The AAA ATPase VPS4/SKD1 Regulates Endosomal Cholesterol Trafficking Independently of ESCRT‐III

The exit of low‐density lipoprotein derived cholesterol (LDL‐C) from late endosomes (LE)/lysosomes (Ly) is mediated by Niemann–Pick C1 (NPC1), a multipass integral membrane protein on the limiting membranes of LE/Ly, and by NPC2, a cholesterol‐binding protein in the lumen of LE/Ly. NPC2 delivers cholesterol to the N‐terminal domain of NPC1, which is believed to insert cholesterol into the limiting membrane for subsequent transport to other subcellular organelles. Few cytoplasmic factors have been identified to govern cholesterol efflux from LE/Ly, and much less is known about the underlying molecular mechanisms. Here we establish VPS4, an AAA ATPase that has a well‐established role in disassembling the ESCRT (endosomal sorting complex required for transport)‐III polymer, as an important regulator of endosomal cholesterol transport. Knocking down VPS4 in HeLa cells resulted in prominent accumulation of LDL‐C in LE/Ly, and disrupted cholesterol homeostatic responses at the endoplasmic reticulum. The level and localization of NPC1 and NPC2 appeared to be normal in VPS4 knockdown cells. Importantly, depleting any of the ESCRT‐III components did not exert a significant effect on endosomal cholesterol transport. Our results thus identify an important cytoplasmic regulator of endosomal cholesterol trafficking and represent the first functional separation of VPS4 from ESCRT‐III.     

BreakTrans: uncovering the genomic architecture of gene fusions

Producing gene fusions through genomic structural rearrangements is a major mechanism for tumor evolution. Therefore, accurately detecting gene fusions and the originating rearrangements is of great importance for personalized cancer diagnosis and targeted therapy. We present a tool, BreakTrans, that systematically maps predicted gene fusions to structural rearrangements. Thus, BreakTrans not only validates both types of predictions, but also provides mechanistic interpretations. BreakTrans effectively validates known fusions and discovers novel events in a breast cancer cell line. Applying BreakTrans to 43 breast cancer samples in The Cancer Genome Atlas identifies 90 genomically validated gene fusions. BreakTrans is available at http://bioinformatics.mdanderson.org/main/BreakTrans     

Targeted Lipidomics in Drosophila melanogaster Identifies Novel 2-Monoacylglycerols and N-acyl Amides

Lipid metabolism is critical to coordinate organ development and physiology in response to tissue-autonomous signals and environmental cues. Changes to the availability and signaling of lipid mediators can limit competitiveness, adaptation to environmental stressors, and augment pathological processes. Two classes of lipids, the N-acyl amides and the 2-acyl glycerols, have emerged as important signaling molecules in a wide range of species with important signaling properties, though most of what is known about their cellular functions is from mammalian models. Therefore, expanding available knowledge on the repertoire of these lipids in invertebrates will provide additional avenues of research aimed at elucidating biosynthetic, metabolic, and signaling properties of these molecules. Drosophila melanogaster is a commonly used organism to study intercellular communication, including the functions of bioactive lipids. However, limited information is available on the molecular identity of lipids with putative biological activities in Drosophila. Here, we used a targeted lipidomics approach to identify putative signaling lipids in third instar Drosophila larvae, possessing particularly large lipid mass in their fat body. We identified 2-linoleoyl glycerol, 2-oleoyl glycerol, and 45 N-acyl amides in larval tissues, and validated our findings by the comparative analysis of Oregon-RS, Canton-S and w1118 strains. Data here suggest that Drosophila represent another model system to use for the study of 2-acyl glycerol and N-acyl amide signaling.     

Stabilizing Exposure of Conserved Epitopes by Structure Guided Insertion of Disulfide Bond in HIV-1 Envelope Glycoprotein

Entry of HIV-1 into target cells requires binding of the viral envelope glycoprotein (Env) to cellular receptors and subsequent conformational changes that culminates in fusion of viral and target cell membranes. Recent structural information has revealed that these conformational transitions are regulated by three conserved but potentially flexible layers stacked between the receptor-binding domain (gp120) and the fusion arm (gp41) of Env. We hypothesized that artificial insertion of a covalent bond will ‘snap’ Env into a conformation that is less mobile and stably expose conserved sites. Therefore, we analyzed the interface between these gp120 layers (layers 1, 2 and 3) and identified residues that may form disulfide bonds when substituted with cysteines. We subsequently probed the structures of the resultant mutant gp120 proteins by assaying their binding to a variety of ligands using Surface Plasmon Resonance (SPR) assay. We found that a single disulfide bond strategically inserted between the highly conserved layers 1 and 2 (C65-C115) is able to ‘lock’ gp120 in a CD4 receptor bound conformation (in the absence of CD4), as indicated by the lower dissociation constant (Kd) for the CD4-induced (CD4i) epitope binding 17b antibody. When disulfide-stabilized monomeric (gp120) and trimeric (gp140) Envs were used to immunize rabbits, they were found to elicit a higher proportion of antibodies directed against both CD4i and CD4 binding site epitopes than the wild-type proteins. These results demonstrate that structure-guided stabilization of inter-layer interactions within HIV-1 Env can be used to expose conserved epitopes and potentially overcome the sequence diversity of these molecules.     

Etoposide Induces Nuclear Re-Localisation of AID

During B cell activation, the DNA lesions that initiate somatic hypermutation and class switch recombination are introduced by activation-induced cytidine deaminase (AID). AID is a highly mutagenic protein that is maintained in the cytoplasm at steady state, however AID is shuttled across the nuclear membrane and the protein transiently present in the nucleus appears sufficient for targeted alteration of immunoglobulin loci. AID has been implicated in epigenetic reprogramming in primordial germ cells and cell fusions and in induced pluripotent stem cells (iPS cells), however AID expression in non-B cells is very low. We hypothesised that epigenetic reprogramming would require a pathway that instigates prolonged nuclear residence of AID. Here we show that AID is completely re-localised to the nucleus during drug withdrawal following etoposide treatment, in the period in which double strand breaks (DSBs) are repaired. Re-localisation occurs 2-6 hours after etoposide treatment, and AID remains in the nucleus for 10 or more hours, during which time cells remain live and motile. Re-localisation is cell-cycle dependent and is only observed in G2. Analysis of DSB dynamics shows that AID is re-localised in response to etoposide treatment, however re-localisation occurs substantially after DSB formation and the levels of re-localisation do not correlate with γH2AX levels. We conclude that DSB formation initiates a slow-acting pathway which allows stable long-term nuclear localisation of AID, and that such a pathway may enable AID-induced DNA demethylation during epigenetic reprogramming.     

Re-establishment of Calluna vulgaris (L.) Hull in an eight-year grazing experiment on upland acid grassland

Upland heathland is an internationally important habitat but a large area in the UK has been degraded to acid grassland by intensive livestock grazing. Re-establishment of dwarf shrubs, particularly Calluna vulgaris (L.) Hull, is a key objective for restoring heathland on these sites. A replicated plot-scale experiment was set up to examine effects of disturbance and seed addition on C. vulgaris establishment in a Nardus stricta L. grassland under three grazing regimes: sheep only (1.5 ewes ha^?1 for 10 months per year); cattle only (0.5 heifers ha^?1 in summer only); and, the cattle regime combined with sheep (1.0 ewes ha^?1 for 10 months per year). Early results of the experiment have been reported previously but it was not known if these results were an indication of the longer-term restoration success. Here we evaluate the success of the restoration methods (disturbance, seeding treatments and grazing regime) eight years after the treatments began. In seeded plots, young C. vulgaris plants had greatest above-ground height, dry weight and shoot length if grazing was excluded or the cattle-only regime was applied. C. vulgaris cover was greatest, and increased most, in plots that had been disturbed, seeded and ungrazed or subjected to the cattle-only regime. The vegetation in these plots also became more similar to reference sites with 50% or more cover of C. vulgaris. The invasive Juncus effusus L. was more frequent in disturbed and grazed plots but less frequent in plots with C. vulgaris established from added seed. Previous results that showed the benefits of disturbance and seeding treatments were still valid but changes in the vegetation composition were still occurring and longer-term studies will be needed to determine when grazing regimes including sheep might be reintroduced.     

Fine Tuning Inflammation at the Front Door: Macrophage Complement Receptor 3-mediates Phagocytosis and Immune Suppression for Francisella tularensis

Complement receptor 3 (CR3, CD11b/CD18) is a major macrophage phagocytic receptor. The biochemical pathways through which CR3 regulates immunologic responses have not been fully characterized. Francisella tularensis is a remarkably infectious, facultative intracellular pathogen of macrophages that causes tularemia. Early evasion of the host immune response contributes to the virulence of F. tularensis and CR3 is an important receptor for its phagocytosis. Here we confirm that efficient attachment and uptake of the highly virulent Type A F. tularensis spp. tularensis strain Schu S4 by human monocyte-derived macrophages (hMDMs) requires complement C3 opsonization and CR3. However, despite a>40-fold increase in uptake following C3 opsonization, Schu S4 induces limited pro-inflammatory cytokine production compared with non-opsonized Schu S4 and the low virulent F. novicida. This suggests that engagement of CR3 by opsonized Schu S4 contributes specifically to the immune suppression during and shortly following phagocytosis which we demonstrate by CD11b siRNA knockdown in hMDMs. This immune suppression is concomitant with early inhibition of ERK1/2, p38 MAPK and NF-κB activation. Furthermore, TLR2 siRNA knockdown shows that pro-inflammatory cytokine production and MAPK activation in response to non-opsonized Schu S4 depends on TLR2 signaling providing evidence that CR3-TLR2 crosstalk mediates immune suppression for opsonized Schu S4. Deletion of the CD11b cytoplasmic tail reverses the CR3-mediated decrease in ERK and p38 activation during opsonized Schu-S4 infection. The CR3-mediated signaling pathway involved in this immune suppression includes Lyn kinase and Akt activation, and increased MKP-1, which limits TLR2-mediated pro-inflammatory responses. These data indicate that while the highly virulent F. tularensis uses CR3 for efficient uptake, optimal engagement of this receptor down-regulates TLR2-dependent pro-inflammatory responses by inhibiting MAPK activation through outside-in signaling. CR3-linked immune suppression is an important mechanism involved in the pathogenesis of F. tularensis infection.     

Identification of Key Hinge Residues Important for Nucleotide-Dependent Allostery in E. coli Hsp70/DnaK

DnaK is a molecular chaperone that has important roles in protein folding. The hydrolysis of ATP is essential to this activity, and the effects of nucleotides on the structure and function of DnaK have been extensively studied. However, the key residues that govern the conformational motions that define the apo, ATP-bound, and ADP-bound states are not entirely clear. Here, we used molecular dynamics simulations, mutagenesis, and enzymatic assays to explore the molecular basis of this process. Simulations of DnaK''s nucleotide-binding domain (NBD) in the apo, ATP-bound, and ADP/P_i-bound states suggested that each state has a distinct conformation, consistent with available biochemical and structural information. The simulations further suggested that large shearing motions between subdomains I-A and II-A dominated the conversion between these conformations. We found that several evolutionally conserved residues, especially G228 and G229, appeared to function as a hinge for these motions, because they predominantly populated two distinct states depending on whether ATP or ADP/P_i was bound. Consistent with the importance of these “hinge” residues, alanine point mutations caused DnaK to have reduced chaperone activities in vitro and in vivo. Together, these results clarify how sub-domain motions communicate allostery in DnaK.