apoptosis, necroptosis & autophagy - novus biologicals · pdf fileapoptosis, necroptosis...
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SIGNALING PATHWAYS & KEY MOLECULAR TARGETS
Apoptosis, Necroptosis & AutophagyNECROPTOSIS AUTOPHAGYAPOPTOSIS
Key Targets in ApoptosisInitiator Caspases (-2, -8, -9, -10): In apoptosis, initiator caspases are involved in the upstream events of death receptor (extrinsic)- or mitochondrion-dependent (intrinsic) signaling pathways. They are responsible for the cleavage of executioner caspases.
Executioner Caspases (-3, -6, -7): Upon activation by initiator caspases, executioner or effector caspases orchestrate cell death by cleaving a large number of cellular proteins. They cleave PARP and inhibit its DNA-nicks repairing potential.
Death receptors and ligands: In the extrinsic pathway, the death ligands TRAIL, FASL and TNF-α bind to the receptors TRAILR1/2 (DR4/DR5), FAS/CD95 and TNFRI respectively and initiate the activation of Caspase-8 and -10.
Bcl-2 Proteins: Intrinsic pathway signals activate the pro-apoptotic Bcl-2 proteins (Bax, Bak, BID, BAD, PUMA, NOXA or BIM) and neutralize the anti-apoptotic Bcl-2 proteins (Bcl-2, Bcl-XL, MCL1) leading to Cytochrome-c/Smac release via disruption of mitochondrial integrity. This is followed by apoptosome-dependent Caspase -9 activation.
Key Targets in AutophagyLC3: LC3, a ubiquitin-like modifier, is critical to the formation of autophagosomes. During autophagy, LC3B-I (cytosolic) is lipidated to form LC3B-II which incorporates into the autophagosomal membranes. Conversion of LC3-I to LC3-II is a gold standard indicator of autophagy induction.
P62/SQSTM1: p62 is a stress-inducible protein with multiple domains/binding partners. In addition to its role as a signaling hub for amino acid sensing and the oxidative stress response, p62 acts as a selective autophagy receptor for degradation of ubiquitinated substrates.
ATG5: During autophagosome biogenesis, ATG5 binds ATG12. Thereafter, the ATG5-ATG12 conjugate binds to ATG16L1 to generate the autophagy elongation complex (ATG5-12/16L1) which regulates lipidation of LC3.
Beclin 1: It is a mammalian ortholog of yeast Atg6 protein. Beclin 1 positively regulates autophagy through activation of Vps34.
Normal Cell Shrinkage & Chromatin Condensation
DNA/Nuclear Fragmentation & Membrane Blebbing Apoptotic Bodies
TNFR1
TRADD TRAF2
FADDDAXX
TLR4TLR4
RIP
Growth Factor
Growth Factor Receptor
Pro-Caspase-8
FLIP
ZVAD-FMK
ActiveCaspase-8
Pro-Caspase-3
ActiveCaspase-3
IκBDegradation
IKK
JNK
Bid
TRIF
IKK
IKK
Apoptosis
PI3K
Erk1/2
AKT
p90RSK
BADHrkDPS
tBid
BAX
HrkDPS
Bcl-XL
Bcl-2
Pro-Caspase-7
ActiveCaspase-7
Noxa
PUMA
Mcl-1p53
Apaf-1
Mule
DNAFragmentation
Ceramide
Sphingomyelin
Acid Sphingomyelinase
Necroptosis
NF-κB
XAP1
IAPHTRA2
SMAC
ARTS
p70S6K
PKC
PKA
Actin
GBS2
D4-GDI
ROCK-1
BAX
AKT
AIF
Pro-Caspase-9
Apoptosis
Fordin
CAD
ICAD
FADD
TRIF
DNA Repair
BAX
BAK
BAK
IκB
DNA Damage
UV, DNADamage
ROS
p38
AKT1
PKB
PDK
c-Myc
BNIP3L
Erg1
MisfoldedProteins
PERK GPR87
GPR87
IRE1 GPR87
GPR87
ATF6
GPR87
GPR87
GPR87
ER Stress R
esponse
Caspase-12
ElF2
ATF6Cleaved
sXBP1
JNK
Annexin V Conjugates
Refold
Unfold
P
Bcl-2
Cytochrome C
CHOP
p58IPK
Chaperones
Restore ERFunction
?
PERK GPR87
FOXO3aPS
PS
PSPS
PS PS
PS
PS
pSIVA-IANBD
TM
PS
PSPS
PS
A
pop
tosi
sGolgi
TLR3TLR3
Fas
NF-kB
PARP
CAD
FOXO3a
c-Jun
FasL
BIM
Bcl-6PUMA
ATF4
sXBP1ATF4
PS
Apoptosis Reversal
Apoptosome
ActiveCaspase-9
αTNF
NFAT1/2
ActiveCaspase-8
GSH SODCAT
m
FasLGrowth Factor
Growth Factor Receptor
PP
Class IIIp13K
Bcl-2/Bcl-XL
mTOR
Induction
Elongation
AutophagosomeMaturation
Autolysosome
ATG16LATG12
ATG5
PE
Pro-LC3
ATG7
LC3-I
LC3-II
LC3-II LC3-II
LC3-II
ATG4
Lysosome
Macroautophagy
Microautophagy
Class Ip13K
Beclin Golgi
Rapamycin
NutrientStarvation
ATG12
ATG7
ATG12
ATG10
ATG12
ATG5
ATG16L
ATG16LATG12
ATG5
Autophagic BodyBreakdown
Hop HipBag-1Hsp70
Hsp40Hsp90
CytosolicProtein
Bag-1HopHip
Hsp70Hsp40Hsp90
CytosolicProtein
Lamp-2a
CP IYS
Hsc70
TLR4TLR4
Lamp-2a
TLR3/7/8
Type IIFN
IFNgTNFa
Recycling ofMacromolecules & Amino Acids
TRAIL DR4/5
CD40L CD40
TCR MHC class II
CD4
MHC class II
Lamp-2a Antigen-MHC class II Transport +
Presentation
CD4+ T Cell
AntigenPresentation &
Recognition
CytosolicProtein
ChaperoneMediated
Autophagy
AKT
Ub
p62
p62Ub
Receptors: Upon binding to their ligands, receptors such as the TNF-alpha receptor superfamily, T-cell receptors (TCRs), interferon receptors (IFNRs) and Toll-like receptors (TLRs) drive necroptosis signaling.
Necrosome: Receptor-interacting protein kinase 1 (RIP1), RIP3 and mixed lineage kinase domain-like protein (MLKL) form the necrosome(RIP1-RIP3-MLKL complex). The necrosome regulates upstream cell death receptors and downstream executing molecules, as well as events including ROS burst, plasma membrane permeabilization, and cytosolic ATP reduction.
RIP1-RIP3-MLKL phosphorylations: RIP1 activitation during necroptosis triggers RIP3 phosphorylation and subsequent phosphorylation of MLKL. Trimerized phospho-MLKL translocates to the plasma membrane for inducing necrotic plasma membrane permeabilization. Some inducers may bypass RIP1 for direct activation of RIP3 or MLKL.
Key Targets in Necroptosis
Normal Induction AutophagosomeFormation
AutophagosomeLysosome FusionNormal Membrane Breakdown Release of Cell Contents Cell Swelling
Process Active, physiological or pathophysiological
Induction stimuli Oxidative stress, death receptor ligands, chemotherapyMorphological changes Nuclear pyknosis, membrane blebbing, generation of apoptotic bodiesMolecular/biochemical changes Cleavege of caspases and PARP, DNA fragmentationClearance Apoptotic bodies phagocytosed by neighboring cells & macrophages
Process Mostly passive, always pathological
Induction stimuli Viral or chemical exposure, radiation, endogenous or pathological factorsMorphological changes Swelling of cells and organelles, loss of membrane integrityMolecular/biochemical changes Acidosis, random DNA degradation, release of cellular proteinsClearance
Process Active, physiological or pathophysiological
Induction stimuli Starvation, hypoxia, chemotherapy, growth factor deprivationMorphological changes Vacuolization, mass degradation of organelles & proteinsMolecular/biochemical changes LC3I lipidation to LC3II, p62/SQSTM1 degradation, lysosomal activityClearance Cells gets cannibalized & the contents recycle for survival of the tissue
DNA DamageRadiation
Chemotherapy
TNFInjury
Chemotherapy
AS Inhibitors
Necroptosis
MnSOD
FHC
Fe++
IkBDegradation
IKK
IKK
IKK
PARP
JNK
PLA2
Ceramide
Caspase-8
FAD
D
TRAF2
ActiveCaspase-8
TRAD
D
TRIF
FADD
RIP
ROS
O2-
Necrostatins(NEC1,2,3)
Apoptosis
AcidCeramide
Sphingomyelin
Acid Sphingomyelinase
Breakdownthe Complex
IkB
ZVAD-FMK
Cycd
Ant
NF-kB
NAD+/ATPi
H2O2
TLR4TLR4
NOX1
RAC1 p22phox
NOXA1NOXO1
TRAD
D
FADD
TRIF
Fas
TLR3TLR3
PARP
αTNF
TNFR1
FasL
αTNF
TNFR1
Pro-Caspase-8
RIP1
RIP2
MLKL
P
P
P
Caspase 8 Inactivation
MLKLP
MLKLP
MLKLP
NF-kB
IRE1
Caspase Independent Cell Death (CICD)
ATG4
Tat Beclin D11& L11 Peptides
Nucleus
Cytoplasm
Nucleus
Cytoplasm
Nucleus
Cytoplasm
MisfoldedProteins
Degraded Organelles, Cytosolic Proteins
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UPR
IRE1
ATF6
PERK
ATF6
ATF4
sXBP1BECN1
sXBP1
XBP1, GRP78
ATG16L, ATG12, ATG3, BECN1, p62, ATG7, ATG10, ATG5, etc.