Phenotypes are established by tight regulation on protein functions. This regulation can be mediated allosterically, through protein binding, and covalently, through post-translational modification (PTM). The integration of an ever-increasing number of PTMs into regulatory networks enables and defines the proteome complexity. Protein PTMs can occur enzymatically and nonenzymatically. Polyphosphorylation, which is a recently discovered PTM that belongs to the latter category, is the covalent attachment of the linear ortho-phosphate polymer called inorganic polyphosphate (polyP) to lysine residues. PolyP, which is ubiquitously present in nature, is also known to allosterically control protein function. To date, lack of reagents has prevented the systematic analysis of proteins covalently and/or allosterically associated with polyP. Here, we report on the chemical synthesis of biotin-modified monodisperse short-chain polyP (bio-polyP8-bio) and its subsequent use to screen a human proteome array to identify proteins that associate with polyP, thereby starting to define the human polyP-ome.

An iterative polyphosphorylation approach is described, which is based on ap hosphoramidite (P-amidite) derived reagent (c-PyPA) obtained from the cyclization of pyrophosphate with areactive diisopropylaminodichlorophosphine.T his type of reagent is unprecedented as it represents ar eactive P-amidite without protecting groups.T he reagent proved to be stable in solution over several weeks.Its utility is described in the context of iterative monodirectional and bidirectional polyphosphorylations.T he ensuing functionalized cyclotriphosphate can be opened with av ariety of nucleophiles providing ready access to diverse functionalized polyphosphate chains of defined length with several tags, including both P-N and P-O labels.Their interaction with exoand endopolyphosphatases is described.

A recently-discovered protein post-translational modification, lysine polyphosphorylation (K-PPn), consists of the covalent attachment of inorganic polyphosphate (polyP) to lysine residues. The nonenzymatic nature of K-PPn means that the degree of this modification depends on both polyP abundance and the amino acids surrounding the modified lysine. K-PPn was originally discovered in budding yeast (Saccharomyces cerevisiae), in which polyP anabolism and catabolism are well-characterized. However, yeast vacuoles accumulate large amounts of polyP, and upon cell lysis, the release of the vacuolar polyP could nonphysiologically cause K-PPn of nuclear and cytosolic targets. Moreover, yeast vacuoles possess two very active endopolyphosphatases, Ppn1 and Ppn2, that could have opposing effects on the extent of K-PPn. Here, we characterized the contribution of vacuolar polyP metabolism to K-PPn of two yeast proteins, Top1 (DNA topoisomerase 1) and Nsr1 (nuclear signal recognition 1). We discovered that whereas Top1-targeting K-PPn is only marginally affected by vacuolar polyP metabolism, Nsr1-targeting K-PPn is highly sensitive to the release of polyP and of endopolyphosphatases from the vacuole. Therefore, to better study K-PPn of cytosolic and nuclear targets, we constructed a yeast strain devoid of vacuolar polyP by targeting the exopolyphosphatase Ppx1 to the vacuole and concomitantly depleting the two endopolyphosphatases (ppn1Δppn2Δ, vt-Ppx1). This strain enabled us to study K-PPn of cytosolic and nuclear targets without the interfering effects of cell lysis on vacuole polyP and of endopolyphosphatases. Furthermore, we also define the fundamental nature of the acidic amino acid residues to the K-PPn target domain.

A tunable chemoenzymatic strategy provides access to magic spot nucleotides and analogues in gram-scale.

Condensed phosphates may exist as linear, cyclic or branched structures. Due to their important role in nature, linear polyphosphates have been well studied. In contrast, branched phosphates (ultraphosphates) remain largely uncharacterised, because they were already described in 1950 as exceedingly unstable in the presence of water, epitomized in the antibranching-rule. This rule lacks experimental backup, since, to the best of our knowledge, no rational synthesis of defined ultraphosphates is known. Consequently, detailed studies of their chemical properties, reactivity and potential biological relevance remain elusive. Here, we introduce a general synthesis of monodisperse ultraphosphates. Hydrolysis half-lives up to days call the antibranching-rule into question. We provide evidence for the interaction of an enzyme with ultraphosphates and discover a rearrangement linearizing the branched structure. Moreover, ultraphosphate can phosphorylate nucleophiles such as amino acids and nucleosides with implications for prebiotic chemistry. Our results provide an entry point into the uncharted territory of branched condensed phosphates.