The molecular response to endolysosomal damage and its regulation by ubiquitylation and VCP/p97

• Applicants: Professor Dr. Christian Behrends, Ph.D.; Professor Dr. Hemmo Meyer
• Subject Area: Cell Biology
• Term: since 2020
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 447112704

Lysosomal membrane permeabilization or full rupture of lysosomes is a common cellular stress condition that is relevant for neurodegeneration, infection and cancer. Lysosomes can either be repaired and regenerated, or cleared by selective macroautophagy, termed lysophagy. A hallmark of the damage response and specifically of lysophagy is the extensive ubiquitination of lysosomal proteins that governs distinct steps in the regeneration and controlled removal of damaged lysosomes. We previously found that the ubiquitin-conjugating enzyme UBE2QL1 and the ubiquitin-directed AAA-ATPase VCP/p97 are essential for efficient lysophagy thus linking lysophagy to VCP/p97-associated neuromuscular degeneration. In the past funding period, we used mass spectrometry to systematically define the ubiquitylated proteome during the lysosomal damage and uncovered a diverse and dynamic set of membrane trafficking and lysophagy regulators. Among the ubiquitylated factors, we focused on CNN2 that we showed to promote phagophore formation through regulating actin dynamics. Of note, we found that CNN2 needs to be ubiquitylated and extracted by p97 in a timely manner for lysophagy to proceed. These findings explain, at least in part, the role of p97 in lysophagy and illustrate the complexity of underlying ubiquitin-related events. However, so far, the identity of the critical ligase(s) that govern lysophagy and other ubiquitylation events in the damage response has remained unclear. Hence, the key regulatory elements in the response to damaged lysosomes are not fully understood. In the next funding period, we therefore wish to focus on the identification and characterisation of the relevant ubiquitin ligase(s) that regulate and coordinate the cellular response to lysosomal damage. To do so, we will use and further refine a lysosome isolation (LysoIP) approach coupled with mass spectrometry that has already yielded a validated candidate. Using ubiquitin remnant mass spectrometry, we will then define the ubiquitylated targets of this and further candidates which will lead us to uncover their exact function in the process. Moreover, we will clarify if and how candidate ubiquitin ligases cooperate with p97 and UBE2QL1. Conversely, we will unravel mechanisms of lysosomal recruitment and activation of critical ubiquitin ligases to understand how they are regulated to coordinate lysophagy. Lastly, while establishing concepts in model cells with experimental perturbation, we will validate our findings regarding diverse types of lysosomal damage and in patho-physiologically relevant cells. Thus, the expected results will reveal how cells manage to deal with damaged lysosomes and coordinate a concerted response that includes regeneration and disposal mechanisms to restore cellular homeostasis and to counteract diverse medical conditions.

• DFG Programme: Research Grants