Cell Signalling And Protein Sorting Flashcards
How many cells are in the body
10^13
How thick is the plasma membrane
2 molecules thick
How do lipophilic substances pass across the plasma membrane (PM)
They dissolve in the PM and so can pass through it
How do channels work
Allow materials to flow downhill into or out of a cell
What does it mean for production if chemical signals are lipophilic
They must be made on demand as they cannot be retained
What do transporters do
Export chemical signals from cytosol across PM
Give 3 ways for cells to receive a signal (ie get across the PM)
Lipophilic pass through the PM to reach intracellular targets
Extracellular messenger may be recognised by a binding site in a channel (to open or close it) and the flux of ions across PM changes
Allosteric PM-spanning protein (where intracellular side of protein changes shape)
3 ways to send our cellular information
Diffusion across PM
transporters
Vesicles
How is the nucleus separated from the cytosol
By a double membrane penetrates by nuclear pores
What is the ER and what does it contain
Endoplasmic reticulum
A lumen where proteins mature and Ca2+ is stored
The ER also contributes to lipid and steroid synthesis
Why is rough ER rough
It is studded with ribosomes
Describe the Golgi apparatus
Stacks of tubules linked to the ER
it has an important role in maturation/ glycosylation of proteins and their dispatch
What are almost all proteins encoded by
What is the exception
Nuclear genes
Those encoded by the small mitochondrial genome
What is the overall process of making proteins
Their DNA is transcribed into mRNA which is processed in the nucleus before export through the nuclear pores. Within the cytosol, mRNA provides the template for synthesis of proteins by ribosomes
How many genes are mitochondrial
37
How can disease caused by defects in mitochondrial genes be avoided
Use an enucleated egg from a surrogate with normal mitochondria to serve as the host for parent’s DNA/nucleus
Where do most proteins begin there life
Then what happens
On a cytosolic ribosome
Address labels in the primary structure dispatch proteins to different destinations
How do fully folded proteins re-enter the nucleoplasm
Nuclear localisation signals allow them to move Through nuclear pores
If the protein does not stay in the cytosol or go to the nucleoplasm, what happens to them
They must cross a membrane and therefore must stay unfolded
What is post translation targeting and where does it occur
When the protein has been fully translated but doesn’t fully fold until it has crossed the membrane
Mitochondria and peroxisomes
What is co-translational targeting
When proteins destined for the ER are dispatched before translation is complete
What is a NLS
A nuclear localisation signal: a stretch of 6 +/ve residues anywhere in the primary sequence, recognised by importin
How can nuclear translocation be regulated
By unmasking a NLS
How do proteins reach the peroxisome
When does this go wrong
The C terminal sequence (Serine-Lysine-Leucine) is recognised by PTS1 receptors and guides it to peroxisome membrane
In Zellweger Syndrome, when the PTS1 is non functional
Why is mitochondrial targeting complex
There are 4 destinations:
Inner and outer membranes, intermembrane space, and matrix
How do cells target for mitochondrial matrix proteins
N-terminal amphipathic helix is recognised by a chaperone protein
Is targeting for peroxisomes and mitochondria reversible
No
It is irreversible
How is co-translational targeting used for proteins to the ER
A hydrophobic signal sequence at the N terminal (for luminal proteins) or internally (for integral membrane proteins) is recognised by a large protein-RNA complex
What are ER proteins from co translational targeting recognised by
Signal Recognition Particle (SRP)
What happens when SRP recognises a protein
The protein’s translation stops so SRP-nascent peptide chain can associate with the SRP receptor on the ER membrane
What does the SRP receptor associate with? What does it do when this has happened?
Translocon (a protein channel in the ER membrane)
Checks the signal sequence of the peptide
What happens once the SNP receptor has checked the peptide
The nascent peptide chain is inserted into the translocon and SRP is released for reuse.
Protein synthesis continues, threading the growing protein through the translocon
What is important when the nascent peptide is inserted into the translocon
It is the right orientation as this cannot be changed later
The translocon continues to survey the growing protein. What is it looking for and what happens if it is found?
Stretches of hydrophobic residue
Retained in the translocon to be formed into membrane spanning domains
What happens when the growing peptide emerges into the ER lumen
It is further scrutinised and a protease cleaves any N terminal signal sequence.
Chaperone proteins pull it into the lumen and help it fold and help form disulphide bonds for example
Give a summary of co-translational targeting of proteins to ER
Recognition of signal peptide by SRP
SRP stops translation
SRP conveys peptide to SRP receptor in ER
SRP is recycled while translation continues and protein is pulled into lumen to be defined and checked
What happens to faulty proteins in the ER
What proportion are faulty
They must be removed as they may aggregate/ clog the system
> 30% of all ER proteins are sent for degradation
How are faulty proteins removed from the ER
ERAD allows them to be sent back to the cytosol through a pore associated with ubiquitin ligases
How are faulty proteins marked for degradation
Ubiquitin ligase attaches ubiquitin to Lys residues on the protein
What is defective in Parkinson’s disease
Parkin- a subunit of ubiquitin ligase
What is the most common cause of Cystic fibrosis
CFTR is degraded by ERAD before it reaches the PM where it should modulate Cl- transport
How is ΔF508 treated
Drugs help the CFTR avoid ERAD and reach the PM
What does cytomegalovirus do
Hijacks ERAD pathway by expressing a protein that associates with MHC (which usually indicates that a cell is infected)
By targeting MHC for degradation, the virus remains undetected
What happens to proteins that were folded and N- glycosylated in the ER
Collected into COPII vesicles and conveyed to the cis Golgi, where the sugar structures are modified
What happens in the Golgi to proteins destined for lysosomes
The sugars are modified to include mannose-6-phosphate (M6P)
How does all trafficking from the ER occur
In vesicles
What is one of the most important organelle identity label?
Describe it
Rabs
Small G proteins that are active when GTP is bound and inactive with GDP bound
Describe rab cycle
Rab-GDP is recruited by an intracellular membrane but only activated if the membrane has the correct proteins for GDP to swap for GTP.
when another organelle recognises the rab-GTP it will hydrolyse it if it has the correct proteins. This hydrolysis releases the rab and GDP for the cycle to restart
Other than rabs, what else provides identity labels
Lipids
What do COPII proteins do
Bind to a specific Cytosolic sequence of proteins in the ER membrane, collecting them into clusters to be cut off as small vesicles coated in COPII
What happens to COPII vesicles
They travel along microtubules to the cis Golgi and shed the COPII coat to reveal their identity labels
Why must the COPII cost be shed
What do SNARE proteins do
COPII must fuse with cis Golgi but not other organelles
Mediate fusion of 2 organelles by drawing their membranes close together
What do COPI do
COPI vesicles carry cargo from Golgi back to the ER
The cargo is recognised by a KDEL sequence which binds to a KDEL receptor.
What is the sequence after the KDEL binds to the KDEL receptor
Cargo is concentrated, pinched off into vesicles, uncoated for recognition and then the vesicles fuse with the membrane
What sorts protein to different destinations
Trans Golgi
How are proteins destined for lysosomes recognised
When do they detach from this
M6P
When they reach the late endosomes, there is a low pH causing the M6P receptors to dissociate and the cargo passes on to the lysosomes
What happens to the M6P receptor when it is dissociated
Sorted into vesicles and returned to the trans Golgi
What is Gaucher disease
How can it be treated
An enzyme is missing meaning M6P receptors are imperfectly targeted
An M6P modifies form of the enzyme can be given and it will be endocytosed by the M6P receptor and delivered to lysosomes
What are the common themes in protein trafficking
Segregation of cargo and coating
Vesicle formation and transport
Uncoating and recognition
Fusion
What is familial hypercholesterolemia
What causes it
Where plasma cholesterol levels are too high
Mutation in LDL receptors so LDL is trapped in the ER and there is no signal sequence
What is “bad cholesterol “
LDL
How is LDL taken up
LDL receptors bind to ApoB-100 component of LDL
Cytosolic side of receptor interacts with AP2 which interacts with clathrin
Clathrin gathers the LDL receptor and cargo into a clathrin Coated pit which is trafficked to endosomes
In the acidic endosome lumen, LDL dissociates from the receptor and and LDL passes to lysosomes where ApoB-100 is degraded
Where do protein trafficking pathways begin and converge
In the ER
or at the PM
Both converge at the Golgi
What are the only signals that enter the cell to regulate activity
Lipophilic signals
All other signals are specifically recognised at the extracellular surface of the membrane spanning receptor and the signal is transmitted inside the cell
2 ways that extracellular signal recognition transmits a signal to the interior
Receptor forms an ion channel
Allosteric proteins, which transmit conformational changes
What mediates the faster form of intercellular communication
Ion channels (they might allow several million ions to pass downhill each second)
What part of the cell does the ion fluxes of APs affect?
Membrane potential, NOT intracellular activity
How is intracellular activity regulated in a neuron
Membrane potential changes must be transduced into Ca2+ which enters the cell
What are nicotinic ACh receptors in muscle permeable to
What do they respond to
Na+ and K+
ACh
How is myasthenia gravis
Drugs that interact with nicotinic ACh receptors
Where are GABA and glycine receptors found and what are they permeable to?
CNS
Cl- permeable
What js the common structure of voltage gated cation channels
24 membrane spanning regions around a central pore
For Na+ and Ca2+ channels what is the channel formed from
What about for K+
A single protein
4 proteins
Describe the selectivity filter of Ca2+ channels
2 Ca binding sites which both can bind to Ca tightly
When both are occupied, electrostatic repulsion between them causes one to be dislodged
How are Ca channels selective against monovalent cations
The repulsion would not be enough to dislodge the other bound Ca
How do VG Ca channels close
Residues at the Cytosolic tip of the pore linking helix come together like the top of a teepee, occluding the channel
How are VG Ca channels opened
A voltage sensor moves outwards when the membrane depolarises
This pulls on a short Cytosolic helix that links the voltage sensor to the TMD forming the pore
This twists the pore helices, opening up the ‘teepee’
Give some general features of signalling pathways (4)
Allostery
Amplification
All steps are reversible
Integration
What is an easy means of reversibly regulating activity
How often is this used
Phosphorylation
50% of all cellular proteins are phosphorylated
How common is tyrosine phosphorylation
rare, but important (0.05%)
How many tyrosine kinases in man
85
How is receptor tyrosine kinase activated?
What happens after activation ?
Activation begins with binding of dimeric extracellular signal, which causes the receptor to dimerise
Dimerisation activates Cytosolic tyrosine kinase, allowing each subunit of receptor to trans-phosphorylate Tyr residues on the other
This allows the proteins which can bind to phospho-Tyr to dock here and be recruited for signalling complexes
What is the insulin receptor structure?
What happens when insulin binds?
Dimeric
Re-arranges dimer so intracellular tyrosine kinase is activates to cause trans-phosphorylation
What happens after trans-phosphorylation in an insulin receptor
IRS-1 is recruited by phospho-Tyr
IRS-1 is further phosphorylated to provide docking sites for other proteins, such as PI3K
Active PI3K phosphorylates PIP2 to give PIP3 which is the scaffold for further recruitment
What does PIP3 do
Recruits Akt2 and PDK1, which both phosphorylate Ser and Thr
Recruitment to PM, causes Akt2 to change shape and becomes a substrate for PDK1 so at PM PDK1 phosphorylates Akt2
When is Akt2 maximally active
Why
In fed cells stimulated by insulin
It is phosphorylated by PDK1 after insulin binds and is further phosphorylated by mTORC2 which is activated only in the fed state
What does Akt2 do when activated
It causes further phosphorylation, leading to stimulation of glycogen synthesis, inhibition of glucose synthesis, and insertion of glucose transporters into the PM
2 classes of allosteric receptors
GCPRs and tyrosine kinase receptors
How do active GPCRs work
Catalyse activation of G proteins by causing them to release the GDP the have bound in the inactive state and replace it with GTP
The active GTP bound G protein then signals onwards
What features does the sequence from cAMP to activation of glycogen phosphorylase show
Amplification
Integration (cAMP both stimulates glycogen breakdown and inhibits glycogen synthesis)
Protein phosphorylation
What did Rodbell serendipitously show
That both ATP and GTP were required to allow liver membranes to make cAMP
What can be used to treat HIV using GPCRs
Maraviroc binds to the recognition site of CCR5 chemokine receptor so I cant bind to HIV coat proteins
HIV cannot then infect the T cells
What must HIV do before infecting cells
It’s coat proteins are recognised by a co-receptor in the host membrane, comprising a chemokine receptor (a GPCR) and a CD4 receptor (in T lymphocytes)
What does vasopressin do
Controls V2 receptor (a GPCR) which controls water reabsorption in the Kidney
How does the V2 receptor control water reabsorbtion
What does mutation cause
Through cAMP, causing insertion of water channels into the PM of collecting tubules
Congenital Nephrogenic Diabetes Insipidus
What does TSH do
What can mutations cause
Stimulates release of hormones from the thyroid
Constitutive activation of TSH receptor leading to hyperthyroidism
Is GDP dissociation from inactive G protein slow
Yes very slow
How do GPCRs assist G protein activation
Active GPCRs speed up GDP dissociation so GTP can bind instead
They are exchange catalysts
Why is the dissociation of GDP an important step in G protein activation
Where can this be seen
Rate limiting step
Boys with mutated G protein (in αs ) so GDP binds weakly, leading to spontaneous activation -> cool testes -> precocious puberty
Unless GDP dissociation is slow there is nothing for the GPCR to control
How are GPCRs exchange catalysts
Reduce AE of GDP dissociation from G protein for activation
Can activate many G proteins in its life time
How are G proteins deactivated
Intrinsic GTPase acts on a molecular clock. It will hydrolyse the GTP to deactivate the G protein
What contributes to reward effects of alcohol consumption in alcoholics
How do they signal
Stimulation of dopamine and GABAb receptors (GPCRs)
Through G protein Gi
What is the i for in Gi
Inhibit (inhibits cAMP formation)
What does RGS6 do
Enhances the GTPase activity of Gi
How are alcoholic mice different
What does this mean
RGS6 is up regulated so more intense stimulation is required for same effect
What treatment may help alcoholics
RGS6 knockout mice do not become alcoholic so drugs which inhibit RGS6 might treat alcoholics
Structure of G proteins activated by GPCRs
Trimeric
βγ subunits are inseparable and anchored to PM
α subunits bind and hydrolyse GTP
GDP is held by an inactive α in a pocket so GDP cannot escape
What does cholera do
Modifies αs to block GTPase activity resulting in over production of cAMP in gut endothelium
How does whooping cough occur
Pertussis toxin covalently modifies αi uncoupling G protein from GPCR
How do GCPRs promote G protein activity
Opening deep pocket containing GDP
What dictates whether a G protein is active
The 3rd P group on the guanine pushes apart Thr and Gly
What are the consequences of GTP binding and the trimeric G protein dissociating
GPCR is separated and can now activate another protein
The α-GTP and βγ can now regulate their effectors (α-GTP acts GTPase)
How is ACh related to GPCRs ?
ACh stimulates GPCRs in the heart
The response is mediated by the dissociation of subunits of Gi that slows heart rate and reduces force of contraction
What is the structure of a GPCR
What happens when activated
Extra cellular N terminus
7 TMDs
Cytosolic C terminus
Extracellular signal stimulates N and a cleft opens between Cytosolic ends of TMDs 3,5,6, and 7 where the α subunit of the G protein can insert, causing the GDP to be released
Are intracellular messengers usually restricted?
Yes they are usually restricted to the cell in which they were made
Different GCPRs converge to regulate intracellular activity through a limited repertoire of intracellular messengers
How is cAMP made
What is its most important target
Made from ATP by adenylyl Cyclases (AC) and inactivated by phosphodiesterases (PDEs)
Protein kinase A (PKA)
How many forms of AC are there
How are they activated and respond
9
Stimulated by G protein sub unit αs-GTP
different responses to other intracellular signals (including Ca2+)
What are most PDEs inhibited by
Caffeine and theophylline (used to treat severe asthma)
What is the target for Ciloztazol,
PDE3 to treat obstructed peripheral arteries
What does PDE3 do
It breaks down cAMP to AMP
It is found in vascular smooth muscle and is stimulated by PKA (cAMP therefore stimulates its own breakdown)
Give the structure of PKA
Tetramer: 2 Regulatory subunits which each bind 2 cAMP molecules
2 catalytic subunits which phosphorylate protein substrates
Each regulatory subunit contains a pseudo-substrate domain
How do PKA molecules work
How is their work undone
When R binds cAMP the subunits fall apart and the blocked active site (from the pseudo substrate) is revealed allowing the real protein to be phosphorylated
Protein phosphatases
What are AKAPs
Scaffold proteins that anchor R subunit on PKA
A Kinase Anchoring Protein
Give the structure of cGMP
Made by guanylyl cyclases
Degraded by PDEs and is mediated by PKG
What is the target of viagra
PDE5 which selectively degrades cGMP
How does Viagra work (in relation to cGMP)
Prevents degradation of cGMP in blood vessels of the penis, causing dilation and accumulation of blood
What is the substrate in the PLC pathway
What intracellular messengers does it provide
PIP2
DAG
IP3
PIP2 itself regulates ion channels so decrease in [PIP2] after PLC is a signalling mechanism
How is DAG an intracellular messenger
How is it deactivated
Remains in PM to activate PKC
Phosphorylation
What are phorbol esters
Why
Tumour promoting molecules
Look like DAG and activate PKC but cannot be degraded
Describe IP3
How is it deactivated and what is the final step of this degradation
Water soluble
Enters cytosol and stimulates Ca release from ER
By dephosphorylation
IP3 into inositol
What happens to deactivated products of PLC
Reunited and synthesised back into PIP2
What is used to treat bipolar? Why?
Lithium ions
Blocks degradation of IP1 to inositol
Blocking this reduces PLC signalling in over active brain areas by preventing reformation of PIP2
How is PLC (phospholipase 3) pathway activated
By GCPRs or PTKs
What did Roger Tsien develop
Ca indicators
How are Ca signals developed
How does this change as stimulus increases
As Ca spikes
Frequency increases
What are Ca spikes good for?
Protect cells from damaging effects of excessive increases in Cytosolic [Ca]
Allow digital signalling
Spatially organised Ca signals allow Ca from different sources to be delivered to different targets
Adipose tissue does not have glycerol kinase. What does this mean?
If glucose is low then glycerol-3-phosphate will be low. FFA cannot be re esterified and will be exported
Name an enzyme adipose releases
AMPK
What causes RAS to exchange GDP for GTP
SOS
Are λ phage vectors only used with cDNA libraries
No
Name a protein that recognises specific DNA sequences using its α helices
Fos- Jun
Name a monoclonal antibody that binds to EGFR
Cetuximab
What is linkage
When a pair of genes do not demonstrate independent segregation
Describe what happens when insulin binds to its receptor (up to PIP3)
Insulin receptor is a dimer and when insulin binds the dimer rearranges so trans phosphorylation of the tyrosine residues can occur.
Now IRS1 can bind to the receptor and be phosphorylated. IRS1 is now the docking site
PI3K binds to IRS1. PI3K is now active and can phosphorylate PIP2 to PIP3
Which subunit of PI3K allows it to bind to IRS1
it’s p85 adaptor subunit
After insulin has bound to its RTK and PIP3 has been made, what happens
PIP3 recruits Akt2 and PDK1 to the PM.
At the PM, Akt2 changes shape to become a substrate for PDK1. Akt2 is thus phosphorylated by PDK1 and mTORC2
Now that Akt2 is activate, it can recruits GLUT4 to the PM and increase glycogen synthesis
What are the actions of Akt2
Recruit GLUT4 to PM
Increase FOXO activity which inhibits gluconeogenesis
Reduce activity of glycogen synthase kinase, thereby increasing glycogen synthesis
Where does the G protein insert Into the 7 TMDs of the GPCR
Between the cytosolic ends of TMDs 3,5,6
Which G protein subunit has GTPase activity
α
Name 2 diseases related to Rab
Charcot Marie Tooth disease (rab7 mutation)
Legionnaire’s Disease (bacterial modifies rab1 to divert ER to a vacuole)
Which domain in GRB2 binds to the activates RTK
AH2 domain
What does RAF do
It is a protein kinase that is activated by binding to a small G protein
Name a amino acid directly involved in the urea cycle
Arginine
Give 4 glucogenic and 4 ketogenic amino acids
Ketogenic: tyrosine, leucine, tryptophan, isoleucine
Glucogenic: arginine and glutamine (these both make glutamate, which makes α ketoglutarate); methionine (which makes succinyl CoA) and phenylalanine (which makes fumarate)
How is HSL activated
phosphorylation by PKA activated by adrenaline
What is AKAP
Scaffold proteins that associate with PKA
What does PLC do
Phospholipase C
Hydrolysis PIP2 into IP3 and DAG
Which amino acid forms an isopeptide bond between ubiquitin and target protein
What is the ubiquitin ligase
Internal lysine
E3 enzyme
What is trisomy 18
Edward’s syndrome
What causes Leber Hereditary Optic neuropathy
A mitochondrial disease with matrilineal heritage
What is KDEL
C terminal sequence that ensures nascent soluble proteins are retained in the ER
K- lysine
D - aspartic acid
E - glutamate
L - leucine
