MLKL-USP7-UBA52 Signaling Is Indispensable for Autophagy in Brain through Maintaining Ubiquitin Homeostasis

Individuals with genetic elimination of MLKL exhibit an increased susceptibility to neurodegenerative diseases like Alzheimer's disease. To elucidate the mechanism by which MLKL, primarily a cytosolic protein, regulates the protein levels of Beclin1 and ULK1, we performed a proteomic screening of MLKL-associated proteins and identified UBA52 as the binding partner under physiological conditions. Loss of MLKL induces a decrease in ubiquitin levels by preventing UBA52 from producing ubiquitin and the C-terminal ribosomal protein L40. Furthermore, we demonstrated that in the brain, the deubiquitinase USP7 mediates the processing of UBA52, which is regulated by MLKL. Knockdown of USP7 or UBA52 showed similar effects on autophagy and ubiquitin levels as MLKL KD/KO. Moreover, our results indicated that the reduction of Beclin1 and ULK1 upon MLKL loss is attributed to a decrease in their lysine 63-linked polyubiquitination. Additionally, single-nucleus RNA sequencing revealed that the loss of MLKL resulted in the disruption of multiple neurodegenerative disease-related pathways, including those associated with AD.