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Molecular Basis of Disease SBS929 > 4.2 Ubiquitin > Flashcards

4.2 Ubiquitin Flashcards

1
Q

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

A

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

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2
Q

Ubiquitin and protein trafficking

What are we talking about today?

A

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

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3
Q

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?

A

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)

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4
Q

Ubiquitin and protein trafficking

What is the pathway for the formation of the CVV

A

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

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5
Q

Ubiquitin and endocytosis

What do ubiquitylation of a plasma membrane protein result in?

What can it be thought of as?

A

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

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6
Q

Ubiquitin and endocytosis

What has no role in this process?
Where are unwanted proteins trafficked too?
What is the common error in papers?

A

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’

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7
Q

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?

A

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

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8
Q

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?

A

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

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9
Q

Ubiquitin and hypertension

Low blood pressure peptide cascade

A
  1. Start with angiotensinogen when low blood pressure is sensed it is hydrolysed by renin
  2. It is then further hydrolysed by ACE (angiotensin conversion enzyme)
  3. Forming angiotensin-2 - which is the active hormone
  4. It is then degraded
  5. 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

  1. It causes vasoconstriction by binding to arteriole, releasing Ca
  2. Angiotensin-2 binds to adrenal coretex wich releases second hormone called Aldosterone which releases sodium and increases blood volume and this blood pressure
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10
Q

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?

A

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

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11
Q

Liddle syndrome

Early onset hereditary hypertension

How many known familial forms?

Where do all the genes map onto?
Where to mutations map?

A

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

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12
Q

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?

A

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

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13
Q

Liddle Syndrome and the ENaC

When blood pressure is low

  1. When blood pressure is low, body wants to increase blood pressure, so it transports sodium through the ENaC transporter into the blood
  2. Ub-ligase is phorsphorylated by PKA (protein kinase A) & Sgk1, these are regulating kinases
  3. 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

A

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

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14
Q

Liddle Syndrome and the ENaC

What inhibitors dont work?

What inhibitors do work?

A

Inhibitors that target Angiotensin-2 production or that are Aldosterone antagonists dont work in Liddles patients

Inhibitors that directly inhibit ENaC do alleviate symptoms

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15
Q

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?

A

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

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16
Q

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?

A

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

17
Q

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

A
18
Q

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?

A

Inflammation response:

  1. Mediated by cytokines interleukin-1 (IL-1)
  2. 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

19
Q

NF-kB signaling and ubiquitin

Chen and Chen 2013 - read it

A

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

20
Q

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?

A

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

21
Q

TNF signaling and autoimmune disease

They cultured fibroblasts what was shown to be reduced?

In contrast in cultured monocytes what was shown?

A

Cultured fibroblasts from these patients showed reduced IKK activation

In contrast, cultured monocytes showed increased inflammatory cytokines resulting from increased TNF signaling

22
Q

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

A
23
Q

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?

A

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

24
Q

Shigella and Ubiquitin

What is shigella?

What do they invade? What do they produce?
What secretion system do they use?

A

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

25
Q

Shigella and ubiquitin

Uses a syringe like mechanims to inject toxins into host cell membrane

A

Tip pushes its way

26
Q

Shigella and ubiquitin

What does Nod1 (protein) recognise?

What does Nod1 then stimulate?

A

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

27
Q

Shigella and ubiquitin

What is IpaH9.8?

A

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

28
Q
A
29
Q

Mitophagy and ubiquitin

What does ubiqitin regulate the stability of?

What is the mitophagy process?

What are mitophagic processes also involved in?

A

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

30
Q

Mitophagy and ubiquitin

What is involved in:

  • Removal of unwanted but undamaged mitochondria?
  • Degradation of damaged mitochondria
A

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

31
Q

Mitophagy and ubiquitin

Mitochondrial quality control

A
32
Q

Parkinson’s disease

A

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

33
Q

Juvenile Parkinson’s disease

What is the mutation that cases J.P

A

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

34
Q

Juvenile Parkinson’s disease

What do the Parkin and Pink1 genes do?

What do damaged mitochondira cause?

A

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

35
Q

What does Pink1 do?

What is it recognised by?

A

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

36
Q

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

A

Molecular Basis of Disease SBS929 (14 decks)

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