Browsing by Subject "OXYSTEROL-BINDING-PROTEIN"

Sort by: Order: Results:

Now showing items 1-7 of 7
  • Motazacker, Mahdi M.; Pirhonen, Juho; van Capelleveen, Julian C.; Weber-Boyvat, Marion; Kuivenhoven, Jan Albert; Shah, Saundarya; Hovingh, G. Kees; Metso, Jari; Li, Shiqian; Ikonen, Elina; Jauhiainen, Matti; Dallinga-Thie, Geesje M.; Olkkonen, Vesa M. (2016)
    Background and aims: Among subjects with high-density-lipoprotein cholesterol (HDL-C) below the 1st percentile in the general population, we identified a heterozygous variant OSBPL1A p.C39X encoding a short truncated protein fragment that co-segregated with low plasma HDL-C. Methods: We investigated the composition and function of HDL from the carriers and non-carriers and studied the properties of the mutant protein in cultured hepatocytes. Results: Plasma HDL-C and apolipoprotein (apo) A-I were lower in carriers versus non-carriers, whereas the other analyzed plasma components or HDL parameters did not differ. Sera of the carriers displayed a reduced capacity to act as cholesterol efflux acceptors (p <0.01), whereas the cholesterol acceptor capacity of their isolated HDL was normal. Fibroblasts from a p.C39X carrier showed reduced cholesterol efflux to lipid-free apoA-I but not to mature HDL particles, suggesting a specific defect in ABCA1-mediated efflux pathway. In hepatic cells, GFP-OSBPL1A partially co-localized in endosomes containing fluorescent apoA-I, suggesting that OSBPL1A may regulate the intracellular handling of apoA-I. The GFP-OSBPL1A-39X mutant protein remained in the cytosol and failed to interact with Rab7, which normally recruits OSBPL1A to late endosomes/lysosomes, suggesting that this mutation represents a loss-of-function. Conclusions: The present work represents the first characterization of a human OSBPL1A mutation. Our observations provide evidence that a familial loss-of-function mutation in OSBPL1A affects the first step of the reverse cholesterol transport process and associates with a low HDL-C phenotype. This suggests that rare mutations in OSBPL genes may contribute to dyslipidemias. (C) 2016 Elsevier Ireland Ltd. All rights reserved.
  • Kentala, Henriikka; Koponen, Annika; Kivelä, Annukka M.; Andrews, Robert; Li, ChunHei; Zhou, You; Olkkonen, Vesa M. (2018)
    ORP2 is implicated in cholesterol transport, triglyceride metabolism, and adrenocortical steroid hormone production. We addressed ORP2 function in hepatocytes by generating ORP2-knockout (KO) HuH7 cells by CRISPR-Cas9 gene editing, followed by analyses of transcriptome, F-actin morphology, migration, adhesion, and proliferation. RNA sequencing of ORP2-KO cells revealed >2-fold changes in 579 mRNAs. The Ingenuity Pathway Analysis (IPA) uncovered alterations in the following functional categories: cellular movement, cell-cell signaling and interaction, cellular development, cellular function and maintenance, cellular growth and proliferation, and cell morphology. Many pathways in these categories involved actin cytoskeleton, cell migration, adhesion, or proliferation. Analysis of the ORP2 interactome uncovered 109 putative new partners. Their IPA analysis revealed Ras homolog A (RhoA) signaling as the most significant pathway. Interactions of ORP2 with SEPT9, MLC12, and ARHGAP12 were validated by independent assays. ORP2-KO resulted in abnormal F-actin morphology characterized by impaired capacity to form lamellipodia, migration defect, and impaired adhesion and proliferation. Rescue of the migration phenotype and generation of typical cell surface morphology required an intact ORP2 phosphoinositide binding site, suggesting that ORP2 function involves phosphoinositide binding and transport. The results point at a novel function of ORP2 as a lipid-sensing regulator of the actin cytoskeleton, with impacts on hepatocellular migration, adhesion, and proliferation.-Kentala, H., Koponen, A., Kivela, A. M., Andrews, R., Li, C., Zhou, Y., Olkkonen, V. M. Analysis of ORP2-knockout hepatocytes uncovers a novel function in actin cytoskeletal regulation.
  • Weber-Boyvat, Marion; Trimbuch, Thorsten; Shah, Saundarya; Jäntti, Jussi; Olkkonen, Vesa M.; Rosenmund, Christian (2021)
    OSBP-homologous proteins (ORPs, Oshp) are lipid binding/transfer proteins. Several ORP/Oshp localize to membrane contacts between the endoplasmic reticulum (ER) and the plasma membrane, where they mediate lipid transfer or regulate lipid-modifying enzymes. A common way in which they target contacts is by binding to the ER proteins, VAP/Scs2p, while the second membrane is targeted by other interactions with lipids or proteins. We have studied the cross-talk of secretory SNARE proteins and their regulators with ORP/Oshp and VAPA/Scs2p at ER-plasma membrane contact sites in yeast and murine primary neurons. We show that Oshp-Scs2p interactions depend on intact secretory SNARE proteins, especially Sec9p. SNAP-25/Sec9p directly interact with ORP/Osh proteins and their disruption destabilized the ORP/Osh proteins, associated with dysfunction of VAPA/Scs2p. DeletingOSH1-3in yeast or knocking down ORP2 in primary neurons reduced the oligomerization of VAPA/Scs2p and affected their multiple interactions with SNAREs. These observations reveal a novel cross-talk between the machineries of ER-plasma membrane contact sites and those driving exocytosis.
  • Koponen, Annika; Arora, Amita; Takahashi, Kohta; Kentala, Henriikka; Kivelä, Annukka M.; Jääskeläinen, Eeva; Peränen, Johan; Somerharju, Pentti; Ikonen, Elina; Viitala, Tapani; Olkkonen, Vesa M. (2019)
    ORP2 is a sterol-binding protein with documented functions in lipid and glucose metabolism, Akt signaling, steroidogenesis, cell adhesion, migration and proliferation. Here we investigate the interactions of ORP2 with phosphoinositides (PIPs) by surface plasmon resonance (SPR), its affinity for cholesterol with a pull-down assay, and its capacity to transfer sterol in vitro. Moreover, we determine the effects of wild-type (wt) ORP2 and a mutant with attenuated PIP binding, ORP2(mHHK), on the subcellular distribution of cholesterol, and analyze the interaction of ORP2 with the related cholesterol transporter ORP1L. ORP2 showed specific affinity for PI(4,5)P-2, PI(3,4,5)P-3 and PI(4)P, with suggestive K-d values in the mu M range. Also binding of cholesterol by ORP2 was detectable, but a K-d could not be determined. Wt ORP2 was in HeLa cells mainly detected in the cytosol, ER, late endosomes, and occasionally on lipid droplets (LDs), while ORP2(mHHK) displayed an enhanced LD localization. Overexpression of wt ORP2 shifted the D4H cholesterol probe away from endosomes, while ORP2(mHHK) caused endosomal accumulation of the probe. Although ORP2 failed to transfer dehydroergosterol in an in vitro assay where OSBP is active, its knock-down resulted in the accumulation of cholesterol in late endocytic compartments, as detected by both D4H and filipin probes. Interestingly, ORP2 was shown to interact and partially co-localize on late endosomes with ORP1L, a cholesterol transporter/sensor at ER-late endosome junctions. Our data demonstrates that ORP2 binds several phosphoinositides, both PI(4)P and multiply phosphorylated species. ORP2 regulates the subcellular distribution of cholesterol dependent on its PIP-binding capacity. The interaction of ORP2 with ORP1L suggests a concerted action of the two ORPs. (C) 2018 Elsevier B.V. and Societe Francaise de Biochimie et Biologie Moleculaire (SFBBM). All rights reserved.
  • Koponen, Annika; Pan, Guoping; Kivelä, Annukka M.; Ralko, Arthur; Taskinen, Juuso H.; Arora, Amita; Kosonen, Riikka; Kari, Otto K.; Ndika, Joseph; Ikonen, Elina; Cho, Wonhwa; Yan, Daoguang; Olkkonen, Vesa M. (2020)
    Oxysterol-binding protein-related protein 2 (ORP2), a cholesterol-PI(4,5)P(2)countercurrent transporter, was recently identified as a novel regulator of plasma membrane (PM) cholesterol and PI(4,5)P(2)content in HeLa cells. Here, we investigate the role of ORP2 in endothelial cell (EC) cholesterol and PI(4,5)P(2)distribution, angiogenic signaling, and angiogenesis. We show that ORP2 knock-down modifies the distribution of cholesterol accessible to a D4H probe, between late endosomes and the PM. Depletion of ORP2 from ECs inhibits their angiogenic tube formation capacity, alters the gene expression of angiogenic signaling pathways such as VEGFR2, Akt, mTOR, eNOS, and Notch, and reduces EC migration, proliferation, and cell viability. We show that ORP2 regulates the integrity of VEGFR2 at the PM in a cholesterol-dependent manner, the depletion of ORP2 resulting in proteolytic cleavage by matrix metalloproteinases, and reduced activity of VEGFR2 and its downstream signaling. We demonstrate that ORP2 depletion increases the PM PI(4,5)P(2)coincident with altered F-actin morphology, and reduces both VEGFR2 and cholesterol in buoyant raft membranes. Moreover, ORP2 knock-down suppresses the expression of the lipid raft-associated proteins VE-cadherin and caveolin-1. Analysis of the retinal microvasculature in ORP2 knock-out mice generated during this study demonstrates the subtle alterations of morphology characterized by reduced vessel length and increased density of tip cells and perpendicular sprouts. Gene expression changes in the retina suggest disturbance of sterol homeostasis, downregulation of VE-cadherin, and a putative disturbance of Notch signaling. Our data identifies ORP2 as a novel regulator of EC cholesterol and PI(4,5)P(2)homeostasis and cholesterol-dependent angiogenic signaling.
  • Olkkonen, Vesa M.; Koponen, Annika; Arora, Amita (2019)
    Oxysterol-binding protein (OSBP)-related proteins (ORPs) constitute a family of intracellular lipid-binding/transport proteins (LTPs) in eukaryotes. They typically have a modular structure comprising a lipid-binding domain and membrane targeting determinants, being thus suited for function at membrane contact sites. Among the mammalian ORPs, ORP2/OSBPL2 is the only member that only exists as a 'short' variant lacking a membrane-targeting pleckstrin homology domain. ORP2 is expressed ubiquitously and has been assigned a multitude of functions. Its OSBP-related domain binds cholesterol, oxysterols, and phosphoinositides, and its overexpression enhances cellular cholesterol efflux. Consistently, the latest observations suggest a function of ORP2 in cholesterol transport to the plasma membrane (PM) in exchange for phosphatidylinositol 4,5-bisphosphate (PI4,5P(2)), with significant impacts on the concentrations of PM cholesterol and PI4,5P(2). On the other hand, ORP2 localizes at the surface of cytoplasmic lipid droplets (LDs) and at endoplasmic-reticulum-LD contact sites, and its depletion modifies cellular triglyceride (TG) metabolism. Study in an adrenocortical cell line further suggested a function of ORP2 in the synthesis of steroid hormones. Our recent knock-out of ORP2 in human hepatoma cells revealed its function in hepatocellular PI3K/Akt signaling, glucose and triglyceride metabolism, as well as in actin cytoskeletal regulation, cell adhesion, migration and proliferation. ORP2 was shown to interact physically with F-actin regulators such as DIAPH1, ARHGAP12, SEPT9 and MLC12, as well as with IQGAP1 and the Cdc37-Hsp90 chaperone complex controlling the activity of Akt. Interestingly, mutations in OSBPL2 encoding ORP2 are associated with autosomal dominant non-syndromic hearing loss, and the protein was found to localize in cochlear hair cell stereocilia. The functions assigned to ORP2 suggest that this protein, in concert with other LTPs, controls the subcellular distribution of cholesterol in various cell types and steroid hormone synthesis in adrenocortical cells. However, it also impacts cellular TG and carbohydrate metabolism and F-actin-dependent functions, revealing a bewildering spectrum of activities.