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Systematic proteomic analysis of oxygen mediated DNA damage

Mcdonald, Sara

[Thesis]. Manchester, UK: The University of Manchester; 2015.

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Abstract

Blood cell numbers are maintained constant due to an interplay between haemopoiesis and the loss of mature cells. Haemopoiesis is a complex process that generates large numbers of all the blood cells in the body from a relatively small number of haemopoietic stem cells (HSCs). HSCs continually replenish cells that are turned over, or lost by injury or trauma, and must also self-renew to maintain themselves over the lifetime of the organism. HSCs are protected from reactive oxygen species induced DNA damage by the fact that they possess low levels of mitochondrial oxidative metabolism and are enclosed in a protective niche microenvironment. However, DNA damage can occur, which ultimately leads to the expression of mutations in genes such as those encoding protein tyrosine kinases, resulting in dysfunctional haemopoiesis which can lead to diseases such as leukaemias. Discovery phosphoproteomics was used to compare the changes in protein phosphorylation in cells in different phases of haemopoiesis when exposed to hydrogen peroxide (H2O2), which causes DNA-damage via ROS. We identified and relatively quantified thousands of phosphopeptides, with a few hundred being potentially modulated in the presence of H2O2. Novel phosphorylation sites differentially expressed between the different populations were identified, such as UHRF1, in mature cells, and HMGA1, in primitive cells. Bioinformatic analyses have identified pathways and interacting proteins of these targets, multiple reaction monitoring confirmed the identified phosphosites. The same method was utilised to analyse the role of CD45. CD45 has a role in the response to oxidative status, is activated by H2O2 treatment, and is reduced in its levels by oncogenic PTKs in the pathway initiated by CXCL12 that results in cell motility. Thousands of phosphopeptides were identified and quantified, and bioinformatics analysis was used to identify their interactors and the pathways in which they are involved. The data was also compared to data from several other studies. Our investigation revealed multiple targets which help unravel the relationship between ROS, the CD45 pathway and leukaemogenic PTKs to further understand normal and leukaemic haemopoiesis.

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Published articles:The use of proteomics for systematic analysis of normal and transformed haemopoietic stem cells. Cadeco S, Williamson AJ, Whetton AD. Curr Pharm Des. 2012;18(13):1730-50. Review.A specific PTPRC/CD45 phosphorylation event governed by stem cell chemokine CXCL12 regulates primitive haemopoietic cell motility. Williamson AJ, Pierce A, Jaworska E, Zhou C, Aspinall-O'Dea M, Lancashire L, Unwin RD, Abraham SA, Walker MJ, Cadecco S, Spooncer E, Holyoake TL, Whetton AD. Mol Cell Proteomics. 2013 Nov;12(11):3319-29. doi: 10.1074/mcp.M112.024604.