Introduction
Inorganic pyrophosphatases (PPases) catalyze the hydrolysis of one
molecule of pyrophosphate (PPi) to two molecules of phosphates (Pi).
PPases are ubiquitous enzymes present in all cell types. PPases include
membrane-bound V-PPases (vacuolar H+-translocating PPases) and soluble
PPases. V-PPases use pyrophosphate (instead of ATP) as an energy source
to transport H+ or Na+ across membranes in plants and some bacteria,
archaea, and parasitic protists [1, 2]. Soluble PPases are divided
into three families that differ in their sequences and structures
[3-5]. PPases from families II and III are only found in
prokaryotes, whereas Family I PPases exist in all kingdoms of life.
Family I PPases are single-domain, Mg2+ dependent
enzymes that exist as hexamers in prokaryotes and dimers in eukaryotes.
PPi is a metabolic product that is generated by many biosynthetic
reactions for the synthesis of various important biomolecules, including
proteins, nucleic acids, polysaccharides, and fatty acids [6]. The
conversion of PPi to Pi by soluble PPases is an essential cellular
process. On the one hand, the reaction is critical for maintaining
cellular PPi homeostasis; on the other hand, the reaction provides a
thermodynamic pull for the PPi-generating biosynthetic reactions because
enzymatic hydrolysis of PPi is highly
exergonic.
Soluble PPases are essential enzymes for growth and development. In E.
coli, the ppa gene for PPase is essential for cell growth [7]. In
fermenting yeast, PPase defects lead to cell cycle arrest and autophagic
cell death [8]. In C. elegans, the PPase enzyme PYP-1 is required
for larval development and intestinal function [9, 10]. In mammals,
the PPase enzyme PPA1 has been reported to have the potential to
regulate neurite growth in mouse neuroblastoma cells [11], to induce
type I collagen synthesis and stimulate calcification by osteoblasts
[12]. Increased expression and activity of cytosolic PPA1 in the
liver of rat and mouse has been correlated with aging [13, 14].
Human possess two Family I PPases, PPA1 (289 aa) and PPA2 (305 aa)
[15]. PPA2 is localized to the mitochondrion. The two human PPases
share ~70% homology. PPA1 is a ubiquitous enzyme found
in all tissues. It is found that PPA1 acts largely as a housekeeping
gene [15], presumably due to its essential role in energy
metabolism. More recently, the role
of PPA1 in tumorigenesis has emerged [16-19]. Upregulated expression
of PPA1 has been linked to many human malignant tumors including
colorectal cancer [20-22], diffuse large B-cell lymphoma (DLBCL)
[23], lung adenocarcinoma [17, 24], prostate cancer [25],
hepatocellular carcinoma [19], breast cancer [26], gastric
cancer[27, 28], intrahepatic cholangiocarcinoma (ICC) [29], and
ovarian cancer [24, 30, 31]. In laryngeal squamous cell carcinoma
(LSCC), metastases tumors showed an intense increase of PPA1 compared to
primary tumors [32]. These data indicate that PPA1 protein is a
potential prognostic biomarker for certain cancers.
The exact mechanism for the involvement of PPA1 overexpression in
tumorigenesis is not fully understood. Dysregulated metabolism is an
important hallmark of cancer cells [33]. Metabolic alterations
caused by PPA1 overexpression may provide the extra energy and
biomaterials for the cancer cells to maintain their high proliferation
rate. In addition, it is found that PPA1 can directly dephosphorylate
phosphorylated JNK1 (c-Jun N-terminal kinase) [22]. Functioning as a
JNK phosphatase, PPA1 may regulate the activities of downstream
effectors (including p53, β –catenin, Bcl-2, and Caspase-3) that
control cell proliferation and apoptosis. In consistence with this
speculated mechanism, we previously found that PPA1 overexpression in
epithelial ovarian carcinoma (EOC) promoted the dephosphorylation and
translocation of β-catenin [31]. In another study on EOC [30],
it was found that PPA1 overexpression caused the decrease in p53 and
increase in β-catenin expression level. Conversely, PPA1 knockdown led
to the increase of p53 and decrease of β-catenin level.
While PPA1 overexpression enhances migration, invasion and proliferation
of cancer cells [27], PPA1 knockdown (by RNAi mediated silencing) is
shown to induce apoptosis and decrease proliferation in various cancer
cells [22-24, 30, 31]. Results from these studies suggest that
strategies for PPA1 down-regulation may have therapeutic potential for
the treatment of certain cancers. PPA1 down-regulation might be achieved
by mRNA knockdown through RNAi or RNA-targeting CRISPR systems.
Alternatively, the PPA1 protein might represent a valuable target for
the development of specific small-molecule inhibitors as anti-cancer
drugs. Efforts had been made to target PPases from pathogenic organisms
for small-molecule drug development. Anti-PPase small molecules were
shown to have promising antibiotic activity against drug resistant
strains of Staphylococcus aureus [34]. Small molecule inhibitors of
the VSP1 (vacuolar soluble pyrophosphatase 1) protein from Trypanosoma
brucei, the causative agent of African trypanosomiasis (HAT), provided a
40% protection from death in a mouse model of T. brucei infection
[35]. Inhibition of PPase activity could also be achieved by
targeting allosteric sites, as demonstrated by the discovery of
allosteric and selective inhibitors of PPase from Mycobacterium
tuberculosis [36].
In spite of the biological and physiopathological importance of human
PPA1, there is no detailed study on the structure and catalytic
mechanisms of this enzyme. Extensive structural and biochemical studies
had been reported on Family I PPases from Escherichia coli (E-PPase)
[37-39] and Saccharomyces cerevisiae (Y-PPase) [40-45]. X-ray
crystallographic studies had also been carried out on soluble PPases
from other species, including Thermus thermophilus (T-PPase) [46],
Pyrococcus
horikoshii (Pho-PPase) [47], human parasites Plasmodium falciparum
(Pf-PPase) and Toxoplasma gondii (Tg-PPase) [48], Mycobacterium
tuberculosis (Mt-PPase) [36, 49], Trypanosoma brucei brucei (also
called vacuolar soluble protein TbbVSP1) [50], and S. japonicum
(Sj-PPase, not published).
In this paper, we present the crystal structure of human PPA1. We also
carried out modeling studies of PPA1 in complex with JNK derived
peptides containing phosphotyrosine and phosphothreonine residues.