Reversible protein phosphorylation, catalysed by protein kinases and phosphatases, is a fundamental regulatory mechanism of numerous cellular processes. Post-translational modification of proteins with phosphate residues can alter their cellular fates, such as modulating their activity or changing their subcellular distribution. Protein kinases add phosphate residues primarily on serine, threonine and tyrosine residues of target proteins, whilst phosphatases remove them. Yet kinases outnumber phosphatases by approximately ten to one. The extent and specificity of protein phosphorylation are tightly regulated in normal cellular processes, and often go awry in disease. Protein phosphatases form holoenzymes composed of a promiscuous catalytic subunit and various regulatory domains to achieve substrate specificity. This project aims to harness the promiscuity of phosphatases to redirect their activity to dephosphorylate phospho-proteins of interest in a targeted manner, akin to targeted protein degradation by harnessing the cellular ubiquitin proteasome system.
Previously, our lab has developed a nanobody-directed target protein degradation tool, dubbed Affinitiy-directed PROtein Missile system (AdPROM), in which an E3 ligases tethered to the nanobody is brought into close proximity with the target protein bound by the nanobody to cause ubiquitylation and degradation of the target protein. Excitingly, we have also been able to demonstrate that the AdPROM technology can be harnessed to deliver the catalytic subunits of some protein phosphatases to specific phospho-proteins for targeted dephosphorylation. These results open up exciting opportunities for exploring targeted dephosphorylation of specific phospho-proteins as a fundamentally novel way of understanding the function of target protein phosphorylation and potentially applying this as an approach for therapeutic intervention in diseases that are caused by constitutive phosphorylation of target proteins.
By initially exploiting CRISPR/Cas9 genome editing and AdPROM technologies, this project aims to develop tools for achieving ligand-inducible dephosphorylation of select phospho-protein targets in cell lines. Subsequently, once the best phosphatases that can be redirected to dephosphorylate target phospho-proteins are identified, small molecule ligands against these phosphatases will be developed and harnessed to develop bivalent molecules that might induce targeted dephosphorylation of specific phospho-proteins in cells and tissues.
Although many protein kinase inhibitors have found success in many biological and therapeutic applications, they suppress phosphorylation of all of their substrates, potentially leading to undesired consequences and thereby limiting the use of some very selective kinase inhibitors in the clinic. Targeted dephosphorylation of specific phospho-proteins would potentially overcome this by achieving intervention at a specific phospho-substrate level.
The successful PhD student will be based within the Sapkota lab at the MRC Protein Phosphorylation and Ubiquitylation Unit (MRC-PPU) (http://www.ppu.mrc.ac.uk/). The student will employ cutting-edge CRISPR/Cas9 genome editing and mass-spectrometric technologies as well as state-of-the-art molecular, biochemical and cell biology methodologies. MRC-PPU collaborates with three leading pharmaceutical companies (Boehringer Ingelheim, GlaxoSmithKline, Merck Serono) through the long-running Division of Signal Transduction Therapy (DSTT) collaboration. As this project is expected to uncover a completely novel way of targeting the dephosphorylation of specific phospho-protein targets, this collaboration will enable us to expedite the translation of our findings and tools into potential drug discovery projects.
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