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Title: Elucidation of the DFNA5 protein through the study in yeast and human cell lines.
Other Titles: Opheldering van de rol van het DFNA5 eiwit via modelsystemen in gist en human cellijnen.
Authors: Van Rossom, Sofie
Issue Date: 16-Dec-2014
Abstract: In 1997, DFNA5 was originally identified as a gene responsible for an autosomal dominant non-syndromic form of hearing loss (HL). Today, already eight families with HL have been reportedwith mutations in DFNA5. Four of these mutations differ at genomic level, but they all result in skipping of exon 8 leading to a truncated immature protein. Skipping of exon 8 causes a frameshift of the open reading frame, leading to a shortened version of the unmutated wild-type DFNA5 protein (wtDFNA5), namely mutant DFNA5 (mutDFNA5). In addition to HL, wtDFNA5 has also been correlated with different forms of cancer, such as breast, colorectal, gastric and melanoma cancer. In these tumours, DFNA5 is epigenetically inactivated by hypermethylation. Furthermore, overexpression of DFNA5 in tumour cell lines resulted in reduced tumour growth and colony size which ledto the hypothesis that DFNA5 is a tumour suppressor gene. Gregan et al. (2003) were the first to demonstrate that mutDFNA5 induced a growth defect in fission yeast, but the exact function of DFNA5 remained unknown for a long time. Recently however, functional studies on <span style="mso-bidi-font-style:normal">DFNA5 <span style="mso-bidi-font-style:normal">identified the gene as an apoptosis-inducing gene in human cell lines. As already demonstrated by Gregan <span style="mso-bidi-font-style:normal">et al., the DFNA5-induced cell death mechanism seems to be at least partially conserved between yeast and human cell lines. Therefore, the first part of this thesis was performed using Saccharomyces cerevisiae as a model organism. A DFNA5-humanised yeast model was developed in the BY4741 background strain, transformed with either wtDFNA5 or mutDFNA5, and validated by several cell<span style="mso-bidi-font-style:normal"><span style="mso-bidi-font-style:normal"> d<span style="mso-bidi-font-style:normal"><span style="mso-bidi-font-style:normal">eath assays to determine the value of yeast as a model organism for DFNA5. <span style="mso-bidi-font-style:normal"><span style="mso-bidi-font-style:normal">Next, an apoptosis deletion strain collection, a collection of eighteen yeast strains deleted in one particular gene related to apoptosis, was screened to identify modulators of the DFNA5 cell death<span style="mso-bidi-font-style:normal"><span style="mso-bidi-font-style:normal">mechanisms in yeast. Growth profiles of every yeast strain and several apoptotic stainings were performed and compared to the background strain. This identified several mitochondrial proteins related to DFNA5-induced cell death. The Fis1, Por1, and, Aac1 and Aac3 proteins were characterised as respectively inhibitors and inducers of DFNA5-related cell death. These proteins were related to either the mitochondrial dynamics or the permeabilisation process, revealing for the first time an important role for the mitochondria. <span style="mso-bidi-font-style:normal"><span style="mso-bidi-font-style:normal">Additionally, wtDFNA5 was subject to normal protein degradation in yeast, whereas mutDFNA5 seemed to escape this protein quality control. As the endoplasmic reticulum (ER) is involved in protein<span style="mso-bidi-font-style:normal"><span style="mso-bidi-font-style:normal">folding and degradation, these results could suggest an additional role for the ER. As both organelles, the mitochondria and the ER, are tightly correlated, interplay between the ER and the mitochondria could be important for<span style="mso-bidi-font-style:normal"><span style="mso-bidi-font-style:normal"> DFNA5-induced cell death in yeast. <span style="mso-bidi-font-style:normal"><span style="mso-bidi-font-style:normal"><span style="mso-bidi-font-style:normal"><span style="mso-bidi-font-style:normal">Later on, an Agilent microarray study performed in yeast confirmed the role of the mitochondria as several gene ontology mitochondria-related processes, such as ATP-coupled electron transport<span style="mso-bidi-font-style:normal"><span style="mso-bidi-font-style:normal">and respiration, were up-regulated upon mutDFNA5 transformation in yeast. Additionally, processes related to protein folding and the ER were down-regulated upon mutDFNA5 transformation. These data validated the former results indicating a potential role for the ER. <span style="mso-bidi-font-style:normal"><span style="mso-bidi-font-style:normal"><span style="mso-bidi-font-style:normal"><span style="mso-bidi-font-style:normal"><span style="mso-bidi-font-style:normal"><span style="mso-bidi-font-style:normal"><span style="mso-bidi-font-style:normal"><span style="mso-bidi-font-style:normal">T<span style="mso-bidi-font-style:normal"><span style="mso-bidi-font-style:normal">he second part of this thesis wasperformed in human HEK293T cells. A former Illumina microarray experiment was re-evaluated by an additional analysis investigating the gene <span style="mso-bidi-font-style:normal"><span style="mso-bidi-font-style:normal">ontology-enriched annotations. This microarray experiment identified two main features, namely the up-regulation of the MAPK pathways and the down-regulation of the protein folding process upon mutDFNA5 transfection. The up-regulation of the MAPK pathway was characterised by phosphorylation of JNK and ERK. Specific inhibition of JNK partially abolished the mutDFNA5-induced cell death, indicating the importance of this pathway in the DFNA5-associated cell death mechanism. Down-regulation of gene ontology processes related to protein folding again revealed a potential role for the ER in DFNA5-associated mechanisms. <span style="mso-bidi-font-style:normal"><span style="mso-bidi-font-style:normal"><span style="mso-bidi-font-style:normal"><span style="mso-bidi-font-style:normal">Because the prior research in yeast identified several mitochondrial proteins, several mitochondrial aspects were additionally investigated in human HEK293T cells. This revealed partial loss of the mitochondrial membrane potential, increased ROS production, release of both cytochrome c and the matrix citrate synthase. These events were independent on caspases. The mitochondrial network was further investigated by fluorescence microscopy and revealed the accumulation of damaged mitochondria in mutDFNA5-transfected HEK293T cells. This <span style="mso-bidi-font-style:normal"><span style="mso-bidi-font-style:normal">was demonstrated by the presence of spherical aggregates and the absence of a tubular integer mitochondrial structure which was observed in the control samples. These results indicate the presence of dysfunctional mitochondria upon expression of mutDFNA5 leading to mitochondrial collapse and eventually cell death. The link between the mitochondria, the MAPK pathway and the ER in DFNA5-related cell death, however, remains unknown at this moment. Additional research is needed to further investigate DFNA5-related processes, which will lead to new insights for DFNA5-associated HL and, more in general, for HL related to mitochondrial cell death. <span style="mso-bidi-font-style:normal"><span style="mso-bidi-font-style:normal"><span style="mso-bidi-font-style:normal"><span style="mso-bidi-font-style:normal"><span style="mso-bidi-font-style:normal"><span style="mso-bidi-font-style:normal"><span style="mso-bidi-font-style:normal"><span style="mso-bidi-font-style:normal"><span style="mso-bidi-font-style:normal"><span style="mso-bidi-font-style:normal"><span style="mso-bidi-font-style:normal"><span style="mso-bidi-font-style:normal"><span style="mso-bidi-font-style:normal"><span style="mso-bidi-font-style:normal"><span style="mso-bidi-font-style:normal"><span style="mso-bidi-font-style:normal"><span style="mso-bidi-font-style:normal"><span style="mso-bidi-font-style:normal"><span style="mso-bidi-font-style:normal"><span style="mso-bidi-font-style:normal"><span style="mso-bidi-font-style:normal"><span style="mso-bidi-font-style:normal"><span style="mso-bidi-font-style:normal"><span style="mso-bidi-font-style:normal"><span style="mso-bidi-font-style:normal"><span style="mso-bidi-font-style:normal"><span style="mso-bidi-font-style:normal"><span style="mso-bidi-font-style:normal"><span style="mso-bidi-font-style:normal"><span style="mso-bidi-font-style:normal"><span style="mso-bidi-font-style:normal"><span style="mso-bidi-font-style:normal"><span style="mso-bidi-font-style:normal"><span style="mso-bidi-font-style:normal"><span style="mso-bidi-font-style:normal"><span style="mso-bidi-font-style:normal"><span style="mso-bidi-font-style:normal"><span style="mso-bidi-font-style:normal"><span style="mso-bidi-font-style:normal"><span style="mso-bidi-font-style:normal"><span style="mso-bidi-font-style:normal"><span style="mso-bidi-font-style:normal"><span style="mso-bidi-font-style:normal"><span style="mso-bidi-font-style:normal"><span style="mso-bidi-font-style:normal">
ISBN: 978-90-8649-787-4
Publication status: published
KU Leuven publication type: TH
Appears in Collections:Molecular Physiology of Plants and Micro-organisms Section - miscellaneous

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