Mechanisms and regulation of nucleotide signalling pathways in humans

• Applicant: Dr. Indra Bekere
• Subject Area: Biochemistry ; Cell Biology
• Term: since 2023
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 526258235

The cyclic GMP-AMP synthase (cGAS)- Stimulator of Interferon Genes (STING) pathway is a recently discovered double-stranded DNA (dsDNA) sensing pathway, which activates cellular signalling, such as interferon and inflammatory responses as well as autophagy and cell death. cGAS is a nucleotidyltransferase (NTase), which is activated by binding to out of context dsDNA, e.g. from pathogens and damaged cells, and synthesizes a cyclic dinucleotide (CDN) second messenger, 2´-5´/ 3´-5´ cyclic GMP-AMP (cGAMP). In the recent years it has become clear that aberrant cGAS-STING signalling causes autoinflammatory and autoimmune diseases. At the same time, the cGAS-STING axis is beneficial in other contexts such as cancer immunotherapy and cGAMP analogues are being actively developed as therapeutic treatments. However, mechanisms that regulate cGAMP levels directly inside the cells are so far unknown. Moreover, phosphodiesterases that cleave intracellular CDNs are widespread across species but no such enzymes have been discovered in humans yet. Therefore, my objective is to identify an intracellular cGAMP phosphodiesterase using mass spectrometry coupled with cellular thermal shift assay (MS-CETSA). Furthermore, I aim to biochemically characterize cGAMP phosphodiesterase and to determine how such phosphodiesterase is regulated and how it affects cGAS-STING signalling in cells. Furthermore, cGAMP is the only known CDN in humans. This is in strong contrast to bacteria, which possess large structural and functional diversity of nucleotides and even the cGAS-STING pathway is conserved in bacteria. Overall, this suggests that more cyclic nucleotide signalling pathways exist in humans but have not been discovered yet. Therefore, my aim is to discover novel nucleotide second messengers in humans and identify cellular players that are involved in these nucleotide signalling pathways. For this I will treat various cell types with a unique nucleotide library and monitor induction of signalling by various cellular assays. Potential enzymes that synthesize novel nucleotide signals may be the cGAS-like Mab21 NTases, which are so far uncharacterized for their cellular function, catalytic activity, activating ligands and nucleotide-based products, but have been implicated in genetic diseases and cancer. Therefore, I aim to identify cellular function of Mab21 NTases and determine whether they are active and generate nucleotide-based products like cGAS. To accomplish this, I will analyse cellular phenotypes upon NTase knockout or overexpression and biochemically characterize NTase ligands and nucleotide products. Overall, the aim of my proposal is to expand our knowledge of nucleotide signalling pathways in humans by detecting mechanisms that directly regulate cGAMP levels inside cells and by identifying novel nucleotide signalling pathways and function of the cGAS-like Mab21 NTases.

• DFG Programme: WBP Position