Arginyltransferase 1 modulates p62-driven autophagy via mTORC1/AMPk signaling.

BACKGROUND: Arginyltransferase (Ate1) orchestrates posttranslational protein arginylation, a pivotal regulator of cellular proteolytic processes. In eukaryotic cells, two interconnected systems-the ubiquitin proteasome system (UPS) and macroautophagy-mediate proteolysis and cooperate to maintain quality protein control and cellular homeostasis. Previous studies have shown that N-terminal arginylation facilitates protein degradation through the UPS. Dysregulation of this machinery triggers p62-mediated autophagy to ensure proper substrate processing. Nevertheless, how Ate1 operates through this intricate mechanism remains elusive. METHODS: We investigated Ate1 subcellular distribution through confocal microscopy and biochemical assays using cells transiently or stably expressing either endogenous Ate1 or a GFP-tagged Ate1 isoform transfected in CHO-K1 or MEFs, respectively. To assess Ate1 and p62-cargo clustering, we analyzed their colocalization and multimerization status by immunofluorescence and nonreducing immunoblotting, respectively. Additionally, we employed Ate1 KO cells to examine the role of Ate1 in autophagy. Ate1 KO MEFs cells stably expressing GFP-tagged Ate1-1 isoform were used as a model for phenotype rescue. Autophagy dynamics were evaluated by analyzing LC3B turnover and p62/SQSTM1 levels under both steady-state and serum-starvation conditions, through immunoblotting and immunofluorescence. We determined mTORC1/AMPk activation by assessing mTOR and AMPk phosphorylation through immunoblotting, while mTORC1 lysosomal localization was monitored by confocal microscopy. RESULTS: Here, we report a multifaceted role for Ate1 in the autophagic process, wherein it clusters with p62, facilitates autophagic clearance, and modulates its signaling. Mechanistically, we found that cell-specific inactivation of Ate1 elicits overactivation of the mTORC1/AMPk signaling hub that underlies a failure in autophagic flux and subsequent substrate accumulation, which is partially rescued by ectopic expression of Ate1. Statistical significance was assessed using a two-sided unpaired t test with a significance threshold set at P<0.05. CONCLUSIONS: Our findings uncover a critical housekeeping role of Ate1 in mTORC1/AMPk-regulated autophagy, as a potential therapeutic target related to this pathway, that is dysregulated in many neurodegenerative and cancer diseases.

Effect of Chlorpyrifos on human extravillous-like trophoblast cells.

An increased risk of pregnancy disorders has been reported in women and animal models exposed to organophosphate pesticides. However, less information is available on impacts to human placental function. Here, we addressed the impact of chlorpyrifos (CPF) on extravillous cytotrophoblasts (evCTB) employing HTR8/SVneo cells as an in vitro model. Cell proliferation, migration and invasion were not affected by CPF under conditions where cell viability was not compromised; however, we observed reduced expression of genes for vascular endothelial growth factor receptor 1, hypoxia-inducible factor 1-alpha, peroxisome proliferator activated receptor gamma, and the ß-subunit of human chorionic gonadotropin. These results are the first effects reported by organophosphate pesticide in evCTB cells and show altered expression of several genes important for placental development that could serve as potential biomarkers for future research.