Browsing by Author "Panfilova, Aleksandra"

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    Cln2-based phosphodegron tags for the regulation of protein stability
    (Tartu Ülikool, 2019) Panfilova, Aleksandra; Shtaida, Nastassia, supervisor; Kivi, Rait, supervisor
    In English: There are several levels of the regulation of protein expression. Protein phosphorylation is a common way of signal transduction in cell signaling pathways and it provides a fast response. Some proteins possess so-called phosphodegron sequences, which being phosphorylated send protein for destruction. In this study, phosphodegron from Saccharomyces cerevisiae cyclin Cln2 was used to generate a series of protein tags to affect EGFP fluorescence level. As it was shown by FACS, a fusion of EGFP with degron tags containing either 4 or 5 phosphorylation sites, resulted in 30% decrease in EGFP fluorescence, without significant difference between 4 and 5 phosphosites. Surprisingly, the addition of Cln2 docking motif from Sic1 protein to the degron led to increasing of EGFP fluorescence, in comparison to alanine mutant or non-tagged control. These results require further investigation. In comparison to the fast response from protein phosphorylation, regulation of protein expression at the promoter level is slow. Combination of an inducible promoter, which activity can be tightly regulated, with phosphodegron tag can provide additional control lever for fine-tuning of protein expression. To do that, yeast strains carrying EGFP under control of inducible pLexAt promoter was created using CRISPR/Cas9-mediated transformation. These strains will be further used in the laboratory for tagging with phosphodegrons. Eesti keeles: Rakus kontrollitakse valgutasemeid mitmel erineval moel. Signaaliülekandes on levinud meetod valkude fosforüülimine, mis võimaldab väga kiiret vastust keskkonnas toimunud muutustele. Mõnedes valkudes on olemas niiöelda fosfodegronid – valgujärjestused, mille fosforüülimise tagajärjel suunatakse valk lagundamisele. Antud töös kasutati S. cerevisiae Cln2 valgust pärineva fosfodegroni erinevaid mutante, mis seoti GFP külge ja jälgiti, kuidas GFP fluorestsents selle tagajärjel muutub. FACSiga mõõtmised demonstreerisid, et 4 või 5 fosfosaidiga degronid vähendasid GFP fluorestsentsi 30%. Üllatava tulemuseni viis Sic1 valgust pärineva Cln2 seondumissaidi lisamine degroni järjestusele, mille tagajärjel võis näha valgu degradatsiooniefektiivsuse vähenemist. Saadud tulemused väärivad edasist uurimistööd. Võrreldes valkude fosforüülimisega on promootori tasemel valguekspressiooni kontroll suhteliselt aeglane. Kombineerides aga valguekspressiooni indutseeritava promootori alt fosfodegronitega, saame tekitada lisakontrolli valguekpressiooni väga täpsel reguleerimisel. Sellise süsteemi loomiseks viidi CrispR/Cas9 meetodi abil pärmi genoomi eGFP koos indutseeritava pLexA promootoriga. Saadud tüve kasutatakse edasiste katsete käigus, kus plaanitakse GFP-d fosfodegronitega modifitseerida.
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    Optimization of Sic1-Cln2-based phosphodegron tag for inducible protein degradation
    (Tartu Ülikool, 2021) Panfilova, Aleksandra; Tartu Ülikool. Loodus- ja täppisteaduste valdkond; Tartu Ülikool. Tehnoloogiainstituut
    Precise regulation of protein abundance in the cell would be instrumental in both research and industry. A variety of tools exists for the regulation of protein expression at the transcriptional level. However, there is a shortage of tools that would allow control over already expressed proteins. In this study, we exploit the native cell cycle regulation system of Saccharomyces cerevisiae to develop a system for targeted inducible protein degradation. Using sequence elements that modulate cyclin-dependent kinase (CDK) substrate specificity, we generated a set of phosphodegron tags that have diverse effects on degradation rates of the tagged proteins. The control over the system is provided by conditional expression of the stabilized version of cyclin Clb3, which drives CDK-dependent phosphorylation of the tag, and F-box protein Grr1, which recognizes phosphorylated tag and label it for further degradation. In the course of this work, we have designed a set of phosphodegron tags that can be used for the regulation of the protein abundance in the cell and its maintenance at desirable levels that can be useful for the industrial production of value-added products. In addition, we discovered that Cks1 priming effect on the multisite phosphorylation is not only distance-, but, likely, also context-dependent which might be of interest for fundamental research.