1. Analysis Of Microarray Data By Harsha Nilakantan & Mihir Solanki

2. Problem Ubiquitin pathway has become one of the most researched pathways in the recent years. One significant aspect is to identify ‘substrates’ for different E3 ligases, which leads to ubiquitination. Some E3 ligases have become targets for anti-cancer drugs. IpaH9.8 implicated in MAPK pathway. Here, we attempt to find ‘IpaH9.8’ substrates from the human proteome.

3. Background Ubiquitin is a small regulatory protein found in all eukaryotic cells. 76 Amino acids, 8.5 kDa. Ubiquitination is one of the most well characterized Post Translational Modifications. 7 lysine groups play an important role in ubiqitination. Human and yeast ubiquitin are 96% similar MQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIP PDQQRLIFAGKQLEDGRTLSDYNIQKESTLHLVLRL RGG

4. http://en.wikipedia.org/wiki/Ubiquitin Ubiquitination of a protein

5. Covalent attachment of single or multiple Ubiquitin moieties to a protein. Concerted action of 3 enzymes: E1 - Ubiquitin activating enzyme E2 - Ubiquitin conjugating enzyme E3 – Ubiquitin ligase High energy thiol ester is formed between C-terminal Gly of ubiqutin and a Cys in the E 1 active site (ATP/AMP) Ub is then transferred to a Cys of E 2 forming a new thiol ester Finally, Ub forms on isopeptide bond between C-terminal Gly of Ub and ε-amino group of Lys on a target protein

6. Substrate-Ub bond Ubiquitin forms covalent bond with the ε-amine group of the Lys residue of substrate. Poly Ub is formed when C-terminal Gly of one Ub forms a bond with the Lys residue of another Ub. Since Ub has 7 lysine residues, a variety of linkages can be formed. Functions of few linkages are known till date. sub K ub GG K K ub GG K K …and so on MQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIFAGKQLEDGRTLSDYNIQKESTLHLVLRLRGG 6 11 27 29 33 48 63

7. Why is ubiquitination important? Proteasomal degradation of proteins in the cell Receptor endocytosis Cellular signaling Protein trafficking DNA transcription and repair Cell differentiation and repair

8. Different Linkage Functions http://www.biomol.de/wiki/index.php/Ubiquitin_Chains

9. Forms of Ubiquitination Biochemical society transactions (2009) 37,937-953-David Komander

10. Experimental Design Control ArrayActive Array Y Y Y YY Y Y Y Protein on array Ubiquitin Primary antibody (mouse anti-ubiquitin) Sec. antibody (anti-mouse) conjugated to fluorophore (Cy5, Cy3 etc.) E1 + E2 + Free Ub E1 + E2 + E3 + Free Ub Y Y Y Y Y Y Y

11. GUI

12. FLOWCHART Control ArrayActive Array Intensity Background N.I. = (I-B)/B Int. Ratio = |log(NI A /NI C )| Display proteins with highest Int. Ratio Ubiquitinated!!! Hence, substrates for IpaH9.8

13. Functions Analyzedata(filename) - ▫Filename is the excel file (control or active) ▫The output - datamatrix is 9600x2, where the 1 st column is a name of the protein and 2 nd is the normalized intensity. Microarrayanalysis(ctrl,active) ▫The input files are control data and active data excel files. ▫This function calls analyzedata. GUI script which has GUI related functions and also includes - ▫Heat map ▫Scatter plot

14. Data Formats Two input files - large excel spreadsheets of 23,232x10. ▫Containing the names of Proteins and inherent controls, intensities, backgrounds, microarray - blocks, rows and spots. ▫Each data set contains duplicates of Proteins. Output is a cell array of 9600x2. ▫Containing names of Proteins and their normalized intensities. In manipulating the data in the input files, we used a lot of cell arrays.

15. Challenges & Unsolved Inherent controls showed a lot of variability in intensity. In plotting a heat map, we faced some difficulty in manipulating the raw data to bring out a data set to plot the heat map. Working on bringing the heat map on to its allotted axis. Working on displaying Highest intensities also within the same scatter plot.

16. The End