Biophysical and proteomic characterization of the voltage-dependent anion-selective channel of Neurospora crassa
| dc.contributor.author | Ferens, Fraser | |
| dc.contributor.examiningcommittee | Oresnik, Ivan (Microbiology) | en_US |
| dc.contributor.examiningcommittee | Stetefeld, Jorg (Chemistry) | en_US |
| dc.contributor.examiningcommittee | O'Neil, Joe (Chemistry) | en_US |
| dc.contributor.examiningcommittee | Prive, Gil (University of Toronto) | en_US |
| dc.contributor.supervisor | Court, Deborah (Microbiology) | en_US |
| dc.date.accessioned | 2019-04-08T17:49:07Z | |
| dc.date.available | 2019-04-08T17:49:07Z | |
| dc.date.issued | 2019 | en_US |
| dc.date.submitted | 2019-03-20T16:10:54Z | en |
| dc.degree.discipline | Microbiology | en_US |
| dc.degree.level | Doctor of Philosophy (Ph.D.) | en_US |
| dc.description.abstract | The oligomeric state of the voltage-dependent anion-selective channel (VDAC) and the stability of the VDAC-hexokinase complex play integral roles in mitochondrially mediated apoptotic signaling. Various small to large assemblies of VDAC have been observed in mitochondrial outer membranes but they do not predominate in detergent-solubilized VDAC samples. In this work, a cholesterol analogue, cholesteryl-hemisuccinate (CHS), was shown induce the formation of Neurospora crassa VDAC multimers in detergent solution. The oligomeric states of VDAC in the absence and presence of CHS were deciphered through an integrated biophysics approach using microscale thermophoresis, analytical ultracentrifugation and size exclusion chromatography-small angle X-ray scattering. It was determined that CHS stabilizes the interaction between VDAC and hexokinase (Kd of 27 ± 6 µM). Thus, sterols such as cholesterol in higher eukaryotes or ergosterol in fungi may regulate the VDAC oligomeric state and may provide a potential target for the modulation of apoptotic signaling by effecting VDAC-VDAC and VDAC-hexokinase interactions. In a separate study, the physiological effects of a small 5 amino-acid deletion in the Neurospora crassa VDAC sequence (238porin) were examined using proteomic and biochemical analysis of a strain expressing only this variant. 238porin was determined to be assembled in the mitochondrial outer membrane by a protease resistance assay; however, the level of expression of 238porin was only 3% of that of the wild-type protein. 238Por displayed respiratory defects; however, it exhibited an almost wild-type linear growth rate. Analysis of mitochondrial proteomes from a VDAC wild-type strain, a strain lacking VDAC (ΔPor-1) and 238Por revealed that the expression of 238poin partially compensates for respiratory defects caused by VDAC knockout. The deletion (ΔPor-1) or depletion (238Por) of VDAC was found to increase the expression of a Rubredoxin-NAD+ reductase, a homologue of the higher eukaryotic apoptosis inducing factor. The studies in this work examined the behavior of isolated VDAC in vitro and the effects of VDAC dysfunction in vivo and provide a better understanding of VDAC behaviors involved in apoptotic signaling and in addition, the integrated biophysical approach described provides a powerful platform for the study of membrane protein complexes solubilized in detergent solutions. | en_US |
| dc.description.note | May 2019 | en_US |
| dc.identifier.citation | Ferens F.G., Spicer V., Krokhin O.V., Motnenko A., Summers W.A.T., Court D.A. 2017. A deletion variant partially complements a porin-less strain of Neurospora crassa. Biochemistry and Cell Biology, 95(2): 318-327 | en_US |
| dc.identifier.citation | Ferens F.G., Patel T.R., Oriss G., Court D.A., Stetefeld J. 2019. A Cholesterol Analog Induces an Oligomeric Reorganization of VDAC. Biophysical Journal, 116(5):847-859, DOI: 10.1016/j.bpj.2019.01.031 | en_US |
| dc.identifier.uri | http://hdl.handle.net/1993/33840 | |
| dc.language.iso | eng | en_US |
| dc.rights | open access | en_US |
| dc.subject | Biophysics | en_US |
| dc.subject | Channel | en_US |
| dc.subject | Biochemistry | en_US |
| dc.subject | Apoptosis | en_US |
| dc.subject | VDAC | en_US |
| dc.subject | Membrane protein | en_US |
| dc.subject | Oligomeric state | en_US |
| dc.subject | Proteomics | en_US |
| dc.title | Biophysical and proteomic characterization of the voltage-dependent anion-selective channel of Neurospora crassa | en_US |
| dc.type | doctoral thesis | en_US |