Baumeister mTOR Signaling

Target of Rapamycin

Drug used as immunosupressiva (organ transplants) fast developement of huge research field

It tells us it must be a very potent central regulator complex that can influence the whole system of the organism. Reseach has shown influences on aging and longevity, stress response, neurodegenerative diseases, cancer, metabolic disorders (diabetes) and its influence on cell growth and the metabolism

Target Specific Components:

• mTORC1: Target RAPTOR

• mTORC2: Target RICTOR

Methods:

RNA Interference (RNAi):

• Use RNAi to specifically knock down RAPTOR (mTORC1) or RICTOR (mTORC2)

• Inducible RNAi systems (e.g., tetracycline-inducible) for temporal control.

Protein-Protein Interaction Inhibitors:

• small molecules or peptides that disrupt the interaction between mTOR and RAPTOR or RICTOR

Proteolysis Targeting Chimeras (PROTACs):

• selectively degrade RAPTOR or RICTOR to down-regulate mTORC1 or mTORC2.

CRE/LOX System:

• Employ CRE/LOX for conditional knockout of RAPTOR or RICTOR.

Chemical Inhibition:

• Use Rapamycin or Rapalogs to specifically inhibit mTORC1 by binding to FKBP12.

CRISPR/Cas9 Gene Editing:

• conditional knockout of RAPTOR (mTORC1) or RICTOR (mTORC2) using CRISPR/Cas9.

Target Downstream Effectors:

• mTORC1: Target S6K (ribosomal protein S6 kinase).

• mTORC2: Target SGK-1 (serum- and glucocorticoid-induced kinase 1).

Subcellular Localization:

• mTORC1: Target lysosomal membrane-related processes.

• mTORC2: Target endosomal or ER-related processes.

Target Negative Regulators of mTORC1 & 2:

• Overexpress inhibiting proteins like TSC1/2

mTORC1 Activity:

4EBP1:

• Mechanism: mTORC1-mediated phosphorylation of 4E-BP1 at multiple sites releases eIF4E, promoting translation initiation.

S6K (Ribosomal Protein S6 Kinase):

• Function: Promotes protein synthesis upon phosphorylation by mTORC1.

ULK1 (Unc-51 Like Autophagy Activating Kinase 1):

• Function: mTORC1 inhibits ULK1, thereby suppressing autophagy.

mTORC2 Activity:

AKT:

• Mechanism: mTORC2 phosphorylates AKT at Ser473, which is critical for its full activation.

SGK1 (Serum- and Glucocorticoid-Inducible Kinase 1):

• Mechanism: mTORC2 phosphorylates SGK1 at Ser422.

Methods:

• Western Blot: Use phospho-specific antibodies to detect phosphorylated ULK1, S6K, 4E-BP1, AKT

• GFP-Tagging: Expression of target genes via GFP tagging changes in fluorescence localization or intensity.

• RT-PCR: Measure transcript levels of downstream genes (4EBP1, S6K, ULK1, AKT, and SGK1) to see how mTORC1 and mTORC2 activities impact gene expression.

• Phenotypical analysis - e.g autophagy related changes

Metformin: prescribed to manage Type II Diabetes for lowering blood glucose levels, improving Insulin sensitivity & weight control by lowering appetite

AMPK Activation:

Metformin activates AMPK (like growth factors as IGF/insulin),

-> increased glucose uptake

-> increased fatty acid oxidation reducing lipid accumulation

-> inhibiting gluconeogenesis (glucose production) in the liver lowering blood sugar levels

Activated AMPK activates TSC1/2 which inhibits Rheb (GTPase that activates mTORC1)

RICTOR (Rapamycin-Insensitive Companion of mTORC2) and RAPTOR (Regulatory-Associated Protein of mTORC1)

• direct interaction with TOR, they make the key difference in the 2 complexes

• Important for research to target only one of the pathways

• Biologically important to mediate the effects of the TOR activity (membrane binding and complex structure)

• depending on how much Raptor or Rictor is present they can determine which complex is formed or by forming more of only one complex change the cells fate

  • more Raptor = more mTORC2 -> AKT activation -> cell proliferation, and survival

  • more mTORC1 -> S6K and 4EBP1 -> enhanced protein synthesis but also -> ULK phosphorylation

    -> inhibiting autophagy

C. elegans name of TOR = let-363 meaning it is lethal

Raptor = daf-15 which stands for dauer formation in this case it is dauer constitutive

Dauer allows the worm to survive adverse conditions —> increased survival 6 lifespan longevity

C. elegans homologue of Rheb is RHEB-1

RHEB-1 is derived directly from the human homolog "Rheb" (Ras homolog enriched in brain). This straightforward naming suggests that the gene was identified based on sequence homology to its mammalian counterpart Rheb = rheb-1 —> homology driven

On the contrary DAF-15 reflects its role in dauer formation. The discovery and naming of DAF-15 are based on genetic screens related to the dauer developmental stage in C. elegans —> function driven

• availability of TOR protein for Construction of mTORC2 complexes with Rictor (needs to be available on high level)

• Wouldn`t per se affect the Activity of the Complex because of the availability of the interactors would increase the activity of the complex

Strategy:

• test targets of mTORC2 for their activity in comparison to when both complexes are active

Otherwise no direct interaction from mTORC1 to mTORC2 the other way round there is a connection through AKT & sgk-1 e.g. AKT inhibition will also inhibit mTORC1

Ways to test the effect of raptor deletion would simply be the use of Rapamycin or Rapalogs that only act on Raptor, or using conditional knockouts / CrisprCas or Raptor mutants and then monitoring and comparing mTORC2 activity with reporters or phospho Antibodys for sgk-1 or AKT or maybe even phenotypical analysis