Matthews Lecture 2_final 140115

Proteins pull down assays co immunoprecipitations two

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Unformatted text preview: va/on 21 Different Modes of Regula/on Canonical – direct DNA- binding CoR Non- Canonical – tethering ep PTMs CBP SRC p300 ER ER TIF2 ER ER ERE RNA TBP Pol-II Sp1 Gene Expression c-Jun c-Fos NF-κB NR- mediated mechanism of ac/on is complex cont’d P P P Ac/vated in the absence of ligand - post transla/onal modifica/on (PTM) - phosphoryla/on, sumolya/on, acetyla/on, ubiqui/nyla/on NR Tissue specific expression Species differences (rodent vs human) PM NR Regulated through nuclear import/export and ligand/ac/va/on mediated proteoly/c degrada/on Ub Ub NM Ub proteosome 23 Protein-Protein Interactions Identifying proteins that interact with NRs; Coregulator proteins •  pull-down assays •  Co-immunoprecipitations •  Two-hybrid assays •  Proteomics (mass spectrometry) 24 Pull- down assay 25 Co-Immunoprecipitation Incubate Mixture of radiolabeled proteins An/body 1 Beads An/body binds to specific protein Dissociate proteins by boiling An/gen- an/body complexes Gel bound to beads electrophoresis An/body 2 Migra/on Expose to film to detect radio- labeled protein 26 Co- Immunoprecipita/on – actual data agonist Protein X FLAG- NRα FLAG- NRβ Interaction of two proteins within an intact cell x = coregulator or protein that is hypothesized to interact with NRs y = nuclear receptor 28 Mammalian two-hybrid assay for the detection of CBP-E2 and p300-E2 interactions. ER Lee D et al. J. Biol. Chem. 2000;275:7045-7051 29 Gal4- Mammalian two- hybrid assay – Iden/fica/on of LxxLL mo/f in NR interac/ng proteins luciferase Gal4 LxxLL Gal4 VP- 16 ER- LBD = VP- 16 LxxLL ER- LBD Gal4 Gal4- Mammalian two- hybrid assay – Iden/fica/on of LxxLL mo/f in NR interac/ng proteins ESTROGEN Agonist 4-hydroxytamoxifen antagonist Helix12 contains sequence that interacts with LxxLL Proper posi/oning is necessary for interac/on with coac/vators Helix12 is re- posi/oned in the antagonist bound form 32 SERMs Display Tissue Selective Pharmacology (Selective Estrogen Receptor Modulators) Estradiol breast + uterus + bone + Genistein OH-TAM ICI 164,384...
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