Identification of Biochemically Distinct Properties of the Small Ubiquitin-related Modifier (SUMO) Conjugation Pathway in Plasmodium falciparum

Small ubiquitin-related modifiers (SUMOs) are post-translationally conjugated to other proteins and are thereby essential regulators of a wide range of cellular processes. Sumoylation, and enzymes of the sumoylation pathway, are conserved in the malaria causing parasite, Plasmodium falciparum. However, the specific functions of sumoylation in P. falciparum, and the degree of functional conservation between enzymes of the human and P. falciparum sumoylation pathways, have not been characterized. Here, we demonstrate that sumoylation levels peak during midstages of the intra-erythrocyte developmental cycle, concomitant with hemoglobin consumption and elevated oxidative stress. In vitro studies revealed that P. falciparum E1- and E2-conjugating enzymes interact effectively to recognize and modify RanGAP1, a model mammalian SUMO substrate. However, in heterologous reactions, P. falciparum E1 and E2 enzymes failed to interact with cognate human E2 and E1 partners, respectively, to modify RanGAP1. Structural analysis, binding studies, and functional assays revealed divergent amino acid residues within the E1-E2 binding interface that define organism-specific enzyme interactions. Our studies identify sumoylation as a potentially important regulator of oxidative stress response during the P. falciparum intra-erythrocyte developmental cycle, and define E1 and E2 interactions as a promising target for development of parasite-specific inhibitors of sumoylation and parasite replication.     

Suppressor Analysis Reveals a Role for SecY in the SecA2-Dependent Protein Export Pathway of Mycobacteria

All bacteria use the conserved Sec pathway to transport proteins across the cytoplasmic membrane, with the SecA ATPase playing a central role in the process. Mycobacteria are part of a small group of bacteria that have two SecA proteins: the canonical SecA (SecA1) and a second, specialized SecA (SecA2). The SecA2-dependent pathway exports a small subset of proteins and is required for Mycobacterium tuberculosis virulence. The mechanism by which SecA2 drives export of proteins across the cytoplasmic membrane remains poorly understood. Here we performed suppressor analysis on a dominant negative secA2 mutant (secA2 K129R) of the model mycobacterium Mycobacterium smegmatis to better understand the pathway used by SecA2 to export proteins. Two extragenic suppressor mutations were identified as mapping to the promoter region of secY, which encodes the central component of the canonical Sec export channel. These suppressor mutations increased secY expression, and this effect was sufficient to alleviate the secA2 K129R phenotype. We also discovered that the level of SecY protein was greatly diminished in the secA2 K129R mutant, but at least partially restored in the suppressors. Furthermore, the level of SecY in a suppressor strongly correlated with the degree of suppression. Our findings reveal a detrimental effect of SecA2 K129R on SecY, arguing for an integrated system in which SecA2 works with SecY and the canonical Sec translocase to export proteins.     

Identification of Small Molecule Inhibitors of Jumonji AT-rich Interactive Domain 1B (JARID1B) Histone Demethylase by a Sensitive High Throughput Screen

JARID1B (also known as KDM5B or PLU1) is a member of the JARID1 family of histone lysine demethylases responsible for the demethylation of trimethylated lysine 27 in histone H3 (H3K4me3), a mark for actively transcribed genes. JARID1B is overexpressed in several cancers, including breast cancer, prostate cancer, and lung cancer. In addition, JARID1B is required for mammary tumor formation in syngeneic or xenograft mouse models. JARID1B-expressing melanoma cells are associated with increased self-renewal character. Therefore, JARID1B represents an attractive target for cancer therapy. Here we characterized JARID1B using a homogeneous luminescence-based demethylase assay. We then conducted a high throughput screen of over 15,000 small molecules to identify inhibitors of JARID1B. From this screen, we identified several known JmjC histone demethylase inhibitors, including 2,4-pyridinedicarboxylic acid and catechols. More importantly, we identified several novel inhibitors, including 2-4(4-methylphenyl)-1,2-benzisothiazol-3(2H)-one (PBIT), which inhibits JARID1B with an IC₅₀ of about 3 μm in vitro. Consistent with this, PBIT treatment inhibited removal of H3K4me3 by JARID1B in cells. Furthermore, this compound inhibited proliferation of cells expressing higher levels of JARID1B. These results suggest that this novel small molecule inhibitor is a lead compound that can be further optimized for cancer therapy.     

The Biosynthetic Origin of Irregular Monoterpenes in Lavandula: ISOLATION AND BIOCHEMICAL CHARACTERIZATION OF A NOVEL cis-PRENYL DIPHOSPHATE SYNTHASE GENE,LAVANDULYL DIPHOSPHATE SYNTHASE*

Lavender essential oils are constituted predominantly of regular monoterpenes, for example linalool, 1,8-cineole, and camphor. However, they also contain irregular monoterpenes including lavandulol and lavandulyl acetate. Although the majority of genes responsible for the production of regular monoterpenes in lavenders are now known, enzymes (including lavandulyl diphosphate synthase (LPPS)) catalyzing the biosynthesis of irregular monoterpenes in these plants have not been described. Here, we report the isolation and functional characterization of a novel cis-prenyl diphosphate synthase cDNA, termed Lavandula x intermedia lavandulyl diphosphate synthase (LiLPPS), through a homology-based cloning strategy. The LiLPPS ORF, encoding for a 305-amino acid long protein, was expressed in Escherichia coli, and the recombinant protein was purified by nickel-nitrilotriacetic acid affinity chromatography. The approximately 34.5-kDa bacterially produced protein specifically catalyzed the head-to-middle condensation of two dimethylallyl diphosphate units to LPP in vitro with apparent K_m and k_cat values of 208 ± 12 μm and 0.1 s⁻¹, respectively. LiLPPS is a homodimeric enzyme with a sigmoidal saturation curve and Hill coefficient of 2.7, suggesting a positive co-operative interaction among its catalytic sites. LiLPPS could be used to modulate the production of lavandulol and its derivatives in plants through metabolic engineering.     

Ontogenetic shifts in phenotype–environment associations in Nile perch,Lates niloticus (Perciformes: Latidae) from Lake Nabugabo,Uganda

Habitat‐associated trait divergence may vary across ontogeny if there are strong size‐related shifts in selection pressures. We quantified patterns of phenotypic divergence in Nile perch (Lates niloticus) from ecologically distinct wetland edge and forest edge habitats in Lake Nabugabo, Uganda, and we compared patterns of divergence across three size classes to determine whether trends are consistent through Nile perch ontogeny. We predicted that inter‐habitat variation in biotic (e.g. vegetation structure) and abiotic (e.g. dissolved oxygen concentration) variables may create divergent selective regimes. We compared body morphology using geometric morphometrics and found substantial differences between habitats, although not all trends were consistent across size classes. The most striking aspects of divergence in small Nile perch were in mouth orientation, head size, and development of the caudal region. Medium‐sized Nile perch also showed differences in mouth orientation. Differences in large individuals were related to eye size and orientation, as well as caudal length. The observed patterns of divergence are consistent with functional morphological predictions for fish across divergent trophic regimes, high and low predation environments, and complex and simple habitats. Although this suggests adaptive divergence, the source of phenotypic variation is unknown and may reflect phenotypic plasticity and/or genetic differences. © 2013 The Linnean Society of London, Biological Journal of the Linnean Society, 2013, 110 , 449–465.     

Latitude drives diversification in Madagascar's endemic dry forest rodent Eliurus myoxinus (subfamily Nesomyinae)

Numerous hypotheses have been proposed for the historical processes governing the rich endemism of Madagascar's biodiversity. The ‘watershed model’ suggests that drier climates in the recent geological past have resulted in the contraction of forests around major watersheds, thereby defining areas of endemism. We test whether this hypothesis explains phylogeographical patterns in a dry forest‐dependent rodent, Eliurus myoxinus, an endemic species widely distributed through western Madagascar. We sequenced the mitochondrial cytochrome b locus and nuclear introns of the β‐fibrinogen and the growth hormone receptor genes for E. myoxinus. Using a parametric bootstrapping approach, we tested whether the mitochondrial gene tree data fit expectations of local differentiation given the watershed model. We additionally estimated population differentiation and historical demographic parameters, and reconstructed the spatial history of E. myoxinus to highlight spatial and temporal patterns of differentiation. The data do not support the watershed model as a clear explanation for the genetic patterns of diversity within extant E. myoxinus populations. We find striking patterns of latitudinal genetic structure within western Madagascar, and indicate possible roles for environmental and ecological gradients along this axis in generating phylogeographical diversity. © 2013 The Linnean Society of London, Biological Journal of the Linnean Society, 2013, 110 , 500–517.     

PATTERNS OF OSSIFICATION IN SOUTHERN VERSUS NORTHERN PLACENTAL MAMMALS

Consensus on placental mammal phylogeny is fairly recent compared to that for vertebrates as a whole. A stable phylogenetic hypothesis enables investigation into the possibility that placental clades differ from one another in terms of their development. Here, we focus on the sequence of skeletal ossification as a possible source of developmental distinctiveness in “northern” (Laurasiatheria and Euarchontoglires) versus “southern” (Afrotheria and Xenarthra) placental clades. We contribute data on cranial and postcranial ossification events during growth in Afrotheria, including elephants, hyraxes, golden moles, tenrecs, sengis, and aardvarks. We use three different techniques to quantify sequence heterochrony: continuous method, sequence‐ANOVA (analysis of variance) and event‐paring/Parsimov. We show that afrotherians significantly differ from other placentals by an early ossification of the orbitosphenoid and caudal vertebrae. Our analysis also suggests that both southern placental groups show a greater degree of developmental variability; however, they rarely seem to vary in the same direction, especially regarding the shifts that differ statistically. The latter observation is inconsistent with the Atlantogenata hypothesis in which afrotherians are considered as the sister clade of xenarthrans. Interestingly, ancestral nodes for Laurasiatheria and Euarchontoglires show very similar trends and our results suggest that developmental homogeneity in some ossification sequences may be restricted to northern placental mammals (Boreoeutheria).     

Towards a metabolic engineering strain “commons”: An Escherichia coli platform strain for ethanol production

In the genome‐engineering era, it is increasingly important that researchers have access to a common set of platform strains that can serve as debugged production chassis and the basis for applying new metabolic engineering strategies for modeling and characterizing flux, engineering complex traits, and optimizing overall performance. Here, we describe such a platform strain of E. coli engineered for ethanol production. Starting with a fully characterized host strain (BW25113), we site‐specifically integrated the genes required for homoethanol production under the control of a strong inducible promoter into the genome and deleted the genes encoding four enzymes from competing pathways. This strain is capable of producing >30 g/L of ethanol in minimal media with <2 g/L produced of any fermentative byproduct. Using this platform strain, we tested previously identified ethanol tolerance genes and found that while tolerance was improved under certain conditions, any effect on ethanol production or tolerance was lost when grown under production conditions. Thus, our findings reinforce the need for a metabolic engineering “commons” that could provide a set of platform strains for use in more sophisticated genome‐engineering strategies. Towards this end, we have made this production strain available to the scientific community. Biotechnol. Bioeng. 2013; 110: 1520–1526. © 2013 Wiley Periodicals, Inc.     

Scribble is required for normal epithelial cell–cell contacts and lumen morphogenesis in the mammalian lung

During lung development, proper epithelial cell arrangements are critical for the formation of an arborized network of tubes. Each tube requires a lumen, the diameter of which must be tightly regulated to enable optimal lung function. Lung branching and lumen morphogenesis require close epithelial cell–cell contacts that are maintained as a result of adherens junctions, tight junctions and by intact apical–basal (A/B) polarity. However, the molecular mechanisms that maintain epithelial cohesion and lumen diameter in the mammalian lung are unknown. Here we show that Scribble, a protein implicated in planar cell polarity (PCP) signalling, is necessary for normal lung morphogenesis. Lungs of the Scrib mouse mutant Circletail (Crc) are abnormally shaped with fewer airways, and these airways often lack a visible, ‘open’ lumen. Mechanistically we show that Scrib genetically interacts with the core PCP gene Vangl2 in the developing lung and that the distribution of PCP pathway proteins and Rho mediated cytoskeletal modification is perturbed in Scrib^Crc/Crc lungs. However A/B polarity, which is disrupted in Drosophila Scrib mutants, is largely unaffected. Notably, we find that Scrib mediates functions not attributed to other PCP proteins in the lung. Specifically, Scrib localises to both adherens and tight junctions of lung epithelia and knockdown of Scrib in lung explants and organotypic cultures leads to reduced cohesion of lung epithelial cells. Live imaging of Scrib knockdown lungs shows that Scrib does not affect bud bifurcation, as previously shown for the PCP protein Celsr1, but is required to maintain epithelial cohesion. To understand the mechanism leading to reduced cell–cell association, we show that Scrib associates with β-catenin in embryonic lung and the sub-cellular distribution of adherens and tight junction proteins is perturbed in mutant lung epithelia. Our data reveal that Scrib is required for normal lung epithelial organisation and lumen morphogenesis by maintaining cell–cell contacts. Thus we reveal novel and important roles for Scrib in lung development operating via the PCP pathway, and in regulating junctional complexes and cell cohesion.