The Functional Proximal Proteome of Oncogenic Ras Includes mTORC2

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Linkage and pyranosyl ring twisting in cyclodextrins

Acylated beta-cyclodextrins (beta-CDs) were studied to gain perspective on maltose octapropanoate, the crystal structure of which was reported in the preceding paper in this issue. Acylated beta-CDs are distorted so we looked at other CDs and gained increased understanding of distortion in CDs and possibly, shapes in starch. Classic CDs have six to eight glucose residues in a doughnut shape that is stabilized by a ring of inter-residue O3,,,O2' hydrogen bonds. On a phi,psi energy map for a maltose analog that does not form hydrogen bonds, classic CD linkages have higher energies than structures that are stabilized by the exo-anomeric effect. In distorted beta-CDs, which lack hydrogen bonding, some linkages attain low-energies from the exo-anomeric effect and acyl stacking. Those linkages result in left-handed helical geometry so other linkages are forced by the CD macrocycle to have counter-balancing right-handed character. Permethylated gamma-CDs have two 'flipping' linkages as do some larger native CDs. Flipping linkages allow two left-handed segments to join into a macrocycle, thus avoiding the higher-energy, right-handed forms. Some glucose rings in derivatized beta-CDs have substantial positive twists of the pseudo torsion angle O1-C1...C4-O4, adding right-handed character to balance the left-handed linkages. In substituted gamma-CD, all residues have negative twists, giving extra left-handed character to the short, pseudo-helical segments. In non-macrocyclic molecules the twists ranged from -14 degrees to +2 degrees , averaging -6.1 degrees. In these beta- and gamma-CDs, the twists ranged from -22 degrees to +16 degrees for (4)C(1) rings, and the (O)S(2) ring in acetylated beta-CD has a twist of +34 degrees . Glucose residues in other CDs were less twisted.     

CSF-1 and TPA stimulate independent pathways leading to lysosomal degradation or regulated intramembrane proteolysis of the CSF-1 receptor

The CSF-1 receptor is a protein-tyrosine kinase that has been shown to undergo regulated intramembrane proteolysis, or RIPping. Here, we have compared receptor downregulation and RIPping in response to CSF-1 and TPA. Our studies show that CSF-1 is a relatively poor inducer of RIPping and that CSF-1-induced receptor downregulation is largely independent of RIPping. TPA is a strong inducer of RIPping and TPA-induced receptor downregulation is mediated by RIPping. We further found that RIPping is dependent on TACE or a TACE-like protease, that CSF-1 and TPA use independent pathways to initiate RIPping, and that the intracellular domain is targeted for degradation through ubiquitination.     

Human Ikaros function in activated T cells is regulated by coordinated expression of its largest isoforms

The Ikaros gene is alternately spliced to generate multiple zinc finger proteins involved in gene regulation and chromatin remodeling. Whereas murine studies have provided important information regarding the role of Ikaros in the mouse, little is known of Ikaros function in human. We report functional analyses of the two largest human Ikaros (hIK) isoforms, hIK-VI and hIK-H, in T cells. Abundant expression of hIK-H, the largest described isoform, is restricted to human hematopoietic cells. We find that the DNA binding affinity of hIK-H differs from that of hIK-VI. Co-expression of hIk-H with hIk-VI alters the ability of Ikaros complexes to bind DNA motifs found in pericentromeric heterochromatin (PC-HC). In the nucleus, hIK-VI is localized solely in PC-HC, whereas the hIK-H protein exhibits dual centromeric and non-centromeric localization. Mutational analysis defined the amino acids responsible for the distinct DNA binding ability of hIK-H, as well as the sequence required for the specific subcellular localization of this isoform. In proliferating cells, the binding of hIK-H to the upstream regulatory region of known Ikaros target genes correlates with their positive regulation by Ikaros. Results suggest that expression of hIK-H protein restricts affinity of Ikaros protein complexes toward specific PC-HC repeats. We propose a model, whereby the binding of hIK-H-deficient Ikaros complexes to the regulatory sequence of target genes would recruit these genes to the restrictive pericentromeric compartment, resulting in their repression. The presence of hIK-H in the Ikaros complex would alter its affinity for PC-HC, leading to chromatin remodeling and activation of target genes.     

Early Cambrian record of failed durophagy and shell repair in an epibenthic mollusc

Predation is arguably one of the main driving forces of early metazoan evolution, yet the fossil record of predation during the Ediacaran-Early Cambrian transition is relatively poor. Here, we present direct evidence of failed durophagous (shell-breaking) predation and subsequent shell repair in the Early Cambrian (Botoman) epibenthic mollusc Marocella from the Mernmerna Formation and Oraparinna Shale in the Flinders Ranges, South Australia. This record pushes back the first appearance of durophagy on molluscs by approximately 40Myr.     

Isolation and characterisation of polymorphic microsatellite loci in the vulnerable spectacled flying fox, Pteropus conspicillatus

The spectacled flying fox, Pteropus conspicillatus, is listed as vulnerable in Australia and is under threat from numerous impacts. Primers to amplify eight co-dominant microsatellite loci were designed for Pteropus conspicillatus, based on an enriched genomic library. Four loci were monomorphic in this species while the remaining four loci were highly polymorphic with 16–23 alleles. Two of the four monomorphic loci were found to be polymorphic in Pteropus alecto, a closely related congener. All but one of the six polymorphic loci were in Hardy Weinberg equilibrium. Additionally, six microsatellite loci isolated for Pteropus rodricensis were tested against individuals of P. conspicillatus with all loci amplifying reliably. These loci will be used to investigate population genetic structure in the vulnerable spectacled flying fox.     

Increased insulin-stimulated Akt pSer473 and cytosolic SHP2 protein abundance in human skeletal muscle following acute exercise and short-term training.

The purpose of the present study was to determine in human skeletal muscle whether a single exercise bout and 7 days of consecutive endurance (cycling) training 1) increased insulin-stimulated Akt pSer(473) and 2) altered the abundance of the protein tyrosine phosphatases (PTPases), PTP1B and SHP2. In healthy, untrained men (n = 8; 24 +/- 1 yr), glucose infusion rate during a hyperinsulinemic euglycemic clamp, when compared with untrained values, was not improved 24 h following a single 60-min bout of endurance cycling but was significantly increased ( approximately 30%; P < 0.05) 24 h following completion of 7 days of exercise training. Insulin-stimulated Akt pSer(473) was approximately 50% higher (P < 0.05) 24 h following the acute bout of exercise, with this effect remaining after 7 days of training (P < 0.05). Insulin-stimulated insulin receptor and insulin receptor substrate-1 tyrosine phosphorylation were not altered 24 h after acute exercise and short-term training. Insulin did not acutely regulate the localization of the PTPases, PTP1B or SHP2, although cytosolic protein abundance of SHP2 was increased (P < 0.05; main effect) 24 h following acute exercise and short-term training. In conclusion, insulin-sensitive Akt pSer(473) and cytosolic SHP2 protein abundance are higher after acute exercise and short-term training, and this effect appears largely due to the residual effects of the last bout of prior exercise. The significance of exercise-induced alterations in cytosolic SHP2 and insulin-stimulated Akt pSer(473) on the improvement in insulin sensitivity requires further elucidation.