B07: Protein targeting and safeguarding upon oxidative unfolding stress

Oxidative stress is a major cause of transcriptional and translational fidelity decline, resulting in the generation of erroneous and potentially aggregation-prone proteins which endanger the integrity of the entire cell. In addition, oxidative damage of proteins can cause protein unfolding and contribute to the accumulation of mis- or unfolded proteins (Lévy et al. 2019). Since oxidative stress also leads to a drop in cellular ATP levels, the function of ATP-dependent chaperones and proteasomal degradation, which normally serve to cope with mis- and unfolded proteins, will be impaired (Winter et al. 2005, Reichmann et al. 2018). Under these conditions, ATP-independent chaperones that function as “holdase” for unfolding protein intermediates become activated to mitigate the severe risk of protein aggregation (Ulrich 2023). The highly conserved dual-function protein ASNA1 (yeast GET3) switches from its ATP-dependent targeting function for tail-anchored membrane proteins into a general, ATP-independent chaperone (Fig. B07.1). Loss of this switch results in an increased sensitivity towards oxidative stress (Ulrich et al. 2022). Our preliminary work showed that ASNA1 is essential to prevent irreversible protein aggregation and ensure rapid recovery of human cells from proteotoxic stresses. As an ATP-independent holdase, ASNA1 is not capable of refolding client proteins. Instead, proteins need to be transferred to ATP-dependent systems for refolding or degradation (Ulrich et al. 2020). The crosstalk of chaperone-active ASNA1 with the cellular proteostasis network, however, remains elusive as well as the client proteins which rely on the stress-induced chaperone function. Using proximity labeling combined with mass spectrometry (MS), we firstly will identify dynamic changes in the ASNA1 interactome depending on its reversible functional switch induced by H2O2 treatment of HEK293 cells. Secondly, we will identify proteins that are prone to aggregate and rely on ASNA1’s chaperone function upon H2O2 stress by employing limited proteolysis coupled with mass spectrometry (LiP-MS). We further will expand our studies to the role of ASNA-1 in maintaining organismal proteostasis in Caenorhabditis elegans and address the systemic function of a chaperone and targeting factor in safeguarding proteins sensitive towards stress- as well as age-associated decline in protein fidelity.