Cholesterol transfer via endoplasmic reticulum contacts mediates lysosome damage repair.

Lysosome integrity is essential for cell viability, and lesions in lysosome membranes are repaired by the ESCRT machinery. Here, we describe an additional mechanism for lysosome repair that is activated independently of ESCRT recruitment. Lipidomic analyses showed increases in lysosomal phosphatidylserine and cholesterol after damage. Electron microscopy demonstrated that lysosomal membrane damage is rapidly followed by the formation of contacts with the endoplasmic reticulum (ER), which depends on the ER proteins VAPA/B. The cholesterol-binding protein ORP1L was recruited to damaged lysosomes, accompanied by cholesterol accumulation by a mechanism that required VAP-ORP1L interactions. The PtdIns 4-kinase PI4K2A rapidly produced PtdIns4P on lysosomes upon damage, and knockout of PI4K2A inhibited damage-induced accumulation of ORP1L and cholesterol and led to the failure of lysosomal membrane repair. The cholesterol-PtdIns4P transporter OSBP was also recruited upon damage, and its depletion caused lysosomal accumulation of PtdIns4P and resulted in cell death. We conclude that ER contacts are activated on damaged lysosomes in parallel to ESCRTs to provide lipids for membrane repair, and that PtdIns4P generation and removal are central in this response.

ORP2 couples LDL-cholesterol transport to FAK activation by endosomal cholesterol/PI(4,5)P2 exchange.

Low-density lipoprotein (LDL)-cholesterol delivery from late endosomes to the plasma membrane regulates focal adhesion dynamics and cell migration, but the mechanisms controlling it are poorly characterized. Here, we employed auxin-inducible rapid degradation of oxysterol-binding protein-related protein 2 (ORP2/OSBPL2) to show that endogenous ORP2 mediates the transfer of LDL-derived cholesterol from late endosomes to focal adhesion kinase (FAK)-/integrin-positive recycling endosomes in human cells. In vitro, cholesterol enhances membrane association of FAK to PI(4,5)P2 -containing lipid bilayers. In cells, ORP2 stimulates FAK activation and PI(4,5)P2 generation in endomembranes, enhancing cell adhesion. Moreover, ORP2 increases PI(4,5)P2 in NPC1-containing late endosomes in a FAK-dependent manner, controlling their tubulovesicular trafficking. Together, these results provide evidence that ORP2 controls FAK activation and LDL-cholesterol plasma membrane delivery by promoting bidirectional cholesterol/PI(4,5)P2 exchange between late and recycling endosomes.

Oculocerebrorenal syndrome of Lowe (OCRL) controls leukemic T-cell survival by preventing excessive PI(4,5)P2 hydrolysis in the plasma membrane.

T-cell acute lymphoblastic leukemia (T-ALL) is one of the deadliest and most aggressive hematological malignancies, but its pathological mechanism in controlling cell survival is not fully understood. Oculocerebrorenal syndrome of Lowe is a rare X-linked recessive disorder characterized by cataracts, intellectual disability, and proteinuria. This disease has been shown to be caused by mutation of oculocerebrorenal syndrome of Lowe 1 (OCRL1; OCRL), encoding a phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] 5-phosphatase involved in regulating membrane trafficking; however, its function in cancer cells is unclear. Here, we uncovered that OCRL1 is overexpressed in T-ALL cells, and knockdown of OCRL1 results in cell death, indicating the essential role of OCRL in controlling T-ALL cell survival. We show OCRL is primarily localized in the Golgi and can translocate to plasma membrane (PM) upon ligand stimulation. We found OCRL interacts with oxysterol-binding protein-related protein 4L, which facilitates OCRL translocation from the Golgi to the PM upon cluster of differentiation 3 stimulation. Thus, OCRL represses the activity of oxysterol-binding protein-related protein 4L to prevent excessive PI(4,5)P2 hydrolysis by phosphoinositide phospholipase C ß3 and uncontrolled Ca2+ release from the endoplasmic reticulum. We propose OCRL1 deletion leads to accumulation of PI(4,5)P2 in the PM, disrupting the normal Ca2+ oscillation pattern in the cytosol and leading to mitochondrial Ca2+ overloading, ultimately causing T-ALL cell mitochondrial dysfunction and cell death. These results highlight a critical role for OCRL in maintaining moderate PI(4,5)P2 availability in T-ALL cells. Our findings also raise the possibility of targeting OCRL1 to treat T-ALL disease.

Human visceral adipose tissue microvascular endothelial cell isolation and establishment of co-culture with white adipocytes to analyze cell-cell communication.

Communication between adipocytes and endothelial cells (EC) is suggested to play an important role in the metabolic function of white adipose tissue. In order to generate tools to investigate in detail the physiology and communication of EC and adipocytes, a method for isolation of adipose microvascular EC from visceral adipose tissue (VAT) biopsies of subjects with obesity was developed. Moreover, mature white adipocytes were isolated from the VAT biopsies by a method adapted from a previously published Membrane aggregate adipocytes culture (MAAC) protocol. The identity and functionality of the cultivated and isolated adipose microvascular EC (AMvEC) was validated by imaging their morphology, analyses of mRNA expression, fluorescence activated cell sorting (FACS), immunostaining, low-density lipoprotein (LDL) uptake, and in vitro angiogenesis assays. Finally, we established a new trans filter co-culture system (membrane aggregate adipocyte and endothelial co-culture, MAAECC) for the analysis of communication between the two cell types. EC-adipocyte communication in this system was validated by omics analyses, revealing several altered proteins belonging to pathways such as metabolism, intracellular transport and signal transduction in adipocytes co-cultured with AMvEC. In reverse experiments, induction of several pathways including endothelial development and functions was found in AMvEC co-cultured with adipocytes. In conclusion, we developed a robust method to isolate EC from small quantities of human VAT. Furthermore, the MAAECC system established during the study enables one to study the communication between primary white adipocytes and EC or vice-versa and could also be employed for drug screening.

Protrudin regulates FAK activation, endothelial cell migration and angiogenesis.

During angiogenesis, endothelial cells form protrusive sprouts and migrate towards the angiogenic stimulus. In this study, we investigate the role of the endoplasmic reticulum (ER)-anchored protein, Protrudin, in endothelial cell protrusion, migration and angiogenesis. Our results demonstrate that Protrudin regulates angiogenic tube formation in primary endothelial cells, Human umbilical vein endothelial cells (HUVECs). Analysis of RNA sequencing data and its experimental validation revealed cell migration as a prominent cellular function affected in HUVECs subjected to Protrudin knockdown. Further, our results demonstrate that knockdown of Protrudin inhibits focal adhesion kinase (FAK) activation in HUVECs and human aortic endothelial cells (HAECs). This is associated with a loss of polarized phospho-FAK distribution upon Protrudin knockdown as compared to Protrudin expressing HUVECs. Reduction of Protrudin also results in a perinuclear accumulation of mTOR and a decrease in VEGF-mediated S6K activation. However, further experiments suggest that the observed inhibition of angiogenesis in Protrudin knockdown cells is not affected by mTOR disturbance. Therefore, our findings suggest that defects in FAK activation and its abnormal subcellular distribution upon Protrudin knockdown are associated with a detrimental effect on endothelial cell migration and angiogenesis. Furthermore, mice with global Protrudin deletion demonstrate reduced retinal vascular progression. To conclude, our results provide evidence for a novel key role of Protrudin in endothelial cell migration and angiogenesis.

GOLM1 depletion modifies cellular sphingolipid metabolism and adversely affects cell growth.

Golgi membrane protein 1 (GOLM1) is a Golgi-resident type 2 transmembrane protein known to be overexpressed in several cancers, including hepatocellular carcinoma (HCC), as well as in viral infections. However, the role of GOLM1 in lipid metabolism remains enigmatic. In this study, we employed siRNA-mediated GOLM1 depletion in Huh-7 HCC cells to study the role of GOLM1 in lipid metabolism. Mass spectrometric lipidomic analysis in GOLM1 knockdown cells showed an aberrant accumulation of sphingolipids, such as ceramides, hexosylceramides, dihexosylceramides, sphinganine, sphingosine, and ceramide phosphate, along with cholesteryl esters. Furthermore, we observed a reduction in phosphatidylethanolamines and lysophosphatidylethanolamines. In addition, Seahorse extracellular flux analysis indicated a reduction in mitochondrial oxygen consumption rate upon GOLM1 depletion. Finally, alterations in Golgi structure and distribution were observed both by electron microscopy imaging and immunofluorescence microscopy analysis. Importantly, we found that GOLM1 depletion also affected cell proliferation and cell cycle progression in Huh-7 HCC cells. The Golgi structural defects induced by GOLM1 reduction might potentially affect the trafficking of proteins and lipids leading to distorted intracellular lipid homeostasis, which may result in organelle dysfunction and altered cell growth. In conclusion, we demonstrate that GOLM1 depletion affects sphingolipid metabolism, mitochondrial function, Golgi structure, and proliferation of HCC cells.

Insulin-inducible THRSP maintains mitochondrial function and regulates sphingolipid metabolism in human adipocytes.

BACKGROUND: Thyroid hormone responsive protein (THRSP) is a lipogenic nuclear protein that is highly expressed in murine adipose tissue, but its role in humans remains unknown. METHODS: We characterized the insulin regulation of THRSP in vivo in human adipose tissue biopsies and in vitro in Simpson-Golabi-Behmel syndrome (SGBS) adipocytes. To this end, we measured whole-body insulin sensitivity using the euglycemic insulin clamp technique in 36 subjects [age 40 ± 9 years, body mass index (BMI) 27.3 ± 5.0 kg/m2]. Adipose tissue biopsies were obtained at baseline and after 180 and 360 min of euglycemic hyperinsulinemia for measurement of THRSP mRNA concentrations. To identify functions affected by THRSP, we performed a transcriptomic analysis of THRSP-silenced SGBS adipocytes. Mitochondrial function was assessed by measuring mitochondrial respiration as well as oxidation and uptake of radiolabeled oleate and glucose. Lipid composition in THRSP silencing was studied by lipidomic analysis. RESULTS: We found insulin to increase THRSP mRNA expression 5- and 8-fold after 180 and 360 min of in vivo euglycemic hyperinsulinemia. This induction was impaired in insulin-resistant subjects, and THRSP expression was closely correlated with whole-body insulin sensitivity. In vitro, insulin increased both THRSP mRNA and protein concentrations in SGBS adipocytes in a phosphoinositide 3-kinase (PI3K)-dependent manner. A transcriptomic analysis of THRSP-silenced adipocytes showed alterations in mitochondrial functions and pathways of lipid metabolism, which were corroborated by significantly impaired mitochondrial respiration and fatty acid oxidation. A lipidomic analysis revealed decreased hexosylceramide concentrations, supported by the transcript concentrations of enzymes regulating sphingolipid metabolism. CONCLUSIONS: THRSP is regulated by insulin both in vivo in human adipose tissue and in vitro in adipocytes, and its expression is downregulated by insulin resistance. As THRSP silencing decreases mitochondrial respiration and fatty acid oxidation, its downregulation in human adipose tissue could contribute to mitochondrial dysfunction. Furthermore, disturbed sphingolipid metabolism could add to metabolic dysfunction in obese adipose tissue.

OSBP-Related Protein 5L Maintains Intracellular IP3/Ca2+ Signaling and Proliferation in T Cells by Facilitating PIP2 Hydrolysis.

Phospholipase C (PLC) isoforms play central roles in signaling cascades by cleaving PIP2 into the second messengers IP3 and DAG. In this study, to our knowledge, we uncover that ORP5L interacts physically with PLCγ1 in T cells, extracts PIP2 from the plasma membrane via its ORD domain (OSBP-related domain), presents it to PLCγ1 (enabling IP3 generation), and eventually maintains intracellular Ca2+ homeostasis. Through this mechanism, ORP5L promotes T cell proliferation in a Ca2+-activated NFAT2-dependent manner. To our knowledge, our study uncovers a new key function of ORP5L as a critical cofactor for PLCγ1 catalysis and its crucial role in human T cell proliferation.

ORP/Osh mediate cross-talk between ER-plasma membrane contact site components and plasma membrane SNAREs.

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. Deleting OSH1-3 in 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.

ORP2, a cholesterol transporter, regulates angiogenic signaling in endothelial cells.

Oxysterol-binding protein-related protein 2 (ORP2), a cholesterol-PI(4,5)P2 countercurrent transporter, was recently identified as a novel regulator of plasma membrane (PM) cholesterol and PI(4,5)P2 content in HeLa cells. Here, we investigate the role of ORP2 in endothelial cell (EC) cholesterol and PI(4,5)P2 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)P2 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)P2 homeostasis and cholesterol-dependent angiogenic signaling.

ORP4L couples IP3 to ITPR1 in control of endoplasmic reticulum calcium release.

Oxysterol-binding protein-related protein (ORP) 4L acts as a scaffold protein assembling CD3-ε, G-αq/11, and PLC-ß3 into a complex at the plasma membrane that mediates inositol (1,4,5)-trisphosphate (IP3)-induced endoplasmic reticulum (ER) Ca2+ release and oxidative phosphorylation in T-cell acute lymphoblastic leukemia cells. Here, we offer new evidence that ORP4L interacts with the carboxyl terminus of the IP3 receptor type 1 (ITPR1) in Jurkat T cells. ORP4L enables IP3 binding to ITPR1; a truncated construct that lacks the ITPR1-binding region retains the ability to increase IP3 production but fails to mediate IP3 and ITPR1 binding. In association with this ability of ORP4L, it enhances Ca2+ release from the ER and subsequent cytosolic and mitochondrial parallel Ca2+ spike oscillations that stimulate mitochondrial energetics and thus maintains cell survival. These data support a novel model in which ORP4L is a cofactor of ITPR1, which increases ITPR1 sensitivity to IP3 and enables ER Ca2+ release.-Cao, X., Chen, J., Li, D., Xie, P., Xu, M., Lin, W., Li, S., Pan, G., Tang, Y., Xu, J., Olkkonen, V. M., Yan, D., Zhong, W. ORP4L couples IP3 to ITPR1 in control of endoplasmic reticulum calcium release.

The PNPLA3-I148M variant increases polyunsaturated triglycerides in human adipose tissue.

BACKGROUND & AIMS: The I148M variant in PNPLA3 is the major genetic risk factor for non-alcoholic fatty liver disease (NAFLD). The liver is enriched with polyunsaturated triglycerides (PUFA-TGs) in PNPLA3-I148M carriers. Gene expression data indicate that PNPLA3 is liver-specific in humans, but whether it functions in adipose tissue (AT) is unknown. We investigated whether PNPLA3-I148M modifies AT metabolism in human NAFLD. METHODS: Profiling of the AT lipidome and fasting serum non-esterified fatty acid (NEFA) composition was conducted in 125 volunteers (PNPLA3148MM/MI , n = 63; PNPLA3148II , n = 62). AT fatty acid composition was determined in 50 volunteers homozygous for the variant (PNPLA3148MM , n = 25) or lacking the variant (PNPLA3148II , n = 25). Whole-body insulin sensitivity of lipolysis was determined using [2 H5 ]glycerol, and PNPLA3 mRNA and protein levels were measured in subcutaneous AT and liver biopsies in a subset of the volunteers. RESULTS: PUFA-TGs were significantly increased in AT in carriers versus non-carriers of PNPLA3-I148M. The variant did not alter the rate of lipolysis or the composition of fasting serum NEFAs. PNPLA3 mRNA was 33-fold higher in the liver than in AT (P < .0001). In contrast, PNPLA3 protein levels per tissue protein were three-fold higher in AT than the liver (P < .0001) and nine-fold higher when related to whole-body AT and liver tissue masses (P < .0001). CONCLUSIONS: Contrary to previous assumptions, PNPLA3 is highly abundant in AT. PNPLA3-I148M locally remodels AT TGs to become polyunsaturated as it does in the liver, without affecting lipolysis or composition of serum NEFAs. Changes in AT metabolism do not contribute to NAFLD in PNPLA3-I148M carriers.

Metabolomic Signature of Angiopoietin-Like Protein 3 Deficiency in Fasting and Postprandial State.

Objective- Loss-of-function (LOF) variants in the ANGPTL3 (angiopoietin-like protein 3) have been associated with low levels of plasma lipoproteins and decreased coronary artery disease risk. We aimed to determine detailed metabolic effects of genetically induced ANGPTL3 deficiency in fasting and postprandial state. Approach and Results- We studied individuals carrying S17X LOF mutation in ANGPTL3 (6 homozygous and 32 heterozygous carriers) and 38 noncarriers. Nuclear magnetic resonance metabolomics was used to quantify 225 circulating metabolic measures. We compared metabolic differences between LOF carriers and noncarriers in fasting state and after a high-fat meal. In fasting, ANGPTL3 deficiency was characterized by similar extent of reductions in LDL (low-density lipoprotein) cholesterol (0.74 SD units lower concentration per LOF allele [95% CI, 0.42-1.06]) as observed for many TRL (triglyceride-rich lipoprotein) measures, including VLDL (very-low-density lipoprotein) cholesterol (0.75 [95% CI, 0.45-1.05]). Within most lipoprotein subclasses, absolute levels of cholesterol were decreased more than triglycerides, resulting in the relative proportion of cholesterol being reduced within TRLs and their remnants. Further, ß-hydroxybutyrate was elevated (0.55 [95% CI, 0.21-0.89]). Homozygous ANGPTL3 LOF carriers showed essentially no postprandial increase in TRLs and fatty acids, without evidence for adverse compensatory metabolic effects. Conclusions- In addition to overall triglyceride- and LDL cholesterol-lowering effects, ANGPTL3 deficiency results in reduction of cholesterol proportion within TRLs and their remnants. Further, ANGPTL3 LOF carriers had elevated ketone body production, suggesting enhanced hepatic fatty acid ß-oxidation. The detailed metabolic profile in human knockouts of ANGPTL3 reinforces inactivation of ANGPTL3 as a promising therapeutic target for decreasing cardiovascular risk.

OSBP-related protein 4L promotes phospholipase Cß3 translocation from the nucleus to the plasma membrane in Jurkat T-cells.

Phosphoinositide phospholipases C (PLCs) are a family of eukaryotic intracellular enzymes with important roles in signal transduction. In addition to their location at the plasma membrane, PLCs also exist within the cell nucleus where they are stored. We previously demonstrated that OSBP-related protein 4L (ORP4L) anchors cluster of differentiation 3ϵ (CD3ϵ) to the heterotrimeric G protein subunit (Gαq/11) to control PLCß3 relocation and activation. However, the underlying mechanism by which ORP4L facilitates PLCß3 translocation remains unknown. Here, using confocal immunofluorescence microscopy and coimmunoprecipitation assays, we report that ORP4L stimulates PLCß3 translocation from the nucleus to the plasma membrane in Jurkat T-cells in two steps. First, we found that ORP4L is required for the activation of Ras-related nuclear protein (RAN), a GTP-binding nuclear protein that binds to exportin 1 and eventually promotes the nuclear export of PLCß3. Second, we also observed that ORP4L interacts with vesicle-associated membrane protein-associated protein A (VAPA) through its two phenylalanines in an acidic tract (FFAT) motif. This complex enabled PLCß3 movement to the plasma membrane, indicating that PLCß3 translocation occurs in a VAPA-dependent manner. This study reveals detailed mechanistic insight into the role of ORP4L in PLCß3 redistribution from storage within the nucleus to the plasma membrane via RAN activation and interaction with VAPA in Jurkat T-cells.

Analysis of ORP2-knockout hepatocytes uncovers a novel function in actin cytoskeletal regulation.

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., Kivelä, A. M., Andrews, R., Li, C., Zhou, Y., Olkkonen, V. M. Analysis of ORP2-knockout hepatocytes uncovers a novel function in actin cytoskeletal regulation.

OSBP-related protein-2 (ORP2): a novel Akt effector that controls cellular energy metabolism.

ORP2 is a ubiquitously expressed OSBP-related protein previously implicated in endoplasmic reticulum (ER)-lipid droplet (LD) contacts, triacylglycerol (TG) metabolism, cholesterol transport, adrenocortical steroidogenesis, and actin-dependent cell dynamics. Here, we characterize the role of ORP2 in carbohydrate and lipid metabolism by employing ORP2-knockout (KO) hepatoma cells (HuH7) generated by CRISPR-Cas9 gene editing. The ORP2-KO and control HuH7 cells were subjected to RNA sequencing, analyses of Akt signaling, carbohydrate and TG metabolism, the extracellular acidification rate, and the lipidome, as well as to transmission electron microscopy. The loss of ORP2 resulted in a marked reduction of active phosphorylated Akt(Ser473) and its target Glycogen synthase kinase 3ß(Ser9), consistent with defective Akt signaling. ORP2 was found to form a physical complex with the key controllers of Akt activity, Cdc37, and Hsp90, and to co-localize with Cdc37 and active Akt(Ser473) at lamellipodial plasma membrane regions, in addition to the previously reported ER-LD localization. ORP2-KO reduced glucose uptake, glycogen synthesis, glycolysis, mRNA-encoding glycolytic enzymes, and SREBP-1 target gene expression, and led to defective TG synthesis and storage. ORP2-KO did not reduce but rather increased ER-LD contacts under basal culture conditions and interfered with their expansion upon fatty acid loading. Together with our recently published work (Kentala et al. in FASEB J 32:1281-1295, 2018), this study identifies ORP2 as a new regulatory nexus of Akt signaling, cellular energy metabolism, actin cytoskeletal function, cell migration, and proliferation.

The Sec1/Munc18 Protein Groove Plays a Conserved Role in Interaction with Sec9p/SNAP-25.

The Sec1/Munc18 (SM) proteins constitute a conserved family with essential functions in SNARE-mediated membrane fusion. Recently, a new protein-protein interaction site in Sec1p, designated the groove, was proposed. Here, we show that a sec1 groove mutant yeast strain, sec1(w24), displays temperature-sensitive growth and secretion defects. The yeast Sec1p and mammalian Munc18-1 grooves were shown to play an important role in the interaction with the SNAREs Sec9p and SNAP-25b, respectively. Incubation of SNAP-25b with the Munc18-1 groove mutant resulted in a lag in the kinetics of SNARE complex assembly in vitro when compared with wild-type Munc18-1. The SNARE regulator SRO7 was identified as a multicopy suppressor of sec1(w24) groove mutant and an intact Sec1p groove was required for the plasma membrane targeting of Sro7p-SNARE complexes. Simultaneous inactivation of Sec1p groove and SRO7 resulted in reduced levels of exocytic SNARE complexes. Our results identify the groove as a conserved interaction surface in SM proteins. The results indicate that this structural element is important for interactions with Sec9p/SNAP-25 and participates, in concert with Sro7p, in the initial steps of SNARE complex assembly.

ORP4L Facilitates Macrophage Survival via G-Protein-Coupled Signaling: ORP4L-/- Mice Display a Reduction of Atherosclerosis.

RATIONALE: Macrophage survival within the arterial wall is a central factor contributing to atherogenesis. Oxysterols, major components of oxidized low-density lipoprotein, exert cytotoxic effects on macrophages. OBJECTIVE: To determine whether oxysterol-binding protein-related protein 4 L (ORP4L), an oxysterol-binding protein, affects macrophage survival and the pathogenesis of atherosclerosis. METHODS AND RESULTS: By hiring cell biological approaches and ORP4L-/- mice, we show that ORP4L coexpresses with and forms a complex with Gαq/11 and phospholipase C (PLC)-ß3 in macrophages. ORP4L facilitates G-protein-coupled ligand-induced PLCß3 activation, IP3 production, and Ca2+ release from the endoplasmic reticulum. Through this mechanism, ORP4L sustains antiapoptotic Bcl-XL expression through Ca2+-mediated c-AMP responsive element binding protein transcriptional regulation and thus protects macrophages from apoptosis. Excessive stimulation with the oxysterol 25-hydroxycholesterol disassembles the ORP4L/Gαq/11/PLCß3 complexes, resulting in reduced PLCß3 activity, IP3 production, and Ca2+ release, as well as decreased Bcl-XL expression and increased apoptosis. Overexpression of ORP4L counteracts these oxysterol-induced defects. Mice lacking ORP4L exhibit increased apoptosis of macrophages in atherosclerotic lesions and a reduced lesion size. CONCLUSIONS: ORP4L is crucial for macrophage survival. It counteracts the cytotoxicity of oxysterols/oxidized low-density lipoprotein to protect macrophage from apoptosis, thus playing an important role in the development of atherosclerosis.

USF1 deficiency alleviates inflammation, enhances cholesterol efflux and prevents cholesterol accumulation in macrophages.

BACKGROUND: The focus of studies on high-density lipoproteins (HDL) has shifted from HDL-cholesterol (HDL-C) to HDL function. We recently demonstrated that low USF1 expression in mice and humans associates with high plasma HDL-C and low triglyceride levels, as well as protection against obesity, insulin resistance, and atherosclerosis. Here, we studied the impact of USF1 deficiency on HDL functional capacity and macrophage atherogenic functions, including inflammation, cholesterol efflux, and cholesterol accumulation. METHODS: We used a congenic Usf1 deficient mice in C57Bl/6JRccHsd background and blood samples were collected to isolate HDL for structural and functional studies. Lentiviral preparations containing the USF1 silencing shRNA expression vector were used to silence USF1 in human THP-1 and Huh-7 cells. Cholesterol efflux from acetyl-LDL loaded THP-1 macrophages was measured using HDL and plasma as acceptors. Gene expression analysis from USF1 silenced peritoneal macrophages was carried out using Affymetrix protocols. RESULTS: We show that Usf1 deficiency not only increases HDL-C levels in vivo, consistent with elevated ABCA1 protein expression in hepatic cell lines, but also improves the functional capacity of HDL particles. HDL particles derived from Usf1 deficient mice remove cholesterol more efficiently from macrophages, attributed to their higher contents of phospholipids. Furthermore, silencing of USF1 in macrophages enhanced the cholesterol efflux capacity of these cells. These findings are consistent with reduced inflammatory burden of USF1 deficient macrophages, manifested by reduced secretion of pro-inflammatory cytokines MCP-1 and IL-1ß and protection against inflammation-induced macrophage cholesterol accumulation in a cell-autonomous manner. CONCLUSIONS: Our findings identify USF1 as a novel factor regulating HDL functionality, showing that USF1 inactivation boosts cholesterol efflux, reduces macrophage inflammation and attenuates macrophage cholesterol accumulation, linking improved macrophage cholesterol metabolism and inflammatory pathways to the antiatherogenic function of USF1 deficiency.

MicroRNA-192* impairs adipocyte triglyceride storage.

We investigated the expression of miR-192* (miR-192-3p) in the visceral adipose tissue (VAT) of obese subjects and its function in cultured human adipocytes. This miRNA is a 3' arm derived from the same pre-miRNA as miR-192 (miR-192-5p) implicated in type 2 diabetes, liver disease and cancers, and is predicted to target key genes in lipid metabolism. In morbidly obese subjects undergoing bariatric surgery preceded by a very low calorie diet, miR-192* in VAT correlated negatively (r=-0.387; p=0.046) with serum triglyceride (TG) and positively with high-density lipoprotein (HDL) concentration (r=0.396; p=0.041). In a less obese patient cohort, the miRNA correlated negatively with the body mass index (r=-0.537; p=0.026). To characterize the function of miR-192*, we overexpressed it in cultured adipocytes and analyzed the expression of adipogenic differentiation markers as well as cellular TG content. Reduced TG and expression of the adipocyte marker proteins aP2 (adipocyte protein 2) and perilipin 1 were observed. The function of miR-192* was further investigated by transcriptomic profiling of adipocytes expressing this miRNA, revealing impacts on key lipogenic genes. A number of the mRNA alterations were validated by qPCR. Western analysis confirmed a marked reduction of the lipogenic enzyme SCD (stearoyl coenzyme A desaturase-1), the fatty aldehyde dehydrogenase ALDH3A2 (aldehyde dehydrogenase 3 family member A2) and the high-density lipoprotein receptor SCARB1 (scavenger receptor B, type I). SCD and ALDH3A2 were demonstrated to be direct targets of miR-192*. To conclude, the present data identify miR-192* as a novel controller of adipocyte differentiation and lipid homeostasis.