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Structural and functional studies of MICAL signalling in cytoskeletal dynamics
Rožová, Dominika ; Rozbeský, Daniel (advisor) ; Sulimenko, Vadym (referee)
The main focus of this project was chicken protein MICAL1, which is involved in the semaphorin-plexin signalling pathway and has a significant effect on the rearrangement of the cytoskeleton. The prominent role of the MICAL1 protein is primarily associated with axon guidance, as it destabilizes actin filaments through its oxidative activity. We focused on elucidating the molecular mechanisms of chicken MICAL1 autoinhibition using molecular and structural biology methods together with new protein structure prediction methods. Chicken MICAL1 was produced in Sf9 insect cells using a baculovirus expression system and we produced both full-length and truncated versions of chicken MICAL1 protein. We kinetically characterized the protein and determined its oligomeric state in solution. We made great efforts to solve the protein structure using crystallography, electron microscopy and protein structure prediction in Alphafold 2. Based on the results of these experiments and assays, we conclude that MICAL1 proteins are regulated through their C terminal domain, which interacts with the monooxygenase domain. The part of this interaction is the autoinhibition of chicken MICAL1. We excluded the possibility that chicken MICAL1 is regulated by changing its oligomeric state. The results of this master's thesis...
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Structural and functional studies of signaling molecules in axon guidance
Knapp, Kryštof ; Rozbeský, Daniel (advisor) ; Novák, Petr (referee)
This work aims to determine a model of the autoinhibition mechanism of MICAL proteins using biochemical, biophysical, and bioinformatical approaches. MICAL proteins are a group of flavin monooxygenases that play a key role in various cellular processes, as they facilitate the reorganization of the actin cytoskeleton. MICAL-1 has long been known for its vital role in axon guidance as an effector of repulsive signaling through oxidative destabilization of actin filaments. However, recent findings indicate that MICAL-1 can also serve as a sig- naling molecule, using localized hydrogen peroxide production to regulate other downstream effectors. Despite the consensus that MICAL-1 activity must be strictly regulated, the exact molecular mechanism of this regulation has not yet been described. In this work, we provide a novel model of MICAL-1 autoinibiton mechanism based on a comparison of steady-state kinetic experiments and molecular dynamics simulations between full-length MICAL-1 from Coturnix japonica and its truncated form lacking the C-terminal domain. In our model, we conclude that changes in MICAL-1 activity are the result of intramolecular protein interac- tions between the C-terminal and the monooxygenase domain. Furthermore, we rule out the role of MICAL-1 oligomerization in its activity...
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