A search for ceramide binding proteins using bifunctional lipid analogs yields CERT-related protein StarD7

Svenja Bockelmann, John G. M. Mina, Sergei Korneev, Dina G. Hassan, Dagmar Mueller, Angelika Hilderink, Hedwich C. Vlieg, Reinout Raijmakers, Albert J. R. Heck, Per Haberkant, Joost C. M. Holthuis

Research output: Contribution to journalArticle

Abstract

Ceramides are central intermediates of sphingolipid metabolism with dual roles as mediators of cellular stress signaling and mitochondrial apoptosis. How ceramides exert their cytotoxic effects is unclear and their poor solubility in water hampers a search for specific protein interaction partners. Here, we report the application of a photoactivatable and clickable ceramide analog, pacCer, to identify ceramide binding proteins and unravel the structural basis by which these proteins recognize ceramide. Besides capturing ceramide transfer protein (CERT) from a complex proteome, our approach yielded CERT-related steroidogenic acute regulatory protein D7 (StarD7) as novel ceramide binding protein. Previous work revealed that StarD7 is required for efficient mitochondrial import of phosphatidylcholine (PC) and serves a critical role in mitochondrial function and morphology. Combining site-directed mutagenesis and photoaffinity labeling experiments, we demonstrate that the steroidogenic acute regulatory transfer domain of StarD7 harbors a common binding site for PC and ceramide. While StarD7 lacks robust ceramide transfer activity in vitro, we find that its ability to shuttle PC between model membranes is specifically affected by ceramides. Besides demonstrating the suitability of pacCer as a tool to hunt for ceramide binding proteins, our data point at StarD7 as a candidate effector protein by which ceramides may exert part of their mitochondria-mediated cytotoxic effects.
Original languageEnglish
Pages (from-to)515-530
Number of pages16
JournalJournal of Lipid Research
Volume59
Issue number3
DOIs
Publication statusPublished - 1 Mar 2018

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Ceramides
Carrier Proteins
Proteins
Lipids
Phosphatidylcholines
steroidogenic acute regulatory protein
Binding Sites
Mutagenesis
Sphingolipids
Mitochondria
Proteome
Ports and harbors
Site-Directed Mutagenesis
Metabolism
Solubility
Labeling

Cite this

Bockelmann, S., Mina, J. G. M., Korneev, S., Hassan, D. G., Mueller, D., Hilderink, A., ... Holthuis, J. C. M. (2018). A search for ceramide binding proteins using bifunctional lipid analogs yields CERT-related protein StarD7. Journal of Lipid Research, 59(3), 515-530. https://doi.org/10.1194/jlr.M082354
Bockelmann, Svenja ; Mina, John G. M. ; Korneev, Sergei ; Hassan, Dina G. ; Mueller, Dagmar ; Hilderink, Angelika ; Vlieg, Hedwich C. ; Raijmakers, Reinout ; Heck, Albert J. R. ; Haberkant, Per ; Holthuis, Joost C. M. / A search for ceramide binding proteins using bifunctional lipid analogs yields CERT-related protein StarD7. In: Journal of Lipid Research. 2018 ; Vol. 59, No. 3. pp. 515-530.
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Bockelmann, S, Mina, JGM, Korneev, S, Hassan, DG, Mueller, D, Hilderink, A, Vlieg, HC, Raijmakers, R, Heck, AJR, Haberkant, P & Holthuis, JCM 2018, 'A search for ceramide binding proteins using bifunctional lipid analogs yields CERT-related protein StarD7', Journal of Lipid Research, vol. 59, no. 3, pp. 515-530. https://doi.org/10.1194/jlr.M082354

A search for ceramide binding proteins using bifunctional lipid analogs yields CERT-related protein StarD7. / Bockelmann, Svenja; Mina, John G. M.; Korneev, Sergei; Hassan, Dina G.; Mueller, Dagmar; Hilderink, Angelika; Vlieg, Hedwich C.; Raijmakers, Reinout; Heck, Albert J. R.; Haberkant, Per; Holthuis, Joost C. M.

In: Journal of Lipid Research, Vol. 59, No. 3, 01.03.2018, p. 515-530.

Research output: Contribution to journalArticle

TY - JOUR

T1 - A search for ceramide binding proteins using bifunctional lipid analogs yields CERT-related protein StarD7

AU - Bockelmann, Svenja

AU - Mina, John G. M.

AU - Korneev, Sergei

AU - Hassan, Dina G.

AU - Mueller, Dagmar

AU - Hilderink, Angelika

AU - Vlieg, Hedwich C.

AU - Raijmakers, Reinout

AU - Heck, Albert J. R.

AU - Haberkant, Per

AU - Holthuis, Joost C. M.

PY - 2018/3/1

Y1 - 2018/3/1

N2 - Ceramides are central intermediates of sphingolipid metabolism with dual roles as mediators of cellular stress signaling and mitochondrial apoptosis. How ceramides exert their cytotoxic effects is unclear and their poor solubility in water hampers a search for specific protein interaction partners. Here, we report the application of a photoactivatable and clickable ceramide analog, pacCer, to identify ceramide binding proteins and unravel the structural basis by which these proteins recognize ceramide. Besides capturing ceramide transfer protein (CERT) from a complex proteome, our approach yielded CERT-related steroidogenic acute regulatory protein D7 (StarD7) as novel ceramide binding protein. Previous work revealed that StarD7 is required for efficient mitochondrial import of phosphatidylcholine (PC) and serves a critical role in mitochondrial function and morphology. Combining site-directed mutagenesis and photoaffinity labeling experiments, we demonstrate that the steroidogenic acute regulatory transfer domain of StarD7 harbors a common binding site for PC and ceramide. While StarD7 lacks robust ceramide transfer activity in vitro, we find that its ability to shuttle PC between model membranes is specifically affected by ceramides. Besides demonstrating the suitability of pacCer as a tool to hunt for ceramide binding proteins, our data point at StarD7 as a candidate effector protein by which ceramides may exert part of their mitochondria-mediated cytotoxic effects.

AB - Ceramides are central intermediates of sphingolipid metabolism with dual roles as mediators of cellular stress signaling and mitochondrial apoptosis. How ceramides exert their cytotoxic effects is unclear and their poor solubility in water hampers a search for specific protein interaction partners. Here, we report the application of a photoactivatable and clickable ceramide analog, pacCer, to identify ceramide binding proteins and unravel the structural basis by which these proteins recognize ceramide. Besides capturing ceramide transfer protein (CERT) from a complex proteome, our approach yielded CERT-related steroidogenic acute regulatory protein D7 (StarD7) as novel ceramide binding protein. Previous work revealed that StarD7 is required for efficient mitochondrial import of phosphatidylcholine (PC) and serves a critical role in mitochondrial function and morphology. Combining site-directed mutagenesis and photoaffinity labeling experiments, we demonstrate that the steroidogenic acute regulatory transfer domain of StarD7 harbors a common binding site for PC and ceramide. While StarD7 lacks robust ceramide transfer activity in vitro, we find that its ability to shuttle PC between model membranes is specifically affected by ceramides. Besides demonstrating the suitability of pacCer as a tool to hunt for ceramide binding proteins, our data point at StarD7 as a candidate effector protein by which ceramides may exert part of their mitochondria-mediated cytotoxic effects.

U2 - 10.1194/jlr.M082354

DO - 10.1194/jlr.M082354

M3 - Article

VL - 59

SP - 515

EP - 530

JO - Journal of Lipid Research

JF - Journal of Lipid Research

SN - 0022-2275

IS - 3

ER -