Abstract
Misfolding mutations in the DNA-binding domain of p53 alter its conformation, affecting the efficiency with which it binds to chromatin to regulate target gene expression and cell cycle checkpoint functions in many cancers, including glioblastoma. Small molecule drugs that recover misfolded p53 structure and function may improve chemotherapy by activating p53-mediated senescence. We constructed and optimized a split Renilla luciferase (RLUC) complementation molecular biosensor (NRLUC-p53-CRLUC) to determine small molecule-meditated folding changes in p53 protein. After initial evaluation of the biosensor in three different cells lines, we engineered endogenously p53P98L mutant (i.e. not affecting the DNA-binding domain) Ln229 glioblastoma cells, to express the biosensor containing one of four different p53 proteins: p53wt, p53Y220C, p53G245S and p53R282W. We evaluated the consequent phenotypic changes in these four variant cells as well as the parental cells after exposure to PhiKan083 and SCH529074, drugs previously reported to activate mutant p53 folding. Specifically, we measured induced RLUC complementation and consequent therapeutic response. Upon stable transduction with the p53 biosensors, we demonstrated that these originally p53P98L Ln229 cells had acquired p53 cellular phenotypes representative of each p53 protein expressed within the biosensor fusion protein. In these engineered variants we found a differential drug response when treated with doxorubicin and temozolomide, either independently or in combination with PhiKan083 or SCH529074. We thus developed a molecular imaging complementation biosensor that mimics endogenous p53 function for use in future applications to screen novel or repurposed drugs that counter the effects of misfolding mutations responsible for oncogenic structural changes in p53.
Highlights
Proteins catalyze, facilitate and regulate all the biological processes that occur in living systems [1]
Identification of a complementation biosensor based on split-Renilla luciferase as being optimal for measuring structural folding changes in p53 protein carrying structural mutations in its DNA-binding domain
We found significant complementation assisted luciferase signal from all constructs, but the biosensor containing Firefly luciferase (FLUC) fragments showed no significant difference between constructs, while the Renilla luciferase (RLUC) biosensor with a split site at residue 110 led to a low level of signal with wt-p53 compared to mutants (Supplementary Figure 3)
Summary
Facilitate and regulate all the biological processes that occur in living systems [1]. Human www.oncotarget.com cancers may arise as a phenotypic consequence of accumulated somatic mutations that result either from loss of a group of proteins, or, loss- or gain-of-function consequent to protein structure perturbations [5]. P53, the guardian of the genome, is considered an important tumor suppressor protein in cells; the functional loss of this protein in part or as a whole can create significant cellular changes leading to cancer. Drugs that stabilize p53 by preventing its ubiquitination or its interaction with human double minute 2 homolog (HDM2) protein is a possible approach to enhance chemosensitivity in cancers that are endogenously single allele deletion/mutant for p53 expression [7]. Approaches that could enable real-time noninvasive monitoring of this rescue process by detecting changes in protein folding within intact cells would be especially useful for identifying and validating new drugs that improve cancer therapy
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
