Expression of cFLIPS: disulfure bridges and activity

master's thesis

• Author: Nikolina Trstenjak
• Mentor: Irena Landeka Jurčević (mentor)
• Granter: University of Zagreb; Faculty of Food Technology and Biotechnology; (Department of Food Quality Control); (Laboratory for Food Chemistry and Biochemistry); Zagreb
• Defense date and country: 2014-07-04, Croatia
• Scientific / art field, discipline and subdiscipline: BIOTECHNICAL SCIENCES; Food Technology

Abstract

c-FLIPS and FADD are two human proteins involved in apoptosis. Binding of c-FLIPS to FADD over DED domains prevents binding of caspase-8 and activation of apoptosis. As c-FLIPS has been found to be highly expressed in different tumor cells, it has become an interesting target for cancer treatments. Revealing the structure of c-FLIPS and understanding its molecular interaction with FADD could help in developing more specific and effective drugs. The aim of the project was to assess the hypothesis that c-FLIPS exists as a dimer in vitro, to determine the amino acids involved in that process and to develop a binding assay to monitor the activity of c-FLIPS. Mutations of cysteine 25 and 155 were successfully introduced and binding assay with FADD as a bait protein was developed. The results demonstrated that the active form in vitro is monomer, but it also exists as an inactive dimer and as an inactive form with an intramolecular disulfide bridge. Cysteines 25 and 155 are responsible for disulfide linkage formation. These results open a new perspective for the expression of c-FLIPS in vitro and they have to be considered when experiments and predictions are done with heterologously expressed protein.

Abstract (english)

Les protéines humaines c-FLIPS et FADD sont impliquées dans l‟apoptose : leur interaction via leur domaine DED empèche la fixation de la pro-caspase-8 et l‟activation de la mort cellulaire. Comme c-FLIPS est surexprimée dans différentes cellules tumorales, cette protéine est devenue une cible intéressante pour le traitement anti-cancer. Etudier la structure de c-FLIPS et comprendre son interaction moléculaire avec FADD sera d‟une grande aide pour développer des médicaments à plus haute spécificité et activité.
Le but de ce stage a été de valider l‟hypothèse que c-FLIPS existait sous forme de dimère in vitro, d‟identifier les acides aminés impliqués dans ce processus, et de développe un test d‟interaction pour mesurer l‟activité de c-FLIPS. La mutation des cystéines 25 et 155 a été réalisée, et le test d‟interaction de c-FLIPS utilisant FADD comme sonde a été développé. Les résultats ont montré que la forme active in vitro est un monomère, mais également que c-FLIPS existait sous forme de dimère et de monomère inactifs. Les cystéines 25 et 155 sont impliquées dans la formation des ponts disulfures des formes inactives (inter- et intra-
moléculaires, respectivement). Ces résultats ouvrent des perspectives quant à l‟utilisation de c-FLIPS sous forme recombinante dans des tests d‟activité.

• Keywords: apoptosis; c-FLIPS; FADD; dimerisation; disulfide linkage
• Keywords (english): apoptose; c-FLIPS; FADD; dimère; pont disulfure
• Language: english
• URN:NBN: urn:nbn:hr:159:232742
• Study programme: Title: Molecular Biotechnology; Study programme type: university; Study level: graduate; Academic / professional title: magistar/magistra inženjer/inženjerka molekularne biotehnologije (magistar/magistra inženjer/inženjerka molekularne biotehnologije)
• Type of resource: Text
• File origin: Born digital
• Access conditions: Access restricted to students and staff of home institution
• Created on: 2017-05-02 09:04:36