mTOR/Cell growth reg

Question 1

Cell growth vs proliferation

Answer 1

growth - size increase - can happen w/out division

proliferation = Growth + division together

Question 2

mTOR basics

Answer 2

part of mTORC1 complex
integrates info about external and internal environment
makes decisions on how cell should grow
regulates processes of cell growth in response to environment

Question 3

Rapamycin and FK506

Answer 3

Rapamycin - drug inhibits proliferation of rapidly proliferating cells

FK506 - aling with rapamycin - has immunosuppresant effects

both bind same site on FKBP12 protein
but inhibit later and earlier stages of T cell development respectively
and inhibit each other

binding FKBP12 creates toxic complex that inhibits different things depending on drug bound

Question 4

mTOR discovery - yeast genetic screens + crosses

Answer 4

rapamycin resistant mutagenesis screen

18 found
use mating/sporulation to parse out interesting ones
eg FKBP12 mutants were resistant - so want to filter these out as this is not the interesting target

15/18 mutants were recessive
mate with known FKBP12 mutants
progeny resistant
so these mutants were allelic with FKBP12 - mapped to same location in genome
filter these out

3/18 non-allelic w FKBP12 (and dominant)
interesting ones as mutations are in genes other than the known actor FKBP12
these 3 mapped to Tor1 and Tor2 (Target of rapamycin)

Question 5

Tor1 and Tor2 plasmid library experiment

Answer 5

purify genomic DNA from these 3
clone into plasmid library
transform into WT yeast

if the WT yeast is transformed w/ the Tor1 DNA - transformant gains resistance (dominant mutation)

found Tor1 and Tor2 genes this way (as can look at DNA on the plasmid the de novo resistant transformant received)

both encoded PI3K related kinases
the genetic targets of rapamycin

Question 6

mTOR discovery - biochemical purification

Answer 6

express FKBP12 w/ GST tag
affinity purification w Glutathione bound beads
pulls down GST-FKBP12 and any associated proteins

in presence of Rapamycin
Tor protein will associated w/ Rapamycin/FKBP12
for control do w/out Rapamycin

can pull down via Glutathione bound beads
Can tell which is rapamycin target as it will only be pulled down in the Rapamycin+ experiment and no the control
take protein from this band

Edam sequencing
-partial protein sequence
-map this to DNA sequence in genome
-also mapped to PI3K related kinase w high conservation to yeast Tor1 and 2

Question 7

mTOR discovery overall

Answer 7

yeast Biochemical and genetic approached converged on same conserved PI3K like kinase gene

mTOR = mammalian target of rapamycin
is the conserved mammalian version of this

Question 8

mTOR in complex

Answer 8

is the catalytic subunit of larger heterodimeric kinase complexes

FKBP12/Rapamycin toxic complex binds right next to kinase active site and prevents mTOR function

mutants that gained resistance were mutated right next to this point

Question 9

mTOR growth reugulation process basic

Answer 9

regulates 2 important processes
>protein synthesis
>autophagy

mTORC1 activity
promotes anabolic processes (macromolecule synthesis)
inhibits catabolic reactions (macromolecule breakdown)

poor nutrients:
-stressed
-Low mTORC1 activity
-Catabolism favoured (breakdown of macromolecules, autophagy)

good nutrients:
-unstressed
-high mTORC1 activity
-anabolism favoured (synthesis, ie translation)

basically mTORC1 activity promotes global protein synthesis
so Rapamycin treatment inhibits global protein synthesis

Question 10

how to test if rapamycin based protein synth inhibition is dependent on TOR

Answer 10

TOR1-1 rapamycin resistant mutant used as control
see no drop in synth with rapamycin treatment (but see drop w Cyclohexamide control)
tells us that Rapamycin based protein synthesis inhibition is dependent on interacting w/ TOR

Question 11

elF4E and translation

Answer 11

is the first thing to bind the mRNA in translation
binds 5’ cap
recruits initiation factors

results in recruitment of 43s complex
has small subunit of ribosome
+the firts Methionine tRNA

1st methionine addition allows recruitment of 60s large ribosomal subunit

eEFs then promote elongation to make polypeptide

Question 12

Polysomes

Answer 12

many ribosomes on same mRNA

will sink firther in sucrose gradient (more ribosomes = heavier/denser)

Question 13

mTORC1 activity: protein synthesis promotion responses

Answer 13

slower response:
targets TFs that affect Pol I and Pol III transcription
activates them
increases rRNA levels
get higher ribosome numbers in cell

faster response:
phosphorylates 4E-BP (elF4E binding proteins) and S6K1

Question 14

4E-BP phosphorylation effects - mTORC1 fast response

Answer 14

rich nutrients
mTOR active
phosphorylates 4E-BP
prevents its interaction w/ elF-4E
Frees elF-4E to interact with proteins involved in recruiting ribosomes to mRNA

increases translation

Question 15

experiment to prove that 4E-BP is main target in fast response

Answer 15

knockdown 4E-BP in cells
inhibit mTOR with Torin
measure protein amounts in cell after short 2hr Torin treatment

get 70% reduction in non-knockdown control
only get 10% change in 4E-BP Knockdown cell

indicates that 4E-BP is important in the fast response

also see this same pattern with polysomes in sucrose sedimentation gradients
4E-BP non-knockdown = see big change in signal for further sedimented ribosomes
4E-BP knockdown - see much smaller change

Question 16

finding mRNAs in transcriptome differentially affected by mTOR inhibition

Answer 16

RiboSeq
freeze (cross link?) ribosomes to mRNA
isolate mRNA from cell (polyTs?)
treat w nuclease cocktail
RNA left over after treatment represents ribosome footprints
sequence the footprints to see where in transcriptome the ribosomes are bound

mTOR controls ALL protein synthesis
BUT
allowed the finding of mRNAs whose translation is differentially reduced in mTOR inhibition
ie mRNAs more sensitive to mTOR regulation
due to TOP motif
on transcripts involved in translational machinery - so mTOR inhibition gave greater decrease in ribosome association on these transcripts

TOP motif - makes transcripts more sensitive to 4E-BP

Question 17

TOP motif molecular mechanism

Answer 17

terminal oligopyrimidine (Cs and Us)

makes transcripts more sensitive to 4E-BP regulation
as in absence of elF46
these sequences cause elF-4E (the first recruiter in translation) to dissociate from the mRNA

Question 18

Autophagy

Answer 18

autophagosomes form around and engulf cytoplasmic components

matures
seals off
fuses w lysosome
breaks down components in autophagosome
recycle them

Question 19

autophagy regulation complexes

Answer 19

2 complexes

Beclin-1
>phosphorylates lipids on early autophagosome stages
>recruits factors needed to regulate following autophagosome maturation and digestion steps

ULK1:
-phosphorylates Beclin-1 and upregulates its lipid phosphorylation activity

Question 20

mTOR regulation of autophagy

Answer 20

Fast response:
mTOR phosphorylates ULK1 and inhibits it

low nutrients
=low mTOR activity
=low ULK1 phosphorylation
=high ULK1 activity
=high Beclin-1 lipid phoshporylation activity
=high autophagy

high nutrients reverse
low autophage (low catabolisis)

Slow response:
mTOR phosphorylates and inhibits TFEB and TFE3
TFs needed for lysosome formation
gives more chronic adjusted state of cell with lower lysosome activity/lower catabolisis

Question 21

Upstream signals integration for mTORC1

Answer 21

by 2 regulators
Rheb - responds to growth factors
Rag complex - responds to Amino acids

molecular AND gate - mTOR only on when both of these are presnt
rag and rheb work synergistically

Question 22

direct regulation of Rag and Rheb

Answer 22

GTPases - active in GTP-bound form
inactive when GTP is hydrolysed to GDP

GAPs - promote hydrolysis
inhibit the GTPases other activity

GEFs - promote GDP->GTP exchange
promotes its other activity

Question 23

Rheb and growth factors

Answer 23

Rheb is specific target of TSC complex (TSC1/2)
TSC is known GAP

Growth factor signalling causes Akt and Erk phosphorylation
phosphorylate and inhibit TSC complex
TSC can no longer promote GTP hydrolysis in Rheb
Rheb can stay active
and therefore can promote mTOR activity

Question 24

active Rheb mTOR promotion

Answer 24

binds mTOR only in the GTP-bound form
distal from active site
causes conformational change that turns on mTOR activity

no growth factors
=no Akt/Erk phosphorylation
=no TSC phosphorylation
=TSC able to promote Rheb GTP hydrolysis
=Rheb in GDP-bound state so cannot bind and activate mTOR
=mTOR activity suppressed

Question 25

TSC ko mutant

Answer 25

mTOR no longer needs growth factors for high mTOR activity
but will still respond to amino acid levels
so amino acid levels regulate it independently

Question 26

mTOR/Rag-GTP binding experiement

Answer 26

IP
Rag-FLAG protein
can pull down w anti-Flag Ab

Myc tagged mTOR pulled down with it when Rag in GTP form
when Rag in GDP form - mTOR was not pulled down - but was present in lysate control lane

Question 27

mTOR localisation in presence of AAs

Answer 27

mTOR localises to lysosomes in presence of AAs
is diffuse in cytoplasm in absence of AAs

add AAs
in 10 mins mTOR recruited to lysosomes
KO Rag - this doesnt happen - so Rag required for lysosome localisation of mTOR

express Rag locked in GTP state
mTOR always localised to lysosomes independent of AA levels

Question 28

mTOR output assay

Answer 28

can western blot from cell lysate measuring S6 kinase phosphorylation (mediated by mTORC1 kinase)

Question 29

Rag-GTP mediated mTOR lysosome localisation

Answer 29

Rag-GTP binds mTOR
localises it to lysosome vie Regulator complex

this puts it in proximity to Rheb (lysosome localised)
so can be activated by Rheb-GTP (if it is present/TSC is inactive in presence of growth factors)

mTOR in cytoplasm cannot be activated by Rheb-GTP

Question 30

Amino acid level mediated regulation of Rag GTPase

Answer 30

Rag GTPase has a GAP
GATOR1
inhibits Rag-GTP form

GATOR2 inhibits GATOR1
GATOR2 has 2 inhibitors:
-CASTOR1 - binds Arginine
-Sesn1 - binds Leucine

low AA levels
-Castor1 and Sesn1 are unbound/free
-can bind GATOR2 and inhibit it
-so GATOR1 cant be inhibited by GATOR2
-GATOR1 is able to promote Rag-GTP->GDP
-Rag-GDP form promoted
-Rag-GDP cannot bind and localise mTOR to the lysosome
-mTOR cant be activated by Rheb on lysosome (lysosome surface used as signalling platform)

mTOR activity inhibits lysosome activity/autophagy
so in low AA conditions
mTOR activity inhibited
autophagy promoted (Ulk1 unphosphorylated - active to phosphorylate Beclin-1 - promotes lysosome maturation/autophagy)

Question 31

mTORC1 and cancer

Answer 31

cancer
disease of uncontrolled growth and division

normal mTORC1 signalling ensures cells only grow when appropriate
dysregulation of this can be part of cancer

many cancer cells evolve to bypass these controls
rarely mutate mTOR directly
but in the regulators
-tumour suppressors that inhibit mTOR activity normally
-oncogenes that work to activate mTOR

TSC v common tumour suppressor mutation

mTOR inhibitors in cancer treatment?

Question 32

mTOR and ageing

Answer 32

decline in aspects of cell biology as organism gets older
cellular damage (accumulation of unfolded proteins and organelle damage)

may result in cell size increase as age

mTORC1 implicated in these processes
eg high mTORC1 activity leads to less catabolism - less digestion and recycling of proteins - more accumulation of unfolded/misfolded proteins

Question 33

Rapamycin ageing study

Answer 33

mice
treat w rapamycin at 600 days old (pretty old) with otherwise normal diet
lifespan extended significantly over untreated control

Question 34

dietary restriction and mTOR ageing stuff

Answer 34

dietary restriction could help extend lifespan
restriction -> less activatoey signalling to mTOR
->increased autophagy
shown to extend lifespan in model organisms

high nutrients
mTOR more often in high activity state
cell is in anabolic state more often
accumulating damaged stuff more than recycling

restrict nutrients
lower mTOR activity
cell in catabolic state more often
get more recycling and repair of cellular damage

Question 35

haematopoietic stem cell size and ability to repopulate irradiated recipient

Answer 35

as organisms age
purify HSCs
size increases over lifespan
and are less proliferative (less functional in forming colonies)

is this size increase causative

take donor mouse
purify its HSCs
irradiate recipients
transplant donor HSCs into diff recipients based on their size (diff sizes from same donor)

see how size of HSC affects ability to repopulate irradiated mouse’s haematopoietic sustem

small/medium ones did wee
larger worse

treats the HSCs w rapamycin
decreases cell size
this mTOR inhibition makes them better at repopulating

cellular enlargement causative decline in function related to aeging