Description: The draft genome of the pear ( Pyrus bretschneideri ) using a combination of BAC-by-BAC and next-generation sequencing is reported. A 512.0-Mb sequence corresponding to 97.1% of the estimated genome size of this highly heterozygous species is assembled with 194 x coverage. High-density genetic maps comprising 2005 SNP markers anchored 75.5% of the sequence to all 17 chromosomes. The pear genome encodes 42,812 protein-coding genes, and of these, ~28.5% encode multiple isoforms. Repetitive sequences of 271.9 Mb in length, accounting for 53.1% of the pear genome, are identified. Simulation of eudicots to the ancestor of Rosaceae has reconstructed nine ancestral chromosomes. Pear and apple diverged from each other ~5.4–21.5 million years ago, and a recent whole-genome duplication (WGD) event must have occurred 30–45 MYA prior to their divergence, but following divergence from strawberry. When compared with the apple genome sequence, size differences between the apple and pear genomes are confirmed mainly due to the presence of repetitive sequences predominantly contributed by transposable elements (TEs), while genic regions are similar in both species. Genes critical for self-incompatibility, lignified stone cells (a unique feature of pear fruit), sorbitol metabolism, and volatile compounds of fruit have also been identified. Multiple candidate SFB genes appear as tandem repeats in the S -locus region of pear; while lignin synthesis-related gene family expansion and highly expressed gene families of HCT , C3'H , and CCOMT contribute to high accumulation of both G-lignin and S-lignin. Moreover, alpha-linolenic acid metabolism is a key pathway for aroma in pear fruit.

Description: Phospholipase C (PLC) isozymes are important signaling molecules, but few small molecule modulators are available to pharmacologically regulate their function. With the goal of developing a general approach for identification of novel PLC inhibitors, we developed a high-throughput assay based on the fluorogenic substrate reporter WH-15. The assay is highly sensitive and reproducible: screening a chemical library of 6280 compounds identified three novel PLC inhibitors that exhibited potent activities in two separate assay formats with purified PLC isozymes in vitro. Two of the three inhibitors also inhibited G protein-coupled receptor-stimulated PLC activity in intact cell systems. These results demonstrate the power of the high-throughput assay for screening large collections of small molecules to identify novel PLC modulators. Potent and selective modulators of PLCs will ultimately be useful for dissecting the roles of PLCs in cellular processes, as well as provide lead compounds for the development of drugs to treat diseases arising from aberrant phospholipase activity.

Description: Autophagy has been shown to facilitate replication or production of avian reovirus (ARV); nevertheless, how ARV induces autophagy remains largely unknown. Here, we demonstrate that the nonstructural protein p17 of ARV functions as an activator of autophagy. ARV-infected or p17-transfected cells present a fast and strong induction of autophagy, resulting in an increased level of autophagic proteins Beclin 1 and LC3-II. Although autophagy was suppressed by 3-methyladenine or shRNAs targeting autophagic proteins (Beclin 1, ATG7, and LC3) as well as by overexpression of Bcl-2, viral transcription, σC protein synthesis, and virus yield were all significantly reduced, suggesting a key role of autophagosomes in supporting ARV replication. Furthermore, we revealed for the first time that p17 positively regulates phosphatase and tensin deleted on chromosome 10 (PTEN), AMP-activated protein kinase (AMPK), and dsRNA dependent protein kinase RNA (PKR)/eIF2α signaling pathways, accompanied by down-regulation of Akt and mammalian target of rapamycin complex 1, thereby triggering autophagy. By using p53, PTEN, PKR, AMPK, and p17 short hairpin RNA (shRNA), activation of signaling pathways and LC3-II levels was significantly suppressed, suggesting that p17 triggers autophagy through activation of p53/PTEN, AMPK, and PKR signaling pathways. Furthermore, colocalization of LC3 with viral proteins (p17 and σC), p62 with LAMP2 and LC3 with Rab7 was observed under a fluorescence microscope. The expression level of p62 was increased at 18 h postinfection and then slightly decreased 24 h postinfection compared with mock infection and thapsigargin treatment. Furthermore, disruption of autophagosome-lysosome fusion by shRNAs targeting LAMP2 or Rab7a resulted in inhibition of viral protein synthesis and virus yield, suggesting that formation of autolysosome benefits virus replication. Taken together, our results suggest that ARV induces formation of autolysosome but does not induce complete autophagic flux.

Description: The Drosophila melanogaster Genetic Reference Panel (DGRP) is a community resource of 205 sequenced inbred lines, derived to improve our understanding of the effects of naturally occurring genetic variation on molecular and organismal phenotypes. We used an integrated genotyping strategy to identify 4,853,802 single nucleotide polymorphisms (SNPs) and 1,296,080 non-SNP variants. Our molecular population genomic analyses show higher deletion than insertion mutation rates and stronger purifying selection on deletions. Weaker selection on insertions than deletions is consistent with our observed distribution of genome size determined by flow cytometry, which is skewed toward larger genomes. Insertion/deletion and single nucleotide polymorphisms are positively correlated with each other and with local recombination, suggesting that their nonrandom distributions are due to hitchhiking and background selection. Our cytogenetic analysis identified 16 polymorphic inversions in the DGRP. Common inverted and standard karyotypes are genetically divergent and account for most of the variation in relatedness among the DGRP lines. Intriguingly, variation in genome size and many quantitative traits are significantly associated with inversions. Approximately 50% of the DGRP lines are infected with Wolbachia , and four lines have germline insertions of Wolbachia sequences, but effects of Wolbachia infection on quantitative traits are rarely significant. The DGRP complements ongoing efforts to functionally annotate the Drosophila genome. Indeed, 15% of all D. melanogaster genes segregate for potentially damaged proteins in the DGRP, and genome-wide analyses of quantitative traits identify novel candidate genes. The DGRP lines, sequence data, genotypes, quality scores, phenotypes, and analysis and visualization tools are publicly available.

Description: The oncogenic roles of PDGF-D and its proteolytic activator, matriptase, have been strongly implicated in human prostate cancer. Latent full-length PDGF-D (FL-D) consists of a CUB domain, a growth factor domain (GFD), and the hinge region in between. Matriptase processes the FL-D dimer into a GFD dimer (GFD-D) in a stepwise manner, involving generation of a hemidimer (HD), an intermediate product containing one FL-D subunit and one GFD subunit. Although the HD is a pro-growth factor that can be processed into the GFD-D by matriptase, the HD can also act as a dominant-negative ligand that prevents PDGF-B-mediated β-PDGF receptor activation in fibroblasts. The active GFD-D can be further cleaved into a smaller and yet inactive form if matriptase-mediated proteolysis persists. Through mutagenesis and functional analyses, we found that the R340R341GR343A (P4–P1/P1′) motif within the GFD is the matriptase cleavage site through which matriptase can deactivate PDGF-D. Comparative sequence analysis based on the published crystal structure of PDGF-B predicted that the matriptase cleavage site R340R341GR343A is within loop III of the GFD, a critical structural element for its binding with the β-PDGF receptor. Interestingly, we also found that matriptase processing regulates the deposition of PDGF-D dimer species into the extracellular matrix (ECM) with increased binding from the FL-D dimer, to the HD, and to the GFD-D. Furthermore, we provide evidence that R340R341GR343A within the GFD is critical for PDGF-D deposition and binding to the ECM. In this study, we report a structural element crucial for the biological function and ECM deposition of PDGF-D and provide molecular insight into the dynamic functional interplay between the serine protease matriptase and PDGF-D.

Description: Transcription factors (TFs) containing the basic leucine zipper (bZIP) domain are widely distributed in eukaryotes and display an array of distinct functions. In this study, a bZIP-type TF gene (MBZ1) was deleted and functionally characterized in the insect pathogenic fungus Metarhizium robertsii. The deletion mutant (ΔMBZ1) showed defects in cell wall integrity, adhesion to hydrophobic surfaces, and topical infection of insects. Relative to the WT, ΔMBZ1 was also impaired in growth and conidiogenesis. Examination of putative target gene expression indicated that the genes involved in chitin biosynthesis were differentially transcribed in ΔMBZ1 compared with the WT, which led to the accumulation of a higher level of chitin in mutant cell walls. MBZ1 exhibited negative regulation of subtilisin proteases, but positive control of an adhesin gene, which is consistent with the observation of effects on cell autolysis and a reduction in spore adherence to hydrophobic surfaces in ΔMBZ1. Promoter binding assays indicated that MBZ1 can bind to different target genes and suggested the possibility of heterodimer formation to increase the diversity of the MBZ1 regulatory network. The results of this study advance our understanding of the divergence of bZIP-type TFs at both intra- and interspecific levels.

Description: tert-Butoxy-(4-phenyl-quinolin-3-yl)-acetic acids (tBPQA) are a new class of HIV-1 integrase (IN) inhibitors that are structurally distinct from IN strand transfer inhibitors but analogous to LEDGINs. LEDGINs are a class of potent antiviral compounds that interacts with the lens epithelium-derived growth factor (LEDGF) binding pocket on IN and were identified through competition binding against LEDGF. LEDGF tethers IN to the host chromatin and enables targeted integration of viral DNA. The prevailing understanding of the antiviral mechanism of LEDGINs is that they inhibit LEDGF binding to IN, which prevents targeted integration of HIV-1. We showed that in addition to the properties already known for LEDGINs, the binding of tBPQAs to the IN dimer interface inhibits IN enzymatic activity in a LEDGF-independent manner. Using the analysis of two long terminal repeat junctions in HIV-infected cells, we showed that the inhibition by tBPQAs occurs at or prior to the viral DNA 3′-processing step. Biochemical studies revealed that this inhibition operates by compound-induced conformational changes in the IN dimer that prevent proper assembly of IN onto viral DNA. For the first time, tBPQAs were demonstrated to be allosteric inhibitors of HIV-1 IN displaying a dual mode of action: inhibition of IN-viral DNA assembly and inhibition of IN-LEDGF interaction.

Description: α-Actinins (ACTNs) are a family of proteins cross-linking actin filaments that maintain cytoskeletal organization and cell motility. Recently, it has also become clear that ACTN4 can function in the nucleus. In this report, we found that ACTN4 (full length) and its spliced isoform ACTN4 (Iso) possess an unusual LXXLL nuclear receptor interacting motif. Both ACTN4 (full length) and ACTN4 (Iso) potentiate basal transcription activity and directly interact with estrogen receptor α, although ACTN4 (Iso) binds ERα more strongly. We have also found that both ACTN4 (full length) and ACTN4 (Iso) interact with the ligand-independent and the ligand-dependent activation domains of estrogen receptor α. Although ACTN4 (Iso) interacts efficiently with transcriptional co-activators such as p300/CBP-associated factor (PCAF) and steroid receptor co-activator 1 (SRC-1), the full length ACTN4 protein either does not or does so weakly. More importantly, the flanking sequences of the LXXLL motif are important not only for interacting with nuclear receptors but also for the association with co-activators. Taken together, we have identified a novel extended LXXLL motif that is critical for interactions with both receptors and co-activators. This motif functions more efficiently in a spliced isoform of ACTN4 than it does in the full-length protein.

Description: HoxA10 is a homeodomain transcription factor that is involved in maintenance of the myeloid progenitor population and implicated in myeloid leukemogenesis. Previously, we found that FGF2 and CDX4 are direct target genes of HoxA10 and that HOXA10 is a Cdx4 target gene. We also found that increased production of fibroblast growth factor 2 (Fgf2) by HoxA10-overexpressing myeloid progenitor cells results in activation of β-catenin in an autocrine manner. In this study, we identify novel cis elements in the CDX4 and HOXA10 genes that are activated by β-catenin in myeloid progenitor cells. We determine that β-catenin interacts with these cis elements, identifying both CDX4 and HOXA10 as β-catenin target genes in this context. We demonstrate that HoxA10-induced CDX4 transcription is influenced by Fgf2-dependent β-catenin activation. Similarly, Cdx4-induced HOXA10 transcription is influenced by β-catenin in an Fgf2-dependent manner. Increased expression of a set of Hox proteins, including HoxA10, is associated with poor prognosis in acute myeloid leukemia. Cdx4 contributes to leukemogenesis in Hox-overexpressing acute myeloid leukemia, and increased β-catenin activity is also associated with poor prognosis. The current studies identify a molecular mechanisms through which increased expression of HoxA10 increases Cdx4 expression by direct CDX4 activation and by Fgf2-induced β-catenin activity. This results in Cdx4-induced HoxA10-expression, creating a positive feedback mechanism.

Description: The apoptotic protease-activating factor 1 (Apaf-1) controls the onset of many known forms of intrinsic apoptosis in mammals. Apaf-1 exists in normal cells as an autoinhibited monomer. Upon binding to cytochrome c and dATP, Apaf-1 oligomerizes into a heptameric complex known as the apoptosome, which recruits and activates cell-killing caspases. Here we present an atomic structure of an intact mammalian apoptosome at 3.8 Å resolution, determined by single-particle, cryo-electron microscopy (cryo-EM). Structural analysis, together with structure-guided biochemical characterization, uncovered how cytochrome c releases the autoinhibition of Apaf-1 through specific interactions with the WD40 repeats. Structural comparison with autoinhibited Apaf-1 revealed how dATP binding triggers a set of conformational changes that results in the formation of the apoptosome. Together, these results constitute the molecular mechanism of cytochrome c - and dATP-mediated activation of Apaf-1.