We aim to understand the molecular mechanism of synaptic vesicle endocytosis (SVE), an important method of nerve cell signalling, by understanding how phosphorylation (the addition of a phosphate molecule to a protein) affects larger protein-protein interactions. Dynamin I has two splice variants, long or short. We discovered that the calcineurin protein selectively docks with the dynamin I short splice variant to regulate bulk endocytosis, which is used during periods of intense nerve cell activity, like epileptic seizures.
Phosphorylation of particular sites within dynamin I (called Ser-774 and 778) blocks recruitment of syndapin, but not endophilin, which binds to the same site. We mapped the endophilin-binding sites and, surprisingly, found that dynamin I actually has two endophilin binding sites: a minor binding site at the previously known site, but also a major binding site in a different position in the dynamin I long variant. Each interacts with endophilin independently, yet endophilin can only engage one site at a time. This is important information for designing drugs to selectively interfere with these interactions, which may allow us to more finely tune the treatments we are developing for kidney disease and other conditions.
The nerve terminal phosphoproteome
Since SVE is a calcium-triggered process, we proposed that the fastest route to discovering which proteins are important to this process and how they function is to identify the phosphorylation sites (aka phosphosites) in each of the SVE proteins that rapidly respond to calcium stimulation. So far, we have identified >400 unique phosphosites on over 160 proteins using our novel pull-down procedure to capture the major SVE and related proteins. In addition, we successfully revealed the calcium-sensitive subset of phosphosites. Bioinformatics has helped to reveal exciting new findings, providing us a handle on the different signalling pathways. Furthermore, we completed an extensive analysis of the phosphorylated SVE proteins from cdk5-knockout mice using mass spectrometry. Cdk5 is an important signalling molecule involved in many cellular processes, but has recently been implicated in a range of neurodegenerative diseases like Parkinson’s and Alzheimer’s. We found a subset of proteins that are potential cdk5 substrates, which will help us understand how cdk5 is involved in these disorders and potentially lead to new avenues of treatment.
Dynamin function in actin dynamics
In order for dynamin to perform its role in endocytosis, it needs to form into a helical structure around the necks of endocytic vesicles, which are basically extensions of the cell membrane. In addition to forming helices, dynamin can also self-assemble into rings that are 40 nanometres in diameter (about 30 molecules) of unknown function. Ring Stabilizer compounds are small molecules that affect dynamin’s activity that we have discovered and patented. These compounds have two unique actions compared with dynamin inhibitors: they stimulate dynamin activity by promoting ring assembly, and they prevent rings from disassembling. This produces prolonged and sustained activation of dynamin. We aim to uncover the cellular mechanisms underlying our novel Ring Stabilizers and develop them as future therapeutics for treatment of proteinuric kidney diseases.
Dynamin drug discovery
We have developed a drug discovery program with Professor Adam McCluskey at the University of Newcastle. In collaboration with our research partners, we designed compounds that inhibit dynamin (dynamin inhibitors) and published many new classes which we call the dynoles, the iminochromenes the pthaladyns and the dyngos. Each of these compounds block endocytosis in cells, but we also demonstrated two additional uses. Firstly, they block the uptake of certain viruses into cells, suggesting they may be of future use to treat infectious diseases. Secondly, we showed that they block cell proliferation and cause cell death in human cancer cells. This series of exciting studies demonstrates their potential to be developed into novel anti-cancer drugs in the long term. We look forward to being able to generate many new and more potent dynamin inhibitors, with the hope that we can distinguish between dyn I and dyn II activity to individually target compounds for epilepsy or glioblastoma treatments.