Disease mutations reveal residues critical to the interaction of P4-ATPases with lipid substrates

Abstract Phospholipid flippases (P4-ATPases) translocate specific phospholipids from the exoplasmic to the cytoplasmic leaflet of membranes. While there is good evidence that the overall molecular structure of flippases is similar to that of P-type ATPase ion-pumps, the transport pathway for the “giant” lipid substrate has not been determined. ATP8A2 is a flippase with selectivity toward phosphatidylserine (PS), possessing a net negatively charged head group, whereas ATP8B1 exhibits selectivity toward the electrically neutral phosphatidylcholine (PC). Setting out to elucidate the functional consequences of flippase disease mutations, we have identified residues of ATP8A2 that are critical to the interaction with the lipid substrate during the translocation process. Among the residues pinpointed are I91 and L308, which are positioned near proposed translocation routes through the protein. In addition we pinpoint two juxtaposed oppositely charged residues, E897 and R898, in the exoplasmic loop between transmembrane helices 5 and 6. The glutamate is conserved between PS and PC flippases, whereas the arginine is replaced by a negatively charged aspartate in ATP8B1. Our mutational analysis suggests that the glutamate repels the PS head group, whereas the arginine minimizes this repulsion in ATP8A2, thereby contributing to control the entry of the phospholipid substrate into the translocation pathway

CEP128 Localizes to the Subdistal Appendages of the Mother Centriole and Regulates TGF-β/BMP Signaling at the Primary Cilium

Summary: The centrosome is the main microtubule-organizing center in animal cells and comprises a mother and daughter centriole surrounded by pericentriolar material. During formation of primary cilia, the mother centriole transforms into a basal body that templates the ciliary axoneme. Ciliogenesis depends on mother centriole-specific distal appendages, whereas the role of subdistal appendages in ciliary function is unclear. Here, we identify CEP128 as a centriole subdistal appendage protein required for regulating ciliary signaling. Loss of CEP128 did not grossly affect centrosomal or ciliary structure but caused impaired transforming growth factor-β/bone morphogenetic protein (TGF-β/BMP) signaling in zebrafish and at the primary cilium in cultured mammalian cells. This phenotype is likely the result of defective vesicle trafficking at the cilium as ciliary localization of RAB11 was impaired upon loss of CEP128, and quantitative phosphoproteomics revealed that CEP128 loss affects TGF-β1-induced phosphorylation of multiple proteins that regulate cilium-associated vesicle trafficking. : Mönnich et al. show that CEP128 localizes to the subdistal appendages of the mother centriole and basal body of the primary cilium. CEP128 regulates vesicular trafficking and targeting of RAB11 to the primary cilium. CEP128 loss leads to impaired TGF-β/BMP signaling, which, in zebrafish, is associated with defective organ development. Keywords: primary cilium, basal body, centriole, subdistal appendage, centrosome, transforming growth factor β, TGF-β, bone morphogenetic protein, BMP, zebrafish, phosphoproteomics, CEP12

Identification of the CRE-1 Cellulolytic Regulon in Neurospora crassa

Background: In filamentous ascomycete fungi, the utilization of alternate carbon sources is influenced by the zinc finger transcription factor CreA/CRE-1, which encodes a carbon catabolite repressor protein homologous to Mig1 from Saccharomyces cerevisiae. In Neurospora crassa, deletion of cre-1 results in increased secretion of amylase and b-galactosidase. Methodology/Principal Findings: Here we show that a strain carrying a deletion of cre-1 has increased cellulolytic activity and increased expression of cellulolytic genes during growth on crystalline cellulose (Avicel). Constitutive expression of cre-1 complements the phenotype of a N. crassa Dcre-1 strain grown on Avicel, and also results in stronger repression of cellulolytic protein secretion and enzyme activity. We determined the CRE-1 regulon by investigating the secretome and transcriptome of a Dcre-1 strain as compared to wild type when grown on Avicel versus minimal medium. Chromatin immunoprecipitation-PCR of putative target genes showed that CRE-1 binds to only some adjacent 59-SYGGRG-39 motifs, consistent with previous findings in other fungi, and suggests that unidentified additional regulatory factors affect CRE-1 binding to promoter regions. Characterization of 30 mutants containing deletions in genes whose expression level increased in a Dcre-1 strain under cellulolytic conditions identified novel genes that affect cellulase activity and protein secretion

Lymphadenectomy in surgical stage I epithelial ovarian cancer

Abstract Objective. To identify the extent of lymphadenectomy performed in women presenting with epithelial ovarian cancer macroscopically confined to the ovary. Furthermore, the effect of lymphadenectomy on overall survival is evaluated

IL2/PHA PBMCs fail to induce type I IFN responses and pro-inflammatory cytokines upon DNA transfection.

<p>(<b>A–D</b>) IL2/PHA PBMCs were transfected with HIV-derived ssDNA and dsDNA (2 µg/mL), or infected with SeV (MOI 0.5). Total RNA was isolated after 6 hours for RT-qPCR measurements of (<b>A</b>) IFNβ, (<b>B</b>) CXCL10, (<b>C</b>) ISG56, and (<b>D</b>) Viperin. (<b>E–H</b>) IL2/PHA PBMCs were transfected with ssDNA and dsDNA (2 µg/mL), or infected with SeV (MOI 0.5). Supernatants were harvested after 24 hours and analyzed for CXCL10, TNFα, MIP-1α, and IL6 protein levels. (<b>I–J</b>) PMA (100 nM) treated THP-1 cells were transfected with ssDNA and dsDNA (2 µg/mL). Total RNA was harvested after 6 hours for RT-qPCR measurements of IFNβ and CXCL10 mRNA. (<b>K</b>) Primary human monocyte-derived macrophages (MDM)s were transfected with ssDNA and dsDNA (2 µg/mL). Total RNA was harvested after 6 hours for RT-qPCR measurements of IFNβ. (<b>L</b>) Un-stimulated PBMCs were isolated and immediately transfected with ssDNA and dsDNA (2 µg/mL), or infected with SeV (MOI 0.5). Supernatants were harvested after 24 hours and analyzed for CXCL10 by ELISA. Both PCR and ELISA data are shown as means of triplicates +/− SD. Similar results were obtained in three independent experiments. Mock, Lipofectamine.</p

Transfected DNA co-localizes with the DNA sensor IFI16 in activated T cells.

<p>(<b>A, C</b>) IL2/PHA PBMCs transfected with 2 µg/mL of FITC-labeled DNA or 0.5 µM ODN2216 as indicated for 2 hours were fixed and stained with anti-IFI16 antibody and visualized by confocal microscopy. Presented cells stained positive for CD3. IFI16 is shown in red and DNA in green. (<b>B, D</b>) Percentage colocalization of cytoplasmic spots positive for IFI16 and DNA. Data is based on quantification of IFI16/DNA spots in more than 100 cells per donor in 3 different donors. Data is shown as means +/− SD. Scale bar, 5 µm.</p