TSC2 N-terminal lysine acetylation status affects to its stability modulating mTORC1 signaling and autophagy

Area of Science:

• Cellular Biology
• Molecular Biology
• Metabolism

Background:

• Protein acetylation is increasingly recognized for its role in metabolic regulation.
• Sirtuins, a class of NAD+-dependent deacetylases, are crucial for maintaining cellular homeostasis by activating autophagy.
• The tuberous sclerosis complex 2 (TSC2) protein is a key regulator of the mTORC1 pathway, influencing cell growth and proliferation.

Purpose of the Study:

• To investigate the role of lysine acetylation of TSC2 in regulating mTORC1 signaling, autophagy, and cell proliferation.
• To elucidate the interplay between TSC2 acetylation, ubiquitination, and protein stability.
• To examine the impact of sirtuin activity on TSC2 acetylation and downstream signaling pathways.

Main Methods:

• Utilized chemical inhibitors (Nicotinamide, Resveratrol, EX527) and genetic manipulations (SIRT1 knockout cells, TSC2 silencing) to modulate sirtuin activity and TSC2 levels.
• Analyzed TSC2 acetylation and ubiquitination status using biochemical assays.
• Assessed mTORC1 signaling activation, autophagy induction, and cell proliferation rates in response to experimental manipulations.
• Investigated the functional consequences of specific TSC2 lysine mutants found in Tuberous Sclerosis Complex (TSC) patients.

Main Results:

• Nicotinamide (NAM) increased TSC2 acetylation and ubiquitination, leading to mTORC1 activation and cell proliferation.
• Resveratrol (RESV), a sirtuin activator, reduced TSC2 acetylation, inhibited mTORC1 signaling, and promoted autophagy.
• Deacetylated TSC2 was protected from ubiquitination, suggesting acetylation promotes TSC2 degradation.
• SIRT1 deficiency or inhibition resulted in hyperacetylated TSC2, rendering mTORC1 signaling unresponsive to NAM or RESV.
• Modulation of SIRT1 activity on mTORC1 signaling was abolished upon TSC2 silencing.
• TSC2 mutants (K599M and K106Q) derived from TSC patients differentially affected basal and NAM-induced mTORC1 signaling.

Conclusions:

• TSC2 lysine acetylation status is directly linked to its protein stability and ubiquitination.
• Acetylation of TSC2 promotes its ubiquitination and subsequent mTORC1 pathway activation.
• Sirtuin-mediated deacetylation of TSC2 stabilizes the protein, inhibits mTORC1 signaling, and induces autophagy.
• This study uncovers a novel acetylation-dependent mechanism controlling TSC2 stability and mTORC1 pathway activity, relevant to Tuberous Sclerosis Complex pathogenesis.