OSBP-related Proteins Research Articles

FvOshC Is a Key Global Regulatory Target in Fusarium verticillioides for Fumonisin Biosynthesis and Disease Control.

Fusarium verticillioides has a substantial impact on maize production, commonly leading to maize ear rot and the production of fumonisin, a mycotoxin that poses health risks to both humans and animals. Currently, there is a lack of molecular targets for preventing the disease and controlling the toxin. The biological functions of oxysterol-binding proteins (OSBP) in filamentous fungi remain unclear. In this research, 7 oxysterol-binding protein-related proteins were identified in F. verticillioides, and these proteins were obtained through prokaryotic expression and purification. FvOshC was identified as the specific protein that binds to ergosterol through fluorescence titration. Gene knockout complementation techniques confirmed that FvOSHC plays a positive role, establishing it as a novel global regulatory protein involved in the pathogenicity and FB1 biosynthesis in F. verticillioides. Additionally, the interaction between FvOshC and FvSec14 was identified using yeast two-hybrid techniques. Moreover, computer-aided drug design technology was utilized to identify the receptor molecule Xanthatin based on FvOshC. The inhibitory effect of Xanthatin on the growth of F. verticillioides and the synthesis of FB1 was significantly demonstrated. These findings provide valuable insights that can aid in the management of mycotoxin pollution.

Abstract 5778: Determining the structure and function relationship of the oxysterol-binding protein (OSBP) to guide drug development

Abstract The human Oxysterol Binding Protein Structure and function relationship remains elusive, yet the anti-cancer and antiviral molecules library targeting OSBP and OSBP-Related proteins (ORPs) continues to grow. OSBP is a ubiquitously expressed protein in most eukaryotes, functioning as a master lipid sensor and cholesterol regulator in cells. OSBP and its closely related protein ORP4 are also reported to be essential in several human diseases, including viral infection and cancer cell proliferation. Our lab has shown that the natural product OSW-1 has potential anti-viral and anti-cancer activity through targeting OSBP and ORP4, as confirmed with protein-ligand interactions that showed binding with high affinity. Moreover, several anti-cancer molecules targeting the ORPs provoke different protein conformation changes and, therefore, lead to varying OSBP functions. However, the structure of full-length OSBP is still unknown, which leaves a gap in knowledge regarding the compounds’ mechanism of action or how OSBP could be established as a molecular anti-cancer target. Our goal is to determine the structure and function relationship of OSBP with emphasis on the effect of small molecule ligands on cellular OSBP levels and function. In pursuit of this research goal, we aim to determine the in vitro activity of OSW-1 analogs (OA) isolated in our lab, as well as determine their interaction with OSBP and ORP4 proteins in multiple cancer cell lines. We mainly seek to determine the structure and ligand interaction with the OSBP-ligand binding domain (LBD) and full-length OSBP to shed light on the conformation changes that occur upon ligand binding. Preliminary data indicate that the tested OA compounds are active in multiple cancer cell lines with reduced OSBP levels effect, suggesting a similar mechanism of action to OSW-1. A transient expression of full-length OSBP and LBD showed that the truncated OSBP (LBD) was mostly found in the pellet, which could imply low solubility, while the full-length OSBP showed sufficient solubility. Our efforts are currently focused on purifying full-length OSBP. We are also working towards purifying full-length OSBP with a cleavage site that allows cutting out the LBD, thus solving the insolubility issue. Once obtained, the full-length OSBP and the ligand binding domain will be used for both binding and structure determination studies. Citation Format: Ruth Fiona Bayimenye, Jorge L. Berrios-Rivera, Susan Nimmo, Christina R. Bourne, Anthony W. Burgett. Determining the structure and function relationship of the oxysterol-binding protein (OSBP) to guide drug development [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5778.

Reconstitution of ORP-mediated lipid exchange coupled to PI4P metabolism

Oxysterol-binding protein-related proteins (ORPs) play key roles in the distribution of lipids in eukaryotic cells by exchanging sterol or phosphatidylserine for PI4P between the endoplasmic reticulum (ER) and other cell regions. However, it is unclear how their exchange capacity is coupled to PI4P metabolism. To address this question quantitatively, we analyze the activity of a representative ORP, Osh4p, in an ER/Golgi interface reconstituted with ER- and Golgi-mimetic membranes functionalized with PI4P phosphatase Sac1p and phosphatidylinositol (PI) 4-kinase, respectively. Using real-time assays, we demonstrate that upon adenosine triphosphate (ATP) addition, Osh4p creates a sterol gradient between these membranes, relying on the spatially distant synthesis and hydrolysis of PI4P, and quantify how much PI4P is needed for this process. Then, we develop a quantitatively accurate kinetic model, validated by our data, and extrapolate this to estimate to what extent PI4P metabolism can drive ORP-mediated sterol transfer in cells. Finally, we show that Sec14p can support PI4P metabolism and Osh4p activity by transferring PI between membranes. This study establishes that PI4P synthesis drives ORP-mediated lipid exchange and that ATP energy is needed to generate intermembrane lipid gradients. Furthermore, it defines to what extent ORPs can distribute lipids in the cell and reassesses the role of PI-transfer proteins in PI4P metabolism.

Orthogonal Targeting of SAC1 to Mitochondria Implicates ORP2 as a Major Player in PM PI4P Turnover.

Oxysterol-binding protein (OSBP)-related proteins (ORPs) 5 and 8 have been shown to deplete the lipid phosphatidylinositol 4-phosphate (PI4P) at sites of membrane contact between the endoplasmic reticulum (ER) and plasma membrane (PM). This is believed to be caused by transport of PI4P from the PM to the ER, where PI4P is degraded by an ER-localized SAC1 phosphatase. This is proposed to power the anti-port of phosphatidylserine (PS) lipids from ER to PM, up their concentration gradient. Alternatively, ORPs have been proposed to sequester PI4P, dependent on the concentration of their alternative lipid ligand. Here, we aimed to distinguish these possibilities in living cells by orthogonal targeting of PI4P transfer and degradation to PM-mitochondria contact sites. Surprisingly, we found that orthogonal targeting of SAC1 to mitochondria enhanced PM PI4P turnover independent of targeting to contact sites with the PM. This turnover could be slowed by knock-down of soluble ORP2, which also has a major impact on PM PI4P levels even without SAC1 over-expression. The data reveal a role for contact site-independent modulation of PM PI4P levels and lipid antiport.

Getting to Grips with the Oxysterol-Binding Protein Family - a Forty Year Perspective.

This review discusses how research around the oxysterol-binding protein family has evolved. We briefly summarize how this protein family, designated OSBP-related (ORP) or OSBP-like (OSBPL) proteins, was discovered, how protein domains highly conserved among family members between taxa paved the way for understanding their mechanisms of action, and how insights into protein structural and functional features help to understand their versatility as lipid transporters. We also discuss questions and future avenues of research opened by these findings. The investigations on oxysterol-binding protein family serve as a real-life example of the notion that science often advances as a collective effort of multiple lines of enquiry, including serendipitous routes. While original articles invariably explain the motivation of the research undertaken in rational terms, the actual paths to findings may be less intentional. Fortunately, this does not reduce the impact of the discoveries made. Besides hopefully providing a useful account of ORP family proteins, we aim to convey this message.

Silencing of GhORP_A02 enhances drought tolerance in Gossypium hirsutum

BackgroundORP (Oxysterol-binding protein-related proteins) genes play a role in lipid metabolism, vesicular transferring and signaling, and non-vesicular sterol transport. However, no systematic identification and analysis of ORP genes have been reported in cotton.ResultIn this study, we identified 14, 14, 7, and 7 ORP genes in G. hirsutum, G. barbadense, G. arboreum, and G. raimondii, respectively. Phylogenetic analysis showed that all ORP genes could be classified into four groups. Gene structure and conserved motif analysis suggest that the function of this gene family was conserved. The Ka/Ks analysis showed that this gene family was exposed to purifying selection during evolution. Transcriptome data showed that four ORP genes, especially GhORP_A02, were induced by abiotic stress treatment. The cis-acting elements in the ORP promoters were responsive to phytohormones and various abiotic stresses. The silenced plants of GhORP_A02 were more sensitive to drought stress when compared to control.ConclusionThe major finding of this study shed light on the potential role of ORP genes in abiotic stress and provided a fundamental resource for further analysis in cotton.

LIPID transfer proteins regulate store-operated calcium entry via control of plasma membrane phosphoinositides

The ER-resident proteins STIM1 together with the plasma membrane (PM)-localized Orai1 channels constitute the molecular components of the store-operated Ca2+ entry (SOCE) pathway. Prepositioning of STIM1 to the peripheral ER close to the PM ensures its efficient interaction with Orai1 upon a decrease in the ER luminal Ca2+ concentration. The C-terminal polybasic domain of STIM1 has been identified as mediating the interaction with PM phosphoinositides and hence positions the molecule to ER-PM contact sites. Here we show that STIM1 requires PM phosphatidylinositol 4-phosphate (PI4P) for efficient PM interaction. Accordingly, oxysterol binding protein related proteins (ORPs) that work at ER-PM junctions and consume PI4P gradients exert important control over the Ca2+ entry process. These studies reveal an important connection between non-vesicular lipid transport at ER-PM contact sites and regulation of ER Ca2+store refilling.

The lipid transporter proteins ORP5 and ORP8 in Ca2+signaling

Oxysterol-binding protein (OSBP)-related proteins (ORPs), a family of lipid transfer proteins (LTPs), mediate the exchange of the lipids PI(4)P and phosphatidylserine (PtdSer) between the plasma membrane (PM) and the endoplasmic reticulum (ER). ORP5 and ORP8 are located and function at membrane contact sites (MCSs), including the ER/PM junctions. MCSs between the ER and PM are essential platform for Ca2+ signaling complexes and within them the ER Ca2+ sensor STIM1 and the PM Ca2+ influx channel Orai1.

Molecular dynamics study of binding mechanism of a peripheral membrane protein in a single and dual membrane environment

Lipid transfer proteins namely, oxysterol-binding protein-related proteins regulate trafficking of lipids across cellular compartments to maintain diverse functional and structural heterogeneity of organelles. The peripheral membrane protein, Osh4, is the abundantly expressed oxysterol binding protein in yeast. However, the functional mechanism of Osh4 in lipid transport is still not well understood. Several experimental studies have suggested that yeast Osh proteins operate at membrane contact sites (MCSs) and shuttles lipid across membranes in a direct fashion or via diffusing through cytoplasm.

Fungal oxysterol-binding protein-related proteins promote pathogen virulence and activate plant immunity.

Oxysterol-binding protein-related proteins (ORPs) are a conserved class of lipid transfer proteins that are closely involved in multiple cellular processes in eukaryotes, but their roles in plant-pathogen interactions are mostly unknown. We show that transient expression of ORPs of Magnaporthe oryzae (MoORPs) in Nicotiana benthamina plants triggered oxidative bursts and cell death; treatment of tobacco Bright Yellow-2 suspension cells with recombinant MoORPs elicited the production of reactive oxygen species. Despite ORPs being normally described as intracellular proteins, we detected MoORPs in fungal culture filtrates and intercellular fluids from barley plants infected with the fungus. More importantly, infiltration of Arabidopsis plants with recombinant Arabidopsis or fungal ORPs activated oxidative bursts, callose deposition, and PR1 gene expression, and enhanced plant disease resistance, implying that ORPs may function as endogenous and exogenous danger signals triggering plant innate immunity. Extracellular application of fungal ORPs exerted an opposite impact on salicylic acid and jasmonic acid/ethylene signaling pathways. Brassinosteroid Insensitive 1-associated Kinase 1 was dispensable for the ORP-activated defense. Besides, simultaneous knockout of MoORP1 and MoORP3 abolished fungal colony radial growth and conidiation, whereas double knockout of MoORP1 and MoORP2 compromised fungal virulence on barley and rice plants. These observations collectively highlight the multifaceted role of MoORPs in the modulation of plant innate immunity and promotion of fungal development and virulence in M. oryzae.

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.

Research advances in oxysterol-binding protein-related proteins

Research advances in oxysterol-binding protein-related proteins

Functions of Oxysterol-Binding Proteins at Membrane Contact Sites and Their Control by Phosphoinositide Metabolism.

Lipids must be correctly transported within the cell to the right place at the right time in order to be fully functional. Non-vesicular lipid transport is mediated by so-called lipid transfer proteins (LTPs), which contain a hydrophobic cavity that sequesters lipid molecules. Oxysterol-binding protein (OSBP)-related proteins (ORPs) are a family of LTPs known to harbor lipid ligands, such as cholesterol and phospholipids. ORPs act as a sensor or transporter of those lipid ligands at membrane contact sites (MCSs) where two different cellular membranes are closely apposed. In particular, a characteristic functional property of ORPs is their role as a lipid exchanger. ORPs mediate counter-directional transport of two different lipid ligands at MCSs. Several, but not all, ORPs transport their lipid ligand from the endoplasmic reticulum (ER) in exchange for phosphatidylinositol 4-phosphate (PI4P), the other ligand, on apposed membranes. This ORP-mediated lipid “countertransport” is driven by the concentration gradient of PI4P between membranes, which is generated by its kinases and phosphatases. In this review, we will discuss how ORP function is tightly coupled to metabolism of phosphoinositides such as PI4P. Recent progress on the role of ORP-mediated lipid transport/countertransport at multiple MCSs in cellular functions will be also discussed.

Structure of human ORP3 ORD reveals conservation of a key function and ligand specificity in OSBP-related proteins.

Human ORP3 belongs to the oxysterol-binding protein (OSBP) family of lipid transfer proteins and is involved in lipid trafficking and cell signaling. ORP3 localizes to the ER-PM interfaces and is implicated in lipid transport and focal adhesion dynamics. Here, we report the 2.6-2.7 Å structures of the ORD (OSBP-related domain) of human ORP3 in apo-form and in complex with phosphatidylinositol 4-phosphate. The ORP3 ORD displays a helix grip β-barrel fold with a deep hydrophobic pocket which is conserved in the OSBP gene family. ORP3 binds PI(4)P by the residues around tunnel entrance and in the hydrophobic pocket, whereas it lacks sterol binding due to the narrow hydrophobic tunnel. The heterologous expression of the ORDs of human ORP3 or OSBP1 rescued the lethality of seven ORP (yeast OSH1-OSH7) knockout in yeast. In contrast, the PI(4)P-binding site mutant of ORP3 did not complement the OSH knockout cells. The N-terminal PH domain and FFAT motif of ORP3 are involved in protein targeting but are not essential in yeast complementation. This observation suggests that the essential function conserved in the ORPs of yeast and human is mediated by PI(4)P-binding of the ORD domain. This study suggests that the non-vesicular PI(4)P transport is a conserved function of all ORPs in eukaryotes.

Fluorescence-Based Measurements of Phosphatidylserine/Phosphatidylinositol 4-Phosphate Exchange Between Membranes

Several members of the evolutionarily conserved oxysterol-binding protein (OSBP)-related proteins(ORP)/OSBP homologs (Osh) family have recently been found to represent a novel lipid transfer protein (LTP) group in yeast and human cells. They transfer phosphatidylserine (PS) from the endoplasmic reticulum (ER) to the plasma membrane (PM) via PS/phosphatidylinositol 4-phosphate (PI(4)P) exchange cycles. This finding allows a better understanding of how PS, which is critical for signaling processes, is distributed throughout the cell and the investigation of the link between this process and phosphoinositide (PIP) metabolism. The development of new fluorescence-based protocols has been instrumental in the discovery and characterization of this new cellular mechanism in vitro at the molecular level. This paper describes the production and the use of two fluorescently labelled lipid sensors, NBD-C2Lact and NBD-PHFAPP, to measure the ability of a protein to extract PS or PI(4)P and to transfer these lipids between artificial membranes. First, the protocol describes how to produce, label, and obtain high-purity samples of these two constructs. Secondly, this paper explains how to use these sensors with a fluorescence microplate reader to determine whether a protein can extract PS or PI(4)P from liposomes, using Osh6p as a case study. Finally, this protocol shows how to accurately measure the kinetics of PS/PI(4)P exchange between liposomes of defined lipid composition and to determine lipid transfer rates by fluorescence resonance energy transfer (FRET) using a standard fluorometer.

<i>In Silico</i> Study on Interaction and Preliminary Toxicity Prediction of <i>Eleutherine americana</i> Components as an Antifungal and Antitoxoplasmosis Candidate

Red bulbs of Eleutherine americana (Aubl.) Merr. ex K. Heyne has been known for its high content of naphthoquinones that have antifungal and antiparasitic activities. In this research, in silico interaction study was performed between 31 compounds reported to be found in E. americana with the selected target proteins for antifungal and antitoxoplasmosis activity using the molecular docking method. An ORPs (OSBP-related proteins), Osh4 (PDB ID: 1ZHX), and N-myristoyltransferase (Nmt, PDB ID: 1IYL) were used as the antifungal target proteins. Toxoplasma gondii purine nucleoside phosphorylase (TgPNP, PDB ID: 3MB8) and calcium-dependent protein kinase-1 (TgCDPK1, PDB ID: 4M84) were used as antitoxoplasmosis target proteins. Three-dimensional structures of the test compounds were made and optimized using GaussView 6.0 and Gaussian 09W. The target proteins were prepared using the Discovery Studio 2016 Program. Aquatic toxicity prediction as the preliminary assessment of the safety of the compounds was performed using ECOSAR v2.0. The results suggest that the compound having both the smallest free binding energy compared with positive control and other test compounds and low predicted toxicity is β-sitosterol with a free binding energy of ‒11.55 and ‒11.18 kcal/mol towards Osh4 and Nmt and ‒8.06 and ‒10.29 kcal/mol towards TgPNP and TgCDPK1, respectively.

Membrane Contact Sites and Organelles Interaction in Plant Autophagy.

Autophagy is an intracellular trafficking and degradation system for recycling of damaged organelles, mis-folded proteins and cytoplasmic constituents. Autophagy can be divided into non-selective autophagy and selective autophagy according to the cargo specification. Key to the process is the timely formation of the autophagosome, a double-membrane structure which is responsible for the delivery of damaged organelles and proteins to lysosomes or vacuoles for their turnover. Autophagosomes are formed by the closure of cup-shaped phagophore which depends on the proper communication with membrane contributors. The endoplasmic reticulum (ER) is a major membrane source for autophagosome biogenesis whereby the ER connects with phagophore through membrane contact sites (MCSs). MCSs are closely apposed domains between organelle membranes where lipids and signals are exchanged. Lipid transfer proteins (LTPs) are a large family of proteins including Oxysterol-binding protein related proteins (ORP) which can be found at MCSs and mediate lipid transfer in mammals and yeast. In addition, interaction between autophagosomes and other organelles can also be detected in selective autophagy for selection and degradation of various damaged organelles. Selective autophagy is mediated by the binding of a receptor or an adaptor between a cargo and an autophagosome. Here we summarize what we know about the MCS between autophagosomes and other organelles in eukaryotes. We then discuss progress in our understanding about ORPs at MCSs in plants and the underlying mechanisms of selective autophagy in plants with a focus on receptors/adaptors that are involved in the interaction of the autophagosome with other cytoplasmic constituents, including the Neighbor of BRCA1 gene 1 (NBR1), ATG8-interacting protein 1 (ATI1), Regulatory Particle Non-ATPase 10 (RPN10), and Dominant Suppressor of KAR2 (DSK2).

ORP3 phosphorylation regulates phosphatidylinositol 4-phosphate and Ca2+ dynamics at plasma membrane-ER contact sites.

Oxysterol-binding protein (OSBP)-related proteins (ORPs) mediate non-vesicular lipid transfer between intracellular membranes. Phosphoinositide (PI) gradients play important roles in the ability of OSBP and some ORPs to transfer cholesterol and phosphatidylserine between the endoplasmic reticulum (ER) and other organelle membranes. Here, we show that plasma membrane (PM) association of ORP3 (also known as OSBPL3), a poorly characterized ORP family member, is triggered by protein kinase C (PKC) activation, especially when combined with Ca2+ increases, and is determined by both PI(4,5)P2 and PI4P After activation, ORP3 efficiently extracts PI4P and to a lesser extent phosphatidic acid from the PM, and slightly increases PM cholesterol levels. Full activation of ORP3 resulted in decreased PM PI4P levels and inhibited Ca2+ entry via the store-operated Ca2+ entry pathway. The C-terminal region of ORP3 that follows the strictly defined lipid transfer domain was found to be critical for the proper localization and function of the protein.

Role of oxysterol-binding protein-related proteins in malignant human tumours.

The oxysterol-binding protein-related protein (ORP) family is a group of proteins that mediate oxysterol metabolism and bioactivity in cells. ORPs constitute a large family of lipid transfer proteins. Much of the current evidence indicates that certain members of the family of oxysterol-binding proteins (OSBPs) can lead to cancer. Many studies have revealed the putative roles of OSBPs in various cancer types. However, the exact effects and mechanisms of action of members of the OSBP/ORP family in cancer initiation and progression are currently unclear. This review focuses on ORP family members that can accelerate human tumour cell proliferation, migration, and invasion. The mechanisms and functions of various ORPs are introduced in detail. We also attempt to identify the roles of these proteins in malignant tumours with the ultimate aim of determining the exact role of the OSBP/ORP family in human tumour cells.

ORP1L, ORP1S, and ORP2: Lipid Sensors and Transporters

Lipid transfer proteins are crucial for intracellular cholesterol trafficking at sites of membrane contact. In the OSBP/ORPs (oxysterol binding protein and OSBP-related proteins) family of lipid transfer proteins, ORP1L, ORP1S and ORP2 play important roles in cholesterol transport. ORP1L is an endosome/lysosome-anchored cholesterol sensor which may also move cholesterol bidirectionally at the interface between the endoplasmic reticulum and the endosome/lysosome. ORP2 delivers cholesterol to the plasma membrane, driven by PI(4,5)P2hydrolysis. ORP1S may also transport cholesterol to the plasma membrane, although it is unclear if phosphoinositides are involved. The source of cholesterol delivered to the plasma membrane by ORP1S and ORP2 remains unclear. This review summarises the roles of these proteins in maintaining cellular cholesterol homeostasis and in human disease.