Biochemical and Spectroscopic Characterization of the Human Mitochondrial Amidoxime Reducing Components hmARC-1 and hmARC-2 Suggests the Existence of a New Molybdenum Enzyme Family in Eukaryotes

The mitochondrial amidoxime reducing component mARC is a newly discovered molybdenum enzyme that is presumed to form the catalytical part of a three-component enzyme system, consisting of mARC, heme/cytochrome b₅, and NADH/FAD-dependent cytochrome b₅ reductase. mARC proteins share a significant degree of homology to the molybdenum cofactor-binding domain of eukaryotic molybdenum cofactor sulfurase proteins, the latter catalyzing the post-translational activation of aldehyde oxidase and xanthine oxidoreductase. The human genome harbors two mARC genes, referred to as hmARC-1/MOSC-1 and hmARC-2/MOSC-2, which are organized in a tandem arrangement on chromosome 1. Recombinant expression of hmARC-1 and hmARC-2 proteins in Escherichia coli reveals that both proteins are monomeric in their active forms, which is in contrast to all other eukaryotic molybdenum enzymes that act as homo- or heterodimers. Both hmARC-1 and hmARC-2 catalyze the N-reduction of a variety of N-hydroxylated substrates such as N-hydroxy-cytosine, albeit with different specificities. Reconstitution of active molybdenum cofactor onto recombinant hmARC-1 and hmARC-2 proteins in the absence of sulfur indicates that mARC proteins do not belong to the xanthine oxidase family of molybdenum enzymes. Moreover, they also appear to be different from the sulfite oxidase family, because no cysteine residue could be identified as a putative ligand of the molybdenum atom. This suggests that the hmARC proteins and sulfurase represent members of a new family of molybdenum enzymes.     

Cyclic AMP potentiates Ca2+-dependent exocytosis in pancreatic duct epithelial cells

Exocytosis is evoked by intracellular signals, including Ca²⁺ and protein kinases. We determined how such signals interact to promote exocytosis in exocrine pancreatic duct epithelial cells (PDECs). Exocytosis, detected using carbon-fiber microamperometry, was stimulated by [Ca²⁺]_i increases induced either through Ca²⁺ influx using ionomycin or by activation of P2Y2 or protease-activated receptor 2 receptors. In each case, the exocytosis was strongly potentiated when cyclic AMP (cAMP) was elevated either by activating adenylyl cyclase with forskolin or by activating the endogenous vasoactive intestinal peptide receptor. This potentiation was completely inhibited by H-89 and partially blocked by Rp-8-Br-cAMPS, inhibitors of protein kinase A. Optical monitoring of fluorescently labeled secretory granules showed slow migration toward the plasma membrane during Ca²⁺ elevations. Neither this Ca²⁺-dependent granule movement nor the number of granules found near the plasma membrane were detectably changed by raising cAMP, suggesting that cAMP potentiates Ca²⁺-dependent exocytosis at a later stage. A kinetic model was made of the exocytosis stimulated by UTP, trypsin, and Ca²⁺ ionophores with and without cAMP increase. In the model, without a cAMP rise, receptor activation stimulates exocytosis both by Ca²⁺ elevation and by the action of another messenger(s). With cAMP elevation the docking/priming step for secretory granules was accelerated, augmenting the releasable granule pool size, and the Ca²⁺ sensitivity of the final fusion step was increased, augmenting the rate of exocytosis. Presumably both cAMP actions require cAMP-dependent phosphorylation of target proteins. cAMP-dependent potentiation of Ca²⁺-induced exocytosis has physiological implications for mucin secretion and, possibly, for membrane protein insertion in the pancreatic duct. In addition, mechanisms underlying this potentiation of slow exocytosis may also exist in other cell systems.     

The Small Subunit AroB of Arsenite Oxidase: LESSONS ON THE [2Fe-2S] RIESKE PROTEIN SUPERFAMILY

Here, we describe the characterization of the [2Fe-2S] clusters of arsenite oxidases from Rhizobium sp. NT-26 and Ralstonia sp. 22. Both reduced Rieske proteins feature EPR signals similar to their homologs from Rieske-cyt b complexes, with g values at 2.027, 1.88, and 1.77. Redox titrations in a range of pH values showed that both [2Fe-2S] centers have constant E_m values up to pH 8 at ∼+210 mV. Above this pH value, the E_m values of both centers are pH-dependent, similar to what is observed for the Rieske-cyt b complexes. The redox properties of these two proteins, together with the low E_m value (+160 mV) of the Alcaligenes faecalis arsenite oxidase Rieske (confirmed herein), are in line with the structural determinants observed in the primary sequences, which have previously been deduced from the study of Rieske-cyt b complexes. Since the published E_m value of the Chloroflexus aurantiacus Rieske (+100 mV) is in conflict with this sequence analysis, we re-analyzed membrane samples of this organism and obtain a new value (+200 mV). Arsenite oxidase activity was affected by quinols and quinol analogs, which is similar to what is found with the Rieske-cyt b complexes. Together, these results show that the Rieske protein of arsenite oxidase shares numerous properties with its counterpart in the Rieske-cyt b complex. However, two cysteine residues, strictly conserved in the Rieske-cyt b-Rieske and considered to be crucial for its function, are not conserved in the arsenite oxidase counterpart. We discuss the role of these residues.     

Fine-mapping and candidate gene analysis of bovine spinal muscular atrophy

Bovine spinal muscular atrophy (SMA), an autosomal recessive neurodegenerative disease, has been mapped at moderate resolution to the distal part of Chromosome 24. In this article we confirm this location and fine-map the SMA locus to an interval of approximately 0.8 cM at the very distal end of BTA24. Despite remarkable similarity to human SMA, the causative gene SMN can be excluded in bovine SMA. However, the interval where the disease now has been mapped contains BCL2, like SMN an antiapoptotic factor, and shown to bind to SMN. Moreover, knockout mice lacking the BCL2 gene show rapid motor neuron degeneration with early postnatal onset, as observed in bovine SMA. A comparative cattle/human map of the distal end of BTA24, based on the emerging bovine genome sequencing data, shows conserved synteny to HSA18 with hints of a segmental duplication and pericentic inversion just after the last available bovine marker DIK4971. This synteny lets us conclude that SMA is in immediate vicinity of the telomere. Candidate gene analysis of BCL2, however, excludes most of this gene, except its promoter region, and draws attention to the neighboring gene VPS4B, part of the endosomal protein-sorting machinery ESCRT-III which is involved in several neurodegenerative diseases. Stefan Krebs and Ivica Medugorac contributed equally to this work and agreed to be considered as first authors.     

Myosin heavy-chain kinase A from Dictyostelium possesses a novel actin-binding domain that cross-links actin filaments

Myosin heavy-chain kinase A (MHCK A) catalyses the disassembly of myosin II filaments in Dictyostelium cells via myosin II heavy-chain phosphorylation. MHCK A possesses a 'coiled-coil'-enriched domain that mediates the oligomerization, cellular localization and actin-binding activities of the kinase. F-actin (filamentous actin) binding by the coiled-coil domain leads to a 40-fold increase in MHCK A activity. In the present study we examined the actin-binding characteristics of the coiled-coil domain as a means of identifying mechanisms by which MHCK A-mediated disassembly of myosin II filaments can be regulated in the cell. Co-sedimentation assays revealed that the coiled-coil domain of MHCK A binds co-operatively to F-actin with an apparent K(D) of approx. 0.5 muM and a stoichiometry of approx. 5:1 [actin/C(1-498)]. Further analyses indicate that the coiled-coil domain binds along the length of the actin filament and possesses at least two actin-binding regions. Quite surprisingly, we found that the coiled-coil domain cross-links actin filaments into bundles, indicating that MHCK A can affect the cytoskeleton in two important ways: (1) by driving myosin II-filament disassembly via myosin II heavy-chain phosphorylation, and (2) by cross-linking/bundling actin filaments. This discovery, along with other supporting data, suggests a model in which MHCK A-mediated bundling of actin filaments plays a central role in the recruitment and activation of the kinase at specific sites in the cell. Ultimately this provides a means for achieving the robust and highly localized disruption of myosin II filaments that facilitates polarized changes in cell shape during processes such as chemotaxis, cytokinesis and multicellular development.     

Choosing SNPs using feature selection

A major challenge for genomewide disease association studies is the high cost of genotyping large number of single nucleotide polymorphisms (SNPs). The correlations between SNPs, however, make it possible to select a parsimonious set of informative SNPs, known as "tagging" SNPs, able to capture most variation in a population. Considerable research interest has recently focused on the development of methods for finding such SNPs. In this paper, we present an efficient method for finding tagging SNPs. The method does not involve computation-intensive search for SNP subsets but discards redundant SNPs using a feature selection algorithm. In contrast to most existing methods, the method presented here does not limit itself to using only correlations between SNPs in local groups. By using correlations that occur across different chromosomal regions, the method can reduce the number of globally redundant SNPs. Experimental results show that the number of tagging SNPs selected by our method is smaller than by using block-based methods. Supplementary website: http://htsnp.stanford.edu/FSFS/.     

Molybdenum and tungsten in biology

Molybdenum is the only second-row transition metal that is required by most living organisms, and the few species that do not require molybdenum use tungsten, which lies immediately below molybdenum in the periodic table. Because of their unique chemical versatility and unusually high bioavailability these two transition metals have been incorporated into the active sites of enzymes over the course of evolution. Enzymes that contain molybdenum or tungsten continue to be discovered and several crystal structures have become available recently. This new structural information has been complemented by spectroscopic and kinetic methods, as well as computational approaches. Together, these studies provide an increasingly detailed view of the reaction mechanisms and the correlation between the electronic structure of the active site and catalytic function, one of the fundamental goals in metallobiochemistry.     

Polar auxin transport: an early invention

In higher plants, cell-to-cell polar auxin transport (PAT) of the phytohormone auxin, indole-3-acetic acid (IAA), generates maxima and minima that direct growth and development. Although IAA is present in all plant phyla, PAT has only been detected in land plants, the earliest being the Bryophytes. Charophyta, a group of freshwater green algae, are among the first multicellular algae with a land plant-like phenotype and are ancestors to land plants. IAA has been detected in members of Charophyta, but its developmental role and the occurrence of PAT are unknown. We show that naphthylphthalamic acid (NPA)-sensitive PAT occurs in internodal cells of Chara corallina. The relatively high velocity (at least 4-5 cm/h) of auxin transport through the giant (3-5 cm) Chara cells does not occur by simple diffusion and is not sensitive to a specific cytoplasmic streaming inhibitor. The results demonstrate that PAT evolved early in multicellular plant life. The giant Chara cells provide a unique new model system to study PAT, as Chara allows the combining of real-time measurements and mathematical modelling with molecular, developmental, cellular, and electrophysiological studies.