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BIOPHYSICAL STUDIES OF THE MECHANISM OF CERAMIDE CHANNEL DESTABLIZATION BY BCL-XL IN APOPTOSIS AND THE USE OF RECTIFICATION TO PROBE THE STRUCTURE AND DYNAMICS OF A NOVEL ESCHERICHIA COLI CHANNEL

dc.contributor.advisorColombini, Marcoen_US
dc.contributor.authorChang, Kai-Tien_US
dc.date.accessioned2016-02-06T06:44:18Z
dc.date.available2016-02-06T06:44:18Z
dc.date.issued2015en_US
dc.identifierhttps://doi.org/10.13016/M2X43J
dc.identifier.urihttp://hdl.handle.net/1903/17308
dc.description.abstractCeramide forms a novel type of channel in the mitochondrial outer membrane and these channels are involved the release of intermembrane space proteins from mitochondria, a decision-making step in the apoptotic process. An antiapoptotic protein, Bcl-xL, regulates the apoptotic process and inhibits the formation of ceramide channels. However, there is no precedent to indicate how a protein regulates a lipid channel. We investigated the mechanism of this regulation and identified the hydrophobic groove of the Bcl-xL as the binding site by which Bcl-xL binds to the channel. This was demonstrated by using a combination of experimental and modeling methods, including site-directed mutagenesis, a fluorescence quenching assay, a mitochondrial outer membrane permeability assay, and molecular dynamic simulations. Interestingly, the hydrophobic groove serves to inhibit another channel former, Bax. We found that the binding sites for Bax and ceramide on Bcl-xL are distinct but overlapping. We used that fact to generate mutants that have differential abilities to inhibit one or the other of these channels. These are useful because although ceramide is important in apoptosis, it is still controversial that whether ceramide channels result in apoptosis in vivo. To probe the relative importance of these two channels in apoptosis, Bcl-xL mutant proteins were expressed in Bcl-xL deficient cells. Weakening the inhibitory potency of Bcl-xL on either Bax or ceramide channels resulted in cells being more sensitive to the induction of apoptosis. This is the first evidence for the role of ceramide channels in the apoptotic process in vivo. In a separate investigation, a novel voltage-gated channel unit was found in E. coli extracts. The unit is consistent with three channels forming the functional triplet. These channels are highly voltage gated and highly cooperative. Those results indicated that one of the channels is oriented in an antiparallel fashion compared to the rest. This arrangement is very rare in protein channels. Rectification of the current flowing through the channels was used to identify the orientation of the channels to provide evidence for or against the antiparallel hypothesis. The results favor the antiparallel hypothesis but also reveal an unexpected asymmetry in the transmembrane electrostatics.en_US
dc.language.isoenen_US
dc.titleBIOPHYSICAL STUDIES OF THE MECHANISM OF CERAMIDE CHANNEL DESTABLIZATION BY BCL-XL IN APOPTOSIS AND THE USE OF RECTIFICATION TO PROBE THE STRUCTURE AND DYNAMICS OF A NOVEL ESCHERICHIA COLI CHANNELen_US
dc.typeDissertationen_US
dc.contributor.publisherDigital Repository at the University of Marylanden_US
dc.contributor.publisherUniversity of Maryland (College Park, Md.)en_US
dc.contributor.departmentBiochemistryen_US
dc.subject.pqcontrolledBiophysicsen_US
dc.subject.pqcontrolledBiochemistryen_US
dc.subject.pquncontrolledApoptosisen_US
dc.subject.pquncontrolledBcl-2en_US
dc.subject.pquncontrolledMitochondriaen_US
dc.subject.pquncontrolledRectificationen_US
dc.subject.pquncontrolledSphingolipiden_US
dc.subject.pquncontrolledVoltage-gated channelen_US


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