4.2 Ubiquitin Flashcards
Ubiquitin and protein trafficking
Which pathway do proteins that are going to be secreted pass through?
What proteins can used this pathway in reverse
What pathway do proteins destined for the lysosome use
Proteins that are destined to be secreted pass through the secretory pathway in transport vesicles
Endocytosed plasma membrane proteins can also use this pathway secretory pathway in reverse
Proteins destined for the lysozome also use the secretory pathway to get to their final destination i.e to the lysosome
Ubiquitin and protein trafficking
What are we talking about today?
We are talking about membrane proteins, secretory proteins, from the ER through the golgi apparatus, where they undergo further post translational modifications they can do directly through to be endocytosed by the plasma membrane, also go through things in lysosome – also looking at things that are in the reverse
Ubiquitin and protein trafficking
What does the attachment of mono-Ub and K63 linked chains recruit?
What do GGA receptors in Golgi apparatus bind to? What does that result in?
Attachment of mono-Ub and K63 linked chains recruits ESCRT machinery (has Ub receptors in them) which directs the formation of clathrin coated vesicles (CCV)
GGA receptors in Golgi apparatus binds to ubiquinated proteins and direct them to MultieVesicularBodies (late endosomes)
Ubiquitin and protein trafficking
What is the pathway for the formation of the CVV
This is what has happened in terms of the formation of the CCV, effectively we have the membrane model, in this case the protein is a EGFR it has Ub attached to it via Ub-ligase called Cbl, this then recruits Hrs complex, which then recruits the ESCRT machinery which pinches of a portion of the plasma membrane and then also you get it released from the plasma membrane into a transport vesicle which contains ubiquinated form
Ubiquitin and endocytosis
What do ubiquitylation of a plasma membrane protein result in?
What can it be thought of as?
Ubiquitylation of a plasma membrane protein results in them being removed from the plasma membrane into vesicle
It represents a method for removing unwanted/damaged proteins
It can be considered an active regulatory system e.g transporter protein can no longer work if it is not at the plasma membrane
Ubiquitin and endocytosis
What has no role in this process?
Where are unwanted proteins trafficked too?
What is the common error in papers?
Proteasome has no role in the endocytosis process, the degradation part is happening in the lysosome
Unwanted proteins are trafficked to the lysosome for degradation
Common error in papers is where the use of a ‘proteasome inhibitor prevents degradation’
Ubiquitin and hypertension
What is hypertension?
What causes excess pressure on arteries and veins?
What is it a major risk factor for?
What are the risk causes?
Hypertension is high blood pressure
Increased pressure on arteries and veins is caused by increased blood volume or narrowing of blood vessels
It is a major risk factor for heart attack and death
The risk causes are both environmental (e.g diet) and genetic risk causes
Ubiquitin and hypertension
What timescales is blood pressure regulated over?
What is longer term regulation of blood pressure controlled by?
What is low blood pressure sensed by?
Blood pressure is regulated over both long and short timescales
For longer term regulation blood pressure is controlled by Renin-angiotensin system (RAS)
Low blood pressure is sensed by specialized receptor cells in the kideny, that activates peptide signalling cascade
Ubiquitin and hypertension
Low blood pressure peptide cascade
- Start with angiotensinogen when low blood pressure is sensed it is hydrolysed by renin
- It is then further hydrolysed by ACE (angiotensin conversion enzyme)
- Forming angiotensin-2 - which is the active hormone
- It is then degraded
- This is why it is called the renin-angiotensin system (RAS)
The active hormone: angiotensin-2
It acts in two ways to increase blood pressure
- It causes vasoconstriction by binding to arteriole, releasing Ca
- Angiotensin-2 binds to adrenal coretex wich releases second hormone called Aldosterone which releases sodium and increases blood volume and this blood pressure
The reason that we know ubiquitin is involved in blood pressure regulation is because of a disease called…?
What does it cause?
What patients is present it, what are they resistant too?
What drugs can be used to treat it?
Liddle syndrome
Early onset hereditary hypertension
Presents in patients that are resistant to standard front line therapies such as ACE inhibitors (Angiotensin-converting enzyme)
It is treatable with drugs such as Triamterene that directly inhibit sodium transport into blood stream
Liddle syndrome
Early onset hereditary hypertension
How many known familial forms?
Where do all the genes map onto?
Where to mutations map?
Around 30 familial forms are known
All map to genes encoding subunits of Epithelial sodium (Na) channel (ENaC)
All mutations map to cytoplasmic regulatory domains
Liddle Syndrome and the ENaC
When blood pressure is high
What is the amounts of ENaC in the epithelial cells?
How are channel numbers kept low?
What does ubiquitin ligase Nedd4-2 bind onto on the ENaC subunit? By adding Ub, what does it initiate the formation off?
When blood pressure is low
What does angiotensin 2 activate? what does this result in?
When blood pressure is high
The amounts of ENaC in the epithelial cells is low - therefore sodium transport is low
Channel numbers kept low via Ub-mediated endocytosis
Ub ligase Nedd4-2 binds to C-terminus of ENaC subunits, add Ub initiating formation of CCVs (initial endocytosis event)
When blood pressure is low
Angiotensin 2 activates kinases that phosphorylate Nedd4-2 and thereby inactivate it
This results in more ENaC, more Na transport and ultimately leads to increased blood pressure
Liddle Syndrome and the ENaC
When blood pressure is low
- When blood pressure is low, body wants to increase blood pressure, so it transports sodium through the ENaC transporter into the blood
- Ub-ligase is phorsphorylated by PKA (protein kinase A) & Sgk1, these are regulating kinases
- Ub-ligase becomes phosphorylated it then becomes a substrate for inhibitor - which the 14.3.3 protein binds to the phosphorylated form of the ligase and stops it from functioning
Lots of transporter into your blood - water follow due osmotic gradient
Liddle Syndrome and the ENaC
When blood pressure is high
The phosphorylation doesnt happen
The ligase then free to interact with regulator
Initiates endocytosis
Less sodium transport - not increasing
With Liddly patietns
All mutations map to this cytoplasmic regulation region
What happens under high blood pressure there is no regulation because the ligase cant interact with the regulation protein
What this means that if you try to inhibit it using normal ihibitors it wont make a difference
Things like teranterin will work because it binds directly
Liddle Syndrome and the ENaC
What inhibitors dont work?
What inhibitors do work?
Inhibitors that target Angiotensin-2 production or that are Aldosterone antagonists dont work in Liddles patients
Inhibitors that directly inhibit ENaC do alleviate symptoms
NF-kB signaling and ubiquitin
What is NF-kB?
What do NF-kB pathways regulate?
Ubiquitylation is key to NF-kB signaling, what does it involve?
NF-kB is a family of dimeric transcription factors
NF-kB pathways regulate a range of physiological processes including: inflammation, immune response and cell survival
Ubiquitylation is key to multiple parts of the NF-kB signaling pathway and it involves both proteasomal and non-proteasomal processes
NF-kB signaling and ubiquitin
In unstimulated cells where do the NF-kB transcription factors bind?
What does binding to IkB sequestes to NF-kB result in?
On activation of the signaling cascade was is IkB phosphorylated by? then ubiquitylated by? and then ligase and degraded by?
What does the NF-kB transcription factor then enter - and do?
In unstimulated cells the NF-kB transcription factors binds to members of the inhibitory IkB family
Binding to IkB sequestes to NF-kB results in cytoplasm preventing activity
On activation of the signaling cascade IkB is phosphorylated by the IKK complex, subsequently ubiquitylated by the SCF-bTrCP E3 ligase and degraded by the 26S proteasome
The NF-kB transcription factor then enters the nucleus and activates target gene expression
NF-kB signaling and ubiquitin
Illustration
We have
an unstimulated cell
NF-kB transcription factor bound to inhibitor
On activation of signalling cascade — the inhibitor is phosphorylated- it then has Ub attached to it
it gets degraded
the NF-kB moves into the nucleus and you get gene expression
NF-kB signaling and ubiquitin
The role of ubiquitn is far more complex than simple proteasome degradation
What are the two best studied pathways in the inflamation response?
Inflammation response:
- Mediated by cytokines interleukin-1 (IL-1)
- And the Tumor Necrosis Factor alpha (TNFa)
Both recognized by distinct receptors (Il-1R and TNFR) and have distinct signaling cascades that merge on TAK1 the complex that phosphorylates the IKK complex which in turn activates its kinase activity of IkB
NF-kB signaling and ubiquitin
Chen and Chen 2013 - read it
What happens is that receptor proteins binds to cytokines
Case phorsphorylation of kinase complex
Kinase - Kinase - Kinase cascade
What happens is with both the receptros the activated receptors activats Ub-ligases - that are Ub chains on the row
The IL-receptor generates K63 linked chains
Linear Ub assembly complex LUBAC - generates Ub linked chains
this chains are acting as a scafflod
TNF signaling and autoimmune disease
Recent paper identified three patients with loss of funcation mutations in HOIL-1 - which is part of the LUBAC complex
What did the mutations result in?
What was strange about this?
What did they show?
Mutations result in loss of activity of LUBAC complex
Oddly patients have complex symptoms associated with both reduced and elevated TNF activity
They show immunodeficiency, autoinflammation and amylopectinosis - formation of glycogen inclusions
TNF signaling and autoimmune disease
They cultured fibroblasts what was shown to be reduced?
In contrast in cultured monocytes what was shown?
Cultured fibroblasts from these patients showed reduced IKK activation
In contrast, cultured monocytes showed increased inflammatory cytokines resulting from increased TNF signaling
NF-kB signaling and ubiquitin
Exact targerts for linear ubiquitin chains in TNF signaling still unclear
Likely to be complex and cell-type specific
Boisson et al 2012
Pathogens and Ubiquitin
Ubiqutin is Eukaryotic
However there are many examples of pathogens that are both viral and bacterial and use the Ub-mediated endocystosis system to do what?
It uses the Ub-mediated endocystosis system to eneter cells
Many examples of ubiquitin cycle enzymes that are expressed by bacteria that function in the host cell
Shigella and Ubiquitin
What is shigella?
What do they invade? What do they produce?
What secretion system do they use?
Shigella are Gram-negative pathogenic bacteria that cause dysentery
Shigella invade via the gut and produce shiga toxin that destroys the epithelial cells of the intestinal mucosa
Shigella uses a Type 3 secretion system - a tye of biological syringe that injects bacterial proteins into the host cell facilitating invasion
Shigella and ubiquitin
Uses a syringe like mechanims to inject toxins into host cell membrane
Tip pushes its way
Shigella and ubiquitin
What does Nod1 (protein) recognise?
What does Nod1 then stimulate?
As part of the host cells defense mechanism to infection peptoglycans produces by the bacteria are recognised by Nod1
Nod1 stimulates teh NF-kB signaling cascade resulting in an inflammatory response
Shigella and ubiquitin
What is IpaH9.8?
IpaH9.8 is a virulence factor injected by Shigella into the host cell
IpaH9.8 is a ubiquitin ligase that binds to and ubiquitylates the NEMO component of the IkB complex
This ubiquitylation recruits a Ub binding protein ABIN-1 which ultimately leads to the proteasome degradation of NEMO
Since NEMO is required for deactivation of IkB this attenuates the inflammatory response making it easier for shigella to take over the host cell
Mitophagy and ubiquitin
What does ubiqitin regulate the stability of?
What is the mitophagy process?
What are mitophagic processes also involved in?
Mitochondria
Mitophagy is the process by which damaged mitochondria are removed
Also used to remove mitochondria from developing erythrocytes and paternal mitochondria from a fertilized oocyte
Mitophagy and ubiquitin
What is involved in:
- Removal of unwanted but undamaged mitochondria?
- Degradation of damaged mitochondria
Removal of unwanted but undamaged mitochondria (RBCs and oocytes) invovles formation of autophagosome which then fuses with lysozome
Degradation of damaged mitochondria involves ubiquitin, proteasome and lysozome although exact details are still unclear
Mitophagy and ubiquitin
Mitochondrial quality control
Parkinson’s disease
Parkinsons disease is a common neurodegenerative disorder
Caused by the loss of specific dopomine producing membranes in the brain
Most cases are spordadic - there are environmnetal and genetic cases
Juvenile Parkinson’s disease
What is the mutation that cases J.P
It is autosomal recessive form of PD
Early onset from less than 65 years old
Mutations that casue J. PD found in either Parkin or Pink1
18% of all early onset forms of PD have mutation in Parkin
Juvenile Parkinson’s disease
What do the Parkin and Pink1 genes do?
What do damaged mitochondira cause?
Parkin and Pink1 are major component of system that senses and removed damaged mitochondira
Mutation tha tprevent this from happening leads to sever cell stress and ultimatel the activation of apoptotic pathways
It is the apopotosis of dopoaminergic neurons that leads to Parkinson’s Disease
What does Pink1 do?
What is it recognised by?
Pink1 is the signal that used to determine whether or not mitochondria is healthy or not
Damaged mitochondira
Transport process doesnt work properly and so PINK1 isnt transported properly and so it shows mitochondira isnt working
The Pink1 is then recognised by Parkin wich releases Ub outside of the mitochondira
Parkin initially interacts with Pink1 but also
Milton - Kinesin - other things that allow microtubules to interact
When the proteins are ubiquinated
stops moving around
More complex thing is the Ub on the outter surface of the Mt acts as a signal
