Universiteit Leiden

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LED3 Lecture - Elucidating inositol pyrophosphate signaling with chemical tools

Friday 9 September 2022
Gorlaeus Building
Einsteinweg 55
2333 CC Leiden
Havinga Lecture Hall


Cells are able to modulate a range of processes according to the external environment and internal sources, by integrating signaling cues with their metabolic status. The diphosphoinositol polyphosphates (PP-InsPs) are a unique group of highly phosphorylated messengers known to control insulin signaling, energy homeostasis, cell migration, and much more, and provide an important link between signaling and metabolic networks. It is thought that PP-InsPs mediate these pleiotropic responses by accessing several different modes of action, including allosteric regulation and protein pyrophosphorylation. To provide a mechanistic picture of PP-InsP signaling – so that this network can be rationally exploited for therapeutic purposes in the long run – new chemical and biochemical tools are much needed.

The common theme among PP-InsP signaling mechanisms is that they all require an interaction between the PP-InsP and the protein target(s). We thus set out to comprehensively annotate the mammalian PP-InsP interactome using affinity reagents, in which the small molecules are immobilized in different ways. Application of the affinity reagents identified between 300 and 400 putative mammalian interacting proteins. Interestingly, we found several examples of proteins or protein complexes that preferentially interact with one of the structurally closely related metabolites over another. Using complementary surface-based PP-InsP probes, these specific interactions were also characterized biophysically.

In parallel, we developed new strategies to detect protein pyrophosphorylation, an understudied protein modification thought to be mediated by PP-InsPs, using mass spectrometry. Analysis of a set of synthesized, site-specifically modified peptides enabled us to develop an “on-the-fly” detection method for reliable identification of pyrophosphopeptides in mammalian cell lysates.  The triggered MS method allowed for the definitive assignment of over 150 sites. The modified proteins span a range of processes and included known in vitro targets (i.e. nucleolar and coiled-body phosphoprotein 1; NOLC1), as well as many new pyrophosphoproteins, such as nucleoside diphosphate kinase 1 (NME1). Using chemical strategies, we are beginning to elucidate the biochemical and structural consequences of these novel phosphorylation patterns at the protein level.

Overall, these new analytical and chemical tools, combined with improved biochemical approaches to probe inositol phosphate interconversion, are now providing the molecular details underlying the pleiotropic functions of PP-InsPs messengers in eukaryotic cells.

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