Larry Marnett Cellular Responses to Aldehydic Products of Lipid Peroxidation

Aldehyde Mediators of Oxidative Damage

Diffusible Electrophiles from Lipid Peroxidation Ox Red -H 2 O Ox

Lipid Peroxidation-Induced DNA Damage

M 1 dG  T M 1 dG  A frameshifts OH-Hexyl-dG  T OH-Hexyl- dG  A Mutagenic Spectrum of Lipid Peroxidation-Induced Adducts

M 1 dG  T M 1 dG  dA frameshifts OH-Hexyl-dG  T OH-Hexyl- dG  dA  dA  dG Mutagenic Spectrum of Lipid Peroxidation-Induced Adducts

HNE and ONE Protein Adducts CRT,8, 284, (1995); CRT, 16, 1598 (2003) plus unstable Michael adducts ONE is 100x more reactive than HNE

Enedials from Other Sources

HNE Induction of Apoptosis and Necrosis CRT, 14, 1090 (2001)

Toxicity of Peroxidation-Derived Aldehydes CRT, 17, 453 (2004)

Depletion of GSH by HNE

Apoptosis Induced by HPNE and ONE in RKO Cells CRT, 17, 453 (2004)

membranereceptor? Cas-3 Procas-3 Cas-8 Cell membrane Mitochondria Bcl-2 Cas-9 Apaf-1 Cyt c PARP  -fodrin DFF45/35

Protein Synthesis is Required for HNE-Mediated Apoptosis What transcriptional pathways are altered by HNE?

Reduced Apoptosis in p53 -/- Cells Exposed to HNE

NF  B Signaling Li et al, Mol.Immunol., 41, 701 (2004)

JBC, 276, (2001) HNE Inhibits NF  B Translocation and DNA Binding

HNE Inhibits NF  B-Dependent Transcription

HNE Modification of I  B Kinase

SHHSSH HS Structure and Regulation of I  B Kinase

Oxidative Stress and Its Consequences IKIK

Doses 5, 20, and 60 µM HNE Array TypeAffymetrix 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)

µM HNE20 µM HNE 60 µM HNE all genes Genes Up-regulated at 6 h Using Microarray

µM HNE60 µM HNE all genes Genes Up-regulated by HNE at 24 h

Known Genes Upregulated by HNE HNE Stimulates Many Different Transcriptional Pathways

HNE Induces Gene Expression via Many Signaling Pathways Response Transcription Factors Induced Genes Antioxidant Response Nrf2HMOX1, NQO1, GCLM, X c – Misfolded/Damaged Protein Response; Amino Acid Starvation HSFs, ATF4, ATF6, XBP1 DnaJA4, HspA6, Hsp70B, Gadd34, ASNS DNA Damage Response p53Mdm2, cyclin G2, p21 General Stress/ Unknown Other/Unknown Gadd45 , Clusterin, CYP39A1 Stress-Activated Kinase Signaling AP-1stress-responsive genes

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

Induction of CYP39A1 by HNE

Increase in Protein Levels Following HNE Treatment

Activation of Stress-Responsive Transcription Factors by HNE

Activation of NRF2 CRT, 18, 1779 (2005)

Knock-down of Nrf2 by siRNA

 -tubulin (50 kDa) Nrf2 (68 kDa) siRNA MG-132 (5  M) none none control control Nrf2 Nrf nuclear protein blot Knock-down of Nrf2 by siRNA

HNE-treatment

Activation 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 Proteins in Alcoholic Liver Disease Microsomal Mitochondrial

Protein Labeling with Azido-HNE Aaron Jacobs

Analysis of Proteins Modified in RKO cells 0  M100  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/lane 4-20% SDS-PAGE gradient gel Nitrocellulose (0.2  m) Probe with S/A-HRP Std Andy Vila

Putative Targets of Electrophilic Lipid Peroxidation Products Low level stress  Protection High level stress  Apoptosis

Not All Aldehydes Are Created Equal HNE Extensive changes in gene expression and signaling p53-Dependent-Apoptosis MDA Changes in gene expression and signaling p53-Dependent G1/S and G2/M block No Apoptosis  -Substituted-MDA R = alkyl/aryl No signaling changes No Apoptosis/No Necrosis

Induction of p53-Dependent Cell Cycle Arrest and Apoptosis by QD-MDA

Aldehyde Mediators of Oxidative Damage

Induces apoptosis in A549 cells at nM Uhlson et al, Chem.Res.Toxicol. 15, 896, 2002

Aldehyde Mediators of Oxidative Damage Protein Targets, Biological Effects?

Chuan Ji James West Aaron Jacobs Andy Vila CRT, 19, 173 (2006)

Carmelo Rizzo Alan Brash Ned Porter Jianxin Ji National Foundation for Cancer Research National Institutes of Health Acknowledgements

Sowell et al, PNAS, 101, (2004) 1.3 µM in human plasma HNE-Ascorbate Conjugate Found In Vivo