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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 (Ca 2+ ) signaling. ORP5/8 have been shown to interact with phosphatidylinositol-4,5-bisphosphate (PIP 2 ) in the PM, and to modulate mitochondrial respiration and morphology. PIP 2 is the direct precursor of inositol trisphosphate (IP 3 ), a key second messenger responsible for Ca 2+ -release from the intracellular Ca 2+ stores. Further, mitochondrial respiration is linked to Ca 2+ transfer from the ER to the mitochondria. Hence, we asked whether ORP5/8 would affect Ca 2+ signaling in these cell compartments, and employed genetically engineered aequorin Ca 2+ probes to investigate the effect of ORP5/8 in the regulation of mitochondrial and caveolar Ca 2+ . Our results show that ORP5/8 overexpression leads to increased mitochondrial matrix Ca 2+ as well as to increased Ca 2+ concentration at the caveolar subdomains of the PM during histamine stimulation, while having no effect on the cytoplasmic Ca 2+ . Also, we found that ORP5/8 overexpression increases cell proliferation. Our results show that ORP5/8 regulate Ca 2+ signaling at specific MCS foci. These local ORP5/8-mediated Ca 2+ signaling events are likely to play roles in processes such as mitochondrial respiration and cell proliferation. [ABSTRACT FROM AUTHOR]

Remarkably good results have been achieved in the treatment of atherosclerotic cardiovascular diseases (CVD) by using statin, ezetimibe, antihypertensive, antithrombotic, and PCSK9 inhibitor therapies and their proper combinations. However, despite this success, the remaining CVD risk is still high. To target this residual risk and to treat patients who are statin-intolerant or have an exceptionally high CVD risk for instance due to familial hypercholesterolemia (FH), new therapies are intensively sought. One pathway of drug development is targeting the circulating triglyceride-rich lipoproteins (TRL) and their lipolytic remnants, which, according to the current view, confer a major CVD risk. Angiopoietin-like protein 3 (ANGPTL3) and apolipoprotein C-III (apoC-III) are at present the central molecular targets for therapies designed to reduce TRL, and there are new drugs emerging that suppress their expression or inhibit the function of these two key proteins. The medications targeting these components are biological, either human monoclonal antibodies or antisense oligonucleotides. In this article, we briefly review the mechanisms of action of ANGPTL3 and apoC-III, the reasons why they have been considered promising targets of novel therapies for CVD, as well as the current status and the most important results of their clinical trials. [ABSTRACT FROM AUTHOR]

Non-cholesterol sterols are present in our body at very low concentrations as compared to cholesterol. Small changes in the structure of sterol molecules confer them highly distinct biological activities. The best-known example are steroid hormones derived from cholesterol. During the past decade, our knowledge of also other biomolecules related to or derived from cholesterol, particularly plant sterols, biosynthetic precursors of cholesterol, and oxysterols, has expanded rapidly. In this review article we recapitulate the latest insights into the properties and physiological activities of these non-cholesterol sterols, as well as their importance in disease processes and potential as diagnostic biomarkers. [ABSTRACT FROM AUTHOR]

Cholesterol is crucial for neuronal synaptic transmission, assisting in the molecular and structural organization of lipid rafts, ion channels, and exocytic proteins. Although cholesterol absence was shown to result in impaired neurotransmission, how cholesterol locally traffics and its route of action are still under debate. Here, we characterized the lipid transfer protein ORP2 in murine hippocampal neurons. We show that ORP2 preferentially localizes to the presynapse. Loss of ORP2 reduces presynaptic cholesterol levels by 50%, coinciding with a profoundly reduced release probability, enhanced facilitation, and impaired presynaptic calcium influx. In addition, ORP2 plays a cholesterol-transport-independent role in regulating vesicle priming and spontaneous release, likely by competing with Munc18-1 in syntaxin1A binding. To conclude, we identified a dual function of ORP2 as a physiological modulator of the synaptic cholesterol content and a regulator of neuronal exocytosis. [Display omitted] • ORP2 is a physiological modulator of synaptic cholesterol content • ORP2 loss coincides with reduced release probability and presynaptic calcium influx • ORP2 regulates vesicle priming in a cholesterol-transport-independent mode • ORP2 competes with Munc18-1 for syntaxin1A binding Weber-Boyvat at al. show that the lipid transfer protein ORP2 interacts with exocytic t-SNARES and localizes to the synapse in murine hippocampal neurons. They identify a dual function of synaptic ORP2 as a physiological modulator of synaptic cholesterol content and a direct regulator of vesicle priming in neuronal exocytosis. [ABSTRACT FROM AUTHOR]

Mitochondria are dynamic organelles essential for cell survival whose structural and functional integrity rely on selective and regulated transport of lipids from/to the endoplasmic reticulum (ER) and across the mitochondrial intermembrane space. As they are not connected by vesicular transport, the exchange of lipids between ER and mitochondria occurs at membrane contact sites. However, the mechanisms and proteins involved in these processes are only beginning to emerge. Here, we show that the main physiological localization of the lipid transfer proteins ORP5 and ORP8 is at mitochondria-associated ER membrane (MAM) subdomains, physically linked to the mitochondrial intermembrane space bridging (MIB)/mitochondrial contact sites and cristae junction organizing system (MICOS) complexes that bridge the two mitochondrial membranes. We also show that ORP5/ORP8 mediate non-vesicular transport of phosphatidylserine (PS) lipids from the ER to mitochondria by cooperating with the MIB/MICOS complexes. Overall our study reveals a physical and functional link between ER-mitochondria contacts involved in lipid transfer and intra-mitochondrial membrane contacts maintained by the MIB/MICOS complexes. [Display omitted] • The endogenous ORP5 and ORP8 mainly localize and interact at MAM • ORP5 and ORP8 interact with the mitochondrial MIB/MICOS complexes • ORP5 and ORP8 mediate non-vesicular transport of PS from the ER to the mitochondria In this study, Monteiro-Cardoso et al. reveal that the lipid transfer proteins ORP5/ORP8 are mainly localized at ER subdomains in contact with mitochondria. At these sites, ORP5/ORP8 interact and cooperate with the mitochondrial MIB/MICOS complexes to mediate non-vesicular transport of phosphatidylserine from the ER to mitochondria. [ABSTRACT FROM AUTHOR]

Abstract: Oxysterol-binding protein (OSBP) and OSBP-related proteins (ORPs) constitute a family of sterol and phosphoinositide binding proteins conserved in eukaryotes. The mechanisms of ORP function have remained incompletely understood. However, several ORPs are present at membrane contact sites and control the activity of enzymatic effectors or assembly of protein complexes, with impacts on signaling, vesicle transport, and lipid metabolism. An increasing number of protein interaction partners of ORPs have been identified, providing clues of their involvement in multiple aspects of cell regulation. The functions assigned for mammalian ORPs include coordination of sterol and sphingolipid metabolism and mitogenic signaling (OSBP), control of ER-late endosome (LE) contacts and LE motility (ORP1L), neutral lipid metabolism (ORP2), cell adhesion (ORP3), cholesterol eggress from LE (ORP5), macrophage lipid homeostasis, migration and high-density lipoprotein metabolism (ORP8), apolipoprotein B-100 secretion (ORP10), and adipogenesis (ORP11). The anti-proliferative ORPphilin compounds target OSBP and ORP4, revealing a function of ORPs in cell proliferation and survival. The Saccharomyces cerevisiae OSBP homologue (Osh) proteins execute multifaceted functions in sterol and sphingolipid homeostasis, post-Golgi vesicle transport, as well as phosphatidylinositol-4-phosphate and target of rapamycin complex 1 (TORC1) signaling. These observations identify ORPs as coordinators of lipid signals with an unforeseen variety of cellular processes. [Copyright &y& Elsevier]

Background: The rs73 8409 C —G single nucleotide polymorphism in the patatin-like phospholipase domain-containing 3 (PNPL43; adiponutrin) leads to a missense mutation (Il48M), which is associated with increased liver fat but not insulin resistance. The Il48M mutation impedes triglyceride hydrolysis in vitro, and its carriers have an increased risk of developing severe liver disease. Objective: We explored whether the rs73 8409 PNPLA3 G allele influences the ability of weight loss to decrease liver fat or change insulin sensitivity. Design: We recruited 8 subjects who were homozygous for the rs738409 PNPL43 G allele (PNPLA3-148MM) and 10 who were homozygous for the rs738409 PNPIA3 C allele (PNPLA3-14811). To allow comparison of changes in liver fat, the groups were matched with respect to baseline age, sex, body mass index, and liver fat. The subjects were placed on a hypocaloric low-carbohydrate diet for 6 d. Liver fat content (proton magnetic resonance spectroscopy), whole- body insulin sensitivity of glucose metabolism (euglycemic clamp technique), and lipolysis ([2H5]glycerol infusion) were measured before and after the diet. Results: At baseline, fasting serum insulin and C-peptide concentra- tions were significantly lower in the PNPLA3- 148MM group than in the PNPLA3-14811 group, as predicted by study design. Weight loss was not significantly different between groups (PNPLA3- 148MM: -3.1 ± 0.5 kg; PNPLA3- 14811: -3.1 ± 0.4 kg). Liver fat decreased by 45% in the PNPLA3-148MM group (P< 0.001 and by 18% in the PNPLA3- 148II group (P <0.01). Conclusion: Weight loss is effective in decreasing liver fat in subjects who are homozygous for the rs738409 PNPLA3 G or C allele. [ABSTRACT FROM AUTHOR]

Abstract: Oxysterols are oxidized derivatives of cholesterol or by-products of cholesterol biosynthesis with multiple functions. Even though they are heterogeneous in their biological activities, they have the common property of transferring between membranes orders of magnitude faster than cholesterol, due to higher polarity and poorer membrane packing. Depending on the nature and location of the oxygen substitution, oxysterols have distinct impacts on the biophysical properties of membranes, including the formation of liquid ordered domains. This is suggested to explain differences in the cytotoxic potential of various oxysterols. Besides the effects of oxysterols on membrane biophysical properties, the endogenous cellular oxysterols are suggested to execute important functions via interactions with receptor proteins. Increasing evidence suggests that oxysterols act as ligands of liver X receptors, transcription factors with key roles in lipid metabolism. Oxysterols were also shown to interact with the Insig (insulin-induced gene) proteins, revealing a mechanism by which they regulate the transport and maturation of sterol-regulatory element binding proteins as well as the stability of a rate-limiting sterol biosynthetic enzyme. Furthermore, a number of other cellular receptors for oxysterols involved in cell signaling, lipid metabolism, and vesicle transport have been discovered, enhancing the interest in these compounds in several branches of biomedical research. [Copyright &y& Elsevier]

[Display omitted] Oxysterol-binding protein -related proteins (ORPs) form a large family of intracellular lipid binding/transfer proteins. A number of ORPs are implicated in inter-organelle lipid transfer over membrane contacts sites, their mode of action involving in several cases the transfer of two lipids in opposite directions, termed countercurrent lipid transfer. A unifying feature appears to be the capacity to bind phosphatidylinositol polyphosphates (PIPs). These lipids are in some cases transported by ORPs from one organelle to another to drive the transfer of another lipid against its concentration gradient, while they in other cases may act as allosteric regulators of ORPs, or an ORP may introduce a PIP to an enzyme for catalysis. Dysregulation of several ORP family members is implicated in cancers, ORP3, −4, −5 and −8 being thus far the most studied examples. The most likely mechanisms underlying their associations with malignant growth are (i) impacts on PIP-mediated signaling events resulting in altered Ca2+ homeostasis, bioenergetics, cell survival, proliferation, and migration, (ii) protein–protein interactions affecting the activity of signaling factors, and (iii) modification of cellular lipid transport in a way that facilitates the proliferation of malignant cells. In this review I discuss the existing functional evidence for the involvement of ORPs in cancerous growth, discuss the findings in the light of the putative mechanisms outlined above and the possibility of employing ORPs as targets of anti-cancer therapy. [ABSTRACT FROM AUTHOR]

• OSBP mediates PI4P removal from LE/Lys and establishes a Lys cholesterol pool necessary for mTORC1 activation. • ORP1L transfers cholesterol over ER-LE/Lys membrane contact sites, possibly in both directions. • ORP2 transfers cholesterol from LE to RE and PI(4,5)P 2 to LE. • ORP2 regulates FAK activity and PI(4,5)P 2 generation on RE, focal adhesion dynamics and LE/Lys tabulation. Oxysterol-binding protein (OSBP) homologues, designated ORP or OSBPL proteins, constitute one of the largest families of intracellular lipid-binding/transfer proteins (LTP). This review summarizes the mounting evidence that several members of this family participate in the machinery facilitating cholesterol trafficking in the late endocytic pathway. There are indications that OSBP, besides acting as a cholesterol/phosphatidylinositol 4-phosphate (PI4P) exchanger at the endoplasmic reticulum (ER)- trans -Golgi network (TGN) membrane contact sites (MCS), also exchanges these lipids at ER-lysosome (Lys) contacts, increasing Lys cholesterol content. The long isoform of ORP1 (ORP1L), which also targets ER-late endosome (LE)/Lys MCS, has the capacity to mediate cholesterol transport either from ER to LE or in the opposite direction. Moreover, it regulates the motility, positioning and fusion of LE as well as autophagic flux. ORP2, the closest relative of ORP1, is mainly cytosolic, but also targets PI(4,5)P 2 -rich endosomal compartments. Our latest data suggest that ORP2 transfers cholesterol from LE to recycling endosomes (RE) in exchange for PI(4,5)P 2 , thus stimulating the recruitment of focal adhesion kinase (FAK) on the RE and cell adhesion. FAK activates phosphoinositide kinase on the RE to enhance PI(4,5)P 2 synthesis. ORP2 in turn transfers PI(4,5)P 2 from RE to LE, thus regulating LE tubule formation and transport activity. [ABSTRACT FROM AUTHOR]

• ORP2 is a mammalian oxysterol-binding protein homologue with affinity for cholesterol, oxysterols and phosphoinositides. • ORP2 is suggested to mediate cholesterol transport to the plasma membrane in exchange for PI4,5P 2. • ORP2 knock-out in hepatoma cells resulted in defective triglyceride synthesis and storage. • ORP2 knock-out impaired cellular glucose uptake and metabolism, co-indicent with defective Akt signaling. • ORP2 manipulations impact on F-actin, cell adhesion, migration and proliferation. Oxysterol-binding protein (OSBP)-related proteins (ORPs) constitute a family of intracellular lipid-binding/transport proteins (LTPs) in eukaryotes. They typically have a modular structure comprising a lipid-binding domain and membrane targeting determinants, being thus suited for function at membrane contact sites. Among the mammalian ORPs, ORP2/OSBPL2 is the only member that only exists as a 'short' variant lacking a membrane-targeting pleckstrin homology domain. ORP2 is expressed ubiquitously and has been assigned a multitude of functions. Its OSBP-related domain binds cholesterol, oxysterols, and phosphoinositides, and its overexpression enhances cellular cholesterol efflux. Consistently, the latest observations suggest a function of ORP2 in cholesterol transport to the plasma membrane (PM) in exchange for phosphatidylinositol 4,5-bisphosphate (PI4,5P 2), with significant impacts on the concentrations of PM cholesterol and PI4,5P 2. On the other hand, ORP2 localizes at the surface of cytoplasmic lipid droplets (LDs) and at endoplasmic-reticulum-LD contact sites, and its depletion modifies cellular triglyceride (TG) metabolism. Study in an adrenocortical cell line further suggested a function of ORP2 in the synthesis of steroid hormones. Our recent knock-out of ORP2 in human hepatoma cells revealed its function in hepatocellular PI3K/Akt signaling, glucose and triglyceride metabolism, as well as in actin cytoskeletal regulation, cell adhesion, migration and proliferation. ORP2 was shown to interact physically with F-actin regulators such as DIAPH1, ARHGAP12, SEPT9 and MLC12, as well as with IQGAP1 and the Cdc37-Hsp90 chaperone complex controlling the activity of Akt. Interestingly, mutations in OSBPL2 encoding ORP2 are associated with autosomal dominant non-syndromic hearing loss, and the protein was found to localize in cochlear hair cell stereocilia. The functions assigned to ORP2 suggest that this protein, in concert with other LTPs, controls the subcellular distribution of cholesterol in various cell types and steroid hormone synthesis in adrenocortical cells. However, it also impacts cellular TG and carbohydrate metabolism and F-actin-dependent functions, revealing a bewildering spectrum of activities. [ABSTRACT FROM AUTHOR]

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 PIP 2 from the plasma membrane and presents it to PLCβ3, enabling IP 3 generation and subsequent Ca2+-dependent bioenergetics. LYZ-81 binds ORP4L competitively with PIP 2 and blocks PIP 2 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. Graphical Abstract Highlights • ORP4L is highly expressed in LSCs and essential for LSC survival • ORP4L extracts and presents PIP 2 from the plasma membrane for PLCβ3 catalysis in LSCs • LYZ-81 is identified as a specific inhibitor of ORP4L • Targeting ORP4L by LYZ-81 selectively eradicates LSCs Zhong et al. report that abnormal expression of ORP4L is essential for leukemia stem cell survival; it enables IP 3 generation by extracting and presenting PIP 2 from the plasma membrane to PLCβ3 for hydrolysis. The compound LYZ-81, which blocks this process via targeting ORP4L, selectively eradicates leukemia stem cells. [ABSTRACT FROM AUTHOR]

[Display omitted] Adipose tissue (AT) expansion through hyperplasia or hypertrophy requires vascular remodeling that involves angiogenesis. There is quite some evidence that obese white AT (WAT) displays altered vasculature. Some studies suggest that this is associated with hypoxia, which is thought to play a role in inducing inflammatory activation of the excessively expanding WAT. Increasing evidence, based on genetic manipulations or treatments with inhibitory or activator pharmaceuticals, demonstrates that AT angiogenesis is crucial for AT metabolic function, and thereby for whole body metabolism and metabolic health. Despite some contradiction between studies, disturbance of WAT angiogenesis in obesity could be an important factor driving WAT dysfunction and the comorbidities of obesity. Endothelial cells (ECs) contribute to healthy WAT metabolism via transport of fatty acids and other plasma components, secretory signaling molecules, and extracellular vesicles (EVs). This communication is crucial for adipocyte metabolism and underscores the key role that the AT endothelium plays in systemic energy homeostasis and healthy metabolism. Adipocytes communicate towards the neighboring endothelium through several mechanisms. The pro-inflammatory status of hypertrophic adipocytes in obesity is reflected in ECs activation, which promotes chronic inflammation. On the other hand, adiponectin secreted by the adipocytes is important for healthy endothelial function, and adipocytes also secrete other pro- or anti-angiogenic effector molecules and a wealth of EVs – however, their detailed roles in signaling towards the endothelium are yet poorly understood. To conclude, targeting AT angiogenesis and promoting the healthy communication between adipocytes and ECs represent potentially promising strategies to treat obesity and its comorbidities. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Background & Aims: Animal studies suggest that endocannabinoids could contribute to the development of nonalcoholic fatty liver disease (NAFLD). In addition, NAFLD has been shown to be associated with multiple changes in lipid concentrations in liver biopsies. There are no data on splanchnic free fatty acid (FFA), glycerol, ketone body, endocannabinoid, and lipid fluxes in vivo in subjects with NAFLD. Methods: We performed hepatic venous catheterization studies in combination with [2H2]palmitate infusion in the fasting state and during a low-dose insulin infusion in 9 subjects with various degrees of hepatic steatosis as determined using liver biopsy. Splanchnic balance of endocannabinoids and individual lipids was determined using ultra performance liquid chromatography coupled to mass spectrometry. Results: Concentrations of the endocannabinoid 2-arachidonoylglycerol were higher in arterialized (91 ± 33 μg/L basally) than in hepatic venous (51 ± 19 μg/L; P < .05) plasma. Fasting arterial (r = 0.72; P = .031) and hepatic venous (r = 0.70; P = .037) concentrations of 2-arachidonoylglycerol were related positively to liver fat content. Analysis of fluxes of 85 different triglycerides showed that the fatty liver overproduces saturated triglycerides. In the plasma FFA fraction in the basal state, the relative amounts of palmitoleate and linoleate were lower and those of stearate and oleate were higher in the hepatic vein than in the artery. Absolute concentrations of all nontriglyceride lipids were comparable in arterialized venous plasma and the hepatic vein both in the basal and insulin-stimulated states. Conclusions: The human fatty liver takes up 2-arachidonoylglycerol and overproduces triacylglycerols containing saturated fatty acids, which might reflect increased de novo lipogenesis. [ABSTRACT FROM AUTHOR]

An abstract of the article "Assays for Interaction between Rab7 and Oxysterol Binding Protein Related Protein 1L (ORP1L)," by Marie Johansson and Vesa M. Olkkonen is presented.