1H NMR metabolomics reveals increased glutaminolysis upon overexpression of NSD3s or Pdp3 in Saccharomyces cerevisiae

NSD3s, the proline-tryptophan-tryptophan-proline (PWWP) domain-containing, short isoform of the human oncoprotein NSD3, displays high transforming properties. Overexpression of human NSD3s or the yeast protein Pdp3 in Saccharomyces cerevisiae induces similar metabolic changes, including increased growth rate and sensitivity to oxidative stress, accompanied by decreased oxygen consumption. Here, we set out to elucidate the biochemical pathways leading to the observed metabolic phenotype by analyzing the alterations in yeast metabolome in response to NSD3s or Pdp3 overexpression using ¹H nuclear magnetic resonance (NMR) metabolomics. We observed an increase in aspartate and alanine, together with a decrease in arginine levels, on overexpression of NSD3s or Pdp3, suggesting an increase in the rate of glutaminolysis. In addition, certain metabolites, including glutamate, valine, and phosphocholine were either NSD3s or Pdp3 specific, indicating that additional metabolic pathways are adapted in a protein-dependent manner. The observation that certain metabolic pathways are differentially regulated by NSD3s and Pdp3 suggests that, despite the structural similarity between their PWWP domains, the two proteins act by unique mechanisms and may recruit different downstream signaling complexes. This study establishes for the first time a functional link between the human oncoprotein NSD3s and cancer metabolic reprogramming.     

A non‐canonical role of the p97 complex in RIG‐I antiviral signaling

RIG‐I is a well‐studied sensor of viral RNA that plays a key role in innate immunity. p97 regulates a variety of cellular events such as protein quality control, membrane reassembly, DNA repair, and the cell cycle. Here, we report a new role for p97 with Npl4‐Ufd1 as its cofactor in reducing antiviral innate immune responses by facilitating proteasomal degradation of RIG‐I. The p97 complex is able to directly bind both non‐ubiquitinated RIG‐I and the E3 ligase RNF125, promoting K48‐linked ubiquitination of RIG‐I at residue K181. Viral infection significantly strengthens the interaction between RIG‐I and the p97 complex by a conformational change of RIG‐I that exposes the CARDs and through K63‐linked ubiquitination of these CARDs. Disruption of the p97 complex enhances RIG‐I antiviral signaling. Consistently, administration of compounds targeting p97 ATPase activity was shown to inhibit viral replication and protect mice from vesicular stomatitis virus (VSV) infection. Overall, our study uncovered a previously unrecognized role for the p97 complex in protein ubiquitination and revealed the p97 complex as a potential drug target in antiviral therapy.     

Kin of IRRE-like Protein 2 Is a Phosphorylated Glycoprotein That Regulates Basal Insulin Secretion

Direct interactions among pancreatic β-cells via cell surface proteins inhibit basal and enhance stimulated insulin secretion. Here, we functionally and biochemically characterized Kirrel2, an immunoglobulin superfamily protein with β-cell-specific expression in the pancreas. Our results show that Kirrel2 is a phosphorylated glycoprotein that co-localizes and interacts with the adherens junction proteins E-cadherin and β-catenin in MIN6 cells. We further demonstrate that the phosphosites Tyr^595–596 are functionally relevant for the regulation of Kirrel2 stability and localization. Analysis of the extracellular and intracellular domains of Kirrel2 revealed that it is cleaved and shed from MIN6 cells and that the remaining membrane spanning cytoplasmic domain is processed by γ-secretase complex. Kirrel2 knockdown with RNA interference in MIN6 cells and ablation of Kirrel2 from mice with genetic deletion resulted in increased basal insulin secretion from β-cells, with no immediate influence on stimulated insulin secretion, total insulin content, or whole body glucose metabolism. Our results show that in pancreatic β-cells Kirrel2 localizes to adherens junctions, is regulated by multiple post-translational events, including glycosylation, extracellular cleavage, and phosphorylation, and engages in the regulation of basal insulin secretion.     

Phytosterolemia on the island of Kosrae: founder effect for a novel ABCG8 mutation results in high carrier rate and increased plasma plant sterol levels

Screening of 932 adults on the Pacific island of Kosrae for plasma plant sterol levels disclosed three subjects, two of them asymptomatic, with phytosterolemia. Sequencing the ATP binding cassette subfamily G member 8 (ABCG8) gene revealed a novel exon 2 mutation that causes a change in codon 24 from glutamine to histidine and a frame shift followed by a premature stop codon, precluding the formation of a functional ABCG8 protein. Genotyping of 1,090 Kosraens revealed 150 as carriers, a 13.8% carrier rate. DNA sequencing of 67 carriers revealed the same mutation as in the probands. In carriers, plasma campesterol and sitosterol levels were 55% and 30% higher, respectively, than in noncarriers. Moreover, compared with noncarriers, carriers showed 21% lower plasma levels of lathosterol, a surrogate marker for cholesterol biosynthesis. There was no difference between the groups in plasma total cholesterol, triglycerides, apolipoprotein B, or apolipoprotein A-I levels. In summary, on the island of Kosrae, a strong founder effect of a mutant ABCG8 allele results in a large number of carriers with increased plasma plant sterol levels and decreased lathosterol levels. The latter finding suggests that heterozygosity for a mutated ABCG8 allele results in a modest increase in dietary cholesterol absorption and a decrease in cholesterol biosynthesis.     

Four-dimensional growth response of mature Larix decidua to stem burial under natural conditions

This paper illustrates the effects of abrupt stem burial (burial depth ~0.5 m) on tree growth in mature (46 ± 8 years) European larch (Larix decidua Mill.) trees. In contrast to the previous research, which was mostly carried out with saplings and on experimental sites where regular aggradation occurred through the transport of sand, this work focuses on the impact of natural, abrupt burial of mature trees with rocks contained in a sandy-silty matrix typical for debris flows in mountainous headwater catchments. The effect of burial is assessed radially and axially within the stem and over almost three decades after burial. The analysis of growth disturbances and their intensity was based on the 143 cross sections (572 growth series) taken at 10-cm intervals from 6 Larix decidua Mill. The results show quite clearly that abrupt burial causes massive suppression of radial growth as compared to pre-event conditions (mean 77 %, min 38 %, max 92 %, SD 7.2 %). The trees sampled were unable to resume pre-burial growth rates even after 25 years, but recovered to reference growth conditions (as measured in undisturbed, local reference trees) after 15 years (min 3 years, max 25 years, SD 9 years). The results differ only insignificantly between different heights along the tree axis and suppression is equally well expressed at different radial positions within the stem.     

An approach to the functional analysis of lecithin-cholesterol acyltransferase. Activation by recombinant normal and mutagenized apolipoprotein AI

Apolipoprotein AI (apo AI) of human serum high-density lipoprotein functions as an activator of lecithin-cholesterol acyltransferase (LCAT) and therefore plays an important role in reversed cholesterol transport. The mechanism of the acyltransfer, the activating polypeptide domains of apo AI and the active site of LCAT in this transesterification are not yet known. Synthetic peptides of the apo AI sequence have been designed to determine the activating structure, but did not yet lead to conclusive results. This also applies to spontaneous apo AI mutants. We therefore used the method of site-directed mutagenesis of apo AI cDNAs using the overlap extension approach by the polymerase chain reaction. These constructs were cloned into the procaryotic vector pET8c and expressed under the inducible T7 promoter. The engineered apo AI polypeptides were isolated and purified by affinity chromatography and assayed for their activator activity. The essentials of this approach to the structure and function of activators in general have successfully been exemplified for the LCAT activation by engineering apo AI mutant polypeptides a) by the deletion of two adjacent amphipathic helices (amino acid residues 146-186) and b) by introducing a point mutation (Glu111----Gln).     

Mapping quantitative trait loci (QTLs) for fatty acid composition in an interspecific cross of oil palm

Background  

Marker Assisted Selection (MAS) is well suited to a perennial crop like oil palm, in which the economic products are not produced until several years after planting. The use of DNA markers for selection in such crops can greatly reduce the number of breeding cycles needed. With the use of DNA markers, informed decisions can be made at the nursery stage, regarding which individuals should be retained as breeding stock, which are satisfactory for agricultural production, and which should be culled. The trait associated with oil quality, measured in terms of its fatty acid composition, is an important agronomic trait that can eventually be tracked using molecular markers. This will speed up the production of new and improved oil palm planting materials.     

3D analysis of anatomical reactions in conifers after mechanical wounding: first qualitative insights from X-ray computed tomography

The ability of trees to recover from damage beyond the last-formed periderm as well as the drivers and nature of associated wound reactions have been studied for more than two centuries using macroscopic (desiccation, aeration or discoloration of wood) and microscopic approaches (anatomical and chemical reactions). However, no studies currently exist which address large-scale macroscopic and microscopic reactions surrounding wounds in the tangential, axial, and radial directions over continuous segments of tree stems. This note explores the potential of 3D X-ray computed tomography in assessing effects of wounding under natural conditions in European conifers (Abies alba, Larix decidua, Picea abies). We present results from a pilot study and qualitatively evaluate the potential of the approach used in assessing and illustrating the formation and spread of de-differentiated xylem parenchyma cells, xylem decay compartmentalization, resin ducts, and stabilizing compression wood cells.