The Genome's Guardian Angel: A Structural Exploration of p53 in Cancer
Restricted (Penn State Only)
- Author:
- Solares Bucaro, Maria
- Graduate Program:
- Molecular, Cellular, and Integrative Biosciences
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- July 14, 2023
- Committee Members:
- Melissa Rolls, Program Head/Chair
Mark Hedglin, Outside Unit Member
Susan Hafenstein, Major Field Member
Justin Pritchard, Outside Field Member
Deb Kelly, Chair & Dissertation Advisor - Keywords:
- p53
tumor suppressor
structural biology
cryo-em
cancer
molecular modeling - Abstract:
- In the war against cancer, scientists have uncovered a vast number of molecular culprits. Among them is tumor suppressor protein TP53 (p53). Identified as the “guardian of the genome,” p53 is involved in complex signaling cascades vital to the everyday workings of a cell. In fact, this tetrameric protein is involved in DNA repair, cell cycle arrest, and apoptosis. It is of no surprise that p53 is highly deregulated in cancer, where 50% of all disease incidences contain gene mutation. Unfortunately, research into p53 has been stalled by a lack of structural information. The primary sequence of a monomer (~48 kDa) can be broadly described in three broad regions: an N-terminal domain (NTD), a core or DNA-binding Domain (DBD), and a C-terminal domain (CTD). Most of the research efforts have been focused on the DBD domain due to its stability. Further, this domain also plays an integral role in binding DNA and transcribing critical genes. However, this tunnel vision has caused a major oversight of the available structural and biological information on NTD and CTD. Studies with NMR and X-ray crystallography have yielded limited results due to the disorganized and flexible nature of these regions. However, neglecting them has led to conflicting theories regarding their regulatory roles. Cryo-electron microscopy (Cryo-EM) is revolutionizing our understanding of the macromolecular world. This technology has advanced well from its “blobology days.” With the development of better TEMs, direct electron detectors, and advanced computer software it is possible to determine structures of flexible proteins. Yet, a reconstruction is only as good as its sample. By limiting the field to only reconstructing recombinant protein models, critical information like post-translational modifications (PTMs) and mutations are understudied. The lack of translational models only ends up stalling therapeutic development. In this thesis, I present the work done on reforming sample preparation methods to yield models of natively-sourced proteins. I developed a highly reproducible protein purification method that yields high concentrations of p53 molecules for cryo-EM analysis. This was done by exploiting properties of p53 such as its innate ability to bind to metal cations. I also used functionalized silicon nitride (SiN) microchips to further enrich samples and prevent the formation of air-water interface particles. By coupling both methods, we were able to reconstruct full-length p53 monomer, full-length dimer, and a partial-length tetramer while preserving PTMs and native conformations. These biological snapshots yielded new information on mechanisms of action and PTM identity. Finally, my monomer model served as a template to create computational models of the most frequent mutations in cancer. These initial modeling proposed altered local and global structures, electrochemical changes, and different molecular dynamics. This data corroborates the destabilization of coordinating zinc ions alongside DNA-binding pockets, and posts these as reasons for mutation-originated toxic effects. As the second leading cause of cancer in the United States, it is imperative to cure cancer. Define new subtyping strategies could aid in ensuring patients have access to appropriate treatments. Yet, this future may only be achieved through understanding patient-derived structures of cancer-related proteins through the field of structural oncology. I believe that my research will provide new avenues for further investigation, both structural and biochemical, that could aid in overcoming the myth of an undruggable p53.