Oxysterol-binding Protein Research Articles

Structural Basis of Lipid Exchange in the Oxysterol-Binding Protein Homologue (OSH) Family

Oxysterol-binding proteins, long known to be involved in the non-vesicular transport of sterol, have recently been shown to also transfer glycerophospholipids, such as phosphatidyl-inositol-4-phosphate (PI4P) and phosphatidyl-serine (PS) between membranes.Using a combination of atomistic molecular dynamics simulations and in vitro lipid transport assays, we characterized the structural determinants that regulate the extraction and release of different lipids by the various Osh proteins.Our results define how distinct lipid-binding modes govern the ability of Osh proteins to transport or exchange lipids between organelles.

Partial genetic suppression of a loss-of-function mutant of the neuronal ceroid lipofuscinosis-associated protease TPP1 in Dictyostelium discoideum.

Neuronal ceroid lipofuscinosis (NCL) is the most common childhood-onset neurodegenerative disease. NCL is inevitably fatal, and there is currently no treatment available. Children with NCL show a progressive decline in movement, vision and mental abilities, and an accumulation of autofluorescent deposits in neurons and other cell types. Late-infantile NCL is caused by mutations in the lysosomal protease tripeptidyl peptidase 1 (TPP1). TPP1 cleaves tripeptides from the N-terminus of proteins in vitro, but little is known about the physiological function of TPP1. TPP1 shows wide conservation in vertebrates but it is not found in Drosophila, Caenorhabditis elegans or Saccharomyces cerevisiae. Here, we characterize ddTpp1, a TPP1 ortholog present in the social amoeba Dictyostelium discoideum. Lysates from cells lacking ddTpp1 show a reduced but not abolished ability to cleave a TPP1 substrate, suggesting that other Dictyostelium enzymes can perform this cleavage. ddTpp1 and human TPP1 localize to the lysosome in Dictyostelium, indicating conserved function and trafficking. Cells that lack ddTpp1 show precocious multicellular development and a reduced ability to form spores during development. When cultured in autophagy-stimulating conditions, cells lacking ddTpp1 rapidly decrease in size and are less viable than wild-type cells, suggesting that one function of ddTpp1 could be to limit autophagy. Cells that lack ddTpp1 exhibit strongly impaired development in the presence of the lysosome-perturbing drug chloroquine, and this phenotype can be suppressed through a secondary mutation in the gene that we name suppressor of tpp1− A (stpA), which encodes a protein with some similarity to mammalian oxysterol-binding proteins (OSBPs). Taken together, these results suggest that targeting specific proteins could be a viable way to suppress the effects of loss of TPP1 function.

Adipocyte-derived factors impair insulin signaling in differentiated human vascular smooth muscle cells via the upregulation of miR-143

Cardiovascular complications are common in patients with type 2 diabetes. Adipokines have been implicated in the induction of proliferative and pro-atherogenic alterations in human vascular smooth muscle cells (hVSMC). Other reports demonstrated the importance of the miRNA cluster miR-143/145 in the regulation of VSMC homeostasis and insulin sensitivity. Here we investigated whether the detrimental effects of adipokines on hVSMC function could be ascribed to alterations in miR-143/145 expression. The exposure of hVSMC to conditioned media (CM) from primary human subcutaneous adipocytes increased the expression of smooth muscle α-actin (SMA), and the miR-143/145 cluster, but markedly impaired the insulin-mediated phosphorylation of Akt and its substrate endothelial nitric oxide synthase (eNOS). Furthermore, CM promoted the phosphorylation of SMAD2 and p38, which have both been linked to miR-143/145 induction. Accordingly, the induction of miR-143/145 as well as the inhibition of insulin-mediated Akt- and eNOS-phosphorylation was prevented when hVSMC were treated with pharmacological inhibitors for Alk-4/5/7 and p38 before the addition of CM. The transfection of hVSMC with precursor miR-143, but not with precursor miR-145, resulted in impaired insulin-mediated phosphorylation of Akt and eNOS. This inhibition of insulin signaling by CM and miR-143 is associated with a reduction in the expression of the oxysterol-binding protein-related protein 8 (ORP8). Finally, the knock-down of ORP8 resulted in impaired insulin-mediated phosphorylation of Akt in hVSMC. Thus, the detrimental effects of adipocyte-derived conditioned media on insulin action in primary hVSMC can be ascribed to the Alk- and p38-dependent induction of miR-143 and subsequent downregulation of ORP8.

A vertebrate model for the study of lipid binding/transfer protein function: Conservation of OSBP-related proteins between zebrafish and human

Oxysterol-binding protein (OSBP) and OSBP-related (ORP) or OSBP-like (OSBPL) proteins constitute a family of lipid-binding/transfer proteins (LTPs) present in eukaryotes from yeast to man. The mechanisms of ORP function have remained incompletely understood. However, several ORPs are present at membrane contact sites and act as either lipid transporters or sensors that control lipid metabolism, cell signaling, and vesicle transport. Zebrafish, Danio rerio, has gained increasing popularity as a model organism in developmental biology, human disease, toxicology, and drug discovery. However, LTPs in the fish are thus far unexplored. In this article we report a series of bioinformatic analyses showing that the OSBPL gene family is highly conserved between the fish and human. The OSBPL subfamily structure is markedly similar between the two organisms, and all 12 human genes have orthologs, designated osbpl and located on 11 chromosomes in D. rerio. Interestingly, osbpl2 and osbpl3 are present as two closely related homologs (a and b), due to gene duplication events in the teleost lineage. Moreover, the domain structures of the distinct ORP proteins are almost identical between zebrafish and man, and molecular modeling in the present study suggests that ORD liganding by phosphatidylinositol-4-phosphate (PI4P) is a feature conserved between yeast Osh3p, human ORP3, and zebrafish Osbpl3. The present analysis identifies D. rerio as an attractive model to study the functions of ORPs in vertebrate development and metabolism.

The amyotrophic lateral sclerosis 8 protein, VAP, is required for ER protein quality control

A familial form of Amyotrophic lateral sclerosis (ALS8) is caused by a point mutation (P56S) in the vesicle-associated membrane protein associated protein B (VapB). Human VapB and Drosophila Vap-33-1 (Vap) are homologous type II transmembrane proteins that are localized to the ER. However, the precise consequences of the defects associated with the P56S mutation in the endoplasmic reticulum (ER) and its role in the pathology of ALS are not well understood. Here we show that Vap is required for ER protein quality control (ERQC). Loss of Vap in flies shows various ERQC associated defects, including protein accumulation, ER expansion, and ER stress. We also show that wild type Vap, but not the ALS8 mutant Vap, interacts with a lipid-binding protein, Oxysterol binding protein (Osbp), and that Vap is required for the proper localization of Osbp to the ER. Restoring the expression of Osbp in the ER suppresses the defects associated with loss of Vap and the ALS8 mutant Vap. Hence, we propose that the ALS8 mutation impairs the interaction of Vap with Osbp, resulting in hypomorphic defects that might contribute to the pathology of ALS8.

Oligo‐astheno‐teratozoospermia in mice lacking ORP4, a sterol‐binding protein in the OSBP‐related protein family

Oligo-astheno-teratozoospermia (OAT), a condition that includes low sperm number, low sperm motility and abnormal sperm morphology, is the commonest cause of male infertility. Because genetic analysis is frequently impeded by the infertility phenotype, the genetic basis of many of OAT conditions has been hard to verify. Here, we show that deficiency of ORP4, a sterol-binding protein in the oxysterol-binding protein (OSBP)-related protein family, causes male infertility due to severe OAT in mice. In ORP4-deficient mice, spermatogonia proliferation and subsequent meiosis occurred normally, but the morphology of elongating and elongated spermatids was severely distorted, with round-shaped head, curled back head or symplast. Spermatozoa derived from ORP4-deficient mice had little or no motility and no fertilizing ability in vitro. In ORP4-deficient testis, postmeiotic spermatids underwent extensive apoptosis, leading to a severely reduced number of spermatozoa. At the ultrastructural level, nascent acrosomes appeared to normally develop in round spermatids, but acrosomes were detached from the nucleus in elongating spermatids. These results suggest that ORP4 is essential for the postmeiotic differentiation of germ cells.

OSBP-Related Proteins: Liganding by Glycerophospholipids Opens New Insight into Their Function

Oxysterol-binding protein (OSBP) and its homologs designated OSBP-related (ORP) or OSBP-like (OSBPL) proteins constitute a conserved family of lipid binding/transfer proteins (LTP) in eukaryotes. The mechanisms of ORP function have remained incompletely understood, but they have been implicated as intracellular sterol sensors or transporters. A number of studies have provided evidence for the roles of ORPs at membrane contact sites (MCS), where endoplasmic reticulum is closely apposed with other organelle limiting membranes. ORPs are postulated to either transport sterols over MCSs or control the activity of enzymatic effectors or assembly of protein complexes with functions in signaling and lipid metabolism. Studies of yeast Saccharomyces cerevisiae ORPs Osh4p, Osh3p, Osh6p and Osh7p have revealed that ORPs do not exclusively bind sterols within their OSBP-related ligand-binding domain (ORD): The Osh4p ORD accommodates either sterols or phosphatidylinositol-4-phosphate (PI4P), and the Osh3p ORD was shown to specifically bind PI4P, the binding cavity being too narrow for a sterol to fit in. Most recently, Osh6p and Osh7p were demonstrated to show specific affinity for phosphatidylserine (PS), and to play a role in the intracellular transport of this glycerophospholipid; Additionally, two mammalian ORPs were shown to bind PS. Thus, the term frequently used for ORPs/OSBPLs, oxysterol-binding proteins, is a misnomer. While a number of ORPs bind oxysterols or cholesterol, other family members appear to interact with phospholipid ligands to regulate lipid fluxes, organelle lipid compositions and cell signaling. As a conclusion, ORPs are LTPs with a wide ligand spectrum and marked functional heterogeneity.

The Antiviral Effector IFITM3 Disrupts Intracellular Cholesterol Homeostasis to Block Viral Entry

(Cell Host & Microbe 13, 452–464; April 17, 2013) In Figure 2A of the above paper, the VECTOR panel was duplicated during the arrangement of the figure panels. Please refer to the corrected, revised figure (Figure 2). We regret any inconvenience this has caused. The Antiviral Effector IFITM3 Disrupts Intracellular Cholesterol Homeostasis to Block Viral EntryAmini-Bavil-Olyaee et al.Cell Host & MicrobeApril 17, 2013In BriefVesicle-membrane-protein-associated protein A (VAPA) and oxysterol-binding protein (OSBP) regulate intracellular cholesterol homeostasis, which is required for many virus infections. During entry, viruses or virus-containing vesicles can fuse with endosomal membranes to mediate the cytosolic release of virions, and alterations in endosomal cholesterol can inhibit this invasion step. We show that the antiviral effector protein interferon-inducible transmembrane protein 3 (IFITM3) interacts with VAPA and prevents its association with OSBP, thereby disrupting intracellular cholesterol homeostasis and inhibiting viral entry. Full-Text PDF Open Archive

A Four-Step Cycle Driven by PI(4)P Hydrolysis Directs Sterol/PI(4)P Exchange by the ER-Golgi Tether OSBP

Several proteins at endoplasmic reticulum (ER)-Golgi membrane contact sites contain a PH domain that interacts with the Golgi phosphoinositide PI(4)P, a FFAT motif that interacts with the ER protein VAP-A, and a lipid transfer domain. This architecture suggests the ability to both tether organelles and transport lipids between them. We show that in oxysterol binding protein (OSBP) these two activities are coupled by a four-step cycle. Membrane tethering by the PH domain and the FFAT motif enables sterol transfer by the lipid transfer domain (ORD), followed by back transfer of PI(4)P by the ORD. Finally, PI(4)P is hydrolyzed in cis by the ER protein Sac1. The energy provided by PI(4)P hydrolysis drives sterol transfer and allows negative feedback when PI(4)P becomes limiting. Other lipid transfer proteins are tethered by the same mechanism. Thus, OSBP-mediated back transfer of PI(4)P might coordinate the transfer of other lipid species at the ER-Golgi interface.

A Twoferase for Lipid Transfer at ER-Golgi Contact Sites

Reporting in Cell, Mesmin etal. (2013) show how a single molecule of oxysterol binding protein, which has a lipid binding domain that solubilizes both sterol and PI4P, might integrate its two specificities to drive the two lipids in opposite directions across endoplasmic reticulum-Golgi membrane contact sites.

Oxysterol-binding protein (OSBP) is required for the perinuclear localization of intra-Golgi v-SNAREs

Oxysterol-binding protein (OSBP) and OSBP-related proteins (ORPs) have been implicated in the distribution of sterols among intracellular organelles. OSBP regulates the Golgi cholesterol level, but how it relates to Golgi function is elusive. Here we report that OSBP is essential for the localization of intra-Golgi soluble vesicle N-ethylmaleimide-sensitive fusion attachment protein receptors (v-SNAREs). Depletion of OSBP by small interfering RNA causes mislocalization of intra-Golgi v-SNAREs GS28 and GS15 throughout the cytoplasm without affecting the perinuclear localization of Golgi target-SNARE syntaxin5 and reduces the abundance of a Golgi enzyme, mannosidase II (Man II). GS28 mislocalization and Man II reduction are also induced by cellular cholesterol depletion. Three domains of OSBP-an endoplasmic reticulum-targeting domain, a Golgi-targeting domain, and a sterol-binding domain-are all required for Golgi localization of GS28. Finally, GS28 mislocalization and Man II reduction in OSBP-depleted cells are largely restored by depletion of ArfGAP1, a regulator of the budding of coat protein complex (COP)-I vesicles. From these results, we postulate that Golgi cholesterol level, which is controlled by OSBP, is essential for Golgi localization of intra-Golgi v-SNAREs by ensuring proper COP-I vesicle transport.

Inhibition of HCV Replication by Oxysterol-Binding Protein-Related Protein 4 (ORP4) through Interaction with HCV NS5B and Alteration of Lipid Droplet Formation

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.

Adenovirus RIDα uncovers a novel pathway requiring ORP1L for lipid droplet formation independent of NPC1

Niemann-Pick disease type C (NPC) is caused by mutations in NPC1 or NPC2, which coordinate egress of low-density-lipoprotein (LDL)-cholesterol from late endosomes. We previously reported that the adenovirus-encoded protein RIDα rescues the cholesterol storage phenotype in NPC1-mutant fibroblasts. We show here that RIDα reconstitutes deficient endosome-to-endoplasmic reticulum (ER) transport, allowing excess LDL-cholesterol to be esterified by acyl-CoA:cholesterol acyltransferase and stored in lipid droplets (LDs) in NPC1-deficient cells. Furthermore, the RIDα pathway is regulated by the oxysterol-binding protein ORP1L. Studies have classified ORP1L as a sterol sensor involved in LE positioning downstream of GTP-Rab7. Our data, however, suggest that ORP1L may play a role in transport of LDL-cholesterol to a specific ER pool designated for LD formation. In contrast to NPC1, which is dispensable, the RIDα/ORP1L-dependent route requires functional NPC2. Although NPC1/NPC2 constitutes the major pathway, therapies that amplify minor egress routes for LDL-cholesterol could significantly improve clinical management of patients with loss-of-function NPC1 mutations. The molecular identity of putative alternative pathways, however, is poorly characterized. We propose RIDα as a model system for understanding physiological egress routes that use ORP1L to activate ER feedback responses involved in LD formation.

Interactome map uncovers phosphatidylserine transport by oxysterol-binding proteins

The internal organization of eukaryotic cells into functionally specialized, membrane-delimited organelles of unique composition implies a need for active, regulated lipid transport. Phosphatidylserine (PS), for example, is synthesized in the endoplasmic reticulum and then preferentially associates--through mechanisms not fully elucidated--with the inner leaflet of the plasma membrane. Lipids can travel via transport vesicles. Alternatively, several protein families known as lipid-transfer proteins (LTPs) can extract a variety of specific lipids from biological membranes and transport them, within a hydrophobic pocket, through aqueous phases. Here we report the development of an integrated approach that combines protein fractionation and lipidomics to characterize the LTP-lipid complexes formed in vivo. We applied the procedure to 13 LTPs in the yeast Saccharomyces cerevisiae: the six Sec14 homology (Sfh) proteins and the seven oxysterol-binding homology (Osh) proteins. We found that Osh6 and Osh7 have an unexpected specificity for PS. In vivo, they participate in PS homeostasis and the transport of this lipid to the plasma membrane. The structure of Osh6 bound to PS reveals unique features that are conserved among other metazoan oxysterol-binding proteins (OSBPs) and are required for PS recognition. Our findings represent the first direct evidence, to our knowledge, for the non-vesicular transfer of PS from its site of biosynthesis (the endoplasmic reticulum) to its site of biological activity (the plasma membrane). We describe a new subfamily of OSBPs, including human ORP5 and ORP10, that transfer PS and propose new mechanisms of action for a protein family that is involved in several human pathologies such as cancer, dyslipidaemia and metabolic syndrome.

Cholesterol-binding molecules MLN64 and ORP1L mark distinct late endosomes with transporters ABCA3 and NPC1

Cholesterol is an essential lipid in eukaryotic cells and is present in membranes of all intracellular compartments. A major source for cellular cholesterol is internalized lipoprotein particles that are transported toward acidic late endosomes (LE) and lysosomes. Here the lipoprotein particles are hydrolyzed, and free cholesterol is redistributed to other organelles. The LE can contain over half of the cellular cholesterol and, as a major sorting station, can contain many cholesterol-binding proteins from the ABCA, STARD, and ORP families. Here, we show that metastatic lymph node 64 (MLN64, STARD3) and oxysterol-binding protein-related protein 1L (ORP1L) define two subpopulations of LE. MLN64 is present on a LE containing the cholesterol transporter ABCA3, whereas ORP1L localizes to another population of LE containing Niemann Pick type C1 (NPC1), a cholesterol exporter. Endocytosed cargo passes through MLN64/ABCA3-positive compartments before it reaches ORP1L/NPC1-positive LE. The MLN64/ABCA3 compartments cycle between LE and plasma membrane and frequently contact "later" ORP1L/NPC1-containing LE. We propose two stages of cholesterol handling in late endosomal compartments: first, cholesterol enters MLN64/ABCA3-positive compartments from where it can be recycled to the plasma membrane, and later, cholesterol enters ORP1L/NPC1 endosomes that mediate cholesterol export to the endoplasmic reticulum.

A Protein Pair with PIPs Inside

Oxysterol binding protein (OSBP) and many of its homologs transfer sterol invitro, but invivo, they are not major sterol transporters. In this issue of Structure, Tong and colleagues find that the yeast OSBP homolog, Osh3, binds PI(4)P but not sterol, supporting the view that PI(4)P regulation, not sterol transport, is the key activity for OSBP homologs.

Activin A impairs insulin action in cardiomyocytes via up-regulation of miR-143

Enhanced activin A release from epicardial adipose tissue (EAT) has been linked to the development of cardiac dysfunction in type 2 diabetes (T2D). This study examined whether the inhibition of insulin action induced by epicardial adipokines in cardiomyocytes can be ascribed to alterations in miRNA expression. Expression levels of miRNAs were assessed by real-time PCR in primary adult rat cardiomyocytes (ARC) exposed to conditioned media generated from EAT biopsies (CM-EAT) from patients with and without T2D. CM-EAT-T2D altered the expression of eight miRNAs in ARC vs. CM-EAT from patients without T2D. Of these, only expression of the miR-143/145 cluster was affected by activin A in the same direction as CM-EAT-T2D. Accordingly, activin A neutralizing antibodies prevented the induction of the miR-143/145 cluster by CM-EAT-T2D. Subsequently, the impact of the miR-143/145 cluster on insulin action was investigated. Transfection of HL-1 cells with precursor-miR-143 (pre-miR-143), but not pre-miR-145, blunted the insulin-mediated phosphorylation of Akt and its substrate proline-rich Akt substrate of 40 kDa (PRAS40), and reduced insulin-stimulated glucose uptake. Also lentivirus-mediated expression of pre-miR-143 in ARC reduced insulin-induced Akt phosphorylation. These effects were ascribed to down-regulation of the miR-143 target and regulator of insulin action, the oxysterol-binding protein-related protein 8 (ORP8) in both ARC and HL-1 cells. Finally, LNA-anti-miR-143 protected against the detrimental effects of CM-EAT-T2D on insulin action in ARC. Activin A released from EAT-T2D inhibits insulin action via the induction of miR-143 in cardiomyocytes. This miRNA inhibits the Akt pathway through down-regulation of the novel regulator of insulin action, ORP8.

Structure of Osh3 Reveals a Conserved Mode of Phosphoinositide Binding in Oxysterol-Binding Proteins

The oxysterol-binding protein (OSBP)-related proteins (ORPs) are conserved from yeast to humans, and implicated in the regulation of lipid homeostasis and in signaling pathways. Saccharomyces cerevisiae has seven ORPs (Osh1-Osh7) that share one unknown essential function. Here, we report the 1.5-2.3Å structures of the PH domain and ORD (OSBP-related domain) of yeast Osh3 in apo-form or in complex with phosphatidylinositol 4-phosphate (PI[4]P). Osh3 recognizes PI(4)P by the highly conserved residues in the tunnel of ORD whereas it lacks sterol binding due to the narrow hydrophobic tunnel. Yeast complementation tests suggest that PI(4)P binding to PH and ORD is essential for function. This study suggests that the unifying feature in all ORP homologs is the binding of PI(4)P to ORD and sterol binding is additional to certain homologs. Structural modeling of full-length Osh3 is consistent with the concept that Osh3 is a lipid transfer protein or regulator in membrane contact sites.

Synthesis and structure of 16,22-diketocholesterol bound to oxysterol-binding protein Osh4

We have synthesized 16,22-diketocholesterol, a novel ligand for oxysterol-binding protein Osh4, and determined X-ray structure of the diketocholesterol in complex with Osh4. The X-ray structure shows that α7 helix of Osh4 assumes open conformation while the rest of Osh4, closed conformation, implying this diketocholesterol–bound Osh4 structure may represent a structural intermediate between the two conformations.

Abstract 22: OSBPL6 is a Novel miR-33 Target Gene Involved in Cellular Cholesterol Efflux

Cellular lipid homeostasis is fundamental to human health. Recent studies indicate that microRNAs (miRNAs) play important roles in the post-transcriptional regulation of lipid metabolism genes. This is illustrated by the discovery, by our group and others, of miR-33 as a central regulator of multiple aspects of lipid metabolism, including cholesterol efflux, fatty acid oxidation, and plasma levels of HDL cholesterol and VLDL triglycerides. In a study of miR-33 antagonism in non-human primates, we identified the most highly de-repressed miR-33 target gene as OSBPL6 (oxysterol binding protein like 6). A member of the OSBP family of cytosolic proteins that bind cholesterol/oxysterols, OSBPL6 contains an ER-targeting FFAT motif as well as a predicted plasma membrane-targeting pleckstrin homology domain, suggesting a putative role for OSBPL6 in vesicular traffic between these compartments. Notably, the OSBPL6 gene resides in a chromosome 2 locus associated with a predisposition to CAD in two major human heart disease studies (Family Heart Study, Framingham Heart Study). We therefore sought to further understand the regulation and function of OSBPL6 in cellular lipid homeostasis. Results We show that OSBPL6 is induced in macrophages and hepatocytes in response to cholesterol loading in vitro, as well as in the livers of LDLR-/- mice and non-human primates fed a high fat diet. Overexpression of miR-33 reduces OSBPL6-3’UTR activity by 40%, and this repression is relieved by mutation of the putative miR-33 binding site in the 3’UTR, confirming that OSBPL6 is a direct target of miR-33. Accordingly, transfection of HEPG2 cells with miR-33 mimic decreases mRNA expression of Osbpl6, but not the related family member Osbpl1. Notably, siRNA-mediated knockdown of OSBPL6 in cholesterol-loaded HEPG2 cells or THP-1 macrophages impairs cholesterol efflux to both apoA-I and HDL, and conversely, OSBPL6 overexpression enhances cholesterol efflux to these acceptors. Conclusion These data identify OSBPL6 as a novel miR-33 target gene in mice and humans that regulates cellular cholesterol trafficking and efflux via the ABC transporters, further highlighting the coordinated regulation by miR-33 of pathways that promote cellular cholesterol clearance.