Investigation of selected autophagic proteins and the effect of tPS1-GFP expression.

ELAZZOUZI, Mowafag F. (2015). Investigation of selected autophagic proteins and the effect of tPS1-GFP expression. Doctoral, Sheffield Hallam University (United Kingdom)..

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Abstract

Normally, the balance between the production and degradation of cellular proteins is necessary for the cell to exist. One of the most important pathways by which the largest number of cytosolic and misfolded proteins is degraded is the autophagy-lysosome pathway. A defect in this process may result in accumulation and aggregation of proteins leading to cellular toxicity and subsequently neurodegenerative diseases. Previous work by Anderson (2003) with a truncated presenillin 1 construct, in HEK293 cell line showed structural changes in the cell in the early stages of autophagy. However, it seems that the autophagic process had failed to clear the presenilin-1 aggregates as would be expected in the normal state and appeared to lead to cell death. The work presented in this thesis primarily attempts to investigate, in term of subcellular localization, the influence of truncated PS1, expressed in HEK293 cells, on selected autophagy regulators (mTOR, raptor and LC3 proteins) and subsequently on the autophagy process. To investigate this, many different commercial antibodies were characterized against whole cell lysates from three different cell lines (NRK, MCF-7 and HEK293) using western blotting to obtain specific antibodies for mTOR, raptor and LC3. Then, the selected antibodies were used in dual label immunocytochemistry with mitochondrial antibody and wheat germ agglutinin (as a Golgi marker) to determine the subcellular localizations of mTOR, raptor and LC3 in non-autophagic HEK293 cells. To study the behaviour of the proteins of interest during autophagy of untransfected cells, HEK293 cells were rapamycin treated or serum starved. To compare the observed results with the behaviour of the proteins after the transfection, a truncated PS1-GFP construct was transiently transfected into HEK293 cells. Also, the subcellular localizations of the proteins were determined by dual label ICC.The data obtained suggests that in non-autophagic HEK293 cells, mTOR appears to be localized to mitochondria, raptor to the cytoplasm and LC3 to Golgi apparatus. Rapamycin treatment and serum starvation have the same influence on the behaviour of these proteins. In both cases, mTOR remained localized to mitochondria (no effect), raptor protein partially moved from the cytoplasm to the perinuclear area (similar to mitochondrial distribution) and some of the LC3 protein diffused to the cytoplasm, while most of it remained localized to the Golgi apparatus. Following the transfection, the observable data suggest that interactions between truncated PS1 and mTOR, raptor and LC3 might be occurring. It seems that truncated PS1 has no influence on the subcellular localizations of mTOR and raptor proteins (similar to mitochondrial distribution). However, in the case of LC3 protein, it seems that the protein has partially moved from the Golgi apparatus to interact with truncated PS1 in a new subcellular localization which is similar to the subcellular localizations of mTOR and raptor. The suggested interactions between truncated PS1 and mTOR, raptor and LC3 may dysregulate mTOR signalling pathway, prevent recruitment of mTOR substrates by raptor or block the fusion between autophagosomes and lysosomes via LC3. The results of this thesis indicate that further investigations into the interactions between PS1 and the proteins of interest especially LC3 are warranted.

Item Type: Thesis (Doctoral)
Additional Information: Thesis (Ph.D.)--Sheffield Hallam University (United Kingdom), 2015.
Research Institute, Centre or Group: Sheffield Hallam Doctoral Theses
Depositing User: EPrints Services
Date Deposited: 10 Apr 2018 17:19
Last Modified: 10 Apr 2018 17:19
URI: http://shura.shu.ac.uk/id/eprint/19678

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