Understanding the Molecular Biology of Atherosclerosis

The Future of Prevention and Intervention in Heart disease

Paul N. Hopkins, MD, MSPHCardiovascular Genetics

University of Utah

A few pearls regarding signaling (1)

• Extracellular ligands bind to:  transmembrane cognate receptors, co‐receptors, integrins, some adhesion molecules – Conformation change of intracellular domain(s) 

• Receptor tyrosine kinase activation• Non‐receptor tyrosine kinase recruitment and activation• Receptor serine/threonine activation

– Less common– TGFβ receptor family phosphorylates SMADs (ancient)

• Altered adaptor binding, oligomerization– TNF family receptors

• Proteolytic activity– NOTCH signaling

Cell Signalling Biology, Michael J. Berridgehttp://www.biochemj.org/csb/default.htm

A few pearls regarding signaling (2)

• Receptor tyrosine kinases (58 in human genome*)– Activation of intrinsic tyrosine kinase domain– Examples:  insulin receptor, EGFR, PDGFR, VEGFR, etc.– Evolutionarily among most recent signaling molecules

• Non‐receptor tyrosine kinases (32 in genome*)– Recruited to many receptors, integrins, etc. without intrinsic tyrosine kinase activity

– Examples:  JAKs, Src family, Syk, ZAP70, FAK, others

* Robinson DR, et al.  Oncogene 2000; 19:5548 

Cell Signalling Biology, Michael J. Berridge

Copyright © 2011 by Saunders, an imprint of Elsevier Inc.Abbas, Lichtman, and Pillai. Cellular and Molecular Immunology, 7th edition. Copyright © 2012 by Saunders, an imprint of Elsevier Inc.

Early Signaling Events in T cell Activation (1)

Fig. 7‐10A

Copyright © 2011 by Saunders, an imprint of Elsevier Inc.Abbas, Lichtman, and Pillai. Cellular and Molecular Immunology, 7th edition. Copyright © 2012 by Saunders, an imprint of Elsevier Inc.

Early Signaling Events in T cell Activation (2)

Fig. 7‐10B

Copyright © 2011 by Saunders, an imprint of Elsevier Inc.Abbas, Lichtman, and Pillai. Cellular and Molecular Immunology, 7th edition. Copyright © 2012 by Saunders, an imprint of Elsevier Inc.

Early Signaling Events in T cell Activation (3)

Fig. 7‐10C

A few pearls regarding signaling (3)

• Tyrosine kinases typically create a binding site for adaptor / docking / scaffold proteins

• Adaptor/scaffold proteins recruit multiple downstream signaling cassettes  interdependent, frequentlyredundant, signaling networks that include:– Serine/threonine kinases– Phospholipases (e.g. PLCγ)– Phosphatidyl inositol kinases (e.g. PI3K)– G‐proteins = small GTPase’s (off/on switches)– Ubiquitin ligases

• Phosphatases generally inhibit the above signaling– Phosphatases must be inhibited by ROS for normal signaling

Cell Signalling Biology, Michael J. Berridge

nucleus

Grb2Sos

(Shc)

•O2-

O2

NOX1/2Other?

ClC3

SOD1

INSR

RasGTP

eNOSactivation,

anti-apoptoticeffects

PTPDAG

p47phoxPTEN

IRS1/2

PLCγ

PIP2

PIP3PIP3

STAT5STAT3 PKB, Bcl-XL, VEGF, ↓ p53

STAT3

RasGTP

insulin

SH2B1 RACK1

MKP1

PTP

RasGDP

NOX activation

Src IRS1/2SHP2

H2O2

? GEF

Rac1GDP

Rac1GTP

JAK2

PTEN

SHP2

PHLPPOther effects

Other effects

PI3Kα

MAPK pathway

Other adapters, and targets

CCND1, claudin 5, NF-κB, eNOSFOXO1,3

S6Rheb S6K1

IRS1/2, iNOS, p21, p27Kip1, FASL, BIM, Noxa, MnSOD, GADD45, Egr1FOXO1,3

FOXO1,3

eIF2B eIF2

FOXO1,3: ↓ cell cycle progression, ↓ inflammation, ↑ apoptosis, ↑ antioxidants

eIF4E4E-BP

autophagy

p53, LKB1, p66Shc

AMPK14-3-3

protein synthesiscell growth

Other effects

PIP3

PIP3PDK

PKCζ

mTORTSC2TSC1

•O2-

INSR

insulin

CCND1

Gab1PI3Kα

peripheral actin assemblyadherens junction

barrier stabilization

Rac1GTP

mTORC1GSK3βOther targetsAkt1

IP3R

ER⬆Ca++

IP3MTM1

Src

PKC

SIRT1

nucleus

Grb2Sos

(Shc)

•O2-

O2

NOX2Other?

ClC3

SOD1

INSR

RasGTP

eNOSactivation,

anti-apoptoticeffects

PTPDAG

p47phoxPTEN

IRS1/2

PLCγ

PIP2

PIP3PIP3

STAT5STAT3 Akt, Bcl-XL, VEGF, ↓ p53

STAT3

RasGTP

insulin

SH2B1 RACK1

MKP1

PTP

RasGDP

NOX activation

Src IRS1/2SHP2

H2O2

? GEF

Rac1GDP

Rac1GTP

JAK2

PTEN

SHP2

PHLPPOther effects

Other effects

PI3Kα

MAPK pathway

Other adapters, and targets

CCND1, claudin 5, NF-κB, eNOSFOXO1,3

S6Rheb S6K1

IRS1/2, iNOS, p21, p27Kip1, FASL, BIM, Noxa, MnSOD, GADD45, Egr1FOXO1,3

FOXO1,3

eIF2B eIF2

FOXO1,3: ↓ cell cycle progression, ↓ inflammation, ↑ apoptosis, ↑ antioxidants

eIF4E4E-BP

autophagy

p53, LKB1, p66Shc

AMPK14-3-3

protein synthesiscell growth

Other effects

PIP3

PIP3PDK

PKCζ

mTORTSC2TSC1

•O2-

INSR

insulin

CCND1

Gab1PI3Kα

peripheral actin assemblyadherens junction

barrier stabilization

Rac1GTP

mTORC1GSK3βOther targetsAkt1

IP3R

ER⬆Ca++

IP3MTM1

Src

PKCβ

SIRT1

Small G‐proteins or small GTPases are off‐on switches

About 150 in 5 families

Loirand G, et al.  Physiol Rev 2013; 93:1659

Loirand G, et al.  Physiol Rev 2013; 93:1659

Cell Signalling Biology, Michael J. Berridge

A few pearls regarding signaling (4)

• Serine/threonine kinases (over 420 in genome) have much more diverse roles than tyrosine kinases– Activate/inhibit enzymes, ion channels, other signaling molecules

– Modify binding affinity (including transcription factors)– Target or block ubiquitination, degradation– Examples:  PKA, Akt/PKB, PKCs, PDK, SGK1, many, many others

– MAPK pathway a special example

AP1 MCP1, ET-1, IL-8, ICAM-1, HO-1 etc.

AP-1

Egr1 ICAM-1, TF, PDGF

Grb2

EGFR

Sos(Shc)

HB-EGF

MSK1

RBD Activation loop

PKA,

S43

Raf1 N

PKA,

S23

3PK

A&B,

S25

9PK

C, S

338

Src,

Y341

Raf1

, T49

1

Raf1

, S49

4

PAK,

S62

1

CRD

14-3-314-3-3

MAP3K

MAP2K

MAPK

MEKK2/3

MEK5

Raf1

MEK1/2 MKK4/7

ASK1 TAK1

MKK3/6

ADAM17

RasGDP

RasGTP

cytoprotection

JunElk1 Fos

ATF2 RelA

Grb2Sos

Shc

INSR

RasGTP

insulin

RasGDP

JNK p38 ERK5

mRNA stabilization

p47phox

MK2

NOX5

p47phox

ERK1/2

Fos RSK1

Ca++

Vav2Rac1 GDP

Rac1 GTP

AP1 MCP1, ET-1, IL-8, ICAM-1, HO-1 etc.

AP-1

Egr1 ICAM-1, TF, PDGF

Grb2

EGFR

Sos(Shc)

HB-EGF

MSK1

RBD Activation loop

PKA,

S43

Raf1 N

PKA,

S23

3PK

A&B,

S25

9PK

C, S

338

Src,

Y341

Raf1

, T49

1

Raf1

, S49

4

PAK,

S62

1

CRD

14-3-314-3-3

MAP3K

MAP2K

MAPK

MEKK2/3

MEK5

Raf1

MEK1/2 MKK4/7

ASK1 TAK1

MKK3/6

ADAM17

RasGDP

RasGTP

cytoprotection

JunElk1 Fos

ATF2 RelA

Grb2Sos

Shc

INSR

RasGTP

insulin

RasGDP

JNK p38 ERK5

inputs

MAPK

activ

ity

many inputs

many outputs

MAPKfilter

mRNA stabilization

p47phox

MK2

NOX5

p47phox

ERK1/2

Fos RSK1

Ca++

Vav2Rac1 GDP

Rac1 GTP

A few pearls regarding signaling (5)

• Ubiquitin E3 ligases are highly diverse– 617 putative E3 ubiquitin ligases in human genome* – K48 ligation can mark for proteosomal degradation– Can also alter binding/function of proteins, enzymes, transcription factors

– K63 or end to end ligation builds dynamic, transient signaling scaffold

• Ubiquitin editing enzymes or deubiquitinases– Important modifiers of inflammatory signaling

• Sumo similar to ubiquitin

* Li W, et al.  PLoS ONE 2008; 3:e1487

Cell Signalling Biology, Michael J. Berridge

ICAM-1, VCAM-1, MCP1, IL-1, IL-2,IL-6, IL-8, Groβ, TNFα, E-selectin, P-selectin, LOX-1, VEGF, IL-10, eNOS, cFLIP, MKPs, COX2, IκBα, cIAP1, XIAP, A20, Cezanne, MnSOD, …

NF-κBC38

Ox, SNOY66NO2

Y152NO2

S276P

K310Ac

S536P

RHD TADp65 (RelA)

S311P

K221Ac

Rac1

•O2-

•O2-

PKCζ

•O2-

ClC3

SOD1

Sos

PKCζ14-3-3

MSK1 PKAc PKCζ

IKK1/2

p52

RelA:p50 RelB:p52

NIK

PAK1/2

A20

NIK

Ras

PKCζp62

MEKK3

NOX1

/2

TNFR1

SODD

LUBACcIAP1/2

RFK

Grb2MADD•O2

-

TRAF2TRADD

TNFR2

TNFα (membrane bound)K63-linked or linear ubiquitin chainsK48-linked ubiquitin chains

BMX

NIK

ceramide

ASM FAN RACK1 EED nSMase

O2

p53

FAK

AKIP1

•O2-

TRAF

3

cIAP1/2TRAF2

TNFα (soluble)

26S

•O2-

NEMONEMO

26S

26S

GEF-H1Rho

ROCK

cIAP1/2TRAF

3

NIK

IRS-1

IκBα p100

sphingosine

S1P SPHK1

S1P PKCδ/ε

lipid raft fusion

IKK2ERK1/2

SIRT1 CBP

p38

RasTAB1 TAB2

TAK1

JNK

H2O2 ERK5

RSK1

IKK1

Src RIP1

Oxidative post-translational modifications on cysteine – a normal part of signaling!

Heaseung S. Chung et al. Circulation Research. 2013;112:382‐392

MAP3K

MAP2K

MAPKERK5

MEKK2/3

MEK5

Raf1

MEK1/2 MKK4/7

ASK1 TAK1

MKK3/6

PKCζ

SOK1

MEKK1

CezanneSENP1

Src

GSTπ

PTPSS

PTPSHSH

TRP14

LC8S

LC8S

LC8SH

PKCζ

S S

Trx

Trx

ERK1/2 JNK p38

TRADDRIP1 TRAF2AIP1

PP2A SOD1

GSTπ

26S

NF-κBIκBα

PKAc

LC8SH

Ras

•O2−

NO

active

ASK1S

S

14-3

-3

ASK1SH

SH

PDK1

inactive

RSK1/2

active

inactive

Syk IKK2

Examples of Cysteine Thiol Switches

RSK1

GSH

eNOS eNOSO2

‐

ROS

NO

ONOO•−

H2O2

Trx

TrxR

Srx

Prdx1

Prdx1 Prdx1

Prdx1

PTENactive

PTEN

inactive

JNK inactive

Prdx1

JNK

active

other chaperone functions

Prdx1Prdx1

Low oxidative stress

Higher oxidative stress

The Peroxiredoxin Oxidative Switch

Garlic as an arterial H2S donor

• Low levels of H2S function analogously to glutathione to protect cysteine sulfhydryl groups

• Organic polysulfides (e.g. allicin = diallyl thiosulfinate) in garlic (GSH in RBC, other cells)  H2S release vasodilation, anti‐inflammatory signaling– Benavides GA, et al. PNAS 2007; 104: 17977– Polhemus DJ, Lefer DJ.  Circ Res 2014; 114:730

• 2 randomized, controlled trials with garlic were positive without effects on standard risk factors.– Koscielny J, et al.  Atherosclerosis 1999; 144:237– Budoff MJ, et al.  Prev Med 2009; 49:101

Endothelial mechanotransduction and signaling explain the distribution 

of atherosclerosis

internal carotid

common carotid

external carotid

flowdivider

SLOW, mildly oscillating flow with LOW shear stress (NOT TURBULENCE!!) predisposes to atherosclerosis

RAPID, high sheer stress, laminar, unidirectional flow is highly protective

Dai G, et al.  PNAS 2004; 101:14871

-100

1020304050

0 500 1000 1500 2000 2500 3000 3500 4000

Shea

r Stre

ss (d

yn/sq

-cm

)

Time (ms)

Atherosclerosis‐prone waveform

0.01 0.1 1 10 100 1000

Infla

mm

ator

y Res

pons

e

Hours

↑ G

PC

R, C

a, K

cha

nnel

s↑

Ras

GTP

(ver

y tra

nsie

nt)

↑ P

GI 2

& N

O re

leas

e↑

PE

CA

M-1

pho

spho

ryla

tion

↓ R

ho G

TP, ↑

Rac

1, C

dc42

↑ B

MP

-4↑

NA

D(P

)H O

x↑

supe

roxi

de, R

OS

↑ N

F-κB

, ER

K1/

2↑

Erk,

JN

K,

↑ R

ho G

TP↑

tyro

sine

kin

ases

↑ IC

AM

-, VC

AM

-1↑

MC

P-1

rele

ase

↑ TL

R-2

↑ ap

opto

sis

↑ m

itosi

sSh

orte

ned

glyc

ocal

yx↑

perm

eabi

lity

↑ M

onoc

yte

adhe

sion

↑ Fi

bron

ectin

syn

thes

is

↑ S

usce

ptib

ility

to

athe

rosc

lero

sis

0

10

20

30

40

50

0 500 1000 1500 2000 2500 3000 3500 4000

Shea

r Stre

ss (d

yn/sq

-cm

)

Time (ms)

Athero‐protective waveform

0.01 0.1 1 10 100 1000 10000

Infla

mm

ator

y Res

pons

e

Time (hours)

↑ N

O re

leas

e

↑ R

ho, ↓

Rac

1, C

dc42

Org

aniz

atio

n of

act

in

cyto

skel

eton

, al

ignm

ent w

ith fl

ow↓

perm

iabi

lity

↑ N

F-κB

con

tinue

s ↑

Nrf2

, Nrf1

↑ A

RE

gen

es (H

O-1

)↑

MK

P-1

, TR

AF3

↑ K

LF2,

KLF

4↑

CO

X-2

, PG

IS↑

PG

I 2R

OS

, NF-κB

, JN

K,

othe

r pro

-infla

mm

ator

y si

gnal

s re

turn

to

base

line

or lo

wer

↑ an

ti-ox

idan

t gen

esC

yclin

s de

crea

seA

ther

o-pr

otec

tion

Onset of flow or directional change in shear stress

caveola

extracellular matrix

Gly

pica

n-1

CD

44

TRPV4 & TRPC1B2

GPCR

eNOScaveolin-1

calmodulin

Kir2.1

MnSOD●O2- H2O2

Arg

●O2- ONOO•-

NO

p47phox, p22phox,

eNOS,integrins,

VE-cadherin

vasodilation

cilium

PKD1

PKD2

PGI2cPLA2

mitochondria

RKIP

COX2

PC

AA

sGC

PKG

P2X4

ATPRas

RasGRP

PAF

PAF acetylase

Ral

vWFANGPT2

P-selectin

WPb

PLD1Ral GDS

Raf1PKCβ

MNK1

PLCβ

DAGIP3

PKG

inhibition, activation of MAPK & IKK

CYP2C/J

EETs

PGIS

LPC

ATX

LPA

LPC

Inflammationthrombosis

COX1

αq

⬆Ca++IP3R RyR

ER

PLD

PKG⬇Vmcalmodulin

CaMKII

PDK1

PYK2

PKCδ

DAG

PI3Kγ

βγ

PYK2

cGMP

The endothelial glycocalyx: an impressive flow transducer

van den Berg BM, et al.  Circ Res 2003; 92:592

Onset of flow

syndecan-1

actin cortical web

Denseperipheralactin bands

extracellular matrixsyndecan-4

actin stress fibers

VEGFR2

PECAM-1

VE-cadherin

fibronectin, fibrinogen, osteopontin

Cx43

hyaluronan

tight junction:occludinsclaudins

JAMs

HSP6

0

ZO-1 linkers

α β

linkers

Src

Cdc42

Integrins α4β1, α5β1, or αVβ3

catenins, vinculin

NFκB

Rho

heparansulfatefibers

ASK1MKKJNK, p38

myosin

SMADsBMP4ZO-1

Shcta

lin

paxil

linta

lin

Shc

p130CASCrk

Dock180βPIX Grb2 Sos

Src

α2β1

PKA

collagen

PLCγ

PI3Kpaxil

lin

FAK

Git

Grb2

Rac1

RhoGDI

Src

PAK1

Nck1

Ras

MAP4K4 NIK

•O2-

O2

xanthine urate

α β

PKCα

XDH

XO

ROSROSPKCζ

Rac1

Shc

talin

Src

PI3Kγ

adherens junction

RhoPIP2

m-calpain

Dbl

Src

MYPT1PPI

NFκB

ROCK2

Raf1MEK1/2ERK1/2

Raf1

MLCK

FAKFAKAck1

filam

in

extracellular matrix

VEGFR2

PECAM-1

VE-cadherinRaf1

MEK2ERK1/2

actin Fyn

Shc

SHP2

Shc Csk

PI3Kγ

Grb2Sos

B2

PP

P

P

P

P

P

integrins αVβ3 or α5β1

P P

PP P P

P

P

talin talinShcP

activated integrinstalin, kindlin bound

endothelial cell

1

2

3

kindl

in

kindl

in

filam

infil

amin

α β

Inhibitory filamin binding

βγαq

?

ADAM15

P

Src

Yes

γβ

αδ

catenins

Arf6ARNO

endosomal removal of VE-cadherinSrc activationSH3 SH2

P

SH3 SH2 kinase

SHP2

SHP2Csk

RasTyrK

P

targets

P

SH3 SH2 kinase

targ

et sP

targ

ets

Pro

PIP2/3

PI3KαPLCγ

PKCβ

Src

100

1000

10000

100000

0 5 10 15 20

Perc

ent o

f bas

elin

e

Hours of Flow

Integrin β3 mRNA Integrin αV mRNA p65 mRNA

0

50

100

150

200

250

300

350

0 1 2 3 4 5 6

Perc

ent o

f bas

elin

e

Hours of Flow

P-FAK P-p38

P-MSK1 P-p65

1248

163264

128256512

10242048

0 2 4 6 8 10 12 14 16 18

Perc

ent o

f bas

elin

e

Hours of Flow

IL8 low flow IL8 high flowGRO-β low flowGRO-β high flow

Prolonged laminar flow

NF-κB

eNOS

Caveolin-1

SIRT1

Grb2

PI3Kα

SHP2

H2O

Prdx,Gpx,Cat

HSP90

IκBα

Akt

MKK4/7

ASK1

JNK

PAK

Ras

MKP1

⬆anti-oxidant genes, Ets-1Maf Nrf2

Keap1Nrf2

Keap1Cul3

NO, electrophiles

Arg

CaMKII

ONOO • -

⬆anti-proliferation, CD59, TM, eNOS, Tie2

CBPPCAF

⬆ Nrf2; ⬇ inflammation, ET-1, TF, vWF, PAI-1, PAR1, ANGPT2, ACE, PAK1

KLF2/4

Grb2 Gab1

PI3Kα

Grb2

SrcPI3K PLCγ

CblGrb2-Gab1

●O2- H2O2lipoic acid

NO, electrophiles p66Shc

•O2-

O2

NOX1/2

•O2-

Bad

NO

Bcl2

Pr-SH, H20

Trx

Pr-SOH, Pr-SS Pr-SSG

Pr-SH + GSH

Gadd45β

TrxTrxR

NO, electrophiles

GrxGSR

H2O2

IKK

β-catenin⬆NOTCH signaling, vascular quiescence

GSK3β

⬆DLL4

⬆ANGPT2

P

PSrc

ABIN2A20

mDia

CBP

PI3Kα

Rho

NO

TXNIP

VEGFR2signaling

Tie2 signaling

SOD1 SOD2

SOD3

15d-PGJ2

PPARγSMRT

p38

TAK1

MKK3/6

Raf1

MEK1/2

ERK1/2

MEKK2/3

MEK5Gab1Sos

PKA

HDAC5/7

HDAC5/7P Foxo1 P

Foxo1

KLF2/4MEF2A/CPCAF

miR-92a

AMPK

CBP

HDAC3

HDAC3P

TXNIP

ERK5

Altered response: ⬆ eNOS,NF-κB

⬆anti-apoptosis genes, ⬇ Inflammation genes

CBP

SIRT1

Akt

0

200

400

600

800

1000

1200

1400

1600

1800

2000

0 2 8 24

Perc

ent o

f bas

elin

e pr

oduc

tion

Hours of laminar shear stress

Nitric oxideSuperoxide

Exercise endothelial sheer stress

Ca++ influx, NOX + mitochondrial production of O2−

H2O2 burst (small, controlled) + electrophile formation (?)

eNOS, PPARγ, Akt, HSP90, SIRT1, altered NF‐κB signaling

block Keap1,  nuclear Nrf2, antioxidant genes induced  

eNOS NO production, anti‐inflammatory NO signaling

Oral antioxidants block beneficial adaptations to exercise in humans

Ristow M, et al.  PNAS 2009; 106:8665

vit C 1000 mg, vit E 400 IU dailyvs placebo

Nitro‐oleate and other nitro‐fatty acids as protective substances

• Reactive nitrogen species + oleic acid  nitro‐oleate– Also formed as termination product of radical reaction + NO

• Low dose nitro‐oleate forms reversible protein adducts with anti‐inflammatory effects:– Covalently binds and potently activate PPARγ– Binds and inactive IKK1 and IKK2– Binds and decrease activity of NF‐κB– Releases Keap1, activates Nrf2 – upregulates anti‐oxidant defense gene transcription– Upregulate heat shock proteins (HSP70)– Can inhibit AT1R, others

• Reduces atherosclerosis in Apo E KO mice• Occurs naturally in extra‐virgin olive oil

Delmastro‐Greenwood M, et al.  Annu Rev Physiol 2014; 76:79

Aging increases vascular susceptibility to atherosclerosis

• Start of western diet in old vs young LDLR‐/‐ mice– Much faster progression of athero in older mice (same lipid levels)– Older mice failed to upregulate antioxidant enzymes as much

• Transplant of old vs young vessels into hyperlipidemic animals– Older vessels have 2x increased rate of atherosclerosis progression with same level of hyperlipidemia.

• Possible reasons for greater susceptibility– 5‐fold greater TNFR1 expression in older arteries– Fibronectin, AGE accumulation– Mitochondrial DNA damage, replication limitation– Increased progerin accumulation (?)

Hopkins PN.  Physiol Rev 2013; 93:1317 

Substrate, product. Translocation

Signaling, activation. Generally by phosphorylation.

Signaling, activation. Multiple steps or unknown intermediates..

Signaling, inhibition by phosphorylation or other..

GPCR (G-protein coupled receptor)

Adapter or scaffold protein

MAP3K (e.g. Raf1, ASK1, TAK1, MEKK1, MEKK3, NIK)

GEF (guanine exchange factor) – activates small G-proteins

Small G-protein or GTP-ase

Phospholipase (e.g., PLC, PLD)

Lipid signaling intermediate (e.g. DAG, IP3)

PI3K isoform (phosphatidyl inositol 3 kinase)

Serine/threonine kinase (e.g. PKA, PKB, PKC, PKG, MAP4K)

Tyrosine kinase (e.g. Src, Src family kinases, Syk)

MAP2K (e.g. MEK1/2, MEK5, MKK3/4/6/7)

MAPK (e.g. ERK1/2, p38, JNK)

Protein tyrosine phosphatase

E3 ubiquitin ligase (e.g. TRAF2, TRAF6)

Ubiquitinase or ubiquitin editing enzyme (e.g. A20)

Other enzymes

Cation channels (Na+, K+, Ca++)

Anion channels (chloride, superoxide)

Connexins, pannexins

Transcription factors (e.g., NFκB, AP-1, etc.)

Co-activators (e.g., p300, CBP, HATs)

K48-linked, K63-linked or linear ubiquitin respectively

GAP (GTPase activating protein) – inactivates small G-proteins

Co-repressors (e.g., HDACs, SIRT1)