Pseudogymnoascus destructans, the fungus that causes white-nose syndrome in hibernating bats, has spread across eastern North America over the past decade and decimated bat populations. The saprotrophic growth of P. destructans may help to perpetuate the white-nose syndrome epidemic, and recent model predictions suggest that sufficiently reducing the environmental growth of P. destructans could help mitigate or prevent white-nose syndrome-associated bat colony collapse. In this study, we screened 301 microbes from diverse environmental samples for their ability to inhibit the growth of P. destructans. We identified 145 antagonistic isolates, 53 of which completely or nearly completely inhibited the growth of P. destructans in co-culture. Further analysis of our best antagonists indicated that these microbes have different modes of action and may have some specificity in inhibiting P. destructans. The results suggest that naturally-occurring microbes and/or their metabolites may be considered further as candidates to ameliorate bat colony collapse due to P. destructans. Pseudogymnoascus destructans Minnis & D.L. Lindner is the fungus that causes a deadly disease in hibernating bats known as white-nose syndrome (WNS) [P. destructans is believed to have been introduced to North America from Europe and was first discovered in New York in 2006 [P. destructans has caused extensive local extinctions and ten-fold reductions in affected North American bat populations [P. destructans has rapidly spread to 32 U.S. states and 5 Canadian provinces [me (WNS) . P. dest in 2006 . Over thulations . FurtherP. destructans causes WNS by colonizing the skin of hibernating bats, creating lesions and increasing the frequency with which bats emerge from torpor. This is often lethal as the increased energy demands of disrupted torpor can result in dehydration and emaciation before water or food is available [P. destructans infection [P. destructans [P. destructans persists in hibernacula by growing saprotrophically when bats are absent [P. destructans in the white-nose syndrome epidemic remains unclear, model predictions have suggested that under certain circumstances, reducing the growth of P. destructans in hibernacula may mitigate or prevent WNS-associated colony collapse [P. destructans in hibernacula may be an important part of managing the WNS epidemic.vailable . Furthernfection . After ttructans ,10 and qtructans . Howevere absent ,13, it ie absent ,14. Whilcollapse . Taken tP. destructans to identify potential biocontrol candidates and microbially-derived natural products that reduce the growth of P. destructans in a low temperature (hibernaculum-like) environment.In this study, we isolated a diversity of microbes from Ontario and Quebec, Canada, and screened for ones that inhibit P. destructans and classified each isolate based on an ability to inhibit P. destructans growth 14 days after inoculation , considerable (50% to 85% inhibition), or complete/nearly complete (greater than 85% inhibition), as summarized in P. destructans, and over 15% completely or nearly completely inhibited growth of P. destructans. Most of the isolates that inhibited P. destructans did so by creating a zone of inhibition surrounding themselves where P. destructans did not grow. However, other modes of inhibition were also evident. For example, some fast-growing filamentous fungi grew over P. destructans colonies and induced a highly vacuolized appearance to P. destructans hyphae that was suggestive of programmed cell death , Pantoea (3 strains), Streptomyces (3 strains), Pseudomonas (2 strains), and 1 strain each from Rahnella, Arthrobacter, and Sphingobium. The 23 most inhibitory filamentous fungi were primarily from the genera Penicillium (11 strains) and Trichoderma (7 strains), and the genera Oidiodendron, Boeremia, Botrytis, and Phoma each had 1 representative. One strongly inhibitory antagonistic filamentous fungus (isolate RW3A2Pa) could not be identified. Yeast belonging to Cystofilobasidium (2 strains) were also among the most inhibitory antagonists. All antagonists of P. destructans were preserved as frozen glycerol stocks at -80°C.Isolates that inhibited the growth of cies see . The 28 P. destructans acted through volatile compounds by inoculating P. destructans separately from antagonists in a shared airspace (see P. destructans for 6 to 10 days after inoculation. Volatiles from 2 of these antagonists (Oidiodendron sp. PCA20P and Pantoea sp. OA1I3M) caused considerable (50–85%) inhibition and 2 (Pantoea ananatis RFA4P2 and Cystofilobasidium capitatum RW3I1a) caused complete or nearly complete (greater than 85%) inhibition of P. destructans at 14 days after inoculation  to identify volatile compounds produced by the antagonists that inhibited P. destructans. All four compounds tested  completely inhibited P. destructans growth for the 14-day experimental period when 100 μl of compound was added. Only propanoic acid was completely inhibitory when 10 μl of compound was applied. These compounds appear to be fungicidal since completely inhibited P. destructans did not resume growth after the compounds were removed. The ethanol control (carrier solvent for 10 μl 1-pentanol assay) also mildly affected P. destructans growth such that after 14 days, the average colony diameter was approximately 30% smaller than in the water control.Compounds tentatively identified through GC-MS were screened in bioassays to determine their ability to inhibit P. destructans against two additional strains of P. destructans and representatives of the closely related species, Pseudogymnoascus pannorum and Pseudogymnoascus roseus. Of the 36 antagonists tested in bioassays, 35 inhibited both additional strains of P. destructans, with 29 causing complete or nearly complete inhibition in both strains. Only 16 antagonists inhibited growth of P. roseus and/or P. pannorum, with 3 of these causing complete or nearly complete inhibition in one or both strains  amended with 15% sterile glycerol at -80°C.tructans . Pseudog-1 cycloheximide to isolate bacteria and select against fungi [-1 Rose Bengal to isolate slow-growing fungi [-1 ampicillin, 50 mg L-1 chloroamphenicol, and 75 mg L-1 streptomycin sulphate to isolate fungi without bacteria, and PDA alone to culture microbes non-specifically. Antagonists were also isolated from decomposing wood, birch bark, and hay. Additional antagonists were obtained from foam taken from the surface of the Rideau River  and by leaving Petri plates open to the air. Further antagonists were isolated from compost and compost tea [Bacillus and Trichoderma were from the culture collection of M.L. Smith. [Antagonists used in this study were from various sources and locations as listed in st fungi , PDA witng fungi , PDA witpost tea ,40 and f. Smith. ,42.P. destructans cultures, macerated with a Waring blender and stored at -80°C in PDB with 15% glycerol, were thawed at room temperature and diluted in PDB to 5000 CFU/ml in a sterile Eppendorf multichannel pipette reagent trough. A 50 × 75-mm flame-sterilized glass microscope slide was dipped edgewise into the trough and used to stamp a narrow line of P. destructans inoculum onto the surface of PDA in a Petri plate. Perpendicular lines were stamped on PDA at an appropriate distance from the centre in a 55-mm (for assaying slow growing antagonists) or 90-mm (for fast growing antagonists) Petri plate so that a given antagonist, inoculated in the centre of the plate, would contact P. destructans at about day seven. Control plates were created by stamping P. destructans inoculum without an antagonist.Aliquots of P. destructans using a glass slide as described above to create two parallel lines of inoculum. Three days after P. destructans was inoculated, 7-μl aliquots of a log phase antagonist culture in LB (bacteria) or PDB (yeast) were streaked in duplicate through both lines of P. destructans. LB broth and PDB were streaked through P. destructans on replicate plates as controls.PDA in 55-mm diameter Petri plates was pre-inoculated with P. destructans inoculation. Inhibition was quantified using image analysis to calculate the area of P. destructans. Images were scaled based on the size of the Petri plates and antagonist diameter was measured using the measure feature in ImageJ [P. destructans from background features, images were cropped to exclude as much background as possible and only one line of P. destructans was considered. Ilastik version 1.2.0 [P. destructans colonies from antagonist colonies and background. Simple segmentations for each image were exported to ImageJ where the greyscale image was thresholded with a value of 2 for each parameter. P. destructans colony area was calculated using the analyze particles feature of ImageJ with no parameters specified for size or circularity. Thresholded images were manually checked to ensure P. destructans was fully and exclusively detected and the reported area for P. destructans was the area of one streak multiplied by 2 or 3, for plates stamped in duplicate or triplicate, respectively. Inhibition scores for each antagonist were reported as percent inhibition of P. destructans, relative to a no-antagonist control. Percent inhibition was calculated as treatment refers to the area of P. destructans in the presence of the antagonist and Areacontrol refers to the area of P. destructans in the no-antagonist control. The percent inhibition for each antagonist, along with the respective day 14 area of P. destructans and antagonist diameter are provided in P. destructans by an antagonist were ranked as 0 = negligible or no (less than 50%) inhibition of P. destructans; 1 = considerable (between 50% and 85%) inhibition of P. destructans; or 2 = complete or nearly complete (greater than 85%) inhibition of P. destructans. Additionally, if applicable, antagonistic ranks were qualified with: A = P. destructans inhibited the antagonist; B = the antagonist grew over P. destructans such that P. destructans colonies were no longer visible; and/or C = P. destructans colonies were present, but considerably smaller than colonies on the control plates. A representative of each of these classifications and a control plate is shown in Assay plates from antagonist screenings were photographed 14 days after n ImageJ . To aid on 1.2.0  was usedP. destructans were identified, including all isolates that caused greater than 85% inhibition, by sequences of ITS rDNA (fungi), 16S (bacteria) rDNA, and beta-tubulin (Penicillium sp.) DNA. Morphological identification was used to augment sequence-based identifications of filamentous fungi.Isolates that inhibited DNA was extracted using a modified form of the protocol outlined by Lõoke and colleagues . AntagonITS region of filamentous fungi and yeast was amplified using ITS5 (5’-GGAAGTAAAAGTCGTAACAAGG-3’) or ITS9mun (5’-TGTACACACCGCCCGTCG-3’) forward primers and ITS4 (5’-TCCTCCGCTTATTGATATGC-3’) reverse primer [16S region of bacterial samples was amplified using Bakt_341F (5’-CCTACGGGNGGCWGCAG-3’) forward and Bakt_805R (5’-GACTACHVGGGTATCTAATCC-3’) reverse primer [beta-tubulin gene of Penicillium antagonists was amplified using Bt2a (5’-GGTAACCAAATCGGTGCTGCTTTC-3’) forward primer and Bt2b (5’-ACCCTCAGTGTAGTGACCCTTGGC-3’) reverse primer [2, 2 μl of 10 mM dNTPs , 1.25 units of Taq (New England BioLabs), approximately 100 ng of template DNA, and sterile Milli-Q water to 50 μl. For filamentous fungal and yeast ITS, the PCR schedule was 5 minutes at 94°C, then 35 cycles each with 30 seconds at 94°C, 56°C, and 72°C, then 7 minutes at 72°C. For bacterial 16S, the schedule was 10 minutes at 94°C, then 35 cycles each with 60 seconds at 94°C, 57°C, and 72°C, then 10 minutes at 72°C. The beta-tubulin PCR schedule was 3 minutes at 95°C, then 35 cycles each with 30 seconds at 95°C, 60°C, and 72°C, then 7 minutes at 72°C. PCR products were purified using a Geneaid PCR DNA fragments extraction kit and sent to Génome Québec  for Sanger sequencing . Forward and reverse sequences were aligned using ExPASy ClustalW and the NCBI nucleotide BLAST database was used to identify the microbes. DNA sequences were submitted to GenBank and accession numbers are given in The e primer –48. The e primer ,50. The e primer . StandarP. destructans in bioassays acted through volatiles. A strip approximately 1 cm wide was cut out of the centre of a 90-mm PDA plate to create two separated pieces of agar. A thawed stock of P. destructans was diluted in PDB and approximately 1.3 × 104 CFUs were spread onto agar on one side of the plate. A small amount of antagonist  was suspended in 250 μl of liquid medium  and 200 μl of this suspension was spread onto the agar surface opposite of P. destructans. The plates were sealed with Parafilm and incubated at 13 ± 1°C. The growth of P. destructans was monitored and compared to a control without an antagonist from 6 to 14 days after inoculation. The day 14 area of P. destructans and respective inhibition scores for each antagonist were calculated using ilastik [P. destructans was scored as negligible, considerable, or complete/nearly complete, as described above.A shared-airspace experiment was performed to assess if any of the antagonists that completely or uniformly inhibited  ilastik  and Imag ilastik , as abovP. destructans after 14 days. Pantoea ananatis RFA4P2, Oidiodendron sp. PCA20P, Pantoea sp. OA1I3M, Cystofilobasidium capitatum RW3I1a, and a no-antagonist (blank) control were inoculated on 3-ml PDA slants inside headspace jars and grown for 5 days at 13 ± 1°C. The headspace jars were covered with a double layer of sterile foil for the incubation period and sealed approximately 10 minutes before performing GC-MS. The GC-MS was done with an Agilent Technologies 7697A headspace sampler coupled to an Agilent Technologies 7820A gas chromatography system and an Agilent Technologies 5977E mass spectrometer detector. The vials were sampled at 33.9°C, the loop temperature was 45°C and the transfer line was 80°C. Samples were injected for gas chromatography in splitless mode. A 30 m × 250 μm × 0.5 μm DB-WAXetr column was used with helium carrier gas at a constant flow rate of 1 ml/min. The oven temperature was held at 50°C for 2 min and then increased at a rate of 10°C/min to 235°C, where it was held for 5.5 minutes. Mass-spectrometry was performed with electron ionization, and identification of volatile compounds was performed by comparison to version 5 of the National Institute of Standards and Technology (NIST) spectra database.Headspace gas chromatography mass spectrometry (GC-MS) was used to analyze volatiles produced by the four microbes that had contact-independent inhibition of P. destructans in bioassays. Approximately 2.6 × 104 CFUs of P. destructans were inoculated onto the surface of 15 ml of PDA in a 90-mm diameter Petri dish. Petri dishes were inverted and a 2.5-cm diameter sterile Whatman 3 paper disc was placed on the lid of each dish. The filter paper was saturated with 10 or 100 μl of 2-methyl-1-propanol , 2-methyl-1-butanol , propanoic acid, or 1-pentanol . The 10 μl aliquots were diluted to 100 μl in sterile distilled water, except for 1-pentanol, which was diluted in 95% ethanol. Separate assays were done using 100 μl/disc of water or ethanol as carrier controls. Plates were sealed with Parafilm and incubated inverted at 13 ± 1°C for 14 days and growth of P. destructans was assessed. To test whether the inhibition of P. destructans was fungistatic or fungicidal, the paper discs were removed on day 14 and the lid was dried with a sterile Kimwipe. Plates were sealed with Parafilm and incubated at 13 ± 1°C for an additional 14 days, after which growth of P. destructans was assessed.Compounds identified by GC-MS were tested for inhibitory activity against P. destructans. We used spent culture media from a subset of antagonists of P. destructans US-15 and screened these against P. destructans strains US-15, SH-864, and SH-991, P. roseus S8A2CN, P. pannorum S8A5ACS1, and Saccharomyces cerevisiae strain S288C. Thirty-five antagonists were each grown stationary in 250 ml flasks with 50 ml of PDB (fungi) or LB (bacteria) for 4 weeks at 13 ± 1°C. After this time, the culture medium was harvested and passed through a 0.2-μm syringe filter and then 10× concentrated following lyophilisation. To assess inhibition of Pseudogymnoascus spp. and yeast, 50 μl of cell-free filtrate was 1:1 serially diluted in 50 μl of PDB (for Pseudogymnoascus spp.) or YPD  in a 96-well microtiter plate before the addition of 150 μl of PDB with approximately 100 CFUs Pseudogymnoascus sp. or YPD with approximately 150 yeast cells. Ten-times concentrated medium  was used as a carrier control for antagonist filtrates. Inhibition was assessed visually after 14 days of growth at 13 ± 1°C (Pseudogymnoascus sp.) or 3 days of growth at 30°C (S. cerevisieae) and was defined as the lowest concentration of spent medium at which no growth was visible.We screened antagonists for production of antimicrobial compounds that inhibit P. destructans, we repeated bioassays using 36 of our top antagonists against two additional strains of P. destructans (SH-864 and SH-991) and two close relatives of P. destructans (P. roseus S8A2CN and P. pannorum S8A5ACS1). The bioassays were conducted as described above for P. destructans US-15, however the concentration of Pseudogymnoascus spp. inoculum stamped onto the plates was adjusted for each strain so that after 14 days a continuous line of fungal mycelium was visible for each strain. The concentrations were approximately 11,300 CFU/ml P. destructans SH-864, 10,000 CFU/ml P. destructans SH-991, 11,100 CFU/ml P. roseus S8A2CN, and 8,700 CFU/ml P. pannorum S8A5ACS1. Inhibition scores were calculated and ranked as described for P. destructans bioassays.To test the specificity of the inhibition of S1 FigP. destructans was pre-inoculated for one week on a PDA-coated microscope slide before the slide was also inoculated with Penicillium crustosum BWA2P. After 4 days of antagonist growth, P. destructans hyphae had a healthy appearance on the colony side away from the antagonist (A), but had a vacuolized appearance suggestive of programmed cell death near antagonist hyphae (B). Scale bar represents 100 μm.(TIF)Click here for additional data file.S1 TableP. destructans by an antagonist. Isolates were ranked as follows: 0 = negligible (< 50%) inhibition, 1 = considerable (50% to 85%) inhibition, 2 = complete or nearly complete (> 85%) inhibition. Additionally, if applicable, ranks were qualified with: A = growth of the antagonist is limited by P. destructans, B = the antagonist grew over P. destructans such that affected P. destructans colonies were no longer visible, C = P. destructans colonies were present, but uniformly smaller than in the control plate. P. destructans area and isolate diameter refer to sizes 14 days after P. destructans was inoculated. Genbank accession numbers refer to sequences of 16S rDNA (bacteria), ITS rDNA (fungi), and beta-tubulin DNA (Penicillium sp.).Isolates are sorted by percent inhibition, which reflects the degree of inhibition of (XLSX)Click here for additional data file.S2 TableP. destructans and an antagonist were inoculated on separate pieces of agar within a single plate and incubated at 13 ± 1°C. Inhibition was assessed at 6, 10 and 14 days after inoculation and percent inhibition was calculated on day 14 as (1 − Areatreatment/Areacontrol) × 100. The average P. destructans area in no-antagonist controls was 2043.85 mm2. Antagonists were ranked as 0 = negligible (< 50%) inhibition, 1 = considerable (50% to 85%) inhibition, or 2 = complete or nearly complete (> 85%) inhibition, based on the area of P. destructans in the presence of the antagonist (treatment) compared to in the no-antagonist control. Asterisks (*) indicate cases where more pronounced inhibition of P. destructans was evident at day 6 and 10 but decreased by day 14.(XLSX)Click here for additional data file.S3 TableP. destructans in further assays.Antagonists were inoculated on 3-ml PDA slants inside of headspace jars and volatiles were identified. Also listed is the corresponding retention time, molecular formula, match factor (MF), reverse match factor (RMF), probability of match (Prob), and in library (InLib) score for each proposed identification. Italicized entries were tested against (XLSX)Click here for additional data file.S4 TableP. destructans and one strain each of P. roseus and P. pannorum were used. Percent inhibition was calculated as  × 100. Pseudogymnoascus spp. areas refer to the total area of Pseudogymnoascus spp. 14 days after inoculation. Antagonists were ranked as: 0 = negligible (< 50%) inhibition, 1 = considerable (50%–85%) inhibition, 2 = nearly complete/complete (> 85%) inhibition, relative to a no-antagonist control. Additionally, if applicable, ranks were qualified with: A = growth of the antagonist is limited by Pseudogymnoascus spp., B = the antagonist grew over colonies such that affected Pseudogymnoascus colonies were no longer visible, C = Pseudogymnoascus colonies were present, but uniformly smaller than in the control plate.Two strains of (XLSX)Click here for additional data file.S5 TablePseudogymnoascus destructans and one strain each of P. roseus, P. pannorum, and Saccharomyces cerevisiae. Inhibitory concentrations were recorded after 14 days of Pseudogymnoascus growth or 3 days of S. cerevisiae growth and are expressed relative to the original concentration of the day 28 antagonist medium. NI indicates that complete inhibition was not evident even at highest concentration of 2.5× antagonist medium and ND indicates that inhibitory concentration was not assessed.Antagonists were grown at 13 ± 1°C for 28 days and screened against three strains of (XLSX)Click here for additional data file.


We show that activation of protein kinase C (PKC) caused phosphorylation of S313, S319 and S1169. Activation of growth factor receptors induced phosphorylation of S1169 through a mechanism that was independent of PKC, indicating that distinct kinases and mechanisms control the phosphorylation of P-Rex1 at different regulatory serines. Genetic and biochemical studies confirmed that the PKC isoform PKCδ was able to directly phosphorylate P-Rex1 at S313. Functional studies using cells with very low endogenous P-Rex1 expression, transfected with wild type P-Rex1 or a mutant form in which S313 was substituted by alanine, indicated that phosphorylation at that residue negatively regulated P-Rex1 exchange activity. We suggest that control of P-Rex1 activity depends on a highly dynamic interplay among distinct signalling routes and its multisite phosphorylation is controlled by the action of different kinases.P-Rex proteins are guanine nucleotide exchange factors (GEFs) that act on the Rho/Rac family of GTP binding proteins. The activity of P-Rex proteins is regulated by several extracellular stimuli. In fact, activation of growth factor receptors has been reported to activate a phosphorylation/dephosphorylation cycle of P-Rex1. Such cycle includes dephosphorylation of serines 313 and 319 which negatively regulate the GEF activity of P-Rex1, together with phosphorylation of serines 605 and 1169 which favour P-Rex1 GEF activity. However, the kinases that regulate phosphorylation at these different regulatory sites are largely unknown. Here we have investigated the potential regulatory action of several kinases on the phosphorylation of P-Rex1 at S PREX1 and PREX2 have been described, each of them coding for three different isoforms [−/−P-Rex1 mice are resistant to metastatic spreading of the melanoma cells [PREX2 have also been reported [P-Rex proteins belong to the family of guanine nucleotide exchange factors (GEFs) which act on the Rho/Rac family of GTP binding proteins , 2. In aisoforms . While kisoforms –8, severisoforms , 4, 9–17isoforms . In melaisoforms . Furtherma cells . In pancreported .Several structural domains have been identified in P-Rex proteins , 20 Fig. The N-t313 and Ser319) accompanied by phosphorylation of other residues (Ser605 and Ser1169). Mutagenesis as well as functional studies showed that under resting conditions P-Rex1 is phosphorylated at Ser313 and Ser319 and such phosphorylations inhibit P-Rex1 GEF activity [313 and Ser319 are included in the PH domain of P-Rex1, a region whose deletion augments the GEF activity of P-Rex1 in vitro [605 and particularly Ser1169 increases the GEF activity of P-Rex1 [1169 increases, and this facilitates the GEF activity of P-Rex1 towards Rac [Phosphorylation of P-Rex represents an important regulatory mechanism. Various reports have demonstrated that P-Rex1 is phosphorylated at multiple sites , 22, 23,activity . Of notein vitro . Therefof P-Rex1 . Under rards Rac . TherefoAdditional evidence that phosphorylation of P-Rex1 may control its GEF activity has been reported upon activation of β-adrenergic receptors . Agonist313, S319, S605 and S1169 were included among those sites and we centered our attention on those residues because of their reported regulatory role on P-Rex1 GEF activity [313 (score: 0.92) and S605 (score: 0.64) of P-Rex1  and S1169 (score: 0.58). The latter residue was also expected to be a target for casein kinase II (CKII) (score: 0.55).In an attempt to identify P-Rex1 phosphorylating kinases, we performed bioinformatic analyses using the NetPhosK 1.0 online tool, which allows predictions of kinase specific eukaryotic protein phosphorylation sites . Upon anactivity . These s1 Figure . In addi313 was identified by the program as a target for kinases of the PKC family  as compared to other predicted phosphorylation sites whose surface availability was below 3.To further explore P-Rex1 phosphorylating kinases in silico, we also used the Scansite 3 beta online tool. Scansite searches for specific motifs within proteins that may be phosphorylated by mammalian protein kinases or bind to domains such as SH2 domains, 14-3-3 domains or PDZ domains . A totalC family . In cont313, S319, S605 and S1169 sites. This, together with previously published data indicating that PKA activity may phosphorylate P-Rex1 [313, S319, S605 and S1169. To this end, MCF7 breast cancer cells were treated with the PKC agonist Phorbol 12-myristate,13-acetate (PMA), or with forskolin, which is known to stimulate PKA activity by augmenting cAMP levels. These cells, which mainly express isoform 1 of P-Rex1, were treated with these drugs for 15 minutes and the phosphorylation of S313, S319, S605 or S1169 was analyzed by Western blotting using antibodies which specifically recognize those residues when phosphorylated [313 -regulated GEF . Becausenhibitor  also fai9 Figure . As expe3 Figure  and S6 , and sustained for several hours  showed a substantial reduction in PKCδ levels  as well as cell lines of the HER2+ subtype  expressed P-Rex1, while HCC1954 which also belongs to the HER2+ subtype did not express P-Rex1  involved in the regulation of such phosphorylation upon activation of growth factor receptors. Since the inhibitor studies failed to define the S1169 kinase, the use of alternative approaches, e. g. kinome targeting with siRNA libraries, may help in the identification of such kinase. These results were taken to indicate the highly regulated nature of P-Rex1 phosphorylation, and the fact that distinct kinases/signaling routes control multisite phosphorylation of P-Rex1.Phosphorylation of P-Rex1 at S313. Moreover, in vitro kinase experiments confirmed that this kinase could directly phosphorylate P-Rex1 at S313. However, our studies cannot exclude that kinases other than PKCδ may phosphorylate P-Rex1 at S313. While in vivo activation of PKC resulted in phosphorylation of P-Rex1 at S1169, the in vitro studies failed to demonstrate direct PKCδ phosphorylation of that residue. It is therefore possible that such failure may be caused by the requirement of another component absent in the in vitro kinase assay. Another possibility is that a PKC isoform other than PKCδ may be involved in the phosphorylation of S1169.Genetic studies, which included gene expression profiling as well as knockdown experiments identified PKCδ as an important kinase in the regulation of P-Rex1 phosphorylation at S313 and S319 of P-Rex1 are located within the β3-β4 loop of the PH domain of P-Rex1, a region that is not expected to directly interact with Rac. However, those studies have shown that the β3-β4 loop is endowed with a high degree of mobility and surface availability. This property may facilitate accessibility for interaction with other regulatory regions of P-Rex1 or with other proteins, such as kinases or phosphatases that may regulate the GEF action of P-Rex1. The high surface availability of S313 could favor its phosphorylation, causing a conformational change that could facilitate phosphorylation of other P-Rex sites. In fact, the delayed time course of phosphorylation of other residues with respect to S313 could be due to such phenomenon, or could be indirect. Phosphorylation of P-Rex1 at S313 has been formerly reported to negatively regulate the GEF function of P-Rex1 against Rac [313 was substituted by non-phosphorylatable alanine. While informative, those studies had limitations, since the knockdown of P-Rex1 was not complete, and some endogenous protein remained. To better assess the relevance of the phosphorylation of P-Rex1 at S313 we analyzed several breast cancer cell lines which belong to the three major clinicopathological subtypes. These studies informed that expression of P-Rex1 was very low in the cell lines representative of the triple negative subgroup. We took advantage of the lack of substantial expression of P-Rex1 in these cell lines to explore the relevance of P-Rex1 phosphorylation at S313 on Rac activity. When expressed at similar levels, the mutated form of P-Rex1 which contained a non phosphorylatable residue at position 313 had a stronger effect on Rac activity than wild type P-Rex1. This is consistent with the concept that phosphorylation of P-Rex1 at S313 restricts P-Rex1 GEF activity towards Rac. We observed that expression of wild type P-Rex1 form in the three cell lines tested substantially augmented resting Rac activity, indicating that overexpression of P-Rex by itself is sufficient to raise Rac activity. This conclusion is relevant, since elevated Rac activity may contribute to progression of diseases such as cancer. Moreover, the expression of elevated levels of P-Rex1 has been linked to a worse patient outcome in breast cancer, particularly in the luminal type [Crystallographic analyses indicated that Rac interacts with the DH domain of P-Rex . Residuenal type  suggestenal type  indicateCell culture media, puromycin, PMA , dbcAMP 473) antibody was from BD Biosciences. The antibodies against P-Rex1 (anti-pan-P-Rex) and phospho P-Rex1  have been described [The antibody against GAPDH was purchased from Santa Cruz Biotechnology. The anti-PKC antibodies were from BD Transduction Laboratories, Inc . The anti-pAKT S73 antibo2 and 95% air. Cells were grown in DMEM or in RPMI medium containing high glucose concentration  and antibiotics  and supplemented with 10% FBS. Cell lines were provided by Drs. J. Losada and A. Balmain  [All cell lines were cultured at 37°C in a humidified atmosphere in the presence of 5% COsources) . Cell idKnockdown of PKCδ in MCF7 cells was performed by infection with lentiviral particles. The lentiviral vectors containing short hairpin RNA (shRNA) for PKCδ were obtained from Thermo Scientific . A minimum of 5 different shRNA sequences were tested and the two that produced higher knockdown levels were used. Preparation of lentiviral vectors was performed as described previously .313A, cells were transfected with pCMV3-myc-P-Rex1-WT and pCMV3-myc-P-Rex1-S313A using JetPEI. Transfections were performed according to the instructions provided by the manufacturer. The expression of exogenous P-Rex1 was analyzed by Western blotting with anti-P-Rex antibodies.To express exogenous P-Rex1-WT and P-Rex1-S2HPO4, 8 mM; KH2PO4, 1.5 mM) and lysed in ice-cold lysis buffer . Lysates were centrifuged at 10,000 xg at 4°C for 10 minutes and supernatants were transferred to new tubes with the corresponding antibody and protein A-Sepharose. Immunoprecipitations were performed at 4°C for at least 2 hours. Immune complexes were recovered by a short centrifugation at 10,000 xg for 15 seconds, followed by three washes with 1 mL ice-cold lysis buffer. Samples were then boiled in electrophoresis sample buffer and placed on SDS-PAGE gels at varying acrylamide concentrations, depending on the molecular weight of the proteins to be analyzed. After electrophoresis, the separated proteins in the gel were transferred to polyvinylidene difluoride membranes (PVDF) . Membranes were blocked in TBST  containing 1% BSA or 5% skimmed milk for 1-3 hours and then incubated with the corresponding antibody for 2-16 hours. After washing three times with TBST during 10 minutes, membranes were incubated with HRP-conjugated anti-mouse or anti-rabbit secondary antibodies for 30 minutes. After the secondary antibody, the membranes were washed three times with TBST and the bands were visualized by using enhanced chemiluminescence [Cultured cells were washed with phosphate-buffered saline (PBS)  following the manufacturer's instructions. Data are plotted as mean±s.d of duplicates from an experiment which was repeated at least twice.313A) was generated as described [The pCMV3 plasmid containing wild type human P-Rex1 tagged with myc was provided by Dr. Heidi C. E. Welch . The P-Rex1 phosphorylation mutant ; distilled water until 60 μl.The PKCδ kinase assay was performed as described in the assay procedure in Sigma, Product Code P 6862, with slight modifications. The reaction mix was as follows: HEPES (pH 7.4), 20 mM; MgClAs substrate of the kinase (PKCδ), P-Rex1 immunoprecipitated from MCF7 cells was used. MCF7 cells were lysed and 1 mg of protein was immunoprecipitated with the anti-pan-P-Rex1 antibody. The immunocomplex were washed three times with lysis buffer and twice with water. 60 μl of the reaction mix was added into each immunocomplex and these were placed at 30°C. Then it was added 2 μl of diluted enzyme  to each assay tube and the reaction was continued up to 10 minutes. The reaction was stopped by adding loading buffer 4X containing β-mercaptoethanol (20 μl). The reaction of kinase assay was evaluated in Western blot using the anti-phospho-P-Rex1 antibodies.Gene expression studies were performed as described . Brieflyhttp://www.cbs.dtu.dk/services/NetPhosK/). The NetPhosK program produces neural network predictions of kinase specific eukaryotic protein phosphorylation sites. Currently NetPhosK covers the following kinases: PKA, PKC, PKG, CKII, Cdc2, CaM-II, ATM, DNA PK, Cdk5, p38 MAPK, GSK3, CKI, PKB, RSK, INSR, EGFR and Src [http://scansite3.mit.edu/#home) [The sequence in FASTA format of P-Rex1 was introduced in the NetPhos web page ( and Src . Similaru/#home) .t test. Differences were considered to be statistically significant when p-values were less than 0.05. Statistical data are presented as the mean±s.d. All data were analyzed using the statistical software SPSS 21.0 .Comparisons of continuous variables between two groups were performed using a two-sided Student's


The underlying mechanism was investigated and it appeared that the integrase active site was required in order for GCN2 to target the integrase residue S255. Moreover, various integrases from other retroviruses  were also recognized as a substrate by GCN2. In cells, HIV-1 lentiviral particles harboring mutation at integrase position 255 were affected in their replication. Preventing phosphorylation resulted in an increase in infectivity that correlated with an increase in viral DNA integration. Infectivity of MLV was also higher in cells knocked-out for GCN2 suggesting a conserved mechanism to control viral replication. Altogether, our data suggest that GCN2 may constitute a general guardian of genome stability by regulating foreign DNA integration and as such be part of the antiviral armamentarium of the cell.GCN2 is a serine/threonine kinase involved in cellular stress response related to amino acid starvation. Previously, we showed that GCN2 interacts with HIV-1 integrase and is activated during HIV-1 infection. Herein, we identified HIV-1 integrase as a previously unknown substrate of GCN2  IN associates itself with the viral DNA to form the intasome, composed of at least a tetramer of enzyme with two viral DNA ends. From this oligomer  with a H12H16C40C43 motif and a C-terminal domain containing a SH3 fold . The active site is encompassed within the central domain , and is composed of a catalytic triad D64D116E152 that coordinates two magnesium ions . Accumulated biochemical and structural data give an overview of the enzymatic reaction taking placemer Fig. , only twons Fig. .Figure 13. While IN alone is sufficient to integrate viral DNA mimics in vitro, the cellular process involves the formation of a pre-integration complex or PIC composed of viral and cellular proteins. Various studies focused on identifying proteins able to interact with IN3. These cofactors have multiple roles, including stabilization of the complex, tethering to the integration site and regulation of IN activity4. This regulation also involves post-translational modifications of the intasome and IN has been shown to undergo acetylation, phosphorylation, SUMOylation and ubiquitination  enables its binding to Pin1 (a peptidyl prolyl-isomerase) and seems to interfere with ubiquitination6. Interaction with Pin1 increases IN stability and ultimately is required for efficient viral replication in activated T cells.Regarding catalysis, IN first hydrolyzes the viral DNA from the 3′-ends  and uses the newly created 3′-OH extremities to attack the phosphodiester backbone of the target DNA ion Fig. 5. Intere8. This protein kinase is conserved from yeast to human and is part of the four eIF2α’s kinases found in mammalian cells along with PERK, PKR and HRI. Human GCN2 is a 187 kDa protein sensor involved in cellular stress response related to amino acid starvation  which interaction with the kinase domain  is important in the activation process. The C-terminal portion is homologous to histidyl-tRNA synthetase (HisRS), contains a ribosome binding motif and is also involved in homodimerization12. When the level in amino acid reaches a critical limit in the cell, uncharged tRNAs bind to the HisRS-like domain and induce a conformational change resulting in GCN2 autophosphorylation at conserved threonine residues (pGCN2)10. Similarly, interaction of the GCN1/GCN20 complex to the N-terminal domain of GCN2 facilitates binding of uncharged tRNA and subsequent activation.Using yeast two-hybrid and co-immunoprecipitation experiments, we identified GCN2 , another serine/threonine protein kinase as a novel IN interacting cellular factor. Interestingly, we found GCN2 to be activated upon HIV-1 infection which, in turn, decreased HIV-1 replicationion Fig. 9, 10. It13. On a biochemical point of view, GCN2 recognizes a conserved 79KGYID83 motif on eIF2α and transfers the γ-phosphoryl group of an ATP molecule to residue S51 of eIF2α. A strong sequence homology has been found with the vaccinia virus protein K3L14  leading to stabilization of the inactive GDP complex. As a result, eIF2-GDP is unable to load methionyl-tRNA onto the ribosomeL14 Fig. . However8. Because IN binds GCN2 through its pseudo-kinase domain15, IN might interfere with the YKD/KD interactions required during the kinase activation process. Thus, interferences observed in GCN2 signaling could be the result of IN being recognized as a pseudo-substrate, similarly to K3L14.Interestingly, our previous study showed that direct delivery of recombinant HIV-1 IN protein into amino acids starved cells prevented GCN2 phosphorylation and tended to reduce the protein synthesis arrestin vitro the mechanism by which HIV-1 IN could interfere with GCN2 kinase activity and showed that HIV-1 IN, as well as IN from other retroviruses were indeed substrates for GCN2. Point mutations were designed to impede phosphorylation and were introduced in the IN coding sequence of lentiviral particles. Infectivity of such mutant viruses was compared to wild-type (WT) HIV-1 and the viral step affected by the mutation was determined.In the present work, we studied 16. IN expressed in yeast was purified as described by Faure et al., in ref. 18. The HIV-1 IN isolated CTD was expressed in bacteria as a fusion protein with a N-terminal GST. After cell lysis , the soluble fraction was applied to a His SpinTrap column and retained proteins were further purified on a GST SpinTrap™ column . Elution was performed using steps at 500 mM imidazole and 20 mM reduced glutathione, respectively. HIV-1 IN harboring mutation Q168L and Q188Q were generous gifts from Dr. S. Emiliani . Vectors for the bacterial expression of eIF2α and exotic INs (Ty3 and chimers) were kind gifts from Dr. Sandmeyer. The Ty3 retrotransposon IN and the chimeric protein HPH (containing a CCD corresponding to PFV IN surrounded by the NTD and CTD of HIV-1 IN) were purified according to previous publication19. Cellular eIF2α was expressed and purified as previously reported20. Briefly, eIF2α was expressed in BL21 cells from a pET15b vector with a N-terminal 6xHis tag and a C-terminal deletion (lacking amino acids 200–304). The resulting ≈28 kDa protein was purified through a nickel affinity column using an AKTÄ purifier . Proteins purity was evaluated by Coomassie staining of a polyacrylamide gel electrophoresis and concentration was determined using a NanoDrop 2000 (ThermoScientific).GCN2 was purchased from SignalChem (cat# E12-11G). This recombinant protein was expressed in insect cells as a truncated hyper-activated mutant (192–1024) with an N-terminal GST tag (132 kDa). HIV-1 IN WT and phosphorylation mutants  were expressed and purified as previously described2, 7.5 mM MnCl2, 10 mM dithiothreitol (DTT), 0.05% Nonidet P-40, 100 µM ATP, 50 µCi/µl of [γ-32P]-ATP . Phosphorylation was performed at 30 °C for 60 minutes unless indicated otherwise. Reaction (20 µl) was stopped by adding 10 µl of loading buffer . Samples were then loaded onto a denaturating SDS-PAGE. After protein separation (120 V for 90 minutes) the gel was stained using Coomassie Brilliant Blue R-250. Autoradiography was performed using an imaging plate (Fujifilm) and images were obtained with a FLA-5000 Imaging System (Fujifilm).Phosphorylation of target proteins was achieved by incubating the substrate  with GCN2 at 24 nM in a reaction buffer containing a mix of 10 mM Tris /20 mM HEPES [4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid] buffered at pH 7.5, 50 mM NaCl, 15 mM MgCl21. Briefly, WT IN or mutant proteins (400 nM) were mixed with 20 nM radiolabeled DNA substrates in a buffer containing 50 mM MOPS pH 7.2, 7.5 mM MgCl2, 14 mM 2-mercaptoethanol at 37 °C. Reactions were stopped after 10, 20, 30, 45, 60, 90 or 120 minutes by addition of an equal volume of loading buffer . Reaction products were separated in 16% poly-acrylamide denaturing sequencing gels. Image of the gels was obtained after autoradiography using a FLA-5000 (Fujifilm). Densitometric analyses were performed using ImageQuant 5.1 software from GE Healthcare.Kinetic of 3′-P and ST activities were performed as previously described4HCO3 and 10% ACN), rehydrated at 4 °C for 10 min, and finally incubated overnight at 37 °C. Gel pieces were then incubated for 15 min in 40 mM NH4HCO3 and 10% ACN at room temperature. The supernatant was collected, and the gel slices were bathed in an H2O/ACN/HCOOH (47.5:47.5:5) extraction solution for 15 min. The extraction step was repeated twice. The supernatants were dried in a vacuum centrifuge and resuspended in 100 µL of 0.1% HCOOH.Bands were cut from the SDS-PAGE gel and were unstained in 25 mM ammonium bicarbonate 50% ACN, rinsed twice in ultrapure water and shrunk in acetonitrile (ACN) for 10 min. After ACN removal, gel pieces were dried at room temperature, covered with trypsin solution (10 ng/µl in 40 mM NH18 PepMapTM trap column (LC Packings) at a flow rate of 20 µl/min in buffer A . Peptides were eluted from the trap column onto an analytical 75-mm id × 15-cm C18 Pep-Map column (LC Packings) with a 2–40% linear gradient of solvent B  in 108 min (200 nl/min). The mass spectrometer operated in positive ion mode at a 1.8-kV needle voltage. Data were acquired using the Xcalibur 2.0.7 software in a data-dependent mode. MS scans (m/z 300-2000) were recorded in the Orbitrap cell at a resolution of R = 60 000 (@ m/z 400) and an AGC target of 5 × 105 ions collected within 500 ms. Dynamic exclusion was set to 30 s and the top 6 ions were selected from fragmentation in Collision Induced Dissociation (CID) mode. MS/MS scans with a target value of 1 × 104 ions were collected in the linear ion trap with a maximum fill time of 200 ms. Additionally, unassigned and +1 charge state ions were rejected for fragmentation. Others settings were as follows: spray voltage, 1.8 kV, no sheath nor auxiliary gas flow, heated capillary temperature, 200 °C; normalized collision energy of 35% and an isolation width of 2 m/z.The peptide mixture was analyzed on a Ultimate 3000 nanoLC system  coupled to a LTQ Orbitrap XL mass spectrometer . Ten microliters of peptide digests were loaded onto a 300-µm-inner diameter × 5-mm C22, and only peptides with a 1% false positive rate at peptide level were considered of “high confidence” and subsequently retained. Phosphosite precise localization was determined using PhosphoRS 3.1 node embedded in Proteome Discoverer23.Spectra originating from peptides with molecular weight below 350 and above 5000 Da were discarded. Selected peptides were then searched by SEQUEST through Proteome Discoverer 1.4 (Thermo Fisher Scientific Inc.) against the Human Reference Proteome Set (UniProt) database in which homemade sequences corresponding to IN were added. Mass accuracy of the monoisotopic peptide precursor and of the peptide fragments were set to 10 ppm and 0.6 Da, respectively. Only b- and y-ions were considered for mass calculation. The oxidation of methionine residues (+16 Da) and phosphorylation of serine, threonine and tyrosine residues (+80 Da) were considered as variable modification while carbamidomethylation of cysteine residues (+57 Da) as fixed modification. Two missed trypsin cleavages were allowed. Peptide validation was performed using the Percolator algorithmDpnI digestion for 1 hour at 37 °C (1 µl/50 µl reaction). DH5α chemically competent cells were transformed with the digestion product. After plasmid extraction (standard miniprep from Macherey-Nagel following manufacturer’s instructions), the presence of the desired mutations and the integrity of the IN sequence were verified by DNA sequencing (MWG Eurofins).Site-directed mutagenesis was performed on the pET-21b-IN, pCMVΔR8.91, and pEGFP-C2-INs plasmids following a standard PCR-based protocol using degenerated primers obtained from MWG Biotech. The amplification reaction was performed using a step at 98 °C for 30 seconds, followed by 20 cycles of 10 seconds at 98 °C, followed by 45 seconds at 60 °C and 1 minute per kb at 72 °C; and finally a step of 10 minutes at 72 °C. Amplification products were subjected to 24. 293 T cells were plated in a 6-well culture plate  and maintained in DMEM Glutamax® medium  supplemented with 10% (v/v) heat-inactivated fetal calf serum  and 50 µg/ml gentamicin (complete DMEM) at 37 °C with 5% CO2. After 24 h, cells were washed and transfected with 1 µg of pEGFP-C2-INs plasmid and 1 µl Lipofectamine 2000 reagent (Invitrogen) in 100 µl of optiMEM for 4 h. OptiMEM containing 20% FCS was added (100 µl/well) and cells were incubated for 20 h. After trypsinization, cells were lysed by incubation for 20 min at 4 °C in lysis buffer .The plasmid for the expression of HIV-1 IN fused to the EGFP (pEGFP-C2-INs) was a gift from Dr. Z. Debyzer (KU Leuven)2HPO4, 1.5 mM KH2PO4 pH 7.4), retained proteins were separated by SDS-PAGE and stained with silver nitrate.For immunoprecipitation, cell lysates were sonicated twice at 40 W for 20 seconds before the addition of the anti-EGFP antibody  in the presence of 50 µl of magnetic beads conjugated to sheep anti-mouse antibody  and incubated overnight at 4 °C. After 3 washes with phosphate-buffered saline  at a dilution of 1/1000 with overnight incubation. Binding of the primary antibody was revealed by a secondary HRP-conjugated anti-mouse antibody  used at a dilution of 1/2000 in PBS with 0.1% BSA and incubated for 1 hour at room temperature. Anti-actin primary antibody from Sigma was also used as loading control at a dilution of 1/1000.For microscopy experiments, cells were grown on a glass coverslip, transfected as described above for 24 h, fixed with 4% paraformaldehyde. Coverslips were mounted onto glass slides using VECTASHIELD Antifade Mounting Medium with DAPI . Representative images were obtained using an AxioImager Z1 (Zeiss).Quantification of IN expression through the EGFP fluorescence was performed as described in the following “Transduction assay” section.−/−) and their relative parental cells (GCN2+/+) were a kind gift from Dr. P. Fafournoux, . Cells were maintained at 37 °C in a humidified atmosphere containing 5% CO2 in complete DMEM medium  and supplemented with 55 µM β-mercaptoethanol for MEF cells.Human 293 T cells were obtained from the ATCC while MEF cells knock-out (KO) for GCN2 . Cells (5 × 106) were plated on 10-cm dishes and transfected the following day by calcium phosphate DNA precipitation. After 48 h, the viral supernatant was collected, filtered through 0.22-µm filters, concentrated by ultracentrifugation, treated with DNase I (Promega) at 40 U/ml for 1 h at 37 °C, and stored at −80 °C. Viral titration was determined by ELISA through p24 quantification .Single-round vectors were produced by transfection of 293 T cells with three different plasmids for HIV-1 based lentiviral vectors  and loaded on a 2% agarose gel in TAE buffer stained with crystal violet. DNA fragments with a length of 200 to 500 base pairs (bp) were extracted and sequenced using the 454 Junior sequencer (Roche).Determination of the integration sites was performed according to previous publication28. To be included in the analysis, BLAT output sequences must be aligned against only once on the host genome (unique integration site) with a score of at least 98%. To account for a potential bias due to the use of the MseI restriction enzyme during sample preparation, we generated theoretical integration sites using in-house Perl scripts  that play the role of control sets, similarly to previous publication28. These theoretical sites were generated according to two criteria: (i) they have to be randomly distributed along the genome and (ii) they must be located at a distance equal to the distance between the restriction enzyme site and the related experimental site29. For each identified unique site, 10 in silico MRC were generated. Correlation between the integration sites and various genomic features  was tested using statistical analysis previously described in ref. in silico generated MRC set using a chi-square test.Reads from nested PCR that matched against the primer sequence were cleaned using an in-house Perl script (available on request) that removes both, PCR primers and viral sequences. Resulting sequences correspond to the genomic portion of the host localized immediately after the 3′-LTR. Sequences of 50 bp in length and above were then aligned against the reference human genome (hg38) using BLAT (Version 34)15. Later on, we confirmed that human GCN2 interacts with HIV-1 IN, as observed by co-immunoprecipitation, and that GCN2 could act as a restriction factor as silencing of GCN2 by siRNA increased viral replication8. To investigate the mechanism by which this cellular protein kinase can affect HIV-1 replication, we first conducted in vitro phosphorylation experiments to determine the impact of HIV-1 IN on GCN2 activity and whether IN could be phosphorylated by GCN2.In a previous study, we identified GCN2 as a potential cellular partner of HIV-1 IN using the two-hybrid approachvitro phosphorylation experiments, HIV-1 IN was expressed in bacteria to ascertain the absence of pre-existing phosphorylation . GCN2 incubated with [γ-32P]-ATP did undergo autophosphorylation and a signal could be observed only at the protein size corresponding to GCN2 . Using increasing concentrations of recombinant eIF2α also led to a proportional labeling of the protein in the presence of GCN2 30. E85D is a conservative mutation that should not drastically impact the capability of IN to form this salt-bridge interaction. On the opposite, E85A should abolish this interaction and increase the hydrophobic property of this region. Under native conditions (BN-PAGE), mutation E85A increased the proportion of high molecular weight complexes of IN, thus favoring oligomerization/aggregation (data not shown). In the phosphorylation assay, the E85D mutant was less phosphorylated compared to WT whereas the E85A mutant showed an increased level of phosphorylation compared to the E85D or the WT IN  such as the Q168L and K188Q were used in the phosphorylation assay31. Neither the Q168L nor the K188Q mutant were an efficient substrate for GCN2 , and two with double mutations (IN S24A/S255A and IN S24D/S255D). As expected, mutating simultaneously the two identified sites to alanine (S24A/S255A) resulted in a weak level of phosphorylation catalyzed by GCN2 Fig. . Thus, ITo confirm this major contribution of S255 in the phosphorylation of the IN, the different truncated IN-proteins CCD (50–212), NTD-CCD (1–212) and CCD-CTD (50–288) were analyzed in a phosphorylation assay. Compared to the full-length IN, only close to background signal was observed at the size of the isolated CCD domain Fig. , lane 5.19 and the HIV-1 IN CTD (residues 221–288). In this context, GCN2 was able to phosphorylate the chimeric protein  was used that harbors the HIV-1 IN NTD (residues 1–56) fused to the PFV IN CCD (residues 116–278 corresponding to HIV-1 IN residues 57–209)ein Fig. , lane 2.ein Fig. , lane 3 Orthoretrovirinae subfamily.Because PFV IN is somewhat divergent from HIV-1 IN, out of the highly conserved active site, recombinant IN from various other viruses were used to investigate the role of the CTD in the phosphorylation process Fig. . UnsurprS. cerevisiae and purified in a manner similar to that of the protein expressed in bacteria was phosphorylated at the same residues targeted by GCN2 in vitro, namely S24 and S255 (data not shown). Thus in yeast, GCN2 interacted with IN and recognized it as a substrate leading to its phosphorylation. Moreover, phosphorylation of residues S24 and S255 was also detected by mass spectrometry when IN, fused to EGFP, was over-expressed in 293 T cells . Because the interaction of IN with GCN2 was initially uncovered during a yeast two-hybrid assay, we first investigated whether this interaction could led to an effective phosphorylation of IN in the yeast cell. HIV-1 IN expressed in versus 20%). Hence, preventing phosphorylation at IN residue S255 enhanced infectivity of the corresponding virus with no to little impact of the phosphorylation status of S24. Because depletion of GCN2 in cells induced a similar increase in HIV-1 replication8, it seems that GCN2 might be the sole cellular factor involved in the phosphorylation of IN residues S24 and S255 during HIV-1 replication leading to viral restriction.To evaluate the effect of IN phosphorylation during viral replication, lentiviral particles carrying mutated IN at residues S24 and S255 were produced. Infectivity of mutant vectors was then compared to the WT control by transducing cells with equivalent amounts of p24 and by measuring the EGFP expression by flow cytometry. Transduction with the WT vector led to around 20% of the cells detected as EGFP positive Fig. . Interes32, the different populations of viral nucleic acids were quantified. No significant differences in the amount of total viral cDNA were observed between the WT control and the mutant viruses  in Gm12878 cells and at least not favored in K562 cells. The same result was obtained with all our IN mutants except for the S255A which wasn’t disfavored in Gm12878 cells  were used. Utilization of GCN2−/− and of a WT parental line as control (GCN2+/+) recapitulated the effect of siRNA knockdown during HIV-1 infection . In addition, sequencing of the integration sites obtained in these two cell lines revealed a similar pattern with integration occurring preferentially in the body of genes (data not shown).GCN2 was able to phosphorylate IN from HIV-1 but also from other retroviruses such as MLV Fig. . To addrion Fig. . Transdu+/+ MEF cells was tested. In this cellular context, vectors carrying the aspartic acid mutations  infected the cells similarly to the WT vector with 9.8, 8.2 and 7.5% of EGFP positive cells, respectively. Conversely, an increase in transduction efficiency with viruses harboring the S255A or S24A/S255A IN mutations ,  retained the capability to be phosphorylated by GCN2. Comparing the primary sequence of HIV-1 IN and eIF2α showed that the eIF2α phosphorylatable residue S51 could correspond to HIV-1 IN residue S57, located at approximately 17 Å from 82GYIE85 in the model of the HIV-1 IN structure36. Then, S57 showed to be phosphorylated by JNK6 may have been a good candidate to serve as the phosphorylation site for GCN2. However, the work by Manganaro et al.6 showed that phosphorylation of IN on residue S57 increased the protein stability leading to an increase in infectivity in activated T cells. In our study, infectivity was increased when phosphorylation of IN was prevented, either by mutating IN or by knocking out GCN2. These opposite phenotypes tend to argue that two different regulation mechanisms are involved in the work by Manganaro and our work.One of the new challenges in HIV research is to better understand the integration process at the cellular level including the mechanisms governing its regulation. While several post-translational modifications of HIV-1 IN have already been describedUsing various truncated versions of HIV-1 IN showed that only the CCD-CTD two domain fragment was an appropriate substrate for GCN2. Because neither the isolated CCD nor the CTD were efficiently phosphorylated by GCN2, this indicates that i) the interaction IN-GCN2 and the phosphorylation site are located at the CCD-CTD junction or distributed within the two domains, or ii) GCN2 recognizes IN under a specific oligomeric organization that can only be formed in the context of this two-domain fragment and not with the isolated domains. Using point mutations that affected the oligomerization status of IN also affected the phosphorylation of the mutant. This data is in favor of the second hypothesis where GCN2 would recognize a specific oligomer of IN. Similarly, using the PFV CCD with the HIV CTD enabled a phosphorylation of the chimeric protein at a level comparable to that of the full-length HIV-1 IN showing that the CCD of PFV can be used as a substitute to that of HIV-1. Thus, these data indicate that an IN CCD, either from PFV or HIV, was required to catalyze the GCN2 mediated phosphorylation. While we cannot rule that sequences conserved between the two orthologous proteins might be involved, it seems that the CCD might play a structural role required for GCN2 recognition. On the other side, the CCD of PFV IN only enabled phosphorylation by GCN2 of the CTD of HIV-1 IN and not of the native PFV IN CTD. Thus, it seems that the CTD of PFV IN is lacking an important feature for GCN2 activity. When comparing IN from HIV-1 and PFV, only 2 phosphorylatable residues serine or threonine are present in the CTD of HIV-1 IN and absent in that of PFV IN. Namely, HIV-1 IN harbors a serine in positions 255 and 283 while PFV IN harbors a glycine and histidine at the corresponding position, respectively. On the other hand, IN from HIV-2, ASV and MLV also sustained phosphorylation by GCN2. All these three proteins present a threonine residue at position 255 or position 359 in the case of MLV. Mass spectrometry confirmed that HIV-1 IN is indeed phosphorylated on two highly conserved serine residues, S24 and S255. Additional biochemical data obtained with mutants INs pinpointed to S255 as being the main phosphorylation site. Thus, it seems that GCN2 recognizes a specific 3D arrangement of the IN oligomeric complex as well as a specific sequence/residue located in the CTD of retroviral integrases.8. But, having identified IN residues that were phosphorylated by GCN2, the corresponding mutations were introduced in the viral context and resulted in an increase in viral infectivity. Conversely, infectivity of viruses carrying an aspartic acid at IN residue 255, that mimics a phosphorylation, was decreased in GCN2−/− cells compared to the WT. Although reverse transcription was not affected, viruses carrying mutations of GCN2’s phosphorylation site exhibited an increase in integrated DNA copy number. Together, these results suggest that GCN2 might decrease viral infectivity by limiting viral integration through the phosphorylation of IN residue S255.In the present study, the infectivity of HIV-1 was increased in cells knocked out for GCN2. This suggested that when it is present, GCN2 can restrict viral replication. A similar phenotype was observed in our previous study, where cellular expression of GCN2 was decreased by RNA silencing38. Here, no difference could be observed in term of stability, cellular expression or subcellular localization between the WT and mutant HIV-1 IN proteins over-expressed in 293 T cells. Thus, IN phosphorylation seems to restrict viral integration and replication without affecting IN bioavailability.Phosphorylation and proteolysis have already been linked in the case of enzymes that as IN belong to the family of the polynucleotidyl transferases, namely transposases. Additionally, it has been shown that IN from an avian retrovirus was phosphorylated on its CTD, on residue S282 and that phosphorylation could control the IN proteolytic processinget al. showed that the Ty5 IN phosphorylation is required for the interaction of IN with the heterochromatin cellular protein Sir4, which tethers the integration into telomeres39. In the case of HIV-1, it is known that integration occurs mainly in the body of highly transcribed genes and that this selectivity is mainly driven by the interaction of IN with the cellular cofactor LEDGF. Sequencing of integration sites revealed that phosphorylation of HIV-1 IN doesn’t affect tethering of the pre-integration complex to the integration site. IN mutations did not affect the integration profile, in terms of genomic region (body of genes) or specific histone modifications. Yet, it should be pointed out that the comparison was performed using data from K562 and Gm12878 cells for the epigenetic marks map while our integration map was obtained in 293 T cells. Also, the S255D single mutant could not be analyzed because for an unknown reason we could not generate enough data.Still, the observed decrease in integration when IN is phosphorylated may arise from an interference with the interaction between IN and a cellular co-factor. This hypothesis is particularly attractive as a precedent has already been reported in the literature. In the case of the yeast Ty5 retrotransposon, Dai In vitro, various assays were performed to evaluate the potential effect of IN mutations on the 3′-P activity and the ST activity. While no significant differences were observed between non-phosphorylatable proteins and the WT enzyme, mutation S255D in combination or not with S24D induced a more profound catalytic defect. This is in agreement with the decreased level of integrated DNA observed with the S255D mutant virus and the corresponding increase obtained in the cells lacking GCN2. Altogether, the data showed that phosphorylating IN at position S255 might decrease its activity leading in the cell to lower level of proviral integration.Finally, the observed increase in viral infectivity when blocking IN phosphorylation might be the resulting of a direct effect of IN capability to catalyze integration. in vitro phosphorylation of IN from other orthoretrovirinae such as MLV and ASV, GCN2 seemed to also restrict MLV infection. Similarly to what was observed in the case of HIV-1, infectivity of MLV-based viruses was enhanced in GCN2−/− cells compared to a parental cell line, with a 2-fold increase in EGFP positive cells. Accordingly, it seems possible for GCN2 to control the replication of retroviruses for which the IN is phosphorylated by the kinase. Because retroviral vectors are actively studied for gene therapy, the fact that their efficiency may be directly linked to the capacity of GCN2 to phosphorylate the IN protein used in such a vector is of interest.As first suggested by the 40 and yeast INs39, has been reported. Further studies will establish if GCN2 may exert a general control regulating DNA mobility in the cell and might consequently contribute to the general genome stability and innate immune response.In conclusion, phosphorylation of enzymes involved in DNA mobility such as retroviral INs, eukaryotic transposasesSupplemental Table S1


O-glycosylated at the threonine side chains with β-d-galactosyl-(1 → 3)-α-N-acetyl-d-galactosamine. Due to their interesting properties and high antifreeze activity, they have many potential applications, e.g., in food industry and medicine. Current research is focused towards understanding the relationship between the structural preferences and the activity of the AFGPs, as well as developing time and cost efficient ways of synthesis of this class of molecules. Recent computational studies in conjunction with experimental results from NMR and THz spectroscopies were a possible breakthrough in understanding the mechanism of action of AFGPs. At the moment, as a result of these findings, the focus of research is shifted towards the analysis of behaviour of the hydration shell around AFGPs and the impact of water-dynamics retardation caused by AFGPs on ice crystal growth. In the field of organic synthesis of AFGP analogues, most of the novel protocols are centered around solid-phase peptide synthesis and multiple efforts are made to optimize this approach. In this review, we present the current state of knowledge regarding the structure and activity of AFGPs, as well as approaches to organic synthesis of these molecules with focus on the most recent developments.Antifreeze glycopeptides (AFGPs) are a class of biological antifreeze agents found predominantly in Arctic and Antarctic species of fish. They possess the ability to regulate ice nucleation and ice crystal growth, thus creating viable life conditions at temperatures below the freezing point of body fluids. AFGPs usually consist of 4–55 repetitions of the tripeptide unit Ala–Ala–Thr that is  O-glycosylated at the threonine side chains. The number and type of AFGPs in the blood sera of polar fish vary with habitat temperature and depth (Fields and DeVries Antifreeze glycopeptides (AFGPs) are a class of biological antifreeze agents found in certain Arctic and Antarctic ectotherm species. These unique compounds have the ability to regulate ice nucleation and ice crystal growth, thus creating viable conditions at temperatures below the freezing point of body fluids. Antifreeze glycopeptides typically consist of 4–55 tripeptide units of Ala–Ala–Thr that are AFGPs are assumed to act through an adsorption–inhibition process in which the AFGP molecule irreversibly binds to the surface of an ice crystal, resulting in a freezing point depression through the Kelvin effect. A characteristic change in the morphology from spherical to hexagonal–bipyramid-shaped crystals occurs upon binding of AFGPs to specific ice crystal surfaces. AFGPs can also inhibit ice recrystallization, an Ostwald ripening process, in which larger crystals grow at the expense of smaller ones.There are several possible applications of AFGPs due to their unique properties. These applications include use in ice-slurries and in the food industry , as well as for biomedical purposes for the long-term cryopreservation of cells and organs or tissues  and forApplying these compounds for biomedical and industrial purposes requires significant amounts of AFGPs in a pure form and at reasonable costs. The isolation of AFGPs from natural sources is both expensive and labour intensive. Therefore, it is necessary to develop an efficient method of producing a wide range of homogeneous and native AFGPs.The previous major reviews on this topic Ben  focused This review aims to summarize the current state of knowledge together with the trends and approaches in research regarding the structure and mechanism of action of AFGPs, as well as the most recent advances in techniques for their synthesis, along with a potential application of these methods for technical purposes.To survive under subzero temperature conditions that are present in the harsh environment of the Arctic and Antarctic seas, many marine organisms developed an evolutionary means of survival—the expression of antifreeze agents. AFGPs prevent the uncontrolled growth of ice in vivo by artificially lowering the freezing point below that of the surrounding seawater, which is approximately −1.8 °C.O-glycosylated with the disaccharide fragment β-d-galactosyl-(1 → 3)-α-N-acetyl-d-galactosamine  and nuclear magnetic resonance spectroscopy (NMR). Recent achievements in this field include the introduction of more elaborate analytical methods, such as atomic force microscopy (AFM).1H and 13C NMR experiments at variable temperatures, as well as nuclear overhauser effect (NOE) measurements. In the case of low-molecular-weight AFGPs, the results showed that the overall structure is temperature dependent. At higher temperatures, the AFGPs adopt the conformation of a flexible coil, but lowering the temperature results in the formation of a threefold left-handed PPII helix. However, AFGPs1–4 were determined to be flexible rods with low structure ordering. Rao and Bush -α-d-N-acetylgalactosamine. In the first step, they prepared the monomer in 17% yield from commercially available 1,3,4,6-tetra-O-acetyl-2-azido-2-deoxy-α/β-d-galactopyranose. Then, the solution-phase method was used to polymerize the monomer. As a result, a mixture of polymers with an estimated mass of 6000–7300 Da (n = 10–12) was obtained.Tsuda and Nishimura  were theLater, Tachibana et al.  reportedN-acetyl groups. Tseng et al.  to achieve better control over the peptide sequence and length. The first solid-phase approaches to the synthesis of the AFGP analogues were reported by Filira et al.  and Meldg et al.  reportedg et al.  publisheThe synthesis of AFGP is divided into two main parts. The first step is the preparation of a glycosylated threonine building block. In the subsequent step, different amino acids are coupled with the peptide sequence via SPPS. The preparation of a suitable building block is crucial in the synthesis of antifreeze glycopeptides. A simple and highly efficient method of synthesizing a glycosylated amino-acid block would improve the efficiency of the entire process.d-galactosyl-(1 → 3)-β-N-acetyl-d-galactosamine attached to the side chain required fewer synthetic steps compared with the method reported by Tseng et al.  due to the poor reactivity of sugar amino-acid derivatives  is a simple and efficient method of preparing mono-disperse antifreeze glycopeptide oligomers. The procedure remains the same as that used for normal SPPS and consists of two steps. A schematic view for the preparation of the antifreeze glycopeptides using SPPS and MW SPPS is presented in Fig. The best results in semi-automated peptide synthesis were achieved with an optimized microwave irradiation procedure with a maximum temperature of 40 °C and using a 2-chlorotrityl resin loaded with Fmoc–Ala–OH -N-acetylgalactosamine attached to the peptide backbone. Interestingly, the unglycosylated Ala–Hyp–Ala repeated sequence exhibits significant thermal hysteresis and the ability to change the shape of the ice crystal, which leads to the conclusion that it could bind to the ice surface . Improper reaction conditions were shown to cause massive decomposition and resulted in the peptide deglycosylation (Heggemann et al. Note that the latest studies focus on the preparation of simplified forms of AFGPs while maintaining the antifreeze activity. The studies of simplified AFGP analogues could explain the exact mechanism of action of these compounds. Furthermore, non-toxic and structurally less complicated AFGP analogues, which can be obtained in a simpler and more efficient manner, provide an opportunity for the application of these materials in the food industry and in biomedicine.


We mapped the cation binding site close to the anion binding site and established a specific mechanism of inhibition occurring only in the closed conformation of the enzyme when the catalytic cysteine is phosphorylated and the catalytic aspartate moves into the active site. We discuss this dual inhibition by anions and cations here for PTP1B, the most thoroughly investigated protein tyrosine phosphatase. The significance of the inhibition in phosphorylation signaling is becoming apparent only from the functions of PTP1B in the biological context of metal cations as cellular signaling ions. Zinc ion signals complement redox signals but provide a different type of control and longer lasting inhibition on a biological time scale owing to the specificity and affinity of zinc ions for coordination environments. Inhibitor design for PTP1B and other PTPs is a major area of research activity and interest owing to their prominent roles in metabolic regulation in health and disease, in particular cancer and diabetes. Our results explain the apparent dichotomy of both cations (Zn2+) and oxyanions such as vanadate inhibiting PTP1B and having insulin-enhancing (“anti-diabetic”) effects and suggest different approaches, namely targeting PTPs in the cell by affecting their physiological modulators and considering a metallodrug approach that builds on the knowledge of the insulin-enhancing effects of both zinc and vanadium compounds.A new paradigm in metallobiochemistry describes the activation of inactive metalloenzymes by metal ion removal. Protein tyrosine phosphatases (PTPs) do not seem to require a metal ion for enzymatic activity. However, both metal cations and metal anions modulate their enzymatic activity. One binding site is the phosphate binding site at the catalytic cysteine residue. Oxyanions with structural similarity to phosphate, such as vanadate, inhibit the enzyme with nanomolar to micromolar affinities. In addition, zinc ions (Zn Clearly, metal ions activate apoenzymes to form active holoenzymes, i.e. metalloenzymes, but this aspect of metallobiochemistry is not considered part of regulation. It is estimated that about 50% of all enzymes require a metal ion, and it is assumed that metalloenzymes are 100% metallated and that the metal ion remains bound during the lifetime of the protein. However, a paradigm for the opposite situation is emerging, namely an active apoenzyme without a metal ion and an inactive holoenzyme with a bound metal ion 2+ and metal anions such as vanadate extremely tightly and with concomitant inhibition. The fact that zinc ion concentrations in the cell are in the range where such enzyme inhibition is observed strongly supports the idea that zinc(II) modulates protein tyrosine phosphatase activity in vivoOver sixty years ago, the late Bert L. Vallee suggested that “[metal ions] may accelerate or inhibit reactions selectively. With reference to the latter, one can point out that about 50 percent of all known enzymes require free sulfhydryl groups for activity: it is well known that metal ions can react with sulfhydryl groups, thereby inhibiting these enzymes. It is completely unknown whether the body avails itself for inhibiting, activating, or both actions of metal – or neither – to control enzyme reactions” Zinc(II) is now known to be a cellular signaling ion, but targets have not been confirmed. PTPs are likely targets as phosphorylation states of many signaling proteins depend on changes in cellular zinc(II). It appears that we have missed a major area of metabolic regulation, in particular a role of zinc ions in the most significant way of signal transduction, namely phosphorylation signaling, and that the number of enzymes that interact with zinc(II) is significantly larger than the already impressive estimate that about 3000 human proteins may require zinc(II) as a cofactor 1.1Protein tyrosine phosphorylation is a dynamic process that regulates many cellular processes through the activities of kinases and phosphatases. Dysregulation of either one of these enzymes is linked to multiple diseases. The human genome contains 107 PTPs divided into several subgroups PTP1B dephosphorylates several substrates including the insulin and leptin receptors, hence having an important role in metabolism. PTP1B knock-out (KO) mice are more sensitive to insulin and have lower serum glucose and insulin levels; when put on a high fat diet, they are resistant to weight gain and insulin. Moreover, the phosphorylation of the insulin receptor (IR) in both skeletal muscle and liver is increased in KO compared to control mice PTP activity is tightly regulated by a plethora of mechanisms including oxidation, phosphorylation, sumoylation, and dimerization 1.2181 to act as a general acid/base. The side chain of Arg221 changes orientation and assists the closure of the WPD loop. Interactions between the re-positioned Arg221, which stabilizes the phosphate group, and the Trp179 and Pro180 residues stabilize the WDP loop in the closed conformation. The sulfhydryl group of Cys215 is the catalytic residue. It has a characteristically low pKa of 5.5 181 transfers a proton to the tyrosine oxygen group of the leaving substrate. In the second step, the resulting phospho-cysteine intermediate is hydrolyzed. Gln262 aligns a water molecule for the nucleophilic attack on the phospho-cysteine intermediate while Asp181 now functions as a general base accepting a proton , electronic absorption  or radioactivity  50 values relative to the substrate concentration employed in the assays are obtained. The true Ki values can be significantly lower. For example, an IC50 value of 98 pM was estimated for receptor protein tyrosine phosphatase β (PTPRB), but the Ki value is actually 21 pM after a full kinetic analysis was performed PTP activity is measured with several enzymatic assays; most of them involve the use of a small molecule or a peptide. Dephosphorylation of these substrates can be detected by fluorescence , dithiothreitol (DTT), or other metal ion chelating agents that compete with the protein for metal ion binding. The presence of these agents will influence the free metal ion concentrations. Therefore, when assays are performed in the absence of a metal ion buffer, the reported inhibition constants may need to be re-evaluated as the inhibition is likely to be stronger.in vitro is not any different from the situation in vivo, where all metal ions are buffered in specific ranges by proteins and small molecules.In our experiments, we employ metal ion buffers that control the free metal ion concentrations. The choice of the chelating agent as a metal ion buffer depends on the desired buffering range, and for the measurement of PTPs it is nitrilotriacetic acid (NTA), as it buffers metal ions such as Zn(II), Cd(II) and Cu(II) in the range where inhibition is observed. Moreover, we measure the total amount of metal present in the solutions by ICP-MS. Using software such as MaxChelator 2Metal ions bind to proteins through interaction with the donors of amino acid side chains, the ligands. The cellular concentrations of the different metal ions vary substantially, and for some metal ions participating in cell signaling, the concentrations fluctuate and depend on the status of the cell.e.g., charges, size) and the ligands . Coordination numbers have been analyzed by employing the Cambridge Structural Database The coordination number is governed by the properties of both the metal ions  and vanadate ions (VO43−) were originally described as inhibitors of PTP1B The enzymatic activity of PTPs is affected by both metal cations and  oxyanions. Among those, zinc ions (Zn2.2We interrogated the Protein Data Bank (PDB) for metal cations (Cd(II), Cu(II), Zn(II), Fe(II)) bound to human PTP1B. None of the 129 crystal structures deposited contains any of these metal ions near the catalytic site. This finding, however, does not mean that metal ions are unable to bind to PTP1B.2.2.150 or Ki values Zinc(II) has been known as an inhibitor of PTPs since the early 1980s 2+Zinc ions also inhibit PTPs in cultured cells. When glioma cells were incubated with zinc ion concentrations in the presence of the zinc(II) ionophore pyrithione, a time-dependent increase in protein tyrosine phosphorylation was observed 2CM2), the enzyme in the closed conformation with the phospho-cysteine intermediate (PDB id: 1A5Y) and the dephosphorylated enzyme in the closed conformation, which was obtained by removing vanadate from the second transition state structure (PDB id: 3I80). For each of the structures, we found several zinc ion-binding sites at the periphery of the enzyme . Because the dephosphorylated substrate is released in the first catalytic step, the retention of 50% activity suggests that the enzyme is inhibited in the second catalytic step when it is still in the phosphorylated form. This observation would indeed explain the failure to see complete inhibition even at high zinc ion concentrations e enzyme .181 has moved into the active site . In the dephosphorylated form of the enzyme (obtained by removing vanadate from the structure), zinc(II) is in a coordination sphere surrounded by Asp181, Arg221, Gln262 and Cys215  (262 coordination of zinc(II) is not shown in 262 is re-introduced in the position in silico, the coordination of the docked zinc ion is very strong with a distance of about 1.5 Å. If Tyr46 changes its position, it may become a ligand as well. The open conformation fails to accommodate zinc(II) in the catalytic pocket. The idea that zinc(II) binds to the catalytic site is supported by the observations that zinc(II) is a competitive inhibitor of PTPRB 262 and Asp181 become ligands of zinc(II) in the closed conformation, Gln262 no longer aligns a water molecule for catalysis and Asp181 can no longer serve as the catalytic base for the dephosphorylation of the phospho-cysteine intermediate in the second step of the reaction. Hence zinc(II) traps the enzyme in the phospho-cysteine form. The coordination of zinc(II) in this form is exclusively from oxygen donors.Remarkably, zinc ion binding to the catalytic site was observed only when the conformational change of the WPD loop has occurred and the catalytic residue Aspd Cys215 A. In thezinc(II) B. The cr266 (1 oxygen), Asp181 (1–2), phosphate (1–2), and Gln262 (1) could all contribute oxygen ligands. Gln has been identified as a ligand of zinc(II) in proteins e.g. the phosphorylated insulin receptor. Zinc(II) would not be an insulin-mimetic as the insulin receptor would not function in signaling if PTP were to remain bound to it. We cannot rule out, however, that zinc(II) locks the enzyme in a closed conformation once the second substrate, i.e. the phosphate group, has left the catalytic site. At present, there does not seem to be a precedent for this mechanism as the cleavage of the phospho-cysteine intermediate is coupled to the transition of the enzyme from the closed to the open conformation.Though the zinc(II) site in the closed conformation of PTP1B is unlike typical zinc(II) coordination in zinc metalloenzymes, it is not without precedence. The number of oxygen donors is sufficient to provide a strong binding site as zinc(II) binds to the magnesium(II) site in phosphoglucomutase and inhibits the enzyme with picomolar affinity 2.2.22+ inactivates recombinant PTPs  in a concentration-dependent manner 2O2), the interaction of H2O2 and Fe2+ with PTP1B was also examined. Physiological concentrations of Fe2+  to this PTP Mononuclear di-citrate (cit) iron complexes interact with PTPs and inhibit their activities ∼500 nM,  not only2.2.350 values of 100 nM and 1 μM for VHR and PTP1B, respectively. However, enzymatic activity was not fully recovered by adding EDTA. Only the presence of reducing agents reactivated the enzymes completely, suggesting that copper(II) oxidizes the catalytic cysteine 2+ and a metal buffering system, we found a much lower IC50 value of 0.3 pM .Inhibition of PTPs has been explored with copper(II) complexes. The inhibition constants vary between low nM to low μM values 2.2.42+ inhibits other phosphatases, there is no report on its effect on PTPs 2+ in an NTA-containing buffer, an apparent inhibition constant of ≈5 nM was obtained , very similar to the one for zinc(II) (5.6 nM), based on concentrations of free cadmium ions calculated as indicated above for zinc.Although Cd2.3Ki value of ∼17 mM Ki (IC50) values of 1.5 μM (vanadate), 9 μM (heptamolybdate), 210 μM (tungstate), 200 μM (arsenate), and 54 mM (nitrate) were measured . The different inhibition constants may reflect the different ionization states of the anion at the pH of investigation, the propensity of vanadate to form a covalent intermediate analogous to the phospho-intermediate in contrast to the other anions, which form Michaelis-like complexes, and geometric factors, such as in the case of nitrate, which has a planar geometry  and 7.2 for dihydrogen phosphate. Thus there is comparatively less di-anion (HVO42−) at pH 7.0. Only the oxidation state +5 of the non-metal phosphorous is relevant for mammalian biology, whereas minimally two oxidation states of the metal vanadium, +4 and +5, are important. Thus phosphorous is redox-inert whereas vanadium is redox-active in biology. Vanadate was discovered as a strong inhibitor of (Na/K)-ATPase in a commercial ATP preparation (Ki = 40 nM in the presence of 25 mM Mg2+) Ki of 0.38 ± 0.02 μM (pH 7.3) Ki of 1.0 ± 0.6 μM (pH 7.5) and forms a trigonal bipyramidal (tbp) SN2 transition state τ-values, the ratio of the angle of the axial vs the equatorial ligands in tbp geometry divided by 60, i.e., τ = (β − α)/60, which, for an ideal geometry with β = 180° and α = 60°, gives a τ-value of 1 τ-values ranging from 0.83 (PDB id: 3I7Z) to 0.90 (PDB id: 3I80) and structures described best as umbrella-like inversions with deviations of ligands from the equatorial plane in exploded transition states, a term referring to increased bond lengths of the breaking and forming bonds. Further investigations employing shape analysis – continuous symmetry measures 215; the equatorial oxygen atoms bind to the amide groups of the P-loop  and to the guanidinium group of Arg221. Gln262 in the first transition state is rotated compared to the position in the second transition state, where it binds the apical oxygen in the trigonal vanadate moiety 262 is critically important as it aligns a water molecule as a nucleophile for releasing the second product, phosphate, from the intermediate. Together the structures of complexes with different oxyanions, i.e., phosphate, vanadate, and tungstate were instrumental in formulating the reaction pathway of PTP1B  ion bound in the active site 4S covalent intermediate 446 and Gln450, to a water molecule, and to the amino group of Lys447  2O)5]2+ with a square pyramidal geometry undergoes consecutive hydrolysis when the pH increases to form anionic species at nanomolar to submicromolar concentrations: [VO(H2O)4(OH)]−, [(VO)2(OH)5]−, and [VO(OH)3]−. Thus, there are anionic and cationic species in equilibrium with insoluble {VO(OH)2}n and with [(VO)2(OH)5]− being a prominent species at neutral pH 2]+, which binds to proteins such as transferrin Vanadate undergoes multiple chemical reactions, such as complexation with many commonly employed buffers, reduction, and olation to form polyoxymetallates 50 values between 30 nM and about 1 μM but rarely do they show pronounced selectivity for a particular phosphatase In addition to inorganic vanadate(V) and oxidovanadium(IV), many organic vanadium(V) and vanadium(IV) compounds were investigated as inhibitors of PTP1B owing to their potential as antidiabetic agents and for many other therapeutic indications (see below). Vanadium complexes inhibit PTP1B with IC2+, oxidovanadium(IV). VO(acac)2 (acac = acetylacetonate) is an uncompetitive inhibitor of PTP1B when assayed with a phosphotyrosine peptide that stimulates the epidermal growth factor receptor (EGFR) 50 of 0.86 ± 0.02 μM. In this case, the inhibition is mixed with Ki values of 1.2 and 2.1 μM for the enzyme and the enzyme/substrate (p-nitrophenyl phosphate) complex, respectively Vanadium compounds receive a great deal of interest as antidiabetic agents and, more recently, as potential anticancer drugs 2.3.2Yersinia PTP (YopH) with tungstate indicates that tungstate behaves like other phosphate mimetics, namely closing the WPD loop 216, Ile219 and Gly220. Arg221 also coordinates with one of the tungstate oxygens. Tungstate binding to the catalytic site of PTP1B causes conformational changes in the enzymes: the side chains of both Cys215 and Arg221 re-position towards the oxyanion. However, the WPD loop remains in the open conformation. The fact that the crystals were obtained by soaking PTP1B in the presence of high concentrations of tungstate together with the fact that the WPD loop is closed in the Yersinia PTP structure, suggests that binding of tungstate in the open conformation of PTP1B is an artifact of the crystallization condition.The crystal structure of 2.3.3There are no available structures of PTP1B with molybdate. There is, however, a structure of the bovine low molecular weight PTP co-crystallized with molybdate 2.3.43−) has on PTP activity. Hence we determined its inhibition. The IC50 value is 54 mM . Crystal structures of PTP1B in the presence of nitrate have been solved 221, Asp181, Ser216, Ala217, and Gly220 . There is no crystal structure of PTP1B with arsenate. However, arsenate reductase belongs structurally to the family of PTPs and several crystal structures have been solved Arsenate inhibits PTP1B with a 3Both metal cations and oxyanions inhibit the activity of PTP1B. Although several metal ion complexes have been investigated with regard to inhibiting PTPs and shown promises as metallodrugs, and in the case of vanadium compounds entered clinical trials, none have developed into human therapeuticals. Given the inhibition of PTP1B by free metal ions, investigations of the interaction of metal ion complexes with PTPs need to consider the stability of the metal ion complexes in order to determine whether the inhibition is due to the metal ion complex itself or the cation dissociating from the complex. Understanding the interaction of free metal ions and/or oxyanions with PTP1B, therefore, is important in order to improve the design of potential therapeutic agents.3VO4 to diabetic patients decreased glycosuria in vitro and in vivo. Of interest was the discovery that Na3VO4 normalized hyperglycemia in an animal model of diabetes i.e. vanadium undergoing 1-electron redox cycling, thus accounting for the formation of organic radicals and the toxicity of vanadium compounds. Among vanadium compounds, bisoxidovanadium(IV) (BEOV), showed encouraging results and was in clinical trials as a prodrug for the treatment of type 2 diabetes 2+, oxidovanadium(IV)) chelate and the corresponding acetylacetonate have been investigated in detail structurally and with regard to their synergism with insulin in cells Investigation of vanadate as an insulin-mimetic dates back to 1899, when it was observed that oral administration of NaSimilar to vanadate, tungstate alters glucose metabolism when added to rat hepatocytes Molybdate has also been widely used as a PTP inhibitor e.g. vanadate, ∼1 μM) inhibit PTPs. Vanadium concentrations in human blood are 0.2–15 nM (median 50 ng/L in 108 human subjects) Ki of 17 mM while the cellular phosphate concentrations are in the range of 0.5–5 mM.It is unclear whether the concentrations at which vanadate and other oxyanions inhibit the activity of PTP1B occur under physiological conditions. Relatively low metal oxyanions concentrations  and at least nine out of the ten members of transporters (ZnT/SLC30A) transport zinc ions into or out of the cytosol SLC39A7) is located on the ER membrane and responsible for transporting zinc ions into the cytosol. Stimulation of cells with EGF in the presence of high intracellular Ca2+ activates casein kinase 2 (CK2) that is consequently recruited to the ER, where it phosphorylates Zip7 on Ser275 and Ser276Cellular zinc ion homeostasis is tightly controlled. It is fairly well established now that the concentration of free zinc ions in the cytosol is in the range of a few hundred pM 2O2 in both hepatocytes and adipocytes 2O2 production in adipocytes while silencing of Nox4 inhibited the insulin signaling cascade 2O2 are PTPs, in particular PTP1B, which is reversibly inhibited, thus enhancing insulin signaling pathways 2O2 with MTs. Oxidation of MTs results in release of tightly bound zinc ions, which also inhibit PTP1B 2O2 production and PTP oxidation Increases of cellular free zinc ions can also arise from the production of reactive oxygen species (ROS) and oxidation of metallothioneins (MTs) . Insulin3.1.2The inhibition of PTP1B by metal ions other than zinc ions does not appear to be physiologically significant but it may have significance under conditions where tissues and cells are exposed to toxic metal ion concentrations 3.2215 in PTP1B) and abrogate its nucleophilic function. Oxidation is reversible as the sulfenic acid form of the oxidized cysteine reacts with the adjacent serine side chain (Ser216) to form a cyclic sulfenamide with the purpose of avoiding further oxidation and facilitating reduction to restore the active PTP 46 (in PTP1B), normally buried in the structure, become more solvent-exposed.Oxidation/reduction (redox) is a key regulatory event in PTP function. Intracellular signals resulting in the production of ROS increase tyrosine phosphorylation due to inhibition of PTPs 215Oxidation can also lead to the formation of sulfinic or sulfonic acid derivatives of Cys3.3Given the role of PTP1B in the pathogenesis of type 2 diabetes and its involvement in cancer signaling, immense research efforts are made to pharmacologically targeting the enzyme There are two main challenges when designing PTP inhibitors: cell membrane permeability and selectivity Since diabetes and other diseases are associated with changes in zinc status, one would assume that an approach targeting intracellular control of zinc is worth exploring for pharmacological intervention.4regulating phosphorylation signaling and they are regulated themselves. We demonstrate additional principles of the regulation of PTPs, namely reversible inhibition by metal cations that inhibit differently from metal anions such as vanadate. Oxyanions bind to the enzymes in the open conformation but cations inhibit only if an oxyanion such as phosphate (or vanadate) is already present and new interactions for cation binding are made possible in the closed conformation of the protein when the catalytic aspartate moves into the active site.Phosphatases are the jockeys while kinases are the racehorses in cellular regulation. Phosphatases perform the task of Metal cation inhibition modulates PTPs differently from established redox modulation. Metal cations bind when phosphate (or another oxyanion) blocks the cysteine in the phospho-intermediate, and thus the sulfur is no longer available for redox modification. Possibly, the combined anion/cation inhibition protects the enzyme from redox modification.Metal modulation has been ignored in virtually all discussions of the biology of PTPs. The inhibition of PTPs by metal ions was recognized when these enzymes were initially characterized Transition metal ions are redox active and influence the redox state of the catalytic cysteine in PTPs. In contrast, zinc(II) is redox-inert in biology and cannot change the redox state of the cysteine. However, ROS release zinc ions from metallothionein and other protein sites and thus increase free zinc ion concentrations. Therefore, some of the effects of ROS on PTPs may be indirect, namely in providing zinc ions as a specific inhibitor of the enzymes. In as much as redox species are ER localized, so is zinc ion release, and specific pathways of zinc ion release support the physiological relevance. The physiological context of dual modulation of PTPs by metal ions and redox is likely another level of control of these important enzymes .i.e. inhibition occurs during enzymatic turnover while redox modulation targets the free enzyme before (or after) it has been enzymatically active.The interplay of anions, cations and redox agents provides a very rich chemistry for affecting the active site of PTPs and consequently their biological control. On a more speculative note, the zinc ion inhibition of the phosphorylated form relates the activity of the PTPs to the phosphorylation state of their substrates, Metal ion interactions with PTPs extend the role of metal ions in phosphatase action and phosphorylation signaling. Alkaline phosphatases use a binuclear zinc site and a regulatory magnesium ion, acid phosphatases a binuclear iron site, and protein phosphatases such as calcineurin a mixed binuclear zinc/iron site. Thus, in addition to using metalloenzymes in the control of phosphorylation signaling, biology has availed itself to using metal ions such as zinc and calcium for biological regulation of phosphatases.in vivo. Such an approach appears to be attractive since the zinc and vanadium inhibition of PTP1B provides a mechanistic basis for the rather copious literature on the pharmacology of insulin-mimetic and anti-diabetic effects of zinc and vanadium complexes It is remarkable that billions of dollars have been spent on drug design and clinical trials of inhibitors of PTPs without appreciating these structural and biological principles of regulation. The new insights provide a different perspective and may give another impetus for rational drug design and for inhibiting PTPs more specifically by modulating zinc metabolism