larry marnett cellular responses to aldehydic products of lipid peroxidation

Larry MarnettCellular Responses to Aldehydic Products of Lipid

Peroxidation

NOO2–

H2O2

HO•

NO

O–O

hydroperoxides, endoperoxides,isoprostanes

MDA, HNE, ONE

NN

N NN

O

gene expression

apoptosis

replication blockmutationdamage signaling

degradation

Proteins

oxidativemetabolism

GSHconjugation

PLA2

hydrolysis

Fe2+ or

ascorbate

p53cell cycle arrest

reduction

+

Aldehyde Mediators of Oxidative DamageAldehyde Mediators of Oxidative Damage

R C5H11Lip. Perox.

O2

R C5H11

H

OC5H11

OOH

H

OC5H11

OH

H

O OH

HPNE

ONE HNE

OOH

H

O O

H

H

OC5H11

O

MDA

Diffusible Electrophiles from Lipid PeroxidationDiffusible Electrophiles from Lipid Peroxidation

Ox

Red-H2O

Ox

Lipid Peroxidation-Induced DNA DamageLipid Peroxidation-Induced DNA Damage

H

OC5H11

OH

HN

N N

N

O

H2N

N

N N

N

O

NH

OH

HO

C5H11

H

OC5H11

O

N

N N

N

NH2

N

N N

N

NH

O

H11C5

+

+

H-εdA

OH-Hexyl-PdG

O

H

OH

HN

N N

N

O

H2N

N

N N

N

O

N

M1dG

+

N

N N

N

NH

O

H11C5

H-εdA

N

N N

N

O

NH

OH

HO

C5H11

OH-Hexyl-PdG

N

N N

N

O

N

M1dG

M1dGTM1dGAframeshifts

OH-Hexyl-dGTOH-Hexyl- dGA

Mutagenic Spectrum of Lipid Peroxidation-Induced AdductsMutagenic Spectrum of Lipid Peroxidation-Induced Adducts

M1dGTM1dGdAframeshifts

OH-Hexyl-dGTOH-Hexyl- dGdA

N

N N

N

NH

εdA

N

N N

N

O

NH

OH

HO

C5H11

OH-Hexyl-PdG

N

N N

N

O

N

M1dG

εdA dG

Mutagenic Spectrum of Lipid Peroxidation-Induced AdductsMutagenic Spectrum of Lipid Peroxidation-Induced Adducts

HNE and ONE Protein AdductsHNE and ONE Protein Adducts

CRT,8, 284, (1995); CRT, 16, 1598 (2003)

plus unstable Michael adducts

ONE is 100x more reactive than HNE

H

OC5H11

OH

OHO

XR

R = Cys, His, Lys

OHN

XR

Lys

N

C5H11 C5H11

C5H11

LysH

NC5H11

OH

Lys

Cys, His, Lys

Lys

Lys –H2O

H

OC5H11

O

ArgN

N

H2N

NH3+

CO2H

ONE

HNE

C5H11O

Enedials from Other SourcesEnedials from Other Sources

H

OC5H11

O

ONE

P-450

O2 OO

HO

OH

OO O

O

O

R

P-450

O2

HO O

H

R

OH

ROH

HNE Induction of Apoptosis and NecrosisHNE Induction of Apoptosis and Necrosis

CRT, 14, 1090 (2001)

H

OC5H11

OOH

H

OC5H11

OH

H

OC5H11

O

H

OC5H11

OH

HPNE ONE (R)-HNE (S)-HNE

Toxicity of Peroxidation-Derived AldehydesToxicity of Peroxidation-Derived Aldehydes

CRT, 17, 453 (2004)

Depletion of GSH by HNEDepletion of GSH by HNE

Apoptosis Induced by HPNE and ONE in RKO CellsApoptosis Induced by HPNE and ONE in RKO Cells

CRT, 17, 453 (2004)

membranemembranereceptor?receptor?

Cas-3Cas-3

Procas-3Procas-3 Cas-8Cas-8

Cell membraneCell membrane

MitochondriaMitochondriaBcl-2Bcl-2

Cas-9Cas-9

Apaf-1Apaf-1

Cyt cCyt c

H

O

OH

4-HNE

PARP-fodrin

DFF45/35

Protein Synthesis is Required forProtein Synthesis is Required forHNE-Mediated ApoptosisHNE-Mediated Apoptosis

What transcriptional pathways are altered by HNE?What transcriptional pathways are altered by HNE?

Reduced Apoptosis in p53Reduced Apoptosis in p53-/--/- Cells Exposed to HNE Cells Exposed to HNE

NFNFB SignalingB Signaling

Li et al, Mol.Immunol., 41, 701 (2004)

. .

/TPA IM (30 )- /HNE ' TPA IM

0 5 15 30 0 5 15 30

Supershift

-NF B

1 2 3 4 5 6 7 8 9 10 11

( )min

JBC, 276, 18223 (2001)

HNE Inhibits NFHNE Inhibits NFB Translocation and DNA BindingB Translocation and DNA Binding

HNE Inhibits NFHNE Inhibits NFB-Dependent TranscriptionB-Dependent Transcription

.

32P-IB-GST ( )IKK activity

( )HNE µM - - - 30 60

Samples

1 2 3 4 5

IKK

-GST IB

220 K

97 K

220 K

97 K

HNE modified IKK ( -IB with anti IKK)

HNE modified IKK ( - )IB with anti HNE

HNE Modification of IHNE Modification of IB KinaseB Kinase

SH HSSH HS

Structure and Regulation of IStructure and Regulation of IB KinaseB Kinase

NOO2–

H2O2

HO•

NO

O–O

hydroperoxides, endoperoxides,isoprostanes

MDA, HNE, ONE

NN

N NN

O

gene expression

apoptosis

replication blockmutationdamage signaling

degradation

Proteins

oxidativemetabolism

GSHconjugation

PLA2

hydrolysis

Fe2+ or

ascorbate

p53cell cycle arrest

reduction

+

Oxidative Stress and Its ConsequencesOxidative Stress and Its Consequences

IK

Doses

5, 20, and 60 µM HNE

Array Type

AffymetrixAffymetrix

U133 Plus 2.0 Chip~55,000 Probes

~47,000 Transcripts

Times

6 and 24 h

Microarray Experiments:Dose Response, Time Course, and Chip Specifications

CRT, 18, 1641 (2005)

4 22

115

0

0 11

3

5 µM HNE 20 µM HNE

60 µM HNE

54520

all genes

Genes Up-regulated at 6 h Using MicroarrayGenes Up-regulated at 6 h Using Microarray

2 742

20 µM HNE 60 µM HNE

54597

all genes

Genes Up-regulated by HNE at 24 hGenes Up-regulated by HNE at 24 h

6 h 24 hiological Response/Gene Acc. No. 20 µM HNE 60 µM HNE 60 µM HNEntioxidant Responseeme oxygenase 1 (HMOX1) NM_002133 8.4 9.1 7.4utamate-cysteine ligase, modifier subunit (GCLM)† NM_002061 3.4 3.1 –AD(P)H-dependent quinone oxidoreductase (NQO1)† NM_000903 2.5 – –lute carrier family 7, member 11 (SLC7A11, xCT)† NM_014331 8.7 6.1 –ioredoxin reductase 1 (TXNRD1) NM_003330 2.4 9.3 5.9

eat Shock ResponsenaJ homolog, subfamily A, member 4 (DnaJA4)† NM_018602 –§ 4.4 3.4naJ homolog, subfamily B, member 1 (DnaJB1)† NM_006145 – 4.3 4.4naJ homolog, subfamily B, member 2 (DnaJB2) NM_006736 – 4.6 3.6naJ homolog, subfamily B, member 4 (DnaJB4)† NM_007037 – 5.7 2.1eat shock 70kDa protein 1A (HSPA1A) NM_005345 – 3.0 3.6eat shock 110kDa protein family (APG-1) NM_014278 – 2.6 –eat shock 105/110kDa protein 1 (HSPH1)† NM_006644 – 9.6 12.1

R Stress/Amino Acid/Nutrient Sensing Responseomocysteine, ER stress-inducible protein 1 (HERP) NM_014685 – 4.9 –tivating transcription factor 3 (ATF3) NM_001674 – 5.1 –lute carrier family 3, member 2 (SLC3A2) NM_002394 – 2.7 –paragine synthetase (ASNS) NM_001673 – 2.8 –

ystathionase (CTH)† NM_001902 – 3.9 –owth arrest, DNA damage-inducible 34 (Gadd34)† NM_014330 – 3.4 3.2bbles homolog 3 (TRB3)† NM_021158 – 3.3 –

ell Signalingsulin-induced gene 1 (INSIG1)† NM_005542 – 2.3 –AP kinase kinase kinase 8 (MAP3K8) NM_005204 – 3.5 2.0DC-like kinase 1 (Clk1) NM_004071 – 3.8 3.2acental growth factor (PGF) NM_002632 – 3.5 4.9

onnective tissue growth factor (CTGF) NM_001901 5.2 4.5 3.5gnal-induced proliferation-associated 1-like 2 (SIPA1L2)† NM_020808 3.8 3.6 –drenomedullin (ADM) NM_001124 – 3.5 4.0TP binding protein expressed in skeletal muscle (GEM) NM_005261 – 4.9 5.5belson leukemia viral oncogene homolog 2 (ABL2) NM_005158 – 3.0 –broblast growth factor receptor substrate 2 (FRS2) NM_006654 – – 2.3AS-like without CAAX1 (RIT1) NM_006912 – 2.6 –

poptotic Regulationowth arrest, DNA damage-inducible 45β (Gadd45β)† NM_015675 – 5.6 3.8usterin (Clus)† NM_001831 – 3.2 3.9urine double minute 2, hum anhomolo g(HDM2)† NM_002392 – – 2.5

ell Cycle Regulationstrin 2 (SESN2)† NM_031459 – 5.4 –yclin G2 (CCNG2)† NM_004354 3.2 2.8 –gulato -r of Gprotein signalin g2 (RGS) NM_002923 – 3.7 3.7ycli -n depende ntkina -selike kina 3 (se CDKL3) NM_016508 – 2.6 –urine-rich bindin g element protein (A PURA) NM_005859 – 3.2 3.7SP -Y lik e2 (TSPYL2)IO kinase 3 (RIO 3K ) NM_003831 – – 2.4

ip id Metabolis man dTr ansportytochro meP450 39A1 (CYP39 1A ) NM_016593 6.1 7.3 –xystero l bindin g protei -n lik 1 (e AOSBPL1A) NM_018030 – 2.3 –

NA Processin /gNuclea r Transportolyadenylatio n element binding protein 3 (CPEB3) NM_014912 – 4.3 –ownregulate din ovaria ncan cer1 (DO 1C ) NM_014890 – 3.2 –eath effector domain, -DNA binding domain 2 (DEDD2) NM_133328 – 3.0 2.2olyadenylatio n element binding protein 4 (CPEB4)† NM_030627 – 3.0 2.6ucleoporin like 1 (NUPL1) NM_014089 – – 2.1un -o like 6, RNA binding prote in(BRUNOL6) NM_052840 – – 3.3

ytoskeletal Function/Structural Maintenanceocollagen-proline 4-hydroxylase (P4HA2)† NM_004199 – 3.3 –tin related protein M1 (ARPM1) NM_032487 – 9.2 4.1ldesmon 1 (CALD1) NM_004342 2.4 – –

ansgelin (TAGLN) NM_003186 2.3 – –rine protease inhibitor H1 (SERPINH1) NM_001235 – – 2.6

ranscriptional Regulationdult retina protein (LOC153222)† NM_153607 – 2.1 –rly growth response 1 (EGR1) NM_001964 – 2.6 3.2nc finger and BTB containing protein 20 (ZBTB20) NM_013360 – 2.9 3.5-box protein 3 (TBX3) NM_005996 – 3.1 –MB-box transcription factor 1 (HBP1) NM_012257 – 2.7 –nc finger protein 10 (ZNF10) NM_015394 – 3.2 –nc finger protein 227 (ZNF227) NM_182490 – 2.5 –nc finger protein 267 (ZNF267) NM_003414 – 2.2 –

AMP response element binding protein 3-like 4 (CREBP3L4) NM_130898 – 2.6 –AX dimerization protein 1 (MAD) NM_002357 – 3.1 –nc finger protein 554 (ZNF554) NM_152303 – 2.4 2.3maf oncogene homolog f (MAFF)† NM_012323 – 3.7 –nc finger protein 12 (ZNF12) NM_033204 – – 2.2ING1 and YY1 binding protein (RYBP) NM_012234 – – 2.5rin (PIR) NM_003662 2.5 – 2.5ADS box transcription enhancer factor 2A (MEF2A) NM_005587 – – 2.2nc finger protein 277 (ZNF277) NM_021994 – – 3.1nc finger protein 79 (ZNF79) NM_007135 – – 3.3nc finger protein 556 (ZNF556) NM_024967 – – 4.2monji domain containing 1C (JMJD1C) NM_004241 – 2.4 –ixed lineage leukemia-trithorax homolog 2 (MLLT2) NM_005935 – 2.9 –x-determining region Y-b ox 18 (SOX18) NM_018419 – – 21.8

n Homeostasis and Cellular Transporta2+-transporting ATPase, type 2C, member 1 (ATP2C1) NM_014382 – 2.9 –holinergic receptor, nicotinic, alpha polypeptide 5 (CHRNA5) NM_000745 – 2.7 –daptor-related protein complex 4, β1 subun it (AP4β1) NM_006594 – 2.2 –ucleosid e diphosphat -e linked moie ty -X typ 9 (e NUDT9) NM_024047 – 2.4 –lut e carrie r family 16, member 14 NM_152527 2.4 – –

NA Repairbiquitin-conjugatin g enzym eE2 B (RAD6 homolo ) (g UBE2B) NM_003337 – 4.0 –RC -A 1-interacting prote in C-terminal helica 1 (se BRIP1) NM_032043 2.3 – –

istonesstone 1, H3 (c HIST1 3H E) NM_003532 – 2.7 –stone 1, H2b (k HIST1 2H BK) NM_080593 – – 2.5stone 1, H2b (g HIST1 2H BG) NM_003518 – – 9.6stone 2, H2a a(HIST2 2H AA) NM_003516 – – 2.6

rotein Tr anslation/Amino Acid Metabolismukaryoti c translatio ninitiatio nfactor 5 (EIF5)† NM_001969 – – 2.5itochondria l ribosomal protein L18 (MRPL18) NM_014161 – 2.2 –oleucin -e tRNA synthetase (IARS) NM_002161 – 4.9 3.5hydrofolate reduct -aselik 1 (e DHFRL1) NM_176815 3.5 – –rosina -se relat edprotein 1 (TYRP1) NM_000550 3.7 – –

6 h 24 hiological Response/Gene Acc. No. 20 µM HNE 60 µM HNE 60 µM HNE

Known Genes Upregulated by HNEKnown Genes Upregulated by HNE

HNE Stimulates Many DifferentHNE Stimulates Many DifferentTranscriptional PathwaysTranscriptional Pathways

HNE Induces Gene Expression via Many Signaling PathwaysHNE Induces Gene Expression via Many Signaling Pathways

ResponseTranscription

FactorsInduced Genes

AntioxidantResponse

Nrf2 HMOX1, NQO1, GCLM, Xc–

Misfolded/DamagedProtein Response;

Amino Acid Starvation

HSFs, ATF4,ATF6, XBP1

DnaJA4, HspA6, Hsp70B,Gadd34, ASNS

DNA DamageResponse

p53 Mdm2, cyclin G2, p21

General Stress/Unknown

Other/Unknown Gadd45β, Clusterin, CYP39A1

Stress-ActivatedKinase Signaling

AP-1 stress-responsive genes

Induction of ARE Target Genes by HNEInduction of ARE Target Genes by HNE(Real Time RT-PCR)(Real Time RT-PCR)

Induction of CYP39A1 by HNEInduction of CYP39A1 by HNE

Increase in Protein Levels Following HNE TreatmentIncrease in Protein Levels Following HNE Treatment

Activation of Stress-ResponsiveActivation of Stress-ResponsiveTranscription Factors by HNETranscription Factors by HNE

Activation of NRF2Activation of NRF2

CRT, 18, 1779 (2005)

Knock-down of Nrf2 by siRNA

Nrf2 siRNA transfection

0

2

4

6

8

10

12

0 MG132, 5 uM 0 MG132, 5 uM 0 MG132, 5 uM

post-transfection treatment

fmol per ug RNA

(x10

-5)

83% 82%

percent reduction (to neg control siRNA)

NO transfection Negative Control siRNA Nrf2 siRNA

β-tubulin (50 kDa)

Nrf2 (68 kDa)

siRNA

MG-132 (5 M)

none none control control Nrf2 Nrf2

- + - + - +

nuclear protein blot

Knock-down of Nrf2 by siRNA

Knock-down of Nrf2 by siRNA

HNE-treatment

HO-1 mRNA (effect of Nrf2 knock-down)

0

5

10

15

20

25

30

0 HNE 15uM HNE 45uM 0 HNE 15uM HNE 45uM 0 HNE 15uM HNE 45uM

post-transfection treatment

fmol per ug RNA

(x10

-4)

NO transfection Negative Control siRNA Nrf2 siRNA

45%

46%

64%

percent expression(to neg control siRNA)

Activation of Heat-Shock Signaling by Hsp90 ModificationActivation of Heat-Shock Signaling by Hsp90 Modification

CRT, 19, 173 (2006)

JPET, 315, 8 (2005); Chem.Res.Toxicol.18, 1324 (2005)

Immunochemical Detection of HNE-Adducted ProteinsImmunochemical Detection of HNE-Adducted Proteinsin Alcoholic Liver Diseasein Alcoholic Liver Disease

Microsomal Mitochondrial

HN NH

S

HN

O

O

O

HN

O

PPh2

O

CH3O

3

Protein N3 +HN NH

S

HN

O

O

O

HN

O

PPh2

NH

O

3

Protein

O

O

H

OH

N3

Protein Labeling with Azido-HNEProtein Labeling with Azido-HNE

Aaron Jacobs

Analysis of Proteins Modified in RKO cellsAnalysis of Proteins Modified in RKO cells

0 M 100 M50 M-FBS+FBS -FBS +FBS

Results:Protein labeling more prominent in cells incubated with media lacking FBS

Most labeled proteins are high Mol. Wt.

Protein: 5 g/lane4-20% SDS-PAGE gradient gelNitrocellulose (0.2 m)Probe with S/A-HRP

Std

Andy Vila

Putative Targets of Electrophilic Lipid Peroxidation Products

HPNE, ONE, HNE

gene expression

GSH

enhanced oxidantsensitivity

IB Kinase

sensitivity to apoptosis

Protein Phosphatases

DNA

Keap1

antioxidantresponse

mutationsdamagesignaling

enhanced kinaseactivation

Ca2+-ATPase

ER Stress

Low level stress ProtectionHigh level stress Apoptosis

Not All Aldehydes Are Created EqualNot All Aldehydes Are Created Equal

H

OC5H11

OH

HNEExtensive changesin gene expression

and signalingp53-Dependent-Apoptosis

H

O OH

MDAChanges

in gene expressionand signaling

p53-DependentG1/S and G2/M block

No Apoptosis

H

O OH

R

-Substituted-MDAR = alkyl/aryl

No signaling changesNo Apoptosis/No Necrosis

H

O OH

N

H

O OH

N

N

Induction of p53-Dependent Cell Cycle Arrest andInduction of p53-Dependent Cell Cycle Arrest andApoptosis by QD-MDAApoptosis by QD-MDA

Aldehyde Mediators of Oxidative DamageAldehyde Mediators of Oxidative Damage

NOO2–

H2O2

HO•

NO

O–O

hydroperoxides, endoperoxides,isoprostanes

MDA, HNE, ONE

Fe2+ orascorbate

reduction

+

(DIFFUSIBLE ELECTROPHILES)

O

O

OP

O

O

O

NHCl

H31C15

O

(CH2)3CO2H

O

O

O

OP

O

O

O

NH31C15

O

O

O

H

PGPC POPC

O

O

OP

O

O

O

NH31C15

O

O

OH

H

O

HOOA-PC

O

O

OP

O

O

O

NH31C15

O

O

OO

OH

C5H11

PEIPC

O

O

OP

O

O

O

NH31C15

O

O

O

H

Induces apoptosis in A549 cells at 100-200 nM

Uhlson et al, Chem.Res.Toxicol. 15, 896, 2002

Aldehyde Mediators of Oxidative DamageAldehyde Mediators of Oxidative Damage

NOO2–

H2O2

HO•

NO

O–O

hydroperoxides, endoperoxides,isoprostanes

MDA, HNE, ONE

Fe2+ orascorbate

reduction

+

(DIFFUSIBLE ELECTROPHILES)

O

O

OP

O

O

O

NHCl

H31C15

O

(CH2)3CO2H

O

O

O

OP

O

O

O

NH31C15

O

O

O

H

PGPC POPC

O

O

OP

O

O

O

NH31C15

O

O

OH

H

O

HOOA-PC

O

O

OP

O

O

O

NH31C15

O

O

OO

OH

C5H11

PEIPC

Protein Targets, Biological Effects?Protein Targets, Biological Effects?

Chuan Ji

JamesWest

AaronJacobs

Andy Vila

CRT, 19, 173 (2006)

Carmelo Rizzo

Alan Brash

Ned Porter

Jianxin Ji

National Foundation for Cancer Research

National Institutes of Health

AcknowledgementsAcknowledgements

Sowell et al, PNAS, 101, 17964 (2004)

1.3 µM inhuman plasma

HNE-Ascorbate Conjugate Found In VivoHNE-Ascorbate Conjugate Found In Vivo