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

www.novusbio.com

UPR

IRE1

ATF6

PERK

ATF6

ATF4

sXBP1BECN1

sXBP1

XBP1, GRP78

ATG16L, ATG12, ATG3, BECN1, p62, ATG7, ATG10, ATG5, etc.