CMB Flashcards
GPCR SIGNALLING
- 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
GPCR signalling inactivation
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
Receptor tyrosine kinase function
Control cell proliferation, differentiation, survival, metabolism, migration
Receptor tyrosine kinase signalling
- 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
Serine threonine kinase signalling
BMP
- 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
Absence of Hedgehog signalling
- 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
Prescence of Hedgehog signalling
- 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
Canonical Wnt Signalling
- 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
Presence of Wnt Signalling
- 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
Intracellular signalling protein Dishevelled in PCP pathway
Dishevelled –> Rho GTPases in cytoplasm – activate proteins that modulate cytoskeletal proteins – affect actin myosin cytoskeleton – affect shape/polarity of cell
Intracellular signalling protein Dishevelled in Ca2+ pathway
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.
Wnt antagonists
Sfrps
Dkk1
Wif1
Sfrps
structure like frizzled - bind to Wnt - sequester it away from frizzled. Can bind to frizzled and interrupt wnt binding.
Dkk1
binds to LRP, sequester it away from frizzled and Wnt – cannot recruit LRP – pathway not activated
Wif1
Bind to Wnt and sequester them
Notch active signalling
- 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
How is Cap structure formed
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)
How Polyadenylation occurs
- 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
Steps of Initiation in protein synthesis
- Assembly of 43S pre-initiation complex
- Binding of 43S complex
- Assembly of 80S initiation complex
How is 43S pre-initiation complex formed
- eIF2/GTP have affinity for Met-tRNA –> ternary complex
- 40S + eIF3 = 43S subunit – cannot reassociate with 60S
- 43S + ternary complex = 43S pre-initiation complex
Structure of eIF2
- 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
How does mRNA bind to 43S complex
- 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
CAP independent translation
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
Steps in Elongation
- 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
4E-BP1
Tumour suppressor molecule.
• Found in cytoplasm in highly phosphorylated state
• Dephosphorylation = inhibit protein synthesis
EIF2
Active in dephosphorylated state = initiate protein synthesis
Phosphorylation = inhibit protein synthesis
EIF4G
cleaved by Caspase 3 = inhibit protein synthesis
- Because there is no longer a bridge between EIF4G and 40S
Fate 1 - post translational protein targeting
- Synthesis of protein occurs in cytoplasm and is released into cytoplasm
- Transported to other organelles
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
Nuclear import of proteins
- Complex forms - cargo protein + NLS associate with importin α and importin β –> trimeric complex
- Can interact with nuclear pore and enter nucleus. Inside lumen - interact with protein Ran – Ran in nucleus is complex with GTP
- 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
- The products are exported from nucleus back into cytoplasm
- Once in cytoplasm Ran hydrolyses GTP into GDP –> conformational change – Ran cannot interact with importin β, and complex dissociates – so undergo another round
- Ran important in nucleus so transported back into nucleus – switch from GDP form to GTP form
Protein import into mitochondria
- Signal sequence on mitochondrial protein must interact with receptor
- 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
- Protein inserted into membrane by TOM complex - protein threaded through channel –> transverse outer membrane
- Then interacts with complex in inner membrane = TIM23
- Once it enters the matrix the signal sequence on the protein is cleaved off by signal peptidase
- Mature mitochondrial protein can do its work/function
3 sources of free energy in mitochondria
Cytosolic Hsp70
Membrane potential in inner membrane
Mitochondrial Hsp70
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
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
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
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
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
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
Secretory pathway of proteins - co-translational protein targeting to the ER
- MRNA translation begins. Ribosome encounters the translation initiation codon
- 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
- 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 - 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 - Ribosome has docked – SRP can dissociate and undergo a new cycle of interaction – dissociation requires the hydrolysis of GTP into GDP
