CMB Flashcards
1
Q
GPCR SIGNALLING
A
- Ligand –> receptor –> conformational change
- GDP exchanged for GTP in α unit –> α unit dissociate from βγ unit
- GTP α unit target protein –> relay signal to other components of signal cascade
- α unit hydrolyse GTP –> GDP = inactivate subunit – this is accelerated by RGS
- Inactive α unit reforms with βγ complex – turn off downstream events
2
Q
GPCR signalling inactivation
A
Arrestin – inactivates receptor by preventing receptor interactions with G proteins.
- Prolonged stimulation –> receptors inactivate – receptor kinase (GRK) phosphorylates receptor –> when phosphorylated arrestin binds – inactivates receptor, no interaction with G proteins.
- Receptors into endosomes and be degraded by lysosomes
3
Q
Receptor tyrosine kinase function
A
Control cell proliferation, differentiation, survival, metabolism, migration
4
Q
Receptor tyrosine kinase signalling
A
- Ligand bind to receptor –> dimerization
- Intracellular tyrosine kinase domains activated –> phosphorylate each other
- Phosphorylated tails recruit adapter protein – Grb-2 & Ras GEF
- Activate Ras protein at the plasma membrane –> ser/thr cascade of phosphorylation
- Phosphorylate MAPKKK –> MAPKK –> MAPK –> phosphorylate bunch of effector proteins
- Proliferation/cell fate determination
5
Q
Serine threonine kinase signalling
BMP
A
- Ligand bind to receptor in cell membrane –> receptor subunit 2 to phosphorylate unit 1
- Subunit 1 phosphorylate intracellular smad protein –> bind to another smad protein –> transcriptional regulatory complex
- Enters nucleus –> activate or represses target genes
6
Q
Absence of Hedgehog signalling
A
- Membrane protein Patched inhibits the membrane protein Smoothened
- Inhibition of smoothened –> transcription factor Ci being held in cytoplasm
- Ci held in 2 protein complexes. 1 with smoothened, and other with protein suppressor of fused (Su(fu)) - Ci in smoothened complex – phosphorylated by 3 protein kinases – PKA, GSK-3, CK1
- Phosphorylation –> cleavage of Ci –> truncated CiRep
- CiRep –> nucleus and repress Hedgehog target gene
7
Q
Prescence of Hedgehog signalling
A
- Hedgehogs bind to patched membrane protein – remove inhibition and block CiRep prod.
- Smoothened is phosphorylated by PKA and CK1
- Ci is released from both complexes in cytoplasm –> enter nucleus – act as gene activator
- Genes activated: wingless, decapentaplegic, and engrailed
8
Q
Canonical Wnt Signalling
A
- Wnt absent, β-catenin bound by GSK-3β, CK1-γ (Destruction complex)
- protein kinase phosphorylates β-catenin – target for ubiquitination & degradation in proteasome = no β-catenin free to move into nucleus
- Absence of β-catenin = transcriptional co-repressors bind to TCF transcription factor
- Prevent expression of certain genes
9
Q
Presence of Wnt Signalling
A
- Wnt bind to GPCR Frizzled
- Signal across membrane by Frizzled and LRP, activating them
- Activation of Frizzled and LRP cause kinases, GSK-3β, CK1-γ to associate with membrane
- Protein kinases phosphorylate the tail of the activated LRP
- Intracellular signalling protein Dishevelled and protein Axin are recruited to the cytoplasmic tails of LRP and Frizzled.
- Prevent formation of destruction complex –> β-catenin accumulate in the cytoplasm
- β-catenin –> nucleus and bind to TCF, displacing co-repressors –> genes expressed
10
Q
Intracellular signalling protein Dishevelled in PCP pathway
A
Dishevelled –> Rho GTPases in cytoplasm – activate proteins that modulate cytoskeletal proteins – affect actin myosin cytoskeleton – affect shape/polarity of cell
11
Q
Intracellular signalling protein Dishevelled in Ca2+ pathway
A
Dishevelled –> control molecules willing to release Ca2+ in the cytoplasm – pathway modulating activity of lots of proteins that combine to Ca2+ depends on Ca2+ for activity.
12
Q
Wnt antagonists
A
Sfrps
Dkk1
Wif1
13
Q
Sfrps
A
structure like frizzled - bind to Wnt - sequester it away from frizzled. Can bind to frizzled and interrupt wnt binding.
14
Q
Dkk1
A
binds to LRP, sequester it away from frizzled and Wnt – cannot recruit LRP – pathway not activated
15
Q
Wif1
A
Bind to Wnt and sequester them
16
Q
Notch active signalling
A
- Ligand binds to extracellular domain – then extracellular domain cleaved
- Cleavage of Notch intracellular domain by enzymes of the Presenilin complex – then released
- Intracellular domain translocated to nucleus
- Bind to CSL complex –> activated, releasing repressor
- Co-activator protein Mastermind is recruited, alongside other co-activator and they bind to complex –> target genes expressed
17
Q
How is Cap structure formed
A
7 methyl guanosine - protect from nuclease degradation.
- Removal of 5’ terminal phosphate (triphosphate)
- Addition of 5’ terminal GMP (Guanyl transferase)
- Methylation of guanine base (Guanine 7 methyl transferase)
- Methylation of ribosome (in some cases)
18
Q
How Polyadenylation occurs
A
- Recognition of AUAAA seq. by specificity components RNA cleavage by cleavage factors
- Initial poly(A) polymerisation by poly(A) polymerase – then binding of PABP
- More poly(A) polymerisation and binding of more PABP
19
Q
Steps of Initiation in protein synthesis
A
- Assembly of 43S pre-initiation complex
- Binding of 43S complex
- Assembly of 80S initiation complex
20
Q
How is 43S pre-initiation complex formed
A
- eIF2/GTP have affinity for Met-tRNA –> ternary complex
- 40S + eIF3 = 43S subunit – cannot reassociate with 60S
- 43S + ternary complex = 43S pre-initiation complex
21
Q
Structure of eIF2
A
- GTP binding site – on γ subunit
- Phosphorylation site – on α subunit, ser 51 – (point of regulation)
- K boxes – on β subunit, involved in interaction of eIF2B and eIF5
22
Q
How does mRNA bind to 43S complex
A
- eIF4E – recognises 5’ CAP on mRNA
- eIF4G – recognise eIF4E and binds to eIF4E, eIF4A and eIF3 – and bind to PABP
- eIF4A – help unwind secondary structure in 5’ end of mRNA
- eIF3 – bridge between eIF4G and 40S
- 4e-bp1 = regulator
23
Q
CAP independent translation
A
Internal ribosome entry site (IRES) - viral mRNA
Close to start codon.
Binding sites for 43S pre-initiation complex - direct binding of 43S complex to mRNA without need for CAP recognition of eIF4E
24
Q
Steps in Elongation
A
- AA-tRNA binding – catalysed by eEF1, requires GTP
- Peptide bond formation – catalysed by ribosome
- Translocation – catalysed by eEF2, requires GTP - movement between A, E, P sites
- Amino acyl site encounter stop codon – recognised by eRF (release factor) - allow formation of last peptide bond and chain released
- Dissociation of 2 ribosomal subunits from mRNA
25
4E-BP1
Tumour suppressor molecule.
• Found in cytoplasm in highly phosphorylated state
• Dephosphorylation = inhibit protein synthesis
26
EIF2
Active in dephosphorylated state = initiate protein synthesis
Phosphorylation = inhibit protein synthesis
27
EIF4G
cleaved by Caspase 3 = inhibit protein synthesis
| - Because there is no longer a bridge between EIF4G and 40S
28
Fate 1 - post translational protein targeting
* Synthesis of protein occurs in cytoplasm and is released into cytoplasm
* Transported to other organelles
29
Fate 2 - post translational protein targeting
* Subset of proteins will carry an ER signal to surface of endoplasmic reticulum
* Synthesis resumes proteins and insert them into ER – depending on destination, will remain in ER or transported to Golgi Apparatus and destinations in secretory pathways
30
Nuclear import of proteins
1. Complex forms - cargo protein + NLS associate with importin α and importin β --> trimeric complex
2. Can interact with nuclear pore and enter nucleus. Inside lumen - interact with protein Ran – Ran in nucleus is complex with GTP
3. Interaction with Ran causes complex to dissociate and cargo protein is released and enter the nucleus - Ran has high affinity for importin β so displace importin α
4. The products are exported from nucleus back into cytoplasm
5. Once in cytoplasm Ran hydrolyses GTP into GDP --> conformational change – Ran cannot interact with importin β, and complex dissociates – so undergo another round
6. Ran important in nucleus so transported back into nucleus – switch from GDP form to GTP form
31
Protein import into mitochondria
1. Signal sequence on mitochondrial protein must interact with receptor
2. Receptor on outer membrane of mitochondria – forms part of TOM complex – protein interacts with receptor and is handed over to channel component of TOM complex
3. Protein inserted into membrane by TOM complex - protein threaded through channel --> transverse outer membrane
4. Then interacts with complex in inner membrane = TIM23
5. Once it enters the matrix the signal sequence on the protein is cleaved off by signal peptidase
6. Mature mitochondrial protein can do its work/function
32
3 sources of free energy in mitochondria
Cytosolic Hsp70
Membrane potential in inner membrane
Mitochondrial Hsp70
33
How is free energy produced from cytosolic Hsp70
* When mitochondria proteins are 1st synthesised in cytoplasm they associate with protein – Cytosolic Hsp70
* Cytosolic Hsp70 = ATPase – for protein to enter TOM complex – Hsp70 must associate – requires hydrolysis of ATP
34
How is free energy produced from membrane potential in the inner membrane
* Protons are pumped from matrix into intermembrane – gradient is used to harvest/generate ATP
* Gradient is also important to Import the transport of the protein to the inner membrane – when this occurs ATP can’t be produced at the same time
35
How is free energy produced from mitochondrial Hsp70
* Binds to protein as it emerges in the matrix – goes through the process of binding and dissociating
* Dissociating requires hydrolysis of ATP
36
Insertion of proteins into inner membrane - mechanism 1
a. Signal sequence emerged from matrix = cleaved off
b. The downstream there is a hydrophobic domain and acts as a transmembrane domain
c. Protein laterally exit TIM23 complex – and anchors protein onto inner membrane
37
Insertion of proteins into inner membrane - mechanism 2
a. Protein anchored on inner membrane
b. Protein inserted into matrix - protein interacts with OXA complex
c. OXA complex mediates the insertion into the intermembrane after reaching matrix
38
Insertion of proteins into inner membrane - mechanism 3
a. Protein inserted in the intermembrane space and then inserted into TIM22 complex on the inner membrane
39
Secretory pathway of proteins - co-translational protein targeting to the ER
1. MRNA translation begins. Ribosome encounters the translation initiation codon
2. Sequence at the end of the terminus of ER proteins that determine the subsequent localisation – 1st part emerges from ribosome when synthesis begins – signal peptide emerge causes translation to stalls till interact with SRP
3. Signal recognition particle (SRP) interact with ribosome complex and mediate the docking of complex onto complex of proteins that sit on the ER - will include an SRP receptor
a. Ribosome dock on channel = peptide translocation complex
4. Ribosome dock – translation resumes – protein is then inserted through channel into the lumen of ER
a. Once protein is in the lumen of ER – signal peptidase cleaves off the signal sequence
5. Ribosome has docked – SRP can dissociate and undergo a new cycle of interaction – dissociation requires the hydrolysis of GTP into GDP
