Architecture of the Tuberous Sclerosis Protein Complex

Ramlaul, Kailash; Fu, Wencheng; Li, Hua; Garrido, Natàlia de Martín; He, Lin; Trivedi, Manjari; Cui, Wei; Aylett, C.H.S.; Wu, Geng

doi:10.1016/j.jmb.2020.166743

Cited by 22 publications

(28 citation statements)

References 31 publications

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“…Recombinant human pTSC forms flexible 40 nm filamentous structures: Consistent with results from two other groups [7,8], we detected ~10 × 40 nm highly flexible particles in NSEM analysis of human recombinant pTSC overexpressed under the control of CMV promoters in human cells (Fig. 1).…”

Section: Resultssupporting

confidence: 90%

“…We found that recombinant human pTSC co-purifies with heat shock protein 70 (Hsp70), as observed in other studies [7,13]. However, no density could be found for the heat shock protein in the recent cryoEM maps indicating that the heat shock proteins most likely binds misassembled or partially unfolded pTSC.…”

Section: Resultssupporting

confidence: 80%

“…Both TSC2 and TBC1D7 were detected with a clear western blot signal in the S3 fraction before and after passage through the Fab-column, indicating that the Fab was capturing only a small fraction of the complexes from S3. Presence of the TBC1D7 in the elution fractions indicates the presence of TSC1 because the small 35 kDa accessory protein interacts directly with TSC1 in pTSC and not TSC2 as observed in the cryoEM structure of the human complex [7,8]. This mode of interaction is supported by X-ray crystallographic studies of truncated TSC1 with TBC1D7 [13,17].…”

Section: Resultsmentioning

confidence: 76%

“…It is also possible that their formation is differentially regulated in rat and pig species upon fasting of the animals. The recent cryoEM models of recombinant human pTSC suggests a head-to-tail oligomerization potential [7]. This hypothesis was supported by a concentration dependent observation of a head-to-tail association of individual pTSC complexes in the cryoEM micrographs and shifting of the elution profile of the recombinant protein toward large molecular weights by increasing concentration [7].…”

Section: Discussionmentioning

confidence: 81%

“…Two recent studies have reported near-complete cryoEM density maps of recombinant human pTSC at near-atomic resolutions [7,8]. Both studies report a rod-shaped architecture that is highly flexible.…”

mentioning

confidence: 99%

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Polymeric assembly of endogenous Tuberous Sclerosis Protein Complex

Dai

Hasan

Woollard

et al. 2021

Preprint

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Tuberous Sclerosis protein complex (pTSC) nucleates a proteinaceous signaling hub that integrates information about the internal and external energy status of the cell in regulation of growth and energy consumption. Biochemical and electron cryomicroscopy (cryoEM) studies of recombinant pTSC have revealed the structure and stoichiometry of the pTSC and have hinted at the possibility that the complex 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 up to 1 μm in length in linear and web-like organizations prepared from pig brain. These observations suggest polymerization of endogenous pTSC into filamentous super-structures.

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Section: Resultssupporting

confidence: 90%

Section: Resultssupporting

confidence: 80%

Section: Resultsmentioning

confidence: 76%

Section: Discussionmentioning

confidence: 81%

mentioning

confidence: 99%

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Polymeric assembly of endogenous Tuberous Sclerosis Protein Complex

Dai

Hasan

Woollard

et al. 2021

Preprint

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Design of negative‐regulating proteins of Rheb/mTORC1with much‐reduced sizes of the tuberous sclerosis protein complex

2023

Protein Science

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The mTORC1 signaling pathway regulates cell growth and metabolism in a variety of organisms from yeast to human, and inhibition of the mTORC1 pathway has the prospect to treat cancer or achieve longevity. The tuberous sclerosis protein complex (TSCC) is a master negative regulator of the mTORC1 signaling pathway through hydrolyzing the GTP loaded on the small GTPase Rheb, which is a key activator of mTOR. However, the large size (~700 kDa) and complex structural organization of TSCC render it vulnerable to degradation and inactivation, thus limiting its potential application. In this work, based on thorough analysis and understanding of the structural mechanism of how the stabilization domain of TSC2 secures the association of TSC2‐GAP with Rheb and thus enhances its GAP activity, we designed two proteins, namely SSG‐MTM (short stabilization domain and GAP domain‐membrane targeting motif) and SSG‐TSC1N, which were able to function like TSCC to negatively regulate Rheb and mTORC1, but with much‐reduced sizes (~1/15 and ~ 1/9 of the size of TSCC, respectively). Biochemical and cell biological assays demonstrated that these designed proteins indeed could promote the GTPase activity of Rheb to hydrolyze GTP, inhibit the kinase activity of mTORC1, and prevent mTORC1 from down‐regulating catabolism and autophagy.

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