Exam 2- Chapter 18- Apoptosis

Question 1

“Silent” cell death

Answer 1

Cell death that is not inflammatory or does not involve the immune system. This includes apoptosis, eryptosis, and cornification. During apoptosis, the cell shrinks, the nucleus and other organelles break apart, and eventually the entire cell ends up breaking apart into pieces called apoptotic bodies

Question 2

“Alarming” cell death

Answer 2

These pathways do induce an immune response and cause inflammation. This includes necroptosis, programmed necroptosis, erythrocyte necroptosis, pyroptosis, ferroptosis, and parthanatos

Question 3

Importance of apoptosis

Answer 3

Senescent (aging) cells undergo apoptosis so they can be removed from the body. It is also used for the elimination of damaged cells. This could be due to injury or infection, sometimes to the advantage of the pathogen. Apoptosis is also common in developmental biology as embryos/fetuses are developing. This is one of the reasons why these processes need to be non-inflammatory- it prevents damage to developing organs

Question 4

Defining features of apoptosis (6)

Answer 4

1. Silent- not inflammatory
2. Apoptotic bodies
3. Nuclear fragmentation, DNA and organelle breakdown occurs in a specific way
4. Cytochrome c is released from the mitochondria into the cytoplasm
5. Phosphatidylserine rearrangement and detection
6. Caspase-dependent

Question 5

Apoptotic bodies

Answer 5

When the cell breaks apart into pieces during apoptosis. The cell does not lyse, everything is neatly packaged. Apoptotic bodies have phosphatidylserine exposed so they can be recognized by phagocytic cells

Question 6

Phosphatidylserine

Answer 6

A membrane lipid on the cytoplasmic half of the membrane in a healthy cell. It is flipped out to the external side ONLY when apoptosis occurs. Macrophages and other phagocytes have phosphatidylserine receptors, so they are able to recognize apoptotic cells/bodies and remove them

Question 7

Caspases

Answer 7

Cysteine-aspartate proteases-they are the key signaling proteins of apoptosis. They are proteases and therefore cleave other molecules to pass the signal along. The caspases themselves also have to be cleaved to be activated, as they are made in an inactive procaspase form. Caspases can be cleaved at one or two specific aspartate residues- cysteine is the active part of the caspase that is cleaving the aspartate component

Question 8

DNA fragmentation

Answer 8

DNA breaks apart during apoptosis- this can be measured experimentally. The DNA is extracted and run on an agarose gel. As time goes on, more and more of a laddering pattern appears on the gel. This laddering pattern is broken down DNA, indicating DNA fragmentation

Question 9

Phosphatidylserine externalization

Answer 9

In healthy cells, phosphatidylserine is always found on the inner half of the membrane. During apoptosis, phosphatidylserine is flipped to the outer surface of the membrane. This helps phagocytic cells to recognize apoptotic cells

Question 10

Caspase cascade

Answer 10

Once it is active, the caspase can go cleave other caspases at aspartate residues. This helps to pass the signal along in the cell

Question 11

Procaspase to caspase conversion

Answer 11

The inactive procaspase can have multiple aspartate cleavage sites. During cleavage, procaspase’s prodomains (domains that keep it inactive) are removed. A second cleavage event can then occur, creating a large subunit and a small subunit of the caspase. The 2 subunits bind to one another to form a dimer, and 2 of these dimers come together to form the active caspase tetramer. It can now go cleave other caspases to activate them

Question 12

Subclasses of caspase (3)

Answer 12

1. Initiator- initiates the apoptosis process
2. Executioner
3. Inflammatory- this class is not responsible for apoptosis since it is a noninflammatory process

Question 13

Death effector domain (DED)

Answer 13

An extra domain on the initiator class of caspases. This domain is found in caspases 8 and 10 and is responsible for the association of other proteins with the caspase

Question 14

Caspase recruitment domain (CARD)

Answer 14

An extra domain on the initiator class of caspases. This domain is found in caspases 9 and 2 and is important for binding to other proteins. It is necessary to form protein complexes that activate the caspase

Question 15

Initiator caspases

Answer 15

These caspases kick off the entire apoptosis pathway. Cleave/activate executioner caspases

Question 16

Executioner caspases

Answer 16

Cleave/activate other executioner caspases. They chop proteins and are therefore responsible for all of the changes cells experience during apoptosis. Cleave target proteins- nuclear lamins (leading to fragmentation of the nucleus), inhibitor of caspase activated Dnase (ICAD- DNA fragmentation), cytoskeletal proteins (formation of apoptotic bodies), or cell adhesion proteins

Question 17

Extrinsic pathway of apoptosis

Answer 17

Stimulated from the outside of the cell. Depends on receptor-ligand interactions at the cell membrane. TNF cytokines can also initiate apoptosis when they bind to their receptors

Question 18

Intrinsic pathway of apoptosis

Answer 18

Stimulated from the inside of the cell and is stress or injury induced. It involves release of cytochrome C from the mitochondria to the cytoplasm. Intrinsic and extrinsic pathways may also be occurring at the same time

Question 19

Death-inducing signaling complex (DISC)

Answer 19

The extrinsic pathway is induced by cytokines, especially TNF cytokines- TNF-α, Fas ligand (FasL), TRAIL. They create the DISC complex when they bind to their receptors- there can be one or two DISC complexes depending on the cytokine involved. For example, the Fas ligand only creates one death complex containing caspase 8. TNF creates 2 DISC complexes- one that is connected to the receptor, and one that is further internalized into the cell.

Question 20

Fas/FasL system (3 steps)

Answer 20

1. Fas ligand and its receptor are both originally membrane proteins. Fas ligand is released from cells and membrane vesicles. Once it is released, it can bind to its receptor on the same cell or a neighboring cell
2. When this occurs, the death inducing signaling complex (DISC) forms- this includes FADD and activated caspase 8
3. Caspase 8 can then activate the executioner caspases and lead to extrinsic apoptosis

Question 21

In the Fas/FasL system, how is caspase 8 activated?

Answer 21

The Fas receptor is a trimeric receptor- it has 3 different subunits. Each of these subunits on the cytoplasmic side has a death domain, which allows it to associate with the FADD protein. The FADD protein also has a death domain itself, allowing death domain-death domain reactions to occur. Additionally, a death effector domain is located on the end of the FADD protein that extends into the cytoplasm. Procaspase 8 also has a death effector domain that can bind to the death effector domain on FADD. There are 3 procaspases due to the trimeric structure of the Fas receptor, and they are brought close enough that they undergo proximity activation, and all of the caspases are activated to caspase 8. Caspase 8 cleaves the executioner caspase and causes apoptosis

Question 22

Proximity activation

Answer 22

When neighboring molecules of caspase 8 are brought so close together that they cleave each other. The caspases are subsequently activated

Question 23

How are TNF-α and the Fas ligand similar?

Answer 23

TNF-α is another inducer of extrinsic apoptosis. It is similar to the Fas ligand in that both TNF-α and the TNF receptor are membrane proteins and are trimeric

Question 24

TNF-α apoptosis (7 steps)

Answer 24

1. TNF-α is a membrane protein that has to be released in a membrane vesicle. It binds to its TNF receptor (also in the membrane) and a death inducing signaling complex is formed- TRADD, RIP1, and cIAP1/2
2. RIP1 has a ubiquitin tag that must be removed by CYLD, a deubiquitylase, so apoptosis can occur. Once the tag is removed, the cell is on a committed track toward cell death
3. FADD and inactive caspase 8 join the complex. The TNF receptor is trimeric, so there will be 3 copies of this complex.
4. Caspase 8 is activated through proximity activation
5. Active caspase 8 cleaves the executioner caspases- 3, 6, and 7. The executioner caspases go produce the cellular changes necessary for apoptosis

Question 25

Duality of TNF signaling

Answer 25

TNF is interesting because it is capable of inducing a variety of signaling pathways. It can induce a pro-survival, pro-inflammatory pathway, but can also induce apoptosis. It can induce extrinsic apoptosis, a canonical NFkB pathway, and necroptosis

Question 26

Pro-inflammatory TNF pathway (4 steps)

Answer 26

1. In this situation, the ubiquitin tag remains bound to RIP1 in the cytoplasm
2. This allows for a protein called TGF beta-activated kinase 1 (TAK1) to bind to RIP1
3. TAK1 binding proteins (TAB) 2 and 3 can also associate with these proteins
4. This complex then promotes the activation of NF-kappa-B and transcription of pro-survival genes

Question 27

How does the protein complex in the pro-inflammatory TNF pathway promote gene expression? (5 steps)

Answer 27

1. From the protein complex, they will pass the signal over to the IKK complex- the complex is activated as it is phosphorylated
2. NF-kappa-B, a transcription factor, is constitutively bound to an inhibitor called I-kappa-B. The inhibitor prevents NFKB from going to the nucleus and acting as a transcription factor
3. However, once the signal is passed over, it goes to the inhibitor (IKB)
4. The signal marks the inhibitor for degradation, IKB is ubiquitylated and degraded
5. NFKB is then free to migrate to the nucleus, where it induces the expression of pro-inflammatory and pro-survival genes

Question 28

What induces intrinsic apoptosis?

Answer 28

Stress or injury to the cell- nutrient starvation, lack of survival signals, lack of O2, DNA damage

Question 29

Bcl2 family proteins

Answer 29

Involved in intrinsic apoptosis. They can be anti- or pro-apoptotic. This family contains anti-apoptotic Bcl2 proteins, which contain 4 domains (BH 1-4). There are also pro-apoptotic BH123 proteins (only have BH 1-3 domains) and BH3-only proteins

Question 30

How do BH123 proteins work?

Answer 30

They oligomerize and form pores in the mitochondrial outer membrane. This helps to induce the release of cytochrome C and other proteins into the cytoplasm. BH123 proteins include Bak and Bax- Bak is always in the outer membrane and Bax is recruited to the outer membrane from the cytoplasm so both of these proteins can oligomerize. BH123 proteins are pro-apoptotic

Question 31

How do Bcl2-type proteins work?

Answer 31

These proteins are anti-apoptotic because they essentially lock the BH123 proteins in place to prevent them from forming pores. Bcl2 proteins on the inner face of the outer mitochondrial membrane

Question 32

How do BH3-only proteins work?

Answer 32

These proteins are pro-apoptotic- they are produced or activated in response to an apoptotic stimulus. Then, BH3 proteins bind to or inhibit Bcl-2 proteins. This allows pores to form

Question 33

Formation of the apoptosome (7 steps)

Answer 33

1. Once the pores form in the mitochondrial outer membrane, cytochrome C is released from the mitochondria into the cytoplasm
2. At this point, released cytochrome C binds apoptotic protease-activating factor 1 (Apaf1)
3. Apaf1 then goes through a conformational change- it opens up and exposes its caspase recruitment (CARD) domain
4. Multiple molecules of cytochrome C bound Apaf1 associate with each other in a “pinwheel” fashion, via their CARD domains
5. The oligomerized Apaf1 complex recruits inactive procaspase-9 (forming the apoptosome)
6. Multiple molecules of caspase-9 are close to each other, so they are subject to proximity activation
7. Then, they can activate the executioner caspases

Question 34

Regulation of intrinsic apoptosis

Answer 34

Activation of intrinsic apoptosis, which is dependent on BH123 pores in the outer mitochondrial membrane. This is thanks to BH3 proteins, which inactivate Bcl-2 proteins. Once the pores form in the mitochondrial outer membrane, cytochrome C is released from the mitochondria into the cytoplasm

Question 35

Apoptosome definition

Answer 35

The initiator signaling complex for intrinsic apoptosis

Question 36

Extrinsic-intrinsic connection

Answer 36

Extrinsic apoptosis can be induced, with intrinsic apoptosis also occurring downstream. Caspase 8 is active in extrinsic apoptosis, but it can also cleave a BH3 only protein called bit- once this protein has been cleaved, it is activated. The protein can then go on to inhibit Bcl2 and allow BH123 proteins to form pores in the outer mitochondrial membrane. This activates intrinsic apoptosis downstream

Question 37

“Downstream” apoptosis

Answer 37

Refers to what executioner caspases go do around the cell

Question 38

Mammalian sterile-20 kinase MST1

Answer 38

This is the active form of the protein, which will be activated when cleaved by executioner caspases. The active active MST1 translocates to nucleus, and phosphorylates histone H2B

Question 39

Histone H2B

Answer 39

Responsible for chromatin condensation during apoptosis

Question 40

Condensation of chromatin (3 steps)

Answer 40

1. The executioner caspase cleaves MST1 and activates it
2. Active MST1 then enters the nucleus and interacts with the histone cores of DNA
3. MST1 phosphorylates histone H2B, which leads to the condensation of chromatin

Question 41

What causes DNA fragmentation?

Answer 41

Due to the executioner caspases cleaving an inhibitor of a DNase. The inhibitor, ICAD, is destroyed, creating the active form, CAD (caspase activated DNA). CAD catalyzes inter-nucleosomal DNA cleavage. This means that CAD cuts the linker DNA between different nucleosomes

Question 42

PARP1

Answer 42

A DNA repair protein that is cleaved by executioner caspases during apoptosis. This is because DNA is not supposed to be repaired during apoptosis, it is supposed to break down in a specific way.

Question 43

PARP1 mechanism (3 steps)

Answer 43

1. The PARP1 protein will dimerize with another PARP1 when DNA is damaged
2. PARP adds poly ADP-ribose to nuclear proteins (PARylation), leading to the relaxation of chromatin (decondensation).
3. Since PARP1 plays such an important role in DNA repair, it is cleaved an inactivated during apoptosis

Question 44

Decondensation

Answer 44

Relaxation of chromatin, which allows for greater accessibility of DNA repair enzymes

Question 45

How does nuclear fragmentation occur?

Answer 45

It occurs due to caspase mediated cleavage of nuclear lamins. Weakening of nuclear lamina = nuclear fragmentation. Executioner caspases cleave the nuclear lamins that make up the nuclear lamina- a meshwork of nuclear intermediate filaments. This causes the fragmentation of the nucleus.

Question 46

Fragmentation of the Golgi during apoptosis

Answer 46

Caspases cleave the Golgi-stacking protein GRASP65, resulting in Golgi fragmentation. When executioner caspases cleave GRASP65, there is nothing to help the Golgi stack properly. At this point, the Golgi begins to fragment

Question 47

Scramblase

Answer 47

A membrane channel that actively externalizes PS during eryptosis- allows PS to be flipped to the outer half of the cell membrane. Scramblase also acts as a channel for lipids in the presence of calcium- lipids flow down their concentration gradient. Calcium elevation can activate scramblase- the presence of calcium in the cytoplasm also acts as a danger signal for cell death

Question 48

Scramblase mechanism

Answer 48

A rise in calcium activates the scramblase. The channel has hydrophilic and hydrophobic portions in order to allow for the transfer of phospholipids. Phosphatidylserines are moved to the outer face, allowing for externalization. Other kinds of lipids can move through the channel, along with PS

Question 49

Formation of apoptotic bodies

Answer 49

Proteolysis (cleavage by caspases) of the Rho effector protein ROCK1 causes contraction of the actin cytoskeleton and membrane blebbing. This is the initial part of apoptotic body formation. Caspases also cleave all of the proteins making up the cytoskeleton- tubulins, microtubule associated and motor proteins. This step officially leads to the formation of apoptotic bodies, and the cell breaks into small pieces

Question 50

Mechanism of apoptotic body formation (5 steps)

Answer 50

1. Executioner caspases cleave and activate ROCK1
2. ROCK1 phosphorylates parts of the cytoskeleton
3. This causes the actin skeleton to slightly contract, causing some membrane blebbing
4. Then, executioner caspases begin cleaving all of the structural proteins of the cytoplasm, like actin
5. Once the membrane is no longer anchored, it will form blebs and apoptotic bodies

Question 51

General process of apoptosis (5 steps)

Answer 51

1. Nuclear condensation and fragmentation
2. Organelle fragmentation
3. Phosphatidylserine is externalized by scramblase
4. The entire cell breaks apart into apoptotic bodies, due to executioner caspases
5. Macrophages with PS receptors remove the apoptotic bodies before they can lyse. This makes apoptosis a non-inflammatory process