Yossef Baidi, a senior majoring in chemistry at Johns Hopkins University, validated a novel PRMT5 interface for its application to MTAP-null cancers.
The gene for methylthioadenosine phosphorylase (MTAP) is deleted in 15% of cancers, including several with high unmet medical need. At the Broad Institute, I was surrounded by driven scientists doing amazing work. Having the opportunity to meet so many leaders in their field, and see how supportive they are of fellow scientists, was inspiring. This supportive environment motivated me in working on my project and helped me get through the challenges and setbacks I encountered. The BSRP staff and cohort were an amazing support system as well.These cancer cells are sensitive to knockdown of the protein arginine methyltransferase 5 (PRMT5)/WD repeat-containing protein 77 (WDR77) heterooctameric complex, which symmetrically dimethylates arginine residues of substrates in many biological processes. The Broad Institute identified a novel protein-protein interface between PRMT5 and another protein, which is also required for MTAP-null cell viability. The mechanism for this interaction was determined by X-ray crystallography. To validate this structure, we produced a mutant form of PRMT5 that is predicted to be incapable of interaction. We generated the mutant form of PRMT5 using a co-expression system in insect cells. The PRMT5/WDR77 complex was purified using affinity chromatography followed by size exclusion chromatography. Binding affinity was determined between the interaction partner and either wildtype or mutant PRMT5 complex using surface plasmon resonance (SPR) and fluorescence polarization (FP) techniques. These experiments showed that point mutations at the crystal structure interface do indeed disrupt this protein-protein interaction. In further cellular studies, the mutant PRMT5 construct will be used to determine whether this specific interface is required for MTAP-null cell viability.
Project: Validating a novel PRMT5 interface for application to MTAP-null cancers
Mentors: Brian McMillan, Center for the Development of Therapeutics