The Role of Progesterone Receptor Membrane Component 1 (PGRMC1) in Cell Biology

    Research output: ThesisDoctoral Thesis

    Abstract

    PGRMC1 is a multifunctional evolutionarily conserved protein. PGRMC1 is associated with many intracellular functions such as, progesterone regulation, cholesterol synthesis, heme binding, cell cycle regulation, and drive many signalling pathways. In cancer, PGRMC1 plays a vital role in the suppression of apoptosis, tumour progression, and chemotherapy resistance. PGRMC1 contains binding sites for SH2 and SH3 domain-containing proteins. PGRMC1 phosphorylation was noticed in a proteomics study on human breast cancer tissue. Furthermore, PGRMC1 has serine and tyrosine phosphorylation sites. This in vitro project was designed to assess the role of PGRMC1 phosphorylation in cell biology, as well as searching the potential of protein-protein interaction with σ2 receptor and translocator protein (TSPO). This study also considered the role of evolutionarily conserved residues of PGRMC1 in mitochondrial cholesterol synthesis. The project was mainly carried out in the National Life Science Hub, Charles Sturt University, Wagga Wagga, Australia.
    Mutant stable cell lines were established by removing putative phosphorylation sites at serine and tyrosine residues of hemagglutinin-tagged PGRMC1 (PGRMC1-HA). Those mutations caused significant changes in the cellular proteome and signaling pathways, Cell morphology, and migration. To further investigate those changes, vinculin and ERR1 were elected to determine their
    roles in PGRMC1-HA wild type (WT) and S57A/S181A double mutant (DM) phenotypes, respectively. Also, western blot analysis was performed to confirm the role of PGRMC1 phosphorylation in signaling pathways such as Akt and MAPK and determine the expression of endogenous PGRMC1 and detection of exogenous PGRMC1-HA expression levels. The elevated migratory behavior of DM cells was investigated by generating confocal microscopy videos. 3D
    microscope imaging was utilised to visualise mitochondrial morphology and cholesterol droplets.
    Removal of Y180 influences genomic instability, regulates nicotinamide N-methyltransferase (NNMT)-1-methylnicotinamide (1-MNA) pathway, and genomic CpG methylation levels. The present study tested PGRMC1 phosphorylation status manipulation in genomic stability by whole genomic sequencing. NNMT knockdown was performed by shRNA and metabolomics profiling to confirm Y180 contribution to NNMT pathway activity. Methylation assessment reveals that all cell lines exhibited significantly different and specific patterns of genomic CpG methylation.
    PGRMC1 has been linked to sigma-2 (σ2) receptor in many studies. σ2 receptor activity was known as an unidentified 18-21 kDa membrane protein which is a potential biomarker of proliferative status of solid tumors. σ2 receptor activity was recently identified for the protein TMEM97. The unknown nature of σ2 receptor and its association with PGRMC1 persuaded us to attempt to investigate their potential interaction. Utilising SW120, a σ2 receptor fluorescence probe, flow cytometry and proximity ligation assays were performed to determine their possible interaction. The present study concluded that despite PGRMC1 colocalisation with σ2 receptor, PGRMC1 is not colocalised with TMEM97 in proximity ligation assay in this cell system. However, TSPO was clearly colocalised with TMEM79.
    Original languageEnglish
    QualificationDoctor of Philosophy
    Awarding Institution
    • Charles Sturt University
    Supervisors/Advisors
    • Cahill, Mike, Principal Supervisor
    • Weston, Leslie, Co-Supervisor
    • Hill, Rodney, Co-Supervisor
    • Gurusinghe, Saliya, Co-Supervisor
    Thesis sponsors
    Award date29 Mar 2021
    Place of PublicationAustralia
    Publisher
    Publication statusPublished - 2021

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