Deficiencies of the oxidative phosphorylation system (OXPHOS) that consists of five enzyme
complexes (I-IV) lead to a diversity of cellular consequences. This includes altered Ca2+
homeostasis, reduced ATP production and increased ROS (reactive oxygen species) production.
One of the secondary consequences of such deficiencies is the adaptive transcriptional responses
of several mitochondrial- and nuclear-encoded genes involved in OXPHOS biogenesis.
Additionally, several other genes that are involved in several other functions, such as
metallothioneins (MTs), are differentially expressed. In this study we investigated two hypotheses:
firstly, that in complex I deficient cells the increased expression of MTs, specifically MT1B and
MT2A, has a protective effect against ROS-related consequences of a complex I deficiency. The
second hypothesis stated that genes involved in mitochondrial replication and transcription are
differentially expressed in OXPHOS deficient cell lines.
Firstly, the expression and role of metallothioneins (MTs) in an in vitro complex I deficient model
was investigated. The increased expression of different MT isoforms in the presence of the
complex I inhibitor rotenone in HeLa cells was confirmed. In this complex I deficient model overexpression
of MT1B and especially MT2A isoforms also protected against ROS, mtPTP opening,
apoptosis and ROS-induced necrosis. This data supports the hypothesis that increased expression
of MT2A has a protective effect against the death-causing cellular consequences of rotenonetreated
HeLa cells.
Secondly, we investigated the differential expression of selected mitochondrial- and nuclear genes
involved in OXPHOS function and regulation. Two experimental in vitro models were developed
and utilized in the study. Firstly, a transient siRNA knockdown model of the NDUFS3 subunit of
complex I in 143B cells was developed, characterized and introduced. Then the effect of the
knockdown on several biochemical parameters (ROS and ATP levels), mtDNA copy number, total
mtRNA levels, and RNA levels of several nuclear- and mitochondrial-encoded transcripts encoding
structural as well as functional proteins was determined. Additionally, to investigate the effect of
stable OXPHOS deficiency, stable shRNA knockdown models of the NDUFS3 subunit of complex
I, as well as the Rieske subunit of complex III were introduced and characterized.
The second hypothesis about the effect of OXPHOS deficiencies on mtDNA replication and
transcription could not, without a doubt, be supported or contradicted by the data. It was
determined from the data that an OXPHOS deficiency, which does not result in increased ROS
levels, does not significantly affect the regulation of mtDNA replication/transcription or nuclear
OXPHOS gene transcription. However, when OXPHOS deficiency was accompanied by increased
ROS levels, some structural mitochondrial-encoded transcripts and regulatory nuclear-encoded
transcripts were up-regulated, specifically ND6, D-loop, DNApol and TFB2M. Nonetheless,
increased ROS production in the presence of OXPHOS deficiency is probably not exclusively
responsible for responses of all regulatory proteins involved in mtDNA replication/transcription in
vitro. Additionally, this compensatory regulation might be more dependent on mtDNA transcription
than mtDNA copy number, and the data showed that TFB2M might be a key regulatory protein
involved early in this mechanism before any other regulatory proteins are affected. / Thesis (Ph.D. (Biochemistry))--North-West University, Potchefstroom Campus, 2010.
| Identifer | oai:union.ndltd.org:NWUBOLOKA1/oai:dspace.nwu.ac.za:10394/4240 |
| Date | January 2010 |
| Creators | Reinecke, Fimmie |
| Publisher | North-West University |
| Source Sets | North-West University |
| Language | English |
| Detected Language | English |
| Type | Thesis |
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