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Larry Marnett

Larry Marnett. Cellular Responses to Aldehydic Products of Lipid Peroxidation. Aldehyde Mediators of Oxidative Damage. Diffusible Electrophiles from Lipid Peroxidation. Ox. Ox. -H 2 O. Red. Lipid Peroxidation-Induced DNA Damage. Mutagenic Spectrum of Lipid Peroxidation-Induced Adducts.

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Larry Marnett

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  1. Larry Marnett Cellular Responses to Aldehydic Products of Lipid Peroxidation

  2. Aldehyde Mediators of Oxidative Damage

  3. Diffusible Electrophiles from Lipid Peroxidation Ox Ox -H2O Red

  4. Lipid Peroxidation-Induced DNA Damage

  5. Mutagenic Spectrum of Lipid Peroxidation-Induced Adducts M1dGT M1dGA frameshifts OH-Hexyl-dGT OH-Hexyl-dGA

  6. Mutagenic Spectrum of Lipid Peroxidation-Induced Adducts M1dGT M1dGdA frameshifts OH-Hexyl-dGT OH-Hexyl-dGdA dA dG

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

  8. Enedials from Other Sources

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

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

  11. Depletion of GSH by HNE

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

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

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

  15. Reduced Apoptosis in p53-/- Cells Exposed to HNE

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

  17. HNE Inhibits NFB Translocation and DNA Binding JBC, 276, 18223 (2001)

  18. HNE Inhibits NFB-Dependent Transcription

  19. HNE Modification of IkB Kinase

  20. SH HS SH HS Structure and Regulation of IB Kinase

  21. IK Oxidative Stress and Its Consequences

  22. Doses Times Array Type Affymetrix U133 Plus 2.0 Chip ~55,000 Probes ~47,000 Transcripts 5, 20, and 60 µM HNE 6 and 24 h Microarray Experiments: Dose Response, Time Course, and Chip Specifications CRT, 18, 1641 (2005)

  23. 5 µM HNE 20 µM HNE 4 22 0 3 0 11 115 54520 all genes 60 µM HNE Genes Up-regulated at 6 h Using Microarray

  24. 20 µM HNE 60 µM HNE 2 2 74 54597 all genes Genes Up-regulated by HNE at 24 h

  25. Known Genes Upregulated by HNE HNE Stimulates Many Different Transcriptional Pathways

  26. Transcription Factors Response Induced Genes Antioxidant Response Nrf2 HMOX1, NQO1, GCLM, Xc– Stress-Activated Kinase Signaling AP-1 stress-responsive genes Misfolded/Damaged Protein Response; Amino Acid Starvation HSFs, ATF4, ATF6, XBP1 DnaJA4, HspA6, Hsp70B, Gadd34, ASNS DNA Damage Response p53 Mdm2, cyclin G2, p21 General Stress/ Unknown Other/Unknown Gadd45b, Clusterin, CYP39A1 HNE Induces Gene Expression via Many Signaling Pathways

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

  28. Induction of CYP39A1 by HNE

  29. Increase in Protein Levels Following HNE Treatment

  30. Activation of Stress-Responsive Transcription Factors by HNE

  31. Activation of NRF2 CRT, 18, 1779 (2005)

  32. Knock-down of Nrf2 by siRNA

  33. Knock-down of Nrf2 by siRNA - + - + - + MG-132 (5 mM) none none control control Nrf2 Nrf2 siRNA Nrf2 (68 kDa) b-tubulin (50 kDa) nuclear protein blot

  34. Knock-down of Nrf2 by siRNA HNE-treatment

  35. Activation of Heat-Shock Signaling by Hsp90 Modification CRT, 19, 173 (2006)

  36. Immunochemical Detection of HNE-Adducted Proteins in Alcoholic Liver Disease Microsomal Mitochondrial JPET, 315, 8 (2005); Chem.Res.Toxicol.18, 1324 (2005)

  37. Protein Labeling with Azido-HNE Aaron Jacobs

  38. Analysis of Proteins Modified in RKO cells 0 M 50 M 100 M Protein: 5 g/lane 4-20% SDS-PAGE gradient gel Nitrocellulose (0.2 m) Probe with S/A-HRP -FBS +FBS -FBS +FBS Std Results: Protein labeling more prominent in cells incubated with media lacking FBS Most labeled proteins are high Mol. Wt. Andy Vila

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

  40. 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 Not All Aldehydes Are Created Equal

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

  42. Aldehyde Mediators of Oxidative Damage

  43. Induces apoptosis in A549 cells at 100-200 nM Uhlson et al, Chem.Res.Toxicol. 15, 896, 2002

  44. Aldehyde Mediators of Oxidative Damage Protein Targets, Biological Effects?

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

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

  47. HNE-Ascorbate Conjugate Found In Vivo 1.3 µM in human plasma Sowell et al, PNAS, 101, 17964 (2004)

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