Lecture 2: Cell Death (Apoptosis)

Importance of Cell Death

• Important in normal tissue function

  • Shapes bodies as we develop      

• MILLIONS of cells die in you every SECOND.

  • replaces damaged/old cells killed by the immune system with new cells – vital to everyday health

Why May Cells Die

• They reach the end of their natural life

• They become damaged or acquire mutation

• They are infected and killed by the immune system

Turnover

• cells die at the same rate as they are produced

How Does Apoptosis Act As Quality Control

• in development it removes abnormal and non-functional cells

Diseases Caused By Defects in Cell Death Regulation

• Degenerative disease - excess cell death (e.g. neurone/ cell loss)

• Immune system can kill own cells - type 1 diabetes - cells recognised as foreign

• Cancer Cells - oncogenic mutation = resistant to apoptosis = tumour

• Ischemia/ reperfusion - death of surrounding cells = extensive damage following stroke and heart attack

Type of Cell Death: Necrosis

• Swelling of cell

• Loss of plasma membrane integrity

  • Plasma membrane has burst and released its content into surroundings      

• Release of contents into surrounding tissue causes an inflammatory response - lysis

  • Problem if uncontrolled

Type of Cell Death: Apoptosis

• Shrinkage of cell  

• Plasma membrane integrity intact

  • Contents not released into surroundings

• Signals are released into surroundings – initiate a response    

• Cell forms apoptotic blebs that are phagocytosed by macrophages

Type of Cell Death: Autophagy

• Maintenance of plasma membrane integrity      

• Cell creates large vesicles containing its contents and then digests it  

  • Associated with cells survive when nutrients are scarce/ can also kill off cells      

• Organelles are broken down and reused as nutrients

  • And may not be cell death.. 

Cell Response to Death

• Cell receives a signal e.g. UV damage/ T-cells recognising infected cells

• Cell recognises signal and causes signalling pathway proteins to activate cell death

• This leads to the execution phase = cell death

Execution Phase

• Cell dies and releases signals that affect how the body responds

• It is specific to the type of cell death - specific to the initial signal and cell type

Importance/ Benefit of Apoptosis

• It protects from infected cells, damaged cells, or unwanted cells

• Apoptosis will minimise collateral damage to the tissue     

  • It achieves this as apoptotic cells are phagocytosed by other cells

  • Controlled removal - is an active process regulated by proteinase

Process of Apoptosis

• Active process regulated by proteinases enzyme in the signalling pathway

• Membrane blebs which break into apoptotic bodies - plasma membrane containing fragments

  • Small enough to be engulfed

• Cell surface alters to attract phagocytes

• Pathway occurs quickly

• These changes require energy – apoptosis uses ATP      

• Once the cell commits to dying it does

• The cell must have everything it requires to carry this process at any time

Proteinases

• Enzymes that control a predictable set of morphological changes in cells regardless of cell type

  • Cell shrinkage

  • Cytoskeleton collapses  

  • Loss of nuclear membrane  

  • Chromatin condenses and DNA is cleaved into fragments

Signal Transduction Pathway

• a signal (e.g., hormone or ligand) binds to a receptor, which activates a signalling enzyme.

• This enzyme transmits the signal downstream to proteins that carry out the biological effect.

  • e.g. kinases that phosphorlyate substrates such as transcription factors to activate gene expression; this can be deactivated by phosphatase to remove the PO4^-

Why is Apoptosis Said to Be A One Way Pathway

• Once the signalling pathway is activated, and the signalling proteinase enzymes are activated and cut the protein substrate, changing its activites, the substrate can’t join back together

  • Pathway can’t be turned off

Caspases

•  Endopeptidases that cleave within a protein

• They are Cysteinyl aspartate proteinases

  • Cystine present in the active site      

  • Cysteine proteases that cleave after an aspartate in the substrate -unusual      

• They cleave specific substrates/sequences and specific sites

  • Cuts 1 or twice to change function – no degradation      

• Not all of these enzymes are involved in apoptosis - e.g. ICE (interleukin-1 converting enzyme)/caspase-1      

• Those involved in apoptosis are present as pre-cursors in the cytosol

• Their activation is the on/off point for apoptosis

Cellular Substrates of Caspases

• They dismantle the cell

• There are many of these substrates present, each recognised by a caspase due to a specific sequence present

• When cleaved and cut, it results in the activation of their function, where these substrates act as a signalling molecule to turn on downstream pathways

Caspase Activated DNase (CAD)

• A caspase within apoptotic cells that cuts DNA to package and digested in phagocytic cells

• It is kept inactive by an inhibitor

• When the inhibitor is cleaved by caspases during apoptosis, it releases DNASE, which then forms a dimer to cut DNA between histone complexes.

• It is activated by caspase signalling, which then acts on downstream effectors.

Phospholipase Release of Signals From Apoptotic Cells

• An enzyme that is constitutively activated following the cleavage of caspases, and converts phosphatidylcholine into lysophosphatidylcholine (soluble) and allows it to diffuse away and signal to and recruit phagocytic cells in apoptosis

  • Allows the release of signals from apoptotic cells via the modification of lipids in the plasma membrane

XKr8

• A lipid scramblase that when activated flips phospholipids between the inner and outer leaflet of the plasma membrane

  • phosphatidylserine normally only on the inner leaflet

• The scramblase is cleaved and activated by Caspase-3, which causes PS to be flipped into the outer leaflet of the membrane and acts as a receptor for phagocytic cells

Classification of Caspases

• Grouped based on structure and activation into 2 types

  • Executioner caspases have small pro–domain–cleave substrates

  • Initiator caspases - large pro-domain on N-terminus – turn on and cleave executioner caspases

Process of Caspase Activation

• They are expressed as inactive proenzymes and become activated during apoptosis   

• A signal activates a receptor, turning on initiator caspases which cleaves and activates the executioner caspases that will cleave substrates

Executioner Caspases

• Includes Caspase 3, 6 and 7

• They are activated by the proteolytic cleavage by initiator caspases

• Their pro-enzyme exists as an insoluble dimer dimer, with 2 active sites made up of cystine, histidine and arginine

  • a loop is present to prevent amino acids from forming an active site

Activation of Executioner Caspases

• Cleavage of the loop between the amino acids cystine, histidine and arginine by initiator caspases to form the active site and activate the catalytic activity of the caspase

How Do Initiator Caspase Result in An Amplification of the Caspase Cascade:

• They activate many copies of one or more executioner caspases resulting in the amplification of the cascade

• This leads to irreversible, destructive effects, such as:

  • Cleavage of nuclear lamins by caspase-6

  • Activation of endonucleases by caspase-3 to cut DNA

Initiator Caspases

• Includes Caspase 8 and 9

• Their pro-enzyme exists as inactive monomers that are activated via induced dimerisation via the pro-domain

• K_d for dimerization  ~ 50 µM, thus cytosolic caspase 9 is an inactive monomer

  • The concentration that they present in cytosol, means they are unable to dimerise spontaneously

• The function is to activate the executioner caspases – carry out apoptosis

• They use one of 2 pathways to be activated

  • the extrinsic or the intrinsic mitochondrial pathway

Activation of Initator Caspases

• Activated by induced dimerisation via the pro-domain

• The pro-domain at the  N-terminus will allow for an apoptotic signal to result in dimerisation

• This triggers the assembly and activation of specific adaptor protein complexes that recruit identical initiator caspases (monomer) to form a larger activation complex to be activated in response to a signal that will then activated apoptosis

Large Pro-domain of Initator Caspases

• They are protein-protein interaction domains that will bind to another domain in a different protein

  • a protein interaction module that allows recruitment to the activating adaptor

• There are 2 types

  • CAD – found on Caspase 9

  • DED – found on Caspase 8

Apoptosome

• A large multimeric complex that results in the activation of caspase 9

• It is composed of APAF-1 which binds to Cytochrome C at the Wd-40 domain, allowing its activation and the subsequent formation of a heptameric complex, with APAF-1s CARD domains exposed in the centre which then binds to Capsase 9 resulting in its activation to the interaction of its CARD domains with that of APAF-1 which recruits many caspase 9 molecules and results in a locally high concentration allowing for its induced dimerisation and activation

• It also contains

  • NOD domain binds ATP/ dATP and can hydrolyse to ADP/dADP

APAF-1

• A cytosolic monomer is activated by binding to cytochrome C

• This causes it to assemble into a heptameter

  • When activated its CARD domain is able to bind to the CARD domain of caspase 9

Cytochrome C

• A small membrane protein in the intermembrane space that has a distinct role in life and death

• Involved in electron transport and apoptosis

• It is sequestered in the mitochondria to prevent APAF-1 activation and cell death

• When bound to APAF-1, it activates APAF-1 and causes it to undergo a conformational change to form a heptameric ring structure H

Importance of CARD Domains of Heptameric APAF-1

• The CARD Domains of APAF-1 are exposed to the centre of the complex, to allow it to bind to the caspase 9s CARD domains and thus allow the recruitment of Caspse 9 to the ‘apoptosome complex

• This allows a high concentration of Caspase 9 locally allowing the dimerisation of caspase 9, in response to Cytochrome C

APAF-1 in Non-Apoptotic Cells

• It is folded in on itself so that the WD domain is kept in an autoinhibited state

• It is unable to bind to dATP or Caspase 9 as the CARD domain is not exposed

Unfolding of APAF-1

• Occurs in the presence of cytochrome c following the interaction at the Wd-40 domain

• This induces a conformational change that results in the hydrolysis of dATP to ADP and causing the exposure and unfolding of APAF-1

• Once unfolded it loses ADP, to bind to more dATP allowing APAF-1 to oligomerise into a heptagonal structure where the CARD domains are clustered to facilitate the recruitment of and interaction with Caspase 9’s CARD domains to allow for induced dimerisation and activation of caspase

Activated Caspase 9

• Once activated it cleaves itself to be released from the apoptosome in an active structure as a dimer to activate and cleave executioner caspases (3,7) which rapidly process substrates that drive apoptosis

BCL-2 Family Proteins

• They control apoptosis by mediating mitochondrial outer membrane permeabilisation

• These proteins sense and respond to cellular damage by forming holes in the outer mitochondrial membrane

• They are located in the mitochondria and tightly regulate the intrinsic pathway to ensure cells kill themselves when suitable

  • Some activate apoptosis, while others inhibit it to keep the cells alive

Mitochondria

• A key signalling nexus for activating apoptosis due to the presence of cytochrome C

  • It is important in the sequestration of this substrate that it is vital for the activation but also the inhibition of apoptosis, as the cell contains all the components for apoptosis, and so they must be kept separate

• Functions include:

  • Aerobic respiration

  • Lipid synthesis (fatty acids, steroids, membrane biosynthesis)

  • Amino acid metabolismm

  • Oxidative phosphorylation → ATP synthesis

Genome of the Mitochondrial Matrix

• It has its own genome and contains 37 genes

  • 20 tRNA,

  • 2 rRNA,

  • 13 proteins in the inner membrane     

• Produces 1500-2000 proteins in total

Mutation in APAF-1 Binding Site

• Does not alter respiration but does block apoptosome formation

Cytochrome C Release

• Occurs immediately before caspase activation from the mitochondrial and is the point of no return for a cells → initation of Apoptosis

• Its release is controlled by the BCL-2 family proteins

• It involves the intrinsic pathway of apoptosis

Follicular Lymphoma

• A cancer of B-cells that involves BCL2

• Chromosomal translocation present → t14;16

  • Part of chromosome 18 (containing BCL2 gene) is swapped onto chromosome 14 (IgH gene)

• This reciprocal translocation places BCL2 (18q21) near the IgH heavy chain region (14q32), causing an overproduction of BCL2 in lymphoma cells

  • Key oncogenic event - with BCL2 prevention apoptosis, but does not cause cellular proliferation

    • B-Cell 2 lymphoma gene was swapped with the gene that synthesised

• Defective VDJ rearrangements during B-cell development contribute to this event.

BCL-2

• The first identified protein of the BCL-2 family

• Prevents apoptosis → anti-apoptotic member of the protein family

Bax

• Bcl-2 associated X protein) functions to promote cell death and antagonise Bcl-2 → Activates apoptosis and is a proapoptotic member of the BCL-2 family proteins

  • Opposite function to BCL2

BCL-2: Bax Ratio

• Ratio of activity determines whether the cell lives or dies

Structure of BCL2 Family Proteins

• Many types of proteins, each having a shared domain structure

• They all share a common amino acid structure → BCL2 homology/BH Domains

Pro-Apoptotic Members of BCL-2 Family Proteins

• Proteins that form holes/ pores in the lipid membranes

  • promote OMM permeablisation

• They allow cytochrome c release from mitochondria to kill the cell

Anti-Apoptotic Members of BCL-2 Family Proteins

• Proteins that can directly bind to pro-apoptotic members and stop pore formation

  • Block OMM permeablisation

BH-3 Only Proteins

• The 3rd group of BCL-2 proteins

• Have a distinct domain and regulate anti- and pro-apoptotic members of the BCL-2 protein family → share a small region of sequence similarity

  • Can either inhibit or activate either of the members

• They are activated by a range of damage signals received by the cell and in response determine how the anti- and pro-apoptotic signals interact

  • Detect DNA damage or insufficient growth factors

• Bind to and activate Bax and Bak - initiate apoptosis

• Bind to antiapoptotic signal to block apoptosis → block binding Bax and Bak

• Important in drug formulation

BH-3 only Proteins in Response to DNA Damage/ Insufficent Growth Factors

• Detection of this damage can initiate the proteins expression and in response can turn off anti-apoptotic signals and the subsequent release of cytochrome C

Interaction Between Pro- and Anti-Apoptotic BCL2 Proteins (&BH3)

• They interact on the OMM to regulate MOMP

  • A balance between the 3 sub groups determines the permeabilisation of the membrane

• Bax and Bak are located on the outer mitochondrial membrane (MOM).

• They interact with BCL-2 to prevent pore formation that drives MOMPS and apoptosis.

BH-3 Domain (Structure)

• A short amphipathic a-helix that binds to other BCL-2 proteins

Activation of BH-3 Only Proteins

• Occurs in response to DNA damage and is sequestered by anti-apoptotic BCL-2 proteins.

• If damage persists, more BH3 is activated and inhibits BCL-2, leading to Bax/Bak activation and incudes a conformational change that results in outer mitochondrial membrane permeabilisation and subsequent cell death

Consequence of t14; 18 Chromosomal Translocation

• Results in the overexpression of BCL-2 that prevents BH3 from inhibiting it, and makes the cell resistant to apoptosis.

• The threshold for apoptosis is shifted, and the cell avoids MOMPS due to a failure in exceeding said threshold.

  • Cells unable to reach the level of damage where pore formation and apoptosis occurs

p53 Mutation

• An example of a mutation within a signalling pathway that responds to stress and the activation of BH3 proteins

• Normally, it activates BH3-proteins (e.g., PUMA) in response to DNA damage.

• Mutations prevent this response, so no BH3 is activated, and apoptosis is blocked.

  • no MOMPs

BH-3 Mimetics

• Drugs that mimic BH3 proteins and that can bind to and inhibit anti-apoptotic BCL-2, resetting the apoptosis threshold.

• This allows chemotherapy-induced damage to activate Bax/Bak and trigger cell death in cells over expressing BCL-2

Consequences of Mutation in Oncogenes and TSGS

• They shift the threshold for BCL-2 regulation of MOMPs

  • By understanding this can allow for the development of novel anti-cancer drugs

BAD

• BH-3 proteins that is regulated  by growth factor receptor tyrosine kinase signalling

• If a growth factor receptor e.g. epidermal growth factor turns on, it activates a signalling pathway involving protein kinase that phosphorylates the BH-3 protein

  • The site of phosphorylation in the protein binds to 14-3-3, which sequesters the protein and keeps it away from the mitochondrial and keeps the pathway off so that there is no apoptosis/ cell death

Consequences of a Cell Loosing Growth Factor Signalling

• The pathway is turned off and BAD is dephosphorylated and can bind to other mitochondrial proteins

• This leads to the permeabilising of the mitochondria and the activation of apoptosis

How do oncogenic mutations and imbalances in Bcl-2 proteins contribute to cancer cell survival?

• They can alter the balance between pro- and anti-apoptotic Bcl-2 proteins, promoting cancer cell survival.

• In cancers like HER2, excess receptor kinases lead to constant growth factor receptor signaling, preventing dephosphorylation of proteins and inhibiting cell death.

• In lymphoma, overproduction of anti-apoptotic proteins removes dephosphorylated BAD, preventing BAX activation, and keeping the cell alive by disrupting the apoptotic balance.

p53

• A tumour suppressor

• It is mutated in 50% of cancers,

• It is involved in complex events downstream of DNA damage

• in normal cells is activated by PK responding to damage – is phosphorylated and turned on

• if mutated – downstream functions are stopped

• It acts as a transcription factor of BH3 proteins (PUMA) in apoptosis

Consequence of Non-Functional p53 in Cancer Cells

• Cells can’t die

  • Genomically unstable due to an incorrect respones to DNA damage

• Chemotherapy drugs used to activate BH-3 only proteins would not be useful in these case

When Is PUMA Transcriptionally Upregulated

• DNA damage activates PUMA transcriptionally.

• High levels of DNA damage = p53 activation → up-regulation of PUMA.

• PUMA activates BAX and inactivates BCL2.

• This leads to Cytochrome C release and cell death.

BH-3 Binding Domain

• It can either activate the pro-apoptotic activity of Bax and Bak or be sequestered by binding the anti-apoptotic proteins like Bcl-XL and Bcl-2.      

• e.g. BAD-BH3 a-helix domain of BAD binds to the hydrophobic groove on the BclXL surface

  • potential to design drugs that mimic BAD-BH3 to bind to the groove and activate pro-apoptotic activity. 

How was structure used to design compounds mimicking the BH3-domain?

• NMR spectroscopy identified small molecules binding to the same region as the BH3-domain.

• Structure-based optimization was used to increase specificity.

• Two low-affinity compounds were combined to create a high-affinity binder.

• Lead compound (ABT737), a novel BH3-mimetic, binds strongly to 3 anti-apoptotic BCL2 proteins.

• Inhibition of these proteins leads to tumour regression.

• Further modification is conducted to increase target specificity and reduce side effects → create an orally active variant

Ventoclasx

• A drug that inhibits BCL2 and is used for leukaemia with mutations that make them susceptible to BCL2 inhibition.

• It acts as a BH3 protein, encouraging cell death and restoring the balance between pro- and anti-apoptotic protein

Intrinsic Mitochondrial Pathway of Apoptosis

• It involves 3 main components

  • Signalling

  • Integration

  • Execution

Intrinsic Mitochondrial Pathway of Apoptosis: Signalling

• Signals, both internal and external to the cell, trigger the apoptotic pathway. 

  • Signals come from various sources, e.g. growth factors, hormones, or cellular stress.   

Intrinsic Mitochondrial Pathway of Apoptosis: Integration

• Cell receives and processes apoptotic signals and assesses whether the cell should undergo apoptosis based on the balance of pro-apoptotic and anti-apoptotic signals

• Regulated by BcL-2 family proteins

  • Bcl-2 can inhibit it, while others e.g. Bax and Bak can promote it.  

Intrinsic Mitochondrial Pathway of Apoptosis: Execution

• If the decision is made to proceed with apoptosis, the execution phase begins

• It involves a cascade of events that lead to the breakdown of the cell.   

Sensor Proteins

• Proteins that detect apoptotic signals and initate the process

Nuclear Fragmentation and Chromatin condensation

• Visible signs of apoptosis - nucleus breaks down and the DNA condenses

Phagocyte Recognition and Protein Degredation

• Once the cell is broken down, its remnants are cleared away by specialized cells called phagocytes

Regulation of The Apoptsome

• Dimerisation activates initiator caspases, not cleavage.

• Activated caspase 9 cleaves itself within the apoptosome to release and activate other caspases.

• Cleavage of caspase 9 stabilizes it and releases it from the apoptosome, which is required to trigger apoptosis.

Apoptotic or Mitochondrial Priming

• The differing sensitivity of cells to apoptotic signals.

• This sensitivity is due to differential levels of BCL2 expression in cells.

• The degree of apoptotic priming influences how cancer cells respond to chemotherapy.

• It can also affect the severity of toxic side effects from chemotherapy and radiotherapy.