Both thioredoxin 2 and glutaredoxin 2 contribute to the reduction of the mitochondrial 2-Cys peroxiredoxin Prx3

The proteins from the thioredoxin family are crucial actors in redox signaling and the cellular response to oxidative stress. The major intracellular source for oxygen radicals are the components of the respiratory chain in mitochondria. Here, we show that the mitochondrial 2-Cys peroxiredoxin (Prx3) is not only substrate for thioredoxin 2 (Trx2), but can also be reduced by glutaredoxin 2 (Grx2) via the dithiol reaction mechanism. Grx2 reduces Prx3 exhibiting catalytic constants (K(m), 23.8 μmol·liter(-1); V(max), 1.2 μmol·(mg·min)(-1)) similar to Trx2 (K(m), 11.2 μmol·liter(-1); V(max), 1.1 μmol·(mg·min)(-1)). The reduction of the catalytic disulfide of the atypical 2-Cys Prx5 is limited to the Trx system. Silencing the expression of either Trx2 or Grx2 in HeLa cells using specific siRNAs did not change the monomer:dimer ratio of Prx3 detected by a specific 2-Cys Prx redox blot. Only combined silencing of the expression of both proteins led to an accumulation of oxidized protein. We further demonstrate that the distribution of Prx3 in different mouse tissues is either linked to the distribution of Trx2 or Grx2. These results introduce Grx2 as a novel electron donor for Prx3, providing further insights into pivotal cellular redox signaling mechanisms.     

Voltage-dependent Anion Channels (VDACs) Recruit Parkin to Defective Mitochondria to Promote Mitochondrial Autophagy

Mutations in the ubiquitin ligase Parkin and the serine/threonine kinase PINK1 can cause Parkinson disease. Both proteins function in the elimination of defective mitochondria by autophagy. In this process, activation of PINK1 mediates translocation of Parkin from the cytosol to mitochondria by an unknown mechanism. To better understand how Parkin is targeted to defective mitochondria, we purified affinity-tagged Parkin from mitochondria and identified Parkin-associated proteins by mass spectrometry. The three most abundant interacting proteins were the voltage-dependent anion channels 1, 2, and 3 (VDACs 1, 2, and 3), pore-forming proteins in the outer mitochondrial membrane. We demonstrate that Parkin specifically interacts with VDACs when the function of mitochondria is disrupted by treating cells with the proton uncoupler carbonyl cyanide p-chlorophenylhydrazone. In the absence of all three VDACs, the recruitment of Parkin to defective mitochondria and subsequent mitophagy are impaired. Each VDAC is sufficient to support Parkin recruitment and mitophagy, suggesting that VDACs can function redundantly. We hypothesize that VDACs serve as mitochondrial docking sites to recruit Parkin from the cytosol to defective mitochondria.     

Thioredoxin 1 Is Inactivated Due to Oxidation Induced by Peroxiredoxin under Oxidative Stress and Reactivated by the Glutaredoxin System

The mammalian cytosolic thioredoxin system, comprising thioredoxin (Trx), Trx reductase, and NADPH, is the major protein-disulfide reductase of the cell and has numerous functions. Besides the active site thiols, human Trx1 contains three non-active site cysteine residues at positions 62, 69, and 73. A two-disulfide form of Trx1, containing an active site disulfide between Cys-32 and Cys-35 and a non-active site disulfide between Cys-62 and Cys-69, is inactive either as a disulfide reductase or as a substrate for Trx reductase. This could possibly provide a structural switch affecting Trx1 function during oxidative stress and redox signaling. We found that two-disulfide Trx1 was generated in A549 cells under oxidative stress. In vitro data showed that two-disulfide Trx1 was generated from oxidation of Trx1 catalyzed by peroxiredoxin 1 in the presence of H₂O₂. The redox Western blot data indicated that the glutaredoxin system protected Trx1 in HeLa cells from oxidation caused by ebselen, a superfast oxidant for Trx1. Our results also showed that physiological concentrations of glutathione, NADPH, and glutathione reductase reduced the non-active site disulfide in vitro. This reaction was stimulated by glutaredoxin 1 via the so-called monothiol mechanism. In conclusion, reversible oxidation of the non-active site disulfide of Trx1 is suggested to play an important role in redox regulation and cell signaling via temporal inhibition of its protein-disulfide reductase activity for the transmission of oxidative signals under oxidative stress.     

Respiration-dependent H2O2 Removal in Brain Mitochondria via the Thioredoxin/Peroxiredoxin System

Mitochondrial reactive oxygen species (ROS) play an important role in both physiological cell signaling processes and numerous pathological states, including neurodegenerative disorders such as Parkinson disease. While mitochondria are considered the major cellular source of ROS, their role in ROS removal remains largely unknown. Using polarographic methods for real-time detection of steady-state H₂O₂ levels, we were able to quantitatively measure the contributions of potential systems toward H₂O₂ removal by brain mitochondria. Isolated rat brain mitochondria showed significant rates of exogenous H₂O₂ removal (9–12 nmol/min/mg of protein) in the presence of substrates, indicating a respiration-dependent process. Glutathione systems showed only minimal contributions: 25% decrease with glutathione reductase inhibition and no effect by glutathione peroxidase inhibition. In contrast, inhibitors of thioredoxin reductase, including auranofin and 1-chloro-2,4-dinitrobenzene, attenuated H₂O₂ removal rates in mitochondria by 80%. Furthermore, a 50% decrease in H₂O₂ removal was observed following oxidation of peroxiredoxin. Differential oxidation of glutathione or thioredoxin proteins by copper (II) or arsenite, respectively, provided further support for the thioredoxin/peroxiredoxin system as the major contributor to mitochondrial H₂O₂ removal. Inhibition of the thioredoxin system exacerbated mitochondrial H₂O₂ production by the redox cycling agent, paraquat. Additionally, decreases in H₂O₂ removal were observed in intact dopaminergic neurons with thioredoxin reductase inhibition, implicating this mechanism in whole cell systems. Therefore, in addition to their recognized role in ROS production, mitochondria also remove ROS. These findings implicate respiration- and thioredoxin-dependent ROS removal as a potentially important mitochondrial function that may contribute to physiological and pathological processes in the brain.     

Accumulation of newly synthesized F1 in vivo in arabidopsis mitochondria provides evidence for modular assembly of the plant F1Fo ATP synthase

F(1) subcomplex in mitochondrial samples is often considered to be a breakage product of the F(1)F(O) ATP synthase during sample handling and electrophoresis. We have used a progressive (15)N incorporation strategy to investigate the plant F(1)F(O) ATP synthase assembly model and the apparently free F(1) in plant mitochondria which is found in both the inner membrane and matrix. We show that subunits within F(1) in the inner membrane and matrix had a relatively higher (15)N incorporation rate than corresponding subunits in intact membrane F(1)F(O). This demonstrates that free F(1) was a newer pool with a faster turnover rate consistent with it being an assembly intermediate in vivo. Import of [(35)S]Met-labeled F(1) subunit precursors into Arabidopsis mitochondria showed the rapid accumulation of F(1) assembly intermediates. The different (15)N incorporation rate in matrix F(1), inner membrane F(1) and intact F(1)F(O) demonstrates these three represent different protein populations and are likely step by step intermediates during the assembly process of plant mitochondrial ATP synthase. The potential biological implications of in vivo accumulation of enzymatically active F(1) in mitochondria are discussed.     

Structural Basis for Antibody Recognition in the Receptor-binding Domains of Toxins A and B from Clostridium difficile

Clostridium difficile infection is a serious and highly prevalent nosocomial disease in which the two large, Rho-glucosylating toxins TcdA and TcdB are the main virulence factors. We report for the first time crystal structures revealing how neutralizing and non-neutralizing single-domain antibodies (sdAbs) recognize the receptor-binding domains (RBDs) of TcdA and TcdB. Surprisingly, the complexes formed by two neutralizing antibodies recognizing TcdA do not show direct interference with the previously identified carbohydrate-binding sites, suggesting that neutralization of toxin activity may be mediated by mechanisms distinct from steric blockage of receptor binding. A camelid sdAb complex also reveals the molecular structure of the TcdB RBD for the first time, facilitating the crystallization of a strongly negatively charged protein fragment that has resisted previous attempts at crystallization and structure determination. Electrospray ionization mass spectrometry measurements confirm the stoichiometries of sdAbs observed in the crystal structures. These studies indicate how key epitopes in the RBDs from TcdA and TcdB are recognized by sdAbs, providing molecular insights into toxin structure and function and providing for the first time a basis for the design of highly specific toxin-specific therapeutic and diagnostic agents.     

Endosomal targeting of the phosphoinositide 3-phosphatase MTMR2 is regulated by an N-terminal phosphorylation site

MTMR2 is a member of the myotubularin family of inositol lipid phosphatases, a large protein-tyrosine phosphatase subgroup that is conserved from yeast to humans. Furthermore, the peripheral neuromuscular disease Charcot-Marie Tooth disease type 4B has been attributed to mutations in the mtmr2 gene. Because the molecular mechanisms regulating MTMR2 have been poorly defined, we investigated whether reversible phosphorylation might regulate MTMR2 function. We used mass spectrometry-based methods to identify a high stoichiometry phosphorylation site on serine 58 of MTMR2. Phosphorylation at Ser(58), or a phosphomimetic S58E mutation, markedly decreased MTMR2 localization to endocytic vesicular structures. In contrast, a phosphorylation-deficient MTMR2 mutant (S58A) displayed constitutive localization to early endocytic structures. This localization pattern was accompanied by displacement of a PI(3)P-specific sensor protein and an increase in signal transduction pathways. Thus, MTMR2 phosphorylation is likely to be a critical mechanism by which MTMR2 access to its lipid substrate(s) is temporally and spatially regulated, thereby contributing to the control of downstream endosome maturation events.     

Mycothiol/Mycoredoxin 1-dependent Reduction of the Peroxiredoxin AhpE from Mycobacterium tuberculosis

Mycobacterium tuberculosis (M. tuberculosis), the pathogen responsible for tuberculosis, detoxifies cytotoxic peroxides produced by activated macrophages. M. tuberculosis expresses alkyl hydroxyperoxide reductase E (AhpE), among other peroxiredoxins. So far the system that reduces AhpE was not known. We identified M. tuberculosis mycoredoxin-1 (MtMrx1) acting in combination with mycothiol and mycothiol disulfide reductase (MR), as a biologically relevant reducing system for MtAhpE. MtMrx1, a glutaredoxin-like, mycothiol-dependent oxidoreductase, directly reduces the oxidized form of MtAhpE, through a protein mixed disulfide with the N-terminal cysteine of MtMrx1 and the sulfenic acid derivative of the peroxidatic cysteine of MtAhpE. This disulfide is then reduced by the C-terminal cysteine in MtMrx1. Accordingly, MtAhpE catalyzes the oxidation of wt MtMrx1 by hydrogen peroxide but not of MtMrx1 lacking the C-terminal cysteine, confirming a dithiolic mechanism. Alternatively, oxidized MtAhpE forms a mixed disulfide with mycothiol, which in turn is reduced by MtMrx1 using a monothiolic mechanism. We demonstrated the H₂O₂-dependent NADPH oxidation catalyzed by MtAhpE in the presence of MR, Mrx1, and mycothiol. Disulfide formation involving mycothiol probably competes with the direct reduction by MtMrx1 in aqueous intracellular media, where mycothiol is present at millimolar concentrations. However, MtAhpE was found to be associated with the membrane fraction, and since mycothiol is hydrophilic, direct reduction by MtMrx1 might be favored. The results reported herein allow the rationalization of peroxide detoxification actions inferred for mycothiol, and more recently, for Mrx1 in cellular systems. We report the first molecular link between a thiol-dependent peroxidase and the mycothiol/Mrx1 pathway in Mycobacteria.     

Post-translationally Abnormal Collagens of Prolyl 3-Hydroxylase-2 Null Mice Offer a Pathobiological Mechanism for the High Myopia Linked to Human LEPREL1 Mutations

Myopia, the leading cause of visual impairment worldwide, results from an increase in the axial length of the eyeball. Mutations in LEPREL1, the gene encoding prolyl 3-hydroxylase-2 (P3H2), have recently been identified in individuals with recessively inherited nonsyndromic severe myopia. P3H2 is a member of a family of genes that includes three isoenzymes of prolyl 3-hydroxylase (P3H), P3H1, P3H2, and P3H3. Fundamentally, it is understood that P3H1 is responsible for converting proline to 3-hydroxyproline. This limited additional knowledge also suggests that each isoenzyme has evolved different collagen sequence-preferred substrate specificities. In this study, differences in prolyl 3-hydroxylation were screened in eye tissues from P3h2-null (P3h2^n/n) and wild-type mice to seek tissue-specific effects due the lack of P3H2 activity on post-translational collagen chemistry that could explain myopia. The mice were viable and had no gross musculoskeletal phenotypes. Tissues from sclera and cornea (type I collagen) and lens capsule (type IV collagen) were dissected from mouse eyes, and multiple sites of prolyl 3-hydroxylation were identified by mass spectrometry. The level of prolyl 3-hydroxylation at multiple substrate sites from type I collagen chains was high in sclera, similar to tendon. Almost every known site of prolyl 3-hydroxylation in types I and IV collagen from P3h2^n/n mouse eye tissues was significantly under-hydroxylated compared with their wild-type littermates. We conclude that altered collagen prolyl 3-hydroxylation is caused by loss of P3H2. We hypothesize that this leads to structural abnormalities in multiple eye tissues, but particularly sclera, causing progressive myopia.     

Peptide Modulation of Class I Major Histocompatibility Complex Protein Molecular Flexibility and the Implications for Immune Recognition

T cells use the αβ T cell receptor (TCR) to recognize antigenic peptides presented by class I major histocompatibility complex proteins (pMHCs) on the surfaces of antigen-presenting cells. Flexibility in both TCRs and peptides plays an important role in antigen recognition and discrimination. Less clear is the role of flexibility in the MHC protein; although recent observations have indicated that mobility in the MHC can impact TCR recognition in a peptide-dependent fashion, the extent of this behavior is unknown. Here, using hydrogen/deuterium exchange, fluorescence anisotropy, and structural analyses, we show that the flexibility of the peptide binding groove of the class I MHC protein HLA-A*0201 varies significantly with different peptides. The variations extend throughout the binding groove, impacting regions contacted by TCRs as well as other activating and inhibitory receptors of the immune system. Our results are consistent with statistical mechanical models of protein structure and dynamics, in which the binding of different peptides alters the populations and exchange kinetics of substates in the MHC conformational ensemble. Altered MHC flexibility will influence receptor engagement, impacting conformational adaptations, entropic penalties associated with receptor recognition, and the populations of binding-competent states. Our results highlight a previously unrecognized aspect of the “altered self” mechanism of immune recognition and have implications for specificity, cross-reactivity, and antigenicity in cellular immunity.     

Human 2-Oxoglutarate Dehydrogenase Complex E1 Component Forms a Thiamin-derived Radical by Aerobic Oxidation of the Enamine Intermediate

Herein are reported unique properties of the human 2-oxoglutarate dehydrogenase multienzyme complex (OGDHc), a rate-limiting enzyme in the Krebs (citric acid) cycle. (a) Functionally competent 2-oxoglutarate dehydrogenase (E1o-h) and dihydrolipoyl succinyltransferase components have been expressed according to kinetic and spectroscopic evidence. (b) A stable free radical, consistent with the C2-(C2α-hydroxy)-γ-carboxypropylidene thiamin diphosphate (ThDP) cation radical was detected by electron spin resonance upon reaction of the E1o-h with 2-oxoglutarate (OG) by itself or when assembled from individual components into OGDHc. (c) An unusual stability of the E1o-h-bound C2-(2α-hydroxy)-γ-carboxypropylidene thiamin diphosphate (the “ThDP-enamine”/C2α-carbanion, the first postdecarboxylation intermediate) was observed, probably stabilized by the 5-carboxyl group of OG, not reported before. (d) The reaction of OG with the E1o-h gave rise to superoxide anion and hydrogen peroxide (reactive oxygen species (ROS)). (e) The relatively stable enzyme-bound enamine is the likely substrate for oxidation by O₂, leading to the superoxide anion radical (in d) and the radical (in b). (f) The specific activity assessed for ROS formation compared with the NADH (overall complex) activity, as well as the fraction of radical intermediate occupying active centers of E1o-h are consistent with each other and indicate that radical/ROS formation is an “off-pathway” side reaction comprising less than 1% of the “on-pathway” reactivity. However, the nearly ubiquitous presence of OGDHc in human tissues, including the brain, makes these findings of considerable importance in human metabolism and perhaps disease.     

Structural Basis of G Protein-coupled Receptor-Gi Protein Interaction: FORMATION OF THE CANNABINOID CB2 RECEPTOR-Gi PROTEIN COMPLEX*

In this study, we applied a comprehensive G protein-coupled receptor-Gα_i protein chemical cross-linking strategy to map the cannabinoid receptor subtype 2 (CB₂)- Gα_i interface and then used molecular dynamics simulations to explore the dynamics of complex formation. Three cross-link sites were identified using LC-MS/MS and electrospray ionization-MS/MS as follows: 1) a sulfhydryl cross-link between C3.53(134) in TMH3 and the Gα_i C-terminal i-3 residue Cys-351; 2) a lysine cross-link between K6.35(245) in TMH6 and the Gα_i C-terminal i-5 residue, Lys-349; and 3) a lysine cross-link between K5.64(215) in TMH5 and the Gα_i α₄β₆ loop residue, Lys-317. To investigate the dynamics and nature of the conformational changes involved in CB₂·G_i complex formation, we carried out microsecond-time scale molecular dynamics simulations of the CB₂ R*·Gα_i1β₁γ₂ complex embedded in a 1-palmitoyl-2-oleoyl-phosphatidylcholine bilayer, using cross-linking information as validation. Our results show that although molecular dynamics simulations started with the G protein orientation in the β2-AR*·Gα_sβ₁γ₂ complex crystal structure, the Gα_i1β₁γ₂ protein reoriented itself within 300 ns. Two major changes occurred as follows. 1) The Gα_i1 α5 helix tilt changed due to the outward movement of TMH5 in CB₂ R*. 2) A 25° clockwise rotation of Gα_i1β₁γ₂ underneath CB₂ R* occurred, with rotation ceasing when Pro-139 (IC-2 loop) anchors in a hydrophobic pocket on Gα_i1 (Val-34, Leu-194, Phe-196, Phe-336, Thr-340, Ile-343, and Ile-344). In this complex, all three experimentally identified cross-links can occur. These findings should be relevant for other class A G protein-coupled receptors that couple to G_i proteins.     

Carboxyl Group Footprinting Mass Spectrometry and Molecular Dynamics Identify Key Interactions in the HER2-HER3 Receptor Tyrosine Kinase Interface

The HER2 receptor tyrosine kinase is a driver oncogene in many human cancers, including breast and gastric cancer. Under physiologic levels of expression, HER2 heterodimerizes with other members of the EGF receptor/HER/ErbB family, and the HER2-HER3 dimer forms one of the most potent oncogenic receptor pairs. Previous structural biology studies have individually crystallized the kinase domains of HER2 and HER3, but the HER2-HER3 kinase domain heterodimer structure has yet to be solved. Using a reconstituted membrane system to form HER2-HER3 kinase domain heterodimers and carboxyl group footprinting mass spectrometry, we observed that HER2 and HER3 kinase domains preferentially form asymmetric heterodimers with HER3 and HER2 monomers occupying the donor and acceptor kinase positions, respectively. Conformational changes in the HER2 activation loop, as measured by changes in carboxyl group labeling, required both dimerization and nucleotide binding but did not require activation loop phosphorylation at Tyr-877. Molecular dynamics simulations on HER2-HER3 kinase dimers identify specific inter- and intramolecular interactions and were in good agreement with MS measurements. Specifically, several intermolecular ionic interactions between HER2 Lys-716-HER3 Glu-909, HER2 Glu-717-HER3 Lys-907, and HER2 Asp-871-HER3 Arg-948 were identified by molecular dynamics. We also evaluated the effect of the cancer-associated mutations HER2 D769H/D769Y, HER3 E909G, and HER3 R948K (also numbered HER3 E928G and R967K) on kinase activity in the context of this new structural model. This study provides valuable insights into the EGF receptor/HER/ErbB kinase structure and interactions, which can guide the design of future therapies.     

Characterization of Natural Human Nucleotide-binding Oligomerization Domain Protein 1 (Nod1) Ligands from Bacterial Culture Supernatant for Elucidation of Immune Modulators in the Environment

Nucleotide-binding oligomerization domain protein 1 (Nod1) is an intracellular protein involved in recognition of the bacterial component peptidoglycan. This recognition event induces a host defense response to eliminate invading pathogens. The genetic variation of Nod1 has been linked to several inflammatory diseases and allergies, which are strongly affected by environmental factors. We have found that many of the bacteria that contain DAP-type peptidoglycan release Nod1 ligands into the environment. However, the structures of natural Nod1 ligands in the environment are not well understood. Herein, we report the isolation and structural elucidation of natural human Nod1 (hNod1) ligands from the Escherichia coli K-12 culture supernatant. The supernatant was fractionated with reversed-phase high performance liquid chromatography (RP-HPLC), resulting in the isolation of several hNod1 stimulatory fractions. Structural characterization studies demonstrated that the molecular structure of the most active fraction was the native hNod1 ligand GlcNAc-(β1–4)-(anhydro)MurNAc-l-Ala-γ-d-Glu-meso-DAP. We also found other peptidoglycan fragments using the 7-(diethylamino)coumarin-3-carbonyl labeling method to enhance sensitivity in mass spectroscopy studies. These results suggested that DAP-containing bacteria release certain hNod1 ligands to the environment, and these ligands would accumulate in the environment and regulate the immune system through Nod1.     

Basis of Miscoding of the DNA Adduct N2,3-Ethenoguanine by Human Y-family DNA Polymerases

N²,3-Ethenoguanine (N²,3-ϵG) is one of the exocyclic DNA adducts produced by endogenous processes (e.g. lipid peroxidation) and exposure to bioactivated vinyl monomers such as vinyl chloride, which is a known human carcinogen. Existing studies exploring the miscoding potential of this lesion are quite indirect because of the lability of the glycosidic bond. We utilized a 2′-fluoro isostere approach to stabilize this lesion and synthesized oligonucleotides containing 2′-fluoro-N²,3-ϵ-2′-deoxyarabinoguanosine to investigate the miscoding potential of N²,3-ϵG by Y-family human DNA polymerases (pols). In primer extension assays, pol η and pol κ replicated through N²,3-ϵG, whereas pol ι and REV1 yielded only 1-base incorporation. Steady-state kinetics revealed that dCTP incorporation is preferred opposite N²,3-ϵG with relative efficiencies in the order of pol κ > REV1 > pol η ≈ pol ι, and dTTP misincorporation is the major miscoding event by all four Y-family human DNA pols. Pol ι had the highest dTTP misincorporation frequency (0.71) followed by pol η (0.63). REV1 misincorporated dTTP and dGTP with much lower frequencies. Crystal structures of pol ι with N²,3-ϵG paired to dCTP and dTTP revealed Hoogsteen-like base pairing mechanisms. Two hydrogen bonds were observed in the N²,3-ϵG:dCTP base pair, whereas only one appears to be present in the case of the N²,3-ϵG:dTTP pair. Base pairing mechanisms derived from the crystal structures explain the slightly favored dCTP insertion for pol ι in steady-state kinetic analysis. Taken together, these results provide a basis for the mutagenic potential of N²,3-ϵG.     

Sulfiredoxin protein is critical for redox balance and survival of cells exposed to low steady-state levels of H2O2

Sulfiredoxin (Srx) is an enzyme that catalyzes the reduction of cysteine sulfinic acid of hyperoxidized peroxiredoxins (Prxs). Having high affinity toward H2O2, 2-Cys Prxs can efficiently reduce H2O2 at low concentration. We previously showed that Prx I is hyperoxidized at a rate of 0.072% per turnover even in the presence of low steady-state levels of H2O2. Here we examine the novel role of Srx in cells exposed to low steady-state levels of H2O2, which can be achieved by using glucose oxidase. Exposure of low steady-state levels of H2O2 (10-20 μm) to A549 or wild-type mouse embryonic fibroblast (MEF) cells does not lead to any significant change in oxidative injury because of the maintenance of balance between H2O2 production and elimination. In contrast, loss-of-function studies using Srx-depleted A549 and Srx-/- MEF cells demonstrate a dramatic increase in extra- and intracellular H2O2, sulfinic 2-Cys Prxs, and apoptosis. Concomitant with hyperoxidation of mitochondrial Prx III, Srx-depleted cells show an activation of mitochondria-mediated apoptotic pathways including mitochondria membrane potential collapse, cytochrome c release, and caspase activation. Furthermore, adenoviral re-expression of Srx in Srx-depleted A549 or Srx-/- MEF cells promotes the reactivation of sulfinic 2-Cys Prxs and results in cellular resistance to apoptosis, with enhanced removal of H2O2. These results indicate that Srx functions as a novel component to maintain the balance between H2O2 production and elimination and then protects cells from apoptosis even in the presence of low steady-state levels of H2O2.     

Probing conformational changes in human DNA topoisomerase IIα by pulsed alkylation mass spectrometry

Type II topoisomerases are essential enzymes for solving DNA topological problems by passing one segment of DNA duplex through a transient double-strand break in a second segment. The reaction requires the enzyme to precisely control DNA cleavage and gate opening coupled with ATP hydrolysis. Using pulsed alkylation mass spectrometry, we were able to monitor the solvent accessibilities around 13 cysteines distributed throughout human topoisomerase IIα by measuring the thiol reactivities with monobromobimane. Most of the measured reactivities are in accordance with the predicted ones based on a homology structural model generated from available crystal structures. However, these results reveal new information for both the residues not covered in the structural model and potential differences between the modeled and solution holoenzyme structures. Furthermore, on the basis of the reactivity changes of several cysteines located at the N-gate and DNA gate, we could monitor the movement of topoisomerase II in the presence of cofactors and detect differences in the DNA gate between two closed clamp enzyme conformations locked by either 5'-adenylyl β,γ-imidodiphosphate or the anticancer drug ICRF-193.     

Altering O-Linked β-N-Acetylglucosamine Cycling Disrupts Mitochondrial Function

Mitochondrial impairment is commonly found in many diseases such as diabetes, cancer, and Alzheimer disease. We demonstrate that the enzymes responsible for the addition or removal of the O-GlcNAc modification, O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA), respectively, are critical regulators of mitochondrial function. Using a SILAC (stable isotope labeling of amino acids in cell culture)-based proteomics screen, we quantified the changes in mitochondrial protein expression in OGT- and OGA-overexpressing cells. Strikingly, overexpression of OGT or OGA showed significant decreases in mitochondria-localized proteins involved in the respiratory chain and the tricarboxylic acid cycle. Furthermore, mitochondrial morphology was altered in these cells. Both cellular respiration and glycolysis were reduced in OGT/OGA-overexpressing cells. These data demonstrate that alterations in O-GlcNAc cycling profoundly affect energy and metabolite production.