title: Ceramide channels: destabilization by Bcl-xL and role in apoptosis.
abstract: Ceramide is a bioactive sphingolipid involved in mitochondrial-mediated apoptosis. Our data suggest that ceramides directly regulate a key initiation step in apoptosis: mitochondrial outer membrane permeabilization (MOMP). MOMP allows release of intermembrane space proteins to the cytosol, inducing the execution of the cell. Ceramides form channels in planar phospholipid membranes and outer membranes of isolated mitochondria, channels large enough to facilitate passage of proteins released during MOMP. Bcl-xL inhibits MOMP in vivo and inhibits the formation of ceramide channels in vitro. However the significance of Bcl-xL's regulation of ceramide channel formation within cells was untested. We engineered Bcl-xL point mutations that specifically affect the interaction between ceramide and Bcl-xL to probe the mechanism of ceramide channel regulation and the role of ceramide channels in apoptosis. Using these mutants and fluorescently-labeled ceramide, we identified the hydrophobic groove on Bcl-xL as the critical ceramide binding site and regulator of ceramide channel formation. Bcl-xL mutants with weakened interaction with ceramide also have reduced ability to interfere with ceramide channel formation. Some mutants have similar altered ability to inhibit both ceramide and Bax channel formation, whereas others act differentially, suggesting distinct but overlapping binding sites. To probe the relative importance of these channels in apoptosis, Bcl-xL mutant proteins were stably expressed in Bcl-xL deficient cells. Weakening the inhibition of either Bax or ceramide channels decreased the ability of Bcl-xL to protect cells from apoptosis in a stimulus-dependent manner. These studies provide the first in vivo evidence for the role of ceramide channels in MOMP.
authors.affiliation: Department of Biology, University of Maryland, United States.
authors.forename: Kai-Ti
authors.initials: KT
authors.lastname: Chang
authors.affiliation: Department of Biology, University of Maryland, United States.
authors.forename: Andriy
authors.initials: A
authors.lastname: Anishkin
authors.affiliation: Department of Pharmacology and Toxicology, University of Louisville, United States.
authors.forename: Gauri A
authors.initials: GA
authors.lastname: Patwardhan
authors.affiliation: Department of Pharmacology and Toxicology, University of Louisville, United States; James Graham Brown Cancer Center, University of Louisville, United States; Department of Medicine, University of Louisville, United States.
authors.forename: Levi J
authors.initials: LJ
authors.lastname: Beverly
authors.affiliation: Department of Pharmacology and Toxicology, University of Louisville, United States; James Graham Brown Cancer Center, University of Louisville, United States.
authors.forename: Leah J
authors.initials: LJ
authors.lastname: Siskind
authors.affiliation: Department of Biology, University of Maryland, United States. Electronic address: colombini@umd.edu.
authors.forename: Marco
authors.initials: M
authors.lastname: Colombini
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000502:physiology; D001665:Binding Sites; D002463:Cell Membrane Permeability / Q000502:physiology; D002478:Cells, Cultured; D002518:Ceramides / Q000737:chemistry* / Q000378:metabolism*; D006801:Humans; D051379:Mice; D008930:Mitochondria, Liver / Q000502:physiology* / Q000648:ultrastructure; D051336:Mitochondrial Membranes / Q000502:physiology* / Q000648:ultrastructure; D056004:Molecular Dynamics Simulation; D011485:Protein Binding; D051381:Rats; D017207:Rats, Sprague-Dawley; D051020:bcl-X Protein / Q000737:chemistry* / Q000378:metabolism*
chemical_list: C494813:Bcl2l1 protein, rat; D002518:Ceramides; D051020:bcl-X Protein
keywords: Apoptosis; Bax; Bcl-2; Mitochondria; Outer membrane; Sphingolipid