1 / 30

carleton/~kbstorey

LIVING WITHOUT OXYGEN. www.carleton.ca/~kbstorey. LITTORINA LITTOREA. Marine gastropod (periwinkle) Found on Atlantic coast of Europe & N. America One of just a few intertidal species in the north Intertidal zone highly variable environment. Intertidal zone. METABOLISM IN ANOXIA.

petula
Download Presentation

carleton/~kbstorey

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  1. LIVING WITHOUT OXYGEN www.carleton.ca/~kbstorey

  2. LITTORINA LITTOREA • Marine gastropod (periwinkle) • Found on Atlantic coast of Europe & N. America • One of just a few intertidal species in the north • Intertidal zone highly variable environment Intertidal zone

  3. METABOLISM IN ANOXIA • mRNA synthesis • Translation • Fuel use (incl. CHO) • BioSyn. / Degrad. • Cell cycle ATP turnover to <5% of normal

  4. [32P] UTP incorporation (cpm x 103/μg nuclear protein) L. littorea Incubation time (min) mRNA synthesis [32P] UTP incorporation (cpm x 103/μg nuclear protein/h) a

  5. In vitro Protein Synthesis

  6. a P A254 M 1 2 3 4 5 6 7 8 9 10 Polysome Analysis

  7. P A254 M Normoxia 1 2 3 4 5 6 7 8 9 10 P Anoxia A254 M 1 2 3 4 5 6 7 8 9 10 Recovery P A254 M 1 2 3 4 5 6 7 8 9 10 Polysome Analysis 30% Linear Sucrose Gradient 15%

  8. PRINCIPLES OF METABOLIC ARREST 1. Metabolic rate reduction 2. Control by protein kinases(SAPKs, 2nd messenger PKs) 3. Most GENES OFF ! 4. Selective gene activation

  9. PRINCIPLES OF METABOLIC ARREST 1. Metabolic rate reduction 2. Control by protein kinases(SAPKs, 2nd messenger PKs) 3. Most Genes OFF 4. Selective gene activation

  10. Ca+2 GENES [ i + e Factors] Nucleus mRNAs GENES ON/OFF CHO PROTEINS [Trans.F] Na ATP K PATHWAYS AA SAPK P PROT ? SMW FAT ADP ATP KINASES (2nd) MITO ETC

  11. Ca+2 GENES [ i + e Factors] Nucleus mRNAs GENES ON/OFF CHO PROTEINS [Trans.F] Na ATP K PATHWAYS AA SAPK P PROT ? SMW FAT ADP ATP KINASES (2nd) MITO ETC

  12. PATHWAY CONTROL IN MR DEPRESSION Phospho / de-Phospho • Glycolysis (GP, GS, PFK, PK) • Fat synthesis (ATP-CL, ACC) • CHO fuel use (PDH) • Translation (eIF2α, eEF2) • Ion pumps (NaK-ATPase, Ca-ATPase) Estivation: Storey & Storey 2012. J. Exp. Biol. 215:1425-33 Hibernation: Storey & Storey 2010. Adv Clin Chem 52:77-108 Hypometabolism: Storey & Storey 2007. J. Exp. Biol. 210, 1700-14

  13. PATHWAY CONTROL IN MR DEPRESSION Phospho / de-Phospho NOVEL DISCOVERIES • CHO: Hexokinase, G6PDH, Aldolase • Key DH’s: GlutDH, IsocitDH, LDH(!) • Energy: AMPD, Arginine Kinase • Signaling: AMPK, GSK-3 • AOE: SOD, catalase, GST, GPox

  14. NOVEL P/deP ENZYME CONTROL GlutDH ARG-K G6PDH LDH

  15. ANOXIA INDUCED CHANGES • Protein Synthesis slows to 1% • Pumps & channels closed • Energy Production slows to 5% • Energy Utilization slows to 2% • Few ‘SAP’ kinases activated • Gene ‘inactivation’ ( mRNA ) • Few Genes activated

  16. TURNING OFF GENES:Role of Epigenetics Epigenetics: Stable changes in gene activity that do not involve changes in DNA sequence. Common mechanisms: - DNA methylation - Histone modification / histone variants - Regulatory non-coding RNAs Hibernators! Frogs! Littorines ?

  17. MICRO RNA • Size ~22 nucleotides • Highly conserved across species • Bind to 3’ UTR of mRNAs • Exact repression mechanism(s) not yet clearbut seems to include: - Block translation of mRNA - Help bind mRNA into stress granules - Target mRNA for degradation

  18. MICRO RNA: Drosha & Dicer Cuellar TL, McManus MT. J Endocrinol. 187(3):327-332, 2005.

  19. Micro RNAs in Littorina littoreaFOOT MUSCLE:Up-regulated by Freezing & Anoxia • miR-1a-1 & miR-133a- myocyte proliferation & differentiation • regulate Mef2a and Gata4, Tfs that promote muscle maintenance • miR-2a • - anti-apoptotic action by targeting the pro-apoptotic protein, Reaper * * * * * * Biggar, Kornfeld & Storey, 2011. Anal. Biochem. 416, 231-3. Biggar, Kornfeld, Maistrovski & Storey, 2012. Genom. Proteom. Biotech. in press

  20. Micro RNAs in Littorina littoreaHEPATOPANCREAS:Up-regulated by Freezing & Anoxia • Major changes: • - miR-1a-1 up in freeze & anoxia (like in foot) • miR-210 up in anoxia • miR-29b up in freeze

  21. DICER ENZYME IN L. littorea TISSUES Dicer proteinincreased in both freezing& anoxia(immunoblots) Elevated miRNAprocessing

  22. ANOXIA INDUCED CHANGES • Few ‘SAP’ kinases activated • Gene ‘inactivation’ ( mRNA) • Few Genes activated

  23. - Antioxidant Enzymes - Shock proteins (GRP, HSP) - Low oxygen Shock (HIF) - Metallothionein & Ferritin - Unknown: SARP, KVN, LRD - Transcription factors Trans.factors ~1-2% GENE CHANGES IN Anoxic L. littorea Larade & Storey. 2009. Curr. Genomics 10, 76-85

  24. OXIDATIVE STRESS • Central theme in oxygen metabolism • Linked with cancer, aging, diabetes, hypoxia, etc. • Antioxidant enzymes as indicators of oxidative stress

  25. ANTIOXIDANT DEFENSE Iron storage: - Ferritin (H & L chains) - Transferrin receptor 2 Antioxidant enzymes - SOD (1) - GST (M5, A2) - GPX (1, 4) - Peroxiredoxin 1 Estivation: Storey & Storey 2012. J. Exp. Biol. 215:1425-33 Cold hardiness: Storey & Storey 2012. Can. J. Zool. 90: 456–475 Marine: Freire et al. 2011. In: Oxidative Stress in Aquatic Ecosystems. pp. 41-57

  26. GLUTATHIONE S -TRANSFERASE Multiple subfamilies incl: Alpha, Kappa, Mu, Pi, Sigma, Theta Some forms controlled by the antioxidant response element (ARE) via the Nrf2 transcription factor

  27. L. littorea FOOT MUSCLE: 20 h anoxia & 4 h recovery at 10°C:GST responses Sigma class GSTs are prominent in marine molluscs

  28. L. littorea HEPATOPANCREAS 20 h anoxia & 4 h recovery at 10°C:GST responses

  29. PRINCIPLES OF HYPOXIA SURVIVAL 1. Metabolic rate depression 2. Kinase / phosphatase action 3. Reversible P-enzymes 4. Global suppression of transcription & translation 5. Selected genes up-regulated

  30. Invertebrate Anaerobiosis • J. STOREY • S. BROOKS • W. WILLMORE • J. DUNCAN • M. HERMES-LIMA • S. GREENWAY • T. ENGLISH • E. RUSSELL • T. PANNUNZIO • J. MACDONALD • K. LARADE • R. WHITWAM • S. KORYCAN • B. MICHAELIDIS • R. BELL • J. LAMA • B. LANT • O. ANOZIE • K. BIGGAR • N. DAWSON www.carleton.ca/~kbstorey Funded by NSERC Canada

More Related