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2012 YCU Science Summer Program

2012.08.15. Lecture #6. 2012 YCU Science Summer Program. Proteomic Approach to Protein Alterations Implicated in Aging and Geriatric Diseases. Yokohama City University Advanced Medical Research Center. Tosifusa Toda. Genome, Transcriptome and Proteome. Individual.

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2012 YCU Science Summer Program

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  1. 2012.08.15 Lecture #6 2012 YCU Science Summer Program Proteomic Approach to Protein Alterations Implicated in Aging and Geriatric Diseases Yokohama City University Advanced Medical Research Center Tosifusa Toda

  2. Genome, Transcriptome and Proteome Individual Genome: entire set of genes (ca. 23,000 genes/individual) Cell DNA Transcription Transcriptome: a set of mRNA (> 3,000 messages/cell) Proteome: > 10,000 proteins/cell mRNA Translation Ribosome Folding Polypeptides tRNA Post-translational modification mRNA PDB

  3. Posttranslational modification of protein 1. Physiological (enzymatic) modification 1.1. Phosphorylation 1.2. Glycosylation 1.3. Acetylation 1.4. Methylation 1.5. Ubiquitination 1.6. Deimination (Citrullination)

  4. Posttranslational modification of protein 1. Physiological (enzymatic) modification 1.1. Phosphorylation 1.2. Glycosylation 1.3. Acetylation 1.4. Methylation 1.5. Ubiquitination 1.6. Deimination (Citrullination) 2. Non-physiological (non-enzymatic) modification 2.1. Oxidation 2.2. Glycation

  5. Posttranslational modification of protein 1. Physiological (enzymatic) modification Met 1-1. Phosphorylation Gly Asn Leu Ser P Thr P Lys Tyr 2-1. Oxidation 2-2. Glycation P Arg Cys Met Ala

  6. Posttranslational modification of protein 1. Physiological (enzymatic) modifications 1-2. Glycosylation

  7. Posttranslational modification of protein 1. Physiological (enzymatic) modifications 1-3. Acetylation Interact with “Bromodomain” of DNA binding proteins

  8. Posttranslational modification of protein 1. Physiological (enzymatic) modifications 1-4. Methylation Arginine

  9. Posttranslational Modification of Protein 1. Physiological (enzymatic) modifications 1.6. Deimination (Citrullination) 2. Non-physiological (non-enzymatic) modifications 2-1. Oxidation 2-2. Glycation

  10. Multiple posttranslational modifications(A typical example: Nucleosomal Histone H3) Citrulline Citrulline

  11. Oxidative stress: The most suspicious cause of aging

  12. Oxidative Protein Modifications Implicated in Aging Carbonylation

  13. Oxidative Protein Modifications Implicated in Aging Tyrosine Nitration Methionine Sulfoxidation

  14. How do we analyze so many proteins comprehensively including various post-translational modifications? Proteomic Approach Proteomics: A strategy for analyzing many proteins in a sample at a time 1. Two-dimensional gel electrophoresis-based Proteomics 2. Liquid chromatography-based Shot-gun Proteomics

  15. 1. Two-dimensional gel electrophoresis-based Proteomics Protein mixture in a sample Separation by 2-DE Image analysis Spot picking In-gel digestion Mass spectrometry Identification、 PTM analysis Database search

  16. Aging-related Protein Alterations Detected in Mouse Hippocampus by 2-DE-Based Proteome Analysis

  17. Matrix Assisted Laser Desorption/Ionization Time-of-Flight (MALDI-TOF) Mass Spectrometry

  18. Oxidation of Calmodulin Detected by Spectrometry

  19. Aging-Related Increase in Methionine Sulfoxide Level in Mouse Hippocampus

  20. Functional impairment in oxidized Calmodulin Native recombinant calmodulin Kd=34.67μM Oxidized calmodulin Kd=178.89 μM

  21. Multiple Functions of CaM

  22. Oxidative Stress Senescence accelerating factor Oxidative Stress Implicated in Aging and Geriatric Diseases Aging Development / maturation Geriatric diseases Genetic background Genome DNA (ca.23,000 genes) Aging-related physical deterioration Epigenetic regulation Transcription mRNA (ca. 3,000 messages/cell) Functional network of normal proteins Translation Interfering Proteomic phenotype Primary translation products (ca. 3,000 proteins/cell） Enzymatic modification Post-translationally modified proteins Oxidative modification Reductase Oxidized proteins (Ox calmodulin etc)

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