Stress-dependent conformational changes of artemin: Effects of heat and oxidant

Abstract

Artemin is an abundant thermostable protein in Artemia embryos and it is considered as a highly efficient molecular chaperone against extreme environmental stress conditions. The conformational dynamics of artemin have been suggested to play a critical role in its biological functions. In this study, we have investigated the conformational and functional changes of artemin under heat and oxidative stresses to identify the relationship between its structure and function. The tertiary and quaternary structures of artemin were evaluated by fluorescence measurements, protein cross-linking analysis, and dynamic light scattering. Based on the structural analysis, artemin showed irreversible substantial conformational lability in responses to heat and oxidant, which was mainly mediated through the hydrophobic interactions and dimerization of the chaperone. In addition, the chaperone-like activity of heated and oxidized artemin was examined using lysozyme refolding assay and the results showed that although both factors, i.e. heat and oxidant, at specific levels improved artemin potency, simultaneous incubation with both stressors significantly triggered the chaperone activation. Moreover, the heat-induced dimerization of artemin was found to be the most critical factor for its activation. It was suggested that oxidation presumably acts through stabilizing the dimer structures of artemin through formation of disulfide bridges between the subunits and strengthens its chaperoning efficacy. Accordingly, it is proposed that artemin probably exists in a monomer–oligomer equilibrium in Artemia cysts and environmental stresses and intracellular portion of protein substrates may shift the equilibrium towards the active dimer forms of the chaperone.

Cryo-EM structure of the diapause chaperone artemin

“…Ever since artemin’s first report in 1980 ( Slobin, 1980 ), a growing body of reports have elucidated the role of artemin as a molecular chaperone ( Shirzad et al, 2011 ; Hassani and Sajedi, 2013 ; Takalloo et al, 2016 ), but structural information about the protein has been limited to in silico modelling and some spectroscopic studies to date ( Takalloo et al, 2020a ). We sought to determine the full-length structure of artemin experimentally using single particle cryo-EM.…”

“…The key to surviving such harsh conditions has been tracked to the brine shrimp’s ability as a cyst to enter a state of metabolic hypoactivity called diapause. In this state, the cyst can survive desiccation, high and low temperatures, radiation, and years of anoxia ( Takalloo et al, 2020a ). A complement of stress tolerance proteins have been reported in Artemia during diapause including p26, artemin and hsc70 ( Clegg and Gajardo, 2009 ).…”

Cryo-EM structure of the diapause chaperone artemin

“…In contrast to artemin, upon exposure to increasing amount of stress, 2-Cys Prxs forms higher molecular weight assemblies (10 or 12 mers). The most drastic difference between artemin and other holdases is the irreversible structural changes that occur on exposure to stresses like heat or H 2 O 2 whereas other redox regulated holdases are reversible 13 . Artemin therefore appears to be a holdase-like chaperone with unique properties; especially since it acts as both a protein and RNA chaperone.…”

“…Based on homology models and biochemical data, a mechanism of action for the chaperoning activity of artemin has been suggested to rely on the activation through a cysteine redox switch in response to environmental stressors. This leads to the 24mer breaking down into oligomers of which dimers are believed to be most abundant and the functional chaperone 13 . The stable dimer putatively interacts with the target protein through the C-terminal helices to stabilize the target protein and prevent either denaturing or unfolding or both.…”