Characterization of Inorganic Polyphosphate in Mammalian Cells

Abstract

Inorganic polyphosphate (polyP) is a linear chain of three to around one thousand orthophosphates linked by phosphoanhydride bonds. Not only is polyP considered as a “molecular fossil” that existed on the primordial earth, but it is also universally found in all living species tested so far. Yet, unlike many other conserved molecules in biology, the pathways by which polyP is metabolized are not conserved, and the polyP synthesizing and degrading machineries in mammalian cells are still completely unknown. This lack in knowledge substantially hampers progress in polyP research since it renders reverse genetics powerless in deciphering the physiological functions of polyP in these species. Moreover, it raises two very important questions in the field: 1) why did evolution give rise to vastly different mechanisms to generate such a conserved molecule, and 2) what are the roles of this ancient polymer in modern species and time? In my thesis work, I used a multipronged approach to address these questions. First of all, I detected an enrichment of polyP in the nucleolus of HeLa cells using an immunofluorescence probe derived from the polyP-binding domain of Escherichia coli exopolyphosphatase. This fraction of polyP is highly dynamic to environmental stress – it rapidly accumulates and relocates to distinct foci in the nucleolus during cisplatin-mediated apoptosis. The extent of polyP accumulation in such foci positively correlates with the intrinsic susceptibility of different ovarian cancer cells to cisplatin. And indeed, when we supplemented HeLa cells and ovarian cancer cells with exogenous polyP, they became significantly albeit slightly sensitized to the chemotherapeutic drug. To take this one step further, I revealed the identities of cisplatin-triggered polyP foci with fluorescence colocalization analysis and reported polyP in the light nucleolar cap, fibrillar cap and Cajal body. Formation of these polyP-rich compartments is inflicted by impaired ribosome RNA synthesis, which underlies cisplatin toxicity. Intriguingly, I also discovered inositol hexakisphosphate kinase, a polyP-related protein, as a novel component of the light nucleolar cap. These results prompted me to hypothesize that polyP, a highly negatively charged polymer, might play a role in regulating the structural dynamics of this phase-separated compartment. However, to illustrate the molecular mechanisms of polyP functions relies on the identification of polyP metabolizing enzymes, in particular, polyphosphate kinase in mammalian cells. Therefore, I conducted a whole genome siRNA screen for genes involved in the up- and down-regulation of polyP, using cisplatin-induced polyP accumulation as a reporter. I proposed several promising candidates, GRIN3B, P2RY1, ATP5F1E, PANK4 and AP3M1, which might be responsible for signaling polyP synthesis or catalyzing the reaction itself. Furthermore, I discovered a potential connection between inositol phosphate metabolism and polyP regulation. Meanwhile, I also uncovered genes involved in the following pathways: nucleotide excision repair, the ubiquitin-proteasome system, and cell cycle progression, to be essential regulators of cisplatin toxicity and potential targets for optimizing the current platinum-based therapeutic interventions. In summary, my work has revealed an important aspect of polyP function in nucleolar stress response and begun to explore the molecular mechanisms of this phenomenon. Moreover, I have laid the foundation for future work aimed at characterizing the polyP regulatory network in mammalian cells, especially the long-sought polyphosphate kinase.