Crystal structures of the naturally fused CS and cytochrome b reductase (bR) domains of Ncb5or reveal an expanded CS fold, extensive CS-bR interactions and productive binding of the NAD(P) nicotinamide ring.

Ncb5or (NADH-cytochrome b oxidoreductase), a cytosolic ferric reductase implicated in diabetes and neurological diseases, comprises three distinct domains, cytochrome b (b) and cytochrome b reductase (bR) domains separated by a CHORD-Sgt1 (CS) domain, and a novel 50-residue N-terminal region. Understanding how interdomain interactions in Ncb5or facilitate the shuttling of electrons from NAD(P)H to heme, and how the process compares with the microsomal b (Cyb5A) and bR (Cyb5R3) system, is of interest. A high-resolution structure of the b domain (PDB entry 3lf5) has previously been reported, which exhibits substantial differences in comparison to Cyb5A.

Protein Targets of Isoniazid-Reactive Metabolites in Mouse Liver in Vivo.

Isoniazid (INH) has been a first-line drug for the treatment of tuberculosis for more than 40 years. INH is well-tolerated by most patients, but some patients develop hepatitis that can be severe in rare cases or after overdose. The mechanisms underlying the hepatotoxicity of INH are not known, but covalent binding of reactive metabolites is known to occur in animals and is suspected in human cases.

A SUMO-acetyl switch in PXR biology.

Post-translational modification (PTM) of nuclear receptor superfamily members regulates various aspects of their biology to include sub-cellular localization, the repertoire of protein-binding partners, as well as their stability and mode of degradation. The nuclear receptor pregnane X receptor (PXR, NR1I2) is a master-regulator of the drug-inducible gene expression in liver and intestine. The PXR-mediated gene activation program is primarily recognized to increase drug metabolism, drug transport, and drug efflux pathways in these tissues.

Endoplasmic Reticulum-resident Heat Shock Protein 90 (HSP90) Isoform Glucose-regulated Protein 94 (GRP94) Regulates Cell Polarity and Cancer Cell Migration by Affecting Intracellular Transport.

Heat shock protein 90 (HSP90) is a molecular chaperone that is up-regulated in cancer and is required for the folding of numerous signaling proteins. Consequently, HSP90 represents an ideal target for the development of new anti-cancer agents. The human HSP90 isoform, glucose-regulated protein 94 (GRP94), resides in the endoplasmic reticulum and regulates secretory pathways, integrins, and Toll-like receptors, which contribute to regulating immunity and metastasis.

SUMOylation and Ubiquitylation Circuitry Controls Pregnane X Receptor Biology in Hepatocytes.

Several nuclear receptor (NR) superfamily members are known to be the molecular target of either the small ubiquitin-related modifier (SUMO) or ubiquitin-signaling pathways. However, little is currently known regarding how these two post-translational modifications interact to control NR biology. We show that SUMO and ubiquitin circuitry coordinately modifies the pregnane X receptor (PXR, NR1I2) to play a key role in regulating PXR protein stability, transactivation capacity, and transcriptional repression.

Isolated RING2 domain of parkin is sufficient for E2-dependent E3 ligase activity.

The E3 ubiquitin ligase activity of the parkin protein is implicated in playing a protective role against neurodegenerative disorders including Parkinson's, Huntington's, and Alzheimer's diseases. Parkin has four zinc-containing domains: RING0, RING1, IBR (in-between ring), and RING2. Mutational analysis of full-length parkin suggests that the C-terminal RING2 domain contains the catalytic core.

Apoptosis in differentiating C2C12 muscle cells selectively targets Bcl-2-deficient myotubes.

Muscle cell apoptosis accompanies normal muscle development and regeneration, as well as degenerative diseases and aging. C2C12 murine myoblast cells represent a common model to study muscle differentiation. Though it was already shown that myogenic differentiation of C2C12 cells is accompanied by enhanced apoptosis in a fraction of cells, either the cell population sensitive to apoptosis or regulatory mechanisms for the apoptotic response are unclear so far.

Protein targets of thioacetamide metabolites in rat hepatocytes.

Thioacetamide (TA) has long been known as a hepatotoxicant whose bioactivation requires S-oxidation to thioacetamide S-oxide (TASO) and then to the very reactive S,S-dioxide (TASO2). The latter can tautomerize to form acylating species capable of covalently modifying cellular nucleophiles including phosphatidylethanolamine (PE) lipids and protein lysine side chains. Isolated hepatocytes efficiently oxidize TA to TASO but experience little covalent binding or cytotoxicity because TA is a very potent inhibitor of the oxidation of TASO to TASO2.

Decreases in plasma membrane Ca²⁺-ATPase in brain synaptic membrane rafts from aged rats.

Precise regulation of free intracellular Ca(2+) concentrations [Ca(2+) ](i) is critical for normal neuronal function, and alterations in Ca(2+) homeostasis are associated with brain aging and neurodegenerative diseases. One of the most important proteins controlling [Ca(2+) ](i) is the plasma membrane Ca(2+) -ATPase (PMCA), the high-affinity transporter that fine tunes the cytosolic nanomolar levels of Ca(2+) . We previously found that PMCA protein in synaptic plasma membranes (SPMs) is decreased with advancing age and the decrease in enzyme activity is much greater than that in protein levels.

Liver protein targets of hepatotoxic 4-bromophenol metabolites.

The hepatotoxicity of bromobenzene (BB) is directly related to the covalent binding of both initially formed epoxide and secondary quinone metabolites to at least 45 different liver proteins. 4-Bromophenol (4BP) is a significant BB metabolite and a precursor to reactive quinone metabolites; yet, when administered exogenously, it has negligible hepatotoxicity as compared to BB. The protein adducts of 4BP were thus labeled as nontoxic [Monks, T.

Covalent modification of lipids and proteins in rat hepatocytes and in vitro by thioacetamide metabolites.

Thioacetamide (TA) is a well-known hepatotoxin in rats. Acute doses cause centrilobular necrosis and hyperbilirubinemia while chronic administration leads to biliary hyperplasia and cholangiocarcinoma. Its acute toxicity requires its oxidation to a stable S-oxide (TASO) that is oxidized further to a highly reactive S,S-dioxide (TASO(2)).

Identification of protein targets of reactive metabolites of tienilic acid in human hepatocytes.

Tienilic acid (TA) is a uricosuric diuretic that was withdrawn from the market only months after its introduction because of reports of serious incidents of drug-induced liver injury including some fatalities. Its hepatotoxicity is considered to be primarily immunoallergic in nature. Like other thiophene compounds, TA undergoes biotransformation to a S-oxide metabolite which then reacts covalently with cellular proteins.

I-domain-antigen conjugate (IDAC) for delivering antigenic peptides to APC: synthesis, characterization, and in vivo EAE suppression.

The objectives of this work are to characterize the identity of I-domain-antigen conjugate (IDAC) and to evaluate the in vivo efficacy of IDAC in suppressing experimental autoimmune encephalomyelitis (EAE) in mouse model. The hypothesis is that the I-domain delivers PLP(139-151) peptides to antigen-presenting cells (APC) and alters the immune system by simultaneously binding to ICAM-1 and MHC-II, blocking immunological synapse formation. IDAC was synthesized by derivatizing the lysine residues with maleimide groups followed by conjugation with PLP-Cys-OH peptide.

Direct infusion mass spectrometry of oxylipin-containing Arabidopsis membrane lipids reveals varied patterns in different stress responses.

Direct infusion electrospray ionization triple quadrupole precursor scanning for three oxidized fatty acyl anions revealed 86 mass spectral peaks representing polar membrane lipids in extracts from Arabidopsis (Arabidopsis thaliana) infected with Pseudomonas syringae pv tomato DC3000 expressing AvrRpt2 (PstAvr). Quadrupole time-of-flight and Fourier transform ion cyclotron resonance mass spectrometry provided evidence for the presence of membrane lipids containing one or more oxidized acyl chains. The membrane lipids included molecular species of phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, digalactosyldiacylglycerol, monogalactosyldiacylglycerol, and acylated monogalactosyldiacylglycerol.

A methodology for simultaneous fluorogenic derivatization and boronate affinity enrichment of 3-nitrotyrosine-containing peptides.

We synthesized and characterized a new tagging reagent, (3R,4S)-1-(4-(aminomethyl)phenylsulfonyl)pyrrolidine-3,4-diol (APPD), for the selective fluorogenic derivatization of 3-nitrotyrosine (3-NT) residues in peptides (after reduction to 3-aminotyrosine) and affinity enrichment. The synthetic 3-NT-containing peptide, FSAY(3-NO(2))LER, was employed as a model for method validation. Furthermore, this derivatization protocol was successfully tested for analysis of 3-NT-containing proteins exposed to peroxynitrite in the total protein lysate of cultured C2C12 cells.

The host outer membrane proteins OmpA and OmpC are associated with the Shigella phage Sf6 virion.

Assembly of dsDNA bacteriophage is a precisely programmed process. Potential roles of host cell components in phage assembly haven't been well understood. It was previously reported that two unidentified proteins were present in bacteriophage Sf6 virion (Casjens et al, 2004, J.

Fluorogenic Tagging of Peptide and Protein 3-Nitrotyrosine with 4-(Aminomethyl)-benzenesulfonic Acid for Quantitative Analysis of Protein Tyrosine Nitration.

Protein 3-nitrotyrosine (3-NT) has been recognized as an important biomarker of nitroxidative stress associated with inflammatory and degenerative diseases, and biological aging. Analysis of protein-bound 3-NT continues to represent a challenge since in vivo it frequently does not accumulate on proteins in amounts detectable by quantitative analytical methods. Here, we describe a novel approach of fluorescent tagging and quantitation of peptide-bound 3-NT residues based on the selective reduction to 3-AT followed by reaction with 4-(amino-methyl)benzenesulfonic acid (ABS) in the presence of K(3)Fe(CN)(6) to form a highly fluorescent 2-phenylbenzoxazole product.

Inactivation of rabbit muscle glycogen phosphorylase b by peroxynitrite revisited: does the nitration of Tyr613 in the allosteric inhibition site control enzymatic function?

There is increasing evidence that sequence-specific formation of 3-nitrotyrosine (3-NT) may cause functional changes in target proteins. Recently, the nitration of Tyr residues in glycogen phosphorylase b (Ph-b) was implicated in the age-associated decline of protein function [Sharov et al., Exp.

Age-associated changes in synaptic lipid raft proteins revealed by two-dimensional fluorescence difference gel electrophoresis.

Brain aging is associated with a progressive decline in cognitive function though the molecular mechanisms remain unknown. Functional changes in brain neurons could be due to age-related alterations in levels of specific proteins critical for information processing. Specialized membrane microdomains known as 'lipid rafts' contain protein complexes involved in many signal transduction processes.

Protein targets of reactive metabolites of thiobenzamide in rat liver in vivo.

Thiobenzamide (TB) is a potent hepatotoxin in rats, causing dose-dependent hyperbilirubinemia, steatosis, and centrolobular necrosis. These effects arise subsequent to and appear to result from the covalent binding of the iminosulfinic acid metabolite of TB to cellular proteins and phosphatidylethanolamine lipids [ Ji et al. ( 2007) Chem.

High-affinity and cooperative binding of oxidized calmodulin by methionine sulfoxide reductase.

Methionines can play an important role in modulating protein-protein interactions associated with intracellular signaling, and their reversible oxidation to form methionine sulfoxides [Met(O)] in calmodulin (CaM) and other signaling proteins has been suggested to couple cellular redox changes to protein functional changes through the action of methionine sulfoxide reductases (Msr). Prior measurements indicate the full recovery of target protein activation upon the stereospecific reduction of oxidized CaM by MsrA, where the formation of the S-stereoisomer of Met(O) selectively inhibits the CaM-dependent activation of the Ca-ATPase. However, the physiological substrates of MsrA remain unclear, as neither the binding specificities nor affinities of protein targets have been measured.

Site-specific arylation of rat glutathione s-transferase A1 and A2 by bromobenzene metabolites in vivo.

The hepatotoxicity of bromobenzene (BB) derives from its reactive metabolites (epoxides and quinones), which arylate cellular proteins. Application of proteomic methods to liver proteins from rats treated with a hepatotoxic dose of [14C]-BB has identified more than 40 target proteins, but no adducted peptides have yet been observed. Because such proteins are known to contain bromophenyl- and bromodihydroxyphenyl derivatives of cysteine, histidine, and lysine, the failure to observe modified peptides has been attributed to the low level of total covalent binding and to the "dilution" effect of multiple metabolites reacting at multiple sites on multiple proteins.

Characterization of the metal-binding site of human prolactin by site-specific metal-catalyzed oxidation.

Site-specific metal-catalyzed oxidation (MCO) was applied to characterize the metal-binding site (MBS) of recombinant human prolactin (hPRL), which belongs to the hematopoietic cytokine family. Copper and ascorbate of various concentrations were used to initiate the oxidation of hPRL, and the oxidation-sensitive motifs were characterized and quantitated by mass spectrometry. Based on the results obtained with 10 microM Cu(2+) and 0.

Tertiary structural rearrangements upon oxidation of Methionine145 in calmodulin promotes targeted proteasomal degradation.

The selectivity underlying the recognition of oxidized calmodulin (CaM) by the 20S proteasome in complex with Hsp90 was identified using mass spectrometry. We find that degradation of oxidized CaM (CaMox) occurs in a multistep process, which involves an initial cleavage that releases a large N-terminal fragment (A1-F92) as well as multiple smaller carboxyl-terminus peptides ranging from 17 to 26 amino acids in length. These latter small peptides are enriched in methionine sulfoxides (MetO), suggesting a preferential degradation around MetO within the carboxyl-terminal domain.

Age-associated tyrosine nitration of rat skeletal muscle glycogen phosphorylase b: characterization by HPLC-nanoelectrospray-tandem mass spectrometry.

We identified age-dependent post-translational modifications of skeletal muscle glycogen phosphorylase b (Ph-b), isolated from F1 hybrids of Fisher 344 x Brown Norway rats. Ph-b isolated from 34 months old rats showed a statistically significant decrease in specific activity compared to 6 months old animals: 13.8+/-0.