B30.2) in two crystal forms at resolutions of 1.6 Å and 1.8 Å. TRIM7B30.2 exhibits the typical B30.2 domain fold, consisting of two antiparallel β-sheets of seven and six strands, arranged as a distorted β-sandwich. Furthermore, two long loops partially cover the concave face of the β-sandwich defined by the β-sheet of six strands, thus forming a positively charged cavity. We used sequence conservation and mutational analyses to provide evidence of a putative binding interface for GN1. These studies showed that Leu423, Ser499, and Cys501 of TRIM7B30.2 and the C-terminal 33 amino acids of GN1 are critical for this binding interaction. Molecular dynamics simulations also revealed that hydrogen bond and hydrophobic interactions play a major role in the stability of a modeled TRIM7B30.2-GN1 C-terminal peptide complex. These data provide useful information that could be used to target this interaction for the development of potential therapeutic agents.Tripartite motif (TRIM)7 is an E3 ubiquitin ligase that was first identified through its interaction with glycogenin-1 (GN1), the autoglucosyltransferase that initiates glycogen biosynthesis. A growing body of evidence indicates that TRIM7 plays an important role in cancer development, viral pathogenesis, and atherosclerosis and, thus, represents a potential therapeutic target. TRIM family proteins share a multidomain architecture with a conserved N-terminal TRIM and a variable C-terminal domain. Human TRIM7 contains the canonical TRIM motif and a B30.2 domain at the C terminus. To contribute to the understanding of the mechanism of action of TRIM7, we solved the X-ray crystal structure of its B30.2 domain (TRIM7 Tripartite motif (TRIM) proteins constitute a family of over 80 members encoded in the human genome  that parin vitro almost 4-fold , the autoglucosyltransferase responsible for the initiation of glycogen biosynthesis, and was therefore named GNIP1 (glycogen interacting protein isoform 1 ). The TRt 4-fold . TRIM7 hlization . Consistlization . Moreovelization . However6K1 axis . TRIM7 i6K1 axis . Through6K1 axis . Moreovexpressed . The relxpressed . Moreovexpressed  and the xpressed . In addixpressed .As these evidences suggest, TRIM7 is emerging as a promising therapeutic target. In this context, its B30.2 domain may be particularly important because specificity determinants of many TRIM proteins lie in this domain . Thus, tB30.2 from now on; the same convention will be used in all other B30.2 domains) has been solved in two different crystal forms (4N7I (A). Even though the packing of the molecules in the two crystal forms is different, the structure of the four independent molecules is very similar, showing backbone root mean square deviations (RMSD) smaller than 0.5 Å for all pairwise comparisons. The structure of chain A at the highest resolution  was used for the general description of TRIM7B30.2 and subsequent analysis.The structure of the human TRIM7 B30.2 domain  as a sems  and six  strands, arranged as a distorted β-sandwich (B and C). In addition, there is a short 310 helix (residues 428–430) located immediately before β6 strand. N- and C-terminal ends are close to each other in the seven-stranded β-sheet. This β-sheet (β-sheet B) forms a slightly convex surface at one side of the β-sandwich, whereas the six-stranded β-sheet (β-sheet A) forms a concave surface on the other side, which is partly covered by the two long loops that connect β2–β3 strands (loop 3) and β5 strand-310 helix (loop 6). The high structural similarity between the four molecules from the two crystal forms and the relatively low B-factor values for the residues of these loops suggest that their conformation is quite stable, most likely because of interactions (hydrogen bonds and to a lesser extent hydrophobic contacts) within and between one another and with residues from loops 1 and 12 and the β12 strand.TRIM7sandwich , B and CB30.2, the PRY domain encompasses residues 344 to 392 (β1-β3 strands), whereas the SPRY domain includes residues 396 to 509 (β4-β13 strands) (C).Sequence homology and evolutionary studies indicate that the B30.2 domain is a fusion of two subdomains, named PRY and SPRY, forming a single structural module. According to the Pfam database assignment, in TRIM7strands) C.B30.2 was BTN3A1B30.2 . Among human proteins, BTN3A1B30.2 also exhibits a high sequence similarity with TRIM7B30.2 showing 41.3% identity. The other top matches from the Dali server analysis were the B30.2 domains of mouse TRIM21 , which residues A. Among  = 1.3 Å ), human  = 1.7 Å ), human  = 1.9 Å ).Figure B30.2, which would be able to form dimers (B30.2 is monomeric both in solution on the basis of gel filtration chromatography (A). The structure of the full-length intracellular domain of BTN3A1 exhibits two potential dimer interfaces . Among the latter are Trp350, Tyr352, and Trp391  which would be particularly important for the interaction because they participate in stable contacts as revealed by MD simulations , the exB30.2 binding region by comparison with other B30.2 domain sequences and structures. Given the high structural homology between TRIM B30.2 domains, the conserved residues might be presumably important in determining the domains’ general conformation, whereas their different binding specificity would be related to differences in their sequences. In this respect, Song has described the existence of four regions in B30.2 domains that exhibit significant variations both in their length and in their amino acid composition  was performed using the program Clustal Omega (A and B). The analysis revealed that most of the conserved residues are clustered on one face of the β-sandwich comprising β-sheet B and loops 1, 2, 4, 10, and 11 (A). In contrast, the vast majority of the nonconserved residues map on the opposite side and are located on loops 3, 6, 7, 8, and 13 and the solvent-exposed region of β-sheet A. Invariant residues corresponding to β-strands 3, 5, 6, and 12 of β-sheet A are mostly hydrophobic, with their side chains buried at the interface between the β-sheets, and are covered by loop 6 and to a lesser extent by loop 3. Regions of high variability corresponding to six loops, most of them analogous to those found to be variable in TRIM7B30.2, have also been described as part of the binding interface in the structure of the TRIM21B30.2 in complex with IgG Fc . TRIM21 residues Asp355, Trp381, Trp383, and Phe450 are essential for IgG Fc binding . This loop is longer in TRIM21 and in addition to the mentioned amino acids contains the bulky residue Phe450; consequently, it constitutes a ridge that separates the two pockets that bind each of the two IgG Fc domains . This ridge is not present in TRIM7, and therefore, only the pocket centered on the nonconserved face, which is shallower than that of TRIM21, can be observed . All these sequence and localized structural divergences between both B30.2 domains suggest that the conformation of the TRIM7B30.2 binding partner and the characteristics of the TRIM7B30.2/partner interaction must be quite different from those observed in the TRIM21B30.2/IgG Fc complex.Because the main function of the B30.2 domains appears to be to mediate interactions with different targets, we sought to define the potential TRIM7position , 35. Usial Omega . The resal Omega . Conserval Omega , A and B, and 11 A. In conh IgG Fc . MoreoveM21B30.2 , A and BB30.2 pocket revealed that it is defined by amino acids from loops 3, 6, 8, and 13 and β-strands 5, 6, and 7, most of them variable residues. During the refinement process, electron density that fitted well to a malonate ion from the crystallization solution was observed in a positively charged cavity found on one side of the pocket, in all four TRIM7B30.2 molecules from both crystal forms (A and B). The most external carboxylate of malonate also forms a bidentate salt bridge with the guanidinium group of Arg487 from a symmetry-related protein molecule. In the fourth molecule, this carboxylate group does not interact with a symmetry-related molecule and consequently exhibits a different orientation. A malonate ion was also observed in a topologically equivalent pocket in BTN3A1B30.2  that constitute a highly positive patch on the pocket surface (A and C), which also interacts with hydroxy-methyl-butyl-pyrophosphonate (cHDMAPP) a synthetic analog of the phosphoantigen hydroxy-methyl-butyl-pyrophosphate (HMBPP)  which are crucial for the binding of acidic peptides  but at  surface , A and C (HMBPP) . In the B30.2 regions spanning residues 460 to 464, 472 to 476, 488 to 494, and 504 to 506, which are equivalent to the regions corresponding to residues 345 to 349 (L1), 357 to 361 (L2), 373 to 379 (L3), and 389 to 391 (L4) of TRIM7B30.2, respectively. The first three regions have approximately 40% sequence identity between both proteins . The first criterion for the choice of these residues was their variability according to the ConSurf server analysis described above (A and B and B30.2 to retinoic acid–inducible gene-I and its ubiquitination (B). Asn438 is equivalent to Trp383 of human TRIM21, which is essential for IgG Fc binding –intein and immobilized in chitin beads. As Figure 6B shows, TRIM7B30.2 Glu368Ala and Asn438Ala mutants binding to GN1 is similar to that of the WT domain. In contrast, the replacement of TRIM7B30.2 Ser499 by alanine abrogates the interaction with GN1. As a control, the clarified extracts of the bacteria overexpressing all the TRIM7B30.2 variants were also analyzed to confirm that they contained similar quantities of the proteins (B).WT TRIM7 protein . Pull-doproteins B.B30.2 crystal structure showed, the β-mercaptoethanol included in the purification and crystallization to prevent precipitation at a high concentration, reacted with three of its four cysteines without significantly affecting the structure as revealed by CD analysis -thiocysteine by the β-mercaptoethanol derivatization could cause the loss of interaction with GN1, a detail to be taken into account when seeking to obtain co-crystals with a binding partner. Based on these results, additional TRIM7B30.2 single mutants were generated by replacing three amino acids located in the vicinity of Ser449  with alanine. As in the case of the first mutants, they were analyzed by CD to verify that the mutations did not produce significant changes in the structure of TRIM7B30.2  had no significant effect on the interaction with GN1. All these results not only indicate that Leu423, Ser499, and Cys501 participate in the interaction with GN1 but also confirm that the binding site is located in the concave face of TRIM7B30.2.As the TRIM7analysis . Given iIM7B30.2 . These nB30.2, C-terminal truncated versions of the enzyme lacking the last 63 (GN1ΔC63) and 33 amino acids (GN1ΔC33) (A) and fused to CBD–intein were tested for their ability to bind TRIM7B30.2 in pull-down experiments. As shown in Figure 7B, the deletion of the last 33 amino acids of GN1 results in the loss of interaction. More importantly, the same result was obtained when pull-down assays were performed using full-length TRIM7 (C). These results indicate that the region of interaction of GN1 with TRIM7 lies within residues 301 to 333.To define the region of GN1 responsible for its interaction with TRIM7GN1ΔC33) A and fusth TRIM7 C. These B30.2 and GN1, we combined protein–protein docking and all-atom MD simulations. As mentioned, our results indicate that at least some of the last 33 residues of GN1 are critical for the interaction with TRIM7B30.2. However, the three-dimensional structure of full length GN1 (333 amino acids long) has not yet been solved by any experimental technique, and most of the available structures of the enzyme correspond to a 31 kDa truncated form lacking the last 71 amino acids. The only known crystal structure of the C-terminal domain is that of a peptide comprising the last 36 amino acids of Caenorhabditis elegans glycogenin in complex with glycogen synthase  that was used to predict the structure of a complex with TRIM7B30.2 using HDOCK algorithm for protein–protein docking .To get insights into the stability of the complex between TRIM7 ID 4QLB ). The ab docking  . The α-helix composed of residues 299 to 311 (α-helix A) achieved convergence after 350 ns, while the other α-helix (α-helix B), formed by the last 12 residues, converged to a position that is close to that of the docking result after 100 ns, with an average RMSD value of approximately 3 Å (B). In this case, a peak in RMSD was observed in the interval from 200 ns to 350 ns, attributed to a transient conformational change of the last 2 residues of GN1-Cter, that then remained stable for the rest of the simulation. A comparison between the converged (snapshot at 700 ns) and the starting conformation (docking result) of GN1-Cter shows that α-helix B undergoes a slight change in its original position with respect to TRIM7B30.2, whereas α-helix A experiences a major conformational change before stabilization occurs (A). To identify the most important interactions between GN1-Cter and TRIM7B30.2, a contact map was calculated using the converged structure of the complex as a reference. Figure 8C shows the contact pattern  mapped onto TRIM7B30.2 surface using a representative coloring scheme. TRIM7B30.2 residues displaying the highest contact time values are Cys501, Leu423, and Arg354, whereas in GN1-Cter, the interactions mainly involve Trp308, Asp319, Phe321, Ile324, Lys327, Leu328, and Tyr331 (D). GN1-Cter Phe321 locates inside the TRIM7B30.2 central pocket interacting with Cys501 and, to a lesser extent, with Ala410, Asn436 and Gln438. This result is more clearly seen in the analysis of the distances between the side chains of these TRIM7B30.2 residues with GN1-Cter Phe321 (A). In the crystal structure, TRIM7B30.2 Leu423 is highly exposed to the solvent, with a solvent accessible surface area (SASA) of approximately 270 Å2. In the complex with GN1-Cter, the SASA value of Leu423 decreases to about 50 Å2, and during MD simulations, this value finally converges to approximately 20 Å2 (B). The reduction of the SASA value suggests that Leu423 participates in the complex formation, and a detailed analysis of the structure reveals that it is surrounded by Ile324, Lys327, Leu328, and Tyr331 of GN1-Cter (D). All of these residues remain mostly at about 4 Å from Leu423 during the simulation, with the exception of Tyr331 (C). However, the most important interactions seem to involve Arg354 of TRIM7B30.2. This residue establishes a cation-π interaction with Trp308 of GN1-Cter from 200 ns and stable hydrogen bonding interactions with Asp319 after 100 ns of MD simulation (D). The analysis of the distance to Trp308 is consistent with the decrease in the SASA of Arg354 (B). The hydrogen bond interaction with Asp319 is the strongest interaction established with GN1-Cter, because it exhibits the highest occurrence frequency (95% of the simulation time) among all the observed contacts (D). Other important hydrogen bonding interactions involve Arg369 and Asp407 of TRIM7B30.2 and Glu326 and Tyr332 of GN1-Cter, respectively, with approximately 40% occurrence (not shown).Using the complex TRIM7y a loop A. The α-tely 3 Å B. In thin occurs A. To ided Tyr331 D. GN1-Ctr Phe321 A. In thely 20 Å2 B. The reGN1-Cter D. All off Tyr331 C. Howevemulation D. The anf Arg354 B. The hycontacts D. Other Members of the TRIM family of proteins are involved in a great variety of cellular processes. In particular, TRIM7 was initially identified through its interaction with GN1, the protein that initiates glycogen biosynthesis, hence its name GNIP. In addition to enhancing the activity of GN1, TRIM7 has E3 ubiquitin ligase activity , and thrB30.2 also participates in the interaction with other proteins and in this way mediates the different effects related to TRIM7. Among these proteins are probably RACO-1 , and an30.2/IgG , TRIM65B0.2/MDA5 , BTN3A1/0.2/MDA5 , and Ash0.2/MDA5 ).B30.2, one of the known binding partners is GN1, and therefore, this protein was used in the binding assays with mutants of TRIM7B30.2 to define potentially important residues for the interaction. This study revealed that mutations Leu423Ala and Ser499A interfered with the interaction between the proteins, whereas the substitution of Arg354 produced an increase in binding affinity. The replacement of the nearby Cys501 by a serine, which is modified by β-mercaptoethanol in the crystal structure, had a similar effect to that observed in Leu423Ala and Ser499Ala on the binding to GN1, indicating that it is also a critical residue for the interaction. Together, these data suggest that the interaction with the enzyme involves the concave surface of TRIM7B30.2 and that the pocket in which malonate is bound (B) could play an important role in such interaction. This pocket would be a good candidate as a binding site for inhibitors of the interaction with GN1 and perhaps with other substrates as well. Moreover, the presence of Cys501 could even be exploited for the binding of cysteine-targeted irreversible inhibitors.In the case of TRIM7is bound B could pB30.2. This portion of the protein is also involved in the interaction with glycogen synthase  participate in the interaction with GN1-Cter. As expected, the positive pocket mentioned above is involved in the binding, and in this region, the most important interaction is the thiol-π-type interaction between Cys501 and Phe321 of GN1-Cter. Leu423 forms hydrophobic interactions with several amino acids, whereas Arg354 interacts strongly with Asp319 and Trp308 of GN1-Cter. If this were the case, the enhanced binding to GN1 when Arg354 is replaced by alanine could be explained by the appearance of new interactions in the absence of this bulky residue and to the formation of hydrophobic interactions involving alanine, for example, with Trp308. On the other hand, the side chain of Ser499 forms a strong hydrogen bond with the main chain amide nitrogen of Thr384, which is also located within the central pocket of TRIM7B30.2. Therefore, the replacement of Ser499 by an alanine could alter the structure of the pocket and, consequently, hinder the hydrophobic interactions between GN1-Cter and TRIM7B30.2.Our findings indicate that the final 33 amino acids of GN1 are important for the binding to TRIM7synthase  from whisynthase , which sAs mentioned before, TRIM7 is also able to interact with RACO-1 and more specifically with its C-terminal end. However, the region of TRIM7 involved in this interaction is unknown. Interestingly, the sequence alignment of the C-terminal ends of GN1 and RACO-1 revealed that they exhibit 18.4% identity and 68.5% similarity . This, tB30.2 and the information related to its interaction with target proteins could be relevant for the development of potential therapeutic agents.During protein ubiquitination, E3 ubiquitin ligases have the crucial role of determining the substrate specificity of the reaction. For this reason, together with the fact that they are implicated in a wide variety of human diseases, they have become attractive therapeutic targets. Whereas the E3 ubiquitin ligase function of TRIM7 and other TRIM proteins depends on the presence of the RING domain, which interacts with the E2 ubiquitin-conjugating enzyme, the specificity of the ubiquitination reaction is determined by the C-terminal domain (B30.2 domain in the case of TRIM7). Modulators of these enzymes targeting the B30.2 domain are therefore expected to have more selectivity and less side-effects. In this context, because TRIM7 is involved in carcinogenesis and viral pathogenesis, the three-dimensional structure of TRIM7Escherichia coli strain Rosetta(DE3) and vector pET-15b were purchased from Novagen. pTYB11 vector and chitin agarose beads were obtained from New England Biolabs. The codon optimized cDNA encoding human TRIM7 was synthesized by GenScript (GenScript Inc). Protease Inhibitor Mix, HisTrap HP, and Superdex 200 10/300 GL columns were from GE Healthcare. Primary anti-His6 tag antibody was from Invitrogen, and dye-labeled secondary antibody was from LI-COR. Except where indicated, all other reagents were from Sigma-Aldrich.B30.2 (amino acids 338–511) was subcloned into the pET-15b vector downstream of the His6-tag encoding sequence of the plasmid. The resulting construct was used as template to generate the mutants TRIM7B30.2 Arg354Ala, TRIM7B30.2 Glu368Ala, TRIM7B30.2 Thr384Ala, TRIM7B30.2 Leu423Ala, TRIM7B30.2 Asn438Ala, TRIM7B30.2 Ser499Ala, and TRIM7B30.2 Cys501Ser by site-directed mutagenesis utilizing the QuickChange site-directed mutagenesis kit (Agilent Technologies). The cDNA of human GN1 was also subcloned in pET-15b to obtain the N-terminal His6-tagged protein. The cDNA of full-length human TRIM7 was subcloned into the pGEX-4T-1 downstream of the GST encoding sequence of the plasmid.The cDNA of human TRIM76-human GN1, His6-human TRIM7B30.2, and its mutants were expressed in the E. coli Rosetta(DE3) strain. Protein expression was induced at OD600 0.6 to 1.0 overnight with 0.15 mM isopropyl β-D-thiogalactopyranoside at 18 °C. The proteins were purified from clarified cell lysates by affinity chromatography on HisTrap HP columns (1 ml), and TRIM7B30.2 was further subjected to gel-filtration chromatography on a Superdex 200 column in 20 mM Tris-HCl buffer pH 7.5, 150 mM NaCl, and 10 mM β-mercaptoethanol. Fractions containing the protein of interest were pooled and desalted with 20 mM Tris-HCl buffer pH 7.5, 10 mM β-mercaptoethanol. Protein concentration was determined based on calculated extinction coefficients at 280 nm of 56,380 M−1 cm−1 and 33,460 M−1 cm−1 for GN1 and TRIM7B30.2, respectively.HisB30.2 was concentrated to 5 mg/ml in 20 mM Tris-HCl buffer pH 7.5, 10 mM β-mercaptoethanol, and 1 mM tris (2-carboxyethyl) phosphine for the crystallization setup. Initial crystallization trials were performed by the hanging-drop vapor diffusion method at 10 °C using commercially available crystallization screens from Qiagen . Small crystals were observed within a week in 1.4 M sodium malonate pH 6.0. During optimization, larger crystals were obtained in the same condition but using the sitting-drop vapor diffusion method and by mixing equal volumes of the protein solution at 10 mg/ml and 1.4 M sodium malonate pH 6.0 supplemented with glycerol 3% using the hanging-drop vapor diffusion method.Purified TRIM7Crystals were cryoprotected by soaking in a solution containing 85% mother liquor and 15% glycerol before being flash-frozen. Data were collected at the Macromolecular Crystallography beamline MX2 (wavelength 1.4586 Å) of the Laboratório Nacional de Luz Síncrotron  at 100 K.4N7I, chain A) as a search model was done using PHASER (http://www.pymol.org), and electrostatic surface calculations were performed using the APBS plugin for PyMOL  were collected using a JASCO J810 spectropolarimeter (Jasco) in 0.2 cm path-length quartz cuvettes. The spectra represent the average of five scans recorded in the far-ultraviolet region (190–260 nm) with a bandwidth of 2.0 nm, a step size of 0.2 nm, and a response time of 8 s. The measurements were corrected by subtracting signals from the buffer control.CD spectra of TRIM7E. coli Rosetta(DE3) strain and immobilized on chitin beads from the bacterial clarified lysate followed by three washes with phosphate-buffered saline (PBS buffer). Full-length TRIM7, TRIM7B30.2, and all the mutants  were obtained in the same E. coli strain. Even though their expression levels were very similar, to ensure the use of equivalent amounts of all the TRIM7B30.2 variants, the clarified extracts were analyzed by immunoblot with an anti-His6 tag antibody, and the relative intensity of the bands was quantified using ImageJ software. Twenty microliter of the chitin beads carrying similar molar amounts of intein or intein–GN1 variants were incubated at 4 °C for 2 h with 50 μl of the extracts  prepared in PBS buffer supplemented with a protease inhibitor mix. The beads were subsequently washed three times with PBS buffer, and the proteins were eluted by boiling at 100 °C for 5 min with SDS-PAGE sample buffer. Eluted TRIM7B30.2 variants were detected by immunoblot analysis on a polyvinylidene fluoride membrane with a primary anti-His6 tag antibody and IRDye 800 goat anti-mouse IgG secondary antibody, using the Odyssey Infrared Imaging System (LI-COR). In the case of the pull-down assay using TRIM7, the chitin beads carrying similar molar amounts of intein or intein–GN1 variants were incubated at 4 °C for 2 h with 50 μl of the extract prepared in 20 mM Tris-HCl buffer pH 7.5, 150 mM NaCl, 0.5% Triton X-100, 100 μM PMSF, 5 mM β-mercaptoethanol, and 10% glycerol  were obtained by introducing a stop codon in the corresponding position by site-directed mutagenesis. Intein used as control was prepared from the empty plasmid. Intein and the different intein–GN1 variants were expressed in the glycerol  and washC. elegans glycogenin C-terminal peptide (residues 394–428) in complex with glycogen synthase  and the modeled C-terminal peptide of human GN1 was predicted using HDOCK docking algorithm (http://hdock.phys.hust.edu.cn/).Human GN1 C-terminal peptide structure was modeled with MODELLER , using a chain E ). The thB30.2 and GN1 C-terminal peptide was placed into a truncated octahedral box of TIP3P water molecules, with a distance of 15 Å between the border of the box and the closest atom of the solute. The total system is composed of 207 protein residues, with 11,707 water molecules, giving a total of 38,403 atoms. The AMBER14SB force field was chosen to assign the parameters of all protein residues. All the systems were optimized with an energy minimization step consisting of 10,000 cycles using the steepest descent algorithm and 10,000 cycles with conjugate gradient minimization. The temperature was increased from 0 to 10 K in a 10 ps constant volume MD with a 0.1 fs time step, and a harmonic restraint potential of 10 kcal mol−1 Å−2 applied over all protein residues in the complex. Thereafter, the temperature was increased from 10 to 300 K in a 50 ps constant volume MD with a 0.5 fs time step, applying a force constant of 5 kcal mol−1 Å−2 to the protein backbone atoms. After the samples had been heated, the density was equilibrated with a 100 ps MD simulation at constant temperature and pressure with a time step of 1 ps and applying a force constant of 1 kcal mol−1 Å−2 to the protein backbone atoms. To control the temperature, a Langevin thermostat was used, whereas a Berendsen barostat was chosen to adjust the pressure to 1 bar (both regulated every 1 ps). For production MD, 750 ns simulations in the NTP ensemble were conducted, with a time step of 2 fs. All the simulations were performed under periodic boundary conditions , cpptraj (The complex between TRIM7nditions  using thnditions  to keep  cpptraj  from AMB cpptraj .The atomic coordinates and structure factors have been deposited in the Protein Data Bank under accession numbers 6UMA (1.6 Å resolution structure) and 6UMB (1.8 Å resolution structure). All remaining data are available in the main text and the supporting information or from the corresponding author upon request.This article contains The authors declare that they have no conflicts of interest with the contents of this article.


The crystal structure of CYP105D18 and its unique structural features for papaverine N-oxidation are presented. 2O2-mediated CYP105D18 from Streptomyces laurentii as the first bacterial CYP for N-oxidation. The catalytic efficiency of CYP105D18 for papaverine N-oxidation was 1.43 s−1 µM−1. The heme oxidation rate (k) was low (<0.3 min−1) in the presence of 200 mM H2O2. This high H2O2 tolerance capacity of CYP105D18 led to higher turnover prior to heme oxidation. Additionally, the high-resolution papaverine complexed structure and substrate-free structure of CYP105D18 were determined. Structural analysis and activity assay results revealed that CYP105D18 had a strong substrate preference for papaverine because of its bendable structure. These findings establish a basis for biotechnological applications of CYP105D18 in the pharmaceutical and medicinal industries.The bacterial CYP105 family is involved in secondary metabolite biosynthetic pathways and plays essential roles in the biotransformation of xenobiotics. This study investigates the newly identified H These enzymes catalyze diverse reactions, including hydroxylation, alcohol and carbonyl oxidation, epoxidation, demethylation, N-oxidation, ring cleavage, coupling, and formation of a vast number of endogenous and exogenous compounds, such as steroids, fatty acids, xenobiotics and other drugs  and organic surrogates such as cumene hydroperoxide or iodosylbenzene for efficient catalysis of different substrates  enzymes comprise a large superfamily of heme 2O2 has been demonstrated for many human CYPs and bacterial CYPs. Human CYP1A2 supports H2O2 shunting for efficient oxidation of aromatic amines  to 1,25-dihydroxy vitamin D3, a hormonally active form, via two sequential hydroxylations . CYP105D1 was well characterized for biotransformation of isoquinoline alkaloids . CYP105D18 was cloned, overexpressed and purified. The theoretical molecular weight was 44.3 kDa, and a single band at ∼50 kDa (with a His-tag from pET28) was obtained by sodium dodecyl sulfate-polyacrylamide gel electrophoresis [Fig. 1a)]. Purified CYP105D18 showed a Soret peak at 418 nm in an oxidized form. The carbon-monoxide-bound and dithionite-reduced form of CYP105D18 exhibited maximum absorption at a wavelength of 449 nm, one of the important spectral characteristics of CYP450 [Fig. 1b)]. The purity of a CYP can be evaluated from its zR value, calculated as the ratio of absorbance (A) at λmax of the Soret band to the A value at 280 nm. The purified enzyme had an zR value of 1.45, indicating high purity . CYP105D18 supported the N-oxidation of papaverine and hydroxylation of tetrahydropalmatine using the oxygen surrogate H2O2. High-performance liquid chromatography (HPLC) analysis of papaverine revealed product formation at a retention time of 18.2 min, equivalent to hydroxylation/N-oxidation in liquid chromatography-mass spectrometry (LC-MS) analysis [Fig. 2a)]. The HPLC results showed an additional peak at a retention time of 22.2 min, which could not be identified because of the poor degree of ionization by LC-electrospray ionization-MS. The major product was purified and characterized by nuclear magnetic resonance analysis as papaverine N-oxide . Similarly, the tetrahydropalmatine product catalyzed by CYP105D18 in the presence of H2O2 showed a single peak at a retention time of 13.5 min in the HPLC analysis [Fig. 2b)]. The LC-MS data revealed the mono-hydroxylation  of tetrahydropalmatine. The maximum product conversion for tetrahydropalmatine under optimum conditions was less than 12%; therefore, we did not purify the product for structural analysis. The results showed no support from H2O2 as an oxygen surrogate partner for biotransformation of berberine and palmatine by CYP105D18.Phylogenetic tree analysis of CYP105D18 revealed that it had the closest relationships with functionally characterized CYP105D7 (71.1%) and CYP105D1 . Even though THP was catalyzed by CYP105D18 using H2O2 as co-substrate, there was no spin-shift observed. The binding of 4-(pyridine-3-yl) benzoic acid and 4-(pyridine-2-yl) benzoic acid to CYP199A4 exhibits type II UV–vis spectral changes showing a greater shift in the Soret wavelength (422 versus 424 nm) . Unexpectedly, the rate constant ‘k’ was low, even in the presence of higher concentrations of H2O2, suggesting the high tolerance capacity of CYP105D18 for H2O2 –3(g). The well established P450 peroxygenase P450s SPα (CYP152B1 from Sphingomonas paucimobilis), BSβ (CYP152A1 from Bacillus subtilis) and OleT  have been reported to replace the acid–alcohol amino acid pair, which is used for proton relay to iron–oxo species to facilitate the catalytic cycle  have also been reported to utilize H2O2. The steroid hydroxylase CYP154C8 surprisingly functioned in the presence of a high concentration of H2O2, as reported in our previous work . A product conversion rate of 70% was observed with 5 mM H2O2. The product conversion was more than 95% between 40 and 200 mM H2O2. The heme dissociation constant was less than 0.014 min−1, and product conversion was greater than 97% using 40 mM H2O2 within 30 min. Therefore, 40 mM H2O2 was used as the optimum concentration to evaluate the enzyme kinetics. The product conversion decreased sharply when the the H2O2 concentration was greater than 200 mM, and more than 10% conversion was observed in the presence of 800 mM H2O2.The heme oxidation rate in vitro conversion of papaverine N-oxide by CYP105D18 is shown in Fig. 4a). The Km and kcat values for papaverine were estimated to be 213 ± 26 µM and 5 ± 0.3 min−1, respectively [Fig. 4b)]. Similarly, Km and kcat values for tetrahydropalmatine were 75 ± 15 µM and 0.13 ± 0.01 min−1, respectively [Fig. 4c)]. The catalytic efficiency (kcat/Km) for papaverine was much higher (1.43 s−1 µM−1) in comparison with tetrahydropalmatine (0.11 s −1 µM−1).Time-dependent 2.3.C2 diffracting to 1.7 Å. A final model was obtained with R factor and Rfree values of 0.16 and 0.21, respectively, and a Molprobity score of 1.48 ]. The asymmetric unit had one molecule of CYP105D18. Consistent with this observation, size exclusion chromatography analysis of CYP105D18 revealed that the protein existed as a monomer in solution . The coordinates were deposited in the Protein Data Bank under entry 7di3 (substrate-free structure). Data collection and refinement statistics are summarized in Table S4 of the supporting information.Structural investigation of CYP105D18 was conducted to obtain detailed structural information related to the active site. Crystallization experiments for full-length CYP105D18 yielded crystals in the space group a)]. A long αI was located in the middle between the two units and connected two units. CYP105D18 contained a heme surrounded by loops from the β-sheet-containing unit and αC, αI and αL from the helix-rich unit. The last C-terminus long loop starting from Lue366 covered the concave active site. As shown in the phylogenetic tree, CYP105D18 had the highest homology with the CYP105D subfamily, including CYP105D4, CYP105D7, CYP105D8 and CYP105D1. Consistent with this, structural similarities generated from the DALI server  showed high similarity with structures of CYP105D7 and CYP105D6 (PetD) from S. avermitilis ], indicating that these regions are essential for substrate selectivity for the CYP family. A previous study of CYP105D7 indicated that the arginine residues  in the B/C loop and αG of the substrate-binding pocket are conserved in the CYP105 family and may be important residues for substrate recognition  to support papaverine N-oxidation and understand the substrate-binding mode and structural changes that occur upon substrate binding. The papaverine complex structure was superimposed with the substrate-free structure and showed an overall Cα relative mean squared deviation of 0.196 Å. Structural comparisons between the substrate-free and complex forms of CYP105D18s revealed that conformation changes occurred mainly in the B/C loop region. A flexible, less structured loop was observed in the B/C loop, and electron density for residues 73–84 was absent from the substrate-free structure, indicating that this region had a high degree of flexibility. However, the papaverine complex structure had a well defined electron density for the B/C loop and formed a short helix named αB′, which has been previously observed in the diclofenac complex CYP105D7 structure , whereas the electron density for Phe68 and Pro69 showed a sharp and a good fit with a low B-factor . Taken together, these findings show that the B/C loop in the active site of the CYP105D18 structure was flexible and remained in an open confirmation without substrate. Once the substrate bound to CYP105D18, the B/C loop formed a helix (αB′) with a closed conformation. This region is thought to be responsible for substrate binding and specificity ]. The dimethodyisoquinoline group of papaverine was closely situated in the center of the heme, and dimethodyisoquinoline nitrogen was located toward the iron of the heme group. An electron-density blob corresponding to a water molecule between the isoquinoline nitrogen and heme iron was found . The distance between the water and nitrogen was 3.2 Å, and that between the water and iron was 2.1 Å, indicating that the binding mode represented the true mode for N-oxidation (Fig. 8Papaverine was buried within the internal hydrophobic cavity of CYP105D18 and was surrounded by residues Ala85, Leu87, Val231, Ala282, Val234 and Ile386, with no hydrophilic residues [Fig. 7on Fig. 8. The dimTo further support this observation, a molecular docking simulation was conducted with bendable substrates and non-bendable substrates. First, we used the papaverine model to check the reliability of the docking simulation. The simulated papaverine binds to the substrate-binding pocket of CYP105D18 with a similar binding mode to that of the co-crystallized structure Fig. 9. The sim3.2O2-mediated N-oxidation of papaverine by this CYP and its high tolerance to H2O2 for papaverine biotransformation. In addition, we investigated the crystal structure of CYP105D18, which suggested that high structural similarity is shared by the CYP105 family. Structural comparison of substrate-free CYP105D18 with a substrate-bound structure revealed high flexibility at the active site entrance comprising the B/C loop. In a papaverine-bound structure, papaverine adopted a bent structure that enabled correct alignment with the heme molecule for the N-oxidation reaction of papaverine. This finding explains why CYP105D18 had strong papaverine activity but weak or no activity for other isoquinoline alkaloids with non-bendable structures in our data. Taken together, our findings showed that this novel CYP exhibited high tolerance for H2O2 and unique substrate specificity, warranting studies of substantial drug modifications for biotechnological applications.In this study, we reported a novel cytochrome P450 enzyme annotated as CYP105D18. We observed H4.supporting information: Bhattarai et al. (2013et al. (2018et al. (2004et al. (2010et al. (2011The following references are cited in the  al. 2013; DeLano  al. 2013; Kumar e al. 2018; Omura & al. 2018; Purdy e al. 2004; Roy et  al. 2010; Winn et al. 2011; Zhang ( al. 2011.10.1107/S2052252521005522/jt5056sup1.pdfExperimental procedures, tables and figures. DOI: CYP105D18 complex with papaverine, 7dlsPDB reference: apo-structure of CYP105D18, 7di3PDB reference:


Long non-coding RNAs (lncRNAs) are important epigenetic regulators, which play critical roles in diverse physiological and pathological processes. However, the regulatory mechanism of lncRNAs in lung carcinogenesis remains elusive. Here, we characterized a novel oncogenic lncRNA, designated as Lung Cancer Associated Transcript 3 (LCAT3).We predicted and validated LCAT3 by analyzing RNA-sequencing (RNA-seq) data of lung cancer tissues from TCGA. Methylated RNA immunoprecipitation was performed to assess m6A modification on LCAT3. The LCAT3-FUBP1-MYC axis was assessed by dual-luciferase reporter, RNA immunoprecipitation and Chromatin immunoprecipitation assays. Signaling pathways altered by LCAT3 knockdown were identified using RNA-seq. Furthermore, the mechanism of LCAT3 was investigated using loss-of-function and gain-of-function assays in vivo and in vitro.LCAT3 was found to be up-regulated in lung adenocarcinomas (LUAD), and its over-expression was associated with the poor prognosis of LUAD patients. LCAT3 upregulation is attributable to N6-methyladenosine (m6A) modification mediated by methyltransferase like 3 (METTL3), leading to LCAT3 stabilization. Biologically, loss-of-function assays revealed that LCAT3 knockdown significantly suppressed lung cancer cell proliferation, migration and invasion in vitro, and inhibited tumor growth and metastasis in vivo. LCAT3 knockdown induced cell cycle arrest at the G1 phase. Mechanistically, LCAT3 recruited Far Upstream Element Binding Protein 1 (FUBP1) to the MYC far-upstream element (FUSE) sequence, thereby activating MYC transcription to promote proliferation, survival, invasion and metastasis of lung cancer cells.Taken together, we identified and characterized LCAT3 as a novel oncogenic lncRNA in the lung, and validated the LCAT3-FUBP1-MYC axis as a potential therapeutic target for LUAD.The online version contains supplementary material available at 10.1186/s13045-021-01123-0. Lung cancer is the most commonly diagnosed cancer and the leading cause of cancer-related death worldwide . It is cLong non-coding RNAs (lncRNAs) are a subclass of transcripts that are longer than 200 nucleotides and that have limited or no protein coding potential. In recent years, lncRNAs are emerging as important regulators in gene expression networks via their control of nuclear architecture and transcription in the nucleus, and via their modulation of mRNA stability, translation and post-translational modifications in the cytoplasm –8. SeverIn the present study, we reported the identification of a novel lncRNA, designated as Lung Cancer Associated Transcript 3 (LCAT3), that is remarkably upregulated in lung cancer tissues with positive association of poor prognosis. LCAT3 upregulation is attributable to N6-methyladenosine (m6A) modification mediated by methyltransferase like 3 (METTL3), leading to LCAT3 stabilization. Functional assays demonstrated that LCAT3 promotes proliferation, survival, migration and metastasis of lung cancer cells both in vitro cell culture and in vivo xenograft models. Mechanistic analysis reveals that LCAT3 interacted with Far Upstream Element Binding Protein 1 (FUBP1), leading to transactivation of c-MYC expression. Taken together, our results demonstrate that LCAT3 plays a pivotal role in enhancing malignant phenotypes of lung cancer cells, which may serve as an attractive therapeutic target for lung cancer.2 and at 37 °C.The human LUAD cell lines  and a human embryonic kidney cell line (HEK-293T) were purchased from the American Type Culture Collection . A549, Calu1 and Hop62 cells were cultured in Roswell Park Memorial Institute 1640 Media . HEK-293T cells were cultured in Dulbecco's Modified Eagle Medium . All medium was supplemented with 10% fetal bovine serum  and 1% penicillin/streptomycin (Gibco). All cell lines were cultured in a humidified atmosphere with 5% CO−△△CT method.Total RNA from cell culture or tissue samples was isolated using TRIzol reagent  following the manufacturer’s instructions. 1 µg of total RNA was reverse transcribed using HiScript II Q RT SuperMix for qPCR (+gDNA wiper) . Real-time PCR was performed on the cDNA using ChamQ Universal SYBR qPCR Master Mix (Vazyme). β-Actin was used as an endogenous control for normalization. All primer sequences are listed in Additional file A PARIS Kit  was used to isolate the cytoplasmic and nuclear fraction following the manufacturer's protocol. Briefly, cells were lysed using a cell fractionation buffer and incubated on ice for 10 min. By centrifuging at 500 g for 5 min, the supernatant was collected as the cytoplasmic fraction, and the remaining nuclear pellets were lysed with cell disruption buffer as the nuclear fraction. The cytoplasmic and nuclear RNA was then extracted and analyzed by qRT-PCR.RACE PCR products were obtained using a SMARTer RACE 5′/3′ Kit  and separated on a 1.2% agarose gel. The gel products were extracted using a gel extraction kit , cloned into a T5-Zero vector  and sequenced. The specific 3′ RACE and 5′ RACE primers are listed in Additional file http://crispr.mit.edu/) and cloned into lenti-gRNA-puro. The lentivirus was collected 48 h after co-transfection of the lenti-gRNA, pCMV-vsvg, and pSPAX2 vector into HEK293T cells using Lipofectamine 3000 transfection reagent. Calu1 cells were infected with filtered lentivirus plus 8 μg/mL polybrene  for 24 h and then treated with 5 μg/mL puromycin  for more than 7 days to obtain stably transfected cells. Sequences of siRNAs, shRNAs, sgRNAs and primers used in the plasmid construction are shown in Additional file For knockdown of LCAT3 and FUBP1, specific small interference RNAs (siRNAs) synthesized from GenePharma  were transfected into cells with GeneMute™ reagent  at a final concentration of 50 nM. To stably knockdown LCAT3 in lung cancer cells, two small hairpin RNAs (shRNAs) were synthesized and cloned into a lentiviral vector pLKO.1. For the overexpression assay, a full-length human LCAT3 sequence was obtained by PCR and subcloned into the pCDNA 3.1 vector by Lipofectamine 3000 (Invitrogen) to establish cells that stably overexpressed LCAT3. For the METTL3 knockout assay, two single guide RNAs (sgRNAs) were designed using the guide design tool (https://imagej.net/).The cell proliferation assay was performed using a cell counting kit-8 . First, cells were plated at a density of 1000 cells/well in 96-well plates containing 100 µL of culture medium. The OD450 was measured for 2 h after adding 10 µL of CCK8 solution. For the colony-formation assay, cells were seeded onto a 12-well plate at a density of 1000 cells/well. After one week of culture, cells were treated with methanol and stained with 0.1% crystal violet. The number of visible colonies was counted by ImageJ software  following the manufacturer’s instructions. Briefly, 5000 cells were seeded into 96-well plates and incubated with 100 μL of 50 μM EdU per well for 2 h at 37 °C. Next, cells were fixed with 4% paraformaldehyde and fixation was stopped by the addition of glycine. Subsequently, cells were stained with 1X Apollo solution and 1X Hoechst33342 solution. Finally, the cells were visualized under the Zeiss fluorescence photomicroscope.4 cells were seeded into the upper chamber of each insert with 300 μL serum-free medium and 500 μL medium with 10% FBS added into the lower chambers. After incubating at 37 °C for 18 h, the cells were fixed with methanol and stained with 0.1% crystal violet. The cells on the top side of the membrane were removed using cotton swabs and the cells that had invaded to the underside of the membrane were then counted and imaged under a microscope .Cell invasion and migration assays were performed using Transwell chambers . 3 × 10The total protein of cells was lysed with RIPA buffer  containing 1 mM PMSF (Beyotime). The concentration of protein was quantified using the Pierce™ BCA Protein Assay Kit . 30 μg of protein samples were separated by 10% SDS-PAGE and transferred to a PVDF membrane . The membrane was blocked in 5% non-fat milk for one hour at room temperature and incubated with diluted primary antibody at 4 °C overnight. Next, the membrane was incubated with horseradish peroxidase (HRP)-labeled secondary antibody for one hour at room temperature. The signal was visualized using ECL reagent and photographed by ChemiDoc Touch Imaging System . Information of antibodies for FUBP1, cMyc, METTL3, Cyclin A2, B1 and D1, and GAPDH can be found in Additional file 6 cells were collected and fixed with 70% ethanol overnight at 4 °C. After washing with PBS, cells were resuspended in 500µL of PI/RNase Staining Buffer  and incubated for 15 min at room temperature whilst being protected from light. A Cytoflex S flow cytometer  was used to analyze cell cycle distribution.For cell cycle analysis, 10n = 6/group). Calu1 cells (4 × 106) that had been stably transfected with sh-LCAT3 or scramble were implanted subcutaneously into the nude mice. Tumor growth was measured after one week, and tumor volumes were calculated with the following formula: Volume (cm3) = (length × width2)/2. After four weeks, the mice were euthanized, and the tumors were collected and weighed. For the in vivo tumor invasion assay, 1.2 × 106 scramble or shLCAT3 cells were injected intravenously into the tail vein of nude mice (n = 6/group). 1.5 mg luciferin  was administered once a week for 4 weeks, to monitor metastases using an IVIS@ Lumina II system . All experiments were performed in accordance with the Guide for the Care and Use of Laboratory Animals , with the approval of Zhejiang University, Hangzhou, China.For the in vivo tumorigenicity assay, female BALB/c nude mice (ages 4–5 weeks) were randomly divided into two groups (n = 3). mRNA libraries were prepared with 5 μg of total RNA using a TruSeq RNA Sample Preparation Kit from Illumina as we have described previously [Total RNA was isolated from Calu1 cells transfected with LCAT3 siRNA  or negative control (NC) siRNA . After UV crosslinking, the membrane was blocked with 5% non-fat milk for 1 h at room temperature, incubated with anti-m6A antibody  overnight at 4 °C and subsequently incubated with secondary antibody for 1 h at room temperature. After washed by PBST, signals were detected using a chemiluminescence system (Bio-Rad). The membrane was stained with 0.02% methylene blue in 0.3 mol/L sodium acetate (pH 5.2) as a loading control.https://bioconductor.org/packages/release/bioc/html/wiggleplotr.html).mRNA was purified using a polyA Spin™ mRNA Isolation Kit . Methylated RNA immunoprecipitation (MeRIP) was performed according to the instructions of the Magna MeRIP™ m6A Kit . Briefly, 18 μg of mRNA was fragmented into approximately 100-nt oligonucleotides using a fragmentation buffer. Then the post-fragmentation size distribution was validated using an Agilent 2100 Bioanalyzer with an Agilent RNA 6000 Kit. The Magna ChIP Protein A/G Magnetic Beads were incubated for 1 h at room temperature with m6A-specific antibody in immunoprecipitation buffer. The mixture was then incubated with the MeRIP reaction mixture overnight at 4 °C. Eluted RNA was then prepared for RNA-seq libraries as described above  #1-#2 were synthesized by Life Technologies . The RNA pull-down assay was performed using a Pierce™ Magnetic RNA-Protein Pull-Down Kit  according to the manufacturer’s instructions. Briefly, 100 pmol biotin labeled anti-sense RNA probes, sense RNA probes and negative control probes were incubated with streptavidin beads for one hour at room temperature. Subsequently, equal amounts of lysate derived from Calu1 cells were incubated with probe-beads complex overnight at 4 °C. After the pull down, the proteins were eluted by 1 × SDS loading buffer. The eluate was then separated by 4–12% sodium dodecyl sulfate polyacrylamide gel electrophoresis and stained with silver staining.g for 10 min. The proteins were immunoprecipitated with an anti-FLAG antibody (Sigma-Aldrich). The RNA/protein immunoprecipitations were boiled in loading buffer, and proteins were detected by western blotting analysis.pcDNA3.1-MS2 plasmid was a gift of Professor Huai Qiang Ju . MCP-3xFLAG plasmid was obtained from OBiO Technology . Calu1 cells were cotransfected with pcDNA3.1-LCAT3-MS2 and MCP-3xFLAG plasmids. After 48 h, the cells were cross-linked with 37% formaldehyde for 10 min at room temperature, followed by 1.25 M glycine quenching for 5 min. Then, the cells were lysed on ice for 10 min and centrifuged at 14,000×7 cells were lysed with 115 μL RIP Lysis Buffer for per immunoprecipitation. 5 µg of the antibody of interest was incubated with Protein A/G Magnetic Beads for 30 min at room temperature. 100 µL of RIP lysate was added to each bead-antibody complex in a RIP Immunoprecipitation Buffer and incubated overnight at 4 °C. Each immunoprecipitant was resuspended in proteinase K buffer and incubated at 55 °C for 30 min. RNA was isolated by phenol, chloroform and isoamyl alcohol according to the manufacturer’s instructions, and was detected by qRT-PCR.The RIP assay was performed using an EZ-Magna RIP Kit  according to the manufacturer’s instructions. Briefly, approximately 2 × 10A549 and Calu1 cells were treated with 5 µg/mL actinomycin D (Sigma-Aldrich) to inhibit transcription. Cells were then collected at various time intervals. LCAT3 and MYC mRNA levels were assessed using qRT-PCR.A549 and Calu1 cells were treated with 100 µg/mL cycloheximide (CHX) for the indicated times and harvested. Protein expression of c-Myc was then determined by western blot analysis.The MYC promoter containing the far-upstream element (FUSE) sequence or lacking the FUSE sequence was sub-cloned and inserted into the pGL3-basic vector . 293T cells were seeded onto 24-well plate and transfected with LCAT3 or FUBP1 siRNA. After incubation for 48 h, the cells were lysed in 1 × Passive lysis. A Dual-Luciferase Reporter Assay System (Promega) was applied to measure the firefly luciferase activity, with Renilla luciferase serving as a transfection control.6 cells were fixed with 1% formaldehyde (Sigma-Aldrich) for 10 min at room temperature. Fixation was stopped by the addition of glycine and further incubation was conducted for 5 min. Chromatin was digested to 150–900 bp using micrococcal nuclease and a sonicator . 10–20 µg of digested, cross-linked chromatin was used per immunoprecipitation. The antibody–chromatin complex was captured with Pierce Protein A/G Magnetic Beads. ChIP DNA was extracted and analyzed by qPCR.Briefly, approximately 5 × 10P < 0.05. All statistical analyses were implemented in the statistical package R (https://www.r-project.org/).Data are presented as mean ± standard deviation of three independent experiments. Two-sided Student’s t test was used to analyze the differences between groups. KEGG pathways that were enriched for differentially expressed genes (DEGs) were detected using hypergeometric tests. Gene Ontology (GO) terms that were enriched for DEGs were evaluated using Fisher's exact tests. Kaplan–Meier curves were plotted to reveal survival differences between two groups of patients according to their expression levels. A log-rank test was used to evaluate statistical differences in survivals. Statistical significance was assigned at To characterize novel lncRNAs deregulated in lung cancer, we analyzed the expression profiles in 485 LUAD tissues and 56 adjacent normal tissues from The Cancer Genome Atlas (TCGA). We identified 93 lncRNAs that were differentially expressed between lung tumors and adjacent normal tissues, and were also associated with prognosis in LUAD patients . Our preLCAT3 is located on human chromosome 3q13.31 and has one transcript with five exons. The 5′ and 3′ cDNA RACE assays revealed that LCAT3 is a long non-coding RNA of 1541 bp in length  data of LUAD in TCGA as previously described . There wOn the other hand, m6A modification is the most prevalent posttranscriptional modifications of mRNA and lncRNA, and regulates it translation, stability, etc. , 20. OurTo investigate the biological function of LCAT3 in lung cancer cells, we silenced LCAT3 in Calu1 and Hop62 lung cancer cells using siRNAs, which sufficiently reduced the expression of LCAT3    analysis Fig. A. ProteiA series of binding region assays were then performed to explore the binding sites between LCAT3 and FUBP1. Briefly, the secondary structure of LCAT3 was analyzed with RNAfold , erased by demethylases (erasers), and recognized via m6A-sepecific binding proteins (readers) , 36. METLncRNAs typically exert their biological functions by physically interacting with regulatory proteins or acting as a miRNA sponge. For instance, glycolysis-associated lncRNA of colorectal cancer (GLCC1) stabilizes c-Myc from ubiquitination degradation by direct interaction with the HSP90 (HSP90AA1) chaperon, thereby reprograming the glycolytic metabolism for colorectal cancer . On the FUBP1 is a member of FUSE-binding proteins (FBPs) family, which recognize single-stranded DNA and RNA , 43. OurIn conclusion, we identified and characterized LCAT3, a novel m6A-regulated lncRNA in lung cancer. Our study fits the following working model. METTL3 is overexpressed by a yet-to-be identified mechanism, which then stabilizes LCAT3 via m6A modification. Overexpressed LCAT3 then recruit FUBP1 to c-MYC promoter to transactivate c-MYC expression, leading to enhanced proliferation, survival, migration/invasion and metastasis of lung cancer cells Fig. G. Our stAdditional file 1:Table S1: Sequences of primers, sgRNAs, siRNA, and shRNA used in the study. Table S2: Information of antibodies used in the study. Table S3: Quality control metrics of the RNA-seq libraries. Table S4: Quality control metrics of the MeRIP-seq libraries. Table S5: Proteins identified by Mass Spectrometry analysis of RNA pull-down fractions. Figure S1: The full sequence of LCAT3 was confirmed by 3’ RACE and 5’ RACE. (A) 3’ RACE assay of LCAT3. (B) 5’ RACE assay of LCAT3. (C) Full sequence of LCAT3 determined by Sanger sequencing. Figure S2: LCAT3 is upregulated by METTL3 through m6A modification. (A) No significant difference in LCAT3 CNV between LUAD tumor and noncancerous normal tissues in TCGA. (B) No significant association of LCAT3 CNV with its expression in LUAD tumor and noncancerous normal tissues. (C) The ENCODE regulation track of UCSC genome browser did not display any regulation elements in LCAT3 locus. (D–F) Expression of LCAT3 after treatment with 5-Aza-2ʹ-Deoxycytidine (5-Aza-dC) in A549, Calu1 and Hop62 cells at 48 hours and different doses. (G) Dot blotting assays detect the m6A modification level in A549 and Calu1 cells. Figure S3: Overexpression of LCAT3 promotes proliferation of lung cancer cells. (A) qRT-PCR analysis of LCAT3 expression in A549 and Calu1 cells expressing a control empty vector (pcDNA3.1) or LCAT3-OE vector. (B) Cell proliferation assays for A549 and Calu1 cells expressing a control empty vector (pcDNA3.1) or LCAT3-OE vector. (C) Cell colony formation assays for A549 and Calu1 cells expressing a control empty vector (pcDNA3.1) or LCAT3-OE vector. *P < 0.05; **P < 0.01; ***P < 0.001. Figure S4: Stable knockdown of LCAT3 inhibits proliferation, invasion, and migration of lung cancer cells. (A) qRT-PCR analysis of LCAT3 expression in Calu1 and Hop62 cells expressing control (Ctrl) or LCAT3 shRNAs. (B) Cell proliferation assays for Calu1 and Hop62 cells expressing control (Ctrl) or LCAT3 shRNAs, using CCK8 assay.  Transwell invasion and migration assays were performed in stable LCAT3 knockdown and control lung cancer cells. Representative images (left) and statistical analysis (right) are shown. *P < 0.05; **P < 0.01; ***P < 0.001. Figure S5: mRNA expression profile of in LCAT3-silenced cells. (A) Hierarchical clustering of 1,221 genes that exhibited significantly altered expression in LCAT3-silenced Calu1 cells as compared with vector-transfected control cells. The color bar indicates the fold change (log2). Some cell cycle related genes  were pointed out.  qRT-PCR analysis of expression of cell cycle related genes in A549 (B) and Calu1 (C) cells transfected with LCAT3-siRNA or NC-siRNA. *P < 0.05; **P < 0.01; ***P < 0.001. Figure S6: LCAT3 physically interacts with FUBP1. (A) Western blotting analysis of FUBP1, DDX5, FUBP3 and HNRPL in sense (S) and antisense (AS) LCAT3 pull-down fractions. GAPDH served as a negative control. (B) RNA secondary structure of LCAT3 predicted by RNAfold. The 208–342 nt of LCAT3 forms a stem-loop structure. (C) Interaction profile (208–342 nt) of LCAT3 and FUBP1 was predicted by catRAPID. X axis represents the RNA sequence distribution, while Y axis indicates the protein interaction score. The 208–342 nt of LCAT3, which forms a stem-loop structure, is the core sequence binding to FUBP1. (D) Calu1 cells were co-transfected with pcDNA3.1-MS2/pcDNA3.1-LCAT3-MS2/pcDNA3.1-208–342 nt-MS2 and MCP-3xFLAG plasmids. FLAG-MCP-MS2 pull-down and western blotting assays were performed to detect binding between LCAT3 and FUBP1. Figure S7: LCAT3 and FUBP1 co-regulate the expression of c-Myc.  Western blot assays detecting the protein expression of FUBP1 in A549 and Calu1 cells transfected with LCAT3 siRNA (A) or LCAT3-OE vector (B).  qRT-PCR (C) and western blot (D) analysis of FUBP1 overexpression efficiency in A549 and Calu1 cells expressing a control empty vector (PLVX-IRES-zsGreen1) or FUBP1-OE vector. (E) qRT-PCR detecting the mRNA expression of MYC in lung cancer cells with FUBP1 overexpression. (F) qRT-PCR detecting the mRNA expression of MYC in lung cancer cells with LCAT3 overexpression. Figure S8: LCAT3 has no effect on MYC mRNA stability as well as c-Myc protein stability.  Half-life of MYC mRNA in A549 (A) and Calu1 (C) cells transfected with LCAT3 siRNA or control siRNA.  Half-life of c-Myc protein in A549 (B) and Calu1 (D) cells transfected with LCAT3 siRNA or control siRNA.  Half-life of MYC mRNA in A549 (E) and Calu1 (G) cells transfected with FUBP1 siRNA or control siRNA.  Half-life of c-Myc protein in A549 (F) and Calu1 (H) cells transfected with FUBP1 siRNA or control siRNA. Figure S9: LCAT3 recruits FUBP1 to activate MYC expression and regulate expression of its downstream genes.  Fragmented DNA samples were separated by electrophoresis on a 1% agarose gel, corresponding to Figure 8A–8C. The majority of chromatin was digested to 1 to 5 nucleosomes in length (150 to 900 bp).  qRT-PCR analysis of expression of MYC downstream genes in A549 (D) and Calu1 (E) cells transfected with FUBP1-siRNA or NC-siRNA.


Heliothis virescens and Heliothis subflexa have been used as a model for deciphering the mechanisms underlying differentiation in pheromone communication, but no information exist regarding the functions of the pheromone receptors (PRs) of H. subflexa. Here, we functionally characterized all candidate PRs of H. subflexa, and found that only the response profile of OR6 differed between the two species. Through domain swapping and site-directed mutation followed by functional characterization, we identified a critical amino acid in OR6 caused a one-way alteration in specificity. This result suggests HsubOR6 evolved from an ancestral OR6 gene with a HvirOR6-like function and implies that the evolutionary direction of the receptor specificity was from the H. virescens-like pattern to H. subflexa-like pattern.The sex pheromone processing system of moths has been a major focus of research on olfaction and speciation, as it is highly specific and closely related to reproductive isolation. The two noctuid moths   •Heliothis subflexaSix pheromone receptors (PRs) were functionally characterized in •H. subflexa and H. virescensFunction of one orthologous PR-OR6 differed between •A critical amino acid residue caused one-way alteration of OR6 specificity•This suggests HsubOR6 evolved from an ancestral gene with a HvirOR6-like function Ecology; Biological sciences; Zoology; Neuroscience; Sensory neuroscience Most insects have a highly sensitive olfactory system, enabling adaptive behaviors in mating, hosts location, oviposition sites selection, and natural avoidance for enemies . The sexSex pheromones play an essential role in species-specific reproductive communication in moths . The comThe preference and sensitivity of male moths to female sex pheromones are controlled by olfactory circuitry, as reviewed in many previous works . The speHeliothis virescens and Heliothis subflexa (Lepidoptera: Noctuidae) are closely related moth species based on studies using morphological characters  analyses of hybrids and populations generated by backcrosses of the two species  that resulted in the functional changes between HvirOR6 and HsubOR6. Moreover, this change is direction dependent: a change in this amino acid in HvirOR6 changed the function of H. virescens to that of H. subflexa, but the reverse mutation did not change the function of H. subflexa to that of H. virescens.In this study, we cloned and functionally characterized six candidate PRs from H. subflexa and H. virescens showed that the orthologous PRs in these two insects shared high sequence identities, ranging from 81.2% (OR6) to 96.5% (OR11)  . Six odotic tree . The 43 tic tree . In conttic tree .Figure 1H. subflexa were individually co-expressed with HsubOrco in Xenopus oocytes followed by testing of the electrophysiological responses elicited by a panel of H. subflexa and H. virescens pheromone compounds. The oocytes co-expressing HsubOR13 and HsubOrco specifically responded to the major sex pheromone component Z11-16:Ald  and Z9-16:Ald , with a specifically higher response to Z9-14:Ald, at a concentration of 10−4 M; the response of HvirOR6 to Z9-14:Ald was 1,541.96 nA, about 8.23 times higher than that to Z9-16:Ald (187.44 nA)  B and 3C.th and 5th transmembrane domains (TMDs)  ; S2. To s (TMDs) B.Figure Functional analysis of the chimeric genes showed that region 2 of OR6 (V2 and S2) has no significant influence on the response to Z9-14:Ald or Z9-16:Ald . If we rBesides the differences in region 2 of OR6, HsubOR6 and HvirOR6 differ at 72 amino acid sites ; S2. To −5 M, which was similar to that of HsubOR6 (4.629 × 10−5 M)  to Z9-14:Ald and Z9-16:Ald with those of HvirOR6 and HsubOR6. The results showed that the response profile of VM321 was exactly the same as that of HsubOR6 A and 7B. 10−5 M) C.Figure −5 M) was similar to those of HvirOR6 (2.476 × 10−5 M) and HsubOR6 (4.629 × 10−5 M) (As one site-directed mutation (VM321) led to a complete change in the function of HvirOR6 to that of HsubOR6, we next mutated this site (V321L) in HsubOR6 (SM321) to clarify whether a change in this site could also lead to a change in the function of HsubOR6 to that of HvirOR6. SM321 did not change the function of HsubOR6, as the response of SM321 to Z9-16:Ald increased, whereas, the response to Z9-14:Ald remained unchanged A and 7B. 10−5 M) C. These H. virescens and H. subflexa are generally accepted as ideal models to study the mechanisms underlying the differentiation in pheromone communication between moth species  have different sex pheromones as ligands . However, we found that this region had no significant effect on the function of OR6, thus demonstrating that it is not critical in determining the specificity of ORs. Based on the results of mutation studies and further functional validation of single-site mutants, we identified a key site in TMD5  causing functional changes between HvirOR6 and HsubOR6. As in HvirOR6, the mutant VM321 lost its response to Z9-14:Ald. The experimental results indicated that the mutation of many sites could lead to changes in HvirOR6 function, especially the loss of the response to Z9-14:Ald . HoweverH. virescens and H. subflexa are closely related species, which are separated by about 2.5 Mya .Further information and requests should be directed to the Lead Contact, Guirong Wang . All animals were maintained in a recirculating tank system at a temperature of 18–20°C. Frogs were fed with adult Xenopus irradiated diet . All dissection procedures are performed under anesthesia. The protocols used in our work were approved by the Institutional Animal Care and Use Committee of Chinese Academy of Agricultural Sciences, Beijing, China.2 years old mature female Z)-11-hexadecenal (Z11-16:Ald), (Z)-9-tetradecenal (Z9-14:Ald), (Z)-9-hexadecenal (Z9-16:Ald), (Z)-11-hexadecenyl acetate (Z11-16:OAc), (Z)-9-hexadecenyl acetate (Z9-16:OAc), (Z)-7-hexadecenyl acetate (Z7-16:OAc), and (Z)-11-hexadecen-1-ol (Z11-16:OH) were purchased from Nimrod Inc. , all with minimum purity of 95%. Stock solutions (1 M) were dissolved in dimethyl sulfoxide (DMSO) and stored at −20°C. Before experiments, the stock solution was diluted in 1 × Ringer's buffer  to working concentration of 10−4 M. 1 × Ringer's buffer containing 0.1% DMSO was used as a negative control.. All the candidate PR and Orco sequences were further confirmed by RACE and PCR amplification.The PR and Orco sequences of HsubOR6, HsubOR11, HsubOR13, HsubOR14, HsubOR15 and HsubOR16), HvirOR6, HsubOrco and HvirOrco were first cloned into pENTR/D-TOPO entry vectors  and then subcloned into pSP64T (converted from pSP64T-Oligo) destination vectors by means of the Gateway LR reaction. The primers used for vector constructed were listed in The full-length cDNA sequences of 6 HsubPRs (http://web.expasy.org/translate/). The amino acid sequences of 6 PRs in H. virescens and 6 PRs in H. subflexa were aligned using DNAMAN software (version 8). The TMDs (transmembrane domains) were predicted using TMHMM server version 2.0 (http://www.cbs.dtu.dk/services/TMHMM/). The 43 OR amino acid sequences used to construct a phylogenetic tree were aligned by using MUSCLE software. The maximum likelihood method was employed to construct the phylogenetic tree by MEGA X software with Jones-Taylor-Thornton amino acid substitution model (JTT). Bootstrap analysis was performed base on 1,000 replicates.The ORFs (Open reading frames) of all PR and Orco genes were predicted by using ExPASy  server version  to synonymous (dS) substitution rate (ω) by the maximum likelihood method (where ω > 1 is considered evidence of positive selection and ω < 1 evidence of purifying selection). Considering functional divergence among gene paralogs or species orthologues, the selective pressures may vary among branches in the tree. So the selective pressures of 8 OR orthologs  in these 6 species were tested separately. For each lineage, the codon sequences were aligned using MUSCLE-Code and a maximum likelihood phylogenetic tree was reconstructed with Maximum Composite Likelihood model by MEGA X software. The substitution rate (ω) of each lineage was calculated in codeml procedure with Site model Model 0: one-ratio.According to the sequence alignment of HvirOR6 and HsubOR6, we can divide the OR6 amino acid sequence of the two species into three fragments. Fragment 1 of the two species (V1 and S1) contains 260 amino acids (aa). Fragment 2 is species-specific and it contains 9 aa or 2 aa in HvirOR6 (V2) or HsubOR6 (S2) separately . FragmenThe site-directed mutants were generated into the HvirOR6 and HsubOR6 pSP64T vectors using the Phusion Site-Directed Mutagenesis Kit  following the manufacturer's instruction. A pair of phosphorylated primers contained the mutation sites were designed and used for amplifying each mutation .Escherichia coli competent cells . After cultivating on Ampicillin (50 mg/mL) LB (Luria-Bertani) agar overnight, individual colonies were cultured in liquid Ampicillin LB and sequenced to check for the right mutations. All the premiers used to prepare mutations were listed in Then the product was used to transform Trans-T1 2+-free washing buffer  for about 1 h at room temperature. After being cultured overnight at 18°C, oocytes were then microinjected with 27.6 ng of Orco cRNA and 27.6 ng of ORs cRNA. Oocytes microinjected with 55.2 ng of Orco were used as negative controls. Injected oocytes were incubated for 4–7 days at 18°C in 1 × washing buffer  supplemented with 5% dialysed horse serum, 50 mg/mL tetracycline, 100 mg/mL streptomycin and 550 mg/mL sodium pyruvate.The vectors contain the full ORF of all ORs were linearized to prepare the templates and the cRNA was synthesized with mMESSAGE mMACHINE SP6 (Ambion). Stage V to VII oocytes were treated with 2 mg/mL collagenase I  in 1 × CaWhole-cell currents were recorded with a two-electrode voltage clamp from the injected oocytes. Odorant-induced currents were recorded with an OC-725C oocyte clamp  at a holding potential of −80 mV. During the recording, oocytes were exposed to pheromones in ascending order of concentrations with an interval between exposures that allowed the current to return to baseline. Data acquisition and analysis were carried out by using Digidata 1440A and pCLAMP 10.2 software .t-test with SAS v8 for windows. Error bars indicate SEM. Statistical significance was determined at α = 0.05 level.Dose-response curves and EC50 value in


The inhibition of osteogenic differentiation is a major factor in glucocorticoid-induced bone loss, but there is currently no effective treatment. Dopamine, a major neurotransmitter, transmits signals via five different seven-transmembrane G protein-coupled receptors termed D1 to D5. Although the relevance of the neuroendocrine system in bone metabolism has emerged, the precise effects of dopamine receptor signaling on osteoblastogenesis remain unknown.In vitro, western blotting and immunofluorescence staining were used to observe the expression of dopamine receptors in MC3T3-E1 and BMSCs cells treated with dexamethasone (Dex). In addition, Alizarin red S (ARS) and alkaline phosphatase (ALP) staining and western blotting were used to evaluate the effect of D1R activation on osteogenic differentiation in Dex-induced MC3T3-E1 cells via the ERK1/2 signaling pathway. In vivo, micro-CT and hematoxylin and eosin (H&E), toluidine blue and immunohistochemical staining were used to determine the effect of D1R activation on Dex-induced bone loss.We demonstrated that the trend in D1R but not D2-5R was consistent with that of osteogenic markers in the presence of Dex. We also demonstrated that the activation of D1R promoted Dex-induced osteogenic differentiation by activating the ERK1/2 pathway in vitro. We further demonstrated that a D1R agonist could reduce Dex-induced bone loss, while pretreatment with a D1R inhibitor blocked the effect of a D1R agonist in vivo.Activation of D1R promotes osteogenic differentiation and reduces Dex-induced bone loss by activating the ERK1/2 pathway. Hence, D1R could serve as a potential therapeutic target for glucocorticoid-induced osteoporosis.The online version contains supplementary material available at 10.1186/s10020-022-00453-0. Glucocorticoids (GCs) have been widely used in the clinic, especially in alleviating inflammatory diseases. It has been indicated that the excessive use of GCs can lead to osteoporosis and osteonecrosis. GC-induced osteoporosis (GIOP) is a serious consequence leading to fractures, but there is currently no effective treatment  was inhibited by dexamethasone (Dex) dose-dependently in vivo and in vitro. In addition, our results demonstrated that a D1R agonist prevented Dex-induced osteoporosis by enhancing bone formation. Mechanistically, the D1R agonist protected osteoblasts from Dex by activating ERK1/2 signaling. Our results demonstrated the therapeutic potential of D1R in the treatment of GIOP  were incubated under osteogenic conditions  until reaching confluence and were then treated with or without 0.01–100 μM Dex  for 12, 24 or 48 h or 3, 14 or 21 days, as indicated. The medium was changed every 3 days. To investigate whether D1R was responsible for the effect on Dex-mediated osteoblast differentiation, the first antagonist (0.2 nM SCH23390) was added for 10 min, then agonist (10 μM SKF38393) for 10 min before Dex in this experiment. To verified whether the ERK1/2 pathway was involved in the protective effect of activation of D1R to Dex-mediated osteoblast differentiation, the first antagonist (1 μM SCH772984) was added for 10 min, then agonist (10 μM SKF38393) for 10 min before Dex. The medium was changed once at 3 days, and the cells were allowed to differentiate for 14 or 21 days. After mineralized nodules had formed, ALP and ARS staining were performed.BMSCs cells and MC3T3-E1 cells  were maintained in α-MEM with 10% fetal bovine serum in an incubator at 5% CO3 cells/well) were plated in 96-well plates. The MC3T3-E1 were cultured in induction medium for 24 h and then treated with various concentrations of Dex, SKF38393, SCH23390 or SCH772984 for 1, 2, or 3 days. Ten microliters of CCK-8  buffer was added to each well. After incubation at 37 °C and 5% CO2 for 1 h, absorbance was measured using a microplate reader  at a wavelength of 450 nm.MC3T3-E1 cells  in the dark for 30 min. The staining outcomes were observed under a microscope.5 cells per well in differentiation medium for 21 days. The cells were fixed with 4% paraformaldehyde at room temperature for 10 min. Then, the wells were rinsed three times with distilled water, and the cells were dyed with 1% ARS  at 37 °C for 30 min. Finally, the cells were washed thoroughly with distilled water three times. The staining outcomes were observed under a microscope.To examine the matrix mineralization of osteoblasts, BMSCs and MC3T3-E1 cells were plated in 12-well plates at a concentration of 1 × 10MC3T3-E1 cells were cultured on 24-well plates, washed with PBS three times, fixed with 4% paraformaldehyde for 30 min and then permeabilized with Triton X-100 for 10 min. The cells were stained with primary antibodies against D1R  at 4 °C overnight, washed and incubated with secondary Alexa Fluor 555 antibodies  and Molecular Probes Alexa Fluor 488 phalloidin  for 1 h in the dark. After the cells were stained with DAPI for 10 min, the cells were imaged using a fluorescence microscope.Lentivirus packaging was made by OBiO Technology . Lentiviral shRNAs targeting D1R were obtained from Gene pharma and shRNA overexpression and silencing sequences are listed follow: pSLenti-CMV-Drd1-3FLAG-PGK-Puro (D1R): CMV-F: CGCAAATGGGCGGTAGGCGTG; MSCV-rev: CAGCGGGGCTGCTAAAGCGCATGC; pLKD-CMV-Puro-U6-shRNA (Drd1)_shDrd#1-TargetSeq: GGACTTTGTCTGTTCTCAT; pLKD-CMV-Puro-U6-shRNA(Drd1)_shDrd#2-TargetSeq: CCATTTCATCCTCCCTCAT; pLKD-CMV-Puro-U6-shRNA(Drd1)_shDrd#3-TargetSeq: CCACCACAGGTAATGGAAA. The MC3T3-E1cells were infected with the lentivirus at a MOI of 100 for 72 h following which stably transfected cell lines were obtained by culturing in medium containing puromycin according to the manufacturer's instructions.BMSCs and MC3T3-E1 cells were collected with 0.5% trypsin . The cells were lysed in radioimmunoprecipitation assay  for 30 min at 4 °C, and then the supernatant was collected by centrifugation at 14,800 rpm at 4 °C. The supernatant was purified, and the total protein concentration was quantified using a bicinchoninic acid (BCA) protein kit . Twenty micrograms of total protein were separated using SDS-PAGE  and then transferred to a PVDF membrane . After being blocked with QuickBlock™ blocking buffer , the membranes were incubated with the appropriate primary antibodies overnight at 4 °C. Antibodies against runt-related transcription factor 2 , osterix (OSX) , ALP , D1R , D2R , D3R , D4R , D5R , total ERK1/2 , p-ERK1/2 , JNK , p-JNK , p38 , p-p38 , and β-actin  were used. The membranes were rinsed 3 times with TBS-Tween and then incubated at room temperature with secondary antibodies for 1 h. Enhanced chemiluminescence  was used to visualize the protein bands, and the relative gray level was analyzed using ImageJ.Eight-week-old C57BL/6 J mice were purchased from JOINN Laboratories . Animal experiments were approved by the Animal Ethics and Welfare Committee (AEWC) of Zhangjiagang TCM Hospital Affiliated to Nanjing University of Chinese Medicine . Briefly, the mice were randomly divided into five groups: (1) control (Ctrl) group, (2) vehicle group, (3) Dex + D1R agonist group, (4) Dex + D1R inhibitor + D1R agonist group and (5) Dex + D1R inhibitor group (n = 6 mice for each group). Mice in the vehicle group were injected with 20 mg/kg Dex. Mice in the Dex + D1R agonist group were administered 20 mg/kg Dex and 1 mg/kg SKF38393, and mice in the Dex + D1R inhibitor + D1R agonist group were administered 20 mg/kg Dex, 1 mg/kg SKF38393 and 1 mg/kg SCH23390. Mice in the Dex + D1R inhibitor group were administered 20 mg/kg Dex and 1 mg/kg SCH23390  of the mice were measured by high-resolution micro-CT . The femurs were scanned at 9 μm per layer, and the X-ray parameters were set at a voltage of 80 kV and a current of 100 mA. Bone mass density (BMD), bone volume per tissue volume (BV/TV), bone surface/bone volume (BS/BV), bone surface/total volume (BS/TV), trabecular number (Tb.N), trabecular thickness (Tb.Th) and connectivity density (Conn.Dn) were measured by the program CTAn . The relevant three-dimensional (3D) images were analyzed after processing.After micro-CT analysis, the femurs were preserved at 4 °C for histological and immunohistochemical analyses. All femurs were decalcified with 15% ethylenediaminetetraacetic acid  for 4 weeks, embedded in paraffin blocks and sectioned at a thickness of 5 μm by a microtome. Hematoxylin and eosin (H&E), toluidine blue and immunohistochemical (IHC) staining were performed. Images were captured with an Olympus microscope.All experiments were performed at least 3 times. All values are expressed as the mean ± standard deviation (M ± SD). Fold changes for each treatment were compared with those of the respective control group at each designated time point. SPSS 25.0 software was used for statistical analysis in this study. One-way analysis of variance (ANOVA) was used for statistical comparisons among more than two groups. The post hoc Newman-Keuls test was suitable for comparisons of two groups. When the p value was less than 0.05, the difference was considered statistically significant.To gain insight into Dex-induced osteogenesis inhibition, we examined the DR signaling pathway. To assess the effects of Dex on the proliferation of osteoblasts, the CCK-8 assay was used, and MC3T3-E1 cells were incubated with 0–100 μM Dex. The results indicated that exposure of MC3T3-E1 cells to Dex at concentrations ranging between 0 and 0.01 µM led to an increase in osteoblast proliferation, and when the concentration of Dex was greater than 0.01 μM, osteoblast proliferation was restrained in a dose-dependent manner  and SCH23390 (a specific D1R inhibitor) were used. As Additional file The mitogen-activated protein kinase (MAPK) pathway plays an important regulatory role in the pathogenesis of osteoporosis, and previous studies indicated that Dex inhibited osteoblast differentiation and proliferation by negatively regulating MAPK signaling , western blot analysis showed that the expression of ERK1/2 in osteoblasts was decreased, and the proliferation of osteoblasts was not inhibited , BV/TV , BS/BV , BS/TV , Tb.Th , Tb.N , and Conn.Dn  (Fig. Next, we further investigated the effects of D1R on Dex-induced osteoporosis in vivo. Representative μCT images of trabecular microstructures in the five groups of mice showed that Dex treatment induced an obvious loss of bone mass compared with that of mice in the control groups, but bone mass in mice in the agonist group was increased. In addition, pretreatment with the D1R inhibitor blocked the effects of the D1R agonist Fig. A. QuantiConsistent with the μCT results, H&E staining of the femur tissue indicated that the D1R agonist alleviated Dex-induced bone mass loss Fig. B. QuantiToluidine blue staining was used to determine whether the D1R agonist attenuated Dex-induced bone loss through the activation of bone formation. The results showed that Dex inhibited the rate of bone formation, and the addition of the D1R agonist enhanced bone formation. This effect was significantly inhibited with the addition of the D1R inhibitor Fig. A and D. In conclusion, these results suggest that D1R activation could promote bone formation and protect against Dex-induced bone loss.Long-term GCs have been widely used in clinical practice although a large number of studies have shown that it may be the main cause of bone loss (Ronchetti et al. Osteoblasts have specific receptors and high affinity for Dex, and a hyperphysiological dose of GCs inhibits the proliferation, differentiation and function of osteoblasts (Rauch et al. Dopamine is an important catecholamine neurotransmitter that is widely found in peripheral tissues and the central nervous system Berke . ThroughMAPKs belong to the serine-threonine protein kinase family, mainly include the ERK1/2, JNK, and P38 pathways, and can guide cellular responses to various stimuli, regulating a variety of cellular functions, including proliferation, differentiation. Xie et al.  found thHowever, this study has the following limitations. Bone metabolism includes two major processes: osteoblast-mediated bone formation and osteoclast-mediated bone absorption, which are not independent and are regulated by the interaction and influence of osteoblasts and osteoclasts (Ping et al. In summary, we found that after Dex administration, mice lost bone mass, bone density decreased, osteoblast differentiation and function were affected, and osteoblast D1R expression was inhibited. Studies have shown that Dex inhibits the expression of D1R but not D2-5R in osteoblasts by reducing the phosphorylation level of the ERK1/2 pathway, thus inhibiting the differentiation of osteoblasts and affecting the function of osteoblasts. D1R agonists can activate the ERK1/2 pathway and significantly reduce the inhibitory effect of Dex on osteoblasts, while ERK1/2 pathway inhibitors can inhibit the protective effect of D1R agonists on osteoblasts. Thus, understanding the direct regulation of D1R on osteoblasts and its mechanism will help to provide a new therapeutic strategy for GC-induced bone loss and provide new ideas to better understand the relationship between neuropsychiatric diseases and osteoporosis.Additional file 1: Figure supplements. Figure S1. Dex inhibited the differentiation of osteoblasts and reduced D1R expression in Rat BMSCs cells. Figure S2. Modulates the expression of D1R in MC3T3-E1 cells. Figure S3. Activation of D1R alleviated Dex-induced inhibition of osteoblast differentiation in Rat BMSCs cells. Figure S4. ERK1/2 mediated the protective effect of D1R against Dex-mediated inhibition of osteoblast differentiation in Rat BMSCs cells. Figure S5. The JNK pathway has no synergistic effects with ERK1/2 in the protective effect of activation of D1R to Dex-mediated osteoblast differentiation. Figure S6. The p38 pathway has no synergistic effects with ERK1/2 in the protective effect of activation of D1R to Dex-mediated osteoblast differentiation. Figure S7. H&E staining of the liver and kidney after treatment in vivo.