Biology subjects

Denoncourt, A.

Publications and source records attributed to Denoncourt, A..

6 recordsLinked to original sources

bioRxiv2026

Regulation of polyphosphate homeostasis in S. cerevisiae by the Sky1 protein kinase

Eukaryotic cells store inorganic phosphate in the form of long chains called polyphosphates (polyP) that can reach many hundreds of residues in length. In the budding yeast Saccharomyces cerevisiae, polyP is synthesized by the VTC complex using ATP as a substrate and sequestered at high concentrations in the vacuole. Here, polyP chain length and turnover are regulated by the polyphosphatase enzymes Ppn1 and Ppn2. In the vacuole lumen, polyP plays a critical role in phosphate and ion homeostasis. Beyond the vacuole, minor populations of polyP are thought to impact various cellular functions, including in the nucleus, in part by binding to target proteins. We speculated that polyP metabolism must be tightly coupled to mechanisms of homeostatic control operating elsewhere in the cell. In this work, we report that cells lacking Sky1, a serine-arginine repeat protein kinase, have very low levels of polyP, an effect that is exacerbated by alkaline stress. Under these conditions sky1{triangleup} mutants also display increased expression of a subset of genes from the PHO regulon. Genetic analysis suggests that both phenotypes are linked to an increase in potassium influx via the Trk1 potassium transporter. Loss of Npl3, a known target of Sky1, partially rescues the phosphate homeostasis defects in sky1{triangleup} mutants, although this occurs independently of a well characterized Sky1 phosphorylation site on Npl3. Finally, the loss of polyP in sky1{triangleup} mutants depends on the Ppn1 and Ppn2 polyphosphatases and the Pep4 protease located in the vacuole. Altogether, our work provides novel insights into how polyP homeostasis in the vacuole is coordinated with events occurring throughout the cell.

molecular biology↗

bioRxiv2025

Identification of polyphosphate-binding proteins in E. coli uncovers targets involved in translation control and ribosome biogenesis

In many bacteria, polyphosphate kinase (PPK) enzymes use ATP to synthesize polyphosphate (polyP) in response to cellular stress. These chains of inorganic phosphates are joined by high-energy bonds and can reach hundreds of residues in length. PolyP plays diverse functions in helping bacteria adjust to changing environmental conditions. However, the molecular mechanisms underlying these functions are poorly understood. In eukaryotic cells, polyacidic serine- and lysine-rich (PASK) motifs of proteins can mediate binding to polyP chains. Whereas PASK motifs are relatively common in yeast and human cells, we report that these sequences are rare in bacteria commonly used for polyP research. Thus, to identify novel polyP-binding proteins in Escherichia coli, we carried out an untargeted screen and identified 7 novel targets with links to translation control and ribosome biogenesis. For two targets, the GTPase activating protein YihI and the ribonuclease Rnr, we mapped the regions of polyP interaction to non-PASK sequences and identified lysine residues critical for binding. We found that deletion of rnr suppressed the slow growth phenotype of {Delta}ppk mutants grown on minimal media. Conversely, ppk deletion resulted in decreased Rnr protein expression. These phenotypes were dependent on the polyP binding region of Rnr but independent of polyP binding itself, suggesting a complex interplay between PPK and Rnr function in E. coli. Overall, our work provides new insights into the scope of polyP binding proteins and extends the connections between polyP and the regulation of protein translation in E. coli.

molecular biology↗

bioRxiv2023

Ionic polyphosphorylation of histidine repeat proteins by inorganic polyphosphate

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) that 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 uncovered 30 human and yeast histidine repeat proteins that are specifically modified by polyP. This polyP modification is histidine-dependent and non-covalent in nature, though remarkably, it withstands harsh denaturing conditions--a hallmark of covalent PTMs. We have termed this interaction ionic histidine polyphosphorylation (iH-PPn) to describe its unique PTM-like properties. Importantly, we show that iH-PPn disrupts phase separation and phosphorylation activity of the human protein kinase DYRK1A, and inhibits the activity of the transcription factor MafB, highlighting iH-PPn as a potential hitherto unrecognized regulatory mechanism. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/536149v1_ufig1.gif" ALT="Figure 1"> View larger version (49K): org.highwire.dtl.DTLVardef@1ed34b7org.highwire.dtl.DTLVardef@200259org.highwire.dtl.DTLVardef@17a2954org.highwire.dtl.DTLVardef@12f5d04_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGRAPHICAL ABSTRACTC_FLOATNO C_FIG

biochemistry↗

bioRxiv2022

Ddp1 cooperates with Ppx1 to counter a stress response initiated by non-vacuolar polyphosphate

In diverse cells from bacterial to mammalian species, inorganic phosphate is stored in long chains called polyphosphates (polyP). These near universal polymers, ranging from 3 to thousands of phosphate moieties in length, are associated with molecular functions including energy homeostasis, protein folding, and cell signaling. In many cell types, polyphosphate is concentrated in subcellular compartments or organelles. In the budding yeast S. cerevisiae, polyP synthesis by the membrane-bound VTC complex is coupled to its translocation into the lumen of the vacuole, a lysosome-related organelle, where it is stored at high concentrations. In contrast, ectopic expression of bacterial polyphosphate kinase, PPK, results in the toxic accumulation of polyP outside of the vacuole. In this study, we used label-free mass spectrometry to investigate the mechanisms underlying this toxicity. We find that PPK expression results in the activation of a stress response mediated in part by the Hog1 and Yak1 kinases, and Msn2/Msn4 transcription factors. This response is countered by the combined action of the Ddp1 and Ppx1 polyphosphatases that function together to counter polyP accumulation and downstream toxicity. In contrast, ectopic expression of previously proposed mammalian polyphosphatases did not impact PPK-mediated toxicity in this model, suggesting either that these enzymes do not function directly as polyphosphatases in vivo or that they require co-factors unique to higher eukaryotes. Our work provides a mechanistic explanation for why polyP accumulation outside of lysosome-related organelles is toxic. Further, it serves as a resource for exploring how polyP may impact conserved biological processes at a molecular level.

molecular biology↗

bioRxiv2020

A broad response to intracellular long-chain polyphosphate in human cells

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.

molecular biology↗

bioRxiv2019

Proteins required for vacuolar function are targets of lysine polyphosphorylation in yeast

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.

molecular biology↗