Remodelling of Cellular Protein Homeostasis by Enhanced ER-Mitochondrial Tethering

Area of Science:

• Cell Biology
• Molecular Biology
• Biochemistry

Background:

• Endoplasmic reticulum (ER)-mitochondrial interactions are crucial for cellular homeostasis.
• Alterations in these interactions are linked to neurodegenerative diseases and protein homeostasis (proteostasis) dysregulation.
• The specific impact of enhanced ER-mitochondrial tethering on cellular proteostasis remains largely unknown.

Purpose of the Study:

• To investigate how experimentally enhanced ER-mitochondrial tethering affects cellular proteostasis and function.
• To determine the impact of different degrees of ER-mitochondrial proximity on protein synthesis, ER stress, and metabolic pathways.
• To explore the potential role of altered ER-mitochondrial tethering in the pathogenesis of neurodegenerative diseases.

Main Methods:

• Overexpression of synthetic ER-mitochondrial linkers (EMLs) to stabilize ER-mitochondrial distance at ~5 nm and ~10 nm in HeLa cells.
• Assessment of cell growth, metabolic activity, ATP levels, and global protein synthesis.
• Analysis of unfolded protein response (UPR) markers (PERK, eIF2α, ATF4, ATF6).
• Shotgun mass spectrometry proteomics and bioinformatic analysis of protein expression changes.
• Lipidomic analysis to assess lipid homeostasis.

Main Results:

• Enhanced ER-mitochondrial tethering reduced metabolic activity and total ATP levels without affecting cell growth.
• Global protein synthesis was significantly reduced, accompanied by altered levels of key protein synthesis regulators and UPR markers.
• Proteomic analysis revealed distinct effects of ~5 nm and ~10 nm tethering on RNA processing, splicing, proteasomal degradation, and protein translation.
• Both tethering conditions impacted proteins involved in mitochondrial function, oxidative stress defense, ER homeostasis, signaling, and secretion.
• Lipidomic analysis indicated differential effects of EMLs on lipid homeostasis.

Conclusions:

• Enhanced ER-mitochondrial tethering profoundly remodels cellular protein homeostasis, impacting multiple cellular pathways.
• The observed derangement of protein synthesis and induction of ER stress suggest a link to neurodegenerative conditions.
• Differential effects of tethering distance highlight the sensitivity of cellular proteostasis to ER-mitochondrial proximity.
• These findings provide insights into the molecular mechanisms underlying neurodegenerative diseases associated with ER-mitochondrial dysfunction.