MET·HCl = 0.66), we observed a slight enhancement in the dissolution properties compared with pure HTZ (1.21-fold). The same analysis for the solid forms of MET·HCl-CTZ (χMET·HCl = 0.33 and 0.5) showed an enhancement in the dissolved amount of CTZ accompanied by a slight improvement in solubility. From these dissolution profiles and saturation solubility studies and by comparing the thermodynamic parameters (ΔHfus and ΔSfus) of the pure drugs with these new solid forms, it can be observed that there was a limited modification in these properties, not modifying the free energy of the solution (ΔG) and thus not allowing an improvement in the dissolution and solubility properties of these solid forms.The mechanochemical synthesis of drug–drug solid forms containing metformin hydrochloride (MET·HCl) and thiazide diuretics hydrochlorothiazide (HTZ) or chlorothiazide (CTZ) is reported. Characterization of these new systems indicates formation of binary eutectic conglomerates, i.e., drug–drug eutectic solids (DDESs). Further analysis by construction of binary diagrams (DSC screening) exhibited the characteristic V-shaped form indicating formation of DDESs in both cases. These new DDESs were further characterized by different techniques, including thermal analysis (DSC), solid state NMR spectroscopy (SSNMR), powder X-ray diffraction (PXRD) and scanning electron microscopy–energy dispersive X-ray spectroscopy analysis (SEM–EDS). In addition, intrinsic dissolution rate experiments and solubility assays were performed. In the case of MET·HCl-HTZ (χ Pharmaceutical cocrystals are multicomponent crystalline entities composed of an active pharmaceutical ingredient (API) and a coformer in a defined stoichiometric ratio ,2,3. TheReplacement of the coformer by another API yields the formation of drug–drug cocrystals (DDCs) ,7, speciThe benefits found in the preparation of DDCs ,7, DDESsThus, when monotherapy is no longer efficient for T2D, the simultaneous utilization of two or more drugs depending on the clinical profile of the patient seems to be a more appropriate approach ,41. Metfint 12.5-fold more than HTZ crystalline and 2.2-fold better than the PM HTZ-atelonol [Within the family of thiazide diuretics, chlorothiazide (CTZ) is a poor water-soluble drug (0.2 and 0.4 g/L at pH 4 and 7), exhibiting a plasma half-life of 45–120 min . Furtheratelonol .Taking this into account, we are interested in the preparation of drug–drug solid forms containing MET·HCl  , in the TM  or Toronto Research ChemicalsTM  and were used as received. The solvents were purchased from TecsiquimTM  and were used as received.All the pharmaceutical reagents were purchased from Tokyo Chemical Industry AmericaTM 7 Fritsch  device. MET·HCl  was ball-milled with HTZ  or CTZ  in a 1:1 stoichiometric ratio. For every LAG experiment 100 μL of solvent were added. The solvents used were, hexane, acetone, acetonitrile, and water. Stainless steel bowls of 20 mL containing 10 stainless steel balls (10 mm diameter) were used. The NG or LAG were carried out at 600 rpm for 30 min. The physical mixtures (PM) were prepared by mechanical shaking MET·HCl  with HTZ  or CTZ  in a 1:1 stoichiometric ratio for 5 min using a Vortex Genie 2 Scientific IndustriesTM  (velocity of shaking 5).NG or LAG solvent-screening for the preparation of the solid forms was performed using a Planetary Micro Mill PulverisetteA simultaneous thermal analyzer Netzsch STA 449 F3 Jupiter was used. The samples were placed (2–4 mg) in sealed non-hermetic aluminum pans and scanned at a heating rate of 10 °C/min from 30–400 °C under a dry nitrogen atmosphere.TM 7 Fritsch at 600 rpm for 2 h. The DSC determinations proceeded at a heating rate of 10 °C/min using the thermal analyzer Netzsch STA 449 F3 Jupiter. Binary phase diagrams were constructed by plotting the melting temperatures from the different compositions (onset), and the second endothermic event as the liquidus (Tonset) in function of the mole fraction of MET·HCl. Tammann diagrams were constructed by plotting ΔHfusion from the different stoichiometric ratios as a function of the mole fraction of MET·HCl. Some samples were occasionally analyzed at a heating rate of 2 or 5 °C/min to improve the viewing accuracy on some thermal events.The determination of the eutectic points was made by means of construction of binary phase and Tammann diagrams ,65. Diffositions , considePXRD experiments were carried out in a Bruker D8 Advance diffractometer with Bragg-Bretano geometry, Cu Kα radiation (1.54060 Å) and Linxeye detector. Each sample was measured by a continuous scan between 5–60° in 2θ, with step time 151.19°/min and step size of 0.0198°. The Rietveld refinements were carried out using the program Fullprof suite to calculate the final crystal lattices of the different outcomes . Deposit1H 300 MHz, 13C 75 MHz and 15N 30 MHz). SSNMR measurements were carried out on a 4 mm rotor double resonance CP-MAS probe at 5–6 kHz spinning rate with a cross-polarization contact time of 2 ms and delay of 5 s. In addition, HMBC and HSQC experiments were carried out in a Bruker Avance III 500 . Solution NMR measurements were carried out on a 4 mm broadband probe with two channels; the heteronuclear channel can be tuned 31P (202 MHz) until 107Ag (27 MHz), with Z-axis gradients. CTZ and HTZ were dissolved in d6-DMSO for the HMBC and HSQC experiments (15N experiments we used NH3(l) δ = 0 ppm as internal reference and glycine (δ = 38 ppm) as secondary standard. The chemical shifts assignment in the 15N-SSNMR experiments presented in 1H, 13C and 15N [6-DMSO and D2O for MET·HCl have been previously reported [15N-SSNMR spectra for MET·HCl we noted the presence of 4 signals as well as the reported NMR solution [13C CP-MAS assignation of CTZ [Solid-state NMR (SSNMR) spectra were recorded in a Bruker Avance II 300 spectrometer  and MET·HCl-HTZ 1:1 (χMET·HCl = 0.5), A JEOL  scanning electron microscope (SEM) model JSM-6510LV was employed to examine the morphology of the solid forms (MET·HCl-CTZ 1:1 (χ2. The dissolution rates were determined with a Wood’s apparatus according to the USP XLI. Dissolution profiles were made in 0.1 N HCl for each batch. Experiments were carried out in triplicate at 37 °C under constant stirring (50 rpm) in a constant volume of 900 mL. For CTZ and the different MET·HCl-CTZ compositions, IDR determinations were made using a HPLC Agilent 1100 with an automatic injector (DE116471) under the following chromatographic conditions: mobile phase of H3PO4 0.025 M and acetonitrile (80:20) with a flow of 1 mL/min and using a Zorbax SB-C18 column with dimensions of 4.6 mm × 150 mm with particle size of 5 µm at a wavelength of 272 nm. For HTZ and the different MET·HCl-HTZ compositions, IDR determinations were performed using a UV-VIS spectrophotometer ) at the wavelength of 272 nm.We determined the IDR constants under physiological conditions. The experiments were performed using tablets, prepared with a hydraulic press at a total force of 180–200 kg/cmAn excess amount of powder (eutectics or pure drug) was weighed (approximately 33.3 mg) and dissolved in a vial with a fixed volume of water (1 mL 0.1 N HCl). The vial was magnetically stirred for 72 h at 37 °C. After the equilibrium time, an aliquot was passed through a 0.45 µm filter and properly diluted and quantified through HPLC (Agilent 1260 infinity II) using a calibration curve. The experiments were made in triplicate.The preparation of the solid forms was performed by ball-milling of MET·HCl with CTZ or HTZ (1:1) using neat grinding (NG) or liquid assisted grinding (LAG) ,62,63,64Analysis by PXRD  of the aP21/c) and CTZ , indicating the coexistence of two solid-phases as a conglomerate of separated components, both APIs keeping their own lattice, being more like a non-continuous single-phase than a eutectic solid. According to Cherukuvada and Nangia, “eutectic solids lack a distinct unique lattice arrangement from the individual components and retain the cohesive interactions in solid solutions” [P21/c) and HTZ , both constituents retaining their own lattice. Hence, no effect of the polarity of the solvent was found for the LAG solvent-screening in both solid forms, the quantitative percentage ratios for the lattice structures between the APIs remaining constant in all cases.Based on the Rietveld refinements  for MET·lutions” ,20. Simifus (197.8–203.2 °C) compared with the parent APIs. In addition, for the PM the endothermic event exhibits two overlapped peaks (209.5 and 218.28 °C), clearly indicating that the different products obtained on the LAG solvent-screening are not PMs, since they only exhibit a single endothermic peak and not separated melting events for the individual components [The products obtained through the LAG solvent-screening were also analyzed by DSC. PMs were prepared combining MET·HCl with HTZ or CTZ in a 1:1 stoichiometric ratio, and analyzed by the same technique. mponents .fus compared with the pure MET·HCl or HTZ  of MET·HCl-CTZ 1:1 or MET·HCl-HTZ 1:1, SSNMR experiments were performed. Atom numbering for MET·HCl, CTZ and HTZ for 13C and 15N nuclei are shown in So far, we have seen that according to PXRD results, homosynthons predominate over the heterosynthons. In this regard, Cherukuvada and Nangia explained that “through different spectroscopical and PXRD analysis, eutectic solid phases and solid solutions have close similarity with their pure constituents” . Thus, u13C-SSNMR experiments, it can be observed that there are no significant Δδ of the pure APIs compared with the new solid forms MET·HCl-CTZ 1:1 or MET·HCl-HTZ 1:1  compared with MET·HCl (232.9 °C) [13C is observed compared with the original APIs (2NH2) catemer chain of HTZ cannot be interrupted by the amide group of ATL. In addition, for MET·HCl-CTZ 1:1 or MET·HCl-HTZ 1:1 apparently the non-self-complementarity (shape mismatch) of the initial components promotes the lack of formation of heterosynthons [Through -HTZ 1:1 . The mins in CTZ . This is32.9 °C) . A similnal APIs . Once agsynthons ,21,82.13C-SSNMR spectra of pure CTZ , lacking signals for C5′, C9′ and C10′ which were not observed, probably due to lack of efficient cross polarization 1H-13C.The pure CTZ  exhibit 15N-SSNMR experiments, for the APIs MET·HCl, CTZ or HTZ and the solid forms of MET·HCl-CTZ 1:1 or MET·HCl-HTZ 1:1  against the mole fraction of MET·HCl (χMET·HCl) at all examined compositions (fusion) against the mole fraction of MET·HCl [Thus, binary diagrams b were coositions . Tammann MET·HCl ,31. For The depression in the melting point, giving place to the characteristic V-shape in the binary diagram, indicates that a true eutectic composition has been found ,64,86. CMET·HCl = 0.5 (molar ratio 1:1), the formation of a true eutectic composition with a Teut = 198.78 °C occurs. Further, the Tammann’s triangle confirmed that at χMET·HCl = 0.5 is a true eutectic point  (MET·HCl = 0.66 (molar ratio 2:1), a higher value  was observed, with this composition not a genuine eutectic point, due to the appearance of a second peak (liquidus) caused by the presence of unreacted or excess components.A typical V-shaped graphic for MET·HCl-CTZ b indicatsolidus) , only exMET·HCl = 0.5 (molar ratio 1:1), with Teut = 169.9 °C since a single endotherm was observed. Once again, according to Tammann’s triangle the highest value of ΔHfus  (MET·HCl = 0.66 (molar ratio 2:1). However, this is an inaccurate determination because both endotherms (solidus and liquidus) almost form a single peak. Thus, composition χMET·HCl = 0.5  gives place to the true eutectic composition.In the case of MET·HCl-HTZ b, the trsolidus)  was for In addition, PXRD experiments  and RietMET·HCl = 0.5 were analyzed by scanning electron microscopy energy–dispersive X-ray spectroscopy SEM–EDS (MET·HCl = 0.5) was EDS Cl: 11.49 and S: 9.00 and calculated Cl: 15.37 and S: 13.90 (MET·HCl = 0.5) it was EDS Cl: 14.49 and S: 14.97 and calculated Cl: 15.37 and S: 13.90  and MET·HCl-HTZ (χMET·HCl = 0.5) (MET·HCl = 0.5) or MET·HCl-HTZ (χMET·HCl = 0.5) are not a continuous single crystalline solid and the APIs are physically separated [Furthermore, the EDS mapping shows the distribution of O and S atoms in the region of interest for both MET·HCl-CTZ (χl = 0.5) , where ieparated . This pheparated , when sueparated .According to the SEM micrographs, the morphology and texture observed in both solid forms are as solid phases with low crystallinity .MET·HCl = 0.5) and MET·HCl-HTZ (χMET·HCl = 0.5) solidified in a faceted manner (irregular microstructure) [By itself, binary eutectic solids exhibit a microstructure-level periodicity different of the original pure crystalline components ,91. Durionstant) ,93 obserregular) . In our J/mol·K)  (Table 2ructure) ,93.fus(excess) with the experimental ones can be used to differentiate whether a eutectic mixture or a PM has been obtained [component = mole fraction of either component):Furthermore, a comparison of the determined values of ΔHobtained . Thus, tfusion(excess) values were observed for both solids MET·HCl-CTZ (χMET·HCl = 0.5) and MET·HCl-HTZ (χMET·HCl = 0.5): −84.90 J/g and −110.05 J/g, thus indicating that DDESs were formed instead of PMs [Based on the above, negative ΔHd of PMs .int values for both thiazides. Thus, IDR experiments were carried out only for the solid phases MET·HCl-CTZ and MET·HCl-HTZ with compositions of 1:1 (χMET·HCl = 0.5), 1:2 (χMET·HCl = 0.33) and 2:1 (χMET·HCl = 0.66), since these proportions are closer to the true eutectic points determined. int values found and Due to the fact that CTZ and HTZ are the limited-water soluble drugs, in this study we only focused on the Kint value determined (0.076 mg/min·cm2) is close to that reported for crystalline HTZ (0.098 mg/min·cm2) [int value of MET·HCl [MET·HCl = 0.66 showed an increase of 1.21-fold compared with pure HTZ, however the compositions χMET·HCl = 0.33 and 0.5 exhibited a decrease in the dissolution rate (0.76- and 0.77-fold). This decrease in the release behavior of HTZ can be attributed to the great excess of HTZ compared with MET·HCl, limiting any probable enhancement during dissolution [MET·HCl = 0.33 and 0.5 showed an increase in the amount of CTZ dissolved (1.31 and 1.65-fold) compared with pure CTZ. However, in the case of MET·HCl-CTZ χMET·HCl = 0.66, it exhibited an increase of 6.06-fold. This should be taken with caution, since this Kint value presented a high standard deviation  was observed, suggesting an enhancement in the amount dissolved of the thiazides. However, there was also a slight increase in the ΔHfus compared with the original components, which affected the ΔG, leading to a limited modification in the amount dissolved, because ΔG tends to be more positive. In the case of the solid forms with composition χMET·HCl = 0.33, both parameters remain almost unchanged, reflecting a limited modification in the amount dissolved: ΔHfus (limited modification in the intermolecular interactions) and ΔSfus (poor dispersibility). On the other hand, in the case of the DDES HTZ:atelonol (0.3:0.7), a 10-fold improvement of the % release of HTZ (phosphate buffer and pH = 7.4) was observed [fus values for both solid forms MET·HCl-CTZ and MET·HCl-HTZ led us to conclude that a limited modification in the intermolecular interactions occurred after the solidification of the DDESs compared with their parent components.The Kmin·cm2) . In a pr MET·HCl . Specifisolution . In addieviation . This isnd ΔHfus . Usuallylubility ,97. Thuslubility . Globallobserved . Haneef MET·HCl = 0.33, 0.5 and 0.66 improved 1.51-, 1.48- and 1.44-fold respectively, and for MET·HCl-HTZ χMET·HCl = 0.33, 0.5 and 0.66 increases in the solubility were 1.11-, 1.13- and 1.36-fold, respectively. Considering again the DDES HTZ:atelonol (0.3:0.7), an improvement in the solubility of HTZ by 14-fold was reported. The poor modifications in the solubility observed in the solid forms MET·HCl-CTZ χMET·HCl = 0.33, 0.5 and 0.66 and MET·HCl-HTZ χMET·HCl = 0.33, 0.5 and 0.66 is attributed to the limited alteration of ΔG in the solution.According to the saturation solubility studies , all theMET·HCl = 0.5 for both solid forms. In general, it has been reported that DDESs have a lower melting point than their original components, and that these solid forms have high free energy, greater molecular mobility and weaker intermolecular interactions [fus and ΔSfus, compared with the original constituents, despite the fact that a considerable decrease in Tfus values is observed.Thus, this paper describes the mechanochemical preparation of the DDESs MET·HCl-CTZ and MET·HCl-HTZ by NG or LAG. Analyses by means of binary diagrams (DSC screening) allowed the determination of the true eutectic points at the composition of χractions ,91,93,99Hence, this study represents a thorough characterization of two DDESs containing MET·HCl and CTZ or HTZ. This shows that despite an active research history describing organic solid eutectics , when re


Glucoamylase is an important industrial enzyme in the saccharification of starch into glucose. However, its poor thermostability and low catalytic efficiency limit its industrial saccharification applications. Therefore, improving these properties of glucoamylase is of great significance for saccharification in the starch industry.TlGa15B from the thermophilic fungus Talaromyces leycettanus JCM12802 was cloned and expressed in Pichia pastoris. The optimal temperature and pH of recombinant TlGa15B were 65 ℃ and 4.5, respectively. TlGa15B exhibited excellent thermostability at 60 ℃. To further improve thermostability without losing catalytic efficiency, TlGa15B-GA1 and TlGa15B-GA2 were designed by introducing disulfide bonds and optimizing residual charge–charge interactions in a region distant from the catalytic center. Compared with TlGa15B, mutants showed improved optimal temperature, melting temperature, specific activity, and catalytic efficiency. The mechanism underlying these improvements was elucidated through molecular dynamics simulation and dynamics cross-correlation matrices analysis. Besides, the performance of TlGa15B-GA2 was the same as that of the commercial glucoamylase during saccharification.In this study, a novel glucoamylase-encoding gene TlGa15B-GA2 make it a good candidate for industrial saccharification applications.We provide an effective strategy to simultaneously improve both thermostability and catalytic efficiency of glucoamylase. The excellent thermostability and high catalytic efficiency of The online version contains supplementary material available at 10.1186/s13068-021-02052-3. Glu211, and Glu433 was predicted to be the catalytic residues that served as the proton donor and acceptor, respectively. Moreover, five highly conserved residues associated with substrate binding in the GH15 family were identified in the catalytic domain of TlGa15B, including Arg86, Asp87, Leu209, Trp210, Glu212, and Arg338 [TlGa15B was constructed using the glucoamylase from Penicillium oxalicum  as template with the help of SWISS-MODEL. Meanwhile, the Ramachandran plot was used to evaluate the quality of the structural model. There were only two amino acid residues observed in disallowed region  was constructed via PCR-mediated site-directed mutagenesis. As shown in Additional file TlGa15B mutants formed disulfide bonds in the target sites. In addition, the enzyme thermal stability system (ETSS) was used to calculate the total interaction energy (Eij) between charged amino acids of TlGa15B [Eij = 27.3 kJ/mol) was mutated to a neutral amino acid alanine , exhibiting the most significant decline in the Eij value. Subsequently, the mutant TlGa15B-GA2 (S132C-Y492C/L548C-A562C/Q108E) was generated via site-directed mutagenesis using TlGa15B-GA2 as the template.It was reported that the construction of disulfide bonds and the optimization of residual charge–charge interactions were efficient strategies to improve the thermostability of industrial enzymes . In this TlGa15B . 6 non-cTlGa15B and its mutants TlGa15B-GA1 and TlGa15B-GA2. By comparison, the optimal temperature and thermostability of mutants both significantly improved. The optimal temperatures of TlGa15B-GA1 and TlGa15B-GA2 were 70 ℃ and 75 ℃, which were 5 ℃ and 10 ℃ higher than that of TlGa15B, respectively , the Tm values of TlGa15B-GA1 and TlGa15B-GA2 showed a significant increase of 8 °C (80 ℃) and 9 °C (81 ℃), respectively.As shown in Fig. TlGa15B-GA1 and TlGa15B-GA2 also exhibited higher catalytic activity and efficiency than TlGa15B  was an important parameter to reflect the rigidity of the protein structure, which correlated with protein thermostability [TlGa15B-GA1 and TlGa15B-GA2 significantly decreased compared with that of TlGa15B, indicating that the overall structures of the mutants were more thermostable  for TlGa15B-GA2 and GA-LD reached 96.4% and 96.7%, respectively, indicating no significant difference between TlGa15B-GA2 and the commercial glucoamylase GA-LD. Moreover, the DX value of TlGa15B-GA2 was in accordance with the industrial requirement for the production of glucose with a DX value above 96%. These results suggested that TlGa15B-GA2 had the same saccharification effect as industrial glucoamylases for the enzymatic saccharification process of starch.In order to evaluate the performance of industrial saccharification applications, the best mutant TlGa15B, derived from thermophilic fungus T. leycettanus JCM12802, exhibited high optimal temperature and excellent thermostability above 60 ℃. By introducing disulfide bonds and optimizing residual charge–charge interactions in a region distant from the catalytic center, TlGa15B-GA1 and TlGa15B-GA2 showed improvements in optimal temperature, melting temperature, specific activity, and catalytic efficiency. The mechanism underlying the improved thermostability and catalytic efficiency of glucoamylase was elucidated by MD simulation and DCCM analysis. The performance of TlGa15B-GA2 was similar to that of the commercial glucoamylase during the saccharification process of starch. These properties of TlGa15B-GA2 make it a promising candidate for industrial saccharification applications.T. leycettanus JCM12802 was purchased from the Japan Collection of Microorganisms RIKEN BioResource Center  and was maintained at 4 ℃ on a potato dextrose agar plate. Soluble starch, amylose, glycogen, α-cyclodextrin, β-cyclodextrin, and γ-cyclodextrin were purchased from Sigma-Aldrich. All other chemicals were of analytical grade and commercially available.The thermophilic fungus T. leycettanus JCM12802 was cultured in the potato dextrose broth (PDB) medium at 40 ℃ for 3 days. Total RNA was extracted using the TRIZOL reagent  according to the operation manual. The first strand cDNA was synthesized from the total RNA using the FastKing RT Kit with gDNase . Based on the 5′ and 3′-end sequences of the TlGa15B structural gene, the TlGa15B-encoding gene devoid of the signal peptide sequence was amplified with gene-specific primers  with the plasmid pPIC9-TlGa15B as the template.TlGa15B, pPIC9-TlGa15B-GA1, and pPIC9-TlGa15B-GA2 were linearized with BglII and transformed into P. pastoris GS115 competent cells, respectively. The positive transformant with the highest glucoamylase activity was inoculated into YPD medium at 30 °C for 2 days with shaking at 200 rpm and used as the inoculum of 300 mL BMGY medium. The cultures were grown at 30 °C for 2 days, and cells were then harvested and resuspended in 200 mL BMMY medium for 2 days growth at 30 °C. The crude enzyme was collected by centrifugation at 12,000g for 10 min at 4 °C, followed by concentration through a 10 kDa cut-off centrifuge filter. The crude enzyme was dialyzed in citrate–phosphate buffer  at 4 °C overnight using a 3 kDa dialysis tube , and run through a HiTrapTM Q Sepharose XL anion exchange column  for purification. The purified recombinant TlGa15B and mutants were verified by SDS-PAGE using a 10% polyacrylamide gel.The recombinant plasmid PIC9-TlGa15B and mutants was determined using soluble starch as the substrate. Reactions containing 900 μL of 1% soluble starch in citrate–phosphate buffer (pH 4.5) and 100 μL of appropriately diluted enzyme solution were incubated at 60 °C for 30 min, using the 3,5-dinitrosalicylic acid (DNS) method to detect the amount of reducing sugar in the reaction. One unit (U) of enzyme activity was defined as the amount of enzyme that released 1 μmol of glucose per minute.The glucoamylase activity of TlGa15B and mutants were determined in glycine–HCl (pH 1–3), citric acid-Na2HPO4 (pH 3–8), and glycine–NaOH (pH 8–12) buffers at 37 ℃. To determine pH stability, the purified TlGa15B and mutants were incubated in different pH (1–12) for 1 h at 37 ℃. The effect of temperature on activity was measured in citric acid-Na2HPO4 buffer (pH 4.5) at 20–90 ℃. To evaluate thermostability, the purified TlGa15B and mutants were incubated at 75 ℃ for different time periods (0–60 min).The effects of pH on the activity of purified 2HPO4 buffer (pH 4.5) at 55 ℃ using 1.0–10.0 mg/mL soluble starch as the substrates. The Lineweaver–Burk plot method was used to calculate the Km and Vmax values of the purified TlGa15B and mutants. The substrate specificities of TlGa15B were studied in terms of the hydrolysis of six different substrates, including soluble starch, amylose, glycogen, α-cyclodextrin, β-cyclodextrin, and γ-cyclodextrin in a citric acid-Na2HPO4 buffer (pH 4.5).Kinetic studies were performed in a citric acid-NaTm) of TlGa15B and mutants were determined using MicroCal™ VP-Capillary DSC . The purified TlGa15B and mutants were dissolved in a 10 mM phosphate buffer (pH 7.4) as an infusion, and the protein concentration was controlled at 0.50 mg/mL. The temperature program was run in the range of 50–100 ℃, with a rising rate of 1 ℃/min and a scanning rate of 1℃/min.The melting temperatures  of the saccharification solution.To evaluate the industrial application potential of Additional file 1: The Ramachandran plot of homology modeling for TlGa15B.Additional file 2: SDS-PAGE analysis of the recombinant TlGa15B and mutants. Lane 1, 3, 5, the culture supernatant of transformants TlGa15B, TlGa15-GA1, and TlGa15-GA2; lane 2, 4, 6, the purified TlGa15B, TlGa15-GA1, and TlGa15-GA2.Additional file 3: The thermostability of the purified recombinant TlGa15B.Additional file 4 LC-MS/MS analysis of the formation of disulfide bonds in the TlGa15B mutants. a: the disulfide-linked peptide SNPSGGLCT/SASGPCA (132C/492C); b: the disulfide-linked peptide PLWYCIV/SAIPCSA (548C/562C).Additional file 5: The modeled structure of TlGa15B-GA2. Six candidate mutated sites away from the catalytic center were indicated in blue balls. Two disulfide bonds were indicated as yellow sticks.Additional file 6: The specific activity of TlGa15B and its mutants in the temperature range from 20 to 90 ℃.Additional file 7: Primers used in this study.


SLC16A2 gene that encodes thyroid hormone transporter. AHDS has been rarely reported in China.Allan-Herndon-Dudley syndrome (AHDS) is an X-linked recessive neurodegenerative disorder caused by mutations in the SLC16A2 gene in an 18-month-old male patient with AHDS. The patient was born to non-consanguineous, healthy parents of Chinese origin. He passed new-born screening for hypothyroidism, but failed to reach developmental milestones. He presented with hypotonia, severe mental retardation, dysarthria and ataxia. Genetic analysis identified a novel splicing mutation, NM_006517.4: c.431-2 A > G, in the SLC16A2 gene inherited from his mother. The patient received Triac treatment, (triiodothyroacetic acid), a thyroid hormone analogue for 3 months. Triac treatment effectively reduced serum TSH concentrations and normalized serum T3 concentrations in the patient.This study reported a novel splicing mutation in the SLC16A2 gene in AHDS patients.This study reported the first case of AHDS treated by Triac in China. And the study expanded the mutational spectrum of the The online version contains supplementary material available at 10.1186/s12887-022-03259-5. SLC16A2 gene (Solute Carrier Family 16 Member 2) [SLC16A2 gene is located in Xq13.2, formerly called monocarboxylate transporters 8 (MCT8) [SLC16A2 variant causes AHDS characterized by mental and motor developmental delay, and thyroid functional abnormalities of high serum T3, reduced T4, and normal or mildly increased Thyroid stimulating hormone (TSH) [SLC16A2 gene were reported [SLC16A2 gene in a Chinese patient with AHDS and the hormonal effects of Triac in this patient.Allan-Herndon-Dudley syndrome (AHDS) is an X-linked neurodevelopmental disorder, first described at 1944 by Allan et al. [8 (MCT8) . As one ne (TSH) . To datereported , 8–12. TThe patient, an eighteen-month-old Chinese boy, was the first child born to healthy parents after uneventful pregnancy and delivery. His birth weight was 3 kg, birth height was 50 cm, and birth head circumstance was 34 cm. He passed hearing test and newborn screening for hypothyroidism after birth. He experienced feeding difficulty since his birth. At 6-month-old he presented with global hypotonia and developmental delay. He had never been able to hold up his head and could not sit. Physical examination revealed hypotonia in the trunk and hypertonia in the extremities. He had elongated face with bifrontal narrowing and flat nose, otherwise didn’t show dysplastic ears, pectus excavatum, scoliosis, flat feet, or lateral deviation of great toe etc. Thyroid function test in local medical center was performed as part of routine evaluation of global developmental delay, demonstrating increased free triiodothyronine (FT3) of 9.49 pg/ml , decreased free thyroxine (FT4) of 0.49 pg/dl  and mildly increased thyroid-stimulating hormone concentration (TSH) of 4.93 µIU/ml  , decreased FT4 of 0.94 pg/dl , and normal TSH of 0.47 µIU/ml . Euthyroxol was prescribed with methimazole to the patient for 3 months and then stopped due to no effect.When the patient was 12-month-old, he was not able to hold up his head, and could not sit and stand. He presented with severe global developmental delay, and generally could not reach any milestones. Physical examination revealed hypotonia in the trunk and hypertonia in the extremities. He had elongated face with bifrontal narrowing and flat nose. His height (75.4 cm) just reached − 2 SD (73 cm) and his weight (7 kg) was below − 2 SD (8.2 kg) on growth chart. The head circumference was 45 cm (-2 SD 44.2 cm) and chest circumference was 44 cm. Thyroid function test revealed increased FTSLC16A2 gene was applied to coding sequences of all exons of the SLC16A2 gene (NM_006517.4.) with exon/intron boundaries when the patient was 12-month-old. DNA was extracted using QIAamp DNA Blood Mini Kit (250) . DNA as a template was amplified by PCR with primers designed by Primer 5.0 software program . Genetic analysis revealed the presence of a novel splicing variant (NM_006517.4: c.431-2 A > G). By using online programs FATHMM, NetGene2, Human Splicing Finder 3.1 and Mutation Taster, the pathogenicity of the variant c.431-2 A > G was predicted to be deleterious by causing splice acceptor site change. The variant c.431-2 A > G may result in splicing change at protein level. Pedigree analysis revealed that this variant was inherited from his heterozygous healthy mother, but was not found in his maternal grandparents , decreased FT4 of 0.68 pg/dl , and normal TSH of 5.33 µIU/ml   and normal TSH of 0.854 µIU/ml . And no abnormality was found in EEG  and B-ultrasound of abdominal organs.Triac, a T3 analog, has been reported as a promising candidate to normalize serum T3 levels and thus alleviate the thyrotoxicosis in the patient with MCT8 deficiency. With a written and signed informed consent, Triac treatment has been initiated to treat the patient and maintained for 3 months. Triac dose started with 10 µg/kg (0.0875 mg) per day, doubling the dose (0.17 mg per day) after 2 weeks. After Triac treatment with the dose of 0.17 mg per day for 3 months, his height (82.4 cm) and weight (8.7 kg) were still mildly below − 2 SD (83 cm and 9.6 kg) on growth chart. The head circumference (48 cm) was in normal range and chest circumference reached 46.5 cm. Thyroid function test showed normal FT3 of 7.35 pmol/L , decreased FTSLC16A2 gene have been reported, one of which carried a variant in the same intron with the case in this study [According to Human Gene Mutation Database (HGMD), a total of 5 splicing variants in the is study . To furtSLC16A2 gene never reported so far. The patient exhibited severe development delay, thyroid function abnormalities of elevated FT3 and decreased FT4, and delayed brain myelination, indicating the novel splicing variant c.431-2 A > G in the SLC16A2 gene is associated with severe phenotype of AHDS. To date, of 159 variants in the SLC16A2 gene, the splicing variants were reported in only few AHDS cases [SLC16A2 gene has been reported [SLC16A2 gene presented with severe intellectual and motor developmental delay, delayed myelination of the white matter and elevated serum FT3 level. These results indicate the splicing site of c.430–431 in the SLC16A2 gene may be the key point for splice acceptor site. Further functional study is needed.We describe a Chinese patient diagnosed with AHDS, carrying a new splicing variant in the DS cases , 9, 12. reported . An 8-mo3 and TSH were back to normal.The T3 analog Triac has been reported as a promising candidate to normalize serum T3 levels and thus alleviate the thyrotoxicosis, and restore thyroid hormone signaling in the brain . There wBecause of reduced FT4 and mildly increased TSH levels, some patients with AHDS have been initially suspected as hypothyroidism and treated with levothyroxine, just like the patient in the current study . AlthougSLC16A2 gene is pathogenic, which is associated with typical phenotype of AHDS. Triac treatment effectively reduced serum TSH concentrations and normalized serum T3 concentrations in the patient. This is the first case of MCT8 deficiency treated by Triac in China.The study supports that the novel splice acceptor site variation c.431-2 A > G in Additional file 1.


LRRK2 phosphorylates Rab8a at T72 (pT72) of its switch 2 α-helix. This post-translational modification enables recruitment of RILPL2, an effector that regulates ciliogenesis in model cell lines. A newly identified RBD motif of RILPL2, termed the X-cap, has been shown to recognize the phosphate via direct interactions between an arginine residue (R132) and pT72 of Rab8a. Here, we show that a second distal arginine (R130) is also essential for phospho-Rab binding by RILPL2. Through structural, biophysical, and cellular studies, we find that R130 stabilizes the primary R132:pT72 salt bridge through favorable enthalpic contributions to the binding affinity. These findings may have implications for the mechanism by which LRRK2 activation leads to assembly of phospho-Rab complexes and subsequent control of their membrane trafficking functions in cells.Parkinson’s-disease-associated LRRK2 is a multidomain Ser/Thr kinase that phosphorylates a subset of Rab GTPases to control their effector functions. Rab GTPases are the prime regulators of membrane trafficking in eukaryotic cells. Rabs exert their biological effects by recruitment of effector proteins to subcellular compartments via their Rab-binding domain (RBD). Effectors are modular and typically contain additional domains that regulate various aspects of vesicle formation, trafficking, fusion, and organelle dynamics. The RBD of effectors is typically an  Parkinson’s-disease-associated LRRK2 kinase phosphorylates Rab8a and Rab10 to subvert their membrane trafficking functions in the formation of primary cilia. Rab8a is phosphorylated at a threonine (pT72) and subsequently binds to a phosphospecific effector (RILPL2) via its C-terminal RH2 domain. Here, we show that recognition of pT72 involves a dual arginine motif involving R130 and R132 from the RH2 domain of RILPL2. Thermodynamic and structural studies suggest that the phosphospecific interactions are highly sensitive to the upstream region, which includes the N-terminal RH1 domain that binds to myosin Va. The dual arginine motif may facilitate the dynamic nature of membrane trafficking processes involving phosphorylation of Rabs.γ-phosphate of GTP. In recent years, several Rab GTPases have been shown to be targets of protein kinases , the X-cap enables projection of an Arg residue (R132RL2) from each monomer toward a phosphorylated threonine residue of Rab8a (pT72R8). In cells, the kinase LRRK2 phosphorylates Rab8a and Rab10 at conserved threonine residues in the switch 2 region, and the post-translational modification tunes the strength of Rab interactions with their binding partners (We have recently described a novel X-cap motif that enables the RILP family of effectors to bind to phosphorylated Rab GTPases . The N-t117) at 1.9-Å resolution. The structure includes residues preceding the X-cap that were not mapped in our previous work. In addition, we have determined the structure of the phosphomimetic T72E mutant of Rab8a (Rab8TE) in complex with the minimal RBD of RILPL2  at 1.7-Å resolution. These two structures are analyzed in comparison to the previously published structure of pRab8a in complex with the minimal RBD of RILPL2 . We find that the overall structures are identical in all three complexes with no significant differences in intermolecular contacts. However, there are dramatic conformational changes localized to two arginine residues  in the X-cap of RILPL2. The complex with the extended RBD (RL2117) has two well-ordered stacked arginines in which R130 appears to stabilize the conformation of R132, which directly forms a salt bridge to the pT72R8. The other two complexes display various levels of disorder, suggesting an influence of the upstream residues of RILPL2 on pRab8a recognition. Calorimetry reveals that RL2117 has a significantly higher affinity for pRab8a relative to RL2129. We conclude that a robust interaction between the RILP family of effectors with cognate phospho-Rabs is dependent on a dual arginine recognition motif. Mutation of distal R130 to lysine/glutamine is sufficient to abolish interactions with pRab8a in cells. The apparent sensitivity of the dual arginine motif to the upstream region of RILPL2 suggests a mechanism by which myosin:RILPL2 complex formation may regulate its recruitment to phospho-Rab8a membranes.Here, we have determined the structure of pRab8a in a complex with an extended RBD of RILPL2 , as previously described (NdeI/BamHI cloning sites and provides a hexahistidine tag that is cleavable by thrombin. The cDNA for human RILPL2117−165 was amplified by PCR from a human full-length RILPL2 construct in pET28a (129−165 was made using the following primers: 5′-C CAG GGA GCA GCC TCG AAC CGT CCG CGT TTC ACC C-3′ (forward); 5′-GC AAA GCA CCG GCC TCG TTA ACC GCT TTT GTA GCA TTG CAG-3′ (reverse). The resulting DNA was cloned using ligation-independent methods into the pLIC-MBP vector (E. coli BL21(DE3) for expression as His6-MBP-(TEV)-RILPL2117−165.The μg/mL kanamycin or 100 μg/mL ampicillin) at 37°C. Media, chemicals, and antibiotics were obtained from FORMEDIUM. At an OD600 of 0.6, the culture was cooled to 18°C for ∼1 h and induced with 0.5 mM IPTG, after which cells were grown overnight at 18°C. Expression of His6-MBP-RILPL2117-165 was carried out in LB (Miller) medium supplemented with 100 μg/mL ampicillin (FORMEDIUM) at 37°C. At an OD600 of 0.6–0.8, the culture was induced with 0.5 mM IPTG. Cells were grown for an additional 4 h at 37°C. After induction, cells were harvested by centrifugation, and the pellets were resuspended in His-tag extraction buffer ; +5 mM MgCl2 for Rab constructs). Cells were lysed by sonication, and the cell lysate was centrifuged at 26,000 × g for 45 min at 4°C to remove cellular debris. The supernatants were loaded onto an Ni2+-agarose resin (Qiagen). The resin was washed with a 10-fold excess of extraction buffer followed by washing with a fivefold excess using the same buffer supplemented with 40 mM imidazole before elution of the bound proteins using extraction buffer supplemented with 200 mM imidazole. The eluted proteins were dialyzed against 20 mM Tris-HCl, 100 mM NaCl, 5 mM MgCl2, and 1 mM DTT (pH 7.5). Overnight incubation at 4°C with thrombin  or TEV protease was used to remove the N-terminal hexahistidine or hexahistidine-MBP tags from each protein. After cleavage, the proteins were run through a second Ni2+-agarose column. The flow-through fractions were collected, while the uncut proteins remained bound to the resin. The peptide corresponding to residues 129–165 of RILPL2 was synthesized with an N-terminal hexahistidine tag (GenScript). The peptide was solubilized in aqueous buffer ) before crystallization trials.Expression of Rab8a was carried out in LB (Miller) broth supplemented with antibiotic ). The protein was then loaded onto a MonoS cation exchange column . The column was subjected to a gradient from low-salt buffer into a high-salt buffer ) over a 30-mL volume. The collected fractions were analyzed by SDS-PAGE to ensure purity of RL2117.The second nickel procedure was repeated to minimize the amount of uncleaved His2, 1 mM DTT (pH 7.5)). The peak fraction containing pure protein was collected and concentrated before crystallization and biophysical experiments. The phosphorylation of Rab8a by MST3 kinase and its specificity to T72 have been described in detail . Phosphorylation took place at room temperature overnight. The phosphorylation mixture was dialyzed against low-salt buffer and loaded to a MonoS  column. Phosphorylated Rab8a was separated from unphosphorylated Rab8a by ion exchange chromatography by a 50% gradient from a 10 mM to 1 M salt buffer ). The phosphorylation of Rab8a was confirmed by PhosTag gel electrophoresis before subsequent experiments.After removal of the hexahistidine tag, Rab8a was further purified by running the protein through a Superdex 75 16/60) gel filtration column  equilibrated in column buffer , 10% PEG 4000, and 10% 2-propanol. Plate-like crystals were harvested in precipitant supplemented with 25% glycerol and stored frozen in liquid nitrogen. X-ray data were collected under a cryogenic nitrogen stream at 100 K . Crystals of pRab8(Q67L):RL2117 complex were obtained in a 1:1 molar ratio of protein/peptide at a total concentration of 5 mg/mL. Crystals were grown in 150 mM DL-Malic acid supplemented with 20% PEG3350. Rod-like crystals were harvested in precipitant supplemented with 25% glycerol and stored frozen in liquid nitrogen. X-ray data were collected under a cryogenic nitrogen stream at 100 K .Crystals of Rab84lhw) (6rir) as search models. Because the Rab8TE:RL2129 crystals are isomorphous to the previously determined pRab8a:RL2129 structure  bound to GTP and two molecules of the RILPL2 . The structure of pRab8a:RILPL2117−165 was solved and refined in a similar manner. However, the space group is different, and the asymmetric unit consists of one molecule of pRab8 and one molecule of RL2117. Details of data collection, structure refinement, and deposited PDB files are shown in α atoms of (p)Rab8a in the various structures typically aligned with a root mean-square displacement (RMSD) of 0.4–0.6 Å.Native diffraction data were reduced using XDS and Aimless, followed by structure determination using the Phaser software in the PHENIX package ,13. Stru4lhw)  and RILP4lhw) rir as setructure . The asyμM each) were mixed together in 1.5-mL centrifuge tubes with 25 μL Ni2+- agarose resin in a final volume of 1 mL of binding buffer (20 mM Tris (pH 8.0), 300 mM NaCl, 20 mM imidazole, 5 mM MgCl2, 10 mM β-mercaptoethanol). The reaction mixture was mildly shaken for 15 min followed by gentle centrifugation (1000 rpm). The resin was washed three times with 1 mL of the binding buffer. After release of proteins from resin with 50 μL elution buffer (20 mM Tris-Cl (pH 8.0), 300 mM NaCl, 200 mM imidazole), samples were subjected to SDS-PAGE and visualization with 0.5% Coomassie Brilliant Blue.For in vitro pulldowns, hexahistidine-tagged full-length RILPL2 (1–211) or hexahistidine-MBP-tagged truncated RILPL2 constructs were used. Rabs and RILPL2 constructs (10 6-RILPL2129−165), or recombinantly expressed RILPL2117−165, was dialyzed together with pRab8a/Rab8TE ). Protein concentrations were calculated based on their A280 using an ND-1000 NanoDrop spectrophotometer (Thermo Scientific). Samples were centrifuged at 13,200 rpm for 10 min before the determination of protein concentration and ITC analyses. Injection of Rab8TE/pRab8a into RILPL2 was performed, rather than the other way around. RILPL2 injections into buffer revealed a large heat of dilution, which complicated estimates of binding enthalpy and Kd. The concentrations of proteins for injections were between 400 and 600 μM Rab8a and 40–60 μM RILPL2. Data were processed using Origin 7.0 with the ITC plug-in.Calorimetry experiments were performed in triplicate. The N-terminal polyhistidine-tagged RILPL2 peptide (Hishttps://mrcppureagents.dundee.ac.uk/reagents-proteins/overview): GFP-empty pcDNA5 FRT/TO (DU13156); Flag-LRRK2 Y1699C pCMV (DU13165); HA-Rab8a WT pCMV (DU35414); RILPL2-GFP WT pcDNA5D FRT/TO (DU27481); RILPL2-GFP P128A pcDNA5D FRT/TO (DU68411); RILPL2-GFP D127A pcDNA5D FRT/TO (DU68427); RILPL2-GFP D127N pcDNA5D FRT/TO (DU68428); RILPL2-GFP T126A pcDNA5D FRT/TO (DU68412); RILPL2-GFP D124N pcDNA5D FRT/TO (DU68429); RILPL2-GFP D124A pcDNA5D FRT/TO (DU68413).The plasmids used for coimmunoprecipitation experiments were acquired from MRC PPU Reagents and Services (μg/mL), anti-LRRK2 C-terminal , and anti-α-tubulin . Secondary antibodies used were LI-COR IRDye for 800CW goat anti-rabbit (925–32211), goat anti-mouse (926–32210), and 680LT goat anti-rat (925–68029) and goat anti-mouse (926–68020), all used at 1:10,000 dilution in TBS-T.Antibodies used in this study were diluted in 5% w/v bovine serum albumin in TBS supplemented with 0.1% Tween-20 (TBS-T) and 0.03% w/v sodium azide. The rabbit monoclonal antibody for total LRRK2 (N-terminus) was purified at the University of Dundee . Anti-GFμg/mL streptomycin. Transient transfections were performed 40–48 h before cell lysis using polyethylenimine PEI (Polysciences) at around 60–70% confluence. Transfections for coimmunoprecipitation experiments were done in 10-cm round cell culture dishes using 3 μg of Flag-LRRK2 Y1699C, 1 μg of HA-Rab8a, and 1 μg of GFP control or RILPL2-GFP cDNA construct per dish diluted in 1 mL of OPTIMEM media and supplemented with 20 μg of PEI, incubated for 20 min before being added to the cell media. At 90 min before lysis, cells were treated with 500 nM of MLI-2 inhibitor or 0.1% DMSO control.HEK293 cells were cultured in Dulbecco’s modified Eagle medium  supplemented with 10% fetal bovine serum (Sigma), 100 U/mL penicillin, and 100 β-glycerophosphate, 50 mM sodium fluoride, 5 mM sodium pyrophosphate × 10H2O, 0.27 M sucrose—and supplemented fresh before lysis with 1% v/v Triton-x100, one tablet of cOmplete Mini (EDTA-free) protease inhibitor  per 10 mL of buffer, 0.1 μg/mL of microcystin, and 1 μM of sodium orthovanadate. Lysates were clarified by centrifugation at 17,000 × g for 10 min. For GFP immunoprecipitation, lysates were incubated with nanobody α-GFP binder Sepharose from MRC PPU Reagents and Services for 1 h at 4°C (15 μL of packed resin/0.5 mg of lysate). Bound complexes were recovered by washing the beads three times with wash buffer (50 mM Tris-HCl (pH 7.5), 150 mM NaCl) before eluting with 2× SDS-PAGE sample buffer supplemented with 1% v/v 2-mercaptoethanol. The samples were denatured at 70°C for 10 min, and the resin was separated from the sample by centrifugation through a 0.22-μm Spinex column . Samples were subjected to immunoblotting.Cells were washed with phosphate-buffered saline and lysed in lysis buffer—50 mM Tris-HCl (pH 7.5), 1 mM EGTA, 10 mM sodium μm nitrocellulose; GE Healthcare) at 90 V for 90 min on ice in transfer buffer . Transferred membranes were blocked with 5% w/v nonfat dry milk dissolved in TBS-T (20 mM Tris/HCl (pH 7.5), 150 mM NaCl, and 0.1% v/v Tween 20) at room temperature for 1 h. Membranes were then incubated with primary antibodies overnight at 4°C. After washing membranes in TBS-T 3 × 15 min, membranes were incubated with secondary antibodies at room temperature for 1 h. After washing membranes in TBS-T 3 × 15 min, membranes were scanned using LI-COR Odyssey CLx. Protein band intensities were analyzed using LI-COR Image Studio Lite software.Samples were run on gels consisting of a 4% w/v acrylamide stacking gel , 0.2% v/v tetramethylethylenediamine, and 0.08% w/v ammonium persulfate (APS)) and 10% w/v acrylamide separating gel , 1% v/v tetramethylethylenediamine, and 0.05% w/v APS) in MOPS buffer  at 90–120 V. Proteins were electrophoretically transferred onto nitrocellulose membranes , in which the asymmetric unit was the biologically relevant heterotetramer . This stable arrangement of the dual arginines may enable formation of a symmetric twofold crystallographic axis. In the ensuing discussions, stacking will refer to planar π-π interactions of the guanidino side chains of arginines. Crystallographic details are shown in The structure of GTP-bound pRab8Q67L) in complex with RL2h RL2129 B, in wh7L in com R132RL2 C. This 129 complex). There are no additional interactions with pRab8a, and upon superposition, equivalent backbone residues have a root mean-square deviation of 1.44 Å. The only significant difference between the complexes is the conformation of R130RL2, which is discussed in more detail below. As a further comparison, the structure of a phosphomimetic mutant Rab8a-T72E (Rab8TE) in complex with RILP129 has also been determined. The complex Rab8TE:RILP129 crystallizes in the same space group as pRab8a:RILP129  of the previous structure . In contrast, the key pT72R8:R132RL2 interaction in the pRab8a:RILP129 complex is well ordered, suggesting a stronger salt bridge with phosphothreonine (B). There is also partial stacking of the R132/R130 side chains in the two complexes with the minimal (RILP129) RBD. R130RL2 in the complex pRab8:RILP129 reveals better stacking relative to Rab8TE, but there remains considerable flexibility as evidenced by electron density maps (B).The three complexes afford an excellent opportunity to investigate the structural and thermodynamic details of phospho-Rab binding by effectors. Electron density maps reveal that one side of the E72geometry A. In cohreonine B. Thereity maps B.Figure117 reveals an ordered R130 side chain that forms strong stacking interactions with the guanidino group of R130 (C). As a measure of the close contacts, the distance between R130(Nε) and R132(NH2) is 3.4 Å. In contrast to the other complexes, pRab8a:RILP117 forms a symmetric twofold axis down the length of the coiled coil. Therefore the two salt bridges between pT72 and R132 are identical by symmetry in the biological heterotetramer. Rab8TE:RL2129 and pRab8a:RL2129 assemble as heterotetramers in the lattice. As suggested previously, it is possible that conformational flexibility in R130/R132 may prevent a symmetric twofold axis from forming in the other two complexes. Previously, we reported that R130RL2 was essential for complex formation in cells. In the context of full-length RILPL2, even a mutation to lysine (R130K) significantly reduced the interaction with pRab8a  cannot substitute for this interaction, thus providing a rationale for the inability of a positive charge to maintain binding to pRab8a.Strikingly, the newly determined structure of pRab8a:RL2 of R130 C. As a h pRab8a . HoweverA). These experiments suggested that full-length RILPL2 (1–211) has less affinity to pRab8a relative to the truncated variant RL2117. Although the in vitro reduction in affinity appears to be modest, the finding is surprising because effectors are modular and RBD interactions with Rabs are generally independent of other domains . As discussed previously, comparisons of the three complexes—Rab8TE:RL2129, pRab8:RL2129, and pRab8:RL2117—reveal no significant differences apart from the side chain of R130. Therefore, the increased enthalpic contribution to the binding affinity can be attributed to a stronger salt bridge between pT72R8 and R132RL2 (C). Although the distal R130RL2 is relatively far from the nearest phosphate oxygen (5.6 Å), it may also contribute to the enthalpy through long-range electrostatic interactions. The bar graph of free-energy signatures  also indicates that the entropic term contributes favorably toward formation of all of the complexes. However, the enthalpic gains are countered by a reduced entropic contribution, which can partially be attributed to ordered guanidino side chains. The complexes Rab8TE:RL2129 and pRab8:RL2129 have a similar Kd, but favorable enthalpy for the pT72:R132 interaction is offset by a reduction in the entropic gains . The side-chain carboxylate of D127RL2 is within 3 Å of the backbone NH groups of N129RL2 and R130RL2. Although the geometry is not ideal, these interactions may nevertheless contribute to stabilization of the backbone conformation. It would also explain why D127NRL2 does not have the same defect as D127ARL2 (A). The effects of mutations are mapped as hotspots on a sequence alignment of the predicted X-cap of effector proteins (B and C). There are modest sequence similarities in the upstream region preceding R130 within the RILP family. Red circles denote hotspots in which any mutation abolishes binding in cells , whereaα-helix of Rab GTPases is a hotspot for phosphorylation, and its modification is likely to influence subsequent interactions with GAPs, GEFs, and effector proteins. Here, we show that the conformation of arginine residues from RILPL2 involved in pRab8a recognition is sensitive to the region preceding the RBD. In particular, distal R130RL2 is flexible, without a stabilizing influence from upstream residues. However, the full-length effector comprising both RH1/RH2 domains dramatically reduces the enthalpy of complex formation, suggesting that the distal R130 is disordered. The structural and thermodynamic studies also suggest that the enthalpic gain from the salt bridge between R132/pT72 is offset by a reduction in favorable entropy arising from the ordering of the guanidino side chains of R130/R132. A video is shown with the three complexes as a trajectory (Rab8TE:RILP129→ Rab8TE:RILP129→pRab8:RILP117) using the Morph application in Chimera . In contrast, the RILPL2 interaction with the phosphorylated switch 2 helix is more peripheral (C). This more subtle interaction motif in the pRab8a:RILPL2 complex may be influenced by the upstream hinge region that connects the RH1 domain to the RH2 domain. We have shown previously that myosin Va binding to the RH1 domain enhances the affinity of the RH2 domain to pRab8a (The structures of numerous signaling complexes involving phosphorylated peptides have been determined. In general, the peptides adopt an extended conformation, and the phosphorylated side chains are deeply embedded, with interactions on both sides of the phosphate moiety , A and Bripheral C. This o pRab8a . An attrD.W. and E.P. analyzed data, performed the research, and wrote the article. A.R.K. designed the project, analyzed data, performed research, and wrote the article.


Bombyx mori. SPINK7 contains three Kazal domains and has 6 conserved cysteine residues in each domain. Quantitative real-time PCR analyses revealed that SPINK7 was exclusively expressed in hemocytes and was upregulated after infection with two fungi, Saccharomyces cerevisiae and Candida albicans. Enzyme activity inhibition test showed that SPINK7 significantly inhibited the activity of proteinase K from C. albicans. Additionally, SPINK7 inhibited the growth of three fungal spores, including S. cerevisiae, C. albicans, and Beauveria bassiana. The pathogen-associated molecular patterns (PAMP) binding assays suggested that SPINK7 could bind to β-D-glucan and agglutinate B. bassiana and C. albicans. In vitro assays were performed using SPINK7-coated agarose beads, and indicated that SPINK7 promoted encapsulation and melanization of agarose beads by B. mori hemocytes. Furthermore, co-localization studies using immunofluorescence revealed that SPINK7 induced hemocytes to aggregate and entrap the fungi spores of B. bassiana and C. albicans. Our study revealed that SPINK7 could recognize fungal PAMP and induce the aggregation, melanization, and encapsulation of hemocytes, and provided valuable clues for understanding the innate immunity and cellular immunity in insects.Serine protease inhibitors of Kazal-type (SPINKs) were widely identified in vertebrates and invertebrates, and played regulatory roles in digestion, coagulation, and fibrinolysis. In this study, we reported the important role of SPINK7 in regulating immune defense of silkworm,  Activation of innate immunity by microorganisms is initiated by pathogen-associated molecular patterns (PAMPs), including peptidoglycan (PGN), lipopolysaccharide (LPS), and β-1,3-glucan, which are bacterial and fungal cell wall and membrane components. In the silkworm Bombyx mori, PAMPs are recognized by pattern-recognition receptors (PRRs) (The insect innate immune system mainly functions s (PRRs) . For celEscherichia coli, Bacillus bombysepticus, Beauveria bassiana, and Bombyx mori nuclear polyhedrosis virus, respectively  —were cultured on potato dextrose agar medium for 15 days at 30°C. Fungal conidia were washed in distilled water containing 0.05% Tween-80 (v v-1), filtered with sterile absorbent cotton, washed three times with Milli Q water, and diluted to 1 × 105 conidia mL-1 with 20 mM phosphate-buffered saline (PBS) buffer (pH 7.5).g at 4°C to separate hemocytes from the serum. Seven larval tissues were collected on day 7 of the fifth instar, including head, integument, fat body, hemocyte, gonad, midgut, and silk gland. All samples were stored at -80°C until use.Fungal spore liquid (10 µL) was injected into each larva on day 3 of the fifth instar. Larvae injected with sterile PBS were used as the control group. The hemolymph of 10 silkworms was collected at 6, 12, 18, and 24 h after injection. Then, the hemolymph was centrifuged for 10 min at 1,000http://web.expasy.org/protparam/) and ProtScale . Prediction of signal peptide was carried out using SignalP 4.1 Server . Multiple alignments of the Kazal domains were conducted using the program CLUSTAL-W. The active site of individual domain was determined through the alignments of the homolog sequence.The SPINK7 sequence was downloaded from the NCBI database. Forecast analysis of protein amino acid composition, molecular weight and isoelectric point information were using online software Protparam  was PCR-amplified using a sense primer (5′- CGC GGA TCC TAT CCA CCA AGC TGT GCG TG -3′) and an antisense primer (5′- ATT TGC GGC CGC CAG GGG TCG TAT TCC GTG GT -3′). The purified PCR product was inserted into the pET28a vector and transformed into E. coli strain BL21 . The E. coli cells were induced with 0.1 mM IPTG and then lysed in binding buffer  by sonication. After centrifugation at 12,000g for 25 min, the supernatants were loaded onto a nickel affinity chromatography column, which was equilibrated with binding buffer. The bound protein was eluted with gradient elution buffer (0-500 mM imidazole in binding buffer) and then loaded onto a HiLoad 16/60 Superdex 200 column  pre-equilibrated with 20 mM PBS (pH 7.5). The purified SPINK7 protein was separated by 12% SDS-PAGE and the protein sample was stored at -80°C.The 2, pH 7.5) for 30 min at 37°C. Then, 100 μL FITC-casein substrate buffer  was added and incubated at 37°C in the dark for 1 h. The fluorescence was measured at 485/535 nm (excitation/emission). Protease inhibition by SPINK7 was assessed using the following formula: % residual activity =  × 100. Three independent replicates were performed for each experiment. Student’s t test was used to evaluate statistical significance. These data were analyzed by GraphPad Prism 5 software.Assay for inhibitory activity against protease was performed as previously described with modification –27. TrypSPINK7 and sw22934  (SPINK7 expression (Cr) was then calculated using the following formula: Cr=2-△(Ct−Csw). These data were analyzed by GraphPad Prism 5 software.The qPCR was performed with qTOWER 2.2 PCR Thermal Cycler  using SYBR Premix Ex Taq II  and SPINK7-specific primers . The qPCR program was performed as follows: initial denaturation at 95°C for 30s, followed by 40 cycles of denaturation at 95°C for 20 s, annealing at 60°C for 60 s, and extension at 72°C for 35 s. Relative expression was calculated from Serum samples were separated using 12% SDS-PAGE followed by electrotransfer to PVDF membrane . The membrane was blocked with 5% BSA in TBST buffer  for 2 h, and then incubated with rabbit anti-SPINK7 or anti-storage protein 2 (SP2) for 1.5 h at 25°C. After washing three times with TBST, the membranes were incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG  at 25°C for 1h and detected using ECL reagent .5 conidia mL-1. The culture medium contained chloramphenicol (25 μg mL-1) to prevent bacterial infection. The protein concentrations of SPINK7 used in this section were 0.1 mg mL-1 and 0.5 mg mL-1, respectively. PBS and BSA were used as negative controls, while EDTA was used as a positive control. Microplates were incubated at 30°C with shaking at 45 rpm. Fungal growth was observed by monitoring the absorbance at 595 nm after culturing for 0, 12, 24, 36, and 48 h. Student’s t test was used to evaluate statistical significance. Three independent replicates were performed for each experiment. These data were analyzed by GraphPad Prism 5 software.Growth inhibition assays were carried out in 96-well plates . Each well was added with 200 μL potato liquid medium containing fungal conidia at a final concentration of 1 × 10-1) was preincubated with 450 μL B. bassiana or C. albicans spore solution in a final concentration of 1 × 105 conidia mL-1 for 2 h at room temperature. Then, 12.5 μL amphotericin B (100 μg μL-1) was added to 487.5 μL spore solution in a final concentration of 1 × 105 conidia mL-1 and incubated for 2 h at room temperature. 50 μL PBS was used instead of SPINK7 solution as the control group. Then, fungal cells were stained with 1:1000 PI fluorescence (1 mg/mL) in the dark for 10 min. Finally, 10 μL solution was mounted on a microscope slide and coverslipped. The slides were observed under a confocal microscope . Three independent replicates were performed for each experiment.Amphotericin B can bind to sterols in the fungal cell membrane and rupture the cell membrane. Further, propidium iodide (PI) can bind to the DNA of dead cells and emit red fluorescence, but cannot pass through healthy cell membranes. Thus, PI fluorescence can indicate whether SPINK7 causes fungal death. 50 μL SPINK7 solution  and incubated with blocking buffer  for 30 min at 37°C. After washing the 96-well plate with PBS, 0 mg mL-1, 0.1 mg mL-1, 0.3 mg mL-1, and 0.5 mg mL-1 SPINK7 in PBS were added and incubated at 25°C for 3 h. After washing the 96-well plate 3 times (10 min each) with PBST (PBS containing 0.1% Tween), 1:1000 rabbit anti-SPINK7  in PBS containing 1% BSA was added and incubated at 37°C for 1 h. The wells were washed three times for 10 min each with PBST. Next, 1:1000 anti-rabbit IgG (H+L)  was added and incubated at 37°C for 1 h. Then, 100 µL TMB  was added to quench the reaction. Absorbance was measured at OD450 using a microplate reader. Non-immune rabbit serum was used as a negative control, and empty wells were used as blanks. Each experiment was repeated in triplicate. Samples with (Psample - Bblank)/(Nnegative - Bblank) > 2.1 were considered positive  and mannan  were dissolved in coating solution , added to a 96-well microtiter plate, and incubated at 4°C overnight. The uncoated PAMPs in the 96-well plate was washed three times with PBS (10 mM Napositive . Student-1) was preincubated with 200 μL β-D-glucan solution (80 μg mL-1 in PBS) or mannan solution (80 μg mL-1 in PBS) for 1 h at room temperature. Then, each sample was incubated with 200 μL C. albicans spore solution in a final concentration of 1 × 105 conidia mL-1 for 1 h at room temperature. 20 μL PBS was used instead of SPINK7 solution as the control group. The spores were incubated with blocking buffer (1% BSA in PBS) at 25°C for 1 h, and then incubated with 1:1000 rabbit anti-SPINK7  in the blocking buffer for 1.5 h at 25°C. Then, samples were washed three times in PBS, and incubated with 1:1000 Cy3-labeled goat anti-rabbit IgG  in blocking buffer. Finally, after washing twice with PBS, the spores were observed with a fluorescence microscope . Three independent replicates were performed for each experiment.20 μL SPINK7 solution (5 μg μLIn vitro encapsulation and melanization assays were performed as previously described (2. Recombinant SPINK7 or tubulin (as a control protein) was incubated overnight with agarose beads in a 1.5 mL tube at 4°C with shaking. Additional protein was added until agarose bead binding was saturated (excess proteins were detected in the remaining supernatant). Generally, 200 μL agarose beads can bind up to 1 mg recombinant SPINK7. Protein-coated beads were washed four times for 5 min with PBS and suspended in PBS at 80-100 beads per microliter (about 1 μg protein per microliter beads).escribed , 30, wit-1) FBS . The diluted hemolymph was added to each well of the agarose-coated plate. Hemocytes were allowed to settle for at least 10 min. Then, 1 μL (80-100 beads) protein-coated agarose beads was added to each well, and the plate was incubated at 25°C. Encapsulation and melanization of the agarose beads were observed after 6 and 24 h incubation, respectively. For the recombinant protein, assay was performed in three different wells.A 48-well cell culture plate  was treated with 1% agarose (ISC Bioexpress). Hemolymph collected from 10 silkworm larvae was combined with 200 μL Grace’s Insect Medium  supplemented with 10%  were placed in a microcentrifuge tube containing 50 μL 1:500 rabbit anti-SPINK7. Then, the samples were incubated overnight at 4°C with shaking. The beads were washed with PBS 3 times and resuspended in 5 μL PBS. The plate was observed with a fluorescence microscope . Three independent replicates were performed for each experiment.To test whether 3 hemocytes collected in phenylthiourea onto poly-L-lysine-treated glass slides. About 10 μL protein sample (5 mg mL-1) and 10 μL heat-killed spores (1×105 conidia mL-1) labeled with Calcofluor White M2R  were added to the hemocyte monolayer and incubated at 25°C for 2 h (-1 BSA and 5 μg μL-1 hdSPINK7 (heat denatured SPINK7) to replace SPINK7 as a control experiment. Then, the hemocytes were washed once with PBS and fixed with 4% paraformaldehyde at 25°C for 5 min. After washing twice with PBS, cells were permeabilized with 0.1% Triton X-100 in PBS. After blocking with 1% BSA and 10% normal goat serum in PBS at 25°C for 1 h, 1:500 mouse anti-tubulin antibody  and 1:500 anti-SPINK7 rabbit antibody  in PBS containing 1% BSA were incubated with the cells for 1.5 h at 25°C. The cells were washed three times in PBS and incubated with 1:500 Cy3-Labeled goat anti-rabbit IgG and 1:500 Alexa Fluor 488-labeled goat anti-Mouse IgG (H+L)  in PBS containing 1% BSA for 1 h at 25°C. Finally, after washing twice with PBS, the slides were observed with a fluorescence microscope . Three independent replicates were performed for each experiment.Overlay experiments were performed on hemocyte monolayers prepared by seeding 5×10 for 2 h . We usedSPINK7 gene encodes a 194-amino acid protein. The predicted signal peptide of SPINK7 is the 19 amino acid N-terminal sequence, indicating that SPINK7 might be extracellularly secreted. The mature protein has a theoretical molecular weight (MW) of 18.8 kDa and a isoelectric point (pI) of 4.7. SPINK7 was predicted to have three Kazal domains, each of which contains 6 conserved cysteine residues, which are responsible for intramolecular disulfide bridge formation  after treatment by amphotericin B  as the control. The results showed that BSA and hdSPINK7 did not bind to hemocytes and could not promote hemocyte aggregation  can be found in the article/ZD, QX, and PZ contributed to conception and design of the study. ZD and LA wrote the manuscript. ZD, LA, ML, MT, HC, XH, YH, GS, XZ, and YZ performed the research and analyzed the data. All authors contributed to the article and approved the submitted version.This work was supported by the Natural Science Foundation of Chongqing (cstc2020jcyj-cxttX0001), and Chongqing Research Program of Basic Research and Frontier Technology (cstc2019jcyj-msxmX0272).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. 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Top7sm2-I68R can be a useful model protein due to its convenient structure determination.Top7 is a de novo designed protein whose amino acid sequence has no evolutional trace. Such a property makes Top7 a suitable scaffold for studying the pure nature of protein and protein engineering applications. To use Top7 as an engineering scaffold, we initially attempted structure determination and found that crystals of our construct, which lacked the terminal hexahistidine tag, showed weak diffraction in X-ray structure determination. Thus, we decided to introduce surface residue mutations to facilitate crystal structure determination. The resulting surface mutants, Top7sm1 and Top7sm2, crystallized easily and diffracted to the resolution around 1.7 Å. Despite the improved data, we could not finalize the structures due to high R values. Although we could not identify the origin of the high R values of the surface mutants, we found that all the structures shared common packing architecture with consecutive intermolecular β-sheet formation aligned in one direction. Thus, we mutated the intermolecular interface to disrupt the intermolecular β-sheet formation, expecting to form a new crystal packing. The resulting mutant, Top7sm2-I68R, formed new crystal packing interactions as intended and diffracted to the resolution of 1.4 Å. The surface mutations contributed to crystal packing and high resolution. We finalized the structure model with the R/R Top7 is a de novo designed protein consisting of two α-helices and five β-strands . BecauseWe had started a protein engineering project using Top7, and we tested the structure determination process of Top7 by X-ray crystallography, which reported previously . UnfortuTo determine the molecular structure of Top7, we chose the high-entropy surface residues Lys42, Gln43, and Lys46 to apply the surface entropy reduction method. We introduced K42A, Q43A, and K46A mutations and the resulting mutant was named Top7sm1  was performed to measure the size of the protein samples c. All su221 space group, Top7sm2 with the P3221 space group, and Top7sm2 with the space group P21, respectively. Two P3221 data points were isomorphous with that of the original Top7 structure (PDBID 1QYS), and one P21 datum of Top7sm2 contained six molecules in an asymmetric unit . Considering their resolution, these values were beyond the acceptable range of structure refinement. Although we have tested several refinement strategies, we could not improve the R values. The R values for the Top7sm2 P21 mutant were also a bit high, even considering its resolution . Due to the high R values, we could not finalize the structure models to fulfill the deposit criteria for the Protein Data Bank (PDB). We analyzed these incomplete models to evaluate the reason for the high R values. PDB files of these mutants (Top7sm1 and Top7sm2) can be found in the We successfully crystallized Top7sm1 and Top7sm2 and obtained diffraction data at a resolution of 1.7 Å, 1.65 Å, and 2.5 Å for TOP7sm1 with the P3ric unit a. Since  mutants a. Howeve221 space group showed isomorphous crystals with the original Top7 structure. It appears that the surface mutations did not alter the crystal packing. It is possible that removal of positive charges of lysine residues may contribute to the improved resolutions, electrostatically. Top7sm2 structure with the P21 space group contained six molecules within the asymmetric unit. Interestingly, these six molecules formed a consecutive string via an intermolecular β-sheet  values of Cα atoms below 0.8 Å. The Top7sm1 and Top7sm2 structures with the P3 β-sheet a. Such cerations b. Thus, To obtain the different crystal packing of Top7 variants, we mutated the intermolecular residue to disrupt the consecutive β-sheet observed in all Top7 structures. We scrutinized the crystal packing of Top7sm2 and found Ile68 at β-strand-4 to be a promising site for the mutation. Ile68 is located where an intermolecular β-strand and a symmetry-related molecule interact a. We mut1 space group, containing two molecules in the asymmetric unit (mol-A and mol-B). The two molecules had very similar structure with an RMSD value of Cα atom as 0.36 Å. As intended, the mutant formed new crystal packing without forming the continuous β-sheet string that was observed in the other Top7 structures (free: 0.20/0.24) were dropped to a relatively decent range for the resolution. The structure was deposited to PDB with a PDBID of 7FAO.We successfully determined the crystal structure of Top7sm2-I68R with a resolution of 1.4 Å . The cryructures c, and thTwo molecules in the asymmetric unit were positioned in a side-by-side manner with a pseudo-twofold symmetry b. Arg68 It has been reported that the resolution improvement can be achieved by the surface engineering of protein. For example, RhoGDI and IGFRK-0P structures received improved resolutions from 2.0 Å to 1.2 Å and 2.7 Å to 1.5 Å, respectively, via surface mutations ,12. The We successfully constructed the Top7 surface mutant (Top7sm2-I68R) that easily forms a single crystal and has a different crystal packing structure from the original Top7. We could expand the resolution from 2.5 Å of the original Top7 to 1.4 Å by surface engineering. The original Top7 structure model (PDBID 1QYS) lacks several side chain atoms, while the Top7sm2-I68R structure contains all side chain atoms because of the high-resolution data. Easy structure determination and the resolution improvement are important for structural analysis investigating the molecular detail of further engineered Top7.We noticed that the molecular surface of Top7 has a relatively high number of charged bulky residues (Lys and Glu), possibly resulting from the de novo designing procedure. Thus, surface engineering for de novo designed proteins may be a feasible approach for general crystal structure determination.E. coli BL21(DE3) strain using Studier’s autoinduction medium [A synthetic gene of Top7 was purchased from Eurofins genomics  and subcloned into a pET28 vector. Expression vectors of the Top7 variants were constructed by a PCR-based mutagenesis using a PrimeSTAR DNA polymerase . All protein samples were expressed by n medium . After oAnalytical SEC was performed using a Superdex200 10/300 column  equilibrated with a 50 mM phosphate pH 7.0, 150 mM NaCl buffer by monitoring 280 nm absorbance.CD spectra were measured using a CD spectrometer J-820  with 10 μM protein samples in a 50 mM phosphate pH 7.0, 150 mM NaCl buffer, and 25 °C. A 1 mm quartz cell was used.www.pymol.org; accessed on 20 October 2021).Initial crystallization conditions were screened using Crystal screen 1/2 and JCSG+ . Hanging drop vapor diffusion was used for crystallization. Protein crystals were formed in conditions of 20% PEG3350, 0.25 M ammonium formate (pH 6.6) for Top7sm1 (26 mg/mL protein concentration), 12% PEG8000, 12% glycerol, 0.16 M calcium acetate, 0.1 M cacodylic acid (pH 6.5) for Top7sm2 (25 mg/mL protein concentration), and 15% PEG8000, 0.1 M magnesium acetate, and 0.1 M cacodylic acid (pH 6.5) for Top7sm2 I68R (25 mg/mL protein concentration). As a cryoprotectant, glycerol was added to the crystallization buffer at concentrations of 15~30%. We collected diffraction data at Photon Factory beamline BL-5A and processed with xds . For the


The purpose of this study was to investigate the effects of homo sapiens solute carrier family 4 member 4 (SLC4A4), which encodes the electrogenic NaThe expression levels of SLC4A4 in PCa and normal prostate tissues were evaluated by immunohistochemistry. The SLC4A4 knockdown cell model was structured by lentiviral infection, and the knockdown efficiency was validated by RT-qPCR and Western blotting. The effects of SLC4A4 knockdown on cell proliferation, apoptosis and cycle, migration, and invasion were detected by Celigo cell counting assay and CCK-8 assay, flow cytometry analysis, wound-healing, and Transwell assay, respectively. Tumor growth in nude mice was surveyed by in vivo imaging and Ki-67 staining. Furthermore, underlying mechanism of SLC4A4 silence induced inhibition of PCa progression was explored by human phospho-kinase array.Our results revealed that SLC4A4 expression was up-regulated in PCa tissues and human PCa cell lines. High expression of SLC4A4 in tumor specimens was significantly correlated with disease progression. SLC4A4 knockdown inhibited cell proliferation, migration and invasion, while facilitated apoptosis, which was also confirmed in vivo. Moreover, SLC4A4 promoted PCa progression through the AKT-mediated signalling pathway.The results of this study indicated that SLC4A4 overexpression was closely associated with the progression of PCa; SLC4A4 knockdown suppressed PCa development in vitro and in vivo. SLC4A4 acts as a tumor promotor in PCa by regulating key components of the AKT pathway and may therefore act as a potential therapeutic target for PCa treatment. Prostate cancer (PCa) is one of the most common cancers in male genitourinary system worldwide and still ranks the second leading cause of cancer-related deaths among men in Western countries . Based o+/HCO3− cotransporter, which is mainly involved in the secretion and absorption of sodium bicarbonate [Homo sapiens solute carrier family 4 member 4 (SLC4A4) is a family member of the solute carrier family and encodes an electrogenic Naarbonate . This prarbonate . For insarbonate . Accumularbonate , 12. SLCarbonate . Moreovearbonate , suggestarbonate , 16, itsTumour tissues and adjacent paired non-tumour tissues were gathered from patients who were diagnosed with PCa and underwent surgical excision in Renmin Hospital of Wuhan University  between June 2018 and January 2020. PCa and normal prostate tissues were gathered from 74 patients; the age range of the patients was between 26 and 87.Immunohistochemistry (IHC) was used to detect the expression of SLC4A4 in these tissues. Paraffin-embedded sections were dewaxed, the antigen was retrieved, and then the paraffin sections were incubated using primary antibody against SLC4A4  (1:200) and secondary antibody goat anti-rabbit  (1:400). After staining, ten fields (× 100 magnification) were chosen to be captured and analyzed with the optical microscope  for each section. The SLC4A4 staining intensity was scored on a range from 0 (negative), 1 (weak), 2 (positive + +) and 3 (positive +  + +). Median IHC score was used to distinguish the high/low expression of SLC4A4.2. The cell culture media were supplemented with 10% fetal bovine serum (FBS) and 1% sodium penicillin G/streptomycin sulfate (P/S). The normal prostate epithelial cell line RWPE-1 was purchased from the Cell Bank of Chinese Academy of Sciences and cultivated in keratinocyte serum-free medium (K-SFM) containing 0.05 mg/mL bovine pituitary extract (BPE), 5 ng/mL epidermal growth factor (EGF) and 1% P/S.Human PCa cell lines DU 145, LNCaP and PC-3 were purchased from the Cell Bank of Chinese Academy of Sciences and cultured in RPMI-1640 medium at 37 °C in a humidified incubator containing 5% COSLC4A4 gene was identified as 5'- TTATTCTTCAGCTGGTCCTTC-3'. Meanwhile, the target sequence of the negative control shRNA was identified as 5'-TTCTCCGAACGTGTCACGT-3'. Oligomers were annealed and ligated to BR-V121 lentiviral vector  through Age I/EcoR I restriction site to produce Lv-shSLC4A4 and Lv-shCtrl. Lastly, the sequencing was performed to validate the construct results.To knock down the expression of SLC4A4 in PCa cell lines, the short hairpin RNA (shRNA) sequence targeting the human The modified BR-shRNA plasmid and pMD2.G and pSPAX2 helper plasmids were transfected three times with Lipofectamine 2000 into HEK-293 T cells to obtain lentiviruses. Next, the lentiviral particles were gathered, filtered, and preserved. The knockdown efficiency of SLC4A4 was evaluated by RT-qPCR and Western blotting.7 TU/ml of the lentivirus containing shRNA interfering with SLC4A4 (shSLC4A4) or shRNA for negative control (shCtrl), and the cells were then incubated at 37 °C for three days. The expression of green fluorescent protein  was observed by fluorescent microscope (EMD Millipore), and the ratio of fluorescent cells to total cells (viewed in white light) was used to evaluate the transfection efficiency.PCa cells were respectively transfected with 1 × 10Premix Ex Taq™ II, Forward and Reverse primers (Sangon Biotech), reverse transcription products and RNase-free H2O. The thermal cycling conditions were: pre-denaturation at 95 °C for 30 s, denaturation at 95 °C for 15 s, annealing at 60 °C for 10 s for a total of 42 cycles, and 72 °C for 5 min (extension). GAPDH was used as an internal reference. The relative expression levels of genes were calculated as the method of the 2−ΔΔCt [PCa cells were gathered and centrifuged. Total RNA was extracted with the TRIzol reagent. Complementary DNAs (cDNAs) was synthesized with the PrimeScript™ RT reagent Kit (Takara). The qPCR reaction system was prepared using a real-time quantitative PCR instrument according to product specification. The reaction system was composed of the following reagents: TB Green® e 2−ΔΔCt . The seqPCa cells were washed twice with ice-cold PBS, lysed with cell lysis buffer (Beyotime) containing protease inhibitors, and incubated on ice for 15 min. The supernatant was harvested by centrifugation, and the protein content was measured with the BCA Protein Assay Kit . Equal amounts of proteins were separated and transferred onto polyvinylidene difluoride (PVDF) membranes (0.45 μM). Subsequently, the membranes were blocked with TBS + 0.1% Tween-20 (TBST) buffer containing 5% skim milk and incubated with various primary antibodies. Following being washed with TBST, the membranes were blotted with HRP-conjugated secondary antibodies. The target bands were visualized with immobilon Western Chemiluminescent HRP Substrote . GAPDH was used as an internal reference.The antibodies used in this experimental study were BAX , BCL-2 , CDK4 , FAS , AKT , p-AKT , GAPDH , horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG , goat anti-mouse , donkey anti-goat .Following infection with shRNA lentivirus, PCa cells were trypsinized, resuspended, counted and then inoculated in 96-well plates. Each group underwent a minimum of 3 duplicate wells. From the second day, the plates were tested by Celigo Imaging Cytometry System once a day for 5 consecutive days. The number of green fluorescent cells in each scan plate was precisely calculated by adjusting the input parameters of the analysis setup; the data were then plotted statistically, and the cell proliferation curve for 5 days was drawn.3 cells per well into a 96-well plate and pre-incubated for 24 h after transfection. Treated or un-treated cells were cultured as appropriate. 10 µl of CCK-8 solution was added to each well, and the cells were incubated for 2 h at 37 °C. The absorbance at 450 nm was recorded with a TECAN infinite M200 Multimode microplate reader . All detections were conducted in triplicate.CCK-8  assay was used to determine the number of living cells. The cell suspension was inoculated with 2 × 105–1 × 106 cells) were stained by 10 µl Annexin V-APC and protected from light for 15 min [Apoptosis was detected by the flow cytometry analysis. The infected PCa cells were cultured until the cell density reached 85%. The cells were trypsinized and resuspended, centrifuged for 5 min. The supernatant was discarded, and cell precipitates were washed with D‑Hank's solution (pH = 7.2–7.4) precooled at 4 °C. The cells were washed with 1 × binding buffer, centrifuged, and resuspended. The cell suspensions  were inoculated in the upper chambers by resuspension with basal serum-free medium, and the lower chambers was added with medium containing 20% FBS to attract cells to penetrate the membrane. Following incubation for 24 h, those cells on the outside surface of the chambers, having invaded through the membrane, were fixed using 4% paraformaldehyde (500 µl per chamber) for 20 min and stained using 0.1% crystal violet for 20 min. Cells on the upper surface and remaining Matrigel were wiped off by cotton swabs. At last, the numbers of stained cells in five different views per chamber were counted.Diluted Matrigel  was added to transwell upper chambers 12 h prior to the experiment and placed at 37 °C for solidification. The PCa cells . Those 20 mice were reared for 31 days, during which length and width of the tumors in mice were measured five times using the Vernier caliper. On day 31, the mice were injected intraperitoneally with D-Luciferin, anesthetized with an intraperitoneal injection of 0.7% pentobarbital sodium and placed under the animal multispectral living imaging system for imaging. Next, the mice were sacrificed with cervical dislocation, and tumors autopsied from the mice were weighed and photographed. Tumor volume in mm3 (V) was calculated based on the formula: V = π/6 × L × W × W, where L represents length and W represents width of the tumor.After a week of acclimatization in Animal Experiment Center of Renmin Hospital of Wuhan University, 20 mice were randomly divided into the control group (shCtrl) and the test group (shSLC4A4) (n = 10 mice/group). Since LNCaP was an androgen-dependent cell line, the tumorigenic rate of subcutaneous inoculation in nude mice alone was very poor . DU 145,Paraffin sections of tumor tissues taken from mice were dewaxed, rehydrated in a decreasing ethanol gradient, and incubated with the anti-Ki-67 antibody . After washing with PBS, the paraffin sections were incubated with secondary antibody goat anti-rabbit , counterstained with hematoxylin, and Ki‑67 expression was observed under an optical microscope. Ten fields of each section were captured for analysis, and this experiment was repeated three times.To explore the potential downstream signal pathways and functional targets of SLC4A4 in PCa, Human Phospho-Kinase Array Kit  was employed. PCa cells transfected with shCtrl or shSLC4A4 were lysed. Meanwhile, 8 nitrocellulose membranes  were closed with 2 ml of Array Buffer 1 (block buffer) for 1 h. Then, the samples were piped to wells and incubated overnight. After washing, 1 × biotinylated antibody cocktail was added to each well and incubated. Afterwards, diluted Streptavidin-HRP was pipetted into each well and incubated. Followed by the washing of membranes, any excess Wash Buffer was blotted off, and 500 µl of Chemi Reagent Mix  was added to each membrane. In the end, the signal density was measured with the chemiluminescence imaging system and analysed with the ImageJ. This experiment was performed in duplicate.SPSS 23.0 and Graphpad Prism 8 were used for data analysis. The quantitative data were presented as the mean ± standard deviation (SD). Chi-squared tests were performed to compare the differences in SLC4A4 expression among PCa patients. Spearman rank correlation analysis was used to analyse the correlation between SLC4A4 expression and clinicopathological features. The histograms of SLC4A4-related signal molecules in carcinoma cell were plotted by SignaLink 2.0 analysis. P-value < 0.05 was considered a statistically significant difference.For the purpose of determining the effect of SLC4A4 on the development of PCa, the expression of SLC4A4 in clinical PCa and normal prostate tissues was examined by IHC. As demonstrated in Fig. The relationship between the levels of SLC4A4 expression and the clinicopathological characteristics in PCa patients was assessed by statistical analysis. The high/low SLC4A4 groups were divided depending on median of IHC scores of all tissue specimens. The results demonstrated that the levels of SLC4A4 expression were remarkably different between patients at different clinical stages, T Infiltrate and lymphatic metastasis  was reduced by 16% (P < 0.01) in DU 145 cells and 68% (P < 0.001) in LNCaP cells  were calculated by in vivo imaging. In contrast to shCtrl group, the bioluminescence intensity of shSLC4A4 group was lowered by 93% , CREB (S133), Chk-2 T68), c-jun (S63), GSK-3α/β (S21/S9), GSK-3β (S9), p53 (S46), JNK1/2/3 , p38α (T180/Y182), PDGF Rβ (Y751), PLC-γ1 (Y783), Src (Y419), PYK2 (Y402), Yes (Y426), STAT1 (Y701) and STAT3 (S727) were assayed to be a significant down-regulation upon SLC4A4 knockdown compared with the shCtrl-infected cells  in the mouse gastrointestinal tract. Mouse ICCs was in charge of the production of slow electrical waves. Moreover, SLC4A4 transcripts expressed in human gastrointestinal smooth muscle cells and mouse ICCs are modifiable isomers. Scott K. Parks et al. [3− transports [The acidic and hypoxic tumor environment requires intracellular pH regulation to facilitate tumor development. S. J. Gibbons et al.  reporteds et al.  demonstransports  and tumoGao et al.  indicateAnalysis of clinicopathological data indicated that SLC4A4 was a free-standing prognostic factor of PCa that was meaningfully associated with T Infiltrate, lymphatic metastasis and clinical stage. In other words, high expression of SLC4A4 predicted high malignancy in PCa patients. These results confirmed that the growth, migration and invasion of PCa cells were inhibited in vitro and in vivo after knockdown of SLC4A4. Moreover, our study emphasized that SLC4A4 knockdown induced apoptosis in PCa cells and raised the BAX/BCL-2 ratio, suggesting that SLC4A4 may have an inhibitory effect on apoptosis. Furthermore, the regulatory functions of SLC4A4 in PCa cells were elucidated by a series of gain-of-function analyses.AKT is an effector molecule of phosphoinositide 3-kinase (PI3K) in the PI3K/AKT/mTOR signalling pathway . ElevateSome limitations of this study exist, such as the insufficient number of clinical specimens. Besides, prognostic implications of SLC4A4 in PCa and effects of SLC4A4 on different PCa cell lines need to be further investigated. The SLC4A4/NBCe1 has five multiple splice variants, in which expression of the B splice variant in mouse kidney cortical proximal tubule has been presented , 39. DesIn conclusion, the present study makes the first attempt to link SLC4A4 to human PCa progression. SLC4A4 could be an oncogene to predict tumour malignancy and survival in PCa patients. All these results of this study identified that SLC4A4 knockdown inhibited the occurrence and progression in PCa. SLC4A4 acts as a tumor promotor that accelerates tumor growth, inhibits apoptosis and arrests cell cycle progression among PCa by regulating key elements of the AKT pathway. Thus, SLC4A4 is a promising potential therapeutic target in the treatment of PCa.


This domain is crucial for the stabilisation of the tight quiescent form of STIM1 as well as for activating the ORAI calcium channel by direct contact, in the extended active form.The protein stromal interaction molecule 1 (STIM1) plays a pivotal role in mediating store-operated calcium entry (SOCE) into cells, which is essential for adaptive immunity. It acts as a calcium sensor in the endoplasmic reticulum (ER) and extends into the cytosol, where it changes from an inactive (tight) to an active (extended) oligomeric form upon calcium store depletion. NMR studies of this protein are challenging due to its membrane-spanning and aggregation properties. Therefore follow the divide-and-conquer approach, focusing on individual domains first is in order. The cytosolic part is predicted to have a large content of coiled-coil (CC) structure. We report the The online version contains supplementary material available at 10.1007/s12104-021-10042-7. Ca2+ controls a variety of cellular processes including gene expression, cell division, neuronal signaling, muscle contraction, fertilization and apoptosis. Besides voltage-dependent and ligand-activated calcium channels, store-operated calcium entry (SOCE) is of major importance for cellular calcium entry  domains denoted as CC1, CC2, CC3 with CC1 subdivided into three alpha helices   also known as CRAC activating domain (CAD) which enables STIM1 to couple to and activate Orai1  overnight at 37 °C. The bacterial stock was used to inoculate 250 mL of LB medium (1:100 dilution), the culture was further cultivated at 37 °C until optical density (OD at 580 nm) reached 0.8. The cells were gently centrifuged  and resuspended in M9 minimal medium with (15NH4)2SO4 (1.5 g/L) and 13C6-glucose (3 g/L) as sole nitrogen and carbon sources, respectively and enriched with BME vitamins (Sigma Aldrich). The culture was incubated at 37 °C for another 30 min and then isopropyl β-D-1-thiogalactopyranoside (IPTG)  was added and the temperature lowered to 28 °C before overnight cultivation. After centrifugation, the cell pellet was suspended in 20 mM Tris-HCl, 100 mM NaCl, 5 mM β-mercaptoethanol (βME), 10 % Glycerol, 0.1 % Triton X-100, pH 7.5 adding 4-(2-Aminoethyl)benzenesulfonyl fluoride hydrochloride (AEBSF)  as protease inhibitor. After sonication on ice (6 cycles of 1 min), an existing purification protocol  fragment (amino acids 388–430) was amplified by PCR and cloned into a pGEX 4T-1 vector using the Xho1 and BamH1 restriction sites. The pGEX 4T-1 vector includes a GST tag along with a thrombin cleavage site. The vector was transformed into competent BL21 2O, pH 6.0 and 550 µL (protein concentration 0.5 mM) transferred to 5 mm NMR tubes (Wilmad 535PPT). Following previous studies, 2,2,2-trifluoroethanol (TFE) buffer additive at various concentrations was tested to suppress aggregation and obtain sufficient resolution in 1H–15N HSQC spectra CO, HNCO, HNCA, H(CCO)NH and CC(CO)NH . Backbone dihedral angles and secondary structure propensities were predicted using TALOS-N  at 20 °C using a quartz cuvette with 0.1 cm path length at sample concentration of 50 µM and processed with Jasco spectra manager (v. 2.14). In order to evaluate the impact of TFE on the CC3 secondary structure, the spectra were measured in 20 mM Bis-Tris, pH 6.0 with and without addition of 17.5 % v/v TFE. The coiled-coil index was determined from the global and local minima at 208 nm and 220 nm. The ratio r = [CD]Aggregation properties of isolated STIM1 CC3 were probed by dynamic light scattering (DLS) using a Zetasizer Nano ZSP 2 . Measurements were carried out in 15 mm centre height UV-micro cuvettes and were performed by averaging 6 runs (with automatic optimized repetitions of 10 s measurements) for both CC3 in 20 mM Bis-Tris and CC3 in 20 mM Bis-Tris, 17.5 % v/v TFE at 20 °C. The analysis was performed using the Zetasizer Software version 8.01 .1H–15N Heteronuclear single quantum coherence spectroscopy (HSQC) and transverse relaxation-optimized spectroscopy (TROSY-HSQC) experiments were first run at 298 K in 20 mM Bis–Tris at pH 6.0. Extensive signal overlap presumably due to coiled-coil aggregation of the domain , and sample instability made it impossible to find a sufficient number of assignable resonances. In order to reduce peak overlap and increase resolution in the HSQC-type spectra, measurements at lower protein concentration as well as pH screening including various buffer components  were carried out. However, there was no significant improvement in the spectral resolution as documented in the Supplementary Data Fig. S2 a). Only with the increasing concentration of TFE the number of observable resonances increased . Finally, the aggregation tendency of the CC3 domain was overcome upon addition of 17.5 % v/v 2,2,2-TFE at 310 K. These conditions had successfully been used in a previous NMR study of STIM1 coiled coil fragments  with accession number 50683. Under the final conditions the 2D 13Cα, 13Cβ, 13CO, 1Hα, 1HN and 15N shifts the secondary structure propensities using TALOS-N web-based software were obtained . The population was classified as polydisperse with the major mass maximum around 443 kDa. The calculated radius was 7.84 nm at 20 °C for the aggregated sample in the absence of TFE. Repetition of the same experiment with addition of 17.5% v/v TFE resulted in much smoother correlation decay curve. However, the exact determination of the protein size and weight was hindered by the presence of the TFE co-solvent itself   interrupts the α-helix close to the N-terminus. These data will serve as a basis for further structural and in vivo functional studies of the STIM1 activation mechanism, for which coiled-coil interaction moieties in the CC3 domain are crucial Below is the link to the electronic supplementary material.