Oxysterol-binding Protein-related Protein Research Articles

RILP Induces Cholesterol Accumulation in Lysosomes by Inhibiting Endoplasmic Reticulum-Endolysosome Interactions.

Endoplasmic reticulum (ER)-endolysosome interactions regulate cholesterol exchange between the ER and the endolysosome. ER-endolysosome membrane contact sites mediate the ER-endolysosome interaction. VAP-ORP1L (vesicle-associated membrane protein-associated protein- OSBP-related protein 1L) interaction forms the major contact site between the ER and the lysosome, which is regulated by Rab7. RILP (Rab7-interacting lysosomal protein) is the downstream effector of Rab7, but its role in the organelle interaction between the ER and the lysosome is not clear. In this study, we found RILP interacts with ORP1L to competitively inhibit the formation of the VAP-ORP1L contact site. Immunofluorescence microscopy revealed that RILP induces late endosome/lysosome clustering, which reduces the contact of endolysosomes with the ER, interfering with the ER-endolysosome interaction. Further examination demonstrated that over-expression of RILP results in the accumulation of cholesterol in the clustered endolysosomes, which triggers cellular autophagy depending on RILP. Our results suggest that RILP interferes with the ER-endolysosome interaction to inhibit cholesterol flow from the endolysosome to the ER, which feedbacks to trigger autophagy.

OSBPL2 compound heterozygous variants cause dyschromatosis, ichthyosis, deafness and atopic disease syndrome

PurposeIn this study, we identified and diagnosed a novel inherited condition called Dyschromatosis, Ichthyosis, Deafness, and Atopic Disease (DIDA) syndrome. We present a series of studies to clarify the pathogenic variants and specific mechanism. MethodsExome sequencing and Sanger sequencing was conducted in affected and unaffected family members. A variety of human and cell studies were performed to explore the pathogenic process of keratosis. ResultsOur finding indicated that DIDA syndrome was caused by compound heterozygous variants in the oxysterol-binding protein-related protein 2 (OSBPL2) gene. Furthermore, our findings revealed a direct interaction between OSBPL2 and Phosphoinositide phospholipase C-beta-3 (PLCB3), a key player in hyperkeratosis. OSBPL2 effectively inhibits the ubiquitylation of PLCB3, thereby stabilizing PLCB3. Conversely, OSBPL2 variants lead to enhanced ubiquitination and subsequent degradation of PLCB3, leading to epidermal hyperkeratosis, characterized by aberrant proliferation and delayed terminal differentiation of keratinocytes. ConclusionsOur study not only unveiled the association between OSBPL2 variants and the newly identified DIDA syndrome but also shed light on the underlying mechanism.

Gene editing with an oxathiapiprolin resistance selection marker reveals that PuLLP, a loricrin-like protein, is required for oospore development in Pythium ultimum

Oomycetes, such as Pythium species, contain numerous devastating plant pathogens that inflict substantial economic losses worldwide. Although CRISPR/Cas9-based genome editing is available, the selection markers available for genetic transformation in these species are limited. In this study, a mutated version of the Phytophthora capsici oxysterol-binding protein-related protein 1 (PcMuORP1), known to confer oxathiapiprolin resistance, was introduced into the CRISPR/Cas9 system for in situ complementation in Pythium ultimum. We targeted PuLLP, which encodes a loricrin-like protein, and showed significant downregulation when the Puf RNA-binding protein-encoding gene PuM90 was knocked out. The PuLLP knockout mutants could not produce oospores, indicating a similar biological function as PuM90. The reintroduction of PuLLP into the knockout mutant using PcMuORP1 as a selection marker restored oospore production. Further comparisons with the conventional selection marker NPTII indicated that PcMuORP1 could be applied at a lower concentration and cost, resulting in a higher screening efficiency. Successive subculturing in the absence of selective pressure showed that PcMuORP1 had little long-term effect on the fitness of transformants. Hence, it could be reused as an alternative selection marker. This study demonstrates the successful implementation of the PcMuORP1 gene as a selection marker in the genetic transformation of Py. ultimum and reveals the loricrin-like protein PuLLP as a sexual reproduction-related factor downstream of the Puf RNA-binding protein PuM90. Overall, these results will help accelerate the functional genomic investigation of oomycetes.

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 β3 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.

Lipid transport protein ORP2A promotes glucose signaling by facilitating RGS1 degradation.

Heterotrimeric GTP-binding proteins (G proteins) are a group of regulators essential for signal transmission into cells. Regulator of G protein signaling 1 (AtRGS1) possesses intrinsic GTPase-accelerating protein (GAP) activity and could suppress G protein and glucose signal transduction in Arabidopsis (Arabidopsis thaliana). However, how AtRGS1 activity is regulated is poorly understood. Here, we identified a knockout mutant of oxysterol binding protein-related protein 2A, orp2a-1, which exhibits similar phenotypes to the arabidopsis g-protein beta 1-2 (agb1-2) mutant. Transgenic lines overexpressing ORP2A displayed short hypocotyls, a hypersensitive response to sugar, and lower intracellular AtRGS1 levels than the control. Consistently, ORP2A interacted with AtRGS1 in vitro and in vivo. Tissue-specific expression of 2 ORP2A alternative splicing isoforms implied functions in controlling organ size and shape. Bioinformatic data and phenotypes of orp2a-1, agb1-2, and the orp2a-1 agb1-2 double mutant revealed the genetic interactions between ORP2A and Gβ in the regulation of G protein signaling and sugar response. Both alternative protein isoforms of ORP2A localized in the endoplasmic reticulum (ER), plasma membrane (PM), and ER-PM contact sites and interacted with vesicle-associated membrane protein-associated protein 27-1 (VAP27-1) in vivo and in vitro through their two phenylalanines in an acidic track-like motif. ORP2A also displayed differential phosphatidyl phosphoinositide binding activity mediated by the pleckstrin homology domain in vitro. Taken together, the Arabidopsis membrane protein ORP2A interacts with AtRGS1 and VAP27-1 to positively regulate G protein and sugar signaling by facilitating AtRGS1 degradation.

Structure-Activity Relationships of Ligand Binding to Oxysterol-Binding Protein (OSBP) and OSBP-Related Protein 4.

Oxysterol-binding protein (OSBP) and OSBP-related protein 4 (ORP4) have emerged as potentially druggable targets in antiviral and precision cancer drug development. Multiple structurally diverse small molecules function through targeting the OSBP/ORP family of proteins, including the antiviral steroidal compounds OSW-1 and T-00127-HEV2. Here, the structure-activity relationships of oxysterols and related compound binding to human OSBP and ORP4 are characterized. Oxysterols with hydroxylation at various side chain positions (i.e., C-20, C-24, C-25, and C-27)─but not C-22─confer high affinity interactions with OSBP and ORP4. A library of 20(S)-hydroxycholesterol analogues with varying sterol side chains reveal that side chain length modifications are not well tolerated for OSBP and ORP4 interactions. This side chain requirement is contradicted by the high affinity binding of T-00127-HEV2, a steroidal compound lacking the side chain. The binding results, in combination with docking studies using homology models of OSBP and ORP4, suggest multiple modes of steroidal ligand binding to OSBP and ORP4.

Alpha lipoic acid treatment in late middle age improves cognitive function: Proteomic analysis of the protective mechanisms in the hippocampus

Alpha lipoic acid (ALA), a powerful antioxidant, has the potential to relieve age-related cognitive impairment and neurodegenerative disease. Clinical randomized controlled studies have demonstrated the cognitive improvement effects of lipoic acid in Alzheimer's disease (AD). In the present study, we examined the effects of ALA on cognitive function in ageing mice and its protective mechanisms. Eighteen-month-old male C57BL6/J mice received ALA or normal saline for 2 months. The Morris water maze test revealed improved cognitive function in animals that received ALA. Furthermore, tandem Mass Tags (TMT) based liquid chromotography with mass spectrometry/mass spectrometry (LC-MS/MS) was established to identify the target proteins. The results showed that 10 proteins were changed significantly. Gene Ontology (GO) analysis indicated that the upregulated proteins were enriched in terminal bouton, synaptic transmission and lipid transporter activity while the down-regulated proteins were involved in nuclear transcription factor-κB binding, apoptosis and mitogen-activated protein kinase binding. Based on the GO results, two upregulated proteins oxysterol-binding protein-related protein 10 (OSBPL10) and oligophrenin 1 (OPHN1), and one downregulated protein, CDK5 regulatory subunit-associated protein 3 (CDK5rap3), were validated through Western blotting. The results were consistent with the proteomic results. Modulation of synaptic transmission, lipid transporter activity and neuroinflammation appears to be the mechanisms of ALA in the aged brain.

Arabidopsis ORP2A mediates ER–autophagosomal membrane contact sites and regulates PI3P in plant autophagy

Autophagy is an intracellular degradation system for cytoplasmic constituents which is mediated by the formation of a double-membrane organelle termed the autophagosome and its subsequent fusion with the lysosome/vacuole. The formation of the autophagosome requires membrane from the endoplasmic reticulum (ER) and is tightly regulated by a series of autophagy-related (ATG) proteins and lipids. However, how the ER contacts autophagosomes and regulates autophagy remain elusive in plants. In this study, we identified and demonstrated the roles of Arabidopsis oxysterol-binding protein-related protein 2A (ORP2A) in mediating ER-autophagosomal membrane contacts and autophagosome biogenesis. We showed that ORP2A localizes to both ER-plasma membrane contact sites (EPCSs) and autophagosomes, and that ORP2A interacts with both the ER-localized VAMP-associated protein (VAP) 27-1 and ATG8e on the autophagosomes to mediate the membrane contact sites (MCSs). In ORP2A artificial microRNA knockdown (KD) plants, seedlings display retarded growth and impaired autophagy levels. Both ATG1a and ATG8e accumulated and associated with the ER membrane in ORP2A KD lines. Moreover, ORP2A binds multiple phospholipids and shows colocalization with phosphatidylinositol 3-phosphate (PI3P) invivo. Taken together, ORP2A mediates ER-autophagosomal MCSs and regulates autophagy through PI3P redistribution.

Hepatocyte-Derived Prostaglandin E2-Modulated Macrophage M1-Type Polarization via mTOR-NPC1 Axis-Regulated Cholesterol Transport from Lysosomes to the Endoplasmic Reticulum in Hepatitis B Virus x Protein-Related Nonalcoholic Steatohepatitis.

Lipid metabolic dysregulation and liver inflammation have been reported to be associated with nonalcoholic steatohepatitis (NASH), but the underlying mechanisms remain unclear. Hepatitis B virus x protein (HBx) is a risk factor for NASH. Based on metabolomic and transcriptomic screens and public database analysis, we found that HBx-expressing hepatocyte-derived prostaglandin E2 (PGE2) induced macrophage polarization imbalance via prostaglandin E2 receptor 4 (EP4) through in vitro, ex vivo, and in vivo models. Here, we revealed that the M1-type polarization of macrophages induced by endoplasmic reticulum oxidoreductase-1-like protein α (ERO1α)-dependent endoplasmic reticulum stress was associated with the HBx-related hepatic NASH phenotype. Mechanistically, HBx promoted Niemann–Pick type C1 (NPC1)/oxysterol-binding protein-related protein 5 (ORP5)-mediated cholesterol transport from the lysosome to the endoplasmic reticulum via mammalian target of rapamycin (mTOR) activation. This study provides a novel basis for screening potential biomarkers in the macrophage mTOR–cholesterol homeostasis–polarization regulatory signaling pathway and evaluating targeted interventions for HBx-associated NASH.

A phosphoinositide signalling pathway mediates rapid lysosomal repair.

Lysosomal dysfunction has been increasingly linked to disease and normal ageing1,2. Lysosomal membrane permeabilization (LMP), a hallmark of lysosome-related diseases, can be triggered by diverse cellular stressors3. Given the damaging contents of lysosomes, LMP must be rapidly resolved, although the underlying mechanisms are poorly understood. Here, using an unbiased proteomic approach, we show that LMP stimulates a phosphoinositide-initiated membrane tethering and lipid transport (PITT) pathway for rapid lysosomal repair. Upon LMP, phosphatidylinositol-4 kinase type 2α (PI4K2A) accumulates rapidly on damaged lysosomes, generating high levels of the lipid messenger phosphatidylinositol-4-phosphate. Lysosomal phosphatidylinositol-4-phosphate in turn recruits multiple oxysterol-binding protein (OSBP)-related protein (ORP) family members, including ORP9, ORP10, ORP11 and OSBP, to orchestrate extensive new membrane contact sites between damaged lysosomes and the endoplasmic reticulum. The ORPs subsequently catalyse robust endoplasmic reticulum-to-lysosome transfer of phosphatidylserine and cholesterol to support rapid lysosomal repair. Finally, the lipid transfer protein ATG2 is also recruited to damaged lysosomes where its activity is potently stimulated by phosphatidylserine. Independent of macroautophagy, ATG2 mediates rapid membrane repair through direct lysosomal lipid transfer. Together, our findings identify that the PITT pathway maintains lysosomal membrane integrity, with important implications for numerous age-related diseases characterized by impaired lysosomal function.

The involvement of oxysterol-binding protein related protein (ORP) 6 in the counter-transport of phosphatidylinositol-4-phosphate (PI4P) and phosphatidylserine (PS) in neurons

Oxysterol-binding protein (OSBP)-related protein (ORP) 6, a member of subfamily III in the ORP family, localizes to membrane contact sites between the endoplasmic reticulum (ER) and other organelles and functions in non-vesicular exchange of lipids including phosphatidylinositol-4-phosphate (PI4P) in neurons. In this study, we searched for the lipid counter-transported in exchange for PI4P by using molecular cell biology techniques. Deconvolution microscopy revealed that knockdown of ORP6 partially shifted localization of a phosphatidylserine (PS) marker but not filipin in primary cultured cerebellar neurons. Overexpression of ORP6 constructs lacking the OSBP-related ligand binding domain (ORD) resulted in the same shift of the PS marker. A PI4KⅢα inhibitor specifically inhibiting the synthesis and plasma membrane (PM) localization of PI4P, suppressed the localization of ORP6 and the PS marker at the PM. Overexpression of mutant PS synthase 1 (PSS1) inhibited transport of the PS marker to the PM and relocated the PI4P marker to the PM in Neuro-2A cells. Introduction of ORP6 but not the dominant negative ORP6 constructs, shifted the localization of PS back to the PM. These data collectively suggest the involvement of ORP6 in the counter-transport of PI4P and PS.

Liver X receptor-agonist treatment rescues degeneration in a Drosophila model of hereditary spastic paraplegia

Hereditary spastic paraplegias (HSPs) are a group of inherited, progressive neurodegenerative conditions characterised by prominent lower-limb spasticity and weakness, caused by a length-dependent degeneration of the longest corticospinal upper motor neurons. While more than 80 spastic paraplegia genes (SPGs) have been identified, many cases arise from mutations in genes encoding proteins which generate and maintain tubular endoplasmic reticulum (ER) membrane organisation. The ER-shaping proteins are essential for the health and survival of long motor neurons, however the mechanisms by which mutations in these genes cause the axonopathy observed in HSP have not been elucidated. To further develop our understanding of the ER-shaping proteins, this study outlines the generation of novel in vivo and in vitro models, using CRISPR/Cas9-mediated gene editing to knockout the ER-shaping protein ADP-ribosylation factor-like 6 interacting protein 1 (ARL6IP1), mutations in which give rise to the HSP subtype SPG61. Loss of Arl6IP1 in Drosophila results in progressive locomotor deficits, emulating a key aspect of HSP in patients. ARL6IP1 interacts with ER-shaping proteins and is required for regulating the organisation of ER tubules, particularly within long motor neuron axons. Unexpectedly, we identified physical and functional interactions between ARL6IP1 and the phospholipid transporter oxysterol-binding protein-related protein 8 in both human and Drosophila model systems, pointing to a conserved role for ARL6IP1 in lipid homeostasis. Furthermore, loss of Arl6IP1 from Drosophila neurons results in a cell non-autonomous accumulation of lipid droplets in axonal glia. Importantly, treatment with lipid regulating liver X receptor-agonists blocked lipid droplet accumulation, restored axonal ER organisation, and improved locomotor function in Arl6IP1 knockout Drosophila. Our findings indicate that disrupted lipid homeostasis contributes to neurodegeneration in HSP, identifying a potential novel therapeutic avenue for the treatment of this disorder.

Matching mouse models to specific human liver disease states by comparative functional genomics of mouse and human datasets

Omics has broadened our view of transcriptional and gene regulatory networks of multifactorial diseases, such as metabolism associated liver disease and its advanced stages including hepatocellular carcinoma. Identifying liver disease biomarkers and potential treatment targets makes use of experimental models, e.g. genetically engineered mice, which show molecular features of human pathologies but are experimentally tractable. We compared gene expression profiling data from human to our studies on transgenic mice with hepatocyte deletion of Cyp51 from cholesterol synthesis with the aim of identifying the human liver disease state best matched by the Cyp51 knockout model. Gene Expression Omnibus was used to identify relevant human datasets. We identified enriched and deregulated genes, pathways and transcription factors of mouse and human disease samples. Analysis showed a closer match of the Cyp51 knockout to the female patient samples. Importantly, CYP51 was depleted in both mouse and female human data. Among the enriched genes were the oxysterol-binding protein-related protein 3 (OSBPL3), which was enriched in all datasets, and the collagen gene COL1A2, which was enriched in both the mouse and one human dataset. KEGG and Reactome analyses revealed the most enriched pathway to be ECM-receptor interaction. Numerous transcription factors were differentially expressed in mice of both sexes and in the human female dataset, while depleted HNF4α and RXRα:PPARα-isoform1 were a hallmark in all cases. Our analysis exposed novel potential biomarkers, which may provide new avenues towards more personalized approaches and different targets in females and males. The analysis was only possible because of availability of open data resources and tools and broadly consistent annotation.

Osh6 Revisited: Control of PS Transport by the Concerted Actions of PI4P and Sac1 Phosphatase.

Osh6, a member of the oxysterol-binding protein–related protein (ORP) family, is a lipid transport protein that is involved in the transport of phosphatidylserine (PS) between the endoplasmic reticulum (ER) and the plasma membrane (PM). We used a biophysical approach to characterize its transport mechanism in detail. We examined the transport of all potential ligands of Osh6. PI4P and PS are the best described lipid cargo molecules; in addition, we showed that PIP2 can be transported by Osh6 as well. So far, it was the exchange between the two cargo molecules, PS and PI4P, in the lipid-binding pocket of Osh6 that was considered an essential driving force for the PS transport. However, we showed that Osh6 can efficiently transport PS along the gradient without the help of PI4P and that PI4P inhibits the PS transport along its gradient. This observation highlights that the exchange between PS and PI4P is indeed crucial, but PI4P bound to the protein rather than intensifying the PS transport suppresses it. We considered this to be important for the transport directionality as it prevents PS from returning back from the PM where its concentration is high to the ER where it is synthesized. Our results also highlighted the importance of the ER resident Sac1 phosphatase that enables the PS transport and ensures its directionality by PI4P consumption. Furthermore, we showed that the Sac1 activity is regulated by the negative charge of the membrane that can be provided by PS or PI anions in the case of the ER membrane.

Cortactin knockdown results in disruption of basal TBCs and alters turnover of Sertoli cell ESs in Rattus norvegicus

Here we explore the prediction that long-term knockdown of cortactin (CTTN), a component of tubulobulbar complexes (TBCs), disrupts TBCs in Sertoli cells and alters the turnover of basal ectoplasmic specializations (ESs). In rats, intratesticular injections of siRNA targeting CTTN (siCTTN) in one testis and nontargeting siRNA (siControl) in the contralateral testis were done on days 0, 2, 4, 6, and 8. The experiment was terminated on day 9 and testes were analyzed by either western blotting, or by stimulated emission depletion (STED), electron and/or conventional fluorescence microscopy. Levels of CTTN were successfully knocked down in experimental testes compared to controls. When cryo-sections were labeled for actin filaments, or CTTN, and oxysterol binding protein-related protein 9 (ORP9) and analyzed by STED microscopy, TBCs were "less distinct" than in tubules of the same stages from control testes. When analyzed by electron microscopy, redundant clumps of basal actin filament containing ESs were observed in experimental sections. Using labeling of actin filaments in ESs, thresholding techniques were used to calculate the number of pixels above threshold per unit length of tubule wall in seminiferous tubules at Stage VII. Median values were higher in experimental testes relative to controls in the four animals analyzed. Although we detected subtle differences in ES turnover, we were unable to demonstrate changes in spermatocyte translocation or in the levels of junction proteins at the sites. Our results are the first to demonstrate that perturbation of basal TBCs alters the turnover of actin-related junctions (ESs).

Oxysterol-binding protein-like 2 contributes to the developmental progression of preadipocytes by binding to \u03b2-catenin

Oxysterol-binding protein-like 2 (OSBPL2), also known as oxysterol-binding protein-related protein (ORP) 2, is a member of lipid transfer protein well-known for its role in regulating cholesterol homeostasis. A recent study reported that OSBPL2/ORP2 localizes to lipid droplets (LDs) and is associated with energy metabolism and obesity. However, the function of OSBPL2/ORP2 in adipocyte differentiation is poorly understood. Here, we report that OSBPL2/ORP2 contributes to the developmental progression of preadipocytes. We found that OSBPL2/ORP2 binds to β-catenin, a key effector in the Wnt signaling pathway that inhibits adipogenesis. This complex plays a role in regulating the protein level of β-catenin only in preadipocytes, not in mature adipocytes. Our data further indicated that OSBPL2/ORP2 mediates the transport of β-catenin into the nucleus and thus regulates target genes related to adipocyte differentiation. Deletion of OSBPL2/ORP2 markedly reduces β-catenin both in the cytoplasm and in the nucleus, promotes preadipocytes maturation, and ultimately leads to obesity-related characteristics. Altogether, we provide novel insight into the function of OSBPL2/ORP2 in the developmental progression of preadipocytes and suggest OSBPL2/ORP2 may be a potential therapeutic target for the treatment of obesity-related diseases.

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

Oxysterol-binding protein-related protein 2 (ORP2), a cholesterol-PI(4,5)P2 countercurrent transporter, was recently identified as a novel regulator of plasma membrane (PM) cholesterol and PI(4,5)P2 content in HeLa cells. Here, we investigate the role of ORP2 in endothelial cell (EC) cholesterol and PI(4,5)P2 distribution, angiogenic signaling, and angiogenesis. We show that ORP2 knock-down modifies the distribution of cholesterol accessible to a D4H probe, between late endosomes and the PM. Depletion of ORP2 from ECs inhibits their angiogenic tube formation capacity, alters the gene expression of angiogenic signaling pathways such as VEGFR2, Akt, mTOR, eNOS, and Notch, and reduces EC migration, proliferation, and cell viability. We show that ORP2 regulates the integrity of VEGFR2 at the PM in a cholesterol-dependent manner, the depletion of ORP2 resulting in proteolytic cleavage by matrix metalloproteinases, and reduced activity of VEGFR2 and its downstream signaling. We demonstrate that ORP2 depletion increases the PM PI(4,5)P2 coincident with altered F-actin morphology, and reduces both VEGFR2 and cholesterol in buoyant raft membranes. Moreover, ORP2 knock-down suppresses the expression of the lipid raft-associated proteins VE-cadherin and caveolin-1. Analysis of the retinal microvasculature in ORP2 knock-out mice generated during this study demonstrates the subtle alterations of morphology characterized by reduced vessel length and increased density of tip cells and perpendicular sprouts. Gene expression changes in the retina suggest disturbance of sterol homeostasis, downregulation of VE-cadherin, and a putative disturbance of Notch signaling. Our data identifies ORP2 as a novel regulator of EC cholesterol and PI(4,5)P2 homeostasis and cholesterol-dependent angiogenic signaling.

Downregulation of ORP3 Correlates with Reduced Survival of Colon Cancer Patients with Advanced Nodal Metastasis and of Female Patients with Grade 3 Colon Cancer.

Genome instability is an essential hallmark in tumor development, including colorectal cancer. We have recently identified the oxysterol binding protein-related protein 3 (ORP3), also known as oxysterol binding protein-like 3 (OSBPL3), as a novel ploidy-control gene, whose knock-out leads to aneuploidy induction and promotes tumor formation, indicating that ORP3 is a bona fide tumor suppressor protein. Here we analyzed expression of ORP3 in a cohort (n = 206) of colon cancer patients in relation to patient survival. We show that low ORP3 mRNA levels correlate with reduced survival of patients with advanced nodal metastasis (N2). While patient survival does not associate with grading when the whole cohort is evaluated, importantly, low ORP3 mRNA levels associate with worse survival of female patients with grade 3 colon cancer. Similarly, low ORP3 mRNA levels associate with worse survival of grade 3 colon cancer patients 70 years of age and younger while low ORP3 mRNA levels seem to be beneficial for colon cancer patients with a T2 tumor size. Together, the data show that ORP3 expression is downregulated during colon cancer progression, which correlates with reduced patient survival. Thus, ORP3 mRNA levels may be a prognostic marker for better stratification of colon cancer patients.

The crystal structure of ORP3 reveals the conservative PI4P binding pattern

Oxysterol-binding protein (OSBP) and its related protein (ORP) constitute a conserved family of lipid transfer proteins (LTPs). ORPs have been implicated as intracellular lipid exchanger and sensor in recent years, which regulate the lipid homeostasis and signal pathway. OSBP-related protein 3 plays key role in controlling cell adhesion and migration and could be developed as the drug target for cancer therapy. Here, we report the crystal structures of human ORP3 ORD to 2.1 Å and ORD-PI4P complex to 3.2 Å. The binding assay in vitro confirms the ORP3 has the capability of PI4P binding. This study further verifies that the PI4P is the common ligand of all ORPs and ORPs should be the lipid exchanger in membrane contact sites(MCS).

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

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