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Ge Zhang, Vadim Baidin, Karanbir S Pahil, Eileen Moison, David Tomasek, Nitya S Ramadoss, Arnab K Chatterjee, Case W McNamara, Travis S Young, Peter G Schultz, Timothy C Meredith, Daniel Kahne
New drugs are needed to treat gram-negative bacterial infections. These bacteria are protected by an outer membrane which prevents many antibiotics from reaching their cellular targets. The outer leaflet of the outer membrane contains LPS, which is responsible for creating this permeability barrier. Interfering with LPS biogenesis affects bacterial viability. We developed a cell-based screen that identifies inhibitors of LPS biosynthesis and transport by exploiting the nonessentiality of this pathway in Acinetobacter We used this screen to find an inhibitor of MsbA, an ATP-dependent flippase that translocates LPS across the inner membrane...
May 7, 2018: Proceedings of the National Academy of Sciences of the United States of America
Toshihide Kobayashi, Anant K Menon
The plasma membrane is a ∼4 nm thick phospholipid bilayer that defines the boundary of a cell, segregating internal content from the external environment. Its hydrophobic interior presents a barrier to the exchange of ions and polar solutes between the inside and outside of the cell, as well as to the spontaneous reorientation of lipids between the two leaflets of the bilayer. Specific transport systems, e.g. ion channels and lipid flippases, are needed to enable the passage of these molecules across the plasma membrane at physiologically relevant rates...
April 23, 2018: Current Biology: CB
Jasmine M Gardner, Cameron F Abrams
Molecular dynamics simulations of a solvent-free coarse-grained lipid model are used to characterize the mechanisms by which lipid-bilayer hemifusion diaphragm (HD) intermediates relax, across a range of global compositions of negative intrinsic curvature (NIC) lipids and neutral-curvature lipids. At low concentrations of NIC lipids, rapid fission produces a double bilayer end state through a lateral diffusion-based mechanism enabled by spontaneous rim-pore defects. At moderately higher NIC lipid concentrations, rim pores are absent and stable leaflet three-junctions persist, revealing an HD relaxation mechanism entirely reliant on lipid flip-flop, and end states that are either stable fusion pores or stable HD's...
April 20, 2018: Biochimica et Biophysica Acta
Xuhua Yu, Jiajun Xu, Wenwu Liu, Weigang Xu
BACKGROUND/AIMS: Intravascular bubbles can exert pleiotropic detrimental effects, partly by inducing endothelial microparticles (EMPs) production, which play critical roles in cell communication and vascular inflammation cascades. However, the underlying mechanisms remain unclear. This study aimed to delineate the possible mechanisms involving bubble-induced EMPs formation. METHODS: Human umbilical vein endothelial cells (HUVECs) were contacted by bubbles and EMPs level in supernatant were quantified by flow cytometry...
April 13, 2018: Cellular Physiology and Biochemistry
Wenyi Sun, Xiaobing Yang, Xueying Wang, Xiang Jiao, Sufang Zhang, Yushi Luan, Zongbao K Zhao
OBJECTIVES: To establish a recombinase flippase (FLP) and flippase recognition target (FRT) system-mediated protocol for post-integration excision of exogenous DNA fragments in the oleaginous yeast Rhodosporidium toruloides. RESULTS: Binary vectors were constructed to harbor FLP expressing cassette together with the hygromycin-resistance marker. Results showed that R. toruloides transformants produced FLP, but failed to mediate removal of the bleomycin-resistance marker within two FRT sites...
March 31, 2018: Biotechnology Letters
Naoto Takada, Tomoki Naito, Takanari Inoue, Kazuhisa Nakayama, Hiroyuki Takatsu, Hye-Won Shin
P4-ATPases are phospholipid flippases that translocate phospholipids from the exoplasmic/luminal to the cytoplasmic leaflet of biological membranes. All P4-ATPases in yeast and some in other organisms are required for membrane trafficking; therefore, changes in the transbilayer lipid composition induced by flippases are thought to be crucial for membrane deformation. However, it is poorly understood whether the phospholipid-flipping activity of P4-ATPases can promote membrane deformation. In this study, we assessed membrane deformation induced by flippase activity via monitoring the extent of membrane tubulation using a system that allows inducible recruitment of Bin/amphiphysin/Rvs (BAR) domains to the plasma membrane (PM)...
March 29, 2018: EMBO Journal
Birke J Benedikter, Antje R Weseler, Emiel F M Wouters, Paul H M Savelkoul, Gernot G U Rohde, Frank R M Stassen
Extracellular vesicles (EVs), including microvesicles and exosomes, are emerging as important regulators of homeostasis and pathophysiology. During pro-inflammatory and pro-oxidant conditions, EV release is induced. As EVs released under such conditions often exert pro-inflammatory and procoagulant effects, they may actively promote the pathogenesis of chronic diseases. There is evidence that thiol group-containing antioxidants can prevent EV induction by pro-inflammatory and oxidative stimuli, likely by protecting protein thiols of the EV-secreting cells from oxidation...
March 28, 2018: Cellular and Molecular Life Sciences: CMLS
Ning Li, Yeming Yang, Cailing Liang, Qiang Qiu, Cong Pan, Mengyuan Li, Shengyong Yang, Lijuan Chen, Xianjun Zhu, Yiguo Hu
The fundamental structure of eukaryotic cell plasma membrane is the phospholipid bilayer, which contains four major phospholipids. These phospholipids are asymmetrically distributed between the outer and inner leaflets. P4-ATPase flippase complexes play essential roles in ensuring this asymmetry. Here we showed that conditional deletion of Tmem30a, the beta subunit of P4-ATPase flippase complex, caused pancytopenia in mice. Tmem30a deficiency resulted in depletion of lineage-committed blood cells in the peripheral blood, spleen, and bone marrow...
March 21, 2018: American Journal of Pathology
Pei-Tseng Lee, Jonathan Zirin, Oguz Kanca, Wen-Wen Lin, Karen L Schulze, David Li-Kroeger, Rong Tao, Colby Devereaux, Yanhui Hu, Verena Chung, Ying Fang, Yuchun He, Hongling Pan, Ming Ge, Zhongyuan Zuo, Benjamin E Housden, Stephanie E Mohr, Shinya Yamamoto, Robert W Levis, Allan C Spradling, Norbert Perrimon, Hugo J Bellen
We generated a library of ~1,000 Drosophila stocks in which we inserted a construct in the intron of genes allowing expression of GAL4 under control of endogenous promoters while arresting transcription with a polyadenylation signal 3' of the GAL4. This allows numerous applications. First, ~90% of insertions in essential genes cause a severe loss-of-function phenotype, an effective way to mutagenize genes. Interestingly, 12/14 chromosomes engineered through CRISPR do not carry second-site lethal mutations. Second, 26/36(70%) of lethal insertions tested are rescued with a single UAS- cDNA construct...
March 22, 2018: ELife
Sean D Workman, Liam J Worrall, Natalie C J Strynadka
Undecaprenyl pyrophosphate phosphatase (UppP) is an integral membrane protein that recycles the lipid carrier essential to the ongoing biosynthesis of the bacterial cell wall. Individual building blocks of peptidoglycan are assembled in the cytoplasm on undecaprenyl phosphate (C55-P) before being flipped to the periplasmic face, where they are polymerized and transferred to the existing cell wall sacculus, resulting in the side product undecaprenyl pyrophosphate (C55-PP). Interruption of UppP's regeneration of C55-P from C55-PP leads to the buildup of cell wall intermediates and cell lysis...
March 20, 2018: Nature Communications
Frederick A Rubino, Sujeet Kumar, Natividad Ruiz, Suzanne Walker, Daniel Kahne
MurJ, the flippase that exports the bacterial cell wall mon-omer Lipid II to the periplasm, is a target for new antibiot-ics, which are desperately needed to treat Gram-negative infections. Quantitative methods to monitor MurJ activity are required to characterize inhibitors but are challenging to develop because the lipid-linked substrate is not chemi-cally altered in a flippase reaction. Here we show that MurJ inhibition can be quantified by measuring the accu-mulation of intracellular Lipid II using a biotin-tagging strategy...
March 20, 2018: Journal of the American Chemical Society
Meriem El Ghachi, Nicole Howe, Chia-Ying Huang, Vincent Olieric, Rangana Warshamanage, Thierry Touzé, Dietmar Weichert, Phillip J Stansfeld, Meitian Wang, Fred Kerff, Martin Caffrey
As a protective envelope surrounding the bacterial cell, the peptidoglycan sacculus is a site of vulnerability and an antibiotic target. Peptidoglycan components, assembled in the cytoplasm, are shuttled across the membrane in a cycle that uses undecaprenyl-phosphate. A product of peptidoglycan synthesis, undecaprenyl-pyrophosphate, is converted to undecaprenyl-phosphate for reuse in the cycle by the membrane integral pyrophosphatase, BacA. To understand how BacA functions, we determine its crystal structure at 2...
March 14, 2018: Nature Communications
Tetsuo Mioka, Konomi Fujimura-Kamada, Nahiro Mizugaki, Takuma Kishimoto, Takamitsu Sano, Hitoshi Nunome, David E Williams, Raymond J Andersen, Kazuma Tanaka
Phospholipid flippase (type 4 P-type ATPase) plays a major role in the generation of phospholipid asymmetry in eukaryotic cell membranes. Loss of Lem3p-Dnf1/2p flippases leads to the exposure of phosphatidylserine (PS) and phosphatidylethanolamine (PE) on the cell surface in yeast, resulting in sensitivity to PS- or PE-binding peptides. We isolated Sfk1p, a conserved membrane protein in the TMEM150/FRAG1/DRAM family, as a multicopy suppressor of this sensitivity. Overexpression of SFK1 decreased PS/PE exposure in lem3Δ mutant cells...
May 15, 2018: Molecular Biology of the Cell
Jani Reddy Bolla, Joshua B Sauer, Di Wu, Shahid Mehmood, Timothy M Allison, Carol V Robinson
Translocation of lipid II across the cytoplasmic membrane is essential in peptidoglycan biogenesis. Although most steps are understood, identifying the lipid II flippase has yielded conflicting results, and the lipid II binding properties of two candidate flippases-MurJ and FtsW-remain largely unknown. Here we apply native mass spectrometry to both proteins and characterize lipid II binding. We observed lower levels of lipid II binding to FtsW compared to MurJ, consistent with MurJ having a higher affinity...
March 2018: Nature Chemistry
João M Monteiro, Ana R Pereira, Nathalie T Reichmann, Bruno M Saraiva, Pedro B Fernandes, Helena Veiga, Andreia C Tavares, Margarida Santos, Maria T Ferreira, Vânia Macário, Michael S VanNieuwenhze, Sérgio R Filipe, Mariana G Pinho
Peptidoglycan is the main component of the bacterial wall and protects cells from the mechanical stress that results from high intracellular turgor. Peptidoglycan biosynthesis is very similar in all bacteria; bacterial shapes are therefore mainly determined by the spatial and temporal regulation of peptidoglycan synthesis rather than by the chemical composition of peptidoglycan. The form of rod-shaped bacteria, such as Bacillus subtilis or Escherichia coli, is generated by the action of two peptidoglycan synthesis machineries that act at the septum and at the lateral wall in processes coordinated by the cytoskeletal proteins FtsZ and MreB, respectively...
February 22, 2018: Nature
Robert Holz, Andreas E Kremer, Dieter Lütjohann, Hermann E Wasmuth, Frank Lammert, Marcin Krawczyk
Benign recurrent intrahepatic cholestasis (BRIC) is a peculiar familial disease caused by mutations of the genes encoding hepatocanalicular flippase for phosphatidylserine (ATP8B1; BRIC type 1) or the bile salt export pump (ABCB11; BRIC type 2). Here, we report on a patient with nasobiliary drainage-refractory BRIC type 2 who improved under plasma separation and anion absorption therapy. We also suggest that nasobiliary drainage might be an ineffective approach in carriers of severe loss-of-function mutations of the bile salt export pump ABCB11...
February 2018: Hepatology Communications
Zonera Hassan, Christian Schneeweis, Matthias Wirth, Christian Veltkamp, Zahra Dantes, Benedikt Feuerecker, Güralp O Ceyhan, Shirley K Knauer, Wilko Weichert, Roland M Schmid, Roland Stauber, Alexander Arlt, Oliver H Krämer, Roland Rad, Maximilian Reichert, Dieter Saur, Günter Schneider
BACKGROUND: Although the mechanistic target of rapamycin (MTOR) kinase, included in the mTORC1 and mTORC2 signalling hubs, has been demonstrated to be active in a significant fraction of patients with pancreatic ductal adenocarcinoma (PDAC), the value of the kinase as a therapeutic target needs further clarification. METHODS: We used Mtor floxed mice to analyse the function of the kinase in context of the pancreas at the genetic level. Using a dual-recombinase system, which is based on the flippase-FRT (Flp-FRT) and Cre-loxP recombination technologies, we generated a novel cellular model, allowing the genetic analysis of MTOR functions in tumour maintenance...
February 6, 2018: British Journal of Cancer
Christopher Bystroff
CatSper is a voltage-dependent calcium channel located in the plasma membrane of the sperm flagellum and is responsible for triggering hyperactive motility. A homology model for the transmembrane region was built in which the arrangement of the subunits around the pseudo-four-fold symmetry axis was deduced by the pairing of conserved transmembranal cysteines across mammals. Directly emergent of the predicted quaternary structure is an architecture in which tetramers polymerize through additional, highly conserved cysteines, creating one or more double-rows channels extending the length of the principal piece of the mammalian sperm tail...
March 2018: Reproductive Biology
Katharina B Beer, Jennifer Rivas-Castillo, Kenneth Kuhn, Gholamreza Fazeli, Birgit Karmann, Jeremy F Nance, Christian Stigloher, Ann M Wehman
Cells release extracellular vesicles (EVs) that mediate intercellular communication and repair damaged membranes. Despite the pleiotropic functions of EVs in vitro, their in vivo function is debated, largely because it is unclear how to induce or inhibit their formation. In particular, the mechanisms of EV release by plasma membrane budding or ectocytosis are poorly understood. We previously showed that TAT-5 phospholipid flippase activity maintains the asymmetric localization of the lipid phosphatidylethanolamine (PE) in the plasma membrane and inhibits EV budding by ectocytosis in Caenorhabditis elegans However, no proteins that inhibit ectocytosis upstream of TAT-5 were known...
January 24, 2018: Proceedings of the National Academy of Sciences of the United States of America
Marcus W Stepp, Mark A Doll, Samantha M Carlisle, J Christopher States, David W Hein
Arylamine N-acetyltransferase 1 (NAT1) expression is reported to affect proliferation, invasiveness, and growth of cancer cells. MDA-MB-231 breast cancer cells were engineered such that NAT1 expression was elevated or suppressed, or treated with a small molecule inhibitor of NAT1. The MDA-MB-231 human breast cancer cell lines were engineered with a scrambled shRNA, a NAT1 specific shRNA or a NAT1 overexpression cassette stably integrated into a single flippase recognition target (FRT) site facilitating incorporation of these different genetic elements into the same genomic location...
April 2018: Molecular Carcinogenesis
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