Structural characterisation of the p53 binding domain of RBBP6 and its mechanism of interaction with p53

Abstract

Worldwide, cancer is a major health issue, causing millions of deaths every year. Retinoblastoma binding protein 6 (RBBP6) is thought to facilitate the interaction between p53 and its prototypical negative regulator, MDM2. RBBP6 has been found to be overexpressed in several cancers and its ability to interact with p53 and cause its degradation, makes it a prospective biomarker and drug target in cancer therapy. In order to target this interaction, a better understating of the p53 binding domain of RBBP6 is needed. In this thesis, expression trials were undertaken for the recombinant p53 binding domain of RBBP6 (namely RBBP6 p53BD) under several conditions. These included different bacterial cell lines, inducer concentrations and post-induction growth time, as well as the domain with and without a polyhistidine tag. The best expression conditions found were NiCo21 (DE3) cells, 37 °C, 0.1 M IPTG and 16 hours post-induction growth. A three-step purification protocol is presented for the polyhistidine-tagged RBBP6 p53BD, utilising hydrophobic interaction and nickel IMAC chromatography. RBBP6 p53BD was purified to approximately 95 % homogeneity. The purified recombinant domain was shown to have structure and be functional as it could bind endogenous p53. The domain was characterised using clear native PAGE and far-UV CD and was found to exist in a single monomer form. Its secondary structure was predicted to be largely intrinsically disordered with 59 % random coil, 19 % alpha-helices, 9 % beta-strands, and 13 % turns. The stability of the domain was also investigated using far-UV CD when the protein was exposed to increasing temperature or known denaturants. The spectrum produced by RBBP6 p53BD with increasing temperature showed a gain in secondary structure and high percentage recovery of its native state after returning to starting temperature. Greater structural changes were seen in the presence of denaturants, with the greatest structural change and lowest percentage recovery seen in the presence of guanidinium chloride. High purity and stability are important for future investigations into the structure of RBBP6 p53BD and drug interaction studies. Localisation images produced from immunocytochemistry experiments are presented here that show different subcellular localisation of RBBP6 and p53 in the cancer cell lines A549, MCF7 and MDA-MB-231 and compared to the normal cell line, HEK293 T. With normal cells having the highest localisation of RBBP6 in the nucleus and negligible localisation in the cytoplasm. Cancer cells still showed the high localisation in the nucleus but higher localisation in the cytoplasm than normal cells. Colocalisation images of p53 and RBBP6 in cancer cell lines are also presented, indicating that p53 and RBBP6 localise in similar cell regions. Using the plugin JACoP from ImageJ, the Manders coefficient of p53 to RBBP6 was predicted to be 0.761 in MCF7 cells and 0.784 in A549 cells. The Manders coefficient of RBBP6 to p53 was found to be 0.904 for MCF7 and 0.834 for A549 cells. The Pearson’s coefficient was found to be 0.759 for MCF7 and 0.630 for A549 cells. This Indicates positive colocalisation of p53 and RBBP6 in both MCF7 and A549 cells, suggesting that these proteins could interact in vivo. Co-immunoprecipitation assays were performed and showed the ability of endogenous p53, RBBP6 and MDM2 to interact and form a complex in vitro in cancer and normal cell lines. Thus supporting RBBP6 as a facilitator to p53 and MDM2’s interaction in human cells as well as supporting RBBP6 as a potential cancer therapy drug target.

Kanker is wêreldwyd 'n groot gesondheidskwessie, wat jaarliks miljoene sterftes veroorsaak. Daar is gevind dat retinoblastoombindende proteïen 6 (RBBP6) ooruitgedruk word in verskeie kankers. Daar word vermoed dat RBBP6 die interaksie tussen p53 en sy belangrikste negatiewe reguleerder, MDM2, fasiliteer. RBBP6 se vermoë om met p53 te reageer en die agteruitgang daarvan te veroorsaak, maak dit 'n voornemende biomerker en geneesmiddelteiken in kankerterapie. Eerstens, in hierdie tesis, is uitdrukkingsproewe onderneem vir die rekombinante p53-bindingsdomein van RBBP6 (naamlik RBBP6 p53BD) onder verskeie toestande. Bevindinge toon dat die beste uitdrukkingstoestande was die domein met 'n polihistidienmerker in NiCo21 (DE3) selle, 37 °C, 0.1 M IPTG en 16 uur na-induksie groei. 'n Drie-stap suiweringsprotokol word aangebied vir die polihistidien-gemerkte RBBP6 p53BD, deur gebruik te maak van hidrofobiese interaksie en nikkel IMAC chromatografie. Daar is getoon dat die gesuiwerde rekombinante domein struktuur het en funksioneel is aangesien dit endogene p53 kon bind. Die domein is gekarakteriseer deur gebruik te maak van clear native-PAGE en ver-UV circular dichroism daar is gevind dat dit in 'n enkele monomeervorm bestaan en 'n groot hoeveelheid intrinsieke versteuring bevat. Die stabiliteit van die domein is ook ondersoek deur gebruik te maak van ver-UV CD wanneer die proteïen aan toenemende temperatuur of bekende denaturante blootgestel is, en daar is gevind dat dit relatief min verandering in struktuur en 'n goeie hoeveelheid herstel onder alle toestande het. Hoë suiwerheid en stabiliteit is belangrik vir toekomstige ondersoeke rondom die struktuur van RBBP6 p53BD en geneesmiddelinteraksiestudies. Tweedens word lokaliseringsbeelde wat uit immunositochemie-eksperimente geproduseer word hier aangebied wat verskillende uitdrukkingsvlakke van RBBP6 en p53 in die kanker- en normale sellyne toon. Wanneer die kolokaliseringsbeelde bestudeer word, het die gegenereerde Manders en Pearson se koëffisiënte gedui op positiewe kolokalisering tussen p53 en RBBP6 in beide MCF7 en A549 selle, wat daarop dui dat hierdie proteïene in vivo interaksie kan hê. Ko-immunopresipitasietoetse is uitgevoer en dit het die vermoë van interaksie tussen endogene p53, RBBP6 en MDM2 getoon en om 'n kompleks in vitro te vorm in kanker en normale sellyne. Dit ondersteun dus RBBP6 as 'n fasiliteerder in p53 en MDM2 se interaksie in menslike selle, asook RBBP6 as 'n potensiële kankerterapie-teiken.

Emhlabeni wonke, umdlavuza uyinkinga enkulu yezempilo, ebulala izigidi zabantu minyaka yonke. I-Retinoblastoma ebopha amaprotheni 6 (RBBP6) kutholakale ukuthi iningi kakhulu kumangqamuzana omdlavuza. I-RBBP6 kucatshangwa ukuthi yenza kube lula ukusebenzisana phakathi kwe-p53 nesilawuli sayo esikhulu i-MDM2. Ikhono le-RBBP6 lokusebenzelana ne-p53 futhi libangele ukuwohloka kwayo liyenza iRBBP6 ibe i-biomarker kanye nokusetshenziswa yemithi ekwelapheni umdlavuza eqondiswe kuyo. Okokuqala, kule thesis, ngiqale ngezivivinyo zokukhiqiza esibophezelayo se-p53 se-RBBP6 (okungukuthi i-RBBP6 p53BD) ngaphansi kwezimo ezimbalwa. Izimo ezinhle kakhulu zokukhiqhiza iRBBP6 p53BD ezitholiwe kwakuyisizinda esinomaka we-polyhistidine kumangqamuzana e-NiCo21 (DE3), ezingeni lokushisa i-37 °C, kanye ne 0.1 M IPTG kumahora angu-16 okukhula kumangqamuzana. Iphrothokholi yokukhishwa kwe-RBBP6 p53BD okulandela ukukhiqhizwa kwayo enezinyathelo ezintathu, kusetshenziswa ukusebenzisana kwe-hydrophobic kanye ne-nickel IMAC chromatography. I-RBBP6 p53BD ekhiqhiziwe ibonakale inesakhiwo futhi siyasebenza njengoba singahlanganisa ne-p53 etholakala kumangqamuzana. Ukuzinza kwe-RBBP6 p53BD ekhiqhiziwe kuphenyisiswe nge-PAGE kanye ne-CD ye-UV ekude futhi kwatholakala ukuthi ikhona kufomu elilodwa le-monomer futhi iqukethe inani elikhulu lokuphazamiseka kwangaphakathi. Ukuzinza kwesizinda (i-RBBP6 p53 BD) kuphinde kwaphenywa kusetshenziswa i-CD ye-UV ekude kwatholakala ukuthi kunoshintsho oluncane kwisakhiwo se-RBBP6 p53BD ezingeni lokushisa elikhulayo noma ama-denaturants aziwayo ahloliwe. Ukuhlanzeka okuphezulu nokuzinza kubalulekile ophenyweni oluzayo mayelana nesakhiwo se-RBBP6 p53BD kanye nezifundo zokusebenzisana nemithi. Okwesibili, izithombe ezibonisa amazinga ahlukene we-RBBP6 kanye ne-p53 kumdlavuza kanye kumangqamuzana avamile zikhiqizwe ekuhlolweni kwe-immunocytochemistry. Lapho utadisha izithombe ze-colocalisation ama-coefficients ka-Manders kanye ne-Pearson akhiqizwa abonisa ukuhlangana okuhle phakathi kwe-p53 ne-RBBP6 kuwo womabili amangqamuzana omdlavuza e-MCF7 kanye ne-A549, okuphakamisa ukuthi lawa maprotheni angasebenzisana emzimbeni. Ukuhlolwa kwe-Co-immunoprecipitation kwenziwa futhi babonisa ikhono le-endogenous p53, i-RBBP6 ne-MDM2 ukusebenzisana nokwenza i-complex kumangqamuzana omdlavuza kanye navamile. Ngakho-ke imiphumela isekela i-RBBP6 njengomgqugquzeli ekusebenzisaneni kwe-p53 ne-MDM2 kumangqamuzana omuntu kanye nokusekela i-RBBP6 njengethagethi yemithi esingaba khona yokwelapha umdlavuza.