Endosomal transport of cargos, as receptors, is tightly regulated by large protein complexes, including CCC and Retriever. Using computational approaches (e.g. AlphaFold), cryo-EM, and biochemical analyses, EndoConnect ESRs revealed important insights about their structural organization and assembly. These findings significantly advanced our understanding of how disease-associated mutations affect the function of these complexes, and provide essential insights for developing strategies to restore complex assembly in patients. Furthermore, the ESRs identified specific cargos and adaptor proteins, as SNX17, that interact with these complexes.
Targeting proteins for degradation is a promising therapeutic strategy for treating diseases such as cancer. One EndoConnect ESR developed innovative tools to selectively degrade GFP-tagged endosomal proteins and additional ones to study key receptors as EGFR and c-MET, which are regulated by the endosomal sorting system and play crucial roles in cancer progression. These tools open new avenues for therapeutic intervention.
Aberrant lipid homeostasis is a key contributor to cardiometabolic diseases as atherosclerosis and fatty liver disease. EndoConnect ESRs used omics approaches,cellular and in vivo models to investigate components of the endosomal protein sorting machinery involved in lipid metabolism. While OMICs identified ERICH4 as potential regulator in lipid metabolism, mice studies did not confirm this. Yet, SMLR1 was identified as critical regulator of the biogenesis of apoB-containing lipoproteins, including VLDL and chylomicrons. A mouse model was developed to investigate intracellular trafficking of apoB-containing lipoprotein. While SMLR1 appears to be involved in VLDL trafficking from the endoplasmic reticulum to the trans-Golgi network (TGN), TGN46 potentially regulated VLDL transport from the TGN to the membrane. In vitro studies demonstrated that TGN46 loss led to a reduction in apoB secretion. Mouse models partially confirm these findings. These novel insights into VLDL metabolism can help develop therapeutic approaches aimed at lowering plasma cholesterol, reducing cardiovascular risk, without excessive accumulation of cholesterol in the liver.
Moreover, mouse models and liver organoids revealed that the endosomal sorting complex retromer plays an essential role in lysosomal cholesterol handling in hepatocytes. Loss of retromer function led to elevated cholesterol levels in the liver and plasma due to impaired lysosomal cholesterol egress. Intriguingly, EndoConnect also uncovered novel roles for the retromer complex in liver cancer and regeneration. Since the retromer complex can be pharmacologically stabilized using specific compounds, it is worth exploring if these compounds enhance cholesterol handling without promoting liver cancer development, andif they could improve liver regeneration in living liver donors.
Mutations in lysosomal protein NPC1 cause Niemann-Pick disease type C (NPC), a severe genetic disorder characterized by the accumulation of cholesterol in multiple organs including the brain and liver. There is no cure available. Two EndoConnect ESRs investigated the role of the HOPS complex in intracellular trafficking of NPC1 to the lysosome. The results are submitted for scientific publication. Understanding NPC1 folding and its trafficking to lysosomes may enable new targeted therapies for restoring cholesterol homeostasis in NPC patients.
Non-alcoholic fatty liver disease (NALFD) significantly increases the risk of cardiovascular disease, liver failure, and liver cancer. To understand the mechanisms driving the progression from NALFD to liver damage and cancer, ESRs employed omics approaches to identify new molecular players. Organelle-specific proteomic analyses revealed distinct roles for mitochondrial subpopulations. In addition, gene expression analyses of fatty liver disease models uncovered new components of the endo-lysosomal trafficking pathway associated with hepatic lipid accumulation. In vitro studies supported this, and follow-up experiments using mouse models are underway. In addition, in vitro models to culture primary hepatocytes from mice and humans were optimized to better study NALFD and glucose metabolism. These models provide a valuable platform for investigating novel therapeutic targets, testing new drugs for fatty liver disease and improving insulin sensitivity.
The endo-lysosomal system plays a critical role in determining the efficacy of RNA-based therapeutics, as endosomal escape is essential for their function. To better understand this, various assays and model systems were developed within the consortium. Among these, the Gal9 assay was successfully established as a tool to evaluate endosomal escape. This assay is further optimized to enhance the therapeutic efficacy of RNA-based treatments.
