larry marnett cellular responses to aldehydic products of lipid peroxidation
TRANSCRIPT
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