Role of the PEST Domains in Proteasomal Degradation of Rett Protein: MeCP2
Date
2024
Authors
Kalani, Ladan
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Abstract
Located on the X-chromosome is the gene encoding the nuclear protein Methyl CpG binding protein 2 (MeCP2). The instability of this protein causes pleiotropic neurological abnormalities, including the debilitating neurodevelopmental disease Rett syndrome (RTT). MeCP2, an epigenetic regulator abundant in neurons, is involved in pleiotropic molecular interaction. Many deleterious mutations of MeCP2 impact its mRNA or protein levels. Neuron maturation and dendritic arborization are compromised when MeCP2 levels are out of the homeostatic range. The mechanisms the cell uses to maintain MeCP2 levels within a tight range have yet to be fully understood. Several hypotheses addressed the homeostatic mechanisms of MeCP2, which involve miRNAs, N-terminal degradation signals or N-degrons, and the PEST domains that act as degradation switches upon post-translational modifications (PTMs). Our lab hypothesized the involvement of MeCP2 PEST-mediated degradation as a mechanism of its homeostatic regulation; however, this hypothesis has yet to be experimentally proven. I experimentally tested the PEST-mediated degradation of MeCP2 with Rett-causing mutations by integrating MeCP2 constructs that have an altered or deleted PEST domain and used microscopy, FRAP analysis and western blotting to characterize in vitro how these constructs behave relative to WT and mutated MeCP2. MeCP2 has Rett-causing mutations that cause lower protein levels, such as T158M; the PEST motif expedites its degradation as deleting it results in higher protein levels. Moreover, mutations that result in higher levels of MeCP2, such as R294X, show stronger DNA binding relative to WT, as assessed by NaCl fractionation. For the first time, we report that the Ct-PEST domain of MeCP2 plays a role in its degradation.
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Keywords
Epigenetics, Rett syndrome, RTT, Neurodevelopmental disease, methyl reader protein, intellectual disability, epilepsy, biochemistry, chromatin, genetic disease