Exploring Nanobodies Interrupting the Interaction between p53 and MDM4

Sanem SARIYAR
Molecular Biology, Genetics and Bioengineering, MSc Thesis, July 2019

Thesis Jury

Prof. Batu Erman (Thesis Advisor), Assoc. Prof. Christopher Mayack,     

Assoc. Prof. Nazlı Keskin

Date & Time: 17 th July 2019 –  11 AM

Place: FENS G032

Keywords : Keywords: p53/MDM4 interaction, nanobodies, nanobody purification,

fluorescent two-hybrid assay, surface plasmon resonance

Abstract

p53 protein is considered as the guardian of the genome thanks to its important tumor suppressor roles such as cell-cycle arrest, apoptosis and senescence. Because these roles are extremely vital, this p53 pathway should be strictly regulated. During unstressed conditions, p53 levels are kept in control by both ubiquitination of p53 protein and inhibition of its transcriptional activity via MDM2 and MDM4 proteins, respectively. Although MDM2 is the main modulator of p53 activity, there is a collaboration between MDM2 and MDM4 proteins to enable the control of p53 so MDM4 protein is as important as MDM2 protein in this mechanism. In human cancers, there is either mutation in p53 or overexpression of its negative regulators, due to this, targeting p53-MDM2-MDM4 interplay is one of the main aims of cancer therapeutics. Also, in some cancers, where there is overexpression of negative regulators, the use of inhibitors for only MDM2 is not enough to activate p53 protein. For this reason, exploring inhibitors for MDM4 become important. In this study, we aimed to optimize purification of in silico designed nanobodies against MDM4/ p53 interaction and test their binding via surface plasmon resonance (SPR) and Fluorescent Two- Hybrid (F2H) assay.