Using high-content subcellular proteomics to identify x-ray-induced protein trafficking

Abstract

X-ray radiation is a common form of ionising radiation (IR) that naturally occurs at low levels in the atmosphere and Earth, which pose minimal threat to organisms as cells have multiple mechanisms to prevent damage or death. However, high levels of IR can overwhelm these mechanisms causing a cascade of events such as oxidative stress, DNA damage, cell cycle changes and cell death. The latter outcome is why IR has been used for radiotherapeutic treatments of highly proliferative cells in cancer for many decades. As well as this, it is often used as a method to induce DNA damage in research to study cell survival mechanisms. Targeting DNA damage repair (DDR) mechanisms in cancer cells has been of particular interest, as genomic instability is a key hallmark of cancers. Therefore, providing a therapeutic window for targeting cancerous tissue over healthy tissue. However, resistance to both radiotherapy and DDR-targeting therapeutics impose a big threat to the effectiveness of these onco-therapeutics. A lot of the recent advances in treatments of disease and understanding of resistance have been aided by omics research (i.e. genomic, transcriptomic and proteomic), which offer high-throughput measurements of cellular components and processes. Though, these methods mainly provide information of the changes in abundance or post-transcriptional modification status of molecules without the spatial context of where these molecules are located within the cell. Many proteins have multiple roles and these functions can be dependent on their subcellular localisation. More traditional methods for measuring subcellular localisation of proteins, such as microscopy are lower throughput, are biased/targeted and can be prone to artefacts. In this thesis, a subcellular proteomics technique, known as Localisation of Organellar Proteins using Isobaric Tagging with differential centrifugation (LOPIT-DC), was coupled with a semi-supervised Bayesian mixture model to address these technical limitations and screen for protein localisation events in an untargeted and cell-wide manner. First, protein expression was measured within the lung carcinoma A549 cell line over several time points, between 0 and 24 hrs, post-irradiation with x-rays using quantitative proteomics, and 2 and 12 hr time points were subsequently selected for further investigation. As phosphorylation is one of the most abundant post-translational modification and drives a lot of protein trafficking events, quantitative phosphoproteomics was also performed. Then, LOPIT-DC used to assess the immediate and delayed protein trafficking responses at 2 and 12 hrs post-IR. Using this approach, 504 and 829 proteins, respectively, were found to differentially localise upon IR with associated biological pathways that included cell cycle checkpoints, transcription by RNA polymerase II, immune system and iron metabolism. Within the 12hr timepoint, several proteins involved in the ferroptosis pathway, a type of programmed iron-dependent cell death, had changed localisation, 3 including a key ferroptosis suppressor. Immunofluorescence microscopy was used to validate several of these differentially localised ferroptosis proteins, AIFM2, TFRC, FTH1 and FTL. Collectively, the subcellar changes of these proteins indicated a potential suppression of ferroptosis, thus a radioresistance mechanism. However, this would require further validation with functional assays. According to our orthogonal proteomics data, changes in subcellular localisation of the proteins appeared to be relatively independent of protein expression or phosphorylation state, indicating that these potentially key cellular responses may be overlooked in other assays. These high-content datasets provide a powerful resource for further investigating the functional significance of such x-ray-dependent differential localisations, potentially finding novel targets associated with oxidative stress and DNA repair that play key roles in disease and radioresistance.