Background:The function of oxysterol-binding protein-related protein (ORP4) is unknown. Results: Silencing ORP4L or all ORP4 isoforms triggers growth arrest and apoptosis, which is suppressed by H-Ras and involves the C-terminal lipid binding domain. Conclusion: ORP4 is a sterol and PI-4P-binding protein required for survival and proliferation of immortalized and transformed cells. Significance: This is the first study to identify a mammalian ORP with growth regulatory activity.

Oxysterol-binding protein (OSBP) and OSBP-related proteins (ORPs) constitute a large eukaryotic gene family that transports and regulates the metabolism of sterols and phospholipids. The original classification of the family based on oxysterol-binding activity belies the complex dual lipid-binding specificity of the conserved OSBP homology domain (OHD). Additional protein- and membrane-interacting modules mediate the targeting of select OSBP/ORPs to membrane contact sites between organelles, thus positioning the OHD between opposing membranes for lipid transfer and metabolic regulation. This unique subcellular location, coupled with diverse ligand preferences and tissue distribution, has identified OSBP/ORPs as key arbiters of membrane composition and function. Here, we will review how molecular models of OSBP/ORP-mediated intracellular lipid transport and regulation at membrane contact sites relate to their emerging roles in cellular and organismal functions.

Objectives: During the advancement of atherosclerosis, plaque cellularity is governed by the influx of monocyte-derived macrophages and their turnover via apoptotic and nonapoptotic forms of cell death. Previous reports have demonstrated that programmed necrosis, or necroptosis, of plaque macrophages contribute to necrotic core formation. Knockdown or inhibition of the necrosome components RIPK1 (receptor-interacting protein kinase 1) and RIPK3 (receptor-interacting protein kinase 3) slow atherogenesis, and activation of the terminal step of necroptosis, MLKL (mixed lineage kinase domain-like protein), has been demonstrated in advanced human atherosclerotic plaques. However, whether MLKL directly contributes to lesion development and necrotic core formation has not been investigated. Approaches and Results: MLKL expression was knocked down in atherogenic Apoe -knockout mice via the administration of antisense oligonucleotides. During atherogenesis, Mlkl knockdown decreased both programmed cell death and the necrotic core in the plaque. However, total lesion area remained unchanged. Furthermore, treatment with the MLKL antisense oligonucleotide unexpectedly reduced circulating cholesterol levels compared with control antisense oligonucleotide but increased the accumulation of lipids within the plaque and in vitro in macrophage foam cells. MLKL colocalized with the late endosome and multivesicular bodies in peritoneal macrophages incubated with atherogenic lipoproteins. Transfection with MLKL antisense oligonucleotide increased lipid localization with the multivesicular bodies, suggesting that upon Mlkl knockdown, lipid trafficking becomes defective leading to enhanced lipid accumulation in macrophages. Conclusions: These studies confirm the requirement for MLKL as the executioner of necroptosis, and as such a significant contributor to the necrotic core during atherogenesis. We also identified a previously unknown role for MLKL in regulating endosomal trafficking to facilitate lipid handling in macrophages during atherogenesis.

Rationale: Atherosclerosis is characterized by an accumulation of foam cells within the arterial wall, resulting from excess cholesterol uptake and buildup of cytosolic lipid droplets (LDs). Autophagy promotes LD clearance by freeing stored cholesterol for efflux, a process that has been shown to be atheroprotective. While the role of autophagy in LD catabolism has been studied in macrophage-derived foam cells, this has remained unexplored in vascular smooth muscle cell (VSMC)-derived foam cells that constitute a large fraction of foam cells within atherosclerotic lesions. Objective: We performed a comparative analysis of autophagy flux in lipid-rich aortic intimal populations to determine whether VSMC-derived foam cells metabolize LDs similarly to their macrophage counterparts. Methods and Results: Atherosclerosis was induced in GFP-LC3 transgenic mice by PCSK9 (proprotein convertase subtilisin/kexin type 9)-adeno-associated viral injection and Western diet feeding. Using flow cytometry of aortic digests, we observed a significant increase in dysfunctional autophagy of VSMC-derived foam cells during atherogenesis relative to macrophage-derived foam cells. Using cell culture models of lipid-loaded VSMC and macrophage, we show that autophagy-mediated cholesterol efflux from VSMC foam cells was poor relative to macrophage foam cells, and largely occurs when HDL (high-density lipoprotein) is used as a cholesterol acceptor, as opposed to apoA-1 (apolipoproteinA-1). This was associated with the predominant expression of ABCG1 in VSMC foam cells. Using metformin, an autophagy activator, cholesterol efflux to HDL was significantly increased in VSMC, but not in macrophage, foam cells. Conclusions: These data demonstrate that VSMC and macrophage foam cells perform cholesterol efflux by distinct mechanisms, and that autophagy flux is highly impaired in VSMC foam cells, but can be induced by pharmacological means. Further investigation is warranted into targeting autophagy specifically in VSMC foam cells, the predominant foam cell subtype of advanced atherosclerotic plaques, to promote reverse cholesterol transport and resolution of the atherosclerotic plaque.

Oxysterol binding protein (OSBP)-related protein 4L (ORP4L, also known as OSBPL2), a closely related paralogue and interacting partner of OSBP, binds sterols and phosphatidylinositol 4-phosphate [PI(4)P] and regulates cell proliferative signalling at the plasma membrane (PM). Here, we report that ORP4L also interacts with the -Golgi network (TGN) in an OSBP-, sterol- and PI(4)P-dependent manner. Characterization of ORP4L lipid and VAP binding mutants indicated an indirect mechanism for translocation to ER-Golgi contact sites in response to 25-hydroxycholesterol that was dependent on OSBP and PI(4)P. shRNA silencing revealed that ORP4L was required to maintain the organization and PI(4)P content of the Golgi and TGN. In contrast, the interaction of ORP4L with the PM was not dependent on its sterol, PI(4)P or VAP binding activities. At the PM, ORP4L partially localized with a genetically encoded probe for PI(4)P but not with a probe for phosphatidylinositol 4,5-bisphosphate. We conclude that ORP4L is differentially localized to the PM and ER-Golgi contacts sites. OSBP-, lipid- and VAP-regulated interactions of ORP4L with ER-Golgi contact sites are involved in the maintenance of Golgi and TGN structure.