Coelastrella terrestris (Chlorophyta) was collected from red mucilage in a glacier foreland in Iceland. Its morphology showed characteristic single, ellipsoidal cells with apical wart-like wall thickenings. Physiological characterization revealed the presence of the rare keto-carotenoid adonixanthin, as well as high levels of unsaturated fatty acids of up to 85%. Initial screening experiments with different carbon sources for accelerated mixotrophic biomass growth were done. Consequently, a scale up to 1.25 L stirred photobioreactor cultivations yielded a maximum of 1.96 mg·L−1 adonixanthin in free and esterified forms. It could be shown that supplementing acetate to the medium increased the volumetric productivity after entering the nitrogen limitation phase compared to autotrophic control cultures. This study describes a promising way of biotechnological adonixanthin production using Coelastrella terrestris.A novel strain of  They ars growth . Additios growth ,5. For ms growth ,7.The natural product portfolio of microalgae is extremely versatile. Whole biomass can be used as a human nutraceutical food source since it is rich in vitamins, essential amino acids and polyunsaturated fatty acids (FA) . It can Haematococcus lacustris, commonly known as Haematococcus pluvialis. Another microalgal SC with high potential in industry is adonixanthin [Secondary carotenoids (SC) can accumulate in certain cell compartments, e.g., cytosolic lipid bodies or in plastoglobuli after stress induction ,16. The ixanthin . It is aixanthin . Adonixaixanthin ,22. HoweCoelastrella terrestris  was characterized. Strain WP154.1 was sampled in Iceland and isolated from red mucilage in a small brook close to a glacier. The species was first described by Reisigl (1964) in the Austrian Alps and initially named Scotiella terrestris [Coelastrella were screened for pigment and lipid production. Hu et al. (2013) identified the pigment composition of the thermotolerant strain Coelastrella sp. F50 grown in glass bottles [Coelastrella sp. isolates and conducted Erlenmeyer flask cultures for pigment and lipid measurements [Coelastrella [C. rubescens NAMSU R1 and identified the carotenoids present after Erlenmeyer flask cultivations [Coelastrella was published by Minyuk et al. (2017) [C. rubescens Erlenmeyer flask experiments with inorganic CO2 or organic acetate as a carbon source (C-source) in combination with low-pH stress for SC production. However, they did not find any correlation of C-source and SC productivity, and mainly focused on the identification of the accumulated SC and lipids rather than the potential of biotechnological production. In this study a novel strain of the green alga rrestris . In rece bottles ; Minhas urements ; Wang etastrella ; Goecke astrella ; and Zayivations . However. (2017) . The groC. terrestris WP154.1 in terms of morphology and physiology, pigment content and fatty acid composition. The potential of this novel strain as alternative source of natural pigments, especially adonixanthin, was investigated for future biotechnological production. Thus, we performed screening experiments to find adequate C-sources for enhanced mixotrophic biomass formation compared to autotrophic growth. Subsequently, this knowledge was used for scaling up to 1.25 L PBR experiments. This study describes a biotechnological adonixanthin production process by C. terrestris. Our goal was to characterize strain C. terrestris WP154.1 appeared ellipsoidal or lemon-shaped, both apices showed wart-like wall thickenings. Their cell size ranged between 9 to 13 µm length and 7 to 10 µm width  are shown in For the quantification of the FA composition of −1 FAME were measured, of which 127.1 mg·g−1 (54.1%) were polyunsaturated (PUFA), 73.7 mg·g−1 (31.4%) monounsaturated (MUFA), and only 34.2 mg·g−1 (14.5%) of all FA were fully saturated . In total, 234.9 mg∙gaturated a. The moll FAMEs b. The se600) for each of the substrates including an autotrophic control culture were tracked for 37 d to identify the substrate for fastest mixotrophic growth (max) between the start of the experiment and day 10 were calculated. This time range was chosen, since all cultures still contained nitrate and supplemented C-source, and thus growth was not limited by nutrient starvation (data not shown).Erlenmeyer flask experiments were done to determine suitable C-sources with the major focus to find accelerated mixotrophic biomass growth compared to an autotrophic reference process. The investigated C-sources were the hexoses fructose and glucose, the pentoses ribose and xylose, as well as acetate and glycerol. Growth curves expressed by optical density values at 600 nm . Thus, subsequent mixotrophic cultivations were conducted with (i) glucose, (ii) acetate, which is a common C-source in algae cultivations, and (iii) an autotrophic control.The culture supplemented with glucose was growing fastest and resulted in a µfructose . The autC. terrestris, externally illuminated stirred PBRs were used. The experiments were conducted at two different nitrate levels of Bold’s Basal Medium (BBM) with standard BBM and BBM with doubled nitrate content (2N-BBM). Both media were supplemented with glucose or acetate as mixotrophic C-sources. For comparison, autotrophic controls were done resulting in a total of six controlled and scalable PBR cultivations. The algal biomass trends expressed as OD600 for these cultivations are shown in To scale up the cultivations of max) for BBM and 2N-BBM were highest in cultures containing glucose. The average µmax was 0.72 d−1 and 0.76 d−1, respectively. These were closely followed by the autotrophic controls with 0.63 d−1 and 0.68 d−1 for BBM and 2N-BBM, respectively. Both bioreactors containing acetate showed reduced µmax of 0.56 d−1 and 0.55 d−1. After a process time of 7 d, the nitrate was completely consumed in all cultivations, which led to reduced growth rates . 2N-BBM runs had more volumetric SC content with the exception of BBM glucose, which was overall third highest with 5.53 mg·L−1 SC. Lowest SC accumulating culture was BBM containing acetate (1.99 mg·L−1). Concerning adonixanthin, the maximum concentration of 1.96 mg·L−1 was detected in 2N-BBM with acetate, followed by the 2N-BBM control (1.64 mg·L−1). Relative ratios of the SC astaxanthin, adonixanthin, canthaxanthin and echinenone of all cultivations were calculated as 46 ± 5%, 28 ± 2%, 23 ± 5% and 2 ± 2%.Extracted pigments were individually quantified and subsequently grouped in the three categories Chl, PC and SC . PBR cul content b, the 2NSC for the 2N-BBM acetate culture with 0.43 mg·L−1·d−1. For adonixanthin, the highest volumetric (radonixanthin) and specific production rates (qadonixanthin) observed were 0.13 mg·L−1·d−1 and 0.06 mg·g−1·d−1 in the 2N-BBM acetate culture after stress induction.For evaluating the productivities of all SC and in particular adonixanthin, the corresponding volumetric (r) and specific production rates (q) were calculated . After 7Coelastrella terrestris  by molecular means. Light microscopical examinations confirmed that cytomorphological characteristics were in agreement with previous descriptions [Coelastrella [The green algal strain WP154.1 was identified as riptions ,30. The astrella . C. terrestris WP154.1 for both, scalable adonixanthin and polyunsaturated FA production. Due to its climate, Iceland seems to be a promising origin of microbial strains useful for biotechnology, as recently shown for another green microalga [This initial physiological characterization indicates great potential for future industrial use of icroalga .Axenic cultivation experiments started with a screening for suitable C-sources regarding accelerated mixotrophic biomass growth compared to autotrophic controls. Mixotrophy could solve the bottleneck for a future economically feasible process . The C-smax, which was 48% and 34% higher than the autotrophic control. All other C-sources caused reduced growth in relation to the control. Acetate, a commonly used C-source for algae, was lowering µmax by 40%. Ribose, xylose and glycerol were not metabolized by the cells, despite reports showing consumption by other green algal species [The hexoses glucose and fructose performed best concerning the growth rate µ species ,33,34. T2 in the used stirred PBR [−1 total SC and 1.96 mg·L−1 (31%) adonixanthin. Adonixanthin was approx. a factor of 11 higher compared to a recently published study of the green alga T. minimum, accumulating adonixanthin to a maximum volumetric content of 0.179 mg·L−1 [−1·d−1 and a factor of 6.9 to 0.06 mg·L−1·d−1, respectively. This increase was much higher compared to glucose and the autotrophic control and again ascribed to addition of acetate in the culture which applied osmotic stress. Furthermore, this explains why acetate is often the stress factor of choice, e.g., for SC production processes for astaxanthin [Coelastrella rubescens was not affected by osmotic stress caused by acetate [The PBR cultivation processes were designed to autoinduce SC production after nitrate in the medium was consumed. Growth rates in PBR were essentially higher compared to Erlenmeyer flask experiments. Comparing the BBM autotrophic cultures in both scales, the growth rate of the PBR cultivation was raised by a factor of 2.7. This was reasoned to be due to the improved light availability and enhanced mass transfer of gaseous COrred PBR ,35. In trred PBR . After prred PBR . During rred PBR . Althoug9 mg·L−1 . After eaxanthin . Additioaxanthin . This mi acetate .C. terrestris WP154.1 revealed high content of the rare keto-carotenoid adonixanthin which has reportedly great potential to treat cancer or protect against brain damage [Haematococcus lacustris; however, this cell stage has not been reported for any Coelastrella species yet [Microalgal biomass or extracted components are regarded as high-value products for human health and nutrition . Specifin damage ,44. To fn damage . Anothercies yet ,46.−2·s−1 (14 h light·d−1) on 1.6% agar plates with Bold’s Basal Medium (BBM), which contained 250 mg·L−1 NaNO3, 75 mg·L−1 MgSO4·7H2O, 25 mg·L−1 NaCl, 75 mg·L−1 K2HPO4, 175 mg·L−1 KH2PO4, 25 mg·L−1 CaCl2·2H2O, and 1 mL·L−1 of 1000× trace metal mix with a composition of 8.82 g·L−1 ZnSO4·7H2O, 1.44 g·L−1 MnCl2·4H2O, 0.71 g·L−1 MoO3, 1.57 g·L−1 CuSO4·5H2O, 0.49 g·L−1 Co(NO3)2·6H2O, 11.4 g·L−1 H3BO3, 50.0 g·L−1 Na2EDTA·2H2O, and 4.98 g·L−1 FeSO4·7H2O [The microalga was collected in Iceland in the foreland of Sölheimjökull glacier from a small brook where macroscopic mucilaginous mats of reddish cyanobacteria dominated. The GPS position was N63°31.890 W19°22.081 at approximately 120 m above sea level. The permission for collection in Iceland was granted by the National Energy Authority Orkustofnun, leyfisnúmer OS-2017_L020-01. Cells were isolated from field material by autotrophic cultivation at 15 °C and 30–40 µmol PAR·mSO4·7H2O . The unialgal culture WP154.2 was established by mechanical isolation of cell colonies with a sterile loop. Axenic cultures were established by a workflow utilizing fluorescence-activated cell sorting combined with plate spreading .C. terrestris WP154.2 (accession number OM574907.1) was done by molecular means with an 18S rDNA marker. The primers used for this were 18F2 (5’-AAC CTG GTT GAT CCT GCC AGT-3’) and 18R2 (5’-TGA TCC TTC TGC AGG TTC ACC TAC G-3’), as described by [Coelastrella terrestris strain CCALA 476 (accession JX513882.1) and Coelastrella terrestris strain KZ-5-4-9 (accession number MK231276.1). Both hits showed 100% identity at a query cover of 99%. The resulting phylogenetic tree was created by the NCBI tool via the neighbor joining method [Coelastrella. Its closest neighbors were Coelastrella terrestris strain CCALA 476 (accession JX513882.1) and Coelastrella terrestris strain KZ-5-4-9 (accession number MK231276.1).The strain identification of g method . It showFor light microscopical observations, a CKX41 inverted microscope  with 1000x total magnification, was equipped with an EOS 250D camera . The camera was operated via EOS utility software .a and b were analyzed photometrically [The whole procedure of pigment extraction, identification and quantification is described in detail elsewhere . Brieflytrically . The workflow for quantifying fatty acids as methyl esters was done according to . About 5−1 NaNO3 at pH 7.0. Additional C-sources fructose, glucose, ribose, xylose, acetate and glycerol were added to the final concentration of 1.0 g·L−1 before autoclaving. After sterilization, the vitamins were added via 0.22 µm syringe filter. All flasks were inoculated with 2 mL preculture grown in BBM in the middle of the exponential growth phase with OD600 about 0.5. The flasks were placed in a Minitron incubation shaker  at 20 °C, 150 rpm (25 mm amplitude) with 3% CO2-enriched atmosphere. The provided illumination expressed as photon flux density (PPFD) was 30 µmol PAR·m−2·s−1 for 14 h light·d−1. Samples were taken after 10 and 21 d. The biomass content was estimated in 1 mL cuvettes on a Nanodrop One photometer  by absorption measurements at 600 nm wavelength. Then, 1 mL aliquots were centrifuged, and the supernatant was stored separately for subsequent C-source and nitrate concentration measurements. Every cultivation experiment was carried out in biological duplicates and the respective average values were used for data evaluation.The experiments were conducted in 250 mL Erlenmeyer flasks. Every flask contained 50 mL standard BBM with 250 mg·LFor cultivations in stirred PBR, a Ralf multi-bioreactor system  was used . The gla−1 carbon in form of glucose (C6H12O6) or acetate (CH3COONa). Then, a 1.25 L prepared medium was filled into the PBR prior to autoclaving. After that, vitamins were injected aseptically via 0.22 µm syringe filter and a septum. The gas was mixed to 3.0% CO2-enriched air and provided at a rate of 0.1 vvm. Cultivation temperate was 20 °C and the stirrer agitated with 300 rpm. The provided PPFD in the center of the reactor was about 170 µmol PAR·m−2·s−1 during the first 72 h of the process and then changed to 850 µmol PAR·m−2·s−1, measured by an US-SQS/L sensor  in a cell-free medium. The light was automatically simulating a 14 h light·d-1 cycle. The pH was set to 6.3 ± 0.05 via automatic addition of 1 mol·L−1 Na2CO3 prior to inoculation. Roughly 20 mL preculture, also grown in BBM, was added via a syringe and septum. The final OD600 after inoculation was between 0.02 and 0.04. The experiment was started when the respective culture reached OD600 of 0.10 ± 0.01 to avoid possible preculture differences. Regular samples for OD600 ) and nitrate quantification were drawn on days 0, 1, 2, 3, 4, 5, 6, 7, 9, 11, 13, 15 and 21. Then, 20 mL biomass samples for pigment quantification were harvested by centrifugation and lyophilized on days 7, 11, 15 and 21. Specific exponential growth rate (µmax) was calculated with OD600 at a certain process time  and compared to the initial value  (Equation (1)):Standard BBM, or BBM with double nitrate content (2N-BBM), was directly used or supplemented with 33.3 mmol·LrP) between two time points (t1 and t2) was calculated after product concentration measurements of the product of interest at the same time points :qP), the biomass (X) was considered additionally (Equation (3)):The average volumetric production rate of a product   by an Aminex HPX-87 H column . The method was recently described in detail . CalibraThe nitrate content in the cultures were determined in the supernatant after centrifugation of the samples. The exact method was published elsewhere . A Dione


XRD studies and diffuse reflectance UV–vis spectral analysis confirmed the formation of single, tetragonal scheelite-type phases with space group I41/a and a direct optical band gap above 3.5 eV. Magnetic and electrical measurements showed insulating behavior with n-type residual electrical conductivity, an almost perfect paramagnetic state with weak short-range ferromagnetic interactions, as well as an increase of spin contribution to the magnetic moment and an increase in the power factor with increasing gadolinium ions in the sample. Broadband dielectric spectroscopy measurements and dielectric analysis in the frequency representation showed a relatively high value of dielectric permittivity at low frequencies, characteristic of a space charge polarization and small values of both permittivity and loss tangent at higher frequencies.A series of Co A number of these compounds have been developed in the field of scintillation detectors 0.50(WO4)0.50  showed a weak p-type electrical conductivity and the paramagnetic state with a change in the short-range interactions from ferromagnetic to antiferromagnetic as well as an increase in the orbital contribution to the magnetic moment with increasing Mn2+ content [3+-doped lead molybdato-tungstates with the chemical formula of Pb1−3xxGd2x(MoO4)1−3x(WO4)3x  showed a paramagnetic state with characteristic superparamagnetic-like behavior. Moreover, they revealed faster and slower relaxation processes in the loss spectra with various time scales for the gadolinium-poorer samples and no signs of any relaxation processes for the gadolinium-richer ones [d transition metal ions or 4f rare earth ones significantly affect the electrical and magnetic properties of molybdato-tungstates.Recently, studies of manganese doped calcium molybdato-tungstates with the chemical formula of Ca content . Next, s1−xMnx(MoO4)0.50(WO4)0.50  showed a weak p-type electrical conductivity and the paramagnetic state with a change in the short-range interactions from ferromagnetic to antiferromagnetic as well as an increase in the orbital contribution to the magnetic moment with increasing Mn2+ content [3+-doped lead molybdato-tungstates with the chemical formula of Pb1−3xxGd2x(MoO4)1−3x(WO4)3x  showed a paramagnetic state with characteristic superparamagnetic-like behavior. Moreover, they revealed faster and slower relaxation processes in the loss spectra with various time scales for the gadolinium-poorer samples and no signs of any relaxation processes for the gadolinium-richer ones [d transition metal ions or 4f rare earth ones significantly affect the electrical and magnetic properties of molybdato-tungstates.Recently, studies of manganese doped calcium molybdato-tungstates with the chemical formula of Ca content . Next, sf shells and are paramagnetic ions. Gadolinium ion  has a high magnetic moment due to its isotropic electronic ground state of 7/2S and excellent magnetic resonance imaging effect used as a common contrast agent [d-electron metal ions with unfilled 3d-shells, such as Co2+, have a bigger capacity for charge accumulation [x and y ions is limited by the phase nature of the compound. The motivation for this work is to search for materials useful in electronics and technology. For this reason, the selection of appropriate elements is essential. Rare earth ions have strong electron screening on 4st agent . In turnmulation ,10. The d and 4f ions. New calcium molybdato-tungstates doped with cobalt and gadolinium ions with the chemical formula of Ca1−3x−yCoyxGd2x(MoO4)1−3x(WO4)3x (CCGMWO), where x = 0.0050, 0.0098, 0.0238, 0.0455, 0.0839, 0.1430, 0.1667, and 0.2000; y = 0.02 and denotes vacancies, were successfully synthesized via high-temperature annealing of ternary CoMoO4/Gd2(WO4)3/CaMoO4 mixtures with various content of initial reactants. The concentration range of Gd3+ ions in materials under study was selected to cover the homogeneity range of new solid solution, i.e., the range when 0 < x ≤ 0.2000 and 0 < y ≤ 0.02. The doped materials adopt the tetragonal scheelite-type structure with space group I41/a. That is why we propose electrical and magnetic investigations of new molybdato-tungstates containing both 33, CoCO3, MoO3, Gd2O3, and WO3 . Calcium molybdate (CaMoO4), cobalt molybdate (CoMoO4), and gadolinium tungstate (Gd2(WO4)3) were obtained according to the procedure used in our previous studies [4, Gd2(WO4)3, and CaMoO4. The concentration of cobalt molybdate was constant in each initial CoMoO4/Gd2(WO4)s/CaMoO4 mixture, taking the value of 3.00 mol%. The content of Gd2(WO4)3 was variable and ranged from 0.50 to 33.33 mol%. The initial mixtures, not compacted to pellets, were heated at temperatures in the range of 1173–1473 K in air, in several 12 h annealing stages. After each sintering period, obtained samples were cooled down to ambient temperature, weighed, ground in a porcelain mortar, and followed by examination for their composition by XRD method. We observed a slight mass loss for each obtained material. This loss did not exceed the value of 0.25%. This observation shows that a synthesis of doped samples runs practically without their mass change and a process between initial reactants occurred according to the general equation: (1−3x−y)·CaMoO4 + x·Gd2(WO4)3 + y·CoMoO4 = Cax−y)(1−3CoyxGdx2(MoO4)x)(1−3(WO4)3, where homogeneity range of this solid solution is 0 < x ≤ 0.2 and 0 < y ≤ 0.02. When initial CoMoO4 content was y = 0.02, the chemical reaction of initial reactants can be described as follows: (0.98−3x)·CaMoO4 + x·Gd2(WO4)3 + 0.02·CoMoO4 = Cax)(0.98−3Co0.02xGdx2(MoO4)x)(1−3(WO4)x3.Microcrystalline samples of new solid solution were obtained by two-step synthesis. In both steps, high-temperature sintering of appropriate materials was applied. The following reactants were used in the first step of synthesis: CaCO studies ,12. In tΘ range of 10–100° on an EMPYREAN II diffractometer  with CuKα1,2 radiation . High-Score Plus 4.0 software was used to analyze collected XRD patterns. Lattice parameters were calculated using DICVOL04 software [X-ray diffraction (XRD) patterns of obtained powder samples were recorded within the 210, D50, and D90 which indicate the volume fraction of the sample under a certain size (µm). Particle size and size distribution were measured using a laser particle size analyzer Mastersizer 3000 , dispersing the particles in water by an ultrasonic bath. For each sample, 10 readings were accounted to allow the statistical analysis. The collected data were processed with the equipment’s software, which calculates particle size and size distribution per percentage of volume. The size distribution was estimated using DUV–vis reflectance spectra were recorded within the spectral range of 200–1000 nm using a JASCO-V670 spectrophotometer  equipped with an integrating sphere.σ(T) was measured by the DC method using KEITHLEY 6517B Electrometer/High Resistance Meter  in the temperature range of 300–400 K. The thermoelectric power S(T) was measured in the temperature range of 300–600 K using Seebeck Effect Measurement System . Dielectric properties of the solid solution under study were measured using a Broadband Dielectric Spectrometer  equipped with an Alpha Impedance Analyzer with Active Sample Cell and Quatro Cryosystem temperature control. Investigations were performed when a temperature decreased from 373 K to 173 K with the T-step of 5 K. The calculations of the effective moment, Landé factor using the Brillouin procedure and the imaginary part of the complex permittivity using the Kramers–Kronig transform are described in detail in [The static dc magnetic susceptibility was measured in the temperature range of 2–300 K. Magnetization isotherms were measured at 2, 10, 20, 40, 60, and 300 K using a Quantum Design MPMS-XL-7AC SQUID magnetometer  in applied external fields up to 70 kOe. The electrical conductivity etail in ,16,17,184 and CCGMWO solid solution with different content of Gd3+ ions, i.e., when x = 0.0050, 0.0098, 0.0283, 0.0455, 0.0839, 0.1430, 0.1667, 0.2000, and the constant concentration of Co2+, i.e., y = 0.02 are shown in 2+-doped and Gd3+-co-doped calcium molybadato-tungstates consisted of diffraction lines. These lines can be attributed to scheelite-type framework. No impurity phases, i.e., initial reactants, metal oxides, and other gadolinium tungstates or molybdates were observed with increasing gadolinium ions concentration only up to x = 0.2 (33.33 mol% of Gd2(WO4)3 in initial CoMoO4/Gd2(WO4)3/CaMoO4 mixtures when y is constant and equals 0.02, i.e., 3.00 mol% of CoMoO4). The observed diffraction lines attributed to the scheelite-type structure kept their position or shifted slightly towards the lower 2Θ angle with increasing Gd3+ content. All registered peaks were successfully indexed to the pure tetragonal scheelite-type structure with space group I41/a . This fact confirmed the formation of a new solid solution of CoMoO4 and Gd2(WO4)3 in CaMoO4 matrix. The diffraction patterns of samples comprising initially above 33.33 mol% Gd2(WO4)3 in initial CoMoO4/Gd2(WO4)3/CaMoO4 mixtures (not shown in our manuscript) revealed simultaneously presence of peaks attributed to the saturated solid solution (when x = 0.2 and y = 0.02) as well as the diffraction lines characteristic Gd2(WO4)3. It means that the solubility limit of gadolinium tungstate in CaMoO4 crystal lattice when the initial concentration of CoMoO4 was 3.00 mol% is not higher than 33.33 mol%.The powder XRD patterns of pure CaMoO4 and samples of new solid solution. Both unit cell parameters of doped materials change non-linearly with increasing Gd3+ concentration. The observed changes of a and c lattice constants are not identical  in CaMoO4 matrix were simultaneously substituted by much smaller Co2+ (0.90 Å for CN = 8) and Gd3+ (1.053 Å for CN = 8) ones [3+-doped and vacancied molybdates, i.e., Cd1−3xxDy2xMoO4 [4 , Mo6+ and W6+ ions are tetrahedrally coordinated by O2− ones and their ionic radii are 0.41 and 0.42 Å, respectively [6+ ions by W6+ ones observed while forming new solid solution did not cause any significant changes in both lattice parameters. We also calculated the lattice parameter ratio c/a  for x = 0.1430. It means that in the whole homogeneity range of CCGMWO solid solution we have observed the deformation of the tetragonal scheelite-type cell of each sample in comparison to the pure matrix’s tetragonal cell. 2+/Co2+/Gd3+/ and Mo/W6+ ions occupy the S4 sites, but the oxygen ions are at C1 sites. Each Mo/W6+ ion is surrounded by four oxygen ions (CN = 4), but each Ca2+/Co2+/Gd3+ or vacancy site is surrounded by eight oxygen ions (CN = 8). The substituted ions and vacancies are randomly distributed in CaMoO4 matrix for low Gd3+ concentration, i.e., when 0 < x ≤ 0.1430 and statistically distributed for x ≥ 0.1667. The experimental density values determined for samples under study are given in 3+ content.dentical . Initialsolution . This is 8) ones . We haveDy2xMoO4 . In scheectively . Thus, tatio c/a . This pa3+ concentrations, and this distribution is typical for annealing processes. The maximum values of particle size, D90, increases with increasing Gd3+ concentration in new Co2+-doped and Gd3+-co-doped calcium molybdato-tungstates from ~90 µm  to ~120 µm for the saturated solid solution .The particle size distribution of doped powder materials in volume is shown in 4 or CaWO4 have a typical optical absorption process characterized by a direct electronic transition occurring in maximum energy states near or in the valence band to minimum energy states located below or in the conduction band [Eg). This value is associated with a degree of order and disorder structural these materials. Optical properties of Co2+-doped and Gd3+-co-doped calcium molybdato-tungstates were studied by UV–vis diffused reflectance spectroscopy. Using the Kubelka–Munk method, the recorded UV–vis reflectance spectra were converted into absorption ones [Eg) using the following Equation (1) [α is absorption coefficient, A is a constant that depends on the transition nature, and hν is photon energy (in eV). The optical band gap was determined by plotting a graph between (αhν)2 versus hν and extrapolation of straight to (αhν)2 = 0 , Gd (1.20), and Co (1.88) as well as between Mo (2.16) and W (2.36). The differences in electronegativity values shift the valence band towards the conduction band and lead to a decrease in a band gap with doping. On the other hand, the samples with more significant Gd3+ ions concentration, i.e., when x > 0.1430 direct band gap systematically increase. This means that a consistently growing number of defects in the crystal lattice of CCGMWO solid solution are ordered. This structure is rearranged so as to be stable and regular.Divalent metal molybdates and tungstates with a scheelite-type structure such as CaMoOion band ,22. Due ion ones . Figure tion (1) ,25,26:(αhν)2 = 0 b. Its vaEU) characterizes the width of located states, and the empirical Urbach’s relation express it [α is an experimentally determined absorption coefficient and α0 is a constant. Urbach energy can be obtained from a slope of the straight line of plotting ln(α) against photon energy (hν). α) versus photon energy of the sample when x = 0.0455. EU values for samples under study were calculated from reciprocal of the straight line slopes, as shown in Eg and EU variations as a function of Gd3+ concentration shows that both Eg and EU values correlate very well. It is very clearly seen from Eg (3.51 eV) we observed for the highest Urbach energy, i.e., when EU = 0.578 eV.The incorporation of doping ions into the structure of some materials often reveals a formation of band tailing in the band gap due to a formation of localized states. The band tail energy  are always present in the lattice, even in the crystal which is ideal in other respects. A necessary condition for free energy minimalization gives n ≅ Nexp(-Ev/kBT) for n << N, where N is the number of atoms in the crystal and Ev is the energy required to transfer the atom from the bulk of the crystal on its surface [3+-doped lead molybdato-tungstates [3+-doped tungstates or molybdates: R2WO6  [2W2O10  [2W2O9  [x1–Gdx(WO4)2  [x1–3Gdx2xMoO4 (0.0005 ≤ x ≤ 0.0455) [The results of the electrical conductivity  and ther surface . Similarngstates , RE3+-do Dy, Ho) , CdRE2W2, Gd–Er) , RE2W2O9, Sm–Gd) , AgY1–xG0, 1.00) , and Cd1 0.0455)  were obs2σ on temperature T. The power factor has a very small value of a few fW/(cmK2). However, its value will significantly increase with the increase in temperature for samples richer in gadolinium ions. Usually, a low value of the power factor suggests rather the hopping transport, magnon scattering, and lower covalence of the studied materials compared to classical thermoelectric semiconductors [2σ) are usually heavily doped semiconductors, such as Bi2Te3 [3+ ions have a large ion radius [nductors . Materias Bi2Te3 . The abon radius .x = 0.0050 (a), 0.0455 (b), 0.0839 (c), and 0.2000 (d) (x = 0.0050) the collected spectra indicated the presence of the relaxation process . However, calculated data from the Kramers–Kronig transform [x = 0.0050 have tanδ ~0.01, in which the relaxation process is inhibited with increasing gadolinium content in the sample  [2W2O9 ceramics  [Broadband dielectric spectroscopy measurements of the solid solution under study showed both small values of the real part (ε′) and imaginary part (ε″) of complex dielectric permittivity strongly decreasing with frequency and temperature a–d. Diel2000 (d) . Only inransform  of ε′ die sample . Both sm0, 0.15)  and in R, Sm-Gd) . It is wn-type residual conductivity. Broadband dielectric spectroscopy measurements and dielectric analysis in the frequency representation showed the space charge polarization and small values of both the real and imaginary part of permittivity and a lack of dipole relaxation. Such small values of the loss tangent of the materials under study give them potential applications for the producing of lossless capacitors. A significant achievement of these studies is the observation of an increase in the thermoelectric power factor with an increase in the gadolinium ions content in the sample.In summary, the samples of CCGMWO solid solution were characterized by optical, magnetic, electrical, and dielectric spectroscopy measurements. They have shown perfect paramagnetic state and superparamagnetic behavior as well as insulating properties with the


These synthesized derivatives were screened for hemolysis, thrombolysis and anticancer therapeutic potential against an A549 lung cancer cell line using an MTT assay. Derivatives 7b (0.1%) and 5e (0.5%) showed the least toxicity against RBCs. Hybrid 7f showed excellent thrombolysis activity (61.4%) when compared against reference ABTS. The highest anticancer activity was displayed by the 5d structural hybridwith cell viability 27.49 ± 1.90 and IC50 6.3 ± 0.7 μM values, which were considerably lower than the reference drug crizotinib (IC50 8.54 ± 0.84 μM). Conformational analysis revealed the spatial arrangement of compound 5d, which demonstrated its significant potency in comparison with crizotinib; therefore, scaffold 5d would be a promising anticancer agent on the basis of cytotoxicity studies, as well as in silico modeling studies.Ultrasound- and microwave-assisted green synthetic strategies were applied to furnish benzofuran–oxadiazole  In recent years, natural- and synthetic furan-based  based chHeterocyclic benzofurans are the basic structural units of biologically active families of natural products such as egonol, homoegonol and moracin . NaturalCancer is a notably complex, prominent and lethal disease which poses a serious human health problem; ithasresulted in 7.6 million deaths globally, and this number is expected to reach 13 million by 2030 ,20,21. NContinuing our previous research work towards the synthesis of benzofuran-based oxadiazole and triazole hybrids using a conventional approach , here, oUltrasonic irradiation and microwave-assisted experiments were performed, respectively, in an ultrasonic cleaner bath  with a mechanical timer and heater switch and at a frequency of 47 kHz, and with microwave apparatus (Model EA-180M) witha frequency of 2450 Hz and power consumption of 1150 watt. Analytical-grade solvents and reagents were used; these were purchased from Merck, Alfa Aesar and Sigma-Aldrich through local suppliers. The reaction progress was monitored using pre-coated silica gel plates. The synthesized scaffolds were purified using the column chromatographic technique and by the process of recrystallization in ethanol and methanol solvents.Method A: Ultrasound-assisted method . The substituted S-alkylated oxadiazole- and triazole-based benzofuran derivatives were afforded by dissolving 5-(benzofuran-2-yl)-1,3,4-oxadiazole-2-thiol 3  and 5-(benzofuran-2-yl)-4-phenyl-4H-1,2,4-triazole-3-thiol 6  inacetonitrile (15 mL). Pyridine (0.213 mmol) was added, and the reaction mixture was stirred for 15 min at 0 °C. The substituted bromoacetanilides 4a–g (0.24 mmol) were added, and the reaction mixture was sonicated at 40 °C for 30 min, as shown in Method B: Microwave-assisted method ,32,33. Benzofuran–oxadiazole hybrid 3  and 5-(benzofuran-2-yl)-4-phenyl-4H-1,2,4-triazole-3-thiol 6  were dissolved in DMF (25 mL). Pyridine (0.213 mmol) was added to the reaction mixtureand stirred for 15 min at 0 °C. The substituted bromoacetanilide derivatives 4a–g (0.24 mmol) werethen added, and the reaction mixture was irradiated in a microwave oven for 60–70s, respectively, as depicted in Oxadiazole- and triazole-based benzofuran derivatives were evaluated for hemolysis, thrombolysis and anticancer activity.A fresh blood sample (5 mL) from a healthy donor was collected in an EDTA tube. The blood was transferred to microcentrifuge tubes and centrifugedfor 5 min at 1000 rpm to obtain the red blood cells (RBCs). The supernatant was then discarded, and the RBCpellet was washed three times with phosphate-buffered saline (PBS). The RBC pellet was collected after washing and 20µL of sample solution in DMSO was added. The tubes were incubated at 37 °C for 60 min. Having been removed from the incubator, the tubes were recentrifuged at 13,000 rpm for 5 min. The supernatant was collected and diluted with chilled PBS solution. Absorbance was recordedat 517 nm. ABTS was used as a positive control, while DMSO was employed as a negative control in this protocol. The experiment wasconducted in triplicate, and the RBC lysis percentage was calculated using this formula :% Age heThe thrombolytic assay was performedaccording tomethodologyin the literature . A blood2 (carbon dioxide) at 37 °C in a moistened atmosphere. The A549 cell line was treated with synthesized compounds in a final concentration of DMSO (dimethyl sulfoxide) of less than 1%.The human lung cancer cell line A549 was cultured in Dulbecco’s modified Eagle medium (DMEM), composed of 10% FBS , 100 units/mL of penicillin (1%) and 100 μg/mL of streptomycin (1%), with 5% COThe cytotoxic therapeutic potential of synthesized structural hybrids was evaluated usinga standard MTT -2,5-diphenyltetrazolium bromide) assay. The cells of theA549 cell line were grown, overnight, in a microculture of 96-well plates. The A549 cells were supplemented with different concentrations of compounds for 48 h and further incubated with 20 μL of MTT mixture (5 mg/mL) for 4 h at 37 °C. Following this, 150 μL of control DMSO was added to formazan crystals.The percentage of cell viability was determined viaquantified absorbance in a microplate readerat 490 nm wavelength .5d with a higher pharmacological therapeutic potential was subjected to in silico studies to delineate the mechanism of anticancer activity against lung cancer A549cell lines. The molecular docking studies of these synthesized compounds at a targeted protein were carried out using auto dock Vina 1.1.2 software intermitted with PyRx. The runs of docking were achieved with grid box coordinates of −7.65, −28.27 and 43.34 for x, y and z, respectively. Considering the important role of signaling parameters in cancer, receptors of ALK (anaplastic lymphoma kinase) present an established target to evaluate the anticancer therapeutic potential of synthesized compounds [crizotinib has been nominated as a potential therapeutic moiety against ALK targets;the structure of the synthesized compound 5d was selected because of its marked experimental results. The structures were drawn on Chemdraw 20 professional software (chemoffice) and saved as a mol file [The compound ompounds . To accomol file ,46. FollThe statistical data analysis was performed by using prism. All the measurements were carried out in triplicate, and the results are depicted as mean ±SD.3 was coupled with substituted bromoacetanilide derivatives 4a–h to synthesize corresponding S-alkylated products of benzofuran–oxadiazole 5a–g in good to excellent yields, as depicted in 5d was achieved withthe maximum yield, while the derivative 5f was obtained withthe least yield via ultrasonication and microwave irradiation approaches, as mentioned in Ultrasound- and microwave-assisted green reactions are environmentally friendly, time saving, economical and good yield methodologies, and were adopted as depicted in 7a–h) were obtained in low to moderate (38–79%) yields in 36 h by utilizing conventional methodology [H-1,2,4-triazole-3-thiol 6 was coupled with substituted bromoacetanilide derivatives 4a–h to synthesize corresponding S-alkylated benzofuran–triazole derivatives 7a–h in good to excellent yield at 40 °Cwithin 30 min. Similarly, the microwave approach was even more efficient in that the S-alkylated benzofuran–triazole structural motifs were obtained in maximum yield (68–96%) within the very short duration of 70 s.The benzofuran–triazole derivative 7h was achieved in the maximum yield (90% and 96%) in ultrasound- and microwave-assisted synthetic approaches, while the derivative 7c was obtained in the least yield(60% and 68%) in ultrasound-assisted andmicrowaveirradiation approaches, as shown in Benzofuran–triazole derivatives were synthesized via green synthetic methodologies, namelyultrasound- and microwave-assisted protocols. Benzofuran–triazole derivatives  among all the synthesized derivatives, whereas benzofuran–triazole derivative 7g (23.4%) and benzofuran–oxadiazole 5b (22.12%) showed the highest toxicity, as depicted in 5d (5.02%), 5g (4.86%) andbenzofuran–triazole scaffolds such as 7a, 7d and 7f displayed moderate results (6.13–15.7%). Benzofuran-oxadiazole hybrids 5a, 5c, 5e and 5f showed 0.5–3.7% hemolysis. The data from 7b (0.1%), 7c (2.15%) and 7e (3.11%) showed the least cytotoxicity among all the benzofuran–triazole derivatives against the positive control. The results demonstrate that the nature and position of the functional groups have a significant effect on the hemolytic potential of synthesized derivatives.Benzofuran–triazole derivative 7f showed the highest and best thrombolysis (61.4%) among all the synthesized hybrids when compared against the reference ABTS. The benzofuran–oxadiazole derivative 5a exhibited significantly good lysis activity (56.8%) among all the benzofuran–oxadiazole scaffolds. The benzofuran–oxadiazole derivative 5g and benzofuran–triazole derivative 7h showed the least lysis (48.3% and 48.1%), respectively. Other derivatives such as 5b (50.7%), 5c (52.8%), 5d (53.5%), 5e (52.4%), 5f (56.5%), 7a (52.2%), 7b (52.5%), 7c (54.0%), 7d (56.2%), 7e (59.1%) and 7g (49.06%) showed a mild thrombolytic effect, as described in All the synthesized analogues were evaluated for thrombolytic potential. Most of the benzofuran–oxadiazole and benzofuran–triazole derivatives displayed a mild to moderate thrombolytic effect when compared with the positive control ABTS. The benzofuran-triazole derivative 5a–g and 7a–h was evaluated against lung cancer cell line A549 in comparison with reference standard drugs crizotinib [5d, with cell viability 27.49 ± 1.90 and IC50 6.3 ± 0.7 μM, which was lower than the other derivatives. The benzofuran–triazole scaffold 7h had slightly less cytotoxic potential, with a cell viability of 29.29 ± 3.98 and IC50 10.9 ± 0.94 μM compared with the derivative 5d. The benzofuran–oxadiazole and triazole derivatives 5e, 7d and 7g, with cell viabilities 34.47 ± 2.19, 39.12 ± 2.21 and 36.26 ± 0.41, respectively, displayed good inhibition potential against the A549 cancercell line. The benzofuran structural hybrids 5b, 5c, 5f, 5g, 7a, 7c and 7e  showed moderate anticancer therapeutic potential against lung cancer cells. The target benzofuran derivatives 5a and 7b exhibited the least cytotoxic potential, with cell viability 64.1 ± 1.72 and 57.62 ± 4.94, respectively. The benzofuran derivative 7f was found to be inactive against the A549 lung cancer cell line with a high value of cell viability (99.1 ± 5.04), as depicted in The cytotoxic perspective of all the synthesized target derivatives izotinib ,52,53,54izotinib ,58,59,60N-(substituted-phenyl)-acetamide. The electron-donating ethoxy group atthe para position of the phenyl ring in benzofuran–oxadiazole compound 5e showed 0.5% toxicity against RBCs, whereas benzofuran–triazole derivative 7b exhibited 0.1% toxicity, which was significantly lower than the other derivatives. On the other hand, compounds 5b and 7g (22.12% and 23.4%) proved to be the most toxic compounds among the benzofuran–oxadiazole and benzofuran–triazole series, respectively  compared withdiethyl amine moiety containing the 5g benzofuran–oxadiazole hybrid. The phenyl group increased the thrombolytic potential of 5a, while the diethylamine resulted in a decrease in thrombolytic activity (48.3%) of the 5g hybrid. The benzofuran–triazole hybrid 7f was the most active thrombolytic agent among all the screened derivatives of benzofuran–oxadiazole and benzofuran–triazole, displaying the highest thrombolysis potential (61.4%) due to the presence of two methyl groups adjacently attached on the phenyl ring at ortho para positions. The benzofuran derivative 7h showed the least thrombolytic potential (48.1%) compared with all thetested compounds. The thrombolysis activity of 7h was significantly decreased due to the presence of two chloro EWD groups adjacently attached on the phenyl ring at the ortho and para positions, as depicted in In the case of thrombolytic activity, the benzofuran–oxadiazole scaffold 5d exhibited excellent anticancer activity (cell viability 27.49 ± 1.90% and IC50 6.3 ± 0.7 μM) compared withall the screened derivatives, due to the presence of a methoxy group at the meta position on the phenyl ring;because of the presence of the ethoxy group at the para position on the phenyl ring, however, the benzofuran–oxadiazole derivative 5e displayedmoderate anticancer activity (cell viability 34.47 ± 2.19% and IC50 17.9 ± 0.46 μM) in comparison with the reference drugs crizotinib and cisplatin (28.22 ± 3.88 and 15.34 ± 2.98 μM), respectively. The structural hybrid 5d is more potent than crizitonib and showed less cytotoxic potential than cisplatin. Due to the presence of two chloro EWD groups adjacently attached on the phenyl ring at the ortho and para positions, benzofuran–triazole scaffold 7h exhibited slightly less potency (cell viability 29.29 ± 3.98% and IC50 10.9 ± 0.94 μM) against lung cancer cell line A549 compared with 5d scaffold, which was the most active compound among all the screened derivatives. The benzofuran–triazole hybrid 7f was an inactive compound (cell viability 99.1%) against the A549 cell line due to the presence of two methyl groups adjacently attached on the phenyl ring at the ortho and para positions (The benzofuran–oxadiazole derivative ositions c.5d (B) and co-crystallized ligand crizotinib were docked against the anaplastic lymphoma kinase (ALK) receptors to understand better the mechanisms of attachment and inhibition. The compound-docked conformations were interpreted by binding interactions, energies, polar bonding and ligand–receptor binding. The synthesized compound 5d (B) is more prominent as it produces excellent results with binding energies of −9.925 Kcal/mol in the best binding mode. This exceeds the average energy threshold of −8.985 Kcal/mol given by crizotinib, thereby commending the improved affinity of synthesized compound 5d (B) against the ALK receptor (The molecular comprehension of docking experimentssuggest that compound receptor .5d (B) displayed more effective binding at these active amino acid AA residues, representing more effective working of this synthesized chemical moiety. The binding of compound 5d (B) and crizotinib with receptors are elaborated in 5d (B) showed π–sigma interaction with LEU A: 1256 and VAL A: 1130, while the NH group gave conventional hydrogen bonds with GLY A: 1201. Another prominent interaction appeared with GLU A: 1210 through π–anion linkage.The active binding site of an ALK receptor prominently consists of ALA: 1252, ARG: 1253, ASN: 1254, CYS: 1255, LEU: 1256, LEU: 1257, THR: 1258, CYS: 1259 and PRO: 1260. Crizotinib was found to have noticeable contacts with these active site residues, but compound 5d(B) was docked to determine its interaction with the subject protein and exhibited interesting results. It gave the negative binding energies of −9.925 Kcal/mol in its best binding mode. The prominent amino acid residues were VAL A: 1130, ALA A: 1148, GLY A: 1201, ASP A: 1203, GLU A: 1210, LEU A: 1256 and PRO A: 1260, as shown in Compound 5d presents excellent interaction inside the binding pocket of a receptor, showing its better anticancer potential as compared withthe co-crystallized ligand crizotinib. These computational studies are reliable and appropriate for performing binding interaction prediction. The conformational analysis revealed thespatial arrangement of compound 5d, justifying it to be more potent, in comparison with crizotinib. Interestingly, compound 5d interactions were more realistic, with better results. Therefore, scaffold 5d would be a promising anticancer agent on the basis of both cytotoxicity and in silico studies.Evaluation through docking studies provides good evidence that synthesized compound S-alkylated structural motifs of benzofuran–oxadiazole and –triazole were synthesized in good to excellent yields by utilizing ultrasound- and microwave-assisted green synthetic protocols. The compounds were evaluated for anticancer, hemolytic and thrombolytic activities, and it wasrevealed that most of tested derivatives displayed significant hemolytic, thrombolytic and anticancer activities. The hemolysis assay indicated that benzofuran–triazole derivative 7b and benzofuran–oxadiazole 5e proved to be the least toxic compounds among all the synthesized derivatives. The benzofuran–oxadiazole hybrid 5d with cell viability 27.49 ± 1.90 and IC50 (6.3 ± 0.7 μM) values was recognized as the most potent anticancer candidate against lung cancer cell line A549 compared with the reference drugs crizotinib (IC50 8.54 ± 0.84 μM) and cisplatin with (IC50 3.88 ± 0.76 μM). The docking studies explored the mode of action and inhibition mechanism of compound 5d againstcancer cells; the results of the comprehensive docking analysis of compound 5d are consistent and in agreement with biological diagnostic findings. Overall, the current studies suggest that benzofuran-linked oxadiazole and triazole hybrids are capable of being established as lead compounds in cancer therapy; in particular, scaffold 5d could be a promising anticancer agent on the basis of cytotoxicity andin silico studies. The SAR studies suggested that more modifications to oxadiazole and triazole derivatives of benzofuran may lead to advanced anticancer candidates in cancer therapy.The target


II-family signal-transducing effectors are in this matter key regulators of the nitrogen metabolism. Their interaction with their various targets is governed by the cellular nitrogen level and the energy charge. Structural studies on GlnK, a PII-family inhibitor of the ammonium transporters (Amt), showed that the T-loops responsible for channel obstruction are displaced upon the binding of 2-oxoglutarate, magnesium and ATP in a conserved cleft. However, GlnK from Methanocaldococcus jannaschii was shown to bind 2-oxoglutarate on the tip of its T-loop, causing a moderate disruption to GlnK–Amt interaction, raising the question if methanogenic archaea use a singular adaptive strategy. Here we show that membrane fractions of Methanothermococcus thermolithotrophicus released GlnKs only in the presence of Mg-ATP and 2-oxoglutarate. This observation led us to structurally characterize the two GlnK isoforms apo or in complex with ligands. Together, our results show that the 2-oxoglutarate binding interface is conserved in GlnKs from Methanococcales, including Methanocaldococcus jannaschii, emphasizing the importance of a free carboxy-terminal group to facilitate ligand binding and to provoke the shift of the T-loop positions.Protein inhibition is a natural regulatory process to control cellular metabolic fluxes. P II-family proteins are key players in the regulation of nitrogen metabolism in bacteria, archaea and in the plastids of plant/algae [2-fixation inhibition by interfering with the dinitrogenase reductase [II proteins are not limited by protein level regulation and can control gene transcription by interacting with transcriptional regulators involved in nitrogen assimilation [2-fixation and sensing of fluctuating inorganic carbon levels in cyanobacteria [II proteins are regulated by the binding of two effectors: 2-oxoglutarate (2OG), whose concentration depends on the cellular carbon and nitrogen status  and three protruding loops called B, C and T [Pnt/algae . Their fnt/algae ,3, ammonnt/algae ,5 and N2eductase ,7. PII pmilation ,9. Theirbacteria ,11 and in status , and ADPnization . The mon C and T . In the  C and T ,15. The  C and T .II-family protein GlnK and its target, the ammonium channel Amt. First described in Escherichia coli [Detailed studies highlighted the molecular mode of interaction between the Phia coli ,3, crysthia coli . ATP andMethanocaldococcus jannaschii suggested that methanogenic archaea have a different type of regulation [1 (MjGlnK1) in which the 2OG was found to be bound at the top of the T-loop instead of the inter-subunit cleft  and performed structural analyses on all present GlnK isoforms (MtGlnK1 and MtGlnK2) as well as GlnK1 from Methanocaldococcus jannaschii without a C-terminal tag (MjGlnK1woT). By combining biochemistry studies with structural analyses, we showed that GlnKs from Methanococcales bind 2OG in the same binding site as and with the same mechanism of typical proteins in the PII-family.To verify this hypothesis, we studied the behavior of GlnK–Amt in membrane extracts from M. thermolithotrophicus  molecule was modelled with high confidence  as well as high translational non-crystallography symmetry (56%) and the poorly defined extra electron density restrained us from placing a model that seems to be an MtGlnK2 homodimer.An extra electron density for nucleotides does not exist in the ligand-binding site of the MtGlnK1-dADP, MjGlnK1woT-Mg-ATP/2OG and MtGlnK2-Mg-ATP/2OG. Modelled T-loops are, however, still more flexible compared to the core of the protein due to their high local B-factors. The T-loop’s tip positions might be influenced due to their participation in the crystal packing  and MtAmtB2 (WP_018153777) based on an E. coli structure (PDB: 2NUU) to visualize the surface charges of the binding interface between MtGlnKs and their transporters  and was cultivated at 65 °C in a minimal mineral medium. For one liter of medium the compounds listed in 2O (dH2O). The pH was set to 6 with NaOH pellets. The media was filled up to a final volume of 1 L by the addition of dH2O.2O under magnetic stirring. The pH was adjusted to 6.2 by adding NaOH pellets. The components listed in 2O.Trace element composition: A 100-fold-concentrated trace element solution was prepared by first dissolving 1.36 g nitrilotriacetic acid (7.1 mM) in 800 mL dHM. thermolithotrophicus was grown under strict anaerobic condition in a 10 L fermenter containing 7 L of medium gassed with up to 50 kPa H2/CO2 and 50 kPa N2 under 500 rpm stirring at 65 °C. The fermenter was inoculated with 600 mL preculture cultivated in the same medium. The cells were grown until late exponential phase (OD600 of 1.35) and then immediately transferred in an anaerobic tent (N2/CO2 atmosphere at a ratio of 90:10). Cells were harvested by anaerobic centrifugation for 30 min at 4500× g at 18 °C. 7 L of culture yielded 19.5 g of cells (wet weight). The cell pellet was transferred in a sealed bottle, gassed with 30 kPa with N2, flash frozen in liquid N2 and stored at −80 °C.M. thermolithotrophicus cells were lysed by the addition of 50 mL lysis buffer  followed by sonication  and centrifuged at 45,000× g for 45 min at 20 °C. The cell lysate was transferred and centrifuged at 150,000× g for 1 h at 25 °C in an ultracentrifuge  to isolate the membrane fraction. The supernatant was discarded, and the membrane fraction was resuspended in the washing buffer  followed by ultracentrifugation at 150,000× g for 1 h at 25 °C. This washing procedure was performed four times. The pellet was resuspended in the lysis buffer and split evenly in 10 aliquots of 10 mL. The following metabolites were then added: (1) no effector; (2) 2 mM MgCl2, 2 mM ADP; (3) 2 mM MgCl2, 2 mM ATP; (4) 2 mM MgCl2, 10 mM 2OG ; (5) 2 mM MgCl2, 2 mM ATP, 10 mM 2OG; (6) 2 mM MgCl2, 2 mM ADP, 10 mM 2OG; (7) 2 mM MgCl2, 2 mM ADP, 2 mM ATP, 10 mM 2OG; (8) 2 mM MgCl2, 2mM ADP, 20 mM ATP, 10 mM 2OG; (9) 2 mM ATP, 10 mM 2OG; (10) 2 mM MgCl2, 2 mM GTP, 10 mM 2OG. All conditions were incubated for 30 min at room temperature, then centrifuged at 150,000× g for 1 h at 25 °C. The supernatant was concentrated to 100 µL in a 10-kDa cut-off concentrator  and analyzed by SDS-PAGE  pH 8.25) for 90 min at 90 Volts. The gel was stained with InstantBlueTM .To investigate the interaction behavior of Amt-GlnK, membrane fractions were isolated natively. Approximately 7 g of Selected Coomassie stained bands were excised, and the proteins within were reduced by DTT, alkylated by iodoacetamide and digested overnight with trypsin. The resulting peptides were extracted twice by 5% formic acid and the extracts were pooled together and dried down. The samples were then reconstituted in 25 µL of 5% formic acid containing 5 fmol/μL retention time standard peptides  and 5 µL were taken for LC-MS/MS analysis.m/z range was 350–1600 and dynamic exclusion time was 15 s. The lock-mass function was set to recalibrate MS1 scans using the background ion (Si(CH3)2O)6 at m/z 445.12. Acquired spectra were searched by MASCOT software (version 2.2.04) against M. thermolithotrophicus protein sequences in NCBI database  and genome (6-frames translation). Mass tolerance was set to 5 ppm and 0.025 Da for precursor and fragment ions respectively; two miscleavages; variable modifications—cysteine carbamidomethyl, propionamide; methionine oxidized. The results were evaluated by Scaffold software , minimum number of matched peptides was set to two; peptide and protein false discovery rate (FDR) was below 1%.LC-MS/MS analysis was performed by MS Facility at the MPI-CBG Dresden, Germany, on a nano-UPLC Ultimate 3000 interfaced on-line to an Orbitrap HF hybrid mass spectrometer . The UPLC system was equipped with Acclaim™ PepMap™ 100, 75 µm × 2 cm trapping column  and 50 cm μPac micro Pillar Array separating column . Peptides were separated using 75 min linear gradients 0% to 65% of acetonitrile . Spectra were acquired using Top20 data-dependent acquisition method, precursor MtGlnK1 and MtGlnK2 were 346 and 784, respectively for the Mg-ATP/2OG condition  vector by Genscript.MjGlnK1, NdeI and XhoI restriction sites were added in the 5′- and 3′-extremities, respectively. A stop codon was also added upstream of the XhoI site in 3′. The DNA synthesized was cloned in the expression vector pET-24b(+) at the NdeI and XhoI restriction enzyme digestion sites by Genscript.For Escherichia coli strain BL21(DE3) cultivated in Lysogeny Broth supplemented with a final concentration of 50 µg/mL kanamycin. The cultures were incubated at 37 °C, being shaken, until an OD600 of 0.6–0.8 was reached. Induction was performed by adding Isopropyl ß-D-1-thiogalactopyranoside at a final concentration of 0.75 mM and the cells were incubated for another hour at 37 °C, being shaken. The cells were harvested by centrifugation at 5000× g for 20 min at 21 °C. Subsequently, the cell pellet was washed in phosphate buffered saline solution and centrifuged for 20 min at 4000× g at room temperature. The washed cell pellets were flash-frozen in liquid nitrogen.Each construct was expressed in g at room temperature for 40 min. The supernatant was then heated up to 70 °C for 30 min followed by centrifugation for 20 min at 45,000× g. The supernatant was filtered through 0.2 µm  and placed at 4 °C overnight, standing.The frozen cell pellets were thawed and resuspended in the lysis buffer  at a ratio of 4 mL lysis buffer per gram of wet weight. The cells were disrupted by sonication  on ice. The lysate was centrifuged at 45,000× MtGlnK1 and MtGlnK2, a white precipitate appeared overnight and the sample was centrifuged at 45,000× g for 30 min at 4 °C. The pellets, corresponding to the protein of interest, were resuspended in 4 mL of gel filtration buffer  and the aggregates were dissolved by heating at 70 °C for 15 min followed by centrifugation 13,000× g for 5 min RT. The supernatants were immediately injected on a HiLoad® 16/600 Superdex® 200 pg . The protein was eluted at a flow rate of 1 mL/min at room temperature. MtGlnK1 eluted as a single gaussian peak at 82 mL, from which the fractions of interest were pooled. MtGlnK2 major peak eluted at 86.6 mL. Both pools were concentrated with a 10-kDa cut-off concentrator  at room temperature and centrifuged at 13,000× g for 5 min to remove aggregates.For MjGlnK1, no precipitate appeared after the overnight incubation at 4 °C. The sample was filtered through 0.2 µm  and immediately injected on a HiLoad 16/600 Superdex 200pg . The sample eluted at a flow rate of 1 mL/min in the gel filtration buffer and four peaks were observable at elution volumes of 62, 66, 73 and 80 mL. The four different pools were concentrated with a 10-kDa cut-off concentrator  at room temperature and centrifuged at 13,000× g for 5 min to remove aggregates.For All purification steps were systematically checked by SDS-PAGE. The protein concentration was determined via Bradford assay. All GlnKs passed on SDS-PAGE exhibited an extra band at 25-kDa, in addition to the expected monomeric molecular weight (circa 12 kDa), as observed in other GlnK studies .2. Since MtGlnK1 and MtGlnK2 with ligands aggregated strongly at 18 °C, the protein sample was first heated to 70 °C and centrifuged at 13,000× g for 5 min at room temperature to remove remaining aggregates before spotting.All purified proteins were immediately crystallized at 18 °C. Initial screening was performed by using the sitting drop method on 96-Well MRC 2-Drop Crystallization Plates in polystyrene  containing 90 µL of crystallization solution in the reservoir. 0.6 µL sample with 0.6 µL crystallization solution were spotted. For all three GlnKs, the protein sample was crystallized with and without 2 mM ATP, 2 mM 2OG and 2 mM MgClMtGlnK1 at a concentration of 33 mg/mL co-crystallized with ligands generated few crystals but none of them diffracted to an exploitable resolution. However, hexagonal rods instantly appeared for MtGlnK1 at 33 mg/mL without ligands in 35% w/v Pentaerythritol propoxylate (17/8 PO/OH), 100 mM MES; pH 6.5 and 200 mM ammonium sulfate.MtGlnK2 at a concentration of 30 mg/mL without ligand crystallized as small squares in 35% w/v Pentaerythritol ethoxylate (15/4 EO/OH) and 100 mM HEPES pH 7.5. When MtGlnK2 at 30 mg/mL was crystallized with ligands, high-quality shard shaped crystals appeared after minutes in 25% w/v Pentaerythritol ethoxylate (3/4 EO/OH) and 100 mM MES pH 6.5.MjGlnK1 were obtained after gel filtration. The best crystals were obtained from the pool containing the protein eluted at 66 mL on the gel filtration. This pool, concentrated to 11.2 mg/mL, was co-crystallized with ligands and high-quality cube-shaped crystals appeared overnight in 20% w/v PEG 3350, 100 mM Bis-Tris propane pH 8.5 and 200 mM sodium nitrate.As stated above, four pools of purified MjGlnK1 crystals were soaked in their crystallization solutions supplemented with 20% v/v glycerol as a cryo-protectant prior to being frozen in liquid nitrogen. MtGlnK2 crystals with ligands were soaked in the crystallization solution supplemented with 60 mM Tb-Xo4  for 3 min 30 s prior to being frozen in liquid nitrogen. The Tb-Xo4 had no effect on the diffraction quality and was not detected in the electron density.X-ray diffraction was collected at 100 K at the Synchrotron Source optimisée de lumière d’énergie intermédiaire du LURE (SOLEIL), PROXIMA-1 beamline; the Swiss Light Source (SLS), X06DA and X10SA beamlines and at PETRA-III, P11 beamline. The best datasets were obtained at the Swiss Light Source (SLS), X06DA and X10SA beamlines.autoPROC  [PHENIX package (Version 1.19.2-4158) [MjGlnK1 (PDB 2J9D) as a template. All models were then manually rebuilt with COOT (Version 0.8.9.2) [PHENIX. MtGlnK2 and MjGlnK1 with ligands were refined by considering all atoms anisotropic. MtGlnK2 apo was refined with applied translational-liberation screw and twinning refinement, considering a twin fraction of 0.39 with the following twin law: h, k, -l. MtGlnK1-dADP was also refined by applying translational-liberation screw. For all models, hydrogens were added in riding position for the last refinement cycles except for MtGlnK2 apo. Hydrogens were omitted for the final deposited model.Data were processed and scaled with dge, UK) . The str.2-4158) , using M0.8.9.2)  and furthttp://molprobity.biochem.duke.edu, accessed on 8 July 2021) [All models were validated through the MolProbity server (ly 2021) . Data cohttps://www.ebi.ac.uk/Tools/msa/clustalo/, accessed on 6 July 2021) [1 and AmtB2 from Methanothermococcus thermolithotrophicus based on E. coli structure (PDB 2NUU) was performed by the Phyre2 server  [MtGlnK2-Mg-ATP/2OG structure was performed with the ConSurf server  [Sequence alignment was performed with Clustal Omega (ly 2021)  and anally 2021) . The homly 2021) . All figly 2021) ,26.Our work emphasized the structural conservation among GlnKs and the importance of the free carboxy group at the C-terminal to optimize ligand coordination. Therefore, adding a tag at this position should be avoided in future studies on the close members of this protein family to avoid unwanted artefacts.Methanococcales do not structurally and mechanistically differ from previously characterized GlnKs, another group of PII proteins present in diazotrophic methanogens might have different properties. These two genes coding for NifI1 and NifI2 are always co-located within the nitrogenase operon and regulate N2-fixation [II-family.While our results show that GlnKs from fixation . They ar


Arabidopsis thaliana, phot1 and phot2. Phots, plant UVA/blue light photoreceptors, regulate growth responses and fast movements aimed at optimizing photosynthesis, such as phototropism, chloroplast relocations and stomatal opening. Sumoylation is a posttranslational modification, consisting of the addition of a SUMO (SMALL UBIQUITIN-RELATED MODIFIER) protein to a lysine residue in the target protein. In addition to affecting the stability of proteins, it regulates their activity, interactions and subcellular localization. We examined physiological responses controlled by phots, phototropism and chloroplast movements, in sumoylation pathway mutants. Chloroplast accumulation in response to both continuous and pulse light was enhanced in the E3 ligase siz1 mutant, in a manner dependent on phot2. A significant decrease in phot2 protein abundance was observed in this mutant after blue light treatment both in seedlings and mature leaves. Using plant transient expression and yeast two-hybrid assays, we found that phots interacted with SUMO proteins mainly through their N-terminal parts, which contain the photosensory LOV domains. The covalent modification in phots by SUMO was verified using an Arabidopsis sumoylation system reconstituted in bacteria followed by the mass spectrometry analysis. Lys 297 was identified as the main target of SUMO3 in the phot2 molecule. Finally, sumoylation of phot2 was detected in Arabidopsis mature leaves upon light or heat stress treatment.The disruption of the sumoylation pathway affects processes controlled by the two phototropins (phots) of  Arabidopsis thaliana: PHOT1 (PHOT2 (Phototropins (phots) are plant blue/UVA photoreceptors, which control growth responses and fast movements, such as stomatal opening and chloroplast relocations . Two phoa: PHOT1  and PHOT1  domains, which carry flavin mononucleotide chromophores. The C-terminal part contains a light activated Ser/Thr kinase . In darkNPH3. Polyubiquitination of phot1 by CRL3NPH3 promotes its degradation in the 26S proteasome  motif at the C-terminus. SUMO terminal carboxyl group is conjugated to the ε-amine lysine group of the target protein. The sumoylation consensus motif consists of ΨKXE/D, where Ψ is a large hydrophobic residue: X, any amino acid; E/D, glutamate or aspartate  called SUMO (SMALL UBIQUITIN-RELATED MODIFIER) to a target protein. Expressed sequence tags of four SUMO genes were found in Arabidopsis: bstrates . The douc lethal . SUM1 aneedlings . The train roots . SUM5 mRspartate . SUMO isee SCE1a . The scec lethal . Finallyc lethal , which ic lethal  and MMS2c lethal , named ac lethal ], are noc lethal . SUMO E4c lethal . Apart fc lethal ., were assessed after the irradiation of dark-adapted leaves with continuous blue light of increasing intensities of 0.4, 1.6, 4, 20, 40, 80, and 120 µmol m−2 s−1  and that of avoidance was smaller (significantly at 20 µmol m−2 s−1) than in the wild type  are shown in siz1 mutation were observed. The amplitudes of transient chloroplast avoidance and accumulation  was observed in the cytoplasm adjacent to the plasma membrane of pavement cells co-transformed with phots fused with C-terminal GFP (cGFP) and either nGFP-SUMO1, nGFP-SUMO2 or nGFP-SUMO3 (Bimolecular fluorescence complementation (BiFC) assay was used to investigate the formation of complexes between phots and SUMO proteins or E3 ligases in transiently transformed FP-SUMO3 . In leavFP-SUMO3 . The fluFP-SUMO3 . In leavFP-SUMO3  in a subFormation of complexes between phots and components of the sumoylation pathway were confirmed using the split-ubiquitin-based membrane yeast two hybrid (MYTH) system . Native in bacteria according to Escherichia coli together with N-terminal parts of PHOT1 or PHOT2. In some experiments, the SIZ1 or MMS21 E3 ligase was also added -based sumoylation site mapping. The identified peptides are shown in in bacteria. The Lys 297 to Arg mutation diminished modification of the N-terminal phot2 fragment by native SUMO1, SUMO2, SUMO3 and SUMO5; however, the pattern differed between repetitions . MS analysis showed that numerous lysine residues of the N-terminal PHOT2 fragment were modified by SUMO3 when co-expressed with proteins of the Arabidopsis sumoylation pathway in E. coli. Lys 297 was dominant among the identified residues. Mutational analysis indicated that this residue was modified by SUMO1, 2, 3 and 5  and primers listed in −2 s−1 of light supplied by fluorescent lamps .The following L_77_G06 , phot1 SK_088841 , phot2 n2 npl1-1  and a tr−2 s−1 and continuous blue light of increasing intensity 0.4, 1.6, 4, 20, 40, 80 and 120 µmol m−2 s−1 (50 min for each light regime) were determined. Photometric curves were analyzed using a custom-written Mathematica  package. The amplitudes (ΔT) with respect to the dark level and the maximal velocities (dT/dt) of transmittance changes were calculated. Chloroplast arrangements in mesophyll cells were analyzed as in −2 s−1 for 50 min. Images were recorded with the objective LD LCI Plan-Apochromat 25×/0.8. Chlorophyll fluorescence was excited with the 633 nm He–Ne laser and emission was collected in the range of 661–721 nm. Maximum intensity projections were calculated from Z-stacks, which spanned whole depth of the epidermis and palisade parenchyma, starting from the leaf upper surface.Chloroplast movements were assessed with the photometric method, as described in −2 s−1. Irradiance (light intensity) was measured with a LI-190R sensor .Arabidopsis wild-type and mutant seeds were sown on square agar plates, kept at 4°C for 2 d and irradiated with white light for 2 h. Seedlings were grown in darkness on vertically positioned plates for 3 d. Phototropic bending experiment was performed as in −2 s−1  for 3 h. Dark-adapted leaves collected at the same time served as a control. Each sample contained material from two plants. Three-day-old etiolated seedlings were illuminated with blue light  of either 0.01 or 5 µmol m−2 s−1 for 12 h. Etiolated seedlings kept in darkness were collected at the same time as the control. Each sample contained material from 50 seedlings. Samples were frozen in liquid nitrogen immediately after treatments.Phot expression analysis at mRNA and protein levels was performed in five biological replicates for Arabidopsis etiolated seedlings and mature leaves. Leaves of 4-week-old plants were dark-adapted overnight and irradiated with blue light of 120 µmol mPHOT1 and PHOT2 and in UBC, PDF2 and SAND. Each sample was quantified in three technical replicates. The mean value of Ct for samples from all experimental groups quantified simultaneously was subtracted from individual Ct values, for the purpose of inter-run calibration. Expression levels were then normalized using factors calculated by geNorm v3.4 , diluted 1:25,000, were incubated at room temperature for 1 h. Signal detection was performed with a Clarity Western ECL Blotting Substrate  by the BioSpectrum Imaging System . Intensities of the chemiluminescent signal were normalized to actin levels in each sample. For this, membranes were stripped with Restore Plus Western Blot Stripping Buffer (Thermo Fisher Scientific) and probed with anti-actin antibody , diluted 1:2,500, at room temperature for 1 h, followed by secondary antibody incubation and ECL detection. Densitometric quantification was performed using ImageJ.Proteins were extracted as in N. benthamiana leaves was performed as described in MultiSite Gateway system (Thermo Fisher Scientific) was used to prepare constructs for BiFC analysis. PHOT1 and PHOT2 constructs were described in ∼20 µmol m−2 s−1, 455 nm 1 W LEDs, epiLED) for 4 d. Each experiment was repeated three times.Protein interactions were tested in yeast using the split-ubiquitin-based MYTH system , with introduced Gateway cloning sequences . Bait anE. coli was performed as in MMS21 sequence with primers 5′GGAATTCCATATGGCGTCGGCGTCCTCGTCTG3′ and 5′CGGGATCCCTAATCTTCATCCACATCTTCTG3′. The sequence was cloned into pET15b using the NdeI and BamHI sites. Bacteria were transformed with either pET28a:c-Myc-6xHis-PHOT1N (amino acids 1–662) or pET28a:c-Myc-6xHis-PHOT2N (amino acids 1–574). pET28a was modified by inserting the sequence coding for c-Myc into the NcoI site. The c-Myc sequence was obtained by annealing overlapping oligonucleotides 5′CATGGAACAGAAACTGATCTCTGAAGAAGACCTGGC3′ and 5′CATGGCCAGGTCTTCTTCAGAGATCAGTTTCTGTTCC3′. PHOT1N amplified with 5′TACTGGATCCATGGAACCAACAGAAAAACCATCG3′ and 5′TACTAAGCTTATGCTTCAAACCAATCGGTTCACC3′ was cloned into the modified pET28acMyc vector using BamHI and NdeI restriction sites. PHOT2N was amplified using primers 5′TACTGTCGACTTATAGTCCCACTGTTTCTCCACTC3′ and 5′TACTCATATGGAACAGAAACTGATCTCTGAAGAAGACCTGATGGAGAGGCCAAGAGCCC3′, which contained the c-Myc coding sequence and cloned into standard pET28a using NdeI and SalI sites. To introduce the Lys 297 to Arg mutation in the PHOT2N fragment, Quik Change II XL Site-Directed Mutagenesis Kit, 200521 (Agilent Technologies), was used with primers 5′GCAATGACACTATGGTAAGACCTGATAGTTCTACTACACC3′ and 5′GGTGTAGTAGAACTATCAGGTCTTACCATAGTGTCATTGC3′. Bacteria were grown at 37°C in LB medium containing chloramphenicol (25 mg l−1), kanamycin (50 mg l−1) and spectinomycin (100 mg l−1). Ampicillin (100 mg l−1) was also added when pET15b:MMS21 or pMAL:SIZ1 was present. After reaching OD600 of 0.6, temperature was decreased to 20°C, protein expression was induced in cultures with 0.2 mM IPTG , and bacteria were grown for 20 h. Further procedures were performed at 4°C. Cells were centrifuged at 5,000× g for 15 min, resuspended in 1 ml of protein extraction buffer , sonicated for 5 min, with 5 s pulses and 10 s intervals and centrifuged at 35,000× g for 20 min. Twenty microliters of Ni-NTA agarose  was added to the cell lysate and incubated for 1 h with constant rotation. Unbound proteins were removed by washing three times with 1 ml of extraction buffer containing 20 mM imidazole for 5 min. Bound proteins were eluted with 20 µl of extraction buffer containing 250 mM imidazole, mixed with SDS–PAGE loading buffer and denatured at 100°C for 5 min. Samples were separated by SDS–PAGE using 12% gels and transferred onto a PVDF membrane (0.2 µm) . The membrane was blocked in 5% milk in PBS  with 0.5% Tween 20 for 30 min and incubated for 2 h with a monoclonal anti-c-Myc antibody (clone 9E10), diluted 1:20,000 (Merck). Then, it was washed three times with 5% milk in PBS with 0.5% Tween 20 for 5 min and incubated for 1 h with a goat anti-mouse IgG–horseradish peroxidase conjugate antibody, diluted at 1:20,000 (Merck). After three washes in PBS with 0.5% Tween 20, chemiluminescent detection was performed using a Clarity Western ECL Blotting Substrate (Bio-Rad) by the BioSpectrum Imaging System.Reconstitution of the sumoylation system in In silico analysis of potential sumoylation sites in phot molecules was performed using GPS-SUMO ], sonicated for 30 s, with 0.5 s pulses in 0.5 s intervals and centrifuged at 30,000× g for 20 min. Twenty-five microliters of Myc-Trap Magnetic Agarose  suspended in 250 µl of c-Myc protein extraction buffer was added to the cell lysate and incubated for 90 min with constant rotation. Unbound proteins were removed by washing four times with 1 ml of c-Myc protein extraction buffer without lysozyme and protease inhibitors for 5 min. Beads were suspended in SDS–PAGE loading buffer and denatured at 95°C for 5 min. Samples were separated by SDS–PAGE using 4–20% gradient Mini-PROTEAN TGX Stain-Free Precast Gel (Bio-Rad) and stained with colloidal Coomassie. Protein bands corresponding to sumoylated phot fragments were excised from the gel and subjected to standard procedure of trypsin digestion. Proteins were reduced with 10 mM DTT for 30 min at 56°C, alkylated with iodoacetamide in darkness for 45 min at room temperature and digested overnight with 10 ng µl−1 trypsin. The resulting peptide mixtures were concentrated and desalted on a RP-C18 pre-column . Further peptide separation was achieved on a nano-Ultra Performance Liquid Chromatography RP-C18 column  using a 160-min linear acetonitrile gradient in the presence of 0.1% formic acid. Column outlet was directly coupled to the ion source of the Orbitrap Elit mass spectrometer , working in the regime of data dependent MS to MS/MS switch. A blank run ensuring absence of cross-contamination from previous samples preceded each analysis. The acquired MS/MS data were pre-processed with Mascot Distiller software . A search was performed with the Mascot Search Engine  against the TAIR10 database . To reduce mass errors, the peptide and fragment mass tolerance settings were established separately for individual LC–MS/MS runs after a measured mass recalibration , mixed with 20 µl of GFP-Trap Agarose (ChromoTek) and incubated for 60 min at 4°C with constant rotation. Unbound proteins were removed by washing once with 1 ml of a 1:1 mixture of Pi-RIPA and dilution buffer and twice with 1 ml of 10 mM Tris, 150 mM NaCl and 0.5 mM EDTA for 5 min each. Beads were suspended in SDS–PAGE loading buffer and denatured at 95°C for 5 min. Samples were split in two parts, separated by SDS–PAGE in 8% gels and transferred to a PVDF membrane using a wet transfer system (Bio-Rad) followed by immunodetection with anti-PHOT2 (as described above) or anti-SUMO1  antibodies. The membrane was blocked in 5% milk in PBS with 0.1% Tween 20 for 1 h and incubated overnight at 4°C with anti-SUMO1 antibody, diluted at 1:2,000. Then, it was washed three times with 5% milk in PBS with 0.1% Tween 20 for 5 min and incubated for 1 h with a goat anti-rabbit IgG–horseradish peroxidase conjugate antibody, diluted at 1:50,000 (Agrisera). After three washes in PBS with 0.1% Tween 20, chemiluminescent detection was performed using a SuperSignal™ West Pico PLUS Chemiluminescent Substrate (Thermo Fisher Scientific) and a Carestream MXBE film .For the analysis of protein sumoylation in planta, 5-week-old HOT2-GFP  were useThe Polish National Science Centre . Confocal microscopy was carried out thanks to the Polish Innovation Economy Operational Program from the European Regional Development Fund . The open-access publication of this article was funded by the Priority Research Area BioSunder the program ‘‘Excellence Initiative –Research University” at the Jagiellonian University in Krakow.No conflicts of interest are declared.pcab027_SuppClick here for additional data file.


Membrane and secretory proteins are synthesized in the endoplasmic reticulum (ER). Perturbations to ER function disrupts protein folding, causing misfolded proteins to accumulate, a condition known as ER stress. Cells adapt to stress by activating the unfolded protein response (UPR), which ultimately restores proteostasis. A key player in the UPR response is ATF6α, which requires release from ER retention and modulation of its redox status during activation. Here, we report that ER stress promotes formation of a specific ATF6α dimer, which is preferentially trafficked to the Golgi for processing. We show that ERp18 regulates ATF6α by mitigating its dimerization and trafficking to the Golgi and identify redox-dependent oligomerization of ATF6α as a key mechanism regulating its function during the UPR. The unfolded protein response (UPR) maintains cellular proteostasis during stress by activating sensors located to the endoplasmic reticulum (ER) membrane. A major sensor for this response, ATF6α, is activated by release from ER retention and trafficking to the Golgi, where it is cleaved to generate a bZIP transactivator to initiate a transcriptional response. The reduction of a disulfide in monomeric ATF6α is thought to be necessary for release from retention, trafficking, and proteolysis. Here we show that, following ER stress, ATF6α undergoes a redox switch to form a disulfide bonded dimer, which traffics to the Golgi for cleavage by the S1P protease. Additionally, we find that overexpression of ERp18 attenuates dimer formation thereby limiting Golgi trafficking. Our results provide mechanistic insight into activation of the ATF6α pathway, revealing an unexpected role for redox-dependent oligomerization prior to Golgi trafficking. The lumen of the endoplasmic reticulum (ER) contains numerous proteins and enzymes that ensure timely and efficient folding of proteins entering the secretory pathway . CellulaIRE1 is the most conserved of the UPR pathways. IRE1 activation promotes its cytosolic kinase and ribonuclease activities , 6, whicThe mechanism of ATF6α activation is less well understood. During ER stress, ATF6α traffics to the Golgi apparatus, where it is sequentially cleaved by site-1 protease (S1P) and site-2 protease (S2P) to release a soluble bZIP transcription factor, ATF6-N . ATF6-N In unstressed cells, ATF6α exists as three redox forms, a monomer and two interchain disulfide-stabilized dimers. These redox forms are maintained by the presence of two highly conserved cysteines in the luminal domain, C467 and C618 . These cSI Appendix, Fig. S1) even at 10-fold lower concentrations of added TG, indicating the effects were not specific to ectopically expressed protein or to the TG concentration used to induce the UPR.The ATF6α luminal domain contains two cysteine residues (C467 and C618) that either form an intrachain disulfide (C467-618) in the monomer or interchain disulfide bonded dimers (C467-467 or C618-618)  Fig. 1AAFig. 1A.Previous reports have shown that hyperglycosylation of ATF6α occurs during ER stress, which results in reduced SDS/PAGE mobility , 16. We SI Appendix, Fig. S2). Here we used a relatively low concentration of DTT to minimize ATF6α disulfide reduction but still allow a robust UPR. Even in the presence of the reducing agent, the main species that becomes hyperglycosylated is 467D.To examine the redox changes to ATF6α 467D following ER stress induced by different reagents, we carried out time courses in the presence of dithiothreitol (DTT), which disrupts protein disulfide formation, or tunicamycin, a glycosylation inhibitor. After 30 min of DTT treatment, we did not detect any significant changes in the levels of ATF6α monomer and 467D; however, we detected the appearance of the hyperglycosylated form of 467D D, lane 2In tunicamycin-treated cells, we also detected a decrease in the ATF6α monomer with a concomitant increase in the 467D, although the accumulation of 467D in these cells exhibited a slower appearance compared to the TG-treated cells compare B and E. SI Appendix, Fig. S3). Immunoblot analyses of endogenous ATF6α in these cells revealed the presence of two forms, monomer and 467D, with the 618D, barely detectable, mirroring that seen with exogenously expressed ATF6α , suggesting that accumulation of this protein in the ER is sufficient to induce a change in the redox status of ATF6α. The expression of misfolded LDLR activated the IRE1 pathway, as indicated by the splicing of XBP1-u to produce XBP1-s, thereby confirming a UPR response . In addition, levels of BiP increased after induction of misfolded LDLR in the absence of S1Pi . This increase was attenuated in the presence of S1Pi . Importantly, the predominant form of ATF6α during ER stress produced by proteotoxic stress is the 467D, which becomes hyperglycosylated indicating selective transport to the Golgi. These results are consistent with our finding with chemotoxic stressors and confirm that the redox changes seen with exogenously expressed ATF6α also occur with endogenous ATF6α.The commonly used UPR inducers, DTT, TG, and tunicamycin exert multiple effects on cellular homeostasis, in addition to activating the UPR pathways. It is apparent that chemical induction of the UPR confers different genetic and protein expression signatures in comparison to the UPR induced by expression of misfolded proteins in the ER . To inveed ATF6α A and F. ed ATF6α F, lane 5The ER stress induced hyperglycosylation of ATF6α 467D would suggest that it is transported from the ER to the Golgi during the UPR. To further elucidate the location of ATF6α 467D hyperglycosylation, we analyzed the sensitivity of ATF6α monomer and dimers to endoglycosidase H (endo H) digestion. Cells pretreated with S1Pi were left untreated or treated with TG, to induce ER stress and cell lysates incubated with or without endo H. In unstressed cells, ATF6α monomer and dimers were sensitive to endo H digestion, as demonstrated by their increased SDS/PAGE mobilities A, lane 2d-galactopyranoside (BADAP) . Treatment with BADAP resulted in increased SDS/PAGE mobilities of the hyperglycosylated ATF6α before or after endo H treatment suggesting inhibition of O-linked glycosylation  , 29. Cel (BADAP)  . These results indicate stress-induced trafficking of ATF6α 467D from ER fractions to lighter fractions consistent with the early Golgi compartment.Previously, it was suggested that only monomeric ATF6α traffics to the Golgi based upon stress induction with DTT or tunicamycin, followed by fractionation of the ER and Golgi by discontinuous sucrose gradient. We also fractionated lysates from unstressed and stressed cells in a discontinuous sucrose density gradient, to partially separate ER and Golgi components. In unstressed cells, all forms of ATF6α were detected in the heavier fractions . SucroseSI Appendix, Fig. S6A), suggesting that inhibition of lysosomal degradation . This band was absent when the samples were immunoblotted with an anti-HA antibody  whose epitope is located at the N-terminal cytosolic region of ATF6α. We confirmed this band to be a result of cleavage by S1P, as it was undetected in the presence of S1Pi  and migrated at about 45 kDa when samples were analyzed under reducing SDS/PAGE  by immunoblot analyses  and by comparing the induction of BiP expression upon TG-induced ER stress with wild-type cells . Cell lysates from either the C467A or C618A cells were treated with or without TG and separated under nonreducing SDS/PAGE  of endogenous ATF6α in these cells  C and D. The presence of the S1P-generated luminal domain dimer with wild-type and C618A mutant, but not the C467A mutant of ATF6α, could be caused by the inability of S1P to cleave the 618D. To determine whether this was the case, we treated cells with brefeldin A and determined the appearance of the ATF6α luminal domain before or after ER stress . BrefeldWe have previously shown that ERp18 and protein disulfide isomerase-related (PDIR) are catalytically active toward ATF6α . We demoGiven the ability of ERp18 to reduce the 467-467 disulfide, we questioned whether overexpression of this oxidoreductase would lead to an attenuation of ATF6α activation. Our previous work had highlighted the role of ERp18 in regulating trafficking of ATF6α . DeletioThe process by which ATF6α becomes activated allowing trafficking to the Golgi is known to involve the dissociation from BiP along with recruitment to transport vesicles . Our datAs ATF6α resides in the ER in predominantly an intrachain disulfide-bonded monomer , the redThe specific trafficking of the 467D to the Golgi raised the question of S1P specificity, which we tested by relocating the protease to the ER. Our results indicate that S1P cleaves both the 467D as well as reduced monomer but cannot cleave the intrachain disulfide bonded monomer or the 618D. Previous studies have shown that S1P can cleave monomeric ATF6α that traffics to the Golgi following treatment of cells with high concentrations of DTT . Indeed,The regulation of Ire1 and PERK by reversible thiol oxidation and the redox-dependent dimerization and trafficking of ATF6α would suggest that redox switches provide a level of regulation for the UPR. Indeed, a change to the ER redox conditions during ER stress has been noted using a redox-sensitive GFP reporter  and corr2 incubator.All HEK293T, HT1080, and the Flp-In T-Rex doxycycline-inducible HeLa cells were maintained in DMEM supplemented with 10% FBS and 100 units/mL penicillin and 100 µg/mL streptomycin at 37 °C in a 5% COThe pcDNA 3.1-ATF6α plasmid containing an N-terminal HA-tag and a C-terminal V5-tag followed by a KDEL sequence and stop codon, and the single cysteine to alanine mutations in the luminal domain of ATF6α (ATF6 C467A and ATF6 C618A) have been described previously . The pcDHEK293T and HT1080 were either singly or cotransfected with plasmid DNA at 80 to 90% confluence using MegaTran (Origene) transfection reagent. To generate stable overexpression cells, the transfected cells were placed on antibiotic selection for ∼3 to 4 wk until colonies appeared. Screening for positive clones was carried out by Western blotting.g for 5 min and then washed twice with ice-cold PBS (Gibco). The cells were then resuspended in cell lysis buffer (CLB) , incubated on ice for 10 min, followed by centrifugation at 16,100 × g to obtain the postnuclear supernatant. Prior to immunoisolation the postnuclear supernatant was precleared by incubating with protein A Sepharose beads (Generon) for 30 min at 4 °C. The mixture was then cleared by centrifugation at 16,100 × g for 1 min and the supernatant incubated with protein A Sepharose beads and the appropriate antibody for 16 h at 4 °C. Immunoisolates were washed three times in CLB. Samples were then boiled at 95 °C for 5 min in 2× Laemmli sample buffer  prior to SDS/PAGE either under reducing (treated with 50 mM DTT) or nonreducing conditions.Cells were harvested by treatment with trypsin-EDTA (Gibco – 25300-054) or by scrapping using a rubber policeman . They were collected by centrifugation at 250 × For Western blotting, proteins were transferred to nitrocellulose membrane (Li-Cor Biosciences), which were blocked in 5% (wt/vol) nonfat dried skimmed milk in TBST  for at least 60 min. Primary antibodies were diluted in TBST and incubations were carried out for ∼16 h at either 4 °C or at room temperature. Li-Cor IRDYE fluorescent secondary antibodies were used for detection, typically at 1:5,000 dilutions. Blots were scanned using an Odyssey SA imaging system (Li-Cor Biosciences).Confluent HEK/ATF6α cells were pretreated with 30 µM PF429242 (S1Pi) for 60 min before treatment with various ER stressors including DTT (at either 1 mM or 10 mM), 5 µM TG, or 5 µg/mL tunicamycin. Whole-cell lysates were subjected to nonreducing SDS/PAGE, followed by Western blotting. Quantification of band intensities were carried out with the LiCor Image Studio v4 software and histograms prepared using Origin 6.0 professional software.The Flp-In T-Rex doxycycline-inducible HeLa/HA-LDLR C646Y cell line was generated by Invitrogen Flip-in T-REx kit according to the manufacturer’s protocol. Briefly, pOG44 and pcDNA3.1/HA-LDLR vector were cotransfected into the cells and selected for by hygromycin B (200 μg/mL) and blasticidin (4 μg/mL). The successfully transfected cells were induced by 2 μg/mL doxycyclin.N-glycosidase F (PNGase). Samples were separated under reducing SDS/PAGE conditions and ATF6α detected using rabbit anti-HA.Cells were treated with 30 µM S1Pi, and either left untreated or treated with 2 mM BADAP for 60 min. The cells were then treated with 10 mM DTT for 30 min. Whole-cell lysates were prepared and incubated either without or with endoH and Peptide: 2, 1 mM EDTA, and 1 mM EGTA) supplemented with protease inhibitor and left on ice for 10 min. Cells were lysed by passing through a 23-gauge needle  attached to a 1-mL syringe, 20 times, followed by centrifugation at 1,000 × g, for 7 min at 4 °C. Next, 200 µL of the resulting supernatant were laid on top a discontinuous sucrose gradient consisting of eight layers, each containing 200 µL of distinct sucrose densities of 20%, 25%, 30%, 35%, 40%, 45%, 50%, and 60%, from top to bottom, in a polycarbonate tubes . The samples were centrifuged at 214,000 × g for 60 min at 4 °C, in a TLS 55 rotor using a Beckman Coulter Optima MAX-XP ultracentifuge. Nine fractions of 200 µL each were carefully taken from the top using a pipette. Samples were analyzed on either reducing or nonreducing SDS/PAGE, followed by Western blotting.HEK/ATF6 cells in 15-cm dishes were either untreated or treated with the S1Pi and ER stressors . Cells were harvested and washed twice in 1 mL ice-cold PBS/NEM , followed by centrifugation at 1,400 rpm for 5 min. PBS was completely removed, and cell pellet was resuspended in 400 µL of ice-cold buffer A  or treated (+) with either 30 µM S1Pi (60 min), 20 mM ammonium chloride (45 min), 100 nM bafilomycin A1 (4 h), or 5 µg/mL brefeldin A (60 min) treatment, before addition of 5 µM TG to induce ER stress. After treatment, whole-cell lysates were prepared and separated under reducing or nonreducing SDS/PAGE, followed by Western blotting.Wild-type or ERp18 KO HEK293T/ATF6α overexpression cells in 6-cm dishes were either left untreated (−) or treated (+) for at least 1 h with 30 µM S1Pi, before addition of 10 mM DTT or 5 µM TG to induce ER stress. For the brefeldin A (5 µg/mL) treatment, cells were incubated with the drug for at least 1 h prior to harvest.g for 5 min, and supernatant completely removed. The cell pellets were resuspended in 1 mL ice-cold buffer A  supplemented with protease inhibitor, transferred to 1.5-mL tube, and left on ice for 10 min. Cells were lysed by passing through a 23-gauge needle attached to a 1-mL syringe, 20 times, followed by centrifugation at 1,000 × g, for 7 min at 4 °C to obtain the nuclear pellet. The pellet was then resuspended in 100 μL buffer B  supplemented with protease inhibitor, followed by incubation for 60 min on a 4 °C rocker. The samples were centrifuged at 100,000 × g for 30 min, 4 °C, to obtain a supernatant containing the nuclear extract. The samples were separated on reducing SDS/PAGE gels, followed by Western blotting.Isolation and analyses of nuclear ATF6α, ATF6-N, has been described previously , 37. BriConfluent cells in 6-cm dishes were either left untreated or treated with 1 µM TG for 16 h. Whole-cell lysates were subjected to reducing SDS/PAGE, followed by Western blotting. Quantification of band intensities were carried out with the LiCor Image Studio v4 software and histograms prepared using Origin 6.0 professional software.HEK/ATF6 wild-type cells were harvested and washed in PBS then lysed in CLB. Endo H and PNGase reactions were set up according to the manufacturer’s protocol (New England Biolabs). The digests were carried out overnight at 37 °C and samples were resolved on either reducing or nonreducing SDS/PAGE.Total RNA from TG-treated cells was extracted using TRIzol Reagent (Ambion), according to the manufacturer’s protocol. RT-PCR was carried out using the SuperScript II Reverse Transcriptase (Invitrogen) with oligo dTs (Invitrogen), according to the manufacturer’s protocol. cDNA for endogenous XBP1 was amplified using the following primers pairs: 5′- GAATGAAGTGAGGCCAGTGG -3′, 5′- GGGGCTTCCTATATATGTGG -3′. Actin cDNA was amplified using the following primer pairs: 5′-CCACACCTTCTACAATGAGC-3′, 5′-ACTCCTGCTTGCTGATCCAC-3′. PCR parameters include an initial denaturation step at 95 °C for 5 min; then 35 cycles of 95 °C for 45 s, an annealing step of 60 °C for 45 s, and 72 °C for 45 s; followed by a final elongation step of 72 °C for 10 min.Using the Integrated DNA Technologies (IDT) Alt-R CRISPR-Cas 9 System, the predesigned guide RNA (gRNA) Hs.Cas9.ATF6.1.AB was selected. We used a single gRNA (cRNA) duplexed with a tracrRNA  and Alt-R S.p. HiFi Cas9 Nuclease V3  for genome editing. To generate an ATF6 KO, Cas9 nuclease was added to a duplex of 1AB crRNA:tracrRNA in Optimem to form the RNP complex , which was then transfected into HT1080 cells using Lipofectamine CRISPRMAX transfection reagent . After 48 h, cells were harvested for genomic DNA using QuickExtract DNA Extraction Solution . Genomic PCR using primers designed for Exon 1 of ATF6α was performed to create template DNA for the T7 Endonuclease assay . Briefly, the wild-type and 1.AB DNA were hybridized slowly and then digested using T7 endonuclease, to detect any cleavage events by mismatching of the DNA via genome editing events. Samples were analyzed on an agarose gel and by DNA sequencing. After cleavage was confirmed, cells were transferred to 15-cm dishes until colonies appeared (about 10 to 12 d later). Positive ATF6α KO cells were identified by Western blotting using the mouse anti-ATF6 antibody.Supplementary File