Functional Pdgfra fibroblast heterogeneity in normal and fibrotic mouse lung.

Aberrant fibroblast function plays a key role in the pathogenesis of idiopathic pulmonary fibrosis, a devastating disease of unrelenting extracellular matrix deposition in response to lung injury. Platelet-derived growth factor α-positive (Pdgfra+) lipofibroblasts (LipoFBs) are essential for lung injury response and maintenance of a functional alveolar stem cell niche. Little is known about the effects of lung injury on LipoFB function. Here, we used single-cell RNA-Seq (scRNA-Seq) technology and PdgfraGFP lineage tracing to generate a transcriptomic profile of Pdgfra+ fibroblasts in normal and injured mouse lungs 14 days after bleomycin exposure, generating 11 unique transcriptomic clusters that segregated according to treatment. While normal and injured LipoFBs shared a common gene signature, injured LipoFBs acquired fibrogenic pathway activity with an attenuation of lipogenic pathways. In a 3D organoid model, injured Pdgfra+ fibroblast-supported organoids were morphologically distinct from those cultured with normal fibroblasts, and scRNA-Seq analysis suggested distinct transcriptomic changes in alveolar epithelia supported by injured Pdgfra+ fibroblasts. In summary, while LipoFBs in injured lung have not migrated from their niche and retain their lipogenic identity, they acquire a potentially reversible fibrogenic profile, which may alter the kinetics of epithelial regeneration and potentially contribute to dysregulated repair, leading to fibrosis.

Cryo-EM reveals the architecture of the PELP1-WDR18 molecular scaffold.

PELP1 (Proline-, Glutamic acid-, Leucine-rich protein 1) is a large scaffolding protein that functions in many cellular pathways including steroid receptor (SR) coactivation, heterochromatin maintenance, and ribosome biogenesis. PELP1 is a proto-oncogene whose expression is upregulated in many human cancers, but how the PELP1 scaffold coordinates its diverse cellular functions is poorly understood. Here we show that PELP1 serves as the central scaffold for the human Rix1 complex whose members include WDR18, TEX10, and SENP3. We reconstitute the mammalian Rix1 complex and identified a stable sub-complex comprised of the conserved PELP1 Rix1 domain and WDR18. We determine a 2.7 Å cryo-EM structure of the subcomplex revealing an interconnected tetrameric assembly and the architecture of PELP1's signaling motifs, including eleven LxxLL motifs previously implicated in SR signaling and coactivation of Estrogen Receptor alpha (ERα) mediated transcription. However, the structure shows that none of these motifs is in a conformation that would support SR binding. Together this work establishes that PELP1 scaffolds the Rix1 complex, and association with WDR18 may direct PELP1's activity away from SR coactivation.

Serum Buprenorphine Concentrations and Behavioral Activity in Mice After a Single Subcutaneous Injection of Simbadol, Buprenorphine SR-LAB, or Standard Buprenorphine.

Buprenorphine, an analgesic commonly used in rodent surgery, requires repeated dosing every 4 to 6 h in order to provide adequate analgesia. However, redosing requires repeated handling, which may itself cause stress. Buprenorphine SR-LAB, which reportedly maintains serum levels of buprenorphine greater than 1 ng/mL for 48 to 72 h, is commercially available. However, the viscosity of the product and small dosing volumes make accurate dosing a challenge. Simbadol is a concentrated formulation of buprenorphine hydrochloride labeled for use in cats with recommended dosing frequency of every 24 h. We measured serum concentrations over time after a single injection of this product in C57BL/6NCrl mice and compared it to standard buprenorphine (Buprenex) and Buprenorphine SR-LAB. Male and female mice were injected subcutaneously with one of the 3 buprenorphine formulations at a dose of 1 mg/kg at time 0. Groups of mice (n = 8) were euthanized at 1, 4, 8, 12, 16 h for all groups and 24 h for the Simbadol and the Buprenorphine SR-LAB. Liquid chromatography-mass spectrometry (LC-MS/MS) was used to determine concentrations of buprenorphine in each serum sample. High concentrations were observed in both Simbadol and standard buprenorphine groups one hour after injection (>50 ng/mL). These groups had similar buprenorphine concentration curves, including rates of decline. The standard buprenorphine group had mean concentrations less than 1 ng/mL by 12 h and the Simbadol group by 16 h. In contrast, the Buprenorphine SR-LAB group remained above the 1 ng/mL therapeutic threshold throughout the 24 h. In addition, clinical signs, including increased activity, that lasted for up to an hour after the injection in the Simbadol and standard buprenorphine groups. We conclude that Simbadol does not offer dosing advantages over the standard buprenorphine formulation when given at 1 mg/kg. Buprenorphine SR-LAB maintained a steady concentration of buprenorphine above 1 ng/mL for at least 24 h, and as such is a superior choice for providing long-term analgesia.

HNF4α regulates sulfur amino acid metabolism and confers sensitivity to methionine restriction in liver cancer.

Methionine restriction, a dietary regimen that protects against metabolic diseases and aging, represses cancer growth and improves cancer therapy. However, the response of different cancer cells to this nutritional manipulation is highly variable, and the molecular determinants of this heterogeneity remain poorly understood. Here we report that hepatocyte nuclear factor 4α (HNF4α) dictates the sensitivity of liver cancer to methionine restriction. We show that hepatic sulfur amino acid (SAA) metabolism is under transcriptional control of HNF4α. Knocking down HNF4α or SAA enzymes in HNF4α-positive epithelial liver cancer lines impairs SAA metabolism, increases resistance to methionine restriction or sorafenib, promotes epithelial-mesenchymal transition, and induces cell migration. Conversely, genetic or metabolic restoration of the transsulfuration pathway in SAA metabolism significantly alleviates the outcomes induced by HNF4α deficiency in liver cancer cells. Our study identifies HNF4α as a regulator of hepatic SAA metabolism that regulates the sensitivity of liver cancer to methionine restriction.

Bacteria Boost Mammalian Host NAD Metabolism by Engaging the Deamidated Biosynthesis Pathway.

Nicotinamide adenine dinucleotide (NAD), a cofactor for hundreds of metabolic reactions in all cell types, plays an essential role in metabolism, DNA repair, and aging. However, how NAD metabolism is impacted by the environment remains unclear. Here, we report an unexpected trans-kingdom cooperation between bacteria and mammalian cells wherein bacteria contribute to host NAD biosynthesis. Bacteria confer resistance to inhibitors of NAMPT, the rate-limiting enzyme in the amidated NAD salvage pathway, in cancer cells and xenograft tumors. Mechanistically, a microbial nicotinamidase (PncA) that converts nicotinamide to nicotinic acid, a precursor in the alternative deamidated NAD salvage pathway, is necessary and sufficient for this protective effect. Using stable isotope tracing and microbiota-depleted mice, we demonstrate that this bacteria-mediated deamidation contributes substantially to the NAD-boosting effect of oral nicotinamide and nicotinamide riboside supplementation in several tissues. Collectively, our findings reveal an important role of bacteria-enabled deamidated pathway in host NAD metabolism.

An obesity-associated gut microbiome reprograms the intestinal epigenome and leads to altered colonic gene expression.

BACKGROUND: The gut microbiome, a key constituent of the colonic environment, has been implicated as an important modulator of human health. The eukaryotic epigenome is postulated to respond to environmental stimuli through alterations in chromatin features and, ultimately, gene expression. How the host mediates epigenomic responses to gut microbiota is an emerging area of interest. Here, we profile the gut microbiome and chromatin characteristics in colon epithelium from mice fed either an obesogenic or control diet, followed by an analysis of the resultant changes in gene expression. RESULTS: The obesogenic diet shapes the microbiome prior to the development of obesity, leading to altered bacterial metabolite production which predisposes the host to obesity. This microbiota-diet interaction leads to changes in histone modification at active enhancers that are enriched for binding sites for signal responsive transcription factors. These alterations of histone methylation and acetylation are associated with signaling pathways integral to the development of colon cancer. The transplantation of obesogenic diet-conditioned microbiota into germ free mice, combined with an obesogenic diet, recapitulates the features of the long-term diet regimen. The diet/microbiome-dependent changes are reflected in both the composition of the recipient animals' microbiome as well as in the set of transcription factor motifs identified at diet-influenced enhancers. CONCLUSIONS: These findings suggest that the gut microbiome, under specific dietary exposures, stimulates a reprogramming of the enhancer landscape in the colon, with downstream effects on transcription factors. These chromatin changes may be associated with those seen during colon cancer development.

Elevated plasma 8-iso-prostaglandin F2α levels in human smokers originate primarily from enzymatic instead of non-enzymatic lipid peroxidation.

It is widely accepted that free radicals in tobacco smoke lead to oxidative stress and generate the popular lipid peroxidation biomarker 8-iso-prostaglandin F2α (8-iso-PGF2α). However, 8-iso-PGF2α can simultaneously be produced in vivo by the prostaglandin-endoperoxide synthases (PGHS) induced by inflammation. This inflammation-dependent mechanism has never been considered as a source of elevated 8-iso-PGF2α in tobacco smokers. The goal of this study is to quantify the distribution of chemical- and PGHS-dependent 8-iso-PGF2α formation in the plasma of tobacco smokers and non-smokers. The influences of gender and hormonal contraceptive use were accounted for. The distribution was determined by measuring the 8-iso-PGF2α/prostaglandin F2α (PGF2α) ratio. When comparing smokers (n = 28) against non-smokers (n = 30), there was a statistically significant increase in the 8-iso-PGF2α concentration. The source of this increased 8-iso-PGF2α was primarily from PGHS. When stratifying for gender, the increase in 8-iso-PGF2α in male smokers (n = 9) was primarily from PGHS. Interestingly, female smokers on hormonal contraceptives had increased 8-iso-PGF2α in both pathways, whereas those not on hormonal contraceptives did not have increased 8-iso-PGF2α. In conclusion, increased plasma 8-iso-PGF2α in tobacco smokers has complex origins, with PGHS-dependent formation as the primary source. Accounting for both pathways provides a definitive measurement of both oxidative stress and inflammation.

Tripping up Trp: Modification of protein tryptophan residues by reactive oxygen species, modes of detection, and biological consequences.

Proteins comprise a majority of the dry weight of a cell, rendering them a major target for oxidative modification. Oxidation of proteins can result in significant alterations in protein molecular mass such as breakage of the polypeptide backbone and/or polymerization of monomers into dimers, multimers, and sometimes insoluble aggregates. Protein oxidation can also result in structural changes to amino acid residue side chains, conversions that have only a modest effect on protein size but can have widespread consequences for protein function. There are a wide range of rate constants for amino acid reactivity, with cysteine, methionine, tyrosine, phenylalanine, and tryptophan having the highest rate constants with commonly encountered biological oxidants. Free tryptophan and tryptophan protein residues react at a diffusion-limited rate with hydroxyl radical and also have high rate constants for reactions with singlet oxygen and ozone. Although oxidation of proteins in general and tryptophan residues specifically can have effects detrimental to the health of cells and organisms, some modifications are neutral, whereas others contribute to the function of the protein in question or may act as a signal that damaged proteins need to be replaced. This review provides a brief overview of the chemical mechanisms by which tryptophan residues become oxidized, presents both the strengths and the weaknesses of some of the techniques used to detect these oxidative interactions, and discusses selected examples of the biological consequences of tryptophan oxidation in proteins from animals, plants, and microbes.

Free radical-operated proteotoxic stress in macrophages primed with lipopolysaccharide.

The free-radical-operated mechanism of death of activated macrophages at sites of inflammation is unclear, but it is important to define it in order to find targets to prevent further tissue dysfunction. A well-defined model of macrophage activation at sites of inflammation is the treatment of RAW 264.7 cells with lipopolysaccharide (LPS), with the resulting production of reactive oxygen species (ROS). ROS and other free radicals can be trapped with the nitrone spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO), a cell-permeable probe with antioxidant properties, which thus interferes with free-radical-operated oxidation processes. Here we have used immuno-spin trapping to investigate the role of free-radical-operated protein oxidation in LPS-induced cytotoxicity in macrophages. Treatment of RAW 264.7 cells with LPS resulted in increased ROS production, oxidation of proteins, cell morphological changes and cytotoxicity. DMPO was found to trap protein radicals to form protein-DMPO nitrone adducts, to reduce protein carbonyls, and to block LPS-induced cell death. N-Acetylcysteine (a source of reduced glutathione), diphenyleneiodonium (an inhibitor of NADPH oxidase), and 2,2'-dipyridyl (a chelator of Fe(2+)) prevented LPS-induced oxidative stress and cell death and reduced DMPO-nitrone adduct formation, suggesting a critical role of ROS, metals, and protein-radical formation in LPS-induced cell cytotoxicity. We also determined the subcellular localization of protein-DMPO nitrone adducts and identified some candidate proteins for DMPO attachment by LC-MS/MS. The LC-MS/MS data are consistent with glyceraldehyde-3-phosphate dehydrogenase, one of the most abundant, sensitive, and ubiquitous proteins in the cell, becoming labeled with DMPO when the cell is primed with LPS. This information will help find strategies to treat inflammation-associated tissue dysfunction by focusing on preventing free radical-operated proteotoxic stress and death of macrophages.

CD34 Antigen: Determination of Specific Sites of Phosphorylation In Vitro and In Vivo.

CD34, a type I transmembrane glycoprotein, is a surface antigen which is expressed on several cell types, including hematopoietic progenitors, endothelial cells, as well as mast cells. Recently, CD34 has been described as a marker for epidermal stem cells in mouse hair follicles, and is expressed in outer root sheath cells of the human hair follicle. Although the biological function and regulation of CD34 is not well understood, it is thought to be involved in cell adhesion as well as possibly having a role in signal transduction. In addition, CD34 was shown to be critical for skin tumor development in mice, although the exact mechanism remains unknown.Many proteins' functions and biological activities are regulated through post-translational modifications. The extracellular domain of CD34 is heavily glycosylated but the role of these glycans in CD34 function is unknown. Additionally, two sites of tyrosine phosphorylation have been reported on human CD34 and it is known that CD34 is phosphorylated, at least in part, by protein kinase C; however, the precise location of the sites of phosphorylation has not been reported. In an effort to identify specific phosphorylation sites in CD34 and delineate the possible role of protein kinase C, we undertook the identification of the in vitro sites of phosphorylation on the intracellular domain of mouse CD34 (aa 309-382) following PKC treatment. For this work, we are using a combination of enzymatic proteolysis and peptide sequencing by mass spectrometry. After which the in vivo sites of phosphorylation of full-length mouse CD34 expressed from HEK293F cells were determined. The observed in vivo sites of phosphorylation, however, are not consensus PKC sites, but our data indicate that one of these sites may possibly be phosphorylated by AKT2. These results suggest that other kinases, as well as PKC, may have important signaling functions in CD34.

Site-specific radical formation in DNA induced by Cu(II)-H2O2 oxidizing system, using ESR, immuno-spin trapping, LC-MS, and MS/MS.

Oxidative stress-related damage to the DNA macromolecule produces a multitude of lesions that are implicated in mutagenesis, carcinogenesis, reproductive cell death, and aging. Many of these lesions have been studied and characterized by various techniques. Of the techniques that are available, the comet assay, HPLC-EC, GC-MS, HPLC-MS, and especially HPLC-MS/MS remain the most widely used and have provided invaluable information on these lesions. However, accurate measurement of DNA damage has been a matter of debate. In particular, there have been reports of artifactual oxidation leading to erroneously high damage estimates. Further, most of these techniques measure the end product of a sequence of events and thus provide only limited information on the initial radical mechanism. We report here a qualitative measurement of DNA damage induced by a Cu(II)-H2O2 oxidizing system using immuno-spin trapping (IST) with electron paramagnetic resonance (EPR), MS, and MS/MS. The radical generated is trapped by DMPO immediately upon formation. The DMPO adduct formed is initially EPR active but subsequently is oxidized to the stable nitrone, which can then be detected by IST and further characterized by MS and MS/MS.

Structural characterization of the conformational change in calbindin-D28k upon calcium binding using differential surface modification analyzed by mass spectrometry.

Calbindin-D28k is a calcium binding protein with six EF hand domains. Calbindin-D28k is unique in that it functions as both a calcium buffer and a sensor protein. It is found in many tissues, including brain, pancreas, kidney, and intestine, playing important roles in each. Calbindin-D28k is known to bind four calcium ions and upon calcium binding undergoes a conformational change. The structure of apo calbindin-D28k is in an ordered state, transitioning into a disordered state as calcium is bound. Once fully loaded with four calcium ions, it again takes on an ordered state. The solution structure of disulfide-reduced holo-calbindin-D28k has been determined by NMR, while the structure of apo calbindin-D28k has yet to be determined. Differential surface modification of lysine and histidine residues analyzed by mass spectrometry has been used in this study to identify, for the first time, the specific regions of calbindin-D28k undergoing conformational changes between the holo and apo states. Using differential surface modification in combination with mass spectrometry, EF hands 1 and 4 as well as the linkers before EF hand 1 and the linkers between EF hands 4 and 5 and EF hands 5 and 6 were identified as regions of conformational change between apo and holo calbindin-D28k. Under the experimental conditions employed, EF hands 2 and 6, which are known not to bind calcium, were unaffected in either form. EF hand 2 is highly accessible; however, EF hand 6 was determined not to be surface accessible in either form. Previous research has identified a disulfide bond between cysteines 94 and 100 in the holo state. Until now, it was unknown whether this bond also exists in the apo form. Our data confirm the presence of the disulfide bond between cysteines 94 and 100 in the holo form and indicate that there is predominantly no disulfide bond between these residues in the apoprotein.

Glycosylation profiles of epitope-specific anti-beta-amyloid antibodies revealed by liquid chromatography-mass spectrometry.

Alzheimer's disease (AD) is the most prevalent form of age-related neurodementia. The accumulation of beta-amyloid polypeptide (Abeta) in brain is generally believed to be a key event in AD. The recent discovery of physiological beta-amyloid autoantibodies represents a promising perspective for treatment and early diagnosis of AD. The mechanisms by which natural beta-amyloid autoantibodies prevent neurodegeneration are currently unknown. The aim of the present study was to analyze the N-linked glycosylation of a plaque-specific, monoclonal antibody (clone 6E10) relevant for immunotherapy of AD, in comparison with the glycosylation pattern of an Abeta autoantibody isolated from an IgG source. Liquid chromatography in combination with tandem mass spectrometry was used to analyze the glycopeptides generated by enzymatic degradation of the antibodies reduced and alkylated heavy chains. The oligosaccharide pattern of the 6E10 antibody shows primarily core-fucosylated biantennary complex structures and, to a low extent, tri- and tetragalactosyl glycoforms, with or without terminal sialic acids. The glycans associated with the serum anti-Abeta autoantibodies are of the complex, biantennary-type, fucosylated at the first N-acetyl glucosamine residue of the trimannosyl chitobiose core and contain zero to two galactose residues, and zero to one terminal sialic acid, with or without bisecting N-acetyl glucosamine. Glycosylation analysis of the Abeta-autoantibody performed at the peptide level revealed all four human IgG subclasses, with IgG(1) and IgG(2) as the dominant subclasses.

Immuno-spin trapping of protein and DNA radicals: "tagging" free radicals to locate and understand the redox process.

Biomolecule-centered radicals are intermediate species produced during both reversible (redox modulation) and irreversible (oxidative stress) oxidative modification of biomolecules. These oxidative processes must be studied in situ and in real time to understand the molecular mechanism of cell adaptation or death in response to changes in the extracellular environment. In this regard, we have developed and validated immuno-spin trapping to tag the redox process, tracing the oxidatively generated modification of biomolecules, in situ and in real time, by detecting protein- and DNA-centered radicals. The purpose of this methods article is to introduce and update the basic methods and applications of immuno-spin trapping for the study of redox biochemistry in oxidative stress and redox regulation. We describe in detail the production, detection, and location of protein and DNA radicals in biochemical systems, cells, and tissues, and in the whole animal as well, by using immuno-spin trapping with the nitrone spin trap 5,5-dimethyl-1-pyrroline N-oxide.

Immuno-spin trapping of a post-translational carboxypeptidase B1 radical formed by a dual role of xanthine oxidase and endothelial nitric oxide synthase in acute septic mice.

Post-translational modification of proteins due to exposure to radicals and other reactive species are markers of metabolic and inflammatory oxidative stress such as sepsis. This study uses the nitrone spin-trap DMPO and a combination of immuno-spin trapping and mass spectrometry to identify in vivo products of radical reactions in mice. We report the detection of dose-dependent production of DMPO-carboxypeptidase B1 (CPB1) adducts in the spleens of mice treated with lipopolysaccharide (LPS). Additionally, we report significant detection of DMPO-CPB1 adducts in mice experiencing normal physiological conditions. Treatments with inhibitors and experiments with knock-out mice indicate that xanthine oxidase and endothelial nitric oxide synthase are important sources of the reactive species that lead to CPB1 adduct formation. We also report a significant loss of CPB1 activity following LPS challenge in conjunction with an increase in CPB1 protein accumulation. This suggests the presence of a possible mechanism for CPB1 activity loss with compensatory protein production.

Procainamide, but not N-acetylprocainamide, induces protein free radical formation on myeloperoxidase: a potential mechanism of agranulocytosis.

Procainamide (PA) is a drug that is used to treat tachycardia in postoperative patients or for long-term maintenance of cardiac arrythmias. Unfortunately, its use has also been associated with agranulocytosis. Here, we have investigated the metabolism of PA by myeloperoxidase (MPO) and the formation of an MPO protein free radical. We hypothesized that PA oxidation by MPO/H 2O 2 would produce a PA cation radical that, in the absence of a biochemical reductant, would lead to the free radical oxidation of MPO. We utilized a novel anti-DMPO antibody to detect DMPO (5,5-dimethyl-1-pyrroline N-oxide) covalently bound to protein, which forms by the reaction of DMPO with a protein free radical. We found that PA metabolism by MPO/H 2O 2 induced the formation of DMPO-MPO, which was inhibited by MPO inhibitors and ascorbate. N-acetyl-PA did not cause DMPO-MPO formation, indicating that the unsubstituted aromatic amine was more oxidizable. PA had a lower calculated ionization potential than N-acetyl-PA. The DMPO adducts of MPO metabolism, as analyzed by electron spin resonance spectroscopy, included a nitrogen-centered radical and a phenyl radical derived from PA, either of which may be involved in the free radical formation on MPO. Furthermore, we also found protein-DMPO adducts in MPO-containing, intact human promyelocytic leukemia cells (HL-60). MPO was affinity-purified from HL-60 cells treated with PA/H 2O 2 and was found to contain DMPO using the anti-DMPO antibody. Mass spectrometry analysis confirmed the identity of the protein as human MPO. These findings were also supported by the detection of protein free radicals with electron spin resonance in the cellular cytosolic lysate. The formation of an MPO protein free radical is believed to be mediated by free radical metabolites of PA, which we characterized by spin trapping. We propose that drug-induced free radical formation on MPO may play a role in the origin of agranulocytosis.

Global changes in and characterization of specific sites of phosphorylation in mouse and human histone H1 Isoforms upon CDK inhibitor treatment using mass spectrometry.

Global changes in the phosphorylation state of human H1 isoforms isolated from UL3 cells have been investigated using mass spectrometry. Relative changes in H1 phosphorylation between untreated cells and cells treated with dexamethasone or various CDK inhibitors were determined. The specific cyclin-dependent kinase consensus sites of phosphorylation on the histone H1 isoforms that show changes in phosphorylation were also investigated. Three sites of phosphorylation on histone H1.4 isoforms have been identified.

Phosphorylation site analysis of the anti-inflammatory and mRNA-destabilizing protein tristetraprolin.

Tristetraprolin (TTP) is a member of the CCCH zinc finger proteins and is an anti-inflammatory protein. Mice deficient in TTP develop a profound inflammatory syndrome with erosive arthritis, autoimmunity and myeloid hyperplasia. TTP binds to mRNA AU-rich elements with high affinity for UUAUUUAUU nucleotides and causes destabilization of those mRNA molecules. TTP is phosphorylated extensively in vivo and is a substrate for multiple protein kinases in vitro. A number of approaches have been used to identify its phosphorylation sites. This article highlights the recent progress and different approaches utilized for the identification of phosphorylation sites in mammalian TTP. Important but limited results are obtained using traditional methods, including in vivo labeling, site-directed mutagenesis, phosphopeptide mapping and protein sequencing. Mass spectrometry (MS), including MALDI/MS, MALDI/MS/MS, liquid chromatography/MS/MS, immobilized metal ion affinity chromatography (IMAC)/MALDI/MS/MS and multidimensional protein identification technology has led the way in identifying TTP phosphorylation sites. The combination of these approaches has identified multiple phosphorylation sites in mammalian TTP, some of which are predicted by motif scanning to be phosphorylated by several protein kinases. This information should provide the molecular basis for future investigation of TTP's regulatory functions in controlling proinflammatory cytokines.

Electron transfer between a tyrosyl radical and a cysteine residue in hemoproteins: spin trapping analysis.

We investigated electron transfer between a tyrosyl radical and cysteine residue in two systems, oxyhemoglobin (oxyHb)/peroxynitrite/5,5-dimethyl-1-pyrroline N-oxide (DMPO) and myoglobin (Mb)/hydrogen peroxide/DMPO, using a combination of techniques including ESR, immuno-spin trapping (IST), and ESI/MS. These techniques show that the nitrone spin trap DMPO covalently binds to one or more amino acid radicals in the protein. Treating oxyHb with peroxynitrite and Mb with H2O2 in the presence of a low DMPO concentration yielded secondary Cys-DMPO radical adduct exclusively, whereas in the presence of high DMPO, more of the primary Tyr-DMPO radical adduct was detected. In both systems studied, we found that, at high DMPO concentrations, mainly tyrosyl radicals (Hb-Tyr42/Tyr24 and Mb-Tyr103) are trapped and the secondary electron-transfer reaction does not compete, whereas in the presence of low concentrations of DMPO, the secondary reaction predominates over tyrosyl trapping, and a thiyl radical is formed and then trapped (Hb-Cys93 or Mb-Cys110). With increasing concentrations of DMPO in the reaction medium, primary radicals have an increasing probability of being trapped. MS/MS was used to identify the specific Tyr and Cys residues forming radicals in the myoglobin system. All data obtained from this combination of approaches support the conclusion that the initial site of radical formation is a Tyr, which then abstracts an electron from a cysteine residue to produce a cysteinyl radical. This complex phenomenon of electron transfer from one radical to another has been investigated in proteins by IST, ESR, and MS.

Top-down and bottom-up mass spectrometric characterization of human myoglobin-centered free radicals induced by oxidative damage.

In an effort to determine the utility of top-down mass spectrometric methodologies for the characterization of protein radical adducts, top-down approaches were investigated and compared to the traditional bottom-up approaches. Specifically, the nature of the radicals on human myoglobin induced by the addition of hydrogen peroxide and captured by the spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO) was investigated. The most abundant ion observed in the electrospray mass spectrum of this reaction mixture corresponds in mass to the human myoglobin plus one DMPO molecule. In addition, a second ion of lower abundance is observed, which corresponds to a second DMPO molecule being trapped on myoglobin. Top-down analyses using Fourier transform ion cyclotron resonance mass spectrometry can be used to characterize proteins and, thus, were performed on several different charge-state ions of both the native and the mono-DMPO nitrone adduct of human myoglobin. Data produced from the top-down analyses are very complex yet information rich. In the case of DMPO-modified human myoglobin, the top-down data localized the DMPO spin trap to residues 97-110 of the myoglobin. The observation of the y43+5 fragment ion arising from C-terminal cleavage to the cysteine-110 residue in the MS/MS spectrum of DMPO-modified myoglobin and not in the unmodified myoglobin implicates a change to this residue, specifically, DMPO adduction. On the other hand, using the traditional bottom-up approach of peptide mapping and MS sequencing methodologies, two DMPO radical adducts on human myoglobin were identified, Cys-110 and Tyr-103. The bottom-up approach is more proven and robust than the top-down methodologies. Nonetheless, the bottom-up and top-down approaches to protein characterization are complementary rather than competitive approaches with each having its own utility.