Nickel Ions Inhibit Histone Demethylase JMJD1A and DNA Repair Enzyme ABH2 by Replacing the Ferrous Iron in the Catalytic Centers

Iron- and 2-oxoglutarate-dependent dioxygenases are a diverse family of non-heme iron enzymes that catalyze various important oxidations in cells. A key structural motif of these dioxygenases is a facial triad of 2-histidines-1-carboxylate that coordinates the Fe(II) at the catalytic site. Using histone demethylase JMJD1A and DNA repair enzyme ABH2 as examples, we show that this family of dioxygenases is highly sensitive to inhibition by carcinogenic nickel ions. We find that, with iron, the 50% inhibitory concentrations of nickel (IC₅₀ [Ni(II)]) are 25 μm for JMJD1A and 7.5 μm for ABH2. Without iron, JMJD1A is 10 times more sensitive to nickel inhibition with an IC₅₀ [Ni(II)] of 2.5 μm, and approximately one molecule of Ni(II) inhibits one molecule of JMJD1A, suggesting that nickel causes inhibition by replacing the iron. Furthermore, nickel-bound JMJD1A is not reactivated by excessive iron even up to a 2 mm concentration. Using x-ray absorption spectroscopy, we demonstrate that nickel binds to the same site in ABH2 as iron, and replacement of the iron by nickel does not prevent the binding of the cofactor 2-oxoglutarate. Finally, we show that nickel ions target and inhibit JMJD1A in intact cells, and disruption of the iron-binding site decreases binding of nickel ions to ABH2 in intact cells. Together, our results reveal that the members of this dioxygenase family are specific targets for nickel ions in cells. Inhibition of these dioxygenases by nickel is likely to have widespread impacts on cells (e.g. impaired epigenetic programs and DNA repair) and may eventually lead to cancer development.     

Structural and functional analyses of the human Toll-like receptor 3. Role of glycosylation

Toll-like receptors (TLRs) play critical roles in bridging the innate and adaptive immune responses. The human TLR3 recognizes foreign-derived double-stranded RNA and endogenous necrotic cell RNA as ligands. Herein we characterized the contribution of glycosylation to TLR3 structure and function. Exogenous addition of purified extracellular domain of TLR3 (hTLR3 ECD) expressed in human embryonic kidney cells was found to inhibit TLR3-dependent signaling, thus providing a reagent for structural and functional characterization. Approximately 35% of the mass of the hTLR3 ECD was due to posttranslational modification, with N-linked glycosyl groups contributing substantially to the additional mass. Cells treated with tunicamycin, an inhibitor of glycosylation, prevented TLR3-induced NF-kappaB activation, confirming that N-linked glycosylation is required for bioactivity of this receptor. Further, mutations in two of these predicted glycosylation sites impaired TLR3 signaling without obviously affecting the expression of the protein. Single-particle structures reconstructed from electron microscopy images and two-dimensional crystallization revealed that hTLR3 ECD forms a horseshoe structure similar to the recently elucidated x-ray structure of the protein expressed in insect cells using baculovirus vectors (Choe, J., Kelker, M. S., and Wilson, I. A. (2005) Science 309, 581-585 and Bell, J. K., Botos, I., Hall, P. R., Askins, J., Shiloach, J., Segal, D. M., and Davies, D. R. (2005) Proc. Natl. Acad. Sci. U. S. A. 102, 10976-10980). There are, however, notable differences between the human cell-derived and insect cell-derived structures, including features attributable to glycosylation.     

Invasive pulmonary pseudallesheriosis in a cross-breed calf

Pulmonary pseudallescheriosis was diagnosed in a two-months old calf. Pneumonic lungs with yellow-white nodules on the surfaces revealed granulomatous lesions microscopically. Septate, pleomorphic hyphae were present in the central caseated core with a bright eosinophilic periphery surrounded by polymorphonuclear cells and macrophages followed by a zone of epithelioid cells admixed with lymphocytes and plasma cells. The fungal agent was demonstrated by Grocott's silver methenamine staining. On isolation, morphologically it was found to be indistinguishable from that ofPseudallescheria boydii. It appears to be first report of fatal mycotic pneumonia in a calf due toP. boydii. The emphasis is given for further detailed investigation on this aspect in veterinary mycopathology.     

Genetic Dissection of Component Traits for Salinity Tolerance at Reproductive Stage in Rice

Plant Molecular Biology Reporter - Rice is highly sensitive to salt stress at flowering stage. With the objective of detection of quantitative trait loci (QTLs) in multi-environment for this stage,...     

Kinesin-2 differentially regulates the anterograde axonal transports of acetylcholinesterase and choline acetyltransferase in Drosophila

Choline acetyltransferase (ChAT) and acetylcholinesterase (AChE) are involved in acetylcholine synthesis and degradation at pre- and postsynaptic compartments, respectively. Here we show that their anterograde transport in Drosophila larval ganglion is microtubule-dependent and occurs in two different time profiles. AChE transport is constitutive while that of ChAT occurs in a brief pulse during third instar larva stage. Mutations in the kinesin-2 motor subunit Klp64D and separate siRNA-mediated knock-outs of all the three kinesin-2 subunits disrupt the ChAT and AChE transports, and these antigens accumulate in discrete nonoverlapping punctae in neuronal cell bodies and axons. Quantification analysis further showed that mutations in Klp64D could independently affect the anterograde transport of AChE even before that of ChAT. Finally, ChAT and AChE were coimmunoprecipitated with the kinesin-2 subunits but not with each other. Altogether, these suggest that kinesin-2 independently transports AChE and ChAT within the same axon. It also implies that cargo availability could regulate the rate and frequency of transports by kinesin motors.     

A metadynamic approach to understand the recognition mechanism of the histone H3 tail with the ATRXADD domain

The binding affinity between the histone 3 (H3) tail and the ADD domain of ATRX (ATRX_ADD) increases with the subsequent addition of methyl groups on lysine 9 on H3. To improve our understanding of how the difference in methylation state affects binding between H3 and the ATRX_ADD, we adopted a metadynamic approach to explore the recognition mechanism between the two proteins and identify the key intermolecular interactions that mediate this protein-peptide interaction (PPI). The non-methylated H3 peptide is recognized only by the PHD finger of ATRX_ADD while mono-, di-, and trimethylated H3 is recognized by both the PHD and GATA-like zinc finger of the domain. Furthermore, water molecules play an important role in orienting the lysine 9 anchor towards the GATA-like zinc finger, which results in stabilizing the lysine 9 binding pocket on ATRX_ADD. We compared our computational results against experimentally determined NMR and X-ray structures by demonstrating the RMSD, order parameter S² and hydration number of the complex. The metadynamics data provide new insight into roles of water-bridges and the mechanisms through which K9 hydration stabilizes the H3K9me3:ATRX_ADD PPI, providing context for the high affinity demonstrated between this protein and peptide.     

Changes in bronchial and pulmonary arterial blood flow with progressive tension pneumothorax

Carvalho, Paula, Jacob Hildebrandt, and Nirmal B. Charan.Changes in bronchial and pulmonary arterial blood flow with progressive tension pneumothorax. J. Appl.Physiol. 81(4): 1664-1669, 1996.We studied theeffects of unilateral tension pneumothorax and its release on bronchialand pulmonary arterial blood flow and gas exchange in 10 adultanesthetized and mechanically ventilated sheep with chronicallyimplanted ultrasonic flow probes. Right pleural pressure (Ppl) wasincreased in two steps from 5 to 10 and 25 cmH₂O and then decreased to 10 and5 cmH₂O. Each level of Pplwas maintained for 5 min. Bronchial blood flow, right and leftpulmonary arterial flows, cardiac output(T),hemodynamic measurements, and arterial blood gases were obtained at theend of each period. Pneumothorax resulted in a 66% decrease inT, bronchialblood flow decreased by 84%, and right pulmonary arterial flowdecreased by 80% at Ppl of 25 cmH₂O(P < 0.001). At peak Ppl, themajority ofT was due toblood flow through the left pulmonary artery. With resolution ofpneumothorax, hemodynamic parameters normalized, although abnormalitiesin gas exchange persisted for 60-90 min after recovery and wereassociated with a decrease in total respiratory compliance.

     

Interaction between sulphur mobilisation proteins SufB and SufC: evidence for an iron-sulphur cluster biogenesis pathway in the apicoplast of Plasmodium falciparum

The plastid of Plasmodium falciparum, the apicoplast, performs metabolic functions essential to the parasite. Various reactions in the plastid require the assembly of [Fe-S] prosthetic groups on participating proteins as well as the reductant activity of ferredoxin that is converted from its apo-form by the assembly of [Fe-S] clusters inside the apicoplast. The [Fe-S] assembly pathway involving sulphur mobilising Suf proteins has been predicted to function in the apicoplast with one component (PfSufB) encoded by the plastid genome itself. We demonstrate the ATPase activity of recombinant P. falciparum nuclear-encoded SufC and its localisation in the apicoplast. Further, an internal region of apicoplast SufB was used to detect PfSufB-PfSufC interaction in vitro; co-elution of SufB from parasite lysate with recombinant PfSufC on an affinity column also indicated an interaction of the two proteins. As a departure from bacterial SufB and similar to reported plant plastid SufB, apicoplast SufB exhibited ATPase activity, suggesting the evolution of specialised functions in the plastid counterparts. Our results provide experimental evidence for an active Suf pathway in the Plasmodium apicoplast.     

Closely related oxidized phospholipids differentially modulate the physicochemical properties of lipid particles

Oxidation of glycerophospholipids results in the formation of large variety of oxidized phospholipid products that differs significantly in their chemical compositions and molecular structures. Biological activities of these oxidized products also differ considerably. Here we report the comparisons of the physicochemical properties of non-oxidized phospholipid particle containing two closely related tOx-PLs: 1-palmitoyl-2-(5-keto-6-octendioyl)-sn-glycero-3-phosphocholine (KOdiA-PC) and 1-palmitoyl-2-(9-keto-10-dodecendioyl)-sn-glycero-3-phosphocholine (KDdiA-PC). DMPC (1,2-dimyristoyl-sn-glycero-3-phosphocholine) was used as a model membrane non-oxidized phospholipid. Physicochemical properties of the lipid particles were characterized by using fluorescence spectroscopy, native polyacrylamide gel and agarose gel electrophoresis. Our result shows that the presence of closely related tOx-PLs, which differ only in the chemical composition of the oxidized fatty acyl chains at the sn-2 position, exerts considerably different effect on the physicochemical properties of non-oxidized phospholipid particles containing them.