Biochemical and Structural Investigations of the Retinoblastoma Protein, Its Binding Partners, and the BRG1 Protein - a Subunit of Human SWI/SNF Remodeling Complexes
Author | : |
Publisher | : |
Total Pages | : |
Release | : 2005 |
ISBN-10 | : OCLC:696262237 |
ISBN-13 | : |
Rating | : 4/5 (37 Downloads) |
Book excerpt: Two projects were undertaken in this thesis. The first project involved functional investigations on the retinoblastoma protein (pRb) and its interaction with binding partners. The second project involved the characterization of functional domains of the human BRG1 protein including the 3D structure determination of BRG1 bromodomain. The retinoblastoma tumor suppressor protein (pRb) is a key negative regulator of cell proliferation that is frequently deregulated in human cancer. More than 130 proteins have been reported to bind to pRb directly. Many of these interactions were reported with the pRb small pocket, which is the major focus of tumorigenic mutations in pRb, and comprises the A and B cyclin - like domains (amino acids 379-578 and 641-791, respectively). However, results found in the literature are ambiguous and contradictory. We have investigated some of these interactions using purified proteins and in-vitro biophysical and biochemical methods, which included, nuclear magnetic resonance (NMR) spectroscopy, mass spectrometry, isothermal titration calorimetry (ITC), the affinity chromatography pull-down assays, and gel filtration chromatography. The interactions of pRb with the following proteins were checked using this approach: MyoD, Id-2, E2F1, HDAC1, PAI2, HPV E7, SV40 large T antigen, BRG1, and gankyrin. Myogenic differentiation promoting, MyoD and inhibiting, Id-2, factors were reported to interact with the pRb pocket domain to coordinate the cell growth and differentiation during the process of myogenesis. There have been contradictory reports in the literature regarding the direct interaction between pRb and MyoD, pRb and Id-2 proteins. We probed these interactions using our multimethod in-vitro approach. Using this approach and we were able to document interactions between pRb and HPV-E7, pRb and SV40 large T antigen, MyoD and DNA, and MyoD and Id-2. However using the same approach, we could unambiguously show that there is no direct protein-protei.