Exploring the structure and function of NUDT5 in de novo purine synthesis

ongoing

Proliferation of tumour cells requires continuous DNA synthesis and energy supply. Purine nucleotides are essential building blocks for DNA. Recent research has highlighted the role of NUDIX hydrolase 5 (NUDT5) catalytic activity in nuclear ATP metabolism in breast cancer. Notably, NUDT5 has also been suggested as a more general prognostic marker for various cancers, and NUDT5 depletion leads to suppression of tumour cell proliferation in a variety of model systems. Strikingly, the host lab has recently discovered that NUDT5 possesses an unexpected non-enzymatic, likely scaffolding role in de novo purine synthesis. This observation is unprecedented for a catalytically active member of the NUDIX family. In this project, I aim to dissect the molecular mechanism and reveal the structural basis of NUDT5-regulated purine metabolism by combining Affinity Purification Mass Spectrometry (AP-MS) proteomics and metabolomics with X-ray crystallography and cryogenic Electron Microscopy (cryo-EM) with three main goals: (1) Unravel the molecular mechanism and role of NUDT5 in purine synthesis using a comparative metabolomics and proteomics approach using CRISPR knock out (KO) cell lines, small molecule inhibitors and degraders of NUDT5 in relevant cell line models. (2) Identify and define the NUDT5 interactome via AP-MS, and validate candidate NUDT5 interactors in biophysical and functional assays using purified proteins and cellular models. (3) Explore NUDT5 protein complexes using X-ray crystallography and cryo-EM to dissect the molecular interaction between NUDT5 and complex partners for regulation of purine synthesis. This work will offer novel fundamental insights into the molecular mechanism of NUDT5-mediated purine metabolism, and the scaffolding role of this enzyme to provide new perspectives on cellular metabolism and potential anti-tumour drug development.