RESUMEN
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.
RESUMEN
An important step in elucidating the function of protein post-translational modifications (PTMs) is gaining access to site-specifically modified, homogeneous samples for biochemical characterization. Protein pyrophosphorylation is a poorly characterized PTM, and here a chemical approach to obtain pyrophosphoproteins is reported. Photo-labile phosphorimidazolide reagents were developed for selective pyrophosphorylation, affinity-capture, and release of pyrophosphoproteins. Kinetic analysis of the reaction revealed rate constants between 9.2 × 10-3 to 0.58 M-1 s-1, as well as a striking proclivity of the phosphorimidazolides to preferentially react with phosphate monoesters over other nucleophilic side chains. Besides enabling the characterization of pyrophosphorylation on protein function, this work highlights the utility of phosphoryl groups as handles for selective protein modification for a variety of applications, such as phosphoprotein bioconjugation and enrichment.
RESUMEN
The peptide hormone calcitonin is intimately connected with human cancer development and proliferation. Its biosynthesis is reasoned to proceed via glycine-, α-hydroxyglycine-, glycyllysine-, and glycyllysyllysine-extended precursors; however, as a result of the limitations of current analytical methods, until now, there has been no procedure capable of detecting these individual species in cell or tissue samples. Therefore, their presence and dynamics in cancer had not been established. Here, we report the first methodology for the separation, detection, and quantification of calcitonin and each of its precursors in human cancer cells. We also report the discovery and characterization of O-glycosylated calcitonin and its analogous biosynthetic precursors. Through direct and simultaneous analysis of the glycosylated and nonglycosylated species, we interrogate the hormone biosynthesis. This shows that the cellular calcitonin level is maintained to mitigate effects of biosynthetic enzyme inhibitors that substantially change the proportions of calcitonin-related species released into the culture medium.
