Principle investigator: Christopher Horst Lillig
Co-supervisors: M. Gellert, C. A. Helm, S. Bekeschus

Background: The mechanisms that lead to the formation of regulatory protein disulfides (thiol switches) in the cytosol are still unresolved [1]. MICAL proteins are FAD-dependent monooxygenases that act as effector proteins in cell signalling cascades. For instance, the semaphorin (Sem) pathway, that controls neuronal development and cell migration. Sem3A binds to its receptor on the cell surface, that consists of neuropilin 1 and plexin A. This signal is transferred through the membrane and leads to the activation of MICALs. Activated MICAL can produce H₂O₂, (see Fig. 1 reactions 1→6→5) [2], however a direct oxidation of methionyl residues of the protein beta-actin has also been described [3]. 

Preliminary results: In a previous project (DFG Li 984/3-1), we have demonstrated that MICAL may also directly oxidize proteins of the peroxiredoxin family (Prx, see Fig. 1, reaction 3).

Hypothesis: Here, we propose the following model for the Sem3A-induced oxidation of protein thiols at the cell membrane (Fig. 1). MICAL with reduced FADH₂ reacts with O₂ to form a peroxy-flavin (reaction 1). This peroxy-group is attacked by the catalytic thiol in the active site of the Prx (2) to form a covalently linked intermediate. This complex dissociates to form H₂O and a sulfenic acid in the active site of the Prx (reaction 3). By attack of a resolving cysteine of a second Prx molecule, this is reduced to H₂O, leaving a disulfide between the Prx molecules (7). The flavin is regenerated in two steps (4→5) by NADPH. The disulfide on the Prx is subsequently transferred to the target protein in a thiol-disulfide exchange reaction (8→9).

Plan of work: The first work package is a detailed analysis of the reaction proposed in Fig. 1 in vitro. We will use recombinant proteins and apply enzyme kinetic measurements and spectroscopic analysis (UV-vis, fluorescence, aerobic/anaerobic, and stopped flow analysis) as well as electrophoretic separation of the proteins to identify each individual intermediate species. The second work package aims at confirming this redox-signalling relay in vivo, i.e. in cell cultures. Next to confirming the sequence of events, we will use the analysis of cell migration by live cell analysis as biological read out. This program requires the support of other RTG groups  and collaboration partners, e.g. Bekeschus, Bornscheuer, and Helm.

Literature

[1] M. Deponte, C.H. Lillig, Enzymatic control of cysteinyl thiol switches in proteins, Biol. Chem. 369 (2015) 401-13.

[2]   E.F. Schmidt, S.-O. Shim, S.M. Strittmatter, Release of MICAL autoinhibition by semaphorin-plexin signaling promotes interaction with collapsin response mediator protein, J. Neurosci. Off. J. Soc. Neurosci. 28 (2008) 2287–97.

[3]   M. Gellert, E.-M. Hanschmann, K. Lepka, C. Berndt, C.H. Lillig, Redox regulation of cytoskeletal dynamics during differentiation and de-differentiation, Biochim. Biophys. Acta BBA - Gen. Subj. 58 (2014) 1575-87.