Extensive protein pyrophosphorylation revealed in human cell lines.

Reversible protein phosphorylation is a central signaling mechanism in eukaryotes. Although mass-spectrometry-based phosphoproteomics has become routine, identification of non-canonical phosphorylation has remained a challenge. Here we report a tailored workflow to detect and reliably assign protein pyrophosphorylation in two human cell lines, providing, to our knowledge, the first direct evidence of endogenous protein pyrophosphorylation. We manually validated 148 pyrophosphosites across 71 human proteins, the most heavily pyrophosphorylated of which were the nucleolar proteins NOLC1 and TCOF1. Detection was consistent with previous biochemical evidence relating the installation of the modification to inositol pyrophosphates (PP-InsPs). When the biosynthesis of PP-InsPs was perturbed, proteins expressed in this background exhibited no signs of pyrophosphorylation. Disruption of PP-InsP biosynthesis also significantly reduced rDNA transcription, potentially by lowering pyrophosphorylation on regulatory proteins NOLC1, TCOF1 and UBF1. Overall, protein pyrophosphorylation emerges as an archetype of non-canonical phosphorylation and should be considered in future phosphoproteomic analyses.

One Scaffold, Two Conformations: The Ring-Flip of the Messenger InsP8 Occurs under Cytosolic Conditions.

Inositol poly- and pyrophosphates (InsPs and PP-InsPs) are central eukaryotic messengers. These very highly phosphorylated molecules can exist in two distinct conformations, a canonical one with five phosphoryl groups in equatorial positions, and a "flipped" conformation with five axial substituents. Using 13C-labeled InsPs/PP-InsPs, the behavior of these molecules was investigated by 2D-NMR under solution conditions reminiscent of a cytosolic environment. Remarkably, the most highly phosphorylated messenger 1,5(PP)2-InsP4 (also termed InsP8) readily adopts both conformations at physiological conditions. Environmental factors-such as pH, metal cation composition, and temperature-strongly influence the conformational equilibrium. Thermodynamic data revealed that the transition of InsP8 from the equatorial to the axial conformation is, in fact, an exothermic process. The speciation of InsPs and PP-InsPs also affects their interaction with protein binding partners; addition of Mg2+ decreased the binding constant Kd of InsP8 to an SPX protein domain. The results illustrate that PP-InsP speciation reacts very sensitively to solution conditions, suggesting it might act as an environment-responsive molecular switch.

Identification of sortase substrates by specificity profiling.

Sortases catalyze the attachment of surface proteins to the peptidoglycan layer of gram-positive bacteria and further represent powerful tools of protein chemistry. During catalysis sortases cleave a donor substrate containing the LPxTG (x=any amino acid) sorting motif under formation of an enzyme-bound thioester and ligate this intermediate to an acceptor protein containing an N-terminal glycine residue. In addition to the well-established sortase A of Staphylococcus aureus several homologs of this enzyme have been identified in the genomes of gram-positive bacteria. We have profiled the specificity of seven sortases of Staphylococci and Streptococci origin and observed that sortases of the latter class displayed a more relaxed specificity for donor and acceptor substrates than their Staphylococci counterparts. Streptococci sortases prefer an LPKLG donor substrate sequence compared to the canonical sorting motif LPKTG. These findings might facilitate the use of Streptococci sortases as tools of protein chemistry.