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

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

Advantages of protein profiling vs. Nucleic acid Microarrays mRNA misses many features of proteins that affect function. - Protein “activities” determine phenotype, not mRNA level or protein levels. - mRNA 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 pI 2. Second dimension: - SDS electrophoresis - Separation according to the MW The 2-D electrophoresis gel resolves thousands of protein spots each time. 1. First dimension: - Denaturing isoelectric focusing - Separation according to the pI 2. Second dimension: - SDS electrophoresis - Separation according to the MW The 2-D electrophoresis gel resolves thousands of protein spots each time.

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, Mix equal amount of the labelled extracts Normal Disease Label protein extract with Cy3 Label protein extract with Cy5 DIA: Difference in gel analysis 2D-gel/Mass Spectrometry-based Proteomics

Poteomics 분석 방법 단백질 분리펩타이드 분자량 측정단백질 동정 ElectrophoresisMass spectrometryDatabase search

>10 6 Dynamic range Proteomic methods in Cells ~2,000 ~13,000 Number of Proteins Proteomic methods in Cells Challenges for Proteomic Analysis  Inherent complexity of biological samples  Wide dynamic range of protein expression

The cell map

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

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

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

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

Regulation by long-range intramolecular signaling DNA damage activates Cdc2 to phosphorylate Ser315 of p53, which generates a potential docking site for the Polo-box domain of Plks and facilitates Ser20 phosphorylation of p53. This modification then stimulates CBP association for Lys382 acetylation. Similarly, Ser46 phosphorylation stimulates the same acetylation. Yang, XJ. Oncogene (2005) 24, 1653–1662

Proteomic approach for addiction research

Lysine-Acetylation & Histone deacetylases (HDACs) 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) N O O NH ~ ~ H N NH 3 + O ~~ H HATs HDACs Loss of charge Extended length Gain hydrophobic contact area

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)

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.

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

 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 Multi-disciplinary Approaches for Biology Studies

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