Specific Fluorescent Probe for Protein Histidine Phosphatase Activity

Abstract

Protein histidine phosphorylation plays a vital role in cell signaling and metabolic processes, and phosphohistidine (pHis) phosphatases such as protein histidine phosphatase 1 (PHPT1) and LHPP have been linked to cancer and diabetes, making them novel drug targets and biomarkers. Unlike the case for other classes of phosphatases, further studies of PHPT1 and other pHis phosphatases have been hampered by the lack of specific activity assays in complex biological mixtures. Previous methods relying on radiolabeling are hazardous and technically laborious, and small-molecule phosphatase probes are not selective toward pHis phosphatases. To address these issues, we herein report a fluorescent probe based on chelation-enhanced fluorescence (CHEF) to continuously measure the pHis phosphatase activity of PHPT1. Our probe exhibited excellent sensitivity and specificity toward PHPT1, enabling the first specific measurement of PHPT1 activity in cell lysates. Using this probe, we also obtained more physiologically relevant kinetic parameters of PHPT1, overcoming the limitations of previously used methods.

Supramolecular Tandem Assay for Pyridoxal‐5′‐phosphate by the Reporter Pair of Guanidinocalix[5]Arene and Fluorescein

“…The obtained results have feasible implications in disease diagnosis, in which PLP is an important biomarker. Moreover, the present reporter pair shows potential application in activity assays of various protein/peptide kinases and phosphatases involved in phosphorylation and dephosphorylation, such as protein tyrosine kinase‐7, T4 polynucleotide kinase, polo‐like kinase 1, protein tyrosine phosphatase, and protein histidine phosphatase …”

Quest for the Crypto‐phosphoproteome

“…We can expect a similar scenario for more labile crypto‐phosphosites. As an example, PHPT1 has been reported as an efficient phosphoramidate hydrolase towards diverse substrates harboring pHis or pLys in vitro [37] . Indeed, Eyers and co‐workers analyzed PHPT1‐knockdown HeLa cell lysates to find numerous pHis and pLys sites as well as pArg, pGlu, pAsp, and pCys sites [34] .…”

“…However, such non‐specific substrates can give misleading results, as in the miscategorization of YwlE (see above). To address this, we developed chelation‐enhanced fluorescence (CHEF) based fluorescent probes for PHPT1, which enabled specific detection of PHPT1 activity in cell lysates [37] . We also developed similar fluorescent probes for Arg phosphorylation to study McsB and YwlE [40] .…”

Combining free energy calculations with tailored enzyme activity assays to elucidate substrate binding of a phospho-lysine phosphatase

“…Similar ndings have been reported recently for LHPP activity on histidine phosphorylations by Choi et al as mentioned above. 18 Next, we evaluated the kinetics for the hydrolysis of pLys peptides with and without LHPP addition for peptides 2a, 3a and 3j, which included the substrates with the highest and lowest hydrolysis yields aer 90 min ( Fig. 5 and Table 1).…”

“…16 Although the enzymatic activity was shown to be higher for pLys, all the later ndings described LHPP in the role of a phosphoramidate hydrolase selective for pHis. 17,18 Although the phosphorylation of lysine residues was described for the rst time more than 45 years ago, its biological role has been, to date, only rudimentarily elucidated. Initially described to occur as an acid-labile group in histone H1, 19,20 pLys could be detected in rat liver in vivo later.…”