The mitogen-activated protein kinase ERK1/2  plays important roles in a wide spectrum of cellular processes and have been implicated in many disease states. The spatiotemporal regulation of ERK activity has been extensively studied. However, scarce information has been available regarding the quality control of the kinases to scavenge malfunctioning ERKs. Using site-specific mutagenesis and mass spectrometry, we found that the disruption of the conserved H-bond between Y210 and E237 of ERK1 through point mutation at or naturally occurring nitration on Y210 initiates a quality control program dependent on chaperon systems and CHIP -mediated ubiquitination and degradation. The H-bond is also important for the quality control of ERK2, but through a distinct mechanism. These findings clearly demonstrate how malfunctioning ERKs are eliminated when cells are in certain stress conditions or unhealthy states, and could represent a general mechanism for scavenging malfunctioning kinases in stress conditions. The mitogen-activate protein kinase ERKs (extracellular-regulated protein kinases) play a central role in various cellular processes, including proliferation, migration, apoptosis, and stress response . More thIn resting cells, ERKs mainly localize in the cytoplasm and anchored by MEKs (mitogen-activated protein kinase kinase) , and douPhosphorylation plays a critical role in regulating ERK activity and localization. It has long been established that TEY motif phosphorylation is important for ERK nuclear translocation . The recβγ-induced autophosphorylation on T188 promotes its nuclear localization to phosphorylate nuclear targets known to cause cardiac hypertrophy [in vitro kinase assay [202TEY204 activation motif, Y210F mutation was shown to repress ERK1 activity [In addition to the TEY and the SPS motifs, ERK activation and subcellular localization could also be affected by post-translational modifications (PTMs) on the other residues. Site-specific mutations of potentially phosphorylatable residues close to the TEY motif revealed that ERK activities were significantly affected in these point mutants, probably due to the abolished ability in accepting modification moieties . For ERKertrophy . For ERKertrophy , and a rertrophy . Both T1activity , yet itsactivity ,13. ThusUsing site-specific mutagenesis, we mutated many potentially phosphorylatable Ser, Thr, and Tyr residues proximal to the TEY motif to their unphosphorylatable counterparts and phosphomimetic residues. We found that Y210F mutation drastically inhibited ERK1 phosphorylation and nuclear translocation. We also found that ERK1 Y210 can be tyrosine nitrated and uncovered a novel tyrosine nitration-induced CHIP-dependent ERK1 degradation pathway, which could serve as an important quality control mechanism for the important kinase when it becomes malfunctioning.−), Leptomycin B (LMB), and MG132 were from CalBiochem . Cycloheximide (CHX) was obtained from Sigma . Phosphor-ERK (T202/Y204) antibody, anti-HSP90, and anti-GAPDH were obtained from Cell Signaling Technology . Anti-ubiquitin and anti-NitroTyr antibodies were from Millipore . Anti-ERK1 antibody was purchased from Santa Cruz Biotechnology . Anti-GFP antibody was from Abcam . Secondary antibodies including a horseradish peroxidase (HRP)-labeled goat anti-rabbit antibody and a HRP-labeled goat anti-mouse antibody were purchased from CW Biotech . Protein A Sepharose was from GE Healthcare Bioscience (Sweden). DAPI and ProLong Gold antifade reagent were from Invitrogen .Peroxynitrite  and Lipofectamine 2000  were used for transfecting plasmids and siRNA, respectively.HEK293T cells were cultured in high-glucose Dulbecco's modified Eagle's medium  supplemented with 10% fetal bovine serum (FBS), 1 mM sodium pyruvate, 100 units/ml penicillin and 100 µg/ml streptomycin  at 37°C with 5% COAll site-directed mutagenesis was performed using the QuikChange II XL site-directed mutagenesis system . The mutations were then confirmed by DNA sequencing. In total, 19 site-specific mutants were generated for the current study (Supplementary Table S1).Transfected 293T cells were plated on coverslips with ∼50–60% confluence and allowed to attach and spread overnight in the starvation medium. The cells were then either treated with 10% FBS for 10 min to activate ERKs or untreated. After the treatment, the cells were washed with ice-cold PBS, fixed with 4% paraformaldehyde for 15 min, permeabilized with 0.1% Triton X-100 for 10 min, and then blocked with 10% BSA in PBS containing DAPI in a closed chamber at 37°C for 1 h. The coverslips were subsequently washed, mounted on glass slides using ProLong Gold antifade reagent, and visualized by Leica fluorescence microscopy .− to the reaction system, and the reaction was allowed to proceed at 37°C for 30 min [4HCO3 (pH 7.4) for mass spectrometry (MS) analysis.Ectopically expressed GST-ERK1 was pulled down from the WCL of transfected HEK293T cells by GST.Bind Resin  at 4°C for 2 h. The beads bound with GST-ERK1 were then incubated with the freshly collected HEK293T cell lysate. Nitration of GST-ERK1 was stimulated by adding 500 µM ONOOr 30 min . After tThe three highly conserved and potentially phosphorylatable residues T198, T207, and Y210 were chosen for site-specific mutagenesis , which replaces the targeted residues with structurally similar but unphosphorylatable ones. Thus, we obtained T198A, T207A, and Y210F mutants in which Ser or Thr residues were replaced with Ala and Tyr was replaced with Phe [To answer whether Y210F mutation affects ERK1 nuclear translocation, 293T cells and HeLa cells expressing N-terminal GFP fusion of ERK1 or Y210F were examined for nuclear localization. While ERK1 was still in nucleus in response to 10% FBS stimulation as expected, Y210F was almost exclusively retained in the cytoplasm in both serum-starved and FBS-stimulated cells  and induActivated ERK1 nuclear translocation requires double phosphorylation of the two Ser residues on the consensus SPS motif ,7. Y210Fper se but the unique structure of the bulky side chain of the Y210 residue that is critical for the optimal ERK1 activation and localization.To investigate if the observed phenotype of Y210F is owing to the loss of phosphorylation potential of the Y210 residue, we generated three other site-specific mutants Y210E, Y210S, and Y210T. Y210E is a possible phosphomimetic mutation, and Y210S and Y210T could retain the phosphorylation potential at this particular residue. Again, all mutations significantly inhibited ERK1 TEY motif phosphorylation and completely abolished ERK1 nuclear translocation in both quiescent and serum-stimulated cells . Apparently, it is not the lost potential of phosphorylation To answer why the Y210 residue of ERK1 is so critical for maintaining optimal ERK1 activity and nuclear localization, the resolved structure of ERK1 was downloaded from Protein Data Bank (PDB) and analyzed with the software Wincoot. We found that there is an H-bond between the phenolic hydroxyl group of Y210 and the carboxyl group on the side chain of E237 . Structu− treatment  from the whole cell lysates (WCL), and the co-IPed proteins were separated by SDS–PAGE and visualized with silver staining. We found a protein, which was subsequently identified as the heat shock protein 90 (Hsp90) by MS , specifiBinding of Hsp90 could have a causal effect on the abolished Y210F nuclear localization due to size increase in forming the complex. Indeed, in cells partially depleted of Hsp90 by siRNA, Y210F regained the ability for nuclear localization . Thus, HBinding of Hsp90 strongly suggests that Y210F mutant is abnormally folded with two possible and opposing consequences, i.e. refolding and degradation, and both could be mediated by Hsp90 as observed for other client proteins –22. BecaTo find proteins that potentially facilitate Y210F degradation, total proteins co-IPed with ERK1 and Y210F were identified by LC–MS. The quantitative analysis based on the normalized peptide spectral counts revealed that substantially more amount of Hsp90, Hsp70, Hsc70, Cdc37, and CHIP were identified to interact with Y210 than with ERK1 . Hsp90 aTo further answer whether CHIP is also the major E3 ubiquitin ligase for ERK1, 293T cells depleted of CHIP or MEKK1 were treated with CHX to inhibit protein synthesis and examined for the stability of GFP-ERK1. In contrast with GFP-Y210F, GFP-ERK1 degradation can be significantly inhibited by depletion of either E3 ligase, though the extent of inhibition is slightly greater in the CHIP-depleted cells . In addiAfter establishing CHIP as the major E3 ubiquitin ligase for the degradation of a subpopulation of abnormally folded ERK1 represented by Y210F, it is interesting to know which sites on ERK1 are ubiquitinated and important for ERK1 degradation. Using MS, many ubiquitination sites were identified from Y210F including K220, K287, K294, and K317 . IntriguTo investigate if the two ubiquitination sites are also important for the degradation of ERK1, site-specific mutants ERK1-K294R, ERK1-K317R, and ERK1-K294R-K317R were examined for the ubiquitination level. The result shows that the double mutations, but not the single mutation, significantly reduce ubiquitination of ERK1 . NeverthTo investigate if ERK2, the isoform of ERK1, follows the same mechanism to degrade as ERK1 does, site-specific mutations were also generated on ERK2 Y193 and E220, the two residues homologous to ERK1 Y210 and E237, respectively, and forming an H-bond as well. The N-terminal GFP fusions of ERK2 and Y193F were expressed in HEK293 cells. Indeed, the disruption of the H-bond by the Y193F mutation significantly represses ERK2 TEY phosphorylation and promotes ERK2 ubiquitination and degradation , confirmSpatiotemporal regulation of ERK activity has been extensively studied. However, little information regarding the quality control of ERKs has been available. Here, we described a nitration-induced and CHIP-dependent quality control mechanism for ERK1, as illustrated by the diagramed model . We showin vitro or under physiological conditions, and the nitration facilitates the breakage of the H-bond between Y210 and E237 as we recently showed using molecular dynamics simulation [The results from the site-specific mutations firmly establish that correct formation of the H-bond between Y210 and E237 is critical for optimal ERK1 activity and localization. Thus, disruption and formation of the H-bond, which is decided by the modification status of Y210, could act as a molecular switch for the quality control of ERK1. Here, we show that Y210 can be nitrated mulation . Proteinmulation . In addimulation –33. In hmulation  and coulmulation ,13, depemulation . Howevermulation ,36.In addition to the nitration identified in the current study, phosphorylation is the other potential PTM reported to occur on ERK1 Y210 . Phosphobona fide client of Hsp90 [Binding of Hsp90 is a strong indicator of abnormal folding of Y210F. Hsp90 has been shown to indirectly regulate ERK activity positively or negatively in different scenarios ,38. Thouof Hsp90 . Here, wPrevious evidence has established that CHIP is important in clearance of Hsp70/Hsp90 substrates such as the glucocorticoid receptor CFTR and the ErbB2 receptor ,40. ConsThe current study clearly demonstrates the importance of the H-bond between Y210 and E237 in regulating ERK1 conformation and activity. However, for ERK2, the H-bond is less important . ERK1 an


Aldose reductase (AR) is an enzyme devoted to cell detoxification and at the same time is strongly involved in the aetiology of secondary diabetic complications and the amplification of inflammatory phenomena. AR is subjected to intense inhibition studies and dimethyl sulfoxide (DMSO) is often present in the assay mixture to keep the inhibitors in solution. DMSO was revealed to act as a weak but well detectable AR differential inhibitor, acting as a competitive inhibitor of the L-idose reduction, as a mixed type of non-competitive inhibitor of HNE reduction and being inactive towards 3-glutathionyl-4-hydroxynonanal transformation. A kinetic model of DMSO action with respect to differently acting inhibitors was analysed. Three AR inhibitors, namely the flavonoids neohesperidin dihydrochalcone, rutin and phloretin, were used to evaluate the effects of DMSO on the inhibition studies on the reduction of L-idose and HNE. In addition, as it can be mixed with water as well as with a number of organic solvents, it has been used as a vehicle for the delivery of various molecules in cultured cells and in ,,Aldose reductase (AR), since its involvement in the onset of diabetic complications, has been the subject of intense study aimed at finding valuable inhibitors to control its activityThis study on ARI shows evidence of a differential inhibitory action exerted by DMSO on the AR activity and examines its influence on the kinetic characterisation of AR inhibitors.Bovine serum albumin (BSA), D,L-dithiothreitol (DTT), D,L-glyceraldehyde (GAL), DMSO, EDTA, were purchased from Sigma-Aldrich . NADPH and L-idose were supplied by Carbosynth ; YM10 ultrafiltration membranes were obtained from Merck-Millipore ; neohesperidin dihydrochalcone (NHDC), rutin and phloretin were obtained from Extrasynthese . All other chemicals were of reagent grade.ε340 = 6.22 mM−1·cm−1) through a Biochrom Libra S60 spectrophotometer . The standard assay mixture contained a 0.25 M sodium phosphate buffer pH 6.8, 0.18 mM NADPH, 0.4 M ammonium sulphate, 0.5 mM EDTA and 4.7 mM GAL. One unit of enzyme activity is the amount that catalyses the conversion of 1 µmol of substrate/min in the above assay conditions. These assay conditions were also adopted to assess the effectiveness of inhibitors when L-idose, trans-4-hydroxy-2,3-nonenal (HNE), or 3-glutathionyl-4-hydroxynonenal (GSHNE) was used as a substrate instead of GAL.The AR activity was determined at 37 °C as previously describedhAR) was expressed and purified as previously describedhAR was 5.3 U/mg. The purified enzyme was stored at −80 °C in a 10 mM sodium phosphate buffer pH 7.0 containing 2 mM DTT and 30% (w/v) glycerol. Before use, the enzyme was extensively dialysed against a 10 mM sodium phosphate buffer pH 7.0.The human recombinant AR .,,i (dissociation constant of the EI complex) of 235 ± 17 mM was determined, while for HNE a Ki of 266 ± 7 mM and a Ki′ (dissociation constant of the ESI complex) of 378 ± 24 mM were measured. Although the overall inhibitory power on the two different reactions appears to be essentially the same, DMSO basically behaves as a differential inhibitor, since it discriminates between the two different substrates undergoing reduction. The effect of DMSO was also tested on GSHNE, an HNE derivative recognised as a substrate by AR, whose reduction product is known to elicit the inflammation response through the activation of the NF-kB cascadeThe ability of DMSO to affect AR activity was evaluated using L-idose and HNE, two of the substrates routinely used to define the inhibitory features of ARDIsIn order to evaluate the possible influence of DMSO in identifying ARDIs, the possibility that an ARI acts differently on the reduction of different substrates was also considered. Thus, three different ARIs, namely the flavonoids neohesperidin dihydrochalcone (NHDC), rutin and phloretin, were used to evaluate the effect of DMSO in the assay mixture when the inhibition features of these molecules were evaluated in the reduction of either L-idose or HNE. This experimental approach was possible due to the solubility of the above inhibitors in 0.7% (v/v) methanol (approximately 0.17 M). At this concentration, the methanol in the enzyme assay mixture did not affect the AR activity (an inhibition less than 5% was observed) in the range of substrate concentrations of 0.4–4 mM and 40–110 µM for L-idose and HNE, respectively.i and Ki′ of the binary (enzyme:inhibitor) and the ternary (enzyme:substrate:inhibitor) complexes, respectively, for NHDC, used as an inhibitor of the reduction of both L-idose and HNE. The same analysis was performed with phloretin and rutin (data not shown). i and Ki′ values of the three inhibitors measured for the reduction of both L-idose and HNE. While phloretin showed essentially the same inhibitory activity towards both substrates, rutin and NHDC exerted a modest, differential inhibitory action on L-idose reduction with respect to HNE reduction. In fact, both rutin and NHDC behave as mixed inhibitors of AR in the presence of L-idose, and as uncompetitive inhibitors in the presence of HNE. While for rutin, the ability to interact with the AR:L-idose complex prevailed, NHDC appeared to preferentially bind the free enzyme.i and Ki′ are again reported in i values, while Ki′ values remained essentially unchanged for all the three inhibitors. However in terms of HNE reduction, DMSO caused an increase in Ki′ values  for rutin and NDHC, and a similar increase in both kinetic inhibition constants for phloretin.To evaluate the effect of DMSO on the inhibitory action of the three flavonoids, the same analytical approach was performed for the three inhibitors acting on both L-idose and HNE reduction, with DMSO in different concentrations in the assay mixture. The values of the apparent KIn order to explain the effects of the observed interference of DMSO on the kinetic characterisation of differently acting ARIs, a kinetic analysis of different inhibition models was considered. Thus the effects were analysed of a competitive inhibition or a mixed type of inhibition  on a competitive, uncompetitive, and mixed type of inhibition of the molecules under investigation.The analysis was performed according to a previously reported double inhibition approachThus, the general equation for the initial reaction rate as a function of the substrate concentration:MK stands for the Michaelis constant. This equation, after simple algebra, can be represented in the usual form of a rectangular hyperbola.and the mass balance for the enzymeThis kinetic equation can be then simplified for different combinations of inhibition models referring both to DMSO and to the tested inhibitors (see Appendix). On the basis of Through this approach, using different DMSO concentrations , it was possible to estimate the theoretical overall effect of DMSO on the apparent efficiency and on the inhibitors model of action , and comThese results highlight the need to keep the solvent rigorously constant in the enzyme assay mixture, and more importantly, suggest that the contribution of the DMSO used as solvent in AR inhibition studies may be not negligible in determining both the binding efficiency and the targeting model of the inhibitor. Regarding the importance of the DMSO effect as a solvent in inhibition studies, it is worth considering that when the effectiveness of the inhibitor under investigation is high, the low concentration needed to define the inhibition parameters may minimise the interference of the solvent. This is both due to mechanistic reasons, linked to the comparison of the kinetic parameters of the solvent and the inhibitor, and because the reduced level of the solvent needed to maintain the inhibitor in solution at low concentrations. However, when a significant level of the solvent is required to keep the molecule in solution, especially when the strength of the inhibitor is not extraordinarily high, the effect of the solvent cannot be disregarded. This is possibly the case in the search and characterisation of AR differential inhibitorsMore concerns may arise when considering the possible participation of the solvent in the targeting event of the enzyme by the inhibitor. In fact, the features of the inhibitor may be strongly affected by the kinetics and thermodynamic restrictions of the desolvation step of the inhibitor from DMSO to the enzyme interactive site. This kind of unavoidable problem, which generally occurs in poorly water-soluble ligand studies, has no easy solution. Only a comparative study of the specific inhibitor in different solvents or solvent cocktails might provide an insight into the role of DMSO in the effectiveness and inhibition mode of an inhibitory molecule.


Specificity within protein kinase signaling cascades is determined by direct and indirect interactions between kinases and their substrates. While the impact of localization and recruitment on kinase–substrate targeting can be readily assessed, evaluating the relative importance of direct phosphorylation site interactions remains challenging. In this study, we examine the STE20 family of protein serine–threonine kinases to investigate basic mechanisms of substrate targeting. We used peptide arrays to define the phosphorylation site specificity for the majority of STE20 kinases and categorized them into four distinct groups. Using structure-guided mutagenesis, we identified key specificity-determining residues within the kinase catalytic cleft, including an unappreciated role for the kinase β3–αC loop region in controlling specificity. Exchanging key residues between the STE20 kinases p21-activated kinase 4 (PAK4) and Mammalian sterile 20 kinase 4 (MST4) largely interconverted their phosphorylation site preferences. In cells, a reprogrammed PAK4 mutant, engineered to recognize MST substrates, failed to phosphorylate PAK4 substrates or to mediate remodeling of the actin cytoskeleton. In contrast, this mutant could rescue signaling through the Hippo pathway in cells lacking multiple MST kinases. These observations formally demonstrate the importance of catalytic site specificity for directing protein kinase signal transduction pathways. Our findings further suggest that phosphorylation site specificity is both necessary and sufficient to mediate distinct signaling outputs of STE20 kinases and imply broad applicability to other kinase signaling systems. Protein kinases, which catalyze the transfer of phosphate from ATP to substrate proteins, are important enzymes in cellular signal transduction pathways mediating responses to extracellular cues. In order to function properly in signal transmission, each kinase must phosphorylate only a limited number of proteins among the thousands present within the cell. One way that kinases choose their substrates is by recognition of amino-acid sequence motifs surrounding the site of phosphorylation, but kinases can also recruit substrates through docking interactions occurring outside of the catalytic cleft. For most kinases, the relative contribution of catalytic site and docking site interactions to substrate selection is not known. Here, we investigated the phosphorylation site specificity of a group of 30 human protein serine–threonine kinases called the STE20 family. Guided by x-ray crystal structures of kinase-peptide complexes, we re-engineered the catalytic clefts of two members of this group, PAK4 and MST4, to exchange their phosphorylation site motifs. In cells, the re-engineered form of PAK4 was unable to phosphorylate substrates of wild-type PAK4 or to carry out its normal function in reorganizing the actin cytoskeleton. In contrast, it was able to function in place of MST4 and related kinases in the growth-controlling Hippo signaling pathway. Overall, these studies suggest that catalytic site interactions can be both necessary and sufficient for kinase function. Protein kinases are key enzymes in signal transduction networks critical to essentially all aspects of cellular regulation. The human genome encodes over 500 protein kinases that function in distinct processes yet share a structurally conserved catalytic domain . The uniOne prominent example of a closely related, yet functionally distinct, group of kinases is the STE20 family. The 30 serine–threonine kinases in this group have been classified into 10 subfamilies based on similarity within the catalytic domain and overall domain architecture  8]. Per. Per8]. Prior studies have largely emphasized the contribution of subcellular localization and noncatalytic site interactions to selective substrate targeting by STE20 kinases. For example, PAKs are localized and activated through interactions with small GTPases, producing an active pool of the kinase in a spatially restricted manner that likely limits its substrate repertoire. Multiple STE20 kinases, including some PAKs and GCKs, harbor proline-rich motifs that bind to Src homology 3 (SH3) domains present in substrates, activators, and/or adaptor proteins –22. At lDespite the established importance of noncatalytic site interactions, differences in phosphorylation site sequence motifs among STE20 kinases may also be functionally important. For example, the established catalytic site motif of PAKs corresponds well to sequences found at known sites of phosphorylation in protein substrates , albeit In this study, we probe the catalytic site specificity of the STE20 family and discover previously unknown features within the kinase domain that help determine selective substrate targeting. Exploiting these features, we engineer kinase mutants that fully exchange phosphorylation site specificity between subfamilies. These reprogrammed kinases provide an unprecedented opportunity to dissect the unique contribution of catalytic site interactions to the signaling output of a protein kinase. We show that phosphorylation site specificity has a predominant role in mediating substrate targeting in STE20-kinase–mediated signaling networks.To better understand how STE20 kinases mediate selective substrate targeting, we performed positional scanning peptide array (PSPA) screens across the entire family. We used a previously reported set of 198 peptide mixtures in which nine positions flanking a central phosphorylation site are systematically substituted with each of the 20 unmodified amino-acid residues, as well as phosphothreonine (pThr) and phosphotyrosine (pTyr) , 39. In Using the semiautomated PSPA protocol, we profiled most of the STE20 kinases not previously analyzed. Together with previously published results, we have collected PSPA data in total for 20 of the 28 active STE20 kinases  displayed increased activity on peptides with a −2 Arg residue. We note that mutation of the −2 interaction pocket of both kinases was accompanied by a decrease in overall catalytic rate. This effect was particularly pronounced for PAK4, resulting in an approximately 1,000-fold decrease in the rate of phosphorylation of the parental PAKtide. While PAK4S443A/D444A did phosphorylate the PAKtide −2Y variant faster than did WT PAK4, a large component of the change in selectivity appears to be conferred by loss of activity on the parental peptide. Overall, these assays confirm that the KxxN residues largely confer specificity at the −2 position in STE20 kinases.Kinases target specific phosphorylation site sequences through complementary interactions within the catalytic cleft. While some insight into the structural basis for kinase–substrate recognition has been obtained from crystallographic studies and site-directed mutagenesis, only a few bona fide specificity-determining residues have been experimentally validated –48. To ianalysis . Furtherrthologs . To asseposition . Similar58–66→RKQQRREL) led to loss of selectivity for basic residues at these positions in the PSPA and caused a 70-fold decrease in the rate of phosphorylation of MSTtide. Furthermore, the activity of this mutant was not impaired by neutralizing the +2 and +3 basic residues of MSTtide  that conforms well to our defined PAK4 consensus motif, including basic residues at the −3 and −2 positions. To assess the importance of PAK4 catalytic site interactions, we mutated this phosphorylation site to a sequence preferred by GCKi–v kinases (KGYNTVRRK). Similar to our analysis of peptide substrates, in steady-state kinetic assays, we found that this mutation reduced β-catenin phosphorylation by PAK4 to background levels . PSPA analysis showed that this mutant had significantly reduced basic preferences at multiple positions and preferred aromatic hydrophobic residues at the +2 position  family select basic residues downstream of the phosphorylation site, primarily at the +2 position. Though PKC isozymes have low sequence similarity to STE20 kinases, their β3–αC loops are also rich in acidic residues. To determine whether this region might also confer selectivity for basic residues to PKCs, we examined a PKCβ mutant in which three acidic residues in the loop were mutated to Ala   ATP. The reaction plate was sealed, centrifuged briefly, and incubated at 30°C for 2 h. Reactions were spotted onto a streptavidin membrane , which was washed, dried, and imaged as described previously  ATP . For steady-state reactions, the PAK4 concentration was 10 nM, and β-catenin concentration was 5 μM. Samples were taken at 5, 10, and 15 min. For single turnover reactions, PAK4 concentration was 500 nM, β-catenin substrates were 1.5 μM, and samples were taken at 5, 10, 15, 20, 30, 60, 90, 120, 150, 240, and 300 seconds. For both steady-state and pre-steady–state experiments, aliquots taken at each time point were quenched with SDS-PAGE loading buffer, boiled, and fractionated by SDS-PAGE. Gels were stained with Coomassie brilliant blue, dried, and exposed overnight to a phosphor storage screen . Screens were scanned using a phosphorimager (Bio-Rad), and band intensities were quantified using Quantity One software (Bio-Rad).Full-length PAK4 and full-length β-catenin were assayed in PAK4 buffer and initiated by addition of [γ-Panc1 pancreatic cancer cells harboring inducible shRNAs were generated by lentiviral transduction as follows. Lentiviral particles containing inducible shRNA were produced in low-passage HEK293T cells by PEI co-transfection of dR8.91, VsV-G, and PAK4-targeting pTRIPZ #395103  or luciferase-targeting control vector in a 10:1:10 ratio. Supernatants were collected at 24 and 36 hours post-transfection. Panc1 cells were infected with lentivirus at an MOI of approximately 1 in the presence of 8 μg/mL polybrene. After 24 hours, infected cells were selected with 2 μg/mL puromycin. After 64 hours selection, cells were maintained in 1 μg/mL puromycin, and knockdown was induced with 500 ng/mL doxycycline at least 7 days prior to the experiments below.To assess phosphorylation of PAK4 substrates, Panc1 cells expressing either shPAK4 or shLuc or HEK293A cells were transfected with the indicated PAK4 expression plasmids either alone or in combination with a 3-fold excess of the indicated substrate expression vector (unless otherwise indicated) using Lipofectamine 2000 as recommended by the manufacturer. Media were exchanged 6 hours later, and cells were incubated for an additional 20 hours (HEK293A cells) or 40 hours (Panc1 cells). For Panc1 cells, media was then aspirated, and cells were washed once with serum-free media and incubated in media containing 0.1% FBS for 2 hours prior to lysis. Cells were transferred to ice, washed once with cold PBS, and extracted into mammalian cell lysis buffer (defined above) lacking DTT. Following incubation for 10 min on ice, lysate was cleared by microcentrifugation . Cleared lysates were analyzed by BCA protein assay , and equal amounts of protein were analyzed by SDS-PAGE followed by immunoblotting. FLAG-β-catenin was isolated from HEK293A cell lysates as described above for kinase purification, and relative quantities of recovered protein were determined by immunoblotting samples with FLAG antibody . Samples containing equal amounts were reanalyzed by immunoblotting with the indicated antibodies. Antibodies used were raised against the following: GEF-H1 pSer886 , GEF-H1 , Pacsin1 pSer346 (Millipore #ABS39), Myc epitope , β-catenin pSer675 , vinculin (Sigma #V9131), and PAK4 . Membranes were imaged using fluorophore-conjugated secondary antibodies  on an Odyssey CLx imager .4Cl, 0.1% Triton X100 in PBS) for 1 hour and then incubated with an anti-vinculin antibody  diluted 1:5,000 in PMZ buffer  for 1 h. After three PBS washes, coverslips were incubated with Alexa Fluor 568 goat anti-mouse antibody  and Alexa Fluor 647 phalloidin  in PMZ buffer for 30 min in a dark, humidified chamber. Coverslips were washed three times with PBS and mounted onto slides using ProLong Diamond mounting media containing 5 μg/mL 4′,6-diamidino-2-phenylindole  and dried overnight in the dark. Cells were imaged at 100× and 40× magnification using a Nikon Eclipse Ti-S microscope , and at least 80 fields with individual cells were captured for each cell line. The entire procedure was performed a total of three times with two different infections. Images were randomly selected from each condition and image set, and blindly scored for actin disassembly using a 7-point scale, with lower values indicating a less extensive stress fiber network. The number of fibers per cell was also quantified in blinded images using SFEX for automated fiber identification [Lentivirus expressing PAK4 variants was produced by co-transfecting HEK293T cells with the corresponding pLX304 vector, as well as psPAX2 and VsV-G packaging and envelope plasmids. NIH-3T3 cells were infected by treatment with lentivirus with 8 μg/mL polybrene for 24 h. Cells were then exchanged into fresh media and selected with blasticidin (5 μg/mL for 8 days and in 2.5 μg/mL thereafter). To visualize actin fibers, cells were blinded and sparsely plated onto coverslips in 24-well plates . After 24 hours, cells were washed twice with PBS and fixed in 4% paraformaldehyde for 15 min at room temperature (RT). Cells were washed three times with PBS and permeabilized with 0.1% Triton X100 for 30 minutes at RT. Coverslips were blocked in blocking buffer  and anti-FLAG  antibodies. Coverslips were imaged at 40× and 100× using a Nikon Eclipse Ti-S microscope (Nikon), with at least 20 FLAG-positive fields captured for each transfection. Images were also analyzed for YAP localization using CellProfiler software [For YAP localization experiments, 2.5 × 10software , using DS1 FigAlternative names and subfamily notation as originally proposed by Dan and colleagues  are show(TIF)Click here for additional data file.S2 Fig2 transformed, normalized values for MST4 analyzed using the fully manual PSPA method [2 transformed values from two separate analyses of cyclic PKA using the semiautomated PSPA method. (C) Correlation between normalized values in two replicate PSPA assays for each STE20 kinase analyzed using the semiautomated method. Average R2 values for all replicates were 0.75 ± 0.14, with most showing R2 > 0.6. For the outlier kinase with the lowest correlation (MST3), two additional PSPA assays were performed. Error bars indicate 95% CIs. Numerical values used to generate all graphs are provided in (A) Comparison of the LogA method  and the (TIF)Click here for additional data file.S3 FigHeat maps and sequence logos were generated from PSPA data averaged from at least two experiments as in (TIF)Click here for additional data file.S4 Fig2 transformed , and Ser/Thr data were removed from all but the 0 position. Logos were generated from positive PSPA selections using enoLOGOS [Single linkage hierarchical clustering of PSPA data was performed using Spearman rank correlation (cluster 3.0). Before clustering analysis, PSPA data were LogenoLOGOS . PSPA, p(TIF)Click here for additional data file.S5 FigMST, Mammalian sterile 20 kinase; PAK, p21-activated kinase.(TIF)Click here for additional data file.S6 FigKinase assay with full-length WT and +1W (V676W) β-catenin under (A) Michaelis–Menten and (B) single turnover conditions. Data used to generate graphs are provided in (TIF)Click here for additional data file.S7 FigM4. (B) Bacterially expressed PAK4M4 catalytic domain has reduced activation loop phosphorylation relative to its WT counterpart. (C) Peptide kinase assays on respective preferred substrates show that introduction of the activation loop phosphomimetic S474E mutation improves the activity of full-length PAK4M4 relative to WT PAK4 . Data used to generate the graphs are provided in (A) The PAK4 activation loop phosphorylation site sequence conforms to the WT PAK4 recognition motif but is likely to be disfavored by PAK4(TIF)Click here for additional data file.S8 FigPanc1 cells expressing inducible shRNA directed to PAK4 or luciferase were co-transfected in 6-well plates with an expression plasmid for Myc–Pacsin1 and increasing amounts of plasmids expressing the indicated PAK4 mutant. Each well received 3 μg of Pacsin1 plasmid and 1 μg total of empty vector mixed with PAK4 expression vector. Quantities of PAK4 vector ranged from 0.2 μg to 1.0 μg. After incubation for 40 hours and serum starvation, cell lysates were prepared and immunoblotted with the indicated antibodies. Pacsin1, Protein kinase C and casein kinase substrate in neurons protein 1; PAK, p21-activated kinase; shRNA, short hairpin RNA.(TIF)Click here for additional data file.S9 FigS474E (Chim) or PAK4M4/S474E (ChimM4). (B) As in t test . Error bars indicate SD. Data used to generate the graphs are provided in (A) Schematic representation of chimeric kinases. The MST1 kinase domain was exchanged with either PAK4(TIF)Click here for additional data file.S10 FigSE and MST1 constructs. Cells lysates were immunoblotted for total and phospho-AKT (pS473), phospho-S6K (pT389), and phospho-Erk (pT202/pY204). The phosphorylation index  was normalized to the 293A parental value for each experiment. Error bars represent SD, and each experiment was performed at least four times. Data used to generate graphs are provided in Changes in the phosphorylation states of established growth pathways in HEK293A parental and MM-8KO cells upon transfection with PAK4(TIF)Click here for additional data file.S1 DataPSPA data were quantified, background subtracted, then normalized by position as previously reported . Replica(XLSX)Click here for additional data file.S2 Data(XLSX)Click here for additional data file.S3 Data(XLSX)Click here for additional data file.S1 Table(PDF)Click here for additional data file.


Tau protein aggregation into neurofibrillary tangles in the central nervous system contributes to the etiology of certain neurodegenerative disorders, including Alzheimer’s disease (AD). Though the mechanism of tau destabilization is not fully understood yet, tau protein has been found to be hyperphosphorylated in tau aggregates. To investigate this further, we developed a highly sensitive and specific mass spectrometry (MS) method using parallel reaction monitoring (PRM) to identify tau phosphorylation sites. This method enables us to compare the abundance of phosphorylation sites in tau proteins in the brain and cerebrospinal fluid (CSF) in humans with and without AD. We detected 29 distinct phosphorylated tau (p-tau) sites in full-length tau from soluble human brain lysate and 12 sites on truncated tau in CSF, mainly in the mid-domain. Brain soluble tau phosphorylation sites are localized on three domains including a proline-rich mid-domain, the C-terminus, and a cluster on the N-terminal projection domain not previously characterized. Some phosphorylation sites increased in CSF, while others decreased compared to brain. Notably, phosphorylation on T205 and S208, recognized by AT8 antibody defining Braak stages of brain tau aggregation, were not detected in normal brain soluble tau but were found in the CSF. Comparison of the p-tau rates from the brain and the CSF indicated that the abundance of phosphorylated sites varied in a site-specific manner. CSF tau proteins from non-AD participants were significantly hyperphosphorylated on T111, T205, S208, T217 and T231. In AD CSF, hyperphosphorylation on these sites was exacerbated, and phosphorylation on T153 and T175 specifically were detected. This supports the hypothesis that tau hyperphosphorylation could be a physiological process amplified by AD pathology. Conversely, we found that S202 was hypophosphorylated in CSF and was not hyperphosphorylated in AD, demonstrating that p-tau isoforms could have different metabolisms depending on which sites are phosphorylated. These site-specific p-tau rates are independent of tau concentration and distinct of current CSF tau and p-tau assays measuring tau isoforms levels. Targeted MS multiplexing ability and high-throughput capacity lets us envision the use of these new p-tau measurements as promising biomarkers for AD diagnosis and tracking therapeutic responses. Tau protein aggregation in the brain is one of the hallmarks of neurodegenerative diseases called tauopathies, including Alzheimer disease (AD), some frontotemporal dementia (FTD), progressive supranuclear palsy (PSP), Pick’s disease, and corticobasal degeneration (CBD). Normally a soluble protein, there is a growing body of evidence associating insoluble tau accumulation in the brain with cognitive decline  in which the concentration of tau is three orders of magnitude lower than in the brain  and amyloid positive and CDR > 0 AD patients . Five and seven pools of 500 μL CSF aliquots were generated from the control and AD groups, respectively. At the time of initial collection, CSF was spun down at 1,000× g for 10 min to remove cell debris and immediately frozen at −80°C. Protease inhibitor cocktail was added during experiments. Tau was immunoprecipitated and desalted as previously described with some modifications  controls  was fitted with HSS T3 75 μm × 100 μm, 1.8 μm column and a flow rate of 0.5 μL/min of a gradient of solution A and B was used to separate the peptides. Solution A was composed of 0.1% formic acid in MS grade water and solution B was composed of 0.1% formic acid in acetonitrile. Peptides were eluted from the column with a gradient of 2%–20% of solution B in 28 min, then 20%–40% solution B for another 13 min before ramping up to 85% solution B in another 3 min to clean the column. The Orbitrap Fusion Lumos was equipped with a Nanospray Flex electrospray ion source . Peptide ions sprayed from a 10 μm SilicaTip emitter  into the ion source were targeted and isolated in the quadrupole. These were then fragmented by HCD and ion fragments were detected in the Orbitrap . Monitoring of hydrophilic peptides  for peptide profiling was performed on a HSS T3 300 μm × 100 μm, 1.8 mm column at a flow rate of 4 μl/min with an elution occurring with a 2%–12% solution B gradient and a spray operating on a 30 mm SilicaTip emitter.in silico and refined using biophysical properties measured for corresponding unmodified peptides that are much more abundant in the sample. For detecting phosphorylated peptides, the quadrupole is set to select a mass window including the hypothesized precursor mass. After precursor collision, all generated fragments are simultaneously measured in the Orbitrap over the time of chromatographic elution. Potentially phosphorylated peptides can be searched in post analysis of the generated data. Skyline software  was used to extract LC-MS/MS data. The hypothetical peptide being screened is detected as a LC-MS/MS fingerprint constituted by the strict co-elution of the extracted masses from predicted MS/MS fragments . Fill time for PRM screening on each investigated phosphorylated peptides was typically set to 1 s.Fill time is also limited by the risk of trap saturation. This occurs when too many ions are sampled within the same scan and transferred to the Orbitrap, leading to inaccurate mass measurement due to space charge effects. To avoid such saturation, narrow precursor isolation (0.7 Da) together with appropriate sample purification (immuno purification) enriching the target over matrix were chosen to decrease the contribution of potential near-isobaric interferences.Specificity of the PRM discovery experiment depends on the resolving power of the LC-Q Orbitrap system. Resolving power can be improved by different analytical parameters, with the ultimate goal to obtain interference-free LC-MS/MS fingerprints for the targeted peptides. This limits the risk of ambiguous fragment assignment due to false positive signals. Sample purification preferentially enriching p-tau can also improve the specificity in the discovery of minor p-tau peptides. High chromatographic peak capacity and resolution limit the likeliness of co-elution. A narrow quadrupole isolation window for the precursor decreases the probability of interference on the MS/MS spectrum together with limiting the risk of trap saturation as described above. High Orbitrap resolution and analyzer calibration allow the accurate extraction of mass fragments during data processing limiting, the risk of transitions interference  phosphorylated peptides are normalizedwith the non-phosphorylated peptide as reference. LC-MS/MS transition specific to each phosphorylated peptide is compared to the corresponding transition from the non-phosphorylated peptide. Each phosphorylated site ratio obtained can be compared across the protein sequence. This strategy may be biased by the difference in fragmentation efficiency between the non-phosphorylated and the phosphorylated peptides. This method cannot be applied when the phosphorylated sites are part of a tryptic missed cleavage; and (3) absolute quantitation using internal synthetic labeledstandards  for each phosphorylated and non-phosphorylated peptide. Signals from phosphorylated and non-phosphorylated standards are used to define an internal ratio. This strategy takes into account the fragmentation specificity of each compared peptide but requires peptides synthesis for each monitored species.To quantitatively assess the relative abundance of phosphorylation of specific sites in tau from the brain and CSF, we measured the extent of phosphorylation on each site detected. We used three methods for this purpose: (1) In this study, except for those including missed trypsin cleavage, the second method was utilized to calculate phosphorylation rates. We also applied the third method  on a limited set of phosphorylated sites found in both brain and CSF extracts when synthetic phosphorylated peptides were available  and one site found only in brain S404; . For AQUpost hoc analyses (Tukey test) were performed for comparing tau phosphorylation rates. Additional statistical analysis was completed using GraphPad version 8.0.1 (244) from GraphPad Software Inc. Statistical significance between relevant groups was determined with a two-tailed, unpaired, Mann-Whitney t-test. Significance was evaluated at the 0.01 and 0.05 level.Data are represented as mean ± SD unless otherwise specified. After confirming the normal distribution of the data, one-way ANOVA followed by In order to determine phosphorylation sites of normal soluble brain tau, extracts from healthy controls were purified by immuno capture using Tau-1 and HJ8.5 tau antibodies concentrating both phosphorylated and unphosphorylated tau. The tryptic digestion of brain tau isoforms generated 27 unmodified peptides that were long and hydrophobic enough to be detected by LC-MS  revealed multiple p-tau peptides eluting into overlapping complex LC-MS/MS patterns –6. We id revealedPhosphorylations at residues S113, T111 and T123 were confirmed on peptide sequences 68–126 (1N) and 88–126 (2N). In all cases, the S113 signal was the most abundant of the three with a phosphorylation rate of 0.2%–0.5% . PhosphoInduction of trypsin missed cleavage was also considered for the screening of phosphorylated residues located after a tryptic site as reported previously . Peptides were detectable to a lesser extent from the N-terminus, and almost no sequence was detectable from the microtubule binding repeat (MTBR) domain or the C-terminus of tau , S199, S202 and T217 . A low sTo increase the probability of detecting additional phosphorylated sites in CSF tau, we analyzed CSF pools from AD patients with mild to moderate dementia. We expected AD CSF pools would contain increased concentrations of tau as well as increased levels of p-tau. The same phosphorylated residues found in normal CSF  were detected in AD CSF. Additionally, signals corresponding to pS113 and pT175 previously found in tau from the brain but not in the normal CSF were detected in AD CSF , 9. A LCIn the brain, S404 was the most highly phosphorylated of the examined phosphorylation sites . Thus, more than half of the brain tau was phosphorylated at S404 compared to other highly phosphorylated sites at S202 and T181 . 1.3% of S199 was phosphorylated but this could be underestimated due to the inability of the Tau1 antibody used for extraction to bind its corresponding phosphorylated epitope , pS202 , and pS214 . Conversely, CSF tau phosphorylation was significantly higher at sites pT217 , pT231  and pT111 from 0N (16-fold increase). PT181 abundance was similar in the CSF and the brain (~10%). When measured in AD CSF, pT175 remained low (0.1%–0.2% compared to 0.1% in brain).In addition to the unique detection of pT205 and pS208 in CSF tau but not brain, the relative phosphorylation abundance of certain other phosphorylation sites detected in brain tau was altered in comparison to control CSF tau . CompareBrain and CSF tau have different truncation patterns: brain tau isoforms are mainly full length while CSF tau isoforms are truncated. Brain and CSF tau isoforms and corresponding peptides recovered after IP depend on the antibody used for the immunoprecipitation  before autopsy can decrease tau phosphorylation measured in brain extracts, we investigated the impact of the PMI on phosphorylation rates commonly found in CSF and brain extracts. Ten brains samples  without tau pathology collected from participants with PMI ranging from 5 to 16 h were analyzed. None of the brain extracts originally analyzed had a PMI of greater than 16 h. We did not find significant association  between PMI and phosphorylation rates measured on T181, S199, S202, S214, T217, T231 and S404 sites.amount, but the rate of phosphorylation is key to understand. pT181, pT217, pT231, pT205, pS208 and pS214 were hyperphosphorylated in AD CSF as was 0N-specific pT111 (p = 0.010), pS214 (p = 0.005), and pT217 (p = 0.003). pT181, pS214, and pT217 showed approximately 1.2-fold, 1.6-fold, and 4-fold increases in phosphorylation respectively. Interestingly, not all the monitored sites were hyperphosphorylated: pS199/S199 was not significantly changed and pS202/S202 was significantly lower (p = 0.030) with an approximately 1.2-fold decrease. Robustness of phosphorylation ratios was assessed by incubating CSF for 16 h. Incubation did not significantly affect tau phosphorylation ratio differences observed between non-AD and AD CSF  the global increase of tau regardless of its phosphorylation status; and (2) increased hyperphosphorylation at specific sites. Normalizing p-tau signal or level using non-phosphorylated tau allows for quantifying changes in phosphorylation stoichiometry  occurring at specific sites independently from the global change in t-tau. The use of “hyper- and hypo- phosphorylation” refers to a change in the ptau/tau ratio in comparison to a ratio measured for a selected reference. For example, we describe hyperphosphorylation of normal CSF in comparison to brain measurements or hyperphosphorylation of AD CSF in comparison to normal CSF. As we recently demonstrated, soluble tau production is increased in AD and correlates with amyloid plaques. Therefore, controlling for not just the ic pT111 . PT153 ad AD CSF . MoreoveWe report the most comprehensive qualitative and quantitative analyses of p-tau in normal brain tissue and CSF to date. Our approach contrasts with previous DDA studies on tau phosphorylation which may not have captured potential minor phosphorylation sites. However, our approach uses highly sensitive targeted-MS in PRM mode to detect and quantify minor phosphorylation. Phosphorylation site identification depends on the careful manual interpretation of LC-MS/MS patterns to identify co-elution of specific ion fragments for each examined phosphopeptide. In prior studies, brain tau phosphorylation has been mainly investigated in insoluble extracts enriched in hyperphosphorylated tau from PHF and, to date, the most detailed study has reported nine phosphorylation sites in normal human tau protein  across the protein, in different compartments (intracellular brain vs. extracellular CSF), and in different pathological conditions (AD vs. non-AD).Some caveats of the PRM screening process are the low throughput for discovery and the risk of missing p-tau species or other post-translational modification (PTM) not hypothesized in the study. Identifications from MS data can be further used in a larger validation or clinical cohort to design scheduled LC-MS methods, increasing multiplexing and throughput  from getting close to each other , and not due to an increase in the relative phosphorylation rate itself. Phosphorylation rate measurements from this study enabled for the first time the comparison of the degree and distribution of phosphorylation rate changes (hyper- vs. hypo-phosphorylation) across the protein, in different compartments (intracellular brain vs. extracellular CSF), and in different pathological conditions (AD vs. non-AD). This method could be used in the future to look at modifications of AD brain phosphorylations on numerous sites across brain regions and Braak stages as recently performed using immunochemistry (Neddens et al., in vivo and in vitro and may also provide a promising tool to assess new drugs targeting abnormal tau metabolism.In this study, we demonstrated that tau in AD CSF is generally hyperphosphorylated in comparison to non-AD CSF. However, the degree of hyperphosphorylation is site dependent. T111, T205, S208 and T217 were more hyperphosphorylated than T181, which is the most commonly measured target used as a p-tau biomarker for AD (Fagan et al., NB conceived the project and designed the study. NB, CS, PM, and NM performed experiments, analyzed the data, and interpreted the data. NB, CS, NM, and RB wrote the article.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Mycobacterium tuberculosis, the causative agent of TB, is among the reasons for delayed diagnosis by bacteriological methods. Previously, it has been shown that mycobacterial resuscitation-promoting factors (RPFs), a family of proteins secreted by actively growing bacteria into the media, are capable of activating the growth of dormant bacteria, thus enhancing the detection of bacilli in the sputum of confirmed TB cases. However, the variability in bacterial resuscitation by RPF in the sputum of suspected pulmonary TB cases that showed differential smear and/or culture positivity during diagnosis has not been fully explored. Here, we report the presence of non-replicating bacteria in the sputum of suspected TB cases that show differential growth response to RPF treatment. Using crude and recombinant RPF treatment, we show improved sensitivity and reduced time to detect bacilli in the sputum samples of smear-positive/culture-negative or smear-negative/culture-negative cases. We also report the phenotypic heterogeneity in the RPF responsiveness among Mtb strains using an in vitro dormancy model. Our findings have implications for improving the bacteriological diagnostic modalities currently used to diagnose TB in endemic countries.Tuberculosis (TB) remains a leading killer among infectious diseases of humans worldwide. Delayed diagnosis is a crucial problem in global TB control programs. Bacteriological methods currently used to diagnose TB in endemic countries take up to 8 weeks, which poses a significant delay in starting antibiotic therapy. The presence of a heterogeneous population of  Mycobacterium tuberculosis (Mtb) is a leading killer among infectious diseases that accounts for 1.5 million deaths and about 10 million new cases worldwide in 2017. Nearly 1.7 billion (23%) of the global population have been estimated to harbor asymptomatic latent Mtb infection (LTBI)1. One of the key characteristics that make Mtb a successful intracellular pathogen is its ability to survive amid various stresses, including hypoxia, reactive oxygen, and nitrogen species (ROS and RNS) and nutrition starvation that prevails in the infected host (Tuberculosis (TB) caused by ted host . Besidested host . Importated host . Howeverin vivo.The efficiency of the standard multidrug antibiotic regimen currently used for TB treatment is closely linked with the nature of Mtb in the infected host. Clinical and experimental studies have demonstrated the presence of various Mtb phenotypes to co-exist in the same sample than it was previously thought . While ain vitro and in the sputum of patients with TB (Resuscitation-promoting factors (RPFs), a group of five proteins (Rpf A–E) secreted by actively replicating virulent Mtb into the culture media, have been demonstrated to reactive at growth of dormant bacteria  with TB . Althoug with TB . For exa with TB .Moreover, the RPF dependency of Mtb was lost after the bacteria were isolated from the patient sputum. Interestingly, during TB chemotherapy, the proportion of RPF-dependent Mtb increased relative to the actively growing bacterial population that form colonies on standard growth media . Thus, cMtb 16S rRNA gene, indicating that either the improved sensitivity of the assay to detect actively growing bacilli or its ability to detect non-replicating persistor bacilli ultimately increased the diagnostic potential of culture-based assays. In this study, we provide more evidence for the presence of non-replicating persistors in the sputum specimen of suspected TB cases that showed negative result in the conventional microbiological diagnosis. Using RPF-containing culture filtrates of Mtb to treat sputum samples of suspected TB cases, we have improved the sensitivity and rapidity of standard culture-based diagnosis. We also report the phenotypic heterogeneity in RPF-responsive Mtb in vitro using Wayne’s dormancy model.Previously, using a diagnostic luciferase reporter phage assay that can detect non-replicating bacilli in patient sputum, we have identified 30 additional positives samples, which failed to grow on standard Lowenstein–Jensen (LJ) agar medium . The prePrevious studies have evaluated the presence of RPF-dependent Mtb in smear and/or culture-positive sputum samples from active TB patients . In our From each patient, two sputum samples were collected; one collected at home and the other collected at the clinic. Both samples were separately processed by modified Petroff’s method, as mentioned in the manuscript. There was no discrepancy in the results, such as smear positivity rating or culture results obtained between these two sputum samples for any of the patients. We used the sputum collected in the clinic for consistency in collection and processing methodology between patients. The sterility of the collection container and proper collection of samples were ensured by medical personnel. The sputum samples were stored at −80°C until further processing. Sputum samples were processed by modified Petroff’s method, and smear positivity was assessed by sputum microscopy using auramine phenol staining . The totM. tuberculosis , Mycobacterium smegmatis, and Escherichia coli DH5α were grown and maintained as per standard procedures in LJ slants, Middlebrook 7H9 broth, and Middlebrook 7H11 agar media supplemented with oleic acid dextrose and catalase enrichment (OADC) (BD Biosciences) and glycerol or in Luria-Bertani (LB) media (for E. coli)2. In all the experiments, bacterial cultures at the mid-log phase (OD600 = 0.6) were used unless stated otherwise.Strains of M. tuberculosis and M. smegmatis were grown in Middlebrook 7H9 media supplemented with OADC and E. coli were grown in LB media to an OD600 = 0.4. Bacterial cultures were centrifuged at 12,000 × g for 20 min at 10°C, and the supernatant was filtered through a 0.2-μm membrane filter and immediately diluted with an equal volume of 7H9 broth. The bacterial cultures were treated with RPF protein at a 1:10 ratio. The culture was plated on Middlebrook 7H11 agar media. At 7 days post-treatment, the colony-forming units (CFU) were enumerated.Crude RPF-containing culture filtrate from bacterial cultures was prepared as described previously . Brieflyn = 251) and smear-negative/culture-positive (n = 17) sputum samples, were used to determine the presence of resuscitable Mtb (RCs). Bacterial growth induction was confirmed by both the increase in the optical density (OD600) of broth culture and growth of RC on Middlebrook 7H11 agar plates. The results for each sample were compared to their corresponding diagnostic smear and/or culture status from the clinic.The impact of RPF in increasing the sensitivity of Mtb detection and the presence of non-replicating persistors was tested on the sputum samples of suspected TB patients. A total of 268 samples, including smear/culture-negative  assay was used as described previously . BrieflyTo test whether RPF can reduce the TTD Mtb, we analyzed 55 smear- and culture-positive sputum specimens. The sputum-smear gradation of smear-positive/culture-positive samples used to evaluate TTD Mtb is presented in E. coli system was a kind gift from Tom H. Ottenhoff of Leiden University Medical Center, Netherlands  or crude RPF or rRPF, respectively. Treated samples were inoculated onto LJ slants, incubated at 37°C, and observed till 8 weeks for Mtb colonies. The number of Mtb colonies and yield of positivity were noted and compared between the groups.Recombinant RPF (rRPF) protein from Mtb that was heterologously expressed using an herlands . The rRPherlands . These therlands . The rRPin vitro anaerobic culture system developed by 600 = 0.1 into 7H9 broth supplemented with OADC and glycerol in an airtight container and incubated at 37°C with very gentle stirring. As reported previously, at 45 days post-inoculation, a hypoxic, non-replicating persistent stage (NRP) was achieved under these conditions, as indicated by discoloration of methylene blue indicator (M. smegmatis), or E. coli DH5α as mentioned above. The culture exposed to RPF or control broth was serially diluted and spotted onto 7H11 agar plates, incubated at 37°C, and observed regularly for up to 8 weeks for the appearance of Mtb colonies.The potential of RPF in resuscitating Mtb from dormancy to metabolically active form was tested using an ndicator . At thist test, and multiple comparisons were calculated by one-way ANOVA using GraphPad Prism. P value < 0.05 was considered statistically significant.Statistical significance for pairwise comparisons was performed using paired Student’s In the agar plating method, among the crude RPF-treated samples, 20 out of 268 (7.5%) yielded bacterial CFU and were positive for RCs. These samples were previously classified as negative by conventional smear/culture-based diagnostic methods. Among these 20 samples that showed positive CFU with RPF, treatment with sterile 7H9 broth (placebo) resulted in Mtb CFU, and positive RCs for only five samples (25%) . Thus, tP = 0.036) increase in the number of positive samples was noted between crude RPF-treated and placebo broth-treated samples  (P = 0.0163), which clearly shows that RPF significantly induces bacterial growth  . We perfl growth . The staP ≤ 0.001) -treated samples (≤ 0.001) . Therefo≤ 0.001) .10 and 2 log10 higher positivity rates, respectively, compared to placebo broth treatment. Out of 13 positive cultures observed with rRPF treatment, seven yielded RCs only in the presence of rRPF and not with either crude RPF or placebo broth treatment. Similarly, 18 samples yielded positive RCs only in the presence of crude RPF, while two samples were positive exclusively for placebo broth treatment , while 6 samples (6.1%) showed positive results for Mtb when treated with placebo broth and 13 samples (13.2%) showed positivity for Mtb after treatment with rRPF. Four out of the six positives noticed in placebo-treated samples showed a higher culture yield when treated with crude RPF or rRPF. Most of the positives obtained by crude RPF treatment were reported negative by placebo broth treatment. Among the tested samples, the Mtb culture yield in crude RPF and rRPF treatment showed 4 logreatment . These dM. smegmatis, as well as E. coli were tested for their potency to reactivate clinical Mtb strains (MTB01 and MTB02) grown in vitro to dormancy by oxygen depletion. Clinical Mtb isolates MTB01 and MTB02 were grown to dormancy and treated with the crude RPF obtained from various bacterial species , 1338 , Erdman, and avirulent mycobacteria such as H37Ra,  species . Results species . The lat species . While cin vivo and the conversion of latent infection to active TB remains poorly understood  and harbor the bacteria in a dormant form. These cases have a 5–10% lifetime risk for reactivation to symptomatic TB . Althougderstood . RPFs, aderstood . Sputum derstood . In clinderstood . FurtherSimilarly, biological samples, other than sputum, from patients with extrapulmonary TB were shown to contain differentially culturable Mtb by their ability to grow only in the presence of RPF-containing culture filtrate . These r600) and growth of viable bacilli (viable cells – VC) on conventional agar media as described previously  from actively growing mycobacteria or recombinant RPF to treat sputum specimens. Results clearly show improved detection of viable but non-culturable (VBNC) Mtb, based on growth in liquid media . Importantly, these two patients completed the full course of antibiotic treatment and showed negative-culture conversion, which was indicative of a good clinical prognosis. However, in the sputum samples of these patients, the presence of the RPF-dependent NRP bacterial population was evident from our assays. Therefore, we predict that the presence of such RPF-dependent NRP is responsible for disease relapse in these patients. Taken together, the presence of resuscitable/viable Mtb even after successful completion of treatment provides a piece of crucial evidence for extending the current treatment duration and for testing the drug susceptibility pattern of infecting Mtb.M. tuberculosis were able to produce increased growth of viable cells among the cells maintained in the stationary phase of growth ]. These studies suggest that RPFs do not mimic the role of just being a growth supplement or nutrient; instead, these proteins play a vital mechanistic role as a growth stimulant, based on the requirement of picomolar level concentration in the medium to reactivate bacterial growth. Moreover, Turapov et al. concluded that the potential of both recombinant RPF and the culture filtrate supernatant are equivocal; although reports from other groups contradicted this observation . Therefore, our observations could be relatable and extended to MGIT conditions. Nonetheless, this claim warrants further evaluation with actual experiments to test the beneficial effect of RPF on MGIT assays, which will be the focus of our future research.In conclusion, we demonstrate the presence of viable Mtb in the clinical specimen of patients with pulmonary TB who had negative results in conventional microbiological tests. We show that crude RPF, as well as recombinant RPF proteins, are capable of resuscitating dormant Mtb and improve the bacillary load as well as reduce the duration to detect Mtb in a clinical specimen. The mechanistic basis for the heterogeneity among Mtb in their response to RPF needs further investigation. Our findings have clinical implications since the treatment of patient sputum samples with RPFs has the potential to improve the current microbiological diagnosis of TB.All datasets generated for this study are included in the manuscript/The studies involving human participants were reviewed and approved by Institutional Ethical Committee of the National Institute for Research on Tuberculosis, a body of Indian Council of Medical Research. The patients/participants provided their written informed consent to participate in this study.AD and SS conceived the concept, designed the experimental studies, wrote and edited the manuscript. AD, MB, GS, SP, and AB performed the experiments. SS, KT, AD, CN, RS, RM, and KT analyzed the data. AH and GR assisted with sample procurement and documentation. All authors read and agreed to publish.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The results showed that the electrostatic binding free energy are correlated with values of constant inhibition (r2 = 0.97).Thus, the model obtained here could contribute to future studies of this important system and, therefore, eventually facilitate development of tyrosinase inhibitors.Tyrosinase is a key enzyme in melanin synthesis and widely distributed in plants and animals tissues. In mammals, this enzyme is related to pigment production, involved in wound healing, primary immune response and it can also contribute to catecholamines synthesis in the brain. Consequently, tyrosinase enzyme represents an attractive and selective target in the field of the medicine, cosmetics and bio-insecticides. In this paper, experimental kinetics and computational analysis were used to study the inhibition of tyrosinase by analogs of Kojic acid. The main interactions occurring between inhibitors-tyrosinase complexes and the influence of divalent cation (Cu Several kinetic and computational studies on tyrosinase inhibition mechanism have been published [Excessive accumulation of melanin, due to the overexpression of enzyme, leads to skin disorders such as age spots, freckles and malignant melanoma ,11. Tyroublished ,17,18,19ublished ,21.aspergillus genera. This natural product presents a wide range of pharmacological profile such as skin-whitening, antioxidant, anti-tirosinase and anti-tumor [A number of tyrosinase inhibitors have been discovered and majority of them consist of the a phenol structure or of metal chelating agents ,24. Thesti-tumor ,24,25. Rti-tumor . In thisKm, and Vmax, the kinetic analysis was determined from the equation of the Lineweaver-Burke plot. The inhibition constant (Ki), which is the concentration of inhibitor required to KA analogs  to inhibit 50% of reaction.In order to show Michaelis constants Vmax of the reaction was unchanged, while the apparent affinity Km was increased to INH2 e INH4 inhibitors  were 64 μM and 145 μM, respectively. These results match the values of affinity enzyme-inhibitor (Ki) and the values of free interaction energy. It is likely that these inhibitors present those kinetics parameters for showing similar features derived of inhibitors with chelanting and competitive properties [The kinetic parameters showed that the maximum velocity hibitors . Kineticoperties .Ki = 1 mM, while the inhibitor INH3, it did not show any kinetic inhibition, suggesting that methyl group of this inhibitor shows less interactions with the amino acid near the enzyme site.The inhibitor INH1 , showed AbTYR, as proposed here, revealed an excellent agreement for the previous theoretical models obtained by molecular docking. In this way, the molecular docking was used to determine the position and conformation of KA and its analogues in the receptor-binding pocket of the AbTYR.The re-docking results show that tropolone conformation obtained by docking is completely superimposed in tropolone experimental conformation. These results are shown in AbTYR catalytic site.Previous molecular docking studies have been applied to elucidate the interactions occurring in Tyrosinase complex with isophthalic acid , hespereVan der Waals interactions are promoted by Met280 and Asn260 residues, these interactions occurring when these residues are contacting to compounds that are founded in tyrosinase catalytic site [Van der Waals interactions could be founded, the CH3S group of Met280 interacts with carbonyl group in all KAD compounds and the carbamoyl group of Asn260 interacts with apolar groups in para position of all KA analogs.Previous computational studies showed that Met280 and Asn260 residues are involved in the first stage of inhibitors recognition and their fixation during docking and MD simulations ,28, resptic site ,9,20,21.2+ A).On the other hand, the inhibitor INH1, which plays a role in preventing the entry of the substrate in the active site, exerting a mixed inhibitory action, presented distance greater than 2 Å concerning the Asn260.About the interactions highlighted previously, we can highlight the one between Asn260 residue and the active compounds INH2 and INH4 with distances less than 2 Å. 2+ A). These data indicate that the region occupied by the inhibitors is similar to that found in previous studies [i.e., inhibition by competition with the substrate L-DOPA in the cavity of the active site. studies ,28, i.e.As explained in theoretical section, all inhibitors, including crystal inhibitor (Tropolone), were subjected to molecular dynamics simulations for a period of 2 ns. During the MD simulation, the inhibitors remained in the active site by performing interactions with copper ions and the amino acid residues constituting the active site. The proposed inhibitors keep similar interactions to those found in previous studies ,21,28 wiThe ligands INH2 and INH4 interacts directly with the copper ion coordinated by His259, His264 and His296, causing a break in the link between the two copper ions, providing chelation, causing the copper ion which had an average distance of 2.07 Å change to a distance of 3.50 Å of His residues . The inhibitor INH1 only positioned in the catalytic cavity of the active site, making interactions only with residues Asn260 and Met280, with no visible interaction with either copper ions. Previous studies recognize compound structures for hydroxyl groups as powerful inhibitors of tyrosinase ,22,29,30−1, getting a difference of 0.14 kcal·mol−1 from the experimentally calculated value which was −5.62 kcal·mol−1. Moreover, the affinity of the complex INH2-tyrosinase was experimentally calculated to −5.23 kcal·mol−1, getting a difference of 1.17 kcal·mol−1 from the value calculated by the LIE methodology .The enzymatic complexes containing ligand-enzyme, as well as ligand in solution only, were subjected to the molecular dynamics calculations described above, allowing the free energy (FE) to be calculated. The complex tyrosinase-INH4 had a calculated energy by LIE method of −5.76 kcal·mol−1 to −0.16 kcal·mol−1, respectively, which is satisfactory, compared to previous works [The values of binding affinity, derived from molecular dynamics trajectories (MD) by LIE method, are shown in us works ,33.−1, respectively) are in agreement with the experimental values . On the other hand, the absolute binding calculated for the INH2 inhibitor using the same methodology  reproduces the experimental affinities  with an error of 1.17 kcal·mol−1. In general, the calculations of bind affinities from MD trajectories through the LIE method are in excellent agreement with experimental data , experimentally it was not possible to identify the reason of this lower affinity; however, our MD simulations revealed that this inhibitor tends to leave the enzyme catalytic site, what can justify its weak binding. From comparison between the theoretical predictions and the experimentally measured kinetic data for all molecules, it seems that the free energy calculations are reliable for practically all analogs of KA in complex with the AbTYR enzyme. The coefficient of determination obtained for the linear fit of predicted values versus the observed contributions was good (r2 =0.97).These results suggest that the standard parameterization of LIE is a robust method that reproduces the experimental affinities.The absolute binding calculated for the Tropolone and Kojic acid inhibitors using LIE  and its analogues (2-Ethyl-3-hydroxy-4H-pyran-4-one (INH1); 5-Hydroxy-4-oxo-4H-pyran-2-carboxylic acid (INH2); 3-Hydroxy-2-methyl-4-pyrone (INH3) and 3-Hydroxy-1,2-dimethyl-4(1H)-pyridone (INH4), L-DOPA and enzyme mushroom tyrosinase were obtained from Sigma-Aldrich. These reagents for anti-tyrosinase assay were of AR grade. Tyrosinase-inhibition activity of the analogues INH1, INH2, INH3 and INH4 were performed by using L-DOPA as a substrate according to Kubo et al.  with sliAll solutions were prepared in Phosphate Buffered Saline (PBS) pH 7.2. The reaction was initiated by addition of enzyme to all wells simultaneously. The change in absorbance due to the formation of dopachrome  was assessed during the first 5 min in the microplate reader with 490 nm filter.Ki was determined with the GraphPad Prism® 5.0 software, according to the equations follows:Competitive Inhibition:Mixed Inhibition:where Y, X and I denotes average absorbance change per minute, concentration of l–DOPA and concentration of KA analog, respectively. The parameter α determines mechanism, its value determines the degree to which the binding of inhibitor changes the affinity of the enzyme for substrate.The mode of inhibition on the enzyme was assayed by Lineweaver-Burk plot. The assay varied the concentracion of L-DOPA  and inhibitor . The kinetic parameter A. bisporus Tyrosinase (AbTYR) was obtained from the Protein Data Bank (access code 2Y9X), a repository for structural data of large biological molecules, such as proteins and nucleic acids [All molecular docking calculations were performed using as staring point the 3D structure of ic acids . The cryic acids ,36. The et al. [et al. [The MVD program  used to et al. ,38 and f [et al. .score) is defined by the following energy terms:inter is the ligand-protein interaction energy and Eintra are the internal energy of the ligand. The Einter is determined by follow equation:The MolDock Score function  term between atoms and the other stronger potential for hydrogen bonds [intra is calculated by the following equation:The Een bonds . The EinEclash assigns a penalty of 1000 if the distance between two atoms (more than two bonds apart) is less than 2.0 Å. Thus, the Eclash term punishes infeasible ligand conformations.The double summation calculates all the energy terms involving pairs of atoms of the ligand, except those connected by two bonds. The second summation calculates the torsional energy, where θ is the torsional angles of the bond. The average of the torsional energy term contributions is used if several torsions can be determined. The last term, The empirical scoring function used by MVD program is fast to be performed; it treats contributions to the binding free energy, e.g., entropy and solvation, in a very approximate fashion, which makes it difficult to obtain accurate predictions. Then, we decided to perform molecular dynamics simulations in order to accurately describe receptor flexibility and salvation around each inhibitor and to determine their binding free energy.2+ ions for enzyme system, a constrain was applied using a force constant of 600.0 kcal·mol−1.Å2 during all MD simulation steps.Molecular dynamics simulations and LIE calculations were performed with the package Q . The MD l − s) were extracted from the collection phase MD trajectories, and binding free energies were calculated according to:vdW) and electrostatic (el) interaction energies. The parameter α has been empirically determined to 0.181 [The LIE method was used to calculate binding free energies from the MD simulations ,48. The to 0.181  and β isto 0.181 . The samKi values, which were converted into experimental free energy values using the equation:Finally, binding free energy estimates were compared to experimentally measured Ki, it seems that the ligand affinity obtained through LIE calculations is in excellent agreement with experimental data. Thus, a combination of inhibition kinetics and computational modeling may facilitate the testing of potential tyrosinase inhibitors and the prediction of their inhibitory mechanisms.In this report, experimental kinetic assays were coupled with computational study, applying molecular docking, molecular dynamics simulations and binding free energy to provide insights into the activity of inhibitor against tyrosinase enzyme. Kinetic studies showed that inhibitors INH2 and INH4 are competitive, while the inhibitor INH1 showed a mixed-type of inhibition. In addition, the inhibitor INH3 did not show any kinetic inhibition. The computational simulation supports these findings. The analysis of individual interactions between the inhibitor and the amino acids of the enzyme active site reveals how the influence of Met280, Asn260, His61, His85, His94, His259, His263 and His294 residues seems to be crucial, being especially important the interactions established between the inhibitor and theses residues. Finally, comparing the theoretical predictions on complexes with the experimentally measured kinetic data,