Abstract
Cardiovascular disease (CVD) is primarily caused by atherosclerosis that ultimately leads to a heart attack or stroke. The complex aetiology of atherosclerosis is linked to various risk factors including high-density lipoprotein (HDL). HDL plays an atheroprotective role by removing cholesterol from arteries and other peripheral tissues. The atheroprotective potential of HDL is associated with HDL cholesterol (HDL-C) level in the blood that directly depends upon the cholesterol efflux mediated by a cellular membrane protein, ATP-binding cassette A1 (ABCA1). The ABCA1 transporter is a transmembrane protein that exports cellular cholesterol to form HDL. The regulation of ABCA1 is influenced by genetic as well as environmental factors. Many mutations in ABCA1 disrupt trafficking to the plasma membrane and reduce its function.
A chemical chaperone, 4-phenylbutyrate (4-PBA) was investigated for its potential to assist ABCA1 trafficking and cholesterol efflux function. 4-PBA is an FDA approved drug. It is a short-chain fatty acid that acts as a histone deacetylase inhibitor and is known to upregulate many transcription factors. The major aim of this study was to determine the impact of 4-PBA on chaperone or trafficking proteins that are functionally relevant to ABCA1. Two mammalian cell lines, HEK293 and HepG2 were used as model systems. The wild type and N1800H mutant ABCA1 expression were introduced to HEK293 cells via transient transfection. The HepG2 cells models were developed by a stable transfection approach to integrate wild type and N1800H mutant ABCA1. The cell lysates of 4-PBA treated and control cells were analysed by proteomic analyses. A discovery proteomics approach, SWATH-MS was used to identify differentially regulated proteins in response to 4-PBA treatment.
The first objective was the determination of differentially regulated proteins by 4-PBA in HEK293 cell models. The assessment of ABCA1 protein level and efflux function established that 4-PBA has the potential to improve ABCA1-mediated cholesterol efflux in HEK293 cells. The proteomic analysis of HEK293 cell models suggested that the regulation of transcription, translation and downstream signalling processes is a conventional effect of 4-PBA based on its histone deactylase inhibitor (HDACi) action. The chaperone action of 4-PBA implied multiple chaperones and trafficking proteins in transfected HEK293 cells. Two classical chaperone proteins, calnexin and calreticulin were downregulated in ABCA1 expressing HEK293 cells. To investigate the functional impact of regulated proteins, a physiologically relevant cell line i.e. HepG2 was selected. The proteomic analysis of HepG2 cell models showed the regulation of a different set of proteins. Calnexin and calreticulin were not regulated by 4-PBA in stably transfected HepG2 cell models. Moreover, ABCA1 protein levels were reduced in 4-PBA treated HepG2 cells. These results motivated us to investigate the effects of 4-PBA on ABCA1 protein in a relevant and less manipulated cellular system.
The proteomics results of untransfected HepG2 cells showed the strong manipulation of trafficking proteins compared to previous proteomic analyses. Three trafficking proteins, Rab10, Sec61β and sortilin were selected for further analysis based on their functional relevance to ABCA1. The immunofluorescence microscopy indicated a potential colocalisation of ABCA1 with all three selected proteins. Further siRNA knockdown showed that only Rab10 has a functional correlation with ABCA1. The siRNA knockdown of Rab10 significantly increased ABCA1-mediated cholesterol efflux in HepG2 cells. This result suggests that Rab10 has the potential to retard ABCA1 trafficking towards the plasma membrane.
Lastly, this thesis has examined the effect of 4-PBA on cell surface expression of ABCA1 in HepG2 cells. The reduced ABCA1 protein level but unaffected ABCA1-mediated cholesterol efflux from 4-PBA treated HepG2 cells encouraged further investigation of cell surface ABCA1. A targeted proteomic analysis, Parallel Reaction Monitoring-Mass Spectrometry (PRM-MS) successfully identified ABCA1 in a surface proteins-enriched fraction of 4-PBA treated cell lysates. The cell surface expression of ABCA1 was unaffected by 4-PBA treatment as assessed by heavy isotope labelled peptides used as internal standards. This result further confirms that 4-PBA does not alter the cholesterol efflux function of ABCA1 in HepG2 cells.
In conclusion, this PhD thesis has progressed our understanding of 4-PBA effects on ABCA1 trafficking and function. A potential association between ABCA1 and Rab10 has been shown for the first time. The cell line dependent regulation of ABCA1 by 4-PBA was determined. These new findings of 4-PBA effects on multiple biological processes may help to discover new therapeutical potentials of 4-PBA.