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.

One Scaffold, Two Conformations: The Ring-Flip of the Messenger InsP 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 C-labeled InsPs/PP-InsPs, the behavior of these molecules was investigated by 2D-NMR under solution conditions reminiscent of a cytosolic environment.

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.