1. ANALYSIS OF SHOTGUN PROTEOMICS DATASETS Federica Montanaro Center for Gene Therapy September 13, 2011

2. Dystrophin – 427kDa Membrane anchoring Signaling pathways Actin filaments Lipid interactions

3. Diseases associated with dystrophin  Duchenne Muscular Dystrophy  Childhood onset  Progressive loss of muscle function  Cardiomyopathy  Becker Muscular Dystrophy  Teen/Adult onset  Progressive loss of muscle function  Cardiomyopathy  X-Linked Dilated Cardiomyopathy  Teen/Adult onset  Severe cardiomyopathy

4. Modeling spectrin repeat structure Repeat A Repeat B Helix 2Helix 1 Helix 2 Folded

5. Modeling of mutations Hinge 3 New hinge INOUT 0 10 20 30 40 50 60 70 Age (years) Spectrin Repeat Phasing 25.5 years 36.5 years P = 0.002

6. Revisiting dystrophin’s interactome

7. Piecing the puzzle together Ahnak Vinculin Cypher Mia3 Mek Citro Cryab Tfg Desmin Vimentin ?

8. Study 1- Genotype-phenotype  Aim: Identify domains of the dystrophin protein that when mutated give rise to an early manifestation of dilated cardiomyopathy  Patients: Becker patients expressing a dystrophin protein with small domains missing (small deletions)

9. Mapping of new dystrophin interactions  Why do some dystrophin mutations affect only the heart?  Is dystrophin interacting with different proteins in heart versus skeletal muscle?  Approach: Combine immunoprecipitation with protein identification by shotgun proteomics