Targeting the Anti-Target: From Structure to Drug in the heart Kv11.1 channel

Projektinformationen

HeartAtaK

ID Finanzhilfevereinbarung: 708051

DOI

10.3030/708051

Projekt abgeschlossen

EK-Unterschriftsdatum 16 März 2016

Startdatum 1 September 2016

Enddatum 31 August 2018

Finanziert unter

• EXCELLENT SCIENCE - Marie Skłodowska-Curie Actions

Gesamtkosten

€ 183 454,80

EU-Beitrag

€ 183 454,80

Koordiniert durch

UNIVERSITY OF LEEDS
United Kingdom

Periodic Reporting for period 1 - HeartAtaK (Targeting the Anti-Target: From Structure to Drug in the heart Kv11.1 channel)

Berichtszeitraum: 2016-09-01 bis 2018-08-31

Zusammenfassung vom Kontext und den Gesamtzielen des Projekts

One of the world’s biggest secrets is still the functioning of the human brain. In particular, the development of synapses and neuronal activity is a fundamental process about which we still know very little. In terms of their malfunctioning in neurological disorders, our knowledge is even more scarce as the clinical presentation can be very diverse and patients can display very different symptoms even though they have the same diagnosis as for example very often seen for autism spectrum disorder (ASD). Symptoms of ASD can include, in different levels of severity, deficits in verbal and non-verbal communication, mental retardation, lack of social interaction and patterns of stereotypical behaviour. Whole genome sequencing of patients having ASD revealed that 1% of the patients have a mutation in a gene called patched domain-containing protein 1 (ptchd1). Vice versa, 40% of people having a mutation in ptchd1 develop ASD. Thus, ptchd1 is the important marker gene for mental retardation. However, so far very little is known about the function of ptchd1.
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.

Arbeit, die ab Beginn des Projekts bis zum Ende des durch den Bericht erfassten Berichtszeitraums geleistet wurde, und die wichtigsten bis dahin erzielten Ergebnisse

The limiting factor in structural biology of membrane proteins is the production and purification of stable protein, as membrane proteins tend to be rather unstable and need special stabilisators which again complicates their purification. However, the problem starts often in the production of sufficient protein amounts, as simply the space in the membrane is limited and already crowded with the cells own membrane proteins. Producing proteins is also a costly expense for cells, stressing them so much that the cell will stop the production halfway or degrade the protein to regain energy and nutrients. Even when a protein is produced, it has to be verified that the cell transports the protein to its correct destination compartment, as sometimes specific signal sequences can be ‘misread’ by the producing cell and the membrane protein belonging into the cell membrane end up in the nucleus membrane.
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.

Fortschritte, die über den aktuellen Stand der Technik hinausgehen und voraussichtliche potenzielle Auswirkungen (einschließlich der bis dato erzielten sozioökonomischen Auswirkungen und weiter gefassten gesellschaftlichen Auswirkungen des Projekts)

Only recently, ptchd1’s crucial role in autism spectrum disorder and mental retardation was identified. Since that, it has been shown that non-functional ptchd1 induces a neurodevelopmental disorder with symptoms like attention-deficit and hyperactivity. However, even though ptchd1 has undoubtedly an important role in brain development, information about ptchd1 is scarce. With smart construct design, expression system and detergent screening I can now produce enough protein of high quality for on-going structural studies.
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.

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