Destination cilium: towards selective probing and perturbation of ciliary signaling

Project Information

DestCilia

Grant agreement ID: 948750

DOI

10.3030/948750

Project closed

EC signature date 19 November 2020

Start date 1 January 2021

End date 31 May 2026

Funded under

• EXCELLENT SCIENCE - European Research Council (ERC)

Total cost

€ 1 408 776,00

EU contribution

€ 1 408 776,00

Coordinated by

UNIVERSITE DE GENEVE
Switzerland

Periodic Reporting for period 3 - DestCilia (Destination cilium: towards selective probing and perturbation of ciliary signaling)

Reporting period: 2024-01-01 to 2025-06-30

Summary of the context and overall objectives of the project

The primary cilium is a small cellular organelle that receives signals from the environment from the cell. These signals are then propagated in the cells leading to a cellular response. It is essential that this organelle functions well, as there are many diseases associated with its malfunctioning, collectively called ciliopathies. In this project, we aim to generate chemical tools to 1) better understand the function of the primary cilium and 2) be able to probe what happens to the proteins inside the primary cilium. More specifically, we use organic synthesis to create new molecules with a specific biological function and then we test them in both healthy and ciliopathy cells. The knowledge gained will be instrumental in designing new treatments for ciliopathies in the future.

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

In the first 30 months of the project, we have synthesised a variety of probes to meet our objectives, and we have started to evaluate the biological activities of the synthesised probes. For the first work package, we have synthesised peptide sequences that we hypothesise should localise specifically to the cilium. In order to be able to validate this, we have implemented a new fluorescence microscopy based assay for the study of ciliary uptake of small molecules. Using this assay, we have been able to validate some of our constructs, and current work is aimed at proving the mechanism by which the peptides are transported. For this, we have generated cells that lack an essential protein for transport, and we are now studying if there is indeed hampered ciliary uptake of our peptides in these mutant cells. In addition, we have expressed this protein in vitro so that we can study the binding of the peptides using fluorescence polarization assays.
For the second work package, we have synthesised many different peptides that can be used in a reaction with an enzyme to label proteins. We have used these peptides successfully to label a model protein (green fluorescent protein) in vitro. We have cloned all the constructs to express the enzyme in cells, and our next steps are to perform the reaction in cells. As a requirement for this is that the synthetic peptides reach the cytoplasm, we are also evaluating their cell permeability properties, and adjusting these when needed by the incorporation of cell penetrating motifs.
For the final work package we have established a successful synthetic route towards modified coenzyme A constructs that we want to use to label cellular structures. We have generated substrate peptides, expressed the enzyme and are now studying enzymatic activity using our peptides. Our preliminary results show that the enzyme does not have much room to accommodate the synthetic cofactors, and so we are currently moving into the generation of enzyme mutants with expanded binding pockets. As a second objective, we have successfully generated bisubstrate inhibitors of the enzyme, consisting of a covalently linked coenzyme A and substrate peptide, which are currently being evaluated in vitro.

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

It is very difficult to study the function of proteins in the primary cilium because the organelle is very small. The content of proteins in the cilium is very well controlled by the cellular machinery. In principle, small molecules, such as drugs, should be able to freely diffuse into the cilium, but there are no good tools to study this. Our new assay to study ciliary uptake will therefore be of great use to prove the entry of drugs into the cilium. This also allows us to study if the peptides we have designed, work as expected. We anticipate to generate so called 'cilium trackers', fluorescent molecules that can be used to 'light up' the cilium when studied under the microscope. Our new assay is also used to study the uptake the peptides that are synthesised in the framework of WP2. We there aim to be able to tag proteins in the cells (and in the cilium), by using an enzyme that recognizes both the protein we want to label, as well as our synthetic peptide that carries the label and fuses them together. A major challenge is for the synthetic peptides to be taken up by the cells, but with our new assay we have made good progress in understanding what the properties are that lead to efficient cellular (and ciliary) uptake. We have also made progress in the expression of the necessary components in cells, so that once we have the right synthetic construct, we can quickly move to intracellular labelling studies. For the final work package, we have made good progress in the establishment of the necessary synthetic routes, the expression of the enzyme, and the conditions necessary for the enzymatic labelling studies. Moreover, we now have the first covalent inhibitor of the enzyme in hand, and are currently evaluating its potency in different settings. We anticipate to have a full toolbox available to probe tubulin post-translational modification by the end of this project.

graphical representation of the primary cilium and our different strategies