Stevia rebaudiana. These molecules comprise an invariant steviol aglycone decorated with variable glycans and could widely serve as a low-calorie sweetener. However, the most desirable steviol glycosides Reb D and Reb M, devoid of unpleasant aftertaste, are naturally produced only in trace amounts due to low levels of specific β (1–2) glucosylation in Stevia. Here, we report the biochemical and structural characterization of OsUGT91C1, a glycosyltransferase from Oryza sativa, which is efficient at catalyzing β (1–2) glucosylation. The enzyme’s ability to bind steviol glycoside substrate in three modes underlies its flexibility to catalyze β (1–2) glucosylation in two distinct orientations as well as β (1–6) glucosylation. Guided by the structural insights, we engineer this enzyme to enhance the desirable β (1–2) glucosylation, eliminate β (1–6) glucosylation, and obtain a promising catalyst for the industrial production of naturally rare but palatable steviol glycosides.Steviol glycosides are the intensely sweet components of extracts from  Stevia rebaudiana are already used as lowcalorie sweeteners, but the most abundant naturally occurring compounds have a bitter aftertaste. Here, the authors characterize and engineer rice glycosyltransferase OsUGT91C1 to facilitate the large-scale production of naturally rare but palatable glycosides Reb D and Reb MSteviol glycosides from the plant  However, the excessive intake of high-calorie sugars such as glucose, fructose, and sucrose is associated with obesity, diabetes, high blood pressure, and cancer3. Therefore, public health would benefit substantially from reducing the dietary intake of high-calorie sugars, which can be achieved through the increasing adoption of low-calorie sweeteners that retain the appeal of sweetness with lower risks to health4.In addition to serving as an energy source, sugars act as sweeteners that stimulate pleasure neurotransmitters. It is unlikely that humans will abandon the consumption of sweet foodsS. rebaudiana Bertoni, which are ~200 times sweeter than sucrose but have negligible calorific content. They have been used as sweeteners by local populations for centuries5. The individual steviol glycoside species comprise an invariant diterpenoid steviol aglycone and two variable glycans. One glycan is attached to the C13-hydroxyl, and the other to the C19-carboxylate of the steviol aglycone5. The aglycone is an elongated molecule. The C13-hydroxyl and C19-carboxylate are at two ends of the long axis of the molecule , and rebaudioside A (Reb A)9 are highly sweet  and M (Reb M) eet Fig.  but are  M) Fig.  are inteS. rebaudiana14. They are all annotated as GT1 family members in the Carbohydrate-Active enZymes (CAZy) Database (www.cazy.org)15 and utilize uridine diphosphate-activated glucose (UDP-glucose) as the sugar donor. In S. rebaudiana, the enzyme UGT85C2 adds the first glucose to the C13-hydroxyl group (R1 end), while UGT74G1 adds the first glucose to the C19-carboxylate moiety (R2 end)12 . UGT76G1 transfers glucose to make a β (1–3) glycosidic bond with the 3-hydroxyl of glucose 1-R1  Fig.  located )12 Fig. . These g)12 Fig. . They pr 16 Fig. . UGT91D2 16 Fig. 14. The n-R1 Fig. . The new-R1 Fig. . The new-R1 Fig. . The add14. The extremely limited addition of glucose 2-1-R2 by UGT91D2 is therefore highly likely to be responsible for the trace production of Reb D and Reb M in the native plant. Therefore, an efficient enzymatic route to the installation of glucose 2-1-R2 is needed to convert the dominant ST into Reb E and Reb A into Reb D. A glycosyltransferase of O. sativa (rice), OsUGT91C1 17 was recently identified, which shares 40% identity (56% similarity) to UGT91D2 and shows potential for adding glucose 2-1-R2 to steviol glycosides. UGT76G1 then could follow as a steviol β (1–3) glycosyltransferase to ultimately produce Reb D and Reb M  glucosylation at the R2 end  in Reb D and Reb M is essential for the absence of bitter notes in their flavor profiles. However, native UGT91D2 has been reported to catalyze β (1–2) glucosylation almost exclusively at the R1 end but is deficient at the R2 endb M Fig. .17 in synthesizing steviol glycosides. OsUGT91C1 is able to catalyze the addition of both glucose 2-1-R1 and glucose 2-1-R2 with roughly equal efficiency. It also catalyzes a third reaction, β (1–6) glucose addition, to create previously uncharacterized compounds. Structural analysis reveals molecular rationales for its catalytic promiscuity and guides the modification of the enzyme to eliminate the unwanted β (1–6) glucosylation and enhance β (1–2) glucosylation on steviol glycoside substrates. The modified enzyme would synthesize more desirable products from the less desirable but abundant steviol glycosides, leading to considerably enhanced yields of Reb D and Reb M.In this work, we show the biochemical and structural characterization of OsUGT91C16 identified two ester-linked glucose units at the R2 end of Rub-X  with UDP-glucose as the donor and commercially available potential substrates as acceptors. Simple glucose is not a substrate for OsUGT91C1, suggesting that the steviol aglycone is required for glucosylation. Rubusoside (Rubu), which contains both glucose 1-R1 and glucose 1-R2 Fig. , was incb-X Fig. .Fig. 2β A five-fold increase in the enzyme concentration led to the detection of a third product. This product was confirmed, by comparison with an authentic standard Fig. , to be RReb A, which has a tri-saccharide at the R1 end and glucose 1-R2 at the other end Fig. , would b12121 with one monomer in the asymmetry unit. PISA analysis18 indicated no higher-order oligomer in the crystals, and gel filtration suggested OsUGT91C1 remains monomeric in solution.Based on our above findings that OsUGT91C1 could add glucose 2-1-R1 and 2-1-R2 to both ends of the steviol glycoside substrates, we sought to determine what characteristics of its active site enabled this promiscuity. We determined the crystal structure of OsUGT91C1 in (1) the apo form, (2) complex with UDP and Reb E, (3) with UDP and ST, (4) with UDP and STB, and (5) the H27A mutant in complex with UDP and Reb D , and the C-terminal domain has six β strands (Cβ1–6). In each Rossmann-like domain, these β strands form a central parallel β-sheet that is sandwiched by the surrounding α-helices and loops  at the N- and C-termini, characteristic of a typical GT-B fold glycosyltransferaseops Fig. . Residueops Fig. .Fig. 3StUDP is bound identically in all four complexes and surrounded by the helices Nα1, Cα4, and the loops at the C-terminal side of β-strands Cβ1, Cβ3, and Cβ4 Fig. . The 2- 21. The cluster of water molecules (or glycerol) forms hydrogen bonds with the side chains of Asp381, Gln382, and Trp360  is also hydrogen-bonded to a cluster of waters (or to a glycerol molecule in some structures) Fig. . The volIn complex structures, the steviol glycoside substrates bind in two different orientations. In one orientation, the R2 end is at the active site with the R1 end at the “out” site on the protein surface Fig. , and in Three complexes, native OsUGT91C1 with UDP and Reb E, native OsUGT91C1 with UDP and ST, and the H27A mutant with UDP and Reb D, bind steviol compounds with the R2 end at the active site. The positions of the common atoms of Reb E Fig.  and ST  are esse19. In this mechanism, His27 acts as the general base in a relay with Asp128 to deprotonate the 2-hydroxyl of the accepting glucose at the active site, creating the nucleophile for the SN2 reaction with UDP-glucose  glucosylation at either end of any previously assayed substrate  glycosidic bond results from enzyme activity, we incubated OsUGT91C1 with Reb E and UDP in solution. LC-MS confirmed that Reb E was converted to both ST and Rub-X, and eventually, these compounds were transformed to Rubu , Reb A (only modifiable at the R2 end), and S13G (only modifiable at the R1 end) Fig.  were useGuided by structure, we constructed three mutants H93A, H93W, and F379A, to suppress β 1–6) glucosylation by eliminating the cryptic glucose pocket seen for glucose 2-1-R1 in the STB complex Fig. . H93A de–6 glucoskcat/Km for Rubu, S13G, and Reb A as the wild-type enzyme. F379A/F208M showed a 3-fold increase in kcat/Km for S13G, a 4-fold increase in kcat/Km for Reb A, and an almost 6-fold increase in kcat/Km for Rubu with a preferred addition at the R2 end  glucosylation activity  glucosylation to the R1 end but shows very little catalysis to the R2 end. This is a drawback as β (1–2) glucosylation to the R2 end is desirable to create more valuable steviol glycosides. OsUGT91C1 from rice is a homolog of UGT91D2 and has a more relaxed specificity, which can catalyze β (1–2) glucosylation to both glucose 1-R1 and glucose 1-R2 and potentially transform the abundant steviol glycosides to superior ones with quality taste.Steviol glycosides are a class of low-calorie sweet compounds. They are formed by glucosylation on both the R1 and R2 ends of the steviol aglycone. The assembly of the glycans requires several glycosyltransferases Fig. . The end28, it is often difficult to obtain structural information on the different substrate recognition modes, hindering promiscuity analysis and insight30. OsUGT91C1 is a rare case that multiple structures were obtained to describe the substrate in different poses and rationalize promiscuity and regiospecificity directly. In addition, β (1–6) glucosylation would not be noted unless the complex structure with STB and Reb E were available. The steviol glycoside substrate in OsUGT91C1 exhibits two motions for promiscuity, R1/R2 rotational swap of steviol aglycone and planar flip of glucose 1-R1. When these two motions are combined, the steviol aglycone and the linked glucose are concertedly recognized in three distinctive modes so that three specific hydroxyl groups of the steviol glycoside substrate can be delivered towards the activation zone of the catalytic His27, then activated to accept the glucose from the sugar donor  glucosylation at the R1 end Fig. . This se26. Other exceptions to the rule are known, including the well-studied UDP-Gal:GlcNAcβ-R β1-4 galactosyltransferase , which can transfer galactose to both the 4-hydroxyl of the terminal glucosamine of a protein glycan and the 4-hydroxyl of free glucose when bound to α-lactalbumin31. The UDP-dependent O-glucosyltransferases involved in flavonoid decoration33 are other well-known systems that break the simple rule. Here we have experimentally visualized how the promiscuity of OsUGT91C1 arises from two distinct structural features (the flexible aglycone site and the cryptic glucose pocket) and have demonstrated that we can control the promiscuity to create an engineered OsUGT91C1 that does not add β (1–6) glucose but has improved catalytic efficiency of the desirable β (1–2) glucosylation at the R2 end of steviol glycoside substrates. This engineered enzyme would relieve the catalytic bottleneck that makes only trace amounts of the most desirable products in the native plant. As a result, an inexpensive, biological source of low-calorie sweeteners with a desirable taste profile would be readily available and have more beneficial impacts on human health.OsUGT91C1 shows unusual catalytic promiscuity in that it catalyzes three distinct glucosylation reactions at the same active site, β (1–2) to the R1 end and R2 end, β (1–6) to the R1 end Fig. . OsUGT91O. sativa was de novo synthesized and subcloned into the expression vector pET28b. The constructed plasmid was transformed into Escherichia coli BL21 (DE3) (Novagen) for overexpression. For native OsUGT91C1, 0.5 mM IPTG was used to induce the expression for 4 h at 37 °C. For selenomethionine (SeMet)-labeled protein, E. coli cells were initially incubated in M9 minimal media at 37 °C until the cell density reached 1.0 at 600 nm. SeMet and IPTG were added, and the cells were cultivated for another 18 h at 16 °C. The E. coli cells were harvested by centrifugation and then lysed by sonication. The supernatant was loaded on a 5 ml HisTrap affinity column (GE Life Sciences), and His-tagged OsUGT91C1 was eluted with 20 mM Tris-HCl buffer pH 7.8, 0.5 M NaCl, and 250 mM imidazole, followed by immediate gel filtration in 10 mM HEPES-NaOH buffer pH 7.2, 150 mM NaCl, and 2 mM DTT. The protein was finally concentrated to 20 mg ml−1 and stored at −80 °C in aliquots. Site-directed mutants of OsUGT91C1 were generated using the QuickChange PCR mutagenesis protocol.The encoding region of OsUGT91C1 of −1) or the protein sample mixed with 1 mM UDP and 5 mM of the various steviol glycoside substrates in DMSO solution were crystallized using the sitting-drop vapor-diffusion method. After initial screening and optimizing the crystalization conditions, the diffractive crystals were obtained in 100 mM HEPES-NaOH buffer pH 6.5 and 20% PEG 4000. The crystals were flash-frozen and stored in liquid N2 after soaking in the cryoprotectant, containing 25% glycerol and the original crystalization composition.The apo protein (20 mg ml34 (version 3.0) in the CCP4i235 package (version 1.0). The crystals were inclined to deteriorate in the cryoprotectant. Only one crystal of SeMet-labeled OsUGT91C1 survived the cryo-treatment and gave a 3.45 Å single-wavelength anomalous diffraction (SAD) dataset. A partial model was generated with CRANK236 (version 2.0) and then used as a molecular replacement search model in Phaser37 (version 2.8) for a higher-resolution native dataset where the complete model can be built. The apo structure acted as the search model to solve the other complex structures. Manual model building and subsequent refinement were performed using Coot38 (version 0.8) and Refmac539 (version 5.8). The restraint libraries for all the steviol compounds were visually built in Coot and then idealized by ProDrg40 (version 2.5) and Acedrg41 (version 217) of the CCP4i package 7.1. The geometry of each steviol glycoside after refinement was validated by Privateer42 (version MKIII) of the CCP4i2 package (version 1.1). The statistics of data collection and refinement are summarized in Supplementary Table X-ray diffraction data were collected at beamline BL18U1 or BL19U1 at Shanghai Synchrotron Radiation Facility  using a Pilatus detector at a wavelength of 0.97853 Å. The data were indexed, integrated, and scaled using DIALSg for 10 min, the upper butanol layer was collected, evaporated, and resuspended in 25% acetonitrile, which was subjected to liquid chromatography-mass spectrometry (LC-MS) or LC-MS/MS.The in vitro biochemical assays of OsUGT91C1 and the mutants were performed at least in triplicate. 0.3 mM of each steviol glycoside substrate, including Rubu, Reb A, STB, Reb E, Reb D, and ST, was added to 200 µl of the reaction mix consisting of 20 mM Tris-HCl buffer pH 7.2 and 1 mM UDP-glucose. The reactions were initialized by two different concentrations of purified OsUGT91C1 or the mutants  and incubated at 25 °C. In all, 60 µl of respective aliquots were sampled at 0, 2, and 18 h of reaction and mixed with an equal volume of 1-butanol to terminate the reaction and extract the hydrophobic steviol glycoside substrate and products. After centrifugation at 17,000×m/z 641.3, m/z 803.3, m/z 965.4, m/z 1127.5) were captured and fragmented under collision energies ranging from 20 to 60 eV. The chromatographic and mass spectrometry data were collected and analyzed in Analyst 1.6.2 software . Authentic steviol glycoside standards were purchased .LC-MS and LC-MS/MS analyses were performed on a Shimadzu Ultra-fast liquid chromatography system  coupled with an AB SCIEX Qtrap 5500 mass spectrometer, which is equipped with a Turbo Spray ion source. Chromatographic separation was achieved on a Waters ACQUITY UPLC BEH C18 column  by a mobile phase consisting of water and acetonitrile, running at a flow rate of 0.5 ml/min and three elution steps that included 5% acetonitrile for 0–1 min, a linear gradient of 5–95% acetonitrile for 1–10 min, and 95% acetonitrile for 10–12 min. The column and autosampler were maintained at 40 °C and 15 °C, respectively. In all, 1 µl of the sample was injected and detected at a wavelength of 210 nm for steviol compounds and simultaneous MS or MS/MS in negative ionization mode. The mass spectrometric parameters were optimized as follows: ion spray voltage at −4500 V, declustering Potential at −90 V, and the temperature set to 500 °C. Q1 scan was scanned in the range of 200–1200 Da. In the MS/MS product ion mode, the parent ions of steviol glycosides (TM Glycosyltransferase Assay Kit (Promega). The sugar transfer of the glycosyltransferase turns UDP-glucose to UDP, which is converted to ATP and then quantified using a luciferase/luciferin reaction. The luminescence intensity linearly correlates to the concentration of UDP when it is <25 µM, thus reflecting UDP production during the reaction. Skanlt software 2.4.3 RE controlled Varioskan™ Flash multimode microplate reader and recorded the luminescence intensity . A standard linear plot between the luminescence intensity and UDP concentrations was drawn for each microplate measurement (R2 > 0.98).Steady-state kinetic assays of those substrates were performed using the UDP-Glon = 3). Steady-state kinetic parameters of the steviol glycoside substrate were determined by nonlinear fitting with shared values  to all the triplicate datasets according to the Michaelis–Menten equation when UDPG was at 200 μM, which was ~10 times its Km. The original data are provided as a Source Data file.After the reaction was started by adding the enzyme, four individual aliquots were taken every 1 min and mixed with an equal volume of the UDP detection reagent, terminating the reaction and quantifying the velocity of UDP production. The blank turnover of UDPG to UDP in the absence of the steviol glycoside substrate was subtracted from each velocity calculation. The appropriate concentrations of the enzyme were tested to ensure that the consumption of both steviol glycoside substrate and UDPG was <10% of the initial concentration during the assay. The steviol glycoside substrates Reb A, S13G, and Rubu were used in steady-state kinetic assays. Reb A or S13G respectively accepts a single β (1–2) glucose transfer at the R2 or R1 end during the assay. S13G may accept β (1–6) glucose transfer after completing β (1–2) glucose transfer. During the assay, the consumption of S13G is <10% of the initial concentration, and β (1–6) glucosylation can be ignored. Since Rubu participates in two β (1–2) sugar transfers, we cannot distinguish the production of UDP at individual ends. The kinetic data of Rubu reflect the combination of dual β (1–2) glucose transfers at both the R1 and R2 ends. All assays were performed in technical triplicate (Further information on research design is available in the Supplementary InformationPeer Review FileReporting Summary


Saccharomyces cerevisiae exists as two isoforms that contain either Ost3p or Ost6p, both noncatalytic subunits. These two OST complexes have different protein substrate specificities in vivo. However, their detailed biochemical mechanisms and the basis for their different specificities are not clear. The two OST complexes were purified from genetically engineered strains expressing only one isoform. The kinetic properties and substrate specificities were characterized using a quantitative in vitro glycosylation assay with short peptides and different synthetic lipid-linked oligosaccharide (LLO) substrates. We found that the peptide sequence close to the glycosylation sequon affected peptide affinity and turnover rate. The length of the lipid moiety affected LLO affinity, while the lipid double-bond stereochemistry had a greater influence on LLO turnover rates. The two OST complexes had similar affinities for both the peptide and LLO substrates but showed significantly different turnover rates. These data provide the basis for a functional analysis of the Ost3p and Ost6p subunits.Oligosaccharyltransferase (OST) catalyzes the central step in N-linked protein glycosylation, the transfer of a preassembled oligosaccharide from its lipid carrier onto asparagine residues of secretory proteins. The prototypic hetero-octameric OST complex from the yeast  N-glycosylation) of proteins is one of the most common covalent posttranslational protein modifications in eukaryotes. Homologous processes are found in archaea and bacteria (N-acetylglucosamine (GlcNAc) residue of the oligosaccharide. High-resolution structures and biochemical studies on the bacterial OST, PglB from Campylobacter lari, propose mechanisms for peptide and LLO binding, amide activation, and catalysis , archaea , and eukaryotic kinetoplastids   . Many re brucei) , 14. The brucei) , 10.2Man9Glc3 oligosaccharide, but smaller glycan substrates exist  is required for efficient glycosylation of a larger subset of proteins than the Ost6p-containing complex (OST6 complex)  . Furthercomplex) , 36, 37 in vitro , 39.in vitro. We found that the two complexes had similar affinities to short, synthetic peptide and LLO substrates but differed significantly in their catalytic efficiency.To understand the differences between the OST3 and OST6 complexes, we characterized the substrate specificities and enzyme kinetics of the two OST isoforms OST6 or OST3 and overexpressing the desired subunit  to ensure the assembly of complete complexes in the cell (A). Both complexes also ran as single monodispersed complexes in size-exclusion chromatography and had a very similar size (B).The two isoforms of the yeast OST complex that contain either of the functional homologs, Ost3p or Ost6p, were purified separately from strains expressing only one type of complex by deleting either the cell , 23. Insthe cell . Silver the cell A. Both clar size B.Figure A). Analysis of the glycosylation site occupancy of the OST complex glycoproteins Stt3p, Wbp1p, and Ost1p by mass spectrometry confirmed that the OST6 complex was generally less efficiently glycosylated than the OST3 complex, but the Stt3p N539 glycosite was fully glycosylated in both OST complexes (C). Both Wbp1p glycosylation sites were fully modified in the OST3 complex, but their site occupancy was reduced in the OST6 complex, particularly that of Wbp1p N60 (reduced by 50%). Regarding the Ost1p glycosylation sites, only Ost1p N99 was fully glycosylated in the OST3 complex and the occupancies of Ost1p N99 and N217 were only slightly reduced in the OST6 complex, but not to the same extent as that observed for the Wbp1p glycosites.Aside from the presence of either Ost3p or Ost6p, the only other difference observed between the purified OST isoforms was that the OST6 complex contained hypoglycosylated subunits A. Analysomplexes C. Both WD). In agreement with our understanding of the yeast glycan modification pathway in the ER and Golgi .The glycan structures at each glycosylation site were determined by mass spectrometry. We found glycan heterogeneity at each site analyzed, but the site-specific glycan structure profile was very similar in both OST3 and OST6 complexes D. In agrnd Golgi , the most3p N539 . The glyse units D.A). We detected and quantified the formation of glycopeptide product using reverse phase UPLC (ultra performance liquid chromatography) . The peak corresponding to the earlier-eluting glycopeptide increased with reaction time, and peak integration allowed an accurate and sensitive quantification of product formation.OST activity was determined using a short peptide labeled with the TAMRA (tetramethylrhodamine) fluorophore and synthetic LLO analogs with two GlcNAc residues (chitobiose) as the sugar moiety, as used previously , 42. IncFig. 2ography)  instead in vitro was first performed using the fluorescently labeled hexapeptide TAMRA-DANYTK-NH2 as the acceptor peptide (C). The data were fitted according to Michaelis–Menten kinetics and apparent KM and kcat values were derived  inhibited the reaction by 50% (D). To directly show that this peptide sequence was indeed a better acceptor substrate than TAMRA-DANYTK, we measured the initial turnover of TAMRA-labeled Peptide 5 (TAMRA-ADTYANATSDVL) and a shorter version of Peptide 5 of only six amino acid residues (TAMRA-YANATS). Fluorophore-labeled Peptide 5 was glycosylated 1.5 times faster than TAMRA-DANYTK and the short version was glycosylated 14 times faster (E) revealed a 3.5-fold lower apparent KM and a 3.5-fold higher maximal initial turnover rate . Quantitative analysis of the LLO analog substrate specificity for the yeast OST3 complex revealed variable substrate specificity, similar to the single subunit kinetoplastid OST, TbStt3A . The apparent KM values shown in cat/KM = 0.02 min−1 μM−1) than LLO C20 and LLO C25, with both a significantly lower turnover rate and affinity to the OST3 complex. It is important to note that LLO C15 also had an unsaturated C2 bond, which was saturated in LLO C20 and LLO C25. Although the apparent maximal turnover rates (kcat) increased with LLO lipid length, the highest turnover rates were observed with the second longest LLO, LLO C20, despite it exhibiting a lower affinity than LLO C25. LLO C20 was about 1/3-fold more specific than LLO C25 under our experimental conditions, suggesting a role of lipid conformation and isoprenoid chain length in substrate binding. Indeed, LLO C20 (ZZ) had a more similar double-bond stereochemistry to the natural yeast dolichol (Zn) than LLO C25 (ZEE), which may have accounted for its higher turnover rate, despite lower affinity when compared with the longer LLO C25.Yeast OST activity was measured using small chemically synthesized LLO analogs composed of a short polyprenyl chain linked east OST , 46. Theeast OST A. Quanti TbStt3A  . Ib  than that reported for TbStt3A . With an IC50 value of 44 μM, Ia was a more potent inhibitor of the yeast OST3 complex than Ib  than the reactive hydrolyzable substrates (lower KM), which was in agreement with the observations of the study on TbStt3A  inhibitFig. 4B, , in cont TbStt3A .Figure 4in vitro using synthetic chitobiose-containing LLOs and the TAMRA-YANATS peptide, we found the OST6 complex activity to be drastically reduced as compared with the OST3 complex (A). The OST6 complex activity was also reduced compared with the OST3 complex using the TAMRA-DANYTK peptide (data not shown). To investigate whether the two OST complexes differed in substrate specificity, we tested all active LLO analogs in combination with the TAMRA-YANATS peptide and found a five- to eightfold reduction in peptide glycosylation activity for the OST6 complex compared with the OST3 complex (B). Both OSTs had the same trend in LLO analog specificity, with the highest turnover rates observed for LLO C20 and the lowest reaction rates for LLO C15. Quantitative analysis of the kinetic parameters for the OST6 complex with LLO C20 confirmed that the OST6 complex did indeed have a fivefold lower maximal turnover rate compared with the OST3 complex, despite very similar KM values . Importantly, this hypoglycosylation of the OST3 complex did not affect the turnover rate of the enzyme (F). Therefore, the difference in glycosylation efficiency of the two OST isoforms appeared to depend on the presence of either the Ost3p or Ost6p subunit within the OST complex and not on the hypoglycosylation of subunits in the OST6 complex.To determine whether hypoglycosylation of the OST subunits observed in the OST6 complex affected OST activity, we generated a hypoglycosylated OST3 complex by expressing it in a strain with a deletion in the LLO biosynthesis gene henotype , 48. The complex E. Importe enzyme F. TherefC). To evaluate whether or not OST subunit hypoglycosylation affected OST activity as measured in vitro, we expressed the OST3 complex in an alg6 mutant strain. Due to the incomplete assembly of the LLO substrate in this strain, a similar hypoglycosylation of Wbp1p as in an Ost6p-only strain was observed, but this hypoglycosylation did not affect OST activity in vitro (F). Therefore, we concluded that differential activities of the recombinant OST complexes were not due to differences in the concomitant changes of OST subunit glycosylation but rather reflected the effect of the OST3/6 subunit on OST function.To obtain the two OST complex isoforms, we genetically tailored yeast strains. This modification of OST by expressing only one of the two alternative subunits allowed for the analysis of pure enzyme, but it also affected the glycosylation of proteins. Since some of the OST subunits are glycoproteins themselves, altering OST composition directly affected glycosylation of OST: we observed hypoglycosylation of Wbp1p in cells only expressing Ost6p, but site occupancy was not affected for the glycosylation sites of Ost1p and Stt3p, the two other glycoproteins of OST C. To evain vitro F. Theref8GlcNAc2  (data not shown), excluding the incomplete processing by ER hydrolases and confirming a transport of OST to early Golgi compartments. Indeed, the C-terminal, cytoplasm-localized KKXX signal of Wbp1p has been shown to mediate retrieval to the ER , close to the catalytic center, seems to be a property of multi-subunit eukaryotic OSTs. This glycosylation site is highly conserved , and an M values determined in this study were within range of those reported previously using tripeptide substrates and a dolichol-linked disaccharide LLO  and in vertebrates  and the OST6 complex strain (MAT α his3Δ1 leu2Δ0 lys2Δ0 ura3Δ0 arg4Δ0 ost3::LEU2MX6 OST4-1D4::kanMX6 YEp352-OST6) were constructed so that either Ost3 or Ost6 is expressed. Using standard genetic techniques, the OST3 or OST6 genes were disrupted by replacement with the Kluyveromyces lactis LEU2 gene (OST4 by homologous recombination of a 1D4-kanMX6 cassette amplified by PCR from the pYM-1D4 plasmid using S2 and S3 primers (OST4 is RTLQVDGGSGGS-LEVLFQGP-TETSQVAPA (linker region followed by a PreScission cleavage site underlined and the 1D4 epitope in bold). Correct tag integration was confirmed by PCR and DNA sequencing. Overexpression plasmids pOST3  and pOST3 (OST6 ) were tr plasmid  using th600 2–4 by centrifugation at 4000g for 10 min at 4 °C, and all steps hereafter were conducted at 4 °C. The cells were washed with cold ddH2O and resuspended in cold lysis buffer containing protease inhibitors , 20 tablets/L cOmplete EDTA-free protease inhibitor cocktail (Roche Diagnostics)). Glass beads (0.5 mm) were added and the cells were lysed in a bead beater  at 400 power, 3× 4 min with 1 min pause between rounds. The lysate was separated from the beads using a 50 ml syringe, and then the unbroken cells were removed by centrifugation at 1500g for 15 min. Membranes were pelleted by centrifugation at 50,000g for 45 min, resuspended by douncing 1:1 (g:ml) in membrane storage buffer  glycerol) and shock frozen in liquid nitrogen for storage at –80 °C.OST was expressed and purified based on the protocol described previously , with so2, 1 mM MnCl2, 10% (v/v) glycerol, 1 mM PMSF, 20 tablets/L cOmplete EDTA-free protease inhibitor cocktail (Roche Diagnostics), 0.05 mg/ml DnaseI (from bovine pancreas), 1% (w/v) n-dodecyl-β-D-maltopyranoside (DDM) (Anatrace), and 0.2% (w/v) cholesteryl hemisuccinate (CHS)) in a ratio of 1:10 (membranes (g): buffer (ml)) for 1.5 h at 1.25 rpm on a rotation wheel. The insoluble fraction was removed by centrifugation at 50,000g for 45 min and the soluble supernatant was mixed with sepharose beads (CNBr activated sepharose 4B) coupled to Rho-1D4 antibody (University of British Columbia) equilibrated in purification buffer  glycerol, 0.03% (w/v) DDM, and 0.006% (w/v) CHS). After at least 3 h of incubation at 1.25 rpm on a rotation wheel, the 1D4 antibody-coupled beads were transferred onto a Protino filter column (Macherey-Nagel) and washed two times with ten column volumes of purification buffer. To elute the bound OST, the beads were incubated with purification buffer supplemented with 0.5 mg/ml 1D4 peptide (GenScript Corp.) for at least 2 h on the filter before being collected. The beads were washed with one column volume purification buffer. The eluted protein was concentrated at 3000g on an Amicon Ultra-15 Centrifugal Filter Device with a 100 kDa cutoff before purification by size-exclusion chromatography on a Superose 6 Increase 10/300 Gl column  to remove aggregates using purification buffer at a flow rate of 0.3 ml/min. The peak eluting at ∼14 ml was collected and concentrated again on an Amicon Ultra-15 ml Centrifugal Filter Device with a 100 kDa cutoff to a concentration of 2–4 mg/ml. The protein concentration was determined by BCA assay . The protein was either stored at 4 °C for a few days or shock frozen in liquid nitrogen and stored in aliquots at –80 °C. Gel samples were prepared by diluting the protein in 1× Lämmli supplemented with 1.5 M urea and incubated at 37 °C for 20 min. Gel samples were loaded on 14% acrylamide gels and SDS-PAGE and subsequent silver staining were performed using standard procedures.Membranes were solubilized in solubilization buffer  per 100 μg protein sample in 0.05 M NH4HCO3 and incubated for 16 h at 37 °C. The peptides were eluted by centrifugation and dried. For site-occupancy analysis, the peptides were dissolved in 50 mM NaOAc, pH 5.2, and incubated with 1 μl of endoglycosidase H  for 3 h at 37 °C. To ensure complete removal of the N-glycan, the reaction was boosted by addition of a subsequent 1 μl of EndoH and incubation for 16 h at 37 °C. The reaction was acidified to 0.5% formic acid (FA) and desalted using C18 ZipTips (Millipore) for mass spectrometric analysis. For glycoproteomic analysis, the peptides were directly dissolved in 2.5% acetonitrile (ACN)/0.1% FA and desalted using C18 ZipTips (Millipore).Purified OST samples collected after size-exclusion chromatography were prepared for mass spectrometry experiments using the filter-assisted sample preparation protocol : 50–100 Samples were analyzed on a calibrated Q Exactive mass spectrometer (Thermo Fischer Scientific) coupled to a Waters nanoACQUITY UPLC System (Waters) with a Picoview nanospray source 500 model (New Objective). The tryptic samples were dissolved in 2.5% ACN/0.1% FA, loaded onto an Acclaim PepMap 100 trap column  and separated on a nanoACQUITY UPLC BEH130 C18 column , at a constant flow rate of 300 nl/min, with a column temperature of 50 °C and a linear gradient of 1 − 35% ACN/0.1% FA in 42 min, followed by a sharp increase to 98% acetonitrile in 2 min and then held isocratically for another 10 min. For site-occupancy analysis, one scan cycle comprised of a full-scan MS survey spectrum, followed by up to 12 sequential higher-energy collisional dissociation (HCD) scans based on the intensity. For site-occupancy analysis, full-scan MS spectra (400–2000 m/z) were acquired in the FT-Orbitrap at a resolution of 70,000 at 400 m/z, while HCD MS/MS spectra were recorded in the FT-Orbitrap at a resolution of 35,000 at 400 m/z. HCD MS/MS spectra were performed with a target value of 1e5 by the collision energy setup at a normalized collision energy (NCE) 25. For glycosylation profiling analysis, full-scan MS spectra (800–2000 m/z) were acquired in the FT-Orbitrap at a resolution of 70,000 at 400 m/z, while HCD MS/MS spectra were recorded in the FT-Orbitrap at a resolution of 35,000 at 400 m/z. HCD MS/MS spectra were performed with a target value of 5e5 by the collision energy setup at a NCE 22.N-acetylhexosamine (HexNAc) at asparagine. The monoisotopic masses of 2+ or more charged peptides were searched with a peptide tolerance of 10 ppm and an MS/MS tolerance of 0.03 Da for fragment ions. Only peptides with a maximum of two missed cleavage sites were allowed in database searches. The false discovery rate (FDR) was 1%. The MS/MS validation was performed manually via Xcalibur 4.1. The list of masses of the peptides with and without HexNAc detected and analyzed is found in MS and MS/MS data were processed into the Mascot generic format (mgfs) files and searched against the Swissprot database (version 202004) through Mascot engine (version 2.2) with the consideration of carbamidomethylation at cysteine, oxidation at methionine, and P and Peak areaP + HexNAc are the areas under the curve measured for the unmodified peptide (P) and the peptide modified with HexNAc (P + HexNAc), respectively. The raw data, mgf, and results files were uploaded to the ProteomeXchange Consortium . In brief, all raw data were loaded into Byonic and the search parameters were a peptide tolerance of 10 ppm and an MS/MS tolerance of 0.03 Da for fragment ions with the consideration of carbamidomethylation at cysteine, oxidation at methionine, and high mannose structures (Hex4-Hex12). Only peptides with a maximum of two missed cleavage sites and also semi-trypsin were allowed in database searches. The FDR was 1%. All assignments were confirmed manually with its corresponding MS/MS spectrum using Xcalibur 4.1. The list of masses of all glycoforms that were detected and analyzed is found in S)-nerylcitronellyl phosphate were prepared according to the previously reported procedure (Ia (GlcNAc2-(OH)CPP-(S)-citronellylneryl (C20)) was also performed according to the reported procedure. In brief, benzyl and ethyl groups of the protected chitobiose diethyl hydroxy-phosphonate were removed by catalytic hydrogenation and treatment with excess TMSBr, respectively, to give deprotected chitobiose hydroxy-phosphonate . CDI-activation of (S)-nerylcitronellyl phosphate as phosphoroimidazolidate and a coupling reaction with deprotected chitobiose hydroxy-phosphonate allowed the isolation of the desired hydroxy-phosphonate nonhydrolyzable LLO analog Ia  after acetyl deprotection and purification by flash chromatography. Compound Ia (minor diastereomer) was obtained in pure form as a colorless lyophilisate , as confirmed by NMR spectroscopy and HRMS. Spectroscopic data to Ia: 1H NMR  δ = 5.11–5.14 , 4.49 , 4.34-4.37 , 4.30 , 4.16-4.19 , 3.99-4.03 , 3.90 , 3.78-3.81 , 3.63-3.69 , 3.58 , 3.46-3.51 , 3.39 , 3.32-3.35 , 1.99-2.09 , 1.96-1.98 , 1.68 , 1.62 , 1.34-1.47 , 1.11-1.21 , 0.93 . 13C NMR  δ = 173.9, 173.7 , 136.2 , 135.8 , 132.3 , 126.7 , 126.2 , 125.4 , 103.1 , 82.6 , 78.1 , 76.7 , 75.8 , 73.3 , 72.1 , 71.9 , 71.9 , 65.6 , 62.6 , 62.6 , 57.6 , 54.2 , 38.9 , 38.8 , 38.8 , 33.2 , 30.8 , 30.6 , 27.7 , 26.4 , 26.0 , 23.8 , 23.7 , 23.2 , 22.8 , 19.7 , 17.8 . 31P NMR  δ = 9.7 , -8.8 . ESI-HRMS (-) m/z calculated 871.3764 (M-[H+]), found 871.3786 for C37H65N2O17P2−.The chemical synthesis of all reactive LLO analogs and nonreactive LLO Ib was previously described in detail . Protectrocedure . The synin vitro glycosylation (see below) by titrating varying amounts of LLO against a known amount of fluorescently labeled peptide. Concentrations of nonreactive LLO analogs were determined by in vitro glycosylation by titrating varying amounts of nonreactive LLO while using known amounts of both fluorescently labeled peptide and active LLO analog.Concentrations of reactive LLO analogs were determined by 2O for reactions.TAMRA-labeled peptides with 5-TAMRA at the N-terminus and an amide group at the C-terminus were synthesized by WatsonBio Sciences. Unlabeled peptides  were syn2, and purified OST after size-exclusion chromatography in purification buffer  glycerol, 0.03% (w/v) DDM, and 0.006% (w/v) CHS). The concentration of OST was calculated using the molecular weight of 280 kDa. The reaction mixture was preincubated at 30 °C for 5 min before adding the peptide to start the reaction that proceeded at 30 °C. Time points were taken such that the reaction was in the linear phase. At each time point, 1 μl reaction mixture was taken and the reaction was stopped by dilution in stop solution  such that the final peptide concentration was 2 μM.Reactions were performed in a total volume of 10 μl containing the specified amount of TAMRA-labeled peptide, synthetic LLO analog, 10 mM MnCl4, pH 8.0 buffer so that the glycopeptide and peptide are fully separated and elute within 3 min at a constant flow rate of 0.7 ml/min, with a column temperature of 45 °C. The peptide and glycopeptide were detected through TAMRA fluorescence .Samples were analyzed by reverse-phase chromatography using a UPLC Dionex UltiMate 3000 with an Accucore 150-C18 100 × 2.1 mm 2.6 μm column (Thermo Fisher Scientific). One microliter sample (2 pmol of peptide) was injected and measured by isocratic elution using a certain proportion of ACN  in 10 mM KPOThe UPLC profiles were analyzed by integrating the peaks corresponding to the glycopeptide and peptide using Chromeleon 7 (Dionex) and calculating the amount of glycopeptide formed using the following equation, as previously described :[GP]=PeaGP/P is the area under the curves measured for the glycopeptide or peptide, respectively, and [P] is the concentration of peptide at the start of the reaction. Time points were taken such that the reaction was in the linear phase and the data were fitted using GraphPad Prism 8 by linear regression to determine initial rates of reaction. Unless specified otherwise, turnover rates were determined from the initial rates of reaction by taking into account the enzyme concentration in the reaction. For determination of KM and kcat values, the data were fitted by nonlinear regression using the Michaelis–Menten equation using GraphPad Prism 8.[GP] is the concentration of glycopeptide, Peak areaFor quantification of the kinetic parameters for peptide TAMRA-DANYTK, reactions were performed with 2.8 μg purified extract containing OST3 complex before size exclusion (corresponds to approximately 1 μM OST3 complex), 150 μM LLO C20, and varying concentrations (10–400 μM) of the peptide. For TAMRA-YANATS, reactions were performed with 0.1 μM purified OST3 complex, 100 μM LLO C20, and varying concentrations (10–500 μM) of the peptide. For quantification of the kinetic parameters for the different LLO analogs, reactions were performed with 0.1 μM purified OST3 complex, 25 μM peptide TAMRA-YANATS, and varying concentrations of the different synthetic LLO analogs . Turnover rates and kinetic parameters were determined as described above.For the peptide competition assay to screen for a better peptide substrate, reactions were performed with 0.4 μM purified OST3 complex, 150 μM LLO C20, 25 μM TAMRA-DANYTK, and 25 μM unlabeled peptide (Genscript Corp.). The amount of glycosylated TAMRA-DANYTK peptide formed after 30 min was measured and inhibition was determined as a percentage relative to a control reaction without unlabeled peptide added.50 values.For quantification of inhibition by synthetic nonreactive LLO analogs, reactions were performed with 0.1 μM purified OST3 complex, 25 μM peptide TAMRA-YANATS, 50 μM LLO C20, and varying concentrations of the different synthetic nonreactive LLOs . Initial reaction rates were determined as described above and inhibition was determined as a percentage relative to the reaction rate of a control reaction without nonreactive LLO added. Data were fitted by nonlinear regression using GraphPad Prism 8 to determine the ICalg6Δ cells, reactions were performed with 100 μM peptide TAMRA-YANATS, 50 μM LLO C20, and varying concentrations of purified OST complex (0.1–0.3 μM). Turnover rates were determined as the slope from the linear regression of the initial rates of reaction measured across the enzyme concentrations of 0.1–0.3 μM for each OST. For quantification of the kinetic parameters for the OST6 complex, reactions were performed with the same conditions as for the OST3 complex, except that 0.5 μM purified OST6 complex was used in reactions for determining the kinetic parameters for LLO C20. For comparison of synthetic LLO preferences, reactions were performed with 25 μM peptide TAMRA-YANATS, 50 μM of the indicated LLO , and either 0.09 μM of purified OST3 complex or 0.5 μM OST6 complex. Turnover rates were determined as described above and the enzyme concentration was taken into account.For comparison of OST3 complex and OST6 complex activity, reactions were performed with 25 μM peptide TAMRA-YANATS and 50 μM LLO C20. In total, 0.1–2 μM of purified OST3 complex or OST6 complex was added to the reaction by adding the amount of protein in microgram that corresponds to the desired enzyme concentration, using the same molecular weight of 280 kDa for both complexes. Turnover rates were determined as the slope from the linear regression of the initial rates of reaction measured across the enzyme concentrations of 0.01– 0.1 μM for OST3 complex and 0.1–0.5 μM for OST6 complex. For comparison of OST3 complex, OST6 complex, and OST3 complex purified from via the PRIDE partner repository with the dataset identifier PXD024590 and 10.6019/PXD024590.All data are contained within the article and the This article contains The authors declare that they have no conflicts of interest with the contents of this article.


The dynamic changes of mRNA and protein levels were examined after x-ray of 8 Gy by qRT-PCR and Western blotting, respectively. Immunoprecipitation (IP) was used to explore the interaction between proteins. Luciferase assay was used to analyze the transcriptional regulation effect of ESR1 on SLC7A11. BODIPY C11 and trypan blue dyes were used to determine lipid peroxidation and cell death, respectively. The result showed that the ferroptosis-related gene SLC7A11 was higher in breast cancer tissues compared with normal tissues and associated with poor survival. A positive correlation exists between ESR1 and SLC7A11 expression. ESR1 promoted SLC7A11 expression at the early stage after IR. ESR1/SLC7A11 knockdown significantly enhanced IR-induced ferroptosis in ER-positive cells. At 12 h after IR, the IP data showed the interaction between E3 ubiquitin ligase NEDD4L and SLC7A11 increased, followed by the ubiquitylation and degradation of SLC7A11. Thus, SLC7A11 expression was regulated by both ESR1 and NEDD4L, in opposite ways. For the first time, we elucidated that ESR1 and NEDD4L functioned together after radiation treatment and finally induced ferroptosis in breast cancer cells, which provides novel insight into the guidance of clinical treatment of breast cancer.Radiotherapy is one of the most important treatments for breast cancer. Ferroptosis is a recently recognized form of regulated cell death that is characterized by lipid peroxidation. However, whether ionizing radiation (IR) could induce ferroptosis in breast cancer and how it works remain unknown. Bioinformatics analysis were performed to screen ferroptosis-related genes differentially expressed in breast tumor tissue and normal tissue. Then, breast cancer cell lines with different estrogen receptor (ER) phenotypes were used for studies  ERα andand PR−) . It has and PR−) . Under tand PR−) . ER is aand PR−) .Ferroptosis is a novel type of programmed cell death distinct from apoptosis, necrosis, and autophagy, which is an iron-dependent form of regulated cell death involving accumulation of reactive oxygen species (ROS) and overwhelming lipid peroxidation . SLC7A11Ionizing radiation (IR) can directly interact with water in human bodies, and generate ROS, which could be amplified by Fenton reaction and generate an extremely reactive hydroxy radical . The hydhttps://gdc-portal.nci.nih.gov) by the TCGA biolinks R package (version 2.20.0) (https://xenabrowser.net/).The gene expression profiles ( raw counts and FPKM-UQ) were downloaded from the TCGA database ( 2.20.0) . Data frMonoclonal anti-FLAG antibody and anti-β-actin were obtained from Sigma . Rabbit polyclonal anti-SLC7A11, and rabbit polyclonal anti-ESR1 were from Cell Signaling Technology . Protein G Plus-agarose was from Sangon Biotech . RNAiso Plus kits, reverse transcription kits, and real-time PCR kits were from Takara Biomedical Technology . Ferrostatin-1 (Fer-1), Erastin, and carbobenzoxy-Leu-Leu-leucinal (MG132) were all purchased from MedChemExpress . The expression plasmids for MYC-Ub were from Dr. Haishan Huang’s lab . SLC7A11 and ESR1 genes were obtained by PCR using MCF-7 cDNA as a template and then inserted into pcDNA3.1-FLAG vectors.mycoplasma. The cell lines were grown in high-glucose DMEM medium  supplemented with 10% fetal bovine serum and 1% penicillin and streptomycin in a 37 C incubator maintained at 5% CO2. For transfection, when the cells had grown to 70%–80% confluence in six-well plates, the plasmid (1 μg) or siRNA (100 μM) was transfected using Lipofectamine 2000  according to the manufacturer's instructions. The siRNAs were synthesized by GenePharma , and the sequences were listed as follows: siRNA for ESR1, CTACAGGCCAAATTCAGATAA; NEDD4L, CGCCTTGACTTACCTCCATAT.The human-source cell lines MCF-7, ZR-75-1, and MDA-MB-231 were obtained from the Cell Bank of Chinese Academy of Sciences and periodically tested for the absence of Cell death and lipid peroxidation were assessed by flow cytometry with trypan blue  and BODIPY™ 581/591 C11  following the manufacturer’s instruction, respectively. In brief, cells were pretreated with 10 µM of Erastin or 10 µM of Fer-1 for 1 h and then exposed to IR. Cells were collected at 48 h for lipid peroxidation or 72 h for cell death testing after X-ray exposure. Cells were resuspended in 400 μl of serum-free medium that contained BODIPY™ 581/591 C11 (5 µM) at 37°C in the dark for 30 min, or in 200 μl of PBS containing 0.04% of trypan blue, respectively to detect lipid peroxidation and cell death. The data were analyzed using the NovoExpress software , and a minimum of 10,000 cells were analyzed per condition.For Western blot, cells were lysed in ice-cold RIPA Lysis Buffer. Lysates were centrifuged at 12,000 rpm for 15 min at 4°C. The supernatant was collected, and the protein content was determined using the BCA Protein Assay Kit  according to the manufacturer’s instructions. Equal amounts of total protein (20 μg) were separated by 10% or 12% SDS–PAGE and then transferred to an equilibrated PVDF membrane . β-actin was used as a loading control. For IP, MCF-7 cells were transfected with expression vectors for FLAG-SLC7A11, and were exposed to IR at 48 h post-transfection. At 24 h after IR, cells were lysed in 1% NP-40 lysis buffer. Whole cell lysates (WCL) were incubated with the specified antibodies overnight at 4°C with constant rotation. Then, protein G Plus-agarose was added for 2 h under rotation at 4°C. After incubation, immunocomplexes were washed five times with IP wash buffer  and subsequently resolved by SDS-PAGE followed by Western blot analysis.Cells were transiently transfected with MYC-tagged ubiquitin (UB), and FLAG-tagged SLC7A11 with or without si-NEDD4L for 48 h, and were exposed to IR for 24 h. At 24 h after IR, cells were pretreated with MG132 (10 μM) for 4 h before harvest. The lysates were subjected to immunoprecipitation with FLAG antibodies overnight at 4°C. Ubiquitinated proteins were determined by immunoblotting with anti-MYC antibodies .Total RNA was isolated from cultured cells using RNAiso Plus kits according to the manufacturer’s instructions. mRNAs were first reverse transcribed into cDNA and quantitative reverse transcription-PCR (qPCR) was performed as the following thermal conditions: 95°C for 30 s followed by 40 cycles of 95°C for 5 s and 60°C for 30 s. qPCR was performed on QuantStudio 3 , and the expression of target genes was normalized to that of GAPDH. The following primers were used: GAPDH forward CCATGGGTGGAATCATATTGGA and reverse TCAACGGATTTGGTCGTATTGG; SLC7A11 forward GCGTGGGCATGTCTCTGAC and reverse GCTGGTAATGGACCAAAGACTTC; and ESR1 forward GCTTACTGACCAACCTGGCAGA and reverse GGATCTCTAGCCAGGCACATTC.Double luciferase–reporter gene determination was conducted to determine whether SLC7A11 the downstream target gene of ESR1. Reporter plasmid of SLC7A11 promoter double luciferase was synthesized from Generalbiol  and inserted into the pGL3-Basic vector. pGL3-Basic SLC7A11 promoter was co-transfected into cells with ESR1 overexpression vector or control vector by Lipofectamine 2000. After transfection for 48 h, luciferase activity was determined on a Centro LB 960 Luminometer  and the activity of renilla luciferase was used as a standardized control.http://www.r-project.org/) and GraphPrism 5.0 software . The differentially expressed gene (DEG) analyses were done with R package edgeR (version 3.34.0)  to balance the counts among the different samples (2 transformed fold change (log2FC) ≥ 1.5 and the false discovery rate (FDR) < 10-10. The FPKM-UQ normalized gene expression data were log-transformed for further analyses. The R package survminer (version 0.4.9) was used to separate patients into higher and lower expression groups by gene expression. The relationship between SLC7A11 expression status and clinicopathologic characteristics of breast cancer was analyzed by Chi-square test. The association with prognosis of breast cancer patients was determined by Kaplan–Meier survival analysis with Log-rank test. A Cox proportional hazard regression model was conducted to perform a univariate and multivariate analysis. All experiments were performed in triplicate, and replicate data were used to calculate means ± standard deviation (SD). ANOVA plus subsequent Dunnett post hoc tests were performed for multiple comparisons. If not specified above, a p-value less than 0.05 was considered to be statistically significant, and p-values were two-tailed.Statistical analysis was performed using R version 4.1.1 ( 3.34.0) . Accordi samples . Genes wBreast cancer data were collected from TCGA. Based on the screening principles, 1191 TP and 112 NT samples were selected into this study. DEGs were reflected in volcano plot . AccordiIn univariate Cox regression for overall survival (OS) model, we included variables that correlated with SLC7A11. The following multivariate analyses confirmed SLC7A11 expression, age, and N classification as independent prognostic factors after adjusting other prognostic indicators . AnalysiThen, we further define the relationship between ferroptosis and ER-positive cancers, and eight ferroptosis-related genes were analyzed according to The IR-induced ferroptosis in different breast cancer cell lines were evaluated in our previous study . Fer-1 wReferring to the above results, we considered that the relationship between SLC7A11 expression and breast cancer survival was probably based on ESR1. Furthermore, a positive correlation of ESR1 with SLC7A11 was found in ER-positive cancers  and the Then, we designed siRNA targeting ESR1 to verify the function of ESR1. To determine whether ESR1 could interact with SLC7A11, an IP experiment was carried out. Our result suggested that there was no direct protein–protein interaction . In ordeIn addition, the mRNA levels of SLC7A11 and ESR1 were evaluated by qRT-PCR based on time course. After exposure to X-ray, there was an increase in the mRNA levels of SLC7A11 and ESR1 . The proLipid peroxidation and cell death were assayed by flow cytometry using the fluorescent probes C11-BODIPY and trypan blue, respectively. ESR1 knockdown rendered ER-positive cells susceptible for ferroptosis and lipid peroxidation accumulation induced by IR. When ESR1 was knockdown, the increase of SLC7A11 induced by IR was shown to significantly attenuate in MCF7  and ZR-7On the basis of these findings, a rescue assay was conducted to confirm the role of ESR1 in SLC7A11-mediated IR-induced ferroptosis. As shown in For further verification of the ESR1 effect, we overexpressed ESR1 in ER-negative cells. The data showed that the upregulation of ESR1 could increase SLC7A11  and reschttp://ubibrowser.ncpsb.org.cn/ubibrowser/)  . Our datrowser/) . We thenrowser/) , suggestrowser/) . Taken tFerroptosis is a form of programmed cell death driven by cellular metabolism and iron-dependent lipid peroxidation, but its role in breast cancer remains unclear. Our study found that SLC7A11 was correlative to survival in breast cancer and high expression implied poor prognosis. Moreover, in multivariate analysis, high expression of SLC7A11 was an independent prognostic factor for OS of breast cancer. The prognostic analysis of SLC7A11 in subtypes showed that SLC7A11 is specifically associated with OS in ER-positive cancers. It is reported that SLC7A11 plays an important role in the protection of cells from oxidative stress and ferroptosis by promoting the uptake of cystine and the synthesis of GSH . Our resFerroptosis is characterized by the overwhelming iron-dependent accumulation of lipid hydroperoxides. Many reports provide definitive evidence for IR-induced lipid peroxidation, and ferroptosis is as important as other forms of cell death . IR induIn this study, we further investigated the mechanism of SLC7A11 degradation induced by IR. NEDD4L is an E3 ubiquitin ligase, containing four WW domains for substrate interaction . To the via two-way regulation of ESR1/NEDD4L to SLC7A11, and ESR1 might be a critical factor responsible for conferring the resistance to radiotherapy and tumor malignancy. We propose that targeting ESR1 in combination with radiotherapy could enhance the radiotherapeutic efficacy of ER-positive cancers.In conclusion, the downregulation of ESR1 leads to a decrease in SLC7A11 expression, resulting in acceleration of IR-induced ferroptosis. Our findings reveal a novel insight that IR-induced ferroptosis was


A central characteristic of Alzheimer’s disease (AD) and other tauopathies is the accumulation of aggregated and misfolded Tau deposits in the brain. Tau-targeting therapies for AD have been unsuccessful in patients to date. Here we show that human polymerase δ-interacting protein 2 (PolDIP2) interacts with Tau. With a set of complementary methods, including thioflavin-T-based aggregation kinetic assays, Tau oligomer-specific dot-blot analysis, and single oligomer/fibril analysis by atomic force microscopy, we demonstrate that PolDIP2 inhibits Tau aggregation and amyloid fibril growth in vitro. The identification of PolDIP2 as a potential regulator of cellular Tau aggregation should be considered for future Tau-targeting therapeutics.  Aging-related neurodegenerative diseases are a worldwide problem with limited therapeutical solutions. Even for the most commonly diagnosed neurodegenerative diseases, such as Alzheimer’s disease (AD) and Parkinson’s disease (PD), there are no effective therapies available that would directly target the cause of the disease. Amyloid β (Aβ), α-synuclein and Tau  are aggregation-prone proteins that play an important role in disease development ,2,3,4. Tau is a natively unstructured protein and is required for stable microtubule function. Under certain conditions, Tau is dissociated from the microtubules and self-aggregates to form soluble oligomers ,5. ThesePolymerase δ-interacting protein 2  has recently been identified as a specific binding partner of Tau monomers, but not Tau oligomers or fibrils . PolDIP2Here, we studied Tau aggregation in the absence and presence of PolDIP2 using thioflavin T (ThT)-based aggregation assays, Tau oligomer-specific dot-blot analysis, and single oligomer/fibril analysis by atomic force microscopy (AFM). We show that PolDIP2 inhibits aggregation and amyloid fibril development of the Tau protein. E. coli ArcticExpress (DE3) cells. An overnight culture was grown at 30 °C, and used for inoculation of 1.5 L terrific broth (TB) supplemented with 50 µg/mL ampicillin that was incubated at 30 °C until A600 0.8. The temperature was then reduced to 12 °C and protein expression induced by addition of 0.4 mM IPTG. After 42 h of incubation, cells were collected by centrifugation at 5000× g for 10 min at 4 °C. Pellets were washed with ice-cold PBS and then resuspended in lysis buffer A . Cells were lysed by pulse-sonication of cell suspensions on ice, and cleared by centrifugation at 25,000× g for 30 min at 4 °C. Cell lysates were supplemented with 10 mM imidazole and incubated with Ni-Sepharose excel resin  for 2 h at 4 °C. The beads were washed with buffer B  with 20 mM imidazole, buffer B with 50 mM imidazole, and the proteins were eluted with buffer B containing 500 mM imidazole. Elution fractions containing PolDIP2 were incubated with TEV protease at 4 °C and dialyzed into buffer B with 20 mM imidazole. The cleaved His peptides were removed by passing the protein solution though the Ni-Sepharose column once more. Then the flow through fraction from the Ni-Sepharose column was concentrated using Vivaspin 20 (30 kDa cut-off) and further purified by gel filtration using a Superdex 200 Increase 10/300 GL column using buffer C . Protein samples were frozen in liquid N2 and stored at −80 °C.For the purification of human PolDIP2, a DNA fragment encoding PolDIP2 amino acids 59 to 355 was cloned in the pET His_1a plasmid, obtaining a TEV-cleavable N-terminally 6xHis-tagged PolDIP2. pET His_1a-PolDIP2 was used to transform E. coli BL21(DE3) OneStar cells and cultured on a petri plate with LB agar supplemented with 100 µg/mL ampicillin and incubated overnight at 37 °C. A colony was inoculated into 50 mL LB supplemented with 100 µg/mL ampicillin and grown overnight at 37 °C. An overnight culture was used for 100-fold inoculation of 3.6 L ZYM-5052 autoinduction medium supplemented with 100 µg/mL ampicillin that was incubated at 37 °C until A600 0.8–1.0. The temperature was then reduced to 25 °C and the culture was incubated for 12 h total. Cells were collected by centrifugation at 6000× g for 20 min at 4 °C. Cell pellets were homogenized in buffer A  and lysed by sonication of cell homogenate on wet ice. The lysed cell suspension was then supplemented with 5 mM MgCl2 and incubated with DNAse I for 30 min with constant mixing at 4 °C. The suspension was cleared by centrifugation at 30,000× g for 35 min at 4 °C. The supernatant was then filtered (0.45 μm pore size syringe filter) and loaded on the BabyBio Ni-NTA column . The column was washed with a 2% gradient of buffer B , and the Sumo-Tau protein was eluted with a 20% buffer B gradient. Elution fractions containing the target protein were pooled, imidazole was removed by loading fractions onto a HiTrap Desalt column  and washed with buffer C . Desalted Sumo-Tau protein fractions were mixed with ULP1 Sumo protease , purified in a single step with Ni-NTA column) to a 1000:1 ratio and incubated for 3 h at 4 °C. Cleaved His-Sumo peptides and ULP1 protease were removed by loading the protein mixture on a HisTrap™ FF Column  and washing with a buffer C and 4% buffer D  gradient. Collected fractions containing Tau were concentrated using Amicon® Ultra-15, 10 kDa , and further purified by gel filtration using a Superdex 200 Tricorn 10/300 column  with buffer E . Fractions containing the target protein were pooled and desalted again using a HiTrap Desalt column  with buffer F . Protein fractions were collected, lyophilized and stored at −20 °C.For the purification of the human Tau protein (isoform 2N4R), a Champion™ pET302/NT-His plasmid carrying a DNA fragment encoding the Tau sequence fused with a SUMO protein  was ordeReaction buffer PBS-TCEP, pH 6.7, was prepared by diluting 0.5 mM TCEP in PBS, pH 7.4, filtered through a sterile 0.22 μm pore size PES membrane filter, and stored at −80 °C. The ThT solution was prepared as described previously ,11. HepaThe mixture (500 µL) of 10 µM PolDIP2 and/or 10 µM Tau were prepared in PBS-TCEP buffer supplemented with 5.3 µM heparin, and incubated at 37 °C for 5 min. Proteins were then separated using a Superdex 200 Increase 10/300 GL column with PBS-TCEP buffer, and the fraction were analyzed by SDS-PAGE and staining using PageBlue™ Protein Staining Solution .The reaction solution was prepared in 1.5 mL tubes at room temperature as previously described , and conhttps://imagej.nih.gov/ij/, accessed 31 October 2020).The reaction solution was prepared in a 1.5 mL tube at room temperature. First, Tau and/or PolDIP2 were diluted in PBS-TCEP buffer. Second, freshly prepared heparin solution was added to a final concentration of 8 µM. After centrifugation, the samples were transferred to a 96-well plate . The plate was sealed and placed into a Tecan Infinite F200 fluorescence microplate reader and incubated at 37 °C with orbital shaking (3 mm amplitude and 5 s duration). At each time point , 1 µL aliquots were withdrawn from the reaction mixture and spotted on an Amersham Protran 0.45 NC nitrocellulose Western blotting membrane . Next, the membrane was dried and blocked with 5% milk solution and incubated overnight at 4 °C with rabbit anti-Tau oligomer antibody T22 (a gift from Prof. Rakez Kayed) ,14. The 2O and dried overnight at room temperature. AFM imaging was performed on a PicoPlus AFM  in tapping mode in air, using a 100-µm scanner. We used a resonance frequency in the 170–190 kHz range, a scan rate of 1 Hz and a resolution of 512 × 512 pixels. We also used a BioScope Catalyst AFM , in peak force tapping mode in air. The scan rate was 0.51 Hz, scan size 5 µm and resolution 512 × 512 pixels. Bruker MSLN and SLN cantilevers were used in all measurements [Each sample was diluted 5 times with PBS-TCEP, and 2 µL of the solution was then spotted on the surface of freshly cleaved mica, followed by incubation for >30 s. The surface was washed 3 times with 100 µL of dHurements .The reaction mixture, containing 5 μM PolDIP2, 0/8/40 μM heparin and PBS-TCEP in a total volume of 50 μL, was incubated for 30 min at 37 °C with intermittent mixing. As a positive control, human polymerase γ subunit B was used in place of PolDIP2 in the assay. After the incubation, heparin sepharose beads were pelleted, and 3 μL of the supernatant, containing the unbound protein, was removed for SDS-PAGE analysis.A cell-based screen found that human PolDIP2 interacts with Tau monomers , and sugSeveral specific peptides or antibodies have been identified with the capability to inhibit or modulate Tau aggregation to decrease toxic oligomers or fibrils formation ,17,18; hAs observed in We also observed that PolDIP2 by itself increased the ThT signal , suggestIn conclusion, the ThT experiments suggest that PolDIP2 specifically interacts with Tau and inhibits its aggregation. To validate our ThT-based assays, we in parallel used a Tau oligomer-specific (T22) antibody-based approach B 13,14,,14,23. ITo characterize the fibril structures formed by Tau in the presence and absence of PolDIP2, we performed a single oligomer/fibril analysis by AFM . AFM imaIn agreement with the dot-blot and ThT assays, AFM analysis revealed that Tau fibril formation was severely inhibited in the presence of PolDIP2 and “globular oligomers”  in the presence of heparin, which gradually develop into longer fibrils (fibril growth) . PolDIP2275VQIINK and 306VQIVYK, which have the highest β-propensity at the beginning of the 2nd and 3rd repeat domains in Tau. Pir et al. showed that the hexapeptides VQPINK and VQPVYK decrease the rate of Tau aggregation in a concentration-dependent manner [277VQLRER) (277VQLRER) could be used in future experiments to test this idea. It also would be interesting to test whether PolDIP2 can inhibit the aggregation of Tau, which is known to occur via the liquid–liquid phase separation (LLPS) process. In this process, the intrinsically disordered regions of Tau form local droplet structures via LLPS, which could also play a role in neuronal Tau aggregation [Previous studies identified specific antibodies or peptides that inhibit Tau aggregation through Tau interaction ,17,18. T7VQLRER) , making regation ,26.Recent studies using cryo-EM  showed t


The three members  of the growth arrest and DNA damage-inducible 45 (GADD45) protein family are involved in a myriad of diversified cellular functions. With the aim of unravelling analogies and differences, we performed comparative biochemical and biophysical analyses on the three proteins. The characterization and quantification of their binding to the MKK7 kinase, a validated functional partner of GADD45β, indicate that GADD45α and GADD45γ are strong interactors of the kinase. Despite their remarkable sequence similarity, the three proteins present rather distinct biophysical properties. Indeed, while GADD45β and GADD45γ are marginally stable at physiological temperatures, GADD45α presents the Tm value expected for a protein isolated from a mesophilic organism. Surprisingly, GADD45α and GADD45β, when heated, form high-molecular weight species that exhibit features (ThT binding and intrinsic label-free UV/visible fluorescence) proper of amyloid-like aggregates. Cell viability studies demonstrate that they are endowed with a remarkable toxicity against SHSY-5Y and HepG2 cells. The very uncommon property of GADD45β to form cytotoxic species in near-physiological conditions represents a puzzling finding with potential functional implications. Finally, the low stability and/or the propensity to form toxic species of GADD45 proteins constitute important features that should be considered in interpreting their many functions. The growth arrest and DNA damage-inducible 45 (GADD45) gene family encodes three strictly related proteins, denoted as GADD45α, GADD45β, and GADD45γ ,2,3,4,5.Recombinant GADD45α, GADD45β, and GADD45γ were obtained in amounts sufficient for performing all subsequent characterizations. The proteins were initially purified by affinity chromatography exploiting the presence of the histidine tag located at the N-terminus. SEC was used to remove aggregates or residual contaminants. All proteins had purities higher than 95% as estimated from by SDS–PAGE analysis . The molIn order to assess their folding state, the three proteins were analyzed by far-UV CD spectroscopy. The resulting CD spectra of GADD45α, GADD45β, and GADD45γ clearly indicate that all of them were well folded a. Indeed−9 M, a value in line with the one previously determined by SPR (6.0 × 10−9 M) [−8 and 1.5 × 10−8, respectively. These values indicate a significant reduced affinity of this isoforms for MKK7_KD compared to GADD45β. These findings indicate that all GADD45 isoforms share the ability to bind the MKK7 kinase domain, although GADD45α and GADD45γ present a 10-fold reduced affinity compared to GADD45β.Once assessed that recombinant GADD45α, GADD45β, and GADD45γ were properly folded, we comparatively evaluated their ability to bind the kinase domain of MKK7, so far described only for GADD45β ,18. BindThe analysis of the stability against temperature was performed by a CD signal at 222 nm in the temperature range 20–100 °C. In all cases, structural transitions were observed at temperatures much lower than 100 °C. Moreover, the strong CD signals detected after the transition indicates that all proteins remained soluble in the explored temperature interval. Despite these analogies, the thermal denaturation experiments highlighted significant differences among GADD45 proteins a. IndeedInterestingly, the analysis of the structural properties of GADD45α, GADD45β, and GADD45γ states obtained upon temperature treatment unravels peculiar and sequence-specific behaviors. As expected, the CD spectra of the three proteins recorded at 100 °C b are radA number of experiments were designed and performed to investigate the structural features of GADD45α/GADD45β states generated by the thermal treatment. To gain insights into the structural features of the β-rich aggregates formed by GADD45α and GADD45β, we evaluated their ability to interact with ThT, a probe widely used to assess the formation of amyloid-like assemblies . ThT emiSince amyloid-like aggregates are frequently cytotoxic, we monitored the effects exerted by exposing human neuroblastoma (SHSY-5Y) and hepatoma (HepG2) cells to GADD45α/GADD45β aggregates and to the three proteins in the native states. Possible dose–response effects were evaluated at different protein concentrations  monitoring cell viability at 24 and 48 h. As shown in In its traditional version, the function–structure paradigm states that proteins acquire their functionality upon folding in a well-defined three-dimensional state. However, fundamental discoveries made in the last decade have progressively reshaped this concept. Indeed, it has been demonstrated that proteins frequently assume, in their functional form, an ensemble of states rather than a single structure. Moreover, it was found that significant portions of proteins are intrinsically disordered, and operate dynamically . FinallyThe findings emerged from the biophysical characterization of the proteins are particularly surprising as they demonstrate that GADD45β and GADD45γ are only marginally stable at physiological temperature, whereas GADD45α presents a Tm value expected for a protein isolated from a mesophilic organism. Even more intriguing are the structural properties of the species formed at high temperatures. While GADD45γ retains a significant level of secondary structure at high temperature and is able to significantly regain the original folding upon cooling, GADD45α and GADD45β when heated form aggregated species enriched in β-structure, despite the remarkable content of α-helix in their native structures. Moreover, the spectroscopic characterization of these aggregates clearly indicates that they possess amyloid-like features as they bind the dye ThT and present the characteristic intrinsic UV/blue fluorescence emission ,23,24. AThe concomitant occurrence in GADD45β of a very limited thermal stability and of a remarkable propensity to form cytotoxic amyloid-like assemblies may appear a puzzling observation. Based on these observations, we hypothesize that the toxic species formed by GADD45β at physiological temperatures might represent a protection mechanism toward the pro-survival role played by this protein in several tumor tissues . SimilarThe rare propensity of GADD45β to form potentially cytotoxic species in near-physiological conditions may also be connected to some other functional features of the protein. In particular, it can be hypothesized that the observed propensity of GADD45β to establish biological partnerships and its limited expression levels may be effective ways to avoid the formation amyloid-like toxic assemblies. Alternatively, its tendency to form aggregates might represent a signal for its clearance when it is no longer needed or when its concentration exceeds harmful levels. Indeed, intracellular protein aggregates can be efficiently removed by a number of distinct mechanisms that include autophagy or secretion outside of the cells . In conclusion, the low stability and/or the propensity to form toxic species, as here revealed though biophysical/biochemical studies might represent crucial properties of GADD45 proteins that must be considered in interpreting their many biological functions. The present finding also represent a stimulus for further investigations aimed at detecting the potential aggregation of these proteins in vivo. Therefore, the analysis of the GADD45β aggregation, which could also be modulated using inhibitors of amyloid-like aggregation ,30,31, iE. coli BL21(DE3) cells, which were grown at 37 °C until 0.6–0.8 OD. Protein expression was induced by the addition of 0.5 mM isopropyl-D-thiogalactoside for 16 h at 22 °C. The cultures were harvested by centrifugation for 15 min at 4 °C and 6000 rpm. The pellets were re-suspended in lysis buffer  and sonicated for 20 min . Supernatants were harvested by centrifugation, for 30 min at 4 °C at 16,500 rpm. Soluble fractions were loaded on a Ni2+- NTA resin  previously equilibrated with lysis buffer. Proteins were eluted increasing the imidazole concentration. GADD45α, GADD45β, and GADD45γ were loaded onto a Superdex 200 16/60 column  connected to a AKTA Purifier system . The column was equilibrated in a buffer containing 20 mM Tris-HCl pH 7.5, 100 mM NaCl, 5 mM DTT, 5% glycerol. Yield and the purity of proteins were assessed by 15% SDS-PAGE analysis. Concentration was determined by the Bradford assay using BSA as reference. The recombinant kinase domain of human MKK7 (hereafter MKK7_KD) was obtained as His-fusion protein as previously reported [The genes of human GADD45α and GADD45γ were purchased from Sigma Aldrich and cloned into the pETM-13 expression vector using Bam-H1 and XhoI enzymes. Human GADD45β (hereafter only GADD45β) was cloned into a pET-28a+ vector as previously reported. GADD45α, GADD45β, and GADD45γ were expressed using reported ,33.v/v) 5% glycerol as running buffer, at a flow rate of 0.5 mL/min. Data were recorded and analyzed using the Astra software . Molecular size dispersion as a function of the hydrodynamic radius (Rh) and the aggregation status were determined by DLS. These experiments were performed at 25 °C using a Zetasizer Nano ZS  equipped with a 173° backscatter detector. Measurements were performed in triplicate using recombinant proteins at a concentration of 10 μM before and after thermal denaturation. Data were analyzed using the OMNISIZE software . Light scattering measurements were performed using both a size exclusion chromatography coupled with light scattering (SEC-LS) and dynamic light scattering (DLS). SEC-LS was conducted using a semi-preparative SEC column  coupled to a light scattering detector  and to a differential refractive index detector . The purification was conducted loading 0.5–1.0 mg of homogeneous sample on the column equilibrated in 20 mM Tris–HCl pH 7.5, 100 mM NaCl, 5 mM DTT, .Far-UV circular dichroism (CD) spectra of the proteins were recorded on a J-810 spectropolarimeter equipped with a Peltier temperature control system . In all experiments, the protein concentration was 10 μM. Measurements were carried out in the 198–260 nm range at 20 °C using a 0.1 cm optical path length cell with the following parameters: 4 s as time constant, a 2 nm bandwidth, and a scan rate of 20 nm min® RED96 System (AlfaTest) equipped with an array of eight independent probing BLI needles. Briefly, GADD45 proteins were dialyzed in 0.01 M HEPES pH 7.4, 0.15 M NaCl, 3 mM EDTA, 0.005% v/v Surfactant P20, 2 mM DTT (running buffer) and opportunely biotinylated using EZ-Link Sulfo-NHS-LC-Biotinylation Kit  following the manufacturer instructions. Subsequently, the biotinylated proteins were efficiently immobilized on the super streptavidin chip  achieving a similar immobilization level on all sensors (2.5 nm). Dose–response assays were simultaneously carried out using MKK7_KD solutions at concentrations ranging between 9 and 300 nM, opportunely diluted in the running buffer. Data were analyzed using the Octet Data Analysis software and fitted by a 1:1 binding model.Bio-Layer Interferometry (BLI) was used to quantify the affinity and the selectivity of binding of the GADD45 proteins to MKK7_KD. The BLI experiments were carried out using an OctetThioflavin T (ThT) fluorescence assays were carried out using a Varian Cary Eclipse spectrofluorometer . The protein samples were placed in a quartz cell of 10 mm path-length at 10 μM concentration. A 440 nm radiation was used for the excitation while the emission was recorded in the interval 450–600 nm.2 in the Dulbecco’s modified medium (DMEM) supplemented with 10% fetal bovine serum, 1% glutamine, 100 U/mL penicillin, and 100 µg/mL streptomycin . For the MTT -2,5-diphenyltetrazolium bromide) assays 104 SH-SY5Y and HepG2 cells were seeded in 96-well plates. Pre-heated GADD45α, GADD45β, and GADD45γ were added at 37 °C to the cells at different concentrations  for either 24 or 48 h. Cell viability was assessed by the MTT reduction assay [v/v) Triton-X100 and 8% (v/v) of a 1 M HCl solution. Absorbance of blue formazan was determined at 570 nm. Cell viability was expressed as the percentage of MTT reduction in treated cells compared to that observed in untreated cells.Human SH-SY5Y neuroblastoma cells and human HepG2 hepatocellular carcinoma cells  were grown at 37 °C in a humidified atmosphere with 5% COon assay . In brie


Cytosolic sulfotransferases (SULTs) catalyze the transfer of a sulfonate group from the cofactor 3’-phosphoadenosine 5’-phosphosulfate to a hydroxyl (OH) containing substrate and play a critical role in the homeostasis of endogenous compounds, including hormones, neurotransmitters, and bile acids. In human, SULT2A1 sulfonates the 3-OH of bile acids; however, bile acid metabolism in mouse is dependent on a 7α-OH sulfonating SULT2A8 via unknown molecular mechanisms. In this study, the crystal structure of SULT2A8 in complex with adenosine 3’,5’-diphosphate and cholic acid was resolved at a resolution of 2.5 Å. Structural comparison with human SULT2A1 reveals different conformations of substrate binding loops. In addition, SULT2A8 possesses a unique substrate binding mode that positions the target 7α-OH of the bile acid close to the catalytic site. Furthermore, mapping of the critical residues by mutagenesis and enzyme activity assays further highlighted the importance of Lys44 and His48 for enzyme catalysis and Glu237 in loop 3 on substrate binding and stabilization. In addition, limited proteolysis and thermal shift assays suggested that the cofactor and substrates have protective roles in stabilizing SULT2A8 protein. Together, the findings unveil the structural basis of bile acid sulfonation targeting 7α-OH and shed light on the functional diversity of bile acid metabolism across species. Bile acids play an essential role in intestinal absorption of lipids and function as signaling molecules and activators of nuclear receptors that regulate lipid, cholesterol, glucose, energy, and bile acid metabolism , 4, 5, 63−) group from the universal cofactor 3’-phosphoadenosine-5’-phosphosulfate (PAPS) to a hydroxyl (OH) group of the substrate to generate a sulfonated product (Cytosolic sulfotransferases (SULTs) are a large family of enzymes catalyzing the sulfonation of both xenobiotics and endogenous compounds, including hormones, neurotransmitters, and bile acids , 16, 17. product , 19, 20. product . Protein product , 26, 27. product , 30, 31, product , 33, 34.Sult2a genes (mSult2a1-mSult2a7) have been previously identified, their transcriptional expression is suppressed or silenced in adult mice except mSult2a7 , hSULT2A1 is the sole isoform of SULT2A subfamily possessing a broad substrate reactivity toward numerous endogenous hydroxysteroids and bile acids , 36. hSUmSult2a7 , and themSult2a7 , 39, 40.ale mice , indicatale mice . In addise liver . Recent se liver , and these liver . Our in gated CA , a predogated CA . TogetheAccumulating studies have demonstrated the mechanism of 3-OH sulfonation , 48, 49;S-transferase (GST)-tagged SULT2A8. This recombinant plasmid was also used for site-directed mutagenesis to generate various SULT2A8 mutants for biochemical assays as indicated in the text. All the plasmids were confirmed by Sanger sequencing and transformed into Escherichia coli BL21(DE3) for protein expression. Cells were grown in lysogeny broth medium at 37°C, and protein expression was induced with 0.2 mM isopropyl β-d-1-thiogalactopyranoside for 16 h at 20°C.The truncated form of SULT2A8 encoding residues 4–282 was amplified from pRSET-SULT2A8 fusion plasmid previously described  using thN-lauroylsarcosine at room temperature for 30 min. Cell debris was pelleted by centrifugation at 20,000 g for 20 min prior to brief sonication. The supernatant was added with 2% Triton-X100 and 20 mM CHAPS before binding to Glutathione Sepharose 4B  at room temperature for 30 min. The beads were washed with the same buffer, and GST tag was cleaved with PreScission protease at 4°C for overnight. SULT2A8 protein was purified by a HiLoad 16/60 Superdex 75 prep grade column  with 20 mM Hepes, pH 8.0, 150 mM NaCl, and 4 mM 1,4-dithiothreitol. Fractions were pooled and concentrated for crystallization and activity assays.SULT2A8 was purified from inclusion bodies as previously described with slight modifications . Briefly2). Crystals were grown with the sitting-drop vapor diffusion method at 16°C and cryoprotected by soaking in reservoir buffer containing 20% glycerol prior to flash freezing. Diffraction data were collected using in-house FR-E+ SuperBright generator (Rigaku) equipped with an R-AXIS IV++ IP detector (Rigaku). Data were processed and integrated using iMosflm (3F3Y) as a searching model. mSULT2A8 model was built and refined with Coot . The initial hit (condition G10 in Index screen) was optimized with grid screening and additive . Higher quality crystals were obtained by 2:1 mixing of ligands and 3% 1,4-dioxane-incubated SULT2A8 protein with reservoir buffer  at a flow rate of 1.0 ml/min on a reverse phase XBridge BEH C18 column  (Waters). The column temperature was 30°C, and peaks were detected at 254 nm wavelength (HPLC assays were performed with a 1260 Infinity II LC System equipped with a 1290 Infinity II Diode Array Detector (Agilent). Injection volume was 25 μl, and analytes were separated with methanol-water  containing 75 mM KHvelength . The peavelength .2, and 4 mM 1,4-dithiothreitol. The reactions were adjusted to a final volume of 20 μl and incubated at room temperature for 30 min. Digestion was terminated with SDS-PAGE loading dye and heated. The digestion reactions were then analyzed by SDS-PAGE and Coomassie blue staining.Limited proteolysis of SULT2A8 was performed in the presence/absence of 500 μM PAP and/or sodium chenodeoxycholate (Na CDC). In brief, 20 μg of purified SULT2A8 was incubated with different concentrations of trypsin  in digestion buffer 90 mM Tris, pH 8.5, 2 mM CaClTm). Briefly, 500 μg/ml of protein in 20 mM Hepes, pH 8.0, 150 mM NaCl, and 4 mM 1,4-dithiothreitol was incubated with 250 mM of PAP and/or tested compounds for 20 min on ice. This was followed by mixing 20 μl of prepared protein with 5 μl of 500-fold diluted JBS Thermofluor Dye (Jena Bioscience) in the same buffer. Control experiments were set using buffer and dye mixture without proteins. Sample mixtures were initially incubated at 4°C for 2 min, followed by heating in a ramp rate of 1°C/min starting from 4 to 95°C with a CFX96 Touch Real-Time PCR Detection System (Bio-Rad). The excitation wavelength was set at 483 nm, whereas emission wavelength was 568 nm. Nonlinear fit was solved, and Tm was calculated by a Boltzmann sigmoidal equation with XLfit (IDBS).The effects of bile acids and steroids on SULT2A8 protein stability were examined by a fluorescent-based thermal shift assay . The prot-test using GraphPad Prism (GraphPad Software). P < 0.05 is considered as statistically significant, and P values were indicated categorically: ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, and ∗∗∗∗P < 0.0001.Biochemical results are expressed as mean ± standard deviation, and differences were compared with the unpaired Student's 2 with two copies of SULT2A8 per asymmetric unit and Matthews coefficient of 2.51 Å3Da−1. Analysis of the integrated data with Xtriage in Phenix suggested a possibility of twinning with twinning fraction of 0.44  and a twin law . After phase determination by molecular replacement, this twin law was applied in the refinement. The model of SULT2A8 was finally refined to Rwork/Rfree value of 23.85%/27.15% with both PAP and CA successfully built in each chain. The two chains of SULT2A8 are almost identical with root mean square deviation of Cα atoms of 0.486. There is no interpretable electron density for residues 227–231 in chain A and residues 90–93, 128–141, 229–238, and 277–280 in chain B, indicating that these regions are flexible. The structural analysis reported here is mainly based on the more complete chain A model.In our earlier efforts, full-length His-tagged SULT2A8 was cocrystallized with PAP or both PAP and Na CDC; however, the crystals were only diffracted to resolutions of 3.6 and 7.9 Å, respectively. A new SULT2A8 expression plasmid with a cleavable GST fusion tag was therefore constructed. This new SULT2A8 construct that has the first three residues from the N terminus removed, when cocrystallized with both PAP and CA, resulted in needle-like crystals. Grid screening on pH produced cube-shaped crystals from pH 4.9 to 5.8. A 2.5 Å complete dataset was collected from a crystal obtained under pH 5.1 . SULT2A8The overall structure of SULT2A8 is a classical SULT α/β fold comprised of a central four-stranded parallel β-sheet flanked by 14 α-helixes A. SULT2A3F3Y) (3) (3)   across a3F3Y) A. In mSU3F3Y) . In addiF3Y) (3) B compare (3) (3) C.Fig. 2CFo-Fc electron density map for the substrate CA was clear that the 3α-, 7α-, and 12α-OH ends of CA could be well modeled into the map , a bile acid lacking both 7α- and 12α-OH groups C, D, fur4IFB) . The conventionally accepted mechanism is that residue His99 acts as a catalytic base for the deprotonation of the target OH of the substrate with subsequent nucleophilic attack of the sulfur atom of PAPS . The hyd4IFB) A. Of intet al.  were added. However, incubating SULT2A8 with both PAP and nonsubstrates, such as LCA, DHEA, androsterone, or pregnanolone, showed comparable thermal denaturation profile to that of PAP only. Compared with apo WT protein, all Tm shifts were statistically significant. These results suggest that both cofactor and substrate protect SULT2A8 from unfolding.To further explore the roles of cofactor and substrates on SULT2A8 stability, thermal stability shift assays were carried out B supple. AdditioTm values of these mutants were significantly increased when PAP was included. When both PAP and native substrates were present, further increase of Tm was observed in mutants H48T, L99V, and L99T. These thermal stability profiles are similar to those of the WT enzyme. Remarkably, the presence of PAP did not enhance the thermal stability in mutant K44A, further confirming that conserved Lys44 is critical for cofactor binding. Interestingly, although the Tm value of N72W was increased when PAP was added, no further changes in Tm were detected upon coincubation with substrates (native or non-native). For mutations on loop 3, mutants C232S and H239G in apo form exhibited similar Tm as that of WT, whereas mutant E237A showed a drastic reduction of Tm. This result suggests that Glu237 plays a critical role in stabilizing SULT2A8 structure. In addition, incubating E237A with native or non-native substrates failed to improve the protein stability. These results further indicate that interaction of the enzyme with bile acids is mediated by Glu237 on loop 3.Thermal denaturation transition profiles of mutants K44A, H48T, N72W, L99V, and L99T were also obtained. Similar to the WT enzyme, the KR value of histidine (pKR = 6.0), His48 would be in a protonated state at pH 5.5. This would then disagree with the role of His48 as a base to deprotonate 7α-OH of bile acids. However, the pKR value of His48 in apo SULT2A8 model calculated with PROPKA , indicating that the binding of cofactor and substrate modulate the actual pKR of catalytic histidine for the deprotonation of bile acids.Excessive accumulation of bile acids can lead to hepatobiliary change like cholestasis . SULT-meh PROPKA  program 3F3Y), this hydrophobic residue locates at the hinge of loop 3 and may be involved in maintaining the conformation of the loop. However, specifically how Ile236 in loop 3 modulates the substrate specificity is unclear. In our study, higher SULT2A8 activities toward taurine-conjugated bile acids compared with their nonconjugated forms were observed. We speculate that the relaxed conformation of loop 2 in SULT2A8 may facilitate the recognition of the side chain-conjugated bile acids. To test this, molecular docking of T-CA to SULT2A8 structure was performed by AutoDock Vina  deposited in Worldwide PDB (http://www.wwpdb.org/) with a generated validation report on the structure quality will be released upon the acceptance of the present article.The authors confirm that the data supporting the findings of this study are available within the article and its supplemental data. Protein structure (PDB ID: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this article.