Periodic Reporting for period 1 - HeartAtaK (Targeting the Anti-Target: From Structure to Drug in the heart Kv11.1 channel)
Reporting period: 2016-09-01 to 2018-08-31
The protein is supposed to be a receptor in the hedgehog signalling pathway, a pathway best known for its involvement in the prenatal development and the distinction of body patterns. Thus, a defect gene in this signalling pathway has severe consequences for the individual. During this fellowship, I want to investigate how the signal is received by the receptor and how it is transferred to downstream effectors. To achieve this, one aim of this project is to solve the structure of ptchd1 by cryo-EM. The three-dimensional structure of a protein provides the basis for its function. Understanding the protein’s structure allows us to deduce where the incoming signal will bind and how its three-dimensional structure changes upon signalling. However, the signal seems not to be transferred directly from protein to protein but via a small molecule of still unknown identity. As structures provide only static information, I also want to investigate ptchd1’s biophysical behaviour and its interaction with other proteins in order to understand the whole process of signal transduction.
This project will provide the first structural information about an important signalling pathway involved in human prenatal development. The information obtained will further shed light a unique signal transfer mechanism.
As very little is known about ptchd1, I used bioinformatics tools to predict regions within the protein that might be highly flexible and so decrease the stability of the protein. Additionally, I identified special motifs in the protein’s sequence that might guide the protein to the cell’s degradation machinery. To enhance the probability of producing ptchd1, I generated various different constructs of the protein omitting these sequence motifs and flexible regions. Additionally, I linked ptchd1 to a green fluorescent protein, which allows me to visualize the expression and location of the protein in living cells with a fluorescent microscope. So far, I was able to produce ptchd1 recombinantly in sufficient amounts for structural studies and verified its translocation into its correct compartment within the cell. Further, I established a protein purification protocol that provides stable, highly pure protein of good quality for structural studies via single-particle cryo electron microscopy. These experiments are currently on-going.
I presented my research findings at the prominent, interdisciplinary Gordon Research Conference on ‘Convergence and Divergence between fragile X chromosome and autism spectrum disorders’ and made valuable contacts to experts in the neurodevelopmental community.
This project provides first insights into the molecular mechanism of ptchd1 in the brain, a crucial requirement to further understand the molecular basis of synapse development. Additionally, it has been shown that mutations in postsynaptic scaffolding protein interacting with ptchd1 are associated with psychiatric disease. Thus, this research has the potential, in the long term, to have a huge impact on the health of society providing general information about synapse architecture, which can lead to new therapeutic strategies in drug development for neurodevelopmental disorders and psychiatric diseases.