INVESTIGATING PROTEIN PYROPHOSPHORYLATION WITH CHEMICALLY SYNTHESIZED PYROPHOSPHOPEPTIDES

Abstract

Protein pyrophosphorylation is a minimally characterized post-translational modification that has been implicated in several diseases, including diabetes, obesity, and cancer. To date, only in vitro evidence of this modification has been found using radiolabeling and biochemical methods. A main challenge to understanding the biological significance of this PTM is that no methods exist for detecting this modification in cell lysates. Therefore, a robust synthetic methodology was developed to access high yields of pure pyrophosphopeptides to use as tool compounds. This methodology was efficient in polar, protic solvents and tolerant of functionally diverse peptide substrates. These results suggested a higher degree of phosphoserine reactivity than previously anticipated and opened the door for a wide range of applications.

This methodology was then used to prepare a series of biologically relevant pyrophosphopeptides to study the chemical and biochemical stability of the PTM. The pyrophosphate moiety was found to be relatively chemically inert but was removed by alkaline phosphatases in vitro. Most importantly, enzyme-dependent dephosphorylation of the pyrophosphopeptides was observed in mammalian and yeast cell lysates, thus providing initial evidence for the reversibility of pyrophosphorylation in a cellular context. Given the lability of the pyrophosphoryl group in lysates, a stable bisphosphate pyrophosphoserine analog incorporable into peptides and proteins was also synthesized. The bisphosphonate-containing peptides exhibited superior chemical, cell lysate, and plasma stability compared to the native diphosphate group to facilitate future pyrophosphoserine antibody generation and affinity purification of endogenous pyrophosphatases.

The ability to prepare synthetic pyrophosphopeptides has also aided in the development of a novel pyrophosphoproteomic strategy. Unique fragmentation patterns for the pyrophosphoryl group were observed by CID and ETD MS/MS ionization techniques. Using these diagnostic fragmentation patterns in conjunction with a selective pyrophosphopeptide affinity reagent previously developed in our lab, several putative in vivo targets of protein pyrophosphorylation from S. cerevisiae have been identified. Taken together, the ability to generate synthetic pyrophosphopeptides and peptides containing a stabilized analog of the pyrophosphate has provided key insights into the chemical properties of this modification which will assist in unveiling the regulatory role of protein pyrophosphorylation and its impact on cellular signaling networks.