Proteotoxic Stress Hijacks MTORC1 to Trigger Cellular Cleanup Pathways
New research reveals how misfolded protein buildup activates autophagy and lysosome production via an unexpected MTORC1-independent mechanism.
Summary
When cells accumulate misfolded proteins—a condition called proteotoxic stress—they activate autophagy and lysosomal biogenesis as a defense. This study shows that proteasome inhibitors, puromycin, and mutant huntingtin protein all drive nuclear entry of transcription factors TFEB and TFE3, master regulators of cellular cleanup. Surprisingly, this activation bypasses the canonical nutrient-sensing pathway (TSC2/ATF4) and instead disrupts the RRAGC GTPase–TFEB interaction, preventing TFEB from reaching lysosomes where MTORC1 normally phosphorylates and inactivates it. Additionally, proteasome inhibition triggers non-canonical autophagy, and deletion of ATG16L1 or ATG5 partially blunts TFEB activation, linking Atg8-family protein lipidation to TFEB nuclear translocation. These findings reveal a novel cellular stress-response circuit with implications for neurodegenerative and proteinopathy diseases.
Detailed Summary
Proteotoxic stress—arising from misfolded protein accumulation due to proteasome dysfunction, translation errors, or aggregation-prone proteins—is a hallmark of numerous diseases including neurodegeneration and cancer. Cells counteract this stress partly through the autophagy-lysosomal pathway (ALP), but the precise mechanisms linking protein misfolding to ALP activation have remained unclear. This study systematically investigates how proteotoxic stress engages TFEB and TFE3, the primary transcriptional regulators of lysosomal biogenesis and autophagy.
The researchers used multiple complementary models of proteotoxic stress: proteasome inhibitors bortezomib (BTZ), carfilzomib (CFZ), and MG132; the translation-disrupting agent puromycin; and expression of polyglutamine-expanded huntingtin (HTT), relevant to Huntington's disease. In all models, TFEB and TFE3 underwent robust nuclear translocation and activated downstream target genes. Critically, the team found that this activation was independent of TSC2 (a canonical MTORC1 suppressor) and ATF4 (an ER stress-linked transcription factor), ruling out previously assumed pathways.
Instead, proteotoxic stress operates through a non-canonical mechanism targeting RRAG GTPases. Under normal conditions, RRAGC recruits TFEB to the lysosomal surface where active MTORC1 phosphorylates TFEB, retaining it in the cytoplasm. The study demonstrates that proteotoxic stress disrupts the RRAGC–TFEB protein interaction, preventing lysosomal recruitment and thereby escaping MTORC1-mediated phosphorylation. Confirming this mechanism, expression of a constitutively active RRAGC mutant rescued impaired lysosomal TFEB localization and blocked nuclear accumulation under stress. Conversely, co-overexpression of FLCN and FNIP2 (a GTPase-activating protein complex for RRAGC) partially restored TFEB dephosphorylation, further validating the RRAGC axis.
An additional and notable finding is that proteasome inhibition also activates non-canonical autophagy—a form of autophagy that proceeds without classical Atg8-family lipidation. Paradoxically, deletion of ATG16L1 or ATG5, which blocks Atg8-family protein lipidation and sequesters the FLCN-FNIP2 complex, partially abolished proteotoxic stress-induced TFEB dephosphorylation and nuclear accumulation. This suggests that Atg8-family lipidation contributes to canonical TFEB activation by modulating the availability of FLCN-FNIP2 for RRAGC regulation.
Collectively, this work establishes a dual-arm stress response: proteotoxic stress simultaneously triggers non-canonical autophagy and, via RRAGC-mediated MTORC1 bypass, activates TFEB/TFE3-driven canonical lysosomal biogenesis and autophagy. These insights have therapeutic implications for diseases involving protein aggregation, suggesting that targeting the RRAGC–TFEB interaction or FLCN-FNIP2 availability could modulate the cellular response to proteotoxic stress.
Key Findings
- Proteotoxic stress drives TFEB and TFE3 nuclear accumulation independently of TSC2 and ATF4 pathways.
- Stress disrupts RRAGC-TFEB binding, preventing lysosomal recruitment and MTORC1-mediated TFEB phosphorylation.
- Constitutively active RRAGC mutant rescues lysosomal TFEB localization and blocks nuclear entry under stress.
- Proteasome inhibition activates non-canonical autophagy alongside TFEB-mediated canonical autophagy.
- ATG16L1/ATG5 deletion partially suppresses TFEB dephosphorylation, linking Atg8 lipidation to TFEB activation.
Methodology
The study employed multiple in vitro proteotoxic stress models including pharmacological proteasome inhibitors (bortezomib, carfilzomib, MG132), puromycin-induced translation errors, and polyglutamine-expanded huntingtin expression in cell lines. Genetic approaches included constitutively active RRAGC mutants, FLCN/FNIP2 overexpression, and ATG16L1/ATG5 knockout to dissect mechanistic pathways. Readouts included TFEB/TFE3 nuclear localization, phosphorylation status, lysosomal recruitment assays, and downstream target gene expression.
Study Limitations
The study is primarily conducted in cell-based models; in vivo validation in animal models of neurodegeneration is needed to confirm physiological relevance. The exact molecular mechanism by which proteotoxic stress disrupts the RRAGC–TFEB interaction remains to be fully characterized at a structural level. The contribution of non-canonical autophagy relative to canonical TFEB-driven autophagy in disease contexts requires further quantification.
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