Signalling Flashcards
How many mitochonDrial proteins are there?
13
Is targeting to the nuceloplasm via importin reversible?
Yes
What happens to protein going to the mitochondria and peroxisomes?
Will stay there
What do chaperones do?
Recognise signal sequences and convey the proteins to specific destinations either before or after ER translation
How do proteins get to the nuceloplasm?
Travels with importin then binds to recognition sequences called nuclear localisation sequences
How do proteins get to peroxisomes?
Travels with a targeting signal called SKL to a peroxisome targeting signal 1 receptor
What does vesicle transport allow proteins to do?
Retain their topology
What signals for transport to lysosome?
Mannose-6-phosphate
Which side of the Golgi is in and which is out?
In = cis-Golgi and out = trans-golgi
What is defective in Parkinson’s disease?
Ubiquitin tagging and protein degradation by proteasome in defective proteins
What does SRP do?
Co-translocational recognition of signal peptide, arrests translation, conveys ribosome/nascent peptide to SRP receptor in ER, associates with sec61 protein pore translocon, then translation resumes and SRP is recycled
What happens after the SRP is recycled?
Topology defined and checked, N-terminal signal peptide cleaved, folded and (Asparagine N-linked) glycosylation, integral proteins pass into bilayer
Why is the N-linked glycosylation in the ER for?
Address label to Golgi
What happens to the CF- ion channel?
Enters ERAD pathway
What is the coat protein II complex for?
ER > golgi
What is the COPI complex for?
golgi > ER
What are rabs proteins?
Small GTPases recognised by tethering proteins on the target membrane to make sure vesicle fuses with the correct membrane, anchored by lipid tail to membrane
What does Legionnaires bacteria do?
Produces rab-modifying enzyme that prevents phagosome-lysosome fusion so bacteria grows and isn’t degraded
Where are the V snares and T snares?
V snare on vesicle, T snare on target membrane
How do ER resident proteins get back from the golgi to the ER?
Have a KDEL sequence and golgi has a KDEL receptor so they are coated with COPI and sent back to ER
How are proteins at ER released in vesicles?
COPII and cargo proteins form localised accumulation and membrane shape change, coated vesicle goes along microtubules, rabs tethering proteins then SNAREs
How does late endosome have acidic lumen?
Protons pumped in
How does proteins get to the lysosome?
Modified glycosylation (M6P), M6P receptor, recognised by AP-1 and GGA adaptor proteins (adaptins), clathrin binds to the adaptors and coats outside of vesicles
What is the structure of the M6P receptor?
Transmembrane
What is lysosomal storage disease?
Lysosome enzymes don’t work so you get accumulation of undigested molecules
What is enzyme replacement therapy?
Add M6P to the broken enzyme and give it intravenously and it will be transported to lysosome as it will bind to M6P receptor on the surafce
What disease is treated by enzyme replacement therapy?
Gaucher
What are clathrin-coated vesicles important for?
Endocytosis - ingestion/invasion of pathogens, uptake of nutrients eg Fe2+, cholesterol, internalisation of PM proteins
What is familial hypercholesterolaemia?
Where cells can’t take up cholestrol from plasma so large amount in plasma, mutations in LDL receptor (no protein, trapped in ER, fails to bind LDL, defective signal sequence)
How does cholestrol uptake by clathrin-coated pits work?
RECEPTOR MEDIATED ENDOCYTOSIS - LDL > LDL receptor > internalised in clathrin-coated vesicle > fuse with endosome - acidic so LDL and receptor dissociate > receptor recycled and LDL goes to lysosomes to release free cholesterol
What can Ca2+ channels treat? How?
Angina, cardiac arrhythmias and high BP - prevent overstimulation
What does diazepam do?
Inhibits GABA receptor function (GABA is receptor for an inhibitory neurotransmitter)
WHat’s the difference between VG Ca and Na pore size?
The same but only Ca is selective
How does the Ca2+ channel work?
Pass in single file, one dislodged and passes through pore when Ca2+ binds alongside it, the negative glutamates have high affinity to Ca2+
How does the Ca2+ channel open when the membrane depolarises?
Electrostatic respulsion - a charged voltage centre in the 4th transmembrane helix of each of the four domains
How does the beta adrenoceptor increase HR?
B-adrenoceptor > G protein > adenyl cyclase > cAMP > PKA > phosphoproteins > increased HR
How does receptor tyrosine kinase recruit signalling proteins with SH2 domains?
Activated RTK > dimerises > allosteric activation of kinase domain > phosphorylation of receptor on tyrosine residues > recruit signalling proteins with SH2 domains
Which kinase do RTKs act through?
PI3 kinase
What does PI3 kinase do?
Phosphorylates PIP2 at 3 position of inositol ring to make PIP3
How many receptor subunits does insulin activate?
2
How does PIP3 coordinate responses?
Via Ser/Thr protein kinase akt - less GSK3 to increase glycogen synthesis, FOXO (transcription factor) inhibits gluconeogenesis GluT4
Membrane + glucagon + ATP + GTP = ?
cAMP
How do you see ATP > cAMP?
Use radioactive ATP
What does GPCR do?
Catalyses G protein activation by promoting GDP release - it’s an enzyme which stabilises transition state
What is the rate-limiting step in a GPCR?
GDP release
What are some properties of allosteric receptors?
Selective recognition of stimulus, transmembrane signalling, recruitment of intracellular signalling proteins, amplifying and integrating
What happens in male precocious puberty?
GDP dissociates rapidly so G proteins too active
What are the three subunits in a G protein?
Alpha which comes apart from beta-gamma
Which subunit has the GTP/GDP?
Alpha
What does alpha-s do?
Stimulates adenyl cyclase
What does alpha-i do?
Inhibits adenyl cyclase
What does alpha-q do?
Stimulates phospholipase C
What does alpha-12 do?
Regulates cytoskeleton
What does cholera do?
Blocks GTPase of alpha-s so persistent GTP and lots of cAMP
What does pertussis toxin do?
Block receptor coupling to alpha-i
How does the guanine nucleotide switch work?
Has channel for binding - when GTP binds the switch clamps gamma phosphate and produces conformational change
What happens to the beta-gamma when GTP binds?
Dissociates and unmasks face of beta-gamma that can interact with effectors
What can beta-gamma subunit stimulate and inhibit?
Stimulate PLC and K+ channel, inhibit Ca2+ channels
How does alpha-GTP and beta-gamma coordinate a decreased heart rate?
alpha-i-GTP causes decreased cAMP which inhibits Ca2+ channel and beta-gamma stimulates K+ and Ca2+
What opens the GDP cavity?
Change in alpha subunit
Where does G protein insert?
Binding of stimulus opens hydrophobic cleft between cytosolic ends of membrane spanning helices into which G protein inserts
How many transmembrane helices are there?
7 (N on outside, C on inside), membrane topolgy conserved e.g. rhosopsin, mACh receptors, adrenaline, glucagon, odour receptor
WHy is adenyl cyclase regulated by several interellular signals?
So can assimilate different signals and return outcome as cAMP
What does increased Ca2+ do to the different AC classes?
Inhibits AC1, activates AC5, no effect on AC2
How does alpha-s GTP affect the adenyl cyclase classes?
Activates them all
alpha-s + no Ca2+ = ?
AC1
alpha-s = ?
AC2
alpha-s + Ca2+ = ?
AC5
What does cAMP target?
cAMP-dependent PKA
What converts cAMP to AMP?
Cyclic nucelotide phosphodiesterase
What effects does AMP cause?
Protein kinase phosphorylates ion channel to activate
What inhibit phosphodiesterases?
Caffeine and theophylline in inhalers
What activates and inhibits PDE3?
Inhibited by cilostazol (to treat peripheral vascular disease), activates by PKA
How is PKA activated?
WHen cAMP causes pseudosubstrate domain to swing out of catalytic site and allow substrate access
WHat does PKA phosphorylate?
Ser or Thr
Is protein phosphatase regulated?
No - on all the time
What activates a protein by phosphorylation?
PKA
What deactivates a protein by dephosphorylation?
Protein phosphatase
What are AKAPs?
Scaffold proteins that organise cAMP signalling proteins
What does AKAP stand for?
A kinase anchoring protein
What do AKAPs do?
Bind component protein kinases of a signalling cascade so they can only interact with each other
What do AKAPs have interaction sites for?
Additional signalling proteins
What activates PLC?
G protein
What does DAG activate?
PKC
How does GTP make GMP?
Like cAMP - GTP > guanyl cyclase > cGMP > PDE > GMP
PIP2 > ?
IP3 + DAG using PLC
IP3 > ?
Inositol + phosphatidate
What happens to the phosphatidate?
Recycled to PIP2
What/where is PIP2?
Minor lipid in plasma membrane
What catalyses PIP2 hydrolysis?
PLC
Why is Ca2+ a good signalling molecule?
Versatile, robust, safe, no cytotoxicity, reliable digital signal, directed to specific targets
What regulates Ca2+?
IP3R and IP3 - so regenerative propogation
Endomembrane system = ?
ER + Golgi + Lysosomes
How does IP3 stimulate Ca2+ release?
Through an ion channel (the IP3 receptor)
What does the chaperone do?
Prevents aggregation and keeps protein unfolded for translocation through import pore before or after complete translation
What controls the “on” and “off” reaction?
Ca2+ = off reaction, IP3 receptor = on reaction
What happens as protein emerges from ribosome?
Recognised by signal recognition particle, undergoes conformational change to stop translation, protein moves to SRP receptor in ER
What cleaves the N terminal signal peptide?
Signal peptidase
At the SRP receptor, what does the protein associated with?
Protein pore called a translocon at sec61, SRP released and recycled
Where is the protein topology defined?
ER lumen
When translation resumes, where does the N terminus of the protein go?
Into ER lumen
What recognises the pre-protein signal sequence at the N terminal?
Chaperone
What is the N terminal signal sequence?
Hydrophobic core with hydrophilic residues, forms alpha helix in non polar environment
WHat is the address to Golgi?
Asparagine (N)-linked glycosylation
What do COPII coat proteins bind to?
Diacidic or dihydrophobic sequences
What are SNAREs?
Long helices which forms leucine zippers
What do hydrophobic sorting signals on proteins for the inner membrane do?
Stops translocation in the inner membrane
What happens at TIM 23?
Matrix proteins and IMS proteins with N terminal cleavable sequences
What happens at TIM22?
Hydrophobic inner membrane proteins with internal targeting sequences - either released laterally or imported into matrix using PAM
Are proteins folded or unfolded through TOM and TIM?
Unfolded
What is PAM?
Presequence translocase-assisted motor
What happens in Huntington’s disease?
Mutant Htt associates with TIM23 complex and inhibits protein transport into brain mitochondria
How does the mitochondrial sequence bind to chaperones?
Alpha helix and hydrophobic on one side
What does the mitochondrial protein chaperone do?
Chaperone = Hsp70, requires ATP hydrolysis to release protein, has targeting signal which binds to receptors on mitochondrial surface then directed to subcompartment
What is the sequence for mitochondira?
+ve, hydroxylated amino acid
WHat happens if protein is destined for matrix?
Targeting sequence is cleaved and mitochondrial Hsp70 (chaperone) helps to refold it into matrix
What do lysosomal proteins do?
Bind to M6P receptor and forms clathrin coated receptor
What is clathrin?
Trimer made of three light and three heavy chains, the three legged stucture is a triskelion
What does the M6PR sorting signal in tail region do?
Recognised by AP-1 and GGA adaptin proteins, clathrin binds to these and coats vesicles, targets vesicles to late endosomes
What is mucolipidosis?
Lack of sorting of proteins to lysosomes, mutation in NAG phototransferase which adds M6P tag, hydrolases secreted intead of going to lysosomes, lysosomes of connective tissue has inclusions of GAGs and glycolipids so not degraded
What are the two layers on secretory vesicles?
Inner is AP (adaptor protein), outer is clathrin
What happens in Parkinsons?
Mutation in ERAD meaning incorrect proteins aren’t stabilised which accumulate and damage cells, a mutation in Parkin gene which acts as a UB ligase and prevents accumulation/aggregation of misfolded protein
What do malfunctions in Hydrolase Acid Alpha Glucosidase do?
Usually does glycogen to glucose, malfunctions cause glycogen buildup which damages cells
Is the N-terminus ER localisation signal hydrophilic or hydrophobic?
Hydrophobic
When is the N-terminus ER localisation signal recognised by SRP?
While still being synthesized (co-translational transport)
What blocks translation in the cytoplasm?
SRP and ribosome-protein-SRP complex > SRP receptor
What happens in the ER lumen?
Signal sequence cleaved, protein folded by chaperones and disulphide bonds form if secreted or external facing, N-linked glycosylation for external/secreted
What happens during N-linked glycosylation?
Sugars added to Ash side chains
What happens during the ERAD pathway?
Misfolded proteins exported by retro-translocation into cytoplasm, Ub-ligase adds ubiquitin to ligase, cytoplasmic proteasome recognises Ub tag and degrades
WHich sorting signals does AP-1 recognise?
Tyr-X-X-bulky hydrophobic
Whoch sorting signals does GGA recognise?
D-X-X-LL
What does the N terminal signal for mitochondrial targeting contain?
Positive and hydroxylated AA
WHy are there M6P receptors on the plasma membrane?
To catch proteins for lysosome if they’ve entered secretory pathway
What is an EPAC?
A guanine nucleotide exchange factor which activates G proteins (becomes active by binding to cAMP). Swaps GDP for GTP and activates Rap which can then activate PLC
