Biochemistry of Apoptosis Signal-regulating Kinase 1 Regulation and Hydrogen Peroxide Signalling

Abstract

All cells within an organism must respond to their environment. Responses are regulated by intracellular signalling pathways and signalling molecules. One such molecule is hydrogen peroxide (H2O2). H2O2 is required in many cell signalling pathways, however, too much H2O2 can be toxic. As a result, organisms have evolved various systems to sense, and respond to, H2O2.

How H2O2 can act as a signalling molecule is paradoxical, as it is rapidly metabolised to water by proteins called Peroxiredoxins (Prdxs). Prdxs—including mammalian Prdx1—are proposed to act as mediators of H2O2 signalling by oxidising protein targets—such as the protein kinase, apoptosis signal-regulating kinase 1 (ASK1)—via disulfide-relays. The overall contribution of Prdxs in H2O2 signalling is unknown, as there are few examples of Prdx disulfide-relays.

ASK1 is an apical protein kinase that is activated in response to stressors (including H2O2), ultimately regulating responses such as apoptosis and inflammation. ASK1 is a large (154 kDa) protein with extensive regulatory regions, both N- and C-terminal to a central kinase domain. Domains within the ASK1 N terminus are known to regulate ASK1, but the precise mechanisms remain unclear, as we lack molecular detail. The oxidoreductase protein, Thioredoxin 1 (Trx1), is purported to directly inhibit ASK1 by binding within an N-terminal regulatory region—described as the thioredoxin binding domain (TBD). The release of Trx1 from ASK1 is required for H2O2-induced activation of ASK1, but how this process is governed is inconclusive.

This Thesis explores various aspects of ASK1 regulation and H2O2 signalling using in vitro methods and proteomics. Recombinant human ASK1 protein constructs were used to investigate ASK1 regulation in vitro. Different domains within the N terminus of ASK1 were seen to be able to both inhibit, and enhance ASK1 kinase activity. A model is proposed whereby conformational changes within the ASK1 TBD can regulate access of substrate proteins to the catalytic kinase domain. Once bound to ASK1, substrate proteins are proposed to undergo a conformational change, making them a better substrate for phosphorylation.

Using recombinant human Trx1 and ASK1, it was seen that Trx1 can interact with ASK1 in vitro. Trx1 and ASK1 are able to form an inter-molecular disulfide bond, for which ASK1-Cys250—within the ASK1 TBD—is required. Surprisingly, Trx1 was not able to inhibit ASK1 invitro, suggesting that Trx1 is not a direct inhibitor of ASK1.

In order to examine the role of Prdx1 in H2O2 signalling, and identify potential Prdx1 signalling-partners other than ASK1, a pilot quantitative proteomics method was tested. Oxidised protein cysteine residues from cell lysates were labelled, and analysed using mass spectrometry. Whilst labelled peptides were detected, the method requires further work to investigate the full cellular contribution of Prdx1 in H2O2 signalling.

Overall, this work advances our knowledge of ASK1 regulation, highlighting mechanisms that are likely conserved throughout metazoa. The proteomics work provides the foundations for further studies, that will advance our understanding of the role of Prdx1 in H2O2 signalling.