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mTOR Flashcards

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1
Q

What does mTOR stand for?

A

mammalian Target Of Rapamycin

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2
Q

What is mTOR?

A

Master regulator of cellular growth in eukaryotic cells. Integrates information about a cell’s internal and external environment to decide how much a cell should grow (protein synthesis vs autophagy). The catalytic subunit of larger heterodimeric kinase complexes (mTORC1).

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3
Q

What is cell growth?

A

An increase in cell biomass / size.

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4
Q

What is cell proliferation?

A

Cell growth + cell division.

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5
Q

What is rapamycin?

A

A molecule that inhibits proliferation in rapidly proliferating cells (yeast, WBCs, cancers). Kills the cells.

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6
Q

How was rapamycin named?

A

Rapa after Rapa Nui (Easter Island) where it was found by a drug discovery team in the 60s.
Mycin for anti fungal acitivity.

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7
Q

What is rapamycin’s clinical use?

A

Immunosuppressant (suppresses WBCs).

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8
Q

What was rapamycin compared to in order to identify mTOR?

A

Another immunosuppressant called FK506.

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9
Q

How are rapamycin and FK506 similar?

A
  • Bind FKBP12 to form a complex that inhibits a protein in the T-cell activation pathway
  • Ultimately inhibit T-cell activation.
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10
Q

How are rapamycin and FK506 different?

A
  • Inhibit different steps of T-cell activation
  • Antagonise each other’s action
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11
Q

How were yeast genetics used to identify the rapamycin target?

A

Screened for rapamycin resistant yeast mutants (weren’t killed by it). Mutation in target or target pathway.

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12
Q

Which mutants had to be eliminated from the yeast screen?

A

FKBP12 - this would also inhibit rapamycin function as the complex couldn’t form. Found these using complementation - isolated rapamycin resistant clones allelic with FKBP12.

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13
Q

What does ‘allelic with’ mean?

A

At the same place in the genome (ie within the gene).

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14
Q

How was it determined if a rapamycin resistant clone was allelic with FKBP12?

A

When crossed with FKBP12 mutants, all spores were rapamycin resistant (no complementation).

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15
Q

How were the TOR1 and TOR2 genes mapped?

A

Linkage analysis of the (3) mutants that provided rapamycin resistance but were not allelic to FKBP12.

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16
Q

How was the TOR1 gene sequenced from yeast?

A

Rapamycin resistant clone had its genomic DNA fragmented and a plasmid library including these fragments was generated. Rapamycin resistant plasmids were observed and sequenced (contained fragment conferring resistance).

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17
Q

What do TOR1 and TOR2 genes encode?

A

PI3kinase-related proteins.

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18
Q

How were mammalian biochemical approaches used to identify the rapamycin target?

A

GST pull down of target proteins with FKBP12-GST (binds glutathione). The FKBP12 binds rapamycin which binds its targets. Centrifugation isolates the proteins bound to the glutathione beads.

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19
Q

What was the control of of the GST pull down?

A

Same process but without rapamycin. Any other proteins present are clearly binding other elements of the system.

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20
Q

How were TOR1 and TOR2 genes sequenced from mammals?

A

SDS PAGE gels were run from the GST-pull down, and proteins only present in the presence of rapamycin were partially sequenced and mapped back to DNA.

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21
Q

How did we discover the TOR proteins are highly conserved between yeast and mammals?

A

Very similar proteins were isolated through genetic and biochemical approaches in each organism.

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22
Q

What is mTORC1?

A

mTOR complex 1; a heterodimeric kinase complex of which mTOR is the catalytic subunit.

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23
Q

What proteins does mTORC1 bind?

A

RAPTOR and LST8 (also 2 copies in the mTORC1 heterodimer along with mTOR).

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24
Q

Where does FKBP12-rapamycin bind to mTOR?

A

Immediately adjacent to the kinase active site - blocks mTOR action.

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25
Q

What is the function of mTORC1?

A
  • Promotes synthesis of macromolecules (anabolic processes)
  • Inhibits breakdown of macromolecules (catabolic processes) (autophagy)
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26
Q

How is mTORC1 activity controlled?

A

Nutrient levels: poor nutrients = low activity = catabolism, high nutrients = high activity = anabolism.

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27
Q

How did we discover mTOR promotes protein synthesis?

A

Yeast treated with rapamycin alone had inhibited protein synthesis, but TOR1 mutant yeast (rapamycin resistant) could maintain protein synthesis. Demonstrates WT TOR allows rapamycin to stop protein synthesis.

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28
Q

Why does protein synthesis need to be regulated?

A

It uses up huge amounts of energy (ATP) which might not always be available.

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29
Q

What do they different types of RNA polymerases synthesise?

A

RNA pol I = most rRNA
RNA pol II = mRNA
RNA pol III = tRNA and some rRNA.

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30
Q

What is the first step of translation?

A

Recruitment of initiation factor eIF4E.

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31
Q

What is the role of eIF4E?

A

Binds 5’ cap of mRNA. Recruits other eIFs which recruit the 43s complex.

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32
Q

What is the 43s complex?

A
  • Ribosome small subunit
  • eIF3
  • Initiator tRNA (methionine bound)
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33
Q

What is the 48s complex?

A

The 43s complex on the mRNA with extra eIFs (but not eIF4E).

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34
Q

What does the 48s complex do once recruited to mRNA?

A

Scans the mRNA for a start codon then recruits the large (60s) ribosome subunit. Then translation initiates.

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35
Q

What are eEFs?

A

eukaryotic elongation factors. Recruited as the ribosome moves along the mRNA until the polypeptide is terminated.

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36
Q

Can eIF4E be reused?

A

Yes - as long as it remains bound to the mRNA cap and the ribosomes continue to move away it can continue to recruit more ribosomes.

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37
Q

What is a polysome?

A

mRNA with multiple ribosomes bound to it.

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38
Q

What is polysome profiling?

A

Using polysome separation by MW on a sucrose gradient as a measure of translation (heavier = more ribosomes bound = more translation).

39
Q

How does mTORC1 promote protein synthesis?

A
  • Fast response: direct phosphorylation of 4E-BP (and S6K1) ultimately results in recruitment of ribosomes to the 5’ mRNA cap.
  • Slow response: regulation of how many ribosomes a cell has via regulation of rRNA transcription factors.
40
Q

What is 4E-BP?

A

A protein that binds and inhibits eIF4E from recruiting other eIFs to the 5’ cap of mRNA. Phosphorylation of it causes dissociation so eIF4E can recruit eIFs.

41
Q

What happens globally when mTOR is inhibited (4E-BP present)?

A

Protein synthesis is reduced by ≈ 70% in 2 hours.

42
Q

What happens globally when mTOR is inhibited (4E-BP KO)?

A

Protein synthesis is only reduced by ≈ 10% in 2 hours. 4E-BP essential for mTOR regulation process.

43
Q

What proportion of the fast response is 4E-BP phosphorylation?

A

≈85%

44
Q

What proportion of the fast response is S6K1 phosphorylation?

A

≈15%

45
Q

What does ribo-seq tell us?

A

The types of transcripts being translated (gene specific), not just the global translation.

46
Q

How does ribo-seq work?

A

RNAses are used to degrade RNA not protected by ribosome binding. The transcripts that were ribosome bound are deep sequenced and quantified so we see which genes were preferentially expressed.

47
Q

Which specific transcripts does mTOR target (inhibited when mTOR was inhibited)?

A

≈250 mRNAs encoding translational machinery; they have a TOP motif.

48
Q

What is a TOP motif?

A

Terminal oligopyrimidine (4-14 C/U after the m7G cap). Causes eIF4E to dissociate in the absence of eIF4G (when 4E-BP is bound / not phosphorylated).

49
Q
A
50
Q

How does 4E-BP cause eIF4E mRNA dissociation?

A

Once 4E-BP binds to eIF4E, other eIFs can’t be recruited and eIF4E dissociates due to the TOP sequence.

50
Q

What is autophagy?

A

Cellular recycling process. Autophagosome engulfs particles too big for the proteasome and fuses to lysosome. Amino acids released back into cytoplasm.

51
Q

How is autophagy regulated?

A
  • Ulk1 kinase complex phosphorylates / activates Beclin-1.
  • Beclin-1 kinase complex phosphorylates autophagosome membrane lipids which recruits other autophagosome components.
52
Q

How does mTOR inhibit autophagy?

A
  • Fast response: phosphorylates and inhibits the ULK1 complex, and therefore autophagosome formation.
  • Slow response: phosphorylates and inhibits TFEB and TFE3 TFs which promote lysosomal gene transcription i.e. fewer lysosomes produced.
53
Q

Is mTOR active with high growth factors or amino acids?

A

No (low S6K phosphorylation). Activation is NOT additive.

54
Q

Is mTOR active with high growth factors and amino acids?

A

Yes (high S6K phosphorylation). Activation is synergistic (need both) - GFs and AAs are a molecular ‘AND’ gate.

55
Q

Which amino acids is mTOR particularly responsive to?

A

Arginine and leucine.

56
Q

Is mTORC signalling a linear pathway or a network?

A

A (complex) network.

57
Q

Which protein alters mTOR activity in response to growth factors?

A

Rheb

58
Q

Which protein complex alters mTOR activity in response to amino acids?

A

Rag

59
Q

What action do Rag and Rheb have?

A

They are GTPases (bind and hydrolyse GTP). Bound by GTP = active or GDP = inactive. mTOR is regulated by changing status.

60
Q

What is the function of GAP proteins?

A

Promote GTP hydrolysis of GTPases (inactivate GTPase).

61
Q

What is the function of GEF proteins?

A

Promote GDP exchange for GTP in GTPases (activate GTPase).

62
Q

What is the growth factor signalling pathway?

A
  • Growth factors inhibit TSC.
  • Therefore TSC cannot convert Rheb to GDP bound form.
  • Rheb is active and activates mTORC1.
63
Q

What is TSC?

A

The GAP for Rheb - inactivates it (-> Rheb-GDP).

64
Q

How does Rheb-GTP activate mTORC1?

A

It binds to mTOR distally from the kinase active site and causes a global conformational change that realigns amino acids inside the active site, accelerating catalysis.

65
Q

How do growth factors inhibit TSC?

A

Akt and Erk are cytoplasmic kinases that are phosphorylated and activated by upstream signalling (growth factors binding to growth factor receptors). They phosphorylate TSC to inactivate it. Rheb remains GTP bound.

66
Q

How was it discovered that amino acid signalling does not involve TSC?

A

Cells were still responsive to amino acid changes with TSC knocked out.

67
Q

How was it discovered that Rag was a GTPase that interacted with mTORC1?

A

FLAG tagged RagGTP and RagGDP were in cell lysate containing myc tagged mTORC1 proteins. Anti FLAG IPs were carried out, and it was observed that myc-mTOR and myc-raptor proteins were only pulled down by (interacting with) RagGTP.

68
Q

How are stable RagGTP and RagGDP established?

A

Mutations lock them in these states.

69
Q

Where does mTOR localise in the presence of amino acids?

A

The lysosome surface.

70
Q

How was it proven Rag is necessary for mTOR lysosome localisation in response to amino acids?

A

Localization doesn’t happen in presence of amino acids but absence of Rag.

71
Q

How was it proven Rag is sufficient for mTOR lysosome localisation?

A

Hyperactive Rag (locked in GTP bound state) causes localisation even in the absence of amino acids.

72
Q

Does the RagGTP/GDP status regulate mTORC1 activity (S6K phosphorylation) in vivo in response to amino acids?

A

Yes - mTORC1 activity is higher when locked in the RagGTP bound status, than in the WT Rag.

73
Q

Is RagGTP/GDP bound status dependent on amino acid levels?

A

Yes - Rag changes status in response to changing amino acid availability. Locked RagGTP continues to activate mTORC1 even in low amino acids (usually RagGDP -> mTORC1 repression).

74
Q

Where are Rag and Rheb constitutively localised?

A

The lysosome surface.

75
Q

How is Rag localised to the lysosome surface?

A

It binds the Ragulator complex (in the lysosome membrane).

76
Q

How does Rag activate mTORC1?

A

It binds and recruits mTORC1 to the lysosomal membrane where it is in close proximity to RhebGTP which can bind and activate it.

77
Q

What is GATOR?

A

The GAP for Rag (inactivates in -> RagGDP).

78
Q

What is the pathway leading Arginine and Leucine to activate mTORC1?

A
  • R and L inhibit CASTOR1 and Sesn2 respectively (aa sensors in the cytoplasm so sense cell’s internal environment).
  • CASTOR1 and Sesn2 are blocked from inhibiting GATOR2.
  • GATOR2 is active and inhibits GATOR1.
  • GATOR1 cannot inhibit Rag with GAP activity.
  • Rag is GTP bound (active) and activates mTORC1.
79
Q

Why can mutations in proteins in the mTORC1 pathway lead to cancer?

A

mTORC signalling is disrupted - increased mTORC activity means cell growth is promoted at inappropriate times.

80
Q

Which proteins are usually mutated in cancers?

A

Those in the mTORC1 signalling pathway but not mTORC1 itself.

81
Q

Which mTORC1 pathway proteins are tumour suppressors?

A

mTORC1 inhibitors e.g. TSC, GATOR1 - when inactivated facilitate tumorigenesis.

82
Q

Which mTORC1 pathway proteins are oncogenes?

A

mTORC1 activators - when activated promote tumorigenesis.

83
Q

Which signalling pathway (growth factor or amino acid) is more commonly mutated in cancers?

A

Growth factor.

84
Q

Where does drug development using mTORC1 knowledge hold the most promise?

A

Treatment of the effects of AGEING. Not been much success with cancer!

85
Q

What is cellular ageing?

A

A complex state with several separate and connected aspects of cell biology that change or decline as an organism gets older.

86
Q

What are classic hallmarks of cellular ageing that are associated with mTORC1?

A
  • Breakdown in nutrient sensing pathways (!)
  • Proteotoxic stress (accumulation of proteins due to damage (forms aggregates) or overexpression).
  • Accumulation of cellular damage e.g. mitochondrial dysfunction.
  • Increased cell size (more growth).
87
Q

Why did mTORC1 study become popular?

A

It was observed that mTORC1 inhibition using rapamycin treatment can extend the lifespan of yeast, worms, flies and mice in lab conditions.
Similar results to dietary restriction but less invasive.

88
Q

Why is treatment of humans with rapamycin (mTORC1 inhibition) not feasible to extend lifespan?

A

Rapamycin is an immunosuppressant so in environments outside the lab it increases risk of (fatal) disease.

89
Q

Why does the Western diet cause an accumulation of cellular damage?

A

It provides excess nutrients, which leads to high mTORC1 activity, which leads to anabolism and accumulation of (damaged) macromolecules.

90
Q

How does the size of hematopoietic stem cells (HSCs) change as we age?

A

The HSCs increase in size.

91
Q

How is HSC size correlated with ability to reconstitute the blood system (in mice)?

A

Larger HSCs are less efficient at reconstituting the blood system (less fit).

92
Q

How does treatment with rapamycin affect HSCs?

A

It reduces HSC size and increases their fitness. This indicates a reversible ageing effect.

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