55 research outputs found
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Kinetic Analysis of BCL11B Multisite PhosphorylationâDephosphorylation and Coupled Sumoylation in Primary Thymocytes by Multiple Reaction Monitoring Mass Spectroscopy
Transcription factors with multiple post-translational modifications (PTMs) are not uncommon, but comprehensive information on site-specific dynamics and interdependence is comparatively rare. Assessing dynamic changes in the extent of PTMs has the potential to link multiple sites both to each other and to biological effects observable on the same time scale. The transcription factor and tumor suppressor BCL11B is critical to three checkpoints in T-cell development and is a target of a T-cell receptor (TCR)-mediated MAP kinase signaling. Multiple reaction monitoring (MRM) mass spectroscopy was used to assess changes in relative phosphorylation on 18 of 23 serine and threonine residues and sumoylation on one of two lysine resides in BCL11B. We have resolved the composite phosphorylationâdephosphorylation and sumoylation changes of BCL11B in response to MAP kinase activation into a complex pattern of site-specific PTM changes in primary mouse thymocytes. The site-specific resolution afforded by MRM analyses revealed four kinetic patterns of phosphorylation and one of sumoylation, including both rapid simultaneous site-specific increases and decreases at putative MAP kinase proline-directed phosphorylation sites, following stimulation. These data additionally revealed a novel spatiotemporal bisphosphorylation motif consisting of two kinetically divergent proline-directed phosphorylation sites spaced five resides apart.Keywords: SRM, T-cell receptor, Post-translational modification, Signal transduction, Sumoylation, MRM, BCL11
Translation Control of Swarming Proficiency in \u3cem\u3eBacillus subtilis\u3c/em\u3e by 5-amino-pentanolylated Elongation Factor P
Elongation factor P (EF-P) accelerates diprolyl synthesis and requires a posttranslational modification to maintain proteostasis. Two phylogenetically distinct EF-P modification pathways have been described and are encoded in the majority of Gram-negative bacteria, but neither is present in Gram-positive bacteria. Prior work suggested that the EF-P-encoding gene (efp) primarily supports Bacillus subtilis swarming differentiation, whereas EF-P in Gram-negative bacteria has a more global housekeeping role, prompting our investigation to determine whether EF-P is modified and how it impacts gene expression in motile cells. We identified a 5-aminopentanol moiety attached to Lys32 of B. subtilis EF-P that is required for swarming motility. A fluorescent in vivo B. subtilis reporter system identified peptide motifs whose efficient synthesis was most dependent on 5-aminopentanol EF-P. Examination of the B. subtilis genome sequence showed that these EF-P-dependent peptide motifs were represented in flagellar genes. Taken together, these data show that, in B. subtilis, a previously uncharacterized posttranslational modification of EF-P can modulate the synthesis of specific diprolyl motifs present in proteins required for swarming motility
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Identification of an Atypical Calcium-Dependent Calmodulin Binding Site on the C-terminal domain of GluN2A
N-methyl-D-aspartate (NMDA) receptors are calcium-permeable ion channels
assembled from four subunits that each have a common membrane topology. The
intracellular carboxyl terminal domain (CTD) of each subunit varies in length, is least
conserved between subunits, and binds multiple intracellular proteins. We defined a
region of interest in the GluN2A CTD, downstream of well-characterized membraneproximal
motifs, that shares only 29% sequence similarity with the equivalent region of
GluN2B. GluN2A (amino acids 875-1029) was fused to GST and used as a bait to
identify proteins from mouse brain with the potential to bind GluN2A as a function of
calcium. Using mass spectrometry we identified calmodulin as a calcium-dependent
GluN2A binding partner. Equilibrium fluorescence spectroscopy experiments indicate
that CaÂČâș/calmodulin binds GluN2A with high affinity (5.2 ± 2.4 nM) in vitro. Direct
interaction of CaÂČâș/calmodulin with GluN2A was not affected by disruption of classic
sequence motifs associated with CaÂČâș/calmodulin target recognition, but was critically
dependent upon Trp-1014. These findings provide new insight into the potential of
CaÂČâș/calmodulin, previously considered a GluN1-binding partner, to influence NMDA
receptors by direct association.Keywords: NMDA, Calmodulin, Calcium, GlutamateKeywords: NMDA, Calmodulin, Calcium, Glutamat
Cyclic Rhamnosylated Elongation Factor P Establishes Antibiotic Resistance in \u3cem\u3ePseudomonas aeruginosa\u3c/em\u3e
Elongation factor P (EF-P) is a ubiquitous bacterial protein that is required for the synthesis of poly-proline motifs during translation. In Escherichia coli and Salmonella enterica, the posttranslational ÎČ-lysylation of Lys34 by the PoxA protein is critical for EF-P activity. PoxA is absent from many bacterial species such as Pseudomonas aeruginosa, prompting a search for alternative EF-P posttranslation modification pathways. Structural analyses of P. aeruginosa EF-P revealed the attachment of a single cyclic rhamnose moiety to an Arg residue at a position equivalent to that at which ÎČ-Lys is attached to E. coli EF-P. Analysis of the genomes of organisms that both lack poxA and encode an Arg32-containing EF-P revealed a highly conserved glycosyltransferase (EarP) encoded at a position adjacent to efp. EF-P proteins isolated from P. aeruginosa ÎearP, or from a ÎrmlC::acc1 strain deficient in dTDP-l-rhamnose biosynthesis, were unmodified. In vitro assays confirmed the ability of EarP to use dTDP-l-rhamnose as a substrate for the posttranslational glycosylation of EF-P. The role of rhamnosylated EF-P in translational control was investigated in P. aeruginosa using a Pro4-green fluorescent protein (Pro4GFP) in vivo reporter assay, and the fluorescence was significantly reduced in Îefp, ÎearP, and ÎrmlC::acc1 strains. ÎrmlC::acc1, ÎearP, and Îefp strains also displayed significant increases in their sensitivities to a range of antibiotics, including ertapenem, polymyxin B, cefotaxim, and piperacillin. Taken together, our findings indicate that posttranslational rhamnosylation of EF-P plays a key role in P. aeruginosa gene expression and survival
Proteomic characterization reveals that MMP-3 correlates with bronchiolitis obliterans syndrome following allogeneic hematopoietic cell and lung transplantation
Improved diagnostic methods are needed for bronchiolitis obliterans
syndrome (BOS), a serious complication after allogeneic hematopoietic cell
transplantation (HCT) and lung transplantation. For proteins candidate
discovery, we compared plasma pools from HCT transplantation recipients with:
BOS at onset (n=12), pulmonary infection (n=16), chronic graft-versus-host
disease without pulmonary involvement (n=15), and no chronic complications
post-HCT (n=15). Pools were labeled with different tags [isobaric Tags for
Relative and Absolute Quantification (iTRAQ)], and two software tools identified
differentially expressed proteins (â„1.5-fold change). Candidate proteins
were further selected using a six-step computational biology approach. The
diagnostic value of the lead candidate, matrix metalloproteinase-3 (MMP-3), was
evaluated by ELISA in plasma of a verification cohort (n=112) with and without
BOS following HCT (n=76) or lung transplantation (n=36). MMP-3 plasma
concentrations differed significantly between patients with and without BOS
(AUC=0.77). Thus, MMP-3 represents a potential non-invasive blood test for
diagnosis of BOS
Biomarker Panel for Chronic Graft-Versus-Host Disease
PURPOSE:
To identify diagnostic and prognostic markers of chronic graft-versus-host disease (cGVHD), the major cause of morbidity and mortality after allogeneic hematopoietic cell transplantation (HCT).
PATIENTS AND METHODS:
Using a quantitative proteomics approach, we compared pooled plasma samples obtained at matched time points after HCT (median, 103 days) from 35 patients with cGVHD and 18 without cGVHD (data are available via ProteomeXchange with identifier PXD002762). Of 105 proteins showing at least a 1.25-fold difference in expression, 22 were selected on the basis of involvement in relevant pathways and enzyme-linked immunosorbent assay availability. Chemokine (C-X-C motif) ligand 9 (CXCL9) and suppression of tumorigenicity 2 (ST2) also were measured on the basis of previously determined associations with GVHD. Concentrations of the four lead biomarkers were measured at or after diagnosis in plasma from two independent verification cohorts (n = 391) to determine their association with cGVHD. Their prognostic ability when measured at approximately day +100 after HCT was evaluated in plasma of a second verification cohort (n = 172).
RESULTS:
Of 24 proteins measured in the first verification cohort, nine proteins were associated with cGVHD, and only four (ST2, CXCL9, matrix metalloproteinase 3, and osteopontin) were necessary to compose a four-biomarker panel with an area under the receiver operating characteristic curve (AUC) of 0.89 and significant correlation with cGVHD diagnosis, cGVHD severity, and nonrelapse mortality. In a second verification cohort, this panel distinguished patients with cGVHD (AUC, 0.75), and finally, the panel measured at day +100 could predict cGVHD occurring within the next 3 months with an AUC of 0.67 and 0.79 without and with known clinical risk factors, respectively.
CONCLUSION:
We conclude that the biomarker panel measured at diagnosis or day +100 after HCT may allow patient stratification according to risk of cGVHD
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Proteomics-driven Antigen Discovery for Development of Vaccines Against Gonorrhea
Expanding efforts to develop preventive gonorrhea vaccines is critical because of the dire possibility of untreatable gonococcal infections. Reverse vaccinology, which includes genome and proteome mining, has proven very successful in the discovery of vaccine candidates against many pathogenic bacteria. However, progress with this approach for a gonorrhea vaccine remains in its infancy. Accordingly, we applied a comprehensive proteomic platformâisobaric tagging for absolute quantification coupled with two-dimensional liquid chromatography and mass spectrometryâto identify potential gonococcal vaccine antigens. Our previous analyses focused on cell envelopes and naturally released membrane vesicles derived from four different Neisseria gonorrhoeae strains. Here, we extended these studies to identify cell envelope proteins of N. gonorrhoeae that are ubiquitously expressed and specifically induced by physiologically relevant environmental stimuli: oxygen availability, iron deprivation, and the presence of human serum. Together, these studies enabled the identification of numerous potential gonorrhea vaccine targets. Initial characterization of five novel vaccine candidate antigens that were ubiquitously expressed under these different growth conditions demonstrated that homologs of BamA (NGO1801), LptD (NGO1715), and TamA (NGO1956), and two uncharacterized proteins, NGO2054 and NGO2139, were surface exposed, secreted via naturally released membrane vesicles, and elicited bactericidal antibodies that cross-reacted with a panel of temporally and geographically diverse isolates. In addition, analysis of polymorphisms at the nucleotide and amino acid levels showed that these vaccine candidates are highly conserved among N. gonorrhoeae strains. Finally, depletion of BamA caused a loss of N. gonorrhoeae viability, suggesting it may be an essential target. Together, our data strongly support the use of proteomics-driven discovery of potential vaccine targets as a sound approach for identifying promising gonococcal antigens.This is the publisherâs final pdf. The published article is copyrighted by The American Society for Biochemistry and Molecular Biology and can be found at: http://www.mcponline.org/content/15/7/233
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Characterization of UV-crosslinked protein-nucleic acid interfaces by Maldi MS and ESI MS/MS
Data from: Quantitative proteomics reveals key roles for post-transcriptional gene regulation in the molecular pathology of FSHD
DUX4 is a transcription factor whose misexpression in skeletal muscle causes facioscapulohumeral muscular dystrophy (FSHD). While DUX4's transcriptional activity has been extensively characterized, the DUX4-induced proteome remains undescribed. Here, we report concurrent measurement of RNA and protein levels in DUX4-expressing cells via RNA-seq and quantitative mass spectrometry. DUX4 transcriptional targets were robustly translated, confirming the likely clinical relevance of proposed FSHD biomarkers. However, a multitude of mRNAs and proteins exhibited discordant expression changes upon DUX4 expression. Our dataset revealed unexpected proteomic, but not transcriptomic, dysregulation of diverse molecular pathways, including Golgi apparatus fragmentation, as well as extensive post-transcriptional buffering of stress response genes. Key components of RNA degradation machineries, including UPF1, UPF3B, and XRN1, exhibited suppressed protein, but not mRNA, levels, explaining the build-up of aberrant RNAs that characterizes DUX4-expressing cells. Our results provide a resource for the FSHD community and illustrate the importance of post-transcriptional processes to DUX4-induced pathology
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