Section on Molecular Medicine

Wake Forest, NC, United States

Section on Molecular Medicine

Wake Forest, NC, United States
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Reisz J.A.,Section on Molecular Medicine | Bechtold E.,Wake forest University | Bechtold E.,Tufts University | King S.B.,Wake forest University | Furdui C.M.,Section on Molecular Medicine
FEBS Journal | Year: 2013

Cellular exposure to reactive oxygen species induces rapid oxidation of DNA, proteins, lipids and other biomolecules. At the proteome level, cysteine thiol oxidation is a prominent post-translational process that is implicated in normal physiology and numerous pathologies. Methods for investigating protein oxidation include direct labeling with selective chemical probes and indirect tag-switch techniques. Common to both approaches is chemical blocking of free thiols using reactive electrophiles to prevent post-lysis oxidation or other thiol-mediated cross-reactions. These reagents are used in large excess, and their reactivity with cysteine sulfenic acid, a critical oxoform in numerous proteins, has not been investigated. Here we report the reactivity of three thiol-blocking electrophiles, iodoacetamide, N-ethylmaleimide and methyl methanethiosulfonate, with protein sulfenic acid and dimedone, the structural core of many sulfenic acid probes. We demonstrate that covalent cysteine -SOR (product) species are partially or fully susceptible to reduction by dithiothreitol, tris(2-carboxyethyl)phosphine and ascorbate, regenerating protein thiols, or, in the case of ascorbate, more highly oxidized species. The implications of this reactivity on detection methods for protein sulfenic acids and S-nitrosothiols are discussed. © 2013 FEBS.

Cao Q.,Georgia State University | Cui X.,Georgia State University | Wu R.,Georgia State University | Zha L.,Georgia State University | And 4 more authors.
Diabetes | Year: 2016

Macrophage inflammation marks all stages of atherogenesis, and AMPK is a regulator of macrophage inflammation. We therefore generated myeloid α1AMPK knockout (MAKO) mice on the LDL receptor knockout (LDLRKO) background to investigate whether myeloid deletion of α1AMPK exacerbates atherosclerosis. When fed an atherogenic diet, MAKO/LDLRKO mice displayed exacerbated atherosclerosis compared with LDLRKO mice. To determine the underlying pathophysiological pathways, we characterized macrophage inflammation/chemotaxis and lipid/cholesterol metabolism in MAKO/LDLRKO mice. Myeloid deletion of a1AMPK increased macrophage inflammatory gene expression and enhanced macrophage migration and adhesion to endothelial cells. Remarkably, MAKO/LDLRKO mice also displayed higher composition of circulating chemotaxically active Ly-6Chigh monocytes, enhanced atherosclerotic plaque chemokine expression, and monocyte recruitment into plaques, leading to increased atherosclerotic plaque macrophage content and inflammation. MAKO/LDLRKO mice also exhibited higher plasma LDL and VLDL cholesterol content, increased circulating apolipoprotein B (apoB) levels, and higher liver apoB expression. We conclude that macrophage a1AMPK deficiency promotes atherogenesis in LDLRKO mice and is associated with enhanced macrophage inflammation and hypercholesterolemia and that macrophage α1AMPK may serve as a therapeutic target for prevention and treatment of atherosclerosis. © 2016 by the American Diabetes Association.

Pollard R.D.,Section on Molecular Medicine | Sorci-Thomas M.G.,Medical College of Wisconsin | Thomas M.J.,Medical College of Wisconsin
Biochemistry | Year: 2016

The first step in removing cholesterol from a cell is the ATP-binding cassette transporter 1 (ABCA1)-driven transfer of cholesterol to lipid-free or lipid-poor apolipoprotein A-I (apoA-I), which yields cholesterol-rich nascent high-density lipoprotein (nHDL) that then matures in plasma to spherical, cholesteryl ester-rich HDL. However, lipid-free apoA-I has a three-dimensional (3D) conformation that is significantly different from that of lipidated apoA-I on nHDL. By comparing the lipid-free apoA-I 3D conformation of apoA-I to that of 9-14 nm diameter nHDL, we formulated the hypothetical helical domain transitions that might drive particle formation. To test the hypothesis, ten apoA-I mutants were prepared that contained two strategically placed cysteines several of which could form intramolecular disulfide bonds and others that could not form these bonds. Mass spectrometry was used to identify amino acid sequence and intramolecular disulfide bond formation. Recombinant HDL (rHDL) formation was assessed with this group of apoA-I mutants. ABCA1-driven nHDL formation was measured in four mutants and wild-type apoA-I. The mutants contained cysteine substitutions in one of three regions: the N-terminus, amino acids 34 and 55 (E34C to S55C), central domain amino acids 104 and 162 (F104C to H162C), and the C-terminus, amino acids 200 and 233 (L200C to L233C). Mutants were studied in the locked form, with an intramolecular disulfide bond present, or unlocked form, with the cysteine thiol blocked by alkylation. Only small amounts of rHDL or nHDL were formed upon locking the central domain. We conclude that both the N- and C-terminal ends assist in the initial steps in lipid acquisition, but that opening of the central domain was essential for particle formation. © 2016 American Chemical Society.

McCall C.E.,Wake forest University | Yoza B.,Section on Molecular Medicine | Yoza B.,Wake forest University | Liu T.,Section on Molecular Medicine | El Gazzar M.,Section on Molecular Medicine
Journal of Innate Immunity | Year: 2010

Inflammation is a fundamental biologic process that is evolutionally conserved by a germ line code. The interplay between epigenetics and environment directs the code into temporally distinct inflammatory responses, which can be acute or chronic. Here, we discuss the epigenetic processes of innate immune cells during serious infections with systemic inflammation in four stages: homeostasis, incitement, evolution, and resolution. We describe feed-forward loops of serious infections with systemic inflammation that create gene-specific silent facultative heterochromatin and active euchromatin according to gene function, and speculate on the role of epigenetics in survival. © 2010 S. Karger AG, Basel.

Furdui C.M.,Section on Molecular Medicine | Poole L.B.,Medical Center Boulevard
Mass Spectrometry Reviews | Year: 2014

Orchestration of many processes relying on intracellular signal transduction is recognized to require the generation of hydrogen peroxide as a second messenger, yet relatively few molecular details of how this oxidant acts to regulate protein function are currently understood. This review describes emerging chemical tools and approaches that can be applied to study protein oxidation in biological systems, with a particular emphasis on a key player in protein redox regulation, cysteine sulfenic acid. While sulfenic acids (within purified proteins or simple mixtures) are detectable by physical approaches like X-ray crystallography, nuclear magnetic resonance and mass spectrometry, the propensity of these moieties to undergo further modification in complex biological systems has necessitated the development of chemical probes, reporter groups and analytical approaches to allow for their selective detection and quantification. Provided is an overview of techniques that are currently available for the study of sulfenic acids, and some of the biologically meaningful data that have been collected using such approaches. © 2013 Wiley Periodicals, Inc.

Devarie-Baez N.O.,Section on Molecular Medicine | Lopez E.I.S.,Section on Molecular Medicine | Furdui C.M.,Section on Molecular Medicine
Free Radical Research | Year: 2016

Selective modification of proteins at cysteine residues by reactive oxygen, nitrogen or sulfur species formed under physiological and pathological states is emerging as a critical regulator of protein activity impacting cellular function. This review focuses primarily on protein sulfenylation (-SOH), a metastable reversible modification connecting reduced cysteine thiols to many products of cysteine oxidation. An overview is first provided on the chemistry principles underlining synthesis, stability and reactivity of sulfenic acids in model compounds and proteins, followed by a brief description of analytical methods currently employed to characterize these oxidative species. The following chapters present a selection of redox-regulated proteins for which the -SOH formation was experimentally confirmed and linked to protein function. These chapters are organized based on the participation of these proteins in the regulation of signaling, metabolism and epigenetics. The last chapter discusses the therapeutic implications of altered redox microenvironment and protein oxidation in disease. © 2015 Taylor & Francis.

Wani R.,Section on Molecular Medicine | Tsang A.W.,Section on Molecular Medicine | Furdui C.M.,Section on Molecular Medicine
Cell Cycle | Year: 2011

Phosphorylation has long been recognized as the key mediator of protein signaling. New modes of signaling regulation are emerging with the development of specific chemical probes and application of high-throughput mass spectrometry technologies. Using biotin- tagged chemical probes for protein oxidation, mass spectrometry and functional assays, our group has recently reported isoform-specific oxidation of Akt2 in response to PDGF signaling. The studies included here investigate the functional consequence of oxidation on Akt2-mediated cell migration and cell cycle. Akt2-KO MEFs transduced with WT and Cys124Ser Akt2 were used as the model system for these studies. The implications of these findings on disease pathology are discussed. © 2011 Landes Bioscience.

Reisz J.A.,Section on Molecular Medicine | Bansal N.,Section on Molecular Medicine | Qian J.,Section on Molecular Medicine | Zhao W.,Section on Molecular Medicine | Furdui C.M.,Section on Molecular Medicine
Antioxidants and Redox Signaling | Year: 2014

Significance: The detrimental effects of ionizing radiation (IR) involve a highly orchestrated series of events that are amplified by endogenous signaling and culminating in oxidative damage to DNA, lipids, proteins, and many metabolites. Despite the global impact of IR, the molecular mechanisms underlying tissue damage reveal that many biomolecules are chemoselectively modified by IR. Recent Advances: The development of high-throughput "omics" technologies for mapping DNA and protein modifications have revolutionized the study of IR effects on biological systems. Studies in cells, tissues, and biological fluids are used to identify molecular features or biomarkers of IR exposure and response and the molecular mechanisms that regulate their expression or synthesis. Critical Issues: In this review, chemical mechanisms are described for IR-induced modifications of biomolecules along with methods for their detection. Included with the detection methods are crucial experimental considerations and caveats for their use. Additional factors critical to the cellular response to radiation, including alterations in protein expression, metabolomics, and epigenetic factors, are also discussed. Future Directions: Throughout the review, the synergy of combined "omics" technologies such as genomics and epigenomics, proteomics, and metabolomics is highlighted. These are anticipated to lead to new hypotheses to understand IR effects on biological systems and improve IR-based therapies. Antioxid. Redox Signal. 21: 260-292. © Mary Ann Liebert, Inc. 2014.

Furdui C.M.,Section on Molecular Medicine
Antioxidants and Redox Signaling | Year: 2014

While chemotherapy and radiation therapy have been integral components of cancer management for decades, the issues of local recurrence, clinical resistance, and toxicities resulting from these treatment modalities have increased the interest in novel therapeutic approaches that could attenuate tumor progression and prevent recurrences. This Forum highlights current research focused on elucidation of the mechanisms of response to radiation treatment and the development of out-of-the-box therapeutic strategies for cancer treatment with radiation. Experts in the field of radiation research contribute with review articles describing the current knowledge on DNA damage response mechanisms, regulation of signaling involved in the DNA damage response by miRNA, the function of tumor hypoxia in tumor response to radiation, and the role of stem cells in protection of normal tissue against radiation damage. Antioxid. Redox Signal. 21, 218-220. © Mary Ann Liebert, Inc. 2014.

Devarie Baez N.O.,Section on Molecular Medicine | Reisz J.A.,Section on Molecular Medicine | Furdui C.M.,Section on Molecular Medicine
Free Radical Biology and Medicine | Year: 2015

Mass spectrometry (MS) has become a powerful and widely utilized tool in the investigation of protein thiol chemistry, biochemistry, and biology. Very early biochemical studies of metabolic enzymes have brought to light the broad spectrum of reactivity profiles that distinguish cysteine thiols with functions in catalysis and protein stability from other cysteine residues in proteins. The development of MS methods for the analysis of proteins using electrospray ionization (ESI) or matrix-assisted laser desorption/ionization (MALDI) coupled with the emergence of high-resolution mass analyzers has been instrumental in advancing studies of thiol modifications, both in single proteins and within the cellular context. This article reviews MS instrumentation and methods of analysis employed in investigations of thiols and their reactivity toward a range of small biomolecules. A selected number of studies are detailed to highlight the advantages brought about by the MS technologies along with the caveats associated with these analyses. © 2014 Elsevier Inc. All rights reserved.

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