This rate was diminished by the presence of Bcl-xL in the medium

This rate was diminished by the presence of Bcl-xL in the medium. the hydrophobic groove on Bcl-xL as the crucial ceramide binding site and regulator of ceramide channel formation. Bcl-xL mutants with weakened conversation 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 evidence for the role of ceramide channels in MOMP. apoptotic process. 2. MATERIALS AND METHODS 2.1 Isolation of rat liver mitochondria Mitochondria were isolated from the liver of male Sprague Dawley rats as described originally (33) and as modified (29). The animal use protocols were approved by the Institutional Animal Care and Use Committee. The animals were euthanized by a procedure consistent with the Panel on Euthanasia of APG-115 the AVMA (American Veterinary Medical Association). The animal facility used to house the animals is usually accredited by AAALAC (Association for Assessment and Accreditation of Laboratory Animal Care). 2.2 Purification of recombinant proteins The construct of full-length human Bcl-xL (Bcl-xL wild-type), Bax and t-Bid were gifts from Dr. Marie Hardwick, Dr. Richard Youle and Dr. Donald Newmeyer, respectively. All single mutations of Bcl-xL were introduced using QuickChange Site-Directed Mutagenesis kit (Stratagene) and verified by DNA sequencing. Recombinant Bcl-xL and its mutants were purified as previously described (34) and as altered (29). In brief, GST-tagged Bcl-xL and GST-tagged Bid were produced in BL21(DE3) pLysS. The cells were induced with 10 M IPTG for 2 hours at 37C for GST-tagged Bcl-xL and the cells were induced with 0.4 mM IPTG for 3 hours at 37C for GST-tagged Bid, resuspended in PBS with 1.25 kU/L cells APG-115 of lysozyme and 35 M PMSF and lysed using a French press. After removal of cell fragments, the GST-tagged Bcl-xL and GST-tagged APG-115 t-Bid were purified using glutathione agarose beads and the GST tag was cleaved with 5 U biotinylated thrombin which was removed using streptavidin beads. Recombinant human Bax was purified using a chitin column (New England Biolabs Inc.) and dialyzed with a 12,000 MW cut-off dialysis membrane to remove dithiothreitol (17, 35, 36). All purified proteins were supplemented with glycerol to 10 %10 % and filtered sterilized through a 0.2 m filter prior to being rapidly shell-frozen in ethanol and dry ice and stored at ?80 C. The concentration of proteins was determined with a MicroBCA Protein Kit (Pierce Chemical). 2.3 Binding of ceramide to Bcl-xL nine microliters of fluorescently-labeled ceramide (C11 TopFluor ceramide; Avanti Polar Lipids) dissolved in isopropanol at 42 g/ml was dispersed to 590 l made up of 1.8 M Bcl-xL wild-type or its mutants in 20 mM Tris, pH 7.4, 10% sucrose. The dispersal was performed by slow delivery of the ceramide answer into the protein answer while it was being vortexed. Entrainment of air was carefully avoided. In order to remove ceramide micelles, 50 l of 20 mM Tris, pH 7.4, 5% sucrose was applied to the top of the mixture to produce a density gradient. Then the gradient was spun in MLA-130 at 55,000 rpm at 25C for 30 min. Ceramide micelles float into the upper layer and so a portion of the solution in the 10% sucrose phase was removed, excited by 490 nm incident light and the emission spectrum from 495 to 520 nm was recorded in a FluoroMax-4. The fluorescence was not detected in the non-protein controls, so all fluorescence was due to ceramide associated with protein. The fluorescent quantum yield of the bound ceramide varied depending on the protein.The animal use protocols were approved by the Institutional Animal Care and Use Committee. 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 evidence for the role of ceramide channels in MOMP. apoptotic process. 2. MATERIALS AND METHODS 2.1 Isolation of rat liver mitochondria Mitochondria were isolated from the liver of male Sprague Dawley rats as described originally (33) and as modified (29). The animal use protocols were approved by the Institutional Animal Care and Use Committee. The animals were euthanized by a procedure consistent with the Panel on Euthanasia of the AVMA (American Veterinary Medical Association). The animal facility used to house the animals is usually accredited by AAALAC (Association for Assessment and Accreditation of Laboratory Animal Care). 2.2 Purification of recombinant proteins The construct of full-length human Bcl-xL (Bcl-xL wild-type), Bax and t-Bid were gifts from Dr. Marie Hardwick, Dr. Richard Youle and Dr. Donald Newmeyer, respectively. All single mutations of Bcl-xL were introduced using QuickChange Site-Directed Mutagenesis kit (Stratagene) and verified by DNA sequencing. Recombinant Bcl-xL and its mutants were purified as previously described (34) and as altered (29). In brief, GST-tagged Bcl-xL and GST-tagged Bid were produced in BL21(DE3) APG-115 pLysS. The cells were induced with 10 M Rabbit Polyclonal to ARNT IPTG for 2 hours at 37C for GST-tagged Bcl-xL and the cells were induced with 0.4 mM IPTG for 3 hours at 37C for GST-tagged Bid, resuspended in PBS with 1.25 kU/L cells of lysozyme and 35 M PMSF and lysed using a French press. After removal of cell fragments, the GST-tagged Bcl-xL and GST-tagged t-Bid were purified using glutathione agarose beads and the GST tag was cleaved with 5 U biotinylated thrombin which was removed using streptavidin beads. Recombinant human Bax was purified using a chitin column (New England Biolabs Inc.) and dialyzed with a 12,000 MW cut-off dialysis membrane to remove dithiothreitol (17, 35, 36). All purified proteins were supplemented with glycerol to 10 %10 % and filtered sterilized through a 0.2 m filter prior to being rapidly shell-frozen in ethanol and dry ice and stored at ?80 C. The concentration of proteins was determined with a MicroBCA Protein Kit (Pierce Chemical). 2.3 Binding of ceramide to Bcl-xL nine microliters of fluorescently-labeled ceramide (C11 TopFluor ceramide; Avanti Polar Lipids) dissolved in isopropanol at 42 g/ml was dispersed to 590 l made up of 1.8 M Bcl-xL wild-type or its mutants in 20 mM Tris, APG-115 pH 7.4, 10% sucrose. The dispersal was performed by slow delivery of the ceramide answer into the protein answer while it was being vortexed. Entrainment of air was carefully avoided. In order to remove ceramide micelles, 50 l of 20 mM Tris, pH 7.4, 5% sucrose was applied to the top of the mixture to produce a density gradient. Then the gradient was spun in MLA-130 at 55,000 rpm at 25C for 30 min. Ceramide micelles float into the upper layer and so a portion of the solution in the 10% sucrose phase was removed, excited by 490 nm.