The effect of hypoxia on the expression of 150 kDa oxygen-regulated protein (ORP 150) in HeLa cells.

Correct protein folding is an important factor, for the translocation of newly synthesised proteins to specific subcellular compartments, extracellular matrix or to biological fluids. This process is regulated by a group of specific proteins, referred to as chaperones. Many stress conditions, such as oxygen or glucose deprivation, slow down the folding process and cause accumulation of unfolded/misfolded proteins in the cell. Molecular chaperones are induced in these conditions; with some named as oxygen-regulated proteins (ORPs). These bind to unfolded / misfolded proteins to facilitate correct assembly. ORP 150 is the subject of this study. Hypoxia results in an enhancement of ORP 150 expression in several tumour cell lines cultured in vitro. HeLa cells grown in hypoxic conditions (despite an intensive expression of ORP 150) demonstrate higher rates of apoptosis in comparison to those cultured in normoxic conditions. Furthermore, the inhibition of ORP 150 synthesis by transfection of these cells with a specific siRNA resulted in an intensification of apoptosis, as indicated by specific markers of this process; the enhancement of poly ADP-ribose protein cleavage and the increase in Bim protein expression. We conclude from our study that the increase in ORP 150 synthesis protects the cells against the proapoptotic effect of hypoxia.     

Role of ORPs in sterol transport from plasma membrane to ER and lipid droplets in mammalian cells

In this study, we investigated the mechanisms of sterol transport from the plasma membrane (PM) to the endoplasmic reticulum (ER) and lipid droplets (LDs) in HeLa cells. By overexpressing all mammalian oxysterol-binding protein-related proteins (ORPs), we found that especially ORP1S and ORP2 enhanced PM-to-LD sterol transport. This reflected the stimulation of transport from the PM to the ER, rather than from the ER to LDs. Double knockdown of ORP1S and ORP2 inhibited sterol transport from the PM to the ER and LDs, suggesting a physiological role for these ORPs in the process. A two phenylalanines in an acidic tract (FFAT) motif in ORPs that mediates interaction with VAMP-associated proteins (VAPs) in the ER was not necessary for the enhancement of sterol transport by ORPs. However, VAP-A and VAP-B silencing slowed down PM-to-LD sterol transport. This was accompanied by enhanced degradation of ORP2 and decreased levels of several FFAT motif-containing ORPs, suggesting a role for VAPs in sterol transport by stabilization of ORPs.     

Oxygen regulated 80 kDa protein and glucose regulated 78 kDa protein are identical

Ischemic stress of cells within solid tumors arises from inadequate perfusion of regions of the tumor and results in microenvironments which are hypoxic and deficient in nutrient delivery and waste product removal. Stressed cells within these microenvironments show growth inhibition and synthesize unique sets of proteins referred to as glucose and oxygen regulated proteins (GRPs and ORPs respectively). The commonality of proteins induced by glucose-starvation and hypoxia has not been proven. To this end, ORPs were induced in Chinese hamster ovary cells in the presence of high glucose concentration in the media and ORP 80 isolated from two dimension gels. Eleven tryptic peptides of the 80 kDa ORP were sequenced and found to be identical to GRP 78 sequences. The data demonstrate that GRP 78 and ORP 80 have the same primary amino acid sequence and suggest that glucose-starvation and hypoxia can induce the same cellular responses.     

Abundant expression of 150-kDa oxygen-regulated protein in mouse pancreatic beta cells is correlated with insulin secretion

The 150-kDa oxygen-regulated protein (ORP150) is a member of glucose-regulated proteins (GRPs), which are induced by stressful conditions such as oxygen or glucose deprivation. Here we investigated the highly abundant expression of ORP150 in mouse pancreas and its relationship with insulin secretion. Immunohistochemical analysis revealed that ORP150 expression was restricted to islets, especially to beta cells. The beta cell-specific expression was also observed in a mouse insulinoma cell line, MIN6, which secretes insulin in response to increased glucose concentration. Furthermore, ORP150 in islets dramatically diminished by fasting, concomitant with reduction of the serum insulin level. These results strongly suggest the role for ORP150 in insulin secretion.     

Oxygen-regulated protein-150 prevents calcium homeostasis deregulation and apoptosis induced by oxidized LDL in vascular cells

Oxidized LDLs (oxLDLs) induce apoptosis, which contributes to the pathogenesis of atherosclerosis. The 150 kDa oxygen-regulated protein (ORP150), an endoplasmic reticulum (ER)-resident chaperone, is upregulated by hypoxia and prevents ischemia-induced cell death. The aim of this work was to investigate whether and how ORP150 can prevent apoptosis induced by oxLDLs in vascular cells. OxLDLs induced ORP150 expression in the ER of human microvascular endothelial cell line (HMEC-1). ORP150 expression was blocked by antioxidants, by the permeant calcium chelator BAPTA-AM, and by inhibitors of the inositol-1,4,5 trisphosphate (IP3) receptors, 2-aminoethyl diphenylborinate (2-APB) and xestospongin C. ORP150 silencing by siRNA-enhanced oxLDL-induced apoptosis, while forced ORP150 expression increased the resistance of cells via an inhibition of the oxLDL-induced calcium rise, and of subsequent calpain activation, cytochrome c release, caspase 3 activation and apoptosis. A similar protective effect was achieved by BAPTA-AM, 2-APB and xestospongin C. Altogether, these data indicate that (i)ORP150 inhibits oxLDL-induced apoptosis by blocking calcium signaling and subsequent apoptosis, (ii)calcium released from ER stores through IP3 channels is involved in the oxLDL-induced calcium rise and apoptosis, and is inhibited by ORP150. Finally, ORP150 is expressed in advanced atherosclerotic lesions, where it may locally participate to reduce the apoptotic effect of oxLDLs and the subsequent risk of plaque rupture.     

The mammalian oxysterol-binding protein-related proteins (ORPs) bind 25-hydroxycholesterol in an evolutionarily conserved pocket

OSBP (oxysterol-binding protein) homologues, ORPs (OSBP-related proteins), constitute a 12-member family in mammals. We employed an in vitro [3H]25OH (25-hydroxycholesterol)-binding assay with purified recombinant proteins as well as live cell photo-cross-linking with [3H]photo-25OH and [3H]photoCH (photo-cholesterol), to investigate sterol binding by the mammalian ORPs. ORP1 and ORP2 [a short ORP consisting of an ORD (OSBP-related ligand-binding domain) only] were in vitro shown to bind 25OH. GST (glutathione S-transferase) fusions of the ORP1L [long variant with an N-terminal extension that carries ankyrin repeats and a PH domain (pleckstrin homology domain)] and ORP1S (short variant consisting of an ORD only) variants bound 25OH with similar affinity (ORP1L, K(d)=9.7x10(-8) M; ORP1S, K(d)=8.4 x10(-8) M), while the affinity of GST-ORP2 for 25OH was lower (K(d)=3.9x10(-6) M). Molecular modelling suggested that ORP2 has a sterol-binding pocket similar to that of Saccharomyces cerevisiae Osh4p. This was confirmed by site-directed mutagenesis of residues in proximity of the bound sterol in the structural model. Substitution of Ile249 by tryptophan or Lys150 by alanine markedly inhibited 25OH binding by ORP2. In agreement with the in vitro data, ORP1L, ORP1S, and ORP2 were cross-linked with photo-25OH in live COS7 cells. Furthermore, in experiments with either truncated cDNAs encoding the OSBP-related ligand-binding domains of the ORPs or the full-length proteins, photo-25OH was bound to OSBP, ORP3, ORP4, ORP5, ORP6, ORP7, ORP8, ORP10 and ORP11. In addition, the ORP1L variant and ORP3, ORP5, and ORP8 were cross-linked with photoCH. The present study identifies ORP1 and ORP2 as OSBPs and suggests that most of the mammalian ORPs are able to bind sterols.     

Glucose deficiency inhibits glycosaminoglycans synthesis in fibroblast cultures

We decided to study the effect of glucose deprivation on glycosaminoglycan (GAG) synthesis and degradation in fibroblast cultures, vitality of these cells and a correlation of these processes with the expression of oxygen/glucose-regulated proteins (ORP150/GRP170). The incorporation of [³H]-glucosamine into both newly synthesised hyaluronic acid and sulphated GAGs and [³⁵S]-sulphate into GAGs was used as an index of glycosaminoglycan synthesis. Quantitative evaluation of newly synthesised GAGs degradation was determined by pulse-chase experiments. We demonstrated that fibroblasts incubated in high glucose medium synthesised significant amounts of GAGs. Most of them were secreted into the culture medium. The shortage of glucose resulted in about 40% reduction in synthesis of GAGs, both those secreted into culture medium and remaining in the cell layer. The pulse-chase experiments demonstrated that the reduced amount of newly synthesised glycosaminoglycans was protected against intracellular degradation. Proportionally less GAGs were degraded in cultures incubated in low glucose than in high glucose media. These phenomena were accompanied by an increase in the expression of chaperon – ORP150 in cultures growing in low glucose medium. We suggest that the increased expression of ORP150 is a factor which prolongs the cell vitality and protects glycosaminoglycans against intracellular degradation induced by glucose deprivation.     

Oxysterol-binding Protein (OSBP)-related Protein 4 (ORP4) Is Essential for Cell Proliferation and Survival

Oxysterol-binding protein (OSBP) and OSBP-related proteins (ORPs) comprise a large gene family with sterol/lipid transport and regulatory activities. ORP4 (OSBP2) is a closely related paralogue of OSBP, but its function is unknown. Here we show that ORP4 binds similar sterol and lipid ligands as OSBP and other ORPs but is uniquely required for the proliferation and survival of cultured cells. Recombinant ORP4L and a variant without a pleckstrin homology (PH) domain (ORP4S) bind 25-hydroxycholesterol and extract and transfer cholesterol between liposomes. Two conserved histidine residues in the OSBP homology domain ORP4 are essential for binding phosphatidylinositol 4-phosphate but not sterols. The PH domain of ORP4L also binds phosphatidylinositol 4-phosphate in the Golgi apparatus. However, in the context of ORP4L, the PH domain is required for normal organization of the vimentin network. Unlike OSBP, RNAi silencing of all ORP4 variants (including a partial PH domain truncation termed ORP4M) in HEK293 and HeLa cells resulted in growth arrest but not cell death. ORP4 silencing in non-transformed intestinal epithelial cells (IEC)-18 caused apoptosis characterized by caspase 3 and poly(ADP-ribose) polymerase processing, DNA cleavage, and JNK phosphorylation. IEC-18 transformed with oncogenic H-Ras have increased expression of ORP4L and ORP4S proteins and are resistant to the growth-inhibitory effects of ORP4 silencing. Results suggest that ORP4 promotes the survival of rapidly proliferating cells.     

氧调节蛋白150在人肝细胞癌中的过度表达及其对凋亡的作用

在前期研究中发现,氧调节蛋白150(ORP150)是与肝细胞癌相关的糖蛋白．进一步研究了ORP150的表达水平与肝细胞癌的相关性．免疫印迹、细胞免疫化学和定量PCR分别在蛋白质水平和mRNA水平检测了ORP150的表达．运用RNA干扰技术检测了其对凋亡和肝细胞癌侵袭性的影响．发现：无论是蛋白质水平还是mRNA水平,与正常肝细胞相比,ORP150在肝细胞癌中表达明显上调;经RNA干扰后,肝细胞癌的凋亡明显增加,但肿瘤细胞的侵袭性无改变．肝细胞癌中,ORP150表达上调,它可能抑制肿瘤细胞的凋亡而促进其生长．ORP150有可能成为肝细胞癌的治疗靶点．     

PI4P/PS countertransport by ORP10 at ER–endosome membrane contact sites regulates endosome fission

Membrane contact sites (MCSs) serve as a zone for nonvesicular lipid transport by oxysterol-binding protein (OSBP)-related proteins (ORPs). ORPs mediate lipid countertransport, in which two distinct lipids are transported counterdirectionally. How such lipid countertransport controls specific biological functions, however, remains elusive. We report that lipid countertransport by ORP10 at ER–endosome MCSs regulates retrograde membrane trafficking. ORP10, together with ORP9 and VAP, formed ER–endosome MCSs in a phosphatidylinositol 4-phosphate (PI4P)-dependent manner. ORP10 exhibited a lipid exchange activity toward its ligands, PI4P and phosphatidylserine (PS), between liposomes in vitro, and between the ER and endosomes in situ. Cell biological analysis demonstrated that ORP10 supplies a pool of PS from the ER, in exchange for PI4P, to endosomes where the PS-binding protein EHD1 is recruited to facilitate endosome fission. Our study highlights a novel lipid exchange at ER–endosome MCSs as a nonenzymatic PI4P-to-PS conversion mechanism that organizes membrane remodeling during retrograde membrane trafficking.     

Bis(monoacylglycero)phosphate regulates oxysterol binding protein-related protein 11 dependent sterol trafficking

Bis(Monoacylglycero) Phosphate (BMP) is a unique phospholipid localized in late endosomes, a critical cellular compartment in low density lipoprotein (LDL)-cholesterol metabolism. In previous work, we demonstrated the important role of BMP in the regulation of macrophage cholesterol homeostasis. BMP exerts a protective role against the pro-apoptotic effect of oxidized LDL (oxLDL) by reducing the production of deleterious oxysterols. As the intracellular sterol traffic in macrophages is in part regulated by oxysterol binding protein (OSBP) and OSBP-related proteins (ORPs), we investigated the role of ORP11, localized at the Golgi-late endosomes interface, in the BMP-mediated protection from oxLDL/oxysterol cytotoxicity. Stably silencing of ORP11 in mouse RAW264.7 macrophages via a shRNA lentiviruses system had no effect on BMP production. However, ORP11 knockdown abrogated the protective action of BMP against oxLDL induced apoptosis. In oxLDL treated control cells, BMP enrichment was associated with reduced generation of 7-oxysterols, while these oxysterol species were abundant in the ORP11 knock-down cells. Of note, BMP enrichment in ORP11 knock-down cells was associated with a drastic increase in free cholesterol and linked to a decrease of cholesterol efflux. The expression of ATP-binding cassette-transporter G1 (ABCG1) was also reduced in the ORP11 knock-down cells. These observations demonstrate a cooperative function of OPR11 and BMP, in intracellular cholesterol trafficking in cultured macrophages. We suggest that BMP favors the egress of cholesterol from late endosomes via an ORP11-dependent mechanism, resulting in a reduced production of cytotoxic 7-oxysterols.     

Comprehensive proteomic analysis of the effects of purine analogs on human Raji B-cell lymphoma

Cladribine (CdA) and fludarabine (FdAMP) are purine analogs that induce apoptosis in chronic lymphocytic leukemia and non-Hodgkin's lymphoma, but the mechanisms are undefined. The effects of CdA and fludarabine nucleoside (FdA) on the cytosolic, mitochondrial, and nuclear proteomes in human Raji lymphoma cells have been determined using two-dimensional fluorescence difference gel electrophoresis (DIGE) and mass spectrometry. Differentially abundant proteins have provided new insights into CdA- and FdA-induced apoptosis. Treatment with these purine analogs induced changes in proteins involved with intermediary metabolism, cell growth, signal transduction, protein metabolism, and regulation of nucleic acids. Differentially abundant mitochondrial 39S ribosomal protein L50, mTERF domain-containing protein 1, Chitinase-3 like 2 protein, and ubiquinone biosynthesis protein COQ9 have been identified in cells undergoing apoptosis. Up-regulation of several stress-associated proteins found in the endoplasmic reticulum (ER) including GRP78, ERp57, and ORP150 suggests that purine analog-induced apoptosis may result from ER stress and unfolded protein response. While mitochondria-dependent apoptosis has been associated with purine analog cytotoxicity, the likely involvement of the ER stress pathway in CdA- and FdA-induced apoptosis has been shown here for the first time.     

Oxysterol-binding proteins: Sterol and phosphoinositide sensors coordinating transport,signaling and metabolism

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.     

Thank ORP9 for FFAT: With endosomal ORP10, it’s fission accomplished!

Heterogeneity in endosomal membrane phospholipid content is emerging as a regulator of endocytic trafficking pathways. Kawasaki et al. (2021. J. Cell. Biol. https://doi.org/10.1083/jcb.202103141) demonstrate exchange of endosomal PI4P for PS by ORP10 at ER–endosome contact sites, with the consequent recruitment of endosomal fission factors.

Most cellular lipids are synthesized in the ER, often undergoing rapid redistribution to other cellular membranes, thereby maintaining low concentrations at the ER. Consequently, lipids exiting the ER may need to be transported against their concentration gradient. Lipid flow along a gradient to the ER can drive countertransport of ER-derived lipid to membranes with a higher lipid concentration. This nonvesicular lipid exchange occurs at membrane contact sites (MCS), where different organelles are closely apposed, providing a platform for lipid transport proteins including oxysterol-binding protein (OSBP)-related proteins (ORPs). Lipid specificity, which varies between ORPs, is defined by the OSBP-related domain (ORD). The ORD of ORP10 shares phosphatidylinositol-4-phosphate (PI4P) and phosphatidylserine (PS) binding residues with ORP5/8 and can bind and extract PS from liposomes (1), suggesting a potential role in PI4P-PS counter transport, analogous to that of ORP5/8 at ER–plasma membrane MCS (2). ORPs are targeted to specific organelles by interaction between their PH domain and membrane phospholipids. Most ORPs also possess a FFAT motif (two phenylalanines in an acidic tract), which simultaneously targets the ORP to ER-localized VAMP-associated proteins (VAPs) at MCS between the ER and other organelles. ORP10, however, lacks a FFAT motif, yet was found to stabilize ER–Golgi MCSs (Fig. 1 A) for PI4P transport to the ER (2). Kawasaki et al. have now uncovered a novel function for ORP10 in PI4P–PS lipid exchange at the ER–endosome interface (Fig. 1 B), with downstream effects on endosomal fission and retrograde transport (3).Open in a separate windowFigure 1.Regulation of retrograde and secretory traffic by ORP10-mediated lipid exchange. (A) ORP10 interacts with VAP-bound ORP9 at ER–endosome and ER–Golgi MCSs, with downstream effects on retrograde transport of mannose 6-phosphate receptor (M6PR). Boxed region (detailed in B) depicts ORP10 at the ER–endosome interface. (B) ORP10 functions in lipid exchange between the ER and endosomes, transporting endosomal PI4P to the ER in exchange for ER-derived PS. Production of PI4P in endosomes by PI4KIIα-dependent phosphorylation of phosphatidylinositol (PI), coupled with its consumption in the ER by ER-localized Sac1, generates a PI4P concentration gradient from the endosome to the ER. Low membrane PS concentrations in the ER are maintained by PS inhibition of PS synthesis from phosphatidylcholine (PC) by Pss1 or from PE by Pss2, with PS synthesis at ER–endosome contact sites promoting rapid PS export from the ER in yeast (not yet known if a similar mechanism operates in mammalian cells). ORP10 mediates PI4P transport along its gradient to the ER, driving countertransport of PS by ORP10 against its concentration gradient to the endosome. PS enrichment at the endosome leads to recruitment of the ATPase EHD1 to facilitate endosome fission for retrograde transport. (C) Depletion of ORP10 prevents lipid exchange at ER–endosome contact sites, resulting in a loss of retrograde transport of M6PR. Additionally, ER–Golgi MCSs are diminished, and secretion of ApoB-100 is increased.The PH domain of ORP10 selectively binds PI4P and is required for ORP10 recruitment to the TGN (2) and endosomes (3), both home to PI4KIIα, a PI4P-producing kinase. Rapid PI4P degradation at the ER by the phosphatase Sac1 generates a PI4P gradient at the ER–endosome or TGN interface, with PI4P flow to the ER driving countertransport of PS to the endosome (as also predicted for the Golgi). Activity of endosomal PI4P phosphatase Sac2 (4) may hamper formation of an endosome–ER PI4P gradient, but since ORP10 did not colocalise well with Sac2 (3), they likely function at different endosome populations.PS synthesis at MCSs may also contribute to ORP10-mediated lipid exchange. Targeting PS synthase to ER:mitochondria contacts in yeast was found topromote PS transport out of the ER to mitochondria (5). Similarly, ER to endosome PS transport was increased when PS synthase was targeted to ER:endosome MCS. Localized PS gradients from PS synthesis in the ER at MCSs, coupled with rapid decarboxylation of PS to phosphatidylethanolamine (PE) in mitochondria/endosomes by yeast PS decarboxylases Psd1/Psd2, could contribute to lipid exchange. In mammalian cells, though, since no endosomal decarboxylase has been identified, ORP10-mediated lipid exchange is likely to be primarily driven by the PI4P gradient. Whether this process is facilitated by localized activation of PS synthase at MCS has not yet been demonstrated. Since PS synthase activity is negatively regulated by PS, exit from the ER is a key factor in its biogenesis. Recruitment of specific ORPs to endosomes/TGN by PI4P for ER tethering and consequent lipid exchange provides an elegant regulatory pathway for PI4P–PS homeostasis in cellular membranes.ORP10 shares functional similarities with ORP11: both proteins comprise an N-terminal PH domain and a C-terminal ORD, with a linker region in between harboring a coiled-coiled domain. Unlike other ORPs, ORP10 and ORP11 possess neither a FFAT motif nor a membrane spanning domain to enable ER interaction, but heterotypic interaction with ORP9, which does contain a FFAT motif, has been demonstrated for both proteins. Kawasaki et al. identified an ORP9-ORP10 interaction at ER–endosome MCSs that is dependent on the ORP10 linker region. ORP9 was also implicated in ORP10-mediated lipid exchange at the TGN, where it may play a redundant role with OSBP in maintaining ER contact. Similarly, ORP11 is also recruited to the TGN and, to a lesser extent, the endosome, by ORP9, with the interaction depending on the linker region of both proteins (6).The finely tuned regulation of PI4P/PS is emerging as an important determinant of endocytic traffic. Previous studies have shown that endosomal PI4P accumulation inhibits retrograde transport from endosomes to the TGN (7), while endosomal PS regulates endosome to Golgi retrograde traffic. As depicted in Fig. 1 B, Kawasaki et al. have built on this to show that through interaction with VAP-bound ORP9, ORP10 mediates lipid countertransport at ER–endosome MCSs, removing PI4P from, and supplying PS to, the endosome, with consequent recruitment of the membrane scission protein EHD1 to control endosomal fission and retrograde transport (3). Spatial and temporal regulation of endosome fission by ER–endosome MCSs involves recruitment of the ER membrane protein TMCC1 to the budding endosome by the actin regulator Coronin 1C, stabilizing the MCS (7), but the mechanism by which MCS might effect scission has remained elusive. The findings of Kawaski et al. present an explanation: by providing a platform for lipid exchange, MCS promote the recruitment of EHD1, which belongs to a conserved class of ATPases that can oligomerise in ring-like structures around tubules to mediate fission (8). VAP interaction with OSBP at ER–endosome MCSs is also required for retrograde transport (7), but potential redundancy between ORP9/OSBP in ORP10-mediated lipid exchange, or if ORP10 functions at Coronin 1C/TMCC1-regulated MCS is not yet established.Interestingly, ORP10 function at the TGN has been implicated in regulating ApoB-100 secretion (Fig. 1 C), with hypersecretion reported in ORP10-depleted cells (9). FFAP1, which promotes PI4P consumption by Sac1 at ER:TGN contacts, also negatively regulates ApoB-100 exit from the TGN in a PI4KIIIβ-dependent manner, suggesting direct regulation of ApoB-100 secretion by PI4P at the TGN (9). Could PI4P coordinate nutrient sensing with cargo sorting and secretion at the TGN? PI4P has been described as lipid biosensor of cytosolic pH, with protonation of its head group regulating protein interactions (10). The influence of cytosolic pH on ORP10-PI4P interaction may provide an additional layer of regulation of lipoprotein secretion in response to changes in cellular energy/pH.How ORP10 function is coordinated at Golgi and endosomal membranes and the significance of potential redundancy with ORP11 remains unclear. The regulation of Sac2 activity and how it relates to ER-endosome lipid exchange is also intriguing. While questions still remain, an important role for ORP10 is emerging in maintaining homoeostasis between endosome maturation, retrograde traffic and secretory transport.     

VAMP-associated protein-A regulates partitioning of oxysterol-binding protein-related protein-9 between the endoplasmic reticulum and Golgi apparatus

We recently showed that oxysterol-binding protein (OSBP), one of twelve related PH domain containing proteins with lipid and sterol binding activity, interacts with VAMP-associated protein (VAP)-A on the endoplasmic reticulum (ER). In addition to OSBP, seven OSBP-related proteins (ORPs) bind VAP-A via a conserved E-F/Y-F/Y-DA 'FFAT' motif. We focused on this interaction for ORP9, which is expressed as a full-length (ORP9L) or truncated version missing the PH domain (ORP9S). Mutation analysis showed that the interaction required the ORP9 FFAT motif and the N-terminal conserved domain of VAP. Endogenous ORP9L displayed Golgi localization, which was partially mediated by the PH domain based on limited localization of OPR9-PH-GFP with the Golgi apparatus. When inducibly overexpressed, ORP9S and ORP9L colocalized with VAP-A and caused vacuolation of the ER as well as retention of the ER-Golgi intermediate compartment marker ERGIC-53/p58 in the ER. ORP9L mutated in the VAP-A binding domain (ORP9L-FY-->AA) did not localize to the ER but appeared with giantin and Sec31 on large vesicular structures, suggesting the presence of a hybrid Golgi-COPII compartment. Normal Golgi localization was also observed for ORP9L-FY-->AA. Results show that VAP binding and PH domains target ORP9 to the ER and a Golgi-COPII compartment, respectively, and that ORP9L overexpression in these compartments severely perturbed their organization.     

An oxysterol-binding protein family identified in the mouse

Oxysterols are oxygenated derivatives of cholesterol. They have been shown to influence a variety of biological functions including sterol metabolism, lipid trafficking, and apoptosis. Recently, 12 human OSBP-related genes have been identified. In this study, we have identified a family of 12 oxysterol-binding protein (OSBP)-related proteins (ORPs) in the mouse. A high level of amino acid identity (88-97%) was determined between mouse and human ORPs, indicating a very high degree of evolutionary conservation. All proteins identified contained the conserved OSBP amino acid sequence signature motif "EQVSHHPP," and most contained a pleckstrin homology (PH) domain. Using RT-PCR, each mouse ORP gene was found to exhibit a unique tissue distribution with many showing high expression in testicular, brain, and heart tissues. Interestingly, the tissue distribution of ORP-4 and ORP-10 were the most selective within the family. Expression of the various ORP genes was also investigated, specifically in highly purified populations of hemopoietic precursor cells defined by the lin(-) c-kit(+) Sca-1(+) (LKS(+)) and lin(-) c-kit(+) Sca-1(-) (LKS(-)) immunophenotype. Most ORP genes were expressed in both LKS(+) and LKS(-) populations, although ORP-4 appeared to be more highly expressed in the primitive, stem-cell enriched LKS(+) population, whereas ORP-10 was more highly expressed by maturing LKS(-) cells. The identification of a family of ORP proteins in the mouse, the frequently preferred animal model for in vivo studies, should further our understanding of the function of these proteins and their interactions with each other.     

Oxysterol binding proteins (OSBPs) and their encoding genes in Arabidopsis and rice

Cell wall deposition, biosynthesis of steroid hormones, and maintenance of membrane composition and integrity, are some of the crucial functions of sterols in plants. Followed by their synthesis in the endoplasmic reticulum, the sterols accumulate in the plasma membrane. The concept of sterol trafficking in plant cell is not well understood. The oxysterol binding proteins are implicated in sterol transport in non-plant systems. In the study, the oxysterol binding proteins in Arabidopsis and rice are described and classified. The Arabidopsis genome encodes 12 oxysterol binding proteins-related proteins (ORPs) as compared to 6 oxysterol binding proteins (OSBPs/ORPs) in rice. The protein alignment studies reveal that amino acid sequences for oxysterol binding proteins are relatively well conserved in Arabidopsis and rice. The rice OSBPs are classified based on their phylogenetic relationship with Arabidopsis ORPs. The sequence LOGO built on LOC_Os03g16690 indicated presence of fingerprint region of amino acids “EQVSHHPP” for Arabidopsis and rice OSBPs/ORPs. The organization of pleckstrin homology domain is identified in several OSBPs/ORPs in Arabidopsis and rice. The Arabidopsis oligonucleotide array data is explored to understand the expression patterns of ORPs under 17 different experimental conditions. The analysis showed the expression of ORPs in Arabidopsis is necessarily under the control of biotic stress, chemical, elicitor, hormone, light intensity, abiotic stress, and temperature conditions. The linear mean signal values for Arabidopsis ORPs revealed their relative expression patterns in different developmental stages. The genes for ORP3C and ORP3B are highly expressed in all developmental stages that were analyzed. The present study thus indicates crucial functional role of the individual members of this gene family in different environmental stress conditions.     

Brefeldin A, thapsigargin, and AIF4- stimulate the accumulation of GRP78 mRNA in a cycloheximide dependent manner, whilst induction by hypoxia is independent of protein synthesis.

The glucose regulated proteins (GRPs) are major structural components of the endoplasmic reticulum (ER) and are involved in the import, folding, and processing of ER proteins. Expression of the glucose regulated proteins (GRP78 and GRP94) is greatly increased after cells are exposed to stress agents (including A23187 and tunicamycin) which inhibit ER function. Here, we demonstrate that three novel inhibitors of ER function, thapsigargin (which inhibits the ER Ca(2+)-ATPase), brefeldin A (an inhibitor of vesicle transport between the ER and Golgi) and AIF4-, (which inhibits trimeric G-proteins), can increase the expression of both GRP78 and 94. The common characteristic shared by activators of GRP expression is that they disrupt some function of the ER. The increased levels of GRPs may be a response to the accumulation of aberrant proteins in the ER or they may be increased in response to structural/functional damage to the ER. The increased accumulation of GRP78 mRNA after exposure of cells to either thapsigargin, brefeldin A, AIF4-, A23187, or tunicamycin can be blocked by pre-incubation in cycloheximide. In contrast, accumulation of GRPs after exposure to hypoxia was independent of cycloheximide. In addition, the protein kinase inhibitor genistein blocked the thapsigargin induced accumulation of GRP78 mRNA, whereas the protein phosphatase inhibitor okadaic acid caused increased accumulation of GRP78 mRNA. The data indicates that there are at least 2 mechanisms for induced expression of GRPs, one of which involves a phosphorylation step and requires new protein synthesis (e.g., thapsigargin, A23187) and one which is independent of both these steps (hypoxia).     

Bax inhibitor-1 regulates endoplasmic reticulum stress-associated reactive oxygen species and heme oxygenase-1 expression

The Bax inhibitor-1 (BI-1) is an anti-apoptotic protein that is located in endoplasmic reticulum (ER) membranes and protects cells from ER stress-induced apoptosis. The ER is associated with generation of reactive oxygen species (ROS) through oxidative protein folding. This study examined the role of BI-1 in the regulation of ER stress-induced accumulation of ROS and expression of unfolded protein response-associated proteins. BI-1 reduced the expression levels of glucose response protein 78, C/EBP homologous protein, phospho-eukaryotic initiation factor 2alpha, IRE1alpha, XBP-1, and phospho-JNK and inhibited the cleavage of ATF-6alpha p-90, leading to the inhibition of ROS. Although ROS scavengers offer some protection against ER stress-induced apoptosis, the expression of pro-apoptotic ER stress proteins was not affected. This study shows that the response of unfolded proteins is followed by ROS accumulation under ER stress, which is regulated in BI-1 cells. The mechanism for these BI-1-associated functions involves the expression of heme oxygenase-1 (HO-1) through nuclear factor erythroid 2-related factor 2. In BI-1 cells, the transfection of HO-1 small interfering RNA completely abolished the BI-1-induced protection. The endogenous expression of HO-1 through ER stress-initiated ROS is believed to be as a protection signal. In conclusion, these observations suggest that BI-1 can inhibit the ER stress proteins as well as the accumulation of ROS, thereby protecting the cells. Moreover, HO-1 plays an important role in the BI-1-associated protection against ER stress.