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Title:Mechanisms of polyphosphate degradation
Author(s):Hebbard, Carleigh Fredda Frances
Director of Research:Morrissey, James H
Doctoral Committee Chair(s):Morrissey, James H
Doctoral Committee Member(s):Grosman, Claudio; Kranz, David M; Olsen, Gary J
Department / Program:Biochemistry
Discipline:Biochemistry
Degree Granting Institution:University of Illinois at Urbana-Champaign
Degree:Ph.D.
Genre:Dissertation
Subject(s):polyphosphate
polyP
Nudt2
Nudt3
pNP
p-nitrophenol
4-nitrophenol
Nudix
phosphoanhydride
phosphoester
chromogenic substrate
fluorogenic substrate
phosphatase substrate
spectrophotometry
DAPI
4-methylumbeliferone
dinucleotide polyphosphate
diadenosine polyphosphate
phosphate
phosphatase
phosphoanhydrase
phosphodiesterase
alkaline phosphatase
acid phosphatase
endopolyphosphatase
exopolyphosphatase
calcium
acidocalcisome
volutin granule
azurophilic granule
chitin
Chs2
ScChs2
cell wall
polysaccharide
oligosaccharide
calcium phosphate sink
ocean phosphate
serum
GlcNAc
UDP-GlcNAc
platelet
clotting
blood
coagulation
coagulation cascade
processive
polymer
TLC
carbohydrate
primer
TCA-insoluble
YO1531
inositol
inositol phosphate
ApnA
Ap3A
Ap4A
Ap6A
NpnA
diadenosine tetraphosphate
diadenosine pentaphosphate
diadenosine hexaphosphate
phytic acid
metaphosphate
phosphate melt
inositol hexaphosphate
5-phosphoribosyl 1-pyrophosphate
PNPP
p-nitrophenol phosphate
DIPP
diphosphoinositol polyphosphate phosphohydrolase
Ddp1p
YOR163w
Saccharomyces cerevisiae
GlcN
GalNAc
ManNAc
Glc
GlcNAc2
GlcNAc3
N-propanoylglucosamine
GlcNPr
N-butanoylglucosamine
GlcNBu
N-glycolylglucosamine
GlcNGc
Abstract:The goal of my research has been to investigate the mechanisms by which inorganic polyphosphate (polyP) is degraded in vivo. PolyP is a linear chain of phosphate moieties linked through high-energy phosphoanhydride bonds and can range in size from three-phosphates-long to thousands. Naturally, these molecular chains are stored in cells within organelles called acidocalcisomes—metal-filled acidic cellular compartments. Human platelets, for example, store polyP of 60-100mer in dense granules (a type of acidocalcisome); upon activation, platelets release the procoagulant molecule into blood. Once in blood, polyP presumably decays. Previous studies indicated that polyP incubated in human serum has a half-life of about 90 minutes. Our hypothesis, therefore, was that native serum endo- and exopolyphosphatases degraded polyP. To study the degradation of polyP in vivo and identify putative polyphosphatases, we needed to develop a high-throughput method for measuring polyphosphatase activity. The study of polyP long has been hampered by the paucity of high-throughput methods for detecting and quantifying rates of polyphosphate degradation. Adapting carboxylic acid chemistry to esterify polyP's terminal phosphates with alcohols, we created chromogenic and fluorogenic polyphosphatase substrates that allow one to measure real-time activity of either endo- or exopolyphosphatases, depending upon assay configuration. We confirmed the products' identities through 1D and 2D 31P, 1H, and 13C NMR analyses. In proof-of-principle experiments we showed that the substrates were useful for spectrophotometrically monitoring the activities of commercially-available polyphosphatases in real time. Utilizing these substrates, we identified a new function for the clinically significant enzyme, Nudt2. The chemistry and substrates developed would be not only applicable for synthetic and clinical applications, but also for identifying putative human sera polyphosphatases. In attempting to identify the putative sera polyphosphatases, we found that the majority of polyphosphate degradation in serum was metal-mediated, rather than enzyme-mediated. Using a 96-well plate format assay, we tested a variety of conditions. PolyP degradation in serum had the following characteristics: resistance to canonical phosphatase inhibitors, resistance to heat, a calcium dependency, and a pH dependency. Our results show that at physiological pH (7.4) and above, calcium concentrations near and above those in serum (x ≥ 1.25 mM) can hydrolyze polyphosphate chains. Most unicellular organisms and many human cells (e.g. mast cells, platelets) store polyP with divalent metals in acidocalcisomes, and the dissolution of acidocalcisome polyP in response to alkaline stress occurs in various organisms and in situ marine mineral sedimentation. Until now, the reigning hypothesis has been that this polyphosphate degradation is enzyme-catalyzed. Our findings raise a question, though, as to whether or not calcium ions alone may be sufficient to facilitate polyP degradation during times of cellular alkaline stress. If so, then the acidification of acidocalcisomes may be an evolutionary cellular adaption that prevents calcium-mediated polyphosphate degradation during times of non-stress and additional work may be needed to investigate this possibility.
Issue Date:2016-04-22
Type:Thesis
URI:http://hdl.handle.net/2142/91597
Rights Information:Copyright 2016 Carleigh Fredda Frances Hebbard
Date Available in IDEALS:2016-09-09
Date Deposited:2016-05


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