Unravelling signalling pathways controlling proteasome homeostasis under stressful conditions

Misfolded and damaged proteins accumulate with age and this progressive collapse of the proteome is defined as one of the hallmarks of ageing and contributes to various human diseases such as cancer and neurodegeneration. The Ubiquitin-Proteasome system (UPS) by clearing these faulty proteins is an essential component to maintain the integrity of the proteome. Our work focuses on better understanding signalling pathways that control proteasome assembly and activity. This is a very fundamental and important question with strong links to understanding healthy aging and human disease. We have recently shown that the kinase Mpk1/ERK5 controls proteasome homeostasis upon various proteotoxic stresses. Thus, we are particularly interested in identifying Mpk1/ERK5 substrates which are required for the regulation of proteasome homeostasis. This study will provide insights on how phosphorylation regulates proteasomal degradation and survival upon stressful conditions. As cancer cells are often addicted to high levels of proteasomes, it will also be exciting to uncover whether any of the key identified substrates are mutated in cancers. With the central function of the proteasome in clearing misfolded and unwanted proteins, this proposal might also help developing new strategies to increase proteasome capacity with the idea that this could be beneficial to treat neurodegenerative diseases.

Increasing evidence suggests that alterations and mutations in the Ubiquitin-Proteasome System (UPS) give rise to various human diseases, such as cancer and neurodegenerative disorders. Then my central interest for this programme is to understand how the activity of the 26S proteasome is regulated in cells so that accumulation of unwanted, misfolded, or damaged proteins can be cleared before they become deleterious. We recently discovered that TORC1 inhibition, which occurs upon various stresses, increases proteasome assembly and activity in both yeast and human cells. This adaptive response relies on the kinase Mpk1/ERK5 which coordinates the induction of all 19S Regulatory particle assembly chaperones (RPACs) to control proteasome assembly and viability upon various stresses. Mpk1 is therefore essential to adjust proteasome capacity to meet the cell requirements. My first research objective is to unravel how Mpk1 regulates proteasome homeostasis under stressful conditions. We will work with Houijiang Zhou and undertake phosphoproteomic analyses of mpk1? cells re-expressing a wild-type or a kinase-dead version of Mpk1. The aim of this project is to identify Mpk1 substrates that are selectively phosphorylated under conditions increasing proteasome assembly and map the Mpk1 phosphorylation sites. We will then test whether in vivo mutagenesis of the identified Mpk1 phosphorylation sites inhibits 26S proteasome assembly and impedes stress resistance. The potential evolutionary conservation of characterized Mpk1/ERK5 substrates that control proteasome assembly will be then further analysed using human cells.
We have also found that RPACs are regulated at the translation levels in a Mpk1-dependent manner upon stressful conditions. This increased translation of RACs likely plays a role in enabling cells to withstand stresses. My second project is aimed at uncovering the mechanism by which RPACs are selectively translated under stress conditions. To this end we will generate deletion mutants of the 5’ and 3’ UTR of RPAC mRNAs to identify the minimal sequence required to efficiently translate the RACs under stressful conditions and establish whether we can identify factors that specifically bind to these sequences and whether these are Mpk1 substrates. These studies should advance our understanding of how selective translation is controlled and provide new insights into how cells respond to stress conditions. Depending on the nature of the targets identified this research has potential to lead to the identification of new therapeutic targets.
Overall, this research programme will help understanding the biology of the 26S proteasome with the emphasis on how signalling pathways control proteasome assembly and activity in cells, especially under stressful conditions. Identification and characterization of such pathways should enable us to develop new strategies to either increase or decrease protein degradation capacity in cells which may have important therapeutic potential.