Studies on polyphosphate kinase 2 mechanism and inorganic polyphosphate function

Abstract

Inorganic polyphosphate (PolyP) is a linear polymer of orthophosphate and is ubiquitous throughout the biological world. Although this ancient molecule was discovered in cells over 100 years ago, its importance was not revealed until recent decades. Studies have shown that polyP can help to maintain the hemostasis of the intracellular environment and is crucial to the survival of cell under stringent and stress conditions, especially in prokaryotes and lower eukaryotes. It is also important in higher prokaryotes like mammals, as research has indicated that it plays an important role in multiple systems and processes, including bone development, immune response, and even in tumors. All evidence shows that polyP is a key molecule in life that has remained throughout evolution. Here, polyP was investigated from two perspectives: one was the perspective of polyP metabolism by an important prokaryotic enzyme polyphosphate kinase 2 (PPK2), and one was the perspective of polyP function by investigating the effect of extracellular polyP on mammalian osteoblast-like SaOS-2 cells.

In order to study the mechanism of PPK2,Mycobacterium tuberculosisPPK2 (MTB

PPK2) was investigated. From the protein structure, there is a basic tunnel in PPK2 which may be important to the enzyme activity. I hypothesized that polyP requires the basic tunnel as a positively charged surface for the polyp polyanion to bind through electrostatic interactions. Sixsingle aminoacids around this tunnelwere mutated to alanine (F184A, R193A, K196A, D201A, K237A and R241A)to investigate the importance of this tunnel for PPK2 activity. These six mutant plasmids were successfully constructed. Three mutants did not fold correctly (F184A, K196A and R241A), implying the importance of these amino acids on protein folding while three others did fold (R193A, D201A, and K237A) and could be expressed and purified using E.coli, together with the wild-type MTB PPK2. R193A and K237A showed reduced activities compared to the wild-type whileD201A showed no detectable activity. This indicated that this basic region is important to the activity of PPK2.Furtherexperiments can focus on the binding between the mutated PPK2and the substrates by applying different bio-physical methods, like isothermal titration calorimetry (ITC) or surface plasmon resonance (SPR).

In the second perspective of this thesis, I investigated the influence of polyP in SaOS-2 osteoblast-like cells. Previous experiments in our laboratory showed polyP triggered upregulation ofinterleukin-11 (IL-11)at the RNA level. My hypothesis was that protein level of IL-11 was upregulated. Enzyme-linked immunosorbent assay (ELISA) revealed the upregulation of IL-11 protein only between three to six hours after exposure of cells to polyP. PolyP was also shown to induce apoptosis in SaOS-2 cells. Future investigations can focus on the signal pathway triggered by polyP in osteoblast-like cell, thus elucidating the mechanisms by which polyP functions in bone development.