Investigation of the function of the proteins GFOD1 and GFOD2 in mammalian cells.

Details

Supervisors

Bommer, Guido

Faculty

Faculté de pharmacie et des sciences biomédicales

Degree label

Master [120] en sciences biomédicales, à finalité approfondie

Abstract

Glucose-fructose oxidoreductase domain contains proteins 1 and 2 (GFOD1 and GFOD2) are highly conserved human proteins that resemble bacterial enzymes acting on sugars or related molecules. In my master’s thesis, I aimed to help understand the function of these proteins. This work proceeded along four lines of investigation: 1. Identification of endogenous interaction partners: we had planned to use a cell line where an affinity purification tag had been knocked into the endogenous locus of GFOD1. Unfortunately, this cell line turned out to be not stable. Thus, the clonal population carrying this tag was lost and the investigation was stopped. 2. Modulation of cellular signaling pathways: Previous investigations had revealed an interaction between overexpressed GFOD1/2 and a major regulator of the NF-κB signaling pathway. I tested how modulation of GFOD1/2 levels affects the activation status of this pathway but did not find any reproducible changes. 3. Improving expression of soluble proteins in bacteria: Previous efforts had revealed that these proteins were largely contained in the insoluble fraction of bacterial lysates. This made purification of recombinant protein almost impossible. Based on the previous observation that GFOD1 and 2 interact with each other and that bacteria resembling protein formed tetrameric complexes, we hypothesized that the co-expression of both proteins might facilitate the production of soluble proteins. Using a bicistronic bacterial expression system, we observe that the soluble fraction of GFOD1 indeed increases upon co-expression. 4. Role of GFOD1 and GFOD2 in cellular metabolism. I used previously established knockout and double knockout cell lines, as well as cell lines in which I expressed GFOD1 and GFOD2 in a defined stoichiometry using a self-cleaving P2A peptide. The abundance of known and unknown metabolites was assessed by liquid chromatography (LC) and gas chromatography (GC) coupled to mass spectrometry (MS). For the analysis of these datasets, I developed an R language script that allows me (and other members of the lab) to rapidly gain an overview of metabolic changes between experimental groups. In my cells, I observed that N-acetylthreonine reproducibly decreases upon GFOD1/2 overexpression.