Modified and unmodified PSM counts for each AA position with a C-terminal +129 Da modification across all timepoints. The plots show unmodified (green) and +129 Da modified (purple) PSM counts across all nine timepoints (x-axis) for the... more
Modified and unmodified PSM counts for each AA position with a C-terminal +129 Da modification across all timepoints. The plots show unmodified (green) and +129 Da modified (purple) PSM counts across all nine timepoints (x-axis) for the C-terminal position of the two proteins that have at least one PSM identified by MODa as containing a C-terminal +129 Da modification. Counts represent the average of the three biologcial replicates. (PDF 14 kb)
Zip file containing several data tables in tab-separated format, as well as a readme file that explains the contents of each data file. (ZIP 175 kb)
Modified and unmodified counts across timepoints for the top 15 exponential-enriched AA positions with a +16 Da modification to tryptophan. The plots show unmodified (green) and +16 Da modified (orange) tryptophan PSM counts across all... more
Modified and unmodified counts across timepoints for the top 15 exponential-enriched AA positions with a +16 Da modification to tryptophan. The plots show unmodified (green) and +16 Da modified (orange) tryptophan PSM counts across all nine timepoints (x-axis) for the protein and position indicated. Plots in columns correspond to the three biological replicates 1 (left column), 2 (center column), and 3 (right column). Counts represent the average of the three biologcial replicates. Plots are ordered from top to bottom by the mean p value of the Fisherâ s exact test for preferential modification (see text), with the most significant protein at the top. (PDF 25 kb)
Modified and unmodified counts across timepoints for the top 10 exponential-enriched AA positions with a +16 Da modification to methionine. The plots show unmodified (green) and +16 Da modified (magenta) methionine PSM counts across all... more
Modified and unmodified counts across timepoints for the top 10 exponential-enriched AA positions with a +16 Da modification to methionine. The plots show unmodified (green) and +16 Da modified (magenta) methionine PSM counts across all nine timepoints (x-axis) for the protein and position indicated. Plots in columns correspond to the three biological replicates 1 (left column), 2 (center column), and 3 (right column). Counts represent the average of the three biologcial replicates. Plots are ordered from top to bottom by the mean p value of the Fisherâ s exact test for preferential modification (see text), with the most significant protein at the top. (PDF 25 kb)
Amino Acid sequence logos generated using WebLogo [92] for a +/- 5 AA window around the MODa-called site of modification for the top 50 most abundant +1Da modifications localized at Asparagine residues (A) and at all other residue types... more
Amino Acid sequence logos generated using WebLogo [92] for a +/- 5 AA window around the MODa-called site of modification for the top 50 most abundant +1Da modifications localized at Asparagine residues (A) and at all other residue types combined (B). Asparagine residues show a preferential enrichment of Glycine, Serine, and Asparagine AAs at the +1 position not observed for non-Asn modifications. (PDF 347 kb)
Modified and unmodified PSM counts for each AA position with a significantly stationary-phase biased +1 Da modification to asparagine. The plots show unmodified (green) and +1 Da modified (brown) PSM counts across all nine timepoints... more
Modified and unmodified PSM counts for each AA position with a significantly stationary-phase biased +1 Da modification to asparagine. The plots show unmodified (green) and +1 Da modified (brown) PSM counts across all nine timepoints (x-axis) for the 10 asparagine residues with the most significant p-values across all three biological replicates. Counts represent the average of the three biologcial replicates. Plots are ordered by the mean p value of the Fisherâ s exact test for preferential modification from left-to-right within each row, and from top-to-bottom across rows, with the most significant position at the top left. (PDF 20 kb)
Fraction of total peptides across timepoints with an N-terminal alanine possessing a +42 Da modification. The plots show unmodified (green) and +42 Da modified (blue) PSM counts across all nine timepoints (x-axis) for the N-terminal... more
Fraction of total peptides across timepoints with an N-terminal alanine possessing a +42 Da modification. The plots show unmodified (green) and +42 Da modified (blue) PSM counts across all nine timepoints (x-axis) for the N-terminal position of all proteins that have both (i) at least one PSM identified by MODa as containing an N-terminal +42 Da modification and (ii) having a penultimate Alanine (AA position 2; i.e. the N-terminal residue following N-terminal methionine cleavage). Counts represent the average of the three biologcial replicates. Plots are ordered from top to bottom by the mean p value of the Fisherâ s exact test for preferential modification (see text) from top to bottom, with the most significant protein at the top. (PDF 17 kb)
Temporally variable modification for individual proteins with an N-terminal threonine possessing a +42 Da modification. The plots show unmodified (green) and +42 Da Modified (blue) PSM counts across all nine timepoints (x-axis) for the... more
Temporally variable modification for individual proteins with an N-terminal threonine possessing a +42 Da modification. The plots show unmodified (green) and +42 Da Modified (blue) PSM counts across all nine timepoints (x-axis) for the N-terminal position of all proteins that have both (i) at least one PSM identified by MODa as containing an N-terminal +42 Da modification and (ii) having a penultimate threonine (AA position 2; i.e. the N-terminal residue following N-terminal methionine cleavage). Counts represent the average of the three biological replicates. Plots are ordered from top to bottom by the mean p value of the Fisherâ s exact test for preferential modification (see text) from top to bottom, with the most significant protein at the top. (PDF 17 kb)
Modified and unmodified counts across timepoints for all AA positions with a +16 Da modification to methionine, pooled by biological replicate. The plots show the total unmodified (green) and +16 Da modified (magenta) PSM counts across... more
Modified and unmodified counts across timepoints for all AA positions with a +16 Da modification to methionine, pooled by biological replicate. The plots show the total unmodified (green) and +16 Da modified (magenta) PSM counts across all nine timepoints (x-axis) for methionine residues that have at least one +16 Da modification at any time point in any replicate. The three panels show counts for each of the three biological replicates, replicate 1 (A), replicate 2 (B) and replicate 3 (C). Note that the y-axis is plotted on a logarithmic (base 10) scale due to the high number of total counts relative to modified counts. (PDF 16 kb)
To exploit vulnerabilities of tumors, it is urgent to identify associated defects in genome maintenance. One unsolved problem is the mechanism of regulation of DNA double-strand break repair by REV7 in complex with 53BP1 and RIF1, and its... more
To exploit vulnerabilities of tumors, it is urgent to identify associated defects in genome maintenance. One unsolved problem is the mechanism of regulation of DNA double-strand break repair by REV7 in complex with 53BP1 and RIF1, and its influence on repair pathway choice between homologous recombination and non-homologous end-joining. We searched for REV7-associated factors in human cells and found FAM35A, a previously unstudied protein with an unstructured N-terminal region and a C-terminal region harboring three OB-fold domains similar to single-stranded DNA-binding protein RPA, as novel interactor of REV7/RIF1/53BP1. FAM35A re-localized in damaged cell nuclei, and its knockdown caused sensitivity to DNA-damaging agents. In a BRCA1-mutant cell line, however, depletion of FAM35A increased resistance to camptothecin, suggesting that FAM35A participates in processing of DNA ends to allow more efficient DNA repair. We found FAM35A absent in one widely used BRCA1-mutant cancer cell l...
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An increasing number of web resources are available for aiding in proteomics research. Databases contain repositories of proteins and associated information. A recent article by Chen et al. (Genomics Proteomics Bioinformatics 13(1):36-39,... more
An increasing number of web resources are available for aiding in proteomics research. Databases contain repositories of proteins and associated information. A recent article by Chen et al. (Genomics Proteomics Bioinformatics 13(1):36-39, 2015) evaluates a number of MS-based proteomics repositories containing MS and expression data, including repositories devoted to cataloguing high confidence post-translational modifications. Many sites have tools developed by research labs that are shared with the community and online tutorials and videos for learning how to use the tools. This chapter contains a selection of web sites useful for proteomics analyses but is by no means comprehensive. Using a search engine such as Google is the easiest way to find the sites using the name given below.
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Research Interests: Chemistry, Technology, Proteomics, Medicine, Biological Sciences, and 13 moreMolecular chaperones, Humans, Pancreas, Peptides, Proteins, Heat Shock Proteins, Protein Conformation, Two-Dimensional Gel Electrophoresis, Amino Acid Sequence, Two dimensional Gel Electrophoresis, Isoelectric point, Molecular Sequence Data, and Medical and Health Sciences
The Hedgehog (HH) signaling pathway is essential for the maintenance and response of several types of stem cells. To study the transcriptional response of stem cells to HH signaling, we searched for proteins binding to GLI proteins, the... more
The Hedgehog (HH) signaling pathway is essential for the maintenance and response of several types of stem cells. To study the transcriptional response of stem cells to HH signaling, we searched for proteins binding to GLI proteins, the transcriptional effectors of the HH pathway in mouse embryonic stem (ES) cells. We find that both GLI3 and GLI1 bind to the pluripotency factor NANOG. The ectopic expression of NANOG inhibits GLI1-mediated transcriptional responses in a dose-dependent fashion. In differentiating ES cells, the presence of NANOG reduces the transcriptional response of cells to HH. Finally, we find that Gli1 and Nanog are co-expressed in ES cells at high levels. We propose that NANOG acts as a negative feedback component that provides stem cell-specific regulation of the HH pathway.
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The molecular mechanisms underlying excessive alcohol consumption are not fully understood. Several recent reports from the Integrative Neuroscience Initiative on Alcoholism (INIA) consortium show that alcohol administration induces... more
The molecular mechanisms underlying excessive alcohol consumption are not fully understood. Several recent reports from the Integrative Neuroscience Initiative on Alcoholism (INIA) consortium show that alcohol administration induces changes in the expression levels of a number of genes involved in synaptic transmission. These changes likely affect the expression levels of the corresponding proteins, which in turn, alter their ability to establish protein-protein interactions required for normal regulation of brain function. Thus, we hypothesize that clusters of genes that change in response to alcohol perturbation may predict protein complexes that underlie alcohol-related phenotypes. In order to better understand the cellular and molecular events that could contribute to alcohol dependence in mice, the objective of our study was to first identify novel protein complexes of key brain signaling systems and, then, to determine the effect of excessive alcohol consumption on the identif...
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Elevated homocysteine levels have long been associated with various disease states, including cardiovascular disease and birth defects, including neural tube defects (NTDs). One hypothesis regarding the strong correlation between these... more
Elevated homocysteine levels have long been associated with various disease states, including cardiovascular disease and birth defects, including neural tube defects (NTDs). One hypothesis regarding the strong correlation between these various disorders and high levels of homocysteine is that a reactive form of this small molecule can attach to mammalian proteins in a phenomenon known as homocysteinylation. These posttranslational modifications may become antigenic or may even directly disrupt certain protein function. It remains to be determined whether dietary influences that can cause globally increased levels of circulating homocysteine confer negative effects maternally, or may otherwise negatively and materially impact the metabolic balance in developing embryos. Herein we present the application of a chemical method of determination of N-homocysteinylation to a set of neural tube closure stage mouse embryos and their mothers. We explore the uses of this newly described techni...
Research Interests: Nutrition and Dietetics, Biology, Folic acid, Medicine, Mice, and 11 moreFemale, Animals, Homocysteine, Post Translational Modifications, Nutritional Biochemistry, Embryonic Development, Food Sciences, Neural Tube Defects, Congenital abnormalities, Biochemistry and cell biology, and folic acid deficiency
We previously reported a protein expression profiling experiment conducted on human pancreatic tissues using 2D gel electrophoresis and mass spectrometry. Here, 18 spots that were identified in the gel at molecular weights more than 10... more
We previously reported a protein expression profiling experiment conducted on human pancreatic tissues using 2D gel electrophoresis and mass spectrometry. Here, 18 spots that were identified in the gel at molecular weights more than 10 kDa lower than ...
Research Interests: Chemistry, Technology, Proteomics, Medicine, Biological Sciences, and 13 moreMolecular chaperones, Humans, Pancreas, Peptides, Proteins, Heat Shock Proteins, Protein Conformation, Two-Dimensional Gel Electrophoresis, Amino Acid Sequence, Two dimensional Gel Electrophoresis, Isoelectric point, Molecular Sequence Data, and Medical and Health Sciences
Ataxia-telangiectasia mutated (ATM) is a cellular damage sensor that coordinates the cell cycle with damage-response checkpoints and DNA repair to preserve genomic integrity. However, ATM also has been implicated in metabolic regulation,... more
Ataxia-telangiectasia mutated (ATM) is a cellular damage sensor that coordinates the cell cycle with damage-response checkpoints and DNA repair to preserve genomic integrity. However, ATM also has been implicated in metabolic regulation, and ATM deficiency is associated with elevated reactive oxygen species (ROS). ROS has a central role in many physiological and pathophysiological processes including inflammation and chronic diseases such as atherosclerosis and cancer, underscoring the importance of cellular pathways involved in redox homeostasis. We have identified a cytoplasmic function for ATM that participates in the cellular damage response to ROS. We show that in response to elevated ROS, ATM activates the TSC2 tumor suppressor via the LKB1/AMPK metabolic pathway in the cytoplasm to repress mTORC1 and induce autophagy. Importantly, elevated ROS and dysregulation of mTORC1 in ATM-deficient cells is inhibited by rapamycin, which also rescues lymphomagenesis in Atm -deficient mic...
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Research Interests:
Research Interests: Gene Silencing, Cell Division, Cell and Molecular Biology, Biological Sciences, Humans, and 15 moreKeratinocytes, Mutation, Mice, Animals, DNA methylation, CpG islands, Proteins, Oral Squamous Cell Carcinoma (OSCC), Histones, Signaling, Genomic instability, Base Sequence, Skin Neoplasms, Molecular Sequence Data, and Medical and Health Sciences
Overexpression of insulin-like growth factor-1 (IGF-1) has been associated with a number of human tumors, including breast, colon, lung, and prostate cancers. In previous studies, we found that mice overexpressing human IGF-1 in the basal... more
Overexpression of insulin-like growth factor-1 (IGF-1) has been associated with a number of human tumors, including breast, colon, lung, and prostate cancers. In previous studies, we found that mice overexpressing human IGF-1 in the basal layer of the epidermis (BK5.IGF-1 mice) developed skin tumors following treatment with the skin tumor initiator, 7,12-dimethylbenz[a]anthracene, indicating that IGF-1 can act as a skin tumor promoter. In the present study, we employed a proteomics approach of two-dimensional (2-D) gel electrophoresis and mass spectrometry to profile differentially expressed proteins in skin epidermis between BK5.IGF-1 transgenic and nontransgenic littermates. Two-D gels from each of three transgenic and three age/sex matched wild-type littermates were compared at two different pH ranges. Differentially expressed protein spots were identified by Bio-Rad's PDQuest image analysis, in-gel digested, and analyzed on a MALDI-TOF MS system. A total of 23 proteins were identified as differentially expressed, 17 of them overexpressed in transgenic mice. These proteins included 14-3-3 sigma, galectin-7, an apoptosis-related protein, three heat shock proteins, four calcium binding proteins, three proteases or protease inhibitors, one actin regulatory capping protein, and translation initiation factor 5A. The differential expression of GRP78, alpha enolase, and galectin-7 was verified by 1-D western blot analysis. Two-D western blot analyses of alpha enolase and galectin-7 further revealed that alpha enolase had more than one protein spot dependent on charge. The current data suggest that some of the differentially expressed proteins may play a role in the tumor promoting action of IGF-1 in mouse skin.
Research Interests: Mass Spectrometry, Proteomics, Apoptosis, Transgenic Mice, Molecular, and 15 moreHumans, Mice, Female, Animals, Male, Proteins, Epidermis, Dimensional, MOLECULAR CARCİNOGENESİS, Heat Shock Proteins, Gel electrophoresis, Heat Shock Protein, Two dimensional Gel Electrophoresis, Gene Expression Regulation, and Differential expression
The degenerate four wave mixing (DFWM) spectrum of the A(1A2)← X(1A1) and B(1B1)← X(1A1) transitions of SO2 in the 299.5–305 nm region is presented. It has been found that the DFWM signal intensities are proportional to the cube of laser... more
The degenerate four wave mixing (DFWM) spectrum of the A(1A2)← X(1A1) and B(1B1)← X(1A1) transitions of SO2 in the 299.5–305 nm region is presented. It has been found that the DFWM signal intensities are proportional to the cube of laser intensity and the square of SO2 pressure. The DFWM signal increases dramatically with the pressure of N2 as a buffer gas. The enhancement of the DFWM signal can be mainly attributed to the thermal grating contribution.
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Exposure of murine skin to tumor-promoting agents such as 12-O-tetradecanoyl-phorbol-13-acetate (TPA) causes up-regulation of cyclooxygenase-2 (COX-2) and increased prostaglandin (PG) synthesis. Pharmacological inhibition of COX-2... more
Exposure of murine skin to tumor-promoting agents such as 12-O-tetradecanoyl-phorbol-13-acetate (TPA) causes up-regulation of cyclooxygenase-2 (COX-2) and increased prostaglandin (PG) synthesis. Pharmacological inhibition of COX-2 significantly reduces skin tumor development. However, we previously demonstrated that K14.COX-2 transgenic (TG) mice that overexpressed COX-2 in the epidermis were unexpectedly resistant to tumor development under the classical 7,12-dimethylbenz[a]anthracene-TPA protocol. In the present study, we employed a proteomic approach of 2-dimensional gel electrophoresis (2-DE) and mass spectrometry to profile differentially expressed proteins in the epidermis of K14.COX-2 TG and wild-type control mice. Various 2-DE approaches were used to identify the maximum number of differentially expressed proteins: 20 for untreated samples, 3 for acetone-treated samples, and 22 for TPA-treated samples. These proteins include 14-3-3 sigma, numerous actin fragments, actin filament related proteins cofilin-1 and destrin, galectin-3, galectin-7, prohibitin, S100A6, S100A9, and many others. The differential expression of galectin-3, galectin-7, S100A9 was validated by Western blot analysis and/or immunohistochemical analysis. The current data suggest that some of the differentially expressed proteins might increase apoptosis and cell cycle arrest, which, in turn, may provide insight into the role of COX-2 in skin tumorigenesis.
Research Interests: Mass Spectrometry, Proteomics, Apoptosis, Transgenic Mice, Biological Sciences, and 15 moreMice, Animals, Skin, Epidermis, Galectin, CHEMICAL SCIENCES, Dimensional, Protein Expression, Gel electrophoresis, Two-Dimensional Gel Electrophoresis, Amino Acid Sequence, Calcium Binding Proteins, Two dimensional Gel Electrophoresis, Proteome, and Molecular Sequence Data
Reactive oxygen species (ROS) play an important role in normal biological functions and pathological processes. ROS is one of the driving forces for oxidizing proteins, especially on cysteine thiols. The labile, transient, and dynamic... more
Reactive oxygen species (ROS) play an important role in normal biological functions and pathological processes. ROS is one of the driving forces for oxidizing proteins, especially on cysteine thiols. The labile, transient, and dynamic nature of oxidative modifications poses enormous technical challenges for both accurate modification site determination and quantitation of cysteine thiols. The present study describes a mass spectrometry-based approach that allows effective discovery and quantification of irreversible cysteine modifications. The utilization of a long reverse phase column provides high-resolution chromatography to separate different forms of modified cysteine thiols from protein complexes or cell lysates. This Fourier transform mass spectrometry (FT-MS) approach enabled detection and quantitation of ataxia telangiectasia mutated (ATM) complex cysteine sulfoxidation states using Skyline MS1 filtering. When we applied the long column ultra high pressure liquid chromatography (UPLC)-MS/MS analysis, 61 and 44 peptides from cell lysates and cells were identified with cysteine modifications in response to in vitro and in vivo H2O2 oxidation, respectively. Long column ultra high pressure liquid chromatography pseudo selected reaction monitoring (UPLC-pSRM) was then developed to monitor the oxidative level of cysteine thiols in cell lysate under varying concentrations of H2O2 treatment. From UPLC-pSRM analysis, the dynamic conversion of sulfinic (S-O2H) and sulfonic acid (S-O3H) was observed within nucleoside diphosphate kinase (Nm23-H1) and heat shock 70 kDa protein 8 (Hsc70). These methods are suitable for proteome-wide studies, providing a highly sensitive, straightforward approach to identify proteins containing redox-sensitive cysteine thiols in biological systems.
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While studying Bim, a BH3-only proapoptotic protein, we identified an ∼36 kDa protein, which was abundantly expressed in all five strains of primary normal human prostate (NHP) epithelial cells but significantly reduced or lost in seven... more
While studying Bim, a BH3-only proapoptotic protein, we identified an ∼36 kDa protein, which was abundantly expressed in all five strains of primary normal human prostate (NHP) epithelial cells but significantly reduced or lost in seven prostate cancer cell lines. The ∼36 kDa protein was subsequently identified as annexin II by proteomic approach and confirmed by Western blotting using an
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We measured the emission spectra of CHI and CDI excited to a predissociative Rydberg state (E,1){2} near 193.3 nm and tabulated emission line positions with their assignments. The emission spectrum of CHI both provides previously... more
We measured the emission spectra of CHI and CDI excited to a predissociative Rydberg state (E,1){2} near 193.3 nm and tabulated emission line positions with their assignments. The emission spectrum of CHI both provides previously unobserved energies of ground state ...
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Research Interests: Chemistry, Mass Spectrometry, Biology, Biological Chemistry, Medicine, and 15 moreBiological Sciences, Mutagenesis, Sequence alignment, Peptides, Phosphorylation, Arabidopsis, CHEMICAL SCIENCES, Substrate Specificity, Amino Acid Sequence, Triticum, Protein Binding, Translation initiation, Protein Biosynthesis, Molecular Sequence Data, and Medical and Health Sciences
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The ataxia-telangiectasia mutated (ATM) protein kinase is activated by DNA double-strand breaks (DSBs) through the Mre11-Rad50-Nbs1 (MRN) DNA repair complex and orchestrates signaling cascades that initiate the DNA damage response. Cells... more
The ataxia-telangiectasia mutated (ATM) protein kinase is activated by DNA double-strand breaks (DSBs) through the Mre11-Rad50-Nbs1 (MRN) DNA repair complex and orchestrates signaling cascades that initiate the DNA damage response. Cells lacking ATM are also hypersensitive to insults other than DSBs, particularly oxidative stress. We show that oxidation of ATM directly induces ATM activation in the absence of DNA DSBs and the MRN complex. The oxidized form of ATM is a disulfide-cross-linked dimer, and mutation of a critical cysteine residue involved in disulfide bond formation specifically blocked activation through the oxidation pathway. Identification of this pathway explains observations of ATM activation under conditions of oxidative stress and shows that ATM is an important sensor of reactive oxygen species in human cells.
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Research Interests: Mass Spectrometry, Biology, Medicine, Brain, Mice, and 15 moreEndocytosis, Animals, Male, Central Nervous System, Immunoprecipitation, Protein Complex Detection, Protein Interaction, Exocytosis, Proteome analysis, Protein Binding, Attachment Protein Receptor, BK channels, Dynamin, Biochemistry and cell biology, and Immunoblotting
Research Interests: Inorganic Chemistry, Organic Chemistry, Biology, Oxidative Stress, Medicine, and 13 moreHumans, Kidney, Animals, Trypsin, Cytoprotection, Proteins, Protein Sequence Analysis, Liquid Chromatography / Electrospray Ionization Mass Spectrometry, Swine, Amino Acid Sequence, Food and Chemical Toxicology, Protein Biosynthesis, and Molecular Sequence Data
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The incidence of non-melanoma skin cancer (NMSC) is equivalent to that of all other human cancers combined, and genetic factors contribute to risk of the disease. Using genetic crosses of skin tumor promotion sensitive DBA/2 mice with... more
The incidence of non-melanoma skin cancer (NMSC) is equivalent to that of all other human cancers combined, and genetic factors contribute to risk of the disease. Using genetic crosses of skin tumor promotion sensitive DBA/2 mice with relatively resistant C57BL/6 mice, loci that modify susceptibility to tumor promotion by 12-O-tetradecanoylphorbol-13-acetate (TPA) have been mapped to chromosomes (chr) 1, 2, 9, and 19. Here, we show that Glutathione S-transferase alpha 4 (Gsta4), which maps to skin tumor promotion susceptibility locus Psl1.2 on chr 9, was expressed at significantly different levels in the epidermis of C57BL/6 versus DBA/2 mice following treatment with diverse promoting agents. Gsta4 expression was dramatically upregulated in the epidermis of C57BL/6 mice, but not DBA/2 mice, following treatment with TPA, okadaic acid, chrysarobin, or ultraviolet light. Gsta4 deficient mice were more susceptible to skin tumor development in the two-stage skin carcinogenesis protocol providing compelling evidence that Gsta4 underlies the effect of Psl1.2 on susceptibility to skin tumor promotion by TPA. A number of polymorphisms were detected in the putative promoter region of Gsta4 in C57BL/6 versus DBA/2 mice, and ongoing studies are addressing the genetic basis of strain-specific expression of Gsta4 during tumor promotion. Finally, inheritance of polymorphisms in GSTA4 was associated with risk of NMSC in human populations, further supporting Gsta4/GSTA4 as a gene that modifies susceptibility to skin tumor development. Supported by NIH grant ES016623. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4210.
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Various soil bacteria use 1,3-dichloropropene, a component of the commercially available fumigants Shell D-D and Telone II, as a sole source of carbon and energy. One enzyme involved in the catabolism of 1,3-dichloropropene is... more
Various soil bacteria use 1,3-dichloropropene, a component of the commercially available fumigants Shell D-D and Telone II, as a sole source of carbon and energy. One enzyme involved in the catabolism of 1,3-dichloropropene is trans-3-chloroacrylic acid dehalogenase (CaaD), which converts the trans-isomers of 3-bromo- and 3-chloroacrylate to malonate semialdehyde. Sequence analysis suggested a relationship between the heterohexameric CaaD and the bacterial isomerase, 4-oxalocrotonate tautomerase (4-OT), thereby distinguishing CaaD from a number of dehalogenases whose mechanisms proceed through an alkyl- or aryl-enzyme intermediate. In this study, the genes for the alpha- and beta-subunits of CaaD have been synthesized using a polymerase chain reaction-based strategy, cloned into separate plasmids, and the proteins expressed and purified as the functional heterohexamer. Subsequently, the product of the reaction was confirmed to be malonate semialdehyde by (1)H and (13)C NMR spectroscopy, and kinetic constants were determined using a UV spectrophotometric assay. In view of the proposed hydrolytic nature of the CaaD-catalyzed reaction, three acetylene compounds were investigated as substrates for the enzyme. One compound, 2-oxo-3-pentynoate, a potent active site-directed irreversible inhibitor of 4-OT, is a substrate for CaaD, and was processed to acetopyruvate with kinetic constants similar to those determined for the trans-isomers of 3-bromo- and 3-chloroacrylate. The remaining two compounds, 3-bromo- and 3-chloropropiolic acid, were transformed into potent irreversible inhibitors of CaaD. The inactivation observed for 3-bromopropiolic acid is due to the covalent modification of Pro-1 of the beta-subunit. The results provide evidence for a hydratase activity and set the stage to use the 3-halopropiolic acids as ligands in inactivated CaaD complexes that can be studied by X-ray crystallography.
Research Interests: Biochemistry, Kinetics, Carbon, Mass Spectrometry, Magnetic Resonance Spectroscopy, and 14 moreMedicine, Macromolecular X-Ray Crystallography, Pseudomonas, Trypsin, Spectrophotometry, Plasmids, Acetylene, Isomerases, Malonates, Glycosyl Hydrolases, Biochemistry and cell biology, Aldehydes, Ultraviolet Rays, and Medical biochemistry and metabolomics
The gene encoding the cis-3-chloroacrylic acid dehalogenase (cis-CaaD) from coryneform bacterium strain FG41 has been cloned and overexpressed, and the enzyme has been purified to homogeneity and subjected to kinetic and mechanistic... more
The gene encoding the cis-3-chloroacrylic acid dehalogenase (cis-CaaD) from coryneform bacterium strain FG41 has been cloned and overexpressed, and the enzyme has been purified to homogeneity and subjected to kinetic and mechanistic characterization. Kinetic studies show that cis-CaaD processes cis-3-haloacrylates, but not trans-3-haloacrylates, with a turnover number of approximately 10 s(-1). The product of the reaction is malonate semialdehyde, which was confirmed by its characteristic 1H NMR spectrum. The enzyme shares low but significant sequence similarity with the previously studied trans-3-chloroacrylic acid dehalogenase (CaaD) and with other members of the 4-oxalocrotonate tautomerase (4-OT) family. While 4-OT and CaaD function as homo- and heterohexamers, respectively, cis-CaaD appears to be a homotrimeric protein as assessed by gel filtration chromatography. On the basis of the known three-dimensional structures and reaction mechanisms of CaaD and 4-OT, a sequence alignment implicated Pro-1, Arg-70, Arg-73, and Glu-114 as important active-site residues in cis-CaaD. Subsequent site-directed mutagenesis experiments confirmed these predictions. The acetylene compounds, 2-oxo-3-pentynoate and 3-bromo- and 3-chloropropiolate, were processed by cis-CaaD to products consistent with an enzyme-catalyzed hydration reaction previously established for CaaD. Hydration of 2-oxo-3-pentynoate afforded acetopyruvate, while the 3-halopropiolates became irreversible inhibitors that modified Pro-1. The results of this work revealed that cis-CaaD and CaaD have different primary and quaternary structures, and display different substrate specificity and catalytic efficiencies, but likely share a highly conserved catalytic mechanism. The mechanism may have evolved independently because sequence analysis indicates that cis-CaaD is not a 4-OT family member, but represents the first characterized member of a new family in the tautomerase superfamily that probably resulted from an independent duplication of a 4-OT-like sequence. The discovery of a fifth family of enzymes within this superfamily further demonstrates the diversity of activities and structures that can be created from 4-OT-like sequences.
Research Interests: Biochemistry, Molecular Evolution, Kinetics, Biology, Enzyme Inhibitors, and 15 moreMedicine, Proline, Molecular cloning, Protein Secondary Structure Prediction, Substrate Specificity, Corynebacterium, Arginine, Amino Acid Sequence, Isomerases, Protons, Glutamic Acid, Glycosyl Hydrolases, Biochemistry and cell biology, Molecular Sequence Data, and Medical biochemistry and metabolomics(Medicine, Proline, Molecular cloning, Protein Secondary Structure Prediction, Substrate Specificity, Corynebacterium, Arginine, Amino Acid Sequence, Isomerases, Protons, Glutamic Acid, Glycosyl Hydrolases, Biochemistry and cell biology, Molecular Sequence Data, and Medical biochemistry and metabolomics)
(Medicine, Proline, Molecular cloning, Protein Secondary Structure Prediction, Substrate Specificity, Corynebacterium, Arginine, Amino Acid Sequence, Isomerases, Protons, Glutamic Acid, Glycosyl Hydrolases, Biochemistry and cell biology, Molecular Sequence Data, and Medical biochemistry and metabolomics)
Research Interests: Biochemistry, Molecular Evolution, Kinetics, Magnetic Resonance Spectroscopy, Mutation, and 7 morePolymerase Chain Reaction, Liquid Chromatography / Electrospray Ionization Mass Spectrometry, Glycosyl Hydrolases, Biochemistry and cell biology, Molecular Structure, Isomerism, and Medical biochemistry and metabolomics
We report the first direct observation of the self-association behavior of the Staphylococcus aureus sortase A (SrtA) transpeptidase. Formation of a SrtA dimer was observed under native conditions by polyacrylamide gel electrophoresis and... more
We report the first direct observation of the self-association behavior of the Staphylococcus aureus sortase A (SrtA) transpeptidase. Formation of a SrtA dimer was observed under native conditions by polyacrylamide gel electrophoresis and fast protein liquid chromatography (FPLC). Subsequent peptide mass fingerprinting and protein sequencing experiments confirmed the dimeric form of the SrtA protein. Furthermore, SrtA can be selectively cross-linked both in vitro and in Escherichia coli. Multiple samples of enzyme were subjected to analytical sedimentation equilibrium ultracentrifugation to obtain an apparent Kd for dimer formation of about 55 microM. Finally, enzyme kinetic studies suggested that the dimeric form of SrtA is more active than the monomeric enzyme. Discovery of SrtA dimerization may have significant implications for understanding microbial physiology and developing new antibiotics.
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Research Interests: Genetics, Mass Spectrometry, Cancer, Immunohistochemistry, Risk assessment, and 15 moreHumans, Mice, Animals, Polymerase Chain Reaction, Risk factors, Genotype, Single Nucleotide Polymorphism, Odds ratio, Risk Factors, Risk Assessment, Glutathione Transferase, Genetic Variability, Case Control Studies, Skin Neoplasms, and Aldehydes
PRMT3 (protein arginine methyltransferase 3) is one of four type I arginine methyltransferases that catalyse the formation of asymmetric dimethylarginine. PRMT3 is unique in that its N-terminus harbours a C2H2 zinc-finger domain that is... more
PRMT3 (protein arginine methyltransferase 3) is one of four type I arginine methyltransferases that catalyse the formation of asymmetric dimethylarginine. PRMT3 is unique in that its N-terminus harbours a C2H2 zinc-finger domain that is proposed to confer substrate specificity. In addition, PRMT3 is the only type I enzyme that is restricted to the cytoplasm. Known in vitro substrates for PRMT3 include GST–GAR (a glutathione S-transferase fusion protein containing the glycine- and arginine-rich N-terminal region of fibrillarin), Sam68 (Src-associated substrate during mitosis 68 kDa) and PABP-N1 [poly(A)-binding protein-N1; PABP2]. Here we report the identification of an in vivo substrate for mammalian PRMT3. We found that FLAG-tagged PRMT3 can ‘pull down’ a protein with a molecular mass of 30 kDa from HeLa cell extracts. MS identified this PRMT3-interacting protein as rpS2 (ribosomal protein S2). In vitro studies showed that the zinc-finger domain of PRMT3 is necessary and sufficient for binding to rpS2. In addition, rpS2 is methylated by PRMT3 in vitro and is also methylated in cell lines. Deletion analysis of the rpS2 amino acid sequence identified a N-terminal Arg-Gly repeat as the methylation site. Furthermore, both PRMT3 and rpS2 co-sediment with free ribosomal subunits. These studies implicate PRMT3 in ribosomal function and in the regulation of protein synthesis.
Research Interests: Protein synthesis, Biological Sciences, Cell line, Humans, Mice, and 15 moreMethylation, Animals, Glutathione, Biochemical, Enzyme, CHEMICAL SCIENCES, HeLa cells, Arginine, Molecular Mass, PRMT, Amino Acid Sequence, Fusion Protein, Ribosomal Protein, Protein Biosynthesis, and Medical and Health Sciences
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Various soil bacteria use 1,3-dichloropropene, a component of the commercially available fumigants Shell D-D and Telone II, as a sole source of carbon and energy. One enzyme involved in the catabolism of 1,3-dichloropropene is... more
Various soil bacteria use 1,3-dichloropropene, a component of the commercially available fumigants Shell D-D and Telone II, as a sole source of carbon and energy. One enzyme involved in the catabolism of 1,3-dichloropropene is trans-3-chloroacrylic acid dehalogenase (CaaD), which converts the trans-isomers of 3-bromo- and 3-chloroacrylate to malonate semialdehyde. Sequence analysis suggested a relationship between the heterohexameric CaaD and the bacterial isomerase, 4-oxalocrotonate tautomerase (4-OT), thereby distinguishing CaaD from a number of dehalogenases whose mechanisms proceed through an alkyl- or aryl-enzyme intermediate. In this study, the genes for the alpha- and beta-subunits of CaaD have been synthesized using a polymerase chain reaction-based strategy, cloned into separate plasmids, and the proteins expressed and purified as the functional heterohexamer. Subsequently, the product of the reaction was confirmed to be malonate semialdehyde by (1)H and (13)C NMR spectroscopy, and kinetic constants were determined using a UV spectrophotometric assay. In view of the proposed hydrolytic nature of the CaaD-catalyzed reaction, three acetylene compounds were investigated as substrates for the enzyme. One compound, 2-oxo-3-pentynoate, a potent active site-directed irreversible inhibitor of 4-OT, is a substrate for CaaD, and was processed to acetopyruvate with kinetic constants similar to those determined for the trans-isomers of 3-bromo- and 3-chloroacrylate. The remaining two compounds, 3-bromo- and 3-chloropropiolic acid, were transformed into potent irreversible inhibitors of CaaD. The inactivation observed for 3-bromopropiolic acid is due to the covalent modification of Pro-1 of the beta-subunit. The results provide evidence for a hydratase activity and set the stage to use the 3-halopropiolic acids as ligands in inactivated CaaD complexes that can be studied by X-ray crystallography.
Research Interests: Biochemistry, Kinetics, Carbon, Mass Spectrometry, Magnetic Resonance Spectroscopy, and 14 moreMedicine, Macromolecular X-Ray Crystallography, Pseudomonas, Trypsin, Spectrophotometry, Plasmids, Acetylene, Isomerases, Malonates, Glycosyl Hydrolases, Biochemistry and cell biology, Aldehydes, Ultraviolet Rays, and Medical biochemistry and metabolomics
Pancreatic cancer is a rapidly fatal disease, and there is an urgent need for early detection markers and novel therapeutic targets. The current study has used a proteomic approach of two-dimensional (2D) gel electrophoresis and mass... more
Pancreatic cancer is a rapidly fatal disease, and there is an urgent need for early detection markers and novel therapeutic targets. The current study has used a proteomic approach of two-dimensional (2D) gel electrophoresis and mass spectrometry (MS) to identify differentially expressed ...
Research Interests: Mass Spectrometry, Cancer, Immunohistochemistry, Pancreatitis, Pancreatic Cancer, and 15 moreGene expression, Extracellular Matrix, Signal Transduction, Humans, Copper, Chronic Disease, Superoxide Dismutase, Antioxidant enzyme, Early Detection, Protein Expression, Calcium Binding Protein, Early Diagnosis, Extracellular Matrix Proteins, Differential expression, and Pancreatic neoplasms
While studying Bim, a BH3-only proapoptotic protein, we identified an 36 kDa protein, which was abundantly expressed in all five strains of primary normal human prostate (NHP) epithelial cells but significantly reduced or lost in seven... more
While studying Bim, a BH3-only proapoptotic protein, we identified an 36 kDa protein, which was abundantly expressed in all five strains of primary normal human prostate (NHP) epithelial cells but significantly reduced or lost in seven prostate cancer cell lines. The 36 kDa ...
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Research Interests: Apoptosis, Biological Sciences, Caspases, Cell line, Humans, and 14 moreCell, Cytochrome C, Proteins, Caspase, Lysine, Protein Conformation, Protein Quaternary Structure, Cell free System, Cell Survival, Nucleotides, Apoptotic Protease Activating Factor-1, binding sites, Medical and Health Sciences, and In Vitro Techniques
In every synapse, a large number of proteins interact with other proteins in order to carry out signaling and transmission in the central nervous system. In this study, we used interaction proteomics to identify novel synaptic protein... more
In every synapse, a large number of proteins interact with other proteins in order to carry out signaling and transmission in the central nervous system. In this study, we used interaction proteomics to identify novel synaptic protein interactions in mouse cortical membranes under native conditions. Using immunoprecipitation, immunoblotting, and mass spectrometry, we identified a number of novel synaptic protein interactions involving soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs), calcium-activated potassium channel (BKCa) alpha subunits, and dynamin-1. These novel interactions offer valuable insight into the protein-protein interaction network in intact synapses that could advance understanding of vesicle trafficking, release, and recycling. Copyright (c) 2010 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
Research Interests: Mass Spectrometry, Biology, Medicine, Brain, Mice, and 15 moreEndocytosis, Animals, Male, Central Nervous System, Immunoprecipitation, Protein Complex Detection, Protein Interaction, Exocytosis, Proteome analysis, Protein Binding, Attachment Protein Receptor, BK channels, Dynamin, Biochemistry and cell biology, and Immunoblotting
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Identification of multiple and novel posttranslational modifications remains a major challenge in proteomics. The present approach uses comparative analysis by matrix-assisted laser/desorption ionization (MALDI) MS of proteolytic digests... more
Identification of multiple and novel posttranslational modifications remains a major challenge in proteomics. The present approach uses comparative analysis by matrix-assisted laser/desorption ionization (MALDI) MS of proteolytic digests from control and treated proteins to target differences due to modifications, without initial assumption as to type or residue localization. Differences between modified and unmodified digest MS spectra highlight peptides of interest for subsequent tandem mass spectrometry (MS/MS) analysis. Targeted HPLC-electrospray ionization (ESI)-MS/MS is then used to fragment peptides, and manual de novo sequencing is used to determine the amino acid sequence and type of modification. This strategy for identifying posttranslational modifications in an unbiased manner is particularly useful for finding modifications produced by exogenous chemicals. Successful characterization of chemically induced posttranslational modifications and novel chemical adducts is given as an example of the use of this strategy. Histone H4 from butyrate-treated LLC-PK1 cells is separated on a gel into bands representing different overall charge state. Bands are analyzed by comparative MALDI-MS and LC-MS/MS to identify the sites of methylation and acetylation. Previous attempts to identify chemically adducted proteins in vivo have been unsuccessful in part due to a lack of understanding of the final adduct form. Cytochrome c is adducted in vitro with benzoquinone, an electrophilic metabolite of benzene capable of interacting with nucleophilic sites within proteins. De novo sequencing identifies a novel cyclized diquinone adduct species as the major reaction product, targeting Lys and His residues at two specific locations on the protein surface. This unpredicted reaction product is characterized using our unbiased methods for detection and demonstrates the important influence of protein structure on chemical adduction.
Research Interests: Inorganic Chemistry, Organic Chemistry, Biology, Medicine, Cell line, and 10 moreMethylation, Animals, Horses, High Pressure Liquid Chromatography, Integrated Approach, Histones, Liquid Chromatography / Electrospray Ionization Mass Spectrometry, Acetylation, Food and Chemical Toxicology, and Benzoquinones
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Protein identification by MS is well-established. Mixtures of proteins from cell extracts are separated by either one- or two-dimensional gel electrophoresis, and specific bands or spots are subjected to in-gel digestion and subsequent... more
Protein identification by MS is well-established. Mixtures of proteins from cell extracts are separated by either one- or two-dimensional gel electrophoresis, and specific bands or spots are subjected to in-gel digestion and subsequent analysis by MS. The two most common types of ionization used in MS are electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI). When ESI is used, the sample is typically analyzed by inline HPLC-ESI-MS/MS with fragmentation of individual digest peptides, followed by database comparison between theoretical and experimental fragmentation patterns. MALDI-MS analysis is based on peptide mass mapping, with mass measurements of the digest peptides searched against a database of theoretical digests. We give here the results of a comparison between ESI-ion trap and MALDI-TOF (time-of-flight) analysis of 11-deoxy,16,16-dimethyl prostaglandin E(2) (DDM-PGE(2)) inducible proteins. Individual peptides identified by the two techniques differed, in general, but the resulting protein identification was the same. Slightly higher coverage of each protein was obtained by MALDI-TOF, but the MS/MS data were more definitive by requiring fewer peptides to assign a positive identification. Both methods effectively identified two proteins in the same gel band. The samples here are derived from a renal epithelial cell line (LLC-PK(1)) established from the New Hampshire minipig, a species poorly represented in the current database, and strategies and limitations for analyzing such species are discussed.