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Human T-cell leukemia virus 1 (HTLV-1) causes adult T-cell leukemia (ATL), but the mechanism underlying its initiation remains elusive. In this study, ORP4L was expressed in ATL cells but not in normal T-cells. ORP4L ablation completely blocked T-cell leukemogenesis induced by the HTLV-1 oncoprotein Tax in mice, whereas engineering ORP4L expression in T-cells resulted in T-cell leukemia in mice, suggesting the oncogenic properties and prerequisite of ORP4L promote the initiation of T-cell leukemogenesis. For molecular insight, we found that loss of miR-31 caused by HTLV-1 induced ORP4L expression in T-cells. ORP4L interacts with PI3Kδ to promote PI(3,4,5)P3 generation, contributing to AKT hyperactivation; NF-κB–dependent, p53 inactivation-induced pro-oncogene expression; and T-cell leukemogenesis. Consistently, ORP4L ablation eliminates human ATL cells in patient-derived xenograft ATL models. These results reveal a plausible mechanism of T-cell deterioration by HTLV-1 that can be therapeutically targeted.

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.

Leukemia stem cells (LSCs) propagate leukemia and are responsible for the high frequency of relapse of treated patients. The ability to target LSCs remains elusive, indicating a need to understand the underlying mechanism of LSC formation. Here, we report that miR-31-5p is reduced or undetectable in human LSCs compared to hematopoietic stem progenitor cells (HSPCs). Inhibition of miR-31-5p in HSPCs promotes the expression of its target gene FIH , encoding FIH [factor inhibiting hypoxia-inducing factor 1α (HIF-1α)], to suppress HIF-1α signaling. Increased FIH resulted in a switch from glycolysis to oxidative phosphorylation (OXPHOS) as the predominant mode of energy metabolism and increased the abundance of the oncometabolite fumarate. Increased fumarate promoted the conversion of HSPCs to LSCs and initiated myeloid leukemia-like disease in NOD-Prkdc scid IL2rg tm1 /Bcgen (B-NDG) mice. We further demonstrated that miR-31-5p inhibited long- and short-term hematopoietic stem cells with a high frequency of LSCs. In combination with the chemotherapeutic agent Ara-C (cytosine arabinoside), restoration of miR-31-5p using G7 poly (amidoamine) nanosized dendriplex encapsulating miR-31-5p eliminated LSCs and inhibited acute myeloid leukemia (AML) progression in patient-derived xenograft mouse models. These results demonstrated a mechanism of HSC malignant transformation through altered energy metabolism and provided a potential therapeutic strategy to treat patients with AML.

Oxysterol-binding protein–related protein (ORP) 4L acts as a scaffold protein assembling CD3-e, G-αq/11, and PLC-β3 into a complex at the plasma membrane that mediates inositol (1,4,5)-trisphosphat...

Hsp90 is a new promising target for cancer treatment. Many inhibitors have been discovered as therapeutic agents, and some have passed Phase I and II. However, no one is approved by FDA yet. Novel and druggable Hsp90 inhibitors are still demanding. Here, we report a new way to discover high potent Hsp90 inhibitors as antinasopharyngeal carcinoma agents through assembling fragments. With chemotyping analysis, we extract seven chemotypes from 3482 known Hsp90 inhibitors, screen 500 fragments that are compatible with the chemotypes, and confirm 15 anti-Hsp90 fragments. Click chemistry is employed to construct 172 molecules and synthesize 21 compounds among them. The best inhibitor 3d was further optimized and resulted in more potent 4f (IC50 = 0.16 μM). In vitro and in vivo experiments confirmed that 4f is a promising agent against nasopharyngeal carcinoma. This study may provide a strategy in discovering new ligands against targets without well-understood structures.

https://doi.org/10.1096/fj.202000202R 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.

Summary: Leukemia stem cells (LSCs) are a rare subpopulation of abnormal hematopoietic stem cells (HSCs) that propagates leukemia and are responsible for the high frequency of relapse in therapies. Detailed insights into LSCs’ survival will facilitate the identification of targets for therapeutic approaches. Here, we develop an inhibitor, LYZ-81, which targets ORP4L with high affinity and specificity and selectively eradicates LCSs in vitro and in vivo. ORP4L is expressed in LSCs but not in normal HSCs and is essential for LSC bioenergetics and survival. It extracts PIP2 from the plasma membrane and presents it to PLCβ3, enabling IP3 generation and subsequent Ca2+-dependent bioenergetics. LYZ-81 binds ORP4L competitively with PIP2 and blocks PIP2 hydrolysis, resulting in defective Ca2+ signaling. The results provide evidence that LSCs can be eradicated through the inhibition of ORP4L by LYZ-81, which may serve as a starting point of drug development for the elimination of LSCs to eventually cure leukemia. : Zhong et al. report that abnormal expression of ORP4L is essential for leukemia stem cell survival; it enables IP3 generation by extracting and presenting PIP2 from the plasma membrane to PLCβ3 for hydrolysis. The compound LYZ-81, which blocks this process via targeting ORP4L, selectively eradicates leukemia stem cells. Keywords: leukemia stem cell, OSBP-related protein 4L, PIP2 hydrolysis, Ca2+ signaling, energy metabolism, therapy

Oxysterol-binding protein related-protein 5 and 8 (ORP5/8) localize to the membrane contact sites (MCS) of the endoplasmic reticulum (ER) and the mitochondria, as well as to the ER-plasma membrane (PM) MCS. The MCS are emerging as important regulators of cell signaling events, including calcium (Ca2+) signaling. ORP5/8 have been shown to interact with phosphatidylinositol-4,5-bisphosphate (PIP2) in the PM, and to modulate mitochondrial respiration and morphology. PIP2 is the direct precursor of inositol trisphosphate (IP3), a key second messenger responsible for Ca2+-release from the intracellular Ca2+ stores. Further, mitochondrial respiration is linked to Ca2+ transfer from the ER to the mitochondria. Hence, we asked whether ORP5/8 would affect Ca2+ signaling in these cell compartments, and employed genetically engineered aequorin Ca2+ probes to investigate the effect of ORP5/8 in the regulation of mitochondrial and caveolar Ca2+. Our results show that ORP5/8 overexpression leads to increased mitochondrial matrix Ca2+ as well as to increased Ca2+ concentration at the caveolar subdomains of the PM during histamine stimulation, while having no effect on the cytoplasmic Ca2+. Also, we found that ORP5/8 overexpression increases cell proliferation. Our results show that ORP5/8 regulate Ca2+ signaling at specific MCS foci. These local ORP5/8-mediated Ca2+ signaling events are likely to play roles in processes such as mitochondrial respiration and cell proliferation.

Immunochromatographic strips (ICSs) are inexpensive, simple, portable, and robust, and therefore have many uses in the medicinal, agricultural, and environmental industries. For detection of small molecules, current ICSs are competitive format (competitive ICSs, CICSs), which only offer a turn-off readout mode, and therefore lead to low sensitivity when evaluating results by the naked eye. To overcome this problem, we report a turn-on CICSs that relies on the ability of gold nano-stars (AuNSs) quenching the signal of quantum dots (QDs). This turn-on CICSs device was applied to detect cadmium ions (Cd2+). The linear detection range (LDR) of the turn-on CICSs was 0.25ng/mL-8ng/mL, and the detection of limit (LOD) was 0.18ng/mL. Compared with traditional turn-off CICSs, the sensitivity of the turn-on CICSs was enhanced by 32 times. The turn-on CICSs also has a high specificity and high recovery for the detection of Cd2+ in Pearl River (95-112%) and tap water samples (103.5-116.67%). Therefore, we believe the turn-on CICSs offers great potential for the detection of other small molecules in clinical diagnostics, food safety investigations, and environment pollution monitoring.

Oxysterol-binding protein-related protein (ORP) 4L acts as a scaffold protein assembling CD3-ε, G-α

Phospholipase C (PLC) isoforms play central roles in signaling cascades by cleaving PIP

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 Gα q/11 and phospholipase C (PLC)-β3 in macrophages. ORP4L facilitates G-protein–coupled ligand-induced PLCβ3 activation, IP 3 production, and Ca 2+ release from the endoplasmic reticulum. Through this mechanism, ORP4L sustains antiapoptotic Bcl-XL expression through Ca 2+ -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/Gα q/11 /PLCβ3 complexes, resulting in reduced PLCβ3 activity, IP 3 production, and Ca 2+ 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.

Oxysterols are 27-carbon oxidized derivatives of cholesterol or by-products of cholesterol biosynthesis that can induce cell apoptosis in addition to a number of other bioactions. However, the mechanisms underlying this cytotoxicity are not completely understood. ORP8 is a member of the oxysterol binding protein-related protein (ORP) family, implicated in cellular lipid homeostasis, migration, and organization of the microtubule cytoskeleton. Here, we report that 25-hydroxycholesterol (OHC) induced apoptosis of the hepatoma cell lines, HepG2 and Huh7, via the endoplasmic reticulum (ER) stress response pathway, and ORP8 overexpression resulted in a similar cell response as 25-OHC, indicating a putative functional relationship between oxysterol cytotoxicity and ORP8. Further experiments demonstrated that ORP8 overexpression significantly enhanced the 25-OHC effect on ER stress and apoptosis in HepG2 cells. A truncated ORP8 construct lacking the ligand-binding domain or a closely related protein, ORP5, was devoid of this activity, evidencing for specificity of the observed effects. Importantly, ORP8 knockdown markedly dampened such responses to 25-OHC. Taken together, the present study suggests that ORP8 may mediate the cytotoxicity of 25-OHC.

Metabolic pathways are reprogrammed in cancer to support cell survival. Here, we report that T-cell acute lymphoblastic leukemia (T-ALL) cells are characterized by increased oxidative phosphorylation and robust ATP production. We demonstrate that ORP4L is expressed in T-ALL but not normal T-cells and its abundance is proportional to cellular ATP. ORP4L acts as an adaptor/scaffold assembling CD3ɛ, Gαq/11 and PLCβ3 into a complex that activates PLCβ3. PLCβ3 catalyzes IP3 production in T-ALL as opposed to PLCγ1 in normal T-cells. Up-regulation of ORP4L thus results in a switch in the enzyme responsible for IP3-induced endoplasmic reticulum Ca2+ release and oxidative phosphorylation. ORP4L knockdown results in suboptimal bioenergetics, cell death and abrogation of T-ALL engraftment in vivo. In summary, we uncovered a signalling pathway operating specifically in T-ALL cells in which ORP4L mediates G protein-coupled ligand-induced PLCβ3 activation, resulting in an increase of mitochondrial respiration for cell survival. Targeting ORP4L might represent a promising approach for T-ALL treatment., Lymphocytic leukaemia cells are characterized by high respiratory rates. Here, the authors report that the oxysterol-binding protein ORPL4 sustains mitochondrial respiration in T-cell acute lymphoblastic leukaemia cells by regulating Ca2+ release from the endoplasmic reticulum.

// Ji-Wei Li 1, 2, * , Yan-Lin Xiao 1, 2, * , Chao-Feng Lai 1, 2, * , Ning Lou 3 , Hong-Ling Ma 1, 2 , Bi-Ying Zhu 1, 2 , Wen-Bin Zhong 1, 2 , Dao-Guang Yan 1, 2 1 Department of Biotechnology, Jinan University, Guangzhou, 510632, China 2 The Key Laboratory of Functional Protein Research of Guangdong Higher Education Institutes, Jinan University, Guangzhou, 510632, China 3 State Key Laboratory of Oncology in Southern China, Collaborative Innovation Center of Cancer Medicine, Guangzhou, 510060, China * These authors have contributed equally to this work Correspondence to: Dao-Guang Yan, email: tydg@jnu.edu.cn Wen-Bin Zhong, email: winbeyz@163.com Keywords: ORP4L, Ca 2+ homeostasis, NFAT, IP 3 R1, proliferation Received: February 22, 2016 Accepted: August 09, 2016 Published: August 29, 2016 ABSTRACT Oxsterol binding protein-related protein 4 (ORP4) is essential for cell proliferation, but the underlying mechanism is unclear. ORP4 is expressed as three variants, ORP4L, ORP4M and ORP4S. Here, we reported that silencing of ORP4L with specific small interfering RNA (siRNA) inhibited the proliferation of human cervical cancer cell lines C33A, HeLa and CaSki, the reverse effect being observed in ORP4L overexpressing cells. For molecular insight, we found that ORP4L maintained intracellular Ca 2+ homeostasis. Through this mechanism, ORP4L activated nuclear factor of activated T cells (NFAT) activity and thus promoted expression of a gene cluster which supported cell proliferation. Of note, ORP4L sustained inositol-1,4,5-trisphosphate receptor 1 (IP 3 R1) expression at both mRNA and protein levels via Ca 2+ -dependent NFAT3 activation, which offered a mechanic explanation for the role of ORP4L intracellular Ca 2+ homeostasis. Furthermore, ORP4L knockdown markedly inhibited tumor growth in a C33A cell xenograft mouse model. To conclude, our results reveal that ORP4L promotes cell proliferation through maintaining intracellular Ca 2+ homeostasis.

Leukemia stem cells (LSCs) are a rare subpopulation of abnormal hematopoietic stem cells (HSCs) that propagates leukemia and are responsible for the high frequency of relapse in therapies. Detailed insights into LSCs' survival will facilitate the identification of targets for therapeutic approaches. Here, we develop an inhibitor, LYZ-81, which targets ORP4L with high affinity and specificity and selectively eradicates LCSs in vitro and in vivo. ORP4L is expressed in LSCs but not in normal HSCs and is essential for LSC bioenergetics and survival. It extracts PIP

The enhanced expression of miR-31 has been observed in many human malignancies including lung cancer, and this microRNA regulates several aspects of oncogenesis. However, the role of miR-31-5p in energy metabolism remains elusive. Here, we confirm that H1299 and A549 cells, 2 lung cancer cell lines, relay on aerobic glycolysis as main source of ATP. Inhibition of miR-31-5p leads to decreased glycolysis and ATP production, while miR-31-5p overexpression increases them. Hypoxia inducible factor 1 (HIF-1) up-regulates the expression of glycolytic enzymes, and the HIF-1α inhibitor (FIH) inhibits HIF-1 activity. Because FIH is a direct target of miR-31-5p, inhibition of miR-31-5p results in enhanced FIH expression and suppression of HIF-1 signaling, while overexpression of miR-31-5p has the opposite effects. Via this mechanism, miR-31-5p up-regulates aerobic glycolytic genes and maintains energy homeostasis. To further validate the mechanism of miR-31-5p in glycolysis regulation, we show that overexpression or knockdown of FIH rescued the effects of miR-31-5p or miR-31-5p inhibitor on HIF activation and its target gene expression, respectively. Finally, by means of an A549 cell xenograft mouse model, we demonstrate that the miR-31-5p promotes cell proliferation via enhancing glycolysis. In summary, this study reveals that miR-31-5p promotes the Warburg effect via direct targeting of FIH.-Zhu, B., Cao, X., Zhang, W., Pan, G., Yi, Q., Zhong, W., Yan, D. MicroRNA-31-5p enhances the Warburg effect via targeting FIH.

Human hepatoma (HCC) has been reported to be strongly resistant to Fas-mediated apoptosis. However, the underlying mechanisms are poorly understood. In this study the function of oxysterol-binding protein-related protein 8 (ORP8) in human hepatoma cells apoptosis was assessed. We found that ORP8 is down-regulated, whereas miR-143, which controls ORP8 expression, is up-regulated in clinical HCC tissues as compared with liver tissue from healthy subjects. ORP8 overexpression triggered apoptosis in primary HCC cells and cell lines, which coincided with a relocation of cytoplasmic Fas to the cell plasma membrane and FasL up-regulation. Co-culture of HepG2 cells or primary HCC cells with Jurkat T-cells or T-cells, respectively, provided further evidence that ORP8 increases HCC cell sensitivity to Fas-mediated apoptosis. ORP8-induced Fas translocation is p53-dependent, and FasL was induced upon ORP8 overexpression via the endoplasmic reticulum stress response. Moreover, ORP8 overexpression and miR-143 inhibition markedly inhibited tumor growth in a HepG2 cell xenograft model. These results indicate that ORP8 induces HCC cell apoptosis through the Fas/FasL pathway. The role of ORP8 in Fas translocation to the plasma membrane and its down-regulation by miR-143 offer a putative mechanistic explanation for HCC resistance to apoptosis. ORP8 may be a potential target for HCC therapy.

It is well established that diabetes mellitus accelerates atherosclerotic vascular disease. Endothelial injury has been proposed to be the initial event in the pathogenesis of atherosclerosis. Endothelium not only acts as a semi-selective barrier but also serves physiological and metabolic functions. Diabetes or high glucose in circulation triggers a series of intracellular responses and organ damage such as endothelial dysfunction and apoptosis. One such response is high glucose-induced chronic endoplasmic reticulum stress in the endothelium. The unfolded protein response is an acute reaction that enables cells to overcome endoplasmic reticulum stress. However, when chronically persistent, endoplasmic reticulum stress response could ultimately lead to endothelial dysfunction and atherosclerosis. Herein, we discuss the scientific advances in understanding endoplasmic reticulum stress-induced endothelial dysfunction, the pathogenesis of diabetes-accelerated atherosclerosis and endoplasmic reticulum stress as a potential target in therapies for diabetic atherosclerosis.

ORP8 is an oxysterol/cholesterol binding protein anchored to the endoplasmic reticulum and the nuclear envelope, and is abundantly expressed in the macrophage. We created and characterized mouse RAW264.7 macrophages with ORP8 stably silenced using shRNA lentiviruses. A microarray transcriptome and gene ontology pathway analysis revealed significant alterations in several nuclear pathways and ones associated with centrosome and microtubule organization. ORP8 knockdown resulted in increased expression and altered subcellular distribution of an interaction partner of ORP8, nucleoporin NUP62, with an intranuclear localization aspect and association with cytoplasmic vesicular structures and lamellipodial edges of the cells. Moreover, ORP8 silenced cells displayed enhanced migration, and a more pronounced microtubule cytoskeleton than controls expressing a non-targeting shRNA. ORP8 was shown to compete with Exo70 for interaction with NUP62, and NUP62 knockdown abolished the migration enhancement of ORP8-silenced cells, suggesting that the endogenous ORP8 suppresses migration via binding to NUP62. As a conclusion, the present study reveals new, unexpected aspects of ORP8 function in macrophages not directly involving lipid metabolism, but rather associated with nuclear functions, microtubule organization, and migration capacity.

Cytoplasmic oxysterol-binding protein homologues, designated OSBP-like (OSBPL) or OSBP-related (ORP) proteins, constitute a family of lipid binding/transfer proteins present in a wide spectrum of eukaryotes. In mammals these proteins are encoded by 12 genes expressed ubiquitously but at different quantities in different tissue and cell types. Their unifying feature is a β-barrel-like ligand-binding domain that has been shown to accommodate sterol ligands: oxysterols, cholesterol, or both, while a recent report shows that also phospholipid can be bound. Certain family members have the capacity to mediate or facilitate intracellular sterol transport or lipid exchange, while others are implicated as sterol sensors involved in cell regulation: lipid homeostatic control, signal transduction, organelle motility, or vesicle transport. Several ORPs have been localized to membrane contact sites, at which endoplasmic reticulum membranes are closely apposed with other organelles—indicating functions in the non-vesicular transport of ions or small molecules, or in regulation of enzymatic components active at the contact sites. OSBPL gene polymorphisms are associated with cardiovascular risk factors, and altered ORP expression levels occur in certain cancers, providing the first indications of ORP involvement in human disease. In this review we summarize and analyze the latest findings concerning the function of OSBPL/ORP proteins.

ORP7 is a member of oxysterol-binding protein (OSBP) family, the function of which has remained obscure. In this study, we identified by yeast two-hybrid screening an interaction partner of ORP7, GATE-16, which (i) regulates Golgi SNARE of 28 kDa (GS28) function and stability, and (ii) plays a role in autophagosome biogenesis. The interaction was confirmed by bimolecular fluorescence complementation (BiFC) assay in living cells. The interacting regions were delineated within aa 1–142 of ORP7 and aa 30–117 of GATE-16. ORP7 knock-down in 293A cells resulted in a 40% increase of GS28 protein while ORP7 overexpression had the opposite effect (25% decrease of GS28). We show evidence that the regulation of GS28 by ORP7 does not occur at the level of transcription, but involves degradation of GS28 on proteasomes. Truncated ORP7 that lacks the GATE-16 binding region failed to affect GS28 stability, evidencing for specificity of the observed effect. Similar to ORP7 overexpression, treatment of cells with 25-hydroxycholesterol (25-OH) resulted in GS28 destabilization, which was potentiated by excess ORP7 and inhibited by ORP7 silencing. Overexpression of ORP7 led in most cells to formation of vacuolar structures positive for RFP-LC3, thus representing autophagic elements. Also GATE-16 was found in the vacuolar ORP7-positive elements, suggesting that excess ORP7 increases entrapment of GATE-16 in autophagosomes. Taken together, our results suggest that ORP7 negatively regulates GS28 protein stability via sequestration of GATE-16, and may mediate the effect of 25-OH on GS28 and Golgi function.

Rational : AMP-activated protein kinase (AMPK) is an energy sensor and ubiquitously expressed in vascular cells. Recent studies suggest that AMPK activation improves endothelial function by counteracting oxidative stress in endothelial cells. How AMPK suppresses oxidative stress remains to be established. Objective : The aim of this study is to examine the effects of AMPK in regulating NAD(P)H oxidase, oxidative stress, and endothelial function. Methods and Results : The markers of oxidative stress, NAD(P)H oxidase subunit expression (gp91 phox , p47 phox , p67 phox , NOX1 to -4), NAD(P)H oxidase–mediated superoxide production, 26S proteasome activity, IκBα degradation, and nuclear translocation of nuclear factor (NF)-κB (p50 and p65) were examined in cultured human umbilical vein endothelial cells and mouse aortas isolated from AMPKα2 deficient mice. Compared to the wild type, acetylcholine-induced endothelium-dependent relaxation was significantly impaired in parallel with increased production of oxidants in AMPKα2 −/− mice. Further, pretreatment of aorta with either superoxide dismutase (SOD) or tempol or apocynin significantly improved acetylcholine-induced endothelium-dependent relaxation in AMPKα2 −/− mice. Analysis of aortic endothelial cells from AMPKα2 −/− mice and human umbilical vein endothelial cells expressing dominant negative AMPK or AMPKα2-specific siRNA revealed that loss of AMPK activity increased NAD(P)H oxidase subunit expression (gp91 phox , p47 phox , p67 phox , NOX1 and -4), NAD(P)H oxidase–mediated superoxide production, 26S proteasome activity, IκBα degradation, and nuclear translocation of NF-κB (p50 and p65), whereas AMPK activation by AICAR or overexpression of constitutively active AMPK had the opposite effect. Consistently, we found that genetic deletion of AMPKα2 in low-density lipoprotein receptor knockout (LDLr −/− ) strain markedly increased 26S proteasome activity, IκB degradation, NF-κB transactivation, NAD(P)H oxidase subunit overexpression, oxidative stress, and endothelial dysfunction, all of which were largely suppressed by chronic administration of MG132, a potent cell permeable proteasome inhibitor. Conclusions : We conclude that AMPKα2 functions as a physiological suppressor of NAD(P)H oxidase and ROS production in endothelial cells. In this way, AMPK maintains the nonatherogenic and noninflammatory phenotype of endothelial cells.

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 HDL(2). 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.

Protein families characterized by a ligand-binding domain homologous to that of oxysterol binding protein (OSBP) are present in eukaryotes ranging from yeast to man. The OSBP-related proteins (ORP) have been implicated in the regulation of various cellular functions. However, their exact mechanisms of action remain elusive, and it is evident that any single model for their function is insufficient. Recent data from our, and other laboratories, suggest that a unifying theme may be the binding of sterol ligands. Work with the yeast Saccharomyces cerevisiae suggests a function for ORPs in the nonvesicular transport of sterols. It is unclear whether this could also apply to mammals having a larger number of ORP genes that encode a variety of structurally complex ORP proteins. The existing evidence on mammalian ORP function points in many different directions, such as integration of sterol and sphingomyelin metabolism, regulation of neutral lipid metabolism, control of signaling cascades and regulation of the ...

Objective— Plasma phospholipid transfer protein (PLTP) is involved in the metabolism of HDL and apolipoprotein B (apoB)-containing lipoproteins. Atherosclerosis susceptibility is decreased in mice with PLTP deficiency that is associated with decreased liver production of apoB-containing lipoproteins and increase in their antioxidant. To investigate additionally the effect of PLTP on the development of atherosclerosis, we overexpressed PLTP in mice. Methods and Results— PLTP was overexpressed in apoE knockout mice using an adenovirus-associated virus (AAV)-mediated system. Plasma PLTP activity was 1.3- to 2-fold higher in mice injected with AAV-PLTP than in mice injected with control AAV-GFP, and PLTP levels were sustained during the experiment period (4 months). We show that 2-fold increased PLTP activity results in (1) a decrease in HDL cholesterol, HDL phospholipid, and apoAI levels; (2) a decrease in vitamin E contents in total plasma and in individual lipoprotein fractions; (3) an increase in lipoprotein oxidizability as assessed by copper-induced formation of conjugated dienes; (4) an increase in autoantibodies against oxidized apoB-containing particles; and (5) an increase in atherosclerosis lesions in proximal aorta. Conclusions— These observations indicate that elevated plasma PLTP levels constitute a novel, long-term risk factor for atherosclerosis.

Visualization of protein-protein interactions in vivo offers a powerful tool to resolve spatial and temporal aspects of cellular functions. The bimolecular fluorescence complementation (BiFC) makes use of nonfluorescent fragments of green fluorescent protein or its variants that are added as "tags" to target proteins under study. Only upon target protein interaction is a fluorescent protein complex assembled, and the site of interaction can be monitored by microscopy. In this chapter, we describe the method and tools for the use of BiFC in the yeast Saccharomyces cerevisiae and in mammalian cells.

Visualization of protein-protein interactions in vivo offers a powerful tool to resolve spatial and temporal aspects of cellular functions. The bimolecular fluorescence complementation (BiFC) makes use of nonfluorescent fragments of green fluorescent protein or its variants that are added as "tags" to target proteins under study. Only upon target protein interaction is a fluorescent protein complex assembled, and the site of interaction can be monitored by microscopy. In this chapter, we describe the method and tools for the use of BiFC in the yeast Saccharomyces cerevisiae and in mammalian cells.

Background Nuclear receptor Rev‐erbα plays important roles in circadian clock timing, lipid metabolism, adipogenesis, and vascular inflammation. However, the role of Rev‐erbα in atherosclerotic lesion development has not been assessed in vivo. Methods and Results The nuclear receptor Rev‐erbα was knocked down in mouse haematopoietic cells by means of sh RNA ‐lentiviral transduction, followed by bone marrow transplantation into LDL receptor knockout mice. The Rev‐erbα protein in peripheral macrophage was reduced by 70% as compared to control mice injected with nontargeting sh RNA lentivirus‐transduced bone marrow. A significant increase in atherosclerotic lesions was observed around the aorta valves as well as upon en face aorta analysis of Rev‐erbα knock‐down bone marrow recipients ( P Conclusions These observations identify hematopoietic cell Rev‐erbα as a new modulator of atherogenesis in mice.

Acute myelogenous leukemia (AML) is one of the deadliest hematological malignancies and there is at present no efficient strategy to defeat it. New detailed insight into AML leukemia stem cells (LSCs) survival will facilitate the identification of targets for the development of new therapeutic approaches. Recent work has provided evidence that LSCs are defective in their ability to employ glycolysis, but are highly reliant on oxidative phosphorylation, and the maintenance of mitochondrial function is essential for LSCs survival. It is increasingly clear that Ca2+ released constitutively from endoplasmic reticulum (ER) is taken up by mitochondria to sustain optimal bioenergetics and cell survival. Here we report three striking findings: 1) oxysterol-binding protein (OSBP)-related protein 4 (ORP4L) is expressed in LSCs but not in normal hematopoietic stem cells (HSCs). 2) ORP4L is essential for LSC bioenergetics; It forms a complex with PLCβ3 and IP3 receptor 1 (ITPR1) to control Ca2+ release from the ER and subsequent cytosolic and mitochondrial parallel Ca2+ spike oscillations that sustain pyruvate dehydrogenase (PDH) activation and oxidative phosphorylation. 3) ORP4L inhibition eradicates LSCs in vitro and in vivo through impairment of Ca2+-dependent bioenergetics. These results suggest a novel role of ORP4L in governing Ca2+ release to sustain mitochondrial function and survival of LSCs and identify ORP4L as a putative new oncoprotein and therapeutic target for LSCs elimination. Disclosures No relevant conflicts of interest to declare.

Oxysterol-binding (OSBP)-related proteins (ORPs) constitute a family of sterol and phosphoinositide binding/transfer proteins in eukaryotes from yeast to man. While their functions have mainly been addressed in cellular lipid metabolism or sterol transport, increasing evidence points to more versatile regulatory roles in a spectrum of cellular regimes. In fact ORPs do not appear to be robust controllers of lipid homeostasis. Several ORPs localize at membrane contacts sites (MCS), where endoplasmic reticulum (ER) is apposed with other organelle limiting membranes. Apparently, ORPs have the capacity to control the formation of MCS or activity of enzymatic machineries at these sites. Thereby, ORPs most likely affect organelle membrane lipid compositions, with impacts on signaling and vesicle transport, but also cellular lipid metabolism. Moreover, an increasing number of protein interaction partners of ORPs have been identified, connecting these proteins with various aspects of cell regulation. Small molecular anti-proliferative compounds, ORPphilins, were recently found to target two members of the ORP family, OSBP and ORP4, revealing an essential function of ORPs in cancer cell proliferation and survival. Further functions assigned for ORPs include regulation of extracellular signal regulated kinase (ERK) activity (OSBP), control of ER-late endosome MCS and late endosome motility (ORP1L), regulation of β1-integrin activity (ORP3), modulation of hepatocyte insulin signaling and macrophage migration (ORP8), as well as post-Golgi vesicle transport, phosphatidylinositol-4-phosphate and target of rapamycin complex 1 signaling and nitrogen sensing (Saccharomyces cerevisiae Osh4p). These and other recent observations shed light on the ORPs as integrators of lipid signals with an unforeseen variety of vital cellular processes.

We characterize here ORP11, a member of the oxysterol-binding protein family. ORP11 is present at highest levels in human ovary, testis, kidney, liver, stomach, brain, and adipose tissue. Immunohistochemistry demonstrates abundant ORP11 in the epithelial cells of kidney tubules, testicular tubules, caecum, and skin. ORP11 in HEK293 cells resides on Golgi complex and LE, co-localizing with GFP-Rab9, TGN46, GFP-Rab7, and a fluorescent medial-trans-Golgi marker. Under electron microscopic observation, cells overexpressing ORP11 displayed lamellar lipid bodies associated with vacuolar structures or the Golgi complex, indicating a disturbance of lipid trafficking. N-terminal fragment of ORP11 (aa 1-292) localized partially to Golgi, but displayed enhanced localization on Rab7- and Rab9-positive LE, while the C-terminal ligand-binding domain (aa 273-747) was cytosolic, demonstrating that the membrane targeting determinants are N-terminal. Yeast two-hybrid screen revealed interaction of ORP11 with the related ORP9. The interacting region was delineated within aa 98-372 of ORP9 and aa 154-292 of ORP11. Overexpressed ORP9 was able to recruit EGFP-ORP11 to membranes, and ORP9 silencing inhibited ORP11 Golgi association. The results identify ORP11 as an OSBP homologue distributing at the Golgi-LE interface and define the ORP9-ORP11 dimer as a functional unit that may act as an intracellular lipid sensor or transporter.

Protein families characterized by a ligand binding domain related to that of oxysterol binding protein (OSBP) have been identified in eukaryotic species from yeast to humans. These proteins, designated OSBP-related (ORP) or OSBP-like (OSBPL) proteins, have been implicated in various cellular functions. However, the detailed mechanisms of their action have remained elusive. Data from our and other laboratories suggest that binding of sterol ligands may be a unifying theme. Work with Saccharomyces cerevisiae ORPs suggests a function of these proteins in the nonvesicular intracellular transport of sterols, in secretory vesicle transport from the Golgi complex, and in the establishment of cell polarity. Mammals have more ORP genes, and differential splicing substantially increases the complexity of the encoded protein family. Functional studies on mammalian ORPs point in different directions: integration of sterol and sphingomyelin metabolism, sterol transport, regulation of neutral lipid metabolism, control of the microtubule-dependent motility of endosomes/lysosomes, and regulation of signaling cascades. We envision that during evolution, the functions of ORPs have diverged from an ancestral one in sterol transport, to meet the increasing demand of the regulatory potential in multicellular organisms. Our working hypothesis is that mammalian ORPs mainly act as sterol sensors that relay information to a spectrum of different cellular processes.

Protein families characterized by a ligand binding domain related to that of oxysterol binding protein (OSBP) have been identified in eukaryotic species from yeast to humans. These proteins, designated OSBP-related (ORP) or OSBP-like (OSBPL) proteins, have been implicated in various cellular functions. However, the detailed mechanisms of their action have remained elusive. Data from our and other laboratories suggest that binding of sterol ligands may be a unifying theme. Work with Saccharomyces cerevisiae ORPs suggests a function of these proteins in the nonvesicular intracellular transport of sterols, in secretory vesicle transport from the Golgi complex, and in the establishment of cell polarity. Mammals have more ORP genes, and differential splicing substantially increases the complexity of the encoded protein family. Functional studies on mammalian ORPs point in different directions: integration of sterol and sphingomyelin metabolism, sterol transport, regulation of neutral lipid metabolism, control of the microtubule-dependent motility of endosomes/lysosomes, and regulation of signaling cascades. We envision that during evolution, the functions of ORPs have diverged from an ancestral one in sterol transport, to meet the increasing demand of the regulatory potential in multicellular organisms. Our working hypothesis is that mammalian ORPs mainly act as sterol sensors that relay information to a spectrum of different cellular processes.

Objective— The purpose of this study was to assess the role of macrophage OSBP-related protein 1L (ORP1L) in the development of atherosclerosis. Methods and Results— C57BL/6 mice overexpressing human ORP1L in macrophages driven by scavenger receptor A promoter were generated. Bone marrow (BM) of the mice was transplanted into LDLr −/− animals, and aortic root lesion area in the recipients was determined after Western-type diet feeding. The recipients of ORP1L BM displayed 2.1-fold increase ( P ORP1L BM recipients showed a decrease in ABCG1 and APOE mRNAs and proteins, and an increase in PLTP message; also the plasma PLTP activity was elevated. The effect of ORP1L on cholesterol efflux was assessed using macrophages loaded with [ 3 H]cholesterol oleate-acLDL or labeled with [ 3 H]cholesterol. The ORP1L transgenic macrophages displayed 30% reduction ( P 2 , but not to apoA-I. ORP1L was shown to bind 25- and 22(R)-hydroxycholesterol, identifying it as an oxysterol binding protein. Furthermore, ORP1L attenuated the response of ABCG1 mRNA to 22(R)-hydroxycholesterol, the effect on ABCA1 being less pronounced. Conclusions— The results demonstrate that macrophage ORP1L can act as a modulator of atherosclerotic lesion development and provide clues to the underlying mechanism.

Background— Oxysterol binding protein (OSBP) has previously been implicated as a sterol sensor that regulates sphingomyelin synthesis and the activity of extracellular signal-regulated kinases (ERK). Methods and Results— We determined the effects of adenovirus-mediated hepatic overexpression of OSBP and its homologues ORP1L and ORP3 on mouse serum lipids. Whereas ORP1L and ORP3 had no effect on serum lipids, OSBP induced a marked increase of VLDL triglycerides (TG). Also, the liver tissue TG were elevated in the AdOSBP-injected mice, and their TG secretion rate was increased by 70%. The messenger RNAs for enzymes of fatty acid synthesis and their transcriptional regulator, SREBP-1c, as well as the Insig-1 mRNA, were upregulated two-fold in the OSBP-expressing livers. No change occurred in the messages of liver X receptor target genes ABCA1 , ABCG5 , and CYP7A1 , and the Insig-2a mRNA was reduced. The phosphorylation of ERK was decreased in AdOSBP-infected liver and cultured hepatocytes. Importantly, silencing of OSBP in hepatocytes suppressed the induction of SREBP1-c by insulin and resulted in a reduction of TG synthesis. Conclusion— Our results demonstrate that OSBP regulates hepatic TG metabolism and suggest the involvement of OSBP in the insulin signaling pathways that control hepatic lipogenesis.

Acute myeloid leukemia (AML) is a heterogeneity disease initiating from a rare population of cells known as leukemia stem cells (LSCs), which have been a major hurdle for the success of acute myeloid leukemia chemotherapy. Therefore, new drugs targeting LSCs is urgently needed. Disulfiram (DS) has been used clinically as a safe anti-alcoholism drug for over 6 decades. Recent studies demonstrated that disulfiram combined with cooper (DS/Cu) have the antitumor activity in a wide range of cancer cell lines. CD34+ CD38- KG1a cells were previous found to have the characteristics of stem cells. Here, we explored whether DS/Cu could induce acute myeloid leukemic stem-like KG1a cells apoptosis and further investigated the molecular mechanism. CD34+ CD38- KG1a cells were sorted from KG1a cell lines by magnetic activated cell sorting (MACS). Flow cytometry (FCM) analysis confirmed the percentage of CD34+ CD38- KG1a cells was 95.37±1.84%. To determine the effect of DS/Cu induced apoptosis in LSC-like KG1a cells, CD34+ CD38- KG1a cells were exposed to DS (5nM) with or without Cu (0.5nM) for 24h. FCM analysis showed the apoptotic proportion of CD34+ CD38- KG1α cells exposed to DS was 11.87±1.30%, while 27.43±1.65% to DS/Cu (P=0.00). To explore the role of ROS in DS/Cu induced apoptosis, ROS levels were detected using DCFH-DA after KG1a cells being treated with DS and DS/Cu for 24h. DS and DS/Cu could induce ROS acumulation by 1.39±0.115 fold and 2.81±0.109 fold, respectively (P To explore the upstream pathway of ROS accumulation induced by DS and DS/Cu in CD34+CD38-KG1α cells, We examined the expression of apoptosis-related moleculars include TNF-a,CD40, TNFRSF11B, TNFRSF1B, Hrk in LSC-like KG1a cells exposed to DS, DS/ Cu and DS/Cu+NAC for 24h. DS and DS/Cu up-regulated TNF-a, CD40, TNFRSF11B, TNFRSF1B, Hrk expression at the mRNA level (P To further investigate the role of TNF-a in DS/Cu induced accumulation of ROS, CD34+ CD38- KG1α cells were pretreated with neutralizing antibody TNF-a mAb (2μg/mL) , control antibody IgG (2μg/mL) for 2h, then cells were exposed to DS/Cu for 24h. ROS accumulation induced by DS/Cu was inhibited by pretreatment with TNF-a mAb (2.78±0.25 vs. 1.28±0.17 folds, P=0.00), while pretreatment with IgG had no impact on ROS accumulation induced by DS/Cu (P=0.23). TNF-a mAb and IgG alone had no effect on ROS accumulation in KG1a cells ( P>0.05). These results confirmed that TNF-a is upstream molecular of ROS induced by DS/Cu in leukemia stem-like KG1a cells. In conclusion, our data demonstrated that DS/Cu could induce leukemia stem-like KG1a cells apoptosis, and the underlying mechanisms may be related with TNF-a/ROS pathway. Disclosures No relevant conflicts of interest to declare.