Apoptosis and Necrosis

Question 1

apoptosis

Answer 1

programmed cell death

process by which cells can control the time of their own death

Normal part of development; and maintenance and development of the animal body

distinguish this from necrosis, in which the cell is violently killed, and membrane bursts

Question 2

Initial events of apoptosis

Answer 2

Plasma membrane receptors Fas and TNF-R1 receive intercellular death signals from the fas ligand or TNF respectively

both receptors foster an 80 aa sequence death domain that binds to FADD on Fas, and TRADD on TNF-R1

Question 3

FADD activates

Answer 3

a cytocolic protease caspase 8 that proteolytically activates other cellular proteses

caspase 8 releases cytochrome c

Question 4

TRADD activates

Answer 4

proteases. The resulting proteolysis is a primary factor in cell death

Question 5

Role of cytochrome C in apoptosis

Answer 5

cytochrome c is a small soluble mitochondrial protien located in the intermembrane space. It carries electrons between complex III and IV during respiration.

It also acts as a trigger for apoptosis by stimulating the activation of a family of proteases called caspases

Question 6

Formation of the apoptsomes

Answer 6

cytochrome C binds with ATP and induced Apaf-1 to form an apoptosome

Question 7

role of apoptosome

Answer 7

causes the dimerization of procaspase 9 creating and active caspase 9 dimers

these dimers catalyze the proteolytic cleavage of caspase 3 and caspase 7 for their activation

these caspases lead to the death and resoption of the cell without the release of pro-inflammatory DAMPS

Question 8

Roles of apoptosis for homeostasis and immunity

Answer 8

important in cellular, tissue and organ development

degredation cells that produce self antibodies

death of DNA damaged cells

Question 9

inappropriate apoptosis

Answer 9

large implications in neurodegenerative diseases

beta amyloid plaques, bypass autophagy and straight to apoptosis

Question 10

necrosis

Answer 10

type of cell death cause by trauma (lack of oxygen, temperature extremes, mechanical failure)

cells explode, DAMPs are releases

the cells swell because the membranes are leaky

lytic enzymes leak out and digest cell components leading to eventual lysis of cell

cellular debris and cytokine are released, to induce and inflammatoy response, damaging surrounging cells

Question 11

Contrast apoptosis from necrosis

Answer 11

apoptosis begins with loss of intercellulaar contact with apperently healthy cells followed by cell skrinkage, condensation of chromatin at the nulcear periphery, collapse of the cytoskeleton, dissolution of the nuclear envelope and fragmentation of its DNA

there is then blebbing of hte plasma membrane to dissosiate into apoptotic bodies which are engulfed by surrounding macrophages

inflammatory response is not induced, cell moleuclar components are conserved

mitochondria are important in triggering apoptosis

Question 12

TNF as a intercellular stressor

Answer 12

from cells of the immune system, interacts with specific TNF receptors

TNFRs bind TNF on the outside and transmit death signal through membrane to TRADD in the cytosol

Question 13

caspases

Answer 13

a family of proteases that participate in apoptosis

all synthesized as inacitive pro-enzymes

all have a critical cys residue at the active site

all hydrolyze their target protiens at the carboxyl terminal side of specific asp residues

caspase 8 = initiator caspase of the apoptosome

Question 14

caspase 8 activation

Answer 14

activated by signal from FADD then self activatees by cleaving its own pro-enzyme form

active caspase 8 targets the mitochondria

leads to increased permeability of outer mitochondria membrane

this allows cytochrome c to escape into the cytosol, and can thus interact with Apaf-1 causing formation of the apoptosome

Question 15

Apaf-1 and the apoptosome

Answer 15

7 (Apaf-1) + 7 (Cytc) => apoptosome

this is the platform on which the procaspase 9 is activated to caspase 9

Question 16

caspase 9

Answer 16

catalyzes the whole-sale destruction of cellular proteins and leads to cell death

involving the effector (executioner) caspase-3

the monomeric products aa and nucleotides are released in apoptosic bodies, ie a controlled fashion

components can be reused by surrounding cells

Question 17

Protein structure

Answer 17

structural diversity is necessary for the different functions of globular proteins

Question 18

denaturation

Answer 18

loss fo 3D structure

Question 19

heat denaturation

Answer 19

disrupts weak interactions especially H bonds

Question 20

extremes of pH

Answer 20

alters net charge, so electrostatic repulsion occurs

Question 21

Organic miscible solvents and denaturation

Answer 21

alcohol, acetone

disrupt hydrophobic interactions which stabilize the core of the protein

Question 22

Chaotropic agents and denaturation

Answer 22

aka solutes like urea, guanidinium chloride

effects same as solvents that disrupt hydrophobic interactions that stabalize the core of the protein

Question 23

detergetns

Answer 23

disrupt hydrophobic, stabalizing interactions of the protein

Question 24

denaturation by gradual heating

Answer 24

is cooperative, and yields a transition midpoint (Tm)

reversible

Question 25

aa sequence determine terriary strucutre

Answer 25

evidence from the renaturation of globular protiens after denaturation

from the classic case of denaturation (to a random coil) and renaturation (to full activity) of ribonuclease A

Question 26

renaturation experiment with ribonuclease a

Answer 26

urea to denature protein

beta mercaptoethanol to reduce disulfide bonds to thiols

see figure 6-38 in viot

Question 27

molecular chaperones

Answer 27

assist in folding

interact with partially unfolded or imporoperly folded polupeptides

facilitate the correct folding pathways or, provide a microenviroment for folding

ex heat shock proteins

Question 28

heat shock proteins

Answer 28

hsp70
abundent in hihg temperature stressed cells

binds to extended hydrophobic regions and prevents premature aggregation

Question 29

chaperonins

Answer 29

principal type in E.coli GroEL-GroES complex

required for folding of cellular proteins that do not fold spontaneously

sequester partially folded molecules from one another and from extraneous interactions

Question 30

proteins disulfide isomerase PSI

Answer 30

interchange or shuffling of -S-S- until bonds of the native protein form

Question 31

diseases due to misfolding

Answer 31

alzheimers

huntingtons

parkinsons

cataract

amyotrophil lateral sclerosis (Lou Gehrigs)

protein is secreted in misfolded state, then converted to amyloid = insoluable fibres = amyloidosis

Question 32

Proteasome

Answer 32

supramoleuclar assembly of proteolytic enzymes

(opposite of the ribosome, on which proteins are synthesized)

degrades proteins not needed by the cell in eukaryotes

1) protiens after their job is done, life growth factors that regualte the cell cycle
2) a damaged protein like to oxidative stress
3) misfolded proteins
4) defective proteins from a mutated gene

Question 33

proteins targeted for destruction via the proteasome

Answer 33

is first covalently tagged by ubiquitin by the enzyme ubiquitin ligase

then, several more ubiquitin moleucles are tagged on, providing the signal for the proteolytic enzyme complex the proteasome to degrade the target protein

after degradation to smaller nontoxic peptides, the amino acid products can then be recycled

Question 34

overrapid protein degredation

Answer 34

seen in alzheimers and perkinsons diseases

cystic fibrosis from the too rapid degredation of the chloride ion channel causing loss of funciton

Question 35

Amyotropic lateral sclerosis = ALS = Lou gehrigs

Answer 35

a fatal neurodegenerative disease

characterized by the progressive loss of selected populations of motor neurons

ther e si the presence of insoluable material, containg proteins involved in the major intracellular degradative pathways and the human DNA-binding protein TDP-43

it is generally accepted that their formation is linked to protien misfolding and may alter motor neuronal functions, impacting on their survival

ie these protein aggregations may cause and or facilitate other cascades of events responsible for motor neuronal cell death in ALS

Question 36

SOD and ALS

Answer 36

some of teh ALS cases have been asociated to Cu/Zn SOD1 gene mutations

SOD dismantles O2* to H2O2 and oxygen

Cu, Zn SOD1 is the most abundant in the eukaryotic sytosol, and is a very efficient enzymes with near diffusion controlled kinetics

intriguingly SOD1 also has a large number of other enzymic effects and thus catalyzes other types of redox reactions that may play subtle but key roles in the life of the cell

Question 37

SOD1 and NOS reactions

Answer 37

affects rxn by scavenging nitroxyl (No-) both aerobically and anaerobically, not by scavenging O2-

therfore, the biochemical reactions catalyzed by SOD1 and the cellualr implications of these reactions may be more complicated

Question 38

SOD1 in ALS leads to enhanced oxidatice stress

Answer 38

due to accumulation of ROS in ALS, and may partly be the cause of the neurodegeneration associated with the disease

in additon it has been postulated that ALS is a consequence of acquisition of toxic properties by the mutant SOD1 protein and rather than due to the loss of SOD activity

Question 39

How does mutant SOD1 work

Answer 39

the mutation results in structural/ conformational inadequacies in the protein and these lead to manifestation of toxic properties by the mutated protein

these toxic properties could be problems associated with the subtle protein-protein and protein-growth factor interactions, involved in the intricate networks in cellular signal transduction pathways

just as in the case of prion protiens which are misfolded forms of regularly functioning proteins

Question 40

how do we know protein misfolding is to blame for disorders

Answer 40

induction of chaperones like HspB8=Hsp22 and Hsp counteracts these effects,

moreover, inhibition of proteasome function induces ubiquitin ligase

releavs that the optimal destruction of hte misfolded protiens is key to the survival of the cell in misfolded protein stress situations

Question 41

The main strucutral changes in protein misfolding diseases PMDs

Answer 41

increase of beta sheet structure, oligomerization

formation of fibrillar amyloid-like polymers

as a result, protein agregates become insoluable, resistant to proteolysis, and resilient to cellular clearance mechanisms

Question 42

common features of PMDs

Answer 42

their apperance late in life

progression and chronic nature of the disease, and tissue deposititon of misfolded protein aggregates

the misfolding and aggregation mechanisms and the structural intermediates are very similar in all PMDs

Question 43

The mechanism of aggregation follows a seeding nucleation process

Answer 43

the critical step is the generation of a misfolded seed

this acts a sa nucleus to catalyze additional aggregation leading to the formation of oligomeric and fibrillar species

before folding is comples, the beta sheet regions of one polypeptide associate with the same region in another polypeptide, forming the nucleus of an amyloid

Question 44

Amyloidosis

Answer 44

a soluble protein, normally secreted from the cells is secreted in a misfolded state and is converted into a insoluable extracellular amyloid fibre

the fibres are hihgly ordered and unbranched

7-10nm

high degree of beta sheet structure

beta strands orientate dperpendicular to the axis of the fibre