Inhibicija proteina toplinskog stresa Hsp90 pomoću male interferirajuće RNA

master's thesis

• Title (english): Inhibition of heat shock protein Hsp90 with small interfering RNA
• Author: Hrvoje Knežević
• Mentor: Jerka Dumić (mentor)
• Committee member: Jerka Dumić
• Committee member: Sandra Šupraha Goreta
• Committee member: Ana Mornar Turk
• Granter: University of Zagreb; Faculty of Pharmacy and Biochemistry; (Department of biochemistry and molecular biology); Zagreb
• Defense date and country: 2015-09-21, Croatia
• Scientific / art field, discipline and subdiscipline: BIOMEDICINE AND HEALTHCARE; Pharmacy; Pharmacy

Abstract

Proteini toplinskog stresa su ubikvintarna zaštitna skupina proteina iznimno važna u regulaciji brojnih staničnih funkcija. Pod tim uključujemo i posredno osiguravanje pravilnog smatanja i sprečavanja agregacije proteina ključnih u prelasku faza staničnog ciklusa iz jedne u drugu ulaskom u interakciju s njima sa ili bez potrošnje ATP-a. Proteini toplinskog stresa podijeljeni su po svojoj molekulskoj masi u brojne skupine, a za ovaj rad su najbitniji Hsp90α, Hsp90β, Hsp70 i Hsp27 koji spada u posebnu skupinu malih proteina toplinskog stresa. Proteine koje pravilno smata određena skupina proteina toplinskog stresa nazivamo njegovim proteinima klijentima. Slično kao i kod drugih Hsp proteina, proteini klijenti šaperona Hsp90 različiti su transkripcijski faktori i polimeraze, proteinske kinaze i skupina ostalih klijenata od kojih su svi veoma važni u radu kompleksne mreže unutarstanične signalizacije koja među ostalim kontrolira upalne procese, tumorigenezu, razvitak rezistencije na protutumorske lijekove, apoptozu, prelazak stanice kroz različite faze staničnog ciklusa, diferencijaciju itd. U kontroli staničnog ciklusa najznačajniji su nastanak kompleksa različitih proteina klijenata Hsp90, tzv. ciklin-ovisnih kinaza sa pripadajućim ciklinima. Samim time inhibitori Hsp90 proteina kao što je analog geldanamicina 17-DMAG ili siRNA molekule koje vežu sebi komplementarnu mRNA i onemogućavaju translaciju (tj. RNA interferencija) pokazali su obećavajući potencijal za uspješnu terapijsku primjenu. U ovom radu tretirane su stanice THP-1 stanične linije akutne monocitne leukemije siRNA molekulama te se pratila ekspresija proteina Hsp90α i β, Hsp70 i Hsp27 te aktivnost njihovih proteina klijenata Cdk1, Cdk2 i [pTyr15] Cdk1. Pokazalo se da je ekspresija Hsp90α izoforme jedina značajnije smanjena ovisno o vremenu za razliku od β izoforme čija je ekspresija neznatno smanjena. Ekspresija Hsp70 i Hsp27 je također nepromjenjena i skoro ne opada s vremenom. Ekspresija proteina klijenata Hsp90 je ostala istom što se može pripisati kompenzatornom mehanizmu stanice da kod inhibicije Hsp90 proteina drugi proteini toplinskog stresa preuzmu njegovu šaperonsku aktivnost i osiguraju pravilno smatanje njegovih proteina klijenata.

Abstract (english)

Heat shock proteins are ubiquitous group of protector proteins which are extremely important in regulation of many cellular functions. That includes indirect ensuring of proper folding and preventing aggregation of proteins critical in cell cycle transition from one phase to another by joining to interact with them, with or without ATP consumption. Heat stress proteins are divided by their molecular mass in a number of groups, and the ones discussed in this paper are Hsp90α, Hsp90β, Hsp70 and Hsp27, which belongs to a special group of small heat shock proteins. Proteins that are properly folded by a particular group of heat shock proteins are called its client proteins. Similar to other Hsp proteins, client proteins of Hsp90 chaperone are different kinds of transcription factors and polymerases, protein kinases and a group of other clients of which all are very important in the work of a complex network of intracellular signaling that among other things controls inflammation, tumorigenesis, development of resistance to anti-tumor drugs, apoptosis, cells transition through different phases of the cell cycle, differentiation and so on. In controling the cell cycle the most important is formation of complexes of Hsp90 client proteins called cyclin-dependent kinases with their appropriate cyclins. Therefore Hsp90 inhibitors such as geldanamycin analogue 17-DMAG or siRNA molecules that bind to the complementary mRNA and prevent translation (that is RNA interference) showed promising potential for a successful therapy. In this paper, THP-1 cell line of acute monocytic leukemia was treated with siRNA molecules and the expression of Hsp90α and β, Hsp70 and Hsp27 proteins and the activity of clients Cdk1, Cdk2 and [pTyr15] Cdk1 was observed afterwards . It has been shown that expression of only one isoform, Hsp90α, was significantly reduced depending on time as opposed to β isoform whose expression is insignificantly reduced. The expression of Hsp70 and Hsp27 was also unchanged and it barely decreases with time. The expression of the Hsp90 client proteins remained the same, which is attributable to a compensatory mechanism of cell to inhibition of Hsp90 proteins where other heat stress proteins take its chaperone activity and ensure proper folding of its client proteins.

• Keywords: Hsp90 proteini; siRNA molekule; RNA interferencija; 17-DMAG
• Keywords (english): Hsp90 proteins; siRNA molecules; RNA interference; 17-DMAG
• Language: croatian
• URN:NBN: urn:nbn:hr:163:262532
• Study programme: Title: Pharmacy; Study programme type: university; Study level: integrated undergraduate and graduate; Academic / professional title: magistar/magistra farmacije (magistar/magistra farmacije)
• Type of resource: Text
• File origin: Born digital
• Access conditions: Open access
• Created on: 2016-02-17 11:31:24