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

Cytosolic DNA does not affect HIV-1 BaL replication in IL2/PHA PBMCs.

<p>(<b>A</b>) IL2/PHA PBMCs were infected with HIV-1 BaL at an MOI of 0.002, and p24 levels were measured in the supernatants after 24, 48, and 72 hours of infection. (<b>B</b>) IL2/PHA PBMCs were pretreated with AZT at increasing doses 30 min before infection with HIV-1 BaL at an MOI of 0.002. Levels of p24 were measured in the supernatants 72 hours post infection by ELISA. (<b>C, D</b>) IL2/PHA PBMCs were transfected with ssDNA (2 µg/mL) (<b>C</b>) 4 hours before or (<b>D</b>) 24 hours after infection with HIV-1. Supernatants were harvested 72 hours post infection and p24 levels measured by ELISA. Data are shown as mean of triplicates +/− SD. Similar results were obtained in three or more independent experiments. Mock, Lipofectamine.</p

Expression of DNA signaling pathway molecules in IL2/PHA PBMCs and CD4+ T cells.

<p>(<b>A</b>) Whole cell lysates of IL2/PHA PBMCs from 2 donors were analyzed for expression of IFI16, STING, TBK1, IRF3 and β-actin by Western Blotting. (<b>B, C</b>) Whole cell lysates from IL2/PHA-treated PBMCs and CD4+ T cells were stimulated with ssDNA, dsDNA (both 2 µg/mL) or lipofactamine for 2 h of IL2/PHA PBMCs were analyzed for levels of IFI16 and cGAS by Western Blotting. Similar results were obtained with two independent donors.</p