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Protein structure in molecular systems biology

Protein structure in molecular systems biology. Dr DAVID SHEEHAN, PROTEOMICS RESEARCH GROUP, DEPT. BIOCHEMISTRY, UNIVERSITY COLLEGE CORK, IRELAND. THE ECOTOXICOLOGY TRIANGLE. POLLUTANTS. TOXICOLOGY BIOTRANSFORMATION BIOCONCENTRATION. SPECIATION DISTRIBUTION. BIOTA.

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Protein structure in molecular systems biology

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  1. Protein structure in molecular systems biology Dr DAVID SHEEHAN, PROTEOMICS RESEARCH GROUP, DEPT. BIOCHEMISTRY, UNIVERSITY COLLEGE CORK, IRELAND McKim Conference on Predictive Toxicology, Duluth MN, Sept. 25-27th 2007

  2. THE ECOTOXICOLOGY TRIANGLE POLLUTANTS TOXICOLOGY BIOTRANSFORMATION BIOCONCENTRATION SPECIATION DISTRIBUTION BIOTA ENVIRONMENT SEASONALITY CLIMATE McKim Conference on Predictive Toxicology, Duluth MN, Sept. 25-27th 2007

  3. SENTINEL SPECIES IN ECOTOXICOLOGY • CHOSEN AS REPORTERS OF STATUS • ROBUST • EASILY-RECOGNISED • ABUNDANT • SESSILE RATHER THAN MOBILE • WIDE GEOGRAPHICAL DISTRIBUTION McKim Conference on Predictive Toxicology, Duluth MN, Sept. 25-27th 2007

  4. SENTINELS - ADVANTAGES • HISTORY OF USE • MULTI-ORGAN MODEL SYSTEMS • PREDICT FOR TOXICITY IN HUMANS • OFTEN BIOCONCENTRATE POLLUTANTS McKim Conference on Predictive Toxicology, Duluth MN, Sept. 25-27th 2007

  5. SENTINELS - DISADVANTAGES • MAY HAVE UNIQUE UPTAKE CHARACTERISTICS • MUCH SIMPLER SYSTEMS THAN MAMMALS • POORLY-REPRESENTED IN SEQUENCE D-BASES McKim Conference on Predictive Toxicology, Duluth MN, Sept. 25-27th 2007

  6. MUSSELS – GENUS MYTILUS • FILTER FEEDERS • SESSILE • ABUNDANT • WIDELY-DISTRIBUTED • RESILIENT TO POLLUTION • HISTORY OF USE • “MUSSELWATCH” PROGRAMMES McKim Conference on Predictive Toxicology, Duluth MN, Sept. 25-27th 2007

  7. PROTEIN-PROTEIN INTERACTIONS GLYCOPROTEOME MODIFIED PROTEINS REDOX PROTEOME SYSTEMS BIOLOGY PROTEOME METABOLOME DNA mRNA PROTEIN METABOLITES TRANSCRIPTOME GENOME PHOSPHOPROTEOME McKim Conference on Predictive Toxicology, Duluth MN, Sept. 25-27th 2007

  8. MYTILUS IS POORLY-REPRESENTED IN D-BASES McKim Conference on Predictive Toxicology, Duluth MN, Sept. 25-27th 2007

  9. Beta-1,4-D-Endoglucanase Cel45a From Blue Mussel Mytilus Edulis X-Ray Structure Of Beta-Mannanase From Blue Mussel Mytilus Edulis Crystal Structure Of Phosphoenolpyruvate Mutase – 6 str.s Mediterranean Mussel Defensin Mgd-1 McKim Conference on Predictive Toxicology, Duluth MN, Sept. 25-27th 2007

  10. SOME IMPLICATIONS OF POOR D-BASE REPRESENTION • SPOT-MATCHING FOR MS IS DIFFICULT • LACK OF STRUCTURAL KNOWLEDGE THROUGH PROTEIN HIERARCHY • DIFFICULT TO LOCATE PROTEIN MODIFICATIONS (e.g. OXIDATION) • PROTEIN-PROTEIN INTERACTION? McKim Conference on Predictive Toxicology, Duluth MN, Sept. 25-27th 2007

  11. OVERVIEW OF THIS PRESENTATION • SEQUENCE-INDEPENDENT PROBES FOR REDOX LESIONS IN PROTEINS • SEQUENCE SIMILARITY IN SPOT MATCHING • USE OF ON-LINE HOMOLOGY MODELS McKim Conference on Predictive Toxicology, Duluth MN, Sept. 25-27th 2007

  12. SEQUENCE-INDEPENDENT PROBES FOR REDOX LESIONS IN PROTEINS McKim Conference on Predictive Toxicology, Duluth MN, Sept. 25-27th 2007

  13. .OOR H202 .O2- OH- .OH GSH Oxidative stress CAT GPX SOD ANTIOXIDANT DEFENCE O2+ ROS McKim Conference on Predictive Toxicology, Duluth MN, Sept. 25-27th 2007

  14. PROTEIN EXPRESSION SIGNATURE APPROACH pH 3 10 IMAGE ANALYSIS SOFTWARE 70 kDa Mr UP-REGULATED 17 kDa NO CHANGE McKim Conference on Predictive Toxicology, Duluth MN, Sept. 25-27th 2007 DOWN-REGULATED

  15. PROTEIN OXIDATION COMPLICATES THE PROTEOME PROTEINS ABSORB ~ 70% OF ROS CARBONYLATION (R → ALDEHYDE/KETONE) UBIQUITINATION NITROSYLATION CYS OXIDATION …. Dowling & Sheehan D. (2006) Proteomics 6, 5597-5604. Sheehan D. (2006) Biochemical and Biophysical Research Communications 349, 455-462. McKim Conference on Predictive Toxicology, Duluth MN, Sept. 25-27th 2007

  16. PROTEIN CARBONYLATION:ANIMALS EXPOSED TO PRO-OXIDANTS 21.5 21.5 31 31 45 66 45 66 pI pI 4 7 4 7 Mr (x10-3) 4 3 2 1 ANTI-DNP + HYDRAZINE - DNP HYDRAZONE - DNP C = O BLOT MYTILUS EDULIS 2-D CARBONYLS __________________________ McDonagh & Sheehan, 2006 Aquat. Toxicol. 79, 325-333 McKim Conference on Predictive Toxicology, Duluth MN, Sept. 25-27th 2007

  17. REDOX LESIONS: UBIQUITINYLATION Ub Ub ANTI-Ub Ub BLOT Ub Ub Ub __________________________ McDonagh & Sheehan, 2006 Aquat. Toxicol. 79, 325-333 McKim Conference on Predictive Toxicology, Duluth MN, Sept. 25-27th 2007

  18. REDOX LESIONS: NITROSOTHIOLS AND THE BIOTIN SWITCH ASSAY NITROSOTHIOLS ARE REDUCED BY ASCORBIC ACID BUT DISULPHIDES ARE NOT – SPONTANEOUSLY HYPERTENSIVE RAT KIDNEY MEDULLA SNO NEM - BIOTIN SH SS SS ASCORBIC ACID S-NEM-BIOTIN SS STREPTAVIDIN McKim Conference on Predictive Toxicology, Duluth MN, Sept. 25-27th 2007

  19. REDOX LESIONS: SULPHENIC ACID (SOH) PROTEINS NEM - BIOTIN McKim Conference on Predictive Toxicology, Duluth MN, Sept. 25-27th 2007

  20. TOXICOLOGY OF NANOPARTICLES IN E. COLI – GOLD AS ANTIOXIDANT -SH- IODOACETAMIDE FLUORESCEIN PROTEIN GOLD MENADIONE CONTROL McKim Conference on Predictive Toxicology, Duluth MN, Sept. 25-27th 2007

  21. REDOX LESIONS: NITROSOTYROSINES ANTI -3NITROTYROSINE YNO SS YNO SS LC-TANDEM MS IDENTIFIED 22 PROTEINS Tyther et al., 2007 Proteomics – Clinical Applications. IN PRESS McKim Conference on Predictive Toxicology, Duluth MN, Sept. 25-27th 2007

  22. SEQUENCE SIMILARITY IN SPOT MATCHING McKim Conference on Predictive Toxicology, Duluth MN, Sept. 25-27th 2007

  23. Cork harbour PROTEINS 7 days DISSECT GILL 24 hr +/- 1 mM menadione McKim Conference on Predictive Toxicology, Duluth MN, Sept. 25-27th 2007

  24. ON OXIDATION…. hsp gp96 PDI hmb calreticulin Protease Serine 1 beta tubulin alpha 2 tubulin PDI GDP diss. inhibitor gelsolin enolase GST Pi RNA binding protein transferrn -SH + IAF  -S-IAF A -SH “DISAPPEAR” 45 kDa B -S-S “APPEAR” 45 kDa Control Menadione McKim Conference on Predictive Toxicology, Duluth MN, Sept. 25-27th 2007

  25. SPOT IDENTIFICATION TRYPTIC DIGEST MS 1 MS 2 HPLC TANDEM MS (i.e. MS/MS) PROTEIN PEPTIDES PEPTIDE FRAGMENT 1 m/z n m/z SCREEN SEQ D-BASES AMINO ACID SEQUENCE SPOT ID. McKim Conference on Predictive Toxicology, Duluth MN, Sept. 25-27th 2007

  26. McDonagh & Sheehan, 2007, Proteomics, 7, 3395-3403 McKim Conference on Predictive Toxicology, Duluth MN, Sept. 25-27th 2007

  27. USE OF ON-LINE HOMOLOGY MODELS McKim Conference on Predictive Toxicology, Duluth MN, Sept. 25-27th 2007

  28. PROCEDURE FOR HOMOLOGY MODELLING TARGET SEQUENCE TEMPLATE STRUCTURE SCRs IDENTIFY STRUCTURALLY CONSERVED REGIONS LOOPS CREATE DATABASE OF LOOPS FROM ELSEWHERE IN PDB ENERGY MINIMISE HOMOLOGY MODEL McKim Conference on Predictive Toxicology, Duluth MN, Sept. 25-27th 2007

  29. ON-LINE HOMOLOGY MODELLING WITH Geno3D COMBET et al., “Geno3D an automated protein modelling web server, BIOINFORMATICS, 2002, 18, 213-214 http://pbil.ibcp.fr/htm/ McKim Conference on Predictive Toxicology, Duluth MN, Sept. 25-27th 2007

  30. ON-LINE HOMOLOGY MODELLING WITH Geno3D • START AT PROTEIN SEQUENCE DATABASE • AMINO ACID SEQUENCE IS INPUT FOR ON-LINE HOMOLOGY MODELING PROGRAM e.g. GENO3D. • SELECT TEMPLATE (PSI-BLAST) • RESULTS SENT VIA e-MAIL. McKim Conference on Predictive Toxicology, Duluth MN, Sept. 25-27th 2007

  31. CATALASE FROM M. Californianus TEMPLATE: HUMAN ERYTHROCYTE CATALASE PDB 1F4J McKim Conference on Predictive Toxicology, Duluth MN, Sept. 25-27th 2007

  32. CATALASE FROM M. Californianus McKim Conference on Predictive Toxicology, Duluth MN, Sept. 25-27th 2007

  33. MODEL QUALITY McKim Conference on Predictive Toxicology, Duluth MN, Sept. 25-27th 2007

  34. STRUCTURALANALYSIS MAIN CHAIN PARAMETERS ARE COMPARED TO A STRUCTURE OF 2Å RESOLUTION McKim Conference on Predictive Toxicology, Duluth MN, Sept. 25-27th 2007

  35. RMSD =  x2 n ROOT MEAN SQUARE DEVIATION a-Cs TEMPLATE x4 x2 x1 X6 .. x3 x5 MODEL McKim Conference on Predictive Toxicology, Duluth MN, Sept. 25-27th 2007

  36. RMSD IS ONLY LOOSELY RELATED TO % SEQ. ID. McKim Conference on Predictive Toxicology, Duluth MN, Sept. 25-27th 2007

  37. AMIDE PLANE C PHI PSI N SER ARG RAMACHANDRAN ANALYSIS McKim Conference on Predictive Toxicology, Duluth MN, Sept. 25-27th 2007

  38. TEMPLATE MODEL LACTOPEROXIDASE: OVERLAY OF MODEL ON TEMPLATE (PyMOL) MOVIE!! McKim Conference on Predictive Toxicology, Duluth MN, Sept. 25-27th 2007

  39. OTHER MODELS METHIONINE ADENOSYLMETHYL TRANSFERASE CYTOCHROME C OXIDASE HEAVY METAL B.P. LYSOZYME MDR ASSOC.D PROTEIN P-450 CYP4Y1 P-GLYCO PROTEIN SUPEROXIDE DISMUTASE TWITCHIN PHOSPHOFRUCTOKINASE McKim Conference on Predictive Toxicology, Duluth MN, Sept. 25-27th 2007

  40. RELEVANCE OF HOMOLOGY MODELS • SIGNAL TRANSDUCTION -> “NETWORKS OF PROTEINS” • GUIDE FOR STRUCTURE-FUNCTION • MODEL SOLVENT ACCESSIBILITY • PROTEIN-PROTEIN INTERACTIONS • CAVEATS!! • MODELS ARE NOT STRUCTURES • SIDE-CHAINS McKim Conference on Predictive Toxicology, Duluth MN, Sept. 25-27th 2007

  41. CONCLUSIONS • PROTEIN MODIFICATIONS NEED TO BE MODELLED • MORE SEQUENCE DATA ARE REQUIRED FOR NON-STANDARD SPECIES • THREE-DIMENSIONAL MODELS ARE POSSIBLE WHERE 3D STRUCTURE ARE LACKING McKim Conference on Predictive Toxicology, Duluth MN, Sept. 25-27th 2007

  42. FUNDING/ACKNOWLEDGEMENTS Prog. Res. Third Level Institutions; IRCSET; EPA; Health Research Board Collaborators: Dr Tim Veenstra (NCI, NIH); Drs Ian Davidson, Phil Cash (Aberdeen); Prof Maria Bebiano (Faro) Drs. Vera Dowling, Brian McDonagh, Ray Tyther Sara Tedesco, Ebenezer Kanagaraj, Hu Wentao McKim Conference on Predictive Toxicology, Duluth MN, Sept. 25-27th 2007

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