Heintze, JM; (2016) The role of Ribose-5-phosphate isomerase A in the regulation of autophagy. Doctoral thesis , UCL (University College London).

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Abstract

Macroautophagy (hereafter: autophagy), cell signalling and cellular metabolism are tightly linked processes. Multiple examples of metabolites and metabolic enzymes have recently been found to regulate signalling pathways and autophagy. Metabolic reprogramming is one of the hallmarks of cancer and often mediated via aberrant signalling pathways, such as receptor tyrosine kinase (RTK) signalling. Growth factor receptor-bound protein 2 (Grb2) is a key RTK signalling adaptor and is involved in a number of downstream signalling cascades, such as mitogen-activated protein kinase (MAPK) or phosphoinositide 3-kinase (PI3K) pathways, which in turn regulate metabolism through alterations in gene expression or by directly modifying enzymatic activity. This study investigates the role of ribose-5-phosphate isomerase (RPIA), a key metabolic enzyme in the pentose phosphate pathway (PPP), in the regulation of autophagy and Grb2-mediated signalling. RPIA expression induces Grb2 translocation from an even cytoplasmic distribution to unknown sub-cellular structures, possibly through direct protein-protein interaction. Interestingly, this effect is independent of the catalytic activity of RPIA, suggesting a non-canonical role in signalling. Neither RPIA over-expression, transient knockdown or deletion by CRISPR/Cas9 genome editing resulted in differences in metabolic activity or the MAPK pathway as tested by extracellular signal–regulated kinases (ERK) 1/2 phosphorylation and MTT assay. Furthermore, knockdown of RPIA by shRNA or genomic deletion resulted in an increase of LC3 processing and LC3-positive autophagosomes, suggesting that endogenous RPIA is an inhibitor of basal autophagy. Data fromSaccharomyces cerevisiae, mass spectrometry of sugar phosphates and pharmacological treatment assays suggest that RPIA may inhibit autophagy through a non-canonical function. Although the molecular mechanisms by which RPIA acts on Grb2 signalling and how it contributes to the regulation of autophagy are currently not fully understood, this study presents some interesting observations that may have implications in the development of therapeutics that target cancer metabolism or aim to modulate autophagy.

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