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Unique TNFα lysine myristoylation; elucidating its function, regulation and integration into inflammation

Periodic Reporting for period 1 - TNFaLysMyr (Unique TNFα lysine myristoylation; elucidating its function, regulation and integration into inflammation)

Reporting period: 2018-09-01 to 2020-08-31

Tumour Necrosis Factor-α (TNFα) is a pro-inflammatory cytokine with major roles in immunity and cellular homeostasis. Deregulated TNFα signalling is implicated in many autoimmune and inflammatory diseases, including sepsis, multiple sclerosis, rheumatoid arthritis, inflammatory bowel disease, type I diabetes and cancer. Blocking TNFα signalling with antibody drugs is a successful treatment strategy but has only temporary effects and clinical responses vary in different diseases, owing to the complexity of TNFα signalling. To improve and expand therapeutic strategies, a better understanding of TNFα and the many other cytokines secreted from cells, is crucial. It was very recently shown that TNFα is myristoylated on a strictly conserved lysine residue (KMyr) – a unique post-translational modification – suggesting an important but yet unexplored role for lysine myristoylation in cytokine biology.
The aim of the project is to investigate the impact of KMyr on TNFα signalling, and its wider role in secretion and signalling of other cytokines, using a unique multi-disciplinary approach combining state-of-the-art chemical biology tools complemented with molecular cell biology, proteomics and biochemistry.
Metabolic labelling of TNFa-producing macrophages with YnMyr, a myristic acid analogue wielding a bio-orthogonal labelling tag that can be functionalised with a fluorophore for visualisation, we confirmed the initial findings that TNFa is myristoylated. Concomitantly blocking canonical N-myristoylation with inhibitors specific for the NMT1 and NMT2 N-myristoyltransferases did not abolish TNFa myristoylation, suggesting a non-canonical myristoylation pathway.

However, the amounts of myristoylated TNFa are exceptionally small. After an extensive period of optimisation, less than 0.1% of the total amount of TNFa was found to be myristoylated. Due to this low abundance, it was not possible to confirm whether this PTM occurs under physiological conditions on TNFa or other proteins.

Concomitantly, studies on lysine acylation (including myristoylation) of R-Ras2 and K-Ras4a were published by others, showing lysine myristoylation could only be identified by chemical proteomics when using large quantities of cell lysate (50 mg total protein), combined with R-Ras2/K-Ras4a overexpression, knockdown of (potential) demyristoylases while also performing the metabolic labelling with high concentrations of YnMyr for extended periods of time. Even then, the modification was not significantly identified. In light of these results, it is unlikely that lysine myristoylation has biological relevance.

Instead, the project turned towards unravelling which PTMs occur and change during the polarization of macrophages, such as in pro-inflammatory macrophages that secrete TNFa and in anti-inflammatory macrophages during wound-healing. By using a similar multi-disciplinary approach as for the initial project, multiple targets have been identified. The insights yielded by this investigation are currently being patented, as well as form the basis for several publications. At the time of writing, one manuscript has been published, one is under peer-review and several manuscripts are being prepared.

Alongside the aforementioned investigation, an in-depth analysis was performed on the compounds regularly applied by the lipidation field to inhibit N-myristoyltransferases in living cells. On-target, cell-active chemical probes are of fundamental importance in chemical and cell biology, whereas poorly characterized probes often lead to invalid conclusions. Only two out of five compounds delivered complete and specific inhibition of N-myristoylation, in the absence of off-target cytotoxicity. This study enables the selection of appropriate on-target probes for future studies and suggests the need for reassessment of previous studies that used off-target compounds.
The project yielded highly interesting results, which are currently being incorporated in a patent. At the time of writing, two manuscripts have been published, two are under peer-review and several manuscripts are being prepared.

Notably, the study on validation and invalidation of chemical probes for the human N-myristoyltransferases allows the scientific community to select the appropriate on-target probes for future studies (Cell Chemical Biology, 2019, 26, 892; see also https://www.cell.com/cell-chemical-biology/pdfExtended/S2451-9456(19)30083-2).
Graphical abstract of Kallemeijn et al (Cell Chemical Biology, 2019)