Walid Maaty | University of Kansas (original) (raw)

Papers by Walid Maaty

PloS one, Jan 16, 2009

To avoid molecular damage of biomolecules due to oxidation, all cells have evolved constitutive a... more To avoid molecular damage of biomolecules due to oxidation, all cells have evolved constitutive and responsive systems to mitigate and repair chemical modifications. Archaea have adapted to some of the most extreme environments known to support life, including highly oxidizing conditions. However, in comparison to bacteria and eukaryotes, relatively little is known about the biology and biochemistry of archaea in response to changing conditions and repair of oxidative damage. In this study transcriptome, proteome, and chemical reactivity analyses of hydrogen peroxide (H(2)O(2)) induced oxidative stress in Sulfolobus solfataricus (P2) were conducted. Microarray analysis of mRNA expression showed that 102 transcripts were regulated by at least 1.5 fold, 30 minutes after exposure to 30 microM H(2)O(2). Parallel proteomic analyses using two-dimensional differential gel electrophoresis (2D-DIGE), monitored more than 800 proteins 30 and 105 minutes after exposure and found that 18 had sig...

Frontiers in microbiology, 2012

The origin and evolutionary relationship of viruses is poorly understood. This makes archaeal vir... more The origin and evolutionary relationship of viruses is poorly understood. This makes archaeal virus-host systems of particular interest because the hosts generally root near the base of phylogenetic trees, while some of the viruses have clear structural similarities to those that infect prokaryotic and eukaryotic cells. Despite the advantageous position for use in evolutionary studies, little is known about archaeal viruses or how they interact with their hosts, compared to viruses of bacteria and eukaryotes. In addition, many archaeal viruses have been isolated from extreme environments and present a unique opportunity for elucidating factors that are important for existence at the extremes. In this article we focus on virus-host interactions using a proteomics approach to study Sulfolobus Turreted Icosahedral Virus (STIV) infection of Sulfolobus solfataricus P2. Using cultures grown from the ATCC cell stock, a single cycle of STIV infection was sampled six times over a 72 h period...

Virology, 2008

Fuselloviridae are ubiquitous crenarchaeal viruses found in high-temperature acidic hot springs w... more Fuselloviridae are ubiquitous crenarchaeal viruses found in high-temperature acidic hot springs worldwide. The type virus, Sulfolobus spindle-shaped virus 1 (SSV1), has a double-stranded DNA genome that contains 34 open reading frames (ORFs). Fuselloviral genomes show little similarity to other organisms, generally precluding functional predictions. However, tertiary protein structure can provide insight into protein function. We have thus undertaken a systematic investigation of the SSV1 proteome and report here on the F112 gene product. Biochemical, proteomic and structural studies reveal a monomeric intracellular protein that adopts a winged helix DNA binding fold. Notably, the structure contains an intrachain disulfide bond, prompting analysis of cysteine usage in this and other hyperthermophilic viral genomes. The analysis supports a general abundance of disulfide bonds in the intracellular proteins of hyperthermophilic viruses, and reveals decreased cysteine content in the membrane proteins of hyperthermophilic viruses infecting Sulfolobales. The evolutionary implications of the SSV1 distribution are discussed.

Journal of General Virology, 2009

The vinegar fly, Drosophila melanogaster, is a popular model for the study of invertebrate antivi... more The vinegar fly, Drosophila melanogaster, is a popular model for the study of invertebrate antiviral immune responses. Several picorna-like viruses are commonly found in both wild and laboratory populations of D. melanogaster. The best-studied and most pathogenic of these is the dicistrovirus Drosophila C virus. Among the uncharacterized small RNA viruses of D. melanogaster, Drosophila A virus (DAV) is the least pathogenic. Historically, DAV has been labelled as a picorna-like virus based on its particle size and the content of its RNA genome. Here, we describe the characterization of both the genome and the virion structure of DAV. Unexpectedly, the DAV genome was shown to encode a circular permutation in the palm-domain motifs of the RNA-dependent RNA polymerase. This arrangement has only been described previously for a subset of viruses from the double-stranded RNA virus family Birnaviridae and the T54 single-stranded RNA virus family Tetraviridae. The 8 Å (0.8 nm) DAV virion structure computed from cryo-electron microscopy and image reconstruction indicates that the virus structural protein forms two discrete domains within the capsid. The inner domain is formed from a clear T53 lattice with similarity to the b-sandwich domain of tomato bushy stunt virus, whilst the outer domain is not ordered icosahedrally, but forms a cage-like structure that surrounds the core domain. Taken together, this indicates that DAV is highly divergent from previously described viruses.

Journal of Biological Chemistry, 2006

Journal of Biological Chemistry, 2013

Background: N-Formylated bacterial/mitochondrial peptides in infected/injured tissues are GPCR ch... more Background: N-Formylated bacterial/mitochondrial peptides in infected/injured tissues are GPCR chemoattractant agonists for neutrophil FPRs. Results: C-terminal tail FPR phosphopeptides were identified by LC/MS/MS in tryptic digests of FPRs immunopurified from human blood neutrophils. Conclusion: FPR1 but not FPR2 is monophosphorylated at any one of seven C-terminal tail Ser/Thr residues after fMLF stimulation. Significance: Decoding of human neutrophil FPR phosphorylation may be important for controlling inflammation. Accumulation, activation, and control of neutrophils at inflammation sites is partly driven by N-formyl peptide chemoattractant receptors (FPRs). Occupancy of these G-proteincoupled receptors by formyl peptides has been shown to induce regulatory phosphorylation of cytoplasmic serine/threonine amino acid residues in heterologously expressed recombinant receptors, but the biochemistry of these modifications in primary human neutrophils remains relatively unstudied. FPR1 and FPR2 were partially immunopurified using antibodies that recognize both receptors (NFPRa) or unphosphorylated FPR1 (NFPRb) in dodecylmaltoside extracts of unstimulated and N-formyl-Met-Leu-Phe (fMLF) ؉ cytochalasin B-stimulated neutrophils or their membrane fractions. After deglycosylation and separation by SDS-PAGE, excised Coomassie Blue-staining bands (ϳ34,000 M r) were tryptically digested, and FPR1, phospho-FPR1, and FPR2 content was confirmed by peptide mass spectrometry. C-terminal FPR1 peptides (Leu 312-Arg 322 and Arg 323-Lys 350) and extracellular FPR1 peptide (Ile 191-Arg 201) as well as three similarly placed FPR2 peptides were identified in unstimulated and fMLF ؉ cytochalasin B-stimulated samples. LC/MS/MS identified seven isoforms of Ala 323-Lys 350 only in the fMLF ؉ cytochalasin B-stimulated sample. These were individually phosphorylated at Thr 325 , Ser 328 , Thr 329 , Thr 331 , Ser 332 , Thr 334 , and Thr 339. No phospho-FPR2 peptides were detected. Cytochalasin B treatment of neutrophils decreased the sensitivity of fMLF-dependent NFPRb recognition 2-fold, from EC 50 ‫؍‬ 33 ؎ 8 to 74 ؎ 21 nM. Our results suggest that 1) partial immunopurification, deglycosylation, and SDS-PAGE separation of FPRs is sufficient to identify C-terminal FPR1 Ser/Thr phosphorylations by LC/MS/MS; 2) kinases/phosphatases activated in fMLF/cytochalasin B-stimulated neutrophils produce multiple C-terminal tail FPR1 Ser/Thr phosphorylations but have little effect on corresponding FPR2 sites; and 3) the extent of FPR1 phosphorylation can be monitored with C-terminal tail FPR1phosphospecific antibodies.

Environmental Microbiology, 2011

Arsenic (As) is the most common toxic element in the environment, ranking first on the Superfund ... more Arsenic (As) is the most common toxic element in the environment, ranking first on the Superfund List of Hazardous Substances. Microbial redox transformations are the principal drivers of As chemical specia-tion, which in turn dictates As mobility and toxicity. Consequently, in order to manage or remediate environmental As, land managers need to understand how and why microorganisms react to As. Studies have demonstrated a two-component signal trans-duction system comprised of AioS (sensor kinase) and AioR (response regulator) is involved in regulating microbial AsIII oxidation, with the AsIII oxidase structural genes aioB and aioA being upregulated by AsIII. However, it is not known whether AsIII is first detected directly by AioS or by an intermediate. Herein we demonstrate the essential role of a peri-plasmic AsIII-binding protein encoded by aioX, which is upregulated by AsIII. An DaioX mutant is defective for upregulation of the aioBA genes and consequently AsIII oxidation. Purified AioX expressed without its TAT-type signal peptide behaves as a monomer (MW 32 kDa), and Western blots show AioX to be exclusively associated with the cytoplasmic membrane. AioX binds AsIII with a K D of 2.4 mM AsIII; however, mutating a conserved Cys108 to either alanine or serine resulted in lack of AsIII binding, lack of aioBA induction, and correlated with a negative AsIII oxidation phenotype. The discovery and characterization of AioX illustrates a novel AsIII sensing mechanism that appears to be used in a range of bacteria and also provides one of the first examples of a bacterial signal anchor protein.

Bioconjugate Chemistry, 2014

Biochimica et Biophysica Acta (BBA) - General Subjects, 2014

Background: The current paradigm of intracellular redox chemistry maintains that cells establish ... more Background: The current paradigm of intracellular redox chemistry maintains that cells establish a reducing environment maintained by a pool of small molecule and protein thiol to protect against oxidative damage. This strategy is conserved in mesophilic organisms from all domains of life, but has been confounded in thermophilic organisms where evidence suggests that intracellular proteins have abundant disulfides. Methods: Chemical labeling and 2-dimensional gel electrophoresis were used to capture disulfide bonding in the proteome of the model thermophile Sulfolobus solfataricus. The redox poise of the metabolome was characterized using both chemical labeling and untargeted liquid chromatography mass spectrometry. Gene annotation was undertaken using support vector machine based pattern recognition. Results: Proteomic analysis indicated the intracellular protein thiol of S. solfataricus was primarily in the disulfide form. Metabolic characterization revealed a lack of reduced small molecule thiol. Glutathione was found primarily in the oxidized state (GSSG), at relatively low concentration. Combined with genetic analysis, this evidence shows that pathways for synthesis of glutathione do exist in the archaeal domain. Conclusions: In observed thermophilic organisms, thiol abundance and redox poise suggest that this system is not directly utilized for protection against oxidative damage. Instead, a more oxidized intracellular environment promotes disulfide bonding, a critical adaptation for protein thermostability. General significance: Based on the placement of thermophilic archaea close to the last universal common ancestor in rRNA phylogenies, we hypothesize that thiol-based redox systems are derived from metabolic pathways originally tasked with promoting protein stability.

Journal of Proteome Research, 2012

Where there is life, there are viruses. The impact of viruses on evolution, global nutrient cycli... more Where there is life, there are viruses. The impact of viruses on evolution, global nutrient cycling, and disease has driven research on their cellular and molecular biology. Knowledge exists for a wide range of viruses, however, a major exception are viruses with archaeal hosts. Archaeal virushost systems are of great interest because they have similarities to both eukaryotic and bacterial systems and often live in extreme environments. Here we report the first proteomics-based experiments on archaeal host response to viral infection. Sulfolobus Turreted Icosahedral Virus (STIV) infection of Sulfolobus solfataricus P2 was studied using 1D and 2D differential gel electrophoresis (DIGE) to measure abundance and redox changes. Cysteine reactivity was measured using novel fluorescent zwitterionic chemical probes that, together with abundance changes, suggest that virus and host are both vying for control of redox status in the cells. Proteins from nearly 50% of the predicted viral open reading frames were found along with a new STIV protein with a homolog in STIV2. This study provides insight to features of viral replication novel to the archaea, makes strong connections to well described mechanisms used by eukaryotic viruses such as ESCRT-III mediated transport, and emphasizes the complementary nature of different omics approaches.

Bioconjugate Chemistry, 2013

The CyDye family of fluorescent dyes is currently the overwhelming choice for applications in pro... more The CyDye family of fluorescent dyes is currently the overwhelming choice for applications in proteomic analysis, using two-dimensional difference gel electrophoresis (2D-DIGE). Protein labeling with CyDyes is hampered by protein precipitation and gel smearing when used above minimal labeling. The solubility of labeled protein may be improved by introducing water solubilizing groups on the dye such as cysteic acids. However, addition of a negatively charged functionality will have the undesired effect of shifting the pI in relation to the unlabeled protein. These limitations have been addressed through the synthesis of highly water-soluble and pI balancing zwitterionic CyDye fluorophores (Z-CyDyes). The new dyes feature a cysteic acid motif, a titratable amine functionality and a NHS activated ester group. In side by side 2D-DIGE comparisons of Z-CyDyes and CyDyes, the new dyes significantly enhanced protein spot volume and the number of spots that were detected. Z-CyDyes have the potential to enhance the depth of proteome coverage and provide a general strategy for improving the performance of protein tagging reagents.

Background: Rotaviruses are known to modulate the innate antiviral defense response driven by IFN... more Background: Rotaviruses are known to modulate the innate antiviral defense response driven by IFN. The purpose of this study was to identify changes in the cellular proteome in response to rotavirus infection in the context of the IFN response. We also sought to identify proteins outside the IFN induction and signaling pathway that were modulated by rotavirus infection. Methods: 2D-DIGE and image analysis were used to identify cellular proteins that changed in levels of expression in response to rotavirus infection, IFN treatment, or IFN treatment prior to infection. Immunofluorescence microscopy was used to determine the subcellular localization of proteins associated with the unfolded protein response (UPR). Results: The data show changes in the levels of multiple proteins associated with cellular stress in infected cells, including levels of ER chaperones GRP78 and GRP94. Further investigations showed that GRP78, GRP94 and other proteins with roles in the ER-initiated UPR includi...

Particle bombardment has been used to transform maize genotype Hi-II. Immature embryos were co-tr... more Particle bombardment has been used to transform maize genotype Hi-II. Immature embryos were co-transformed with a plasmid containing the selectable and scorable marker genes (bar and uidA, respectively) and a plasmid containing the high-molecular-weight glutenin subunit Dy10 gene. Eight transgenic events (T0) were recovered from 1000 bombarded scutella (transformation efficiency thus 0.8%). T1 generation was produced by cross pollination between T0 plants and nontransgenic plants of the Egyptian inbred line Sd63. Integration of trangenes has been confirmed in the genome of T0 plants by PCR and Dot blot hybridization analysis. Expression of marker genes was detected in T0 plants by leaf painting and histochemical staining for bar and uidA genes, respectively.

Journal of the American Chemical Society, Jan 3, 2016

There is considerable interest in the discovery of peptide ligands that bind to protein targets. ... more There is considerable interest in the discovery of peptide ligands that bind to protein targets. Discovery of such ligands is usually approached by screening large peptide libraries. However, the individual peptides must be tethered to a tag that preserves their individual identities (e.g., phage display or one-bead one-compound). To overcome this limitation, we have developed a method for screening libraries of label-free peptides for binding to a protein target in solution as a single batch. The screening is based on decreased amide hydrogen exchange by peptides that bind to the target. Hydrogen exchange is measured by mass spectrometry. We demonstrate the approach using a peptide library derived from the Escherichia coli proteome that contained 6664 identifiable features. The library was spiked separately with a peptide spanning the calmodulin binding domain of endothelial nitric oxide synthase (eNOS, 494-513) and a peptide spanning the N-terminal 20 residues of bovine ribonuclea...

Plant physiology and biochemistry : PPB / Societe francaise de physiologie vegetale, Jan 14, 2016

Senescence is the last developmental phase of plant tissues, organs and, in the case of monocarpi... more Senescence is the last developmental phase of plant tissues, organs and, in the case of monocarpic senescence, entire plants. In monocarpic crops such as barley, it leads to massive remobilization of nitrogen and other nutrients to developing seeds. To further investigate this process, a proteomic comparison of flag leaves of near-isogenic late- and early-senescing barley germplasm was performed. Protein samples at 14 and 21 days past anthesis were analyzed using both two-dimensional gel-based and label-free quantitative mass spectrometry-based ('shotgun') proteomic techniques. This approach identified >9000 barley proteins, and one-third of them were quantified. Analysis focused on proteins that were significantly (p < 0.05; difference ≥1.5-fold) upregulated in early-senescing line '10_11' as compared to late-senescing variety 'Karl', as these may be functionally important for senescence. Proteins in this group included family 1 pathogenesis-related pr...

Bioconjugate Chemistry, 2013

The CyDye family of fluorescent dyes is currently the overwhelming choice for applications in pro... more The CyDye family of fluorescent dyes is currently the overwhelming choice for applications in proteomic analysis, using two-dimensional difference gel electrophoresis (2D-DIGE). Protein labeling with CyDyes is hampered by protein precipitation and gel smearing when used above minimal labeling. The solubility of labeled protein may be improved by introducing water solubilizing groups on the dye such as cysteic acids. However, addition of a negatively charged functionality will have the undesired effect of shifting the pI in relation to the unlabeled protein. These limitations have been addressed through the synthesis of highly water-soluble and pI balancing zwitterionic CyDye fluorophores (Z-CyDyes). The new dyes feature a cysteic acid motif, a titratable amine functionality and a NHS activated ester group. In side by side 2D-DIGE comparisons of Z-CyDyes and CyDyes, the new dyes significantly enhanced protein spot volume and the number of spots that were detected. Z-CyDyes have the potential to enhance the depth of proteome coverage and provide a general strategy for improving the performance of protein tagging reagents.

Where there is life, there are viruses. The impact of viruses on evolution, global nutrient cycli... more Where there is life, there are viruses. The impact of viruses on evolution, global nutrient cycling, and disease has driven research on their cellular and molecular biology. Knowledge exists for a wide range of viruses, however, a major exception are viruses with archaeal hosts. Archaeal virushost systems are of great interest because they have similarities to both eukaryotic and bacterial systems and often live in extreme environments. Here we report the first proteomics-based experiments on archaeal host response to viral infection. Sulfolobus Turreted Icosahedral Virus (STIV) infection of Sulfolobus solfataricus P2 was studied using 1D and 2D differential gel electrophoresis (DIGE) to measure abundance and redox changes. Cysteine reactivity was measured using novel fluorescent zwitterionic chemical probes that, together with abundance changes, suggest that virus and host are both vying for control of redox status in the cells. Proteins from nearly 50% of the predicted viral open reading frames were found along with a new STIV protein with a homolog in STIV2. This study provides insight to features of viral replication novel to the archaea, makes strong connections to well described mechanisms used by eukaryotic viruses such as ESCRT-III mediated transport, and emphasizes the complementary nature of different omics approaches.

PloS one, Jan 16, 2009

To avoid molecular damage of biomolecules due to oxidation, all cells have evolved constitutive a... more To avoid molecular damage of biomolecules due to oxidation, all cells have evolved constitutive and responsive systems to mitigate and repair chemical modifications. Archaea have adapted to some of the most extreme environments known to support life, including highly oxidizing conditions. However, in comparison to bacteria and eukaryotes, relatively little is known about the biology and biochemistry of archaea in response to changing conditions and repair of oxidative damage. In this study transcriptome, proteome, and chemical reactivity analyses of hydrogen peroxide (H(2)O(2)) induced oxidative stress in Sulfolobus solfataricus (P2) were conducted. Microarray analysis of mRNA expression showed that 102 transcripts were regulated by at least 1.5 fold, 30 minutes after exposure to 30 microM H(2)O(2). Parallel proteomic analyses using two-dimensional differential gel electrophoresis (2D-DIGE), monitored more than 800 proteins 30 and 105 minutes after exposure and found that 18 had sig...

Frontiers in microbiology, 2012

The origin and evolutionary relationship of viruses is poorly understood. This makes archaeal vir... more The origin and evolutionary relationship of viruses is poorly understood. This makes archaeal virus-host systems of particular interest because the hosts generally root near the base of phylogenetic trees, while some of the viruses have clear structural similarities to those that infect prokaryotic and eukaryotic cells. Despite the advantageous position for use in evolutionary studies, little is known about archaeal viruses or how they interact with their hosts, compared to viruses of bacteria and eukaryotes. In addition, many archaeal viruses have been isolated from extreme environments and present a unique opportunity for elucidating factors that are important for existence at the extremes. In this article we focus on virus-host interactions using a proteomics approach to study Sulfolobus Turreted Icosahedral Virus (STIV) infection of Sulfolobus solfataricus P2. Using cultures grown from the ATCC cell stock, a single cycle of STIV infection was sampled six times over a 72 h period...

Virology, 2008

Fuselloviridae are ubiquitous crenarchaeal viruses found in high-temperature acidic hot springs w... more Fuselloviridae are ubiquitous crenarchaeal viruses found in high-temperature acidic hot springs worldwide. The type virus, Sulfolobus spindle-shaped virus 1 (SSV1), has a double-stranded DNA genome that contains 34 open reading frames (ORFs). Fuselloviral genomes show little similarity to other organisms, generally precluding functional predictions. However, tertiary protein structure can provide insight into protein function. We have thus undertaken a systematic investigation of the SSV1 proteome and report here on the F112 gene product. Biochemical, proteomic and structural studies reveal a monomeric intracellular protein that adopts a winged helix DNA binding fold. Notably, the structure contains an intrachain disulfide bond, prompting analysis of cysteine usage in this and other hyperthermophilic viral genomes. The analysis supports a general abundance of disulfide bonds in the intracellular proteins of hyperthermophilic viruses, and reveals decreased cysteine content in the membrane proteins of hyperthermophilic viruses infecting Sulfolobales. The evolutionary implications of the SSV1 distribution are discussed.

Journal of General Virology, 2009

The vinegar fly, Drosophila melanogaster, is a popular model for the study of invertebrate antivi... more The vinegar fly, Drosophila melanogaster, is a popular model for the study of invertebrate antiviral immune responses. Several picorna-like viruses are commonly found in both wild and laboratory populations of D. melanogaster. The best-studied and most pathogenic of these is the dicistrovirus Drosophila C virus. Among the uncharacterized small RNA viruses of D. melanogaster, Drosophila A virus (DAV) is the least pathogenic. Historically, DAV has been labelled as a picorna-like virus based on its particle size and the content of its RNA genome. Here, we describe the characterization of both the genome and the virion structure of DAV. Unexpectedly, the DAV genome was shown to encode a circular permutation in the palm-domain motifs of the RNA-dependent RNA polymerase. This arrangement has only been described previously for a subset of viruses from the double-stranded RNA virus family Birnaviridae and the T54 single-stranded RNA virus family Tetraviridae. The 8 Å (0.8 nm) DAV virion structure computed from cryo-electron microscopy and image reconstruction indicates that the virus structural protein forms two discrete domains within the capsid. The inner domain is formed from a clear T53 lattice with similarity to the b-sandwich domain of tomato bushy stunt virus, whilst the outer domain is not ordered icosahedrally, but forms a cage-like structure that surrounds the core domain. Taken together, this indicates that DAV is highly divergent from previously described viruses.

Journal of Biological Chemistry, 2006

Journal of Biological Chemistry, 2013

Background: N-Formylated bacterial/mitochondrial peptides in infected/injured tissues are GPCR ch... more Background: N-Formylated bacterial/mitochondrial peptides in infected/injured tissues are GPCR chemoattractant agonists for neutrophil FPRs. Results: C-terminal tail FPR phosphopeptides were identified by LC/MS/MS in tryptic digests of FPRs immunopurified from human blood neutrophils. Conclusion: FPR1 but not FPR2 is monophosphorylated at any one of seven C-terminal tail Ser/Thr residues after fMLF stimulation. Significance: Decoding of human neutrophil FPR phosphorylation may be important for controlling inflammation. Accumulation, activation, and control of neutrophils at inflammation sites is partly driven by N-formyl peptide chemoattractant receptors (FPRs). Occupancy of these G-proteincoupled receptors by formyl peptides has been shown to induce regulatory phosphorylation of cytoplasmic serine/threonine amino acid residues in heterologously expressed recombinant receptors, but the biochemistry of these modifications in primary human neutrophils remains relatively unstudied. FPR1 and FPR2 were partially immunopurified using antibodies that recognize both receptors (NFPRa) or unphosphorylated FPR1 (NFPRb) in dodecylmaltoside extracts of unstimulated and N-formyl-Met-Leu-Phe (fMLF) ؉ cytochalasin B-stimulated neutrophils or their membrane fractions. After deglycosylation and separation by SDS-PAGE, excised Coomassie Blue-staining bands (ϳ34,000 M r) were tryptically digested, and FPR1, phospho-FPR1, and FPR2 content was confirmed by peptide mass spectrometry. C-terminal FPR1 peptides (Leu 312-Arg 322 and Arg 323-Lys 350) and extracellular FPR1 peptide (Ile 191-Arg 201) as well as three similarly placed FPR2 peptides were identified in unstimulated and fMLF ؉ cytochalasin B-stimulated samples. LC/MS/MS identified seven isoforms of Ala 323-Lys 350 only in the fMLF ؉ cytochalasin B-stimulated sample. These were individually phosphorylated at Thr 325 , Ser 328 , Thr 329 , Thr 331 , Ser 332 , Thr 334 , and Thr 339. No phospho-FPR2 peptides were detected. Cytochalasin B treatment of neutrophils decreased the sensitivity of fMLF-dependent NFPRb recognition 2-fold, from EC 50 ‫؍‬ 33 ؎ 8 to 74 ؎ 21 nM. Our results suggest that 1) partial immunopurification, deglycosylation, and SDS-PAGE separation of FPRs is sufficient to identify C-terminal FPR1 Ser/Thr phosphorylations by LC/MS/MS; 2) kinases/phosphatases activated in fMLF/cytochalasin B-stimulated neutrophils produce multiple C-terminal tail FPR1 Ser/Thr phosphorylations but have little effect on corresponding FPR2 sites; and 3) the extent of FPR1 phosphorylation can be monitored with C-terminal tail FPR1phosphospecific antibodies.

Environmental Microbiology, 2011

Arsenic (As) is the most common toxic element in the environment, ranking first on the Superfund ... more Arsenic (As) is the most common toxic element in the environment, ranking first on the Superfund List of Hazardous Substances. Microbial redox transformations are the principal drivers of As chemical specia-tion, which in turn dictates As mobility and toxicity. Consequently, in order to manage or remediate environmental As, land managers need to understand how and why microorganisms react to As. Studies have demonstrated a two-component signal trans-duction system comprised of AioS (sensor kinase) and AioR (response regulator) is involved in regulating microbial AsIII oxidation, with the AsIII oxidase structural genes aioB and aioA being upregulated by AsIII. However, it is not known whether AsIII is first detected directly by AioS or by an intermediate. Herein we demonstrate the essential role of a peri-plasmic AsIII-binding protein encoded by aioX, which is upregulated by AsIII. An DaioX mutant is defective for upregulation of the aioBA genes and consequently AsIII oxidation. Purified AioX expressed without its TAT-type signal peptide behaves as a monomer (MW 32 kDa), and Western blots show AioX to be exclusively associated with the cytoplasmic membrane. AioX binds AsIII with a K D of 2.4 mM AsIII; however, mutating a conserved Cys108 to either alanine or serine resulted in lack of AsIII binding, lack of aioBA induction, and correlated with a negative AsIII oxidation phenotype. The discovery and characterization of AioX illustrates a novel AsIII sensing mechanism that appears to be used in a range of bacteria and also provides one of the first examples of a bacterial signal anchor protein.

Bioconjugate Chemistry, 2014

Biochimica et Biophysica Acta (BBA) - General Subjects, 2014

Background: The current paradigm of intracellular redox chemistry maintains that cells establish ... more Background: The current paradigm of intracellular redox chemistry maintains that cells establish a reducing environment maintained by a pool of small molecule and protein thiol to protect against oxidative damage. This strategy is conserved in mesophilic organisms from all domains of life, but has been confounded in thermophilic organisms where evidence suggests that intracellular proteins have abundant disulfides. Methods: Chemical labeling and 2-dimensional gel electrophoresis were used to capture disulfide bonding in the proteome of the model thermophile Sulfolobus solfataricus. The redox poise of the metabolome was characterized using both chemical labeling and untargeted liquid chromatography mass spectrometry. Gene annotation was undertaken using support vector machine based pattern recognition. Results: Proteomic analysis indicated the intracellular protein thiol of S. solfataricus was primarily in the disulfide form. Metabolic characterization revealed a lack of reduced small molecule thiol. Glutathione was found primarily in the oxidized state (GSSG), at relatively low concentration. Combined with genetic analysis, this evidence shows that pathways for synthesis of glutathione do exist in the archaeal domain. Conclusions: In observed thermophilic organisms, thiol abundance and redox poise suggest that this system is not directly utilized for protection against oxidative damage. Instead, a more oxidized intracellular environment promotes disulfide bonding, a critical adaptation for protein thermostability. General significance: Based on the placement of thermophilic archaea close to the last universal common ancestor in rRNA phylogenies, we hypothesize that thiol-based redox systems are derived from metabolic pathways originally tasked with promoting protein stability.

Journal of Proteome Research, 2012

Where there is life, there are viruses. The impact of viruses on evolution, global nutrient cycli... more Where there is life, there are viruses. The impact of viruses on evolution, global nutrient cycling, and disease has driven research on their cellular and molecular biology. Knowledge exists for a wide range of viruses, however, a major exception are viruses with archaeal hosts. Archaeal virushost systems are of great interest because they have similarities to both eukaryotic and bacterial systems and often live in extreme environments. Here we report the first proteomics-based experiments on archaeal host response to viral infection. Sulfolobus Turreted Icosahedral Virus (STIV) infection of Sulfolobus solfataricus P2 was studied using 1D and 2D differential gel electrophoresis (DIGE) to measure abundance and redox changes. Cysteine reactivity was measured using novel fluorescent zwitterionic chemical probes that, together with abundance changes, suggest that virus and host are both vying for control of redox status in the cells. Proteins from nearly 50% of the predicted viral open reading frames were found along with a new STIV protein with a homolog in STIV2. This study provides insight to features of viral replication novel to the archaea, makes strong connections to well described mechanisms used by eukaryotic viruses such as ESCRT-III mediated transport, and emphasizes the complementary nature of different omics approaches.

Bioconjugate Chemistry, 2013

The CyDye family of fluorescent dyes is currently the overwhelming choice for applications in pro... more The CyDye family of fluorescent dyes is currently the overwhelming choice for applications in proteomic analysis, using two-dimensional difference gel electrophoresis (2D-DIGE). Protein labeling with CyDyes is hampered by protein precipitation and gel smearing when used above minimal labeling. The solubility of labeled protein may be improved by introducing water solubilizing groups on the dye such as cysteic acids. However, addition of a negatively charged functionality will have the undesired effect of shifting the pI in relation to the unlabeled protein. These limitations have been addressed through the synthesis of highly water-soluble and pI balancing zwitterionic CyDye fluorophores (Z-CyDyes). The new dyes feature a cysteic acid motif, a titratable amine functionality and a NHS activated ester group. In side by side 2D-DIGE comparisons of Z-CyDyes and CyDyes, the new dyes significantly enhanced protein spot volume and the number of spots that were detected. Z-CyDyes have the potential to enhance the depth of proteome coverage and provide a general strategy for improving the performance of protein tagging reagents.

Background: Rotaviruses are known to modulate the innate antiviral defense response driven by IFN... more Background: Rotaviruses are known to modulate the innate antiviral defense response driven by IFN. The purpose of this study was to identify changes in the cellular proteome in response to rotavirus infection in the context of the IFN response. We also sought to identify proteins outside the IFN induction and signaling pathway that were modulated by rotavirus infection. Methods: 2D-DIGE and image analysis were used to identify cellular proteins that changed in levels of expression in response to rotavirus infection, IFN treatment, or IFN treatment prior to infection. Immunofluorescence microscopy was used to determine the subcellular localization of proteins associated with the unfolded protein response (UPR). Results: The data show changes in the levels of multiple proteins associated with cellular stress in infected cells, including levels of ER chaperones GRP78 and GRP94. Further investigations showed that GRP78, GRP94 and other proteins with roles in the ER-initiated UPR includi...

Particle bombardment has been used to transform maize genotype Hi-II. Immature embryos were co-tr... more Particle bombardment has been used to transform maize genotype Hi-II. Immature embryos were co-transformed with a plasmid containing the selectable and scorable marker genes (bar and uidA, respectively) and a plasmid containing the high-molecular-weight glutenin subunit Dy10 gene. Eight transgenic events (T0) were recovered from 1000 bombarded scutella (transformation efficiency thus 0.8%). T1 generation was produced by cross pollination between T0 plants and nontransgenic plants of the Egyptian inbred line Sd63. Integration of trangenes has been confirmed in the genome of T0 plants by PCR and Dot blot hybridization analysis. Expression of marker genes was detected in T0 plants by leaf painting and histochemical staining for bar and uidA genes, respectively.

Journal of the American Chemical Society, Jan 3, 2016

There is considerable interest in the discovery of peptide ligands that bind to protein targets. ... more There is considerable interest in the discovery of peptide ligands that bind to protein targets. Discovery of such ligands is usually approached by screening large peptide libraries. However, the individual peptides must be tethered to a tag that preserves their individual identities (e.g., phage display or one-bead one-compound). To overcome this limitation, we have developed a method for screening libraries of label-free peptides for binding to a protein target in solution as a single batch. The screening is based on decreased amide hydrogen exchange by peptides that bind to the target. Hydrogen exchange is measured by mass spectrometry. We demonstrate the approach using a peptide library derived from the Escherichia coli proteome that contained 6664 identifiable features. The library was spiked separately with a peptide spanning the calmodulin binding domain of endothelial nitric oxide synthase (eNOS, 494-513) and a peptide spanning the N-terminal 20 residues of bovine ribonuclea...

Plant physiology and biochemistry : PPB / Societe francaise de physiologie vegetale, Jan 14, 2016

Senescence is the last developmental phase of plant tissues, organs and, in the case of monocarpi... more Senescence is the last developmental phase of plant tissues, organs and, in the case of monocarpic senescence, entire plants. In monocarpic crops such as barley, it leads to massive remobilization of nitrogen and other nutrients to developing seeds. To further investigate this process, a proteomic comparison of flag leaves of near-isogenic late- and early-senescing barley germplasm was performed. Protein samples at 14 and 21 days past anthesis were analyzed using both two-dimensional gel-based and label-free quantitative mass spectrometry-based ('shotgun') proteomic techniques. This approach identified >9000 barley proteins, and one-third of them were quantified. Analysis focused on proteins that were significantly (p < 0.05; difference ≥1.5-fold) upregulated in early-senescing line '10_11' as compared to late-senescing variety 'Karl', as these may be functionally important for senescence. Proteins in this group included family 1 pathogenesis-related pr...

Bioconjugate Chemistry, 2013

The CyDye family of fluorescent dyes is currently the overwhelming choice for applications in pro... more The CyDye family of fluorescent dyes is currently the overwhelming choice for applications in proteomic analysis, using two-dimensional difference gel electrophoresis (2D-DIGE). Protein labeling with CyDyes is hampered by protein precipitation and gel smearing when used above minimal labeling. The solubility of labeled protein may be improved by introducing water solubilizing groups on the dye such as cysteic acids. However, addition of a negatively charged functionality will have the undesired effect of shifting the pI in relation to the unlabeled protein. These limitations have been addressed through the synthesis of highly water-soluble and pI balancing zwitterionic CyDye fluorophores (Z-CyDyes). The new dyes feature a cysteic acid motif, a titratable amine functionality and a NHS activated ester group. In side by side 2D-DIGE comparisons of Z-CyDyes and CyDyes, the new dyes significantly enhanced protein spot volume and the number of spots that were detected. Z-CyDyes have the potential to enhance the depth of proteome coverage and provide a general strategy for improving the performance of protein tagging reagents.

Where there is life, there are viruses. The impact of viruses on evolution, global nutrient cycli... more Where there is life, there are viruses. The impact of viruses on evolution, global nutrient cycling, and disease has driven research on their cellular and molecular biology. Knowledge exists for a wide range of viruses, however, a major exception are viruses with archaeal hosts. Archaeal virushost systems are of great interest because they have similarities to both eukaryotic and bacterial systems and often live in extreme environments. Here we report the first proteomics-based experiments on archaeal host response to viral infection. Sulfolobus Turreted Icosahedral Virus (STIV) infection of Sulfolobus solfataricus P2 was studied using 1D and 2D differential gel electrophoresis (DIGE) to measure abundance and redox changes. Cysteine reactivity was measured using novel fluorescent zwitterionic chemical probes that, together with abundance changes, suggest that virus and host are both vying for control of redox status in the cells. Proteins from nearly 50% of the predicted viral open reading frames were found along with a new STIV protein with a homolog in STIV2. This study provides insight to features of viral replication novel to the archaea, makes strong connections to well described mechanisms used by eukaryotic viruses such as ESCRT-III mediated transport, and emphasizes the complementary nature of different omics approaches.