Lecture 18. Programmed Cell Death

Question 1

What are the features of classical necrosis?

Answer 1

External trigger (non-specific)
Moderate chromatin condensation
Cytoplasmic, mitochondrial and ER swelling
Rupture of the plasma membrane
Collateral damage to surrounding tissues

Question 2

What is necrosis?

Answer 2

Cells die because of a response to trauma: they release their contents into the extracellular space – stimulating inflammatory responses. Uncontrolled, passive. Collateral damage

Question 3

What is apoptosis?

Answer 3

A sequence of genetically programmed responses. Cell contents not released, no immune sequelae. Cells absorbed by neighbours. Controlled, active.
Apoptosis also occurs in plant cells where no inflammation takes places

Question 4

Are apoptosis and necrosis deemed different?

Answer 4

Nom, no longer deemed different

Question 5

What does programmed cell death lead to in unisexual flowers?

Answer 5

PCD lads to abortion or developmental arrest: of carpel primordia in male flowers and stamen abortion in female flowers

Question 6

What is the role of programmed cell death (PCD) during sporogenesis?

Answer 6

PCD occurs in supporting sporophytic tissues including the anther tapetum (supportive tissue for microspores) and the nucellus in the developing ovules. After meiosis, non-functional megaspores degenerate

Question 7

What are the five genetically programmed stages in trichome development?

Answer 7

Rapidly dividing protodermal cells (cells on leaf)
Trichome cell selcetion
Mitosis replaced by endoreduplication
Genetically controlled branching
Death programme activated (stinging trichome), stops spread of pathogen

Question 8

How is PCD used for developmental changes in animals?

Answer 8

Mouse hand formation
Almost all larval structures are destroyed during Drosophila metamorphosis e.g. Salivary glands (SG), Muscles (M), midgut (MG) and hindgut (HG)
Almost all larval cells die, with larval tissue being replaced by adult tissue

Question 9

How is PCD used to control cell numbers in mammals?

Answer 9

Deletion of cells that fail to partner controls cell number
Elimination of dangerous and abnormal cells such as autoreactive lymphocytes
Neural deletion adjusts number of nerve cells to target size

Question 10

What is the overview of how apoptotic signals trigger apoptosis?

Answer 10

1. Apoptotic signal (can have multiple signals)
2. Signals assembly of an activating complex with initiator procaspases (there are multiple activating complexes)
3. Activation of initiator caspases by proximity, autocleavage and rearrangement into fully active form (multiple caspases)
4. Activation of executioner caspses by initiator caspase cleavage and rearrangement (links with development and inflammation)

Question 11

What is intrinsic apoptosis?

Answer 11

Regulated cell death initiated by multiple signals and marked by mitochondrial outer membrane permeabilisation
Release the contents of the inner membrane space into the cytosol

Question 12

What are examples of the multiple signals that can cause intrinsic apoptosis?

Answer 12

DNA damage, ER stress, ROS burden, replication stress, microtubular changes, mitotic abnormalities and more

Question 13

How does initiation of intrinsic apoptosis occur?

Answer 13

1. Apoptotic stimulus triggers the release of cytochrome c from the mitochondrial IMS by a membrane channel formed by Bax
2. cyt c ais captured by and activates apoptotic protease activating factor-1 (Apaf-1)
3. Apad-1 forms a heptameric apoptosome
4. The heptameric apoptosome recuits procaspase 9 via caspase activation and recruitment domain (CARD) interactions: CARDs are members of the death domain (DD) family
5. Caspase cascade leads to apoptosis

Question 14

How is the intrinsic apoptotic pathway initiation regulated?

Answer 14

BCL-2 gene which inhibits the release of cytochrome c following an apoptotic stimulus
BCL-2 inhibits the pro-apoptotic gene (Bax like proteins) which from the channels in the outer membrane of the mitochondria and stimulates the release of cyt c
BH3-only proteins inhibit BCL-2

Question 15

How is the intrinsic apoptotic pathway regulated in healthy cells?

Answer 15

Pro-apoptotic Bax family proteins circulate between the cytosol (TM domain hidden) and the mt OM (TM unmasked), inhibited when interact with the membrane
Anti-apoptotic Bcl-2 family members heterodimerise with membrane bound Bax family members and inhibit them

Question 16

How is the intrinsic apoptotic pathway regulated in apoptotic cells?

Answer 16

Pro-apoptotic BH3-only proteins inhibit Bcl-2 family proteins, allowing membrane oligomerisation of Bax family proteins
Bax family proteins form a channel that releases cytochrome c into the cytosol: mitochondrial outer membrane permeability (MOMP)

Question 17

How do BH3-only proteins inhibit Bcl-2 family members?

Answer 17

It is displaced by the BH3 domain of the BH3-only family proteins, blocking the hydrophobic groove: Bcl-2 family members can no longer heterodimerise with membrane-bound Bax family members. Thus, Bad proteins permit Bax protein family channel formation

Question 18

How are BH3-only proteins tightly controlled?

Answer 18

BH3-only proteins are regulated post-transcriptionally by both survival stimuli (trophic signals) and apoptotic stimuli
Survival stimuli (trophic signals): Phosphorylation on S₇₅ and S₉₉ which promotes phosphorylation of S₁₁₈ (and S₁₃₄). S₁₁₈ phosphorylation disrupts the BH3 domain, and so releases Bcl-XL
Apoptotic stimuli: Methylation of R₉₄ and R₉₆ inhibits phosphorylation of S₉₉

Question 19

How is PUMA (p53 upregulated modulator of apoptosis) expression controlled?

Answer 19

By transcriptional regulation as well as by phosphorylation

Question 20

What happens when caspase 9 is activate by procaspase 9?

Answer 20

Activated by proximity
Caspase 9 activates an executioner called procspase 3, activating to caspase 3
Caspase 3 activates procasapse 6 and 7
These target thousands of substrates

Question 21

What is the role of p53 in intrinsic apoptosis and development?

Answer 21

p53 activates apoptosis program - guardian of the genome - responds to genomic stress by signalling apoptotic signals to that cell

Question 22

How does p53 function?

Answer 22

If p53 responds to much stress, it will trigger the transcription of PUMA
PUMA is closely related to Bad which inhibits Bcl-2, promoting the release of cytochrome c, promoting apoptosis

Question 23

What can p53 create if it’s stressed in the right way?

Answer 23

PIDDOsome (p53 induce death domain), which activates CASp2 that deletes webbing between fingers, carves tissues into organs, organ genesis and development

Question 24

What are the important parts of the PIDDOsome?

Answer 24

There is a p53 induced death domain protein (PIDD-CC)
There is an adaptor that has a death domain that heterodimerises with PID that has a CARD domain (RAIDD)
And that can recruit the initiator CARD bearing procaspase-2

Question 25

What is the role of trophic factors in intrinsic apoptosis?

Answer 25

P75 neurotrophic receptor (p75NTR) is an important receptor for the role of neurotrophins (NGF, nerve growth factors) in modulating brain plasticity and
apoptosis

Question 26

What is extrinsic apoptosis?

Answer 26

Death receptor-mediated apoptosis

Question 27

What do death domains trigger?

Answer 27

Apoptosis and inflammation (intimately connected)

Question 28

What to tumour-necrosis factor receptors trigger?

Answer 28

TNF-R1 triggers apoptosis and inflammation as bound to death domain
TNF-R2 triggers proliferation and releases TNF as a cytokine

Question 29

What does CD95 stimulate?

Answer 29

Apoptosis when bound to CD95L (CD95 ligand)

Question 30

How are death effector filaments created in the extrinsic apoptotic pathway?

Answer 30

CD95 (FAS) binds to CD95L (FSL) which results in the activation of the death domains bound to CD95
The death domains recruit FADD (FAS-associated death domain proteins) which then recruit either procaspace 8 or 10
Whole chains of procaspase 8 and 10 build up and CASP8 and 10 are brought together by dimerisation

Question 31

What is the role of c-FLIP in the extrinsic apoptotic pathway?

Answer 31

Heterodimerises and inhibits recruitment of procaspases 8 or 10 and stops activation

Question 32

How is the extrinsic pathway regulated by ligand interactions?

Answer 32

Soluble decoy receptor 3 (DcR3) competes with CD95: the strength of the apoptotic signal depends on the expression/activity of DcR3
Cell-specific receptors (most cells trigger TNFR1, but some trigger TNFR2, proliferation of endothelial and epithelial tissues)
Responses by ligands include death, inflammation, stress and proliferation. But death always requires interaction with death domain containing receptors

Question 33

How is the extrinsic pathway regulated by the expression of c-FLIP proteins?

Answer 33

c-FLIPs interfere with dimerisation of procaspase-8 and pro-caspase 10 and therefore reduce CASP8 and CASP10 activity
Over-expression reduced apoptosis, so apoptosis is regulated by the expression levels of the c-FLIP proteins

Question 34

What are c-FLIP expression levels regulated by?

Answer 34

Ubiquitylation and phosphorylation

Question 35

What happens with c-FLIP in cancers and viruses?

Answer 35

Many cancers over-express c-FLIPs and some viruses express v-FLIPs

Question 36

How does Beclin interact with c-FLIP?

Answer 36

c-FLIP expression up-regulated Beclin which upregulates Bcl-2 and Bcl-XL activity

Question 37

Do plants have caspases?

Answer 37

Plants do not express caspases, but instead express some distantly related metacaspases
Metacaspases are arginine/lysine-specific, in contrast to caspases, which are aspartate-specific

Question 38

What is PANoptosis?

Answer 38

Inflammatory cell death where three major pathways of programmed cell death, pyroptosis, apoptosis, and ecroptosis, become activated

Question 39

How can TNF-R1 activation stimulate both apoptosis and an inflammatory necrotic cell death through cell destruction mechanisms?

Answer 39

Activation of TFN-R1 deubiquitylates and activates RIPK1 (rest in peace kinase 1)
A ripoptosome forms, activating CASP 8 and leading to apoptosis
The RIPK1-RIPK3 necrosome activates mixed lineage kinase-like domain (MLKL), which causes loss of plasma membrane integrity

Question 40

What is pyroptosis and what does it activate?

Answer 40

Caused principally by bacterial infection
An inflammatory signal (eg from TLR4 on the cell surface) triggers the assembly of: NLRP3, ASC and procaspase 1 which all form an inflammasome
Activation of procaspase 1 to give CASP1
Different mechanism activates CASP 4 /5