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.

Respiratory syncytial virus (RSV) is a leading cause of mortality in infants and young children. Despite the RSV disease burden, no vaccine is available, and treatment remains nonspecific. New drug candidates are needed to combat RSV. Toward this goal, we screened over 2,000 compounds to identify approved drugs with novel anti-RSV activity. Cardiac glycosides, inhibitors of the membrane-bound Na/K-ATPase, were identified to have anti-RSV activity. Cardiac glycosides diminished RSV infection in human epithelial type 2 cells and in primary human airway epithelial cells grown at an air-liquid interface. Digoxin, a U.S. Food and Drug Administration-approved cardiac glycoside, was also able to inhibit infection of primary nasal epithelial cells with community isolates of RSV. Our results suggest that the antiviral effects of cardiac glycosides may be dependent on changes in the intracellular Na and K composition. Consistent with this mechanism, we demonstrated that the ionophoric antibiotics salinomycin, valinomycin, and monensin inhibited RSV in human epithelial type 2 cells and primary nasal epithelial cells. Our data indicate that the K/Na-sensitive steps in the RSV life cycle occur within the initial 4 hours of viral infection but do not include virus binding/entry. Rather, our findings demonstrated a negative effect on the RSV transcription and/or replication process. Overall, this work suggests that targeting intracellular ion concentrations offers a novel antiviral strategy.

Polyphosphates (polyP) are long chains of inorganic phosphates that can be attached to lysine residues of target proteins as a non-enzymatic post-translational modification. This modification, termed polyphosphorylation, may be particularly prevalent in bacterial and fungal species that synthesize and store large quantities of polyP. In this study, we applied a proven screening strategy to evaluate the polyphosphorylation status of over 200 candidate targets in the budding yeast S. cerevisiae. We report 8 new polyphosphorylated proteins that interact genetically and physically with a previously identified network of targets implicated in ribosome biogenesis. The expanded target network includes vacuolar proteins Prb1 and Apl5, whose modification with polyP suggests a model for feedback regulation of polyP synthesis, while raising additional questions regarding the location of polyphosphorylation in vivo.. CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under

Nicotinamide is both a reaction product and an inhibitor of the conserved sirtuin family of deacetylases, which have been implicated in a broad range of cellular functions in eukaryotes from yeast to humans. Phenotypes observed following treatment with nicotinamide are most often assumed to stem from inhibition of one or more of these enzymes. Here, we used this small molecule to inhibit multiple sirtuins at once during treatment with DNA damaging agents in the Saccharomyces cerevisiae model system. Since sirtuins have been previously implicated in the DNA damage response, we were surprised to observe that nicotinamide actually increased the survival of yeast cells exposed to the DNA damage agent MMS. Remarkably, we found that enhanced resistance to MMS in the presence of nicotinamide was independent of all five yeast sirtuins. Enhanced resistance was also independent of the nicotinamide salvage pathway, which uses nicotinamide as a substrate to generate NAD+, and of a DNA damage-induced increase in the salvage enzyme Pnc1. Our data suggest a novel and unexpected function for nicotinamide that has broad implications for its use in the study of sirtuin biology across model systems.KEYWORDS nicotinamide; sirtuins; DNA damage; checkpoint; Pnc1; NAD+ T HE DNA damage checkpoint is a highly conserved signaling cascade initiated in response to DNA lesions. In the budding yeast Saccharomyces cerevisiae, checkpoint activation begins with the exposure of single-stranded DNA (ssDNA), either from exonuclease-resected DNA doublestrand breaks (DSBs), or from stalled replication forks during S phase. Resected DNA coated by the ssDNA binding protein RPA is thought to act as a landing pad for Mec1-Ddc2 complexes (Melo and Toczyski 2002;Gobbini et al. 2013;Edenberg et al. 2014a). Mec1 is a sensor kinase that, in concert with adaptor proteins such as Rad9 or Mrc1, phosphorylates downstream checkpoint targets, including the Rad53 and Chk1 transducing kinases (Melo and Toczyski 2002;Gobbini et al. 2013; Bastos de Oliveira et al. 2015). Following autophosphorylation and release from adaptors, Rad53 is thought to move throughout the cell to phosphorylate targets that promote cell cycle arrest, the inhibition of late-firing origins of replication, and a global transcriptional response (Melo and Toczyski 2002;Jaehnig et al. 2013;Edenberg et al. 2014a). While the DNA damage response is traditionally associated with phosphorylationbased signaling cascades, it has recently emerged that other post-translational modifications including ubiquitylation, sumoylation, and acetylation play prominent roles in the response in both yeast and other eukaryotes (Downey and Durocher 2006a;Psakhye and Jentsch 2012;Panier and Durocher 2013;Edenberg et al. 2014a;Elia et al. 2015).Acetylation of lysine residues is catalyzed by histone acetyltransferases (HATs) and reversed by histone deacetylases (HDACs). Despite their names, these enzymes also have nonhistone targets that play critical roles in maintaining cellular homeostasis in organisms from ba...

Polyphosphates (polyP) are long chains of inorganic phosphates that can be attached to lysine residues of target proteins as a non-enzymatic post-translational modification. This modification, termed polyphosphorylation, may be particularly prevalent in bacterial and fungal species that synthesize and store large quantities of polyP. In this study, we applied a proven screening strategy to evaluate the polyphosphorylation status of over 200 candidate targets in the budding yeast S. cerevisiae. We report 8 new polyphosphorylated proteins that interact genetically and physically with a previously identified network of targets implicated in ribosome biogenesis. The expanded target network includes vacuolar proteins Prb1 and Apl5, whose modification with polyP suggests a model for feedback regulation of polyP synthesis, while raising additional questions regarding the location of polyphosphorylation in vivo.

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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