Structural basis of calcineurin inhibition by FATZ-1Sahir, Dunia (Avtor) Djinović Carugo, Kristina (Mentor) Muscle fiber remodellingsarcomereZ-diskcalcineurin-NFAT signalling pathwayFATZ-1 inhibitionα-actinin-3 deficiencyVoluntary movement in humans and other vertebrates is the result of an intricate mechanism involving hundreds of precisely organized and coordinated skeletal muscle cells. Falling into the category of striated muscles, this highly ordered cell apparatus has the primary function of generating force and contraction in order to support respiration, locomotion, and posture. Exercise, growth, aging and illness change the contraction properties of skeletal muscles, and nevertheless, they persist in their restless contraction to gift us movement for a lifetime. Nevertheless, understanding the underlying mechanisms and reasons through which these changes emerge is an important undertaking, since much is still to be uncovered. Structural and molecular studies of striated muscles allowed us to appreciate the details of the basic organization of proteins that make up the sarcomere, which repeats over and over again as the primary structural unit through the muscle. The highly dynamic and complex structure of proteins composing the sarcomere is in constant flux for regeneration, but at the same time provides high stability and flexibility. Failure of any component to complete its role can result in illness, the malfunction of the contractile units or generally cause phenotypic deviations. The point of encounter between adjacent sarcomeres, namely the Z-disk, is an intricate assembly of over 40 proteins that, working in tight collaboration, coordinate forces and give rise to a controlled, unidirectional contraction. α-actinin maintains the integrity of the Z-disk while controlling the mechanical strength generated at the sarcomere, so it is not a surprise that mutations in the hActn1, hActn2 and hActn3 genes are linked to myopathies and changes in muscle differentiation. A premature stop codon polymorphism in hActn3 is common in humans, but α-actinin-3 is dispensable and most common in fast-twitch muscle fibers, so the consequences seem not to be too harmful, since it seems that α-actinin-2 may be able to compensate its role. α-actinin-3 deficiency primarily affects muscle differentiation seems to be linked to an increase of endurance performance in athletes. The present study offers a launch pad and builds the foundation to inquire into the possible mechanisms underlying this change in cellular phenotype in case of a hActn3 malfunction. Protein interactions are the key to most biological processes, mediating structural and functional regulation. This master thesis thus undertakes a wholistic approach to understand the molecular mechanisms of regulation of calcineurin (CN) by the interaction with skeletal muscle Z-disk protein FATZ-1/ calsaricin-2/ myozenin-1. The calcium and calmodulin-dependent protein phosphatase CN is involved in signalling that controls both cardiac hypertrophy as well as slow muscle fiber gene expression and thus skeletal muscle differentiation, that seems to be controlled by the hActn 3 genotype. FATZ-1 has been shown to bind and inhibit the activity of CN while at the same time interacting with several other components of the Z-disk, including α-actinin. As a result, it acts as a center for the interaction of important proteins and plays a key role in myofibril genesis and the association of CN with the contractile apparatus. Our enzymatic experiments show that FATZ-1 is a linear competitive inhibitor of CN, while a combination of enzymatic assays, binding experiments and structural analysis narrowed down the binding site to the C-terminal region (CTR) of FATZ-1. Understanding the details of the interaction between CN and FATZ-1 may shed a light on the activation and regulation of the CN signalling pathway and lead to an insight into skeletal muscle differentiation as well as the causes for cardiac myopathies.20232023-11-23 09:45:01Magistrsko delo/naloga152394VisID: 18741COBISS_ID: 176648707sl