Priprava mutant proteina toplotnega šoka 90β in določitev vezavne afinitete zaviralcev s termoforezo na mikroskali

Mencin, Amadeja (author), Pečar Fonović, Urša (mentor), Cingl, Jernej (comentor)

Rakava obolenja sodijo med vodilne vzroke prezgodnje umrljivosti, pri čemer demografski trendi napovedujejo nadaljnje povečanje njihove pojavnosti. To poudarja potrebo po odkrivanju novih tarčnih učinkovin in potrjevanju že znanih na novih tarčah. Pri ohranjanju celične stabilnosti ima pomembno vlogo sistem za uravnavanje celične proteostaze, v katerem delujejo molekulski šaperoni, zlasti proteini toplotnega šoka. Eden najpomembnejših je protein Hsp90β, ki zaradi svoje vloge pri vzdrževanju tumorskega fenotipa predstavlja obetavno tarčo v razvoju novih protirakavih zdravil. Do sedaj so raziskave večinoma temeljile na razvoju zaviralcev N-končne domene Hsp90, vendar so zaradi toksičnosti in indukcije odziva na toplotni šok vse bolj v ospredju alternativne strategije, usmerjene na C-končno domeno. Ko-kristalna struktura kompleksa nekovalentnega zaviralca C-končne domene s Hsp90 še ni bila eksperimentalno določena, kar ovira nadaljnji razvoj. Kot ključno interakcijo za vezavo v predlaganem vezavnem žepu so identificirali ionsko interakcijo med bazičnim centrom spojine in aminokislinskim ostankom glutaminske kisline na mestu 489 v zaporedju proteina. Z mestnospecifično mutagenezo s prekrivajočima nukleotidoma smo vnesli dve mutaciji, s katerima smo spremenili omenjeno interakcijo. Za kloniranje smo uporabili ekspresijski plazmid pET28 z zapisom za heksahistidinsko oznako in sev E. coli TOP10 One ShotTM, za izražanje pa ekspresijski sev NiCo21 (DE3). Proteine smo izolirali in očistili na sistemu ÄKTA z uporabo kolon HisTrap in SEC, njihovo prisotnost in čistost pa potrdili s prenosom western. Afiniteto spojin (novobiocina in UL-Hsp90-1) do mutant Hsp90β smo ovrednotili z metodo termoforeze na mikroskali. Pri testiranjih mutant z novobiocinom smo potrdili afiniteto vezave v podobnem velikostnem razredu kot pri nemutiranem rekombinantnem Hsp90β, kar nakazuje, da mutaciji bistveno ne vplivata na vezavo novobiocina, oziroma da ostanek glutaminske kisline na 489. mestu ni ključen za interakcijo. V celotnem koncentracijskem območju spojine UL-Hsp90-1 pri nobeni od mutant nismo zaznali vezave na Hsp90β, zato predvidevamo, da ima spojina UL-Hsp90-1 drugačno vezavno mesto na Hsp90β kot novobiocin. Dokončna potrditev bi bila možna šele z določitvijo ko-kristalne strukture.

• Language: Slovenian
• Keywords: proteini toplotnega šoka, Hsp90β, mutageneza, zaviralci C-končne domene, termofereza na mikroskali
• Work type: Master's thesis/paper
• Typology: 2.09 - Master's Thesis
• Organization: FFA - Faculty of Pharmacy
• Year: 2025
• PID: 20.500.12556/RUL-176283
• COBISS.SI-ID: 258919427
• Publication date in RUL: 26.11.2025

Secondary language

• Language: English
• Title: Cloning of heat-shock protein 90β mutants and determination of inhibitor binding affinity by microscale thermophoresis

Abstract

Cancer is one of the leading causes of premature mortality, and demographic trends predict a further increase in its incidence. This highlights the need to discover new target molecules and to validate existing ones on novel targets. The cellular proteostasis network, in which molecular chaperones—particularly heat shock proteins—play a key role, is essential for maintaining cell stability. Among them, Hsp90β is one of the most important, as its involvement in sustaining the tumor phenotype makes it a promising target for the development of new anticancer drugs. To date, research has mainly focused on inhibitors of the N-terminal domain of Hsp90; however, due to their toxicity and induction of the heat shock response, alternative strategies targeting the C-terminal domain are gaining increasing attention. The co-crystal structure of a non-covalent C-terminal domain inhibitor complexed with Hsp90 has not yet been experimentally determined, which hinders further development. The key interaction within the proposed binding pocket has been identified as an ionic bond between the basic center of the compound and the glutamic acid residue at position 489 in the protein sequence. Using site-directed mutagenesis by overlap extension', two mutations were introduced to alter this interaction. Cloning was performed using the pET28 expression plasmid with a His-tag sequence and E. coli strain TOP10 One Shot™, while expression was carried out in the NiCo21 (DE3) strain. Proteins were isolated and purified on an ÄKTA system using HisTrap and SEC columns, and their presence and purity were confirmed by western blotting. The affinities of novobiocin and UL-Hsp90-1 for mutant Hsp90β were evaluated using microscale thermophoresis (MST). Binding assays with novobiocin confirmed affinities in a similar range to those observed for recombinant wild-type Hsp90β, indicating that the mutations do not significantly affect novobiocin binding and that the E489 residue is not crucial for the interaction. Across the entire concentration range of UL-Hsp90-1, no binding to Hsp90β mutants was detected, suggesting that UL-Hsp90-1 interacts with a different binding site on Hsp90β than novobiocin. Definitive confirmation would require determination of the corresponding co-crystal structure.

• Keywords: heat shock proteins, Hsp90β, mutagenesis, C-terminal domain inhibitors, microscale thermophoresis