Polyphosphates (polyP) are chains of inorganic phosphates found in all cells. Previous work has implicated these chains in diverse functions, but the mechanism of action is unclear. A recent study reports that polyP can be non-enzymatically and covalently attached to lysine residues on yeast proteins Nsr1 and Top1. One question emerging from this work is whether so-called "polyphosphorylation" is unique to these proteins or instead functions as a global regulator akin to other lysine post-translational modifications. Here, we present the results of a screen for polyphosphorylated proteins in yeast. We uncovered 15 targets including a conserved network of proteins functioning in ribosome biogenesis. Multiple genes contribute to polyphosphorylation of targets by regulating polyP synthesis, and disruption of this synthesis results in translation defects as measured by polysome profiling. Finally, we identify 6 human proteins that can be modified by polyP, highlighting the therapeutic potential of manipulating polyphosphorylation in vivo.

Highlights d Multiple cell compartments can support accumulation of high levels of polyPs d Over 350 transcripts and 100 proteins show mis-regulation with polyP accumulation d Intracellular polyP results in selective signaling via MAPK effectors d Targets of lysine polyphosphorylation change localization with polyP accumulation

Polyphosphates (PolyP) are composed of long chains of inorganic phosphates linked together by phosphoanhydride bonds. They are found in all kingdoms of life, playing roles in cell growth, infection, and blood coagulation. A resurgence in interest in polyP has shown links to diverse aspects of human disease. However, unlike in bacteria and lower eukaryotes, the mammalian enzymes responsible for polyP metabolism are not known. Many studies have resorted to adding polyP to cell culture media, but it is not clear if externally applied polyP enters the cell to impact signaling events or whether their effect is mediated exclusively by extracellular receptors. For the first time, we use RNA-seq and mass spectrometry to define a broad impact of polyP produced inside of mammalian cells via ectopic expression of the E. coli polyP synthetase Ppk1. RNA-seq demonstrates that Ppk1 expression impacts expression of over 350 genes enriched for processes related to transcription and cell motility. Analysis of proteins via label-free mass spectrometry identified over 100 changes with functional enrichment in cell migration. Follow up work suggests a role for internally-synthesized polyP in promoting activation of mTOR and ERK1/2-EGR1 signaling pathways implicated in cell growth and stress. Finally, fractionation analysis shows that polyP accumulated in multiple cellular compartments and was associated with the relocalization several nuclear/cytoskeleton proteins, including chromatin bound proteins DEK, TAF10, GTF2I and translation initiation factor eIF5b. Our work is the first to demonstrate that internally produced polyP can activate diverse signaling pathways in human cells.Significance StatementFor many years following its discovery in 1890, polyphosphates (polyP) were dismissed as evolutionary fossils. Best understood for its role in bacteria and yeast, our understanding of polyP in mammals remains rudimentary because the enzymes that synthesize and degrade polyP in mammalian systems are currently unknown. In our work, we carried out large-scale transcriptome and proteome approaches on human cells designed to accumulate internally produced polyP via ectopic expression of a bacterial polyP synthetase. Our work is the first to systematically assess the impact of increased intracellular polyP.

Polyphosphates (polyP) are long chains of linked inorganic phosphates ranging from 3–1000s of residues in length. They are found in all organisms and play critical roles in a range of functions including blood clotting and bacterial virulence. Given its diverse roles, polyP is an attractive target for novel therapies. Recently, it was shown that polyP can be non‐enzymatically added to protein targets within poly‐acidic, serine, and lysine rich (PASK) motifs as a lysine post‐translational modification termed polyphosphorylation. This modification was described for two yeast proteins, Nsr1 and Top1. In yeast, polyP is initially synthesized by a polyP synthetase (VTC4) and subsequently added as a PTM non‐enzymatic. Our lab has developed a screen to assess polyphosphorylation of yeast substrates wherein we uncovered 25 novel substrates including a conserved network of proteins functioning in ribosome biogenesis. Disruption of this polyP synthesis through vtc4D deletion results in translation defects as measured by polysome profiling. Finally, we found that expression of E. coli polyP synthetase (EcPPK1) in vtc4D mutant yeast results in extreme sensitivity to rapamycin and cycloheximide, despite its ability to restore polyphosphorylation of multiple yeast targets. We propose a model wherein polyphosphorylation of unique targets regulates multiple aspects of cell growth. Moreover, regulation of polyphosphorylation in space and time is critical to preserve essential cell functions, such as ribosome function.Support or Funding InformationThe funding agencies that support this research are: University of Ottawa and CIHRThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

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Polyphosphates are long chains of phosphates attached to one another via high‐energy phosphoanhydride bonds. The literature reports their implication in a variety of medically related functions, such as apoptosis, blood coagulation, and inflammation. The mechanisms involved with polyphosphate and its functions have been studied in bacteria and in yeast, but the synthesis pathway of polyphosphate in mammals is still unknown. Moreover, the concentrations of polyphosphate in mammalian cells (<1mM) are much lower than the ones found in yeast (100–200mM). This makes it challenging to study polyphosphate biology and polyphosphorylation in mammalian cell lines. Recently, Azevedo, et al. (2015) identified a new PTM in yeast called polyphosphorylation and two targets of this PTM: Nsr1 and Top1. It consists of the non‐enzymatic covalent attachment of polyphosphate chains to lysine residues found in a poly‐acidic serine and lysine‐rich (PASK) motif. Our lab has since identified 24 novel yeast targets being polyphosphorylated. Our next aim was to test if human proteins can also be polyphosphorylated. In order to modulate the concentrations of polyphosphate in the cell, we transfected mammalian cells with PPK1, an E. coli enzyme that synthesizes polyphosphate. This ectopic expression allowed us to perform a small “screen” and identify 6 human proteins that can be polyphosphorylated: nucleolin, hNop56, Mesd chaperone, DEK, eIF5B and UPF3B. We will present our latest work focused on determining the molecular function of polyphosphorylation in mammalian cells.Support or Funding InformationThis project is funded by the Canadian Institutes of Health Research (CIHR).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.