Oxysterol-binding Protein Homologues Research Articles

The management of grapevine downy mildew: from anti-resistance strategies to innovative approaches for fungicide resistance monitoring

AbstractEfficient grapevine downy mildew control necessitates the implementation of anti-resistance strategies to ensure the ongoing efficacy of available substances and optimal disease control. With the gradual disappearance of multi-site fungicides from the market, reliance on single-site fungicides poses a long-term risk of selecting strains resistant to multiple modes of action. Challenges in disease management encompass selecting optimal spray programs and monitoring field population sensitivity. This study evaluated the efficacy of anti-resistance strategies, including two single-site fungicides (mandipropamid and oxathiapiprolin), on disease control and fungicide sensitivity through a combination of field trials and laboratory tests for the biological and molecular characterization of the pathogen populations over a three-year period (2019–2021). Mandipropamid, a cellulose synthase inhibitor, is used since a long time for downy mildew control, while oxathiapiprolin, an OxySterol Binding Protein homologue Inhibitor, was introduced recently. Field trials demonstrated effective disease control, even in the presence of mandipropamid-resistant strains (with G1105S/V mutations in PvCesA3) and revealed a pronounced selection and spread of resistance to both fungicides in the vineyard where disease pressure was higher. Characterizing pathogen strains remained a significant obstacle in sensitivity monitoring, hindering precise determination of resistance frequencies related to fungicide programs. Traditional techniques, in fact, lack the resolution required for high-throughput isolation and characterization of resistant individuals. To address this challenge, we propose utilizing flow cytometry and fluorescence-activated cell sorting on field sporangia populations, a method able to determine both the number of resistant isolates and isolate pathogen strains in a single assay.

Osh-dependent and -independent Regulation of PI4P Levels During Polarized Growth of Saccharomyces cerevisiae.

Polarized secretion facilitates polarized cell growth. For a secretory vesicle to dock at the plasma membrane, it must mature with a progressive association or dissociation of molecules that are, respectively, necessary for or inhibitory to vesicle docking, including an exchange of Rab GTPases. In current models, oxysterol-binding protein homologue 4 (Osh4p) establishes a phosphatidylinositol 4-phosphate (PI4P) gradient along the secretory trafficking pathway such that vesicles have higher PI4P levels after budding from the trans-Golgi relative to when vesicles arrive at the plasma membrane. In this study, using the lipid-binding domain P4M and live-cell imaging, we show that secretory vesicle-associated PI4P levels remain constant when vesicles traffic from the trans-Golgi to the plasma membrane. We also show that deletion of OSH4 does not alter vesicle-associated PI4P levels, though loss of any individual member of the OSH family or complete loss of OSH family function alters the intracellular distribution of PI4P. We propose a model in which the Rab GTPases Ypt32p and Sec4p remain associated with a secretory vesicle during trafficking, independent of PI4P levels and Osh4p. Together these data indicate the necessity of experiments revealing the location and timing of events required for vesicle maturation.

A functional genomic screen in Saccharomyces cerevisiae reveals divergent mechanisms of resistance to different alkylphosphocholine chemotherapeutic agents.

The alkylphosphocholine (APC) class of antineoplastic and antiprotozoal drugs, such as edelfosine and miltefosine, are structural mimics of lyso-phosphatidylcholine (lyso-PC), and are inhibitory to the yeast Saccharomyces cerevisiae at low micromolar concentrations. Cytotoxic effects related to inhibition of phospholipid synthesis, induction of an unfolded protein response, inhibition of oxidative phosphorylation, and disruption of lipid rafts have been attributed to members of this drug class, however, the molecular mechanisms of action of these drugs remain incompletely understood. Cytostatic and cytotoxic effects of the APCs exhibit variability with regard to chemical structure, leading to differences in effectiveness against different organisms or cell types. We now report the comprehensive identification of S. cerevisiae titratable-essential gene and haploid nonessential gene deletion mutants that are resistant to the APC drug miltefosine (hexadecyl-O-phosphocholine). Fifty-eight strains out of ∼5600 tested displayed robust and reproducible resistance to miltefosine. This gene set was heavily enriched in functions associated with vesicular transport steps, especially those involving endocytosis and retrograde transport of endosome derived vesicles to the Golgi or vacuole, suggesting a role for these trafficking pathways in transport of miltefosine to potential sites of action in the endoplasmic reticulum and mitochondrion. In addition, we identified mutants with defects in phosphatidylinositol-4-phosphate synthesis (TetO::STT4) and hydrolysis (sac1Δ), an oxysterol binding protein homolog (osh2Δ), a number of ER-resident proteins, and multiple components of the eisosome. These findings suggest that ER-plasma membrane contact sites and retrograde vesicle transport are involved in the interorganelle transport of lyso-PtdCho and related lyso-phospholipid-like analogs to their intracellular sites of cytotoxic activity.

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.

A PhotoClick cholesterol-based quantitative proteomics screen for cytoplasmic sterol-binding proteins in Saccharomyces cerevisiae.

Ergosterol is a prominent component of the yeast plasma membrane and essential for yeast cell viability. It is synthesized in the endoplasmic reticulum and transported to the plasma membrane by nonvesicular mechanisms requiring carrier proteins. Oxysterol-binding protein homologues and yeast StARkin proteins have been proposed to function as sterol carriers. Although many of these proteins are capable of transporting sterols between synthetic lipid vesicles in vitro, they are not essential for ergosterol transport in cells, indicating that they may be functionally redundant with each other or with additional-as yet unidentified-sterol carriers. To address this point, we hypothesized that sterol transport proteins are also sterol-binding proteins (SBPs), and used an in vitro chemoproteomic strategy to identify all cytosolic SBPs. We generated a cytosol fraction enriched in SBPs and captured the proteins with a photoreactive clickable cholesterol analogue. Quantitative proteomics of the captured proteins identified 342 putative SBPs. Analysis of these identified proteins based on their annotated function, reported drug phenotypes, interactions with proteins regulating lipid metabolism, gene ontology, and presence of mammalian orthologues revealed a subset of 62 characterized and nine uncharacterized candidates. Five of the uncharacterized proteins play a role in maintaining plasma membrane integrity as their absence affects the ability of cells to grow in the presence of nystatin or myriocin. We anticipate that the dataset reported here will be a comprehensive resource for functional analysis of sterol-binding/transport proteins and provide insights into novel aspects of non-vesicular sterol trafficking.

The anti-cancer compound Schweinfurthin A targets Osh2 and disrupts lipid metabolism in the yeast model.

Schweinfurthin A (Sch A) is a natural product with a selective and strong anti-cancer effect. Although it is known to target oxysterol binding proteins, the detailed mode of action is not well understood. Here, we provide strong evidence that yeast cells can be used as a eukaryotic model system to decipher the molecular modes of Sch A. We show that Sch A (100µM) targets Osh2 (a yeast oxysterol binding protein homolog) genetically and taking advantage of computational chemistry indicate that the tetrahydro-2H-xanthene portion of Sch A forms H-bonds with residues Ser105, Val113, and Lys201, while its isoprenoid side chain is placed in a hydrophobic pocket lined by the side chains of Leu41, Leu45, Leu58, Met56, and Phe174 in Osh2. This model suggests that Sch A occupies the same binding pocket in Osh2 which is occupied by its natural substrate, ergosterol. Osh proteins transport sterol and PI(4)P in a cyclic manner between two membranes. Therefore, we suggest that Sch A interferes with this function of Osh2. In support of this hypothesis, we show that Sch A toxicity rate changes upon manipulating the enzymes that modify the levels of sterol and PI(4)P. This approach also informs how Sch A exerts its toxic effect in yeast cells. These enzymes include Coq1, Sac1, Plc1, Stt4, Pik1, and Mss4. We demonstrate that Coq1 an enzyme required for coenzyme Q synthesis (also involved in sterol metabolism indirectly), Sac1, and Stt4 the enzymes governing PI(4)P level modify Sch A toxicity and finally propose Sch A disrupts sterol/PI(4)P exchange between membranes by occupying the sterol/PI(4)P binding pocket in Osh2.

Advances in understanding of the oxysterol-binding protein homologous in yeast and filamentous fungi.

Oxysterol-binding protein is an important non-vesicular trafficking protein involved in the transportation of lipids in eukaryotic cells. Oxysterol-binding protein is identified as oxysterol-binding protein-related proteins (ORPs) in mammals and oxysterol-binding protein homologue (Osh) in yeast. Research has described the function and structure of oxysterol-binding protein in mammals and yeast, but little information about the protein's structure and function in filamentous fungi has been reported. This article focuses on recent advances in the research of Osh proteins in yeast and filamentous fungi, such as Aspergillus oryzae, Aspergillus nidulans, and Candida albicans. Furthermore, we point out some problems in the field, summarizing the membrane contact sites (MCS) of Osh proteins in yeast, and consider the future of Osh protein development.

Mutations in ORP1 Conferring Oxathiapiprolin Resistance Confirmed by Genome Editing using CRISPR/Cas9 in Phytophthora capsici and P. sojae.

Oxathiapiprolin is a novel fungicide that was recently registered in a number of countries to control plant-pathogenic oomycetes such as Phytophthora capsici. In our previous study, point mutations G770V and G839W in oxysterol binding protein-related protein 1 (ORP1) were detected in oxathiapiprolin-resistant P. capsici isolates (PcORP1). Here, we used the CRISPR/Cas9 system to verify the effects of these two point mutations on P. capsici phenotypes. Transformants containing heterozygous G770V and G839W mutations in PcORP1 showed high levels of oxathiapiprolin resistance. The G770V transformants showed otherwise similar phenotypes compared with the wild-type isolate BYA5, including sporangia and zoospore production, cyst germination, and pathogenicity. However, two independent transformants with heterozygous G839W mutations in PcORP1 could not produce sporangia. Three transformants with an unexpected point mutation in PcORP1 (ΔN837) showed high oxathiapiprolin resistance, and either similar or significantly reduced fitness compared with BYA5. The same deletion (ΔN837) was confirmed to confer oxathiapiprolin resistance in P. sojae by using CRISPR/Cas9. These homozygous P. sojae mutants also showed either similar or strongly reduced fitness compared with the wild-type parent isolate P6497. These results improve our understanding of oxathiapiprolin resistance in Phytophthora spp., and will be useful for the development of novel oxysterol-binding protein homolog inhibitor fungicides.

Oxysterol-binding protein homologs mediate sterol transport from the endoplasmic reticulum to mitochondria in yeast

Sterols are present in eukaryotic membranes and significantly affect membrane fluidity, permeability, and microdomain formation. They are synthesized in the endoplasmic reticulum (ER) and transported to other organelles and the plasma membrane. Sterols play important roles in the biogenesis and maintenance of mitochondrial membranes. However, the mechanisms underlying ER-to-mitochondrion sterol transport remain to be identified. Here, using purified yeast membrane fractions enriched in ER and mitochondria, we show that the oxysterol-binding protein homologs encoded by the OSH genes in the yeast Saccharomyces cerevisiae mediate sterol transport from the ER to mitochondria. Combined depletion of all seven Osh proteins impaired sterol transport from the ER to mitochondria in vitro; however, sterol transport was recovered at different levels upon adding one of the Osh proteins. Of note, the sterol content in the mitochondrial fraction was significantly decreased in vivo after Osh4 inactivation in a genetic background in which all the other OSH genes were deleted. We also found that Osh5-Osh7 bind cholesterol in vitro We propose a model in which Osh proteins share a common function to transport sterols between membranes, with varying contributions by these proteins, depending on the target membranes. In summary, we have developed an in vitro system to examine intracellular sterol transport and provide evidence for involvement of Osh proteins in sterol transport from the ER to mitochondria in yeast.

Lipid-dependent regulation of exocytosis in S. cerevisiae by OSBP homolog (Osh) 4.

Polarized exocytosis is an essential process in many organisms and cell types for correct cell division or functional specialization. Previous studies established that homologs of the oxysterol-binding protein (OSBP) in S. cerevisiae, which comprise the Osh protein family, are necessary for efficient polarized exocytosis by supporting a late post-Golgi step. We define this step as the docking of a specific sub-population of exocytic vesicles with the plasma membrane. In the absence of other Osh proteins, yeast Osh4p can support this process in a manner dependent upon two lipid ligands, PI4P and sterol. Osh6p, which binds PI4P and phosphatidylserine, is also sufficient to support polarized exocytosis, again in a lipid-dependent manner. These data suggest that Osh-mediated exocytosis depends upon lipid binding and exchange without a strict requirement for sterol. We propose a two-step mechanism for Osh protein-mediated regulation of polarized exocytosis by using Osh4p as a model. We describe a specific in vivo role for lipid binding by an OSBP-related protein (ORP) in the process of polarized exocytosis, guiding our understanding of where and how OSBP and ORPs may function in more complex organisms.

Phosphatidylserine translocation at the yeast trans-Golgi network regulates protein sorting into exocytic vesicles.

Sorting of plasma membrane proteins into exocytic vesicles at the yeast trans-Golgi network (TGN) is believed to be mediated by their coalescence with specific lipids, but how these membrane-remodeling events are regulated is poorly understood. Here we show that the ATP-dependent phospholipid flippase Drs2 is required for efficient segregation of cargo into exocytic vesicles. The plasma membrane proteins Pma1 and Can1 are missorted from the TGN to the vacuole in drs2∆ cells. We also used a combination of flippase mutants that either gain or lose the ability to flip phosphatidylserine (PS) to determine that PS flip by Drs2 is its critical function in this sorting event. The primary role of PS flip at the TGN appears to be to control the oxysterol-binding protein homologue Kes1/Osh4 and regulate ergosterol subcellular distribution. Deletion of KES1 suppresses plasma membrane-missorting defects and the accumulation of intracellular ergosterol in drs2 mutants. We propose that PS flip is part of a homeostatic mechanism that controls sterol loading and lateral segregation of protein and lipid domains at the TGN.

Functional Analysis of Sterol Transporter Orthologues in the Filamentous Fungus Aspergillus nidulans.

Polarized growth in filamentous fungi needs a continuous supply of proteins and lipids to the growing hyphal tip. One of the important membrane compounds in fungi is ergosterol. At the apical plasma membrane ergosterol accumulations, which are called sterol-rich plasma membrane domains (SRDs). The exact roles and formation mechanism of the SRDs remained unclear, although the importance has been recognized for hyphal growth. Transport of ergosterol to hyphal tips is thought to be important for the organization of the SRDs. Oxysterol binding proteins, which are conserved from yeast to human, are involved in nonvesicular sterol transport. In Saccharomyces cerevisiae seven oxysterol-binding protein homologues (OSH1 to -7) play a role in ergosterol distribution between closely located membranes independent of vesicle transport. We found five homologous genes (oshA to oshE) in the filamentous fungi Aspergillus nidulans. The functions of OshA-E were characterized by gene deletion and subcellular localization. Each gene-deletion strain showed characteristic phenotypes and different sensitivities to ergosterol-associated drugs. Green fluorescent protein-tagged Osh proteins showed specific localization in the late Golgi compartments, puncta associated with the endoplasmic reticulum, or diffusely in the cytoplasm. The genes expression and regulation were investigated in a medically important species Aspergillus fumigatus, as well as A. nidulans. Our results suggest that each Osh protein plays a role in ergosterol distribution at distinct sites and contributes to proper fungal growth.

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

ORP3 is an R-Ras interacting oxysterol-binding protein homolog that regulates cell adhesion and is overexpressed in several cancers. We investigated here a novel function of ORP3 dependent on its targeting to both the endoplasmic reticulum (ER) and the plasma membrane (PM). Using biochemical and cell imaging techniques we demonstrate the mechanistic requirements for the subcellular targeting and function of ORP3 in control of R-Ras activity. We show that hyperphosphorylated ORP3 (ORP3-P) selectively interacts with the ER membrane protein VAPA, and ORP3–VAPA complexes are targeted to PM sites via the ORP3 pleckstrin homology (PH) domain. A novel FFAT (two phenylalanines in an acidic tract)-like motif was identified in ORP3; only disruption of both the FFAT-like and canonical FFAT motif abolished the phorbol-12-myristate-13-acetate (PMA) stimulated interaction of ORP3-P with VAPA. Co-expression of ORP3 and VAPA induced R-Ras activation, dependent on the interactions of ORP3 with VAPA and the PM. Consistently, downstream AktS473 phosphorylation and β1-integrin activity were enhanced by ORP3–VAPA. To conclude, phosphorylation of ORP3 controls its association with VAPA. Furthermore, we present evidence that ORP3–VAPA complexes stimulate R-Ras signaling.

Structural Basis of Lipid Exchange in the Oxysterol-Binding Protein Homologue (OSH) Family

Oxysterol-binding proteins, long known to be involved in the non-vesicular transport of sterol, have recently been shown to also transfer glycerophospholipids, such as phosphatidyl-inositol-4-phosphate (PI4P) and phosphatidyl-serine (PS) between membranes.Using a combination of atomistic molecular dynamics simulations and in vitro lipid transport assays, we characterized the structural determinants that regulate the extraction and release of different lipids by the various Osh proteins.Our results define how distinct lipid-binding modes govern the ability of Osh proteins to transport or exchange lipids between organelles.

A Protein Pair with PIPs Inside

Oxysterol binding protein (OSBP) and many of its homologs transfer sterol invitro, but invivo, they are not major sterol transporters. In this issue of Structure, Tong and colleagues find that the yeast OSBP homolog, Osh3, binds PI(4)P but not sterol, supporting the view that PI(4)P regulation, not sterol transport, is the key activity for OSBP homologs.

Osh proteins regulate COPII-mediated vesicular transport of ceramide from the endoplasmic reticulum in budding yeast

Lipids synthesized at the endoplasmic reticulum (ER) are delivered to the Golgi by vesicular and non-vesicular pathways. ER-to-Golgi transport is crucial for maintaining the different membrane lipid composition and identities of organelles. Despite their importance, mechanisms regulating transport remain elusive. Here we report that in yeast coat protein complex II (COPII) vesicle-mediated transport of ceramide from the ER to the Golgi requires oxysterol-binding protein homologs, Osh proteins, which have been implicated in lipid homeostasis. Because Osh proteins are not required to transport proteins to the Golgi, these results indicate a specific requirement for the Osh proteins in the transport of ceramide. In addition, we provide evidence that Osh proteins play a negative role in COPII vesicle biogenesis. Together, our data suggest that ceramide transport and sphingolipid levels between the ER and Golgi are maintained by two distinct functions of Osh proteins, which negatively regulate COPII vesicle formation and positively control a later stage, presumably fusion of ceramide-enriched vesicles with Golgi compartments.

OSBP-Related Proteins (ORPs) in Human Adipose Depots and Cultured Adipocytes: Evidence for Impacts on the Adipocyte Phenotype

Oxysterol-binding protein (OSBP) homologues, ORPs, are implicated in lipid homeostatic control, vesicle transport, and cell signaling. We analyzed here the quantity of ORP mRNAs in human subcutaneous (s.c.) and visceral adipose depots, as well as in the Simpson-Golabi-Behmel syndrome (SGBS) adipocyte cell model. All of the ORP mRNAs were present in the s.c and visceral adipose tissues, and the two depots shared an almost identical ORP mRNA expression pattern. SGBS adipocytes displayed a similar pattern, suggesting that the adipose tissue ORP expression pattern mainly derives from adipocytes. During SGBS cell adipogenic differentiation, ORP2, ORP3, ORP4, ORP7, and ORP8 mRNAs were down-regulated, while ORP11 was induced. To assess the impacts of ORPs on adipocyte differentiation, ORP3 and ORP8, proteins down-regulated during adipogenesis, were overexpressed in differentiating SGBS adipocytes, while ORP11, a protein induced during adipogenesis, was silenced. ORP8 overexpression resulted in reduced expression of the aP2 mRNA, while down-regulation of adiponectin and aP2 was observed in ORP11 silenced cells. Furthermore, ORP8 overexpression or silencing of ORP11 markedly decreased cellular triglyceride storage. These data identify the patterns of ORP expression in human adipose depots and SGBS adipocytes, and provide the first evidence for a functional impact of ORPs on the adipocyte phenotype.

Modification of the lipidome in RAW264.7 macrophage subjected to stable silencing of oxysterol-binding proteins

Oxysterol-binding protein homologs (ORPs) are implicated in lipid metabolism, vesicle transport and cell signaling. In this study we generated RAW264.7 cells with ORP1L, ORP3, or ORP8 silenced using shRNA lentiviruses. The lipidome of the cells under basal serum-free culture conditions or as treated with oxidized LDL (oxLDL), enzymatically modified LDL (E-LDL), or lipopolysaccharide (LPS) was analyzed by mass spectrometry. Reduction in each ORP resulted in distinct and complex effects on macrophage lipidome. Under basal conditions, ORP1L silencing had strongest effects on phosphatidylinositols (PI,increase), free cholesterol (FC, increase), and cholesteryl esters (CE, increase). ORP3 silencing affected most the glucosyl ceramides (GluCer, decrease) and PE-plasmalogens (PE-pl, decrease), while ORP8 silencing increased FC and CE, and decreased GluCer and PE-pl. Upon LPS treatment, the ORP effects were modified: under these conditions ORP1L silencing caused increase of Cer, ORP3 silencing decrease of PI, and ORP8 silencing decrease of PI and increase of PE, not detectable under basal conditions. The lipid species data were subjected to multivariate statistical analysis of principal components, revealing numerous specific alterations upon ORP silencing. The cells cultured in basal conditions or treated with LPS showed qualitatively different responses. However, in LPS-stimulated cells silencing of any of thethree ORPs decreased the relative amount of arachidonic acid-containing PI species, increased the corresponding PE species, and favored 16-carbon sphingomyelin (SM) species at the expense of the 24-carbon ones. As a conclusion, the present study reveals the distinct and sophisticated roles of different ORP proteins as regulators of macrophage lipid composition, with implications for inflammatory signaling.

Oxysterols and their cellular effectors.

Oxysterols are oxidized 27-carbon cholesterol derivatives or by-products of cholesterol biosynthesis, with a spectrum of biologic activities. Several oxysterols have cytotoxic and pro-apoptotic activities, the ability to interfere with the lateral domain organization, and packing of membrane lipids. These properties may account for their suggested roles in the pathology of diseases such as atherosclerosis, age-onset macular degeneration and Alzheimer’s disease. Oxysterols also have the capacity to induce inflammatory responses and play roles in cell differentiation processes. The functions of oxysterols as intermediates in the synthesis of bile acids and steroid hormones, and as readily transportable forms of sterol, are well established. Furthermore, their actions as endogenous regulators of gene expression in lipid metabolism via liver X receptors and the Insig (insulin-induced gene) proteins have been investigated in detail. The cytoplasmic oxysterol-binding protein (OSBP) homologues form a group of oxysterol/cholesterol sensors that has recently attracted a lot of attention. However, their mode of action is, as yet, poorly understood. Retinoic acid receptor-related orphan receptors (ROR) α and γ, and Epstein-Barr virus induced gene 2 (EBI2) have been identified as novel oxysterol receptors, revealing new physiologic oxysterol effector mechanisms in development, metabolism, and immunity, and evoking enhanced interest in these compounds in the field of biomedicine.

Long term induction by pterostilbene results in autophagy and cellular differentiation in MCF-7 cells via ROS dependent pathway

This study shows the effect of pterostilbene on intracellular neutral lipid accumulation in MCF-7 breast cancer cells leading to growth arrest and autophagy. On exposing the breast cancer cells with 30μM pterostilbene for 72h there was almost 2-folds increase in neutral lipids and triglycerides. Also the phytochemical caused a 4-folds increase in the expression of adipogenic differentiation marker c/EBPα. Further, pterostilbene inhibited 3β-hydroxylsterol-Δ7-reductase, the enzyme which catalyzes the last step conversion of 7-dehydrocholesterol to cholesterol, and thereby causes the intracellular accumulation of the former sterol. These results were associated with over-expression of oxysterol binding protein homologue and liver X receptor (LXR) by ∼7-folds. Pterostilbene also caused a simultaneous increase in the expression autophagic marker proteins Beclin 1 and LC3 II (microtubule-associated protein 1 light chain 3) by approximately 6-folds, which leads to an alternative pathway of autophagy. These effects were observed in association with the loss of mitotic and metastatic potential of MCF-7 cells which was abolished in the presence of catalase (ROS scavenger) or 3MA (autophagic inhibitor). Thus the present data shows that the long term exposure to pterostilbene causes growth arrest in MCF-7 cells which may be due to differentiation of the mammary carcinoma cells into normal epithelial cell like morphology and activation of autophagy.