Project Outline

The neuronal ceroid lipofucscinoses are a group of inherited neurodegenerative disorders with primarily childhood onset. The most common form, also known as Batten disease or CLN3 disease, is caused by recessively inherited mutation in the CLN3 gene. This encodes a multispan protein that appear to regulate vesicular trafficking but its exact functions are not well understood.

The CLN3 gene is predicted to encode a multi-spanning transmembrane protein but it does not conform to any known structure: potentially it represents a novel protein fold. If the structure was to be solved, it would indicate likely mechanisms of action and disease-causing mutations identified in Batten disease patients could be mapped to the 3D structure to highlight the key functional regions of the protein. This project aims to solve the structure and elucidate the function of CLN3.

The student will express and purify CLN3 from human cells. CLN3 will be engineered to include affinity tags in predicted loops, based on preliminary studies. The protein will then be purified from cells and characterised by a battery of analytical tools, including analytical ultracentrifugation and circular dichroism, before structural analysis by cryoEM.

Key mutations will be incorporated into the CLN3 gene by site-directed mutagenesis, these mutations, based on structure/known naturally occurring mutations, will provide key information on how the protein functions.

To complement the structural studies, we will employ proximity labelling to identify the protein partners of CLN3 in complexes within the membranes of living cells. This will shed significant light on how CLN3 operates in cells. Crucially, proximity labelling allows protein complexes to be identified in situ while the cells are intact and living. There is no need to lyse the cells with detergents, which is likely to disrupt protein complexes. We have used it successfully for other membrane proteins, including another neuronal ceroid lipofuscinosis protein, CLN7.

In this project, the student will use CRISPR/Cas9 gene editing to incorporate a proximity labelling enzyme into the CLN3 gene at a site unlikely to disrupt the structure in cultured human cells. This will by-pass the need to overexpress the CLN3 gene and prevent excessive labelling and false identification of non-CLN3-interacting proteins. Cell biology and protein biochemistry methods will then be used to isolate the proteins found directly adjacent to CLN3 in the membranes of live cells and mass spectrometry to identify the protein partners.

That mutations in CLN3 that cause Batten disease have been known for more than 25 years yet the function of the CLN3 protein is still not properly understood is astounding. Together, this two-pronged approach will provide key structural understanding of the Cln3 protein, potentially highlighting a novel protein fold, but also help to elucidate the function of this protein within the cell and identifying any partners it may bind to.

Techniques

Cell biology, structural biology, cryoEM, gene editing, protein biochemistry, mass spectrometry.