2012 YCU Science Summer Program2012 YCU Science Summer Program

Lecture #6

Proteomic Approach to Protein Alterations Implicated in Aging and Geriatric Diseases

Tosifusa Toda

Yokohama City UniversityAdvanced Medical Research Center

2012.08.15

Genome, Transcriptome and ProteomeGenome, Transcriptome and Proteome

Genome: entire set of genes(ca. 23,000 genes/individual)

DNA

Folding

Post-translationalmodification

PDB

Proteome: > 10,000 proteins/cell

Transcriptome: a set of mRNA (> 3,000 messages/cell)

Transcription

mRNA

Translation

Ribosome

tRNAPolypeptides

mRNA

Cell

Individual

Posttranslational modification of protein Posttranslational modification of protein

1. Physiological (enzymatic) modification

1.1. Phosphorylation1.2. Glycosylation1.3. Acetylation1.4. Methylation1.5. Ubiquitination1.6. Deimination (Citrullination)

Posttranslational modification of protein Posttranslational modification of protein

1. Physiological (enzymatic) modification

2. Non-physiological (non-enzymatic) modification

1.1. Phosphorylation1.2. Glycosylation1.3. Acetylation1.4. Methylation1.5. Ubiquitination1.6. Deimination (Citrullination)

2.1. Oxidation2.2. Glycation

Posttranslational modification of protein Posttranslational modification of protein

1. Physiological (enzymatic) modification

1-1. Phosphorylation

2-1. Oxidation2-2. Glycation

MetMet

GlyGly

AsnAsn

LeuLeu

SerSer

ThrThr

LysLys

TyrTyr

ArgArg

CysCys

MetMet

AlaAla

P

P

P

Posttranslational modification of proteinPosttranslational modification of protein1. Physiological (enzymatic) modifications

1-2. Glycosylation

Posttranslational modification of protein Posttranslational modification of protein

1. Physiological (enzymatic) modifications

1-3. Acetylation

Interact with “Bromodomain” of DNA binding proteins

Posttranslational modification of protein Posttranslational modification of protein

1. Physiological (enzymatic) modifications

1-4. Methylation

Arginine

Posttranslational Modification of Protein Posttranslational Modification of Protein1. Physiological (enzymatic) modifications

2. Non-physiological (non-enzymatic) modifications

1.6. Deimination (Citrullination)

2-1. Oxidation2-2. Glycation

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

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

Citrulline

Citrulline

Oxidative stress: The most suspicious cause of aging

Oxidative Protein Modifications Implicated in Aging

Carbonylation

Oxidative Protein Modifications Implicated in Aging

Tyrosine Nitration

Methionine Sulfoxidation

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

Protein mixturein a sample

Mass spectrometry

Database searchIdentification、

PTM analysis

Image analysis

Spot picking

1. Two-dimensional gel electrophoresis-based Proteomics

Separation by 2-DE

In-gel digestion

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

Analysis

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

Oxidation of Calmodulin Detected by Spectrometry

Aging-Related Increase in Methionine Sulfoxide Level in Mouse Hippocampus

Native recombinant calmodulin

Oxidized calmodulin

Kd=34.67μM

Kd=178.89 μM

Functional impairment in oxidized Calmodulin

Functional impairment in oxidized Calmodulin

Multiple Functions of CaM

Development / maturation

Genome DNA (ca.23,000 genes)

mRNA (ca. 3,000 messages/cell)

Primary translation products (ca. 3,000 proteins/cell ）

Post-translationally modified proteins

Proteomic phenotype

Genetic background

Functional network of normal proteins

Oxidative Stress Implicated in Aging and Geriatric Diseases

Epigenetic regulation

Translation

Transcription

Enzymatic modification

Senescence accelerating factor

Oxidative Stress

Oxidized proteins (Ox calmodulin etc)

Aging

Aging-related physical deterioration

Geriatric diseases

Oxidative modification

Interfering

Reductase