mechanisms of cell death notes

8/8/2019 Mechanisms of Cell Death Notes

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APOPTOSIS inAPOPTOSIS in C.elegans C.elegans

C.elegans genome: 19099 genes (790 seven-passtransmembrane receptors, 480 zinc finger proteins, and410 protein kinases)The life cycle of C. elegans from egg to sexual maturity

(and new eggs) is about 3 daysced-1, -3, -4, and -9 (Ce ll d eath determining) proteins inC.elegans are closely related to mammalian apoptosis-regulating genes

The adult hermaphrodite consists of exactly 959somatic cells of precisely determined lineage andfunction. Individual cells are named and their relationships to their neighbors are known

Overall, the 959 somatic cells of adult C.elegans arisefrom 1090 original cells; exactly 131 somatic cellsundergo programmed cell death in the wild type worm

Of the 1090 cells, 302 are neurons, and many of theprogrammed deaths also lie in the neuronal lineage

www.chembio.uoguelph.ca


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11/30Nature Immunology 4, 416 - 423 (2003)

Autophagic cell death (type II programmed cell death) meaning thatthe cytoplasm is actively destroyed long before nuclear changes becomeapparent;Classical apoptotic cell death meaning that the chromatin marginatesand the cell and nucleus fragment before morphological changes are seenin intracellular organelles

)


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Nuclear alterations in different forms of programmed cell deathThe use of chromatin condensation as a criterion to distinguish apoptosis from apoptosis-like PCD hasbeen inconsistent in the scientific literature, and the potential for overlapping definitions and errors islarge. The following examples of classical apoptosis ( c,e ) and apoptosis-like PCD ( b ,d ,f ,g i) mightprovide a general guideline. Examples of control chromatin ( a ), and caspase-independent chromatinmargination triggered directly by microinjection of AIF ( b ). Caspase-dependent strong chromatincompaction ( c ) versus caspase-independent, AIF-driven lumpy chromatin condensation ( d ) in PCD of mouse embryonic stem cells. ( e ) Caspase-dependent chromatin compaction to crescent shaped massesat the nuclear periphery and chromatin fragmentation to two compact spheres ( f ) or caspase-independent lumpy chromatin condensation without nuclear fragmentation in colchicine-inducedneuronal cell death. Incomplete, lumpy chromatin condensation (compare with b ,d ) in caspase-independent apoptosis-like PCD triggered by Hsp70 depletion ( g ) or the active form of vitamin D ( i), andin caspase-dependent TNF-induced apoptosis-like PCD ( i) in caspase-3 deficient MCF-7 cells.

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13/30Nature Reviews Cancer 2; 647-656 (2002)

APOPTOSISAPOPTOSISSIGNALSSIGNALS

Mitochondria-dependentapoptosis

Caspase-dependentapoptosis

Death Receptor-dependentapoptosis

Caspase-dependentapoptosis

Caspase-independent

apoptosis

Caspase-independent

necrosis


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The target sequence for Ced-3 and caspases(Cys catalytic Asp targeting prote ases )

consists of a tetrapeptide with C-terminal Asp (D) .


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Procaspase-1 can be a substrate for Caspase-1 , and autocatalyticactivation is common among caspases. Thus activation shows positivefeedback characteristics consistent with a binary on-off regulation.

Ectopic expression of caspases in mammalian cells induces apoptosis .This is the strongest evidence for proteolytic mediation of apoptosis. The keyintracellular event appears to be caspase activation by proteolytic cascade.

Multiple caspases control different apoptotic pathways and providefunctional redundancy. Caspase-1/ICE is one of a family of relatedproteases, which are coexpressed, and cDNAs of all caspases can be

recovered from a single cell line. This appears to provide redundancy for animportant function and circumvents what was once an embarrassment, thatcaspase-1 knockout mice still undergo apoptosis.

The full range of apoptotic processes in mammalian cells involve several

caspases, some of which have specialized purposes; a whole subgroup isinvolved in cytokine activation rather than apoptosis. Some caspases areinitiators , i.e. their targets are downstream effector caspases. The targetsequences of several caspases resemble the activating sequences of other caspases. In many cases, caspases can target their own activatingsequences, suggesting autocatalytic positive feedback.



16/30Earnshaw et al. (1999)

Mammalian CaspasesMammalian Caspases


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DNA dependent protein kinase, protein kinase C, CAMkinase, focal adhesion kinase, MAP and ERK kinases, Raf1,Akt1/protein kinase B, ROCK I.

Protein kinases

-catenin, Bcl-2Cytoplasmic

actin, gelsolin, spectrin, keratinCytoskeleton

MCM3, Repair enzymes including Rad51, poly-ADP-ribosepolymerase (PARP), topoisomerase, inhibitor of caspase

activated DNase, ( iCAD/DFF45)

DNA related

Lamins, nucleoplasmin, the SR protein 70K U1, hnRNP C,

RNA Pol I upstream binding factor, the p53 regulator MDM2,pRB, p27 Kip and p21 Cip

Nuclear

In vivo In vivo substrates of effector caspasessubstrates of effector caspases



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Mitochondria play a central roleMitochondria play a central rolein mediating the apoptotic signalin mediating the apoptotic signal

Mitochondria-free cytoplasm would not induce apoptosis in vitro

Cytochrome c-neutralizing antibodies block apoptosis

Cytochrome c is an abundant protein of the mitochondrial inner membrane, and acts as an electron transport intermediate.

a and b type cytochromes are inaccessible components of largecomplexes, but cytochrome c is monomeric, freely diffusible inthe inner membrane, and in equilibrium between inner membrane, inter-membrane space and cristae.

The events of apoptotic activation lead to alterations inpermeability of the mitochondrial membrane pore proteins andrelease of cytochrome c.

Initial release of cytochrome c occurs by a highly specificprocess, involving proteins of the Bcl-2 family



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Death receptors of the TNFR family, as well as

various oxidants, detergents andchemotherapeutic drugs, induce the release of active cathepsins from the lysosomalcompartment. These cathepsins cleave Bid,which can then mediate cathepsin-induced MPT.Disruption of the cytoskeleton leads to therelease of the BH3 domainonly proteins Bimand Bmf. DNA damage induced by radiation or various chemotherapeutic drugs induces thep53-mediated transcription of genes encodingBax, BH3 domainonly proteins (Noxa or Puma),

proteins involved in ROS generation andcathepsin D. ER stress results in the release of calcium, which may cause direct mitochondrialdamage or activate Bax through calpain-mediated cleavage. Various death stimuli,mediated through death receptors, trigger the

production of lipid second messengers (such asganglioside (GD3), arachidonic acid (AA) andceramide) that are involved in MPT andmitochondrial damage. Depending on thestimulus and the type of cell, as well as themetabolic status of the cell, MPT leads to either caspase-mediated apoptosis or caspase-independent PCD.

Nature Immunology 4, 416 - 423 (2003)

Signaling pathways leading toSignaling pathways leading to MMitochondriaitochondria PP ermeabilityermeability TTransitionransition


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BclBcl --2 family: Pro2 family: Pro --Life and ProLife and Pro --Death factionsDeath factions

Bcl-2 and its closest relatives Bcl-X L, Bcl-w and Ced-9 are a-helical proteins having all four

BH domains and are pro-survival . They suppress cytochrome c release, and are oncogenicwhen overexpressed. However, Bcl-X S , a splice variant of Bcl-X L having BH4 but lacking BH1and BH2 is pro-apoptotic.

Bax and Bak lack the BH4 domain, and are pro-apoptotic . Bax expression is stimulated byp53, a mechanism for pro-apoptotic action of p53. Ectopic or overexpression of Bax inducescytochrome c release and apoptosis, and addition of Bax to mitochondria in vitro inducescytochrome c release.

The BH3-only sub group are strongly pro-apoptotic , and include Bim , Bik and Egl-1 ,which only have the 18-residue BH3 and the transmembrane region, while Bad and Bid only

have BH3. The helical BH3 element allows for homo- and heterodimerization between familymembers. The non-homologous regions of BH3-only proteins could provide links to apoptoticsignaling systems.



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Caspase-9

Effector caspases


Vertebrate Apaf-1 activation occurs throughcytochrome c binding. Bcl-2 and Bcl-X L appearsto act by dimerizing with pro-apoptotic agonistssuch as Bax or Bak.

Normally, the balance is in favor of Bcl-2 or Bcl-XL, but the BH3-only factors appear to act totitrate out the Bcl2/Bcl-X L, tipping the balance infavor of Bax/Bak.

Bax can oligomerize in the membrane to form apermeability channel able to transportcytochrome c .

BH3-only factors have been reported to induce

reorganization of the cristae. Alternative modelssuggest that Bid/Bad/Bak like factors act to openpermeability channels such as the permeabilitytransition pore , by disrupting the membranepotential, and affecting the voltage-dependentanion channel VDAC and ATP/ADP exchangetransporter.

BclBcl --2 family: Pro2 family: Pro --Life and ProLife and Pro --Death factionsDeath factions


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Survival mechanisms downstream of cytochrome cSurvival mechanisms downstream of cytochrome c

1. Sequestration by heat shock proteins:

Apaf1 interacts with heat shock proteins hsp70 and hsp90. Hsp70 directly sequesters CARD, and blockscaspase-9 recruitment, and possibly assembly of the oligomeric apoptosome as well. Hsp90 alsoassociates with the monomeric Apaf1, and may represent a significant fraction of the normal autoinhibitedstate. Hsp90 appears to compete with cytochrome c for binding, suggesting action at an earlier step thanhsp70.

2. Direct inhibition of the caspase catalysis by Inhibitor of Apoptosis Proteins (IAPs):

Inhibitor of apoptosis proteins (IAPs) represent thefinal line of defense against apoptosis, and act bybinding directly to the substrate site of caspases

Smac/DIABLO: the mitochondrial answer to IAPs:

Mitochondria initiate the apoptosis cascade byreleasing cytochrome c, but this effect could benullified if IAP were allowed to maintain their inhibitionof caspases. The apoptotic signal is instead sustainedby the release of Smac/DIABLO (s econd m itochon-drial a ctivator of c aspase/ d irect IAP b inding protein of lo w pI), which binds to and antagonizes the IAPs.



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Mitochondrial damage leads to the release of numerous mitochondrial proteins that mediatePCD.

Release of cytochrome c triggers caspaseactivation and classic apoptosis.

Smac (also known as Diablo) and Omi assistcytochrome c induced caspase activation bycounteracting caspase inhibitory factors ( IAPs ).

AIF triggers a caspase-independent deathpathway that culminates in DNA fragmentationand chromatin condensation characteristic of

apoptosis-like PCD. EndoG cleaves DNA and induces chromatin

condensation The serine protease activity of Omi can mediate

caspase-independent cellular rounding andshrinkage without changes in the nuclear morphology

Calcium and ROS can lead to severemitochondrial dysfunction and necrosis-likePCD either directly or through autophagy of damaged mitochondria. Autophagy also may be

associated with cathepsin activation and so canresult in apoptosis-like PCD.


Mitochondria permeability transition can trigger caspaseMitochondria permeability transition can trigger caspase --dependent dependent and caspaseand caspase --independent programmed cell death (PCD):independent programmed cell death (PCD):


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DEATH RECEPTORS:DEATH RECEPTORS:Pathways linking external signal receptors to caspasePathways linking external signal receptors to caspase --88

A variety of cell surface receptors related to TNF-R (tumor necrosisfactor receptor) interact with the apoptotic activation system. Theintracellular portion of the receptor carries a specific proteininteraction domain called the death domain, DD . The DD isactivated by proximity, brought about when bound extracellular ligand induces receptor oligomerization. Activation can also beinduced in absence of ligand by artifical cross-linking of thereceptor.

Clustered receptor DDs recruit a variety of DD-containing adapters,

of which FADD , Fas- a ssociated d eath d omain protein (also knownas MORT1) bridges to a second protein interaction domain, DED ,or d eath e ffector d omain. The cluster of FADD-DEDs recruitsprocaspase-8, which also carries DEDs at its N-terminus(corresponding to the CARDs on Procaspase-9).

Procaspase-8 is activated to Caspase-8 by proximity-induced self-cleavage. Procaspase-10 is the only other caspase with DEDboxes, and may substitute for Caspase-8 in some cases.

In some cells, TNF receptors associate with adaptors linked to cellproliferation or inflammatory signalling pathways, and may induceanti-apoptotic c-IAPs.



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decoy receptor, ligandsequestrationnoneFas-L/APO-1LDcR3

decoy receptor, ligandsequestration

noneTRAIL/APO-2LDcR2

decoy receptor, ligandsequestrationnoneTRAIL/APO-2LDcR1

Procaspase-8, apoptosisFADDTRAIL/APO-2LDR5

Procaspase-8, apoptosisFADDTRAIL/APO-2LDR4

Procaspase-8, apoptosisFADD?APO-3LDR3/APO-3

MEKK, Jun/Ap1, cell proliferation,IKK, NF- B, inflammation, c-IAPs

TRAF2+TRAF1TNF TNF-R2/CD40

MEKK, Jun/Ap1, cell proliferation,IKK, NF- B, inflammation, c-IAPs

TRADD+RIP1+TRAF2TNF TNF-R1

Procaspase-8, apoptosisTRADD+FADDTNF TNF-R1

Procaspase-8, apoptosisFADD/MORT1Fas-L/APO-1LFas/CD95/APO-1

TargetAdaptor LigandReceptor


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The death receptor is stimulated byligand-induced activation of the receptor trimer. The receptor death domains (DDs)of Fas then recruit FADD and RIP1 to thereceptor complex. After recruitment to

FADD through interactions between their death effector domains (DEDs), caspase-8 and caspase-10 are activated andtrigger effector caspases, either directly or through a Bid-mediated mitochondrialpathway (activation of Apaf-1 andcaspase-9).FADD and RIP initiate a caspase-independent necrotic pathwaymediated by the formation of, mostprobably, mitochondrion- or cPLA2-

derived ROS. TNFR1 signaling differsfrom Fas signaling in the following steps:first, binding of FADD and RIP to thereceptor complex requires the adaptor protein TRADD; and second, the RIP1-mediated necrotic pathway is inhibited by

FADD and activated caspase-8

Death receptorDeath receptor triggered caspasetriggered caspase --dependent dependent and caspaseand caspase --independent pathwaysindependent pathways


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Detection of apoptotic changes in DNA:Detection of apoptotic changes in DNA:

Nucleic acid staining nuclear morphology

Detection of nuclear DNA fragmentation

TUNEL staining(terminal deoxynucleotidyl transferasemediated dUTP nick end-labeling)

Single-cell electrophoresis (Comet assay)

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Detection of changes in membrane integrity:Detection of changes in membrane integrity:

Membrane permeability

Phospholipid symmetry (Annexin V staining)

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Detection of apoptotic changes in mitochondria:

Caspase Protease Assays (individual caspases):

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MPTMorphology


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Detection of pro- and anti-apoptosis proteins, Fas-ligand, cytokines, etc.

Detecting changes in gene expression for pro- and anti-apoptosis genes

mechanisms of cell death notes

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