scholarly journals Molecular and cellular dissection of the oxysterol-binding protein cycle through a fluorescent inhibitor

2020 ◽  
Vol 295 (13) ◽  
pp. 4277-4288 ◽  
Author(s):  
Tiphaine Péresse ◽  
David Kovacs ◽  
Mélody Subra ◽  
Joëlle Bigay ◽  
Meng-Chen Tsai ◽  
...  
Keyword(s):  
Binding Protein ◽  
Lipid Binding ◽  
Lipid Transfer ◽  
Intracellular Lipid ◽  
Membrane Reconstitution ◽  
Bioactive Natural Products ◽  
Oxysterol Binding Protein ◽  
Binding Cavity ◽  
Phosphatidylinositol 4

ORPphilins are bioactive natural products that strongly and selectively inhibit the growth of some cancer cell lines and are proposed to target intracellular lipid-transfer proteins of the oxysterol-binding protein (OSBP) family. These conserved proteins exchange key lipids, such as cholesterol and phosphatidylinositol 4-phosphate (PI(4)P), between organelle membranes. Among ORPphilins, molecules of the schweinfurthin family interfere with intracellular lipid distribution and metabolism, but their functioning at the molecular level is poorly understood. We report here that cell line sensitivity to schweinfurthin G (SWG) is inversely proportional to cellular OSBP levels. By taking advantage of the intrinsic fluorescence of SWG, we followed its fate in cell cultures and show that its incorporation at the trans-Golgi network depends on cellular abundance of OSBP. Using in vitro membrane reconstitution systems and cellular imaging approaches, we also report that SWG inhibits specifically the lipid transfer activity of OSBP. As a consequence, post-Golgi trafficking, membrane cholesterol levels, and PI(4)P turnover were affected. Finally, using intermolecular FRET analysis, we demonstrate that SWG directly binds to the lipid-binding cavity of OSBP. Collectively these results describe SWG as a specific and intrinsically fluorescent pharmacological tool for dissecting OSBP properties at the cellular and molecular levels. Our findings indicate that SWG binds OSBP with nanomolar affinity, that this binding is sensitive to the membrane environment, and that SWG inhibits the OSBP-catalyzed lipid exchange cycle.

scholarly journals Molecular and cellular dissection of the OSBP cycle through a fluorescent inhibitor

2019 ◽  
Author(s):  
Tiphaine Péresse ◽  
David Kovacs ◽  
Mélody Subra ◽  
Joëlle Bigay ◽  
Meng-Chen Tsai ◽  
...  
Keyword(s):  
Lipid Binding ◽  
Cellular Level ◽  
Lipid Transfer ◽  
Intracellular Lipid ◽  
Oxysterol Binding Protein ◽  
Cholesterol Levels ◽  
Lipid Exchange ◽  
Binding Cavity ◽  
Direct Binding ◽  
First Time

ABSTRACTORPphilins, natural molecules that strongly and selectively inhibit the growth of some cancer cell lines, are proposed to target intracellular lipid-transfer proteins of the Oxysterol-binding protein (OSBP) family. These conserved proteins exchange key lipids, such as cholesterol and phopsphatidylinositol-4-phosphate (PI(4)P), between organelle membranes. Among ORPphilins, molecules of the schweinfurthin family interfere with intracellular lipid distribution and metabolism, but their functioning at the molecular level is poorly understood. We report here that cell line sensitivity to schweinfurthin G (SWG) is inversely proportional to cellular level of OSBP. By taking advantage of the intrinsic fluorescence of SWG, we follow its fate in cell cultures and show that its incorporation at the TGN depends on OSBP cellular abundance. We report that SWG inhibits specifically the lipid exchange cycle of OSBP. As a consequence, post-Golgi trafficking, membrane cholesterol levels and PI(4)P turnover are affected. Finally, we demonstrate the direct binding of SWG into OSBP lipid-binding cavity by intermolecular FRET. Collectively these data describe for the first time a specific and intrinsically fluorescent pharmacological tool to dissect OSBP properties at the cellular and molecular levels.

scholarly journals Functional analyses of phosphatidylserine/PI(4)P exchangers with diverse lipid species and membrane contexts set unanticipated rules on lipid transfer

2021 ◽  
Author(s):  
Souade Ikhlef ◽  
Nicolas-Frédéric Lipp ◽  
Vanessa Delfosse ◽  
Nicolas Fuggetta ◽  
William Bourguet ◽  
...  
Keyword(s):  
Lipid Transfer ◽  
Oxysterol Binding Protein ◽  
Lipid Exchange ◽  
Exchange Processes ◽  
Lipid Species ◽  
Acyl Chains ◽  
Phosphatidylinositol 4 ◽  
Functional Analyses ◽  
Related Proteins

Several members of the oxysterol-binding protein-related proteins (ORPs)/oxysterol-binding homology (Osh) family exchange phosphatidylserine (PS) and phosphatidylinositol 4-phosphate (PI(4)P) at the endoplasmic reticulum/plasma membrane (PM) interface. It is unclear whether they preferentially exchange PS and PI(4)P with specific acyl chains to tune the features of the PM, whether they use phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) instead of PI(4)P for exchange processes and whether their activity is influenced by the association of PS with sterol in the PM. Here, we measured in vitro how the yeast Osh6p and human ORP8 transported diverse PS and PI(4)P subspecies, including major cellular species, between membranes. We established how their activity is impacted by the length and unsaturation degree of these lipids. Surprisingly, the speed at which they individually transfer these ligands inversely depends on their affinity for them. To be fast, the transfer of high-affinity ligands requires an exchange with a counterligand of equivalent affinity. Besides, we determined that Osh6p and ORP8 cannot use PI(4,5)P2 for intracellular lipid exchange, as they have a low affinity for this lipid compared to PI(4)P, and do not transfer more PS into sterol-rich membranes. This study provides insights into PS/PI(4)P exchangers and sets unanticipated rules on how the activity of lipid transfer proteins relates to their affinity for ligands.

scholarly journals The oxysterol-binding protein superfamily: new concepts and old proteins

2012 ◽  
Vol 40 (2) ◽  
pp. 469-473 ◽  
Author(s):  
Michelle L. Villasmil ◽  
Vytas A. Bankaitis ◽  
Carl J. Mousley
Keyword(s):  
Lipid Metabolism ◽  
Membrane Trafficking ◽  
Transcriptional Control ◽  
Binding Protein ◽  
Lipid Binding ◽  
Oxysterol Binding Protein ◽  
New Concepts ◽  
Endosomal Membranes ◽  
Phosphatidylinositol 4 ◽  
Golgi Network

The Kes1 OSBP (oxysterol-binding protein) is a key regulator of membrane trafficking through the TGN (trans-Golgi network) and endosomal membranes. We demonstrated recently that Kes1 acts as a sterol-regulated rheostat for TGN/endosomal phosphatidylinositol 4-phosphate signalling. Kes1 utilizes its dual lipid-binding activities to integrate endosomal lipid metabolism with TORC1 (target of rapamycin complex 1)-dependent proliferative pathways and transcriptional control of nutrient signalling.

New molecular mechanisms of inter-organelle lipid transport

2016 ◽  
Vol 44 (2) ◽  
pp. 486-492 ◽  
Author(s):  
Guillaume Drin ◽  
Joachim Moser von Filseck ◽  
Alenka Čopič
Keyword(s):  
Molecular Mechanisms ◽  
Lipid Binding ◽  
Phosphoinositide Metabolism ◽  
Lipid Transfer ◽  
Cellular Lipid ◽  
Transport Pathways ◽  
Oxysterol Binding Protein ◽  
Membrane Contact Sites ◽  
Associated Proteins ◽  
Phosphatidylinositol 4

Lipids are precisely distributed in cell membranes, along with associated proteins defining organelle identity. Because the major cellular lipid factory is the endoplasmic reticulum (ER), a key issue is to understand how various lipids are subsequently delivered to other compartments by vesicular and non-vesicular transport pathways. Efforts are currently made to decipher how lipid transfer proteins (LTPs) work either across long distances or confined to membrane contact sites (MCSs) where two organelles are at close proximity. Recent findings reveal that proteins of the oxysterol-binding protein related-proteins (ORP)/oxysterol-binding homology (Osh) family are not all just sterol transporters/sensors: some can bind either phosphatidylinositol 4-phosphate (PtdIns(4)P) and sterol or PtdIns(4)P and phosphatidylserine (PS), exchange these lipids between membranes, and thereby use phosphoinositide metabolism to create cellular lipid gradients. Lipid exchange is likely a widespread mechanism also utilized by other LTPs to efficiently trade lipids between organelle membranes. Finally, the discovery of more proteins bearing a lipid-binding module (SMP or START-like domain) raises new questions on how lipids are conveyed in cells and how the activities of different LTPs are coordinated.

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

2021 ◽  
Vol 221 (1) ◽  
Author(s):  
Asami Kawasaki ◽  
Akiko Sakai ◽  
Hiroki Nakanishi ◽  
Junya Hasegawa ◽  
Tomohiko Taguchi ◽  
...  
Keyword(s):  
Membrane Trafficking ◽  
Binding Protein ◽  
Dependent Manner ◽  
Oxysterol Binding Protein ◽  
Membrane Contact Sites ◽  
Contact Sites ◽  
Lipid Exchange ◽  
Membrane Contact ◽  
Phosphatidylinositol 4

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.

scholarly journals Nonvesicular sterol movement from plasma membrane to ER requires oxysterol-binding protein–related proteins and phosphoinositides

2006 ◽  
Vol 173 (1) ◽  
pp. 107-119 ◽  
Author(s):  
Sumana Raychaudhuri ◽  
Young Jun Im ◽  
James H. Hurley ◽  
William A. Prinz
Keyword(s):  
Plasma Membrane ◽  
Binding Protein ◽  
Large Family ◽  
Lipid Binding ◽  
Oxysterol Binding Protein ◽  
Sterol Transport ◽  
Cellular Compartments ◽  
Related Proteins ◽  
Lipid Binding Proteins

Sterols are moved between cellular membranes by nonvesicular pathways whose functions are poorly understood. In yeast, one such pathway transfers sterols from the plasma membrane (PM) to the endoplasmic reticulum (ER). We show that this transport requires oxysterol-binding protein (OSBP)–related proteins (ORPs), which are a large family of conserved lipid-binding proteins. We demonstrate that a representative member of this family, Osh4p/Kes1p, specifically facilitates the nonvesicular transfer of cholesterol and ergosterol between membranes in vitro. In addition, Osh4p transfers sterols more rapidly between membranes containing phosphoinositides (PIPs), suggesting that PIPs regulate sterol transport by ORPs. We confirmed this by showing that PM to ER sterol transport slows dramatically in mutants with conditional defects in PIP biosynthesis. Our findings argue that ORPs move sterols among cellular compartments and that sterol transport and intracellular distribution are regulated by PIPs.

scholarly journals Multisite phosphorylation of oxysterol-binding protein regulates sterol binding and activation of sphingomyelin synthesis

2012 ◽  
Vol 23 (18) ◽  
pp. 3624-3635 ◽  
Author(s):  
Asako Goto ◽  
Xinwei Liu ◽  
Carolyn-Ann Robinson ◽  
Neale D. Ridgway
Keyword(s):  
Binding Capacity ◽  
Binding Protein ◽  
Protein A ◽  
Phosphorylation Site ◽  
Oxysterol Binding Protein ◽  
Transfer Activity ◽  
Individual Site ◽  
Direct Binding ◽  
Phosphatidylinositol 4

The endoplasmic reticulum (ER)-Golgi sterol transfer activity of oxysterol-binding protein (OSBP) regulates sphingomyelin (SM) synthesis, as well as post-Golgi cholesterol efflux pathways. The phosphorylation and ER-Golgi localization of OSBP are correlated, suggesting this modification regulates the directionality and/or specificity of transfer activity. In this paper, we report that phosphorylation on two serine-rich motifs, S381-S391 (site 1) and S192, S195, S200 (site 2), specifically controls OSBP activity at the ER. A phosphomimetic of the SM/cholesterol-sensitive phosphorylation site 1 (OSBP-S5E) had increased in vitro cholesterol and 25-hydroxycholesterol–binding capacity, and cholesterol extraction from liposomes, but reduced transfer activity. Phosphatidylinositol 4-phosphate (PI(4)P) and cholesterol competed for a common binding site on OSBP; however, direct binding of PI(4)P was not affected by site 1 phosphorylation. Individual site 1 and site 2 phosphomutants supported oxysterol activation of SM synthesis in OSBP-deficient CHO cells. However, a double site1/2 mutant (OSBP-S381A/S3D) was deficient in this activity and was constitutively colocalized with vesicle-associated membrane protein–associated protein A (VAP-A) in a collapsed ER network. This study identifies phosphorylation regulation of sterol and VAP-A binding by OSBP in the ER, and PI(4)P as an alternate ligand that could be exchanged for sterol in the Golgi apparatus.

The Oxysterol-Binding Protein Cycle: Burning Off PI(4)P to Transport Cholesterol

2018 ◽  
Vol 87 (1) ◽  
pp. 809-837 ◽  
Author(s):  
Bruno Antonny ◽  
Joëlle Bigay ◽  
Bruno Mesmin
Keyword(s):  
Secretory Pathway ◽  
Lipid Transfer Protein ◽  
Binding Protein ◽  
Asymmetric Distribution ◽  
Lipid Transfer ◽  
Oxysterol Binding Protein ◽  
Transfer Protein ◽  
Lipid Exchange ◽  
Opposite Process ◽  
Phosphatidylinositol 4

To maintain an asymmetric distribution of ions across membranes, protein pumps displace ions against their concentration gradient by using chemical energy. Here, we describe a functionally analogous but topologically opposite process that applies to the lipid transfer protein (LTP) oxysterol-binding protein (OSBP). This multidomain protein exchanges cholesterol for the phosphoinositide phosphatidylinositol 4-phosphate [PI(4)P] between two apposed membranes. Because of the subsequent hydrolysis of PI(4)P, this counterexchange is irreversible and contributes to the establishment of a cholesterol gradient along organelles of the secretory pathway. The facts that some natural anti-cancer molecules block OSBP and that many viruses hijack the OSBP cycle for the formation of intracellular replication organelles highlight the importance and potency of OSBP-mediated lipid exchange. The architecture of some LTPs is similar to that of OSBP, suggesting that the principles of the OSBP cycle—burning PI(4)P for the vectorial transfer of another lipid—might be general.

scholarly journals Nir2 Is an Effector of VAPs Necessary for Efficient Hepatitis C Virus Replication and Phosphatidylinositol 4-Phosphate Enrichment at the Viral Replication Organelle

2019 ◽  
Vol 93 (22) ◽  
Author(s):  
Hongliang Wang ◽  
Andrew W. Tai
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Viral Replication ◽  
Binding Protein ◽  
Lipid Transfer ◽  
Lipid Transfer Proteins ◽  
Oxysterol Binding Protein ◽  
Transfer Protein ◽  
Phosphatidylinositol Transfer ◽  
Phosphatidylinositol 4

ABSTRACT The endoplasmic reticulum (ER)-resident proteins vesicle-associated membrane protein (VAMP)-associated protein A and B (VAPA and VAPB) have been reported to be necessary for efficient hepatitis C virus (HCV) replication, but the specific mechanisms are not well understood. VAPs are known to recruit lipid transfer proteins to the ER, including oxysterol binding protein (OSBP), which has been previously shown to be necessary for cholesterol delivery to the HCV replication organelle in exchange for phosphatidylinositol 4-phosphate [PI(4)P]. Here, we show that VAPA and VAPB are redundant for HCV infection and that dimerization is not required for their function. In addition, we identify the phosphatidylinositol transfer protein Nir2 as an effector of VAPs to support HCV replication. We propose that Nir2 functions to replenish phosphoinositides at the HCV replication organelle to maintain elevated steady-state levels of PI(4)P, which is removed by OSBP. Thus, Nir2, along with VAPs, OSBP, and the phosphatidylinositol 4-kinase, completes a cycle of phosphoinositide flow between the ER and viral replication organelles to drive ongoing viral replication. IMPORTANCE Hepatitis C virus (HCV) is known for its ability to modulate phosphoinositide signaling pathways for its replication. Elevated levels of phosphatidylinositol 4-phosphate [PI(4)P] in HCV replication organelles (ROs) recruits lipid transfer proteins (LTPs), like oxysterol-binding protein (OSBP). OSBP exchanges PI(4)P with cholesterol, thus removing PI(4)P from the HCV RO. Here, we found that the phosphatidylinositol transfer protein Nir2 acts as an LTP and may replenish PI at the HCV RO by interacting with VAMP-associated proteins (VAPs), enabling continuous viral replication during chronic infection. Therefore, the coordination of OSBP, Nir2, and VAPs completes our understanding of the phosphoinositide cycle between the ER and HCV ROs.

scholarly journals Identification and assessment of the role of a nominal phospholipid binding region of ORP1S (oxysterol-binding-protein-related protein 1 short) in the regulation of vesicular transport

2005 ◽  
Vol 387 (3) ◽  
pp. 889-896 ◽  
Author(s):  
Gregory D. FAIRN ◽  
Christopher R. McMASTER
Keyword(s):  
Binding Protein ◽  
Vesicular Transport ◽  
Lipid Binding ◽  
Binding Domain ◽  
Negative Regulator ◽  
Carboxypeptidase Y ◽  
Binding Region ◽  
Phospholipid Binding ◽  
Oxysterol Binding Protein

The ORPs (oxysterol-binding-protein-related proteins) constitute an enigmatic family of intracellular lipid receptors that are related through a shared lipid binding domain. Emerging evidence suggests that ORPs relate lipid metabolism to membrane transport. Current data imply that the yeast ORP Kes1p is a negative regulator of Golgi-derived vesicular transport mediated by the essential phosphatidylinositol/phosphatidylcholine transfer protein Sec14p. Inactivation of Kes1p function allows restoration of growth and vesicular transport in cells lacking Sec14p function, and Kes1p function in this regard can be complemented by human ORP1S (ORP1 short). Recent studies have determined that Kes1p and ORP1S both bind phospholipids as ligands. To explore the function of distinct linear segments of ORP1S in phospholipid binding and vesicular transport regulation, we generated a series of 15 open reading frames coding for diagnostic regions within ORP1S. Purified versions of these ORP1S deletion proteins were characterized in vitro, and allowed the identification of a nominal phospholipid binding region. The in vitro analysis was interpreted in the context of in vivo growth and vesicle transport assays for members of the ORP1S deletion set. The results determined that the phospholipid binding domain per se was insufficient for inhibition of vesicular transport by ORP1S, and that transport of carboxypeptidase Y and invertase from the Golgi may be regulated differentially by specific regions of ORP1S/Kes1p.

Sign in / Sign up

Export Citation Format

Share Document