Tehnoloogiainstituut

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Browsing Tehnoloogiainstituut by Author "Agerova, Alissa"

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    Cell cycle independent signaling by modified Clb5- Cdk1 complex
    (Tartu Ülikool, 2021) Agerova, Alissa; Tartu Ülikool. Loodus- ja täppisteaduste valdkond; Tartu Ülikool. Tehnoloogiainstituut
    A eukaryotic cell contains a robust regulatory network that controls the ordered sequence and timing of distinct cell cycle events. In budding yeast this is mainly regulated by the growing activity of cyclin-dependent kinases (Cdks) during the cell cycle. Post-translational modifications, particularly protein phosphorylation is a powerful mechanism regulating stability, localization, activity and interactions of proteins. Building synthetic circuits based on protein phosphorylation has remained a riddle due to the overlapping specificity of protein kinases. To tackle this issue it was aimed to obtain a cell cycle independent cyclin-Cdk1 input that could target substrates created specifically for this particular cyclin-Cdk1 complex. By manipulating the Cdk1 specificity of phospho-degron and -localization modules it was anticipated to design orthogonal Cdk circuits that respond to the modified Clb5-Cdk1 complex.
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    Design of Clb5-specific Cdk1 substrates for cell cycle independent signalling
    (Tartu Ülikool, 2019) Agerova, Alissa; Örd, Mihkel, supervisor
    In English: Cyclin-dependent kinases (Cdks) are master regulators of the cell cycle with Cdk1 driving most cell cycle events in budding yeast. Phosphorylation enables the regulation of protein stability, localization and interactions, thus, the ability to predictably program phosphorylation regulation could have a strong impact on the design of synthetic pathways. Cdk1 has a complex, but well-studied substrate targeting mechanisms, making it potentially suitable for such applications. In this work a minimal phosphodegron module was created to show, that phosphorylation efficiency can be manipulated. By inducing Clb5 it was possible to enhance phosphorylation, leading to degradation of the phospho-degron modules. Also it was found that the activity of Clb5 can be targeted specifically to the degron module by addition of a cyclin docking motif. Eesti keeles: Tsükliinist sõltuvad kinaasid (Cdk-d) on rakutsükli peamised regulaatorid, mille hulgast vastutab Cdk1 enamiku rakutsükli sündmuste läbi viimise eest. Fosforüleerimine reguleerib valkude stabiilsust, lokalisatsiooni ja interaktsioone, seega, võime programmeerida fosforüleerimist võib avaldada suurt mõju sünteetilisele bioloogiale. Kinaasil Cdk1 on mitmekesine ning hästi uuritud substraatide äratundmismehhanismid, võimaldades kasutamist sünteetilise bioloogia rakendustes. Antud töös loodi minimaalse fosfodegroni moodul ning näidati, kuidas on fosforüleerimise efektiivsusega võimalik manipuleerida. Tsükliini Clb5 indutseerides suurendati fosforüleerimist, millest tulenevalt suurenes ka fosfodegroni moodulite lagundamine. Lisaks leiti, et Clb5 aktiivsust saab suunata degroni mooduli juurde, lisades tsükliinspetsiifilise seondumismotiivi.