Cell Morphology

Question 1

What is programmed necrosis?

Answer 1

Caspase independent cell death
Includes necroptosis pathway
Cellular constituents are leaked (unlike apoptosis)
Get cell rupture (osmotic swelling, energetic catastrophe, lysosomal membranes permeabilise)

Question 2

How is programmed necrosis triggered?

Answer 2

Inflammasome, LPS-mediated (ROS), necrosome

NAD^+ and ATP depletion, calcium overload, many ROS, action of phospholipases

Question 3

What is the difference between necrosis and apoptosis?

Answer 3

Necrosis:
Swelling of cell and organelles as opposed to shrinking
Chromosomes don’t decondense
Doesn’t require executioner caspases
Needs RIP3 and MLKL (dispensable in apoptosis)
Plasma membrane rupture as opposed to blebbing
Inflammation and tissue damage

Question 4

What is the effect of deleting FADD or pro-caspase 8 on cell survival?

Answer 4

RIPK3 is induced, forming complex IIb (the necrosome). This causes phosphorylation of MLKL (by RIPK3). MLKL oligomerises and translocates to the plasma membrane where it disrupts its integrity, leading to necroptotic cell death.

Question 5

What do cIAPs do?

Answer 5

They are negative caspase regulators in TNF signalling
Have an additional role as a ubiquitin ligase.
Conjugates RIPK1 with ubiquitin chains including K63 linked chains. This allows the recruitment of several other molecules, leading to the release of NFkB inhibition. Survival genes are then expressed.
If cIAP is deactivated, get formation of complex IIa, leading to apoptosis
If RIPK1 is unregulated (regulated by FADD) get necroptosis

Question 6

What is the ripoptososme?

Answer 6

RIPK1 and RIPK3 and FADD and caspase 8. Forms when there is no cIAP activity
Leads to either apoptosis or necroptosis dependent on the component composition

Question 7

What is the effect of caspase 8 on necroptosis?

Answer 7

Suppresses necroptosis - deletions in caspase 8 are lethal (also need deletion in RIPK3/ RIPK1 + FADD to survive)
Caspase 8 and FLIP cleave RIPK1

Question 8

What is the effect of RIPK1 knock out?

Answer 8

RIPK1 is a kinase and a scaffold protein
Deletion is lethal
Inserting an inactive kinase is ok - shows that scaffold function is necessary, not kinase function.
Can rescue with a FADD and RIPK3 knock out

Question 9

What is the effect of FADD knock out?

Answer 9

Is lethal. Can reverse by also knocking out RIPK3.

Question 10

What is the function and structure of MLKL?

Answer 10

Oligomerises to disrupt the plasma membrane
Oligomerises through N terminal 4 helix bundle (this then causes translocation to the membrane)
Oligomerisation is induced by RIPK3 phosphorylation, releasing auto inhibition
2 coiled coil domains. CC2 is for oligomerisation and CC1 is for recruitment to membrane

Question 11

How does MLKL disrupt the membrane?

Answer 11

Not completely clear.
Could bind ion channels leading to calcium influx
Could regulate sodium channels to increase osmotic pressure
Could bind membrane lipids by positively charged amino acids, forming pores

Question 12

What is the role of ESCRT in necroptosis?

Answer 12

Mediates plasma membrane shedding

Question 13

What are the functions of MLKL?

Answer 13

Disrupts plasma membrane integrity AND many other things (some below)
Homeostatic level for vesicle trafficking
Rapid calcium influx when activated
Removal of MLKL by ESCRT-III allows production of many cytokines and chemokine
Release of inflammasome products

Question 14

What does the RHIM domain on RIPK1 and RIPK3 do?

Answer 14

Association of RIPK1 and RIPK3 through RHIM-RHIM domain interactions upon induction of necroptotic pathway by death receptors. Leads to phosphorylation of RIPK3 and phosphorylation of MLKL.

Question 15

What is the outcome of TNF signalling?

Answer 15

Normally, get complex I (at membrane) formation, leading to the activation of NFkB and other signalling
Can get formation of complex IIa, leading to apoptosis
If lack of IAPs, get complex IIb formation (ripoptosome like), leading to apoptosis via caspase 8, RIPK1 and RIPK3 platform. If caspase 8 is inhibited, get necroptosis.

Question 16

What is the outcome of Fas or TRAILR signalling?

Answer 16

Get DISC complex triggering caspase 8 mediated apoptosis, independent of RIPK1
If lack of IAP, get RIPK1 recruitment to make the ripoptosome for apoptosis or necroptosis

Question 17

What is the outcome of TLR signalling?

Answer 17

Formation of the necrosome through RHIM adaptor TRIF, resulting in RIPK3 dependent necroptosis.

Question 18

What is the outcome of viral DNA detection?

Answer 18

DAI recognises dsDNA and recruits RIPK3 through its RHIM domain and induces necrosome formation without RIPK1. Get necroptosis.

Question 19

What are the outcomes of JAK/STAT activation?

Answer 19

IFNa/b induce transcription of RIPK1, thus inducing formation of the necrosome.

Question 20

What is the scaffolding function of RIPK1 in vivo?

Answer 20

In intestinal epithelium, knock out of RIPK1 causes apoptosis and loss of many cell types. Suggests RIPK1 has a role in inhibiting apoptosis. TNFR1 pathways seem to be the main cause.

Question 21

How is necroptosis inflammatory?

Answer 21

When the epithelial barrier is damaged (e.g. due to epithelial cell death), get disruption of the barrier. Allows microbes to invade the tissue. Recognition of PAMPs induces expression of cytokines and chemokine that result in inflammation. Some of the immune cell cytokines could further trigger cell death of epithelial cells leading to chronic inflammation.
DAMP release by necroptotic cells strongly triggers inflammation (compared to apoptosis)

Question 22

How do RIPK1 and RIPK3 regulate cytokine production?

Answer 22

Phosphorylation of RIPK3 can lead to inflammatory cytokine production
The NLRP3 inflammasome, formed due to phosphorylation of RIPK3 activates caspase 1 which makes mature IL-1B
Caspase can cleave RIPK1 to inhibit activation of IRF3 by RIG-I

Question 23

What are the pro and anti-cell death functions of RIPK1?

Answer 23

Absence of RIPK1 can trigger cell death by decreasing expression of anti-apoptotic genes/enhancing complex II or necrosome formation.
RIPK1 absence/inactivation of kinase activity can also prevent necroptotic cell death

Question 24

What are the pro and anti-cell death functions of RIPK3?

Answer 24

Is a death inducing protein
But there is evidence that it inhibits apoptosis.
Kinase dead RIPK3 causes increase in apoptosis, dependent on RIPK1 and caspase 8.

Question 25

What is necrostatin-1?

Answer 25

A molecule which blocks RIPK1 kinase activity (along with other targets - bad. Now have more specific ones)
Has been shown to be effective at treating stroke, heart failure and neurological disorders. Reduces ischaemic repercussion injury

Question 26

What are the advantages of targeting necroptosis for therapy?

Answer 26

Can lessen disease severity in mouse models
But there are complexities in regulation of cell death by RIPK1/3, meaning value of inhibiting these is cell and context dependent.

Question 27

How can necroptosis be inhibited?

Answer 27

RIPK1 is dispensable for necroptosis (has a scaffold function that is needed to inhibit cell death)
RIPK3 inhibition can lead to apoptosis
Inhibition of MLKL can lead to necroptotic inhibition

Question 28

What are the advantages of necroptosis?

Answer 28

Contributes to innate immunity to clear pathogens

Alerts the immune system through release of DAMPs

Question 29

What are the disadvantages of necroptosis?

Answer 29

Can lead to many inflammatory diseases if unscheduled
Can be subverted by some cancers
Some species lack MLKL or RIPK3 suggesting selection against necroptosis