Nutrient Sensing and mTOR Signalling Flashcards
In what circumstances is control of mRNA translation important?
In cell growth and proliferation = speeds up protein synthesis to grow faster
Respond to hormones, growth factors and nutrients
Controls synthesis of specific proteins during development
Respond to stresses or energy depletion
What happens to protein synthesis under low intracellular amino acid levels?
Protein synthesis is inhibited
Why is protein synthesis inhibited under low intracellular amino acid levels?
Because protein synthesis is an energy demanding process and requires amino acids
What is needed for full translation in eukaryotes?
5’ CAP = 7-methyl GTP
Poly A tail = role in mRNA splicing, transport, stability and translation
What impairs translation when amino acid levels are low?
4E-BP1 binds euk initiation factor 4E and prevents its interaction with the eIF4G protein.
This interaction blocks the formation of the eIF4F complex, which is crucial for the initiation of translation in eukaryotic cells
What occurs to 4E-BP1 when amino acids are present?
4E-BP1 is phosphorylated by the mTOR pathway
This leads to euk initiation factor 4G binding eIF4E
What does eIF4g function as?
Scaffold proteins that recruits other eIFs and the ribosome to the cap-moiety
What is eIF4A function?
RNA helicase
Unwinds secondary structure in the 5’UTR = allowing ribosome to efficiently scan AUG start codon
What is the TRANSLATION initiation complex?
Together eIF4E, 4G, and 4A = eIF4F translation initiation complex
How does nutrient withdrawal affect translation?
Inhibits eIF4E-dependent translation because 4E-BP1 blocks it
What is the role of mTOR?
Nutrient sensing
When amino acids are present mTOR phosphorylates 4E-BP1 inhibiting it from blocking eIF4E = allowing protein translation to occur
mTOR phosphorylates S6K1 to activate it = results in pS6 being phosphorylated and activated
Leading to cell growth/prolifation and translational control
What are mTOR downstream signalling targets?
4E-BP1 and S6K1
How does 4E-BP1 repress cap-depdenent translation?
Through impairing eIF4G ability to recruit other euk initiation factors to mRNA
Where was Rapamycin disovered and what properties were discovered?
Discovered in soil sample
Used as anti-fungal agen
Discovered Rapamycin has immunosuppressive effects (used as anti-rejection drug for transplants) AND anti-tumour effects
What protein does Rapamycine bind to?
FKBP12
What type of kinase is mTOR?
Serine/Threonine Kinase
What is mTOR master regulator of and how does it perform regulation?
Translational machinery via phoshporylation
What effect does Rapamycin/FKBP12 have on mTOR?
Inhibits mTORC1
What are the components of mTORC1?
PRAS40 and Raptor
Deptor and mLST8
What are the components of mTORC2?
Protor, mSIN1 and Rictor
Deptor and mLST8
What components do mTORC1 and mTORC2 have in common?
Deptor and mLST8
What domains does mTOR have?
HEAT repeats
FAT = focal adhesion targeting domain
FATC
FRB = where Rapa/FKBP12 binds???
Kinase domain
What in the heart does mTOR regulate?
Angiogenesis through VEGF expression
(vascular endothelial growth factor)
What occurs in tuberous sclerosis complex (TSC)?
Autosomal dominant disorder
Characterized by haratomas = benign tumours in many organs
Growths within the brain = calcify with age and become hard or sclerotic (scarred)
What common medical complications occur with TSC?
Epilepsy
Intellectual disability
Behavioural problems
What two TSC-deteremining genes have been identified?
TSC1 encodes HAMARTIN
TSC2 encodes TUBERIN
What happens when hamartin and tuberin proteins bind?
Form functional tumour suppressor heterodimer
What does TSC1/2 regulate?
Regulate cellular growth primarily by acting as a negative regulator of the mTORC1
When TSC1 is mutated (non-functional) = larger fly eye and wing bristles
When TSC1/2 overexpressed = smaller because mTOR being repressed severely
How does insulin affect TSC1/2 activity?
Negative regulator of TSC1/2 activity
What happens when either the insulin receptor or PKB is KO’d?
TSC1/2 is no longer inhibited
So TSC1/2 acts to inhibit mTOR strongly
Smaller cell size
Where are most TSC patient-derived mutations found and what are the mutations?
Mutations can result in premature stop codons which results in the loss of C-terminal regions that contains putative GAP domain
Single point mutations can also be found within this GAP region
What do GAP and GEF stand for?
GAP = GTPase activating protein
GEF = guanin exchange factor
What is TSC2 protein?
GTPase activating protein
Hydrolysis of GTP to GDP
Inhibits Rheb (small G-protein)
What is Rheb?
small G-protein
Potential target of TSC2 GAP acitivty
What is the role of Rheb?
When active = activates mTOR which phoshporylates S6K1
What is the role of TSC1?
TSC2 only funcitons when in a dimer with TSC1
TSC1 is crutial
What phosphorylates TSC1/2?
Akt/PKB
What inhibits PI3K?
Wortmannin
What phophorylation sites does Akt/PKB target on TSC2?
Serine 939
Serine 981
Describe the cascade when insulin binds its receptor resultsing in mTOR activation
Insulin binds it receptor causing PI3K activation
PI3K phosphorylates Akt/PKB which then phosphorylates inhibiting TSC2 at S939 and S981
TSC2 translocates from being associated with membrane to CYTOSOL and interacts with 14-3-3 protein
Rheb is now active and associates with and turns ON mTOR
mTOR phoshporylates S6K1 which then leads to pS6 phosphorylation
What cellular responses do amino acids participate in?
Regulate secretion (insulin/glucagon)
Protein turnover
Gene expression
Cell Signalling (mTOR etc)
Metabolism/biosynthesis (glutamte/glutamine)
What is System A?
A system responsible for transporting neutral amino acids into the cell
What does SNAT stand for?
Sodium-coupled Neutral Amino acid Transporter
SNAT = family within System A
Where are SNAT1, 2, and 4 located?
1 = brain/heart
2 = ubiquitously expressed
Transports all classical System A substrates (amino acids)
4 = liver (low level expression in muscle)
What activates SNAT translocation?
Regulated by many stimuli
Insulin
Growth factors
Osmotic stress
Amino acid availability
What happens when cells don’t have enough amino acids?
Stimulation of System A activity to transport them in
1:1 ratio of imported Na+ and amino acids (SYMPORTER)
What is trans-inhibition in the context of System A?
When one amino acid is abundant in the extracellular space, it can inhibit the transport of other amino acids that need to be transported by the same system.
What is LAT1 aka SLC7A5?
L-type Amino Acid Transporter 1
What is the function of LAT1?
Bidirecitonal transport of large neutral branch-chain amino acids via facilitated diffusion
Na+ independent transporter
What protein does LAT1 need to function?
Forms heterodimer with 4F2hc via disulphide bridge
Why does LAT1 need to form heterodimer?
Needs 4Fhc to function and translocate to plasma membrane
How is LAT1 associated with mTORC1?
Activates mTORC1 by providing amino acids
Under amino acid starvation what is the effect SESTRINS have on mTORC1 signalling?
Inhibit mTORC1
How does Sestrin interacting with AMPK inhibit mTORC1?
Activation of AMPK because of low amino acids in lysosome
TSC1/2 activation causes GTPase acitivty inhibiting Rheb = hydrolysing any GTP
What effect does Sestrin have when it inhibits GATOR2?
Inhibits mTORC1 activity
Inhibiting GATOR2 causes activation of GATOR1
GATOR1 activates GTPase activity keeping Rags inactive
What is sestrins affect on RagA-GDP?
Keps RagA-GDP in this inactive state
