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A Superoxide Dismutase Capable of Functioning with Iron or Manganese Promotes the Resistance of Staphylococcus aureus to Calprotectin and Nutritional Immunity.
Garcia YM, Barwinska-Sendra A, Tarrant E, Skaar EP, Waldron KJ, Kehl-Fie TE
(2017) PLoS Pathog 13: e1006125
MeSH Terms: Animals, Chromatography, Ion Exchange, Disease Models, Animal, Iron, Leukocyte L1 Antigen Complex, Manganese, Mass Spectrometry, Mice, Mice, Inbred C57BL, Polymerase Chain Reaction, Staphylococcal Infections, Staphylococcus aureus, Superoxide Dismutase
Show Abstract · Added April 8, 2017
Staphylococcus aureus is a devastating mammalian pathogen for which the development of new therapeutic approaches is urgently needed due to the prevalence of antibiotic resistance. During infection pathogens must overcome the dual threats of host-imposed manganese starvation, termed nutritional immunity, and the oxidative burst of immune cells. These defenses function synergistically, as host-imposed manganese starvation reduces activity of the manganese-dependent enzyme superoxide dismutase (SOD). S. aureus expresses two SODs, denoted SodA and SodM. While all staphylococci possess SodA, SodM is unique to S. aureus, but the advantage that S. aureus gains by expressing two apparently manganese-dependent SODs is unknown. Surprisingly, loss of both SODs renders S. aureus more sensitive to host-imposed manganese starvation, suggesting a role for these proteins in overcoming nutritional immunity. In this study, we have elucidated the respective contributions of SodA and SodM to resisting oxidative stress and nutritional immunity. These analyses revealed that SodA is important for resisting oxidative stress and for disease development when manganese is abundant, while SodM is important under manganese-deplete conditions. In vitro analysis demonstrated that SodA is strictly manganese-dependent whereas SodM is in fact cambialistic, possessing equal enzymatic activity when loaded with manganese or iron. Cumulatively, these studies provide a mechanistic rationale for the acquisition of a second superoxide dismutase by S. aureus and demonstrate an important contribution of cambialistic SODs to bacterial pathogenesis. Furthermore, they also suggest a new mechanism for resisting manganese starvation, namely populating manganese-utilizing enzymes with iron.
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13 MeSH Terms
Analysis of protein composition using multidimensional chromatography and mass spectrometry.
Link AJ, Washburn MP
(2014) Curr Protoc Protein Sci 78: 23.1.1-23.1.25
MeSH Terms: Chromatography, High Pressure Liquid, Chromatography, Ion Exchange, Chromatography, Reverse-Phase, Mass Spectrometry, Proteins, Proteomics
Show Abstract · Added February 20, 2015
Multidimensional liquid chromatography of peptides produced by protease digestion of complex protein mixtures followed by tandem mass spectrometry can be coupled with automated database searching to identify large numbers of proteins in complex samples. These methods avoid the limitations of gel electrophoresis and in-gel digestions by directly identifying protein mixtures in solution. One method used extensively is named Multidimensional Protein Identification Technology (MudPIT), where reversed-phase chromatography and strong cation-exchange chromatography are coupled directly in a microcapillary column. This column is then placed in line between an HPLC and a mass spectrometer for complex mixture analysis. MudPIT remains a powerful approach for analyzing complex mixtures like whole proteomes and protein complexes. MudPIT is used for quantitative proteomic analysis of complex mixtures to generate novel biological insights.
Copyright © 2014 John Wiley & Sons, Inc.
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6 MeSH Terms
A structure-specific nucleic acid-binding domain conserved among DNA repair proteins.
Mason AC, Rambo RP, Greer B, Pritchett M, Tainer JA, Cortez D, Eichman BF
(2014) Proc Natl Acad Sci U S A 111: 7618-23
MeSH Terms: Adenosine Triphosphate, Animals, Chromatography, Affinity, Chromatography, Agarose, Chromatography, Gel, Chromatography, Ion Exchange, Cloning, Molecular, Crystallization, DNA Helicases, DNA Repair, Hydrolysis, Likelihood Functions, Mice, Models, Molecular, Nucleic Acids, Protein Structure, Tertiary, Scattering, Small Angle, X-Ray Diffraction
Show Abstract · Added May 19, 2014
SMARCAL1, a DNA remodeling protein fundamental to genome integrity during replication, is the only gene associated with the developmental disorder Schimke immuno-osseous dysplasia (SIOD). SMARCAL1-deficient cells show collapsed replication forks, S-phase cell cycle arrest, increased chromosomal breaks, hypersensitivity to genotoxic agents, and chromosomal instability. The SMARCAL1 catalytic domain (SMARCAL1(CD)) is composed of an SNF2-type double-stranded DNA motor ATPase fused to a HARP domain of unknown function. The mechanisms by which SMARCAL1 and other DNA translocases repair replication forks are poorly understood, in part because of a lack of structural information on the domains outside of the common ATPase motor. In the present work, we determined the crystal structure of the SMARCAL1 HARP domain and examined its conformation and assembly in solution by small angle X-ray scattering. We report that this domain is conserved with the DNA mismatch and damage recognition domains of MutS/MSH and NER helicase XPB, respectively, as well as with the putative DNA specificity motif of the T4 phage fork regression protein UvsW. Loss of UvsW fork regression activity by deletion of this domain was rescued by its replacement with HARP, establishing the importance of this domain in UvsW and demonstrating a functional complementarity between these structurally homologous domains. Mutation of predicted DNA-binding residues in HARP dramatically reduced fork binding and regression activities of SMARCAL1(CD). Thus, this work has uncovered a conserved substrate recognition domain in DNA repair enzymes that couples ATP-hydrolysis to remodeling of a variety of DNA structures, and provides insight into this domain's role in replication fork stability and genome integrity.
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18 MeSH Terms
AKAP signaling in reinstated cocaine seeking revealed by iTRAQ proteomic analysis.
Reissner KJ, Uys JD, Schwacke JH, Comte-Walters S, Rutherford-Bethard JL, Dunn TE, Blumer JB, Schey KL, Kalivas PW
(2011) J Neurosci 31: 5648-58
MeSH Terms: A Kinase Anchor Proteins, Amino Acid Sequence, Animals, Behavior, Animal, Blotting, Western, Chromatography, High Pressure Liquid, Chromatography, Ion Exchange, Cocaine-Related Disorders, Cyclic AMP-Dependent Protein Kinases, Male, Molecular Sequence Data, Nerve Tissue Proteins, Neural Networks, Computer, Nucleus Accumbens, Proteomics, Rats, Rats, Sprague-Dawley, Receptors, AMPA, Self Administration, Signal Transduction, Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization, Subcellular Fractions, Synapses
Show Abstract · Added May 27, 2014
To identify candidate proteins in the nucleus accumbens (NAc) as potential pharmacotherapeutic targets for treating cocaine addition, an 8-plex iTRAQ (isobaric tag for relative and absolute quantitation) proteomic screen was performed using NAc tissue obtained from rats trained to self-administer cocaine followed by extinction training. Compared with yoked-saline controls, 42 proteins in a postsynaptic density (PSD)-enriched subfraction of the NAc from cocaine-trained animals were identified as significantly changed. Among proteins of interest whose levels were identified as increased was AKAP79/150, the rat ortholog of human AKAP5, a PSD scaffolding protein that localizes signaling molecules to the synapse. Functional downregulation of AKAP79/150 by microinjecting a cell-permeable synthetic AKAP (A-kinase anchor protein) peptide into the NAc to disrupt AKAP-dependent signaling revealed that inhibition of AKAP signaling impaired the reinstatement of cocaine seeking. Reinstatement of cocaine seeking is thought to require upregulated surface expression of AMPA glutamate receptors, and the inhibitory AKAP peptide reduced the PSD content of protein kinase A (PKA) as well as surface expression of GluR1 in NAc. However, reduced surface expression was not associated with changes in PKA phosphorylation of GluR1. This series of experiments demonstrates that proteomic analysis provides a useful tool for identifying proteins that can regulate cocaine relapse and that AKAP proteins may contribute to relapse vulnerability by promoting increased surface expression of AMPA receptors in the NAc.
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23 MeSH Terms
Alterations in the proteome of the NHERF1 knockout mouse jejunal brush border membrane vesicles.
Donowitz M, Singh S, Singh P, Salahuddin FF, Chen Y, Chakraborty M, Murtazina R, Gucek M, Cole RN, Zachos NC, Kovbasnjuk O, Broere N, Smalley-Freed WG, Reynolds AB, Hubbard AL, Seidler U, Weinman E, de Jonge HR, Hogema BM, Li X
(2010) Physiol Genomics 42A: 200-10
MeSH Terms: Animals, Cadherins, Chromatography, Ion Exchange, Female, Immunoblotting, Immunohistochemistry, Jejunum, Male, Mice, Mice, Inbred C57BL, Mice, Knockout, Microscopy, Electron, Microvilli, Phosphoproteins, Proteome, Proteomics, Sodium-Hydrogen Exchangers, Tandem Mass Spectrometry, Transport Vesicles, beta Catenin
Show Abstract · Added March 5, 2014
Na/H exchanger regulatory factor 1 (NHERF1) is a scaffold protein made up of two PDZ domains and an ERM binding domain. It is in the brush border of multiple epithelial cells where it modulates 1) Na absorption by regulating NHE3 complexes and cytoskeletal association, 2) Cl secretion through trafficking of CFTR, and 3) Na-coupled phosphate absorption through membrane retention of NaPi2a. To further understand the role of NHERF1 in regulation of small intestinal Na absorptive cell function, with emphasis on apical membrane transport regulation, quantitative proteomic analysis was performed on brush border membrane vesicles (BBMV) prepared from wild-type (WT) and homozygous NHERF1 knockout mouse jejunal villus Na absorptive cells. Jejunal architecture appeared normal in NHERF1 null; however, there was increased proliferative activity, as indicated by increased crypt BrdU staining. LC-MS/MS analysis using iTRAQ to compare WT and NHERF1 null BBMV identified 463 proteins present in both WT and NHERF1 null BBMV of simultaneously prepared and studied samples. Seventeen proteins had an altered amount of expression between WT and NHERF1 null in two or more separate preparations, and 149 total proteins were altered in at least one BBMV preparation. The classes of the majority of proteins altered included transport proteins, signaling and trafficking proteins, and proteins involved in proliferation and cell division. Affected proteins also included tight junction and adherens junction proteins, cytoskeletal proteins, as well as metabolic and BB digestive enzymes. Changes in abundance of several proteins were confirmed by immunoblotting [increased CEACAM1, decreased ezrin (p-ezrin), NHERF3, PLCβ3, E-cadherin, p120, β-catenin]. The changes in the jejunal BBMV proteome of NHERF1 null mice are consistent with a more complex role of NHERF1 than just forming signaling complexes and anchoring proteins to the apical membrane and include at least alterations in proteins involved in transport, signaling, and proliferation.
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20 MeSH Terms
Evaluation of strong cation exchange versus isoelectric focusing of peptides for multidimensional liquid chromatography-tandem mass spectrometry.
Slebos RJ, Brock JW, Winters NF, Stuart SR, Martinez MA, Li M, Chambers MC, Zimmerman LJ, Ham AJ, Tabb DL, Liebler DC
(2008) J Proteome Res 7: 5286-94
MeSH Terms: Adenocarcinoma, Biomarkers, Cations, Chromatography, Ion Exchange, Chromatography, Liquid, Colonic Neoplasms, Computational Biology, Gene Expression Regulation, Neoplastic, Humans, Isoelectric Focusing, Mass Spectrometry, Peptides, Proteomics, Reproducibility of Results, Trypsin
Show Abstract · Added March 5, 2014
Shotgun proteome analysis platforms based on multidimensional liquid chromatography-tandem mass spectrometry (LC-MS/MS) provide a powerful means to discover biomarker candidates in tissue specimens. Analysis platforms must balance sensitivity for peptide detection, reproducibility of detected peptide inventories and analytical throughput for protein amounts commonly present in tissue biospecimens (< 100 microg), such that platform stability is sufficient to detect modest changes in complex proteomes. We compared shotgun proteomics platforms by analyzing tryptic digests of whole cell and tissue proteomes using strong cation exchange (SCX) and isoelectric focusing (IEF) separations of peptides prior to LC-MS/MS analysis on a LTQ-Orbitrap hybrid instrument. IEF separations provided superior reproducibility and resolution for peptide fractionation from samples corresponding to both large (100 microg) and small (10 microg) protein inputs. SCX generated more peptide and protein identifications than did IEF with small (10 microg) samples, whereas the two platforms yielded similar numbers of identifications with large (100 microg) samples. In nine replicate analyses of tryptic peptides from 50 microg colon adenocarcinoma protein, overlap in protein detection by the two platforms was 77% of all proteins detected by both methods combined. IEF more quickly approached maximal detection, with 90% of IEF-detectable medium abundance proteins (those detected with a total of 3-4 peptides) detected within three replicate analyses. In contrast, the SCX platform required six replicates to detect 90% of SCX-detectable medium abundance proteins. High reproducibility and efficient resolution of IEF peptide separations make the IEF platform superior to the SCX platform for biomarker discovery via shotgun proteomic analyses of tissue specimens.
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15 MeSH Terms
Enzymatic ligation creates discrete multinanoparticle building blocks for self-assembly.
Claridge SA, Mastroianni AJ, Au YB, Liang HW, Micheel CM, Fréchet JM, Alivisatos AP
(2008) J Am Chem Soc 130: 9598-605
MeSH Terms: Chromatography, High Pressure Liquid, Chromatography, Ion Exchange, DNA Adducts, DNA Ligases, DNA, Single-Stranded, Electrophoresis, Polyacrylamide Gel, Gold, Metal Nanoparticles, Models, Molecular, Scattering, Small Angle, X-Ray Diffraction
Show Abstract · Added April 12, 2013
Enzymatic ligation of discrete nanoparticle-DNA conjugates creates nanoparticle dimer and trimer structures in which the nanoparticles are linked by single-stranded DNA, rather than by double-stranded DNA as in previous experiments. Ligation was verified by agarose gel and small-angle X-ray scattering. This capability was utilized in two ways: first, to create a new class of multiparticle building blocks for nanoscale self-assembly and, second, to develop a system that can amplify a population of discrete nanoparticle assemblies.
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11 MeSH Terms
Phosphorylation and glycosylation of bovine lens MP20.
Ervin LA, Ball LE, Crouch RK, Schey KL
(2005) Invest Ophthalmol Vis Sci 46: 627-35
MeSH Terms: Amino Acid Sequence, Animals, Cattle, Chromatography, Ion Exchange, Electrophoresis, Polyacrylamide Gel, Eye Proteins, Glycosylation, Lens, Crystalline, Membrane Proteins, Molecular Sequence Data, Molecular Structure, Peptide Mapping, Phosphorylation, Serine, Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization, Threonine
Show Abstract · Added May 27, 2014
PURPOSE - Membrane protein 20 (MP20) is the second most abundant integral membrane protein in the lens, yet little is known about its function and post-translational modifications. The purpose of this work was the determination of the primary protein structure of MP20 and the types and sites of in vivo modifications.
METHODS - Bovine MP20 was isolated by anion exchange chromatography or SDS-PAGE followed by digestion with cyanogen bromide (CNBr) or trypsin. The total membrane protein fraction was also digested with trypsin in solution. The CNBr and trypsin peptides were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and matrix-assisted laser desorption ionization (MALDI) mass spectrometry.
RESULTS - Using these approaches to integral membrane protein analysis, complete sequence coverage of MP20 was obtained. The in vivo sites of phosphorylation were identified as Ser-170 and Thr-171, the latter being previously unreported. The most abundant form of MP20 is monophosphorylated at Ser-170, whereas unphosphorylated and diphosphorylated forms were in lower abundance. In addition, two sites of a rare type of glycosylation (C-mannosylation) were identified at tryptophan residues 43 and 61.
CONCLUSIONS - The functional significance of phosphorylated MP20, the predominant form, remains unknown. Glycosylation of tryptophan residues represents a new lens protein modification that can explain galectin-3 interaction and suggests a topology for MP20 in which these peptides are located in an extracellular domain.
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16 MeSH Terms
Purification and characterization of a 7-kDa protein from Clonorchis sinensis adult worms.
Lee HJ, Lee CS, Kim BS, Joo KH, Lee JS, Kim TS, Kim HR
(2002) J Parasitol 88: 499-504
MeSH Terms: Amino Acid Sequence, Animals, Antigens, Helminth, Base Sequence, Blotting, Western, Chromatography, Gel, Chromatography, Ion Exchange, Clonorchiasis, Clonorchis sinensis, DNA, Complementary, Electrophoresis, Polyacrylamide Gel, Humans, Male, Mice, Microscopy, Electron, Molecular Sequence Data, RNA, Helminth, Reverse Transcriptase Polymerase Chain Reaction, Sequence Analysis, DNA
Show Abstract · Added March 14, 2014
A 7-kDa protein was purified from extracts of adult Clonorchis sinensis by a combination of ammonium sulfate precipitation, anion exchange chromatography, cation exchange chromatography, gel-filtration chromatography, and reversed-phase FPLC. The 7-kDa protein exists in the excretory-secretory products of adult C. sinensis, but not in extracts of adult Paragonimus westermani. Also, the 7-kDa protein reacted with the sera of patients with clonorchiasis but not with paragonimiasis or normal human sera. To observe the localization of the 7-kDa protein in the tissue of adult C. sinensis, an immunogold labeling method was followed using anti-7-kDa antibody. The gold particles were observed in the basal layer below the tegumental syncytium, in the interstitial matrix of the parenchyma, and in the content of the uterus. The 7-kDa cDNA was obtained through reverse transcription-polymerase chain reaction using a primer designed from N-terminal sequence analysis. Rapid amplification of cDNA ends (5'-RACE) was used to obtain the complete protein coding sequence. The sequence encodes a 90-amino acid polypeptide. The deduced amino acid sequence of the 7-kDa protein revealed no homology with proteins of different organisms reported so far. These results suggest that the 7-kDa protein is a fluid antigen and may be valuable as a tool for the immunodiagnosis of clonorchiasis.
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19 MeSH Terms
Arrestin: mutagenesis, expression, purification, and functional characterization.
Gurevich VV, Benovic JL
(2000) Methods Enzymol 315: 422-37
MeSH Terms: Amino Acid Substitution, Animals, Arrestin, Blotting, Western, Cell-Free System, Chromatography, Affinity, Chromatography, Ion Exchange, Cloning, Molecular, Cyclic AMP-Dependent Protein Kinases, Electrophoresis, Polyacrylamide Gel, Escherichia coli, Mutagenesis, Site-Directed, Phosphorylation, Protein Biosynthesis, RNA, Messenger, Rabbits, Recombinant Proteins, Reticulocytes, Rhodopsin, Transcription, Genetic, beta-Adrenergic Receptor Kinases
Added December 10, 2013
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21 MeSH Terms