Sirvi Autor "Faustova, Ilona, juhendaja" järgi

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Analysis of different substrate docking pockets on Clb5- and Clb4-Cdk1
(Tartu Ülikool, 2023) Kiselev, Viacheslav; Faustova, Ilona, juhendaja; Örd, Mihkel, juhendaja; Loog, Mart, juhendaja; Tartu Ülikool. Loodus- ja täppisteaduste valdkond; Tartu Ülikool. Tehnoloogiainstituut
Cell cycle is coordinated via temporally resolved protein phosphorylation by cyclin-dependent kinases (CDKs). CDKs form cell-cycle-stage-specific complexes with cyclins. Cyclins in turn recruit substrate proteins through specific binding motifs and direct the kinase to phosphorylate different proteins to trigger cell cycle events. While most of the cyclin-substrate interactions take place via a hydrophobic patch on the cyclin, recent studies have found novel pockets that could participate in substrate recognition. This work investigates the im- portance of two novel substrate binding pockets on Saccharomyces cerevisiae S and G2 phase cyclins Clb5 and Clb4. Structural and conservational analysis supported the presence of multiple substrate pockets on the cyclin surface. Kinase assays revealed that the phosphate-binding pocket and an NPFF motif pocket are critical in mediating phosphorylation of Kar9 by Clb4-Cdk1 complex. A comparison between Clb4-, Clb3-, and Clb5-Cdk1 revealed distinct substrate preferences for these complexes within a panel of 10 Cdk1 substrates of S/G2 phase. Further, assays using mutant cyclin-Cdk1 complexes suggested that the NPFF pocket is used by a small number of substrates. Overall, these findings highlight the im- portance of cyclins as substrate recruitment modules rather than just activators of CDKs.
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Cdk1-regulated diphosphodegron tags for controlled protein expression
(Tartu Ülikool, 2023) Strelchenko, Stepan; Faustova, Ilona, juhendaja; Örd, Mihkel, juhendaja
Cyclin dependent kinases (CDKs) regulate the cell cycle by phosphorylating downstream proteins in an ordered manner. In yeast S. cerevisiae, Cdk1 regulates the cell cycle progression from G1 to M phase. Cdk1 phosphorylation can have various effects on the proteins, for example, they can be activated or targeted for degradation. The latter was used in this work to design a regulatory network for controlled protein expression that could be used in cell factories. For this, Far1-based diphosphodegron tags that are degraded in response to phosphorylation by Cdk1, were used to differentially regulate the levels of the transcriptional repressor TetR. Furthermore, as cell factories require dynamic control of protein levels over the cultivation process, the effect of cell culture density on cell cycle and the degron tags was studied. The work shows that Far1-based degron tags can be used as modular tags to regulate exogenous proteins by switching them on or off in an OD-dependent manner using the endogenous Cdk1 machinery. Finally, it was shown that more complex patterns of regulation can be achieved by inducing expression of cyclins at high OD.
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The analysis of novel Far1 motif important for Cdk1 inhibition
(Tartu Ülikool, 2024) Mukhadze, Juli; Faustova, Ilona, juhendaja; Maljavin, Artemi, juhendaja; Loog, Mart, juhendaja; Tartu Ülikool. Loodus- ja täppisteaduste valdkond; Tartu Ülikool. Tehnoloogiainstituut
The cell cycle progression in yeast is regulated by cyclin-dependent kinases (CDKs), which phosphorylate different substrate proteins to ensure proper order and timing of cell cycle events. In the presence of pheromones, cell division can be interrupted in the G1 phase via activation of the mating mitogen-activated protein kinase (MAPK) pathway. In this pathway, MAP kinase Fus3 phosphorylates Far1 protein. Consequently, phosphorylated Far1 can bind to the CDK complex and suppress its activity. Although T306 phosphorylation of Far1 was shown to be crucial for CDK inhibition, the exact mechanism and main determinants of this process in the Far1 protein sequence remain unclear. In this study, we analysed the previously undescribed amino acid region of Far1 and identified key residues with the strongest impact on CDK activity, which likely points to their importance for protein-protein interactions. Our results suggest that the analysed Far1 region may potentially serve as a CDK docking site. However, further analyses are required to reveal the exact mechanism of inhibition.