Insights into Ubiquitination from the Unique Clamp-like Binding of the RING E3 AO7 to the E2 UbcH5B

RING proteins constitute the largest class of E3 ubiquitin ligases. Unlike most RINGs, AO7 (RNF25) binds the E2 ubiquitin-conjugating enzyme, UbcH5B (UBE2D2), with strikingly high affinity. We have defined, by co-crystallization, the distinctive means by which AO7 binds UbcH5B. AO7 contains a structurally unique UbcH5B binding region (U5BR) that is connected by an 11-amino acid linker to its RING domain, forming a clamp surrounding the E2. The U5BR interacts extensively with a region of UbcH5B that is distinct from both the active site and the RING-interacting region, referred to as the backside of the E2. An apparent paradox is that the high-affinity binding of the AO7 clamp to UbcH5B, which is dependent on the U5BR, decreases the rate of ubiquitination. We establish that this is a consequence of blocking the stimulatory, non-covalent, binding of ubiquitin to the backside of UbcH5B. Interestingly, when non-covalent backside ubiquitin binding cannot occur, the AO7 clamp now enhances the rate of ubiquitination. The high-affinity binding of the AO7 clamp to UbcH5B has also allowed for the co-crystallization of previously described and functionally important RING mutants at the RING-E2 interface. We show that mutations having marked effects on function only minimally affect the intermolecular interactions between the AO7 RING and UbcH5B, establishing a high degree of complexity in activation through the RING-E2 interface.     

Flavin containing monooxygenase 3 exerts broad effects on glucose and lipid metabolism and atherosclerosis

We performed silencing and overexpression studies of flavin containing monooxygenase (FMO) 3 in hyperlipidemic mouse models to examine its effects on trimethylamine N-oxide (TMAO) levels and atherosclerosis. Knockdown of hepatic FMO3 in LDL receptor knockout mice using an antisense oligonucleotide resulted in decreased circulating TMAO levels and atherosclerosis. Surprisingly, we also observed significant decreases in hepatic lipids and in levels of plasma lipids, ketone bodies, glucose, and insulin. FMO3 overexpression in transgenic mice, on the other hand, increased hepatic and plasma lipids. Global gene expression analyses suggested that these effects of FMO3 on lipogenesis and gluconeogenesis may be mediated through the PPARα and Kruppel-like factor 15 pathways. In vivo and in vitro results were consistent with the concept that the effects were mediated directly by FMO3 rather than trimethylamine/TMAO; in particular, overexpression of FMO3 in the human hepatoma cell line, Hep3B, resulted in significantly increased glucose secretion and lipogenesis. Our results indicate a major role for FMO3 in modulating glucose and lipid homeostasis in vivo, and they suggest that pharmacologic inhibition of FMO3 to reduce TMAO levels would be confounded by metabolic interactions.     

Regulation of NF-κB signaling by oxidized glycerophospholipid and IL-1β induced miRs-21-3p and -27a-5p in human aortic endothelial cells

Exposure of endothelial cells (ECs) to agents such as oxidized glycerophospholipids (oxGPs) and cytokines, known to accumulate in atherosclerotic lesions, perturbs the expression of hundreds of genes in ECs involved in inflammatory and other biological processes. We hypothesized that microRNAs (miRNAs) are involved in regulating the inflammatory response in human aortic endothelial cells (HAECs) in response to oxGPs and interleukin 1β (IL-1β). Using next-generation sequencing and RT-quantitative PCR, we characterized the profile of expressed miRNAs in HAECs pre- and postexposure to oxGPs. Using this data, we identified miR-21-3p and miR-27a-5p to be induced 3- to 4-fold in response to oxGP and IL-1β treatment compared with control treatment. Transient overexpression of miR-21-3p and miR-27a-5p resulted in the downregulation of 1,253 genes with 922 genes overlapping between the two miRNAs. Gene Ontology functional enrichment analysis predicted that the two miRNAs were involved in the regulation of nuclear factor κB (NF-κB) signaling. Overexpression of these two miRNAs leads to changes in p65 nuclear translocation. Using 3′ untranslated region luciferase assay, we identified 20 genes within the NF-κB signaling cascade as putative targets of miRs-21-3p and -27a-5p, implicating these two miRNAs as modulators of NF-κB signaling in ECs.     

Genomic and transcriptomic analysis of the endophytic fungus Pestalotiopsis fici reveals its lifestyle and high potential for synthesis of natural products

Background

In recent years, the genus Pestalotiopsis is receiving increasing attention, not only because of its economic impact as a plant pathogen but also as a commonly isolated endophyte which is an important source of bioactive natural products. Pestalotiopsis fici Steyaert W106-1/CGMCC3.15140 as an endophyte of tea produces numerous novel secondary metabolites, including chloropupukeananin, a derivative of chlorinated pupukeanane that is first discovered in fungi. Some of them might be important as the drug leads for future pharmaceutics.

Results

Here, we report the genome sequence of the endophytic fungus of tea Pestalotiopsis fici W106-1/CGMCC3.15140. The abundant carbohydrate-active enzymes especially significantly expanding pectinases allow the fungus to utilize the limited intercellular nutrients within the host plants, suggesting adaptation of the fungus to endophytic lifestyle. The P. fici genome encodes a rich set of secondary metabolite synthesis genes, including 27 polyketide synthases (PKSs), 12 non-ribosomal peptide synthases (NRPSs), five dimethylallyl tryptophan synthases, four putative PKS-like enzymes, 15 putative NRPS-like enzymes, 15 terpenoid synthases, seven terpenoid cyclases, seven fatty-acid synthases, and five hybrids of PKS-NRPS. The majority of these core enzymes distributed into 74 secondary metabolite clusters. The putative Diels-Alderase genes have undergone expansion.

Conclusion

The significant expansion of pectinase encoding genes provides essential insight in the life strategy of endophytes, and richness of gene clusters for secondary metabolites reveals high potential of natural products of endophytic fungi.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-014-1190-9) contains supplementary material, which is available to authorized users.     

Isolation and performance evaluation of halotolerant phosphate solubilizing bacteria from the rhizospheric soils of historic Dagong Brine Well in China

The use of halotolerant phosphate solubilizing bacteria as inoculants to convert insoluble phosphorus of salt-affected soils to an accessible form is a promising strategy to improve the phosphorus ingestion of plants in salt-affected agriculture. A total of four aerobic isolates with biggest clear halos on the 10% NaCl NBRIP medium plate containing tricalcium phosphate were isolated from the rhizospheric soils of native plants growing on the wall of Dagong Ancinet Brine Well, located in Sichuan of China. And these four isolates were classified to the same strain, named QW10-11, and closely related to Bacillus megatherium var. phosphaticum DSM 3228 and B. megaterium ATCC 14581 according to their phenotype and 16S rRNA. However, the Molecular evolutionary evidences of 16S-23S rRNA ISR further suggested that QW10-11, DSM 3228 and ATCC 14581 have respectively fall into the different sub-divisions in intra specific phylogeny. Strain QW10-11 has significantly better ability of tricalcium phosphate solubilization than that of lecithin solubilization. When it grows under pH 4.8–8.0, 24–33°C and 5–10% NaCl, it can exhibit the higher values of solubilized tricalcium phosphate between 59.3 and 71.4 μg ml⁻¹. Furthermore, its tricalcium phosphate solubilizing activity was associated with the release of organic acids. Taken together, our results indicted that QW10-11 from the rhizospheric soils of halobiot of Dagong Ancinet Brine Well is attractive as efficient phosphate solubilizing candidates in the salt-affected agriculture.     

Antihyperglycemic and neuroprotective effects of one novel Cu-Zn SOD mimetic

Increasing evidence supports that OS plays important roles in diabetes mellitus and cerebral ischemia. This suggests that recovering an impaired endogenous superoxide dismutase (SOD) enzyme system induced by OS with a mimetic would be beneficial and protective for these diseases. In present study, one nonpeptidyl small molecular weight compound (D34) was synthesized. Its SOD mimetic activity and the potential therapeutic actions were also evaluated both in vivo and in vitro. The in vitro nitro blue tetrazolium (NBT) assay indicated that D34 presents an SOD mimetic activity. D34 (20 μmol/kg) exhibited significant antihyperglycemic activity in alloxan-diabetic mice. D34 could also ameliorate the cerebral neuronal death in hippocampus of global cerebral ischemia mice. Furthermore, the D34 treatment significantly decreased malondialdehyde (MDA) contents and increased SOD activities in brains or livers of diabetes mice or cerebral ischemic mice. In conclusion, these preliminary findings support that D34 exhibits SOD mimetic activity and possesses significant antihyperglycemic and neuroprotective effects.     

EGID: an ensemble algorithm for improved genomic island detection in genomic sequences

Genomic islands (GIs) are genomic regions that are originally transferred from other organisms. The detection of genomic islands in genomes can lead to many applications in industrial, medical and environmental contexts. Existing computational tools for GI detection suffer either low recall or low precision, thus leaving the room for improvement. In this paper, we report the development of our Ensemble algorithm for Genomic Island Detection (EGID). EGID utilizes the prediction results of existing computational tools, filters and generates consensus prediction results. Performance comparisons between our ensemble algorithm and existing programs have shown that our ensemble algorithm is better than any other program. EGID was implemented in Java, and was compiled and executed on Linux operating systems. EGID is freely available at http://www5.esu.edu/cpsc/bioinfo/software/EGID.     

Different rates of chromosome elimination in symmetric and asymmetric somatic hybridization between <Emphasis Type="Italic">Festuca arundinacea</Emphasis> and <Emphasis Type="Italic">Bupleurum scorzonerifolium</Emphasis>

The protoplasts of tall fescue (Festuca arundinacea Schreb.) were fused with those of Bupleurum scorzonerifolium Willd. The latter were irradiated with UV at an intensity of 380 μW/cm² for 0 s (combination I), 30 s (combination II), and 60 s (combination III) before fusion. Putative hybrid calli, leaves, and shoots were generated from the fusion products. They were recognized as somatic hybrids by a combined analysis of chromosome numbers, isozyme, RAPD, and 5S rDNA spacer sequence. The hybrid calli with morphogenetic ability and leaves/shoots differentiation had the B. scorzonerifolium phenotype, whether they were derived from symmetric fusion (UV 0 s) or asymmetric fusion (UV 30 s/60 s). Cytological tests revealed that these hybrids contained the complete set (12) of B. scorzonerifolium chromosomes and 0–4 partner tall fescue chromosomes. The tall fescue chromosomes were rapidly eliminated in combinations II and III, but gradually lost in combination I. It was noted that the green leaves and shoots were produced earlier, and the differentiation frequency was higher in combinations II and III than in combination I, which corresponded to the speed of elimination of the tall fescue chromosomes in the hybrids. Therefore, UV irradiation can indirectly promote elimination of tall fescue chromosomes and hybrid differentiation. B. scorzonerifolium can repel partner chromosomes with mechanism that differs from UV.