are directed against a specific epitope on a given antigen. A recently emerged example with medical importance may be provided by the differentiation of patients who developed antibodies against distinctly different epitopes on platelet factor 4 (PF4). Whereas some patients showed presence of antibodies that were directed against PF4s heparin binding site and then suffered from vaccine-induced thrombotic thrombocytopenia (VITT), other patients who suffered from related but different heparin-induced thrombocytopenia (HIT) possessed antibodies that were directed against a different but adjacently located epitope on PF4. While development of HIT normally requests administration of heparin as a medical intervention, activation of platelets to contribute to thrombocytopenia and thrombosis in VITT patients is independent from heparin treatment but was triggered by vaccination with adenoviral vector-based vaccines . KnowingBlood tests, i.e., serological testing for diagnosis of allergies, are routinely used for determining presence of allergen-specific immunoglobulins . AccordiImmunological seroconversion does typically not give rise to a single monoclonal antibody but, in fact, the immune response typically yields oligo- and/or polyclonal antibodies. Hence, the use of a monoclonal anti-Ovalbumin antibody that had been spiked into blood serum has its limitations and serves as a surrogate or mimicking experimental approximation of seroconversion. The term seroconversion remains reserved for the immuno-biological process, and the ITEM—SIX methodology will be equally applicable to real seroconverted specimen analysis. Of interest as well, although not shown in this research report, the alternative to epitope extraction, epitope excision, i.e., an immune complex is first created between the intact antigen and the antibody of concern and then is subjected to enzymatic proteolysis, is self-evident. Both epitope extraction and epitope excision have been successfully applied for analyzing sequential (linear) as well as assembled  epitopes , and, heGenerating immunoglobulin preparations from blood serum without the application of affinity chromatography had been developed during World War II ,41 and wEpitope-based serology is considered an important next step for improving diagnostic accuracy , which nStep 1: A volume of 170 µL of serum  was pipetted into a 1.5 mL reaction tube . Then, 30 µL of deionized water  and 600 µL of a 60 mM sodium acetate solution , prepared in de-ionized water, were added. The mixture was vortexed  for 30 s. Then, 20 µL of neat octanoic acid  was slowly added. A white precipitate formed and the suspension was vortexed at room temperature for 30 min [g and at 4 °C for 30 min to separate the white precipitate, P1, from its supernatant solution, S1. P1 was discarded. S1 (volume ca. 0.82 mL) was aspirated and filled into a 2 mL syringe , which was equipped with a PES syringe filter . S1 was filtered into a 5 mL Eppendorf tube. The filter was washed with 180 µL of deionized water and this washing solution was added to S1. The S1 solution (ca. 1 mL) may be kept at 4 °C overnight.r 30 min . SubsequStep 2: To solution S1 (ca. 1 mL) was added 3 mL of chilled acetone  at −20 °C. The mixture was vortexed for 1 minute (no precipitation was visible). Then, the tube was placed into the freezer  and was kept at −20 °C overnight (flocculates appeared the next day). The suspension was centrifuged using the Biofuge Stratos  centrifuge at 10,000× g and −5 °C for 30 min to separate the white precipitate, P2, from its supernatant solution, S2. Supernatant S2 was discarded. Precipitate P2 was suspended in 100 µL of deionized water [g and 4 °C for 30 min. The supernatant solution was again discarded. This washing operation was repeated two times. Then, the precipitate P2 (ca. 3 mg) was suspended in 100 µL of 200 mM ammonium acetate , prepared in LC-MS water , pH 6.7, and was vortexed at room temperature overnight (max. for 10 h). The suspension was centrifuged using the Biofuge Stratos centrifuge at room temperature and at 9000 rpm for 3 min to generate supernatant S3. Then, the residual precipitate was removed by aspirating supernatant S3 (ca. 0.1 mL). Solution S3 may be kept at 4 °C overnight.ed water . Then, tStep 3: A centrifuge ultrafilter unit  with 50 kDa cut-off was filled with solution S3 (ca. 0.1 mL). Then, 300 µL of 200 mM ammonium acetate, pH 6.7, were added. The solution was centrifuged  at room temperature at 9000 rpm for 5 min. The filtrate solution was discarded and the retentate solution, R1, was filled up with 200 mM ammonium acetate solution . Centrifugation, discarding of filtrate solution, and refilling were repeated eight times [ht times . Then, tA volume of 50 µL of the supplier’s original anti-Ovalbumin antibody solution  was transferred into an ultrafilter tube with 50 kDa cut-off (Amicon) and the buffer was exchanged with 0.2 M ammonium acetate, pH 6.7, using the procedure as reported above for IgG extraction (Step 3). The so prepared anti-Ovalbumin antibody solution  in 0.2 M ammonium acetate, pH 6.7, had a protein concentration of 0.28 µg/µL. This solution was kept in the refrigerator for further use.To generate mAb-spiked serum, 170 µL of rabbit serum with a protein concentration of 40.2 µg/µL were added to 10 µL of anti-Ovalbumin antibody solution with 0.98 µg/µL protein concentration . Then, 175 µL of mAb-spiked serum solution were used to perform IgG extraction according to the above described IgG preparation protocol. The retentate R1  in 0.2 M ammonium acetate, pH 6.7, had a protein concentration of 1.41 µg/µL.v/v) and then with 200 µL of the same elution solvent. The eluted volumes were combined in a 1.5 mL reaction tube. Solvent was evaporated using the SpeedVac RVC 2-25 and peptides were re-dissolved in 30 µL of 0.2 M ammonium acetate prepared in LC-MS water, pH 6.7, to yield the digested Ovalbumin peptide solution (solution 2′) with final peptide concentration of 0.63 µg/µL. A volume of 10 µL of original Ovalbumin solution  with protein concentration of 9.66 µg/µL was pipetted into a 0.5 mL reaction tube and mixed with 6.8 µL of 15 M urea  dissolved in 50 mM ammonium bicarbonate solution, pH 8.0 (solution 2). To 17 µL of Ovalbumin solution (urea-containing solution 2) were added 2 µL of 100 mM DTT dissolved in 50 mM ammonium bicarbonate solution, pH 8.0, and the mixture was warmed up to 57 °C for 30 min. Then, 1.5 µL of freshly prepared 300 mM iodoacetamide  dissolved in 50 mM ammonium bicarbonate solution, pH 8.0, were added and the mixture was incubated in the dark at room temperature for 30 min. Then, 114 µL of 50 mM ammonium bicarbonate, pH 8.0, and 20 µL of Trypsin  dissolved in 3 mM TrisHCl, pH 9.3, were added. Incubation was maintained in the dark at 37 °C overnight. Final urea concentration during digestion was 0.66 M. Protein/peptide concentration was 0.63 µg/µL; final volume was 154.3 µL ,52,53,54A volume of IgG-containing solution  that contained between 0.5 µg and 1 µg of protein was filled up with deionized water to reach a total volume of 16 µL. Then, 4 µL of non-reducing sample buffer were added. Alternatively, 4 µL of reducing sample buffer were added to the diluted IgG solutions and these solutions were mixed and heated  at 95 °C for 5 min. To check the tryptic digestion yield, an SDS-PAGE run was performed using a 1.2 µL aliquot from the Ovalbumin digestion mixture (solution 2′) and 3.2 µL of Ovalbumin solution 0.24 µg/µL, prepared by dilution of 2 µL of Ovalbumin solution 9.66 µg/µL with 78 µL of 50 mM ammonium bicarbonate, pH 8.0. SDS-PAGE analysis was performed as described elsewhere . Raw data were exported and graphic files (CorelDraw 17.0 software package) were saved on computer drives. Obtained raw data were used for peptide assignments by the PLGS 3.0 software.A volume of 1 µL of tryptic Ovalbumin peptide mixture (solution 2′) was diluted with 3 µL of 0.2 M ammonium acetate, pH 6.7, to yield a final peptide concentration of 0.28 µg/µL. 2.5 µL of this diluted tryptic Ovalbumin peptide mixture was filled into a gold-coated nanospray needle for offline nanoESI-MS analysis . Offline−1 mbar; quadrupole analyzer vacuum was 3.9 × 10−5 mbar (softer desolvation conditions); the TOF analyzer vacuum was 3.2 × 10−7 mbar [−5 mbar (harsher desolvation conditions) was applied. The mass spectra were collected from m/z 500 to m/z 7500 for ca. 5 min each. Spectra were recorded using the MassLynx 4.0 data system from Waters . Obtained mass spectra were smoothed in 3 cycles with a window of 5 for low mass range spectra and with 50 cycles with a window of 100 for high mass range spectra, respectively, using the Savitzky–Golay algorithm. The MassLynx 4.0 software package was used for data analysis and spectral image preparation in conjunction with the CorelDraw 2017 software package [At this point, 2.5 µL of the retentate R1 (solution 0) or 2.5 µL of anti-Ovalbumin antibody (solution 1a) were transferred into a gold-coated capillary needle and analyzed by offline nanoESI-MS using the Q-TOF II mass spectrometer  . Likewis0−7 mbar . Alterna package .−1 mbar, quadrupole analyzer vacuum was 3.5 × 10−5 mbar, the ToF analyzer vacuum was 4.5 × 10−7 mbar. Transmission of ions below m/z 1450 was completely blocked via respective quadrupole settings: M1 = 2000, dwell time, and ramp time 25%; M2 = 2000, dwell time, and ramp time 25%; and M3 = 2000. Mass spectra were recorded in ranges from m/z 200 to m/z 8000. Collision gas pressure was set to 4 psi and collision cell voltage difference initially was set to 3 V. After 1.5 min of recording, the collision cell voltage difference was increased to 20 V. Recordings lasted again for 1.5 min each using the MassLynx 4.0 data system . Obtained mass spectra were smoothed in 3 cycles with a window of 5 for low mass range spectra and with 50 cycles with a window of 100 for high mass range spectra, respectively, using the Savitzky–Golay algorithm. The MassLynx 4.0 software package was also used for data analysis and spectral image preparation in conjunction with the CorelDraw 17.0 software package.A volume of 10 µL of anti-Ovalbumin antibody solution (solution 1a) with a protein concentration of 0.28 µg/µL; 1.9 pmol/µL) was mixed with 3.1 µL of peptide mixture from digested Ovalbumin solution (solution 2′) with a peptide concentration of 0.055 µg/µL. This Ovalbumin peptide solution was obtained by dilution of 1 µL of digested Ovalbumin solution (peptide concentration 0.28 µg/µL) with 4 µL of 0.2 M ammonium acetate, pH 6.7. The mixture (solution 3a) has a final antibody concentration of 0.21 µg/µL (1.4 pmol/µL) and a final peptide concentration of 0.013 µg/µL (32 pmol/µL). After mixing, solution 3a was incubated at room temperature for at least 1.5 h. A volume of 2.5 µL of solution 3a was filled into a gold-coated nanospray capillary needle. Offline nanoESI-MS ITEM—ONE analysis was performed as described . SettingA volume of 5 µL of digested Ovalbumin solution (solution 2′) with 0.32 µg/µL protein concentration was obtained by dilution with 0.2 M ammonium acetate, pH 6.7. To this diluted Ovalbumin peptide solution (solution 2′) were added 4 µL of IgG extracted from converted rabbit serum . The mixture (solution 3b) has a final volume of 9 µL, a final antibody concentration of 0.63 µg/µL (4.2 pmol/µL), and a final Ovalbumin peptide concentration of 0.18 µg/µL. Solution 3b was incubated at room temperature for 1.5 h. A volume of 2.5 µL of solution 3b was filled into a gold-coated nanospray capillary needle for offline nanoESI-MS ITEM—SIX experiments.IgG extracted from rabbit serum (solution 0) served as negative control and was diluted with 0.2 M ammonium acetate, pH 6.7, to a protein concentration of 0.23 µg/µL (1.6 pmol/µL). A volume of 2.5 µL of digested Ovalbumin peptide solution (solution 2′) with peptide concentration of 0.055 µg/µL (13.7 pmol/µL) was added to 10 µL of diluted IgG from rabbit serum (solution 0) to generate solution 3c. The final volume of solution 3c was 12.5 µL, final IgG concentration 0.19 µg/µL (1.2 pmol/µL), and final Ovalbumin peptide concentration 0.011 µg/µL (34.0 pmol/µL). After mixing, solution 3c was incubated at room temperature for at least 1.5 h. A volume of 2.5 µL of solution 3c was filled into a gold-coated nanospray capillary needle for offline nanoESI-MS ITEM—SIX experiments.−5 mbar (soft desolvation conditions) and 4.4 × 10−5 mbar (inefficient desolvation conditions). Obtained mass spectra were smoothed in 3 cycles with a window of 5 for low mass range spectra and with 50 cycles with a window of 100 for high mass range spectra, respectively, using the Savitzky–Golay algorithm. The mass spectrometry data have been deposited to the ProteomeXchange Consortium via the PRIDE [Offline nanoESI-MS ITEM—SIX analysis was performed as described above for ITEM—ONE, but, this time, the applied capillary needle voltage varied between 1.2 and 1.8 kV and the quadrupole analyzer vacuum was varied between 3.5 × 10he PRIDE  partner v/v) Tween-20 to a 1:2 dilution of Intercept Blocking buffer  with PBS buffer. First, 35 µL of anti-Ovalbumin antibody (solution 1a with 0.017 µg/µL) were added to 3 mL blocking buffer and used as primary antibody solution. Second, 40 µL of R1 (solution 0), i.e., IgG extracted from rabbit blood serum, were used and mixed with 3 mL blocking buffer solution to obtain the negative control solution.Western blot analysis was performed as described elsewhere (see details in The unique combination of (i) targeted pre-analytical methods for rapid analyte preparation and (ii) advanced analytical procedures with high sensitivity and low sample consumption paves the way for cutting-edge serological inspection in an unprecedented way. ITEM—SIX provides the means to precisely learn about presence or absence of an antibody of concern in an individual patient’s/donor’s blood serum with neither the need for chemical immobilization of the antigen nor the use of a labeled reporter molecule for detection. ITEM—SIX exploits the binding motif recognition capability of an antibody, which leads to epitope-based stratification of patient cohorts with unsurpassed precision.


The organic anion transporting polypeptides OATP1B1 and OATP1B3 are membrane proteins that mediate uptake of drugs into the liver for subsequent conjugation and biliary excretion, a key step in drug elimination from the human body. Polymorphic variants of these transporters can cause reduced drug clearance and adverse drug effects such as statin-induced rhabdomyolysis, and co-administration of OATP substrates can lead to damaging drug-drug interaction. Despite their clinical relevance in drug disposition and pharmacokinetics, the structure and mechanism of OATPs are unknown. Here we present cryo-EM structures of human OATP1B1 and OATP1B3 bound to synthetic Fab fragments and in functionally distinct states. A single estrone-3-sulfate molecule is bound in a pocket located in the C-terminal half of OATP1B1. The shape and chemical nature of the pocket rationalize the preference for diverse organic anions and allow in silico docking of statins. The structure of OATP1B3 is determined in a drug-free state but reveals a bicarbonate molecule bound to the conserved signature motif and a histidine residue that is prevalent in OATPs exhibiting pH-dependent activity. A key step of drug metabolism in the human body is the uptake into liver cells, which is mediated by transport proteins of the OATP family. Here, authors report cryo-EM structures of two human OATP proteins, providing insight into their function. Members of the organic anion transporting polypeptide family (OATPs) have a key role in this step8. They are expressed at the basolateral membrane, facing the space of Disse and the portal blood plasma, from where they mediate the uptake of a multitude of endogenous and exogenous compounds  and its conjugates, and unconjugated and conjugated bile acids6. The exogenous substrates include a multitude of drugs including antivirals, chemotherapeutic agents, antidiabetics, or drugs used for treatment of cardiovascular diseases12. The latter class includes statins, which are widely prescribed for lowering plasma cholesterol levels for the reduction of the risk of atherosclerosis13.The liver facilitates vital processes due to its strategic location between the gut and the systemic circulation. It is exposed to most nutrients and xenobiotics, and consequently has a central role in protecting the body from toxic compounds by metabolism and excretion. These processes require the uptake of substances and their metabolites across the plasma membrane and into the cytoplasm of hepatocytesnds Fig. 6,9,10. TSLCO1B1) and OATP1B3 (gene name SLCO1B3) have been found to be of high clinical relevance. Impaired function of OATP1B1 or OATP1B3 due to genetic variations or inhibition is generally associated with adverse clinical events11. For example, genetic variants of the SLCO1B1 gene have been associated with a higher incidence of adverse events in statin therapy, which required dose adjustments in patients11. Furthermore, co-administration of statins with OATP1B inhibitors such as cyclosporine or rifampicin can lead to increased systemic concentration of the victim drug, which can cause side-effects that range in severity from mild myopathy to life-threatening rhabdomyolysis16. Due to the large number of recorded clinical cases of such adverse events associated with reduced OATP1B function, regulatory agencies including the FDA, EMA, and PMDA recommend testing of newly developed drugs for potential OATP-mediated drug-drug interactions20.Among the hepatic OATPs, OATP1B1  shows how the architectural features of the substrate-binding pocket facilitate the recognition of structurally diverse anionic compounds. The structure of OATP1B3 reveals a binding pocket for a bicarbonate ion, which may serve as a counter-ion in the transport cycle. Our results enable us to predict the interaction of statins with OATP1B1 and OATP1B3 and provide a mechanistic explanation why certain OATPs mediate pH-sensitive transport.9. E1S contains pharmacophore features shared among OATP substrates: an anionic head group and a large hydrophobic moiety30. The activity of OATP1B3 was demonstrated by measuring uptake of [3H]-labeled estradiol-17-β-D-glucuronide (E17βG), a conjugated sterol that is a physiological substrate. To assess the interaction of the over-expressed transporters with exogenous compounds, we determined [3H]-E1S uptake by OATP1B1 in the presence of excess amounts of two statins, atorvastatin and pitavastatin. Under these conditions, [3H]-E1S uptake was abolished, demonstrating that these drugs compete with the observed transport activity of OATP1B1  that form stable complexes with OATP1B3. Given that OATP1B1 and OATP1B3 share ~75% of sequence identity  against OATP1B3 from a synthetic Fab library by phage displayUsing single-particle cryo-EM, we determined high resolution structures of OATP1B1 and OATP1B3. This was challenging for the OATP1B1-Fab18 complex because of the pronounced preferential orientation of the particles, which limited the resolution of the 3D reconstruction. We therefore collected additional data by tilting the specimen stage at angles of 20° and 30° and combined it with the data collected without tilting -GAWW-x-G-(F/L)-L, where x denotes any amino acid residue. This motif is present in all OATPs and is therefore termed “OATP signature motif“8, and the two neighboring tryptophan residues were shown to be critical for activity35. The corresponding region was well-resolved in the EM density maps both of OATP1B1 and OATP1B3.At the external side and exposed to the space of Disse, OATP1B1 and OATP1B3 both contain a large extracellular domain (ECD) with a mass of 25 kDa distributed over 4 extracellular loops (ECLs) and containing multiple disulfide bridges Fig. . The lar36, which is in line with our structural observation. The shape and size of the binding pocket rationalize how OATP1B1 can recognize structurally distinct compounds that contain a negatively charged head group and a hydrophobic or aromatic body . However, we observed structural differences at the interface of the N-bundle and the C-bundle as well as in TM7, TM8, and their connecting loop ECL4  transporter MFSD2A41. However, the remote site of bicarbonate binding and the apparent allosteric coupling with the drug-binding pocket in OATP1B3 have not been observed in MFS transporters to date. The key findings in our OATP1B1 structure are the location, shape and chemical nature of the drug binding pocket.Transport proteins of the major facilitator family, to which OATPs belong, are thought to operate by an alternating access mechanism40. The release of bicarbonate, binding of a substrate molecule, and switching to an inward-open conformation would complete the cycle.Given that one of the main functions of OATP1B1 and OATP1B3 is drug uptake, our OATP1B1 structure captured a state prior to cytoplasmic substrate release. We propose that the transport can be thought to involve at least three states. State I, represented by our E1S-bound OATP1B1 structure, features an inward-facing conformation and contains a substrate-filled, polyspecific binding pocket, allowing substrate release into the cytosol. State II, represented by our bicarbonate-bound OATP1B3 structure, features a narrowed substrate-binding pocket, a consequence of TM7 and TM8 adopting a more closed conformation Fig. . These c3− might act as this counter-ion. If the transport is electrogenic, there might still be a role for bicarbonate. However, rather than a transport substrate, HCO3- might act as an allosteric modulator of the transporter , which might cause an increase of the pKa of His115. Consequently, His115 is optimally positioned to accept a proton from the external side of the membrane and provide it to the bound bicarbonate. This would facilitate the conversion of HCO3- into the less stable H2CO3, which likely dissociates into CO2 and H2O. We speculate that release of these degradation products could trigger the conversion of the transporter to the putative outward-open conformation  and OATP1B3 (Uniprot: Q9NPD5) genes were codon-optimized for expression in human cells and purchased from GenScript. The synthetic genes were independently cloned in a pcDNA5 vector with a cleavable C-terminal YFP-1D4 tag. For protein expression, tetracycline-inducible stable cell lines  were generated for each construct. Cells were grown and maintained in Dulbecco’s Modified Eagle Medium  supplemented with 10% fetal bovine serum , 100 μg/mL streptomycin, 100 units/mL penicillin (Thermo Fisher Scientific) at 37 °C with 5% COBefore protein expression, the media was aspirated, the cells were washed once with phosphate-buffered saline (PBS) buffer, and expression medium consisting of fresh phenol red dye-free DMEM (Gibco) supplemented with 2% FBS, 100 μg/mL streptomycin, 100 units/mL penicillin, 1x GlutMAX (Thermo Fisher Scientific), and 1x Sodium Pyruvate (Thermo Fisher Scientific), was added. Protein expression was induced with 1 μg/mL tetracycline (Sigma) at 37 °C. Following 72 h of expression, cells were harvested, washed with PBS, flash-frozen in liquid nitrogen, and stored at −80 °C for a maximum of 6 months.2, 1.1 mM KH2PO4, 0.8 mM MgSO4, 5.3 mM KCl, 11 mM D-glucose, and 20 mM HEPES, pH adjusted with 3 M Tris to pH 7.4). Transport was initiated by the addition into each well of 250 μL of UB containing 1.0125 μM of non-radiolabelled (Sigma) and radiolabelled  estrone-3-sulfate (E1S) for OATP1B1, and 1.01 μM non-radiolabelled (Sigma) and radiolabelled (46.3 Ci/mmol) estradiol-17-β-D-glucuronide (E17βG) for OATP1B3, at a molar ratio of 1:80 or 1:100 hot to cold substrate, respectively. For the competition assays, statins (100 μM atorvastatin or pitavastatin for OATP1B1 and 100 μM pitavastatin or 50 μM telmisartan for OATP1B3) were added to the UB. The uptake assay was stopped after 30 sec by aspirating the UB and immediately washing the cells three times with 500 μL ice-cold UB supplemented with 20 μM non-radiolabelled substrate. Cells were solubilized by adding 500 μL buffer composed of 2% Triton-X-100 and 1 M NaCl for 1 h at 37 °C. Total protein concentration per condition was measured in triplicates for OATP1B1, and duplicates for OATP1B3 using the BCA assay according to the manufacturer’s manual. For OATP1B1 350 μL of lysed cells, and for OATP1B3 450 μL were transferred to a 4 mL Scintillation Vial Ultima Gold (PerkinElmer). Intracellular radioactivity was measured by liquid scintillation counting after 24 h incubation. Data points compromised by technical errors were omitted from the analysis, resulting in an uneven number of replicates for different conditions. Data were processed and analyzed using ordinary one-way ANOVA, multiple comparison test (Dunnett’s test at 95% CI) in GraphPad Prism (version 9.0).Stable cell lines expressing OATP1B1 or OATP1B3 were independently seeded onto a 24-well plate  previously coated with poly-D-lysine. Cells were grown for 18-24 h until they were 60–80% confluent. Before induction with 1 μg/mL tetracycline, the cells were washed with 1 mL of PBS. Afterwards, 0.75 mL/well of expression medium were added. For negative control, tetracycline was omitted. Following 24 h of expression, media was removed, and the cells were washed three times with 500 μL of pre-warmed Uptake Buffer  and the supernatant was incubated with pre-equilibrated Sepharose-coupled Rho-ID4 antibody (University of British Columbia) for 2–3 h. The resin was washed four times with ten column volumes (CV) of purification buffer containing 25 mM HEPES, pH 7.4, 150 mM NaCl, 20% glycerol, 0.025% DDM, and 0.005% CHS, followed by four washes with ten CV of wash buffer containing 25 mM HEPES, pH 7.4, 150 mM NaCl, 0.025% DDM, 0.005% CHS or supplemented with 10% glycerol for OATP1B1. After this step, OATP1B1 was eluted from the resin with 3 CV of wash buffer supplemented with 0.5 mg/mL 1D4 peptide following overnight incubation. OATP1B3 was eluted by incubating the resin with 3 CV of wash buffer containing 3C protease at a 1:50 wt/wt ratio for 1 h at 4 °C.Cell pellets were thawed at room temperature, broken by 25 strokes in a Dounce homogenizer in lysis buffer containing 25 mM HEPES pH 7.4, 150 mM NaCl, 20% (v/v) glycerol, 1 mM PMSF (phenylmethylsulfonyl fluoride), 2 μg/mL DNaseI (Roche), and protease inhibitor cocktail (Sigma). All subsequent steps were performed at 4 °C. Cell lysate was solubilized with 1% DDM , 0.2% (w/v) CHS  for 60–90 min. The solubilized lysate was ultracentrifuged at 140,000 × Pre-mixed lipids  with cholesterol (Avanti) were solubilized with detergent and added to purified OATP1B1 or OATP1B3 and incubated for 10 min at room temperature. Purified membrane scaffold protein MSP1D1 was added to the mixture and incubated for an additional 20 min at room temperature. Bio-Beads SM-2 were pre-washed with HBS  and added before overnight incubation. Nanodisc-reconstituted OATP1B3 was pre-mixed with Fab19 at a molar ratio of 1:1 and the OATP1B3-Fab19 sample was further purified by size-exclusion chromatography in HBS prior to sample freezing. For OATP1B1, the eluted mixture was incubated for 5 h with pre-equilibrated Sepharose-coupled Rho-ID4 antibody and washed four times with 3 CV of HBS to remove empty nanodiscs. The purification tag was removed by incubating for 2 h with 3C protease. Eluted mixture was premixed with 1:1.75 molar excess Fab18 prior to size-exclusion chromatography step  in HBS buffer to remove excess Fab.44. Briefly, the OATP1B3 Avi-tag construct was incubated overnight at a 1:1 molar ratio with in-house purified BirA to protein in buffer supplemented with 0.02% LMNG, 0.004% CHS, 250 μM biotin, 10 mM ATP, 50 mM Bicine, pH 8.3, and 10 mM Mg Acetate. Biotinylation efficiency of the sample was verified by a pull-down assay on streptavidin-coated (SA) magnetic particles (Promega). Biotinylated OATP1B3 was purified by size-exclusion chromatography in HBS supplemented with 0.02% LMNG and 0.004% CHS.An OATP1B3 expression construct containing an Avi-tag sequence (GLNDIFEAQKIEWHE) was generated using PCR-restriction cloning (forward primer 5′-TCG ACG GGT TGA ATG ATA TTT TCG AAG CAC AGA AAA TTG AAT GGC ATG AGG − 3′ and reverse primer 5′-CGG GTT GAA TGA TAT TTT CGA AGC ACA GAA AAT TGA ATG GCA TGA GGT CGA − 3′). Protein expression and purification were performed as described above with the following changes. Cell pellets were lysed and subsequently solubilized with 1% LMNG , 0.2% CHS (w/v). Following elution with 3C protease to remove the purification tag, the sample was enzymatically biotinylated using in-house purified BirA, as described previously45. The selection buffer consisted of 25 mM HEPES, pH 7.4, 150 mM NaCl, 0.02% LMNG, 0.004% CHS, and 0.5% BSA. In the first round, 250 nM of target was immobilized on 250 µL SA magnetic beads. Then, 100 μL of a phage library E were added to the target bound to the SA beads and incubated for 30 min46. The resuspended beads containing bound phages were washed extensively and then used to infect log phase E.coli XL1-Blue cells. Phages were amplified overnight in 2xYT media with 50 µg/mL ampicillin and 109 p.f.u./mL of M13-KO7 helper phage. To obtain binders of high affinity and specificity, four additional rounds of selection were performed with decreasing the target concentration in each round with the final concentration of the target being 25 nM in the 5th round of selection. In each round, the amplified pool of phages of the preceding round was used as the input. From the second round onwards, the bound phages were eluted using 100 mM glycine, pH 2.7. This elution technique often results in the elution of non-specific and Streptavidin binders. To eliminate them, the precipitated phage pool from the second round onwards were negatively selected against 100 µL of SA magnetic beads before adding them to the target. The pre-cleared phage pool was then used as input for the selection.Biotinylated OATP1B3 was used for phage display selection. Phage display selection was performed at 4 °C according to published protocols2CO3 buffer, pH 9.6 and subsequently blocked by 0.5% BSA in PBS. A single-point phage ELISA was used to rapidly screen the binding of the obtained Fab fragments displayed on phage. Colonies of E.coli XL1-Blue harboring phagemids from 5th round of selection were inoculated directly into 500 μL of 2xYT broth supplemented with 100 μg/mL ampicillin, and M13-KO7 helper phage. The cultures were grown overnight at 37 °C in a 96-deep-well block plate. The ELISA buffer was identical to that used in selection. The experimental wells in the ELISA plates were incubated with 50 nM OATP1B3 in ELISA buffer for 15 min. Only buffer was added to the control wells. Overnight culture supernatants containing Fab phage were diluted 10-fold in ELISA buffer. The diluted phage supernatants were then transferred to ELISA plates that were pre-incubated with biotinylated target and washed with ELISA buffer. The ELISA plates were incubated with the phage for another 15 min and then washed with ELISA buffer. The washed ELISA plates were incubated with a 1:1 mixture of mouse anti-M13 monoclonal antibody  and peroxidase conjugated goat anti-mouse IgG  for 30 min. The plates were washed again, developed with TMB substrate, then quenched with 1.0 M HCl, and the absorbance at 450 nm was determined. The background binding of the phage was monitored by the absorbance from the control wells.The ELISA experiments were performed at 4 °C in 96-well plates coated with 50 µL of 2 µg/mL neutravidin in NaE. coli. E. coli C43 (Pro+) cells were transformed with sequence-verified clones of Fab fragments in pRH2.247. Fab fragments were grown in TB autoinduction media with 100 μg/mL ampicillin overnight at 30 °C. Harvested cells were kept frozen at −80 °C until use. Frozen pellets were re-suspended in PBS supplemented with 1 mM PMSF, and 1 μg/mL DNaseI. The suspension was lysed by ultrasonication. The cell lysate was incubated at 65 °C for 30 min followed by centrifugation. The supernatant was filtered through 0.22 µm filter and loaded onto a HiTrap Protein L 5-mL column pre-equilibrated with lysis buffer . The column was washed with 10 column volumes of lysis buffer followed by elution of Fab fragments with elution buffer (100 mM acetic acid). Fractions containing protein were directly loaded onto a Resource S 1-mL column pre-equilibrated with buffer A  followed by washing with 10 column volumes wash with buffer A. Fab fragments were eluted with a linear gradient 0–50% of buffer B . Affinity and ion-exchange chromatography were performed using an automated program on ÄKTA explorer system. Purified Fabs were dialyzed overnight against 20 mM HEPES, pH 7.4, 150 mM NaCl. The quality of purified Fab fragments was analyzed by SDS–PAGE.From phage ELISA, clones  were sequenced at the DNA Sequencing Facility at the University of Chicago. Twelve unique clones were obtained. These were sub-cloned in pRH2.2, an IPTG inducible vector for expression of Fabs in 450) was determined. To determine the affinities, the data were fitted in a dose-response sigmoidal function in GraphPad Prism and EC50 values were calculated.Multipoint ELISA was performed at 4 °C to estimate the affinity of the Fabs to OATP1B3. 25 mM HEPES, pH 7.4, 150 mM NaCl, 0.02% LMNG, and 0.004% CHS supplemented with 0.5% BSA was used as the ELISA buffer. 50 nM of target immobilized on a neutravidin coated ELISA plate was incubated with 3-fold serial dilutions of the purified Fabs starting from 4 μM for 20 min. The plates were washed, and the bound target-Fab complexes were incubated with a secondary HRP-conjugated Pierce recombinant protein L  for 30 min. The plates were again washed, developed with TMB substrate, quenched with 1.0 M HCl, and absorbance (ACryo-EM grids were prepared using a Vitrobot Mark IV (FEI) with an environmental chamber set at 95% humidity and 4 °C. Aliquots of 3.5 μL of purified OATP1B1 in complex with Fab18 in presence of 0.065 mM E1S or OATP1B3:Fab19 complex in presence of 0.05 mM atazanavir at a protein concentration of 0.22 mg/mL and 0.54 mg/mL respectively, were placed onto Quantifoil carbon grids  previously glow-discharged for 45 s with 25 mA using Pelco easiGlow Glow Discharge Cleaning System. Grids were blotted for 3.5 s and flash-frozen in a mixture of liquid ethane and propane cooled by liquid nitrogen.2. The final OATP1B3-Fab19 dataset was composed of 25,887 super-resolution movies collected during two cryo-EM sessions. Movie stacks had identical exposure time, dose, and frame number as the the E1S-bound OATP1B1-Fab18 dataset.Grids were imaged with a Titan Krios (Thermo Fisher Scientific) electron microscope operated at 300 keV, equipped with a Gatan K3 Summit direct electron detector and Gatan Imaging Filter (GIF), with a slit width of 20 eV to remove inelastically scattered electrons. Movies were recorded semi-automatically with EPU2 software (Thermo Fisher Scientific) in super-resolution counting mode with a defocus range of –0.6 to –2.2 μm and a super-resolution pixel size of 0.255 Å/pixel. The final E1S-bound OATP1B1-Fab18 dataset consisted of 21,416 super-resolution movies collected during three separate Titan Krios sessions, including 20 degrees and a 30 degrees tilted datasets containing 1424 and 7041 movies, respectively. Tilted datasets were collected to overcome the problem of particle preferred orientation. Each movie was exposed for 1.5 s with an exposure time of 0.03 s per frame with electron exposure of 10.5 e-/pix/s, resulting in 50 frames per movie and a frame exposure rate of 1.2 e-/Å49. Gctf was used to estimate the parameters of contrast transfer function (CTF)50 The micrographs were automatically sorted in Relion, and only those with an estimated resolution lower than 4 Å (or lower than 5 Å for datasets with tilt stage) were selected for further processing. First, particles were autopicked using Laplacian-of-Gaussian filtering and 2D classified in several rounds, followed by a generation of ab initio models of both OATP1B1-Fab18 and OATP1B3-Fab19 complexes. These models were used accordingly as template for the particles autopicking. Extracted particles were binned by a factor of 4 and used for several rounds of 2D and 3D classifications. From each dataset the 3D classes that most closely represented the expected structure of the complexes were selected.The analyses of data processing are presented in Supplementary Figs. 51 The NU-refinement, followed by the Local CTF refinement and another NU-refinement yielded the final map at 3.67 Å resolution. The half-maps from the last 3D refinement were sharpened and post-processed with DeepEMhancer.52For the OATP1B1-Fab18 dataset, the combined particles from three datasets  were re-extracted (unbinned to 0.51 Å/px) and used for 3D classification. The class with the well-resolved TMD region was selected  and used for another 3D classification. Then the class, in which ICLs were well-resolved, was selected, 3D refined and CTF refined. The particles  were Bayesian polished (first 20 frames), again CTF refined and 3D refined using mask that excluded the density of the nanodisc and the constant domain (Fc) of the Fab18. The final EM density map generated in Relion yielded 3.55 Å resolution. Next, the shiny particles from Relion were transferred to CryoSPARC4.1.For the OATP1B3-Fab19 complex dataset, the combined particles from two datasets  were used for 3D classification. The class with the well-resolved TMD and ECD regions was selected  and the particles were re-extracted (unbinned to 0.51 Å/px), followed by the 3D refinement. Then the particles were 3D classified without alignment and with masking out the nanodisc and constant domain (Fc) of the Fabs. Among the four classes, we did not observe more than a single conformation or any density indicating the presence of the atazanavir molecule. Therefore, the class that showed good connectivity of the signal and the best resolved TMD and ECD regions was selected for further processing. The 75,610 particles were 3D refined, Bayesian polished (first 15 frames), CTF refined, 3D refined and post-processed (B-factor sharpened) yielding the final EM density map at 2.97 Å resolution.53. Both maps were of sufficient resolution to allow de novo building of the transmembrane regions and parts of the extracellular domains of the transporters. The variable domain of the Fab binders were modelled de novo in Coot and the chemical structures for E1S, HCO3− and cholesterol were obtained from the monomer library available in Coot, using the codes FY5, BCT and CLR, respectively. The generated models were refined in Phenix using Real Space Refinement, and the quality was assessed by MolProbity. Further analyses of the EM density map and the model for each structure are presented in Supplementary Figs. 55.Model building for both OATP1B1 and OATP1B3 was performed using Coothttps://zinc15.docking.org/) or PubChem (https://pubchem.ncbi.nlm.nih.gov/). If necessary, the carboxyl group was modified (deprotonated) in JSME Molecular Editor (Zinc15) and the SMILES strings were copied. The SMILES were converted to SDF format in OpenBabel56. The ligand PDBQT files were generated from SDF files using the Meeko python package . The protonation state of the ligands was checked before docking. For molecular docking with flexible side chains, we used the PDBQT ligand files.The chemical structures of the compounds  were taken from the Zinc15 database (https://autodock-vina.readthedocs.io/en/latest/docking_flexible.html#). Briefly, docking was performed with either our current OATP1B1 model (the E1S was removed) or the OATP1B3 homology model based on our OATP1B3 and OATP1B1 structures built in SWISS-MODEL (https://swissmodel.expasy.org/). The models were prepared for the docking using Autodock Vina with prepare_receptor with the default settings; the missing hydrogen atoms  and Gasteiger charges were added to the models.Flexible docking was performed using Autodock Vina as described in the online tutorial . The Vina forcefield was used for the analyses. The center and dimensions of the grid space were defined (in Angstrom) Grid center: X 120 Y 130 Z 110. Grid size: X 30 Y 30 Z 30. An exhaustiveness of 64 was used for a more extensive sampling of poses. Nine poses were generated for each system and classified based on their affinity scores . Autodock Vina wrote the PDBQT output files which were displayed in ChimeraX using the interactive ViewDockX tool.57. Chemical structures were prepared using ChemDraw .Figures were prepared using the programs ChimeraX, PyMOL , and GraphPad. Multiple sequence alignments were generated using Clustal Omega online softwareFurther information on research design is available in the Supplementary InformationPeer Review FileReporting SummarySource Data


DNA hypermethylation at promoter CpG islands (CGIs) is a hallmark of cancers and could lead to dysregulation of gene expression in the development of cancers, however, its dynamics and regulatory mechanisms remain elusive. Bivalent genes, that direct development and differentiation of stem cells, are found to be frequent targets of hypermethylation in cancers.OVOL2 to promote tumorigenesis. Knockdown of OVOL2 in LSD1 knockout HCT116 cells restored the cancer cell phenotype.Here we performed comprehensive analysis across multiple cancer types and identified that the decrease in H3K4me1 levels coincides with DNA hypermethylation at the bivalent promoter CGIs during tumorigenesis. Removal of DNA hypermethylation leads to increment of H3K4me1 at promoter CGIs with preference for bivalent genes. Nevertheless, the alteration of H3K4me1 by overexpressing or knockout LSD1, the demethylase of H3K4, doesn’t change the level or pattern of DNA methylation. Moreover, LSD1 was found to regulate the expression of a bivalent gene In summary, our work identified a universal indicator that can pre-mark DNA hypermethylation in cancer cells, and dissected the interplay between H3K4me1 and DNA hypermethylation in detail. Current study also reveals a novel mechanism underlying the oncogenic role of LSD1, providing clues for cancer therapies.The online version contains supplementary material available at 10.1186/s12864-023-09341-1. OVOL2 (ovo like zinc finger 2), MLH1 and CDKN2A (p16/ARF) were silenced in colorectal tumors [BRCA1 was silenced in ovarian cancer [Cancer cells are featured with epigenetic changes that lead to the gene expression alterations to drive tumorigenesis and metastasis. In particular, tumor cells are characterized by global DNA hypomethylation and focal DNA hypermethylation at promoter CpG islands (CGIs) . Large-sl tumors –7, BRCA1n cancer  and brean cancer . Bivalenn cancer , 11. Pron cancer , 13. Then cancer –16. The n cancer , 18.In addition to DNA methylation, histone modification is another well-studied epigenetic regulation that plays a significant role in cancer development . EvidencThe histone modification H3K4me1 is usually enriched at enhancers, however, recently, increased evidence indicate that H3K4me1 also exists at promoters to regulate gene transcription –30. H3K4LSD1 has been demonstrated to promote the growth and metastasis of a variety of tumor cells –40. The OVOL2, via modifying the H3K4 methylation at promoter regions to inhibit the growth and metastasis of cancer cells. Our study revealed the mechanisms of epigenetic regulation of LSD1 and shed light on the therapeutic potential of LSD1.In this study, we revealed that the decrease in H3K4me1 levels coincides with DNA hypermethylation at the bivalent promoter CGIs in the development of various types of cancers. Therefore, DNA hypermethylation accrual at tumor promoter CGIs can be predicted by H3K4 monomethylation pattern of their tumor-origin tissues. Furthermore, alterations of DNA methylation at bivalent promoter CGIs dramatically affect the distribution pattern of H3K4me1, but the change of H3K4me1 through overexpressing or knockout LSD1 had no effect on the DNA methylation pattern. In addition, we provided evidence that LSD1 plays an oncogenic role in colon cancer through epigenetic regulation. Mechanistically, knockout of LSD1 upregulates the transcription of bivalent genes, such as Alteration of promoter DNA methylation is an epigenetic hallmark of cancer cells. To systematically explore the biological significance of the aberrant DNA methylation pattern in different cancer types, whole-genome bisulfite sequencing (WGBS) data of 10 different tumors including bladder urothelial carcinoma (BLCA), breast invasive carcinoma (BRCA), colon adenocarcinoma (COAD), lung adenocarcinoma (LUAD), lung squamous cell carcinoma (LUSC), rectum adenocarcinoma (READ), stomach adenocarcinoma (STAD), uterine corpus endometrial carcinoma (UCEC), glioblastoma multiforme (GBM), liver hepatocellular carcinoma (LIHC), and tissue normal counterparts, were collected from previous publications , 43 and 2 test, p-value < 2.2e-16, Fig. To further characterize the five groups of genes, we analyzed the expression level, tissue specificity and distribution of the 5 group genes along the genome. Generally, genes of all-hyper group are expressed at a lower level compared with genes in C5 groups Fig. A. GlobalAs elaborated above, DNA methylation and histone methylation correlates with each other , 52, the2 test, p-value < 2.2e-16, Fig. Since enrichment of H3K4me1 at promoter CGIs in tissue normal counterparts correlates with the DNA hypermethylation in cancers, the interplay between DNA methylation and H3K4me1 is further evaluated. WGBS data, H3K4me1/3 and H3K27me3 ChIP-seq data of HCT116 cells lacking two DNA methyltransferases–DNMT1 and DNMT3B (DKO) were downloaded from the previous study  and subjp-value < 8.8e-9, Fig. We further evaluated whether alteration of H3K4me1 influence the DNA methylation at promoter CGIs vice versa. LSD1, the demethylase of H3K4, was overexpressed in NCM460 cell line (LSD1 OE) or ablated in HCT116 cells to manipulate the methylation of H3K4. First, LSD1 was successfully overexpressed , including 96 up- and 61 down-regulated genes, were identified in LSD1 KO cells  of these genes were analyzed. The upregulated genes showed significantly increased H3K4me1/3 at promoters, while the downregulated genes showed similar level of H3K4me1/3 Fig. B, C. ThiOVOL2 is a bivalent gene  and a proximal promoter region 330 bp upstream to the TSS  and LSD1-KO + own Fig. B. Consision Fig. D, E. Colype Fig. F. Taken OVOL2 via regulating the histone modification level of its promoter CGI. Our study systemically established H3K4me1 as a pre-mark for DNA hypermethylation at promoter CGIs across multiple cancer types and dissected the interplay between H3K4me1 and DNA hypermethylation. In particular, a novel molecular mechanism of LSD1 mediated tumor development was identified, deepening the understanding of LSD1’s role in regulating epigenetic modification, gene expression and tumorigenesis.Aberrant DNA hypermethylation happens on the promoter CGIs during the development of many types of cancers , 62. AltDNA hypermethylation at promoters was a general epigenetic feature during tumorigenesis , 63, morDNA hypermethylation at promoter CGIs results in repressed expression of key tumor suppressors or the dysregulated expression of genes governing cell growth and metastasis , 65. TheBivalent genes, whose promoters are marked by both H3K4me3 and H3K27me3, play critical roles in the differentiation of ESCs . RecentlDNA methylation and histone modifications are both critical epigenetic marks regulating tumorigenesis, and they can interplay with each other in cancer cells: removal of DNA methylation at promoters leads to increased histone modifications, including H3K4me1/3 and H3K27me3 . Our stuLSD1 regulates the expression of bivalent genes via mediating H3K4 methylation through its demethylase activity in human ESCs . WhetherOVOL2 is a bivalent gene whose promoter accumulates H3K4me1/3 upon LSD1 knockout in HCT116. Inhibiting OVOL2 in LSD1 KO cells restored the cancer stem cell phenotype of HCT116 cells. In sum, we proposed a novel oncogenic mechanism of LSD1 and confirm it in HCT116 cells, although more experiments involving more cancer cell types are needed in the future to tamp the foundation for this mechanism.LSD1 is previously reported to promote tumor growth and metastasis, however, the underlying mechanisms are not fully understood , 69. BivIn summary, our work systemically explored the data of multiple cancer types and revealed the correlation between H3K4me1 and DNA hypermethylation at the promoter CGIs in cancer cells. Moreover, current research studied the interplay between DNA hypermethylation and H3K4me1 in detail, highlighting a previously unappreciated epigenetic event, reciprocal regulation between H3K4me1 and DNA methylation, in the process of cancer development. The current study also revealed a novel mechanism for the oncogenic role of LSD1. Taken together, this study unveiled a novel mechanism of epigenetic landscape alteration in cancers, providing novel insights into cancer therapy.https://zwdzwd.github.io/pmd, data of normal brain was downloaded from MethBase [http://smithdata.usc.edu/methbase/data/Lister-Brain-2013/Human_FrontCortexFemale64Yr/tracks_hg19). H3K4me1/3 and H3K27me3 ChIP-seq datasets of normal colon tissue and COAD were collected from GSE136889 [https://egg2.wustl.edu/roadmap/data/byFileType/signal/). The RNA-seq datasets of tumors and tissue normal counterparts used in this study were downloaded from http://duffel.rail.bio/recount/v2/TCGA/rse_gene.Rdata and processed by R package recount 2 [https://www.encodeproject.org/). The H3K4me1/3 ChIP-seq data and RRBS data of LSD1 control and overexpressed NCM460 cells, and the H3K4me1/3 ChIP-seq data, RRBS data and RNA-seq data of HCT116 wild type and LSD1 knockout HCT116 cells of our study were available at NCBI Gene Expression Omnibus (GEO) under accession number GSE222612.Processed WGBS data of LIHC and normal liver tissue were collected from GSE70090 . ProcessSE136889 . H3K4me1ecount 2 . H3K4me1ecount 2 . H3K4me3https://genome.ucsc.edu/cgi-bin/hgTables). CGIs overlapped with any TSS of gene transcripts were defined as promoter CGIs and only genes with promoter CGIs were used for the classification analysis. All promoter CGIs were splitted into 20 bins and the DNA methylation level of each bin of CGIs were calculated using the function ‘normalizeToMatrix’ from R package ‘EnrichedHeatmap’. Then based on the distribution of DNA methylation difference between normal tissues and tumors along CGIs, genes with promoter CGIs were classified into 5 groups using the k-means algorithm.The coordinates of CGIs of human genome (hg19) were retrieved using Table Browser (https://github.com/apcamargo/tspex/) based on TPM (transcripts per million) expression data. We downloaded the TPM expression data of representative 10 tissue types from Genotype-Tissue Expression (GTEx)  database. Tissue enrichment analysis was conducted using the R package ‘TissueEnrich’ with default parameters which used Human Protein Atlas (HPA) as reference dataset [To evaluate the tissue specificity of genes in each group, Tau values were calculated with tspex  was performed using the R package ‘clusterProfiler’. Only terms with both 2. All cell lines were authenticated by short tandem repeat (STR) profiling.Human colorectal carcinoma HCT116 cell line was kindly provided by Professor Wenjing Zhao , and cultured in McCoy’s 5A medium supplemented with 10% fetal bovine serum (FBS) and 100 U/mL penicillin/streptomycin . Human colonic epithelial cell line NCM460 was purchased from BeNa Culture Collection  and cultured in DMEM medium  supplemented with 10% FBS  and 100 U/mL penicillin/streptomycin . HEK 293 T cell lines were kindly provided by Professor Wenxue Zhao  and cultured in DMEM medium supplemented with 10% FBS and 100 U/mL penicillin/streptomycin. All cell lines were incubated at 37℃ containing 5% COOVOL2 gene expression. The targeting shRNA sequences are: shOVOL2: CAGGCATTCGTCCCTACAAAT; negative control (GV-NC): TTCTCCGAACGTGTCACGT. HEK 293 T cells were transfected with pCDH-LSD1/GV248 and packaging plasmids psPAX2 (Addgene #12260) and pMD2.G (Addgene #12259) using Lipofectamine3000  according to the manufacturer's instructions. The supernatant containing viruses was collected, filtered, and used for infection after 24 h and 52 h of transfection. NCM 460 cells or LSD1 KO HCT116 cells were infected with supernatant containing lentivirus mixed with complete culture medium at a ratio of 1:1 for 24 h, and then subjected to 1 μg/ml puromycin treatment for 3 days. Immunoblotting or qPCR was subsequently performed to examine the overexpression or knockdown efficiency.LSD1 coding sequence was amplified from the cDNA of HEK 293 T cells and subcloned into the lentiviral vector pCDH (Addgene #72265) for overexpressing LSD1. The primers used for molecular cloning are provided in Supplementary Table SLSD1 was selected as the CRISPR target as previously described [The sequence CGCGGAGGCTCTTTCTTGCG in exon 1 of escribed  and subcThe cells were collected and lysed at 4 °C with RIPA  buffer containing protease and phosphatase inhibitors. Protein lysates were resolved by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and transferred to PVDF membranes . Membranes were incubated in blocking buffer  for 1 h and probed overnight with primary antibodies at 4 °C, followed by incubation with the corresponding HRP-conjugated secondary antibodies. Pierce ECL Plus Western blotting substrate kit . The antibodies were used according to the manufacturer’s recommendations were used. Antibodies used for Western blot analyses were as follows: LSD1 , FLAG , β-actin , Goat anti-rabbit IgG H&L (HRP) , HRP-conjugated Affinipure Goat Anti-Mouse IgG(H + L) .Gapdh was used for qRT-PCR normalization of the samples. All the data were measured in at least three independent experiments. The primer sequences used in this study are shown in Supplementary Table STotal RNA was extracted with a total RNA extraction kit . cDNA was synthesized using RevertAid First Strand cDNA Synthesis Kit . Gene expression was detected by real-time qPCR using Sybrgreen detection systems . 6 cells were cross-linked with 1% formaldehyde for 10 min and quenched by 125 mM glycine for 5 min at room temperature with gentle shaking, and washed twice with PBS. Cells were then pelleted and lysed in ice-cold lysis buffer1  and lysis buffer 2  supplemented with cOmplete protease inhibitors  for 10 min. The cell lysate was sonicated using Covaris M220 with 5% duty factor for 10 min at 4 °C to shear DNA to fragments (200 ~ 1000 bp). Soluble chromatin was diluted in shearing buffer  with 1% Triton X-100 and 150 mM NaCl and incubated with 3 μg ChIP-grade antibody at 4 °C overnight with gentle shaking. 5% of input was stored prior to the de-crosslinking procedure. 30 μl Protein-G magnetic beads  were used for subsequent pull-down of antibody-chromatin complex by incubating for 2 h at 4 °C with gentle shaking. The beads were washed with the following buffers for 2 times each: IP buffer , High Salt Wash Buffer , LiCl Wash Buffer , and TE buffer + 50 mM NaCl  1 time. DNA was eluted with Elution Buffer  at 65℃ for 30 min with shaking at 1,400 rpm. The supernatant was then collected and incubated at 65 °C for overnight to reverse-crosslink the DNA. The day after, the enriched DNA was treated with 4 µL of 20 mg/mL RNase at 37 °C for 2 h and 4 µL of 20 mg/mL Proteinase K  at 55 °C for 2 h. The immunoprecipitation DNA was purified by phenol–chloroform-isoamyl alcohol , washed with ethanol, eluted in nuclease-free water, and used for subsequent experiments. The following antibodies were used in ChIP: H3K4me1 , H3K4me3 , H3K27me3 , LSD1 , IgG . ChIP-seq libraries were constructed and sequenced by the Novogene Bioinformatics Institute . Libraries were sequenced on the Illumina HiSeq 2500 platform.H3K4me1-, H3K4me3- and H3K27me3-ChIP experiments were performed based on the previously described protocol , 75. Briinput—CTsample). The sequences of the primers were in Supplementary Table SFor ChIP-qPCR, after the immunoprecipitated DNA was purified, qPCR was performed to detect the LSD1 protein binding sites of the DNA samples. The calculation of the ChIP signal is % input = 5% × 2 ^  for RRBS. The DNA samples were digested using methylation-insensitive restriction enzyme Mspl. All the cytosines were subjected to methylation-modified sequencing adaptors, DNA fragments with insert lengths within the range of 40–220 base pairs were cut from the gel and bisulfite treatment was carried out using an EZ DNA Methylation Gold Kit . PCR amplification was then performed to obtain the final DNA library. After quality checks of the DNA library, the samples were sent for Illumina HiSeq 2500 sequencing.RNA sequencing libraries were constructed and sequenced by the Novogene Bioinformatics Institute . High-throughput sequencing was performed as paired-end 150 sequencing using a Hiseq 2500 sequencing system.https://zwdzwd.github.io/pmd.For WGBS and RRBS data analysis, raw reads were first trimmed of low-quality reads and adaptor sequences with TrimGalore (version 0.6.6). Then, Bisulfite treated reads were mapped to the hg19 reference genome using Bismark with parameters: “-N 1 –parallel 4 –bowtie2”. Methylation calling of CpG sites was performed using a bismark methylation extractor and merged symmetric CpG sites by coverage2cytosine. The bismark coverage files were imported into R and CpG sites with a minimum of 10 × coverage and were used for further analysis. PMDs and HMDs regions annotations were described in the previous study , the PMDFor ChIP-seq data analysis, FastQC (version 0.11.9) was used to access the reads quality of raw data and TrimGalore (version 0.6.6) was used to trim the adaptor and low-quality reads with parameters “-q 25 –phred33 –length 40 -e 0.1 –stringency 3”. After quality control, the remaining reads were mapped to the reference genome hg19 with bowtie2 (version 2.4.5). Only uniquely mapped reads were kept and duplicates were removed by sambamba (version 0.8.2). Then, we call peaks using MACS2 with the following arguments: ‘‘-f BAM -g hs -q 0.05’’ for narrow peaks. To reduce the background noise, overlapping peaks between repetitions were preserved. Histone modification signal tracks for each sample were generated using the deepTools and were normalized to RPKM for visualization in WashU Epigenome Browser.q value < 0.1, corrected for multiple tests using the Benjamini Hochberg algorithm.For RNA-seq data analysis, FastQC (version 0.11.9) was used to access the reads quality of raw data and Trimmomatic (version 0.39) was used to trim the adaptor and low-quality reads. After quality control. The clean read pairs were mapped to the GENCODE reference transcriptomes (GRCh38.p13 v36 release) and quantified with Salmon. Differential gene expression analysis was performed using R package ‘DESeq2’ (version 1.34.0). Differentially expressed genes were defined with 2 for 48 h. Photographs were captured at 0 h and 48 h.In brief, HCT116 cells were seeded into 6-well plates. A straight line “wound” was made with a scape using a 10 μL pipette tip. After washing twice with PBS to remove debris, FBS-free medium was added and cells were incubated at 37℃ with 5% CO5/mL HCT116 cell suspension in serum-free medium was added into the up chamber of 8.0 µm transwell chambers  in 24-well plates. 500 μL medium supplemented with 10% FBS was added to the lower chamber. The cells were then incubated for 48 h at 37℃ with 5% CO2. Cells that did not migrate through the pores in the upper chambers were gently removed with a cotton swab, while cells that migrated to the underside of the filter were fixed with 4% paraformaldehyde for 30 min and stained with 0.1% crystal violet for 30 min. Subsequently, three random fields of stained cells were photographed by microscope.For transwell migration assay, 200 μL of 2.5 × 102 for 12 days. Cell colonies were fixed with 4% paraformaldehyde for 30 min and stained with 0.1% crystal violet for 30 min. Cell colonies (> 50 cells per colony) were counted and photographed.For colony formation assay, HCT116 cells were seeded onto 6-well plates at a density of 400 cells per well with McCoy’s 5A medium supplemented with 10% FBS and incubated at 37℃ containing 5% COt-test indicated in each figure was used to calculate p values. “ns” represents not significant, p ≥ 0.05, “*” represents p < 0.05, “**” represents p < 0.01, “***” represents p < 0.001, “****” represents p < 0.0001.In this study, each of the experiments was performed with at least two biological replicates unless otherwise specified. Data were presented as the mean ± SD. calculated by GraphPad Prism 6.0 software. Wilcoxon Rank Sum or Student’s Additional file 1: Supplementary Figure S1. Aberrant DNA methylation patterns in different cancers.Additional file 2: Supplementary Figure S2. DNA hypermethylation features of different cancers. A Number of genes in each group in different cancer types. B The overlap of C4 group genes in different cancer types.Additional file 3: Supplementary Figure S3. Gene expression patterns in different cancer types. A Abundance of genes from 5 groups in tumors or tissue normal counterparts. B The distribution of promoter CGIs in 5 groups in PMDs, HMDs and other regions in different cancer types. C The proportion of bivalent and non-bivalent genes in 5 groups in different cancer types. D Tau index of genes in 5 groups in different cancer types.Additional file 4: Supplementary Figure S4. Go analysis of each group of genes in different cancers.Additional file 5: Supplementary Figure S5. Correlations between DNA methylation, histone methylation and gene expression across multiple tissues. Additional file 6: Supplementary Figure S6. Relationship between H3K4me1 and DNA methylation at promoter CGIs. A Distribution pattern and level of H3K4me1 at promoter CGIs and flanked regions in high-H3K4me1 group and low-H3K4me1 group in different normal tissues. B DNA methylation differences  in high-H3K4me1 group and low-H3K4me1 group. Additional file 7: Supplementary Figure S7. Generation of LSD1 OE cell line and genome distribution of H3K4me1 peaks of each group. A Expression of FLAG-LSD1 in NCM460 control (NC) and LSD1 OE cells. B Relative mRNA expression of LSD1 in NCM460 control (NC) and LSD1 OE cells. C The overlap of H3K4me1 peaks in NC and LSD1 OE NCM460 cells. D Genomic distribution of H3K4me1 peaks of each group. Additional file 8: Supplementary Figure S8. Generation of LSD1 KO cell lines and enrichment of H3K27me3 at promoter CGIs. A Expression of LSD1 in WT and LSD1 KO HCT116 cells. B Enrichment of H3K27me3 at promoter CGIs in WT and LSD1 KO cells.Additional file 9: Supplementary Figure S9. The role of LSD1 in regulating multiple epigenetic modifications. A Genome distribution of H3K4me1 peaks and H3K4me3 peaks. B, C DNA methylation and enrichment of H3K4me1/3 at promoters of Up genes and Down genes in WT and LSD1 KO cells. Additional file 10: Supplementary Figure S10. Enrichment of H3K4me3 and H3K27me3 at promoter of OVOL2 in H1 cells.Additional file 11: Supplementary Figure S11. Generation of LSD1 OE and OVOL2 KD cell lines. A Relative mRNA expression of LSD1 and OVOL2 in control (NC) and LSD1 OE HCT116 cells. B Relative mRNA expression of OVOL2 in control (GV-NC) and OVOL2 KD cells. Additional file 12: Supplementary Figure S12. Uncropped blots used in this study. Additional file 13: Supplementary Table S1. Summary of 5 gene groups in different cancer types.Additional file 14: Supplementary Table S2. Primer information used in this study.Additional file 15: Supplementary Table S3. Public datasets used in this study.


The catalyst was constructed by integrating multi-layer thin flake Co3O4 with attached PPy nanofibers, utilizing the rich active sites of Co3O4 and the flexibility and tunability of PPy nanofibers to optimize the catalyst structure. Through comprehensive characterization and performance evaluation, our results demonstrate that the PPy@Co3O4 (0.1 : 1) catalyst exhibits remarkable OER catalytic activity and stability. This research provides new strategies and insights for the development of efficient and stable OER catalysts, holding promising prospects for energy conversion and storage applications in relevant fields.The oxygen evolution reaction (OER) plays a crucial role in energy conversion and storage processes, highlighting the significance of searching for efficient and stable OER catalysts. In this study, we have developed a composite catalyst, PPy@Co 3O4 and PPy nanofibers, our catalyst optimizes structure, harnessing rich Co3O4 sites and PPy flexibility for enhanced OER catalytic performance.Through synergy between multi-layer Co However, their high cost and limited availability restrict further research and application. Therefore, there is an urgent need to develop cost-effective, highly stable, and electrochemically active electrocatalysts.7,8With the rapid development of the fossil fuel industry, such as natural gas, petroleum, and methane, the world is facing severe energy shortages and environmental pollution crises. To propel human society towards further advancement and address challenges of resource scarcity and environmental pollution, the exploration of renewable and recyclable clean energy has become a key research area in this century.3O4 have attracted significant attention due to their excellent oxygen evolution reaction (OER) performance.9 Various catalysts with diverse structures and exposed crystal facets, such as particulate, spherical, bulk, bouquet-like, and network structures, can be obtained by employing different preparation methods, which profoundly influence the catalytic activity in different fields.10–12 However, despite certain progress being made, challenges still exist in current research, necessitating further exploration and optimization of the performance of Co3O4 catalysts to achieve more efficient and stable OER reactions.As a transition metal, metal cobalt and its oxide Co3O4, this study introduces a conductive polymer material, polypyrrole (PPy). As a commonly studied and utilized conducting polymer with alternating single-double bond conjugation, PPy possesses a C–N five-membered heterocyclic conjugated structure and finds extensive applications in electrochemical fields such as supercapacitors, oxygen reduction reactions (ORR), and oxygen evolution reactions (OER).13–15 The incorporation of PPy enhances the conductivity of the nanocomposite, effectively facilitating electron transfer in the OER process and thus improving the electrocatalytic performance of OER.16 In composite materials, the morphology of Co3O4 and its combination with PPy play a crucial role in their performance in the electrocatalysis field. For instance, a hierarchical Co3O4@PPy core–shell nanowire can be prepared by uniformly coating an amorphous PPy layer on the surface of Co3O4 nanowires via a simple hydrothermal method, and this composite material has been employed as an electrode material for supercapacitors.17,18 In this study, a hydrothermal method combined with the in situ polymerization technique using polyvinylpyrrolidone (PVP) as a soft template was employed to synthesize multi-layered flake-like Co3O4, followed by the preparation of PPy@Co3O4 composite material. The catalytic performance of PPy@Co3O4 composite material in the OER reaction was investigated, aiming to provide new insights and theoretical guidance for the development of efficient and stable electrocatalysts.To further enhance the OER catalytic performance of Coii) nitrate hexahydrate (Co(NO3)3·6H2O) was purchased from Xilong Chemical Co., Ltd. Pyrrole was obtained from Macklin Reagent Company. Ammonium persulfate was purchased from Tianjin Kaitong Chemical Reagent Co., Ltd. Urea ((NH2)2CO) and sodium dodecylbenzenesulfonate (SDBS) were obtained from Tianjin Kemiou Chemical Reagent Co., Ltd. Polyvinylpyrrolidone (PVP) was purchased from Tianjin Bodi Chemical Co., Ltd. All chemicals used were of analytical grade and did not require further purification. The ultrapure water used in this experiment had a resistivity of 18.2 MΩ cm.Cobalt(3)3·6H2O and (NH2)2CO were mixed in a molar ratio of 1 : 5 in 70 mL of ultrapure water. While stirring, an appropriate amount of PVP was gradually added to the mixture solution and stirred overnight until complete dissolution and mixing. The solution was transferred to a 100 mL polytetrafluoroethylene hydrothermal reaction vessel and reacted at 120 °C for 6 h. After the reaction, the reaction vessel was cooled to room temperature, and the precipitate was centrifuged. The centrifuged sample was washed three times with alternating ultrapure water and anhydrous ethanol solution. The sample was then placed in a hot air oven and dried at 80 °C for 6 h. After drying, the sample was removed and placed in a high-temperature furnace. The temperature was raised to 400 °C at a heating rate of 5 °C min−1 in air and calcined for 4 hours, resulting in black Co3O4 powder.Co2S2O8) was added dropwise. After stirring for 2 h, the mixture was centrifuged at low speed, and the resulting sample was washed three times with alternating ultrapure water and anhydrous ethanol. The sample was then placed in a hot air oven set at 70 °C and dried for 8 hours to obtain the PPy@Co3O4 composite with a mass ratio of 0.1 : 1, denoted as PPy@Co3O4 (0.1 : 1). For comparison experiments, other PPy@Co3O4 composite materials with mass ratios of 0.02 : 1, 0.05 : 1, and 0.2 : 1 were prepared by adjusting the amount of pyrrole monomer under the same conditions. These samples were denoted as PPy@Co3O4 (0.02 : 1), PPy@Co3O4 (0.05 : 1), and PPy@Co3O4 (0.2 : 1), respectively. The preparation of pure PPy was conducted under the same conditions without the addition of Co3O4.Approximately 0.3 g of the prepared black Co3O4 composite, Fourier-transform infrared spectroscopy (FT-IR) analysis was performed using the Spec drum infrared spectrometer from PerkinElmer, USA. The chemical environment of elements in the PPy@Co3O4 composite was analyzed using the ESCALAB 250Xi X-ray photoelectron spectrometer (XPS) from Thermo Fisher Scientific, USA. The Brunauer–Emmett–Teller (BET) method was employed to determine the specific surface area using a Hitachi Regulus 8100 instrument. The Barrett–Joyner–Halenda (BJH) method was utilized to assess the pore size distribution.To analyze the crystal structure and phase composition of the prepared samples, we employed the Smart Lab X-ray diffractometer (XRD) from Rigaku Corporation, Japan. The microscopic morphology of the materials was observed using the S-3400 scanning electron microscope (SEM) from Hitachi Ltd., Japan. Elemental scanning analysis was carried out with an energy dispersive X-ray spectroscope (EDS). Additionally, we utilized the H-7650 transmission electron microscope (TEM) from Hitachi Ltd. to observe the samples. For the observation of PPy in the PPy@Co6 Hz with an amplitude of 5 mV. The double-layer capacitance (Cdl) is estimated from the cyclic voltammetry (CV) curve. The electrochemical active surface area (ECSA) is calculated as Cdl/Cds, where Cs is 40 μF cm−2.Electrochemical performance testing was conducted using a Shanghai CHI 760E electrochemical workstation in a 0.1 M KOH electrolyte solution. A three-electrode system was employed, with a catalyst-modified glassy carbon electrode as the working electrode, an Ag/AgCl electrode as the reference electrode, and a platinum wire electrode as the counter electrode. Prior to each test, the glassy carbon electrode was carefully polished using aluminum oxide powder with particle sizes of 0.1, 0.3, and 0.05 mm, followed by rinsing with deionized water. Electrochemical impedance spectroscopy (EIS) measurements were performed over a frequency range of 0.1 to 102. Linear sweep voltammetry (LSV) polarization curve measurements were performed using a rotating disk electrode (RRDE) at a rotation speed of 1600 rpm and a scan rate of 5 mV s−1. The potential of the reference electrode Ag/AgCl was converted to the reversible hydrogen electrode (RHE), and all obtained potential values were adjusted according to the established formula ERHE = EAg/AgCl + 0.059 pH + 0.198.19A 5 wt% Nafion solution was diluted 10 times to obtain a 0.5 wt% solution, which was then subjected to 30 min of ultrasonication in a water bath. Subsequently, 15 mg of the catalyst sample was weighed and added to 5 mL of 0.5 wt% Nafion solution, followed by 1 h of ultrasonication in a water bath, resulting in a catalyst ink. Using a microsyringe, 10 μL of the ink was drop-cast onto a glassy carbon electrode with a diameter of 4 mm. The electrode was left undisturbed at room temperature for 1 hour to allow the formation of a film covering the electrode surface. The geometric surface area of the rotating disk electrode (RRDE) was calculated to be 0.1256 cm3O4 nanosheets were synthesized using a simple hydrothermal method. Polyvinylpyrrolidone (PVP) was used as a soft template, and Co(NO3)3·6H2O was employed as the cobalt source to synthesize Co(OH)2 intermediate via urea. Subsequently, the Co3O4 nanosheets were obtained through a short calcination process. The synthesis of Co3O4 nanosheets is chiefly accomplished through the hydrothermal approach. Crucially, the use of PVP as a soft template plays a pivotal role, guiding growth along specific crystallographic planes while simultaneously suppressing other orientations, ultimately resulting in the formation of sheet-like architectures. To load PPy onto the surface of Co3O4 nanosheets, an in situ polymerization method was employed. The specific steps were as follows: SDBS surfactant, pyrrole monomer, and oxidant (NH4)2S2O8 were added into the Co3O4 nanosheets, and the in situ polymerization reaction was carried out at a controlled temperature range of 0–5 ○C. This resulted in the formation of a fibrous network structure of PPy attached to the surface of Co3O4 nanosheets, forming the PPy@Co3O4 composite material. The addition of SDBS surfactant facilitated the formation of stable micelle structures on the surface of Co3O4, ensuring the uniform dispersion of PPy nanoparticles on the Co3O4 surface and promoting the interaction between PPy and Co3O4.As shown in 3O4, PPy, and PPy@Co3O4 catalyst samples with different mass ratios. The Co3O4 black powder exhibits distinct peaks at 18.9°, 31.3°, 36.8°, 44.8°, 55.6°, 59.3°, and 65.2°, which are in good agreement with the standard spectrum of Co3O4 (PDF#74-2120) and correspond to the (111), (220), (311), (400), (442), (511), and (440) crystal planes of Co3O4 product.14 This confirms the successful synthesis of the target product, Co3O4, through the hydrothermal decomposition of the precursor.4 Furthermore, the XRD patterns of the PPy@Co3O4 composite materials with different mass ratios also exhibit characteristic diffraction peaks of Co3O4, further confirming the presence of Co3O4 in the prepared catalyst samples.3O4 catalyst material, Fourier-transform infrared spectroscopy (FT-IR) analysis was conducted. As shown in −1 and 3329 cm−1, which are attributed to the N–H stretching vibrations of aromatic amines.20 The peak at 1164 cm−1 corresponds to the stretching vibration of C–N bonds, while the absorption peak at 1038 cm−1 is attributed to the C–H vibration mode in the pyrrole ring.21 The peak observed at 1614 cm−1 corresponds to the stretching vibration of C PBM data was replaced with SVG by xgml2pxml:<glyph-data id="z.dbd" format="PBM" resolution="300" x-size="8" y-size="10" xml:space="preserve">00000000000000000000000000000000111111110000000011111111000000000000000000000000</glyph-data><svg xmlns="http://www.w3.org/2000/svg" version="1.0" width="13.200000pt" height="16.000000pt" viewBox="0 0 13.200000 16.000000" preserveAspectRatio="xMidYMid meet"><metadata>Created by potrace 1.16, written by Peter Selinger 2001-2019</metadata><g transform="translate scale" fill="currentColor" stroke="none"><path d="M0 440 l0 -40 320 0 320 0 0 40 0 40 -320 0 -320 0 0 -40z M0 280 l0 -40 320 0 320 0 0 40 0 40 -320 0 -320 0 0 -40z"/></g></svg>C bonds, and the peak at 558 cm−1 is attributed to the C–H vibration.22 These spectral peaks further confirm the presence of PPy in the prepared composite catalyst material. Additionally, a peak at 665 cm−1 corresponding to the Co–O bond23 was observed, indicating the presence of Co3O4 in the prepared composite catalyst material. Based on the analysis of the FT-IR characteristic peaks and the previous XRD test results, we can conclude that the prepared catalyst material is a PPy@Co3O4 composite material.To demonstrate the structure and composition of the prepared PPy@Co2 adsorption–desorption isotherm and pore size distribution curve, we observed that within the relative pressure range of 0 to 1.0, the catalyst samples displayed typical characteristics of a Type IV isotherm. As clearly depicted in 3O4 sample was measured to be 21.58 m2 g−1, whereas that of the PPy@Co3O4 (0.1 : 1) was notably higher at 41.83 m2 g−1. 3O4 was approximately 13.26 nm, while that of PPy@Co3O4 (0.1 : 1) was slightly larger, around 13.88 nm. Notably, both materials predominantly exhibit a mesoporous structure. In a comprehensive comparison, PPy@Co3O4 (0.1 : 1) not only possesses a larger surface area but also a richer mesoporous structure, offering it numerous active sites, which are anticipated to significantly enhance its catalytic performance.24–26Based on the results of the N3O4 catalyst surface. In the spectrum shown in 3+, while the binding energies at 796.3 and 780.9 eV can be attributed to Co2+. The energy gap of approximately 15.5 eV between 796.3 eV and 779.5 eV is consistent with the characteristic of Co 2p 1/2 and Co 2p 3/2 orbitals in Co3O4,27,28 further indicating the coexistence of Co2+ and Co3+. Furthermore, the shift of peaks in the Co 2p region towards higher binding energy suggests strong interactions between Co3O4 and PPy, leading to electron transfer from PPy to Co3O4.29 In 3O4. Specifically, the peak at 529.5 eV is attributed to the presence of metal–oxygen bonds, the binding energy at 530.0 eV corresponds to the presence of OH−, and the binding energy at 531.2 eV corresponds to low-coordinated oxygen ions.30Based on XPS analysis, we have confirmed the chemical states of the PPy@Co3O4 (0.1 : 1) shown in 31 The peak at 400.6 eV corresponds to the CN bond in PPy4,32 and the peak at 399.8 eV provides evidence for the formation of a Co–N bond, further substantiating the potential for electron transfer between PPy and Co3O4. Additionally, as observed in Fig. S1(b),3O4 catalysts in high-resolution XPS can be divided into four peaks. The peaks at 284.4, 285.2, 286.5, and 289.1 eV correspond to C–C, C–N, C–O, and CO/CN bonds, respectively.20,33 These results contribute to further characterizing the chemical composition and bonding states of the PPy@Co3O4 composite materials.In the N 1s spectrum of PPy@Co3O4 prepared using PVP as a template exhibits a multi-layered sheet-like structure. This is likely the result of the gradual growth of cobalt nitrate and urea precursor on the surface of the PVP template.34 With prolonged reaction time, the particle mass of the cobalt nitrate and urea precursor product gradually increases, leading to the gradual aggregation of initially independent nanocrystals through weak intermolecular interactions. After high-temperature calcination, they transform into a multi-layered Co3O4 nanosheet structure. This conclusion is further supported by the TEM results shown in 3O4 nanosheet structure. 3O4, clearly showing the distribution of PPy nanofibers on the surface of Co3O4 nanosheets. The corresponding EDS analysis results  catalyst exhibits the best catalytic performance, achieving a potential of 1.794 V vs. RHE at a current density of 10 mA cm−2, while the other PPy@Co3O4 and Co3O4 catalyst samples did not reach the potential for a current density of 10 mA cm−2. Compared to other catalysts, the optimized PPy@Co3O4 (0.1 : 1) catalyst demonstrates exceptional catalytic performance, achieving a potential of 1.794 V vs. RHE at a current density of 10 mA cm−2. Meanwhile, other PPy@Co3O4 and Co3O4 catalyst samples failed to attain this potential at the same current density. This indicates that as the content of PPy increases, its evolving network structure extensively covers the nanoplate surfaces, enhancing electron transport rates.By conducting LSV tests, as shown in 35–37 Furthermore, by comparing the corresponding Tafel slopes shown in 3O4 (0.1 : 1) possesses the lowest slope value of 67.9 mV dec−1 among the catalyst samples, indicating higher catalytic activity and a faster OER reaction rate. Additionally, the catalytic activity of PPy@Co3O4 (0.1 : 1) is comparable to similar catalyst materials reported in the literature,3,4,38,39 as evident from the comparison in However, surpassing an optimal PPy content starts to negate these benefits, as active sites on the nanoplates could be permanently occluded by thick layers of PPy, hindering electron transfer. In short, polypyrrole plays a pivotal role in enhancing conductivity and acting as a conductive binder, but an excess of PPy can suppress the OER performance of the catalyst.3O4 at different mass ratios and pure PPy catalyst, respectively. The Nyquist plot is fitted based on an equivalent circuit model, as depicted in the inset. In this model, Rs represents the solution resistance; R1 corresponds to the electron transfer resistance from the catalyst to the electrode; and Rct denotes the charge transfer resistance at the interface. Typically, a smaller Rct value suggests a faster kinetic response of the catalyst. From the fitting data, it's observed that PPy@Co3O4 (0.1 : 1) has the lowest Rct value of 630 Ω. In comparison, PPy@Co3O4 (0.02 : 1) and PPy@Co3O4 (0.05 : 1) exhibit Rct values of 950 Ω and 1029 Ω, respectively. On the other hand, PPy@Co3O4 (0.2 : 1) displays a relatively higher impedance. This indicates that PPy@Co3O4 at a 0.1 : 1 mass ratio demonstrates the best charge transfer efficiency and enhanced catalytic activity.3O4 composite catalysts at any given ratio. This further confirms that an appropriate amount of PPy can facilitate electron transport between PPy and Co3O4. This synergistic effect leads to a reduced charge transfer resistance at the interface between the electrolyte and the electrode, enhancing the overall charge transport performance and decreasing interfacial resistance.Additionally, as shown in 3O4, cyclic voltammetry (CV) tests were conducted on all the prepared PPy@CoCo3O4 composite materials at varying scan rates  values for the catalysts were calculated as follows: PPy@Co3O4 (0.02 : 1) 1.80 mF cm−2, PPy@Co3O4 (0.05 : 1) 4.35 mF cm−2, PPy@Co3O4 (0.1 : 1) 6.07 mF cm−2, and PPy@Co3O4 (0.2 : 1) 1.77 mF cm−2. Correspondingly, their electrochemical surface areas (ECSA) were 45.00 cm2 cm−2, 108.75 cm2 cm−2, 151.75 cm2 cm−2, and 44.25 cm2 cm−2, respectively. Notably, PPy@Co3O4 (0.1 : 1) exhibited the largest ECSA. ECSA is a pivotal metric in evaluating the performance of electrocatalysts. A larger ECSA often implies that more active sites are exposed to the reaction medium, frequently leading to enhanced OER catalytic activity. Additionally, an increased ECSA provides more pathways and channels for the efficient transport of electrolytes, intermediates, and products, thereby further optimizing catalytic performance.To better elucidate the electrocatalytic activity of PPy@Co2+ to Co3+, highlighting the crucial step for efficient OER activity.1,40 As observed from Fig. S4(a)–(d),3O4 (0.1 : 1) sample, the oxidation peak in the first cycle appears at 1.62 V and shifts to 1.65 V in the second cycle.The pre-oxidation of the electrocatalyst plays a pivotal role in evaluating the active species through CV activation. CV activation promotes the transition from Co3O4, oxidation peaks are observed at 1.64 V (first cycle) and 1.67 V (second cycle). This demonstrates that PPy can facilitate the pre-oxidation of Co2+, accelerating electron transfer.For Co3O4 nanosheets and attached PPy nanofibers, offers advantages such as increased effective reaction interface, abundant active sites, excellent charge transfer performance, and facilitated reactant diffusion. These characteristics collectively contribute to the enhanced OER catalytic activity of the catalyst.16,41,42 To evaluate the practical application potential of the PPy@Co3O4 (0.1 : 1) catalyst, we conducted cyclic stability testing with 2000 cycles of CV measurements, as shown in Based on the above analysis, it is evident that the composite catalyst structure, consisting of multi-layered Co3O4 (0.1 : 1) catalyst. The multilayered thin flake-like Co3O4 provides a substantial surface area, while the attached PPy nanofibers further expand the reaction interface, creating abundant active sites for catalytic reactions. This unique composite structure significantly enhances the catalyst-electrolyte contact area, synergistically promoting catalytic efficiency. Moreover, the PPy nanofibers serve as a protective layer for the multilayered Co3O4 structure. They effectively shield Co3O4 from direct contact with the KOH electrolyte, reducing corrosion and dissolution of Co3O4, thus enhancing catalyst stability and maintaining long-term catalytic activity. Additionally, the flexible and tunable nature of PPy nanofibers mitigates volume changes and stress accumulation. The distinctive microstructural features of the PPy@Co3O4 (0.1 : 1) catalyst enable sustained high catalytic activity during prolonged use.3O4, consisting of multilayered thin-film Co3O4 with PPy nanofibers attached on the surface. The catalytic performance and stability of PPy@Co3O4 in OER were evaluated. Comparative analysis of Co3O4, PPy, and different mass ratios of PPy@Co3O4 catalyst samples revealed that PPy@Co3O4 (0.1 : 1) exhibited the highest OER catalytic activity. It demonstrated a lower overpotential at a current density of 10 mA cm−2, indicating a faster reaction rate in the OER. Tafel slope analysis further confirmed the rapid kinetic response of the PPy@Co3O4 (0.1 : 1) catalyst. Moreover, the CV stability test demonstrated excellent stability performance of the PPy@Co3O4 (0.1 : 1) catalyst with only a 2.3% decay after 2000 cycles. The unique structure of multilayered thin-film Co3O4 and attached PPy nanofibers provided numerous active sites, enhanced charge transfer properties, and a protective layer, effectively promoting the OER while minimizing catalyst corrosion and dissolution. These findings offer new insights and strategies for the development of efficient and stable OER catalysts with potential applications in energy conversion and storage.In summary, we successfully synthesized a composite catalyst, PPy@CoThere are no conflicts to declare.RA-013-D3RA05936G-s001