Molecular Basis for Enzymatic Sulfite Oxidation: HOW THREE CONSERVED ACTIVE SITE RESIDUES SHAPE ENZYME ACTIVITY*S?

Sulfite dehydrogenases (SDHs) catalyze the oxidation and detoxification of sulfite to sulfate, a reaction critical to all forms of life. Sulfite-oxidizing enzymes contain three conserved active site amino acids (Arg-55, His-57, and Tyr-236) that are crucial for catalytic competency. Here we have studied the kinetic and structural effects of two novel and one previously reported substitution (R55M, H57A, Y236F) in these residues on SDH catalysis. Both Arg-55 and His-57 were found to have key roles in substrate binding. An R55M substitution increased K_{m(sulfite)(app)} by 2–3 orders of magnitude, whereas His-57 was required for maintaining a high substrate affinity at low pH when the imidazole ring is fully protonated. This effect may be mediated by interactions of His-57 with Arg-55 that stabilize the position of the Arg-55 side chain or, alternatively, may reflect changes in the protonation state of sulfite. Unlike what is seen for SDH^WT and SDH^Y236F, the catalytic turnover rates of SDH^R55M and SDH^H57A are relatively insensitive to pH (∼60 and 200 s^–1, respectively). On the structural level, striking kinetic effects appeared to correlate with disorder (in SDH^H57A and SDH^Y236F) or absence of Arg-55 (SDH^R55M), suggesting that Arg-55 and the hydrogen bonding interactions it engages in are crucial for substrate binding and catalysis. The structure of SDH^R55M has sulfate bound at the active site, a fact that coincides with a significant increase in the inhibitory effect of sulfate in SDH^R55M. Thus, Arg-55 also appears to be involved in enabling discrimination between the substrate and product in SDH.Sulfite-oxidizing enzymes protect cells against potentially fatal damage to DNA and proteins caused by exposure to sulfite, and consequently they are found in all forms of life (1). In bacteria, sulfite oxidation is often linked to energy-generating processes during chemolithotrophic growth on reduced sulfur compounds (2, 3), whereas both plant and vertebrate sulfite oxidases have been shown to detoxify sulfite arising from the degradation of methionine and cysteine and exposure to sulfur dioxide (4, 5).All known sulfite-oxidizing enzymes belong to the same family of mononuclear molybdenum enzymes. Their active sites contain one molybdopterin unit per molybdenum atom, and these enzymes may also contain heme groups as accessory redox centers (6–9). Examples of different types of sulfite-oxidizing molybdoenzymes are the homodimeric plant sulfite oxidase, which does not contain a heme group and uses oxygen as its preferred electron acceptor (9), the homodimeric chicken and human liver sulfite oxidases (CSO³ and HSO, respectively) (10), which are also able to use oxygen as an electron acceptor, and the bacterial sulfite dehydrogenase (SDH) isolated from the soil bacterium Starkeya novella (11, 12), which cannot donate electrons directly to oxygen. Each monomer of CSO and HSO contains a heme b center in addition to the molybdenum center, and the redox centers are located within separate, flexibly linked domains of the same protein subunit. In contrast, the bacterial enzyme is a heterodimer where each subunit of the enzyme contains one redox center. The molybdopterin cofactor is located in the larger 40.2-kDa SorA subunit, and the c-type heme is located in the smaller, 8.8-kDa SorB subunit (12). The SDH quaternary structure thus differs clearly from that of the human and chicken sulfite oxidases.Crystal structures are available for plant sulfite oxidase, CSO, and the bacterial SDH (10, 11, 13–15) and have revealed molecular details of the sulfite-oxidizing enzymes. In the CSO structure, the mobile heme b domain occupies a position too removed from the molybdenum active site to mediate efficient electron transfer (10), and indeed the kinetics of this enzyme are known to be complicated by domain movements (16). In contrast, the bacterial SDH is a tight complex with strong electrostatic interactions between the subunits, and the close approach of the redox centers (Mo–Fe distance 16.6 Å) allows for rapid electron transfer (11, 17) (Fig. 1, A and B).Open in a separate windowFIGURE 1.Details of the crystal structure of wild type SDH and comparison with CSO. A, ribbon diagram of the SDH heterodimer with the SorA and SorB subunits colored blue and cyan, respectively, and the redox cofactors in space-filling mode with the molybdenum atom colored green and the iron atom colored violet. B, ribbon diagram of a single subunit of CSO with the molybdopterin binding domain in the same orientation as SorA in A. The cytochrome domain of CSO is clearly in a different position with respect to the molybdenum cofactor than is seen for the cytochrome subunit of SDH. C, SDH molybdopterin cofactor demonstrating the geometry of the molybdenum ligands. The thiol ligands donated by the organic component of molybdopterin and the Cys-104 side chain, and the reactive oxygen ligand (O_eq) sit in the equatorial plane with the axial oxygen (O_ax) ligand at the apex of a square pyramid. Atoms are colored as follows: molybdenum (green), sulfur (orange), phosphorous (magenta), oxygen (red), nitrogen (blue), and carbon (yellow in the cofactor and white in the protein). D, hydrogen bonding network around the substrate binding site. The molybdopterin and heme cofactors are shown together with active site residues Cys-104, Arg-55, His-57, Tyr-236, and Gln-33. Figs. 1 and ​and44 were prepared using Pymol (37).Despite the overall structural differences of these proteins, the coordination geometries of the molybdenum active sites of these sulfite-oxidizing enzymes are nearly identical. The oxidized molybdenum center has a square pyramidal conformation, with three sulfur and two oxo ligands (18). Within this molybdenum center, the equatorial oxo ligand is proposed to be catalytically active, whereas the axial oxo ligand is not thought to participate directly in the reaction (Fig. 1C). During catalysis, the equatorial oxo ligand is transformed into a hydroxy/water ligand as a result of the reduction of the molybdenum center (Fig. 2), and it is in this form that it is generally observed in the CSO and SDH crystal structures.Open in a separate windowFIGURE 2.Proposed reaction mechanism for S. novella sulfite dehydrogenase. The reaction is shown in terms of the redox states of the molybdenum and heme centers present in the enzyme. Shown in boldface type and boxed are the stable redox states of the S. novella SDH. Cyt. c, a mitochondrial type cytochrome c₅₅₀ (e.g. horse heart or S. novella cytochrome c₅₅₀) that can act as the external electron acceptor.SDH, CSO, and HSO show similarly high affinities for their substrate, sulfite, and several highly conserved residues surround the substrate-binding and molybdenum active site, namely Tyr-236 (all residues given in SDH numbering (11)), Arg-55, and His-57 (Fig. 1D). Both Arg-55 and Tyr-236 form hydrogen bonds to the catalytically active equatorial Mo-oxo group, whereas His-57 is positioned close to both Arg-55 and Tyr-236 (10, 11) (Fig. 1D). In addition, the crystal structure of the bacterial SDH shows that Arg-55 interacts directly with the second SDH redox center by hydrogen bonding to heme propionate-6 (Fig. 1D) (11).As a result of the similarities in catalytic parameters and the structure of the active site, the bacterial SDH is a very good system for studies of enzymatic sulfite oxidation and especially the molecular basis for catalysis. Since this enzyme does not rely on domain movement for catalysis, it has a less complicated reaction mechanism than the vertebrate enzymes, which facilitates the interpretation of kinetic data, and it can be readily crystallized with both redox centers present in an electron transfer competent conformation. We have previously reported data on the structure, kinetics, EPR, and redox properties of a Y236F-substituted SDH (13). In addition to reduced turnover and substrate affinity, this substitution influences the reactivity of the SDH toward oxygen, turning SDH^Y236F essentially into an (albeit weak) sulfite oxidase. In order to further understand the roles of the conserved amino acids surrounding the molybdenum active site of sulfite-oxidizing enzymes, we have created two novel amino acid substitutions in the Arg-55 and His-57 residues present at the active site and have investigated their effect on catalytic and spectroscopic parameters of the bacterial SDH. We have also solved the crystal structures of the substituted enzymes, which have provided new insights into the conformation and plasticity of the active site of sulfite-oxidizing enzymes and how the conserved active site residues contribute to sulfite oxidation.     

Lipids as Tumoricidal Components of Human α-Lactalbumin Made Lethal to Tumor Cells (HAMLET): UNIQUE AND SHARED EFFECTS ON SIGNALING AND DEATH*

Long-chain fatty acids are internalized by receptor-mediated mechanisms or receptor-independent diffusion across cytoplasmic membranes and are utilized as nutrients, building blocks, and signaling intermediates. Here we describe how the association of long-chain fatty acids to a partially unfolded, extracellular protein can alter the presentation to target cells and cellular effects. HAMLET (human α-lactalbumin made lethal to tumor cells) is a tumoricidal complex of partially unfolded α-lactalbumin and oleic acid (OA). As OA lacks independent tumoricidal activity at concentrations equimolar to HAMLET, the contribution of the lipid has been debated. We show by natural abundance ¹³C NMR that the lipid in HAMLET is deprotonated and by chromatography that oleate rather than oleic acid is the relevant HAMLET constituent. Compared with HAMLET, oleate (175 μm) showed weak effects on ion fluxes and gene expression. Unlike HAMLET, which causes metabolic paralysis, fatty acid metabolites were less strongly altered. The functional overlap increased with higher oleate concentrations (500 μm). Cellular responses to OA were weak or absent, suggesting that deprotonation favors cellular interactions of fatty acids. Fatty acids may thus exert some of their essential effects on host cells when in the deprotonated state and when presented in the context of a partially unfolded protein.     

Anti-inflammatory IgG production requires functional P1 promoter in β-galactoside α2,6-sialyltransferase 1 (ST6Gal-1) gene

The anti-inflammatory properties associated with intravenous immunoglobulin therapy require the sialic acid modification of the N-glycan of the Fc domain of IgG. Sialylation of the Fc fragment is mediated by β-galactoside α2,6-sialyltransferase 1 (ST6Gal-1), acting on the Gal(β4)GlcNAc terminal structure of the biantennary N-glycans on the Fc domain. However, little is known regarding the in vivo regulation of Fc sialylation and its role in the progression of inflammatory processes. Here, we report that decreased Fc sialylation of circulatory IgG accompanies the acute phase response elicited by turpentine exposure or upon acute exposure to either nontypeable Haemophilus influenzae or ovalbumin. However, Fc sialylation was increased 3-fold from the base line upon transition to chronic inflammation by repeated exposure to challenge. The P1 promoter of the ST6Gal-1 gene is critical for Fc sialylation, but P1 does not drive ST6Gal-1 expression in B cells. The Siat1ΔP1 mouse, with a dysfunctional P1 promoter, was unable to produce sialylated Fc in the systemic circulation, despite the presence of Gal(β4)GlcNAc termini on the Fc glycans. The major contribution of P1 action is to synthesize ST6Gal-1 enzymes that are deposited into the systemic circulation. The data strongly indicate that this pool of extracellular ST6Gal-1 in the blood impacts the sialylation of IgG Fc and that defective Fc sialylation is likely a major contributing mechanism for the proinflammatory tendencies previously noted in Siat1ΔP1 animals.     

Regulation of cAMP-dependent Protein Kinases: THE HUMAN PROTEIN KINASE X (PrKX) REVEALS THE ROLE OF THE CATALYTIC SUBUNIT αH-αI LOOP*

cAMP-dependent protein kinases are reversibly complexed with any of the four isoforms of regulatory (R) subunits, which contain either a substrate or a pseudosubstrate autoinhibitory domain. The human protein kinase X (PrKX) is an exemption as it is inhibited only by pseudosubstrate inhibitors, i.e. RIα or RIβ but not by substrate inhibitors RIIα or RIIβ. Detailed examination of the capacity of five PrKX-like kinases ranging from human to protozoa (Trypanosoma brucei) to form holoenzymes with human R subunits in living cells shows that this preference for pseudosubstrate inhibitors is evolutionarily conserved. To elucidate the molecular basis of this inhibitory pattern, we applied bioluminescence resonance energy transfer and surface plasmon resonance in combination with site-directed mutagenesis. We observed that the conserved αH-αI loop residue Arg-283 in PrKX is crucial for its RI over RII preference, as a R283L mutant was able to form a holoenzyme complex with wild type RII subunits. Changing the corresponding αH-αI loop residue in PKA Cα (L277R), significantly destabilized holoenzyme complexes in vitro, as cAMP-mediated holoenzyme activation was facilitated by a factor of 2–4, and lead to a decreased affinity of the mutant C subunit for R subunits, significantly affecting RII containing holoenzymes.     

Diversity of Structure and Function of α1α6β3δ GABAA Receptors: COMPARISON WITH α1β3δ AND α6β3δ RECEPTORS*

δ subunit-containing γ-aminobutyric acid, type A (GABA_A)receptors are expressed extrasynaptically and mediate tonic inhibition. In cerebellar granule cells, they often form receptors together with α₁ and/or α₆ subunits. We were interested in determining the architecture of receptors containing both subunits. We predefined the subunit arrangement of several different GABA_A receptor pentamers by concatenation. These receptors composed of α₁, α₆, β₃, and δ subunits were expressed in Xenopus oocytes. Currents elicited in response to GABA were determined in the presence and absence of 3α,21-dihydroxy-5α-pregnan-20-one (THDOC) or ethanol, or currents were elicited by 4,5,6,7-tetrahydroisoxazolo[5,4-c]-pyridin-3-ol (THIP). Several subunit configurations formed active channels. We therefore conclude that δ can assume multiple positions in a receptor pentamer made up of α₁, α₆, β₃, and δ subunits. The different receptors differ in their functional properties. Functional expression of one receptor type was only evident in the combined presence of the neurosteroid THDOC with the channel agonist GABA. Most, but not all, receptors active with GABA/THDOC responded to THIP. None of the receptors was modulated by ethanol concentrations up to 30 mm. Several observations point to a preferred position of δ subunits between two α subunits in α₁α₆β₃δ receptors. This property is shared by α₁β₃δ and α₆β₃δ receptors, but there are differences in the additionally expressed isoforms.     

SYNTHESIS OF 5-(PYRIDINYL AND PYRIDINIUMYL)-2′-DEOXYURIDINE NUCLEOSIDES: REVERSIBLE ELECTRON TRAPS FOR DNA

The desire to produce reversible electron traps for direct, room temperature studies of excess electron transport in DNA duplexes and hairpins motivated our efforts first to link pyridines to 2′-deoxyuridine (pyridinyl-dU) and then to convert these new conjugates into pyridiniumyl-dU nucleosides. Base sensitivity studies presented here rule out general use of bipyridinediiumyl compounds, but show that pyridiniumyl compounds are suitable for use under the strand cleavage and base deprotection procedures required for automated solid-phase oligonucleotide synthesis. This paper presents the synthesis of four 5′-O-DMT-protected 5-(N-methylpyridiniumyl)-dU conjugates using either ethynyl or ethylenyl linkers to join the pyridiniumyl and dU subunits.     

17β-Estradiol Inhibits Phosphorylation of Stromal Interaction Molecule 1 (STIM1) Protein: IMPLICATION FOR STORE-OPERATED CALCIUM ENTRY AND CHRONIC LUNG DISEASES*

Sex plays a significant role in the development of lung diseases including asthma, cancer, chronic bronchitis, and cystic fibrosis. In cystic fibrosis, 17β-estradiol (E2) may inhibit store-operated Ca²⁺ entry (SOCE) to impinge upon airway secretions, leaving females at greater risk of contracting lung infections. Stromal interaction molecule 1 (STIM1)-mediated SOCE is essential for cell homeostasis and regulates numerous processes including cell proliferation, smooth muscle contraction, and secretion. E2 can signal nongenomically to modulate Ca²⁺ signaling, but little is known of the underlying mechanisms. We found that E2 exposure inhibited STIM1 translocation in airway epithelia, preventing SOCE. This correlated with a decrease in STIM1-STIM1 FRET and STIM1 mobility in E2-exposed HEK293T cells co-expressing estrogen receptor α. We also examined the role of STIM1 phosphorylation in E2-mediated inhibition of STIM1 mobility. STIM1 is basally phosphorylated at serine 575, which is required for SOCE. Exposure to E2 significantly decreased STIM1 serine phosphorylation. Mutating serine 575 to an alanine blocked STIM1 phosphorylation, reduced basal STIM1 mobility, and rendered STIM1 insensitive to E2. These data indicate that E2 can signal nongenomically by inhibiting basal phosphorylation of STIM1, leading to a reduction in SOCE.     

Splice Cassette II of Na+,HCO3? Cotransporter NBCn1 (slc4a7) Interacts with Calcineurin A: IMPLICATIONS FOR TRANSPORTER ACTIVITY AND INTRACELLULAR pH CONTROL DURING RAT ARTERY CONTRACTIONS*

Activation of Na⁺,HCO₃⁻ cotransport in vascular smooth muscle cells (VSMCs) contributes to intracellular pH (pH_i) control during artery contraction, but the signaling pathways involved have been unknown. We investigated whether physical and functional interactions between the Na⁺,HCO₃⁻ cotransporter NBCn1 (slc4a7) and the Ca²⁺/calmodulin-activated serine/threonine phosphatase calcineurin exist and play a role for pH_i control in VSMCs. Using a yeast two-hybrid screen, we found that splice cassette II from the N terminus of NBCn1 interacts with calcineurin Aβ. When cassette II was truncated or mutated to disrupt the putative calcineurin binding motif PTVVIH, the interaction was abolished. Native NBCn1 and calcineurin Aβ co-immunoprecipitated from A7r5 rat VSMCs. A peptide (acetyl-DDIPTVVIH-amide), which mimics the putative calcineurin binding motif, inhibited the co-immunoprecipitation whereas a mutated peptide (acetyl-DDIATAVAA-amide) did not. Na⁺,HCO₃⁻ cotransport activity was investigated in VSMCs of mesenteric arteries after an NH₄⁺ prepulse. During depolarization with 50 mm extracellular K⁺ to raise intracellular [Ca²⁺], Na⁺,HCO₃⁻ cotransport activity was inhibited 20–30% by calcineurin inhibitors (FK506 and cyclosporine A). FK506 did not affect Na⁺,HCO₃⁻ cotransport activity in VSMCs when cytosolic [Ca²⁺] was lowered by buffering, nor did it disrupt binding between NBCn1 and calcineurin Aβ. FK506 augmented the intracellular acidification of VSMCs during norepinephrine-induced artery contractions. No physical or functional interactions between calcineurin Aβ and the Na⁺/H⁺ exchanger NHE1 were observed in VSMCs. In conclusion, we demonstrate a physical interaction between calcineurin Aβ and cassette II of NBCn1. Intracellular Ca²⁺ activates Na⁺,HCO₃⁻ cotransport activity in VSMCs in a calcineurin-dependent manner which is important for protection against intracellular acidification.     

α-2,6唾液酸转移酶(ST6Gal1)的cDNA克隆、原核表达及生物活性研究

从HepG2细胞中提取总RNA,经RT-PCR扩增α-2,6唾液酸转移酶基因,构建于pMD-18T克隆载体中,经序列测定后与GenBank中报道的已知序列比对完全一致,再将已知目的序列插入原核表达载体pET-28a(+)中。将构建正确的原核表达载体转化表达受体菌BL21（DE3）中,IPTG诱导其进行表达并鉴定目的蛋白,对鉴定正确的目的蛋白复性后经Ni2 +亲和层析柱纯化获得高纯度的α-2,6唾液酸转移酶蛋白。然后用霍乱弧菌神经氨酸酶处理健康鸡红细胞,清除鸡红细胞表面所有类型的唾液酸寡糖链;再用表达的α-2,6唾液酸转移酶以 CMP-唾液酸作为底物在霍乱弧菌神经氨酸酶处理的鸡红细胞表面标记上SAα2,6 Gal受体。将标记有SAα2,6Gal受体的鸡红细胞应用微量血凝试验和流式细胞仪进行检测,以验证所表达蛋白的生物活性。结果表明,原核表达的α-2,6唾液酸转移酶具有较好的生物活性。     

1-(3-C-ETHYNYL-β-D-RIBO-PENTOFURANOSYL)CYTOSINE (ECYD,TAS-106)1: ANTITUMOR EFFECT AND MECHANISM OF ACTION

The antitumor activity, cellular metabolism and mechanism of action of the antitumor nucleoside analog, 1-(3-C-ethynyl-β-D-ribo-pentofuranosyl)cytosine (ECyd) are described.     

SYNTHESIS OF UNNATURAL 7-SUBSTITUTED-1-(2-DEOXY-β-D-RIBOFURANOSYL)ISOCARBOSTYRILS†: “THYMINE REPLACEMENT” ANALOGS OF DEOXYTHYMIDINE FOR EVALUATION AS ANTIVIRAL AND ANTICANCER AGENTS

A group of unnatural 1-(2-deoxy-β-D-ribofuranosyl)isocarbostyrils having a variety of C-7 substituents [H, 4,7-(NO₂)₂, I, CF₃, CN, (E)-CH=CH-I, -C═CH, -C═C-I, -C═C-Br, -C═C-Me], designed as nucleoside mimics, were synthesized for evaluation as anticancer and antiviral agents. This class of compounds exhibited weak cytotoxicity in a MTT assay (CC₅₀=10^?3 to 10^?5 M range) with the 4,7-dinitro derivative being the most cytotoxic, relative to thymidine (CC₅₀=10^?3 to 10^?5 M range), against a variety of cancer cell lines. The 4,7-dinitro, 7-I and 7-C═CH compounds exhibited similar cytotoxicity against non-transfected (KBALB, 143B), and HSV-1 TK⁺ gene transfected (KBALB-STK, 143B-LTK) cancer cell lines possessing the herpes simplex virus type 1 (HSV-1) thymidine kinase gene (TK⁺). This observation indicates that these compounds are not substrates for HSV type-1 TK, and are therefore unlikely to be useful in gene therapy based on the HSV gene therapy paradigm.

     

SYNTHESIS AND BIOLOGICAL EVALUATION OF SOME ACYCLIC α-(1H-PYRAZOLO[3,4-d]PYRIMIDIN-4-YL)THIOALKYLAMIDE NUCLEOSIDES

ABSTRACT

The chemical synthesis of some acyclic α-(1H-pyrazolo[3,4-d]pyrimidin-4-yl)thioalkylamide nucleosides (10–12)a–c is described. The treatment of 1H-pyrazolo[3,4-d]pyrimidin-4-thione 1 with compounds 2a–c gave, regioselectively, ethyl α-(1H-pyrazolo[3,4-d]pyrimidin-4-yl)thioalkylates 3a-c, respectively. These heterocycles were alkylated, separately, with alkylating agents 4, 5 and 6 to give, regioselectively, the N₁-acyclic nucleosides (7-9)a-c which were deprotected to afford the desired products (10-12)a-c. All synthetic compounds were characterized on the basis of their physical and spectroscopic properties. The products (10-12)a–c were evaluated for their inhibitory effects against the replication of HIV-1 (III_B), HIV-2 (ROD), various DNA viruses, a variety of tumor-cell lines and M. tuberculosis. No marked biological activity was found.     

SUMMER DECLINE OF ULVA LACTUCA (CHLOROPHYTA) IN A EUTROPHIC EMBAYMENT: INTERACTIVE EFFECTS OF TEMPERATURE AND NITROGEN AVAILABILITY?1

Throughout the summer, abundance of Ulva lactuca L. declined while biomass of Cladophora vagabunda (L.) van den Hoek and Gracilaria tikvahiae McLachlan increased in a New England embayment undergoing eu-trophication (Waquoit Bay, Massachusetts). We investigated the physiological basis for the summer dieback, focusing on temporal variations in photosynthetic performance and tissue nitrogen (N). We also compared photosynthetic and N uptake capabilities of U. lactuca with other abundant species in this eutrophic system. Photosynthetic egiciency and capacity of U. lactuca declined markedly at 25°C, compared with a spring (15°C) peak in photosynthetic performance; P_max was 4.6 ± 0.3 and 1.8 ± 0.6 μmol O₂.m^?2.s^?1 during spring and summer, respectively. Notably, summer p_max of other abundant species of the embayment was 1.5–3 × higher than that measured for U. lactuca. Ulva lactuca showed a signifciant photosynthetic response to dissolved inorganic carbon enrichment during summer, when water-column-dissolved CO₂ levels were 20% of spring values. Although ammonium uptake rates of U. lactuca were extremely high at both subsaturating (15μM) and saturating (75 μM) N concentrations, as predicted by the functional-form hypothesis, tissue N fell to 1% by late summer. We suggest that a carbon imbalance, initiated by rising water temperatures and declining water-column N; thermal stress; and biological factors (competition, grazing) all contribute to the recurrent summer decline of U. lactuca in this shallow, eutrophic embayment. Thus, while the morphology of U. lactuca might be considered a successful strategy for disturbed, or “stressed” (sensu Littler and Littler 1980), habitats, its inability to persist and flourish in this environment emphasizes the complexity of factors at work in natural systems.     

The Role of Mg(II) in DNA Cleavage Site Recognition in Group II Intron Ribozymes: SOLUTION STRUCTURE AND METAL ION BINDING SITES OF THE RNA·DNA COMPLEX*

Group II intron ribozymes catalyze the cleavage of (and their reinsertion into) DNA and RNA targets using a Mg²⁺-dependent reaction. The target is cleaved 3′ to the last nucleotide of intron binding site 1 (IBS1), one of three regions that form base pairs with the intron''s exon binding sites (EBS1 to -3). We solved the NMR solution structure of the d3′ hairpin of the Sc.ai5γ intron containing EBS1 in its 11-nucleotide loop in complex with the dIBS1 DNA 7-mer and compare it with the analogous RNA·RNA contact. The EBS1·dIBS1 helix is slightly flexible and non-symmetric. NMR data reveal two major groove binding sites for divalent metal ions at the EBS1·dIBS1 helix, and surface plasmon resonance experiments show that low concentrations of Mg²⁺ considerably enhance the affinity of dIBS1 for EBS1. Our results indicate that identification of both RNA and DNA IBS1 targets, presentation of the scissile bond, and stabilization of the structure by metal ions are governed by the overall structure of EBS1·dIBS1 and the surrounding loop nucleotides but are irrespective of different EBS1·(d)IBS1 geometries and interstrand affinities.     

A Blood-Brain Barrier (BBB) Disrupter Is Also a Potent α-Synuclein (α-syn) Aggregation Inhibitor: A NOVEL DUAL MECHANISM OF MANNITOL FOR THE TREATMENT OF PARKINSON DISEASE (PD)*

The development of disease-modifying therapy for Parkinson disease has been a main drug development challenge, including the need to deliver the therapeutic agents to the brain. Here, we examined the ability of mannitol to interfere with the aggregation process of α-synuclein in vitro and in vivo in addition to its blood-brain barrier-disrupting properties. Using in vitro studies, we demonstrated the effect of mannitol on α-synuclein aggregation. Although low concentration of mannitol inhibited the formation of fibrils, high concentration significantly decreased the formation of tetramers and high molecular weight oligomers and shifted the secondary structure of α-synuclein from α-helical to a different structure, suggesting alternative potential pathways for aggregation. When administered to a Parkinson Drosophila model, mannitol dramatically corrected its behavioral defects and reduced the amount of α-synuclein aggregates in the brains of treated flies. In the mThy1-human α-synuclein transgenic mouse model, a decrease in α-synuclein accumulation was detected in several brain regions following treatment, suggesting that mannitol promotes α-synuclein clearance in the cell bodies. It appears that mannitol has a general neuroprotective effect in the transgenic treated mice, which includes the dopaminergic system. We therefore suggest mannitol as a basis for a dual mechanism therapeutic agent for the treatment of Parkinson disease.     

Shear-induced Interleukin-6 Synthesis in Chondrocytes: ROLES OF E PROSTANOID (EP) 2 AND EP3 IN cAMP/PROTEIN KINASE A- AND PI3-K/Akt-DEPENDENT NF-κB ACTIVATION*

Mechanical overloading of cartilage producing hydrostatic stress, tensile strain, and fluid flow can adversely affect chondrocyte function and precipitate osteoarthritis (OA). Application of high fluid shear stress to chondrocytes recapitulates the earmarks of OA, as evidenced by the release of pro-inflammatory mediators, matrix degradation, and chondrocyte apoptosis. Elevated levels of cyclooxygenase-2 (COX-2), prostaglandin (PG) E₂, and interleukin (IL)-6 have been reported in OA cartilage in vivo, and in shear-activated chondrocytes in vitro. Although PGE₂ positively regulates IL-6 synthesis in chondrocytes, the underlying signaling pathway of shear-induced IL-6 expression remains unknown. Using the human T/C-28a2 chondrocyte cell line as a model system, we demonstrate that COX-2-derived PGE₂ signals via up-regulation of E prostanoid (EP) 2 and down-regulation of EP3 receptors to raise intracellular cAMP, and activate protein kinase A (PKA) and phosphatidylinositol 3-kinase (PI3-K)/Akt pathways. PKA and PI3-K/Akt transactivate the NF-κB p65 subunit via phosphorylation at Ser-276 and Ser-536, respectively. Binding of p65 to the IL-6 promoter elicits IL-6 synthesis in sheared chondrocytes. Selective knockdown of EP2 or ectopic expression of EP3 blocks PKA- and PI3-K/Akt-dependent p65 activation and markedly diminishes shear-induced IL-6 expression. Similar inhibitory effects on IL-6 synthesis were observed by inhibiting PKA, PI3-K, or NF-κB using pharmacological and/or genetic interventions. Reconstructing the signaling network regulating shear-induced IL-6 expression in chondrocytes may provide insights for developing therapeutic strategies for arthritic disorders and for culturing artificial cartilage in bioreactors.