Cell Death

What can force cells to die?

Severe insults such as ischemia (lack of blood flow) or exposure to poisons.

Why do cells die in a programmed way?

To eliminate cells that pose a danger or are no longer needed — for example, during immune defense or development.

Which immune cells can trigger death of other cells?

Effector/cytotoxic T cells

What is necrosis

A biologically uncontrolled process - accidental cell death

How does regulated cell death differ from necrosis

Give example of regulated cell death

It is controlled by defined signalling cascades and effector mechanisms.

E.g. apoptosis

What necrotic-like cell deaths are regulated - name 3

Necroptosis, pyroptosis, and NETosis.

What normal physiological process involves apoptosis?

Keratinocyte terminal differentiation.

What are the 3 main types of regulated cell dath

Apoptosis

Pyroptosis

Netotic cell death

What happens which leads to apoptosis

DNA fragmentation 

Loss of mitochondria function

What happens which leads to pyroptosis

Membrane rupture

What happens which leads to Netotic cell death

Histone citrullination

List the key morphological changes of apoptosis that occur in a sequential manner

• Blebbing

• Cell shrinkage

• Nuclear fragmentation

• Formation of apoptotic bodies

• Phagocytosis of apoptotic bodies

List key biochemical changes in apoptosis.

• Caspase activation (execution of the cell death programme)

• Degradation of cytoskeletal proteins (blebbing, nuclear fragmentation)

• Exposure of phosphatidyl serine on the cell surface (inducing phagocytosis)

• DNA fragmentation (unrepairable DNA damage)

• Loss of mitochondrial membrane potential (and ATP production)

What enzymes execute apoptosis?

Caspases — cysteine-dependent aspartate-specific proteases

What are the two main types of caspases?

Initiator caspases& executioner caspases

What are the main functional domains of caspases?

Pro-domains (for protein–protein interactions) and catalytic subunits (for enzymatic activity).

Name a protein-protein interaction that would happen on a caspase structure

Apaf-1 – pro-caspase-9 interaction

Name some targets of caspase cleavage (execution of apoptosis by cleaving essential proteins)

Caspase-dependent DNase

Lamin

Bcl-2

Mitochondrial complexes

What is the Bcl-2 protein family?

B cell lymphoma 2 protein 

A family of proteins that regulate mitochondrial apoptosis

Name the three main groups of Bcl-2 proteins

Anti-apoptotic: Bcl-2, Bcl-XL, Mcl-1 (contain 4 BH domains: BH1–4).

Multidomain pro-apoptotic: Bax, Bak (contain 3-4 BH domains: BH1–3). 

BH3-only pro-apoptotic: Bad, Bik, Bim, Bid, Puma, Noxa, Hrk

What stresses can trigger Bcl-2–mediated apoptosis

ER stress, DNA damage, and growth factor deprivation.

What are death receptors?

Single transmembrane domain receptors without enzymatic activity that mediate apoptosis upon ligand binding.

What ligands bind to death receptors?

Members of the TNF ligand family — Fas ligand, TNF, and TRAIL (TNF-related apoptosis-inducing ligand)

What domain do death receptors use to signal apoptosis

The death domain, which recruits adaptor proteins to propagate apoptotic signalling.

What triggers the intrinsic apoptosis pathway?

Signals originating from inside the cell that indicate severe cellular stress and unrepairable cellular damage initiate the intrinsic pathways of apoptosis.

Unrepairable cellular damage e.g. endoplasmic reticulum stress - accumulation of unfolded or misfolded proteins in the ER

What are the steps of the intrinsic apoptosis pathway

1. Cellular stress leads to activation of BH3 pro-apoptotic proteins

2. BH3 inhibits Bcl-2/Bcl-XL & activates Bax & Bak

3. Bax and Bak form megapores in the mitochondrial outer membrane - Mitochondrial outer membrane permeabilization (MOMP)

4. MOMP allows cytochrome c to released from the mitochondria into the cytosol.

5. Cytochrome c + APAF 1 + Pro-caspase-9 assemble to form the apoptosome

6. Pro-caspase-9 is activated in the apoptosome

7. Activated caspase-9 (a-C9) activates executioner caspases (e.g. a-C3) → apoptosis execution

Explain the steps of the extrinsic apoptosis pathway

1. Immune cell presents a death ligand (Fas, Tumor necrosis factor (TNF), TNF-related apoptosis-inducing ligand (TRAIL)) to the receptor (part of tumour necrosis factor receptor family)

2. The receptor recruits adaptor proteins (FADD) and procaspase-8 to form the DISC (Death-Inducing Signalling Complex)

3. Within the DISC, procaspase-8 is activated → caspase-8 (caspase-8 activation platform)

4. Caspase 8 activates caspase 3

5. Steps 3 & 4 with the help if cytochrome c leads to the caspase enzyme cascade (this is the amplification system)

6. Caspase substrate proteins are the substrate of apoptosis

7. Biological response = cell death (or can also be inflammation / cell differentiation)

Cell lysis process

1. Release of perforin 

2. Plasma membrane permeabilisation

3. Loss of osmotic homeostasis

4. Necrosis/cell lysis

TNF receptor 1 belongs to a superfamily of receptors called

Death Receptors

Death domain superfamily includes proteins with what 4 domains

What kind of interactions do domains in the death domain superfamily form

Domains form homotypic interactions, and occasionally heterotypic interactions

Does the death domain superfamily include both receptors & adaptors? Is that all it includes

Receptors

Adaptors

Enzymes

Name a receptor that has a death domain

TNFR1

TNFR1 signalling is mediated by recruitment of which adaptor protein

TRADD

Does TNFR1 have intrinsic enzymatic activity?

No — TNFR1’s intracellular domain lacks intrinsic enzymatic activity.

It contains the DD (death domain) instead

How is TNFR1 signalling initiated?

Homotypic interactions between the DD (death domain) of TNFR1 and DD- containing adaptor protein TRADD activates signalling

What event occurs when TNFR1 binds its ligand (TNF)?

Ligand binding induces trimerisation of the TNFR1 receptor - 3 signalling pathways are activated

What are the 3 major signalling pathways activated by TNFR1?

NF-κB (Nuclear factor–kappaB) and survival pathway

MAPK (JNK) pathway

Cell death (apoptotic) pathwa

What is NF-κB?

A family of homo & heterodimeric transcription factors that regulate genes involved in inflammation, immunity, and cell survival.

What is the most common NF-κB dimer in canonical signalling?

The p50/p65 dimer

How does TNFR1 activation lead to NF-κB activation?

DD of TNFR1 recruits other DD proteins (e.g. TRADD) through homotypic interactions

TRADD acts as a platform for the recruitment of a signalling complex called Complex I that leads to activation of NF-kB

What does TRADD recruit by the TRADD recruitment platform to Complex I

TRADD recruits:

1. RIP1 through homotypic DD interactions (RIP=RIP1=RIPK1)

2. TRAF2 which recruits c-IAP

What is the function of cIAPs in Complex I?

They are E3 ubiquitin ligases that ubiquitinate target proteins to promote downstream signalling.

What does the BIR1 domain of cIAP do?

Mediates interaction with TRAF2.

What does the RING domain of cIAP do?

Provides E3 ligase activity.

What are the 3 main types of ubiquitination that happen to substance K in TNFR1 signalling?

K48-linked ubiquitination → proteasomal degradation.

K63-linked ubiquitination → signalling

Linear (head-to-tail) ubiquitination → signalling proteosomal degradation

What two kinase complexes are recruited to ubiquitin scaffolds in Complex I?

cIAP ubiquitinates itself and RIP1

Role of K63 polyUb chain in complex I

The K63 polyUb chains act as platforms for recruitment of downstream signalling molecules

In recruiting of kinase complexes, what is the role of the RIP1 polyUb chain & the c-IAP polyUb chain

RIP1 polyUb chain acts as a scaffold for TAK1/TAB kinase complex

c-IAP polyUb chain acts as a scaffold for IKK complex (I-kB kinase (IKK) complex comprising NEMO, IKK1, IKK2) & LUBAC (Linear Ub chain assembly complex)

Process of Phosphorylation of I-κB

1. TAK1/TAB kinase complex activates the IKK complex by phosphorylating IKK1

2. IKK phosphorylates I-κB

How is NF-κB kept inactive in the cytosol

Binding to IkB

What happens when IκB is phosphorylated?

It is tagged with K48-linked polyubiquitin and degraded by the proteasome.

What happens after IκB degradation?

NF-κB translocates into the nucleus, where it activates transcription of pro-survival genes.

Name 6 effects of activation of NF-kB

How does TNF-R1 signalling lead to cell death

1. TRADD recruits FADD

2. FADD recruits pro-caspase-8

3. Proximity-induced auto- activation of caspase-8

4. Activation of downstream caspases

5. Cell death

What two key factors determine whether TNF-R1 signalling leads to cell survival or death?

Timing of recruitment of specific components to the complex

Location in the plasma membrane

How does timing influence TNF-R1 signalling outcome?

Early recruitment of pro-survival complexes (Complex I) favours NF-κB activation and cell survival

Delayed or altered recruitment favours formation of pro-death complexes (Complex II) leading to apoptosis.

What in complex I inhibits caspase-8 binding to complex 2

cFLIP

What in complex I inhibits activity of caspases including caspase-3

XIAP

How does receptor location in the plasma membrane affect TNF-R1 signalling?

TNFR clustering within lipid rafts promotes NF-κB activation and survival

Localisation in non-raft regions favours apoptosis and death signalling.

How is TNF signalling implicated in autoimmune diseases like rheumatoid arthritis?

Excess TNF causes chronic inflammation, leading to diseases such as rheumatoid arthritis.

What are 3 examples of anti-TNF therapeutics and their mechanisms

Infliximab: Chimeric human-murine monoclonal antibody that binds and inhibits TNF-α.

Adalimumab: A recombinant human monoclonal antibody that binds and inhibits TNF.

Etanercept (Enbrel): Soluble TNFR-Fc fusion protein that inhibits TNF-α from binding to cell surface TNF receptors.

What are the steps involved in Regulated necrosis: NETosis

1. Pathogen phagocytosis

2. Oxidative stress/reactive oxygen species production

3. DNA de-condensation (dissociation of histones)

4. Gasdermin activation

5. Membrane permeabilization

6. Release of DNA net

7. Capture of extracellular pathogen by DNA

8. pathogen-DNA globule clearance by professional phagocytes (e.g. macrophages

Why might inhibition of pyroptosis components, such as PRRs and gasdermins be used clinically

Pyroptosis may result extensive cytokine and DAMP (damage-associated molecular pattern molecule) release

This results in immune overactivation

Overactivated immune cells attack non-damaged/non-infected cells causing tissue damage

E.g.: sepsis, Autoimmune and inflammatory diseases: Crohn’s disease, automimmune arthritis, colitis, multiple schlerosis

Inhibition of pyroptosis components, such as PRRs and gasdermins can reduce tissue damage and are in current testing for the therapy of chronic inflammatory conditions