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 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 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.

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.

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 The crystal structure of human microsomal triglyceride transfer protein

2019 ◽  
Vol 116 (35) ◽  
pp. 17251-17260 ◽  
Author(s):  
Ekaterina I. Biterova ◽  
Michail N. Isupov ◽  
Ronan M. Keegan ◽  
Andrey A. Lebedev ◽  
Anil A. Sohail ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Structural Information ◽  
Neutral Lipids ◽  
Microsomal Triglyceride Transfer Protein ◽  
Lipid Binding ◽  
Rational Drug Design ◽  
Lipid Transfer ◽  
Very Low Density Lipoproteins ◽  
Transfer Protein ◽  
Binding Cavity

Microsomal triglyceride transfer protein (MTP) plays an essential role in lipid metabolism, especially in the biogenesis of very low-density lipoproteins and chylomicrons via the transfer of neutral lipids and the assembly of apoB-containing lipoproteins. Our understanding of the molecular mechanisms of MTP has been hindered by a lack of structural information of this heterodimeric complex comprising an MTPα subunit and a protein disulfide isomerase (PDI) β-subunit. The structure of MTP presented here gives important insights into the potential mechanisms of action of this essential lipid transfer molecule, structure-based rationale for previously reported disease-causing mutations, and a means for rational drug design against cardiovascular disease and obesity. In contrast to the previously reported structure of lipovitellin, which has a funnel-like lipid-binding cavity, the lipid-binding site is encompassed in a β-sandwich formed by 2 β-sheets from the C-terminal domain of MTPα. The lipid-binding cavity of MTPα is large enough to accommodate a single lipid. PDI independently has a major role in oxidative protein folding in the endoplasmic reticulum. Comparison of the mechanism of MTPα binding by PDI with previously published structures gives insights into large protein substrate binding by PDI and suggests that the previous structures of human PDI represent the “substrate-bound” and “free” states rather than differences arising from redox state.

The role of dynamics in modulating ligand exchange in intracellular lipid binding proteins

2014 ◽  
Vol 1844 (7) ◽  
pp. 1268-1278 ◽  
Author(s):  
Laura Ragona ◽  
Katiuscia Pagano ◽  
Simona Tomaselli ◽  
Filippo Favretto ◽  
Alberto Ceccon ◽  
...  
Keyword(s):  
Ligand Exchange ◽  
Binding Proteins ◽  
Lipid Binding ◽  
Intracellular Lipid ◽  
Lipid Binding Proteins

scholarly journals INTRACELLULAR LIPID-BINDING PROTEINS AND THEIR GENES

1997 ◽  
Vol 17 (1) ◽  
pp. 277-303 ◽  
Author(s):  
David A. Bernlohr ◽  
Melanie A. Simpson ◽  
Ann Vogel Hertzel ◽  
Leonard J. Banaszak
Keyword(s):  
Binding Proteins ◽  
Lipid Binding ◽  
Intracellular Lipid ◽  
Lipid Binding Proteins

scholarly journals An Application of Fit Quality to Screen MDM2/p53 Protein-Protein Interaction Inhibitors

Molecules ◽  
2018 ◽  
Vol 23 (12) ◽  
pp. 3174 ◽  
Author(s):  
Xin Xue ◽  
Gang Bao ◽  
Hai-Qing Zhang ◽  
Ning-Yi Zhao ◽  
Yuan Sun ◽  
...  
Keyword(s):  
Protein Interaction ◽  
Drug Target ◽  
P53 Protein ◽  
Pharmacophore Model ◽  
Cellular Level ◽  
Complex Structures ◽  
Ligand Complex ◽  
Null Cell ◽  
Protein Protein Interaction ◽  
First Time

: The judicious application of ligand or binding efficiency (LE) metrics, which quantify the molecular properties required to obtain binding affinity for a drug target, is gaining traction in the selection and optimization of fragments, hits and leads. Here we report for the first time the use of LE based metric, fit quality (FQ), in virtual screening (VS) of MDM2/p53 protein-protein interaction inhibitors (PPIIs). Firstly, a Receptor-Ligand pharmacophore model was constructed on multiple MDM2/ligand complex structures to screen the library. The enrichment factor (EF) for screening was calculated based on a decoy set to define the screening threshold. Finally, 1% of the library, 335 compounds, were screened and re-filtered with the FQ metric. According to the statistical results of FQ vs activity of 156 MDM2/p53 PPIIs extracted from literatures, the cut-off was defined as FQ = 0.8. After the second round of VS, six compounds with the FQ > 0.8 were picked out for assessing their antitumor activity. At the cellular level, the six hits exhibited a good selectivity (larger than 3) against HepG2 (wt-p53) vs Hep3B (p53 null) cell lines. On the further study, the six hits exhibited an acceptable affinity (range of Ki from 102 to 103 nM) to MDM2 when comparing to Nutlin-3a. Based on our work, FQ based VS strategy could be applied to discover other PPIIs.

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