Biology Theses and Dissertations
Permanent URI for this collectionhttp://hdl.handle.net/1903/2749
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Item Mitochondrial outer membrane permeability to metabolites influences the onset of apoptosis(2007-05-08) Tan, Wenzhi; Colombini, Marco; Biology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Apoptosis is a process in multicellular organisms to signal and induce death of specific cells, while avoiding inflammatory reactions. It is an important way to recycle the materials of unwanted cells and maintain cell balance. The execution phase of apoptosis can be initiated by proteins released from mitochondria (such as cytochrome c). Results reported here are consistent with this release being influenced by changes in the mitochondrial outer membrane permeability to metabolites. Phosphorothioate oligonucleotides induce cell death and block VDAC, a protein in the mitochondrial outer membrane that facilitates metabolite flow. These properties seem to be linked in that both require the phosphorothioate modification, both are enhanced by an increase in oligonucleotide length, and both are insensitive to nucleotide sequence. VDAC reconstituted into planar phospholipid membranes is blocked by phosphorothioate oligonucleotides with a 1:1 stoichiometry. They block the pore of the channel through interacting with the inner wall of the pore. The rate of binding occurs at a 100 μs scale but the binding is usually unstable. However, some conformational change stabilizes the complex resulting in long-term complete blockage of VDAC. In mitochondria, this blockage interferes with metabolite flow and inhibits the respiration of mitochondria. It is very specific for VDAC at sub-micromolar concentrations of phosphorothioate oligonucleotide and under these conditions there is minimal effect on enzymatic processes in the mitochondrial inner membrane. The ability of PorB from Neisseria meningitidis to inhibit apoptosis by moving to the mitochondrial outer membrane, was investigated in light of VDAC's role in apoptosis. PorB is unable to alter VDAC's gating properties but does allow ATP to cross membranes. Thus it may restore metabolite flux when VDAC channels close early in apoptosis. Attempts to test this in yeast were not successful. VDAC gating influences transmembrane Ca2+ flux. The closed states favor calcium permeation and the open state limits calcium flux. In mitochondria this gating could influence the rate of Ca2+-dependent mitochondrial swelling and subsequent cytochrome c release. Thus, the mitochondrial outer membrane permeability regulated by VDAC gating may play an important role in mitochondrial function and control of apoptosis.Item Ceramide Metabolism and Transport: Implications on the Initiation of Apoptosis(2006-12-19) Stiban, Johnny; Colombini, Marco; Biology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Apoptosis is a process by which unwanted cells are eliminated in a controlled manner. Early in apoptosis, ceramide levels rise and the mitochondrial outer membrane becomes permeable to proteins. The permeability of the outer membrane is attributed to the self-assembly of ceramide in form of channels. In the only direct structural study, to date, ceramide channels were visualized in liposomes using transmission electron microscopy. Those channels were of various sizes, averaging 10 nm in diameter. In concert, using electrophysiological techniques, the estimated diameter of ceramide channels was also around 10 nm. These channels are large enough to release all the pro-apoptotic intermembrane space proteins to initiate apoptosis. Dihydroceramide desaturase converts the inactive precursor, dihydroceramide to ceramide. Both long and short chain dihydroceramides inhibit ceramide channel formation in mitochondria. The inhibition is strong as one tenth as much dihydroceramide inhibited the outer membrane permeabilization by 95% (C2) and 51% (C16). Other mitochondrial components are not required for such inhibition as comparable amounts prevented the permeabilization of liposomes. Hence, the apoptogenic activity of ceramide may depend on the ceramide to dihydroceramide ratio perhaps resulting in a more abrupt transition from the normal to the apoptotic state. The location of the desaturase is the endoplasmic reticulum (ER). Only minimal activity was measured in mitochondria. However, newly synthesized ceramide from 14C-C8-dihydroceramide or 3H-sphingosine (in the ER) can transfer rapidly to mitochondria (40 % in 10 min) and permeabilize them to cytochrome c and adenylate kinase. The transfer of sphingolipids is bidirectional and non-specific. The transfer mechanism is consistent with direct membrane contact, since reducing the organellar concentrations by half resulted in a four-fold reduction of the transfer rate. Thus this ceramide exchange obviates the need for a complete ceramide de novo pathway in mitochondria in order for cells to use ceramide to activate mitochondria-mediated apoptosis. These results demonstrate the ability of ceramide to form large channels capable of releasing proteins from mitochondria. Ceramide can rapidly reach mitochondria and there are mechanisms to control the propensity for ceramide channel formation. Clearly ceramide channels play a central role in the decision to undergo apoptosis.Item Bacterial Phenotypes and Molecular Mechanisms of Mechanosensitive Channels(2005-12-09) Shirinian, Lena; Sukharev, Sergei; Biology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)This work presents a functional analysis of mutations in two bacterial mechanosensitive channels, MscL and MscS using bacterial growth phenotyping combined with electrophysiological and structural analyses. The introduction of aromatic caps at the ends of lipid facing helices in MscL compromises the osmotic rescuing function of the channel and changes gating parameters. The characteristic absence of aromatic residues at membrane interfaces is critical for MscL function, as the opening transition is associated with a strong helical reorientation. According to the current model of MscS, the pore-forming TM3 helices are predicted to separate, tilt, and straighten upon channel opening. This dynamic transition has been examined using a cysteine scan of this region and MTS accessibility experiments. Both cell viability assays and electrophysiological data support the hypothesis of a helical separation. Conductance measurements in gate mutants suggest that the pore lumen narrows toward the periplasmic end, consistent with the current model.Item MITOCHONDRIAL VDAC AND BACTERIAL PORA/C1: ION PERMEATION AND SELECTIVITY(2005-07-25) Komarov, Alexander Gennadievich; Colombini, Marco; Biology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)VDAC and PorA/C1 are large diameter channels with properties reminiscent of those found in narrow channels. VDAC, located in the mitochondrial outer membrane, shows high selectivity for ATP over comparably sized ions. VDAC is characterized by a single open state with anionic selectivity and multiple cation-selective closed states. PorA/C1 from Neisseria meningitidis achieves high cationic selectivity and large conductance. VDAC has multiple functions in cellular processes and the most important one is the regulation of metabolite flow across the outer membrane. A variety of functions could be achieved by the existence of different isoforms. In this thesis I summarized the electrophysiological properties of VDAC-like proteins from Drosophila Melanogaster encoded by genes CG17137, CG17139 and CG17140. The ability of these proteins to form channels was tested on planar membranes and liposomes. Channel activity was observed with varying degrees of similarity to VDAC. Two of these proteins (CG17137, CG17140) produced channels with anionic selectivity in the open state. Sometimes channels exhibited closure and voltage gating, but for CG17140 this occurred at much higher voltages than is typical for VDAC. CG17139 did not form channels. The special selectivity of VDAC for large anions was explored using the mutant of the mouse isoform 2. Inserted into planar membranes, mutant channels lack voltage gating, have a lower conductance, demonstrate cationic selectivity and, surprisingly, are still permeable to ATP. The estimated ATP flux through the mutant is comparable to that for the wildtype. Also we determined that the intact outer membrane containing the mutant is permeable to NADH and ADP/ATP. Both experiments support the counterintuitive conclusion that converting a channel from anion to cation preference does not substantially influence the flux of negatively charged metabolites. However, this finding supports the previous proposal that ATP translocation through VDAC is facilitated by a set of specific interactions between ATP and the channel wall. The third part of my thesis represents experimental data supporting the theoretical model for the PorA/C1 structure. This model explains the almost ideal cationic selectivity of the channel and high level of rectification. These properties are proposed to arise from a high density of charges in the channel that results in both high selectivity and high ionic flux.