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.

The lipid transporter ORP2 regulates synaptic neurotransmitter release via two distinct mechanisms.

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.

Reexamination of N-terminal domains of syntaxin-1 in vesicle fusion from central murine synapses.

Syntaxin-1 (STX1) and Munc18-1 are two requisite components of synaptic vesicular release machinery, so much so synaptic transmission cannot proceed in their absence. They form a tight complex through two major binding modes: through STX1's N-peptide and through STX1's closed conformation driven by its Habc- domain. However, physiological roles of these two reportedly different binding modes in synapses are still controversial. Here we characterized the roles of STX1's N-peptide, Habc-domain, and open conformation with and without N-peptide deletion using our STX1-null mouse model system and exogenous reintroduction of STX1A mutants. We show, on the contrary to the general view, that the Habc-domain is absolutely required and N-peptide is dispensable for synaptic transmission. However, STX1A's N-peptide plays a regulatory role, particularly in the Ca2+-sensitivity and the short-term plasticity of vesicular release, whereas STX1's open conformation governs the vesicle fusogenicity. Strikingly, we also show neurotransmitter release still proceeds when the two interaction modes between STX1A and Munc18-1 are presumably intervened, necessitating a refinement of the conceptualization of STX1A-Munc18-1 interaction.

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.

Uncovering oxysterol-binding protein (OSBP) as a target of the anti-enteroviral compound TTP-8307.

The genus Enterovirus (e.g. poliovirus, coxsackievirus, rhinovirus) of the Picornaviridae family of positive-strand RNA viruses includes many important pathogens linked to a range of acute and chronic diseases for which no approved antiviral therapy is available. Targeting a step in the life cycle that is highly conserved provides an attractive strategy for developing broad-range inhibitors of enterovirus infection. A step that is currently explored as a target for the development of antivirals is the formation of replication organelles, which support replication of the viral genome. To build replication organelles, enteroviruses rewire cellular machinery and hijack lipid homeostasis pathways. For example, enteroviruses exploit the PI4KIIIß-PI4P-OSBP pathway to direct cholesterol to replication organelles. Here, we uncover that TTP-8307, a known enterovirus replication inhibitor, acts through the PI4KIIIß-PI4P-OSBP pathway by directly inhibiting OSBP activity. However, despite a shared mechanism of TTP-8307 with established OSBP inhibitors (itraconazole and OSW-1), we identify a number of notable differences between these compounds. The antiviral activity of TTP-8307 extends to other viruses that require OSBP, namely the picornavirus encephalomyocarditis virus and the flavivirus hepatitis C virus.

A loss-of-function variant in OSBPL1A predisposes to low plasma HDL cholesterol levels and impaired cholesterol efflux capacity.

BACKGROUND AND AIMS: Among subjects with high-density-lipoprotein cholesterol (HDL-C) below the 1st percentile in the general population, we identified a heterozygous variant OSBPL1A p.C39X encoding a short truncated protein fragment that co-segregated with low plasma HDL-C. METHODS: We investigated the composition and function of HDL from the carriers and non-carriers and studied the properties of the mutant protein in cultured hepatocytes. RESULTS: Plasma HDL-C and apolipoprotein (apo) A-I were lower in carriers versus non-carriers, whereas the other analyzed plasma components or HDL parameters did not differ. Sera of the carriers displayed a reduced capacity to act as cholesterol efflux acceptors (p < 0.01), whereas the cholesterol acceptor capacity of their isolated HDL was normal. Fibroblasts from a p.C39X carrier showed reduced cholesterol efflux to lipid-free apoA-I but not to mature HDL particles, suggesting a specific defect in ABCA1-mediated efflux pathway. In hepatic cells, GFP-OSBPL1A partially co-localized in endosomes containing fluorescent apoA-I, suggesting that OSBPL1A may regulate the intracellular handling of apoA-I. The GFP-OSBPL1A-39X mutant protein remained in the cytosol and failed to interact with Rab7, which normally recruits OSBPL1A to late endosomes/lysosomes, suggesting that this mutation represents a loss-of-function. CONCLUSIONS: The present work represents the first characterization of a human OSBPL1A mutation. Our observations provide evidence that a familial loss-of-function mutation in OSBPL1A affects the first step of the reverse cholesterol transport process and associates with a low HDL-C phenotype. This suggests that rare mutations in OSBPL genes may contribute to dyslipidemias.

OSBP-Related Protein Family: Mediators of Lipid Transport and Signaling at Membrane Contact Sites.

Oxysterol-binding protein (OSBP) and its related protein homologs, ORPs, constitute a conserved family of lipid-binding/transfer proteins (LTPs) expressed ubiquitously in eukaryotes. The ligand-binding domain of ORPs accommodates cholesterol and oxysterols, but also glycerophospholipids, particularly phosphatidylinositol-4-phosphate (PI4P). ORPs have been implicated as intracellular lipid sensors or transporters. Most ORPs carry targeting determinants for the endoplasmic reticulum (ER) and non-ER organelle membrane. ORPs are located and function at membrane contact sites (MCSs), at which ER is closely apposed with other organelle limiting membranes. Such sites have roles in lipid transport and metabolism, control of Ca(2+) fluxes, and signaling events. ORPs are postulated either to transport lipids over MCSs to maintain the distinct lipid compositions of organelle membranes, or to control the activity of enzymes/protein complexes with functions in signaling and lipid metabolism. ORPs may transfer PI4P and another lipid class bidirectionally. Transport of PI4P followed by its hydrolysis would in this model provide the energy for transfer of the other lipid against its concentration gradient. Control of organelle lipid compositions by OSBP/ORPs is important for the life cycles of several pathogenic viruses. Targeting ORPs with small-molecular antagonists is proposed as a new strategy to combat viral infections. Several ORPs are reported to modulate vesicle transport along the secretory or endocytic pathways. Moreover, antagonists of certain ORPs inhibit cancer cell proliferation. Thus, ORPs are LTPs, which mediate interorganelle lipid transport and coordinate lipid signals with a variety of cellular regimes.

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.

Bimolecular fluorescence complementation (BiFC) technique in yeast Saccharomyces cerevisiae and 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.

Sterol liganding of OSBP-related proteins (ORPs) regulates the subcellular distribution of ORP-VAPA complexes and their impacts on organelle structure.

Oxysterol-binding protein (OSBP) and its homologues (ORPs) are lipid-binding/transfer proteins with affinity for oxysterols, cholesterol and glycerophospholipids. In addition to a ligand-binding domain, a majority of the ORPs carry a pleckstrin homology domain that targets organelle membranes via phosphoinositides, and a motif targeting the endoplasmic reticulum (ER) via VAMP-associated proteins (VAPs). We employed here Bimolecular Fluorescence Complementation (BiFC) to systematically assess the effects of sterol manipulation of HuH7 cells on complexes of established sterol-binding ORPs with their ER receptor, VAMP-associated protein A (VAPA). Depletion of cellular cholesterol with lipoprotein-deficient medium and Mevastatin caused concentration of OSBP-VAPA complexes and Golgi complex markers at a juxtanuclear position, an effect reversed by low-density lipoprotein treatment. A similar redistribution of OSBP-VAPA but not of sterol-binding deficient mutant OSBP(ΔELSK)-VAPA, occurred upon treatment with the high-affinity ligand, 25-hydroxycholesterol (25OHC), which reduced total and free cholesterol. ORP2-VAPA complexes, which localize in untreated cells at blob-like ER structures with associated lipid droplets, were redistributed upon treatment with the ORP2 ligand 22(R)OHC to a diffuse cytoplasmic/ER pattern and the plasma membrane. Analogously, distribution of ORP4L-VAPA complexes between the plasma membrane and vimentin intermediate filament associated compartments was modified by statin or 25OHC treatment. The treatments resulted in loss of vimentin co-localization, and sterol-binding deficient ORP4L(ΔELSR)-VAPA localized predominantly to the plasma membrane. In conclusion, treatment with statin or oxysterol ligands modify the subcellular targeting of ORP-VAPA complexes, consistent with the notion that this machinery controls lipid homeostasis and signaling at organelle interfaces.

Ligand-dependent localization and function of ORP-VAP complexes at membrane contact sites.

Oxysterol-binding protein/OSBP-related proteins (ORPs) constitute a conserved family of sterol/phospholipid-binding proteins with lipid transporter or sensor functions. We investigated the spatial occurrence and regulation of the interactions of human OSBP/ORPs or the S. cerevisiae orthologs, the Osh (OSBP homolog) proteins, with their endoplasmic reticulum (ER) anchors, the VAMP-associated proteins (VAPs), by employing bimolecular fluorescence complementation and pull-down set-ups. The ORP-VAP interactions localize frequently at distinct subcellular sites, shown in several cases to represent membrane contact sites (MCSs). Using established ORP ligand-binding domain mutants and pull-down assays with recombinant proteins, we show that ORP liganding regulates the ORP-VAP association, alters the subcellular targeting of ORP-VAP complexes, or modifies organelle morphology. There is distinct protein specificity in the effects of the mutants on subcellular targeting of ORP-VAP complexes. We provide evidence that complexes of human ORP2 and VAPs at ER-lipid droplet interfaces regulate the hydrolysis of triglycerides and lipid droplet turnover. The data suggest evolutionarily conserved, complex ligand-dependent functions of ORP-VAP complexes at MCSs, with implications for cellular lipid homeostasis and signaling.

OSBP-related protein 3 (ORP3) coupling with VAMP-associated protein A regulates R-Ras activity.

ORP3 is an R-Ras interacting oxysterol-binding protein homolog that regulates cell adhesion and is overexpressed in several cancers. We investigated here a novel function of ORP3 dependent on its targeting to both the endoplasmic reticulum (ER) and the plasma membrane (PM). Using biochemical and cell imaging techniques we demonstrate the mechanistic requirements for the subcellular targeting and function of ORP3 in control of R-Ras activity. We show that hyperphosphorylated ORP3 (ORP3-P) selectively interacts with the ER membrane protein VAPA, and ORP3-VAPA complexes are targeted to PM sites via the ORP3 pleckstrin homology (PH) domain. A novel FFAT (two phenylalanines in an acidic tract)-like motif was identified in ORP3; only disruption of both the FFAT-like and canonical FFAT motif abolished the phorbol-12-myristate-13-acetate (PMA) stimulated interaction of ORP3-P with VAPA. Co-expression of ORP3 and VAPA induced R-Ras activation, dependent on the interactions of ORP3 with VAPA and the PM. Consistently, downstream AktS473 phosphorylation and ß1-integrin activity were enhanced by ORP3-VAPA. To conclude, phosphorylation of ORP3 controls its association with VAPA. Furthermore, we present evidence that ORP3-VAPA complexes stimulate R-Ras signaling.

Oxysterol-binding proteins: functions in cell regulation beyond lipid metabolism.

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 ß1-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.

A conserved regulatory mode in exocytic membrane fusion revealed by Mso1p membrane interactions.

Sec1/Munc18 family proteins are important components of soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex-mediated membrane fusion processes. However, the molecular interactions and the mechanisms involved in Sec1p/Munc18 control and SNARE complex assembly are not well understood. We provide evidence that Mso1p, a Sec1p- and Sec4p-binding protein, interacts with membranes to regulate membrane fusion. We identify two membrane-binding sites on Mso1p. The N-terminal region inserts into the lipid bilayer and appears to interact with the plasma membrane, whereas the C-terminal region of the protein binds phospholipids mainly through electrostatic interactions and may associate with secretory vesicles. The Mso1p membrane interactions are essential for correct subcellular localization of Mso1p-Sec1p complexes and for membrane fusion in Saccharomyces cerevisiae. These characteristics are conserved in the phosphotyrosine-binding (PTB) domain of ß-amyloid precursor protein-binding Mint1, the mammalian homologue of Mso1p. Both Mint1 PTB domain and Mso1p induce vesicle aggregation/clustering in vitro, supporting a role in a membrane-associated process. The results identify Mso1p as a novel lipid-interacting protein in the SNARE complex assembly machinery. Furthermore, our data suggest that a general mode of interaction, consisting of a lipid-binding protein, a Rab family GTPase, and a Sec1/Munc18 family protein, is important in all SNARE-mediated membrane fusion events.

ORP10, a cholesterol binding protein associated with microtubules, regulates apolipoprotein B-100 secretion.

ORP10/OSBPL10 is a member of the oxysterol-binding protein family, and genetic variation in OSBPL10 is associated with dyslipidemias and peripheral artery disease. In this study we investigated the ligand binding properties of ORP10 in vitro as well as its localization and function in human HuH7 hepatocytes. The pleckstrin homology (PH) domain of ORP10 selectively interacts with phosphatidylinositol-4-phosphate, while the C-terminal ligand binding domain binds cholesterol and several acidic phospholipids. Full-length ORP10 decorates microtubules (MT), while the ORP10 N-terminal fragment (aa 1-318) localizes at Golgi membranes. Removal of the C-terminal aa 712-764 of ORP10 containing a predicted coiled-coil segment abolishes the MT association, but allows partial Golgi targeting. A PH domain-GFP fusion protein is distributed mainly in the cytosol and the plasma membrane, indicating that the Golgi affinity of ORP10 involves other determinants in addition to the PH domain. HuH7 cells expressing ORP10-specific shRNA display increased accumulation of apolipoprotein B-100 (apoB-100), but not of albumin, in culture medium, and contain reduced levels of intracellular apoB-100. Pulse-chase analysis of cellular [(35)S]apoB-100 demonstrates enhanced apoB-100 secretion by cells expressing ORP10-specific shRNA. The apoB-100 secretion phenotype is replicated in HepG2 cells transduced with the ORP10 shRNA lentiviruses. As a conclusion, the present study dissects the determinants of ORP10 association with MT and the Golgi complex and provides evidence for a specific role of this protein in ß-lipoprotein secretion by human hepatocytes.

Sec1p and Mso1p C-terminal tails cooperate with the SNAREs and Sec4p in polarized exocytosis.

The Sec1/Munc18 protein family members perform an essential, albeit poorly understood, function in association with soluble n-ethylmaleimide sensitive factor adaptor protein receptor (SNARE) complexes in membrane fusion. The Saccharomyces cerevisiae Sec1p has a C-terminal tail that is missing in its mammalian homologues. Here we show that deletion of the Sec1p tail (amino acids 658-724) renders cells temperature sensitive for growth, reduces sporulation efficiency, causes a secretion defect, and abolishes Sec1p-SNARE component coimmunoprecipitation. The results show that the Sec1p tail binds preferentially ternary Sso1p-Sec9p-Snc2p complexes and it enhances ternary SNARE complex formation in vitro. The bimolecular fluorescence complementation (BiFC) assay results suggest that, in the SNARE-deficient sso2-1 Δsso1 cells, Mso1p, a Sec1p binding protein, helps to target Sec1p(1-657) lacking the C-terminal tail to the sites of secretion. The results suggest that the Mso1p C terminus is important for Sec1p(1-657) targeting. We show that, in addition to Sec1p, Mso1p can bind the Rab-GTPase Sec4p in vitro. The BiFC results suggest that Mso1p acts in close association with Sec4p on intracellular membranes in the bud. This association depends on the Sec4p guanine nucleotide exchange factor Sec2p. Our results reveal a novel binding mode between the Sec1p C-terminal tail and the SNARE complex, and suggest a role for Mso1p as an effector of Sec4p.