Lecture 8 - Death receptors and alternate forms of cell killing

Question 1

Necrotic cell death: is it on purpose like apoptosis and what its the morphological and physiological characteristics?

Answer 1

Accident rather than design - passive, not active

• Cell swells and plasma membrane ruptures
• no condensation of chromatin
• No membrane blebbing

Question 2

Role of ATP in necrosis

Answer 2

ATP is required for the function of ion pumps in the plasma
membrane. Loss of this function leads to the loss of PM
gradients, leading to swelling and rupture

Question 3

Reperfusion

Answer 3

the restoration of blood flow to an organ or tissue after a period of ischemia (lack of blood supply)

Question 4

ROS induced by reperfusion

Answer 4

Decreased pH - ROS generation - DNA damage - necrosis

Question 5

’’

Answer 5

Catalyses the polymerisation of poly-ADP ribose chains which are signals to recruit DNA repair enzymes (e.g. DNA ligase III and DNA polymerase beta).

After DNA repair, the PAR chains are degraded by Poly(ADP-ribose) glycohydrolase.

Answer 6

NAD+ is used by PARP as the source of ADP-ribose. Excessive DNA damage can rapidly deplete the cells available NADH by 90% within minutes. This requires regeneration of NAD+, which uses up ATP.
NAD+ is a cofactor for glycolysis, so loss of NAD+ allows only the ATP consuming part, and reduced pyruvate
Loss of pyruvate leads to reduced mitochondrial NADH
Mitochondria deficient for NADH become depolarised, and ATP-synthase works backward, using ATP to maintain polarisation…

Question 7

Lack of pyruvate generation

Answer 7

Unable to move protons through ETC and maintain membrane potential - use ATP to move protons through ETC

ATP stores get depleted, channel proteins cannot do their job - necrosis generated

Question 8

Increased cytosolic Ca2+ and decreased pH can open the mitochondrial permeability transition pore

Answer 8

PTP is composed of pores in the inner and outer membranes
VDAC (voltage dependent anion channel)
ANT (adenosine nucleotide transporter)
cyclophilin D in the matrix (peptidyl prolyle isomerase)
F1F0-ATPase
Mitochondrial calcium uniporter

Question 9

Signals released from necrotic cells activate inflammatory caspases

Answer 9

DAMPs and PAMPs - detected by receptors
on cells which the initiate inflammation
PAMPs may be bacterial proteins, lipids or viral DNA/RNA
DAMPs may be debris released from necrotic cells

Question 10

The inflammasome activates caspase 1

Question 11

The inflammasome is a large filamentous protein assembly

Answer 11

DNA binding of AIM2 relieves autoinhibitory interaction between the HIN and Pyd domains
This nucleates the recruitment of ASC and its assembly into a filament
This recruits caspase-1 via a CARD/CARD interaction and nucleates assembly of capase-1 filaments

Question 12

IL-1beta and Gasdermin are caspase-1 substrates

Question 13

Gasdermin D

Answer 13

Cleaved by caspase 1 and then enters the plasma membrane and creates a pore for the exit of IL-1β and IL-18 as well as promoting pyroptosis (lytic form of cell death)

Question 14

Gasdermin and Ninjurin1 both drive pyroptosis
- a form of lytic programmed cell death linked to the inflammatory response

Question 15

The TNFr superfamily as an alternate caspase activation platform

Question 16

Role of CD95 and FasL in immune system homeostasis

Answer 16

Acute lymphoproliferative syndrome (ALPS)

• apoptosis usually controls the numbers of lymphocytes
• accumulation of T-lymphocytes
• patients suffer from autoimmune conditions and lymphoid tumours
• found to be caused by inactivating mutations in CD95, FasL or caspase 8

Cytotoxic T-cells express FasL which kills infected target cells by binding to CD95 on the surface

The inappropriate recognition of your own cells can kill them, such as in auto-immune conditions such as Type-1 diabetes

Question 17

Activation of Fas (CD95) by FasL presented on cytotoxic T-cells
- the prototype Death Receptor

Question 18

The two DED in caspase 8 promote assembly of a chain of active caspases

Question 19

Autocatalytic cleavage of caspase 8 stabilises the active dimer

Question 20

Inhibitors of death receptor signalling

Answer 20

These inhibitors can have complex effects on what happens downstream of receptor activation

Question 21

Crosstalk between Death Receptor signalling and mitochondrial apoptosis

Answer 21

Fas mediated caspase 8 activation needs to activate the mitochondrial pathway of apoptosis sometimes

Question 22

Concept #3.
Death receptors can signal in different ways, and not always to induce apoptosis - lessons from TNFR1

Answer 22

TNFR1 initiates a complex set of cell responses depending upon the proteins recruited to it:

• apoptosis via caspase 8
• cell survival and proliferation via NFkB
• programmed necrosis (necroptosis)

Question 23

TNFR1 - a variation on Fas and the DISC

Answer 23

TRADD - an adaptor protein similar to FADD, whch can bind to activated TNFR1. It cannot directly interact with or activate caspase 8

Question 24

NF-kb signalling from TNFR1 keeps cells alive

Answer 24

NF-kb is a transcription factor that activates genes involved in inflammation and survival.

This complex, termed complex I, keeps cells alive and does NOT activate caspase 8

TNFR1 activates apoptosis if NF-kb cannot be activated - The TRAF/TRADD/RIPK1 complex becomes cytosolic, loses its Ub
The now exposed DD of TRADD can recruit FADD vis its DD, thus recruiting and activating caspase 8

Answer 25

TNF-alpha - produced by activated macrophages during inflammation

Question 26

RIP kinases alter how cells respond to TNFr activation

Answer 26

Apoptosis is not the only form of cell death that TNFR can activate Programmed necrosis (called necroptosis) has many features of necrotic cell death caused by cell damage It can also result in inflammatory responses via the release of DAMPs Macrophages release TNFalpha when activated during inflammation, the ligand for TNFR1

Question 27

TNFR1 can also activate necrotic cell death

Question 28

RIPK1 and RIPK3 control necroptosis by TNFR1 activation

Answer 28

Necrostatin - a drug that inhibits RIPK1, can block necroptosis induced by TNFalpha Caspase 8 cleaves the kinase domain of RIPK1, inactivating it and preventing necroptosis Necroptosis requires RIPK kinase activity, as it can be blocked by necrostatin - a RIPK inhibitor

Question 29

RIPK1 and RIPK3 assemble into amyloid like structures that phosphorylate MLKL - a pseudokinase

Answer 29

MLKL - mixed lineage kinase like Binds ATP but is catalytically inactive Phosphorylated on T357 and S358 by RIPK3 (within “kinase” domain) MLKL KO mice show the same resistance to necroptosis as RIP3K KO

Question 30

Phosphorylated MLKL oligomerises and inserts into membranes to disrupt them

Answer 30

Unclear if MLKL is the sole regulator of necroptosis Necroptosis has still unexplained events, such as PS exposure (as in apoptosis) Is MLKL only regulated by RIPK3?

Question 31

Why have necroptosis?

Answer 31

- Some viruses express caspase 8 inhibitors, such as v-FLIP - Necroptosis can therefore act to kill cells where activation of apoptosis has been inhibited - RIPK3 deficient mice succumb to vacinia virus infection - It may also act to increase inflammation in the area of infection, therefore boosting the immune response

Question 32

Summary of learning objectives from lecture three:

Answer 32

Necrosis represents an important form of cell death that both signals to initiate inflammation and may occur as a result (pyroptosis) Understand the causes of necrosis at a molecular level Be able to expalin how signals from necrotic cells activate inflammatory signals Understand how the inflammasome acts to activate IL1b, gasdermin and ninjurin1 to drive lytic cell death and inflammation The TNFr superfamily can initiate a variety of responses in a cell, and are not always pro-apoptotic - Understand how different TNF receptors interact with adapters and caspases to initiate different cellular responses Necroptosis is programmed necrosis in response to specific cirumstances - Understand how RIP kinases initiate programmed necrosis