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Summary: Polyphosphate exhibits a unique post-translational modification-like function, known as histidine polyphosphate modification (HPM), marked by a robust non-covalent interaction with histidine repeat proteins. Here, we present a protocol for detecting HPM of human proteins via maltose-binding protein-tagged expression in E. coli. We describe steps for detecting HPM by observing electrophoretic mobility shifts on NuPAGE gels followed by western blot. We then detail procedures for analyzing the influence of ionic strength and pH on HPM.For complete details on the use and execution of this protocol, please refer to Neville et al.1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Summary: Inorganic polyphosphate (polyP) is a linear polymer of orthophosphate that is present in nearly all organisms studied to date. A remarkable function of polyP involves its attachment to lysine residues via non-enzymatic post-translational modification (PTM), which is presumed to be covalent. Here, we show that proteins containing tracts of consecutive histidine residues exhibit a similar modification by polyP, which confers an electrophoretic mobility shift on NuPAGE gels. Our screen uncovers 30 human and yeast histidine repeat proteins that undergo histidine polyphosphate modification (HPM). This polyP modification is histidine dependent and non-covalent in nature, although remarkably it withstands harsh denaturing conditions—a hallmark of covalent PTMs. Importantly, we show that HPM disrupts phase separation and the phosphorylation activity of the human protein kinase DYRK1A, and inhibits the activity of the transcription factor MafB, highlighting HPM as a potential protein regulatory mechanism.