Trisha Brady

Trish Brady – NIH Trainee 2022-2024

Research progress: I am a PhD student in the Hardy and Vachet labs studying the structural heterogeneity of the apoptotic protease caspase-9. During these past two years I developed my skills in protein expression and purification, native ion mobility-mass spectrometry (IM-MS), collision-induced unfolding (CIU), and scientific writing/presenting. In the last year have made progress on several of the aims of my thesis, as outlined below:  

Uncontrolled cell death underpins neurodegeneration and cancers, necessitating tight regulation. Caspase-9 (casp-9), a key apoptosis protease, lacks a full-length, high-resolution structure, leaving domain interactions and their influence on activity enigmatic. For Aim 1, we are evaluating the effect of substrates and key post-translational modifications on monomer:dimer equilibrium and oligomeric State. Using IM-MS and structural modeling, we probe casp-9’s conformations in various maturation states. We are using collision cross sections (CCSs), coupled with structural modeling, to filter casp-9 models. Casp-9 has a pro-domain, called CARD, a large subunit, and a small subunit, that assemble to form a monomer. WT casp-9 displays several charge state distributions (CSDs), indicating conformational heterogeneity. Three CSDs are associated with the monomeric state, one with the dimeric state, and one each for CARD+large and small. Measured CCS values vary for the three CSDs of casp-9 monomer, with the lowest monomer charge state (12+) having a CCS of 3320 ± 10 Å and the highest (32+) having a CCS of 8230 ± 70 Å. Native MS measurements of ΔCARD, show only one monomer CSD, suggesting that the flexible CARD pro-domain is the primary cause of the conformational heterogeneity of the casp-9 monomer. Interestingly, while the monomer shows three CSDs under native nanospray conditions, only one is observed for dimer, suggesting stabilization. Possibly, structural changes during dimerization prevent further alterations in CARD orientation. More data is required to better understand the role of the CARD in casp-9 structural heterogeneity. We are also investigating dimer formed in the presence of z-VAD-fmk, a substrate mimic which inactivates the complex while forcing dimerization.