Structural Characterization of Endogenous Tuberous Sclerosis Protein Complex Revealed Potential Polymeric Assembly

Area of Science:

• Cellular biology
• Biochemistry
• Neuroscience

Background:

• The tuberous sclerosis protein complex (pTSC) is a key regulator of cell growth and energy consumption.
• Previous studies on recombinant pTSC suggested potential for large oligomer formation.
• Understanding the in vivo structure of pTSC is crucial for elucidating its function.

Purpose of the Study:

• To investigate the native structure and organization of endogenous pTSC in mammalian brains.
• To explore the potential for pTSC polymerization into higher-order structures.

Main Methods:

• Partial purification of endogenous pTSC from fasted mammalian brains (rat and pig) using a specific recombinant antibody fragment (Fab).
• Negative stain electron microscopy (EM) to visualize purified pTSC structures.
• Immunogold labeling to confirm the localization and aggregation of pTSC.

Main Results:

• Successfully purified endogenous pTSC from brain homogenates using Fab-mediated affinity purification.
• EM revealed rod-shaped pTSC particles (10 nm width, variable length) exhibiting conformational flexibility.
• Larger filamentous structures (≤1 μm length, 10 nm width) and weblike organizations were observed.
• Immunogold labeling confirmed linear aggregation of pTSC into filamentous superstructures.

Conclusions:

• Endogenous pTSC can polymerize into filamentous superstructures in mammalian brains.
• These findings suggest a novel structural organization for pTSC with potential implications for cellular signaling and regulation.