Cloning and sequence analysis of cDNAs encoding the cytosolic precursors of subunits GapA and GapB of chloroplast glyceraldehyde-3-phosphate dehydrogenase from pea and spinach

Chloroplast glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is composed of two different subunits, GapA and GapB. cDNA clones containing the entire coding sequences of the cytosolic precursors for GapA from pea and for GapB from pea and spinach have been identified, sequenced and the derived amino acid sequences have been compared to the corresponding sequences from tobacco, maize and mustard. These comparisons show that GapB differs from GapA in about 20% of its amino acid residues and by the presence of a flexible and negatively charged C-terminal extension, possibly responsible for the observed association of the enzyme with chloroplast envelopes in vitro. This C-terminal extension (29 or 30 residues) may be susceptible to proteolytic cleavage thereby leading to a conversion of chloroplast GAPDH isoenzyme I into isoenzyme II. Evolutionary rate comparisons at the amino acid sequence level show that chloroplast GapA and GapB evolve roughly two-fold slower than their cytosolic counterpart GapC. GapA and GapB transit peptides evolve about 10 times faster than the corresponding mature subunits. They are relatively long (68 and 83 residues for pea GapA and spinach GapB respectively) and share a similar amino acid framework with other chloroplast transit peptides

LRP1 Receptor Controls Adipogenesis and Is Up-Regulated In Human and Mouse Obese Adipose Tissue

The cell surface low-density lipoprotein receptor-related protein 1, LRP1, plays a major role in lipid metabolism. The question that remains open concerns the function of LRP1 in adipogenesis. Here, we show that LRP1 is highly expressed in murine preadipocytes as well as in primary culture of human adipocytes. Moreover, LRP1 remains abundantly synthesised during mouse and human adipocyte differentiation. We demonstrate that LRP1 silencing in 3T3F442A murine preadipocytes significantly inhibits the expression of PPARγ, HSL and aP2 adipocyte differentiation markers after adipogenesis induction, and leads to lipid-depleted cells. We further show that the absence of lipids in LRP1-silenced preadipocytes is not caused by lipolysis induction. In addition, we provide the first evidences that LRP1 is significantly up-regulated in obese C57BI6/J mouse adipocytes and obese human adipose tissues. Interestingly, silencing of LRP1 in fully-differentiated adipocytes also reduces cellular lipid level and is associated with an increase of basal lipolysis. However, the ability of mature adipocytes to induce lipolysis is independent of LRP1 expression. Altogether, our findings highlight the dual role of LRP1 in the control of adipogenesis and lipid homeostasis, and suggest that LRP1 may be an important therapeutic target in obesity

Low Density Lipoprotein Receptor-Related Protein 1 Dependent Endosomal Trapping and Recycling of Apolipoprotein E

BACKGROUND: Lipoprotein receptors from the low density lipoprotein (LDL) receptor family are multifunctional membrane proteins which can efficiently mediate endocytosis and thereby facilitate lipoprotein clearance from the plasma. The biggest member of this family, the LDL receptor-related protein 1 (LRP1), facilitates the hepatic uptake of triglyceride-rich lipoproteins (TRL) via interaction with apolipoprotein E (apoE). In contrast to the classical LDL degradation pathway, TRL disintegrate in peripheral endosomes, and core lipids and apoB are targeted along the endocytic pathway for lysosomal degradation. Notably, TRL-derived apoE remains within recycling endosomes and is then mobilized by high density lipoproteins (HDL) for re-secretion. The aim of this study is to investigate the involvement of LRP1 in the regulation of apoE recycling. PRINCIPAL FINDINGS: Immunofluorescence studies indicate the LRP1-dependent trapping of apoE in EEA1-positive endosomes in human hepatoma cells. This processing is distinct from other LRP1 ligands such as RAP which is efficiently targeted to lysosomal compartments. Upon stimulation of HDL-induced recycling, apoE is released from LRP1-positive endosomes but is targeted to another, distinct population of early endosomes that contain HDL, but not LRP1. For subsequent analysis of the recycling capacity, we expressed the full-length human LRP1 and used an RNA interference approach to manipulate the expression levels of LRP1. In support of LRP1 determining the intracellular fate of apoE, overexpression of LRP1 significantly stimulated HDL-induced apoE recycling. Vice versa LRP1 knockdown in HEK293 cells and primary hepatocytes strongly reduced the efficiency of HDL to stimulate apoE secretion. CONCLUSION: We conclude that LRP1 enables apoE to accumulate in an early endosomal recycling compartment that serves as a pool for the intracellular formation and subsequent re-secretion of apoE-enriched HDL particles

Measurement of the cross-section and charge asymmetry of WW bosons produced in proton-proton collisions at s=8\sqrt{s}=8 TeV with the ATLAS detector

This paper presents measurements of the $W^+ \rightarrow \mu^+\nu$ and $W^- \rightarrow \mu^-\nu$ cross-sections and the associated charge asymmetry as a function of the absolute pseudorapidity of the decay muon. The data were collected in proton--proton collisions at a centre-of-mass energy of 8 TeV with the ATLAS experiment at the LHC and correspond to a total integrated luminosity of 20.2~\mbox{fb^{-1}}. The precision of the cross-section measurements varies between 0.8% to 1.5% as a function of the pseudorapidity, excluding the 1.9% uncertainty on the integrated luminosity. The charge asymmetry is measured with an uncertainty between 0.002 and 0.003. The results are compared with predictions based on next-to-next-to-leading-order calculations with various parton distribution functions and have the sensitivity to discriminate between them.Comment: 38 pages in total, author list starting page 22, 5 figures, 4 tables, submitted to EPJC. All figures including auxiliary figures are available at https://atlas.web.cern.ch/Atlas/GROUPS/PHYSICS/PAPERS/STDM-2017-13

Search for chargino-neutralino production with mass splittings near the electroweak scale in three-lepton final states in √s=13 TeV pp collisions with the ATLAS detector

A search for supersymmetry through the pair production of electroweakinos with mass splittings near the electroweak scale and decaying via on-shell W and Z bosons is presented for a three-lepton final state. The analyzed proton-proton collision data taken at a center-of-mass energy of √s=13  TeV were collected between 2015 and 2018 by the ATLAS experiment at the Large Hadron Collider, corresponding to an integrated luminosity of 139  fb−1. A search, emulating the recursive jigsaw reconstruction technique with easily reproducible laboratory-frame variables, is performed. The two excesses observed in the 2015–2016 data recursive jigsaw analysis in the low-mass three-lepton phase space are reproduced. Results with the full data set are in agreement with the Standard Model expectations. They are interpreted to set exclusion limits at the 95% confidence level on simplified models of chargino-neutralino pair production for masses up to 345 GeV

Search for direct stau production in events with two hadronic tau-leptons in root s=13 TeV pp collisions with the ATLAS detector

A search for the direct production of the supersymmetric partners ofτ-leptons (staus) in final stateswith two hadronically decayingτ-leptons is presented. The analysis uses a dataset of pp collisions corresponding to an integrated luminosity of139fb−1, recorded with the ATLAS detector at the LargeHadron Collider at a center-of-mass energy of 13 TeV. No significant deviation from the expected StandardModel background is observed. Limits are derived in scenarios of direct production of stau pairs with eachstau decaying into the stable lightest neutralino and oneτ-lepton in simplified models where the two staumass eigenstates are degenerate. Stau masses from 120 GeV to 390 GeV are excluded at 95% confidencelevel for a massless lightest neutralino

Search for new phenomena in final states with an energetic jet and large missing transverse momentum in pp collisions at √ s = 8 TeV with the ATLAS detector

Results of a search for new phenomena in final states with an energetic jet and large missing transverse momentum are reported. The search uses 20.3 fb−1 of √ s = 8 TeV data collected in 2012 with the ATLAS detector at the LHC. Events are required to have at least one jet with pT > 120 GeV and no leptons. Nine signal regions are considered with increasing missing transverse momentum requirements between Emiss T > 150 GeV and Emiss T > 700 GeV. Good agreement is observed between the number of events in data and Standard Model expectations. The results are translated into exclusion limits on models with either large extra spatial dimensions, pair production of weakly interacting dark matter candidates, or production of very light gravitinos in a gauge-mediated supersymmetric model. In addition, limits on the production of an invisibly decaying Higgs-like boson leading to similar topologies in the final state are presente

LRP1 Regulates Architecture of the Vascular Wall by Controlling PDGFRβ-Dependent Phosphatidylinositol 3-Kinase Activation

Low density lipoprotein receptor-related protein 1 (LRP1) protects against atherosclerosis by regulating the activation of platelet-derived growth factor receptor beta (PDGFRbeta) in vascular smooth muscle cells (SMCs). Activated PDGFRbeta undergoes tyrosine phosphorylation and subsequently interacts with various signaling molecules, including phosphatidylinositol 3-kinase (PI3K), which binds to the phosphorylated tyrosine 739/750 residues in mice, and thus regulates actin polymerization and cell movement.In this study, we found disorganized actin in the form of membrane ruffling and enhanced cell migration in LRP1-deficient (LRP1-/-) SMCs. Marfan syndrome-like phenotypes such as tortuous aortas, disrupted elastic layers and abnormally activated transforming growth factor beta (TGFbeta) signaling are present in smooth muscle-specific LRP1 knockout (smLRP1-/-) mice. To investigate the role of LRP1-regulated PI3K activation by PDGFRbeta in atherogenesis, we generated a strain of smLRP1-/- mice in which tyrosine 739/750 of the PDGFRbeta had been mutated to phenylalanines (PDGFRbeta F2/F2). Spontaneous atherosclerosis was significantly reduced in the absence of hypercholesterolemia in these mice compared to smLRP1-/- animals that express wild type PDGFR. Normal actin organization was restored and spontaneous SMC migration as well as PDGF-BB-induced chemotaxis was dramatically reduced, despite continued overactivation of TGFbeta signaling, as indicated by high levels of nuclear phospho-Smad2.Our data suggest that LRP1 regulates actin organization and cell migration by controlling PDGFRbeta-dependent activation of PI3K. TGFbeta activation alone is not sufficient for the expression of the Marfan-like vascular phenotype. Thus, regulation of PI3 Kinase by PDGFRbeta is essential for maintaining vascular integrity, and for the prevention of atherosclerosis as well as Marfan syndrome

Cholesterol-Dependent Anaplasma phagocytophilum Exploits the Low-Density Lipoprotein Uptake Pathway

In eukaryotes, intracellular cholesterol homeostasis and trafficking are tightly regulated. Certain bacteria, such as Anaplasma phagocytophilum, also require cholesterol; it is unknown, however, how this cholesterol-dependent obligatory intracellular bacterium of granulocytes interacts with the host cell cholesterol regulatory pathway to acquire cholesterol. Here, we report that total host cell cholesterol increased >2-fold during A. phagocytophilum infection in a human promyelocytic leukemia cell line. Cellular free cholesterol was enriched in A. phagocytophilum inclusions as detected by filipin staining. We determined that A. phagocytophilum requires cholesterol derived from low-density lipoprotein (LDL), because its replication was significantly inhibited by depleting the growth medium of cholesterol-containing lipoproteins, by blocking LDL uptake with a monoclonal antibody against LDL receptor (LDLR), or by treating the host cells with inhibitors that block LDL-derived cholesterol egress from late endosomes or lysosomes. However, de novo cholesterol biosynthesis is not required, since inhibition of the biosynthesis pathway did not inhibit A. phagocytophilum infection. The uptake of fluorescence-labeled LDL was enhanced in infected cells, and LDLR expression was up-regulated at both the mRNA and protein levels. A. phagocytophilum infection stabilized LDLR mRNA through the 3′ UTR region, but not through activation of the sterol regulatory element binding proteins. Extracellular signal–regulated kinase (ERK) was up-regulated by A. phagocytophilum infection, and inhibition of its upstream kinase, MEK, by a specific inhibitor or siRNA knockdown, reduced A. phagocytophilum infection. Up-regulation of LDLR mRNA by A. phagocytophilum was also inhibited by the MEK inhibitor; however, it was unclear whether ERK activation is required for LDLR mRNA up-regulation by A. phagocytophilum. These data reveal that A. phagocytophilum exploits the host LDL uptake pathway and LDLR mRNA regulatory system to accumulate cholesterol in inclusions to facilitate its replication