Lysine acetylation has been primarily investigated in the context of transcriptional regulation, but a role for acetylation in mediating other cellular responses has emerged. Multiple studies have described global lysine acetylation profiles for particular biological states, but none to date have investigated the temporal dynamics regulating cellular response to perturbation. Reasoning that lysine acetylation may be altered in response to growth factors, we implemented quantitative mass spectrometry-based proteomics to investigate the temporal dynamics of lysine acetylation in response to growth factor stimulation in cultured carcinoma cell lines. We found that lysine acetylation changed rapidly in response to activation of several different receptor tyrosine kinases by their respective ligands. To uncover the effects of lysine acetylation dynamics on tyrosine phosphorylation signaling networks, cells were treated with an HDAC inhibitor. This short-term pharmacological inhibition of histone deacetylase activity modulated signaling networks involving phosphorylated tyrosine and thereby altered the response to receptor tyrosine kinase activation. This result highlights the interconnectivity of lysine acetylation and tyrosine phosphorylation signaling networks and suggests that HDAC inhibition may influence cellular responses by affecting both types of post-translational modifications. Long thought to be primarily restricted to histone modification, lysine acetylation has recently emerged as a proteome-wide post-translational modification rivaling phosphorylation in scale and biological relevance –2. High-Many oncogenic phenotypes, such as increased migration and invasion, have historically been ascribed to aberrant phosphorylation signaling cascades driven by altered expression, mutation, or activation of protein kinases and phosphatases –16. A plin vitro and in vivo. To explore the effect of RTK activation on lysine acetylation at a network level, we implemented multiplex quantitative mass spectrometry based proteomics to profile temporal acetylation dynamics downstream of RTK activation in cultured carcinoma cells. Our data demonstrate that growth factor stimulation induces rapid changes in lysine acetylation across the acetylproteome, suggesting a link between tyrosine phosphorylation pathways activated by RTKs and altered lysine acetylation. These results provide insight as to how HDACi’s may modulate the tyrosine phosphorylation signaling response to RTK activation.The link between oncogenic signaling from receptor tyrosine kinases and altered lysine acetylation has not been systematically explored, yet such a study may provide insight into the mechanisms by which HDACi’s act to affect phenotypic outcomes both HepG2 (ATCC # HB-8065), A549 (ATCC# CCL-185), and DLD1 (ATCC# CCL-221) cell lines were obtained from ATCC. HepG2 cells were cultured in DMEM with 10% fetal bovine serum, 2mM glutamine, 100 units/ml penicillin, and 100 mg/ml streptomycin. A549 cells were cultured in RPMI with 10% fetal bovine serum, 2mM glutamine, 100 units/ml penicillin, and 100 mg/ml streptomycin. Prior to stimulation, cells were washed with PBS and incubated in serum-free media overnight. For TSA experiments, cells were pre-treated with vehicle or 1 μM TSA for one hour prior to growth factor treatment. A549 cells were stimulated with 100 ng/mL EGF or IGF-1 for 0, 1, 5, and 30 minutes, and HepG2 cells were stimulated with 150 nM insulin for the same times. Concentrated ligand (10 μL) was added to 10mL of cell culture media for stimulation. DLD1 cells were cultured in light (K0 & R0) or heavy (K8 & R10) SILAC media for at least six doublings to achieve efficient stable isotope incorporation. Cells were serum depleted; cells cultured in heavy SILAC were then stimulated with 100 ng/mL EGF for one minute. At appropriate time points, cells were lysed in 8M urea on ice. Lysates for western blotting were prepared identically, with the exception that cells for westerns were lysed in radioimmunoprecipitation (RIPA) buffer.Protein concentration was quantified using a BCA assay. Samples for mass spectrometry were prepared as previously described .iTRAQ labeling was performed as previously described (36) using 4plex iTRAQ for HepG2 analyses and 8plex iTRAQ for A549 analyses. Each timepoint was labeled with a unique iTRAQ label. Samples for a particular time course analysis were combined and concentrated to dryness in a speedvac. Peptides were then dissolved in 400 μL IP buffer  and the pH was adjusted to 7.4 prior to immunoprecipitation.40 μL of beads with preconjugated anti-acetyllysine antibodies  were rinsed four times and then combined with resuspended iTRAQ labeled peptides and incubated overnight at 4°C with rotation. Beads were rinsed once with IP buffer and then four times with rinse buffer , and peptides were eluted into 70 μL of 100 mM glycine pH 2. Eluents were cleared using immobilized affinity metal chromatography (IMAC) . Peptide14 overnight at 4°C. Membranes were washed three times, five minutes each with TBS-T prior to incubation with a secondary antibody for an hour at room temperature. Membranes were again washed three times, five minutes each, prior to incubation with the enhanced chemiluminescent kit.Equal protein amounts of lysate from each of the four time points were separated using SDS gel electrophoresis. Proteins were transferred to nitrocellulose membranes. Membranes were blocked for one hour using 5% bovine serum albumin (BSA) dissolved in Tris-Buffered saline with 0.1% Tween (TBS-T). Membranes were incubated with either a histone H3 or specific antibody against histone H3 Lyshttp://msquant.sourceforge.net/). All resulting MS/MS spectra were searched against an NCBI 2009 Homo sapiens database using Mascot version: 2.1.03 (Matrix Science). Trypsin enzyme specificity was applied with a maximum of nine missed cleavages. Mass tolerance was set at 10 ppm and fragment mass tolerance was set at 0.8 Da. MS/MS spectra searches incorporated fix N-terminal iTRAQ and carbamidomethylation of cysteine while incorporating variable modifications of oxidized methionine, and acetylation of lysine or iTRAQ modification of lysine. For SILAC analyses, MS/MS spectra searches incorporated fixed carbamidomethylation of cysteine, and variable incorporation of SILAC, acetylation, and methionine oxidation. Manual validation of peptides was performed using CAMV, a MATLAB-based tool for both precursor contamination and identification [Raw mass spectrometry data files were converted into the. mgf format using DTASupercharge 1.31  using isobaric tagging for relative and absolute quantification (iTRAQ). Unlike other methods to profile the acetylproteome, this method allowed for the quantification of up to eight timepoints simultaneously. Furthermore, the method developed here saw a reduction in the percent of non-specific binding (60% versus 80%) as reported elsewhere [To visualize the changes in lysine acetylation that had similar temporal responses, we clustered the acetylation profiles . AlthougTo better understand the regulation of acetylation dynamics by growth factor signaling, we selected proteins on which we identified multiple acetylation sites. Comparing the acetylation dynamics across multiple sites on the same protein revealed site-specific temporal dynamics. For example, histone H3 shows a diverse set of responses depending on the site modified , whereasTo determine whether the observed rapid acetylation dynamics occurred in multiple cell lines following stimulation of different RTKs, we measured the acetylation responses of A549 non-small cell lung cancer (NSCLC) cells stimulated with either insulin-like growth factor 1 (IGF-1) or epidermal growth factor (EGF). We stimulated A549 cells with 100 ng/mL IGF-1 or EGF for 0, 1, 5, and 30 minutes and then processed the samples for simultaneous comparison of EGF- or IGF-1-stimulated lysates within the same MS analysis. This analysis resulted in the identification of 84 unique acetyllysine-containing peptides on 61 proteins . Similar194 (unchanged) and the doubly-acetylated histone H4 peptide Lys6 and Lys9 (rapid and sustained decrease). The commonly regulated rapid dynamics on histone H4 suggests that a shared pathway between EGF and IGF-1 signaling may drive these acetylation dynamics. Although high-confidence differential dynamics between EGF and IGF-1 stimulated samples were not identified, it is worth noting that differential acetylation dynamics between these two RTK ligands may exist, and may be revealed through additional analyses.To identify shared temporal acetylation responses to EGF or IGF-1 stimulation, we clustered the dynamic profiles. Several sites showed a rapid decrease in acetylation that was sustained across the entire length of ligand treatment, whereas other sites showed a rapid increase immediately following stimulation followed by a decrease back to basal. The acetylation status of many sites did not change in response to ligand stimulation . Leverag194, recapitulating the results previously generated using iTRAQ and thereby confirming the trends observed. Additionally, we observed acetylation dynamics on a different site on GAPDH Lys263 where EGF stimulation induced a five-fold increase in lysine acetylation. To further confirm the trends of our mass spectrometry based analysis, we quantified histone H3 Lys14 acetylation dynamics in A549 cells stimulated with EGF using western blotting . Heavy isotope labeled DLD1 cells were stimulated with 100 ng/mL EGF for one minute; light isotope labeled DLD1 cells were unstimulated. These cells were then processed for MS analysis. We identified 39 unique acetyllysine-containing peptides on 23 proteins . Rapid ablotting . Western798 on PTPRA has been shown to drive an increase in tyrosine phosphorylation; these effects may either be due to the direct result of reduced PTPRA activity or the altered activity of PTPRA substrates [798 on PTPRA, the function of the phosphorylated tyrosine residues that showed a change in status following TSA treatment is poorly characterized in the literature. Previous studies have demonstrated that acetylation and phosphorylation can act in a coordinated fashion on the same protein, providing a potential explanation for altered tyrosine phosphorylation following short-term TSA treatment. However, the bottom-up proteomics method applied here did not identify acetylated peptides originating from these proteins . In the absence of RTK stimulation, 45 phosphotyrosine-containing peptides on 34 proteins were identified and quantified , along wbstrates . Thus, bproteins . These d354 on TNS3 and Tyr844 on catenin delta 1 (CTNND1), demonstrating large changes in their EGF response (FCR < 0.3 and FCR >1.9). For many other phosphorylation sites in the network, including EGFR phosphorylation sites, TSA pre-treatment did not affect the phosphorylation response to EGF stimulation. Despite a plethora of publications regarding EGFR signaling, many EGF-regulated phosphorylation sites have not been mechanistically connected to the receptor, yet may be functionally relevant. Several of the sites affected by TSA treatment fall into this group of proteins whose connection to EGFR is undefined, but phosphorylation of these proteins might be critical for regulating downstream biological response to therapy. Overall, this data demonstrates that inhibition of lysine deacetylase activity affects the tyrosine phosphorylation of specific components downstream of the receptor within the EGFR network, suggesting that acetylation may be a regulatory input into the EGF pathway.To determine whether altered lysine acetylation affected the phosphorylation signaling network response to RTK activation, we quantified tyrosine phosphorylation in cells pre-treated with TSA for one hour and then stimulated with EGF for 5 minutes. This analysis resulted in the identification and quantification of 82 tyrosine phosphorylation sites on 64 proteins . To quanTo understand the potential connection between phosphotyrosine signaling and lysine acetylation, we quantified the immediate-early temporal dynamics of lysine acetylation in response to growth factor stimulation. To our surprise, many lysine acetylation sites displayed dramatic changes in their amount of modification within one minute of growth factor stimulation. Other network-level studies have demonstrated rapid changes in the phosphorylation signaling network in response to RTK activation –33. WhilPhosphotyrosine profiling of cells treated with HDAC inhibitors suggests a complex relationship between these two dynamic post-translational modifications. Our results demonstrate that short-term treatment with an HDAC inhibitor modulates the response to EGF stimulation, establishing a link between acetylation and the enzymes that regulate tyrosine phosphorylation. We first identified phosphorylation sites that were altered upon short-term TSA treatment. Three of the four proteins in which altered phosphorylation was detected upon TSA treatment are proteins implicated in cell adhesion. This result suggests that perturbation of deacetylases may influence these processes, a result that agrees with reports implicating HDACs such as HDAC6 in regulating cell motility . With bi14 has been identified at active sites of transcription suggesting a functional consequence of lysine acetylation on this site [Lysine acetylation has long been synonymous with transcriptional control as acetylation alters chromatin structure and histone-DNA interactions. To this end, we observed rapid acetylation dynamics on multiple sites on histone proteins within one minute of EGF stimulation. How these changes might affect transcription is yet to be determined, but results from the Yarden lab have demonstrated altered expression of hundreds of genes within 20 minutes of EGF stimulation . The pothis site . In addihis site –4. In ouhis site –39. HereWhile this study has demonstrated that there exists a link between lysine acetylation and tyrosine kinases, the exact mechanism by which ligand stimulation drives acetylation dynamics remains poorly understood. Signaling pathway diagrams of the EGF signaling network rarely include enzymes involved in altering lysine acetylation, although several studies have suggested potential links between the EGF signaling network and various acetyltransferases and deacetylases. For instance, HDAC6 and EGFR have been shown to interact in a modified yeast two-hybrid assay; this same study established a negative feedback loop in which HDAC6 is phosphorylated at Tyr570 and thereby regulates EGFR endocytosis . AnotherThis study has highlighted that tyrosine kinase activation can lead to rapid changes in lysine acetylation. This discovery suggests the existence of novel pathways that link phosphotyrosine signaling with proteins known to regulate lysine acetylation. Furthermore, the temporal dynamics experiments and subsequent experiment demonstrating feedback from HDAC inhibition to phosphotyrosine signaling highlights novel avenues in which therapeutic interventions may have a broader effect on signaling and the subsequent cellular responses. These results suggest future experiments to investigate the link between these two PTMs as well as links between other modifications. For instance, it is conceivable that rapid changes in lysine acetylation may also lead to rapid changes in other lysine post-translational modifications such as methylation and ubiquitylation, since lysine can be the site of a number of different post-translational modifications. Future studies should continue to focus both on the diversity of the perturbations that may alter these post-translational modification networks as well as the time scale over which these responses may be regulated. Further characterization of the specific function of these acetylation and phosphorylation sites will allow us to link the temporal dynamics with a particular cellular response. While the similarities between lysine acetylation and tyrosine phosphorylation dynamics are striking, these results reinforce the complex interactions at play in cellular signaling networks. Taken together, the insight into intracellular signaling gained in this study, coupled with biochemical analysis and computation, will allow us to better understand the mechanisms of a vast array of therapeutics as well as build a better portrait of the complex signaling pathways that integrate to execute and control cellular behavior.S1 FigFalse discovery rate analyses aid in identifying a potential threshold for accepting MS/MS spectra without manual validation. Oftentimes, a particular FDR is selected and a peptide score for which that FDR is met is selected. Here, an FDR analysis was performed as a function of Mascot score. Instead of relying on an FDR analysis where the identity of true positives and true negatives are unknown, we manually validated each MS/MS spectra manually. Each page represents a manually validated MS/MS spectrum.(PDF)Click here for additional data file.S2 FigFalse discovery rate analyses aid in identifying a potential threshold for accepting MS/MS spectra without manual validation. Oftentimes, a particular FDR is selected and a peptide score for which that FDR is met is selected. Here, an FDR analysis was performed as a function of Mascot score. Instead of relying on an FDR analysis where the identity of true positives and true negatives are unknown, we manually validated each MS/MS spectra manually. Each page represents a manually validated MS/MS spectrum.(PDF)Click here for additional data file.S3 FigInstead of relying on an FDR analysis where the identity of true positives and true negatives are unknown, we manually validated each MS/MS spectra manually. Each page represents a manually validated MS/MS spectrum.(PDF)Click here for additional data file.S4 FigInstead of relying on an FDR analysis where the identity of true positives and true negatives are unknown, we manually validated each MS/MS spectra manually. Each page represents a manually validated MS/MS spectrum.(PDF)Click here for additional data file.S5 FigInstead of relying on an FDR analysis where the identity of true positives and true negatives are unknown, we manually validated each MS/MS spectra manually. Each page represents a manually validated MS/MS spectrum.(PDF)Click here for additional data file.S6 FigInstead of relying on an FDR analysis where the identity of true positives and true negatives are unknown, we manually validated each MS/MS spectra manually. Each page represents a manually validated MS/MS spectrum.(PDF)Click here for additional data file.S7 FigThe fold change ratio in response to TSA treatment mapped to a visual representation of proteins involved in kinase signaling, scaffolding, and cell adhesion. Each circle represents a unique phosphorylation site identified on the protein. The sites are color coded to characterize the relative effect of TSA pre-treatment on the EGF response. Proteins are labeled by their gene name. Sites of phosphorylation are labeled.(TIF)Click here for additional data file.S1 Table(XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.S4 Table(XLSX)Click here for additional data file.S5 Table(XLSX)Click here for additional data file.S6 Table(XLSX)Click here for additional data file.


Methanococcus maripaludis S2, the swimming organelle, the archaellum, is composed of three archaellins, FlaB1S2, FlaB2S2 and FlaB3S2. All three are modified with an N-linked tetrasaccharide at multiple sites. Disruption of the N-linked glycosylation pathway is known to cause defects in archaella assembly or function. Here, we explored the potential requirement of N-glycosylation of archaellins on archaellation by investigating the effects of eliminating the 4 N-glycosylation sites in the wildtype FlaB2S2 protein in all possible combinations either by Asn to Glu (N to Q) substitution or Asn to Asp (N to D) substitutions of the N-glycosylation sequon asparagine. The ability of these mutant derivatives to complement a non-archaellated S2ΔflaB2 strain was examined by electron microscopy (for archaella assembly) and swarm plates . Western blot results showed that all mutated FlaB2S2 proteins were expressed and of smaller apparent molecular mass compared to wildtype FlaB2S2, consistent with the loss of glycosylation sites. In the 8 single-site mutant complements, archaella were observed on the surface of Q2, D2 and D4 (numbers after N or Q refer to the 1st to 4th glycosylation site). Of the 6 double-site mutation complementations all were archaellated except D1,3. Of the 4 triple-site mutation complements, only D2,3,4 was archaellated. Elimination of all 4 N-glycosylation sites resulted in non-archaellated cells, indicating some minimum amount of archaellin glycosylation was necessary for their incorporation into stable archaella. All complementations that led to a return of archaella also resulted in motile cells with the exception of the D4 version. In addition, a series of FlaB2S2 scanning deletions each missing 10 amino acids was also generated and tested for their ability to complement the S2ΔflaB2 strain. While most variants were expressed, none of them restored archaellation, although FlaB2S2 harbouring a smaller 3-amino acid deletion was able to partially restore archaellation.In  N-glycosylation is a prevalent protein modification found in all three domains of life in which the attachment of the glycan is via the nitrogen atom in asparagine residues located in the acceptor glycoprotein [N-glycosylation pathways are shared among the three domains [N-glycan precursor is first synthesized on a phosphorylated isoprene-based lipid carrier, either a dolichol derivative in Eukarya and Archaea, or an undecaprenol derivative in Bacteria, via the activities of glycosyltransferases. The assembled lipid-linked glycan is then flipped across a membrane, i.e. to face into the lumen of the endoplasmic reticulum in Eukarya or to the external face of the cytoplasmic membrane in Archaea and Bacteria. A signature enzyme of the pathway, the oligosaccharyltransferase (OST), transfers the completed glycan en bloc from its lipid carrier to select Asn residues in target proteins, although further sugars can still be added to the protein-bound glycan. The Asn residue to which the N-glycan is attached is usually located in an Asn-Xaa-Ser/Thr sequon (Xaa cannot be Pro), although in some Bacteria, i.e. Campylobacter spp., a negatively charged amino acid residue is also needed at the-2 position. Between the two prokaryotic domains, Bacteria and Archaea, N-glycosylation appears to be much more widespread among Archaea, where 166 of 168 sequenced genomes examined contained at least one copy of a gene encoding the archaeal OST AglB [N-glycans [N-glycosylation systems has combined both structural and genetic methods, typically using archaellins and S-layer proteins as reporter proteins. Since the first archaeal glycosylation (agl) genes were identified in 2006 [Methanococcus maripaludis S2, Methanococcus voltae PS, Haloferax volcanii H53 and Sulfolobus acidocaldarius MW001 [N-glycosylation pathway is not essential for any of the three studied euryarchaeotes , as mutants carrying a deletion or insertional inactivation of aglB were readily obtained [S. acidocaldarius MW001, repeated attempts to delete or interrupt aglB were unsuccessful and only the integration of a second copy of aglB into the genome allowed for the deletion of the original aglB [N-glycosylation pathway in Archaea are on S-layer stability, growth of cells at high salinities and on archaellation and motility [oprotein –6. Gener domains –3,5,7,8.OST AglB . A varieOST AglB ), have b-glycans –18. Rece in 2006 ,19, thisus MW001 ,15,16,21obtained ,19,22. Hnal aglB . The keymotility –30.N-linked glycans and interruption of the normal N-glycosylation pathway leads to defects in archaella assembly or function [aglB has been deleted or insertionally inactivated , the cells are unable to make archaella. If the pathway is interrupted at steps that lead to a truncated glycan, there is an impairment in motility, although archaella are still made unless the truncation is too great [M. maripaludis S2 strain, archaella contain 3 archaellins: the major archaellins FlaB1S2 and FlaB2S2 form the filament while the minor archaellin FlaB3S2 comprises the hook region [S2, FlaB2S2 and FlaB3S2 are decorated at multiple positions with a unique tetrasaccharide [agl genes that result in a N-glycan of at least two sugars, but non-archaellated if the deleted agl gene results in a glycan of only a single sugar or prevents N-glycosylation totally (as in an aglB deletion) [The archaellum  is the major motility apparatus found in Archaea . It is afunction ,40. In soo great ,19,22. Ik region . The thrk region . The hypccharide . Cells aeletion) ,43. ThesN-glycosylation of the major archaellin FlaB2S2 for archaellation in M. maripaludis S2. FlaB2S2 has five N-glycosylation sequons, 26NTS28, 66NIT68, 110NLT112, 119NTT121 and 124NWS126. The first sequon 26NTS28 located in the N-terminal conserved region was previously reported to be unoccupied with glycan while the remaining four, located in the hypervariable region, were modified with tetrasaccharide  in all possible combinations . We also generated a series of FlaB2S2 scanning deletions in an attempt to determine regions of the molecule that were essential for assembly of archaella.A major goal of this study was to examine the requirement of M. maripaludis S2 Δhpt (Mm900) [M. maripaludis S2 Δhpt ΔflaB2S2 (ΔflaB2S2 in short hereafter) [M. maripaludis ΔRC (formerly Methanococcus deltae ΔRC [2:H2 (20:80) at 37°C with shaking [Escherichia coli TOP 10 cells (Invitrogen Inc.), used for molecular cloning steps, were cultured at 37°C in Luria Broth (LB) medium with shaking or on LB plates (containing 1.5% w/v agar) in the presence of 100 μg/mL ampicillin for plasmid selection. Strains and plasmids used in this study are listed in  (Mm900) , M. marireafter)  and all ltae ΔRC ,46) were shaking . Cells c shaking . For swa shaking . EscheriflaB2S2 genes, the wildtype flaB2S2 gene was first cloned into the pCR2.1-TOPO-TA vector (Invitrogen Inc.) to create pKJ902. This pCR2.1-TOPO-flaB2S2 and its derivatives were used as template to generate the mutants listed in flaB2S2 gene used in cloning was generated by PCR using the complementation primers listed in To generate mutant flaB2S2 genes that would encode proteins in which N-glycosylation sites were eliminated, the SDM protocol was employed [N-glycosylation sequon Asn residue  gene was amplified by PCR using M. maripaludis ΔRC whole cells as template with the complementation primers in G10 gene whose protein product contains additional glycosylation sites was chemically synthesized by GenScript USA Inc. .The flaB2S2 whose protein products would contain scanning deletions, inverse PCR and overlapping primers was employed, again with pKJ902 as template. The forward primer was designed to contain the flaB2S2 gene sequence flanking the desired in-frame 30 bp deletion. The reverse primer was complementary to the forward primer sequence upstream of the deletion  that was still the same length as the wildtype version. All the mutant versions of flaB2S2 generated were sequenced to confirm the mutations.For construction of the mutant versions of deletion . After DM. maripaludis S2, mutant flaB2S2 genes in the pCR2.1-TOPO vector were PCR amplified using complementation primers with an NsiI restriction site incorporated into the forward primer and an XbaI site into the reverse primer  and then transferred onto an Immobilon-P membrane (Millipore Inc.) [S2 proteins were detected using chicken anti-FlaB2S2 specific primary antibody [Whole cell lysates of complemented cells carrying the various mutant re Inc.) . Mutant antibody . HorseraΔflaB2S2 strains carrying plasmids with mutant flaB2S2 genes encoding proteins having mutations at the various N-glycosylation sites were examined for motility using semi-solid swarm plates [600 of an overnight cell culture was measured and adjusted to 1.0. Five microliters of the adjusted cell culture were inoculated onto semi-solid Balch medium containing 0.25% (w/v) agar using a micropipette in an anaerobic chamber by stabbing the tip into the agar. Plates were incubated in an anaerobic canister at 37°C for 4 or 6 days.Complemented m plates . BrieflyM. maripaludis ΔflaB2S2 cells carrying mutant flaB2S2 genes were collected from an overnight culture by centrifugation at 20 000 g for 1 min, washed with 2% (w/v) NaCl and resuspended in phosphate-buffered saline. Resuspended cells were loaded onto carbon-Formvar-coated copper grids and stained with 2% phosphotungstic acid, pH 7.0. Grids were examined in a Hitachi 7000 electron microscope operating at an accelerating voltage of 75 kV.Complemented N-glycosylation are known to cause severe defects in archaellation and motility [Hfx. volcanii H53, changing the sequence of the major archaellin flgA at any of the 3 examined sequons so that the encoded amino acid changed from Asn to Gln led to mutant forms of the protein that could not rescue the swimming defect of an flgA deletion strain, suggesting that each glycosylation site was necessary for archaellation [M. maripaludis S2. Previous work in this methanogen showed that a strain that had a spontaneous mutation in flaB2S2 which led to the loss of the 2ndN-glycosylation site of the archaellin that is normally decorated with the N-linked tetrasaccharide, was, nonetheless, still archaellated and motile [N-glycosylation site, either alone or in combination with other sites, might have on archaella formation and motility in M. maripaludis S2, various mutant flaB2S2 genes whose products were lacking single to quadruple N-glycosylation sites either by Asn to Gln (N to Q) substitution, or Asn to Asp (N to D) substitution of the N-glycosylation sequon asparagine were generated and cloned into the complementation vector pWLG40 . FlaB2ΔRC and FlaB2S2 share 95% identity, with the differences almost exclusively confined to several N-glycosylation sites . We have found previously that cells carrying mutations in any gene that prevents assembly of archaella (as in the ΔflaB2S2 mutant) often stop transcribing the fla operon after several sub-cultures in the laboratory. This then makes the complementation of the original gene deletion back to an archaellated state impossible [fla operon, was also confirmed by western blot to ensure that the fla operon was still transcribed in the ΔflaB2S2 mutant during the course of the complementation experiments (data not shown) [Western blots were run to detect the expression and stability of the various mutant versions of FlaB2possible . For thit shown) .S2 proteins missing N-glycosylation sites all had a smaller apparent molecular mass than that of wildtype FlaB2S2 when examined by western blotting, with the possible exceptions of D4 and Q4 which ran at very close to wildtype size. The greater the number of N-glycosylation sites eliminated in a particular FlaB2S2 mutant, the faster the mutant proteins migrated, i.e., single-site mutants had the largest apparent molecular mass, and the quadruple-site mutant D1,2,3,4 had the smallest. However, the 8 single-site mutants did not migrate as proteins of the same apparent molecular mass. Of the 8 mutants, D1 and Q1, both of which had the 1stN-glycosylation site eliminated, had the smallest apparent molecular mass, while D4 and Q4 had the largest. Similar results were observed from the double-site and triple-site mutations. In the 6 double-site mutations, FlaB2S2 with D1,2 and D1,3 sites eliminated had the smallest apparent molecular mass, followed by FlaB2S2 with D1,4 and D2,3 sites eliminated, while the archaellin having the D2,4 and D3,4 sites eliminated migrated with the largest apparent molecular mass. In the triple-site mutants, FlaB2S2 with any of the 1stN-glycosylation site eliminated  migrated at the same apparent molecular mass while FlaB2S2 with the other triple combination of sites eliminated  migrated more slowly.In general, mutant FlaB2S2 proteins lacking the same number of N-glycosylation sites have the same number of N-glycans attached but the attachment of N-glycan on some sequons might have effects on the local protein structure so that the glycoprotein is not able to be totally denatured by SDS, thus resulting in an unusual migration pattern. This unusual electrophoretic mobility has been observed in other similar studies and been the suggested explanation. Human erythropoietin (Epo) has 3 N-glycosylation sites, and the 3 single-site mutants generated by N to Q SDM showed uneven migrations on western blot, although all of the 3 mutants had the same theoretical molecular mass but differed only in the position of the N-glycans [N-glycosylation site [One possible explanation for the observed different electrophoretic mobilities is that mutant FlaB2-glycans . Similarion site .stN-glycosylation site might interfere with the cell’s ability to N-glycosylate the remaining sites, resulting in FlaB2S2 where not all the remaining sequons are actually occupied . On the other hand, it may be that elimination of one or more glycosylation sites results in the attachment of glycan to the sequon 26NTS28 that is not glycosylated under our usual growth conditions. This could explain why, for example, the FlaB2S2 mutants that are missing the 4thN-glycosylation site (D4 and Q4) showed a larger apparent molecular mass than the other single mutants and close to wildtype size. If D4 and Q4, missing the 4th glycosylation site, now had N-glycan attached at the normally unused sequon, these mutant proteins would have an identical mass as the wildtype. There is precedent for glycosylation at one sequon influencing what happens at distant sites. For example, it has been reported for rabies virus glycoprotein that N-glycosylation at one sequon can influence processing of the N-glycans at a different site on the protein [M. voltae PS, the archaella are composed of 4 archaellins FlaAMv, FlaB1Mv, FlaB2Mv and FlaB3Mv, which share high sequence similarity in the N-terminal conserved region with FlaB1S2 and FlaB2S2 [Mv and FlaB2Mv including the 26NTS28 sequon, are identical to those of FlaB2S2, but in the case of the M. voltae PS archaellins, the 26NTS28 sequon was found to be occupied with N-linked glycan [26NTS28 sequon in FlaB2S2 might be able to be N-glycosylated in M. maripaludis S2 under different conditions.While local folding effects might explain the altered electrophoretic mobility, another possible explanation for this unusual western blot result is that elimination of the 1 protein . In M. v FlaB2S2 ,55. Inted glycan . ClearlyS2 protein  with additional glycosylation sequons compared to wildtype FlaB2S2 had a larger apparent molecular mass (~32 kDa) than wildtype FlaB2S2 (~27 kDa) in western blots. Since G6 has 6 N-glycosylation sites (excluding the 26NTS28 sequon), its larger apparent molecular mass suggests that at least some, and possibly all, of the extra sequons are, in fact, occupied since the mass of the tetrasaccharide is only 1036 Da [The G6 mutant FlaB2 1036 Da . AlthougS2 proteins having mutations at N-glycosylation sites were examined by transmission electron microscopy for archaellation, and the results are listed in Complemented cells carrying FlaB2ΔflaB2S2 mutant strain was non-archaellated. Archaella were observed when the ΔflaB2S2 strain was complemented with the wildtype version of flaB2S2, but not when the ΔflaB2S2 strain was complemented with the empty vector pWLG40.The archaellation state of the 4 control strains was as expected. Wildtype Mm900 cells (900) were archaellated while the S2 , could restore archaellation. In the 3 archaellated complements, Q2 and D2 had different amino acid substitutions at the same 2ndN-glycosylation site. The N to D amino acid change at the 2nd sequon generated in this study replicates the spontaneous mutation in flaB2S2 that we previously reported [D2 gene generated in this study showed no impairment in archaellation or swarming motility [ndN-glycosylation site alone does not significantly interfere with archaellation. In contrast, complementation of the ΔflaB2S2 strain with flaB2S2 lacking the 4thN-glycosylation site differed markedly depending on what amino acid the original Asn was changed to; cells complemented with the D4 version had archaella under electron microscopy while cells complemented with the Q4 version did not  were co did not .flaB2S2 , the ΔflaB2S2 cells were not able to assemble archaella. These results indicate that in M. maripaludis S2, archaella can be assembled using FlaB2S2 lacking as many as 3 out of the 4 glycosylation sites, as long as the first site remained intact  but not when the archaellin is entirely non-glycosylated .Among the 6 double-site mutation complements, all but the D1,3 version could assemble archaella , while complements with the flaB2S2 genes carrying D1,2, D2,4 and D3,4 had a smaller swarming diameter . For the D1,2 and D3,4 complementations, the smaller swarming distance may be explained by the lower percentage of cells that were observed by electron microscopy to be archaellated, but in the case of the D2,4 all cells examined were archaellated. Interestingly, G6 appeared hyper-motile as it consistently swarmed further than cells complemented with the wildtype version of flaB2S2 (swarming diameter of G6/WT = 1.24±0.10).In general, complemented cells in which archaellation was restored were also motile on swarming plates and all of the complemented cells that were non-archaellated as determined by electron microscopy  were also non-motile on swarm plates, even after an extra 2-day incubation . The unun-motile , Fig. 4AHfx. volcanii H53, none of the 3 FlgA single-site mutation complements showed motility [M. maripaludis S2 cells were still as motile as wildtype cells when the ΔflaB2S2 strains was complemented with flaB2S2 with the D2,3,4 changes, in which archaella were assembled using FlaB2S2 lacking 3 out of the 4 N-glycosylation sites. The structural protein (flagellin) of the functionally analogous bacterial swimming organelle, the flagellum, can also be modified with glycan, especially in Gram-negative bacteria, although the linkage is O-glycosidic rather than N-glycosidic [O-glycan modification in bacterial flagellin can be critical for flagella assembly, stabilization, motility, and even virulence in pathogens [Pseudomonas syringae pv. tabaci, flagellin FliC has 6 O-glycosylation sites, and single-site mutations in any of these sites resulted in various impairments in motility, while a mutant carrying mutations to eliminate all 6 O-glycosylation sites in FliC was non-motile [O-glycosylated [O-glycosylation of type IV pilin resulted in reduced twitching motility in Pseudomonas aeruginosa 1244 and P. syringae pv. tabaci but did not interfere with pili assembly [P. aeruginosa 5196 in which a different O-glycan was attached to the type IV pilin PilA, O-glycosylation played critical roles in both type IV pili assembly and twitching motility [In motility . Howeverycosidic ,57. The athogens ,58–60. In-motile . The strn-motile ,34, can osylated ,62. Elimassembly . Howevermotility .S2 version. Of all the complemented strains, only the G6 complement consistently demonstrated an increased zone of swarming compared to the wildtype. Interestingly, similar results were also observed in regards the O-glycosylation of flagellin in Helicobacter pylori [O-glycosylation of the flagellins FlaA and FlaB with pseudaminic acid is essential for flagella assembly and cell motility [H. pylori mutant defective in deglycosylation of flagellins showed both hyper-O-glycosylation (3 fold more pseudominic acid) of FlaA as well as hyper-motility [The results obtained with the G6 complemented cells indicate that an increase in glycosylation can lead to hyper-motility. The western blots results indicate that the G6 version of FlaB2 is hyper-glycosylated compared to the wildtype FlaB2r pylori  where O-motility ,67. A H.motility . HoweverS2 is essential, nonetheless glycosylation at some site is necessary for archaella formation. In the case of the 4 triple-site mutants it is clear that glycosylation of only site 1 is sufficient for archaellation and motility. However, removal of the 1st site did not always lead to non-archaellated cells as witnessed by the archaellated and motile cells observed in the D1,4 complementation, suggesting that glycosylation of FlaB2S2 at several different combinations of sites could be sufficient for incorporation of the subunits into functional archaella. In some ways, this is reminiscent of a situation in Wzc, a tyrosine autokinase essential for capsule formation in E. coli. Phosphorylation of tyrosine residues in the C-terminus of Wzc are necessary for its function but no single tyrosine is essential for phosphorylation and it was suggested that the overall level of phosphorylation rather than a precise combination of tyrosine residues accessible to phosphorylation is what is important for Wzc activity [The data obtained from the glycosylation site elimination mutants indicates that while no particular single site of glycosylation on FlaB2activity .S2 protein are critical for archaella formation, a series of FlaB2S2 scanning deletion mutants that sequentially lacked 10 amino acids were generated in the complementation vector pWLG40 and transformed into the ΔflaB2S2 mutant. The scanning deletions in flaB2S2 were identified since they migrated slightly faster than the wildtype version of flaB2S2 in 0.8% agarose gels due to the 30 bp deletion (Δ31–40). For the first 10 amino acids, two versions were generated. The first was deleted for amino acids 2–10 (named as Δ2–10), leaving the +1 amino acid which we thought might be important for successful cleavage of the 12 amino acid signal peptide. We also generated a 4–10 amino acid deletion (named as Δ4–10) since the +3 glycine of the mature protein is needed for signal peptide removal in archaellins of the related methanogen M. voltae PS [To determine which regions of the FlaB2oltae PS .S2 proteins from whole cell lysates of the various complemented cells were detected using anti-FlaB2S2 specific antibody on western blot analysis, as shown in S2 scanning deletions, 19 mutant proteins  were readily detected on western blot by anti-FlaB2S2 specific antibody, although the expression level of Δ2–10 and Δ21–31 was relatively lower compared to that of the other mutants. Evidence of some possible protein degradation was observed in the Δ2–10 and Δ161–170 FlaB2S2 as multiple lower molecular mass bands were detected in these two lanes. These results indicate that some of the mutant proteins were either not expressed or were unstable and degraded. Fourteen mutant proteins, including Δ21–30, Δ31–40, Δ41–50, Δ51–60, Δ71–80, Δ81–90, Δ91–100, Δ131–140, Δ141–150, Δ151–160, Δ161–170, Δ171–180, Δ181–190 and Δ194–204, had similar apparent molecular masses, which were smaller than FlaB2S2 from the wildtype strain Mm900 (900) or from the ΔflaB2S2 strain complemented with the wildtype version of flaB2S2. This is consistent with the fact that all these mutation proteins are 10 amino acids shorter than wildtype FlaB2S2. The upper band in the mutant Δ2–10 lane appears slightly larger than the neighboring WT lane. This might be due to the lack of processing of the archaellin signal peptide in this deletion since in M. voltae PS the +3 glycine position was essential for cleavage of the signal peptide by the pre-archaellin peptidase FlaK and this is missing in the Δ2–10 version of FlaB2S2 [N-glycans [S2 would be expected to have a full complement of attached N-glycans as well as the extra amino acids of the signal peptide contributing to make it run slightly larger in the western blots than the processed wildtype version of FlaB2S2.Mutant FlaB2 FlaB2S2 . The pre-glycans  so this S2 mutants Δ61–70, Δ101–110 and Δ111–120 had even smaller apparent molecular masses compared to the FlaB2S2 carrying other scanning deletions. This can be attributed to the fact that these three deletions result in the loss of one N-glycosylation sequon. Surprisingly, the scanning deletion that contains the remaining, 4th, glycosylation sequon, namely Δ121–131, does not follow this pattern. In this lone case, FlaB2S2 migration in western blots is slower than the other three deletion mutants missing N-glycosylation sites. This observation is consistent with the results of the N-glycosylation sequon mutation complementations. In the case of both single 4th sequon mutants (D4 and Q4), FlaB2S2 had a larger apparent molecular mass than the other single-site mutations  even though most of them produced FlaB2S2 detected by western blot. These results suggested that either all regions of the molecule were essential for archaella formation or that a certain critical archaellin length is important for the archaella filament to be assembled.To examine if ΔS2 scanning deletion mutants could restore archaellation, we suspected that the 10-amino acid deletion in the scanning deletions was too long for FlaB2S2 to be assembled into archaella. To address this, we created a shorter 3-amino acid deletion in the hypervariable region of FlaB2S2. This protein was detected by western blot at a similar apparent molecular mass as the wildtype FlaB2S2 , creating a FlaB2S2 hybrid that had amino acids 91–100 replaced with a second copy of amino acids 161–170 so that the resulting length of the FlaB2S2  was wildtype. Both the donor and the acceptor regions are located in the hypervariable region of FlaB2S2, and do not contain N-glycosylation sequons to minimally reduce the effects from disruption of the conserved regions that might be involved in subunit-subunit interaction or in glycosylation. The SUB protein was expressed in the complement cells and showed similar apparent molecular mass as that of WT protein, as expected  and a gold-binding 12 amino acid peptide have been inserted into variable regions of different archaellins and these mutant proteins were still able to be assembled into archaella [We also tried to examine the possible length requirement of archaellins in a different way. In a FlaB2expected . Howeverexpected , suggestolerated . It is kormation . In addiormation . This isrchaella . Howeverrchaella –77.N-glycosylation seems to be a common modification of archaellin, but the relationship between N-glycosylation and archaella assembly is unclear [N-glycosylation sites as well as scanning deletions of the archaellin FlaB2S2 on archaella assembly and function in M. maripaludis S2. In M. maripaludis S2, functional archaella can be assembled using FlaB2S2 lacking as many as 3 out of 4 glycosylation sites , but not when the archaellin is entirely non-glycosylated . A hyper-N-glycosylated version of FlaB2S2 (G6) resulted in hyper-motile M. maripaludis S2 cells. Attempts to define essential and nonessential domains of the archaellin by scanning deletion analysis revealed that no contiguous 10 amino acid stretch could be deleted and still have the archaellin complement a ΔflaB2s2 strain back to an archaellated phenotype.Archaella are unique swimming organelles that are thought to be assembled like bacterial type IV pili but function like bacterial flagella by filament rotation ,33,34,78 unclear ,40. In t


Plasmodium falciparum FIKK8 (PfFIKK8) and its Cryptosporidium parvum orthologue (CpFIKK) – the only member of the family predicted to be cytosolic and conserved amongst non-Plasmodium parasites. Recombinant protein samples of both were catalytically active. We characterized their phosphorylation ability using an enzymatic assay and substrate specificities using an arrayed positional scanning peptide library. Our results show that FIKK8 targets serine, preferably with arginine in the +3 and −3 positions. Furthermore, the soluble and active FIKK constructs in our experiments contained an N-terminal extension (NTE) conserved in FIKK8 orthologues from other apicomplexan species. Based on our results, we propose that this NTE is an integral feature of the FIKK subfamily.FIKKs are protein kinases with distinctive sequence motifs found exclusively in Apicomplexa. Here, we report on the biochemical characterization of  Such a complex life cycle is regulated by a signal transduction system of ∼90 protein kinases (PK) Plasmodium PKs absent from mammalian genomes are likely involved in parasite-specific functions and potential targets of selective drugs. One such group, known as FIKK kinases, is exclusive to the phylum of Apicomplexa and thus, far the subject of few studies.Transmission of malaria, perennially one of the deadliest infectious diseases in the world, is mediated by transmission of Plasmodium falciparum and Plasmodium reichenowiPlasmodium proteins PfFIKK8) which is also the longest member. It is also the only member conserved in all Plasmodium species, as well as in the genomes of Toxoplasma, Cryptosporidium, Neospora and EimeriaThe defining architecture of the FIKK subfamily includes a highly variable N-terminal region followed by a conserved C-terminal kinase domain (KD) where the eponymous quartet of residues, Phe–Ile–Lys–Lys, is located. Two species, namely PfFIKK4.1, PfFIKK9.3, PfFIKK9.6 and PfFIKK12 have been found to be localized in the Maurer’s cleft PfFIKK7.1 or PfFIKK12 PfFIKK4.1 Plasmodium berghei (PbFIKK8) was not successful, suggesting that FIKK8 might play an essential role in cell cycle regulation of Plasmodium parasites FIKK kinases are expressed mostly during the blood stage of the parasitic life cycle PfFIKK12 and PfFIKK4.1 immuno-precipitated from cell lysates have been found to be capable of phosphorylating myelin basic protein (MBP) P. falciparum FIKK8  and its C. parvum orthologue CpFIKK (cgd5_4390 at www.cryptodb.org), and studied their enzymatic properties.Most of the catalytically important residues of typical eukaryotic protein kinases are conserved in FIKK kinases 2PfFIKK8 and CpFIKK . Using an Escherichia coli system previously proven for parasite PKs PfFIKK8 and one of CpFIKK containing the NTE . We then proceeded to assess their auto-phosphorylation behavior and determine their ability to phosphorylate a set of common peptide substrates.We cloned multiple constructs of PfFIKK8l and CpFIKKd samples were incubated with ATP and MgCl2. The samples were then trypsinized and analyzed using LC–MS–MS. We used the maps of the resulting peptides  to identify multiple phospho-serines (pS) on the Plasmodium sample, both phospho-serines and phospho-threonines (pT) on the Crytposporidium sample and one phospho-tyrosine (pY) on each. One of the phosphorylated serines (pS1320) on PfFIKK8l is located in the predicted activation loop. Unfortunately, the corresponding serine on CpFIKKd was not part of any of the detectable peptides. Furthermore, some of the phosphorylated residues were found in the NTE, including a phospho-serine that is conserved on both samples.To assess their auto-phosphorylation behavior, the purified PfFIKK8l and CpFIKKd to be active against a set of standard kinase substrates – bovine casein, bovine MBP and Syntide-2 (PLARTLSVAGLPGKK), with MBP producing the highest level of activity.We also found PfFIKK8l and CpFIKKd in an unbiased manner, we assayed the proteins using a positional-scanning peptide array To systematically determine the sequence preferences of PO) with the sequence RRRAPSFYRK and three variants. The variants featured mutation of Arg to Ala at the +3 (PAR) and −3 (PRA), as well as PT, a truncated version of PO. Using an LDH–PK coupled kinase assay PfFIKK8l, PfFIKK8o and CpFIKKd respectively as catalysts. The two PfFIKK8 constructs behaved very similarly to each other with almost all the peptides  and catalytic efficiency values (kcat) were obtained using PO and PT for all three FIKK samples, with all indicating at least average substrate binding. On the other hand, both mutants (PAR and PRA) resulted in higher Km and lower kcat values, thus, emphasizing the importance of the flanking arginines.To evaluate the contributions of the arginines and other flanking residues to phosphorylation efficiency, we designed an optimized peptide substrate . To investigate the relevance of this phosphoserine to FIKK8 activation, we attempted to express a mutant form of PfFIKK8l with S1320 mutated to alanine; however, the mutated protein did not express. Therefore, a possible regulation mechanism for FIKK kinases mediated by phosphorylation of the activation loop remains to be confirmed.In addition to the NTE, the FIKK motif and the absence of a C-terminal extension, FIKK kinases are also defined by a number of conserved divergences in some common kinase motifs. First, Phe and Gly in the DFG triad are often replaced by other hydrophobic residues. Second, in many FIKK kinases, the activation loop features proline in place of the more common alanine in the APE. Furthermore, the HRD motif in subdomain VI features a leucine in place of arginine. The absence of arginine in this position typically signifies that a kinase does not need auto-phosphorylation of the activation loop to become active. Interestingly, we found S1320 in PfFIKK8l and CpFIKKd is corroborated by the kinetic parameters we obtained, all of which are in the range of active protein kinases with average to above average binding affinities for ATP and the optimized substrates used (PO and PT). Furthermore, sequence alignment (not shown) indicates that the NTE is conserved among available FIKK8 orthologues from apicomplexan parasites and, to a lesser degree, the other FIKK paralogues found in P. falciparum and P. reichenowi . Given that PfFIKK8l and PfFIKK8o behaved nearly indistinguishably in the kinetics study, we propose that PfFIKK8o specifically defines the boundaries of the active FIKK8 domain and that M1049 is the start of the NTE. Identifying the functional significance of this NTE is left for future research; however, the evidence of auto-phosphorylation discussed above suggests the possibility of a regulatory role.The inclusion of the NTE as an integral component of PfFIKK8l. The N-lobe site, namely S1099, has previously been reported in a phospho-proteomics study of P. falciparumCpFIKKd sample  and, significantly, located on the glycine-rich loop of both kinases – implicated in positioning of γ–phosphate in ATP hydrolysis. Previously, a phosphoserine in the same region of yeast ATG1 protein kinase was found to be inhibitory In addition to phosphoserines in the NTE, our auto-phosphorylation experiment also revealed one phosphorylated site in the N-lobe and 2 more in the C-lobe of PfFIKK8l and CpFIKKd  when the shortened substrate PT was used in place of PO, suggesting that this short substrate may be as an ideal tool for assaying FIKK8 activity and screening for small molecule inhibitors.The peptide array study revealed a preference for Arg at the −3 and +3 positions for  CpFIKKd  with botPfFIKK8 and CpFIKK are orthologous and catalytically active protein kinases, both of which featuring an approximately 40-residue long integral N-terminal extension. Future research to determine the function of this extension may reveal the mechanism of FIKK kinases. It is also possible that, in vivo, regions of the proteins not included in our active constructs may play catalytic, regulatory and localization roles.In conclusion, our recombinant samples of


We have described that mitochondrial ERK1/2 phosphorylates StAR and that mitochondrial fusion, through the up-regulation of a fusion protein Mitofusin 2, is essential during steroidogenesis. Here, we demonstrate that mitochondrial StAR together with mitochondrial active ERK and PKA are necessary for maximal steroid production. Phosphorylation of StAR by ERK is required for the maintenance of this protein in mitochondria, observed by means of over-expression of a StAR variant lacking the ERK phosphorylation residue. Mitochondrial fusion regulates StAR levels in mitochondria after hormone stimulation. In this study, Mitofusin 2 knockdown and mitochondrial fusion inhibition in MA-10 Leydig cells diminished StAR mRNA levels and concomitantly mitochondrial StAR protein. Together our results unveil the requirement of mitochondrial fusion in the regulation of the localization and mRNA abundance of StAR. We here establish the relevance of mitochondrial phosphorylation events in the correct localization of this key protein to exert its action in specialized cells. These discoveries highlight the importance of mitochondrial fusion and ERK phosphorylation in cholesterol transport by means of directing StAR to the outer mitochondrial membrane to achieve a large number of steroid molecules  Mitochondria are a key control point for the regulation of steroid hormone biosynthesis. The transport of cholesterol across the intermembrane space from the outer (OMM) to the inner mitochondrial membrane It is well known that cAMP-dependent protein kinase (PKA) activation increases StAR gene transcription Mitochondrial fusion/fission events are critically important for maintaining the integrity of these organelles per unit of StAR protein.In this study, we evaluated the contribution of mitochondrial fusion and ERK phosphorylation in StAR synthesis and mitochondrial localization. We observed that StAR activity depends on the correct activation and localization of mitochondrial ERK in the temporal frame of kinase activity. We showed for the first time that StAR depends on mitochondrial fusion to reach the mitochondria where it is retained through a mechanism involving ERK phosphorylation in the presence of cholesterol. Here, we highlight the fact that StAR site of action is effectively the OMM, which establishes a StAR activity cycle that could in turn enable the metabolism of many molecules of cholesterol Purified hCG was provided by Dr. Parlow . Waymouth MB752/1 cell culture media, acrylamide, bis.acrylamide, agarose, BSA, 8-bromoadenosine 3′∶5′-cyclic monophosphate (8Br-cAMP), H89, 22(R)-OH-cholesterol and carbonyl cyanide m-chlorophenyl hydrazone (CCCP) were purchased from Sigma Chemical Co. . Serum, antibiotics, trypsin-EDTA and Lipofectamine 2000 were from Life Technologies, Inc. . Tri-Reagent was from Molecular Research Center Inc. . Electrophoresis supplies, polyvinylidendifluoride membrane (PVDF) and secondary antibody (horseradish peroxidase conjugated goat antibody) were from Bio-Rad Laboratories Inc. . Antibodies against Mfn2 and StAR FL (full length) were obtained from Santa Cruz Biotechnology Inc. . Anti-OxPhos complex III core 2 subunit (III Complex) was purchased from Invitrogen . M-MLV reverse transcriptase (RT), GoTaq DNA polymerase and other molecular biology reagents were purchased from Promega . PD98509 was obtained from Calbiochem . SYBR Select Master Mix was obtained from Applied Biosystems . Sterile and plastic material for tissue culture was from Orange Scientific . All other reagents were of the highest grade available.3, 20 mM HEPES, 50 µg/ml gentamicin and 15% heat-inactivated horse serum. Cells were maintained at 36°C in a humidified atmosphere containing 5% CO2. Purified hCG with a biological potency of 11900 IU/mg or 8Br-cAMP, a permeable analog of cyclic-AMP, were used to treat the cells for the indicated times.The MA-10 cell line is a clonal strain of mouse Leydig tumor cells that produces progesterone (P4) rather that testosterone as the main steroid Cells were transiently transfected using Lipofectamine 2000 reagent and transfection was performed with pRc/CMVi plasmid either containing StAR wild-type cDNA (StAR wt) or StAR mutated cDNA (StAR S232A), as we used previously invitro using the Rabbit Reticulocyte Lysate System . Plasmids were incubated separately in 70 µl of the master mix, supplemented with L-Methionine as indicated by the manufacturer, plus 300 µg of mitochondrial fraction obtained from control MA-10 cells in import buffer, as previously described 2, 5 mM EGTA, 2×10−4% Tween-20, 100 µM ATP, 1 mM sodium orthovanadate, 1 mM DTT) was added plus 10 µCi of [γ-32P]ATP and 50 µM of cholesterol. The reaction continued for other 30 min at 30°C and was finished after 1 h by the addition of 25 µM FCCP for 10 min. at 4°C. The samples were centrifuged at 20000×g for 20 min and the mitochondrial pellet was resuspended in MSHE buffer, as previously described 1.5 µg of the pRc/CMVi plasmid containing cDNA sequence from StAR wt and StAR S232A were transcribed/translated Cells were treated as indicated in the corresponding figure. Then, subcellular fractionation and measurement of mitochondrial steroidogenesis was performed according to our published procedures Mitochondria were isolated as described previously MA-10 Leydig cells total RNA was extracted using Tri-Reagent following the manufacturer’s instructions. Two µg of total RNA were reverse transcribed using random hexamers and M-MLV Reverse Transcriptase according to the manufacturer's protocol.5′-GAAATCGCCAATGCCAACTC-3′ and reverse, 5′-TCTTAGACCTGCGAGCCTCA-3′ (amplicon size 405-bp) For semi-quantitative RT-PCR, the following specific primers were used: mouse StAR cDNA forward, 5′-GGGACGAAGTGCTAAGTAAGATGG-3′ and reverse, 5′-GGTCAATGTGGACAGTCC-3′ (amplicon size 566-bp); and mouse ribosomal protein L19 cDNA forward, 5′-TTGGGCATACTCAACAACCA-3′ and reverse, 5′-CTTGACATTTGGGTTCCAC-3′5′-ATTCCGATAACGAACGAGACT-3′ and reverse, 5′-AGCTTATGACCCGCACTTACT-3′ . Real-time PCR was performed using Applied Biosystems 7300 Real-Time PCR System. For each reaction, 20 µl of solution containing 5 µl of cDNA, 10 µM forward and reverse primers, and 10 µl of SYBR Select Master Mix was used. All reactions were performed in triplicate. Amplification was initiated by a 2-min preincubation at 50°C, 2-min incubation at 95°C, followed by 40 cycles at 95°C for 15 sec, 55°C for 15 sec and 72°C for 1 min, terminating at 95°C for the last 15 sec. StAR mRNA expression levels were normalized to mouse 18S RNA expression, performed in parallel as endogenous control. Real-time PCR data were analyzed by calculating the 2−ΔΔCt value (comparative Ct method) for each experimental sample.For Real-Time PCR, the following specific primers were used: mouse StAR cDNA forward, t test or analysis of variance (ANOVA) followed by Student-Newman-Kuels test.P4 production in cell culture media was measured by RIA as described previously CCCP mediated-ΔΨm disruption effects on StAR metabolism, mitochondrial import and steroidogenesis after hormone stimulation have been previously shown in MA-10 Leydig cells Next, we analyzed the impact of the two-phase assay on cholesterol metabolism, measured as P4 production. Given that mitochondrial StAR is not enough to reactivate complete steroidogenesis, these results suggest that steroid production mediated by StAR involves the cooperation with (an)other factor(s) in the mitochondria of steroidogenic cells, particularly in Phase I. To adress this particular issue, we analyzed phospho-ERK1/2 (pERK) levels in mitochondria after the two-phase experiment. In order to analyze the consequence of ΔΨm disruption in mitochondrial StAR protein levels, we performed a transient transfection over-expressing StAR , so thatInterestingly, the presence of MG132 (a proteasome inhibitor) in Phase II increases StAR mitochondrial levels two-fold (invitro in serine 232 (Ser232), a crucial event in StAR activity and cholesterol transport. Mitochondrial ERK phosphorylation was followed by progressive decrease of its activity during the first hour of hormonal action StAR is phosphorylated by ERK invitro assay. Plasmids containing StAR wt and StAR S232A sequences were subjected to the import assay in the presence of isolated mitochondria from control cells and recombinant active ERK1. Then, mitochondrial proteins were subjected to SDS-PAGE, autoradiography and immunoblot. In To confirm that ERK phosphorylation of StAR is effectively the key event involved in StAR retention at the OMM, we performed a mitochondrial import and phosphorylation Steroidogenesis requires full activity of ERK and PKA localized at the OMM’s transduceome complex We recently described that Mfn2 is up-regulated by the hCG/cAMP system in MA-10 Leydig cells and postulated a key role of mitochondrial fusion in the hormone stimulation of steroidogenesis StAR gene expression regulation has been extensively studied in steroidogenic and even non-steroidogenic tissues In this study we have demonstrated that steroidogenesis in MA-10 Leydig cells depends on the hormonally-stimulated mitochondrial fusion that regulates StAR mRNA and protein abundance and therefore its translocation to the mitochondria. We have shown for the first time that mitochondrial ERK1/2 activation is an essential step in localization, activity and/or retention of StAR on the OMM.We determined that StAR protein is effectively translocated to the matrix after the ΔΨm is restored, as determined by the detection of the 30 kDa processed StAR. Even though we observed a significant amount of mitochondrial StAR levels after a 2 and 3 h wash-out of CCCP, P4 production in Phase II was not reactivated in any recovery time evaluated. .per se is not sufficient to recover steroidogenesis. It is conceivable that other mitochondrial factors, which are unable to reach the mitochondria when CCCP is added to the culture media, are involved in StAR activation in Phase I. Mitochondrial ERK can be suggested as a good candidate to cooperate with StAR in Phase I to achieve maximal P4 production, since its activation/deactivation cycle correlates with the time-course of Phase I/II respectively. This notion is reinforced by the fact that mitochondrial ERK activation is impared in the presence of CCCP; nevertheless, pERK levels are not restored after CCCP wash-out in Phase II.P4 synthesis rate was not reactivated even when mitochondrial function was normalized and StAR localized at the matrix; then, it appears that StAR presence in the mitochondrial matrix Since CCCP affected StAR mRNA total levels  and overWe showed that over-expression of StAR S232A significantly abolishes the presence of mitochondrial StAR after hCG or cAMP stimulation. ERK phosphorylation affects post-transcriptionally mitochondrial StAR levels,. since expression of transfected StAR is independent of endogenous regulation. In MA-10 cells, a mitochondrial module includes MEK (Mitogen-activated protein kinase kinase), ERK and cholesterol with a direct physical association between StAR and ERK Results depicted in In this work we have clearly shown that mitochondrial fusion through the increase of Mfn2 levels in mitochondria is strictly required for StAR protein synthesis and mitochondrial localization after hormone stimulation by hCG and cAMP. Mitochondrial fusion reduction correlates with a decrease in StAR protein in mitochondria and mRNA levels. The results obtained with Mfn2 knockdown are entirely consistent with those observed with CCCP treatments, suggesting that the effect of CCCP might be partially due to mitochondrial fusion inhibition. In this regard, we have demonstrated here that ERK mitochondrial activity depends on Mfn2 presence in mitochondria, in agreement with the lack of steroidogenic capacity of cells in the presence of CCCP and with previous published work A direct role of Mfn2 on StAR gene expression is unknown to date. In this regard, a previous report shows that over-expression or knockout of Mfn2 modulates and affects mRNA levels of several mitochondrial proteins Briefly, we may conclude that mitochondrial fusion stimulated by steroidogenic hormones, through the cAMP pathway, defines not only StAR mRNA levels in the cell but also the localization of active StAR protein in the mitochondria. Indeed, the StAR phospho/dephosphorylation cycle could be mediated by ERK activity and mitochondrial phosphatases in the context of the OMM, enabling a very small amount of mitochondrial StAR to be responsible for the very large number of steroid molecules produced in a short period of time.Figure S1Effect of CCCP on StAR mRNA levels. Cells were subjected to the two-phase experiment. Total RNA was isolated, reverse transcribed and subjected to semi-quantitative PCR using specific primers for StAR and L19 cDNA, as loading control. PCR products were resolved in ethidium bromide-stained agarose gels. Treatments and corresponding phases are indicated in the figure. A representative gel is shown. For each band, the OD of the expression levels of StAR were quantified (arbitrary units) and normalized to the corresponding L19 abundance. The relative levels of StAR are shown: **p<0.001 vs. Phase I; ¥¥¥ p<0.001 vs. control; ns p>0.05 vs. control CCCP. All the results are expressed as means ± SEM of three independent experiments.(TIF)Click here for additional data file.Table S1Effect of CCCP on P4 production sustained by 22(R)-OH-cholesterol. P4 production was measured by RIA in the culture media in the presence or absence of 22(R)-OH-cholesterol and CCCP, at the indicated times. Data are shown as P4 concentration (ng/ml). *ns p>0.05 without CCCP vs. with CCCP.(TIF)Click here for additional data file.


The results obtained showed that DZ exhibits partial to no inhibition of the isozymes examined while GST inhibited hMAO-B (IC50 of 6.81 μM), and its hMAO-A inhibition was more potent than the standard deprenyl. Furthermore, the reversibility, mode of inhibition kinetics, and tyramine oxidation of GST were examined. GST was a time-independent reversible and competitive hMAO-A and hMAO-B inhibitor with a lower Ki of hMAO-B (1.45 μM) than hMAO-A (4.31 μM). GST also inhibited hMAO-B tyramine oxidation and hydrogen peroxide production more than hMAO-A. Docking studies conducted indicated that the GST reversibility and hMAO-B selectivity of inhibition may relate to C5-OH effects on its orientation and its interactions with the threonine 201 residue of the active site. It was concluded from this study that the natural product GST has competitive and reversible MAOs inhibitions and may be recommended for further investigations as a useful therapeutic agent for Parkinson's disease.Monoamine oxidases inhibitors (MAOIs) are effective therapeutic drugs for managing Parkinson's disease (PD) and depression. However, their irreversibility may lead to rare but serious side effects. As finding safer and reversible MAOIs is our target, we characterized the Neurological disorders such as Parkinson's disease (PD) and major depressive disorder (MDD) are associated with brain neurotransmitters depletion, particularly dopamine (DA), serotonin 5-HT), and norepinephrine (NE). MDD comorbidities may affect 72% of PD patients . However-HT, and MAO exists in two isoforms, MAO-A, and MAO-B . The the2O2) and aldehydes [2O2, and oxidative stress occurs and rises with the presence of iron, aggravating DNA and lipid membrane damage and contributing to glial and neuronal cell death. Hence, MAO inhibition provides a valuable strategy for the prevention of oxidative stress in addition to its ability in maintaining functional neurotransmitters levels.In the brain, the mitochondrial bound flavin-containing amine oxidoreductase  works to catalyze monoamine neurotransmitters oxidative deamination to maintain homeostasis. MAO-A catalyzes NE, DA, and 5-HT, while MAO-B catalyzes DA more specifically . The abnldehydes , 13. GluThe earlier discovered nonselective MAOIs, such as phenelzine, or the current selective MAO-BIs such as DEP have an irreversible inhibitory mechanism . MAO irrFlavonoids as chromone-containing polyphenolic structures are well-known natural antioxidant agents that may affect the catecholamine synthesis , 18. Api recombinant human MAO isozymes (hMAO-A and hMAO-B) inhibition with the objective to understand the mechanism of action and structure-activity relationship. By choosing these two structurally close compounds , any difference in their inhibitory activity can disclose the structure-activity relationship between the extra GST functional group and the MAO-A or MAO-B inhibitions. Comparing another compound  with GST will not enable us to make a clear conclusion. Ultimately, the current study investigates the possibility of identifying safer and reversible natural MAO inhibitors for the possible therapeutic use in the management of PD.Genistein (GST) and daidzein (DZ) are two isoflavone analogs naturally found in traditionally used medicinal plants such as bakuchi, soybeans, and red clover –22. Prev™ Assay kit was obtained from Promega . Genistein (GST) (purity > 98%) and tyramine HCl were obtained from Santa Cruz Biotechnology Inc. . The isozymes hMAO-A and hMAO-B derived from recombinant baculovirus and their aliquoted active units (U) were obtained from Sigma-Aldrich. Each hMAO-A and hMAO-B stock was diluted with 10 mM HEPES in cold Hank's Balanced Salt Solution (HBSS) (pH 7.4) and kept in −80°C until use. Daidzein (DZ) (purity ≥ 98%) and selective standard MAOIs including DEP and clorgyline (CLORG) and other materials were also purchased from Sigma-Aldrich .MAO-Glow hMAO-A and hMAO-B inhibitory effects were tested by measuring the decrease in Arbitrary Light Units (ALU) using MAO-Glow Assay [μM each. Ethanol and DMSO were used (<2%) as solvents. In white opaque 96-well plates, 12.5 μL of each analog was plated with 25 μL of hMAO-A or hMAO-B for a 0.88 U/mL final concentration (0.04 U per reaction). In other wells, reaction buffer substituted each isozyme (to make the blanks) and each analog (to make negative control). After 40 min incubation, 12.5 μL luciferin derivative substrate (LDS) was added for a final concentration of 40 and 4 μM for hMAO-A and hMAO-B, respectively. After 2 h incubation, the reactions were stopped by adding 50 μL of Reporter Luciferin Detection Reagent (RLDR). Plates were incubated for 30 min to produce luminescence signal and then samples were read on Synergy HTX Multi-Reader .GST and DZow Assay  with sli hMAO-A and hMAO-B after the inhibition by GST was measured by a previously reported preincubation and dilution method [50 and 100x IC50 for 40 min at RT (pH 7.4). For positive control, standards at 10x IC50 were simultaneously preincubated (0.03 μM CLORG for hMAO-A and 1.01 μM DEP for hMAO-B). Tests of GST of 4x IC50 with 4x of each isozyme was also simultaneously conducted for extra control. All reactions were subsequently diluted 100-fold when mixed with LDS in a 96-well plate to yield final concentrations of GST of 0.1x IC50 and 1x IC50. The microplates were then incubated for 30, 40, 50, and 60 min. RLDR was added, and luminescence was measured as mentioned above.The recovery of bothn method  with slin method . BrieflyμM for hMAO-A and 0 to 40 μM for hMAO-B. Three GST concentrations for a final of 5, 10, and 20 μM and a buffer (for a negative control) were mixed with each isozyme for a final concentration of 0.88 U/mL (2 : 1 ratio). After 40 min, the reactions were initiated by adding 19 μL of the enzyme and GST (or buffer) mixture to LDS in 96-well plates. After 1.5 h of incubation, RLDR was added, and the luminescence signal was detected as mentioned in the hMAO-A and hMAO-B assay.The modes of MAO inhibitory effects of GST were analyzed by Michaelis-Menten kinetics using MAO-Glow Assay. Standard DEP was used to validate the method. Seven serially diluted LDS concentrations and a buffer (for a negative control) were prepared for a maximum final range from 0 to 150  hMAO-A or hMAO-B inhibited by GST. DEP was used as a standard control for this assay. Briefly, after optimizing tyramine used concentration for hMAO-A, 5x substrate and peroxidase chromogen reagent were mixed (1 : 1) for final of 0.5 mM tyramine HCl. In 96-well plates, 25 μL of GST serial dilutions up to 46 μM final concentrations were mixed with 50 μL isozyme for 0.7 U/mL final concentrations (0.09 U per reaction). After 40 min incubation at RT, 50 μL of the reagent/substrate mix was added to initiate the reaction and to make the final concentrations. The developing color (H2O2 indicator) was monitored over time at 490 nm by the μQuant Microplate Spectrophotometer .A slightly modified continuous spectrophotometric method  was used human MAO-A and MAO-B at their Ligand Binding Domain. HYBRID of OEDocking (v3.0.1.) by OpenEye Scientific Software  was used as the docking method [GST, its analog DZ, and a selective MAO-B standard rasagiline (RAS) were docked as test ligands to the X-ray structure ofg method . Brieflyg method . Using S50 values were determined by nonlinear regression best-fit model of normalized response with variable slope. The inhibitor constant (Ki) values were obtained from the competitive inhibition model. Lineweaver-Burk plot was obtained from Michaelis-Menten data. Relative selectivity (RS) folds were defined by the ratio of hMAO-A IC50 to hMAO-B IC50. The significance of the difference between two groups was determined using an unpaired t-test, between the control and treatments using one-way ANOVA followed by Dunnett's multiple comparisons tests and between two sets of data using two-way ANOVA followed by Sidak's multiple comparisons test.All analyses were performed by GraphPad Prism Software 6.02 . IC hMAOs superiorly more than DZ (p < 0.0001). DZ showed minor or partial inhibitory effect up to 250 μM of the tested concentrations (p < 0.0001). Meanwhile, GST had a sigmoidal inhibitory pattern and potency with an hMAO-B mean IC50 of 6.81 μM to be 1.4% of DEP potency  and 4.3% (DEP), which indicates an unrecovered reduction of MAO activity after incubation with 10x IC50 concentrations. Meanwhile, preincubation of 0.1x IC50 GST with either hMAO-A or hMAO-B allowed a recovery of hMAO-A and hMAO-B catalytic rates to 86.8% and 86.1%, respectively. Their recovered activities measured in the presence of GST at its mean 1x IC50 value were 42.3% (MAO-A) and 47.1% (MAO-B). Both isozymes recoveries from GST were close to 90% or 50%, which is expected from a reversible inhibitor. No significant difference was found between the 100x IC50 and 4x IC50 or between 100x IC50 of both preincubated enzyme-inhibitor mixtures (data not shown). To investigate if the inhibition of activity is time-dependent, as it is known for irreversible inhibitors, we measured the postincubation time recovery in Figures  hMAO-A and hMAO-B, respectively. The results obtained provide more evidence that GST is a reversible inhibitor of hMAO-A and hMAO-B up to the tested 60 min at RT and a maximum of 100x IC50 value.The recovery of the Figures . At as ly-axis and not x-axis indicates a competitive mode of inhibition. Meanwhile, DEP linear regression crossed y-axis with hMAO-A and x-axis with MAO-B, confirming its noncompetitive hMAO-B mode of inhibition (data not shown). Vmax and LDS Km in the presence of GST were further determined and illustrated as folds of change  while LDS Km values increased severalfold with GST increased concentrations (p < 0.002 for hMAO-A and p < 0.0001 for hMAO-B). Interestingly, GST increased LDS Km for hMAO-B more significantly (14.9-fold) than LDS Km for hMAO-A (8.1-fold) by more than 1.8-fold at 20 μM GST concentration.Further investigations on the competitiveness of GST reversible inhibition on both MAOs are illustrated in  Figures . GST shoTo further assess the GST competitiveness, the alpha parameter was determined. The alpha value is used in Prism software to determine the degree at which the inhibitor can change the affinity with increasing substrate concentration. In the mixed model of inhibition analysis, alpha values were “too large” in all GST concentrations and both isozymes. The large alpha value indicates that GST reduced the substrate-binding affinity to the isozyme. The above studies exclude the noncompetitive, uncompetitive, or even mixed mode of GST inhibition to favor a competitive mode of inhibition on both MAOs.Ki) for both isozymes by Prism . After 2 h incubation, GST inhibited the H2O2 production by hMAO-B more selectively (IC50  2.9 ± 0.2 μM) than of hMAO-A (IC50  28.9 ± 0.1 μM) by about 10-fold (p < 0.0001) when using tyramine as a substrate . Secondly, the dose of GST can be more than 54 mg/day as reported in a clinical trial in elderly [The determination of the proper dosage in patients to achieve MAOs inhibitory concentrations of GST based on the current 1.00 μM . We beli 1.00 μM , 50. In  elderly . Thus, b elderly  (greater elderly . Nonethe elderly . Similar hMAO-A and hMAO-B inhibitory activities compared to its analog DZ negligible inhibitory effects. GST MAO-A inhibition potency was more than the standard DEP. However, its affinity and potency to inhibit hMAO-B were higher than hMAO-A. GST valuable MAO inhibitory capacity over DZ and selectivity to inhibit MAO-B may be due to the presence of C5-OH substitution that may have worked as an inhibitory group. The data obtained clearly show that GST is a reversible and competitive MAO-A and MAO-B inhibitor and, consequently, can safely decrease hMAOs toxic H2O2 byproduct, with the least chance to have the cheese effect incidence. Based on the data obtained in this study we recommend further investigations to examine the use of GST as a possible agent for the therapeutic management of PD patients with depression.The obtained results indicate that the isoflavone GST has potent


Actinobacteria species and has been shown to promote the growth of active cells and resuscitate dormant (non-dividing) cells. We previously reported the biological activity of an Rpf protein in Tomitella biformata AHU 1821T, an Actinobacteria isolated from a permafrost ice wedge. This protein is excreted outside the cell; however, few studies have investigated its contribution in environmental samples to the growth or resuscitation of bacteria other than the original host. Therefore, the aim of the present study was to determine whether Rpf from T. biformata impacted the cultivation of other bacteria from the permafrost ice wedge from which it was originally isolated. All experiments used recombinant Rpf proteins produced using a Rhodococcus erythropolis expression system. Dilutions of melted surface sterilized ice wedge samples mixed with different doses of the purified recombinant Rpf (rRpf) protein indicated that the highest concentration tested, 1250 pM, had a significantly (p <0.05) higher number of CFUs on agar plates after 8 d, approximately 14-fold higher than that on control plates without rRpf. 16S rRNA gene sequences revealed that all the colonies on plates were mainly related to Brevibacterium antiquum strain VKM Ac-2118 (AY243344), with 98–99% sequence identity. This species is also a member of the phylum Actinobacteria and was originally isolated from Siberian permafrost sediments. The results of the present study demonstrated that rRpf not only promoted the growth of T. biformata from which it was isolated, but also enhanced colony formation by another Actinobacteria in an environmental sample.Resuscitation-promoting factor (Rpf) is a protein that has been found in a number of different  Actinobacteria (Actinobacteria has been verified using purified Rpf proteins from Micrococcus luteus (Mycobacterium tuberculosis (18), and Corynebacterium glutamicum   . We demoproteins . The RpfActinobacteria in the environment. Based on these findings and also Rpf exhibiting cross species activity to some laboratory isolates, the aims of the present study were to determine whether the purified T. biformata Rpf protein applied to an environmental sample increased the cultivation efficiency of bacteria and also if it had cross species activity. To achieve these objectives, a permafrost ice wedge sample collected from the Fox tunnel, Alaska, USA, from which T. biformata was originally isolated, was used as the environmental source of bacteria. Furthermore, the rpf gene from T. biformata was cloned into an actinobacterial Rhodococcus erythropolis expression system  was produced using the pTip expression vector , washed twice with 2 mL of 10% glycerol, and resuspended in 1 mL of 10% glycerol. The cells were stored at −80°C until later use. One to five micrograms of DNA was mixed with 100 μL of electrocompetent cells. Electroporation was performed under high-voltage conditions  using a Gene Pulser Xcell (Bio-Rad). The R. erythropolis M1218/pTip-rpf-his strain was cultured at 27°C for 12 h in 1 L of W minimal medium (−1). Cultures were then induced using thiostrepton  (0.5 μg mL−1) and incubated for 5 h. The supernatant was harvested by centrifugation at 6,000×g for 20 min (4°C), and the resulting supernatant was passed through a 0.2-μm filter  and further purified using nickel affinity chromatography as described previously . Growth rates were calculated from the slope of the best fitting linear regression of log-transformed exponential growth data plotted against time.The recombinant Rpf protein expressed in n vector  provided2 vector . The resl medium  supplemeeviously . The fileviously . Briefly0, 10−1, 10−2, 10−3, and 10−4) were mixed with each amount of the Rpf protein  being tested for a total of 30 treatment combinations in triplicate. The results from the denatured proteins were not significantly different from the 0 rRpf controls and, therefore, were not shown in the Results section. Mixtures were spread onto R2A agar medium containing antimicrobials (0.05 g nystatin L−1 and 0.01 g cycloheximide L−1) to inhibit fungal growth. Plates were incubated at 15°C for 2 weeks and colony numbers were counted daily. The presence of living cells in the melted ice was confirmed by epifluorescence microscopy after staining using the Live/Dead Baclight staining kit  as previously described . DNA was amplified using the KOD-FX Neo kit (TOYOBO) with the primers 27F (5′-AGAG TTTGATCCTGGCTCAG-3′) and 1492R (5′-TACGGTTACCTTG TTACGACTT-3′) . The dosage effect of rRpf was determined using a least square regression analysis of log transformation rRpf concentrations versus the d of the lag phase or growth rates. Correlations were considered significant if R. erythropolis (rRpf-R) produced two bands, estimated to be 32 and 39 kDa in size, on an SDS-PAGE gel  with increases in the dosage of rRpf (log transformed). However, a correlation was not observed between growth rates and the rRpf concentrations tested . There was also no dosage effect for the resuscitation of non-dividing cells (p <0.01) higher OD values and a lag phase that was shorter (p=0.57) than cultures with higher protein concentrations and the “no protein added” control  did not correlate with the growth rate of colonies appearing on plates (p <0.05) greater number of CFUs was observed on plates dosed with 1250 pM of protein than with all other doses tested after 8 d (The rRpf dosage effect (rn plates . On the fter 8 d . Colony Brevibacterium antiquum strain VKM Ac-2118 (AY243344) with >99% sequence identity. The 11 almost full length sequences that were determined confirmed that the highest identity to a type strain was B. antiquum strain VKM Ac-2118 with >98% sequence identity  and the tentative new species B. antarcticum DVS 5a2 (AJ577724) with >98% identity .A total of 192 colonies were collected after 8 d of incubation and their 16S rRNA gene sequences were determined . Since sT. biformata, the source of the rpf gene used in these experiments, was first isolated. Although T. biformata did not grow from this sample, the colonies were all Brevibacterium or a related genus in the phylum Actinobacteria. Although only one bacterial species grew from this ice wedge, the number of different taxa, especially Actinobacteria, was limited in these samples; therefore, diversity among colonies was not high. The specific activity of Rpf on Actinobacteria species was expected because rpf-like genes are widely distributed among this phylum  increased the number of colonies formed from an environmental sample over that on plates with no or low rRpf additions during the earlier stages of incubation. A melted permafrost ice wedge was chosen as the environmental sample because it was the habitat from which s phylum , 26. Othficities . These fT. biformata in dividing and non-dividing states indicated that rRpf mediated both functions. Colony formation occurred sooner in cultures that had been induced into a non-dividing state and in cultures inoculated with low concentrations of dividing cells. This result was supported by the growth and resuscitation-promoting activities of T. biformata rRpf expressed in E. coli, which we reported previously  were shown to promote growth (T. biformata rRpf (300 pM) also increased colony numbers on agar plates, which had not been reported in earlier studies on Myc. tuberculosis (T. biformata. These differences in activity may have been due to differences in the protein sequence and structure of T. biformata Rpf from other functionally known Rpf (Although the rRpf from rculosis , which w. luteus . Furthere growth . T. biforculosis ; howeverrculosis . Previourculosis , 17, 18.rculosis , 18, theVibrio parahaemolyticus induced into a non-dividing state were found to still contain a subset of dividing cells that started to re-grow when conditions were changed and not because of cell resuscitation (Another factor potentially contributing to differences between these various studies and possibly between replicate experiments using the same rRpf source may be the physiological heterogeneity of cells. Populations of citation . This spcitation , 4. It ccitation . If the T. biformata was required at higher concentrations than other sources of Rpf (Corynebacterium glutamicum and when over-expressed as a recombinant (Although the results of the present study indicated that rRpf from s of Rpf , 18, it s of Rpf , 19. Theombinant  indicateActinobacteria from a permafrost ice wedge, the environment from which T. biformata was originally isolated. Rpf appears to be active against other members of the Actinobacteria, as suggested previously. Higher concentrations of rRpf from T. biformata were needed for growth and resuscitation-promoting activities in the present study than previously reported. These results demonstrate the need for further studies on Rpf from different Actinobacteria in order to understand its role and ecological importance in the environment. Future investigations may also develop a method by which to increase the fraction of cells that can be cultivated from environmental samples.We herein demonstrated that Rpf exhibited cross species activity and increased the colony number of


The phosphorylation sites in dbPPT were manually curated from the literatures, whereas datasets in other public databases were also integrated. In total, there were 82 175 phosphorylation sites in 31 012 proteins from 20 plant organisms in dbPPT, presenting a larger quantity of phosphorylation sites and a higher coverage of plant species in comparison with other databases. The proportions of residue types including serine, threonine and tyrosine were 77.99, 17.81 and 4.20%, respectively. All the phosphoproteins and phosphorylation sites in the database were critically annotated. Since the phosphorylation signaling in plants attracted great attention recently, such a comprehensive resource of plant protein phosphorylation can be useful for the research community.As one of the most important protein post-translational modifications, the reversible phosphorylation is critical for plants in regulating a variety of biological processes such as cellular metabolism, signal transduction and responses to environmental stress. Numerous efforts especially large-scale phosphoproteome profiling studies have been contributed to dissect the phosphorylation signaling in various plants, while a large number of phosphorylation events were identified. To provide an integrated data resource for further investigations, here we present a comprehensive database of dbPPT (database of Phosphorylation site in PlanTs, at Database URL:http://dbppt.biocuckoo.org Throet al.  found th -based identification techniques greatly advanced large-scale detection of phosphorylation events in plants . Early iet al.  publishellenging . In 2008  to provide user-friendly online services for data retrieval and access. The keyword-based search and blast search services were implemented. The browse service was also provided to query information for a specific organism of interest. As an application of the dbPPT database, the most significant sequence motifs for phosphorylation in Arabidopsis were analyzed. Taken together, dbPPT serves as the most comprehensive data resource for phosphorylation in plants, and would be helpful for the research communityIn this study, we provided a large collection of 82 175 phosphorylation sites in 31 012 phosphoproteins that were manually curated from literatures. The proportions for phosphorylated serines, threonines and tyrosines were 77.99, 17.81 and 4.20%, respectively. The phosphoproteins were from 20 plant species, whereas 29 058 phosphorylation sites in 9003 phosphoproteins were from 3DB and PhosPhAt. The literature database of PubMed (http://www.ncbi.nlm.nih.gov/pubmed) was searched to retrieve published plant phosphorylation-related studies through various keywords combinations such as ‘plant(s) phosphoproteomic’, ‘plant(s) phosphoproteome’ and more than 20 plant species names combined with ‘phosphoproteomic’ or ‘phosphoproteome’ like ‘Arabidopsis thaliana phosphoproteomic’, ‘Arabidopsis thaliana phosphoproteome’. To avoid missing data, additional keywords like ‘plant(s) large-scale phosphorylation’, ‘plant(s) MS phosphorylation’ and other related keywords were also employed to search PubMed. The retrieved literatures were manually checked to collect the experimentally identified phosphorylation substrates and sites in plants. The collected sites were cross checked with other databases such as P3DB and PhosPhAt.To construct the most comprehensive database for phosphorylation in plants, we integrated data from multiple different sources including PubMed, PBrassica napus could not be fully mapped to UniProt entries. Therefore, sequence information from other public databases such as Ensembl (Release 75)  database  as the sease 75)  and NCBIease 75)  were empSupplementary Table S2). Obviously, most of the identified phosphorylation sites were phosphoserines, while the number of phosphotyrosine was limited. However, with the advancement of phosphoproteomics techniques, more tyrosine phosphorylation will be identified  of phosphoserine, phosphothreonine and phosphotyrosine were 64 089 (77.99%), 14 632 (17.81%) and 3454 (4.20%), respectively    , while tno acid) A. The toP-X-pT-P B. PrevioP-X-pT-P , which i of CKII , 36, whiactivity . The topArabidopsis (Besides the results of motif analyses, which might be helpful for prediction of phosphorylation sites in plants, actually, PhosphAt database provided prediction service for bidopsis . To evalWith the development of phosphoproteome techniques, more efforts will be contributed to this area. The systematic methods to dissect phosphorylation signaling in yeast and animals such as network-based analyses could be employed in plants , while tSupplementary data are available at Database Online.