Cell cycle

Question 1

What are the phases of cell cycle?

Answer 1

□ INTERPHASE (duplication and growth) – DNA and organelles are duplicated, and protein synthesis increases. □ G1 PHASE (growth phase and checkpoint); S PHASE (synthesis of DNA); G2 PHASE (growth phase and checkpoint). □ G0 phase occurs when cell cycle machinery is dismantled. □ MITOTIC PHASE (mitosis – cell division) – nuclear division and cell division (cytokinesis).

Question 2

Why is mitosis the most vulnerable stage of the cell cycle? (x4)

Answer 2

□ Cells are more easily killed from irradiation, heat shock and chemicals. □ DNA damage cannot be repaired. □ Gene transcription is silenced. □ Metabolism is reduced.

Question 3

What happens in S phase? (x3)

Answer 3

□ DNA replication. □ Protein synthesis: initiation of translation and elongation increased. Capacity for protein production is also increased – more ribosomes. □ Replication of organelles: centrosomes, mitochondria, Golgi etc. In mitochondria, mtDNA is also replicated – this is coordinated with DNA replication.

Question 4

What is the structure of centromeres? What do they do?

Answer 4

□ Consists of two centrioles – each made up of 9 triplet microtubules, arranged into a barrel.

□ The centrioles are 90 degrees from each other.

□ They are MICROTUBULE ORGANISING CENTERS and synthesis mitotic spindle through polymerisation of microtubules.

Question 5

What are the stages of mitosis?

Answer 5

Prophase, prometaphase, metaphase, anaphase, telophase.

Question 6

What happens in prophase?

Answer 6

□ Condensation of chromatin – DNA is coiled around nucleosomes, coiled, scaffolded (folded) and further supercoiled into 30nm chromatin fibres. □ The condensed chromosomes consist of sister chromatids, each with a kinetochore – associated with the CENTROMERE – where the spindle fibres attach in later stages. □ LATE PROPHASE/PROMETAPHASE: Chromosomes are now condensed. CENTROSOMES migrate to opposite sides of the nucleus and organise the assembly of spindle microtubules.

Question 7

What happens in spindle formation (in prophase)?

Answer 7

□ Radial microtubule arrays (ASTERS) form around each centrosome.

□ Where radial arrays meet in the middle of the cell (along the metaphase plate), these GROW, while arrays that have extended in other directions SHORTEN (see photo).

□ RESULT: polar microtubules form.

Question 8

What happens in prometaphase?

Answer 8

□ EARLY PROMETAPHASE: breakdown of nuclear membrane, spindle formation is largely complete, and there is attachment of chromosomes to spindle via the kinetochores (centromere regions of chromosomes). □ LATE PROMETAPHASE: microtubules from opposite poles are captured by sister chromatids (i.e. spindle-kinetochore attachment complete). Chromosomes progress to the middle, sliding along microtubules.

Question 9

What happens in metaphase?

Answer 9

Chromosomes are aligned at the equator of the spindle.

Question 10

What happens in anaphase?

Answer 10

□ Paired chromatids separate to form daughter chromosomes. Anaphase is split into two parts:

□ ANAPHASE A: COHESIN (a protein complex which loosely holds together sister chromatids) is broken down. Microtubules get shorter and daughter chromatids are pulled toward opposite spindle polls.

□ ANAPHASE B: daughter chromosomes migrate towards polls and spindle poles (the centrosomes) migrate apart. Clears a large region of cytoplasm separating the two sets of chromosomes.

Question 11

What happens in telophase?

Answer 11

Nuclear envelope reassembles at each pole; Contractile ring assembles (of actin ang myosin).

Question 12

What happens in cytokinesis?

Answer 12

The contractile ring narrows and produces a cleavage furrow –> contraction of the ring separates cell into two.

Question 13

What is the spindle assembly checkpoint?

Answer 13

Checks that METAPHASE has gone correctly: senses completion of chromosome alignment and spindle assembly by monitoring kinetochore activity. Unattached kinetochores generate checkpoint signals, so when all are attached to spindle, metaphase is permitted to progress into anaphase A.

Question 14

What proteins are involved in the signalling of the spindle assembly checkpoint? (x2)

Answer 14

□ BUB PROTEIN KINASES: dissociate from the kinetochore when chromosomes are properly attached to the spindle – when all dissociate, cell cycle progresses to anaphase. □ CENP-E – a protein that senses tension at kinetochore.

Question 15

What are the different types of aneuploidy from mis-attachment of microtubules to kinetochores? (x4)

Answer 15

□ SYNTELIC: both kinetochores are attached with spindle from the same centrosome, so both chromatids go to the same pole. One cell therefore gains a chromosome, while the other loses one.

□ MONOTELIC: one kinetochore is empty. So, both chromatids go to the same pole.

□ MEROTELIC: spindle from each centrosome is attached to the same kinetochore, so chromosome is lost at cytokinesis and fails to go either way.

□ ABERRANT CENTROSOME DUPLICATION: so more than two centrosomes form. There are therefore more than two spindle poles, so more than two cells are formed, each containing an unpredictable number of chromosomes – see photo.

Question 16

What is the term given when microtubules are attached normally to kinetochores?

Answer 16

Amphelic.

Question 17

How does cancer therapy exploit mechanisms for aneuploids? (x2)

Answer 17

□ CHECKPOINT KINASE INHIBITORS can be used on tumour cells to inhibit attachment-error-correction mechanisms. Therefore, cells are permitted to the next stages of the cell cycle and become aneuploid. Checkpoint kinases (CHKE1 and CHKE2) control serine threonine kinase activation which holds cells in the G2 phase until all is ready. Inhibition of these kinases therefore pushes cells with gross chromosomal abnormalities into mitosis –> aneuploidy and APOPTOSIS. □ TAXANES and VINCA ALKOIDS alter microtubule dynamics and produce unattached kinetochores, leading to mitotic arrest.

Question 18

What happens when something goes wrong in the cell cycle? (x2)

Answer 18

□ E.g. Cell is not big enough, or DNA damage. □ CELL CYCLE ARREST: occurs at checkpoints and can be temporary e.g. cell has mechanism to repair DNA damage, OR: □ PROGRAMMED CELL DEATH (apoptosis): occurs when DNA damage is too great and cannot be repaired, there are chromosomal abnormalities or toxic agents have damaged the cell/DNA.

Question 19

What is the G2 checkpoint?

Answer 19

Checking for DNA damage following DNA replication in S phase, size and favourable environmental factors.

Question 20

What is the G1 checkpoint?

Answer 20

Checks for growth factors and that the environment is favourable.

Question 21

How do tumours affect each cell cycle checkpoint?

Answer 21

Tumours inhibit G2, G0 and metaphase checkpoints (so cells are pushed through the cell cycle despite DNA damage and incorrect chromosome alignment and cannot leave when errors/damage occur). Tumour progression also increases the G1 checkpoint by upregulating the growth factor receptors, and thereby lowering the growth factor threshold required for progression to the next stage of the cycle.

Question 22

What triggers a cell to enter the cell cycle and divide? (x2) What is the normal state of cells?

Answer 22

□ In the absence of a stimulus, cells go into G0 phase – quiescent phase. Most cells in the body which are differentiated are in this phase.

□ Exit from G0 is highly regulated and requires GROWTH FACTORS and INTRACELLULAR SIGNALLING CASCADES.

Question 23

What are the intracellular signalling cascades which put cells into the cell cycle? How do they work? !!!

Answer 23

1. RECEPTOR PROTEIN TYROSINE KINASES (RPTK) exist as monomeric receptors. Dimeric ligands (GROWTH FACTORS) bind to receptors and form RECEPTOR DIMERS (see photo). In a dimer form, the receptors become activated by phosphorylation from amino acids in the kinase domain of both receptors.
2. RECEPTOR ACTIVATION: Phosphorylation occurs in three different amino acids (serine, threonine and tyrosine) on the receptor. The hydroxyl groups of each amino acids are removed, and phosphate group added via ATP. The added NEGATIVE phosphate alters protein function by causing conformational change AND creating docking sites for ADAPTOR PROTEINS.
3. Receptor activation and subsequent action of adaptor proteins activates SMALL G (GTP-BINDING) PROTEIN (Ras).
4. RAS ACTIVATION TRIGGERS kinase cascades. In the kinase cascades, activation of one kinase via phosphorylation results in the phosphorylation and activation of the next –> SIGNAL AMPLIFICATION.
5. Phosphatase reverses the phosphorylation in the kinase cascades and ensures that this amplification is short-lived.
6. Signal integration.
7. Modulation of the signal by other pathways inside the cell.
8. RESULT: signal divergence to multiple targets –> regulation of metabolic pathway, gene expression and cytoskeleton that may bring cell into the cell cycle.
9. NB: RECEPTOR PROTEIN TYROSINE KINASES AND GTP-BINDING RAS ARE THE REGULATORS OF THE KINASE CASCADE.

Question 24

What examples are there of receptor protein tyrosine kinases and growth factors which induce signalling cascades? (x2)

Answer 24

Receptor protein tyrosine kinases include: EPIDERMAL GROWTH FACTOR RECEPTOR (EGFR) and PLATELET-DERIVED GROWTH FACTOR RECEPTOR (PDGFR). Peptide growth factors include: EPIDERMAL GROWTH FACTOR (EGF) and PLATELET-DERIVED GROWTH FACTOR (PDGF).

Question 25

How do changes in gene expression (from intracellular signalling cascades) bring the cell into the cell cycle?

Answer 25

The kinase cascade stimulates transcription of IMMEDIATE EARLY GENE TRANSCRIPTION FACTORS such as c-Myc gene – a transcription factor that stimulates the expression of cell cycle genes. They are transiently upregulated, so process is tightly controlled and not long-lasting. The c-Myc transcription factor stimulates the cell to enter the S-phase of the cell cycle.

Question 26

What happens to gene expression (in the intracellular signalling cascade) in cancer?

Answer 26

Transcription factors such as c-Myc are ONCOGENES and are overexpressed in many tumours, leading to hyperactivation of genes that push cells into the cell cycle.

Question 27

What do adaptor proteins do (in intracellular signalling cascade)? Example?

Answer 27

* When receptors are dimerised and phosphorylated, phosphorylation produces docking sites in the cytosolic domain of the receptor. Here, ADAPTOR PROTEINS bind. * These docking sites and adaptor proteins allow for PROTEIN-PROTEIN INTERACTONS; bringing proteins together. By bringing proteins together, signals can be transmitted inside the cell. * Adaptor proteins contain domains that allow these interactions, because they are involved in MOLECULAR RECOGNITION i.e. have no enzymatic function, but simply bring together other proteins. * For example, Grb2 is an adaptor protein: it has an SH2 and SH3 domains. SH2 domains bind to phosphorylated tyrosines (i.e. binds to the activated receptor protein tyrosine kinases); SH3 domains bind to proline-rich proteins. These proline-rich proteins go on to activate intracellular pathways and cascades.

Question 28

What does Grb2 adaptor protein do in the intracellular signalling cascade?

Answer 28

* GTP-binding Ras protein is an oncoprotein activated by binding to GTP. This activated Ras protein is subsequently inactivated by GTPase ACTIVATING PROTEINS, which de-phosphorylated the bound-GTP into GDP (keeps the activation transient). * SOS, an exchange factor, reactivates the GTP-binding Ras protein by removing the GDP, and adding a new GTP. * SH3 domains of Grb2 binds to the activated receptor protein tyrosine kinases. SH2 domains of Grb2 binds to this SOS EXCHANGE FACTOR. * Grb2-SOS interaction brings the SOS close to Ras proteins attached to the intracellular cell surface membrane, and subsequently ACTIVATES RAS PROTEIN. NB: Ras must be bound to plasma membrane to become activated. * This leads to kinase cascade.

Question 29

How does Herceptin work as a cancer therapy?

Answer 29

Herceptin is an Anti-HER2 antibody and binds to the extracellular domain of the kinase receptor, preventing ligand binding and therefore preventing all the subsequent signalling.

Question 30

What happens to the Ras protein activation by Grb2 in cancer? Examples? (x2)

Answer 30

• Oncogenic Ras forms contains mutations that subverts the normal stimulation and inhibition pathways of Ras. It means that Ras is constantly active, therefore constantly stimulating the kinase cascade. • V12Ras mutation prevents GTPase Activating Proteins (GAP) from binding to Ras and inactivating it. • L61Ras mutation prevents GTP hydrolysis, so GTP cannot be de-phosphorylated and inactivation is difficult.

Question 31

How does Ras activate the protein kinase cascade?

Answer 31

Ras activates the EXTRACELLULAR SIGNAL-REGULATED KINASE (ERK) CASCADE. It converts ATP to ADP as subsequent kinases are phosphorylated and activated. In the cascade, the first kinase is Raf, which is converted into MEK and then ERK. ERK is the mitogen-activated protein kinase (MAPK) that leads to MITOSIS (cell entering the cell cycle).

Question 32

What is MAPK cascade?

Answer 32

It is a general term for the kinase cascades activated by receptor protein tyrosine kinases. It stands for MITOGEN-ACTIVATED PROTEIN KINASE cascade. An example of a MAPK cascade is the ERK cascade which is mediated by Ras.

Question 33

What is the result of the ERK cascade in the cell?

Answer 33

Effects on the cell are BROAD – there are changes in protein activity and changes in gene expression e.g. upregulation of c-Myc.

Question 34

What is the role of cyclin-dependent KINASES (Cdks)? Presence? Regulated by? (x2)

Answer 34

They CONTROL the cell cycle by progressing the cell through different phases of mitosis and interphase. They are present in ALL proliferating cells throughout the WHOLE cell cycle. Its activity is regulated by INTERACTION WITH CYCLINS and PHOSPHORYLATION.

Question 35

What is the property and presence of cyclins? How do they affect Cdks?

Answer 35

Cyclins are transiently expressed at specific points in the cell cycle. They are synthesised and degraded very quickly. Different cyclin-Cdk complexes trigger different events in the cell cycle.

Question 36

What do Cdks need for activation? (x2) What do activated Cdks do?

Answer 36

• Cdks are activated when they are bound to cyclins, because the complex that is subsequently formed produces an ACTIVE SITE. BUT, complete activation occurs when Cdk is phosphorylated as well. • Activated Cdk(-cyclin complexes) phosphorylate proteins on Serine and Threonine amino acids (using their active site). These proteins, when phosphorylated, drive cell cycle progression.

Question 37

What is the role of Cyclins? (x2)

Answer 37

• Activate Cdks. • Cyclins also target Cdks to specific substrates: Cyclins form part of the active site of Cdks, so binding of DIFFERENT cyclins produces slightly different active sites. This allows the same Cdk to work at DIFFERENT points of the cell cycle by using different cyclins.

Question 38

How are Cdks regulated by phosphorylation?

Answer 38

1. Cyclin and Cdk bind to form an INACTIVE cyclin-Cdk complex. 2. Cdk-activating kinase (Cak) and Cdk-inhibiting kinase (Wee1) both phosphorylate Cdk, donating an inhibitory and activating phosphate group. 3. Phosphatase Cdc25 activates Cdk by removing the inhibitory phosphate (DEPHOSPHORYLATION) from Cdk. 4. The activated Cdk positively feedbacks on the Cdc25 phosphatase, to reinforce its activation and therefore drive cell cycle progression.

Question 39

How does cyclin and Cdks regulate mitosis?

Answer 39

* Mitotic Cyclin B and Cdk1 form a complex – called M-PHASE PROMOTING FACTOR. DEPHOSPHORYLATION activates this complex at the end of interphase/beginning of mitosis. * When activated, Cdk1 keeps mitosis ON HOLD, allowing key proteins to be phosphorylated. * Signals from fully attached kinetochores in metaphase causes cyclin B to be degraded (remember, cyclins are only TRANSIENTLY EXPRESSED). Cdk1 is therefore inactivated. * Mitosis progresses.

Question 40

Apart from Cdk1 and Cyclin B in mitosis, what other Cdks and cyclins are activated throughout the cell cycle? (x4) How does activation of each complex link together? !!!

Answer 40

* Cdk2 and Cyclin E in cell G1 to S-phase transition – complex is called the START KINASE and trigger DNA replication machinery. * Cdk2 and Cyclin A in S phase/G2 phase. * Cdk4 and Cyclin D in G1 phase. * Cdk6 and Cyclin D in G1 phase. * LINK: Cdks become SEQUENTIALLY ACTIVATED. When a Cdk and cyclin forms a complex and is activated, it stimulates the synthesis of genes required for activation of the next complex, such that when one cyclin degrades, the other cyclin-Cdk complex is activated = CYCLIC ACTIVATION.

Question 41

How do the actions of Cdk link to the intracellular signalling cascades which put cells into the cell cycle?

Answer 41

In the intracellular signalling cascade, kinase cascades result in transcription of immediate early gene transcription factors such as c-Myc. These transcription factors stimulate transcription of other genes. C-Myc stimulates transcription of CYCLIN D. Cyclin D forms complexes with and activates CDK4 and CDK6, which stimulates synthesis of Cyclin E and transition of G1 into S-phase i.e. growth factors push cells from G0 --\> G1 (into the cell cycle); Cyclins and Cdks regulate the cell once it has been pushed into the cell cycle.

Question 42

What is the role of Rb protein in the cell cycle?

Answer 42

* Active Rb (RETINOBLASTOMA) protein (pRb) is found in G0 cells and binds to the E2F transcription factor, PREVENTING it from transcribing target genes. * Genes regulated by the transcription factor E2F are involved in the cell cycle. * Active pRb therefore puts a BRAKE ON THE CELL CYCLE. * When C-Myc stimulates transcription of cyclin D and there is subsequent activation of Cdk4/6-cyclin D complexes, active Cdk4 and Cdk6 phosphorylate pRb. * This causes the pRb to partially DEACTIVATE, and it therefore releases small amounts of the E2F transcription factor – enough to transcribe Cyclin E. * CDK2-Cyclin E phosphorylates pRb further – this leads to progressive DEACTIVATION of pRb and larger amounts of E2F transcription factor are released --\> transcription of Cyclin A. * CDK2-Cyclin A phosphorylates pRb again and it is eventually deactivated. This leads to activation of other transcription factors which results in transcription of Cyclin B. * Therefore, pRb has a sequential role in the inactivation and progression of the cell cycle.

Question 43

What is the effect of cancer on pRb?

Answer 43

Rb gene is considered a “tumour suppressor”. In cancer where this gene is mutated, the E2F transcription factor is always activated, so factors involved in progression of the cell cycle e.g. Cyclin E are always being produced.

Question 44

What is the role of CDK inhibitors (CKI)?

Answer 44

CDK inhibitors also regulate CDKs. They inactivate CKDs so that cells can PROGRESS through the cell cycle. Inhibitors hold complexes in their inactive form.

Question 45

What are the two CKI families? What do each do?

Answer 45

• INK4 FAMILY: control CDKs at G1 phase by inhibiting CDK4/6 by displacing cyclin D. • CIP/KIP FAMILY: control S-phase CDKs by inhibiting all CDKs by binding to the CDK/cyclin complex.

Question 46

How does p27Kip1 (CDK inhibitor from CIP/KIP family) regulate cell cycle? Outline its own regulation.

Answer 46

It forms a complex with cyclinD-Cdk4 – when this occurs, the inhibitor promotes cell cycle progression. When the inhibitor is phosphorylated, it is inactivated, and the cyclin D-Cdk4 is allowed to put the cell cycle on hold. p27Kip1 is phosphorylated by GSK3b.

Question 47

What happens to levels of CKIs during the cell cycle?

Answer 47

They are degraded in the same way that cyclins are. Therefore, their activity is also CYCLIC.

Question 48

Why are cells stimulated to undergo programmed cell death (apoptosis)?

Answer 48

• Cells are HARMFUL e.g. cells with viral infection or DNA damage. • DEVELOPMENTALLY DEFECTIVE cells e.g. B lymphocytes expressing antibodies against self-antigens. • EXCESS/UNNECESSARY cells e.g. embryonic development in brain to eliminate excess neurons, liver regeneration and sculpting of digits and organs. • OBSOLETE cells e.g. mammary epithelium at the end of lactation. • EXPLOITATION e.g. chemotherapeutic killing of cells.

Question 49

What is the difference between necrosis and apoptosis?

Answer 49

NECROSIS = unregulated cell death associated with trauma, cellular disruption and an INFLAMMATORY response; APOPTOSIS = programmed, regulated cell death, with controlled disassembly of cellular contents with NO inflammatory response.

Question 50

What happens in necrosis?

Answer 50

• Plasma membrane becomes permeable so cell swells (fluid influx) and cellular membrane ruptures. • Release of proteases leads to autodigestion and dissolution of the cellular structures. • Cell lysis and invasion of phagocytic cells to clean up debris, associated with localized inflammation.

Question 51

What are the phases of apoptosis? What happens in each?

Answer 51

* LATENT PHASE: death pathways are activated, but cells appear morphologically the same. * EXECUTION PHASE: loss of microvilli and intercellular junctions, cell shrinkage, loss of plasma membrane asymmetry, chromatin and nuclear condensation, DNA fragmentation, formation of membrane blebs (see photo), and fragmentation into membrane-enclosed apoptotic bodies (so intracellular contents of cell are never released). * Apoptotic bodies are phagocytosed by neighboring cells and roving macrophages. * Plasma membrane remains intact and intracellular contents are never released --\> NO INFLAMMATION.

Question 52

What is meant by ‘loss of plasma membrane asymmetry’ in apoptosis? (x2 points)

Answer 52

Asymmetric lipid and protein distribution in plasma membrane of e.g. intestinal epithelium, is vital to function, but lost in apoptosis. In addition, in many cells, phosphatidylserine lipid is found on intracellular side of plasma membranes but appears in outer leaflet in apoptosis.

Question 53

What happens to DNA during apoptosis?

Answer 53

DNA FRAGMENTS.

Question 54

What is apoptosis-like PCD?

Answer 54

Contains some, but not all, features of apoptosis. For example, they differ from because they display phagocytic recognition molecules before plasma membrane lysis.

Question 55

What is necrosis-like PCD?

Answer 55

Described as aborted apoptosis – variable features of apoptosis occur, before cell lysis (necrosis).

Question 56

What proteins initiate and mediate apoptotic cell death?

Answer 56

Caspases – the executioners.

Question 57

What are caspases? How do they function?

Answer 57

Cysteine-dependent aspartate-directed proteases (Caspases) initiate and carry out apoptosis (they are the executioners). They exist physiologically in inactive forms and are activated by proteolysis. In active form, they are PROTEASES, and carry out their effects by cleaving other proteins/enzymes. There are many different types of caspases which carry out different parts of apoptosis – hence, they are activated via a CASCADE PATHWAY (where one caspase activates the other).

Question 58

What are the two types of caspase?

Answer 58

* INITIATOR – CASPASES 2 AND 9: have a CARD (Caspase Recruitment Domain) which places the Caspase at particular sites in the cell. CASPASES 10 AND 8 have DED (Death Effector Domains) and involved in homotypic protein-protein interactions (interactions with two of the same proteins). * EFFECTOR – CASPASES 3, 6, AND 7 contain no additional domains. * Both contain p20 and p10 domain.

Question 59

What is the process of caspase maturation? How are caspases activated?

Answer 59

Caspases are synthesized as procaspases (zymogens) and activated when needed. Initiator caspases are activated by proteolytic cleavage of their DED/CARD domains and between LS and SS domains. Effector caspases are activated by cleavage between LS and SS domains. Cleavage is followed by folding of 2 LS and 2 SS chains to form an ACTIVE HETERO-TETRAMER – formation of these is how the CASPASE CASCADE works. Caspase 8 and 3 are shown in the photo.

Question 60

What are the steps of the caspase cascade?

Answer 60

1. Initiator caspases 8 and 9 trigger apoptosis by cleaving and activating effector caspases 3 and 7. 2. Effector caspases carry out the apoptotic program. 3. Caspase 3, cleaves and thereby activates Caspase 6, 2 and 1. 2 and 1 are initiator caspases; 6 is an effector caspase. 4. Caspase 6 feedbacks into the cascade by activating Caspase 8 (and 10).

Question 61

What are the roles of the caspase cascade?

Answer 61

Amplification, divergent responses (one reaction leads to many different actions), and regulation (of apoptosis).

Question 62

How do effector caspases carry out the apoptotic program?

Answer 62

1. LOSS OF FUNCTION: Cleave and inactivate proteins. 2. LOSS OF FUNCTION: Cleave and inactivate protein complexes e.g. nuclear lamins, leading to nuclear breakdown. 3. GAIN OF FUNCTION: They activate enzymes incl. protein kinases, nucleases e.g. CAD Caspase Activated DNase (clips DNA during DNA fragmentation) by DIRECT CLEAVAGE. 4. GAIN OF FUNCTION: They activate enzymes by CLEAVAGE OF INHIBITORY MOLECULES.

Question 63

What are the two mechanisms that the caspase cascade is first activated?

Answer 63

• DEATH BY DESIGN: receptor-mediated (extrinsic) pathway. Cell will sense something extracellularly that triggers cell to apoptose. Utilize DEATH RECEPTORS. • DEATH BY DEFAULT: mitochondrial (intrinsic) death pathway. Internal stimulus triggers pathway. Utilize APOPTOSOMES.

Question 64

What is the structure of death receptors?

Answer 64

• Transmembrane receptors and must be in TRIMERIC FORM for activation. They therefore require trimeric ligands for activation. • Have extracellular cysteine-rich domains. • All death receptors have a common INTRACELLULAR DEATH DOMAIN.

Question 65

How do death receptors initiate the caspase cascade? Example?

Answer 65

• USES CASPASE 8 (remember, this has a DED domain) as its initiator caspase. 1. Death receptor undergoes trimerization by trimer ligand. EXAMPLE: Fas death receptor is trimerized by Fas-ligand on lymphocytes. 2. ADAPTOR PROTEIN FADD is recruited to the death receptor: FADD has a (death domain) DD which interacts with the DD on the death receptor. 3. FADD also has a DED domain: FADD recruits and oligomerizes (joins together) procaspase 8 through procaspase 8 DED-FADD DED interaction. This forms a DEATH-INDUCING SIGNALLING COMPLEX (DISC). 4. Three FADDs are recruited to each TRImerized death receptor, and three DISCs are therefore formed and OLIGOMERISED. 5. Initiator procaspases have low intrinsic catalytic activity. Oligomerization of the DISCs at the receptor therefore enables the Procaspase 8s to trans-cleave each other. Others are activated by conformational change on oligomerization. NB: at least 2 procaspases are required to form an active TETRAMER. 6. Activation of Caspase 8 --\> cleaves and activates effector Caspase 3 and 7 – THE CASPASE CASCADE HAS BEGUN.

Question 66

How is death receptor activation of caspase 8 inhibited?

Answer 66

* Death receptors also have FLIP adaptor proteins. * FLIP adaptor proteins have two DED domains – just like procaspase 8 but with no proteolytic activity. * They therefore compete with procaspase 8 for the DED domain on FADDs at trimerized receptors. * When it does access a site on the receptor, it interferes with trans-cleavage; inhibiting activation of procaspase 8.

Question 67

What are the two types of FLIP adaptor protein?

Answer 67

SHORT and LONG FLIPs – LONG FLIPS have p20 and p10 domains; SHORT FLIPS do not.

Question 68

How does the death by default pathway activate caspase cascade in apoptosis?

Answer 68

• REGULATED BY MITOCHONDRIA. 1. When the mitochondria are put under stress e.g. lack of or overstimulation by growth factors, DNA damage (p53), ROS; they respond by losing their membrane potential. 2. Cytochrome C is released with other apoptosis-inducing factors . 3. This triggers the formation of the APOPTOSOME COMPLEX. 4. Apoptosome complex brings together initiator caspase 9s for caspase cascade.

Question 69

What is the structure of the apoptosome complex? How do they form?

Answer 69

1. Apaf1 (Apoptotic activating factor 1) oligomerize to form a heptamer (7 Apaf-1s). Each Apaf-1 has a CARD domain which are found in the center, an ATPase domain, and WD-40 repeats which are found around the outside of the heptamer. 2. When the mitochondria are under stress, they release CYTOCHROME C which interacts with the WD-40 repeats of the Apaf1 proteins. 3. At the center of the heptamer (at the CARD domain), EACH Apaf-1 also attracts an Initiator Procaspase 9 (since they also have CARD domains). 4. When all these proteins form, ATP is required, and they create an APOPTOSOME. 5. Caspase-3 can bind to the Procaspase 9s.

Question 70

What is the role of apoptosomes? How do they exercise this role?

Answer 70

• Apoptosome is a structure that brings in different proteins to carry out apoptosis. • The oligomerization of Apaf-1s mean that multiple procaspase 9s are brought close together. • When this happens, trans-cleavage results in their activation, and they are released as active caspase 9 tetramers. • They active Caspase 3, leading to caspase cascade and apoptosis.

Question 71

How does ATP effect the apoptosome and apoptosis?

Answer 71

The apoptosome requires ATP. Energy levels in the cell may determine whether death is by necrosis or apoptosis.

Question 72

What is the family that modulates apoptosis?

Answer 72

Bcl-2 family.

Question 73

How is the Bcl-2 family of proteins categorized? (x2)

Answer 73

* ANTI-APOPTOTIC: Bcl-2 and Bcl-xL. Found attached to mitochondria. * PRO-APOPTOTIC: Bid, Bad, Bax, Bak. Can move between cytosol and mitochondria.

Question 74

How do the death receptor and mitochondrial pathways of apoptosis activation link together?

Answer 74

Caspase 8 in the death receptor pathway cleaves Bid (a protein from the Bcl-2 family) which enhances release of mitochondrial proteins, thus engaging the intrinsic pathway.

Question 75

How is apoptosis regulated: What pathway inhibits apoptosis? (x4 effects) !!!

Answer 75

1. GROWTH FACTORS stimulate production of PI3’-K – a lipid kinase involved in growth control and cell survival. 2. PI3’-K activates Protein Kinase B (PKB, aka Akt). 3. PKB (aka Akt) (i) phosphorylates Bad (a pro-apoptotic); inactivating it, (ii) phosphorylates and inactivates caspase 9, (iii) inactivates FOXO transcription factors by phosphorylation, which promote expression of apoptosis-promoting genes (when phosphorylated, FOXO is exported from the cell and degraded), (iv) stimulates ribosome production and protein synthesis, and (v) inactivates a kinase called GSK3b which inactivates cyclinD-Cdk4. 4. RESULT: induce cell survival by BLOCKING APOPTOSIS. This pathway is active in all healthy cells.

Question 76

How is apoptosis regulated: What pathways promotes apoptosis?

Answer 76

* When growth factors are absent, Bad is dephosphorylated and released from 14-3-3 protein which keeps Bad in an inactive form. * Bad displaces Bax and Bak from their Bcl-2 and Bcl-XL complexes on the mitochondrial surface, so Bax and Bak become active. * Bax and Bak create a pore in the membrane which allows release of cytochrome C = apoptosis.

Question 77

How can the PI3’-K anti-apoptotic pathway be inactivated?

Answer 77

PTEN is a lipid phosphatase which reverses the reaction of PIP2 to PIP3 (which is a step in the PI3’-K pathways that activates PKB). Reversing the PIP2--\>PIP3 reaction inactivates the pathway = pro-apoptotic. NB: PIPs are LIPIDS (not proteins) that are embedded in the MEMBRANE.

Question 78

What are the roles of Inhibitor of Apoptosis Proteins (IAPs)? (x2 mechanisms)

Answer 78

• This is an extrinsic pathway of inhibiting apoptosis. • IAPs inhibit apoptosis by (i) binding to procaspases and preventing their activation, and (ii) binding to already active caspases and inhibiting their activity.

Question 79

SUMMARY: what are the different mechanisms that inhibit apoptosis? (x5) !!!

Answer 79

• INTRINSIC PATHWAYS: Bcl-2 and Bcl-xL. • EXTRINSIC PATHWAYS: FLIP and IAPs. • Growth factor pathways via PI3’-K and PKB/Akt.

Question 80

What are the therapeutic uses of apoptosis in cancer? (x2)

Answer 80

• Destroy harmful (oncogenic) cells e.g. cells with DNA damage or viral infection. • Chemotherapeutic killing of tumor cells e.g. Dexamethasone stimulates DNA cleavage.