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

Abstract

Tuberous sclerosis protein complex (pTSC) nucleates a proteinaceous signaling hub that integrates information about the internal and external energy status of the cell in the regulation of growth and energy consumption. Biochemical and cryo-electron microscopy studies of recombinant pTSC have revealed its structure and stoichiometry and hinted at the possibility that the complex may form large oligomers. Here, we have partially purified endogenous pTSC from fasted mammalian brains of rat and pig by leveraging a recombinant antigen binding fragment (Fab) specific for the TSC2 subunit of pTSC. We demonstrate Fab-dependent purification of pTSC from membrane-solubilized fractions of the brain homogenates. Negative stain electron microscopy of the samples purified from pig brain demonstrates rod-shaped protein particles with a width of 10 nm, a variable length as small as 40 nm, and a high degree of conformational flexibility. Larger filaments are evident with a similar 10 nm width and a ≤1 μm length in linear and weblike organizations prepared from pig brain. Immunogold labeling experiments demonstrate linear aggregates of pTSC purified from mammalian brains. These observations suggest polymerization of endogenous pTSC into filamentous superstructures.

• Authors: David L Dai, S M Naimul Hasan, Geoffrey Woollard, Yazan M Abbas, Stephanie A Bueler, Jean-Philippe Julien, John L Rubinstein, Mohammad T Mazhab-Jafari
• Journal: Biochemistry (Biochemistry) Vol. 60 Issue 23 Pg. 1808-1821 (06 15 2021) ISSN: 1520-4995 [Electronic] United States
• PMID: 34080844 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)

Chemical References

• Recombinant Proteins
• Tsc2 protein, rat
• Tuberous Sclerosis Complex 2 Protein
• Tumor Suppressor Proteins

Topics

• Animals
• Cryoelectron Microscopy (methods)
• Cytoskeleton (metabolism)
• Humans
• Protein Binding (physiology)
• Rats
• Recombinant Proteins (metabolism)
• Signal Transduction (genetics)
• Swine
• Tuberous Sclerosis (genetics, metabolism)
• Tuberous Sclerosis Complex 2 Protein (chemistry, metabolism, ultrastructure)
• Tumor Suppressor Proteins (metabolism)