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Title: The regulatory role of APC/C-Cdh1 in proliferating and differentiating pre-adipocytes
Author: Kovacs, I.
ISNI:       0000 0004 8503 7688
Awarding Body: UCL (University College London)
Current Institution: University College London (University of London)
Date of Award: 2016
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It is well known that proliferating cells, whether they are normal or malignant, exhibit increased glycolysis and glucose uptake. Breakdown of glucose through the glycolytic cascade not only yields essential precursors for macromolecule biosynthesis but also provides energy in the form of ATP and reducing equivalents in the form of NADH. Acceleration of glycolysis can be achieved by several mechanisms but the most efficient way is to increase the activity of the rate-limiting glycolytic enzyme, phosphofructokinase- 1 (PFK1). PFKFB3 (6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase, isoform 3) is an enzyme that activates glycolysis through the production of fructose-2,6-bisphosphate, an allosteric activator of PFK1. Previously we have demonstrated in various normal and neoplastic cells that the E3 ubiquitin ligase APC/C (anaphase promoting complex/cyclosome)-Cdh1 targets PFKFB3 for degradation during proliferation and thus regulates glycolytic flux. In my doctoral thesis I aim to address two questions: (i) whether the same proteolytic mechanism controls glycolysis in proliferating pre-adipocytes, and (ii) whether nondividing, differentiating pre-adipocytes which also known to become highly glycolytic, likewise regulate glycolytic flux through the proteolytic control of PFKFB3 expression. In the first part of this work I show that in proliferating pre-adipocytes the APC/C-Cdh1 ubiquitin ligase actively degrades the PFKFB3 protein. As growth-arrested pre-adipocytes start to proliferate, Cdh1 protein levels drop and this is associated with an increase in PFKFB3 protein levels and increased lactate production. In the second part of the thesis I show that PFKFB3 undergoes ubiquitination in cells in culture. I also demonstrate that PFKFB3 is ubiquitinated by APC/C-Cdh1 in an in vitro reconstituted ubiquitination assay. In the third part of the thesis I demonstrate that Cdh1 protein levels also fall during preadipocyte differentiation. Similarly to the proliferating pre-adipocytes, the drop in Cdh1 protein levels associate with an increase in PFKFB3 and lactic acid production. Although, the results suggest that it is a consequence of increased PFKFB3 mRNA expression. The results also suggest that differentiating pre-adipocytes deactivate APC/C by phosphorylating Cdh1 and upregulating the APC/C inhibitor Emi1 (early mitotic inhibitor 1). Thus, although the increase of PFKFB3 seems to be the key step in both pre-adipocyte proliferation and differentiation, it seems to be controlled by different mechanisms.
Supervisor: Moncada, S. ; Okorokov, A. Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available