Our previous biochemical data showed that the αγ heterodimer and the holoenzyme can be activated by low concentrations of ATP but inhibited by high concentrations of ATP; however, the molecular mechanism was unknown. Here, we report the crystal structures of the αγ heterodimer with ATP binding only to the allosteric site (αMgγMg+CIT+ATP) and to both the allosteric site and the active site (αMg+ATPγMg+CIT+ATP). Structural data show that ATP at low concentrations can mimic ADP to bind to the allosteric site, which stabilizes CIT binding and leads the enzyme to adopt an active conformation, revealing why the enzyme can be activated by low concentrations of ATP. On the other hand, at high concentrations ATP is competitive with NAD for binding to the catalytic site. In addition, our biochemical data show that high concentrations of ATP promote the formation of metal ion-ATP chelates. This reduces the concentration of free metal ion available for the catalytic reaction, and thus further inhibits the enzymatic activity. The combination of these two effects accounts for the inhibition of the enzyme at high concentrations of ATP. Taken together, our structural and biochemical data reveal the molecular mechanism for the dual regulatory roles of ATP on the αγ heterodimer of human NAD-IDH.Human NAD-dependent isocitrate dehydrogenase (NAD-IDH) is responsible for the catalytic conversion of isocitrate into α-ketoglutarate in the Krebs cycle. This enzyme exists as the α The crystal structures of human cytosolic NADP-IDH  and porcine mitochondrial NADP-IDH  have been determined, and together with biochemical data, these enzymes have been shown to exist and function as homodimers in which both subunits are catalytic6. The activity of human IDH1 is regulated through conformational changes of the active site upon substrate binding, and the other mammalian NADP-IDHs appear to share a similar regulatory mechanism7.The Krebs cycle is a central part of the metabolic pathway to generate ATP through the oxidation of acetyl-CoA derived from the chemical breakdown of carbohydrates, fats and proteins. Separately, the Krebs cycle also provides metabolic intermediates for biosynthetic reactions leading to the de novo synthesis of proteins, lipids and nucleic acids. Isocitrate dehydrogenases (IDHs) are a family of enzymes responsible for the oxidative decarboxylation of isocitrate (ICT) into α-ketoglutarate (α-KG) using NAD or NADP as coenzyme with the concomitant production of NADH or NADPH. In mammals, NAD-dependent IDHs  are localized to the mitochondria and participate in the Krebs cycle. In addition, mammalian cells contain two NADP-dependent IDHs (NADP-IDHs) located to both the cytosol and the mitochondria, which have been demonstrated to play important roles in cellular defense against oxidative damage, detoxification of reactive oxygen species, and synthesis of fat and cholesterol9. The α and β subunits form a heterodimer (αβ) and the α and γ subunits form another heterodimer (αγ), which are assembled into a heterotetramer (α2βγ) and further into a heterooctamer 11. In addition, the activities of mammalian NAD-IDHs can be regulated by many metabolic intermediates and products of the Krebs cycle via feedback activation and inhibition mechanisms. Early biochemical studies showed that the α subunit is the catalytic subunit in the holoenzyme, whereas the β and γ subunits are the regulatory subunits, and the activity of the holoenzyme is positively regulated by citrate (CIT) and ADP and negatively regulated by ATP and NADH15. Recently, we carried out a comprehensive biochemical study on the enzymatic properties of the α2βγ holoenzyme and the αβ and αγ heterodimers of human NAD-IDH16. We found that the αγ heterodimer exhibits similar enzymatic properties as the α2βγ holoenzyme and can be allosterically activated by CIT and ADP, whereas the αβ heterodimer cannot; moreover, both heterodimers can be inhibited by NADH16. In addition, our biochemical data showed that in the holoenzyme, both α subunits have catalytic activity; in contrast, the γ subunit plays a regulatory role, whereas the β subunit plays a structural role to facilitate the assembly of the holoenzyme16. Our detailed structural and biochemical studies of the αγ and αβ heterodimers further revealed the molecular mechanisms by which the αγ heterodimer can be allosterically activated by CIT and ADP and inhibited by NADH, and the αβ heterodimer cannot be regulated by CIT and ADP but can be inhibited by NADH20. Intriguingly, our biochemical data also showed that the activities of the αγ heterodimer and the holoenzyme can be activated by low concentrations of ATP, but inhibited by high concentrations of ATP16. However, the molecular mechanism underlying the dual regulatory roles of ATP on the αγ heterodimer and the holoenzyme remained elusive.Compared to the NADP-IDHs, the composition and regulation of NAD-IDHs are much more complex. Mammalian NAD-IDHs are comprised of three types of subunits in the ratio of 2α:1β:1γ with similar molecular masses of 37 kDa, 39 kDa and 39 kDa, respectively2+ alone or Mg2+ and ATP, and the allosteric site bound with Mg2+, CIT and ATP (αMgγMg+CIT+ATP and αMg+ATPγMg+CIT+ATP). In the αMgγMg+CIT+ATP structure, there is ATP bound to the ADP binding site of the allosteric site, which stabilizes CIT binding through Mg2+-mediated interactions and leads the enzyme to adopt an active conformation. This provides the structural basis for why the enzyme can be activated by low concentrations of ATP. In the αMg+ATPγMg+CIT+ATP structure, there is ATP bound to the NAD binding site of the active site, which hinders NAD binding and thus inhibits the enzymatic activity. In addition, our biochemical data show that high concentrations of ATP facilitate the formation of metal ion-ATP chelates and decrease the concentration of free metal ion available for the catalytic reaction, thus inhibiting the enzymatic activity. The combination of these two effects explains why the enzyme can be inhibited by high concentrations of ATP. Our structural and biochemical data together reveal the molecular mechanism for the dual regulatory roles of ATP on the αγ heterodimer of human NAD-IDH.In this work, we determined two crystal structures of the αγ heterodimer of human NAD-IDH with the active site bound with either Mg2+ and the allosteric site bound with Mg2+, CIT and ATP from a crystallization solution containing CIT and a relatively low concentration of ATP (2 mM). This led to the determination of the structure of the αMgγMg+CIT+ATP heterodimer. Crystallization of the αγ heterodimer in solutions containing CIT and relatively high concentrations of ATP (10–20 mM) yielded crystals of the αγ heterodimer in which there is residual electron density at the NAD binding site of the active site, indicating that the NAD binding site is partially occupied by a ligand . Soaking the crystals of the αMgγMg+CIT heterodimer in a solution containing a very high concentration of ATP (200 mM) yielded crystals of the αγ heterodimer with the active site bound with Mg2+ and ATP and the allosteric site bound with Mg2+, CIT and ATP, which led to the determination of the structure of the αMg+ATPγMg+CIT+ATP heterodimer. Of note, the very high concentration of ATP (200 mM) in the soaking solution is unlikely to be physiologically relevant. Nevertheless, the structure of the αMg+ATPγMg+CIT+ATP heterodimer can provide structural insights into the mechanism by which high concentrations of ATP might compete with NAD to bind at the NAD binding site and thus inhibit the enzymatic activity of the αγ heterodimer. The αMgγMg+CIT+ATP and αMg+ATPγMg+CIT+ATP structures were determined at 2.30 Å and 2.26 Å resolution, respectively  than the inactive αMgγ structure . In particular, the key residues and structure elements at the allosteric site, the active site, and the heterodimer interface all exhibit very similar conformations to those seen in the αMgγMg+CIT+ADP structure, rather than to those seen in the αMgγ structure  at the allosteric site is oriented towards the CIT-binding subsite and forms a hydrogen bond with CIT, and the side chain of Tyr126A at the active site is oriented towards the ICT-binding subsite and in a proper position to interact with ICT. In both the α and γ subunits, the N-terminal region of the α7 helix at the heterodimer interface assumes an α-helical conformation to form a long α7 helix, and the β7 strand bends towards the β5-β6 loop and the α7 helix to form a network of hydrogen bonds. These results indicate that the two structures adopt an active conformation.Our previous structural and biochemical studies demonstrated that the binding of CIT at the allosteric site induces conformational changes at the allosteric site, which are then transmitted to the active site via the heterodimer interface. This leads the active site to assume an active conformation favorable for substrate (ICT) binding, and thus decreases ure Fig. . For exaMgγMg+CIT+ATP and αMg+ATPγMg+CIT+ATP structures, there are Mg2+, CIT and ATP bound at the allosteric site, and the residues involved in the binding of the metal ion and ligands assume almost identical conformations as those seen in the αMgγMg+CIT+ADP structure . Structural analysis shows that ATP occupies the ADP binding site, and particularly the adenine and α-phosphate of ATP make very similar interactions with the surrounding residues as those of ADP. In detail, the adenine of ATP forms two hydrogen bonds with the main-chain amine and carbonyl of Asn285G, and the α-phosphate forms two hydrogen bonds with the main-chain amines of Thr274G and Gly275G. However, unlike the β-phosphate of ADP, which is oriented towards the Mg2+-binding site and forms two hydrogen bonds with the side chains of Asn273G and Thr274G and a coordination bond with the Mg2+, the β- and γ-phosphates of ATP are oriented away from the Mg2+-binding site and make different interactions with the enzyme due to potential steric conflicts with the surrounding residues. Specifically, the β-phosphate of ATP forms two hydrogen bonds with the main-chain amines of Lys276G and Ser277G, and the γ-phosphate forms a hydrogen bond with the side chain of Ser277G. Meanwhile, a water molecule occupies the position of the β-phosphate of ADP and mediates the interaction between the Mg2+ and the α-phosphate of ATP. These results indicate that ATP can mimic ADP to bind to the allosteric site, and this ATP binding stabilizes the CIT and Mg2+ binding and thus the related conformational changes, leading the enzyme to adopt an active conformation. This provides the structural basis for why the αγ heterodimer can be activated by low concentrations of ATP. Since ATP has a larger volume than ADP, the ADP binding site cannot accommodate the three phosphates of ATP, and hence the β- and γ-phosphates of ATP are positioned away from the Mg2+-binding site. As a result, the interaction between ATP and Mg2+ is mediated via a water molecule instead of the direct interaction between ADP and Mg2+, suggesting that the stabilization of the CIT and Mg2+ binding by ATP is weaker than the stabilization by ADP. This explains why ATP has a weaker activatory effect than ADP17.In both the αure Fig. . Structu75GFig. . Howeverg2+Fig. , the β- 77GFig. . MeanwhiMgγMg+CIT+ATP structure, there is Mg2+ bound at the active site, and in the αMg+ATPγMg+CIT+ATP structure, there are both Mg2+ and ATP bound at the active site. Structural comparison shows that in both structures, the key residues at the active site assume very similar conformations, and the Mg2+ maintains identical interactions with the surrounding residues as those seen in the αMgγMg+CIT+ADP structure, rather than those seen in the αMgγ structure . As there is no structure of the αγ heterodimer bound with an NAD at the active site thus far, we compared the αMg+ATPγMg+CIT+ATP structure with the αMg+NADHγNADH structure to dissect the effects of the ATP binding at the active site. The structural comparison shows that ATP binds to the NAD binding site, and the adenine, ribose, and α-phosphate of ATP occupy the positions of the corresponding moieties of NADH in the αMg+NADHγNADH structure and maintain similar interactions with the surrounding residues19. In detail, the adenine of ATP forms two hydrogen bonds with the main-chain amine and carbonyl of Asn276A, the ribose 2′-OH forms a hydrogen bond with the side chain of Asp268A, and the α-phosphate forms two hydrogen bonds with the main-chain amines of Gly264A and Thr265A. On the other hand, the β- and γ-phosphates of ATP occupy a slightly different position from the nicotinamide moiety of NADH and make no interactions with any residues of the enzyme; however, there is a water molecule mediating the interaction between the α- and β-phosphates of ATP. The observation that ATP makes fewer interactions with the enzyme compared to NADH (and NAD) suggests that ATP has a weaker binding with the enzyme than NADH (and NAD). These results indicate that ATP at high concentrations can mimic NADH (and NAD) to bind to the NAD binding site and prevents NAD binding which accounts for in part why high concentrations of ATP can inhibit the enzymatic activity of the αγ heterodimer.In the αure Fig. . As thers19Fig. . In deta2+)23. In cells, about 90% of total intracellular ATP (in the range of 1–10 mM depending on the cell types and determination methods) exists in the form of MgATP, and about 50% of total intracellular ADP (in the range of 0.1–1 mM) exists in the form of MgADP27. Thus, the concentrations of ATP and ADP can affect the proportion of the metal ion that exists in chelate form versus free ion form, thereby modulating the enzymatic activity in many metal dependent catalytic reactions. We speculated that high concentrations of ATP may promote the formation of MnATP or MgATP and decrease the concentration of free Mn2+ or Mg2+ available for the catalytic reaction, leading to the inhibition of the enzyme. To investigate this possibility, we analyzed the dual regulatory roles of ATP on the activity of the αγ heterodimer at different concentrations of Mn2+ or Mg2+  and in the absence of the activators CIT and ADP , ATP has activatory effect at low concentrations but inhibitory effect at high concentrations. Moreover, as the metal ion concentration increases, the ATP concentrations required to reach the optimal activation and the complete inhibition of the enzyme are elevated; additionally, the ATP concentration range for activation is also extended  with other ligand(s), we also analyzed the regulatory effect of ATP on the αγ heterodimer at different concentrations of the substrate ICT, the cofactor NAD, the inhibitor NADH, and the activators CIT and ADP Fig. . As expe19. This might be explained as follows: when the CIT concentration reaches the optimal activation level (2 mM), more CIT would compete with ICT to bind to the active site, and thus inhibit the enzymatic activity. In the presence of lower concentrations of ADP , ATP plays dual regulatory roles, and the ATP concentration required to reach the optimal activation is gradually decreased from 1.5 mM ATP (at 0.5 mM ADP) to 0.5 mM (at 2 mM ADP); meanwhile, the enzymatic activity is also increased accordingly , a further increase in ADP and ATP would compete with NAD to bind to the active site or reduce the concentration of free metal ion, thus inhibiting the enzymatic activity. As expected, in the presence of both CIT and ADP, ATP exhibits a regulatory pattern similar to a combination of the patterns seen in the presence of CIT and ADP alone  to 1 mM ATP in the presence of higher concentrations of CIT (2 mM and 4 mM) Fig. . Intrigugly Fig. . In the one Fig. . In the 17. Intriguingly, while the early biochemical studies showed that ATP has inhibitory effect on the activity of mammalian NAD-IDHs20, our biochemical studies showed that ATP can activate the activities of the αγ heterodimer and the holoenzyme of human NAD-IDH at low concentrations but inhibit the enzymatic activity at high concentrations. To investigate the molecular basis for the dual regulatory roles of ATP on the αγ heterodimer, we determined the crystal structure of αMgγMg+CIT+ATP, which was obtained from crystals grown from a crystallization solution containing a low concentration of ATP (2 mM), and we also determined the crystal structure of αMg+ATPγMg+CIT+ATP, which was obtained from soaking the crystals of αMgγMg+CIT in a crystallization solution containing a high concentration of ATP (200 mM). In addition, we performed biochemical studies to analyze the effects of the concentrations of metal ion and ligands on the dual regulatory roles of ATP on the αγ heterodimer. The structural and biochemical data together reveal the molecular mechanisms of the activation and inhibition of the αγ heterodimer by ATP.In cells, the ATP/ADP ratio is considered to be a key factor for controlling respiration and metabolism in response to energy demand. A low ATP/ADP ratio corresponds to a low energy level of the cell and thus attenuates cellular respiration, whereas a high ATP/ADP ratio corresponds to a high energy level of the cell and thus ramps down cellular respiration. As a rate-limiting step of the Krebs cycle, the activity of NAD-IDH is also regulated by ADP and ATP. Our previous biochemical and structural studies and the early biochemical studies of other groups showed that ADP can positively regulate the activity of mammalian NAD-IDHs via an allosteric mechanismMgγMg+CIT+ATP and αMg+ATPγMg+CIT+ATP structures, there are Mg2+, CIT and ATP bound to the allosteric site. The ATP mimics ADP in binding to the ADP binding site; this ATP binding stabilizes the CIT and Mg2+ binding and the associated conformational changes at the allosteric site, the active site, and the heterodimer interface, leading the enzyme to adopt an active conformation similar to that of αMgγMg+CIT+ADP. As the ADP binding site cannot accommodate the three phosphates of ATP, the β- and γ-phosphates of ATP are oriented away from the Mg2+-binding site, and the stabilization of the CIT and Mg2+ binding by ATP is mediated via a water molecule, which is apparently weaker than the stabilization by ADP. The structural results are consistent with our previous and current biochemical data showing that both ADP and ATP have a synergistic activatory effect with CIT on the αγ heterodimer; however, the activatory effect of ATP is weaker than that of ADP, and thus can be compromised by ADP. The structural and biochemical data together provide the molecular basis to explain why the αγ heterodimer can be activated by low concentrations of ATP.In both the αMg+ATPγMg+CIT+ATP structure, there are Mg2+ and ATP bound to the active site. The ATP mimics NADH in binding to the NAD binding site; this binding prevents the binding of NAD and leads to the inhibition of the enzyme. Nevertheless, although the adenine, the ribose, and the α-phosphate of ATP occupy the same positions as the corresponding moieties of NADH in the αMg+NADHγNADH structure and maintain similar interactions with the surrounding residues19, the β- and γ-phosphates of ATP occupy a slightly different position from the nicotinamide moiety of NADH and make no interactions with the enzyme. This suggests that ATP has a weaker binding with the enzyme compared to NADH (and NAD). These results are also consistent with the structural data demonstrating that ATP can only bind to the NAD binding site at a very high concentration. Moreover, our biochemical data show that increases in the metal ion concentration can increase the ATP concentration required to reach the optimal activation and the complete inhibition of the enzyme, as well as broaden the ATP concentration range for activation. This indicates that increasing the metal ion concentration can compensate for the inhibitory effect of ATP. These results suggest that high concentrations of ATP facilitate the formation of MgATP or MnATP and reduce the concentration of free metal ion available for the catalytic reaction, leading to the inhibition of the enzyme. The combined effects of the competitive binding of ATP to the NAD binding site and the formation of ATP-metal chelate account for the inhibition of the αγ heterodimer by high concentrations of ATP.On the other hand, the inhibition of the αγ heterodimer by high concentrations of ATP occurs through two different mechanisms. In the α16. Briefly, the DNA fragments encoding the α and γ subunits of human NAD-IDH were cloned into co-expression vector pQlinkN with the C-terminal of the γ subunit attached with a TEV protease cleavage site and a His6 tag following the pQlinkN cloning procedure28. The pQlinkN-α-γ-tev-His6 plasmid was transformed into E. coli BL21(DE3) CodonPlus strain (Novagen), and protein expression was induced by 0.4 mM IPTG for 20 hours at 23 °C. The target protein was first purified by affinity chromatography using an Ni-NTA column (Qiagen). The His6-tag of the target protein was cleaved by TEV protease, and the protein mixture was reloaded on an Ni-NTA column and the flow-through fraction containing the target protein was further purified by gel filtration using a Superdex 200 10/300 GL column . The purified protein has high purity (>95%) as analyzed by 12% SDS-PAGE, which was concentrated to about 10 mg/ml and stored in the storage buffer  for the structural and biochemical studies.The αγ heterodimer of human NAD-IDH was prepared as described previouslyMgγMg+CIT+ATP heterodimer were grown from drops consisting of equal volumes (1 μl) of the protein solution supplemented with 0.2 mM Mg2+ and 2 mM ATP, and the reservoir solution containing 0.2 M sodium citrate, pH 8.0, and 20% (w/v) PEG3350. Crystals of the αMgγMg+CIT heterodimer were grown from drops consisting of the protein solution supplemented with 0.2 mM Mg2+, and the reservoir solution containing 0.2 M sodium citrate, pH 5.5, and 24% (v/v) PEG400. Crystals of the αMg+ATPγMg+CIT+ATP heterodimer were obtained by soaking the crystals of αMgγMg+CIT in the crystallization solution supplemented with 200 mM ATP for 10 mins. The pH values of the stock solutions of the metal ion and ATP were adjusted to 7.4, which is the same as that of the protein stock solution, before addition into the crystallization or soaking solutions. Prior to diffraction data collection, the crystals were cryoprotected using the reservoir solution supplemented with 25% ethylene glycol and then flash-cooled into liquid N2. Diffraction data were collected at 100 K at BL17U1 of Shanghai Synchrotron Radiation Facility (SSRF) and BL19U1 of National Facility for Protein Science in Shanghai (NFPSS) and processed with HKL200029. Statistics of the diffraction data are summarized in Table Crystallization was performed using the hanging drop vapor diffusion method at 20 °C. Crystals of the α volumes  μl of thMgγMg+CIT+ATP and αMg+ATPγMg+CIT+ATP structures were solved with the molecular replacement method as implemented in program Phaser30 using the αMgγ structure (PDB code 5GRH) as the search model. Structure refinement was carried out initially with program Phenix31 and finally with program REFMAC532. Manual model building was carried out with program Coot33. Stereochemistry and quality of the structure models were analyzed using programs in the CCP4 suite34. Structure figures were prepared using PyMol35. Statistics of the structure refinement and final structure models are summarized in Table The α16. The standard reaction solution (1 ml) consisted of 33 mM Tris-acetate, pH 7.4, 2 μg enzyme, 0.6 mM ICT, 2 mM Mn2+, and 3.2 mM NAD. The activity is defined as the velocity (V) at the specific conditions, which is expressed as μmol of NADH produced per min per mg of enzyme (μmol/min/mg). To analyze the dual regulatory roles of ATP on the activity, the activity was measured at the standard conditions with varied concentrations of ATP (0–10 mM). The pH value of the ATP stock solution was adjusted to 7.4, which is the same as that of the protein stock solution, before addition into the reaction mixture. The effect of ATP is presented as the ratio (V/Vab) of the activity of the enzyme in the presence of ATP (and regulators) (V) verse that in the absence of ATP and any regulators at the standard conditions (Vab which is defined as 1).Enzymatic activity of the αγ heterodimer was assayed using a method as described previously2+ and Mg2+ on the dual regulatory roles of ATP, the activity was measured at the standard conditions with varied concentrations of Mn2+ or Mg2+ . To analyze the effects of the substrate ICT and the cofactor NAD on the dual regulatory roles of ATP, the activity was measured at the standard conditions with varied concentrations of ICT  or NAD . To analyze the effect of the inhibitor NADH on the dual regulatory roles of ATP, the activity was measured at the standard conditions with varied concentrations of NADH . To analyze the effects of the activators CIT or/and ADP on the dual regulatory roles of ATP, the activity was measured at the standard conditions with varied concentrations of CIT , or ADP , or both CIT and ADP. The pH values of the stock solutions of the metal ion and ligands were adjusted to 7.4, which is the same as that of the protein stock solution, before addition into the reaction mixture. All experiments were carried out at 25 °C and repeated at least twice under the same conditions.To analyze the effects of MnMgγMg+CIT+ATP and αMg+ATPγMg+CIT+ATP structures of human NAD-IDH have been deposited in the Protein Data Bank with accession codes 6L57 and 6L59, respectively.The α


Pyridinium containing sulfonamides have been largely investigated as carbonic anhydrase inhibitors (CAIs), showing interesting selectivity features. Nevertheless, only few structural studies are so far available on adducts that these compounds form with diverse CA isoforms. In this paper, we report the structural characterization of the adduct that a triphenylpyridinium derivative forms with hCA II, showing that the substitution of the pyridinium ring plays a key role in determining the conformation of the inhibitor in the active site and consequently the binding affinity to the enzyme. These findings open new perspectives on the basic structural requirements for designing sulfonamide CAIs with a selective inhibition profile.  In detail, an applied photophysics stopped-flow instrument has been used for assaying the CA catalyzed CO2 hydration activity2SO4 (for maintaining constant the ionic strength), following the initial rates of the CA-catalyzed CO2 hydration reaction for a period of 10–100 s. The CO2 concentrations ranged from 1.7 to 17 mM for the determination of the kinetic parameters and inhibition constants. Six traces of the initial 5–10% of the reaction have been used for determining the initial velocity. The uncatalyzed rate was determined in the same manner and subtracted from the total observed rate. Stock solution of inhibitors (0.1 mM) were prepared in distilled–deionized water and dilutions up to 0.01 nM were done thereafter with the assay buffer. Inhibitor and enzyme solutions were preincubated together for 15 min at room temperature prior to assay, to allow for the formation of the E–I complex. The inhibition constants were obtained by non-linear least-squares methods using PRISM 3  and the Cheng–Prusoff equation and represent the mean from at least three different determinations.Inhibition constants reported in 2 adduct was obtained using a procedure previously described for other hCA II/inhibitor complexes,® 500; Sartorius, Göttingen, Germany). Crystals were obtained at 20 °C using the hanging drop vapour diffusion technique by equilibrating drops containing 1 µL of complex solution and an equal volume of precipitant solution consisting of 1.3 M sodium citrate, 0.1 M TRIS-HCl, pH 8.5, over a reservoir containing 0.5 mL of precipitant solution. Crystals appeared after 3 days. Diffraction data were collected to 1.65 Å resolution, in-house at −180 °C, using a Rigaku MicroMax-007 HF generator producing Cu Kα radiation and equipped with a Saturn 944 CCD detector. Cryoprotection of the crystals was achieved transferring the crystals into the precipitant solution with the addition of 10% (v/v) glycerol. Data were indexed, integrated, and scaled using HKL2000The hCA II/,B-value refinement. The inhibitor molecule was identified from peaks in |Fo| − |Fc| maps and gradually built into the model over several rounds of refinement. Composite simulated-annealing omit maps were used regularly during the building process to verify and correct the model,R-free calculations to monitor the progress of refinement. Topology files of the inhibitor were generated using the PRODRG2 serverA previously solved structure of hCA II (PDB code 5O07)1 and 2 were previously described by our groups, and were obtained by reaction of 4-aminoethyl-benzenesulfonamide with 2,4,6-trisubstituted pyrylium salts,1 is a low nanomolar inhibitor of hCA IX and XII, effectively inhibits hCA II and hCA IV, but it is less effective as hCA I inhibitor, the triphenylpyridinium 2 is a quite effective hCA XII inhibitor, it also inhibits hCA IX, but its affinity for the other isoforms is in the micromolar range  were higher with respect to those observed for the 4-ethylbenzenesulfonamide moiety (11.7 Å2). The binding of the inhibitor within the active site did not cause significant changes in the enzyme structure as demonstrated by the low value of the RMSD calculated by superposing the Cα atoms in the adduct and the non-inhibited enzyme (0.3 Å).Crystals of the hCA II/ran plot . Since tran plot . Accordi2 was bound to the enzyme with its sulfonamide group coordinated to the zinc ion in a tetrahedral geometryAs expected for a benzenesulfonamide inhibitor, compound 2 and 1 when bound to the hCA II active site, showing that even if the benzenesulfonamide groups of the two inhibitors are quite perfectly superimposable, the two trisubstituted pyridinium moieties are oriented toward different regions of the active site. In particular, the 2,4,6-trimethylpyridinium moiety of 1, which was described as perfectly defined in the electron density maps2, although oriented toward the hydrophilic part of the active site, is flexible and does not establish many stabilizing interactions with enzyme’s residues. Interestingly, the only differences between the two inhibitors are the substituents of the pyridinium ring, namely three methyl groups for compound 1 and three phenyl moieties for compound 2, which make it much more bulky and do not allow its accommodation into the hydrophobic pocket defined by residues Ile91, Gln92, and Phe131. Indeed, in this position the substituted ring would strongly clash with residues which delimit the pocket , the triphenyl derivative 2 showed a selective inhibition profile for the tumour over cytosolic isoforms, which represents a valuable feature for compounds to be investigated as antitumor agents. Thus, understanding the detailed interactions between inhibitors belonging to similar structural classes, as 1 and 2 discussed here, may shed new light on the basic structural requirements for designing sulfonamide CAIs with a selective inhibitory profile.The X-ray data presented here explain why the triphenylpyridinium-substituted sulfonamide


Here, we identified the serine residues S2362 and S2364 of the voltage-dependent calcium channel CaV2.1 as targets of CK2 phosphorylation. Furthermore, co-immunoprecipitation experiments revealed that CaV2.1 binds to CK2 in vitro and in vivo. CaV2.1 knockdown experiments showed that the increase in the intracellular Ca2+ concentration, followed by an enhanced insulin secretion upon CK2 inhibition, is due to a Ca2+ influx through CaV2.1 channels. In summary, our results point to a modulating role of CK2 in the CaV2.1-mediated exocytosis of insulin.The regulation of insulin biosynthesis and secretion in pancreatic β-cells is essential for glucose homeostasis in humans. Previous findings point to the highly conserved, ubiquitously expressed serine/threonine kinase CK2 as having a negative regulatory impact on this regulation. In the cell culture model of rat pancreatic β-cells INS-1, insulin secretion is enhanced after CK2 inhibition. This enhancement is preceded by a rise in the cytosolic Ca V1.2 γATP either in the presence of the specific CK2 inhibitor CX-4945 or with DMSO as a solvent control. After phosphorylation, proteins were separated by SDS polyacrylamide gel electrophoresis, blotted to a PVDF membrane and subjected to autoradiography (32P). Subsequently, the same membrane was subjected to an immunoblot analysis with a CaV2.1 antibody and, as loading control, with an anti-tubulin antibody  of zebra fish (Genbank protein ID: NP_001315637.1), mice (AAW56205.1), rats (XP_017456671.1), dogs (XP_013977279.1) and humans  and searched for consensus sequences of protein kinase CK2 phosphorylation sites  or with [32P]γATP alone as a negative control (-). The control peptides showed clear signals that were absent when the serine or threonine residue was replaced by alanine  within the Caanalysis C. Pentad alanine C. We alsV2.1, encompassing the amino acid residues 2177–2369, either as its wild-type or as its double-mutated (S2362A/S2364A) version, in frame with glutathione-S-transferase (GST) tags. The recombinant fusion proteins were expressed in Escherichia coli, purified by affinity chromatography, subjected to in vitro phosphorylation with the CK2 holoenzyme, separated by SDS gel electrophoresis and analyzed by autoradiography (V2.1(2177-2369) fragment at the predicted size of 46 kDa. The fragment was only efficiently phosphorylated in its wild-type but not its double-mutated form, thus confirming the data from the peptide filter and demonstrating that S2362 and S2364 are target sites of CK2 phosphorylation within the C-terminus of CaV2.1.To verify these results in an independent approach, we subcloned the C-terminal fragments of murine Caiography D. The phV2.1 after yeast two-hybrid experiments [V2.1. The individual CK2α and CK2β subunits were labeled with [35S]-methionine by in vitro translation and mixed separately with the GST-tagged CaV2.1(2177-2369) fusion protein in its wild-type and its double-mutated (S2362A/S2364A) forms. Protein complexes were pulled down using a glutathione (GSH) sepharose affinity matrix and analyzed on a SDS polyacrylamide gel. GST fusion proteins were stained with Coomassie Brilliant Blue (CBB) and bound CK2 subunits were visualized by autoradiography , the other given putative sites are not embedded in an acidic environment. In the present paper, we demonstrated that endogenous CaV2.1 from INS-1 cells is phosphorylated by CK2; however, we could not prove that the phosphorylated sites were identical with those from the in vitro experiments. However, both sites were found to be phosphorylated in the course of a phosphoproteome analysis in 3T3-L1 cells γATP  in 1 mL kinase buffer. As a negative control, the mixture without CK2 was tested on a second identical filter. The peptide filters were incubated for 1 h at room temperature with gentle shaking. After incubation, the filter was washed with kinase buffer, air dried and analyzed by autoradiography.For the analysis of the phosphorylation of CaV2.1 proteins were mixed with a recombinant CK2 holoenzyme in 20 µL kinase buffer. The phosphorylation reaction was started by the addition of [32P]γATP . After 30 min at 37 °C, the reaction was stopped by the addition of 10 µL sample buffer  SDS). Proteins were separated on SDS polyacrylamide gel (10% or 12.5%) followed by Coomassie Brilliant Blue staining and autoradiography.Recombinant GST-tagged Cat-test , statistically significant differences were shown as follows: *** p < 0.001, ** p < 0.01 or *p < 0.05.Microsoft Excel 2013 and the software “Quantity One 1-D Analysis” (version 4.6.7) from Bio-Rad Laboratories Inc.  were used to analyze the data. All values were expressed as mean ± SEM of at least three independent experiments. Statistical analysis of the data was performed using the Student’s


PMP22) gene resulting in focal sensorimotor deficits. Our lab has identified a disruption of myelin junctions in excessively permeable myelin that impairs action potential propagation. This mechanism is expected to cause fatigue in patients with HNPP. Therefore, the objective was to characterize fatigue in patients with HNPP and determine the relationship of fatigue to nerve pathology, disability, and quality of life.Hereditary Neuropathy with Liability to Pressure Palsies (HNPP) is caused by a heterozygous deletion of peripheral myelin protein‐22  that captured fatigue ratings five times per day.Participants demonstrated mild neurological impairment (CMTNS: 5.7 ± 2.8), yet reported high fatigue levels (average fatigue intensity over 2 weeks: 5.9 out of 10). Higher fatigue levels were associated with poorer quality of life and more pain. Higher fatigue was associated with significantly greater distal nerve proton density changes on peripheral nerve MRI, which is in line with hyper‐permeable myelin in HNPP.Fatigue is common and severe among patients with HNPP whose disabilities are minimal by conventional outcome measures. Therapeutic interventions targeting fatigue have the potential to improve quality of life and may serve as a robust outcome measure to show longitudinal changes for patients with HNPP. This impairs action potential propagation in the absence of demyelination (stripping myelin off the axon), called functional demyelination,Our laboratory has demonstrated an abnormally increased permeability of myelin in an HNPP mouse model  were recruited through the Wayne State University Charcot‐Marie‐Tooth (CMT) Clinic where the author (JL) serves as the Director. All testing occurred in a single visit. All participants signed an informed consent prior to testing, and all testing procedures were approved by the Wayne State University Institutional Review Board.PMP22.HNPP was diagnosed in all participants by the genetic test (multiplex PRC) showing a heterozygous deletion of NCS were done as previously described.Medical history and neurological exam were collected from each patient. CMTNS is comprised of sensorimotor symptoms, physical findings in limbs as well as nerve conduction studies.i) an interleaved two‐point Dixon three‐dimensional (3D) gradient recalled echo (GRE) scan for muscle fat fraction (FF);ii) a high‐resolution 3D GRE scan for nerve fascicular cross‐section area (fCSA); iii) the strategically acquired gradient echo (STAGE) imagingiv) two 3D GRE scans performed with and without MT pulses for nerve MTR and effective transverse relaxation time (T2star).In addition to the participants with HNPP, we recruited four additional female healthy controls  for qMRI study. These HCs have no known history of neurological disease. All subjects were scanned on the same 3T system  with an eight‐channel knee coil positioned at two boney marker locations: mid‐thigh level proximal to the knee, and mid‐calf level distal to the knee. An established imaging protocol was performed in the axial plane.MRI data were processed using an in‐house developed program in MATLAB  with image alignments via SPM (fil.ion.ucl.ac.uk/spm/).This established processing pipelineParticipants completed the 10‐meter walk test for gait speed assessment, 9‐hole peg test for upper extremity dexterity, as well as a battery of survey measures to examine quality of life (SF‐36), pain , depression  and sleep . Neuro‐QoL and PROMIS measures were developed and validated to evaluate physical, mental, and social effects experienced by adults living with chronic neurological conditions.We defined pathologic fatigueThe NeuroQoL Fatigue Short FormThe NeuroQoL Fatigue was supplemented by Ecological Momentary Assessment (EMA), where participants wore a wrist‐worn device, the ProDiary  that prompted the user to report fatigue levels five times per day  over a 2‐week period following the in‐person visit. Participants were asked to rate both the intensity of fatigue (“what is your level of fatigue?”) and impact of fatigue (“how much is fatigue interfering with your daily activity right now?”) on a 0‐10 numerical rating scale entered directly on the ProDiary. EMA measures have been shown to provide a more reliable and sensitive assay of symptoms compared to traditional recall reportsThe ProDiary device was outfitted with accelerometry for examination of activity levels and mobility throughout the 2‐week period. Average scores for activity were calculated.P < 0.05 as significant, to evaluate differences between HC and HNPP for each qMRI index; (2) linear regression model by fitting to data considering strong correlation effect for 2>=0.7r, moderate effect for 2>=0.50.7> r, and weak (or no) effect for 2 < 0.5r, each with a threshold of P < 0.05 as significant model.SPSS version 26 was used for fatigue data analysis. Spearman correlations were used to determine the relationship among fatigue measures and measures of function. Given the small sample size of persons with HNPP, we evaluated r‐values  for potential impact of relationships between variables. MATLAB was used for statistical analyses on qMRI data and the correlations between qMRI indices and fatigue measurements: (1) Wilcoxon rank‐sum test with a threshold of Anonymized data will be shared by request from any qualified investigator.PMP22.Nine women participated the study . None used any assistant devices, such as ankle or wrist brace. All participants had a genetically confirmed heterozygous deletion of All patients with HNPP reported 1–3 incidents of reversible focal sensory loss and weakness throughout their life, which may or may not be triggered by mechanical compression. Participants reported that these episodes took from weeks to more than a year to recover.NCS showed focally slowed nerve conduction at the sites susceptible to mechanical pressure. For instance, six patients had a prolonged distal latency of the median motor nerve across the wrist  and interference (r = 0.500)]; however, weekly subjective fatigue intensity and interference were strongly related  (Table Eight of nine participants completed fatigue assessments. Subjective report of fatigue at a single time point (NeuroQol‐Fatigue) was moderately correlated with average weekly subjective reports of fatigue [EMA measures of fatigue intensity  suggesting that greater fatigue is associated with poorer quality of life. Greater fatigue intensity was also related to poorer ratings in other domains of quality of life, including more role limitations in physical health (r = 0.486), more role limitations in emotional health (r = 0.433), poorer overall energy quality of life (r = −0.618); poorer emotional well‐being (r = −0.733); poorer social functioning (r = −0.537) and worse pain (r = −0.604).EMA weekly average fatigue intensity and interference scores were strongly related to the general health quality of life subscale of SF‐36 (r = 0.509) on the PROMIS Pain scale. No relationship was noted between fatigue levels and depression, sleep, gait, activity levels, or hand functioning in this cohort.Finally, a moderate relationship was seen between greater fatigue intensity and higher pain ratings  at calf level, but not thigh level (P = 0.61), compared with controls, confirming an axonal loss in a length dependent fashion. In line with muscle FF finding, distal  nerve proton density (PD), but not proximal (sciatic) nerve PD, was significantly elevated (P = 0.008), compared with controls  and interference  measured with EMA  with a moderate and positive effect  nerve PD versus average fatigue severity  was at a median of 3.0 (range 1–5) in these patients,Among all those qMRI indices, PD appears to be the most sensitive measure to detect nerve pathology in patients with HNPP. It is known that the MRI PD is linearly correlated to the tissue water content. We speculate the increased permeability of myelin in HNPP causes increased water content between myelin lamina, but overall myelin density remains unchanged. This is consistent with the MTR data reflecting myelin density that was not significantly different in HNPP compared to controls. Therefore, such conspicuous discrepancy between subtle neurological disabilities and severe fatigue demands explanation in patients with HNPP.internode. The small gap between internodes forms the node of Ranvier where the voltage gated sodium channels (Nav) are concentrated  adjacent to the paranode is the juxtaparanode and is defined by the distribution of voltage gated potassium channels Kv1.1 and Kv1.2.incisures. The incisures are non‐compacted myelin tunnels spiraling around the axon.tight junctions and adherens junctions that separate them from the compact myelin.non‐compact myelin, in contrast to the compact myelin.Schwann cells in the peripheral nervous system (PNS) wrap axons concentrically in consecutive segments, defining the territory of an individual d Figure . SchwannPMP22 in HNPP results in disruption of myelin junctions. The impaired seal of myelin leads to slowed conduction velocity, conduction block, and temporal dispersion in nerve conduction studies. These electrophysiological features recapitulate those seen in acquired demyelinating neuropathies while internodal myelin is still intact in HNPP nerves. We termed this “functional demyelination”.Myelin seals axons, which enables the fastest conduction of action potentials. This seal also requires myelin junctions to obliterate the spaces between each layers of myelin wrap Figure . Our preIn those myelinated nerve fibers with less severe disruption of myelin junctions, conduction block may not occur, but action potential propagation would be at the risk to fail. Intermittent failure of conduction in those nerves would explain fatigue during physical activities. This hypothesis needs to be further tested in the future studies of humans with HNPP.Our findings also raise several critical issues for the future clinical trials against HNPP: Patients with HNPP exhibit two distinct sets of clinical phenotypes. First, HNPP patients may present with transient, multi‐focal sensory loss or weakness that may or may not be associated with physical activities triggering the focal deficits. Second, axonal loss accumulated over time results in a length‐dependent and symmetric polyneuropathy causing distal limb sensory loss, muscle weakness and atrophy in aged patients. Conventional outcome measurements, such as CMT neuropathy score (CMTNS), were designed based on the length‐dependent and symmetric polyneuropathy seen in other types of CMT. CMTNS would measure the second phenotype; but is not suitable for the first phenotype. Those focal deficits in HNPP may be transient and would introduce unacceptable variations of CMTNS scores, depending on when the scores are collected (at the peak versus at the trough of the episodes). While axonal loss may be measured using techniques like the muscle MRI FF, due to the slow progression of axonal loss in patients with HNPP, sample size may have to be large to detect differences, which is difficult to do due to relatively rare prevalence of HNPP.Fatigue changes in patients with HNPP appear to be robust in our study. Therefore, fatigue score may serve as an alternative outcome measure if future longitudinal studies can demonstrate good responsiveness, thereby reducing the sample size.Moreover, in patients with HNPP, fatigue was strongly related to quality of life as measured by the SF‐36 subdomains, including general health, pain, and social functioning. These findings suggest that interventions targeting fatigue have the potential to greatly improve quality of life in patients with HNPP, and are consistent with our prior data in multiple sclerosis showing that fatigue is related to higher pain ratings and poorer quality of life.Because of the fluctuating nature of HNPP phenotype, the EMA captures day to day variations in fatigue and symptoms over time. EMA fatigue levels are reported five times per day over a 2‐week period on a wrist‐worn device. Unless a severe limb paralysis developed, fatigue score is unlikely affected by transient focal episodes. To ensure no complication from these focal episodes, participants were asked to report limb paralysis with a custom diary during the EMA period, which could be used to rectify large variations (though none were present in this study).This work is limited by a small sample size (common in rare diseases). Our sample is unintentionally comprised of females only. Because prior work has shown that sexual hormones play a role in regulating PMP22 expression, a single sex of cohort would improve the consistency of the data.In summary, our study revealed a remarkable level of fatigue in patients with HNPP. This finding is in line with previously reported the impaired safety factor of action potential propagation in PMP22 deficient nerves .N.E. Fritz reports no disclosures. Y. Chen reports no disclosures. L. Waters reports no disclosures. S. Saba reports no disclosures. M. Hackett reports no disclosures. F.C. Mada reports no disclosures. J Li is a member in CMT Association Scientific Advisory Committee, an adhoc reviewer in several NIH study sections and provides consultations to Neurogene and Alnylam.Nora E. Fritz: Conceptualization, methodology, formal analysis, investigation, writing‐original draft, writing‐ review & editing, supervision. Yongsheng Chen: Conceptualization, data curation, methodology, formal analysis, investigation, writing‐ review & editing, visualization. Lauren Waters: Data curation, writing‐review & editing. Sadaf Saba: Investigation, data curation, writing‐review & editing, visualization. Melody Hackett: Investigation, data curation, writing‐ review & editing. Flicia Christina Mada: Investigation, data curation, writing‐ review & editing. Jun Li: Conceptualization, methodology, formal analysis, investigation, resources, writing‐ review & editing, supervision, project administration, funding acquisition.


Serum inhibition of allergen‐specific IgE has been associated with competing IgG4 and non‐specific polyclonal IgE. In allergen immunotherapy, beneficial responses have been associated with high IgG4/IgE ratios. Helminths potentiate antibody class switching to IgG4 and stimulate polyclonal IgE synthesis; therefore, we hypothesized a role for helminth‐associated IgG4 and total IgE in protection against atopic sensitization and clinical allergy (asthma) in tropical low‐income countries.Schistosoma mansoni (Sm)–endemic islands and a mainland urban setting with lower helminth exposure, and among urban asthmatic schoolchildren and non‐asthmatic controls, we measured total, Schistosoma adult worm antigen (SWA)–specific, Schistosoma egg antigen (SEA)–specific and allergen (house dust mite [HDM] and German cockroach)–specific IgE and IgG4 by ImmunoCAP® and/or ELISA. We assessed associations between these antibody profiles and current Sm infection, the rural‐urban environment, HDM and cockroach skin prick test (SPT) reactivity, and asthma.Among community residents of Ugandan rural Sm infection and SPT reactivity were positively associated with allergen‐specific and total IgE responses. SPT reactivity was inversely associated with Schistosoma‐specific IgG4, allergen‐specific IgG4/IgE ratios and total IgE/allergen‐specific IgE ratios. Asthmatic schoolchildren, compared with non‐asthmatic controls, had significantly higher levels of total and allergen‐specific IgE, but lower ratios of allergen‐specific IgG4/IgE and total IgE/allergen‐specific IgE.Total IgE, total IgG4 and SWA‐, SEA‐ and allergen‐specific IgE and IgG4 levels were significantly higher in the rural, compared to the urban setting. In both community settings, both Our immuno‐epidemiological data support the hypothesis that the IgG4–IgE balance and the total IgE–allergen‐specific IgE balance are more important than absolute total, helminth‐ or allergen‐specific antibody levels in inhibition of allergies in the tropics. Moreover, chronic helminth infection is associated with elevated serum IgG4 levels,Studies conducted four decades agoSm‐endemic Ugandan rural fishing villages,Sm‐ and allergen‐specific IgE and IgG4 profiles, and to analyse their associations with current Sm infection, the rural‐urban environment, allergic sensitization and asthma.Large, well‐defined immuno‐epidemiological studies in helminth‐endemic settings are required to better understand population‐level interactions between allergy‐related disease and helminth‐ and allergen‐associated IgG4 and polyclonal IgE profiles. This will contribute to bridging the gap between understanding basic antibody mechanisms and clinical applications. We used the opportunity presented by studies designed to assess the epidemiology of allergy‐related disease in (1) 22.1The current investigation was conducted using samples from participants of cross‐sectional community surveys in ruralSm‐endemic rural fishing villages of Koome islands, Lake Victoria. It was an open cluster‐randomized trial of community‐wide standard versus intensive anthelminthic mass drug administration (MDA).The rural survey was the three‐year outcome survey (September 2015–August 2016) of the Lake Victoria Island Intervention Study on Worms and Allergy‐related diseases .The urban survey (September 2016–September 2017) was designed to collect data for comparison with the rural survey.The asthma case‐control study (May 2015–July 2017)2.2Sm, hookworm (Necator americanus), Ascaris lumbricoides and Trichuris trichiura infections. For each participant, one stool sample (prepared on two slides) was examined under a microscope by two laboratory technologists blinded to each other's result. In the rural and urban surveys, stool was further suspended in 70% ethanol, stored at −80°C and later examined for Sm, Strongyloides stercoralis and hookworm infections using multiplex real‐time PCR.Schistosoma haematobium has not been documented in our study areas.Schistosoma adult worm antigen (SWA)‐ and Schistosoma egg antigen (SEA)–specific IgE and IgG4 by ELISA  technique2.3Dermatophagoides mix (D. pteronyssinus and D. farinae), Blomia tropicalis and Blattella germanica  was assessed using standard methods.D. pteronyssinus, HDM) and German cockroach (B. germanica) extract–specific plasma IgG4 and total IgG4. Total IgE and allergen extract–specific IgE were measured using the ImmunoCAP® IgE test .®. The in‐house assays are described in this article's supplementary information Appendix Wheezing in the previous 12 months was also assessed in the rural and urban surveys using interviewer‐administered standardized paper and video ISAAC questionnaires. Skin prick test (SPT) reactivity to crude extracts from 2.4Statistical analyses were conducted using Stata 15.0 (StataCorp). Graphs were drawn using GraphPad Prism . We accounted for study design in all analyses involving the rural and urban community surveys: Stata “svy” commands were used to allow for the non–self‐weighting clustering by village in the rural survey and for clustering by sub‐ward in the urban survey.Next, participant characteristics were tabulated and compared between urban and rural settings separately, between asthma cases and controls, using logistic and linear regression for categorical and continuous variables, respectively.Schistosoma‐specific and allergen‐specific IgE and IgG4 concentrations; allergen‐specific IgG4/IgE, total IgE/allergen‐specific IgE, total IgG4/total IgE ratios) and Sm infection or SPT reactivity. In the asthma case‐control study, we additionally investigated associations between asthma and antibody concentrations and ratios. Rural‐urban comparisons of antibody profiles were conducted using data from the rural and urban surveys. Most raw antibody concentrations and ratios were skewed, so for all analyses, linear regression models of log‐transformed data were used. ImmunoCAP® allergen–specific IgE concentrations were log10 (+0.001)‐transformed, while all other antibody data were log10 (+1)‐transformed. The results were then back‐transformed to obtain geometric mean ratios (GMRs) and 95% confidence intervals (CIs).Independently for each of the three studies, we conducted crude and age‐ and sex‐adjusted cross‐sectional analyses to investigate the direction and the strength of association between antibody concentrations or ratios  and London School of Hygiene and Tropical Medicine (reference numbers: 6187 and 10709), and the Uganda National Council for Science and Technology . We obtained written informed consent from all participants and/or their legal guardians. Informed assent was obtained from children ≥8 years.33.1Schistosoma‐ or allergen‐specific IgE or IgG4. There was no effect of intensive versus standard anthelminthic MDA on antibody profiles  and male (p = 0.003). Prevalence of SPT reactivity to Dermatophagoides mix and B. tropicalis was higher among urban participants (p = .001). However, rural participants had higher median total IgE levels (p < .001), and higher prevalence of IgE sensitization to crude German cockroach (ImmunoCAP® concentration ≥0.35 kU/L) [p = .001], ELISA‐detectable cockroach‐specific IgE (≥312.5 ng/ml) [p = .001], and infection with Sm (p < .001) and at least one nematode (p < .001). Reported wheeze was rare in both settings.Table Compared to non‐asthmatic controls, asthma cases were on average older, and more likely to be SPT positive, and to have higher total and allergen‐specific IgE levels. Helminth infection prevalence was low in the asthma case‐control study and comparable between cases and controls Table .3.2Sm infection was positively associated with total and SWA‐ and SEA‐specific IgE and IgG4 (p < .001), and cockroach‐specific IgG4 concentrations (p = .002). Cockroach SPT reactivity was positively associated with ImmunoCAP®‐determined cockroach‐specific IgE  and total IgE concentrations . Dermatophagoides (hereinafter HDM) SPT reactivity was positively associated with HDM‐specific IgE , but inversely associated with SWA‐specific IgG4 .In the rural survey Table , Sm infeSm infection was positively associated with total and SWA‐ and SEA‐specific IgE and IgG4 (p < .001), and ELISA‐determined HDM‐specific IgE (p = .037), cockroach‐specific IgE (p = .040) and cockroach‐specific IgG4 concentrations (p = .005). Cockroach SPT reactivity was positively associated with cockroach‐specific IgE , cockroach‐specific IgG4  and total IgE concentrations . Despite a positive association with SEA‐specific IgE , cockroach SPT reactivity was inversely associated with SEA‐specific IgG4 . HDM SPT reactivity was positively associated with HDM‐specific IgE , HDM‐specific IgG4  and total IgE concentrations .In the urban survey Table , Sm infeAkin to observations in the rural and urban surveys, cockroach and HDM SPT reactivity were generally positively associated with total and with cockroach‐ and HDM‐specific IgE concentrations, respectively, among both asthma cases and non‐asthmatic controls Table .3.3Schistosoma mansoni infection was positively associated with total IgE/cockroach‐specific IgE ratios in both community surveys , and with cockroach‐specific IgG4/IgE ratios in the rural survey  , p = .003; asthma study: 0.23 , p < .001) , p < .001; asthma study: 0.03 , p < .001), and HDM‐specific IgG4/IgE ratios  [Figure p < .001) [Figure In all three studies, HDM SPT reactivity was inversely associated with total IgE/HDM‐specific IgE ratios  Figure . HDM SPT) Figure .Assessment of associations between antibody ratios and skin prick test reactivity, independently among asthma cases and non‐asthmatic controls, showed observations akin to the above: cockroach and HDM SPT reactivity were inversely associated with allergen‐specific IgG4/IgE ratios, total IgE/allergen‐specific IgE ratios and total IgG4/total IgE ratios Table .3.4p < .001), cockroach‐specific IgE  and IgG4 (p = .038), total IgE (p < .001), total IgG4 (p = .011), total IgE/cockroach‐specific IgE ratios (p < .001) and total IgE/HDM‐specific IgE ratios (p < .001) [Table ®, p = .038) and IgG4 (p = .001), and HDM‐specific IgG4/IgE ratios (p = .011), were higher in the urban survey.Urban participants, compared with rural survey participants, had lower geometric mean concentrations of SEA‐ and SWA‐specific IgE, IgG and IgG4 (3.5p = .005), cockroach‐specific IgE  and HDM‐specific IgE  [Figure p < .001), total IgE/cockroach‐specific IgE ratios  and HDM‐specific IgG4/IgE ratios  [Figure Asthma was positively associated with total IgE  Figure . Convers) Figure .). Furthermore, HDM SPT–positive asthma cases had lower HDM‐specific IgG4/IgE, total IgG4/total IgE and total IgE/HDM‐specific IgE ratios, compared with both SPT‐negative and SPT‐positive controls (Table ). The same trend was observed for comparisons between cockroach SPT‐positive asthma cases and both SPT‐negative and SPT‐positive controls, albeit without statistical significance  environment were positively associated with total, Schistosoma‐ and allergen‐specific IgE and IgG4, and with allergen‐specific IgG4/IgE ratios and total IgE/allergen‐specific IgE ratios.We assessed total, Sm infection and allergen‐specific IgG4 and IgE among participants of our rural and urban community surveys. Strong positive associations between Sm infection and total IgE are consistent with observations of helminth‐induced polyclonal stimulation of non‐specific IgE.Host responses to helminths exhibit significant similarities with responses to common allergens, owing to related molecular targets.Sm‐infected and SPT‐negative participants irrespective of study setting—in excess of Mitre's threshold for inhibition.Mitre et al.Our observations of inverse associations between allergen‐specific IgG4/IgE ratios and SPT reactivity and asthma strengthen the argument that the IgG4‐IgE balance is also important in protection against allergies. In addition, SWA‐ and SEA‐specific IgG4 concentrations were significantly lower among HDM SPT‐reactive rural survey participants and cockroach SPT‐reactive urban survey participants, respectively. Although IgG4 has been implicated in IgG4‐related disease,There is a counter‐argument that IgG4 might not have a direct mechanistic role in protection against allergy‐related outcomes. Instead, the inverse association between IgG4 levels and IgE effector function may represent an ‘epiphenomenon’, merely reflective of parallel phenomena, such as abundance of IL‐10 (and/or TGF‐β)–producing T regulatory and B regulatory cells.® test, and later, using an in‐house ELISA assay. The latter technique was used to facilitate determination of IgG4/IgE ratios, since, for logistical reasons, we could not measure allergen‐specific IgG4 by ImmunoCAP®. However, patterns of associations with ImmunoCAP®‐ and ELISA‐determined IgE were comparable (except for cockroach‐specific IgE in a few instances). We did not formally adjust for multiple statistical testing. However, our interpretations rely on patterns and magnitude of associations, and consistency and biological credibility of our findings based on other published works. It is noteworthy that our studies were cross‐sectional; therefore, it was not possible to demonstrate causal relationships between antibody profiles and Sm infection, the rural‐urban environment, allergic sensitization and asthma.We measured allergen extract–specific IgE using the standard ImmunoCAPin vitro experiments using human samples. For example, a microarray screening platform could be utilized to identify helminth antigens that provoke robust serum IgG4 and little or no IgE response. Such antigens may be valuable for therapeutic approaches against allergic conditions.In conclusion, we underline the potential role of the total IgE‐allergen‐specific IgE balance and the IgG4‐IgE balance in mitigation of allergic responses, providing correlative evidence from three cross‐sectional population‐based studies. These results build upon previous findings that apportion high IgG4/IgE ratios an important role in allergen‐specific immunotherapy.The authors declare no conflict of interest.AME, GN, HM and ELW contributed to the conception and experimental design of the studies. GN, JN and JK performed the laboratory experiments. AME, HM, RES and MN led and participated in field and clinic procedures. GN analysed the results with significant input from ELW. GN wrote the manuscript, with all authors contributing to the interpretation of the results, and revision and approval of the final manuscript.Appendix S1Click here for additional data file.


Caenorhabditis elegans, a conserved RING finger protein, ZHP-3, is essential for meiotic recombination and localizes to sites of crossover formation. Whether ZHP-3 is regulated to promote recombination remains poorly understood. In vitro analysis identified two putative CHK-1 kinase phosphorylation sites on ZHP-3. However, mutation of the phosphorylation sites identified in vitro had no effect on meiotic recombination or localization of ZHP-3. Thus, these two phosphorylation sites appear to be dispensable for ZHP-3’s role in meiotic recombination or its localization.Meiotic chromosome segregation depends on crossover recombination to link homologous chromosomes together and promote accurate segregation in the first meiotic division. In  Defects in meiotic recombination can lead to gametes that contain an incorrect number of chromosomes (aneuploidy), which can cause infertility, miscarriages, and genetic disorders such as Down Syndrome. Therefore, identifying how recombination is regulated is crucial for understanding multiple aspects of human reproductive health.Haploid sex cells such as sperm and eggs depend on a specialized form of cell division called meiosis. Throughout prophase of meiosis, homologous chromosomes pair, synapse, and recombine to form a physical link between them called a chiasma. Chiasmata are essential for proper segregation . One of these crossover factors, ZHP-3 (Zip homologous protein 3), is highly conserved, has a budding yeast counterpart that acts as a SUMO ligase  and is essential for crossover formation . Therefore, when ZHP-3 is absent, chromosome pairs are not linked by chiasmata. Because sex determination in C. elegans is based on the number of X chromosomes, with hermaphrodites having two X chromosomes while males have one, a high incidence of male (HIM) phenotype is diagnostic for chromosome segregation errors. Loss of ZHP-3 results in a high rate of embryonic lethality and a HIM phenotype , both indicating chromosome segregation errors. We’ve shown that ZHP-3’s localization is dynamic throughout prophase: in mid-pachytene ZHP-3 is found all along the synaptonemal complex (SC) between synapsed homologous chromosomes. During late pachytene, it re-localizes and concentrates at the crossover site . How ZHP-3 achieves this dynamic localization to promote meiotic recombination is currently unknown.Previous research in et al. 2017), designating the short arm of chromosome pairs to promote two step cohesion loss , and activating negative feedback loops in response to defects in synapsis or crossover formation . CHK-1 (Checkpoint Kinase 1) localizes to early and mid-pachytene nuclei , correlating with when ZHP-3 is present along the SC. Similar to SC proteins that are known to be phosphorylated , ZHP-3 also contains an unstructured C-terminal tail containing multiple serine residues . Here we test whether CHK-1 is a potential regulator of ZHP-3’s localization and if phosphorylation of two residues on ZHP-3 is required for its dynamic localization and/or to promote crossover formation.Meiotic proteins are phosphorylated to promote essential steps in meiotic prophase, including stabilizing the SC  to mutate serines 308 and 366 to alanines (S308Azhp-3 and S366Azhp-3) so that they can no longer undergo phosphorylation. Along with generating the single phosphorylation mutant strains, we also created the double phosphorylation mutant  to test for redundancy between these two sites.To determine the significance of these phosphorylation events on ZHP-3’s function and meiotic recombination et al. 2008, S308A, ZHP-3S366A and ZHP-3S308A, S366A colocalized with SYP-1 on the SC . Recombination is lost in zhp-3(jf61) null mutants, resulting in 12 DAPI stained bodies corresponding to non-recombinant univalents  held together by chiasmata  to determine if there were defects in bivalent morphology. HTP-3 localizes to both the long and short arm of bivalents in a cruciform pattern, and HTP-1 localizes to the long arm on wildtype bivalents . In contrast, HTP-3 and HTP-1 are lost on the long arm in zhp-3(jf61) null mutants  null mutants have reported 1.3% viable progeny and ~30% males . We found that both the single phosphorylation mutants and double phosphorylation mutants had 100% viable progeny and 0.03%, 0.2%, and 0.1% male progeny respectively , bivalent formation , or chromosome segregation . How these proteins coordinate to promote recombination and whether they require protein modifications are interesting questions for determining how crossover formation is regulated to promote meiotic chromosome segregation.We conclude that ZHP-3 is either not regulated by these phosphorylation sites or that there is potential redundancy for ZHP-3 regulation by phosphorylation. Whether ZHP-3 is modified at other residues by phosphorylation or other post-translational modifications remain open questions in the field. It has also been recently discovered that there are additional ZHP proteins that play a role in crossover formation in C. elegans strains and genetics:C. elegans strain  was used as the wildtype control for all experiments and as the parent strain for the single phosphorylation mutant CRISPR/Cas9 genome editing experiments. The S366Azhp-3  strain was used as the background to create the S308A ,S366Azhp-3  double phosphorylation mutant.The Bristol N2 Strains were maintained at 20°C under standard conditions for all immunostaining experiments and maintained at 15°C for all CRISPR/Cas9 genome editing experiments.ZHP-3 protein expressionTwo fragments of ZHP-3, the N-terminus (aa 1-173) and the C-terminus (aa 174-387), were first amplified from cDNA, and then inserted into pDONR221 via BP reaction and shuttled into pDEST15, which contains an N-terminal GST tag, via LR reactions using the Gateway Cloning System (Invitrogen). GST-tagged ZHP-3 fragments were expressed in BL21 codon plus cells (Agilent) overnight at 18ºC (14-16 hours) to maintain solubility after induction with 0.2mM IPTG. Cells were lysed in a coffee grinder, resuspended in cold lysis buffer  with protease inhibitor (1mM PMSF), sonicated, and then spun for 1 hour at 35k to clarify lysate. A glutathione agarose column (Sigma) was equilibrated in lysis buffer, the lysate was loaded, the column washed , washed a second time in the same buffer emitting detergent (Tween-20). Protein then was eluted from the column  in 1mL fractions. The fractions containing protein  were combined and dialyzed overnight  and then aliquoted and stored at -80 ºC.In vitro Kinase Assays32P- γ-ATP (Perkin Elmer), 0.5uL of activated Chk1 (Sigma), and 2ug of substrate. Reactions were quenched by adding 20uL 2X sample buffer and boiling for 5 minutes. Reactions were run out in duplicate on two 10% SDS-PAGE gels, one was stained with Coomassie to verify equivalent amounts of substrate were utilized, and one was dried and then exposed to a phosphor screen (GE Life Sciences) for analysis of phosphorylation.Kinase assays were performed at 30ºC in 20uL reactions with 1X kinase buffer  for 30 minutes. Reactions included 0.5uL Mass Spectrometric AnalysisIn vitro assays were performed as above without radiolabeled ATP. Samples were frozen in liquid nitrogen and sent to the QB3/Chemistry Mass Spectrometry Facility at University of California, Berkeley, which performed the desalting and trypsin digest prior to mass spectrometry. Mass spectrometry produced multiple peptides of the protein sequence of the C-terminus and GST tag, indicating good coverage, and identified S308 and S366 as the in vitro phosphorylated residues.Generating phosphorylation mutant strains using CRISPR/Cas9 genome editing:S308A, zhp-3S366A,zhp-3 and S308A, S366Azhp-3 phosphorylation mutant strains were all generated using a CRISPR/Cas9 ribonucleoprotein (RNP) approach  combined with the coconversion genome editing method .The zhp-3 specific crRNA , the dpy-10 coconversion crRNA  , tracrRNA  a zhp-3 specific repair oligo  the dpy-10 coconversion repair oligo , and purified Cas9 protein (40μM stock). F1 roller and dpy progeny from injected P0’s were isolated to individual plates, and non-rolling non dpy siblings from jackpot plates (> 10 transformed worms) were grouped 3-5 F1’s per plate. After F2 progeny were generated, F1’s were screened for the respective phosphorylation mutant allele via PCR and restriction digest. F2’s from a F1 plate that had the confirmed mutant allele were singled to identify homozygotes for each phosphorylation mutation. All phosphorylation mutant strains were confirmed via sequencing. Phosphorylation mutant strains were outcrossed to N2 worms 1-2 times prior to analysis.N2 young adults were injected with a mix that included a S308Azhp-3 strain, the following sgRNA was used: 5’-GGA CAA AAC UAG UAU GUC ACG UUU UAG AGC UAU rGrCrU-3’. The following single stranded oligo was used for the S308A mutation repair template: 5’-GGA CAA AAC TAG TAT GTC ACT CGA AAA TTG GAG GCA AAA TAG AGC GAA TGC ATT CGG AGT GCA TGA TAT GTG AGA TAT TTT CAA AGT ATC TGT GAT TCC TAA TTC-3’.For creating the S366Azhp-3 strain, the following sgRNA was used: 5’– AAA AGA GUG AAU GAC AGA CCG UUU UAG AGC UAU rGrCrU –3’. The following single stranded oligo was used for the S366A mutation repair template: 5′ – GTG CAG CTG GAT TCG ACC GTC AGC AGA TAC AAG AAA TGC GTC GAA TCT CAG CTC AAC CTG GTC TGT CAC CCA CTC TTT TAT TCC TCA ATT TCC CTC GAC CCT CTC ATC –3’.For creating the S308A, S366Azhp-3 strain, the same S308A sgRNA and oligo repair template above were used to create the S308A mutation in the S366Azhp-3 mutant background.For creating the DAPI staining and Immunostaining:et al. 2005). For visualizing bivalents in mature oocytes for quantification, adult hermaphrodites were fixed and stained 48 hours post L4 stage.Adult hermaphrodites were fixed and stained 24-26 hours post L4 larval stage as in , rabbit anti-SYP-1 (1:250) , chicken anti-HTP-3 (1:500) . All secondary antibodies were used at a 1:500 dilution and included: Alexa 488 anti guinea pig (Invitrogen), Cy3 anti rabbit , and Cy5 anti chicken .For assessing ZHP-3 localization, the following primary antibodies were used: guinea pig anti-ZHP-3 (1:250)  and chicken anti-HTP-3 (1:500) . All secondary antibodies were used at a 1:500 dilution and included: Alexa 488 anti rabbit (Invitrogen) and Cy3 anti chicken .For assessing bivalent formation, the following primary antibodies were used: rabbit anti-HTP-1 (1:400)  equipped with a 100× N.A. 1.40 oil-immersion objective (Olympus), resulting in an effective XY pixel spacing of 0.064 or 0.040 μm. Z-stacks were collected at 0.2-μm Quantification of bivalents:Bivalent quantification was performed on worms 48 hours post L4 larval stage. Each genotype included at least 3 germlines for quantification.Viability assay and male counts:zhp-3(jf61),S308A, zhp-3S366A,zhp-3 and S308A, S366A zhp-3late L4 stage worms were isolated to plates and maintained at 20 degrees. F1 embryos and oocytes were scored at 24, 48 and 72 hours post L4 larval stage. Once F1’s reached the late L4 stage, viable and male progeny were scored. The total brood size, percent viability and percent HIM phenotype were calculated for each strain. A two-sided Wilcoxon-Mann-Whitney test was performed to assess statistical significance.N2, Figures and statistics:et al. 2016). For bivalent quantification, viability assay and male counts, a two-sided Wilcoxon-Mann-Whitney test was used to determine statistical significance.Figures were assembled using Adobe Illustrator. All histograms and statistics were performed using R (R core team) with the ggplot2 package (Wickham


The Mitogen-Activated Protein Kinase (MAPK) Slt2 is central to signaling through the yeast Cell Wall Integrity (CWI) pathway. MAPKs are regulated by phosphorylation at both the threonine and tyrosine of the conserved TXY motif within the activation loop (T190/Y192 in Slt2). Since phosphorylation at both sites results in the full activation of MAPKs, signaling through MAPK pathways is monitored with antibodies that detect dually phosphorylated forms. However, most of these antibodies also recognize monophosphorylated species, whose relative abundance and functionality are diverse. By using different phosphospecific antibodies and phosphate-affinity (Phos-tag) analysis on distinct Slt2 mutants, we determined that Y192- and T190-monophosphorylated species coexist with biphosphorylated Slt2, although most of the Slt2 pool remains unphosphorylated following stress. Among the monophosphorylated forms, only T190 exhibited biological activity. Upon stimulation, Slt2 is first phosphorylated at Y192, mainly by the MAPKK Mkk1, and this phosphorylation is important for the subsequent T190 phosphorylation. Similarly, dephosphorylation of Slt2 by the Dual Specificity Phosphatase (DSP) Msg5 is ordered, with dephosphorylation of T190 depending on previous Y192 dephosphorylation. Whereas Y192 phosphorylation enhances the Slt2 catalytic activity, T190 is essential for this activity. The conserved T195 residue is also critical for Slt2 functionality. Mutations that abolish the activity of Slt2 result in a high increase in inactive Y192-monophosphorylated Slt2. The coexistence of different Slt2 phosphoforms with diverse biological significance highlights the importance of the precise detection of the Slt2 phosphorylation status. Saccharomyces cerevisiae possesses five MAPK pathways, which are mediated by the ERK1/2 orthologs Fus3 and Kss1, the p38 ortholog Hog1, the Erk5 ortholog Slt2 , and the non-conventional Smk1 MAPK. These MAPK pathways are involved in the control of mating, invasive and pseudohyphal growth, the osmolarity response, the cell wall integrity (CWI), and ascospore formation, respectively [S. cerevisiae has been successfully employed as a eukaryotic model, providing very valuable information over the years about the regulation of MAPK cascades and how these pathways propagate signals and modulate cellular responses.Mitogen-Activated Protein Kinase (MAPK) pathways are evolutionarily conserved transduction modules in eukaryotes that convert a variety of disparate signals into cellular responses, allowing cells to quickly adapt to changing environmental conditions and promoting cell growth and survival. The canonical MAPK cascade consists of a core module of three protein kinases that are sequentially activated by phosphorylation, termed MAPK kinase kinase , MAPK kinase , and MAPK . Four coIt has long been established that phosphorylation plays a critical role in regulating the timing, duration, and intensity of MAPK signaling. MAPKs share a common posttranslational mechanism of kinase stimulation, characterized by dual phosphorylation of the threonine and tyrosine residues located at their conserved TXY motif within the activation loop. Initial studies showed that MAPKs are only active when both threonyl and tyrosyl residues are phosphorylated ,5. StudiS. cerevisiae Hog1 was found to be essential for its catalytic and biological activity, whereas tyrosine phosphorylation seems to be required to obtain a further increase in activity, only being necessary in extreme conditions [Interestingly, reports on several mammalian and yeast MAPKs have highlighted that threonine phosphorylation confers higher catalytical and biological activity than tyrosine phosphorylation ,13,14,15nditions . In the nditions . In contnditions . Moreovenditions .In addition to TXY, MAPK activity is also affected by post-translational modifications (PTMs) in other residues. Thereby, several phosphosites within MAPKs have been experimentally identified, mainly by phosphoproteomic approaches ,20,21,22Among the budding yeast MAPK pathways, the CWI pathway is mainly involved in sensing and responding to cell wall stress that arises during morphogenetic processes or under cell wall-threatening conditions. The core module consists of a MAPKK Kinase, Bck1, a pair of redundant MAPK Kinases, Mkk1 and Mkk2, and the MAPK Slt2. Phosphorylation of Slt2 in the TEY motif (T190-E-Y192) makes the MAPK active . Dually The aim of this study is to deepen the existing knowledge on the mechanisms regulating the phosphorylation state of the MAPK Slt2. Here, we show the existence and biological relevance of Slt2 monophosphorylated forms, and that both phosphorylation and dephosphorylation of this MAPK are highly ordered, with action on Y192 occurring first. We uncover additional residues within Slt2 that are involved in its activation and biological function.® rabbit mAb  detects endogenous levels of Erk1 and Erk2 when dually phosphorylated and singly phosphorylated at threonine. In turn, the anti-phospho-p44/42 MAPK (Erk1/2) (T202/Y204) (197G2) rabbit mAb  detects Erk1 and Erk2 when dually phosphorylated and individually phosphorylated at tyrosine. In contrast, the anti-MAPK activated (Diphosphorylated ERK-1&2) mouse mAb  reacts specifically with the dually phosphorylated form and it does not recognize the monophosphorylated species of these MAPKs. Because some monophosphorylated MAPKs have been found to display functional roles, we wanted to explore the actual specificity of these antibodies on Slt2. To this end, extracts from slt2∆ cells expressing either the dually phosphorylatable wild type Slt2, the monophosphorylatable mutants Slt2T190A and Slt2Y192F, or the unphosphorylatable double mutant Slt2T190A Y192A, treated with cell wall-altering agent Congo Red (CR) [T190A Y192A. However, #M8159 did not detect any of the monophosphorylatable versions of Slt2, #4370 did not give any signal on Slt2 T190A, and #4377 did not recognize Slt2Y192F, indicating that these reagents are only able to detect dual phosphorylation, dual and T190 monophosphorylation, and dual and Y192 monophosphorylation, respectively. The #M8159, #4370, and #4377 antibodies mentioned above are hereafter referred to in this work as anti-Slt2-pTpY, anti-Slt2-pT/pTpY, and anti-Slt2-pY/pTpY, respectively. Interestingly, a comparison of the signals provided by the distinct antibodies on each of the Slt2 versions revealed that, compared with the wild type, the Slt2T190A mutant displayed higher tyrosine monophosphorylation, whereas Slt2Y192F showed lower monophosphothreonine levels. These results indicate that the use of complementary antibodies with distinct phosphosite specificities allowed the study of the different Slt2 phosphoforms.The activation status of Slt2 has traditionally been monitored by using commercial antibodies that detect the dually phosphorylated forms of mammalian p42/44 (Erk1/Erk2), which share the same activation motif (TEY) . These aRed (CR) , were anTo deepen the characterization of dual phosphorylation dynamics at the TEY motif of Slt2, time-course monitoring of Slt2 T190 and Y192 phosphorylation following Congo Red treatment of yeast cells was performed. As observed in T190A’s interaction with its activating MAPKKs, Mkk1, and/or Mkk2. Similarly, the slight reduction in T190 phosphorylation in the Y192F version could be because of a diminished interaction with the activator kinases. To check this, we carried out copurification experiments of wild type, T190A, and Y192F versions of Slt2 with Mkk1-myc and Mkk2-myc expressed from genomic-tagged MKK1 and MKK2, respectively. As observed in T190A when compared to Slt2, indicating that the increased Y192 phosphorylation of this mutant protein was not caused by a trapping effect by either Mkk1 or Mkk2. Similarly, Slt2Y192F bound the same amount of Mkk1 and Mkk2 as Slt2T190A and Slt2, revealing that these mutations do not result in differences in binding to their activators.The increased Y192 phosphorylation in the absence of T190 could be due to an intensification of Slt2MKK1 deletion severely reduced the CR-induced Y192 phosphorylation of both wild type and T190A Slt2 versions. The lack of Mkk2 also resulted in a similar, although less intense, decrease  Msg5 ,12. Sinclt2T190A A, whereaA  resulted in a partially functional allele that poorly complemented this phenotype. To further ascertain the biological relevance of each of these phosphorylation sites, we first analyzed the ability of these mutants to complement the sensitivity of slt2∆ cells to distinct stresses that trigger activation of the CWI pathway [slt2Y192F showed partial complementation of the sensitivity of slt2∆ cells not only to 38 °C, but also to caffeine, zymolyase, sodium dodecyl sulfate (SDS), tunicamycin, and low concentrations of Calcofluor White (CFW) and CR, which indicates that T190-monophosphorylated Slt2 retains some activity in the absence of Y192 phosphorylation. In contrast, both slt2T190A and the non-phosphorylatable slt2T190A Y192A behaved like the kinase-dead version slt2K54F (slt2KD) [slt2∆ cells in most of the assayed conditions, and thus evidencing the loss of activity caused by these mutations. Curiously, transformants bearing any of these inactive alleles were able to grow slightly more than slt2∆ cells carrying the empty vector in the presence of SDS, tunicamycin, caffeine, and a low CFW concentration. These results suggest that inactive Slt2 could still play a biological role by mediating a response that is strong enough to partially cope with these stresses through a mechanism independent of the phosphorylation of the TEY motif within its activation loop.Initial work by Lee et al. in 1993  showed t pathway  in drop (slt2KD) , not allMLP1 promoter by using a reporter transcriptional assay with lacZ [Y192F showed any activity in one of the analyzed molecular readouts, as revealed by the partial mobility upshift observed for Mkk1-myc  phosphorylate and consequently promote an SDS–PAGE shift in the transcription factor Rlm1  Figure B and theith lacZ   [CD3-expressing cells displayed a wild type phenotype, except when exposed to high concentrations of CR and CFW, in which their ability to grow was slightly reduced. T190 and Y192 phosphorylation of Slt2CD3 was similar to that of wild type Slt2 . An Slt2 mutant version carrying substitutions of the conserved acidic residues D326, D323, and E327 for asparagine within the CD of Slt2 (Slt2CD3)  was analype Slt2 B. Copurih higher E. Furtheh higher B and MLPe levels D were alΔC374). As observed in ΔC374 displayed higher phosphorylation than Slt2 under stress. However, in basal conditions, the C-terminal truncated form only showed higher phosphorylation than wild type Slt2 on T190, and not on Y192 . Phos-tag is a tool based in a functional molecule, which, in the presence of Zn2+, forms complexes with phosphorylated serine, threonine, and tyrosine, inducing mobility shifts in phosphorylated proteins, depending on their phosphorylation status . When SlSDS–PAGE A.T195V Y198F. Immunodetection with anti-Slt2-pY/pTpY  the impact of individual phosphorylation events on functionality, (ii) the order of phosphorylation and dephosphorylation reactions within the TEY motif, (iii) the Slt2 phosphorylation requirements for key activators and down regulators to act on this MAPK, and (iv) the molecular and functional relevance of several key motifs of Slt2.Schizosaccharomyces pombe [Y192F is partially active, whereas Slt2T190A only retained very residual activity, similar to that of catalytically inactive Slt2KD, suggesting that a non-catalytic mechanism underlies the limited effect of the Y192-monophosphorylated form. Furthermore, we have shown that, in contrast to Slt2, the threonine monosphosphorylatable Slt2Y192F mutant was able to retrophosphorylate Mkk1, but not Mkk2, suggesting that the different phosphoforms may have distinct substrate specificities, as observed for p38α [T190A, Slt2KD, and Slt2T195V, correlate with hyperphosphorylation of the Y192. This demonstrates that an increase in the phosphorylation signal does not always correlate with an increase in the activity of the MAPK and shows the importance of discerning the different phosphorylation states of an MAPK by using complementary antibodies, and ideally, the Phos-tag methodology.Activation of the CWI pathway has long been known to correlate with an increased phosphorylation of Slt2 . Therefoes pombe . Here, wfor p38α . IntriguIn this study, we characterized, for the first time, the Slt2 phosphorylation state by using Phos-tag analysis. In other yeast MAPKs, such as Hog1 and Kss1, only dual-phosphorylated forms have been detected ,45. In tS. pombe Pmk1 [T190A mutant version by any of the MKKs, nor by a lack of Msg5 phosphatase activity on the Y192 residue. On the contrary, Msg5 preferentially dephosphorylates the phosphotyrosine residue. Together, these results suggest that dual phosphorylation and dephosphorylation is a highly ordered mechanism in which Y192 phosphorylation can be considered as a stepping-stone for full Slt2 activation/inactivation, while T190 phosphorylation is responsible for providing activity to the MAPK. The same phosphorylation and desphosphorylation order was described in the mammalian Erk1 [S. pombe [S. cerevisiae [Here, we have shown that, following stress, when phosphorylation on Y192 is constrained, T190 is rarely phosphorylated and, in clear contrast, when the phosphorylation of T190 is inhibited, Y192 phosphorylation accumulates, leading to the “clogging” of Y192-monophosphorylated Slt2. Similar behavior has also been described for mbe Pmk1 . Our resian Erk1 ,47,48. Aian Erk1 , a laterS. pombe , and forCD3 mutant protein is still able to signal to Rlm1, suggesting that alternative sites also exist in Slt2 for Rlm1 D domain interaction. Whereas the lack of a functional CD domain reduced Slt2 activity, removal of the C-terminal part of Slt2 rendered it hyperactive. A similar truncated Slt2 version was previously observed to be hyperphosphorylated by using an anti-phospho-ERK antibody that detects Tyr 204 phosphorylation within the TEY motif [ΔC374 also displays hyperphosphorylation at the T190 residue, supporting the idea that the Slt2 tail negatively regulates autophosphorylation on T190 and, ultimately, the activity of the kinase.Our results also allowed us to gain an insight into the impact of residues and domains of Slt2 other than TEY on its activation by phosphorylation and/or activity, reflecting the complexity of its regulation. Although it has been described that the interaction of the Erk CD domain is crucial for binding to D domains of MKPs , in prevEY motif . These aT195V is highly phosphorylated on Y192, as mentioned above for non-active Slt2 proteins, such as Slt2T190A or Slt2KD. This phenomenon could be due to a compensatory increase in the activity of upstream elements along the route, caused by cell wall defects provoked by the absence of a functional CWI pathway. Additionally, inactive Slt2 mutants would be unable to trigger negative feedback loops, such as retrophosphorylation of Mkk1 and Mkk2 [In most MAPKs, modifications other than TEY phosphorylation at the activation loop are essential for regulating kinase activity. T195 and Y198 of Slt2 are equivalent positions to phosphosites T207 and Y210 in mammalian Erk1, which are two highly conserved amino acids found not only in MAPKs, but also in most human protein kinases . Howeverand Mkk2 .MAPKs from yeast to humans have the TXY motif in common, whose dual phosphorylation is required for them to be fully active. Nevertheless, results such as those presented in this study show that monophosphorylated forms can play different biological roles and that conserved residues other than those in the TEY motif influence the activity, even having opposite effects among MAPKs. This underscores the importance of deepening the study of each MAPK in order to provide a broad view of the complexity of this family of kinases, which, although highly conserved, also shows a great variety of regulatory mechanisms.Escherichia coli strain DH5α as the host. All employed oligonucleotides are listed in Plasmids used in this study  were genslt2T190A and pRS316-slt2Y192F, the 1.3 kb HindIII–HindIII fragments from YCp50-slt2T190A, and YCp50- slt2Y192F plasmids were cloned into the 5.8 kb HindIII–HindIII fragment of pRS316-SLT2. The pRS315-SLT2, pRS315-slt2T190A, and pRS315-slt2Y192F plasmids were constructed by inserting the 2.8 kb SacI-BamHI fragment from pRS316-SLT2, pRS316-slt2T190A, or pRS316-slt2Y192F into the pRS315 vector cleaved with the same endonucleases.To obtain pRS316-SLT2-5 and SLT2-3 primers, in combination with mutagenic primers SLT2-MNP5/SLT2-MNP3, SLT2-MSTOP5/SLT2-MSTOP3, and SLT2-CD5/SLT2-CD3, was carried out to construct pRS316-slt2T190A Y192A, pRS316-slt2ΔC374, and pRS316-slt2CD3, respectively, by using pRS316-SLT2 as the template. The HindIII–HindIII digested PCR products were inserted into the 5.8 kb HindIII–HindIII fragment from pRS316-SLT2. The kinase-dead version of SLT2 (pRS316-slt2KD) was obtained by subcloning the EcoRI–SalI 2.2-kb fragment bearing slt2-K54F from pHR3 into EcoRI–SalI sites of pRS316.An overlapping PCR with slt2T195V, pRS316-slt2Y198F, and pRS316-slt2T195V Y198F were obtained by site-directed mutagenesis with primers SLT2-T195V5/SLT2-T195V3, SLT2-Y198F5/SLT2-Y198F3, and SLT2-T195VY198F5/SLT2-T195VY198F3, respectively, using the plasmid pRS316-SLT2 as the template, and followed by DpnI digestion.pRS316-GFP-SLT2 was constructed via a two-step process. First, the fragment ClaI-6xHis-MluI was introduced into the N-terminal region of SLT2 by an overlapping PCR with primers SLT2-PreSalI.5, SLT2-PostEcoRI.3, SLT2-ClaI6×HisMluI.5, and SLT2-ClaI6×HisMluI.3, using pRS316-SLT2 as the template to generate pRS316-6×His-SLT2. Second, GFP was amplified from the pLA10 plasmid with primers GFP-5 and GFP-3, which include ClaI and MluI restriction sites, respectively. The PCR product was then digested and subcloned into ClaI–MluI sites of pRS316-6xHis-SLT2, producing pRS316-GFP-SLT2. Plasmids pRS316-GFP-slt2T195V and pRS316-GFP-slt2T195V Y198F were obtained with the same strategy and primers as pRS316-slt2T195V and pRS316-slt2T195V Y198F, respectively, using pRS316-GFP-SLT2 as the template.The plasmid pRS316-MKK1-6MYC and MKK2-6MYC  into the MKK1 and MKK2 loci of BY4741 slt2Δ (Y00993 strain). The strains YGGR32, YGGR33, and YGGR34 were constructed by amplification of the slt2::NatMx4 cassette from the YSTH4 strain with the primers SLT2-pre121.5 and SLT2-post165.3, and subsequent integration into the BY4741 isogenic kanMx4 deletion mutants mkk1Δ, mkk2Δ, and msg5Δ .All yeast strains used in this study are described in 600) of 0.3 in YPD to achieve the mid-log phase. Cell cultures were then treated with 30 µg/mL of Congo Red  and incubated for an additional 2 h. After that time, cells were collected and frozen immediately in liquid nitrogen for subsequent processing.Yeast cultures were grown in yeast extract–bacto-peptone–dextrose (YPD) medium or in selective synthetic dextrose media (SD) containing 2% glucose and the appropriate amino acids for plasmid selection. All strains were cultured at 30 °C unless stated otherwise. Experiments for analyzing Slt2 phosphorylation under stress were carried out by incubating cells overnight in synthetic dextrose (SD) media and refreshing them to an optical density , and 0.05% ammonium peroxysulfate (PSA). The resolving gel was poured into the cassette, overlayed with isopropanol, and polymerized at room temperature for 1 h. Before adding the stacking gel solution to the cassette, the resolving layer was rinsed five times with 350 mM Bis–Tris (pH 6.8). The stacking gel solution  was polymerized at room temperature for 1 h. Samples to be analyzed by Zn2+–Phos-tag SDS–PAGE were run at a constant current of 20 mA per gel for 150 min. Prior to transfer, the gel was immersed in transfer buffer containing 5 mM ethylenediaminetetraacetic acid (EDTA) . Next, the gel was immersed in blotting buffer without EDTA for 10 min. Proteins were then transferred to the membrane using the tank method in buffer containing 0.1% SDS overnight (18 h), at 20 V and 4 °C. After protein transfer, the membrane was washed in deionized water .Yeast extracts were obtained by trichloroacetic acid precipitation as previously described . Once soribed in . We opti2+–Phos-tag SDS–PAGE gels were cast, run, and transferred using the 1.0-mm Bio-Rad Mini-PROTEAN gel system and a 0.45 µm nitrocellulose blotting membrane . The resulting membranes were blocked by incubation in 5% non-fat dry milk diluted in phosphate-buffered saline (PBS) Tween 0.1% for 1 h at room temperature.Both SDS–PAGE and ZnMembranes were probed with an appropriate primary antibody, all of which are listed in After washing samples with PBS Tween 0.1% , membrane-bound immune complexes were detected by incubation with a 1:5000 dilution of an appropriate infrared dye-labeled secondary antibody—800 CW Goat anti-Rabbit IgG (926-32211), 680LT Goat anti-Rabbit IgG (926-68021), 800 CW Goat anti-Mouse IgG (926-32210), or 680LT Goat anti-Mouse IgG (926-68020) . After washing as described above, immunoblots were analyzed with an infrared imaging system .600 of 0.5 in water. Tenfold serial dilutions of the cell suspensions were spotted onto agar plates containing YPD medium. When indicated, YPD plates were supplemented with a corresponding concentration of any of the following compounds: Congo Red (Merck); caffeine (Sigma-Aldrich); SDS (PanReac Applichem ITW Reagents); tunicamycin (Sigma-Aldrich); Calcofluor White (Sigma-Aldrich); or zymolyase 100T . The plates were imaged after incubation at 30 °C  for 72 h.To perform the growth assays, cultures were grown to saturation overnight in SD media, and adjusted to an ODTM Protein G (Thermo Fisher Scientific) coupled to the Mpk1-E9 antibody overnight at 4 °C. Beads were extensively washed with washing buffer (0.05% Tween 20 and 0.5 M NaCl in PBS) and protein complexes were eluted with elution buffer (50 mM glycine pH 2.8). Then, 5x SDS loading buffer was added; samples were boiled for 5 min, analyzed by SDS–PAGE, and immunoblotted as indicated above (4.3).For protein extraction, cells were resuspended in cold lysis buffer (50 mM Tris/HCl (pH 7.5), 5 mM EDTA (pH 8), 150 mM NaCl, 50 mM NaF, 5 mM sodium pyrophosphate, 10% glycerol, 0.1% NP-40, 50 mM β-glycerol phosphate, and 1 mM sodium orthovanadate) supplemented with 1 mM phenylmethylsulfonyl fluoride (PMSF) and protease inhibitor mixture  and lysed using 0.75–1 mm diameter glass beads. The extracts were then adjusted to the same protein concentration and incubated with Dynabeadsβ-galactosidase activity was determined as described previously . BrieflyFor GFP in vivo fluorescence microscopy, cells were collected by centrifugation at 2500 rpm and prepared for visualization. The microscope used was an Eclipse TE2000U  with an appropriate set of filters. Digital images were acquired with an Orca C4742-95-12ER charge-coupled device camera  and processed with the HCImage software (Hamamatsu).