Apoptosis signal-regulating kinase 1 (ASK1) is a member of the mitogen-activated protein kinase kinase kinase (MAP3K) family that activates c-Jun N-terminal kinase and p38 MAP kinase pathways in response to various stress stimuli, including oxidative stress, endoplasmic reticulum stress, and calcium ion influx. The function of ASK1 is associated with the activation of apoptosis in various cells and plays a key role in the pathogenesis of multiple diseases including cancer, neurodegeneration and cardiovascular diseases. The kinase activity of ASK1 is regulated by many factors, including binding of thioredoxin (Trx) and the 14-3-3 protein that both function as inhibitors of ASK1 [1]. However, the mechanisms by which these binding interactions inhibit ASK1 are still unclear.
To better understand the role of Trx binding in the inhibition of ASK1, we performed structural characterization of the isolated Trx-binding region of ASK1 (ASK1-TBD) and its complex with reduced TRX1 using fluorescence spectroscopy, circular dichroism and small-angle X-ray scattering. It has been shown that ASK1-TBD is a compact monomeric and rigid domain that under reducing conditions forms with TRX1 a stable and well defined complex with 1:1 molar stoichiometry. We showed that Trp31 of TRX1 is directly involved in the interaction. Moreover, TRX1 interacts with the region of ASK1-TBD located in the vicinity of Cys250 [2].
To elucidate the role of cysteine residues of both proteins in the interaction under reducing and oxidative conditions, we will use spectrophotometric assay with Ellman´s reagent.
This work was supported by the Czech Science Foundation (Project 14-10061S) and The Czech Academy of Sciences (Research Projects RVO: 67985823 of the Institute of Physiology).