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ABSTRACT: A recent study by Bromenshenk et al., published in PLoS One (2010), used proteomic analysis to identify peptides purportedly of Iridovirus and Nosema origin; however the validity of this finding is controversial. We show here through re-analysis of a subset of this data that many of the spectra identified by Bromenshenk et al. as deriving from Iridovirus and Nosema proteins are actually products from Apis mellifera honey bee proteins. We find no reliable evidence that proteins from Iridovirus and Nosema are present in the samples that were re-analyzed. This article is also intended as a learning exercise for illustrating some of the potential pitfalls of analysis of mass spectrometry proteomic data and to encourage authors to observe MS/MS data reporting guidelines that would facilitate recognition of analysis problems during the review process.
PLoS ONE 01/2011; 6(6):e20873. · 4.09 Impact Factor
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ABSTRACT: Proteases are a ubiquitous group of enzymes that play key roles in the life cycle of parasites, in the host-parasite relationship, and in the pathogenesis of parasitic diseases. Furthermore, proteases are druggable targets for the development of new anti-parasitic therapy. The subtilisin protease (SUB; Clan SB, family S8) of Leishmania donovani was cloned and found to possess a unique catalytic triad. This gene was then deleted by gene knock-out, which resulted in reduced ability by the parasite to undergo promastigote to amastigote differentiation in vitro. Electron microscopy of SUB knock-out amastigotes revealed abnormal membrane structures, retained flagella, and increased binucleation. SUB-deficient Leishmania displayed reduced virulence in both hamster and murine infection models. Histology of spleens from SUB knock-out-infected hamsters revealed the absence of psammoma body calcifications indicative of the granulomatous lesions that occur during Leishmania infection. To delineate the specific role of SUB in parasite physiology, two-dimensional gel electrophoresis was carried out on SUB(-/-) versus wild-type parasites. SUB knock-out parasites showed altered regulation of the terminal peroxidases of the trypanothione reductase system. Leishmania and other trypanosomatids lack glutathione reductase, and therefore rely on the novel trypanothione reductase system to detoxify reactive oxygen intermediates and to maintain redox homeostasis. The predominant tryparedoxin peroxidases were decreased in SUB(-/-) parasites, and higher molecular weight isoforms were present, indicating altered processing. In addition, knock-out parasites showed increased sensitivity to hydroperoxide. These data suggest that subtilisin is the maturase for tryparedoxin peroxidases and is necessary for full virulence.
Journal of Biological Chemistry 10/2010; 285(41):31120-9. · 4.77 Impact Factor
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ABSTRACT: We have developed a method to enhance fluorescence quantification in two-dimensional gel electrophoresis using the inherent Raman scattering of water as an internal standard. We demonstrate this water internal standard (WIS) method using quantitative comparisons of commercially available protein standards that were either covalently tagged or passively stained with fluorescent tags. Thus, WIS opens up the possibility of enhancing intra- and intergel quantitative comparisons.
Journal of Proteome Research 04/2008; 7(3):1341-5. · 5.11 Impact Factor
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Jan Dvorák,
Susan T Mashiyama,
Simon Braschi,
Mohammed Sajid, Giselle M Knudsen,
Elizabeth Hansell,
Kee-Chong Lim,
Ivy Hsieh,
Mahmoud Bahgat,
Bryony Mackenzie,
Katalin F Medzihradszky,
Patricia C Babbitt,
Conor R Caffrey,
James H McKerrow
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ABSTRACT: Schistosomes are parasitic platyhelminths (flatworms) of birds and mammals. As a parasitic disease of humans, schistosomiasis ranks second only to malaria in global importance. Schistosome larvae (cercariae) must invade and penetrate skin as an initial step to successful infection of the vertebrate host. Proteolytic enzymes secreted from the acetabular glands of cercariae contribute significantly to the invasion process. In this comparative study, we analyzed protease activities secreted by cercariae of Schistosoma mansoni, Schistosoma japonicum and Schistosomatium douthitti. Using protease-family specific, irreversible active-site probes, fluorogenic peptidyl substrates, immuno-histochemistry and high-resolution mass spectrometry, considerable species differences were noted in the quantity and character of proteases. Serine proteases, the most abundant enzymes secreted by S. mansoni cercariae, were not identified in S. japonicum. In contrast, the acetabular gland contents of S. japonicum cercariae had a 40-fold greater cathepsin B-like activity than those of S. mansoni. Based on the present data and previous reports, we propose that cysteine proteases represent an archetypal tool for tissue invasion among primitive metazoa and the use of serine proteases arose later in schistosome evolution. Computational analysis of serine protease phylogeny revealed an extraordinarily distant relationship between S. mansoni serine proteases and other members of the Clan PA family S1 proteases.
Biochimie 03/2008; 90(2):345-58. · 3.02 Impact Factor
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ABSTRACT: Schistosomiasis is a global health problem caused by several species of schistosome blood flukes. The initial stage of infection is invasion of human skin by a multicellular larva, the cercaria. We identified proteins released by cercariae when they are experimentally induced to exhibit invasive behavior. Comparison of the proteome obtained from skin lipid-induced cercariae (the natural activator), a cleaner mechanical induction procedure, and an uninduced proteomic control allowed identification of protein groups contained in cercarial acetabular gland secretion versus other sources. These included a group of proteins involved in calcium binding, calcium regulation, and calcium-activated functions; two proteins (paramyosin and SPO-1) implicated in immune evasion; and protease isoforms implicated in degradation of host skin barriers. Several other protein families, traditionally found as cytosolic proteins, appeared concentrated in secretory cells. These included proteins with chaperone activity such as HSP70, -86, and -60. Comparison of the three experimental proteomes also allowed identification of protein contaminants from the environment that were identified because of the high sensitivity of the MS/MS system used. These included proteins from the intermediate host snail in which cercariae develop, the investigator, and the laboratory environment. Identification of proteins secreted by invasive larvae provides important new information for validation of models of skin invasion and immune evasion and aids in rational development of an anti-schistosome vaccine.
Molecular & Cellular Proteomics 01/2006; 4(12):1862-75. · 7.40 Impact Factor
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ABSTRACT: Catalase-peroxidases (KatG) are bifunctional enzymes possessing both catalase and peroxidase activities. Three crystal structures of different KatGs revealed the presence of a novel Met-Tyr-Trp cross-link that has been suggested to impart catalatic activity to the KatGs. High-performance liquid chromatographic separation of the peptide fragments resulting from tryptic digestion of recombinant Mycobacterium tuberculosis WT KatG identified a peptide with unusual UV-visible spectroscopic features attributable to the Met(255)-Tyr(229)-Trp(107) cross-link, whose structure was confirmed by mass spectrometry. WT KatG lacking the Met-Tyr-Trp cross-link was prepared, making possible studies of its formation under oxidizing conditions that generate either compound I (peroxyacetic acid, PAA) or compound II (2-methyl-1-phenyl-2-propyl hydroperoxide, MPPH). Incubation of this "cross-link-free" WT KatG with PAA revealed complete formation of the Met-Tyr-Trp structure after six equivalents of peracid were added, whereas MPPH was unable to promote cross-link formation. A mechanism for Met-Tyr-Trp autocatalytic formation by KatG compound I is proposed from these studies. Optical stopped-flow studies of WT KatG and KatG(Y229F), a mutant in which the cross-link cannot be formed, were performed with MPPH and revealed an unusual compound II spectrum for WT KatG, best described as (P.)Fe(III), where P. represents a protein-based radical. This contrasts with the oxoferryl compound II spectrum observed for KatG(Y229F) under identical conditions. The structure-function-spectroscopy relationship in KatG is discussed with relevance to the role that the Met-Tyr-Trp cross-link plays in the catalase-peroxidase mechanism.
Journal of Biological Chemistry 07/2005; 280(24):22651-63. · 4.77 Impact Factor
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ABSTRACT: Catalase-peroxidases (KatG) are bifunctional enzymes possessing both catalase and peroxidase activities. Three crystal structures
of different KatGs revealed the presence of a novel Met-Tyr-Trp cross-link that has been suggested to impart catalatic activity
to the KatGs. High-performance liquid chromatographic separation of the peptide fragments resulting from tryptic digestion
of recombinant Mycobacterium tuberculosis WT KatG identified a peptide with unusual UV-visible spectroscopic features attributable to the Met255-Tyr229-Trp107 cross-link, whose structure was confirmed by mass spectrometry. WT KatG lacking the Met-Tyr-Trp cross-link was prepared,
making possible studies of its formation under oxidizing conditions that generate either compound I (peroxyacetic acid, PAA)
or compound II (2-methyl-1-phenyl-2-propyl hydroperoxide, MPPH). Incubation of this “cross-link-free” WT KatG with PAA revealed
complete formation of the Met-Tyr-Trp structure after six equivalents of peracid were added, whereas MPPH was unable to promote
cross-link formation. A mechanism for Met-Tyr-Trp autocatalytic formation by KatG compound I is proposed from these studies.
Optical stopped-flow studies of WT KatG and KatG(Y229F), a mutant in which the cross-link cannot be formed, were performed
with MPPH and revealed an unusual compound II spectrum for WT KatG, best described as (P·)FeIII, where P· represents a protein-based radical. This contrasts with the oxoferryl compound II spectrum observed for KatG(Y229F)
under identical conditions. The structure-function-spectroscopy relationship in KatG is discussed with relevance to the role
that the Met-Tyr-Trp cross-link plays in the catalase-peroxidase mechanism.
Journal of Biological Chemistry 06/2005; 280(24):22651-22663. · 4.77 Impact Factor
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ABSTRACT: The AhpC/AhpD system of Mycobacterium tuberculosis provides important antioxidant protection, particularly when the KatG catalase-peroxidase activity is depressed, as it is in many isoniazid resistant strains. In the absence of lipoamide or bovine dihydrolipoamide dehydrogenase (DHLDH), components of the normal catalytic system, covalent dimers, tetramers, and hexamers are formed when a mixture of AhpC and AhpD is exposed to peroxide. Each of the oligomers contains equimolar amounts of AhpC and AhpD. This oligomerization is reversible because the oligomers can be fully reduced to the monomeric species by dithiothreitol. Using mutagenesis, we confirm here that Cys61 and Cys174 of AhpC as well as Cys133 and Cys130 of AhpD are critical for activity in the fully reconstituted system consisting of AhpC, AhpD, lipoamide, DHLDH, and NADH. A key step in the reduction of oxidized AhpC by reduced AhpD is formation of a disulfide cross-link between Cys61 of AhpC and Cys133 of AhpD. This cross-link can be reduced by intramolecular reaction with either Cys174 of AhpC or Cys130 of AhpD. Cys176 can also, to some extent, substitute for Cys174, providing a measure of redundancy that helps to maintain the efficiency of this antioxidant protective system.
Archives of Biochemistry and Biophysics 08/2004; 427(1):41-7. · 2.93 Impact Factor
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ABSTRACT: Inducible (iNOS) and constitutive (eNOS, nNOS) nitric-oxide synthases differ in their Ca2+-calmodulin (CaM) dependence. iNOS binds CaM irreversibly but eNOS and nNOS, which bind CaM reversibly, have inserts in their reductase domains that regulate electron transfer. These include the 43-45-amino acid autoinhibitory element (AI) that attenuates electron transfer in the absence of CaM, and the C-terminal 20-40-amino acid tail that attenuates electron transfer in a CaM-independent manner. We constructed models of the reductase domains of the three NOS isoforms to predict the structural basis for CaM-dependent regulation. We have identified and characterized a loop (CD2A) within the NOS connecting domain that is highly conserved by isoform and that, like the AI element, is within direct interaction distance of the CaM binding region. The eNOS CD2A loop (eCD2A) has the sequence 834KGSPGGPPPG843, and is truncated to 809ESGSY813 (iCD2A) in iNOS. The eCD2A contributes to the Ca2+ dependence of CaM-bound activity to a level similar to that of the AI element. The eCD2A plays an autoinhibitory role in the control of NO, and CaM-dependent and -independent reductase activity, but this autoinhibitory function is masked by the dominant AI element. Finally, the iCD2A is involved in determining the salt dependence of NO activity at a post-flavin reduction level. Electrostatic interactions between the CD2A loop and the CaM-binding region, and CaM itself, provide a structural means for the CD2A to mediate CaM regulation of intra-subunit electron transfer within the active NOS complex.
Journal of Biological Chemistry 09/2003; 278(34):31814-24. · 4.77 Impact Factor
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ABSTRACT: Five conserved histidine residues are found in the human endothelial nitric-oxide synthase (NOS) heme domain: His-420, His-421,
and His-461 are close to the heme, whereas His-146 and His-214 are some distance away. To investigate whether the histidines
form a non-heme iron-binding site, we have expressed the H146A, H214A, H420A, H421A, and H461A mutants. The H420A mutant could
not be isolated, and the H146A and H421A mutants were inactive. The H214A mutant resembled the wild-type enzyme in all respects.
The H461A mutant had a low-spin heme, but high concentrations of l-Arg and tetrahydrobiopterin led to partial recovery of activity. Laser atomic emission showed that the only significant metal
in NOS other than calcium and iron is zinc. The activities of the NOS isoforms were not increased by incubation with Fe2+, but were inhibited by high Fe2+ or Zn2+ concentrations. The histidine mutations altered the ability of the protein to dimerize and to bind heme. However, the protein
metal content, the inability of exogenous Fe2+ to increase catalytic activity, and the absence of evidence that the conserved histidines form a metal site provide no support
for a catalytic role for a non-heme redox-active metal.
Journal of Biological Chemistry 07/1999; 274(31):21617-21624. · 4.77 Impact Factor
