Načrtovanje fosforilacijske signalne poti v sesalskih celicah

Kodila, Anja (avtor), Lunder, Mojca (mentor), Jerala, Roman (komentor)

Eden izmed pomembnih ciljev sintezne biologije je vnos novih signalnih poti, kot je primer imunoterapije s CAR-T celicami za zdravljenje raka. Zaradi potrebe po hitrejših celičnih odzivih na vhodni signal, se sintezna biologija vedno bolj osredotoča na uravnavanje bioloških sistemov na posttranslacijskem nivoju, kot sta proteoliza in fosforilacija. V magistrskem delu smo načrtovali koncept uravnavanja aktivnosti tarčnega proteina s fosforilacijo. V ta namen smo v poročevalski protein kresničkina luciferaza vstavili zaporedje za SH2 vezavno domeno ter substratni peptid, ki ga ustrezna kinaza prepoznava in fosforilira. Z opisano manipulacijo smo dosegli, da je tarčni protein v osnovnem stanju neaktiven, fosforilacija le-tega z ustrezno kinazo pa mu aktivnost povrne. Za preverjanje najbolj optimalnega razmerja med neaktivnim in aktivnim stanjem kresničkine luciferaze smo pripravili serijo konstruktov, ki so se med seboj razlikovali po drugačnih pozicijah vstavljenih fosfopeptidnih domen in substratnih peptidov, ter različnih dolžinah povezovalnih segmentov. Načrtovali smo dva enomolekulska konstrukta; razcepljeno luciferazo z vstavljeno fosfopeptidno domeno in substratnim peptidom med N in C koncem in razcepljeno luciferazo z vstavljenim substratnim peptidom med N in C koncem, ter fosfopeptidno vezavno domeno vezano na C konec s fleksibilnim povezovalnikom. Uspešnost koncepta smo preverjali s kinazama Lyn in Lck, ki sta članici družine Src nereceptorskih tirozinskih kinaz in sta zaradi svoje vloge v patoloških procesih privlačni terapevtski tarči. V magistrskem delu smo z načrtovanjem od fosforilacije odvisne aktivnosti tarčnega proteina zasnovali temelje za oblikovanje sintetične fosforilacijske kaskade v sesalskih celicah, katera ima dve poglavitni prednosti, in sicer (i) hiter odziv na signale iz okolja ter (ii) reverzibilnost. Sistem je zasnovan modularno, to pomeni, da je razširljiv in omogoča načrtovanje logičnih funkcij, fosforilacijskih kaskad ter pozitivnih in negativnih povratnih zank.

• Jezik: Slovenski jezik
• Ključne besede: fosforilacija, razcepljena kresničkina luciferaza, kinaza Lyn, kinaza Lck, uravnavanje na posttranslacijskem nivoju
• Vrsta gradiva: Magistrsko delo/naloga
• Organizacija: FFA - Fakulteta za farmacijo
• Leto izida: 2021
• PID: 20.500.12556/RUL-127515
• Datum objave v RUL: 11.06.2021

Sekundarni jezik

• Jezik: Angleški jezik
• Naslov: Design of phosphorylation signaling pathways in mammalian cells

Izvleček

One of the important goals of synthetic biology is to introduce new signalling pathways, such as CAR-T cancer immunotherapy. Due to the need for faster cellular responses to the input signals, synthetic biology is increasing its focus on regulating biological systems at the posttranslational level, such as proteolysis and phosphorylation. In the master's thesis, we constructed the concept of regulating the activity of the target protein through phosphorylation. For this purpose, we inserted into the firefly luciferase reporter protein the sequence for the SH2 binding domain and a substrate peptide that is recognised and phosphorylated by the corresponding kinase. The manipulation described above results in the target protein being inactive in its native state and its activity can be restored by phosphorylation with the appropriate kinase. To establish the most optimal relationship between the inactive and active state of firefly luciferase, we prepared a series of constructs with different linker lengths and different positions of inserted phospho-peptide binding domains and substrate peptides. We designed two one-molecular constructs; a split luciferase with an inserted phospho-peptide binding domain and a substrate peptide between N- and C-terminus and a split luciferase with an inserted substrate peptide between N- and C-terminus, and a phospho-peptide binding domain attached to the C-terminus with a flexible linker. We tested the concept with Lyn and Lck kinases, which are members of the Src family of non-receptor tyrosine kinases and are attractive therapeutic targets due to their role in pathological processes. In this thesis, by designing the phosphorylation-dependent activity of the target protein, we have laid the foundations for the design of a synthetic phosphorylation cascade in mammalian cells, which has two major advantages, namely (i) rapid response to environmental signals and (ii) reversibility. The system is designed in a modular way, i.e. it is scalable and allows the design of logic functions, phosphorylation cascades and positive and negative feedback loops.

• Ključne besede: phosphorylation, split firefly luciferase, Lyn kinase, Lck kinase, post-translational regulation