Apoptosis

Question 1

What is apoptosis?

Answer 1

programmed cell death

most common type of cell death

Question 2

what are the two types of cell deaths?

Answer 2

necrosis - accidental death; dirty way of dying; in necrosis, contents spill out of cell

apoptosis - clean way; programmed; in apoptosis, cell shrinks and condenses and contents never leak out

Question 3

what does apoptosis help minimize?

Answer 3

apoptosis is a routine controlled cell death that minimizes spread of damage and/ or inflammation

Question 4

how is apoptosis critical for development?

Answer 4

helps in development of mature forms.

cell death sculpts hands and feet during embryonic development

Question 5

what are some importances of apoptosis?

Answer 5

its important for certain cells: abnormal, non-functional, potentially dangerous (cancer) cells

eliminate lymphocytes after destroying and ingesting microbes

organs can be kept the correct size (liver)

DNA damaged cells - destroyed if damage is irreparable

Question 6

what are some of the phenotypes of apoptosis?

Answer 6

overall shrinkage in volume of cell and its nucleus

loss of adhesion to neighboring cells

formation of blebs on surface

DNA fragmentation

cytoskeleton collapses

nuclear envelope disassembles

Rapid engulfment of dying cell by phagocytosis (macrophages)

Question 7

what are some characteristics of biochemistry of apoptotic cells?

Answer 7

• an endonuclease cleaves DNA not ladder of fragments in distinctive sizes
• cleavages occur in linker regions of nucleosomes; agarose gel shows pattern

Question 8

what is the significance of Cytochrome C?

Answer 8

it is a marker of apoptosis

it is released from mitochondria

Question 9

what is the significance of caspase?

Answer 9

apoptosis is an intracellular proteolytic cascade mediated by the proteases called caspases

activation of caspases is a key event in apoptosis

Question 10

what does caspase stand for and what does it target? what amino acid is in the active site?

Answer 10

caspase = cysteine aspartyl specific protease

it targets proteins and cleaves them in their sequence where an aspartic amino acid residue occurs

cysteine is in the active site

Question 11

where does caspase come from?

Answer 11

it is synthesized first as an inactive precursor - procaspase

becomes activated by protease cleavage

procaspases cleaved at specific sites to form a large and small subunit which form a heterodimer AND caspases activates procaspases

Question 12

what are the two major classes of caspases? what do they do?

Answer 12

initiator caspase (initiates caspase)

executioner caspase: destroys actual targets - executes apoptosis
*does so by: cleaving downstream proteins; cleaves inactive endonuclease; targets cytoskeleton; attacks cell adhesion proteins

Question 13

what are some characteristics of the caspase cascade?

Answer 13

• its irreversible
• there are specific caspases
• machinery for apoptosis (caspases) are always in place
• initiator caspase auto-activates itself (may create problem)
• executioner caspases cleaves cellular targets

Question 14

what are the two apoptotic pathways? what are some characteristics about them?

Answer 14

internal pathway and external pathway; they depend on factors that INDUCE apoptosis (eg: internal, DNA abnormalities; external, removal of survival factors or proteins of TNF family)

internal: mitochondrial dependent
external: mitochondrial independent

Question 15

describe the extrinsic pathway

Answer 15

-extracellular signals (Fas ligand) binds to cell surface death receptors (homotrimer) and triggers extrinsic pathway

then adaptor proteins are recruited: FADD adaptor (fas associated death domain) & procaspase-8 with death effector domain

• this forms DISC: death inducing signal complex
• activates caspase-8 or 10, which activates downstream executioner caspase-3

Question 16

whats one method of controlling or restraining extrinsic pathway?

Answer 16

decoy receptors: FLIP (a protein resembling initiator procaspase with no proteolytic domain; competitive inhibitor against procaspase-8/10

they have ligand binding domain but no death domain; so it does not activate apoptosis

Question 17

what is the intrinsic pathway?

Answer 17

apoptosis that is activated from inside the cell

Question 18

what triggers intrinsic pathway?

Answer 18

in response to injury, DNA damage, lack of oxygen, nutrients or lack of extracellular survival signals

Question 19

what is the key event in intrinsic pathway?

Answer 19

the translocation of cytochrome c from the intermediate space of mitochondrial

Question 20

explain intrinsic pathway

Answer 20

the translocation of cytochrome c from the intermediate space of mitochondria to cytosol leads to it binding to adaptor protein to activate procaspase.

it binds to procaspase-activating adaptor protein: Apaf1

Apaf1 forms apotosome which activates caspase-9; caspase-9 activates executioner caspase-3 (common to both pathways)

Question 21

what does Bcl2 family proteins do?

Answer 21

they regulate intrinsic pathways

Question 22

what does Bcl2 protein itself do?

Answer 22

controls release of cytochrome c into cytosol (Bcl = B cell lymphoma)

Question 23

what are the two types of Bcl2 proteins?

Answer 23

anti-apoptotic = blocks release of cytochrome c (Bcl2); Bcl2 protein has 4 distinctive domains called Bcl homology domains or BH domains

pro-apoptotic = promotes release of cytochrome c; (BH123 protein and BH-3 only protein)

Question 24

what are the 3 classes of Bcl2 proteins?

Answer 24

Bcl2 protein (anti-apoptotic)

BH123 protein (pro-apoptotic)

BH3-only protein (pro-apoptotic)

Question 25

how does the BH123 protein work? is it pro-apoptotic or anti-apoptotic?

Answer 25

it’s pro-apoptotic

• an apoptotic stimulus triggers intrinsic pathway
• BH123 protein becomes activated, forming an aggregation in the mitochondrial outer membrane and induces the release of cytochrome c - (which then goes on to form the apoptosome by binding to Apaf1)

Question 26

what inhibits BH123?

Answer 26

Bcl2 proteins (Bcl2 and Bcl-XL) (both BH1234)

• mainly located on cytosolic surface of outer mito membrane
• these proteins prevent apoptosis by binding to pro-apoptotic proteins (BH123) and prevent aggregation into active form

Question 27

What protein inhibits Bcl2 activity?

Answer 27

BH3-only protein; it’s pro-apoptotic

an active BH3-only protein is cytosolic

translocates to mito after apoptotic signal activates it

*inhibits Bcl2 protein FROM INHIBITING aggregation to release cytochrome c

Question 28

what regulates caspases?

Answer 28

IAPs: Inhibitors of APotosis

they bind and inhibit caspases

some IAPs add ubiquitin to caspases to mark them for destruction by proteasome
others block apoptosis by binding to caspases

*this solves auto-activating caspases

Question 29

What happens when you do need apoptosis to occur though? how do you get around the IAPs?

Answer 29

if there is an apoptotic stimuli or signal, then this causes the release of ANTI-IAPs from the mitochondria. these block activity of IAPs; the executioner caspases can therefore be activated with the IAPs blocked

Question 30

speak on insufficient apoptosis leading to disease in regards to Bcl2

Answer 30

Excess Bcl2 promotes development of cancer by inhibiting apoptosis: DNA-damaged cells will not be programmed for cell death but can go on to cause cancer

Question 31

speak on p53 mutation leading to disease

Answer 31

mutated p53 can no longer cause cell cycle arrest; insufficient and no longer promotes apoptosis. cells with DNA damage stick around and cancer can be generated.

Question 32

what can excessive apoptosis lead to?

Answer 32

heart attacks and strokes