Proteomics in Addiction Systems-wide proteomic technologies for addiction

Proteomics in Addiction: (Systems-wide proteomic technologies for addiction research: the study of post-translational modifications) Sungchan Kim Department of Biochemistry College of Medicine, Hallym University

Proteomics A system-wide screening technology for analysis of proteins’ properties at global scale. State 2 State 1 Proteome 1 v Expression v Modification v Subcellular localization v Protein/protein interactions v Activities Proteome 2

Advantages of protein profiling vs. Nucleic acid Microarrays • m. RNA misses many features of proteins that affect function. - Protein “activities” determine phenotype, not m. RNA level or protein levels. - m. RNA levels frequently do not correlate with protein levels. - Most proteins are post-translationally modified(PTM). - PTMs usually have critically important functional differences. - Function of many proteins in affected by change in subcellular location. - Consequently, the function/dysfunction (in a certain disease) of a specific gene cannot be predicted comprehensively by its sequence only.

Why is Proteomics Important? • Identification of proteins in normal and disease conditions. – Most cancer markers are proteins. – The majority of drug targets are proteins. • Identification of pathogenic mechanisms. – Reveals gene regulation events involved in disease progression • Promise in novel drug discovery via analysis of clinically relevant molecular events. • Contributes to understanding of gene function. Biomarker or drug target

Principle of 2 -D Electrophoresis (Gel-based approach) 1. First dimension: - Denaturing isoelectric focusing - Separation according to the p. I 2. Second dimension: - SDS electrophoresis - Separation according to the MW The 2 -D electrophoresis gel resolves thousands of protein spots each time.

2 D-gel/Mass Spectrometry-based Proteomics DIA: Difference in gel analysis Normal Disease Label protein extract with Cy 3 Label protein extract with Cy 5 Mix equal amount of the labelled extracts Ünlü, M. , Morgan, M. E. , and Minden, J. S. (1997). Difference gel electrophoresis: a single gel method for detecting changes in cell extracts. Electrophoresis, 18, 2071 -2077

2 D-gel/Mass Spectrometry-based Proteomics

Poteomics 분석 방법 Electrophoresis 단백질 분리 Mass spectrometry 펩타이드 분자량 측정 Database search 단백질 동정

Challenges for Proteomic Analysis § Inherent complexity of biological samples § Wide dynamic range of protein expression >106 Dynamic range Number of Proteins ~13, 000 ~2, 000 Proteomic methods in Cells 103 -4 Proteomic methods in Cells

The cell map

Simplifying the Proteome Based on Common Structural Features - modifications O OP O O –Ser –Thr –Tyr O O Phosphorylated –Farnesylation –Geranylgeranylation –Myristoylation –Pamitoylation –GPI anchor Lipid Modified –O-Glc. NAc –O-Fucosylation –O-Glucosylation –Complicated Glycosylated

Known Post-translational Modifications: >200 Protein: Analysis and Design. Angeletti, R. H. (ed). 121 -207 (Academic Press, San Diego, CA, 1998). Diseases Modifications Cellular functions Cardiovascular diseases Hypertension Cancer Phosphorylation Ser Thr Tyr Cell growth Cell division Cell movement Cell memory Cell patterning Cell-cell interaction Apoptosis Signaling transduction Inflammation Stroke Alzheimer Parkinson's disease Schizophrenia Depression Hodgkin’s disease Meningitis Drug addiction Osteoporosis Multiple sclerosis Asthma Arthritis Diabetics Aging Cystic fibrosis Glycosylation O-Glc. NAc Fucosylation Glucosylation O-linked complex glycosylation N-linked complex glycosylation Lipid Modification Farnesylation Geranylgeranylation Myristoylation Palmitoylation GPI-anchor Acylation Acetylation Methylation DNA Replication DNA damage repair Transcription Translation Splicing Protein degradation Protein trafficking Subcellular localization Interactions Energy generation Energy consumption

Mechanism of modifications action of post-translational (from Ole N. Jensen. 2006. Nature Reviews Molecular cell biology. 7: 391 -403)

Agonistic and antagonistic effects of neighboring modifications Yang, XJ. Oncogene (2005) 24, 1653– 1662

Regulation by long-range intramolecular signaling • DNA damage activates Cdc 2 to phosphorylate Ser 315 of p 53, which generates a potential docking site for the Polo-box domain of Plks and facilitates Ser 20 phosphorylation of p 53. • This modification then stimulates CBP association for Lys 382 acetylation. • Similarly, Ser 46 phosphorylation stimulates the same acetylation. Yang, XJ. Oncogene (2005) 24, 1653– 1662

Proteomic approach for addiction research

< Post-translational modification and Addiction Research >

Lysine-Acetylation & Histone deacetylases (HDACs) O O ~N H HATs ~ ~ ~N H NH + NH 3 HDACs O • • • Loss of charge Extended length Gain hydrophobic contact area • Discovered in 1964 • < 90 targets identified to date (histones, transcription factors) • Dynamic and highly regulated modification – ~ 20 Histone Acetyl Transferases (HATs) – 18 Histone Deacetylases (HDACs)

Physiological Implications of Lysine-Acetylation (K-Ac) and Its Regulatory enzymes • Cancer – Histone Deacetylases (HDAC) inhibitors under >40 phase III clinical trials • Heart failure • Neurodegenerative diseases • Addiction • Aging and longevity

** CBP: one of Histone Acetyl Transferases (HAT)

< Post-translational modification and neuroscience research >

< Post-translational modification and neuroscience research >

< Post-translational modification and neuroscience research > • The role of HDAC inhibitors in Alzheimer's disease. First, HDAC inhibitors inhibit Aβ-induced hyperphosphorylation of tau protein. Second, HDAC inhibitors alter the expression of important genes which participate in the learning and memory.

< Post-translational modification and neuroscience research >

< Post-translational modification and cardiovascular disease research >

< Post-translational modification and Addiction Research >

Proteomics for post-translational modification research § Identification of differentially expressed protein. (2 D-gel based/MS approach) §Antibody-based affinity purification ü Lysine Acetylation ü Lysine Methylation (mono, di, tri) ü Tyrosine phosphorylation ü Lysine Propionylation ü Lysine Butyrylation ü Lysine Sumoylation ü… § Tagging-via-substrate approach ü Farnesylation (lipid modification) ü O-Glc. NAc modification ü Sumoylation ü Phosphorylation ü… § Dynamics studies possible

Multi-disciplinary Approaches for Biology Studies § Proteomics is a powerful biochemical techniques that is able to carry out systems-wide screening with high sensitivity and high efficiency. § Proteomics could complement molecular biology and genetics approach for dissection of biological pathways. § Integration of proteomics with molecular biology and genetics makes biological studies really powerful. Proteomics Mol/Cell Biology Genetics/Physiology

감사합니다.