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Sulfenylation of Human Liver and Kidney Microsomal Cytochromes P450 and Other Drug-Metabolizing Enzymes as a Response to Redox Alteration.
Albertolle ME, Phan TTN, Pozzi A, Guengerich FP
(2018) Mol Cell Proteomics 17: 889-900
MeSH Terms: Animals, Biocatalysis, Cysteine, Cytochrome P-450 Enzyme System, Humans, Hydrogen Peroxide, Kidney, Mice, Transgenic, Microsomes, Liver, Oxidation-Reduction, Pharmaceutical Preparations, Recombinant Proteins, Staining and Labeling, Sulfenic Acids, Sulfhydryl Compounds
Show Abstract · Added March 14, 2018
The lumen of the endoplasmic reticulum (ER) provides an oxidizing environment to aid in the formation of disulfide bonds, which is tightly regulated by both antioxidant proteins and small molecules. On the cytoplasmic side of the ER, cytochrome P450 (P450) proteins have been identified as a superfamily of enzymes that are important in the formation of endogenous chemicals as well as in the detoxication of xenobiotics. Our previous report described oxidative inhibition of P450 Family 4 enzymes via oxidation of the heme-thiolate cysteine to a sulfenic acid (-SOH) (Albertolle, M. E. (2017) 292, 11230-11242). Further proteomic analyses of murine kidney and liver microsomes led to the finding that a number of other drug-metabolizing enzymes located in the ER are also redox-regulated in this manner. We expanded our analysis of sulfenylated enzymes to human liver and kidney microsomes. Evaluation of the sulfenylation, catalytic activity, and spectral properties of P450s 1A2, 2C8, 2D6, and 3A4 led to the identification of two classes of redox sensitivity in P450 enzymes: heme-thiolate-sensitive and thiol-insensitive. These findings provide evidence for a mammalian P450 regulatory mechanism, which may also be relevant to other drug-metabolizing enzymes. (Data are available via ProteomeXchange with identifier PXD007913.).
© 2018 by The American Society for Biochemistry and Molecular Biology, Inc.
1 Communities
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15 MeSH Terms
Trapping redox partnerships in oxidant-sensitive proteins with a small, thiol-reactive cross-linker.
Allan KM, Loberg MA, Chepngeno J, Hurtig JE, Tripathi S, Kang MG, Allotey JK, Widdershins AH, Pilat JM, Sizek HJ, Murphy WJ, Naticchia MR, David JB, Morano KA, West JD
(2016) Free Radic Biol Med 101: 356-366
MeSH Terms: Cross-Linking Reagents, Disulfides, Glutathione Peroxidase, Methionine Sulfoxide Reductases, Oxidants, Oxidation-Reduction, Oxidative Stress, Oxidoreductases Acting on Sulfur Group Donors, Peroxiredoxins, Saccharomyces cerevisiae, Saccharomyces cerevisiae Proteins, Sulfhydryl Compounds, Sulfones, Thioredoxins, tert-Butylhydroperoxide
Show Abstract · Added April 24, 2017
A broad range of redox-regulated proteins undergo reversible disulfide bond formation on oxidation-prone cysteine residues. Heightened reactivity of the thiol groups in these cysteines also increases susceptibility to modification by organic electrophiles, a property that can be exploited in the study of redox networks. Here, we explored whether divinyl sulfone (DVSF), a thiol-reactive bifunctional electrophile, cross-links oxidant-sensitive proteins to their putative redox partners in cells. To test this idea, previously identified oxidant targets involved in oxidant defense (namely, peroxiredoxins, methionine sulfoxide reductases, sulfiredoxin, and glutathione peroxidases), metabolism, and proteostasis were monitored for cross-link formation following treatment of Saccharomyces cerevisiae with DVSF. Several proteins screened, including multiple oxidant defense proteins, underwent intermolecular and/or intramolecular cross-linking in response to DVSF. Specific redox-active cysteines within a subset of DVSF targets were found to influence cross-linking; in addition, DVSF-mediated cross-linking of its targets was impaired in cells first exposed to oxidants. Since cross-linking appeared to involve redox-active cysteines in these proteins, we examined whether potential redox partners became cross-linked to them upon DVSF treatment. Specifically, we found that several substrates of thioredoxins were cross-linked to the cytosolic thioredoxin Trx2 in cells treated with DVSF. However, other DVSF targets, like the peroxiredoxin Ahp1, principally formed intra-protein cross-links upon DVSF treatment. Moreover, additional protein targets, including several known to undergo S-glutathionylation, were conjugated via DVSF to glutathione. Our results indicate that DVSF is of potential use as a chemical tool for irreversibly trapping and discovering thiol-based redox partnerships within cells.
Copyright © 2016 Elsevier Inc. All rights reserved.
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15 MeSH Terms
Nanoscale phase segregation of mixed thiolates on gold nanoparticles.
Harkness KM, Balinski A, McLean JA, Cliffel DE
(2011) Angew Chem Int Ed Engl 50: 10554-9
MeSH Terms: Gold, Metal Nanoparticles, Organogold Compounds, Sulfhydryl Compounds
Added January 20, 2015
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1 Members
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4 MeSH Terms
ReCLIP (reversible cross-link immuno-precipitation): an efficient method for interrogation of labile protein complexes.
Smith AL, Friedman DB, Yu H, Carnahan RH, Reynolds AB
(2011) PLoS One 6: e16206
MeSH Terms: Cross-Linking Reagents, Immunoprecipitation, Methods, Multiprotein Complexes, Protein Stability, Sulfhydryl Compounds
Show Abstract · Added December 12, 2013
The difficulty of maintaining intact protein complexes while minimizing non-specific background remains a significant limitation in proteomic studies. Labile interactions, such as the interaction between p120-catenin and the E-cadherin complex, are particularly challenging. Using the cadherin complex as a model-system, we have developed a procedure for efficient recovery of otherwise labile protein-protein interactions. We have named the procedure "ReCLIP" (Reversible Cross-Link Immuno-Precipitation) to reflect the primary elements of the method. Using cell-permeable, thiol-cleavable crosslinkers, normally labile interactions (i.e. p120 and E-cadherin) are stabilized in situ prior to isolation. After immunoprecipitation, crosslinked binding partners are selectively released and all other components of the procedure (i.e. beads, antibody, and p120 itself) are discarded. The end result is extremely efficient recovery with exceptionally low background. ReCLIP therefore appears to provide an excellent alternative to currently available affinity-purification approaches, particularly for studies of labile complexes.
2 Communities
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6 MeSH Terms
A structural mass spectrometry strategy for the relative quantitation of ligands on mixed monolayer-protected gold nanoparticles.
Harkness KM, Hixson BC, Fenn LS, Turner BN, Rape AC, Simpson CA, Huffman BJ, Okoli TC, McLean JA, Cliffel DE
(2010) Anal Chem 82: 9268-74
MeSH Terms: Glutathione, Gold, Hydrophobic and Hydrophilic Interactions, Ligands, Magnetic Resonance Spectroscopy, Mass Spectrometry, Metal Nanoparticles, Polyethylene Glycols, Sulfhydryl Compounds, Surface Properties, Tiopronin
Show Abstract · Added January 20, 2015
It is becoming increasingly common to use gold nanoparticles (AuNPs) protected by a heterogeneous mixture of thiolate ligands, but many ligand mixtures on AuNPs cannot be properly characterized due to the inherent limitations of commonly used spectroscopic techniques. Using ion mobility-mass spectrometry (IM-MS), we have developed a strategy that allows measurement of the relative quantity of ligands on AuNP surfaces. This strategy is used for the characterization of three samples of mixed-ligand AuNPs: tiopronin:glutathione (av diameter 2.5 nm), octanethiol:decanethiol (av diameter 3.6 nm), and tiopronin:11-mercaptoundecyl(poly ethylene glycol) (av diameter 2.5 nm). For validation purposes, the results obtained for tiopronin:glutathione AuNPs were compared to parallel measurements using nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry (MS) without ion mobility separation. Relative quantitation measurements for NMR and IM-MS were in excellent agreement, with an average difference of less than 1% relative abundance. IM-MS and MS without ion mobility separation were not comparable, due to a lack of ion signals for MS. The other two mixed-ligand AuNPs provide examples of measurements that cannot be performed using NMR spectroscopy.
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11 MeSH Terms
Unexpected toxicity of monolayer protected gold clusters eliminated by PEG-thiol place exchange reactions.
Simpson CA, Huffman BJ, Gerdon AE, Cliffel DE
(2010) Chem Res Toxicol 23: 1608-16
MeSH Terms: Animals, Female, Gold, Kidney Tubules, Leukocytes, Metal Nanoparticles, Mice, Mice, Inbred BALB C, Polyethylene Glycols, Sulfhydryl Compounds, Water
Show Abstract · Added January 20, 2015
Monolayer protected clusters (MPCs) are small, metal nanoparticles capped with thiolate ligands that have been widely studied for their size-dependent properties and for their ability to be functionalized for biological applications. Common water-soluble MPCs, functionalized by N-(2-Mercaptopropionyl)-glycine (tiopronin) or glutathione, have been used previously to interface with biological systems. These MPCs are ideal for biological applications not only due to their water-solubility but also their small size (<5 nm). These characteristics are expected to enable easy biodistribution and clearance. In this article, we show an unexpected toxicity is associated with the tiopronin monolayer protected cluster (TMPC), making it incompatible for potential in vivo applications. This toxicity is linked to significant histological damage to the renal tubules, causing mortality at concentrations above 20 μM. We further show how the incorporation of poly ethylene glycol (PEG) by a simple place-exchange reaction eliminates this toxicity. We analyzed gold content within blood and urine and found an increased lifetime of the particle within the bloodstream due to the creation of the mixed monolayer. Also shown was the elimination of kidney damage with the use of the mixed-monolayer particle via Multistix analysis, MALDI-TOF MS analysis, and histological examination. Final immunological analysis showed no effect on white blood cell (WBC) count for the unmodified particle and a surprising increase in WBC count with the injection of mixed monolayer particles at concentrations higher than 30 μM, suggesting that there may be an immune response to these mixed monolayer nanoparticles at high concentrations; therefore, special attention should be focused on selecting the best capping ligands for use in vivo. These findings make the mixed monolayer an excellent candidate for further biological applications using water-soluble nanoparticles.
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11 MeSH Terms
Thiol-based, site-specific and covalent immobilization of biomolecules for single-molecule experiments.
Zimmermann JL, Nicolaus T, Neuert G, Blank K
(2010) Nat Protoc 5: 975-85
MeSH Terms: Binding Sites, Enzymes, Immobilized, Fluorescence, Immobilized Proteins, Maleimides, Microscopy, Atomic Force, Nucleic Acids, Polyethylene Glycols, Succinimides, Sulfhydryl Compounds
Show Abstract · Added June 2, 2013
The success of single-molecule (SM) experiments critically depends on the functional immobilization of the biomolecule(s) to be studied. With the continuing trend of combining SM fluorescence with SM force experiments, methods are required that are suitable for both types of measurements. We describe a general protocol for the site-specific and covalent coupling of any type of biomolecule that can be prepared with a free thiol group. The protocol uses a poly(ethylene glycol) (PEG) spacer, which carries an N-hydroxy succinimide (NHS) group on one end and a maleimide group on the other. After reacting the NHS group with an amino-functionalized surface, the relatively stable but highly reactive maleimide group allows the coupling of the biomolecule. This protocol provides surfaces with low fluorescence background, low nonspecific binding and a large number of reactive sites. Surfaces containing immobilized biomolecules can be obtained within 6 h.
0 Communities
1 Members
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10 MeSH Terms
Characterization of thiolate-protected gold nanoparticles by mass spectrometry.
Harkness KM, Cliffel DE, McLean JA
(2010) Analyst 135: 868-74
MeSH Terms: Gold, Mass Spectrometry, Metal Nanoparticles, Spectrometry, Mass, Electrospray Ionization, Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization, Sulfhydryl Compounds
Show Abstract · Added January 20, 2015
Thiolate-protected gold nanoparticles (AuNPs) are a highly versatile nanomaterial, with wide-ranging physical properties dependent upon the protecting thiolate ligands and gold core size. These nanoparticles serve as a scaffold for a diverse and rapidly increasing number of applications, extending from molecular electronics to vaccine development. Key to the development of such applications is the ability to quickly and precisely characterize synthesized AuNPs. While a unique set of challenges have inhibited the potential of mass spectrometry in this area, recent improvements have made mass spectrometry a dominant technique in the characterization of small AuNPs, specifically those with discrete sizes and structures referred to as monolayer-protected gold clusters (MPCs). Additionally, the unique fragmentation data from mass spectrometry enables the characterization of the protecting monolayer on larger AuNPs. The development of mass spectrometry techniques for AuNP characterization has begun to reveal interesting new areas of research. This report is a discussion of the historical challenges in this field, the emerging techniques which aim to meet those challenges, and the future role of mass spectrometry in the growing field of thiolate-protected AuNPs.
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6 MeSH Terms
Surface fragmentation of complexes from thiolate protected gold nanoparticles by ion mobility-mass spectrometry.
Harkness KM, Fenn LS, Cliffel DE, McLean JA
(2010) Anal Chem 82: 3061-6
MeSH Terms: Gold, Metal Nanoparticles, Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization, Sulfhydryl Compounds
Show Abstract · Added January 20, 2015
Matrix-assisted laser desorption/ionization-ion mobility-mass spectrometry (MALDI-IM-MS) was used to analyze low mass gold-thiolate fragments generated from thiolate-protected gold nanoparticles (AuNPs). This is the first report of using gas-phase structural separations by IM-MS for the characterization of AuNPs, revealing significant structural variation between organic and gold-thiolate ionic species. Through the separation of background chemical noise, gold-thiolate ion species corresponding to fragments from the AuNP surface can be isolated. In the negative ion mode, many of these fragments correlate to capping structural motifs observed in the literature. In the positive ion mode, the fragment ions do not correlate to predicted structural motifs, but are nearly identical to the positive ions generated from the gold-thiolate AuNP precursor complexes. This suggests that energetic processes during laser desorption/ionization induce a structural rearrangement in the capping gold-thiolate structure of the AuNP, resulting in the generation of positively charged gold-thiolate complexes similar to the precursors of AuNP formation by reduction and negatively charged complexes more representative of the AuNP surface.
0 Communities
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4 MeSH Terms
Crystallographic studies of chemically modified nucleic acids: a backward glance.
Egli M, Pallan PS
(2010) Chem Biodivers 7: 60-89
MeSH Terms: Base Pairing, Bridged Bicyclo Compounds, Carbohydrates, Crystallography, X-Ray, Molecular Conformation, Nucleic Acids, Nucleotides, Sulfhydryl Compounds
Show Abstract · Added March 7, 2014
Chemically modified nucleic acids (CNAs) are widely explored as antisense oligonucleotide or small interfering RNA (siRNA) candidates for therapeutic applications. CNAs are also of interest in diagnostics, high-throughput genomics and target validation, nanotechnology and as model systems in investigations directed at a better understanding of the etiology of nucleic acid structure, as well as the physicochemical and pairing properties of DNA and RNA, and for probing protein-nucleic acid interactions. In this article, we review research conducted in our laboratory over the past two decades with a focus on crystal-structure analyses of CNAs and artificial pairing systems. We highlight key insights into issues ranging from conformational distortions as a consequence of modification to the modulation of pairing strength, and RNA affinity by stereoelectronic effects and hydration. Although crystal structures have only been determined for a subset of the large number of modifications that were synthesized and analyzed in the oligonucleotide context to date, they have yielded guiding principles for the design of new analogs with tailor-made properties, including pairing specificity, nuclease resistance, and cellular uptake. And, perhaps less obviously, crystallographic studies of CNAs and synthetic pairing systems have shed light on fundamental aspects of DNA and RNA structure and function that would not have been disclosed by investigations solely focused on the natural nucleic acids.
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8 MeSH Terms