RESUMO
Lysosomal membrane permeabilization (LMP) is an underlying feature of diverse conditions including neurodegeneration. Cells respond by extensive ubiquitylation of membrane-associated proteins for clearance of the organelle through lysophagy that is facilitated by the ubiquitin-directed AAA-ATPase VCP/p97. Here, we assessed the ubiquitylated proteome upon acute LMP and uncovered a large diversity of targets and lysophagy regulators. They include calponin-2 (CNN2) that, along with the Arp2/3 complex, translocates to damaged lysosomes and regulates actin filaments to drive phagophore formation. Importantly, CNN2 needs to be ubiquitylated during the process and removed by VCP/p97 for efficient lysophagy. Moreover, we identified the small heat shock protein HSPB1 that assists VCP/p97 in the extraction of CNN2 and show that other membrane regulators including SNAREs, PICALM, AGFG1, and ARL8B are ubiquitylated during lysophagy. Our data reveal a framework of how ubiquitylation and two effectors, VCP/p97 and HSPB1, cooperate to protect cells from the deleterious effects of LMP.
Assuntos
Macroautofagia , Ubiquitina , Actinas/metabolismo , Adenosina Trifosfatases/metabolismo , Proteínas de Ciclo Celular/metabolismo , Lisossomos/metabolismo , Ubiquitina/metabolismo , Proteína com Valosina/genética , Proteína com Valosina/metabolismoRESUMO
The founding member of the F-box protein family, Cyclin-F, serves as a substrate adaptor for the E3 ligase Skp1-Cul1-F-box (SCF)<sup>Cyclin-F</sup> which is responsible for ubiquitination of proteins involved in cell cycle progression, DNA damage and mitotic fidelity. Missense mutations in <i>CCNF</i> encoding for Cyclin-F are associated with amyotrophic lateral sclerosis (ALS). However, it remains elusive whether <i>CCNF</i> mutations affect the substrate adaptor function of Cyclin-F and whether altered SCF<sup>Cyclin-F</sup>-mediated ubiquitination contributes to pathogenesis in <i>CCNF</i> mutation carriers. To address these questions, we set out to identify new SCF<sup>Cyclin-F</sup> targets in neuronal and ALS patient-derived cells. Mass spectrometry-based ubiquitinome profiling of <i>CCNF</i> knockout and mutant cell lines as well as Cyclin-F proximity and interaction proteomics converged on the HSP90 chaperone machinery as new substrate candidate. Biochemical analyses provided evidence for a Cyclin-F-dependent association and ubiquitination of HSP90AB1 and implied a regulatory role that could affect the binding of a number of HSP90 clients and co-factors. Together, our results point to a possible Cyclin-F loss-of-function-mediated chaperone dysregulation that might be relevant for ALS.