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Neale D Ridgway - One of the best experts on this subject based on the ideXlab platform.
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Phosphorylation of a serine/proline-rich motif in oxysterol binding Protein-related Protein 4L (ORP4L) regulates cholesterol and vimentin binding
2019Co-Authors: Antonietta Pietrangelo, Neale D RidgwayAbstract:The family of oxysterol binding Protein (OSBP) and OSBP-related Proteins (ORPs) mediate sterol and phospholipid transfer and signaling at membrane contact sites (MCS). The activity of OSBP at MCS is regulated by phosphorylation, but whether this applies to ORPs is unknown. Here we report the functional characterization of a unique proline/serine-rich phosphorylation motif (S762SPSSPSS769) in the lipid binding OSBP-related domain of full-length ORP4L and a truncated variant ORP4S. Phosphorylation was confirmed by mass spectrometry and [32P]PO4 incorporation, and in silico and in vitro assays using purified ORP4L identified putative proline-directed kinases that phosphorylate the site. The functional significance of the phospho-site was assessed by mutating serine 762, S763, S766 and S768 to aspartate or alanine to produce phosphomimetic (S4D) and phosphorylation-deficient (S4A) mutants, respectively. Solution binding of 25-hydroxycholesterol and cholesterol by recombinant ORP4L-S4D and -S4A was similar to wild-type but ORP4L-S4D more effectively extracted cholesterol from liposomes. ORP4L homo-dimerization was unaffected by phosphorylation but gel filtration of ORP4L-S4D indicated that the native conformation was affected. Confocal microscopy revealed that ORP4L-S4D also strongly associated with bundled vimentin filaments, a feature shared with ORP4S which lacks the PH and dimerization domains. We conclude that phosphorylation of a unique serine/proline motif in the ORD induces a conformation change in ORP4L that enhances interaction with vimentin and cholesterol extraction from membranes.
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oxysterol binding Protein related Protein 1l regulates cholesterol egress from the endo lysosomal system
2017Co-Authors: Kexin Zhao, Neale D RidgwayAbstract:LipoProtein cholesterol is delivered to the limiting membrane of late endosomes/lysosomes (LELs) by Niemann-Pick C1 (NPC1). However, the mechanism of cholesterol transport from LELs to the endoplasmic reticulum (ER) is poorly characterized. We report that Oxysterol-Binding Protein-related Protein 1L (ORP1L) is necessary for this stage of cholesterol export. CRISPR-mediated knockout of ORP1L in HeLa and HEK293 cells reduced esterification of cholesterol to the level in NPC1 knockout cells, and it increased the expression of sterol-regulated genes and de novo cholesterol synthesis, indicative of a block in cholesterol transport to the ER. In the absence of this transport pathway, cholesterol-enriched LELs accumulated in the Golgi/perinuclear region. Cholesterol delivery to the ER required the sterol-, phosphatidylinositol 4-phosphate-, and vesicle-associated membrane Protein-associated Protein (VAP)-binding activities of ORP1L, as well as NPC1 expression. These results suggest that ORP1L-dependent membrane contacts between LELs and the ER coordinate cholesterol transfer with the retrograde movement of endo-lysosomal vesicles.
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oxysterol binding Protein activation at endoplasmic reticulum golgi contact sites reorganizes phosphatidylinositol 4 phosphate pools
2016Co-Authors: Asako Goto, Mark Charman, Neale D RidgwayAbstract:Oxysterol-Binding Protein (OSBP) exchanges cholesterol and phosphatidylinositol 4-phosphate (PI-4P) at contact sites between the endoplasmic reticulum (ER) and the trans-Golgi/trans-Golgi network. 25-Hydroxycholesterol (25OH) competitively inhibits this exchange reaction in vitro and causes the constitutive localization of OSBP at the ER/Golgi interface and PI-4P-dependent recruitment of ceramide transfer Protein (CERT) for sphingomyelin synthesis. We used PI-4P probes and mass analysis to determine how OSBP controls the availability of PI-4P for this metabolic pathway. Treatment of fibroblasts or Chinese hamster ovary (CHO) cells with 25OH caused a 50-70% reduction in Golgi-associated immunoreactive PI-4P that correlated with Golgi localization of OSBP. In contrast, 25OH caused an OSBP-dependent enrichment in Golgi PI-4P that was detected with a pleckstrin homology domain probe. The cellular mass of phosphatidylinositol monophosphates and Golgi PI-4P measured with an unbiased PI-4P probe (P4M) was unaffected by 25OH and OSBP silencing, indicating that OSBP shifts the distribution of PI-4P upon localization to ER-Golgi contact sites. The PI-4P and sterol binding activities of OSBP were both required for 25OH activation of sphingomyelin synthesis, suggesting that 25OH must be exchanged for PI-4P to be concentrated at contact sites. We propose a model wherein 25OH activation of OSBP promotes the binding and retention of PI-4P at ER-Golgi contact sites. This pool of PI-4P specifically recruits pleckstrin homology domain-containing Proteins involved in lipid transfer and metabolism, such as CERT.
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oxysterol binding Protein osbp related Protein 4 orp4 is essential for cell proliferation and survival
2014Co-Authors: Mark Charman, Terry R Colbourne, Antonietta Pietrangelo, Laurent Kreplak, Neale D RidgwayAbstract:Oxysterol-Binding Protein (OSBP) and OSBP-related Proteins (ORPs) comprise a large gene family with sterol/lipid transport and regulatory activities. ORP4 (OSBP2) is a closely related paralogue of OSBP, but its function is unknown. Here we show that ORP4 binds similar sterol and lipid ligands as OSBP and other ORPs but is uniquely required for the proliferation and survival of cultured cells. Recombinant ORP4L and a variant without a pleckstrin homology (PH) domain (ORP4S) bind 25-hydroxycholesterol and extract and transfer cholesterol between liposomes. Two conserved histidine residues in the OSBP homology domain ORP4 are essential for binding phosphatidylinositol 4-phosphate but not sterols. The PH domain of ORP4L also binds phosphatidylinositol 4-phosphate in the Golgi apparatus. However, in the context of ORP4L, the PH domain is required for normal organization of the vimentin network. Unlike OSBP, RNAi silencing of all ORP4 variants (including a partial PH domain truncation termed ORP4M) in HEK293 and HeLa cells resulted in growth arrest but not cell death. ORP4 silencing in non-transformed intestinal epithelial cells (IEC)-18 caused apoptosis characterized by caspase 3 and poly(ADP-ribose) polymerase processing, DNA cleavage, and JNK phosphorylation. IEC-18 transformed with oncogenic H-Ras have increased expression of ORP4L and ORP4S Proteins and are resistant to the growth-inhibitory effects of ORP4 silencing. Results suggest that ORP4 promotes the survival of rapidly proliferating cells.
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inhibition of hcv replication by oxysterol binding Protein related Protein 4 orp4 through interaction with hcv ns5b and alteration of lipid droplet formation
2013Co-Authors: Neale D Ridgway, Inwoo Park, Jean Ndjomou, Yahong Wen, Ziqing Liu, Cheng C KaoAbstract:Hepatitis C virus (HCV) RNA replication involves complex interactions among the 3’x RNA element within the HCV 3’ untranslated region, viral and host Proteins. However, many of the host Proteins remain unknown. In this study, we devised an RNA affinity chromatography /2D/MASS proteomics strategy and identified nine putative 3’ X-associated host Proteins; among them is Oxysterol-Binding Protein-related Protein 4 (ORP4), a cytoplasmic receptor for oxysterols. We determined the relationship between ORP4 expression and HCV replication. A very low level of constitutive ORP4 expression was detected in hepatocytes. Ectopically expressed ORP4 was detected in the endoplasmic reticulum and inhibited luciferase reporter gene expression in HCV subgenomic replicon cells and HCV core expression in JFH-1-infected cells. Expression of ORP4S, an ORP4 variant that lacked the N-terminal pleckstrin-homology domain but contained the C-terminal Oxysterol-Binding domain also inhibited HCV replication, pointing to an important role of the Oxysterol-Binding domain in ORP4-mediated inhibition of HCV replication. ORP4 was found to associate with HCV NS5B and its expression led to inhibition of the NS5B activity. ORP4 expression had little effect on intracellular lipid synthesis and secretion, but it induced lipid droplet formation in the context of HCV replication. Taken together, these results demonstrate that ORP4 is a negative regulator of HCV replication, likely via interaction with HCV NS5B in the replication complex and regulation of intracellular lipid homeostasis. This work supports the important role of lipids and their metabolism in HCV replication and pathogenesis.
Joelle Bigay - One of the best experts on this subject based on the ideXlab platform.
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A lipid exchange market : vectorial cholesterol transport by the Protein OSBP
2020Co-Authors: Joelle Bigay, Bruno Mesmin, Bruno AntonnyAbstract:Cholesterol is synthesized in the endoplasmic reticulum (RE) and then transported to cellular compartments whose functions require high cholesterol levels. Here, we describe the mechanism by which cholesterol is transported from the RE to the trans-Golgi network (TGN) by the Protein OSBP (Oxysterol-Binding Protein). OSBP has two complementary activities. First, it tethers the RE to the TGN by forming a contact site where the two membranes are about twenty nanometers away. Then, it exchanges RE cholesterol for a TGN lipid, phosphatidylinositol 4-phosphate (PI4P). Eventually, PI4P is hydrolyzed at the RE, making the exchange cycle irreversible. Thus, OSBP is at the center of a lipid exchange market where a transported cholesterol “costs” a PI4P. Antiviral or anti-cancer molecules target OSBP, suggesting the importance of the OSBP cycle in different physiopathological contexts. The general principles of this cycle are shared by other lipid-transfer Proteins
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an intrinsically disordered region in osbp acts as an entropic barrier to control Protein dynamics and orientation at membrane contact sites
2019Co-Authors: Denisa Jamecna, Joelle Bigay, Bruno Mesmin, Joel Polidori, Manuela Dezi, Daniel Levy, Bruno AntonnyAbstract:Summary Lipid transfer Proteins (LTPs) acting at membrane contact sites (MCS) between the ER and other organelles contain domains involved in heterotypic (e.g., ER to Golgi) membrane tethering as well as domains involved in lipid transfer. Here, we show that a long ≈90 aa intrinsically unfolded sequence at the N terminus of Oxysterol-Binding Protein (OSBP) controls OSBP orientation and dynamics at MCS. This Gly-Pro-Ala-rich sequence, whose hydrodynamic radius is twice as that of folded domains, prevents the two PH domains of the OSBP dimer from homotypically tethering two Golgi-like membranes and considerably facilitates OSBP in-plane diffusion and recycling at MCS. Although quite distant in sequence, the N terminus of OSBP-related Protein-4 (ORP4) has similar effects. We propose that N-terminal sequences of low complexity in ORPs form an entropic barrier that restrains Protein orientation, limits Protein density, and facilitates Protein mobility in the narrow and crowded MCS environment.
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poliovirus evolution toward independence from the phosphatidylinositol 4 kinase iii β oxysterol binding Protein family i pathway
2019Co-Authors: Minetaro Arita, Joelle BigayAbstract:Phosphatidylinositol-4 kinase III β (PI4KB) and Oxysterol-Binding Protein (OSBP) family I provide a conserved host pathway required for enterovirus replication. Here, we analyze the role and essentiality of this pathway in enterovirus replication. Phosphatidylinositol 4-phosphate (PI4P) production and cholesterol accumulation in the replication organelle (RO) are severely suppressed in cells infected with a poliovirus (PV) mutant isolated from a PI4KB-knockout cell line (RD[ΔPI4KB]). Major determinants of the mutant for infectivity in RD(ΔPI4KB) cells map to the A5270U(3A-R54W) and U3881C(2B-F17L) mutations. The 3A mutation is required for PI4KB-independent development of RO. The 2B mutation rather sensitizes PV to PI4KB/OSBP inhibitors by itself but confers substantially complete resistance to the inhibitors with the 3A mutation. The 2B mutation also confers hypersensitivity to interferon alpha treatment on PV. These suggest that the PI4KB/OSBP pathway is not necessarily essential for enterovirus replica...
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the oxysterol binding Protein cycle burning off pi 4 p to transport cholesterol
2018Co-Authors: Bruno Antonny, Joelle Bigay, Bruno MesminAbstract:To maintain an asymmetric distribution of ions across membranes, Protein pumps displace ions against their concentration gradient by using chemical energy. Here, we describe a functionally analogous but topologically opposite process that applies to the lipid transfer Protein (LTP) Oxysterol-Binding Protein (OSBP). This multidomain Protein exchanges cholesterol for the phosphoinositide phosphatidylinositol 4-phosphate [PI(4)P] between two apposed membranes. Because of the subsequent hydrolysis of PI(4)P, this counterexchange is irreversible and contributes to the establishment of a cholesterol gradient along organelles of the secretory pathway. The facts that some natural anti-cancer molecules block OSBP and that many viruses hijack the OSBP cycle for the formation of intracellular replication organelles highlight the importance and potency of OSBP-mediated lipid exchange. The architecture of some LTPs is similar to that of OSBP, suggesting that the principles of the OSBP cycle-burning PI(4)P for the vectorial transfer of another lipid-might be general.
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uncovering oxysterol binding Protein osbp as a target of the anti enteroviral compound ttp 8307
2017Co-Authors: Lucian Albulescu, Joelle Bigay, Leen Delang, Cristina M Dorobantu, Hilde M Van Der Schaar, Bishyajit Kumar Biswas, Marion Weberboyvat, Youngsik Jung, Johan Neyts, Vesa M OlkkonenAbstract:The genus Enterovirus (e.g. poliovirus, coxsackievirus, rhinovirus) of the Picornaviridae family of positive-strand RNA viruses includes many important pathogens linked to a range of acute and chronic diseases for which no approved antiviral therapy is available. Targeting a step in the life cycle that is highly conserved provides an attractive strategy for developing broad-range inhibitors of enterovirus infection. A step that is currently explored as a target for the development of antivirals is the formation of replication organelles, which support replication of the viral genome. To build replication organelles, enteroviruses rewire cellular machinery and hijack lipid homeostasis pathways. For example, enteroviruses exploit the PI4KIIIβ-PI4P-OSBP pathway to direct cholesterol to replication organelles. Here, we uncover that TTP-8307, a known enterovirus replication inhibitor, acts through the PI4KIIIβ-PI4P-OSBP pathway by directly inhibiting OSBP activity. However, despite a shared mechanism of TTP-8307 with established OSBP inhibitors (itraconazole and OSW-1), we identify a number of notable differences between these compounds. The antiviral activity of TTP-8307 extends to other viruses that require OSBP, namely the picornavirus encephalomyocarditis virus and the flavivirus hepatitis C virus.
Vesa M Olkkonen - One of the best experts on this subject based on the ideXlab platform.
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uncovering oxysterol binding Protein osbp as a target of the anti enteroviral compound ttp 8307
2017Co-Authors: Lucian Albulescu, Joelle Bigay, Leen Delang, Cristina M Dorobantu, Hilde M Van Der Schaar, Bishyajit Kumar Biswas, Marion Weberboyvat, Youngsik Jung, Johan Neyts, Vesa M OlkkonenAbstract:The genus Enterovirus (e.g. poliovirus, coxsackievirus, rhinovirus) of the Picornaviridae family of positive-strand RNA viruses includes many important pathogens linked to a range of acute and chronic diseases for which no approved antiviral therapy is available. Targeting a step in the life cycle that is highly conserved provides an attractive strategy for developing broad-range inhibitors of enterovirus infection. A step that is currently explored as a target for the development of antivirals is the formation of replication organelles, which support replication of the viral genome. To build replication organelles, enteroviruses rewire cellular machinery and hijack lipid homeostasis pathways. For example, enteroviruses exploit the PI4KIIIβ-PI4P-OSBP pathway to direct cholesterol to replication organelles. Here, we uncover that TTP-8307, a known enterovirus replication inhibitor, acts through the PI4KIIIβ-PI4P-OSBP pathway by directly inhibiting OSBP activity. However, despite a shared mechanism of TTP-8307 with established OSBP inhibitors (itraconazole and OSW-1), we identify a number of notable differences between these compounds. The antiviral activity of TTP-8307 extends to other viruses that require OSBP, namely the picornavirus encephalomyocarditis virus and the flavivirus hepatitis C virus.
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1Lipid insights 2015:8(s1) OSBP-Related Protein Family in Lipid Transport Over Membrane Contact Sites Supplementary Issue: Cellular Anatomy of Lipid Traffic
2016Co-Authors: Vesa M OlkkonenAbstract:ABSTR ACT: Increasing evidence suggests that Oxysterol-Binding Protein-related Proteins (ORPs) localize at membrane contact sites, which are high-capacity platforms for inter-organelle exchange of small molecules and information. ORPs can simultaneously associate with the two apposed membranes and transfer lipids across the interbilayer gap. Oxysterol-Binding Protein moves cholesterol from the endoplasmic reticulum to trans-Golgi, driven by the retrograde transport of phosphatidylinositol-4-phosphate (PI4P). Analogously, yeast Osh6p mediates the transport of phosphatidylserine from the endoplasmic reticulum to the plasma membrane in exchange for PI4P, and ORP5 and-8 are suggested to execute similar functions in mam-malian cells. ORPs may share the capacity to bind PI4P within their ligand-binding domain, prompting the hypothesis that bidirectional transport of a phosphoinositide and another lipid may be a common theme among the Protein family. This model, however, needs more experimental support and does not exclude a function of ORPs in lipid signaling
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osbp related Protein family in lipid transport over membrane contact sites supplementary issue cellular anatomy of lipid traffic
2015Co-Authors: Vesa M OlkkonenAbstract:ABSTR ACT: Increasing evidence suggests that Oxysterol-Binding Protein-related Proteins (ORPs) localize at membrane contact sites, which are high-capacity platforms for inter-organelle exchange of small molecules and information. ORPs can simultaneously associate with the two apposed membranes and transfer lipids across the interbilayer gap. Oxysterol-Binding Protein moves cholesterol from the endoplasmic reticulum to trans-Golgi, driven by the retrograde transport of phosphatidylinositol-4-phosphate (PI4P). Analogously, yeast Osh6p mediates the transport of phosphatidylserine from the endoplasmic reticulum to the plasma membrane in exchange for PI4P, and ORP5 and -8 are suggested to execute similar functions in mammalian cells. ORPs may share the capacity to bind PI4P within their ligand-binding domain, prompting the hypothesis that bidirectional transport of a phosphoinositide and another lipid may be a common theme among the Protein family. This model, however, needs more experimental support and does not exclude a function of ORPs in lipid signaling.
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osbp related Protein 2 is a sterol receptor on lipid droplets that regulates the metabolism of neutral lipids
2009Co-Authors: Riikka Hynynen, Monika Suchanek, Christoph Thiele, Johanna Spandl, Nils Back, Vesa M OlkkonenAbstract:Oxysterol binding Protein-related Protein 2 (ORP2) is a member of the oxysterol binding Protein family, previously shown to bind 25-hydroxycholesterol and implicated in cellular cholesterol metabolism. We show here that ORP2 also binds 22(R)-hydroxycholesterol [22(R)OHC], 7-ketocholesterol, and cholesterol, with 22(R)OHC being the highest affinity ligand of ORP2 (K(d) 1.4 x 10(-8) M). We report the localization of ORP2 on cytoplasmic lipid droplets (LDs) and its function in neutral lipid metabolism using the human A431 cell line as a model. The ORP2 LD association depends on sterol binding: Treatment with 5 microM 22(R)OHC inhibits the LD association, while a mutant defective in sterol binding is constitutively LD bound. Silencing of ORP2 using RNA interference slows down cellular triglyceride hydrolysis. Furthermore, ORP2 silencing increases the amount of [(14)C]cholesteryl esters but only under conditions in which lipogenesis and LD formation are enhanced by treatment with oleic acid. The results identify ORP2 as a sterol receptor present on LD and provide evidence for its role in the regulation of neutral lipid metabolism, possibly as a factor that integrates the cellular metabolism of triglycerides with that of cholesterol.
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osbp related Protein 8 orp8 suppresses abca1 expression and cholesterol efflux from macrophages
2008Co-Authors: Mikko I Mayranpaa, Jenny Wong, Christian Ehnholm, Andrew J Brown, Markku Lehto, Matti Jauhiainen, Julia Perttila, Petri T Kovanen, Vesa M OlkkonenAbstract:Abstract ORP8 is a previously unexplored member of the family of Oxysterol-Binding Protein-related Proteins (ORP). We now report the expression pattern, the subcellular distribution, and data on the ligand binding properties and the physiological function of ORP8. ORP8 is localized in the endoplasmic reticulum (ER) via its C-terminal transmembrane span and binds 25-hydroxycholesterol, identifying it as a new ER Oxysterol-Binding Protein. ORP8 is expressed at highest levels in macrophages, liver, spleen, kidney, and brain. Immunohistochemical analysis revealed ORP8 in the shoulder regions of human coronary atherosclerotic lesions, where it is present in CD68(+) macrophages. In advanced lesions the ORP8 mRNA was up-regulated 2.7-fold as compared with healthy coronary artery wall. Silencing of ORP8 by RNA interference in THP-1 macrophages increased the expression of ATP binding cassette transporter A1 (ABCA1) and concomitantly cholesterol efflux to lipid-free apolipoProtein A-I but had no significant effect on ABCG1 expression or cholesterol efflux to spherical high density lipoProtein HDL2. Experiments employing an ABCA1 promoter-luciferase reporter confirmed that ORP8 silencing enhances ABCA1 transcription. The silencing effect was partially attenuated by mutation of the DR4 element in the ABCA1 promoter and synergized with that of the liver X receptor agonist T0901317. Furthermore, inactivation of the E-box in the promoter synergized with ORP8 silencing, suggesting that the suppressive effect of ORP8 involves both the liver X receptor and the E-box functions. Our data identify ORP8 as a negative regulator of ABCA1 expression and macrophage cholesterol efflux. ORP8 may, thus, modulate the development of atherosclerosis.
Christopher R. Mcmaster - One of the best experts on this subject based on the ideXlab platform.
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a detour for yeast oxysterol binding Proteins
2012Co-Authors: Christopher R. Mcmaster, Christopher T Beh, Keith G Kozminski, Anant K MenonAbstract:Oxysterol binding Protein-related Proteins, including the yeast Proteins encoded by the OSH gene family (OSH1–OSH7), are implicated in the non-vesicular transfer of sterols between intracellular membranes and the plasma membrane. In light of recent studies, we revisited the proposal that Osh Proteins are sterol transfer Proteins and present new models consistent with known Osh Protein functions. These models focus on the role of Osh Proteins as sterol-dependent regulators of phosphoinositide and sphingolipid pathways. In contrast to their posited role as non-vesicular sterol transfer Proteins, we propose that Osh Proteins coordinate lipid signaling and membrane reorganization with the assembly of tethering complexes to promote molecular exchanges at membrane contact sites.
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lipid binding requirements for oxysterol binding Protein kes1 inhibition of autophagy and endosome trans golgi trafficking pathways
2010Co-Authors: Marissa A Leblanc, Christopher R. McmasterAbstract:The Saccharomyces cerevisiae Protein Kes1/Osh4 is a member of the enigmatic family of Oxysterol-Binding Proteins found throughout Eukarya united by a β-barrel structure that binds sterols and oxysterols. In this study, we determined that phosphoinositides are the major determinant in membranes that facilitate Kes1 association both in vitro and in cells. Increased expression of Kes1 in yeast cells decreased the levels of both phosphatidylinositol 4-phosphate (PI4P) and phosphatidylinositol 3-phosphate (PI3P). Phosphoinositide and sterol bindings by Kes1 were necessary for Kes1 to decrease the level of PI4P but not PI3P. Kes1 inhibited vesicular trafficking between the trans-Golgi and plasma membrane as evidenced by accumulation of the vacuolar soluble NSF attachment Protein receptors Snc1 in the cytoplasmic vesicles. Sterol and phosphoinositide binding by Kes1 both contributed to its regulation of Snc1 trafficking. This study also describes a previously unknown role for Kes1 in the regulation of the autophagy/cytoplasm to the vacuole trafficking pathway. The Kes1-mediated regulation of the autophagy/cytoplasm to the vacuole trafficking pathway was prevented by increasing expression of the PI3K Vps34, suggesting that it is the Kes1-mediated decrease in PI3P level that contributes to this regulation.
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emerging roles of the oxysterol binding Protein family in metabolism transport and signaling
2008Co-Authors: Gregory D Fairn, Christopher R. McmasterAbstract:OSBP (Oxysterol-Binding Protein) and ORPs (OSBP-related Proteins) constitute an enigmatic eukaryotic Protein family that is united by a signature domain that binds oxysterols, sterols, and possibly other hydrophobic ligands. The human genome contains 12 OSBP/ORP family members genes, while that of the budding yeast Saccharomyces cerevisiae encodes seven OSBP homologues (Osh). Of these, Osh4 (also referred to as Kes1) has been the most widely studied to date. Recently, three-dimensional crystal structures of Osh4 with and without sterols bound within the core of the Protein were determined. The core consists of 19 anti-parallel β-sheets that form a near-complete β-barrel. Recent work has suggested that Osh Proteins facilitate the non-vesicular transport of sterols in vivo and in vitro, while other evidence supports a role for Osh Proteins in the regulation of vesicular transport and lipid metabolism.This article will review recent advances in the study of ORP/Osh Proteins and will discuss future research issues regarding the ORP/Osh family.
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The oxysterol binding Protein Kes1p regulates Golgi apparatus phosphatidylinositol-4-phosphate function
2007Co-Authors: Gregory D. Fairn, Amy J. Curwin, Christopher J. Stefan, Christopher R. McmasterAbstract:The Saccharomyces cerevisiae phosphatidylcholine/phosphatidylinositol transfer Protein Sec14p is required for Golgi apparatus-derived vesicular transport through coordinate regulation of phospholipid metabolism. Sec14p is normally essential. The essential requirement for SEC14 can be bypassed by inactivation of (i) the CDP–choline pathway for phosphatidylcholine synthesis or (ii) KES1, which encodes an oxysterol binding Protein. A unique screen was used to determine genome-wide genetic interactions for the essential gene SEC14 and to assess whether the two modes of “sec14 bypass” were similar or distinct. The results indicate that inactivation of the CDP–choline pathway allows cells with inactivated SEC14 to live through a mechanism distinct from that of inactivation of KES1. We go on to demonstrate an important biological function of Kes1p. Kes1p regulates Golgi apparatus-derived vesicular transport by inhibiting the function of Pik1p-generated Golgi apparatus phosphatidylinositol-4-phosphate (PI-4P). Kes1p affects both the availability and level of Golgi apparatus PI-4P. A set of potential PI-4P-responsive Proteins that include the Rab GTPase Ypt31p and its GTP exchange factor are described.
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identification and assessment of the role of a nominal phospholipid binding region of orp1s oxysterol binding Protein related Protein 1 short in the regulation of vesicular transport
2005Co-Authors: Gregory D. Fairn, Christopher R. McmasterAbstract:The ORPs (Oxysterol-Binding-Protein-related Proteins) constitute an enigmatic family of intracellular lipid receptors that are related through a shared lipid binding domain. Emerging evidence suggests that ORPs relate lipid metabolism to membrane transport. Current data imply that the yeast ORP Kes1p is a negative regulator of Golgi-derived vesicular transport mediated by the essential phosphatidylinositol/phosphatidylcholine transfer Protein Sec14p. Inactivation of Kes1p function allows restoration of growth and vesicular transport in cells lacking Sec14p function, and Kes1p function in this regard can be complemented by human ORP1S (ORP1 short). Recent studies have determined that Kes1p and ORP1S both bind phospholipids as ligands. To explore the function of distinct linear segments of ORP1S in phospholipid binding and vesicular transport regulation, we generated a series of 15 open reading frames coding for diagnostic regions within ORP1S. Purified versions of these ORP1S deletion Proteins were characterized in vitro, and allowed the identification of a nominal phospholipid binding region. The in vitro analysis was interpreted in the context of in vivo growth and vesicle transport assays for members of the ORP1S deletion set. The results determined that the phospholipid binding domain per se was insufficient for inhibition of vesicular transport by ORP1S, and that transport of carboxypeptidase Y and invertase from the Golgi may be regulated differentially by specific regions of ORP1S/Kes1p.
Minetaro Arita - One of the best experts on this subject based on the ideXlab platform.
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poliovirus evolution toward independence from the phosphatidylinositol 4 kinase iii β oxysterol binding Protein family i pathway
2019Co-Authors: Minetaro Arita, Joelle BigayAbstract:Phosphatidylinositol-4 kinase III β (PI4KB) and Oxysterol-Binding Protein (OSBP) family I provide a conserved host pathway required for enterovirus replication. Here, we analyze the role and essentiality of this pathway in enterovirus replication. Phosphatidylinositol 4-phosphate (PI4P) production and cholesterol accumulation in the replication organelle (RO) are severely suppressed in cells infected with a poliovirus (PV) mutant isolated from a PI4KB-knockout cell line (RD[ΔPI4KB]). Major determinants of the mutant for infectivity in RD(ΔPI4KB) cells map to the A5270U(3A-R54W) and U3881C(2B-F17L) mutations. The 3A mutation is required for PI4KB-independent development of RO. The 2B mutation rather sensitizes PV to PI4KB/OSBP inhibitors by itself but confers substantially complete resistance to the inhibitors with the 3A mutation. The 2B mutation also confers hypersensitivity to interferon alpha treatment on PV. These suggest that the PI4KB/OSBP pathway is not necessarily essential for enterovirus replica...
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Poliovirus Evolution toward Independence from the Phosphatidylinositol‑4 Kinase III β/Oxysterol-Binding Protein Family I Pathway
2019Co-Authors: Minetaro Arita, Joëlle BigayAbstract:Phosphatidylinositol-4 kinase III β (PI4KB) and Oxysterol-Binding Protein (OSBP) family I provide a conserved host pathway required for enterovirus replication. Here, we analyze the role and essentiality of this pathway in enterovirus replication. Phosphatidylinositol 4-phosphate (PI4P) production and cholesterol accumulation in the replication organelle (RO) are severely suppressed in cells infected with a poliovirus (PV) mutant isolated from a PI4KB-knockout cell line (RD[ΔPI4KB]). Major determinants of the mutant for infectivity in RD(ΔPI4KB) cells map to the A5270U(3A-R54W) and U3881C(2B-F17L) mutations. The 3A mutation is required for PI4KB-independent development of RO. The 2B mutation rather sensitizes PV to PI4KB/OSBP inhibitors by itself but confers substantially complete resistance to the inhibitors with the 3A mutation. The 2B mutation also confers hypersensitivity to interferon alpha treatment on PV. These suggest that the PI4KB/OSBP pathway is not necessarily essential for enterovirus replication in vitro. This work supports a two-step resistance model of enterovirus to PI4KB/OSBP inhibitors involving unique recessive epistasis of 3A and 2B and offers insights into a potential evolutionary pathway of enterovirus toward independence from the PI4KB/OSBP pathway
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itraconazole inhibits enterovirus replication by targeting the oxysterol binding Protein
2015Co-Authors: Minetaro Arita, Jeroen R P M Strating, Lonneke Van Der Linden, Lucian Albulescu, Joelle Bigay, Leen Delang, Pieter LeyssenAbstract:Itraconazole (ITZ) is a well-known antifungal agent that also has anticancer activity. In this study, we identify ITZ as a broad-spectrum inhibitor of enteroviruses (e.g., poliovirus, coxsackievirus, enterovirus-71, rhinovirus). We demonstrate that ITZ inhibits viral RNA replication by targeting Oxysterol-Binding Protein (OSBP) and OSBP-related Protein 4 (ORP4). Consistently, OSW-1, a specific OSBP/ORP4 antagonist, also inhibits enterovirus replication. Knockdown of OSBP inhibits virus replication, whereas overexpression of OSBP or ORP4 counteracts the antiviral effects of ITZ and OSW-1. ITZ binds OSBP and inhibits its function, i.e., shuttling of cholesterol and phosphatidylinositol-4-phosphate between membranes, thereby likely perturbing the virus-induced membrane alterations essential for viral replication organelle formation. ITZ also inhibits hepatitis C virus replication, which also relies on OSBP. Together, these data implicate OSBP/ORP4 as molecular targets of ITZ and point to an essential role of OSBP/ORP4-mediated lipid exchange in virus replication that can be targeted by antiviral drugs.
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phosphatidylinositol 4 kinase iii beta and oxysterol binding Protein accumulate unesterified cholesterol on poliovirus induced membrane structure
2014Co-Authors: Minetaro AritaAbstract:Studies on anti-picornavirus compounds have revealed an essential role of a novel cellular pathway via host phosphatidylinositol-4 kinase III beta (PI4KB) and Oxysterol-Binding Protein (OSBP) family I in poliovirus (PV) replication. However, the molecular role for this pathway in PV replication has yet to be determined. Here, viral and host Proteins modulating production of phosphatidylinositol 4-phosphate (PI4P) and accumulation of unesterified cholesterol (UC) in cells were analyzed and the role of the PI4KB/OSBP pathway in PV replication characterized. Virus Protein 2BC was identified as a novel interactant of PI4KB. PI4KB and VCP/p97 bind to a partially overlapped region of 2BC with different sensitivity to a 2C inhibitor. Production of PI4P and accumulation of UC were enhanced by virus Protein 2BC, but suppressed by virus Proteins 3A and 3AB. In PV-infected cells, a PI4KB inhibitor suppressed production of PI4P, and both a PI4KB inhibitor and an OSBP ligand suppressed accumulation of UC on virus-induced membrane structure. Inhibition of PI4KB activity caused dissociation of OSBP from virus-induced membrane structure in PV-infected cells. Synthesis of viral nascent RNA in PV-infected cells was not affected in the presence of PI4KB inhibitor and OSBP ligand; however, transient pre-treatment of PV-infected cells with these inhibitors suppressed viral RNA synthesis. These results suggest that virus Proteins modulate PI4KB activity and provide PI4P for recruitment of OSBP to accumulate UC on virus-induced membrane structure for formation of a virus replication complex.
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oxysterol binding Protein family i is the target of minor enviroxime like compounds
2013Co-Authors: Minetaro Arita, Hirotatsu Kojima, Tetsuo Nagano, Takayoshi Okabe, Takaji Wakita, Hiroyuki ShimizuAbstract:Enviroxime is an antipicornavirus compound that targets host phosphatidylinositol 4-kinase III beta (PI4KB) activity for its antipicornavirus activity. To date, several antipoliovirus (PV) compounds similar to enviroxime that are associated with a common resistance mutation in viral Protein 3A (a G5318A [3A-Ala70Thr] mutation in PV) have been identified. Most of these compounds have a direct inhibitory effect on PI4KB activity, as well as enviroxime (designated major enviroxime-like compounds). However, one of the compounds, AN-12-H5, showed no inhibitory effect on PI4KB and was considered to belong to another group of enviroxime-like compounds (designated minor enviroxime-like compounds). In the present study, we performed a small interfering RNA (siRNA) sensitization assay targeting PI4KB-related genes and identified Oxysterol-Binding Protein (OSBP) as a target of minor enviroxime-like compounds. Knockdown of OSBP and OSBP2 increased the anti-PV activities of AN-12-H5 and a newly identified minor enviroxime-like compound, T-00127-HEV2, and also to T-00127-HEV1 to a minor extent, in the cells. A ligand of OSBP, 25-hydroxycholesterol (25-HC), acted as a minor enviroxime-like compound. Minor enviroxime-like compounds induced relocalization of OSBP to the Golgi apparatus in cells. Treatment of the cells with major or minor enviroxime-like compounds suppressed the expression of genes (HMGCS1 and SQLE) in the SREBP/SCAP regulatory pathway and diminished endogenous phosphatidylinositol 4-phosphate (PI4P) at the Golgi apparatus. Our results suggested that minor enviroxime-like compounds are phenotypically identical to 25-HC and that major and minor enviroxime-like compounds suppress the production and/or accumulation of PI4P in PV-infected cells by targeting PI4KB and OSBP family I activities, respectively.
