The Selective Enrichment of the Selenoproteome

The genome codes for 25 selenoproteins, i.e. proteins containing selenocysteine. It is surprising to note that their precise functions are only partially elucidated due to challenges in their expression, purification and analysis. The selenoproteins with known functions serve important roles in cellular homeostasis and under stress. They also represent promising anticancer drug targets, since they are involved in key detoxification processes and therapy resistance. This project aims to establish selenocysteine-selective probes in order to successfully enrich and subsequently analyse the entirety of selenoproteins in biological samples on the molecular level using mass spectrometry-based proteomics. The proposed research combines synthetic chemistry, analytical chemistry, proteomics and cell culture. By means of these novel selenoprotein-selective probes, it will be possible to map basal expression levels of selenoproteins across different cancer cell types and cancer cell lines. Then, the differential gene expression of selenoproteins will be investigated upon selenium supplementation and chemical stimuli. Finally, this method will be used to characterize the selenoprotein target selectivity of drug candidates. The specific enrichment of selenoproteins from biological systems will thus provide a foundation for their comprehensive analysis and establish the required knowledge to design appropriate therapeutic strategies.

Selenoproteins are a rare but vital class of proteins in the human body that contain the trace element selenium, which plays an essential role in regulating oxidative stress and supporting immune function. However, studying these proteins is challenging. One reason for this is that the selenium-containing amino acid selenocysteine is reactive. In addition, selenoproteins are usually only present in cells in very low amounts, making them difficult to study using conventional laboratory techniques. This project presents a novel approach to overcome these challenges using specially developed gold-based chemical probes. These probes are designed to selectively bind to selenocysteine and allow selenoproteins to be extracted and enriched from complex biological samples. Specifically, a gold(III) complex was prepared that can be chemically immobilized on a resin by a free functional group. Chemoproteomic analyses were performed to identify those proteins from cell extracts that interacted with the gold probe. A typical cell extract thereby contains thousands of proteins. Indeed, the probe showed a strong and selective interaction with thioredoxin reductase 1 (TXNRD1), an important selenoprotein involved in maintaining the redox balance in cells. TXNRD1 contains a unique sequence motif, a reactive combination of cysteine and selenocysteine, which has also been characterized as the binding site of the probe. The interaction with TXNRD1 takes place via a characteristic two-stage mechanism. First, the gold metal coordinates predominantly to selenium-containing sites of the protein. In the next step, the chelating ligand is covalently bound to the protein by reductive elimination. The free, non-immobilized gold probe was also found to inhibit TXNRD1 activity in living cells and activate the NRF2-KEAP1 pathway, a protective cellular response to oxidative stress that cancer cells rely on for survival. A fluorescent derivative of the probe was also successfully produced and shown to accumulate in the nucleus of cancer cells. In summary, this work demonstrates that gold-based probes represent a powerful new method to investigate selenoproteins. Meaningful tools were established to study their functions in health and disease, particularly in cancer, where selenoproteins play a crucial role, and thus can also be targeted in drug discovery campaigns. Strikingly, the novel gold(III) probe showed exceptional selectivity for the selenoprotein TXNRD1 in a whole cell extract. Future work will focus on selenocysteine-specific probes to enrich the entire selenoproteome.