INVESTIGATING THE ROLE OF CELLULAR AUTOPHAGY IN HUMAN MONOCYTIC CELL DEATH BY KINOME ANALYSIS
Cells of the Monocyte / Macrophage lineage are key players in innate and adaptive immunity. They eliminate pathogens through their phagocytic and antimicrobial properties, secretion of inflammatory and immunoregulatory cytokines, as well as their capacity to present foreign antigens to T lymphocytes in lymphoid tissues. The importance of M/Ms in the immune response require them to undergo strict regulation, which occurs, at least in part, through the control of monocytic cell survival. Autophagy is a ubiquitous cellular process by which cells degrade intracellular, cytoplasmic components via a network of interconnected vacuoles to carry out a variety of functions. Autophagy typically functions in maintaining cellular homeostasis and mitigating stresses. However, more recent studies have shown that autophagy may play a role in cell death. Our laboratory has previously found that the cytokine IFNγ can induce cell death in human monocytes in an autophagy-dependent manner. Conversely, IL-10 inhibits both spontaneous and IFNγ-induced cell death, a capacity that ironically, is also associated with the induction of autophagy. We are thus interested in understanding how the autophagy pathway can play a dual role in human monocyte survival. Interestingly, a novel autophagy-inducing antimicrobial peptide (Atg peptide) was recently shown to be capable of inducing an autophagy-dependent form of cell death. In this study, I established an in vitro model for Atg peptide-induced cell death in human monocytic cells. Subsequently, I designed and tested an autophagy- and cell death pathway-centric kinome microarray in order to begin elucidating the molecular mechanisms responsible for monocytic cell death. Kinome analysis identified several interesting phosphorylation events in response to Atg peptide stimulation, including the tumour suppressor protein p53, which was phosphorylated at S9, an Na+,K+-ATPase ATP1A1 which was phosphorylated at Y10, and the transcription factor 4E-BP1 which was phosphorylated at S65. I confirmed these phosphorylation results in part using Western blotting. Finally, I present several hypotheses for the potential molecular mechanisms involved in Atg peptide-induced autophagic cell death in monocytes revealed by kinome analysis, which provide a basis for further exploration into this extremely interesting area.
DegreeMaster of Science (M.Sc.)
DepartmentSchool of Public Health
ProgramVaccinology and Immunotherapeutics
SupervisorKryworuchko, Marko; Tikoo, Suresh
CommitteeGriebel, Philip; Napper, Scott; Carvalho, Carlos
Copyright DateJune 2017