Chapter 29: Apoptosis

Question 1

Necrosis

Answer 1

• Cell death due to some type of acute injury such as trauma or lack of blood supply
  
  • Cells swell, burst, and spill their guts
  • Causes an inflammatory response at the site of injury and potentially a dangerous systemic response

Question 2

Apoptosis

Answer 2

• Programmed cell death: activation of a specific biochemical pathway leading to cell death and removal by a macrophage or neighboring cell
  
  • Apoptosis is inherent to all metazoan cells
  • 300 million cells die in our bodies every minute (blood, intestinal, skin cells)
  • Also occurs with genetically damaged cells
  • These are exactly replaced by cell replication

Question 3

When Apoptosis is a Good Thing

Answer 3

• Development/Regeneration
  
  • body part sculpting during development
  • death to neurons that don’t connect (about 50% of neurons)
  • death to immune cells that are duds or recognize self
  • turnover of intestinal, skin, and bone marrow cells
• Cancer prevention
  
  • death to cells that have incurred heavy DNA damage, are not well fed, or have suffocated

Question 4

When Apoptosis Is Not So Good

Answer 4

• Neurodegenerative diseases (Alzheimer’s disease; Parkinson’s disease; ataxias; etc.)
• AIDs
• Ischemic injury (myocardial infarction; stroke)
• Alcohol-induced liver disease

Question 5

Symptoms of Apoptosis

Answer 5

• Cytoskeleton collapses; cells becomes more compact
• Nuclear envelope disassembles and chromatin condenses
• Nuclear DNA breaks up into nucleosomal-sized fragments**
• Mitochondrial function is lost
• Phosphatidylserine flips from the inside to the outside of the plasma membrane bilayer**
• Plasma membrane is altered (blebbing), allowing dying cell to be engulfed by macrophages via phagocytosis

Question 6

TUNEL (Terminal deoxynucleoidyl transferase dUTP nick end labeling)

Answer 6

Assays add a fluorescent nucleotide to DNA ens. The DNA ends are extended with the template-independent DNA polymerase terminal deoxynucleotidyl transferase (TdT). TdT adds flluorescently labeled dUTPs to the ends of fragmented DNA. Large numbers of DNA fragments leads to bright fluorescent dots in apoptotic cells.

Question 7

Caspases: The Mediators of Apoptosis (and Infllammation)

Answer 7

• Apoptosis is mediated by a cascade of highly specific proteolytic cleavages by a family of proteases known as caspases
• Caspases utilize an active site cysteine residue as the mechanism of hydrolysis
• Caspases cleave the peptide bond at a specific four amino acid sequence on the C-terminal side of an aspartate residue
• Some caspases (caspases 1, 4, and 5) are involved in inflammation rather than apoptosis
• Caspases are synthesized as inactive proenzymes that need to be activated by cleavage to remove an inhibitory pro-peptide segment.

Question 8

Initiator Caspases

Answer 8

• Caspases 2, 8, 9, and 10
• Exist as inactive, soluble monomers in the cytosol before the apoptotic signal
• Function is to activate the executioner caspases

Question 9

Executioner (Effector) Caspases (cut target proteins)

Answer 9

• Caspases 3, 6, and 7
• Exist as inactive dimers in the cytosol before the apoptotic signal

Question 10

CAD (caspase-activated DNase)

Answer 10

• the enzyme responsible for DNA cleavage during apoptosis
  
  • CAD is normally inhibited by an inhibitor (iCAD)
  • Executioner caspases inactivate iCAD by cleaving it
  • This releases an active CAD, which fragments DNA between nucleosomes

Question 11

A Few Examples of Target Proteins Degraded by Executioner Caspases

Answer 11

• CAD
• Executioner caspases cleave nuclear lamins, the structural protein responsible for the nuclear envelope and nuclear integrity
• Executioner caspases degrade Mdm2, the E3 ubiquitin ligase that targets p53 for ubiquitination and thus proteasomal degradation. Thus, without Mdm2, p53 remains high and can signal cells to stop proliferating and to die.

Question 12

Overview of Caspase Activation

Answer 12

Caspases are originally made in an inactive, procaspase form.

• Extrinsic or intrinsic signals trigger the assembly of adaptor proteins with the procaspases.
• Upon binding of the adaptor proteins (FADD or Apaf), the procaspases dimerize and are activated, cleaving the procaspase into a large and small subunit. (The prodomain is degraded.)
• The initiator caspases then clip the executioner caspase dimer, and the dimer rearranges and is active.

Question 13

Caspases Function in Cascades that Can Greatly Amplify the Initial Signal

Answer 13

• Initiator caspases activate the executioner (effector) caspases by clipping off their prodomains.
• The catalytic activation creates an amplification step as one initiator caspase can activate many executioner caspases.

Question 14

2 Pathways to Apoptosis

Answer 14

• All metazoan cells express procaspases and thus express the proteins necessary for their own destruction
• Initiation of apoptosis can occur in response to either external signals (extrinsic pathway) or internal signals (intrinsic pathway)
• In either case, protein:protein interactions mediated by specific modular interaction domains are particularly important to these signaling pathways

Question 15

DD

Answer 15

Death domain

Question 16

DED

Answer 16

Death effector domain

Question 17

CARD

Answer 17

Caspase-associated recruitment domains

Question 18

Extrinsic Apoptotic Pathway

Answer 18

• Activated by something outside the cell
• Death signals:
  
  • TNFalpha
  • Fas ligand (FasL)
  • Both TNFα and Fas ligand are cell surface proteins and they bind to specific receptors on adjacent cells (contact-dependent signaling) that are members of the TNF receptor family (TNF, FAS, TRAIL), also known as the death receptors

Question 19

TNFα (tumor necrosis factor α)

Answer 19

Secreted by macrophages and triggers cell death in chronic inflammatory diseases

Question 20

Fas ligand (FasL)

Answer 20

Cell surface protein produced by activated natural killer cells and cytotoxic T lymphocytes to kill of viruses, foreign graft cells, some tumor cells, etc.

Question 21

Death Receptors

Answer 21

• Transmembrane proteins that have an extracellular ligand binding domain, a single transmembrane domain, and an internal death domain (DD)
• Receptors (and ligands) are homotrimers
• Liganded death receptros cluster and interact with the FADD (Fas-associated death domain) adaptor proteins
• FADD recruits an initiator caspase (caspase 8) with a death effector domain (DED)

Question 22

Death by FasL Is the Best Characterized Extrinsic Pathway

Answer 22

• The extrinsic pathway starts with a ligand trimer binding to a death receptor trimer such as Fas.
• The receptor has a DD that attracts an adaptor protein such as FAAD that has both a DD and a DED.
• The adaptor protein attracts a complex that contains several initiator caspases (typically caspases 8 and/or 10)

Question 23

he Receptor/Adaptor Complex Recruits the Initiator Procaspase

Answer 23

• The aggregate of receptor, adaptor and procaspase is called DISC (death-inducing signaling complex).
• The aggregation of the procaspases causes them to cross-activate each other.

Question 24

The Initiator Caspases Activate the Executioner Caspases, Notably Caspase-3

Answer 24

Once the procaspases are active, then two associate form an active dimer.

This dimer cleaves itself in the region by the DD, which frees up the initiator caspase dimer so it can move around the cytosol and activate executioner caspases.

Question 25

More Notes on the Extrinsic Pathway

Answer 25

* Six different types of receptors that can trigger apoptosis are known; all have a related death domain in their cytosolic region * Procaspases-8 and -10 can activate themselves because the procaspase has a small amount of basal proteolytic activity; when many molecules are brought together, one will activate its neighbor, initiating a chain reaction\*\*\* * The extrinsic pathway is called into play in chronic inflammatory diseases, tissue grafts/transplants, immune response * Many viruses express proteins that bind to the death domains of receptors to inhibit interaction with FADD. Why?\*\*\*

Question 26

The Intrinsic Pathway of Apoptosis Is Activated by the Cell Itself

Answer 26

* The intrinsic (internal) pathway is initiated by a number of different signals including extensive DNA damage, lack of O2, lack of nutrients, or the absence of growth factor signaling. * The intrinsic pathway is activated by release of cytochrome c from inside the mitochondria. * Released cytochrome c binds to the procaspase activating adaptor protein Apaf-1 (apoptotic protease activating factor 1), causing its aggregation into an apoptosome. * Apaf-1 contains the CARD protein interaction domain, which binds the initiator procaspase 9. * Procaspase 9 is activated by the apoptosome.

Question 27

What triggers the release of cytoc

Answer 27

* The Bcl2 family of proteins (15 members) control the release of cytochrome c. * Pro- and anti-apoptotic Bcl2 proteins bind to each other to form hetero-oligomers. * The balance between the activities of the pro- and anti-apoptotic proteins determines whether a cell lives or dies by the intrinsic pathway.

Question 28

Bcl-2 family members come in two functional classes:

Answer 28

–Anti-apoptotic (survival): e.g., Bcl2 –Pro-apoptotic (death): e.g., Bad, Ba

Question 29

Anti-Apoptotic Bcl2 Family Members

Answer 29

* Bcl2 (and other anti-apoptotic family members) reside in the outer mitochondrial membrane * Act mainly by binding to and inhibiting the action of pro-apoptotic family members, i.e. Bcl2 is going to inhibit Bax * Act to prevent inappropriate apoptosis * Five members in mammalian cells; at least one must be expressed for cell survival!

Question 30

Bcl22 Inhibits Bax (BH123 Proteins) in the Absence of an Apoptotic Signal or When RTKS are Activated

Answer 30

* Anti-apoptotic Bcl2 proteins bind to and inhibit the BH123 (Bax) proteins, preventing clustering, cytochrome c release, and thus apoptosis. * The Bcl2 proteins are actually bound to the mitochondrial surface, although not shown here.

Question 31

Pro-Apoptotic BH123 Family Members

Answer 31

* BH123 (Bax) family members are found on mitochondrial membrane or free in the cytoplasm * In response to an apoptotic signal for the intrinsic pathway, Bax (BH123 family) is activated and forms oligomers in the outer mitochondrial membrane * Formation of the oligomers forms a pore that allows release of cytochrome c

Question 32

Examples of Aberrant Apoptosis Contributing to Disease- Too many cells die by apoptosis:

Answer 32

* Neurodegenerative disorders—the progressive loss of neurons * Parkinson’s * Alzheimer’s * •Huntington Chorea * Stroke * Schizophrenia (?) * Ischemic damage (loss of oxygen due to heart attacks or strokes; often includes both necrosis and apoptosis,) * Aids * Developmental disorders: disruption of brain development by blocking apoptosis

Question 33

Examples of Aberrant Apoptosis Contributing to Disease- Too few cells die by apoptosis

Answer 33

* Mutations in FasL or FasR lead to excessive numbers of lymphocytes , which can cause autoimmune diseases (when lymphocytes react against the person’s own cells: rheumatoid arthritis, lupus, multiple sclerosis, myasthenia gravis, etc.) * Cancer: one of the hallmarks of cancer is that damaged cells evade apoptosis.

Question 34

Apoptosis and Cancer

Answer 34

* The normal defense to loss of growth control in a cell is the induction of apoptosis. * However, blocking the normal apoptotic pathway is essential for cancer development and maintenance. * Therefore, many components of apoptotic signaling pathways have been identified as oncogenes or tumor-suppressor genes in human cancers.

Question 35

Bcl2 Is One Example of an Aberrant Anti-apoptotic Protein that Causes Cancer

Answer 35

* Bcl2 = B-cell lymphoma2 (follicular lymphoma) * This gene was first identified in a common type of lymphoma, where a chromosomal translocation between chromosomes 14 and 18 puts the Bcl2 gene under the regulation of the immunoglobulin gene enhancer. The result is that too much Bcl2 is made, and these cells essentially become immortal. * A number of drugs are being developed to interfere with anti-apoptotic Bcl2 family proteins. These drugs bind with high affinity to the hydrophobic groove on these proteins and act as a BH3 (Bax) protein mimetic.

Question 36

Mutations in p53 Also Contribute to Inappropriate Cell Survival

Answer 36

* p53 is a transcriptional activator that accumulates in response to many types of DNA damage * p53 causes cells to arrest the cell cycle in response to DNA damage until the damage is repaired * If DNA damage is too extensive, p53 induces the expression of pro-apoptotic family members (e.g., Bax) to trigger apoptosis * Hence, p53 is pro-apoptotic, and it is the most commonly mutated tumor suppressor gene in humans