Molecular Bio - L19-21

•Delay or arrest in cell cycle progression in response to problems completing a specific step in the cell cycle or in response to other cellular problems

Checkpoints may pause the cell cycle and promote

•repair before continuing. Checkpoints can also induce permanent arrest or apoptosis.

•Many checkpoints are not essential under normal conditions.

Checkpoints are essential for

•protecting against cancer.

•SAC is recruited to unattached kinetochores.

Mad2 component of SAC is recruited to

•kinetochore, activated, released.

Activated Mad2 binds and inhibits APC/Cdc20

Spindle assembly checkpoint - APC/Cdc20 is inhibited in response to

incomplete kinetochore/microtubule attachments, Allows spindle to set up properly before anaphase is initiated.

G1 DNA damage checkpoint - activated if DNA damage is detected in

•G1 à G1 arrest (or G0 arrest).

-Allows cell cycle to pause while DNA is repaired.

-A similar checkpoint operates in G2 and prevents entry into mitosis

Replicative senescence - telomere shortening ->

•G0 arrest (senescence).

- Protects against chromosome fusion.

Oncogene induced senescence - hyperproliferation due to oncogene activation ->

•G0 arrest (senescence).

Other checkpoints detect

hypoxia, loss of cell adhesion and other abnormal cellular conditions

ATR and ATM - kinases are recruited to sites of

DNA damage

ATM - double strand breaks

ATR - exposed single stranded

DNA, other DNA damage

ATM (and ATR) has two functions at these sites:

1) help recruit repair machinery

2. activate a cell cycle checkpoint.

•related kinases recruited to sites of DNA damage

ATM recruited to

double strand breaks

ATR recruited to exposed

single stranded DNA

-Some overlap in activity between ATM and ATR

ATM and ATR phosphorylate Chk1 and Chk2 kinases to

•promote cell cycle arrest (or apoptosis)

ATM and ATR also phosphorylate to activate

proteins involved in DNA repair

Wee1 and Cdc25 also regulate

Cdk2-cyclin A -> control S-phase

•ATR and ATM both recognize different types of DNA damage.

àrecruit and phosphorylate the

Chk1 and Chk2 kinases.

Chk1 and Chk2 phosphorylate Cdc25 phosphatase, targeting it for ubiquitination by

•SCF ubiquitin ligase, and destruction by the proteasome.

•Cdk2 remains phosphorylated by Wee1 (cannot be dephosphorylated)

•Cdk2 is inactive

Therefore cells arrest in

•G1/S.

Essentially the same checkpoint can be activated in G2

Phosphorylation and degradation of

•Cdc25

•Cdk1 remains phosphorylated by Wee1

•Cdk1 is inactive

Therefore cells arrest in

•G2/M

•ATR and ATM both recognize different types of DNA damage.

à recruit and phosphorylate the Chk1 and Chk2 kinases.

•

In case of more severe damage ATM,ATR, Chk1 and Chk2 phosphorylate and activate

p53

p53 is a transcription factor that induces a permanent cell cycle arrest by activating

•p21 expression

-p21 binds and inhibits Cdk2/cyclin complexes

p53 can also induce apoptosis by activating expression of

•pro-apoptotic genes.

In undamaged cells p53 is inactive due to

Mdm2 binding

•Mdm2 inhibits p53 activity

also ubiquitinates p53

leading to proteasomal degradation

Chk1/Chk2 (and ATM/ATR) phosphorylate

p53

phosphorylated p53 is released from

Mdm2

p53 is a

transcription factor, induces p21 expression

p21 binds and inhibits

Cdk2/cyclin E and Cdk2/cyclin A

Cells arrest in G1

Replicative senescence, G0 arrest of cells if

telomeres are too short

•Telomeres - chromosome ends contain multiple copies of a telomere repeat

Telomerase activity required for

•telomere replication

Telomeres Are Protected by telomere binding proteins - sheltrin complex binds to

telomere repeats

Sheltrin complex binding protects telomeres from

DNA repair machinery that repairs double stranded breaks

p53 mediates

replicative senescence

During DNA replication, telomere length is maintained by

telomerase that adds these repeats.

Sheltrin binds to these repeats

Most cell types in humans do not express

telomerase.

As a result telomeres shorten each cell generation

Telomeres shorten every cell division

If telomeres shorten too much it leads to no more

repeat sequences

Sheltrin can no longer bind

Genomic instability resulting from failure to arrest the cell cycle in response to

double stranded DNA breaks.

•Telomere shortening at each cell division eventually leads to loss of all of the telomere repeats.

Sheltrin no longer bind to

•chromosome ends.

The double strand break sensing checkpoint recognizes these chromosome ends and induces

permanent cell cycle arrest - replicative senescence.

•ATM binds to exposed double stranded DNA at chromosome ends.

p53

•stabilization

•p21 expression

•Inactivation of G1/S and S Cdks

If this pathway is not functioning (Replicative senescence),

chromosome fusions

p53 is the

cellular gatekeeper

Apoptosis is often referred to as

programmed cell death or cell suicide.

Apoptosis is cell death via an active cellular response to

•extracellular or intracellular signals.

•Apoptosis as a developmental process

-salamander tail

-removal of interdigital cells during digit formation

development of visual system and other neural circuits - neurons that fail to

-connect undergo apoptosis.

-regulation of tissue size by cell competition

C. elegans development - the ced genes.

Apoptosis as a response to cell cycle and other

cellular checkpoints.

Sidney Brenner- developed C elegans as a model system - early 60s

John Sulston - lineage map - revealed that many cells undergo

programmed cell death - early 70s

Robert Horvitz - screen for genes that when mutated allow these cells to survive - ced genes - early 80s

Apoptosis involves activation of a cell death pathway while necrosis is a

passive process resulting from damage to the cell.

•Features of apoptotic cells:

cytoskeleton collapse, nuclear envelope breaks down, chromatin

-condenses and breaks into fragments, cell surface blebbing, apoptotic bodies

-apoptotic cells signal to phagocytic cells to be phagocytosed.

Necrotic cells swell, burst and their cellular contents illicit

•an inflammatory response.

TUNEL labeling - apoptotic cells have

fragmented chromosomes. Label DNA ends with Terminal deoxynucleotidyl Transferase

Assays for apoptosis, •Plasma membrane loses integrity

Uptake of cell impermeable

DNA dyes - Acridine orange.

•Chromatin breakage

ladder of DNA seen by

-gel electrophoresis

-TUNEL labeling

•Membrane flipping

labeled Annexin5 - Phosphatidylserine flips to outside of membrane

-acts as signal for macrophages).

•Mitochondrial activity is lost

positively charged fluorescent dyes that accumulate in

active mitochondria - rhodamine 123

•Pro-caspase cleavage

Antibodies against

-cleaved caspases

Proteases that mediate apoptosis are called

Caspases

Caspases are made as Pro-caspases - Pro domain keeps it

inactive

Procaspase cleavage may be mediated by another caspase.

Two classes of caspase:

Initiator (activator) caspases and Effector (executioner) caspases

Initiator (activator) caspases - cleave

other caspases

Effector (executioner) caspases - cleave

specific cellular targets to cause apoptosis.

Initiator Caspase activation leads to rapid amplification of a

caspase cascade.

Caspase activation leads to rapid and irreversible

initiation of apoptosis.

•Intrinsic pathway

-cellular response to various stresses, DNA damage, depletion of survival factors.

BH-domain family of proteins regulate release of proteins from

-mitochondrial intermembrane space to activate procaspases.

initiator caspases activated by apoptosome

•Extrinsic pathway

-apoptosis induced via signal from specialized cells - eg cytotoxic T-lymphocytes

cell surface "death receptors" - egFas, TNF, TRAIL respond to

-TNF-related ligands.

-Adaptor proteins interact with receptor and activate procaspases.

Intrinsic Pathway of Apoptosis regulated by

BH-Domain family of proteins

proapoptotic Bax and other BH1,2,3 proteins form mitochondrial channels that promote

-release of cytochrome c and other proteins from intermembrane space of mitochondria.

cytochrome c promotes formation of

apoptosome that in turn stimulates activator caspases.

intrinsic pathway induced by various stresses acting through

-p53, and also can be induced by external signals.

anti-apoptotic protein

Bcl2, prevents pore formation

pro-apoptotic protein

BH123 protein, forms a pore in the mitochondria

pro-apoptotic protein only protein

BH3-only

BH123 proteins

- mitochondrial pore

Bcl2 inhibits aggregation of

BH123 proteins

BH3-only proapoptotic proteins inactivate

Bcl2

BH-3 promotes transcription of

p53

BCL2 is inhibited by

p53

BH123 proteins promotes transcription by

p53

mitogens promote survival by phosphorylation to inactivate

Anti IAPS

Intrinsic pathway activation depends on a family of Bcl2 related proteins (BH-domain proteins)

All have

1 or more Bcl2 Homology Domains (BH1-4)

Three classes of BH-domain protein:

•BH123 - form mitochondrial pore - release Cytochrome C -> proapoptotic

•Bcl2 - inhibit BH123 pore formation - antiapoptotic

•BH3-only - inhibit Bcl2 - proapoptotic.

•regulated by BH-Domain family of proteins

-proapoptotic BH1,2,3 proteins form mitochondrial channels that promote release of cytochrome c and other proteins from intermembrane space of mitochondria.

Cytochrome C promotes formation of

-apoptosome that in turn stimulates activator caspases.

-intrinsic pathway induced by various stresses acting through p53, and also can be induced by external signals.

Cytochrome C release from the mitochondria is a key event in the

intrinsic pathway.

Cytochrome C release - binding to Apaf1 leading to the

CARD domain exposed - multimerization (Apoptosome)

Apoptosome - recruits

procaspases

•depends on CARD domain interactions

•pro-caspase 9 cleaved leads to it being activated

the release of cytochrome c is done by

pore formation by BH-3

Bcl2 inhibits

BH123 aggregation

BH3-only proteins inhibit

Bcl2

Cytochrome C release from

mitochondria

Cytochrome C binds Apaf1 to promote

procaspase 9 binding and apoptosome formation.

procaspase 9 activated by

cleavage - cleaves executioner caspases.

IAPs

prevent spontaneous activation of

•caspases by binding cleaved caspases.

IAPS •first identified as viral proteins that prevent host cell apoptosis.

bind activated

caspases and prevent their activity

Anti-IAPs

compete with caspases for

•IAP binding -> promote caspase activation.

In vertebrates anti-IAPs are released along with cytochrome c from

•mitochondria

•expression is induced in response to apoptotic stimuli (anti-IAP genes are p53 targets).

•Cytochrome C binds Apaf1 to promote procaspase 9 binding and apoptosome formation.

procaspase 9 activated by

cleavage - cleaves executioner caspases.

IAPs block

caspase-9 activity

anti-IAPs promote

-caspase-9 activity