However, as there are other NO3−RR pathways present, selectively guiding the reaction pathway towards NH3 is currently challenged by the lack of efficient catalyst. Here, we demonstrate a novel electrocatalyst for NO3−RR consisting of Au doped Cu nanowires on a copper foam (CF) electrode (Au–Cu NWs/CF), which delivers a remarkable NH3 yield rate of 5336.0 ± 159.2 μg h−1 cm−2 and an exceptional faradaic efficiency (FE) of 84.1 ± 1.0% at −1.05 V (vs. RHE). The 15N isotopic labelling experiments confirm that the yielded NH3 is indeed from the Au–Cu NWs/CF catalyzed NO3−RR process. The XPS analysis and in situ infrared spectroscopy (IR) spectroscopy characterization results indicated that the electron transfer between the Cu and Au interface and oxygen vacancy synergistically decreased the reduction reaction barrier and inhibited the generation of hydrogen in the competitive reaction, resulting in a high conversion, selectivity and FE for NO3−RR. This work not only develops a powerful strategy for the rational design of robust and efficient catalysts by defect engineering, but also provides new insights for selective nitrate electroreduction to NH3.Electrochemical nitrate reduction reaction (NO 3−RR) to synthesize valuable ammonia (NH3) is considered as a green and appealing alternative to enable an artificial nitrogen cycle.Electrochemical nitrate reduction reaction (NO RHE) for the electrocatalytic NO3−RR under neutral conditions. 15N isotopic labelling experiments were performed to confirm the origin of ammonia, which was quantified by both 1H nuclear magnetic resonance (NMR) spectra and colorimetric methods. The XPS analysis and in situ infrared spectroscopy (IR) spectroscopy characterization results indicated that the oxygen vacancies in Au–Cu NWs/CF can weaken the N–O bonding,25 moreover, the electron transfer between Cu and Au interface could inhibit the competitive reaction of the hydrogen evolution reaction (HER),11 resulting in high NH3 yield rate and FE of NO3−RR.Herein, we utilized a facile three-step method to fabricate the Au doped Cu nanowires on a copper foam (CF) (denoted as Au–Cu NWs/CF) electrode for the selective nitrate electroreduction to ammonia. The Au–Cu NWs/CF sample exhibited an exceptional performance with the NH2 NWs/CF was prepared via a facile wet-chemical oxidation method. Subsequently, NWs/CF was directly immersed into 10 mM HAuCl4·3H2O solution for 12 h, dried at 60 °C under vacuum for 4 h, the Au–Cu(OH)2 NWs/CF was annealed under Ar atmosphere to obtain Au–CuO NWs/CF. Finally, the Au–Cu NWs/CF was obtained by in situ electrochemical reduction of the resultant Au–CuO NWs/CF. The scanning electron microscopy (SEM) images of CF , (200) and (220) plane of metallic Cu (JCPDS no. 04-0836), respectively.26–28 While besides of typical diffraction peaks of metallic Cu, the Au–Cu NWs/CF sample also displays the weak characteristic peaks of Au nanoparticles at 2θ = 38.2°, 44.3°, 64.6° and 77.5°, suggesting the formation of fcc Au phase on Cu nanowires with low loading content.29 The actual loading of Au was calculated to be 5.6 wt% by inductively couple plasma atomic emission spectroscopy (ICP-AES). High-resolution TEM  measurement was performed to investigate the surface composition and valence state of Au–Cu NWs/CF. For comparison, we also performed the XPS characterization of CF sample. The XPS survey spectra and high-resolution XPS spectra of Au 4f verified the existence of doped Au in the Au–Cu NWs/CF . The higu NWs/CF , due to  spectra , 530.9 a3−RR performance of the as-fabricated Au–Cu NWs/CF catalysts in a 0.1 M Na2SO4 + 10.0 mM KNO3 solution (pH = 5.6) using a three-electrode configured two-compartment cell. In all experiments, the Au–Cu NWs/CF catalyst was used as working electrode, Ag/AgCl (saturated KCl solution) and Pt mesh were used as the reference electrode and counter electrode, respectively. Colorimetric methods were adopted determine the concentration of NO3−, NO2− and NH4+ , which may be due to the electrochemical reduction of NO3−. Chronoamperometry (CA) measurements of Au–Cu NWs/CF were conducted at different potentials for 2 h with continuous argon gas (Ar) bubbling. Fig. S5avs. RHE). The concentration of NH3 product was measured using indophenol blue method  of 5336.0 ± 159.2 μg h−1 cm−2 and the FE of 84.1 ± 1.0% at −1.05 V (vs. RHE). The selectivity of NH3 (S3NH) and R3NH show the same trend with the increase of potential, and highest S3NH was 90.6 ± 3.2% (vs. RHE) (vs. RHE), the R3NH and S3NH decreased due to the competitive hydrogen evolution reaction (HER).30 Although the electrodynamic potential of NO3− to NO2− is higher than that of NO3− to NH3, NO2− is easily detected an main by-product of NO3−RR.31 As shown in Fig. S62− is detected after electrocataytic reduction at −1.05 V (vs. RHE), further demonstrating the high selective reduction of nitrate to NH3. We also tested R3NH and FE of bare CF with 0.1 M Na2SO4 + 10.0 mM KNO3 solution at −1.05 V (vs. RHE) to exclude the influence of substrate. As shown in Fig. S7R3NH and FE for bare CF were 1777.7 μg h−1 cm−2 and the FE of 49.9%, much lower than Au–Cu NWs/CF. The corresponding equivalent circuit diagrams of bare CF and Au–Cu NWs/CF are shown in Fig. S8Rs) and charge-transfer resistance (Rct) from Au–Cu NWs/CF confirms its high electrical conductivity, which could be an important attribute for the achieved high R3NH and FE.We evaluated the NO6 ± 3.2% . In addivs. RHE) . When th15N isotope labeling with 1H nuclear magnetic resonance (NMR) is usually required in NO3−RR experiments to confirm that the detected NH3 indeed originates from the electrochemical nitrate reduction to rule out contaminations. We carried out chronoamperometry measurement at −1.05 (V vs. RHE) for 2 h in the electrolyte with K15NO3 and K14NO3 as N source, respectively. As shown in 14NO3, the 1H NMR spectra of the obtained products displayed typical peaks of 14NH4+. In contrast, when the K15NO3 was used as nitrogen source, the 1H NMR spectra showed typical double peaks of 15NH4+. Such results indicated that the produced NH3 was entirely derived from the nitrate in the electrolyte, rather than from the contaminations. Meanwhile, the electrolyte without nitrate addition is also tested. The electrochemical measurement in blank 0.1 M Na2SO4 electrolyte produced ignorable NH3 , no noticeable decay in the cathodic current density and UV-vis absorptions  spectroscopy characterization to detect intermediates and monitor the reaction. in situ IR spectra of Au–Cu NWs/CF under various potentials. As shown, without the applied potential, there is no any infrared peak in the in situ IR spectra. In the investigated potential range from −0.7 to −1.1 V (vs. RHE), the new infrared bands at ∼1541 cm−1 was assigned to the −NOx intermediates.33,34 In addition, the bending mode of –NH2 is also found at ∼1457 cm−1.34,35 Clearly, as the applied potential increased, the peak intensity of −NOx intermediates and –NH2 gradually increased (in situ IR measurements for the NO3−RR at −1.05 V (vs. RHE). The IR intensity of the peaks at around 1457 cm−1 and 1541 cm−1, corresponding to –NH2 and −NOx intermediates is increased obviously from 4 to 36 min, implying that the NO3−RR takes place gradually with reaction time under the given electrocatalytic conditions. Evidenced by the in situ IR results, the NH3 synthesis by NO3−RR is successfully achievable  in neutral electrolyte. The in situ IR spectroscopy measurements confirm the successful realization of NH3 synthesis by NO3−RR over Au–Cu NWs/CF. Our work would be helpful to design and develop high-efficiency NO3−RR electrocatalysts for ambient electrosynthesis of ammonia.In conclusion, Au doped Cu nanowires on a copper foam electrode was synthesized There are no conflicts to declare.RA-013-D3RA00679D-s001


In addition, we determined various structures of the murine Elongator complex, including the fully assembled mouse Elongator complex at 5.9 Å resolution. Our results confirm the structural conservation of Elongator and its intermediates among eukaryotes. Furthermore, we complement our analyses with the biochemical characterization of the assembled human Elongator. Our results provide the molecular basis for the assembly of Elongator and its tRNA modification activity in eukaryotes.Transfer RNA (tRNA) molecules are essential to decode messenger RNA codons during protein synthesis. All known tRNAs are heavily modified at multiple positions through post-transcriptional addition of chemical groups. Modifications in the tRNA anticodons are directly influencing ribosome decoding and dynamics during translation elongation and are crucial for maintaining proteome integrity. In eukaryotes, wobble uridines are modified by Elongator, a large and highly conserved macromolecular complex. Elongator consists of two subcomplexes, namely Elp123 containing the enzymatically active Elp3 subunit and the associated Elp456 hetero-hexamer. The structure of the fully assembled complex and the function of the Elp456 subcomplex have remained elusive. Here, we show the cryo-electron microscopy structure of yeast Elongator at an overall resolution of 4.3 Å. We validate the obtained structure by complementary mutational analyses   Together with messenger RNAs (mRNA), they are responsible for the correct transfer of information from the genomic DNA into functional polypeptide chains. tRNAs undergo significant post-transcriptional modifications, which confer stabilization of the folded tRNA, regulate the interaction with other molecules or tune the codon-anticodon pairing during the mRNA translation process within the ribosome . In eukaand Sit4 . Subsequ(mcm5s2) ,8. These(mcm5s2)  and thus(mcm5s2) . Further(mcm5s2) , like fa(mcm5s2) , rolandi(mcm5s2) , intelle(mcm5s2) , amyotro(mcm5s2) , other n(mcm5s2)  and chil(mcm5s2) .Saccharomyces cerevisiae Elp123 (ScElp123) adopts a symmetric ‘moth’-like shape, with two distinguishable lobes that are separated by a cleft and connected by an arch region. In contrast, the structure of the fully assembled Elongator complex seems asymmetric as Elp456 only binds to one lobe of the dimeric Elp123 subcomplex (S-adenosyl-methionine (SAM) cleavage product 5′-deoxyadenosine (5′-dA) and the respective tRNA substrate (S-adenosyl methionine (rSAM) and lysine acetyltransferase (KAT) domains to modify the well-positioned U34 nucleotide  . The combcomplex ,28. Basebcomplex , encompabcomplex . Subsequubstrate . Of noteubstrate ,33. In dcleotide . tRNA bicleotide  and invocleotide .in vitro  and cloned in pairs into three individual vectors with inducible GAL1 and GAL10 promoters to allow for simultaneous expression of two gene products from a single vector  protease cleavage site to facilitate the purification from yeast cells. All sequences were introduced at the 3′ end of the individual ORFs. Mus musculus and Homo sapiens biGBac  were cloned separately into pFastBac1 plasmids. ELP3 was cloned in frame with a Twin-Strep-Tag sequence and ELP6 with FLAG-TEV-Protein A sequence at their 3′ ends. ELP2 genes were cloned into pFastBac-HTA with a 6xHis-tag sequence at its 5′ end. All genes were amplified from the pFastBac plasmids by PCR using primers with predefined 5′ and 3′ overhangs in each gene, followed by Gibson assembly reactions ectively . Mutagenhis3-Δ200, leu2-Δ1, trp1-Δ63, ura3-52), co-transformed with the constructs expressing all six Elongator genes, were grown on synthetic drop-out (SD) media plates (2% w/v agar and 2% w/v glucose) lacking tryptophan, leucine and uracil. Cells were grown shaking overnight at 30°C in the SD media (2% w/v glucose), reinoculated (3:50 ratio) on the next day into fresh SD medium and further incubated overnight at 30°C with shaking. For protein overproduction, cells were inoculated (1:20) in fresh SD media containing 2% w/v raffinose and incubated for 5–7 h at 30°C while shaking. ELP genes expression was induced by addition of galactose to a final concentration of 2% w/v and cells were left shaking for 21 h at 30°C. Cells were harvested by centrifugation, resuspended in lysis buffer (250 mM HEPES (pH 7.5), 100 mM NaCl, 10% v/v glycerol, 0.1% v/v Tween 20, 1 mM sodium orthovanadate, 20 mM sodium fluoride, aprotinin (2 μg/ml), leupeptin (5 μg/ml), 1 μM pepstatin A, and 1 mM PMSF) and small cell suspensions droplets were flash frozen in liquid nitrogen. Droplets were lysed by cryo-milling (Qiagen TissueLyser II) and stored as a powder at −80°C until use. All purification steps were performed at 4°C or on ice. 12 ml of fresh lysis buffer was added per 40 g of dry yeast pellet and thawed for 1 h under agitation. Lysates were clarified by two consecutive centrifugation steps at 21 000 × g for 30 min. Cell extracts were incubated for 30 min with 3 ml of IgG Sepharose™ 6 Fast Flow resin  and washed three times with lysis buffer. Proteins were eluted in 5 ml lysis buffer by TEV protease cleavage (1 mg) in presence of 1 mM dithiothreitol (DTT) for 1 h. Subsequently, the eluent was applied to a 1 ml StrepTrap HP column  and eluted with 20 mM d-desthiobiotin. The complex was concentrated using an Amicon Ultra-15 centrifugal filter (100 kDa cut-off) and the Elongator was further purified on a Superose 6 Increase 10/300 GL column  equilibrated with 20 mM HEPES (pH 7.5), 100 mM NaCl and 3 mM DTT. Selected fractions were pooled and concentrated with an Amicon Ultra-0.5 centrifugal filter (100 kDa cut-off). Aliquots were frozen in liquid nitrogen and stored at −80°C for further use.DS1-2b yeast cells , leupeptin (5 μg/ml), 1 μM pepstatin A, 1 mM phenylmethylsulfonyl fluoride in presence of DNase I by three freeze-thaw cycles and sonication. Lysates were clarified by two consecutive centrifugation steps at 4°C and 63 000 × g for 30 and 60 min. Elp123 subcomplexes were purified from cell extracts using a 5 ml StrepTrap HP column  and eluted with 10 mM d-desthiobiotin. Next, Elp123 subcomplexes were further purified on a 5 ml HiTrap Heparin HP column  and eluted with a linear KCl gradient . The final purification step was carried out on a Superose 6 Increase 10/300 GL column  equilibrated with 20 mM HEPES (pH 7.5), 100 mM NaCl and 5 mM DTT. Mouse and human Elp456 subcomplexes were purified from cell extracts using IgG Sepharose™ 6 Fast Flow resin equilibrated with 50 mM HEPES (pH 7.5), 300 mM NaCl, 1 mM DTT and 5% v/v glycerol. Elution from the resin was accomplished by TEV protease cleavage (1 mg/5 ml). Eluates were concentrated using Amicon Ultra-15 centrifugal filters (100 kDa cut-off) and subjected to a Superose S6 Increase 10/300 GL column  equilibrated with 20 mM HEPES (pH 7.5), 100 mM NaCl and 5 mM DTT. Purity, integrity, protein quality and all complexes stoichiometry were monitored during every step by SDS-PAGE analysis and visualization with Coomassie stain.Mouse and human Elp123 were overproduced in Super Sf9-1, Sf9-3 respectively, while both Elp456 in Hi-5 cells. Cells were cultured in ESF921 medium (Expression Systems) to a density of 800 000 cells/ml for Super Sf9-1/Sf9-3 and 500 000 cells/ml for Hi-5 cells, followed by baculovirus infection with the multiplicity of infection (MOI) of 1 and grown for 3 days at 27°C while shaking. Cell pellets were harvested and lysed in 50 mM HEPES (pH 7.5), 100 mM NaCl for Elp123 and 300 mM NaCl for Elp456, 2 mM MgClS. cerevisiae strains were grown in YPD (Yeast Extract–Peptone–Dextrose) to an OD600 nm of 1, harvested, washed and resuspended in NucleoZOL (Machery Nagel). Cells were lysed by bead beating in 6 cycles each 60 s, 20% v/v volume of chloroform was added and samples were vigorously vortexed for another 60 s. Subsequently, the suspension was incubated for 15 min at 4°C and centrifugated for 30 min at 21 000 × g. The aqueous RNA-containing phase was transferred into a new reaction tube and large RNAs were precipitated for 3 h at −20°C using 2/3 volume of 8 M LiCl. Large RNAs were pelleted for 30 min at 4°C and 21 000 × g. To increase tRNA purity, the LiCl precipitation step was repeated. The tRNA-containing supernatant was precipitated with 10% v/v 3 M sodium acetate pH 5.2 and 2.5 volumes 100% ethanol overnight at −20°C. The tRNA containing pellet was washed twice with 75% v/v ethanol, air dried and resuspended in 10 mM sodium acetate pH 5.2. tRNA concentrations were determined using the Epoch (Agilent BioTek) spectrophotometer and stored at −80°C.To obtain yeast bulk tRNA, 2, 1% TritonX-100 v/v, 0.5% v/v sodium deoxycholate, 0.5 mM DTT) at 4°C. The lysate was mixed with MiliQ water at a 1:1 ratio v/v and the RNA was isolated three times with 1 volume of acid-phenol:chloroform (1:1 ratio v/v), followed by an additional step using 1 volume chloroform. After each step the sample was vortexed and centrifugated for 10 min at 4500 × g at 4°C and the aqueous phases were collected, combined and mixed with 10% volume of 3 M sodium acetate pH 5.5, three volumes of 100% ethanol, 1 μl of glycogen and incubated overnight at −80°C. The precipitate, was centrifuged at 8000 × g for 30 min at 4°C and the supernatant was discarded. The pellet was resuspended in 100 μl equilibration buffer without TritonX-100 (10 mM Bis–Tris (pH 6.3), 15% ethanol v/v, 200 mM KCl). RNA concentration was measured using a NanoDrop spectrophotometer and the sample was stored at −80°C until use. To isolate bulk tRNA 900 μg of total RNA (diluted in 2 ml equilibration buffer without TritonX-100) was loaded on an equilibrated NucleoBond AX100 column (10 mM Bis–Tris (pH 6.3), 15% v/v ethanol, 200 mM KCl, 0.15% v/v TritonX-100) and washed twice with 12 ml washing buffer (10 mM Bis–Tris (pH 6.3), 15% v/v ethanol, 500 mM KCl) tRNA was eluted with 12 ml elution buffer (10 mM Bis–Tris (pH 6.3), 15% v/v ethanol, 800 mM KCl) and mixed with 2.5 volumes of 100% ethanol, 1 μl of glycogen and incubated overnight at −80°C. After precipitation, the sample was centrifuged for 30 min at 8000 × g (4°C), and the supernatant was discarded. The tRNA pellet was washed with 40 ml 80% v/v EtOH by vortexing. Afterward, the tRNA was centrifuged for 30 min at 8000 × g (4°C). Subsequently, the tRNA pellet was air dried and resuspended in 30 μl RNAse free water. tRNA concentration was measured using a NanoDrop spectrophotometer and the sample was stored at −80°C for later use.HEK293T cells cultured on 15-cm Petri dishes were washed with PBS and lysed for 5 min in 250 μl RNA lysis buffer (10 mM Tris–HCl (pH 7.5), 100 mM NaCl, 10 mM MgCl® R 2/1 copper grids (200 mesh) were glow-discharged on a Leica EM ACE 200 glow discharger . 2.5 μl of the sample at 0.4–0.6 mg/ml concentration was plunge-frozen using a Vitrobot Mark IV (Thermo Fisher) set to 100% humidity and 4°C with the following blotting parameters − 15 s wait time, blot force 5 and 5 s blot time. tRNA was used at 3–5 molar excess with 15 min incubation on ice for ScElp123–tRNA and 37°C for mouse Elp123 (MmElp123)–tRNA samples. ScElp123–tRNA micrographs were acquired at 300 kV using a Titan Krios 2  equipped with a Gatan Quantum energy filter and a K2 Summit direct electron detector. 11,080 micrographs were collected with 0.81 Å pixel size and 0.8–2 μm under-focus for a total of 40 frames accumulating 42.45 e−/Å2 dose. The remaining datasets were collected on Titan Krios G3i  equipped with a Gatan Quantum energy filter and a K3 Summit direct electron detector. The used under-focus range was 0.9–3 μm for a total of 40 frames accumulating an overall dose of 40 e−/Å2. 6339, 4716, 6415, 7000 and 5232 micrographs were collected for ScElongator, ScElp456, MmElp123, MmElp123–tRNA and MmElongator, respectively with a pixel size of 1.1 Å for ScElp456 and 0.86 Å for the remaining data sets.QUANTIFOILScElp123–tRNA, ScElp456 and MmElp123; with WARP 1.0.9  was corrected. Blob picking was performed on 10–15% of micrographs to select an initial set of particles for template picking (MmElp123–tRNA) and to train the TOPAZ particle picker  equilibrated with 20 mM HEPES (pH 7.5), 100 mM NaCl and 5 mM DTT. For the pull-down experiments 10 μg of bait and 20 μg of prey were used. Elp123 was immobilized on StrepTactin™ Sepharose™ (Cytiva) beads via Twin-Strep-tagged Elp3 or in case of Elp456 via anti-DYKDDDDK (Pierce™) resin and FLAG-tagged Elp6. The respective subcomplex was added and incubated for 30–60 min at 4°C, followed by three wash steps with 20 mM HEPES (pH 7.5), 100 mM NaCl, 1 mM DTT, 0.05% v/v Tween 20. Elution from StrepTactin or anti-DYKDDDK resin was carried out in the same buffer containing either 5 mM UCU (0.2 μM) or tRNA AlaUGC (0.3 μM) were incubated for 5 min at 37°C with serial dilutions of Elp123, Elp456 or Elongator variants (starting from 10 μM) in MST Buffer (20 mM HEPES (pH 7.5), 100 mM NaCl, 5 mM DTT, 0.0125% v/v Tween 20). Measurements were performed at 60% excitation power in Premium Coated capillaries on the Monolith NT.115 (Nanotemper Technologies) at 37°C. Obtained data were analyzed and dissociation constant values were calculated using MO. Affinity software (Nanotemper Technologies) from at least three independent experiments (n > 3). For gel shift experiments internally Cy5-labled tRNA AlaUGC (0.2 μM) was incubated with 0.05–1 μM wild type Elp123, Elongator or Elp123Elp1Δ1132–1224 variant at 4°C for 15 min. Next samples were separated on NativePAGE™ 4–16% Bis–Tris Protein Gels at 4°C for 90 min and 150 V. Gels were imaged on BIO-RAD ChemiDoc™ MP Imaging System in Cy5 detection mode.The internally Cy5-labeled tRNA ArgThe assay was performed as previously described . In detaELP1, ELP3, ELP4, ELP5 and ELP6 genes were generated in the UMY2893 wild type strain  and 3ELP5-(FLAG) into the genome was facilitated by homologous recombination of the desired tag together with a KlLEU2 (PCR-based deletions of 3 marker  for late3 marker . In ordebination . Mutant escribed . Introdua KlLEU2  or clonNa KlLEU2 . DescripEscherichia coli BL21 (DE3) pRARE strain. Cultures were grown at 37°C in autoinduction media  column, concentrated, and dialyzed into a storage buffer (50 mM Tris–HCl (pH 7.5) at 4°C, 150 mM NaCl, 2 mM DTT).The GST-γ-toxin construct was expressed in the on media  and were5s2U34 modification status of Elongator mutants an in vitro cleavage assay using GST-γ-toxin was applied as previously described (To probe for the mcmescribed . In detain vivo acetyl-CoA carboxylase (Acc1) interaction, co-immunoprecipitation was carried out as described before ). Bound protein was eluted with elution buffer  at 50°C for 15 min and subsequent incubation of 1× Laemmli buffer at 98°C for 5 min. Proteins were separated on a 10% SDS-PAGE and analysed by Western blotting on a 0.45 μM pore sized polyvinylidene difluoride  membrane. Proteins were detected using anti-c-myc (9E10 hybridoma supernatant kindly provided by Prof. Dr M. Maniak), anti-GFP antibody , anti-FLAG antibody  and anti-Cdc19 serum .To probe for d before . 8 mg lyScElongator complex at 2.22 μM concentration was crosslinked with sulfo-SDA  at 1:500 and 1:1000 sample:crosslinker molar ratios. The reaction was incubated at room temperature for 60 min irradiated with UV light at 365 nm using a Luxigen34 LZ1 LED emitter (Osram Sylvania Inc.) for 10 s, and quenched with a final concentration of 50 mM ammonium bicarbonate (ABC). The crosslinked complex was separated from single subunits on a Novex Bis–Tris 4–12% SDS-PAGE gel (Life Technologies). Gel pieces containing the crosslinked complex were excised. The sample was reduced with DTT and free sulfhydryl groups were alkylated using iodoacetamide (85 μg of cetamide . Proteincetamide . The eluFor the liquid chromatography–tandem mass spectrometry (LC–MS/MS) analysis, an Ultimate 3000 RSLCnano system  connected to an Orbitrap Fusion Lumos Tribrid mass spectrometer (Thermo Fisher Scientific) was used. Each SEC fraction was resuspended in 1.6% v/v ACN and 0.1% v/v formic acid before injection onto a 50-cm EASY-Spray C18 LC column . The flow rate during sample loading and separation was 0.3 μl/min. The mobile phase consisted of solvent A (0.1% v/v formic acid) and B . A 2–55% gradient of B was applied over the course of 90–100 min (optimised per SEC fraction) with a final increase to 95% B within 2.5 min. An EASY-Spray source (Thermo Scientific) ionised the eluting peptides before introducing them to the mass spectrometer. For each SEC fraction, two acquisitions were carried out. Data-dependent MS data were acquired in cycles of 2.5 s using the top-speed setting and the full scan mass spectrum was recorded in the Orbitrap with a resolution of 120 000. Ions with a charge state from 3+ to 7+ were selected for fragmentation by stepped higher-energy collisional dissociation  using a decision tree . SpectraMS2 peak lists were generated from the mass spectrometric raw data with the MSConvert module of ProteoWizard (version 3.0.11729). Precursor and fragment m/z values were recalibrated based the average mass error of linear peptide spectrum matches. Additional peptide modifications were identified using FragPipe (version 18.0) with MSFragger  and phiS. cerevisiae Elongator (ScElongator) enabling us to simultaneously overexpress the full-length versions of Elp1, Elp2, Elp3, Elp4, Elp5 and Elp6 in suitable yeast strains , Elp5 (FLAG) in an Acc1 (GFP) background  remain n Figure . We did n Figure , althougn Figure . Charactx Figure  92)..ScElp456in vivo function of Elongator  and Elp4  in CP1 show no measurable effects on the tRNA modification activity of Elongator. Strikingly, all tested mutations in CP2 nearly abolished Elongator's tRNA modification activity. Mutations and deletions of the identified residues in Elp1 , Elp4  and Elp5 (Δ1–20) display tRNA hypo-modification phenotypes that are indistinguishable from their respective gene deletion mutants, in all conducted in vivo and in vitro tRNA modification assays  and Elp6  are resistant to growth inhibition by zymocin. Of note, mutants in Elp123 demonstrate identical phenotypes in all applied assays, whereas individual mutations in Elp456  exhibited a slightly divergent phenotype. These variations mostly affect mutants that display intermediate phenotypes and might occur due to different sensitivity of performed assay formats. These results also support the recent observation that Elp456 function might only be relevant for a subset of cellular tRNAs  analysis of the purified yeast Elongator complex. 638 out of 883 detected cross links are in agreement (30 Å distance) with our structural model . In addiMmElp123 and MmElp456 genes separately in insect cells. The incorporation of affinity tags on Elp3 (TST) and Elp6 (FLAG) facilitated the purification of the individual subcomplexes using standard affinity and size exclusion chromatography. The gel filtration profiles of MmElp123 (∼610 kDa) and MmElp456 (∼220 kDa) show that they all elute at the expected elution volume   of Elp1 is displaced by Elp456 and would not be able to interact with the T-arm of tRNAs  Figure . The sups Figure . This obbcomplex . Of notein vitro transcribed (IVT) tRNAs. Like yeast Elp123 , (ii) the Elp1 binding loop of Elp4 (Elp4Δ133–175) and (iii) the very N-terminus of Elp5 (Elp5Δ1–9). The pull-down assays showed that all mutations moderately affect Elongator reconstitution, with stronger effects for Elp1Δ1132–1224 and Elp4Δ133–175 and less prominent reduction of binding by the Elp5 truncation  might be sufficient to maintain tRNA binding. Finally, tRNA binding might not be affected by the tested mutants as the second Elp456-free lobe of Elp123 could mediate tRNA binding in the presence of Elp456. Nonetheless, the biochemical analyses of human Elongator support our structural findings, and several aspects need to be addressed by additional analyses in the future.We further investigated the importance of these structural rearrangements by complementary biochemical analyses. We used human Elongator subcomplexes produced in insect cells and reconstituted them as we did with the mouse Elongator sample . First, t Elp123 , mouse ay Figure . These dy Figure . Second,y Figure . Initialy Figure . To exclanalyses . Third, n Figure . These dx Figure . This isUGC using MST assays with varying ATP concentrations  in yeast Elp1, which is essential for the modification activity in vivo and seems to support the integrity of the Elp123 subcomplex. As this loop is not visible in any of the previous cryo-EM structures and produces several violating crosslinks, it might become structured only under certain conditions indicating a dynamic component of complex assembly also for the yeast complex.In this work, we developed novel protocols that allow overexpression and purification of the whole Elongator complex from yeast cells for the first time. Furthermore, we managed to overproduce, purify and reconstitute mouse and human Elongator complexes using advanced insect cell expression systems. We have determined several high-resolution cryo-EM structures for yeast and mouse Elongator complexes in the presence and absence of tRNAs. Foremost, the comparison of our cryo-EM structures demonstrates high similarities between yeast and mouse Elongator and their intermediates. Our results reveal how both subcomplexes interact with each other and that in both cases, the Elp456 ring binds to only one Elp123 lobe, leaving the second one unoccupied. As the reconstitutions of the mammalian Elongator complexes were carried out in excess of the smaller subcomplex the possibility existed that both Elp123 lobes would bind one Elp456 ring, resulting in a 1:2 stoichiometry of the subcomplexes. Yet, in our 34 is ideally positioned to be modified, resulting in cm5U34. Moreover, our structures represent snapshots at certain stages of the catalytic reaction. First, the MmElp123 harbors a SAM molecule that is bound to the iron-sulfur cluster, indicating that SAM binding occurs before tRNA binding. Second, the ScElp123 in complex with tRNA harbors the SAM cleavage products, namely 5′-dA and methionine, in its catalytic site. Third, we could not identify any SAM molecule in the full Elongator, suggesting SAM is consumed. Hence, we depict the catalytic reaction as follows: (i) the Elp123 structure with intact SAM represents the initial state prior to tRNA binding and SAM cleavage; (ii) subsequently Elp123 mediates SAM cleavage upon tRNA binding (i.e. the Elp123–tRNA structure) which represents the stage of a radical generation just before its transfer from 5′-dA; (iii) the SAM free-Elongator depicts the post state of the catalytic reaction.Apart from the overall structural similarities between fungal and murine Elongator, the spatial organization of their active sites is highly conserved as well. As the molecular mechanism of the tRNA modification reaction is still not fully characterized, we analyzed the conserved features of the various active sites in all our structures to understand the underlying principles. Selvadurai and colleagues proposed a catalytic reaction mechanism based on the biochemical characterizations of archaeal Elp3. The Elp3 utilizes its rSAM domain to accommodate and cleave SAM to produce a 5′-dA radical. The 5′-dA radical is then used to generate an acetyl radical where the acetyl group comes from the hydrolyzed ACO product or from a covalent acetyl-Elp3 intermediate . An unreThis is the first study to present the interaction of the Elp123 and Elp456 subcomplexes at high-resolution with sufficient structural details to provide pseudo-atomic models. Based on these structure analyses, we further biochemically characterized the role of Elp456 during the Elongator reaction cycle. It is interesting to see that the Elp456 ring occupies a similar region as the tRNA suggesting that Elp456 could spatially compete with a bound tRNA substrate. However, by super-positioning the structures of Elp123–tRNA and Elongator, we had to realize that tRNA still can spatially fit between Elp123 and Elp456. Despite the extensive efforts, we still could not manage to obtain any Elongator–tRNA structure. We further present that the CTD of Elp1 is essential for the catalytic activity and it has dual interacting targets, tRNA and Elp456. It seems that the interaction is discriminated to one or the other. However, the tRNA interaction with CTD is not crucial for tRNA recruitment.in vivo in yeast Elp1 result in the inactivation of the Elongator pathway, but its removal in human Elp123 is not sufficient to diminish tRNA binding to Elp123, clearly proving that tRNA binding mainly occurs through the ASL bound in the active site of Elp3. The mechanistic rational behind tRNA selectivity and detailed intramolecular relay of tRNA remain elusive, but as Elp123 does not require Elp456 to bind tRNAs, we believe that Elp456 is important for the late stages of the modification reaction. Strikingly, we found that at physiologically relevant ATP concentration the affinity of mammalian Elp456 towards tRNAs exceeds the one measured for Elp123, respectively. As neither ATP nor tRNA influenced the interaction of Elp123 and Elp456, we speculate that ATP-binding might trigger the transition of an already modified tRNA to the Elp456 complex, which then releases the tRNA after ATP hydrolysis . All three deletions 5 at U34 by the activity of the Elp123, but some tRNAs additionally require the assistance of Elp456 to be unloaded from the complex after the modification reaction. To release certain modified tRNAs from Elp123, the smaller subcomplex would bind again and displace the loop in the Elp1 CTD allowing the tRNA to be released. We speculate that the affinity of certain tRNAs is simply too high for a release from Elp123 and that the displacement of the loop, facilitates the ATP-dependent transfer of tRNAs to the Elp456 ring. How the transfer from Elp123 to Elp456 exactly happens will require additional studies. In summary, our data provide novel insights into the structural and functional conservation of Elongator in eukaryotes and our work paves the way for further analyses of the intrinsic dynamics of Elongator and the specific differences between its tRNA substrate molecules.Based on previous findings, we conclude that Elongator can bind all types of tRNAs, but only the recognition of specific bases in certain tRNAs triggers ACO hydrolysis in the KAT domain and SAM cleavage in the rSAM domain. In mammals, the binding of tRNAs induces large structural rearrangements in the arch domain, which might confer additional selectivity and create a second contact at the elbow region of the bound tRNA molecule. All modifiable tRNAs would receive cmhttps://repository.jpostdb.org/entry/JPST001914 (ID JPST001914). All other data generated in this study are available from corresponding authors on reasonable request.The models and cryo-EM densities map have been deposited in the EMData Bank (EMDB) and the Protein Data Bank (PDB) – yeast Elongator (PDB ID 8ASV and EMD-15622), mouse Elongator (EMD-15626), yeast Elp456 (PDB ID 8AT6 and EMD-15635), mouse Elp123 (PDB ID 8AVG and EMD-15682), yeast Elp123–tRNA (PDB ID 8ASW and EMD-15623), mouse Elp123–tRNA (EMD-15625). All XL-MS data are available at gkac1232_Supplemental_FileClick here for additional data file.


With the advancements in bioinformatic technology, an increasing number of circular RNAs (circRNAs) have been discovered and their crucial roles in the development and progression of various malignancies have been confirmed through multiple pathways. However, the specific mechanisms involving protein-binding circRNAs in colorectal cancer (CRC) remain largely unexplored.Differential circRNA expression was assessed using a human circRNA microarray in five CRC tissue and paired normal samples. CircGPRC5A expression was then confirmed in the CRC tissues and paired normal samples using qRT-PCR. The biological function of circGPRC5A in CRC were studied in vitro and in vivo. Western blotting, fluorescence in situ hybridization, immunofluorescence, RNA pulldown, mass spectrometry, immunoprecipitation, quantitative phosphoproteomics, and RNA-binding protein immunoprecipitation assays were used to study circGPRC5A.Our analysis revealed that circGPRC5A expression was higher in CRC tissues compared to normal tissues and was associated with tumor size, tumor stage and lymph node status. CircGPRC5A promoted CRC cell proliferation, migration, and metastasis in vitro and in vivo. CircGPRC5A could stabilize PPP1CA protein by inhibiting the binding between UBA1 and PPP1CA, and increasing YAP dephosphorylation.Our study revealed that circGPRC5A plays an essential function in CRC progression by stabilizing PPP1CA protein and enhancing YAP dephosphorylation. CircGPRC5A could act as a novel and potential target for CRC.The online version contains supplementary material available at 10.1186/s13046-023-02915-7. Colorectal cancer (CRC) ranks as the third most common malignancy of the digestive system and is the second major cause of cancer mortality , 2. DespCircular RNAs (circRNAs) lack a 5′-cap or 3′-polyadenylation tail, making them more stable and resistant to exonucleases compared with linear RNAs , 7. BioiProtein Phosphatase 1 Catalytic Subunit Alpha (PPP1CA) encodes PP1A, which is one of the three catalytic subunits of protein phosphatase 1 (PP1) that is specific for serine/threonine , 15. ProThe Hippo signaling pathway protein Yes-associated protein (YAP) is crucial to many biological processes. –21. Ser1GPRC5A gene (has_circRNA_101017 (circBase ID: has_circ_0025506)), circGPRC5A, using a human circRNA array to profile circRNAs expression in CRC. CRC tissue was found to have higher levels of circGPRC5A than adjacent normal tissues. CircGPRC5A promoted CRC progression by competitively inhibiting the binding of UBA1 and PPP1CA, thereby preventing PPP1CA ubiquitination. This caused a low phosphorylation state of YAP, and ultimately could lead to CRC development. Our results suggest that circGPRC5A can contribute to improving the understanding of the roles of circRNA in CRC and can act as a novel and potential therapeutic target for CRC patients.In our study, we focused on a circRNA from the Between October 2021 and March 2022, 80 pairs of human CRC samples and paired adjacent non-tumor tissues were collected from the First Affiliated Hospital of Chongqing Medical University. All samples came from CRC patients who underwent surgical resections, and they were immediately frozen in liquid nitrogen before being moved to − 80 °C for long-term storage. The clinicopathological data of all CRC patients were gathered and examined from electronic medical records. All patients gave their informed consent, which was evaluated and approved by the First Affiliated Hospital of Chongqing Medical University’s ethics committee (NO.2020–358).2.The human CRC cell lines HT29, HCT116, HCT8 and SW480 were purchased from Cell Bank of the Chinese Academy of Sciences . The normal colonic epithelial cell line NCM460 was obtained from the American Type Culture Collection . HT29, HCT116, HCT8, SW480 and NCM460 were cultured in Dulbecco’s Modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum  and 1% penicillin/streptomycin . Cells were grown in an incubator at 37 °C and 5% COSiRNA and plasmids were transfected into CRC cell lines using Lipofectamine 3000 reagent in accordance with the manufacturer’s instructions . Empty vector (EV) and negative control siRNA were used as controls. The siRNA sequences targeting circGPRC5A and UBA1 are shown in Table SFive CRC tissues and paired para-cancerous tissues were compared using a human circRNA microarray. Thermo Fisher Scientific’s NanoDrop 1000 was used to measure the purity as well as the concentration of the total RNA that was extracted using TRIzol . Rnase R  was utilized to process total RNA from each sample to enrich for circular RNA, which was then amplified and turned into fluorescent circRNA using random primers in accordance with the Arraystar Super RNA Labeling methodology. Using an Agilent Hybridization Oven , the tagged circRNAs were hybridized onto the Arraystar Human CircRNA Arrays V2  and cultured for 17 h at 65 °C. After washing, slides were examined using the Agilent Scanner G2505C . Finally, data were collected and analyzed using Agilent Feature Extraction software and the R software limma package.GAPDH. Primer sequences can be found in Table STotal RNA was isolated from CRC tissues and cells using TRIzol reagent . The Cytoplasmic and Nuclear RNA Purification Kit  was used to extracted nuclear and cytoplasmic RNA from CRC cells according to the manufacturer’s guidelines. The concentration and purity of total RNA was determined using the NanoDrop 2000 . cDNA was generate using the RT Reagent Kit  and the TB Green Kit  was used for qRT-PCR. The 2^-ΔΔCt method was used to calculate the relative expression level of circRNA and mRNA, and was normalized to 2, followed by measurement of the absorbance at 450 nm. For colony formation assays, 200 or 1000 transfected CRC cells were seeded into 24-well plates or 6-well plates and cultured for 7 days at 37 °C in 5% CO2. When colonies formed, the plates were preserved with 4% paraformaldehyde and stained with 0.1% crystal violet.Transfected CRC cells were inoculated into a 96-well plate at a density of 1000 cells per well. A total of 10 uL of the Cell Counting Kit-8 (CCK-8) solution  was used to measure cell proliferation at 0, 24, 48, 72, and 96 h after seeding. The plate was cultured for 2 h at 37 °C in 5% CO2. Next, 50 µl/L EdU solution was added to each well, and the plates were cultured at 37 °C in 5% CO2 for 2 h. After three washes in PBS, the cells were then fixed for 30 min at room temperature with 4% paraformaldehyde.The cell proliferation was measured using a 5-ethynyl-2′-deoxyuridine (EdU) Cell Proliferation Kit . Briefly, transfected CRC cells were seeded into 96-well plates and incubated for 24 h at 37 °C in 5% COTriton X-100 (1%) was used to permeabilize the transfected CRC cells. The cells were then stained with Azide 594 and Hoechst 33,342. Confocal laser scanning microscopy was used to image the cells.4) were resuspended in 200 µL serum-free DMEM and plated in the upper chamber , while the lower chamber included DMEM with 15% fetal bovine serum. After 48 h, 4% paraformaldehyde was used to fix the invaded cells and 1% crystal violet was used to stain these cells. Matrigel  was used to coat the upper chamber in the Transwell invasion assay, and cells were treated as previously described.For the Transwell migration assay, transfected CRC cells  was used to purify circRNA. Total RNA (4 µg) was mixed with 10 × reaction buffer (2 µL) and RNase R (3–4 U/µg RNA). Then the reaction was brought to 20 µL using RNase-free Water. The sample was incubated for 10 min at 37 °C. Finally, qRT-PCR was performed to assess the relative expression levels of circRNA and mRNA.For the actinomycin D assay, 2 mg/mL Actinomycin  was used to block gene transcription and to assess the half-life of circRNA. Cells were harvested at 0, 4, 8, and 24 h and the relative expression levels of circRNA and mRNA were assessed using qRT-PCR.RIPA Lysis Buffer , which includes 1% PMSF and 1% phosphatase inhibitor, was used to lyse cells. The BCA Protein Assay Kit  was used to measure total protein concentrations. Identical amounts of protein were separated on an 8% or 10% SDS-PAGE gel and then transferred onto 0.22 um polyvinylidene fluoride membranes . The PVDF membranes were blocked with 5% skim milk or bovine serum albumin for 1.5 h and then incubated with specific antibodies for 12–16 h at 4 °C. The primary antibodies used were PPP1CA , Ub (#3936S CST), UBA1 , FBXO2 , YAP-Ser127 , YAP-Ser109 , YAP-Ser61, YAP , Pan Phospho-Serine/Threonine , and GAPDH . The next day, the PVDF membranes were treated with HRP-conjugated goat anti-rabbit or anti-mouse IgG for 2 h. Membranes were washed with TBST 3X, and analysis was performed using the ECL detection system.A Cy3-labeled circGPRC5A probe  was used for fluorescence in situ hybridization (FISH) . Briefly, circGPRC5A-MS2 and MS2-CP-Flag vectors were transfected into HCT116 cells. Then, MS2-CP-Flag antibodies were combined with the capture protein MS2-CP-Flag to further enrich for circRNA pulldown products. Finally, circGPRC5A pulldown products and the negative control were analyzed using mass spectrometry .The RNA immunoprecipitation (RIP) assay was conducted using a Magna RIP RNA-Binding Protein Immunoprecipitation Kit  to explore the relationship between circGPRC5A and PPP1CA. In brief, CRC cells were lysed and collected using RIP lysis buffer. Target antibody and lysate were mixed and agitated for 6 h at 4 °C. After washing, TRIzol reagent was used to extract the immunoprecipitated (IPed) and enriched circRNA. The enriched circRNA was then subjected to qRT-PCR analysis.Co-immunoprecipitation (co-IP) was conducted using a Co-IP Kit . Briefly, CRC cell lysates were incubated with target antibody for 12 h at 4 °C. The mixture, including antigen and target antibody, were combined with protein A/G magnetic beads for 2 h at room temperature. Next, the A/G magnetic bead and lysate mixture was washed with IP lysis/rinsing buffer. Finally, antigen–antibody complexes were eluted for 10 min at 95 °C. The co-IP products were analyzed using mass spectrometry  and western blotting.Phosphoproteomics was used to analyze the changes of protein phosphorylation levels in the transfected CRC cells. In brief, protein was isolated from the transfected CRC cells and the protein concentration was evaluated using the BCA or Bradford assays. Then, SDS-PAGE electrophoresis analysis was performed with the protein samples to evaluate whether the sample quality met the requirements for subsequent experiments. High-quality sample was reduced and alkylated, and equal amount of each sample were taken for trypsin hydrolysis and liquid chromatography coupled with tandem mass spectrometry analysis. Finally, data statistics and bioinformatics analyses were conducting according to search results obtained from the MaxQuant Software.Tissue samples were fixed with 4% formaldehyde. After embedding in paraffin, tissue blocks were cut into 4um sections. After dewaxing and hydrating, a 3% hydrogen peroxide solution was added to the tissue sections to inhibit endogenous peroxidase activity. Anti-PPP1CA and anti-Ki 67 antibody solutions were added to the sections and incubated on the tissues for 12–16 h at 4 °C with anti-PPP1CA and anti-Ki 67 antibodies. Brown precipitates were produced after 3,3'-diaminobenzidine incubation. Finally, the sections were counter-stained using hematoxylin. Immunohistochemistry (IHC) images were obtained using an Olympus microscope.7 cells were injected into the right subcutaneous axilla (n = 5 per group). The long and short axes of the subcutaneous tumors were measured on days 5, 10, 15, 20, and 25 after injection, and the formula V = (width2 + long)/2 was used to calculate the subcutaneous tumor volume. Mice were euthanized 25 days after tumor injection. The subcutaneous tumors were isolated from the nude mice, the tumors were weighed, and the mass and volumes were measured. Tumors were fixed in a 4% formalin solution for hematoxylin and eosin staining and IHC.The Animal Care Committee of Chongqing Medical University reviewed and approved the animal experimental protocol (NO. IACUC-CQMU-2022–0019). For xenografts in mice, 4–6-week-old female BALB/c nude mice were purchased from Chongqing Ensiweier Biotechnology Ltd. Next, 1 × 106 cells were injected into the spleens of nude mice. Mice were sacrificed after 25 days. Liver tissue sections were stained with hematoxylin and eosin, and tumor colonies were examined under a microscope.We constructed a liver metastasis model to explore the metastatic ability of cancer cells. A total of 3 × 10P < 0.05 was considered statistically significant. The statistical analysis was performed using R software (V4.0.5) and GraphPad Prism 8.All experiments were independently repeated three times and the data are presented as mean ± standard deviation (SD). The Chi-square test or Fisher’s precision probability test was used to analyze the relationship between circGPRC5A and CRC patient clinical characteristics. A Wilcoxon signed-rank analysis was conducted to test non-normal distribution paired samples. A Mann–Whitney test was applied to analyze the data with non-matched and non-normally distributed samples. The comparison of two samples was assessed using a Student’s t-test, and the comparison of three or more samples was performed using a one-way ANOVA analysis. Correlation analyses were conducted using a Spearman’s rank correlation coefficient test. GPRC5A and is located on chr12:13,061,176–13062105. To verify the closed-loop structure of circGPRC5A, we confirmed the presence of a splice junction in the PCR product of circGPRC5A by using Sanger sequencing  was defined. When deltaP_value ≥ 1, it indicates that the phosphorylated peptide in the protein is upregulated, and when deltaP_value ≤  − 1, it demonstrates that the phosphorylated peptide in the protein is downregulated. According to deltaP_value, we found upregulated phosphorylation levels of 383 proteins and downregulated phosphorylation levels of 201 proteins  in HT29 and HCT116 cells  has been reported to be expressed in tumors. One study revealed that circGPRC5A-peptide-GPRC5A can be utilized to target bladder cancer and bladder cancer stem cells . AnotherCircRNA-protein interactions play an essential role in multiple signaling pathways in cancer, such as protein expression, degradation, and biogenesis. Zheng et al. have reported that circLPAR1 exosomal direct interaction with eIF3h inhibited the METTL3-eIF3h interaction and reduced the translation of the oncogene BRD4 . On the Numerous physiological processes, including the cell cycle, differentiation, signal transduction, are tightly regulated by ubiquitination. Proteasomal degradation after ubiquitination mainly involves four processes. First, ubiquitin activating enzyme E1 activates ubiquitin molecules. Second, ubiquitin activating enzyme E1 transfers the activated ubiquitin molecules to ubiquitin binding enzyme E2. Third, ubiquitin ligase E3 transfers ubiquitin molecules binding to ubiquitin binding enzyme E2 to the target protein. Finally, ubiquitinated proteins are eventually broken-down by the proteasome , 44. In Phosphorylation is a kind of post-translational modification and phosphorylated proteins account for about one-third of all proteins . TherefoIn conclusion, the research shows that circGPRC5A functions as a tumor-promoting gene to encourage CRC cell growth and metastasis both in vivo and in vitro. CircGPRC5A is highly correlated with clinicopathological characteristics of CRC patients, such as tumor size, lymph node status, and TNM stage. Mechanistically, circGPRC5A acts as a binding molecule to enhance the levels of PPP1CA protein by preventing the interaction between PPP1CA and UBA1. As a result, it leads to the dephosphorylation of YAP at Ser127 and Ser109. The dephosphorylation of YAP at Ser127 and Ser109 results in YAP entering the cell nucleus and binding to TEADs, leading to CRC progression. Thus, our study revealed that circGPRC5A plays an essential role in CRC progression and can act as a new and potential target for patients with CRC.Additional file 1: Figure S1. CircGPRC5A expression in paired normal tissues and CRC tissues by using a FISH probe. A, B FISH was used to investigate the expressions of circGPRC5A in paired normal tissues and CRC tissues. C Statistical analysis using a bar chart to evaluate relative expression of circGPRC5A.v. Figure S2. The relationship between PPP1CA and circGPRC5A. A, B PPP1CA protein level detection in 40 pairs of cancer and adjacent samples using western blotting. C A positive correlation between PPP1CA and circGPRC5A using correlation analysis. Figure S3. UBA1 is critical for circGPRC5A-mediated promotion of CRC. A–D CCK-8, colony formation, Transwell, and wound healing assays were conducted to investigate the effects of PPP1CA on circGPRC5A-mediated proliferations and migrations of HT29 and HCT116 cells. E –J The experimental results involving the effects of UBA1 on circGPRC5A-mediated proliferation and migration of HT29 and HCT116 cells were quantified and presented using a bar chart. Values are shown as the mean ± SD based on three independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001. Figure S4. CircGPRC5A can cause dephosphorylation of YAP and further lead to changes in the distribution of YAP by stabilizing the PPP1CA protein. A, B Western blotting was used to examine nuclear YAP and total YAP after silencing and overexpressing PPP1CA or circGPRC5A. C–D The effects of YAP distribution via PPP1CA on circGPRC5A-induced changes were detected by western blotting. Values are shown as the mean ± SD based on three independent experiments. *P < 0.05;**P < 0.01; ***P < 0.001. Figure S5. YAP is critical for circGPRC5A/PPP1CA-mediated promotion of CRC. A, B CCK-8 assays were conducted to determine the effects of YAP on circGPRC5A/PPP1CA-mediated cell proliferation. C–J The effects of YAP on circGPRC5A/PPP1CA-induced cell proliferation and migration were measured with a colony formation assay, and migration and invasion were investigated using Transwell and wound healing assays. Values are shown as the mean ± SD based on three independent experiments. *P < 0.05;**P < 0.01; ***P < 0.001. Figure S6. Statistic analysis chart of western blotting for various proteins. A. PPP1CA protein levels were assessed after silencing or overexpressing circGPRC5A. B, C After silencing circGPRC5A in HT29 and overexpressing circGPRC5A in HCT116, PPP1CA was evaluated with or without MG132. D, E Ploy(Ub) for PPP1CA was assessed after silencing or overexpressing circGPRC5A in HT29 or HCT116. F, G UBA1 and FBXO2 protein levels were assessed after silencing or overexpressing circGPRC5A using the IP assay. H-K: PPP1CA and UBA1 were evaluated when silencing circGPRC5A and UBA1 in HT29 or overexpressing circGPRC5A and UBA1 in HCT116 cells. L-M: Ploy(Ub) for PPP1CA were evaluated by IP assays for PPP1CA when silencing circGPRC5A and UBA1 in HT29 or overexpressing circGPRC5A and UBA1 in HCT116 cells. Values from three independent experiments are presented. *P < 0.05; **P < 0.01;***P < 0.001. Figure S7. Statistic analysis chart of western blotting for various proteins. A-E: The protein levels of YAPser127, YAPser109, YAPser61, PPP1CA, and YAPpan ser/thr were assessed after overexpressing and silencing PPP1CA. F-G: The protein levels of YAPser127, YAPser109, YAPser61, PPP1CA, and YAPpan ser/thr were assessed after overexpressing and silencing circGPRC5A. K–O The protein levels of YAPser127, YAPser109, YAPser61, PPP1CA, and YAPpan ser/thr were assessed after silencing circGPRC5A and overexpressing PPP1CA in HT29 cells. P –T The protein levels of YAPser127, YAPser109, YAPser61, PPP1CA, and YAPpan ser/thr were assessed after overexpressing circGPRC5A and silencing PPP1CA in HCT116 cells. Values from three independent experiments are presented. *P < 0.05;**P < 0.01; ***P < 0.001. Figure S8. Statistic analysis chart of Ki-67 and PPP1CA staining and IHC analysis. A Quantitative analysis of liver metastases in mice. B Quantitative analysis of IHC for PPP1CA of subcutaneous xenograft tumors. C Quantitative analysis of IHE for Ki-67 of subcutaneous xenograft tumors.Additional file 2. Additional file 3. Additional file 4: Table S1. shRNA, siRNA target sequences. Table S2. The primer sequences used for real-time quantitative PCR. Table S3. The sequence of probes was used in this study. Table S4. Association between the status of circGPRC5A expression and clinicopathological characteristics of human colorectal cancer.


While the role of endocytosis in focal adhesion turnover-coupled cell migration has been established in addition to its conventional role in cellular functions, the molecular regulators and precise molecular mechanisms that underlie this process remain largely unknown. In this study, we report that proto-oncoprotein hematopoietic PBX–interacting protein (HPIP) localizes to focal adhesions as well as endosomal compartments along with RUN FYVE domain–containing protein 3 (RUFY3) and Rab5, an early endosomal protein. HPIP contains two coiled-coil domains (CC1 and CC2) that are necessary for its association with Rab5 and RUFY3 as CC domain double mutant, that is, mtHPIPΔCC1-2 failed to support it. Furthermore, we show that HPIP and RUFY3 activate Rab5 by serving as noncanonical guanine nucleotide exchange factors of Rab5. In support of this, either deletion of coiled-coil domains or silencing of HPIP or RUFY3 impairs Rab5 activation and Rab5-dependent cell migration. Mechanistic studies further revealed that loss of HPIP or RUFY3 expression severely impairs Rab5-mediated focal adhesion disassembly, FAK activation, fibronectin-associated-β1 integrin trafficking, and thus cell migration. Together, this study underscores the importance of HPIP and RUFY3 as noncanonical guanine nucleotide exchange factors of Rab5 and in integrin trafficking and focal adhesion turnover, which implicates in cell migration. In tns (FAs) , 8, 9. Wvia cytoskeletal remodeling that results in Rac1 activation  is an oncoprotein with an adaptor function that can integrate various signaling proteins in the regulation of cell migration , 23, 24.In this report, we have characterized HPIP and RUN FYVE domain–containing protein 3 (RUFY3) as noncanonical guanine nucleotide exchange factors (GEFs) of Rab5 that regulate endocytosis-coupled FA turnover and cell migration. Through coiled-coil (CC) domains HPIP connects with Rab5 and RUFY3. This trimolecular molecular association triggers FAK activation, FA disassembly, and fibronectin (FN) associated-β1 integrin trafficking, and thus cell migration.A). In addition to these known domains, in silico analysis (COILS server of Expasy) revealed two highly conserved CC domains in HPIP, which are CC1, 270 to 341 aa and CC2, 370 to 415 aa, and they are well conserved across the species (B and S1A). Further protein modeling studies showed that CC1 configures three unequal helices connected by spacers, whereas CC2 forms a single continuous helix . CC proteins are known to be involved in many important biological functions, such as protein–protein interaction, dimerization/oligomerization, and gene expression . Later, the GST tag was removed by digesting the GST-fusion proteins with thrombin treatment . The purified CC1 and CC2 domains were subjected to CD for secondary structural analysis. It revealed that CC1 and CC2 domains comprise 76.9% and 75.9% of α-helix, 13.7% and 14.9% of β-sheet , and 8.10% and 8.2 % of the random coil, respectively, which is in close agreement with previous reports (F and S1B).Our earlier studies defined the role of HPIP in cell migration . In the Fig. 1 species B and S1Apression , 30. To  reports  Figs. 1A. In addG and H and S1C). Earlier reports showed RUFY3 interacting with Rab5 as an early endosomal marker , CC domain–containing 105, and RUFY3 (A). Subsequently, the coimmunoprecipitation (Co-IP) assay demonstrated their association in MDA-MB231 cells (B). Sequence-based coevolutionary signals are used to predict interfacial residues between two proteins . Next, we validated the requirement of CC domains in the interaction of HPIP with RUFY3 and Rab5. To check this, first, we generated HPIP mutants devoid of CC1, CC2 alone, or both domains such as mtHPIPΔCC1, mtHPIPΔCC2, and mtHPIPΔCC1-2, respectively, and subsequently, in vitro pull-down assay was performed using [S35]-labeled HPIP mutants and His-RUFY3 or His-Rab5 (D). The data revealed that RUFY3 could interact with WT HPIP only but not with mtHPIPΔCC1, mtHPIPΔCC2, or double mutant, that is, mtHPIPΔCC1-2, indicating that RUFY3 interacts with HPIP via CC1 and CC2 domains (E). Rab5 could interact with WT HPIP and mtHPIPΔCC2 but neither with mtHPIPΔCC1 nor mtHPIPΔCC1-2, indicating that Rab5 can interact with HPIP via the CC1 domain only (F). To support this data, in vitro GST pull-down assays were performed using GST-tagged CC1 or CC2 by incubating with purified His-tagged Rab5 or RUFY3. The data confirmed the involvement of CC1 and CC2 in interacting with RUFY3, albeit stronger affinity for CC2 than CC1, whereas CC1 alone interacts with Rab5 (G and H). These results indicate that HPIP has two conserved CC domains, which present a unique architecture to the HPIP scaffolding function to directly interact with RUFY3 and Rab5, which may have a role in cellular signaling and function.In order to characterize the functional association among HPIP, RUFY3, and Rab5, we first examined their expression in a panel of breast cancer cells. We found coexpression of HPIP, RUFY3, and Rab5 in most of the cell lines examined, while their relative abundance was more in MDA-MB231 cells and less in MCF10A cells A. Subseq31 cells B. Sequenproteins . Using Cproteins . We pick package . We foun package C. Next, His-Rab5 D. The da domains E. Rab5 cain only F. To supith Rab5 , G and Hin vitro MAN-GTP assay as described earlier (A) and then evaluated the GTP-binding potential of Rab5 by HPIP and RUFY3 as GEFs. Rab5 could bind to GTP with a dissociation constant (Kd) of 1.59 ± 0.013 (A). The GTP binding of Rab5 was significantly elevated by incubating either recombinant His-HPIP (Kd = 0.21 ± 0.004) or His-RUFY3 (Kd = 0.098 ± 0.002) as compared to the control. Further elevation of Rab5 activation was also observed with combined incubation of HPIP and RUFY3, suggesting a synergistic effect on Rab5 activation (Kd = 0.07 ± 0.003) (A). Next, we evaluated Rab5 GEF functions of HPIP and RUFY3 in MDA-MB231 cells by analyzing GTP-bound Rab5 by probing cell extracts with Rab5-GTP antibody. Knockdown of HPIP or RUFY3 in MDA-MB231 cells significantly decreased GTP-bound Rab5 over control cells (B). Since CC domains of HPIP are crucial for its scaffolding activity, we next evaluated their role in Rab5 activation. Although wtHPIP could enhance the Rab5 activation over control cells, CC mutants of HPIP failed to do so (C). Next, we performed a rescue experiment by altering the expression of HPIP by the gain and loss-of-function approach. HPIP depletion significantly decreased GTP-bound Rab5 (active Rab5) over control cells . Conversely, ectopic expression of T7-HPIP in HPIP-silenced cells restored Rab5 activation . Together, these data suggested that HPIP and RUFY3 act as GEFs of Rab5.Activation of early endosomal protein Rab5 is crucial for endosome internalization and maturation, as ectopic expression of Rab5 (Q79L), a GTPase-defective mutant form of Rab5, results in the formation of enlarged early endosomes due to defective budding of the endosomal vesicles . Moreove earlier . We clon earlier A and the ± 0.013 A. The GT± 0.003) A. Next, ol cells B. Since to do so C. Next, ol cells D, lane 2tivation D, lane 3A. A punctate cytoplasmic distribution pattern for HPIP was observed with a significant colocalization with Rab5 (Pearson coefficient = 0.74 ± 0.01). Despite RUFY3 distributes throughout the cytoplasm, a significant colocalization with HPIP was observed in punctuate regions, which represents endosomes (Pearson coefficient = 0.69 ± 0.01) (A and B). Further supporting this observation, biochemical fractionation analysis revealed marked enrichment of HPIP, RUFY3, and Rab5 in an endosomal fraction in MDA-MB231 cells (C).After establishing the role of HPIP and RUFY3 as GEFs of Rab5, we next studied their subcellular localization to unravel the signaling mechanism and its associated function. Rab5 is a small GTPase that predominantly localizes to endosomes, as shown in Figure 4 ± 0.01) , A and B31 cells C.Figure D and E, a significant fraction of HPIP, RUFY3, and Rab5 are localized to FAs . Further, biochemical fractionation analysis showed a marked enrichment of HPIP, Rab5, and RUFY3 in FN-coated cells (F). Though earlier studies reported the FA localization of HPIP (G and H). RUFY3 has three domains: CC, RUN, and FYVE. We next explored which domain of RUFY3 is required for its interaction with HPIP and, thus, possible role in FA localization. RUFY3 domains were cloned, expressed as His-tagged proteins, and used for pull-down assay. In vitro, interaction assay demonstrated the interaction of HPIP with the CC domain of RUFY3 but not with FYVE and RUN domains (I). Together these results indicate that HPIP, RUFY3, and Rab5 localize to FAs as well as endosomes, and RUFY3 recruits HPIP possibly via the CC domain into FAs.It is well established that FN- induced integrin signaling regulates FA disassembly and cell migration . Given ted cells F. Though of HPIP , the fac of HPIP . Given t of HPIP , G and H domains I. Togethvia Rab5 activation. Knockdown of HPIP, Rab5, and RUFY3 alone or combined knockdown  of these components significantly decreased cell migration of MDA-MB231 cells (A and S2A). We next performed rescue studies to check if Rab5-mediated cell migration depends on HPIP or RUFY3 expression. As shown in Figure 5B and Fig. S2B, cell migration was significantly hampered upon HPIP knockdown in MDA-MB231 cells, further over expression of Rab5, RUFY3, or both, however, failed to rescue it. In a similar experiment, over expression of HPIP, Rab5, or both failed to rescue MDA-MB231 cell migration in which RUFY3 was depleted (C and S2C). As we established that the interaction of HPIP with Rab5 and RUFY3 was dependent on CC domains in HPIP, we examined the cell migration ability of HPIP’s CC mutants. While wtHPIP could significantly enhance cell migration, as demonstrated by scratch wound closure assay, HPIP-CC mutants failed to do so (D and S2D). To further strengthen these findings, we performed cell invasion assays using 3D cultures of MDA-MB-231 cells grown in Matrigel. Silencing of HPIP, RUFY3, and Rab5 alone or combined knockdown (HERURA) significantly hampered the cell invasions measured by cellular protrusions (E). Together, these data support that HPIP–RUFY3–Rab5 axis modulate cell migration, which is partly dependent on the CC domains of HPIP.After establishing the cellular localization of HPIP, Rab5, and RUFY3, we next focused on the functional significance of their interaction in cancer cells. Earlier independent studies showed the involvement of these proteins in cell migration , 27, 39.31 cells A and S2Adepleted C and S2Cto do so D and S2Dtrusions E. TogethA–C and via FAK activation, it is unclear whether Rab5-mediated activation of FAK is HPIP- or RUFY3-dependent. We examined FAK activation by altering the expression of HPIP, RUFY3, or Rab5. The data revealed that FAK activation by Rab5 is HPIP-dependent as HPIP depletion significantly abrogated it (D). Similar results were also observed for RUFY3, as it failed to activate FAK upon HPIP knockdown (E). Since HPIP bridges RUFY3 and Rab5, CC deletions in HPIP could also abolish FAK activation (F). Together, these results imply that Rab5-mediated FA disassembly via FAK activation is HPIP- and RUFY3-dependent.We next focused on the cellular mechanism that underlies cell migration mediated by Rab5 in concert with HPIP and RUFY3. Earlier studies have demonstrated the role of HPIP and Rab5, but not RUFY3, in FA turnover , 27. Hengated it D. Similanockdown E. Since tivation F. TogethA and B). Depletion of individual components or together  did not significantly alter the cell surface levels of total β1 integrin measured by ELISA-based assay (A). Conversely, the internalization rates of β1 integrin were significantly decreased in cells transfected with siRNAs for three proteins (B and S4B). Under similar experimental conditions, the internalization of LDL receptor was not altered, indicating the effect was specific to β1 integrin (C). The formation and disassembly of FAs during cell migration are coupled to integrins' recycling or lysosomal degradation via Rab proteins . Together these data suggest that the HPIP and RUFY3 are required for Rab5-mediated FN-associated β1-integrin internalization and recycling mechanism.Earlier studies established that endocytosis regulates cell migration , 2; direed assay A. Converproteins B and S4Bintegrin C. The foysosomes , 42, 43.ysosomes D. TogethThis study characterized HPIP and RUFY3 as novel GEFs of Rab5. Multiple lines of evidence suggest that HPIP and RUFY3 play critical roles in Rab5-mediated FA disassembly, FN-associated β1-integrin internalization, and recycling, which implicates in cell migration.via FAK activation, the underlying cellular mechanism remained elusive   supplemented with 5% Horse serum (Gibco), 100 U/ml penicillin, and 100 μg/ml streptomycin.MDA-MB231, MCF7, SKBR3, BT549, MCF10A, and HEK293T cells were obtained from the National Center for Cell Sciences. Except for MCF10A, all the cell lines were maintained at 37 °C with 5% COGene stable expression or knockdown in MDA-MB231 cells was carried out as described previously . Cells wRUFY3 gene was amplified using primers listed in RUFY3 gene was subcloned into the pET-28a vector through PCR amplification using the primers mentioned in DpnI enzyme, followed by transformation into Escherichia coli DH5α cells, and all clones were confirmed by DNA sequencing.CC1 and CC2 domains of HPIP were generated by PCR amplification using pcDNA3.1A-HPIP (full length) as a template using specific primers . AmplifiE. coli BL21 (DE3) cells harboring pGEX4T1-HPIP, pGEX4T1-HPIP-CC1, pGEX4T1-HPIP-CC2, pET-RUFY3, or pET-Rab5 were cultured and recombinant proteins were induced with IPTG (1 mM). After 3 h of induction, cells were harvested and resuspended in bacteria lysis buffer . The cell lysates were sonicated and separated by centrifugation at 11,000 rpm for 10 min at 4 °C, and the soluble fraction was collected. The supernatant was incubated with nickel beads for His-tag proteins or Glutathione Sepharose beads for GST-tag proteins (Clonetech) for 90 min at 4 °C, followed by three washes with Nonidet P-40 (NP 40) lysis buffer . To check the protein purity, 20 to 30 μl of beads were loaded onto SDS-PAGE.In vitro, transcription and translation of WT HPIP and CC deletion mutants of HPIP were performed using a TNT kit (Promega Scientific), where 1 μg template plasmid DNA was translated in the presence of (35S) methionine in a reaction volume of 50 μl. The GST pull-down assays were performed by incubating equal amounts of His-Rab5 or His-RUFY3 beads with 10 μl of in vitro translated (35S)-labeled protein in binding buffer  for 90 min. Post incubation at 37 °C for 1 h, beads were washed five times with GST-binding buffer, followed by eluting the proteins using 2× SDS buffer and finally separating the proteins on SDS-PAGE. The interacted proteins were then visualized by autoradiography (Typhoon scanner).For the formation of heterodimer on the exchange between differently tagged proteins, His-Rab5 or His-RUFY3 and GST- HPIP-CC1, GST-HPIP-CC2, and GST-HPIP-CC1-2 were incubated together in a 1:2 M ratio at room temperature for 30 min in 500 μl volume of binding buffer . Samples were flash-frozen in liquid nitrogen to capture the resulting association state and then thawed on ice. After thoroughly washing the beads with binding buffer or NP40 buffer, the bound proteins were eluted by 2× SDS sample buffer and subjected to SDS-PAGE.Rab5 activation assay was performed according to the manufacturer protocols (Abcam or New East Biosciences). Forty-eight hours posttransfection of plasmid constructs or siRNA against the client proteins, cells were harvested, and 1 mg of protein extracts were immunoprecipitated using anti-Rab5(GTP) antibody for 1 h at 4 °C, followed by pulled down with AG beads and washed with 1× assay lysis buffer and were analyzed by Western blotting with respective antibodies.A wound closure assay was carried out to determine the cell migration as described previously . Cells gImmunoblotting and Co-IP analysis were performed as described previously , 27. Bri2), and the resulting cleaved Rab5 was collected. This protein was then loaded onto a glutathione Sepharose HR16/10 column, and unbound proteins were buffer exchanged into Hepes-buffered saline . The protein concentration was determined using a Lowry assay (Sigma-Aldrich). To conduct the MANT-GTP assay, various concentrations of Rab5  were mixed with either His-RUFY3 (1 μM), His-HPIP (1 μM), or both HPIP/RUFY3, along with MANT-GTP (750 μM) . The resulting mixture was incubated in a buffer  to a final volume of 200 μl for 2 h at 30 °C in a black-walled 96-well plate. Fluorescence measurements were taken by subtracting the fluorescence at time 0 from the fluorescence at each time point . Data were analyzed by nonlinear regression using GraphPad Prism for curve fitting and Kd (dissociation constant) calculations.The MANT-GTP assay was performed using a previously described protocol . Brieflyhttps://imagej.net/ij/), the outgrowth area was determined in each well by subtracting the area at 0 h from the area of other time points.Briefly, 20 μl of MDA-MB-231 cells  consisting of 1000 cells were grown as hanging drops culture in Petri dishes by inverting the lid for 48 h with proper humidification inside the incubator. In prechilled 96 well-plates, 200 μl of Matrigel was poured and above that hanging-drop spheroids were carefully embedded. The cellular dissemination was monitored by capturing images at 0 h, 12 h, 24 h, 42 h, and 78 h in the mentioned siRNA-treated cells using a bright field microscope . Using Fiji/ImageJ , as previously described . Primaryg centrifugation. The plasma membrane fraction was collected with 10 min 10000g centrifugation. The endosomal and cytoplasmic fractions were collected with 1 h 100,000g centrifugation. All fractionation experiments were performed at 4 °C. The pellets were dissolved in a sample buffer for Western blotting.Subcellular fractionation was performed as described previously . BrieflyFA was isolated, as described previously . MDA-MB22CO3, pH 9.6 at 4 °C) was blocked in PBS containing 0.05% Tween 20 (PBS-T) with 5% BSA for 1 h at room temperature, followed by the overnight incubation of the cell lysate  at 4 °C. After extensive washing with PBS-T to remove unbound materials, wells were incubated with streptavidin-conjugated horseradish peroxidase (Abcam) in PBS-T containing 1% BSA for 1 h at 4 °C. Wells were washed five to ten times with PBS-T and biotinylated integrins were detected by chromogenic reaction, incubation in dark with 100 μl of substrate solution prepared by dissolving ortho-phenylenediamine (0.4 mg/ml) in 0.05 M phosphate-citrate buffer, pH 5 with 30% hydrogen peroxide for 30 min in room temperature. The chromogenic reaction was stopped by adding 50 μl of 2N H2SO4 (stop solution) reading the absorbance at 450 nm (without stop solution) or 492 nm (with stop solution).Biotin-based integrin internalization assays were performed as described previously . MDA-MB-2, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, and supplemented with protease inhibitor cocktail). Cell suspension was collected in a microcentrifuge tube and subjected to mechanical disruption by passing it through a 25 Gauge (G) syringe for ten times, 30 μl suspension taken out as direct lysate. Disrupted cells were centrifuged at 3000 rpm for 10 min, supernatant fraction was carefully collected in a fresh tube, and the pellet containing nuclear fraction was stored for further analysis. To obtain, membrane fraction, the supernatant was centrifuged at 100,000g for 1 h at 4 °C. After centrifugation, pellet was resuspended in 400 μl of subcellular fractionation buffer, disrupted by passing through 25 G syringe ten times and recentrifuged at 1,00,000g for 45 min at 4 °C. After discarding the supernatant, pellet was reconstituted in lysis buffer  and used for Western blot analysis.MDA-MB-231 cells were transfected either with siCtrl or siHPIP, siRAB5A, and siRUFY3. After a period of 36 h post transfection, cells were supplemented with growth factors deprived medium for 1 h. Cells were treated with complete medium for different time intervals . Afterward, cells were scraped in subcellular fractionation buffer .CD spectra of CC domains were recorded with a spectropolarimeter (Model: Jasco J-810), using a quartz cell with a path length of 0.02 cm. Five scans were run at a scan speed of 50 nm minwww.expasy.com/coil) using the HPIP protein sequence. The structure for CC domains was determined by I-TASSER software (https://zhanggroup.org/I-TASSER/) (CC domains for HPIP were identified by running the Coil server (TASSER/) , 67, 68.TASSER/) . We haveTASSER/) , 34.p values were calculated using the Student’s t-test using Sigma Plot or GraphPad Prism.The results are expressed as means ± SD, and differences between groups were analyzed by one-way ANOVA using either Sigma plot or GraphPad Prism. All the khsaratsingh@gmail.com) or V. P. (vasu2168@gmail.com).All data are contained within the manuscript. All the original Western blotting data will be shared upon request and may be contacted by first authors, S. S. K. (This article contains The authors declare that they have no conflicts of interest with the contents of this article.


Since May 2022, mutant strains of mpox (formerly monkeypox) virus (MPXV) have been rapidly spreading among individuals who have not traveled to endemic areas in multiple locations, including Europe and the United States. Both intracellular and extracellular forms of mpox virus have multiple outer membrane proteins that can stimulate immune response. Here, we investigated the immunogenicity of MPXV structural proteins such as A29L, M1R, A35R, and B6R as a combination vaccine, and the protective effect against the 2022 mpox mutant strain was also evaluated in BALB/c mice. After mixed 15 μg QS-21 adjuvant, all four virus structural proteins were administered subcutaneously to mice. Antibody titers in mouse sera rose sharply after the initial boost, along with an increased capacity of immune cells to produce IFN-γ alongside an elevated level of cellular immunity mediated by Th1 cells. The vaccine-induced neutralizing antibodies significantly inhibited the replication of MPXV in mice and reduced the pathological damage of organs. This study demonstrates the feasibility of a multiple recombinant vaccine for MPXV variant strains. The viruses . Due to viruses , 5. Furtviruses . Meanwhiviruses . RecombiOrthopoxvirus and the immunogenicity of the selected protein. Similar to other poxviruses, MPXV also has two forms, namely, extracellular enveloped virus (EEV) and intracellular mature virus (IMV). The most abundant particles are the IMVs, which accumulate in infected cells and can be released as cells die. Meanwhile, EEVs have undergone exocytosis from cells at the plasma membrane to enhance spread within the host and are essentially the IMVs with additional membrane. However, they are highly susceptible to rupture, whereas the EEV membrane bear several important neutralizing epitopes  supplemented with 10% heat-inactivated fetal bovine serum  alongside 50 units/mL penicillin–streptomycin . Meanwhile, the MPXV strain (WIBP-MPXV-001) was cultured in Vero E6 cells. The confluent monolayers Vero E6 cells were infected at a multiplicity of infection (MOI) of 2 for 72 h at 37 °C, followed by three repeated freeze-thaws. Cell debris were then precipitated and removed by centrifugation at 4,000 × g for 5 min. Subsequently, the supernatant was harvested and stored at -80°C as the virulent strain for the MPXV attack challenge for this experiment. All virus culture and challenge were performed in a biosafety level 3 facility .Vero E6 cells were maintained at 37 °C with 5% CO2.2Soluble forms of the MPXV recombinant proteins of A29L, M1R, A35R, and B6R  were marked with a His-tag respectively. QS-21  were purchased from MedChem Express and was used as vaccine adjuvant .2.3ACE2-293T cells transformed with pcDNA 3.1-A29L-His tag, pcDNA 3.1-B6R-His tag, pcDNA 3.1-A35R-His tag, and pcDNA 3.1-M1R-His tag were propagated and harvested, respectively. Cell from cultures were resuspended in Laemelli SDS sample buffer  and heated at 95°C for 10 min. These samples were separated on 12% SDS-PAGE, transferred to PVDF membrane  and blocked with 5% skimmed milk-Tris buffered saline amended with 0.1% Tween 20  overnight at 4°C. Then wash three times, membrane was incubated for 1 h with anti-A29L, anti-B6R, anti-A35R and anti-M1R antibodies  at RT with continuous slow shaking, respectively. Membrane was incubated with 1:3000 anti-rabbit IgG conjugated to horseradish peroxidase  for 30 min at RT. Signals were detected using SuperSignal™ West Femto Maximum Sensitivity Substrate .2.4Here, 5-to-6-week-old female BALB/c mice were supplied by the animal reproduction facilities of the Wuhan Institute of Biological Products (WIBP) and subsequently kept in the animal laboratory of WIBP. All mice were randomly divided into three groups of eight mice each. A mixture of 15 μg each of A29L, M1R, A35R, and B6R was made and then this mixture was mixed with 15 μg of QS-21 in phosphate-buffered saline (PBS) to compose a recombinant protein vaccine. Mice in the vaccine group were injected subcutaneously with this recombinant protein vaccine on days 0, 21, and 42. Meanwhile, the mice in the QS-21 group were injected subcutaneously with 15 μg QS-21, whereas the mice in PBS group were subcutaneously injected with PBS.8 PFU/mL. Additionally, the weight and clinical symptoms were monitored during the experiment.Two weeks after the third immunization, three mice from each group were randomly selected to measured T cell responses, and the remain mice (n = 5 mice/group) were anesthetized with isopentane and inoculated intranasally and intraperitoneally using 20 μL and 480 µL of the MPXV, respectively. MPXV titer was 2.24 × 10 2.5Eight mice from each group had venous blood collected through the infraorbital plexus on days 0, 21, 42, and 56. Three mice from each group were randomly selected to be sacrificed and collected spleens on day 56. Five days after viral challenge, the remaining five mice from each group were euthanized and blood samples, spleens, lungs and ovaries were all collected. Blood was then incubated at 37°C for 1 h, before being maintained overnight at 4°C to shrink the clot, and subsequently centrifuged at a relative centrifugal force of 2,000 × g for 10 min. Sera were then collected and stored at -80°C until later use.2.6Vero E6 cells were inoculated in 24-well plates the day before transfection, with transfection starting when cell density reached 80%. System A was 500 μL OPTI-MEM mixed with 10 μg plasmid pcDNA3.1-His-C  and then left at room temperature (RT) for 5 min. Meanwhile, System B was 500 μL OPTI-MEM mixed with 20 μL Lipofectamine 2000 , and then left at RT for 5 min. Systems A and B were then mixed and allowed to stand for 15 min, before being inoculated into 24-well plates with 100 μL per well. The plates were incubated in the carbon-dioxide incubator at 37°C for 6 h, and the medium was changed to DMEM supplemented with 10% FBS and 50 units/mL penicillin–streptomycin and subsequently incubated for 60 h. Cells were washed with PBS and then incubated in PBS containing 5% BSA  and 1% Triton X-100  at RT for 1 h, followed by incubation overnight at 4°C with primary antibodies. The primary antibody for the positive control was 6 × His Tag Monoclonal Antibody , and for the rest of the group was mouse serum. After washing to remove primary antibody, cells were incubated with FITC-conjugated AffiniPure Goat Anti-mouse IgG(H+L)  at RT for 1 h, and then washed thoroughly. Cell nuclei were stained with DAPI  for 10 min, followed by washing thoroughly again and being photographed under a fluorescent microscope to collect images.2.72CO3 and 3.5 mM NaHCO3) at 4°C overnight. Plates were then washed with PBS-0.05% Tween 20 and blocked with PBS-10% FBS at 37°C for 1 h. The serum was heat-inactivated at 56°C for 30 min prior to analysis. Two-fold serial dilutions of sera in PBS-10% FBS were then added to the blocked plates and incubated at 37°C for 1 h. After washing, horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG antibody  in PBS-10% FBS was added to the wells and then incubated at 37°C for 1 h. After washing, ELISA was performed by adding a 1:1 mixture of tetramethylbenzidine and hydrogen peroxide.  Color development was subsequently stopped with 2 N sulfuric acid, and the absorbance was measured at 450 nm on an Epoch Microplate Reader  with the reference filter set at 630 nm. There were 8 samples from each group, and three replicates were set up for each sample.Polystyrene 96-well round bottom plates  were coated with recombinant proteins A29L, M1R, A35R, and B6R in 0.05 M carbonate buffer  at 37°C for 1 h. The virus-serum mixtures were added onto pre-formed Vero E6 cell monolayers and incubated at 37 °C for 1 h in a 5% CO2 incubator. Following this, the supernatant was removed, and the cell monolayers were covered with methylcellulose overlay . After 96 h, the plates were fixed with an equal volume of 8%  paraformaldehyde for at least 1 h, with the cell monolayers being stained with 1% crystal violet, before the plaques being counted and photographed. Neutralizing antibody titers were defined as the highest serum dilution that resulted in 50% (PRNT50) in the number of virus plaques reduction. The PRNT50 was given a value of 10 when no neutralization was observed. There were 8 serum samples at post-prime, post-initial boost and post-second boost these 3 time points while there were only 5 serum samples after challenge.Vero E6 cells were seeded 1 day prior to the experiment in 12-well plates  at a density of 2 × 102.9g at RT for 30 min. Next, the lymphocyte layer was aspirated, before adding 10 mL R10 and washing upside down. This was then centrifuged at 250 × g at RT for 10 min before collecting the cells. Subsequently, cells were resuspended with R10 and then counted. ELISpot assays were performed using the Mouse IFN-γ ELISpotPLUS kit  in accordance with the manufacturer’s instructions. Subsequently, 400,000 splenocytes were plated into each well and stimulated for 30 h with A29L, M1R, A35R, and B6R, at 20 μg/mL each. R10 and concanavalin A  were used for negative and positive controls respectively. Spots were scanned and quantified by ImmunoSpot CTL reader , and spot-forming unit (SFU) per million cells was calculated by subtracting the negative control wells. There were 3 samples from each group, and three replicates were set up for each sample.Mice spleens were collected separately and ground on a 70 μm cell sieve rinsed with mouse lymphocyte separation medium . Single cell suspensions were subsequently collected in 15 mL centrifuge tubes, with 1 mL RPMI 1640 medium  containing 10% FBS (R10) being added. The samples were then centrifuged at 800 × 2.10g at 4°C for 10 min. Thereafter, 200 μL of the supernatant from the centrifugation was used to extract viral DNA using the DAAN Nucleic Acid Extraction or Purification Kit . Furthermore, qPCR was performed using QuantStudio 5 real-time PCR system  and MPXV nucleic acid detection kit targeting the F3L fragment . There were 5 samples from each group, and three replicates were set up for each sample.Whenever commercial kits were used, the manufacturer’s instructions were followed without modification. Each tissue sample was weighed to 50 mg and then homogenized in 1 mL PBS (pH 7.4). The homogenized samples were subsequently centrifuged at 5,000 × 2.11Tissues were fixed in 4% paraformaldehyde  and then embedded in paraffin, followed by staining with hematoxylin and eosin . Stained slides were subsequently analyzed by veterinary pathologists who were blinded to study groups.2.12All statistical analyses were performed using GraphPad Prism 8 software . Unpaired Student’s t-tests were used to compare these data, with significance being set at p < 0.05.33.1MPXV proteins A29L, M1R, A35R, and B6R were measured using Western blot analysis, which revealed bands approximate to the predicted protein molecular weights of 8.4 kDa, 20.5 kDa, 14.6 kDa, and 29.6 kDa respectively . Wuhan Institute of Biological Products Co., Ltd., Wuhan City, China.XGL, ZW, and JL designed and supervised the study. DT, XKL, HF, XX, KS, RW, CL, DD, and HD performed the experiments. DT, XKL and JL performed the statistical analysis. DT and XKL wrote the manuscript. XGL, ZW, and JL revised the manuscript. All authors contributed to the article and approved the submitted version.