An interrogation into the role of p62 in the pluripotent state

Bullen, L. F. (2021) An interrogation into the role of p62 in the pluripotent state. Doctoral thesis (PhD), Manchester Metropolitan University.

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Abstract

The reprogramming of somatic cells to induced pluripotent stem cells (iPSC) has huge potential for disease modelling, drug screening and regenerative medicine, and is an extremely important area of 13 research and development across the globe. The inefficiency of the reprogramming process from somatic cell to pluripotency using established gold-standard methodologies is a major roadblock to realising the full potential of iPSC in cell therapy. The sequestosome protein p62 is an evolutionarily highly conserved scaffolding protein with roles in a range of crucial cell regulatory processes including autophagy, nutrient sensing and inflammation as well as being involved in the pathology or causation of various diseases. The canonical role of p62 is to aggregate ubiquitinated proteins to form the sequestosome, a precursor to the autophagosome, in the induction of autophagy under conditions of stress or starvation, but it has six known functional domains with a range of binding partners including NRF2, NF-κB, Traf6 and LC3. In addition, p62 directly binds to ubiquitinated proteins involved in the anti-oxidant response and inflammation and mediates mitophagy. There is an emerging role for autophagy in iPSC reprogramming, and a growing body of evidence for the role of p62 in maintaining stemness in cancer stem cells. As yet, the role of p62 in establishing or maintaining pluripotency in iPSCs has not been elucidated. I have created a ‘genetic-manipulation toolbox’ including overexpression, shRNA and functional mutants of p62 in order to assess the mechanistic role of p62 in iPSC reprogramming and maintenance of pluripotency. Utilising p62 null patient fibroblasts, and lentiviral based shRNA to create a stable p62 knock-down cell line, I have established a novel role for p62 in the maintenance of the pluripotent state. Future works will further utilise the tools and cell lines I have created to elucidate the mechanisms by which p62 exerts its effects on the pluripotent state.