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Daniel C Liebler

Publications and source records attributed to Daniel C Liebler.

At least 19 recordsLinked to original sources
PubMed2006

Covalent adduction of human serum albumin by 4-hydroxy-2-nonenal: kinetic analysis of competing alkylation reactions.

The electrophilic lipid oxidation product 4-hydroxy-2-nonenal (HNE) reacts with proteins to form covalent adducts, and this damage has been implicated in pathologies associated with oxidative stress. HNE adduction of blood proteins, such as human serum albumin (HSA), yields adducts that may serve as markers of oxidative stress in vivo. We used liquid chromatography-tandem mass spectrometry (LC-MS-MS) and the P-Mod algorithm to map the sites of 10 adducts formed by reaction of HNE with HSA in vitro. The detected adducts included Michael adducts formed at histidine and lysine residues. The selectivity of HNE in competing adduction reactions was evaluated by analysis of kinetics for HNE Michael adduction at six targeted HSA histidine residues. Reaction kinetics were analyzed by selected reaction monitoring in LC-MS-MS using stable isotope tagging with phenyl isocyanate. Rate constants ranged over 4 orders of magnitude, with the order of reactivity being H242 > H510 > H67 > H367 > H247 approximately K233. The most reactive target, H242, is located in a fatty acid- and drug binding cavity in subdomain IIa of HSA and appears to be a hot-spot for HNE modification. Analysis of adduction kinetics together with HSA structure and target residue pK(a) values suggest that location in the hydrophobic binding cavity and low predicted pK(a) of H242 account for its high reactivity toward HNE. H242 adducts may be preferred products of adduction by lipophilic electrophiles and may comprise a family of biomarkers for oxidative stress.

Aldehydes↗
PubMed2006

Inhibition of protein phosphatase 2A activity by selective electrophile alkylation damage.

Protein serine/threonine phosphatase 2A (PP2A) is a critical regulator of numerous cellular signaling processes and a potential target for reactive electrophiles that dysregulate phosphorylation-dependent signal transduction cascades. The predominant cellular form of PP2A is a heterotrimeric holoenzyme consisting of a structural A, a variable B, and a catalytic C subunit. We studied the modification of two purified PP2A holoenzyme complexes (ABalpha(FLAG)C and ABdelta(FLAG)C) with two different thiol-reactive electrophiles, biotinyl-iodoacetamidyl-3,6-dioxaoctanediamine (PEO-IAB) and the biotinamido-4-[4'-(maleimidomethyl)cyclohexanecarboxamido]butane (BMCC). In vivo treatment of HEK 293 cells with these electrophiles resulted in alkylation of all three PP2A subunits. Electrophile treatment of the immunopurified FLAG-tagged holoenzymes produced a concentration-dependent adduction of PP2A subunits, as observed by Western blot analysis. Although both electrophiles labeled all three PP2A subunits, only BMCC inhibited the catalytic activity of both holoenzymes. Alkylation patterns in the A and B subunits were identical for the two electrophiles, but BMCC alkylated four Cys residues in the C subunit that were not labeled by PEO-IAB. Homology between the catalytic subunits of PP1 and PP2A enabled generation of a comparative model structure for the C subunit of PP2A. The model structure provided additional insight into contributions of specific BMCC-Cys adducts to PP2A enzyme inhibition. The results indicate that site selectivity of protein adduction should be a critical determinant of the ability of electrophiles to affect cellular signaling processes.

Alkylation↗
PubMed2006

N-Terminal amino acid side-chain cleavage of chemically modified peptides in the gas phase: a mass spectrometry technique for N-terminus identification.

Although genome databases have become the key for proteomic analyses, de novo sequencing remains essential for the study of organisms whose genomes have not been completed. In addition, post-translational modifications present a challenge in database searching. Recognition of the b or y-ion series in a peptide MS/MS spectrum as well as identification of the b1 - and yn-1 -ions can facilitate de novo analyses. Therefore, it is valuable to identify either amino-acid terminus. In previous work, we have demonstrated that peptides modified at the epsilon-amino group of lysine as a t-butyl peroxycarbamate derivative undergo free radical promoted peptide backbone fragmentation under low-energy collision-induced dissociation (CID) conditions. Here we explore the chemistry of the N-terminal amino group modified as a t-butyl peroxycarbamate. The conversion of N-terminal amines to peroxycarbamates of simple amino acids and peptides was studied with aryl t-butyl peroxycarbonates. ESI-MS/MS analysis of the peroxycarbamate adducts gave evidence of a product ion corresponding to the neutral loss of the N-terminal side chain (R), thus identifying this residue. Further fragmentation (MS3) of product ions formed by N-terminal residue side-chain loss (-R) exhibited an m/z shift of the b-ions equal to the neutral loss of R, therefore labeling the b-ion series. The study was extended to the analysis of a protein tryptic digest where the SALSA algorithm was used to identify spectra containing these neutral losses. The method for N-terminus identification presented here has the potential for improvement of de novo analyses as well as in constraining peptide mass mapping database searches.

Amines↗
PubMed2006

Redox instability induced by 4-hydroxy-2-nonenal in porcine and bovine myoglobins at pH 5.6 and 4 degrees C.

Myoglobin (Mb) redox stability affects meat color and is compromised by lipid oxidation products such as 4-hydroxy-2-nonenal (HNE). Pork lipids are generally more unsaturated and would be expected to oxidize readily and produce more oxidation products than beef. Supranutritional supplementation of vitamin E improves Mb redox stability of beef but not pork. The present study investigated HNE-induced redox instability in porcine and bovine myoglobins at 4 degrees C and pH 5.6. Oxymyoglobin (OxyMb) was incubated with HNE (0.075 mM porcine OxyMb + 0.5 mM HNE; 0.15 mM bovine OxyMb + 1.0 mM HNE). In porcine Mb, only monoadducts formed via Michael addition were detected after 72 h, whereas in bovine Mb both mono- and diadducts were identified. LC-MS-MS identified four histidine residues (His 36, 81, 88, and 152) of bovine Mb that were readily adducted by HNE, whereas in porcine Mb only two histidine residues (His 24 and 36) were adducted. These results suggested that the primary structure of bovine Mb predisposes it to greater nucleophilic attack by HNE and subsequent adduction than is suffered by porcine Mb.

Aldehydes↗
PubMed2006

Identification of S-nitrosylation motifs by site-specific mapping of the S-nitrosocysteine proteome in human vascular smooth muscle cells.

S-nitrosylation, the selective modification of cysteine residues in proteins to form S-nitrosocysteine, is a major emerging mechanism by which nitric oxide acts as a signaling molecule. Even though nitric oxide is intimately involved in the regulation of vascular smooth muscle cell functions, the potential protein targets for nitric oxide modification as well as structural features that underlie the specificity of protein S-nitrosocysteine formation in these cells remain unknown. Therefore, we used a proteomic approach using selective peptide capturing and site-specific adduct mapping to identify the targets of S-nitrosylation in human aortic smooth muscle cells upon exposure to S-nitrosocysteine and propylamine propylamine NONOate. This strategy identified 20 unique S-nitrosocysteine-containing peptides belonging to 18 proteins including cytoskeletal proteins, chaperones, proteins of the translational machinery, vesicular transport, and signaling. Sequence analysis of the S-nitrosocysteine-containing peptides revealed the presence of acid/base motifs, as well as hydrophobic motifs surrounding the identified cysteine residues. High-resolution immunogold electron microscopy supported the cellular localization of several of these proteins. Interestingly, seven of the 18 proteins identified are localized within the ER/Golgi complex, suggesting a role for S-nitrosylation in membrane trafficking and ER stress response in vascular smooth muscle.

Amino Acid Sequence↗
PubMed2006

Identification of phosphorylation sites in glycine N-methyltransferase from rat liver.

Previous studies have shown that rat glycine N-methyltransferase (GNMT) is phosphorylated in vivo, and could be phosphorylated in vitro on serine residues with a significant increase of enzyme activity, but no phosphorylation sites were identified. In this work the identification of the specific phosphorylation sites of rat GNMT is reported. Three different preparations of rat GNMT were analyzed: (1) purified from liver by standard methods of protein purification, (2) prepared from isolated hepatocytes and from liver tissue by immunoprecipitation, and (3) recombinant protein expressed in Escherichia coli. We measured the molecular weights of protein isoforms using electrospray mass spectrometry and used liquid chromatography-tandem mass spectrometry (LC-MS/MS) of peptides resulting from tryptic and chymotryptic digests. We also performed chemical analysis of phosphoamino acids and protein sequencing. In all samples, the phosphorylated serine residues 71, 182, and 241 were found. In GNMT prepared from liver tissue and hepatocytes an S9 additional residue was found to be phosphorylated. In hepatocytes and in recombinant GNMT S139 was detected. Serine 9 was also identified as a target for cAMP-dependent protein kinase in vitro. The positions of these phosphorylated residues in the tertiary structure of GNMT indicate their possible effect on enzyme conformation and activity.

Amino Acid Sequence↗
PubMed2006

Cytosolic and nuclear protein targets of thiol-reactive electrophiles.

Reactive electrophiles formed from toxic drugs and chemicals and by endogenous oxidative stress covalently modify proteins. Although protein covalent binding is thought to initiate a variety of adaptive and toxic responses, the identities of the protein targets are generally unknown, as are protein structural features that confer susceptibility to modification. We have analyzed the protein targets in nuclear and cytoplasmic proteomes from HEK293 cells treated in vitro with two biotin-tagged, thiol-reactive electrophiles, (+)-biotinyl-iodoacetamidyl-3, 6-dioxaoctanediamine (PEO-IAB) and 1-biotinamido-4-(4'-[maleimidoethylcyclohexane]-carboxamido)butane (BMCC). Biotinylated peptides were captured by affinity enrichment using neutravidin beads, and the adducted peptides were then analyzed by multidimensional liquid chromatography-tandem mass spectrometry. A total of 897 adducts were mapped to different cysteine residues in 539 proteins. Adduction was selective and reproducible, and > 90% of all adducted proteins were modified at only one or two sites. A core group of 125 cysteines (14% of the total) was consistently modified by both electrophiles. Selective modification of several protein domain structures and motifs indicates that certain protein families are particularly susceptible to alkylation. This approach can be extended to studies of other protein-damaging oxidants and electrophiles and can provide new insights into targets and consequences of protein damage in toxicity and disease.

Alkylation↗
PubMed2006

Selectively increased oxidative modifications mapped to detergent-insoluble forms of Abeta and beta-III tubulin in Alzheimer's disease.

Deleterious post-translational modifications (PTMs) to the neuronal cytoskeleton are a proposed mechanistic link between accumulation of amyloid (A) beta peptides and subsequent abnormalities of tau and neurodegeneration in Alzheimer's disease (AD). Here we tested the hypothesis that PTMs on neuronal tubulins selectively accumulate in a pathological protein fraction in AD. We used new software, P-MOD, to identify comprehensively and map PTMs using mass spectral data from soluble (normal) and detergent-insoluble (pathological) protein fractions from AD, as well as total extracts from controls, for selected proteins: Abeta, tau, apolipoprotein (apo) E, glial fibrillary acidic protein (GFAP), alpha-III tubulin, and beta-III tubulin. Our results confirmed direct observations of others by identifying methionine (M) sulfoxides at Abeta position 35 and numerous sites of tau phosphorylation in detergent-insoluble protein from AD, while no PTMs were enriched on primarily astrocyte-derived apoE or GFAP in this fraction. P-MOD mapped several abundant M sulfoxides to neuron-enriched beta-III tubulin but not its heterodimeric partner, neuron-enriched alpha-III tubulin, a result confirmed by selective suppression of CNBr-mediated cleavage of beta-III tubulin. These findings are the first comprehensive assessment of PTMs in AD and point to oxidative modification of beta-III tubulin as a potential contributor to the neuronal cytoskeletal disruption that is characteristic of AD.

Alzheimer Disease↗
PubMed2005

Specific patterns of electrophile adduction trigger Keap1 ubiquitination and Nrf2 activation.

Activation of the transcription factor Nrf2 regulates expression of phase II enzymes and other adaptive responses to electrophile and oxidant stress. Nrf2 concentrations are regulated by the thiol-rich sensor protein Keap1, which is an adaptor protein for Cul3-dependent ubiquitination and degradation of Nrf2. However, the links between site specificity of Keap1 modification by electrophiles and mechanisms of Nrf2 activation are poorly understood. We studied the actions of the prototypical Nrf2 inducer tert-butylhydroquinone (tBHQ) and two biotin-tagged, thiol-reactive electrophiles, N-iodoacetyl-N-biotinylhexylenediamine (IAB) and 1-biotinamido-4-(4'-[maleimidoethyl-cyclohexane]-carboxamido)butane (BMCC). Both IAB and tBHQ induced antioxidant response element (ARE)-directed green fluorescent protein (GFP) expression in ARE/thymidine kinase GFP HepG2 cells, and both initiated nuclear Nrf2 accumulation and induction of heme oxygenase 1 in HEK293 cells. In contrast, BMCC produced none of these effects. Liquid chromatography tandem mass spectrometry (MS-MS) analysis of human Keap1 modified by IAB or BMCC in vitro indicated that IAB adduction occurred primarily in the central linker domain, whereas BMCC modified other Keap1 domains. Treatment of FLAG-Keap1-transfected HEK293 with the Nrf2-activating compounds IAB and tBHQ generated high molecular weight Keap1 forms, which were identified as K-48-linked polyubiquitin conjugates by immunoblotting and liquid chromatography MS-MS. Keap1 polyubiquitination coincided with Nrf2 stabilization and nuclear accumulation. In contrast, BMCC did not induce Keap1 polyubiquitination. Our results suggest that Nrf2 activation is regulated through the polyubiquitination of Keap1, which in turn is triggered by specific patterns of electrophile modification of the Keap1 central linker domain. These results suggest that Keap1 adduction triggers a switching of Cul3-dependent ubiquitination from Nrf2 to Keap1, leading to Nrf2 activation.

Biotin↗
PubMed2005

Sample preparation and digestion for proteomic analyses using spin filters.

We describe the use of commercially available microcentrifugation devices (spin filters) for cleanup and digestion of protein samples for mass spectrometry analyses. The protein sample is added to the upper chamber of a spin filter with a > or = 3000 molecular weight cutoff membrane and then washed prior to resuspension in ammonium bicarbonate. The protein is then reduced, alkylated, and digested with trypsin in the upper chamber and the peptides are recovered by centrifugation through the membrane. The method provides digestion efficiencies comparable to standard in-solution digests, avoids lengthy dialysis steps, and allows rapid cleanup of samples containing salts, some detergents, and acidic or basic buffers.

Bicarbonates↗
PubMed2005

Proteomic identification of ubiquitinated proteins from human cells expressing His-tagged ubiquitin.

A proteomics method has been developed to purify and identify the specific proteins modified by ubiquitin (Ub) from human cells. In purified samples, Ub and 21 other proteins were identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS) spectra using SEQUEST. These proteins included several of the expected carriers of Ub including Ub-conjugating enzymes and histone proteins. To perform these experiments, a cell line coexpressing epitope tagged His(6X)-Ub and green fluorescent protein (GFP) was generated by stably transfecting HEK293 cells. Ubiquitinated proteins were purified using nickel-affinity chromatography and digested in solution with trypsin. Complex mixtures of peptides were separated by reversed phase chromatography and analyzed by nano LC-MS/MS using the LCQ quadrupole ion-trap mass spectrometer. Proteins identified from His(6X)-Ub-GFP transfected cells were compared to a list of proteins from HEK293 cells, which associate with nickel-nitrilotriacetic acid (Ni-NTA)-agarose in the absence of His-tagged Ub. In a proof of principle experiment, His(6X)-Ub-GFP transfected cells were treated with As (III) (10 microM, 24 h) in an attempt to identify substrates increasingly modified by Ub. In this experiment, proliferating cell nuclear antigen, a DNA repair protein and known ubiquitin substrate, was confidently identified. This proteomics method, developed for the analysis of ubiquitinated proteins, is a step towards large-scale characterization of Ub-protein conjugates in numerous physiological and pathological states.

Amino Acid Sequence↗
PubMed2005

Alkylation of cytochrome c by (glutathion-S-yl)-1,4-benzoquinone and iodoacetamide demonstrates compound-dependent site specificity.

The reaction of cytochrome c with the electrophilic compounds (glutathion-S-yl)-1,4-benzoquinone (GSBQ) and iodoacetamide was studied using mass spectrometry. GSBQ is a nephrotoxic quinol-thioether metabolite of benzoquinone, while iodoacetamide is an alkylating agent targeting cysteine thiols. Both chemicals formed covalent adducts with cytochrome c. GSBQ formed adducts with cytochrome c at pH 6 on several histidine and lysine residues. At a pH >7, the initial product rearranged to a disubstituted cyclic quinone species preferentially found at two sites on the protein, Lys25-Lys27 and Lys86-Lys87, via quinol amine linkages. These two sites were previously determined to be the targets of benzoquinone adduct formation [Person et al. (2003) Chem. Res. Toxicol. 16, 598-608]. Cyclic reaction products are preferentially formed at two sites on the protein because of the presence of multiple basic residues in a conformationally flexible region whereas noncyclic products bind to a broad spectrum of available lysine and histidine nucleophiles. Iodoacetamide was a less selective alkylating agent able to form adducts on the majority of the nucleophilic sites of the protein. MS/MS spectra were used to identify signature ions for GSBQ-adducted peptides from the characteristic fragmentation patterns. Neutral losses of the 129 Da gamma-glutamate residue and of the 273 Da glutathione moiety were found in both cysteine thiol- and lysine amine-linked GSBQ adduct MS/MS. Characteristic fragment ions were used in conjunction with the scoring algorithm for spectral analysis to search for adducted species present at low levels in the sample, and the analysis is applicable generally to detection of glutathione conjugates by MS/MS. Parallel analysis using matrix-assisted laser desorption/ionization-MS to compare spectra of control and treated samples allowed identification of peptide adducts formed by direct addition of GSBQ and by the subsequent loss of the glutathione moiety in a pH-dependent cyclization reaction.

Algorithms↗
PubMed2005

Identification of sensor cysteines in human Keap1 modified by the cancer chemopreventive agent sulforaphane.

The chemopreventive agent sulforaphane is an isothiocyanate derived from cruciferous vegetables. Sulforaphane exerts cancer chemopreventive effects by inducing antioxidant/electrophile response element (ARE)-regulated phase 2 enzyme and antioxidant genes through activation of the transcription factor nuclear factor-E2-related factor 2 (Nrf2), which is regulated by the thiol-rich sensor protein Kelch-like ECH-associated protein 1 (Keap1). Sulforaphane is an electrophile that can react with protein thiols to form thionoacyl adducts. We hypothesized that, like other electrophilic Nrf2 activators, sulforaphane activates this system through specific modifications of the Keap1 protein. However, thionoacyl adducts are labile to hydrolysis and transacylation reactions, which complicate the identification of the sulforaphane adduct sites on Keap1. In this study, we characterized the stability of sulforaphane thionoacyl adducts and developed a liquid chromatography-tandem mass spectrometry method to map labile sulforaphane adduct sites formed on Keap1 in vitro. Sulforaphane displays a distinctly different pattern of Keap1 modification than previously studied ARE inducers that modify Keap1 by alkylation. Sulforaphane modified Keap1 most readily in the Kelch domain, rather than in the central linker domain, which is targeted by previously characterized ARE inducers. Also, in contrast to previously studied ARE inducers and as reported recently [Zhang, et al. (2005) J. Biol. Chem. 280, 30091-30099], sulforaphane treatment in vivo does not lead to the accumulation of ubiquitinated Keap1. Our observations suggest a novel mechanism for Nrf2 stabilization by sulforaphane-Keap1 thionoacyl adduct formation.

Amino Acid Sequence↗
PubMed2005

Elucidating mechanisms of drug-induced toxicity.

The early and high-throughput application of assays for non-genetic toxicity is of great interest to the pharmaceutical industry, although few systems have been validated as being of good predictive value. New technologies could enable toxicity to be studied in the context of systems biology. An important factor to be considered is the metabolism of drugs to reactive intermediates. Chemical reactions of these with cell and tissue nucleophiles are relatively well understood, but predicting how biological modifications will affect signalling and regulatory networks remains a challenge. Some of these pathways could be useful as sentinels for toxicity. This article will cover some examples of drug toxicity and the prospects for future technology development.

Animals↗
PubMed2004

Global shifts in protein sumoylation in response to electrophile and oxidative stress.

Human small ubiquitin-like modifier (sumo) proteins include sumo-1 and the less studied, nearly identical sumo-2 and sumo-3 proteins. Whereas the structurally related ubiquitin molecule targets proteins for degradation, sumo provides a distinct, yet poorly understood regulatory signal. Protein sumoylation is sensitive to diverse cellular stresses, yet the targets of sumoylation in stress are unknown. We studied protein sumoylation in HEK293 cells exposed to hydrogen peroxide, alkylating agents, and the lipid oxidation-derived electrophile 4-hydroxynonenal, which is an ubiquitous product of lipid oxidation associated with oxidative stress. Confocal immunofluorescence microscopy indicated that in unstressed cells sumo-1 targeted nuclear proteins, whereas sumo-2/3 targeted proteins in both nuclei and cytoplasm. Western blot analyses revealed changes in sumo-1 and sumo-2/3 targeting patterns with stress. We used immunoaffinity chromatography to harvest sumo-associated proteins from HA-sumo-1- and HA-sumo-3-expressing HEK293 cells both before and after treatment with 4-hydroxynonenal. Multidimensional liquid chromatography-tandem mass spectrometry analyses identified 54 HA-sumo-1-associated proteins and 38 HA-sumo-3-associated proteins. Major protein targets included RNA binding and processing proteins, transcription factors, metabolic enzymes, and cytoskeletal regulators. Treatment with 4-hydroxynonenal caused a near-complete redistribution of sumo-1 and sumo-3 to different protein targets, which included chaperones, antioxidant, and DNA damage signaling proteins. A 10-15% overlap of sumo-1 and sumo-3 targets before and after stress suggests that sumo proteins target distinct protein groups. The results suggest that reactive electrophiles not only directly modify proteins but also lead to indirect changes in endogenous protein modifications that regulate protein functions.

Aldehydes↗