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A functional proteomics approach to extracellular redox signalling

Final Activity Report Summary - REDOX SIGNALING (A functional proteomics approach to extracellular redox signalling)

The Marie Curie excellence team has worked in the emerging field of oxidative signalling and redox regulation. We have investigated pathways by which oxidants and oxidoreductases contribute to the control of cellular behaviour, especially in the context of immune cell function. Over the last years, it has become clear that oxidants play essential roles as cellular messengers, causing reversible oxidative modifications in specific target proteins. Clearly, a more detailed understanding of the relationship between redox processes, regulatory pathways and cell fate is needed. However, the fleeting and labile nature of oxidants, reductants and protein redox states has always been a major limitation for research. Recognising that technical advances are required before substantial progress can be made; the team has focused on the development of new and improved research tools: Firstly, we developed an activity-based proteomics approach (kinetic trapping) which allowed us to identify principal target proteins and pathways regulated by reversible oxidation-reduction processes. Secondly, based on the knowledge we obtained during the first half of the project we developed a genetically encoded biosensor which allows to visualize cellular redox changes in real-time and with unprecedented sensitivity and resolution.

The project was centred on the role of disulfide bonds as regulatory switches for protein function. In extracellular proteins disulfide bonds normally serve to stabilize the protein scaffold. However, some disulfide bridges have a different function, they act as dynamical molecular switches to activate or inactivate protein function. The opening and closing of disulfide bonds is mediated by enzymes termed oxidoreductases. Some of them are released to the cell surface. For instance, Thioredoxin-1 (Trx1) is secreted by lymphocytes and changes their behaviour. To identify the mechanism behind this phenomenon we applied kinetic trapping to the surface of intact lymphocytes (Science's STKE 2007, p. l8). We found that Trx1 prominently targets one particular surface protein, a member of the tumour necrosis factor receptor superfamily, CD30. We found that the redox state of CD30 determines its ability to bind its ligand and to transduce signals. We could show that extracellular Trx1 affects CD30-dependent lymphocyte effector functions (EMBO Journal 26, 3086-3097). We knew from previous experiments that the cell surface peptide receptor MHC-I, while binding antigenic peptides for presentation to T cells, interacts with another oxidoreductase, ERp57. We could successfully address the question of how the opening and closing of a single specific disulfide bridge at the receptor-ligand interface regulates the peptide receptivity of the receptor (Nature Immunology 8, pp. 864-872).

To learn about oxidative processes associated with immune cell stimulation we applied kinetic trapping to identify intracellular proteins undergoing cycles of oxidation and reduction in rapidly proliferating acute lymphoblastic leukaemia cells. This approach led to the first comprehensive overview on Trx-regulated oxidative processes in mammalian cells. Our results strongly support the notion that Trx1 not only plays a role in oxidant scavenging but is also extensively involved in the regulation of intracellular signal transduction, apoptosis and the cell cycle.

Having learned about reversible disulfide formation and its regulation by thiol-dependent oxidoreductases, we realised that the principle of catalysed making and breaking of disulfide bonds can be exploited to create highly sensitive redox biosensors for live imaging. To this end, the redox catalyst glutaredoxin-1 (Grx1) was fused to a disulfide bond-containing variant of green fluorescent protein, which mimics a Grx1 target protein in that it becomes dynamically oxidised and reduced in response to the glutathione redox potential, comparable to physiological Grx1 target proteins. This tool opens new opportunities to analyse the causes and consequences of physiologically relevant redox changes (Nature Methods 5, pp. 553-559).