Regulation of Notch signaling and endocytosis by the Lgl neoplastic tumor suppressor

The evolutionarily conserved neoplastic tumor suppressor protein, Lethal (2) giant larvae (Lgl), plays roles in cell polarity and tissue growth via regulation of the Hippo pathway. In our recent study, we showed that in the developing Drosophila eye epithelium, depletion of Lgl leads to increased ligand-dependent Notch signaling. lgl mutant tissue also exhibits an accumulation of early endosomes, recycling endosomes, early-multivesicular body markers and acidic vesicles. We showed that elevated Notch signaling in lgl⁻ tissue can be rescued by feeding larvae the vesicle de-acidifying drug chloroquine, revealing that Lgl attenuates Notch signaling by limiting vesicle acidification. Strikingly, chloroquine also rescued the lgl⁻ overgrowth phenotype, suggesting that the Hippo pathway defects were also rescued. In this extraview, we provide additional data on the regulation of Notch signaling and endocytosis by Lgl, and discuss possible mechanisms by which Lgl depletion contributes to signaling pathway defects and tumorigenesis.     

Zinc-binding Domain of the Bacteriophage T7 DNA Primase Modulates Binding to the DNA Template

The zinc-binding domain (ZBD) of prokaryotic DNA primases has been postulated to be crucial for recognition of specific sequences in the single-stranded DNA template. To determine the molecular basis for this role in recognition, we carried out homolog-scanning mutagenesis of the zinc-binding domain of DNA primase of bacteriophage T7 using a bacterial homolog from Geobacillus stearothermophilus. The ability of T7 DNA primase to catalyze template-directed oligoribonucleotide synthesis is eliminated by substitution of any five-amino acid residue-long segment within the ZBD. The most significant defect occurs upon substitution of a region (Pro-16 to Cys-20) spanning two cysteines that coordinate the zinc ion. The role of this region in primase function was further investigated by generating a protein library composed of multiple amino acid substitutions for Pro-16, Asp-18, and Asn-19 followed by genetic screening for functional proteins. Examination of proteins selected from the screening reveals no change in sequence-specific recognition. However, the more positively charged residues in the region facilitate DNA binding, leading to more efficient oligoribonucleotide synthesis on short templates. The results suggest that the zinc-binding mode alone is not responsible for sequence recognition, but rather its interaction with the RNA polymerase domain is critical for DNA binding and for sequence recognition. Consequently, any alteration in the ZBD that disturbs its conformation leads to loss of DNA-dependent oligoribonucleotide synthesis.     

A Combination of Broadly Neutralizing HIV-1 Monoclonal Antibodies Targeting Distinct Epitopes Effectively Neutralizes Variants Found in Early Infection

Neutralizing antibody protection against HIV-1 may require broad and potent antibodies targeting multiple epitopes. We tested 7 monoclonal antibodies (MAbs) against 45 viruses of diverse subtypes from early infection. The CD4 binding site MAb NIH45-46W was most broad and potent (91% coverage; geometric mean 50% inhibitory concentration [IC(50)], 0.09 μg/ml). Combining NIH45-46W and a V3-specific MAb, PGT128, neutralized 96% of viruses, while PGT121, another V3-specific MAb, neutralized the remainder. Thus, 2 or 3 antibody specificities may prevent infection by most HIV-1 variants.     

Fine structure analysis of black band disease (BBD) infected coral and coral exposed to the BBD toxins microcystin and sulfide

Black band disease (BBD) of corals is a complex pathogenic polymicrobial mat community that lyses coral tissue as it migrates over an infected colony. Two known toxins are produced by BBD microorganisms - sulfide, produced by sulfate-reducing bacteria, and microcystin, produced by cyanobacteria. Experiments were carried out to determine the effects of exposing healthy coral fragments to variable concentrations of purified microcystin, sulfide at a concentration known to exist in BBD, and a combination of the two. Healthy fragments of the coral Montastraea annularis were placed into experimental chambers with known toxin/s for 18-22.5 h. Fine structural analysis using scanning electron microscopy (SEM) showed that toxin exposure resulted in thinning or removal of the coral epidermal layer coupled with degradation of the gastrodermis. These effects were exacerbated when both toxins were used in combination. Exposure to sulfide and the highest concentration of microcystin caused zooxanthellae to dissociate from the coral tissue and to form clusters on the coral surface. Examination of coral fragments infected with BBD was carried out for comparison. It was determined that the effects of exposure to sulfide and microcystin on coral fine structure were consistent, both quantitatively and qualitatively, with the effects of artificially induced and naturally occurring BBD on M. annularis.     

Nitrogen monoxide (NO) storage and transport by dinitrosyl-dithiol-iron complexes: long-lived NO that is trafficked by interacting proteins

Nitrogen monoxide (NO) markedly affects intracellular iron metabolism, and recent studies have shown that molecules traditionally involved in drug resistance, namely GST and MRP1 (multidrug resistance-associated protein 1), are critical molecular players in this process. This is mediated by interaction of these proteins with dinitrosyl-dithiol-iron complexes (Watts, R. N., Hawkins, C., Ponka, P., and Richardson, D. R. (2006) Proc. Natl. Acad. Sci. U.S.A. 103, 7670-7675; Lok, H. C., Suryo Rahmanto, Y., Hawkins, C. L., Kalinowski, D. S., Morrow, C. S., Townsend, A. J., Ponka, P., and Richardson, D. R. (2012) J. Biol. Chem. 287, 607-618). These complexes are bioavailable, have a markedly longer half-life compared with free NO, and form in cells after an interaction between iron, NO, and glutathione. The generation of dinitrosyl-dithiol-iron complexes acts as a common currency for NO transport and storage by MRP1 and GST P1-1, respectively. Understanding the biological trafficking mechanisms involved in the metabolism of NO is vital for elucidating its many roles in cellular signaling and cytotoxicity and for development of new therapeutic targets.     

The 'porin-cytochrome' model for microbe-to-mineral electron transfer

Many species of bacteria can couple anaerobic growth to the respiratory reduction of insoluble minerals containing Fe(III) or Mn(III/IV). It has been suggested that in Shewanella species electrons cross the outer membrane to extracellular substrates via 'porin-cytochrome' electron transport modules. The molecular structure of an outer-membrane extracellular-facing deca-haem terminus for such a module has recently been resolved. It is debated how, once outside the cells, electrons are transferred from outer-membrane cytochromes to insoluble electron sinks. This may occur directly or by assemblies of cytochromes, perhaps functioning as 'nanowires', or via electron shuttles. Here we review recent work in this field and explore whether it allows for unification of the electron transport mechanisms supporting extracellular mineral respiration in Shewanella that may extend into other genera of Gram-negative bacteria.     

Workshop on the ecosystem and fisheries of the Coral Sea: an Australian perspective on research and management

This report summarizes a workshop on the Coral Sea to determine key research findings and identify the research gaps needed to support sustainable management of a proposed Coral Sea Marine Reserve. Key research questions included determining the connectivity of apex predators with the broader southwest Pacific Ocean, and assessing the regions?? biodiversity in relation to seabed topography and oceanographic processes. The workshop concluded noting the importance of engaging surrounding countries in maintaining the sustainability and uniqueness of the Coral Sea.     

The crystal structure of the extracellular 11-heme cytochrome UndA reveals a conserved 10-heme motif and defined binding site for soluble iron chelates

Members of the genus Shewanella translocate deca- or undeca-heme cytochromes to the external cell surface thus enabling respiration using extracellular minerals and polynuclear Fe(III) chelates. The high resolution structure of the first undeca-heme outer membrane cytochrome, UndA, reveals a crossed heme chain with four potential electron ingress/egress sites arranged within four domains. Sequence and structural alignment of UndA and the deca-heme MtrF reveals the extra heme of UndA is inserted between MtrF hemes 6 and 7. The remaining UndA hemes can be superposed over the heme chain of the decaheme MtrF, suggesting that a ten heme core is conserved between outer membrane cytochromes. The UndA structure has also been crystallographically resolved in complex with substrates, an?Fe(III)-nitrilotriacetate dimer or an Fe(III)-citrate trimer. The structural resolution of these UndA-Fe(III)-chelate complexes provides a rationale for previous kinetic measurements on UndA and other outer membrane cytochromes.     

Mapping, complementation, and targets of the cysteine protease actinidin in kiwifruit

Cysteine proteases (CPs) accumulate to high concentration in many fruit, where they are believed to play a role in fungal and insect defense. The fruit of Actinidia species (kiwifruit) exhibit a range of CP activities (e.g. the Actinidia chinensis variety YellowA shows less than 2% of the activity of Actinidia deliciosa variety Hayward). A major quantitative trait locus for CP activity was mapped to linkage group 16 in a segregating population of A. chinensis. This quantitative trait locus colocated with the gene encoding actinidin, the major acidic CP in ripe Hayward fruit encoded by the ACT1A-1 allele. Sequence analysis indicated that the ACT1A locus in the segregating A. chinensis population contained one functional allele (A-2) and three nonfunctional alleles (a-3, a-4, and a-5) each containing a unique frameshift mutation. YellowA kiwifruit contained two further alleles: a-6, which was nonfunctional because of a large insertion, and a-7, which produced an inactive enzyme. Site-directed mutagenesis of the act1a-7 protein revealed a residue that restored CP activity. Expression of the functional ACT1A-1 cDNA in transgenic plants complemented the natural YellowA mutations and partially restored CP activity in fruit. Two consequences of the increase in CP activity were enhanced degradation of gelatin-based jellies in vitro and an increase in the processing of a class IV chitinase in planta. These results provide new insight into key residues required for CP activity and the in vivo protein targets of actinidin.