Sinteznobiološka orodja v podporo ultrazvočni stimulaciji celic

Varda, Nina (author), Benčina, Mojca (mentor), Gaber, Aljaž (comentor)

Sposobnost ultrazvoka, da neinvazivno prenese mehanske sile do celic, predstavlja atraktivno možnost nadzorovanja celic na daljavo, kar ima izjemen potencial za aplikacije v medicini in biotehnologiji. Velik izziv pri razvoju takšne stimulacije je učinkovita sklopitev mehanskih dražljajev s specifično aktivacijo procesov v celici. Slednja ob ultrazvočni stimulaciji lahko poteka preko vdora kalcijevih ionov v celico. To povzroči aktivacijo fosfataze kalcinevrin, ki defosforilira jedrni dejavnik aktiviranih T-celic (NFAT). Translokacija transkripcijskega dejavnika v jedro aktivira izražanje genov, ki so pod nadzorom transkripcijskega dejavnika NFAT. Sidranje sintetičnega transkripcijskega dejavnika na osnovi regulatorne domene NFAT s sidrnim peptidom KRϕ na membrano pomembno zmanjša aktivacijo transkripcije tarčnih genov v celičnem mirovanju. Analiza je pokazala nepopolno translokacijo transkripcijskega dejavnika v jedro ob aktivaciji, kar pripisujemo močnejši vezavi sidrnega peptida na membrano. V nalogi smo preverili, ali lahko s točkovnimi mutacijami v peptidu KRϕ izboljšamo razmerje aktivacije od NFAT odvisne regulatorne podenote v mirovanju in vzbujenem stanju. Sidranja peptida KRϕ in mutant na membrano smo spremljali z lokalizacijo in aktivacijo od NFAT odvisne regulatorne podenote transkripcijskega dejavnika povezane s peptidom KRϕ. Pripravili smo 16 mutant peptida KRϕ s spremenjenim nabojem in hidrofobnostjo. Peptid z nabojem 9+ in s spremembo F11L je zagotavljal nizko izražanje luciferaze v nestimuliranih celicah in visoko razmerje med izražanjem luciferaze v stimuliranih in nestimuliranih celicah. V drugem delu naloge smo pripravili od kalcija odvisno stikalo za spremljanje delovanja ultrazvočnega stimuliranja celic. To je temeljilo na dveh antiparalelnih obvitih vijačnicah, povezanih z zaporedjem, ki izvira iz motivov EF-dlani, ki ob spremembi koncentracije kalcija spremeni konformacijo. Na koncih vijačnic sta se nahajala segmenta razdeljenega reporterskega proteina. Odzivnost na spremembo koncentracije kalcijevih ionov smo uspeli zaznati z uporabo razdeljene luciferaze ne pa tudi z razdeljenim fluorescirajočim proteinom.

• Language: Slovenian
• Keywords: kalcijeva signalizacija, sidrni peptid KRϕ, od kalcija odvisno stikalo
• Work type: Master's thesis/paper
• Typology: 2.09 - Master's Thesis
• Organization: FKKT - Faculty of Chemistry and Chemical Technology
• Year: 2023
• PID: 20.500.12556/RUL-147396
• COBISS.SI-ID: 160206851
• Publication date in RUL: 04.07.2023

Secondary language

• Language: English
• Title: Synthetic biological tools to support ultrasound stimulation of cells

Abstract

The ability of ultrasound to noninvasively transmit mechanical forces to cells presents an attractive opportunity for controlling cells remotely, which has a remarkable potential for applications in medicine and biotechnology. A major challenge in the development of such stimulation is the efficient coupling of mechanical stimuli with specific activation of processes in the cell. The latter can take place through the influx of calcium ions into the cell. This results in the activation of the phosphatase calcineurin, which dephosphorylates the nuclear factor of activated T-cells (NFAT). Translocation of the transcription factor into the nucleus activates the expression of genes under the control of NFAT transcription factor. Anchoring of a synthetic transcription factor based on the regulatory domain of NFAT with the anchoring peptide KRϕ to the membrane significantly reduces the activation of the transcription of target genes during cellular rest. The analysis showed incomplete translocation of the transcription factor to the nucleus upon activation, which we attribute to stronger binding of the anchoring peptide to the membrane. In this work, we examined whether point mutations in the KRϕ peptide can improve the ratio of activation of the NFAT-dependent regulatory subunit in resting and excited states. Membrane anchoring of the KRϕ peptide and mutants was monitored by localization and activation of the NFAT-dependent regulatory subunit of the transcription factor associated with the KRϕ peptide. We prepared 16 mutants of the KRϕ peptide with altered charge and hydrophobicity. The peptide with 9+ charge ad F11L mutation provided low luciferase expression in unstimulated cells and a high ratio of luciferase expression in stimulated to unstimulated cells. We also prepared a calcium-dependent switch for monitoring the action of ultrasound stimulation of cells. The switch on two antiparallel coiled helices linked by a sequence originating from EF-hand motifs that change conformation when the calcium concentration changes. Segments of the split reporter protein were located at the ends of the helices. We were able to detect the response to the change in the concentration of calcium ions using split luciferase, but not with split fluorescent protein.

• Keywords: calcium signalization, KRϕ anchor peptide, calcium-dependent switch