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Human schistosomiasis is a disease which globally affects over 229 million people. Three major species affecting humans are Schistosoma mansoni, S. haematobium and S. japonicum. Previous treatment of S. mansoni includes the use of oxamniquine (OXA), a prodrug that is enzymatically activated in S. mansoni but is ineffective against S. haematobium and S. japonicum. The OXA activating enzyme was identified and crystallized, as being a S. mansoni sulfotransferase (Sm SULT). S. haematobium and S. japonicum possess homologs of Sm SULT (Sh SULT and Sj SULT) begging the question; why does oxamniquine fail to kill S. haematobium and S. japonicum adult worms? Investigation of the molecular structures of the sulfotransferases indicates that structural differences, specifically in OXA contact residues, do not abrogate OXA binding in the active sites as previously hypothesized. Data presented argue that the ability of SULTs to sulfate and thus activate OXA and its derivatives is linked to the ability of OXA to fit in the binding pocket to allow the transfer of a sulfur group. Image 1 • OXA can kill S. mansoni but not S. haematobium or S. japonicum. • S. mansoni whole worm homogenates activate OXA, while S. haematobium and S. japonicum homogenates do not. • Differences in SULT amino acid contacts do not abrogate OXA binding in S. haematobium but may affect binding in S. japonicum. • The ability of OXA or its derivative to fit in the binding pocket determines whether sulfation takes place and parasite killing results. [ABSTRACT FROM AUTHOR]

In eukaryotic cells, the introns are excised from pre-mRNA by the spliceosome. These introns typically have a lariat configuration due to the 2' - 5' phosphodiester bond between an internal branched residue and the 5' terminus of the RNA. The only enzyme known to selectively hydrolyze the 2' -5' linkage of these lariats is the RNA lariat debranching enzyme Dbr1. In humans, Dbr1 is involved in processes such as class-switch recombination of immunoglobulin genes, and its dysfunction is implicated in viral encephalitis, HIV, ALS, and cancer. However, mechanistic details of precisely how Dbr1 affects these processes are missing. Here we show that human Dbr1 contains a disordered C-terminal domain through sequence analysis and nuclear magnetic resonance. This domain stabilizes Dbr1 in vitro by reducing aggregation but is dispensable for debranching activity. We establish that Dbr1 requires Fe2+ for efficient catalysis and demonstrate that the noncatalytic protein Drn1 and the uncharacterized protein trichothiodystrophy nonphotosensitive 1 directly bind to Dbr1. We demonstrate addition of trichothiodystrophy nonphotosensitive 1 to in vitro debranching reactions increases the catalytic efficiency of human Dbr1 19-fold but has no effect on the activity of Dbr1 from the amoeba Entamoeba histolytica, which lacks a disordered C-terminal domain. Finally, we systematically examine how the identity of the branchpoint nucleotide affects debranching rates. These findings describe new aspects of Dbr1 function in humans and further clarify how Dbr1 contributes to human health and disease. [ABSTRACT FROM AUTHOR]

Abstract: A range of regio- and stereo-selective 1,3-dipolar reactions of imines of α-amino esters, generating polysubstituted prolines, catalysed by silver(I) exchanged zeolites or silver(I) supported on titania, both in combination with DBU, are described. The use of a catalytic amount of silver(I) oxide, Ni(II) complexes and cuprous iodide as catalysts for the cycloaddition reactions are also disclosed. [Copyright &y& Elsevier]

Abstract: We constructed four high-resolution, finite-element thermal models across the Nicaragua – Costa Rica subduction zone to predict the (i) thermal structure, (ii) metamorphic pressure (P)–temperature (T) paths followed by subducting lithosphere, and (iii) loci and types of slab dehydration reactions. These new models incorporate a temperature- and stress-dependent olivine rheology for the mantle–wedge that focuses hot asthenosphere into the tip of the mantle–wedge. At P =3GPa (100km depth), predicted slab interface temperatures are ∼800°C, about 170°C warmer than temperatures predicted using an isoviscous mantle–wedge rheology. At the same pressure, predicted temperatures at the base of 7km thick subducting oceanic crust range from 500°C beneath SE Costa Rica to 400–440°C beneath Nicaragua and NW Costa Rica. The high thermal gradients perpendicular to the slab interface permit partial melting of subducting sediments while the underlying oceanic crust dehydrates, consistent with recent geochemical studies of arc basalts. Hydrous eclogite is predicted to persist to ∼120km depth beneath Nicaragua. This is slightly less than the ∼150km depth extent of a dipping low-seismic-velocity wave guide which may reflect deeper persistence of metastable gabbro. Along-strike variations in the calculated thermal structure are relatively minor compared to variations in the distribution of Wadati-Benioff earthquakes and arc geochemistry, suggesting that regional variations in slab stresses, crustal thickness, incoming sediment load, and the distribution of hydrous minerals in the incoming lithosphere play important roles. [Copyright &y& Elsevier]

The antischistosomal prodrug oxamniquine is activated by a sulfotransferase (SULT) in the parasitic flatworm Schistosoma mansoni. Of the three main human schistosome species, only S. mansoni is sensitive to oxamniquine therapy despite the presence of SULT orthologs in Schistosoma hematobium and Schistosoma japonicum The reason for this species-specific drug action has remained a mystery for decades. Here we present the crystal structures of S. hematobium and S. japonicum SULTs, including S. hematobium SULT in complex with oxamniquine. We also examined the activity of the three enzymes in vitro; surprisingly, all three are active toward oxamniquine, yet we observed differences in catalytic efficiency that implicate kinetics as the determinant for species-specific toxicity. These results provide guidance for designing oxamniquine derivatives to treat infection caused by all species of schistosome to combat emerging resistance to current therapy. [ABSTRACT FROM AUTHOR]

Molecular surveillance provides a powerful approach to monitoring the resistance status of parasite populations in the field and for understanding resistance evolution. Oxamniquine was used to treat Brazilian schistosomiasis patients (mid-1970s to mid-2000s) and several cases of parasite infections resistant to treatment were recorded. The gene underlying resistance ( SmSULT-OR ) encodes a sulfotransferase required for intracellular drug activation. Resistance has a recessive basis and occurs when both SmSULT-OR alleles encode for defective proteins. Here we examine SmSULT-OR sequence variation in a natural schistosome population in Brazil ∼40 years after the first use of this drug. We sequenced SmSULT-OR from 189 individual miracidia (1–11 per patient) recovered from 49 patients, and tested proteins expressed from putative resistance alleles for their ability to activate oxamniquine. We found nine mutations (four non-synonymous single nucleotide polymorphisms, three non-coding single nucleotide polymorphisms and two indels). Both mutations (p.E142del and p.C35R) identified previously were recovered in this field population. We also found two additional mutations (a splice site variant and 1 bp coding insertion) predicted to encode non-functional truncated proteins. Two additional substitutions (p.G206V, p.N215Y) tested had no impact on oxamniquine activation. Three results are of particular interest: (i) we recovered the p.E142del mutation from the field: this same deletion is responsible for resistance in an oxamniquine selected laboratory parasite population; (ii) frequencies of resistance alleles are extremely low (0.27–0.8%), perhaps due to fitness costs associated with carriage of these alleles; (iii) that four independent resistant alleles were found is consistent with the idea that multiple mutations can generate loss-of-function alleles. [ABSTRACT FROM AUTHOR]

The functional and structural significance of the intrasubunit disulfide bond in copper-zinc superoxide dismutase (SOD1) was studied by characterizing mutant forms of human SOD1 (hSOD) and yeast SOD1 lacking the disulfide bond. We determined x-ray crystal structures of metal-bound and metal-deficient hC57S SOD1. C57S hSOD1 isolated from yeast contained four zinc ions per protein dimer and was structurally very similar to wild type. The addition of copper to this four-zinc protein gave properly reconstituted 2Cu,2Zn C57S hSOD, and its spectroscopic properties indicated that the coordination geometry of the copper was remarkably similar to that of holo wild type hSOD1. In contrast, the addition of copper and zinc ions to apo C57S human SOD1 failed to give proper reconstitution. Using pulse radiolysis, we determined SOD activities of yeast and human SOD 1s lacking disulfide bonds and found that they were enzymatically active at ~ 10% of the wild type rate. These results are contrary to earlier reports that the intrasubunit disulfide bonds in SOD1 are essential for SOD activity. Kinetic studies revealed further that the yeast mutant SOD1 had less ionic attraction for superoxide, possibly explaining the lower rates. Saccharomyces cerevisiae cells lacking the sod1 gene do not grow aerobically in the absence of lysine, but expression of C57S SOD1 increased growth to 30-50% of the growth of cells expressing wild type SOD1, supporting that C57S SOD1 retained a significant amount of activity. [ABSTRACT FROM AUTHOR]

Chlamydia trachomatis infection is the most common sexually transmitted bacterial disease. Left untreated, it can lead to ectopic pregnancy, pelvic inflammatory disease, and infertility. Here we present the structure of the secreted C. trachomatis protein Pgp3, an immunodominant antigen and putative virulence factor. The ∼84-kDa Pgp3 homotrimer, encoded on a cryptic plasmid, consists of globular N- and C-terminal assemblies connected by a triple- helical coiled-coil. The C-terminal domains possess folds similar to members of the TNF family of cytokines. The closest Pgp3 C-terminal domain structural homologs include a lectin from Burkholderia cenocepacia, the C1q component of complement, and a portion of the Bacillus anthracis spore surface protein BclA, all of which play roles in bioadhesion. The N-terminal domain consists of a concatenation of structural motifs typically found in trimeric viral proteins. The central parallel triple-helical coiled-coil contains an unusual alternating pattern of apolar and polar residue pairs that generate a rare right-handed superhelical twist. The unique architecture of Pgp3 provides the basis for understanding its role in chlamydial pathogenesis and serves as the platform for its optimization as a potential vaccine antigen candidate. [ABSTRACT FROM AUTHOR]

Mutations in the gene encoding human copper-zinc superoxide dismutase (SOD1) cause a dominant form of the progressive neurodegenerative disease amyotrophic lateral sclerosis. Transgenic mice expressing the human G85R SOD1 variant develop paralytic symptoms concomitant with the appearance of SOD1-enriched proteinaceous inclusions in their neural tissues. The process(es) through which misfolding or aggregation of G85R SOD1 induces motor neuron toxicity is not understood. Here we present structures of the human G85R SOD1 variant determined by single crystal x-ray diffraction. Alterations in structure of the metal-binding loop elements relative to the wild type enzyme suggest a molecular basis for the metal ion deficiency of the G85R SOD1 protein observed in the central nervous system of transgenic mice and in purified recombinant G85R SOD1. These findings support the notion that metal-deficient and/or disulfide-reduced mutant SOD! species contribute to toxicity in SOD1-linked amyotrophic lateral sclerosis. [ABSTRACT FROM AUTHOR]

Because copper is both an essential cofactor and a toxic metal, different strategies have evolved to appropriately regulate its homeostasis as a function of changing environmental copper levels. In this report, we describe a metallochaperone-like protein from Schizosaccharomyces pombe that maintains the delicate balance between essentiality and toxicity. This protein, designated Pccs, has four distinct domains. SOD activity assays reveal that the first three domains of Pccs are necessary and sufficient to deliver copper to its target, copper-zinc superoxide dismutase (SOD1). Pccs domain IV, which is absent in Saccharomyces cerevisiae CCS1, contains seventeen cysteine residues, eight pairs of which are in a potential metal coordination arrangement, Cys-Cys. We show that S. cerevisiae ace1Δ mutant cells expressing the full-length Pccs molecule are resistant to copper toxicity. Furthermore, we demonstrate that the Pccs domain IV enhances copper resistance of the ace1Δ cells by an order of magnitude compared with that observed in the same strain expressing a pccs+I-II-III allele encoding Pccs domains I-III. We consistently found that S. pombe cells disrupted in the pccs+ gene exhibit an increased sensitivity to copper and cadmium. Furthermore, we demonstrate that overexpression of pccs+ is associated with increased copper resistance in fission yeast cells. Taken together, our findings suggest that Pccs activates apo-SOD1 under copper-limiting conditions through the use of its first three domains and protects cells against metal ion toxicity via its fourth domain. [ABSTRACT FROM AUTHOR]