PEROXISOOMDYSFUNCTIE EN AUTOFAGIE: DE KLOOF OVERBRUGGEN

Peroxisomes are ubiquitous cell organelles critical for cellular lipid and H2O2 metabolism. To maintain functionality in response to environmental changes, new organelles need to be formed, and dysregulated organelles must be selectively removed. Emerging evidence suggests that disturbances in peroxisome homeostasis affect intracellular communication and contribute to diseases such as cancer and neurodegenerative disorders. Given their role as key intermediaries in H2O2 metabolism, peroxisomes are expected to have the capacity to modulate the activity of other cellular processes, including autophagy. Conversely, disruptions in autophagy or pexophagy can lead to the accumulation of dysfunctional peroxisomes, causing metabolic imbalances. Over the past years, several pexophagy receptors or adaptors have been identified, which act cooperatively to target peroxisomes for lysosomal degradation. However, the underlying molecular mechanisms are complex and multifaceted and have only recently begun to receive the attention they deserve. This project aimed to gain deeper insights into (i) whether and how peroxisome dysfunction affects the (selective) autophagic process, (ii) how the health of the peroxisome population is maintained, and (iii) how peroxisomes are integrated into intracellular communication networks. Firstly, to investigate the direct relationship between peroxisomal H2O2 emission and pexophagy, we developed and validated a novel pexophagy living cell reporter, po-mKeima. Subsequently, we established po-DD-DAO HEK-293 and HeLa po-mKeima cell lines, allowing precise modulation of peroxisomal H2O2 production in a dose- and time-dependent manner. Our findings indicate that varying levels of po-H2O2, from low to high, cannot induce pexophagy. Moreover, excessive peroxisomal H2O2 release can oxidatively modify redox-sensitive selective autophagy receptors, such as optineurin (OPTN), and core autophagy proteins, such as ATG3, thereby blocking autophagy at late stages. Notably, by the end of the study, we discovered that endogenous OPTN, a well-documented mitophagy receptor, can localize to peroxisomes and, upon overexpression, trigger pexophagy in a cell type-dependent manner. To further explore the molecular mechanisms underlying OPTN-mediated pexophagy, we first conducted proximity labeling proteomics studies. These studies led to the identification of PEX14, a peroxisomal membrane protein, as an interacting partner of OPTN. Combined with FACS and co-immunoprecipitation analysis, we demonstrated that (i) OPTN interacts with the predicted coiled-coil domain of PEX14 through its ubiquitin-binding domain, but independently of ubiquitin, and (ii) the interaction of OPTN with PEX14 is required for its pexophagy-inducing properties. In summary, our findings indicate that PEX14 serves as a docking factor for OPTN at the peroxisomal membrane, thereby facilitating the interaction between peroxisomes and the autophagic membrane scaffold during OPTN-mediated pexophagy. Finally, we examined the changes in autophagy under conditions in which peroxisome biogenesis was defective, both in vivo and in vitro. We established that the loss of functional peroxisomes in mouse liver impairs autophagy through the mTOR/ULK1 signaling pathway. In our in vitro study, we observed an increase in mitochondrial H₂O₂ levels, accompanied by disruptions in autophagic flux in skin fibroblasts derived from a PEX10H310D/E10Gfs patient. Furthermore, by employing the autophagy stimulator Torin-1, we were able to successfully restore autophagic flux in these cells. Lastly, we demonstrated that compared to control cells, PEX10H310D/E10Gfs cells stably expressing EGFP-PTS1 exhibited distinct patterns in FACS analysis, this FACS-based approach may offer researchers with a swift, cell-based method for screening and identifying drugs to treat patients with PEX10 or other peroxin mutations. In summary, our experimental data offer novel insights into the interplay between peroxisome dysfunction and autophagy, indicating that disturbances in peroxisomal metabolism can modulate (selective) autophagy. Although the molecular triggers of pexophagy remain unclear, our identification of a novel pexophagy receptor, OPTN, and its peroxisomal partner, PEX14, marks a significant discovery. This finding paves the way for exploring peroxisome turnover in both normal physiology and human disease from new perspectives.