Investigation of protein-protein interactions involving Retinoblastoma Binding Protein 6 using immunoprecipitation and Nuclear Magnetic Resonance Spectroscopy
Year: 2019
Type: dissertation
Abstract: Retinoblastoma Binding Protein 6 (RBBP6) is a 200 KDa multi-domain protein that has been shown to play a role in mRNA processing, cell cycle arrest and apoptosis.RBBP6 interacts with tumour suppressor proteins such as p53 and pRb and has been shown cooperate with Murine Double Minute 2 (MDM2) protein in catalyzing ubiquitination and suppression of p53.Unpublished data from our laboratory has suggested that RBBP6 and MDM2 interact with each other through their RING finger domains.RBBP6 has also been shown to have its own E3 ubiquitin ligase activity, catalyzing ubiquitination of Y-Box Binding Protein 1 (YB-1) in vitro and in vivo.YB-1 is a multifunctional oncogenic protein that is generally associated with poor prognosis in cancer, tumourigenesis, metastasis and chemotherapeutic resistance.Unpublished data from our laboratory shows that RBBP6 catalyzes poly-ubiquitination of YB-1, using Ubiquitin-conjugating enzyme H1 (UbcH1) as E2 ubiquitin conjugating enzyme.We have furthermore shown that the zinc knuckle of RBBP6 interacts specifically with the Ubiquitin-associated domain (UBA) domain of UbcH1.This thesis reports investigations of two interactions between the RING finger domains of RBBP6 and MDM2 in Chapter 3, and between the zinc finger of RBBP6 and the UBA domain of UbcH1 in Chapter 4. In each case, the previously-reported interaction is first confirmed using GST pulldown assay.Then 15 N-enriched samples of the proteins are prepared and used in Nuclear Magnetic Resonance (NMR) spectroscopy-based chemical shift perturbation assays.The aims of these assays are, firstly, to validate the interaction by determining whether addition of the interacting partner perturbs the 15 N, 1 H-HSQC spectrum of the protein.The second aim is to identify resonances most affected by the interaction, and thereby identify amino acids in the proteins close to the interaction interface, which would serve as useful targets for future mutagenesis studies.GST pull-down studies showed preliminary evidence of the interaction between the RING finger domains of RBBP6 and MDM2.A 15 N-enriched sample of RBBP6-RING was produced in bacteria and a chemical shift perturbation assay carried out using the 500 MHz spectrometer at the University of the Western Cape (UWC).Definite changes were observed on addition of unlabeled MDM2-RING.However, interpretation of the changes was hampered by the previously-reported dilution-dependent changes to the spectrum of RBBP6-RING, resulting from changes to the monomer-homodimer equilibrium.Although there is evidence of changes in addition to the dilution dependent effects, definitive conclusions cannot be reached.The assay will have to repeated, taking steps to minimize the dilution dependent effects.GST pull-down studies also showed preliminary evidence of the interaction between the zinc finger of RBBP6 and the UBA domain from UbcH1.A number of 15 N-enriched samples of UBA were produced in bacteria and three different chemical shift perturbation assays were carried out, using the 500 MHz spectrometer at UWC and the 700 MHz spectrometer in the United Kingdom (UK) equipped with a cryoprobe.The different 15 N, 1 H-HSQC spectra of UBA showed that the domain exists in two different configurations: one showing good spectral dispersion characteristic of a fully folded configuration, the other showing reduced dispersion characteristic of a partially folded configuration.Two separate isoforms for the RBBP6 zinc finger were cloned and expressed; a longer construct which was expected to be partially unstructured, and a shorter construct, centered around the zinc knuckle domain, which was expected to be almost completely structured.However, chemical shift perturbation assays showed that both isoforms stabilized the partially unfolded form of UBA, leading to decay of the spectrum corresponding to the fully folded form, and growth of the spectrum corresponding to the partially unfolded form.This conclusion was supported by the fact that the NMR sample precipitated partially at stoichiometric ratios of 1:1 and higher ([UBA]: [zinc]).The possibility that the UBA unfolded spontaneously, due to the long periods spent in the NMR spectrometer at 25 o C, which was necessitated due to the low sensitivity of the spectrometer at UWC; was addressed by repeating the assay within the course of a single day, using a spectrometer equipped with a cryoprobe at King's College London, thereby negating this possibility.
Author: Po‐An Chen
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: green
OpenAlex ID: W3130682390