The microscopic openings across the walls of many capillaries contain a protein mesh that is formed by the extracellular domain (ECD) of plasmalemma vesicle-associated protein (PLVAP). The three-dimensional structure of one segment of the PLVAP ECD together with biochemical and spectroscopic experiments on this and other segments show that the majority of the PLVAP ECD consists of a dimeric alpha-helical coiled coil, consistent with electron micrographs showing thin fibrous strands arranged across the capillary openings like the spokes of a bicycle wheel. Protein structure determination benefited from the availability of a microfocused long-wavelength X-ray beam, a helical data collection strategy, and an ultrasensitive detector, which together allowed rapid rotation and translation of the crystal during collection of anomalous scattering data from sulfur atoms. In many organs, small openings across capillary endothelial cells (ECs) allow the diffusion of low–molecular weight compounds and small proteins between the blood and tissue spaces. These openings contain a diaphragm composed of radially arranged fibers, and current evidence suggests that a single-span type II transmembrane protein, plasmalemma vesicle-associated protein-1 (PLVAP), constitutes these fibers. Here, we present the three-dimensional crystal structure of an 89-amino acid segment of the PLVAP extracellular domain (ECD) and show that it adopts a parallel dimeric alpha-helical coiled-coil configuration with five interchain disulfide bonds. The structure was solved using single-wavelength anomalous diffraction from sulfur-containing residues (sulfur SAD) to generate phase information. Biochemical and circular dichroism (CD) experiments show that a second PLVAP ECD segment also has a parallel dimeric alpha-helical configuration—presumably a coiled coil—held together with interchain disulfide bonds. Overall, ~2/3 of the ~390 amino acids within the PLVAP ECD adopt a helical configuration, as determined by CD. We also determined the sequence and epitope of MECA-32, an anti-PLVAP antibody. Taken together, these data lend strong support to the model of capillary diaphragms formulated by Tse and Stan in which approximately ten PLVAP dimers are arranged within each 60- to 80-nm-diameter opening like the spokes of a bicycle wheel. Passage of molecules through the wedge-shaped pores is presumably determined both by the length of PLVAP—i.e., the long dimension of the pore—and by the chemical properties of amino acid side chains and N-linked glycans on the solvent-accessible faces of PLVAP. The efficient movement of molecules between the intravascular space and the surrounding tissues is central to vascular function . Movemen1Plvap gene leads to loss of diaphragms from fenestrae, transendothelial channels, and caveolae  analyses of the PLVAP ECD and its fragments showing that these polypeptides are almost entirely alpha-helical, electrophoretic analyses of secreted PLVAP fragments with distinct tags showing that they form parallel dimers, and an atomic resolution X-ray structure of an 89-amino acid fragment of the PLVAP ECD showing that it consists of an extended alpha-helical homodimer, held together by five disulfide bonds. The structure was solved using single-wavelength anomalous diffraction from sulfur-containing amino acids (sulfur SAD). We also demonstrate the utility of combining sulfur SAD with molecular replacement and protein modeling for coiled-coil structure determination. Finally, we map the epitope of MECA-32, an anti-PLVAP monoclonal antibody (mAb) , and detSI Appendix, Fig. S1A)]. Two regions spanning amino acids 141-224 and 272-389 are predicted to be in a coiled-coil (CC) configuration by MARCOIL . To express biophysical quantities of the full-length PLVAP ECD and the individual CC1 and CC2 subregions in a soluble and correctly folded and disulfide-bonded form, PLVAP coding region sequences were inserted downstream of DNA sequences coding for E. coli thioredoxin, an 8xHis tag, and the cleavage site for 3C protease . Protein production in E. coli SHuffle cells, in which the intracellular redox potential favors disulfide bond formation . Following immobilized metal affinity chromatography (IMAC) purification, cleavage with 6xHis-tagged 3C protease, and removal of the His-tagged thioredoxin and 3C protease, the PLVAP ECD and its subregions appeared to be present as disulfide-linked dimers based on their electrophoretic mobilities in reducing and nonreducing gels .The secondary structure predictions for the PLVAP ECD using the Quick2D program  which co MARCOIL  and Deep MARCOIL , and we,ormation , led to SI Appendix, Fig. S1A).To assess the secondary structure content of the PLVAP ECD, CD spectra were obtained from IMAC-purified soluble dimeric ECD segments encompassing residues 52 to 438 (for the full-length ECD), 141 to 229 (for CC1), and 270 to 395 (for CC2) . A derivTo explore the assembly of the PLVAP ECD in a more native context, segments corresponding to its N- and C-terminal halves were expressed as secreted fusions to human growth hormone (hGH) in transiently transfected HEK/293T cells and harvested from serum-free conditioned medium (SFCM). A set of PLVAP ECD N-terminal regions, encompassing amino acids 53-222, 53-235, and 53-257  version of this region, site-directed mutants with the first cysteine changed to alanine (C337A) and the second cysteine changed to alanine (C386A), or both cysteines changed to alanine were produced as hGH fusions  domain-containing versions of constructs D–F. Based on their distinct epitope tags, members of these two sets of constructs can be independently visualized in fluorescent immunoblots. Additionally, the higher molecular weight of constructs L–N lowers their mobility in nonreducing gels. Heterodimers formed between individual members of constructs H–K and constructs L–N would be predicted to exhibit a mobility distinct from and intermediate to the mobilities of the corresponding homodimers. In the immunoblots of nonreducing SDS-PAGE gels shown on the left side of To further explore the disulfide bonding arrangement within PLVAP CC2, we tested the stabilities of heterodimers formed between all pairwise combinations of constructs I and J cotransfected with constructs M and N. In each of these constructs, one of the two cysteines has been mutated to alanine. If the presumptive dimers are aligned in a parallel fashion and if disulfide bonding is intermolecular, we would predict that only the pairs of constructs in which the same cysteine is preserved would form disulfide-linked heterodimers, i.e., I+M and J+N. By contrast, if the presumptive dimers are aligned in an anti-parallel fashion and if disulfide bonding is intermolecular, we would predict that only the pairs of constructs in which different cysteines are preserved would form disulfide-linked heterodimers, i.e., I+N and J+M. As seen in the upper immunoblot on the right side of SI Appendix, Figs. S2 and S3). PLVAP CC2 crystallized in several conditions , and the crystals diffracted to a resolution of 3.65 Å, revealing an I212121 space group with unit-cell parameters 82.6 Å, 98.2 Å, and 413.6 Å. Of note is that PLVAP CC2 crystals revealed a severely anisotropic diffraction pattern, with resolution limits along to a*, b*, and c* axes of 6.6 Å, 5.3 Å, and 3.65 Å. Moreover, each asymmetric unit appears to contain 4 to 5 dimers of PLVAP CC2 based on an analysis of the Matthews coefficient . Because PLVAP CC1 contains only one methionine, we initially produced PLVAP CC1 with selenium-labeled methionine (SeMet) for multi- or single-wavelength anomalous diffraction (MAD or SAD). However, the resulting anomalous signals were too weak to be useful for structure determination.PLVAP CC1 crystallized in the P212121 space group with unit-cell parameters 35.6 Å, 86.2 Å, and 181.6 Å, referred to hereafter as crystal form I. We then collected data at a wavelength of 1.77 Å, using a low-noise and fast readout EIGER X detector, with an exposure time of 0.015 s per 0.2° oscillation. The X-ray beam was 90% attenuated to give ~5 × 1011 photons s−1 with a beam cross-section of 5 μm × 7 μm. The data collection encompassed a total rotation of 1,800° while continuously advancing the crystal 300 μm .PLVAP CC1 contains five cysteines and one methionine, and previous studies have demonstrated that SAD from sulfur-containing residues—sulfur SAD—can be used for experimental phasing and structure determination –26. The l 300 μm A. ConstrSI Appendix, Figs. S5 and S6). Phases derived from the full sulfur SAD dataset revealed electron densities corresponding to ten disulfide bonds and four methionines .To determine how much data are required for sulfur SAD phasing of PLVAP CC1, we indexed and integrated the complete 1,800° dataset and then divided it into five separately scaled datasets, each comprising one 360° rotation A and B. hionines A. The Bit 1.77 Å . SubsequThe past several years have witnessed dramatic advances in protein structure prediction , 28, whi29o (datasets 1 + 2 + 3) = 1,080°, as described in the preceding section . Next, we used the CCFold algorithm . We used this computational model for MR with the 1,080° diffraction dataset to generate an electron density map that exhibited a coiled-coil-like pattern . However, this approach appeared to retain an inherent MR model bias, with the result that the MR-based phases were of insufficient quality to permit structure refinement beyond values of Rwork = 46.8% and Rfree = 50.4% .For this test, we first combined the X-ray diffraction data from the first three crystal rotations corresponding to 3 × 360  section A and B. lgorithm , a proteRwork = 31.7% and Rfree = 37.3% . During the course of this work, the MR–SAD pipeline in PHENIX AutoSol  and four (d) that form the basis of knobs-in-holes packing (a and d are hydrophilic (d), Gln206 (position d), and Gln210 (position a) do not appear to contribute to the coiled-coil configuration  and heavy-chain (VH) segments from the MECA-32 hybridoma cell line  and a human heavy-chain IgG constant region , respectively  on the solvent-accessible faces of PLVAP. The 64% estimate for the helical content of the ~390-amino acid PLVAP ECD—based on the CD spectrum in SI Appendix, Fig. S1B), consistent with the concept that coiled coils act as “molecular rulers” within macromolecular assemblies  reducing air absorption with a vacuum or a helium-filled cone between the crystal and the detector , 55, ii)11 photons s−1 with a beam cross-section of 5 μm × 7 μm) at a wavelength of 1.77 Å, a vector/helical data collection strategy, and an ultrafast shutter and high-sensitivity detector to collect 1,800° of sulfur SAD data from a single crystal in ~2.25 min. For comparison, Weinert et al. reported using a 90 μm × 45 μm beam cross-section with ~1010 photons s−1 at a wavelength of 2.07 Å to collect 2,880° (8 × 360°) of sulfur SAD data from a single crystal . Starting from the N terminus, this vector codes for E. coli thioredoxin (TrxA), an 8xHis tag, and a 3C protease cleavage site . All constructs were confirmed by sequencing. The plasmid was transformed into E. coli SHuffle T7 cells (New England Biolabs) and induced with 0.2 mM isopropyl β-thiogalactopyranoside (IPTG) in terrific broth containing 100 μg/mL ampicillin (MilliporeSigma) at room temperature (~25o C) overnight. For cell disruption, the cells were harvested by centrifugation and resuspended in B-PER bacterial protein extract reagent (ThermoFisher Scientific) supplemented with 50 mM HEPES, pH 7.5, 0.3 M NaCl, 30 mM imidazole, 1 mM MgCl2, 500 U benzonase (MilliporeSigma), 0.2 mg/mL lysozyme, and “cOmplete” Protease Inhibitor Cocktail (MilliporeSigma). The cell lysate was clarified by centrifugation, and the supernatant was filtered using a 0.45 μm Steritop filter (MilliporeSigma). Proteins were purified by immobilized metal affinity chromatography (IMAC) using Ni Sepharose 6 Fast Flow resin (Cytiva). The resin was washed with 20 mM HEPES, pH 7.5, 0.5 M NaCl, 30 mM imidazole, and10% glycerol and eluted in 20 mM HEPES, pH 7.5, 0.15 M NaCl, and 0.5 M imidazole. The eluted protein was dialyzed against 20 mM Tris, pH 7.5, 0.5 M NaCl, and 5% glycerol and treated with His-tagged 3C protease prepared as described previously  in 10 mM Tris, pH 8.0, 0.3 M NaCl  or 10 mM Tris, pH 7.0, and 0.4 M NaCl (pXG61) were dialyzed for 2.5 h against 10 mM Tris, pH 7.4, and 0.15 M NaCl, degassed for 30 min, and then diluted with dialysis buffer to a final protein concentration of 100 μM  or 40 μM (pXG61) for CD measurements.The four PLVAP ECD fragments .For production of hGH fusion proteins in HEK/293T cells (ATCC CRL-11268), DNA segments coding for the PLVAP ECD or its fragments were inserted into the pSGHP1 expression vector  were grown in 0.1 M sodium acetate, pH 5.0, and 15% polyethylene glycol (PEG) 4000 by the hanging drop vapor diffusion method at 21 °C. For sulfur SAD experiments, PLVAP CC1 crystals (space group P212121) were grown in 0.2 M sodium malonate, pH 5.0, and 20% PEG3350 by the sitting drop vapor diffusion method at 21 °C. PLVAP CC2 crystallized in 0.1 M MgCl2, 0.1 M MES, pH 6.0, and 8% PEG 6K at 21 °C. Crystals were transferred into a reservoir solution supplemented with 5 to 10% propane-1,2-diol for PLVAP CC1 and 25 to 30% glycerol for PLVAP CC2 and then cryocooled in liquid nitrogen.PLVAP fragments CC1 and CC2 were concentrated to 10 mg/mL in 10 mM HEPES, pH 7.5, and 0.15 M NaCl and 12 mg/mL in 3 mM Bis-Tris, pH 6.5, and 90 mM NaCl for crystallization trials, respectively. Native crystals of PLVAP CC1  at the Stanford Synchrotron Radiation Light Source at the National Accelerator Laboratory and were processed with the XIA2 system  with Difb server .12 photons s−1 to ~5 × 1011 photons s−1, with a beam cross-section of 5 μm × 7 μm). Images were indexed, integrated, and scaled using the XIA2 system  beamline in the National Synchrotron Light Source II (NSLS II) at the Brookhaven National Laboratory at a wavelength of 1.77 Å using an EIGER X 9M detector (DECTRIS) with the crystal-to-detector distance set to record reflections to 2.4 Å resolution at the corners of the square detector and to 2.6 Å resolution at the center of the detector’s sides. The vector module was used to collect continuous helical data over a 1,800° rotation with an exposure time of 0.015 s per 0.2° . Sulfur identification (substructure site) and initial anomalous signal analysis were conducted in the HKL2MAP interface  using SHg PHASER  at PHENIg PHASER , 35 withg PHASER  and theng PHASER  to generg PHASER  was manug PHASER  and theng PHASER  and PHENg PHASER , with thSI Appendix, Table S1.For MR–SAD, the CCFold protein modeling algorithm  was usedStructure-based multiple sequence alignment was performed using Clustal Omega  and ESPr5 MECA-32 hybridoma cells (H and VL) were PCR amplified from the resulting complementary DNA using the PCR primers described in ref. H : EVQLVESGGGLVQPGRSMKLSCAASGFTFSDYYMAWVRQAPMKGLKWVASISYEGNKTYYGDSVKGRFTISRDNAKSILYLQMNSLKSEDTATYYCARQSYSSYIFDYWGQGVMVTVSS. Mature VL: DIQMTQTPSSMSASLGERVTISCGTSQGVNIFLNWYQQKPDGTIKPLIFFTSHLQSGVPSRFSGSGSGTAYSLTISSLEPEDFAVYYCQQYDSSPPTFGGGTKLYLK. The PCR-amplified VH and VL segments were inserted into expression vectors containing a human heavy-chain IgG constant region  and a human light-chain constant region [CL with a C-terminal rhodopsin 1D4 epitope tag Click here for additional data file.


To address these issues, oxygen-vacancy-rich LaFe0.9M0.1O3−δ  perovskite submicrofibers have been designed from the startingmaterial LaFeO3−δ (LF) by a B-site substitutionstrategy and used as the eNITRR electrocatalyst. Consequently, theLaFe0.9Cu0.1O3−δ (LF0.9Cu0.1) submicrofibers with a stronger Fe–Ohybridization, more oxygen vacancies, and more positive surface potentialexhibit a higher ammonia yield rate of 349 ± 15 μg h–1 mg–1cat. and a Faradaicefficiency of 48 ± 2% than LF submicrofibers. The COMSOL Multiphysics simulations demonstrate that the more positive surfaceof LF0.9Cu0.1 submicrofibers can induce NO3– enrichment and suppress the competinghydrogen evolution reaction. By combining a variety of insitu characterizations and density functional theory calculations,the eNITRR mechanism is revealed, where the first proton–electroncoupling step (*NO3 + H+ + e– → *HNO3) is the rate-determining step with a reducedenergy barrier of 1.83 eV. This work highlights the positive effectof cation substitution in promoting eNITRR properties of perovskitesand provides new insights into the studies of perovskite-type electrocatalyticammonia synthesis catalysts.The electrocatalytic nitrate (NO However, designingadvanced electrocatalysts with high selectivity for transforming NO3– to NH3via the eNITRR remains a challenge because the eNITRR is a complicated8e– transfer reaction involving many intermediates.15 Among the various eNITRR electrocatalysts,Cu-containing catalysts demonstrate superior performance comparedto other catalysts.20 However, the issueof instability in copper-containing catalysts limits their applications,which has also sparked research interest in the reaction mechanismof Cu-containing catalysts and the design of highly stable Cu-containingcatalysts. For instance, extensive research has been conducted onvarious aspects, such as regulating the d-band center of Cu-containingcatalysts,14 the electrochemical restructuringof Cu-containing catalysts,22 and the synergistic effects betweenCu and other metals.24 Nonetheless, addressing the stabilityconcern associated with Cu still poses a significant challenge.Ammonia  areconventionally prepared by ball milling or sol–gel methods,resulting in an uncontrollable and blocked structure with severe particleagglomeration.29 Generally, it has been demonstratedthat exposing accessible active sites of perovskite oxides by constructingspecific geometrical structures, such as SrNb0.1Co0.7Fe0.2O3−δ nanorods,30 BiFeO3 nanosheets,31 NaNbO3 nanocubes,32 and so on, can result in a highly efficient electrocatalytic process.Among them, the cross-linked network woven by one-dimensional (1D)perovskite oxide submicrofibers can not only facilitate reactant diffusionand electron transport during the electrocatalytic process but alsoavoid particle agglomeration to expose the maximum number of activesites for the adsorption and reduction of NO3– ions. In addition to the construction of the unique structure, theactivity of the perovskite is also affected by the electronic environmentaround the active sites.33 As the perovskiteoxides can accommodate ∼90% of metallic elements of the periodictable, their high compositional flexibility enables the incorporationof metal elements to form a B-site bimetallic perovskite. Based onthe molecular orbital theory and band theory, the catalytic performanceof perovskite oxides can be influenced by tailoring the octahedralstructure of their [BO6] units, regulating the hybridizationof B–O bonds, and generating oxygen vacancies (OVs). For example,the covalency of transition metal–oxygen bonds, which reflectsthe adsorption strength of oxygen-related intermediates, was verifiedto be an important descriptor for electrocatalytic activity.34 Therefore, it is rational to optimize the adsorption strength ofintermediates in the eNITRR process by substituting the B-site cationwith other elements. Apart from the variation of the electronic structure,the B-site cation substitution strategy will also affect the workfunction of the catalyst surface, which is accompanied by a changein surface potential.36 The surface potential and correspondingwork function play important roles in facilitating the transport ofelectrons from the catalyst to the reactant.The previous search for cost-effective and efficient eNITRR catalystshas revealed the potential of perovskite oxides owing to their flexibleelectronic structures and chemical versatility.0.9M0.1O3−δ  submicrofibers  were constructed by a B-site substitutionstrategy and acted as the eNITRR electrocatalysts. The LF0.9Cu0.1 submicrofibers showed a higher NH3 yieldrate of 349 ± 15 μg h–1 mg–1cat. and a higher FE of 48 ± 2% than the parent LFsubmicrofibers, which are attributed to the inhomogeneous charge redistributionand abundant OVs on the surface of LF0.9Cu0.1 submicrofibers, as well as its more positive surface potential andlower work function for achieving an enhanced adsorption ability towardNO3– ions. COMSOL Multiphysicssimulations also confirm this discovery from the perspective of atheoretical calculation. The reaction mechanism was investigated indetail by combining operando Fourier transform infrared (FT-IR) spectroscopy,online differential electrochemical mass spectrometry (DEMS), anddensity functional theory (DFT) calculations. In the deoxidation processof the reaction stages, the first proton–electron couplingstep of *NO3 + H+ + e– →*HNO3 is the rate-determining step (RDS). The substitutionof Cu in the B site lowers the barrier energy in this step, therebyfacilitating the reaction.Inspired by theFe active sites in both Haber–Bosch catalysts(Fe-based compounds) and nitrogenase enzymes (mainly containing theFe–Mo cofactor), a series of Fe-rich perovskite oxides of LaFe23 (LF) and LF0.9M0.1  submicrofibers were synthesized via an electrospinning technique followed by calcination, as illustratedin N,N-dimethylformamideto form a homogeneous, viscous solution, which was used for the electrospinningprocess. Subsequently, the electrospun metal salt–polymer submicrofiberswere calcined to generate 1D perovskite oxide submicrofibers . As the scanning electron microscopy (SEM) imagesshow in S1, all these samples consist of 1Dsubmicrofibers without obvious fracture or aggregation. As an example,LF0.9Cu0.1 submicrofibers are stacked layer-by-layerand connected to form three-dimensional networks . Notably,the LF0.9Cu0.1 submicrofiber was generated byconcatenating abundant LF0.9Cu0.1 nanoparticles,with diameters ranging between 50 and 70 nm, into an integrated structure.This is evident from the transmission electron microscopy (TEM) image plane of the perovskite oxide  patterns, as shown in Figure S3.All samples display clear diffraction peaks at 22.6, 25.3, 32.2, 39.7,46.1, 47.6, 52.0, 53.3, 57.4, 67.3, and 76.7°, which can be indexedto the (101), (111), (121), (220), (202), (212), (103), (311), (123),(242), and (204) planes, respectively, of orthorhombic perovskitestructures (JCPDS 88–0641) without any impurity phase. It shouldbe noted that the diffraction peaks would shift slightly after cationicsubstitution, such as the (121) peak, which may imply the latticevariation due to the cation substitution.37 The crystal structures were further confirmed by Rietveld refinementof the XRD patterns  in Table S1. By taking LF0.9Cu0.1 as an example, the decrease in unit cell volume indicates that thesubstitution of Cu caused lattice shrinkage due to the reduced ionicradii of Cu2+ ions. The atomic ratios of Fe/M in LF0.9Co0.1, LF0.9Ni0.1, andLF0.9Cu0.1 submicrofibers were also checkedto be approximately 9:1 by inductively coupled plasma mass spectrometry(ICP–MS) (Table S2). In short, the1D perovskite submicrofibers formed by the directional accumulationof nanoparticles with tunable B-site bimetallic cations were successfullysynthesized by the electrospinning technique and calcination process.The LaFeOnetworks 1b, with networks S2. Notabnetworks S2. Notabnetworks S2. Notab2SO4 with 50 ppm NO3––N (NO3––Nrepresents the concentration of NO3– expressedin terms of the nitrogen content) aqueous solution was used as theelectrolyte. The concentrations of reactant NO3–, reductive product NH3, and byproduct nitrite (NO2–) were detected using colorimetric methods.40 The linearsweep voltammetry (LSV) curves of LF0.9Cu0.1 submicrofibers in 0.5 M Na2SO4 electrolyteswith and without NO3– were recorded toinvestigate the potential window for the eNITRR process. When thepotential is more negative than −0.6 V vs thereversible hydrogen electrode (RHE), the current density for the LSVcurve with NO3– is enhanced significantly in S8, and S9, the FEs for NH3 of thesesamples follow a volcano-type trend as a function of applied potentials.This phenomenon can be attributed to the increased competition fromthe HER at more negative potentials, coupled with a decrease in theavailable charges at more positive potentials. Notably, the LF0.9Cu0.1 submicrofibers exhibit the best eNITRRperformance with a maximal FE value of 48 ± 2% and a high NH3 yield rate of 349 ± 15 μg h–1 mg–1cat. at −0.90 V vs RHE. Additionally, the 1H nuclear magneticresonance method  was alsoconducted as another quantitative measurement to check the NH3 yield rates for all electrocatalysts at a potential of −0.9V vs RHE, which are in good agreement with the resultsby the UV–vis spectroscopy method . The N-selectivity indicates the ratio of NH4+–N in converted NO3––N, and is an important parameter that reflects the degreeof conversion from NO3– to NH4+. By testing the NO3– concentrationsfor various samples after 2 h eNITRR processes at −0.9 V vs RHE, it can be observed that all the perovskite oxidesdisplay high N-selectivities of NH3 of over 80% (3– electroreduction (Table S3). The N-selectivitiesof the four samples for NO2– are closeto 20%, indicating that their main soluble byproduct is NO2– . To evaluatethe eNITRR performance of LF0.9Cu0.1 comprehensively,the NH3 yield rates and FEs at different concentrationswere also measured. The results, as depicted in Figure S13, demonstrate that the catalyst maintains high catalyticactivity even at higher concentrations.The eNITRR performance of various perovskite submicrofiberswasevaluated in an H-type electrolytic cell under ambient conditions,where 0.5 M Naficantly2a, sugge methodsS5–S7.40 e method S10 was a2SO4 electrolyte without NO3–. As shown in Figure S14, no NH3 can be detected, which indicates that the producedNH3 during the eNITRR process originates from the NO3– ions in the Na2SO4 electrolyte. Furthermore, the electrocatalytic and structural stabilitiesof the LF0.9Cu0.1 submicrofibers were evaluatedby 6 consecutive electrolysis cycles at −0.9 V vs RHE. As shown in 3 yield rate and FE value of each cyclefluctuate slightly but remain stable, suggesting the excellent electrocatalyticstability of LF0.9Cu0.1 submicrofibers for potentialpractical applications. The perovskite structure of LF0.9Cu0.1 submicrofibers is also maintained well based onthe XRD pattern of LF0.9Cu0.1/carbon paper afterthe eNITRR process , whichillustrates the excellent structural stability of LF0.9Cu0.1 submicrofibers. ICP–MS was employed for theevaluation of changes in the composition of LF0.9Cu0.1 submicrofibers after the stability test. The results showthat no significant changes have occurred in the composition of thecatalyst, further highlighting its excellent stability (Table S4). Figure S16 shows the Nyquist plots of the LF and LF0.9Cu0.1 submicrofibers. The charge transfer resistance of LF0.9Cu0.1 (28.6 ± 3.9 Ω) is smaller than that ofLF (40.0 ± 3.0 Ω), which indicates that the electron transferrate is faster in LF0.9Cu0.1. To investigatethe influence of active sites on the eNITRR, we calculated the electrochemicallyactive surface areas (ECSAs) of LF and LF0.9Cu0.1. As shown in Figures S17 and S18, thedouble-layer capacitance of LF0.9Cu0.1 is 0.31mF cm–2, which is almost equal to that of LF at0.27 mF cm–2. Based on this, the activity gap betweenLF and LF0.9Cu0.1 was not strongly correlatedwith the ECSA.To exclude the interference from the electrocatalystitself orthe external environment, a comparison test was performed in 0.5 MNa0.9Cu0.1 submicrofibers, and the peaks at 710.2eV (Fe2+ 2p3/2), 712.1 eV (Fe3+ 2p3/2), 723.7 eV (Fe2+ 2p1/2), and 725.5eV (Fe3+ 2p1/2) indicate the coexistence ofFe2+ and Fe3+.42 The Fe3+/Fe2+ ratio increases with the substitution oflow-valent Cu atoms in LF because more Fe3+ ions are requiredto balance the charges (Table S5).43 This phenomenon also illustrates the chargeredistribution in LF as a result of the cation substitution strategy.44 Moreover, the valence state of Fe is influencedby the filling degree of the d orbitals, thereby determining the d-bandcenter of the catalyst. Therefore, the value for the d-band centerof the Fe element becomes more positive with the increase of its valencestate, leading to an enhanced adsorption of NO3– ions.45 The Fe coordination environmentsof LF and LF0.9Cu0.1 were also investigatedby synchrotron-based XAS measurements. The X-ray absorption near-edgestructure (XANES) spectra of LF, LF0.9Cu0.1,standard Fe2O3, standard FeO, and Fe foil areshown in 0.9Cu0.1 are found to be between those of FeO and Fe2O3, suggesting that their average valence states of Fe are both between+2 and +3. Meanwhile, XANES spectra of Fe in LF0.9Cu0.1 has a higher energy than that in LF, indicating that thevalence state of Fe in LF0.9Cu0.1 is slightlyhigher than that in LF,46 which is consistentwith the XPS results. The Fourier transform extended X-ray absorptionfine structure (FT-EXAFS) spectra in R-space forLF and LF0.9Cu0.1 are shown in 0.9Cu0.1 that originate from the scattering paths of theFe–O, La–Fe, and Fe–O–Fe bonds,47 respectively. With partial Fe atoms substitutedby Cu atoms, the length of the Fe–O bond decreases and indicatesan enhanced hybridization of the Fe 3d–O 2p orbitals.48 The wavelet transform (WT)-EXAFS measurementswere also used to provide insights into the coordination structuresof Fe atoms in LF and LF0.9Cu0.1 (S19). The maximum intensity is closely associated with thepath length, which can provide pivotal clues for identifying the coordinationenvironment. Specifically, the scattering path signals at  of  and  are associated with Fe–O bonds in the LF and LF0.9Cu0.1 submicrofibers, respectively. The second set ofWT-EXAFS peaks at  and  can be assigned to theLa–Fe contributions in the LF and LF0.9Cu0.1 submicrofibers, respectively. The third set of WT-EXAFS peaks at and  originate from the Fe–O–Febonds in the LF and LF0.9Cu0.1 submicrofibers,respectively. It is further demonstrated that the bond length of Fe–Oreduces after Cu substitution, which agrees with the results of theFT-EXAFS spectra in R space. The fitting curves atthe R space and k space are consistentwith the FT-EXAFS spectra of LF and LF0.9Cu0.1 . Based on the aboveanalysis, it is evidenced that the B-site substitution strategy withlow-valent metal elements can lead to charge redistribution and curtateFe–O bond distance.49 It can befurther inferred that with the increase of Fe 3d–O 2p orbitalhybridization, the lattice O p bands shift toward the EFermi as the Fe d states shift closer to the lattice Op energy.48 The shift of the lattice O p band lowersthe formation energy of OVs. Subsequently, it facilitates the generationof more OVs on the surface, promoting full contact between the reactantand active B-site transition metals. The OVs in LF and LF0.9Cu0.1 were probed via the XPS spectraof O 1s species and electron paramagnetic resonance (EPR) spectra.The corresponding deconvolution results of O 1s are shown in 2–, a highly oxidative oxygen species (O22–/O–), surface-adsorbed O2 or hydroxylgroups, and surface-adsorbed H2O, respectively.50 Based on the relative areas of peaks (Table S6), the concentrations of surface OVs, which are correlatedwith the O22–/O– species,51 were calculated to be 13.6 and 17.6% for LFand LF0.9Cu0.1, respectively. These resultsindicate that the surface OVs slightly increased with Cu substitution.To further study the variation of OVs, the EPR spectra of LF and LF0.9Cu0.1 submicrofibers were also recorded, as shownin Figure S22. Both samples exhibit EPRsignals at g = 2.004, which are identified as thetrapped electrons in the OVs.52 Furthermore,the EPR signal intensity of LF0.9Cu0.1 is higherthan that of LF, indicating that more OVs have been generated in LF0.9Cu0.1. The chemical state of Cu of the LF0.9Cu0.1 submicrofibers was subsequently checkedby XPS measurements (+ 2p3/2), 934.0 eV (Cu2+ 2p3/2), 952.8 (Cu+ 2p1/2), and 954.5 eV (Cu2+ 2p1/2) suggest that there are two chemical states for the Cu element inLF0.9Cu0.1 submicrofibers (Table S7).53 Considering the potentialstability issues associated with Cu during the eNITRR process, XPSmeasurements of Cu were also conducted after the stability test forLF0.9Cu0.1 . The results demonstrate that except for a slight decrease in theoxidation state, Cu still maintains the same two chemical states (Table S8). Following the bespoke analysis, itcan be concluded that the charge redistribution and increased surfaceOVs induced by the B-site substitution strategy are expected to promotethe eNITRR catalytic activity  and synchrotron-based X-rayabsorption spectroscopy (XAS) techniques. 36 Therefore, Kelvin probeforce microscopy (KPFM) was conducted to investigate the surface potentialof the LF and LF0.9Cu0.1 submicrofibers. Thepotential distributions at the LF0.9Cu0.1 andLF submicrofiber surfaces are shown in 0.9Cu0.1 is roughly 66 mV higher than thatof LF. The above results show that the work function of LF0.9Cu0.1 is about 0.066 eV lower than that of LF accordingto the conversion formula between the work function and surface potential.36 A lower work function of LF0.9Cu0.1 submicrofibers implies a smaller energy barrier for electrontransfer to the reactant (NO3–).54 Notably, the surface potential of LF0.9Cu0.1 submicrofibers becomes more positive, which indicatesthat LF0.9Cu0.1 is more favorable to adsorbingand enriching NO3–. Therefore, it isadvantageous by constructing eNITRR catalysts with more positive surfacepotential in addressing the issue of low activity resulting from weakmass transfer and small concentration gradients near the electroderegion in low-concentration NO3– electrolytes.55 Meanwhile, COMSOL Multiphysicssimulations were used to simulate the ionic distribution after thevariation of the potential on the nanoreactor surface. Since the competitiveHER can inhibit the eNITRR process, the concentration of hydrogenions on the catalyst surface is also an important factor affectingthe eNITRR performance. The variations of ion concentrations are describedthrough the Nernst–Planck equation (see the details in the Supporting Information). Before the simulation,it is assumed that the anions and cations are distributed randomly,without electrostatic force. After the surface potential becomes morepositive, the distributions of the NO3– and H+ ions are shown in S24. TheNO3– concentration increases gradually,indicating a NO3– enrichment. On thecontrary, the H+ concentration at the surface of LF0.9Cu0.1 submicrofibers decreases progressively.These results demonstrate that a surface with more positive potentialwould be beneficial for not only promoting the enrichment of NO3– ions but also reducing the HER process. Before the calculation ofthe total reaction steps, it is crucial to determine the mode of NO3– adsorption and hydrogenation on the LF0.9Cu0.1 surface. The free energies of two differentpathways to form *NO3 and *HNO3 were calculated.As shown in 3– coupling and first proton–electron coupling steps of singleoxygen (O1 mode) on the Fe site are lower than those of dioxygen (O2mode). For example, the free energy values for the first proton–electroncoupling step (*NO3 + H+ + e– → *HNO3) with the O1 and O2 modes are 1.83 and2.67 eV, respectively, which indicate that the O1 mode is a more optimalstructure for the coupling step between the LF0.9Cu0.1 catalyst and NO3– species,as well as the following hydrogenation. It is noted that the byproductNO2(g) can be produced in the side reaction *HNO3 + H+ + e– → *OH + NO2(g). The free energy of this potential intermediate was also calculatedwith a larger free energy of −2.61 eV than that of the stepfor the generation of the *NO2 species (−3.86 eV).Therefore, the side reaction is much less likely to occur than thesecond proton–electron coupling step (*HNO3 + H+ + e– → H2O + *NO2). After other possibilities are excluded, the total reactionpathway is shown as follows. First, the reactant NO3– is chemically absorbed on the catalyst surface toform *NO3 with a decrease of total energy, indicating thedriving force of this reaction step. Then, the N–O bond iscontinuously cleaved by proton-coupled electron transfer to form *NO2 and *NO. During the following hydrogenation processes, the*NO intermediate is converted to *HNO, *H2NO, and *O speciesgradually. In the final active site refreshing process, the *O intermediateis coupled with two other protons and converted to a H2O molecule. To be noted, different intermediates were adsorbed onthe surface of catalysts, and the most stable adsorption models areemployed to describe the eNITRR process , wherethe energy barriers are 2.07 and 1.83 eV for LF and LF0.9Cu0.1, respectively. This indicates that the eNITRR processcan be facilitated on the surface of LF0.9Cu0.1. Combining all experimental results with the theoretical calculations,it can be concluded that the eNITRR performance can be promoted bythe B-site substitution strategy.DFT calculations were also conducted to explorethe relationshipbetween the Cu substitution and improved eNITRR activity. The chargedensity difference of LF processS25. Befo processS25. Befo30.9Cu0.1 submicrofibers canact as a selective and efficientelectrocatalyst to reduce NO3– to valuableNH3. At the optimal potential of −0.9 V, the NH3 yield rate, FE, and selectivity can reach 349 ± 15 μgh–1 mg–1cat., 48 ±2%, and 88 ± 4%, respectively. Synchrotron-based XAS revealedthe charge redistribution on the L0.9Cu0.1 submicrofiberand confirmed the increase of hybridization of the Fe 3d–O2p orbital, which favors the generation of surface OVs. Combined withthe results of KPFM and COMSOL Multiphysics simulations,it is shown that a more positive surface can be induced by the cationsubstitution strategy, which promotes the fixation of more NO3– anions on the catalyst surface. Basedon a variety of in situ characterizations and DFTcalculations, the reaction pathway was deduced. The results showedthat the lower free energy for the RDS (*NO3 + H+ + e– → *HNO3) for LF0.9Cu0.1 led to its optimal NH3 synthesis performance.This work opens new avenues for designing efficient perovskite-typeelectrocatalytic NH3 synthesis catalysts via a cation substitution strategy.In summary, we have demonstratedthat perovskite LF


Oxidative stress (OS) induced by an imbalance of oxidants and antioxidants is an important aspect in anticancer therapy, however, as an adaptive response, excessive glutathione (GSH) in the tumor microenvironment (TME) acts as an antioxidant against high reactive oxygen species (ROS) levels and prevents OS damage to maintain redox homoeostasis, suppressing the clinical efficacy of OS-induced anticancer therapies.2@MnO2) to form a TME stimulus-responsive hybrid nanopharmaceutical  for enhancing oxidative stress. Once exposed to TME, as MnO2 responds and consumes GSH, the released Mn2+ converts endogenous hydrogen peroxide (H2O2) into hydroxyl radicals (·OH), which together with the subsequent release of GAL from SiO2 increases ROS. The “overwhelming” ROS cause OS-mediated mitochondrial malfunction with a decrease in mitochondrial membrane potential (MMP), which releases cytochrome c from mitochondria, activates the Caspase 9/Caspase 3 apoptotic cascade pathway. Downregulation of JAK2 and STAT3 phosphorylation levels blocks the JAK2/STAT3 cell proliferation pathway, whereas downregulation of Cyclin B1 protein levels arrest the cell cycle in the G2/M phase. During 18 days of in vivo treatment observation, tumor growth inhibition was found to be 62.7%, inhibiting the progression of pancreatic cancer. Additionally, the O2 and Mn2+ released during this cascade catalytic effect improve ultrasound imaging (USI) and magnetic resonance imaging (MRI), respectively.A naturally occurring ROS-activating drug, galangin (GAL), is introduced into a Fenton-like catalyst (SiOThis hybrid nanopharmaceutical based on oxidative stress amplification provides a strategy for multifunctional integrated therapy of malignant tumors and image-visualized pharmaceutical delivery.The online version contains supplementary material available at 10.1186/s12951-023-01932-0. Pancreatic cancer causes almost as many deaths as incidences due to its poor prognosis , with a 1O2), superoxide radical (O2‒), hydroxyl radical (·OH) and hydrogen peroxide (H2O2), have made significant advances in physiology and disease as pleiotropic physiological signaling mediators Cl2 (RDPP) functions as a sensitive fluorescence probe that can be quenched by O2 [2O2 . Then, the fluorescence intensity was recorded at 615 nm at various times . As a control, the O2 generation of SG@M (200 µg/mL) and free H2O2 were also measured. Every 6 s, a dissolved oxygen meter  measured the dissolved oxygen content in the above groups.To track Oed by O2 . First, 2 and 37 °C.Human pancreatic cancer cell (PANC-1) was generously offered by the Stem Cell Bank, Chinese Academy of Sciences, and cultured in Dulbecco's modified eagle medium (DMEM) medium containing 10% fatal bovine serum (FBS) and 1% penicillin–streptomycin at 5% CO2, SG, S@M and SG@M for 24 h. After 24 h, each group was introduced to 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA) (10 µM) for 20 min of incubation before images were captured by laser scanning confocal microscopy (LSCM). Control cells were those that received no treatment. With ImageJ software, the fluorescence intensity was quantitatively analyzed.PANC-1 cells were exposed to free GAL, SiO2, SG, S@M or SG@M. All cells were washed with PBS before being stained for 20 min with JC-1 solution (10 µM). LSCM obtained fluorescent images, and flow cytometry measured the ratio of JC-1 aggregates/monomers.The changes in MMP in cells were assayed by JC-1 following various treatments. Briefly, PANC-1 cells were seeded in confocal dishes for 24 h, then incubated for another 24 h with either PBS, free GAL, SiO4 cells per well) were seeded into a 96-well plate and incubated for 24 h, the medium was replaced with new medium containing SiO2, SG, S@M and SG@M at varying concentrations , and further incubated for 24 h. Then, the cell viability was measured by CCK-8 assay. Cells were treated with different concentrations of S@M  for 24 h and 48 h to further investigate the cytotoxicity of CDT. The cell viability was calculated to follow the Eq.  were cultured in a 6-well plate for 24 h. Following several treatment methods employing PBS, SiO2, free GAL (22.18 µg/mL), SG (200 µg/mL), S@M (200 µg/mL) and SG@M (200 µg/mL), cells were collected for flow cytometry.To assess apoptosis, cells , Caspase 9 and Caspase 3 activity were calculated for each group.To assess relative Caspase 9 and Caspase 3 activity5 cells per well), which were then incubated for 24 h. For the flow cytometry experiment, cells were collected and dispersed in a solution of PBS (100 µL) and 75% ethanol (900 µL).To assess cell cycle, PBS, free GAL (22.18 µg/mL), SG (200 µg/mL), S@M (200 µg/mL) and SG@M (200 µg/mL) were added to PANC-1 cells  with protease inhibitors to extract total proteins. The protein samples were then separated by SDS–PAGE and transferred to PVDF membranes . Afterward, the membranes were blocked with 5% BSA solution for 1 h and incubated with primary antibodies against STAT3 , p-STAT3 , p-JAK2 , Bcl-2 , BAX , Cyclin B1 , and GAPDH  overnight at 4 ℃. After washing with TBST, the membranes were incubated with respective secondary antibodies conjugated with horseradish peroxidase for another 1 h at RT. Finally, the protein signals were recorded and visualized using hypersensitive ECL chemiluminescence reagent (Biosharp).Western Blot analysis7 cells) were suspended in a PBS (100 µL) and Matrigel mixture  and subcutaneously transplanted into the right foreleg axilla of each mouse. After approximately 15 days, the tumor volumes of the tumor-bearing mice reached 100 mm3 for in vivo treatment and imaging. .BALB/c nude mice  were obtained from Tengxin Biotechnology Co., Ltd.  and housed in the Laboratory Animal Center of Chongqing Medical University. All animal experiments and procedures were approved by Ethics Committee of the Second Affiliated Hospital of Chongqing Medical University. By co-inoculating of PANC-1 cells with the Matrigel mixture, the pancreatic xenograft subcutaneous transplantation tumor model was established. Briefly, PANC-1 cells  were added to a transparent gel tube and then monitored via a Vevo LAZR imaging system . For in vivo US imaging, SG@M NPs  were intravenously administered into PANC-1 tumor-bearing nude mice (n = 3). Prior to the injection as well as at certain intervals , B-mode and CEUS images were captured using the US system of EPIQ 7 .For in vitro US imaging, various solutions  were distributed in a PBS solution (pH = 6.5) containing GSH (5 mM), and the samples were subsequently imaged by the MRI system. In addition, the same concentrations of SG@M NPs in PBS solution  were utilized as a control. In vivo, SG@M NPs  were intravenously injected into PANC-1 tumor-bearing nude mice (n = 3), and T1-weighted signals were recorded before and after the injection for 2, 4, 6, 8, 12, and 24 h using the MRI scanner.A dose of SG@M  was injected intravenously into PANC-1 tumor-bearing mice (n = 3). Approximately 20 µL of blood was taken from each tail at various times  following injection. The mice were killed at the conclusion of the experiment, and the tumors were collected and weighed. ICP-MS was utilized to determine the quantity of Mn in each sample.3: control (group 1); SiO2 (group 2); GAL (group 3); SG (group 4); S@M (group 5); SG@M (group 6). In the experimental groups, mice were intravenously injected with GAL , equal amounts of SG, S@M and SG@M , and the control group only received saline solution. On a regular basis throughout the course of the treatment, the body weight and tumor volume of the mice in each group were noted. On the 18th day, the mice were sacrificed, and the tumor tissues were dissected and stained with hematoxylin and eosin (H&E), terminal deoxynucleotidyl transferase dUTP nick end (TUNEL), and Ki-67 antibody. Meanwhile, immunofluorescence staining was performed on the harvested tumor sections to assess the expression using HIF-1α, JAK2, and p-STAT3.The mice were randomly assigned to 6 groups (n = 6) when tumor sizes reached 100 mmTo test the toxicity of SG@M NPs in vivo (n = 3), mice were intravenously injected with SG@M NPs . For H&E staining and hematological analysis, the primary organs and blood samples were collected at 0, 2, 4, 10, and 18 days after injection.t-test were used to test the significance of differences among groups. Additionally, the statistical significance difference was shown as *p < 0.05, **p < 0.01, and ***p < 0.001.All data are expressed as the mean ± standard deviation. One-way ANOVA tests and Student’s 2 layer was formed in situ on the surface of SiO2 via a reduction reaction of PEG and KMnO4. Transmission electron microscopy (TEM) images showed the spherical structure of SG NPs  and 641.5 eV (Mn 2p2/3), as illustrated in Fig. 2 and SiO2  [As illustrated in Fig. NPs Fig. B. After NPs Fig. C, with aNPs Fig. : Fig. S1morphous . Additio57 cm‒1) . The abo2 shell to deplete GSH in the TME, we compared the variation in SG@M NPs in different buffers (pH 7.2 and pH 6.5) as well as different concentrations of GSH in the acidic buffer (pH 6.5). The progressive fading of the brown solution, which can be seen as the first sign of the reaction between MnO2 and GSH  compared to a normal environment (pH 7.2), but the degradation could be accelerated with the addition of GSH, which trend was not so pronounced in the solution at pH 7.2 , as shown in Fig. 2+ in the acidic system (pH 6.5 without GSH) slowly increased over time. Therefore, we further explored the cumulative release of Mn2+ over 2 h. More than 80% of Mn2+ was released from SG@M in the pH 6.5 solution containing GSH after 2 h of incubation, and GSH accelerated the release of Mn2+  and autoactivated Caspase 9, cytochrome c can create an apoptosome that will activate Caspase 3 and carry out the apoptotic process . More im2 groups, as shown in Fig. 2 groups exhibited the highest red fluorescence percentages, which was consistent with the LSCM images. More importantly, the percentage of red fluorescence dropped by more than 40% in the SG@M group , with SiO2 and compared the intracellular fluorescence intensity at the same time point with LSCM and flow cytometry. Compared to the red fluorescent spots of concentrated MB observed in the free MB group, only occasional intense fluorescent spots were found in the SiO2-MB group, while a rather diffusive fluorescence background  was higher than that of free GAL, which was reasonably explained by the fact SiOIncreased intracellular ROS can cause DNA damage and decrease the levels of cyclin B1 , which i2O2 in the TME is a prerequisite for SG@M-triggered O2 generation for enhanced US imaging. Considering the remarkable catalytic ability of SG@M for H2O2 in vitro, we further investigated the US imaging potential of SG@M with sustained O2 release capability. We initially measured the O2 production of SG@M in PANC-1 cells also using the RDPP probe, which exhibited intense red fluorescence under hypoxia and was gradually quenched after 12 h and 24 h of coincubation due to O2 production  did not demonstrate any discernible changes. To confirm the US imaging ability in vivo, a mouse model of pancreatic tumors was utilized to observe the signal changes within 24 h after i.v. injection of SG@M. Figure 2 bubbles and generate sufficient echogenic reflectivity to reinforce US signal. Additionally, by utilizing enhanced permeability and retention (EPR) effect-mediated passive, progressive, and relatively selective accumulation at the diseased location over time, CEUS imaging can be used to describe malignancies.Excess H2+ produced by its degradation guided T1-weighted MRI. As usual, we first demonstrated its MRI performance in vitro. Compared with the normal system (pH 7.2), most Mn2+ was released in the acidic (pH 6.5) containing GSH system, which showed the strongest T1 signal , indicating that SG@M has significant MRI properties. Then we investigated whether SG@M could exhibit this performance in vivo in tumor-bearing mice. Figure i.v. injection of SG@M, the tumor location became brighter over time, peaking at 2 h. Interestingly, the variations in signal strength at the liver were identical to those seen in tumors; they peaked at 2 h and remained for a long period, and the signal was still visible at 24 h. This suggested that SG@M not only functioned by aggregating at the tumor site with blood but was also metabolized out of the body through the liver system over time, thus minimizing the side effects on normal organs [In addition to the properties of SG@M for US imaging, the Mndic pH 6. containidic pH 6. containil organs .2+ content of the tumor and blood circulation was detected using ICP-MS to determine the metabolism of SG@M. As shown in Fig. 2+ content in tumors showed that SG@M accumulated in the tumor site at 2 h after injection, and although it slightly decreased at 12 h, it continued to be higher than before administration until 24 h  had the most pronounced inhibitory effect on tumor progression. On the 18th day, the inhibition rate of tumor growth (IRG) [Motivated by the effective ROS-related anticancer activity of SG@M in vitro and the significant tumor accumulation of SG@M in vivo, we explored the oxidative stress-mediated antitumor performance of SG@M in vivo with PANC-1 tumor-bearing mice. As seen in Fig. ves Fig. B, C, theth (IRG)  was 28.22 production capacity of SG@M, IF staining of tumor slices for hypoxia-inducible factor-1α (HIF-1α) was performed to verify whether SG@M could alleviate hypoxia in vivo. Figure 2, the pre four groups showed strong red IF signals accompanied by overexpression of HIF-1α, indicating serious hypoxic levels inside the tumors. The faint red fluorescent signal of HIF-1α revealed that its expression level was drastically downregulated in the final two groups, as predicted. Such a reduction in HIF-1α was attributed to the CAT-like effect of MnO2, suggesting the role of O2 production from SG@M in alleviating tumor hypoxia. Similarly, subject to the JAK2/STAT3-mediated tumor suppressive effect of SG@M in vitro, we explored the effect of SG@M on this pathway in vivo. As seen by the IF results of JAK2 and p-STAT3 from tumor slices, SG@M inhibited this signaling pathway . Figure S2. XPS analysis results of SG@M, and the enlarged elements patterns of O, C and Si. Figure S3. UV−vis absorption spectrum of SG and SG@M and photos of the corresponding solutions (inset). Figure S4. Structural formula of galangal (GAL). Figure S5. Standard curve of GAL based on HPLC and chromatogram of GAL content in SG@M. (n = 3). Figure S6. UV-vis absorption spectra of SG@M in PBS (pH=7.2) containing different concentrations of GSH. Figure S7. Mn2+ release from SG@M in various systems. Figure S8. Degraded Si amount from SG in different systems. Figure S9. Degraded Si amount from SG@M in different systems. Figure S10. The trend of absorbance of MB at 665 nm with SG@M and GSH concentration. Figure S11. LSCM images of cellular uptake of SG@M and corresponding flow cytometry analysis. Scale bar: 50 µm. (DiI labeled SG@M NPs and DAPI labeled cell nuclei). Figure S12. Standard curve of ρNA and relative Caspase 9 activity in various treatment groups. (n = 3). Figure S13. Standard curve of ρNA and relative Caspase 3 activity in various treatment groups. (n = 3). Figure S14. Standard Curve of GSH and the concentration trend of SG@M in cellular GSH levels (n = 3). Figure S15. The concentration and time trend in CDT effect of S@M (n=3). Figure S16. Therapeutic efficiency of synergistic effect (SG@M) compared with SG and CDT (S@M). The additive therapeutic efficiencies of independent SG and CDT treatments were estimated using the relation Tadditive = 100 − (fSG × fCDT) × 100, where f is the fraction of surviving cells after each treatment (n=3). Figure S17. The amount of GAL after incubating the cells with free GAL or SG NPs for 4 h and 24 h, respectively (n=3). Figure S18. The uptake levels of free MB and SiO2-MB. (a) LSCM images of PANC-1 cells treated with free MB or SiO2-MB  and (c) corresponding transmittance images the morphologies of the specific cells . (c) Corresponding flow cytometry analysis (n=3). Figure S19. The uptake levels of free DOX and SiO2-DOX. (a) LSCM images of PANC-1 cells treated with free DOX or SiO2-DOX  and (c) corresponding transmittance images the morphologies of the specific cells . (c) Corresponding flow cytometry analysis (n=3). Figure S20. LSCM images of O2 production detected with RDPP after 12 h and 24 h incubation with SG@M. Scale bars: 50 µm.


However, its RNA binding specificity and functional role in the cell are not well understood. Here we dissected structural and sequence requirements for binding and methylation of its RNA targets. Specificity of tRNA modification by hTRMT2A is achieved by a combination of modest binding preference and presence of a uridine in position 54 of tRNAs. Mutational analysis together with cross-linking experiments identified a large hTRMT2A–tRNA binding surface. Furthermore, complementing hTRMT2A interactome studies revealed that hTRMT2A interacts with proteins involved in RNA biogenesis. Finally, we addressed the question of the importance of hTRMT2A function by showing that its knockdown reduces translation fidelity. These findings extend the role of hTRMT2A beyond tRNA modification towards a role in translation.5-Methyluridine (m  In addi(TRMT2A) ,12.E. coli, although deletion of the whole gene is lethal , use SAM (S-adenosylmethionine) as methyl donor  have revealed that 87% of the cellular hTRMT2A targets are indeed tRNAs  induction overnight at 18°C, cells were harvested . All purification steps were performed at 4°C. For hTRMT2A RBD purification, typically pellets from 6 l of culture were resuspended in lysis buffer-A  supplemented with one tablet of EDTA-free protease inhibitor cocktail (Roche). After sonication , the lysate was clarified by centrifugation . The lysate was loaded onto a 5 ml HisTrap FF column , equilibrated in His-A buffer  and washed with 10 column volumes (CVs) of His-A buffer, followed by a 10 CV wash with His-B buffer . SUMO-tagged protein was eluted with a 10 CV gradient of His-A buffer and His-C elution buffer . For tag cleavage, the eluate was supplemented with 100 μg of PreScission™ protease and dialyzed against dialysis buffer-A  overnight. Cleaved protein was run on a subtractive HisTrap FF column and the protein-containing flow-through was collected and concentrated. Protein was loaded onto a size exclusion chromatography column  equilibrated in SEC-A buffer . Protein concentrations were determined by measurement of the A280. Aliquots were flash-frozen in liquid nitrogen and stored at –80°C.Plasmid containing the hTRMT2A sequence was provided by Aaron Voigt. The hTRMT2A RNA-binding domain , Central domain (amino acids 237–414) and Fusion domain (amino acids 68–240/413–590) were cloned with a cleavable SUMO-tag at the N-terminus and expressed in Rosetta 2 (DE3) in the case of the RBD or Rosetta 2 (DE3) pLyS  For the Central and Fusion domains, lysis buffer-B was used . Moreover, all buffers contained HEPES-NaOH with pH 7.5, and for the final size exclusion chromatography a Superdex 200 column (16/600) and SEC-B buffer were used . The Fusion domain was otherwise purified in the same way as the hTRMT2A RBD. The SUMO-tag of the Central domain was not cleaved due to subsequent protein degradation. Instead, after overnight dialysis in dialysis buffer-B , the protein was loaded onto a HiTrap Heparin column equilibrated with dialysis buffer-B and eluted with Hep-B elution buffer . Protein-containing fractions were processed as described above. Pooled protein peak fractions were concentrated to 2–5 mg/ml with a centrifugal filter (Amicon), flash-frozen in liquid nitrogen and aliquots were stored at –80°C. Primers for cloning and mutagenesis are listed in Supplementary Table S16 cells/ml. The cells were then incubated for 4 days at 27.5°C.The Bac-to-Bac baculovirus expression system (Invitrogen) was employed for expression of hTRMT2A full-length (hTRMT2A FL). The N-terminal SUMO tag-fused hTRMT2A FL and its mutants were cloned into the pFastBac vector. For transfection using the FuGene HD transfection reagent, 1.5 μg of bacmid DNA and 3 μl of transfection reagent were pre-incubated for 30 min in 200 μl of Sf-900 medium at room temperature, then added dropwise to the Sf21 cells pre-seeded on 6-well plates with a density of 0.4 × 106 cells/ml. After 3–4 days of incubation at 27.5°C, insect cells were harvested by centrifugation for 10 min at 4°C at 2000 rpm (Hettich centrifuge Rotina 420R). Virus-containing supernatant P1 was sterile-filtered using a 0.22 μm filter (Merck). To amplify the virus, 1.5 ml of P1 was added to 250 ml of Sf21 shaking cell culture at a density of 0.4 × 106 cells/ml. After another 3–4 days of incubation at 27.5°C, cells were harvested. The virus-containing supernatant P2 was sterile filtered and stored at 4°C. Protein was expressed in Hi5 insect cells by adding 20 ml of P2 to 500 ml of shaking culture at a density of 1 × 106 cells/ml. Proteins were expressed in Hi5 insect cells for 2–3 days at 27.5°C.The supernatant of transfected cells from two 6-well plates was used to infect 10 ml of Sf21 shaking cell culture at a density of 1.4 × 10g, 15 min, 4°C) and resuspended in lysis buffer-C . Next, insect cells were lysed using a Dounce homogenizer. The lysate was clarified by centrifugation . Subsequent purification was performed as for the hTRMT2A Fusion domain with an initial HisTrap FF column, then cleavage of the SUMO tag was followed by a subtractive HisTrap FF column, dialysis overnight with dialysis buffer-A and a final size exclusion chromatography run with a Superdex 200 column (16/600) in SEC-B buffer. hTRMT2A FL for cross-linking and mass spectrometry (MS) experiments was purified with SEC-C buffer , where DTT was exchanged by TCEP. Pooled protein peak fractions were concentrated to 2–5 mg/ml with a centrifugal filter (Amicon), flash-frozen in liquid nitrogen and aliquots were stored at –80°C.Cells were harvested by centrifugation  800, 0.5 mg/ml T7 RNA polymerase and 10× TRX-buffer  were added to the annealed primer pair in a final volume of 5 ml. After incubation for 3 h at 37°C, the precipitate was removed by centrifugation for 10 min at 4°C at 14 000 g. Thereafter the RNA was precipitated with 0.1 volume of 3 M sodium acetate and 3.5 volumes of ethanol for > 30 min at –20°C. RNA pellets were resuspended in 0.5 ml of diethylpyrocarbonate (DEPC)-treated H2O, and 0.5 ml of denaturing loading dye was added . Separation by 8% denaturing Tris-boric acid-EDTA (TBE)–PAGE was followed by visualization of transcripts by UV shadowing and excision from the gel. RNA was extracted by electroelution  at 200 V in 1× TBE according to the manufacturer's instructions. Fractions containing the RNA were pooled, and dialyzed twice against 5 M NaCl and once against DEPC-H2O. RNAs were lyophilized, subsequently resuspended in DEPC-H2O and stored at –20°C. General recommendations for working with RNAs were followed (All RNAs were transcribed from a high-performance liquid chromatography (HPLC)-purified forward primer, which contained the T7 RNA polymerase promoter region and a polyacrylamide gel electrophoresis (PAGE)-purified reverse primer that consisted of the reverse complement DNA template and the T7 RNA polymerase promoter region . In brieIn vitro transcribed RNA was 5′ dephosphorylated using FastAP™ thermosensitive alkaline phosphatase (Thermo Scientific) according to the manufacturer's recommendations. Thereafter, RNA was purified with phenol–chloroform–isoamylalcohol extraction followed by isopropanol precipitation. Purified RNA (13 pmol) was phosphorylated with [γ-32P]ATP  by T4 polynucleotide kinase (NEB) for 30 min at 37°C. The enzyme was deactivated at 75°C for 10 min and labeled RNA was purified with a NucAway Spin column (Ambion). To ensure proper RNA folding after each thawing cycle, RNA was denatured at 98°C for 2 min and snap-cooled on ice prior to use.32P-labeled RNA in the presence of 2.5 ng/μl poly(U) competitor  for 20 min at room temperature in RNase-free EMSA buffer . This results in a final concentration of 5 nM labeled RNA and 0–1.25 μM protein per reaction. Complexes were separated by 4% native TBE–PAGE in TBE running buffer at 80 V. After gel fixation with 30% (v/v) methanol and 10% (v/v) acetic acid solution for 10 min, gels were dried in a vacuum gel dryer . For analysis, phosphor imaging plates were exposed to the radioactive dried gels for >20 min and scanned with a FLA-300 system (Fujifilm). EMSAs were performed in triplicate on different days.For 20 μl EMSA reactions, increasing protein concentrations (0–25 μM) were incubated with 100 nM γ-Km values were performed with increasing substrate concentrations. A final concentration of 5 μM hTRMT2A FL was incubated with a final concentration of 0–6 μM in vitro transcribed tRNA and 20 μM SAM in 1× reaction buffer  for 60 min at room temperature. The reaction was stopped by adding 0.5% trifluoroacetic acid . After 5 min incubation, 6× MTase-Glo reaction solution diluted in MilliQ-H2O was added and incubated for 30 min at room temperature. Finally, MTase-Glo detection solution was added, incubated for 30 min in the dark at room temperature and luminescence was read using a microplate reader (Perkin-Elmer). The data were transformed to display relative luminescence by subtracting the value of a negative control reaction without RNA. Data of triplicate measurements were plotted with R (version 4.1.0) and fitted with the Michaelis–Menten equation to obtain Km values.hTRMT2A FL methylation activity was analyzed with the MTase-Glo™ assay (Promega). The assay was performed according to the manufacturer's instructions and experiments described in  in whitein vitro transcribed tRNAGln and recombinantly purified hTMRT2A FL were reconstituted in reconstitution buffer  in a 1:4 ratio. For the second set of experiments, the complex was co-purified using size exclusion chromatography. Briefly, tRNAGln and hTRMT2A FL were incubated in a 1:1 ratio on ice for 30 min. Then, the complex was separated from single RNA and protein using an S6 (15/150) column. For UV cross-linking, the in vitro reconstituted and co-purified complex was irradiated at 254 nm for 10 min on ice using an in-house-built cross-linking apparatus as described previously . Enriched peptides were dried and dissolved in 2% (v/v) acetonitrile, 0.05% (v/v) TFA. Liquid chromatography–tandem MS (LC-MS-MS) analyses were performed on a Thermo Scientific Orbitrap Exploris 480 mass spectrometer coupled to a nanoflow liquid chromatography system (Thermo Scientific Dionex Ultimate 3000). Peptide separation was performed over 58 min with a flow rate of 300 nl/min using a linear gradient and a buffer system consisting of 0.1% (v/v) formic acid (buffer A) and 80% (v/v) acetonitrile, 0.08% (v/v) formic acid (buffer B). Peptide–nucleotide hetero-conjugates were analyzed in positive mode using a data-dependent top 30 acquisition method. MS1 and MS2 resolution were set to 120 000 and 30 000 FWHM , respectively. AGC targets were set to 106 (MS1) and 105 (MS2), normalized collision energy to 28, dynamic exclusion to 10 s and maximal injection times to 60 ms (MS1) and 120 ms (MS2). MS data were analyzed and manually validated using the OpenMS pipeline RNPxl and Open MS TOPPASViewer  according to the manufacturer's recommendations. SA-chip  was prepared according to the manufacturer's instruction before biotin-labeled RNA diluted in DEPC-H2O was coupled. Analysis of RNA–protein interactions was performed at a flow rate of 30 μl/min in running buffer  at 25°C and an a 10 Hz data collection rate. The analyte of interest was diluted in running buffer and single-cycle concentration series were injected on the chip with 240 s contact time per concentration and a final dissociation time of 900 s. To remove any residual attached protein after the last analyte injection, 2 × 2 min regeneration injections with 0.5% (w/v) sodium dodecylsulfate (SDS) were done in between runs. Concentration series reached from 62.5 nM to 4000 nM for hTRMT2A wild-type (WT) and 15.6–1000 nM for hTRMT2A CDM  protein. Data were analyzed with the BIAevaluation software . Obtained binding curves were double-referenced against the signal in the empty reference channel and a buffer run. At equilibrium of the binding curves, the corresponding response was plotted against the analyte concentration. The KD was determined by fitting this curve to a steady-state affinity model. All experiments were performed in triplicates.SPR studies were performed using a BIACORE S200 system . tRNA7 to 1 × 108 per replicate) were centrifuged for 5 min at 4°C at 300 g. Cells were resuspended in 5 ml of lysis buffer  and incubation for 10 min at 98°C. Finally, tubes were placed onto the magnetic stand and the supernatant was transferred to a new tube for respective MS analysis and analytical western blot analysis. Enriched and depleted proteins are listed in Cell pellets were thawed and resuspended in 1 ml of lysis buffer  supplemented with one tablet of Pierce® Protease Inhibitor (per 100 ml of lysis buffer). The suspension was transferred to a new tube and centrifuged for 10 min at 4°C at 16 100 From BioID and Co-IP experiments, the sample was mixed with SDS loading buffer  and heated for 10 min to 95°C. Cell lysates from hTRMT2A WT and knockdown (KD) cell lines (20–50 μg) were supplemented with Laemmli buffer and boiled for 5 min at 95°C. Samples were run on SDS–PAGE and transferred onto an activated nitrocellulose membrane by semi-dry blotting. The membranes were blocked [5% skimmed milk or 1% casein in TBS-Tween (TBS-T) for 60 min] and incubated with the primary antibody at 4°C overnight. Membranes were washed three times for 10 min in TBS-T. In the case of anti-streptavidin–horseradish peroxidase (HRP) antibody , the blot was washed five times for 5 min and thereafter directly visualized. Other blots were incubated with the secondary HRP-coupled antibody for 1–2 h at room temperature. Subsequently, the membranes were washed three times in TBS-T for 10 min, and the chemiluminescence signal was detected using the Super Signal® West Femto Maximum Sensitivity Substrate (Thermo Scientific) or Pierce®ECL Western blotting detection kit according to the manufacturer's instructions. Chemiluminescence was visualized using the Fusion SL 4 device (Vilber Lourmat) or the Alliance UVltec (Biometra) systems. Primary antibodies used were: rabbit anti-TRMT2A , mouse anti-FLAG , rat anti-H3 , rat anti-FLAG , rabbit anti-PARP1  and mouse anti-GAPDH . Secondary HRP-coupled antibodies used were: goat anti-rabbit , mouse anti-rat , sheep anti-mouse  or donkey anti-rabbit .m/z. From the MS scan, the 15 most abundant peptide ions were selected for fragmentation via HCD with a normalized collision energy of 28, an isolation window of 1.6 m/z and a dynamic exclusion of 30 s. MS/MS spectra were recorded at a resolution of 15 000 with an AGC target of 1e5 and a maximum injection time of 50 ms. Unassigned charges and charges of +1 and >8 were excluded from the precursor selection.Proteins were proteolyzed with LysC and trypsin with the filter-aided sample preparation procedure (FASP) as described ,30. AcidP-value. Peptide assignment was re-imported into the Progenesis QI software and the abundances of all unique peptides allocated to each protein were summed up. The resulting normalized abundances of the individual proteins were used for the calculation of fold changes of protein ratios between conditions. Proteins with a sample/control ratio of at least 4 were considered differentially abundant. Statistical analysis was performed on log2-transformed normalized abundance values using Student's t-test. Changes in protein expression between conditions were considered significant at P < 0.05. Principal component analysis was performed in Progenesis QI software.Raw spectra were imported into Progenesis QI software (version 4.1). After feature alignment and normalization, spectra were exported as Mascot Generic files and searched against the human UniProt database (20 434 sequences) with Mascot  with the following search parameters: 10 ppm peptide mass tolerance and 20 mmu fragment mass tolerance, one missed cleavage allowed, carbamidomethylation was set as fixed modification, methionine oxidation and asparagine or glutamine deamidation were allowed as variable modifications. A Mascot-integrated decoy database search calculated an average false discovery rate (FDR) of < 1% when searches were performed with a mascot percolator score cut-off of 13 and an appropriate significance threshold ‘Biological processes’ (BP) and ‘cellular compartments’ (CC) Gene Ontology (GO) term analysis was performed with data from BioID experiments using the GO enrichment analysis tool from GO employing the PANTHER databank . UniProtFor stable transfection of HEK 293T cells, a piggyBac (PB) transposon plasmid was used ,33. Co-tThe same expression level of the protein of interest was achieved by fluorescence-activated cell sorting (FACS). Protein expression in cells was induced with doxycycline 18 h before the FACS experiment. For FACS, the cells were detached with trpysin, washed twice with PBS for 5 min and resuspended in sterile FACS buffer . Directly prior to the FACS experiment, the cells were passed through a 35 μm cell strainer. The first gating step etracted dead cells and aggregates. A second gate was set to sort the cell population with a low GFP signal. Sorted cells were plated in 6-well plates and propagated with complete medium containing hygromycin B.856s1c1, KD2 = NM_182984.2–1574s1c1; Sigma-Aldrich). Cells with stable integration of short hairpin RNA (shRNA) were selected as puromycin-resistant colonies. hTRMT2A silencing effect was probed by western blotting.Stable RNA interference (RNAi)-mediated KD of hTRMT2A was achieved as described earlier . HEK 2932, 50 mM Tris–HCl pH 7.4). For cell lysis, 0.7% (v/v) NP-40 was added and incubated on ice for 10–15 min. To separate nuclei, the cell lysate was centrifuged for 10 min at 750 g and 4°C. To prepare the fraction without mitochondria, the supernatant was centrifugated for 10 min at 12 500 g and 4°C. Post-mitochondrial fraction (PTM) supernatant was transferred to a new tube and the KCl concentration was adjusted to 500 mM using a 4 M stock solution. The adjusted PTM was carefully layered on top of a 2 ml sucrose cushion . Samples were ultracentrifuged for 2 h at 250 000 g and 4°C. The supernatant was discarded and the translucent ribosome pellet was washed with 200 μl of cold DEPC-treated H2O. Finally, ribosomes were resuspended in 3 × 100 μl buffer C .Ribosome isolation from hTRMT2A WT, KD1 and KD2 HEK 293T cells was performed according to a protocol published earlier . Briefly2O and 15 μl of 3 M NaOAc. Next, 1 volume of phenol–chloroform–isoamylalcohol (25:24:1) was added. After centrifugation for 1 min at 16 100 g, the upper aqueous phase was mixed with 1 volume of chloroform and again centrifuged. The recovered upper phase was precipitated in the presence of 0.5 μl of RNA-grade glycogen and 2 volumes of ethanol for > 20 min at –20°C. RNA was pelleted for > 20 min at 16 100 g and 4°C. The dry pellet was resuspended in 20 μl of DEPC-H2O.For rRNA isolation, 100 μl of ribosomes diluted in buffer C were mixed with 15 μl of DEPC-HFor isolation of 18S and 28S rRNA, purified rRNA was separated on a 0.8% agarose gel for 80 min at 120 V in 1× TAE buffer. The bands for 18S and 28S rRNA were cut from the gel and electroeluted for 200 min at 150 V in 1× TAE buffer. After electroelution, the rRNA was precipitated with 2 volumes of 100% ethanol for 30 min at –20°C and further pelleted for 30 min at 13 200 rpm at 4°C. To increase the yield, 1 μl of RNA-grade glycogen per 1 ml of the solution was added. The pellet was dissolved in water and the samples were frozen in liquid nitrogen for further storage at –80°C. All reagents used were RNase and sodium ion free. The integrity and purity of rRNA were checked with TapeStation analysis done using the Agilent TapeStation 4150 with the Agilent High Sensitivity RNA ScreenTapes according to the manufacturer's instructions.2 (1 mM) containing Tris  buffer. In a final volume of 30 μl, 1 μg of pentostatin, 5 μg of tetrahydrouridine (Merck) and a final concentration of 10 μM butylated hydroxytoluene were added to each reaction for protection against degradation of the released nucleosides. After incubation for 2 h at 37°C, 20 μl of LC-MS Buffer A (see below) was added to the mixture and then filtered through 0.2 μm Supor Natural PP filters  at 3000 g and 4°C for 30 min before measurement by LC-MS/QQQ.From 500 ng to 5 μg of pre-purified rRNA subunits (20 μl of each) were digested to single nucleosides by using 2 U of benzonase nuclease , 0.2 U of phosphodiesterase I (VWR) and 2 U of alkaline phosphatase  in MgCl2 gas temperature of 120°C and N2 gas flow of 11 l/min, sheath gas (N2) temperature of 280°C with a flow of 11 μl/min, capillary voltage of 3000 V, nozzle voltage of 0 V, nebulizer at 60 psi, high-pressure radiofrequency at 100 V and low-pressure radiofrequency at 60 V. The instrument was operated in dynamic MRM mode , and an organic buffer B of 2 mM NH4COOH in 80% acetonitrile . The gradient started at 100% solvent A for 0.5 min, followed by an increase of solvent B to 10% over 5.5 min. From 6.0 min to 8.5 min, solvent B was increased to 20% then to 80% in 1 min and maintained at 80% for 1.5 min before returning to 100% solvent A in 0.5 min and a 2.2 min re-equilibration period. A 10 μl aliquot of each sample was co-injected with 1 μl of stable isotope-labeled internal standard (ISTD) which was aspirated automatically before each injection from the instrument itself. The sample data were analyzed by the quantitative MassHunter Software from Agilent using the integrated calibration function. The calibration solutions ranged from 0.05 to 100 pmol for each canonical nucleoside and from 0.002 to 5 pmol for each modified nucleoside .For quantitative MS, an Agilent 1290 Infinity equipped with a variable wavelength detector (VWD) combined with an Agilent Technologies G6490 Triple Quad LC/MS system with electrospray ionization  was used. Operating parameters: positive ion mode, cell accelerator voltage of 5 V, NMRM mode . For sepThe measured molar amount of each canonical nucleoside (pmol) was divided by their expected occurrence in the respective rRNA subunit reported in the literature [# per molecule from modomics ].18S rRNA: #C = 493, #U = 356, #G = 540, #A = 403 (RNA_Source_ID = J01866)28S rRNA: #C = 1646, #U = 687, #G = 1790, #A = 781 (RNA_Source_ID = K03432)In this way, the measured amount of injected rRNA subunit in pmol is calculated by each of the four canonical nucleosides separately. Those results were averaged and used as normalization for the modifications to determine the number of modifications per rRNA molecule in the respective sample.Immunocytochemistry (ICC) experiments were performed to determine hTRMT2A localization in HEK 293T cells. Cells on coverslips were fixed for 10 min at room temperature using 3.7% formaldehyde solution in PBS. After two PBS washing steps, cells were permeabilized for 5 min with 0.5% Triton X-100 in PBS. Next, cells were blocked for 30 min using blocking buffer (1% goat serum in PBS-T). Primary antibody diluted in PBS was added for 1 h at room temperature. After three washing steps with PBS-T, cells were incubated with secondary antibody diluted in blocking buffer for 1 h at room temperature in the dark. Cells were washed three times with PBS-T. Next, the cell nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI) at a concentration of 0.5 μg/ml DAPI (Thermo) in PBS for 5 min at room temperature and washed twice with PBS. Finally, coverslips were mounted using the ProLong™ Diamond Antifade Mountant (Invitrogen). Cells were imaged with the Leica DMi8 fluorescence microscope and the Leica imaging software. Images were analyzed with the Fiji ImageJ software. The primary antibody used was mouse anti-BirA  and the secondary antibody was goat anti-mouse IgG Alexa Fluor 647 The dual luciferase assay to probe translation fidelity was performed using three Renilla–Firefly fusion protein constructs. In two Renilla sequences, mutations were introduced at residues crucial for luciferase activity .TransIT®-LT1 Transfection Reagent (Mirus Bio) according to the manufacturer’s instructions. After 48 h of incubation at 37°C and 5% CO2, the culture medium was exchanged with fresh growth medium, and luciferase activity was measured with the Dual-Glo® Luciferase Assay Kit (Promega) according to the manufacturer’s instructions using the Tecan Spark® plate reader. Integration time was 10 s. Data represent the mean ± standard deviation (SD) of six biological replicates, in technical triplicates. Statistical analysis was performed with the Wilcoxon rank test and Student's t-test in R.hTRMT2A WT (scrambled control) as well as KD1 and KD2 (scrambled control) were cultured (Gibco™ DMEM + 10% Gibco™ FBS + 1% Gibco™ Antibiotic-Antimycotic) and seeded at a density of 20 000 cells/well in a 96-well plate suitable for luminescence measurements (greiner bio-one #655075). Cells were transfected with Renilla and Firefly fusion constructs  using in vitro characterization of the TRMT2A binding preferences, we prepared recombinant human TRMT2A full-length  as a competitor to preclude non-specific binding. To explore how important the uridine in position 54 of a given tRNA is for binding, we included two tRNAs with U54 (tRNAGln and tRNAPhe) and two tRNAs lacking U54. The latter consisted of tRNAAla with A54 and a mutant version of tRNAGln where U54 had been changed into G (tRNAGln U54G). hTRMT2A FL bound all these tRNAs with apparent equilibrium dissociation constants (KD) in the low nanomolar range -RNA, the binding appeared only as a smear and not as a specific bandshift, indicative of a rather unspecific interaction .,37.Gln Ty Figure . Both ste Figure , which iin vitro stem–loop structured RNAs with modestly lower affinities than tRNAs. While binding affinities are in the expected range  were methylated orders of magnitude less efficiently   Figure . In EMSA) Figure  and henc) Figure . This inGln interaction surface with near-amino acid resolution, we performed NM- and UV radiation-induced cross-linking of the hTRMT2A–tRNAGln complex followed by MS. Experiments were conducted with the in vitro reconstituted protein–RNA complex . Stable complex formation was apparent as a shift of the elution peak to smaller elution volumes (from 17.41 ml to 15.46 ml), corresponding to a higher molecular weight , we consistently found in at least three of them the following amino acids to be cross-linked to RNA . The hTRMT2A CDM was used in comparative RNA binding and enzymatic activity experiments with hTRMT2A WT, the previously reported catalytic base mutant E581A (CBM) and the catalytic nucleophile mutant C538A (CNM)   (Supplem8A (CNM) . In EMSAs Figure . SurprisGln. The experiments indicated rather complex binding events possibly involving multiple interaction sites, which prevented precise determination of binding kinetics. However, in agreement with EMSAs, hTRMT2A WT exhibited high affinity binding with an apparent KD of 306 ± 60 nM, and hTRMT2A CDM showed a similar, albeit slightly lower affinity (apparent KD of 428 ± 71 nM)  Figure . These rs Figure .KM of 0.64 ± 0.06 μM when compared with hTRMT2A WT  . In this2 ratio > 2, –log10P-value > 3), 222 and 177 proteins were enriched in WT and CDM samples over BirA* background control, respectively. Both datasets shared a subset of 115 proteins. Since hTRMT2A WT and CDM displayed similar RNA binding, a large overlap of 115 proteins was expected and constitute candidates for interaction partners. GO term analyses for biological processes and cellular compartments revealed enrichment of rRNA processing and ribosome biogenesis for protein function and suggested a primary localization of the identified proteins in the nucleolus . This revealed enrichment of 74 and 72 proteins in hTRMT2A WT and CDM samples over FLAG background control, respectively  were generated using stable transfection of shRNA. Knockdowns were validated with western blotting and show a clear reduction of TRMT2A protein levels, with a higher reduction in KD1  and the identification of ribosomal proteins such as RPL4 or NCL as possible hTRMT2A interactors motivated us to explore a potential functional link between hTRMT2A-dependent methylation and rRNA processing/ribosome biogenesis. Previous cryoelectron microscopy (cryoEM) studies of the human ribosomes had revealed all chemical modifications in rRNAs . Two repnit rRNA . Moreovenit rRNA . Of noteiscussed . Thus, wn in KD1  . Single amino acid substitutions within the Renilla luciferase (E145Q or D120N) render the enzyme inactive. In a condition with increased translation error rate, the Renilla luciferase would stochastically be synthesized as a functional protein. The dual luciferase set up allowed us to control for protein expression and therefore measure mistranslation accurately.To test this hypothesis, we used a gain-of-function reporter previously described to detect small increases in mistranslation rates . The repThe reporter constructs were transfected into hTRMT2A WT and KD cells, and luminescence was measured. Indeed, for the E145Q Renilla mutation, both KD1 and KD2 cell lines showed significantly increased normalized luminescence ratios, which are in line with an increased mistranslation in hTRMT2A KD over WT. Of note, a higher increase in luminescence ratios was seen for the KD1 cell line, which displayed a stronger reduction of hTRMT2A levels than KD2 in western blot analysis . However5U modification in translation.In this study, we characterized the properties of the human tRNA methyltransferase TRMT2A with respect to RNA binding and catalysis. We also identified its RNA-binding interface. Furthermore, we provide the first unbiased assessment of the protein interaction network of hTRMT2A. These insights suggest novel implications of hTRMT2A and min vivo. In the same study, no further dissection of structural requirements, orthogonal assays or mutational studies of the sequence motif were performed. Thus, we chose positive hits from these experiments to complement available data with our orthogonal in vitro experiments. As an initial result, we observed that the binding affinity of hTRMT2A for tRNAs was in the expected range. The major parameter that increased methylation efficiency was the presence of a uridine positioned in the T-loop of the tRNA. However, in our experimental set-up, binding and methylation were not restricted to tRNA as exemplified by the unrelated ASH1 E3 stem–loop structured RNA containing a hTRMT2A-binding motif A , which sGln U54G and tRNAAla binding and methylation  and methylation (FICC-CLIP) of RNA . Here, in Figure . These ohaperone ,46.in vivo by masking of unspecific methylation sites by other factors or an increase of specificity in RNA binding by cofactors of TRMT2A. Further experiments will be required to clarify whether KCND2 binding and methylation are indeed a physiologically relevant event in vivo.Apart from the consistent results presented here, we reported unspecific binding and methylation of a KCND2 mRNA fragment . Hence, Gln interaction surface to a near-amino acid level. To better understand binding of hTRMT2A to tRNA, we used the AlphaFold homology model of hTRMT2A  and mapped the identified cross-links onto the model. Superposition of this homology model on the crystal structure of TrmA bound to a tRNA T-loop allowed us to confidently superpose a full tRNAPhe (PDB ID: 4TRA) into the homology model  were previously identified in orthogonal studies, which may serve as positive controls and indicate that our combinatory interactome study is robust and reliable . Proteing of SSB ,51. Therg of SSB .5U status of cytosolic rRNA isolated from hTRMT2A WT and hTRMT2A KD cell lines. We observed that rRNA from hTRMT2A WT and KD showed only a minimal fraction of m5U-modified uridines, which was similar to the background levels of modifications that were known to be absent in rRNA ] disease modifier. Reduced levels of hTRMT2A were associated with decreased poly(Q) aggregates and photoreceptor death  aggregation disorders (Together our isorders ,56, thisgkad565_Supplemental_FilesClick here for additional data file.


We show that chloroplast CLPB3 is required for resolving heat-induced protein aggregates containing stromal TRIGGER FACTOR (TIG1) and the small heat shock proteins 22E/F (HSP22E/F) in vivo, and for conferring thermotolerance under heat stress. Although CLPB3 accumulation is similar to that of stromal HSP70B under ambient conditions, we observed no prominent constitutive phenotypes. However, we found decreased accumulation of the PLASTID RIBOSOMAL PROTEIN L1 (PRPL1) and increased accumulation of the stromal protease DEG1C in the clpb3 mutants, suggesting that a reduction in chloroplast protein synthesis capacity and an increase in proteolytic capacity may compensate for loss of CLPB3 function. Under ambient conditions, CLPB3 was distributed throughout the chloroplast, but reorganized into stromal foci upon heat stress, which mostly disappeared during recovery. CLPB3 foci were localized next to HSP22E/F, which accumulated largely near the thylakoid membranes. This suggests a possible role for CLPB3 in disentangling protein aggregates from the thylakoid membrane system.In the cytosol of plant cells, heat-induced protein aggregates are resolved by the CASEIN LYTIC PROTEINASE/HEAT SHOCK PROTEIN 100 (CLP/HSP100) chaperone family member HSP101, which is essential for thermotolerance. For the chloroplast family member CLPB3 this is less clear, with controversial reports on its role in conferring thermotolerance. To shed light on this issue, we have characterized two  Chlamydomonas. Chloroplast CLPB3 resolves heat-induced protein aggregates by organizing into stromal foci near the thylakoid membranes, thereby inducing thermotolerance in  E. coli ClpB can initiate at the N- or C-termini or at internal sites of substrate proteins in protein aggregates, such that entire peptide loops are translocated through the pore (Deville The casein lytic proteinase/heat shock protein 100 (Clp/Hsp100) chaperones belong to the large family of AAA+ proteins  and mitochondrial ClpB (termed CLPB4 or ClpB-m) are both derived from cyanobacterial ClpB Lee . SuppresChlamydomonas reinhardtii has five CLPB genes  and mutant strains clpb3-1 (LMJ.RY0402.250132_1) and clpb3-2 (LMJ.RY0402.104257_1) from the Chlamydomonas Library Project (Chlamydomonas Resource Center (https://www.chlamycollection.org/). Cultures were grown mixotrophically in TRIS-ACETATE-PHOSPHATE (TAP) medium  or photometrically by optical density measurements at 750 nm (OD750). For heat stress experiments, cultures of exponentially growing cells were placed into a water bath heated to 41 °C if not stated otherwise, and incubated under agitation and constant illumination at ~40 μmol photons m–2 s–1 for 1 h, with subsequent recovery at 25 °C for 6 h. To determine survival rates via colony forming units, cultures of wild type and clpb3-1 were grown at 25 °C to a density of 1 × 106 cells ml–1 and split into equal parts. For each strain, one culture was left at 25 °C for 2 h while the other was incubated at 41 °C for 2 h in a water bath. After the treatment, cells were diluted to 1 cell µl–1 and 200 µl were plated out in triplicates on TAP agar plates in four independent experiments. For spot tests, cells were grown to a density of 3–5 × 106 cells ml–1 and diluted in TAP medium or high salt medium (HSM) such that 10 µl contained 104, 103 or 102 cells. Following this, 10 µl of each dilution were spotted onto agar plates containing TAP medium or HSM, and incubated in low light (30 µmol photons m–2 s–1) for 7 d, in high light (600 µmol photons m–2 s–1) for 4 d, in the dark for 7 d, or exposed to three ~24 h heat shock treatments at 40 °C with ≤24 h recovery in between. HSM was prepared according to d method  as descrChlamydomonas genomic DNA, 5 ml of exponentially growing cells were pelleted and resuspended in 250 μl water. Following this, 250 μl of 2× lysis buffer  and 3 μl proteinase K  were added and incubated under agitation at 55 °C for 2 h. The lysate was supplemented with 80.9 μl of 5 M NaCl and mixed by vortexing. After the addition of 70 μl of pre-warmed CTAB/NaCl , lysates were vortexed and incubated under agitation for 10 min at 65 °C. Nucleic acids were extracted by addition of 1 volume phenol: chloroform: isoamylalcohol , mixing the two phases and separating for 5 min at 18 000×g and 4 °C. Phenol/chloroform extraction of the aqueous phase was repeated once. An equal volume of chloroform: isoamylalcohol  was added to the upper phase, and the mixture was centrifuged as above. Recovery of nucleic acids was achieved by precipitating with an equal volume of isopropanol. Finally, the pellet was resuspended in TE-buffer , from which 1 ng of DNA was used for PCR. Validation of the aphVIII cassette insertion site within the genes of the Chlamydomonas Library Project (CLiP) mutant lines was performed using the specific primers listed in For the extraction of EcoRI and HindIII, and cloned into EcoRI-HindIII-digested pETDuet-1 vector  lacking two nucleotides upstream from the BamHI site, producing pMS976. CLPB3 was expressed with an N-terminal hexahistidine tag in E. coli Rosetta cells after induction with 1 mM IPTG for 16 h at 20 °C, and purified by cobalt-nitrilotriacetic acid affinity chromatography, according to the manufacturer’s instructions , including a washing step with 5 mM Mg-ATP. Eluted CLPB3 was gel filtrated using an Enrich SEC650 column. Fractions containing CLPB3 were pooled and concentrated in Amicon® Ultra-4 Centrifugal Filter Units , with a subsequent buffer exchange using 6 M Urea, 50 mM NaCl, 20 mM Tris-HCl, pH 7.5. Protein concentrations were determined via a NanoDrop 2000 . Proteins were frozen in liquid nitrogen and stored at −80 °C. From this, 2.6 mg of the protein were used for the immunization of a rabbit via the 3 month standard immunization protocol of Bioscience bj-diagnostik .The CLPB3 coding region lacking the chloroplast transit peptide was amplified by PCR from EST clone AV631848 , resulting in three level 0 constructs: L0-CLPB3-up containing a 1933 bp fragment (pMBS495), L0-CLPB3-down1 with a 1713 bp fragment (pMBS496), and L0-CLPB3-down2 with a 1010 bp fragment (pMBS497). All three level 0 constructs were combined with plasmids pCM0-020 (HSP70A/RBCS2 promoter + 5’UTR), pCM0-100 (3×HA), and pCM0-119 (RPL23 3’UTR) from the Chlamydomonas MoClo kit ]. Cells were sonicated on ice once for 10 s  and centrifuged for 10 min at 500 × g and 4 °C to remove intact cells and cell debris. Protein concentrations in the supernatant were measured by the Bradford assay , and resuspended in 500 μl ACA buffer . Cells were disrupted by sonication. Intact cells and cell debris were removed by centrifugation for 5 min at 300×g and 4 °C. Whole cell lysates  were solubilized for 20 min with 1% (w/v) β-dodecyl maltoside  on ice in the dark and insolubilized material was precipitated by centrifugation at 18 500×g for 10 min at 4 °C. Following this, supernatants were supplemented with native sample buffer [750 mM ε-aminocaproic acid and 5% (w/v) Coomassie Brilliant Blue G250] and separated on a 4–15% (w/v) blue-native (BN) polyacrylamide gel, followed by immunoblotting.Blue native (BN) PAGE with whole cell proteins was carried out according to published protocols  for at least 30 min at 25 °C. Slides were incubated over night at 4 °C with antisera against HSP22EF and the HA-tag using 1:1000 dilutions, in PBS-BSA. Slides were then washed five times with PBS-BSA at 25 °C followed by incubation in a 1:200 dilution of the tetramethylrhodamine-isothiocyanate-labelled goat anti-rabbit antibody  and fluorescein isothiocyanate-labelled goat anti-mouse antibody  in PBS-BSA for 1.5 h at 25 °C in the dark. Finally, the slides were washed five times with PBS and mounting solution containing 4’,6-diamidino-2-phenylindole  was dispersed over the cells. HSP22E/F and the HA-tag images were captured with a Zeiss LSM880 Axio Observer confocal laser scanning microscope equipped with a Zeiss C-Apochromat 40×/1.2 W AutoCorr M27 water-immersion objective . Fluorescent signals of FITC (excitation/emission 488 nm/491–589 nm) and TRITC (excitation/emission 633 nm/647–721 nm) were processed using the Zeiss software ZEN 2.3 or ImageJ. Light microscopy images were taken with an Olympus BX53 microscope.For immunofluorescence microscopy, cells were fixed and stained as described previously .Chlorophyll fluorescence was measured using a pulse amplitude-modulated Mini-PAM fluorometer  essentially according to the manufacturer’s protocol after 3 min of dark adaptation  of the total cell proteins.The  peptide . The matdine tag  and raisted site . We obseChlamydomonas, we ordered two clpb3 CLiP mutants (clpb3-1) and in intron 4  or to 85% of wild type  to understand whether or not their accumulation was affected by the reduced CLPB3 production in the mutants. Chloroplast chaperones CPN60A and HSP22E/F Bai , C. ProdRies Rütgers clpb3-2c , C. The -1c line , C. clpb3-2 mutant and clpb3-2c only under ambient conditions  or to 1 h 41 °C only (for clpb3-1c). Since we expected a co-localization of HSP22E/F and CLPB3 in aggregates, we employed mouse antibodies against the HA tag to detect transgenic CLPB3, and rabbit antibodies against HSP22E/F, on the same cells. In all cells, HSP22E/F was weakly detectable under ambient conditions, and gave rise to strong signals in the chloroplast after heat shock and recovery  and TIG1  versus the wild type  (We wondered whether the impaired ability of the  between . Under aP=0.025) . Hence, in vitro, and aggregates formed by aggregation-prone DXS in vivo  trl domain ; (iii) tl domain ; (iv) thal CLPB3 , 6; and 2 mutant . Since wt extent , 7, the l domain . Our datThe following supplementary data are available at E. coli and chloroplasts.Fig. S1. Alignment of amino acid sequences of CLPB proteins from E. coli.Fig. S2. Production of recombinant CLPB3 in CLPB3 gene by PCR.Fig. S3. Analysis of the CIB1 integration sites in the CLPB3 promoter.Fig. S4. Putative heat shock elements (HSEs) in the Chlamydomonas clpb3 mutants display no obvious phenotype regarding chloroplast development and PSII activity.Fig. S5. clpb3 mutant lines.Fig. S6. Screening for complemented Table S1. Primers used for cloning and genotyping.erad109_suppl_Supplementary_Table_S1_Figures_S1-S6Click here for additional data file.